C H A P T E R

4
Alkaline Treatment
Raveendran Sindhu, Ashok Pandey, Parameswaran Binod
Centre for Biofuels, Biotechnology Division, CSIR e National Institute
for Interdisciplinary Science and Technology, Trivandrum, India

4.1 INTRODUCTION
Pretreatment of biomass is the first step in the bioethanol process. The purpose of pretreat-
ment is to disorganize the crystalline structure of micro- and macrofibrils and to modify
pores for enzyme access. An effective pretreatment should be an inexpensive process and
should use simple equipment and avoid formation of inhibitors. Pretreatment is an impor-
tant tool for cellulose conversion processes. It is needed to alter the structure of cellulosic
biomass to make cellulose more accessible to the enzymes that convert the carbohydrate
polymers into fermentable sugars. The goal is to break the lignin seal and disrupt the crys-
talline structure of cellulose. Pretreatment has been viewed as one of the most expensive pro-
cessing steps in lignocellulosic biomass to fermentable sugars conversion. Pretreatment also
has great potential for improvement of efficiency and lowering the cost through research and
development.
An effective and proper pretreatment can reduce the overall cost in lignocellulosic conver-
sion. A number of lignocellulosic pretreatment technologies are under intensive investigation
in both laboratory as well as pilot-plant scales. Alkali treatment is considered to be a prom-
ising pretreatment method that alters structural properties, such as accessible surface area
and crystallinity of pretreated substrate, thereby enhancing its enzymatic hydrolysis [1].
Processing of lignocellulosics to ethanol consists of four major unit operations: pretreat-
ment, hydrolysis, fermentation, and product separation and purification. Pretreatment is
required to alter the biomass macroscopic and microscopic size and structure as well as its
submicroscopic chemical composition and structure so that hydrolysis of the carbohydrate
fraction to monomeric sugars can be achieved more rapidly and with greater yields.
An effective pretreatment avoids the need for reducing the size of biomass, preserves the
pentose (hemicellulose) fractions, limits formation of inhibitors (degradation products) that
inhibit growth of fermentative microorganism, minimizes energy demands, and limits cost.
These properties, along with others including low pretreatment catalyst cost or inexpensive

Pretreatment of Biomass
http://dx.doi.org/10.1016/B978-0-12-800080-9.00004-9 51 Copyright Ó 2015 Elsevier B.V. All rights reserved.
52 4. ALKALINE TREATMENT

catalyst recycle and generation of higher-value lignin coproduct form a basis of comparison
for various pretreatment options.
There are several physical, chemical, physicochemical, and biological methods for pre-
treatment of lignocellulosic biomass. Compared with acid, alkali pretreatment is preferred
because of its ability to remove lignin and cause less degradation of carbohydrates. No
washing is required after pretreatment, and the addition of alkali would not result in a corro-
sion problem in the extruder as compared to acid [2].
Alkali pretreatment processes utilize lower temperatures and pressures compared with
other pretreatment technologies. Alkali pretreatment may be carried out at ambient condi-
tions, but pretreatment time is in hours or days rather than minutes or seconds [3]. Unlike
acid-catalyzed pretreatments, a limitation occurs because some of the alkali is converted to
irrecoverable salts or incorporated as salts into the biomass by the pretreatment reactions.
Unlike the use of steam and acid pretreatments, alkaline pretreatments for example,
lime (Ca (OH)2) solubilize lignin and a small percentage of the hemicellulose [4]. During
the alkaline pretreatment, the lignocellulose undergoes two reactionsdsolvation and sap-
onificationdwhich cause the structure of the lignocellulose to swell, decreasing the degree
of polymerization, thus making the lignocellulose components more accessible to enzy-
matic and microbial degradation. It also has been found that alkaline solutions can be
used in the solubilization, redistribution, and condensation of lignin, which also leads to
the modification of the crystalline cellulose. The most common alkaline pretreatments
that are employed make use of sodium hydroxide (NaOH) and Ca (OH)2. The following
sections review the developments in alkali pretreatment of lignocellulosic biomass. Table
4.1 shows the list of different types of alkali used for the pretreatment of lignocellulosic
biomass. The process flow chart for the alkali pretreatment is shown in Figure 4.1.

