Bioresource Technology 199 (2016) 4958

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Review

The role of pretreatment in improving the enzymatic hydrolysis

of lignocellulosic materials
Shaoni Sun a, Shaolong Sun a, Xuefei Cao a, Runcang Sun a,b,
a
b

Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing 100083, China
State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China

h i g h l i g h t s
 The structure and factors constrain the digestibility of biomass were reviewed.
 The features of the most important pretreatments were discussed.
 Combined pretreatments were applied to optimize the utilization of lignocellulose.
 Three combined pretreatments showed great potential for large-scale application.

a r t i c l e

i n f o

Article history:
Received 30 June 2015
Received in revised form 10 August 2015
Accepted 11 August 2015
Available online 21 August 2015
Keywords:
Lignocellulose
Pretreatment
Enzymatic hydrolysis
Digestibility
Cellulosic ethanol

a b s t r a c t
Lignocellulosic materials are among the most promising alternative energy resources that can be utilized
to produce cellulosic ethanol. However, the physical and chemical structure of lignocellulosic materials
forms strong native recalcitrance and results in relatively low yield of ethanol from raw lignocellulosic
materials. An appropriate pretreatment method is required to overcome this recalcitrance. For decades
various pretreatment processes have been developed to improve the digestibility of lignocellulosic biomass. Each pretreatment process has a different specificity on altering the physical and chemical structure of lignocellulosic materials. In this paper, the chemical structure of lignocellulosic biomass and
factors likely affect the digestibility of lignocellulosic materials are discussed, and then an overview about
the most important pretreatment processes available are provided. In particular, the combined pretreatment strategies are reviewed for improving the enzymatic hydrolysis of lignocellulose and realizing the
comprehensive utilization of lignocellulosic materials.
2015 Elsevier Ltd. All rights reserved.

1. Introduction
Bioethanol derived from biomass is considered as one of the
most promising alternatives to fossil fuels due to its clean burning,
higher octane number, broader flammability limits, higher flame
speeds and higher heats of vaporization than gasoline, which can
be blended with gasoline or directly used as neat alcohol in dedicated engines (Kim and Choi, 2010). However, most bioethanol,
especially starch ethanol, is derived from food-related crops. The
large scale production of starch ethanol will cause serious food
issues. Cellulose is a major component of plant cell walls, which
has similar polysaccharide structure to starch. Cellulose cannot
be digested by humans or animals, which makes it an ideal candidate for bioethanol production. However, cellulosic ethanol pro Corresponding author at: Beijing Key Laboratory of Lignocellulosic Chemistry,
Beijing Forestry University, Beijing 100083, China. Tel./fax: +86 10 62336972.
E-mail address: rcsun3@bjfu.edu.cn (R. Sun).
http://dx.doi.org/10.1016/j.biortech.2015.08.061
0960-8524/ 2015 Elsevier Ltd. All rights reserved.

duction involves a much more expensive process than starch

ethanol. Sustainable production of cellulosic ethanol from lignocellulose materials is one of the greatest challenges in biorefinery.
Generally, lignocellulosic biomass is mainly comprised of cellulose (3850%), hemicelluloses (2332%), and lignin (1525%), as
well as small amounts of extractives (McKendry, 2002). Cellulose
and hemicellulose are chain polysaccharides, while lignin is a
heterogeneous, cross-linked three-dimensional phenyl-propane
polymer, which closely associates with cellulose and hemicelluloses (Mussatto et al., 2008). These components are strongly intermeshed and bonded through covalent or non-covalent bonds
forming the lignocellulosic matrix. The structural characteristics
of lignocellulosic materials form strong native recalcitrance to
enzymes and result in relatively low digestibility of raw lignocellulosic materials. An appropriate pretreatment method is required to
overcome this recalcitrance and makes polysaccharides easily
available for enzyme digestion.

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S. Sun et al. / Bioresource Technology 199 (2016) 4958

To date, various pretreatment processes have been developed,

which can be broadly categorized into physical, chemical,
physico-chemical, biological processes and also by some combinations of these processes. Since each pretreatment process has its
advantages and disadvantages, some combination of pretreatment
processes may not only enhance the accessibility of the enzymes to
cellulose but also facilitate the recovery of the associated lignin
and hemicelluloses for producing high value products. This article
first discusses the structure of lignocellulose and factors that constrain the enzymatic hydrolysis of lignocellulosic materials, and
then reviews the most commonly used pretreatment processes in
improving the digestibility of lignocellulosic materials, with a special emphasis on combined pretreatment strategies.

2. Structure of lignocellulose and its major components

Cellulose is a polysaccharide composed of a linear chain of b-1,4
linked D-glucose units with a degree of polymerization ranged from
several hundreds to over ten thousands, which is the most abundant organic polymer on the earth. Because of the polysaccharide
structure of cellulose, a large amount of hydroxyl groups exists
along the cellulose backbone. These hydroxyl groups can form
well-ordered hydrogen bonding networks, resulting in a compact
crystalline structure. Additionally, partial cellulose chains are
arrayed irregularly, resulting in the amorphous region of cellulose.
Hemicelluloses are also polysaccharide polymers but derived
from several sugars and uronic acids. Generally, hemicelluloses
are branched heteropolymers with a degree of polymerization
around 200. The content and chemical structure of hemicelluloses
can be varied with different plants and their parts. Xylan is the
most abundant hemicelluloses in nature, which mainly contains
b-D-xylopyranosyl residues linked by 1,4 glycosidic bonds. Xylan
backbone is usually highly substituted with arabinose, glucuronic
acid, acetyl groups or xylose (Sun et al., 2005). Because of their
branched nature of hemicelluloses, hemicelluloses are likely exist
in plants in amorphous form.
Lignin is a heterogeneous three-dimensional network macromolecule mainly constructed from the oxidative combinatorial
coupling of p-hydroxycinnamyl alcohol monolignols, especially phydroxycinnamyl, coniferyl and sinapyl alcohols (Ralph et al.,
2004). Except the three major monolignols, traces of hydroxycinnamyl aldehydes, acetates, p-coumarates, p-hydroxybenzoates,
and tyramine ferulate were also observed (Boerjan et al., 2003).
These monolignols show different abundances in different plant
types, plant tissues and cell wall layers. Additionally, there are various possible linkages among these monolignols, such as b-O-4, b5, b-1, b-b, 5-5, and 4-O-5 (Sette et al., 2011). Due to the variety of
monolignols and the randomness of linkages among these monolignols, the actual structure of the lignin macromolecule is quite
complex (Ralph et al., 2004).
Cellulose (both crystalline and amorphous), branched hemicelluloses, and lignin network interact closely with each other and
construct the macroscopic plant cell walls. During the formation
of plant cell walls, lignocellulose is not just simply physical blending of cellulose, hemicelluloses, and lignin. These components are
strongly intermeshed and bonded through non-covalent bonds
and covalent cross-linkages (Prez et al., 2002). There are many
possible linkages among these components. The covalent linkages
between hemicelluloses and lignin and the absence of covalent
linkages between hemicelluloses and cellulose have been confirmed, but the covalent linkages between lignin and cellulose
are still uncertain (Jin et al., 2006; Zhou et al., 2010). Generally,
ester and ether linkages between hydroxyl groups of polysaccharides and a-carbanol of phenylpropane subunits in lignin are the
major connections in wood (Jeffries, 1990). In grasses, ferulic and

