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Pretreatment of Lignocellulosic Biomass Toward Biofuel Production

Chapter · November 2018

DOI: 10.1007/978-3-319-67678-4_9

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Pretreatment of Lignocellulosic Biomass
Toward Biofuel Production

Soumya Sasmal and Kaustubha Mohanty

Abstract To improve the competence of cellulose hydrolysis, pretreatment is

required to efficiently break its recalcitrant structure. Pretreatment has been viewed
as one of the most expensive processing steps in cellulosic biomass-to-fermentable
sugars conversion, and it has great potential for improvement of efficiency and
lowering of cost through research and development. Pretreatment is an important
cost-driver of lignocellulose conversion to biofuel and an important step prior to
enzyme hydrolysis. It disrupts the plant cell wall network and partially separates the
major polymer components (lignin, cellulose, and hemicellulose). However, pre-
treatment of lignocellulosic materials may also result in the release of inhibitors and
deactivators of the enzymatic hydrolysis of cellulose. Development of enzyme
processes for hydrolysis of cellulose to glucose must reduce inhibition and deac-
tivation effects in order to enhance hydrolysis and reduce enzyme usage. Therefore,
great attentions have paid in designing pretreatment technologies to split recalci-
trant characteristics of lignocellulose biomass.

Abbreviations
AFEX Ammonia Fiber Explosion/Expansion
IU International Unit
IL Ionic Liquid
LCC Lignin Carbohydrate Complex
FPU Filter Paper Unit

S. Sasmal
Division of Biological Sciences and Engineering,
Netaji Subhas Institute of Technology Delhi, Delhi, India
K. Mohanty (&)
Department of Chemical Engineering,
Indian Institute of Technology Guwahati, Guwahati, India
e-mail: [email protected]

© Springer International Publishing AG 2018 203

S. Kumar and R. K. Sani (eds.), Biorefining of Biomass
to Biofuels, Biofuel and Biorefinery Technologies 4,
https://doi.org/10.1007/978-3-319-67678-4_9
Pretreatment of Lignocellulosic Biomass
Toward Biofuel Production

Soumya Sasmal and Kaustubha Mohanty

Abstract To improve the competence of cellulose hydrolysis, pretreatment is

required to efficiently break its recalcitrant structure. Pretreatment has been viewed
as one of the most expensive processing steps in cellulosic biomass-to-fermentable
sugars conversion, and it has great potential for improvement of efficiency and
lowering of cost through research and development. Pretreatment is an important
cost-driver of lignocellulose conversion to biofuel and an important step prior to
enzyme hydrolysis. It disrupts the plant cell wall network and partially separates the
major polymer components (lignin, cellulose, and hemicellulose). However, pre-
treatment of lignocellulosic materials may also result in the release of inhibitors and
deactivators of the enzymatic hydrolysis of cellulose. Development of enzyme
processes for hydrolysis of cellulose to glucose must reduce inhibition and deac-
tivation effects in order to enhance hydrolysis and reduce enzyme usage. Therefore,
great attentions have paid in designing pretreatment technologies to split recalci-
trant characteristics of lignocellulose biomass.

Abbreviations
AFEX Ammonia Fiber Explosion/Expansion
IU International Unit
IL Ionic Liquid
LCC Lignin Carbohydrate Complex
FPU Filter Paper Unit

S. Sasmal
Division of Biological Sciences and Engineering,
Netaji Subhas Institute of Technology Delhi, Delhi, India
K. Mohanty (&)
Department of Chemical Engineering,
Indian Institute of Technology Guwahati, Guwahati, India
e-mail: [email protected]

© Springer International Publishing AG 2018 203

S. Kumar and R. K. Sani (eds.), Biorefining of Biomass
to Biofuels, Biofuel and Biorefinery Technologies 4,
https://doi.org/10.1007/978-3-319-67678-4_9
204 S. Sasmal and K. Mohanty

1 Introduction

Lignocellulosic biomass materials are readily available in our surrounding habitats

