Biotechnology and Bioprocess Engineering 2009, 14: 606-611

DOI/10.1007/s12257-008-0320-0

Bioethanol Production from Ammonia

Percolated Wheat Straw
Minhee Han1, Se-Kwon Moon1, Yule Kim1, Youngran Kim2, Bongwoo Chung2, and
Gi-Wook Choi1*
1
Changhae Institute of Cassava and Ethanol Research, Changhae Ethanol Co., Ltd, Jeonju 561-203, Korea
2
=
Department of Bioprocess Engineering, Chonbuk National University, Jeonju 561-156, Korea

Abstract This study examined the effectiveness of ammonia percolation pretreatment of wheat straw for ethanol production.
Ground wheat straw at a 10% (w/v) loading was pretreated with a 15% (v/v) ammonia solution. The experiments were
performed at treatment temperature of 50~170°C and residence time of 10~150 min. The solids treated with the ammo-
nia solution showed high lignin degradation and sugar availability. The pretreated wheat straw was hydrolyzed by a cellu-
lase complex (NS50013) and β-glucosidase (NS50010) at 45°C. After saccharification, p~ÅÅÜ~êçãóÅÉë=ÅÉêÉîáëá~É was
added for fermentation. The incubator was rotated at 120 rpm at 35°C. As a result of the pretreatment, the delignification
efficiency was > 70% (170°C, 30 min) and temperature was found to be a significant factor in the removal of lignin than
the reaction time. In addition, the saccharification results showed an enzymatic digestibility of > 90% when 40 FPU/g cel-
lulose was used. The ethanol concentration reached 24.15 g/L in 24 h. This paper reports a total process for bioethanol
production from agricultural biomass and an efficient pretreatment of lignocellulosic material. © KSBB

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INTRODUCTION less expensive and available in large quantities, thus offer a

Bioethanol, as a liquid fuel by the fermentation of renew-
able biomass, is important from the viewpoint of global envi-
ronmental protection. Biomass, which includes animal and
human waste, trees, shrubs, yard waste, wood products,
grasses and agricultural residue, such as wheat straw, corn
stover, rice straw, and cotton stalks, is a renewable resource
that stores energy from sunlight [1]. It can be processed either
chemically or biologically by breaking the chemical bonds to
extract energy in the form of biofuels, such as bioethanol.
Currently, starch and sugar base materials are the primary
raw materials for ethanol production worldwide. They are
easily broken down into glucose, which is then fermented to
ethanol. However, the starch and sugar to ethanol industry
draws its feedstock from a food stream and is quite mature
with little possibility of process improvements [2]. In order
to overcome this problem, there have been extensive techno-
logical developments for bioethanol production from ligno-
cellulosic biomass. Lignocellulosic feedstock has the poten
tial to reduce the production cost of ethanol because they are
*Corresponding author
Tel: +82-63-214-7800 Fax: +82-63-214-7805
e-mail: changrd@chethanol.com
plausible alternative. One promising technology is to convert
this abundant and renewable lignocellulosic biomass to etha-
nol via an enzyme-based process [3]. However, the conversion
of lignocellulosic biomass to ethanol is more challenging due
to the complex structure of the plant cell wall. Pretreatment is
needed to alter the structural and chemical composition of
lignocellulosic biomass to facilitate the rapid and efficient
hydrolysis of carbohydrates to fermentable sugars [4,5].
A variety of physical (comminution, hydrothermolysis),
chemical (acid, alkali, solvents, ozone), physico-chemical
(steam explosion, ammonia fiber explosion), and biological
pretreatment techniques have been developed to improve the
accessibility of enzymes to cellulosic fibers [6]. Acid pre-
treatments involve the use of sulfuric, nitric, or hydrochloric
acids to remove the hemicellulose components and expose
the cellulose for enzymatic digestion [3]. Agricultural resi-
due, such as corncobs and stovers, is particularly well suited
to dilute acid pretreatment [7,8]. Alkali pretreatment refers to
the application of alkaline solutions to remove lignin and
various uronic acid substitutions on hemicellulose that lower
the accessibility of an enzyme to hemicellulose and cellulose
[4]. Generally, alkaline pretreatments are more effective on
agricultural residues and herbaceous crops than on wood
materials [9]. A peroxide pretreatment enhance enzymatic

