Pretreatment Methods for Bioethanol Production

Zhaoyang Xu & Fang Huang

Received: 21 January 2014 / Accepted: 15 June 2014 /
Published online: 28 June 2014
#
Springer Science+Business Media New York 2014
Abstract Lignocellulosic biomass, such as wood, grass, agricultural, and forest residues,
are potential resources for the production of bioethanol. The current biochemical process
of converting biomass to bioethanol typically consists of three main steps: pretreatment,
enzymatic hydrolysis, and fermentation. For this process, pretreatment is probably the
most crucial step since it has a large impact on the efficiency of the overall bioconver-
sion. The aim of pretreatment is to disrupt recalcitrant structures of cellulosic biomass to
make cellulose more accessible to the enzymes that convert carbohydrate polymers into
fermentable sugars. This paper reviews several leading acidic, neutral, and alkaline
pretreatments technologies. Different pretreatment methods, including dilute acid pre-
treatment (DAP), steam explosion pretreatment (SEP), organosolv, liquid hot water
(LHW), ammonia fiber expansion (AFEX), soaking in aqueous ammonia (SAA), sodium
hydroxide/lime pretreatments, and ozonolysis are intensively introduced and discussed.
In this minireview, the key points are focused on the structural changes primarily in
cellulose, hemicellulose, and lignin during the above leading pretreatment technologies.
Keywords Pretreatment
.
Cellulose
.
Hemicellulose
.
Lignin
Abbreviations
AFEX Ammonia fiber expansion
CrI Crystallinity index
CS Combined severity
DAP Dilute acid pretreatment
DP Degree of polymerization
HW Hardwood
HMF 5-Hydroxymethylfurfural
LCC Lignin-carbohydrate complex
Appl Biochem Biotechnol (2014) 174:4362
DOI 10.1007/s12010-014-1015-y
Z. Xu (*)
College of Materials Science and Engineering, Nanjing Forestry University, Jiangsu 210037, Peoples
Republic of China
e-mail: hughxzy@hotmail.com
F. Huang (*)
School of Chemistry and Biochemistry, Institute of Paper Science and Technology, Georgia Institute of
Technology, Atlanta, GA 30332, USA
e-mail: huangfanghg@gmail.com
LHW Liquid hot water
LODP Leveling-off degree of polymerization
NMR Nuclear magnetic resonance
SAA Soaking in aqueous ammonia
SEP Steam explosion pretreatment
SW Softwood
XRD X-ray diffraction
Introduction
In order to cope with the growing demand for energy, the depletion of fossil fuel resources and
environmental concerns raised by fossil fuel use, countries wishing to limit their energy
dependence on petroleum exporting countries are developing alternative energy sources, such
as bioethanol produced from renewable biomass [14]. Cellulosic bioethanol is regarded as
one of the most promising renewable biofuels in the transportation sector for the next coming
few decades [5]. Current production of bioethanol relies on sugars that are obtained from
starch-based agricultural crops by using first-generation conversion technologies [6]. Nowa-
days, bioethanol produced from lignocellulosic biomass using second-generation technologies
has become a promising alternative, mainly because lignocellulosic raw materials do not
compete with food crops or productive agricultural land and they are also less expensive
and more abundant than conventional agricultural feedstocks [7, 8].
The biological process of converting biomass to bioethanol typically consists of three
main steps: pretreatment, enzymatic hydrolysis, and fermentation (Fig. 1). During the
whole process, pretreatment is the most crucial step since it has a large impact on the
efficiency of the overall bioconversion. In lignocellulosic biomass, cellulose and hemi-
cellulose are densely packed together with lignin, which serves several plant functions
including protection against enzymatic hydrolysis [9]. The aim of pretreatment is to
disrupt recalcitrant structures of cellulosic biomass to make cellulose more accessible to
the enzymes that convert carbohydrate polymers into fermentable sugars. During the
pretreatment, the removal of lignin and hemicellulose depends on the pretreatment
technology, process conditions, and severity. For example, acidic chemical pretreatment
removes most of hemicellulose, and lignin is condensed when pretreatment temperature
reaches above 160 C [10]. On the contrary, the ammonia fiber expansion (AFEX)
pretreatment does not significantly remove hemicellulose.
Numerous pretreatment strategies have been developed to enhance the reactivity of cellu-
lose and to increase the yield of fermentable sugars. Typical goals of pretreatment include (a)
producing highly digestible solids to enhance sugar yields during enzyme hydrolysis, (b)
avoiding the degradation of sugars including those derived from hemicellulose, and (c)
minimizing the formation of inhibitors for subsequent fermentation steps [11].
The pretreatment methods can be classified into different categories according to various
criteria [2, 12]. It is common to differentiate pretreatment methods that are efficient at high,
low, or neutral pH. Basically, low pH methods require addition of acids to increase the
hydrolytic capacity while higher pH methods need pH-adjusting agents such as sodium
hydroxide or ammonia. The neutral pH methods, mainly liquid hot water (LHW) pretreatment,
simply apply water in the process. However, the substrate medium in the neutral methods is
weakly acidic due to the release of organic acids from the biomass during the pretreatments.
