Bioresource Technology 102 (2011) 62546259

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

Liquid hot water and alkaline pretreatment of soybean straw for improving
cellulose digestibility
Caixia Wan a, Yuguang Zhou a,b, Yebo Li a,
a
Department of Food, Agricultural, and Biological Engineering, The Ohio State University/Ohio Agricultural Research and Development Center, 1680 Madison
Avenue, Wooster, OH 44691, USA
b
College of Engineering, China Agricultural University, PO Box 50, 17 Qinghua East Road, Beijing 100083, China

a r t i c l e

i n f o

Article history:
Received 16 December 2010
Received in revised form 16 February 2011
Accepted 17 February 2011
Available online 20 February 2011
Keywords:
Soybean straw
Liquid hot water
Alkaline pretreatment
Cellulose digestibility

a b s t r a c t
Soybean straw was pretreated with either liquid hot water (LHW) (170210 C for 310 min) or alkaline
soaking (440 g NaOH/100 g dry straw) at room temperature to evaluate the effects on cellulose digestibility. Nearly 100% cellulose was recovered in pretreated solids for both pretreatment methods. For LHW
pretreatment, xylan dissolution from the raw material increased with pretreatment temperature and
time. Cellulose digestibility was correlated with xylan dissolution. A maximal glucose yield of 70.76%,
corresponding to 80% xylan removal, was obtained with soybean straw pretreated at 210 C for
10 min. NaOH soaking at ambient conditions removed xylan up to 46.37% and the subsequent glucose
yield of pretreated solids reached up to 64.55%. Our results indicated LHW pretreatment was more effective than NaOH soaking for improving cellulose digestibility of soybean straw.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
With soaring increases in world energy consumption and growing concerns about climate change, biomass-based renewable energy, such as cellulosic ethanol, is considered to be one solution
to the energy crisis and global warming. Lignocellulosic biomass,
such as crop residue, woody biomass, and energy crops, is widely
and sufciently available for cellulosic ethanol production. However, the recalcitrance of lignocellulosic biomass to chemical and
enzyme conversion hinders efcient production of cellulosic ethanol. Thus, a suitable pretreatment process is important to reduce
the recalcitrance and to make bioconversion processes more efcient, economic, and environmentally friendly.
Liquid hot water (LHW) pretreatment has been shown to
improve cellulose digestibility of various types of lignocellulosic
biomass. LHW pretreatment differs from other current leading pretreatment methods (e.g., diluted acid, alkaline, steam explosion)
with respect to chemical usage and formation of inhibitory compounds (e.g., HMF and furfural). It is a chemical free process which
hydrolyzes hemicellulose at elevated temperature and pressure.
Acetic acid and other organic acids generated from autohydro-

Corresponding author at: Department of Food, Agricultural, and Biological

Engineering, The Ohio State University/Ohio Agricultural Research and Development Center, 1680 Madison Avenue, Wooster, OH 44691, USA. Tel.: +1 330 263
3855; fax: +1 330 263 3670.
E-mail address: li.851@osu.edu (Y. Li).
0960-8524/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.02.075

lysis can catalyze further hydrolysis (Kim et al., 2009). In the meantime, accumulation of hydrogen ions accelerates degradation of
fermentable sugars. However, compared to steam explosion to
which no chemical catalyst is added, fewer inhibitory compounds
to fermentation are formed during LHW pretreatment (Laser
et al., 2002). The pH controlled, LHW pretreatment can prevent
degradation of fermentable sugars but involves the use of a base
(Mosier et al., 2005a).
Alkaline pretreatment is one of the current leading chemical
pretreatment methods, particularly for dissolving lignin. In addition, acetyl groups and various uronic acid substitutes, which
lower susceptibility of hemicellulose and cellulose to hydrolytic
enzymes, are also removed by alkaline pretreatment (Mosier
et al., 2005b). Agricultural residues and herbaceous crops have
been shown to be more suitable to alkaline pretreatment than
woody biomass (Galbe and Zacchi, 2007). Reaction temperature,
pretreatment time, and alkali loading are critical to alkaline pretreatment (Silverstein et al., 2007). Varga et al. (2002) reported
that more than 95% lignin and about 88% hemicellulose in corn
stover were removed as a result of pretreatment with 10% NaOH
at 120 C. Alkaline soaking at room temperature instead of elevated temperatures was also found to be effective in improving
cellulose digestibility of corn stover (Li et al., 2004). As alkaline
pretreatment does not need a complicated reactor, there is an increased interest in applying alkaline pretreatment on-farm (Digman et al., 2007). The major problem of chemical pretreatments
is washing and/or neutralization of the pretreated slurry. In order
to reduce the environmental impacts of chemical waste disposal,

