Silvio Silvério da Silva

Anuj Kumar Chandel Editors

Biofuels in
Brazil
Fundamental Aspects, Recent
Developments, and Future Perspectives
Biofuels in Brazil
Silvio Silvério da Silva •

Anuj Kumar Chandel

Editors

Biofuels in Brazil
Fundamental Aspects, Recent Developments,
and Future Perspectives

123
Editors
Silvio Silvério da Silva
Anuj Kumar Chandel
Department of Biotechnology,
Engineering School of Lorena
University of São Paulo
Lorena, São Paulo
Brazil

ISBN 978-3-319-05019-5 ISBN 978-3-319-05020-1 (eBook)

DOI 10.1007/978-3-319-05020-1
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The One who gives life the entire universe,
Is Immortal: He is the One Lord of all.

Guru Nanak Dev

Foreword

Biofuels in Brazil: Fundamental Aspects, Recent Developments, and Future

Perspectives has been compiled to cater the needs of graduate and post-graduate
students, researchers in academia and industries, managerial organizations, biotech
business, policy makers, policy analysts and readers at large. This book apprises
the technical updates on bioenergy research of Brazil eventually discussing the
system biology based approached to manufacture bioenergy crops, biomass pre-
treatment, improved microbial strains for the fermentation of sugar solution super
enzyme titers, techno-economic analysis of biofuels production, technical aspects
of biodiesel and other renewable hydrocarbons production, feedstock availability
in Brazil and biofuel policy issues in Brazil.
Among the sustainable biofuels, bioethanol is the most promising and
sustainable alternative to gasoline in the context of Brazil. Brazil has played a key
role in implementation of bioethanol as an alternative of gasoline. Sugarcane juice
is the primary source of ethanol production in Brazil. However, in future definitely
the second generation feedstock like biomass residues need to be taken into
consideration vigorously. Brazilian Ministry of Science and Technology through
the Research and Projects Financing (FINEP) and NIST Bioethanol development
via CNPq, FAPESP has developed a network of more than 20 research institutions
working on the promotion of biofuels production in Brazil. The success rate of
bioethanol policy from Brazil can be followed by other developing nations such as
India, China and others for reducing their dependency on oil import thus saving
foreign exchange reserves.
This book also disseminates the key information on life cycle assessment of
biofuels, arable land and climate changes after implementing bioenergy options.
Essentially, this book focuses on the Brazil Government policies for the promotion
of biofuels in the country. The success of bioenergy program in Brazil is a learning
example for other countries particularly agricultural rich countries for imple-
menting the affordable policies for the commercialization of biofuels. Overall, this
book is a special collection of quality chapters written by the peers of field updating
research/analysis on bioenergy options in Brazil. I am confident in forwarding this

vii
viii Foreword

book to the worldwide readers to learn about the biofuels development in Brazil,
technical aspects of biofuels production and other basic ingredients of biofuel
research in the Brazilian context.

Henrique Duque de Miranda Chaves Filho

Vice Chancellor
Federal University of Juiz de Fora (UFJF)
Juiz de Fora, MG, Brazil
Preface

Life is energy. A sustainable supply of energy is required for the overall human
development. The Sun is the primary source of energy on Earth. Fossil energy, the
major source of energy (80 % of current world power consumption), is the result of
energy entrapped by plants through photosynthesis in past eras. Fossil energy is a
finite source and is likely to be exhausted sooner rather than later due to the fast
pace of urbanization and increased use worldwide. Regular price hikes and
environmental damage caused by excessive use of fossil fuels are the major
alarming concerns. Owing to these geopolitical factors, the time is now to look out
intensively for alternatives to fossil fuels. Renewable or bioenergy is the suitable
answer as it can be produced directly from natural resources.
Bioenergy sources are diverse and broad in range. It can be categorized mainly
as solar, wind, hydrothermal, and biomass-derived. Amongst all the renewable
resources, biomass is one of the most promising answers, particularly for trans-
portation fuels. Brazil is the fourth largest country in the world and is largely
blessed by nature for appropriate fertile land, rain, light, and water. Brazil is
representative of renewable energy program in the world and ranks second in
ethanol production. The government of Brazil has taken appreciable initiatives in
order to make the bioenergy program successful. Sugarcane-juice-derived ethanol,
so-called first-generation ethanol, is the principle component of bioenergy in
Brazil. However, cellulosic ethanol or second-generation ethanol is a prospective
energy source in the near future. There are numerous research programs nation-
wide to make cellulosic ethanol a reality in the near future with financial help from
the Ministry of Science and Technology, Government of Brazil. Many countries,
particularly the developing world, can learn from the success stories of the Bra-
zilian Bioenergy program and implement the policies for their energy security and
betterment of socioeconomic status.
This book aims to disseminate the current advances in the bioenergy program of
Brazil starting from feedstock analysis, availability, chemical composition, tech-
nical aspects, technoeconomic analysis, and government policies. This book is a
fine and unique collection of 19 book chapters written by specialists in the related
research area, who afford critical insights into several topics, review of current
research, and discuss future progress in this area. Broadly, this book intends to

ix
x Preface

provide critical insight and background research analysis on raw materials, pro-
cessing, synthesis, recovery, and application as energy sources, comparative
account on major alternative energy producing countries in addition to feedstock
variation and analysis. In regard to technical updates, this book highlights the
system biology-based approaches for the development of new energy feedstocks,
microorganisms, and enzyme titers. Furthermore, recent technical progress made
toward pretreatment, enzymatic hydrolysis of biomass, and the fermentation of
sugars into ethanol is also mentioned. Similarly, these aspects have also been
discussed for the production of other biofules such as biohydrogen, biodiesel, or
bio oil. An assessment on technological development for capturing, regeneration,
and storage of solar energy, wind energy, and turbines is also made along with
future directions. Comparative technoeconomic and life cycle analysis of various
biofuels have been discussed in the last section along with the commercialization
of cellulosic ethanol and other by-products. Additionally, initiatives taken by the
Brazil Government for implementing effective bioenergy policy for the promotion
of biofuels through research, commercialization, and private investment support
have been apprised to the readers. Apart from researchers and graduate students of
microbial biotechnology, and chemical and industrial engineers, this book will
assist the business community and policy analysts who deal with geopolitical
analysis of bio-based products, bioenergy, and their marketing.
We greatly appreciate the scholarly contribution of authors who added highly
informative chapters in this book. We thank Isabel Ullmann, Hanna Hensler,
Anette Lindqvist, and the production staff of Springer Verlag, Germany for their
timely suggestions and support to publish this book. We would like to thank our
colleagues Andre Ferraz, George J. M. Rocha, M. G. A. Felipe, Adriane M.
F. Milagres, Walter Carvalho, Rita C. L. B. Rodrigues, Ines C. Roberto, and
Messias B. Silva for the completion of this book. We are grateful to FAPESP,
CNPq, CAPES, and University of São Paulo, Brazil for the financial support and
infrastructure setup. We express our sincere thanks to our families for their
unconditional support and cooperation while editing this book. Last but not least,
we welcome the reader’s suggestions to improve the future editions. We think that
Readers’ benefits are the best reward for editors, contributing authors, and
publisher.

Silvio Silvério da Silva

Anuj Kumar Chandel
Contents

1 Techno-Economic Analysis of Second-Generation Ethanol

in Brazil: Competitive, Complementary Aspects
with First-Generation Ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Anuj Kumar Chandel, Tassia Lopes Junqueira,
Edvaldo Rodrigo Morais, Vera Lucia Reis Gouveia, Otavio Cavalett,
Elmer Ccopa Rivera, Victor Coelho Geraldo, Antonio Bonomi
and Silvio Silvério da Silva

2 An Assessment of Brazilian Government Initiatives and Policies

for the Promotion of Biofuels Through Research,
Commercialization, and Private Investment Support . . . . . . . . . . 31
Luís Augusto Barbosa Cortez, Gláucia Mendes Souza,
Carlos Henrique de Brito Cruz and Rubens Maciel

3 Renewable Liquid Transportation Fuels: The Cornerstone

of the Success of Brazilian Bioenergy Program . . . . . . . . . . . . . . 61
Veronica de Araujo Bruno and Adilson Roberto Gonçalves

4 Socio-Economic and Ambient Impacts of Sugarcane

Expansion in Brazil: Effects of the Second Generation
Ethanol Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
André Luis Squarize Chagas

5 Integrated Production of 1G–2G Bioethanol and Bioelectricity

from Sugarcane: Impact of Bagasse Pretreatment Processes . . . . 85
Caliane Bastos Borba Costa, Felipe Fernando Furlan,
Antonio José Gonçalves Cruz, Raquel de Lima Camargo Giordano
and Roberto de Campos Giordano

6 Potential Biomass Resources for Cellulosic Ethanol Production

in Brazil: Availability, Feedstock Analysis, Feedstock
Composition, and Conversion Yields . . . . . . . . . . . . . . . . . . . . . . 97
Boutros F. Sarrouh, Júlio C. dos Santos,
Mário Antônio A. Cunha and Ricardo F. Branco

xi
xii Contents

7 Advances in Methods to Improve the Sugarcane Crop

as ‘‘Energy Cane’’ for Biorefinery: An Appraisal. . . . . . . . . . . . . 125
Francis Julio Fagundes Lopes and Viviane Guzzo de Carli Poelkin

8 The Essential Role of Plant Cell Wall Degrading Enzymes

in the Success of Biorefineries: Current Status
and Future Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Marcos Henrique Luciano Silveira, Matti Siika-aho,
Kristiina Kruus, Leyanis Mesa Garriga and Luiz Pereira Ramos

9 Mapping of Cell Wall Components in Lignified Biomass

as a Tool to Understand Recalcitrance . . . . . . . . . . . . . . . . . . . . 173
André Ferraz, Thales H. F. Costa, Germano Siqueira
and Adriane M. F. Milagres

10 Dilute Acid Pretreatment and Enzymatic Hydrolysis

of Sugarcane Bagasse for Ethanol Production . . . . . . . . . . . . . . . 203
Paula J. Esteves, Celso Santi Jr. and Walter Carvalho

11 Scale-up Pretreatment Studies on Sugarcane Bagasse

and Straw for Second-Generation Ethanol Production . . . . . . . . . 225
George Jackson de Moraes Rocha, Viviane Marcos Nascimento,
Vinicius Fernandes Nunes da Silva and Anuj Kumar Chandel

12 Novel Yeast Strains from Brazilian Biodiversity:

Biotechnological Applications in Lignocellulose Conversion
into Biofuels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Raquel Miranda Cadete, César Fonseca and Carlos Augusto Rosa

13 Trends in Biodiesel Production: Present Status

and Future Directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Victor H. Perez, Euripedes G. Silveira Junior, Diana C. Cubides,
Geraldo F. David, Oselys R. Justo, Maria P. P. Castro,
Marcelo S. Sthel and Heizir F. de Castro

14 Critical Technological Analysis for Enzymatic Biodiesel

Production: An Appraisal and Future Directions. . . . . . . . . . . . . 303
Marcelle Alves Farias and Maria Alice Z. Coelho

15 Critical Analysis of Feedstock Availability and Composition,

and New Potential Resources for Biodiesel Production
in Brazil. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
Betania F. Quirino, Bruno S. A. F. Brasil, Bruno G. Laviola,
Simone Mendonça and João R. M. Almeida
Contents xiii

16 Techno-Economic and Life Cycle Analysis of Biodiesel

Production: Perception of Land Use, Climate Change,
and Sustainability Measurements . . . . . . . . . . . . . . . . . . . . . . . . 351
Donato A. G. Aranda, Cecilia M. Soares and Neyda Om Tapanes

17 Microalgal Feedstock for Bioenergy: Opportunities

and Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
Cristiano Eduardo Rodrigues Reis, Mateus de Souza Amaral,
Carla Cristina Almeida Loures, Patrícia Caroline Molgero da Rós,
Bo Hu, Hélcio José Izário Filho, Heizir Ferreira de Castro,
Sônia Maria Flores Gianesella and Messias Borges Silva

18 Technological Advancements in Biohydrogen Production

and Bagasse Gasification Process in the Sugarcane Industry
with Regard to Brazilian Conditions . . . . . . . . . . . . . . . . . . . . . . 393
Jose Luz Silveira, Celso Eduardo Tuna, Daniel Travieso Pedroso,
Marcio Evaristo da Silva, Einara Blanco Machin,
Lúcia Bollini Braga and Valdisley José Martinelli

19 Nonconventional Renewable Sources in Brazil

and Their Impact on the Success of Bioenergy . . . . . . . . . . . . . . 413
Luís Cláudio Oliveira-Lopes and Cláudio H. Ferreira da Silva
Editors Biography

Prof. Silvio Silvério da Silva is a professor at the Department of Biotechnology,

Engineering School of Lorena, University of São Paulo, São Paulo, Brazil. He
completed his doctorate in Biochemical and Pharmaceutical Technology from the
University of São Paulo (USP) and Gesellschaft Fuer Biotechnologishe Forshung
(GBF), Germany in 1994. Prof. Silva offers consulting services to various scien-
tific journals, ad hoc institutions, and biotechnological industries. He has published
more than 130 papers in peer reviewed international journals and presented more
than 455 papers in international conference proceedings. He has also 18 book
chapters to his credit. He has recorded two patents on technological processes
for xylitol production. Prof. Silva has guided 4 post-doctoral fellows, 11 doctoral
students, 26 masters’ dissertations, and 58 scientific initiation students in the area
of Applied Microbiology, Biochemical Engineering, and Biotechnology. He has
successfully completed 19 research projects funded by Brazilian Government and
private funding agencies, including International Cooperative Projects. He also
received important awards in biotechnology field for his contribution. His research
area is Micro-biotechnology harnessing the potential of lignocellulosic feedstock
for the production of Xylitol and Bioethanol since the last 25 years. He has visited
several international research institutes from various countries for exchange of
scientific knowledge on the various aspects of xylitol and bioethanol production.
Dr. Anuj Kumar Chandel completed his doctorate in 2009 from Jawaharlal
Nehru Technological University, Hyderabad, India. After his master’s from the
Indian Institute of Technology Roorkee in 2000, Anuj joined Dalas Biotech Ltd.,
Bhiwadi, for the large-scale production of penicillin acylase and antibiotic inter-
mediates. Subsequently, he worked at University of Delhi in a research project
funded by the Department of Biotechnology (DBT), Government of India. Later he
joined Celestial Labs Ltd., Hyderabad as a research associate. After this, he did
post-doctoral studies at University of Stellenbosch, South Africa. Anuj worked as
a post-doctoral fellow at Engineering School of Lorena, University of São Paulo,
Brazil on biofuels development in a thematic project funded by Bioen-FAPESP.

xv
xvi Editors Biography

Currently, Anuj is working at the Department of Chemical Engineering, University

of Arkansas, Fayetteville, Arkansas, USA. He is the author of 3 books on
D-xylitol, lignocellulose degradation and Brazilian Bioenergy development. Anuj
has published 51 articles in peer-reviewed journals and 16 book chapters. He has
also recorded one Brazilian patent on biomass pretreatment.
Chapter 1
Techno-Economic Analysis
of Second-Generation Ethanol
in Brazil: Competitive, Complementary
Aspects with First-Generation Ethanol

Anuj Kumar Chandel, Tassia Lopes Junqueira, Edvaldo Rodrigo Morais,

Vera Lucia Reis Gouveia, Otavio Cavalett, Elmer Ccopa Rivera,
Victor Coelho Geraldo, Antonio Bonomi and Silvio Silvério da Silva

Abstract Brazil achieved important success in the implementation of ethanol as a

reality renewable energy source after the inception of the National Alcohol Pro-
gram (PROÁLCOOL) in 1970. Today, ethanol produced from sugarcane replaces
almost 50 % of gasoline in Brazil. More than 448 bioethanol production (first-
generation ethanol) units are functional, which fulfill the 25 % ethanol blending to
gasoline that eventually reduces the import of 550 million oil barrels improving
the socioeconomic status and saving foreign exchange reserves. Brazil has more
than 80 % of its light vehicles running on bioethanol, reducing greenhouse gas
emissions. At present, this demand for ethanol is being met through first-genera-
tion (1G) ethanol which is directly produced from sugarcane juice and molasses.
However, significant research in bioenergy in the last two decades has shown the
possibilities of commercialization of second-generation (2G) ethanol, which can
be produced from sugarcane bagasse (SB) and straw (SS), complementing 1G
ethanol. Nevertheless, both the residues (SB and SS) are an excellent source for
cogeneration of heat and power (CHP) in sugarcane processing units. Process
simulation studies have provided additional source of information on the overall
use of sugarcane for ethanol production and CHP. For the evaluation of the fullest
utilization of sugarcane and its by-products, CTBE (Brazilian Bioethanol Science

A. Kumar Chandel (&)  S. S. da Silva (&)

Department of Biotechnology, Engineering School of Lorena, University of São Paulo,
Estrada Municipal do Campinho, Lorena, SP 12602-810, Brazil
e-mail: anuj.kumar.chandel@gmail.com
S. S. da Silva
e-mail: silvio@debiq.eel.usp.br
T. L. Junqueira  E. R. Morais  V. L. R. Gouveia  O. Cavalett  E. C. Rivera 
V. C. Geraldo  A. Bonomi
Laboratório Nacional de Ciência e Tecnologia do Bioetanol–CTBE, P. O. Box 6170,
Campinas, SP 13083-970, Brazil

S. S. da Silva and A. K. Chandel (eds.), Biofuels in Brazil, 1

DOI: 10.1007/978-3-319-05020-1_1,  Springer International Publishing Switzerland 2014
2 A. K. Chandel et al.

and Technology Laboratory) has developed the Virtual Sugarcane Biorefinery

(VSB), a comprehensive assessment framework to evaluate a sustainability
standpoint (economic, environmental, and social), different biorefinery alterna-
tives. This chapter reviews the important insights made into bioethanol production
in Brazil. Technical configuration for 1G and 2G ethanol production and sus-
tainability of ethanol (economic and environmental assessment) have also been
discussed.

  
Keywords Brazilian bioenergy Fuel ethanol Sugarcane Sugarcane residues 
 
Techno-economic analysis Environmental assessment Bioelectricity Second- 
generation ethanol

1.1 Introduction

Energy encompasses all the important features of the overall growth of human
development. As per the human development index (HDI) set by the United
Nations, nearly 4 kW per capita power consumption is required (Dale and Ong
2012). Developed countries reach this HDI by heavy usage of fossil energy; China,
India, and other developing countries are approaching their increasing energy
needs also with fossil fuels, whereas Brazil is the only exception, depending
heavily on renewable energy. In the present scenario, fossil energy is the major
source of energy (80 % of the world power consumption) in the form of oil
(35.03 %), coal (24.54 %), and gas (20.44 %) (Goldemberg 2007). The use of
fossil energy is considered as one of the most important man-made factor
impacting on global economy and weather (Vertès et al. 2006). It is a widely
accepted fact that fossil energy sources are finite and generally exported from
politically unstable nations. Moreover, continuous huge demand of gasoline, low
and expensive recovery yields, and oil spills are making the situation worse. The
limited sources of fossil fuels in the world may not fulfill the increased demand for
gasoline in the future. Already, experts have claimed that ‘‘peak-oil’’ (conferring
the maximum rate of oil production) has arrived and the oil production rate after
this point must decline (Kerr et al. 2011).
Keeping all the aforementioned points in view, the momentum is shifting toward
the implementation of renewable energy sources. Biomass derived fuels have the
potential to create a transition in the global economy from fossil fuel to a renewable
fuel economy, however, it needs intensive technological and multidisciplinary
efforts (Vertès et al. 2006; Yuan et al. 2008; Herrera 2006; Ohlrogge et al. 2009).
Among the renewable energy sources (constituting around 13.61 %), biomass
derived fuels, particularly ‘‘bioethanol’’ is gaining significant importance due to its
inherent properties. Ethanol produced from cane juice (in Brazil) and corn starch (in
USA) is already an established energy commodity. Brazil and USA are the two
major countries that have successfully implemented bioethanol as an alternative
1 Techno-Economic Analysis of Second-Generation Ethanol in Brazil 3

energy source and have shown the signs of global energy commodity making
ethanol fully competitive with gasoline and suitable for replication in other coun-
tries like India, China, etc. (Goldemberg 2007). Approximately [37.85 billion
liters of ethanol (today this figure has more than doubled) is produced globally per
year from corn, sugarcane, and sugar beet through fully mature and well-estab-
lished processes (Rass-Hansen et al. 2007).

1.2 Brazil and Bioethanol

Sugarcane juice derived ethanol has replaced nearly 50 % of gasoline consumption

in Brazil. Ethanol production and its implementation have achieved unprecedented
success in Brazil as a fuel and gasoline additive. Sugarcane productivity and
technical advancements led the ethanol production increase from 0.6 billion liters
in 1975/1976 to 24 billion liter in 2012/2013 (Goldemberg 2013; Canilha et al.
2013).
In 2012/2013, it is expected that Brazilian sugar–alcohol mills will process
more than 602 million tons of sugarcane, accounting for the production of roughly
39 million tons of sugar and 24 billion liters of ethanol. Experimentally, each ton
of processed sugarcane generates approximately 270–280 kg of bagasse and
140 kg of straw (Canilha et al. 2013). Therefore, taking this value into account, it
can be estimated that Brazilian mills will produce around 163–169 million tons of
sugarcane bagasse and 84 million tons of straw in the 2012/2013 harvest (Canilha
et al. 2013). In addition to sugarcane juice derived ethanol (first-generation), the
exploration of lignocellulosic residues of sugarcane (bagasse and straw) also has a
great potential for ethanol production (Chandel et al. 2012a; Dias et al. 2012a, b).
In Brazil, tremendous research efforts are on the way to develop a robust tech-
nological setup for the cellulosic ethanol production at commercial scale. Sugar-
cane is a primary source of renewable energy in Brazil and can be considered as a
model feedstock for bioethanol production. The net energy balance (ratio of
energy contained in a given volume of ethanol divided by the fossil energy
required for its production) for ethanol production from sugarcane is very high
(8.2–10) compared with other feedstock sources such as corn (1.3), sugar beet, and
wood (approximately 2) (Goldemberg 2008).
The Brazilian National Alcohol Program (PROÁLCOOL) was launched in
1974 to decrease gasoline consumption and thus reduce oil imports. Since then, it
has gained significant success and today there is no more government subsidy to
the producers (Goldemberg 2008). Nowadays, in the Brazilian automobile sector,
more than 90 % of new cars are flex-fueled driven which can run on gasoline as
well as on ethanol. Since 1976, ethanol saved 1.51 billion barrels of gasoline
correspondingly saving 75 billion US$ (BNDES and CGEE 2008). The successful
Brazilian ethanol program can be a learning curve for other countries. Table 1.1
shows the data on ethanol production in various countries and the projected
demand per year of ethanol up to 2020/2022.
4 A. K. Chandel et al.

Table 1.1 Profile of gasoline consumption, ethanol production, and futuristic ethanol demand as
per the mandates up to 2020/2022
Country Gasoline consumption Ethanol production Ethanol demand (on the
in 2007 (billion liters)a in 2008 (billion basis of present mandates
liters)b up to 2020/2022)
USA 530 34 136
European Union 148 2.3 8.51
China 54 1.9 5.4
Japan 60 0.1 1.8
Canada 39 0.9 1.95
United Kingdom 26 0.03 1.3
Australia 20 0.075 2.0
Brazil 25.2 27 19.6
South Africa 11.3 0.12 0.9
India 13.6 0.3 0.68
Thailand 7.2 0.3 0.7
Argentina 5.0 0.2 0.25
The Philippines 5.1 0.08 0.26
Total 943.2 67.3 178.7
Source a OECD/IEA (2010)
b
REN21 (2009) and Goldemberg (2013)

1.3 Critical Analysis of Technological Routes for Cellulosic

Ethanol Production

The production of bioethanol from lignocelullosic materials is assumed to present

the largest potential among the possible alternatives to increase bioethanol pro-
duction in the world without compromising food security, even though it is not yet
a reality in an industrial scale (Kazi et al. 2010; Dias et al. 2012a). Lignocellulosic
materials are abundant and cheap, do not compete with food crops (Ojeda et al.
2011; Dias et al. 2012a), and consequently, have larger potential to be used as
feedstock for the production of sustainable biofuels (Dias et al. 2012a). Ethanol
production from lignocellulosic biomass usually contains four major unit opera-
tions: (1) pretreatment, (2) enzymatic hydrolysis, (3) fermentation of sugars into
ethanol, and (4) ethanol recovery.
The pretreatment is perhaps the single most crucial step as it has a large impact
on all the other steps in the process (Galbe and Zacchi 2012). It is responsible for
removing lignin or hemicellulose from the lignocellulosic material, and allows
cellulose accessibility, enhancing the surface area of substrates for improved
sugars recovery after enzymatic hydrolysis. An ideal pretreatment must meet the
following requirements: minimum chemical requirement, low residence time, low
investment cost, high amount of sugars recovery with less degradation of sugars or
the ability to subsequently form sugars by hydrolysis, and minimum formation of
inhibitory by-products (Kumar et al. 2009; Rocha et al. 2012). The pretreatment
1 Techno-Economic Analysis of Second-Generation Ethanol in Brazil 5

process can be categorized into four major categories: physical, physico-chemical,

chemical, and biological. Each type of pretreatment has inherent specificity in
terms of mechanistic application on cell wall components with the applied con-
ditions. Physicochemical and chemical pretreatment is fast, effective, but non-
specific, and generates hemicellulosic derived inhibitors. Biological pretreatment
methods are used for delignification but hampers by slow reaction rates and
nonselectivity. The pretreated material needs to be submitted to enzymatic
hydrolysis for the utmost sugars recovery (Agbor et al. 2011). The extent of
enzymatic hydrolysis depends on lignin removal and the employed hydrolysis
conditions. Hemicellulosic hydrolysates obtained after acid catalyzed reactions
generally have cell wall derived inhibitors, i.e., furans, furfurals, phenolics, weak
acids, among others. These inhibitors affect the efficiency of microorganisms
employed in fermentation process leading to poor ethanol yields. It is essential to
eliminate these inhibitors prior to microbial fermentation in order to obtain the
desired ethanol yields. Several detoxification methods like evaporation, calcium
hydroxide overliming, use of membranes, ion-exchange resins, activated charcoal,
and enzymatic detoxification using laccases are in practice to overcome these
inhibitors (Chandel et al. 2013a). Simultaneous detoxification of hydolysates and
fermentation (SDF) is also possible for ethanol production using two different
microorganisms (microorganism eliminating inhibitors + microorganism for eth-
anol production).
There are four configuration processes to produce ethanol from lignocellulosic
biomass: separate hydrolysis and fermentation (SHF), simultaneous saccharifica-
tion and fermentation (SSF), simultaneous saccharification and co-fermentation
(SSCF), and consolidated bioprocessing (CBP) (van Zyl et al. 2011). The most
common process is separate (or sequential) hydrolysis and fermentation (SHF),
where hydrolysis of pretreated lignocellulosic material is done first and the
resultant sugar solution is fermented separately into ethanol in different vessel/
reactor. SHF is a lengthy process but has shown optimum sugars production fol-
lowed by their conversion reaching the desired ethanol yields. Both the processes
can be carried out employing the most appropriate conditions. After enzymatic
hydrolysis, the solid material can be used for cogeneration of heat and electricity.
SSF (simultaneous saccharification and fermentation) or SSCF (simultaneous
saccharification and co-fermentation) are other configuration processes where the
enzymatic hydrolysis of pretreated lignocellulosic material and the fermentation of
released sugars or mixture of sugars (pentose + hexose) is carried out simulta-
neously. SSF/SSCF has shown great advantages over SHF in the terms of reducing
processing time and process complexities. However, the temperature difference in
both the reactions is of concern (Olofsson et al. 2008). Enzymatic hydrolysis
usually shows the best results at 50 C, while ethanol fermentation is done at
30 C. Therefore, pre-hydrolysis at 50 C can be inducted to initiate the hydrolysis
for some time followed by the execution of fermentation reaction. To obtain the
maximum ethanol yield, thermotolerant yeast or ethanol producers could be more
relevant as they can grow and produce ethanol at the hydrolysis temperature
(\50 C). Furthermore, risk of contamination can be avoided using thermo
6 A. K. Chandel et al.

Step Key reactions

Pretreatment Cellulase Hydrolysis Hexose Pentose

SHF
production fermentation fermentation

SSF Pretreatment Cellulase Hydrolysis + hexose Pentose

production fermentation fermentation

Cellulase Hydrolysis + fermentation (hexose +

SSCF Pretreatment production pentose sugars)

Pretreatment Hydrolysis + cellulase production + fermentation (hexose +

CBP
pentose sugars)

IBP Pretreatment + cellulase production + hydrolysis + fermentation

(hexose + pentose sugars)

Fig. 1.1 Summary of technical routes for ethanol production from biomass under various
process configurations. Each box represents the specific reaction performed in sequential order.
SHF separate hydrolysis and fermentation, SSF simultaneous saccharification and fermentation,
SSCF simultaneous saccharification and co-fermentation, CBP consolidated bioprocessing, IBP
integrated bioprocessing

tolerant microorganism in SSF. Another advantage of SSF/SSCF configuration is

to avoid the enzyme inhibition by the released glucose as it is simultaneously
converted into ethanol by the microorganism. Moreover, capital cost investment
and the processing time could be minimized by employing SSF/SSCF (Olofsson
et al. 2008). The latest development in process configuration is CBP (consolidated
bioprocessing), wherein the enzyme production and hydrolysis of pretreated lig-
nocellulosic material followed by the fermentation of sugars can be performed in a
single reactor (Olson et al. 2012). The key difference between CBP and the other
strategies of biomass processing is that only one microbial community is employed
both for the production of cellulases and fermentation (Cardona and Sánchez
2007).
Realizing the importance of process integration, IBP (integrated bioprocessing)
could provide an important breakthrough in developing an economic and sus-
tainable platform for cellulosic ethanol production. IBP includes the microbial
assisted pretreatment of biomass followed by enzyme recovery and delignified
biomass saccharification coupled with microbial conversion of released sugars into
ethanol simultaneously in a single reactor (Chandel et al. 2013b). Nevertheless,
ethanol production through IBP has not been tried as yet. In IBP, there is
involvement of at least more than one microorganism (one microorganism for
biodelignification and another microorganism for ethanol production). The
recovered sugars solution from pretreated lignocellulosic biomass can be used for
1 Techno-Economic Analysis of Second-Generation Ethanol in Brazil 7

ethanol production via modified fermentation strategies like fed-batch, recycling

of immobilized cells in continuous fermentation, and semi-continuous processing.
Figure 1.1 shows an overview of the technical configurations required in each
processing routes for 2G ethanol production.

1.4 Process Simulation and Co-products Utilization

Process simulation using computational tools have been increasingly used to

evaluate biorefinery configurations, since it allows the integration of process steps,
technologies, and routes, thus providing critical information to assess technical
feasibility, detecting process bottlenecks and potential advantages. In this context,
CTBE (Brazilian Bioethanol Science and Technology Laboratory), one of the
national laboratories of the Brazilian Center of Research in Energy and Materials
(CNPEM) developed a comprehensive tool—VSB (Virtual Sugarcane Biorefin-
ery)—based on simulation platforms for the evaluation of different technologies
through assessment of their sustainability indicators (economical, environmental,
and social) (CTBE 2012).
The integration of 1G (from sugarcane juice) and 2G (from bagasse and straw)
was assessed using the VSB. The main process configurations and parameters
adopted in the construction of the VSB are described in the following sections.

1.4.1 Process Simulation for 1G Ethanol Production

According to BNDES and CGEE (2008), approximately 70 % of the sugarcane

processing units in Brazil are annexed plants. First-generation ethanol production
from sugarcane takes place in autonomous distilleries or annexed plants. Auton-
omous distilleries produce only ethanol. In an annexed plant, a fraction of the
sugarcane juice is diverted for sugar production and the remaining fraction along
with the molasses (residual solution of sugars that came from sucrose crystalli-
zation) is used for ethanol production. Usually, the annexed plant operates using
half of sugarcane juice for sugar production. Part of the reason for the success of
ethanol production in Brazil is the flexibility of annexed plants to produce more
ethanol or more sugar, depending on market demands.
The sugarcane processing facility is self-sufficient on its energy consumption:
all the thermal and electric energy required for the production process is produced
in combined heat and power generation (CHP) systems using bagasse as a fuel. If
sugarcane straw is recovered from the field, it may also be used as a fuel to
increase energy generation. A scheme of the sugar, ethanol, and electricity pro-
duction process from sugarcane is shown in Fig. 1.2. In an autonomous distillery,
the unit operations related to the sugar production (left side of Fig. 1.2) is not
included in the sugarcane mill.
8 A. K. Chandel et al.

Sugarcane

Straw
Cleaning

Extraction of Combined Heat and

Bagasse
sugars Power generation

Juice treatment Juice treatment Steam, Electricity

Juice Juice
concentration concentration
Molasses

Crystallization Fermentation

Distillation and
Drying
Rectification

Sugar (VVHP) Dehydration Anhydrous Ethanol

Fig. 1.2 Block-flow diagram of the production of sugar, ethanol, and electricity from sugarcane
(CTBE 2012)

1.4.1.1 Process Description and Governing Parameters

of the Sugarcane Processing Facility

The basic configuration of an annexed plant (1G) and the related process
parameters for ethanol and sugar production have been summarized in this section.
The capacity of the sugarcane processing facility has been considered for the
processing of 500 metric tons of sugarcane (TC) per hour, during 167 days/year
and processing a total of 2 million TC/year.

Sugarcane Reception and Cleaning

The sugarcane arrives at mills with dirt and other impurities dragged in the har-
vesting process. Therefore, upon reception in the factory, sugarcane must be
cleaned. The efficiency of dirt removal in sugarcane washing is 90 % (BNDES and
CGEE 2008). Sugarcane cleaning is usually carried out using wash water, which is
recycled to the cleaning process after removal of dirt and other impurities.
1 Techno-Economic Analysis of Second-Generation Ethanol in Brazil 9

The amount of sugar lost during the whole sugarcane washing may be calcu-
lated as 25 % of the losses for the mechanically harvested sugarcane washing
(3.2 kg/TC) as observed by Rein (2007). However, usually no washing is carried
out for mechanically harvested (chopped) sugarcane due to the high sugar losses
that would occur. The same authors consider that the average amount of water
dragged with sugarcane during washing is 7.5 t/100 TC.

Sugarcane Processing and Juice Extraction

After cleaning, sugarcane is fed to the cane preparation system, on which a series
of equipment (shredder, hammers, etc.) are used to cut open the sugarcane
structure and enhance sugar extraction in the following operation. After prepara-
tion, sugarcane passes over a magnet that removes eventual metallic particles
dragged along prior to entering the mills.
Juice extraction is usually done using crushing mills, where sugarcane juice and
bagasse are separated. Water at a rate of 28 wt% of the sugarcane flow (imbibition
water) is used to improve sugars recovery. The imbibition water temperature is
50 C (Ensinas 2008) and the efficiency of sugar extraction in the mills is 96 %
(Walter et al. 2008). Sugarcane juice contains water, sucrose, and reducing sugars, in
addition to impurities such as minerals, salts, organic acids, dirt, and fiber particles,
which must be removed prior to fermentation. A rotary screen is used to remove
solid particles from the juice. The fibers obtained in this screen return to the mills for
further recovery of sugars, while the juice is sent to juice treatment. Efficiency of dirt
and bagasse removal in the screen is around 65 % (Mantelatto 2010).

Juice Treatment

The aim of the juice treatment process is to separate as much as possible the
dissolved and suspended juice impurities without reducing sucrose concentration.
It must be done soon after milling to prevent yeasts and enzymes action. Thus,
following extraction, the juice undergoes chemical treatment to remove other
impurities. This process consists of juice heating from 30 to 70 C, addition of
phosphoric acid or lime followed by a second heating operation, up to 105 C
(Copersucar 1987). Hot juice is flashed to remove dissolved air and after addition
of a flocculant polymer, impurities are removed in a settler, where mud and
clarified juice are obtained. A filter is used to recover some of the sugars carried
along with the mud, and the separated solids are recycled to the process prior to the
second heating operation. Bagasse fines, also called bagacillo, and wash water are
used in the filter to improve recovery of sugars. The clarified juice is fed to the
screens to remove solid particles that were not removed in the clarifier. The
clarified juice, at 98 C, destined for sugar production, contains around 15 wt%
solids (Mantelatto 2010) and it is concentrated on a five-stage multiple effect
10 A. K. Chandel et al.

evaporator (MEE) up to 65 wt% solids. In the annexed distillery, a fraction of the

syrup, as well as final molasses, are used to concentrate the clarified juice destined
for ethanol production up to around 22 wt% solids, which is cooled and fed into
the fermenters.

Sugar Production

The sucrose present in the syrup as sugar crystals is separated from the solution in
equipment called vacuum pans and crystallizers, usually operated under vacuum
and in fed-batch mode. The syrup is fed into the vacuum pans, where water is
removed in a similar way as in the evaporators. The mixture of sugar crystals and
molasses (liquid part) inside the equipment is called massecuite. When the amount
of material reaches the limit of the vacuum pan (at the end of a batch), the
massecuite is transferred to crystallizers and, after an appropriate residence time, it
is sent to centrifuges that separate the crystals and the molasses. It is possible to
exhaust more the molasses (recuperating more sugar) by repeating the process one
or two more times. The washing water temperature (at centrifuges) is 110 C
(Mantelatto 2010).
It is assumed that crystals are separated using the two-boiling system approach,
where two types of sugars are produced (Jesus 2004): grade ‘‘A’’ sugar (final
product) and grade ‘‘B’’ sugar (intermediate sugar that is produced and recycled
inside the process as ‘‘B’’ Magma, a solid–liquid stream rich in sugar crystals). The
Brix of the ‘‘A’’ sugar is 99 (Ribeiro 2003) and purity (VVHP—very very high
polarization) 99.6 % (Bazico 2010). For the ‘‘B’’ sugar, Brix is 98 (Ribeiro 2003)
and purity 88 % (Camargo 1990). The final sugar is dried in a rotary dryer at
100 C (Camargo 1990) and cooled before shipment.

Fermentation for Ethanol Production

After the juice treatment, concentrated juice is mixed with molasses and sent for
ethanol production in the fermenters. A fed-batch fermentation process with cell
recycle is assumed. The temperature of fermentation is 33 C and conversion of
sugars into ethanol is about 89.5 % (Mantelatto 2010), which is slightly lower than
the conversion in an autonomous distillery, due to the presence of molasses from
sugar production. In this process, yeast cells solution is fed to the fermenters prior
to sugarcane juice addition. During fermentation, gases released in the fermenters
are collected and sent to an absorption column where the entrained ethanol is
recovered using water. After the completion of fermentation reaction, the wine is
sent to the centrifuges, where cells are separated from the ethanol solution. Cells
obtained in the centrifuges are treated in a separate reactor by the addition of
sulphuric acid and water, to decrease bacterial contamination. After this treatment,
the cells are recycled to be used in another batch. Some part of the yeast cream,
also known as alcohol distillery yeast extract, is removed before being recycled.
1 Techno-Economic Analysis of Second-Generation Ethanol in Brazil 11

Fig. 1.3 Simplified scheme of the distillation columns (CTBE 2012)

This product is used mostly as protein source for animal feed. The produced wine
is mixed with the alcoholic solution obtained in the absorption process (to recover
ethanol from the CO2 stream) and sent for purification. Ethanol content in the wine
fed to the distillation columns is 8.5GL (Mantelatto 2010).

Distillation

The distillation aims at concentrating the wine until alcoholic content is up near
the azeotropic point for the hydrated ethanol production, with ethanol content
between 92.6 and 93.8 wt% (92.6 and INPM 93.8) (Dias 2008). Wine is sent to a
series of distillation and rectification columns (Fig. 1.3). Distillation columns
comprise two set of columns A, A1 and D, and rectification columns B1 and B,
each located one above the other. Wine is preheated in the condenser of column B
(heat exchanger E) and by exchanging heat with the bottom of column A (heat
exchanger K) before being fed into the top of column A1. Ethanol-rich streams
(phlegm) are obtained on top of column A and at the bottom of column D, and then
fed to column B-B1. Vinasse is produced at the bottom of column A, containing
less than 200 ppm of ethanol, while second grade ethanol is obtained from the top
of column D. Hydrated ethanol is produced on top of column B and nearly pure
12 A. K. Chandel et al.

water (phlegmasse) is obtained at the bottom of column B1. Fusel alcohol, con-
taining most of the higher alcohols, is obtained as a side withdrawal in column B.

Dehydration

The hydrated ethanol must be dehydrated to achieve alcohol content over 99.3 %
(mass) to be blended with gasoline. The ethanol dehydration cannot be made by
conventional distillation due to the azeotropic nature of ethanol solution (95.6 %
mass) at atmospheric pressure. Thus, alternative methods of separation must be
used to produce anhydrous ethanol (Dias 2008). The dehydration process for
anhydrous ethanol production can be carried out considering azeotropic distillation
with cyclohexane or adsorption on molecular sieves. The adsorption on molecular
sieves is a separation process with reduced energy consumption and without sol-
vent if compared to the azeotropic distillation. In this process, a zeolite bed is used
to adsorb water from hydrated ethanol to produce anhydrous ethanol. Usually three
beds are used, one of which is always in regeneration, to remove accumulated
water.

Combined Heat and Power Generation

Traditionally, cogeneration systems (CHP—combined heat and power generation)

used in Brazilian sugarcane mills are based on the Rankine cycle (Fig. 1.4).
During sugarcane processing, the juice is separated from the fibers, which pro-
duces large amounts of bagasse (approximately 140 kg/TC, dry basis). This
bagasse is used as a fuel in the cogeneration system to supply thermal, mechanical,
and electrical demand for sugar and ethanol production process. Formerly, low
efficient boilers (22 kg f/cm2) were used to produce steam and electricity for the
plant. However, the restructuring of the electricity sector in Brazil and the
incentives for energy production from renewable sources have driven to an
increase in investments for the production of surplus electricity in the mills. As a
result, more efficient boilers and turbines have been employed in order to produce
high pressure steams (65 kg f/cm2), and generate large amounts of electricity. As a
consequence, new sugarcane mill projects considering the use of Rankine cycles
with steam at higher levels of temperature and pressure, employing extraction-
condensing steam turbines and burning all bagasse produced in the mills have been
developed. The surplus electricity generated in this new configuration can be sold
to the power grid, improving the revenues of the company.
The amount of electricity produced by the sugarcane processing plant may be
increased significantly when sugarcane straw is collected from the field and
transported to the factory for further processing. Around 140 kg of straw (dry
basis) is produced per ton of sugarcane stalks, but part of the straw is usually left in
1 Techno-Economic Analysis of Second-Generation Ethanol in Brazil 13

Fig. 1.4 Scheme of back-pressure and extraction-condensing turbines based on Rankine cycle
(CTBE 2012)

the field in order to provide for weed and disease control as well as nutrient
recycling (Hassuani et al. 2005). However, removal of 50 % of the straw from the
fields is considered feasible (Dias et al. 2009; Hassuani et al. 2005; Walter and
Ensinas 2010).
Besides being used as a fuel in boilers for the production of steam and elec-
tricity, sugarcane lignocellulosic material (bagasse and straw) may also be used as
feedstock for second-generation ethanol production. Since it is composed basically
of cellulose, hemicellulose, and lignin, it may be converted into fermentable sugars
(hexoses and pentoses) through pretreatment and hydrolysis processes. Never-
theless, the amount of surplus lignocellulosic material used as feedstock depends
on the energy consumption of the whole production process. In this way, reduction
on process steam demand may lead to an increase in the amount of surplus bagasse
and straw, which can be employed as feedstock for second-generation ethanol
production when lignocellulosic material hydrolysis technologies are available.
The residues of the pretreatment and hydrolysis operations (residual cellulose,
lignin, and eventually biogas from pentoses biodigestion) may be used as fuels
increasing the amount of lignocellulosic material available as feedstock for 2G
(Dias et al. 2011, 2012a, b).
Different cogeneration systems were simulated in VSB to represent the inte-
grated 1G and 2G ethanol production processes (Fig. 1.5). The considered alter-
natives for cogeneration systems as well as the main parameters adopted in the
computer simulations are shown in Table 1.2.
14 A. K. Chandel et al.

Fig. 1.5 Block-flow diagram of the integrated first- and second-generation ethanol production
process from sugarcane (CTBE 2012)

Table 1.2 Main parameters adopted in the cogeneration system (CTBE 2012; Dias et al. 2013a)
Parameters Boiler pressure (kg f/cm2)
22 42 65 90
Steam pressure (kg f/cm2) 22 42 65 90
Steam temperature (C) 300 400 485 520
Steam production (kg steam/kg bagasse) 2.50 2.36 2.23 2.18
Boiler efficiency—LHV basis (%) 85.8 87.0 87.2 87.7
Gases outlet temperature (C) 180 160 160 160
Electricity demand—direct drives (kW h/TC) 16 16 – –
Electricity demand—electric drives (kW h/TC) 30 30 30 30
Direct steam drive efficiency (%) 55 55 – –
Steam turbines efficiency (%) 78 78 85 85
Generator efficiency (%) 98 98 98 98

1.4.2 Process Simulation for 2G Ethanol Production

Currently, one of the greatest concerns worldwide is the large-scale production of

alternative forms of energy, such as biofuels, which could reduce greenhouse gases
emissions and improve energy security when compared to their fossil counterparts
1 Techno-Economic Analysis of Second-Generation Ethanol in Brazil 15

(Chavez-Rodriguez and Nebra 2010). In this context, bioethanol has received

special attention, as it is already produced in large scale and used as automotive
fuel (Seabra et al. 2010).
In first-generation plants, sugarcane juice is used for sugar and ethanol pro-
duction, while sugarcane bagasse is used as a fuel in the boilers, providing heat
and power to the plant. However, in the context of expansion of the production and
consumption of ethanol, bagasse is considered as a potential feedstock for 2G
ethanol, since it does not compete with food crops and is less expensive than
conventional agricultural feedstocks (Alvira et al. 2010). In this case, 2G ethanol
production processes can be integrated with 1G ethanol plants, sharing part of the
infrastructure such as juice concentration, fermentation, distillation, cogeneration,
and water cooling systems. Another important residue that may be employed for
bioethanol production in the sugarcane industry is sugarcane straw, which includes
sugarcane leaves and tops, usually burnt or left in the field (Dias et al. 2011;
Macrelli et al. 2012).
For integration of 2G ethanol process with 1G, biomass pretreatment and
hydrolysis are usually considered in the processing of lignocellulosic material,
since it does not contain monosaccharaides readily available to be fermented. In
some cases, fermentation of the pentoses released during the pretreatment step to
ethanol can also be carried out; however, conventional microorganisms employed
in alcoholic fermentation are not able to ferment pentoses.
Preliminary assessments were carried out using VSB considering two levels of
development: current technology—hydrolysis with low yield and low solids
loading and biodigestion of C5 liquor—and a second level, potentially available in
2020 (futuristic scenario)—hydrolysis with higher yield and solids loading, C5
fermentation into ethanol, and lower investment and enzyme cost. Process alter-
natives are shown in Fig. 1.5. Operational conditions and yields are described in
subsequent sections.
Due to the high recalcitrance of biomass, pretreatment process is required to
increase the accessibility of cellulolytic enzymes toward cellulose (Alvira et al.
2010). Although different types of pretreatments were tested in different conditions
over the years, advances are still needed for overall costs to become competitive
(Rabelo et al. 2011; Chandel et al. 2010a). In VSB, steam explosion is defined as
the pretreatment process where most of the hemicellulose is hydrolyzed into
pentoses, with small cellulose loss and no lignin solubilization (Ojeda et al. 2011).
The pretreated solids are separated from the pentoses liquor via filtration. In order
to allow an increase in hydrolysis yield for the futuristic scenario, an alkaline
delignification step of the solid fraction was included after pretreatment, so most of
the lignin is removed, decreasing its inhibitory effects on the following enzymatic
hydrolysis step (Rocha et al. 2012). Table 1.3 presents main operational conditions
and yields for steam explosion pretreatment and alkaline delignification. Cellulose
obtained from pretreatment is converted into glucose after saccharification using
cellulolytic enzymes. Enzymatic hydrolysis allows the process to be carried out in
milder conditions than acid hydrolysis. In addition, enzymes offer the advantage of
producing higher yields of sugars with little degradation (Mussatto et al. 2010;
16 A. K. Chandel et al.

Table 1.3 Parameters adopted in the pretreatment and deliginification processes (CTBE 2012)
Parameter Value
Pretreatment—hemicellulose conversion 70 %
Pretreatment—cellulose conversion 2%
Pretreatment—temperature 190 C
Pretreatment—reaction time 15 min
Alkaline delignification—lignin solubilization 90 %
Alkaline delignification—temperature 100 C
Alkaline delignification—reaction time 1h
Alkaline delignification—solids loading 10 %
Alkaline delignification—NaOH content 1 % (m/V)

Table 1.4 Parameters adopted in the enzymatic hydrolysis and sugars recovery (CTBE 2012)
Parameters Values
Hydrolysis—cellulose conversion (current/future scenarios) 60/70 %
Hydrolysis—hemicellulose conversion (current/future scenarios) 60/70 %
Hydrolysis—solids loading (current/future scenarios) 10/15 %
Hydrolysis—reaction time (current/future scenarios) 72/48 h

Table 1.5 Parameters adopted in C5 biodigestion and fermentation for 2G ethanol production
(CTBE 2012)
Parameters Values
Pentose biodigestion—chemical oxygen demand (COD) removal 70 %
Pentose fermentation to ethanol 80 %

Chandel et al. 2012b). Tables 1.4 and 1.5 show the enzymatic hydrolysis operating
conditions and sugars yields.
Saccharomyces cerevisiae is one of the traditionally used microorganisms in 1G
ethanol production from corn and sugarcane due to its high efficiency in fer-
menting hexose to ethanol, and superior tolerance to low pH (Zhang et al. 2010)
and high ethanol concentration. However, for use in the 2G ethanol production
process, microorganisms that can convert C5 sugars into ethanol are limited and
generally have low ethanol and inhibitors tolerance and take longer incubation
times (Girio et al. 2010). In order to increase sugar yields, efficient conversion and
utilization of hemicellulosic sugars has become an important task and an oppor-
tunity to reduce ethanol production cost (Alvira et al. 2010).
Alternatively, C5 liquor may be biodigested, producing biogas for use as fuel,
increasing the amount of surplus lignocellulosic material. Pentoses biodigestion
1 Techno-Economic Analysis of Second-Generation Ethanol in Brazil 17

Table 1.6 Some examples of the process simulation for analysis of cost incurred for ethanol
production from various feedstock
Raw material Production technology Production Reference
cost of ethanol
(US$/L)
Sugarcane 1G + 2G ethanol 0.39 Dias et al. (2012c)
bagasse
Corn stover Dilute acid pretreatment, 1.36/L of ethanol Kazi et al. (2010)
enzymatic hydrolysis (gasoline
and ethanol fermentation equivalent)
Corn stover SSCF 6 US$/gallon Lynd et al. (2005)
Corn stover CBP 1.11 US cents/L Lynd et al. (2005)
Sugarcane 1G + 2G ethanol 0.40 Macrelli et al. (2012)
bagasse
Corn stover Ion-liquid pretreatment 6 US$/gallon Klein-Marcuschamer
et al. (2011)
Straw 0.73 Gnansounou and
Eucalyptus 0.56 Dauriat (2010)
Poplar 0.76
Switchgrass 0.71
Tall Fescue Dilute acid 0.83 Kumar and
(Festuca Dilute alkali 0.88 Murthy (2011)
arundinacea Hot water 0.81
Schreb) Steam explosion 0.85
Empty fruit Dilute acid hydrolysis, enzymatic 0.49 (with
bunches hydrolysis, and fermentation cogeneration)
with recombinant Zymomonas 0.58 (without
mobilis cogeneration)
Rice husk Dilute acid hydrolysis, enzymatic 0.53 Qunitero et al. (2013)
Coffee cut stems hydrolysis, and fermentation 0.585
Sugarcane with recombinant Z. mobilis 0.684
bagasse

and fermentation parameters are shown in Table 1.5. Other applications of pen-
toses include production of xylitol, lactic acid, 2, 3-butanediol, butanol, furfurals,
and other valuable products (Chandel et al. 2010b; Girio et al. 2010). However,
fermentative production of D-xylitol from hemicellulosic hydrolysate has been
considered one of the most beneficial processes to cater to the needs of various
commercial sectors (Silva and Chandel 2012) (Table 1.6).
18 A. K. Chandel et al.

1.4.3 Screening Method Applied to Analysis

of Technological Parameters in 1G Ethanol
Production Process

Mathematical models are useful tools for development, analysis, and optimization
of industrial processes. Models can be defined as a dataset and abstract ideas used
to explain a phenomenon of interest and relate the parameters of a given process. A
well-adjusted model can predict the parameters behavior so precisely that it
becomes a practical and cheap way to obtain information about the process under
study. Therefore, if the model is improved, it also improves the description of the
reality.
Incorporating mathematical models into computational simulation platforms is
not frequently applied to sugarcane-based biorefineries due to its complexity,
specificity, variability, interaction with environment, and other inherent
characteristics.
In VSB, the simulation for 1G ethanol production is described by variables such
as fermentation yield, steam consumption, steam pressure in boiler, among others.
The variable values used in this simulation were collected initially from the lit-
erature or provided by specialists. In addition, this information was complemented
and validated with data from Brazilian sugar and ethanol mills. However, inspite
of intense efforts in collecting variable values, this process is a difficult task in the
modeling procedure of 1G ethanol production due to its complexity and natural
variability.
In this context, screening methods are presented as useful tools to quantify the
impact of inputs variations on a given model response (Ruano et al. 2012).
Therefore, if a small change in an input variable leads to a large variation in a
certain response parameter of the model, this variable is considered important and
its determination must be as precise as possible (Cangussu et al. 2003). Assuming
that only some input variables contribute significantly to the outcome, screening
methods facilitate data collection by limiting the maximal precision to inputs
considered most important (Rivera et al. 2013).
Besides being used to obtain information about the degree of importance of
each variable, screening methods are frequently used to validate the model itself.
This validation reports whether the model follows (or not) an expected behavior.
In the simulation for 1G ethanol production in VSB, after screening procedure the
ranking of technological parameters can be analyzed to assess if the results agree
with what is expected for the current ethanol production in Brazil. Therefore,
specialized information (practical knowledge) from professionals is extremely
important for analysis and improvements of this model. As a result, screening
methods are also mechanisms to detect and adjust model inadequacies (Cangussu
et al. 2003). Screening procedure can be performed through design of experiments
(DOE) such as central composite design (CCD) (Montgomery 2001) and simu-
lation models.
1 Techno-Economic Analysis of Second-Generation Ethanol in Brazil 19

A study performed in the VSB context illustrates the efficiency and usefulness
of CCD as a method to screen the main variables in 1G ethanol production process.
Initially, the main input variables of the process were identified as: (i) fermentation
yield, (ii) steam consumption, (iii) steam pressure in boiler, (iv) juice extraction
yield, (v) residual ethanol concentration in vinasse, and (vi) alcohol content in
wine. The influence of these variables on ethanol output and surplus electricity has
been studied in the screening procedure. The interpretation of the results was
accomplished from the analysis of the model features and the expected behavior of
the input variables, bearing in mind the knowledge of the current 1G ethanol
production. Therefore, the analysis involved the collaboration among the spe-
cialists in the sugarcane sector and CTBE research team.
Variables under study were ranked by CCD with a significance level of 99 %.
For Ethanol Output, the input variables juice extraction yield and fermentation
yield were shown to be significant. An efficient juice extraction means that a large
amount of sugar will be available to the fermentation process without increasing the
amount of milled cane. The variable fermentation yield is of significance as it
influences directly on the amount of ethanol produced and also in the main
dependent variables such as, fermentation time, volume of fermenter, among others.
The variables steam consumption reduction, resulting from energy integration
in the production process, and steam pressure in boiler were significant for the
surplus electricity parameter. Decreasing the consumption of steam there will be
more steam available for the cogeneration process; therefore, more electricity will
be produced. The boiler steam pressure is directly related to the electricity
cogeneration. More electricity will be produced by the plant with a higher boiler
pressure.
The screening procedure was successfully used to identify the relevant variables
in 1G ethanol production process. In this procedure, the CCD proved particularly
efficient to obtain information about the significance of the input variables. Thus, it
was concluded that through screening methods it is possible to understand the
behavior of the technological parameters and compare it with the current process.

1.5 Techno-Economic Analysis of Sugarcane-Based

Biorefineries

In order to provide a comparison in terms of economic viability, important indi-

cators from Economy Engineering, such as internal rate of return (IRR), produc-
tion costs of products, beyond others, can be estimated considering a set of
scenarios related to different biorefinery alternatives. The principles for the eval-
uation of economic viability are based on a cash flow projected for each techno-
logical scenario to be evaluated, taking into account the investment needed for the
project and all expenses and revenues for an expected project lifetime. The main
operating expenses (OPEX) and revenues might come from mass and energy
20 A. K. Chandel et al.

balances obtained from computer process simulation. The basis for the monetary
values related to the capital expenses (CAPEX) can be obtained from the literature,
consulting with engineering companies, experts, and others.
Several studies were carried out at CTBE following the techno-economic and
environmental aspects of first- and/or second-generation ethanol production from
sugarcane (Dias et al. 2012a, b, 2013a, b; Cavalett et al. 2012; Junqueira et al.
2012). In this section are summarized the most important findings from the pre-
vious studies carried out at CTBE related to the techno-economic analysis and
environmental impacts of 1G ethanol and 2G ethanol productions from various
biorefinery configurations.

1.5.1 1G Ethanol Production Process

Environmental and economic aspects of autonomous distilleries and annexed plants

in Brazil were compared by Cavalett et al. (2012). In addition, optimized tech-
nologies for autonomous distilleries and annexed plants were considered in the
study and benefits of more flexible scenarios for annexed plants were examined. In
such configurations, CAPEX proved to be an important issue, since it increases
from autonomous to annexed plants and from fixed to flexible plant, having sig-
nificant impact on the IRR. Another important observation was that annexed plants
present higher IRR for both flexible (favoring sugar production) and fixed plants.
Although autonomous distillery also presented good results, it is important to take
into account that market oscillations can considerably change and flexibility may be
decisive for maintaining and even improving the sugarcane biorefinery profitability.

1.5.2 Integrated and Stand-Alone 2G Ethanol Production

Processes

Dias et al. (2012c) evaluated different scenarios for integrated and stand-alone 2G
ethanol production from sugarcane bagasse and straw. Five scenarios were
selected to demonstrate the economic and environmental impacts of 2G ethanol
production in comparison to an optimized autonomous 1G ethanol production
plant in Brazil. Results showed that the current integrated 1G and 2G ethanol
production scenario, characterized by higher investment cost in 2G (due to the fact
it will be one of the first plants), higher enzyme cost, lower yield, and lower solids
load in the hydrolysis step presents lower IRR in comparison to the optimized 1G
ethanol production. However, the integrated 1G and 2G ethanol production con-
sidering future scenarios, where target parameters are used for second-generation
processes and ethanol can be also produced from C5 sugars, is more attractive to
the investor than the optimized 1G ethanol production.
1 Techno-Economic Analysis of Second-Generation Ethanol in Brazil 21

1.5.3 Different Process Configurations for 2G Ethanol

Production Process

Junqueira et al. (2012) assessed economic and environmental impacts of different

options for the 2G ethanol production process integrated to the 1G sugarcane
biorefinery. The study evaluated two pretreatment options (steam explosion and
hydrothermal processing), as well as two alternatives for pentose utilization
(biodigestion and fermentation to ethanol). A delignification step was also ana-
lyzed after both pretreatments. Results showed that hydrothermal pretreatment
based on liquid hot water has higher energy consumption than steam explosion;
consequently, larger ethanol production is obtained from steam explosion pre-
treated bagasse. These results are in accordance with economic and environmental
analyses, which shows that the process with steam explosion presents the largest
IRR and lower life cycle environmental impacts.
Further, Dias et al. (2012b) evaluated different configurations for the 2G ethanol
production process (e.g., pretreatment with steam explosion coupled or not with
delignification, pentose biodigestion or fermentation to ethanol, solids loading for
hydrolysis) in an integrated 1G and 2G ethanol production biorefinery. The results
were used to evaluate which process alternatives provide higher ethanol yield,
pointing toward the direction in which research should be oriented. Computer
simulations of integrated 1G and 2G ethanol production process from sugarcane
showed that high hydrolysis yields (that may be achieved using low solids loading
on the hydrolysis reactor) do not lead to the best results in terms of overall ethanol
production. Because the lignocellulosic material (sugarcane bagasse and straw)
used as feedstock in 2G is also used as fuel, low solids loading requires more
steam on the concentration step. Therefore, even though that scenario leads to the
highest 2G ethanol production per lignocellulosic material processed (around 200
and 400 L/t dry lignocellulosic material (LM) for the processes with pentose
biodigestion and fermentation, respectively), lower yields and higher solids
loading lead to larger amounts of ethanol produced per ton of sugarcane (up to
122 L/TC for 20 % solids, as opposed to 116 L/TC for the process with pentose
fermentation and 5 % solids loading in hydrolysis). This study confirmed the
importance of evaluating the whole process to better understand it and to guide
further experiments aiming at the viability of 2G ethanol production process.
Dias et al. (2013a) evaluated different cogeneration systems configurations for
integrated 1G and 2G ethanol production, as well as different destinations for the
pentose (biodigestion or fermentation to ethanol) obtained after pretreatment of the
LM. Economic analyses showed that coupling electricity production with 2G
ethanol production in the integrated process improves its economic results, even
when electricity is produced in relatively low amounts using low efficiency boilers
(22 bar boilers). Another interesting finding of the study is that high pressure
boilers (82 bar) consume more bagasse than low pressure boilers, thus decreasing
final ethanol output. Nevertheless, revenues obtained with the sale of electricity in
the processes employing cogeneration systems with high pressure boilers outweigh
22 A. K. Chandel et al.

the losses in ethanol yield and the increased investment of these cogeneration
systems. Among the evaluated process configurations, the one with 65 bar boilers
presents the lowest environmental impacts in most categories including global
warming potential. In the context of C5 use, pentose fermentation allows a large
increase in the total ethanol production (40–50 % higher than 1G production)
compared to the gains of pentose biodigestion (around 30 %).

1.5.4 Improving 2G Ethanol Production Through

Optimization of 1G Plant

Dias et al. (2012a) evaluated some improvements in the 1G ethanol production

process, aiming at reducing process steam consumption; maximizing surplus LM;
or increasing electricity output. The process improvements analyzed in the study
decreased the steam consumption in the 1G ethanol production in an autonomous
distillery. Results showed that a considerable increase in the amount of surplus LM
can be obtained with the use of efficient cogeneration systems (among other
process improvements) and the recovery of 50 % of the straw. Significant increase
in ethanol production in optimized autonomous sugarcane distilleries integrated
with 2G ethanol production, along with electricity production, can be obtained if
high pressure boilers are employed. Gains on ethanol production in an integrated
1G and 2G ethanol production process are possible when efficient, low pressure
boilers are employed.

1.5.5 Flexibility on 2G Ethanol and Electricity Production

Dias et al. (2013b) evaluated a flexible biorefinery with the ability of diverting a
fraction of the lignocellulosic material (sugarcane bagasse and straw) either for
electricity production or as feedstock in 2G ethanol production. The flexible
sugarcane biorefinery selling surplus electricity in the spot market when prices are
favorable presented better economic returns than the conventional biorefinery
using all surplus lignocellulosic material as feedstock for 2G ethanol production.
The flexible biorefinery and the plant with maximum ethanol production lead to
the highest cut-off in carbon dioxide emissions. However, biorefineries producing
more ethanol present higher environmental impacts per unit of ethanol produced
than the configuration with maximum electricity production due to the high
impacts of chemicals used in the 2G process. The study concluded that even
though flexible biorefinery has a high IRR, changes in ethanol prices affect the IRR
more significantly compared with increases in electricity spot market prices. Thus,
if ethanol prices increase, the fixed biorefinery operating with maximum ethanol
production will be more advantageous in economic terms.
1 Techno-Economic Analysis of Second-Generation Ethanol in Brazil 23

1.6 Life Cycle Assessment of Sugarcane-Based

Biorefineries

Life cycle assessment (LCA) is a recognized method for determining the envi-
ronmental impact of a product (good or service) during its entire life cycle, from
extraction of raw materials through manufacturing, logistics, use, and final dis-
posal or recycling. In LCA, substantially broader environmental aspects can be
covered, ranging from GHG emissions and fossil resource depletion to acidifi-
cation, toxicity, water, and land-use aspects; hence it is an appropriate tool for
quantifying environmental impacts of a product system. The ISO 14040 series
provides a technically rigorous framework for carrying out LCAs (ISO 2006a, b).
The method consists of four main steps: goal and scope definition, inventory
analysis, impact assessment, and interpretation. First, the goal and scope provides
the context for the assessment and explains to whom and how the results are to be
communicated. This step includes detailing of technical information—such as
defining the functional unit, system boundaries, assumptions and limitations of the
study, impact categories, and methods used to allocate environmental burdens in
cases where there is more than one product or function.
Life cycle inventory (LCI) is the methodological step where an overview is
given of the environmental interventions (energy use, resource extraction, or
emission to an environmental compartment) caused by or required for processes
within the boundaries of the studied system. With its translation of the product
system’s environmental flows from the life cycle inventory phase (LCI) into scores
that represent their impacts on environment, life cycle impact assessment (LCIA)
is essential for the interpretation of the results in relation to the questions posed in
the goal definition (Finnveden et al. 2009). The challenge of LCIA is to evaluate
the potential impact of the emitted substances by using a procedure that is ideally
simple, applicable consistently to all substances, uses a common unit of measure,
and gives results that are comparable between impact categories.
A life cycle interpretation is necessary for identifying, quantifying, checking,
and evaluating information from the results of the LCI and/or the LCIA. This
interpretation should also raise significant environmental issues, including an
evaluation of the study considering completeness, sensitivity, and consistency
checks; and limitations.
Regarding the possibilities of using different LCIA methods, Cavalett et al.
(2013) used seven different LCIA methods for a comparative assessment of eth-
anol and gasoline in Brazil. The study provided an updated and comprehensive
LCI for sugarcane ethanol in Brazil considering the stages of agricultural pro-
duction, transport, ethanol production, and its final use. Results showed that the
use of different LCIA methods can lead to different comparative environmental
impacts of ethanol and gasoline, mainly when single-score indicators are applied.
A relative convergence in the results of equivalent environmental impact cate-
gories using different midpoint LCIA methods was observed. Results of the
comparison of the five midpoint LCIA methods showed that ethanol presents
24 A. K. Chandel et al.

better environmental performance than gasoline in important categories such as

global warming, fossil depletion, eco-toxicities, and ozone layer depletion but
worse environmental performance than gasoline in the categories acidification,
eutrophication, photochemical oxidation, and agricultural land use.
Calculated environmental impacts using the LCA methodology presented by
Cavalett et al. (2012) indicate that sugarcane biorefinery optimization technologies
for 1G ethanol production have a great potential for a significant decrease of the
environmental impacts in sugarcane biorefineries (for both autonomous and
annexed plants). Ethanol production in annexed plants presented slightly lower
environmental impacts in comparison to autonomous distilleries mainly due to
allocation rules used in the study. Results also showed that flexibility in annexed
plants produce little effect on the environmental impacts when the entire ethanol
production chain is considered.
Dias et al. (2012c) observed that a current integrated ethanol production plant
(1G and 2G) has potential to decrease the environmental impacts in relation to 1G
ethanol production process. The study identified that the use of high amount of
sodium hydroxide in the alkaline delignification step has strong influence in the
increase of the 2G ethanol environmental impacts. Junqueira et al. (2012) also
found that alkaline delignification contributed to higher environmental impacts in
the 2G ethanol production process. Also, pentose fermentation should be
emphasized in experimental studies instead of biodigestion to produce biogas,
since fermentation to ethanol leads to the best technical, economic, and environ-
mental results in the 2G ethanol production process.
Galdos et al. (2013) showed the importance of including black carbon emis-
sions for the calculation of global warming and human health environmental
indicators. The results quantitatively demonstrated that the technological trends
considering past, present, and future scenarios for ethanol production in Brazil is
showing lower environmental impacts. Avoiding the preharvesting burning of LM
will decrease the emissions of black carbon and greenhouse gases in the sugarcane
production phase. In addition, increase in yield of sugarcane per hectare, and of
ethanol per ton of sugarcane, will eventually decrease the environmental impacts
per unit of biofuel produced. Results showed that 2G ethanol production plays a
key role in increasing the amount of ethanol produced per unit of area. Results also
indicated that the Brazilian sugarcane sector presents a trend of using more effi-
ciently the resources per unit of ethanol produced, as well as promoting good
management practices that reduce its environmental impacts.

1.7 Conclusions and Future Recommendations

Among the renewable energy sources, use of ethanol as transportation fuel has
achieved significant success in countries like Brazil and USA. This review shows
the potential of computer-aided process modeling and simulation, life cycle
1 Techno-Economic Analysis of Second-Generation Ethanol in Brazil 25

assessment, processing technological routes for biochemical ethanol production

(1G + 2G) from sugarcane juice, and lignocellulosic residues of sugarcane.
Brazil is the largest sugarcane producer (625 million tons of sugarcane in 2011)
in the world showing the tremendous potential of sugarcane ethanol as sustainable
energy source governing the economic, strategic policy, and environmental
impacts on the nation. Today, in Brazil, 44 % of energy matrix used is renewable,
and 13.5 % of renewable energy is derived only from sugarcane. In the context of
the Virtual Sugarcane Biorefinery, from CTBE has extensively worked toward
developing rigorous process simulation with the help of in-house derived exper-
imental database to perform mass and energy balances, which is helpful for robust
scale-up, and allows a better understanding of economic and environmental
impacts. Evaluating several scenarios for 1G and 2 G ethanol production (stand-
alone and integrated plants) in the dynamic context of biorefineries using sugar-
cane as a main energy driver, it was concluded that integrating 2G to 1G ethanol
production improves its economic results. Moreover, in the context of C5 use,
pentoses fermentation allows a large increase in the total ethanol production
(40–50 % higher than 1G production) compared to the gains of pentoses biodi-
gestion (around 30 %). Another interesting finding of the study is that high
pressure boilers (82 bar) consume more bagasse than low pressure boilers, thus
final ethanol output is small if very high pressure boilers are used. Furthermore, 2G
ethanol may favorably compete with bioelectricity production when sugarcane
straw is used in addition to the application of improved technologies using low
cost enzymes for biomass hydrolysis. In regard to determining environmental
impacts by LCA methodology, optimized cellulosic ethanol production technol-
ogies could have a great potential for significant decrease in the environmental
impacts of present sugarcane biorefineries (autonomous and annexed plants).
Summarizing all the important features, 2G ethanol production in Brazil seems
promising in the present scenario, which is a learning example for many countries
in order to harness their natural agro resources. This is a fundamental step toward
the development of renewable and sustainable sources of energy.

Acknowledgment The authors are grateful to BIOEN-FAPESP for the financial support.

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Silva SS, Chandel AK (2012) D-xylitol: fermentative production, application and commerciali-
sation. In: Silva SS, Chandel AK (eds) Springer, Heidelberg. ISBN: 978-3-642-31886-3
van Zyl WH, den Haan R, la Grange DC (2011) Developing organisms for consolidated
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intechopen.com/books/biofuel-production-recent-developments-andprospects/developingorganisms-
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24:761–764
Walter A, Ensinas AV (2010) Combined production of second-generation biofuels and electricity
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sustainability analysis of the Brazilian ethanol. A report supported by UK Embassy and
DEFRA. Available online at www.unica.com.br
Yuan JS, Tiller KH, Al-Ahmad H, Stewart NR, Stewart CN Jr (2008) Plants to power: bioenergy
to fuel the Future. Trends Plant Sci 13:421–429
Zhang X, Shen Y, Shi W, Bao X (2010) Ethanolic co-fermentation with glucose and xylose by
the recombinant industrial strain Saccharomyces cerevisiae NAN-127 and the effect of
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Chapter 2
An Assessment of Brazilian Government
Initiatives and Policies for the Promotion
of Biofuels Through Research,
Commercialization, and Private
Investment Support

Luís Augusto Barbosa Cortez, Gláucia Mendes Souza,

Carlos Henrique de Brito Cruz and Rubens Maciel

Abstract This chapter describes some of the scientific and technological

achievements that have contributed to develop sugarcane bioenergy as a major
contributor to the Brazilian energy matrix. Today, modern bioenergy plays a key
role in the Brazilian economy, with 18 % of Brazilian energy usage coming from
sugarcane, with ethanol used as fuel and bagasse to generate electricity. This
chapter also discusses the Brazilian biodiesel opportunities and biofuels for
aviation, which hold promise for the future. The long-term role played by the
Brazilian government in promoting biofuels is considered as a key factor to suc-
cess, particularly with sugarcane ethanol. Government-funded research agencies
have played a strategic role in consolidating knowledge and human capacity to
maintain leadership in the bioenergy sector. Brazil presents exceptional conditions
to expand bioenergy industry (ethanol, biodiesel and biofuels for aviation) and also
bioelectricity and green chemistry. To this end it is necessary to create conditions

L. A. B. Cortez (&)
Faculty of Agriculture Engineering-FEAGRI, State University of Campinas, UNICAMP,
Barão Geraldo, Caixa-postal: 6011, Campinas, SP 13083-970, Brazil
e-mail: luisabcortez@yahoo.com.br
G. M. Souza
Instituto de Química, Departamento de Bioquímica, Universidade de São Paulo,
Av. Prof. Lineu Prestes 748-sala 954, São Paulo, SP 05508-000, Brazil
e-mail: glmsouza@iq.usp.br
C. H. de Brito Cruz
Physics Instutute-IFGW, State University of Campinas, UNICAMP, Barão Geraldo,
Caixa Postal 6011, Campinas, SP 13083-970, Brazil
e-mail: brito@ifi.unicamp.br
R. Maciel
School of Chemical Engineering -FEQ, State University of Campinas, UNICAMP,
Barão Geraldo, Caixa Postal 6011, Campinas, SP 13083-970, Brazil
e-mail: maciel@feq.unicamp.br

S. S. da Silva and A. K. Chandel (eds.), Biofuels in Brazil, 31

DOI: 10.1007/978-3-319-05020-1_2,  Springer International Publishing Switzerland 2014
32 L. A. B. Cortez et al.

for the increase of the private R&D expenditures, as well as governmental actions
to train human resources in the area of bioenergy. With new research centers,
graduate programs have the potential to contribute to increasing competence at all
stages of bioenergy development.

2.1 Introduction

By the end of the nineteenth century, the Brazilian energy matrix was dominated
by traditional bioenergy such as the extraction of firewood. In the early twentieth
century, use of hydroelectricity and fossil fuels (coal and oil) had gained promi-
nence, imparting complexity to the Brazilian energy matrix. However, even
before the end of the Second World War, oil began to dominate the transportation
energy sector and hydroelectricity also gained importance. Nevertheless, both
traditional (firewood and charcoal) and modern (ethanol and bagasse) forms of
bioenergy have remained important. The importance of wood fuel, however, has
diminished in the energy matrix, while the use of sugarcane for energy purposes
has gained momentum, especially since 1975 (Guerra and Cortez 1992).
Today, modern bioenergy plays a key role in the Brazilian economy, with 18 % of
Brazilian energy usage coming from sugarcane, with ethanol used as fuel and
bagasse to generate electricity.
In this chapter, the major scientific and technological achievements that have
contributed to the rising prominence of sugarcane as a bioenergy in the Brazilian
energy matrix are presented. This chapter also presents the Brazilian biodiesel
market and biofuels for aviation, which hold promise for the future. In this paper,
biofuel is defined as a bio-based liquid fuel, but the definition could be extended to
include solid biofuels, such as wood and eucalyptus. However, because Brazil is
experiencing an upsurge in the use of modern biofuels such as bioethanol and
biodiesel, this chapter will be confined to the limited definition.

2.2 Sugarcane Ethanol in Brazil

Brazilian research on sugarcane ethanol as an automotive fuel began in the 1920s

with studies conducted by the National Institute of Technology (INT). The addition
of ethanol to imported gasoline was mandated by law in Brasil (1931). In 1933, the
Brazilian Institute for Sugar and Alcohol (IAA) was created because of a developing
crisis in the sugar sector over the use of sugarcane for ethanol. The IAA’s focus was to
help sugarcane producers while also reducing the consumption of imported gasoline.
In 1938, Law No. 737 was passed, requiring the compulsory addition of sugarcane
ethanol to all gasoline in Brazil. This law remains in effect even today, with different
percentages of ethanol added depending on economic constraints (Fig. 2.1).
2 An Assessment of Brazilian Government Initiatives and Policies 33

25% % ethanol

20%
15%
10%
5%
0%
1925 1945 1965 1985 2005

Fig. 2.1 Average ethanol content in the gasoline in Brazil between 1925 and 2005 (Nogueira 2008)

Fig. 2.2 Phases of Proálcool, 1972–2007 (Datagro 2006; elaborated by ICONE and UNICA)

The first oil crisis in 1973 seriously hurt the Brazilian economy. At that time,
the country imported nearly 80 % of its oil, which represented approximately
50 % of total imports. An immediate solution was required to reduce Brazil’s
dependence on oil. The newly elected President, Ernesto Geisel, who was the
former president of Petrobras, enacted a number of measures for the energy sector.
On November 14th, 1975, by Decree No. 76.593, the Brazilian government created
the National Alcohol Program, also known as ‘‘Proálcool.’’
The Proálcool program has had varying levels of success and failure (Fig. 2.2).
Financed by subsidies and liberalization policies, the program has survived changes
in regimes (from military to democratic), considerable variations in the price of oil
and sugar, and economic and political crises over the past 38 years. The most
critical period occurred in the second half of the 1980s, when the ethanol car (E100)
accounted for 90 % of total sales of new vehicles in the country. In 1989, ethanol
34 L. A. B. Cortez et al.

production did not match the domestic demand, primarily because of a lack of
planning. Many consumers felt aggrieved and lost confidence in the ethanol car,
which dramatically reduced the sales to almost zero in the following years.
The 1990s were marked by a restructuring of the sugar-ethanol sector through
gradual deregulation, which allowed Brazil to become a major exporter of sugar.
The automotive industry had already begun favoring the sale of cars running on
gasoline-ethanol blends (ranging from E20 to E25). With a growing fleet,
domestically consumed ethanol was more anhydrous than hydrous, and the
demand for both types of ethanol made the total demand for ethanol more or less
constant.
By the late 1990s, many consumers had tried using different percentage blends
of ethanol and gasoline, popularly known as a ‘‘cocktail.’’
In the twenty-first century, the rising price of oil has given sugarcane ethanol a
new impetus in Brazil. The automotive industry realized that consumers want a
vehicle with a flexible engine that would work with any proportion of ethanol in
the fuel mixture. The consumer does not want to be at the mercy of price fluc-
tuations, which are common today during the sugarcane inter-harvest period, or be
held hostage to a fuel that could be depleted and thus devalue their assets (cars).
Concurrently, flex-fuel vehicles (FFV) models were being launched in the U.S.,
and in 2002, the Ford Fiesta was introduced in Brazil. In 2003, Volkswagen
launched the GOL as their first Brazilian flex-fuel vehicle.
The ‘‘lambda probe,’’ a sensor developed by Bosch and Magneti Marelli (MM)
to identify blends of fuel, constituted a considerable qualitative development for
Brazilian flex engines operating in bi-fuel vehicles. Today, approximately 90 % of
the new cars sold in Brazil are flex-fuel vehicles that allow the consumer to select
between fuel types, offering greater protection against the fluctuating prices of
ethanol and gasoline.
In Brazil, the successful use of sugarcane ethanol was the result of a learning
trajectory based primarily on incremental innovations (Furtado et al. 2011).
Copersucar (1989), Leite (1990), Magalhães et al. (1991), Moreira and Goldemberg
(1999), Moraes (1999), BNDES (2008), Cortez-Coord (2010) Souza and Macedo
(2010) and Rosillo-Calle et al. (1998, 2000) describe in detail the history of Proá-
lcool and how Brazil created this internationally recognized success story.

2.2.1 Sugarcane Agricultural Research

Brazil became the world leader in the production and use of sugarcane-derived
fuel ethanol and thanks to a successful combination of long-term actions of the
government and private sector, including important agronomic research activities.
When the National Alcohol Program was implemented in 1975, Brazil was already
a major producer of sugarcane (the second largest after India), milling approxi-
mately 100 million TC/year. However, the production of fuel ethanol was modest,
at approximately 600 million liters per year.
2 An Assessment of Brazilian Government Initiatives and Policies 35

Fig. 2.3 Diversification of commercial sugarcane varieties from 1984 to 2010 in Brazil (Source
Costa et al. 2011)

Important research centers were also studying sugarcane, including the Agro-
nomic Institute of Campinas (IAC), which had started its breeding program in
1933 and collaborated with the emerging sugar industry in the State of São Paulo
to generate important information in the areas of plant nutrition and agricultural
practices, forming the basis of existing technologies (IAC and IACSP), and the
IAA breeding program of Campos, RJ Station, which was responsible for the CB
varieties of sugarcane.
In the early 1970s, the National Program for Genetic Improvement of Sugarcane
(Planalsucar) was created by the Brazilian federal government and the Copersucar
breeding program (SP varieties) was created with the funds from the private sector.
Until 1975, Brazil had depended on a few sugarcane varieties (Fig. 2.3),
including the predominant variety NA5679 from Argentina. However, Planalsucar
played an important role in preparing researchers to experiment with sugarcane
varieties and creating a sugarcane ‘‘genetic bank’’ in Alagoas, northeast Brazil.
New findings emerged in soils, herbicides, diseases, and biological control of
sugarcane pests. After the IAA disbanded, Planalsucar resumed its activities in
1990. Its researchers, who were reorganized within federal universities, created the
Institutional Network for the Sugar and Alcohol Segment Development (RIDESA)
to continue its research and discovery on sugarcane. RIDESA consists of ten
federal universities (UFPR, UFSCar, UFV, UFRRJ, UFS, UFAL, UFRPE, UFMT,
UFG, and UFPI), has 34 stations, and is responsible for RB varieties of sugarcane.
In the private sector, the State of São Paulo’s Sugarcane, Sugar & Alcohol
Producers Cooperative—Copersucar was created in 1978, forming the Copersucar
36 L. A. B. Cortez et al.

Fig. 2.4 Increased yield per hectare for selected Brazilian crops (1970–2006) (Elaborated by
M.P. Cunha (CTBE) using data from Brazilian Agricultural Census (IBGE) and from the
Brazilian Agroenergy Yearbook 2009)

Technology Center (CTC). After 2004, the CTC was renamed as the Center for
Sugarcane Technology and then the Sugarcane Research Center (http://www.
ctcanavieira.com.br/). The CTC has played a key role in technology transfer for
both the agricultural and industrial sectors. The CTC produced significant
advances in agricultural management and in areas such as agricultural mechani-
zation, microbiology fermentation, energy and water conservation, and application
of vinasse and filter cake (Burnquist and Landell 2005).
The CTC was established to conduct research and develop new technologies for
application in agricultural activities, logistics and industrial sectors, and to create
new varieties of sugarcane, with the technology provisioned to cooperative mills.
The CTC is responsible for varieties that make up approximately 60 % of the crops
of cooperative units and 45 % of the crops of other producers.
The breeding program of the IAC Sugar Cane Program was reorganized in 1994
by the Cane Center of Ribeirão Preto, which has 128 research units operating in 12
states in Brazil that are committed to the integration of science and technology.
Some of the contributions of the Cane Center were highlighted by Dinardo-Miranda
et al. 2008. This program has the support of APTA units in the cities of Piracicaba,
Jaú, Mococa, Pindorama, Assis and Adamantina, and breeding farms in Goianésia
(GO) and Luis Eduardo Magalhães (BA).
There are currently four active sugarcane programs in Brazil: IAC, CTC,
RIDESA, and Canavialis. The success of the breeding programs is evident from
the evolution of sugarcane agricultural productivity in Brazil (Fig. 2.4). Other
crops such as corn have shown a remarkable increase in agricultural productivity
in recent decades; however, the agro-industrial yield of sugarcane ethanol is
approximately 7,000 L/ha.year in Brazil whereas the yield of ethanol from corn is
approximately 3,500 L/ha.year in the U.S. Although important progress has been
2 An Assessment of Brazilian Government Initiatives and Policies 37

made in developing new cane varieties in Brazil, more research is required, par-
ticularly in developing areas where sugarcane is expanding, such as Central Brazil.

2.2.1.1 Other Crops for Ethanol Production

When the Proálcool program was first implemented, the possibility of using other
raw materials, such as cassava and sorghum, in the production of ethanol was
considered. Production of biodiesel is experiencing a similar growth pattern,
including encouraging different cultures, decentralizing production, and giving
access to small producers. Initiatives of the Brazilian Ministry of Science and
Technology/Secretary of Industrial Technology (MIC/STI) (1980) included
designing a mini-distillery to produce 10,000 L/day of cassava alcohol and
experiments with EMBRAPA to create an SP model using small-scale diffusers
instead of mills and operating with sorghum and sugarcane (Figueiredo et al.
1984). Another important project was led by Petrobras in Curvelo, MG, in 1978,
who attempted to implement a cassava ethanol distillery that was developed by
INT (Motoyama 2004). The project failed primarily because of agronomic diffi-
culties related to large-scale cassava production. Many of the problems associated
with alternative feedstocks are related to a lack of long-term research.

2.2.2 Other Agricultural Issues in Ethanol Production

Numerous researchers and Brazilian institutions have played an important role in

the development of sustainable sugarcane ethanol, with significant contributions to
sugarcane ethanol production originating in Brazilian universities and research
centers. The sections below include some of the most important contributions.

2.2.2.1 The End of Sugarcane Burning

Sugarcane burning prior to harvesting is a major environmental problem that

pollutes the air and increases respiratory diseases in winter. In September 2002, the
legislature of the State of São Paulo passed legislation mandating the gradual
reduction of sugarcane burning in São Paulo until 2017: State Law No. 11.241, of
19 September, 2002, shown in Fig. 2.5. ‘‘Raw’’ sugarcane harvesting (without
burning) is now practiced in more than 80 % of the harvested areas in São Paulo,
which grows over 50 % of Brazil’s sugarcane. Raw cane harvesting is expected to
reach 100 % of harvested areas in São Paulo State as mandated by the law.
However, the harvest of sugarcane without burning introduced a new technological
challenge: mechanically and economically harvesting the entire cane (stalks and
straw) without compacting the soil, consuming high amounts of fuel, losing sug-
arcane, damaging the stumps, and introducing too many impurities to the product.
38 L. A. B. Cortez et al.

100%

Sugarcane harvesting without burning 90%

80%
70%
60%
50%
40%
30%
20%
2005 2007 2009 2011 2013 2015 2017 2019 2021
State law (11.241/2002) Environmental Protocol
Mechanized area Mechanizable area, projection

Fig. 2.5 Evolution of the cessation of sugarcane burning and sugarcane harvesting mechani-
zation in the State of São Paulo from 2001 to 2021 (adapted of Macedo 2007)

The CTBE is developing a mechanization approach for sugarcane harvesting

(Braunbeck et al. 2005) called a ‘‘Controlled Traffic Structure (ETC)’’ which is
specifically designed for low impact mechanization and will introduce no-till
farming and precision agriculture for growing sugarcane. (http://www.bioetanol.
org.br/interna/index.php?chave=baixoimpacto).
Cane harvesting without burning also introduces the problem of eliminating the
cane trash. Burning sugarcane eliminates trash, transforming it into emissions and
ash. However, when sugarcane is harvested without burning, left behind trash
(composed primarily of leaves and tops) makes the harvesting process less effi-
cient. Significant amounts of sugarcane trash left on the fields confers a degree of
agronomical benefit, such as a reduction of soil erosion and moisture loss; how-
ever, certain agricultural practices must be changed to manage soil pests that are
attracted to the trash. Agronomists recommend that no more than 50 % of the trash
be removed from the field, preferably simultaneous with the harvesting process to
keep weeds under control. Although the trash material (primarily fibers) may
represent an opportunity for generating electricity or second-generation ethanol,
there are not enough incentives to make use of the trash economically viable.

2.2.2.2 Recycling Vinasse as Fertilizer (Fertirrigation Technology)

In the early years of Proálcool, vinasse was identified as a major environmental

contaminant because it has a high organic load and was commonly disposed of in
waterways. An Esalq-USP research group studied vinasse application on the
ground, which eventually became a routine practice (Gloria 1975, 1976). Subse-
quently, the CTC developed more the fertirrigation technology. Today, applying
2 An Assessment of Brazilian Government Initiatives and Policies 39

vinasse to the ground helps the sugarcane industry save significant amounts of
potassium (Copersucar 1978, 1980; Freire and Cortez 2000 and Fredo et al. 2008).
In addition, Cetesb regulation controls the inappropriate use of vinasse in fer-
tirrigation to prevent groundwater contamination. This standard establishes criteria
for calculating the maximum amount of vinasse to be used depending on soil-type
and other parameters (Vinhaça–Critérios e Procedimentos para Aplicação no Solo
Agrícola P4.231 Dez/2006 http://www.cetesb.sp.gov.br/tecnologia/camaras/P4_
231.Pdf). Although fertirrigation technology represents a good solution in most
cases, a reduction is still required in the volume of produced vinasse and the GHG
emissions potential.

2.2.3 The Brazilian Industrial Model for Ethanol Production

2.2.3.1 Scale and Model of Ethanol Production in Brazil

When the Proálcool program was first established, the government proposed that a
‘‘standard distillery’’ (capacity of 120,000 L of alcohol/day) be used for production
of ethanol. This plant size, now considered too small, was challenged by many
researchers who believed that smaller units would favor small-scale production and
increase social benefits. The MIC/STI (1981) proposed production models based on
micro-distilleries. A research group from USP—São Carlos proposed a model
based on ‘‘integrated mini-ethanol plants’’ that was capable of producing 20,000 L
of ethanol/day and could, in theory, become a ‘‘more social’’ version of the ethanol
program (Corsini 1992). At the time, there were several companies selling units of
up to 1,000 L/day, but the small-scale distilleries never achieved the levels of
productivity, quality, and economic viability of the large plants. At present, a
standard plant has a production capacity of approximately 1 million liters of eth-
anol/day, although the largest plants in Brazil can produce 4 times that amount.

2.2.3.2 The Brazilian Model of Simultaneous Production

of Sugar and Ethanol

The creation of what is known as the ‘‘Brazilian model’’ of simultaneous pro-

duction of sugar and ethanol was the result of the combined efforts of several
researchers: José Paulo Stupiello, Esalq—USP, who contributed to sugar pro-
duction technology; Young Park, UNICAMP, who contributed to the microbiology
of ethanol and fundamental studies on fermentation using sugarcane and other raw
materials; and Carlos Vaz Rossell and Jaime Finguerut, CTC, who contributed to
optimizing and building the concept of a ‘‘flex plant’’ (Copersucar 1990). These
efforts resulted in significant advancements in the ethanol industry, as evidenced
by the reduction in fermentation time.
40 L. A. B. Cortez et al.

Fig. 2.6 Agro-industrial productivity evolution of sugarcane ethanol through R&D from 1975 to
2005 (Source Brito 2012)

These contributions substantially improved ethanol’s agro-industrial produc-

tivity, which is an indicator based on improvements in agricultural and industrial
productivity (Fig. 2.6).

2.3 Development of Ethanol and Flex Fuel

Engines in Brazil

One of the first publications on the subject of ethanol and flex fuel engines was the
book ‘‘Internal combustion engines and ethanol engines,’’ published in 1937 by
Eduardo Sabino de Oliveira from the IAA. Much later, Urbano Ernesto Stumpf
(CTA) contributed important research on the study of engines fueled by alcohol and
received patent #PI8106855-7 for an ‘‘alcohol-specific carburetor’’ on 23/10/1981
(O’Donnell 2009). Another important contribution was made by Romeo Corsini
(USP—São Carlos), who received a patent (#PI8402740-1) for the ‘‘MAV—pre-
evaporized alcohol engine.’’ Additional recognition should be given to the work of
Fernando Barata de Paula Pinto of Maxion International Motors, who helped to
develop alcohol engines and flex fuel engines. Francisco Nigro (IPT) and Henry
Joseph Jr. (ANFAVEA and Volkswagen) helped to develop the alcohol engine and
flex-fuel ethanol. Although Brazil has achieved important success in using ethanol
fuel, there are still significant challenges to overcome in the design of the engine,
which must undergo major changes for use with hybrid engines. Figure 2.7 shows
the irregular ethanol vehicle production in Brazil since 1979.
2 An Assessment of Brazilian Government Initiatives and Policies 41

Fig. 2.7 Production and sales of ethanol-based automobiles in Brazil (BNDES 2008)

Fig. 2.8 Sugarcane ethanol learning curve (Goldemberg et al. 2008)

2.4 Sustainability of Sugarcane Ethanol

The work of Silva et al. (1978) is also worth mentioning because it is considered as a
reference publication on Proálcool and should be recognized as one of the first to
address energy balance in the production of ethanol for different crops (Nogueira 1987).
A learning curve proposed by Goldemberg et al. (2004) and collaborators
illustrates how production costs have decreased as the quantity of ethanol has
increased (Fig. 2.8).
42 L. A. B. Cortez et al.

Several research institutions have made significant contributions in the area of

sustainability (Camargo 1990), including the IPT (Bonomi 2004), CETESB
(Technical Standard P4.231/2006) and INT (STI/INT 1976). Studies have been
performed on the digestion of vinasse; however, the digestion of vinasse has not
been incorporated in ethanol distilleries, with the exception of Usina São Mart-
inho. UNICA also published an important contribution that summarized 12 major
themes related to ethanol’s energy and environmental sustainability (Macedo-
Coord 2005). Several current ethanol research groups have concentrated their
efforts in trying to understand the socioeconomic and environmental issues arising
from sugarcane ethanol production and use (Macedo et al. 2008).

2.4.1 Impacts of Ethanol Fuel Use on Population Health

The academic community has made important contributions to public health issues
related to vehicle emissions. Saldiva from the Laboratory of Pathology—Medicine
(USP) studied the impact of ethanol fuel usage and the emissions generated by
more than 5–6 million cars (mostly flex-fuel) in the city of São Paulo. According
to Saldiva, replacing gasoline with ethanol fuel is an important public policy
measure that improves the health of populations living in large cities because
ethanol fuel helps to alleviate air pollution (UNICA 2009 and http://www.
worldcat.org/identities/lccn-no00-41804).

2.4.2 Social Science Research Related to Sugarcane Ethanol

Research on the social and economic impacts of sugarcane ethanol production and
use in Brazil has received substantial attention from researchers. Presented below
is a list of important contributions in ethanol production and use.
Balsadi and Borin (2006) applied a ‘‘Quality Index’’ based on earnings, level of
formality, education, and other forms of economic support to analyze the sugar-
cane sector with regard to improvement of employment (both in quantity and
quality) and concluded that each of these indicators had shown significant
improvement over the period studied, from 1990 to 2002.
Moraes (2007, 2009, and 2011a , b) analyzed several aspects of the sugarcane,
sugar, and ethanol labor markets in Brazil, including the evolution of socioeco-
nomic indicators (number of workers, wages, work formalization, and conditions,
etc.), sugarcane worker migration, and income determinants for workers in sug-
arcane plantations and in the sugar and ethanol industries (influence of education,
labor unions, region, etc.).
Moraes (2011a) analyzed the social externalities of fuels and compared indi-
cators between the sugarcane-ethanol and oil industries. They also estimated the
socioeconomic impacts of substituting gasoline with ethanol.
2 An Assessment of Brazilian Government Initiatives and Policies 43

Chagas et al. (2011) analyzed the effects of increased sugarcane production on

municipal revenues in the State of Sao Paulo. Their results suggested that there is a
significant and substantial increase in revenue with increased shares of sugarcane
in the municipal agricultural output.
Hofmann (2006) analyzed the effects of increased ethanol production on the
reduction of poverty in Brazil. The effects of increased ethanol production on the
country’s food security is primarily positive. Lack of food security in Brazil is
strongly associated with poverty, which should diminish with the increase in
employment and income that results from an expansion of sugarcane agribusiness,
thus, compensating for the negative effect of eventual increases in food prices.
Martinelli et al. (2011) compared development indicators in municipalities of
the State of São Paulo. A series of indices were used, including the following: the
human development index (HDI) of the UN, an HDI index based on São Paulo’s
social responsibility index (SRI) and the Rio de Janeiro municipal development
index (MDI). The results showed that the HDI, SRI, and MDI for cattle munici-
palities were significantly lower than for all the other categories, with the highest
results in municipalities with both sugarcane and processing mills, which were
higher than nonrural municipalities.
Assato and Moraes (2011) analyzed the socioeconomic impacts of the expan-
sion of the sugarcane sector in two municipalities of Mato Grosso do Sul State and
found an increase in aggregate income and improvements in education because of
educational programs installed after the expansion of the sugarcane industry.
Satolo and Bacchi (2013) evaluated the effects of the expansion of the sugar-
cane sector on the municipal per capita GDP in São Paulo State. The results from a
dynamic spatial panel indicated a positive impact on per capita GDP.
Moraes (2007) and Oliveira (2009) showed that sugarcane production results in
higher wages than other crops, a greater level of formal relations (meaning legal
protections) and a lower presence of child labor. Sugarcane is a crop with a greater
reliance on the external market and a larger production scale, so labor relations
tend to be more formalized and in line with legislation.
Sallum (2007), Moraes and Pessini (2004), and Moraes (2009, 2011a) analyzed
the institutional and organizational environment of the labor market in the sugarcane
industry and observed that there are clear and specific rules governing the labor
market. The authors also demonstrated that employer associations and labor unions
within the state of São Paulo were strong and highly active and engaged in wage
negotiations for the sugarcane workers at the beginning of each harvest season.

2.4.3 Planning Land Use for Bioenergy in Brazil

The prospects of a rapid expansion of the sugarcane sector for the production of
bioenergy intended for export has had a major impact on land-use planning in
Brazil, such as the Brazilian Land Use Model (BLUM) (Nassar et al. 2009), the
Agro-Ecological Zoning of Sugarcane, which was prepared by the Ministry of
44 L. A. B. Cortez et al.

Fig. 2.9 Sugarcane agro-ecological zoning in Brazil (MMA 2009) http://www.cnps.embrapa.br/

zoneamento_cana_de_acucar/ZonCana.pdf

Environment (MMA) (Fig. 2.9), the Agro-environmental Zoning for the cultiva-
tion of sugarcane, which was released on 18/09/2008 by the government of the
State of São Paulo (Fig. 2.10) and based on the work of the BIOTA FAPESP, and
the IAC work coordinated by Orivaldo Brunini in 2008, which resulted in the
publishing of an agro-climatic suitability map for the state of São Paulo.
2 An Assessment of Brazilian Government Initiatives and Policies 45

Fig. 2.10 Science-based sugarcane agroenvironmental zoning in São Paulo State http://www.
ambiente.sp.gov.br/etanolverde/zoneamento-agroambiental/

2.4.4 The R&D Contribution of the Sugar and Ethanol

Private Sector

The sugar and ethanol private sector has contributed substantially to consolidating
the industry. In agriculture, contributions have been made to research on new
varieties and on planting techniques such as the ‘‘plene’’ planting system, which
was developed by Syngenta (http://www.syngenta.com/COUNTRY/BR/PT/
PRODUTOSEMARCAS/PLENE/Pages/Tecnologia-plene.aspx). (Several indus-
tries have introduced harvesters (e.g., John Deere, Case, Valtra, Santal) and Jacto
is developing a new concept (ETC) with CTBE (http://www.bioetanol.org.br/
noticias/detalhe.php?ID=NDY2).
The New Holland/CTC partnership on raw sugarcane harvesting has developed a
system that simultaneously harvests sugarcane and straw and cleans the straw before
industrial use (http://www.bioetanol.org.br/noticias/detalhe.php?ID=NDY2).
Brazilian industry has also conducted important research on sugarcane ethanol.
Dedini researched the integration of ethanol and biodiesel production and the or-
ganosolv process of hydrolysis of bagasse, known as Dedini Rapid Hydrolysis
(DHR) (Dedini 2008). Dedini and Fermentec developed a process for reducing
amounts of vinasse (http://www.slideshare.net/tabVlae/dedini-fermentec-vinasse-
concentration). Dedini also introduced environmentally sustainable solutions
designed to reduce water consumption in the process of ethanol production (BIO-
WATER) and recycle solid and liquid waste for use as a fertilizer (BIOFOM).
46 L. A. B. Cortez et al.

Additional innovations were developed by Braskem, who produced green

plastics (polyethylene) using ethanol as the raw material (http://www.braskem.
com.br/site.aspx/plastic-green).
In the recent years, significant efforts in increasing ethanol production have
been made by the private sector in conjunction with federal and state agencies,
such as the GranBio project in Alagoas, Brazil where an ethanol plant is being
built and is expected to start operations in 2014 (http://www.novacana.com/n/
etanol/2-geracao-celulose/graalbio-preve-usinas-etanol-2g-210313/%23).

2.4.5 Fostering Bioenergy Research in Brazil

Research funding in Brazil is comprised of federal, state, and private initiatives. In

building the Brazilian research system, public agencies have been instrumental in
improving the quality of research and consolidating graduate programs. Public
agencies have made considerable efforts in stimulating businesses to fund research
programs, supporting innovative R&D for small businesses, and establishing
partnerships for jointly funding research with medium and large companies.
Both the National Research Council—CNPq, which promotes research in
Brazil, and the National Fund for Research and Projects—FINEP, which is ded-
icated to supporting projects with the participation of industry, have been active in
financing bioenergy research since the beginning of Proálcool. CNPq played a key
role in the early stages of the Proálcool program when it published the book
‘‘Rating the technological ethyl alcohol’’ (Anciães 1978), which was a benchmark
in the field of ethanol production. CNPq also acted decisively in offering schol-
arships to graduates within and outside Brazil at a time when national programs
were not yet fully developed.
In the latter half of the 1990s, the federal government created ‘‘sectorial funds’’
(Pacheco 2007). CT-Energ (http://www.mct.gov.br/index.php/content/view/1410/
CT___Energ.html) was designed to stimulate energy research and innovation in
Brazil and addressed all technical aspects of energy except oil, which had its own
specific fund, CT-Petro. Coordinated by ANEEL and ANP together with energy
utility companies, funds were raised for R&D and energy efficiency. Bioenergy
research to promote innovation that results in better yields and more reliable
services has benefited from these resources. However, much more would be gained
if there was greater integration of resources, for example, especially in overcoming
difficulties related to bioelectricity.
The State of São Paulo Research Funding Agency—FAPESP has contributed
considerable resources to bioenergy research (FAPESP 2007). In 1998, the SU-
CEST project, funded by FAPESP as part of its Genome Program, initiated the
sequencing of sugarcane-expressed sequence tags (ESTs). Sequencing the giant
genome of sugarcane, where one gene is represented by an average of 10 alleles, is
a major challenge. At the time, collections of ESTs represented as a fast alternative
2 An Assessment of Brazilian Government Initiatives and Policies 47

Fig. 2.11 Number of

scientific articles on
sugarcane cited at ISI WoS
for main sugar-producing
countries (Source Brito 2012)

for the initial characterization of a genome. SUCEST significantly contributed to

the identification of genes associated with agronomic traits of interest. Approxi-
mately, 43,000 genes were identified (Vettore et al. 2003). This initiative was
instrumental in the formation of a network of genome researchers in Brazil and
placed the country in the lead of sugarcane-indexed international publications
(Fig. 2.11). A survey of research on sugarcane and ethanol funded by FAPESP in
recent years has been published in the book ‘‘Brazil world leader in knowledge and
technology of sugarcane ethanol.’’