Another way to do the grouping is based on the principal mechanism acting during pretreat-
ment. As such, the methods can be classified as physical, physicochemical, and chemical
pretreatments. In this review, mainly the first classification is used. The acidic pretreatments
44 Appl Biochem Biotechnol (2014) 174:4362
include dilute acid pretreatment (DAP), steam explosion pretreatment (SEP), and organosolv
pretreatment. The neutral pretreatment is LHW, and the sodium hydroxide/lime pretreatment
and AFEX are classified into the alkaline pretreatments (Fig. 1). Besides the above methods,
pretreatment of biomass with high pressure ozone (ozonolysis) was recently developed in
bioethanol production [13].
In this review, a brief process description is first given with recent developments for each
pretreatment, followed by discussion of the technologys advantages and disadvantages.
Different feedstocks, including herbaceous/agricultural residues, hardwood and softwood
biomasses, applied in these technologies are highlighted. The key points are focused on the
structural changes primarily in cellulose, hemicellulose, and lignin during the above leading
pretreatment technologies.
Fig. 1 Schematic flow sheet for the bioconversion of biomass to bioethanol
Appl Biochem Biotechnol (2014) 174:4362 45
Lignocellulose Compositions
The main components of the lignocellulosic biomass are cellulose, hemicellulose, lignin, and a
remaining smaller part (extractives and ash). The composition of lignocellulose highly depends
on its source. There is a significant variation of the lignin and (hemi)cellulose content of
lignocellulose depending on whether it is derived from hardwood (HW), softwood (SW), or
herbaceous/agricultural residue biomass. Table 1 summarizes the typical composition of
lignocellulose encountered in the most common sources of biomass.
In the chemical aspects, cellulose is a linear chain homopolymer consisting of (1-
4)--D-glucopyranosyl units with a varying degree of polymerization (DP) up to ~10,000
[17]. The cellulose chain has a tendency to form intramolecular and intermolecular
hydrogen bonds through hydroxyl groups on its glucose units, which promote cellulose
aggregations and lead to a supramolecular structure with crystalline and amorphous
domains. Hemicellulose consists of a broad class of mixed heteroglycans of pentoses
and hexanoses (mainly xylose and mannose) that are linked together and frequently have
branching and substitution groups. Lignin is an irregular polyphenolic biopolymer
constructed of phenylpropanoid monomers with various degrees of methoxylation that
are biosynthesized into a complex and highly heterogeneous aromatic macromolecule.
Generally, the plant cell wall microstructure is regarded to be a matrix of lignin and
polysaccharides intimately associating with each other [18, 19].
Acidic Pretreatment
The processes and mode of action are discussed for different acidic pretreatment methods
including DAP, SEP, and organosolv pretreatment.
Dilute Acid Pretreatment
Process Description
Among the numerous pretreatment techniques, dilute acid pretreatment has been shown as a
leading pretreatment process that is currently under commercial development. Dilute acid
Table 1 Typical lignocellulosic biomass composition (% dry basis)
Cellulose Hemicellulose Lignin Reference
Pine 43.3 20.5 28.3 [5]
Spruce 45.0 22.9 27.9 [5]
Douglas fir 44.0 19.2 30.0 [5]
Poplar 44.7 18.5 26.4 [4]
Eucalyptus 49.5 13.1 27.7 [4]
Corn stover 36.8 30.6 23.1 [5]
Miscanthus 52.1 25.8 12.6 [14]
Wheat straw 44.1 23.8 20.5 [15]
Switchgrass 33.5 26.1 17.4 [16]
46 Appl Biochem Biotechnol (2014) 174:4362
pretreatment can significantly reduce lignocellulosic recalcitrance by disrupting the composite
material linkage, such as the covalent bonds [20]. The most widely used and tested approaches in
DAP are based on dilute sulfuric acid since it is inexpensive and effective [21, 22]. However, nitric
acid [23], hydrochloric acid [24], and phosphoric acid [25] have also been examined. In addition, it
was shown that SO
2
was also an efficient acid catalyst in the dilute acid pretreatment, especially for
softwood [26]. The DAP is usually performed over a temperature range of 120 to 210 C, with acid
concentration typically less than 4 wt%and residence time fromseveral minutes to an hour [27]. In
the DAP pretreatment, the combined severity (CS) is used for an easy comparison of pretreatment
conditions and for facilitation of process control, which relates to the experimental effects of
temperature, residence time, and acid concentration [28]. Lower CS is beneficial for the hemicel-
lulose to hydrolyze to oligomers and monomers while higher CS could further convert these
monomers to furfurals and 5-hydroxymethylfurfural (HMF), which are inhibitors for the subse-
quent enzymatic hydrolysis [29]. In order to maximize the efficiency of pretreatments, several
studies have proposed a two-step procedure for dilute acid pretreatment of softwoods [21, 30]. The
conditions in the first step are less severe and serve to hydrolyze the hemicelluloses resulting in a
high recovery of hemicellulose-derived fermentable sugars in the pretreatment effluent. The solid
material recovered fromthe first step is treated again under more severe conditions which promotes
the enzymatic digestibility of cellulose fibers.
