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Bioresource Technology 99 (2008) 3729–3736

Comparative study on chemical pretreatment methods

for improving enzymatic digestibility of crofton weed stem
a,*
Xuebing Zhao , Lihua Zhang b, Dehua Liu a,*

a
Department of Chemical Engineering, Tsinghua University, Beijing 100084, China
b
Kunming Devision, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming, Yunnan 650223, China

Received 10 May 2007; received in revised form 10 July 2007; accepted 10 July 2007
Available online 20 August 2007

Abstract

In order to utilize and control the invasive weed, crofton weed (Eupatorium adenophorum Spreng), a potential pathway was proposed
by using it as a feedstock for production of fermentable sugars. Three chemical pretreatment methods were used for improving enzymatic
saccharification of the weed stem. Mild H2SO4 pretreatment could obtain a relatively high yield of sugars in the pretreatment (32.89%,
based on initial holocellulose), however, it led to only a slight enhancement of enzymatic digestibility. NaOH pretreatment could obtain a
higher enzymatic conversion ratio of cellulose compared with H2SO4 pretreatment. Peracetic acid (PAA) pretreatment seemed to be the
most effective for improving enzymatic saccharification of the weed stem in the three chemical pretreatment methods under the same
conditions. The conversion ratio of cellulose in the sample pretreated by PAA under the ‘‘optimal’’ condition was increased to 50%
by cellulase loading of 80 FPU/g cellulose for 72 h incubation. A number of empirical quadratic models were successfully developed
according to the experimental data to predict the yield of sugar and degree of delignification.
Ó 2007 Elsevier Ltd. All rights reserved.

Keywords: Crofton weed; Utilization; Control; Pretreatment; Enzymatic digestibility

1. Introduction damaging native ecosystems and causing great economic

Crofton weed (Eupatorium adenophorum Spreng) is a
tufty, semi-shrubby, perennial herbaceous plant of about
1–2 m in height. It is native to Central America, mainly
Mexico, although it is now distributed widely in the United
States of America, Australia, New Zealand and many
countries in Southeast Asia. This exotic weed prefers a
warm and wet environment, but its ability to adapt to dif-
ferent environmental conditions is very strong. It can grow
in environments with temperature ranges from 5 °C to
42 °C on dry and barren hills, and can even grow in cracks
in stone walls and houses (Sun, 2003). Since the 1950s, this
exotic plant has spread rapidly across southwest China,
*
Corresponding authors. Tel.: +86 10 62782654 (X. Zhao), +86 10
62794742 (D. Liu); fax: +86 10 62785974.
E-mail addresses: zhaoxb04@mails.tsinghua.edu.cn (X. Zhao),
dhliu@tsinghua.edu.cn (D. Liu).
losses (Yu et al., 2005). Many farmlands, pasture fields
and forests have been destroyed by the weed. Much atten-
tion has been paid to management of this weed and several
methods have been developed to control it, including man-
ual, chemical and biological control, however, no obvious
progress is made. The weed is still spreading from south-
west to northeast at a rate of 10–60 km per year in China
(Lu et al., 2005).
Compared with simple control processes, utilization of
the weed gives more benefits. According to the published
literatures, the weed can be used as bio-pesticide (Wang
et al., 2002; Liu et al., 2004; Kaushal et al., 2001), organic
fertilizer and feedstuff, feedstock for production of marsh
gas (Patrabansh and Madan, 2000). We determined the
glycan content in the stem of this weed and found that it
was similar to that of some agricultural wastes, such as
corn stover, rice straw and wheat straw. Therefore, in this
article, it was proposed to use the weed as a biomass

