3.30 am - 5.

00 pm 22-09-2020

LECTURE-3

FERMENTATION PROCESSES
 Microbial fermentation is the only method for commercial
production of certain products that are made in substantial
quantities.
 Table-1 of Lecture-1 compiles the production figures for a number
of established fermentation products.
 The antibiotics market alone exceeds US$30 billion and includes
about 160 antibiotics and derivatives.
 Other important pharmaceutical products produced by
microorganisms are cholesterol lowering agents or statins, enzyme
inhibitors, immunosuppressants and antitumor compounds.
 The world market for statins is about US$15 billion.
 The total pharmaceutical market is well in excess of US$400 billion
and continues to grow faster than the average economy.
 Biotechnology processes are involved in making many of these
drugs.
 Novel fermentation production methods for established drugs and
drug precursors are being developed continually.
 One example is the production of cholesterol lowering drug
lovastatin that is also used for producing other semisynthetic statins.
 Various novel bioprocess intensification strategies are being put to
use to substantially enhance productivities and efficiencies of
numerous bioprocesses
 Vitamins are still mainly produced using organic chemistry, but
biotechnology is making increasing contribution to vitamin
production.

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 For example, DSM Nutritional Products replaced the company’s

 The global market for biopharmaceuticals already exceeds US$40
billion, having grown by more than 3-fold compared to only 4 years
ago (Melmer, 2005).
 Market size of selected biopharmaceuticals is shown in Table-2.
Table-2: Market size (2001) of selected biopharmaceuticals (Melmer, 2005)

ENZYMATIC PROCESSES
 Enzymes are increasingly penetrating the chemical industry as
catalysts for numerous reactions.
 The global market of enzymes is estimated at around US$1.5 billion
and is expected to grow by 5–10% annually.
 Table-3 lists major types of industrial enzymes, their substrates and
reactions they catalyze.

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 Millions of years of evolution has provided enzymes with

unparalleled capabilities of facilitating life reactions in ways that are
sustainable.
 Compared with conventional chemical catalysts, enzyme catalysis is
highly specific and it functions under temperatures, pressures and
pHs that are compatible with life.
Table-3: Some Industrial Enzymes and their Applications

 Semisynthetic penicillin and cephalosporin antibiotics derived from

6-aminopenicillanic acid (6-APA) and 7-aminocephalosporanic acid
(7-ACA), respectively, are produced using enzymatic processes.
 Production of numerous other pharmaceuticals relies on enzymatic
biotransformations.
 Cephalexin is a semisynthetic antibiotic derived from cephalosporin
C. DSM Company (www.dsm.com),the Netherlands, is a major
producer of cephalexin.

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 The conventional chemical production of this compound required

up to 10 steps.
 The conventional process generated up to 50 kg of waste per kg of
antibiotic (Table-4) but this was reduced to about 15 kg with
extensive developmental effort.
 Subsequently, a four-step enzymatic process was developed that
further reduced waste and consumption of most resources.
 A direct fermentation rout is now available and this is even better
than the enzymatic route.

 The various processes for production of cephalexin are compared in

Table -4.

Table -4: Comparison of Chemical and Biotechnology Processes for

Producing Cephalexin (Demain, 2000)

 Table-5 compares the conventional chemical and biotechnology

based production of acrylamide.

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 The bioprocess requires milder conditions, achieves greater single-

pass conversion and a higher final concentration of the product in
the bioreactor. The bioprocess uses about 20% as much energy as
the conventional process and produces much less carbon dioxide
than the traditional process (Table-5).
 While enzymatic process can have definite advantages compared to
their chemical alternatives, much research is needed to make them
cost-competitive for use in the broader chemical industry.

Table-5: Chemical Versus Biotechnological Production of Acrylamide

 While enzymatic process can have definite advantages compared to

their chemical alternatives, much research is needed to make them
cost-competitive for use in the broader chemical industry.
 A report entitled New Biocatalysts: Essential Tools for a Sustainable
21st Century Chemical Industry
(www.eere.energy.gov/biomass/pdfs/biocatalysis_roadmap.pdf)
identified the following major objectives for biocatalysts for a
sustainable chemical industry:
1. developing biocatalysts that are better, faster, less expensive
and more versatile than comparable chemical catalysts;

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2. development of biocatalysts that can catalyze an increased

range of reactions, have higher temperature stability an
improved solvent compatibility;
3. developing molecular modeling and other tools to permit
rapid design of new enzyme catalysts.