in different forms. Nature produces about 180 billion metric tons of biomass/year, of
which about 75% is in the form of carbohydrates such as hexose or pentose sugars.
On account of the global annual production of biomass (1
1011 tons) as well as
the specific energy contents of biomass and crude oil, it has been concluded that in
only one decade, Earth’s plants can renew in the form of cellulose, hemicellulose,
and lignin all of the energy stored as conventional crude oils. As lignocellulosic
materials have low nitrogen and sulfur contents, the fuel products (bioethanol,
biobutanol, or biodiesel) obtained from this will be environment friendly. The
vegetal biomass comes from photosynthesis, and the CO2 from biomass does not
represent a net input to the amount of carbon already making part of the carbon
cycle, which displays CO2-neutrality nature of lignocellulose materials. There have
been many attempts by various researchers and government as well as nongovern-
ment initiatives from all over the world to commercialize the production of biofuels
such as ethanol, biodiesel with lignocellulose as the biomass feedstock but the
complex structure of lignocelluloses acts as a protecting barrier of plants cell wall
from attack by potential pathogenic organisms and purveys resistance against
mechanical forces. The digestibility of cellulose or other polysaccharides present in
lignocellulosic biomasses into monomers or the fermentable sugars is hindered by
many chemical, structural, or physiochemical factors. Modern biotechnology
research has designed enzymes or the cocktail of several enzymes to hydrolyze raw
lignocellulosic material to yield monosaccharides but the process is time-consuming
and needs sophisticated supervision as well as operating procedures which makes
the process less economically feasible technology. To accelerate the hydrolysis
process, scientists are interested to incorporate an additional step, which is known as
“pretreatment”. Pretreatment process rapidly disintegrates the lignocelluloses to its
mother components i.e., lignin, cellulose, and hemicellulose. Therefore, the primary
step toward biofuel production from lignocellulosic biomass is pretreatment. This
step facilitates enzymatic hydrolysis process by altering the structural features,
increasing surface areas and porosity of lignocellulosic biomasses. In general, the
basic objectives of any type of pretreatment process are as follows,
1. Production of reactive cellulose fiber, which is suitable for enzymatic hydrolysis
for the production of monomers such as glucose, xylose etc.
2. Reduction of byproducts formation during pretreatment process which reduces
not only sugar yield but also inhibitory chemicals (e.g., furfural) for further
downstream process.
3. Preserve the hemicellulose fraction in biomass.
4. Minimum energy requirement of the entire process.
5. Input of fewer low cost and less hazardous chemicals to initiate process.
6. Reducing the cost of feedstock (lignocellulosic biomass) size reduction.
Pretreatment of Lignocellulosic Biomass Toward … 205

The pretreatment process can roughly be classified into physical, chemical, and
biochemical pretreatment process (Fig. 1).
Cost effective pretreatment of lignocellulosic biomass is a major challenge of
lignocellulose-based biorefinery technology (Krishnan et al. 2010). However, there
is a sound opportunity for reducing the cost of pretreatment process through
extensive research and development for which several pretreatment techniques like
physical (milling and grinding), physicochemical (steam explosion/autohydrolysis,
hydrothermolysis, and wet oxidation), chemical (alkali, dilute acid, oxidizing
agents, and organic solvents), and biological processes have been used for efficient
conversion of the structural carbohydrates to fermentable sugars or other
value-added chemicals.
Each pretreatment process has their own advantages and disadvantages. Table 1
shows the advantages and disadvantages of different pretreatment processes.
Among all the pretreatment processes the chemical pretreatment process results in a
rapid and good yield of sugar after enzymatic hydrolysis. The biological pretreat-
ment sounds good but the slower rate of conversion and cost of enzymes does not
make the process popular. Beside irradiation, other physical pretreatment processes
demand high-energy input to make the pretreatment process successful. The present
study focused on two pretreatment processes to achieve high product yields in
subsequent enzymatic hydrolysis and fermentation operations. The dilute sulfuric
acid pretreatment process and ultrasound assisted lime pretreatment process was
conducted and compared accordingly.
In a broad spectrum, the benefits of lignocellulosic biomass materials can be
accomplished according to two different philosophies, the headmost is to direct

Fig. 1 Different methods of biomass pretreatment

206 S. Sasmal and K. Mohanty

Table 1 Comparison of different pretreatment processes

Pretreatment Advantages Disadvantages References
Chemical Dilute acid Operation time is Acid Recovery and Chen et al.
pretreatment less, High yield of formation of furfural (2012a, b),
process pentose sugar Sindhu et al.
(2011)
AFEX High yield of pentose Recovery of Speers and
sugar; No inhibitory ammonia is not Reguera (2012),
compounds effective. Tao et al. (2011)
Less effective
process with
increasing lignin
content
Lime No inhibitory Operation time is Jin et al. (2012),
compounds more Sierra-Ramírez
et al. (2011)
Organosolvosis High yield of pentose Solvent recovery is Zhao et al.
sugar expensive (2009), Arato
et al. (2005)
Physical Milling Operation time is less The overall yield is Inoue et al.
pretreatment poor. Energy (2008),
process requirement is high Craegveld et al.
(2008)
Irradiation High yield of sugar; Need special design Yong et al.
No inhibitory of equipment and (2011), Khan
compounds process et al. (2006)
High-pressure No Inhibitory Maintaining Valery et al.
compounds high-pressure itself (2011)
is a challenge
Biological Microorganisms Low energy Rate of reaction is Bak et al. (2010),
pretreatment requirement slow Singh and Chen
process No production of (2008)
inhibitory compound
Mild operation
conditions

utilization of the whole biomass, for example, by combustion, pyrolysis, lique-

faction, or gasification. The other mode is known as “fractionation” in which the
major components of the lignocellulosic biomass are separated into “fractions”
made up of compounds with related properties, followed by separate processing of
each fraction for specific purposes. It is expected that operation in biorefineries
should follow the basic principles of green chemistry, for example, regarding an
efficient utilization of raw materials and avoiding the utilization of wastes instead of
performing end-of-pipe waste remediation, enabling the production of end products
to fulfill societal needs, and shortening the dependence on fossil resources such as
Pretreatment of Lignocellulosic Biomass Toward … 207

coal, petroleum etc. In this way, a sustainable industrial and societal development
can be achieved by achieving the needs of the present generation without com-
promising the needs of future generations.