C. Wan et al. / Bioresource Technology 102 (2011) 62546259

extra processes are needed for wastewater treatment and chemical recycling.
As one of the major food and energy crops, soybean plantings
occupy large land areas all over the world. In 2009, nearly 77.5
million acres of soybeans were planted in the U.S. (USDA, 2009).
Soybean straw, left after soybean harvesting, is partially collected
for use as animal feed (Gupta et al., 1978). With the annual demand for soybeans increasing, there is growing interest in soybean
straw as a potential feed stock for cellulosic ethanol production.
However, there is little information related to bioconversion of
soybean straw. The only test to date on pretreatment of soybean
straw used ammonia soaking, which resulted in 51% cellulose
digestibility (Xu et al., 2007). It was our goal to evaluate the efcacy of LHW and alkaline pretreatment on soybean straw in terms
of cellulose digestibility. The degradation of xylan and formation of
inhibitory compounds were also investigated.
2. Methods
2.1. Materials
Soybean straw, collected from the farm of The Ohio Agricultural
Research and Development Center (OARDC) in Wooster, OH, was
dried at 40 C to moisture content less than 5%, ground to pass a
3/16 inch screen, and stored in an airtight container at room temperature prior to use. Raw soybean straw was composed of 34.09%
cellulose, 16.05% hemicellulose(11.43% xylan, 1.83% galactan,
1.00% arabinan, and 1.79% mannan), 21.6% lignin, 11.01% extractives, 5.16% ash, and 12.09% other non-identied compounds
(e.g., protein, pectin, acetyl groups, glucuronic acid substitutes).
Spezyme CP was obtained from Genencor (Palo Alto, CA) and its
activity was determined to be 50 FPU/mL according to NREL
Laboratory Analytical Procedure (LAP) 006 (Adney and Baker,
1996).
2.2. LHW pretreatment
LHW pretreatment was conducted in a 1-L Parr reactor (Parr
Instrument Company, Moline, IL). Forty grams of soybean straw
(dry mass) were evenly mixed with 400 mL of deionized (DI) water
in the stainless steel vessel of the reactor. The temperature and
pressure of the reactor were automatically controlled. The reactor
was heated to the target temperatures of 170, 180, 190, 200, or
210 C from room temperature (21 2 C) within 3045 min and
with continuous agitation (approximately 400 rpm). The corresponding pressures at the target temperatures were 6.9, 9.3, 12.1,
15.2, and 18.6 bar, respectively. After being held at each target
temperature for 3, 5, or 10 min, the reactor was cooled down to
room temperature by turning on the cooling water for approximately 30 min. The pretreated slurry was washed with 1000 mL
DI water and ltered by vacuum ltration using glass ber lters
(1.6 lm). The liquor and retained solid on the lter paper were collected separately for analysis.
2.3. Sodium hydroxide pretreatment
Fifty grams of soybean straw (dry mass) were soaked in 500 mL
NaOH solution in a1-L beaker to obtain 440 g NaOH loading based
on 100 g dry mass. The ratio of solid to liquid was 1:10. The beaker
was covered with plastic wrap and placed at room temperature
(24 1 C) for 24 h. The pretreated slurry was extensively washed
with tap water using a sieve (325 mesh) until the pH reached
around 8 then was further neutralized with HCl to a pH value between 6.5 and 7.0. The neutralized solid was then drained, washed,
and collected for further analysis.

