BT5520

Biorefineries
Krithika Ravi
Course Instructor: Prof. Krithika Ravi (krithikaravi@iitm.ac.in)
TAs:

Mr. Nilesh Kumar (bt23d001@smail.iitm.ac.in)

Mr. Salem Aslam (be20b027@smail.iitm.ac.in)

Slot B: Time-table (BT003)

Mondays (9:00 to 9:50 am)
Tuesdays (8:00 to 8:50 am)
Wednesdays (12:00 noon-12:50 pm)
Fridays (11:00-11:50 am)
Tentative marking scheme: total marks 100
Quiz – 20 marks
Seminar – 10 marks
Project presentation (phase I & II) – 10+20=30 marks
Project report – 20 marks
Report peer review – 10 marks
Attendance – 10 marks
Topics to be covered
• Introduction to Biomass and Biorefinery
• Fractionation and pretreatment strategies
• Thermal/Chemical conversion processes
• Design of microorganisms as cell factories
• Microbial conversion processes

• Downstream processing for separation of biochemicals

• Case studies on the production of some biochemicals
• Process modeling and evaluation - Project
Textbooks for reference:
▪ S. Yang, H.A. El-Enshasy, N. Thongchul, 2013. Bioprocessing
Technologies in Biorefinery for Sustainable Production of Fuels,
Chemicals and Polymers, Wiley.
▪ Donald L. Klass, 2006. Biomass for Renewable Energy, Fuels, and
Chemicals, Academic Press, Elsevier.

▪ P.D. María, 2016. Industrial Biorenewables, 1st ed, Wiley.

▪ S. Yang, 2007. Bioprocessing for Value Added Products from Renewable
Resources, Elsevier.

▪ A.A. Vertes, N. Qureshi, H.P. Blaschek, H. Yukawa, 2010. Biomass to

Biofuels: Strategies for Global Industries, Wiley.
What is a biorefinery?
▪ A biorefinery is a manufacturing facility that uses biomass as
feedstock to produce fuels, power, chemicals, and materials.
▪ It is analogous to petroleum refineries, which use petroleum-based
feedstocks, mainly oil and natural gas, to produce multiple fuels,
commodity chemicals, industrial products, and commercial goods.
Biomass
▪ Biomass includes any organic matter that is available on a
renewable or recurring basis.
(A renewable resource can replenish itself at the rate it is used,
while a nonrenewable resource has a limited supply.)
▪ As biomass is renewable and abundant, it has the potential to
replace fossil fuels and petrochemicals.
Biomass-based Biorefinery
▪ The commercialization of biomass-based biorefinery is
largely dependent on the exploitation of full utilization of
biomass components by producing multiple products, thus
maximizing the value derived from feedstock while minimizing
the waste.
▪ A biorefinery might produce one or several low-volume but
high-value products, such as functional food ingredients and
pharmaceuticals, and low-value but high-volume liquid
transportation fuels, such as bioethanol and biodiesel, while
generating process heat (steam) and electricity for its own
use and perhaps enough for sale.
Why are biorefineries important?
▪ Growing demand for energy, fuel, materials, and chemicals
(growing market)
▪ Finite availability of fossil fuel resources (continued price rises)
▪ Overdependence of many countries on imported resources
(national security)
▪ Reality of climate change and the need to reduce greenhouse
gases (societal demand for eco-production)
▪ Competitiveness within the global economy
▪ Need to stimulate growth within rural economies
Classification of Biorefineries

*Nonedible oilseeds such as the perennials jatropha and pongamia are also used
*Energy Crops
*Domestic waste
Energy crops
▪ Exclusively cultivated to generate biomass for electricity, heat, and
to make biofuels on a renewable basis.
▪ These crops could be incorporated into bioenergy systems to
produce feedstock for liquid (methanol, ethanol, diesel), solid
(chips, pellets, briquettes, logs), and gaseous (biogas, hydrogen)
fuels.

▪ Biomass can be directly converted into electricity or heat through

thermochemical processes (e.g., combustion and gasification).
Energy crops
▪ Ideally, an energy crop should provide high and constant yields,
favorable energy and economic balances, low nutrient
requirements, drought resistant (for specific local conditions), high
water use efficiency, resistant to pests and diseases, minimal
changes in land use and farm machinery, and high quality of
feedstock (e.g., low ash content) with a good adaptability to
handling and storing.
Example
✓ Sugarcane, maize (Bioethanol)
✓ Rapeseed, soybean (Biodiesel)
✓ Poplar, willow, switchgrass, miscanthus, and some other
perennial grasses (Solid biofuels)
Evolution of Biorefineries
Food crops: Corn, wheat,
oil seeds, sugarcane
Lignocellulosic feedstock:
agricultural and forest
residues, municipal and
industrial waste
Algal feedstock: Microalgae,
macroalgae, cyanobacteria
Genetically modified raw
material as feedstock
First-generation
▪ 1G biofuels are produced from specific parts (usually edible) of
oil-based plants and starch and sugar crops.
▪ Initially, 1G biofuels showed a promising capability to reduce fossil
fuel combustion and lower atmospheric levels of CO2, which is
consumed by crops as they grow.
▪ However, it generates competition between food production
vs. fuels, arable lands, and biodiversity loss, in addition to being
responsible for ecological degradation.
Second generation
▪ 2G biofuels, known as “advanced biofuels,” overcome the “food
versus fuels” competition by using inedible raw materials.
▪ The net carbon (emitted–consumed) from combusting secondgeneration biofuels is
neutral.

▪ Examples: agricultural and food processing residues, manure,

used cooking oil, wood/sawdust, food waste, and energy crops.
▪ Lignocellulosic residues of crops – primary candidates due to
its abundance, wide availability throughout the world and at times
of the year, and low cost.
▪ However, the structural heterogeneity in the composition of
these residues requires more complex production processes,
making 2G biofuels not industrially profitable.
Third generation
▪ Third-generation biofuels are produced from aquatic feedstock
(usually algae).
▪ Algae are a promising alternative feedstock due to their high lipid
and carbohydrate content, increased carbon dioxide absorption,
and the possibility of cultivating in wastewater and seawater.
▪ Unproductive drylands and marginal farmlands do not compete
with food crops on arable land or in freshwater environments.
▪ However, it requires a higher initial investment for its production.