2 Overview of Different Bonds in Lignocellulosic Material

Lignocellulosic biomass can be classified into (1) agricultural wastes that arise
mainly from various agricultural cultivations and farming activities, (2) energy
crops that are grown especially for biofuel or electricity production, and (3) forestry
residues from forest logging areas. Lignocellulosic biomass is composed of dif-
ferent layers of plant cell wall. The chemical composition of the cell walls is
dependent on the species of the plants. The principal components of lignocellulosic
biomass material are described briefly in Fig. 2.

2.1 Cellulose

Cellulose is the b-1,4-polyacetal of cellobiose (4-O-b-D-glucopyranosyl-D-glucose).

Cellulose is more commonly considered as a polymer of glucose because cellobiose
consists of two molecules of glucose.
The polymer of cellulose is formed on the basis of two main linkages:
1. The glucosidic linkage is the one that forms the initial polymer chain. More
specifically, it is a 1-4 b D-glucosidic bond that connects the glucose units
together. The glucosidic bond can also be considered as an ether bond since it is,
in fact, the connection of two carbon atoms with an elementary oxygen inter-
fering (Solomon 1988).
2. The hydrogen bond is considered to be responsible for the crystalline fibrous
structure of cellulose. The arrangement of the polymer in long straight parallel

Fig. 2 Schematic diagram of principal components of lignocellulosic biomass

208 S. Sasmal and K. Mohanty

chains together with the fact that the hydroxyl groups are evenly distributed on
both sides of the glucose monomer (see 0 and 0), allow the formation of
hydrogen bond between two hydroxyl groups of different polymer chains
(Faulon et al. 1994). It has been identified that carboxyl groups are also present
in cellulose in a fraction of 1 carboxyl per 100 or 1000 monomer units of
glucose (Krassig and Schurz 2002), although this does not appear obvious from
the main structure of cellulose. As already mentioned, hemicellulose consists of
polysaccharides other than cellulose. Its structure reveals that ether type of
bonds, such as the fructosic and glucosidic bonds, is the main one that forms its
molecule. The main difference with cellulose is that the hydrogen bonds are
absent and that there is a significant amount of carboxyl groups. The carboxyl
groups can be present as carboxyl or as esters or even as salts in the molecule
(Kirk-Otmer 2001).

2.2 Hemicellulose

The most common type of polymers that belongs to the hemicellulose family of
polysaccharides is xylan. The molecule of a xylan involves 1!4 linkages of
xylopyranosyl units with a-(4-O)-methyl-D-glucuronopyranosyl units attached to
anhydroxylose units. The result is a branched polymer chain that is mainly com-
posed of five carbon sugar monomers, xylose, and to a lesser extent six carbon
sugar monomers such as glucose.

2.3 Lignin

Lignin is a three-dimensional polymer of phenyl-propanoid units and amorphous

phenolic macromolecule that can be found only in the secondary cell wall. It
functions as glue, which provides compressive strength to the plant tissue and
individual fibers into it. It also imparts stiffness to the cell wall and resistance
against insects and pathogens.
Three primary units, namely, guaiacyl (G), sinapyl (S), and p-hydroxyphenyl
(H) are the structural unit of lignin. The high level of heterogeneity of lignin is
synthesized through the oxidative coupling of 4-hydrophenylpropanoids during
secondary cell wall deposition, which results in layers of lignin in different monomers.
There are four main types of bonds identified in the lignocellulose complex.
Those are ether type of bonds, ester bonds, carbon-to-carbon bonds, and hydrogen
bonds. These four bonds are the main types of bonds that provide linkages within
the individual components of lignocellulose (intra-polymer linkages), and connect
the different components to form the complex (inter-polymer linkages). The posi-
tion and bonding function of the latter linkages are summarized as follows.
Pretreatment of Lignocellulosic Biomass Toward … 209

2.3.1 Ester Bond Linkages

Ester bonds are identified between lignin and polysaccharides as well as within the
hemicellulose polymer. In the latter case, it is the acetyl group that forms ester bond
with a hydroxyl of the main chain of the polysaccharides. However, with respect to
the linkage of lignin with polysaccharides, there is no definite conclusion whether
the ester bond lies between lignin and cellulose or lignin and hemicellulose, or
between lignin and both cellulose and hemicellulose.

2.3.2 Intra Polymer Linkages

The main types of bonds that connect the building molecules within the lignin
polymer are ether bonds and carbon-carbon bonds (Table 2). Ether bonds may
appear between allylic and aryl carbon atoms, or between aryl and aryl carbon
atoms, or even between two allylic carbon atoms. The total fraction of ether type
bonds in the lignin molecule is around 70% of the total bonds between the
monomer units. The carbon-to-carbon linkages form the remaining 30% of the total
bonds between the units. They can also appear between two aryl carbon atoms or
two allylic carbon atoms, or between one aryl and one allylic carbon atom
(Kirk-Otmer 2001).
In order to determine the linkages that connect the different polymers of the
lignocellulose complex, lignocellulose is broken down and the individual compo-
nents are separated. However, their separation is commonly achieved by methods
that result in alteration of their original structure. As a consequence, the conclusions
on the connecting linkages between the polymers are not definite. However, it has
been identified that there are hydrogen bonds connecting lignin with cellulose and
with hemicellulose, respectively. Furthermore, the existence of covalent bonds
between lignin and polysaccharides are identified. More specifically, it is certain
that hemicellulose connects to lignin via ester bonds. It is also known that there are
ether bonds between lignin and the polysaccharides. It is still not clear though
whether the ether bonds are formed between lignin and cellulose, or hemicellulose.
Hydrogen bonding between hemicellulose and cellulose is also identified. However,
this linkage is not expected to be strong due to the fact that hemicellulose lacks
primary alcohol functional group external to the pyranoside ring (Faulon et al.
1994).
From the above discussion on molecular arrangement of lignocellulosic biomass,
it is envisaged the need of pretreatment, although it is the most energy-expensive
process hence, cost-effective pretreatment of lignocellulosic biomass is a major
challenge of lignocellulose-based bioenergy technology. The present discussion is
an attempt to put an overview of both comparison as well as critical discussion on
existing pretreatment technologies and upcoming promising pretreatment tech-
nologies over lignocellulosic biomass.
210 S. Sasmal and K. Mohanty