Mode of Action
One of the main reactions that occurs during acid pretreatment is the hydrolysis of hemicel-
lulose. Hemicelluloses are hydrolyzed to fermentable sugars during DAP [12]. Solubilized
hemicelluloses (oligomers) can be subjected to hydrolytic reactions producing monomers,
furfural, HMF, and other (volatile) products in acidic environments [31, 32]. Recently, Hu and
Sannigrahi et al. [33, 34] have demonstrated that pseudo-lignin can be generated solely from
carbohydrates without a significant contribution from lignin during DAP especially under high
severity pretreatment conditions. Further analysis indicates that pseudo-lignin is a polymeric
material that is present on the surface of pretreated biomass as spherical droplets and
structurally has carbonyl, aromatic, methoxy, and aliphatic functionalities.
During DAP pretreatments, the hydrolyzation of cellulose and subsequent solubilization of
glucose most often results in an increase of cellulose crystallinity index (CrI) in biomass, as
shown in Table 2. Foston et al. [33, 35] suggested that the majority of the increase of cellulose
crystallinity is primarily due to localized hydrolyzation and removal of cellulose from the
amorphous regions. Cao et al. [36] observed that the CrI of poplar cellulose remained almost
unchanged during the early DA pretreatment (0.35.4 min), then a slight increase during the
DAP. In addition, the partial hydrolyzation of cellulose in DAP also leads to a reduction of
cellulose DP (Table 2) especially at high severity pretreatment conditions, which increases the
enzymatic digestibility of cellulose. The DP of cellulose from different substrates usually
decreases gradually until reaching a nominal value, namely, the leveling-off degree of poly-
merization (LODP) throughout the course of pretreatment [37]. The initial DP reduction phase
period is believed to represent the hydrolysis of the reactive amorphous region of cellulose,
whereas the slow plateau rate phase corresponds to the hydrolysis of the slow reacting
crystalline fraction of cellulose, to some extent.
In contrast, DAP does not lead to significant delignification [44]. Recent studies have
revealed an increase in the degree of condensation of lignin during the dilute acid pretreatment.
The increase in degree of condensation is accompanied by a decrease in -O-4 linkages which
are fragmented and subsequently recondensed during the high temperature acid-catalyzed
reactions [45]. In addition, studies also indicated that lignin balls (or lignin droplets) were
Appl Biochem Biotechnol (2014) 174:4362 47
formed during DAP. These lignin droplets were proposed to originate from lignin and possible
lignin carbohydrates complexes [34, 46, 47].
DAP not only alternates the lignocellulosic biomass chemical structures but also changes
the anatomical structure of plant cell wall, especially the pore structures. The specific surface
area and the mean pore size of the plant cell wall are influential structural features related to
cellulose accessibility to cellulases during the enzymatic hydrolysis [48, 49]. Several studies
have indicated that the breakdown and loosening of the lignocellulosic structure by DAP
increases the specific surface area, pore volume, and pore size of the biomass [50, 51].
Advantage and Disadvantage
DA pretreatment can facilitate high enzymatic deconstruction reaction rates and significantly
improve hemicellulose and cellulose hydrolysis by varying the severity of the pretreatment [3].
A drawback of this method is the formation of many inhibiting by-products and pH neutral-
ization requirements for downstream processes [52]. In addition, the corrosive nature of this
pretreatment mandates expensive materials of construction [53]. Furthermore, most of the
reported work used bioresources with significant size reduction, which consume large amounts
of energy, and future studies will need to examine the reactivity of DAP with larger biomass
chip sizes [53].
Steam Explosion Pretreatment
Process Description
SEP is one of the most common pretreatments applied to fractionate biomass components
and increase its chemical and biological reactivity. In this method, biomass is treated
with high-pressure saturated steam, and then, the pressure is swiftly reduced, which
makes the materials undergo an explosive decompression. Steam explosion is typically
initiated at a temperature of 160260 C (corresponding pressure, 0.694.83 MPa) for
several seconds to a few minutes before the material is exposed to atmospheric pressure
[54]. Addition of an acid catalyst such as SO
2
or preferably H
2
SO
4
because it is
inexpensive to steam explosion can significantly increase its hemicellulose sugar yields
[55]. SEP applies physicochemical pretreatment for lignocellulosic biomass. The
Table 2 Cellulose CrI and DP before and after DAP pretreatments
Substrate Pretreatment
conditions
CrI (%) before
pretreatment
CrI (%) after
pretreatment
DP before
pretreatment
DP after
pretreatment
Corn stover
[38]
180 C, 3 wt%
H
2
SO
4
, 90 s
50.3
a
52.5
a
7,300
b
2,600
b
Poplar [38] 190 C, 2 wt%
H
2
SO
4
, 70 s
49.9
a
50.6
a
3,500
b
600
b
Loblolly
pine [39]
180 C, 1.0 wt%
H
2
SO
4
, 30 min
55.1
c
59.8
c
3,642
d
1,326
d
a
CrI was measured by X-ray diffraction (XRD) method [40]
b
DP was measured by viscometric method [41]
c
CrI was measured by solid-state nuclear magnetic resonance (NMR) technique [42]
d
DP was measured by gel-permeation chromatography (GPC) technique [43]
48 Appl Biochem Biotechnol (2014) 174:4362
mechanical effects are caused by a sudden decompression to separate the fibers in the
biomass [2]. The most important factors affecting the effectiveness of steam explosion
are temperature, residence time, and the combined effect of both temperature (T) and
time (t), which is described by the severity factor (R
0
) as follows [56]:
R
0
texp T100 =14:75
Higher temperatures result in an increased removal of hemicelluloses from the solid fraction
and an enhanced cellulose digestibility; they also promote higher sugar degradation. Previous
studies indicated the optimal conditions for maximum sugar yield with a severity factor (R
0
)
between 3.0 and 4.5 [56].