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2.4. Enzymatic hydrolysis

The enzymatic hydrolysis was conducted following NREL protocol LAP-008 (Dowe and McMillan, 2001). The solid and Spezyme CP
loading were 2.5% (w/w) and 20 FPU/g solid, respectively. The
hydrolysis lasted 48 h at 50 C with an agitation speed of
130 rpm on a rotary shaker (Excella E24, Newbrunswick, NY,
USA). After 48 h, the hydrolysate was boiled for 5 min to deactivate
enzymes and then chilled in an ice bath. The supernatant was ltered through a 0.2 lm nylon membrane lter and solids collected
for sugar analysis.
2.5. Analytical methods
Compositions of untreated and pretreated soybean straw were
determined following NREL protocols (Sluiter et al., 2008). The
sugar concentrations were determined by high performance liquid
chromatography (HPLC) (Agilent 1200 series, MN, USA) with a
BioRad Aminex HPX-87P column (BioRad Inc., Hercules, CA) and
a refractive index detector (RID). The mobile phase was HPLC grade
water, eluting at a ow rate of 0.6 mL/min. The temperatures of the
column and detector were maintained at 80 and 55 C, respectively. HMF and furfural were analyzed by HPLC using a Phenomex
Rezex RFQ Fast Acid column (Phenomex, Torrance, CA). The mobile
phase was 0.005 N sulfuric acid at a ow rate of 0.6 mL/min. The
temperatures of the column and detector were maintained at 55
and 45 C, respectively.
The solid content in the pretreated materials was determined
using a 105 C drying oven. Solid recovery were calculated as the
mass of pretreated solids or water soluble solids in the pretreated
slurry expressed as a percentage of the total mass in the raw material. Total mass recovered was determined by the amount of pretreated solid residue plus the soluble solids in the liquor.
Cellulose or xylan recovery in the solids or liquor was calculated
as the mass of cellulose or xylan in each fraction expressed as a
percentage of total glucan or xylan in the raw material. The sugar
concentration in the liquor was determined according to NREL protocols (Sluiter et al., 2006). Sugar (glucose or xylose) yield by enzymatic hydrolysis was dened as the total mass of sugar produced
by enzymatic hydrolysis as a percentage of its total mass in the
raw material.
The monomeric sugar was degraded into HMF or furfural in severe pretreatment conditions. The converting factors for HMF to
glucose, and furfural to xylose were 180/126 and 150/96, respectively (Zeng et al., 2007). HMF and furfural yields were thus calculated as the amount of corresponding degraded glucose or xylan
expressed as a percentage of total glucan or xylan in the raw
material.
2.6. Statistical analysis
All data reported were the average values of duplicates. A 95%
condence level was applied for statistical analysis and one-way
analysis of variance (ANOVA) was performed with Turkey simultaneous tests (SAS Inc., Cary, NC).
3. Results and discussion
3.1. LHW pretreatment
3.1.1. Biomass degradation
As shown in Table 1, recovery of mass in the solid residue after
pretreatment decreased with pretreatment temperature and time.
In contrast, recovery of solids dissolved in the liquor increased
with pretreatment time and temperature except a signicant

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C. Wan et al. / Bioresource Technology 102 (2011) 62546259

Table 1
Overall mass recovery, composition of pretreated solids, and total sugar yields through pretreatment and enzymatic hydrolysisa.
Mass recovery

a
b

Carbohydrate content in pretreated solids

Sugar yieldb

Temp (C)

Time (min)

Solid residue (%)

Soluble in liquor (%)

Total (%)

Glucan (%)

Xylan (%)

Glucose (%)

Xylose (%)