0960-8524/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2007.07.016
3730 X. Zhao et al. / Bioresource Technology 99 (2008) 3729–3736
Nomenclature
D loading of NaOH or peracetic acid, % (w/w,
based on the raw materials)
L liquid-to-solid ratio, ml/g (v/w)
T pretreatment temperature, °C
t pretreatment time, min
CC cellulose content in the residue solid, % (w/w)
CRC conversion ratio of cellulose by enzymatic
hydrolysis for 72 h, % (w/w, based on the cellu-
lose in the pretreated samples)
resource to produce fermentable sugars for further produc-
tion of biofuel such as ethanol, instead of just simply con-
trolling it.
There are two common ways to convert lignocellulosic
biomass to sugars, namely acid hydrolysis and enzymatic
hydrolysis. Acid hydrolysis has been developed for a long
time for direct saccharification. It is also a pretreatment
method for improving the enzymatic digestibility of bio-
mass. Enzymatic saccharification is a promising way
because of the advantages such as low energy input, less
pollution, no side products and so on, but the low enzy-
matic accessibility of the native cellulose is a key problem
for biomass-to-ethanol processes. Therefore, pretreatment
is an essential element in the bioconversion of lignocellu-
losic substrates. The objective of this article is thus to
investigate the effect of different chemical pretreatment
processes on the enzymatic digestibility of the weed stem,
in order to obtain a high enzymatic conversion ratio of cel-
lulose-to-sugar and preliminarily study the possibility of
utilization of the weed for production of fermentable
sugars.
2. Methods
2.1. Materials and analytical methods
Crofton weed was obtained from Lincang city, Yunnan
province in Southwestern China. Its stem was air dried,
crushed and screened. The fraction collected between 20
and 40 mesh was used in all the experiments. The chemical
compositions were determined according to the corre-
sponding Chinese Standards as follows: moisture content,
GB/T 2677.2-1993; ash, GB/T 2677.3-1993; hot water
extractives, GB/T 2677.4-1993; 1% NaOH extractives,
GB/T 2677.5-1993; benzene–ethanol extractives, GB/T
2677.6-1994; holocellulose, GB/T 2677.10-1995; Klason
lignin, GB/T 2677.8-1994; acid-soluble lignin, GB/T 747-
2003. The cellulose content was determined by nitric
acid–ethanol method.
All the chemicals were purchased locally. Peracetic acid
was prepared by reaction of acetic acid and 30% hydrogen
peroxide with their volume ratio of 1.5:1 at 30 °C for 24 h.
DD degree of delignification, expressed as the per-
centage of Klason lignin removed, %
SY solid yield, weight ratio of residue solid after
pretreatment to the initial solid, %
YRSAH yield of reducing sugar by acid hydrolysis, %
(w/w, based on the initial holocellulose)
1% (w/w) of sulfuric acid was added as catalyst. The deter-
mination of peracetic acid concentration was in accordance
with Chinese standard GB/T 19108-2003.
The cellulase enzyme used in the experiment was Cellu-
lase R-10, which was produced by Yakuh Honsha Co. Ltd.
in Japan. The cellulase activity was determined by the
method recommended by Ghose (1987), and expressed in
filer paper units (FPU). One FPU was defined as the
amount of enzyme capable of producing 1 lM of reducing
sugars in 1 min. The total reducing sugar was determined
by 3,5-dinitrosalicylic acid colorimetry (DNS Method)
(Ghose, 1987). The main monosaccharides in the liquid
phase of acid hydrolysis process were determined by HPLC
using an Aminex HPX-87 H column and RID-10A detec-
tor. The mobile phase was 0.005 M H2SO4 at a flow rate
of 0.8 ml/min.
2.2. Pretreatment process
Pretreatment was carried out in a 250 ml glass flask
immerged in a water bath. Ten grams of screened materials
was packed into the flask and certain volume (ml) of liquid
was added. A polytetrafluoroethylene paddle was used for
intermittent stirring to keep the system as homogeneous as
possible. After pretreatment, the materials were washed
with water until neutrality and dried at 105 °C for 6 h.
The oven-dried samples were stored in valve bags for fur-
ther analysis and enzymatic hydrolysis. The liquid was col-
lected for analysis of sugars. Each of the experimental data
was the average value of at least duplicate tests.
2.3. Enzymatic hydrolysis
The oven-dried samples were digested by a cellulase
loading of 20 FPU/g cellulose. The initial concentration
of cellulase in the liquid phase was 0.15 FPU/ml. The enzy-
matic digestibility tests were conducted as follows: temper-
ature of 50 ± 0.5 °C, pH of 4.8 (0.1 M sodium acetate
buffer), 130 rpm in a air-bath shaker. The digestibility,
denoted as conversion ratio of cellulose (CRC), was
defined as the percentage of cellulose converted to glucose
after 72 h of incubation with the cellulase enzyme.
 X. Zhao et al. / Bioresource Technology 99 (2008) 3729–3736 3731

3. Results and discussion necessary to pretreat the weed to remove the toxic sub-
stances and enhance the enzymatic digestibility.
3.1. Chemical composition of the weed stem