2.4.5.1 Scientific Publications in Sugarcane in Brazil

In Brazil, the Institute of Sugar and Alcohol, created in the 1930s, provided a
major contribution to the study of bioenergy by publishing the magazine Brasil
Açucareiro and Anuário Açucareiro, which was a yearbook-type of publication
that was started in 1935 and discontinued in 1975. The Society of Technical and
Sugar and Ethanol Producers of Brazil—STAB currently represents the only sci-
entific magazine in the country in the sugar-ethanol sector. An inventory of
publications held by Vian and Corrente (2007) allows a better understanding of
how the industry disseminates its knowledge.

2.4.5.2 Scientific Publications in Sugarcane in Indexed Journals

Highlighting the Evolution of Research in Brazil and the State
of São Paulo

The SUCEST project was succeeded by two initiatives led by researchers at USP
and UNICAMP and a ‘‘spin-off’’ biotechnology company. The SUCEST-FUN
project (http://sucest-fun.org), which was started in 2003, focused on the identi-
fication of genes associated with agronomic traits of interest (such as yield,
48 L. A. B. Cortez et al.

tolerance to biotic and abiotic stresses, mineral nutrition, sugar content, and
responses to climate change). The project was based at the Institute of Chemistry,
USP, and was a collaboration of groups from USP, UNICAMP, UFSCar, CTC,
IAC, UFRJ, UFPE, UFRPE, UFAL, and RIDESA. Genes associated with sucrose
content and drought resistance have been patented, and transgenic plants were
developed that should contribute to the improvement of sugarcane. Concurrently, a
project based at CBMEG, UNICAMP, developed molecular markers,
genetic-statistical tools, and a functional genetic map for breeding progenies of the
CTC, IAC, and RIDESA programs. These initiatives allowed the development of
molecular tools that promise to accelerate the release of new cultivars by classical
breeding programs (Cantarella et al. 2012).
Alellyx was found in February 2002, by a group of molecular biologists and
bioinformaticians involved in the SUCEST project or the FAPESP Genome Pro-
gram. Set in Campinas, Alellyx operated in partnership with CanaVialis, which
was found in 2003, and whose focus was sugarcane breeding. Together, Allelyx
and CanaVialis represented one of the most modern sugarcane breeding programs
in the world and had an important influence on the FAPESP Genome Program.
After the initial phase of venture capital investments by Votorantim New Business,
both companies were acquired by Monsanto in what was one of the biggest pur-
chases of a start-up company in Brazil at the time.
In 2008, FAPESP created the FAPESP Bioenergy Research Program BIOEN
(http://bioenfapesp.org). BIOEN is organized in five divisions: Biomass, Biofuel
Technologies, Biorefineries, Engines, and Sustainability and Impacts. FAPESP
establishes partnerships with national and international funding agencies and
businesses in its efforts to articulate and integrate research initiatives. CNPq
resources, for instance, were mobilized under Pronex and INCT joint grants. The
private sector is also represented with BIOEN agreements that involve public
research institutions and companies such as Braskem, Dedini, Oxiteno, Microsoft
Research, ETH, Boeing, BP, PSA, BE-Basic, and Vale, all of which share human,
material, and financial resources. In 2013, the program had grown to more than
400 researchers in over 20 countries. Considering its size, broad research spectrum
and the depth of its goals, BIOEN can be considered as one of the most important
bioenergy research programs in the world. BIOEN was built on a solid base of
exploratory academic research that is generating new knowledge and highly
qualified experts, which are essential for enhancing the industry’s ability to run on
ethanol technologies and increasing internal and external competitiveness.
A strategically important FAPESP project was the Project for Public Policy
Research on Ethanol (PPP Ethanol), developed in partnership with APTA (www.
apta.sp.gov.br/cana). PPP Ethanol promoted a broad discussion of the entire eth-
anol production chain from sugarcane, with researchers from academia and the
private sector. This research produced a technology roadmap for the sector and
resulted in the publication of the book ‘‘Bioethanol from Sugarcane: research &
development productivity and sustainability’’ (Cortez-Coord. 2010).
2 An Assessment of Brazilian Government Initiatives and Policies 49

2.4.6 New Frontier Research on Sugarcane Bioenergy

2.4.6.1 Breeding, Molecular Biology, Genomics, and GMOs

In 2009, BIOEN catalyzed a series of discussions on biotechnological paths for the

improvement of sugarcane, including the development of new sugarcane varieties
with increased yield, tolerance to stress (especially drought), and adaptations to the
soil and climate conditions of areas where sugarcane expansion is taking place
(mid-west, south, and north east) (Hotta et al. 2010). Defined research priorities
included (1) obtaining the sequence of a reference genome of sugarcane (2)
developing molecular markers and genetic maps to assist breeding programs in the
choice of parents and progenies (3) understanding the physiological processes
underlining the partition of carbon, photosynthesis, and use of water (4) discov-
ering genes and functional genomics for identifying genes of interest, and (5)
researching the stable production of transgenic plants. Challenges and bottlenecks
in breeding were recognized as well as the role of high-performance technologies
in overcoming breeding issues.
Improving yield and resilience of sugarcane varieties can be achieved by
integrating agronomic practices, adequate management practices, traditional
breeding and molecular-assisted breeding, and development of transgenic plants.
The discovery of genes associated with agronomical and physiological traits will
provide the necessary knowledge for the development of cultivars dedicated to the
production of bioenergy (energy cane) or to be used as biofactories.
It is expected that the amount of information regarding sugarcane will increase
exponentially in the coming years in response to research incentives. The devel-
opment of integrated databases, such as the SUCEST-FUN Database (http://
sucest-fun.org) is essential for the optimal use of research results. In particular,
collecting large amounts of sequence data will require the implementation of
bioinformatics and databases for information management. This demand will be
similar in systems biology projects designed for an integrated understanding of the
various aspects of plant growth and their adaptive development to different
environments. Project data management will be absolutely crucial for the inte-
gration of heterogeneous data coming from different methodologies and groups.

2.4.6.2 Second-Generation Technologies

Lignocellulosic Ethanol (hydrolysis)

Hydrolysis research in Brazil began with José Carlos Campana Gerez, Institute of
Chemistry, UNICAMP, in the late 1970s with studies on acid hydrolysis. These
works led to the installation of a pilot plant on the UNICAMP campus, but the
project was discontinued in the early 1980s because of a lack of resources.
However, the process resulted in patent #PI8203026-0 (1982).
50 L. A. B. Cortez et al.

A 1979 initiative of the federal government saw the passage of Law No. 6.768
that established the company COALBRA—Coke and Alcohol Wood S/A, which
was based on technology of Russian origin and designed to convert wood into
methanol by acid hydrolysis. This project was coordinated by Sérgio Motta, who
acquired a full-scale industrial unit that was installed in Minas Gerais, near U-
berlândia, but was discontinued because of technical difficulties and a lack of
funding. At the beginning of the 1980s, CESP (Companhia Energética do Estado
de São Paulo) installed a pilot plant to gasify wood and produce methanol from
synthesis gas, which was also discontinued.
Research on cellulosic ethanol production started again in the mid-1990s with
studies by Dedini in partnership with the CTC and FAPESP and coordinated by
Carlos Eduardo Vaz Rossell. The research, based on the organosolv pretreatment
of bagasse, was used in a demonstration plant installed at São Luiz Mill in Pira-
ssununga, SP. The plant had the capacity to produce 5000 L of ethanol/day, taking
advantage of the synergy of an integrated process in the first-generation. Because
of technical problems regarding the sugarcane bagasse supply, the production of
inhibitory compounds for fermentation and a viable use for the fraction of lignin, it
became clear that additional research was required at a smaller scale. However, the
studies indicated important technical challenges that helped to drive future studies
in the area.
In 2005, the Ministry of Science, Technology & Innovation- MCTI, created the
Bioethanol Network, which was coordinated by Rogério Cezar de Cerqueira Leite,
UNICAMP. The network’s activities involved several universities and research
institutions (such as CTC) and attempted to identify necessary skills and contribute
to the development of a technology to produce cellulosic ethanol in Brazil (http://
cenbio.iee.usp.br/projetos/bioetanol.htm). After 3 years of intense work, the Bio-
ethanol Network program was able to identify skills as well as scientific and
technological barriers and served as a foundation for the creation of the hydrolysis
program at CTBE (National Laboratory of Science and Technology of Bioetha-
nol). The hydrolysis program at CTBE is coordinated by Carlos Eduardo Vaz
Rossell and aims to make improvements in the four basic areas of enzymatic
hydrolysis: pretreatment, enzymes, hydrolysis, and fermentation. Facilities were
installed at the CTBE for conducting experiments at laboratory scale and pilot
plant scale (up to 500 L). The pilot plant is a flexible unit, and the cellulosic
ethanol conceptual process is designed to make use of the biorefinery concept,
which may introduce many potential product options in addition to bioethanol
(http://www.bioetanol.org.br/).
CENPES/Petrobras is also developing a program to research cellulosic ethanol,
especially in the area of enzyme production, in association with universities and
domestic and foreign companies. Dedini has also worked with several companies
in search of robust and economically competitive enzymatic hydrolysis processes.
Other groups have also emphasized research work in the area of enzymatic
hydrolysis, such as the CTC and Luiz Ramos at UFPR.
2 An Assessment of Brazilian Government Initiatives and Policies 51

Considering the worldwide efforts in hydrolysis, it is now understood that the

problem is quite complex and offers an opportunity to bring together more basic
research, such as understanding the deconstruction of the cane fiber, and techno-
logical challenges, such as creating efficient processes for the production of
enzymes and obtaining enzymes robust enough to operate in an industrial envi-
ronment at lower prices. Given the highly favorable characteristics of fiber and the
availability of utilities, the 1G plant environment is considered very suitable for
coupling a 2G plant. In addition, ethanol fuel hydrolysis technology will allow the
plants to make use of the biorefinery concept to produce high-value molecules,
such as polymers, and develop innovative products.

The BNDES PAISS Program to Promote Second-Generation Ethanol

The PAISS is a joint initiative of the BNDES and FINEP, which are a selection of
business plans and development projects that include the development, production,
and commercialization of new technologies intended for the industrial processing
of biomass derived from sugarcane. The purpose of PAISS is to organize requests
for financial assistance under the two institutions to allow greater coordination of
actions for development and better integration of available financial support
instruments http://www.bndes.gov.br/SiteBNDES/bndes/bndes_pt/Areas_de_Atuacao/
Inovacao/paiss/.
The PAISS program is prepared to invest R$1 billion (nearly US$400 million)
to install pilot plants and demonstrate innovative technologies in this area. The
company GraanBio (GranBio) is planning a demonstration unit for second-gen-
eration bioethanol from sugarcane bagasse that should be operational by the first
semester of 2014. The mill will have the capacity to produce 82 million liters/year,
and it will use innovative solvent-free processes in the pretreatment step. The
initiative will offer an opportunity to evaluate the technology and obtain process
data and information for the design of large-scale units.

Thermoconversion Technologies: Torrefaction, Gasification, Pyrolysis,

and Combustion

Experimental work on the thermoconversion of biomass (torrefaction, pyrolysis,

gasification, and combustion) was started at the Institute for Technological Research
(IPT) in São Paulo in the 1970s when the combustion laboratory was created.
Two groups stand out in this area: Carlos Luengo at IFGW-UNICAMP, who
conducted basic research into the processes of thermal conversion of biomass
pyrolysis and roasting, and Saul D’Avila at FEQ-UNICAMP, who researched and
formed many frames in gasification and pyrolysis of biomass. Concurrently,
CIETEC of Rio Grande do Sul was conducting research in the area of thermal
biomass conversion. The CTC in the 1990s led first phase of the GEF project with
the support from the World Bank to conduct research and gather information about
52 L. A. B. Cortez et al.

the gasification of straw and bagasse. This project was designed for the eventual
construction of an advanced gasification pilot plant, but was halted because of
funding problems. CHESF, with the participation of Shell and support from GEF
(Global Environmental Facility), attempted to complete the second phase with a
focus on gasification of eucalyptus wood, but because of administrative and
financial difficulties, the second phase of the project was never completed. The IPT
currently continues to work in this area in collaboration with Swedish industries
that research pyrolysis charcoal. The work performed by the UFMG (Maria Emilia
Rezende) resulted in the creation of the company Biocarbo, although it is not
involved in managing sugarcane biomass. As a result of the efforts of Saul
D’Avila, José Cláudio Moura and Themistocles Rocha, the Termoquip company
was started in the region of Campinas and produces biomass gasifiers, including
Petrobras, and has been instrumental in the creation of various reactors used for
research at UNICAMP (FEQ, FEM and AEC) and UNIFEI. A spin-off company
called Bioware produces thermal conversion technology for sugarcane and pro-
duces products such as bio-oil, acids and pyrolytic carbon. A lab-scale pyrolysis
and gasification unit was designed and constructed specifically to process sugar-
cane bagasse and straw and is currently in operation with the focus of obtaining
kinetic data for the reaction and gathering useful information for process scale-up.
The project is coordinated by Rubens Maciel Filho at FEQ/UNICAMP.

2.4.7 New Bioenergy Research Centers and a Graduate

Program in Bioenergy in Brazil

The new generation of bioenergy research centers in Brazil linked to the resurgence
of ethanol have been motivated by events of this century such as the electricity
‘‘blackout’’ in 2002, production of flex-fuel cars, and interest by the United States in
second-generation ethanol research, which is considered more sustainable than the
first-generation when compared to ethanol from corn and other cereals.
In the early 1990s, he National Reference Center for Biomass-CENBIO was
created to develop research activities in conjunction with universities and compa-
nies in the area of bioenergy. CENBIO has made important contributions to gov-
ernment policies at the state level, such as studies conducted by the State Committee
for Bioenergy and coordinated by José Goldemberg (Goldemberg et al. 2008).
In 2005, a project coordinated by Rogério Cerqueira Leite was begun that per-
forms a series of studies with the Center for Strategic Studies and Management in
Science, Technology, and Innovation—CGEE to study issues involved with the
possibility of replacing 10 % of all gasoline consumed worldwide with ethanol from
sugar cane by 2025, which would constitute an increase of approximately 10 times
the ethanol currently produced in a season (Leite 2009 and Leite et al. 2009). This
study allowed Brazil to better understand the importance of producing high-level
2 An Assessment of Brazilian Government Initiatives and Policies 53

research toward the sustainable use of whole-sugarcane resources. Created officially

in 2008, the National Laboratory of Science and Technology of Bioethanol—CTBE
at the CNPEM in Campinas, SP began 5 research programs covering basic research,
mechanization, minimum impact agriculture, hydrolysis, and virtual biorefinery
sustainability.
The federal government created the Agroenergy Embrapa Center in 2006 in
Brasilia research issues related to biodiesel, ethanol, and the energetic use of
agricultural and forest residues.
Petrobras Biofuels has promoted the engagement of CENPES in the research of
biofuels and is primarily studying second-generation ethanol.
The reorganization of IAC sugarcane research that was started in the 1990s led
to the creation of the Centro Cana IAC/APTA in Ribeirão Preto, where labora-
tories were installed to research plant breeding, agricultural entomology, bio-
technology, agribusiness technology, and management of sugarcane varieties. The
research center currently houses a public collection of germplasm of sugarcane.
The creation of the State of São Paulo Bioenergy Research Center was also
important. This center is a consortium of three state universities in São Paulo
(USP, UNICAMP and UNESP) and FAPESP and will have a research budget of
$75 million. The infrastructure is funded by the government of the State of São
Paulo, and the universities are already hiring professors. FAPESP’s role is to fund
high-quality research in bioenergy.
A new Ph.D. program in bioenergy is being jointly implemented by USP, UNI-
CAMP, and UNESP in an attempt to create highly qualified human resources. This
initiative demonstrates the commitment of these universities in promoting long-term
research and innovation in the field of bioenergy in Brazil. The new program will
have a strong international orientation and will collaborate with the best universities
and research centers around the world http://agencia.fapesp.br/en/17301.

2.5 The Use of Vegetable Oils for Biodiesel Production

2.5.1 Brazilian Biodiesel Research from Vegetable Oils

When Proálcool was first begun, the federal government also proposed ‘‘Proóleo,’’
a national program based on the production of vegetable oil fuel to replace diesel.
Unlike the sugar and alcohol sector, which had already organized, Proóleo did
not have a sector or culture to lean on in 1970s. At the time, soy was a nascent
culture in Brazil, and other oilseeds, including palm, were not produced com-
mercially or at scale. Fernando Homem de Melo and Eduardo Giannetti da
Fonseca from USP, writing in ‘‘Proálcool, Energy and Transports,’’ calculated land
requirements for different cultures of oil crops (Melo and Fonseca 1981).
In 1969, Leopold Hartman at FEA—UNICAMP published an important article
on the transesterification of vegetable oils and created the Laboratory of Oils and
54 L. A. B. Cortez et al.

Fats with the support of GTZ (Informativo SBCTA 2005). The first patent for
biodiesel and aviation jet fuel in Brazil (PI8007957) is credited to Expedito Pa-
rente in 1980. Ulf Schuchardt from IQ—UNICAMP patented the use of vegetable
oils for fuel purposes (#PI 8302366-6) and published ‘‘Continuous reactor with
organic heterogenized catalysts for transesterification of vegetable oils’’ in 1982
and ‘‘Process for the preparation of esters with organic catalysts and method of
rapid determination of the composition of oils and fats’’ in 1983.
Although there was available technology to produce biodiesel in Brazil,
problems remained concerning the raw material. Replacing diesel fuel would
require the cultivation of an energy crop for oil, as sugarcane was for ethanol.
Although the cultivation of palm is considered equivalent to sugarcane, an agri-
cultural sector did not exist that was sufficiently organized to realize this potential.
However, recent information indicates that the palm crop is expanding in the State
of Pará.
Biodiesel is popular in Europe, where it is produced with rapeseed, and was
initially considered as an option in Brazil that could meet social goals and alleviate
dependence on diesel. On January 13, 2005, the federal government enacted Law
No. 11.097, which created the National Program for Production and Use of Bio-
diesel—PNPB. Despite initially suggesting the production of biodiesel in family
units and making use of transesterification of ethanol, more than 80 % of biodiesel
in Brazil is currently produced using soybeans and methanol because of the
absence of other oil crops on the scale of soybeans and the technical difficulties in
the transesterification of ethanol.
If Brazil invests in oil crops with energy potential such as oil palm and produces
the equivalent of the biodiesel yield in Malaysia and Indonesia, which is
approximately 5,000 L of oil/ha year, then a sustainable program to replace diesel
with biodiesel can be developed, an example of which has been developed in
Colombia.

2.5.2 Other Routes for Biodiesel Production

The sector of renewable chemicals is also considering of producing new com-

pounds and biofuels via biological routes, which is called synthetic biology.
Synthetic biology is engaged in the construction of new components and biological
systems or redesigning natural systems using evolutionary processes. These arti-
ficial systems can perform new tasks, such as the production of plastics, bio-
kerosene and bio-gasoline. A major initiative is underway by Amyris, a California
company that opened a subsidiary in Brazil for the production of new biofuels such
as biodiesel and aviation fuel from sugarcane sucrose (Amyris 2008). Amyris’s
initial goal was to develop technology that allowed the production of an antima-
larial drug, artemisinin, by microorganisms. The platform is being applied
industrially to the development of yeast with the ability to produce gasoline and
2 An Assessment of Brazilian Government Initiatives and Policies 55

kerosene and to the scheduling of production processes for farnesenes. This

approach must be improved to match production requirements and market prices,
especially with respect to bio-kerosene.
The production of biodiesel from algae should also be mentioned, particularly
for its ability to minimize land use for biofuels. This technology aims to produce
third generation biofuels, which are produced from the use of CO2.
A group led by Franco and Maciel at FEQ-UNICAMP and Ana Teresa Lom-
bardi (UFSCar) have been researching the use of algae to produce biodiesel;
however, production of biodiesel from algae is still somewhat advanced in Brazil,
although it deserves attention, especially because it is so innovative in that it does
not require land and makes use of CO2. Numerous lab-scale studies are evaluating
the use of microalgae as a lipids feedstock for biodiesel, and many interesting
options are under consideration, especially those that integrate CO2 from ethanol
mill fermentation to increase microalgae growth (research project funding by
FAPESP 2008/57873-8).

2.6 Future Perspectives of Biofuels for Aviation in Brazil

In October 2011, Boeing, Embraer and FAPESP formally agreed to investigate

how Brazil could contribute to the production of sustainable biofuels for aviation
in an attempt to reduce CO2 emissions. The result was Sustainable Aviation
Biofuels Brazil, a national assessment of the technological, economic and sus-
tainability challenges, and opportunities associated with the development and
commercialization of sustainable aviation biofuel in Brazil.
Multiple possible pathways to produce biofuels for aviation were identified
during the project. Certification requirements for use in commercial aviation are
established internationally according to ASTM D7566, which contains one special
annex for each approved alternative jet fuel production process. Figure 2.12 pre-
sents an overview of all identified pathways pertinent to Brazil, including the
denomination and status of the ASTM approval process. As depicted, two of the
final jet fuel production processes are already approved (green boxes in Fig. 2.12),
and several others are still under analysis in ASTM’s Emerging Fuels Committee.
The project concludes that Brazil has exceptional conditions to develop and
produce sustainable biofuels for aviation. However, additional research is required
to develop the identified pathways and additional effort is required to improve the
transportation infrastructure to lower raw material costs. More information can be
found here: http://www.fapesp.br/publicacoes/flightpath-to-aviation-biofuels-in-brazil-
action-plan.pdf.
Recently, the Brazilian National Agency for Oil, Natural Gas and Biofuels-
ANP approved resolutions for biofuels for aviation in Brazil (http://www.
petronoticias.com.br/archives/31531).
56 L. A. B. Cortez et al.

Fig. 2.12 Identified pathways for the production of sustainable jet biofuel in Brazil [Note HEFA
Hydroprocessed Esters and Fatty Acids; CH Catalytic Hydrothermolysis; DSHC Direct
fermentation of Sugars to Hydrocarbons; ATJ Alcohol to Jet; FT Fischer-Tropsch hydropro-
cessed-synthesized paraffinic kerosene; HDCJ Hydrotreated Depolymerized Cellulosic to Jet]
(Boeing et al. 2013)

2.7 Conclusions

Brazil has a prosperous future in the field of bioenergy. The participation of

sugarcane in the Brazilian energy matrix has grown 1 % per year since 2002,
reaching 19 % in 2010. Although the potential is much larger, not only for sug-
arcane but for eucalyptus, palm tree, and innumerous other crops, the country has
already built a significant history in the bioenergy area. However, much more can
be done, particularly in adding combined value to products and utilizing concepts
that produce sustainable solutions to improve the industry in the new agricultural
frontiers of Brazil.
The long-term role played by the Brazilian government in promoting biofuels is
considered as a key factor to success, particularly with sugarcane ethanol. Gov-
ernment-funded research agencies have played a strategic role in consolidating
knowledge and human capacity to maintain leadership in the bioenergy sector.
However, investments in research and development of human resources in the
area of bioenergy must also grow proportionately, particularly in the private sector.
With new research centers, graduate programs have the potential to contribute to
increasing competence at all stages of bioenergy development.
2 An Assessment of Brazilian Government Initiatives and Policies 57

Acknowledgments Special thanks to Antonio Bonomi, Carlos Eduardo Vaz Rossell, Cylon
Gonçalves, Isaías Macedo, José Luiz Olivério, José Goldemberg, Luiz Augusto Horta Nogueira,
Manoel Regis Lima Verde Leal, Márcia Azanha de Moraes, Marco Aurélio Pinheiro Lima,
Marcos Landell, Oscar Antonio Braunbeck, Paulo Soares, Rogério Cezar de Cerqueira Leite, and
Terezinha de Fátima Cardoso for their contributions in this text.

References

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Chapter 3
Renewable Liquid Transportation
Fuels: The Cornerstone of the Success
of Brazilian Bioenergy Program

Veronica de Araujo Bruno and Adilson Roberto Gonçalves

Abstract This chapter presents the historical evolution, together with social and
economical aspects concerning biofuels in Brazil. The country plays an important
role due to its large land area and the tropical climate, favoring sugarcane and
soybean cultures. In respect to ethanol, Brazil has reached process and technology
maturity, the production is rising and the market will grow in the coming decades,
drived especially by flex-fuels engines. Currently, attention is also focused on
second-generation ethanol, obtained from lignocellulosic materials. Networks for
ethanol production are much more structured, integrated, and developed than those
for the production of biodiesel. Addition of 2 % biodiesel from soybean to regular
diesel, contributed significantly to increase domestic production of this biofuel,
pushing Brazil to a global context. In 2010, this percentage increased to 5 % and is
forecasted to reach 20 % in 2020. When anhydrous ethanol from sugarcane is
mixed with gasoline at a 25 % ratio, 1900 kg CO2 eq/m3 of bioethanol is avoided.
The use of biodiesel to replace diesel fuel reduces 90 % emissions of burning gas
and 78 % of smoke emissions.

3.1 Introduction

During the 1970s OPEC decided to raise the oil price by 70 %. Countries
depending on this fuel were forced to develop new sources of energy. As one of
those countries, Brazil began the intensification of programs supporting the energy
matrix diversification, oil crisis being a driver for the biomass fuel’s development.

V. de Araujo Bruno
Engineering College at Lorena, University of São Paulo, Lorena, Brazil
A. R. Gonçalves (&)
Brazilian Bioethanol Science and Technology Laboratory (CTBE),
R. Giuseppe Máximo Scolfaro, 10000—Polo II de Alta Tecnologia,
Cx. Postal 6192, Campinas, SP CEP 13.083-970, Brazil
e-mail: adilson.goncalves@bioetanol.org.br

S. S. da Silva and A. K. Chandel (eds.), Biofuels in Brazil, 61

DOI: 10.1007/978-3-319-05020-1_3,  Springer International Publishing Switzerland 2014
62 V. de Araujo Bruno and A. R. Gonçalves

However, the national interest in biofuels dates back much earlier. Since 1920,
Brazil produces vegetable oils and the first official push for the production of
renewable fuels was in 1938: law 737 ruling the blending of ethanol in gasoline
nationwide (Goldemberg et al. 2004).
Currently, research in renewable fuels is focused on reducing greenhouse gas
emissions and on energy security supply (Masieiro and Lopes 2008). Brazil plays
an important role due to its large land area and the tropical climate, favoring
sugarcane and soybean cultures. The energy agriculture could be incorporated in
the Brazilian economy without competing with agriculture for food, as well as be
possible to perform multiple cultures within the year.
In respect to ethanol, Brazil has reached process and technology maturity, the
production is rising and the market will grow in the coming decades (Brazilian
Government 2011). Brazilian ethanol production can be divided into four stages.
The first was from the beginning to the 1970s, when production was intended
mainly to manufacturing beverages, considering ethanol as a by-product of sugar
production. The second stage began with the creation of Proalcool in 1975 which
determined that ethanol be mixed with gasoline in cars. Already in 1979 the
second phase of the program was initiated, the production reached 3.4 million m3,
and the goal of the program became the incentive for the production of cars that
moved entirely on ethanol. At that time, there was great expansion in the sector
due to the large number of autonomic distilleries. In 1985, the production of
Brazilian ethanol accounted for about 80 % of world production. The program,
however, lived the end of its apogee with the drastic drop in oil prices, reducing
the attractiveness. Recently, after almost 20 years, ethanol production has entered
a new stage with automotive flex fuel engines that operate with fractions of gas-
oline and ethanol in any proportion (Milanez et al. 2008).
Currently, attention is also focused on second-generation ethanol, obtained
from lignocellulosic materials. Ethanol from straw and sugarcane bagasse is
promising and a sustainable alternative to the by-products from the sugarcane
industry and does not imply the need for an additional plantation area. However,
technical difficulties are at play for the extraction of sugars from lignocellulosic
matrix for subsequent fermentation. Hydrolytic enzymes together with chemical
and physical treatments are necessary to break the biomass structure, allowing the
cellulose be accessible (Masiero and Lopes 2008).
While studies of renewable fuels started in Brazil in the 1920s, only in 1975 the
use of vegetable oils for energy purposes was actually proposed. This occurred
through the Pro-Óleo program, which aimed to sell surplus of vegetable oil,
including a blend of 30 % vegetable oil in diesel and increase up to 100 % in long-
term use. Decrease in oil price in 1986 decreased research incentives. In 2004,
biodiesel production was resumed with the National Program for Production and
Use of Biodiesel (PNPB), which included the addition of 2 % biodiesel to regular
diesel, contributing significantly to increase domestic production, pushing Brazil
to a global context. In 2010, this percentage increased to 5 % and is forecasted to
reach 20 % in 2020 (ANP 2011).
3 Renewable Liquid Transportation Fuels 63

In Brazil, ethanol is responsible for the majority percentage (16 %) of energy

from biomass, while less than 3 % is derived from biodiesel. According to the
Ministry of Mines and Energy in 2010–2011, about 28 million m3 of ethanol, and
2.4 million m3 biodiesel (MME 2011) were produced.

3.2 Sugarcane and Soybeans: The Foundations

of the Brazilian Biofuel

Brazil has 152.5 million ha of total available arable land (17.9 % of the territory),
and 62.5 million ha (7.3 %) are already under use (Safras e Mercado 2012).
Currently in Brazil, 90 % of vegetable oil is produced from soy and 80 % of
industries produce biodiesel using soybean oil as feedstock. The remaining cor-
responds to animal fat (15 %) and other oilseeds (5 %) (Castellanelli 2008).
According to Embrapa (2011), the 2010/2011 national harvest produced
75 million tons of soybean, making Brazil the second largest producer in the
world, standing just behind the United States. Soybean emerges as the main raw
material for the production of domestic biodiesel (Embrapa 2011).
Sugarcane occupies the first position in agricultural production (675 million tons
in 2011), making Brazil the world’s largest sugarcane producer, but the second
ethanol producer, United States being the first.
In Brazil there are two crops of sugarcane, depending on rainfall patterns: one
occurs in the South-Central region (April to December) and the other in the North-
Northeast (October to March). Thus an integration of crops occurs, allowing
ethanol supply throughout the year (Gorren 2009).
The ideal climate provides a long, hot season with high solar irradiation and
moisture from rainfall, and another season reasonably dry but sunny and cool,
frost-free for the ripening and cultivation. The total amount of water decreases in
the cane growing, going from 83 % in young up to 71 % in mature plants.
However, the sucrose content varies from 10 to 45 % (Embrapa 2009).
Besides, the natural conditions of the Brazilian territory are favorable to
growing sugarcane for obtaining ethanol; other advantages are also found com-
pared to alcohol from corn. Planting corn uses high amounts of pesticides from
fossil fuels; processing the alcohol from corn emits more CO2 and the productivity
is low compared to ethanol from sugarcane (Schaeffer 2007).
Although Brazilian ethanol still finds obstacles for its export, the country
configures worldwide as a protagonist in the production of clean, renewable
energy, and recognized due to its large potential in the field (Rached 2011).
64 V. de Araujo Bruno and A. R. Gonçalves

3.2.1 Ethanol

There are two types of ethanol, hydrated and anhydrous. The alcohol resulting from
biological fermentation of sucrose is hydrated, a colorless liquid whose composi-
tion is approximately 5 % water. To obtain anhydrous ethanol, a further dehy-
dration step is needed to decrease the percentage of water to 0.5 % (Gorren 2009).
Distilleries are those capable of producing ethanol and sugar in varied pro-
portions, while independent distilleries are those that are dedicated exclusively to
the production of alcohol.
Production of alcohol is a series of interconnected unit operations, with the
main objective of converting sugar into alcohol, consisting basically of two main
steps, fermentation and distillation.
After grinding, the sugar syrup (molasses) is adjusted regarding sugar con-
centration, acidity, nutrients, and antiseptics. Yeast is added and, after fermenta-
tion time, the wine is separated from the yeast that is recovered for a new use
cycle. The wine is transferred to decanters and after other cleaning processes
distilled to obtain ethanol (Castro 2011). The most widely used process in the
ethanol industry is Melle-Boinot-Almeida, consisting of a batch process with cell
recycling.

3.2.2 Biodiesel

Biodiesel is a synthetic fuel made from vegetable oils, animal fats, algae, or fungi.
The most common way of producing this fuel is through transesterification and
esterification of vegetable or animal fats and oils (Krawczyk 1996). Brazilian
biodiesel is derived mostly from soy. In 2010, they produced in Brazil approxi-
mately 680,000 tons of soybean oil (CONAB 2012).
The first step in the biodiesel production process is the preparation of raw
materials. The oil received is stored in a tank and later pumped to a decanter which
separates the materials in suspension and then limed. Oil and alcohol are then
pumped into the reactor, where the transesterification occurs.
The next step is the separation of coproducts. The mixture is transferred to a
separator, where the esters are glycerin and stored in separate storage tanks.
Finally, purification of the esters is performed, and distillation of biodiesel and
glycerin.
A major problem in obtaining biodiesel is the large quantities of glycerin
formed as a by-product. The production of 90 m3 of biodiesel generates 10 m3 of
glycerin (SBQ 2009). New uses for glycerin have been investigated such as a
composite for reducing friction in oil rigs.
3 Renewable Liquid Transportation Fuels 65

Table 3.1 Adjustments percentage of anhydrous ethanol added to gasoline (MAPA 2011)
Regulation (Brazilian nomenclature) Scope Percentage (%) of ethanol
added to gasoline
Decreto 19.717—Feb, 20 1931 Brazil 5
Decreto 59.190—Sept, 8 1966 Brazil 25
Portaria CNP 94—July, 1 1976 Pernambuco (NE) 10
Portaria CNP 88—May, 19 1977 São Paulo 20
Portaria CNP 245—June, 30 1981 Center–South 12
Portaria CNP 142—Nov, 16 1989 Brazil 13
Portaria MAPA 278—Nov, 10 2006 Brazil 23
Portaria MAPA 7—Jan, 11 2010 Brazil 25
Portaria MAPA 678—Aug, 31 2011 Brazil 20
Portaria MAPA 105—Feb, 28 2013 Brazil 25

Fig. 3.1 Production of anhydrous and hydrated ethanol million liters over the years (IBP 2012)

3.3 Brazilian Strategies

In Brazil, the use of the mixture of anhydrous ethanol in gasoline, dating back to
the 1930s, has been subject to adjustments, discussions, and energy policies.
Percentages of anhydrous ethanol range depending on the region and the economic
policy of the time. Different regulations have been applied over the years, as
observed in Table 3.1.
Figure 3.1 shows the production of anhydrous and hydrated ethanol in Brazil.
66 V. de Araujo Bruno and A. R. Gonçalves

Fig. 3.2 Evolution of car emissions in Brazil (IBAMA 2006)

The exhaust gases produced by combustion reactions inside motors released

into the atmosphere became one of the major reasons for concern about the pol-
lution of the environment, specifically the atmospheric air. A well-known fact is
that burning ethanol, or their mixture with gasoline, releases smaller amounts of
carbon monoxide, sulfur oxides, hydrocarbons, and other polluting compounds.
Figure 3.2, compiled from data from IBAMA, presents the reduced greenhouse
gas emissions in Brazil over the years.
More recently, in 2003, official rule determined the evaluation of Brazilian
biodiesel production to understand its current situation, availability, as well as
advantages and disadvantages. With satisfactory results in hand, the Federal
Government decided to immediately take the necessary measures to ensure that
biodiesel became representative in the Brazilian energy matrix and thus created the
National Program for Production and Use of Biodiesel (NPPB) in December 2004.
The main objectives of the program were the stimulus to the formation of a
national biodiesel market, a definition of tax model, creating lines of funding and
development of farmers organizations for both counting on the collaboration of the
main industries involved in producing this biofuel chain.
Biodiesel can replace diesel oil, obtained in the fractionation of oil in diesel
cycle engines, for example, present in trucks, buses, airplanes, and tractors or can
be added to it in high proportions. The Act 11.097/2005 made compulsory the
addition of 2 % biodiesel to diesel from 2008 throughout the national territory.
A new adjustment increased this percentage to 5 % in 2010 and, currently, a
regulatory mark was sent to the National Congress to increase this percentage to
7 % by 2014 and to 10 % by 2020, which would mean 7.5 billion liters of bio-
diesel produced and consumed in the country. Studies show that biodiesel can be
added up to 20 % without compromising the efficiency and without being nec-
essary adjustments to the engine.
3 Renewable Liquid Transportation Fuels 67

3.4 Biofuels and the Environment

Modern environmental concepts rule every industrial process, sustainability being

inserted and prioritized in order to use from nature and return to it in the same
proportion. The development of biofuels has confirmed to be the most viable
solution to the energy problems, especially when biofuels replace petroleum,
mitigating greenhouse gas emissions. Any CO2 generated by burning biofuel is
incorporated again to the carbon cycle through photosynthesis. However, biofuel
production, from cultivation to the final consumer, uses fossil fuels.
Currently, the production of anhydrous ethanol from sugarcane generates
440 kg CO2 eq/m3 of bioethanol, while ethanol from corn generates 1700 kg
CO2 eq/m3. This is mainly because other fractions of maize as a source of energy
are not used. When anhydrous ethanol from sugarcane is mixed with gasoline at a
25 % ratio, 1900 kg CO2 eq/m3 of bioethanol is avoided (Macedo et al. 2008).
Like ethanol, biodiesel has smaller net greenhouse gas launched in the atmo-
sphere than burning different fossil fuels. The use of biodiesel to replace diesel fuel
reduces 90 % emissions of burning gas and 78 % of smoke emissions.
These rates are measured from the entire lifecycle of biofuels production, soil
preparation, use of pesticides and fertilizers, harvesting, manufacturing, storage,
distribution, and use as fuel.

3.5 Perspectives and Conclusions

Brazil not only sets up as one of the most developed countries regarding the use of
renewable energy sources, but also because this sector is constantly expanding.
Climate and territorial conditions were always favorable to agriculture in the
country, and as a result the production of biofuels from plant biomass became
especially extremely viable in a global scenario which seeks to mitigate the use of
fossil fuel origin.
Ethanol from sugarcane and biodiesel produced by soybeans are the most
significant liquid biofuels, a scenario constructed over the years as a combination
of a number of factors that favored such production forms. The current Brazilian’s
moment shows that networks for ethanol production are much more structured,
integrated, and developed than those for the production of biodiesel. However,
despite its more recent history, soybean biodiesel has grown quickly and studies
aimed to its development prove to be promising.
By analyzing some aspects regarding biofuels, we conclude that they have
undoubtedly great environmental advantages over fossil fuels. But there is a
notable difference between these two biofuels. While sugarcane is planted mostly
to meet the fuel market, to be the most advantageous among the crops for this
purpose, soybean is planted with the aim of meeting the food market. Biodiesel
production using soybean becomes a consequence of its large crop in the country,
but among other oilseeds soybean does not have greater benefits in all aspects.
68 V. de Araujo Bruno and A. R. Gonçalves

Biofuels question has long ceased to be purely energetic and has achieved
social, political, and economic sphere, becoming a government policy.

References

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2011. http://www.anp.gov.br/?pg=57890#Se__o_4. Accessed 23 Sept 2012
Brazilian Government (2011) Matriz energética. http://www.brasil.gov.br/cop/panorama/o-que-
o-brasil-esta-fazendo/matriz-energetica. Accessed 2 Sept 2012
Castro HF (2011) Indústria alcooleira. https://www.alunos. Accessed 17 Sept 2012
Castellanelli CA (2008) Estudo da viabilidade da produção de biodiesel, obtido através do óleo de
fritura usado, na cidade da Santa Maria. 2008, 111f. Dissertation (master in production
engeneering)—Universidade Federal de Santa Maria, Santa Maria
CONAB Companhia Nacional de Abastecimento (2012) Boletim da cana-de açúcar. www.conab.
gov.br/…/12_04_10_09_19_04_boletim_de_cana.pdf. Accessed 15 Sept 2012
Embrapa (2009) Clima favorável a produção de cana-de-açúcar. http://www.agencia.cnptia.
embrapa.br/Repositorio/clima_para_cana_000fhc5hpr702wyiv80efhb2aul9pfw4.pdf. Accessed
17 Sept 2012
Embrapa (2011) Soja em números (safra 2010/2011). http://www.cnpso.embrapa.br/index.php?
cod_pai=1&op_page=294. Accessed 11 Sept 2012
Goldemberg J, Coelho ST, Plinio MN, Lucond O (2004) Ethanol learning curve—the Brazilian
experience. Biomass Bioenergy 26:301–304
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Estados Unidos e Alamanha. 2009, 132 f. Dissertation (Master—Programa Interunidades de
Pós-Graduação em Energia)—EP/FEA/IEE/IF, Universidade de São Paulo, São Paulo
IBAMA (2006) www.licenciamento.ibama.gov.br. Accessed 10 Sept 2013
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UF. Accessed 10 Sept 2013
Krawczyk T (1996) Biodiesel—alternative fuel makes inroads but hurdles remain. Inform
7:801–829
Macedo IC, Seabra JEA, Silva JEAR (2008) Green house gases emissions in the production and
use of ethanol from sugarcane in Brazil: the 2005/2006 averages and a prediction for 2020.
Biomass Bioenergy 32:582–595
MAPA (2011) http://www.agricultura.gov.br/arq_editor/file/Desenvolvimento_Sustentavel/Agroen
ergia/Orientacoes_Tecnicas/01-Mistura%20etanol%20anidro-gasolina-CRONOLOGIA(Atualiz_
02_09_2011).pdf. Accessed 10 Sept 2013
Masiero G, Lopes H (2008) Etanol e biodiesel como recursos energéticos alternativos: perspectivas
da América Latina e da Ásia. Revista Brasileira de Políticas Internacionais 51:60–79
Milanez A, Favert Filho P, Rosa S (2008) Perspectivas para o etanol brasileiro. http://www.bndes.
gov.br/SiteBNDES/export/sites/default/bndes_pt/
MME, Ministério de Minas e Energia (2011). Boletim mensal dos combustíveis renováveis. http://
www.mme.gov.br/portalmme/opencms/spg/galerias/arquivos/publicacoes/boletim_mensal_
combustiveis_renovaveis/Boletim_DCR_nx_042__junho_de_2011.pdf. Accessed 5 Sept 2012
Rached AZ (2011) Barreiras á exportação do etanol brasileiro. 2011, 113 f. Dissertation
(Master—Programa Interunidades de Pós-Graduação em Energia)—EP/FEA/IEE/IF, Univer-
sidade de São Paulo, São Paulo
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22 Sept 2012
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de glicerina. http://www.uff.br/sbqrio/novidades/Novidades2009/Biodiesel%20Glicerina%20
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Schaeffer R (2007) As vantagens do etanol de cana. Revista Abre Aspas, Maio
Chapter 4
Socio-Economic and Ambient Impacts
of Sugarcane Expansion in Brazil: Effects
of the Second Generation Ethanol
Production

André Luis Squarize Chagas

Abstract The growing demand for clean energy sources to replace petroleum has
substantially expanded the use of biofuels—fuels produced from agricultural
products. For Brazil, instead of representing a hindrance to growth because of the
need for changes in the country’s energy mix, this represents a great opportunity to
generate value and income, since the country has clear comparative advantages in
producing these fuels from renewable sources. The main biofuel in the country is
ethanol, made from sugarcane. The country’s cane growing sector has been
undergoing intense transformations, with the attraction of foreign capital, opening
of new distilleries and intensification of mergers and acquisitions. However,
doubts have been raised about the socioeconomic effects of the spread of sugar-
cane growing, such as the effects on the environment, labor market, social con-
ditions and food prices, among others. This work reviews the papers that discuss
these impacts. The results suggest that the expansion in recent years helps to
improve the capital-labor relationship; the sugarcane growing is not the cause of
increased land and food prices; the environmental indicators in sector is better than
fossil fuel sector, or other relevant concurrent; the sector has no significant effects
(positive or negative) on social conditions in cane growing regions, and that the
sector can contribute positively by increasing local tax revenue.

Keywords Sugarcane 
Social impacts  Environmental impacts  Second
generation ethanol production

A. L. S. Chagas (&)
Department of Economics, and CNPq (Proc. 481027/2011-4),
University of São Paulo, São Paulo, Brazil
e-mail: achagas@usp.br

S. S. da Silva and A. K. Chandel (eds.), Biofuels in Brazil, 69

DOI: 10.1007/978-3-319-05020-1_4,  Springer International Publishing Switzerland 2014
70 A. L. S. Chagas

4.1 Introduction

The growing demand for clean energy sources to replace fossil fuels has caused
great expansion of bioenergy, fuels produced from agricultural products, mainly
ethanol and biodiesel. These products can be obtained from different sources of
raw materials. In the case of ethanol, we highlight sugarcane and corn, and in the
case of biodiesel, soybeans, canola, palm oil, castor oil, among other various
sources. The new needs provide increasing prominence to bioenergy, and repre-
sents greatest potential to generate income for countries that have comparative
advantage in the production of these goods.
Ethanol production from renewable sources places Brazil at the forefront of the
search for clean energy, along with the strong presence of hydroelectric energy
matrix of the country. Brazil is the only country that has a large-scale program of
vehicles with engines that use clean and renewable energy sources. The compet-
itiveness of ethanol produced from sugarcane in Brazil is significantly higher than
that of other producers, especially in relation to U.S. corn ethanol, both in the
matter of production costs, such as energy balance (BNDES and CGEE 2008).
In recent years, the sector invests in second generation of ethanol, to increase
the production. The production of second generation ethanol, or cellulosic ethanol,
consists of a first stage of pre-treatment, hydrolysis for liberating the molecules, a
second steps in degradation of sugars enzymatic or chemical means, and the last
fermentation stage, obtaining as ethanol product (Lee 1997; Chandrakant and
Bisaria 1998; Lin and Tanaka 2006; Cardona and Sánchz 2007; Soccol et al.
2010). In contrary than other places, in Brazil, the second generation of ethanol
will make deepening the current mode of production (Chagas 2013).
The main raw material to cellulosic ethanol, in Brazil, is the sugarcane bagasse
obtained as waste in the process of juice extraction plant. Bagasse is typically
fibrous, with high content of lignocellulosic material. Currently, the bagasse is
used for cogeneration of electricity, but in future, it can be used to ethanol pro-
duction (Pandey et al. 2000; Macrelli et al. 2012).
The ethanol production based on sugarcane bagasse is favored, in Brazil,
because the production process can be attached to the already existent plants,
requiring lower investments, infrastructure, logistics and energy supply. Besides,
the bagasse is generated at the industrial units, and with lowest transportation costs.
This is a promising scenario because from each 10 million tons of dry biomass, 600
million gallons of ethanol could be produced, considering the use of its cellulosic
part only (Soccol et al. 2010). Including the bagasse in production, the ethanol
production can increase at least 30 % reaching to 100 % (Santos et al. 2012).
For Brazil, the sugarcane expansion represents a great opportunity to increase
the value and income, since the country has clear comparative advantage in the
production of renewable fuels (Hoffmann 2006). Nevertheless, there are contro-
versies about the indirect effects of ethanol from sugarcane on the environment,
market labor, and social conditions in producer regions (Chagas et al. 2008, 2011;
Chagas 2009).
4 Socio-Economic and Ambient Impacts of Sugarcane Expansion in Brazil 71

The purpose of this article is review recent studies that analyze the impact of
the production of sugarcane, especially in producers regions. In Sect. 4.2, I
comment the work that analyses the sugarcane market labor in recent years. The
Sect. 4.3 reviews the food versus bioenergy discussion, and Sect. 4.4 discusses the
climate impacts of sugarcane production. In the next section (Sect. 4.5), I discuss
the social impact of sugarcane production and other impacts, I report in Sect. 4.6.
The last section presents the conclusion.

4.2 Labor Market

The most part of the studies about the sugarcane market labor relates to health
problems associated to the harvest manual, accidents in job, and energy expen-
diture and repetitive activity. The main idea associated to the sector workforce is
the strong labor-unskilled presence and the temporary labor for manual harvest.
This characteristic results in intense migratory flows to producer regions in harvest
periods. Additionally, the work is unhealthy and requires high physical exertion,
resulting in a number of severe accidents and mortality (Alessi and Navarro 1997;
Scopinho 2000; Alves 2006, 2007; Baccarin et al. 2008).
Alves (2006) calls attention to the extreme physical exertion required of
workers in the sector, especially those engaged in manual harvesting. Although
this aspect is still a problem at present, legislation in the most relevant producing
areas has changed to make mechanical harvesting mandatory in the next few years.
Other chapters study the economic relationship between the cane cutter and the
sugar mill (Basaldi 2007; Silva 2005). There are also some studies that show the
evolution and profile of labor in sugarcane, evidencing the changes in labor
relations (Goza 1997; Moraes 2007), and the implications of the process of
mechanization in sugarcane sector (Ramos 2007; Staduto et al. 2004).
Some recent studies show, however, that the wage level in sugarcane cultiva-
tion is higher than in other cultures. Of course, the highest wage in sector may be
consequence of the effort of work. But, other indicators are also better in sugarcane
sector than others sector, when is higher the degree of formalization of labor
relations (formal signed contract), the presence of child labor is lower. Possibly
because it is a culture whose product has greater integration in foreign markets,
and larger scale production, its working relationships are more formalized and in
accordance with the law (Toneto-Jr and Liboni 2008).
Moraes (2007) analyzed the impact of the end of the burn during the harvest
process. The conclusion is that there was an increase in mechanization and
changes in the profile of agricultural labor. Additionally, the new planting areas
tend to be mainly mechanized (Toneto-Jr and Liboni 2008). The introduction of
machines in harvest process is not damaging to employment since it occurs while
the sector is expanding. Thus, there is not job destruction. The recent transfor-
mation still helps to deepen a feature of the sector. The wages in sector tends to
increase with the mechanization.
72 A. L. S. Chagas

The situation of labor in the sector tends to improve with increasing mecha-
nization, which will tend to eliminate the aspect in which the sector indicators are
worse: the low-skill of work force and the high effort. In addition, mechanization
reduce the weight of the primary employment, which are the most common
complaints related to fatigue and the intensity of work (Toneto-Jr and Liboni 2008;
Hoffmann and Oliveira 2008). Thus, it appears to be unfounded the concerns about
the deterioration of working conditions, due to a significant expansion of the
sector, due the second generation ethanol, mainly when considering that the
expansion of the sector will be with increased mechanization.

4.3 Food Versus Bioenergy

The capacity of expansion of production is a concern that follows the discussions

on the sugarcane sector in Brazil, repeatedly. The introduction of 5 % ethanol in
gasoline, in developed countries, should demand about 90 billion l/year of ethanol.
Given the magnitude of these numbers is possible to think of failure of land, which
would generate pressure on land and food prices (FAO 2008). With some changes,
this is a recurrent debate on economic since Malthus (Abramovay 2010).
There are significant trade-offs, however, involved in the massive expansion of
the production of sugarcane and other crops for fuel. Chief among these would be
a shift of major amounts of the world’s food supply to fuel use when significant
elements of the human population remains ill-fed (Avery 2006).
The main criticism argues that the increase in sugarcane production would lead
to increased competition for land use, with an increase in land rent. With higher
land prices, increase agricultural production costs, impacting food prices (Chagas
et al. 2008). If this argument is true, should be a long-term relationship between
the sugarcane production, land rent and food prices series.
Chagas et al. (2008) tests the existence in long-term relationship. The results for
Granger causality test showed that there is no temporal precedence of sugarcane
production on the land rent, but rather the opposite, that is, the price of land which
causes (in the Granger sense) the production of sugarcane. The long-term rela-
tionship identified establishes a common trend between these two variables, but
not statistically significant. Since the coefficient of short-term adjustment to the
price of land is not statistically different from zero, with the result of Granger
causality test, it is concluded that the price of land is exogenous with respect to the
production of sugarcane and the price of food. In other words, the order of
causality identified did not show that an increase in the production of sugarcane
positively impact the price of land, although the variables walk in the same
direction.
4 Socio-Economic and Ambient Impacts of Sugarcane Expansion in Brazil 73

With respect to the price of food, the long-term relationship with production of
sugarcane follows opposite direction to what would be expected. This result is
robust if the change range used for measuring the cost of food to consumers.1
Chagas et al. (2008) concludes that the growth in agricultural demand explains
the incorrect association between increase production of sugarcane and food price.
In fact, the effect of China’s demand tends to pressure the international com-
modities price while pressing the production too.

4.4 Ethanol and Environmental Impacts

Considering the environment impact of sugarcane production, the main concern

refers to the risk of contamination of soil, water use, shifting other crops to forest
regions, fires, use of protected areas (springs, riverbanks, mountain tops, etc.),
among others. Several studies were undertaken in order to estimate the amount of
fossil energy expended in the production of sugarcane in the Brazilian conditions.
Among which may be mentioned: Macedo (1998), Macedo et al. (2008), Urquiaga
et al. (2005), Pimentel and Patzek (2008), Oliveira et al. (2005), Oliveira (2008).
The conclusion of these studies is that the energy balance of sugarcane (ratio of
total energy contained in the fuel produced and fossil energy invested in its pro-
duction) is quite varied. Studies undertaken by researchers in Brazil estimate this
relationship between 8 and 9, and can reach to 12, under certain conditions.
However, studies performed abroad indicate far less expressive numbers, around
3.7 and 1.1. The main reason for this divergence of findings refers to the
assumptions adopted in the calculation.
The studies carried out abroad (Pimentel and Patzek 2008; Oliveira et al. 2005;
Oliveira 2008) assume very outdated technology in field operations, resulting in a
consumption of fossil energy much higher than what would be reasonable.
Pimentel and Patzek (2008) estimated consumption of 2,596 Mcal (approximately
10,640 MJ) per a thousand liters of ethanol, due to energy use in the preparation
stages of cleaning and crushing of sugarcane in conveyor belts, filters and cen-
trifuges and heating the juice for fermentation. These values of power consump-
tion account for about a half of all the energy contained in the ethanol. However,
Brazilian mills produce all the energy they consume these processes from burning
bagasse in high pressure boilers, whose steam generated drives turbines that
produce electricity cogeneration unit. So it is not correct to assume that these
energy costs come from fossil source (Chagas 2013).
Soares et al. (2009) presents a comprehensive review of available data and
factors of fossil energy consumption in the production of sugarcane in the

1
The argument, however, seems valid with respect to the price of food at wholesale. This
difference between the consumer price and wholesale price, is possibly due to the fact that
wholesale prices closely follow production decisions, while consumer prices also depend on
industrial dynamics and technological innovations, that can dampen the effects of any
reallocation of land use.
74 A. L. S. Chagas

Brazilian conditions.2 Whereas a liter of ethanol produces 21.45 MJ of energy, a

hectare of sugarcane can produce 6,510 l of ethanol per year, generating
139,639 MJ of energy, approximately nine times the fossil energy invested in
agricultural operations.
Thus, from an environmental point of view, the sugarcane sector has very
positive results, due to use of a renewable feedstock for the production of cleaner
fuels, enabling both the use of by-products and generate a less polluting energy to
country. Additionally, the sector complements the hydroelectric power supply,
because the harvest of sugarcane in Midwest and South occurs in the dry season
and low in the reservoirs (Chagas 2013).
Nevertheless, there are other environmental concerns related to the production
of ethanol and sugarcane, like environmental impacts due new investments, legal
reserves, harvest’s burning, by-products generated in the production process.
To control the environmental impacts due new investments, all business plans
should obtain an environmental license and submit the Environmental Impact
Assessment and Environmental Impact Report (EIA-RIMA, in Portuguese acro-
nym) requires study prior to any activity that may potentially cause environmental
degradation. Actually, three types of licenses are required: preliminary permit
approving the location and design of the project and establishes the requirements for
obtaining licenses following; installation permit and operating license. The latter is
3 years for the production of sugar and 2 years for ethanol and should be requested
renewal before expiration. Licensing is the responsibility of the state environmental
authority, except in cases where venture beyond the limits of the state.
In relation to land use, it is stipulated the requirement for a legal reserve of
approximately 20 % of the total area that cannot be used beyond the preservation of
permanent protection areas (riparian forests, river springs, etc.). This is a problem
in the sector, since historically the sugarcane producers with advanced plantations
in all areas, including the areas of permanent protection.3 The intensification of
monitoring has led the implementation of significant programs of riparian forests
restoration and protection to the sources. It is noticed that there was a significant
progress in the areas of sugarcane plantation on protected areas, mainly in the past.
In many regions these problems are being repaired. Increased supervision and
greater control of compliance have led to recovery of protected areas.4

2
The estimate of total fossil energy used in field operations, including the transportation of cane
to the mill and the supply of inputs, is 12329.7 MJ/ha/year. Already tickets fossil energy
associated with the material used in the construction and equipment of plants representing
2,611 MJ/ha/year, totaling 14940.8 MJ/ha/year.
3
In São Paulo state, 8.1 % of the cane area refers to riparian forests. Of this area, 3.4 % have
natural forest and 0.8 % was reforested (Chagas 2013).
4
There is a discussion regarding the responsibility for the preservation of protected areas and
legal reserves. The plants produce using own cane, sugarcane produced on land owned and leased
areas, and third-party sugarcane, obtained from the suppliers. In relation to land owned and third-
party suppliers, there is no doubt about the responsibility, but in relation to leased areas is
doubtful.
4 Socio-Economic and Ambient Impacts of Sugarcane Expansion in Brazil 75

In the past, many rivers in Brazil were contaminated by stillage discharged by

the mills. Currently, the water withdrawn for industrial process is almost entirely
treated and reused in the plant itself, causing low water uptake; industrial systems
are virtually closed. The sugarcane produced in the traditional regions of Midwest
and Southwest regions uses virtually no irrigation, depending basically of rains.
The capture of water in surface and/or groundwater is controlled by the state and
depends on the granting of grants by the environmental agency (Department of
Water and Power, in the case of São Paulo). In several watersheds of the São Paulo
state the capture, consumption and disposal of effluents are charged, which should
induce a reduction in funding and better treatment of wastewater.
Currently, almost all by-products are utilized in the production process of the
sugarcane mills. The stillage (vinasse) is used for fertilization in the field in a
process called fertirrigation5. Another by-product used as fertilizer is the filter
cake.
Also in relation to the consumption of fungicides (practically zero) and pesti-
cides, these are lower than the other crops. The borer control (major pest) and
leafhopper is made by biological means. Only the control of ants, beetles, and
termites are made by chemical means. The use of pesticides (fungicides, herbi-
cides, and insecticides) is also regulated by federal law and controlled by state or
federal agency depending on the state.6
The burning of harvest is a public health problem, as will comment below,
because it causes respiratory problems. This practice tends to occur with greater
intensity during the dry season, which intensified their negative effects. Burning is
a practice designed to facilitate manual harvesting of sugarcane. The law prohibits
certain types of fires in certain areas and times. The controlled burning of sugar-
cane is regulated by specific federal law (Decree 2661/98), and in the State of São
Paulo has a specific law more restrictive (State Law 11.241/02). The trend is that
this practice be ended in a few years, both by regulatory pressures to reduce the
emission of pollutants and their harmful effects, as by the economic stimulus
resulting from full use of the cane (sugarcane juice, straw, leaves and bagasse).
The issues related to the labor market, formalization of hand labor and workforce
enhancement contribute too.7
The advance in mechanization could contain the burning process. Harvest
mechanization of sugarcane reached 65.2 % of the harvested area in the state of
São Paulo, in 2011/2012 season. Data from the Environmental Protocol Sugarcane
Industry shows that sugarcane production in the state has been fulfilling the targets

5
As the vinasse is a valuable organic fertilizer and a source of replacement water into the soil,
your use reduces the need for fertilizers and water. There exist rigorous restrictions on the amount
of vinasse used by area, so there is no problem of contamination of the soil, and the plants.
6
The limits of use are determined and monitored by specific department, and the producers are
required to return the packaging used.
7
In São Paulo, the Environmental Protocol signed between the plants, sugarcane producers and
the government establishes the order of the burned areas for mechanization in 2014 and in all
areas in 2017.
76 A. L. S. Chagas

set for reducing burned. Since 2007, when the proposal was signed between the
sugarcane industry and the Government of São Paulo State, mechanization
increased from 34.2 to 65.2 % of the harvested area.

4.5 Social Conditions and Sugarcane Production

Piketty et al. (2008) have shown that the sugarcane culture has not played a
significant role in reducing poverty and inequality in the country. Indeed, for the
state of São Paulo (Brazil’s main cane producing state), the authors concluded that
the sector contributed to the concentration of income.
Camargo-Jr and Toneto-Jr (2008) have found a positive association between
sugarcane growing and sugar and alcohol production and socioeconomic indica-
tors. In general, municipalities with strong involvement in the sugar-alcohol sector
perform better on socioeconomic indicators, and in some cases even outperform
the greater São Paulo Metropolitan Region (SPMR), the state’s main region in
economic terms. Silva (2008) also found the same positive impact when no cross-
effects on other variables are considered.
However, when consideration is made for the fact that the sector’s presence can
affect local human development through its impact on other variables, he found
that the situation is reversed and the sector’s presence has net negative impacts.
The above studies do not take into consideration the full heterogeneity of
producing regions, treating regions with different aptitudes for distinct crops as the
same. A more reasonable assessment must compare similar places with and
without sugarcane, which is clearly impossible to do. To overcome this difficulty,
Chagas et al. (2011) apply matching methods to estimate the impact of determined
treatments on treated subjects, as explained in the following section.
Chagas et al. (2011) implements a spatial propensity score matching test, an
original contribution to this type of study. This methodology is useful because it
deals with the fact that one cannot immediately compare average indicators of
cane producing regions with those of nonproducing ones, since the probability
of production is not a random variable. Thus, spatial factors need to be considered
to control for the probability of producing or not.8
Although there are arguments in favor and against the sector’s impacts on local
social conditions in growing regions, Chagas et al. (2011) indicates that the

8
To calculate the spatial propensity score were considered neighborhood effects, as the
proximity of county to sugar mill and a dummy variable for those located in states dense
sugarcane production. The spatial effects capture both the fact that in a region whose neighbors
are producers, the probability of producing sugarcane is higher (dependence or spatial
autocorrelation), as well as the specific soil and climate of each region. The second part seeks
to control the probability of production take place in regions near the plants (potential plaintiffs
production). The last one captures state effects specific, such as legislation, ease of flow of
production, access to tax incentives, etc.
4 Socio-Economic and Ambient Impacts of Sugarcane Expansion in Brazil 77

presence of sugarcane is not relevant to determine their social conditions, whether

for better or worse. It is thus likely that public policies, especially those focused
directly on improving education, health and income generation/distribution, have
much more noticeable effects on the municipal HDI than sugarcane production.
Possibly public policies, especially those aimed directly at improving education,
health, production and income distribution, have greater impacts on the HDI-M.
However, the same is not true when consider the healthy impact of sugarcane
production, mainly to respiratory problems that it associate to burning of sugar-
cane. The burning is meant to increase the productivity of workers. It facilitates
the harvest, easing access to the plants and reducing work hazards (dry leaves are
harmful, there might be poisonous insects and snakes). It takes place in the
beginning of harvest, which coincides with the dry season. Many studies relate
sugarcane burning to increases in fine particulate matter, coarse particulate matter
and black carbon concentration, especially in burning hours (Lara et al. 2005).
Allen et al. (2004) observe increases in the concentration of substances as nitrite,
sulfite, oxide of carbon, and others. The sugarcane is harvested by unskilled
workers, mostly manually. The literature also indicates that exposition to classical
pollutants (matter, sulfite, nitrite, oxide carbon, etc.) can affect negatively human
health (Sicard et al. 2010), especially for young, elderly and woman people (Braga
et al. 1999; Roseiro 2002; Gonçalves et al. 2005).
The burning of sugarcane generates a massive quantity of smoke that spreads in
the region, reaching cities and becoming a potential threat to the human health.
Few studies have linked the burning of sugarcane straws with respiratory diseases
in the producing regions. Although the pollution from sugarcane burning may be
as harmful as the pollution from traffic and manufacturing Mazzoli-Rocha et al.
2008, many studies relate its impact on health, for specific municipalities or for
larger regions: Arbex et al. (2000, 2004), Cançado et al. (2006), Arbex et al.
(2007), Ribeiro (2008), Uriarte et al. (2009), Carneseca et al. (2012). These studies
consider only the local, or short-distance, effects of burning to respiratory health,
ignoring the effects on neighboring municipalities.
Chagas et al. (2013) proposes to measure the impact of burning on respiratory
problems of children, teenagers, and elderly people, working with a balanced
panel of 644 municipalities, from 2002 to 2011, and using a spatial difference-
in-difference technique, to control for the effect of sugarcane burning on non-
producing regions in the vicinity of producing regions. The paper concludes that
sugarcane burning significantly increases the incidence of respiratory problems in
producing regions. The use of a spatial diff-in-diff model allowed the authors to
find out that the effect on nonproducing nearby regions is also significant and
quantitatively relevant, at least 66 % of the effect on producing regions. The
results suggest that this method makes it possible to better identify the impact, not
only in the treated regions, but on the nontreated too.
78 A. L. S. Chagas

4.6 Other Impacts

The expansion of sugarcane growing has speeded up substantially in recent years

in the state of São Paulo, because of increasing demand for both sugar and alcohol.
This expansion has been accompanied by debate over the costs and benefits of this
expansion. Chagas et al. (2010) verify the effects of the increased production of
sugarcane specifically on municipal revenues.
To do this, the authors gathered a panel data, to control for possible specific
effects of each municipality. Given the time persistence of revenue, the traditional
fixed- and random-effects models can generate biased estimators, so, they consider
a dynamic panel model to be better. Moreover, in view of the geographic
dimension of the phenomenon studies, they also introduced spatial controls.
The results of the tests suggest that for all the revenue categories studied there is
a significant and substantial increase in revenue with the expanded participation of
sugarcane in the municipal agricultural output. This effect can be from the direct
and indirect effects of the sector on the local economy and its relation with
neighboring municipalities.
The value of agricultural production of sugarcane is greater per hectare than for
most other crops, indicating a greater value of agricultural income. Cane also
employs more workers per hectare (considering both agricultural and industrial
workers), once again showing greater generation of income. The greater geo-
graphic integration between the agricultural and industrial phases and the demand
for services at plants should cause greater impacts on generation of urban income,
meaning a greater multiplying effect on local economic activity. Therefore, even
though it brings some negative effects, the growing and processing of sugarcane
appears to have significant impacts on generation of income, which is partly
captured by municipal tax revenues, as suggested by the results of this study. In
other words, if on the one hand the expansion of sugarcane creates pressures on
municipal spending on public services for migratory workers and greater health
expenditures because of the deleterious effects of burn-off, among others, these
municipalities appear to have higher revenues that offset the higher spending
pressures.

4.7 Final Remarks

The need to reduce the use of fossil fuels in global energy poses risks and
opportunities. The discussion of alternatives is urgent and necessary. Biofuels—
fuels produced from agricultural products—are on a possible alternative. Their
environmental, social, and economic impacts need to be scaled. In general, the
studies suggest that the net effects are positive when compared with fossil fuels.
For Brazil, this process appears to have more beneficial consequences than neg-
atives. The country has very large share of renewable energy as inputs, such as
hydropower and ethanol from sugarcane.
4 Socio-Economic and Ambient Impacts of Sugarcane Expansion in Brazil 79

The expansion of the production of sugarcane for ethanol production seems

inevitable and desirable. Brazil has indisputable comparative advantage in the
production of ethanol: sector productivity per land use is increased and costs are
significantly lower. Nevertheless, doubts prevail about the impacts of the sector in
the economy as a whole, the social welfare and on the other sectors. This consider
a relevant literature about these issues.
The expansion in recent years has helped to improve the capital-labor rela-
tionship, due the change in production technic (mechanization). This change will
tend to eliminate the aspect in which the sector indicators are worse: the low-skill
of work force and the high effort. In addition, mechanization reduce the weight of
the primary employment, which are the most common complaints related to
fatigue and the intensity of work.
Regarding the conflict between land uses for energy production versus food
production, the results do not support their existence. The long-term relationship
identified establishes common path between the production of sugarcane and the
price of land, but this relationship is not statistically significant. Indeed, there are
indications that the price of land is not determined by the production of sugarcane,
although this result may be compromised by sample period and for the errors of
measurement of the variable price of land available.
With regard to the price of food, there are apparently long-term relationship
between this variable and the production of sugarcane, but in the opposite direction
to what would be expected if it were valid the argument that the production of
sugarcane increases the price of food.
This result is robust to number of price change used to measure the price of food
to consumers. The conflict, however, seems valid with respect to the price of food
at wholesale. The eventual realization that the increased production of sugarcane
can push the price of land is, possibly, a result of a combination of several factors
that contribute to increase agricultural production in general, with the increase of
demand for these products, due to the increase in world income (especially poor
and populous countries, like China and India). If this is true, it is possible to get in
the short term, expanding the production of sugarcane and increase the price of
food. Clearly, future work will test the robustness of this hypothesis.
In terms of strategic option for the country, it must be examined whether the
increased production of other commodities, with lower value added and with much
weaker links with other productive sectors, it is more advantageous to invest in the
production of commodity that can provide advantages comparative energy to the
country.
The use of ethanol as fuel generates positive environmental impact when
compared to oil, which has made many countries to adopt measures to encourage
its production and use. Moreover, there has been a massive investment to
discovery of new energy sources and development of new technologies for the
production of ethanol from different materials. The main focus, in developed
countries, is the development of the process of hydrolysis, which will enable the
production of ethanol from cellulosic materials.
80 A. L. S. Chagas

The utilization of all by-products generated in the production process increase

the environmental benefits of sector. The recent regulatory acts contribute to make
more transparent the actions of the sector on the carbon savings. Additionally, the
sector complements the hydroelectric power supply, because the harvest of sug-
arcane in Midwest and South occurs in the dry season and low in the reservoirs.
Another aspect discussed refers to the impacts of the industry on the social
conditions of the locations which concentrate the production of sugarcane. Aside
from the potential benefits that the industry can bring to the country (such as
employment generation, income, and revenue), it may be that in the regions
producing the burden is greater than the bonuses, considering the pressure for local
public services, urban infrastructure, etc.
The results, however, suggest that the presence of the industry in one location is
not relevant to determine their social conditions, for better or for worse. Possibly,
there are public policies which should be much more obvious impacts on the HDI,
particularly those related directly to the improvement of the conditions of edu-
cation and health as well as to improve production and income distribution. Future
works could be done by testing this hypothesis.
With respect to health impact, however, it seems that the burning of harvest has
a negative impact over the respiratory cases in producers region, impacting non-
producers neighbors regions too. The deepening of technical change, with greater
adoption of mechanization, can have a positive impact in this regard.
Finally, it seems that low agricultural sectors generate direct revenue, especially
for city government, due to the low incidence of taxes on such activity in Brazilian
federative system. But, it is also true that agricultural production is inevitable,
much more municipalities. In this case, it may be that the multiplier effects of the
production of sugarcane are larger than that of competing products, so that their
expansion will increase the revenue of municipalities.
The results corroborate this hypothesis. For all categories of income is con-
sidered significant and substantial increase in revenue with the expansion of the
production of sugarcane. This effect may possibly be outdated. An increase in
sugarcane production this year will have impact on the share from transfer from
the next period. Thus, the revenue gains obtained in the counties producing sug-
arcane may offset any spending pressures that arise due to the characteristics of the
sugarcane industry. The expansion of sugarcane in the counties, replacing other
agricultural activities, appears to result in increased tax revenues.
In summary, the results suggest that (i) the sugarcane expansion seems help to
improve the relationship between sugarcane mill and workers, (ii) is not the
expansion of the production sector the main reason to push the price of food, (iii)
the environmental indicators in sector is better than fossil fuel sector, or other
relevant competitor, (iv) the sector has no significant effects (positive or negative)
on social conditions in cane growing regions, (v) the mechanization may con-
tribute to reduce the sector impact in people’s health, and (vi) the sector can
contributes to public policies, to the extent that generates indirect effects on the
rest of the economy, increasing the local revenue.
4 Socio-Economic and Ambient Impacts of Sugarcane Expansion in Brazil 81

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Chapter 5
Integrated Production of 1G–2G
Bioethanol and Bioelectricity
from Sugarcane: Impact of Bagasse
Pretreatment Processes

Caliane Bastos Borba Costa, Felipe Fernando Furlan,

Antonio José Gonçalves Cruz,
Raquel de Lima Camargo Giordano
and Roberto de Campos Giordano

Abstract The industrial plant for production of second-generation (2G) ethanol

from sugarcane bagasse will most probably be integrated to the already existing
facilities for first-generation (1G) ethanol from sugarcane juice. This will allow
lower investment costs, since the former would be able to take advantage of the
existing infrastructure, setup for the later. Nevertheless, the exploitation of
sugarcane bagasse as raw material must take into account that this biomass is also
used as boiler fuel in order to produce steam to meet process demands. Addi-
tionally, steam demand is highly dependent on the pretreatment used. In this
context, five pretreatments were chosen and an ethanol production process was
proposed for each of them. Steam demand was calculated and used to determine
the maximal bagasse that could be diverted from steam production. Among the
pretreatments considered, the alkaline one presented the higher increase in ethanol
production (5.7 L/tonne of sugarcane). This was due the almost complete cellulose
hydrolysis and the lower steam demand of this process. On the other hand,
pretreatment and hydrolysis reactor volumes were first and second higher,
respectively, for this pretreatment. This suggests that, from an economic
perspective, steam explosion (with a 2G ethanol production of 2.8 L/tonne of
sugarcane) might be a better option.

C. B. B. Costa  F. F. Furlan  A. J. G. Cruz  R. de Lima Camargo Giordano 

R. de Campos Giordano (&)
Department of Chemical Engineering, Federal University of São Carlos, UFSCar, Rodovia
Washington Luiz km 235, São Carlos, SP 13565-905, Brazil
e-mail: roberto@ufscar.br

S. S. da Silva and A. K. Chandel (eds.), Biofuels in Brazil, 85

DOI: 10.1007/978-3-319-05020-1_5,  Springer International Publishing Switzerland 2014
86 C. B. B. Costa et al.

5.1 Introduction

The main liquid biofuel, nowadays and in the foreseeable future, is bioethanol, and
the fermentation of carbohydrates coming from different crops is the dominant
technology for its production. In Brazil, sugarcane is the raw material for this
biofuel and the consolidated production process uses sugarcane juice (essentially
sucrose) as substrate for ethanolic fermentation by Saccharomyces cerevisiae. This
production process, known as first generation (1G), utilizes sugarcane bagasse as
fuel to the boiler, which is important, in the cogeneration system, both to produce
steam to the process and to cogenerate electric energy, in the turbines coupled to
electric generators. The electric energy produced is used in-house and, when
integration with the electricity companies’ lines is possible, it is sold to the grid.
The production of bioethanol can be increased if the carbohydrates present in
sugarcane bagasse are used in the fermentation steps as well, giving rise to the so-
called second-generation (2G) ethanol. Since the main constituents of bagasse are
polymers of carbohydrates (around 45 % of cellulose and 27 % of hemicellulose,
dry basis), they can, in principle, be separated from the other components of
bagasse (lignin, ashes), and then hydrolyzed in order to make their sugars
accessible to be fermented. Since bagasse is already at the 1G plant site, it is
natural to think of a 2G plant integrated to the 1G one, with common pieces of
equipment, like the cogeneration system (combined heat and power system) and
distillation columns.
The production of 2G ethanol from bagasse, nevertheless, has two points of
concern. First of all, not all bagasse can be used to produce 2G ethanol, since
bagasse is necessary as fuel to the boiler, to produce steam and electric energy both
to 1G and to the new 2G process. Second, bagasse must be pretreated to make
cellulose more accessible to enzymes in the enzymatic hydrolysis (the dominant
technology for industrial application), turning this step more efficient. Several
alternatives of pretreatment of bagasse have been studied and, from the process
point of view, they differ in many aspects: in the ratio of water used, in which
component (cellulose, hemicellulose, lignin) is mostly attacked, in the recovery of
their different constituents, in the crystallinity of obtained cellulose, etc. Of course,
since these different options of pretreatment use different ratios of water and
produce different qualities of constituents, they impact differently in the integrated
production of 1G–2G bioethanol (and bioelectricity). It is important to bear in
mind that many 1G plants have long-term contracts to sell their surplus of electric
energy. And the diversion of bagasse for production of 2G ethanol could change
the surplus of generated electric energy by the industry, due to the less amount of
bagasse burnt and to the higher steam consumption of the integrated 1G–2G
bioethanol and bioelectricity production plant. Of course, part of the so-called crop
trash (leaves, straw, tips, etc.) might be used for bioelectricity production. This
surplus feed for the boiler would certainly be important for an economic assess-
ment, but it does not change the relative performances of the different pretreatment
processes.
5 Integrated Production of 1G–2G Bioethanol and Bioelectricity from Sugarcane 87

Here, different pretreatment options reported in the literature are revised and
some of them are chosen for a study of their impact on bioethanol and bioelec-
tricity production in 1G–2G autonomous distilleries (i.e., no sugar production is
considered). Since it is not the aim of this chapter to assess the economic feasi-
bility of different pretreatments, the use of sugarcane trash was not considered in
our analysis, for the sake of simplicity.

5.2 Bagasse Pretreatment

A number of different pretreatments for biomass, including bagasse, have been

investigated over the past decades. These include milling, steam explosion,
ammonia fiber expansion (AFEX), organosolv extraction, the use of microwaves,
of acid, alkaline, or oxidative mediums, of liquid hot water, or the use of ionic
liquids. Many studies consider just one pretreatment for bagasse and some of them
use two or more pretreatments in sequence in order to evaluate their combined
effects, but an evaluation of the overall integrated process is still lacking.
Milling is a purely physical pretreatment, used to enhance the access of the
enzymes cocktail to the fibers. Furthermore, it has the advantage of not producing
fermentation inhibitors (Silva et al. 2010).
Among the physicochemical options of pretreatment, steam explosion of
bagasse (Chen et al. 2010; Dias et al. 2011b; Kaar et al. 1998) applies a sudden
decrease in pressure in order to expose the material fibers. Hemicellulose is
autohydrolyzed at high temperatures prior to the decompression, while lignin
remains almost intact, retained with the exposed cellulose (yielding the so-called
cellulignin). AFEX uses anhydrous ammonia to break down lignin–carbohydrate
linkages. In opposition to steam explosion, hemicellulose is not removed in AFEX
as a separate liquid stream (Krishnan et al. 2010). Liquid hot water pretreatment
(Hernandez et al. 2012; Wang et al. 2012; Yu et al. 2013) uses liquid water at high
pressure and temperature (150–230 C) in order to hydrolyze mainly the hemi-
cellulose fraction, which is removed as a liquid stream.
Chemical pretreatments make use of acid, alkali, oxidant, or solvent in order to
attack different constituents of lignocellulosic material. Weak acid has been
investigated by many authors (Pietrobon et al. 2011; Rocha et al. 2011;
Vasconcelos et al. 2013; Zhao et al. 2007) in order to make hemicellulose soluble,
and to facilitate the enzymatic hydrolysis of cellulignin (Canilha et al. 2012). Very
different process conditions are reported in the literature (with respect to tem-
perature, space time, the acid that is used and its concentration). These conditions
will influence the transformation of the formed monosaccharides, through unde-
sired reactions, into other compounds, that are inhibitors to fermentation process.
Alkaline pretreatment aims to remove lignin from the biomass and has also been
studied by many researchers lately (Fuentes et al. 2011; Hernandez et al. 2012;
Rabelo et al. 2009; Wu et al. 2011). It requires lower temperatures than other
chemical pretreatments, but, on the other hand, is a slower pretreatment, requiring
88 C. B. B. Costa et al.

longer space times (Canilha et al. 2012). Addition of surfactant to alkaline medium
has been also investigated (Cao and Aita 2013) in order to promote a better
removal of hydrophobic compounds (lignin) due to the decrease in surface tension.
Oxidation pretreatment (Cheng et al. 2008; Martín et al. 2007; Martín et al. 2008;
Rabelo et al. 2011) uses pure oxygen or air combined with water or alkaline
solutions (alkaline-oxidative pretreatment) to attack lignin structure. According to
Martín et al. (2007), toxic furaldehydes and phenol aldehydes have their formation
diminished with the use of the alkaline-oxidative alternative. The use of solvents to
solubilize lignin in the pretreatment characterizes the so-called organosolv pre-
treatment (Mesa et al. 2010; Wolf 2011). The action of the solvent solution on the
biomass is often catalyzed by acid or alkali. Compared to alkaline pretreatments,
organosolv is supposed to have the advantage of solvent recovery and less use of
water.
The combination of established pretreatments is commonly found in the liter-
ature. For example, Giese et al. (2012) studied the effect of acid pretreatment
followed by alkaline delignification on the enzymatic hydrolysis of sugarcane
bagasse, and showed that a decrease in enzyme loading costs could be achieved. A
combination of steam explosion and alkaline pretreatments is also found in the
literature, in order to remove lignin from the solid fraction after steam explosion,
leading to a fourfold increase of ethanol production (Wanderley et al. 2013). Zhao
et al. (2011) combined alkaline and peracetic acid pretreatments and their results
show a better digestibility by cellulases. The combination of weak acid with
organosolv pretreatment with NaOH was the focus of Mesa et al. (2011), who
concluded that this combination was very efficient, increasing the glucose con-
centration of the enzymatic hydrolyzed stream.
Other types of pretreatments, not so extensively investigated in the literature,
are the ones that make use of microwaves or ionic liquids. The former is used as an
alternative source of heat, aiming to achieve the temperatures required by different
pretreatments, to which the microwave is combined (Binod et al. 2012). Ionic
liquids, by their turn, may dissolve cellulose. Zhu et al. (2012) applied them to
promote the performance of the enzymatic hydrolysis.

5.3 Selected Conditions for the Different Pretreatment

Analysis

As it can be seen in Sect. 5.2, the effect of several pretreatments in the composition
and hydrolysis susceptibility of sugarcane bagasse has been reported. Since not all
options of pretreatment are in equal state of development, only five of them were
chosen for an analysis of their influence on the performance of the integrated 1G–2G
bioethanol-bioelectricity production process: steam explosion, organosolv, liquid
hot water, weak acid, and alkaline ones.
5 Integrated Production of 1G–2G Bioethanol and Bioelectricity from Sugarcane 89

Table 5.1 Pretreatment conditions, according to each selected literature article

Pretreatment Pretreatment conditions
Steam explosion (Warderley et al. 2013) Temperature: 200 C
Reaction time: 7 min
Organosolv (Wolf 2011) Temperature: 190 C
Reaction time: 90 min
Solid/liquid ratio: 9.1 % (m/v)
Reagent: ethanol/water solution (50 %, m/v)
Liquid hot water (Yu et al. 2013) Temperature: 180 C
Reaction time: 20 min
Solid/liquid ratio: 5 % (m/v)
Weak acid (Rocha et al. 2011) Temperature: 190 C
Reaction time: 10 min
Solid/liquid ratio: 9.1 % (m/v)
Reagent: 1 % (m/v) of H2SO4
and 1 % (m/v) of acetic acid
Alkaline (Yu et al. 2013) Temperature: 110 C
Reaction time: 1 h
Solid/liquid ratio: 14.3 % (m/v)
Reagent: 0.18 % (m/v) of NaOH

In the literature, it is possible to find a great number of papers for each one of
these selected pretreatments. Nevertheless, only a few provide enough information
for a complete process analysis. The main criterion for our selection of pretreat-
ment conditions was the existence of complete reported information in the liter-
ature. For complete reported information, one can cite sugarcane bagasse
composition (both before and after the pretreatment is applied), mass yield, pre-
treatment conditions (temperature, pressure, reactants concentration, reaction time,
solid/liquid ratio), and enzymatic hydrolysis conditions (reaction duration and
attained conversion, load of solids, enzyme concentration and temperature).
Cellulose recovery and sugar yields were also considered, since pretreatment
conditions that led to higher recovery and yields were preferred.
Table 5.1 shows the articles selected from the literature and, based on them, the
pretreatment conditions that were considered.
In order to standardize the results and facilitate their comparison, a unique
composition for sugarcane bagasse was used. This value was calculated as the
average of the compositions reported in the literature. For the sake of simplicity,
the references for natural bagasse composition were suppressed, but the values are
shown in Table 5.2. The compositions of bagasse after each pretreatment, as
reported in the selected articles, were adapted for this specific bagasse. It was
assumed that the percentage solubilized of each component remained the same as
described in the article, despite the differences in bagasse composition. The
composition of the pretreated biomass, after each pretreatment, is shown in
Table 5.2.
90 C. B. B. Costa et al.

Table 5.2 Bagasse mass composition before and after pretreatments

Pretreatment In natura Steam Organosolv Liquid hot Weak acid Alkaline
bagasse explosion water
Reference Warderley Wolf Yu et al. Rocha et al. Yu et al.
et al. (2013) (2011) (2013) (2011) (2013)
Cellulose 44.9 50.2 64.2 59.6 59.6 60.7
Hemicellulose 27.7 9.5 17.6 4.5 2.7 29.2
Lignin 23.6 34.9 11.8 29.8 31.9 3.6
Ash 3.8 5.4 6.3 6.0 5.8 6.5
Solid mass 68.0 60.1 63.5 65.1 58.5
yield

The yield of enzymatic cellulose hydrolysis is exceedingly dependent on the

pretreatment used, and cannot be inferred by composition of the biomass after the
procedure. As stated beforehand, hydrolysis conditions and the resulting conver-
sion of cellulose were explicitly reported on all articles that were used as base for
our analysis of the processes, i.e., all these articles also reported experimental
results of enzymatic hydrolysis of the material. It is worth mentioning that none of
these publications optimized the hydrolysis conditions. Since the pretreated
bagasse has different digestibility after each pretreatment, it is expected that
process conditions will be distinct. The conditions used in each study are shown in
Table 5.3.
Neither ethanol nor biogas production from the hemicellulose fraction was
considered in this analysis. Except for the steam explosion pretreatment, the
pentoses concentration in the liquid fractions obtained was too low for direct use as
raw material for these processes and a concentration step would increase steam
demand and decrease the bagasse available for 2G ethanol production. This might
be circumvented by a heat integration of the process, which is outside the scope of
this chapter.
An integrated 1G–2G bioethanol and bioelectricity production plant was con-
sidered in the process analysis. In this scenario, bagasse is diverted from the
combined heat and power system to the 2G sector (set of unit operations necessary
for production of 2G bioethanol). Since process steam demand (1G and 2G) is met
by the bagasse combustion, there is a limit to the amount of bagasse that can be
diverted to 2G sector. A typical 1G process was considered, based on the simu-
lations performed by Dias et al. (2012).
Based on the data provided by the selected literature articles, a process was
proposed for each of the pretreatments chosen. Solvent recovery was only
implemented for the organosolv pretreatment, as shown in Fig. 5.1. The other four
pretreatments shared the same basic process, shown in Fig. 5.2. It is worth men-
tioning that the processes proposed do not take full advantage of the possible heat
and mass integration for the combined 1G and 2G process.
5 Integrated Production of 1G–2G Bioethanol and Bioelectricity from Sugarcane 91

Table 5.3 Cellulose hydrolysis conditions, according to each selected literature article
Pretreatment Hydrolysis condition
Steam explosion (Warderley et al. 2013) Cellulose to glucose conversion: 32 %
Reaction time: 24 h
Solid/liquid ratio: 8 % (m/v)
Enzyme load: 10 FPU/g of cellulose
Organosolv (Wolf 2011) Cellulose to glucose conversion: 52 %
Reaction time: 42 h
Solid/liquid ratio: 9.1 % (m/v)
Enzyme load: 20 FPU/g of lignocellulosic material
Liquid hot water (Yu et al. 2013) Cellulose to glucose conversion: 52.3 %
Reaction time: 72 h
Solid/liquid ratio: 5 % (m/v)
Enzyme load: 15 FPU/g of lignocellulosic material
Weak acid (Rocha et al. 2011) Cellulose to glucose conversion: 75 %
Reaction time: 48 h
Solid/liquid ratio: 2 % (m/v)
Enzyme load: 15 FPU/g of lignocellulosic material
Alkaline (Yu et al. 2013) Cellulose to glucose conversion: 99 %
Reaction time: 72 h
Solid/liquid ratio: 5 % (m/v)
Enzyme load: 15 FPU/g of lignocellulosic material

Fig. 5.1 Organosolv Solvent

makeup
Mixer
pretreatment simplified block Vapor
fraction
diagram
Bagasse Liquid Liquid To effluent
Pretreatment Flash Filter_1
fraction fraction treatment

Solid fraction
Water and enzymes
Hydrolysis

To the Liquid fraction

concentration Filter_2
step
Solid fraction

To the boiler

5.4 Results

The results for the organosolv pretreatment show that the proposed solvent
recovery step was not adequate and more ethanol was lost in this step than pro-
duced by the fermentation of the glucose produced in the hydrolysis of cellulose.
Therefore, a more complex system must be implemented, including a distillation
column for the liquid fraction of Filter_1 (Fig. 5.1). Since it is not the scope of this
chapter to make such a detailed simulation, the organosolv pretreatment was not
further considered.
92 C. B. B. Costa et al.

Fig. 5.2 Steam explosion, Bagasse Liquid To effluent

Pretreatment Filter_1
liquid hot water, weak acid, fraction treatment
and alkaline pretreatments
Solid fraction
simplified block diagram
Water and enzymes
Hydrolysis

To the Liquid fraction

concentration Filter_2
step
Solid fraction

To the boiler

Table 5.4 Main results for the process simulations

Pretreatment Steam Liquid hot Weak Alkaline
explosion water acid
Specific production of 2G bioethanol(L/TdB) 104.2 94.0 198.0 239.0
Specific steam consumption of 2G sector (kg of 3.9 13.3 22.4 6.9
steam/kg of dry bagasse)
Maximal amount of bagasse diverted to 2G 27.3 15.0 10.0 23.7
production (kg of dry bagasse/TC)
Decrease in electric energy production (kW/TC) 34.5 19.0 12.6 29.9
Increase in ethanol production (L/TC) 2.8 1.4 2.0 5.7
TdB ton of dry bagasse, TC tonne of sugarcane

Table 5.4 shows the main results of the simulations for each pretreatment. The
specific ethanol production was estimated considering the cellulose hydrolysis
yields provided by the references and assuming 90 % of the theoretical fermen-
tation yield. No inhibition effect by the byproducts of the hydrolysis was con-
sidered, since it was supposed that glucose would be concentrated and fermented
together with the sugarcane juice from first-generation process, in the same bio-
reactor, thus diluting the inhibitors. Specific steam consumption, as shown in
Table 5.4, takes into account the steam produced by burning the residual solids
from Filter_2. Since the steam demand for both first- and second-generation eth-
anol must be provided by the combustion of these residual solids and bagasse, the
steam consumption determines the amount of bagasse available for 2G ethanol
production. Considering a production of dry bagasse of 140 kg per ton of sugar-
cane (Dias et al. 2011b), a steam production of 4.5 kg per ton of dry bagasse
(based on information from Dias et al. 2011a) and a steam consumption of 360 kg
per tonne of sugarcane in the 1G process (Seabra and Macedo 2011), it is con-
cluded that 1G process is responsible for the consumption of 57.3 % of the
5 Integrated Production of 1G–2G Bioethanol and Bioelectricity from Sugarcane 93

Table 5.5 Volumes of the main pieces of equipment as a function of the amount of bagasse
processed
Pretreatment Pretreatment reactor volume Hydrolysis reactor volume
(m3/TdB) (m3/TdB)
Steam explosion 0.35 243
Liquid hot water 7.01 569
Weak acid 1.84 1665
Alkaline 7.01 917
TdB tonne of dry bagasse

bagasse. Therefore, approximately 60 kg of bagasse per ton of sugarcane would be

available for 2G ethanol production. Table 5.4 shows the maximal amount of
bagasse that could be diverted to 2G ethanol production considering each
pretreatment.
From Table 5.4, it is possible to determine that it is necessary, only for the 2G
sector, 37.4 kg of steam per each liter of 2G bioethanol produced for the steam
explosion pretreatment. For the other pretreatments these values are 141.5, 113.1,
and 28.9 for, respectively, liquid hot water, weak acid, and alkaline. The 1G plant
has a consumption around 4.0 kg of steam per each liter of 1G bioethanol pro-
duced (Seabra and Macedo 2011). This suggests that the requirement of thermal
energy for producing 2G bioethanol is still high. Furthermore, liquid hot water and
weak acid pretreatments have intense steam consumption and would certainly
benefit from process heat integration.
The decrease in electric energy production shown in Table 5.4 considers only
the bagasse diverted to 2G ethanol. It does not consider the decrease related to
steam extractions at high pressures to meet the 2G steam demand (between 12 and
16 bar). If the selling of electric energy to the grid were considered, its market
price could be used to estimate the minimal ethanol selling price for 2G ethanol.
Reactor volumes were also considered, as an approximation of the investment
costs for the comparison among the pretreatments. Table 5.5 shows the volume of
both the pretreatment reactor and the hydrolysis reactor as a function of the
bagasse flow into the system. Weak acid presented the highest total volume,
mainly due to its low solid loads in the hydrolysis reactor. This is also the reason
for the large volume of the hydrolysis reactor for the alkaline pretreatment. This
suggests that it is important to improve solid loads in the enzymatic hydrolysis of
cellulose.
Among the considered pretreatments, alkaline obtained the higher increase in
ethanol production. This was due both to its higher conversion of cellulose to
glucose in the hydrolysis reactor and to its low steam consumption. On the other
hand, its reactor volumes were the second higher, which suggests that steam
explosion might be a better option when the overall investment costs are con-
sidered, despite its lower ethanol production.
94 C. B. B. Costa et al.

5.5 Conclusion

The production of 2G ethanol from sugarcane bagasse, integrated to the 1G pro-

cess, for five different pretreatments were analyzed from a process perspective.
Considering the experimental results reported in the literature and used in this
study, ethanol production could be increased by 5.7 L/TC for the best pretreatment
option (alkaline). For the same process, electric energy production is decreased by
29.9 kW/TC. Its market value could be used to determine ethanol minimum selling
price in an economic analysis. If investment costs are considered, the results for
the demanded volumes for the pretreatment and hydrolysis reactors suggest that
steam explosion might be a better option, despite its lower 2G ethanol production.
As a general case, an increase in solid loads both in the pretreatment and in the
hydrolysis reactor could greatly benefit the 2G ethanol process feasibility.

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Chapter 6
Potential Biomass Resources for Cellulosic
Ethanol Production in Brazil: Availability,
Feedstock Analysis, Feedstock
Composition, and Conversion Yields

Boutros F. Sarrouh, Júlio C. dos Santos, Mário Antônio A. Cunha

and Ricardo F. Branco

Abstract Due to economic, geopolitical, and environmental issues, the world’s

attention turns to alternative energy sources, especially for second-generation
ethanol. In addition to economic considerations, other factors such as energy
security, greenhouse gas emissions, and global climate change are boosting sci-
entific researches concerning alternative bioenergy. According to the literature
projections, more than 10 % of all gasoline used in the world can be replaced by
biofuels over the next 15–20 years. Currently, Brazil is faced with the prospect of
a significant increase in demand for ethanol. This prediction holds up in some
market realities, as increasing domestic consumption of hydrous ethanol by the
successful introduction of the alternative flex fuel vehicle market in automotive
lightweight and expansion of Brazilian ethanol exports due to the increasing global
interest in mixing alcohol with gasoline. In this context, all the potential sources
for ethanol production must be considered wherein cellulosics stands out as an
important alternative. In this chapter, we discuss about the potential to produce
ethanol from lignocellulosic materials, a renewable source largely available in the

B. F. Sarrouh
Department of Chemistry, Biotechnology and Bioprocess Engineering, Federal University
of São João Del Rei, São João Del Rei, Brazil
e-mail: bsarrouh@yahoo.es
J. C. dos Santos
Department of Biotechnology, Engineering School of Lorena, São Paulo University,
São Paulo, Brazil
e-mail: jsant200@usp.br
M. A. A. Cunha (&)
Department of Chemistry, Technological Federal University of Paraná, Curitiba, Brazil
e-mail: mcunha@utfpr.edu.br
R. F. Branco
Institute of Exact Science, Fluminense Federal University, Niterói, Brazil
e-mail: ricardobranco@puvr.uff.br

S. S. da Silva and A. K. Chandel (eds.), Biofuels in Brazil, 97

DOI: 10.1007/978-3-319-05020-1_6,  Springer International Publishing Switzerland 2014
98 B. F. Sarrouh et al.

world. Particularly, some aspects of potential sources in Brazil are described. First,
however, brief comments on the composition of these materials and some ana-
lytical methods used to characterize them are exposed.

6.1 Introduction

The agro-industrial waste can contain many substances of high value. If appro-
priate technology is used, this material can be converted into commercial products
or raw materials for secondary processes using biotechnological processes. It is
therefore important to develop new techniques aimed at using these residues to
obtain products useful to mankind, and with this goal, the use of fermentation
processes have been extensively studied (Baudel et al. 2005; Delgenes et al. 1998).
In Brazil there is a great potential to exploit renewable sources with competitive
costs in international terms. When searching in Brazil for production of low cost
energy, the choice falls on a renewable alternative. Among the larger countries, it
is a unique case. The analysis of Brazilian resources to be exploited commercially
by merit of maintaining lower cost indicates a power supply with a large share of
renewables, with the same mix adopted in recent decades: use of energy derived
from sugarcane and exploitation of the hydroelectric potential of the country.
Brazil is the world’s largest producer of ethanol derived from sugarcane and as
a consequence there is generation of large amounts of lignocellulosic biomass
from this industrial sector which can be converted into second-generation ethanol.
Additionally, as the country has a robust agro-industrial park, with industries using
different vegetal feedstocks, several residues with potential for alcohol production
are generated. There are however some bottlenecks that need to be overcome to
make the production of second-generation ethanol competitive and viable eco-
nomically compared to the first generation.
The use of the full potential of any cellulosic feedstock is associated to a deep
knowledge of its composition. This, before any discussion on potential feedstocks
to second-generation production in Brazil, some comments will be carried out on
analytical methods used for the compositional characterization of the material.
Particularly, some brief comments will be put on the new approaches related to
analytical strategies used for lignocellulosics.

6.2 Feedstock: Composition-Advanced Analytical

Techniques for Characterization

Wastes from vegetable biomass are composed mainly of macromolecular fractions

from plant cell wall: cellulose, hemicellulose, and lignin.
Cellulose is a polysaccharide composed of glucose molecules linked through
intermolecular interactions, which result in a crystalline structure which gives high
6 Potential Biomass Resources for Cellulosic Ethanol Production in Brazil 99

strength and insolubility of the molecule to the action of chemicals. Nevertheless,

the pulp is prone to chemical or enzymatic hydrolysis.
The hemicellulose, xylan, unlike cellulose, comprises various types of sugars
being considered, therefore, a heteropolymer. It consists of pentoses (D-xilose,
b-and a-L-arabinose), hexoses (b-D-manose, b-D-glucose and a-D-galactose) and/
or uronic acids (a-D-glucuronic acid, a-D-4-O-a-metilgalacturonicoacid, and
D-galacturonic acid). This polysaccharide is more prone to hydrolysis. Xylan is the
second most polysaccharide found in nature.
Lignin is formed by a complex structure which gives the plant a higher
mechanical strength. This is a compound highly resistant to chemical and enzy-
matic degradation and as overlying other polysaccharides, is a problem when
trying to use the crushed sugarcane to produce ethanol because it prevents access
to the other tissues. However, there are microorganisms that can carry out their
degradation.
Other substances that can be extracted from the plant material are waxes,
alcohols, lipids, steroids, fatty acids, hydrocarbons, and flavonoids. Some of these
compounds can be toxic extractives. These molecules may vary according to the
species examined and the type of processing to which the material is subjected.
The remaining materials are considered nonextractive substances, mainly ashes.
These inorganic compounds are known as salts or minerals such as potassium,
silicon, manganese, sodium, calcium, among others. The composition of nonex-
tractive substances depends on soil conditions, climate, and environment.
Due to biomass heterogeneity, robust analytical methods are needed to support
and enable biomass conversion processes. The chemical composition of a biomass
feedstock varies as a function of many factors including plant genetics, growth
environment, harvesting method, and storage (Hames et al. 2003).
Detailed analysis of the chemical composition of herbaceous and woody biomass
feedstocks is labor-intensive, expensive, and time-consuming. Rapid methods of
analysis would enable timely estimates of biomass quality, and may provide a tool
for evaluating the biomass composition as it enters the conversion plant (Sanderson
et al. 1996).
Different techniques are used to quantify these compounds, including High
Performance Liquid Chromatography (HPLC) and Gas Chromatography-mass
Spectrometry (GC/MS). However, these techniques, although largely used due to
its high accuracy, require processing samples in the laboratory, which are time and
cost consuming. In this sense, currently, the use of other techniques in qualitative
and quantitative analysis of solid, liquid, and gas is already widely recognized. The
most widespread ones are absorption infrared (NIR–NIR and FTIR-absorption
infrared Fourier transform), Raman scattering, fluorescence, and photoacoustic. In
particular, infrared spectroscopy methods combined with statistical analysis of
spectra have been successfully used in various areas such as food, environment,
and pharmaceuticals. Recently, the use of Near Infrared (NIR) official method was
accepted as recognized by agencies such as the American Society for Testing and
Materials (ASTM) and the United States Pharmacopoeia (Haack 2010).
100 B. F. Sarrouh et al.