p-coumaric acids are likely to form inter-molecular ester-ether

bridges between hemicelluloses and lignin (Jeffries, 1990).
3. Factors affect the enzymatic hydrolysis of lignocellulosic
materials
The enzymatic hydrolysis of lignocellulosic materials can be
influenced not only by the effectiveness of the enzymes but also
by the physical, chemical, and morphological characteristics of
the lignocellulosic materials. Most factors that affect the enzymatic
hydrolysis of lignocellulosic materials can be primarily divided in
two groups: enzyme-related and substrate-related factors (Alvira
et al., 2010). Enzyme-related factors are mainly focused on improving the enzyme activity, including endproduct inhibition, thermal
stability, synergism, and adsorption (Zhao et al., 2009a).
Substrate-related factors are mainly concentrated in improving
the accessibility of enzymes to cellulose. The accessibility of cellulose can be affected by many factors, such as cellulose crystallinity,
contents and distributions of lignin and hemicelluloses, and available surface area (Alvira et al., 2010). These substrate-related factors and the inhibitors originated from lignocellulosic materials
degradation are discussed.
3.1. Cellulose
Regardless of the interaction of hemicelluloses and lignin with
cellulose, the crystallinity of cellulosic substance is an important
factor in determining its digestibility. Generally, a considerable
amount of cellulose exists in the form of crystalline state, which
is compact and highly resistant to chemical and biological hydrolysis. Because of the big size of cellulase (318 nm), cellulase can
only digest the cellulose crystal from its surfaces, resulting in a relatively low enzymatic hydrolysis rate (Zhao and Chen, 2013). However, amorphous cellulose can be easily hydrolyzed by enzymes.
Regenerated cellulose with low crystallinity showed much higher
enzymatic hydrolysis than the cellulose samples with high crystallinity. The initial enzymatic hydrolysis rate of cellulose is closely
associated with the crystallinity index of cellulose.
3.2. Hemicelluloses and lignin
Because hemicelluloses and lignin are closely associated with
cellulose through non-covalent and covalent linkages, the content
and distribution of hemicelluloses and lignin also show obvious
effect on the enzymatic hydrolysis of lignocellulose (Alvira et al.,
2010). The existence of hemicelluloses and lignin largely impedes
the contact of enzymes with cellulose, resulting in a very low enzymatic hydrolysis rate of raw materials (Pan et al., 2005). Furthermore, the non-productive binding of enzymes to lignin also
restricts the hydrolysis of cellulose (Pan et al., 2005). Removal of
lignin and hemicelluloses can increase the pore size and accessible
surface of the lignocellulosic materials and reduce the nonproductive adsorption of enzymes to lignin (Mussatto et al.,
2008; Taherzadeh and Karimi, 2008). Therefore, the removal of
hemicelluloses and lignin can significantly improve the digestibility of lignocellulosic materials (Mussatto et al., 2008). Many pretreatment processes are aimed to reduce the recalcitrance of
lignocellulosic materials by removing hemicelluloses and/or lignin.
3.3. Accessible specific surface area
The accessible specific surface area is a crucial factor that affects
the enzymatic hydrolysis of lignocellulosic materials. The particle
size and pore volume (or porosity) show great influence on the
accessible specific surface area of lignocellulosic materials. A rela-

S. Sun et al. / Bioresource Technology 199 (2016) 4958

tively high enzymatic hydrolysis rate and glucose yield can be

achieved with the reduction of the lignocellulose particle size
(Yeh et al., 2010). As lignocellulosic materials are dispersed into
nanoscale, the accessible specific surface area of lignocellulose will
increase dramatically. Most cellulose is exposed to the enzymes,
resulting in almost complete digestion of cellulose within a short
time (Martin-Sampedro et al., 2012). Previous studies also suggested that the pore volume of lignocellulosic materials is linearly
correlated with its initial enzymatic hydrolysis rate (Grethlein,
1985). The digestibility of lignocellulose can be significantly
enhanced when the pores of the substrate are large enough to
accommodate both large and small enzyme components to maintain the synergistic action of the cellulase enzyme system.
3.4. Inhibitors of enzymes
Previous works reported that the activity of cellulase can be
inhibited by many substances, such as cellubiose, xylooligosaccharides, glucose, and some degradation products of lignocellulose formed during pretreatment, such as weak acids, furfural,
5-hydroxymethyl furfural, and soluble phenolic compounds
(Andric et al., 2010; Zhao et al., 2004). Cellobiose combined with
tryptophan can locate near the active site of cellobiohydrolase
forming steric hindrance to prevent cellulose chains reaching the
active sites of cellulase (Zhao et al., 2004). The addition of bglucosidase during the enzymatic hydrolysis can reduce the inhibition of cellubiose to some degree (Sun and Cheng, 2002). However,
a high content of glucose in the hydrolysate inhibit both the activities of b-glucosidase and cellulase (Andric et al., 2010). Xylooligosaccharides decrease the initial hydrolysis rate and final glucose yield even at a low concentration (Qing et al., 2010). Weak
acids, furfural, HMF, and soluble phenolic compounds are probably
released during the steam explosion and hydrothermal pretreatment, which can inhibit the enzymatic hydrolysis of lignocellulosic
materials, as well as the subsequent fermentation (Jonsson et al.,
2013).
4. Pretreatment of lignocellulosic materials
Since the raw lignocellulosic materials exhibit strong recalcitrance to enzymes, a suitable pretreatment process is required to
facilitate the access of enzymes to the plant polysaccharides. Based
on the structure of lignocellulosic materials and the factors that
constrain the enzymatic hydrolysis of lignocellulosic materials, a
good pretreatment method should weaken the factors that constrain the enzymatic hydrolysis of lignocellulose as much as possible on the premise of economy. In the past few decades, different
pretreatment processes have been developed to improve the
digestibility of lignocellulosic materials (Alvira et al., 2010). In
the following sections, the most important pretreatment processes
are reviewed to discuss their impact on lignocellulosic materials
and their effects on improving the enzymatic hydrolysis.
4.1. Physical pretreatments
Physical pretreatments are aimed to increase the accessible
specific surface area of lignocellulosic materials to enzymes by
reducing their particle size or disrupting their structural regularity.
Different types of physical processes, such as mechanical comminution (chipping, grinding, or milling) and irradiation (gamma
rays, electron beam or microwave) have been used to improve
the digestibility of lignocellulosic materials (Taherzadeh and
Karimi, 2008).
Mechanical comminution can ground lignocellulosic materials
into different size (ranging from lm to mm) (Yeh et al., 2010).

51

Among these mechanical comminution processes, chipping or/

and grinding is required to disperse massive lignocellulosic materials into small pieces and facilitate the following pretreatment
processes. After chipping or/and grinding, various milling methods
(ball milling, two-roll milling, hammer milling, colloid milling, and
vibro energy milling) can be used to mill lignocellulosic biomass
into fine powder (Taherzadeh and Karimi, 2008). Milling can significantly reduce the particle size and degree of crystallinity of lignocellulosic materials, and consequently improve their enzymatic
hydrolysis by extending the milling time (Yeh et al., 2010). Previous work reported that lignocellulosic nanofibrils obtained from
a high-pressure fluidizer almost completely hydrolyzed by
enzymes within short time (Martin-Sampedro et al., 2012). However, the energy consumption of mechanical comminution is closely related to the final particle size of lignocellulosic materials.
Therefore, a relatively high energy input is required to achieve a
high enzymatic hydrolysis rate and fermentable sugar yield, which
makes milling not economically feasible.
Irradiation also has been applied to improve the enzymatic
hydrolysis of lignocellulosic materials. Since irradiation cannot
remove hemicelluloses or lignin from the lignocellulosic materials
or reduce their particle size, irradiation is generally used as assistance to other pretreatment processes. It was reported that microwave assisted chemical pretreatments are more effective than the
conventional heating chemical pretreatments (Moretti et al., 2014).
Electron beam or gamma rays combined with mechanical crushing,
hydrothermal, or chemical pretreatments can further enhance the
enzymatic hydrolysis of lignocellulosic materials as compared with
no electron beam or gamma rays assistance (Duarte et al., 2012).
Previous literatures reported that after pretreatment with electron
beam (50 kGy), the following hydrothermal or dilute acid pretreatment significantly enhanced the digestibility of sugarcane bagasse,
which increased the enzymatic hydrolysis yield of cellulose by 20%
than the hydrothermal alone and by 30% than the dilute acid pretreatment alone (Duarte et al., 2012). Although irradiation pretreatment showed a positive effect on improving the digestibility
of lignocellulosic materials, the installation cost is too expensive
for industrial applications (Taherzadeh and Karimi, 2008). Therefore, except preliminary chipping or/and grinding, other physical
processes are rarely used due to their high costs.
4.2. Chemical pretreatments
4.2.1. Alkali pretreatment
Alkali pretreatment is the most commonly used method to
remove lignin and hemicelluloses from lignocellulosic materials
and disperse bulk lignocellulosic materials into lignocellulosic
fibers. During the alkali pretreatment, the ester linkages in hemicelluloses and lignin are easily broken down under alkaline conditions. The ether linkages in hemicelluloses and lignin can also be
destroyed at relatively high temperature. The cleavages of these
linkages significantly promote the solubilization of hemicelluloses
and lignin, resulting in the exposure of cellulose to enzymes. Various alkaline reagents, such as NaOH, Ca(OH)2, KOH, Na2CO3 and
aqueous ammonia, have been used to pretreat many lignocellulosic
materials. Among these alkaline reagents, NaOH and Ca(OH)2 are
the most commonly used.
Alkali pretreatment can alter the structure of lignocellulosic
matrix via two approaches: (1) swelling cellulose; and (2) removing lignin and hemicelluloses. Cellulose can be swelled or even dissolved in aqueous NaOH or NaOH/urea solutions at cold
temperature (around 15 C) (Cai and Zhang, 2005). After swelling,
the crystalline structure of cellulose is interrupted, leading to the
significant decrease of the crystallinity of lignocellulosic biomass.
Zhao et al. (2008) investigated the enzymatic hydrolysis behaviors
of spruce pretreated with aqueous NaOH and aqueous NaOH/urea