The composition of the weed stem used in this study is
presented in Table 1. Some typical agricultural residues
are also listed in the table for comparison. It can be seen
that the holocellulose and cellulose content in the weed
stem were similar to those of corn stover and wheat straw,
but much higher than that of cotton stalk. The weed also
had a much higher content of benzene–ethanol extractives.
According to some reports, these extractives contain some
allelochemicals that can inhibit the germination and
growth of other plants (Song et al., 2000), which makes it
have a strong ability to invade any land. The Klason lignin
content of the weed stem was a little lower than that of
corn stover and wheat straw. The acid-soluble lignin was
not listed in the table, because that it was found that the
water extractives of the weed stem contained some chemi-
cals that also had a maximum absorption at near 205 nm.
Therefore, the conventional method for determination of
acid-soluble lignin by measuring the absorbance of the
solution at 205 nm was not accurate for the weed.
Comparing the weed stem with the listed agricultural
wastes, we can see that the weed stem is also similar to corn
stover and wheat straw in other chemical compositions
such as ash and 1% NaOH extractives. Recently, using
the agricultural residues such as wheat straw and corn stalk
to produce fermentable sugars for further production of
ethanol has attracted much interest. The weed has fast-
growth, large biomass and strong adaptability characteris-
tics. It is estimated that the biomass yield of the weed in
south of China can be as much as 48 ton per hectare.
Therefore, it is possible to utilize the weed as a potential
biomass feedstock for bioenergy production. However,
the weed contains some toxic chemicals which may inhibit
the enzyme activity and growth of microorganisms, so it is
3.2. Effect of sulfuric acid pretreatment
Dilute acid pretreatment of lignocellulosic biomass has
received considerable research attention over the years
(Sun and Cheng, 2002; Mosier et al., 2005). It predomi-
nantly removes hemicelluloses, which increases the accessi-
ble surface area of the biomass. There are primarily two
types of dilute acid pretreatment processes: high tempera-
ture (T greater than 160 °C), continuous-flow process for
low solids loading (5–10% [weight of substrate/weight of
reaction mixture]), and low temperature (T less than
160 °C), batch process for high solids loading (10–40%)
(Sun and Cheng, 2002). In our study, we used dilute sulfu-
ric acid to treat the weed stem at a temperature below
120 °C, so it was called a ‘‘mild’’ sulfuric acid pretreatment
process. The factors affecting sulfuric acid pretreatment
mainly include particle size (D), temperature (T), reaction
time (t), sulfuric acid concentration (C), and liquid-to-solid
ratio (L) and so on. We have found that too small liquid-
to-solid ratio could lead to ununiformity of the system.
Therefore, a relatively high liquid-to-solid ratio (8:1, v/w)
was selected in our experiments and an L9(34) orthogonal
experimental design was used for optimization of the other
parameters. The experimental conditions and results are
shown in Table 2. It can be seen that mild H2SO4 pretreat-
ment can obtain much higher yield of reducing sugar by
acid hydrolysis (YRSAH) than that by enzymatic hydroly-
sis (CRC). The highest YRSAH was obtained by test 8, but
no obvious differences between YRSAH and CRC were
Table 2
L9(34) experiment of mild H2SO4 pretreatment
Test No. Parameters SYd (%) CCe (%) YRSAHf (%) CRCg (%)
Ta tb Cc

Table 1 1 T1 t1 C1 73.14 51.98 21.56 2.50

Chemical composition of crofton weed stem and comparison with some 2 T1 t2 C2 65.57 56.64 25.56 3.50
agricultural residues 3 T1 t3 C3 58.00 57.74 31.19 4.17
4 T2 t1 C2 64.00 58.95 28.34 3.34
Crofton Sugarcane Corn Wheat Cotton 5 T2 t2 C3 58.57 61.72 31.90 3.74
weed stem bagasse stovera strawa stalkb 6 T2 t3 C1 67.14 53.76 25.86 5.22
Holocellulose (%) 66.22 76.76 65.30 66.00 41.80 7 T3 t1 C3 51.86 61.83 32.89 4.66
Cellulose (%) 37.14 44.98 37.68 40.40 31.10 8 T3 t2 C1 53.86 61.14 32.89 4.90
Klason lignin (%) 16.42 18.45 18.38 22.34 27.90 9 T3 t3 C2 51.57 63.60 33.04 4.79
Ash (%) 4.52 1.38 4.66 6.04 6.00 a
T: pretreatment temperature, T1, T2, T3 denote 100, 110, 120 °C,
1% NaOH 47.54 34.2 45.62 44.56 respectively.
extractives (%) b
t: pretreatment time, t1, t2, t3 denote 90, 120, 150 min, respectively.
Cold water 18.16 2.15 10.65 5.36 c
C: concentration of sulfuric acid, C1, C2, C3 denote 1, 2, 3% (w/v),
extractives (%) respectively.
Hot water 18.12 5.16 20.40 23.15 d
SY: solid yield, % (weight ratio of residue solid after pretreatment to
extractives (%) the initial solid).
Benzene–ethanol 8.86 3.17 0.56 0.51 e
CC: cellulose content in the residue solid, %.
extractives (%) f
YRSAH: yield of reducing sugar by acid hydrolysis (based on the
a
Data from Yang (2001). initial holocellulose).
b g
Data from Silverstein et al. (2007); all the composition percentages are CRC: conversion ratio of cellulose by enzymatic hydrolysis for 72 h
on a dry-weight basis. (base on the initial cellulose in the sample).
3732 X. Zhao et al. / Bioresource Technology 99 (2008) 3729–3736