conversion through oxidative delignification and a decrease
in cellulose crystallinity [10]. Increased lignin solubilization
and cellulose availability were observed after a peroxide
pretreatment of wheat straw [11], Douglas fir [12], and oak
[13]. Ozonation is another attractive pretreatment method
that does not leave strong acidic, basic, or toxic residues in
the treated material [14]. The effects of ozone pretreatment
are essentially limited to lignin degradation. Hemicellulose is
attacked slightly, while cellulose is barely affected [15].
Ozonation is used widely to reduce the lignin content of both
agricultural and forestry waste [14].
Wheat grows up rapidly on crude land and contains abun-
dant nutrients, and there has been an increase in wheat plan-
tation in Korea. Wheat straw can serve as a low-cost feed-
stock to increase the production of fuel ethanol through
proper pretreatment, hydrolysis, and fermentation.
In order to utilize wheat straw as a feedstock for ethanol
production, a pretreatment is necessary to render the cellu-
lose fibers more amenable to the action of hydrolytic en-
zymes. In this study, ammonia percolation using wheat straw
as a lignocellulosic biomass, enzymatic saccharification, and
bioethanol fermentation were carried out.
MATERIALS AND METHODS
Materials
Wheat straw, cultivated in June 2008, was obtained from the
Honam Agricultural Research Institute, Korea. Prior to com-
position analysis, the biomass was ground to a particle size < 1
mm in a cutter mill and stored in sealed plastic bags at room
temperature until used for the experiment. The enzymes were
provided by NOVO. The cellulase complex (NS50013) has an
activity of 70 filter paper unit (FPU)/g cellulose. The β-
glucosidase, NS 50010, is a 250 cellobiase unit (CbU)/g. All
reagents used for this study were of analytical grade.
Pretreatment
Ammonia solution (15% (v/v)) was used to pretreat 0.8 g
of ground wheat straw at solid loading of 10% (w/v). The
treatments were carried out at 50~170oC using an oil bath
and residence time of 10~150 min. The treated solids were
washed with deionized water until the pH was approximately
7.0 and used for analysis.
Enzymatic Hydrolysis
The pretreated wheat straw at a 1% solid loading (1 g dry
weight/100 mL) in 50 mM acetate buffer (pH 4.8) containing
1 mL of a 2% sodium azide solution (to avoid microbial con-
tamination) were preincubated in a shaking incubator at
50oC and 150 rpm for 10 min. Hydrolysis was carried out at
a cellulase activity of 10~70 FPU/g cellulose and a β-
glucosidase activity of 30 CbU/ g, respectively. One millili-
ter aliquots were taken at the termination of enzymatic hy-
drolysis after 72 h, chilled immediately on ice, and centri-
Biotechnol. Bioprocess Eng. 607=
Table 1. Composition of untreated wheat straw
Component Percentage (%)
a
Holocellulose 62.3
Cellulose 37.6
Xylose 22.9
Arabinose 1.8
Acid-insoluble lignin 17.8
Acid-soluble lignin 1.8
Ash 2.5
Others 15.6
a
Composition percentages are on a dry-weight basis.
fuged at 5,000 rpm for 10 min. The supernatant was re-
moved and the sugar content was analyzed [16]. The per-
centage cellulose conversion was calculated as follows:
%GH
% Cellulose conversion = × 100 (1)
%GP
where GH is the dry wt% of glucose in the enzymatic hy-
drolysis supernatant (g glucose/g solids hydrolyzed) and GP
is the dry wt% cellulose in the pretreated solids (g glucose/g
solids pretreated).
pK=ÅÉêÉîáëá~É Fermentation
After saccharification, 10 g/L of the yeast extract and 20
g/L of peptone were added to supply the nitrogen source and
minor nutrient. The bottles were capped and autoclaved for
15 min at 121oC. After cooling, the bottles were inoculated
with S. cerevisiae and the solid caps were replaced with caps
containing silicone septa, through which 22 g needles had
been pierced to exhaust the CO2 released by fermentation.
The temperature of the shaker/incubator was reset to 35oC
and the bottles were returned. The bottles were sampled pe-
riodically for the next 72 h and samples were stored at −20oC.
The S. cerevisiae inoculum was prepared by growing the
strain CHY 1011 on a solid YPD medium, containing per
liter: 10 g, yeast extract; 20 g, protease peptone; and 10 g,
dextrose supplemented with 15 g bacto agar. The solid cul-
ture was incubated at 32oC for 48 h. A single colony was
transferred to a 50 mL Erlenmeyer flask containing 10 mL of
YPD and grown at 32oC with agitation (120 rpm) for 12 h.
This culture was used to inoculate the seed culture, which
consisted of an Erlenmeyer flask (500 mL) containing 200
mL of YPD for 12 h [17].
Analytical Methods
The total solids, acid-soluble lignin, and acid-insoluble
lignin content of the untreated wheat straw as well as the
solid fraction remaining after pretreatment were determined
using the National Renewable Energy Laboratory (NREL)
Standard Biomass Analytical Procedures [18]. The carbohy
drate content of the untreated wheat straw and pretreated
solids was determined by measuring the hemicellulose (xy-
lose, galactose, and arabinose) and cellulose (glucose) de-
608