PLASTICS AND OTHER POLYMERS

 Occurrence of biodegradable plastics such as polyhydroxyalkanoic
acids (PHA) in bacteria has been known since the 1920s.
 Expense of producing bioplastics and the availability of versatile
low-cost petrochemicals-derived plastics led to bioplastics being
ignored for a long time.
 Concern over persistence of petrochemical plastics in the
environment is a renewing interest in biologically derived polymers.
 The Japan Institute of Physics and Chemical Research engineered a
microorganism to produce up to 96% of its dry weight as
biodegradable plastic.
 Many diverse plastic and nonplastic biopolymers are now available.
 Even though they remain relatively expensive, their production and
use are environmentally sustainable.
 Substantial effort is underway in developing improved production
of polyhydroxyalkanoates (PHAs) and other biodegradable,
renewable, biopolymers.
 Biopolymers with enhanced properties and microbial strains for
producing them are being developed. More efficient fermentation
and product recovery processes are being investigated.
 The use of mixed cultures and inexpensive substrates can
substantially reduce the production cost of PHAs.

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 The conversion of acrylonitrile to acrylic acid for the production of

anionic polyacrylamides is an example of a large-scale
biotransformation with significant commercial and environmental
benefits.
 Ciba Specialty Chemicals (www.cibasc.com) manufactures a range
of polymers based on acrylamide and acrylic acid using biological
technologies.
 The conventional method for producing acrylic acid was a
hazardous, multistep, energy-intensive process that required high
concentrations of toxic acrylonitrile, operated at an elevated
temperature and produced hazardous emissions.
 Ciba’s biotransformation route is claimed to have the following
benefits: a simple, one-step process that is cost-
effective and provides a product of good quality; production at
ambient temperature and atmospheric pressure; low concentration
of hazardous acrylonitrile throughout manufacture; few by-
products; and near quantitative conversion.
 As another example, the Mitsubishi Rayon’s bioprocess for
producing acrylamide has already been mentioned.
 Acrylamide is then polymerized to the conventional plastic
polyacrylamide.
 In the UK, Baxenden Company (www.baxchem.co.uk)
manufactures polyesters, acrylic polymers and emulsions and other
specialty chemicals using biocatalytic processes that have reduced
the reaction temperature to 60 8C compared to 200 8C for
equivalent chemical processing.
 There are number of other examples where bioprocesses are
involved in manufacturing of biopolymers.

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COSMETICS, TOILETRIES, SOAPS AND DETERGENTS

 The cosmetics and toiletries industry has traditionally been a major
user of biologically sourced materials and fine chemicals.
 Enzymes are finding use in cosmetics; for example, laccase is used in
hairdyeing products.
 The soaps and detergents industry uses biomass-derived feedstock
and enzymes.
 Most soaps are produced from oils and fats derived from plants and
animals.
 Although biotechnology per se does not appear to be used in
processing of soaps and detergents, most washing detergents contain
enzymes.
 Lipases and proteases are added to help in removing oil and protein
stains, respectively.
 In addition, cellulases are added to help prevent pilling of cotton.
 These enzymes are increasingly produced by using genetically
modified microorganisms.
 Detergent formulations typically contain less than 1% enzyme by
volume, but the enzymes contribute about 8% to the cost of the
detergent.
 Biotechnological production of enzymes of course consumes
resources, but reduced severity of washing regimens as a result of
their use can produce overall benefits.
 Clothes laundered with enzyme-containing detergents tend to be
much cleaner compared to clothes washed with traditional
phosphate-containing detergents.
 Compared with traditional detergents, enzyme-containing
detergents may be formulated with less phosphate, to greatly reduce
the release of this eutrophication agent to the environment.