observed among test 7–9. HPLC analysis indicated that the of test 8 as the ‘‘optimal’’ condition for mild H2SO4 pre-
reducing sugar obtained by acid hydrolysis mainly con- treatment. Under this condition, approximately 46% of
sisted of xylose, glucose and arabinose. Xylose and glucose the raw material was dissolved in the pretreatment;
accounted for about 70% and 20% of the total reducing YRSAH was 32.89%; and the cellulose content was
sugar, respectively (see Table 3). It indicated that the increased to 61.14%. However, the content of Klason lignin
hydrolysis of glycan in mild acid pretreatment mainly in the pretreated sample was also increased to 26%, and
occurred in hemicellulose. only 14% of the Klason lignin was removed. Perhaps using
A further orthogonal analysis was used to study the higher temperatures and pressures or concentrations of
optimal level of each parameter for YRSAH and CRC, H2SO4 can further remove hemicelluloses and increase
as shown in Table 4. According to the R values, the signif- the accessible surface area that will further improve the
icance of each parameter affecting YRSAH and CRC enzymatic digestibility. Nevertheless, the poor delignifica-
showed the following order: T > C > t for YRSAH and tion ability of sulfuric acid is still a limit of mild acid pre-
T > t > C for CRC. It also can be known that SY decreased treatment, because lignin is believed to be a major
with temperature, time and sulfuric acid concentration due hindrance in enzymatic hydrolysis (Chang and Holtzapple,
to part removal of hemicelluloses and extractives. Con- 2000; Kim and Holtzapple, 2006).
versely, CC, YRSAH and CRC increased with these
parameters, however, it was found that time and concen- 3.3. Effect of sodium hydroxide pretreatment
tration of sulfuric acid did not affect YRSAH and CRC
significantly (p > 0.05). Table 4 indicates that the optimum Although acid pretreatment could obtain a relatively
condition for YRSAH was T3t2C3, but we did a comple- high YRSAH, the enzymatic digestibility of the materials
mentary experiment using the combination of the optimum was not significantly improved. Using sodium hydroxide
levels and found the CRC was 4.82% and the YRSAH was to pretreat lignocellulosic materials is an alternative to
just 28.7%, which was lower than the results of test 7–9. It sulfuric acid pretreatment. The main effect of sodium
was probably because that more reducing sugar was lost hydroxide pretreatment on lignocellulosic biomass is delig-
when the acid concentration was increased or reaction time nification by breaking the ester bonds cross-linking lignin
was prolonged. Therefore, we selected the parameter levels and xylan, thus increasing the porosity the of biomass
(Sun and Cheng, 2002; Mosier et al., 2005; Silverstein
et al., 2007). Dilute NaOH treatment of lignocellulosic
Table 3
Yield of some monosaccharide (based on initial holocellulose) in mild materials also causes swelling, leading to an increase in
H2SO4 pretreatment internal surface area, a decrease in the degree of polymer-
Test Yield of xylose Yield of glucose Yield of Others ization and crystallinity (Sun and Cheng, 2002). It seems
No. (%) (%) arabinose (%) (%) that sodium hydroxide pretreatment is more effective to
1 15.34 4.77 0.87 0.59 enhance the enzymatic digestibility than acid pretreatment,
2 17.12 4.70 1.14 2.60 since alkali has stronger delignification ability. Therefore,
3 22.56 5.21 1.42 2.00 we used sodium hydroxide to pretreat the weed stem in
4 20.14 5.53 1.66 1.01 order to improve the enzymatic saccharification. Our pre-
5 23.89 5.55 1.58 0.88
experiment using a single-factor test showed that the opti-
6 16.99 4.75 1.21 2.91
7 23.52 5.12 1.10 3.14 mal liquid-to-solid ratio for NaOH pretreatment was 6:1.
8 23.06 6.30 1.28 2.24 A liquid-to-solid ratio lower or higher than 6:1 led to
9 23.19 6.00 1.46 2.39 decrease of CRC. This may be because that a smaller
liquid-to-solid ratio made the system less homogeneous,
but increasing liquid-to-solid ratio led to a decrease of
Table 4 alkali concentration in the liquid phase when the loading
Analysis of L9(34) experiment results for YRSAH and CRC in H2SO4 of NaOH was fixed. Therefore, for the NaOH pretreat-
pretreatment ment, the liquid-to-solid ratio of 6:1 was selected.
YRSAH CRC A L9(34) orthogonal experimental design was subse-
T t C T t C quently used to optimize the parameters including temper-
K1 78.31a 82.79 80.31 10.18 10.50 12.63 ature (T), time (t) and loading of sodium hydroxide (D,
K2 86.10 90.35 86.94 12.30 12.14 11.63 based on raw materials). The experimental conditions
K3 98.82 90.09 95.98 14.35 14.19 12.57 and results are shown in Table 5. Compared with mild
k1 26.10b 27.60 26.77 3.39 3.50 4.21 H2SO4 pretreatment, NaOH pretreatment enhanced CRC
k2 28.70 30.12 28.98 4.10 4.05 3.88
more effectively. Analysis of the experiment results for
k3 32.94 30.03 31.99 4.78 4.73 4.19
R 6.84c 2.52 5.22 1.39 1.23 0.33 CRC, shown in Table 6, indicated that the significance of
Optimal level T3 t2 C3 T3 t3 C1 each parameter affecting CRC followed the order of
a
K Ti ¼ the sum of YRSAH or CRC at T i . T > D > t. However, according to experimental results,
b
k Ti ¼ K Ti =3. temperature and time had no significant effects on SY
c
   