3 Different Pretreatment Process

3.1 Dilute Acid Pretreatment Process

Acid pretreatment method includes application of dilute or concentrated acids to the

lignocellulosic biomass material, which facilitates breaking the rigid lignocellulosic
matrix. The most commonly used acid is dilute sulfuric acid (H2SO4). The dilute
acid pretreatment method may be defined as exposure to high temperature (e.g.,
180 °C) during a short period of time; or at a lower temperature (e.g., 120 °C) for
longer retention time (30–90 min) to the lignocellulosic materials (Alvira et al.
2010). At present, the dilute sulfuric acid treatment is receiving substantial attention
over concentrated sulfuric acid (Kobayashi et al. 2011). Recent articles have
reviewed that the dilute sulfuric acid pretreatment process facilitates enzymatic
hydrolysis of lignocellulosic biomass (Himmel et al. 2007; Kumar et al. 2009).
Dilute sulfuric acid pretreatment process has been successfully applied with dif-
ferent types of lignocellulosic biomasses such as Switch grass (Digman et al. 2010),
Corn stover (Xu et al. 2009), and Poplar (Wyman et al. 2009). Olive tree biomass
was pretreated with 1.4% H2SO4 at 210 °C resulting in 76.5% of hydrolysis yields
and cashew apple bagasse pretreated with diluted H2SO4 at 121 °C for 15 min
yielded ethanol as high as 0.47 g g−1 glucose (Cara et al. 2008; Rocha et al. 2009).
Dilute acid hydrolysis has many advantages compared with concentrated acids such
as less corrosion, shorter reaction times, and higher reaction rate (Huang et al.
2009). Lignin is known as a nuisance material for ethanol makers as it retards
enzymatic hydrolysis procedure. Beside lignin, the native crystalline structure
(cellulose I) of cellulose is considered to be one of the major factors limiting its
potential in terms of cost-competitive lignocellulosic biofuel production (Cheng
et al. 2011). Xuebin et al. (2009) used Rapeseed straw as a lignocellulosic material
which has 60% carbohydrate. A central composite design of response surface
method was used to optimize H2SO4 catalyzed hydrothermal pretreatment of
rapeseed straw, with respect to acid concentration (0.5–2%), treatment time (5–
20 min), and solid content (10–20%) at 180 °C. Enzymatic hydrolysis and fer-
mentation were also measured to evaluate the optimal pretreatment conditions for
maximizing ethanol production. The results showed that acid concentration and
treatment time was more significant than solid content for optimization of xylose
release and cellulose recovery. Pretreatment with 1% sulfuric acid and 20% solid
content for 10 min at 180 °C was found to be the most optimal condition
for pretreatment of rapeseed straw for ethanol production. This study has demon-
strated that hemicellulose in the rapeseed straw can be removed efficiently by
H2SO4-catalyzed hydrothermal pretreatment at high solid loading (20%). The
concentration of inhibitory product like furfural were reported less in this process
(Xuebin et al. 2009).
Noureddini and Byun (2010) studied distiller’s grain and corn fiber as the
biomass for pretreatment process. The total carbohydrate content of distillers’
grains and corn fiber were 57.7 ± 2.0 and 77.0 ± 1.0 wt%, respectively. In this
Pretreatment of Lignocellulosic Biomass Toward … 211