Fig. 6.1 Representative near infrared spectra of various biomass feedstocks. Reproduced from
Sanderson et al. (1996)

The near infrared spectroscopy has been used to determine the quality
parameters as soluble solids, polarizable sugars, and reducing sugars present in
sugarcane. Likewise, infrared spectroscopy Fourier Transform is also used in the
investigation of solid bagasse from sugarcane (Thermo Nicolete Corp 2001). Near
infrared reflectance spectroscopy (NIRS) has been used commercially as a rapid
and effective analysis tool to estimate lignocellulosic composition (Sanderson
et al. 1996).
NIRS is characterized as a nondestructive technique, with an easy sample
preparation and management (no reagents are required), rapid (1 min per spec-
trum) and inexpensive. Near infrared radiation (700–2,500 nm) is absorbed by
various bonds, such as C–H, C–C, C = C, C–N, and O–H, characteristics of
organic matter, as shown in Fig. 6.1 (Ludwig and Khanna 2001).
Similarly, Raman scattering is extremely efficient and of high sensitivity widely
used in the identification of organic compounds, including those of interest in
lignocellulosic: aliphatic acids, furans, and phenolic compounds. The advantage of
Raman spectroscopy with respect to the infrared absorption lies in its greater
sensitivity. Moreover, one should consider the complementary character of the
techniques, since no active compounds, Raman and infrared active (Lupoi and
Smith 2012).
Another promising technique used for lignin structure analysis is known as
pyrolysis Gas Chromatography–Mass Spectrometry (Py–GC–MS). This analytical
method utilizes a microscale quartz reactor inserted into a platinum wire probe
6 Potential Biomass Resources for Cellulosic Ethanol Production in Brazil 101

capable of heating to high temperatures at extremely fast rates. This pyroprobe is

directly coupled to a GC–MS instrument through a transfer line, allowing rapid
analysis. Py–GC–MS revealed a variety of pyrolytic products, including methoxy-
phenols and other aromatic compounds derived from the monomeric units coumaryl,
coniferyl, and sinapyl alcohols within the lignin structure (Mendu et al. 2011).

6.3 Potential Feedstocks

6.3.1 Sugarcane Bagasse

In Brazil, studies have been conducted systematically for biofuel production (high
environmental value) and biomolecules (high added value) for organic conversion,
mainly using sugarcane bagasse as feedstock. Deployment of ethanol technology
from sugarcane bagasse in Brazil is favored because the production process can be
attached to existing units of sugar and alcohol industries, requiring lower invest-
ment, infrastructure, logistics, and energy supply. Furthermore, bagasse is gener-
ated in industrial plants, and as such, free transportation costs.
Brazil is the largest producer of sugar in the world. Therefore, it is one of the
countries that generates waste bagasse from sugarcane for which new techniques
of exploitation are always needed. For the season 2013/2014, the culture of sug-
arcane is expected to continue to expand. The prediction is that Brazil will attain
an increase of 408 ha of planted area, which is equivalent to 4.8 % compared
to the 2012/2013 crop. São Paulo, Minas Gerais, Goiás, and MatoGrosso do
Sul should be the states with the largest increase in planted areas with 141.400,
106.100, 101.100, and 43.500 ha, respectively. This growth is due to the expansion
of new planting areas for the sugar-alcohol industries already in operation. The
total cultivated area with sugarcane in 2013/2014 is estimated at 8.933.00 ha,
distributed in all producing states according to their characteristics.
The forecast for total sugarcane to be ground is 653.81 million tons, an increase
of 11.0 % compared to the 2012/2013 crop, which was 588.92 million tons,
meaning that the amount to be ground to be 64.89 million tons more than in the
previous harvest.
The forecast for sugarcane production in 2012/2013 was approximately 196
millions of tons, according to the proportion indicated by Procknor (2000).
Sugarcane bagasse is composed of the fibrous material obtained after sugarcane
is crushed to extract the juice. Much of the bagasse is used by the industry itself as
an energy source, and the plants themselves use up to 80 % of bagasse as an
energy source to replace fuel oil in the heating process of the boilers and for the
generation and sale of electricity (Teixeira et al. 2007). There are, however, non-
energy uses for sugarcane bagasse, some of them already made viable commer-
cially. Bagasse plays an important role as a raw material in the paper industry and
cardboard manufacturing clusters, as alternative materials in construction, animal
102 B. F. Sarrouh et al.

feed, and microbial biomass production, acoustical, fodder for agriculture, xylitol,
ethanol, hydroxy methyl furfural, alkaloids, and enzymes (Sarrouh and Silva 2008;
Carvalho et al. 2008; Neureiter et al. 2004; Howard et al. 2003; Pandey et al.
2000). However, there is still a surplus (10–20 %) of this waste which is not used,
causing serious environmental pollution and storage (Teixeira et al. 2007).

6.3.2 Sweet Sorghum Straw and Bagasse

Sorghum is able to produce more than 2,500 L of ethanol per hectare during off-
season of sugarcane. The development of this culture will generate an improve-
ment in the productivity of sugarcane by providing a favorable period for its
harvest (Monsanto 2013).
Sweet sorghum can also be produced between May and December, being
economically more competitive than sugarcane in marginal areas or potentially in
rotation for ethanol production and cogeneration for electricity production (Ceres
2013).
The sorghum plant’s culture became popular in many parts of the world due to
its ability to adapt to a wide range of environments, especially under water stress
conditions (Williams et al. 1999). Table 6.1 shows the chemical composition of
sweet sorghum plant.
As sorghum plants mature more quickly than canesugar and achieve an opti-
mum level of sugar in different periods of the year, sugar obtaining by the ethanol
industries can extend the operating season for up to 60 days or more. Figure 6.2
shows that by extending the operational season of the plant the price of ethanol
becomes more competitive.
In the 1980s, sweet sorghum plant (Sorghum bicolor) was introduced in Brazil
as an alternative to ethanol production during the off-season of canesugar. The
initiative was promoted by Pro-alcohol program developed by the Federal Gov-
ernment to encourage the use of alcohol and other energy sources as an alternative
to gasoline in a period of global oil crisis. However, at the beginning, the large-
scale production of this raw material was not successful because there were no
adequate hybrids suitable for the Brazilian regional planting peculiarities. Today,
30 years later, sweet sorghum has become a commercial reality due to techno-
logical advances in the industry, especially breeding technologies for sugarcane
and sweet sorghum. Figure 6.3 shows the main differences between sugarcane and
sweet sorghum, especially in terms of productivity and cultivation cycle.
Sorghum has great potential as an annual energy crop. While primarily grown
for its grain, sorghum can also be grown for animal feed and sugar. Sorghum is
morphologically diverse, with sorghum grain being of relatively short stature
grown for the grain, while forage and sweet sorghums are tall and grown primarily
for their biomass.
The main product obtained from sweet sorghums is the fermentable sugar-rich
juice that is produced and accumulated in the stalks in a similar fashion as
6 Potential Biomass Resources for Cellulosic Ethanol Production in Brazil 103

Table 6.1 Chemical characterization of the sweet sorghum grains, juice, and bagasse
Grains (% wet base) Juice (% wet base) Bagasse (% dry base)
Starch 70.1 Soluble solids 18 Cellulose 38.5
Proteins 11.2 Sucrose 8.5–12.4 Hemicellulose 21.4
Humidity 11.6 Glucose 2.1 Lignin 17.6
Fibers 1.82 Fructose 1.2 Protein 1.1
Lipids 3.54 Starch 0.5 Extractives 13.7
Ashes 1.8 Water 84 Ashes 3.7
Barcelos (2012), Rossell (2011), Panagiotopoulos et al. (2010), Wu et al. (2007)

Fig. 6.2 Relation between

additional operational days
by using sweet sorghum and
the price of 1 L of ethanol
expressed in real
(R$1 = US$0.5).
Reproduced from Ceres
(2013)

sugarcane. The extracted sweet juice is mainly composed of sucrose, glucose, and
fructose, and thus can be directly fermented into ethanol with efficiencies of more
than 90 % (Wu et al. 2010).
Considering two crop harvests a year, sorghum can yield about 15.62 ton/ha of
biomass which can be exploited to produce second-generation ethanol. In addition,
15.6 ton/ha of panicles with a high value for silage or as direct feed is produced
(Cardoso et al. 2013).
Current interest in bioethanol research is focusing on how to efficiently liberate
sugar molecules from lignocellulosic feedstock for increased bioethanol production.
Therefore, integration of the sugars from sweet sorghum bagasse (cellulosic residue
after cane extraction) with sugar derived from the stem/cane will further increase
ethanol yield and also make bioethanol affordable (Aleke 2011).
104 B. F. Sarrouh et al.

Fig. 6.3 Comparison

between sugarcane and sweet
sorghum. Reproduced from
Globo (2013)

6.3.3 Corn Stover

Corn is a grain that is produced and known worldwide due to its rich starch
content. In Brazil, the production this year (2013) was estimated as 79 million
tons, which is approximately 10 % more than the last harvest 2012, this increase
was a reflect of an increase in the planted area and culture efficiency due to
demand and governmental incentives (CONAB 2013). The main production
regions are the Center-West and South, representing, respectively 42 and 33 % of
6 Potential Biomass Resources for Cellulosic Ethanol Production in Brazil 105

the national production, being the estates of MatoGrosso (Center-West) and Paraná
(South), the top two producers (CONAB 2013).
However, corn culture is one of the agricultural activities that most generate
residues, because the corncob represents little considering the whole plant.
Therefore, after the harvest the stem, leaf (straw), and cob are left in the field;
these residues are known as corn stover (Gil et al. 2013). Figure 6.4 shows the
residues of corn culture after the harvest. According to Lindstrom et al. (1981)
(quoted in many scientific articles) the ratio between corn harvested and stover
above ground is 1:1 (16 % moisture), much greater than sugar bagasse, mentioned
before). Brazil alone in (2013) produced around 66 million tons (dry weight) of
corn stover. Nevertheless, only part of this amount can be removed and used, an
average of 40 % can, on a sustainable basis, be removed (Kadam and McMillan
2003; Walsh et al. 2000). It is advised to leave some stover on the field in order to
prevent soil erosion. The removal optimization of corn stover from the field has
been studied by Gil et al. 2013. Therefore, considering these data, in 2013 Brazil
generated 26.4 million tons (dry weight) of corn stover available as raw material.
Corn stover can be used for many purposes as listed below:
(1) Farm/animal uses: It is a potential feed for dairy cattle (Adams 1998). How-
ever, it is not a high-quality feed, its biggest disadvantage is its physical
character.
(2) Fuels: It can be used as a fuel, after milling, in a boiler furnace, as most
lignocellulosic material.
(3) Biobased materials: Particleboard has been produced from bagasse and wheat
straw as well as other types of fibers (Karr and Sun 2000; Karr et al. 2000).
Building panels have been made from several crop residues including wheat
straw, rice straw, and bagasse, and can also be made from corn stover. A
drawback to manufacturing particleboard using these fibers, like corn stover, is
the need for expensive resin binders (Kadam and McMillan 2003). Pulp and
paper: Corn stover possesses cellulose, as other agricultural residues, therefore
corn stover-based pulp and paper production is a viable alternative (Wagner
et al. 2000). Using corn stover has advantages as environmental benefit (di-
oxins are less generated), and according to Kadam and McMillan (2003) the
lower lignin content requires less bleach than that needed for wood pulp.
(4) Chemicals: Corncobs can be feedstock for producing furfural (Foley and
Vander Hooven 1981; Riera et al. 1991).
(5) Miscellaneous: Hog manure and corn stover can compose potting soil (Adams
1998). Corn stover can also be put along the roadsides to prevent soil erosion
and can be similarly used for slope stability (Zinkand 2000).
Although about 26 dry tons per year of corn stover is available in Brazil, based
on the above recent estimates and many uses without much transformation, if one
looks at corn stover chemical composition its real potential will be noted.
106 B. F. Sarrouh et al.

Fig. 6.4 Residue of corn

cultures, known as corn
stover (stem, leaf and cob).
Reproduced from Stephanie
Chen Design Lab (2013)

Corn stover is a lignocellulosic material, hence there are three parts (cellulose,
hemicellulose, and lignin) as mentioned before. In 6.2 is presented the compo-
sition of corn stover (Kim and Holtzapple 2005).
It must be pointed out the amount of glucan (cellulose), xylan, and arabinan
(hemicellulose) in corn stover, according to Table 6.2 sums up 60 % of the
material. Considering that these polymers are composed of fermentable sugar
(glucose, xylose, and arabinose), one can affirm that most of the corn stover is a
sugar source, which makes this material ideal for sustainable processes.

6.3.4 Soybean Waste

The Global production of soybeans in 2011/2012 was 236.38 million tons (USDA
2013) and in this scenario, Brazil is the second largest producer, surpassed only by
the United States. The Brazilian soybean production in 2012 was 65.7 million tons
(almost 30 % of the global production) and its estimated production is 81.1 mil-
lion tons for 2013 (IBGE 2013).
Soybean (Fig. 6.5) has a wide field of industrial applications including the
production of vegetable oil, meal for animal feed, margarine, protein extracts,
polymers, pharmaceuticals, soaps, printing inks, fertilizers, and others. The oil and
meal are the main derived products from soybeans and also in the beneficiation
process of the grain are generated wastes such as shells and soy molasses.
6 Potential Biomass Resources for Cellulosic Ethanol Production in Brazil 107

Table 6.2 Corn stover Composition % mm-1 (dry weight)

chemical composition. Kim
and Holtzapple (2005) Glucan 36.1
Xylan 21.6
Arabinan 3.6
Klason lignin 17.2
Acid-soluble lignin 3.6
Protein 3.5
Acetyl 3.2
Ash 6.9
Others (mannan, galactan, uronic 6.1
acid and non-structural sugar)

6.3.4.1 Soybean Hulls

Soybean hulls are a residue obtained from the rupture of the grains in the early
stages of the process and also is the main by-product of this oilseed processing
industry. This biomass represents 8–10 % of the total weight of the grain (Lia et al.
2011; Gnanasambandan and Proctor 1999). Considering the Brazilian soybean
crop in 2012, around 5.9 million tons of bark were generated in the country and, in
the same period, produced 21.3 million tons in the world. The high production
volume of this residue can become attractive for Biorefining and production of
high-value products in countries such as Brazil and the United States.
The chemical composition of soybean hulls may vary depending on growing
conditions, grain growth, and efficiency of the extraction process. The method of
removing the bark is done with greater intensity when it is desired a soybean meal
with higher protein content that can interfere with the chemical composition of the
bark. In Table 6.3 is described the chemical composition of this biomass according
to Cassales et al. (2011), Yoo et al. (2011) and Mielenz et al. (2009).
Glucose is the main component of the polysaccharide fraction (39.7 %), fol-
lowed by xylose (19.6 %) and arabinose (5.9 %), respectively. Other components
are present as acetic and glucuronic acid (2.6 %), cellobiose (1.6 %), lignin
(9.1 %), protein (1.13 %), ash (0.6 %), and extractives (3.2 %) (Cassales et al.
2011). The authors suggested this residue as a potential biomass for bioethanol
production due to the presence of fermentable sugars associated with low lignin
content. The lignin present in soybean hulls is lower than found in other wastes
like sugarcane bagasse (22.8 %), corn stover (16.3 %), wheat straw (18–20 %),
corncob, among others (Merali et al. 2013; Siqueira et al. 2013; Cybulska et al.
2012). The degradation of lignin produces compounds toxic to microorganisms
and the smaller content of this macromolecule in the lignocellulosic wastes are
desirable for use in processes of bioconversion. Note that soybean hulls have lower
amounts of lignin hydrolyzate and contain minor amounts of toxic phenolic
derivatives than hydrolysates of other waste. Soy hulls also contain appreciable
quantities of minerals (P, K, Mn, Ca, Fe, Cu, S, and Zn), which can facilitate
fermentative processes, since some may participate as enzymatic cofactors and the
auxiliary microbial metabolic activity.
108 B. F. Sarrouh et al.

Fig. 6.5 Soybean grain (a) and soybean hulls (b)

Table 6.3 Chemical composition (mass fraction % on a dry basis) of soybean hull
Composition Mielenz et al. (2009) (%) Cassales et al. (2011) (%) Yoo et al. (2011) (%)
Cellulose 29–51 39.7 35.4
Hemicellulose 10–20 25.5 17.2
Lignin 1–4 9.1 2.3
Pectin 6–15 – –
Protein 9–14 13.1 –
Ash 1–4 0.6 –

6.3.4.2 Soybean Molasses

Soy molasses is a by-product of soy protein concentrate production. After the oil is
removed from the crushed soybean, the defatted flakes (white flakes) or soybean
meal is washed with 70–90 % aqueous ethanol to remove the carbohydrates and
concentrate the protein. The washed solids have a protein content of at least 65 %
(dry matter basis) and are known as soybean protein concentrate (Long and
Gibbons 2013). The sugars present in the soybeans are extracted by ethanol–water
mixture, and after recovery of ethanol is sourced molasses with a moisture content
of around 50 % (Fig. 6.6).
Soybean molasses is composed mainly of carbohydrates, lipids, protein, fiber,
and ash. The most abundant carbohydrates are sucrose, raffinose, and stachyose,
present at percentages of 28.4, 18.6, and 9.7, respectively, based on dry weight
(Silva et al. 2012). Reducing sugars and sucrose are obtained by enzymatic
hydrolysis of the stachyose and raffinose oligosaccharides using a-galactosidase
and invertase (Silva et al. 2012). In Table 6.4 is described the chemical compo-
sition of this biomass according to Long and Gibbons (2013), Silva et al. (2012),
Siqueira et al. (2008) and Knudsen et al. (2007).
6 Potential Biomass Resources for Cellulosic Ethanol Production in Brazil 109

Fig. 6.6 Production process of the soybean protein-concentrate. Reproduced from Siqueira et al.
(2008)

6.3.5 Eucalyptus and Forest Residues

The world forest area totals over 4 billion hectares in the world, covering 31 % of
the total land area of the earth. The five most forest-rich countries are the Russian
Federation, Brazil, Canada, the United States of America, and China, which
account for more than half of the total forest area (53 %) (FAO 2010).
Forests are fundamental from an ecological viewpoint, but are also very
important economically, with the world trade in 2007 reaching about
US$10 trillion (BSS 2008). Products from forest resources are among the top ten
main internationally traded ones, corresponding to 3 % of global trade (about
US$300 million). In this context, silvicuture is an important tool that allows the
economical use of these resources preventing further deforestation of native
vegetation and contributing to reforestation or afforestation (i.e., planting of trees
on land that was not previously forested) (FAO 2010; BSS 2008).
Since 2000, Brazilian silvicuture has surpassed forest extraction in production
value. The sector’s gross production value of forest-based sector associated with
forestry in Brazil corresponded to about 30 billion US dollars in 2012, repre-
senting an important social activity, with the producing chain generating about 4.4
110 B. F. Sarrouh et al.

Table 6.4 Chemical composition (mass fraction % on a dry basis) of soybean molasses
Composition Knudsen et al. Siqueira et al. Silva et al. Long and Gibbons
(2007) (2008) (2012) (2013)
Total carbohydrate – 57.3 – –
Glucose – 0.243 – 4.67
Fructose – 0.127 – 2.96
Galactose – 0.254 –
Sucrose 35.3 21.3 25.99 18.5
Raffinose 3.7 9.7 11.74 25.5
Stachyose 18.9 18.6 15.50 34.2
Proteins 8.4 9.4 6.44 11.7
Lipids 15.5 21.2 15.60 4.91
Fibers 5.7 – –
Ash 6.9 6.4 7.88 21.9

million jobs and resulting in an investment of about 75 millions US dollars in

social improvement, education, and environmental programs, benefiting 1.3 mil-
lion people and approximately one thousand communities located around the
companies. The largest forest plantations in Brazil are found in the southern and
south-eastern regions, especially in the states of Minas Gerais, São Paulo, and
Parana. The sector involves a number of industrial segments such as Pulp and
Paper, Industrialized Wooden Panels, Mechanically Processed Wood, Charcoal-
fired Steelworks, and Biomass, among others (ABRAF 2013; IBGE 2012;
Gonçalves et al. 2008).
The main species of Brazilian forestry correspond to Eucalyptus. Indigenous to
the Australasian region, this angiosperm from the genus Eucalyptus includes about
800 species, with mainly the following cultured in Brazil: Eucalyptus grandis,
Eucalyptus citriodora, Eucalyptus camaldulensis, Eucalyptus saligna, Eucalyptus
urophilla, among others, including hybrids as Eucalyptus urograndis, obtained from
E. urophilla and E. grandis (Ministério da Agricultura, Pecuária e Abastecimento
2010; Coppen 2002). The last is the mostly clonally propagated and the most
planted, owing to its excellent adaptation to regions with low to moderate water
deficiency and low fertility soils (Gonçalves et al. 2008).
According to ABRAF (2013), the Eucalyptus planted area in Brazil in 2012
reached 5.10 ha, 53 % of which is located in southeast Brazil, with the northern
region of the Brazilian Amazon representing only 6.2 % of this area. Eucalyptus
corresponds to 70.8 % of total area of forest plantations. Plantations are harvested
at short rotation periods of about 7 years before coppice and the productivity is in
the range of 35–55 m3 per hectare per year (BSS 2008). The main use of this genus
of tree in Brazil is in the industrial segment of Pulp and Paper.
Another important group of species is pine, which corresponded to 22 % of the
total area of forest plantations in Brazil in 2012. The remaining 7.8 % of planted
area is used with species of Acacia, Araucaria, Populus, Teak, Rubber Tree, and
Paricá.
6 Potential Biomass Resources for Cellulosic Ethanol Production in Brazil 111

Forest harvest practices or forest product processing results in a large quantity

of residues, a part of which remains in land and contributes to fertilize the soil, but
much of it is wasted and can pile up, resulting in environmental problems and
representing a loss of valorous raw material. For instance, besides previously
discussed for agricultural sources, wood residues are suitable lignocellulosic raw
material to be used in second-generation ethanol production.
According to Ferreira-Leitão et al. (2010), forestry wastes correspond to parts
of trees not profited for cellulose production, such as tips and branches, which
contribute to soil fertility upon degradation. Also, according to these authors, these
wastes are by nature heterogeneous in size, composition, and structure, including
mainly small pieces of wood, as tree bark, corresponding to about 71 % of total
waste, and sawdust about 22 % of the total.
Industrial waste wood can be grouped into sawdust, wood shavings, solids from
wood, bark, and others and are generated from the transportation of the roundwood
industry, to its handling and processing. The residues can be from sawmills and
plywood manufacturers, or from industries like pulp and cellulose, wood panel, or
furniture. Besides, there are urban residues from civil construction, municipal and
urban landscaping, or wood packaging (Wiecheteck 2009).
In an early work, Ferreira-Leitão et al. (2010) estimated the total potential of
generation of forest residues in Brazil in 52.8 9 106 tons (dry mass). Concerning the
waste available in Brazil to energy production, another simple estimative of the
magnitude of total mass can be carried out by considering only the residue generated
after processing of timber logs and taking into account that, in this case, about 50 %
of the wood mass is wasted (Coelho et al. 2012). According to IBGE (2012),
125.9 9 106 m3 of timber logs were produced in Brazil in 2011. Using the corre-
spondence of 0.68 t to each m3 (BSS 2008; Coelho et al. 2012), 75.54 9 106 tons of
timber logs were produced, generating about 37.77 9 106 tons of residues in the
country in 2011. As this mass was generated only during timber processing, residues
that were left in the field were not taken into consideration.
Besides the total biomass production, the composition of the material is funda-
mental information to be considered, because ethanol production is obtained by
carbohydrate fraction, but lignin content is an important parameter in initial path-
ways of the process of production of second-generation ethanol. The composition is
variable and depends on the wood species of origin of waste. Trees are generally
classified into two broad categories known as ‘‘softwoods’’ (gymnosperms) and
‘‘hardwoods’’ (angiosperms or dicotyledonous angiosperms) (Álen 2000). Soft-
woods are also referred to as coniferous wood, and include species of pines, e.g.,
Hardwoods, in turn, include species of eucalyptus, oak, and poplar, among others.
Table 6.5 shows the range of variation of main components for hardwood and
softwood species.
As shown in Table 6.5, although the cellulose content is not so different for
hardwood and softwood, in general, hardwoods have comparatively higher tenor of
hemicellulose and lower tenor of lignin. However, there are exceptions to this rule:
tropical hardwood has higher lignin content compared to softwood.
112 B. F. Sarrouh et al.

Table 6.5 Percentage of the Composition Percentage of content (% dry mass)

main components of woods
(Álen 2000) Softwood Hardwood
Cellulose 40–45 40–45
Hemicellulose 25–30 30–35
Lignin 25–30 20–25
Extractives 2–5 2–5

Specifically in relation to Eucalyptus, Emmel et al. (2003) related the following

composition for industrial chips of E. grandis: 44.65 % of cellulose, 25.77 % of
lignin, 15.33 % of hemicellulose (xylan), 3.25 % of extractives in benzene:ethanol
(2:1, v/v) and 11 % of unidentified components which included 4-O-methyl-glu-
curonic acid and acetyl groups present in heteroxylans.

6.3.6 Other Potential Raw Materials

There are a number of other potential raw materials to be used to produce ethanol in
Brazil. The use of a specific one is dependent on factors as regional availability and
logistic aspects. Following, two examples of important possibilities are commented.

6.3.6.1 Cassava Residues

Another important crop in Brazil with great potential for ethanol production is
Cassava (Manihotesculenta, Crantz), a woody widely cultured in the tropical
regions of Africa, Asia, and Latin America (Wanapat and Khampa 2007; Boonnop
et al. 2009; Ferreira-Leitão et al. 2010). An annual crop, this plant of the family
Euphorbiacea represents the third most important source of calories in the tropics,
after rice and maize (FAO 2013; Silva et al. 2001). The root is composed almost
entirely of carbohydrate which can be used as an important food source.
The annual world production of cassava root in 2011 was about 252 millions
tons, with Brazil producing about 25 million tons (FAO 2013). Its tuber contains
70 % starch by dry weight and has been used as a promising feedstock for fuel
ethanol (Huang et al. 2010; Sanchez and Cardona 2008). It is possible to develop a
system to produce ethanol from the whole plant using both starch and cellulose
available. This is a true mainly considering residues of the cassava crop, estimated
to weigh 144–257 % of the root weight, and residues from processing, such as
bran from the root peeling, cassava waste liquor from pressing and waste fibers
generated in industrial production of starch and cassava flour (Ferreira-Leitão et al.
2010). If an ethanol producing process from starch cassava is taken into account,
massive amount of residues will be produced as by-products, nearly half a ton of
cassava residues for producing 1 ton of ethanol, cellulose accounts for nearly one-
quarter of the dry residue weight (Zhang et al. 2011, 2013).
6 Potential Biomass Resources for Cellulosic Ethanol Production in Brazil 113

6.3.6.2 Peanut Hulls

In Brazil, peanut hulls represent another vegetable biomass that can be used for
second-generation ethanol production. The world peanut production is approxi-
mately 29 million metric tons per year, with the U.S. being the world’s third
largest producer, after China and India. Brazil occupies the 17th rank of peanut
producers (Soyatech 2013). Brazil produced 226.5 million metric tons of peanuts
in the 2010/2011 agricultural season in a land area of 84.100 ha. Several value-
added products have been obtained from peanut as peanut oil and butter, peanut
flour, and roasted peanuts.
Peanut shells are abundant lignocellulosic residues that could be considered as
raw materials for ethanol production in China, India, and United States. Although
Brazil is the 17th peanut producer, its production is relatively significant and
therefore large quantities of shells are generated per year. This biomass has high
contents of cellulose as can be seen in Table 6.6.
Cellulose from peanut shells can be chemically or enzymatically hydrolyzed to
glucose and subsequently converted into ethanol. A potential barrier for the
hydrolysis of this biomass could be its high content of lignin. Boonmee (2012)
reports lower total sugar yield (22.8 g/100 g dry weight) after acid hydrolysis
compared to other biomass such as bagasse, rice hull, leaf, and stalk of sugarcane
(43.8–49.6 g/100 g dry weight). The high lignin content could contribute to
obtaining hydrolyzate rich in compounds toxic to microbial cell. However, the
employment of appropriate detoxification systems associated with the use of
adapted cells could overcome this problem.

6.4 Potential for Second-Generation Ethanol Production

6.4.1 Sugarcane Bagasse

Sugarcane bagasse is a fibrous by-product resulting from grinding of the cane for
sucrose extraction and can have many uses, from producing energy by combustion
in boilers to soil incorporation or as part of the bovine diet. Even after extraction of
sucrose and other nutrients, bagasse still contains a lot of organic matter, thus
being a possible source of more energy and other fine chemicals. The alcohol
obtained from bagasse is known as second-generation ethanol (lignocellulosic
ethanol). However, due to the complexity of its fibrous components, many studies
are still needed to improve the efficiency of ethanol production. The crushed
vegetal material is rich in polysaccharides (complex sugars) such as cellulose and
hemicellulose, compounds commonly found in the cell walls of plant cells. Lignin
is also contained in this organic mass. These three materials together constitute
more than 75 % of the biomass and confer mechanical strength to the plant. The
remaining biomass is composed of substances such as proteins, vegetable, and
mineral oils.
114 B. F. Sarrouh et al.

Table 6.6 Chemical composition (mass fraction % on a dry basis) of peanut shell
Composition Al-Masria and Boonmee Riville et al. Kuprianov and
Guenther (1999) (%) (2012) (%) (2012) (%) Arromdee (2013) (%)
Cellulose 42.1 22.1 40.5 51.3
Hemicellulose 11.5 12.1 14.7 10.7
Lignin 37.4 35.2 26.4 45.5
Protein 5.7 – – –
Ash 2.8 2.2 – 6.3

The deployment of the technology of second-generation ethanol from sugarcane

bagasse in Brazil is favored because the production process can be attached to
existing units of sugar and alcohol industries, requiring lower investment, infra-
structure, logistics, and energy supply. Furthermore, the bagasse is generated in
industrial plants, and as such, free transportation costs. This is promising because
from every 10 million tons of dry biomass, 600 million gallons of ethanol can be
produced considering the use of only a part of cellulosic fibers (Soccol et al. 2010).

6.4.2 Sweet Sorghum Straw and Bagasse

During the processing of sweet sorghum by industries, only stems are used in the
manufacture of alcohol. The pulp and seeds are discarded and in some cases are
used for animal production. Sorghum bagasse is normally employed in furnaces at
power plants as energy source. Nowadays and after the development of innovative
technologies, sorghum bagasse is used for the production of ethanol as a source
renewable energy (Oliveira et al. 2009).
According to Barcelos (2012) the composition of sweet sorghum’s juice and
bagasse is similar to sugarcane, as well as the efficiency of bagasse for cogene-
ration (2.150 kcal kg-1 for sugarcane bagasse and 2.200 kcal kg-1 for sorghum
bagasse).
In comparison with other feedstocks, the characteristics of sorghum cellulosic
fiber are similar to that of other nonwoody sources, such as cotton stalks and corn
stover (Reddy and Yang 2005), and sorghum has lower lignin levels than many
woody and nonwoody fiber sources (Godin et al. 2010).
The sweet sorghum bagasse presents a Brix of 16.5 %, differing from sugarcane
bagasse. According to Silva et al. (2007) sugarcane bagasse presents a Brix of
20 %. This difference between sweet sorghum bagasse and sugarcane bagasse is
probably due to the method of juice extraction. Since, sugarcane has thicker stems
than sorghum, the grinder can better extract the juice from the cane. The same
authors confirm that, the density of sorghum bagasse is lower than that of sug-
arcane bagasse, thus requiring reactors with larger capacity for the development of
the hydrolysis processes.
6 Potential Biomass Resources for Cellulosic Ethanol Production in Brazil 115

Sorghum fiber hydrolyzates are liquors rich in both hexoses and pentoses,
therefore production of bioethanol from these matrixes is possible only with the
use of osmotolerant and pentose fermenting yeast or bacterial strains. Ballesteros
et al. (2004) obtained 16.2 g ethanol/L when hydrolyzates obtained from sweet
sorghum bagasse were fermented with Kluyveromyces marxianus.
According to Saldívar-Serna et al. (2012) experimental data obtained from
sweet sorghums cultivated in Central Mexico indicated that these materials are
capable of yielding 6.38 tons of sugar/ha/cut. Consequently, when adequately
bioconverted, they have the potential of producing 4.1 L ethanol. Regarding lig-
nocellulosic fraction, if 15.3 ton of bagasse/ha is obtained containing 29 % cel-
lulose and hemicellulose and 5.4 % of remaining unextracted soluble sugars, up to
2,400 L of ethanol can be obtained.
According to Hess et al. (2007) the logistic cost, including the harvesting,
collecting, preprocessing, transporting, and handing of the raw materials, has an
important share in the whole cost of ethanol production. Thus, if sweet sorghum
bagasse (SSB) could be effectively utilized for ethanol production integrating with
juice fermentation, the overall cost of refining ethanol from sweet sorghum would
be reduced by sharing the co-logistic cost.

6.4.3 Corn Stover

As abovementioned, corn stover is a potential sugar source that can be used for
many purposes, therefore this material can also be used as sugar source for second-
generation ethanol. Since it was already discussed before, in this part it will not be
described pretreatments and treatments, and fermentation details, however studies
have been conducted in this area in order to optimize these processes (Kim and
Holtzapple 2005; Gáspár et al. 2007).
In order to calculate more accurately the corn stover potential to produce
ethanol one must make some consideration: (a) Both cellulose and hemicellulose
will be converted to ethanol; (b) Considering sugar extraction and purification
efficiency of 50–70 %; (c) Fermentation for ethanol of both cellulose and hemi-
cellulose of 40–45 % and; (d) 10 % of the corn stover will be used for energy
(furnaces) and animal feed. These estimates are based on experimental observation
and on the literature (Mosier et al. 2005; Kaar and Holtzapple 2000; Kadam and
McMillan 2003). After these considerations, it is possible to calculate ethanol
production. In Brazil, in 2013 alone it was possible to generate, from
19.80,000 million tons of corn stover 3.96,000–6.23,700 million tons of ethanol
(4.96,000–7.8,200 millions of L), which is 250–394 L of ethanol per ton of corn
stover (dry weight). Other countries, for example the U.S.A., which has a large
corn plantation area, could produce 14.00,000–20.00,000 millions of L of ethanol
per year (Sokhansanj et al. 2002).
116 B. F. Sarrouh et al.

6.4.4 Soybean Residues

6.4.4.1 Soybean Hulls

Pretreatment processes are are necessary for the use of soybean hulls in the pro-
duction process of bioethanol. The objective of pretreatment is to break the pro-
tective barrier of lignin and disrupt the crystalline structure of cellulose, thus
making more accessible carbohydrate enzymes to increase the yield of fermentable
sugars. There have been few reports of studies on processes for pretreatment and
hydrolysis of soybean hulls in the literature.
Thermo-mechanical extrusion pretreatment of soybean hulls followed by
enzymatic hydrolysis was described by Yoo et al. (2011) as a feasible way for
cellulose to glucose conversion. Thermo-mechanical extrusion was shown to be a
feasible pretreatment method for lignocellulosic ethanol production. Values of
cellulose to glucose conversion of until 95 % were obtained. Cellulose conversion
from extrusion pretreatment of soybean hulls was comparable or better than that
obtained from traditional chemical pretreatments utilizing acid and alkali.
Soybean hulls were evaluated as a substrate for production of ethanol by fer-
mentation with Saccharomyces cerevisiae D5A and simultaneous enzymatic sac-
charification by Mielenz et al. (2009). The authors obtained ethanol concentrations
of 25–30 g/L, while under these conditions corn stover, wheat straw, and switch
grass produced 3–4 times lower ethanol yields.
Since there is a valuable market for soybean hulls as animal feed, little attention
has been given to this biomass for the production of second-generation ethanol. In
Brazil this biomass is not competitive for the production of ethanol fuel yet,
especially when compared with the sugarcane bagasse due to the large quantities
of bagasse generated by numerous ethanol plants in the country. However, con-
sidering that ethanol is used in industrial plants producing soy protein in the form
of alcohol solution with a concentration of 70 %, the deployment unit producing
ethanol from soybean hulls associated with producing plant protein concentrate
can be feasible and economically attractive.
Similarly, ethanol is also used in the production of biodiesel from soybean oil
obtained by the transesterification method. In the transesterification of vegetable
oils, a triglyceride reacts with an alcohol in the presence of strong acid or base to
produce a mixture of fatty acids alkyl esters and glycerol. For a complete stoi-
chiometrically transesterification a 3:1 molar ratio of alcohol per triglyceride is
required.
Soy is the crop most used in Brazil for the production of biodiesel and in this
sense, industrial complexes consisting of units associated with the production of
ethanol and biodiesel derived from soybean residues may become a promising
strategy in the future.
6 Potential Biomass Resources for Cellulosic Ethanol Production in Brazil 117

6.4.4.2 Soybean Molasses

Conversion of the carbohydrates from soybean molasses into ethanol through

simultaneous saccharification and fermentation using commercial enzymes (cellu-
lase, b-glucosidase, and pectinase) and S. cerevisiae NRRL Y-2034 or Scheffer-
somyces stipitis NRRL Y-7124 was examined by Long and Gibbons (2013). S.
cerevisiae and S. stipitis produced about 12.5 and 6.00 g/L ethanol, respectively, on
molasses.
Letti et al. (2012) reports 78.3 % maximum theoretical yields and 24.2 g/L of
ethanol in flasks fermentation using Zymomonas mobilis NRRL 806 and 96.0 %
maximum theoretical yields, with productions of 29.3 g/L of ethanol in bioreactor
fermentation. Bioreactor fermentation using S. cerevisiae LPB1 was also studied
and it was reached 89.3 % of the theoretical maximum value.
Silva et al. (2012) studied the production of ethanol from natural soybean
molasses by fermentation with S. cerevisiae, the enzymatic hydrolysis of soybean
molasses by a-galactosidase and the subsequent fermentation with S. cerevisiae
(HF) and ethanol production via simultaneous hydrolysis and fermentation (SHF)
of soybean molasses. The results showed that, although the fermentation of natural
soybean molasses provided a fermentation yield of 72.9 % under optimized con-
ditions, hydrolyzed soybean molasses provided a fermentation yield 7.6 % higher
using the HF process and 8.2 % higher using SHF. Both fermentation processes
resulted in lower concentrations of residual sugar. Comparing the processes of
hydrolysis and subsequent fermentation (HF) and SHF, it appears that the pro-
duction of ethanol (54.0 g/L) were higher in HF when compared to the values for
the simultaneous process (49.2 g/L ethanol).
In Araucária Town (Paraná state) is located the world’s first company to pro-
duce ethanol from soybean molasses. The company can produce up to 10,000 L of
hydrous ethanol per day and it has license from Brazilian National Agency of
Petroleum to produce and commercialize ethanol fuel for cars.
Ethanol production from soybean molasses on industrial scale for use as fuel
still is not economically advantageous compared to other biomasses such as
sugarcane bagasse. However, the transformation of biomass into ethanol by the
company generating the waste can become an attractive prospect, especially when
alcohol is used as solvent for the industry itself or used in power generation or such
fuel for the company’s fleet.

6.4.5 Forest Residues

With relation to forest residues, the main drawback to produce ethanol can be
related to the high degree of difficulty to liberate fermentable sugars from lignin
seal that composes the macromolecular net of the material. This is true mainly for
eucalyptus or hardwoods (Mcintosh et al. 2012).
118 B. F. Sarrouh et al.

Different conversion yields for the process have been related, in dependence of
evaluated conditions and use of hexoses or pentoses in fermentation. Mcintosh et al.
(2012), evaluated conditions for dilute acid pretreatment of eucalypt (Eucalyptus
dunnii) and spotted gum (Corymbiacitriodora) forestry thinning residues for bio-
ethanol. In their work, the authors observe that in the optimized conditions, an
enzyme cellulose hydrolysis yield of 74 % is theoretical. S. cerevisiae efficiently
fermented hexoses from crude E. dunnii cellulosic hydrolysate within 30 h, yielding
18 g/L ethanol, representing a glucose to ethanol conversion rate of 0.475 g/g
(92 %). In another work, Silva et al. (2011), besides fermentation of hexoses from
cellulose with S. cerevisiae, have also evaluated the pentoses fermentation from
hemicellulosic fraction of residual wood chips of cellulose industry. Dilute acid
pretreatment was used to produce hemicellulosic hydrolysate; its fermentation was
carried out using a flocculating strain of Pichia stipitis. The process resulted in
15.3 g/L of ethanol in 40 h of fermentation, corresponding to a yield of 0.32 g/g.
Still considering that work, the solid fraction generated after pretreatment was
subjected to enzymatic hydrolysis, which was carried out simultaneously with
glucose fermentation (SSF: Simultaneous Saccharification and Fermentation Pro-
cess), using a strain of S. cerevisiae and resulting in 28.7 g/L of ethanol in 55 h.
According to the authors, the global yield of the ethanol production process was
100 L of ethanol/ton of eucalyptus wood chips.
The global yield of ethanol production from Eucalyptus biomass was consid-
ered by Gonzales et al. (2011) as equivalent to those in corn stover. In that work,
software aided simulation was carried out for technical and financial performance
of high yield Eucalyptus biomass in a cocurrent dilute acid pretreatment followed
by enzymatic hydrolysis process. The authors have considered an ethanol yield per
ton of dry Eucalyptus biomass of 347.6 L of ethanol, with average carbohydrate
content in the biomass of 66.1 %.

6.5 Future Trends

According to Saldívar-Serna et al. (2012) the genetic improvement of crops is one of

the most promising research priorities in agricultural production with high economic
relevance. In the case of sorghum and sugarcane for fuels there are important
advances in the development of biomass, sweet and high yielding grain varieties and
hybrids, but is yet one of the most important and critical research topics. New
cultivars should be adapted to marginal lands and they must be resistant to pests,
other phytopathogens, and stable facing water stress (Saldívar-Serna et al. 2012).
According to Reddy and Yang (2005) the main obstacle in creating new hybrids
for ethanol production is the ‘‘non additive’’ character of their relevant traits, such
as plant height, total soluble solids, juice production, and lignin:cellulose:hemi-
cellulose ratio. On the other hand according to Turhollow et al. (2010), the genetic
mapping combined with its relatively fast hybridization and field tests, can
facilitate the design and development of dedicated bioenergy cultivars.
6 Potential Biomass Resources for Cellulosic Ethanol Production in Brazil 119

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Chapter 7
Advances in Methods to Improve
the Sugarcane Crop as ‘‘Energy Cane’’
for Biorefinery: An Appraisal

Francis Julio Fagundes Lopes and Viviane Guzzo de Carli Poelkin

Abstract Plant biomass is a source of renewable energy and biomolecules ame-

nable to feed environmentally sustainable biorefineries. Chemistry, biotechnology,
and process engineering advances will make biorefineries feasible in technical and
cost aspects. Efforts have been concentrated in assessing plant biodiversity and
crop potentialities for manipulation of physiological responses such as carbon
fluxes toward soluble, storage, and structural sugars, waxes, oils, phenolics, and
many other products. Thanks to advances in the ‘‘omics’’ field by the use of model
plants, these issues have been addressed, allowing for a better comprehension of
the general plant metabolism with concomitant inferences to important crops, like
sugarcane. Plant cell walls are one of the most abundant, renewable, and useful
biomaterial on the earth. However, wall polymers are entrapped in an imbricated
structural organization. Thus, the viability of using such feedstock in a bio-based
economy will greatly depend on the integration of ‘‘green’’ and ‘‘white’’ tech-
nologies in the production processes to efficiently extract and use molecules and
energy stored in biomass. In this chapter, we discuss some principles underlying
biorefination and bottlenecks under the crop physiology aspects—including Sac-
charum. Correlations between biomass yield and properties with environmental
factors are revisited.

F. J. F. Lopes (&)
Instituto de Ciências Biológicas, Campus Samambaia, Universidade
Federal de Goiás, Goiânia, GO, Brazil
e-mail: fagundesufg@gmail.com
V. G. de Carli Poelkin
Centro de Ciências Agrárias, Ambientais e Biológicas, CCAAB, Universidade
Federal do Recôncavo da Bahia, Cruz das Almas, BA, Brazil
e-mail: vivianedecarli@gmail.com

S. S. da Silva and A. K. Chandel (eds.), Biofuels in Brazil, 125

DOI: 10.1007/978-3-319-05020-1_7,  Springer International Publishing Switzerland 2014
126 F. J. F. Lopes and V. G. de Carli Poelkin

7.1 Photosynthesis and the Potential Biomass Yield

of Primary Production

Photosynthesis is responsible for all plant biomass entering the biosphere. After
incorporated into the first and simple trioses-phosphate sugars, carbon is then
partitioned toward different classes of substances, such as structural and soluble
carbohydrates, lignin, protein, and many other biomolecules.
The primary plant productivity (Pn) is under influence of many environmental
factors such as competition with weeds, diseases, nutrient scarcity, mineral tox-
icity, temperature, and water availability. The primary productivity is usually
taken to describe the physiological yield potential (Yp) parameter,
Y p ¼ g  Pn
where Yp represents the product of the harvest index (g) by the primary produc-
tivity (Pn). The harvest index (g) means the partition of the biomass produced by
photosynthesis toward effectively harvested products, while the primary produc-
tivity (Pn) denotes the total biomass accumulated during the growing season as a
result of photosynthetic activity (Evans and Fischer 1999).
The primary productivity (Monteith and Moss 1977) for a culture is a parameter
associated with climatic conditions, photosynthesis efficiency, and allocation of
biomass. Therefore, the occurrence of stresses greatly impacts the primary pro-
ductivity, since it affects intrinsic photochemical and biochemical performances of
photosynthesis. The primary productivity (Pn) is expressed as

Pn ¼ St  ei  ec  K 1
where:
St = Annual Integral of the Incident Sun Light Energy (MJ m-2)
ei = Light harvesting efficiency
ec = Efficiency conversion of absorbed light into biomass
K = Energy content of the total biomass (17.5 MJ Kg-1) or total carbohydrates
(15.9 MJ Kg-1).

Countries located in the intertropical regions receive a great amount of solar

energy during the entire year. The annual mean daily horizontal global solar
radiation in any region of Brazil during a decade of study (1995–2005) was much
higher (1500–2500 kWh m-2) than that for the majority of countries located
in Europe (Pereira et al. 2006): Germany (900–1250 kWh m-2), France
(900–1650 kWh m-2), or Spain (1200–1850 kWh m-2). In Brazil, the Northeast
and Central regions receive the largest amount of daily global solar radiation
(about 5.6 kWh m-2 or 20.16 MJ m-2) in the fall and winter seasons (between
June and September), when clear sky days are usual.
The primary productivity depends on the photosynthetically active radiation
(PAR), since it represents the range of the visible spectrum (approximately 40 %)
7 Advances in Methods to Improve the Sugarcane Crop 127

effectively available for photosynthesis. Taking into consideration the Northern

and Central regions of Brazil, the annual mean for the daily PAR is estimated as
2.4 kWh m-2. This is the same as 8.64 MJ m-2 or 8.64 9 104 MJ ha-1 day-1.
This energy is in part converted by plants into chemical potential energy (C–C
bounds) used for respiration or storage. It is known that a C4 plant, like sugarcane,
will convert, the best as possible, only 6 % of the total incident radiation into
carbohydrates, due to losses such as reflection, transmission, heat convection, and
metabolic costs (Zhu et al. 2008). This is equivalent to 1.21 9 104 MJ ha-1 day-1
being converted into structural or storage carbohydrates. For each Kg of C4 pro-
duced biomass, 15–17.5 MJ of energy equivalent is required (Lorimer et al. 2010).
In this sense, 1.21 9 104 MJ h-1 day-1 may potentially render 690 kg of bio-
mass ha-1 day-1 or 251.8 ton ha-1 year-1. This represents an estimate for the
annual mean of the sugarcane primary productivity (total biomass).
On the basis of ecophysiological and agronomic studies (Larcher 2003), the
sugarcane harvest index (economic yield/biomass production yield) ranges from
0.6 to 0.85, which allow us to estimate the yield potential (Yp) for sugarcane in the
Northeast and Central Brazilian regions, which is about 182 ton ha-1 year-1 (or
18 kg m-2 year-1). However, in practice sugarcane aboveground harvestable
biomass is reported (Larcher 2003) to be around 6–8 kg m-2 year-1 (60–80
ton ha-1 year-1). Hence, the exploitation of lignocellulosic residues for second-
generation biofuels has the potential to increase Yp as a function of the increment
of g.
Despite high yield potential estimated for cane in Brazil, the average produc-
tivity was 69.4 ton ha-1 in 2012. In 2011 and 2012, the Northeast region expe-
rienced a severe drought that greatly affected the ratoon crop, that did not sprout. A
great variation in sugarcane productivity in the different regions was registered in
this period. The North/Northeast region averaged 49.7 ton ha-1, whereas the
Midwest/South region harvested 72.4 ton ha-1. Acre, a hot and humid north state,
registered 95 ton ha-1 in 2012, one of the best records for the period of 2012 (da
Silva 2013). Since 2011, climatic instabilities have been causing huge productivity
losses. In 2011, about 571 million tons of cane were harvested, 10 % less than the
year before.
For many crops abnormal seasons may cause atypical flowering and more
propensity to diseases. In this sense, biotic and abiotic stresses are the main causes
of great losses in productivity every year in the world. In nematode infested areas,
yield loss up to 50 % may occur, and the costs for managing weed infested areas
are 15–30 % higher (Barela and Christoffoleti 2006).

7.2 Basic Concepts on Biorefinery

Biorefineries are a set of processes to produce energy and products from renewable
feedstocks, such as plant, algae, animal, or bio-based wastes, with minimal
environmental impact. Biorefineries will potentially mitigate the indiscriminate
128 F. J. F. Lopes and V. G. de Carli Poelkin

use of ancient carbon reserves that produce greenhouse effects. Biorefination will
also allow the development of new products and processes that will aggregate
value to biomass. On the social and political scenarios, it could also open
opportunities for adoption of policies to allow the participation of a global market
involving land use, technology transfer, and employments. Conceptually, it needs
to operate with minimal cost and time, produce low or zero environmental
impacts, to be large-scale operated, and must generate social benefits.
The US Department of Energy defined that a biorefinery is an overall concept of
a processing plant where biomass feedstocks are converted and extracted into a
spectrum of valuable products (DOE 2014). Also, the American National
Renewable Energy Laboratory (NREL 2009) stated that: ‘‘A biorefinery is a
facility that integrates biomass conversion processes and equipments to produce
fuels, power and chemicals from biomass. The biorefinery concept is analogous to
today’s petroleum refineries, which produce multiple fuels and products from
petroleum. Industrial biorefineries have been identified as the most promising route
to the creation of a new domestic biobased industry’’ (NREL 2009). A more
general definition is: ‘‘Biorefining is the transfer of the efficiency and logic of
fossil-based chemistry and substantial converting industry as well as energy pro-
duction onto the biomass industry’’ (Kamm et al. 2012).
The great development recently experienced in biotechnology, chemistry, and
process engineering points out limitless possibilities for biorefination. For a long
time, microorganisms have been used to produce or transform many biomolecules
of interest (drugs, food, textile, recombinant enzymes), much like a micro-biore-
finery plant. The biological systems may be considered as high-end biofactories
(Fig. 7.1) that one scarcely knows how to operate, but keep huge capabilities to be
exploited through genetic engineering.
Cell wall polymers, which represent a rich resource of important biomolecules
for biorefination, are entrapped in an imbricated structural organization in the wall.
Therefore, the viability of using such feedstock in the bio-based economy will
greatly depend on development and proper integration of ‘‘green’’ and ‘‘white’’
technologies in the production processes. At the ‘‘green side’’ there is a need to
improve crop performance and biomass traits and at the ‘‘white side’’ industry has
been challenged to deal with efficient conversion of biomass into products and
energy (Vanholme et al. 2013). In the recent years, many forestry (Liu 2010),
agriculture (Mariano et al. 2013), aquaculture (Demirbas and Demirbas 2011), and
waste feedstocks (Vanholme et al. 2013; Weiland 2010) have been assessed for
their potential use as biomass resources (Vanholme et al. 2013).
Three stages of the biorefineries development can be identified (Kamm et al.
2012): (a) Generation I biorefinery: Still limited biomass/feedstock utilization,
basically, a dry milling ethanol plant using grains as raw material. (b) Generation
II biorefinery: A more flexible mill that uses wet technology to produce different
types of end products from grains (oil, syrup, ethanol, starch). (c) Generation III
biorefinery: Uses agricultural or forest biomass to produce ethanol, chemical, and
plastics. Reviews on possible industrial processes and products can be found at
(Jong et al. 2008). Concerning biomass feedstock, two generations can be
7 Advances in Methods to Improve the Sugarcane Crop 129

Fig. 7.1 Parallel between a traditional refination and the biorefination

identified: The first-generation feedstocks consist of crops with potential use as

food or fuel, such as crop for bio-oils, sucrose, and starch, and therefore raise many
debates about impacts on food prices and plantation area, mainly in countries with
limited agricultural land. The second-generation feedstocks refer to the possible
use of any part of the plant, such as the plant cell wall fibers, increasing the
potential utilization of the whole harvestable biomass (Kamm et al. 2012).
Concerning biorefinery implementation, two main approaches are objects of
study (Jong et al. 2008): (a) A value chain approach, by which interesting com-
pounds are identified and isolated directly from biomass or through (bio)conver-
sion steps. By this approach, a great and concomitant development in separation
and bioconversion of products is required, and (b) Integrated process chain
approach, by which universal biomass substrates are first transformed into com-
mon building blocks, also used by the petrochemical refinery. This facilitates the
integration with the current feasible and economically viable petrochemical
facilities. Here, the challenge is to improve the transformation of biomass into
these common universal building blocks.

7.2.1 The Plant Biomass and Its Potential Use as Biorefinery

Feedstock

7.2.1.1 Plant Cell Wall

The plant cell wall is a rich resource of biopolymers and monomers for biore-
fineries. It is classified as primary or secondary, according to its composition and
structural organization.
Primary walls contain cellulose, structural proteins, and a hydrated polysac-
charide matrix consisting of hemicelluloses and pectin. The primary walls are
130 F. J. F. Lopes and V. G. de Carli Poelkin

Fig. 7.2 The stepwise use of different cell wall degrading enzymes to fractionate the sugarcane
biomass (Source Adapted from Souza et al. 2012)

usually classified as type I or type II. Type I walls are present in dicots and
noncommelinoid monocots. Xyloglucan is the major hemicellulose found in type I
walls, which also contains abundant amounts of pectic polysaccharides. Type II
walls, found in commelinoid monocots, are abundant in cellulose and only neg-
ligible amounts of pectin and proteins are found (Carpita 1996; Pauly and Keegstra
2008). In Poales, such as sugarcane, arabinoxylan is the predominant hemicellu-
lose (Souza et al. 2012).
Secondary walls are thicker than primary walls and may be deposited in dif-
ferent layers (S1–S3) according to the microfibrils orientation (Higuchi 1996).
Bamboo has cell walls with much more layers (Parameswaran and Liese 1976).
The deposition of secondary wall ceases cell enlargement. The secondary walls
contain cellulose and arabinoxylan and/or glucomannans as hemicellulose (Pauly
and Keegstra 2010). In secondary walls, pectin is replaced by lignin, which makes
them very impenetrable to solutes and enzymes (the so-called recalcitrance).
In order to use lignocellulosics as feedstock in biorefineries, the plant fibers
need to be first fractionated. This involves chemical, physical, and/or enzymatic
processes to disrupt the native fibers configuration. The cell wall fractionation
through enzymatic methods is clean and preserves the chemical identity of the
original polymer in the fragments released (Fig. 7.2).
The main derivatives of C5 and C6 sugars with great economic potential were
reported by the US-DOE to be organic acids such as lactic and succinic acids,
sugar alcohols (sorbitol), and ethanol (Bozell and Petersen 2010). In addition, fine
7 Advances in Methods to Improve the Sugarcane Crop 131

chemicals (enzymes, vaccines) are examples of products that will be facilitated by

biorefineries.
Next, we briefly discuss the potential use of plant biomolecules as substrates for
biorefination, with reviews and original papers indicated for each topic.
Cellulose: Cellulose, when hydrolyzed, releases glucose, which can be readily
fermented to produce second-generation ethanol (2G). Second-generation ethanol
will be commercially produced in Brazil from 2014. The use of lignocellulosics
from different crops could enter the process chain to increase ethanol yields.
Second-generation ethanol from lignocellulosics on its maturity could increase the
current Brazilian production by 50 % without expanding the current sugarcane
agricultural frontiers. The first commercial Brazilian plant for the 2G ethanol
estimates the initial production of 82 million liters ethanol per year. Today, about
22 billion liters of ethanol is consumed in Brazil, but until 2020 a demand of 47–68
billion litters is expected (Viana 2013).
Hemicelluloses: Hemicellulose from type II primary walls or secondary walls
releases mainly pentoses when hydrolyzed. It is a source of C5 sugars, such as
xylose. Xylose can be converted into xylitol and furfural. Xylitol is a sweetener
with antimicrobial, remineralization, and teeth hardening activities and therefore
enters the formulation of chewing gums and toothpaste (Roberto et al. 1999;
Sarrouh and da Silva 2013). The first report of furfural production was in 1831, by
Dübereiner, who reported the distillation of bran with diluted sulfuric acid.
Industrial technology for furfural production from pentose was developed by
Quaker Oats in the 1900s. DuPont has established the production of nylon-6.6
since 1960 from furfural, despite the plant furfural has been replaced by fossil-
based substrates since then. Furfural is also used in the manufacture of phenol
plastics, varnishes, and pesticides (Montané et al. 2002).
Pectin: Pectin is a complex polysaccharides composed of four main pectic
compounds: homogalacturonan (HG), rhamnogalacturonan-I (RG-I), rhamnoga-
lacturonan-II (RG-II), and xylogalacturonan (XGA). XGA exhibits the basic HG
core with xylosil substituents attached to it, while RG-II, the more complex pectic
component, is the modification of HG with four different side chains exhibiting a
great diversity of sugar linkages (Atmodjo et al. 2013; Carpita and McCann 2009).
Pectin is synthesized in Golgi apparatus and recently, it has been reported that
many glicosyltransferases physically interact to form large supramolecular com-
plexes responsible for the synthesis of pectic compounds (Atmodjo et al. 2011).
Pectin enters the secretory pathway and is deposited in the apoplast in a highly
methyl-esterified form (Driouich et al. 2012) that is subsequently modified (de-
methyl-esterified) by the action of Pectin Methyl Esterases (PME), cross-linking of
divalent cations, usually Ca2+ or RG-II dimerization through borate-diester link-
ages between two RG-II (Carpita and McCann 2009). These listed modifications
are thought to increase the cell wall stiffness, although the modification of the cell
wall status by pectin seems to depend on the organ type and situation, thus
complex effects on plant development and growth might be expected (Derbyshire
et al. 2007; Peaucelle et al. 2012). The overexpression of a PME inhibitor in
Arabidopsis provoked biomass and saccharification efficiency increases, indicating
132 F. J. F. Lopes and V. G. de Carli Poelkin

the potential of the biotechnological modification of pectin metabolism for 2G

ethanol (Lionetti et al. 2010). A pectin-based biorefinery includes its use as gelling
agent, thickener, stabilizer in jams and drinks, and gelatin. The complex pectin
structure reflects its many potential functions and applications. Pectin may act as a
signaling molecule in plant defense responses (Davis and Hahlbrock 1987), may
serve for many medical and pharmaceutical applications such as in drug delivery
or gene delivery systems and tissue engineering. The multitude of ways by which
the complex pectic structure can be further chemically modified offers opportu-
nities to discover novel anti-cancer and anti-metastatic drugs (Jackson et al. 2007;
Munarin et al. 2011; Munarin et al. 2012).
Lignin: The presence of lignin in plant biomass has been mainly associated with
the recalcitrance of biomass to saccharification. Lignin impacts the yield of fer-
mentable sugars and fermentation efficiency of lignocellulosics in different man-
ners. It hinders the access of carbohydrate degrading enzymes to C6 and C5 sugars
in the cell wall. Lignin inhibits cellulase activity by adsorbing them, increasing the
need for higher enzyme loads during saccharification (Jørgensen and Olsson 2006;
Berlin et al. 2006). Concerning lignin applications, many aromatic products might
be produced from it. Vanillin and gallic acid are examples of building blocks that
have attracted great interest (Walton et al. 2003). Vanillin can be used as basic
monomeric unit for the production of herbicides, anti-foaming agents or drugs,
such as papaverine, L-dopa, and trimethoprim (Loureiro et al. 2011).

7.2.1.2 Plant Oils

Plant oils and animal fats are sustainable alternatives to mineral oils in the pro-
duction of lubricants, fuel (biodiesel), surfactants, cosmetics, and emulsifiers.
Many hydrophobic substances depend today basically on the petroleum. The
market potential for the bio-based oils is immense and the development of oils
with different properties will depend on the assessment of different biomass
resources.
Edible and nonedible plants like soybean, rapeseed, sunflower or castor beans
oils, and animal fats like fish oil and tallow are sources of lipids. The richness of
oils with still unknown properties is huge, reaching up to a thousand types if plant
and animal lipids are combined. The lipid source depends on the region, season,
and knowledge of plants (Chou 2011).
The Brazilian Agroenergy Plan (Oliveira and Ramalho 2006) recommends the
search for new raw materials with higher energy content. It also advises for the
cultivation of oleaginous plants according to the particularities of each State or
Region, preferably where they are already introduced and consumed. Species with
high potential for oil-based biorefineries are Jatropha curcas, L. (pinhão-manso),
Acrocomia aculeata, Jacq (macaúba), Astrocaryumm urumuru, Mart (tucumã),
Orbignya phalerata, Mart. (babaçu), and Maximiliana maripa (inajá).
7 Advances in Methods to Improve the Sugarcane Crop 133

The assessment of natural diversity of native plants has much to offer due to the
infinity of bio-oils with different characteristics that can be found. However, the
domestication of native plant species is still in its infancy, concerning the
exploitation of their genetic potentials. Biotechnology will be helpful to manip-
ulate quantitatively and qualitatively the lipid profile in plants or algae biomass.
The biotechnological advances on lipid metabolism will greatly depend on the
knowledge of how fatty acid biosynthesis is regulated in the oleaginous crops.
Equally important is the definition of new routes to the desired products and public
policies for the regional development. Technical support for local producers will
be important for the implementation of standard production methods to keep the
quality control of the raw material, associated with sustainable production
practices.

7.2.1.3 Waxes and Suberin

Another class of plant substances with potential application in biorefineries are

waxes and suberin, which are produced and deposited outside the epidermis,
forming a water repellent barrier to protect the plants against biotic and abiotic
stresses.
Waxes are composed of very long fatty acids produced by the endoplasmic
reticulum of epidermal cells. Many years of genetic studies on molecular aspects
of waxes biosynthesis in Arabidopsis have led to the characterization of fatty acid
elongating enzymes and waxes transporters (Bernard and Joubès 2013). In
agreement with their protective function against exacerbated water loss, the wax
biosynthesis is upregulated at the transcription level by water deficit. In addition,
abscisic acid (ABA)—a plant hormone that plays a major role in water stress
signaling, increases wax synthesis and decreases cutin permeability (Kosma et al.
2009). The MYB96 transcription factor has been implicated in the regulation of a
set of genes in an ABA dependent manner in Arabidopsis plants undergoing water
stress, including those related with wax metabolism (Seo et al. 2011).
In addition, waxes protect external plant tissues avoiding the establishment of a
prolonged humid environment that could propitiate pathogens colonization. Indeed,
anti-feeding function has also been attributed to the inner layer of the waxes (in-
tracuticular compartment) that contains aromatic and triterpenoid compounds that
could counteract herbivory (Eigenbrode and Espelie 1995). The aromatic com-
pounds present in the wax are also known to absorb UV-B and UV-C radiation, and
the cuticle concentrations of these constituents were shown to provide moderate
UV protection in some plant species (Krauss and Markstädter 1997).
In this scenario, plant waxes have potential applications as raw material for
production of inseticides, cosmetics, sealing agents, and much more, based on their
natural properties already identified.
Suberin constitutes the periderm layer that is deposited during secondary
growth of many plant species—the best known is Quercus suber, the cork oak tree.
The apoplastic deposition of suberin protects polysaccharide cell walls from
134 F. J. F. Lopes and V. G. de Carli Poelkin

decomposition, as evidenced by the slow chemical decay of cork bark in soil

(Vane et al. 2006). Suberin incrustation in the apoplastic space makes the cell
walls a highly selective barrier against water, solutes, and gases. Although still a
topic of debate (Naseer et al. 2012), plants use suberin along lignin to build the
Casparian band in root endodermis and exodermis to restrict the transport of
substances and microorganism through the apoplastic pathway, conferring addi-
tional absorption selectivity for the roots in soil.
The fine structure of suberin is still uncertain due to difficulties on the substance
isolation. Nonetheless, some compositional analyses reveal that suberin can be
chemically described as a bio-polyester, mainly comprising x-hydroxy acids;
a,x-dicarboxylic acids (diacids) with low amounts of fatty acids and alcohols. The
carbon chains length ranges from C16 to C32. Glycerol and minor amounts of
aromatic phenylpropanoids may also be part of the aliphatic suberin polyester
(Franke et al. 2012).
Suberin has the potential to be source of new hydrophobic plant-derived
polyesters. Industrial cork by-products may also render antioxidants, triterpenes,
and other lipophilic compounds (Santos et al. 2013; Sousa et al. 2006). The cork
stopper and agglomerate industries generate considerable amounts of suberin-rich
residues. The exploitation of such biomass resource is therefore in agreement with
the biorefineries concepts (Sousa et al. 2011).
The state of the art on suberin and cuticular waxes biosynthesis, deposition, and
regulation is reported by (Buschhaus and Jetter 2011; Franke et al. 2012; Samuels
et al. 2008) and (Bernard and Joubès 2013).

7.2.1.4 Rubber

Rubbers are natural polymers with unique properties and are mainly obtained from
‘‘Seringueira’’ (Hevea brasiliensis). Nonetheless, guayule (Parthenium argentatum
Gray), a xerophytic shrub growing mainly in the arid regions of Mexico, has been
pointed as a good source of natural rubber. To make exploitation of guayule rubber
feasible, domestication programs must be conducted, especially because this
species can grow in arid and semiarid areas around the world, bringing economic
importance to these regions (Thompson and Ray 1989).
For medical purposes, rubber must be hypoallergenic and the research field of
rubber-associated proteins is of great importance. The rubber genetic breeding
program has been conducted in Brazil since the 1930s, for yield and resistance
against Microcyclus ulei P. Henn (Filho and de Resende 2000). Interspecific
crosses among Hevea brasiliensis, H. benthamiana and H. pauciflora are the basis
of the ‘‘seringueira’’ genetic breeding program. However, little is known about the
genes participating in the biosynthesis and traits of rubber, and how they are
regulated at molecular level.
7 Advances in Methods to Improve the Sugarcane Crop 135

7.2.1.5 Cogeneration of Energy

Cogeneration is the autonomous production of energy in a mill by burning of crop

residues. The thermal energy generated this way can supply the plant demand for
energy and generate a surplus that can be exported, benefiting other industrial,
home, or commercial installations nearby. Combustion processes using high effi-
ciency, multi-pass, steam turbines to produce electricity can currently achieve an
overall efficiency of 35–40 % (McKendry 2002). Cogeneration makes the pro-
cessing of biomass independent of other energy sources, eliminating the need for
electric substations, and does not increase the carbon budget in the atmosphere
since it displaces the use of fossil fuels to generate energy.
In the past, the burning of exceeding sugarcane trashes was a common practice,
but today much of the trash is used as soil cover and the remaining for cogene-
ration. With the foreseen second-generation ethanol from lignocellulosics, via-
bility studies will be needed to decide how much trash will be partitioned to
cogeneration, fermentation, and soil cover.
Any biomass crop is expected to give the same calorific power when burnt
(*17–21 MJ kg-1) (McKendry 2002). Thus, the possibility of decreasing the
calorific energy output in benefit of increasing the yield of fermentable sugars by
traditional genetics or molecular breeding aggregates more value to the plant
material than increasing calorific energy, since ethanol is used by vehicles and can
also be an exportation product. Considering the integration of lignocellulosics into
the process chain, fiber content and composition in bagasse and crop trashes (tops
and leaves) should receive attention by breeders in order to decrease biomass
recalcitrance. Hence, it would be expected that new varieties improved for bio-
mass yield and fiber composition for better saccharification are preferred over
cogeneration purpose.

7.3 A Few Considerations on Plant Molecular Physiology

Aspects Influencing Biomass Yield and Quality

It is well documented that biomass yield and quality begins to be regulated since
the cell cycle (Francis 2011; Ng et al. 2013) until the last steps accomplished by
enzymes in the metabolic pathways leading to plant products of interest (Gray
et al. 2012). Subtle changes introduced in the final raw material might affect the
feasibility to obtain the monomers or polymer of interest. The best known example
is how lignin imposes recalcitrance to the cell wall enzymatic degradation (Jung
et al. 2013).
136 F. J. F. Lopes and V. G. de Carli Poelkin

Fig. 7.3 Carbon flux under

demand

7.3.1 Carbon Partition and Allocation

7.3.1.1 Sink and Source Relations

The carbon fixed through photosynthesis may flow through primary and secondary
plant metabolism. Respiration is responsible for production of energy and meta-
bolic precursors at the expense of high losses of carbon fixed by photosynthesis.
The rates by which carbon flows toward the synthesis of different classes of
compounds is highly dictated by the sink and source relations (Fig. 7.3).
It is accepted that the increase in sink strength leads to a higher demand for
photoassimilates, upregulating the functioning of the photosynthetic machinery
(McCormick et al. 2006). For instance, if plants are attacked by herbivores, the
remaining leaves seems to compensate for the loss of photosynthetic area by
improving the functional efficiency of photosynthesis in the remaining leaves
(Thomson et al. 2003). Under normal developmental conditions, the cell prolif-
eration and expansion in active growing regions must be accompanied with higher
C inputs provided by source leaves through phloem. Apical regions of root and
stems, sprouts, sugar loading on storage tissues, seeds, and grains are some
examples of high C demanding organs.
In sugarcane, invertase activity is highly required by the young developing in-
ternodes, which exhibits a high C turnover for the synthesis of metabolic interme-
diates (Rae et al. 2005; Rose and Botha 2000; Whittaker and Botha 1997). During
the internode elongation, a high sink strength mediated by invertase, which can
convert sucrose into glucose and fructose, is established in these tissues, favoring for
sucrose delivery in the young expanding internode cells (Rose and Botha 2000). On
this agreement, metabolomic studies have shown that as the internodes elongate
(become older), invertase activity and hexose accumulation decreases, favoring the
increase in sucrose content (storage) in culm parenchyma, which assumes a sucrose
storage function (de carli Poelking 2012). Sugarcane can store up to 25 % of its fresh
weight as sucrose in parenchyma (Moore and Maretzki 1996).
In sugarcane, the mechanism of sucrose phloem unloading to the vascular
parenchyma is not well understood. In the sugarcane stem, the vascular bundles are
surrounded by a layer of cells that become lignified as the internodes mature (de
carli Poelking 2012). This lignification forms a barrier that difficults the apoplastic
mechanism for the phloem unloading (Jacobsen et al. 1992), if only the apoplastic
pathway is used. A sucrose transporter in sugarcane (ShSUT1) with homology to
7 Advances in Methods to Improve the Sugarcane Crop 137

the SUT/SUC family of plant sucrose transporters was identified (Rae et al. 2004).
ShSUT1 was expressed predominantly in mature leaves that were exporting
sucrose and in stem internodes actively accumulating sucrose. They also found
that a simplastic tracer dye can move from phloem into the vascular parenchyma
cells and then, through the first lignified cell layer of the parenchyma cells which
surround the vascular bundle. This suggests that sucrose may be able to enter the
storage parenchyma through symplastic connections.
The downregulation of pyrophosphate: fructose 6-phosphate 1-phosphotrans-
ferase (PFP), a key enzyme in the primary C metabolism operating at the gly-
colysis level, led to increased sucrose content in immature internodes (Groenewald
and Botha 2007). These data confirm that respiratory activity is intense in
immature internodes, and that the impairment in the conversion of the fructose 6-
phosphate and pyrophosphate (PPi) into fructose 1,6-bisphosphate plus inorganic
phosphate (Pi) bottlenecks the C flow through respiration, promoting sucrose
accumulation. Interestingly, these authors also reported that some field grown
transgenic lines had high fiber content. This is an evidence that it is possible to
manipulate the C metabolism toward high sucrose and fiber content, creating
mixed-purpose sugarcanes that could produce juice for sugar and 1G ethanol and
fiber for 2G ethanol production. However, these authors did not report whether the
transgenic lines were more susceptible to diseases, since a higher sucrose content
would also mean more resources for plant pathogens.

7.3.1.2 Tillering Response

When sugarcane is cultivated for high sucrose yield purposes, the number of culms
per cultivated area is often associated to cane yield and used as a criteria for the
sugarcane payment. In a work accessing the inheritance of yield-related traits in
sugarcane, 227 individuals from a cross between the Australian variety Q165 and a
Saccharum officinarum accession were evaluated during three years for stalk
weight, stalk diameter, stalk number, stalk length, and total biomass (Aitken et al.
2008). In this work, the authors mapped two alleles of a candidate gene showing
homology with the teosinte branched 1 (tb1) gene from maize. This gene in maize
has a prominent role in regulating branching, although the authors reported a
minor effect in sugarcane (Aitken et al. 2008).
The teosinte branched 1 (tb1) gene belongs to the TCP gene family. The
members of this family encode putative basic helix-loop-helix DNA-binding
proteins that may play a role in organ growth. The tb1 related genes may encode
negative regulators of branching and their function has been recently investigated
in rice tillering. The overexpression or RNAi suppression of a maize tb1 gene in
rice plants reduced or increased, respectively, the number of tillers and panicles of
transgenic rice (Choi et al. 2012). This effect was less pronounced for plants
growing in paddy fields than for those growing in greenhouse, suggesting that
environment also plays a role in controlling this trait.
138 F. J. F. Lopes and V. G. de Carli Poelkin

Tillering involves perception and transduction of environmental clues by plant

hormones. Strigolactone is a class of carotenoid derived plant hormones partici-
pating in the control of tillering in monocots and dicots (Shinohara et al. 2013).
Besides controlling branching, strigolactones are also involved in processes like
root branching, hyphae branching in arbuscular mycorrhiza, and seed germination
of parasitic weed by the exudates of the host plant roots (Matusova et al. 2005).
Tillering may change the sink/source relation in plants. An evidence that til-
lering imposes a high sink strength in plant development is that in barley, cyclic
crossing and selections led to high head numbers and tiller mortality, low kernels
numbers per head, low kernel weight, and high susceptibility to lodging (Benb-
elkacem et al. 1984). Thus, tillering may exert negative impacts in grains pro-
ductivity (reproductive development) due to sinks competition, mainly if
branching occurs during seed filling. In this circumstances, neither reproductive
nor vegetative development can be supported by adequate water and nutrient
supply. In the case of sugarcane, the number of culms (vegetative development)
against low flowering and grain production (reproductive development) is a
desirable trait to be selected by breeding or produced by genetic engineering.
The tillering intensity in sugarcane is variable. In order to increase the number
of culms in sugarcane, induction of sprouting in the initial phases of the plant
development has been recommended (Silva et al. 2007). The highest number of
tillers (10–20) may occur after 4 months of cultivation and then decrease as a
result of probably tillers competition for resources, such as water, light, and
nutrients (Castro and Christoffoleti 2005). Limited resources may cause some
tillers to abort with possible increase of the diameter of the remaining ones.
Increased stalk diameter suggests resources availability for the crop. Therefore,
increased tillering may also need to be supported by better crop management
practices.
In the ratoon crop, sprouting is highly dependent on water availability and
drought stress greatly impacts the success of the culture in ratoons. Design of
canes tolerating large variations in water soil potential, for regions experiencing
remarkably drought in defined seasons is an important strategy to increase cane
yields.
Despite tillering being an important component of yield, high plant densities may
inhibit tillering by the initiation of a shading avoidance response, mediated by
phytochromes, particularly the PHY B type, which senses the decrease of red:far-red
light ratio in the transmitted light under shading condition. Under shading,
branching is inhibited, plants grow taller, produce less tillers and reproductive
structures, leaf expansion is inhibited and higher mortality of young vegetative
tillers may also be found (Casal et al. 1986). The signaling pathway by which
strigolactones operates in tillering or shading avoidance responses seems to be down
stream of the PHY B light quality perception and probably crosstalks with auxin
signaling pathways to control the developmental responses (Brewer et al. 2013).
7 Advances in Methods to Improve the Sugarcane Crop 139

7.3.1.3 Lignin Accumulation

Ligning is a complex polymer from the plant secondary metabolism and plays an
important role in the plant growth and development. It is the second most abundant
polymer on the earth, after celulose. About 30 % of atmospheric CO2 is fixed as
lignin (Boerjan et al. 2003) which constitutes 10–40 % of the total plant dry matter
(Sederoff et al. 1999).
Lignin biosynthesis is a considerable sink of carbon fixed by plants. Since
plants cannot degrade it to recover the carbon invested in its synthesis, there might
be a fine control of spatial and temporal lignin deposition in the cell wall (Rogers
et al. 2005).
Lignin biosynthesis is influenced either by internal (genetics and physiology) or
environmental factors. Environmental stimuli and developmental cues regulate the
carbon flux toward lignin, and this is also accomplished by a complex network of
tissue-specific transcriptional factors—TFs (Rogers and Campbel 2004; Bonawitz
and Chapple 2010). The identification of regulatory cis-elements targeted by
different TF’s families in the promoter region of many genes from the monolignol
biosynthesis pathways is helping to understand the transcriptional regulation of
lignin genes (Zhao and Dixon 2011).
Conserved motifs, such as AC elements, which are targeted by MYB TF’s are
ubiquitous to regulatory region of genes encoding enzymes participating in the
phenylpropanoid and monolignol biosynthesis (Bugos et al. 1991; Sablowski et al.
1994). MYB recognized elements are present in the promoter of PAL, 4CL, C3H,
CCoAMT, CCR, C4H, COMT, and CAD genes (Zhou et. al. 2009; Zhao and Dixon
2011) and can regulate the lignin genes in a positive or negative manner. Ligni-
fication must also be controlled in a tissue-specific manner in order to avoid
improper lignin deposition. Repressors of lignin biosynthesis genes should work in
this occasions. For instance, AtMYB32 and KNOX are well known TFs repressing
lignin accumulation. AtMYB32 is highly expressed in flowers but not in lignified
tissues, whereas KNOX keeps the meristematic cells in shoot apical meristem in
an undifferentiated and unlignified state (Tsiantis et al. 1999; Zhao and Dixon
2011). Conversely, the lignin deposition may occur through the inactivation of
repressors instead the induction of activators, as exemplified for PAL, CCR, and
F5H, which are not usually expressed in epidermis, but under attack of pathogens,
their transcripts substantially increases (Bhuiyan et al. 2009).
TFs have a central role in the transduction of plant intrinsic signals leading to
alterations in the metabolism of lignin. However, the post-translational regulation
of these proteins and their possible interacting partners are still not well known.
Moreover, lignin biosynthesis related TF’s might also crosstalk with hormonal
pathways, as exemplified by AtMYB32, which is strongly activated after auxin
treatment (Preston et al. 2004).
The potential of general regulators of lignin biosynthesis might be exploited in
order to reduce biomass recalcitrance, but selective downregulation of lignin
biosynthesis genes should be preferred since many developmental traits may be
affected by general regulators due to lignin biosynthesis network crosstalks with
140 F. J. F. Lopes and V. G. de Carli Poelkin

hormonal signaling, which controls many aspects of plant development. For

instance, the down-regulation of the enzyme hydroxycinnamoyl CoA:shikimate
hydroxycinnamoyl transferase (HCT) in Arabidopsis thaliana and Medicago
sativa significantly reduced lignin levels and cell wall recalcitrance to sacchari-
fication, but also impacted plant growth due to enhanced accumulation of salicilic
acid (Gallego-Giraldo et al. 2011). Salicilic acid content increased conversely to
lignin content in HCT-down-regulated M. sativa. Also, other lignin metabolism
related genes were down-regulated, exemplifying that a single gene alteration can
exert a great impact in the metabolism due to the interconnected signaling path-
ways (Lee et al. 2011).
Many genes involved in lignin biosynthesis are under light, circadian cycle, and
sugar levels control. It has been shown that C4H, COMT, CCoAOMT and CCR
expression oscillates according to the circadian clock (Rogers et al. 2005). Since
carbon fluxes also exhibit oscillation—starch is usually accumulated during the
day and hydrolyzed at night—there might be a control through sugar signaling and
circadian rhythm to optimize the metabolic fluxes of C toward different pathways
(Rogers et al. 2005; Zhao and Dixon 2011). Then, a question arises: Which
environment diverts C toward phenolics or carbohydrates? Stresses are a driving
force guiding the plant metabolism to surveillance. Light, temperature, and water
stresses are a few examples of conditions that can saturate the photosynthetic
electron transfer chain leading to oxidative stress. Plants are usually adapted to a
range of environmental conditions. Under acclimated condition, the metabolism
can make its shifts properly. However, plants usually dispose of more energy than
they, in fact, can use in the environment and the synthesis of aromatic compounds
such as lignin and flavonoids may serve as a protective mechanism to channel the
excess of reducing power in the photosynthetic electron transport chains toward
the high energy-consuming phenylpropanoid biosynthesis. Aromatic phenolics
exhibit a remarkably abundance of Pi electrons involved in aromaticity, conferring
them a high reductional state and thus, a remarkably antioxidant characteristic.
Stoichiometry analysis of mass-energy equivalence confirms that in order to
produce 1 g of lignin, 2.7–3.0 g of glucose may be required, since lignin has 30 %
more energy than carbohydrates, in average (Novaes et al. 2010).
The accumulation of phenolics is a typical response to elevated visible irradi-
ance or UV-A/B in the environment (Guo et al. 2008; Matus et al. 2009; Shin et al.
2007). Under clear skies, the abundance of visible and UV light is high and plants
need to protect themselves. UV-B perception has always been a puzzle in the field
of plant photomorphogenesis until the identification of a plant UV-B receptor in
Arabidopsis (Rizzini et al. 2011). The characterization of UVR8 impaired plants
has shown that UVR8 is involved in the regulation of a set of photomorphogenic
responses, including: inhibition of hypocotyl elongation, leaf expansion regulation,
stomatal differentiation, and accumulation of phenolics (Morales et al. 2013;
Wargent et al. 2009). UVR8 has been implicated in the regulation of a set of genes
involved in protection against oxidative stress and hormone signaling. Interest-
ingly, PAL—the key enzyme of the phenylpropanoid pathway entrance, was
positively regulated by UV-B light in an UVR8-dependent manner. Thus, the
7 Advances in Methods to Improve the Sugarcane Crop 141

regulation of PAL encoding genes is an evidence that lignin and other phenyl-
propanoids synthesis are much influenced by environmental factors such as light
and injuries caused by mechanical stress and pathogens (Rogers and Campbel
2004). Not surprisingly, sites of pathogen penetration may exhibit accumulation of
phytoalexins.
Since PAL is the first enzyme of the phenylpropanoid pathway, it drives the C
metabolism to the synthesis of these compounds (Sewalt et al. 1997). Concomi-
tantly, C must also be diverted toward cellulose and hemicellulose synthesis.
However, the precise control levels by which these shifts operate are not thor-
oughly understood. At least in Arabidopsis, the biogenesis of secondary wall
shares a common TF network that also regulate lignin biosynthesis genes. The
NAC, SND1, with their homologs—NST1, NST2, VND6, and VND7 belong to
this network and may regulate cellulose, xylan, and lignin biosynthesis related
genes (Zhong and Ye 2009). SND1 and NST1 double knockout lines exhibited a
complete loss of secondary wall thickening, suggesting their relation with multiple
cell wall components biosynthesis (Zhong and Ye 2009; Boejan et al. 2010).
Moreover, it has been proposed that SND1 and MYB46 are on top of the signaling
cascade controlling lignin, cellulose, and xylan deposition (Zhong and Ye 2009;
Zhao and Dixon 2011).
There are evidences that the flux toward secondary metabolism might be lim-
ited by the primary metabolism precursors. An evidence is that the Arabidopsis
mutant sex1 is defective in starch degradation and as a consequence, many genes
involved in the monolignol biosynthesis exhibit low transcript levels and the plants
accumulate less lignin (Rogers et al. 2005). When grown in dark with supple-
mented sucrose, lignin accumulation is restored in sex1. This finding supports the
idea that sucrose availability through starch degradation may stimulate lignin
accumulation.
When starch is normally degraded but cellulose synthesis is impaired, then
lignification can be invoked, including defense responses, as reported by (Delgado
et al. 2003). On this agreement, defense responses seem to be activated in detri-
ment of energy invested in vegetative growth. The competition of C partitioning
toward lignin or polysaccharides in plants have been confirmed by direct and
forward genetics. A Pinus taeda CAD mutant exhibited negative correlation
between biomass yield and lignin content. Down-regulated 4CL plants also
exhibited lignin reduction, which was counterbalanced by increase in cellulose and
hemicellulose contents, on agreement with a reduced C flux toward lignin might be
compensated by cellulose and hemicellulose accumulation (Hu et al. 1999).
Therefore, the carbon flux toward cellulose and hemicellulose often correlates with
growth and yield while lignin deposition marks the cessation of cell growth and
proliferation.
The lignin profile has been associated with its reactivity. In the Kraft process,
the noncondensed ether bonds (b-O-4-) are more amenable to the delignification,
whereas C–C bonds (b-b, b-1, b-5 e 5-5) are more resistant to chemical degra-
dation. Modifications of key steps of the lignin metabolism may render plants less
recalcitrant for pulping and saccharification purposes (Baucher et al. 1998,
142 F. J. F. Lopes and V. G. de Carli Poelkin

Lapierre et al. 2000, Baucher et al. 2003). The Shikimate pathway is the beginning
of phenylpropanoid biosynthesis (Schmid and Amrhein 1995). This pathway
provides prephenate, which is formed by the combination of phosphoenolpiruvate
(PEP), produced by glycolysis, and erytrose-4-phosphate, a precursor derived from
the Calvin-Benson cycle or pentose phosphate oxidative pathway—PPOP (Amthor
2003). The synthesis of monolignols takes place after the phenylalanine deami-
nation, successive hydroxylations, and o-methylations, that modify the aromatic
ring of the cinnamic acids produced. The latter steps reduce the cinnamic acids
into the monolignols, which are then incorporated into lignin. The p-coumaril,
coniferil, and synapil alcohols are referred as H, G, and S type lignin, respectively.
The relative amount of H, G, and S units is temporally and tissue-specific con-
trolled and may also receive influence of the environment (Boerjan et al. 2003;
Bonawitz and Chapple 2010). However, only minor qualitative differences in H,
G, and S composition were detected in bagasse from different varieties of sugar-
cane bagasse (Lopes et al. 2011).
Lignin polymer is formed by the action of many enzymes leading to the
sequential deposition of p-hydroxyphenil (H), guayacil (G), and then syringil (S)
(Boerjan et al. 2003; Donaldson 2001). Transgenic plants down-regulated for
single genes of the lignin biosynthetic pathway exhibited phenotypical alterations
that allowed for the functional characterization of the particular lignin related
genes in the plant growth and development (Table 7.1).
As an example of the potential manipulation of these enzymes, in a recent
study, transgenic sugarcane expressing low levels of COMT exhibited increased
yield of fermentable sugars with much less enzyme loads (Jung et al. 2013). In the
same work, authors reported that reduction of lignin content by 6 % improved
saccharification efficiency by 19–23 % without significant changes in agronomic
traits, such as plant height, tillering, brix, or stalk diameter. Nevertheless, lignin
decreases of above 8 % increased saccharification in more than 28 %, but
impacted biomass yield. Combining metabolism manipulations could alleviate the
negative side effects due to drastic lignin reductions through other compensatory
effects.

7.3.2 The Water Status

Stomatal responses to root water status is triggered by the transmission of a

chemical signal in xylem sap, suggesting that root water status directly affects CO2
assimilation. In sugarcane, pots, and field grown crops exhibited stomatal and root
hydraulic conductance correlation result in homeostatic regulation of leaf water
potential (Smith et al. 2005).
Water deficit negatively affects the cell expansion and, therefore, cell prolif-
eration and biomass yield. In addition, all the basic physiological processes are
affected. The turgor pressure governs the cell expansion process as expressed by
the growth rate equation:
7 Advances in Methods to Improve the Sugarcane Crop 143

Table 7.1 Effects of downregulation of phenylpropanoid genes on lignin content and profile
Enzyme Impact on lignin profile References
PAL—phenylalanine The low expression of PAL decreases the G (Boerjan et al.
ammonia-lyase unit content and the overall lignin content 2003)
C4H—cinnamate The low expression of C4H decreases the S (Boerjan et al.
4-hydroxylase unit content in lignin and the overall lignin 2003)
content
COMT—caffeic acid/5- The down-regulation of COMT decreases the (Zhong et al. 2008)
hydroxyferulic acid lignin content up to 30 % in some plants. S
3/5-O-methyltransferase lignin type is reduced and occurs
incorporation of 5-hydroxyconiferyl
alcohol into lignin
CCoAOMT—caffeoyl CoA Reduced CCoAOMT activity results in lower (Parvathi et al.
O-methyltransferase content of lignin and increased S/G ratio 2001)
due to reduction of G units
F5H (FAH-1)— Arabidopsis F5H mutants have depletion of (Franke et al.
coniferaldehyde/ferulic S lignin, with concomitant presence of 2000)
acid 5-hydroxylase dibenzodioxins and phenylcoumaran. The
overexpression of F5H in Arabidopsis
markedly increases S lignin content
4CL—4-coumarate: CoA The down-regulation of the 4CL in some (Boerjan et al.
ligase plants reduces lignin content and increases 2003)
the cell wall-bound hydroxycinnamic
acids
CCR—cinnamoyl-CoA CCR down-regulated plants exhibit increase (Boerjan et al.
reductase in the S/G ratio due to a decrease in G 2003; Ruel
units. However, the lignin becomes more et al. 2009)
condensed. In Arabidopsis, CCR1 mutants
are dwarf and exhibit collapsed xylem due
to reduced lignin content in the cell wall
CAD—Cinnamyl alcohol CAD deficient plants exhibit minor changes in (Boerjan et al.
dehydrogenase lignin content. In tobacco and poplar, the 2003; Ralph
down-regulation of CAD leads to the et al. 2001)
incorporation of sinapaldehyde and
coniferaldehyde into the lignin polymer

GR ¼ mðwP   Þ:
GR is the growth rate, m is the cell wall extensibility, wP is the turgor pressure
(the pressure that the wall exerts upon the symplast) and ! is the yield threshold
(the pressure value in which the cell wall resists to plastic deformation). If
wP = !, then GR = 0. Under normal hydration conditions, wP is slightly higher
than ! (0.1–0.2 MPa), that means the cell expansion may be affected by even low
decreases in water content. Besides affecting the wP, the soil water deficit also
causes structural changes in the wall, since the xylem sap becomes more alkaline.
Wall extensibility coefficient (m) decreases due to the alkalinity of the apoplastic
144 F. J. F. Lopes and V. G. de Carli Poelkin

fluid, which inhibits the activation of the expansions, whose activity is required for
the acidic growth mechanism mediated by auxins. Another possible explanation
for the decrease of expansibility, and thus growth rate (GR) under long-term water
deficit condition is that ! may also be affected due to biochemical changes in the
cell wall that may not be easily reverted if (Radin et al. 2010).

7.3.3 The Important Role of Roots

Much attention is given to the improvement in harvestable aerial plant organs. The
ability to better explore the soil for water and minerals, properly responding to
stress signals, will make the whole plant perform better in the environment. Roots
are strong carbohydrate sinks in the plants, which respond to a variety of stresses.
In drought stressed roots of sunflower, decrease in respiration rates were detected
(Burton et al. 1998; Hall et al. 1990). On the other hand, during grain filling, root
respiration rates increase significantly (Hall et al. 1990) in order to support the
aerial parts with water, minerals, and hormones. Therefore, the impact that drought
causes in root respiration rates also down-regulate photosynthesis. On the other
hand, it has been recently reported that salt stressed rice roots increased the
cyanide-resistant respiration, through alternative oxidase (AOX), which probably
mediated cell death in the tissues (Feng et al. 2013).
In sugarcane, the root system is continuously renewed, since the older roots lose
their absorptive function and die by a mechanism still unknown. The absorptive
function is assumed by the new roots produced. The ratoon is known to be sensible
to water deficit, probably due to inhibition of aerobic respiration, responsible for
the turnover of carbon stored in culms for the new sprouts. In such situation the
root system may also play an important role for the success of the ratooning.
Root performance should be addressed to improve the use of soil available
resources and increase aboveground biomass. It is possible that improvements in
yield through breeding have come at the expense of roots performance for water
and nutrient uptake (Smith et al. 2005). To investigate this, old and new cultivars
should be revisited in order to address this question.
In the biorefinary scenario, a potential use of roots as bio-factory is discussed by
(Skarjinskaia et al. 2013). Recombinant proteins, such as vaccines, could be
produced in the hairy root system of edible plants for biomedical and pharma-
ceutical applications. If therapeutical molecules are produced this way, there will
be no cost with expensive protein extractions and purifications and the biomole-
cule could be kept active improving its recovery. The plant cell wall would retain
and gradually release the bioactive substances as the plant tissues goes through the
gastrointestinal tract.
7 Advances in Methods to Improve the Sugarcane Crop 145

7.4 Final Considerations

Biorefinery is a broad and recent concept that complies the appeal for a cleaner and
sustainable way to produce energy and commodities from biomass, although it still
must find its ways to be cost competitive.
The transition of the current oil refineries to the total ‘‘green’’ refineries is
challenging and may not be implemented in the short term. It is expected that
integration of biomass and petrochemical platforms could allow for the use of
basic building blocks, more flexibility, and initial cost reduction. In Brazil, the flex
fuel car is an example of adaptation of platforms to a transition market demanding
alternative fuels.
The 2G ethanol technology still faces the challenging task to be efficient, cheap,
and clean and the hardest mission is to efficiently deconstruct the lignocellulosic
biomass. It is accepted that the achievement of higher yields of fermentable sugars
might occur through the route of reduction of biomass recalcitrance by genetic
breeding and transgenics, allied to the use of recombinant microbial enzymes mix
cheaply produced. Associated with less aggressive biomass pretreatments, these
approaches would allow for higher recovery of fermentable sugars and enzymes.
In spite of being a prominent energy crop, faster advances in sugarcane genetic
breeding is hampered by a high ploidy and complex genome structure. Molecular
breeding is thus expected to speed up the generation and release of new lines. How
then should the future cane be? There are speculations that breeding for sucrose
decreases fiber content and vice versa. However, recent experiments have shown
that either directions are possible but not mutually exclusive. Regarding the still
little exploited sugarcane genetics and molecular physiology, it is not risky to say
that Saccharum genus still reserves a huge breeding potential. An exciting time for
biorefineries is emerging as new findings and concepts in the field of ‘‘green’’ and
‘‘white’’ technologies arise.

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Chapter 8
The Essential Role of Plant Cell Wall
Degrading Enzymes in the Success
of Biorefineries: Current Status
and Future Challenges

Marcos Henrique Luciano Silveira, Matti Siika-aho, Kristiina Kruus,

Leyanis Mesa Garriga and Luiz Pereira Ramos

Abstract The viability of cellulosic ethanol depends on the optimal use of bio-
mass component through the biorefinery concept and this requires the integration
of unit operations that are involved in the production of fuel and chemicals. In this
regard, enzymes are important tools to improve the efficiency and sustainability of
a biorefinery process. Therefore, a comprehensive approach and full understanding
of the structure and function relationships that are involved in the enzymatic
hydrolysis of lignocellulosic materials is a fundamental step toward the optimi-
zation of these bioconversion processes.

8.1 Introduction

The viability of cellulosic ethanol depends on the optimal use of biomass com-
ponent through the biorefinery concept and this requires the integration of unit
operations that are involved in the production of fuel and chemicals. In this regard,
enzymes are important tools to improve the efficiency and sustainability of a
biorefinery process. Therefore, a comprehensive approach and full understanding
of the structure and function relationships that are involved in the enzymatic
hydrolysis of lignocellulosic materials is a fundamental step toward the optimi-
zation of these bioconversion processes.

M. H. L. Silveira  L. M. Garriga  L. P. Ramos (&)

Research Centre in Applied Chemistry (CEPESQ), Department of Chemistry, Federal
University of Paraná, P.O. Box 19081, Curitiba, Brazil
e-mail: luiz.ramos@ufpr.br
M. H. L. Silveira  M. Siika-aho  K. Kruus
VTT Technical Research Centre of Finland, P.O. Box 1000, 02044 VTT, Espoo, Finland
L. M. Garriga
Centro de Análisis de Procesos Facultad de Química-Farmacia, Universidad Central ‘‘Marta
Abreu’’ de Las Villas, Santa Clara, Cuba

S. S. da Silva and A. K. Chandel (eds.), Biofuels in Brazil, 151

DOI: 10.1007/978-3-319-05020-1_8,  Springer International Publishing Switzerland 2014
152 M. H. L. Silveira et al.

Biomass conversion may be performed by chemical or biochemical routes. For

many reasons ranging from process efficiency to environmental issues, most of
these pathways are ideally performed by biochemical catalysts (enzymes) such as
polysaccharide hydrolases (Mussatto et al. 2010). However, the relatively high
cost of enzymes and the complexity of carrying out enzymatic hydrolysis in large
scale are still limiting the implementation of biorefineries for fuels and chemicals
based on lignocellulosic materials. Besides glycoside hydrolases, it is also widely
known that other enzymes have an important role in the deconstruction of the plant
cell wall. These include oxidases that are involved not only in lignin degradation
but also in the chemical modification of carbohydrates. Hence, a full spectrum of
enzymes is required to deal with the wide diversity of chemical linkages and
chemical environments that are found in the plant cell wall. This chapter attempts
to describe the essential role of plant cell wall degrading enzymes in the success of
biorefineries, particularly with regard to the use of lignocellulosic materials for
fuels and chemicals.

8.2 Plant Cell Wall

The physical and chemical association of the three main components of the plant
cell wall, cellulose, hemicelluloses, and lignin, has been the subject of many
reviews that are found in the specialized literature (Higuchi 1985; Matthews et al.
2006; Coughlan and Hazlewood 1993). In short, linear chains of b-(1 ? 4)-glu-
cans (cellulose) interact with one another by hydrogen bonding to produce well-
organized crystalline regions that are regularly interrupted by less-organized or
‘‘amorphous’’ regions in which these chains are more randomly oriented. These
ribbons of polysaccharide chains are embedded in a matrix of hemicelluloses and
lignin, whose distribution and close association defines the outstanding physical
and chemical properties of this natural composite (Fig. 8.1).
A short review of the chemical and structural properties of the main plant cell
wall macromolecular components is presented below. However, important but
minor components such as pectic materials were not included in this chapter.
Details about this class of compounds may be found in reviews that are already
available in the literature (Jayani et al. 2005).

8.3 Cellulose

Cellulose is a linear homopolysaccharide composed of anhydro-D-glucopyranose

units joined together by b-(1 ? 4) glycosidic linkages (Fig. 8.2). The equatorial
orientation of the anomeric hydroxyl of the b-D-glucopyranosyl units confers
linearity to the cellulose chains, which interact with one another to produce
aggregates of great molecular order whose supramolecular structure is
8 The Essential Role of Plant Cell Wall Degrading Enzymes 153

Fig. 8.1 Structural representation of a lignin-carbohydrate complex, in which cellulose and

lignin are interconnected by their chemical interaction with arabinoferuloyl xylan units

Fig. 8.2 Cellulose structure. a Cellobiose unit. b Inter- and intramolecular interactions among
cellulose chains, with hydrogen bonds (HB) shown in different colors: red for intermolecular HB
involving C6 and C3; black for intermolecular HB between C2 and C6; blue for intramolecular
HB involving C3 and the hemiacetalic oxygen atom

architecturally stabilized by a network of intra- and intermolecular hydrogen

bonding (Zhang and Lynd 2004). As a result, adjacent cellulose chains are held
together as flat layers, allowing the more hydrophobic faces of the ribbons to stack
(Matthews et al. 2006). However, there is a thermodynamic limit beyond which
this molecular order is gradually lost, characterizing a transition to less-organized
regions in which the cellulose chains are more randomly oriented (amorphous
regions). Hence, depending on its ‘‘amorphous character,’’ the whole structure
154 M. H. L. Silveira et al.

presents cavities or pores that are able to hold relatively large amounts of water by
capillarity (Mihranyan et al. 2004).
Cellulose chains may organize themselves in different ways, forming allo-
morphs that are known as cellulose I, II, III, or IV. The natural cellulose form is
the metastable cellulose I, which contains two coexisting phases, Ia (triclinic) and
Ib (monoclinic), and the ratio between them varies depending of its origin, being
the type Ia commonly found on algae and bacteria, while type Ib is primarily found
in higher plants. The main difference between celluloses Ia and Ib lies on the
displacements of the sheets relative to one another and cellulose Ia can be con-
verted to Ib by bending during microfibril formation (Jarvis 2000). For cellulose Ia,
the chains are regularly displaced from each other in the same direction, whereas
for cellulose Ib, this displacement is found in alternating directions. This differ-
ence leads to different water adsorption profiles as well as different chemical
accessibilities for conversion (Matthews et al. 2006).
The other cellulose allomorphs are not natural. Cellulose II is generally
obtained either by mercerization or by regeneration of cellulose in organic solvents
and ionic liquids (Jhonson 1969; Okano and Sarko 1985). Cellulose III can be
produced by treatment with liquid ammonia or in the presence of some amines
(e.g., ethylene diamine). This way, cellulose IIII derived from cellulose I while
cellulose II leads to cellulose IIIII. Finally, cellulose III can be treated with
glycerol at high temperature to produce cellulose IV and, by doing so, cellulose
IVI and IVII can be obtained from cellulose IIII and IIIII, respectively (Loeb and
Segal 1954; Tsuda and Mukoyama 1957).
For many years, cellulosic materials have been extensively studied as a source
for ethanol production. In this case, conversion of lignocellulose to fermentable
sugars (mostly glucose and xylose) may be carried out by acid or enzymatic
hydrolysis (Caes et al. 2013; Yabushita et al. 2013). However, the use of acid
hydrolysis may lead to lower sugar yields due to the use of more drastic reaction
conditions, in which an array of both hydrolysis and fermentation inhibitors are
usually produced (Ramos 2003). Due to its higher specificity and lower environ-
mental impact the enzymatic hydrolysis of cellulose has received much more
attention from the international scientific community as well as from the industry.
The enzymatic conversion of cellulose to glucose is primarily performed by the
synergic/concerted action of three main classes of hydrolases, which are usually
referred to as the cellulolytic complex or cellulases: endo-b-(1 ? 4)-glucanases
(EC 3.2.1.4) (EnG), exo-b-(1 ? 4)-glucanases (EC 3.2.1.91) (ExG), and b-(1 ?
4)-glucosidases (EC 3.2.1.21) (bG). Many EnG and ExG enzymes are able to
adsorb on the substrate surface through a carbohydrate-binding module (CBM),
which is connected to the catalytic domain by a linker peptide (Notenboom et al.
2001). Several researches have shown that CBMs increase the performance of
cellulases and other hydrolases. The role of CBM in hydrolysis was recently
shown by Várnai et al. (2013). These authors were able to show that more than
60 % of cellulase genes do not have a CBM or any alternative protein structure
linked to them (dockerins) (Várnai et al. 2013). Furthermore, the effect of CBM
was more pronounced at low total solids (1 wt%, dry basis), being more important
8 The Essential Role of Plant Cell Wall Degrading Enzymes 155

Fig. 8.3 Hydrolytic and nonhydrolytic enzymes on the synergic action of the cellulose
conversion

for ExGs than EnGs of Trichoderma reesei. The results suggest that CBMs would
not be required at high total solids because these conditions would already promote
enough enzyme-to-substrate interactions for hydrolysis to occur.
EnG enzymes have a catalytic domain with a cleft shape active site that is able
to break down glycosidic bonds along the cellulose chain, acting mainly at the
less-organized ‘‘amorphous’’ regions (Rabinovich et al. 2002). This reaction leads
to the formation of two new chain ends triggering off the so-called endo-exo
synergism. ExG enzymes have a tunnel-shaped catalytic site through which the
cellulose chains must penetrate prior to eliciting its catalytic activity, releasing
mostly cellobiose. These enzymes need to adsorb on to the cellulose surface in
order to facilitate this process (Beckham et al. 2010). Once captured by cellulase
enzymes, the cellulose chain is forced to unglue/unbind from the surface and its
gradual solubilization starts processively by ExG enzymes. Finally, cellobiose and
other low molecular mass oligomers are converted to glucose by the action of bG
enzymes. Figure 8.3 shows a pictorial representation of the enzymatic hydrolysis
of cellulose.
T. reesei is the most widely studied organism for the production of cellulases.
Wild-type T. reesei strains are able to secret at least four EnGs (TrCel5A,
TrCel12A, TrCel7B TrCel45A), two ExGs (TrCel7A, TrCel6A), at least one xy-
loglucanase (TrCel74A, with EnG activity), and several bGs (Foreman et al.
2013). However, it is known that TrCel7A, TrCel6A, and TrCel5A are the pre-
dominant enzymes in the enzymatic pools of T. reesei (Nidetzky and Claeyssens
1994). Therefore, considering that the expression levels of bG by T. reesei are
enough for the growing cells but insufficient for industrial applications, enzymes
from other fungi such as Aspergillus spp. must be used to supplement this enzyme
component. In addition, besides being more tolerant to end-product inhibition, the
bG enzymes from Aspergillus spp. are able to act not only on cellooligosaccha-
rides (COS) but also on insoluble COS with an average degree of polymerization
of 20 (Sakamoto et al. 1985).
156 M. H. L. Silveira et al.