▪ The biofuel produced from algae is less stable, mainly because the
oil generated by the algae is highly unsaturated, which is more
volatile at high temperatures.
▪ Furthermore, the high water quantity is also a problem, which
requires dewatering via either centrifugation or filtration before
extracting lipids.
Fourth generation
▪ These biofuels, which are still in the developmental stage, use
bioengineered microorganisms such as microalgae, yeast,
fungi, and cyanobacteria.
▪ Genetically altered crops are used to consume more CO2 from
the environment than they emit.
First generation Biorefineries
Production quantity of major crops
Biorefineries using corn, soybeans, and
sugarcane (whole-crop biorefinery)
▪ As the oil price continued to rise in the last few decades, traditional
agricultural crops have been increasingly used to produce fuel
ethanol and biodiesel.
▪ Current commercial biorefineries are using traditional sugar- and
starch-based feedstocks such as corn, soybeans, and sugarcane
to produce value-added products for food and feed applications,
and fuel ethanol and specialty chemicals.
Biorefineries using corn, soybeans, and
sugarcane (whole-crop biorefinery)
▪ Traditional agricultural processing companies (e.g., Cargill, ADM,
Tate & Lyle) are gradually transforming into a fully integrated
biorefineries with more fuels and chemicals, often partnering with
large chemical (e.g., DuPont, Dow Chemical) and oil companies
(e.g., Shell, British Petroleum).
▪ Almost all of the current biofuels (mainly ethanol, butanol, and
biodiesel) and bio-based chemicals (lactic acid, itaconic acid, 1,3propanediol
[1,3-PDO], etc.) are produced in this type of
biorefineries.
Corn Refinery
▪ In addition to corn oil, starch, and feed products, various
bioproducts including fuel ethanol, organic acids (mainly citric,
lactic, and itaconic acids), amino acids (e.g., lysine, threonine),
and
biopolymers
such
as
xanthan
gum
and
polyhydroxyalkanoates (PHAs) are currently produced by
microbial fermentation in corn refinery.
▪ In addition, new processes to produce butanol, 1,3-PDO (BioPDO), and other
platform chemicals such as succinic acid, 3hydroxyl propionic acid, adipic acid,
and acrylic acid that can be
converted to various polymers (plastics) have also been
developed.
Major Components in Corn Grains and
Products Derived from Them
Integrated corn biorefinery
Corn Refinery
HFCS, high-fructose corn syrup;
PHA, polyhydroxyalkanoate;
PGA, poly-γ-glutamate;
1,3-PDO, 1,3-propanediol.
▪ Xylose can be converted to xylitol via a biological reaction.
▪ Corn fiber contains starch (∼24%),
hemicellulose (∼35%), protein, and oil.

cellulose

(∼15%),

▪ Corn fiber can be treated by hot water, dilute acid hydrolysis,

and enzymatic hydrolysis to convert starch, cellulose, and
hemicellulose into glucose, xylose, and arabinose, which can
be used for ethanol production.
Corn Refinery
▪ Corn gluten meal consists of proteins (∼60%) and hydrophobic
amino acids (∼10% leucine), with the remaining components
mainly being moisture, fiber, and lipids.
▪ Corn gluten can be used for animal feed, food, pharmaceuticals,
and industrial products.
▪ Value-added biodegradable high-performance engineering
plastics and composites can be produced using corn gluten by
plasticizing with glycerol/ethanol and blending with commercial
polymers.
▪ Corn gluten was also used as substrate in solid-state fermentation
to produce enzymes. Corn proteins extracted from gluten can be
used to produce protein-based films and coatings in the food
industry.
Corn Refinery
▪ CSL containing approximately 47% protein has been widely used
as a nutrient and nitrogen source in fermentation to produce
protease, lactic acid, and ethanol.
▪ Lactic acid can be converted to polylactic acid and used as
bioplastics for packaging and textile fibers.
▪ Lactic acid and ethanol can react to form ethyl lactate ester, which
can be used as an industrial “green” solvent, replacing the
petroleum-based solvents currently used in the semiconductor
industry.

▪ 1,3-PDO and succinic acid are chemical building blocks that can
be produced from corn dextrose.
Corn Refinery
▪ Improved production of platform chemicals and biofuels could be
achieved by engineering the microorganisms.
▪ A genetically engineered Escherichia coli was developed by
DuPont to produce high-level 1,3-PDO (up to 130 g/L) from
glucose.
▪ Using global transcription machinery engineering (gTME),
Saccharomyces cerevisiae strain with improved tolerance to high
concentrations of glucose and ethanol to produce high-level
ethanol from high-concentration glucose was developed.
Soybean Biorefinery
▪ More than 50% of soybeans are processed to produce vegetable
oils and soybean meal.
▪ Due to the rising oil price, biodiesel production in the United
States, which is mainly from soybean oil, has increased rapidly
over the last 20 years.
Major Components of Soybeans and
Products Derived from Them
The biodiesel cycle
Comparison of Different
Technologies for
Biodiesel Production
from Soybean Oil
▪ Different methods for biodiesel production from soybean oil,
includes non-catalytic process (supercritical alcohol technology)
and catalytic processes using alkali, acid, and enzyme as
catalysts.
▪ In general, the alkali process is the most efficient of all processes
and has a high reaction rate.

▪ It is the only process currently used in biodiesel production at an

industrial scale.
▪ However, enzymatic and supercritical processes are more
environmentally friendly and have also shown promising
applications, although further optimization of these processes,
such as continuous operation, and scale up and economic
evaluations are needed.
Integrated soybean biorefinery for biodiesel
production from soybean oil and chemical production
from glycerol and other soybean by products.
Soybean Biorefinery
SPC, soy protein concentrate;
SPI, soy protein isolate;
PHA, polyhydroxyalkanoate;
PHB, poly(3-hydroxybutyric acid);
1,3-PDO, 1,3-propanediol.
▪ About 10% (w/w) of glycerol is generated in biodiesel production.

▪ The large amounts of glycerol produced in the biodiesel industry

have surpassed the market demand and driven down the crude
glycerol price.
▪ Converting the abundant and low-cost glycerol generated in the
biodiesel industry to higher-value products is essential to achieve
economic viability by offsetting the relatively high cost of
soybeans and other oilseeds.
Crude glycerol
▪ Methanol, soap, catalysts, salts, non-glycerol organic matter, and
water impurities usually are contained in the crude glycerol.
▪ For example, crude glycerol from sunflower oil biodiesel
production had the following composition (w/w): 30% glycerol,
50% methanol, 13% soap, 2% moisture, approximately 2-3%
salts (primarily sodium and potassium), and 2-3% other impurities

Value-added opportunities for crude glycerol

▪ Animal feedstuff
▪ Chemicals produced via bioconversion (1,3-propanediol, PHA,
citric acid, succinic acid, Docosahexaenoic acid, etc.)
▪ Chemicals
produced
via
conventional
catalysis
(Hydrogen/syngas, oxygenated chemicals, monoglyceride, etc.)
Soybean Biorefinery
▪ Many microorganisms can use glycerol as carbon source to
produce various chemicals.
▪ For example, both pure and crude glycerol present in biodiesel
wastes can be used for the production of PHA and 3hydroxypropionaldehyde.
▪ Compared with glucose, glycerol as carbon source in succinic
acid fermentation can give a higher product yield and
concentration with lower production of the by-product acetic acid.
▪ Other products that can be biologically produced from glycerol
include 2,3 butanediol, n-butanol, dihydroxyacetone (DHA),
glyceric acid, citric acid, oxalic acid, lactic acid, and polyols
(mannitol, arabitol, and erythritol).
Sugarcane Biorefinery
▪ Sugarcane contains about 70−75% water, 11−16% sucrose, and
10−16% fiber.
▪ Sugarcane processing begins with the extraction of cane juice by
mill tandems, leaving behind bagasse, the fibrous material that is
sent to the lignocellulosic processing to produce ethanol or
chemicals, or sent to the boiler house to generate electricity or
steam.
▪ Most of the sugar juice is used to produce sugar by purification
and crystallization.
▪ The molasses by-product from sugar processing and some of the
sugar juice are used to produce ethanol.
Integrated sugarcane biorefinery for fuel and chemical
production from cane juice, molasses, and bagasse.
Schematic diagram of biorefinery processes from sugar and starch crops.
Third generation Biorefineries
Aquacultures and algae biorefinery
▪ Microalgae offer another promising resource for biofuels,
especially biodiesel, production.