R = max k Ti  min k Ti . and CC. Increasing loading of NaOH resulted in an
 X. Zhao et al. / Bioresource Technology 99 (2008) 3729–3736 3733

Table 5 needed. Peracetic acid (PAA) can be used to pretreat the

L9(34) experiment results of NaOH pretreatment weed stem in order to obtain higher degree of delignifica-
Test No. Parameters SY (%) CC (%) CRC (%) tion (DD). PAA has been recognized as a powerful oxidiz-
T a
t b
Dc
ing agent and is quite selective toward the lignin structure.
1 T1 t1 D1 69.14 59.37 13.18 It oxidizes aromatics in lignin generating dicarboxylic acid
2 T1 t2 D2 64.14 60.93 11.79 and their lactones (Teixeira et al., 2000). The enzymatic
3 T1 t3 D3 61.43 61.39 11.83 digestibility of PAA-pretreated or PAA pre-pretreated bio-
4 T2 t1 D2 62.43 61.69 15.39 mass was effectively enhanced (Ando et al., 1988; Teixeira
5 T2 t2 D3 59.29 62.24 12.80
6 T2 t3 D1 68.57 58.95 14.68
et al., 1999; Teixeira et al., 2000). According to previous
7 T3 t1 D3 62.71 64.31 12.81 experiments, the factors affecting PAA pretreatment
8 T3 t2 D1 70.71 58.15 18.53 mainly included temperature (T), time (t), loading of
9 T3 t3 D2 63.29 61.21 15.68 PAA (D, based on raw materials) and liquid-to-solid ratio
a
T: pretreatment temperature, T1, T2, T3 denote 90, 100, 110 °C, (L). A L16(45) orthogonal experimental design was used to
respectively. optimize the parameters in PAA pretreatment. The pre-
b
t: pretreatment time, t1, t2, t3 denote 90, 120, 150 min, respectively. treatment conditions and results are shown in Table 7. It
c
D: loading of NaOH, D1, D2, D3 denote 10, 14, 18% (w/w),
respectively.
shows that PAA pretreatment can remove lignin effectively
and cause degradation of some hemicelluloses, which made
the cellulose exposed. Analysis of the experiments (Table 7)
showed that the significance of each parameter affect-
Table 6
Analysis of L9(34) experiment results for CRC in NaOH pretreatment ing YRSAH and CRC followed the same order of
D > T > L > t. Loading of PAA is the most important fac-
T t D
tor for all the response variables.
K1 36.80 41.38 46.38
Fig. 1 shows that increasing temperature resulted in a
K2 42.86 43.12 42.86
K3 47.02 42.18 37.44 decrease of SY and increase of CC, DD, YRSAH and
k1 12.27 13.79 15.46 CRC. It was due to the fact that more lignin and carbohy-
k2 14.29 14.37 14.29 drates were dissolved at a higher temperature, which led to
k3 15.67 14.06 12.48 decrease of SY and increase of YRSAH and CRC. How-
R 3.41 0.58 2.98
ever, temperature over 80 °C had no significant influence
Optimal level T3 t2 D1