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S. Sun et al. / Bioresource Technology 199 (2016) 4958

at different temperatures ( 15 C, 23 C, and 60 C). Results

showed that the enzymatic hydrolysis efficiency of spruce pretreated at 15 C was much higher than those pretreated at high
temperatures, suggesting that the high swelling ability of cellulose
in aqueous NaOH or aqueous NaOH/urea at a low temperature was
likely to lead to the high accessibility of lignocellulosic materials to
enzymes and further cause a relatively high enzymatic hydrolysis
efficiency. Alkali pretreatment at a high temperature breaks down
lignin and hemicelluloses into soluble fragments and makes more
cellulose exposed to enzymes. Barley straw pretreated with aqueous NaOH (0.52.0%) at 105 C for 10 min resulted in a maximum
84.8% removal of lignin and 79.5% removal of hemicelluloses,
meanwhile, a maximum reducing sugar yield of 86.5% was
achieved during the following enzymatic hydrolysis process
(Haque et al., 2012).
Ca(OH)2, called lime as well, has been applied to pretreat various lignocellulosic materials for further enzymatic hydrolysis. As
compared with aqueous NaOH pretreatment, lime pretreatment
has a lower cost and better environmental benefit due to its easy
recovery by reaction with CO2 (Alvira et al., 2010). Because of the
low solubility of lime at a high temperature, lime pretreatment is
usually performed at a relatively low temperature for a long reaction time (Kim and Holtzapple, 2005). During the lime pretreatment, lignin is gradually removed over the whole pretreatment
process. Non-oxidative lime pretreatment of corn stover at 25
55 C for 16 weeks removed 43.648.4% of the original lignin in
corn stover, while oxidative lime pretreatment removed up to
57.887.5% of the initial lignin (Kim and Holtzapple, 2005). The
enzymatic digestibility of corn stover was significantly improved
after lime pretreatment under the oxidative condition due to the
removal of most lignin and acetyl groups. Additionally, green
liquor that mainly consisted of Na2CO3 and Na2S for kraft pulping
has also been applied for alkali pretreatment. About 45% of lignin
in corn stover was removed after green liquor pretreatment and
about 70% of polysaccharides were converted into fermentable
sugars in the subsequent enzymatic hydrolysis (Gu et al., 2012).

Except mineral acids, some organic acids, such as formic, acetic,

propionic, fumaric, maleic and oxalic acids, have also been applied
to pretreat lignocellulosic materials. Because of their relatively low
acidities and high solubility for lignin, relatively high concentrations of organic acids are usually applied to pretreat lignocellulosic
materials. Miscanthus x giganteus pretreated in a formic acid/acetic
acid/water ratio of 40/40/20% solution at 107 C for 3 h resulted in
79.6% removal of lignin with a maximum cellulose conversion of
75.3% (Vanderghem et al., 2012).

4.2.2. Acid pretreatment

Since the glucosidic bonds of hemicelluloses and cellulose are
susceptible to acid, acid pretreatment can be applied to solubilize
partial hemicelluloses from lignocellulosic materials and further
improve the accessibility of enzymes to cellulose. Both concentrated and diluted acids have been used to pretreat various lignocellulosic materials. However, the concentrated acid pretreatments
are likely to cause severe degradation of cellulose, high concentration of inhibitors and serious equipment corrosion, thus the concentrated acids are less attractive (Alvira et al., 2010).
Various diluted mineral acids, such as H2SO4, HCl, H3PO4, and
HNO3, have been applied to pretreat different lignocellulosic materials (Alvira et al., 2010). Plenty of studies investigated the effects
of dilute H2SO4 pretreatment conditions on the recovery of both
xylose and glucose during the pretreatment and subsequent enzymatic hydrolysis processes (Garcia et al., 2014; Hernandez et al.,
2013). Dilute H2SO4 pretreatment is generally performed at temperatures from 100 to 200 C with a H2SO4 concentration of 0.5
2.5%. Residual empty pods of Moringa oleifera pretreated with
dilute H2SO4 led to above 87% of overall cellulose recovery with
24.750.2% of xylan recovery (Hernandez et al., 2013). Jatropha curcas fruit shells pretreated with 0.52.5% H2SO4 at 110150 C for
1545 min resulted in above 80% cellulose conversion in the following enzymatic hydrolysis or SSF process (Garcia et al., 2014).
However, the mineral acid pretreatment is not conducive to the
removal of lignin. After pretreated with mineral acids, lignocellulosic residues with high contents of lignin are usually obtained. A
following delignification process is always required to further
improve the digestibility of the lignocellulosic residues.

4.2.4. Organosolv process

In organosolv process, organic or aqueous organic solvents are
mainly used to extract lignin from lignocellulosic materials, which
can increases the pore volume and accessible surface area of lignocellulosic materials and significantly reduces their lignin contents.
Many organic solvents, such as ethanol, methanol, acetone, organic
acid, organic peracid, acetone, and ethylene glycol, have been utilized to pretreat various lignocellulosic materials (Zhao et al.,
2009b). Among these possible solvents, ethanol is the most favorable solvent because of its low toxicity and easy recovery.
Since most organic solvents (except organic acids) are too mild
to destroy the linkages and structure of lignocellulosic materials,
organosolv process with the assistance of various catalysts (acids,
salts, and alkalis) is usually recommended to pretreat different lignocellulosic materials (Huijgen et al., 2011; Park et al., 2010).
Huijgen et al. (2011) investigated the organosolv process of willow
wood and wheat straw in ethanol under the catalysis of H2SO4, HCl,
and MgCl2. It was reported that the addition of catalysts improved
the fractionation of both willow wood and wheat straw. Among
these catalysts, acids can significantly promote the removal of
hemicelluloses. The maximum enzymatic cellulose conversion
was 87% for willow wood and 99% for wheat straw as compared
with 74% (willow wood) and 44% (wheat straw) for the noncatalytic process. Park et al. (2010) evaluated the effect of NaOH
catalytic ethanol organosolv process on improving the digestibility
of pitch pine. Organosolv pretreatment with 2% NaOH significantly
reduced the lignin content from 26.17% to 11.72%, whereas lignin
content was almost unchanged without the addition of NaOH.
Comparing with less than 10% of enzymatic cellulose conversion

4.2.3. Oxidative delignification

Oxidative delignification is a commonly used method applied in
pulp bleaching. Generally, O3, O2, H2O2, ClO2, NaClO, and Cl2 can be
used as oxidation reagents. During the oxidative delignification
process, oxidation reagents release a large number of free radicals,
resulting in remarkable oxidative fragmentation and removal of
lignin from lignocellulosic matrix. Wheat and rye straw pretreated
with O3 could significantly reduce their lignin content, while little
degradation of hemicelluloses and negligible loss of cellulose were
observed during the oxidative delignification process (GarcaCubero et al., 2009). Enzymatic hydrolysis yields of up to 89%
and 57% were achieved for O3 pretreated wheat and rye straw, as
compared with 29% and 16% for wheat and rye straw without pretreatment, respectively. Oxidative delignification is usually performed at relatively mild conditions and inhibitory compounds
are hardly produced during the oxidation process (Sun and
Cheng, 2002). With the assistance of oxidation reagents, almost
all lignin can be removed from lignocellulosic materials with the
remaining of most cellulose and hemicelluloses, which is conducive to maximize the recovery of fermentable sugars (Qi et al.,
2009). However, since the cost of oxidative delignification is much
higher than traditional alkali or acid pretreatments, oxidative
delignification is usually used as an assistance of other pretreatments, especially alkali pretreatments, to remove residual lignin
in lignocellulosic materials (Qi et al., 2009).