Untreated
170
180
190
200
210
170
180
190
200
210
170
180
190
200
210

3
3
3
3
3
5
5
5
5
5
10
10
10
10
10

87.57
82.70
76.20
70.58
67.24
84.36
81.04
75.29
69.17
66.68
82.22
79.54
71.63
68.38
65.19

12.09
15.78
18.39
20.61
21.15
15.10
15.95
18.85
20.00
19.96
15.63
16.25
20.41
20.95
16.96

99.65
98.48
94.59
90.94
88.39
99.46
96.99
94.14
88.87
84.13
97.84
95.79
92.04
89.33
82.15

34.09
38.63
40.72
45.13
47.37
50.71
39.30
41.99
45.39
49.20
50.87
42.28
42.86
48.50
50.29
50.60

11.43
13.06
12.60
10.69
8.56
4.82
12.90
12.02
10.50
7.57
4.20
12.84
10.96
9.26
5.54
3.47

38.47
49.02
49.92
56.74
63.92
67.42
51.15
52.39
58.09
65.74
69.11
51.39
54.42
60.61
67.37
72.52

16.57
41.62
57.84
81.01
66.29
53.20
50.11
70.00
80.50
58.86
36.77
56.61
78.10
78.95
50.30
23.05

Data reported as an average of two replicates.

Sugar yield (% of theoretical yield in raw soybean straw) includes sugar recovery in liquor and sugar yield of pretreated solids by enzymatic hydrolysis.

decrease was observed with the test condition of 210 C and

10 min. The overall mass recovery was higher than 90% except
for those with severe pretreatment conditions (200210 C for all
pretreatment times). The lost mass might disappear as gaseous
compounds formed during degradation of soybean straw by LHW
pretreatment (Turn et al., 1998).
The glucan recovery in the solid fraction was nearly 100% and
did not change signicantly with pretreatment conditions (Fig.

Fig. 1. Glucan or xylan recovery during LHW pretreatment of soybean straw: (a)
sugar recovery in pretreated solid and (b) sugar recovery in liquor.

1a). A maximum amount of glucan (3.00%) was solubilized at

210 C and 10 min (Fig. 1b), of which 0.97% of glucan was degraded
into HMF (Fig. 2). The glucan content in the solid residue was up to
51%, much higher than that in raw soybean straw (34.09%), which
also revealed that most of the glucan was preserved in the pretreated solid fraction (Table 1).
Xylan recovery in the solid fraction decreased gradually with
an increase in pretreatment temperature (Table 1). At a given
temperature, the xylan recovery at the pretreatment time of 3
and 5 min was similar but was higher than that for 10 min. In
contrast to xylan recovery in the solid residue, xylan recovery
in the liquor increased rapidly with an increase in temperature
from 170 to 190 C, irrespective of pretreatment time. When
the temperature further increased, the xylan recovery in the
liquor decreased rapidly except for tests with a pretreatment
time of 3 min. More than 90% of dissolved xylan existed as oligomers at pretreatment temperatures lower than 190 C (data not
shown), thus preventing xylan degradation to furfural. As a
result, full recovery of xylan in the pretreated slurry (solid and
liquor fraction) was observed at temperatures lower than
190 C for all pretreatment times. However, the xylan recovery
decreased under more severe conditions, inversely proportional
to xylan dissolution (Fig. 3). For example, the most severe degra-

Fig. 2. HMF and furfural formation during LHW pretreatment of soybean straw.

C. Wan et al. / Bioresource Technology 102 (2011) 62546259

6257

Fig. 3. Total xylan recovery as a function of xylan solubilization after LHW

pretreatment of soybean straw.