Table 2. Effect of the reaction time and temperature on the composition in ammonia percolation pretreatmenta

b
Solid composition
Temperature and treatment time (min) SR (%) Delignification (%)
Cellulose (%) Hemicellulose (%)
Untreated 100.00 0 37.63 22.91
10 81.00 21.65 35.50 11.95
30 80.76 24.93 36.08 12.33
60 78.87 31.00 35.40 11.93
50°C
90 78.50 37.64 36.10 12.69
120 78.00 41.52 36.02 12.14
150 77.64 44.79 35.37 12.27
10 77.82 32.01 35.00 11.24
30 75.32 38.41 35.24 11.49
60 73.93 45.51 36.01 11.41
80°C
90 71.77 54.62 35.19 11.42
120 70.85 56.76 35.44 11.05
150 70.03 59.52 35.84 10.62
10 73.09 41.11 34.95 12.05
30 70.49 48.00 34.89 12.67
60 69.23 52.81 35.11 12.14
110°C
90 68.55 59.00 35.21 12.10
120 66.02 61.73 35.33 11.56
150 63.98 63.58 34.94 11.17
10 63.41 55.08 36.25 12.00
30 63.34 58.11 36.50 11.99
60 62.77 58.92 36.12 11.45
140°C
90 62.28 61.00 35.97 10.38
120 61.59 63.50 36.49 10.44
150 61.02 65.03 35.96 10.14
10 60.01 63.39 35.74 11.69
30 59.26 65.13 36.00 11.75
60 58.99 65.50 35.98 11.39
170°C
90 58.71 66.21 35.94 11.30
120 58.61 66.99 36.14 10.97
150 58.51 67.39 35.85 10.49
a
Data in the table are based on the untreated biomass.
Pretreatment conditions: 10 (w/v)% biomass concentration, 15 (v/v)% ammonia solution.
b
SR stands for solid remaining after reaction.

rived sugars. The composition of the hydrolysate from en-
zymatic hydrolysis was determined by measuring the glu-
cose and xylose level using high performance liquid chroma-
tography (HPLC). The HPLC system was equipped with a
Bio-Rad Aminex HPX-87P column, a guard column, an
automated sampler, a gradient pump, and a refractive index
detector. The mobile phase used was deionized water at a
flow rate of 0.6 mL/min and 85oC. Before HPLC injection,
all samples (derived from solids and hydrolysate) were neu-
tralized with calcium carbonate, centrifuged at 5,000 g for 10
min, and filtered through a 0.2 µm syringe filter. The con-
centration of impurities and ethanol were determined using a
Density/Specific Gravity Meter (DA-510, KEM Co., Ltd.,
Japan) and gas chromatography (GC) using a Supelco 6.6%
CARBOWAX 20 M column, Agilent (USA), respectively.
RESULTS AND DISCUSSION
Characteristics of Wheat Straw
The chemical composition of wheat straw varies according
to the growing location, season, harvesting methods, and
analysis procedures [19]. Table 1 lists the composition of
wheat straw used in this study. HPLC carbohydrate analysis
showed that the sugar fraction was 62.3% of the dry biomass.