TrCel7B is the major endo-acting enzyme from T. reesei, showing 6–10 % of

its total cellulase production (Ståhlberg 1991; Nidetzky and Claeyssens 1994).
TrCel7B has been reported as catalytically active on both soluble (modified cel-
lulose such as CMC) and insoluble cellulosic substrates as well as on xylans and
glucomannans (Shoemaker et al. 1983). On the other hand, the TrCel5A is not able
to act on xylans but it is also active on soluble and insoluble cellulosic substrates
including mannans (Henrissat et al. 1985; Macarron et al. 1996; Karlsson et al.
2002). Unlikely the major EGs from T. reesei, minor enzyme components, such as
TrCel12A and TrCel45A, can also act on both soluble and insoluble substrates
including glucomannans.
Other cellulolytic enzyme systems have been investigated in their performance
to hydrolyze cellulosic substrates, such as the proteome of Neurospora crassa
(Phillips et al. 2011), Penicillium cellulases (Marjamaa et al. 2013), Cel7A pro-
teins from different thermophilic fungi (Voutilainen et al. 2008) and several EnG
enzymes from GH families 5, 6, 7, 9, 12, and 45 (Vlasenko et al. 2010), among
others. A thorough description about fungal enzymes that are able to degrade
lignocellulosic materials can be found elsewhere (Dashtban et al. 2009).
Cellulolytic enzymes represent one of the most important enzymes for the
development of biorefineries. However, ancillary proteins have also been identi-
fied as important auxiliary tools to achieve high conversion rates in cellulose
saccharification (Arantes and Saddler 2010; Ekwe et al. 2013) such as expansins,
swollenins, and lytic polysaccharide monooxygenases (LPMO). Cellulose binding
proteins can promote the deagglomeration of the cellulose chains at crystalline
regions causing amorphogenesis and this seems to be a critical step toward the
development of high accessibilities (Din et al. 1991; Chen et al. 2010). Interest-
ingly, Reese et al. (1950) suggested about 60 years ago that cellulolytic enzymes
may require the action of nonhydrolytic proteins in order to promote the disruption
of the substrate polymer packing.
The presence of expansins in plant tissues have been originally described by
Cosgrove and co-workers (Cosgrove 1999; Cosgrove 2000a, b). Expansins are
proteins of 25–27 kDa of molecular mass and their mechanism of action consists
on break the noncovalent bonds between cell wall polysaccharides, thereby
inducing the plant cell wall extension and swelling (Cosgrove 2000a; Lee et al.
2001). Also, Yuan et al. (2001) proposed that some cellulases such as TrCel12 may
have expansin-like properties in addition to its hydrolytic activity.
Likewise expansins, swollenins can also break down the physical interactions
among cellulose chains. Jäger et al. (2011) expressed the T. reesei swollenin
protein in a recombinant Kluyveromyces lactis strain and studied the effect of this
recombinant swollenin on cellulosic substrates. In general, treatment with swol-
lenin led to a decrease in both substrate particle size and crystallinity while
increasing the extent of cellulase adsorption on cellulose. As a result, high cel-
lulose hydrolysis rates were obtained. Gourlay et al. (2013) showed that T. reesei
swollenin affected especially xylan of pretreated corn stover substrate, enhancing
the production of sugars in hydrolysis. More recently, Kang et al. (2013) char-
acterized a novel recombinant swollenin from Penicillium oxalicum with regard to
8 The Essential Role of Plant Cell Wall Degrading Enzymes 157

its ability to facilitate cellulose hydrolysis. This new swollenin consists of a family
1 CBM connected to a family 45 endoglucanase-like domain by a linker.
In 2005, Vaaje-Kolstad et al. (2005) identified a novel bacterium able to secret a
chitin binding domain (CBP21) that is able to break down chitin while increasing
the substrate accessibility to chitin hydrolases. Based in this, CBP21 was classified
as a family 33 carbohydrate-binding module (Cantarel et al. 2009). This study
revealed that CBP21 cleaves glycosidic bonds in chitin by oxidation, leading to the
generation of a terminal gluconic acid residue and a normal nonreducing chain
end. These and other authors have also demonstrated that CBP21 is able to
increase the accessibility of cellulose to cellulolytic enzymes (Harris et al. 2010;
Eijsink et al. 2008; Vaaje-Kolstad et al. 2005) but the mechanism of CBP21 action
was only clarified by Vaaje-Kolstad et al. (2010).
New studies with CelS2, a CBM33 protein from Streptomyces coelicolor,
showed that it produces aldonic acids on the cellulose surface. Like other oxidative
enzymes, CelS2 also depends on the presence of divalent metal ions. Westereng
et al. (2011) revealed that these enzymes are copper-dependent monooxygenases.
Interestingly, CBM33 was also characterized as a copper-dependent lytic enzyme
(Vaaje-Kolstad et al. 2012).
Recently, a new type of fungal protein was discovered and classified as family
61 Glycoside Hydrolases (GH61, LPMO) (Harris et al. 2010; Quinlan et al. 2011;
Beeson et al. 2012). Likewise CBP21, this enzyme catalyses the oxidative
cleavage of polysaccharides, generating new chain ends while modifying the
charge distribution of the cellulosic substrate surface. The activity of these oxi-
dative enzymes depends upon the presence of a divalent metal ions and an electron
donor. Also, unlike ExG enzymes, their activity on crystalline cellulose does not
require the pull-out of a cellulose chain from the surface of the crystalline matrix
(Vaaje-Kolstad et al. 2010). These and other authors have shown that oxidative
enzymes such as those belonging to LPMO and which are abundant in fungal
genomes increase the rate of conversion of cellulosic materials by enzymatic
hydrolysis. Figure 8.4 shows one hypothesis for the action of LPMO. Oxidized
cellulose chain ends are partially converted to aldonic acid and this highly sol-
vated-opened structure forces these chains to pull out from the surface, leading to a
gradual disaggregation of the cellulose structure and to an increase in the avail-
ability of new reaction sites for both ExG and EnG.
Anaerobic microorganisms are also able to produce multi-enzymatic complexes
called cellulosomes that are able to deconstruct the structural organization of plant
polysaccharides (Fontes and Gilbert 2010). In this system, several types of cel-
lulolytic and hemicellulolytic enzymes are assembled in scaffolding subunits that
are connected to the whole cell by protein-to-protein noncovalent interactions
involving docking and anchoring protein models that are referred to as docherins
and cohesins, respectively (Bayer et al. 1994). Like most fungal cellulases, the
cellulosome systems have a CBM in mainly their anchoring protein in order to
bind to the cellulose surface (Bayer et al. 1994). Furthermore, recently the cel-
lulosomal enzymes had showed synergistic action on the presence of cellulases
(Resch et al. 2013).
158 M. H. L. Silveira et al.

Fig. 8.4 Release of a single chain from the crystalline region after the enzyme-mediated
oxidation of cellulose

8.4 Hemicelluloses

Hemicelluloses are heteropolysaccharides that are strongly associated with cellu-

lose by hydrogen bonds as well as van der Waals forces. Their primary structure
ranges from linear to highly branched polymeric chains with varying degrees of
substitution which, upon acid hydrolysis, may release different types of mono-
saccharides such as D-mannose, D-galactose, D-xylose, D-glucose, glucuronic acid,
4-O-methyl-D-glucuronic acid, and L-arabinose addition of L-rhamnose and
D-galacturonic acid present in rhamnogalacturonans of pectin materials (Bon et al.
2008). Compared to cellulose, these heteropolysaccharides have lower thermal and
chemical stabilities probably due to their lower crystallinity index and lower
degree of polymerization, reasons for what they are much more susceptible to both
acid and alkaline hydrolysis (Ramos 2003).
The main hemicellulose components of dicotyledonous angiosperms are xylans
and these usually correspond to about 20 wt% of plant dry mass (Singh et al.
2003). However, in monocotyledonous plants, xylans are no more than 2 wt% of
plant dry mass. The main backbone of these polysaccharides is composed of
anhydro-D-xylopyranosyl residues that are linked together by b-(1 ? 4) glycosidic
bonds in which substituents are usually found such as a-L-arabinofuranosyl, a-D-4-
O-methylglucuronosyl and O-acetyl groups (Sunna and Antranikian 1997). In
angiosperms, 10 % of the D-xylopyranosyl residues are substituted on C-2 position
by O-acetyl (Coughlan and Hazlewood 1993). Figure 8.5a shows a theoretical
model of the xylan structure as well as the enzymes involved on its degradation.
Most xylanases are classified in the hydrolase families 10 and 11 (Biely et al.
1997). The main difference between these two families is addressed to their
8 The Essential Role of Plant Cell Wall Degrading Enzymes 159

Fig. 8.5 a Xylan and b glucomannan structures and the main enzymes involved in their
enzymatic hydrolysis

catalytic properties. Therefore, they usually display a greater catalytic versatility,

particularly in the hydrolysis of highly substituted xylans. Likewise cellulases,
some xylanases have a CBM in their structure, bring either a xylan- or a cellulose
binding module (Shareck et al. 1991; Sakka et al. 1993).
Generally, endoxylanases act on xylans releasing mainly xylobiose, xylotriose,
and branched xylooligomers up until xylopentaose. In addition, most endoxylan-
ases hydrolyze nonsubstituted xylans more efficiently and their tolerance to the
presence of side chain varies from one enzyme to another.
Considering the high degree of substitution of xylans, endo-acting enzymes are
dominant to the exo-mode. However, although xylans are mainly composed of
b-(1 ? 4) linkage, some xylanases are able to hydrolyze b-(1 ? 3) linkages.
Furthermore, exo-acting enzymes show great affinity for polymeric xylan, how-
ever, b-xylosidase rather to act on the xylooligosaccharides. As described earlier,
glucomannans may be present as one hemicellulose component of the plant cell
wall. They have a primary backbone composed of anhydro-D-mannose and
anhydro-D-glucose linked together by b-(1 ? 4) glycosidic bonds and this may be
furnished by side chain groups such as acetyl and anhydro-D-galactosyl groups.
Therefore, like other polysaccharides, different enzymes are required for their total
hydrolysis. Figure 8.5b shows the theoretical model of a (galacto)glucomannan
fragment as well as the enzymes involved on its degradation.
The main backbone of both glucomannans and galactoglucomannans is
hydrolyzed primarily by b-(1 ? 4)-endomannanases (EC 3.2.1.78). One of the
160 M. H. L. Silveira et al.

problems relies on the fact that some mannanases are able to hydrolyze not only
the b-(1 ? 4) linkage between two mannose residues but also the b-(1 ? 4)
linkage between glucose and mannose residues (Kusakabe et al. 1988; Tenkanen
et al. 1997). Glucomannans are also efficiently hydrolyzed by endoglucanases
(Mikkelson et al. 2013).
As the concentration of oligomers builds up as a result of hydrolysis, other
enzymes such as b-mannosidase (EC 3.1.1.25) and b-glucosidase assume their role
in converting these substrates in the monomeric constituents. These enzymes are
able to remove mannose or glucose from the nonreducing end of mannooligomers.
Furthermore, T. reesei b-xylosidases and Aspergillus niger b-mannosidases may
also catalyze the removal of xylose and mannose units from the chain ends of
xylans and mannans, respectively (Margolles-Clark et al. 1996; Ademark et al.
1999). Also, some endoglucanases are able to hydrolyze not only the internal
glycosidic linkages of cellulose but also those found in other polysaccharides such
as xyloglucans due to the cleft shape of their catalytic domain. In addition, these
enzymes are also able to act on mixed b-(1 ? 3, 1 ? 4)-glucans.
Endo-(1 ? 3)-b-D-glucanases are able to catalyze the hydrolysis of b-(1 ? 3)
linkages; however, these enzymes show limited activity on the mixed glucans. On
the other hand, endo-(1 ? 3, 4)-b-D-glucanases are able to hydrolyze both
(1 ? 3) and (1 ? 4) b-linkages. Furthermore, some exo-glycosyl hydrolases are
able to cleave b-(1 ? 3) linkages in glucans by a processive action from the
nonreducing end, releasing glucose as its main end-product.
Figure 8.5 shows the average side groups that have been already found in
xylans and glucomannans. Therefore, the enzymes required to unfurnish these
polysaccharides are clearly different from those involved in the hydrolysis of the
main chain. The main enzymes involved in the removal of these side chains are a-
glucuronosidases, a-D-galactosidases, a-arabinofuranosidases, acetyl xylan ester-
ases, and ferulic acid esterases. For instance, a-glucuronosidases (EC 3.2.1.139)
carry out the partial hydrolysis of heteroxylans releasing both of glucuronic and 4-
O-methylglucuronic acid residues.
a-D-Galactosidases has not been as thoroughly studied as other enzymes but
their specific activity is critical for the complete hydrolysis of softwood mannans.
The role of this enzyme is to catalyze the hydrolysis of a-D-galactosyl side groups
that are covalently linked to the O-6 position of the anhydro-D-mannose backbone
residues (Puls 1997).
In the case of a-arabinofuranosidases, besides being active on the removal of
side chains from xylans, some of these enzymes have been reported as catalytically
active in the hydrolysis of pectins, arabinans, and arabinoxylans (Hata et al. 1992;
Saha 2000; Ximenes et al. 1996). Besides, these enzymes are particularly
important for the deconstruction of the plant cell wall because arabinose units are
connected to ferulic acid residues in lignin carbohydrate complexes.
As mentioned earlier, acetyl groups are present in several types of hemicellu-
loses such as xylans and galactoglucomannans. In hardwood and herbaceous xy-
lans, the level of acetyl groups is much higher than in the case of softwoods.
However, acetyl groups can be removed from these polysaccharides by the action
8 The Essential Role of Plant Cell Wall Degrading Enzymes 161

of acetyl xylan esterases (AXEs) and, like other enzymes already described in this
work, AXEs’ specificity depends on the nature of the substrate and its degree of
polymerization. Furthermore, AXEs can also show synergism with other enzymes
such as xylanases (Poutanen et al. 1990; Bartolome et al. 1997). For biorefinery
processes, the use of AXEs must be carefully planned because the release of acetyl
groups from the hemicellulose structure decreases the pH and this may be not
favorable to some fermenting microorganisms (de Mancilha and Karim 2003;
Martin and Jonsson 2003; Lima et al. 2004).
Non-saccharide side chains can also be found in hemicelluloses, such as in the
case of ferulic acid in herbaceous and hardwood xylans. Ferulic acid is normally
esterified at the C-2 position of an arabinosyl residue (Fig. 8.5a) and its role is
apparently associated to the three-dimensional stability of the polymer network
(Mathew and Abraham, 2004). Basically, ferulic acid units may be involved in the
crosslinking of adjacent xylan backbones by ether linkages forming diferulate
bridges, and may also play an important role in linking hemicelluloses directly to
the lignin component (Bartolome et al. 1997) (Fig. 8.1). Ferulic acid esterases
(FAEs) are responsible for removing ferulic acid decorations from xylans and
some of these enzymes are also effective in releasing coumaric acid from similar
chemical environments (Donaghy and McKay 1997). Likewise, some FAEs may
differ from each other by the affinity to the substrates that they act upon, either
polysaccharides (xylans and pectins) or substituted xylan oligomers (de Vries and
Visser 1999). Furthermore, new studies have demonstrated the presence of syn-
ergism between xylanases and FAEs, and also an enhanced catalytic activity in
FAE/xylanase fusion proteins (Faulds et al. 1995; de Vries et al. 2000; de Vries
and Visser 2001; Yu et al. 2003). However, likewise AXEs, the activity of FAEs
may lead to the release of aromatic compounds that are inhibitory to fermentation
microorganism.
The use of a specific ratio of hollocellulose degrading enzymes, including
EnGs, ExGs, bGs, xylanases, b-xylosidases, mannases, and b-mannases, is a
critical step toward the completed hydrolysis of lignocellulosic materials and this
ratio must be in agreement with the pretreatment technology applied in the process
(Várnai et al. 2011). In the case of hemicelluloses, both debranching and de-
polymeration enzymes are required to improve the extent by which these poly-
saccharides are hydrolyzed. However, different criteria may apply when the
desired products are oligosaccharides with special properties for special uses.
The synergistic action among debranching and depolymeration enzymes with
different specificities has already been extensively reported. For instance, the
synergism between a-glucuronosidases and endoxylanases in the hydrolysis of
wheat xylans led to the highest release of 4-O-methylglucuronic acid (de Vries
et al. 2000). Therefore, a-arabinofuranosidases can act synergistically with many
different enzymes such as xylanases, acetyl xylan esterases, and ferulic acid
esterases (Kroon and Williamson 1996; Coutinho and Henrissat 1999; de Vries
et al. 2000; Puls 1997; Bachmann and McCarthy 1991).
The factors affecting the performance of hydrolytic enzymes in biorefinery
processes are diverse and originate from enzyme characteristic, process conditions,
162 M. H. L. Silveira et al.

and substrates. The high catalytic efficiency of individual proteins and optimal ratio
of mixture components are the first prerequisites for efficiency. Thermal stability
has been shown to be beneficial for enzymes, due to better stability, higher con-
version rates, and flexibility in terms of process design (Viikari et al. 2007).
Nonproductive adsorption on biomass, especially on lignin reduces the availability
of enzymes for hydrolysis, and results in enzyme inactivation especially in high
temperature (Rahikainen et al. 2011). Enzyme inhibition has been extensively
studied, and it can be caused by several compounds, such as sugars and oligosac-
charides, various chemical compounds being often degradation products of bio-
mass, and also by each other on biomass surfaces. The inhibitory environment can
be improved by milder pretreatment conditions and by intelligent design of the
process. The behavior of enzymes in high dry matter conditions, applied in
industrial conditions differ clearly from that in laboratory conditions which are in
most cases used for screening and evaluation studies. High dry matter has conse-
quences in the performance of enzymes (e.g., Jørgensen et al. 2007) as well as to
fundamental features such as the effect of CBMs in hydrolysis (Várnai et al. 2013).

8.5 Lignin

Lignin is the most abundant polyphenolic compound in nature, reaching 20–30 %

of the lignocellulosic biomass produced worldwide (Fengel and Wegener 1989).
Its hydrophobic and complex structure is mainly formed by the following units:
4-(3-hydroxyprop-1-enyl)-phenol, 4-(3-hydroxyprop-1-enyl)-2-methoxyphenol
and 4-(3-hydroxyprop-1-enyl)-2, 6-dimethoxyphenol. Like hemicelluloses, the
lignin type and distribution depends on the plant species and varies from one tissue
to another. Besides, the chemical characteristics of isolated lignin depend largely
on the method used for extraction.
Unlike cellulose and hemicelluloses, the lignin building blocks or monomeric
units are not disposed in order and their crosslink includes ether linkages between
aromatic rings and aliphatic chains (b-O-40 and O-a 40 ) and different carbon-to-
carbon bonds involving aliphatic chains (b-b0 , a-a0 , and a-b0 ), aliphatic chains and
aromatic rings (b-50 , b-10 , a-10 , and b-60 ), and aromatic rings (5-50 ) (Higuchi,
1985). According to Lee (1997), the most important linkages in the lignin structure
are the b-1 and b-O-4 types, the latter of which corresponding to more than 50 %
of its polyphenolic structure. Figure 8.6 shows the model structure of a lignin
fragment derived from P. albis (Higuchi 1985) in close association with a feru-
loylated arabinoxylan, forming a lignin-carbohydrate complex.
Some microorganisms are able to produce lignin-degrading enzymes such as
lignin peroxidase (LiP) and manganese peroxidase (MnP), which are extracellular
heme proteins (Shin et al. 2005; Sharma et al. 2011). In a general, LiP catalyses the
conversion of aromatic compounds in the presence of H2O2 to their corresponding
aldehydes or ketones, and the hydroxylation of benzylic methylene groups. On the
other hand, MnP may behave as an oxidase or a peroxidase (Singh et al. 2011).
8 The Essential Role of Plant Cell Wall Degrading Enzymes 163

Fig. 8.6 Lignin fragments connected to an arabinoferuloylxylan residue

MnP acts by oxidating Mn2+ to Mn3+ with H2O2 in order to convert aromatic
compounds to polycyclic aromatic hydrocarbons (Steffen et al. 2002; Shin et al.
2005). Therefore, LiP and MnP are known as primary enzymes for degradation of
lignin. Besides the LiP and MnP, laccases are also known to degrade lignin to a
certain extent (Youn et al. 1995; Eggert et al. 1997). Several studies have dem-
onstrated the use of laccases in the detoxification of aromatic compounds but its
role in lignin degradation has not been well established as yet. Furthermore, it is
known that mushrooms can grow on lignocellulosic materials using plant carbo-
hydrates as the carbon source while secreting lignin-degrading enzymes.
In general, oxidative enzymes require the presence of cofactors such as
metallic ions and H2O2 and for this reason it is very difficult to carry out a
bioprocess with simultaneous use of lignin-degrading enzymes and carbohydrate-
degrading enzymes. Therefore, for the biorefinery processes development based
on the use of the lignocellulosic materials, these enzymes are mainly useful for
the biological pretreatment of the substrate such as in the case of biopulping
(Aguiar and Ferraz 2012).
164 M. H. L. Silveira et al.

8.6 The Role of Enzymes in the Biorefinery

Apart from improvements in the development viable enzyme technologies for

converting biomass to fuels and chemicals, insights on the pretreatment technol-
ogies of the lignocellulosic materials also represent a key factor for the industrial
biorefinery based on agroindustrial wastes. In other words, pretreatment is crucial
for the technical and economic viability of the overall process.
There are several pretreatment technologies already available for separating the
main plant cell wall components in different streams: steam explosion with and
without the use of an exogenous catalyst (Ramos 2003), dilute acid hydrolysis
(Larsson et al. 1999), liquid hot water (Laser et al. 2002; Mosier et al. 2005), wet
oxidation (Martin et al. 2007), ammonia fiber expansion (Balan et al. 2009;
Chundawat et al. 2010), alkali extraction (Gupta and Lee 2010), alkaline hydrogen
peroxide (Xiang and Lee 2000), organosolv extraction (Araque et al. 2008; Obama
et al. 2012), and treatment with ionic liquids (Li et al. 2010), among others. When
removed, hemicelluloses and lignin can be utilized in direct applications or as
precursors for a wide range of industrial chemicals and materials. For instance,
lignin can be directly used as a fuel (Menon and Rao 2012) or be converted
to many value-added products including activated carbon (Demirbas 2004),
binders (Dizhbite et al. 1999), dispersants, emulsifiers, and sequestrants (Suhas
and Ribeiro 2007; Adler, 1997), vanillin and polyurethanes (Borges da Silva et al.
2009). Lignin can also be used in blends with polyhydroxyalkanoates (Ghosh et al.
2000) and polylactides and polyglycolides (Doherty et al. 2011), in epoxy resins
(Wang et al. 1992) and as antioxidant in asphalts (Pan 2012). By contrast, he-
micelluloses such as xylans can be converted to furfural (Montané et al. 2002),
hydrogen (Caye et al. 2008), succinic acid (Nghiem, 2005), xylitol (Felipe et al.
1997), and xylooligosaccharides (Vazquez et al. 2000). Finally, apart from its
more classical uses, cellulose can be converted to glucose to produce ethanol
(Wyman 1994; Sun and Cheng 2002), lactic acid (Hofvendahl and Hahn-Häger-
dahl 2000), succinic acid (Wang et al. 2011), and acetic acid (Wang et al. 2013) by
fermentation, or used in pharmaceutical applications (Cherian et al. 2011) and as
reinforcing agent in nanocomposites (Alves et al. 2013).
Figure 8.7 shows a simplified scheme for a biorefinery based on lignocellulosic
materials. This biorefinery involves a multistep process in which the first step is
the pretreatment of the biomass to render its macromolecular components ame-
nable for further processing. The outputs of this process could be used as it is or be
converted into chemical building blocks for further processing into polymers,
chemicals, fuels, energy, and composite materials.
According to the applied pretreatment technology, different substrates are
produced and their chemical composition would require a different enzyme
composition for optimal enzymatic hydrolysis. In fact, this is a major challenge for
commercial enzymes because none of them can be claimed as universal in their
application to substrates with different compositional analysis and physical
8 The Essential Role of Plant Cell Wall Degrading Enzymes 165

Fig. 8.7 Conceptual schematic biorefinery to the technology of system integration energy

properties such as degree of crystallinity, degree of polymerization, particle size,

available surface area, and pore volume distribution.
Acid pretreatments tend to remove most of the hemicelluloses as water-soluble
mono- and oligosaccharides (the so-called hemicellulose hydrolysate), leaving a
lignocellulosic material whose hydrolysis would require an enzyme cocktail that
is less susceptible to hydrophobic interactions and to the inhibitory effects of
aromatic compounds such as phenolic acids derived from lignin. The immediate
consequence of this pretreatment option is the possibility of using the hemicel-
lulose hydrolysate for a variety of applications including ethanol production after
partial detoxification. Also, by enzymatic hydrolysis of acid-pretreated materials,
glucose is obtained as the main product and this could be one important issue for
the desired integration of cellulosic ethanol into the currently existing first-gen-
eration ethanol producing technologies. Finally, the lignin-rich residue obtained
after enzymatic hydrolysis could be used for co-generation or bioelectricity and
also for other applications in the fuel and chemical platforms.
Alkaline pretreatments are able to extract the lignin component of plant
biomass and depending on the extent of lignin extraction, the resulting fibrous
166 M. H. L. Silveira et al.

material may be classified as holocellulose. In this case, higher hemicellulase

activities would be required in the enzyme cocktail to achieve complete hydrolysis
of the delignified cellulosic material. Alternatively, the hemicellulose component
could be extracted from these substrates in its poly- or oligomeric form, allowing
its use as a polyelectrolyte, sizing agents, food additives, thickeners, films, and as a
component of natural composites. Also, the lignin component can be obtained in
higher molecular mass and with a lower degree of condensation, opening a venue
of possible industrial applications in resins, emulsions, adsorbents, carbon fiber,
films, polymers, adhesives, and composites.
With the abundance of biomass wastes, the development of new technologies
that will make use of biomass for materials production beyond biofuels represents
an important opportunity to fully utilize the resources. Development of efficient
techniques to fractionate lignocellulosic biomass into its core components will
facilitate research on the production of specific biomass-derived sugars, building
block chemicals, and ultimately value-added commodity chemicals while pre-
serving the concept of the biorefinery approach by promoting effective utilization
of all feedstock fractions.

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Chapter 9
Mapping of Cell Wall Components
in Lignified Biomass as a Tool
to Understand Recalcitrance

André Ferraz, Thales H. F. Costa, Germano Siqueira

and Adriane M. F. Milagres

Abstract Lignocelulosic biomass is recalcitrant to enzymatic digestion because

terrestrial plants develop an efficient manner to grow upward and resist the
microbial degradation of the polysaccharides contained in their cell walls. The
complex cell ultrastructure, varied tissues, and the composite characteristic of the
cell walls are among the several factors explaining the recalcitrance of lignified
plants. Mapping the macromolecular components in the cell walls has proved to be
useful to understand the varied recalcitrance of different biomass tissues. Available
data indicate that lignin and hemicellulose greatly affect the final digestibility of
the lignocellulosic materials. Removal of these components from the cell walls
with varied pretreatments or even using lignin- and/or hemicellulose-depleted
plants indicate that a critical characteristic of the cell wall to be digestible is to
present most as possible available cellulose. This chapter revises some basic
information on cell wall structure and advance in the knowledge compiling
information on the mapping of cell wall components by several techniques and
showing that the removal of cellulose encapsulating components is a key factor to
increase cell wall porosity and digestibility by hydrolytic enzymes.

9.1 Introduction

Lignocelulosic biomass is recalcitrant to the enzymatic digestion because terrestrial

plants develop an efficient manner to grow upward and resist the microbial deg-
radation of the polysaccharides contained into their cell walls. Indeed, only a small
group of organisms is able to digest the lignified cell walls in natural environments.
They comprise soft-, brown-, and white-rot fungi, which use an intricate extra-
cellular system to decompose the lignified cell wall macromolecules into small

A. Ferraz (&)  T. H. F. Costa  G. Siqueira  A. M. F. Milagres

Departamento de Biotecnologia, Escola de Engenharia de Lorena, Universidade
de São Paulo, Lorena, SP 12602-810, Brazil
e-mail: aferraz@debiq.eel.usp.br

S. S. da Silva and A. K. Chandel (eds.), Biofuels in Brazil, 173

DOI: 10.1007/978-3-319-05020-1_9,  Springer International Publishing Switzerland 2014
174 A. Ferraz et al.

compounds that can pass across the cell membrane integrating the intracellular
metabolism (Sanchez 2009; Nilson 2009). The complex cell ultrastructure and the
composite characteristic of the cell walls are among the several factors explaining
the recalcitrance of lignified plants. In this chapter, an overview of lignocellulosic
biomass ultrastructure and some key characteristics of the plant cell walls are
revised to show the origins of the recalcitrance in lignified plants. Understanding
the origins of the recalcitrance is fundamental for the development of the future
industry involved with the biomass conversion to monomeric sugars. Mapping the
macromolecular components in the cell walls, determining how the chemither-
momechanical pretreatments affect these components distribution and how the
pretreated materials respond to the enzymatic digestion was revisited. The aim was
to delineate the correlations between the changes occurred during the removal of
cell wall components with the subsequent efficiency in the enzymatic hydrolysis of
the polysaccharides. Emphasis is given on the recalcitrance of sugarcane, which is
the main lignocellulosic substrate for the polysaccharides conversion into mono-
meric sugars for use in the second-generation biofuel industries in Brazil.

9.2 Fundamental Aspects of Biomass Recalcitrance

9.2.1 Ultrastructure and Cell Types in Lignified Biomass

Revisited

The ultrastructure of wood and nonwood biomass is well known based on

microscopic studies of these materials. Complete revisions on this subject have
been published in wood chemistry textbooks (Fengel and Wegener 1989; Thomas
1991; Wiedenhoeft and Miller 2005; Daniel 2009) and in book chapters dedicated
to the study of grass monocotyledons such as sugarcane (Moore 1987). In this
chapter, a brief review on key aspects of the ultrastructure and cell types in
lignified biomass was revisited to show the cell diversity in these plants and the
importance of secondary walls for the processes involved in biomass conversion.
Wood biomass contains axial and radial cell systems. The axial cells are
oriented along the longitudinal direction of the trunk whereas radial cells are
orientated perpendicularly to the axial cells. The amount of each cell system varies
according to the wood classification and among wood species. The gymnosperms
(conifers, also referred as softwoods) and the angiosperms (deciduous or broad-
leaf trees, also referred as hardwoods) produce the solid material known as wood
biomass. The gymnosperms present a relatively simple ultrastructure where the
cells named tracheids dominate the secondary xylem (Fig. 9.1). The tracheids are
part of the axial cell system of gymnosperms, presenting conduction and
mechanical support functions. The tracheids are long cells with approximate
dimensions of 20–65 lm wide and 1.4–4.6 mm long (Fengel and Wegener 1989).
In gymnosperms, the reserve materials of the plant, such as starch, are stored
mainly in ray parenchyma cells that are part of the radial cell system.
9 Mapping of Cell Wall Components in Lignified Biomass 175

Fig. 9.1 Scanning electron microscopy of a gymnosperm showing all available faces. X cross-
section (transversal) showing tracheids in early wood (wide, thin-walled cells) and late wood
(narrow, thick-walled cells); T tangential surface with ray parenchyma cells (indicated by arrows)
viewed from the tangential cut; and R Radial surface with the ray parenchyma (indicated by
arrows) viewed from the radical cut (Reproduced with modifications from N.C. Brown Center for
Ultrastructure Studies at SUNY-ESF, Syracuse, NY, http://www.esf.edu/scme, previously pub-
lished in Thomas 1991)

The angiosperms present a more complex anatomy where the fibers have the
support function similar to that of the tracheids in gymnosperms. However,
together with the fibers, the angiosperms present vessels as an integral part of the
axial cell system (Fig. 9.2). The presence of vessels represents the main difference
between angiosperms and gymnosperms. The vessel elements are cells connected
each other along the longitudinal axes of the tree forming a cell system (vessels)
with the main function of water conduction. The fibers are narrower and shorter
cells than the tracheids of gymnosperms with approximate dimensions of
15–40 lm wide and 0.6–1.6 mm long (Fengel and Wegener 1989). In addition to
fibers and vessels, the angiosperms present abundant parenchyma cells that can
occur in the ray tissue of the radial cell system, but also as longitudinal paren-
chyma, making part of the axial cell system. As in gymnosperms, the main
function of the parenchyma cells is the storage of reserve materials for the tree.
In monocotyledons such as sugarcane, the cell distribution along the axial axes
of the plant is different from that observed in gymnosperms and angiosperms. The
internodes of monocotyledons present vascular bundles surrounded by parenchyma
cells as illustrated in Fig. 9.3. Each vascular bundle contains a small phloem (sieve
176 A. Ferraz et al.

Fig. 9.2 Scanning electron microscopy of an angiosperm showing all available faces. X cross-
section (transversal) showing fibers (narrow cells) and vessels (wide cells); T tangential surface with
multi-cell ray parenchyma (indicated by arrows); and R Radial surface showing vessel elements in
details E (Reproduced with modifications from N.C. Brown Center for Ultrastructure Studies at
SUNY-ESF, Syracuse, NY, http://www.esf.edu/scme, previously published in Thomas 1991)

Fig. 9.3 Transversal cut of a

monocotyledon (sugarcane
hybrid, Saccharum sp.)
visualized through optical
microscopy illustrating
mature vascular bundles
surrounded by parenchyma
cells. V Vessels; Ph Phloem
cells; P Thin-walled
parenchyma cells; and
(Fibers) Thick-walled fibers
in the vascular bundle
(Micrograph provided by the
authors)

elements and companion cells), whereas most of the bundle area is composed of
vessels and fiber cells that present similar functions as described for angiosperms.
The fibers are similar to angiosperms fibers with approximate dimensions of
15–25 lm wide and 0.6–1.7 mm long (SanJuan et al. 2001). The parenchyma cells
surrounding the vascular bundles have the main function of storage of reserve
materials, which in the case of sugarcane is composed mainly of sucrose.
9 Mapping of Cell Wall Components in Lignified Biomass 177

Table 9.1 Abundance of Biomass origin (%) Cell type

different cell types in biomass
from gymnosperms, Fibers Vessels Ray parenchyma
angiosperms, and Gymnosperms
monocotyledons (Chum et al. Volume 90–95 absent 5–10
1985; Fengel and Wegener Dry mass 95–98
1989) Angiosperms
Volume 45–65 10–40 10–30
Dry mass 70–85 10–15 4–8
Monocotyledons
Volume 20–60 1–10 30–70

In monocotyledons such as sugarcane, the thickness of the cell walls in different

cell types is relevant when biomass conversion is under scrutiny because the
parenchyma cells can represent up to 70 % of the internodes’ volume. However,
most of the parenchyma cells present very thin cell walls that contrast with vessels
and especially fibers, which develop an extensive deposition of thick secondary
walls. Therefore, the secondary walls of fibers are the most important process
material in biomass conversion, since the secondary wall represents most of the
dry matter of the mature plant. Table 9.1 illustrates the volume and mass pro-
portion of different cell types in gymnosperms, angiosperms, and monocotyledons.
There is a great variation inside each plant group, but a general trend is that the
gymnosperms tracheids are responsible for more than 95 % of the secondary
xylem dry mass. In angiosperms, the fibers account for 70–85 % of the dry matter
and the vessels represent 10–15 %. In both cases, the parenchyma cells present
only a minor participation in the secondary xylem dry mass (Chum et al. 1985;
Fengel and Wegener 1989).
The average thickness of the different wall layers is variable according to the
wood classification and the cell type. In general, the primary wall and the middle
lamella are very thin and difficult to be distinguished each other by microscopic
techniques. The thickness for the pair primary wall plus middle lamella (also
referred as compound middle lamella) is in the range of 0.05–0.1 lm, indepen-
dently on the origin of the lignified biomass. In contrast, the three layers of the
secondary walls together (S1, S2, and S3, when present) comprise approximately 2
and 4.2 lm in early and late wood tracheids of gymnosperms, respectively (Fengel
and Wegener 1989). In angiosperms, the secondary wall of fibers presents the
approximate thickness of 3 to 5 lm, whereas in longitudinal and radial paren-
chyma it can reach 0.7 and 1.3 lm, respectively (Daniel 2009). In the sugarcane
monocotyledon, the fiber cell walls were reported to present an average thickness
of 4 lm, whereas vessel and parenchyma cell walls present average values of
2.7 and 1.7 lm, respectively (SanJuan et al. 2001). The average value of 1.7 lm
for the parenchyma cell walls suggests that secondary wall deposition occurs at
least in some parenchyma cells of sugarcane.
178 A. Ferraz et al.

Table 9.2 Dry mass contents Internode region Dry biomass content (%)
of four different regions
dissected from sugarcane Sugarcane cultivars
internodes (Costa et al. 2013) A B C
Pith 7 6 6
Interface pith-rind 18 19 20
Rind 50 63 58
Outermost fraction 25 12 16

In monocotyledons, the volume occupied by parenchyma cells can vary from

30–70 % of the internode, but it should represent less than 10–20 % of the in-
ternodes’ dry mass. An example of the dry mass significance of each cell type in
sugarcane can be obtained by dissection of the internodes into several regions. In a
detailed study of three different cultivars of sugarcane, Costa et al. (2013) dis-
sected the sugarcane internodes into four different fractions between the pith and
the outermost part of the stalk. These fractions corresponded to the pith, the pith-
rind interface, the rind, and the outermost fraction, which composed approximately
10, 29, 49, and 12 % of the internodes’ volume, respectively. Micrographs of
transverse cuts from each internode region showed that wide parenchyma cells
predominated in the pith, while a small number of vascular bundles containing
thin-walled-large-lumen fibers were also observed. In contrast, vascular bundles
containing numerous thick-walled fibers were observed in the rind fraction. The
pith–rind interface was comparable to the pith, whereas the outermost fraction
contained epidermis cells, a ring of thick-walled cortical cells and small parts of
the peripheral vascular bundles. These observations confronted with the mass
proportion of the regions (Table 9.2) indicated that the central region of the
internode (pith plus the interface pith-rind), in which the parenchyma cells
predominated, accounted to a maximum of 25 % of the dry material. In contrast,
the rind and outermost fractions, in which the vascular bundles predominated,
contained the remaining 75 % of the dry matter of the internode.

9.2.2 Composite Characteristics of Secondary Walls Limiting

the Enzyme Access to the Cellulose Chains

Some characteristics of the lignocellulosic substrates are critical to limit the

enzymatic hydrolysis of cellulose and hemicellulose present in the lignified cell
wall. Besides the anatomical tissue complexity of lignified biomass, the secondary
cell walls of lignocellulosic materials are true composites since the strands of
cellulose microfibrils are embedded with an amorphous matrix composed of lignin
and hemicelluloses (Fengel and Wegener 1989; Daniel 2009). In some grass
monocotyledons, usually consumed by ruminants, hydroxycinnamic acids make
bridges between hemicellulose chains or between lignin and hemicelluloses and
9 Mapping of Cell Wall Components in Lignified Biomass 179

Fig. 9.4 Surface area 1000

accessible to dextran
molecules of different sizes in

Total accessible area (m 2/g)

800
untreated and 4-h delignified
sugarcane bagasse. The lignin Approximate size of cellulolytic enzymes
content in each material was 600
21 and 6 %, respectively
(Reproduced with
400
modifications from Santi Jr. untreated
2011) 4h-delignified
200

0
0 100 200 300 400 500
Pore size accessible to dextran molecules (Angstron)

are also important for the formation of this type of embedding matrix (Grabber
et al. 2002; Lam et al. 1994). In all cases, the cell walls of these materials exhibit
porosity of molecular scale dimensions, which limits the permeability of the
enzymes through the cell wall composite. The low porosity of the cell walls is,
therefore, one of the main causes of lignocellulose recalcitrance. For example, in
untreated and 4-delignified sugarcane bagasse, the area accessible to molecules of
approximately 50 Angstrons, which is the approximate size of cellulolytic
enzymes (Rollin et al. 2011), increases from 56 m2/g to 172 m2/g (Fig. 9.4),
indicating that lignin removal let voids in the cell wall matrix. This change in the
material porosity caused by lignin removal presented a remarkable effect in both,
cellulose and xylan conversion to monomeric sugars by commercial cellulases,
since they were limited to 20 and 9 % in untreated material, and increased to 96
and 85 %, respectively, in the 4-h delignified material (Santi Jr. 2011).
The effect of the matrix composed by hemicellulose and lignin on enzymatic
hydrolysis of cellulose has been investigated for several lignocellulosic materials
(Liao et al. 2005; Mussatto et al. 2008; Lee et al. 2009; Mendes et al. 2011;
Siqueira et al. 2011). All these works indicate that lignin and hemicellulose
removal from each material enhances the hydrolysis ability of the lignocellulosic
material. However, it is noteworthy that the complete removal of these compo-
nents is not necessary to achieve cellulose hydrolysis levels higher than 80 %.
Thus, a comprehensive study of the structural features that cause the major effects
on enzymatic hydrolysis of lignocellulose is still needed to finally determine a
correlation between lignin and/or hemicellulose removal and digestibility.
Pan et al. (2005) suggested that lignin reduces the cellulose hydrolysis by two
distinct mechanisms: by forming a physical barrier that impedes or prevents
enzyme access to the cellulose and by unproductively binding cellulolytic
enzymes. In fact, lignin irreversibly adsorbs the cellulase enzymes, preventing
their action on cellulose. A consequence of both phenomena is that biomass
samples with high lignin content are poorly hydrolyzed by celulases. Otherwise,
180 A. Ferraz et al.

Table 9.3 Extracted components from sugarcane bagasse treated with sodium chlorite and the
corresponding initial rates of enzymatic hydrolysis of cellulose in the treated samples (measured
after 4 h of reaction) (Siqueira et al. 2013)
Treatment time with Extracted Extracted Extracted Initial hydrolysis
sodium chlorite (h) lignin (%) hemicellulose (%) cellulose (%) rate (% h-1)
0 0 0 0 2.8
1 41.0 0 0 7.8
2 63.2 0 0 10.2
3 72.2 9.2 0 10.4
4 76.2 10.3 0 12.3

for samples with low lignin content, many enzymes can adsorb onto cellulose,
which is effectively and rapidly digested (Chang and Holtzapple 2000).
Many studies have been conducted to access the effects of the lignin removal in
the enzymatic hydrolysis of cellulose. Some chemical treatments are able to
selectively remove this component, providing information about the role of lignin
in limiting the enzymes action. The challenge on the selective removal of cell wall
components is to remove one of them without changing the structure or chemical
properties of the others. However, all the treatments, even the selective ones, affect
the remaining components, making it difficult to study a single effect. Lignin can
be removed using specific ionic liquids, such as 1-ethyl-3-methylimidazolium
acetate, which solubilizes mainly lignin, preserving the other cell wall components
(Lee et al. 2009). Oxidative methods, such as chlorite treatment, are widely studied
and have given some information about restriction of enzymatic hydrolysis by
lignin (Siqueira et al. 2013; Várnai et al. 2010; Kumar et al. 2013). The chlorite
treatment consists of the hourly addition of sodium chlorite and acetic acid to the
biomass at a reaction temperature of 70 C. During the reaction, chlorine dioxide
is formed as the main product, which can efficiently oxidize lignin (Browning
1968; Gellerstedt 2009).
Siqueira et al. (2013) studied the effect of lignin removal with sodium chlorite
in the subsequent enzymatic hydrolysis of sugarcane bagasse, one of the typical
grass monocotyledons used in biomass conversion processes. The bagasse was
treated for 4 h and biomass samples were collected after each hour of treatment,
generating four substrates with different lignin contents. The chlorite delignifica-
tion was selective up to 2 h of treatment, removing 60 % of the initial lignin from
the sugarcane bagasse. After that reaction time, part of the hemicellulose was also
extracted from the samples (approximately 10 %). However, even up to 4 h of
treatment, the cellulosic fraction was not solubilized (Table 9.3). Considering the
difficulty to selectively remove a single component, the chlorite treatment has been
considered one of the best treatments to study the effect of lignin in the enzymatic
hydrolysis. The data presented in Table 9.3 corroborate that the enzymatic
hydrolysis rates are determined by the accessibility to the substrate. Removing
lignin from the substrate provided more accessible cellulose, resulting in higher
hydrolysis rates. However, during the enzymatic hydrolysis, the reaction rate
9 Mapping of Cell Wall Components in Lignified Biomass 181

Fig. 9.5 Cellulose 100

conversion after 72 h of

Cellulose conversion (%)

enzymatic hydrolysis as a 80
function of extracted lignin,
cellulases with b-glucosidase 60
(filled circle) and cellulases
without b-glucosidase (filled 40
diamond) (Reproduced from
Siqueira et al. 2013) 20

0
0 20 40 60 80
Lignin removal (%)

decreases mainly because the more accessible cellulose is hydrolyzed first,

whereas the residual material is enriched in recalcitrant substrate (Arantes and
Saddler 2011).
One of the goals of selective removal of lignin is to access how much of lignin
has to be removed to achieve good hydrolysis levels (above 80 %), either to design
better pretreatments or to develop plants with lower lignin contents. Studies have
shown that removing between 40 and 60 % is enough to reach good hydrolysis
(Lee el al. 2009; Siqueira et al. 2013). For sugarcane bagasse, Siqueira et al. (2013)
showed that removing 63 % of the initial lignin, more than 90 % of the cellulose
was converted into glucose, while all cellulose was hydrolyzed after removing
72 % of the lignin (Fig. 9.5). The authors also combined the effect of lignin
removal and the addition of b-glucosidases to the reaction medium. With the
addition of b-glucosidase, less lignin needs to be removed to achieve similar
hydrolysis levels. In the example of the Fig. 9.5, setting 80 % hydrolysis as a goal,
63 % of the lignin has to be removed if b-glucosidase is not added to the system.
However, supplementing the enzyme mixture with b-glucosidase, the same
hydrolysis level is achieved if 48 % of the lignin is removed.
Hemicellulose also exerts a great influence on the enzyme attack to the cellulose
chains because this component is closely related to cellulose into the cell walls,
covering part of the microfibrils. In the same manner, as illustrated for selective
lignin removal from the lignocellulose matrix, some published work successfully
removed hemicellulose from brewer’s spent grain (86.5 % removal of hemicellu-
lose and only 14 % removal of lignin) and evaluated the digestibility of the resulting
solid material (Mussatto et al. 2008). In this case, the enzymatic conversion of
cellulose to glucose was 3.5 times increased compared to the untreated material,
attaining a value of 78 %. However, the real effect caused by the presence of
hemicellulose in the enzymatic cellulose hydrolysis is not conclusive since the
acidic removal of hemicellulose also change the content of crystalline cellulose in
the sample as well as can diminish the cellulose degree of polymerization. In
addition, some recent reports indicate that the presence of xylan in the substrate
can facilitate its swelling creating more accessible surface area for the interaction of
the enzymes with the cellulose present in the substrate (Ju et al. 2013).
182 A. Ferraz et al.

An example of multivariate effects occurring during hemicellulose removal can

be assessed in some reports indicating that despite the hemicellulose removal
facilitate cellulose hydrolysis by increasing the porosity of the lignocellulosic
ultrastructure, the content of crystalline cellulose increases after the acid
pretreatments owing to the coupled removal of amorphous cellulose from the lig-
nocellulosic material (Kim and Holtzapple 2006). The lignocellulose crystallinity
(also referred as crystallinity index, CrI) has been considered as a significant
parameter that affects negatively the biomass digestibility despite the different cell
wall composition of samples (Lee et al. 2009; Xu et al. 2012). However, some
contrary data have indicated that CrI did not affect the efficiency of the hydrolysis.
This controversy remains, mainly if complex substrates, instead of pure cellulose
such as cotton fibers, are under scrutiny. Several factors, substrate and enzyme
related, may confound the isolated effects caused by CrI. Residual lignin and
hemicellulose are among the materials that can interfere in the data interpretation
related to the enzymatic hydrolysis of complex lignocellulosic substrates, since the
action of cellulases is also affected by irreversible adsorption of enzymes onto these
components as discussed below (Palonen et al. 2004; Rahikainen et al. 2011).
Mixtures of pure cellulose substrates and purified enzymes indicate that the specific
enzymes can attack both amorphous and crystalline cellulose simultaneously, with
no accumulation of crystalline cellulose (Hall et al. 2010). Consequently, CrI alone
may not adequately explain differences in observed hydrolysis rates and should be
considered just one of several parameters that affect the enzymatic hydrolysis of
cellulose in a complex biomass sample (Pu et al. 2013).

9.2.3 Unproductive Binding of Cellulolytic Enzymes

on Lignin

As mentioned before, lignin has an important negative effect on the enzymatic

hydrolysis of cellulose, mainly because its presence in the cell wall matrix limits
the accessibility of the enzymes to the cellulose polymer. However, this is not the
single inhibitory role that lignin plays in the conversion of cellulose to glucose.
The enzymatic hydrolysis of cellulose is a heterogeneous reaction, which requires
the adsorption of the enzymes to the cellulosic chains. This adsorption depends on
the pairing of aromatic amino acid residues from the enzyme with the glycoside-
exposed surface of the cellulose. This enzyme-substrate interaction occurs mainly
due to the presence of tyrosine residues in the enzyme helped by hydrogen bonds
between glutamine residues and cellulose hydroxyls (Linder et al. 1995). The
coupling is not specific and the celluloytic enzymes can bind to other molecules
such as lignin, which reduces the amount of available enzymes to act on cellulose.
The adsorption of cellulolytic enzymes on molecules that differs from cellulose is
usually named as unproductive binding. The phenomenon was observed in a
variety of different biomasses subjected to a range of different pretreatments, and
is considered an important inhibitory effect of lignin during the enzymatic
9 Mapping of Cell Wall Components in Lignified Biomass 183

hydrolysis of cellulose (Chernoglazov et al. 1988; Palonen et al. 2004; Berlin et al.
2005; Nakagame et al. 2010; Rahikainen et al. 2011).
The extent of enzymes binding depends on the lignin structural features (Linder
et al. 1995; Palonen et al. 2004). This aspect is relevant, because during the pre-
treatment employed to increase the efficiency of the enzymatic hydrolysis of bio-
mass, chemical modifications in lignin can change its affinity for the cellulolytic
enzymes. For example, some pretreatments that increased the phenolic hydroxyl
contents of residual lignin also increased the capacity of the lignin to bind to
proteins (Sewalt et al. 1997; Rahikainen et al. 2013). In contrast, pretreatments that
turned lignin more hydrophilic (mostly by generation of acid groups) were effective
to diminish the unproductive binding of cellulases on the pretreated material
(Nakagame et al. 2011a; Lou et al. 2013). The main effect of introducing acid
groups in lignin is that, at the pH of the enzymatic hydrolysis (usually from
4.8–5.0), at least part of these acid groups are ionized, giving to lignin a negative
charge. The cellulases with isoelectric point below 4.8 (most of them present iso-
eletric poins between 3.6 and 8.5) are also negatively charged at pH 4.8–5.0, which
causes repulsion of the enzyme to the lignin moieties, diminishing the unproductive
binding (Nakagame et al. 2011b). Because of this, some authors suggest the use of
higher pH values for enzymatic hydrolysis (Lou et al. 2013). In addition to the
pretreatment effect on the lignin capacity to bind cellulases, different biomasses
also differ on unproductive binding properties. For example, Nakagame et al.
(2010) demonstrated that the lignin from wood biomass binds more to cellulases
than lignin from monocotyledon agricultural residues.
The cellulolytic complex is a mixture of several enzymes that differ on molar
mass, isoelectric point, and hydrophobicity. Because of this, unproductive binding
studies have been conducted with the whole cellulase mixture and also with some
purified enzymes. For example, comparing the major enzymes involved in the
cellulose breakdown, it is apparent that the cellobiohydrolase Cel7A from T. reesei
binds more strongly to lignin than endoglucanase Cel5A produced by the same
fungus. The binding affinity of Cel7A was also 3 times higher than Cel5B, despite
the lignin tested (Palonen et al. 2004).
Another interesting aspect of unproductive binding is the role of cellulose
binding modules (CBMs). Cel7A, which contains a CBM, binds faster and to a
larger extent to lignin films compared to Cel7A lacking the CBM (Rahikainen
et al. 2013). It is suggested that the three aligned tyrosines (Y5, Y31, and Y32)
present in the CBM are important for the hydrophobic interactions that drive the
CBM-cellulose binding (Linder et al. 1995). However, it is probable that these
amino acid residues are also important for the unproductive binding of the protein
to hydrophobic lignin surfaces.
Unproductive binding is a problem for the enzymatic hydrolysis of cellulose not
only because it decreases the amount of available protein, but also because of the
thermal inactivation of the enzymes. Most of the adsorption studies are conducted
at low temperature (4 C) to avoid structural changes in the substrates. At this
temperature, the bound enzymes can be recovered with almost the same activity.
However, at hydrolysis temperatures of 45 C or more, the protein–lignin
184 A. Ferraz et al.

interactions are intensified and the proteins lose their native structure, becoming
denatured and irreversibly bound to lignin (Rahikainen et al. 2011).
Adsorption is a concentration-dependent phenomenon, with the available sur-
faces becoming saturated as the protein concentration increases. Because of this,
the unproductive binding can be overcome if the enzyme loading is relatively high
(Nakagame et al. 2010; Kumar et al. 2012). Making the cellulose more accessible
is another way to avoid unproductive binding because the cellulases bind faster to
cellulose than to lignin (Tu et al. 2009; Kumar et al. 2012). However, overcoming
unproductive binding at low enzyme loadings is still a challenge.
As the unproductive binding is not a specific interaction, the addition of other
proteins prior to the addition of cellulases can reduce the amount of cellulases bound
to the lignin. For example, Yang and Wyman (2006) added bovine serum albumin
(BSA) to the reaction mixture and measured the cellulase activity in the supernatant
during the course of hydrolysis. After 72 h, the cellulose activity in the supernatant
was 20 % of the initial activity if BSA was not added to the reaction. In contrast,
when BSA was added 1.5 h before cellulases addition, 50 % of the initial activity
remained in the liquid fraction after 72 h. Another way to decrease the unproductive
binding is to add surfactants to the reaction mixture. The presence of surfactants
can increase the desorption rates, reducing the amount of lignin-bound enzymes
(Eriksson et al. 2002).

9.3 Topochemical Distribution of Cell Wall Components

and Its Correlation with Varied Recalcitrance
in Different Cell Types

9.3.1 Lignin and Hydroxycinnamic Acids

Several microscopic techniques have been used to detect lignin and other aromatic
compounds directly into the cell layers of lignified plants. The most traditional
technique involves the UV absorption of lignin moieties that enables the direct
assessment of lignin contents in each cell layer (Fergus et al. 1969; Koch and
Kleist 2001). Based on this technique, fine details on lignin deposition into cell
walls, middle lamella, and cell corner have been revealed. Textbooks on wood
chemistry present classical data for some wood species indicating that most of the
lignin contained in the lignocellulosic materials is located in the secondary walls
simply because the lignified secondary walls are the thickest layers and represent
most of the dry matter in wood biomass. An overall view of lignin distribution in
the cell wall layers of wood biomass is summarized in Table 9.4. The highest
concentration of lignin is always found in the cell corners, followed by the middle
lamella and then secondary walls. However, 65–75 % of the total lignin available
in the gymnosperms is located into de tracheid secondary walls. In angiosperms,
approximately 60 % of the lignin is in the secondary walls of fibers, 20 % is in the
9 Mapping of Cell Wall Components in Lignified Biomass 185

Table 9.4 Approximate lignin distribution and concentration in the several cell layers of
gymnosperms and angyosperms (adapted with modifications from Fengel and Wegener 1989;
Henriksson 2009)
Wood Cell Cell layer Contribution to the total Lignin concentration in
type type lignin content (%) the layer (%)
Gymnosperms early wood
Tracheid Secondary wall (S1–S3) 65 24
Middle lamella plus 21 49
primary wall
Cell corner 14 64
Gymnosperms late wood
Tracheid Secondary wall (S1–S3) 75 22
Middle lamella plus 14 51
primary wall
Cell corner 11 78
Angiosperms wood
Fiber Secondary wall (S1–S2) 60 19
Middle lamella plus 9 40
primary wall
Cell corner 9 85
Vessel Secondary wall 9 25
Middle lamella plus 2 40
primary wall
Ray cells Secondary wall 11 25

cell walls of other cell types, and the rest is distributed in the middle lamella and
the cell corners (Table 9.4).
In the last decades, the UV microspectrophotometry evolved to 0.25 lm2 of
geometrical resolution and appropriate softwares translate the absorption intensi-
ties of the spots in the cell layers into multicolored pixels to illustrate the lignin
distribution in the biomass tissues. An example of this mapping technique is shown
in Fig. 9.6 for the early wood tracheids from the gymnosperm Pinus taeda.
In monocotyledons, there are fewer studies related to the lignin distribution in
the cell layers and cell types. However, the general trend of the major proportion
of lignin in the secondary walls is valid, with the highest concentrations also
observed in the cell corners and middle lamella. In addition to lignin, the cell walls
of monocotyledons can also present UV absorption assigned to the presence of
hydroxycinnamic acids. For example, the distribution of lignin and hydroxycin-
namic acids in different cell types of sugarcane was formerly studied by He and
Terashima (1990, 1991) using microautoradiography and UV microspectropho-
tometry. These authors demonstrated that the lignification of vessels occurred in
the early cell maturation stage followed by lignification of fibers. In contrast, the
UV absorption spectra of the parenchyma cell walls suggested the predominance
of hydroxycinnamic acids instead of lignin. More recently, Siqueira et al. (2011)
mapped the lignin occurrence in different cell types of mature sugarcane samples.
186 A. Ferraz et al.

Fig. 9.6 UV micrograph of and early wood tracheid from the angiosperm Pinus taeda with
0.25 lm2 of geometrical resolution. Appropriate software translates the absorptions intensities at
278 nm (shown in the left of the image) into multiple colors to illustrate the lignin distribution in
the biomass tissues. The image clearly indicates the cell corners as the region with the highest
absorption (colored with black), followed by the middle lamella (colored with gray to light blue)
and by the secondary cell wall (colored with pink to dark blue) (Micrography provided by the
authors and previously published in Mendonça et al. 2004)

The highest UV absorbance was detected in the cell walls of vessels followed by
fibers and then parenchyma. UV spectra of fiber and vessel cell walls presented
bands near to 278 nm and 315 nm (Fig. 9.7). The band at 278 nm was assigned to
the aromatic rings of lignin, whereas the strong band at 315 nm was assigned to
hydroxycinnamic acids linked to the lignin and/or arabino-methylglucurono-xylan
backbones often found in grasses (He and Terashima 1991; Lybeer et al. 2006).
The spectra from the parenchyma cell walls revealed the lowest absorbance values,
the band at 278 nm was not resolved, and the most intense absorption appeared at
315 nm, which is consistent with the predominance of hydroxycinnamic acids as
the main UV absorbing compounds in these cell walls. The parenchyma cells
found in the central part of the internode (pith) presented even lower absorbance
values as seen in Fig. 9.7.
Selected areas of the sugarcane fibers scanned at the geometrical resolution of
0.25 lm2 corroborated previous studies with wood tissues. The most intense
absorbance values were observed in the cell corners and the middle lamella fol-
lowed by the cell walls (Fig. 9.8). It is noteworthy that the absorption intensities at
278 nm (proportional to the lignin concentrations) in the cell corners and middle
lamella of the sugarcane fibers are lower than those observed in the fibers of the
gymnosperm Pinus taeda (Fig. 9.7).
Confocal Raman microscopy also has been used to map the lignocellulose
components into the cell layers (Agarwal 2006; Gierlinger and Schwanninger
2006). In this case, an especially set Raman spectrometer is attached to the
microscope enabling the spectrum record in defined areas of the cell layers sim-
ilarly to the described before for UV microspectrophotometry. Usually the Raman
band intensities at the regions of 1,519–1,712 and 978–1,178 cm-1 are used to
9 Mapping of Cell Wall Components in Lignified Biomass 187

0.6 0.6
vessel
0.5 vessel
fiber 0.5
fiber
0.4 parenchyma parenchyma
0.4

Absorbance
Absorbance

0.3 0.3

0.2 0.2

0.1 0.1

0 0

-0.1 -0.1
220 240 260 280 300 320 340 360 380 400 220 240 260 280 300 320 340 360 380 400
Wavelenght (nm) Wavelenght (nm)

Fig. 9.7 UV spectra recorded from 1 lm2 areas selected in the cell walls of different cell types
excised from the rind (left) and the pith (right) region of mature sugarcane (UV spectra provided
by the authors and previously published in Siqueira et al. 2011)

Fig. 9.8 UV micrograph of fiber cells from the monocotyledon Saccharum sp with 0.25 lm2 of
geometrical resolution. Appropriate software translates the absorptions intensities at 278 nm
(shown in the left of the image) into multiple colors to illustrate the lignin distribution in the
biomass tissues. The image clearly indicates the cell corners as the region with the highest
absorption (colored with light- and dark green), followed by the middle lamella (colored with
light green to pink) and by the secondary cell wall (colored with pink to dark blue) (Micrography
provided by the authors and previously published in Siqueira et al. 2011)

map lignin and cellulose distribution, respectively. Stimulated Raman scattering

(SRS) microscopy was recently used to map the cell wall components in maize
(Ding et al. 2013). With this promising technique, the signal intensity at
1600 cm-1 (aromatic breathing modes, primary assigned to lignin moieties) was
used to reveal the lignin localization and abundance into the cell walls, similarly to
the discussed before for the UV microspectrophotometry. In addition, the Raman
signal intensity at 2900 cm-1 (C–H stretch, primarily assigned to polysaccharides)
188 A. Ferraz et al.

Fig. 9.9 Transversal cuts of maize internodes treated with the rumen biota for 24 and 96 h of
digestion. Note that a group of parenchyma cells was completely removed (digested) from the
samples, whereas some other parenchyma and all vascular bundles resisted to the digestion even
after 96 h of treatment (Reproduced with modifications from Jung and Cassler 2006)

was used to reveal the localization and abundance of polysaccharides into the cell
walls. The technique has been applied as a two-color SRS microscopy of fresh
samples where the polysaccharides and lignin absorptions were related to two
different colors in the produced image.
With the advance in microscopic techniques for mapping the lignocellulosic
components into the cell walls, some attempts to correlate the topochemical dis-
tribution of lignin and hydroxycinnamic acids with the in vitro recalcitrance of the
biomass material to hydrolytic enzymes have been reported. In the case of
untreated grass monocotyledons, the recalcitrance varies according to the cell type
and maturation stage (Siqueira et al. 2011; Zeng et al. 2012; Jung and Casle 2006).
Some reports indicate that the parenchyma cells from the maize internode are
promptly hydrolyzed by commercial cellulases or by the rumen biota (Fig. 9.9).
This occurs because these cells are not extensively lignified and contain a limited
amount of hydroxycinnamic acids as compared to other cells in the biomass
material (Costa et al. 2013; Zeng et al. 2012; Jung and Casler 2006; Ding et al.
2013; Siqueira et al. 2011). In contrast, the rind region of the monocotyledon
internodes contains highly lignified vessels and fibers arranged in the vascular
bundles that are very recalcitrant to enzymatic hydrolysis (Fig. 9.9). Similar
results were obtained with sugarcane samples as described by Costa et al. (2013).
The authors evaluated the digestibility of the same sugarcane cultivars and in-
tenode regions previously described in the Table 9.2 of this chapter. As illustrated
in the Fig. 9.10 (for the cultivar C from Table 9.2), the outermost fraction and the
rind regions from the sugarcane internodes were very recalcitrant, whereas the
pith–rind interface and the pith were significantly less recalcitrant.
As already discussed, several publications related to the enzymatic digestion of
wood and nonwood substrates indicate that the selective lignin removal from the
9 Mapping of Cell Wall Components in Lignified Biomass 189

Fig. 9.10 Cellulose

conversion to glucose 80
catalyzed by commercial

Cellulose conversion (%)

cellulases acting on different
regions of untreated
sugarcane internodes. 60
(Reproduced with
modifications from Costa
et al. 2013) 40

20

0
0 24 48 72
Hydrolysis time (h)

lignocellulosic material promptly diminishes its recalcitrance (Lee et al. 2009;

Siqueira et al. 2011; Siqueira et al. 2013; Ding et al. 2013). In the case of sugarcane,
a detailed UV microspectrophotometric evaluation of the lignin removal from fiber
cell walls corroborated that the highly lignified fibers from the rind region of the
internode become less recalcitrant as a function of the lignin and hydroxycinnamic
acids removal (Siqueira et al. 2011). In contrast, even untreated parenchyma cells
from the pith region (characterized by low UV absorbance of the cell walls) were
promptly hydrolyzed by commercial cellulases (Fig. 9.10). The data indicated that
the action of the cellulolytic enzymes was not restrained by the aromatics occurring
in the pith parenchyma, but it was strongly controlled by the high lignin content
present in the fiber cell walls from the rind region of the internode. The chlorite
treatment (delignification) of the pith region, rich in parenchyma cells, did not
enhance cellulose conversion, whereas the application of the same treatment to rind
cells, rich in vascular bundles, led to significant removal of hydroxycinnamic acids
and lignin, resulting in a significant enhancement of the cellulose conversion by
commercial cellulases.

9.3.2 Hemicellulose

As stated before, there is a clear correlation between lignin removal from the fiber
cell walls and the increased efficiency in the subsequent cellulose hydrolysis of the
delignified residue induced by enzymes. However, the important role of hemicel-
lulose on the recalcitrance should not be ruled out. For example, several pretreat-
ment processes are developed under acidic conditions resulting in hemicellulose
removal. In most cases, the pretreated residue with reduced hemicellulose content is
190 A. Ferraz et al.

less recalcitrant to the enzymatic digestion by commercial cellulases (see previous

chapters in this book). Therefore, the hemicellulose encapsulating the cellulose
microfibrils can also help to explain part of the recalcitrance of the cell walls of
lignified biomass.
Hemicelluloses are synthesized from varied monosaccharides including
D-glucose, D-galactose, D-mannose, D-xylose, L-arabinose, D-glucuronic acid, and
4-O-methyl-D-glucuronic acid. Most of the hemicelluloses are branched and
present varied levels of acetyl groups substituting free hydroxyls in the monomers
of the polysaccharide backbone (Fengel and Wegener 1989; Teleman 2009). The
hemicelluloses are found in both, primary and secondary cell walls, as well as in the
middle lamella at low concentrations. In primary cell walls, pectin is also of great
importance because it is a key component of immature cells and parenchyma cell
walls. Xyloglucan, arabinoxylan, and pectic polysaccharides are the major
polysaccharides present in plant primary cell walls besides cellulose. In grass
monocotyledons, the primary cell walls may also present high levels of hydroxy-
cinnamates and structural proteins (Carpita 1996; Vogel 2008). Xyloglucan is in
close association with cellulose microfibrils and may be the main barrier to
cellulose access in primary cell walls (Hayashi and Kaida 2011). In secondary cell
walls, the hemicelluloses generally represent 25–35 % of the dry matter and are
composed mainly by galactoglucomannans and arabinoglucuronoxylans in gym-
nosperms. 4-O-methyl-glucuronoxylans and glucomannans are the most frequent
hemicelluloses in the secondary cell walls of angiosperms. In grass monocotyle-
dons, 4-O-methyl-glucuronoarabinoxylan is the main hemicellulose in the sec-
ondary cell walls (Teleman 2009; Masarin et al. 2011).
Once the hemicelluloses may cooperate with recalcitrance of lignocellulosic
materials, and its contents vary in different tissues and cell types, the evaluation of
the topochemical distribution of this component becomes relevant in the biomass
conversion studies. A singular past work on mapping hemicelluloses in the trac-
heids of gymnosperms was presented by Hoffmann and Parameswaran (1976). The
authors carefully delignified spruce tissues with sodium chlorite and then oxidized
the reducing ends of the hemicelluloses. The carboxyl end groups were then
contrasted with colloidal iron, silver, or lead. Imaging ultrathin cuts of these
samples by electron transmission microscopy revealed that the highest concen-
tration of hemicelluloses was found in the S1 layers with the concentration
decreasing toward the secondary wall zones composed by the S2. The authors also
assigned the dense contrast observed in the cell corners and the compound middle
lamella (middle lamella plus the primary cell walls) to the presence of hemicel-
luloses and pectin, which naturally present acid groups.
One of the recent strategies used to map the hemicelluloses into the cell walls
involves the immunolabeling of the polysaccharide chains of interest with
monoclonal antibodies. The detection is performed by adding a secondary anti-
body labeled with gold particles or fluorescent compounds followed by the anal-
yses of the cell walls under electron or fluorescent microscopy, respectively (Kim
and Daniel 2012a, b; Petersen et al. 2012). Figure 9.11 illustrates fiber and vessel
cell walls of mature xylen from aspen wood based on the fluorescence detection of
9 Mapping of Cell Wall Components in Lignified Biomass 191

Fig. 9.11 Example o immunofluorescence localization of substitute xylans in fiber F and vessel
V cell walls of mature xylen from apen wood. LM11 denotes the antibody used to bind to
substituted xylan structures. The bar size corresponds to 10 lm. Note that the cell corners and
middle lamella were poorly labeled whereas secondary walls were strongly labeled with the LM
11 antibody indicating the regions where substitute xylan predominates in the mature xylem
(Reproduced from Kim et al. 2012)

antibodies selected to bind to substituted xylan structures (Kim et al. 2012). The
differentiation between low- and highly substituted xylans is also possible by using
two different specific antibodies (McCartney et al. 2005). Using these techniques,
Kim et al. (2010) mapped the xylan distribution in the gymnosperm Cryptomeria
japonica. During the tracheid maturation, the xylan deposition started in the corner
of the S1 layers of tracheids. Using two different antibodies (LM10 that binds to
low-substituted xylans and LM11 that binds highly substituted xylans) the authors
suggested that structurally different types of xylans may be deposited in the
tracheid cell layers according to the development stage.
The topochemical distribution of xylan was also assessed in an angiosperm
hybrid (Populus tremula L. and P. tremuloides Michx) (Kim et al. 2012). Xylan
immunolocalization in differentiating xylem cells indicated that the xylan depo-
sition begins in the fibers (at the cell corner of the S1 layer as in the tracheids of
gymnosperms), followed by vessels and ray cells. Xylan was not immunodetected
in the cambial and radial zones because these tissues present cells with mostly
primary cell walls and then low xylan content. However, in mature xylem, xylan
was strongly detected in all cell types and layers, including the middle lamella.
In bamboo, an important fast grow grass monocotyledon, the immunofluores-
cence technique has revealed that the xylan deposition in the cell walls increased
along maturation of a growing plant. The authors confirmed that in mature tissues,
there was a higher xylan content in the vascular bundles, especially in thickened
secondary cell walls of the fibers (Chang et al. 2013). In sugarcane, the immu-
nolocalization of xylan has not been attempted up-to-date.
Another method to map hemicellulose in lignocellulosic materials involve
synthetic or natural special peptides called ‘‘carbohydrate binding modules’’ that
192 A. Ferraz et al.

may bind to specific carbohydrates of plant tissues, such as xylan. These binding
modules may be labeled with fluorescent compounds or immunotargeted with
modified antibodies in order to detect the signals by microscopy. Through this
technique xylan chains were detected in pulp fibers, wood sections, and tobacco
(Hervé et al. 2009; Filonova et al. 2007).
As reported for lignin, the heterogeneous distribution of hemicelluloses also has
been associated with the varied recalcitrance of different tissues of lignocellulosic
materials. For example, xyloglucan topochemistry indicated that this polysac-
charide is in close association with cellulose microfibrils and may be the main
barrier to cellulose access in primary cell walls (Hayashi and Kaida 2011).
Nonetheless, xyloglucan immunofluorescence is generally associated with that of
pectin and xylan and it is difficult to differentiate these polysaccharides in the thin
primary cell walls.
The spatial distribution of hemicelluloses in different internode regions and
tissues of some grasses such as sugarcane and maize may also differ significantly.
Such fact permits to evaluate how the hemicelluloses may affect recalcitrance in
each case. For example, sugarcane and maize present different hemicelluloses
contents (mostly assigned to 4-O-methylglucuronoarabinoxylans with some cross-
link with hydroxycinnamates bridges) in different anatomical regions of their
internodes. Recent studies showed that the total hemicellulose content in these
grasses increases from pith toward rind (Costa et al. 2013; Bairros-Rios et al. 2012;
Zeng et al. 2012; Siqueira et al. 2011). The same studies showed that untreated
rind tissues are very recalcitrant whereas the pith region can be easily hydrolyzed
by commercial cellulases. These reports suggest that the different hemicellulose
contents in the each region are in some way correlated with the recalcitrance of the
material. In fact, the study of Costa et al. (2013) indicated that the sum of
hemicellulose and lignin (the cellulose embedding components) in each sugarcane
region was a key factor to explain the varied recalcitrance of the different tissues.

9.4 Pretreatments Affecting the Cell Wall Components

Distribution and the Effects on Enzymatic Hydrolysis
of Polysaccharides

As discussed in the previous sessions, lignin and/or hemicellulose removal from

the cell walls can improve the access of the cellulolytic enzymes to the cellulose
chains. In fact, the partial removal of these components is the basis for the pre-
treatment processes. Mechanical disruption of the cell wall ultrastructure,
including cell wall rupture, can also enhance the enzyme access to the cellulose
chains; however, pure mechanical pretreatments are extremely energy consuming
and will not be considered in this chapter.
The technological basis of several biomass pretreatments were considered in
other chapters of this book. Briefly, the pretreatments are necessary to increase the
porosity of the cell wall, through the removal of physical barriers caused by lignin
9 Mapping of Cell Wall Components in Lignified Biomass 193

and/or hemicellulose, and allow the enzymatic hydrolysis of lignocellulosics. The

focus in this chapter is on the mapping of the lignin and/or hemicellulose removal
or redistribution in some technical pretreatments with the aim to show how the cell
wall become more accessible to the enzymes after the pretreatment.
To remove part of the lignin in technological processes various chemicals such
as alkaline salts, sulfites, ammonia, and organic solvents have been exploited
(Mosier et al. 2005; Hendriks and Zeeman 2009). The alkaline pretreatments also
affect the cellulose degradation by enzymes because, in some reaction conditions,
cellulose I arrangement is transformed in cellulose II after neutralization (Hall
et al. 2011; Hendriks and Zeeman 2009).
An example of lignin removal from spruce wood tracheids during the alkaline/
sulfite treatment can be observed in Fig. 9.12 (Koch et al. 2003). In alkaline
reaction media, the lignin is progressively removed from the lumen toward the
middle lamella. Therefore, lignin is first removed from the secondary walls and
only at advanced delignification stages it is removed from the middle lamella
(Jayme and Torgersen 1967; Procter et al.1967; Goring 1981; Koch et al. 2003).
This is a relevant observation when delignification pretreatments are involved
because only the internal surfaces of the cell walls become accessible to the
enzymes owing to the increased porosity. The external surfaces of the cell walls
continue capped by a middle lamella layer and are not accessible to the enzymes.
A simple demonstration of this phenomenon was showed during the evaluation of
the alkaline and alkaline/sulfite pretreatment of sugarcane bagasse (Mendes et al.
2011). Fibers released from the vascular bundles after the pretreatment were
observed by light microscopy before and after 96 h of enzymatic digestion by
commercial cellulases (Fig. 9.13). The action of the enzymes on the internal
surfaces of the fibers can be observed as cavities caused in the secondary cell
walls. In the untreated fibers only small cavities are observed and the cellulose
conversion to glucose was limited to 20 % (Fig. 9.13a and d). The alkaline pre-
treated material becomes less recalcitrant and the cavities were more dispersed
along the internal surfaces of the fibers (Fig. 9.13b, e). In this case, the cellulose
conversion reached 50 %. Most of the alkaline/sulfite pretreated material was
susceptible to the enzymatic hydrolysis, since 85 % of cellulose conversion was
obtained. The few resistant fibers, after 96 h of enzymatic digestion, still presented
the cavities in the cell walls starting from the internal surfaces of the fibers
(Fig. 9.13c, f). In all cases, a middle lamella layer outside of the nondigested fibers
can be visualized as a dark contrasting layer that have restrained the permeation of
the enzymes from the external surface of the fibers.
Besides lignin removal, the residual lignin in the alkaline/sulfite pretreated
materials is modified by the incorporation of sulfonic groups (Gellerstedt 2009).
The sulfonic acid groups turn the residual lignin less hydrophobic allowing an
increased capacity of water retention by the fibers (Mendes et al. 2013). This
characteristic is critical because the swollen fibers become more porous, facili-
tating the enzyme permeation toward the secondary walls of the pretreated
material. The cellulotytic enzymes apparently do not adsorb irreversibly to the
lignin in this type of pretreated material, which diminishes the enzyme load
194 A. Ferraz et al.

Fig. 9.12 UV micrographs of late wood tracheids from spruce wood treated under alkaline/
sulfite processes after incipient delignification, 30 min (a), and advanced delignification, 120 min
(b). The absorption intensities at 278 nm (shown in the left of the image) are translated into
multiple colors to illustrate the lignin distribution in the biomass tissues. Pink, light blue, and
green, as well as yellow and black correspond to mid to strong UV absorptions indicating high
lignin concentrations. Blue to brown colors indicate low UV absorption intensities and low lignin
concentrations. The images clearly indicate that the cell corners and the middle lamella remain
present even after a long treatment time when the lignin originally present in the secondary walls
were significantly removed (Reproduced with modifications from Koch et al. 2003)

required for efficient hydrolysis as well as turn the enzymes recycling more fea-
sible (Zhu et al. 2009; Liu and Zhu 2010). These combined effects have been
claimed to bring low enzyme consumption and costs in this type of process.
9 Mapping of Cell Wall Components in Lignified Biomass 195

Fig. 9.13 Light micrographs

of selected fibers from
sugarcane bagasse treated
under alkaline and alkaline/
sulfite processes, before
(a–c) and after enzymatic
digestion with commercial
cellulases (d–f). a and
d Untreated fibers; b and
e Fibers pretreated with the
alkaline process; c and
f Fibers pretreated with the
alkaline/sulfite process.
(Micrographs provided by the
authors, previously published
in Mendes et al. 2011)

Some pretreatments performed under acidic conditions, such as steam explo-

sion and hydrothermal processing, cause partial removal of hemicelluloses (Ramos
2003). In these treatments, acetyl groups linked to the hemicelluloses are cleaved
and, therefore, act as hydrolysis catalysts. The removal of the hemicelluloses
increases the porosity and the internal surface area of the pretreated material,
facilitating the accessibility of the enzymes for the subsequent cellulose hydrolysis
(Himmel et al. 2007). Lignin, on the other hand, is removed to a limited extent
from the material but it is rather redistributed on the internal cell surfaces owing to
the softening occurred at temperatures above 130 C. A former work presenting
this phenomenon was published by Michalowicz et al. (1991), which studied the
ultrastructural changes in poplar cell wall during steam explosion treatment by
transmission electron microscopy of ultrathin cross-sections of the xylem. The
authors described the ‘‘melting’’ of the lignin, which agglomerated as droplets
196 A. Ferraz et al.

Fig. 9.14 Scanning electron

micrographs (SEM) of corn
stover fiber surfaces treated
with 0.8 % sulfuric acid at
150 C for 20 min. a Control
sample and b and c treated
samples at different
magnifications. The bars
represents 2 lm (a and b),
and 0.5 lm (c). Note that the
lignin droplets diffuse from
the secondary cell walls
toward the internal surfaces
of the lumen (Reproduced
from Donohoe et al. 2008)

inside the secondary wall and diffused toward the lumen. Donohoe et al. (2008)
demonstrated a similar redistribution of lignin in the fibers of corn stover treated
under acidic conditions as illustrated in Fig. 9.14.
9 Mapping of Cell Wall Components in Lignified Biomass 197

Similar to that reported for lignin, part of hemicelluloses present in secondary

cell walls of lignocellulosic materials can also migrate during dilute acid pre-
treatment. This phenomenon was demonstrated using fluorescence microscopy and
scanning electron microscopy based on semiquantitative analysis of xylan anti-
body signals detected in the cell walls (Brunecky et al. 2009). An interesting
aspect of these techniques was that the pixel intensities were quantified along the
cell wall to identify the distribution and the removal of xylan occurring during the
dilute acid pretreatment. The authors observed that a decrease in the average signal
intensity detected in the inner cell walls of fibers from corn stover closely matched
the progressive loss of xylan determined by chemical analysis.

9.5 Concluding Remarks

The complex cell ultrastructure and the composite characteristic of the cell walls
are among the several factors explaining the recalcitrance of lignified plants.
Understanding the origins of this recalcitrance is fundamental for the development
of the future industry involved with the biomass conversion to monomeric sugars.
Mapping the macromolecular components in the cell walls has proved to be useful
to understand the varied recalcitrance of different biomass tissues, as well as how
the removal of individual components can affect the final digestibility of the
pretreated material. Data available to date indicate that parenchyma cells of
monocotyledons are significantly less recalcitrant than fibers and vessels. However,
even in parenchyma rich materials such as sugarcane bagasse, this cell type
represents a minor fraction of the dry biomass. Consequently, pretreatments are
necessary to remove some of the cellulose embedding components (lignin and
hemicellulose) in order to enhance the cell wall digestibility. The information
revised in this chapter indicates that lignin removal from the cell walls significantly
enhance the digestibility of the material by commercial enzymes. Hemicellulose
removal can also help on some extent and the general trend is that the diminished
recalcitrance in pretreated materials or in different tissues of the biomass is obtained
when the cellulose become more accessible to the enzymes. In this subject, some
recent data support that the available cellulose can be estimated in the biomass
materials as the content of cellulose divided by the sum of the embedding com-
ponents, hemicellulose, and lignin.

Acknowledgments The authors received financial support for research on the subject of this
chapter from Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP (contract number
08/56256-5 and 11/50535-2) and from Conselho Nacional de Desenvolvimento Científico
e Tecnológico—CNPq.
198 A. Ferraz et al.

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Chapter 10
Dilute Acid Pretreatment and Enzymatic
Hydrolysis of Sugarcane Bagasse
for Ethanol Production

Paula J. Esteves, Celso Santi Jr. and Walter Carvalho

Abstract Many efforts have been dedicated to understanding and refining

different technologies to promote the conversion of the sugars contained in the
sugarcane bagasse into ethanol. One of the promising strategies include the pre-
treatment of the bagasse with dilute sulfuric acid followed by the saccharification
of the remaining polysaccharides with enzymes, and by the fermentation of the
generated monosaccharides (both hexoses and pentoses) with yeasts. In the present
chapter, data regarding the characterization and conditioning of the raw material as
well as its pretreatment, saccharification, and fermentation are disclosed to illus-
trate that the sugarcane bagasse, like many other agroindustrial residues, consists
of a heterogeneous material and that its different constituent sugars, not necessarily
only plant cell wall polysaccharides, may need to be recovered under different
experimental conditions if high conversion yields are to be achieved using the
proposed technology.

10.1 Introduction

10.1.1 Sugarcane and Sugarcane Bagasse

Brazil is one of the large producers of sugarcane in the world. Sugarcane repre-
sents one of the most important agroindustrial cultures in the country, with
plantations concentrated in the center-south and northeast regions; mainly in the
state of São Paulo (Conab 2013).

P. J. Esteves  C. Santi Jr.

Department of Biotechnology, University of São Paulo, Engineering College
of Lorena, São Paulo, Brazil
W. Carvalho (&)
Escola de Engenharia de Lorena, Estrada Municipal do Campinho s/n8,
Campinho Lorena, SP, CEP 12602-810, Brazil
e-mail: carvalho@debiq.eel.usp.br

S. S. da Silva and A. K. Chandel (eds.), Biofuels in Brazil, 203

DOI: 10.1007/978-3-319-05020-1_10,  Springer International Publishing Switzerland 2014
204 P. J. Esteves et al.

Each metric ton of sugarcane processed in the mills for extraction of the juice,
used in the production of sugar and/or ethanol, generates about 280 kg of moist
bagasse; a lignocellulosic material, usually burned for power generation (Cenbio
2013).
The sugarcane bagasse, as any other lignocellulosic material, is composed of
three major constituents: cellulose, hemicellulose, and lignin. Cellulose, a poly-
saccharide formed exclusively by glucose units, represents the main component. In
the plant cell wall, the elementary fibrils of cellulose are formed by the association
of linear cellulose chains that are maintained by intra and intermolecular hydrogen
bonds; which explains, at least in part, its resistance to microbial degradation
(Bragatto et al. 2012). Hemicellulose is a general name given to a family of
heteropolysaccharides (Ebringerová 2006), whose structures may include hexoses
(D-mannose, D-glucose, and D-galactose) and pentoses (D-xylose and L-arabinose)
as well as small amounts of deoxihexoses (L-fucose and L-rhamnose) and uronic
acids (glucuronic acid, galacturonic acid, and methylglucuronic acid). Properties
such as low degree of polymerization and amorphous structure make the hemi-
celluloses less stable to biological degradation than cellulose (Ek et al. 2009).
Lignin, on the other hand, is a non-carbohydrate macromolecule that confers
stability to the plant cell wall due to the formation of a composite, together with
the polysaccharides (cellulose and hemicellulose), highly resistant to microbial
degradation. Its structure derives from three primary precursors (coniferyl, sinapyl,
and coumaryl alcohols), but the biological synthesis generates complex structures
with different types of linkages (mainly b-O-4, a-O-4, b-1, b-5, 5-5, and b-b)
oriented towards all the spatial directions (Fengel and Wegener 1989).
In addition to the major constituents, lignocellulosic materials exhibit varying
amounts of other substances, sometimes referred to as minor constituents. Such
constituents include organic and inorganic compounds, and are divided into two
classes: the first comprise materials known as extractives, being extractable with
water or neutral organic solvents, or volatilized in the presence of steam; the
second refer to non-extractable materials, including certain inorganic salts and
proteins (Browning 1967). The qualitative and quantitative compositions of these
so-called minor constituents, however, depend on several factors, including the
origin and the age of the material (Kai 1991).

10.1.2 Production of Ethanol from Sugarcane Bagasse

The structural sugars contained in the sugarcane bagasse, including cellulose and
hemicellulose, represent potential substrates that could be used for increasing the
ethanol production by the sugarcane processing mills (Carvalho et al. 2007). The
challenge consists of quantitatively recovering the constituent sugars, ideally in
their monomeric forms, and in efficiently converting them into ethanol; in an
economical way (Wyman 1994).
10 Dilute Acid Pretreatment 205

In order to overcome the bottlenecks associated with the conversion of the

sugars contained in the sugarcane bagasse into ethanol, many efforts have been
dedicated to the understanding and refinement of different technologies able to
achieve the above mentioned targets. One of the promising strategies include the
pretreatment of the bagasse with dilute sulfuric acid, followed by the saccharifi-
cation of the remaining polysaccharides with enzymes and by the fermentation of
the generated monosaccharides with yeasts (Canilha et al. 2009).

10.1.2.1 Pretreatment with Dilute Sulfuric Acid

Pretreatment of lignocellulosic materials with dilute sulfuric acid is one of the

most effective methods for solubilizing hemicelluloses and generating solids
highly reactive to enzymatic saccharification (Canilha et al. 2011).
Depending of the pretreatment severity, the solubilized sugars can undergo
degradation reactions and give rise to compounds like furfural and hydroxymeth-
ylfurfural (HMF) which inhibit the bioconversion (Palmqvist and Hahn-Hagerdal
2000; Carvalho et al. 2004a). Similar behavior is observed for the small fraction of
lignin soluble in acid medium, which may reveal itself as composed by strong
inhibitors of both the enzyme saccharification of polysaccharides (Ximenes et al.
2010) and the yeast fermentation of monosaccharides (Carvalho et al. 2004b); not to
mention the inhibitory effect of acetic acid, released from certain hemicelluloses
(Han et al. 2006).
As the pretreatment conditions that maximize the recovery of xylose in the
hemicellulosic hydrolysate are generally different from the pretreatment conditions
that maximize the recovery of glucose during the enzymatic hydrolysis of the
pretreated solids, a two-step pretreatment can be employed (Nguyen et al. 2000;
Söderstrom et al. 2003). The approach consists of hydrolyzing the hemicellulose
under milder conditions and, then, in conditioning the resulting pretreated solids
under more drastic conditions.

10.1.2.2 Saccharification with Enzymes

The enzymatic hydrolysis of pretreated lignocellulosic materials is specific and

carried out under mild conditions of pH and temperature, which avoids the gen-
eration of inhibitors and leads to high sugar yields (Taherzadeh and Karimi 2007).
Cellulose is hydrolyzed into glucose by the coordinated action of endoglu-
canases, exoglucanases, and b-glucosidases (Alvira et al. 2010). Depending on the
pretreatment conditions, residual hemicellulose may remain in the pretreated
solids and hinder the action of cellulases; in these cases, the use of hemicellulases,
in association with cellulases, improves the efficiency of saccharification (Öhgren
et al. 2007a).
Besides compounds such as furans, phenolics, and organic acids, originated
or released during the pretreatment, the sugars released during the enzymatic
206 P. J. Esteves et al.

saccharification of the pretreated solids can themselves act as inhibitors of the

enzymes: glucose inhibits the action of b-glucosidases; cellobiose, the activity of
exo and endoglucanases (Andric et al. 2010). Such inhibition, however, can be
overcome by integrating the saccharification and fermentation operations in a
process configuration like the simultaneous saccharification and fermentation
(SSF), in which the glucose released during the enzymatic saccharification of the
cellulose is simultaneously converted into ethanol by an appropriate microor-
ganism; which prevents the inhibition of the enzymes (Öhgren et al. 2007b).

10.1.2.3 Fermentation with Yeasts

The sugars generated in the pretreatment of the lignocellulosic material with dilute
sulfuric acid and/or in the saccharification of the pretreated solids with enzymes
must be quantitatively converted into ethanol (Kuhad et al. 2011); for process
economy and environmental sustainability.
Although Saccharomyces cerevisiae, the yeast long used by humankind for
ethanol production in hexoses-based media, can be genetically engineered to
produce ethanol from pentoses like xylose and arabinose (Bettiga et al. 2009),
there are also a number of yeast species able to naturally produce ethanol from
such substrates; among them: Pachysolen tannophilus, Candida shehatae, and
Scheffersomyces (formerly Pichia) stipitis (Jeffries and Kurtzman 1994).
Among the yeasts that naturally produce ethanol from pentoses, S. stipitis
stands out, due to its high substrate-to-product conversion efficiency and versatility
towards the consumption of different substrates; even though it may present
stringent oxygen requirements for the production of ethanol from pentoses, and in
spite of its susceptibility towards inhibiting compounds (Agbogbo and Coward-
Kelly 2008).

10.2 Case Study

In the following, a case study that reports results of an ongoing project that deals
with the conversion of the sugars contained in the sugarcane bagasse into ethanol
by pretreatment with dilute sulfuric acid followed by enzyme saccharification and
yeast fermentation is presented.
Data regarding the characterization and conditioning of the raw material as well
as its pretreatment, saccharification, and fermentation are disclosed to demonstrate
that the sugarcane bagasse is a heterogeneous material and that its different con-
stituent sugars may need to be recovered under different experimental conditions if
high conversion yields are to be achieved during the conversion as a whole.
10 Dilute Acid Pretreatment 207

10.2.1 Materials and Methods

Figure 10.1 presents a schematic representation of the ongoing project; the main
results of which, achieved up to now, are hereafter presented.

Sugarcane bagasse

Dilute acid
pretreatment

Hemicellulosic Pre-treated
hydrolysate sugarcane bagasse
Slurry fractionation

Fermentation
Ethanol

Enzyme
saccharification
Fermentation

Cellulosichydrolysate

Fig. 10.1 Schematic representation of the study

10.2.1.1 Raw Material, Characterization, and Conditioning

Two samples of sugarcane bagasse, acquired from different mills located in São
Paulo state, hereafter nominated as sugarcane bagasses A and B, were used in the
study. Bagasse A was milled in a hammer mill and the fines were removed, with
the help of compressed air, by using a device consisting of a plastic box in which
the bottom was replaced by two intertwined 20 mesh sieves (Fig. 10.2). For the
fractionation, 150 g of sample were transferred into the box; next, through a lateral
opening, compressed air was injected during 40 s. The fractions of bagasse
retained (bagasse without fines) and not retained (fines) by the device were
characterized regarding the distributions of particle sizes and chemical composi-
tions. In addition, samples of the raw bagasses A and B were knife-milled to pass
through a 20 mesh sieve. The milled bagasses were packed into cellulose thimbles
(4.5 g) and submitted to solid–liquid extraction in a Soxhlet extraction system.
208 P. J. Esteves et al.

Fig. 10.2 Photos of the device used to fractionate the sugarcane bagasse with the help of
compressed air: a (top view); b (bottom view); c (front view, with the entrance for injection of
compressed air)

Water, ethanol or water followed by ethanol were used as extracting solvents.

After adding 800 mL of the solvent into the receiving flask, the Soxhlet apparatus
was assembled and the heating mantle was turned on. Two extractions in each
solvent were performed, each one lasting 12 h. At the end of the extractions, the
solids were quantitatively transferred into weighing bottles which were then oven-
dried at 105 C until constant weight. The chemical compositions of both
extracted and non-extracted samples were determined in the sequence.
The distributions of particle sizes were analyzed by determining the mass
fractions selectively retained on a set of standard sieves with openings of 3.35,
1.70, 0.85, 0.42, and 0.21 mm, respectively. For each analysis, 20 g of sample was
transferred into a magnetic stirrer in which the sieves were stacked in order of
decreasing apertures; afterwards, the system was set to vibrate for 1 h. The
chemical composition of solid materials were determined according to the tradi-
tional two-step acid hydrolysis procedure of Klason, employing one of two
methods (Sluiter et al. 2010; Gouveia et al. 2009) previously shown to lead to
similar results (Canilha et al. 2011). Sugars were quantified by HPLC using a
refraction index detector and a Biorad Aminex HPX-87H column at 45 C. Sul-
furic acid 0.01 N at a flow rate of 0.6 mL/min was used as eluent, and the injection
volume was 20 lL. Furfural and hydroxymethylfurfural concentrations were also
10 Dilute Acid Pretreatment 209

determined by HPLC, using a UV–VIS detector at 280 nm and a Hewlett-Packard

RP18 column at 25 C. Acetonitrile: water (1:8) supplemented with 1 % acetic
acid was used as eluent at flow rate of 0.8 mL/min. The injection volume was
20 lL. The profiles of UV light absorption of the aqueous and alcoholic extracts
were determined in an UV–VIS spectrophotometer, after appropriate dilution. The
results were expressed in terms of relative absorbances, which were calculated by
multiplying the absorbance values at the different wavelengths by the dilution
factors used in each determination (Carvalho et al. 2008). The content of sugars in
the extracts were determined by the phenol–sulfuric acid method (Dubois et al.
1956), using glucose as standard.

10.2.1.2 Pretreatment of the Sugarcane Bagasse with Dilute Sulfuric

Acid

Sugarcane bagasse A, as received from the mill, was used in the pretreatments,
which were performed in a pilot reactor (total capacity of 100 L) heated by direct
steam. In each experiment, an initial mass of 20 kg, including bagasse (15 % dry
mass basis), sulfuric acid, and water was used. The temperature, the acid con-
centration, and the time of pretreatment at the target temperature (heating and
cooling were fast and, consequently, not considered) were varied according to the
experimental design shown in Table 10.1. After the pretreatments, the slurries
were separated into liquid (hemicellulosic hydrolysates) and solid (pretreated
solids; pretreated sugarcane bagasse in Fig. 10.1) fractions; the pretreated solids
were exhaustively washed with hot water and dried at room temperature, while the
hemicellulosic hydrolysates were maintained frozen. The chemical compositions
of the pretreated solids and of the hemicellulosic hydrolysates were determined as
described previously.

10.2.1.3 Enzymatic Saccharification of the Pretreated Solids

The 20 pretreated solids obtained under the different pretreatment conditions were
subjected to enzymatic hydrolysis using a mixture of enzymes characterized
previously (Santos et al. 2011). The assays were performed in 125 mL Erlenmeyer
flasks containing 12.5 mL of sodium citrate buffer (100 mM, pH 4.8) supple-
mented with sodium azide (0.02 % w/v), 10 % of solids, and 0.025 g of Tween 20
and 10 FPU of cellulases per gram of bagasse; the final volume of each assay was
25 mL, completed with distilled water. The experiments were performed at 45 C
in a rotatory shaker at 150 rpm. Samples were withdrawn in 24 h intervals, boiled
for 5 min and centrifuged for 30 min at 12,000 g. The supernatants were analyzed
by HPLC.
210 P. J. Esteves et al.

Table 10.1 Real and coded values of the independent variables according to the 23 central
composite full factorial design with 6 central points
Exp Temperature Acid concentration Time Temperature Acid Time
(C) (% w/w) (min) (coded) concentration (coded)
(coded)
1 140 1 20 -1 -1 -1
2 160 1 20 1 -1 -1
3 140 3 20 -1 1 -1
4 160 3 20 1 1 -1
5 140 1 40 -1 -1 1
6 160 1 40 1 -1 1
7 140 3 40 -1 1 1
8 160 3 40 1 1 1
9 150 2 30 0 0 0
10 150 2 30 0 0 0
11 150 2 30 0 0 0
12 131.91 2 30 -1.81 0 0
13 168.09 2 30 1.81 0 0
14 150 0.19 30 0 -1.81 0
15 150 3.81 30 0 1.81 0
16 150 2 11.90 0 0 -1.81
17 150 2 48.09 0 0 1.81
18 150 2 30 0 0 0
19 150 2 30 0 0 0
20 150 2 30 0 0 0

10.2.1.4 Fermentation of the Hemicellulosic Hydrolysates

Scheffersomyces stipitis DSM 3651, previously used by Canilha et al. (2010), was
employed in the fermentation assays. The inoculum was grown by transferring
cells from a malt extract agar slant into a 500-mL Erlenmeyer flask containing
200 mL of synthetic medium consisting of xylose (30 g/L), yeast extract (3 g/L),
malt extract (3 g/L), and peptone (5 g/L). The flasks were incubated in a rotatory
shaker at 30 C and 200 rpm for 24 h, and the cells were collected by a 30 min
centrifugation at 2,000 g; followed by suspension in sterile distilled water.
The 20 hemicellulosic hydrolysates obtained under the different pretreatment
conditions had their pHs adjusted to 6 with NaOH and, after removal of the
precipitates, were sterilized by autoclaving at 111 C for 15 min.
The fermentations were carried out in 125-mL Erlenmeyer flasks containing
50 mL of medium and inoculated with 3 g/L cells (dry weight). The fermentation
media were composed by the autoclaved hydrolysates supplemented with yeast
extract (3 g/L), malt extract (3 g/L), and peptone (5 g/L). The flasks were main-
tained in a rotatory shaker at 30 C and 200 rpm for 120 h. Samples were peri-
odically collected to determine the concentrations of sugars and ethanol, by HPLC.
10 Dilute Acid Pretreatment 211

10.2.1.5 Statistical Analysis of the Results

In order to evaluate the effects of the pretreatment conditions on the composition

and enzymatic saccharification of the pretreated solids, as well as on the com-
position and fermentation of the hemicellulosic hydrolysates, empirical models
(Eq. 10.1) were adjusted to the experimental data generated according to the
experimental design.
X
n X
n n1 X
X n
Yi ¼ b 0 þ bi x i þ bii x2i þ bij xi xj ð10:1Þ
i¼1 i¼1 i¼1 j¼iþ1

where Yi represents the dependent variable, b0, bi, bii and bij represent the
regression coefficients, and xi and xj represent the independent variables. The
significance of the regression coefficients kept in the models was evaluated con-
sidering, as statistically significant coefficients, those that, in general, exceeded the
confidence level of 95 %.

10.2.2 Results and Discussion

10.2.2.1 Raw Material, Characterization, and Conditioning

Figure 10.3 presents the particle size distributions determined for bagasses A and B.
The profiles observed for the two bagasses showed that they consist of heter-
ogeneous materials that present polydisperse distributions of particle sizes. Con-
sidering this heterogeneity, reduction, and/or fractionation of the raw material
before conversion is a strategy described in the literature (Gámez et al. 2006;
Hernández-Salas et al. 2009; Pietrobon et al. 2011). Small particle sizes have
increased surface areas (Driemeier et al. 2011); moreover, the diffusion of
chemical reagents such as dilute H2SO4 has been shown to be optimized for
smaller particles (Kim and Lee 2002).
Table 10.2 presents the chemical compositions determined for bagasses A and
B, before and after the extractions with water followed by ethanol.
Considerable differences were observed in the compositions of the two mate-
rials, with bagasse A being poorer in cellulose and richer in ash. Moreover, the
extraction with solvents removed a considerable portion of ‘‘pseudo-lignin’’, due
to the fact that some extractives can condense and precipitate during the compo-
sitional analysis (Hatfield and Fukushima 2005). A similar behavior was already
observed for other lignocellulosic materials (Grohmann et al. 1986; Nguyen et al.
2000); and, to remove such interfering compounds, the National Renewable
Energy Laboratory (NREL/USA) recommends successive extractions with water
and ethanol (Sluiter et al. 2008).
Further analysis of the effects promoted by extraction of the raw material with
solvents, performed with bagasse B, showed that the water extraction solubilized
212 P. J. Esteves et al.