Mode of Action
During the SEP pretreatment, the steam penetrates the plant cell wall, causing hemicellulose
degradation and lignin transformation due to high temperature, thus increasing the potential of
cellulose hydrolysis [27]. The external acid (H
2
SO
4
or SO
2
) or the acetic acid released from
acetylated hemicellulose serves to catalyze and further hydrolyze hemicellulose in the biomass.
In addition, water itself also possesses certain acid properties at high temperature, thereby
contributes to the hydrolysis hemicellulose during the SEP [57]. In general, the hemicelluloses
are dissolved as oligosaccharides after acid hydrolysis, which are partially further hydrolyzed
to monosaccharides. Under high temperature and acid conditions, these monosaccharides can
be degraded into inhibitor compounds (i.e., furfural and HMF) for the subsequent enzymatic
hydrolysis [58]. In addition, the acids also lead to the acidolysis of lignin and the cleavages of
-O-4 linkage, which are similar to the LHWand DAP pretreatments [59]. As steam explosion
pretreatment conditions get more severe, the condensation and repolymerization reactions will
take place between the decomposition products of hemicelluloses, leading to the formation of
pseudo-lignin, as observed in DAP pretreatment [60, 61].
During the SEP pretreatment, the structure changes of cellulose are mainly on the
degree of polymerization and degree of crystallinity. Asada et al. [62] observed that the
DP of cellulose decreased significantly as the steaming time increased until reaching a
minimum value of about 700 when examining the bagasse SEP pretreatment, which was
due to the depolymerization of cellulose at a relatively short steaming time. The steam
explosion pretreatment also affects cellulose crystallinity. After the SEP pretreatment, the
increase of cellulose crystallinity was reported by several researchers in the literature [60,
63, 64]. Generally, this increase was attributed to the selective hydrolysis of amorphous
portions [63] and transformation of some amorphous portions into crystalline portions in
the cellulose structure [65]. Furthermore, cellulose can be also degraded and form HMF
during SEP; the mechanism of HMF formation in steam explosion pretreatment is similar
to that in DAP [34].
In addition, after the SEP pretreatment, the accessible pore volume and accessible surface
area of the pretreated fibers are progressively increased, which are probably the result of
hemicellulose extraction and lignin redistribution [66].
Advantage and Disadvantage
SEP pretreatment is an attractive process because it makes limited use of chemicals and it does
not result in excessive dilution of the resulting sugars. Moreover, it requires low energy input.
The disadvantage of SEP is its incomplete destruction of lignin-carbohydrate matrix resulting
Appl Biochem Biotechnol (2014) 174:4362 49
in the risk of condensation and precipitation of soluble lignin components making the biomass
less digestible. Other limitation of this process is the formation of fermentation inhibitors at
higher temperatures [67].
Organosolv Pretreatment
Process Description
In the organosolv pretreatment, numerous organic or aqueous solvent mixtures can be
utilized, including methanol, ethanol, acetone, ethylene glycol, and tetrahydrofurfuryl
alcohol, in order to solubilize lignin and hemicellulose, providing treated cellulose
suitable for enzymatic hydrolysis [68]. Although several organic solvents can be applied
in the organosolv pretreatments, the low molecular weight alcohols such as ethanol and
methanol are favored solvents mainly due to their lower boiling points. The preferred
conditions of organosolv process is generally in the following ranges: a cooking tem-
perature of 180195 C, a cooking time of 3090 min, an ethanol concentration of 35
70 % (w/v), and a liquor to solid ratio ranging from 4:1 to 10:1. The pH of the liquor
ranges from 2 to 4. In some studies, these mixtures are combined with acid catalysts
(HCl, H
2
SO
4
, oxalic, or salicylic) to break hemicellulose bonds and lignin linkages [69].
A high yield of xylose can usually be obtained with the addition of acid. However, this
acid addition can be avoided for a satisfactory delignification by increasing process
temperature (above 185 C) [70]. Usually, in the organosolv pretreatment, high lignin
removal (>70 %) and minimum cellulose loss (less than 2 %) could be achieved [71].