▪ Many microalgae have a high lipid content (as high as 70% of dry
weight) that can be extracted and used to produce biodiesel.
▪ It has been estimated that microalgae have the highest biodiesel
production efficiency based on the land use (12,000−98,500 L
/ha/year) that is up to 220-fold of oil crops (soybean: 446;
sunflower: 952; rapeseed: 1190; jatropha: 1892; oil palm: 5950).
Aquacultures and algae biorefinery
▪ The current oil-producing crops would not be able to supply more
than 50% of our current energy demand even if they were
cultivated on all the arable land on the Earth.
▪ In contrast, the area required for microalgae cultivation for
supplying global oil demand would be 2.5−20.5% of global arable
land based on the biomass yield of 10−50 g/m2 day with a
30−50% content of triacylglycerides.
▪ Aquacultures of microalgae, either in outdoor ponds or closed
bioreactors, can be situated on non-arable land and thus would
not have any negative effect on the arable land for food.
▪ Therefore, algal oil production can supply the so-called thirdgeneration biofuels
in the future
Integrated microalgae biorefinery for production of
algal oil (biodiesel) and other value-added products.
PUFA, polyunsaturated fatty acid.
Aquacultures and algae biorefinery
▪ Current microalgae cultivation technologies are still away from
economical biofuel production.
▪ In general, outdoor cultivation has a lower initial capital cost but
is prone to contamination and low CO2 efficiencies.
▪ Closed bioreactor systems, including plate, tubular, and airlift
bioreactors are expensive to operate and difficult to scale up.
▪ The slow cell growth, low cell density (usually less than 1−10
g/L), and large water content (>90% cell weight) of cell biomass
make microalgae cultures expensive to justify the relatively high
production costs for the relatively low-priced biofuels.
Aquacultures and algae biorefinery
▪ Therefore, the microalgae biorefinery must utilize all of the cell
components and extract the high-value products such as
carotenoids (e.g., lutein and astaxanthin) and polyunsaturated
fatty acids (PUFAs) (e.g., eicosapentaenoic acid and
docosahexaenoic acid).
▪ Some microalgae are rich in carbohydrate and can be harvested
and used as feedstock for fermentation to produce ethanol and
other chemicals.
Macroalgae biorefinery
▪ In addition to microalgae, macroalgae from marine cultures have
also been proposed as a renewable resource for the “third
generation” ethanol (and other biofuels) production.
▪ Many marine macroalgae are rich in carbohydrates
(polysaccharides of galactan, mannan, etc.) with little or no lignin,
and can be mass cultivated in oceans and harvested, hydrolyzed
to monosaccharides, and then used as feedstock for fermentation
to produce fuels (e.g., ethanol and butanol) and chemicals.

▪ The macroalgae thus can provide another alternative and

promising biorefining platform for fuel and chemical production.
Second generation Biorefineries
Lignocellulosic Biorefinery
▪ Today’s bioethanol and biodiesel represent the first-generation
biofuels produced from readily processable bioresources such
as sucrose, starch, and plant oils from grains.
▪ Research attention has shifted toward the next-generation
biofuels from lignocellulosic biomass such as agricultural
residues (e.g., corn stovers, corn fiber, and wheat straw), woody
biomass, and municipal solid wastes.

▪ Lignocellulosic biomass is well-suited feedstock for the

production of biofuels and biochemicals because of its low cost,
large-scale availability, and environmentally benign production.
▪ Particularly bioenergy production and utilization cycles based on
lignocellulosic biomass have near-zero greenhouse gas
emission
Lignocellulosic Biomass
▪ Lignocellulose consists of three major components: cellulose,
hemicellulose, and lignin.
▪ Their compositions vary greatly, depending on the type of plant,
cultivation conditions, and the age of the plant.
▪ In general, Lignocellulosic biomass contains cellulose
(39−49%, w/w), hemicellulose (21−25%, w/w), and lignin
(20−28%, w/w) as major components and proteins and
minerals (4−10%, w/w) as minor components, depending on its
source.
▪ Cellulose and hemicellulose are carbohydrates, and lignins are
polyphenolic compounds.
Organic Components of Some Lignocellulosic Biomass
Typical composition of fibers across cell wall
Structure and composition of lignocellulosic biomass
Cellulose
▪ Cellulose provides strength and flexibility to the plant cell wall and
the fibers.
▪ Cellulose is a linear polymer chain of β-D-glucopyranose units.
These glucose units are linked together by β-1,4-glycosidic
linkages.
▪ Strictly speaking, the cellobiose unit is the repeating unit of the
cellulose chain.
The degree of polymerization
(DP) of native cellulose is in
the range of 500–15,000.
Glucose
Cellulose
▪ Bundles of cellulose molecules are aggregated together in the
form of microfibrils.
▪ Microfibrils build up fibrils and cellulose fibers.
▪ Cellulose molecules are completely linear and have a strong
tendency to form intra- and intermolecular hydrogen bonds.

Cellulose microfibrils.
▪ Each cellulose chain approximates to a flat ribbon, with alternate
glucose units facing in opposite directions.

▪ They are locked in this position by a hydrogen bond between a

hydroxyl group (O3–H) of one glucose unit and the ring oxygen
(O5′) of the next.
Intra and inter-molecular hydrogen bonds of
cellulose molecules
Hemicellulose
▪ Hemicelluloses are heterogeneous polysaccharides that are
formed through biosynthesis.
▪ Similar to cellulose, hemicellulose is one of the supporting
materials in the cell walls.
▪ They consist of five different major sugar units, including glucose,
xylose, galactose, arabinose, and mannose plus small amounts of
rhamnose, glucuronic acid, 4-O-methyl–glucuronic acid, and
galacturonic acid.
▪ Among these sugars, xylose (a pentose or five-carbon sugar) is
the major component of hemicellulose in a wide variety of
lignocellulosic biomass species.
Hemicellulose
▪ The amount of hemicellulose in lignocellulosic biomass is typically
20–35% of biomass weight.
▪ Hemicelluloses have a DP of only about 200, which is much lower
than that of cellulose.
▪ In order to achieve high overall biomass-to-ethanol (or bio-based
products) process yield, hemicellulose utilization is necessary
since the contribution of hemicellulose is 40–50% of total
carbohydrates in lignocellulosic biomass.
Backbone: β-1,4
Sidechain: α-1,6
Backbone: β-1,4
Backbone: β-1,4
Sidechain: α-1,3
Backbone: β-1,4
Sidechain: α-1,2

Backbone: β-1,3
Sidechain: α-1,6

The types and simplified structures of the major

hemicelluloses in wood
Hemicellulose
▪ Hemicelluloses are typically less ordered; that is, more
amorphous than cellulose, and consequently are more easily
hydrolyzed by acids or enzymes to monomers.
▪ Under common pretreatment conditions, in particular with acids,
it can easily be degraded into decomposition products, including
furfural, which is known as inhibitory to fermentation.
▪ Hemicelluloses have highly branched structures and are a
mixture of furanose and pyranose forms.
▪ This highly branched structure of hemicelluloses is responsible
for the high solubility in water. It also has many applications in
food, pharmaceutical, and paper industries, such as thickeners,
emulsifiers, and gels.
Lignin
▪ Lignin is a polyphenolic compound made up of three
(phenylpropanoid) monolignol units: coniferyl, sinapyl, and pcoumaryl alcohol.
▪ Lignin molecules are highly cross-linked to each other and are
a high-molecular-weight natural product.
▪ Lignin is present in amounts ranging from 12% to 33% by
weight in lignocellulosic biomass.
▪ One of its functions in plants is to hold cellulosic fibers together
and to impart strength to the lignocellulosic bio-composite.
Lignin
▪ The two main types of lignins are (1) the guaiacyl lignins, in
which alcohol precursors with one methoxy substituent
predominate (characteristic of softwood), and (2) the guaiacylsyringyl lignins, in
which both monomethoxy- and dimethoxy
substituted alcohols are present.