TABLE 4.1 List of Commonly Used Alkali for Pretreatment of Various

Lignocellulosic Biomass

Type of Alkali Biomass References

Sodium hydroxide Switch grass [5]

Rice straw [6]

Sugarcane bagasse [7]

Cotton postharvest residue [8]
Dendrocalamus [9]
Sugarcane tops [10]
Potassium hydroxide Switch grass [11]
Lime Switch grass [4]

Corn stover [12]

Poplar wood [13]
Ammonia Corn stover [14]

A. LIGNOCELLULOSIC BIOMASS
 4.2 TYPES OF ALKALI 53

Lignocellulosic biomass

Milling

Size reduction

Addition of water and alkali

Slurry preparation

Pretreatment

Centrifugation to separate solids and liquids

Supernatant
Pretreated residue

Lignin and alkali

Neutralization

Enzymatic saccharification

Saccharomyces cerevisiae

Fermentation

Fuel ethanol

FIGURE 4.1 Process flow chart for alkali pretreatment of lignocellulosic biomass.

4.2 TYPES OF ALKALI

The common alkalis used for pretreatment of lignocellulosic biomass are NaOH, KOH,
NH3, and Ca (OH)2. The cheapest alkali commonly used for pretreatment is Ca (OH)2, which
removes lignin and acetyl groups that in turn increase the rate of enzymatic saccharification
[15]. The rate of lignin removal from the biomass during Ca (OH)2 pretreatment depends on
the type of biomass treated as well as the quantity of lignin present in the sample. This pre-
treatment is less effective against woody plants when compared with herbaceous and

A. LIGNOCELLULOSIC BIOMASS
54 4. ALKALINE TREATMENT

agricultural plants because of the higher quantity of lignin present in woody plants.
Although there are certain limitations, there are certain advantages of Ca (OH)2 pretreatment,
which include low cost of the pretreatment agent, nontoxicty, ubiquitousness, and ability to
be recovered and recycled [16].
Kraft process is one of the well-known processes of alkaline chemical pulping of lignocel-
lulosic biomass. The delignification of biomass was carried out with NaOH and Na2S as a
delignifying agent [17]. During the kraft process, lignocellulosic biomass is cooked with
NaOH and NaS to produce NaHS and H2S. These sulfur derivatives react with lignin com-
ponents and produce thiolignins.
NaOH commonly is used in the chemical pretreatment of lignocelluloses because of its
ability to delignify biomass. But the large-scale production of bioethanol using NaOH as a
pretreatment agent may not be cost-effective [18e21]. This treatment causes swelling of
lignocellulosic biomass, which leads to an increase in the internal surface area, reduces
cellulose crystallinity, and disrupts lignin structure, thereby enhancing the reactivity of the
remaining carbohydrate [22]. The schematic representation of the process involved in
NaOH pretreatment of lignocellulosic biomass is shown in Figure 4.2. During alkali pretreat-
ment, saponification of ester bonds takes place, which results in the swelling of wood and
enhances the enzyme penetration into the cell wall fine structure [23].
Other pretreatments commonly employed use NH3, which improve the digestibility of
cellulose by decreasing crystallinity of the fibrils. At high temperature, NH3 depolymerizes
the lignin to release the polysaccharide matrix [24]. In ammonia fiber explosion (AFEX) the

FIGURE 4.2 Effect of NaOH pretreatment of lignocellulosic biomass.