Fig. 10.3 Distributions of 30.0

Bagasse mass (% w/w)

particle sizes for bagasses A
25.0
and B, determined by sieving
20.0

15.0
Bagasse A
10.0 Bagasse B

5.0

0. 0
3.35 1.70 0.85 0.42 0.21 0
Sieve opening (mm)

Table 10.2 Chemical compositions determined for bagasses A and B, before and after the
extractions with water followed by ethanol
Component (% w/w) Before extraction After extraction
Bagasse A Bagasse B Bagasse A Bagasse B
Cellulose 38.8 ± 0.1 46.4 ± 0.3 38.3 ± 0.3 45.0 ± 0.1
Hemicellulose 26.6 ± 0.0 27.2 ± 0.2 27.8 ± 0.0 25.8 ± 0.1
Lignin 27.9 ± 0.1 24.8 ± 1.0 22.5 ± 2.6 19.1 ± 0.2
Ash 6.7 ± 0.0 1.6 ± 0.2 5.3 ± 0.2 1.0 ± 0.0
Extractives 6.1 9.1

6.0 % of solids, while ethanol extraction led to a similar content of extractives

(5.7 %). The sequential extraction with both solvents, however, reduced the dry
weight of the raw material in 9.1 %, thus showing that the solvents dissolved, at
least in part, structurally different compounds (Table 10.3).
Figure 10.4 shows that the aqueous extracts presented peaks of absorbance at
200 and 280 nm (A), while the alcoholic extracts exhibited peaks at 220 and
315 nm, with a shoulder at 290 nm (B). Although, for the same solvent, 2nd
extractions removed much lower amounts of soluble compounds than 1st extrac-
tions (A and B), previous extractions of the raw bagasse with water did not seem to
have affected the qualitative and quantitative profiles of light absorption by con-
secutive ethanolic extracts (C).
Considering that peaks of maximum absorbance (kMAX) near 280 nm are typ-
ical of lignin-derived aromatics and that phenolic acids, originally ester-linked to
carbohydrates, show a bathochromic shift from 280 nm to a shoulder near 290 nm
with kMÁX near 320 nm (Akin 2007), it is supposed that the aqueous and alcoholic
extracts were rich in aromatics and phenolic acids, respectively.
As measured by the phenol–sulfuric acid method, the amount of sugars
recovered in the aqueous extracts (73.3 mg total sugars/g bagasse) was higher than
that recovered in the alcoholic extracts (49.4 mg total sugars/g bagasse), which can
be explained by the comparatively higher solubility of sugars in water (Alves et al.
2007). Moreover, the sequential extraction with both solvents increased the
recovery of sugars to 99.2 mg total sugars/g bagasse, an unusually high value
10 Dilute Acid Pretreatment 213

Table 10.3 Chemical compositions determined for bagasse B, before and after the extractions
with water, ethanol, and water followed by ethanol
Component (% w/w) Before extraction After extraction
Water Ethanol Water ? Ethanol
Cellulose 46.4 ± 0.3 44.6 ± 2.0 46.2 ± 0.4 45.0 ± 0.1
Hemicellulose 27.2 ± 0.2 26.2 ± 1.5 27.8 ± 0.3 25.8 ± 0.1
Lignin 24.8 ± 1.0 22.0 ± 0.6 19.3 ± 0.2 19.1 ± 0.2
Ash 1.6 ± 0.2 1.2 ± 0.1 1.0 ± 0.0 1.0 ± 0.0
Extractives 6.0 5.7 9.1

(a) 18 (b)
18
Relative absorbance (-)

Relative absorbance (-)

Water (1st) Ethanol (1st)
15 15
Water (2nd) Ethanol (2nd)
12 12
9 9
6 6

3 3

0 0
200 250 300 350 400 200 250 300 350 400
Wavelenght (nm) Wavelenght (nm)

(c) 18
Water (1st)
Relative absorbance (-)

15 Water (2nd)
Ethanol (3rd)
12 Ethanol (4th)

0
200 250 300 350 400
Wavelenght (nm)

Fig. 10.4 UV-light absorption spectra of the extracts prepared from sugarcane bagasse B.
Extracting solvents: a (water); b (ethanol); c (water followed by ethanol)

compared to the content of sucrose expected to be found in this industrial

byproduct, 0.2–5.0 % (Tewari and Malik 2007).
Figure 10.5 shows the distributions of particle sizes determined for bagasse A
before and after hammer-milling.
As can be seen, the strategy of milling was effective in reducing the proportion
of particles with larger sizes, thereby contributing to homogenization by commi-
nuting the material. More than grinding their sugarcane bagasse sample, Gámez
et al. (2006) selected particles smaller than 0.5 mm before the pretreatment with
dilute H3PO4; Hernández-Salas et al. (2009), on the other hand, selected the
fraction which passed through a 1.68 mm sieve but was retained by a 0.149 mm
sieve.
214 P. J. Esteves et al.

Fig. 10.5 Distributions of 40.0

particle sizes for bagasse

Bagasse mass (% w/w)

35.0
A before and after hammer-
30.0
milling, determined by
25.0
sieving
20.0
Raw bagasse
15.0
Milled bagasse
10.0
5.0
0.0
3.35 1.70 0.85 0.42 0.21 0
Sieve opening (mm)

As already mentioned, after milling, bagasse A was fractionated by using a

device designed to accomplish the removal of fines with the help of compressed
air. As illustrated in Fig. 10.6, the fraction of fines, in fact, exhibited smaller
particles in comparison to the milled bagasse. In spite of this, the fractionation was
not selective; the major part of the whole mass consisted in particles retained by
sieves with openings bigger than or equal to 0.21 mm.
Samples of both fractions were extracted with water followed by ethanol, and,
subsequently, submitted to compositional analysis. The relative contents of some
constituents are presented in Table 10.4.
Significant differences (p \ 0.05) were observed for the contents of water
extractives, xylan, insoluble lignin, acetyl, and ash. While the fines presented
higher contents of water extractives and ash, the bagasse without fines exhibited
higher contents of xylan, insoluble lignin, and acetyl groups. In turn, the total
content of sugars determined in the extract prepared from the fraction of fines was
higher than that determined in the extract prepared from the fraction without fines,
of 2.5 % (dry mass basis). Sanjuán et al. (2001) also demonstrated that, when the
sugarcane bagasse was extracted with hot water, the amount of extractives
depended on the nature of the sample in terms of constituent cell types. While
10.3 % of solids were solubilized from pith cells, the amount of compounds
extracted from vascular bundles did not exceed 1.1 %. When ethanol was used as
solvent, the contents of extractives were of 3.4 and 1.1 %, respectively.
Considering the aforementioned, it is important to point out that the production
of goods from lignocellulosic feedstocks by biochemical means can be strongly
influenced by the nature and content of both structural and nonstructural constit-
uents. For example: the extractives can be either toxic compounds (Venalainen
et al. 2004) or fermentable carbohydrates that can undergo degradation reactions
under high temperatures and low pHs (Bower et al. 2008); a high content of ash
imply in higher consumption of acid during the pretreatment (Linde et al. 2006); a
reduced content of lignin can make the structural carbohydrates more accessible to
10 Dilute Acid Pretreatment 215

(a)
40.0

Bagasse mass (% w/w)

35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0
3.35 1.70 0.85 0.42 0.21 0
Sieve opening (mm)

(b) (c)
45.0 50.0
Bagasse mass (% w/w)

Bagasse mass (% w/w)

40.0 45.0
35.0 40.0
30.0 35.0
30.0
25.0
25.0
20.0
20.0
15.0 15.0
10.0 10.0
5.0 5.0
0.0 0.0
3.35 1.70 0.85 0.42 0.21 0 3.35 1.70 0.85 0.42 0.21 0
Sieve opening (mm) Sieve opening (mm)

Fig. 10.6 Distributions of particle sizes for bagasse A before and after fractionation, determined
by sieving: a (milled bagasse); b (milled bagasse, fraction without fines); c (milled bagasse,
fraction of fines)

Table 10.4 Chemical compositions determined for the fractions of bagasse A, generated by
using the device shown in Fig. 10.2
Component (% w/w) Fraction without fines Fraction of fines
Extractives (water) – 12.2 ± 1.0
Extractives (ethanol) – 2.6 ± 1.6
Glucan – –
Xylan – 14.3 ± 0.1
Arabinan 1.6 ± 0.1 –
Soluble lignin 3.7 ± 0.1 –
Insoluble lignin – 11.8 ± 1.2
Acetyl 3.5 ± 0.3 –
Ash 1.7 ± 0.4 –

enzymatic hydrolysis (Yang et al. 2009; Siqueira et al., 2011); and so on.
Therefore, appropriate selection of the material that will be used as raw material
during the conversion may be advantageous.
216 P. J. Esteves et al.

10.2.2.2 Effects of the Pretreatment Conditions on the Composition

and Enzymatic Saccharification of the Pretreated Solids,
and on the Composition and Fermentation
of the Hemicellulosic Hydrolysates

The compositions of the different pretreated solids and the corresponding cellu-
lose-to-glucose conversions with enzymes, as well as the concentrations of xylose
and furfural in the respective hemicellulosic hydrolysates and the corresponding
sugars-to-ethanol conversions with yeasts, are presented in Table 10.5.
Regarding the compositions of the pretreated solids, the hemicellulose content
showed the greatest variation in function of the experimental conditions employed
during the pretreatment, with relative contents varying from 0.14 to 17.62 % (dry
mass basis). Cellulose and lignin were much less solubilized, and their relative
contents varied from 52.38 to 65.81 % and from 21.25 to 35.48 %, respectively.
The effects of the pretreatment conditions on the content of hemicellulose and
on the efficiency of cellulose saccharification after 24 h of hydrolysis were ana-
lyzed statistically (Table 10.6).
Acid concentration was the variable that affected the hemicellulose content the
most, followed by temperature and time. Hsu et al. (2010) also observed that
temperature and H2SO4 concentration were the major variables influencing the
hemicellulose content in rice straw pretreated with dilute H2SO4.
In turn, cellulose conversion into glucose was influenced mostly by tempera-
ture, followed by acid concentration. A similar behavior was observed by Cai et al.
(2012) when studying the pretreatment of corncobs with dilute H2SO4.
The empirical model proposed to explain the effects of the pretreatment vari-
ables on the hemicellulose content (coefficients shown in Table 10.6) was reduced
by setting the time of pretreatment at maximum, giving rise to Eq. 10.2. For
predicting the cellulose conversion after 24 h of hydrolysis as a function of the
pretreatment conditions, the values of the regression coefficients shown in
Table 10.6 were used directly (Eq. 10.3).

Hc ¼ 2:28  2;95 A  3;06 B þ 1;51 B2 ð10:2Þ

Cc ¼ 49:09 þ 5:97 A þ 4:08 B  3:23 B2 ð10:3Þ

where Hc is the hemicellulose content and CC is the efficiency of cellulose con-
version after 24 h of hydrolysis, both obtained as functions of the coded values of
temperature (A) and acid concentration (B).
The response surfaces corresponding to the above mentioned models are shown
in Fig. 10.7.
As can be seen, the increase in the acid concentration and in the temperature
used during the pretreatment with dilute H2SO4 led to pretreated solids with lower
hemicellulose contents and, thus, exhibited improved cellulose saccharification
efficiencies. In practice, the maximal efficiency of cellulose saccharification
(63.76 % after 24 h; 87.72 % after 72 h) was achieved in experiment 8 (pretreated
10 Dilute Acid Pretreatment 217

Table 10.5 Chemical compositions and yields of cellulose saccharification after 24 h of

hydrolysis (pretreated solids) as well as xylose and furfural concentrations and yields of sugars
conversion into ethanol (hemicellulosic hydrolysates) determined in the experiments of the sta-
tistical design (Table 10.1)
Std Pretreated solids Hemicellulosic hydrolysates
Cellulose Hemicellulose Lignin CC (%) Xylose Furfural YP/S
(% w/w) (% w/w) (% w/w) (g/L) (g/L) (g/g)
1 53.56 14.51 23.51 30.99 13.04 0.08 0.48
2 57.97 5.93 26.96 55.81 17.29 1.28 0.36
3 62.35 4.61 28.92 49.20 19.53 1.09 0.38
4 63.94 0.79 31.77 54.82 13.52 3.73 0
5 59.41 9.05 29.18 44.37 16.59 0.48 0.39
6 64.01 1.82 29.85 53.28 14.08 3.53 0
7 61.64 5.38 30.21 44.90 20.53 1.66 0
8 59.76 0.14 35.48 63.76 7.02 4.40 0
9 62.02 5.47 29.55 49.76 11.71 1.02 0.40
10 61.62 5.88 29.28 42.69 19.61 1.40 0.36
11 60.86 5.48 29.46 52.05 19.71 1.32 0.36
12 55.31 11.13 23.43 41.45 18.27 0.34 0.36
13 64.04 1.23 30.05 57.30 1.43 4.68 0
14 52.38 17.62 21.25 25.35 8.61 0.11 0.47
15 61.31 4.28 26.83 42.69 20.13 1.20 0.25
16 58.11 8.78 29.41 39.55 17.94 0.59 0.33
17 65.81 3.04 30.47 45.11 17.07 1.68 0
18 60.28 1.56 30.21 50.30 21.05 1.68 0.26
19 60.34 5.18 30.04 46.34 20.50 2.04 0.30
20 62.13 5.38 28.27 46.17 20.08 1.74 0.32

Table 10.6 Values of the regression coefficients maintained in the models proposed to predict
the content of hemicelulose and the efficiency of cellulose saccharification within 24 h of
hydrolysis (pretreated solids)
Variable Hemicellulose content Cellulose saccharification
Coefficient S. Error p Coefficient S. Error p
Constant 4.76 0.53 – 49.09 1.59 –
A -2.95 0.50 \0.0001 5.97 1.49 0.0010
B -3.06 0.50 \0.0001 4.08 1.49 0.0146
C -1.37 0.50 0.0153 – – –
B2 1.51 0.44 0.0035 -3.23 1.31 0.0252
Model \0.0001 0.0006
Lack of fit 0.3133 0.0894
R2 0.85 0.65
A Temperature; B Acid concentration; C Time
218 P. J. Esteves et al.

Fig. 10.7 Response surfaces showing the effects of acid concentration and temperature on the
content of hemicellulose (a) and on the efficiency of cellulose conversion into glucose after 24 h
hydrolysis (b); pretreated solids

solids exhibiting a hemicellulose content of only 0.14 % w/w), which confirms

that the enzymatic conversion of cellulose into glucose depends on the chemical
composition of the pretreated solids, which, in turn, depends on the conditions
employed during the pretreatment with dilute sulfuric acid.
Back into the data presented in Table 10.5, regarding the compositions of the
hemicellulosic hydrolysates, it can be seen that the concentration of xylose ranged
from 1.43 to 21.05 g/L; the concentration of furfural, from 0.08 to 4.68 g/L. In
turn, the yield of conversion of the major sugars (xylose ? glucose) into ethanol
varied from 0 to 0.48 g/g.
Table 10.7 shows the values of the regression coefficients of the models pro-
posed to predict the concentrations of xylose and furfural as functions of the levels
of the independent variables used during the pretreatment with dilute sulfuric acid.
Regarding carbohydrates, the concentration of xylose was heavily dependent of
the temperature used during the pretreatment; the acid concentration was kept in
the model because the interaction between temperature and acid concentration was
also significant. Regarding the sugar dehydration product, all the three independent
variables influenced the furfural concentration significantly (p \ 0.05); tempera-
ture was the most influential variable, followed by acid concentration and time of
pretreatment, respectively.
Neureiter et al. (2002) found that the acid concentration, and not the temper-
ature, was the most important variable impacting the xylose yield from sugarcane
bagasse, although temperature had a strong influence on furfural generation.
Aguilar et al. (2002), on the other hand, observed that both temperature and acid
concentration influenced the kinetics of xylose generation from xylan and of
xylose degradation into furfural.
The model proposed to explain the effects of the pretreatment conditions on the
concentration of xylose in the hemicellulosic hydrolysate is described by Eq. 10.4.
10 Dilute Acid Pretreatment 219

Table 10.7 Values of the regression coefficients maintained in the models proposed to predict
the concentrations of xylose and furfural (hemicellulosic hydrolysates)
Variable Xylose concentration Furfural concentration
Coefficient S. Error p Coefficient S. Error p
Constant 18.61 1.18 – 1.40 0.15 –
A -3.32 0.91 0.0030 1.20 0.14 \ 0.0001
B 1.41 0.91 0.1467 0.51 0.14 0.0026
C – – – 0.40 0.14 0.0128
A2 -2.55 0.81 0.0076 0.42 0.13 0.0044
B2 -1.17 0.81 0.1723 – – –
AB -2.66 1.23 0.0496 – – –
Model 0.0036 \ 0.0001
Lack of fit 0.4664 0.1200
R2 0.70 0.87
A Temperature; B Acid concentration; C Time

For predicting the concentration of furfural, the respective model (coefficients

shown in Table 10.7) was reduced by setting the time of pretreatment at maximum
(Eq. 10.5).

CX ¼ 18:61  3:32 A þ 1; 41 B  2:55 A2  1:17 B2  2:66 AB ð10:4Þ

CF ¼ 2:12 þ 1:20 A þ 0:51 B þ 0:42 A2 ð10:5Þ

where CX and CF are the concentrations of xylose and furfural in the hemicellu-
losic hydrolysate, both obtained as functions of the coded values of temperature
(A) and acid concentration (B).
The response surfaces corresponding to the abovementioned models are shown
in Fig. 10.8.
As can be seen, the use of high acid concentration in association with low
temperature during the pretreatment with dilute sulfuric acid increased the xylose
concentration in the hemicellulosic hydrolysate; high temperatures, however,
caused degradation of xylose into furfural.
Table 10.8 shows different combinations of the pretreatment variables, encoded
at their maximum and minimum levels, and the respective predictions for the
content of hemicellulose and the enzymatic conversion of cellulose into glucose
after 24 h of saccharification, as well as for the xylose and furfural concentrations
in the correspondent hemicellulosic hydrolysates.
As illustrated, the conditions of pretreatment of the sugarcane bagasse with
dilute H2SO4 that maximize the concentration of xylose in the hemicellulosic
hydrolysate and the enzymatic saccharification of cellulose in the pretreated solids
are different. To maximize the xylose concentration (23.70 g/L), the pretreatment
needs to be carried out with maximum acid concentration associated with minimal
temperature, regardless of time of pretreatment. On the other hand, to achieve
maximum cellulose saccharification (56.79 %), the pretreatment needs to be
220 P. J. Esteves et al.

Fig. 10.8 Response surfaces showing the effects of acid concentration and temperature on the
concentrations of xylose (a) and furfural (b); hemicellulosic hydrolysates

Table 10.8 Predictions of the levels of the dependent variables (CX, CC, HC and CF) as functions
of the levels of the independent variables (A, B and C)
A B C CX CC HC CF
(C) (% w/w) (min) (g/L) (%) (%) (g/L)
-1.81 -1.81 -1.81 1.20 20.34 23.12 0
1.81 6.61 41.96 12.47 3.27
-1.81 1.81 23.70 35.17 12.04 0.79
1.81 0 56.79 1.39 5.14
-1.81 -1.81 1.81 1.20 20.34 18.18 0.38
1.81 6.61 41.96 7.54 4.73
-1.81 1.81 23.70 35.17 7.10 2.24
1.81 0 56.79 0 6.59
A Temperature; B Acid concentration; C Time; CX and CF xylose and furfural concentrations in
the hemicellulosic hydrolysate; HC Hemicellulose content in the pretreated solids; CC Enzymatic
conversion of cellulose into glucose within 24 h of hydrolysis

carried out with maximum acid concentration associated with maximum temper-
ature, also regardless of time. This behavior may be due to the fact that, when the
pretreatment is conducted at high temperature, the extent of hemicellulose removal
is high, which improves the cellulose saccharification in the pretreated solids.
Under such severe conditions of pretreatment, however, xylose is dehydrated to
furfural, diminishing the xylose concentration in the hemicellulosic hydrolysate.
A similar behavior was observed for rice straw (Hsu et al. 2010) and switchgrass
(Shi et al. 2011).
As a real example obtained in the present study: a high concentration of furfural
(4.40 g/L) and a low concentration of xylose (7.02 g/L) were observed in exper-
iment 8; which, however, as already pointed out, led to solids highly digestible.
10 Dilute Acid Pretreatment 221

Table 10.9 Values of the regression coefficients maintained in the model proposed to predict the
yield of sugars (xylose ? glucose) conversion into ethanol (hemicellulosic hydrolysates)
Variable Coefficient S. Error p
Constant 0.280 0.03 –
A -0.110 0.03 0.0010
B -0.086 0.03 0.0049
C -0.098 0.03 0.0019
A2 -0.046 0.02 0.0636
Model 0.0002
Lack of fit 0.0384
R2 0.75
A Temperature; B Acid concentration; C Time

Fig. 10.9 Response surface

showing the effects of acid
concentration and
temperature on the yield of 0.040
sugars (xylose ? glucose) 0.100
0.4 0.180
conversion into ethanol;
above
hemicellulosic hydrolysates
0.3

0.2

0.1

131.91
0.19

2 150

3.81 168.09

Last, but not least, Table 10.9 shows the values of the regression coefficients of
the model proposed to predict the yield of sugars (xylose ? glucose) conversion
into ethanol as a function of the levels of the independent variables used during the
pretreatment with dilute sulfuric acid.
As can be seen, all the three independent variables influenced significantly
(p \ 0.05) the bioconversion of the major sugars contained in the hemicellulosic
hydrolysate into ethanol; temperature, again, was the most influential variable.
In order to elaborate the response surface shown in Fig. 10.9, the complete
model that correlates the level of the dependent variable with the levels of the
independent variables was simplified by setting the time of pretreatment at max-
imum (Eq. 10.6).
222 P. J. Esteves et al.

YP=S ¼ 0:10  0:11 A  0:09 B  0:05 A2 ð10:6Þ

where YP/S is the yield of sugars (xylose ? glucose) conversion into ethanol,
obtained as function of the coded values of temperature (A) and acid concentration
(B).
The data show that the efficiency of converting sugars into ethanol exhibited by
the yeast Scheffersomyces stipitis in the hemicellulosic hydrolysate is optimized
when the pretreatment is carried out under conditions of low severity, due to the
low content of inhibitory compounds. This conclusion is supported by the man-
uscript written by Scordia et al. (2010), which reports data obtained when pre-
treating Saccharum spontaneum with oxalic acid and fermenting a selected
hemicellulosic hydrolysate with Scheffersomyces stipitis CBS 6054.

Acknowledgments The financial support from Fapesp, CNPq, CAPES and USP is acknowl-
edged. W. Carvalho is thankful to the many who have contributed to the evolution of the study.

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Chapter 11
Scale-up Pretreatment Studies
on Sugarcane Bagasse and Straw
for Second-Generation Ethanol
Production

George Jackson de Moraes Rocha, Viviane Marcos Nascimento,

Vinicius Fernandes Nunes da Silva and Anuj Kumar Chandel

Abstract Sugarcane juice-derived ethanol (1G ethanol) has been the major
renewable energy source in Brazil after the inception of National Alcohol Program
in 1970. The remaining part, after the processing of sugarcane and extraction of
juice (sugarcane bagasse-SB and straw-SS), are the promising sugar feedstock for
cellulosic ethanol (2G ethanol) due to their abundant availability round the year and
high energy content. However, sugar recovery from lignocellulosic biomass is not
easy and needs intensive processing. Pretreatment to overcome the recalcitrance of
these feedstocks and sugar recovery constitute almost 30 % cost of 2G ethanol
production. Several pretreatment methods have been studied recently aiming to
either lignin removal or hemicellulose from SB/SS for the subsequent enzymatic
hydrolysis for fermentable sugar production. However, steam explosion and dilute
sulfuric acid have been emerged out as two successful options for the pretreatment
of SB/SS. Pilot level studies at our institute (Laboratório Nacional de Ciência e
Tecnologia do Bioetanol—CTBE, Campinas, Brazil), for the pretreatment of SB/SS
considering steam explosion and dilute acid pretreatment, have shown the prom-
ising results. Both the pretreatment strategies are scalable and reproducible at the
commercial level. This chapter deals with the experiments made on SB/SS for the
steam explosion and dilute acid hydrolysis and the sugar recovery after enzymatic
hydrolysis. Furthermore, process configurations for saccharification of pretreated
biomass and the conversion of released sugars into ethanol have also been discussed.

Keywords Sugarcane bagasse 

Sugarcane straw  Scale-up pretreatment 

Enzymatic hydrolysis Ethanol production

G. J. de Moraes Rocha (&)  V. M. Nascimento  V. F. N. da Silva

Laboratório Nacional de Ciência e Tecnologia do Bioetanol, P. O. Box 6170,
Campinas, São Paulo 13083-970, Brazil
e-mail: george.rocha@bioetanol.org.br
A. K. Chandel
Department of Biotechnology, Engineering School of Lorena, University of São Paulo,
Estrada Municipal do Campinho-Caixa, P.O. Box 116, Lorena, São Paulo 12.602.810, Brazil

S. S. da Silva and A. K. Chandel (eds.), Biofuels in Brazil, 225

DOI: 10.1007/978-3-319-05020-1_11,  Springer International Publishing Switzerland 2014
226 G. J. de Moraes Rocha et al.

11.1 Introduction

Contemporary industrial developments increased energy demands, and rapid pace

of urbanization have necessitated for the search of economic and environmentally
sustainable energy sources. Ethanol made from either sugar from sugarcane, corn,
sugar beet (1G ethanol) is a well-established process in countries like USA, Brazil,
China, India, and others have shown a promising option for transportation fuel.
However, the food versus fuel concerns have alarmed the researchers and policy
makers to implement the ethanol production from the second-generation feedstocks
like sugarcane bagasse, corn stover, grasses, and dedicated energy crops. Biomass-
derived cellulosic ethanol may provide unique environmental, economic strategic
benefits, and can be considered as a safe and cleanest liquid fuel alternative to fossil
fuels (Goldemberg 2008). Brazil and USA are the major ethanol producing coun-
tries in the world from sugarcane juice and corn grains, respectively. Brazilian mills
is likely to produce around 163–169 million tons of sugarcane bagasse and
84 million tons of straw in the 2012/13 harvest (Canilha et al. 2012). Undoubtedly,
SB is a preferred source of co-generation of heat and power in sugarcane processing
industries for the sugars and first-generation ethanol production. However, the
remaining amount of SB or SS can be used for the 2G ethanol production for the
fullest valorization of biomass (Chandel et al. 2012a). For the effective conversion
of lignocellulosic material into ethanol, there are three major steps involved first,
thermochemical pretreatment—a preprocessing step that improves enzyme access
to the cellulose; second enzymatic saccharification—use of cellulases and hemi-
cellulases; and third, fermentation of released sugars by specialized organisms.
Pretreatment is an important tool for practical cellulose conversion processes,
and has a strong impact on ethanol production from lignocellulosics following the
biorefinery concept (Galbe and Zacchi 2002). The goal of pretreatment is either to
break the lignin seal or hemicellulose removal and disrupt the crystalline structure
of cellulose for the maximum sugar recovery after enzymatic action (Taherzadeh
and Karimi 2007; Yang and Wyman 2008). Several pretreatment technologies
have been investigated for the pretreatment of SB/SS (Chandel et al. 2012b).
However, steam explosion and dilute acid hydrolysis of SS/SB have presented the
most promising results in terms of improved sugar recovery from the substrates
upon cellulase-mediated action.
During steam explosion pretreatment, biomass is heated with saturated steam,
followed by a sudden decompression of the pressurized system. Thus, steam
penetrates into the lignocellulosic matrix and condensates to form liquid water
inside the fibers which is rapidly evaporated causing the explosion of fibers. Acetyl
groups are hydrolyzed and the released acetic acid mechanistically acts on
hemicellulose into monomeric products (Hu and Ragauskas 2012). Additionally,
partial degradation of lignin or delocalization of lignin moities is also possible
increasing the accessibility of cellulases enzymes toward the substrate for the
hydrolysis of cellulose into glucose (Chen et al. 2007).
11 Scale-up Pretreatment Studies on Sugarcane Bagasse and Straw 227

Dilute acid hydrolysis is another method which is particularly well suited for the
pretreatment of SB/SS. Dilute mineral acids such as sulfuric acid effectively solu-
bilize the hemicellulose fraction of the cell wall at high temperature eventually
increasing the accessibility of cellulolytic enzymes action to the carbohydrates
present in the pretreated substrate (Santos et al. 2011). The remaining cell wall
fraction after dilute acid hydrolysis is called cellulignin which have shown 60 %
sugars recovery after enzymatic hydrolysis. The hemicellulose fraction of SB/SL
after steam explosion or dilute acid hydrolysis is depolymerized primarily into
pentose sugars (xylose and arabinose) and hexose sugars (glucose, galactose, man-
nose, etc.) along with inhibitory compounds (Canilha et al. 2012). The effectiveness
of steam explosion and dilute acid hydrolysis as pretreatment for SB and SS has been
shown in laboratory- and pilot scale experiments (Rocha et al. 2011, 2012a, b).
The enzymatic hydrolysis is a promising and environmentally feasible method
for saccharification of lignocellulosics to sugars. Further, in order to develop
integrated process configurations, enzymatic hydrolysis and fermentation of
released sugars may be combined in a single vessel, the so-called simultaneous
saccharification and fermentation (SSF), enzymatic hydrolysis, and co-fermenta-
tion of pentose and hexose sugars by single or mixed microorganisms (SSCF). In
the line to advance the process intensification, process like consolidated biopro-
cessing (CBP) have been emerged wherein the enzyme production, enzymatic
hydrolysis, and co-fermentation of released sugars into ethanol is possible in single
vessel (Lynd et al. 2005). Microbial delignification has given the new idea in order
to develop highly integrated process for ethanol production. It is possible for
pretreatment of biomass, enzyme production, enzyme hydrolysis, and co-fer-
mentation of released sugars into ethanol in a single vessel. This process may be
called as integrated bioprocessing (IBP). The idea of IBP is in nascent stage and is
subjected to multidisciplinary research efforts for its realization.
This chapter entails about the experimental outcome of pilot scale studies
concerned with steam explosion and dilute acid hydrolysis of sugarcane bagasse
and straw. Various process configurations for second-generation ethanol produc-
tion have also been discussed.

11.2 Pretreatment by Steam Explosion

The steam explosion pretreatment of SB or SS is probably one of the most

commonly applied methods among the physical–chemical methods. This tech-
nology can achieve high reaction rates has a high potential in many industrial
fields: the paper and textile industry, extraction and fermentation biotechnology,
fine chemicals, and biodegradable polymer production (Focher et al. 1988;
Hendriks and Zeeman 2009; Mosier et al. 2005; Hu and Ragauskas 2012).
Pretreatment by steam explosion has been proposed as one of the most prom-
ising methods in the separation of the main components of lignocellulosic mate-
rials: cellulose and hemicellulose. When subjected to high steam pressures for
228 G. J. de Moraes Rocha et al.

certain period of time, this material suffers a process known as self-hydrolysis

whereas when it is subjected to high temperatures the links owned by the biomass
become weak and brittle in some parts. This process allows, after the decom-
pression, the defibration, and reduction of the material to smaller particles,
meaning that the hemicelluloses are hydrolyzed in soluble sugars and the lignin is
partially modified, increasing its susceptibility for enzymes and chemical reagents
(Martín et al. 2002, 2008; Rocha et al. 2011).
Several works have been reported in the literature showing the technological
advances of this technique of pretreatment for plants biomass, aiming a wide range
of applications of the main components of this material.

11.2.1 Reaction in a 200L Pilot Reactor

Rocha et al. (2013) studied the pretreatment by steam explosion with sugarcane
bagasse for 20 reactions in a 200L pilot reactor with 1.3 MPa pressure, equivalent
to 13 kgfcm-2 at 190 C, and a period of 15 min. The results showed an excellent
reproducibility, resulting in an average yield in mass of 66.1 % and a standard
deviation of 0.8 %. The average results of the main components of these reactions
were 57.5 ± 1.6 % in cellulose, 6.6 ± 1.5 % in hemicellulose, and 32.5 ± 2.4 %
in lignins. The solubilization of hemicellulose was an average of 82.7 % with a
standard deviation of 4.3 %.
The cellulignin fractions obtained in pretreatment was submitted to an alkaline
delignification in a pilot scale. The steam explosion pretreated bagasse was reacted
with a NaOH solution 1.0 % (w/v). The delignification reactions were made in a
stainless steel 316L reactor with a 350L capacity, fitted with mixing and heating
systems, using a solid–liquid ratio 1:10 (w/v). The operation was carried out at
100 C for 1 h.
The amount of cellulose increased to an average of approximately 87 % and the
removal of hemicellulose and lignin exceeded 90 %, showing an excellent
removal of lignin from the biomass. It solubilized a 92.7 % average with a stan-
dard deviation of 3.9 %. The hemicellulose hydrolysis was 94.7 % with standard
deviation of 0.9 %. The process hydrolyzed 31.1 % of cellulose with a deviation of
3.5 %. It evidences that even in milder conditions, the steam explosion pretreat-
ment and alkaline delignification processes causes a substantial cellulose loss.
Figure 11.1 shows a flowchart of the separation processes for the main compo-
nents of SB, with the pretreatment of steam explosion followed by alkaline
delignification.
The micrographs of the SB pretreated by steam explosion and delignification
are shown in Fig. 11.2.
The micrographs with magnification of 200 times reveals aggregated fibers, due
to the complex cellulose-hemicelluloses-lignin-extractives. A high content of
marrow flakes is observed which evidences an element ringed at the top that
probably came from a xylem vase during the grinding of sugarcane.
11 Scale-up Pretreatment Studies on Sugarcane Bagasse and Straw 229

Fig. 11.1 Flowchart of the separation processes for the main components of sugarcane bagasse
(Rocha et al. 2013)

Fig. 11.2 Micrographs of bagasse in natura (a), pretreated by steam explosion (b), and bagasse
pretreated by steam explosion and delignified (c)

The fiber characteristic of the SB pretreated by steam explosion showed a

structural disintegration and disaggregation, resulting in fiber damage. The surface
structure of fibers is apparent from the highest enlargement.
After the removal of main components of vegetable biomass by the processes of
delignification of SB pretreated by steam explosion, is observed a total disag-
gregation of the fibers of bagasse rich in cellulose, evidencing a largest surface of
contact and accessibility to chemical or biological attacks, such as enzymes cel-
lulolytic in processes of saccharification.

11.2.2 Steam Explosion Reaction in a 2.5 m3 Reactor

Industrial-scale steam explosion pretreatment of SS for enzymatic hydrolysis of

cellulose for production of second-generation ethanol was studied by Oliveira
et al. (2013a). Pretreatment was conducted in a 2.5 m3 reactor for 15 min at 180,
190, and 200 C, respectively. The flowchart process is shown in Fig. 11.3.
The chemical composition of raw straw, after the pretreatment and the
pretreatment and delignification process, respectively, are shown in Table 11.1.
230 G. J. de Moraes Rocha et al.

Fig. 11.3 Schematic representation of the processing of sugarcane straw by steam explosion
pretreatment, alkaline delignification, and enzymatic hydrolysis

11.2.3 Steam Explosion Reaction in a 5 m3 Industrial

Reactor

Oliveira et al. (2013b) studied the pretreatment of bagasse carried out in the
industrial reactors of the Mill. This company has three 5 m3 reactors of Caldema
for steam explosion pretreatment of SB or SS, as shown in Fig. 11.4. The pre-
treatment was performed under the 15.5 kgfcm-2 pressure at temperature near a
200 C for 7 min.
The cellulignin fractions obtained in the pretreatment applied a pilot scale
alkaline delignification. The steam explosion pretreated bagasse was reacted with a
NaOH solution 1.0 % (w/v). The delignification reactions were made in a stainless
steel 316 L reactor with a 350L capacity, fitted with mixing and heating systems,
using a solid–liquid ratio 1:10 (w/v). The operation was carried out at 100 C for 1 h.
The chemical compositions of the pretreated and pretreated and delignified SB
are shown in Table 11.2.
Pretreatment indicated a mass yield of 68 %, approximately 78 % hydrolyses of
hemicelluloses and solubilizing 20 % of the cellulose without a significant reduc-
tion in lignin content. The alkaline delignification had a mass yield of 59.1 %, while
lignin content was reduced by 90 %, and hemicelluloses decreased by 95 %.
Currently, major advances have been achieved as the pretreatment by steam
explosion, especially as new equipment, such as continuous reactors, which allows
to obtain kinetic and thermodynamic pilot scale seeking an extension to an
industrial scale (Fig. 11.5).
That justifies the technique as one of the most promising for obtaining second-
generation ethanol.

11.3 Hydrothermal Pretreatment

Hydrothermal pretreatment is gaining attention as an environmentally friendly

solvent and an attractive reaction media for a variety of applications. In this
process, at 150–230 C range temperatures, lignocellulosic materials undergo
hydrolysis reactions in the presence of the hydronium ions generated by water
auto-ionization, which act as catalysts.
 11

Table 11.1 Chemical composition of in natura, pretreated, and delignified sugarcane straw samples
Steam explosion pretreatment
Components (%) Raw sugarcane straw PTS 180 C PTSD PTS 190 C PTSD PTS 200 C PTSD
Mass yield 100 58 59 57 55 56 54
Cellulose 39.8 ± 0.3 47.8 ± 0.2 73.0 ± 0.2 48.6 ± 0.7 74.8 ± 0.3 48.7 ± 0.5 74.6 ± 0.5
Hemicellulose 28.6 ± 0.2 16.2 ± 0.2 9.5 ± 0.7 7.3 ± 0.2 6.1 ± 0.6 3.7 ± 0.1 5.9 ± 0.4
Lignin 22.5 ± 0.1 32.5 ± 0.1 8.8 ± 0.4 38.1 ± 0.2 10.3 ± 0.3 41.8 ± 0.2 14.0 ± 0.4
Ash 2.4 ± 0.3 3.5 ± 0.1 7.1 ± 0.8 5.2 ± 0.2 8.5 ± 0.8 5.2 ± 0.1 7.0 ± 0.7
Extractives 6.2 ± 0.3 NA NA NA NA NA NA
Total 99.6 ± 1.2 100.0 ± 0.7 99.3 ± 2.2 99.1 ± 1.3 99.7 ± 2.0 99.33 ± 1.0 101.3 ± 0.7
PTS Pretreated material with steam explosion
PTSD Pretreated and delignified material
NA Not available
Scale-up Pretreatment Studies on Sugarcane Bagasse and Straw

Percentage Mean of three replicate analyses (Oliveira 2012)

231
232 G. J. de Moraes Rocha et al.

Fig. 11.4 Set reactors of pretreatment by steam explosion with 5 m3 capacity to treat 3400 kg
of bagasse per hour (a), and sugarcane bagasse before and after the pretreatment by steam
explosion (b)

Table 11.2 Chemical compositions of the raw, pretreated, and delignified sugarcane bagasse
samples
Steam explosion pretreatment
Components (%) Raw sugarcane PTS PTSD
Mass yield 100.0 68.0 59.1
Cellulose 43.8 ± 1.1 51.7 ± 0.6 90 ± 2
Polyoses 25.8 ± 0.8 8.9 ± 0.1 3.4 ± 0.3
Lignins 22.1 ± 0.8 34.3 ± 0.3 5.5 ± 0.2
Ash 1.4 ± 0.2 5.5 ± 0.2 1.4 ± 0.1
Extractives 6.1 ± 0.3 NA NA
Total 99.2 ± 0.8 100.3 ± 0.4 99.9 ± 0.5
PTS Pretreated material with steam explosion
PTSD Pretreated and delignfied material
NA Not available

Fig. 11.5 Continuous

reactor of steam explosion
AdvanceBio LLC-USA with
a capacity of 75 kg.h-1
11 Scale-up Pretreatment Studies on Sugarcane Bagasse and Straw 233

Fig. 11.6 Rotatory reactor,

REGMED AU/E-20

11.3.1 Reaction in 20L Reactor

Hydrothermal pretreatment of sugarcane bagasse for production of second-gen-

eration ethanol was studied by Silva (2009). The pretreatment was conducted in a
20L reactor (REGMED AU/E-20) indicated on Fig. 11.6, for 10 min at 180, 190,
and 195 C, respectively.
The chemical composition of raw sugarcane, after the hydrothermal pretreat-
ment and after the pretreatment and delignification process, are shown in
Table 11.3.
This pretreatment indicated a mass yield of 62.1, 51.7, and 49.6 % for hydro-
thermal pretreatment performed at 180, 190, and 195 C, respectively. Approxi-
mately, 89 % of hemicellulose was hydrolyzed during this pretreatment at 195 C.
The heterocyclic ether bonds of hemicelluloses are the most susceptible to this
type of reaction, leading both to generation of oligosaccharides and to splitting of
the acetyl groups from the hemicellulosic fraction of the raw materials. In further
reaction stages, the hydronium ions generated from acetic acid auto-ionization also
act as catalysts in the degradation of polysaccharides.
The hydrothermal pretreatment solubilizes almost 25 % of the cellulose con-
tent, without a significant reduction in lignin content, however, the cellulose could
be largely preserved during hydrothermal pretreatment and the dissolution is low,
occurs an enhancement in the cellulose digestibility in the enzymatic hydrolysis,
due to the solubilization of the hemicellulose.
The alkaline delignification step hydrolyses almost 80 % of the lignin content,
for the cellulignin pretreated in 195 C, with a mass yield of 59.7 %. Lignin is not
significantly solubilized during autohydrolysis, but during the pretreatment a
 234

Table 11.3 Chemical composition of in natura, hydrothermal pretreated and delignified sugarcane bagasse samples
Hydrothermal pretreatment
Components (%) Raw sugarcane bagasse PTH 180 C PTHD PTH 190 C PTHD PTH 195 C PTHD
Mass yield 100.0 62.1 75.1 51.7 68.6 49.6 59.7
Cellulose 42.8 ± 0.3 54.3 ± 0.3 65.3 ± 0.6 60.8 ± 0.9 73.1 ± 0.6 63.4 ± 1.1 79.2 ± 0.6
Hemicellulose 25.9 ± 0.3 15.4 ± 0.2 12.3 ± 0.1 8.9 ± 0.4 7.1 ± 0.1 5.9 ± 0.1 3.7 ± 0.2
Lignin 22.1 ± 0.2 26.2 ± 0.1 19.8 ± 0.7 24.9 ± 0.7 17.3 ± 0.9 28.5 ± 1.2 14.2 ± 0.3
Ash 1.4 ± 0.1 4.1 ± 0.6 2.9 ± 0.9 5.4 ± 0.1 2.6 ± 0.0 2.1 ± 0.1 3.6 ± 0.4
Extractives 6.1 ± 0.1 NA NA NA NA NA NA
Total 98.3 ± 1.0 100.0 ± 1.2 100.3 ± 2.3 100.0 ± 2.1 100.1 ± 1.6 99.9 ± 1.5 100.7 ± 1.5
PTH Pretreated material with hydrothermal pretreatment
PTHD Hydrothermic pretreated and delignfied material (NaOH 1.0 % (m/v), 100 C for 1 h)
NA Not available
Percentage Mean of three replicate analyses (Silva 2009)
G. J. de Moraes Rocha et al.
11 Scale-up Pretreatment Studies on Sugarcane Bagasse and Straw 235

redistribution of the lignin occurs, which causes the lignin to cease acting as a
steric barrier to enzymatic hydrolysis (Rohowsky et al. 2013).
The hydrothermal pretreatment is known to form only minor amount of fer-
mentation inhibitor products as long as the pH is kept between 4 and 7. Other
fractions of lignocellulosic materials different from hemicelluloses can also react
in the presence of water; for example, cellulose and lignin can be partially de-
polymerized by similar hydrolysis reactions (Garrote et al. 1999; Silva et al. 2011).

11.4 Dilute Acid Pretreatment

Among different types of pretreatment on lignocellulosic materials, the hydrolysis

using diluted acid is the most used technical on the removal of hemicellulose and
has been applied to various agricultural wastes, using a wide range of catalysts
such as: sulfuric, hydrochloric, phosphoric, and nitric acid (Ramos 2003; Seabra
2008; Mussatto and Teixeira 2010). This pretreatment process is conducted under
high temperature and pressure, and has a reaction time in the range of seconds or
minutes, which facilitates continuous processing.

11.4.1 Dilute Acid Pretreatment in a 350L Reactor

Silva (2009) performed a diluted sulfuric acid pretreatment of sugarcane straw for
production of second-generation ethanol in 350L reactor for 10 min at 120 C
(Fig. 11.7). The chemical composition of raw straw, after the diluted sulfuric acid
and after the pretreatment and delignification process are shown in Table 11.4.
The objective of this process is the hydrolysis of sugars present in hemicellu-
loses: xylose, arabinose, and others, which are water soluble, rendering the cel-
lulose fraction more amenable for a further enzymatic treatment (Hendriks and
Zeeman 2009; Gírio et al. 2010), once that the presence of hemicellulose and
lignin in lignocellulosic biomass are responsible for reduction of enzymatic sac-
charification efficiency (Mussatto et al. 2008).
According to Table 11.4, the diluted acid pretreatment performed with sugar-
cane straw showed almost 22.7, 67.0, and 32 % of cellulose, hemicellulose, and
lignin solubilization, respectively. The delignification step improves the lignin
solubilization to 67.8 %, summarizing almost 78 % of solubilization in both
processes (pretreatment and delignification). The micrographs of the raw sugar-
cane straw, pretreated by diluted sulfuric acid and the pretreated and delignified
are shown in Fig. 11.8.
Micrographs of sugarcane straw prior to pretreatment and delignification pro-
cess exhibited the recalcitrant external surface. It is necessary to treat the material
before the enzymatic or acid hydrolysis to breakdown the cell and tissues, resulting
236 G. J. de Moraes Rocha et al.

Fig. 11.7 Picture of the 350L reactor, were a diluted sulfuric acid pretreatment of sugarcane
straw were performed for production of second-generation ethanol

Table 11.4 Chemical composition of raw sugarcane straw pretreated by diluted sulfuric acid and
pretreated and delignified
Diluted sulfuric acid Pretreatment
Components (%) Raw sugarcane straw PTA 120 C PTAD
Mass yield 100.0 % 56.8 % 63.1 %
Cellulose 38.1 ± 0.2 51.9 ± 0.1 74.2 ± 0.2
Hemicellulose 29.2 ± 0.3 17.0 ± 0.1 9.1 ± 0.2
Lignin 24.2 ± 0.2 29.0 ± 0.1 14.9 ± 0.2
Ashes 2.4 ± 0.1 1.9 ± 0.0 1.0 ± 0.0
Extractives 5.9 ± 0.2 – –
Total 99.8 ± 1.0 99.8 ± 0.3 99.2 ± 0.6
PTA Pretreated material with diluted sulfuric acid
PTAD Material pretreated by diluted sulfuric acid and delignfied with NaOH 1.0 % (m/v), 100 C
for 1 h

Fig. 11.8 Micrographs of raw (a), pretreated by diluted sulfuric acid (b) and pretreated and
delignified (c) sugarcane straw

in ‘‘free’’ cellulose fibers (C). The micrographs also reveal the remotion of
parenchyma cells of sugarcane straw after the acid pretreatment.
The acid pretreatment can also be conducted with concentrated acid to increase
the solubility of hemicellulose, however, requires intensive care because these
11 Scale-up Pretreatment Studies on Sugarcane Bagasse and Straw 237

reagents are toxic, corrosive, favor higher solubilized lignin precipitation,

increased formation of hydroxymethylfurfural and furfural (degradation products
of cellulose and hemicellulose, respectively), and increased release of acetic acid
by acetylated hemicellulose, and these compounds act as inhibitors of the fer-
mentation process for ethanol production (Fengel and Wegener 1989; Shevchenko
et al. 1999; Liu and Wyman 2003; Ramos 2003). Of all chemical pretreatments,
historically dilute sulfuric acid (0.5–1.5 %, T above 160 C) has been most
favored for industrial application, because it achieves reasonably high sugar yields
from hemicellulose: at least xylose yields of 75–90 % (Hamelinck et al. 2005;
Seabra 2008), and also improves the subsequent process which is the enzymatic
hydrolysis of cellulose (Yu et al. 2008).
After the processes of removing or altering the recalcitrant lignocellulosic
biomass structures, via pretreatment, the substrate dramatically changes and
become more susceptible to acid and/or enzymatic hydrolysis. Many pretreatments
act differently on the cell wall structure, with varied results as cellulosic micro-
fibrils exposure and lignin removal (Joshi et al. 2011; Iranmahbooba et al. 2002).
Usually, defined by the cleavage of chemical bonds by the addition of water
(Eq. 11.1), the main step of the hydrolysis process is to generate fermentable
monomeric sugars from biomass cellulose content by the following reaction
(Wyman et al. 2005).
ðC6 H10 O5 Þn þ nH2 O ! nC6 H12 O6 ð11:1Þ
Thus, each glucose unit in the long chain combines with a water molecule, and
180 mass units of glucose are released from 162 mass units of glucan and 18 mass
units of water, an 11.1 % mass gain (Wyman et al. 2005).
The cellulose hydrolysis is carried out by cellulase enzymes which are com-
posed mainly by (1) endoglucanase, (2) exoglucanase or cellobiohydrolase, and (3)
b-glucosidase. The endoglucanases cleaves the ‘‘middle’’ b-1, 4-glycosidic bonds
on cellulose chains to form glucose, attacking randomly; the exoglucanases or
cellobiohydrolases attack the nonreducing end of cellulose chain to form the
cellobiose units, cellobiose are the unit formed by 2 glucose linked by a glycosidic
linkage, and the b-glucosidases or cellobiase converts cellobiose into D-glucose
(Joshi et al. 2011; Zhang and Lynd 2004; Liu et al. 2009). In general, the enzy-
matic hydrolysis consists of three steps: adsorption of enzymes onto the surface of
the substrate, the biodegradation of cellulose to fermentable sugars, and desorption
of the enzymes (Fig. 11.9) (Sun and Cheng 2002).
Table 11.5 shows the values of enzymatic conversion, for pretreated and pre-
treated and delignified samples of sugarcane straw and sugarcane bagasse.
The biomass recalcitrance, due to the presence of hemicellulose and/or lignin in
the sample, affects directly the cellulose conversion. The enzymatic conversion of
sugarcane straw pretreated by steam explosion showed at 200 C the high cellu-
lose yield conversion. As noticed for all pretreatments the enzymatic conversion
proportionally increases with the raise of pretreatment temperature, due to the
presence of different contents of hemicellulose and/or lignin in the sample which
238 G. J. de Moraes Rocha et al.

Fig. 11.9 Proposed mechanism for cellulose depolymerization by cellulases (Arantes and
Saddler 2010)

affects directly the cellulose conversion. The sugarcane bagasse and straw cellu-
lignin obtained from steam explosion pretreatment performed at 200 C showed
52 and 80 % of cellulose conversion, respectively.
According to Santos et al. (2012), the enzymatic hydrolysis efficiency, from
hardwoods, was correlated to the wood chemical composition and lignin character-
istics, with lignin content, enzyme adsorption on substrate and, the ratio of syringyl/
guaiacyl of the substrate as the most important key features. The lignin content cannot
explain a correlation with enzymatic hydrolysis, but several studies showed several
changes in cellulose conversion, resulted from lignin removal (Kooa et al. 2012).
The delignification step of the sugarcane straw pretreated by steam explosion
and diluted sulfuric acid load to a significant enhancement of the enzymatic
hydrolysis of the material pretreated at 180 C. However, the effect of delignifi-
cation was not similar for all the pretreatment conditions. Differently for the
cellulignin yield conversion, the improvement of the enzymatic convertibility after
delignification decreased with the delignification process temperature.
 11

Table 11.5 Cellulose conversion by enzymatic hydrolysis, for pretreated, and delignified samples of sugarcane straw and sugarcane bagasse
Enzymatic conversion
Hydrolysis conditions 15 FPU/g of celluclast and 10 UI/g of b-glicosidase
Pretreatment Steam explosion Diluted sulfuric acid Hydrothermal
Samples Sugarcane strawa Sugarcane bagasseb Sugarcane strawc Sugarcane bagassec
PT conditions 1 2 3 4 5 6 7 8
Raw material 16.0 ± 1 15.0 ± 0.3 7.7 ± 1.3 6.0 ± 0.3
PT 58.8 ± 1 69.7 ± 2 80.0 ± 2 52 ± 2 51.4 ± 3 37.4 ± 0.5 56.9 ± 0.7 69.2 ± 2.6
PTD 85.1 ± 4 73.0 ± 1 71.5 ± 4 76.0 ± 0.7 85.0 ± 0.5 73.0 ± 0.7 82.3 ± 0.6 89.2 ± 2.2
1 PTS in a 2.5 m3 reactor for 15 min at 180 C; 2 PTS in a 2.5 m3 reactor for 15 min at 190 C; 3 PTS in a 2.5 m3 reactor for 15 min at 200 C; 4 PTS in
5 m3 industrial reactor at 200 C for 7 min, 5 PTA in H2SO4 1 % (m/v), 120 C, 10 min, 6 PTH 180 C in a 20L reactor for 10 min; 7 190 C in a 20L
reactor for 10 min; 8 195 C in a 20L reactor for 10 min
a
Oliveira et al . 2012
b
Oliveira et al. 2012, 2013a
Scale-up Pretreatment Studies on Sugarcane Bagasse and Straw

c
Silva et al. 2009
239
240 G. J. de Moraes Rocha et al.

Concerning the low enzymatic rate, many hypotheses have been proposed, such
as lignin content, enzymatic activity, temperature, pH, enzyme source and enzyme
concentration, the linkage between lignin and carbohydrates, the hydrophobic
interaction between lignin and enzyme, enzyme inactivation, substrate accessi-
bility and reactivity cellulose crystallinity enzyme synergism, surface obstacles,
low porosity, and fractal nature of the substrate (Caminal et al. 1985; Monney et al.
1998; Chen et al. 2007; Xu and Ding 2007; Kumar and Wyman 2009; Park et al.
2010; Jalak and Väljamäe 2010; Wada et al. 2010; Kurasin and Väljamäe 2011;
Paul and Teli 2011; Bansal et al. 2012). The important key enzymatic hydrolysis
development is to identify the main causes for the slowdown enzymatic rate: key
factors that have remained challenging (Bansal et al. 2012).
According to Santos et al. (2012), the enzymatic hydrolysis efficiency, from
hardwoods, was correlated to the wood chemical composition and lignin character-
istics, with lignin content, enzyme adsorption on substrate, and the ratio of syringyl/
guaiacyl of the substrate as the most important key features (Santos et al. 2012). The
lignin content cannot explain a correlation with enzymatic hydrolysis, but several
studies showed several changes in cellulose conversion, resulted from lignin removal
(Kooa et al. 2012).
The Crystallinity index of cellulose has been reported as one of the most
important structural parameter and the major limiting factor during enzymatic
hydrolysis (Chang and Holtzapple 2000; Park et al. 2010), although the overall
carbohydrate conversion of enzymatic hydrolysis did not showed a correlation
with lignin removal and sample crystallinity index (Yu et al. 2011; Ioelovich and
Morag 2011).
Another important affecting factor of the enzymatic hydrolysis is drying of the
wet sample. The difference between the enzymatic conversion of the nondried and
dried lignocellulosic (cellulose) samples is due to the fact that drying process can
cause an irreversible collapse of the pore structure, decreasing the hydrolysability
and the cellulose access (Ioelovich and Morag 2011).
The major limitations in the commercialization of second-generation ethanol
biofuel, by breakdown of cellulose with enzymes, are their high cost, low-specific
activity, and slow rates of hydrolysis (Bansal et al. 2012). The enzymes contribute
in about 25 % of the biomass conversion process, excluding feedstock cost, to
obtain biofuels (NREL 2012).
Because of its specificity, a lot of research groups and biotechnology companies
are focused on the improvement of the enzymatic hydrolysis process, by protein
engineering (Himmel et al. 2007) and substrate engineering, to make the ligno-
cellulosic substrate less recalcitrant to enzymatic action, and provide enzymes with
more hydrolyzation capability (Ragauskas et al. 2006; Bansal et al. 2012). It is
shown in Table 11.6 the enzymatic hydrolysis of different biomass as the
respective yields of conversion.
Table 11.6 Enzymatic hydrolysis of vegetal biomass
11

Hydrolysis of biomass cellulose

Lignocellulosic Type of hydrolysis and conditions Catalyser dosage Sugar conversion (%) References
subtrate
Sugarcane Enzymatic hydrolysis substrate condition: 25 FPU Accellerase 1500 and 50 The total cellulose conversion increases Rezende et al. (2011)
bagasse sugarcane bagasse pretreated by acid UI of Beta-Glucanase per significantly from 22.0 % (value for the
followed by the step with NaOH 1 % gram of biomass untreated bagasse) to 72.4 %
Sunflower Enzymatic hydrolysis substrate condition: 23–31 FPU celuclast, 15 IU 90 % glucose conversion can be obtained after Diaz et al. (2011)
stalks sunflower stalks pretreated by b-glucosidase per g of 72 h enzyme action on pretreated sunflower
hydrothermal pretreatment pretreated substrate stalks at 220 C
Corn stover Enzymatic hydrolysis substrate condition: 15 FPU cellulase per g cellulose One-stage hydrolysis reach a yield of 62.8 % Yang et al. (2010)
pretreated corn sttover (72 h), 70.2 % with enzyme recycling and
76.1 % with the supplement of fresh
enzyme to eliminate enzyme recovery
procedure, were obtained in 24 h
Bagasse pulp Enzymatic hydrolysis substrate condition: 15 IU/g of Fusarium oxysporum 82.38 % sugar yield Xiea et al. (2013)
bagasse pulp prepared from the treatment enzyme extract
process with active oxygen and MgO-based
solid alkali
Wood Enzymatic hydrolysis substrate condition: 25 FPU celluclast and 123 CBU First: continuous addition of substrate during Jacquet et al. (2012)
cellulose fiber from wood pulp Novozym 188 continuous hydrolysis—increase by 50 % the
additions every 2 h concentration hydrolyzed products second:
continuous and simultaneous addition of
enzyme and substrate during hydrolysis
Scale-up Pretreatment Studies on Sugarcane Bagasse and Straw

step led to very high concentrated

hydrolysates (170 g/l)
Sugarcane straw Enzymatic hydrolysis substrate condition: 15 FPU.mL celluclast, 10 IU g Delignification increase the enzymatic Oliveira et al. (2013b)
sugarcane straw pretreated by steam Novozym 188 conversion (from 58.8 % in the cellulignin
explosion at 180, 190 and 200 C for to 85.1 % in the delignificated pulp)
15 min and pretreated and delignified straw
by sodium hydroxide
241
242 G. J. de Moraes Rocha et al.

11.5 Microorganism for Cellulosic Ethanol Production:

An Asset in Biorefinery

The sugar syrup obtained after thermochemical or enzymatic hydrolysis of ligno-

cellulosic materials is used for ethanol fermentation. Lignocellulose hydrolysates
contain a variety of sugars, i.e., glucose, xylose, cellobiose, xylose, mannose, and
others, however, glucose and xylose are the principle sugars in hydrolysates com-
prising more than 90 % (Hahn-Hägerdal et al. 2007). In order to obtain the desired
ethanol yields and productivities, it is necessary to convert maximum amount of
sugars into ethanol. Therefore, the ideal organism for the production of ethanol from
lignocellulosic hydrolysate would be the one, which can utilize various forms of
sugars generated by lignocellulose hydrolysis. The ability to ferment pentoses along
with hexoses is not wide spread among microorganisms (Chandel et al. 2011).
Conventional ethanol producers in industries such as Saccharomyces cerevisiae
and Zymomonas mobilis are capable of converting only hexose sugars to ethanol.
Yeasts such as Candida shehatae, Pichia stipitis, and Pachysolen tanophillus have
shown abilities for the conversion of xylose and glucose into ethanol (Hahn-
Hägerdal and Pamment 2004). However, their low ethanol and substrate tolerance
and poor ethanol productivities make them a limited choice for cellulosic ethanol
production at industrial scale. Of the various xylose-fermenting yeasts, P. stipitis
has shown greater ethanol production than C. shehatae. This was due to the
increased uptake of xylose, glucose, mannose, cellobiose, and galactose (Chandel
et al. 2011). Commercial exploitation of these yeasts for ethanol production from
xylose is restricted mainly by their low ethanol tolerance, slow rates of fermen-
tation, difficulty in controlling the rate of oxygen supply at the optimal level plus
sensitivity to inhibitors generated during pretreatment, and hydrolysis of ligno-
cellulosic substrates (Hahn-Hägerdal et al. 2007).
Nevertheless, xylose can be converted to xylulose using the enzyme xylose
isomerase and traditional yeasts can ferment xylulose to ethanol although the
process is not cost-effective. Arabinose and other pentose sugars are often present
in hemicellulosic hydrolysates depending on the source, but only a few yeast
strains can barely ferment arabinose to ethanol, thus no naturally occurring yeast
can ferment all these sugars to ethanol.
The Table 11.7 shows the substrate range of various yeasts. Genetically engi-
neered organisms are now being employed for ethanol fermentation, these can
greatly improve ethanol production efficiency and reduce the cost of operation
(Dien et al. 2000). The recombinant strains of Escherichia coli with the genes from
Zymomonas mobilis for the conversion of pyruvate to ethanol have been con-
structed. The recombinant plasmids with xylose reductase and xylitol dehydro-
genase genes from P. stipitis and xylulokinase gene from S. cerevisiae have been
transformed into Saccharomyces sp. for the co-fermentation of glucose and xylose
(Hahn-Hägerdal and Pamment 2004). Though new technologies have greatly
improved bioethanol production, yet there are still a lot of problems that have to be
solved. The major problems include maintaining a stable performance of
11 Scale-up Pretreatment Studies on Sugarcane Bagasse and Straw 243

Table 11.7 Native yeast and fungal species capable of fermenting xylose to ethanol
Microorganism Sugar utilization pattern References
C. shehatae Has both active and positive transport system for xylose Jeffries and
uptake: produces moderate amount of xylitol does not grow Shin (1999)
anaerobically requires biotin and thiamine
C. boidinii Produces large amount of xylitol: oxidizes methanol Ko et al. (2008)
P. stiptis Ferment all sugars found in wood some strains ferment xylan Nigam (2002)
F. oxysporum Ferments 20 different carbon sources including xylitol; does Suihko et al.
not use xylan or cellulose: converts xylose to ethanol, (1983)
acetic acid, carbon dioxide
Mucor species Ferment pentoses Sharifia et al.
(2008)
P. tannophilus Ferment xylose glucose and glycerol metabolize xylose Zhao et al.
anaerobically produce large amount of xylitol (2008)

genetically engineered yeast in commercial scale fermentation operations (Dien

et al. 2000), developing more efficient pretreatment technologies for lignocellu-
losic biomass, and integrating optimal components into an economic ethanol
production system (Hahn-Hägerdal et al. 2007).

11.5.1 Process Routes for Cellulosic Ethanol Production

In the past, all the three major fermentation process (Batch, Fed-batch, and Con-
tinuous) have been employed for biomass conversion into ethanol. Nevertheless
batch fermentation has been the preferred choice for cellulosic ethanol production
due to its simplicity and fast conversion rates. The desired choice of fermentation
strategies usually depend upon the kinetics of fermenting microorganism, type of
hydrolysate, and process economics. Generally, batch fermentation has some
limitations like the capacity which in turn reflects into low productivity and labor
intensive (Dien et al. 2000). In general, fed-batch fermentation is not successful for
biomass to ethanol production. In reality, fed batch fermentation is more suitable
production process where the product is biomass associated. In such cases for
getting the higher concentration of required product, more biomass is needed. But
ethanol production does not relate directly to cell mass as it is not intracellular or
even periplasmic originated metabolite. Fed-batch operation can be more useful
where lignocellulose hydrolysate contains a high concentration of inhibitors so by
feeding the hydrolysate with slow rate, the effect of inhibitors can be minimized to
microorganism which can give a high concentration of ethanol with a considerable
good yield but at the helm of more time consumption, which will result in low
productivities (Olsson and Hahn-Hägerdal 1996).
There are two methods to increase cell density—Immobilization and recycling
of cell mass which leads to higher productivity and ultimately the requirement of
fermenter size and therefore the capital cost becomes lower.
244 G. J. de Moraes Rocha et al.

By supplying fresh lignocellulose hydrolysate to fermenting medium and

simultaneously withdrawing spent broth containing cells and ethanol, it becomes
continuous mode of cultivation. Both types of continuous systems—closed (where
cells are retained) and open (cell withdrawal) were applied for bioethanol fer-
mentation. The main problem in continuous cultivation is that it takes a long start
up time to establish steady state. The continuous process eliminates much of the
unproductive time associated with cleaning, recharging adjustment of media and
sterilization (Chandel et al. 2009). Prolonged continuous operation with the same
yeast can result in generation of a culture that becomes well adapted to the par-
ticular feed and processing conditions.

11.5.2 Separate Hydrolysis and Fermentation

In Separate hydrolysis and fermentation (SHF) process, biomass hydrolysis and

fermentation is performed separately. The recovered sugar solution from ligno-
cellulosic biomass either by thermochemical methods or enzymatic action contains
a variety of sugars: glucose, xylose, arabinose, mannose, cellobiose, and others.
This sugar solution can be fermented into ethanol by a suitable ethanol producer.
The fermentation of sugars into ethanol have several options to be adopted such as
batch, fed-batch, and continuous. The SHF process has been studied extensively in
the laboratories. During the SHF process, both yeast and enzymes can work at their
optimal temperature, but an accumulation of end products can reduce the effi-
ciency of hydrolysis (Margeot et al. 2009). Table 11.8 summarizes the examples of
ethanol production from lignocellulosic biomass employing SHF.
The SHF is a lengthy, cumbersome process which requires more process steps
and equipment/vessels making process overall more costly. The recovered sugar
solution can be concentrated by vacuum evaporation and subsequently can be
employed for fermentation process to obtain high ethanol concentration. Further-
more, the yeast cell mass can also be recovered and conditioned for the use in next
fermentation reaction. The solid lignocellulosic biomass recovered after thermo-
chemical hydrolysis or enzymatic hydrolysis can be used in co-generation for the
heat and electricity production. These features may be very useful to economize the
cellulosic ethanol production process following SHF process configuration.

11.5.3 Simultaneous Saccharification and Fermentation

The SSF have been investigated as a method of lignocellulosic conversion to

economic ethanol production (Takagi et al. 1977; Olofsson et al. 2008). Hydrolysis
and fermentation of released hydrolysate with ethanol producing microorganisms
can be performed simultaneously, which is referred to as SSF (Ohgren et al. 2006;
Rudolf et al. 2005).
Table 11.8 Recent reports on the cellulosic ethanol production from lignocellulosic raw materials adopting various process configurations
11

Raw material Type of pretreatment Process Microorganism used Ethanol production (g/l) or ethanol References
configuration yield (g/g or %)
Mixture of NA Fed-batch Scheffersomyces 40.7 g/L Unrean and Nguyen (2013)
glucose and (Pichia) stipitis
xylose
Saccharum Soaking in aqueous ammonia Recycling of Saccharomyces 21.66 ± 0.62 g/L (yield, Chandel et al. (2009)
spontaneum cells cerevisiae VS3 0.434 ± 0.021 g/g)
Cassava mash NA Continuous S. cerevisiae 86.1 g/L, and 91 % ethanol yield Moon et al. (2012)
CHFY0321
Triploid poplar Biodegradation (Fungal SSF S. cerevisiae 5.16 g/L Wang et al. (2013)
pretreatment with Trametes
velutina D10149)
Wheat straw Dilute acid SHF Recombinant 41.1 ± 1.1 g ethanol/L Saha et al. (2011)
Escherichia coli
strain FBR5
Sugarcane Ammonia fiber expansion SHF Recombinant S. 34–36 g/L of ethanol with 92 % Krishnan et al. (2010)
bagasse (AFEX) cerevisiae (424A theoretical yield
LNH-ST)
Switch grass AFEX SSCF Recombinant S. Jin et al. (2010)
cerevisiae (424A
LNH-ST)
Scale-up Pretreatment Studies on Sugarcane Bagasse and Straw

Spruce Steam pretreatment Fed-batch S. cerevisiae 68.9 % Hoyer et al. (2010)

SSCF
Rice straw Dilute acid SHF Pichia stipitis 0.44 ± 0.02 Lin et al. (2012)
Miscanthus Liquid hot water SSF Active dry yeast S. 98.27 % Li et al. (2013)
giganteus cerevisiae
Sugarcane Ball milling SHF S. cerevisiae 91.8 % da Silva et al. (2010)
straw
(continued)
245
Table 11.8 (continued)
246

Raw material Type of pretreatment Process Microorganism used Ethanol production (g/l) or ethanol References
configuration yield (g/g or %)
Water hyacinth Microbial pretreatment with SHF S. cerevisiae 0.192 g/g of dry matter Ma et al. (2010)
white rot fungus,
Echinodontium taxodii
Sorghum straw NA Deep-bed solid Thermotolerant 0.25 g-ethanol/g-dry stalk Kwon et al. (2011)
state Issatchenkia
fermentation orientalis IPE
100
Jerusalem CBP S. cerevisiae DQ1 128.7 g/L Guo et al. (2013)
artichoke
tuber
Corn stover AFEX Continuous Recombinant S. 80 % glucose-to-ethanol Jin et al. (2013)
SSCF cerevisiae (424A conversion and 47 % xylose-
LNH-ST) to-ethanol conversion
NA Not available
G. J. de Moraes Rocha et al.
11 Scale-up Pretreatment Studies on Sugarcane Bagasse and Straw 247

For the maximum conversion of released sugars (pentoses and hexoses) into
ethanol, mixture of yeasts can be used in the fermentation reaction, so-called
simultaneous saccharification and co-fermentation (SSCF). In general, SSF has
been pivotal to achieve fast hydrolysis reaction rates with low enzyme loadings
and high ethanol yields in addition to saving manifold processing time and min-
imizing the equipment usage and capital cost (Chandel et al. 2010).
In contrast to a set-up where hydrolysis and fermentation are separated (SHF),
glucose released during enzymatic cellulose hydrolysis, is simultaneously fer-
mented in an SSF set-up (Olofsson et al. 2008). Thus, glucose or any other released
sugars will not accumulate during SSF and enzyme inhibition due to glucose can be
avoided. This was confirmed in SSF of steam pretreated corn stover with S. cere-
visiae TMB3400 (Ohgren et al. 2006). If SSF or SSCF process performed effi-
ciently, ethanol could be produced at prices competitive with that of petroleum fuel.
The decrease in capital investment has been estimated to be more than 20 %
(Wingren et al. 2003). SHF and SSCF economics was also analyzed using cellu-
lase enzymes in both configurations with SSF being less expensive by about 10 %;
and estimated the ethanol production cost of 0.56–0.67 $/L (Wingren et al. 2003).
According to the National Renewable Energy Laboratory (NREL, Colorado, USA)
estimations, ethanol production cost of 20 cents per liter is possible in another
15 years from lignocellulose biomass employing designer cellulases and SSF
(Wingren et al. 2003). However, there are also disadvantages of SSF such as the
optimum temperature (45–50 C) for enzymatic hydrolysis of cellulosics is usually
higher than microbial fermentation of hydrolysates into ethanol (30 C). There-
fore, the thermotolerant ethanol producing microorganisms will be a desired
choice to be incorporated in SSF or SSCF to avoid the ethanol yield loss.

11.5.4 Consolidated Bioprocessing

The CBP is a consolidated technological platform summarizing all the critical steps
of bioethanol production, i.e., cellulase production, substrate hydrolysis, and fer-
mentation of released sugars into ethanol in one step. CBP can effectively save the
processing time, processing costs, energy while reducing the number of involved
steps (Lynd et al. 2005). However, it is very difficult to find such microorganism
that can perform all these reactions. CBP can save the bioethanol production cost
drastically due to elimination of requirement of enzymes addition from outside and
separate hydrolysis (Olson et al. 2012). The need of hour is to develop such
microorganism which can perform all these steps simultaneously. Several tech-
nological developments have been attempted aiming to develop the microbial traits
for the incorporation in CBP platform (van Zyl et al. 2011; Olson et al. 2012).
Goyal et al. (2011) developed a yeast consortium showing endoglucanase,
exoglucanase, and b-glucosidase enzyme titers aiming to utilize cellulose for
growth coupled with hydrolysis and ethanol production in one vessel (1.25 gL-1,
87 % of theoretical value). Jin et al. (2012) studied the ethanol production from
248 G. J. de Moraes Rocha et al.

Fig. 11.10 Schematic paradigm of major process configurations for cellulosic ethanol produc-
tion from lignocellulosic biomass

delignified corn stover (ammonia pretreated) using CBP approach. They found
48.9 % glucan conversion and 77.9 % xylan conversion after 264 h with 7 gL-1
ethanol production by Clostridium phytofermentans ATCC 700394.

11.5.5 Integrated Bioprocessing

All the four essential steps of biomass conversion into ethanol could be performed in a
single vessel sequentially first time termed as IBP, which is distinguished from other
less highly integrated configurations in that it does not involve a dedicated process
step for pretreatment. All necessary steps can be done in the same vessel in order to
combine the overall process steps. During the microbial pretreatment of LB,
microorganisms secrete a cocktails of plant cell wall degrading enzymes which can be
recovered (on-site enzyme production) and subsequently can be used for the sac-
charification of pretreated biomass alone or with the supplementation of necessary
enzymes from outside. There is involvement of at least two microorganisms in IBP
(first for delignification and second for ethanol production from released sugars from
pretreated cellulosic biomass). IBP may provide a unique breakthrough for cheap
cellulosic ethanol production due to economic advantages and time savings. How-
ever, there is no practical report using IBP for ethanol production as yet (Fig. 11.10).

11.6 Conclusions

As can be drawn from the above discussion, the success of next-generation bio-
fuels, such as cellulosic ethanol will depend on the efforts in reducing capital costs,
financial support during scale up, establishing feedstock supply arrangements, and
11 Scale-up Pretreatment Studies on Sugarcane Bagasse and Straw 249

overcoming blend wall constraints (Coyle 2010). There are a number of compo-
nents that affect the cellulosic ethanol production cost, estimating 14.5 % for
enzymes, 36.4 % for feedstock, 20 % of capital, and 29.1 of other components
such as pretreatment and fermentation steps (Coyle 2010).
The survey collected showed that in 2012, the cost of cellulosic ethanol pro-
duction was $0.94 per liter, around 40 % higher than the $0.67 per liter (L) cost of
producing ethanol from corn (Isola 2013). According to the world’s leading pro-
ducer of enzymes, the cost of enzymes for cellulosic ethanol had been reduced
significantly in the last 2 years to about 50 cents per gallon, reducing total pro-
duction costs in the near term to about $2 per gallon (Novozymes 2010). The costs
of cellulosic ethanol, that have fallen significantly, are expected to decline more as
companies scale-up production, but further advance in these technologies are
required to turn this process into a competitive fuel with first-generation ethanol
and gasoline (Ziolkowska et al. 2011; Isola 2013). Dilute acid hydrolysis and
steam explosion are the successful pretreatment technologies used for the sugar-
cane bagasse and straw which can be applicable in industrial-scale operations.

Acknowledgments This work was supported by Escola de Engenharia de Lorena da Univer-

sidade de São Paulo (EEL-USP), Laboratório Nacional de Ciência e Tecnologia do Bioetanol
(CTBE) belonging to Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), De-
partamento de Antibioticos da Universidade Federal de Pernambuco (UFPE), Débora Lee Simões
Corso a Graduate Student in Chemical Technology from the Universidade Estadual Paulista
(UNESP), Fundacão de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desen-
volvimento Científico e Tecnológico (CNPq).

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Chapter 12
Novel Yeast Strains from Brazilian
Biodiversity: Biotechnological
Applications in Lignocellulose Conversion
into Biofuels

Raquel Miranda Cadete, César Fonseca and Carlos Augusto Rosa

Abstract The bioprospection of novel biochemical traits from world biodiversity

is far underexploited. Brazil is one of the richest megadiverse countries, and a
source of new species and strains with potential application to biotechnological
processes. Among the organisms of interest, yeasts capable of fermenting sugars
from lignocellulosic biomass have particular interest for the development of
efficient fermentative technologies in the production of biofuels, like second-
generation ethanol, and other chemicals, like xylitol. In this chapter, recent studies
performed with novel Brazilian D-xylose- and/or cellobiose-fermenting yeasts are
highlighted. The new isolates from the genus Scheffersomyces and Spathaspora
represent an important contribution of new species and strains to yeast taxonomy
and ecology, and their characterization a first screening for potential biotechno-
logical applications. These yeasts species and strains represent a new set of
biological material that can be used directly in the conversion of lignocellulosic
biomass into value-added bioproducts, or a source of genetic material for the
improvement of the fermentative capacity of industrial microorganisms, like the
yeast Saccharomyces cerevisiae, toward the production of second-generation
biofuels.

R. M. Cadete (&)  C. A. Rosa

Departamento de Microbiologia, Universidade Federal de Minas Gerais,
Belo Horizonte, MG, Brazil
e-mail: raquelcadete@gmail.com
C. A. Rosa
e-mail: carlrosa@icb.ufmg.br
C. Fonseca
Unidade de Bioenergia, Laboratório Nacional de Energiae Geologia,
I. P., Lisbon, Portugal
e-mail: cesar.fonseca@lneg.pt

S. S. da Silva and A. K. Chandel (eds.), Biofuels in Brazil, 255

DOI: 10.1007/978-3-319-05020-1_12,  Springer International Publishing Switzerland 2014
256 R. M. Cadete et al.

12.1 Introduction

Yeast biotechnology embraces a variety of processes involving these microor-

ganisms, their enzymes, and metabolites for the production of fermented foods and
beverages, fuels, chemicals, and pharmaceuticals. Traditional attributes of yeasts
include their primary roles in fermentation processes, particularly in alcoholic
fermentation. The use of Saccharomyces cerevisiae in ethanol production
processes is well documented and has been exploited for centuries, namely in the
production of alcoholic beverages. The application of ethanol from fermentative
origin as a partial or total substitute of gasoline in spark-ignition internal com-
bustion engines is continuously increasing and has been significantly used for
decades in Brazil and USA—first-generation (1G) bioethanol. The interest in
bioethanol as a renewable energy source, especially as fuel for transportation, has
been fluctuating substantially during most of the last century mainly in response to
oil crises.
The growing concern about the environmental consequences of the intensive use
of fossil fuels have been promoting innumerous policy actions toward the reduction
of greenhouse gas (GHG) emissions, including incentives for the use of renewable
sources with low impact in environment for the production of fuels and chemicals.
Lignocellulosic materials from forestry, agriculture, agro-industry, or municipal
solid waste constitute the most abundant renewable raw feedstock in the world. The
composition of lignocellulosic materials varies among plant species but generally
consists of *25 % lignin and *75 % carbohydrate polymers (cellulose and
hemicellulose) (Zaldivar et al. 2001). The cellulosic and hemicellulosic fractions
can be separated from the lignin and depolymerized by physical, chemical, and
biochemical processes (pretreatment and enzymatic hydrolysis) to obtain their
constituent sugars, mainly hexoses (glucose) from cellulose and pentoses (usually
D-xylose) from hemicellulose, which, in turn, can be converted to ethanol by
biological processes (fermentation). The economic viability of ethanol produced
from lignocellulosic materials—second-generation (2G) bioethanol—is dependent
on the complete utilization of the carbohydrate fraction, including hemicellulose
(van Vleet and Jeffries 2009; Gírio et al. 2010). However, S. cerevisiae lacks the
ability to ferment a number of saccharides derived from cellulose and hemicellu-
lose. It cannot readily utilize cellulose and hemicellulose, nor the disaccharides
cellobiose and xylobiose (Lynd et al. 2002), neither can ferment the pentose sugars
D-xylose and L-arabinose (Hahn-Hägerdal et al. 2007; van Vleet and Jeffries 2009;
Gírio et al. 2010). On the other hand, among yeasts other than Saccharomyces spp.,
referred to as nonconventional yeast (NCY) (Boekhout and Kurtzman 1996) are
those that consume the pentose sugars D-xylose (Skoog and Hahn-Hägerdal 1988),
L-arabinose (Dien et al. 1996; Fonseca et al. 2007) and lignocellulose-derived
di- and trisaccharides (Freer and Detrov 1983; Parekh and Wayman 1986; Golias
et al. 2002; Ryabova et al. 2003). The so-called NCYs provide alternative
biocatalysts for metabolic engineering of S. cerevisiae toward the utilization of
all the sugars present in lignocellulosic materials (Hahn-Hägerdal et al. 2007).
12 Novel Yeast Strains from Brazilian Biodiversity 257

The increased exploration of NCYs including detailed characterization of their

physiology, metabolism, and genomics are inevitably leading to a wide range of
useful biotechnological and industrial applications (Wolf et al. 2003; Buzzini and
Vaughan-Martini 2006).

12.2 Yeast Biotechnology and Biodiversity

Yeasts are unicellular fungi characterized by a widespread distribution throughout

all biomes in the world in association with sugary substrates like flowers, fruits,
tree exudates, leaves, and mushrooms. These organisms also occupy a diverse
variety of micro-ecosystems and are well adapted to a wide range of weathers,
altitudes, substrates, and geographical locations, being found in glaciers, high
salinity lakes, water, soil, air, and gut of a variety of vertebrates and invertebrates
(Rosa and Péter 2006; Starmer and Lachance 2011).
Yeasts have benefitted humankind for millennia. These microorganisms have
wide-ranging fundamental and industrial importance in scientific, food, medical,
and agricultural disciplines. Traditional industrial attributes of yeasts include their
primary roles in many food fermentations and other long-standing industrial
processes, like the production of fuel ethanol, single cell protein (SCP), feeds and
fodder, industrial enzymes and small molecular weight metabolites, and as host for
the heterologous expression of proteins of interest. Several of these processes and
products have reached commercial scale, while others are still under development
(Johnson and Echavarri-Erasun 2011).
Once that microbial biodiversity is the source of innovation in biotechnology
(Bull et al. 1992), the exploration and isolation of yeasts from various habitats is a
growing field where novel species and its physiological abilities are potentially
useful in the search of new products by means of direct application in industrial
processes or metabolic engineering approaches of industrial microorganisms, like
the yeast S. cerevisiae (Barriga et al. 2011). The number of fungi present on Earth
is estimated between 1.5 and 3.3 million species (Hawksworth 1991, 2012) and
about 100,000 species have already been described (Hibbett et al. 2011). Cur-
rently, nearly 1,500 yeast species among 149 genera are recognized (Kurtzman
et al. 2011). Therefore, it is estimated that the vast majority of the potential
biodiversity of yeasts is still unknown, which supports a need for increasing efforts
to study the biological diversity of these microorganisms, especially in mega
diverse countries from the tropical regions of the planet. To date, most of yeast
species cataloged has been discovered in countries from the Northern hemisphere.
Relatively few studies dedicated to yeast biodiversity have been done in tropical
zones of the planet and in Southern hemisphere countries that embrace abundant
and diverse ecosystems. Particularly, South America is a region that offers great
potential in terms of biodiversity (Barriga et al. 2011), being Brazil, the largest
country located in this continent, considered one of the world’s richest megadi-
versity countries (Mittermeier et al. 2005).
258 R. M. Cadete et al.

12.2.1 Brazilian Biodiversity as a Potential Source

of New Yeasts

Brazil spans 8.5 million km2. Its geographic space presents a great diversity of
climate types, physiognomy, soils, and vegetation. These great ecological varia-
tions led to the formation of distinct biogeographical zones or biomes within the
country: Amazon, the world’s biggest rainforest (which spans 49 % of the Bra-
zilian territory); Pantanal (1.7 %), the biggest flood plain; Cerrado (23.9 %), with
savannahs and woods; Caatinga (9.9 %), with semiarid forests; Pampas’ meadows
(2 %); and the Atlantic rainforest (13 %) (IBGE 2010). Two of these biomes—the
Atlantic rainforest and the Cerrado—are classified as hotspot regions, areas with
high biodiversity, elevated levels of endemism, and great anthropic pressure.
Moreover, the Atlantic rainforest is considered as one of the five leading biodi-
verse hotspots of the planet (Myers et al. 2000). These relevant ecosystems,
together with the Amazonian forest, also embraces ecoregions, defined as a
relatively large unit of land or water containing a characteristic set of natural
communities that share a large majority of their species, dynamics, and environ-
mental conditions (Dinerstein et al. 1995; Olson and Dinerstein 1998). Ecoregions
function effectively as conservation units at regional scales because they hold
similar biological communities and because their boundaries roughly coincide
with the area over which key ecological processes most strongly interact (Orians
1993; Noss 1996).
Forest ecosystems are an attractive site for the collection of yeasts (Morais et al.
2006). Approximately 2.3 million km2 of Brazil—27 % of its total area and
almost 17 % of the world’s global stock—comprises tropical moist forests, making
it the third highest ranked country in terms of remaining frontier forest and the first
in plant biodiversity among frontier forest nations. Its tropical forest endowment
and its importance to global biodiversity are unparalleled in the world (Lele et al.
2000; Morais et al. 2006). Ecosystems such as forests are considered a mosaic of
patchy habitats for organisms, consisting of soil, litter, tree stems, trunks, canopy,
flowers, and fruits, a feature that supports a huge biodiversity of microorganisms
and represents different niches for colonization of yeasts (Morais et al. 2006). Until
now, the studies on yeast from Brazil’s ecosystems have focused mainly on
Atlantic rainforest (Morais et al. 1992, 1995a, 1996; Prada and Pagnocca 1997;
Abranches et al. 1998; Araújo et al. 1998; Ruivo et al. 2004, 2005, 2006; Rosa
et al. 2007a; Barbosa et al. 2009; Cadete et al. 2009; Pimenta et al. 2009; Santos
et al. 2011; Morais et al. 2013a, b). Few studies have been conducted on Cerrado
ecosystem (Morais et al. 2004; Rosa et al. 2007b, 2009, Canelhas et al. 2011;
Barbosa et al. 2012; Safar et al. 2013) and Amazonian forest sites (Mok et al.
1984; Morais et al. 1994, 1995b; Vital et al. 2002, Cadete et al. 2012a, b, 2013),
which, in association with the frequent discovery of new yeast species regardless
of the sampling area, increases the impact of the rarity of studies on yeast from
12 Novel Yeast Strains from Brazilian Biodiversity 259

Brazilian biomes. In this chapter, the recognition of these ecosystems as potential

locals for research on yeast biodiversity toward its biotechnological application for
biomass conversion to biofuels (2G bioethanol) and other bioproducts is shown.

12.3 Fermentation of Lignocellulosic Sugars

Lignocellulose is a complex and chemically rich material represented by the

physical–chemical interaction of cellulose, a linear glucose polymer, with hemi-
cellulose, a highly branched sugar heteropolymer, and lignin, a high molecular
weight and cross-linked aromatic macromolecule (Ferreira-Leitão et al. 2010). The
pretreatment of lignocellulosic materials by hydrothermal or other acidic methods
generates a liquid fraction also named hemicellulosic hydrolysate. The conversion
of the hemicellulosic hydrolysate is a challenge on ethanol production from
lignocellulose, due to the presence of inhibitors of microbial metabolism (Almeida
et al. 2007) and to the heterogeneity of sugars usually found in this fraction—
oligosaccharides like xylooligosaccharides (XOS), and monosaccharides like
D-xylose L-arabinose, D-galactose, D-glucose and D-mannose. The identification
or development of microbial strains able to efficiently ferment these sugars is
mandatory for the successful industrial production of ethanol (Hahn-Hägerdal
et al. 2007; Fukuda et al. 2009; van Vleet and Jeffries 2009; Gírio et al. 2010;
Ferreira et al. 2011).
The desired properties of strains required for fermenting lignocellulosic
hydrolysates are: the efficient utilization of hexoses and pentoses; high ethanol
titers, yields and productivities; high tolerance to ethanol, fermentation inhibitors,
low pH and high temperature; high viability and vitality; and others process-
specific characteristics like sugar co-consumption and appropriate flocculation
properties (Hahn-Hägerdal et al. 2007; Pasha et al. 2007). The yeast S. cerevisiae
is the most commonly used microorganism in traditional industrial fermentations,
including current sucrose-, starch-, and cellulose-based bioethanol production.
Saccharomyces cerevisiae is also generally recognized as safe (GRAS) and can
ferment efficiently simple hexose sugars, such as D-glucose, D-mannose and
D-galactose, and disaccharides like sucrose and maltose, reaching ethanol con-
centrations as high as 20 % (v/v) (Gírio et al. 2010). Moreover, this species has a
relatively good tolerance to lignocellulose-derived inhibitors and to high osmotic
pressure (Almeida et al. 2007). The major inconvenience to the use of S. cerevisiae
for lignocellulosic fermentation is its lack of natural ability to utilize the pentose
sugars D-xylose and L-arabinose (Hahn-Hägerdal et al. 2007; van Vleet and
Jeffries 2009; Gírio et al. 2010). Contrary to S. cerevisiae, other yeast species, and
also bacteria and filamentous fungi can ferment pentoses. However, despite the
existence of pentose-fermenting microorganisms and the innumerous efforts on
metabolic engineering of S. cerevisiae, it is still challenging to reach high ethanol
productivities from pentose sugars while simultaneously withstanding fermenta-
tion inhibitors (Hahn-Hägerdal et al. 2007; Chandel et al. 2011). The identification
260 R. M. Cadete et al.

and/or development of new yeast strains which ferment hemicellulosic sugars will
improve prospects for lignocellulosic ethanol production (Jeffries and Kurtzman
1994; Hahn-Hägerdal et al. 2007; van Vleet and Jeffries 2009). The exploitation of
biodiversity through the identification of novel microorganisms and their unique
traits and the use of adaptation strategies and/or metabolic and evolutionary
engineering approaches are contributing to the development of novel cell factories
for the production of bioethanol, other biofuels, and biochemicals from lignocel-
lulosic materials (van Maris et al. 2006; Sanchez et al. 2010; Fonseca et al. 2011;
Nielsen et al. 2013).

12.3.1 D-xylose-Fermenting Yeasts

Pentose (C5) sugars can constitute up to 70 % of the fermentable sugars in

hydrolysates (Gírio et al. 2010). Once that high ethanol yields are required for the
development of economically feasible second-generation ethanol processes, the
conversion of C5 sugars is a prerequisite for a cost-effective lignocellulosic
ethanol production (van Vleet and Jeffries 2009; Gírio et al. 2010). As the major
pentose in hemicelluloses from hardwood, cereals, and other herbaceous crops,
D-xylose is the second most abundant sugar component in lignocelluloses and the
second most abundant carbohydrate in nature after glucose (Jeffries 2006;
Watanabe et al. 2007). This feature makes D-xylose the C5 sugar most studied in
lignocellulose fermentation processes. Several bacteria, yeasts, and filamentous
fungi naturally ferment D-xylose to ethanol (Jeffries 1983). Yeasts have advan-
tages over bacteria for commercial fermentations due to larger size, thicker cell
wall, better growth at low pH, less stringent nutritional requirement, higher
tolerance to fermentation products, and greater resistance to contamination
(Jeffries 2006), whereas presenting higher rates of sugar consumption and product
formation than filamentous fungi (Skoog and Hahn-Hägerdal 1988).

12.3.1.1 D-xylose Metabolism

The pentose phosphate pathway (PPP) is the biochemical route for D-xylose
metabolism. This pathway is found in virtually all cellular organisms providing
D-ribose for nucleic acid biosynthesis, D-erythrose-4-phosphate for the synthesis
of aromatic amino acids, and NADPH for anabolic reactions. The PPP consist of
two parts. The oxidative part converts the hexose D-glucose-6-phosphate into the
pentose D-ribulose-5-phosphate, plus CO2, and NADPH. The nonoxidative part
converts D-ribulose-5-phosphate into D-ribose-5-phosphate, D-xylulose-5-
phosphate, D-sedoheptulose-7-phosphate, D-erythrose-4-phosphate, D-fructose-
6-phosphate, and D-glyceraldehyde-3-phosphate. D-glyceraldehyde-3-phosphate
and D-fructose-6-phosphate can be converted to pyruvate in the Embden-
Meyerhof-Parnas pathway (glycolysis). Pyruvate can either be decarboxylated and
12 Novel Yeast Strains from Brazilian Biodiversity 261

reduced to ethanol or can enter the tricarboxylic acid cycle. To enter the central
carbon metabolism, D-xylose must first be converted to the intermediate
compound of the PPP, D-xylulose-5-phosphate, and, essentially, two different
pathways are available in nature for the conversion of D-xylose into D-xylulose:
reduction/oxidation-based pathways and isomerization-based pathways (Bettiga
et al. 2008). In D-xylose-utilizing yeasts, aerobic fungi, and other eukaryotes, this
proceeds via a two-step reduction and oxidation mediated by xylose reductase
(XYL1, Xyl1p, XR) and xylitol dehydrogenase (XYL2, Xyl2p, XDH), respectively
(Kötter et al. 1990; Jeffries 2006). D-xylose is first reduced by XR to xylitol which
is then oxidized to D-xylulose through XDH. In bacteria and some anaerobic
filamentous fungi, the D-xylose is directly converted into D-xylulose by a xylose
isomerase (xylA, XI) (Walfridsson et al. 1996; Kuyper et al. 2003; Jeffries 2006).
After D-xylose conversion to D-xylulose through XR/XDH or XI, the metabolism
proceeds via phosphorylation of D-xylulose, a reaction catalyzed by xylulokinase
(XKS1 or XYL3, Xks1p or Xyl3p, XK) (Jeffries 2006).
The D-xylose-oxido-reductase pathway found in yeasts faces cofactor
requirements—NAD(P)(H)—by XR and XDH, which has great impact in xylitol
and ethanol yields from D-xylose fermentation under oxygen-limited conditions
(Bruinenberg et al. 1983). The relevance of this topic led to an extensive char-
acterization of these enzymes with respect to enzymatic activity, specificity, and
cofactor requirement in yeasts grown under different experimental conditions. The
existence of XRs strictly NADPH-dependent or showing dual cofactor specificity,
with preference for NADH or NADPH has been shown (Bruinenberg et al. 1983,
1984a; Yablochkova et al. 2003; Hou 2012). XDH activities are virtually NAD+-
dependent. Indeed, no correlation was observed between the ability to ferment
D-xylose and the activity of NADP+-linked XDH (Bruinenberg et al. 1984a).
Although the existence of NAD(P)H-utilizing XRs, NADPH is still the preferred
cofactor in most of known D-xylose-fermenting yeasts. Therefore, the different
cofactor requirement of XR and XDH (NADPH and NAD+, respectively) leads to
the accumulation of NADP+ and NADH. In addition, the absence of transhydro-
genase in yeast prevents cofactor interconversion (Bruinenberg et al. 1985;
Dellomonaco et al. 2010). Whereas NADP+ can be reduced through recycling
D-fructose-6-phosphate (via D-glucose-6-phosphate) in the oxidative PPP during
pentose metabolism (Bruinenberg et al. 1983; Fonseca et al. 2008), NADH is
mainly oxidized to NAD+ through oxygen in the respiratory chain. Under oxygen
limitation, NAD+ is not efficiently regenerated, and xylitol is accumulated (Gírio
et al. 2010). Thus, yeasts harboring strictly NADPH-dependent XR produce xylitol
as the major product of D-xylose fermentation under oxygen-limited conditions
(Bruinenberg et al. 1984b; Gírio et al. 1994; Silva et al. 1996; Fonseca et al. 2007).
Yeasts producing a XR with dual cofactor specificity can oxidize NADH to NAD+
in this step, thereby reducing xylitol formation and allowing D-xylose fermenta-
tion to proceed under oxygen-limited conditions (Bruinenberg et al. 1983).
A direct relationship between the dual cofactor dependence of XR with regard to
NADH-linked activities and the ability to ferment D-xylose to ethanol with high
262 R. M. Cadete et al.

efficiency by naturally or mutated D-xylose-fermenting yeasts has already been

demonstrated (Bruinenberg et al. 1984a; Watanabe et al. 2007; Bengtsson et al.
2009; Runquist et al. 2010; Hou 2012).

12.3.1.2 Ecology, Taxonomy, and Phylogenetic Relationships

of D-xylose-Fermenting Yeasts

The first researches focused on D-xylose conversion to ethanol by yeasts emerged

in the 1980s. These studies attempt to isolate and screen D-xylose-fermenting
yeasts (Nigam et al. 1985; du Preez and Prior 1985), to demonstrate the fermen-
tation of this pentose by several strains (Gong et al. 1983; Toivola et al. 1984), or
by particular species (Jeffries 1981; Schneider et al. 1981; du Preez and van der
Walt 1983; Dellweg et al. 1984), and to verify the impact of several parameters in
this process, like the oxygen supply (Delgenes et al. 1986; Skoog and Hahn-
Hägerdal 1990), mixtures of hemicellulosic sugars (du Preez et al. 1986; Jeffries
and Sreenath 1988), high D-xylose concentrations (Slininger et al. 1985), the
nitrogen sources, and the pH (Jeffries 1985). Studies addressing the transport of
D-xylose were also initiated in this decade (Kilian and Uden 1988). In common,
these reports revealed as best D-xylose-fermenting yeasts producing ethanol
the species Pachysolen tannophilus, Scheffersomyces (Pichia) stipitis, and
Scheffersomyces (Candida) shehatae, although the recognition of other D-xylose-
fermenting species. Indeed, until a few years ago, the majority of studies con-
cerning D-xylose conversion to ethanol have been conducted with these three yeast
species, being Sc. stipitis considered the best D-xylose-fermenting yeast and
the source of genes for metabolic engineering of S. cerevisiae for D-xylose
fermentation (Kötter et al. 1990). More recently, the isolation of new D-xylose-
fermenting species and strains especially from the Spathaspora clade in particular
habitats, among which Brazilian ecosystems stands out for its important contri-
bution, came to cause upheavals in this scenario (Nguyen et al. 2006; Barbosa
et al. 2009; Cadete et al. 2009, 2012a, b, 2013; Morais et al. 2013b).
D-xylose-fermenting yeasts exhibit as a common feature the association with
vegetal biomass sources (Fig. 12.1). These microorganisms have been isolated
from tree exudates (Nigam et al. 1985), wood-boring insects (Toivola et al. 1984;
Suh et al. 2003; Nguyen et al. 2006; Urbina et al. 2012, 2013), decaying wood
(Toivola et al. 1984), rotten fruit, and tree bark (Rao et al. 2008). This behavior can
be explained by the role of yeast ecology in these substrates and by the fact that
biomass materials contain D-xylose in their structures. Typically, yeasts are con-
sidered as primarily decomposers among the earlier colonizers of nutrient rich
substrates, where they are followed by a succession of organisms that degrade dead
organic matter. Land plant tissues (stems, flowers, and fruits) are rich in organic
compounds and moisture, and consequently provide a favorable environment for
yeast growth. Likewise, exudates of leaves, roots, flowers, and tree trunks are good
habitats in which yeasts flourish. Many yeast species that are found in live or
decaying plant parts are associated with insects that also use these habitats as
12 Novel Yeast Strains from Brazilian Biodiversity 263

Fig. 12.1 Sampling of rotting wood in an Atlantic rainforest site (Nova Friburgo, RJ, Brazil) to
isolate D-xylose-fermenting yeasts (on left) and budding yeast cell and asci of Spathaspora
passalidarum NRRL Y-27907 (type strain) cultured on diluted V8 agar after 5 days at 20 C
(on right)

feeding or breeding sites. In general, these three-part associations (insect-yeast-

plant) are maintained by reliance on reciprocal benefits exchanged by the insect-
yeast partners. Often the yeast supplies essential nutrients or beneficial supplements
to the insect while the insect provides transportation of the yeast to new habitats
(Starmer and Lachance 2011).
Besides being linked to common substrates, D-xylose-fermenting yeasts display
close phylogenetic relationships. Although these microorganisms appear scattered
throughout the subphylum Saccharomycotina (Kurtzman et al. 2011; Urbina et al.
2012), the species that have been reported to exhibit the highest rates of D-xylose
fermentation and ethanol production are members of the clades Scheffersomyces
(Suh et al. 2006; Jeffries et al. 2007; Kurtzman and Suzuki 2010; Urbina et al.
2012), and Spathaspora (Nguyen et al. 2006; Wohlbach et al. 2011; Hou 2012;
Long et al. 2012; Cadete et al. 2013). Both clades belong to the same family,
Debaromycetaceae, and are phylogenetically closed to each other in relation to
other clades of this family (Kurtzman et al. 2011). The ascosporic species assigned
to the genus Scheffersomyces were initially identified within a polyphyletic genus,
Pichia, whose classification based on phenotypic similarities, such as formation of
hat-shaped ascospores and inability to assimilate nitrate as a sole source of
nitrogen resulted in the placement of phylogenetically distant species within the
same genus (Kurtzman and Suzuki 2010; Kurtzman 2011). The combined analyses
of sequences of the D1/D2 domains of the large subunit and the nearly complete
small subunit rRNA genes showed that the species described as Pichia stipitis,
P. segobiensis, and P. spartinae were distantly related to Pichia membranifaciens,
the type species of the genus Pichia (Kurtzman and Robnett 1998), but phylo-
genetically close to each other, leading to the propose of the genus Scheffer-
somyces (Kurtzman and Suzuki 2010) to the reclassification of these species.
Currently, the genus Scheffersomyces is represented by 16 species (Cadete et al.
2012b; Urbina et al. 2012, 2013) including the asexual species previously
264 R. M. Cadete et al.

described as Candida (C. amazonensis, C. coipomoensis, C. ergatensis, C.

gosingica, C. insectosa, C. lignicola, C. lignosa, C. queiroziae, and C. shehatae)
and now identified as Scheffersomyces (Sc. amazonensis, Sc. coipomoensis and so
on) and by the species Sc. stipitis (type species) Sc. segobiensis, Sc. spartinae,
Sc. quercinus, Sc. illinoinensism, Sc. virginianus, and Sc. cryptocercus. In addition
to the update of the genus, the Scheffersomyces clade was divided into three
subclades:
(1) the early diverging S. spartinae and S. gosingicus (C. gosingica) subclade,
being this last species able to ferment cellobiose;
(2) the cellobiose-fermenting Sc. ergatensis subclade, comprising the species Sc.
amazonensis, Sc. coipomoensis, Sc. ergatensis, Sc. lignicola, and Sc. queiroziae;
(3) the largest, D-xylose-fermenting Sc. stipitis subclade presenting the remaining
species. Within the Scheffersomyces clade, Brazilian isolates have so far
contributed with new Sc. stipitis and Sc. shehatae strains (Ferreira et al. 2011;
Cadete et al. 2012a; Martiniano et al. 2013a, b) and mainly to the description
of the new species Sc. amazonensis (Cadete et al. 2012b) and Sc. queiroziae
(Santos et al. 2011).
The Spathaspora clade was first described to harbor the species Sp. passal-
idarum, the first teleomorphic species of the genus (Fig. 12.1), and the anamorphic
species C. jeffriesii. Both species were isolated from wood-boring beetles col-
lected, respectively, in Louisiana (USA) and Chiriqui (Panama) (Nguyen et al.
2006). Today, the Spathaspora clade is represented by the teleomorphic species
Sp. passalidarum, Sp. arborariae, Sp. brasiliensis, Sp. roraimanensis, and Sp.
suhii, and by the anamorphic species previously described as belonging to the
genus Candida, Sp. jeffriesii, Sp. lyxosophila, Sp. materiae, Sp. insectamans, Sp.
subhashii, and Sp. xylofermentans (Nguyen et al. 2006; Barbosa et al. 2009;
Cadete et al. 2009, 2012a, 2013). Among these species, six were described from
isolates associated to rotting wood sampled in Brazilian biomes: Sp. materiae
(Barbosa et al. 2009), Sp. arborariae (Cadete et al. 2009), and Sp. brasiliensis, Sp.
roraimanensis, Sp. suhii and Sp. xylofermentans (Cadete et al. 2013). Also, six new
Sp. passalidarum strains, a species described from a single isolate and recently
shown as the best ethanol from D-xylose-producing yeast ever reported (Hou
2012; Long et al. 2012) were obtained from a forest reserve located in the
Brazilian Amazonian forest (Cadete et al. 2012a).