▪ In general, plant materials can be classified into three types

which are hardwood, softwood, and grass species.
▪ Hardwoods and grasses contain guaiacyl-syringyl lignins and
also acids, such as p-hydroxybenzoic acid, esterified to the
lignin macromolecular core.
Spermatophytes (Seed
bearing plants)

Gymnosperm
(Naked seeds)
Conifers,
Evergreen,
Softwood
All woody

Angiosperms
(Covered seeds)

Monocotyledonous
Non woody

Dicotyledonous
Woody or
Non-woody
Relative distribution of syringyl (S), guaiacyl (G),
and p-hydroxyphenyl (H) lignin subunits in different
types of biomass.
Example linkages between lignin monomers
Lignin
▪ In its natural state in the wood cell wall, lignin is referred to as
"protolignin" or “native lignin” and is markedly thermoplastic.
▪ It is also much less hydrophilic than cellulose or hemicellulose,
almost to the point of being hydrophobic.
▪ Monomers after lignin breakdown will depend on the type of
treatment method.
▪ The utilization of lignin is an increasingly important field. The field
of lignin utilization includes primarily
✓ lignin as fuel (due to its high energy content of 11,300 Btu/lb);
✓ lignin as polymeric products;
✓ lignin as a source of high-value low molecular weight
compounds.
Extractives
▪ A large variety of wood components are soluble in neutral
organic solvents or water, which are called extractives.
▪ The extractives comprise an extraordinarily large number of
individual compounds of both lipophilic and hydrophilic types.
▪ The content of extractives varies but usually is less than 10%.
▪ These substances can be extracted from the fiber wall with
either water or various organic solvents (alcohols, ethers,
acetone, and others), the choice of solvents varying with the
nature of the extractive.
Extractives
▪ Extractives analysis typically consists of two steps, which
determine water extractives and ethanol extractives.
▪ Water soluble materials may include inorganic materials,
nonstructural sugars, and nitrogenous materials, among
others.
▪ Ethanol-soluble material includes chlorophyll, waxes, or other
minor components.
Major Classes of Wood Extractives
1.

ALIPHATIC
•Fats
•Waxes
•Sterols

2.

TERPENES/TERPENOIDS
•Volatile monoterpenes
•Terpenoid
•Resin acids
•Other (high melting point)

3.

PHENOLIC COMPOUNDS
•Simple phenols
•Lignans
•Stilbenes
•Tropolones
•Polyphenols
•Hydrolyzable tannins
•Flavonoids
•Condensed tannins
Inorganic components (Ash)
▪ ∼ 1% of dry weight.
▪ Ash originates from a variety of salts deposited in the cell walls
and lumina.
▪ Ash consists of the metallic ions of sodium, potassium, calcium,
and the corresponding anions of carbonate, phosphate, silicate,
sulfate, chloride, etc. remaining after the controlled combustion
of wood.
PRETREATMENT OF LIGNOCELLULOSIC BIOMASS
▪ The hydrolysis of lignocelluloses to fermentable sugars remains
the greatest challenge in the development of economical plant
biomass feedstock for the biorefinery industry.
▪ In order to produce fuels and chemicals from lignocellulosic
biomass, cellulose and hemicellulose in this feedstock should be
hydrolyzed to produce various monomeric sugars either by
acid/alkali or enzymes.
PRETREATMENT OF LIGNOCELLULOSIC BIOMASS
PRETREATMENT OF LIGNOCELLULOSIC BIOMASS
▪ Major difficulties in the bioconversion scheme are the
heterogeneous composition of the polysaccharides in plant
cell walls and the recalcitrant nature of the cellulosic part of the
substrates; that is, cellulosic fibers are highly crystalline and
are thus very resistant to enzyme-catalyzed hydrolysis.

▪ Today’s lignocellulosic biorefinery comprises of three main

sections to convert lignocellulose into Biofuels or bio-based
products:
✓ Thermochemical pretreatment,
✓ Enzymatic hydrolysis, and
✓ Sugar fermentation to fuels/chemicals.
Schematic diagram of the lignocellulosic biomass
to ethanol (biofuels) conversion.
Enzymatic hydrolysis can
produce high yields of relatively
pure glucose syrups without
generating glucose degradation
products, and utility costs are
low since the hydrolysis occurs
under mild reaction conditions
PRETREATMENT OF LIGNOCELLULOSIC BIOMASS
▪ Lignocellulosic material requires pretreatment in order to open
up the rigid structure and to enhance the susceptibility of the
biomass to the enzyme.
▪ Development of effective pretreatment method is one of the
most feasible ways which can reduce the production cost of
bio-based products by contributing to reductions of the use of
enzymes as well as the pretreatment processing costs,
including capital and operating costs.
PRETREATMENT OF LIGNOCELLULOSIC BIOMASS
The common effects of pretreatment include the following:
▪ Decrease of lignin, hemicellulose, and extraneous components
▪ Increase of surface area, porosity, and pore size
▪ Reduction of the crystallinity of cellulose
▪ Enhancement of the accessibility of enzyme to the cellulosic
substrate.
Why Is Pretreatment Important?
Factors Affecting Cellulose Hydrolysis by Enzymes
PRETREATMENT OF LIGNOCELLULOSIC BIOMASS
▪

The choice of pretreatment technology must take into account

sugar-release patterns and solid concentrations for each
pretreatment in conjunction with their compatibility with the
overall process, feedstock, enzymes, and organisms to be
applied.

▪ A successful pretreatment must meet the following requirements:

1. improve sugar yield or the ability to subsequently release
sugars by hydrolysis,
2. avoid degradation or loss of carbohydrate,
3. avoid formation of by-products inhibitory to subsequent
hydrolysis and fermentation processes, and
4. be cost effective.
Different pre-treatment methods
Many low-cost pretreatment technologies have been developed to realize
high sugar yields from both cellulose and hemicellulose. They can be
categorized as
Physical pre-treatment
Biomass size reduction
▪ Various mechanical size reduction methods are employed to
increase the digestibility of lignocellulosic biomass such as
chipping, shredding, grinding, coarse size reduction and
milling.

▪ These pretreatment methods decrease the cellulose crystallinity

and the degree of polymerization as well as increase the specific
surface area.
▪ More reduction of biomass particles i.e. smaller than 0.4 mm has
no significant effect on yield and rate of hydrolysis.
▪ The energy demand for mechanical comminution of lignocellulosic
material depends on the biomass features and final particle size.
Physical pre-treatment
Biomass size reduction
▪ The milling method increases enzymatic hydrolysis and
digestibility and includes hammer milling, disk milling, ball
milling, and vibratory milling
▪ All the mill pretreatment methods do not produce any toxic
compounds e.g. levulinic acid and hydroxymethyl furfuraldehyde
(HMF).
▪ It makes mill pretreatment a good choice of initial pretreatment
for a broad range of lignocellulosic feedstocks.
Chemical methods
Alkaline pre-treatment
▪ This method is one of the most reliable pretreatments due to it
strong effect and the relatively simple process.
▪ Selective removal of lignin without losing reducing sugar and
carbohydrates, enhancing porosity and surface area of biomass,
and therefore improving enzymatic hydrolysis are the advantages
of alkaline method.
▪ However, the main disadvantage of this method compared to the
other pretreatments methods is longer reaction times.
Chemical methods
Alkaline pre-treatment
▪

The major role of this method is improving the enzymatic

digestibility through delignification of biomass.