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 4.2 TYPES OF ALKALI 55
lignocellulosic biomass was pretreated with liquid NH3 under pressure. The rapid release of
pressure results in decrystallization of cellulosic components and hydrolyzes traces of lignin
as well as alters the lignin structure [25,26]. Although AFEX pretreatment does not remove
substantial quantity of hemicelluloses and lignin, the enzymatic saccharification rate in-
creases, which may be due to decrystallization of cellulose, which in turn increases enzyme
accessibility [20,25]. Ammonia recycle percolation (ARP) pretreatment is another process that
makes use of ammonia. During ARP, ammonia solution is passed though a reactor packed
with lignocellulose at temperatures between 80 and 180
C, and the ammonia present in
the effluent is recycled.
Enhanced enzymatic hydrolysis of spruce by alkaline pretreatment at low temperature was
reported by Zhao et al. [27]. It was observed that enzymatic hydrolysis rate and efficiency can
be improved significantly by this pretreatment. At low temperature, NaOH can slightly
remove lignin, hemicelluloses, and cellulose in the lignocellulosic biomass. This treatment dis-
rupts connections between hemicelluloses, celluloses, and lignin and alters the structure of
treated biomass to make cellulose more accessible to enzymatic saccharification.
Alkali pretreatment of switch grass by radio frequency (RF)-based dielectric heating was
reported by Hu et al. [5]. Because of unique features of RF like volumetric heat transfer and
deep-heat penetration of samples, this pretreatment is effective with high-solid loading.
When the solid content exceeded 25%, conventional heating could not handle this high-
solid sample because of the loss of fluidity, poor mixing, and heat transfer of the samples.
As a result, there was a significantly lower sugar yield, but the sugar yield of the RF-based
pretreatment process still was maintained at high levels. The optimal particle size and alkali
loading in the RF pretreatment was 0.25e0.50 mm and 0.25 g NaOH/g biomass, respectively.
At alkali loading of 0.20e0.25 g NaOH/g biomass, heating temperature of 90
C, and solid
content of 20%, the glucose, xylose, and total sugar yield from the combined RF pretreatment
and the enzymatic hydrolysis were 25.3, 21.2, and 46.5 g/g biomass, respectively.
Lime pretreatment has been reported for pretreatment of different biomasses, such as
switch grass [4], wheat straw [15], corn stover [12], and poplar wood [13]. Rabelo et al.
[28] evaluated the effectiveness of lime pretreatment in improving sugarcane bagasse suscep-
tibility to enzymatic hydrolysis. The effect of lime pretreatment on digestibility was studied
through analyses using central composite design (response surface), considering pretreat-
ment time, temperature, and lime loading as factors. The study revealed that the optimum
conditions of pretreatment were with 0.15 g/g of biomass, pretreatment temperature of
86.8
C, and 65.6 h of pretreatment time.
Cheng et al. [6] evaluated high-solid-loading alkaline pretreatment of rice straw. The
biomass was pretreated with NaOH and lime. The study revealed that alkaline loading
and reaction time have positive effects on delignification. Treatments at higher temperature
improved the delignification rate. Although Ca (OH)2 pretreatment yielded the highest
glucose conversion ratios and glucose yields, higher temperatures were employed in com-
parison with NaOH pretreatment. The requirement for washing Ca (OH)2-pretreated rice
straw may make this source of alkali unfavorable on a large scale.
Park and Kim [29] compared the efficacy of soaking and percolation pretreatments with
alkaline solutions for lignocellulosic biomass. Various biomasses (such as rice straw and
barley) were pretreated by soaking processes in various alkaline solutions, including sodium
hydroxide, potassium hydroxide, aqueous ammonia, and sodium carbonate. The enzymatic

A. LIGNOCELLULOSIC BIOMASS
56 4. ALKALINE TREATMENT

digestibility of rice straw and barley pretreated by soaking in aqueous ammonia was 85%
and 95%, respectively. Eucalyptus residue, Larix leptolepis and Pinus rigida, exhibited relatively
low enzymatic digestibility in the range of 3.2e8.3%. The enzymatic digestibility of pre-
treated Eucalyptus residue was increased by five times compared with native biomass. In
the pretreatment of Eucalyptus residue, delignification was more than doubled with percola-
tion pretreatment compared with soaking pretreatment. The enzymatic digestibility of the
percolation pretreated Eucalyptus residue was increased 12 times.
Macarie et al. [30] reported the use of alkaline lyophilization process for lignocellulosic
biomass pretreatment. The study revealed that alkaline lyophilization of the biomass signif-
icantly improves the yield in free sugars. The major advantages of this pretreatment are that it
reduces consumption of the reagents; pretreatment is achieved in one stage as well as
simplicity and efficiency.
A novel soaking pretreatment of corn stover using NaOH and aqueous ammonia for
delignification for improved enzymatic saccharification rate was evaluated by Zuo et al.
[14]. The results revealed 63.6% of lignin removal while reserving most of the carbohydrates.
The optimum conditions of pretreatment were 1% NaOH þ 8% NH4OH with a solid liquid
ratio of 1:10 and pretreatment temperature of 50
C for 48 h.
Kuttiraja et al. [9] reported alkali pretreatment of Indian bamboo variety Dendrocalamus sp.
Diluted alkali pretreatment of the biomass resulted in the efficient removal of lignin, effec-
tively increasing the concentration of cellulose from 46.7% to 63.1%. The pretreatment
method could recover 64.31% of the total sugar polymers and a hydrolysis efficiency of
82.36% was achieved.
The effect of KOH as a pretreatment agent for switch grass was evaluated by Sharma et al.
[11]. The pretreatment condition of 0.5% KOH, 24 h, 121
C was determined to be the most effec-
tive, as it utilized the least amount of KOH while generating 582.4 mg sugar/g raw biomass for a
corresponding percent carbohydrate conversion of 91.8%. The study revealed the requirement
for a post-pretreatment washing step to generate maximum sugars from the pretreated biomass.
A high sugar yield with the 0.5% KOH, 12 h, 121
C pretreatment indicates that even very low
concentrations of KOH can be effective in generating high sugars during hydrolysis.
Alkaline pretreatment of sugarcane tops was reported by Sindhu et al. [10]. The reducing
sugar yield from NaOH pretreated biomass was seven times higher when compared with
untreated sugarcane tops. The study demonstrated that enzymatic hydrolysis was improved
significantly due to lignin removal by alkali pretreatment.