S. Sun et al. / Bioresource Technology 199 (2016) 4958

of raw material, the conversion of these catalytic pretreated samples was enhanced up to 60% (Park et al., 2010).
Comparing to other chemical pretreatments, organosolv process
has many advantages, such as easy recovery of solvent by distillation, low environmental impact, and recovery of high quality lignin
as by-product (Pan et al., 2006). The high quality lignin can be further applied in many fields such as antioxidants, biodispersants,
polyurethane foams, and epoxy resins (Sun et al., 2014b). Although
most solvents used in the organosolv process can be recycled from
the reactor to reduce the operational cost, the high price and
potential hazards of handling large volumes of organic solvents
limit the utilization of organosolv process.
4.2.5. Ionic liquids (ILs) pretreatment
Ionic liquids (ILs) are salts typically composed of large organic
cations and small inorganic anions. The properties of ILs can be
varied by adjusting the constituents of the cations and anions
(Zavrel et al., 2009). Some ILs are powerful solvents for lignocellulosic materials, which can dissolve cellulose or lignocellulosic biomass forming homogeneous solutions (Zavrel et al., 2009). Li et al.
(2010) investigated the effect of various ILs pretreatment on enzymatic hydrolysis of corn cob. It was found that ILs containing phosphate or chloride groups enhanced the yield of reducing sugar
significantly. Theoretically, the accessible specific surface area of
lignocellulosic biomass is extensively enlarged after dissolution,
thus the enzymes can contact and hydrolyze polysaccharides much
easily. However, because enzymes hardly survive in ILs, the enzymatic hydrolysis of cellulose cannot be performed directly in ILs.
Therefore, the homogeneous solutions containing lignocellulose
are generally regenerated in the anti-solvents of ILs to prepare
regenerated lignocellulosic biomass. Lignocellulosic materials
regenerated from ILs always show much lower crystallinity and
higher accessibility to enzymes than the untreated materials
(Zhao et al., 2009a). The cellulose in the regenerated lignocellulose
can be efficiently hydrolyzed into glucose by cellulase. Additionally, lignocellulosic materials regenerated in appropriate buffer
solutions can form swelling lignocellulosic hydrogel and the enzymatic hydrolysis rate of the regenerated lignocellulose can be
improved by several times to tens of times as compared with the
raw sample (Cao et al., 2014b).
For lignocellulosic materials containing lignin, due to the components are strongly intermeshed and bonded, more severe pretreatment conditions are required to dissolve these
lignocellulosic materials in ILs. Meanwhile, most ILs are very
expensive. Therefore, further research is required to adjust the solubility of ILs for lignocellulosic materials, improve the economics
of ILs pretreatments and construct effective IL recovery procedures.
4.3. Physico-chemical pretreatments
4.3.1. Steam explosion
Steam explosion is one of the most commonly used physicochemical methods to pretreat lignocellulosic materials. During
the steam explosion, chipped lignocellulosic materials are subjected to high pressure saturated steam at high temperature for
several seconds to several minutes, and then the pressure is
instantly reduced to atmospheric pressure. The bulk lignocellulosic
materials will be exploded into separated fibers with the rapid
release of pressure. During this process, hemicelluloses and lignin
can be decomposed and removed from the lignocellulosic materials in different degrees (Pan et al., 2005). Steam explosion shows
several attractive features, such as low environmental influence,
less hazardous process chemicals, and high energy efficiency
(Alvira et al., 2010).
Steam explosion can be directly applied to chipped lignocellulosic materials without the addition of other chemicals. Wheat

53

straw samples pretreated with steam at 170220 C were used to

explore their enzymatic hydrolysis behaviors (Horn et al., 2011).
Results showed that the maximum enzymatic hydrolysis yield
was obtained after steam exploded at 210 C for 10 min. Although
similar glucose yield could also be achieved at more severe conditions, inhibitors such as aromatic compounds and dehydration byproducts formed under harsh steam explosion conditions, which
further impacted the subsequent fermentation process. Therefore,
a washing step for detoxification is required. Water washing can
effectively reduce the inhibitor contents in the steam exploded lignocellulosic materials, which is beneficial to the following enzymatic hydrolysis and fermentation processes.
Since a large number of enzymatic hydrolysis and fermentation
inhibitors formed under severe steam explosion conditions, different impregnating reagents have been applied to improve the isolation of hemicelluloses at relatively mild steam explosion
conditions. Among a variety of impregnating reagents, SO2 and
H2SO4 are the most widely explored. Boussaid et al. (2000) investigated the chemical component changes and enzymatic hydrolysis
behaviors of SO2 impregnated wood chips pretreated under different steam explosion conditions. Although the enzymatic digestibility of the steam exploded wood chips increased with the increment
of the steam exploded severity, a high steam explosion severity
resulted in an excessive loss of hemicelluloses-derived sugars.
Medium steam explosion severity not only improved the enzymatic digestibility of the solids but also maximized the recovery
of total sugars (Boussaid et al., 2000). Martin et al. (2002) explored
the enzymatic hydrolysis and fermentability of SO2 and H2SO4
impregnated sugarcane bagasse pretreated by steam explosion at
205 C for 10 min. Higher yields of xylose and total sugar were
obtained for the SO2-impregnated sugarcane bagasse, while higher
glucose yield but lower total sugar yield were observed for the
H2SO4-imprgnated bagasse. H2SO4 accelerated the hydrolysis of
polysaccharides and catalyzed the formation of enzymatic hydrolysis and fermentation inhibitors. However, relatively low contents
of inhibitors were observed in the hydrolyzates from SO2impregnated and non-impregnated sugarcane bagasse. Similar
results were also observed in H2SO4- and SO2-impregnated corn
stover and aspen chips (De Bari et al., 2007; Varga et al., 2004).
4.3.2. Hydrothermal pretreatment
Generally, the major components of lignocellulosic materials
show different thermal stabilities. The decomposition of hemicelluloses is usually easier than cellulose and lignin. Since most hemicelluloses and partial lignin can be removed before the degradation
of cellulose under hydrothermal condition, hydrothermal pretreatment has been applied on various lignocellulosic materials to
improve their enzymatic digestibility. Because severe degradation
of cellulose occurs at temperature higher than 240 C, the
hydrothermal temperatures are usually in a wide range of 160
240 C (Cao et al., 2014a; Sun et al., 2014a). Xiao et al. (2014) investigated the chemical composition changes of bamboo at different
hydrothermal pretreatment conditions (at 140200 C for 10
120 min) and further explored their enzymatic hydrolysis behaviors. Most hemicelluloses were gradually removed from bamboo
with the increment of hydrothermal intensity, while only a small
amount of cellulose and lignin were degraded. After pretreatment
at 200 C for 120 min, a maximum glucose conversion of 75.7% was
achieved comparing to 15.7% of the untreated sample (Xiao et al.,
2014). The hydrothermal liquids rich in hemicelluloses or
hemicelluloses-derived sugars can be further converted into furfural or other chemicals. Additionally, hydrothermal pretreatment
significantly increased the pore volume and specific surface area of
beech wood, resulting in the increase of enzymatic digestibility of
beech wood from less than 7 wt.% of the raw material to 70 wt.%
of the pretreated residues (Nitsos et al., 2013). Hydrothermal pre-

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treatment is very attractive because of its outstanding advantages,