12.3.1.3 Studies with Brazilian Yeasts

In the past few years, the Brazilian biodiversity has contributed with new
D-xylose-fermenting yeast species and strains, and studies conducted with these
organisms regarding physiology, biochemistry, molecular biology, and genomics
have already been published or are still in progress. All these research efforts show
potential results concerning the bioconversion of D-xylose to ethanol or xylitol
under different culture conditions (Table 12.1).
Table 12.1 Main fermentation product (ethanol or xylitol) yield [Yp/s (g.g-1)] and productivity [Qp (g.l-1.h-1)] achieved in defined or hydrolysate
12

fermentation media by Brazilian D-xylose-fermenting strains

Clade Species Strain Fermentation Main fermentation Yp/s (g.g-1) Qp (g.l-1.h-1) Reference
medium product
Scheffersomyces Sc. stipitis UFMG-XMD-15.2 YPXa Ethanol 0.28 0.51 Cadete et al. (2012a)
UFMG-HMD-32.1 0.22 0.23
UFMG-XMD-15.2 ScBHHb 0.34 0.20
UFMG-IMH-43.2 0.19 0.13 Ferreira et al. (2011)
Sc. shehatae BR6-2AI YPX 0.45 0.35 Martiniano et al. (2013a)
CG8-8BY 0.47 0.37
PTI-1BASP 0.44 0.36
BR6-2AY 0.48 0.37
CG8-8BY ScBHH 0.3 0.15
BR6-2AY 0.21 0.11
CG8-8BY ScBHH 0.33 0.21 Martiniano et al. (2013b)
CG8-8BY ScBHH 0.2 0.12
UFMG-HM-52.2 ScBHH 0.35 0.13 Chandel et al. (2013)
UFMG-HM-52.2 ScBCHc 0.28 0.20
Novel Yeast Strains from Brazilian Biodiversity

Scheffersomyces Sc. amazonensis UFMG-XMD-24.1 YPX Xylitol 0.59 – Cadete et al. (2012a)
UFMG-XMD-26.2 0.58 –
UFMG-HMD-26.3 0.57 –
UFMG-XMD-40.2 0.55 –
UFMG-XMD-40.3 0.56 –
Spathaspora Sp. passalidarum UFMG-HMD-1.1 Ethanol 0.36 0.75
UFMG-HMD-1.3 0.35 0.72
UFMG-HMD-2.1 0.31 0.62
UFMG-HMD-10.2 0.33 0.69
UFMG-HMD-14.1 0.37 0.68
UFMG-HMD-16.2 0.33 0.64
UFMG-HMD-1.1 ScBHH 0.2 0.09
UFMG-HMD-14.1 0.18 0.10
(continued)
265
Table 12.1 (continued)
266

Clade Species Strain Fermentation Main fermentation Yp/s (g.g-1) Qp (g.l-1.h-1) Reference
medium product
Spathaspora Sp. arborariae UMFG-HM-19.1A YPX Ethanol 0.50 – Cadete et al. (2009)
G20X20Ad10 0.46 0.21 Cunha-Pereira et al.
RHHe 0.45 0.16 (2011)
ScBHH 0.14 0.04 Martiniano et al. (2013b)
Sp. brasiliensis UMFG-HMD-19.3 YPX Xylitol 0.16 – Cadete et al. (2012a)
Sp. roraimanensis UFMG-XMD-23.2 Ethanol 0.26 0.21
Sp.suhii UFMG-XMD-16.2 0.33 0.27
UFMG-HMD-16.3 0.27 0.22
Sp. xylofermentans UFMG-HMD-23.3 0.18 0.10
UFMG-HMD-25.5 Xylitol 0.22 –
Sp. roraimanensis UFMG-XMD-23.2 ScBHH 0.61 –
Sp. suhii UFMG-XMD-16.2 0.57 –
a
YPX = D-xylose, peptone and yeast extract
b
ScBHH = sugarcane bagasse hemicellulosic hydrolysate
c
ScBCH = sugarcane bagasse cellulosic hydrolysate
d
G20X20A10 = glucose, D-xylose and arabinose
e
RHH = rice hull hydrolysate
R. M. Cadete et al.
12 Novel Yeast Strains from Brazilian Biodiversity 267

The species Sc. stipitis and Sc. shehatae have been the D-xylose-fermenting
yeasts better described in the past decades and the source of genes for metabolic
engineering of S. cerevisiae (Bruinenberg et al. 1984a; Verduyn et al. 1985; du
Preez et al. 1986; Ligthelm et al. 1988; Prior et al. 1989; Skoog and Hahn-
Hägerdal 1990; Kötter et al. 1990). It is expected that the screening, identification,
and characterization of new and unrelated species from biomes harboring high
biodiversity would even be more beneficial for yeast biotechnology, once the
access to that genetic diversity will certainly conduce to the identification of new
traits (Lachance 2006). Also, it is well known that there are variations within
strains from the same species, and some metabolic abilities/disabilities are not
necessarily linked to the species but result rather from strain variability (Barriga
et al. 2011). Thus, the bioprospection toward the identification of new yeasts able
to convert lignocellulosic sugars would generate a portfolio of species, strains, and
varieties suitable for exploitation purposes to the conversion of lignocellulose into
value-added products, like 2G bioethanol, other advanced fuels, and chemicals.
In the past years, a strong effort has been made in Brazil for the identification of
novel yeasts able to ferment lignocellulosic sugars (e.g., D-xylose and cellobiose).
Scheffersomyces stipitis strains have been isolated from the gut of wood-boring
insects collected in a natural reserve of Atlantic rainforest (Cadete 2009; Ferreira
et al. 2011) and from rotting wood sampled in forest reserves of Amazonian forest
(Cadete et al. 2012a). Scheffersomyces shehatae strains have been isolated from
rotting wood of Atlantic rainforest (Cadete 2009; Chandel et al. 2013) and
different natural habitats within Brazilian forests, like bromeliads, mushroom, and
palm tree (Martiniano et al. 2013a, b).
The new D-xylose-fermenting strains have been recently tested for D-xylose
fermentation under different conditions, including define medium and hemicellu-
losic hydrolysates. The production of ethanol from D-xylose by Sc. stipitis UFMG-
IMH-43.2 was evaluated in a hemicellulosic hydrolysate obtained by dilute-acid
hydrolysis of sugarcane bagasse (Ferreira et al. 2011). The supplementation of the
fermentation medium (with MgSO47H2O, yeast extract and/or urea) was required,
and yeast extract was reported as favoring ethanol production (Ferreira et al.
2011). Also, initial D-xylose concentration and inoculum load showed significant
(p \ 0.05) influence on ethanol production. The best results (ethanol yield and
productivity of 0.19 g.g-1 and 0.13 g.l-1.h-1, respectively) were obtained using
the hydrolysate containing an initial D-xylose concentration of 30 g.l-1, supple-
mented with 5.0 g.l-1 yeast extract and inoculated with an initial cell concentra-
tion of 2.0 g.l-1 (Ferreira et al. 2011).
Two Sc. stipitis strains isolated from the Brazilian Amazonian forest were
tested in complex medium (YPX) with D-xylose as sole carbon source and peptone
and yeast extract as nitrogen sources and in detoxified sugarcane bagasse hydro-
lysate (Cadete et al. 2012a). Great differences were observed in the behavior
of each strain during the fermentation assay. In the complex medium, strain
UFMG-XMD-15.2 showed the best ethanol production results, yielding 0.28 g.g-1
ethanol, with productivity equal to 0.51 g.l-1.h-1, whereas strain UFMG-
HMD-32.1 presented yield and productivity of 0.22 g.g-1 and 0.23 g.l-1.h-1,
268 R. M. Cadete et al.

respectively. Due to its good ethanol production achieved in the complex medium,
Sc. stipitis UFMG-XMD-15.2 was evaluated together with several NCY strains
and species using hemicellulosic hydrolysate. Among the microorganisms tested,
this strain was the best D-xylose-fermenting, reaching ethanol yield of 0.34 g.g-1
and productivity equal to 0.20 g.l-1.h-1.
Scheffersomyces shehatae UFMG-HM-52.2 was assayed in batch fermentations
of hemicellulosic and cellulosic hydrolysates prepared from sugarcane bagasse
pretreated with oxalic acid (OA) and detoxified using calcium hydroxide over-
liming or subjected to enzymatic hydrolysis after OAFEX pretreatment (Chandel
et al. 2013). In detoxified hemicellulosic acid hydrolysate, this strain reached
an ethanol yield and productivity of 0.35 g.g-1 and 0.13 g.l-1.h-1, respectively.
When the cellulosic fraction was fermented after enzymatic hydrolysis, an ethanol
yield of 0.28 g.g-1 and productivity equal to 0.20 g.l-1.h-1 were obtained.
Additionally, Sc. shehatae UFMG-HM-52.2 showed a similar growth pattern in
both hydrolysates, being more than 80 % of the sugars utilized within 24 h of
incubation. To compare the behavior of different strains from the same species,
four Sc. shehatae strains (BR6-2AI, CG8-8BY, PT1-1BASP, BR6-2AY) were
evaluated under the same D-xylose fermentation conditions (Martiniano et al.
2013a). These strains were grown in YPX medium and detoxified hemicellulosic
hydrolysate from dilute-acid pretreatment of sugarcane bagasse. All the strains
showed high ethanol yields when cultured in complex medium. Scheffersomyces
shehatae BR6-2AY presented the maximum ethanol yield (0.48 g.g-1) followed
by the strains CG8-8BY (0.47 g.g-1), BR6-2AI (0.45 g.g-1), and PT1-1BASP
(0.44 g.g-1). The productivities ranged from 0.35 to 0.37 g.l-1.h-1. Among all
these four strains, CG8-8BY and BR6-2AY were selected for ethanol production
from hemicellulosic hydrolysate due to their high ethanol production yields in
defined media. The fermentation performances of both strains were lower using
the hydrolysate as culture medium, due to the presence of undesired toxic com-
pounds (e.g., acetic acid) in this substrate even after detoxification. Scheffer-
somyces shehatae CG8-8BY and BR6-2AY showed ethanol yields and
productivities of 0.30 g.g-1, 0.15 g.l-1.h-1, 0.21 g.g-1, and 0.11 g.l-1.h-1,
respectively. As the best Sc. shehatae isolate selected in this study, the strain CG8-
8BY was further characterized (Martiniano et al. 2013b). Two different media
formulations were used for inoculum preparation and fermentation medium, using
yeast extract and rice bran extract (RBE) as nitrogen sources supplementing a
detoxified hemicellulosic hydrolysate from dilute-acid pretreatment of sugarcane
bagasse. This strain showed an ethanol yield of 0.33 g.g-1 and productivity equal
to 0.21 g.l-1.h-1 using a fermentation medium supplemented with RBE. On
the contrary, the same strain, when grown in hydrolysate supplemented with
yeast extract, exhibited an ethanol yield and productivity of 0.20 g.g-1 and
0.12 g.l-1.h-1, respectively. All these results demonstrate the influence of several
fermentation conditions and the intraspecific variability among strains from the
same species in the performance of D-xylose conversion to ethanol.
Apart from the importance of the isolation and identification of the new
D-xylose-fermenting strains of the Scheffersomyces clade from natural habitats in
12 Novel Yeast Strains from Brazilian Biodiversity 269

Brazil, the new six isolates belonging to the species Sp. passalidarum reported as
associated with rotting wood in the Brazilian Amazonian forest are the most
relevant finding (Cadete et al. 2012a). This occurrence is relevant not only because
before the isolation of these strains, this species was represented by a single
isolate, the type strain, but also due to the recent reports highlighting Sp. pas-
salidarum as the major naturally ethanol producer from D-xylose (Hou 2012; Long
et al. 2012). The first demonstration of D-xylose fermentation under ‘‘anaerobic’’
conditions by Sp. passalidarum (NRRL Y-27907, type strain) resulted in high
ethanol production yield, fast cell growth, and rapid sugar consumption with
D-xylose being consumed after glucose depletion (Hou 2012). In this work, it was
further demonstrated that for this species, D-xylose conversion takes place by
means of NADH-preferred xylose reductase and NAD+-dependent xylitol dehy-
drogenase. Thus, the capacity of Sp. passalidarum to utilize D-xylose under
‘‘anaerobic’’ conditions was proved to be possible due to the balance between the
cofactor’s supply and demand through its XR–XDH pathway. It has also been
shown that this species simultaneously assimilate glucose and D-xylose aerobi-
cally and simultaneously co-ferment glucose, cellobiose, and D-xylose with an
ethanol yield of 0.42 g.g-1 and productivity of 0.53 g.l-1.h-1, exhibiting a specific
ethanol production rate on D-xylose more than three times that of the corre-
sponding rate on glucose (Long et al. 2012). Moreover, in this work, an adapted
strain of Sp. passalidarum produced ethanol from a nondetoxified hardwood
hydrolysate with yield of 0.34 g.g-1. Metabolome analysis of Sp. passalidarum
before onset and during the fermentations of glucose and D-xylose showed that the
flux of glycolytic intermediates is significantly higher on D-xylose than on glu-
cose. High affinity of its xylose reductase activities for NADH and D-xylose
combined with allosteric activation of glycolysis probably account in part for its
unusual capacities (Long et al. 2012). So far, the performance of the Brazilian Sp.
passalidarum strains was evaluated in YPX medium and in detoxified hemicel-
lulosic hydrolysate from dilute-acid pretreatment of sugarcane bagasse (Cadete
et al. 2012a). In this study, all the strains were responsible for the highest ethanol
production in complex medium, yielding from 0.31 to 0.37 g.g-1 ethanol, with
productivities of 0.62 to 0.75 g.l-1.h-1, which are far above those found in Sc.
stipitis and Sc. shehatae. When the hemicellulosic hydrolysate was used as fer-
mentation medium, the production of ethanol by the strains Sp. passalidarum
UFMG-HMD-1.1 and UFMG-HMD-14.1 was detected, but with lower yields
(0.20 and 0.18 g.g-1, respectively) when compared to the results in complex
medium. However, this production can be enhanced through evolutionary engi-
neering (Long et al. 2012) or protoplast fusion (Hou and Yao 2012).
New D-xylose-fermenting yeast species are important contributions to a better
understanding about the evolution process and the metabolism of this pentose
among such microorganisms. Although described and characterized as a cellobiose-
fermenting yeast (Cadete et al. 2012b, Urbina et al. 2012), Sc. amazonensis is also
able to ferment D-xylose, but with a remarkably xylitol yield (0.55 to 0.59 g.g-1)
and, consequently, low ethanol yields (0.07 to 0.08 g.g-1) in YPX medium (Cadete
et al. 2012a). The recently discovered species from the Spathaspora clade,
270 R. M. Cadete et al.

Sp. brasiliensis, Sp. roraimanensis, Sp. suhii, and Sp. xylofermentans, are capable of
producing ethanol and xylitol from D-xylose at different concentrations (Cadete
et al. 2012a, 2013). Under aerobic conditions in YP medium with 2 % of D-xylose
(Cadete et al. 2013), Sp. xylofermentans UFMG-HMD-25.1 reached the maximum
ethanol yield (0.34 g.g-1) followed by Sp. roraimanensis UFMG-XMD-23.2
(0.29 g.g-1), Sp. suhii UFMG-XMD-16.2 (0.14 g.g-1) and Sp. brasiliensis UFMG-
HMD-19.3 (0.12 g.g-1). When the fermentation process was shift to a less oxy-
genated condition conducted in complex medium with D-xylose (Cadete et al.
2012a), Sp. suhii UFMG-XMD-16.2 and UFMG-HMD-16.3 produced more ethanol
(0.33 and 0.27 g.g-1) than xylitol (0.21 and 0.17 g.g-1). The production of ethanol
by Sp. roraimanensis UFMG-XMD-23.2 was also higher (0.26 g.g-1) than xylitol
(0.19 g.g-1). Inversely, Sp. brasiliensis UFMG-HMD-19.3 produced similar
amounts of both products (0.13 g.g-1 and 0.16 g.g-1 of ethanol and xylitol yields,
respectively). The strains Sp. xylofermentans UFMG-HMD-25.1 and UFMG-HMD-
23.3 exhibit different behaviors in this assay. Whereas UFMG-HMD-25.1 showed a
higher yield of xylitol (0.22 g.g-1 against 0.14 g.g-1 ethanol yield), the reverse was
observed for UFMG-HMD-23.3 (0.18 g.g-1 ethanol yield and 0.13 g.g-1 xylitol
yield). In this study, two of these four new species were cultured in detoxified
hemicellulosic hydrolysate from dilute-acid pretreatment of sugarcane bagasse.
Spathaspora suhii UFMG-XMD-16.2 and Sp. roraimanensis UFMG-XMD-23.2
achieved the highest xylitol yields (0.57 and 0.61 g.g-1) and the lowest ethanol
yields (0.23 and 0.22 g.g-1), respectively.
Spathaspora arborariae has been the most studied new D-xylose-fermenting
yeast from Brazilian ecosystems, as denoted by the significant number of studies
published with this species (Cadete et al. 2009; Cunha-Pereira et al. 2011; Hickert
et al. 2013; Martiniano et al. 2013b). The type strain, UFMG-HMD-19.1A, is
capable of producing ethanol and xylitol from D-xylose, being ethanol the main
fermentation product. This yeast showed ethanol yields equal to 0.50 g.g-1 in
batch D-xylose fermentation (Cadete et al. 2009), 0.45 g.g-1 in nondetoxified rice
hull hydrolysate (Cunha-Pereira et al. 2011) and 0.14 g.g-1 in detoxified sugar-
cane bagasse hemicellulosic hydrolysate supplemented with RBE (Martiniano
et al. 2013b). When co-cultured with S. cerevisiae ICV D254 in nondetoxified rice
hull hydrolysate in bioreactor cultures under oxygen limitation (Hickert et al.
2013), hexoses and pentoses from the hydrolysate were converted to ethanol and
xylitol, with yields of 0.48 and 0.39 g.g-1, respectively. Regarding the ability of
this yeast to produce ethanol from D-xylose, a major influence of the fermentation
media was revealed.

12.3.2 Cellobiose-Fermenting Yeasts

Cellulose is the most abundant biopolymer on Earth and has great potential as a
renewable energy source. The enzymatic hydrolysis of cellulose, followed by
fermentation to ethanol, is a promising green alternative for the production of
12 Novel Yeast Strains from Brazilian Biodiversity 271

transportation fuels (Lynd et al. 2002). However, its crystalline structure makes
this polymer insoluble and inaccessible to cellulolytic enzymes, and therefore a
pretreatment step is required for its biochemical conversion processing (Gray et al.
2006; Olofsson et al. 2008; Chauve et al. 2010).
In nature, cellulose is degraded mostly by fungi and bacteria, which excrete a
number of hydrolytic and oxidative enzymes (Lynd et al. 2002; Horn et al. 2012),
including cellulases, hemicellulases, and enzymes involved in lignin breakdown.
Cellulases are divided into endoglucanases (EGs), cellobiohydrolases (CBHs), and
b-Glucosidases (BGs). Endoglucanases (EGs) attack cellulose chains at random
positions generating cello-oligosaccharides. CBHs are exo-acting enzymes that
release cellobiose units from cellulose chain ends. The hydrolysis of cellulose is
completed by b-Glucosidases (BGs), which hydrolyze cellobiose and soluble
cello-oligosaccharides to glucose (Singhania et al. 2013). Cello-oligosaccharides
and cellobiose are potent inhibitors of endoglucanases and cellobiohydrolases. The
catalytic activity of the BGs is rate limiting in the saccharification of cellulose.
b-Glucosidases not only determine the rate but also the extent of cellulose
hydrolysis by relieving end product inhibition of CBHs and EGs (Lynd et al. 2002;
Olofsson et al. 2008). In addition, the produced glucose also inhibits b-Glucosidase
and exerts feedback inhibition (Krogh et al. 2010).
To be economically feasible, the hydrolysis of cellulose must be conducted at a
high dry matter concentration, which inevitably results in a high concentration of
hydrolysis endproducts and makes the product inhibition of enzymes a major
challenge in rate limiting for lignocelluloses hydrolysis in high-solid conditions
and enzyme engineering (Kristensen et al. 2009; Olofsson et al. 2008; Teugjas and
Väljamäe 2013). To minimize the end product inhibition, the most often applied
setup development is a process called simultaneous saccharification and fermen-
tation (SSF), whereby glucose is constitutively removed by fermentation to ethanol
due to the addition of a fermenting organism in parallel with hydrolytic enzymes
(Olofsson et al. 2008). However, the rate of ethanol production during SSF can be
limited by degradation of cellobiose to glucose because Saccharomyces cerevisiae
cannot directly use cellobiose and cello-oligosaccharides (Lee et al. 2013).
Cellulases preparations with sufficient b-Glucosidase activity are expensive to
produce. To bypass the use of BGs and lessen the need of these hydrolytic
enzymes, researchers have investigated the use of cellobiose itself as a fermentable
sugar (van Rooyen et al. 2005). Using a yeast capable of fermenting both glucose
and cellobiose in a coupled system may have several advantages, like circum-
venting the endproducts inhibition of the cellulase complex and increasing the
effective activity of the cellulolytic enzymes (Freer and Detrov 1983), thereby
enhancing the ethanol production.
Following the same reasoning adopted for studies with D-xylose-fermenting
yeasts, studies toward the use of native cellobiose-fermenting yeast strains for SSF
can be direct either as an alternative or co-culture usage with S. cerevisiae or to
generate yeast strains capable of fermenting cellobiose and cello-oligosaccharides.
However, studies on bioprospecting yeasts capable of fermenting cellobiose are
scarce. Most works in this area are focused on screening the ability to ferment
272 R. M. Cadete et al.

cellobiose within species from culture collections (Maleszka et al. 1982; Freer and
Detrov 1983; Gondé et al. 1982; Toivola et al. 1984; Morikawa et al. 1985) or to
demonstrate the property of one or few yeast strains to convert cellobiose to
ethanol (Blondin et al. 1983; Parekh and Wayman, 1986; Spindler et al. 1992;
Golias et al. 2002; Ryabova et al. 2003). Therefore, studies of bioprospecting
facing such microorganisms are innovative and of interest.

12.3.2.1 Studies with Brazilian Yeasts

Although limited to a few studies, the discovery of new Brazilian cellobiose-

fermenting yeast species and strains open a range for future researches in this field.
Both Sc. queiroziae, a new species described from six isolates related to rotting
wood and wood-boring insects collected in areas of Atlantic rainforest (Santos
et al. 2011; Morais et al. 2013b), and Sc. amazonensis, a new species described
from five isolates related to rotting wood from the Brazilian Amazonian forest
(Cadete et al. 2012a, b) are able to ferment cellobiose. An ethanol yield from
cellobiose (0.32 g.g-1) obtained with two Sc. queiroziae strains, UFMG-CLM-5.1
and UFMG-IMX-6.1, is in the same range as those obtained during glucose fer-
mentation by these yeasts in YP medium with 2 % of cellobiose or glucose (Santos
et al. 2011). Moreover, both UFMG-IMX 6.1 and UFMG-CLM 5.1 lack (or have
very low) periplasmic b-Glucosidase activity, with rates of cellobiose hydrolysis
of less than *5 U.g-1 dry yeast cells at pH 5.0 or 7.0. When the yeast cells were
permeabilized, a significant BG activity could be verified at pH 5.0 (29–47 U.g-1
dry yeast cells), and especially at pH 7.0 (167–230 U.g-1 dry yeast cells), which is
consistent with an intracellular b-Glucosidase as being responsible for cellobiose
hydrolysis by this species (Santos et al. 2011). Recently, the conversion of
D-xylose to ethanol was also demonstrated by new Sc. queiroziae strains (Morais
et al. 2013b), a feature that enhances the potential of this species in fermentative
process of biomass conversion.
Scheffersomyces amazonensis has also the ability of ferment D-xylose, with a
remarkable production of xylitol (Cadete et al. 2012a). In a fermentation assay
performed in complex medium with D-xylose as sole carbon source, all the strains
belonging to this species achieved the highest xylitol yields (0.55–0.59 g.g-1)
accomplished with a low ethanol production (0.07–0.08 g.g-1) when compared to
the other strains tested (Cadete et al. 2012a). Once that xylitol is one of the most
expensive polyol sweeteners in the world market and has been the subject of
specific health claims (Saha 2003), the biotechnological production of xylitol
using microorganisms is of economic interest, and yeasts demonstrating this
capacity can be used in processes of bioconversion of hemicellulosic hydrolysates
to this high-added value product.
12 Novel Yeast Strains from Brazilian Biodiversity 273

12.4 Final Remarks and Future Perspectives

Brazilian biodiversity has allowed the isolation, identification and characterization

of novel yeasts species and strains able to ferment lignocellulosic sugars. In view
of its efficient D-xylose/cellobiose fermentation, these yeasts are a new source of
genes coding enzymes (including sugar transporters) to engineer industrial strains
for the production of 2G bioethanol and other advanced biofuels and chemicals.
Several studies performed with Sp. passalidarum in the past years makes this
species the most attractive microorganism within this field. The recent genome
sequencing of Sp. passalidarum type strain (Wohlbach et al. 2011) is an important
tool to elucidate the remarkable conversion of D-xylose to ethanol attained by this
yeast and to develop unprecedented D-xylose-fermenting S. cerevisiae strains.
Under this scenario, Brazilian Sp. passalidarum strains arise as a great set of
‘‘tools’’ to be explored and exploited for industrial production of lignocellulosic
ethanol and other advanced biofuels.

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Chapter 13
Trends in Biodiesel Production: Present
Status and Future Directions

Victor H. Perez, Euripedes G. Silveira Junior, Diana C. Cubides,

Geraldo F. David, Oselys R. Justo, Maria P. P. Castro,
Marcelo S. Sthel and Heizir F. de Castro

Abstract The use of renewable fuels, an alternative that reduces the generation of
greenhouse gases, is one proposal to mitigate the effects that contribute to global
warming. Brazil is the fourth largest producer of biodiesel in the world, and growth
expectations of the productive capacities have generated huge technological and
environmental challenges. In this context, this chapter discusses some of the rel-
evant aspects of biodiesel production in Brazil, including sustainability of raw
materials, conventional technology limitations, and further presents technological
alternatives as strategies that will guide the future directions which can result in
processes with greater environmental and economic returns.

13.1 Introduction

Global warming is a serious environmental problem these days (Kerr 2013).

Consequently, substantial climate changes have been observed, causing major
socioeconomic and environmental impacts to society and biodiversity. The emission

V. H. Perez (&)
UENF/CCTA/LTA, Av. Alberto Lamego 2000. Pq California,
Campos dos Goytacazes-RJ 28013-602, Brazil
e-mail: victorh@uenf.br
V. H. Perez  E. G. Silveira Junior  D. C. Cubides  G. F. David
Food Technology Department, State University of the North Fluminense,
Campos dos Goytacazes-RJ, Brazil
O. R. Justo
Estácio de Sá University, Campos dos Goytacazes-RJ, Brazil
M. P. P. Castro  M. S. Sthel
Physical Sciences Department, State University of the North Fluminense,
Campos dos Goytacazes-RJ, Brazil
H. F. de Castro
Engineering School of Lorena, University of São Paulo, São Paulo-SP, Brazil

S. S. da Silva and A. K. Chandel (eds.), Biofuels in Brazil, 281

DOI: 10.1007/978-3-319-05020-1_13,  Springer International Publishing Switzerland 2014
282 V. H. Perez et al.

of greenhouse gases from the use of fossil fuels on a large scale after the industrial
revolution is seen as the main cause of this phenomenon (Beck 2013).
Most of the energy consumed in the world comes from fossil oil, natural gas,
and coal. Fossil fuels are widely used as a transportation and machinery energy
source due to their high heating power, availability, and quality combustion
characteristics (Hassan and Kalam 2013). However, as foreseen, fossil fuel
resources will inevitably be depleted, while demand for energy is increasing due to
population growth, technological progress, and urbanization. Thus, estimates for
2100 suggest that worldwide energy demand will be five times greater than today
(Hossain and Davies 2013). At the same time, a report of the Intergovernmental
Panel on Climate Change (IPCC) pointed out the use of renewable fuels as an
alternative to mitigate the emission of greenhouse gases (IPCC 2007).
Several countries have investigated, developed, or are considering the intro-
duction of biofuels in their national energy programs. Particularly, Brazil has
developed programs that use biofuels and other renewable energy sources, for both
transport and power generation. In 2002, the Incentive Program for Alternative
Sources of Energy (PROINFA) was implemented with the purpose of developing
alternative and renewable sources of energy for electricity production, taking into
account the characteristics and potential region, aimed at reducing emissions of
greenhouse gases. More recently, the National Program for Production and Use of
Biodiesel (PNPB) was launched in 2004 to regulate the production and distribution
of Brazilian biodiesel from various sources of raw materials, in a sustainable way,
both technically and economically, with a focus on social inclusion and regional
development. Thus, small farms can cultivate oilseeds according to regional
characteristics in order to produce biodiesel. However, although soybean is the
main raw material marketed by the program, other crops such as sunflower,
peanut, sesame, castor, and soybean oil itself have a higher market value.
Therefore, some of the oil can be sold to the program, but can also be used for
manufacturing products with higher commercial value.
Biodiesel production in Brazil is an overcoming history when compared with
the ethanol production, since ethanol has a consolidated technology in relation to
biodiesel, which is still incipient (Sallet and Alvim 2011). The methods for bio-
diesel production are well known (Basha et al. 2009). However, the chemical
transesterification using methyl alcohol or ethyl alcohol, in some cases by one or
two reaction steps, has been adopted as a conventional route for its production at
an industrial scale. In general, biodiesel production can be considered as a simple
process. However, production on a large scale presents challenging technological
and production cost problems. In addition, as the raw materials used in the pro-
duction of biodiesel fuel are sometimes the source of food for humans and/or
animal consumption, controversy, and competition between biofuels and food,
sustainability and limited land for use, and deforestation have been generated
(Elbehri et al. 2013).
This chapter discusses some of the aspects of biodiesel production in Brazil,
including feedstock used, technological routes established, and alternative
13 Trends in Biodiesel Production 283

processes such as unconventional methods of characterization as well as the

challenges that must be met to make this process more competitive and attractive
in terms of industrial and environmental concerns.

13.2 Raw Materials for Biodiesel Production

For biodiesel production, the raw materials frequently used around the world,
according Pahl (2008), are rapeseed oil (59 %), soybean oil (25 %), palm oil
(10 %), sunflower oil (5 %), and other sources (1 %) which include: coconut,
jatropha sp, camelina, peanut, safflower, mustard, hemp, corn (maize), waste
frying oil, animal fat, and algae. For Brazil, Fig. 13.1 shows the profile of use of
raw materials in the production of biodiesel based on the average values of the first
half of 2013, according to data reported by the National Agency of Petroleum,
Natural Gas and Biofuels (ANP). Brazil is the second largest soybean producer in
world, and as can be seen, soybean biodiesel represents more than 72 % compared
to other raw materials, followed by beef tallow and cotton which are approxi-
mately 20 and 3 %, respectively. The explanation for this national scene is due to
investments in the production chain over decades, which has resulted in the
development of new varieties of species, genetic improvement, and plague control
and, consequently, a higher rate of productivity compared to other oilseeds,
resulting in a relatively lower cost of soybean production.
However, other crops have been gaining ground as sources of raw materials
(Fig. 13.2). Furthermore, it is predicted that over the years, this growth will
become more significant insofar as technological advances in agriculture are
reached, especially for those oilseeds with higher energy density than soybeans
(Table 13.1), i.e., the higher oil content of seeds. In fact, this may be possible
because of the geographic characteristics of Brazil, which is basically a tropical
country that has a large territory, important water resources, regular rainfall, high
biodiversity, and well-developed agricultural technologies, therefore, having great
potential for bioenergy production (Sthel et al. 2009).
Many studies have demonstrated the potential of some of these oilseeds in
biodiesel production. Macedo et al. (Macedo et al. 2011a) studied the thermal
properties of biodiesel obtained from oiticica oil, while Andrade et al. (2012a)
produced biodiesel through moriche palm oil (Buriti oil) to evaluate thermal
behavior in blends with diesel. Other studies investigated the potential of using
different raw materials such as macaw palm oil (Ferrari and de Azevedo Filho
2012), babassu oil (Freitas et al. 2009; Nascimento et al. 2009), Pequi oil (Macedo
et al. 2011b), in the production of biodiesel.
Although there is still much to be done for its implementation on an industrial
scale, microalgae has been identified as third-generation biodiesel and presents
several benefits over other raw material resources, such as land use, potential
cultivation in nonfertile locations, and especially its faster growth and high lipid-to-
biodiesel yield (Torres et al. 2013). According to Demirbas and Demirbas
284 V. H. Perez et al.

Raw material for Biodiesel production (%)

Fig. 13.1 Raw materials
80
used for Brazilian biodiesel
production during the first 70

6 month of 2013 (ANP 2013) 60

50

40

30

20

10

l
l

p
w

t
t
t
oi

oi
oi

oi

fa
fa
fa

ni
llo
an

ng
n

rs
en
k

tu
Ta

to

or

al

e
be

yi

ck
ot

ge

th
P

P
Fr
oy

hi

O
ra
C
S

Fo
Raw material for biodiesel production (%)

Fig. 13.2 Profile of the use

of raw materials used in 90
biodiesel production in Brazil 2009
2010
between 2008 and 2013 (ANP 2011
2013) 2012
60 2013

30

0
cids

oil
oil
low

t
il

n fa
k fa
no

ton

ing
Tal
ea

ty a

icke
Por
yb

Cot

Fry
Fat
So

Ch

(Demirbas and Demirbas 2011), high oil species of microalgae cultured in

optimized growth conditions in photobioreactors have the potential to yield
19.000–57.000 L of microalgal oil per acre per year; consequently, the yield of oil
from algae is over 200 times the yield from the best-performing plant vegetable
oils. More than three thousand species of algae have been identified;thus the choice
of highly producing strains of oils for industrial application will probably depend on
genetic improvement. The oil content of some microalgae is very attractive for
biodiesel production, since it can be higher than 70 %. For example, the oil content
of Schizochytrium sp. and Botryococcus braunii are 50–77 and 25–75 %, respec-
tively (Chisti 2007). Similarly, single cell oils (SCOs) accumulated by oleaginous
microorganisms have recently emerged as a potential feedstock for production of
biodiesel. This is a very interesting raw material because the biomass can be pro-
duced in conventional fermenters using low-cost substrates as carbon sources, such
as sugar cane bagasse (Kamat et al. 2013), glycerol generated from biodiesel
Table 13.1 Several oilseeds with potential to produce biodiesel from vegetable oil in Brazil
13

Oilseed type Oil Oil yield Brazilian regiona Refs.

content (ton/ha)
(%)
Babassu palm (Orbignya 60–68 0.12b Largest producer is the State of Maranhão (Freitas et al. 2009)
phalerata)
Canudo-de-pito (Mabea fistulifera) 40 – Minas Gerais, Rio de Janeiro, and São Paulo (CENBIO 2013)
Crambe (Crambe abyssinica 35 0.45–2.5b Plantations extend through central warmer regions (Vargas-Lopez et al. 1999)
Hochst) of Brazil
Cream nut or monkey pot 54.80 – Ceará to Rio de Janeiro, South Bahia, and North (CENBIO 2013)
(Lecythis pisonis Camb.) Espírito Santo
Trends in Biodiesel Production

Linseed (Linum usitatissimun L.) 33–43 0.4–1.45 Brazil South Region, especially Rio Grande do Sul (CENBIO 2013)
Macaw palm (Acronomia 20–25 1.5–5.0 Midwest and North, Minas and São Paulo (do Amaral et al. 2011)
aculeata)
Maraja (Bactris tomentosa Mart.) 28 – Maranhão and Pará (CENBIO 2013)
Monguba (Pachira aquática Aubl) 56–58 – Entire Amazon region to Maranhão (CENBIO 2013)
Moriche palm (Mauritia flexuosa) 29 – Acre, Amazonas, Bahia, Ceará, Goiás, Tocantins, (CENBIO 2013)
Maranhão, Pará, Piauí, São Paulo
Palm (Opuntia cochenillifera) 22 2.0–8.0b Amazonas and North Region (Queiroz et al. 2012)
Jatropha (Jatropha curcas L) 50 1.2–1.5b Goiás, Minas Gerais and in the Northeast. (CENBIO 2013)
Rapeseed (Brassica napus L. var. 34–40 0.8 Goiás, Mato Grosso do Sul, Paraná, Rio Grande do (CENBIO 2013)
oleifera) Sul
Safflower (Carthamus tinctorius) 30–45 0.7 A very promising planting in the semiarid region in (CENBIO 2013)
Brazil
Sessame (Sesamum indicum L.) 50–60 0.24 Goiás, Mato Grosso and Southeast (mainly in São (CENBIO 2013)
Paulo)
Sunflower (Helianthus annus L.) 40–47 0.774b Alagoas, Ceará, Goiás, Mato Grosso, Mato Grosso (Bergmann et al. 2013)
do Sul, Paraná, Rio Grande do Norte, Rio Grande
do Sul, Sergipe
(continued)
285
Table 13.1 (continued)
286

Oilseed type Oil Oil yield Brazilian regiona Refs.

content (ton/ha)
(%)
Tucuma palm (Astrocaryum 30–50 – Acre, Amapá, Amazonas, Pará, and Rondônia (CENBIO 2013)
aculeatum)
Tung-oil tree (Aleurites 35–40 0.790 Rio Grande do Sul (CENBIO 2013)
moluccanus)
Turnip forage (Raphanus sativus 30 2.2 Mato Grosso, Mato Grosso do Sul, Minas Gerais, (CENBIO 2013)
L.) and São Paulo
Ucuúba (Virola surinamensis) 58–60 – Amazon region to Maranhão and Pernambuco (CENBIO 2013)
Western soapberry (Sapindus 30 – Amazon region to Goiás and Mato Grosso (CENBIO 2013)
saponaria L.)
a
IBGE-Brazilian Institute of Geography and Statistic (http://www.ibge.gov.br/english/)
b
Yield oil (Bergmann et al. 2013)
V. H. Perez et al.
13 Trends in Biodiesel Production 287

production (Xu et al. 2012), biomass pyrolytic sugars (levoglucosan) (Lian et al.
2013), and aqueous fractions rich in organic short chain (C1–C4) obtained in
thermochemical conversion processes of biomass (Lian et al. 2012).
The choice of suitable feedstocks must also answer technical questions. Thus,
parameters such as flash point, viscosity, density, acid value, cetane number, and
oxidative stability, among others, must be observed for both biodiesel and diesel/
biodiesel blends. Depending on the chemical composition of the raw material,
some properties of the produced biodiesel may be undesirable. In these cases, the
use of additives may be required to attenuate these effects (Focke et al. 2012; Ali
et al. 2013). Thus, the production of biodiesel from oils with high iodine value, for
example, can result in a product susceptible to oxidation. Similarly, raw materials
with high content of saturated fatty acids result in biodiesel which tends to have
solidification problems with temperature variations (Knothe et al. 2005).

13.3 Process of Biodiesel Production: Trends

and Alternatives

In 2010, Brazil became the second world producer of biodiesel with a production
of 2.4 billion of liters, approximately, second only to Germany. However, in 2011,
both the United States and Argentina increased production, and now Brazil is the
fourth world producer of biodiesel, as shown in Fig. 13.3.
At present, 69 Brazilian plants are authorized for biodiesel production, corre-
sponding to a total capacity of 22,334.06 m3/day. Among these, 11 plants are
allowed to expand their production capacity, while 3 other new plants should be
built, providing an increase of 9 % in the current production capacity of biodiesel
in Brazil (ANP 2013).
Figure 13.4 shows the values of biodiesel production in Brazil in relation to the
overall production. The global average annual growth rate over the period from the
end of 2005 through 2011 was approximately 37 %. Compared with global pro-
duction, the Brazilian profiles were similar; biodiesel production increased from
70 million liters in 2006 to 2.7 billion liters in 2011. Thus, the Brazilian average
annual growth rate over the period from 2008 to 2011 was 33 %. In 2012, bio-
diesel production continued to expand, but at much lower rate, nearly 1.7 %, while
the global production was just 0.4 %. However, the Brazilian biodiesel market is
not open, and the ANP itself, which currently regulates sales through public
auctions, gives preference to companies with the ‘‘Social Fuel Label.’’
Industrially, biodiesel production is achieved by chemical transesterification of
oils/fats using methanol or ethanol in some cases, by one or two reaction steps in
the presence of a homogeneous alkaline catalyst such as KOH or NaOH or its
corresponding alkoxide (Na+CH3O-or K+CH3O-). These alkoxides can be pro-
duced in a very simple way, dissolving sodium hydroxide in alcohol, before its
addition to the reaction medium. Basically, in this process one mole of oil reacts
with three moles of alcohol, but in industrial practice alcohol is used in excess to
288 V. H. Perez et al.

Fig. 13.3 Global ranking of Canada

China
biodiesel production for 2012 Colombia
(REN21 2013) Austria

Producer countries
Belgium
Netherlands
Spain
Thailand
Indonesia
France
Brazil
Germany
Argentina
Unite States
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
Biodiesel production (Billion liters/year) Biodiesel production in 2012 (Billion litres/ year)

Fig. 13.4 Biodiesel 25

production record by years.
Symbols: Global 20
production (REN21 2013);
Brazilian production (ANP
15
2013)
10

0
00

01

02

03

04

05

06

07

08

09

10

11

12
20

20

20

20

20

20

20

20

20

20

20

20

20
displace the equilibrium of the reaction and promote the formation of the product.
As a result, a mixture of alkyl esters of fatty acids of a long chain and glycerin as
byproduct is obtained (Fig. 13.5).
From a technological point of view, this is a relatively simple process which
can be conducted at atmospheric pressure and under moderate conditions of
temperature (50–60 C), resulting in high conversion rates at relatively low
reaction times (Meher et al. 2006). When low-cost oils and fats (waste frying oil,
etc.) are used as raw material, they cannot be converted to biodiesel using alkaline
catalyst because of their large amounts of free fatty acids. Therefore, two-step
processes are required. First, the free fatty acids are converted to fatty acid methyl
esters by an acid catalyst; in the second step, transesterification is completed using
an alkaline catalyst.
A general description for a conventional process is synthesized in Fig. 13.6, in
which a mixture of oil, methanol, and catalyst is fed to a system of two stirred
tanks reactors; after reaction in the first reactor, the glycerol is removed from the
first reactor before being fed into the second reactor. The subsequent sections
allow separation of biodiesel as the light phase from glycerol, and it is purified by
washing, neutralization, and then vacuum dried. Meanwhile, the glycerol is sep-
arated from the residual fraction, neutralized and the excess of methanol is
removed by evaporation and recycled in the process (Knothe et al. 2005).
13 Trends in Biodiesel Production 289

Fig. 13.5 Simplified scheme for biodiesel production by chemical transesterification

Fig. 13.6 Flow sheet for conventional biodiesel production

The presence of free fatty acids and water in the reaction facilitates soap for-
mation; the need to neutralize the catalyst after the reaction; the impossibility of
catalyst retrieval; as well as requirements for treatment of effluents generated
during the washing steps are some drawbacks that currently require attention in the
process. Furthermore, when free fatty acid content is very high, this process can
also be carried out using homogeneous acid catalysts despite the alkali, but
reaction times are longer, requiring much higher oil molar ratio oil: alcohol and
reaction temperatures.
On the other hand, transesterification processes in situ have been reported for a
diversity of oils using methanol (Abo El-Enin et al. 2013). In these processes, the
ground oilseed is mixed directly, instead of purified oils, with alcohol and catalyst,
to produce alkyl fatty acid esters, as shown in the generic scheme (Fig. 13.7). The
molar ratio oil: alcohol higher than the value calculated according to stoichiometry
has been tested alkaline and acid catalyst. The simplicity of the process will pave
290 V. H. Perez et al.

Fig. 13.7 Pilot scale for in situ transesterification from rapeseeds (Modified from Abo El-Enin
et al. 2013)

the way for oil seed growers to move away from overdependence on crushing and
solvent extraction plants.
Emphasis can be also placed on the technology developed by PETROBRAS
(Brazil) for the biodiesel production known as Grain route. In the process, biodiesel
is obtained directly from oilseed plant grain using ethanol, which works not only as
an acyl acceptor, but also as a solvent in the oil extraction. This process eliminates
steps such as oil extraction and refining; its feasibility on an industrial scale was
assessed in a small plant in Rio Grande do Norte (Brazil) (Sauer et al. 2006).
The production of biodiesel from algae is also a process that is under discussion.
A variety of high value-added byproducts can be produced from algae; therefore, it
is likely that in the future production of biodiesel from algae will be the least
important application. Basically, bioreactors for algal culture consist of open ponds,
photobioreactors, and closed systems. Open pond systems are shallow ponds in
which algae are cultivated, while photobioreactors include different types of tanks
or closed systems. The biodiesel from algal oil is similar to biodiesel produced from
vegetable oils. The biggest challenges of this process are the choice or development
of bioreactors, the definition of the best choice to formulate nutrients, and methods
for biomass processing to extract the oil, which involves costly steps such as
concentration, separation, drying, and oil extraction. After obtaining the oil, the
following steps involve transesterification and purification of biodiesel, alcohol
recovery and separation of glycerin, as in the conventional process. Economic
evaluation studies have been reported comparing different alternatives (Gallagher
2011; Nagarajan et al. 2012). Closed systems are relatively expensive compared to
open ponds due to the required infrastructure costs. However, open systems are
more vulnerable to bacterial contamination. Thus, the tubular photo bioreactor
seems to be the most satisfactory choice for producing algal biomass on the scale
needed for biodiesel production (Demirbas and Demirbas 2011).
13 Trends in Biodiesel Production 291

Another common issue in this process that must be addressed relates to the use
of methanol for biodiesel production. Ethyl esters proved to be a viable alternative
to diesel fuel being more sustainable than methyl esters (Brunschwig et al. 2012).
Methanol is a product known to be toxic and although it can be obtained from
synthesis gas (syngas) of biomass, it is usually produced from fossil materials,
such as natural gas. In anyway, in Brazil lacks self-sufficiency in the CH3OH
production. In this sense, the use of ethanol instead of methanol fossil is a very
attractive alternative. A technological condition in Brazil that is quite atypical with
respect to most countries engaged in biofuels production, especially for being the
second largest ethanol producer in the world, reaching in 2012 a production of 21.6
million liters of ethanol. But either way it would be necessary to increase the
production levels to meet demand for biodiesel in the next years.
Technological advances have made possible the construction of industries
which are able to process raw materials from various sources, using both methylic
and/or ethylic routes, in batch or continuous processes (Table 13.2). Stand out
Barralcool plant, designed with Dedini-Balestra technology and built-in integrated
way with a sugarcane industry. In addition, processes that use centrifuges instead
of decanters to separate biodiesel from glycerol, such as Westfalia technology,
improve the separation phases, but production cost can be increased as a conse-
quence of high energy consumption. On the other hand, some Brazilian research
technologies that can modify the conventional reaction step are summarized in
Table 13.3. Several of these emerging technologies seem to be cost-effective and
environmentally friendly operations in comparison with conventional biodiesel
production technologies. In some cases, the conventional processes without great
modification can be adapted, e.g., ultrasound reactors when the mechanical stirrer
is replaced with ultrasound equipment. Furthermore, the use of packed-bead
reactors, which are well known in other industrial processes, are limited by the
lack of optimization studies applied to biodiesel production.
On the other hand, extensive research activity has been observed to use het-
erogeneous catalysts as alternative to the use of conventional homogeneous cat-
alysts (Table 13.4). In these systems are required typical reaction temperatures and
molar ratios of oil: alcohol more higher, however, probably one of the most
important advantages is that these catalysts do not produce soap, can be recovered
and consequently, their use results in processes with lower environmental impact.
Heterogeneous catalysts may be chemical or enzymatic, the latter consisting of
enzymes and cells (whole cells), either free or immobilized. Particularly, in the
case of chemical heterogeneous catalysts, many studies have focused on both
alkaline and acid catalysts, as well as the reaction mechanisms and their physi-
cochemical properties that influence biodiesel yields (Islam et al. 2013; Semwal
et al. 2011; Endalew et al. 2011). There is still no consensus, however, as to
whether alkaline catalysts are a better choice than acid in terms of reaction rate and
biodiesel productivity. One disadvantage of the use of a solid catalyst is the
formation multiphasic system, which leads to diffusion limitations that decrease
the reaction rate (Semwal et al. 2011).
 292

Table 13.2 Some examples of Brazilian industries designed to produce biodiesel production by methylic and/or ethylic routes (BiodieselBR 2013)
Industry Feedstock Process Technological Production
route capacity
(106 L/year)
Araguassu Soybeans (80 %), sunflower, cotton, castor beans Own technology/continuous Methylic/ethylic 36
Barralcool Soybeans Dedini-Balestra batch/ Methylic/ethylic 60
continuous
Bigfrango Animal fat, recycled oil Own technology/batch Methylic/ethylic 2
Bio Petro Soybeans Own technology/continuous Methylic/ethylic 70
Biopar Parecis Animal fat Methylic/ethylic 36
Bioverde Soybeans (40 %), cotton (50 %), recycled oil (10 %) Own technology/batch Methylic/ethylic 181
Cooperbio Soybeans, sunflower, cotton, animal fat, recycled oil Own technology/continuous Methylic/ethylic 166
Delta Biocombustíveis Soybeans, cotton, crambe, and beef tallow Methylic/ethylic 108
Fertibom Soy, Sunflower, jatropha, animal fat, recycled oil, peanut Own technology/batch Ethylic 120
Granol (Anapolis city, Soybeans (90 %), cotton (90 %) Dedini-Balestra Methylic/ethylic 372
GO) Forage turnip, animal fat, recycled oil continuous
Granol Soybeans (90 %), cotton, forage turnip, animal fat, Westfalia/continuous Methylic/ethylic 336
recycled oil
SP Bio Soybeans (80 %), animal fat, recycled oil Own technology/batch Methylic/ethylic 25
V. H. Perez et al.
13 Trends in Biodiesel Production 293

Table 13.3 Unconventional technologies for biodiesel production

Type of reactor Characteristics Refs.
Microwave Palm oil using ethyl alcohol with Pseudomonas (Da Rós et al.
irradiation fluorescens immobilized. Conversion 97.56 %, 12 h, 2013)
43 C, productivity of 64.2 mg ethyl esters g-1h-1
Macaw acid oil, ethanol (1:9), and commercial enzymes, (Nogueira et al.
use of microwave increased about one order the 2010)
biocatalyst activity
Adapted domestic microwave oven with babassu (Nascimento
coconut oil, methanol and KOH, 70 s, et al. 2009)
conversion [90 %
Heterogeneous fixed Soybean oil ethanol 3:1, lipase from B. cepacia, (Salum et al.
bed conversion of 95 %, 46 h, 50 C, alcohol added in 2010)
two steps (0 and 7 h) and 1 % (m/m) of water.
Column of 17 mm (ID) and 100 mm high,
recirculation (1.5 mL/min) bottom– top
Pellets of mixed oxides in a reactor 30 cm long column (Suarez and da
flowed with soybean oil (168 g/h) and methanol or Silva 2012)
ethanol (89 g/h) at 100 C reached 80 % yield
(methanol) and 40 % (ethanol). At 180 C,
ethanolysis reach yields up to 90 %
Ultrasonic cavitation Using commercial immobilized enzymes, mild (Batistella et al.
irradiation power supply (100 W), 60 C in 4 h, 90 2012)
wt% of conversion
Soybean oil with ethanol (1:24) and potassium (Brito et al.
hydroxide (1.5 %) using low-frequency ultrasound 2012)
(20 kHz), reaction mixtures between 39 and 52 C.
With three-step reaction, the yield was of
approximately 98 % after 6 min
Soybean oil, ethanol and NaOH at room temperature, for (Rodrigues
30 min produce yield of 91.8 % et al. 2009)
Using a frequency of 24 kHz beef tallow, methanol (Teixeira et al.
(6:1), NaOH (0.5 %), 70 seg, 60 C, conversion 2009)
92 %
Reactive distillation Simulation and experimental reaction of soybean oil, (Da Silva et al.
column ethanol, catalyzed with NaOH, attained a conversion 2012)
of 99.84 wt% after 6 min
Reactive extraction Simulation of batch and continuous processes (Dussan et al.
demonstrated the possibility of applying biocatalyst 2010)
system (C. rugosa immobilized in tetraethyl
orthosilicate with magnetite) in the reactive zone
using external magnetic fields
2 step reaction by Two step transesterification procedure which starts with (Guzatto et al.
transesterification a basic catalysis, followed by an acidic catalysis. 2011)
97 % conversion for waste cooking oil and soybean
oil and 98 % for linseed oil were achieved
(continued)
294 V. H. Perez et al.

Table 13.3 (continued)

Type of reactor Characteristics Refs.
Supercritical Soybean oil, supercritical ethanol (1:20), catalyst-free, (Trentin et al.
microtube reactor carbon dioxide as co-solvent (0.2:1 mass). 598 K, 2011)
20 MPa, oil to ethanol molar ratio of 1:20 and using
a CO2 to substrate mass ratio of 0.2:1
Soybean oil and macaw oil with supercritical methyl (Doná et al.
acetate (1:5), without catalyst, at 20 MPa, with 2013)
45 min at 350 C obtain yield of 44 % for soybean
oil and with macaw oil 83 % of yield
Ultra-shear reactor Equipment with rotor–stator mixing with high speed and (Da Silva de
intense shear frequency. Soybean, ethanol, NaOH, et al. 2011)
1:6:1.35, conversion of 99.26 wt%, with 12 min,
78 C and constant agitation of 7900 rpm

The use of a co-solvent can help to solve this problem, but in industrial practice
this method should not be used to avoid increasing cost production. In addition,
another important aspect that must be observed concerns the particle size of these
catalyst systems, which are usually synthesized as very small particles or fine
powder. Conceptually, high reaction rates should be expected when catalysts with
high surface area are used (Levenspiel 1999). However, this may result in the
formation of clusters due to the physiochemical properties of the reaction medium
oil: alcohol. Consequently, on one hand it affects the performance of the catalysts
and on the other side, more complex and expensive downstream steps are required.
Bifunctional heterogeneous catalysts have also been studied as a potential
alternative means to simultaneously develop biodiesel production by esterification
and transesterification reactions (Borges and Díaz 2012; Farooq et al. 2013), but to
attain a catalyst bifunctional with adequate surface area, size, and porous volume
and high activity, as well as being inexpensive, more investigations are required.
However, Axens has commercialized a process for the production of biodiesel via
heterogeneous catalysis at elevated temperatures (180–220 C) and consequently
higher pressures, known as Esterfip-H process. The transesterification reaction
makes use of rapeseed oil and methanol and as catalyst a spinel, e.g., one co-mixes
the alumina support material with zinc (Bournay et al. 2005).
In addition, Albemarle Corporation (www.albermarle.com), a leader in the
market of heterogeneous catalysts, has a pilot plant demonstration (BECON Pilot
Plant) in Iowa (USA) with a capacity of 300,000.00 gal/year for the production of
biodiesel via heterogeneous catalysis, using the catalyst known as GoBio T300.
This process uses vegetable and algae oils and operates at pressures and temper-
atures similar to conventional homogeneous catalysis process.
In a similar way, large efforts have been made to investigate enzymatic
transesterification (Tan et al. 2011; Gog et al. 2012). Lipases are the most studied
enzymes and they show great potential for enzyme immobilized on organic or
inorganic supports. Basically, the high biochemical specificity of lipases, which
allows conducting the reactions under mild conditions of temperature, as well as
the ease of biocatalyst reuse in several reaction cycles, are some of the major
 13

Table 13.4 Some study cases of Biodiesel production using heterogeneous catalysts
Catalysts Oil, alcohol (molar ratio % cat)/Temperature/Reaction time/ Refs.
Conversion
HUSY and Ce/HUSY zeolites Soybean oil, ethanol (30:1: 0.001 mol)/200 C under constant (Borges et al. 2013)
stirring (1000 rpm) and autogenous pressure (20 bar)/
24 h/ [97 % for barium
Alkaline compounds of strontium Babassu, methanol (1:6:1.0 %)/65 C/ 1 h/ [95 %/reusability (de Carvalho et al. 2013)
SrCO3 ? SrO ? Sr(OH)2 6 times
Trends in Biodiesel Production

Ni0.5Zn0.5Fe2O4 ferrites doped with Cu Soybean oil, methanol (1:20:4 %)/160 C/ 2 h/ [42 % (Dantas et al. 2013)
Sn(IV) complexes: Butyl stannoic acid, di-n-butyl- Soybean FFAs, methanol (simultaneous transesterification/ (Brito et al. 2012)
oxo-stannane and dibutyl tin dilaurate esterification) (4:1:0.01)/160 C/ 1 h/ [90 %
Alumina impregnated with potassium iodide Bran oil, methanol (15:1: 5 %)/–/92 h/95.2 % (Evangelista et al. 2012)
Iodide potassium incorporated on mesoporous Sunflower oil, methanol (1:15:1 e 2 %)/60 C/4 h/*85 % (de Galvão et al. 2012)
molecular sieves (SBA-15 and MCM-41)
Lewis acid/surfactant rare earth trisdodecylsulfate Waste cooking soybean oil with 8.8 wt.% of free fatty acids, (de Mattos et al. 2012)
ethanol (1:6:10 %)/100 C/1 h/76–86 %
Mesoporous silica active phase (La50SBA-15) Soybean oil, ethanol (20:1:1 %), at inert atmosphere (N2)/ (Quintella et al. 2012)
343 K/6 h/[80 %
Prepared from the waste material, Amazon flint Esterification of distillate produced by deodorization of palm (do Nascimento et al. 2011)
kaolin and activated with 4 M sulfuric acid oil and methanol (1:60)/160 C/4-h/92.8 %
H3PW12O40 (HPA) Oleic acid, methanol (esterification) (1:1:0,1 g)/25 C/10 h/ (Sepulveda et al. 2011)
80 %
Mixed oxides (Al2O3)0.8(SnO)0.2-x(ZnO)x Soybean oil, methanol/100 C/3 h/[80 %/200 h (Suarez and da Silva 2012)
(0.2 \ x \ 0)
295
296 V. H. Perez et al.

attractions of this alternative. Novozym 435 has been reported to be an effective

biocatalyst for biodiesel production (Da Rós et al. 2012a). Also, some successful
strategies combine the use of enzymes with unconventional reactors, resulting in
considerable improvements in the productivity of biodiesel production, as in the
case of a microwave reactor used for the transesterification of beef tallow by
Burkholderia cepacia lipase immobilized on silica-PVA, where full conversion
was achieved only at 8 h reaction (Da Rós et al. 2012b, 2013). More recently, the
preparation of biocatalysts with magnetic properties has motivated the scientific
community, and some work has been applied to the production of biodiesel (Wang
et al. 2011; Ngo et al. 2013; Liu et al. 2012). These systems allow the separation of
the magnetic biocatalyst by applying an external magnetic field at the end of the
reaction. However, the main obstacles to the use of immobilized enzymes are the
high cost and low conversion rates as a function of the reaction time (Jang et al.
2012). Thus, whole cells are an attractive alternative because they can be obtained
at much lower cost than purified enzymes (Gog et al. 2012; Andrade et al. 2012b),
but the long reaction times required and low conversion rate are still undesirable.
In these cases, diffusional limitations imposed by cellular walls certainly con-
tribute to lower conversion rates. Therefore, to further reduce the cost of bio-
catalysts in biodiesel production, new immobilization procedures with higher
activity and stability still need to be explored.

13.4 Characterization of Biodiesel Through

Unconventional Techniques

The growth of biodiesel production simultaneously demands research for the

development and implementation of analytical techniques to evaluate the quality
of biodiesel, as well as of diesel/biodiesel blends. Basically, biodiesel quality is
determined through the analysis of chemical composition and several physical
properties, which have been extensively reviewed (Knothe et al. 2005; Knothe
2006; Monteiro et al. 2008).
Nevertheless, there is demand for new procedures and, in this scenario,
photothermal techniques arise as alternatives and unconventional methodologies
for the characterization of biodiesel. Photothermal methods include various
techniques and phenomena based on the absorbed optical energy into heat con-
version. Basically, these techniques consist of detecting very small variations in
temperature resulting from the absorption of the given modulated radiation, and
therefore it is possible to determine physical properties such as conductivity and
thermal diffusivity of the raw material, biodiesel, and blends with diesel. Among
the different types of phothermical techniques, two have been shown to be sen-
sitive to the study and characterization of the thermal biodiesel properties:
(a) photopyroelectric, and (b) thermal lens. Lima et al. (2009) conducted a study
with soybean samples to show the influence of waste and antioxidants in the
thermal properties of the samples. Castro et al. (2011) reported a study which
13 Trends in Biodiesel Production 297

observed a correlation between the iodine number and the thermal diffusivity for
biodiesel samples attained from oils of several sources and consequently with
different fatty acid composition. Another study developed by Guimarães et al.
(2009) also presents measures of diffusivity, conductivity and effusivity for mix-
tures of diesel/biodiesel. Furthermore, Ventura et al. (2012) conducted a study in
which the thermal lens technique was applied to biofuel samples to test their
potential to distinguish diesel from biodiesel in binary mixtures. More recently,
Crespo (2013) presented a study on the correlation between the NOx emissions of
biodiesel with the thermal and rheological properties. This study may serve as a
new methodology for the characterization of NOx emissions from the combustion
of this type of biofuel.

13.5 Application of Glycerin

Since the beginning of the program of biodiesel production in Brazil there has
always been concern with respect to the glycerol accumulation. Over the years,
increase in biodiesel production has created a scenario that is indeed alarming.
Glycerol, a by-product of the transesterification, represents about 10 % of the total
biodiesel, e.g., just in 2012 approximately 274 million liters were produced in
Brazil. Research focusing on new applications for glycerol is being developed to
improve the economic viability of biodiesel production and its environmental
impact (Quispe et al. 2013). Some of the main uses of refined glycerin include
food, personal care products, and oral hygiene products, which make up approx-
imately 64 % of total consumption (Stelmachowski 2011). Other applications
include its use in the manufacture of pharmaceuticals and cosmetics (Tan et al.
2013). But the range of applications is now much broader, including its use in the
production of chemicals, fuel additives, production of hydrogen, development of
fuel cells, ethanol production, animal feed, co-digestion, and co-gasification
(Leoneti et al. 2012) as well as single cell oil (Xu et al. 2012). In addition, an
economic evaluation study found that attaining acrolein, hydrogen, and 1,2-pro-
panediol from glycerol was feasible from a technological standpoint, with good
profitability. At the same time, the conversion of glycerol into value-added
products such as 1,3-propanediol, PHB, and ethanol (da Silva et al. 2009) also
proved cost-effective and a high margin of difference between the cost of pro-
duction and sale using glycerol was observed (Cardona et al. 2010). As can be
observed, there are many applications, but the current panorama of glycerol
accumulation will be changed only if the technologies to transform it in chemical
products of high value are used on a large scale. A strategy that seems particularly
promising would be to look for applications inside the biodiesel process, as
addictive or as raw material for methanol production, eliminating the need of
acquisition from methanol from natural gas, transforming the biodiesel completely
in a renewable process. However, in some way, glycerin will probably be available
in the market, at low cost, in the next few decades.
298 V. H. Perez et al.

13.6 Concluding Remarks

Brazil has a large potential for bioenergy production derived from plants and
several biomass, as well as other residual sources. The question in debate between
energy versus food may be increasingly attenuated as new raw materials not
competing with the food chain begin to gain more attention in biodiesel produc-
tion. Thus, the expectation of using algae continues to be of great relevance,
although more research is required to reduce processing costs. In general, further
efforts should be made to reduce the cost of biodiesel production. Intense research
activity is occurring with the search for new chemical and biological catalysts,
proposing improvements and alternative processes, but on the industrial scale,
development of new technologies including the imminent use of glycerol to add
value to the process, such that they become more efficient and friendly with the
environment, is imperative. Other aspects no less important that were not
addressed here, but which should be observed, include problems in the stock,
stability, and formation of sediments after the manufacturing process of biodiesel
and even increasing the level of NOx emission with increasing future demands of
blends of diesel/biodiesel B10, B20, etc. Hence, major challenges in scientific and
technological development in this branch of biofuels, and particularly biodiesel,
will be specifically required to achieve these goals in a sustainable way.

Acknowledgments We are grateful to the Rio de Janeiro Research Foundation (FAPERJ) and
The National Council for Scientific and Technological Development (CNPq) for the financial
support.

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Chapter 14
Critical Technological Analysis
for Enzymatic Biodiesel Production:
An Appraisal and Future Directions

Marcelle Alves Farias and Maria Alice Z. Coelho

Abstract Biodiesel has attracted considerable interest in recent years as an

alternative energy source, once the world petroleum and gas resources will soon be
exhausted. Additionally, the biodiesel is a biodegradable and renewable fuel. The
conventional alkaline process for biodiesel production generates undesirable by-
products such as soaps, which make difficult biofuel separation and purification.
This technology becomes less interesting in Brazilian industry, once the significant
amount of raw material, available in Brazil, has as characteristic high acidity
value. In this scenario, to find an alternative technology that could eliminate these
problems is desired by Brazilian biodiesel market. Designed to overcome these
drawbacks, the enzymatic biodiesel production has been studied due to some
relevant advantages over conventional process, such as: glycerol can be easily
recovered without any complex process, free fatty acids contained in the oils can
be completely converted to esters and subsequent wastewater treatment is not
required. Nevertheless, despite the advantages of using enzymatic biodiesel pro-
duction, the enzymatic route is not an industrial-scale reality yet. There are some
challenges that should be overcome before biocatalysts can be made feasible for
biodiesel production, like their higher cost, biodiesel productivity, and enzyme
inhibition.

14.1 Introduction

The global interest in biofuels is growing in Europe, North America, Asia, and
Brazil and its production is expanding faster than conventional oil supply
(Nogueira et al. 2011). Chemical catalysis is a well-established process for

M. A. Farias (&)  M. A. Z. Coelho

Rio de Janeiro, Brazil
e-mail: farias.marcelle@gmail.com

S. S. da Silva and A. K. Chandel (eds.), Biofuels in Brazil, 303

DOI: 10.1007/978-3-319-05020-1_14,  Springer International Publishing Switzerland 2014
304 M. A. Farias and M. A. Z. Coelho

biodiesel production. The homogeneous alkali-catalyzed transesterification pro-

cess has been extensively applied to the large-scale synthesis of alkyl esters,
especially due to the low cost of base catalysts and their efficiency even at low
concentrations. The chemical reaction, however, has some disadvantages as it is
energy-intensive, requires several separation/purification steps and generates sig-
nificant amounts of wastewater to be treated (Feltes et al. 2012). The drawbacks in
the homogeneous alkaline transesterification process have encouraged researchers
and biodiesel industry to look into different biodiesel production methods.
In this sense, particular attention has been dedicated to the use of lipases as
biocatalysts for biodiesel production due to compatibility with quality variations of
the raw material (especially triglycerides with high free fatty acid content), their
favorable conversion rate obtained in gentle conditions and, relatively simple
downstream processing steps for the purification of biodiesel and by-products
(Gog et al. 2012). Additionally, the use of lipase as a biocatalyst can minimize
wastewater treatment and improve the glycerol recovery (Vyas et al. 2010). Other
advantage of enzymatic route is a more favorable life cycle assessment when
compared to chemical catalysis (Harding et al. 2008). Nevertheless, comparatively
to conventional chemical processes, the major obstacles for enzymatic biodiesel
production remain to be the cost of lipases (Yaakob et al. 2013), the relatively
slower reaction rate and lipases inactivation caused by methanol and glycerol and,
in this way, this technology already presents economic disadvantage (Marchetti
et al. 2007). Then, in order to overcome these challenges, new technologies have
been developed aiming at finding alternatives for enzymatic biodiesel production
at industrial scale. These technologies should take into account the enzyme choice
(source or if it is free or immobilized), medium condition that can improve the
productivity, beyond the process design related to efficiency and operational cost.
Undoubtedly, Brazil is a promising country for biotechnological products
development, such as enzymes for the bioenergy industry, once its biodiversity and
environmental characteristics can contribute to this opportunity.

14.2 Lipase as Catalyst

Lipases (triacylglycerol ester hydrolases E.C.3.1.1.3.) are enzymes classified as

hydrolases that can catalyze both the hydrolysis and the synthesis of esters from
glycerol and long-chain fatty acids. The last reaction occurs only in the presence of
water traces (Paiva et al. 2000). These enzymes, under specific conditions, are also
capable to catalyze reversible reactions: interesterification, aminolysis, and
transesterification reactions. The microbial lipases are glycoprotein and its
molecular weight can vary between 19 and 60 kDa, presenting around from 258 to
544 amino acids residues and the majority of them have hydrophobic characteristics
(Jaerger and Reetz 1998).
Figure 14.1 shows the main reactions catalyzed by lipases. The lypolitic
reactions occur in the lipid-water interface. Jaeger et al. (1999) reported two
14 Critical Technological Analysis for Enzymatic Biodiesel Production 305

Fig. 14.1 Reactions catalyzed by lipases (Ribeiro et al. 2011)

different classification criterias to distinguish between a lypolytic enzyme from

‘‘true lipases’’(E.C. 3.1.1.3): (a) It should be activated by the presence of an
interface, i.e., its activity should sharply increase as soon as the triglyceride
substrate forms an emulsion. This phenomenon was termed ‘‘interfacial activa-
tion’’ (b) It should contain a ‘‘lid,’’ which is a surface loop of the protein covering
the active site of the enzyme and moving away on contact with the interface.
Therefore, these hypotheses seem to be unsuitable for classification, mainly
because some enzymes have a lid but not exhibit the interfacial activation (Verger
1977). Although there is no strict definition available for the term ‘‘long-chain,’’
glycerol esters with an acyl chain length of \10 carbon atoms, usually indicates
the presence of an esterase. It should be emphasized that most lipases are perfectly
capable of hydrolyzing these esterase substrates.
The lipases belong to a/b hydrolases family, considering the structural aspect
and, its activity depends on the catalytic triad. In most lipases, the access to
catalytic site is controlled by helicoidal structure called lip, that under specific
conditions, is responsible for covering the catalytic site. Its role is to block the
306 M. A. Farias and M. A. Z. Coelho

Fig. 14.2 Classically

proposed mechanisms of
hydrolysis, esterification, and
alcoholysis reactions
catalyzed by lipases ME
methyl ester, EE ethyl ester,
MeOH methanol, EtOH
ethanol, AG Fat acid
(Vaysse et al. 2002)

active site in the absence of substrate (closed conformation) while an hydrophobic

interface induces a conformational modification of the lid, rendering the active site
of the lipase accessible to the substrate (open conformation). Knowledge of their
three-dimensional structures and the factors that determine their regiospecificity
and enantiospecificity are traditionally essential to tailor lipases for specific
applications (Jaeger and Reetz 1998).
As mentioned, the reactions involving lipases occur in lipid-water interface and,
as consequence, the kinetics cannot be described by Michaelis–Menten equation,
once this model is only valid to homogenous phase catalysis (Jaeger and Reetz 1998).
The reactions catalyzed by lipases usually follow the ‘‘ping-pong bi–bi’’ mechanism
(Haffner et al. 1999).
Vaysse et al. (2002) proposed a general resumed scheme showed in Fig. 14.2.
From this scheme, it is possible to find the main differences between substrates
(S1 and S2) and products (P1 and P2), considering hydrolysis, esterification, and
transesterification reactions catalyzed by lipases, using methanol (MeOH) and
ethanol (EtOH) as acyl acceptors. Regarding the substrates, it is important to note
that S1 is the acyl donor and S2 is the acyl acceptor.
The transesterification reaction is a term that is widely used to describe an
important class of organic reactions, where one ester is converted into another. The
transfer of an acyl group can happen between an ester and an acid (acidolysis), one
ester and another ester (interesterification) or between an ester and an alcohol
14 Critical Technological Analysis for Enzymatic Biodiesel Production 307

(alcoholysis). The alcoholysis reaction catalyzed by lipases, involves two-step

mechanism for each ester bond of the triglyceride molecule. In the first step, the
ester bond is hydrolyzed and the alcohol moiety is released, followed by an
esterification with the second substrate (Kaieda et al. 1999). Some basic differ-
ences have been identified between the transesterification of triacylglycerols
(TAGs) and the esterification of free fat acids (FFAs). According to Freire et al.
(2011), the transesterification is a sequence of three reaction (diacylglycerol and
monoacylglycerol are formed as intermediates), and the esterification involves
parallel reactions of FFAs in order to produce biodiesel. The last reaction is
quicker than the former one. The higher polarity of FFAs, compared to TAGs,
makes the short-chain alcohols more soluble in the reaction medium. Moreover,
water is one of the esterification products and shifts the equilibrium toward
hydrolysis when the concentration exceeds optimal level.

14.3 Process Variable for Enzymatic Biodiesel Production

Production of biodiesel by lipase is critically influenced by various process vari-

ables such as the choice of enzyme (source and kind), temperature, water content,
water activity, enzyme amount, oil to alcohol ratio, addition of organic solvents,
among others. In this sense, the most relevant variables whose affect the enzymatic
biodiesel production will be discussed in this item.

14.3.1 The Choice of Enzyme

The lipases present many advantages when compared to others catalysts. In this
context, it is important to point out the biocompatibility, biodegradability, and
environmental benefits (Marchetti et al. 2007).
Lipases from bacteria and fungi are the most commonly used for transesteri-
fication process and, the choice of lipase will depend on the origin as well as the
formulation of the enzyme. In general, the best enzymes are able to reach con-
versions above 90 %, while reaction temperatures vary between 30 and 50 C.
Reaction time also vary greatly from a low of 8 h for immobilized Pseudomonas
cepacia lipases transesterifying jatropha oil with ethanol, to a high of 90 h for the
same free enzyme transesterifying soybean oil with methanol (Fjerbaek et al.
2009). Thus, besides the source, it becomes necessary to evaluate different raw
material (oil and fat), acyl acceptors, free or immobilized enzymes, among others
variables.
Free enzymes are far cheaper than immobilized ones. They can be purchased in
an aqueous solution composed by enzymes plus nothing more than a stabilizer to
prevent enzyme denaturation (glycerol or sorbitol) and a preservative to inhibit
microbial growth (Freire et al. 2011). However, in some cases, these enzymes can
308 M. A. Farias and M. A. Z. Coelho

loss the activity in the presence of some compounds. Improved immobilization

technology has provided an enhanced level of reusability, operational stability, and
optimum temperature, resulting in higher conversion rates and shorter reaction
time, respectively. Until present, most thoroughly investigated commercial
immobilized lipases are Novozym 435, Lipozyme TL IM, Lipozyme RM IM, and
Lipase PS-C. Different immobilization methods can be applied for lipases used in
biodiesel production: adsorption, cross-linkage, entrapment, encapsulation, and
covalent bonding (Gog et al. 2012).
To overcome the higher costs of enzymes, compared to homogeneous chemical
catalysts, its reutilization is essential (Li et al. 2012; Tan et al. 2010; Yan et al. 2011).
The longer the reuse of the same enzyme, higher productivity will be obtained for a
given batch of enzyme and, as consequence, the biodiesel production price will
decrease. Efficient reuse is dependent upon whether the enzymes can obtain and
maintain a high initial activity without inactivation or inhibition (Fjerbaek et al.
2009). Moreover, the size of immobilized enzyme helps its separation from reaction
medium when it is compared to free enzyme.

14.3.2 Water and Acidy Impact

The water concentration in enzymatic transesterification has an important hole for

enzyme conformation, protecting the protein against structural deformation
(Fjerbaek et al. 2009). For the lipase-catalyzed biodiesel production in predomi-
nantly nonaqueous media, in fact, water plays multiple roles and it has strong
influence on the catalytic activity and stability of the lipase (Gog et al. 2012).
Lipases have a unique feature that consists in catalyze the reaction at interface
between water and organic phase. Generally, the enzyme activity depends on the
interface and, the increasing of interfacial surface is well inclined to enzyme
activity maintenance. However, the excess of water can conduct undesirable
reactions, like hydrolysis during transesterification process. Thus, the water
quantity required to maximize the lipolytic activity will depend on raw-material,
immobilization support, and the solvent used (Tan et al. 2010).
The effect of water concentration in enzymatic biodiesel catalysis by R. oryzae,
C. rugosa, P. fluorescens, Novozym 435, and B. cepacia showed that lipase activity
was lower in water absence. This result demonstrates that a minimum water con-
centration would be required for enzyme activation. With increasing amount of
water added to the medium, there was also an increased formation of ester,
confirming the rise of lipase activity (Al-Zuhair and Emirates 2007). On the other
hand, Shimada et al. (1999) emphasized that the higher water addition in the
reaction, the lower would be the ester formation. Corroborating Shimada et al.
(1999), Azócar et al. (2011) investigated biodiesel production, in anhydrous med-
ium, by lipase from Novozym 435, using frying oil as raw-material and methanol as
acyl acceptor. The low concentration of water allowed the esterification of fatty
14 Critical Technological Analysis for Enzymatic Biodiesel Production 309

acids (already presented in raw material) at the beginning of reaction and, after, the
transesterification reaction producing methyl esters.
Considering the acidity of raw material, comparing the transesterification
process by chemical catalysis to the enzymatic one, besides being a cleaner
technology, the last presents advantages over the alkaline chemical catalysis. In
chemical catalysis, the raw material needs to be previously treated when it has high
concentration of fat acids, in order to diminish the acidity (Freedman et al. 1984;
Kaieda et al. 1999; Zhang et al. 2003). The pretreatment is also important to
reduce the saponification of free fat acids, caused by alkaline neutralization
process that promotes difficulties in the separation of biodiesel and glycerol.
Additionally, it can generate alkaline residual water (Meher et al. 2006; Mittelbach
1990) and can cause environmental impact and higher energy consumption.
Different from alkaline transesterification, the enzymatic technology for bio-
diesel production do not form ‘‘soap’’ (when raw materials with significant amount
of fatty acids are used) and, it can esterify the FFA in one unique step, without
need of subsequent washing process. Thus, enzymes are potential catalysts for
biodiesel production in industrial scale, decreasing the costs with regard to raw
material treatment. The enzymatic technology becomes very attractive because it
will not be necessary to have a raw material with strict specification (acid and
water concentration) being its commercial value lower than the raw material used
for alkaline chemical transesterification.

14.3.3 Organic Solvent

The use of organic solvents in enzymatic biodiesel synthesis improves mutual

solubility of hydrophobic triglycerides and hydrophilic alcohols and also protects
enzymes from denaturation by high concentrations of alcohols (Gog et al. 2012).
Additionally, it reduces the viscosity of the reaction mixture increasing the dif-
fusion rate reducing mass transfer problems around the enzyme. For immobilized
enzymes, nonpolar solvents might maintain the residual water around the enzyme
increasing the water activity locally and solvents might help stabilizing enzymes
(Fjerbaek et al. 2009).
Tert-butanol has been shown as an excellent solvent for maintaining the
enzyme activity (Liu et al. 2011; Chattopadhyay et al. 2011) due to its ramifica-
tion, which is more miscible in triacylglycerol when compared with linear alcohols
with the same carbon number (Azócar et al. 2011). Royon et al. (2007) investi-
gated the biodiesel production using cotton seed oil as raw material, lipase as
biocatalyst from immobilized Candida antartica and tert-butanol as a solvent. The
results showed some advantages when tert-butanol was used: (a) In the presence of
this solvent, high reaction rates and yield were obtained. (b) The quantity of
enzyme needed to catalyze the reaction within a reasonable time periods was lower
than that of other systems. (c) A very simple, one step continuous reactor was used
310 M. A. Farias and M. A. Z. Coelho

for biodiesel production. (d) No catalyst regeneration steps were needed for lipase
reuse. (e) The operational stability of the catalyst was high even at 50 C.
However, according to Kumari et al. (2007) and Soumanou and Bornscheuer
(2003), the use of solvents can greatly reduce the enzyme activity, increasing
investment with reagents and fixed assets, and can increase the reactor volumes to fit
the additional volume of solvent. Then, the enzymatic technology using solvent-free
system becomes very interesting feature for future industrial scale. Nevertheless,
additional efforts are necessary to conquer the reaction time reduction.

14.3.4 Temperature

The enzymatic transesterification is generally performed at lower temperature, when

it is compared to chemical reaction, in order to prevent loss of lipase activity. Opti-
mum temperature determined by various lipases, used for biodiesel synthesis, ranges
between 30 and 55 C (Gog et al. 2012). Strategically, this range of temperature
results from interaction between operational stability and high conversion rate.
Generally, immobilized lipases present greater temperature resistance when
compared to free ones. According to Fjerbaek et al. (2009), the binding to the
carrier material gives stability to the enzyme and promote a decrease of the
thermal effect, avoiding deactivation when compared to the free enzyme.
Table 14.1 presents examples of lipases from different microorganisms and their
respective temperatures.

14.3.5 Acyl Acceptor

Different acyl acceptors have been studied for enzymatic biodiesel production, and
the alcohol is the main chemical molecule chosen. Many alcohols as methanol,
ethanol, 2-propanol, and 2-butanol have been studied as acyl acceptors for enzy-
matic transesterification of triacylglycerols; however, they can affect lipase
activity through different mechanisms. Jech et al. (2003) used different alcohols
aiming at evaluating the lipase inhibition level. Linear alcohols as methanol,
ethanol, propanol, and butanol, and also ramified ones as isopropanol and isobu-
tanol were investigated. All linear alcohols tested were toxic to enzyme. The
inhibition level was inversely proportional to the carbon number presented in
alcohol chain. When the linear chain is compared to the ramified one, the latter is
less impactable to lipase activity (Jech et al. 2003).
Shimada et al. (1999) and Watanabe et al. (1999) proposed a different solution
for this drawback. They reported the gradual addition of alcohol into reaction
medium in order to minimize the enzyme activity loss. Shimada et al. (2002)
recommended methanol addition in steps, once the methanol is more soluble in
14 Critical Technological Analysis for Enzymatic Biodiesel Production 311

Table 14.1 Temperatures used for enzymatic biodiesel production (Fjerbaek et al. 2009)
Temperature (C) Lipase, fatty acid/oil/tallow and alcohol
50–60 P. fluorescens, oleic acid, propanol, and butanol
70 P. fluorescens, oleic acid, propanol, and butanol
20–60 Novozym 435, soybean and rapeseed oils mixture, methanol
25–60 Novozym 435/Lipozyme TL IM/Lipozyme RM IM, soybean oil, methanol

acyl ester than in triacylglycerol. Rodrigues et al. (2010) reported the addition of
ethanol in two steps in order to promote the ethanolysis of soybean oil by
immobilized enzyme of Thermomyces lanuginosus and the best result reached
100 % of conversion.
Depending on acyl acceptor used for biodiesel production, it will influence the
fuel proprieties, it means, the behavior of fluidity (Lee et al. 1995; Wang et al.
2005) and lubricity (Drown et al. 2001) when in contact with different temperature
levels. The alcohols used for enzymatic or chemical process need to have a low
commercial value, in order to reduce the total manufacturing cost. In this sense,
methanol and ethanol appear as good options because of their lower price, when
compared to secondary and tertiary alcohols. Despite the higher price, these last
alcohols are also appropriate to biodiesel production, since these compounds are
responsible to form an ester with low fluidity point. However, according to
Stamenković et al. (2011), the complexity of alcoholysis conditions carry on
economically unviable for secondary and tertiary alcohols.
In an industrial process, the acyl acceptors, besides having a low cost, need to
be commercially available in large scale. Taking into account these considerations,
methanol and ethanol continue to be very interesting alternatives for biodiesel
production, once they present competitive prices and market availability.

14.3.6 Phospholipids

The enzymes showed considerable inhibition by phospholipids presented in the

crude oil, during the biodiesel production (Lai et al. 2005; Wei et al. 2004). The
phospholipids are the main components removed by degumming process and,
because of this limitation, the use of refined oil is relevant for enzymatic biodiesel
production. The best alternative for phospholipids removal would be the use of
phospholipases, followed by enzymatic transesterification reaction. This strategy
was investigated by Jang et al. (2012) and reached conversion up to 89 %.
According to Séverac et al. (2011), the inhibition caused by the presence of
phospholipids, in crude high-oleic sunflower oil, was eliminated by using tert-
butanol. This solvent was chosen, because of its medium polarity. Additionally,
tert-butanol helps to preserve the activity of Novozym 435 as well as improve its
stability in the medium.
312 M. A. Farias and M. A. Z. Coelho

14.3.7 Glycerol Formation

Although glycerol being a by-product formed during the enzymatic transesterifi-

cation, the influence of this compound on enzyme activity is relevant. During
biodiesel production, Soumanou and Bornscheuer (2003) reported that glycerol
could inhibit transesterification reaction by limiting the mass transfer due to its
insolubility in the oil. According to Lee et al. (2011), the effect of glycerol on
enzyme activity was tested using Novozym 435 and Lipozyme RM IM. Both
enzymes were incubated in a mixture of canola oil with glycerol (0–15 % w/w) for
2.5 h prior to activity assay (the immobilized lipases were washed with isopropyl
alcohol, in order to avoid the effect of glycerol adsorbed onto support). The results
obtained by this experiment showed that high glycerol contents did not influence
the enzyme performance and, the lowered performance of lipases by glycerol
during biodiesel synthesis in mainly due to mass transfer limitation rather than
direct inhibition of the enzyme. However, some authors corroborated that the
enzyme deactivation could be caused by glycerol, especially under a solvent-free
system (Robles-Medina et al. 2009; Talukder et al. 2009) and can inhibit the
enzyme activity by forming a hydrophilic coat on its surface that exclude TAG
from the active site (Véras et al. 2011). According to Xu et al. (2011), glycerol
formed in biodiesel synthesis by immobilized lipases can severely reduce the
reaction rate by surrounding the catalyst in a hydrophilic layer, thereby limiting the
mass transfer of substrate to the enzyme.

14.4 Process Design: Technological Trends

The process setup is very important needing to consider the above discussed
technical issues like, reaction/product mixture, solubility of alcohol, enzyme sta-
bility and recovery, among others (Nielsen et al. 2008). Regarding the enzyme
characteristics, it is relevant to include the reuse of this biocatalyst. For free
enzymes, this can be achieved using an ultrafiltration or centrifugation unit and,
for immobilized ones, different techniques and matrix (support) are available for
immobilization.
Considering such features, a reactor configuration for industrial applications has
an important role to make the enzymatic biodiesel production economically fea-
sible. Then, there are several different processes to be considered, in order to
develop a process design: batch, continuous stirred-tank reactors and packed-bed
reactors. Other possible solutions were described in the literature, according to
Fjerdaek et al. (2009): fluid beds, expanding bed, recirculation membrane reactors,
or reactors with static mixers.
According to Nielsen et al. (2008), the batch design is a typical process used in
laboratory scale due to the simple setup. All reagents used in the reaction are
introduced from the start, whereas stepwise addition of alcohol (mainly methanol)
14 Critical Technological Analysis for Enzymatic Biodiesel Production 313

is recommended. On the other hand, this process setup in large scale promotes a
long reaction time and the gradual decline of enzyme activity according to the
number of reuses. As time goes by, the plant capacity will decrease and, even-
tually, becomes unacceptably low.
The continuous stirred-tank reactor (CSTR) consists of a continuous supply of
substrate feed and product withdrawal. The design requires multiples tanks in
series to assure the same degree of conversion for the same reaction. It is important
to note that this process has interesting advantages like: (a) the reaction can hold
enzymes of different age/activities; (b) possibility of introducing separation steps
between the tanks in order to eliminate the glycerol formed as byproduct. In
contrast, Tan et al. (2010) pointed out the stress caused by stirring, once it would
disrupt the enzyme carrier by physical agitation. So, the immobilized enzymes
sometimes might not be reused for a long period.
Nielsen et al. (2011) reported an enzymatic large-scale production of biodiesel
in two different steps using free enzymes and immobilized ones. First, a liquid
formulated lipase is used (CalleraTM Trans) for transesterification and the second
step is the esterification of FFA with the immobilized enzyme (Callera Ultra).
They tested a setup with three CSTRs in series. The FAME content out of the
reactors was 67, 85, and 89 % in reactor 1, 2, and 3, respectively, when the system
was in steady y state. The remaining FFA, inside the tank 3, was converted to
FAME and, in addition, transesterifies the remaining glycerol esters.
Considering the industrial scale of enzymatic biodiesel production, Tan et al.
(2010) reported that Lvming Co. Ltd., in 2007, established an enzymatic production
line with capacity of 10,000 tons in Shanghai, China. The process is carried in
stirred-tank reactor (STR) system and the technique used comes from Beijing
University of Chemical Technology, with immobilized lipase Candida sp. 99–125
as catalyst. A waste cooking oil have been used as raw material. A centrifuge is
used to separate out the glycerol and the water produced during the reaction, and the
yield of FAME has reached 90 % of conversion under optimal conditions. Another
plant that conducts enzymatic catalysis in China is Hainabaichuan Co. Ltd., Hunan
Province. The factory has used the technology of Tsinghua University and
commercial Novozym 435 as catalyst.
Together with STR, the packed-bed reactors (PBR) are the most widely used
reactors for enzymatic biodiesel production. This system consists of a continuous
operation without separation of the catalyst from the reaction product. The PBRs
generally use immobilized enzyme packed in column that allows an easy imple-
mentation of continuous process. In this way, as biodiesel is a chemical com-
modity, its production in continuous-flow systems would certainly reduce the
operational costs of its production (Freire et al. 2011). However, the main dis-
advantage is that the resulted glycerol remains at the bottom of the reactor and
might deposit on the surface of the immobilized lipase, thus decreasing the cat-
alytic efficiency (Gog et al. 2012). So, it is relevant to know that the glycerol
produced in the reaction can be removed between the columns and the inactivation
of the enzyme by addition of methanol/ethanol can be solved by stepwise addition
before each column. In this sense, immobilizing enzymes for this application
314 M. A. Farias and M. A. Z. Coelho

generally has: a positive effect on the operational stability of the catalyst (compared
to free enzymes), an easier handling (compared to free enzyme powder), and allows
operation under low-water conditions (compared to liquid formulated enzymes)
(Nielsen 2008).
Fjerdaek et al. (2009) concluded that for continuous production, it is possible to
achieve longtime enzyme stability in PBRs, with or without solvents. The use of
solvents in itself only increases production costs as they have to be removed and
purified for recycling. On the other hand, the pressure drop caused by the high
viscosity of solvent-free systems could become a problem. For large-scale pro-
duction, PBRs should operate at low flow rates or using larger biocatalyst particle
sizes to minimize such a drop in pressure, once with increasing particle diameter,
the pressure drop decreases.
In Brazil, most of the process design for enzymatic biodiesel production is
based on STR reactors. However, in a recent study from Federal University of
Santa Catarina, Dors et al. (2012) demonstrated the potential of lipase as biocat-
alyst in continuous PBR for biodiesel production using a great variety of raw
material. The best result and conditions for transeterification of this study can be
found in Table 14.2. According to Table 14.2, PBR is a potential technology for
enzymatic biodiesel production. However, a suitable process technology has yet to
be established.

14.5 Enzymatic Biodiesel Production: Brazilian

Experience

14.5.1 Why Brazil is a Potential Country for Enzymatic

Biodiesel Production

There are two interesting arguments that should stimulate technology develop-
ments for enzymatic biodiesel production in Brazil. One of them is the possibility
of using a raw material with low price, that presents high acidity value. Another
reason is the use of agroindustrial residues as substrate, for microbial lipase
production.
Brazil has large diversity of oleaginous cultures which have high potential of
producing biodiesel. Almost all vegetable oils can be used as raw matter for
biodiesel production, which is a promising activity in Brazil due to the potential
growth of sunflower, soybean, castor bean, African palm, babassu, cotton, peanut,
linseed, macauba, pequi, buriti, sesame, canola, and others (Lopes et al. 2011).
Even the great variety of vegetable oils available for biodiesel production, when
these oils have high concentration of fatty acids, it is necessary to previously treat
this raw material, in order to reduce the saponification of free fatty acids. The
pretreatment is indispensable for biodiesel production using alkaline chemical
catalysis technology.
Table 14.2 Enzymatic biodiesel production by different design processes
14

Reactor Lipase source Oil/fat alcohol Condition Conversion Solvent Operation References
(%) time (h)
Packed-bed C. antartica Soybean oil and 1 bioreactor 75 solvent 168 Chang et al.
reactors (Novozym 435) isopropanol continuously free (2009)
operated
(sR = 1 h,
T = 51.5 C).
1:4 oil/alcohol
ratio
Packed-bed Burkholderia cepacia (lyophilized Soybean oil and 1 bioreactor 95 solvent 190 Salum et al.
reactors and delipidated fermented solid) ethanol operated in free (2010)
batch mode
(sR = 46 h,
T = 50 C)
with 2
stepwise
additions of
alcohol and 3:1
alcohol/oil
molar ratio
Batch system The recombinant Rhizopus oryzae Pistaciachinensisbge 1 bioreactor 94 solvent 60 Li et al. (2012)
immobilized on macroporous seed oil (PCO) (T = 37 C) free
resin and anion exchange resin with methanol and methanol
Critical Technological Analysis for Enzymatic Biodiesel Production

to oil molar
ratio 5:1, water
content 20 %
by weight of
oil
(continued)
315
Table 14.2 (continued)
316

Reactor Lipase source Oil/fat alcohol Condition Conversion Solvent Operation References
(%) time (h)
Packed-bed C. antarctica (Novozym 435) Sunflower oil and 1 bioreactor 90 solvent 210 Jachmanián
reactors isopropanol continuously free et al.
operated (2009)
(T = 50 C)
with oil/
alcohol/
isopropyl ester
weight ratioof
35:35:30
Packed-bed C. antarctica (Novozym 435) Cottonseed oil and 1 bioreactor 95 t-butanol 24 Royon et al.
reactors methanol continuously (2007)
operated
(T = 50 C)
with oil/
alcohol/tert-
butanol weight
ratio of 1:2, 4:4
Four-packed- Pseudomonas cepacia Soybean oil and 4 bioreactor over 88 n-hexane 192 Wang et al.
bed (commerciallipase Fe3O4 methanol continuously (2011)
reactors nanoparticlebiocompositecatalyst) operated
(T = 40 C).
The ratio of
the volume of
soybean
oil:distilled
water:
methanol:n-
hexane was
6:3:1:0, 2
M. A. Farias and M. A. Z. Coelho

(continued)
Table 14.2 (continued)
14

Reactor Lipase source Oil/fat alcohol Condition Conversion Solvent Operation References
(%) time (h)
Batch system Crude pancreatic lipase Cottonseed oil and 1 bioreactor 75–80 t-butanol 4 Chattopadhy
methanol (T = 37 C) et al.
and methanol/ (2011)
oil molar ratio
was 15:1 and
water con-
centration of
5 % (wt of oil)
Packed-bed C. antartica Crude high-oleic 1 bioreactor 96 t-butanol 48 Séverac et al.
reactors (Novozym 435) sunflower oil and (T = 60 C) (2011)
butanol and butanol/oil
molar ratio
was 5:1
Batch system Self-developed Burkholderia Olive oil 1 bioreactor (room 70 solvent 12 Liu
immobilized onto hydrophobic temperature) free et al.(2012)
magnetic particles and methanol/
oil molar ratio
was 4:1 and
water con-
centration of
10 % (wt of oil)
Critical Technological Analysis for Enzymatic Biodiesel Production

Batch system Thermomycesl anuginosus was Pomace oil and 1 bioreactor 93 solvent 24 Yücel et al.
immobilized by covalent binding methanol (T = 25 C) free (2011)
onto olive pomace and methanol/
oil molar rate
was 6:1, using
three-step
addition of
alcohol
317

(continued)
Table 14.2 (continued)
318

Reactor Lipase source Oil/fat alcohol Condition Conversion Solvent Operation References
(%) time (h)
Continuous P. fluorescens lipase immobilized on Palm oil and ethanol 1 bioreactor 87.6 t-butanol 4.6 Dors et al.
packed- epoxy-polysiloxane–polyvinyl (T = 50 C) (2012)
bed alcohol composite and methanol/
reactor oil molar rate
was 9:1, 70 %
of the
biocatalyst
activity was
retained even
after
continuous
operation for
almost 48 days
Packed-bed Commercial C. antartica lipase B Rapeseed blended 1 bioreactor 98.6 solvent After 8th Hama et al.
reactors immobilized on macroporous with soybean oil (T = 30 C) free pass (2011)
integeated acrylic resin and methanol and methanol/ (time not
with oil molar ratio informed)
glycerol was 1:2 and 10
separation stepwise
additions of
alcohol
Batch system Lipozyme TL IM Castor and jatropha 1 bioreactor 78.3 solvent 24 Maleki et al.
oil blended (1:5) (T = 45 C) free (2013)
and methanol and methanol/
oil molar ratio
was 1:1, using
single stepwise
of alcohol
M. A. Farias and M. A. Z. Coelho
14 Critical Technological Analysis for Enzymatic Biodiesel Production 319

Another potential raw material for enzymatic biodiesel production could be

tallow and fried oils, once they present low cost and, in many cases, reduction of
environmental problems associated to the final deposition of these materials.
Brazil has the second largest herd in the world, with a cattle herd of 207.2 million
heads in 2007 and is one of the greatest producers and first exporter of beef over
the world. The current production in Brazil is around 700,000 tons of tallow per
year. As regard used fried oil, there is also a good potential of supply estimated in
about 300,000 tons per year (Nogueira 2011).
Take in advance the fact that Brazil generates, annually, thousands of tons of
agricultural and agroindustrial residues, the bioconversion of these residues for
lipase production, as well as, other value-added products would point out Brazil a
prominent position in the future biotechnology developments. Oil cakes of various
residues obtained from extraction of oils have been utilized for fermentative
production of lipases (solid state fermentation). This is because their residual oil
contents serve as inducers for lipase production. Several agricultural residues have
been reported to be effective for lipase production and these include brans (wheat,
rice, soybean, barley), oil cakes (soy, olive, gingelly, babassu), and bagasse
(sugarcane) (Salihu et al. 2012). Additionally, these residues have attracted
increasing attention as abundant and cheap renewable feedstock and can diminish
the environmental impact of biodiesel supply chain. Nowadays, these residues can
be used as animal feed (when it is on specification) or can be discarded in landfill.
Some of the most reported microbial genera that produce lipases using solid
state fermentation (SSF) technique are Aspergillus, Candida, Humicola, Peni-
cillium, Rhizopus, Geotrichum, Mucor, Pseudomonas, and Rhizomucor. In the
last decade, lipases have been increasingly studied as biocatalysts for biodiesel
production either lyophilized or immobilized (Castro and Castro 2012;
Gunasekaran and Das 2005). Additionally, Castilho et al. (2000) compared the
economic viability of lipases production by Penicillium restrictum using SSF and
submerged fermentation strategies. After scale up, authors concluded the
returned unitary production cost was 47 % lower than the practiced selling price
in the period the study was carried out, and the major reason was the use of
low-cost agroindustrial raw materials.
The Brazilian biodiversity and environmental characteristics create an oppor-
tunity to be a major producer of biotechnological products, such as enzymes for
the bioenergy industry (Castro and Castro 2012). It is also important to notice the
great potential of this country to integrate processes, it means, the biocatalyst and
raw material for biodiesel production could come from the unique one oleaginous.
320 M. A. Farias and M. A. Z. Coelho

14.5.2 Enzymatic Transesterification for Biodiesel

Production in Brazil

The transesterification is the most studied process in enzymatic biodiesel

production and this reaction consists in one unique step. In fact, the Brazilian
academic community is looking for a better conversions and economic viability,
considering diversity of raw materials, by-products, and sources of lipases. In this
sense, different strategies for transesterification have been tested to reach better
conversions.
Freitas et al. (2009) studied different commercial lipases for integrated pro-
duction of biodiesel and monoacylglycerol, such as: Candida antartica B (CAL
B), Pseudomonas fluorescens (Lipase AK), Burkholderia cepacia (Lipase PS), and
Penicillum camemberti (Lipase G). All lipases were immobilized on silica–PVA
composite by covalent immobilization. The assays were performed using babassu
oil and ethanol for biodiesel production and glycerol for monoacylglycerol pro-
duction, in solvent free system. For both substrates, lipase from B. cepacia (lipase
PS) was found to be the most suitable enzyme to attain satisfactory yields. For
biodiesel production, the highest transesterification yield was [98 % in 48 h of
reaction at 39 C using an oil-to-ethanol molar ratio of 1:7. For monoacylglycerol
production, the better conditions were oil-to-glycerol molar ratio of 1:15 at 55 C.
This investigation showed the potential integrated process in order to produce one
more product and, as consequence, to increase the revenue of possible future
technologies.
Rodrigues et al. (2008) studied immobilized lipases (Novozym 435, Lipozyme
TL-IM and Lipozyme RM-IM) in enzymatic alcoholysis of three vegetable oils,
soybean, sunflower, and rice bran, using different acyl acceptors (ethanol, propanol
and butanol). The results showed that each lipase displayed the alcoholysis reac-
tions using the three different alcohols. Novozym 435 presented higher activity in
methanolysis, at a 5:1 methanol:oil molar ratio; Lipozyme TL-IM presented higher
activity in ethanolysis, at a 7:1 ethanol:oil molar ratio; and Lipozyme RM-IM
presented higher activity in butanolysis, at a 9:1 butanol:oil molar ratio. The
conversion reached values around 50 % of FAME. The optimal temperature was in
the range of 30–35 C for all lipases and when commercial ones were washed with
n-hexane, approximately 90 % of the enzyme activity remained after seven syn-
thesis cycles.
Sangaletti et al. (2012) investigated other strategy to produce biodiesel, used to
positively contribute to the development of an integrated and environmentally
friendly technology. The authors studied the replacement of hexane by ethanol in
soybean oil extraction process and, in this sense the production of biodiesel from
oil and ethanol micelle (oil ? solvent) by direct transesterification using Novozym
435 as catalyst and ethanol as acyl acceptor was investigated. The best experi-
mental conditions were found using 40 C of temperature, oil:ethanol molar ratio
1:4.5 and catalyst concentration 9.5 % for 24 h, reaching 85.4 % of fat acid ethyl
ester (FAEE) yield. Tert-butanol was used as co-solvent and increased the ethyl
14 Critical Technological Analysis for Enzymatic Biodiesel Production 321

esters yield at 18 %, keeping a high FAEE yield (over 70 %) for more than three
cycles of enzyme reuse.
Another way, to minimize the costs, consists in using a non-commercial
immobilized lipase, in other words, to developed an indigenous technology that
provides the production and immobilization of lipases. Moreira et al. (2007)
reported the transesterification of palm oil with ethanol in a solvent free system
using lipase from different sources (T. lanuginosus, P. fluorescens, B. cepacia,
Penicillium camembertii, and Candida antarctica, porcine pancreatic), immobi-
lized on hybrid support polysiloxane–poly-(vinyl alcohol). The best performance
was attained with the lipase from P. fluorescens that reached almost full con-
version (99.4 %) in less than 24 h of reaction, under established operational
conditions, using 18:1 as molar ratio of ethanol:oil at 58 C. This is an excep-
tional option for the Brazilian biodiesel production, because both palm oil and
ethanol are readily available in this country. In this sense, Rodrigues et al. (2010)
investigated the immobilization and stabilization of lipase from T. lanuginosus
(TLL) on aldehyde-Lewatit (Lew-TLL). Lew-TLL was 10-fold more thermo
stable than the commercial TLL preparation (Lipozyme TL-IM). The stabilized
Lew-TLL was used for the enzymatic transesterification of ethanol and soybean
oil. When n-hexane was used as co-solvent, the transesterification reached 100 %
of conversion after 10 h, while in solvent-free system the yield was 75 %. The
ethanol was added in two steps, using 7.5:1 as ethanol:soybean oil molar ratio.
Aiming at finding new alternatives for conversion improvement, Brazilian
researches have been investigated different mechanisms/processes to increase
enzymatic transesterification yields. Considering this issue, the use of supercritical
CO2 in the production of biodiesel appears to be a very interesting process to be
analyzed. Rodrigues et al. (2011) reported a continuous process for biodiesel
production in supercritical carbon dioxide. This apparatus consisted of two main
sections: a reaction section comprising a high pressure packed-bed enzymatic tubular
stainless steel reactor, and a separation section. The transesterification of virgin
sunflower oil with methanol was carried using Lipozyme TL IM as a biocatalyst.
Fatty acid methyl esters yield exceeded 98 % at 20 MPa and 40 C for a residence
time of 20 s and an oil to methanol molar ratio of 1:24. The authors also informed that
Lipozyme TL IM was less efficient using waste cooking sunflower oil as raw material.
In this case, a combination of Lipozyme TL IM and Novozym 435 afforded FAME
yield nearing 99 %. The use of supercritical carbon dioxide increases both mass and
thermal transfer and leads higher reaction rates (Lee et al. 2009; Lozano et al. 2011).
In order to minimize the enzyme inhibition and maximize the conversion of
triacylglycerol to ethyl ester, Gamba et al. (2008) investigated the biodiesel pro-
duction using lipase from P. cepacia supported in ionic liquid, 1-n-butyl-3-
methylimidazolium bis (trifluorome-thylsulfonyl)imide, as an alternative of
‘‘green’’ method for biodiesel production from the alcoholysis of soybean oil. The
transesterification reaction, catalyzed by this ionic liquid-supported enzyme, was
able to be performed at room temperature, in the presence of water and without the
use of organic solvents. The biodiesel was separated by simple decantation and the
recovered ionic liquid/enzyme catalytic system could be reused at least four times
322 M. A. Farias and M. A. Z. Coelho

without loss of catalytic activity and selectivity. According to this investigation,

96 % of conversion in 48 h was obtained using 8.2 mmol of ionic liquid (as a
support), methanol/water rate of 70:30 at 30 C. In this studied ionic liquid
provides the ideal medium for the stabilization of the enzyme and, additionally,
removes glycerol from the reaction medium, avoiding enzyme inhibition.
Considering the conversion optimization, Batistella and Lindomar (2012)
reported the use of ultrasonication, once this technique reduces the processing and
phase separation time of the transesterification reaction. This work investigated
soybean oil transesterification with ethanol using two commercial immobilized
lipases (Novozym 435 and Lipozyme RM IM) under the influence of ultrasound
irradiation (ultrasonic water bath). Results showed that ultrasound-assisted lipase-
catalysis might be a potential alternative route to conventional alkali-catalyzed
method, once this experiment reached 90 wt.% of FAME. This yield was obtained
at mild irradiation power supply (100 W), and temperature (60 C) in a relatively
short reaction time, around 4 h, using Lipozyme RM IM as catalyst and oil/ethanol
molar rate of 1:3. However, catalyst repeated use under the optimum experimental
condition resulted in decay in both enzyme activity and product conversion after
two cycles. Nogueiraet al. (2010) investigated the biodiesel synthesis from
macauba oil (Acrocomia aculeata) and ethanol using Novozym 435 and Lipozyme
IM, under microwave irradiation. The results showed that the activity of the
enzyme had increased about one order of magnitude due to microwave, however
the conversion of FAME remained low.