Table 3. Characteristics of the enzymes used in this study
a
Enzyme (Name) Activities
NS50013 (Cellulase complex) 700 EGU/g (~70 FPU/g)
NS50010 (β-glucosidase) 250 CbU/g
a
EGU = Endo-Glucanase, FPU = Filter Paper Unit, and CbU = Cellobiase Unit.
Fig. 1. Enzymatic digestibility of cellulose with various enzyme con-
centrations c●c cc ccccg cellulosec ●c cc ccccg cellulosec ●c
3c ccccg cellulosec ▽c 3c ccccg cellulosec ▽c 3c ccccg cel-
lulosec ▀c 6c ccccg cellulosec and ▌c 7c ccccg cellulose).
cellulose, which was derived from both the wheat fiber and
plant cell wall, was the major component at 37.6%. Xylan,
which is the major hemicellulose constituent, comprised
22.9%. Arabinan accounted for only a small portion of the
biomass, while galactan and mannan were not detected.
These can be converted to ethanol by hexose fermenting
organisms. However, it is too difficult to ferment pentose by
microorganisms, so the strategy in this study was to retain
the cellulose in the solid during pretreatment, which can then
be utilized through the fermentation of hexoses derived from
the solid portions. This strategy also eliminates the lignin
that interrupts enzymatic hydrolysis of pentose.
Effect of Ammonia Percolation Pretreatment
Ammonia percolation pretreatment of lignocellulosic
biomass is one of the most effective pretreatments that
mainly affect lignin degradation with little impact on hemi-
cellulose. Table 2 shows the residual solid, delignification,
cellulose, and hemicellulose changes during the ammonia
pretreatment of wheat straw. The solid remaining after the
pretreatment varied from 81% (10 min, 50oC) to 58.51%
(150 min, 170oC). The cellulose and hemicellulose content
ranged from 34.94 to 36.49% and 10.14 to 12.69%, respec-
tively. In addition, delignification, which was based on a
comparison of the initial weight of lignin before pretreatment
with the weight of lignin in the solid remaining after pre
treatment, ranged from 21.65 to 67.39%. This means that the
Biotechnol. Bioprocess Eng. 609=
Density (g/mL) pH ( - ) Temperature (°C)
1.2 4.5~6.5 45~50
1.2 2.5~6.5 45~70
Fig. 2. Cellulose digestibility of pretreated wheat straw at 3c min
with 3c ccccg cellulose and 3c Cbccg c●c no treatmentc ●c
8c°C pretreatmentc ●c ccc°C pretreatmentc ▽c c3c°C pre-
treatmentc and ▽c c7c°C pretreatment).
ammonia pretreatment is effective in removing lignin, which
hinders the approaching enzyme and keeping cellulose, and
can be converted to ethanol easily. Increasing the tempera-
ture had the most pronounced effect on delignification. Al-
though delignification increased with increasing reaction
time at low temperatures, there was little effect on denignifi-
cation at high temperatures. This suggests that temperature is
a more important factor for removing lignin than the reaction
time.
Enzymatic Digestibility of Pretreated Wheat Straw
Table 3 lists the characteristics of the enzymes supplied by
NOVO. The enzymatic hydrolysis experiments were carried
out using α-cellulose reagent (Sigma) to determine the en-
zyme concentration. Fig. 1 shows the level of cellulose con-
version of α-cellulose after 72 h of enzymatic hydrolysis.
The enzymatic digestibility of cellulose to glucose was more
than 90% in 36 h with 30 FPU/g cellulose. The conversion
ratio increased with increasing enzyme dose. However, there
was little difference in enzymatic digestibility, when the
enzyme dosage was > 30 FPU/g cellulose.
Based on these results, an enzyme loading of 40 FPU/g
cellulose was chosen to examine the enzymatic digestibility
of pretreated wheat straw because of its high stability and