such as no catalyst requirement, low reactor cost, relatively low
inhibitor content in pretreated samples, and direct utilization of
wet or fresh lignocellulosic materials.
Catalytic hydrothermal pretreatments have also been developed to assist the removal of hemicelluloses or lignin and optimize
the recovery of both hemicellulosic and cellulosic sugars (Sun et al.,
2014a). Dilute alkali catalyzed hydrothermal pretreatment significantly reduced the lignin content in sugarcane bagasse and
enhanced its enzymatic hydrolysis rate and glucose yield (Miura
et al., 2012). H2SO4-catalyzed hydrothermal pretreatment was also
used to pretreat lignocelluloses to improve their digestibility (Lu
et al., 2009). Hemicelluloses in the rapeseed straw can be efficiently removed by H2SO4-catalyzed hydrothermal pretreatment
at a high solid loading (20%). Under the optimal pretreatment conditions (20% solids content with addition of 1% H2SO4 at 180 C for
10 min), 27.3 g/L xylose was released corresponding to 68.4% of the
theoretical yield and 19 g/L ethanol was produced corresponding
to 67% of the theoretical yield after enzymatic hydrolysis and fermentation (Lu et al., 2009).
4.3.3. Ammonia fiber explosion (AFEX)
In AFEX process, lignocellulosic materials are exposed to liquid
ammonia at temperature around 60100 C and high pressure
(250300 psi) for a period of time, and then the pressure is suddenly released (Alvira et al., 2010). Liquid ammonia causes the
swelling of lignocellulose and the crystal structure change of cellulose from native cellulose I to cellulose III. The rapid release of
pressure would cause the physical disruption and crystallinity
reduction of lignocellulosic materials. AFEX pretreatment removes
little lignin and hemicelluloses from lignocellulosic materials, but
it can improve the enzymatic hydrolysis efficiency of various lignocellulosic materials at low enzyme loadings. It was reported that as
the coastal Bermuda grass was pretreated with AFEX at 100 C for
30 min, its chemical composition almost did not change except
some relocalization of lignin, but as high as 94.8% of sugar yield
was achieved in the following enzymatic hydrolysis process (Lee
et al., 2010). Switchgrass pretreated with liquid ammonia at near
100 C for 5 min resulted in a 93% glucan conversion, as compared
with 16% of the un-pretreated samples (Alizadeh et al., 2005). Similarly, as corn stover was pretreated by AFEX at 90 C for 5 min,
approximately 100% of cellulose and 80% of hemicelluloses were
converted into fermentable sugars in the following enzymatic
hydrolysis process (Teymouri et al., 2005). AFEX is a promising pretreatment process with several advantages, such as environmental
friendly, high energy efficiency, mild reaction temperature, and
negligible the formation of inhibitors. Although most of the ammonia can be recovered and recycled back into AFEX reactor to reduce
the operating cost, liquid ammonia is very expensive for practical
application.
4.3.4. Supercritical CO2 (SC-CO2) pretreatment
SC-CO2 is an important commercial and industrial extraction
solvent with many advantages, such as low cost, non-toxicity, nonflammability, easy recovery, and low environmental impact
(Taherzadeh and Karimi, 2008). SC-CO2 can diffuse through interspaces like gas and dissolve materials like liquid. The size of CO2
molecule is comparable to H2O and NH3, thus SC-CO2 also has been
applied to pretreat lignocellulosic materials. Explosive release of
CO2 pressure consequently increases the accessible surface area
of the lignocellulosic materials (Alvira et al., 2010). Furthermore,
CO2 can form carbonic acid in the presence of moisture, which
favors the hydrolysis of polymers (Alvira et al., 2010; Sun and
Cheng, 2002). The moisture content presented in lignocellulosic
materials is an important factor that significantly influences the
following enzymatic digestibility of the SC-CO2 pretreated ligno-

cellulosic materials. The enzymatic digestibility of SC-CO2 pretreated aspen and southern yellow pine showed an increase
trend with the increase of moisture content, whereas SC-CO2 pretreatment showed no promotion effect on the enzymatic hydrolysis of absolutely dried aspen and southern yellow pine (Kim and
Hong, 2001). Lv et al. (2013) reported that during the SC-CO2 pretreatment addition of waterethanol as co-solvents can also efficiently increase the lignin removal and further improve the
enzymatic hydrolysis of corn stover. Additionally, because cellulase is relatively stable in SC-CO2 at 35 C, simultaneous SC-CO2
pretreatment and enzymatic hydrolysis in one step has been
applied to produce fermentable sugars (Muratov and Kim, 2002).
It was reported that simultaneous SC-CO2 pretreatment and enzymatic hydrolysis resulted in a higher enzymatic hydrolysis rate and
glucose yield than discrete SC-CO2 pretreatment and enzymatic
hydrolysis process (Muratov and Kim, 2002).
4.4. Biological pretreatment
Microorganisms or enzymes have also been applied to pretreat
various lignocellulosic materials before enzymatic hydrolysis.
Microorganisms such as brown-, white- and soft-rot fungi have
been used for selective degradation of lignin and hemicelluloses
(Snchez, 2009). Among these fungi, white-rot fungi are the most
widely used microorganisms (Alvira et al., 2010; Taherzadeh and
Karimi, 2008). Various white-rot fungi such as Phanerochaete
crysosporium, Ceriporia lacerata, Cyathus stercoletus, Ceriporiopsis
subvermispora, Pycnoporus cinnabarinus and Pleurotus ostreatus
have been used to pretreat different lignocellulosic materials
(Hatakka, 1983). Lignin-modifying enzymes, such as laccase, lignin
peroxidases, and manganese peroxidases, produced from white-rot
fungi showed obvious effects on lignin degradation (Kumar and
Wyman, 2009). Rice straw pretreated with Pleurotus ostreatus for
60 d resulted in the degradation of 41% of Klason lignin, 17% of cellulose and 48% of hemicelluloses based on the untreated rice straw
(Taniguchi et al., 2005). In the pretreated rice straw, 52% holocellulose and 44% cellulose were hydrolyzed with a commercial cellulase for 48 h, as compared to 33% holocellulose and 32% cellulose
in the untreated rice straw. After wheat straw was pretreated with
Pleurotus ostreatus for five weeks, 35% of the original wheat straw
was converted into reducing sugars in the following enzymatic
hydrolysis as compared with 12% of the un-pretreated sample
(Hatakka, 1983). Although biological pretreatment has many
advantages, such as less energy intensive, no chemicals requirement and mild pretreatment conditions, a very long pretreatment
time (several weeks) is always required to achieve a relatively high
enzymatic hydrolysis rate, which severely limits its industrial
application.
4.5. Features of different pretreatment processes
For decades, various pretreatment processes have been
described and explored to improve the enzymatic digestibility of
different lignocellulosic materials. All these pretreatment processes are performed to reduce the recalcitrance of lignocellulose
to enzymes mainly by reducing the content of lignin and hemicelluloses, increasing accessible surface area, reducing the crystallinity of cellulose, increasing porosity, or altering lignin
structure. The main effects of these pretreatment processes on
the chemical compositions and structures of lignocellulosic materials are summarized and illustrated in Table 1. Most pretreatment
processes can weaken the recalcitrance of lignocellulosic materials
by many ways.
The advantages and disadvantages of these pretreatment processes for pretreating lignocellulosic materials are also summarized
and shown in Table 2. Among these pretreatment processes,

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Table 1
Effects of different pretreatment processes on the compositions and structures of lignocellulosic materials.

Pretreatment
methods

Removing
lignin

Removing
hemicelluloses

Increasing accessible
area

Decrystallizing
cellulose

Increasing
porosity

Generating
inhibitors

Milling
Alkali
Acid
Oxidative
Organosolv
ILs
Steam explosion
Hydrothermal
AFEX
SC-CO2
Biological

Ha
Ma
H
H
M
La
M
M
L
H

H
H

L
L
H
H
L
L
M

H
H
H

H
H
H
M
H
H
H

H
L

H
M
H
M
H
H
M
H
H
H

L
L
H
L
L
L
H
H
L
L
L

H: high effect; M: medium effect; L: low effect.

Table 2
Major advantages and disadvantages of different pretreatment processes for pretreating lignocellulosic materials.
Pretreatment methods

Major advantages

Major disadvantages

Milling
Alkali
Acid
Oxidative
Organosolv process
ILs pretreatment
Steam explosion
Hydrothermal
AFEX
SC-CO2
Biological

Increases accessible specific surface area and reduces crystallinity

Reduces the content of lignin and hemicelluloses and low cost
Reduces the content of hemicelluloses and low cost
Efficient removal of lignin
Separation and recovery of high quality lignin
Reduces the crystallinity of lignocellulose efficiently
Solubilizes hemicelluloses, alerts the structure of lignin, and cost effective
Solubilizes hemicelluloses and recovers sugars from hemicelluloses
Causes cellulose swell and increases accessible specific surface area
Increases accessible specific surface area
Degrades lignin and hemicelluloses