14.5.3 Other Strategies for Enzymatic Biodiesel Production

in Brazil

In Brazil, beyond the transesterification process, others strategies have been

developed for enzymatic biodiesel production. In this way, enzymatic hydroeste-
rification reactions (hydrolysis followed by esterification step); concomitant
esterification and transesterification reactions; and esterification of high acid raw
materials have been studied as enzymatic biodiesel production alternatives.
Aiming at using high acid oils as raw material for biodiesel production, Corrêa
et al. (2011) studied an alternative route based on esterification of free fatty acids
present in by-products obtained from vegetable oil refining, such as palm oil fatty
acid distillate (PFAD). PFAD is a byproduct of the production of edible palm oil,
which contains 96 wt.% of free fatty acids, becoming quite impossible its use in
conventional alkaline transesterification. In this way, the authors investigated the
biodiesel synthesis via esterification of PFAD, using methanol and ethanol as acyl
acceptors, catalyzed by commercial immobilized lipases (Novozym 435, Lipozyme
RM-IM, and Lipozyme TL-IM), in a solvent-free system. The best result was
reached when Novozym 435 was used as biocatalyst and ethanol as acyl acceptor
(two stepwise additions). The conversion was 93 % after 2.5 h of esterification
reaction using 1.0 wt.% of enzyme at 60 C.
14 Critical Technological Analysis for Enzymatic Biodiesel Production 323

A different strategy to reduce the costs of enzymatic biodiesel production was

proposed by Salum et al. (2010). This group demonstrated a possible strategy
regarding the use of lipase from B. cepacia LTEB11 by solid-state fermentation
and added the lyophilized fermented solid (LFS) directly to the reaction medium to
catalyze esterification and transesterification reactions. Not only is the solid sub-
strate relatively cheap, but also the steps of lipase extraction, purification, and
immobilization are avoided. This fermented solid was packed into a column and
used to catalyze the synthesis of biodiesel through the ethanolysis of soybean oil in
a medium free of co-solvents. The best conversion was 95 % after 46 h, which was
obtained at 50 C, with an alcohol:oil molar ratio of 3:1, alcohol addition in two
steps and the addition of 1 % of (m/m) water to the reaction medium. Fernandes
et al. (2007) also found important results using the same strategy. The main result
of this research was found after 18 h, reaching 94 % of esterification and after
120 h, up to 95 % for transesterification reaction.
With the advent of LFS as an immobilized system for biodiesel production, the
relevance of technology protection becomes to be a very important issue, because
of industrial interest. In this way, the patent PI0704791-6 was deposited in order to
protect the process of esters production, using oleic acid, ethanol, and LFS (from
B. cepacia). This technology has a great potential, once this lipase is immobilized
in the raw material, avoiding subsequent steps for separation and could be reused
for several times.
Beyond these strategies mentioned in this chapter, some researchers have been
directed to hybrid catalysis using enzymatic and chemical reactions. Sousa et al.
(2010) studied the biodiesel in two steps. The first step consisted in Jatropha curcas
oil hydrolysis by vegetable lipase from the own plant and, as result, 98 % of tria-
cylglycerols were converted into fat acids after 2 h. The second step of the process
converted the fat acids into esters, using methanol as acyl acceptor and niobic acid as
catalyst (heterogeneous acid catalysis). The results showed that 97.1 % of methyl
esters (high quality biodiesel) were produced. Cavalcanti-Oliveira et al. (2010) also
investigated the hybrid catalysis for biodiesel production. First, the hydrolysis was
conducted by lipase from T. lanuginosus, using soybean oil as triacylglycerol. In this
study, the conversion rate was 89 % after 48 h of reaction. Following the process,
the fatty acids were transformed into ester, using methanol and ethanol as acyl
acceptors. The results were 92 and 83 % using, respectively, ethanol and methanol
as acyl acceptors. It is important to note that these results were reached after 1 h of
reaction.

14.6 Economic Aspects

The scientific community has published high values of FAME conversion by

enzymatic route (Shah et al. 2007; Royon et al. 2007; Salis et al. 2005).
Undoubtedly, the main challenge of enzymatic biodiesel production is to prove the
324 M. A. Farias and M. A. Z. Coelho

economic feasibility of this process when it is compared to alkaline catalysis. In

both process, the capital and operating costs will depend highly on the chosen
process design and its implications on purification steps, etc. However, in general
terms, processing costs will be a function of factors such as: cost of enzyme, cost
of oil (usage of lower-cost high-FFA); cost of alcohol; cost of preprocessing steps;
process yield; cost of waste product handling; value of glycerol stream and;
posttreatment stages (Nielsen et al. 2008).
Analyzing the costs with raw materials, it constitutes the significant component
of overall production costs, and the soy oil feedstock, for instance, is the biggest
contributing factor, itself constitute 88 % of the overall production cost. These
values are consistent with the results of others costs analysis of biodiesel pro-
duction from refined soy oil (Haas et al. 2006). Zhang et al. (2003) reported that
approximately 70–95 % of the total biodiesel production cost arises from the cost
of raw material; that is, vegetable oil or animal fats. Virgin oil costs approximately
2–3 times more than waste cooking oil indicating that use of virgin oil leads to a
substantial increase in total manufacturing cost. When cooking oils are used as raw
material, the viability of a continuous transesterification process and recovery of
high quality glycerol as a biodiesel by-product is an interesting option to be
considered to lower the cost of biodiesel (Demirbas 2009).
Since there is no detailed data for biodiesel production (alkaline catalysis) costs
in Brazil, Giersdorf (2012) reported that the production costs of this biofuel can
only be estimated by using a process model (Haas et al. 2006). The study consists
mainly of three different steps: the pretreatment of the feedstock, the alkaline
catalysis transesterification, and the purification of biodiesel and co-products. The
total biodiesel cost was estimated to be 0.53 USD/L. Since the vegetable oil alone
represents 87 % of the subtotal costs, it is obvious that a change in the feedstock
and/or feedstock costs/prices will significantly impact the overall production costs.
In this way, the possibility of using an enzyme, that accepts high acidity raw
materials, appears to be very attractive for economic aspects.
Besides raw material cost, the price of enzymes should be brought down if this
biocatalyst intend to compete with the chemical catalyst. Besides the price of
enzyme, it is relevant to choose between free and immobilized ones. The immo-
bilized products have a significantly higher price per ‘‘activity unit’’ compared to
liquid products. It is difficult to make general comparisons between costs of liquid
formulated versus immobilized enzymes, once it will depend very much on the
cost of immobilization process. The immobilized lipase that has been extensively
used (Novozym 435) has a high price per kilogram, meaning that a very high
productivity is required for the process to be cost-effective. One the other hand,
new immobilization technology resulted in a much lower selling price for the
immobilized lipase and was recently successfully introduced for interesterification
(Nielsen et al. 2008). Additionally, immobilized enzymes are required in biodiesel
production due to the easier handling and reuse.
In order to compare the economic potential of enzymatic to chemical process
for biodiesel production, few studies pointed out the productivity (kg biodiesel/kg
enzyme) that can be easily used for cost comparisons. This calculation will also
14 Critical Technological Analysis for Enzymatic Biodiesel Production 325

depend on yield, number of reuse, and enzyme concentration. Nielsen et al. (2008)
reported that the maximum price of the enzyme should be the same as when using
chemical catalysis, 25 USD/ton biodiesel, thus the enzyme prices can vary from 12
to 185 USD/kg, depending on the productivity in the application. Fjerbaek et al.
(2009) also analyzed the comparison between Novozym 435, a price of approxi-
mately 1,000 USD per kg, and NaOH as a chemical catalyst, approx. 0.62 USD
(Haas et al. 2006). The authors reported that lipases have up to 74 times higher
productivity, then considering the productivity, an enzyme cost of 0.14 USD per
kg ester compared to 0.006 USD per kg ester for NaOH. If the enzyme purchase
cost dropped to 44 USD per kg or the enzyme could be reused around 6 years, the
enzymatic route could be competitive based on productivity alone. However, not
only enzyme, but the overall process needs to be considered in the economic
feasibility for enzymatic biodiesel production.
Sotoft et al. (2010) investigated the economic feasibility using current lipase
prices. The authors considered in its study the size and capacity of the plant and
the solvent use to make a comparison between different scenarios. One of these
comparisons, considered the product price of 8 million tons of biodiesel/year. The
literature showed that it can produce biodiesel for 0.55–0.62 €/kg with high quality
raw materials and traditional catalysts, while this study showed that it can be
produced at 0.75–1.49 €/kg, using solvent free system and lipase as catalyst. In this
sense, enzymes showed to be more expensive to use, but if the shelf life and yield
of the lipases could be improved, considering also the improvement in environ-
mental impacts, the enzymatic production of biodiesel will sure to become very
attractive for industrial prospect.

14.7 Conclusions

The choice between the chemical and enzymatic catalysis is an important decision
to make before to invest in biodiesel production. The enzymatic catalysis can offer
many advantages, however the acquisition cost involved with this technology is
still economically not feasible, mainly due to enzyme cost. Du et al. (2008) elu-
cidated there are two ways to be considered in order to reduce the costs of lipase.
One of them would be to reduce the production costs through new lipase devel-
opment, fermentation optimization, and downstream processing improvement.
Another way is to improve/extend the operational life of the lipase, and this can be
achieved through enzyme immobilization, alcoholysis reaction optimization,
among others. This chapter also demonstrated that the raw material is an important
variable to be considered, once it can impact from 70 to 90 % of the total cost
involved in conventional biodiesel production, depending on the raw material
specification. In summary, the major issues to be investigated, for lipase-mediated
alcoholys aiming at biodiesel industrialization, are to reduce the lipase production
cost and to develop new technologies that allow the use of poor quality raw
326 M. A. Farias and M. A. Z. Coelho

material. In this scenario, enzymatic route is a very promising technology to be

developed in Brazil, once it is possible to integrate the variety of raw materials
available and the use of agroindustrial residues to produce low-cost lipase.

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Chapter 15
Critical Analysis of Feedstock Availability
and Composition, and New Potential
Resources for Biodiesel Production
in Brazil

Betania F. Quirino, Bruno S. A. F. Brasil, Bruno G. Laviola,

Simone Mendonça and João R. M. Almeida

Abstract The worldwide demand for renewable energy has increased consider-
ably in the recent years, and the need for biofuels should increase even more,
especially in developing countries. Brazil has 43 % of its energy matrix based on
renewable resources and is a leading country in the production of biofuels. The
Brazilian National Program for Biodiesel Production and Use (PNPB) that started
in 2005 encouraged biodiesel production, leading Brazil to become one of the
world’s top producers with a production of 2,718.48 thousand m3 of biodiesel in
2012. Currently, soybean is the main feedstock used for biodiesel production in
Brazil. However, as the demand for this fuel is constantly increasing, and soybean
has low oil yield and productivity, alternative feedstocks for biodiesel production
have been evaluated. In this review, we discuss the feedstocks that are currently
most used for biodiesel production in Brazil (i.e., soybean, tallow, and cotton), as
well as the more important feedstock alternatives (i.e., oil palm, physic nut, and
microalgae) for the future. In addition, an analysis of oil physical–chemical
properties and their effects on biodiesel production and quality is presented.
Finally, different scenarios for the biodiesel industry in Brazil for the short-,
medium-, and long-terms are discussed.

B. F. Quirino  B. S. A. F. Brasil  B. G. Laviola  S. Mendonça  J. R. M. Almeida (&)

Embrapa Agroenergy, Parque Estação Biológica S/N, Av. W3 Norte (final), Brasília, DF
70770-901, Brazil
e-mail: joao.almeida@embrapa.br
B. F. Quirino
Universidade Católica de Brasília, Genomic Sciences
and Biotechnology Program, Brasília, DF 70790-160, Brazil
B. S. A. F. Brasil
Universidade Federal do Tocantins, Biotechnology Program, Gurupi, TO, Brazil
J. R. M. Almeida
Universidade Estadual do Oeste do Paraná/UNIOESTE, Energy
in the Agriculture Program, Cascavel, PR, Brazil

S. S. da Silva and A. K. Chandel (eds.), Biofuels in Brazil, 331

DOI: 10.1007/978-3-319-05020-1_15,  Springer International Publishing Switzerland 2014
332 B. F. Quirino et al.

15.1 Introduction

Environmental issues and energy prices have prompted worldwide increase in

biofuels’ production in the recent years (Almeida et al. 2012). Biodiesel is a
biofuel made of fatty acid monoalkyl esters derived from biologically produced
oils or fats, including vegetable oils, animal fats, and microalgal oils. It is possible
to produce biodiesel from different vegetable feedstocks and the most appropriate
choice will depend on technical, economical, and socioenvironmental competi-
tiveness. Agronomic aspects of the plant feedstock are also important, and the
following characteristics should be taken into consideration: (a) oil content and
type, (b) productivity (i.e., production per area unit), (c) production systems, (d)
crop cycle (i.e., seasonality), (e) regional adaptation (i.e., preferably broad to meet
different environmental conditions), (f) socio-environmental development, and (g)
oil quality. Given the mentioned factors, different crops have been used as the
main source for biodiesel production in different countries. In the United States,
for example, soybean oil is considered an essential feedstock, however, in tropical
countries such as Malaysia, palm oil is more often used for biodiesel production. In
Germany, rapeseed oil is used in the production of biodiesel, and it is distributed in
a pure form, free of any additives or blending (Singh and Singh 2010; Atabani
et al. 2012). Brazil is currently one of the largest biodiesel producers in the world.
The feedstock of choice is soybeans. Using this feedstock, the production of
biodiesel in Brazil increased from 0.74 thousand m3 in 2005 to 2,718.48 thousand
m3 in 2012 (ANP 2013). Indeed, 80 % of the biodiesel produced in Brazil over the
years is derived from soybean, a commodity that has a well-established production
chain (Fig. 15.1).
The successful establishment of a biodiesel industry in Brazil was only possible
due to investments in biodiesel research and strong public policies mainly estab-
lished by program PNPB (The Brazilian National Program for Biodiesel Produc-
tion and Use) (MME 2013a, b). PNPB was created in December 2004 with the aim
to encourage biodiesel production and its use to replace, partially or completely,
petrodiesel. This would reduce Brazil’s dependence on petrodiesel imports and
promote new sources of renewable energy which are increasingly important in the
country’s energy matrix. Indeed, renewables contributed with 43 % to the energy
matrix in 2012 (EPE 2013). To accomplish this goal, the program brought
incentives to family farmers (small farmers) and to the biodiesel producers. Pro-
ducers buying oil from family farmers benefit from a social seal, which guarantees
fiscal incentives, including federal tax reductions of up to 68 %, and special credit
lines with favorable rates from governmental banks. The family farmers also
benefit from special credit lines. Contracts that established selling oil prices for the
family farmers and delivery dates to the producers brought security to the system
and encouraged structuring in the sector. With this, Brazilian biodiesel production
plants were installed in all five geopolitical regions of the country (Fig. 15.2).
Today, the biodiesel production capacity of Brazil is 7,343 thousand m3/year, but
only 37 % of this capacity is being used. Another important characteristic of the
15 Critical Analysis of Feedstock Availability 333

Soybean Livestock Cotton

11 63 % 37 %
In Natura 51% % 89 %
Fat Defatted carcass Seed Fiber

In Natura 40%
Processed 49%
21% 79%
Other uses
Processed 60%
79% 21%
Bran Oil

20% 80%
63% 37% Oil Bran
Other uses

5% 30% 70%
17%
Other uses
Others
75% (3%)

Biodiesel

Fig. 15.1 Main feedstocks and their relative contribution to Brazilian biodiesel production (pie).
Percentage of each feedstock biomass that is processed in Brazil and their respective oil (or fat for
livestock) content are also presented. For 2012, 100 % biodiesel, soybean, and cotton are
equivalent to 2,718.48 thousand m3, 66.4 and 3.1 million tons, respectively. 11 % fat is
equivalent to 1,560,000 tons/year

PNPB is the incentive to the diversification of feedstocks for biodiesel production,

which focuses on the development and use of oil crops other than soybean to
produce biodiesel in specific regions of the country. This would promote regional
development and allow a more stable supply of oil throughout the year while
decreasing dependence on soybean oil. Brazil holds a large territory and there is a
wide variety of feedstocks that could be exploited for biodiesel production, thus
diversification of the biodiesel feedstock matrix is not only feasible, but very
attractive.
Blending of 2 % of biodiesel into petrodiesel (B2) became mandatory in Brazil
since 2008. The same law (N.11.097/2005) stated that the percentage of biodiesel
blended into petrodiesel should increase to 5 % in 2013. However, considering the
expansion of biodiesel participation in the Brazilian energy matrix, in addition to
the economical, social, and environmental benefits of biodiesel and National
Energy Policies, the government decided to implement the mandatory B5 blend
3 years ahead of schedule, i.e., in 2010 (Resolução CNPE N8 6, de 16.9.2009—
DOU 26.10.2009). Currently, B5 is still in use, but oil and biodiesel producers
expect this percentage to increase soon, since the country’s biodiesel plants are
334 B. F. Quirino et al.

Northeast region production

North region production
100%
100%
50% 32.03
50% 6.88
3.37 0.52 0%
0% Soybean Cotton
Soybean Cotton
Biodiesel cost: US$ 1.04
Biodiesel cost: US$ 1.01

Center West region production Southeast region

100% production
64.43
47.35
50% 100%
50% 6.65 3.00
0% 0%
Soybean Cotton
Biodiesel cost: US$ 0.93

Biodiesel cost: US$ 0.99

South region production

100%

50% 35.76
Biodiesel plants
0.02
0%
Soybean Cotton
Biodiesel cost: US$ 0.90

Fig. 15.2 Soybean and cotton production and cost of biodiesel by region in Brazil. Biodiesel
production plants are indicated by circles in the map

working well below production capacity. The current production of biodiesel from
only soybeans in Brazil would allow mixtures up to B10 levels (Fig. 15.1 and
Table 15.1).
Brazil is a tropical country of continental dimensions with approximately 90
million hectares of available arable land. There are also 210 million hectares of
pasture land (pasture fields) that could be employed in agriculture after mild
recovering. In addition, there are several species able to grow and produce oils in
high amounts and with high productivity in the different regions of the country
(Bergmann et al. 2013). Characteristics of these crops and their relative technical
advantages and disadvantages for biodiesel production were recently reviewed by
Bergmann et al. (2013). Altogether, Brazil has a unique opportunity to increase oil
production, valorize diversification of feedstocks, and regionalize its production,
without the need to expand the production of biodiesel feedstocks into the
remaining areas with native vegetation. However, to further develop the biodiesel
industry, depending on the crop considered, as many as three, of the following
technical-scientific challenges need to be overcome: (i) technological know-how
about the feedstock—techniques to achieve high yields and productivity, solving
agronomic issues such as seeding, growing, and harvesting problems are needed;
(ii) production scale—despite technical know-how, some crops may not be pro-
duced yet at a large enough scale to support biodiesel production for a 5 % blend;
 15

Table 15.1 Technical–economical characteristics of feedstocks for biodiesel production in Brazil

Feedstock Participation in Brazil’s Oil cost Brazilian annual oil Oil destined to Biodiesel Oil Biomass productivity Oil productivity
biodiesel matrix (%)a (US$/ton)b production (t)c production (t) (%) (kg/ha/year) (kg/ha/year)
Soybean 75 972.00 5,450,000 1,993,082 18–21 2,938e 540
Animal 17 845.00 1,560,000 329,274 – – –
fat
Critical Analysis of Feedstock Availability

Cotton 5 1,098.00 300,000 90,175 20 2,168e 360

Oil palm \1 858.00 110,000 \40,000 22 20,000 4,000
Physic – – – – 38 4,500 1,500
not
c
Algae – [1,500.00 – – 40–50d 75,000–230,000f 46,000–110,000f
a c d e
(MME 2013a, b); b (CONAB 2013a, b); (Nunes 2007) and http://www.agencia.cnptia.embrapa.br; (Stephenson et al. 2011); (CONAB 2013a, b);
f
(Stephens et al. 2010)
335
336 B. F. Quirino et al.

(iii) production chain logistics—involves the spatial distribution of feedstock and

biodiesel production plants, transportation costs, and all possible diverse uses of the
feedstock. Critical analysis of these three challenges is essential to allow the
identification of the best candidate crops for the biodiesel industry.
Soybean oil, cotton oil, and beef tallow (Fig. 15.1) are the main feedstocks
currently used for biodiesel production in Brazil. In this chapter, we present how
the production chains for these feedstocks fit with the biodiesel industry in Brazil.
In addition, oil palm (Elaeis guineensis Jacq.), physic nut (Jatropha curcas), and
microalgae are shown as the most important alternative feedstocks for biodiesel
production in Brazil. The main challenges to employ them in the industry are
discussed. The potential of other feedstocks, such as sunflower (Helianthus an-
nuus), coconut (Cocos nucifera), babassu (Attalea speciosa), castor bean (Ricinus
communis L.), rapeseed (Brassica napus), and other exotic oil producing species
found in Brazil have been recently reviewed (Bergmann et al. 2013) and are not
discussed here. An analysis of oil physical–chemical properties and their effects on
biodiesel production and quality are presented. Finally, different scenarios on the
short-, medium-, and long-term perspectives for the biodiesel industry in Brazil are
discussed.

15.2 Feedstocks for Biodiesel Production in Brazil

Soybean oil, animal fat, and cotton oil are the three major feedstocks for biodiesel
production in Brazil (Fig. 15.1, Table 15.1). Despite their importance in the bio-
diesel feedstock matrix, biodiesel can be considered a secondary product obtained
from these feedstocks. It is important to note that the amount of oil and fat used for
biodiesel production represents only a small fraction of the products obtained from
these feedstocks (Fig. 15.1). Indeed, the production of soybeans, cotton, and
animal beef have been developed for many years in Brazil for other uses and only
recently, especially after the PNPB, biodiesel production started to integrate the
value chain of these commodities. The well-established production and distribu-
tion chains of soybean and cotton in the country is one of the main reasons to use
these crop species for biodiesel production, as their oil yield is low, i.e., below
600 kg/ha. Low cost of residual fat from beef production explains its use as a
feedstock for biodiesel production. It is important, however, to further develop the
biodiesel industry, so that feedstock crops have higher yields of oil, increasing the
current yields of only 350–600 kg/ha of cotton and soybean to 5,000 kg/ha, and
potentially contributing to regional development. Here, we summarize the
advantages and disadvantages of the current substrates used for biodiesel pro-
duction in Brazil (soybean, cotton, and animal fat) as well as some alternative
feedstocks with great potential for biodiesel production (oil palm, physic nut, and
microalgae).
15 Critical Analysis of Feedstock Availability 337

15.2.1 Vegetable Oils

Soybean (Glycine max): Brazil is one of the top soybean producers in the world. In
2012 it produced 66.4 million tons of grain. This number should be surpassed in
2013, when soybean production may reach 81.5 million tons. National productivity
reached 2,938 kg/ha and the planted area 27,721.5 thousand hectares, which
represents increments of 10.8 and 10.7 %, respectively, compared to the 2012
numbers (MME 2013a, b).
The Brazilian success in soybean production, processing, and export in the
recent years has made its oleaginous seed the main feedstock for biodiesel pro-
duction in the country. The well-established production chain and distribution
logistics assure the soybean market and value, and allow its inclusion as a reliable
source of oil in the biodiesel feedstock matrix. It should be noted that only a
relative small amount of soybean is used for biodiesel production. Brazil exports
approximately 50 % of its production in natura, while the other half is processed
internally to produce protein bran for animal feed (23.5 million tons in 2012) and
edible oil (5.8 million tons in 2012). From the oil produced, only 25–30 % is used
in the biodiesel industry (Table 15.1). Inspite of the low oil productivity and the
relative small amount of feedstock designated to produce biodiesel, soybean is still
the major feedstock for production of this biofuel, representing at least 75 % of the
feedstock used (Table 15.1, Fig. 15.1).
Cotton (Gosssypiuym hirsutum latifolium Hutch LR): Cotton oil is the third
most important feedstock for biodiesel production in Brazil, with 5 % of partici-
pation in biodiesel feedstock matrix, only behind soybean oil and beef tallow
(Fig. 15.1, Table 15.1) (MME 2013a, b). However, in the Northeast region of the
country, in some months of the year, cotton oil becomes the second main feedstock
for biodiesel production. In Brazil, cotton is produced mainly in the Center-west
and Northeast regions of the country, the states of Mato Grosso and Bahia being
the largest producers. In 2012, 3.1 million tons of cotton seeds were produced.
Like for soybean, the cotton seed oil content is low, approximately 20 %. This fact
together with the relative small area of cotton production (1.4 million hectares)
limits the use of cotton oil for biodiesel production. In addition, the cake obtained
after oil extraction cannot be used for animal feed, because of the presence of toxic
gossypol.
Oil Palm (E. guineensis Jacq.) Oil palm is especially suitable for biodiesel
production due to high yields of biomass and oil, which are approximately 7 times
higher in oil palm than in soybean (Table 15.1). Despite these impressive numbers,
palm oil contributes with less than 1 % of the biodiesel production in Brazil.
Relatively small-scale cultivation of this palm, estimated at 180,000 hectares, and
uses of palm oil in other industries explain its modest contribution to biodiesel
production. In Brazil, oil palm cultivation is restricted to specific soil and climate
conditions mainly found in the North and Northeast regions of the country. Oil
palm has the greatest potential to become the main feedstock for biodiesel pro-
duction in the future. The Brazilian Federal government launched the National
338 B. F. Quirino et al.

Program for Sustainable Production of Oil Palm to stimulate cultivation of this

crop and also to regulate its expansion and establish its agroecological distribution.
In addition, there are oil palm breeding programs aiming to improve oil production
and crop resistance to pathogens. In this context, a genetic hybrid of the African oil
palm, E. guineensis, and the American oil palm, Elaeis oleifera, was released in
2010 by EMBRAPA (Cunha and Lopes 2010). This hybrid is resistant to fatal
yellowing, which has decimated thousands of plants in the North region of the
country. Ongoing R&D programs for the expansion, characterization, and con-
servation of a germplasm bank, genetic improvement of oil palm, and seed pro-
duction programs are occurring. Oil acidity and time-costly and time-consuming
manual harvest of fruit bunches are two important problems to be addressed to
improve industrial performance of this crop. Strategies to solve the former prob-
lem will be discussed in detail in the following section. The latter problem is
expected to be solved with improvements in mechanization of harvesting.
Physic nut (J. curcas L.) is a perennial plant of the Euphorbiaceae family,
probably native of Central America. Jatropha shows potential for high yield of
seeds, which are high in oil content (Table 15.1). Despite the advantages of using
Jatropha for biodiesel production, this species is still under domestication and
there are many challenges to be overcome by research. The Jatropha varieties
currently available in Brazil are not genetically characterized and there is little
information about production levels in the different regions of the country. Ag-
ronomical production systems are not yet completely validated and more infor-
mation about propagation, plant density, maintenance, nutrition, and pest
management are necessary to allow industrial scale production. Currently, there
are many research and developmental efforts to enable biodiesel production with
Jatropha oil in Brazil, from implementation and analysis of germplasm banks to
agronomical studies (Rosado et al. 2010). For instance, population evaluation
allowed the identification of 5-year-old plants able to produce 4,500 kg/ha of
seeds, which result in approximately 1,500 kg/ha of oil (Sotolongo et al. 2007;
Laviola and Alves 2011). Importantly, researchers have identified Jatropha cul-
tivars that do not produce phorbol ester in the seeds. This may allow the devel-
opment of nontoxic commercial cultivars whose cake (i.e., left over from oil
extraction) can be used for animal nutrition.

15.2.2 Animal Fats

Animal fat is currently the second most used feedstock for biodiesel production in
Brazil (Fig. 15.1, Table 15.1). Although poultry fat and lard are also used to make
biodiesel, the vast majority of the fat used to produce biodiesel in Brazil is tallow
(ANP 2012a, b).
Brazil is one of the world’s top meat producers. In 2012, 7.4 million tons of
beef (IBGE 2013), 3.5 million tons of pork, and 11.5 million tons of poultry were
produced (IBGE 2013). Tallow is a by-product of the meat and rendering industry
15 Critical Analysis of Feedstock Availability 339

and it is estimated that 10.9 % of the live slaughter animal weight corresponds to
tallow (Nelson and Schrock 2006). It is produced in a centralized manner in
slaughter/processing facilities and historically it has low-market value (Teixeira
et al. 2010). If not directed to other uses, animal fat can be an environmental
pollutant.

15.2.3 Microalgae

Microalgae are photosynthetic microorganisms that grow in water and convert

CO2 into carbohydrates, protein, and natural oils. They are recognized as one of
the most productive organisms in terms of biomass. In tropical areas, marine
phytoplankton biomass can be produced at a rate of 100 tons/ha/year (Ben-Amotz
and Jinjiikhashvily 2008). In addition, for some microalgae species, as much as
80 % of their mass is composed of lipids, which can be used to produce biodiesel.
Indeed microalgae have the potential to produce oils at quantities up to 110,000 L/
ha/year (Table 15.1) (Stephenson et al. 2011). Furthermore, microalgae which are
not generally used as human food, can be grown in nonarable land using seawater,
brackish water, or even wastewater, and can capture carbon emissions from
industrial plants (Carioca et al. 2009; Stephenson et al. 2011). These character-
istics render microalgae biomass a promising alternative source for biofuels with
minimal problems with direct and indirect land use.
Brazil has great potential for large-scale microalgae production given that the
country possesses a large tropical coastal area, with 10,959 km, has approximately
12 % of the world’s freshwater supply, and receives average insolation levels of
8–22 MJ/m2 day (IBGE 2013). Nonetheless, there are significant technological
challenges to produce economically competitive, algal-derived biofuel (Stephens
et al. 2010). Aiming to reduce production costs (Table 15.1), research efforts on
the isolation, characterization, and domestication of highly productive algal strains
from Brazil’s biodiversity are currently underway. For example, it was shown that
a freshwater strain of Choricystis sp. can provide 115 % more fatty acids per gram
of biomass than soybean grain (Menezes et al. 2013). In another study, Nascimento
and coworkers (Nascimento et al. 2013) screened microalgae strains isolated from
freshwater lagoons from the Northeast region of Brazil based on their lipid pro-
ductivity and fatty acid profiles. The highest values for lipid productivity were
observed for a Chlorella vulgaris strain (i.e., 204.91 mg/L/day) and two Botryo-
coccus strains (i.e., 112.43 and 98.00 mg/L/day for Botryococcus braunii and
Botryococcus terribilis, respectively). Comparable levels have been reported for
the most promising microalgae species isolated from other parts of the world such
as Nannochloropsis gadinata (i.e., 310 mg/L/day), Nannochloropsis salina (i.e.,
170 mg/L/day) and Phaeodactylum tricornutum (i.e., 50 mg/L/day) (Radakovits
et al. 2012).
340 B. F. Quirino et al.

Table 15.2 Physical properties of vegetable oils and its biodiesel

Feedstock Kinematic Cetane Cloud point (C) Flash point Density (g/cm3)
Viscosity at (C)
38 C (mm2/s)
Oil Biodiesel Oil Biodiesel Oil Biodiesel Oil Biodiesel Oil Biodiesel
Cottonseed 33.5 3.8–4.0 41.8 46–52 1.7 234 182 0.914 0.871
Soybean 32.6 4.1–4.5 37.9 45–53 -3.9 1 254 178 0.913 0.865–0.885
Rapeseed 37.0 4.4–4.6a 37.6 51–59 -3.9 -3 to 4 246 127 0.911 0.857–0.882
Palm 39.6 5.7 42.0 62 31.0 13 267 164 0.918 0.867–0.880
Dieselb 2–4.5 49 (min) – 55(min) 0.820–0.860
a
Source (Srivastava and Prasad 2000; Singh and Singh 2010); (Ramos et al. 2009) at 40 C;.
b
EN590:1999

15.3 Biodiesel Composition and Quality

In principle, any vegetable oil can be used directly in diesel engines. In the last two
decades in Brazil, several oils have been directly tested in motors (e.g., babassu,
castor bean, palm oil, Jatropha, macaw palm, and others). However, research has
shown that direct use causes adverse effects on engines, such as problems in
pumping, atomization, gumming, and piston ring sticking. These problems are due
to the high viscosity, density, iodine value, and poor/nonvolatility of oils. Hence, it
is essential to modify these characteristics for better combustion of the vegetable
oils by, for example, a transesterification reaction for biodiesel production (Kumar
et al. 2013). As shown in Table 15.2, this reaction dramatically changes some
physical properties of oil.
The most significant components of the oils and fats (conceptually, the dif-
ference is that oils are in liquid state at room temperature, whereas grease and fats
are in solid state at room temperature; and also that the former are from plant
source) are triglycerides and their physical properties depend on the structure and
distribution of fatty acids. The majority of feedstocks for biodiesel production have
triglycerides composed of 10 different types of fatty acids. These fatty acids have
between 12 and 22 carbons in the chain, with 90 % or more having 16 and 18
carbons. Table 15.3 shows the fatty acid composition of oils/fats currently used for
biodiesel production in Brazil and also of the most promising feedstocks for future
use.
Biodiesel is characterized by physical–chemical properties. Some of these
properties include density (g/cm3), viscosity (mm2/s), cloud and pour points (C),
flash point (C), cetane number, oxidation stability, and distillation range, which
basically depend on the type of feedstock and their fatty acid composition. Other
properties, like acid value (mg KOH/g-oil), ash content (%), copper corrosion,
phosphorus (mg/kg), sulfur content, carbon residue, water content and sediment,
and glycerin (% m/m) are more affected by processing (Atabani et al. 2012). In
Box 1, at the end of this section, explanations of some general properties of
biodiesel are presented. Currently, the properties of biodiesel must comply with
15 Critical Analysis of Feedstock Availability 341

Table 15.3 Fatty acid profile of selected oils and fat used in biodiesel production
Fatty acids Soybean Cotton Palm Physic nut Tallow
C14:0 0 1 1 0 3
C16:0 12 21 43 15 23
C18:0 3 3 5 6 19
C18:1 23 19 41 35 43
C18:2 56 55 10 44 3
C18:3 6 1 0 0 1
C20:0 0 0 0 0 0
C22:1 0 0 0 0 0
Saturated 15 24 48 21 45
Iodine value 130 105 37 101 35–48
Source (Ma and Hanna 1999; Singh and Singh 2010); Mendonça S. (unpublished)

international biodiesel standard specifications established by one of the various

organizations that set fuel standards. Particularly important specifications for
biodiesel fuel (B100) include the ASTM 6751 from the American Standards for
Testing Materials (ASTM 2012) and the EN 14214 from the European Committee
for Standardization (ECN 2008). However, there are other standards available
globally such as those from Germany (DIN 51606), considered to be even stricter
than the European norms, and the Brazilian resolution (ANP 04/2010) (ANP
2012a, b), which is based on ASTM 6751 and EN 14214.
Some parameters for the quality of biodiesel in different countries and a
summary of physical–chemical properties of diesel and biodiesel produced from
different feedstocks are shown in Table 15.4. Biodiesel standards in Brazil and in
the U.S. are applicable for both fatty acid methyl esters (FAME) and fatty acid
ethyl esters (FAEE), whereas the current European biodiesel standard is only
applicable for fatty acid methyl esters (FAME). Also, the standards for biodiesel in
Brazil and in the U.S. are used to describe a product that is a blending component
in conventional hydrocarbon-based diesel fuel, whereas the European biodiesel
standard describes a product that can be used either as a stand-alone diesel fuel or
as a blending component. These differences in technical specifications are pri-
marily related to the origin of the feedstock and the characteristics of the local
markets. Though this currently translates into some significant divergence in
specifications and properties of the derived fuels—which could be perceived as an
impediment to trade—in most cases it is possible to meet the various regional
specifications by blending the various types of biodiesel to the desired quality and
specifications (Tripartite Task Force 2007).
The Cold Filter Plugging Point (CFPP) is very important in colder regions,
where a high CFPP indicates a high likelihood that the fuel will clog up the vehicle
engine. Biodiesel from palm oil and tallow show the poorest performance (highest
temperature points) in terms of CFPP, while biodiesel from rapeseed generally
 342

Table 15.4 Biodiesel standards in different countries and comparison with biodiesel characteristics from diverse feedstocks
Characteristic Brazil European Union USA (ASTM Soybean Rapeseed Cottonseed Palm Jatropha
(ANP14/2012) (EN 14.214) D6751) biodiesel biodiesel biodiesel biodiesel biodiesel
Density at 15 C (g/cm3) 0.850–0.900a 0.860–0.900 – 0.880–0.884 0.879–0.882 0.875 0.864–0.880 0.864–0.880
Kinematic viscosity at 3.0–6.0 3.5–5.0 1.9–6.0 4.0–4.2 4.4 4.1 4.5–5.7 4.2–4.8
40 C (mm2/s)
Cetane number (min.b) Report 51 47 45–58.1 54–59 54 62 51–57
Oxidation stability at 6 6 3 1.3–3.8 6.4–7.6 1.8 11–13 2.3–3.2
110 C: h (min.)
Iodine value -g I2/100 g Report 120 115 128 109 57 104
(max.c)
CFPP (C) (max.c) 19 0 summerd–20 – -5 -20 to 10 1 10–12 0
winter
Sulfur (mg/kg) (max.c) 50 10 15 0.2–0.8 0.2 0.01 0.01 0.3
Flash point (min.) C 100 120 93 160–254 170 150 135–176 163–191
(min.b)
a
at 20 C; b minimum limit; c maximum limit; depends on country and season, the given example is for Germany
Source (Foidl et al. 1996; Demirbas 2008; Ramos et al. 2009; Atabani et al. 2012)
B. F. Quirino et al.
15 Critical Analysis of Feedstock Availability 343

shows the best performance (lowest temperature points). Because of large

geographic and seasonal temperature variations, neither the U.S. nor European
biodiesel standards have strict specifications for these low temperature properties,
though they are among the most important properties in determining the suitability
of biodiesel fuels in-use. In Europe, CFPP values must be established for each
country according to its climate. In the United States, the value of CP (Cloud
Point) is used instead of CFPP, and it is also dependent on the season of the year.
According to the Brazilian Resolution, except for castor bean biodiesel, a maxi-
mum of 19 C for CFPP is applicable for the South, Southeast, Midwest, and the
state of Bahia. For the other Brazilian regions with tropical climate, there is no
recommended value for the cloud point, although it needs to be reported.
The density and viscosity increase with the number of carbons of the fatty acid
chain and are reduced by the presence of double bonds. Triglycerides with satu-
rated fatty acids (SUFA) have higher density and viscosity (more solid at room
temperature) causing problems to SUFA-derived fuels in cold regions, i.e., they
have higher CFPP. Triglycerides rich in polyunsaturated fatty acids present low
oxidative stability and low CN, which may lead to oxidation and thermal poly-
merization, whereas they present better CFPP (low values for CFPP). More sat-
urated triglycerides such as tallow are solid at room temperature. Thus, they are
difficult to use as fuel because of the higher values of CFPP, whereas excessive
carbon deposits in engine are reported when polyunsaturated triglycerides like
rapeseed oil are used as fuel. Vegetable oils are mostly unsaturated and thus more
susceptible to oxidation and thermal polymerization reaction (Kumar et al. 2013).
To achieve a balance between CFPP and oxidative stability, a biodiesel feedstock
should have as high as possible monounsaturated fatty acid content.
The degree of unsaturation may be expressed as iodine value (i.e., amount in
grams of iodine which reacts with the double bonds present in 100 g of the sample)
and can be used to classify oils into three categories: drying (iodine value greater
than 170; e.g., linseed oil), semidrying (iodine value between 100 and 170; e.g.,
soybean and sunflower); nondrying (iodine value less than 100; e.g., palm oil).
Drying oils tend to form films, becoming solid due to polymerization of the chains
in consequence of oxidation. On the other hand, nondrying oils are resistant to
oxidation and will remain liquid for a long time (Meier et al. 2007). This does not
mean that those oils cannot be used for biodiesel production; however, they should
be mixed with biodiesel from other sources to achieve the recommended quality
standard. For example, adding 5 % of tallow biodiesel to rapeseed biodiesel would
increase the cetane number, without significant interference in CFPP. Soybean
biodiesel presents an iodine value above the acceptable range established by the
European Norm (EN) (i.e., 125–140, where the EN14214 limit is 120) and lower
cetane number. The mixture with 20 % of tallow biodiesel could lower the iodine
value allowing export to Europe and at the same time increase the cetane number
improving fuel characteristics. Similarly, 70 % of palm oil biodiesel could be
blended to 30 % Jatropha biodiesel to optimize CFPP of the former and oxidative
stability of the latter.
344 B. F. Quirino et al.

Biodiesel can be produced from beef tallow using the traditional route of NaOH
catalysis and methanol with high yields (i.e., 96.26 %) (Araujo et al. 2010).
Although biodiesel from vegetable oils and tallow have comparable properties,
tallow biodiesel has more saturated fatty esters because beef tallow has more sat-
urated fatty acids (Table 15.3). Among these saturated fatty acids, stearic (C18:0)
and palmitic (C16:0) acids are the most abundant. This in turn has consequences to
fuel quality. Viscosity of tallow biodiesel at 40 C is 4.89 mm2s-1, compared to
4.20 and 3.47 for soybean biodiesel and petrodiesel, respectively (Table 15.4)
(Teixeira et al. 2010). Another fuel property is the cold filter plugging point, which
can be interpreted as the lowest temperature at which the fuel will flow without
problems such as clogging a fuel system. The cold filter plugging point for tallow
biodiesel is 15 C, while for soybean biodiesel it is 4 C and for petrodiesel it is
10 C (Teixeira et al. 2010). Therefore, the properties of tallow biodiesel are not the
most advantageous, particularly for cold weather climates. However, tallow bio-
diesel can still be used successfully if blended to soybean biodiesel or petrodiesel
(Teixeira et al. 2010). The use of tallow biodiesel only becomes a concern in a
scenario of B100, where tallow biodiesel is in high proportion compared to soybean
or other vegetable-oils biodiesel. Given that tallow is a by-product of the meat and
rendering industries, it is unlikely that the amount of tallow to make biodiesel will
increase disproportionately and that this scenario will become a reality.
Microalgae represent a very diverse group and their fatty acid profile varies
drastically depending on the species (Nascimento et al. 2013). In addition, culti-
vation parameters will also affect fatty acid composition (Cabanelas et al. 2013;
Xu et al. 2006). Generally, solar incidence, nitrogen, and carbon limitations/source
will lead to increase/decrease in saturated/insatured fatty acids. Xu et al. (2006)
demonstrated that oil derived from a heterotrofically cultivated strain of Chlorella
protothecoides could be used to produce biodiesel that meets the Brazilian
National Agency of Oil (ANP) standards (Franco et al. 2013). Furthermore, good
quality microalgae biodiesel may also be obtained by using a mixture of oils from
different microalgae species, from other biodiesel feedstocks or by optimizing
microalgae culture conditions.

BOX 1: Parameters for biodiesel quality

Kinematic Viscosity: The viscosity of biodiesel increases with carbon chain
length and degree of saturation and influences the process of fuel burning in
the combustion chamber of the engine. Due to decreased efficiency of
atomization in the combustion chamber, high viscosity causes heterogeneity
in the combustion of biodiesel and residues are deposited in the internal parts
of the engine.
Cloud and Pour Points, Cold Filter Plugging Point (CFPP): At low
temperatures, biodiesel tends to partially lose fluidity or solidify, leading to
fuel flow disruption, clogging of the filtration system, and engine damage
due to inadequate lubrication. This causes problems in starting the engine.
15 Critical Analysis of Feedstock Availability 345

Cloud point refers to the temperature at which the liquid begins to become
turbid, and the pour point is the temperature at which the liquid no longer
flows freely. Both are influenced by feedstock characteristics, and also the
alcohol used in the transesterification reaction. Usually, international stan-
dard specifications are expressed in CFPP, which is correlated to both cited
cloud and pour points and refers to the temperature at which the test filter
starts to plug due to fuel components that have started to gel or crystallize.
Thus, the higher the CFFP point, the higher the chances that a small decrease
in weather temperature will cause problems to the fuel. The CFPP from
biodiesel derived from rapeseed oil is between -7 and -12 C, whereas
from animal fat it is between 15 and -1 C.
Iodine value: It is an indicator of the number of double bonds present in
biodiesel. It does not distinguish double bond location (i.e., the fuels’ oxi-
dation depends not only on the number of double bonds but also on their
proximity to each other), so the iodine value is a weak predictor of bio-
diesel’s oxidation stability or its tendency to form deposits in the engine.
Oxidation and Thermal Stability: There are two types of stability to be
considered: stability during long-term storage (oxidation stability) and at
high temperatures and/or pressure in the engine (thermal stability). The
available data indicate that biodiesel has a good thermal stability, even
producing less coke residues in the engines injectors than conventional
diesel. Biodiesel’s aging or oxidation may lead to high acidity, high vis-
cosity, and formation of gum and sediment that plugs filters. If these latter
properties exceed the limits permitted by ASTM D6751, B100, it is con-
sidered out of specification and should not be used as fuel. The higher the
unsaturation level of the original feedstock, the higher the probability of fuel
oxidation. As a rule, saturated fatty acids (e.g., C14:0 or C16:0) are more
stable than unsaturated fatty acids (e.g., C18:2 or C18:3). For every double
bond added, the fuel’s stability decreases 10 times. Other factors, such as
exposition to oxygen, light, and high temperatures, and also to contaminants
accelerate oxidation. Brazilian and European legislation specify an accel-
erated test for biodiesel’s oxidation stability, called the Rancimat test,
recently also adopted as part of the United States standards.
Cetane Number/Cetane Index: Cetane number is an indicator of the ignition
quality of a fuel for a diesel engine, and it has a direct influence on motor
starting and operation under load. A high cetane number of a fuel indicates
good combustion in a diesel engine. Furthermore, in conventional diesel
engines, high cetane numbers are correlated with lower nitrogen oxide (NOx)
emission. The larger the number of unsaturations and the shorter the chain of
fatty acids that compose the biodiesel, the lower the cetane number is corre-
sponding to a greater the emission of NOx from the fuel (Kumar et al. 2013).
The average cetane number for biodiesel (B100) is 55. For petrodiesel this
index is between 48 and 52 (minimum of 40). This is the reason that bio-
diesel burns much better in a diesel engine than petrodiesel. In terms of legal
346 B. F. Quirino et al.

requirements, the minimum cetane number for biodiesel (B100) is 51 in

Europe and 47 in the United States; the Brazilian legislation does not
establish a minimum, but the cetane number should be reported.

15.4 Critical Analysis

Despite the low oil yield of soybeans, this crop has been the major feedstock for
biodiesel production in Brazil for a number of reasons. First, technological know-
how for soybean cultivation is well established. Brazilian crop breeders made a huge
effort to obtain soybean varieties that were viable in the Cerrado (the Brazilian
Savanna) region of the country. Cerrado is a vast area in the Center-West region of
Brazil with poor and acidic soils that were thought to be inappropriate for agriculture.
Research led to the improvement in soil quality. Furthermore, when new tropical
soybean varieties were introduced, the flat Cerrado topography proved to be ideal for
mechanization and the adoption of an agribusiness model based on technology. The
large-scale production of soybeans is the second reason that it has been used as a
feedstock for biodiesel production. By definition, the demand for energy is con-
stantly increasing and therefore any feedstock used to produce energy needs to be
available in large amounts. Today in Brazil no other feedstock is produced in a large
enough scale to reliably supply the biodiesel production chain. The third reason for
Brazil’s current dependence on soybeans for biodiesel production is that soybeans
are planted in all states of the country. This makes it widely available and contributes
to lower prices of biodiesel in the regions that concentrate the production (Fig. 15.2).
Currently, tallow and cotton oil play important roles as feedstocks for biodiesel
production in Brazil. Tallow is used in the biodiesel industry because it is a low-
cost by-product of the meat and rendering industries (Fig. 15.1). However, its offer
is not related to the biofuel market. In the long term, it is not expected that tallow
will increase its contribution to biodiesel production by more than 20 %, even if
one considers that all tallow generated in Brazil is used to produce biodiesel. This
is due to the increasing production of biodiesel from vegetable oils and to the fact,
as previously discussed, that tallow biodiesel presents quality obstacles that hinder
long-term storage or use in higher levels of blending (Table 15.3), as tallow
biodiesel has high CFPP around 12 C. Thus, tallow should always be considered
as an additional feedstock to be mixed with biodiesel produced from other oils.
Participation of cotton oil in the biodiesel feedstock matrix in Brazil is not
expected to increase considerably in the future and should remain around the
current 5 % (Fig. 15.1). Like for soybean, cotton seed oil content and yield are
low. In addition, as previously mentioned, the cake obtained after oil extraction
cannot be used as animal feed because of the toxic compound gossypol.
Palm oil will probably start to play a more important role as a feedstock for
biodiesel production over the years in Brazil. As with soybeans, the technological
15 Critical Analysis of Feedstock Availability 347

know-how for oil palm cultivation is available. Given that oil production per
hectare is much higher than that of soybeans (Table 15.1), large-scale production
of palm oil should not be a problem. The Brazilian government wants to encourage
a greater participation of oil palm as a biodiesel feedstock, and it is implementing
policies to support this growth in a controlled and organized manner. For instance,
there are governmental incentives for oil palm planting in specific areas. This takes
into consideration not only which areas have the appropriate soil and climatic
conditions, but also restricts planting to areas with a history of anthropic inter-
vention (i.e., agroecological zoning). One of the major problems with oil palm, as
well as other perennial crops, is that the farmer needs to invest money over many
years before any profits are made. To make this viable, there is a need for specific
lines of credit during this period. Another problem that needs to be addressed is
that there are many small farmers producing oil palm and the most advanced
technology is not always available to them to increase productivity.
In the long term, Jatropha may be a good candidate crop for further development
of the Brazilian biodiesel program. It has high productivity of fruits and good
quality oil for biodiesel production (Tables 15.1, 15.3 and 15.4). However, there are
still many challenges to be overcome before large plantings of Jatropha are viable,
since no commercial cultivars, and consequently no production system, are avail-
able. In addition, currently the Jatropha cake cannot be used as animal feed, and
thus is a negative economic impact in the Jatropha-biodiesel production chain.
Biofuel companies are also seeking to achieve commercial production of mic-
roalgae and to design economically viable systems for growing microalgae in Brazil.
An example is Austria’s See Algae Technology (SAT) and the Brazilian JB Group
partnership that aim to produce biodiesel from microalgae in a plant in Brazil’s
Northeast region (http://www.seealgae.com/article32.htm). The US$ 5 million pilot
project is based on SAT-designed solar prisms that concentrate sunlight, through
optical fibers, on microalgae grown in tanks. Another strategy is being used by the U.
S. company Solazyme, which is in partnership with the Brazilian company Bunge in a
US$120 million investment (http://solazyme.com/media/2013-01-16). Solazyme
uses genetically modified algae that produce oil for renewable chemicals and biofuels
cultivated in a closed heterotrophic system. In the microalgae fermentation facility,
which is annexed to Bunge’s Moema unit in São Paulo (Brazil), the feedstock for the
plants will be sugarcane juice. The Brazilian startup company, Algae (http://www.
algae.com.br), is building a pilot plant in São Paulo in partnership with the Federal
University of São Carlos (UFSCAR). In this model, the carbon source is vinasse, a
by-product of sugarcane processing and an environmental pollutant. Also in the
Northeast region—taking advantage of its favorable climate with a high number of
sunny days—in 2012, Petrobras, the main Brazilian petroleum company, began
operating its first microalgae cultivation tanks for the production of biodiesel (http://
www.petrobras.com.br/pt/energia-e-tecnologia/tecnologia-e-pesquisa/diversifican-
do-os-produtos/). In the laboratories of the Federal University of Rio Grande do Norte
(UFRN), the company selected microalgae species that can be grown in water used
for petroleum production.
348 B. F. Quirino et al.

Regardless of feedstock, the Brazilian National Program for Biodiesel

Production and Use needs to set new goals for higher blends of biodiesel into
petrodiesel. The biodiesel industry is currently at half its maximum capacity,
which means it can easily produce enough biodiesel to supply a B10 demand.
However, until the government sets these new goals, the only increase in demand
for biodiesel will come from an increase in diesel B5 use. Government planning,
even if only for the long term, will be very important so that the biodiesel industry
can prepare itself to meet demands, thus avoiding a future shortage of biodiesel.
Clearly established goals will be equally important to ensure the availability of
feedstocks for biodiesel production in the future.

15.5 Conclusion

The Brazilian biodiesel industry was developed based on the soybean, cotton, and
beef production chains. These feedstocks allowed the industry to be established and
made Brazil one of the largest biodiesel producers in the world. However, consid-
ering the increasing demand for renewable fuels, other potential oil crops for bio-
diesel production have been evaluated for usage in the short-, medium- and long-
term perspectives. Soybean produced in Brazil is enough to easily increase biodiesel
mixtures from the current B5 to up to B10 and it should continue to be the main
feedstock for biodiesel production in the short term. Alternative feedstocks like oil
palm, which have much higher productivity and yields of oil, should increase its
participation in the energetic matrix in the medium term. This will help with
diversification and regionalization of feedstocks, especially in the North and
Northeast regions of the country. Nevertheless, substantial financial investments
need to be made in the coming years to support the cultivation of this perennial crop.
Finally, Jatropha and microalgae may become significant components of the bio-
diesel production chain in the long term, after technical challenges are surpassed.
Clear governmental demands for biodiesel usage and establishment of specific goals
for the diversification of biodiesel feedstocks are essential to guarantee the long-
term success of the Brazilian National Program for Biodiesel Production and Use.

Acknowledgments We thank the CNPq (Brazilian National Council for Scientific and Tech-
nological Development), FAP-DF (Fundação de Apoio à Pesquisa do Distrito Federal), FINEP
(Financiadora de Estudos e Projetos), and Embrapa for financial support.

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Chapter 16
Techno-Economic and Life Cycle Analysis
of Biodiesel Production: Perception
of Land Use, Climate Change,
and Sustainability Measurements

Donato A. G. Aranda, Cecilia M. Soares and Neyda Om Tapanes

Abstract Motivation of this study is the strategic importance of bioenergy and

biofuels for sustainable development of the global economy. Brazilian Bioenergy
Program has enabled the consolidation of Brazil within the leading countries in the
production of energy and renewable fuels. Within this program, biodiesel occupies
a prominent position, influenced by significant technological, economic, environ-
mental, and social advantages. This chapter covers issues like the life cycle analysis
for the biodiesel production, allowing the mapping of resources, impacts of this
economic activity, and the premises of sustainability. It also provides market
information by analyzing the demand—production relationship, prices, and product
quality supervision. Finally, it presents technical and economic parameters of the
main technological routes of biodiesel production in Brazil (hydroesterification and
transesterification) using current data and allowing the growing demand for new
approaches and technological advances.

16.1 The Biodiesel Market

The next decade biofuel demand is increasing in Europe. However, non-European

countries must represent more than 60 % of the world demand by 2030 and about
70 % by 2050. China, India, and Latin America will probably be the leaders in this
market (Fig. 16.1).
Brazil holds an important position in the biofuel world scenario. Biodiesel has
the advantage to be used pure or with diesel blends in internal combustion com-
pression engines. In addition to the advantages of being produced locally, biodiesel
has several environmental advantages compared to regular fossil diésel. Moreover,

D. A. G. Aranda (&)  C. M. Soares  N. Om Tapanes

Rio de Janeiro, RJ, Brazil
e-mail: Donato@eq.ufrj.br

S. S. da Silva and A. K. Chandel (eds.), Biofuels in Brazil, 351

DOI: 10.1007/978-3-319-05020-1_16,  Springer International Publishing Switzerland 2014
352 D. A. G. Aranda et al.

Fig. 16.1 Biofuel demand by region, 2010–2050 (IEA 2010)

social benefits and regional development with significant amount of new jobs and
income can be obtained once its production and consumption are promoted in a
non-centralized way with multiple feedstock (Ferreira and Oliveira 2010).
There are many factors that contribute to the increase in investments in
biodiesel in Brazil. It is possible to mention the environmental pressures, world
political instabilities, and uncertainty about the future of oil exploration, the social
stimulus to agriculture, and the dependence on foreign diesel oil, where about
18 % of this fuel comes from.
Diesel oil is the principal fuel used in Brazil, because of the extensive use of
road logistics all over the country that has been estimulated by the Federal
Government since the decade of the 1950s (Fig. 16.2).
Provisional Bill n 214, from 13 September 2004, the Petroleum National
Agency Agência Nacional do Petróleo (ANP) defines technically biodiesel as a
fuel for combustion engines with internal compression ignition, renewable and
biodegradable, derived from vegetable oils or animal fats which can replace
partially or totally fossil diesel (Soares 2008).
In the Brazilian Biodiesel Standard, B100 is defined as a fuel consisting of alkyl
esters of long chain fatty acids, derived from vegetable oils or animal fats. The B2
is a commercial fuel composed of 98 % by volume of diesel fuel, as ANP spec-
ification, and 2 % of biodiesel. The other compositions follow the models of B2
and B100 (Soares 2008).
The Brazilian law 11,097 of 2005 provides for the introduction of biodiesel into
the Brazilian energy matrix, and sets to 5 % in volume its mandatory minimum
starting at 2013.
However, since 1 January 2010, diesel fuel sold in Brazil contains 5 % bio-
diesel. This rule was established by Resolution No. 6/2009 of the National Energy
Policy (CNPE), which increased from 4 to 5 % the mandatory percentage blending
of biodiesel to diesel oil. The continued rise in the percentage of biodiesel added to
16 Techno-Economic and Life Cycle Analysis of Biodiesel Production 353

Fig. 16.2 Final energy

consumption by source in
Brazil, 2011 (Balanço
Energético Nacional 2012)

diesel demonstrates the success of the National Program for Production and Use of
Biodiesel and the experience accumulated by Brazil in the production and use of
biofuels on a large scale (ANP 2013).
It is possible to highlight three groups that have been involved in biofuel
production: those who already have the necessary resources (including agribusi-
ness entrepreneurs, oil companies, plant operators, and small farmers); suppliers of
products and services (including seed companies, engineering and equipment, and
biotechnology), and the market participants (such as farmers, agricultural equip-
ment companies, fertilizer suppliers, and logistics providers) (Caesar 2007 in
Soares 2008).
In 2011, the amount of B100 produced in Brazil reached 2,672,760 m3, against
2,386,399 m3 in the previous year. Thus, there was an increase of 12 % in bio-
diesel available in the national market. In 2011, the percentage of B100 com-
pulsorily added to mineral diesel remained constant at 5 %. The main raw material
was soybean oil (81.2 %) followed by tallow (13.1 %) (Balanço Energético
Nacional 2012).
Since 2005, the Petroleum National Agency performs biodiesel auctions where
refineries buy biodiesel to be blended with fossil diesel. The initial purpose of the
auction was to generate a permanent market and thereby stimulate the production
of biodiesel to meet the national law (ANP 2013).
These auctions were structured for the period between 2005 and 2007, but to
preserve the participation of family agriculture in the supply of raw materials, the
government preferred to keep the systematic purchase through auctions after that
period, rather than direct negotiations between producers and distributors or
refineries, as occurs in the ethanol market (Amaral Mendes and Da Costa 2009).
The evolution of the biodiesel auctions can be evidenced in Fig. 16.3, from the
first auction, which started with 70,000 m3 in November 2005, until the thirtieth
auction in April 2013 that fetched 488,532 m3 (auction for 2 months delivery).
Figures 16.4, 16.5, and 16.6 summarize average prices in each biodiesel auction
as organized by ANP. After several changes in the auction manner, currently, it
involves a direct offering between biodiesel producers and fuel distribution
companies.
354 D. A. G. Aranda et al.

Fig. 16.3 Biodiesel auctioned volume—ANP auctions (ANP 2013)

Fig. 16.4 Auction average prices 1–22 (ANP)

Despite being a new industry, the potential biodiesel offer is much higher than
the mandatory demand. This means a dangerous industrial idling (Fig. 16.6). Until
January, 2013, 64 biodiesel plants obtained operating licenses with a total capacity
of 20,207.76 m3/day. In the next few years, more plants are scheduled to work in
addition to some extended size ones.
Biodiesel Program promotes social inclusion through deals involving biodiesel
producers and small farmers. Agriculture Development Ministry published the
‘‘Instrução Normativa n 1 de 20/06/2011,’’ which currently regulates the ‘‘Social
Fuel Seal.’’ Basically, the biodiesel industries with purchase contracts with small
farmers are included in the ‘‘Programa Nacional de Fortalecimento da Agricultura
Familiar (Pronaf).’’ Once the producers have obtained this seal, they are able to
obtain some tax reductions and special fundings. Producers have to purchase at least
16 Techno-Economic and Life Cycle Analysis of Biodiesel Production 355

Fig. 16.5 Average prices in biodiesel auctions (ANP auction 23–31)

8000
Accumulated Capacity (Authorized by ANP)
7000
Obligatory Biodiesel Demand
6000 Biodiesel Production
Estimated Value
5000

4000

3000

2000

1000

0
2005 2006 2007 2008 2009 2010 2011 2012 2013

Fig. 16.6 Mandatory demand and operating licensed capacity for biodiesel plants in Brazil,
1000 m3 (ANP 2013)

30 % of their feedstocks from Northeast, Southeast, and South region small farmers,
or 15 % in the case of North or West-Central region (BRASIL/MDA 2011).
A part of biodiesel producers are asking for an open market phase instead of the
regulated auctions as is currently done. In this case, ANP activity would be
restricted to quality control as well as the blended biodiesel-diesel regulation.
Biodiesel development occurrs in Brazil due to the mandatory process once its
price becomes historically higher than mineral diesel. Average Price for B5 in
April/2013 was R$2.33/L, 14 % higher than April/2012. This higher price is
basically due to the higher prices of mineral diesel and not due to a more expensive
biodiesel (Fig. 16.7).
356 D. A. G. Aranda et al.

Start of B5

Fig. 16.7 B5 prices (R$/liter) (ANP 2013)

Table 16.1 Brazilian prices for biodiesel and mineral diesel (R$/m3)
Fuel prices (R$/m3) 2012 2013* D%
Biodiesel—Auctions—ANP 2,187.91 2,249.42 2.8
Diesel in refinery 1,372.13 1,538.28 12.1
Diesel to distribution companies 1,816.50 2,002.75 10.3
Diesel in fuel station 2,041.25 2,259.50 10.7
Imported diesel 1,448.32 1,604.67 10.8
Source ANP (2013). Dólar/Brazilian Real Exchange, R$2.00/US$1.00
* 1st quarter of 2013

Average prices for biodiesel purchased in the auctions were between

R$2,553.46/m3 and R$2,213.57/m3 in the first quarter of 2013 (Table 16.1). After
that the price dropped to R$1,981.22/m3. Comparing with the same period in 2012,
prices were 2.8 % higher. At the same time, mineral diesel prices increased twice
(ABIOVE 2013a, b).
It is important to stress that biodiesel price is ascribed to vegetable oil prices.
Actually, biodiesel production cost is about 85 % of vegetable oil. In the Brazilian
case, soybean oil price is relevant. It represents 75 % of the feedstocks to
biodiesel. Another important factor in the biodiesel historical prices is the large
amount of companies offering biodiesel in the auctions. In the ANP auctions, only
the maximum prices are fixed; final prices are based on the competitive edge
(Amaral Mendes and Da Costa 2010).
16 Techno-Economic and Life Cycle Analysis of Biodiesel Production 357

16.2 The Environmental Issue on Biodiesel

Once there is oxygen in its structure, biodiesel is able to promote a more complete
combustion, reducing emissions of carbon monoxide (CO) and particulate matter,
and increasing lubricity, guaranteed by sulfur in diesel, hence improving the life of
engine components. There are also reductions in emissions of sulfur oxides
because biodiesel does not contain sulfur. Furthermore, biodiesel provides a small
increase in emissions of NOx for more than 20 % B20+ (Soares 2008). However,
urea solution additive significantly reduces problems ascribed to NOx emissions in
diesel engines.
The impact of NOx emissions by replacing diesel with biodiesel is not
significant, but the reductions in CO, hydrocarbons, particulate matter, and poly-
aromatics imply significant benefits (Monteiro 2005 in Soares 2008).
In general, it is considered that biodiesel is able to reduce the total greenhouse
gases (GHG) emissions compared to diesel fuel. However, for a more compre-
hensive study of emissions from biofuels, it is necessary to consider some variables
for the production of biodiesel, such as the production technology route, oilseed,
and alcohol used in the process (Soares 2008).

16.3 Life Cycle Analysis of Biodiesel: An Overview

of the Brazilian Case

Life Cycle Assessment (LCA) of a product or process is a management technique

to quantify the mass flow, energy, and emissions assessing the environmental
aspects and potential impacts from its production chain (Soares 2008).
ISO 14040, in the same group of ISO 14000, which establishes guidelines for
corporate environmental management, indicates a methodology to life cycle
assessment of products and services.
In order to estimate the environmental impact of the production and use of
biodiesel, it is proposed a simplified life cycle analysis of its production.
The scope of this life cycle assessment includes two basic steps:
• an initial mapping of the most used vegetable oils in Brazil and potentially
usable main raw materials for the biodiesel production;
• a mapping of biodiesel production key data in Brazil.

Another important issue is the land use. Most countries have a conflict between
the land used for food plantations and the land dedicated to bioenergetic crops. In
the case of Brazil, there are about 850 million ha including forests, cattle pasture,
lakes, cities, etc. If one takes into account just the available land for new crops, the
official number is about 90 million ha. One million hectares with palm plantation
could produce about 5 million mton/year of palm biodiesel. This would mean a
358 D. A. G. Aranda et al.

Table 16.2 Biodiesel production by feedstock (m3 biodiesel)

Feedstock 2008 2009 2010 2011 2012 2013
Soybean oil 801,320 1,250,577 1,960,822 2,152,298 2,042,730 466,588
Beef tallow 206,966 258,035 327,074 357,664 469,215 141,260
Cottonseed oil 18,353 59,631 57,458 84,711 123,325 26,797
Others 140,489 40,206 41,086 78,088 83,683 37,215
Total 1,167,128 1,608,448 2,386,438 2,672,760 2,718,954 671,859
Source/Preparation ANP (2013), ABIOVE 2013a, b—Coordination of economics and statistics

Fig. 16.8 Oilseeds market

share for biodiesel production
(ANP 2013; ABIOVE
2013a, b)

B10 program just with 1/90 of the available land. Thus, land use in Brazil is not a
big issue. On the other hand, most of the Brazilian soybean producers are part of
the Round Table on Responsible Soy Association (RTRS), which tracks the
soybean origin. Soybean produced in a deforested area or using slavery labor
conditions is not allowed.

16.3.1 The Oilseed Producing Biodiesel in Brazil

ABIOVE (Brazilian Association of Vegetable Oil Industries) considers that the

biodiesel market is represented by the following oilseeds (Table 16.2).
These data contribute to the formation of the following market share of oilseeds
for biodiesel production (Fig. 16.8).
It may be noted that soybean oil, beef tallow, and cottonseed oil contribute, on
average, by about 90 % market share for feedstocks producing biodiesel.
It should also be noted that soybean oil is the main feedstock responsible for the
production of fuel, representing more than 60 % of the total oilseed. The expla-
nation for this lies in the fact that soybeans have a role as one of the main items of
Brazilian agricultural production, due to its development observed more sharply
16 Techno-Economic and Life Cycle Analysis of Biodiesel Production 359

since the 1970s, which enabled the crop to meet the biodiesel national market in an
easy way. Brazil holds the second position in the world ranking for soybean
production, behind only to USA.
Based on the soybean oil produced in Mato Grosso state, the farthermost
soybean production center from the main consumption biodiesel places (São Paulo,
Santos port, Paulínia fuel bases) Delta CO2 company made an LCA of the pure
soybean biodiesel (Delta CO2 2013). Results indicated a reduction in the green-
house gases to about 70 % compared to fossil diesel. If one considers the total
amount of biodiesel being produced and consumed so far in Brazil, it means about
21 million m ton of avoided CO2 due to the Brazilian biodiesel experience. It is
important to emphasize that animal fat, the second more used feedstock, was not
considered in this evaluation. Usually, animal fat biodiesel has an even better
environmental performance than soybean.
Beef tallow is the second most widely used feedstock for biodiesel production
and has contributed to almost 500,000 m3 of fuel in 2012. This feedstock is also
justified for the production of biodiesel because livestock is one of the main
economic activities in Brazil. Brazil has the second largest herd in the world,
behind India, currently occupying an area of almost 200 million ha—which is
about three-fourths of the occupied area by the entire agricultural industry in the
country.
Cottonseed oil contributed in recent years by 2–5 % of oilseeds for biodiesel
production in Brazil. Cotton production in the country in 2011/2012 was over
1.8 million tons. Brazil is the fifth largest producer, behind China, India, Pakistan,
and the United States, and is the third largest exporter of this oilseed (Abrapa 2013).
Among the other possible oilseeds that produce biodiesel, those that stand out
as market reality are palm oil, babassu oil, castor bean oil, and sunflower oil.
However, it is expected that the feedstocks used for this fuel would be diversified
with advances in research and development in the Brazilian agricultural sector in
order to reduce competition with food and land use, and optimize production and
implementation costs, providing integration with the use of manpower to the
agricultural industry in a socially dignified way.
The main trend for the future of biodiesel production in the country is the use of
algae because of a significantly higher biomass productivity at current oil, which
reduces the demand for extensive lands, besides the fact that they have carbon
dioxide and light as their main inputs.

16.3.2 Biodiesel Production in Brazil

In general, the biodiesel market in the country still lacks important studies and
research, such as life cycle assessment for the production and use of fuel associated
with the market reality. This was observed through a simplified market research
performed with some of the leading producers of biodiesel in Brazil.
360 D. A. G. Aranda et al.

These data reflect the difficulty to relate the relevant and reliable data on the life
cycle assessment for the production of biodiesel in Brazil. Companies such as
Petrobras, which conducts similar ongoing studies reflects a trend of concern about
the alignment of fuel data to the company’s need. There is also a series of
academic studies focused on life cycle assessment of biodiesel, but each with its
specific limitations and considerations that do not necessarily refer to a reality in
the market.
The factors to be considered and inventoried for a life cycle assessment of the
production and use of biodiesel reflecting the reality of Brazil are:
• Origin and indicators of production processes for the oilseeds employed;
• Distance, type of transport, and logistics, possible loss estimates associated with
the origin of oilseed and its production process;
• Indicators of the production process for biodiesel;
• Distance, type of transport, and logistics possible losses estimate associated with
the oilseed to the biodiesel production process;
• Origin and production process indicators of feedstock, supplies, and utilities for
the biodiesel production processes;
• Distance, type of transport, and logistics possible loss estimates associated with
the feedstocks, supplies, and utilities to the biodiesel production; and
• Indicators of the biodiesel use to the end consumer.
The indicators of the production of biodiesel should also consider the waste-
water generation, solid waste, and gaseous emissions of its productive chain.
Amaral Mendes and Da Costa (2009) define the biodiesel industry as structured
by companies with three distinct classifications in relation to its main feedstock:
integrated, partially integrated, and nonintegrated.
The integrated companies have the cultivation or marketing of oilseeds step in
its supply chain. These companies typically have greater competitiveness in the
market due to greater flexibility of marketing products in accordance with the
stages of its production.
The partially integrated company has the ability to produce, in addition to
biodiesel, vegetable oil, although they do not sell or plant the crop plants.
The nonintegrated companies produce only biodiesel and are vulnerable to the
oilseed price market fluctuations.
In relation to feedstocks, it is observed that besides oilseeds, the market mainly
uses methanol as the reagent alcohol for the transesterification of the oil, despite
the large supply of ethanol in the country. This is also explained by the large use of
ethanol as an automotive fuel, as well as sugar and ethanol feedstock, an important
item in the food market.
The use of ethanol as a feedstock potentially emits less greenhouse gases, since
its production is made from sugarcane, which is renewable and widely exploited in
various regions of the country. Methanol, in turn, has its origin in the petro-
chemical industry and is produced domestically only in the city of Rio de Janeiro,
which is a negative fact for the logistics of the reagent to the producers.
16 Techno-Economic and Life Cycle Analysis of Biodiesel Production 361

The main supply input for biodiesel production is sodium methylate, which is
used as a catalyst in the process.
The necessary utilities in this production are basically electricity and steam.
The National Program for Production and Use of Biodiesel was the major
regulatory milestone for the compulsory and progressive use of the fuel in the
country. However, as already mentioned, it was established by Law 11,097 in
2005, i.e., the growth of this market is still very recent. Therefore, it is still
expected to be built a learning curve for the use of biodiesel in order to exploit
natural resources in a better way to meet the same standards.
The Brazilian biodiesel market is fragmented. There are several producers with
none having a market share larger than 16 %. Petrobras is the largest buyer but
Shell, Exxon Mobil, Repsol, and Ipiranga are also important players. An important
alternative to Brazilian producers could be export. However, the big international
markets, like the European one, have technical restrictions to a pure soybean
biodiesel. The main advantages of biodiesel production are ascribed to social and
environmental issues. First, because it is labor intensive and can be produced from
different types of raw materials and in several regions in the country. Second, it
replaces a very important transportation fuel reducing local polutants like soot,
carbon monoxide, and SOx. In addition, it significantly reduces greenhouse gas
emissions (Amaral Mendes and Da Costa 2010).
The main risks are ascribed to new technology trends both in the case of
different types of feedstocks and the so-called second and third generations, as
microalgae, for instance. A good opportunity is to try to explore high valued
coproducts in this chain.

16.4 Biodiesel Production Technology

As mentioned elsewhere, the price of raw materials has a strong influence on the
final cost of production. In general, more than 80 % of the cost is based on this
price (Shi and Bao 2008). In order to reduce this cost, efforts are based on cheaper
feedstocks and process optimization.

16.4.1 Transesterification

Transesterification is a reaction between a vegetable oil and a short chain alcohol

like methanol or ethanol to produce monoesters and glycerol. This is a reversible
reaction and an excess of alcohol is used to shift the equilibria. Stoichiometrically,
this reaction involves 3 mols of alcohol to each molecule of triglyceride, producing
3 mols of esters and 1 molecule of glicerol. Industrially, at least 6 mols of alcohol
is used for each triglyceride molecule in order to obtain a more complete con-
version (Fukuda et al. 2001).
362 D. A. G. Aranda et al.

From the most studied transesterification catalysts, Brønsted bases and acids are
the main ones, with alcoxides and alkaline hydroxides the prefered ones (Suarez
et al. 2007).
It is clear in the literature that basic catalysis have operating problems when high
amounts of free fatty acids are found in vegetable oil. In this case, soap is produced
reducing the yields with associated emulsions. Similar behavior occurs when
moisture is in the reaction media. Hydrolysis of esters produce fatty acids, which
react with the base catalyst leading to soap and emulsions (Ma and Hanna 1999).

16.4.2 Hydroesterification

Hydroesterification has been presented as a new alternative to biodiesel produc-

tion. Several studies are conducted on kinetics, catalysts, multiple feedstocks,
production costs, and hydroesterification plant instalation (Lima Leão 2007;
Gonçalves 2007; Encarnação 2008; Gomes 2009; Leão 2009).
Hydroesterification means a first reaction of triglyceride hydrolysis producing
fatty acids and glycerol. Secondly, an esterification reaction, where fatty acids plus
methanol or ethanol produce biodiesel and water (Kuss 2012).
Hydrolysis reaction increases the feedstock acidity, thus a fatty acid remotion
is not necessary. Thus, any fatty material (vegetable oil, animal fat, used fried oil,
brown grease, etc.) can be used in this process with any acidity or moisture
content. The ability to use those types of crude feedstocks is the main difference
compared to regular transesterification, which always produces soap and reduces
yields due to a difficult glicerol/biodiesel separation (Encarnação 2008).
Acid hydrolysis promotes a complete transformation of fatty materials in fatty
acids which are converted into biodiesel in the second step. Glycerol does not
suffer any contamination due to alcohol or biodiesel contact since it is removed
during hydrolysis. Esterification produces biodiesel and water which can be reused
in the hydrolysis step (Arceo 2012).
Based on the above mentioned, hydroesterification (hydrolysis plus esterification)
is a promising alternative to conventional biodiesel production (Arceo 2012).

16.4.3 Transesterification and Hydroesterification Costs

A large transesterification biodiesel plant usually presents operating costs of about

US$70/ton (electricity, steam, chemicals, and labor) (Encarnação et al. 2009). In
the hydroesterification process with no homogeneous catalysts and no inorganic
acids in the washing step, total operating cost is about US$35/ton. In a medium to
large size biodiesel plant (100,000 mton/year), this process costs about US$3.5
million/year in operating costs.
16 Techno-Economic and Life Cycle Analysis of Biodiesel Production 363

Table 16.3 Transesterification versus hydroesterification comparison

( /L) Transesterification Hydrolysis ? Esterification
Chemicals 4 1
Energy 1 2
Other operating costs 5 3
Source Cruz and Aranda (2011)

Currently, there are several feedstocks that can be used and transformed into an
international standard biodiesel with high yields (about 98 %). The transesterifi-
cation process cannot be applied efficiently to crude feedstocks. Few hydroeste-
rification studies are found in the literature. Lima Leão (2007), who studied
hydroesterification of soybean and castor oils obtained high conversions for castor
fatty acids esterification (87.24 %) and soybean fatty acids (92.24 %), with nio-
bium-based catalysts (20 %), temperature (200 C). Chenard et al. (2009) studied
the same process using jatropha oil obtaining conversions from 86.60 to 88.35 %.
As about 80 % of biodiesel operating cost is ascribed to feedstock price,
hydroesterification allows a significantly better performance in the feasebility of
a biodiesel project. In Table 16.3, a comparison between operating costs for
hydroesterification and transesterification (in a 50,000 mton/year) is given.
In order to obtain important advances in this growing biofuel demand, new
approaches are necessary. Currently, algae in biodiesel research is considered a
new frontier in this sector presenting superior yield compared to other conven-
tional plantation. Biodiesel expectations are huge because: (i) algae absorb CO2;
(ii) growing rate is fast; (iii) high lipid content; (iv) can be cultivated in pools,
lakes, raceways, and photobioreactors; (v) high yield by acre; and (vi) not con-
sidered as a food item. Nevertheless, Brazil should seek an alternative to soybean
with higher efficiency, not necessarily ascribed to edible oil prices (Amaral
Mendes and Da Costa 2010).

16.5 Conclusions

The approaches in this chapter allow the understanding of the development

strategies of biodiesel production and consumption in Brazil, based on techno-
logical, economical, social, and environment sustainability assumptions.
It is relevant to pay attention to the rapidly changing industrial capacity to
produce biodiesel. Until September 2013, 63 units were authorized to produce this
biofuel, with a total nominal capacity of approximately 8 billion L/year. Over
80 % of this capacity is ascribed to social seal companies that are involved with
small farmers providing their feedstocks. From 2005 to September 2013, Brazil
produced 13 billion L of biodiesel, reducing diesel imports of US$11 billion and
contributing positively to the Brazilian Trade Balance.
364 D. A. G. Aranda et al.

Finally, it should be emphasized that biodiesel relevance in the Brazilian

industry is influenced by a large amount of R&D funding throughout the pro-
duction chain, ranging from the agricultural stage to the industrial production
processes, including coproducts and storage. In this sense, the current tax model
gives Brazilian biodiesel a unique feature in the world supported by a biofuel
policy with social orientation.

Acknowledgments Special thanks to the companies Caramuru, Olfar, Petrobrás, and ABIOVE
for contributions informed by e-mail.

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Jatropha Curcas puro y mezclado com aceite Rícino para la producción de biodiesel, 6o
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de Agosto 2009
Cruz YR, Aranda DG (2011) Relatório de tecnologias para a produção de biodiesel. Laboratório
de Tecnologias Verdes (GREENTEC). Escola de Química. UFRJ
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Soja
Encarnação APG (2008) Geração de Biodiesel pelos Processos de Transesterificação e
Hidroesterificação, Uma Avaliação Econômica-Rio de Janeiro: UFRJ/EQ, 2008.144 f.
Dissertação (Mestrado em Tecnologia de Processos Químicos e Bioquímicos)-Universidade
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econômica-transesterificação e hidroesterificação. Curitiba: Biodieselbr, p. 113
Ferreira MHG, Oliveira DL (2010) Um panorama do biodiesel: novo combustível para o Brasil.
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oleoquímica. Quim Nova 30(3):667–676
Chapter 17
Microalgal Feedstock for Bioenergy:
Opportunities and Challenges

Cristiano Eduardo Rodrigues Reis, Mateus de Souza Amaral,

Carla Cristina Almeida Loures, Patrícia Caroline Molgero da Rós,
Bo Hu, Hélcio José Izário Filho, Heizir Ferreira de Castro,
Sônia Maria Flores Gianesella and Messias Borges Silva

Abstract The utilization of algal feedstock for bioenergy can be considered as

one of the greatest challenges for biosystems engineering in the near future. Some
species of microalgae show high potential for oil accumulation and further utili-
zation of its biomass for biogas generation, pyrolysis, ethanol production, and even
as fertilizer. Microalgae can utilize CO2 as carbon source and can also be grown on
nonagricultural environments, such as wastewater facilities, industrial effluents,
freshwater, and marine water habitats. The vast research field on microalgae
engineering is due to the facts that it can be a source of energy and act as an air and
water pollutants removal. There have been considerable advances in engineering
its growth, in bioreactor designs, and on lipid accumulation due to chemical,
biochemical, and genetic studies. Despite that, there are still some fundamental
processing aspects that are considered challenges, either economical, ecological,
or technical, such as biomass harvesting and the competition with the higher value
products produced from algae, as proteins.

C. E. R. Reis  M. de Souza Amaral  P. C. M. da Rós 

H. J. I. Filho  H. F. de Castro  M. B. Silva (&)
Department of Chemical Engineering, School of Engineering of Lorena,
University of São Paulo, Lorena, São Paulo, Brazil
e-mail: messias@dequi.eel.usp.br
C. C. A. Loures  M. B. Silva
Department of Industrial Engineering, College of Engineering of Guaratinguetá,
São Paulo State University, Guaratinguetá, São Paulo, Brazil
B. Hu
Department of Bioproducts and Biosystems Engineering,
College of Food, Agricultural and Natural Resource Sciences,
University of Minnesota, Saint Paul, MN, USA
S. M. F. Gianesella
Department of Biological Oceanography Oceanographic Institute,
University of São Paulo, São Paulo, Brazil

S. S. da Silva and A. K. Chandel (eds.), Biofuels in Brazil, 367

DOI: 10.1007/978-3-319-05020-1_17,  Springer International Publishing Switzerland 2014
368 C. E. R. Reis et al.

17.1 Introduction

The pursue of alternative sources for energy in the new century is due to the
scarcity of fossil fuels in the near future, i.e., energy security reasons, and also the
concern with the environment. According to the Intergovernmental Panel on
Climate Change (IPCC), the accelerated production of carbon dioxide as a result of
human activity is a major factor which contributes to the greenhouse effect
(Houghton et al. 2001). The history of biofuels, which have been considered a
green alternative for fossil fuels, has been changing much for the past 40 years.
There were an ethanol boom with Brazil and the United States (Ribeiro and
Younes-Ibrahim 2001; Goldemberg et al. 2004) and a huge interest in producing
biodiesel from oleaginous plants in the last decades (Demirbas 2008; Pousa et al.
2007). Despite these being designed as green alternatives, recent studies imply that
ethanol and biodiesel produced from plant feedstocks do not match several criteria
for sustainability (Hoekman 2009). The large acreage of corn for ethanol pro-
duction in the United States, for example, has raised concerns among specialists
regarding pollution from pesticides and fertilizers, reduction of biodiversity, soil
erosion, and a shift on the equilibrium on the food supply chain (Fargione et al.
2008, 2010; Hill et al. 2009).
An alternative showing promising results are known as second-generation
biofuels, i.e., biofuels produced from lignocellulosic residues (Sun and Cheng
2002). These are still being developed and are based on the utilization of sugar
monomers released from agro-residual biomass hydrolysis and on the production
of biogas from biomass controlled combustion (Hendriks and Zeeman 2009).
Despite these efforts are considerable and important for supplying clean energy to
human society, microbes have been considered as one of the new potential sources
of energy harvesting (Xia et al. 2011; Huang et al. 2009; Li et al. 2008; Millati
et al. 2005; Illman et al. 2000; Ratledge and Wilkinson 1988). In the group of
microbes, microalgae have earned much attention from the academic society for a
vast number of reasons. Some of which are: the tendency of producing more
biomass than terrestrial plants per unit of area, they can be produced in marginal
lands, in fresh water, and in salt water ecosystems (Chisti 2007) and their non-
competition with food systems, since they can be produced in areas where there is
no agricultural productivity (Hill et al. 2006). Another characteristic of microalgae
that can make its production more feasible and sustainable is its capacity to uptake
human produced CO2 (Benemann and Oswald 1996) as well as removing certain
water pollutants (Powell et al. 2008; Munoz and Guieysse 2006).
The interest in microalgae is not something new though from 1978 to 1996, the
U.S. Department of Energy funded a program to develop renewable transportation
fuels from algae (Sheehan et al. 1998). Their goal was the production of biodiesel
from high lipid content algae utilizing waste CO2 from coal plants, and throughout
these almost 20 years of research, there was a considerable advance in metabolism
manipulation and bioprocessing engineering for algae growth. In the recent years,
there was a development in the field of investigation on genetic modification for
17 Microalgal Feedstock for Bioenergy: Opportunities and Challenges 369

enhancing lipid production (Rosenberg et al. 2008; Radakovits et al. 2010), as well
as the studies on biochemical engineering regarding optimization of growth.
Factors such as reactor configuration (Vergara-Fernández et al. 2008; Wu and
Merchuk 2002, 2004), nutrient loads (Fabregas et al. 2000; Heredia-Arroyo et al.
2010; 2011), light fluxes, and others are some present in the literature.
Another key aspect regarding algae for bioenergy is the utilization of its dry
biomass for biogas generation (Vergara-Fernández et al. 2008; Bohutskyi and
Bouwer 2013; Mussgnug et al. 2010), for production of other fuels and even as
feedstock for char as potential fertilizer (Johnson et al. 2013). Therefore, it can be
seen that the trend of microalgae research nowadays is mainly focused on the
conversion of algal biomass to fuels and the engineering toward optimization of
cultivation methods and oil and lipid enhancing.
The purpose of this chapter is to present some technologies available in the field
of growing, harvesting, and utilizing microalgae biomass, the chemical and bio-
chemical nature of microalgae biomass, and the basic concepts of biodiesel, bio-
gas, biohydrogen, bioethanol, and other fuels production from microalgae biomass
and lipids. Alongside the technologies, the current challenges and some oppor-
tunities are presented.

17.2 Cultivation of Microalgae

As well as any other microorganism, microalgae grow in environments with its basic
nutritional needs. In lab scale, there have been studies on formulating specific me-
dias for their growth since the nineteenth century (Lourenço 2006). Basically, all the
culture media for microalgae cultivation should be composed of basic macronutri-
ents (C, H, O, N, P, S, K, Mg, Si, and Fe) and micronutrients (Mn, Mo, Co, B, V, Zn,
Cu, Se, Br, and I) (Lobban 1994), as well as light and water. Grobbelaar (2004)
presented the following ratio for some nutrients: CO0.48H1.83N0.11P0.01. There are
several classifications, from which two are more useful in this chapter: into marine
species, which have affinity for high concentration of salt, and freshwater species
(Bilanovic et al. 2009); and the division into autotrophic, heterotrophic, and
mixotrophic species, noticing that some species can grow under two or three of these
regimes (Heredia-Arroyo et al. 2010).
Light administration is a key factor in both indoor and outdoor systems,
affecting especially those microalgae that grow on photoautotrophic regime. For
outdoor systems the most common light source is sunlight while in indoor culti-
vation, artificial light sources are required. Chen et al. (2011) summarized several
artificial light sources, from the conventional one (with a high electricity con-
sumption) to more engineered options, such as LED, Optical fiber excited by
metal-halide lamp (OF-MH), Optical fiber excited by solar energy (OF-solar), and
an option with zero electricity consumption and high operation stability, which is
the LED/OF-solar combined with wind power/solar panel.
370 C. E. R. Reis et al.

Fig. 17.1 Scheme of a

Raceway pond

Producing microalgal biomass nowadays is generally more expensive than

crops, although the culture media are inexpensive (Acién Fernández et al. 1999).
There should also be a temperature control within 20–30 C in most cases (Chisti
2007).

17.2.1 Large Scale Production of Photoautotrophic

Microalgae

According to Chisti (2007), the only practicable ways to produce microalgae in

large scale are in raceway ponds and in photobioreactors.
A raceway pond is a simple and continuous system of microalgae growth. It is
consisted on a closed loop recirculation channel, with an average depth of 0.3 m,
built generally with PVC, concrete, or compacted earth covered with a plastic film
(Chisti 2007; Terry and Raymond 1985). The continuous flow is guided around
bends and it is mixed and circulated by a paddlewheel, used also to prevent
sedimentation. After its retention growth, culture broth is harvested on the com-
pletion of the circulation loop. A simple scheme of a raceway pond can be seen in
Fig. 17.1.
There are also circular ponds and unstirred ponds. The main advantage of a
raceway pond, when compared to a photobioreactor, is its price, having opera-
tional and building costs lower than the other does.
Photobioreactors (PBRs) are closed systems, which have the differential char-
acteristic of enhancing light availability for each microalgae cell in the reactor
(Suali and Sarbatly 2012). These are generally built with transparent materials,
17 Microalgal Feedstock for Bioenergy: Opportunities and Challenges 371

Fig. 17.2 Scheme of a

cylinder photobioreactor

allowing light penetration into the culture. These are usually more expensive than
raceway ponds, however, they show higher productivity (Lourenço 2006). In the
literature, three types of geometry are most cited: the flat-plate, the cylinder, and
the tubular.
Tubular and PBRs are constructed with transparent glass or plastic, and these
sort of PBRs have been gaining attention from the academic community in the past
decades. Geometrically, they can be horizontal, vertical, conical, and even
inclined. Mixing can be done either by air lift or by air pumps (Chen et al. 2010).
A simple scheme of a tubular PBR can be seen in Fig. 17.2.
The flat-plate bioreactors consist of airlift driven columns or rectangular tanks
with a recirculation loop. In this kind of reactor, illumination is provided by an
external light source or a bank of lights (Silva et al. 1987).
372 C. E. R. Reis et al.

17.2.2 Cultivation on Heterotrophic Conditions

The so-called heterotrophic cultivation is that one utilizes organic carbon under
dark conditions, as purpose for carbon sources and energy. There are a large
number of organic substrates that microalgae can assimilate, such as glucose,
acetate, glycerol, fructose, sucrose, lactose, galactose, and mannose, which can be
derived from residue biomass, such as corn stover, wheat straw, and sugarcane
bagasse hydrolysates (Chojnacka 2004).
Heredia-Arroyo et al. (2010) confirmed that heterotrophic growth could result
in a higher biomass concentration, when compared to autotrophic conditions for
Chlorella protothecoides under the conditions they studied; the oil content was
similar between these two conditions though. A drawback of using heterotrophic
cultivation is the possibility of bacterial contamination.

17.2.3 Cultivation of Mixotrophic and Photoheterotrophic

Microalgae

Mixotrophic cultivation is the one when microalgae undergo photosynthesis and

use both organic compounds and CO2 as carbon source (Chen et al. 2011). Cul-
tivating a microorganism under mixotrophic conditions means that this organism
would be able to grow under phototrophic, heterotrophic, or both conditions.
There is also a cultivation condition known as photoheterotrophic, also known
as photoorganitrophic, photoassimilation, and photometabolism. In this sort of
metabolism, light is a requirement to utilize organic compounds as carbon source.
Utilizing mixotrophic and photoheterotrophic conditions to grow microalgae,
with a supply of organic compounds from waste resources with zero or even
negative carbon and economic costs may be a good opportunity. Some mixo-
trophic conditions may enhance lipid accumulation, achieving yields significantly
higher than those in solely autotrophic conditions (Heredia-Arroyo et al. 2011).
A brief comparison of the characteristics of different cultivation conditions is
shown (Chen et al. 2011) in Table 17.1.

17.2.4 Biomass Harvesting Techniques

The recovery of algal biomass consists on a solid–liquid separation process and

may account with costs up to 30 % of the total costs in producing microalgal
derived fuels (Gudin and Thepenier 1986). The traditional biomass harvesting
processes are: flocculation, filtration, flotation, and centrifugal sedimentation. The
most appropriate harvesting process depends on a series of factors, such as culture
density, size, value of the products, and available technology (Brennan and
Owende 2010).
17 Microalgal Feedstock for Bioenergy: Opportunities and Challenges 373

Table 17.1 Comparison among cultivation conditions

Cultivation Energy Carbon Achieved Reactor scale- Cost References
condition source source cell density up
Phototrophic Light Inorganic Low Open pond or Low Chen et al.
PBR (2011)
Heterotrophic Organic Organic High Conventional Medium Chen et al.
fermentor (2011)
Mixotrophic Light and Inorganic and Medium PBR High Chen et al.
organic organic (2011)
Photoheterotrophic Light Organic Medium PBR High Chen et al.
(2011)

Cell flocculation is a process in which cells are aggregated together, in order to

form larger particles for settling, i.e., decanting. There are some process well
established, such as chemical autoflocculation (precipitation of carbonate salts
with algal cells at an alkaline pH) (Chen et al. 2011), chemical coagulation (with
organic and inorganic coagulants) (Grima et al. 1994), and even combined floc-
culation techniques.
Filtration is known as the unit operation of separating solid particles from a
suspension using a screen with a particular pore size, accumulating solids at one
side, and decreasing biomass concentration on the other (Grima et al. 1994). This
technique shows several advantages toward other methods, such as simplicity in
cost and operation, and with some adaptations, it can achieve recovery rates of up
to 89 % (Petrusevski et al. 1995). There are some operation limitations, e.g., the
reduction of effectiveness when biomass is too concentrated and clogging issues
(Chen et al. 2011).
Flotation is based on gravity separation in which air or gas bubbles attach to
solid particles and then carry them to the liquid surface (Chen et al. 2011). Flo-
tation can be divided in two major categories: Dissolved air flotation (DAF) and
Dispersed air flotation. The main difference between these two is the bubble size;
while in the first, the air bubbles are within the diameter range of 10–100 lm, the
second one is based on bubbles of 700–1500 lm formed by a high speed agitator
(Rubio et al. 2002).
Centrifugation is a traditional harvesting method, even though its operational
costs can be high enough to make it unfeasible. It is a quick and effective method,
achieving recovery rates of up to 90 % within 2–5 min (Chen et al. 2011).
Depending on the situation, it may not be adequate, since high shear forces could
damage cell wall.

17.2.5 Oil Extraction Techniques

Oil extraction from microalgal biomass is usually an energy demanding process,

due to, among other factors, the requirement of biomass dewatering. The costs of
harvesting and dewatering can achieve costs up to 30 % of the total algal biodiesel
374 C. E. R. Reis et al.

Table 17.2 Comparison among biomass drying processes

Method Advantages Disadvantages References
Drum-drying Fast, efficient High costs Chen et al. (2010);
Becker (1994)
Spray-drying Fast, efficient High costs Chen et al. (2010);
Becker (1994)
Sun-drying Cheap Slow, weather Chen et al. (2010);
dependent Becker (1994)
Solar-drying Cheap Weather dependent Chen et al. (2010);
Becker (1994)
Cross-flow-drying Moderate rate and costs Electricity costs Chen et al. (2010);
Becker (1994)
Vacuum-shelf-drying Gentle process High costs Chen et al. (2010);
Becker (1994)
Freeze-drying Gentle process Slow, high costs Chen et al. (2010);
Becker (1994)

production (Tampier et al. 2009). The most economically viable way would be
natural drying, with solar and wind energy, however this would be a weather
dependent process, which could make oil production seasonal. There are a few
common drying methods, as well presented by Chen et al. (2010) and Becker
(1994) in Table 17.2.
A widely used method for extracting oil from microalgal biomass is using
organic solvents. It is a common pathway for extracting oil from oleaginous plants
and it has been used for microalgae in most cited cases as well (Grima et al. 1994).
The ideal organic solvent should have some characteristics: it has to match the
lipid polarity in the cells, it should be cheap, easy to remove, present low to zero
toxicity, insoluble in water, recyclable, and efficient in dissolving some targeted
components (Chen et al. 2010).
Even though chloroform has high risks of toxicity and flammability, it is a very
common solvent used in lipid extraction. Chloroform is able to extract hydro-
carbons, carotenoids, chlorophylls (source of the green color of algal oil), sterols,
triacylglycerols, wax esters, fatty alcohols, aldehydes, and free fatty acids (Chen
et al. 2010). A traditional method which combines chloroform with methanol at a
2:1 v/v ratio is one of the most used in the published studies (Folch et al. 1957).
Attempts in utilizing other solvents, such as ethanol, 1-butanol, hexane, isopro-
panol, and hexane have also been studied (Grima et al. 1994; Medina et al. 1998;
Cartens et al. 1996; Nagle and Lemke 1990).
In order to optimize solvent extraction, some mechanical and physic-chemical
approaches have been studied to disrupt microalgal cell wall. These include:
autoclaving, microwave, sonication, bead-beating, osmotic shock, and cell
grinding, i.e., ‘‘blending,’’ freeze-press and enzymatic and chemical lysis. Such
methods will not be discussed in this chapter, but they are very well presented by
Chisti and Moo-Young (1986).
17 Microalgal Feedstock for Bioenergy: Opportunities and Challenges 375

Two new techniques in extracting lipids are the supercritical fluid extraction
(SFE) and the subcritical water extraction (SWE). The idea behind SFE is the
utilization of supercritical thermodynamic properties, such as better diffusivity and
decreased viscosity, which improve diffusion rates through solid materials, aiming
higher extraction efficiencies (Anklam et al. 1998). On the other hand, the principle
of SWE is the utilization of water under subcritical conditions, which decreases its
polarity, improving solubility of nonpolar organic compounds (Chen et al. 2010).