dation occurred at 210 C and 10 min, under which 80% xylan

was solubilized during pretreatment while only 9.58% xylan
was recovered as a form of xylooligomers and xylose from the
liquid fraction.
The yield of furfural was less than 1.5% when the temperature
was lower than 190 C, while the furfural yield increased to
9.13% at 210 C and 10 min (Fig 2). Nevertheless, this furfural yield
did not account for all of the xylan loss during pretreatment, suggesting other decomposed products were also generated from the
degradation of xylose. The unidentied xylan loss partially accounted for total mass lost at the severer conditions. Similar results
in terms of decreased overall xylan recovery and mass balance
were reported by Allen et al. (2001a). In their study, xylan recovery
decreased to about 40% at 100% xylan dissolution and the total
mass recovery also declined to 85%.
3.1.2. Sugar yield
Enzymatic hydrolysis was conducted to evaluate the cellulose
and xylan digestibility of the solid fraction. As shown in Fig. 4a, sugar yield increased with both pretreatment time and temperature.
The highest glucose yield of 70.76% was obtained at the most
severe pretreatment condition (210 C for 10 min), which was
32.29% higher than the glucose yield of raw soybean straw.
Fig. 4b indicates that glucose yield was well correlated to xylan
removal from raw soybean straw (R2 = 0.97). The more xylan
removed, the more cellulose digested. Similar trends have been observed from LHW pretreatment of corn stover (Yang and Wyman,
2004; Zhou et al., 2010), yellow poplar sawdust (Allen et al.,
2001a), and wheat straw (Kabel et al., 2007).The predicted glucose
yield of soybean straw resulting from complete xylan dissolution
was 74.1% (Fig. 4b), which was slightly higher than the obtained
yield of 70.76% with 80% xylan removal at 210 C and 10 min.
These results suggested a limited impact of xylan removal on
cellulose digestibility of soybean straw. Compared to corn stover
pretreated with LHW under the same conditions (Zhou et al.,
2010), the glucose yield of soybean straw was much lower than
that of corn stover. For example, the glucose yield of corn stover
was 85.94% when pretreated with LHW at 210 C for 10 min,
indicating that soybean straw was more recalcitrant to LHW
pretreatment.
The lowest xylose yield of 13.47% was obtained from soybean
straw at 210 C and 10 min which was even lower than that of
raw straw. The decreased xylose yield was mainly due to
signicant removal of xylan in the solids during pretreatment.

Fig. 4. (a) Sugar yield of LHW pretreated soybean straw by enzymatic hydrolysis,
and (b) correlation between cellulose digestibility and xylan removal.

Xylan remaining in the solid fraction, which was most likely hard
to digest, also led to the reduced xylose yield.
The dissolved xylan needs to be recovered to obtain higher
fermentable sugar yields. As presented in Table 1, the total xylose
yield was as high as 81% when xylan recovery in the liquor was
also taken into account. A similar xylose yield (approximately
82%) of corn stover through LHW pretreatment and cellulase
hydrolysis was also reported by Mosier et al. (2005b). The maximum xylan recovery of 42.11% in the liquor was obtained at
190 C and 10 min, at which 95.42% of xylan-derived sugars remained in the oligomeric form. However, severer pretreatment
conditions (at temperatures higher than 190 C), which resulted
in increased cellulose digestibility, adversely affected total xylose
yield from both pretreatment and enzymatic hydrolysis (Table 1).
Liu and Wyman (2005) also determined a maximum xylose yield
of about 46% in batch LHW pretreatment of corn stover. Flow or
partial ow of LHW, used in their study, removed xylose or xylose
oligomers throughout pretreatment and achieved a higher xylose
recovery of 83.5% in the liquor. Thus, LHW pretreatment, conducted at a selected conditions, can be used to produce different
types of enriched end-products (e.g., xylooligmers or digestible bers) for industrial applications.