Alkyl-aryl linkages in lignin are easily broken under alkaline

circumstances.

Different types of alkaline solutions have been used such as

sulfite, sodium hydroxide, ammonium hydroxide, and
lime.

Sodium hydroxide is the most used alkaline solution as it is

effective for delignification and works in various conditions.
Acid pre-treatment
▪

Acid hydrolysis is often used because it can be adapted to suit

a wide variety of feedstocks.

Although there have been numerous attempts to pretreat the

biomass using various acids such as hydrochloric acid,
phosphoric acid, and peracetic acid, dilute sulfuric acid
pretreatment has been the most widely used method because
of cheap chemical cost.

In the dilute acid pretreatment, nearly complete hydrolysis of

hemicellulose and partial hydrolysis of cellulose typically
occur.

In steam explosion pretreatment, sulfuric acid can also be

added to catalyze the hemicellulose hydrolysis reaction.
Acid pre-treatment
▪

Dilute acid pretreatment process is generally carried out at

elevated temperatures (130–240 °C) for various lengths of
time.

Although this is generally inexpensive, acid hydrolysis may

also produce large quantities of degradation by-products and
undesirable inhibitory compounds at higher temperatures.

A large portion of the xylose fraction is easily degraded to

furfural, and at high temperature, glucose is degraded to
hydroxymethylfurfural.

One of the important features in dilute acid pretreatment is that

it can recover hemicellulose sugar in significant quantities
(80–95% of the initial amount) in a liquid form.
Process scheme of dilute acid pretreatment
Some Potential
Inhibitors from
Lignocellulose after
Thermochemical
Pretreatment
Inhibitors and detoxification
▪ The amounts and types of inhibitors vary strongly among raw
materials and depend on the pretreatment method used.
▪ Several methods of detoxification, such as overliming,
extraction with organic solvents, ion exchange, molecular
sieves, and steam stripping, have been explored.
▪ Overliming with Ca(OH)2 is the most commonly used method. A
significant drawback of this method is that calcium salts may
precipitate in the process and contaminate the surfaces of the
distillation column, evaporators, and heat exchangers.

▪ Hence, detoxification should be avoided as much as possible

due to the additional cost and possible loss of fermentable
sugars.
Inhibitors and detoxification
▪ By applying a fed-batch mode of substrate addition with proper
feed protocol and control variables, the inhibitors can be kept at
acceptable levels.
▪ More tolerant strains can be obtained through genetic
modification. Approaches for overexpressing genes that encode
enzymes for resistance against specific inhibitors and altering
cofactor balance in the cell may lead to the production of better
strains.
Common Detoxification Methods and
Their Advantages and Disadvantages
Ionic Liquid Pretreatment
▪

Ionic liquids are salts that are in a liquid state at room

temperature and are stable up to approximately 300 °C.

A new type of nonvolatile solvents, namely ionic liquids,

have recently demonstrated potential as pretreatment
solvents because they dissolve lignocellulosic biomass
and then allow recovery of cellulose for enzyme hydrolysis at
high rates.

The cellulose regenerated from ionic liquid was found

essentially amorphous and porous.

Dissolved cellulose can be precipitated by the addition of an

antisolvent such as water or ethanol.
Ionic Liquid Pretreatment
▪

However, at present, the commercially available ionic liquids

are prohibitively expensive for production of ethanol on a large
scale.

▪
▪
▪

1-butyl-3-methylimidazolium chloride [Bmim][Cl],

1 allyl-3-methylimidazolium acetate [Amim][OAc], and
1-butyl-3-methylimidazolium hydrogen sulphate [Bmim][HSO4]
Organic solvent pre-treatment
▪

The organosolv is one type of organic pretreatment that

utilizes organic or aqueous-organic solvent at temperatures
ranging from 100 to 250 °C.

The advantages of this method are the separation of high

purity cellulose with only minor degradation and
hemicellulose fractionation with high efficiency.

The high-purity lignin and lignin derivative chemicals

from organic pretreatment are major economic merits of this
method rather than dilute acid pretreatment.

Easy solvent recovery, and solvent reuse.

Organic solvent pre-treatment
▪

A wide range of organic solvents, such as alcohol, phenol,

esters, propionic acid, acetone, formaldehyde dioxane
and amines with and without catalyst, have been used for
the pretreatment of LCBs.

Ethanol and methanol as alcohols with low boiling points

are preferred due to their low cost and ease of recovery.
Supercritical Pre-treatment
▪

Supercritical pre-treatment, mainly using carbon dioxide, was

also developed to increase the enzyme hydrolysis efficiency of
Lignocellulosic biomass.

It has many advantages because CO2 is inexpensive, nontoxic

and environmentally benign, easy to recover after use, and the
reaction temperature is low.

However, in commercialization, high capital costs are projected

due to requirements of high-pressure equipment.

Supercritical CO2 behaves like a solvent.

Supercritical Pre-treatment
▪

At high temperature, supercritical carbon dioxide passes

through a vessel having biomass.

At high-temperature CO2 enter the biomass and forms

carbonic acid which causes hemicellulose hydrolysis.

High-pressure gas disorders the biomass structure and

increase the accessible surface area.

This process is favorable for the production of ethanol,

because it produces low inhibitory products and removes lignin
at more feasible way (non-acidic and non-corrosive).

Moreover, CO2 as the supercritical fluid do not degrade the

required sugar monomers because of its mild environment.
Autohydrolysis (Hot-Water and Steam Explosion)
▪ The autohydrolysis reaction involves the formation of acids from
the solubilization of acidic components in hemicellulose, such as
acetic acid, formic acid, and glucuronic acid.
▪ For example, under hot-water pretreatment conditions, the
hydronium ion initially causes xylan depolymerization and
cleavage of the acetyl group. The autohydrolysis reaction then
follows, in which the acetyl group catalyzes the hydrolysis of the
hemicellulose.
▪ The hydrolysis of glycosidic linkages in hemicellulose and the β –
ether linkages in lignin are catalyzed by acetic acid formed at high
temperature from acetyl groups present in hemicellulose
(autohydrolysis).
▪ Autohydrolysis at temperatures below 230°C, cellulose is not
significantly affected, whereas the lignin fraction can be
depolymerized to give soluble compounds and a nonsoluble fraction
with increased susceptibility toward further processing.
Autohydrolysis (Hot-Water and Steam Explosion)
▪ In steam explosion, the biomass is pressurized with high-pressure steam
(superheated steam) for a certain period of time (typically at 200–450
psig for a period of ∼ 10 minutes) and then the product is explosively
discharged to atmospheric pressure, which results in a sudden
decompression.
▪ The high-pressure steam initiates an autohydrolysis reaction, and the
mechanism involved in steam explosion is similar to that of pretreatment
using hot water or dilute acid.
▪ This explosive discharge changes the biomass into fibrous product by a
combination of mechanical and chemical action.
▪ Steam explosion of biomass increases enzyme and solvent accessibility
of cellulose and renders biomass separable (by fractionation) into
different components.
▪ The parameters controlling the steam explosion process are reaction
temperature and retention time.
Ammonia-related pretreatment
▪ The ARP (ammonia recycled percolation) has been developed
to remove lignins so as to improve susceptibility of biomass to
enzymes.
▪ In this process, typically 10–15% ammonia, ∼ 170°C, and 2.3
MPa are used. > 80% lignin can be removed.