High energy consumption

High pollution and high chemical recovery cost
Chemical recovery problem
High cost of bleaching agents
High price of organic solvents
High cost of ILs
High equipment cost and generation of inhibitors
High equipment cost
High equipment and ammonia costs
High equipment cost
Long pretreatment period

chemical and physico-chemical pretreatments are the most effective and promising processes for industrial applications. Generally,
chemical pretreatments, especially alkali and acid pretreatments,
can effectively remove hemicelluloses and lignin in lignocellulosic
materials with low cost. Physico-chemical pretreatment processes
can solubilize hemicelluloses, disorder the structure of lignocellulose, and further increase the accessible specific surface of lignocellulosic materials to enzymes with little environmental impact.
Although some pretreatment methods, such as milling, organosolv
process, and ILs pretreatment, can also significantly improve the
digestibility of lignocellulosic materials, their high operating costs
severely limit their commercial application.
5. Combined pretreatments
As shown in Table 1, most pretreatment processes can only
weaken partial factors that constrain the enzymatic hydrolysis of
lignocellulosic materials, thus combined pretreatment processes
are considered to improve their digestibility and maximize the utilization of isolated hemicelluloses and lignin. A schematic diagram
of combined pretreatment strategies to maximize the utilization of
lignocellulosic materials is shown in Fig. 1. Acid, alkali, steam
explosion, and hydrothermal pretreatments show obvious effect
on the removal of hemicelluloses, while alkali, oxidative delignification, and biological pretreatment can effectively remove lignin
from lignocellulosic materials. Physical pretreatments such as
steam explosion, AFEX, and supercritical CO2 pretreatment, can
increase the accessible surface area to varying degrees. These pretreatment processes are likely to be combined to improve the
digestibility of lignocellulosic materials and facilitate the recovery
of lignin and hemicelluloses for producing high value products.
Therefore, plenty of combined pretreatments, such as steam explosion or hydrothermal combined with alkaline peroxide (Chen et al.,
2008; Cuevas et al., 2014), SC-CO2 combined with ultrasound or

ionic liquid (Benazzi et al., 2013; Silveira et al., 2015), biological

combined with mild acid (Ma et al., 2010), hydrothermal combined
with alkali pretreatment (Sun et al., 2014a), mild physical or chemical pretreatment combined with biological pretreatment (Yu et al.,
2009), dilute acid combined with alkali pretreatment (Lee et al.,
2015), have been applied to pretreat various lignocellulosic materials. Cuevas et al. (2014) explored the component changes and
enzymatic digestibility of almond-tree prunings pretreated by
hydrothermal combined with alkaline peroxide. About 60% of lignin was removed during the subsequent alkaline peroxide process
and the digestibility of the residues after the combined pretreatment was significantly improved as compared with the residues
after the hydrothermal pretreatment alone.
Most combined pretreatments showed beneficial effect on further improving the enzymatic hydrolysis of lignocellulosic materials, but increased operating cost might be simultaneously involved.
An appropriate combined pretreatment should not only improve
the digestibility of lignocellulosic materials at a relatively low
operating cost but also maximize the utilization of lignocellulosic
components. In order to facilitate the recovery of lignin and hemicelluloses or their derivatives, the approach that separate hemicelluloses and lignin individually from lignocellulosic materials and
retain residues rich in cellulose for enzymatic hydrolysis is likely
an appropriate method to realize the full utilization of lignocellulosic components. Since hemicelluloses are closely associated with
lignin in lignocellulosic materials and most delignification processes are accompanied with the removal of hemicelluloses, hemicelluloses are recommended to be separated in the first step of the
combined pretreatment process. As seen in Table 1, alkali, acid,
steam explosion and hydrothermal pretreatments show relatively
high effect on removing hemicelluloses from lignocellulosic materials, which can be used as the first step of the combined pretreatment. Alkali, oxidative, organosolv and biological pretreatments
are relatively effective on removing lignin. However, the operating

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S. Sun et al. / Bioresource Technology 199 (2016) 4958

Fig. 1. Schematic diagram of combined pretreatment strategies to maximize the utilization of lignocellulosic materials.

costs of organosolv and oxidative delignification processes are

much higher than alkali pretreatment. Furthermore, oxidative
delignification, especially alkali assisted oxidative delignification,
is usually used to prepare chemical pulp with low content of lignin.
Biological pretreatment shows many advantages on removing lignin from lignocellulosic materials, but the low lignin removal efficiency limits its practical application. So far, alkali pretreatments
are still the most attractive and cost-effective methods to remove
lignin from lignocellulosic materials. Based on this view, dilute
acid, hydrothermal and steam explosion combined with alkali pretreatments are likely the most promising combinations for pretreating lignocellulosic materials.
Lee et al. (2015) explored the feasibility of applying dilute
H2SO4 combined with NaOH pretreatment on pretreating corn
stover to improve its enzymatic digestibility. It was reported that
dilute H2SO4 used in the first step hydrolyzed 74.677.3% of xylan,
while NaOH used in the second step removed 85.989.4% of lignin.
After the combined pretreatment, the enzymatic digestibility of
corn stover was significantly enhanced. After the following enzymatic hydrolysis, about 89.197.9% of glucose and 71.075.9% of
xylose were obtained. Sun et al. (2014a) applied hydrothermal
combined with NaOH pretreatment on pretreating thermomechanical fiber from Eucalyptus urophylla and further investigated its enzymatic digestibility. Almost all hemicelluloses were
degraded into the hydrothermal liquid within 30 min during the
first step of the hydrothermal pretreatment (reaction temperatures
>180 C), while the lignin was relatively stable under the
hydrothermal condition. In the following alkali pretreatment,
about 5060% of lignin in the Eucalyptus urophylla fiber was solubilized into the alkaline solution, which significantly improved its
enzymatic hydrolysis. Taking the energy consumption and recovery of hemicelluloses and lignin into consideration, an optimum
66.3% of cellulose was converted into glucose in the final enzymatic hydrolysis process. Additionally, Sun et al. (2014c) also
investigated the feasibility of steam explosion combined with
alkali pretreatment. The exploded residues showed relatively low
xylan (8.3220.85%), while the lignin content decreased slightly
during the steam explosion process. In the following alkali pretreatment, it showed that a higher steam explosion pressure was
conducive to the removal of lignin in the sequential alkaline. The
enzymatic hydrolysis results showed that steam explosion alone
improved the enzymatic hydrolysis rate by 7.933.1%, while the
steam explosion combined with alkali pretreatment enhanced
the rate by 45.763.9%. Generally, the hydrolysate obtained in

the first step pretreatment containing high content of hemicellulosic derivatives (mainly monosaccharides, xylo-oligosaccharides,
and furfural) can further dehydrate under the catalysis of acids
to prepare furfural. The alkaline solutions containing high contents
of lignin fractions can be concentrated and burned to provide
energy and recover chemicals. Alternatively, the lignin fraction
can also be isolated and utilized as feedstocks of high valueadded chemicals or materials to cover the pretreatment cost
(Cherubini, 2010). From this point of view, the combined pretreatments not only improve the digestibility of lignocellulosic materials but also facilitate the recovery of lignin and hemicelluloses for
producing high value products, which show great potential for
large-scale application.
6. Conclusions
Due to the strong native recalcitrance of lignocellulosic materials, various pretreatment processes are comprehensively investigated to overcome this recalcitrance by releasing the factors that
constrain their enzymatic hydrolysis. However, an economically
and ecologically viable pretreatment process for sustainable production of cellulosic bioethanol has not been established. A suitable pretreatment should not only largely improve the
digestibility of lignocellulosic materials but also facilitate the
recovery of lignin and hemicelluloses. The sequential pretreatments are likely to combine their advantages and decrease their
cost in production of cellulosic bioethanol, which show great
potential for large-scale application in biorefinery.
Acknowledgements
The authors are extremely grateful to financial support from the
National Natural Science Foundation of China for the key projects
(31430092, 31110103902) and Program of International S&T Cooperation of China (2015DFG31860).
References
Alizadeh, H., Teymouri, F., Gilbert, T.I., Dale, B.E., 2005. Pretreatment of switchgrass
by ammonia fiber explosion (AFEX). Appl. Biochem. Biotechnol. 121124, 1133
1141.
Alvira, P., Toms-Pej, E., Ballesteros, M., Negro, M.J., 2010. Pretreatment
technologies for an efficient bioethanol production process based on
enzymatic hydrolysis: a review. Bioresour. Technol. 101, 48514861.