Mode of Action
During the organosolv pretreatment, the largest component, cellulose, is partially hydro-
lyzed into smaller fragments that largely remain insoluble. Sannigrahi et al. [72] revealed
the degree of cellulose crystallinity increases and the relative proportion of
paracrystalline and amorphous cellulose decreases after the organosolv pretreatment of
Loblolly pine. The second largest component, hemicellulose, is hydrolyzed mostly into
soluble components, such as oligosaccharides, monosaccharides, and acetic acid. Acetic
acid can act as catalyst for the rupture of lignin-carbohydrate complex [68]. Some of the
pentose sugars are subsequently dehydrated under the operating conditions to form
furfural [54]. The third major polymer component, lignin, is hydrolyzed into lower
molecular weight fragments that dissolve in the aqueous ethanol liquor. In addition,
the depolymerization of lignin occurs primarily through cleavage of -O-4 linkages [73].
Moreover, lignin condensation was also observed in organosolv pretreatment [74]. In
addition, the hydrolysis of hemicellulose and degradation of lignin in organosolv pre-
treatment also lead to the increase of cellulose surface accessibility [75].
Advantage and Disadvantage
Compared with other pretreatments, organosolv pretreatment has some advantages as follows:
(1) organic solvents are always easy to recover by distillation and recycled for pretreatment; (2)
the chemical recovery in organosolv pulping processes can isolate lignin as a solid material
and carbohydrates as a syrup, both of which show promise as chemical feedstocks [7678]. It
seems that organosolv pretreatment is feasible for biorefinery of lignocellulosic biomass,
which considers the utilization of all the biomass components. However, there are inherent
50 Appl Biochem Biotechnol (2014) 174:4362
drawbacks to the organosolv pretreatment. Organic solvents are always expensive, so it should
be recovered as much as possible, but this causes increase of energy consumption. In addition,
organosolv pretreatment must be performed under extremely tight and efficient control due to
the volatility/flammability of organic solvents which increase capital cost. Moreover, removal
of solvents from the system is necessary since the solvents might be inhibitory to enzymatic
hydrolysis and fermentative microorganisms [3].
Neutral Pretreatment
The liquid hot water (LHW) pretreatment is generally regarded as the neutral pretreatment
since the water (pH neutral) is used as pretreatment media.
Process Description
LHW pretreatment, also named as autohydrolysis, is a hydrothermal pretreatment. The
temperatures applied in the pretreatment can range from 160 to 240 C and over lengths
of time ranging from a few minutes up to an hour [79, 80]. LHW pretreatment results in
solubilization of hemicelluloses and increase of cellulose digestibility in the enzymatic
hydrolysis. LHW pretreatment can be performed in batch and flow-through reactors. In
batch reactors, the slurry of biomass and water is heated to the desired temperature and
held at the pretreatment conditions for the desired residence time before being cooled. In
a flow-through reactor, hot water is made to pass over a stationary bed of lignocelluloses
[81]. Generally, the flow-through process is regarded to be the more effective for
removing hemicellulose than the batch technique. This is because, in part, the large
amount of water used in a flow-through reactor quickly dilutes and removes organic
acids, which lowers the organic acid concentrations and minimizes the time for them to
act on the solid hemicellulose.
Mode of Action
During LHW, water acts as a weak acid and releases the hydronium ion, which causes
depolymerization of hemicellulose by selective hydrolysis of glycosidic linkages, liber-
ating O-acetyl group and other acid moieties from hemicellulose to form acetic and
uronic acids. The release of these acids has been postulated to catalyze the hydrolysis of
hemicelluloses and oligosaccharides from hemicelluloses [8285]. LHW pretreatment
results in preserving most of the cellulose in solid form, and the amount of glucan
retained is greater than in DAP [86]. Usually, the extent of cellulose hydrolysis in LHW
is less than in DAP, which is mainly due to the milder acid conditions. Similar to the
DAP pretreatment, several researchers have reported that the CrI of cellulose increased
after LHW pretreatment because the amorphous cellulose is more reactive than crystal-
line cellulose [87, 88]. The degradation of hemicellulose and cellulose will also form
furfural and HMF, respectively, during LHW pretreatment as occurred in DAP. However,
the quantities of sugar degradation products generated are lower than DAP [89, 90] and
would not significantly inhibit the fermentation process if LHW pretreatment is per-
formed under 220 C [83]. Moreover, LHW can maximize the solubilization of the
hemicellulose fraction while minimizing the formation of monosaccharides and the
subsequent sugar degradation products by maintaining the pH between 4 and 7 [91,
92]. Usually, the pH can be controlled by adding some bases (i.e., NaOH or KOH) into
Appl Biochem Biotechnol (2014) 174:4362 51
LHW pretreatment process with its role to maintain the pH value not as a catalyst in
alkaline pretreatment [93, 94]. In addition, the organic acids released from the biomass
also lead to the acidolysis of lignin and decrease the -O-4 linkage content in lignin
during the LHW pretreatment [95]. Moreover, the removal of hemicellulose and the
cleavage of lignin in LHW result in the increase of cellulose accessibility [96].
Advantage and Disadvantage
The major advantage of LHW pretreatment is that no additives such as acid catalysts are
required, minimizing the formation of inhibitory products. This also eliminates the need for
final washing step or neutralization because the pretreatment solvent is water. However, the
LHW pretreatment is regarded as energy demanding because of higher pressures and large
amounts of water supplied to the system.