study, dilute sulfuric acid hydrolysis for the conversion of distillers’ grains and
corn fiber to monomer sugars and the formation of furfural was investigated. The
extent of solubilization of biomass beyond monomer sugars was also monitored.
Biomass loadings in the range of 5–20 wt% at 5% intervals, acid concentrations in
the range of 0.5–1.5 vol% at 0.5% intervals, and temperatures of 120 and 140 °C
were studied. The experimental results confirmed an increasing trend in the for-
mation of monomeric sugars as a function of time. The highest yields of monomeric
sugars were observed at the lower substrate loadings (5 and 10 wt%), higher
concentrations of sulfuric acid (1.0 and 1.5 vol%), and when the temperature was
140 °C [Noureddini and Byun (2010)]. For the majority of the cases under con-
sideration, the most effective period of hydrolysis appeared to be during the initial
20–30 min of the reaction. Formation of furfural during the course of hydrolysis
was significantly lower at 120 °C and also lower for the distillers’ grains samples
compared with the corn fiber samples. The total amount of the solubilized matter
during the hydrolysis was significantly higher than the amount of the monomeric
sugars. Shi et al. investigated dilute sulfuric acid pretreatment of corn straw and rice
straw, and enzymatic hydrolysis of cellulose (Shi et al. 2012). The straw was
pretreated at 121 °C with different sulfuric acid concentrations (1, 2, 3, 4, and 5%,
vol/vol.) and residence times (30, 60, and 90 min). Pretreatment residence time
plays a key role in increasing the glucose concentration comparing to sulfuric acid
concentration. Cellulose remaining in the pretreated feedstock was highly digestible
by cellulase from Trichoderma viride. The result showed that the saccharification
yield was 72.38 and 82.84% from corn straw and rice straw respectively. The
pretreatment conditions were as follows: acid concentration 2% (vol/vol), temper-
ature 121 °C, and time 60 min. To produce ethanol cost-effectively from herba-
ceous feedstocks such as corn stover, efficient xylan hydrolysis with a good amount
of monomeric xylose yield is required. Dilute acid pretreatment is well-established
as one of the pretreatment technologies for xylan hydrolysis; however, the accu-
mulation of salts, production of toxic byproducts, and the release of acetic acid can
inhibit enzymatic saccharification and fermentation, which leads to decrease the
yield of ethanol. Successful removal of acetyl groups from native corn stover by
alkali de-esterification could potentially increase the yield of monomeric xylose
from pretreatment and enzymatic hydrolysis, improve cellulose digestibility, and
reduce the cytotoxicity of the fermentation broth. Chen et al. (2012a, b), reported
that the dilute acid pretreatment process removed a significant amount of acetyl
groups from corn stover. Dilute acid pretreatment improved saccharification process
of xylan and glucan hydrolysis by 15 and 30% of control samples. In whole slurry
enzymatic hydrolysis, a 30% improvement in cellulose digestibility was obtained
over the control (Chen et al. 2012a, b).

3.1.1 Theory of Acid Hydrolysis Process

The molecular mechanism of acid-catalyzed hydrolysis of cellulose (cleavage of

b-1-4-glycosidic bond) follows the pattern outlined in Fig. 3. The cellulosic
212 S. Sasmal and K. Mohanty

Fig. 3 Mechanism of acid hydrolysis of cellulose

substrate is made up of microcrystalline bundles attached to each other by

para-crystalline regions and surrounded by a charged water boundary layer that
results from dipole–dipole interactions caused by aligning of water molecules
around the polar surface of the cellulose exterior (Roland 1976). Water molecules
and H+ ions (from acid) have to penetrate the cellulose fiber in order to precede the
hydrolysis reaction effectively. Otherwise, hydrolysis takes place only on the sur-
face of the cellulose. Acid catalyzes the breakdown of long cellulose chains to form
shorter chain oligomers and then to sugar monomers that the acid can degrade. The
hydrolysis of cellulose begins with the reaction of acidic proton and oxygen that
bonds two glucose units, forming the corresponding conjugated acid. The cleavage
of this C–O bond and breakdown of the conjugate acid to the cyclic carbonium ion
then takes place, which adopts a half-chair conformation. Free sugar and a proton
are liberated after rapid addition of water. The formation of the intermediate car-
bonium ion takes place more rapidly at the end than in the middle of the
polysaccharide chain.

3.2 Pretreatment with Base

Different types of bases such as caustic soda, lime, ammonium hydroxide were also
used as pretreatment agent. Alkali pretreatment process can be operated under mild
conditions than other pretreatment process besides this most of the alkaline
chemicals directly interact on lignin, therefore, the sugar components become
unaffected which is the prime target for any biofuel production process. The
aforementioned process thus makes alkaline pretreatment process among the
front-runner of all other established pretreatment process. The major reactions
during alkaline pretreatment process include dissociation of lignin from sugar
molecules (cellulose and hemicellulose) by saponification of intermolecular ester
bonds. Alkaline pretreatment preserves more amount of pentose sugar than other
pretreatment processes which lead to more sugar yield.
Pretreatment of Lignocellulosic Biomass Toward … 213

3.3 Ultrasound Assisted Lime Pretreatment

Among the various existing pretreatment technologies, lime pretreatment has pro-
ven to be a useful pretreatment method because it demands less amount of energy
and yields a good amount of fermentable sugar after enzymatic hydrolysis (Xu and
Cheng 2011). Moreover, it is a less expensive and nonhazardous chemical agent
(Chang et al. 1998). The beauty of lime pretreatment lies in the removal of lignin
from biomass without any momentous loss of structural carbohydrate materials e.g.,
glucose, xylose, arabinose etc. It is also reported that alkaline pretreatments remove
acetyl groups from hemicellulose, which improves saccharification by lowering
steric hindrance of enzymes (Falls et al. 2011). The sole hitch of this pretreatment
process is longer process operation time. Few researchers overcome this drawback
by elevating the reaction temperature to curtail operational time (Chang et al. 2001).
However, lime can be more effective as a pretreatment agent at a mild temperature
as lime is more soluble at a lower temperature, which leads to more alkalinity and
may lead to more efficient pretreatment of lignocellulosic materials (Xu et al. 2010).