S. Sun et al. / Bioresource Technology 199 (2016) 4958

Andric, P., Meyer, A., Jensen, P., Dam-Johansen, K., 2010. Effect and modeling of
glucose inhibition and in situ glucose removal during enzymatic hydrolysis of
pretreated wheat straw. Appl. Biochem. Biotechnol. 160, 280297.
Benazzi, T., Calgaroto, S., Astolfi, V., Dalla Rosa, C., Oliveira, J.V., Mazutti, M.A., 2013.
Pretreatment of sugarcane bagasse using supercritical carbon dioxide combined
with ultrasound to improve the enzymatic hydrolysis. Enzyme Microb. Technol.
52, 247250.
Boerjan, W., Ralph, J., Baucher, M., 2003. Lignin biosynthesis. Annu. Rev. Plant Biol.
54, 519546.
Boussaid, A.-L., Esteghlalian, A.R., Gregg, D.J., Lee, K.H., Saddler, J.N., 2000. Steam
pretreatment of Douglas-fir wood chips Can conditions for optimum
hemicellulose recovery still provide adequate access for efficient enzymatic
hydrolysis? Appl. Biochem. Biotechnol. 8486, 693705.
Cai, J., Zhang, L.N., 2005. Rapid dissolution of cellulose in LiOH/urea and NaOH/urea
aqueous solutions. Macromol. Biosci. 5, 539548.
Cao, X.F., Peng, X.W., Sun, S.N., Zhong, L.X., Sun, R.C., 2014a. Hydrothermal
conversion of bamboo: identification and distribution of the components in
solid residue, water-soluble and acetone-soluble fractions. J. Agric. Food Chem.
62, 1236012365.
Cao, X.F., Peng, X.W., Sun, S.N., Zhong, L.X., Wang, S., Lu, F.C., Sun, R.C., 2014b. Impact
of regeneration process on the crystalline structure and enzymatic hydrolysis of
cellulose obtained from ionic liquid. Carbohydr. Polym. 111, 400403.
Chen, H.Z., Han, Y.J., Xu, J., 2008. Simultaneous saccharification and fermentation of
steam exploded wheat straw pretreated with alkaline peroxide. Process
Biochem. 43, 14621466.
Cherubini, F., 2010. The biorefinery concept: using biomass instead of oil for
producing energy and chemicals. Energy Convers. Manage. 51, 14121421.
Cuevas, M., Garcia, J.F., Sanchez, S., 2014. Enhanced enzymatic hydrolysis of
pretreated almond-tree prunings for sugar production. Carbohydr. Polym. 99,
791799.
De Bari, I., Nanna, F., Braccio, G., 2007. SO2-catalyzed steam fractionation of aspen
chips for bioethanol production: optimization of the catalyst impregnation. Ind.
Eng. Chem. Res. 46, 77117720.
Duarte, C.L., Ribeiro, M.A., Oikawa, H., Mori, M.N., Napolitano, C.M., Galvao, C.A.,
2012. Electron beam combined with hydrothermal treatment for enhancing the
enzymatic convertibility of sugarcane bagasse. Radiat. Phys. Chem. 81, 1008
1011.
Garca-Cubero, M.T., Gonzlez-Benito, G., Indacoechea, I., Coca, M., Bolado, S., 2009.
Effect of ozonolysis pretreatment on enzymatic digestibility of wheat and rye
straw. Bioresour. Technol. 100, 16081613.
Garcia, A., Cara, C., Moya, M., Rapado, J., Puls, J., Castro, E., Martin, C., 2014. Dilute
sulphuric acid pretreatment and enzymatic hydrolysis of Jatropha curcas fruit
shells for ethanol production. Ind. Crops Prod. 53, 148153.
Grethlein, H.E., 1985. The effect of pore size distribution on the rate of enzymatic
hydrolysis of cellulosic substrates. Nat. Biotechnol. 3, 155160.
Gu, F., Yang, L.F., Jin, Y.C., Han, Q., Chang, H.M., Jameel, H., Phillips, R., 2012. Green
liquor pretreatment for improving enzymatic hydrolysis of corn stover.
Bioresour. Technol. 124, 299305.
Haque, M.A., Barman, D.N., Kang, T.H., Kim, M.K., Kim, J., Kim, H., Yun, H.D., 2012.
Effect of dilute alkali on structural features and enzymatic hydrolysis of barley
straw (Hordeum vulgare) at boiling temperature with low residence time. J.
Microbiol. Biotechnol. 22, 16811691.
Hatakka, A.I., 1983. Pretreatment of wheat straw by white-rot fungi for enzymic
saccharification of cellulose. Eur. J. Appl. Microbiol. Biotechnol. 18, 350357.
Hernandez, E., Garcia, A., Lopez, M., Puls, J., Parajo, J.C., Martin, C., 2013. Dilute
sulphuric acid pretreatment and enzymatic hydrolysis of Moringa oleifera
empty pods. Ind. Crops Prod. 44, 227231.
Horn, S.J., Nguyen, Q.D., Westereng, B., Nilsen, P.J., Eijsink, V.G.H., 2011. Screening of
steam explosion conditions for glucose production from non-impregnated
wheat straw. Biomass Bioenergy 35, 48794886.
Huijgen, W.J.J., Smit, A.T., Reith, J.H., Uil, H.d., 2011. Catalytic organosolv
fractionation of willow wood and wheat straw as pretreatment for enzymatic
cellulose hydrolysis. J. Chem. Technol. Biotechnol. 86, 14281438.
Jeffries, T., 1990. Biodegradation of lignin-carbohydrate complexes. Biodegradation
1, 163176.
Jin, Z.F., Katsumata, K.S., Lam, T.B.T., Iiyama, K., 2006. Covalent linkages between
cellulose and lignin in cell walls of coniferous and nonconiferous woods.
Biopolymers 83, 103110.
Jonsson, L.J., Alriksson, B., Nilvebrant, N.O., 2013. Bioconversion of lignocellulose:
inhibitors and detoxification. Biotechnol. Biofuels 6, 1625.
Kim, H., Choi, B., 2010. The effect of biodiesel and bioethanol blended diesel fuel on
nanoparticles and exhaust emissions from CRDI diesel engine. Renew. Energy
35, 157163.
Kim, K.H., Hong, J., 2001. Supercritical CO2 pretreatment of lignocellulose enhances
enzymatic cellulose hydrolysis. Bioresour. Technol. 77, 139144.
Kim, S., Holtzapple, M.T., 2005. Lime pretreatment and enzymatic hydrolysis of corn
stover. Bioresour. Technol. 96, 19942006.
Kumar, R., Wyman, C.E., 2009. Effects of cellulase and xylanase enzymes on the
deconstruction of solids from pretreatment of poplar by leading technologies.
Biotechnol. Progr. 25, 302314.
Lee, J.M., Jameel, H., Venditti, R.A., 2010. A comparison of the autohydrolysis and
ammonia fiber explosion (AFEX) pretreatments on the subsequent enzymatic
hydrolysis of coastal Bermuda grass. Bioresour. Technol. 101, 54495458.
Lee, J.W., Kim, J.Y., Jang, H.M., Lee, M.W., Park, J.M., 2015. Sequential dilute acid and
alkali pretreatment of corn stover: sugar recovery efficiency and structural
characterization. Bioresour. Technol. 182, 296301.