Application of Acidic and Neutral Pretreatment
Table 3 shows the applications of different acidic pretreatments on herbaceous/agricultural
residues, hardwood, and softwood biomasses. It should be noted that the cellulose conversion
yields cited in this paper were based on cellulose fraction in the pretreated biomass, as
calculated in the literature [97]. Generally, the acidic pretreatments are effective for the
herbaceous/agricultural residues (i.e., corn stover, wheat straw, and miscanthus) and hardwood
(i.e., poplar and olive tree) while less effective for the softwood (i.e., radiata pine, Loblolly
pine, Douglas fir, and Lodgepole pine), which is mainly due to its high lignin content in the
biomass [34]. Among LHW, DAP, SEP, and organosolv pretreatments, the organosolv pre-
treatment yields the highest cellulose conversion (>80 %).
Alkaline Pretreatment
Alkaline pretreatments have received numerous studies as another major chemical pretreat-
ment technology besides acidic pretreatments. Alkaline pretreatments can be divided into two
groups based on the chemical used: (1) pretreatments that use sodium or calcium hydroxide
and (2) pretreatments that use ammonia [107]. The process description and alternations of
major chemical components for these two types of alkaline pretreatments are discussed in the
following sections.
Sodium Hydroxide and Lime Pretreatment
Process Description
Both sodium hydroxide and lime pretreatments have been shown to be effectively enhancing
cellulose digestibility [108111]. However, lime has received much more attentions than
sodium hydroxide since it is inexpensive (about 6 % cost of sodium hydroxide) [112], has
improved handling, and can be recovered easily by using carbonated wash water [113]. In
comparison with other pretreatment technologies, the sodium hydroxide and lime pretreat-
ments usually use lower temperatures and pressures, even at ambient conditions. Pretreatment
time, however, is recorded in terms of hours or days which are much longer than other
pretreatment processes. In the alkaline pretreatment, the residual alkali could be reused through
the chemical recycle/recovery process, which may make the system more complex due to the
52 Appl Biochem Biotechnol (2014) 174:4362
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Appl Biochem Biotechnol (2014) 174:4362 53
need for chemical recovery [94, 114]. In order to increase the pretreatment efficiency, usually,
the particle size needs to be reduced to 10 mm or less [113].
Mode of Action
The sodium hydroxide and lime pretreatments are basically a delignification process, in which
a significant amount of hemicellulose is solubilized as well. The major effect is the removal of
lignin from the biomass, thus improving the reactivity of the remaining polysaccharides. In
addition, the alkaline pretreatment could swell cell wall and improve cell wall accessibility for
the subsequent enzymatic hydrolysis. The proposed reaction mechanism is believed to be
saponification of intermolecular ester bonds cross-linking hemicellulose and lignin [94]. The
saponification leads to the cleavage of lignin-carbohydrate complex (LCC) linkages, and the
expose of cellulose microfibrils can increase enzymatic digestibility of cellulose. Acetyl
groups and various uronic acid substitutes are also removed by alkali, thereby reducing steric
hindrance of hydrolytic enzymes and increasing the accessibility of carbohydrates to enzymes
[109]. Furthermore, the degraded hemicellulose could also form furfural and HMF in the
hydrolysates, but the amount is typically lower than that with DAP [115]. In addition, alkaline
pretreatment decreases the DP of cellulose (Table 4) and causes swelling of cellulose, leading to
an increase in its internal surface area [116]. This makes cellulose more accessible for enzymes
in the subsequent hydrolysis stage. In terms of cellulose crystallinity change during the
pretreatment, research indicated that the amorphous regions suffered greater peeling reactions
than the crystalline regions, and the occurrence of the peeling actions of the amorphous regions
leads to an increase of cellulose crystallinity (Table 4) [108]. During the alkaline pretreatment,
lignin suffered delignification, which has similarities to soda chemical pulping technologies
[113, 117]. In addition, recent studies indicated that the alkali pretreatment could also increase
the fiber porosity due to the disruption of biomass structures [118, 119].
Advantage and Disadvantage
Compared with DAP, alkaline pretreatments have some practical operational advantages
including lower reaction temperatures and pressures. A significant disadvantage of alkaline
pretreatment is the conversion of alkali into irrecoverable salts and/or the incorporation of salts
into the biomass during the pretreatment reactions so that the treatment of a large amount of
salts becomes a challenging issue for alkaline pretreatment [94]. In addition, during the lime
pretreatment, the precipitation of calcium oxalate on the heat exchanger and process streams is
another issue to be considered since it may cause severe problems in the plants [120].