3.3.1 Theory of Lime Pretreatment

The major effect of alkaline pretreatment is delignification. Alkaline pretreatments

successfully increase the lignocelluloses digestibility without the production of
furfural and methyl furfural (Harmsen et al. 2010). The alkaline material reacts with
biomass in three modes namely,
1. Reaction with lignin
2. Neutralization of organic acids
3. Reaction with resins and waxes of biomass materials
Lignin can be described as three-dimensional macromolecules with high
molecular weight in the range of 100 KD. It originates from phenyl-propanoid
precursors such as coumaryl, coniferyl, and sinapyl alcohol C6–C3 and is present in
vascular plants (Sun and Tomkinson 2002). Depolymerization of lignin using
alkaline materials (e.g., lime) depends on the cleavage of two types of aryl ether
bonds: Caliphatic OCaromatic and Caromatic OCaromatic (ordered from least to most
stable). Oxidative agents prominently improve the effects of alkaline pretreatments.
In alkaline media (pH > 12) oxygen is reduced through the reaction with phenolic
hydroxyl groups to superoxide radical (O2 ). Reactions involved in alkaline
oxidative pretreatments are primarily single-electron (radical) reactions.
Delignification reactions involve the formation of several different acids produced
during the degradation of carbohydrates that introduce hydrophilic groups into the
lignin structure (Klinke et al. 2002). Nucleophilic attack also occurs in some extent
causing ring opening, which promotes further degradation and solubilization. The
schematic diagram (Fig. 4) shows the typical delignification reactions (when OH¯
anions involved).
214 S. Sasmal and K. Mohanty

Fig. 4 Lignin degradation reactions in alkaline conditions involving a and b aryl ether linkages

In highly basic environment, the acidic proton of phenols (in lignin) gave
phenolate ion. Then the phenolate ion eliminates the ether from benzylic position to
produce an intermediate. This intermediate underwent two transformations either
regain the aromaticity or produce highly conjugated arylvinyl-phenol after cleavage
of CH2O group (Fig. 3). In presence of molecular oxygen, phenolate moiety
(Lignin) oxidized to 1,4-dicarboxylic acid which is soluble in basic solution
(Fig. 3). Therefore, lime facilitates delignification steadily in presence of oxygen
without any damage of the carbohydrates.
Rabelo et al. (2011) worked on pretreatment of sugarcane bagasse with lime
(calcium hydroxide) followed by bioethanol production. Experiments were per-
formed using the bagasse as it comes from an alcohol and sugar factory
(non-screened bagasse) and bagasse in the size range from 0.248 to 1.397 mm
(screened bagasse) (12–60 mesh). It was observed that the particle size presented
influence in the release of fermentable sugars after enzymatic hydrolysis using low
loading of cellulase and b-glucosidase (3.5 FPU g−1 dry pretreated biomass and
1.0 IU g−1 dry pretreated biomass, respectively) (Rabelo et al. 2009). Chang et al.
(1998) used lime as a pretreatment agent to enhance the enzymatic digestibility of
two common crop residues: bagasse and wheat straw. A systematic study of
Pretreatment of Lignocellulosic Biomass Toward … 215

pretreatment conditions suggested that for short pretreatment time (1–3 h), required
high temperature (85–135 °C) to achieve high sugar yields, whereas, for long
pretreatment times (e.g., 24 h), low temperatures (50–65 °C) were effective. The
recommended lime loading was 0.1 g Ca(OH)2 g−1 dry biomass. Water loading had
little effect on the digestibility. Under the recommended conditions, the 3-d
reducing sugar yield of the pretreated bagasse increased from 153 to
659 mg Eq. glucose g−1 dry biomass, and that of the pretreated wheat straw
increased from 65 to 650 mg Eq. glucose g−1 dry biomass. A material balance
study on bagasse showed that the biomass yield after lime pretreatment was 93.6%.
No glucan or xylan was removed from bagasse by the pretreatment, whereas 14%
of lignin became solubilized. A lime recovery study showed that 86% of added
calcium was removed from the pretreated bagasse by 10 washings and could be
recoveredReactions involved in alkaline by carbonating the wash water with CO2 at
pH 9.5 (Chang et al. 1998). Mass et al. (2008) reported an integrated pilot-scale
process where lime-treated wheat straw with a high dry-matter content (around 35%
by weight) is converted to ethanol via simultaneous saccharification and fermen-
tation by commercial hydrolytic enzymes and bakers’ yeast (Saccharomyces
cerevisiae). After 53 h of incubation, an ethanol concentration of 21.4 g lit−1 was
detected, corresponding to 48% glucan-to-ethanol conversion of the theoretical
maximum. The xylan fraction remained mostly in the soluble oligomeric form
(52%) in the fermentation broth (Mass et al. 2008). Cheng et al. (2010) used rice
straw as a substrate for alkaline pretreatment. 5 g H2O g−1 straw using sodium
hydroxide (NaOH) and compared to pretreatment at 10 g H2O g−1 straw by
hydrated lime (Ca(OH)2). The reaction temperature was held constant at 95 °C for
lime pretreatment and 55 °C for NaOH pretreatment. The range of delignification
was 13.1–27.0% for lime pretreatments and was 8.6–23.1% for NaOH. Treatment
at a higher temperature also improved delignification; delignification with water
alone ranged from 9.9 to 14.5% for pretreatment at 95 °C, but there was little effect
observed at 55 °C. Post-pretreatment washing of biomass was not necessary for
subsequent enzymatic hydrolysis. Maximum glucose yield was 176.3 mg g−1 dried
biomass (48.5% conversion efficiency of total glucose) in lime-pretreated (un-
washed biomass) and was 142.3 mg g−1 dried biomass (39.2% conversion effi-
ciency of total glucose) in NaOH-pretreated (unwashed biomass) (Cheng et al.
2010).
The carbon-carbon bonds are stable under alkaline conditions, the cleavage of
oxygen-carbon bonds are the most significant reaction in the pretreatment at the
basic condition. This reaction will take place and is producing phenolic hydroxyl
groups from the cleavage of the aryl-alkyl-ether bonds. Several researchers to
explain the phenomena of delignification of lignocellulosic biomass materials (Kim
and Holtzapple 2006) classified three different stages namely initial, bulk, and
residual phases. During the initial delignification stage in alkaline pulping with
sodium hydroxide, phenolic a-O-4-linkages in lignin and some non-phenolic ß-
O-4-linkages are cleaved. In the bulk stage, the major reaction is the cleavage of
non-phenolic ß-O-4-linkages and at the residual delignification stage, carbon–car-
bon linkages in lignin are cleaved and carbohydrates are degraded.
216 S. Sasmal and K. Mohanty