effective reaction. Fig. 2 shows the enzymatic digestibility of
pretreated wheat straw at various temperatures at 30 min
with a 40 FPU/g cellulose enzyme loading. In these experi-
610
Fig. 3. TTTT TTTTTT TT TTTTTTT TTTTTTTTTTTT T●T TTTTTTT TTT ●T
wTTTT TTTTw).
ments, the pretreated biomass at 170oC showed higher en-
zymatic digestibility (> 95%) while the untreated biomass
was < 35%. The pretreatment temperature played a signifi-
cant role in improving the enzymatic digestibility. However,
there is a little difference in enzymatic digestibility between
140 and 170oC. Therefore, the fermentation experiment was
carried out with pretreated biomass at 140oC considering the
energy cost. In addition, for fermentation, saccharification
was carried out to increase the biomass concentration at 10%
(W/V).
Ethanol Production from Pretreated Wheat Straw with
pK=ÅÉêÉîáëá~É
The fermentability of the pretreated materials was evalu-
ated using S. cerevisiae. The pretreated biomass was mixed
with a cellulase complex and β-glucosidase for 96 h at 45oC.
Pectinase and xylanase were not included because S. cere-
visiae does not ferment arabinose or xylose. Fermentation
was carried out at 35oC for 24 h.
The hydrolysate made from lignocellulosic biomass con-
tains a variety of inhibitors, such as furfural, 5-hydroxymethyl
furfural (HMF), acetate, hydroxybenzaldehyde (HBA), syrin-
galdehyde (SGA), and vanillin, which have a negative influ-
ence on the efficiency and fermentation rate during the ethanol
production process [20,21]. Therefore, this study was carried
out to confirm the inhibitor effect using YPD (10 g/L yeast
extract and 20 g/L peptone) medium as a control, which is the
most optimized fermentation. The initial glucose concentra-
tions were 57.51 and 54.96 g/L for the control and wheat
straw hydrolysate, respectively. Fig. 3 shows the level of etha-
nol production from the control and wheat straw hydrolysate.
The final ethanol concentrations for the control and wheat
straw hydrolysate were 26.65 and 25.14 g/L, respectively, and
the ethanol yields were 91.57 and 90.66% of the theoretical
value, respectively. This means that the inhibitors were not
created by Ammonia percolation pretreatment.
Previous studies showed that the ethanol yield were 88%
and 90% of the theoretical for ammonia fiber explosion and
liquid hot-water using dried distillers’ grains with soluble
(DDGS) [22] and 73.6% of the maximum theoretical yield
using barley hull soaked in aqueous ammonia [23].
CONCLUSION
Bioethanol was produced from wheat straw using an am-
monia percolation pretreatment through a basic process. The
pretreatment using ammonia has the advantage and effec-
tiveness to remove lignin, which is a complex material that
acts as a glue to hold the cellulose and hemicellulose to-
gether.
The enzymatic hydrolysis of ammonia percolated biomass
with cellulase and β-glucosidase supplied by NOVO re-
leased almost all the available glucose (> 90%), when 40
FPU/g cellulase and 30 CbU/g β-glucosidase were used. The
final ethanol concentration after fermentation for 24 h at
35oC was 25.14 g/L (90.66% of the theoretical yield) in 24 h.
Overall, an ethanol production process using pretreated
wheat straw by ammonia percolation is effective.
Acknowledgements This study was financially supported
by the Ministry of Education, Science Technology (MEST)
and Korea Industrial Technology Foundation (KOTEF)
through the Human Resource Training Project for Regional
Innovation.
Received January 3, 2009; accepted April 14, 2009
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