17.3 Microalgal Biomass Utilization

The composition of microalgal biomass varies according to several factors:

according to the species, to the growth conditions, temperature, Carbon to
Nitrogen ratio, and others (Volkman et al. 1989). This section will describe some
technologies used in harvesting bioenergy from microalgal biomass.
There are a few most studied species used in the bioenergy field, such as
Chlorella sp. (Illman et al. 2000; Heredia-Arroyo et al. 2011; Wang et al. 2010;
Liang et al. 2009), Dunaliella sp. (Tang et al. 2011; Shuping et al. 2010; Zou et al.
2009; Minowa et al. 1995), Nannochloris sp. (Takagi et al. 2000; Demirbas and
Fatih Demirbas 2011; Hsieh and Wu 2009), Parietochloris incisa (Bigogno et al.
2002), and a few others. Among these, Nannochloris and Dunaliella are marine
microalgae and some Chlorella sp. are as well.

17.3.1 Biodiesel from Microalgal Oils

One of the key points in investing technology to achieve a sustainable biodiesel

production pathway from nonedible microalgal oil is its high productivity,
achieving numbers as high as 5000–100,000 L ha-1 a-1 (Levine et al. 2010).
As well described by Qiul et al. (2011), production of biodiesel from micro-
algae consists of a series of steps: lipid extraction, removal of solvent, catalyzed
transesterification, and purification. There are also a few other processes, such as
hydrolysis followed by esterification (also known as hydroesterification), in situ
transesterification, and supercritical transesterification.
The well-known reaction of lipids transesterification consists of a reaction of a
fatty ester with an alcohol, in order to form fatty acid alkyl esters and glycerol (Ma
and Hanna 1999). This reaction under normal conditions of pressure and moderate
temperatures and reaction times usually requires a catalyst, which can be alkaline,
acid, or enzymatic (Meng et al. 2009).
Microalgal oil has some characteristics that are not desirable in the biodiesel
production: it usually has a high free fatty acid value (Miao and Wu 2006) and it
also contains a high degree of polyunsaturated fatty acids (PUFA) when compared
to vegetable oils (Chisti 2007). A major implication on having a high free fatty
376 C. E. R. Reis et al.

acid value is making the alkali catalysis, which is the cheapest one, unviable
(Lotero et al. 2005). Having a high degree of PUFA makes it more susceptible to
oxidation, thus limiting conditions of storage (Chisti 2007).
There are a few publications about the so-called in situ transesterification. This
process consists of simultaneously extracting and transesterifing the lipids to
produce biodiesel. The biomass has to be dewatered, since water can act as an
inhibitor in this process (Chen et al. 2010). This can be a promising strategy, since
costs are lowered due to the removal of a step in the whole production.
Due to the fact of having high free fatty acid indexes, the alternative of pro-
ducing biodiesel from microalgal oils through hydroesterification has also been
considered. Hydroesterification is consisted of a hydrolysis followed by an
esterification (Diaz et al. 2013; Reyes et al. 2012). Very little has been done yet
utilizing microalgal lipids, even though it may be a good alternative for research.
Reyes et al. (2012) utilized an autoclave reactor at 250 C for the hydrolysis
reaction and niobium powder for the esterification, achieving conversion rates, i.e.,
formation of methyl esters up to 91.7 %.
There has been a trend in this field of study using supercritical conditions. Patil
et al. (2011) studied the optimization of a single-step supercritical process for
simultaneous process for simultaneous extraction and transesterification of wet algal
biomass, using methanol as alcohol. They present some advantages of using
supercritical conditions, such as that they use modest temperatures, the high rate of
production, and the final product price, which, according to the authors, is even
lower than the biodiesel produced from traditional transesterification.
Up-scale processes have been studied as well. Li et al. (2007) presented the
results of utilizing bioreactors with up to 11,000 L at a biodiesel production rate of
6.24 g L-1 and conversions up to 98.15 %. They used immobilized lipase from
Candidia sp. and the microalgal species was C. protothecoides.
Biodiesel from microalgal oils has some advantages when compared to petro-
leum diesel: it can be a totally renewable and biodegradable fuel, a low carbon
footprint, it has low levels of toxicity, and it contains reduced levels of particu-
lates, carbon monoxide, hydrocarbons, and SOx (Brennan and Owende 2010).
Another key point that may lead biodiesel from microalgal oils to a commercial
process is its low freezing point and its high energy densities, making it an
interesting alternative for the aviation industry (Chisti 2010).

17.3.2 Biogas from Microalgal Biomass

In order to make microalgae a more sustainable source for bioenergy, there must be
a use for its residual biomass, i.e., the biomass in which higher value products were
removed, such as lipids and proteins. The high productivities of microalgae may
release high amount of nitrogen and phosphate into the environment, which would
shift the bioenergy harvesting from microalgae toward an unsustainable position. A
process that could solve this issue is the anaerobic digestion, converting biomass to
17 Microalgal Feedstock for Bioenergy: Opportunities and Challenges 377

biogas, recovering more usable energy from cell walls. Anaerobic digestion is the
conversion of organic wastes into biogas, which consists of methane and CO2, with
traces of other compounds, such as H2S (Bridgwater 2008).
Theoretically, there is more energy to be harvested through anaerobic digestion,
producing a mixture rich in methane, than from lipid extraction (Sialve et al.
2009). So far, it is still a research field with very little work done and published.
Only small-scale experiments have been reported achieving efficiencies in the
range of 20–80 % (Zamalloa et al. 2011).
Sialve et al. (2009) identified a few challenges in digesting microalgae: the bio-
degrability can be low depending on the biochemical composition and on the
nature of the cell wal,; which may result in ammonia release leading to toxicity in
cases with high cellular protein content and in inhibition of the process by sodium,
when marine species are considered.
There are a few key points that make microalgal biomass an interesting
opportunity for investing in anaerobic digestion. Besides carbon, nitrogen, and
phosphorus, there are nutrients in lower concentration, such as iron, cobalt, and
zinc, which are able to stimulate methanogenesis (Speece 1996). The theoretical
methane production increases with higher lipid content, since lipids are energy
condensed structures (Angelidaki and Sanders 2004).
Sialve et al. (2009) calculated the methane potential and ammonia release from
anaerobic digestion of several different species of microalgae using data from
(Becker 2004). The results of these researchers are presented in Table 17.3.
The quantity and the quality of biogas generated are dependent upon the bio-
mass composition, pH, temperature, solid retention time, hydraulic retention time,
and loading rate (Singh and Olsen 2011).

17.3.3 Ethanol from Microalgal Biomass

There are three possible pathways for producing ethanol from microalgae. Algae
can assimilate considerable amounts of starch and cellulose, which can be con-
vertible to fermentable sugars. These can be fermented to produce ethanol using a
yeast strain, for example. Some species can also produce ethanol during the dark
fermentation methabolic pathway; the third possible process is to generate genetic
engineering microalgae to produce ethanol directly (John et al. 2011).
Starch is stored in microalgal cells and can be extracted from biomass at regular
intervals from photobioreactors or open ponds through mechanical processes or by
dissolution of cell walls through enzymatic reactions. This starch goes through
solvent extraction and then used for microbial fermentation (John et al. 2011).
Once again, the biomass composition is a key point to achieve high yield on this
sort of fuel. It has been reported that C. vulgaris is a good source for ethanol
fermentation, due to the high starch content, of around 37 % dry weight, achieving
conversion efficiencies of up to 65 % (Hirano et al. 1997). Following well-known
procedures, Harun et al. (2010) investigated the feasibility of producing ethanol
 378

Table 17.3 Biomass composition of several different species of microalgae with CH4 and N–NH3 productivity (VS = Volatile solids)
Species Protein (%) Lipid (%) Carbohydrate (%) CH4 (L g-1 VS) N–NH3 (mg g-1 VS) References
Euglena gracilis 39–61 14–20 14–18 0.53–0.8 54.3–84.9 Sialve et al. (2009)
Chlamydomonas Reinhardtii 48 21 17 0.69 44.7 Sialve et al. (2009)
Chlorella pyrenoidosa 57 2 26 0.8 53.1 Sialve et al. (2009)
Chlorella vulgaris 51–58 14–22 12–17 0.63–0.79 47.5–54.0 Sialve et al. (2009)
Dunaliella salina 57 6 32 0.68 53.1 Sialve et al. (2009)
Spirulina maxima 60–71 6–7 13–16 0.63–0.74 55.9–66.1 Sialve et al. (2009)
Spirulina platensis 46–63 4–9 8–14 0.47–0.69 42.8–58.7 Sialve et al. (2009)
Scenedesmus obliquus 50–56 12–14 10–17 0.59–0.69 46.6–42.2 Sialve et al. (2009)
C. E. R. Reis et al.
17 Microalgal Feedstock for Bioenergy: Opportunities and Challenges 379

from Chlorococum sp. biomass and achieved yields as high as 3.8 g/L using a
10 g/L substrate solution, through fermentation by Saccharomyces bayanus.
Besides starch, microalgae can also accumulate cellulose in their cell walls, as a
structural polysaccharide. This is a common characteristic among green algae
(John et al. 2011). Cellulose can be hydrolyzed into its monomers, i.e., glucose
monosugars, and further fermented to ethanol. A huge advantage when comparing
biomass residues from algae when to plant materials is the inexistence of lignin
in algae, therefore, reducing energy costs and making ethanol production from
cellulose a more feasible process.
A second possible pathway is through the metabolic pathway called dark fer-
mentation. In absence of light and in presence of oxygen, microalgae usually
maintain their life by consuming starch or glycogen; however, if oxygen is also not
available, the oxidative reaction of starch is incomplete, and several other products
are formed, such as hydrogen gas, carbon dioxide, ethanol, lactic acid, formic acid,
etc. (John et al. 2011). A patented process is based on this sort of fermentation
(Ueda et al. 1996), in which microalgal cells contained a large amount of poly-
saccharides, which were catabolized under dark and anaerobic conditions to eth-
anol. This process does not apply to all species of microalgae, but according to
(Ueda et al. 1996), classes Chlorophyceae, Prasinophyceae, Cryptophyceae, and
Cyanophyceae are the ones able to be induced to produce ethanol.
As well explained by John et al. (2011), the algal photosynthesis is based on
Calvin cycle in which ribulose-1,5-bisphosphate (RuBO) combines with CO2 to
produce two 3-phosphoglyceric acid (3-PGA), which is used to produce glucose
and other several metabolites. There is a current attempt trying to redirect the
3-PGA produced to ethanol transformation. This is mainly done by introducing
ethanol producing genes, such as pyruvate decarboxylase and alcohol dehydro-
genase (John et al. 2011). Deng and Coleman (1999) published a work using
modified cyanobacterium (Synechococcus sp.) in order to utilize light, CO2 and
inorganic nutrients to produce ethanol and have it diffused from the cell into the
culture medium.
Ethanol producing from microalgae is, thus, a challenge for biotech companies.
There are a few bottlenecks in these three processes; such as the high cost of
starch/cellulose depolymerizing enzymes for pretreatment of algal biomass and the
competition with higher value fuels.

17.3.4 Biohydrogen from Microalgal Biomass

Microalgae generally have the necessary genetic, metabolic, and enzymatic

characteristics to photoproduce H2 gas. There are two possible pathways for
hydrogen production under anaerobic conditions from eukaryotic microalgae:
either as an electron donor in the process of fixating CO2 or evolved in both light
and dark (Ghirardi et al. 2000; Melis and Happe 2001).
380 C. E. R. Reis et al.

During the process of photosynthesis, microalgae convert water into H+ and

oxygen. H+ can be subsequently converted to H2 through hydrogenase catalyzed
reactions under anaerobic conditions (Melis and Happe 2001; Cantrell et al. 2008).
The key for producing hydrogen in this situation is the utilization of anaerobic
environments, since oxygen is a key inhibitor to hydrogenases (Akkerman et al.
2002). This reaction is reversible, therefore, hydrogen is either produced or con-
sumed by the conversion of protons into hydrogen gas.
Brennan and Owende (2010) cited two fundamental approaches for photosyn-
thetic H2 production from water. The first one is a two-stage photosynthesis
process in which photosynthetic oxygen production and generation of hydrogen
gas are spatially separated. The first stage of this process consists of microalgae
growing in normal conditions; the second one consists of privation of sulfur, which
induces anaerobic conditions, stimulating hydrogen production (Melis and Happe
2001). This is a time-limited process, and hydrogen yields achieve a maximum
after 60 h of production.
The second approach consists of simultaneously producing oxygen and hydro-
gen gases. In this process, the hydrogenase reaction is fed directly with electrons
that are released upon oxidation of water (Ghirardi et al. 2000). There is a con-
siderable higher productivity in this process, when compared to the first, however,
hydrogenases are inhibited after a short time by the oxygen produced. Melis and
Happe (2001) calculated the theoretical maximum yield of hydrogen using the two-
step process and found numbers as high as 198 kg H2 ha-1 day-1.
There is little research yet on biohydrogen production from microalgal derived
routes. The main challenge is to achieve high yields, in order to make this process
feasible, since the theoretical photochemical efficiency of the photoheterotrophic
process is low, of around 10 %. Even achieving high yields, with very high light
intensities, still a large surface would be needed to reach a reasonable hydrogen
production (Melis and Happe 2001). A summary provided by Melis and Happe
(2001) is shown, comparing efficiencies in Photossynthetically active radiation
(PAR) and H2 production, in Table 17.4.

17.3.5 Pyrolysis of Microalgal Biomass

Microalgal biomass can be converted to bio-oil, syngas, and charcoal through

pyrolysis. Pyrolysis processes happen at medium range temperatures (350–700 C)
in the absence of air (Goyal et al. 2008). There are a few operating modes of
pyrolysis, as well described by Bridgwater (2012), which are called: Flash, Fast,
and Slow pyrolysis.
Flash pyrolysis works at moderate temperatures of 500 C, it has a short hot
vapor residence time, of about 1 s, and is deemed to be a viable technique for
future replacement of fossil fuels with biomass derived liquid fuels, since there is a
high biomass-to-liquid conversion ratio. This ratio achieves numbers as high as
95.5 % (Clark and Deswarte 2011; Demirbas 2006). Fast pyrolysis also works at
17 Microalgal Feedstock for Bioenergy: Opportunities and Challenges 381

Table 17.4 Energy conversion efficiencies of green algae for H2 production

Species Absorbed light H2 (nmol h-1) Efficiency References
(lW/cm2) (PAR) %
C. reinhardtii (sup) 2.2 44–61 13–18 Melis and
Happe 2001
C. reinhardtii (UTEX 90) 8.4 78–104 6–8 Melis and
Happe 2001
Chlorella moewusii 9.1 253–337 18–24 Melis and
Happe 2001
The results are based on several different light periods and with a heating value of H2 of 0.23 MJ/mol

the vapor residence time, of around 10–20 s. Slow pyrolysis, on the other hand, is
processed at lower temperatures, of around 400 C and has very long solids res-
idence time. The liquid percentage in these three processes are, respectively, 75,
50, 30 %; the char is known to be around 2, 20, and 35 % and the gas percentage
of 13, 30, and 35 % for flash, fast, and slow pyrolysis, respectively. These numbers
are based on Bridgwater (2012).
Bio-oil from microalgal biomass has higher quality than the one extracted from
lignocellulosic materials (Demirbas 2006), making it a promising area of studies.
Bio-oils have been preferred over the other products of pyrolysis because they have
the potential for being upgraded to liquid transportation fuels (Chen et al. 2010).
Pyrolysis of microalgal biomass converts lipids, starch, protein, and cellulose
into bio-oil, combustible gas, and charcoal (Chen et al. 2010; Ginzburg 1993). It is
interesting to note that the products from heterotrophic and from autotrophic
grown microalgae can be very different; these effects are believed to be due to
different methabolic pathways during their growth (Miao and Wu 2004).
Some current challenges in making pyrolysis from microalgal biomass feasible,
presented by Chen et al. (2010), are: the dewatering process prior to the pyrolysis
itself which is a very high energy requiring step, and the fractioning of the
resulting bio-oil. Bio-oil can achieve high levels of component complexity and
acidity as well. There is a field of studies in testing different conditions and
catalysts to improve bio-oil quality (Wan et al. 2009).
A new approach in microalgal biomass pyrolysis is the utilization of micro-
waves. This technology, developed at the University of Minnesota, provides a few
important advantages toward the conventional processes (Du et al. 2011), such as
easier to control heating, fewer requirements on the feedstock grinding, cleaner
conversion products, produced syngas with a higher heating value, and low cost.

17.3.6 Other Energy Products from Microalgal Biomass

Algal biomass can also be converted to a combustible gas mixture called ‘‘syn-
thesis gas,’’ or simply syngas. These reactions consist of partial oxidation of
biomass in the range of temperatures from 700 to 1100 C (Chen et al. 2010). As
382 C. E. R. Reis et al.

well as pyrolysis, gasification products vary according to the temperature, moisture

content, and other factors. The major applications of syngas are based as source of
thermal energy in gas engines or gas turbines and feedstock for catalytic reforming
and fermentation, in order to produce other chemicals.
Gasification of microalgal biomass has had little interest over the past years, thus
making a broad field of study available for researchers and research groups with
available technology to work on this. Demirbas (2009) aimed at producing H2 from
microalgal biomass, having CO2, CO, and CH4 as byproducts through a gasification
process. Although there are some companies working on syngas production, such as
Ensyn Corp and Plascoenergy Group, both Canadian industries, few research has
been developed using microalgal biomass. Gasification and catalytic reforming of
residue biomass might be an answer for achieving higher sustainability index from
microalgal derived fuels. A new approach is to include algal charcoal, after biomass
utilization, as a new product in the portfolio of algal products (Johnson et al. 2013).
There is also the energetic route known as thermochemical liquefaction, which
aims to produce liquid fuel from wet algal biomass (Patil et al. 2008). The
so-called bio-oil derived from liquefaction is produced at a range of low tem-
peratures, usually from 300 to 350 C, at high pressures, from 5 to 20 MPa, with
the presence of a catalyst and in the presence of hydrogen. The mechanism of bio-
oil production through this process is the high water activity in subcritical ther-
modynamic conditions in order to decompose, i.e., break down biomass to smaller
molecules with a higher energy density (Patil et al. 2008). Dote et al. (1994)
produced bio-oil with a heating value of 45.9 MJ kg-1, with an yield of 64 % at
dry weight of biomass and a positive energy balance of 6.67:1 (output/input).
These numbers, especially the last one, may make thermochemical liquefaction of
algal biomass a promising alternative for further energetic studies.
Hydrothermal processes have several technical and engineering challenges,
such as controlling the ideal heating rate, the residence time, and up-scaling
processes. Most studied processes are still in batch conditions, which may show a
wide range of temperatures and a long time to have the reactor cooled down before
analysis of products. Continuous processes would theoretically show better results,
since these problems are minimized (Chen et al. 2010). Therefore, some of the
engineering challenges would be improving heating rate, making a homogeneous
flow (in order to prevent clogging) and higher-pressure pumps.
A relatively less promising option, which could be used as one of the last steps
in the algal biomass lifecycle, is the direct combustion of biomass. Combustion is
the oxidation reaction at high rates in presence of air and high temperatures, of
800 C or more, at furnaces, boilers, or steam turbines. Not only algal, but any
biomass should have a maximum moisture content of 50 % (McKendry 2002). A
drawback for growing algal biomass solely for combustion is the need for drying,
chopping, and grinding, which raises costs, producing a relatively cheap product.
Therefore, it may not be feasible to burn directly biomass without extracting
higher value products, such as lipids and proteins.
17 Microalgal Feedstock for Bioenergy: Opportunities and Challenges 383

17.4 Challenges in Optimizing Sustainability

Prior to any discussions regarding biomass purification and extraction of products,

it is important to discuss what would be the best way to cultivate microalgae. As
previously shown in this chapter, the utilization of photobioreactors may yield
higher productivity yields; on the other hand, it shows higher costs than other
cultivation methods. The cheapest way to produce microalgae in large scale would
be utilizing lagoons, lakes, or open ponds. In these, however, without any light
administration, there would be a daily loss of biomass of around 25 % due to
overnight respiration (Ratledge and Cohen 2008), not even mentioning the pos-
sibility of contamination from protozoa, bacteria, other algae, and fungi. Yields in
lagoon systems would require up to 2 months for the culture to reach an optimum
biomass density, in order to harvest.
As previously described, collecting and concentration algal biomass are cost
intensive processes and have been subject of study in order to enhance sustainability
from algal biofuels. A study from the 1960s was made comparing several harvesting
techniques, including filtration, flocculation, precipitation, ion exchange, and
ultrasonic vibration (Golueke and Oswald 1965). The authors concluded that only
centrifugation and chemical flocculation were economically viable at the time. With
environmental concerns nowadays, chemical flocculation may have some draw-
backs, since the usage of chemicals in these processes is not incentivized; centri-
fugation may not be the most feasible option as well, because it is an energy
intensive process and may damage cell wall, which can represent yield loss in some
extraction steps. Alternatives, such as the usage of low energy ultrasound waves, are
gaining room in microalgae production. These sort of waves allow cell to aggregate
and settle down once the ultrasonic field is turned off. The main disadvantage,
common among some new techniques, is the high power consumption and low
concentration factors (Bosma et al. 2003).
Up to the date, it is proven that oil extraction from microalgae is an expensive and
difficult process. There is no well-defined and ready to scale-up method available on
the market and most of extractions face challenges with chemical waste and/or high
costs of operation. There are some new technologies, such as nano-dispersion
(promotes dispersion of nano-sized particles), electroporation, and the usage of co-
solvent systems (Chen et al. 2010). Once again, engineering challenges are faced in
the oil extraction step, and whichever method makes itself more sustainable and
economically feasible will definitely attain market interest. Dewatering is also
another key issue previously discussed. Thus, an extensive engineering work must
be done in order to make oil production feasible and reduce the minimum cost of
today production of US$5600–7000/ton (Ratledge and Cohen 2008).
Although with all the required processing steps in order to achieve the oil
extraction step, its characteristics may limit biodiesel production. First of all, even
though some microalgae species are able to accumulate up to 70 wt% of lipids
384 C. E. R. Reis et al.

(Botryococcus braunii) (Chen et al. 2010) under starvation of N, P, and Si, the
overall yield may not be high enough to make it economically feasible. The
development of genomic engineering to map all the pathways in the algal cell,
especially using Chlamydomonas reinharttii, has been done in order to fully
understand lipids production and, obviously, address its optimization afterwards.
Algal oil characteristics are also challenges toward commercialization of fungal
biodiesel, for example. The high free fatty acid value and the presence of unsat-
urated bonds are two drawbacks in the biodiesel industry, since series of pre-
treatments, higher costs with catalysts (since the cheapest and most traditional in
biodiesel plants, NaOH and KOH, cannot be used), and lack of oxidation stability
are faced.
In the 1960s, Japan started to produce Chlorella as a food additive, and since
then, the potential of using microalgae in the food industry has grown enormously.
Today, the most used species in human nutrition are primarily from Chlorella,
Spirulina, and Dunaliella classes (Brennan and Owende 2010). The high content
of beta-carotene in D. salina (up to 14 %) (Moore 2008) and the usage of Chlo-
rella sp. in the pharmaceutical industry make the biofuels industry less advanta-
geous, comparing economically values. Going further, microalgae can also be
source of high value PUFA, such as docosahexaenoic acid (Crypthecodinium and
Schizochytrium spp.), eicosapentanenoic acid (Nannochloropsis, Phaedactylum,
Nitzchia, and Pavlova spp.), c-linolenic acid (Spirulina sp.), and arachidonic acid
(Porphyridium sp.) (Spolaore et al. 2006). In addition, microalgae can be source
for pigments, aquaculture feed, high value fertilizer, and biochemical isotope
chemicals, that have higher value than biofuels (Spolaore et al. 2006). The chal-
lenge of facing these industries could be deviated using a combined platform,
producing these higher value products and also further processing algal biomass,
producing biodiesel, methane, bio-oil, etc.

17.5 Opportunities and Other Applications of Microalgae

17.5.1 Growth in Municipal Leachate

There are a few publications about the usage of microalgae toward landfill leachate
purification. They could utilize organic compounds present in there as carbon and
nitrogen sources (Lin et al. 2007; Cheung et al. 1993). A recent project at the
University of São Paulo, Brazil, aims at the utilization of leachate as culture media
for Chlorella sp. growth. The current stage of this project is the chemical, physical,
and nutritional factor screening toward cell growth.
17 Microalgal Feedstock for Bioenergy: Opportunities and Challenges 385

Table 17.5 Reported values Metal mg g-1 (metal/biomass) References

of heavy metals uptake by
C. vulgaris Au 25.02 Ting et al. (1995)
Cd 12.48 Sandau et al. (1996)
Cu 190.62 Mehta and Gaur (2001)
Ni 205.48 Mehta and Gaur (2001)
Pb 17.2 Sandau et al. (1996)
Zn 6.6 Sandau et al. (1996)

17.5.2 Cocultivation with Pelletized Fungus

A recent study at the University of Minnesota (Hu et al. 2013) is based on a novel
approach of utilizing microalgae and pelletized fungus for a series of advantages.
The coculture enables filamentous fungi, under pelletized morphology, to have
microalgae attached on the pellets; which may drastically decrease harvesting
costs, avoid second pollution from flocculants and the researchers claim that it also
stimulates the algae production (Zhang and Hu 2012).
The fungal pellets (Aspergilus niger), with an average diameter of 2–5 mm, act
as nuclei for microalgal cells to attach. The proposed mechanism for this phe-
nomenon is due to the production of hydrophobins, which are hydrophobic pro-
teins, a family of low molecular weight amphipathic proteins (Linder 2009)
detected hydrophobin on the fungal hyphae, and one of the functions of these
proteins is to coordinate the adherence of hyphae to solid substrates. This study
still needs inputs for larger scale purposes, and may be one of the answers for
enhancing higher sustainability from microalgae-derived fuels.

17.5.3 Metal Sorption

The so-called biosorption is the capability of passive removal of toxic heavy

metals such as Cd2+, Cu2+, Zn2+, Pb2+, Cr3+, and Hg2+ by inexpensive biomaterials
(Davis et al. 2003). Some green algae, such as Chlorella spp., Cladopho-
ra spp., Scenedesmus spp., Chlamydomonas reinhardttii, have been studied due
to their capability of some metals sorption, and the affinity of sorbing a particular
ion is particular to a species and to the physical conditions the cells are grown,
since the cell wall composition may change (Mehta and Gaur 2005).
Chlorella vulgaris, for instance, has a broad spectrum of metal sorption, such as
those reported in Table 17.5. These numbers are based on (Mehta and Gaur 2001;
Sandau et al. 1996; Ting et al. 1995).
386 C. E. R. Reis et al.

17.5.4 Microalgal Biorefinery

The concept of a microalgal-based refinery is based on a traditional petroleum

refinery. Since many products are possible to be produced from microalgal bio-
mass, as well as their utilization as cleaning and depolluting agents, microalgae
would be able to be source of this type of industry. If adequate project and systems
studies are applied, one can achieve maximization on revenues from these
organisms, achieving high economic and environmental benefits.
Unlikely a petroleum refinery, a biorefinery utilizes biomass as feedstock for its
operation, producing a wide range of products from one or more biological
resources (Chen et al. 2010). An integration approach can be applied, making it
possible to produce multiple products from a single biomass feedstock, and this
system can also be self-sufficient in energy, in case of using biomass residues as
energy source.

17.6 Conclusion

Although microalgae have been an interesting field of study in the most diverse
areas of engineering, microbiology, and biochemistry, it still needs further tech-
nical development to make an algal biorefinery something feasible, making
products sufficiently cheap, sustainable, and profitable. As argued by Ratledge
(2008), oil contents of algal cells should be at least 40 % or above to be a starting
material for biodiesel, for example. According to them, producing methane
through anaerobic digestion would yield very little revenue, as well as burning the
residual biomass.
Chen et al. (2010) cited a few key economic concerns of the mass algal pro-
duction systems; which are basically: the cost of the resources for producing
microalgae, the cost of construction and maintenances of the culture system, the
operational costs of harvesting systems, downstream processing and refining. It is
clear that costs vary according to location, solar energy availability, species, etc.
Microalgae does not show any direct competition with the food supply system,
which is an attractive point toward its production. However, the well-established
method for producing Spirulina for consumption is very simple (usage of lakes and
natural lagoons, without mechanical stirring and simple methods of harvesting and
sun drying) and produces a higher value products than, for instance, fuels. Its
biomass is also source of beta-carotene and PUFA, which have a great interest
from food and pharmaceutical industries.
A very favorable point when growing microalgae is the carbon capture issue.
Also according to Chen et al. (2010), for every ton of algal biomass produced,
approximately one ton of carbon dioxide is fixed (assuming 40 wt% of dry algal
biomass as carbon). While most plants capture very dilute CO2 from the
17 Microalgal Feedstock for Bioenergy: Opportunities and Challenges 387

atmosphere, most algae are able to use very concentrated CO2 as carbon source,
allowing it to be part of industrial effluent cleaning processes for example.
Therefore, it is clear that an extensive work must yet be done in order to make
microalgae a major source for energy production a feasible option. The dream of
making a microalgae-derived refinery is far in the near future reality, but it shows
potential of becoming an alternative of supplementing and even replacing non-
renewable fuels. The possibilities of growing microalgae under autotrophic con-
ditions and of utilizing its properties to clean air and wastewater are some unique
advantages that are counting toward its feasibility. Economic studies have shown
also that commodity oils, such as soybean, have doubled and even trebled within
one year (Ratledge and Cohen 2008). Following this trend, there will be an
equivalent point in which commodity oil and algal oil will match in price and from
this point onward, algal oil should be cheaper. Within 10–20 years, there should be
innumerable research advances which will probably derive microalgae as a
potential energy source in the world.

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Chapter 18
Technological Advancements
in Biohydrogen Production and Bagasse
Gasification Process in the Sugarcane
Industry with Regard to Brazilian
Conditions

Jose Luz Silveira, Celso Eduardo Tuna, Daniel Travieso Pedroso,

Marcio Evaristo da Silva, Einara Blanco Machin, Lúcia Bollini Braga
and Valdisley José Martinelli

Abstract Global warming is caused mainly by the excessive use of fossil fuels
(coal, oil, diesel, gasoline, etc.) that emit millions of tons of pollutants into the
environment. Besides, the fact that these fossil fuels are nonrenewable resources
promotes the research in cleaner energy sources. In this chapter are presented two
different technologies that could be introduced in the sugarcane industry to gen-
erate electricity and other kinds of clean fuel (producer gas and hydrogen); the
case of hydrogen production by ethanol steam reforming and biomass gasification,
which appear like promising technologies for energy generation in the sugarcane

J. L. Silveira (&)  C. E. Tuna  D. T. Pedroso  M. E. da Silva 

E. B. Machin  L. B. Braga  V. J. Martinelli
Laboratory of Optmization of Energy Systems—Energy Department,
College of Engineering of Guaratinguetá, São Paulo State University,
Guaratinguetá, Brazil
e-mail: joseluz@feg.unesp.br
C. E. Tuna
e-mail: celso.tuna@feg.unesp.br
D. T. Pedroso
e-mail: danieltravieso@feg.unesp.br
M. E. da Silva
e-mail: mevaristo@feg.unesp.br
E. B. Machin
e-mail: einara@feg.unesp.br
L. B. Braga
e-mail: lucia@feg.unesp.br
V. J. Martinelli
e-mail: valdisley@feg.unesp.br

S. S. da Silva and A. K. Chandel (eds.), Biofuels in Brazil, 393

DOI: 10.1007/978-3-319-05020-1_18,  Springer International Publishing Switzerland 2014
394 J. L. Silveira et al.

industry. Currently, most hydrogen is obtained from natural gas through a process
known as reforming. Other technologic alternatives that may improve the supply
of energy to the sugarcane industry is the use of biomass gasifiers in association
with cogeneration system utilizing combined cycles to produce simultaneously
electricity and heat, a technology known as Biomass Integrated Gasification/Gas
Turbine Combined Cycle (BIG/GTCC). Cogeneration, has been accepted by dif-
ferent industries and has gained great application in the sugarcane industry, where
the thermic and electric demands are favorable to use this type of energy system.
The main fuel used in the process is sugarcane bagasse which is a by-product of
sugar and ethanol production processes; the obtained energy is used in the form of
mechanical power, electric power, and saturated steam in the processes. The
surplus electricity can be sold. Technical, economical, and ecological analyses
were performed for introduction of hydrogen production and BIG/GTCC in the
sugarcane industry, using bagasse as fuel, in order to identify the better scenarios
for electricity and heat generation. The introduction of these technologies will
engender innovations in the sugarcane industry and will promote the sector
development and as main results will increase electricity production with an
economic and ecologic sustainable approach.

18.1 Introduction

The gradual increase in energy demand and environmental pollution caused by

combustion processes of fossil fuels, has made necessary the development of new
technologies employing alternative fuels, thus facilitating the reduction of depen-
dence on fossil fuels oil, natural gas, and coal. Generally, the combustion process of
fossil fuels produce greenhouse gases such as CO2, NOx, and others that increases
the temperature. Energy consumption, mainly in developed countries, has reached
incredible limits, which has led, combined with other factors, the increase in global
warming, with large worldwide implications. Biomass is a renewable resource that
plays a substantial role in the sustainable energy future. Currently, the sensitivities
to environmental issues and energy security have led to the promotion of the use of
endogenous renewable energy sources. Biomass as an energy source covers 10 %
(50 EJ) of the global primary energy source (IEA 2009). Sugarcane is cultivated in
more than 80 countries and the by-products obtained from the sugar production
process represent a great biomass potential. The harvest of sugarcane in the pro-
ducing countries is about 1.2 Gt and potentially its residue can be used for electric
power production of 300 TW h y-1 (Filippis et al. 2004). Besides, nearly 95 % of
hydrogen is produced from fossil-based materials, with steam reforming of methane
being the most used and usually the most economical option. However, in this
process, carbon is transformed into CO2 and released into the atmosphere, leading
18 Technological Advancements in Biohydrogen Production 395

to global climate change (Navarro et al. 2005); thus the main interest is focusing on
alternative methods for the production of hydrogen from renewable energy sources.
These processes are being investigated as long-term solutions, while generation of
hydrogen from biomass has been recognized as a more feasible option for the near-
term solution due to its renewable and carbon-neutral nature (Yang et al. 2006). In
this chapter are presented two different technologies that could be introduced in the
sugarcane industry to generate electricity and other kind of clean fuel (producer gas
and hydrogen); specifically hydrogen production by ethanol steam reforming and
biomass gasification, which are promising technologies for energy generation in the
sugarcane industry.

18.2 Incorporation of Biohydrogen Production

in Sugarcane Industry

Fuel Cell (FC) appears like a promising alternative technology for energy gen-
eration, since it is any efficient system that consists of an electrochemistry process.
In this process, water, electricity, and heat are generated through the combination
of hydrogen and oxygen (Silveira et al. 2009). Hydrogen can be produced from a
variety of sources including water and biomass (Silveira et al. 2008). Currently,
most hydrogen is made from natural gas through a process known as reforming.

18.2.1 The Steam Reforming Process

For hydrogen production, several technologies can be used. Steam reforming is

one of the most common installed in chemical industries. The reforming process
efficiency is a function of physical–chemical properties of feedstock, thermody-
namic conditions (temperature and pressure of reaction), technical configurations
of reformer (dimensions and catalysts), and feedstock and water flows. The
reformer to be used depends on the fuel cell, which will use the reforming
products. The fuel cell technology determines the hydrogen purity required. Steam
reforming occurs in the presence of a catalyst, the syngas produced includes
hydrogen (H2), carbon monoxide (CO), carbon dioxide (CO2), methane (CH4),
among others. Some arrangements to minimize various reactions that can con-
tribute to decrease the hydrogen production are necessary. Since this reaction is
endothermic, heat from external sources is necessary. To minimize losses, several
products of steam reforming, like the nonreacted fraction of reactants, might be
utilized to heat up the reactants (Souza et al. 2006).
396 J. L. Silveira et al.

Fig. 18.1 Inlet and outlet

flows of ethanol steam
reforming process (Silveira
et al. 2009)

18.2.1.1 Ethanol Reforming Reactions

Souza et al. (2006) indicated that this way of reforming can be described through
the following main reactions:
• Global reaction. Ethanol reacts with steam in an endothermic reaction, taking
place in the production of carbon dioxide and hydrogen, as shown in Eq. 18.1:
C2 H5 OH þ 3 H2 O ! 2 CO2 þ 6 H2 ð18:1Þ
Figure 18.1 shows inlet and outlet flows of ethanol steam reforming process, and
Fig. 18.2 shows the prototype developed in São Paulo State University, by
Energetic Systems Optimization Group (www.feg.unesp.br/gose).
• Ethanol steam reforming reaction. Equation 18.2 shows the reaction where the
production of carbon monoxide and hydrogen occurs:
C2 H5 OH þ H2 O ! 2 CO þ 4 H2 ð18:2Þ
• Water Gas Shift Reaction. Since carbon monoxide damages fuel cell catalyst, an
additional process is necessary to remove it. The Water Gas Shift Reaction
(Eq. 18.3), is exothermic, reversible, and occurs at lower temperatures than the
forming reaction:
CO þ H2 O ! CO2 þ H2 ð18:3Þ
• Methanation. Several chemical reactions occur simultaneously. Equation 18.4
shows methane production from carbon monoxide:
CO þ 3 H2 ! CH4 þ H2 O ð18:4Þ
• Bouduard Reaction. This reaction (Eq. 18.5) describes carbon production from
carbon monoxide decomposition:
2 CO ! CO2 þ C ð18:5Þ
18 Technological Advancements in Biohydrogen Production 397

Fig. 18.2 Prototype developed in São Paulo State University

18.2.1.2 Hydrogen Production in Sugarcane Industry

Brazil has the largest and most successful biofuel programs in the world, involving
production of ethanol from sugarcane and has the world’s first sustainable biofuel
economy. Together, Brazil and the United States lead the industrial world in global
ethanol production, accounting for 70 % of the world’s production (Silveira et al.
2009). The Brazilian sugarcane-based industry is far more efficient than the corn-
based industry of USA. In the near future, the sugarcane industry of Brazil could
be modified according to our purpose, as shown in Fig. 18.3. In this case, in
addition to the production of sugar and ethanol, the Brazilian sugarcane industry
will be able to produce biohydrogen.
The goal is innovation in the sugarcane industry production chain through
incorporation of hydrogen production process by steam reforming of ethanol.
It is proposed to incorporate ethanol steam reforming to the traditional sugar-
cane industry process which is composed of extraction, juice treatment, evapo-
ration, cooking, fermentation, and distillation to produce ethanol and sugar as well
as electricity generation through cogeneration system, as shown on Fig. 18.3.
These processes are described below:
• Extraction. In this step the cane is cleaned and milled. The milling consists in
breaking the hard structure of the cane and grinding it. To increase the amount
of juice, water is added. The bagasse obtained from extraction is used in the
boiler of the cogeneration system, as shown in Fig. 18.4 (Pellegrine 2009).
398 J. L. Silveira et al.

Fig. 18.3 Incorporation of steam reforming process to sugarcane industry

• Juice treatment. The juice is first strained to remove large particles. Then it is
treated with chemical substances to modify the pH, coagulate the colloidal
material (greases, proteins, etc.), and precipitate certain impurities (organic
acids, sulfates, etc.). The purification process is chosen according to the sugar
type that is desired to produce. After the addition of chemical substances, the
mixture is heated with water vapor in high pressure. The insoluble particulate
mass (mud) is separated by decantation (Silva 2010). Clarified juice goes to the
evaporators without additional treatment. The mud is filtered and the filter cake
is washed with water.
• Sugar production. According to Castro (2001) the sugar production involves
two steps: evaporation and cooking, as described below.
• Evaporation. In this step the clarified juice is concentrated. First the juice is
passed through heat exchangers to preheat and then to the evaporator stations,
typically a series of five evaporators called multiple-effect evaporators. The
concentrated juice (syrup) follows to the cooking step (Silva 2010).
• Cooking. The syrup goes through the second phase of concentration until it takes
the consistency of honey and begins to form sugar crystals. Once crystallization
is complete, the massecuite is centrifuged and the crystallized sugar and honey
18 Technological Advancements in Biohydrogen Production 399

Fig. 18.4 Cogeneration system (Bernardo 2013)

are separated. The crystals obtained are of good quality and the syrup returns to
the crystallization process. The end honey, or molasses, can be used as raw
material for the fermentation of ethanol. The crystal sugar obtained goes through
the refining process, where it is transformed into amorphous sugar, aiming to
improve the purification and composition (Silva 2010).

Pellegrine (2009) advocates two stages for alcohol obtaining: fermentation and
distillation.
• Fermentation. The mud is diluted to correct the concentration and transferred to
vats where the fermentation process takes place. In this stage are added nutri-
ents, antiseptic, and yeast, mainly responsible for fermentation. After that, wine
is obtained, which goes to the distillation process (Pellegrine 2009).
• Distillation. The wine is directed to a decanter and after that to centrifuge where
the yeast wine is obtained. It is transferred to a wine reservoir where the alcohol
is separated through distillation processes (Silva 2010).
The cogeneration system is shown in Fig. 18.4.
In 18.4, after juice extraction, bagasse is directed to the boilers where it is
burned. The steam from boiler goes to the steam turbine, which is connected to the
electricity generator. In self-sufficient power plants, the surplus electricity can be
sold to grid. As a result of the incorporation and the new configuration of the
sugarcane industry will be produced hydrogen in addition to sugar, ethanol, and
electricity.
400 J. L. Silveira et al.

18.3 Incorporation of Biomass Gasification

in Sugarcane Industry

Traditionally, sugar mills use bagasse and cane trash with high moisture content as
fuel for low pressure boilers to generate steam, using a conventional condensing–
extraction steam-turbine (CEST) technology to provide the plant with heat, elec-
tricity, and mechanical power. The recent years have seen more modern systems
for burning bagasse in suspension that allow to raise the steam pressure and
temperature for the purpose of obtaining a higher electric power cycle cogenera-
tion. The plant thermal efficiency is usually in the 15–30 % range, consequently
the size of conventional combined heat and power generation plants from bagasse
have been limited by these low efficiencies and the amount of fuel within an
economical transportation radius.
The BIG/GTCC technology has been identified by several authors (Babu 1995;
Larson et al. 2001) as an advanced technology with the potential to be cost-
competitive with CEST technology using the biomass by-products of sugarcane
processing as fuel, while dramatically increasing the electricity generated per unit
of sugarcane processed. This type of technology does not require a large invest-
ment demand and can be inserted into the production process of ethanol (Sánchez
Prieto and Nebra 2001).

18.3.1 Biomass Gasification Process

Gasification is a thermochemical process in which a carbonaceous substrate is

transformed into a fuel gas, through a number of reactions that take place at high
temperature in the presence of a gasifying agent (air, oxygen, and/or water vapor).
The gasification process includes the following steps:
• Drying is an endothermic process and to achieve acceptable efficiencies the
maximum amount of moisture in the solid is limited between 20 and 30 % by
weight. The drying process begins at temperatures below 100 C and can be
expressed by the following reaction:
Biomasswet þ Heat $ Biomassdry þ H2 OðgÞ ð18:6Þ

• Pyrolysis is an endothermic process, consists of biomass thermal degradation,

and is developed at temperatures between 200 and 600 C. The pyrolysis
products are carbon, condensable gases (light and heavy hydrocarbons), and
noncondensable gases (methane, water vapor, carbon monoxide, hydrogen, and
carbon dioxide). The reaction can be represented as
Biomassdry þ Heat $ Char þ CO þ CO2 þ H2 þ C2 H4 þ Tar ð18:7Þ
18 Technological Advancements in Biohydrogen Production 401

• Oxidation. The oxidation step is important from the energy point of view, since
it is the exothermic reaction that releases the energy required to develop the
gasification process. The reaction represented by this phase would be:
Pyrolisis products þ O2 $ CO2 þ CO þ H2 OðgÞ þ Heat ð18:8Þ

• Reduction. The reduction step begins to develop significantly when the solid
reaches a temperature around 700 C. Thus, the char reacts with water vapor,
carbon dioxide, and hydrogen, and gases react together to produce the final gas
mixture, obtained as a result of the following reactions:
Char þ H2 O þ O2 $ CO2 þ Heat þ Ash ð18:9Þ

Char þ Heat þ CO2 $ 2CO þ Ash ð18:10Þ

Char þ H2 O þ Heat $ CO þ H2 þ Ash ð18:11Þ

The producer gas is the principal product of gasification, and its lower heating
value (LHV) varies depending on the composition of biomass and the gasifying
agent employee. Using air as the gasifying agent, the LHV of the producer gas is in
the range between 4 and 6 MJ/Nm3 and using water vapor or oxygen the LHV is
between 8 and 20 MJ/Nm3 (Reed et al. 2005).

18.3.2 Gasification in Fluidized Bed Reactors

Fluidized bed reactors are those in which the gasifying agent circulates inside them
at a rate such that a bed is in a state of fluidization, existing inside the gasifier
several conditions that intensify the transfer of energy and material between the
fuel and gas. There are two main categories within these types of gasifiers: bub-
bling and circulating. In the bubbling fluidized beds, the fluidizing velocity–gas-
ifying agent is sufficiently low as there is no significant movement of solid. By
contrast, in the circulating fluidized bed, the velocity of the agent is much higher
resulting in a solids circulation. This solid is recirculated to the reactor by the use
of a cyclone return system to the gasifier. The main advantages of fluidized beds
include better control of temperature and reaction rates, high specific capacity,
potential scaling to larger sizes, and adaptation to changes of biomass. On the
contrary, show moderate–high tars and particulates levels in the exhaust gas and
the fuel conversion are not as high as in the fixed bed gasifiers. A comparison
between bubbling and circulating fluidized bed gasifiers is shown in Table 18.1
(Williams et al. 1995).
402 J. L. Silveira et al.

Table 18.1 Comparison between bubbling and circulating fluidized bed gasifiers
Fluidized Temperature (C) Biomass Feed Gasification Tar
bed reactor agent content
Reaction Exit
Bubbling 700–1000 700–800 Wood chips, leftover Directly in The bottom Medium–
corn cobs, rice the area of the High
husks of the gasifier
bed
Circulating 700–1000 600–800 Sugarcane bagasse, Directly in The bottom Low
wood chips, the area of the
sawdust, rice of the gasifier
hulls bed

18.3.3 Biomass Gasification Technology for Cogeneration

of Heat and Power

Cogeneration study can be divided into three different types of cycles. The con-
ventional cycle in which steam is used at low pressure and temperature and the
steam and power generated is just enough for own consumption of the plant. A
second cycle is the advanced cycle with similar configuration to conventional
cycle but operating with a higher pressure and temperature, which results in sig-
nificantly greater generation of electricity than the needs of the sugar factory; the
excess energy can be sold to external consumers. The third cycle is the BIG/GTCC
and also generates an excess of electricity. The combined cycle system is the
simplest scheme used for cogeneration, shown in Fig. 18.5. It employs a gas
turbine, a heat recovery steam generator without supplementary firing, and steam-
turbine (Silveira et al. 2006).

18.3.4 Sugarcane Bagasse as Biomass

There are several studies on the use of sugarcane bagasse as fuel in gasification
processes. Olivares (1995) studied different types of bagasse with the objective of
introducing it as fuel in a fluidized bed gasifier. Bagasse is a material with high
fiber content and low density and has an extensive range of sizes. It exits the sugar
production process with a moisture content of approximately 50 % (wet basis); for
this reason, a pre-treatment process is necessary that includes drying, crushing, and
others in order to improve their properties and facilitate the feeding process to
fluidized bed reactors.
One of the principal parameters to evaluate the quality of bagasse is the
moisture content because the more humid bagasse will decrease its lower calorific
value (LHV), and therefore it will has less available energy for the same amount of
fuel.
18 Technological Advancements in Biohydrogen Production 403

Fig. 18.5 Gas turbine associated with the steam turbine (Combined Cycle)

Table 18.2 shows the main physical and chemical properties of sugarcane
bagasse reported by Jenkins et al. (1998). Bagasse is classified as a fuel with high
reactivity due to its high content of volatiles and low ash content, making it a good
feedstock for gasification.

18.3.5 Combined Cycle Associated with a Fluidized Bed

Gasifier

In a combined cycle, the fuel combustion provides the mechanical energy to the
electric generator and the exit gases from the combustion are directed to a heat
recovery steam generator to produce steam; this steam will drive a steam turbine
that will be linked the other electricity generator. It is generally employed in this
type of cycle-to-cycle Brayton combination with a Rankine cycle type (Diniz et al.
2013). To have the possibility of entering the gasifier in this cycle, there must be
previous drying since the sugarcane bagasse has relative humidity around 50 % in
natura (Olivares et al. 1995). It is also necessary to clean the producer gas gen-
erated, since it contains a load of particulate and tar, as shown in Fig. 18.6.
Research has shown the potential of BIG/GTCC-based systems to be compet-
itive with, if not superior to, conventional combustion power plants because of
their higher efficiency, superior environmental performance, and competitive cost
(Reed et al. 2005). However, much of the advancements are still under research
and development. BIG/GTCC is a combination of two leading technologies:
gasification and gas turbine combined cycle. The gasification portion of the BIG/
GTCC plant produces a clean gas which fuels the gas turbine. For this system, the
gasification stage is carried out in a fluidized bed. Typical operating temperature of
404 J. L. Silveira et al.

Table 18.2 Main physical Proximate analysis

and chemical properties of
Volatile matter (wt%, dry basis) 85.61
sugarcane bagasse (Jenkins
Fixed carbon (wt%, dry basis) 11.95
et al. 1998)
Ash (wt%, dry basis) 2.44
Higher heating value (MJ. kg-1, dry basis) 18.99
Ultimate analysis (wt%, dry basis)
C 48.65
H 5.87
O (by difference) 42.82
N 0.16
S 0.04
Cl 0.03

Fig. 18.6 Cogeneration combined cycle associated with the fluidized bed gasifier

a fluidized bed is 800–850 C. Air is blown through the bed at a sufficient velocity
to keep the bed materials in a state of suspension. The fuel particles are introduced
at the bottom of the reactor, very quickly mixed with the bed material, and almost
instantaneously heated up to the bed temperature and hence the subsequent pro-
ducer gas generation. After the producer gas has left the fluidized bed chamber, it
goes through a cleaning unit. The gas after the cleaner unit is then led to a boost
compressor that compresses it to the gas turbine combustion chamber pressure
conditions. The exhaust heat from the combustion turbine is recovered in the heat
recovery steam generator to produce steam. This steam then passes through a
steam turbine to power another generator, which produces more electricity. The
combined cycle is more efficient than conventional power generating systems
because it reuses waste heat to produce more electricity (Okure et al. 2006).
18 Technological Advancements in Biohydrogen Production 405

18.3.5.1 Energy Analysis of the Integration of Gasification of Bagasse

in the Sugar and Alcohol Sector

The energy analysis comprises the study based on the first law of thermodynamics,
the law of conservation of energy. This type of study is universally valid and is
used in the mass and energy balances in the gasifier and other components of the
BIG/GTCC system. For realization of this energy, analysis is determined of the
operating parameters and efficiencies of the process and its components. However,
only the use of the first law of thermodynamics proves to be insufficient in sub-
sequent economic evaluation of an energy system, because it does not estimate the
amount of energy available for conversion into work or power. Given this, the
study based on only the first law of thermodynamics gives us an incomplete
analysis of the potential energy of a system.

18.3.5.2 Exergetic Analysis of Integration of Gasification of Bagasse

in the Sugar and Alcohol Sector

Given the limitations of the first law in formulating the quality and quantity of
useful energy in a system, the concept of Exergy was created from the second law
of thermodynamics. According to Tuna (1999), Exergy is that portion of noble
energy that can be completely converted into work reversibly. However, the ex-
ergy can be defined as the maximum useful work that can be obtained by an energy
carrier (Tsatsaronis 1993). The exergy inefficiency of a system consists in a
destruction of exergy associated with irreversibilities. The irreversibility in a
system can be decomposed into internal irreversibility, known as the Second Law
of Thermodynamics as destruction of energy and external irreversibility, which is
the exergy loss to the environment, developing out of the control volume selected
for thermodynamic analysis (Valero et al. 2011). The maximum improvement in
exergy efficiency for a process or system is obviously determined when the exergy
loss or irreversibility is minimized, the latter being determined by the following
equation (Sozen et al. 2002; Utlu et al. 2006):
X X
I¼ Exin  Exout ð18:12Þ

ex1 ¼ ðh1  h0 Þ  T0 ðS1  S0 Þ ð18:13Þ

Ex1 ¼ m  ex1 ð18:14Þ

Exergy analysis or even availability analysis is then drawn in this way to
achieve the goal of a more effective use of energy resource as it enables the
location, cause, and true magnitude of waste and loss. Such information can be
used in the design of efficient energy systems and to increase the performance of
existing systems. Exergy analysis also provides a broader view of the problem
under consideration, avoiding conclusions based purely on the application of the
406 J. L. Silveira et al.

first law of thermodynamics. Tuna (1999) emphasizes that the analysis of first and
second law are not competing, but complementary, and together contribute to a
consistent assessment of the thermal system.

18.4 Economic Analysis

In both technologies the methodology to make the economic analysis is similar

and is based on engineering economics calculations developed by Silva (2010),
who considered the sugarcane industry producing hydrogen using ethanol and
gasification of electricity generated in the gas turbine and steam turbine by use of
producer gas from bagasse gasification. In order to reach this proposal, an eco-
nomic analysis based on the investment of the hydrogen production system and
BIG/GTCC system were developed considering the input costs, operating cost,
maintenance cost, operation period, interest rate, and annuity factor.
The global equation for hydrogen cost is shown in the following equation:
I nvref  f
CH2 ¼ þ COP þ Cman ð18:15Þ
H  E H2
The annual cost of obtaining electricity (Cel), US$/kWh, for each selected
system is given as
 
  Per
Ipl  Ivcr  f Ccomb  E comb  E cr  2
Cel ¼ þ þ CMstg ð18:16Þ
H  Ep Ep

qk  ð q  1Þ
f ¼ ð18:17Þ
qk
where:
k is the amortization period or pay-back, given in years. CH2 —Hydrogen pro-
duction cost (US$/kWh); Cel —Electricity production cost (US$/kWh); Invref—
Reference investment for hydrogen production (9104 US$); f—Annuity factor (1/
year); H—Equivalent period of operation (h/year); EH2 —Energy provided by
Hydrogen (kW); Cop—Operational cost (US$/kWh); Cman—Maintenance cost
(US$/kWh).
Operational cost using bagasse as fuel is shown in Eq. 18.18, and the operational
cost using electricity is shown in Eq. 18.19. According to Kothari et al. (2008), the
maintenance cost of steam reformer was estimated as 3 % of investment.
Efuel  Cfuel EEtOH  CEtOH
C OP ¼ þ ð18:18Þ
E H2 E H2
EElet  CElet EEtOH  CEtOH
C OP ¼ þ ð18:19Þ
EH2 EH2
18 Technological Advancements in Biohydrogen Production 407

where:
Efuel Energy provided by sugarcane bagasse (kW);
Cfuel Fuel cost (sugarcane bagasse) (US$/kWh);
EEtOH Energy provided by ethanol (kW);
CEtOH Ethanol cost (US$/kWh);
EElet Electricity consumed by reformer (kW);
CElet Electricity cost (US$/kWh).

The investment cost (acquisition cost of equipment, installation cost) to produce

steam covers the cost of system gas turbine (compressor, combustion chamber, gas
turbine electric generator, and other accessories), the heat recovery steam gener-
ator is considered as separate module. Thus, the following equation is used to
calculate the investment to be made:
Ipl ¼ Istg þ Ivcr ð18:20Þ
For investment cost of heat recovery steam generator (Ivcr) without supple-
mental fuel burning is used Eq. 18.21, defined as the technique of Boehn (1987)
according to the steam production in (kg/h), with a multiplicative factor of 10 %
related to the cost of installation of the boiler recovery and valid for production
values higher than 800 kg/h and less than 4000 kg/h.
 m 0:81
v
Ivcr ¼ 1:1  160000  ð18:21Þ
1500
The equation final investment cost becomes:
 
Ipl ¼ Istg þ Ivcr  1:3 ð18:22Þ
 m
C ¼ Cr S=Sr ð18:23Þ

where:
C—Equipment cost for an interest capacity S; m—Incidence factor indicating
the economics scale (0.5–1.0); Cr—Equipment cost for a reference capacity Sr.
Silva (2010) has adapted the reference investment for steam reforming process
with hydrogen production range of 1 up to 1500 (Nm3/h), resulting in Eq. 18.24
m 0:5304
H2
Invref ¼ ð18:24Þ
750
The expected annual revenue is calculated as the sum of earnings or annual
benefits due to the installation of a system (Silveira and Tuna 2003, 2004).
408 J. L. Silveira et al.

18.5 Ecological Analysis

At present, practically all known forms of energy production have some kind of
interference in the environment. Due to this fact using biomass gasification
combined with a cogeneration system is a set of recommended alternative energy,
from the environmental point of view.

18.5.1 Ecological Efficiency

The ecological efficiency analysis is based on calculations of equivalent carbon

dioxide [(CO2)e], and pollution indicator (Pg) for determining the ecological
efficiency of the process of hydrogen production by ethanol steam reforming, and
for the BIG/GTCC system.

18.5.2 Determination of Equivalent Carbon Dioxide (CO2e)

The equivalent carbon dioxide depends on the emission of SO2, NOx, and PM, and
can be determined using Eq. 18.25.
CO2e ¼ CO2 þ 80  SO2 þ 50  NOx þ 67  MP ð18:25Þ

18.5.3 Determination of Pollution Indicator ðPP Þ

The pollution indicator ðPP Þ is the ratio between the amount of CO2e in kg and the
power supplied by the producer gas and for the hydrogen production it is shown in
Eq. 18.26 (Silveira et al. 2012).
CO2e
Pp ¼ ð18:26Þ
PCI

18.5.4 Determination of Ecological Efficiency ðeÞ

Ecological efficiency is defined as an indicator for evaluating the performance of a

particular system, considering the emissions of pollutants burning 1 kg of fuel.
Their values vary between 0 and 1, where the higher the vicinity of 0 means the
greater the environmental impact, and if it is proximate to 1, indicates that it is
18 Technological Advancements in Biohydrogen Production 409

nonpolluting (zero environmental impact). The ecological efficiency can be

determined using Eq. 18.27 (Silveira et al. 2012).
"  #0:5
0:204  gsystem  ln 135  Pp
e¼ ð18:27Þ
gsystem þ Pp

18.5.5 Calculation of CO2 Emissions from Combustion

Process of Sugarcane Bagasse

The CO2 emissions from 1 kg of fuel can be calculated according to Eq. (18.28).
ðw1  44  1ÞCO2
M¼ ð18:28Þ
N
where:
MCO2 —CO2 emissions (kgCO2 /kgfuel); Molar mass of fuel (bagasse) (kg/kgmol).
The molar mass of bagasse can be determined based on the elemental com-
position (Table 18.2). Therefore, the molar mass of bagasse can be calculated
through Eq. 18.27.
N ¼ ða1 12Þ þ ðb1 1Þ þ ðc1 16Þ þ ðd1 14Þ þ ðe1 32Þ ð18:29Þ

18.6 Conclusions

Hydrogen, the principal energy carrier to fuel cells, can be produced through
various ways, but ethanol steam reforming is an alternative to guarantee the
volume of production necessary in the Brazilian case. The integration or associ-
ation of hydrogen production with sugar industry, can certainly put Brazil in a
good classification in the ‘‘Hydrogen Era,’’ in the near future. Similarly in terms of
ecological efficiency, the fluidized bed gasifier operating with bagasse is an
environmentally friendly way, with high ecological efficiency to produce energy in
the sugarcane industry. This technology proves that this type of combine cycle is
an excellent alternative to the traditional electric power generation technology,
based on the Rankine cycle, used in this industry for electricity and heat gener-
ation. Thus, these technologies can be inserted with energy and environmental
gains in the sugarcane industry.
410 J. L. Silveira et al.

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Chapter 19
Nonconventional Renewable Sources
in Brazil and Their Impact on the Success
of Bioenergy

Luís Cláudio Oliveira-Lopes and Cláudio H. Ferreira da Silva

Abstract Brazil has abundant natural sources of renewable energy. Existing

renewable sources of energy are discussed and an overview of the energy options
in Brazil is assessed with their current situation and future potential. Given the
great deal of opportunities in terms of energy from wind, solar, and biomass and
their impact in Brazil energy matrix, a diverse and sustainable framework for
producing energy needs to be consolidated. The effectiveness of success of the
Brazilian renewable energy market strongly depends on legislation and country
policies. The growth will be as fast as the country implements energy policy to
support the renewable sources of energy by breaking economic, regulatory, or
institutional barriers. Furthermore, the impact of the nonconventional renewable
energy in the success of bioenergy highly depends of the policymaker initiative on
seeking a variety of renewable energy sources and their incorporation into the
energy matrix of the country.

19.1 Introduction

Brazil is the fifth largest country in the world with an abundant potential for
hydropower, based on several important rivers (Paraná, Tocantins, São Francisco,
Iguaçu, Paranaíba, among others). Hydroelectric capacity is complemented by
conventional thermal and nuclear plants, totaling 107 GW of installed power
capacity (EPE 2009b), of which more than 79 GW is hydropower, 24 GW ther-
moelectric, 2 GW nuclear, and 602 MW wind. The country posses a great variety

L. C. Oliveira-Lopes (&)
Federal University of Uberlândia, Uberlândia, Brazil
e-mail: lcol@ufu.br
C. H. F. da Silva
CEMIG SA, Belo Horizonte, Brazil

S. S. da Silva and A. K. Chandel (eds.), Biofuels in Brazil, 413

DOI: 10.1007/978-3-319-05020-1_19,  Springer International Publishing Switzerland 2014
414 L. C. Oliveira-Lopes and C. H. F. da Silva

of natural resources, but their exploitation may cause significant environmental

impacts. In the case of hydroelectric power, Brazil exploits only 30 % of its
potential, but the remainder is mainly located in environmentally sensitive regions,
like Amazon.
Besides the richness in hydropower potential, Brazil is a large country and
presents different climate zones that open other potential for renewable source of
energy (Meisen and Hubert 2010). While in the central region of the country there
is a dry and sunny climate, which gives a great opportunity for solar energy
production, a large and windy coast allows a wind power production and perhaps,
wave power. In addition to those aspects, Brazil has a large potential of biomass
generation, and with that sources for bioenergy production (bioethanol, biobutanol,
and biohydrogen). Thanks to an ideal climate for sugarcane, Brazil is the second
largest ethanol producer in the world (after the United States). The ethanol from
sugarcane in Brazil is a very competitive and mature industry (Pereira et al. 2012).
In this scenario, Brazil has indicated that it is committed to maintaining a large
share of renewable source in its energy matrix (Fig. 19.1), through Law no 10.438
of 2002, of the Program for Incentive of Alternative Electric Energy Sources
(PROINFA), whose main objectives are to promote the diversification of electric
power generation sources, in order to increase supply security, prioritize action that
exploit regional and local characteristics and potentialities, and the reduction of
greenhouse gas emissions (Pereira et al. 2011). Besides, to promote the use of
renewable technologies (wind, biomass cogeneration, and from small hydroelectric
plants) through incentives and subsidies, PROINFA planned the installation of 144
power plants (with the installed capacity of 3,299.40 MW), distributed among 63
small hydroelectric plants (1,191.24 MW), 54 wind power plants (1,422.92 MW),
and 27 biomass-based power plants (685.24 MW) (MME 2013), with an increase in
the Brazilian renewable share of annual energy consumption to 10 %.
Brazil has an electricity generation matrix based mainly on renewable sources
(44.1 % in 2011). Figure 19.2 shows the internal supply of electricity in Brazil in
2011 (BNE 2012). A total of 466.8 TW h of was produced in 2011.
With an electricity matrix based mostly on renewable sources (88.8 % in 2011),
the country still have many challenges in the future. In fact, a wide variety of
renewable source technologies will be needed to meet the challenges of sustainable
energy development, considering that biomass, biogas, and small hydropower plants
are already competitive compared to traditional generation sources (Ren-21 2013).
Brazil is under strong development and with that it is expected a significant
increase in energy consumption (EPE 2007a, b, 2009a, 2013). The availability of the
energy system should be increased by over 100 % in the next decades. This scenario
represents a large environmental, economical, and social effort for the country.
However, it is a great opportunity for implementing the country vocation in incor-
porating renewable energy to the energy generation matrix (Pottmaier et al. 2013).
In this sense, this chapter addresses nonconventional renewable sources of
energy, their impact, and potentialities in the consumption energy profile in Brazil.
19 Nonconventional Renewable Sources 415

Fig. 19.1 Brazilian energy matrix for 2011 (EPE 2012)

Fig. 19.2 Brazilian internal electricity supply by source in 2011 (EPE 2012)

19.2 Brazilian Potential for Nonconventional Renewable

Sources

Considering the environmental situation, the depletion of natural resources, the

greenhouse gases issue, and also the challenge of energy planning that allows
reconciling supply and demand; it is necessary to move toward a set of solutions
where many options or alternatives need to be used, constituting a contribution to
the construction of a energy system toward low tariffs, high sustainability, and the
use of the local potential thought the country. After all, there exist an important
relationship between society and energy, which may be strongly impacted eco-
nomically and even drive a country perspective of future.
Due to many possibilities of increasing the use of energy renewable sources,
this section will not cover the conventional sources. Nevertheless, a quick over-
view is presented next:
• Hydropower—it is mostly dependent on precipitation and elevation changes;
high precipitation levels and large elevation changes are necessary to generate
416 L. C. Oliveira-Lopes and C. H. F. da Silva

Table 19.1 Summary of the largest hydroelectric plants in Brazil (Source ANEEL 2013)
Dam River MW Reservoir (km2) Status Location
Itaipú Paraná 14000 1350 OP Paraná(BR)/Paraguay
Belo Monte Xingú 11233 516 UC Pará
São Luiz do Tapajós Tapajós 8381 722 PL Pará
Tucuruí Tocantins 8370 2850 OP Pará
Santo Antonio Madeira 3665 271 UC Rondônia
Ilha Solteira Paraná 3444 1195 OP São Paulo
Jirau Madeira 3300 108 UC Rondônia
Xingó São Francisco 3162 60 Alagoas/Sergipe
Paulo Afonso IV São Francisco 2462 12.9 OP Bahia
Jatobá Tapajós 2338 646.3 PL Pará

significant quantities of electricity and irregularity in the wet/dry seasons may

affect the energy production.
In Brazil hydropower is characterized by large reservoirs, located in several
hydrographical basins, which are mostly far from the main consumption centers.
The National Interconnected System is operated by the National System
Operator that optimizes the use of all hydroelectric sources. Brazil has over a
hundred hydroelectric plants. This number almost gets doubled when one
includes small hydroelectric plants (from 1 to 30 MW and a reservoir area up to
3 km2). Table 19.1 presents the major hydroelectric plants in Brazil
(OP = operating, UC = under construction, and PL = planned).
• Ethanol—is a liquid fuel that can be produced from any primary matter which
contains sugar or material that can be transformed into sugars (i.e., starches or
cellulose). Together with the U.S. Brazil produce 87 % of the world’s ethanol.
The vast majority of U.S. produce ethanol from corn, while in Brazil, it is
produced by fermentation of sugars from sugarcane using Saccharomyces ce-
revisiae. Approximately 90 % of Brazilian sugarcane production takes place in
South-Central Brazil with the remainder grown in Northeastern Brazil. Both
production regions are located around 2,500 km (1,550 miles) away from the
Amazon region. In addition to an ideal climate for sugarcane, available land to
produce sugarcane without deforestation, Brazil has the world’s most compet-
itive program of development and ethanol production. Nowadays, the use of flex
fuel vehicles (that can run on either gasoline or hydrous alcohol) account for
90 % of new car sales in Brazil, the reduction of greenhouse gas emission when
using ethanol as a substitute fuel, and the mandatory addition of up to 25 %
(volume) of hydrated alcohol to the all gasoline used in Brazil make ethanol a
highly competitive substitute fuel in Brazil. Table 19.2 presents the global
ethanol production.

Given the vast possibilities and alternatives, the next section will focus on some
options in terms of energy from wind, solar, and biomass and their potential and
impacts on Brazil energy matrix.
19 Nonconventional Renewable Sources 417

Table 19.2 Global ethanol production in billions of gallons

Country 2007 2008 2009 2010 2011 2012
USA 6.521 9.309 10.938 13.298 13.948 13.300
Brazil 5.019 6.472 6.578 6.922 5.573 5.577
Europe 570 734 1.040 1.209 1.168 1.179
China 486 502 542 542 555 555
Canada 211 238 291 357 462 449
Asiaa 132 156 527 244 335 397
South Americab 75 79 83 200 199 223
Mexico and Central America Nac Na Na 364 39 19
Australia 26 26 57 66 87 71
Africa Na Na Na 44 38 42
Other 82 128 247 66 Na Na
WORLD 13.123 17.644 20.303 23.311 22.356 21.812
a
Excluding China; b Excluding Brazil; c Na not available
Source F.O. Licht, cited in Renewable Fuels Association, Ethanol Industry Outlook 2008–2013
reports

19.2.1 Wind Energy

Air moves due to difference in pressure. In the atmosphere it is simply called as

wind, which has kinetic energy (due to its motion). Therefore, wind energy pro-
duction is related to capturing the wind kinetic energy with turbines, which are
designed with a vertical (VAWTs) or a more commonly found horizontal-axis
(HAWTs), to spin a shaft linked to a generator that transfers the rotational energy
to electricity.
Wind energy has been used for 1000 years, it begins with sailing boats, grinding
grains in windmills, water pumping systems, and in the last century has started
being used for electricity generation with turbines operating based on large
aerodynamic turbine systems in a wind farm, which consists of a group of wind
turbines distributed over an area that may be used for agricultural or other purposes
or even be located offshore.
Wind energy has several positive aspects: it does not burn fossil fuels, wind
turbines are scalable and space-saving when compared to other alternatives for
electricity production, and wind energy is an unlimited source for energy pro-
duction. On the other hand, wind energy uses turbines that are quite noisy, requires
several units to produce enough energy compared to other alternatives, has low
reliability and consistency due to the wind nature, brings visual pollution, and it is
still costly to install.
Table 19.3 presents the worldwide current installed wind power capacity,
(282,482 MW), for which less than 2 % are offshore installation.
In Brazil, the use of wind power for electricity started in 1992 with the
installation of a small wind turbine in Fernando de Noronha (PE), followed 2 years
418 L. C. Oliveira-Lopes and C. H. F. da Silva

Table 19.3 Installed wind Rank Country Capacity (MW)

power capacity (MW) in
2012 (GWEC 2013) 1 China 75,564
2 U.S. 60,007
3 Germany 31,332
4 Spain 22,796
5 India 19,564
6 U.K. 8,445
7 Italy 8,144
8 France 7,196
9 Canada 6,200
10 Portugal 4,525
11 Denmark 4,162
12 Sweden 3,745
13 Japan 2,614
14 Australia 2,584
15 Brazil 2,508
16 Poland 2,497
17 Netherlands 2,391
18 Turkey 2,312
19 Romania 1,905
World total 282,482

later by Morro do Camelinho, installed in 1994 in the city of Gouveia—MG.

Brazil wind energy potential is shown in Fig. 19.3, where it can be seen that the
estimated potential for wind energy in Brazil based on the annual mean wind speed
at 70 m above ground level is 143.5 GW (272.2 TW h/yr). However, it was only
with the PROINFA that the wind energy production presented an important
growth. Figure 19.4 shows the historical flowchart for wind energy in Brazil
(MME 2012).
Currently wind energy in Brazil accounts for 2 % of national electricity con-
sumption. In 2012 alone, 40 new wind farms came online, adding more than 1 GW
of new capacity to the Brazilian electricity grid.
Table 19.4 presents some of the most important wind power plant in Brazil.
Most of the plants are located in Rio Grande do Sul, Bahia, Rio Grande do Norte,
and Ceará. Last year, the Alto Sertão-I Wind Complex (with 294 MW installed
wind capacity) and 14 wind farms, was inaugurated, and the Alto sertão-II Wind
Complex (with 386 MW) is currently under construction.
Despite the wind energy growth, Brazil lacks a great deal of investment in
infrastructure to connect all those planned/under construction wind farms to the
Brazilian network of energy. According the Brazil’s National Electric Energy
Agency (ANEEL) there are 197 granted wind-based enterprise (5,253,425 kW), 93
under construction (2,346,866 kW) and 96 operating units (2,109,341 kW). Those
numbers do not contain the information of those units that were constructed, but
still not integrated in the Brazilian distribution system.
19 Nonconventional Renewable Sources 419

Fig. 19.3 Potential for wind energy in Brazil based on the annual mean wind speed at 70 m
above ground level (MME 2012)

Fig. 19.4 Brazil installed wind power capacity (2006–2012) (MME 2012)
420 L. C. Oliveira-Lopes and C. H. F. da Silva

Table 19.4 Largest wind power plants in Brazil by installed capacity

Wind power complex Installed capacity (kW) Local Status
Alto Sertão-I 294,000 Southwest—BA OPI-2012
Alto Sertão-II 386,000 Southwest—BA OPC
Alegria complex 151,650 Guamaré—RN OPP-2011
Praia formosa 105,000 Camocim—CE OPF-2009
Aracati complex 138,500 Aracati—CE OPF-2008
Osório complex 150,000 Osório—RS OPF-2007
Icaraízinho 65,100 Amontada—CE OPC
Elebrás Cidreira 1 70,000 Tramandaí—RS OPF-2011
Vale dos Ventos 48,000 Mataraca—PB OPF-2009
OPI lacks infrastructure for full operation, OPC planned or under construction, OPP partially
operating, OPF fully operational (ANEEL 2013)

19.2.2 Solar Energy

Solar energy refers to the use of the energy from the sun for practical use. It is
known that the Earth receives 174 petawatts of solar radiation at the upper
atmospheres (IPHE 2011), reflecting back around 30 % of it to space. Solar
radiation is spread around the world, but strongly depends on the distance from the
equator.
Solar power is the conversion of sunlight into electricity either directly using
photovoltaics (PV) or indirectly using concentrated solar power (CSP). CSP sys-
tems use lenses or mirrors and tracking systems to focus a large area of sunlight
into a small beam. PV converts light into electric current using the photoelectric
effect.
Brazil has a large majority of its land in the tropics, therefore, the estimated
solar incidence for Brazil ranks among the highest in the world. Solar insolation
levels are relatively high and promising across the country.
The need for high quality information on solar data for Brazil was addressed by
SWERA (Solar and Wind Energy Resource Assessment) Project. SWERA Project
was supported by The United Nations Environment Program (UNEP) and the
Global Environmental Facility (GEF) (Martins et al. 2008) and in Brazil the pro-
ject was coordinated by Centre for Weather Forecast and Climate Studies of the
Brazilian Institute for Space Research (CPTEC/INPE). The solar irradiation data
provided by SWERA was based on the model BRASIL-SR developed by CPTEC/
INPE and LABSOLAR/UFSC (Pereira et al. 2000).
According to the SWERA data, (prepared by using BRASIL-SR radiative
transfer model and satellite database acquired from 1995 to 2005 with a spatial
resolution of 10 km 9 10 km) the maximum daily global solar irradiation value
(6.5 kW h/m2) occurs in the semi-arid climate area of Brazilian Northeastern
region. The lowest daily global solar irradiation (4.25 kW h/m2) occurs on the
shore of Southern region of Brazil. Those values are much greater than those for
the majority of the European countries. BRASIL-SR model was validated through
19 Nonconventional Renewable Sources 421

Fig. 19.5 Annual mean daily

solar irradiation (kW h/m2)
(IPHE 2011)

a comparison with measured values at the ground stations spread throughout the
country with ground data collected by the SONDA (National Organization of
Environmental Data System) network stations and by automatic weather stations
(AWS). Figure 19.5 shows the annual daily solar irradiation for several countries
and Figs. 19.6 and 19.7 show the annual average of daily total global solar irra-
diation and the daily solar irradiation per region of the country, respectively.
PROCEL (National Program for Electricity Conservation) estimates that there
are more than 30 million electric showers installed in Brazil, consuming about 6 %
of all electricity produced in the country, accounting for approximately 18 % of
the peak demand of the national electric system. This means that to heat water,
Brazil is consuming too much resource. This fact indicates that solar thermal can
be a smart move for Brazil’s demand of water heating systems (Martins and
Pereira 2011).
Solar heating is a case of success in all sectors of the Brazilian economy:
residential, commercial, and industrial. Belo Horizonte, capital of the state of
Minas Gerais in 2010 had 1.87 million square meters of solar panels (Companhia
Energética de Minas Gerais 2012), from 1991 to 2010, saving the total of 861,000
TEP (Ton Equivalent Petrol).
Besides using solar energy for water heating, there are a few power plants in
Brazil for electricity production. According to ANEEL, the installed capacity of
photovoltaic power plants is about 7.5 MW (ANEEL). The SER (Sistema de
Energia Renovável) announced plans to build a total of 600 MW of solar power
capacity in Brazil by 2020. Its first project, a 5 MW capacity solar power
422 L. C. Oliveira-Lopes and C. H. F. da Silva

Fig. 19.6 Annual average of daily total global solar irradiation (Pereira et al. 2006)

Fig. 19.7 Daily solar

irradiation in Brazil for each
region (data from Pereira
et al. 2006)
19 Nonconventional Renewable Sources 423

Fig. 19.8 Worldwide

cumulative installed
photovoltaic (PV) Power
2012 (IEA 2012b)

installation, is currently being built. Because domestic solar PV manufacturing

units are in its infancy, the need to import all the components of solar PV systems
adds to the solar energy cost in Brazil. That is likely to change and it is expected
that the new solar power capacity will be competitive as compared to other
electricity sources within 6 years. Figure 19.8 shows the share of all electricity
production from photovoltaic power plants in the world.
The Brazilian’s most important solar power plants (operating or under con-
struction) are: Tauá power plant (SP), with 1 MWp of installed capacity, Tanquinho
(SP) with 1 MWp, Alto do Rodrigues (RN), with 1.1 MW, Itajobi (SP), with 3
MWp, several sport arena (i.e., Mineirão (MG) with 1.5 MWp, Marcanã (RJ), with
3.3 MWp), and Pituaçu (BA) with 404,80 kW. According the Brazil’s ANEEL,
there are just 19 granted solar-based enterprise (6,637 kW) already in operation.
There are many planned CSP plants planned or under construction: Araçuai (MG)
(20 MW), Barra (BA) I and II (60 MW), Belém do São Francisco (PE) I and II
(60 MW), Ceará (CE) I and II (60 MW), Jaguaretama (CE) I and II (60 MW), Dr.
Miguel Arraes (PE) I and II (60 MW), Piauí (PI) I and II (60 MW), Remanso (BA) I
to VIII (240 MW), Sousa (PB) I and II (60 MW), and Xique–Xique (BA) I to VIII
(240 MW). According to the Empresa de Pesquisa Energética (EPE), the energy
plan 2013-2022 plans a solar energy production of 1.400 MW by 2022.

19.2.3 Biohydrogen

About 45 billion kg hydrogen is produced annually (IPHE 2011), but most of it is

used for industrial purposes. The dominant technology for hydrogen production is
steam reforming from hydrocarbons (Fig. 19.9). However, there are many other
production routes including electrolysis, thermolysis, and renewable energy
technologies, such as wind, solar, geothermal, and hydroelectric power. Hydrogen
production is an industry over a hundred billion dollars. Most of the produced
hydrogen is used in oil refining, ammonia, and methanol production.
Biological hydrogen, known as biohydrogen, can be produced through direct
biophotolysis, indirect biophotolysis, photofermentation, and dark fermentation
(considered the rote with greatest potential by many). Table 19.5 presents the most
promising methods and technologies for hydrogen production from renewable
sources.
In Brazil, biohydrogen production has attracted great interest mainly due to the
possibility of using renewable energy sources, as well as the reuse of waste
424 L. C. Oliveira-Lopes and C. H. F. da Silva

Fig. 19.9 Sources of current

worldwide hydrogen
production (IPHE 2011)

Table 19.5 Methods for hydrogen production from renewable sources (IPHE 2011)
Energy source Technology Process Prospect
Wind, solar, geothermal, hydro Renewable electrolysis Electrolytic Near-, Mid- and Long-
term
Biomass Gaseification Thermal Mid-term
Biomass: ethanol, bio-oil Reforming Mid- to Long-term
CSP High temperature water Long-term
splitting
Microorganisms: algae, Photobiological water Photolytic Long-term
cyanobacteria splitting
Semiconductors Photochemical water Long-term
splitting

materials (lignocelluloses or starch materials, glycerol, palm oil mill effluent, food
and dairy wastes, paper mill wastes, among others). In a complementary pro-
duction unit, one can exploit residual streams from first-generation ethanol, sec-
ond-generation ethanol, and biodiesel production. A wide range of feedstock could
be utilized to produce glycerol via transesterification of oils and greases. The
Brazilian annual potential of electricity generation utilizing residues is
318.58 GWh with overall installed potential of 63.72 MW. The potential of
electricity generation of other feedstock (castor bean, peanut, and soybean) is
1587 GWh/year with installed potential of 317.49 MW (Souza and Silveira 2011).
Additionally, utilizing hydrogen produced through steam reforming of glycerol,
high amounts of H-BIO, and biopropane could be produced. Electricity can be
produced in a fuel cell which combines hydrogen and oxygen to produce elec-
tricity, heat, and water.
19 Nonconventional Renewable Sources 425

An intense research effort has been carried out in the entire world to improve the
use of hydrogen as an energy source and also as a carrier of energy. The Brazilian
Ministry of Science, Technology, and Innovation has promoted the development of
initiatives for the hydrogen economy through programs such as ProCaC—Brazilian
Program for Fuel Cells (Centro de Gestão e Estudos Estratégicos 2002) and
ProH2—Brazilian Program of Science, Technology, and Innovation for the
Hydrogen Economy (Centro de Gestão e Estudos Estratégicos 2005, 2010). The
main reasons for the country to develop hydrogen technology are: the availability of
biomass, biogas, and ethanol can put the country on strategic condition for pro-
ducing renewable hydrogen and participate in markets for capital goods and ser-
vices associated with hydrogen.
The Brazilian path to the hydrogen economy is still undefined and many actions
for their development (Centro de Gestão e Estudos Estratégicos 2005, 2010) are
required: (a) creating a market for hydrogen energy from production to con-
sumption; (b) definition of logistics and required associated developments; (c)
implementation of pilot projects, with research and development integrated with
collaborative action for exploiting hydrogen energy; and (d) dissemination of
hydrogen technology.

19.2.4 Biogas

Sometimes referred to as biomethane, waste gas, or renewable gas, biogas refers to

a gas produced by anaerobic digestion with bacteria or fermentation of biode-
gradable materials and it comprises primarily methane (CH4) (50–75 %), carbon
dioxide (CO2) (30–40 %), nitrogen (N2), and may have small amounts of hydrogen
sulfide (H2S), moisture, and siloxanes. Biogas technology (biomethanation), the
generation of a combustible gas from anaerobic biomass digestion is a well-known
technology.
A biogas plant yield depends not only on the type of feedstock, but also on the
plant design, fermentation temperature, and retention time. The most common
form of biogas use is to produce combined heat and power (CHP), including
internal combustion engines, combustion gas turbines, microturbines, fuel cells,
and steam turbines. The largest and most widespread barriers to biogas use are
because they are economic, related to higher priority demands on limited capital
resources or to perceptions that the economics do not justify the investment. There
is also other uses of biogas, non-CHP applications, like injection of purified biogas
into natural gas pipelines and as a vehicle fuel.
Germany is the largest biogas producer in the world. German biogas electricity
production represented 14 % of total renewable electricity production in 2011.
Germany biogas power generation is forecast to increase from 18,244 Gigawatt
hours (GWh) in 2012 to 28,265 GWh in 2025. By comparison, the US, the second
most productive biogas power producer, is expected to increase generation from a
more modest 2012 figure of 9,072–20,936 GWh in 2025. The impressive growth
426 L. C. Oliveira-Lopes and C. H. F. da Silva

and current scale of the German biogas industry provides many important insights
for other countries that are looking to expand biogas resources (Bilek 2011).
There are small- and medium-sized biogas power plants in Brazil, mostly
installed in agroindustrial settings. There also exists biogas production in Brazil
from landfills. The main purpose of these plants (based on waste of animal pro-
duction facility or municipal solid waste) is sanitation and environmental pro-
tection, but also producing gas and electricity. In Brazil, in an energetic context
only, electricity price is not high enough to guarantee a biogas power plant
profitable operation without government incentives.
The growing demand for waste treatment processes and an increased focus on
greenhouse gas mitigation are generating demand on a worldwide basis. According
to the Atlas of GHG Emission and Energy Potential (2013), Brazil produced
approximately 198,000 tons of municipal solid waste (MSW) per day in 2011, of
which 90 % of the total waste produced was collected and of this, 58 % was
disposed of in sanitary landfills, 24 % went to controlled landfills, and 17 % to
dumpsites. Disposal sites have potential to develop greenhouse gas (GHG) miti-
gation projects, as the final product of decomposition of solid waste under confined
oxygen-free conditions is biogas. Of the mitigation projects in Brazil, 50 %
consider of capture/flaring of recovered biogas, and the other half consider the
energy utilization of biogas. The overall installed capacity stated for electricity
generation in the verified Project Design Documents (PDDs) of these projects is
254 MW. The potential of electricity production from biogas from MSW in Brazil
is of 282 MW, with the regional share shown in Fig. 19.10.
From an industrial point of view, a great opportunity in Brazil for Biogas
production from waste refers to the digestion of vinasse, a byproduct of the ethanol
industry, once for each liter of ethanol produced it is also produced 13 liters of
vinasse, there is a significant potential energy to be exploited. The stillage is
currently used in fertigation, but its transformation into biogas constitutes an
economic and environmental benefit. Furthermore, the current implementation
requires appropriate monitoring, since the indiscriminate use of vinasse in ferti-
gation can lead to acidification and leaching processes, with impact on soil pro-
ductivity and contamination of groundwater.
From a commercial livestock industry point of view, Brazil has the potential for
exploiting biogas from animal manure. Today there is a growing interest in biogas
production and utilization, mainly in the states of Mato Grosso, Minas Gerais,
Goiás, Paraná, Santa Catarina, and Rio Grande do Sul, where livestock breeding is
predominant. Biogas is most commonly used on the farm where it is produced,
mostly for electricity.
The Brazilian National Policy on Solid Waste (known by its acronym in Por-
tuguese, PNRS), which was passed on August 2, 2010, provides the key regulatory
framework for the waste sector in the country, and shall hopefully have a positive
impact on mitigation and use of produced biogas.
The use of microalgae has gained attention in Brazil and is presented as a
promising technology for the production of biomass for energy purposes, and may
19 Nonconventional Renewable Sources 427

Fig. 19.10 Brazil biogas-to-

electricity generation
potential (APRELPE 2013)

have application in digestion and consequent production of biogas. Moreover, such

systems may be implemented using exhaust gas heat systems allowing the capture
of carbon dioxide, which in this case happens to be an input to the process of
producing microalgae. The use of microalgae associated with genetics also opens
up the possibility of development of liquid fuel in the form of alcohols and oils.

19.2.5 Biodiesel

The Federal Law No. 11,907 of 2005 defines biodiesel as a new fuel in Brazil’s
energy mix, and since 2008 2 % biodiesel component blended to 98 % diesel oil,
known as B2. In the beginning of 2010, the mix requirement increased to 5 %
(B5), 3 years ahead of the target established by law. There is the possibility for
higher blend percentages up to pure biodiesel (B100) by authorization of the
Brazilian Petroleum, Gas and Biofuels Regulator (ANP), which has regulatory and
fiscal control.
In 2011, the amount of B100 produced in Brazil reached 2,672,760 m3, against
2,386,399 m3 in the previous year. Thus, there was an increase of 12.0 % in
biodiesel available in the national market. Table 19.6 presents the installed
capacity for B100 production in Brazil.
Additionally, in 2011, the percentage of B100 compulsorily added to mineral
diesel remained constant at 5 %. The main raw material was the soybean oil
(81.2 %), followed by tallow (13.1 %). Since the launch of the National Biodiesel
Production and Use Program in December 2004, up to the end of 2011, Brazil
avoided importing 7.9 billion liters of diesel, equivalent to a gain of about US$5.2
billion in the Brazilian trade balance. Nowadays, biodiesel blend is sold in more
than 30,000 service stations around the country. Figure 19.11 shows the effect of
the government action on the biodiesel production in Brazil. According to the
(EPE), if the use of biodiesel in Brazil is kept at the 5 % (B5), the total production
capacity in the country is guaranteed until 2019.
428 L. C. Oliveira-Lopes and C. H. F. da Silva

Table 19.6 Installed Producer Location/State Installed capacity (m3/day)

capacity of B100 production
in 2012 (ANP 2013) ADM Rondonópolis/MT 1,352.0
Agrenco Alto Araguaia/MT 660.0
Bianchini Canoas/RS 900.0
Bionasa Porangatu/GO 653.0
Camera Ijuí/RS 650.0
Caramuru São Simão/GO 625.0
Caramuru Ipameri/GO 625.0
Cargill Três Lagoas/MS 700.0
Granol Cachoeira do Sul/RS 933.3
Granol Anápolis/GO 1,033.0
Olfar Erechim/RS 600.0
Oleoplan Veranópolis/RS 1,050.0
Petrobras Candeias/BA 603.4
Others 10,183.1
Total 20,567.8

Fig. 19.11 Production of

biodiesel (B100) in Brazil
(data from ANP 2013)

There is also biodiesel produced from algae. However, although it allows a high
yield per acre, current technology and the scale of production make the average
cost per liter of biodiesel from algae of 5–10 times greater than the biodiesel plants
such as soy, peanut or sunflower. Nevertheless, Brazil is planning to start the
world’s first algae-based biodiesel plant. The planned capacity is 1.2 million liters
each year. The unit is yet to be approved by Brazil’s National Petroleum Agency
(ANP), but if approved it will be located in the Northeastern Brazilian state of
Pernambuco (PE) and the facility will utilize carbon emissions from an ethanol
producer unit from sugarcane to speed up the photosynthesis process in the sea-
weeds and reduce emissions of pollutant gases.
Figure 19.12 shows an outlook for B100 production in Brazil kept the same
legislation and addition level in the country.
19 Nonconventional Renewable Sources 429

Fig. 19.12 Agricultural

outlook 2013–2022 for
biodiesel production in Brazil
(OECD-FAO 2013)

19.2.6 Other Sources (Biobutanol, Geothermal

and Ocean Energy)

19.2.6.1 Biobutanol

Biobutanol, or biogasoline, is an alcohol that is produced from biomass feedstock,

by a thermochemical route with biomass gaseification or by fast pyrolysis with a
bio-oil steam reform. Biobutanol can be utilized in internal combustion engines
both as a gasoline additive and or a fuel blend with gasoline.
As an automotive fuel, butanol is recognized by its ‘‘drop-in’’ characteristics
and is a superior blend stock that can be blended with gasoline, diesel, biodiesel,
and ethanol (Mariano et al. 2013).
The energy content of biobutanol is 10 % less than that of regular gasoline, but
it can be integrated into regular internal combustion engines easier than ethanol.
Biobutanol has the potential to reduce the carbon emissions by 85 % when
compared to gasoline (Dürre 2007).
Biobutanol is made via fermentation of biomasses and the difference from eth-
anol production is primarily in the fermentation of the feedstock and minor changes
in the separation (Kumar and Gayen 2011). Perhaps the main bottlenecks of bio-
butanol production are: (a) low final biobutanol concentration caused by butanol
toxicity; (b) many by-products, and (c) low biobutanol productivity. However,
efforts are currently underway to improve the existing microorganisms used for
fermentation and the separation costs of biobutanol from fermentation broth.
Through a mixture of genetic engineering and new separation technology (based on
membranes for instance), leads to thinks that biobutanol has a promising future in
the biofuels market.
A promising development in biobutanol production technology has been
recently discovered, and this can impact the biobutanol production in the next
years.
430 L. C. Oliveira-Lopes and C. H. F. da Silva

A possible trend is the purchases of ethanol fermentation plants by biobutanol

companies. These ethanol plants can be retrofitted with advanced separation sys-
tems to allow them to produce biobutanol; since biobutanol has inherently higher
value than bioethanol this trend can change the biobutanol market in a small period
of time. In this context, it is possible to see the formation of biorefinery (Vaz Jr
2011) that is able to integrate biomass conversion processes and equipment to
produce fuels, power, heat, and chemicals with greater value from biomass. In
Brazil, HC Sucroquímica is an example of a sugarcane biorefinery that is currently
producing biobutanol (Mariano et al. 2013).

19.2.6.2 Geothermal

Geothermal energy is thermal energy generated and stored in the Earth. The main
sources for geothermal energy are the heat flow from the earth’s core and mantle
(*40 %), and that generated by the gradual decay of radioactive isotopes in the
earth’s continental crust (*60 %) (IEA 2012a). Bertan (2012) presents the total
installed capacity from worldwide geothermal power plant.
In Brazil, the total capacity of low temperature geothermal systems in use is
estimated at 362 MWt and the annual energy use at 6536 TJ. About a dozen of the
spring systems account for the bulk of this capacity. It is known that regions of very
high geothermal gradients are absent in Brazil. This is a natural consequence of the
fact that there are no areas of young volcanism or active tectonics. The best sites for
extraction of geothermal energy in Brazil are the younger sedimentary basins, which
makes the Paraná Basin a suitable place to tap geothermal energy. Furthermore, most
of the major springs are located in central Brazil (in the state of Goiás) and in the
south (in the state of Santa Catarina). The potential for large-scale exploitation of low
temperature geothermal water for industrial use and space heating may be consid-
ered in southern and southeastern parts of Brazil, but at this date the geothermal
exploitation is quite small and investments needed to convert this energy into
electricity would appear to be too expensive at this time. However, other applications
can be found like residential and commercial systems utilizing hot water.

19.2.6.3 Ocean Energy

Ocean energy can be defined as energy derived from technologies that utilize
seawater as their motive power or harness the water’s chemical or heat potential.
The sources are: (a) wave; (b) tidal range; (c) tidal current; (d) ocean current; (e)
thermal gradient; and (f) salinity gradient. The worldwide resource of wave energy
has been estimated to be greater than 2 TW (Cruz et al. 2008).
According to a workshop leaded by Segen Estefen from Alberto Luiz Coimbra
Institute and published by IEA, the total wave energy potential for Brazil is around
122 GW. The southeast of the country possesses circa of 50 GW of wave energy
potential capacity along its coast, equivalent to three times the capacity of Itaipu
19 Nonconventional Renewable Sources 431

Dam. In Brazil, except for the project in the Port of Pecém (CE), with an installed
capacity of 500 kW, the ocean energy potential has not been exploited.
The technologies for the exploitation of the energy potential of the oceans are
the least mature. Due to the small number of prototypes in operation, there is still
no relevant data on the costs, environmental impacts, strengths, and difficulties of
integration with the distribution systems. This is because, with the exception of
tidal technology with the use of dams, which has some commercial units operating
in the world, all other developments are in precommercial prototype stage.

19.3 Nonconventional Renewable Energy Assessment

Certain aspects need to be considered to assess nonconventional renewable energy

production and their potential use in Brazil. The very first point is the fact that the
country has indicated that it is committed to maintaining a large share of renewable
source in its energy matrix, as shown by the Alternative Sources Incentive Program
(PROINFA). Second, Brazil posses a great variety of natural resources and much of
this potential are still to be better exploited. Additionally, as the economy growth
affects the demand for energy. Furthermore, to examine the competitiveness of
renewable sources advantages such as: GHG emission avoidance, energy cost, job
creation, required public investment, technology know-how, and the country
potential for using it in a sustainable way should be considered (Estefen 2012).
Brazil has an electricity source mostly based on hydroelectric power, however,
a mere 30 % of its potential are exploited, but the remainder potential is mainly
located in the environmentally sensitive Amazon region, far from where energy it
required the most. Additionally, drought greatly affects the country capacity for
producing energy.
There are isolated villages and are difficult to access in the national grid, where
small hydropower through simple domestic applications would be very promising
to develop. According to the Electricity Regulatory Agency (ANEEL) data of
2011, there were 397 SHPs (Small hydroelectric plants) in operation in Brazil
(3500 MW), and a growth rate of 10 % per annum with the total potential of
around 25.9 GW. Therefore, SHPs are one of the main priorities of the ANEEL
and they are ideally suited to meeting the energy demands of small urban centers
and rural areas.
Ethanol production is expected to grow in the next decade (RFA 2013).
However, the main issue with using sugarcane as a biofuel is the rise in price,
which can directly affect consumers. That is why the country must also focus on
second-generation biofuel (from non-food staples) so as to avoid this issue.
The use of wind energy will likely be consolidated to achieve as much success
as ethanol and hydroelectricity for energy production in Brazil. Wind power
requires high initial investments but less important operational costs. The strategy
for structuring the wind energy market through public auctions ensured the
432 L. C. Oliveira-Lopes and C. H. F. da Silva

participation of the local content, encouraged domestic industry, and drove a

reduction by 60 % in the price of wind energy after the PROINFA auction in 2011.
Nowadays, there are at least a dozen manufacturers producing wind turbines in
Brazil making Brazil self-sufficient regarding wind turbines and wind farms
equipments. Nevertheless, there exist certain difficulties in the consolidation pro-
cess of wind energy (Silva et al. 2013). To answer to those points, ANEEL proposed
a Strategic R&D Call No. 17/2013 (ANEEL 2013), whose main objective is to
foster the development of domestic technology in the area of wind energy, seeking
to reduce its dependence on technology of the country and to stimulate the pro-
duction of innovative, high value-added technology adapted to the Brazilian reality.
Solar energy for water heating is by far the most widespread application of solar
energy in Brazil, where solar heating industry is well developed and able to supply
the country market for this resource. An interesting factor impacting the use of
solar energy is that it allows a greater production when there is greater energy
demand (Summer time and midday air-conditioning energy requirements). In
addition, solar power also allows the implementation of mini-grids local operated.
Unfortunately, unlike wind energy, solar energy is not a case of success in
PROINFA results. The photovoltaic market in Brazil represents a promising
market and is expected to grow quite fast in the coming years. Rural electrification
is one of the underserved markets that can benefit from this technology.
Solar PV has also been the subject of intense discussions. In 2011, ANEEL
through the R&D Strategic Call No. 13/2011 released the hiring of 18 projects,
which aims at the development of technical arrangements and commercial power
generation through solar photovoltaic technology, in an integrated and sustainable
way. The action seeks to create conditions for the development of technological
and technical infrastructure and technology for insertion of solar photovoltaic
generation in the national energy matrix (Viana et al. 2011). Hopefully, the results
of these pilot projects contribute to demonstrate the technical and economic via-
bility of solar photovoltaic generation of electricity in the country. As the
implementation period is 36 months, the results of the implementation of these
projects will be available in the next couple of years.
The development of technologies for the production and use of biohydrogen has
limitations mainly due to the low price competing energy. The great contribution
of these programs was to structure research groups and laboratories with human
resources to deal with the technologies associated with biohydrogen in Brazil and
the next decade. Some important initiatives are linked to demo projects of city bus
powered by biohydrogen, which are being developed in the cities of Rio de Janeiro
and São Paulo. Another important step was the normalization by a Special
Committee of Studies (EEC) for biohydrogen.
Biohydrogen production is subject to limitations of low conversion efficiencies.
Furthermore, there are certain technological bottlenecks associated with the bio-
hydrogen economy: (a) the price for hydrogen is established for its use as a
chemical feedstock and this fact does not provide economic viability for energy
use in the economy of today; (b) there are several ways to produce hydrogen, but
with exception of reforming or water electrolysis, all other approaches require
19 Nonconventional Renewable Sources 433

further developments for a wide and advantageous use as a energy source; and (c)
hydrogen use in fuel cells, still requires the development for durability, robustness
and prices compatibles with competing technologies. Therefore, it is still necessary
to compensate many aspects in order to get biohydrogen production economically
feasible. In this scenario, the feasibility of the widespread use of hydrogen is
restricted to certain market niches, such as backup power systems.
In the biogas context, the ANEEL has launched a R&D Call focusing on biogas
(National Agency of Electric Energy 2012). The goal is to define a model of biogas
system applicable to the sanitation sector, producing biogas in sewage treatment
plants and the organic fraction of solid waste. Hopefully, the results of this call for
strategic project contribute to demonstration and improvement of technical and
economic feasibility of generating electricity from biogas derived from waste/
wastewater in the country (MMA 2011). As the implementation period of this
project is 36 months, the expectation of concrete results will occur around 2016.
The National Program for Production and Use of Biodiesel (PNPB) completed
8 years (Pousa et al. 2007). In this short period of time, the program was able to
induce the formation of an industrial park able to meet a demand of about two and
a half billion gallons of biodiesel. Nowadays, the dependence of soybeans and the
difficulties in promoting the social inclusion of family farmers represent major
PNPB challenges.
In all other energy sources covered in this text, there is not a structural
framework and legislation in order to produce a competitive energy. Most of the
initiative are from private groups (as in the case of biobutanol) or research pro-
totype based mostly on universities.
Finally, regional organizations are fostering development of regional energy
trade. Latin America countries could have great mutual benefits in collaborating in
energy production and distribution, but many actions will need to be performed to
make true that scenario.

19.4 Conclusions

Over the last two decades, energy policy in Brazil has sought to reduce the country’s
dependence on foreign energy supply and stimulate the development of domestic
energy sources. However, it had left behind a huge unexploited potential of
renewable energy sources. The effectiveness of success of the Brazilian renewable
energy market strongly depends on legislation and country policies. The growth will
be as fast as the country implement energy policy to support the renewable sources
of energy by breaking economic, regulatory, or institutional barriers. Therefore, the
impact of the nonconventional renewable energy in the success of bioenergy
depends highly on the policy maker initiatives on seeking a variety of renewable
energy sources incorporated into the energy matrix of the country.
The renewable energy future looks very promising in Brazil. However, it is
necessary to develop national technology that requires skilled manpower and
434 L. C. Oliveira-Lopes and C. H. F. da Silva

scientific development, which depends on education and government policies.

Many technologies are already a reality in other parts of the world. However, the
national development of a given technology favors sustainable solutions and local
application.
Country leadership has to be quite alert in order to guarantee the national
development of energy routes that might not be a competitive one in today’s
energy market, but can be promising under different scenarios or with improved
technological solution.

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