Table 4 Cellulose CrI and DP before and after lime pretreatments
Substrate Pretreatment conditions CrI (%) before
pretreatment
CrI (%) after
pretreatment
DP before
pretreatment
DP after
pretreatment
Corn
stover
55 C, 0.5:1 Ca(OH)
2
to
biomass, 4 weeks
50.3
a
56.2
a
7,300
b
3,200
b
Poplar 65 C, 0.5:1 Ca(OH)
2
to
biomass, 4 weeks
49.9
a
54.5
a
3,500
b
1,600
b
From Kumar et al. [38]
a
CrI was measured by X-ray diffraction (XRD) method [40]
b
DP was measured by viscometric method [41]
54 Appl Biochem Biotechnol (2014) 174:4362
Ammonia Fiber Expansion Pretreatment
Process Description
The AFEX pretreatment, also known as ammonia fiber explosion pretreatment, is another
physicochemical process, much like SEP, in which the biomass material is subjected to liquid
anhydrous ammonia under high pressures and moderate temperatures and is then rapidly
depressurized. This swift pressure release leads to a rapid expansion of the ammonia gas that
causes swelling and physical disruption of biomass fibers [121]. The AFEX is usually
performed to treat moist biomass (0.12 g H
2
O/g dry biomass) with liquid ammonia (0.3
2 g NH
3
/g dry biomass) while heating (60100 C) the biomass-water-ammonia mixture for a
period of time (560 min) before rapidly releasing the pressure [122]. AFEX can be carried out
in either a batch or a flow-through reactor. The major parameters influencing the AFEX
process are ammonia loading, temperature, pressure, moisture content of biomass, and resi-
dence time [123]. During the AFEX pretreatment, about 95 % of the ammonia can be
recovered in the gas phase and recycled, with a small amount of ammonia that remains in
the lignocellulosics which might serve as a nitrogen source for the microbes in the fermenta-
tion process [124].
Mode of Action
During the AFEX, the physicochemical treatment induces the disruption in the lignin-
carbohydrate linkage, hemicellulose hydrolysis, and ammonolysis of glucuronic cross-linked
bonds, and partial decrystallization of the cellulose structure, all leading to a higher accessible
surface area for enzymatic attack [123]. In AFEX pretreatment, the removal of acetyl groups
from hemicellulose results in the formation of acetamide and acetic acid, but it is reported that
AFEX removes the least amount of acetyl groups from lignocellulosic material compared to
other leading pretreatment technologies [38]. In addition, the ammonia also causes a series of
ammonolysis (amide formation) and hydrolysis reactions (acid formation) in the presence of
water, which cleave LCC ester linkages [125]. The major effect of AFEX on cellulose is the
decrystallization, which is mainly due to the generation of more amorphous portions in
cellulose during this process [38]. These chemical structural changes lead to the formation
of highly porous structures on the fiber cell wall, which greatly enhance enzyme accessibility
to the cellulosic microfibrils [125].
Advantage and Disadvantage
The important advantages of AFEX pretreatment include lower moisture content, lower
formation of sugar degradation products due to moderate conditions, almost complete recovery
of solid material and high cellulose digestibility in the subsequent enzymatic hydrolysis. One
concern for this process is the cost of ammonia and the need for recycling technologies [94].
Soaking in Aqueous Ammonia Pretreatment
Another type of process utilizing ammonia is soaking aqueous ammonia (SAA), which
appears as an interesting alternative since it is performed at lower temperature (3075 C)
[2]. The main purpose of SAA pretreatment is the removal of lignin, which is the physical and
chemical barrier that inhibits accessibility of enzyme to the cellulose substrate [126]. It is
regarded as a valuable pretreatment methodology due to the retention of the hemicellulose
Appl Biochem Biotechnol (2014) 174:4362 55
fraction and removal of lignin after pretreatment [127]. SAA has been shown to be able to
remove 74 % of the lignin from corn stover while retaining >85 % of the xylan and nearly
100 % of the glucan [128]. This allows easy downstream utilization of sugars in a single co-
fermentation process in which net sugar yield was increased due to the presence of hemicel-
luloses [129]. SAA utilizing lower temperatures and less extreme pHs reduces the associated
chemical and energy costs and may also reduce the formation of carbohydrate degradation
products. However, the SAA pretreated biomass pretreated shows relatively lower enzymatic
digestibility due to its mild pretreatment conditions [130, 131].
Application of alkaline pretreatment
Table 5 shows the applications of different alkaline pretreatments on herbaceous/agricultural
residues, hardwood, and softwood biomasses. Among the numerous types of biomass, soft-
woods are generally recognized as being much more refractory than hardwoods or herbaceous/
agricultural residues in the alkaline pretreatment process. This is, in part, due to the fact that
softwoods have a more rigid structure and contains more lignin [132].