▪ One problem associated with ARP is that only about half of the
hemicellulose remains in the solid, whereas the rest of it is
solubilized along with soluble lignin, which complicates
hemicellulose utilization.
Ammonia-related pretreatment
▪ SAA (Soaking aqueous ammonia or ammonia steeping) is a batch process,
which retains most of hemicellulose and cellulose in solid (> 85% of
hemicellulose and nearly 100% cellulose).
▪ The SAA process still can remove a considerable amount of lignin (> 70%).
▪ Commercial cellulases often contain enough xylanase activity to
concurrently convert both xylan and glucan in SAA-treated solids to ethanol
in the simultaneous saccharification and cofermentation (SSCF) process.
▪ Process: Lignocellulosic biomass typically is soaked in 15-30% ammonia at
30–80°C for 4–24 hours.
▪ These moderate reaction conditions generally produce minimal degradation
products. SAA pretreatment requires relatively longer pretreatment time.
Ammonia-related pretreatment
▪ AFEX (ammonia fiber explosion) utilizes anhydrous liquid ammonia
(can be recovered and recycled).
▪ The pretreatment conditions of the AFEX process are relatively mild: ∼
100°C with liquefied anhydrous ammonia. Rapid pressure release ends
the treatment.

▪ The effects of AFEX process primarily include depolymerization of

hemicellulose and lignin. AFEX minimizes degradation of the sugar in
the biomass.
▪ Although the AFEX process has several advantages it is more
complicated to implement at commercial scale because it requires
much higher pressure in the pretreatment reactor, its reaction involves
phase changes (gas-to-liquid and then liquid-to-gas), thus requiring
expensive equipment and having high operating costs.
Biological pretreatment
▪ For the degradation of lignocellulose biomass, natural
microorganisms possessing enzymes (bacteria, brown-white
and soft rot fungi) are employed that are capable cell wall
deconstruction.
▪ Biological pretreatment method does not produce any unwanted
products as compared to chemical and physical pretreatment.
▪ Additionally, high pressure, acids, alkali, high temperature or any
reactive species are not compulsory for this pretreatment.
▪ White- and soft-rot fungi contain lignin-degrading enzymes like
lignin peroxidases, manganese-dependent peroxidases,
polyphenol oxidases, and laccases which are effective for the
lignin degradation.
▪ Brown-rot fungi mainly attack cellulose.
Biological pretreatment
▪ Low energy and mild environmental conditions are considered
as an advantage. However, the main drawback is the low
hydrolysis rate.
▪ Most lignin-degrading microorganisms also degrade some
cellulose and hemicellulose.
▪ Extensive research is required for the implementation of these
microorganisms on a large commercial scale for biological
pretreatment of lignocellulose biomass.
Various Pretreatment Technologies and Representative Reaction Conditions
Products from lignin?
▪ The use of lignocellulosic biomass is mainly focused on creating
pulp/paper, energy, sugars and bioethanol from the holocellulose
component, leaving behind lignin to be discarded or burned as
waste despite of its highest aromatic carbon and energy content
(22–29 KJ/g).
▪ During the pulping/pretreatment process, lignin undergoes
significant structural changes to yield technical lignin.
▪ For a circular bioeconomy, there is a need to enhance the use of
native lignin for generating more valuable products.
▪ Over the last few years, a new method called ’lignin-first’, or
’reductive catalytic fractionation’ (RCF), has been devised to
generate selective phenolic monomers.
Reductive catalytic fractionation: Lignin-first approach
Reductive catalytic fractionation
▪ Reductive catalytic fractionation (RCF) is the emerging
methodology for the valorization of native lignin present in LCB.

▪ RCF process involves three primary stages

(i) solvothermal extraction of lignin from the lignocellulosic
matrix,
(ii) depolymerization of extracted lignin into monomers, and
(iii) stabilization of monomers.
▪ Hence, this methodology is a one-step process to convert
native lignin into phenolic monomers.
▪ These phenolic monomers can be considered as building blocks
for the production of value-added chemicals, polymers, fuels and
pharmaceuticals.
Reductive catalytic fractionation
▪ The main objective of the RCF process is to maximize the
delignification while preserving the carbohydrates.
▪ The reductive atmosphere used in the RCF process could be
generated from external hydrogen or the use of H2-donor
solvents MeOH, EtOH, iPrOH, alcohol: water, acids or using the
biomass component (hemicellulose) itself for H2 production.
▪ The solvent predominantly controls lignin extraction from
lignocellulosic biomass, irrespective of the catalyst.
▪ In RCF, the catalyst plays a crucial role in depolymerizing lignin
and stabilizing monomers.
Reductive catalytic fractionation
▪ Several studies have shown the effect of catalysts in increasing
monomer yield and also in increasing selectivity towards
particular monomers.
▪ Ru/C displayed 75% selectivity for 4-propyl phenolics (PG/PS)

▪ Pd/C favored the creation of propanol-substituted phenolics

(PG-OH, PS-OH) with 91% selectivity.
(a) Conventional methodology of lignocellulosic biomass valorization
(b) Reductive catalytic fractionation to valorize lignocellulosic biomass.
Reaction chemistry behind lignin valorization in RCF.
Effect of feedstock on phenolic monomers yield in RCF of LCB
Enzymatic hydrolysis (EH)
▪ EH is considered an environmentally friendly and greener alternative
compared to the chemical hydrolysis process (e.g., acid hydrolysis), as it is
less energy-intensive and uses milder and non-corrosive reaction
conditions.
▪ EH is mediated by cellulolytic enzymes (cellulases and hemicellulases)
that act on (hemi-) cellulosic polymers of LCB biomass to generate
fermentable (hexose and pentose) sugars from biomass and requires
many synergistic enzymes.
▪ Cellulase is the main enzyme involved in lignocellulose bioconversion that
attack cellulose in a sequential manner by randomly cleaving low
crystallinity cellulose and thereby, releasing cellobiose and finally glucose.
▪ Cellulase complex is composed of endo-1, 4-β-glucanases, responsible
for breaking 1, 4- β-glucan bonds randomly; exo-1, 4-β-D-glucanases,
which free up the D-glucose and cellobiose and hydrolyze cellobiose
gradually; and, β-D-glucosidase, that generates D-glucose from
cellobiose.
Enzymatic hydrolysis (EH)
▪ Various microbes are used to commercially synthesise cellulases,
however, filamentous fungi like Trichoderma reesei, Acremonium sp.,
Aspergillus niger, Penicillium oxalicum and Thermoascus aurantiacus are
the most superior cellulase producers.
▪ Hemicellulases are involved in breakdown of hemicellulosic polymers and
side chains of hemicellulose. Hemicellulosic enzymes include
arabinofuranosidases, mannanases, galactomannase, esterases,
xylanases, and xylosidases.
▪ Use of accessory hemicellulolytic enzymes including α-glucuronidase,
α-L-arabinofuranosidase, α-galactosidase, acetylxylan esterase, and
ferulic acid esterase often enhances sugar release from hemicellulosic
fractions.
▪ Lytic polysaccharide monooxygenases (LPMOs) are mono-copper
enzymes that catalyze the hydroxylation of glycosidic bonds via
oxidation-assisted cleavage are now essential part of the cellulolytic
enzyme mix
Lignocellulosic bioethanol
▪ Ethanol accounts for the majority of biofuels worldwide; and its
production from lignocellulosic biomass seems very attractive and
sustainable.
▪ The “conventional” process for producing ethanol from lignocellulosic
biomass includes the following four main steps:
1. Pretreatment—breaking down the structure of the lignocellulosic
matrix.
2. Enzymatic Hydrolysis—depolymerizing cellulose to glucose by
means of cellulolytic enzymes.