3.3.2 Theory of Ultrasonication

Ultrasound is sound with a sonic spectrum ranges between 20 and 10 MHz,

therefore, its pitch is above human hearing as the human ear can detect up to
16 kHz only. In practice, three ranges of frequencies are reported for three distinct
uses of ultrasound (i) high frequency, for diagnostic purposes (2–10 MHz), (ii) low
frequency or conventional power ultrasound (20–100 kHz), and medium frequency,
or “Sonochemical-effects” ultrasound (300–1000 kHz) (Ince et al. 2001). There are
two theory exists to explain the phenomena happened during the ultra-sonication
process. Hot spot theory postulates the formation of microbubbles at the site of
nucleation when high-frequency sound waves (50–60 kHz) are applied to the liq-
uid. The bubbles generate due to the decrease of boiling point caused by negative
pressure and it collapses due to positive pressure created by the same wave. This
alternating change of pressure is known as rarefaction. The positive pressure forced
the bubble to implode which leads to the formation of local hot spot with tem-
perature and pressure around 5000 K and 500 atm respectively (Thompson and
Doraiswamy 1999). Therefore, huge pressure and temperature can be achieved
which is required to break the polymeric biomass into their mother components
without designing any high-pressure vessel. This method is not only cost-effective
but also safe to use.
The electrical theory explained the above phenomena of breaking bonds by
means of generating huge electrical charges. The electrical double layer can be
formed in any liquid on the surface of the cavitation bubbles at the zone of cavi-
tation. This process is independent of the method of creation of cavitation (Margulis
and Margulis 2002).
Application of ultrasound technology exists in food processing technology for
depolymerization of biopolymers, emulsification, tanning of vegetables etc. (Sun
and Tomkinson 2002). Ultrasonic pretreatment of corn slurry was reported 20-fold
reduction in corn size particle, which facilitated 30% more glucose yield at the time
of enzymatic hydrolysis (Khanal et al. 2007). Cassava chips were pretreated using
the ultrasonic unit, which had a maximum power output of 20 KW and frequency
of 20 kHz (Nitayavardhana et al. 2008). Ultrasonic assisted (250 W, 30 min)
hydrogen peroxide pretreatment method followed by biological treatment
(Pleurotus ostreatus) was performed on rice hull yielded 31.8 and 32.2% more total
sugar and glucose respectively than that of sole biological treatment (Yua et al.
2009). Considering all the inputs from the literature, it was decided to combine the
lime and ultrasound technique for the pretreatment of biomass.

3.4 Ammonia-Based Pretreatment Process

Ammonia is a commodity chemical widely used in the chemical, pharmaceutical,

and food industries. It is easily recoverable, noncorrosive, and nontoxic chemical
agent and also inexpensive than other pretreatment chemical agent. Its industrial use
Pretreatment of Lignocellulosic Biomass Toward … 217