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 Enzyme-containing washing detergents are more environment-

friendly overall.
 Companies such as Henkel (www.henkel.com) have successfully
incorporated natural enzymes in detergent formulations since the
1970s.
 Genetically engineered enzymes have been added to detergents since
the late 1980s (Maurer and Kottwitz, 1999).
 For example, the development of the Bacillus lentus alkaline
protease (BLAP) is estimated to have reduced environmental
pollution associated with detergents, by more than 65%.
 BLAP-S protease is an example of a genetically modified enzyme
that is used in washing detergents. This enzyme has been produced
since 1995 and is based on the genetically modified BLAP protease.
 There are many other examples too where biotechnology is involved
directly or indirectly.

 AGRICULTURAL CHEMICALS
 Agricultural chemicals, mainly fertilizers and pesticides, are used in
massive amount worldwide to sustain the productivity of land.
 Because of their widespread use agrochemicals are an important
source of pollution, health risk, and consume large amounts of
resources in their production.
 Biotechnology can supply useful products that can replace
conventional agrochemicals, or enhance their effectiveness so that
their overall consumption is reduced.
 In addition, biotechnology can provide animal feeds with enhanced
nutritional and keeping quality, to improve the sustainability of
animal production.

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 BIOPESTICIDES
 Pesticides are used in crop protection, management of weeds,
control of insects, treatment of seeds, control of algae in
swimming pools and preservation of wood and textiles
 A biopesticide is any microscopic biological agent or product
derived from microorganisms, for use in controlling insects,
weeds and rodent pests.
 Packaging, handling, storage and methods of application of
biopesticides are similar to those for traditional pesticides.
 Biopesticides have had some spectacular successes, but there have
been concerns related to their effectiveness.
 Approximately US$160 million worth of biopesticides were sold in
2000.
 Of this, over 90% represented sales relating to Bt products.
 At present, biopesticidescapture less than 2% of the global
pesticides market but this is expected to increase significantly in
the future.
 Biopesticides generally tend to be highly target specific, do not
leave toxic residues, reduce the risk of resistance development in
the target species and produce a lesser overall impact on the
environment than conventional chemical pesticides.
 Biofungicides have been used in both the phylloplane and
rhizosphere to suppress fungal infection in plants.
 Species of Bacillus and Pseudomonas have been successfully used
as seed dressings to control certain soilborne plant diseases.
 Table-6 shows some of the commercial biopesticide products
being marketed for use against soilborne plant pathogens.

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Table-6: Some Commercial Biocontrol Products for use against Soilborne
Crop Diseases

 BIOFERTILIZERS AND SOILINOCULANTS

 Biofertilizers and inoculants are attracting attention as
inexpensive and safe alternative to chemical fertilizers that are
used to deliver nitrogen, phosphorus, potassium, sulfur and
certain other inorganic nutrients required for crop growth.
 The first generation of biological fertilizers was based on nitrogen
fixing rhizobial bacteria found naturally in the root nodules of
legumes.

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 The bacteria fix nitrogen from the air, to provide the plant with
assimilable nitrogen.
 Microbial inoculants may be used to complement conventional
fertilizers, by enhancing their absorption by plants.
 Enhanced use of biofertilizers is expected to contribute
significantly to reducing pollution, energy and resource
consumption associated with the use of conventional fertilizers.
 The US sales of biofertilizers were US$690 million in 2001 and
were expected to grow to US$1.6 billion by 2006
 Some biofertilizers and soil conditioners used currently in
agriculture are shown in Table-7.
Table-7: Biofertilizers and Soil Conditioners used in Agriculture

 FIBER, PULP AND PAPER PROCESSING

 Through biotechnology and improved silviculture, trees and other
bioresources used in papermaking can be specifically tailored to
match the properties required in cellulose fibers for different
product applications.
 This can greatly increase useful paper yield from trees, enhance
product quality and decrease requirements for energy and
chemicals used in papermaking.