3. Fermentation—metabolizing the glucose to ethanol, generally by

yeast strains.
4. Distillation – Rectification – Dehydration – separating and
purifying the ethanol to meet fuel specifications.
Lignocellulosic bioethanol
▪ Four approaches are being studied for the production of liquid biofuels
from biomass with cellulase.
✓ Separate hydrolysis and fermentation (SHF)
✓ Simultaneous saccharification and fermentation (SSF)
✓ Simultaneous saccharification and co-fermentation (SSCF)
✓ Consolidated bioprocessing (CBP)
Simultaneous Saccharification and Fermentation
(SSF)
SSF involves the concurrent breakdown of cellulose (and
hemicellulose, if present) into monomeric sugars, and the
conversion of these sugars into products via fermentation. The
primary focus of the fermentation in SSF is on the conversion
of glucose to the targeted product.

Simultaneous Saccharification and Co-Fermentation

(SSCF)
SSCF focuses on the co-fermentation of the hemicellulosederived pentose sugars
(e.g. xylose and arabinose) alongside the
conventional hexose sugars. Hence, bioprocesses using the SSCF
approach typically incorporate pretreatments where much of
the hemicellulose remains in the solid phase so that it will be coprocessed with
the cellulose in the SSCF stage.
Consolidated Bioprocessing (CBP)
▪ Like SSF, in CBP the hydrolysis, and fermentation steps occur
in the same reactor. However, unlike SSF, the production of
enzymes also takes place in the same reactor.
▪ CBP has the potential to reduce costs and simplify the biofuel
production process significantly. However, the implementation
of CBP is currently challenging because it requires a single
microorganism, or a consortium of microorganisms, that can
efficiently perform all the required functions.

▪ CBP is mainly applied to ethanol production from cellulose

using cellulolytic bacteria such as Clostridium cellulolyticum,
Clostridium
thermocellum,
Thermoanaerobacterium
thermosaccharolyticum,
and
Thermoanaerobacterium
saccharolyticum.
The consolidated bioprocess (CBP) combines enzyme
production, hydrolysis, and fermentation in one operation
step as indicated by the dashed box.
The SSF Concept
▪ SSF offers easier operation and a lower equipment
requirement since no hydrolysis reactors are needed
▪ One of the main advantages of SSF over SHF is the reduction
of end-product inhibition by sugars formed in the hydrolysis.

▪ The formed ethanol also inhibits hydrolysis but to a lesser

extent than cellobiose or glucose.
▪ Several compounds present in pretreatment hydrolyzates,
which inhibit the enzymatic hydrolysis, can be metabolized by
the fermenting microorganism; and this could help increase the
ethanol yield.
The SSF Concept
▪ Incomplete hydrolysis of solid biomass is a major problem in
the SSF process.
▪ Except for inhibition by end products or other components, this
may be due to enzyme deactivation, and unproductive enzyme
adsorption.
▪ One point to be noted in the SSF process is that the enzyme and
yeast cell concentrations should be appropriately balanced in
order to minimize costs for yeasts and enzyme production.
Characteristics of Fermenting Microorganisms.
▪ It should give a high ethanol yield and productivity,

▪ should be able to withstand high ethanol concentration in order to

keep distillation costs low,
▪ should be tolerant to inhibitors,
▪ should have tolerance to temperature,
▪ should have the ability to utilize multiple sugars,
▪ Tolerance toward low pH will minimize the risk of contamination.
Characteristics of Fermenting Microorganisms.
▪ Due to the very attractive properties of Saccharomyces
cerevisiae in industrial fermentations, there have been
significant efforts to design recombinant xylose and arabinose
fermenting strains of this yeast.

▪ Xylose-fermenting strains of S. cerevisiae can be constructed

either by introducing genes encoding xylose isomerase (XI) or
xylose reductase (XR) and xylitol dehydrogenase (XDH).
▪ Also the endogenous gene encoding xylulokinase (XK) must be
overexpressed in order to obtain significant xylose fermentation.
Characteristics of Fermenting Microorganisms.
▪ In contrast to baker’s yeast and Zymomonas mobilis,
Escherichia coli is capable of metabolizing a wide variety
of substrates (including hexoses, pentoses, and lactose), but
the wild-type organism has a mixed fermentative pathway and
is thus a poor ethanol producer.
▪ E. coli can be genetically engineered into an ethanol producer
by overexpression of pdc (encoding pyruvate decarboxylase)
and adhB (encoding alcohol dehydrogenase) from Z. mobilis.
Comparison of SSF Resulting from Various
Lignocellulosic Materials
Optimization of the SSF Process
For an efficient performance of the process, optimal substrate
loading, enzyme loading, and yeast concentration, as well as
optimal temperature and pH, are required.

Substrate Loading
▪ To achieve a high ethanol concentration, a high substrate loading
and, hence, high water-insoluble solids (WIS) are crucial.

▪ When WIS content is increased, the ethanol yield tends to

decrease.
▪ Fed-batch approach can be used. The major advantage of
running in fed-batch mode is that the levels of inhibitors can be
kept lower.
Substrate Loading
▪ Stirring is a significant problem at high WIS contents due to the high
viscosity, which results in mass and heat transfer problems.
▪ This becomes less pronounced with fed-batch SSF, due to the gradual
hydrolysis of added fibers.
▪ An alternative to a fed-batch addition is to make a prehydrolysis, which
is to add enzymes to the bioreactor sometime before the fermenting
organism is added. This can be done at an elevated temperature and
will decrease the initial viscosity at the start of fermentation.

Enzyme Loading
▪

The enzyme loading is very important for the process economy.

It should be optimized on a case-to-case basis.

Yeast Loading
▪ The rate of enzymatic hydrolysis in most of the reported SSF
processes has been rate determining, and the yeast
concentration could therefore be lowered.
▪ Higher yeast concentration in the SSF will result in a lower
overall ethanol yield if the substrate cost for the production
of the yeast is considered.
▪ However, lowering the yeast concentration will lower the
volumetric productivity, which necessitates the optimization
of the yeast loading.
Temperature
▪ In SSF, a compromise between the optimal temperatures
for the cellulolytic enzymes and the yeast is needed.
▪ Thermotolerance is clearly important and thermotolerant
strains (eg. Fabospora fragilis, Saccharomyces uvarum,
Candida brassicae) have been evaluated to allow fermentation
at the optimal temperature for the enzymatic action.
▪ Cellulases work at 40–50°C whereas the fermentation of
hexoses with S. cerevisiae is carried out at 30°C.
▪ The development of microbes that ferment at 50°C can
potentially reduce the cost of the cellulase enzyme by onehalf as an increment of
20°C during the saccharification may
imply a doubling of the cellulose hydrolysis rate.
Simultaneous Saccharification and Co-fermentation
▪ Another promising alternative is the inclusion of pentose
fermentation in the SSF process called SSCF.
▪ If co-culture is been used, it is necessary that both fermenting
microorganisms are compatible in terms of operating pH and
temperature.
▪ A system including the isomerization of xylose and the
fermentation with S. cerevisiae in a simultaneous way can be
utilized.
▪ A natural xylose-isomerase converts the xylose into xylulose,
which can be assimilated by the yeasts implying the
cofermentation of lignocellulosic biomass.
Consolidated Bioprocessing (CBP)
▪ CBP combines enzyme production, saccharification, and
fermentation into a single process step mediated by a single
microorganism or microbial consortium.
▪ This process eliminates the enzyme cost, hence helping to
lower the ethanol cost.
CBP involves four biologically mediated transformations.