4.3 CONDITIONS OF ALKALI PRETREATMENT

The condition of alkali pretreatment varies depending on the type as well as the compo-
sition of biomass used for pretreatment. The most important parameters affecting pretreat-
ment of lignocellulosic biomass are the type of alkali, concentration of alkali, biomass
loading, pretreatment temperature, and pretreatment time. To develop an economically
viable process, it is essential to develop a pretreatment strategy to be carried out at low
temperature and with low concentration of alkali.
The optimum conditions for NaOH pretreatment of sugarcane tops were 3% w/w NaOH
with 15% w/w of biomass loading and pretreatment time of 60 min in a laboratory autoclave

A. LIGNOCELLULOSIC BIOMASS
 4.5 PHYSICOCHEMICAL CHARACTERIZATION OF ALKALI PRETREATED BIOMASS 57
[10]. For cotton postharvest residue, the optimum conditions of pretreatment was 4% w/w of
NaOH with a pretreatment temperature of 180
C for 45 min in a high-pressure reactor [8]. The
optimum conditions for alkali pretreatment of sugarcane bagasse were 5% w/w of biomass
loading with 3% w/w of NaOH, pretreatment time of 60 min in a laboratory autoclave [7].

4.4 MECHANISM OF ALKALI PRETREATMENT

The major effect of alkali pretreatment is delignification of the biomass, which in turn
improves the saccharification efficiency. The mechanism involved in alkaline pretreatment
is cleaving linkages in lignin and glycosidic bonds of polysaccharides and cause a reduction
in degree of polymerization and crystallinity. In addition, this treatment also removes acetyl
and various uronic acid substitutions that lower the accessibility of cellulase [31]. The effec-
tiveness of alkaline pretreatment depends on the physical structure, composition of the
biomass, and the treatment conditions. Generally, alkaline pretreatment is more effective
for hardwood with high lignin content. The commonly used alkalis for pretreatment are
sodium hydroxide, calcium hydroxide, ammonium hydroxide, and potassium hydroxide.

4.5 PHYSICOCHEMICAL CHARACTERIZATION

OF ALKALI PRETREATED BIOMASS
Janu et al. [7] reported morphological and physicochemical changes of NaOH pretreated
sugarcane bagasse by SEM, XRD, and FTIR. Scanning electron micrographs revealed differ-
ences between the native and pretreated samples (Figure 4.3). Native samples exhibited a
rigid, highly compact, and nonporous structure, while the pretreated samples showed an
increase in porosity and greater surface area. This is due to the removal of lignin and hemi-
celluloses, which in turn destroyed the cellulose-hemicellulose-lignin network, leading to the
disruption of the hydrogen bond between the cellulose and becoming more susceptible for
enzymatic hydrolysis. The loose structure as well as an increase in surface area of the alkali
pretreated sugarcane bagasse allows hydrolytic enzymes to penetrate, adsorb, and hydrolyze
the lignocellulosic materials more easily, thus increasing the hydrolysis efficiency.
With alkaline pretreatment, there is 82% reduction in lignin content. Crystallinity intensity
analysis by XRD indicated a considerable increase in crystallinity index (CrI) of alkali

FIGURE 4.3 Scanning electron micrographs of native and pretreated sugarcane bagasse.