Ozonolysis
Ozonolysis of biomass is usually carried out at low temperature (2030 C), and the ozone
flow rate ranged from 0.5 to 0.8 L/min. The ozone consumption (% dry wt. of biomass) is 2
Table 5 Application of alkaline pretreatment for different biomasses
Pretreatment method Substrate Pretreatment
conditions
Cellulose
conversion yield (%)
Enzyme loadings
NaOH or
Ca(OH)
2
pretreatment
Corn
stover [133]
55 C, 7.3 wt%
Ca(OH)
2
, 4 weeks
98 in 96 h 15 FPU/g for cellulase
from Spezyme CP
and 40 CBU/g for
-glucosidase from
Novozyme 188
Birch [134] 80 C, 7.0 wt%
NaOH, 2 h
80 in 96 h 20 FPU/g for cellulase
from Celluclast 1.5 L
and 50 IU/g for
-glucosidase from
Novozyme 188
Spruce [134] 80 C, 7.0 wt%
NaOH, 2 h
24 in 96 h 20 FPU/g for cellulase
from Celluclast 1.5 L
and 50 IU/g for
-glucosidase from
Novozyme 188
AFEX
pretreatment
Corn
stover [49]
90 C, 1:1 ammonia
to biomass loading,
60 % moisture
content, 5 min
76.6 in 72 h 15 FPU/g for cellulase
from Celluclast 1.5 L
and 26.25 CBU/g for
-glucosidase from
Novozyme 188
Poplar [135] 180 C, 2:1 ammonia
to biomass loading,
233 % moisture
content, 30 min
70 in 72 h 15 FPU/g for cellulase
from Celluclast 1.5 L
and 26.25 CBU/g for
-glucosidase from
Novozyme 188
56 Appl Biochem Biotechnol (2014) 174:4362
7 % [115, 136]. Ozone treatment is one way of reducing the lignin content of lignocellulosic
wastes. This results in an increase of the in vitro digestibility of the treated material, and unlike
other chemical treatments, it does not produce toxic residues. Ozone can be used to degrade
lignin and hemicellulose in many lignocellulosic materials such as wheat straw [137], bagasse,
green hay, peanut, pine [138], cotton straw [139], and poplar sawdust [140]. Research
indicated [141] that ozone is highly reactive toward compounds incorporating conjugated
double bonds and functional groups with high electron densities. Therefore, the moiety, most
likely to be oxidized in ozonization of lignocellulosic materials, is lignin due to its high content
of C=C bonds. Thus, during the ozonolysis, the degradation is mainly limited to lignin. Ozone
attacks lignin releasing soluble compounds of less molecular weight, mainly organic acids
such as formic and acetic acids [141]. The main advantages linked to this process are the lack
of any degradation products that might interfere with subsequent hydrolysis or fermentation
and the reactions occurring at ambient temperature and normal pressure. Furthermore, the fact
that ozone can be easily decomposed by using a catalytic bed or increasing the temperature
means that processes can be designed to minimize environmental pollution. A drawback of
ozonolysis is that a large amount of ozone is required, which can make the process expensive
and less applicable [142]. However, recently, Hu et al. [13] demonstrated that a lower charge of
ozone could be used to enhance the enzymatic digestibility of cellulose, if the ozone-treated
biomass was not washed and the in situ generated acids were employed in a subsequent DAP.
Conclusions and Perspectives
Most of the pretreatment technologies that have been described herein are effective on one or
more factors that contribute to lignocellulosic recalcitrance, as shown in Table 6. From the
work that has been presented, it is clear that each pretreatment method has its own merits and
disadvantages and consequences on the enzymatic hydrolysis. Recently, researchers applied
some combinations of different pretreatment methods, such as alkaline pretreatment followed
by steam pretreatment [143, 144] and organosolv pretreatment coupled with steam explosion
[143], to improve the biomass digestibility. Although the results are promising compared with
the single pretreatment, the extra equipment and operation cost will compromise these
Table 6 Effect of different chemical pretreatment technologies on the structure of lignocellulose
Pretreatment
method
Increase of
accessible surface
area
Cellulose
decrystallization
Hemicellulose
solubilization
Lignin
removal
Generation of
inhibitor
compounds
Lignin
structure
alteration
DAP H L H L H H
SEP H L M L H M
Organosolv H L H H H H
LHW H L M L L M
NaOH/
Ca(OH)
2
H L M M L H
AFEX H H L L L H
SAA H L L H L L
Ozonolysis H L M H L H
From Alvira et al., Brodeur et al., and Li et al. [2, 11, 20]
H high effect, M moderate effect, L low effect
Appl Biochem Biotechnol (2014) 174:4362 57
processes. In addition, advanced transgenic techniques were reported to alternate the chemical
structures of lignocellulosic biomass with the aim to reduce their recalcitrance in the pretreat-
ments [145, 146]. However, these techniques are still in development, and significant research
needs to be done before the commercialization.
Despite much research that has been dedicated to understanding the chemistry and the
plant cell wall structure changes during various pretreatment technologies, the insuffi-
cient knowledge of cell wall structure, ultrastructure, and pretreatment effects still limits
the economics and effectiveness of pretreatment. For instance, the biological and chem-
ical properties of plants are very complex in terms of composition, structure, and
ultrastructure [147]. Although researchers have put significant effort into optimizing
the pretreatment effectiveness, the fundamental science behind these optimizations is
still not fully understood. Furthermore, there has been a lack of mechanistic understand-
ing of the ultrastructural and physicochemical changes occurring within the cell wall at
the molecular level and the cellular/tissue scale during various pretreatment technologies.
It is thus essential to understand the effects of pretreatment on plant cell walls at a more
fundamental level, in order to develop a cost-effective pretreatment technology with
maximum fermentable sugar recovery; minimum inhibitor production and energy input;
low demand of post-pretreatment processes; and low capital costs for reactors, water, and
chemicals. In addition, advances in the analytical chemistry would provide useful tools to
investigate the cell wall deconstruction and understand the recalcitrance during the
pretreatment process [148, 149].
Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No.
31300483), Natural Science Foundation of Jiangsu Province of China (Grant No. BK20130971), and the Priority
Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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