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 Producing optimal fibers for papermaking through genetic

engineering is an important long-term objective that requires a
better understanding of fiber biosynthesis in plants.
 Furthermore, use of engineered microorganisms and enzymes can
displace many of the environmentally adverse practices used in pulp
processing.
 Some of these developments are discussed below:
 BIOPULPING
 Biopulping is the treatment of wood chips with lignin-
degrading fungi prior to pulping.
 Biopulping is an experimental technology that has been
researched extensively mostly as a pretreatment prior to
mechanical pulping of wood.
 Prior biopulping greatly eases subsequent mechanical and
chemical pulping by improving penetration and effectiveness
of chemicals during the cooking of wood chips for separating
the cellulose fibers from the lignin.
 Consequently, biopulping reduces the demand for energy and
chemicals, improves paper quality, and decreases the
environmental impact of pulp production.
 ENZYME-AIDED PULP, PAPER AND TEXTILE
PROCESSING
 Enzymes are already well established in processing of pulp and
paper.
 For example, enzymes are used in biobleaching of pulp to
reduce chlorine consumption; pulp dewatering and deinking;
removal of pitch; degradation of dissolved and suspended
organics in concentrated effluents of mills; and enhanced
fibrillation to give stronger paper.

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 Uptake of enzymatic processing has been driven by savings

they generate by reducing the use of chemicals and energy and
the improved quality of the product that can be attained with
there use.
 Energy savings are produced, for example, by elimination of
processing steps, their simplification and reduction of the
severity of treatment that would be required in the absence of
enzymes.
 In kraft pulping, bleaching of the pulp remains one of the most
expensive operations and a prime target for cost reduction.
 Because of the polluting potential of chlorine bleach, pulp mills
in the United States and Canada are mostly moving to using
bleaching methods that do not require elemental chlorine.
 In Canada, about 10% of bleached kraft pulp is now
manufactured with xylanase treatment to reduce the
consumption of chlorine dioxide and associated costs.
 Thermostable microbial xylanases that are free of cellulases
and active under alkaline conditions of pulping are generally
preferred for biobleaching..
 Oxidative enzymes such as laccase provide other promising
options for reducing costs in pulp mills.
 Other processing improvements have been obtained by using
lipases to control deposits of pitch; cellulases to improve rates
of dewatering of pulp; and pectinases for digesting pectins.

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 ATTAINING TOTAL WATER RECYCLING IN PAPER

MILLS
 Production of paper consumes huge amounts of water.
 Extensive research is underway in treating the wastewater
from paper mills, for total recycling.
 Pulp and paper mills in Canada are aiming for total effluent
reuse after secondary and tertiary biotreatment.
 Wastewater recycling potentially saves on the expense of
treating any freshwater entering the mill and greatly reduces
the environmental impact of effluent disposal.

 BIOTECHNOLOGY FOR PAPER RECYCLING

 Market for recycled paper is substantial, global and profitable.
 Recycled newspaper reduces input of new resources in the pulp
and paper industry.
 Recycled newspaper needs to be deinked before it can be used
to make new newsprint and white paper.
 A deinking process involving sodium hydroxide, flocculants,
dispersants and surfactants is used widely currently.
 The alkali can yellow the treated pulp and, consequently,
hydrogen peroxide is used subsequently to bleach the alkali
deinked pulp.
 In addition, alkaline deinking diminishes the strength of the
pulp fiber and the chemicals used contribute to environmental
pollution.
 An enzyme-based biotechnology alternative to chemical
deinking has been developed.

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 Enzymes facilitates dewatering of pulp and removal of
contaminants without reducing the strength of the recycled
pulp fibers.
 Speedier dewatering improves sheet formation and allows faster
processing in paper machine.
 In the enzymatic process, cellulase and hemicellulase enzymes are
mixed with the paper pulp.
 The enzymes hydrolyze some of the surface sugars on the pulp
fiber and this releases the ink particles bound to the fiber.
 Washing and draining of the pulp remove most of the ink.
 Any remaining ink is removed during a conventional flotation
step.
 Treatment with alkali is not used and this eliminates the need for
subsequent bleaching with hydrogen peroxide.
 Any residual enzymes are deactivated during drying of the paper.
 Enzymatic deinking works with old newsprint and office waste
paper.
 Unlike conventional deinking, the enzyme treatment effectively
removes laser printer and photocopier inks that are
mostly found in office wastepaper.