obvious decrease of SY and increase of CC. CRC was Table 7

increased with temperature, but decreased with loading of L16(45) experiment of peracetic acid pretreatment
NaOH. This interesting phenomenon maybe caused by Test Parameters SY CC DDe YRSAH CRC
extractives in the weed stem competitively reacting with No.
Ta
t b
Dc
L d (%) (%) (%) (%) (%)
NaOH and further affecting the kinetics and efficiency of
1 T1 t1 D1 L1 77.00 51.46 44.42 1.02 2.26
NaOH pretreatment. This should be further investigated. 2 T1 t2 D2 L2 72.43 54.40 73.83 5.34 4.31
Table 6 showed that the ‘‘optimal’’ condition for NaOH 3 T1 t3 D3 L3 68.71 56.80 88.21 8.82 11.39
pretreatment was T3t2D1, which was included in the exper- 4 T1 t4 D4 L4 66.71 59.45 90.75 11.33 26.92
iment design. The ‘‘optimal’’ condition for NaOH pretreat- 5 T2 t1 D2 L3 72.14 55.53 58.04 6.31 5.42
ment thus was as follows: temperature of 110 °C, reaction 6 T2 t2 D1 L4 74.43 52.33 47.45 3.72 3.54
7 T2 t3 D4 L1 57.00 70.38 96.25 24.81 31.72
time of 120 min, NaOH loading of 10% and liquid-to-solid 8 T2 t4 D3 L2 65.29 61.25 87.75 13.99 34.50
ratio of 6:1. Under these conditions, approximate 30% of 9 T3 t1 D3 L4 67.14 59.21 83.84 13.44 21.58
the raw material and 25% of the lignin were dissolved in 10 T3 t2 D4 L3 55.43 69.98 97.59 26.82 29.19
the pretreatment, and the cellulose content was increased 11 T3 t3 D1 L2 72.57 54.73 62.51 6.37 3.47
to 58.15%. The CRC was 18.53%, which was greatly 12 T3 t4 D2 L1 64.29 63.28 79.01 16.46 26.10
13 T4 t1 D4 L2 50.00 78.61 95.79 32.20 36.05
enhanced juxtaposed with sulfuric acid pretreatment. 14 T4 t2 D3 L1 52.29 73.76 95.23 30.42 38.06
When cellulase loading was increase to 80 FPU/g cellulose, 15 T4 t3 D2 L4 65.43 60.25 73.77 14.59 8.36
CRC correspondingly increased to 29%. 16 T4 t4 D1 L3 70.14 55.22 64.20 9.15 7.58
a
T: pretreatment temperature, T1, T2, T3, T4 denote 60, 70, 80, 90 °C,
respectively.
3.4. Effect of peracetic acid pretreatment b
t: pretreatment time, t1, t2, t3, t4 denote 60, 90, 120, 150 min,
respectively.
c
It was shown that NaOH pretreatment could increase D: loading of PAA, D1, D2, D3, D4 denote 20, 30, 40, 50% (w/w),
respectively.
the degree of delignification and enhance the enzymatic d
L: liquid-to-solid ratio, L1, L2, L3, L4 denote 4, 5, 6, 7 (v/w),
digestibility of the weed stem, however, the CRC was still respectively.
low. A higher temperature may further increase it, but e
DD: degree of delignification (percentage of the Klason lignin
more energy consumption and a higher pressure will be removed), %.
3734 X. Zhao et al. / Bioresource Technology 99 (2008) 3729–3736

Fig. 1. Effect of temperature, time, loading of PAA and liquid-to-solid ratio on SY, CC, DD, CRC and YRSAH in peracetic acid pretreatment.

on DD. Pretreatment time significantly affected all the
response variables (p > 0.05). DD did not increase obvi-
ously and YRSAH conversely decreased after 90 min,
probably due to the degradation of sugars by oxidation.
Loading of PAA had a very significant effects on all the
response variables (p < 0.001). Increasing PAA loading sig-
nificantly increased CC, DD, YRSAH and CRC, but also
increased the cost. Liquid-to-solid ratio actually affected
the concentration of PAA in the liquid phase, which fur-
ther affected pretreatment. When the loading of PAA was
fixed, increasing liquid-to-solid ratio decreased PAA con-
centration in the liquid phase, leading to decrease of DD
and CRC.
It should be noted that PAA was prepared by acetic acid
and hydrogen peroxide with sulfuric acid as a catalyst. In
the pretreatment these chemicals also caused degradation
of carbohydrates and lignin. Acetic acid is believed to be
a good solvent for lignin and used for pulping or fraction-
ation of lignocellulosic materials (Pan and Sano, 2005).
However, in the acetic acid pulping process, when the con-
centration of acetic acid in the aqueous solution was lower
than 60%, almost no lignin was dissolved at a temperature
below 90 °C. Even at a high temperature (about 110 °C), in
order to obtain a good degree of delignification, the acetic
acid concentration must be equal to or greater than 71.3%
(Ligero et al., 2007). Therefore, the delignification ability of
acetic acid in PAA pretreatment was so insignificant that it
could be negligible. On the other hand, acetic acid and sul-
furic acid could catalyze hydrolysis of hemicelluloses, but
sulfuric acid is the main agent leading to degradation of
hemicelluloses. Hydrogen peroxide could also react with
lignin, but its activity was weakened in an acid condition.
Therefore, PAA plays a major role in delignification.
Table 8 shows that the optimal condition for CRC was
T4t4D4L1. A complementary experiment according to this
combination of the optimum levels gave a DD of 97.2%,
with a CRC of only 28.8%. It has been found that the cel-
lulose crystallinity was increased with DD in PAA pretreat-
ment. Therefore, probably due to the increase of cellulose
crystallinity, the combination of the optimum levels did
not give the highest CRC. It indicated that lignin content
was not the only factor affecting enzymatic hydrolysis.
There are some other factors affecting the enzymatic digest-
ibility of the materials. The details will be discussed in a
continued study. Test 14 was therefore selected as the
‘‘optimum’’ condition for PAA pretreatment. Under this