and recovery procedures are well-established. Ammonia can be easily recovered

and reused because of its high volatility, and it offers versatile processing options
(Kim et al. 2013). Aqueous ammonia is used for pretreatment of biomass materials
for delignification, without significantly affecting the building block carbohydrate
contents in it. It is a very effective pretreatment method especially for substrates that
have low lignin contents such as agricultural residues and herbaceous feedstock. Oil
palm empty fruit bunches were pretreated by aqueous ammonia soaking for ethanol
production was used by Jung et al. (2011). The main effects of ammonia pre-
treatment methods include removal or modification of lignin, an increase of surface
area alone with pore size, and modification of cellulose and hemicellulose struc-
tures. In aqueous form, it causes swelling of the biomass, which brings significant
morphological changes to the entire biomass morphology. It also affects the degree
of crystallinity of the integral biomass, which paves the way to make entire biomass
significantly amenable toward enzymatic hydrolysis.
Ammono-lysis is similar to liquid ammonia hydrolysis process in which
ammonia ion plays the role of water molecules in hydrolysis process. The ammonia
molecule is dissociated producing H+ and NH2 species, which then induce breakage
of chemical bonds in sugar molecules and lignin. The presence of the
lignin-carbohydrate complex in lignocellulosic biomass has been confirmed, and
various chemical linkages have been identified between lignin and the carbohy-
drates present in biomass (Du et al. 2010). The major linkages in biomass include
ether (R–O–R) and ester (R–CO–OR) bonds in LCCs, and ether (C–O–C) bonds in
lignin polymers. Ammonia molecules promote very effective selective cleavage of
these linkages, leading to lignin dissolution and eventually to lignin removal.
The AFEX (ammonia fiber explosion/expansion) process was introduced as a
pretreatment method for lignocellulosic biomass, its effect is similar to that of liquid
ammonia hydrolysis process except that it utilizes anhydrous liquid ammonia.
In this process, biomass is contacted with liquefied anhydrous ammonia at
elevated temperature (60–120 °C). After operation, the pressure is released rapidly.
Due to the expansion of the liquid ammonia trapped in the biomass, its structure is
disrupted, which is a beneficial pretreatment effect. The ammonia used in the
pretreatment is recovered at this stage in the form of a low-pressure gas and reused.
The pretreatment conditions of AFEX are moderate for temperature and high for
pressure. Because most of it is recovered, net consumption of ammonia is low.
During AFEX, neither component dissolution nor weight loss of solids occurs.
AFEX pretreatment has increased glucan and xylan conversions and ethanol
yields for a variety of feedstocks including switchgrass, corn stover, bagasse etc.

3.5 Application of Ionic Liquid as Pretreatment Agent

Ionic liquids or more specifically room temperature ionic liquids are the new
upcoming potential pretreatment agent for lignocellulosic biomass. Ionic liquids
can be defined as those chemical reagents which are liquid at room temperature.
218 S. Sasmal and K. Mohanty

The common characteristic among all the ionic liquids is they are comprised with
inorganic anion and an organic cation. Due to their unique hereto-molecular
structure, polarity, and low volatility (many ionic liquids can be distilled below
200–300 °C). The researcher claimed that ionic liquids can also act as a very good
solvent for lignin and cellulose which facilitates the posttreatment process to obtain
valuable chemicals as well as biofuel without acknowledging the harsh effect of
traditional acid, alkali method.
Ionic liquids can be classified according to their cations: quaternary ammonium
ILs, N-alkyl-pyridinium ILs, N-alkyl-isoquinolinium ILs, and imidazo-lium-based
ILs. Liu et al. (2012). Ionic liquids can be used for a number of chemical or
biochemical processes, such as process for the production of second-generation
biofuels (such as bioethanol or biobutanol) or short-chain organic acids. In these
kinds of bioprocesses, lignocellulosic materials are processed to hydrolyze
polysaccharides (cellulose and/or hemicelluloses) into monomers i.e., sugars, which
are fermented to yield the target products. Presence of strong intermolecular
hydrogen bonding of cellulose, makes cellulose almost insoluble to any conven-
tional solvents except carbon disulfide such as N-methyl morpholine-N-oxide etc.,
but using those abovementioned solvent have some practical usage problems for
bulk usages such as toxicity, undesired side chain reaction etc., therefore those
solvents are not used in biorefineries. The new researches emphasis on using ionic
liquids as a solvent for lignocellulosic biomass. Employing suitable ionic liquids,
cellulose can be easily separated which involves the interaction of hydroxyl groups
in cellulose with both the cation and anion in IL. The oxygen atoms of OH groups
and IL anions together act as strong electron donors (for example, halogen and
pseudo-halogen ions) during the dissolution process of lignocellulosic biomass
materials, whereas hydrogen atoms of hydroxyl groups and IL cations act as
electron acceptors (Wang et al. 2012). The dissolved cellulose can be regenerated
again by addition of antisolvents such as water, alcohols, ethers, or ketones. When
an anti-solvent is added to the IL–cellulose system, the IL ions form hydrogen
bonds with water molecules and are displaced into the aqueous phase, whereas
cellulose (which previously interacted with IL) are expelled and reunites its intra-
and intermolecular hydrogen bonds and is then precipitated from the mixture.

4 Conclusion

Pretreatment is an inevitable step for lignocellulosic biomass pretreatment irre-

spective of the final product i.e., bioethanol, biogas or biodiesel. As pretreatment is
most energy consuming step research has been going on to low cost, low energy
consuming process. The scope of pretreatment is vast and its application also varies
depending on the application and quality of biomass. The pretreatment method for
biogas production is not the same as the pretreatment process for biodiesel or
bioethanol process. The very next step of pretreatment method is saccharification or
enzymatic treatment process. Although enzymatic treatment processes are mild in
Pretreatment of Lignocellulosic Biomass Toward … 219

nature, cost of enzyme is huge. Therefore, recent research aims to reduce the
application of enzyme in biomass-based industries. The various modes of pre-
treatments are described throughout the chapter which shows the recent trends
toward pretreatment process.

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