57

Li, Q., Jiang, X.L., He, Y.C., Li, L.Z., Xian, M., Yang, J.M., 2010. Evaluation of the
biocompatible ionic liquid 1-methyl-3-methylimidazolium dimethylphosphite
pretreatment of corn cob for improved saccharification. Appl. Microbiol.
Biotechnol. 87, 117126.
Lu, X.B., Zhang, Y.M., Angelidaki, I., 2009. Optimization of H2SO4-catalyzed
hydrothermal pretreatment of rapeseed straw for bioconversion to ethanol:
focusing on pretreatment at high solids content. Bioresour. Technol. 100, 3048
3053.
Lv, H., Yan, L., Zhang, M., Geng, Z., Ren, M., Sun, Y., 2013. Influence of supercritical
CO2 pretreatment of corn stover with ethanol-water as co-solvent on lignin
degradation. Chem. Eng. Technol. 36, 18991906.
Ma, F.Y., Yang, N., Xu, C.Y., Yu, H.B., Wu, J.G., Zhang, X.Y., 2010. Combination of
biological pretreatment with mild acid pretreatment for enzymatic hydrolysis
and ethanol production from water hyacinth. Bioresour. Technol. 101, 9600
9604.
Martin-Sampedro, R., Filpponen, I., Hoeger, I.C., Zhu, J.Y., Laine, J., Rojas, O.J., 2012.
Rapid and complete enzyme hydrolysis of lignocellulosic nanofibrils. ACS Macro
Lett. 1, 13211325.
Martin, C., Galbe, M., Nilvebrant, N.-O., Jonsson, L.J., 2002. Comparison of the
fermentability of enzymatic hydrolyzates of sugarcane bagasse pretreated by
steam explosion using different impregnating agents. Appl. Biochem.
Biotechnol. 98100, 699716.
McKendry, P., 2002. Energy production from biomass (part 1): overview of biomass.
Bioresour. Technol. 83, 3746.
Miura, T., Lee, S.H., Inoue, S., Endo, T., 2012. Improvement of enzymatic
saccharification of sugarcane bagasse by dilute-alkali-catalyzed hydrothermal
treatment and subsequent disk milling. Bioresour. Technol. 105, 9599.
Moretti, M.M.D., Bocchini-Martins, D.A., Nunes, C.D.C., Villena, M.A., Perrone, O.M.,
da Silva, R., Boscolo, M., Gomes, E., 2014. Pretreatment of sugarcane bagasse
with microwaves irradiation and its effects on the structure and on enzymatic
hydrolysis. Appl. Energy 122, 189195.
Muratov, G., Kim, C., 2002. Enzymatic hydrolysis of cotton fibers in supercritical
CO2. Biotechnol. Bioprocess Eng. 7, 8588.
Mussatto, S.I., Fernandes, M., Milagres, A.M.F., Roberto, I.C., 2008. Effect of
hemicellulose and lignin on enzymatic hydrolysis of cellulose from Brewers
spent grain. Enzyme Microb. Technol. 43, 124129.
Nitsos, C.K., Matis, K.A., Triantafyllidis, K.S., 2013. Optimization of hydrothermal
pretreatment of lignocellulosic biomass in the bioethanol production process.
ChemSusChem 6, 110122.
Prez, J., Muoz-Dorado, J., de la Rubia, T., Martnez, J., 2002. Biodegradation and
biological treatments of cellulose, hemicellulose and lignin: an overview. Int.
Microbiol. 5, 5363.
Pan, X.J., Gilkes, N., Kadla, J., Pye, K., Saka, S., Gregg, D., Ehara, K., Xie, D., Lam, D.,
Saddler, J., 2006. Bioconversion of hybrid poplar to ethanol and co-products
using an organosolv fractionation process: optimization of process yields.
Biotechnol. Bioeng. 94, 851861.
Pan, X.J., Xie, D., Gilkes, N., Gregg, D.J., Saddler, J.N., 2005. Strategies to enhance the
enzymatic hydrolysis of pretreated softwood with high residual lignin content.
Appl. Biochem. Biotechnol. 121, 10691079.
Park, N., Kim, H.Y., Koo, B.W., Yeo, H., Choi, I.G., 2010. Organosolv pretreatment with
various catalysts for enhancing enzymatic hydrolysis of pitch pine (Pinus rigida).
Bioresour. Technol. 101, 70467053.
Qi, B., Chen, X., Shen, F., Su, Y., Wan, Y., 2009. Optimization of enzymatic hydrolysis
of wheat straw pretreated by alkaline peroxide using response surface
methodology. Ind. Eng. Chem. Res. 48, 73467353.
Qing, Q., Yang, B., Wyman, C.E., 2010. Xylooligomers are strong inhibitors of
cellulose hydrolysis by enzymes. Bioresour. Technol. 101, 96249630.
Ralph, J., Lundquist, K., Brunow, G., Lu, F., Kim, H., Schatz, P., Marita, J., Hatfield, R.,
Ralph, S., Christensen, J., Boerjan, W., 2004. Lignins: natural polymers from
oxidative coupling of 4-hydroxyphenyl-propanoids. Phytochem. Rev. 3, 2960.
Snchez, C., 2009. Lignocellulosic residues: biodegradation and bioconversion by
fungi. Biotechnol. Adv. 27, 185194.
Sette, M., Wechselberger, R., Crestini, C., 2011. Elucidation of lignin structure by
quantitative 2D NMR. Chem.-Eur. J. 17, 95299535.
Silveira, M.H.L., Vanelli, B.A., Corazza, M.L., Ramos, L.P., 2015. Supercritical carbon
dioxide combined with 1-butyl-3-methylimidazolium acetate and ethanol for
the pretreatment and enzymatic hydrolysis of sugarcane bagasse. Bioresour.
Technol. 192, 389396.
Sun, S., Cao, X.F., Sun, S.L., Xu, F., Song, X.L., Sun, R.C., Jones, G.L., 2014a. Improving
the enzymatic hydrolysis of thermo-mechanical fiber from Eucalyptus urophylla
by a combination of hydrothermal pretreatment and alkali fractionation.
Biotechnol. Biofuels 7, 116127.
Sun, S.L., Wen, J.L., Ma, M.G., Sun, R.C., Jones, G.L., 2014b. Structural features and
antioxidant activities of degraded lignins from steam exploded bamboo stem.
Ind. Crops Prod. 56, 128136.
Sun, S.N., Cao, X.F., Zhang, X.M., Xu, F., Sun, R.C., Jones, G.L., 2014c. Characteristics
and enzymatic hydrolysis of cellulose-rich fractions from steam exploded and
sequentially alkali delignified bamboo (Phyllostachys pubescens). Bioresour.
Technol. 163, 377380.
Sun, X.F., Sun, R.C., Fowler, P., Baird, M.S., 2005. Extraction and characterization of
original lignin and hemicelluloses from wheat straw. J. Agric. Food Chem. 53,
860870.
Sun, Y., Cheng, J.Y., 2002. Hydrolysis of lignocellulosic materials for ethanol
production: a review. Bioresour. Technol. 83, 111.
Taherzadeh, M.J., Karimi, K., 2008. Pretreatment of lignocellulosic wastes to
improve ethanol and biogas production: a review. Int. J. Mol. Sci. 9, 16211651.

58

S. Sun et al. / Bioresource Technology 199 (2016) 4958

Taniguchi, M., Suzuki, H., Watanabe, D., Sakai, K., Hoshino, K., Tanaka, T., 2005.
Evaluation of pretreatment with Pleurotus ostreatus for enzymatic hydrolysis of
rice straw. J. Biosci. Bioeng. 100, 637643.
Teymouri, F., Laureano-Perez, L., Alizadeh, H., Dale, B.E., 2005. Optimization of the
ammonia fiber explosion (AFEX) treatment parameters for enzymatic
hydrolysis of corn stover. Bioresour. Technol. 96, 20142018.
Vanderghem, C., Brostaux, Y., Jacquet, N., Blecker, C., Paquot, M., 2012. Optimization
of formic/acetic acid delignification of Miscanthus  giganteus for enzymatic
hydrolysis using response surface methodology. Ind. Crops Prod. 35, 280286.
Varga, E., Reczey, K., Zacchi, G., 2004. Optimization of steam pretreatment of corn
stover to enhance enzymatic digestibility. Appl. Biochem. Biotechnol. 113116,
509523.
Xiao, X., Bian, J., Li, M.F., Xu, H., Xiao, B., Sun, R.C., 2014. Enhanced enzymatic
hydrolysis of bamboo (Dendrocalamus giganteus Munro) culm by hydrothermal
pretreatment. Bioresour. Technol. 159, 4147.
Yeh, A.I., Huang, Y.C., Chen, S.H., 2010. Effect of particle size on the rate of enzymatic
hydrolysis of cellulose. Carbohydr. Polym. 79, 192199.
Yu, J., Zhang, J.B., He, J., Liu, Z.D., Yu, Z.N., 2009. Combinations of mild physical or
chemical pretreatment with biological pretreatment for enzymatic hydrolysis
of rice hull. Bioresour. Technol. 100, 903908.

Zavrel, M., Bross, D., Funke, M., Bchs, J., Spiess, A.C., 2009. High-throughput
screening for ionic liquids dissolving (ligno-)cellulose. Bioresour. Technol. 100,
25802587.
Zhao, H., Jones, C.L., Baker, G.A., Xia, S.Q., Olubajo, O., Person, V.N., 2009a.
Regenerating cellulose from ionic liquids for an accelerated enzymatic
hydrolysis. J. Biotechnol. 139, 4754.
Zhao, J.Y., Chen, H.Z., 2013. Correlation of porous structure, mass transfer and
enzymatic hydrolysis of steam exploded corn stover. Chem. Eng. Sci. 104, 1036
1044.
Zhao, X.B., Cheng, K.K., Liu, D.H., 2009b. Organosolv pretreatment of lignocellulosic
biomass for enzymatic hydrolysis. Appl. Microbiol. Biotechnol. 82, 815827.
Zhao, Y., Wu, B., Yan, B.X., Gao, P.J., 2004. Mechanism of cellobiose inhibition in
cellulose hydrolysis by cellobiohydrolase. Sci. China Ser. C 47, 1824.
Zhao, Y.L., Wang, Y., Zhu, J.Y., Ragauskas, A., Deng, Y.L., 2008. Enhanced enzymatic
hydrolysis of spruce by alkaline pretreatment at low temperature. Biotechnol.
Bioeng. 99, 13201328.
Zhou, Y.P., Stuart-Williams, H., Farquhar, G.D., Hocart, C.H., 2010. The use of natural
abundance stable isotopic ratios to indicate the presence of oxygen-containing
chemical linkages between cellulose and lignin in plant cell walls.
Phytochemistry 71, 982993.