1. the production of saccharolytic enzymes (cellulases and

hemicellulases),
2. the hydrolysis of carbohydrate components present in pretreated
biomass to form sugars,
3. the fermentation of hexose sugars (glucose, mannose, and
galactose), and
4. the fermentation of pentose sugars (xylose and arabinose).
Consolidated Bioprocessing (CBP)
▪ No natural microorganism possesses all the properties desired
for CBP; some bacteria/fungi exhibit some of the needed traits.
▪ Two primary developmental approaches capable of producing
industrially viable CBP microbial strains are

1. Engineering a cellulase producer, such as Clostridium

thermocellum, to be ethanologenic
2. Engineering an ethanologen, such as S. cerevisiae or
Z. mobilis, to be cellulolytic.
Comparison of Potential CBP Microorganisms
Thermal and catalytic conversion of
biomass to products
Thermochemical conversion
▪ Thermochemical conversion of biomass is a feasible and
frequently explored route to convert biomass to biofuels.

▪ Biomass thermochemical conversion processes (combustion,

torrefaction, liquefaction, pyrolysis, and gasification) can produce
heat, biochar, bio-oil and syngas.
Combustion
▪ Biomass combustion simply means burning organic material.

▪ Combustion is a reaction of a fuel with oxygen in air to release

heat
▪ Biomass can be incinerated directly as an alternative to coal to
produce heat and electricity in power plants.
▪ However, the high contents of O and the moisture of the
biomass lead to it having a low calorific value.
Torrefaction (high-temperature drying or lowtemperature pyrolysis)
▪ Torrefaction is a thermochemical process that aims to decrease
the water and volatiles contents from the biomass.
▪ The tenacity and fibrous structure of biomass are damaged by
carboxylation and dehydration reactions, which help to remove
oxygen and water from the feedstock.
▪ The torrefied biomass (biochar) presents improved properties
compared with ordinary biomass: it has higher energy and mass
density, higher heating value, better hydrophobicity, and
improved flowability, giving it somewhat similar properties to coal.
Pyrolysis
▪ Pyrolysis is the thermal decomposition of biomass occurring in
the absence of oxygen.
▪ The products of biomass pyrolysis include biochar, bio-oil and
gases including methane, hydrogen, carbon monoxide, and
carbon dioxide.

▪ Depending on the thermal environment and the final

temperature, pyrolysis will yield mainly biochar at low
temperatures, less than 450 °C, when the heating rate is quite
slow, and mainly gases at high temperatures, greater than
800 °C, with rapid heating rates.
▪ At an intermediate temperature and under relatively high
heating rates, the main product is bio-oil.
Liquefaction
▪ Converts biomass into bio-oil (target product, 40–60%), biochar
(10–12%), and gases (35–45%).

▪ It is often performed in a solvent, such as water, ethanol,

acetone, glycerol, phenols, and tetralin, at relatively low
temperature (200–600 °C) and under high pressure (5–25
MPa).
▪ The product compositions depend primarily on the biomass
type, reaction temperature, pressure, residence time, solvent,
catalyst, etc.
▪ To achieve efficient and high-quality production of bio-oil,
optimisation of process parameters is necessary.
Gasification
▪ Gasification is an endothermic process where biomass is
partially oxidised at higher temperatures (600–1300 °C) with
the help of gasification agents like air/O2, and steam.
▪ The gaseous products consist of CO, CO2, H2, CH4, and
hydrocarbons.
▪ Steam is the optimal gasification agent to improve the calorific
values and compositions of gasification products because it
introduces an H resource (steam) and offsets the drawback of
high O contents of biomass to some extent.
Catalytic conversion of biomass to products
Chemocatalytic vs. Enzymatic Production of Ethanol
Mechanism of Ethanol Formation from Glucose
Catalytic Pathways of Producing Renewable Ethylene from Biomass
Mechanistic Pathways for the Catalytic Transformation
of Glycerol into Propylene
Synthesis of p-Xylene from Biorenewable sources

2,5-dimethylfuran
Methanol Production from Biomass via Syngas and Its
Downstream Applications

Methanol
to olefin

Methanol
to gasoline
Catalytic conversion of biomass to products
Primary petrochemicals:
▪ Ethylene, propylene, butadiene, BTX (benzene, toluene, xylenes), and
methanol, produced from petroleum and other fossil resources, can
produce practically all petrochemicals and synthetic organic polymers
used in industries.
▪ These must be sourced from renewable carbon feedstock to achieve
sustainability in chemical manufacturing.
Design of Microorganisms as cell factories
▪ Two main approaches can be taken to carry out strain/protein
engineering: rational design and directed evolution.
▪ Rational design involves performing chosen point mutations,
insertions or deletions in the coding sequence, and mutation
choice is typically based on structural and functional
information about the target biomolecule.
▪ Nonetheless, the sequence–structure– function relationship is
often difficult to predict accurately, particularly at the single
residue level.
▪ Additionally, reliable structural information is frequently not
available for the protein of interest
Directed evolution
▪ Directed evolution is a robust method to design proteins with
desirable functions.
▪ Directed evolution bypasses the need to determine specific
mutations a priori by mimicking the process of natural evolution
in the laboratory.
▪ In nature, mutations which are beneficial for individuals are
iteratively selected through numerous generations. In directed
evolution, this process takes place on a much shorter timescale,
and generates biomolecules that suit human-defined
applications.
▪ Unlike rational methods, directed evolution generates random
mutations in the gene of interest and requires no protein
structure information.
Random mutagenesis techniques
Random mutagenesis techniques
Recombination-based mutagenesis techniques
Adaptive laboratory evolution
▪ Adaptive laboratory evolution (ALE) is an innovative approach
for the generation of evolved microbial strains with desired
characteristics, by implementing the rules of natural selection
▪ During microbial ALE, a microorganism is cultivated under
clearly defined conditions for prolonged periods of time, in the
range of weeks to years, which allows the selection of improved
phenotypes.
Adaptive laboratory evolution of Y. lipolytic for enhanced tolerance to vanillic
acid. The black squares
represent the cell growth without vanillic acid addition and were used as a
control. The red triangles
represent the cell growth of evolved strain cultured in the YNBXI medium with the
addition of vanillic
acid. The whole adaptive evolution had lasted for 130 days with Y. lipolytica
transferred for 66 times
and generating approximately 188 generations: first stage included 7 passages and
22 generations;
second stage included 5 passages and 15 generations; third stage included 14
passages and 41
generations; last stage included 40 passages and 110 generations.