Table 8
Analysis of L16(45) experiment results for DD, YRSAH and CRC in peracetic acid pretreatment
YRSAH CRC
T t D L T t D L
K1 26.50 52.97 20.26 72.70 44.89 65.31 16.85 98.15
K2 48.83 66.29 42.69 57.90 75.19 75.10 44.19 78.33
K3 63.08 54.59 66.66 51.10 80.33 54.94 105.52 53.57
K4 86.36 50.93 95.16 43.08 90.05 95.10 123.89 60.40
k1 6.63 13.24 5.07 18.18 11.22 16.33 4.21 24.54
k2 12.21 16.57 10.67 14.47 18.80 18.77 11.05 19.58
k3 15.77 13.65 16.66 12.77 20.08 13.74 26.38 13.39
k4 21.59 12.73 23.79 10.77 22.51 23.78 30.97 15.10
R 14.96 3.84 18.72 7.41 11.29 10.04 26.76 11.14
Optimal level T4 t2 D4 L1 T4 t4 D4 L1
 X. Zhao et al. / Bioresource Technology 99 (2008) 3729–3736
condition, 30.42% of the glycan was converted to reducing
sugar in the pretreatment, with xylose yield of 21.2% and
glucose yield of 6.1%. When the pretreated sample
was digested by cellulase loading of 20 FPU/g cellulose,
38.06% of the cellulose was hydrolyzed to glucose in
72 h. The CRC was increased to 50% when increasing cel-
lulase loading to 80 FPU/g cellulose.
3.5. Comparison of the three chemical pretreatment methods
under the same condition
PAA pretreatment can obtain a high degree of delignifi-
cation, but compared with mild H2SO4 pretreatment and
NaOH pretreatment, the loading of PAA is relatively high.
The ‘‘optimal’’ conditions for these chemical pretreatments
were also different, so it is not suitable to compare the effec-
tiveness of different pretreatment methods when the condi-
tions were different. PAA can effectively remove lignin at a
low temperature, and we selected the same mild condition
(temperature of 90 °C, time of 150 min, loading of chemical
of 50% based on raw materials and liquid-to-solid ratio of
4:1) to compare the effects of the three chemical pretreat-
ment methods on the enzymatic digestibility of the weed
stem. The results are shown in Table 9. Mild H2SO4 pre-
treatment can hydrolyze hemicellulose, but its delignifica-
tion ability is poor. Therefore, the enzymatic digestibility
of H2SO4 treated samples was still very weak. NaOH pre-
treatment can increase the removal of lignin and enhance
the enzymatic conversion of cellulose compared with
H2SO4 pretreatment. PAA pretreatment gave the highest
degree of delignification and also degraded part of hemicel-
luloses, so the CRC was also greatly enhanced. It indicated
that PAA was effective for improving the enzymatic digest-
ibility of the weed stem at a low temperature. On the other
hand, the decomposition product of PAA is mainly acetic
acid. It can be easily recovered by distillation and reused.
Moreover, acetic acid can be formed by deacetylation of
hemicelluloses in the pretreatment, which is a complement
of acetic acid. However, an economic study of the three
pretreatment methods at their optimum conditions should
be done in future for a further comparison.
Table 9
Comparison of the three chemical pretreatments under the same condition
Control H2SO4 NaOH
(Untreated) treated treated
samples samples
Solid yield (%) 100 56.86 53.00
Cellulose 37.14 63.13 66.64
content (%)
Klason Lignin 0 14.10 27.30
removed (%)
Carbohydrates 0 32.10 30.25
dissolved
(%)
CRC after 72 h 0.80 2.61 7.40
(%)
3735
We also found that the CRC of the untreated and trea-
ted weed stem were lower than those of some agricultural
residues. The CRC of PAA-pretreated bagasse with lignin
content of less than 6% could reach 80% in 72 h when
the same loading of cellulase was used. It demonstrated
that the weed stem was harder to pretreat and digest by cel-
lulase enzyme than common agricultural residues.
3.6. Modeling of the pretreatment processes
Some empirical quadratic models were developed with