 BIOENERGY AND FUELS

 Biotechnology-based production of fuels continues to attract much
attention.
 Bioethanol, firewood, biogas, biodiesel and biohydrogen are
examples of biofuels.
 Except for biohydrogen, commercial or pilot experimental use of the
other biofuels is already established or emerging.

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 Although bioconversion of lignocellulosic biomass to sugars for

fermentation to ethanol has been extensively studied, it remains
intractable.
 More successful and widely used is the bioconversion of starch to
sugars for producing bioethanol.
 Similarly, fuel ethanol produced from residues of cane and beet
sugar processing has been in use for several decades.
 Anaerobic digestion of organic waste to methane is another widely
used technology.
 Modern biotechnology has already greatly impacted the traditional
production of bioethanol.
 For example, the higher yielding genetically modified corn reduces
cost of the main feedstock; the starch in gene engineered corn is
more amenable to enzymatic bioconversion to sugars, than natural
corn starch; microbial enzymes have been engineered for enhanced
stability and ability to rapidly convert starch to fermentable sugars;
microorganisms have been engineered to withstand higher levels of
toxic ethanol and achieve rapid fermentation.
 These and other future improvements will make bioethanol more
economic than it is today.
 Similar advances are being targeted for enhancing anaerobic
digestion technologies.
 Blending of gasoline with bioethanol directly reduces consumption
of fossil fuels and environmental pollution (e.g. volatile organic
compounds, nitrous oxides, benzene and particulates) associated
with combustion of unblended gasoline.
 Similarly, biodiesel is significantly less polluting than petrodiesel.

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 Conversion of biomass to energy is highly attractive.

 Although in energy terms annual land production of biomass is
about five times the global energy consumption, only 1% of
commercial energy originates from biomass at present.
 Organic waste from landfill sites and farms is converted to
combustible biogas (approximately 55% methane and 45% carbon
dioxide) through anaerobic digestion.
 Liquid hydrocarbon fuels are being produced from plant, animal
and microbial oils.

 BIOPROCESSING OF BIOMASS TO PRODUCE

INDUSTRIAL CHEMICALS
 Nearly US$24 billion worth of hydrocarbon feedstocks are used
annually in the chemical industry.
 Hydrocarbon purchases represent the major share of the industry’s
raw materials costs.
 As reserves of high-quality fossil fuels are depleted, other renewable
sources will need to be found for any hydrocarbon feedstocks that
cannot be substituted.
 These resources include renewable vegetable, animal and microbial
matter.
 A change of feedstocks from fossil hydrocarbons to plant-derived
matter will dramatically restructure chemical manufacture to
enable sustainable production.
 Local agricultural production would provide the feedstocks.

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 Local availability of feedstock, reduced energy demand for

processing, less need for waste disposal and efficient production
would mean that small production facilities located close to markets
would become economically viable, particularly for high-value
products.
 Net decreases in emissions of greenhouse gases is achieved without
compromising the current quality of life.
 In fact, until the 1930s, most bulk chemicals were produced from
biomass such as corn, potatoes, wood and plant oils by chemical and
fermentation processes.
 Modern biotechnology is greatly expanding the scope of what is
possible and the capability of traditional biomanufacturing.
 Primary resources are already providing a remarkable diversity of
industrial and consumer goods (Table-8).
Table-8: Common Products from Biomass

 ENVIRONMENTAL BIOTECHNOLOGY
 Treatment of municipal wastewater by activated sludge method was
perhaps the first major use of biotechnology in bioremediation
applications.

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 Activated sludge treatment remains a workhorse technology for
controlling pollution of aquatic environment.
 Similarly, aerobic stabilization of solid organic waste through
composting has a long history of use.
 Both these technologies have undergone considerable improvement.
 More recently, microorganisms and enzymes have been successfully
used in diverse bioremediation.
 Effective and controlled bioremoval of nitrate and phosphate
contamination from wastewater has become.
 Biotechnology is already playing a major role in maintaining a clean
environment and this role will expand substantially as methods are
developed and deployed for bioremediation of all kinds of industrial
effluents.
 Rapid and highly specific detection of numerous pollutants has
become possible by using biosensors.

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