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C. Wan et al. / Bioresource Technology 102 (2011) 62546259

3.2. Alkaline pretreatment

3.2.1. Recovery of solid and sugar after pretreatment
The pretreatment liquor with remaining alkali is generally reused for pretreatment. Therefore, neutralization of the liquor for
further analysis was neglected in this study. Only the solid fraction
of soybean straw after pretreatment was analyzed. The solid and
sugar recovery from the NaOH pretreated slurry was similar to that
pretreated by LHW. As shown in Fig 5a, solid recovery decreased
from 88.66% to 78.62% when the alkali loading was increased from
4% to 40% (w/w). In the recovered solids, nearly all glucan was retained at all NaOH concentrations while xylan recovery gradually
decreased with increased alkali loading. More than 50% xylan
was lost at 40% alkali loading. Lignin degradation increased with
alkali loading. Lignin degradation of 14.78% was observed at 40%
NaOH loading, which was almost twice that obtained at 4% NaOH.
3.2.2. Enzymatic hydrolysis
The solid fraction recovered from alkaline pretreatment was
subjected to enzymatic hydrolysis after washing and
neutralization. The glucose yield increased with alkaline loading
due to a synergistic effect of degradation of hemicellulose and lignin. The highest glucose yield of 64.55% was obtained at 40% alkali
loading (Fig 5b), about a 26.08% improvement over that of raw soybean straw, but 6% lower than the highest sugar yield obtained
from LHW pretreatment. The xylose yield was improved by alkaline pretreatment, to more than twice that of raw straw. However,

a slight decrease in xylose yield at higher alkali loading was

observed, probably due to decreased xylan content in the solid
fraction. Considering the amount of xylan removal and the resulting sugar yield, cellulose digestibility of alkaline-pretreated straw
was comparable to that of LHW pretreated solids.
3.3. Comparison between LHW and alkaline pretreatments
The degree to which digestibility of feedstock is improved generally depends on both substrate and pretreatment methods. Allen
et al. (2001b) found LHW resulted in less formation of inhibitory
compounds and higher cellulose conversion of corn ber than
steam explosion. For pretreatment of corn stover and poplar, the
sugar yield resulting from pH-controlled LHW was higher than that
by ammonia ber explosion (AFEX) but lower than by other chemical pretreatments (i.e., dilute acid, lime and SO2) (Kumar et al.,
2009). However, Dien et al. (2008) reported that sugar yields of
AFEX-treated distillers grains were higher than that of LHWpretreated ones.
Compared to LHW treatment of soybean straw, NaOH soaking
dissolved a smaller amount of hemicellulose and also resulted in
a lower cellulose digestibility in the pretreated solids. However,
at room temperature and similar alkali loading, cellulose digestibility of NaOH treated soybean straw was similar to that of ammonia treated (Xu et al., 2007). In contrast, NaOH pretreatment at
elevated temperatures can improve cellulose digestibility due to
more degradation of lignin and hemicelluloses (Varga et al.,
2002). In this study, LHW was more effective for fractionation of
soybean straw compared to low temperature alkaline pretreatment. Although alkaline pretreatment has disadvantages, such as
high alkali loading, long pretreatment time, and generation of a
large volume of waste water from washing, it is suitable for onfarm pretreatment at high solid loading as neither complicated
reactors nor heating is required for the pretreatment process.
LHW pretreatment with a catalyst such as acid is suggested for
the pretreatment of soybean straw to obtain a higher glucose yield.
4. Conclusions
LHW and NaOH pretreatments of soybean straw were investigated for their impact on recalcitrance reduction and cellulose
digestibility improvement. Both pretreatment methods achieved
full cellulose recovery in pretreated solids but led to increasing xylan dissolution with increased pretreatment severity. The cellulose
digestibility was remarkably improved by both pretreatment
methods, while NaOH pretreatment at ambient conditions was less
effective than LHW pretreatment. However, the glucose yields of
LHW pretreated soybean straw were much lower than that of corn
stover at the same pretreatment conditions (pretreatment temperature and time).
Acknowledgements
This work was supported by funding from North Central Sun
Grant Program (No. GRT00013735), USDA NIFA 1890 Capacity
Building Program, and Ohio Agricultural Research and Development
Center Seeds Program. The authors wish to thank Mrs. Mary Wicks
(Department of Food, Agricultural and Biological Engineering,
OSU) for reading through the manuscript and providing useful
Suggestions and Drew Martin for help on the experiments.
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