A. LIGNOCELLULOSIC BIOMASS
 58 4. ALKALINE TREATMENT

105 FIGURE 4.4 FTIR spectrum of

native and alkali pretreated sugar-
100 cane tops.
% Transmiance
95
90
85
80
75
70
500 1000 1500 2000 2500 3000 3500 4000
Wavenumber (/cm)

Alkali pretreated SCT Nave SCT

pretreated sugarcane bagasse when compared with a native sample. Crystallinity of lignocel-
luloses is a major factor that influences enzymatic hydrolysis. The increase in CrI indicates
that alkali pretreatment is more effective for amorphous zone than the crystalline zone,
which in turn increases the CrI.
The structural modifications of native and pretreated biomass were analyzed using FTIR
spectroscopy. FTIR spectra of lignocellulosic materials were influenced by three main
polymersdcellulose, hemicelluloses and lignin. FTIR spectra of native and alkali pretreated
sugarcane tops showed difference in the absorption spectra (Figure 4.4). The carbonyl band at
1735/cm was weakened on pretreated sugarcane bagasse indicating removal of hemicellu-
lose. The peaks corresponding to aromatic ring stretch at 1590/cm also were weakened indi-
cating delignification.

4.6 PROSPECTS AND CONSEQUENCES

The major advantage of using alkali during pretreatment is that it utilizes lower temper-
ature and pressure compared with other pretreatment strategies. Because pretreatment is car-
ried out at lower temperature, there will be considerably low concentrations of primary
degradation products that will affect subsequent fermentation by yeast. The advantage of
this pretreatment technology lies in the fact that it would create a washed clean substrate
that is highly digestible and rich in cellulose and xylan. Another advantage of using alkali
over dilute acid is that the corrosion rate of alkali is slower when compared with acid.
Alkaline pretreatment has a unique application in many integrated biorefineries where
value added products, other than ethanol, are produced from lignocellulosics. After enzy-
matic hydrolysis, a clean sugar stream could be obtained at high yield and low enzyme
dose. For processes that are highly sensitive to impurities (inhibitors, salts), alkaline pretreat-
ment is certainly a better choice [32].
One of the major limitation in alkali pretreatment is the generation of irrecoverable salts or
incorporated as salts in the biomass during the pretreatment [3]. Pretreatment using dilute

A. LIGNOCELLULOSIC BIOMASS
 REFERENCES 59
acid yields a separate pentose stream and a pretreated residue rich in hexoses, while alkaline
pretreatment does not provide any separation of pentose and hexoses. Most of the pilot-plant
operations utilize dilute acid pretreatment because hexoses and pentoses are separated.

4.7 COMMERCIALIZATION ASPECTS

Pilot-plant and commercial-scale facilities for converting lignocellulosic biomass have
been in existence since 1900. Majority of the early plants used acids for hydrolysis of cellulose
to ethanol. According to previous reports by the National Renewable Energy Laboratory, the
cost of lignocellulosic ethanol plant using dilute alkali pretreatment was higher when
compared with lignocellulosic ethanol plant using dilute acid. From an economic point of
view, an alkali-based biorefinery is less economically attractive. The key factors that play sig-
nificant role in the economic viability of a process are pretreatment temperature, and water
requirements will directly affect the cost of equipment, operational energy, and wastewater
treatment. The specific alkali used and concentration affect not only the cost of materials but
also the alkali recovery operations. Thus, a technoeconomic analysis is necessary for devel-
oping the best alkaline pretreatment process on a large scale.

4.8 CONCLUSION
Various parameters that contribute resistance for biomass enzymatic saccharification
include accessible surface area, crystallinity of cellulose, protection by lignin, and hemicellu-
loses. To develop an economically viable process, it is essential to focus on reducing the con-
centration of pretreatment agent, pretreatment time, and pretreatment temperature as well as
the production of value added products, such as organic acids, amino acids, and biopoly-
mers. The efficacy of pretreatment depends strongly on the type of feedstock rather than
the type of pretreatment adopted. To achieve this, one should have a thorough knowledge
about the unique characteristics about the different types of biomass and the conditions
for efficient fractionation of lignocelluloses.

Acknowledgement
The authors acknowledge the financial support for the Centre for Biofuels by the TIFAC, New Delhi and MNRE,
New Delhi. RS acknowledge DBT Bio-CARe programme for financial support.

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