the corresponding variables using JMP 5.1 software to pre-
dict the yield of sugars and/or degree of delignification for
mild sulfuric acid pretreatment and peracetic acid pretreat-
ment from the following expression:
X
n X
n X
n
Y ¼ a0 þ bi X i þ ci X 2i þ d ij X i X j þ    ð1Þ
i¼1 i¼1 i¼1; j¼1
where Y is the response variable. Xi–Xn denote the vari-
ables including temperature (T), time (t), concentration of
sulfuric acid (C), loading of PAA (D) and liquid-to-solid
ratio (L); n is the number of variables; a0, bi, ci, . . . are cor-
responding regression parameters. After eliminating the
insignificant terms (p > 0.05) from the model based on
the p-values from JMP analytic results, the reduced empir-
ical quadratic model used to quantify the yield of reducing
sugar by acid hydrolysis (YRSAH, based on initial holocel-
lulose, %) in the liquid phase was
YRSAHjMSAP ð%Þ ¼ 81:9348 þ 0:8020T þ 0:03355t
þ 7:6593C þ 0:0086ðT  111:74Þ
 ðt  90Þ  1:0298ðC  1:522Þ
 ðT  111:74Þ  0:1366ðC  1:522Þ
2
 ðt  90Þ þ 0:0514ðT  111:74Þ ð2Þ
The subscript ‘MSAP’ denotes ‘mild sulfuric acid pretreat-
ment’. The square of the correlation coefficient (R2) was
0.94. The yield of xylose and glucose in sulfuric acid pre-
treatment can be estimated by the following expressions:
Yield of xylose ð%Þ ¼ 0:7  YRSAHjMSAP ð3Þ
Yield of glucose ð%Þ ¼ 0:2  YRSAHjMSAP ð4Þ
Similarly, we obtained the models for estimation YRSAH
and degree of delignification (DD) in PAA pretreatment
PAA shown as expression (5) and (6).
treated
DDjPAAP ¼ 25:3024 þ 0:06918T þ 0:1046t
samples
þ 1:3895D  1:7285L  0:0094ðD  35Þðt  105Þ
44.43
88.87  0:02545ðD  35Þ2 R2 ¼ 0:97 ð5Þ
YRSAHjPAAP ¼ 34:3138 þ 0:454T þ 0:0259t
97.2
þ 0:6217D  1:8486L  0:0763ðD  35ÞðL  5:5Þ
34.92 þ 0:01359ðT  75ÞðD  35Þ R2 ¼ 0:98 ð6Þ
We also found that the solid yield (SY) and cellulose con-
28.8 tent (CC) in the treated samples had a good linear relation
(see Fig. 2) as follows:
3736 X. Zhao et al. / Bioresource Technology 99 (2008) 3729–3736
Fig. 2. Relations between solid yield and cellulose content in the
pretreated samples.
CC ¼ 0:4943 SY þ 88:331ðR2 ¼ 0:92Þ
for H2 SO4 pretreatment ð7Þ
2
CC ¼ 0:3096 SY þ 80:55ðR ¼ 0:89Þ
for NaOH pretreatment ð8Þ
2
CC ¼ 0:8822 SY þ 119:87ðR ¼ 0:92Þ
for PAA pretreatment ð9Þ
These empirical models could successfully predict the
experimental data and may be used in the development
of better estimation tools.
4. Conclusions
The stem of crofton weed had a content of glycan simi-
lar to those of corn stover and wheat straw. It is possible to
utilize the weed as a feedstock to produce fermentable sug-
ars for further production of biofuel such as ethanol. Mild
sulfuric acid pretreatment could obtain a relatively high
yield of sugars in the pretreatment, but it led to only a
slight enhancement of enzymatic digestibility. NaOH pre-
treatment could obtain a higher enzymatic conversion ratio
of cellulose compared with H2SO4 pretreatment. Peracetic
acid pretreatment seemed to be the most effective for
improving enzymatic saccharification of the weed stem in
the three chemical pretreatment methods under the same
conditions. The conversion ratio of cellulose in the sam-
ple pretreated by PAA under the ‘‘optimal’’ condition
was increased to 50% when using cellulase loading of
80 FPU/g cellulose to digest the materials for 72 h.
Some empirical quadratic models were successfully
developed according the experimental data to predict the
yield of sugar and degree of delignification. In addition,
this study can serve as a step towards the further optimiza-
tion of pretreatment of the weed to utilize and control it.
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