Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 82:1115–1121 (2007)

Effect of several factors on peracetic

acid pretreatment of sugarcane bagasse
for enzymatic hydrolysis
Xue-bing Zhao, Lei Wang and De-hua Liu∗
Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China

Abstract

BACKGROUND: Lignocellulose should undergo pretreatment to enhance its enzymatic digestibility before being
saccharified. Peracetic acid (PAA) is a strong oxidant that can remove lignin under mild conditions. The sulfuric
acid in the PAA solution also can cause degradation of hemicelluloses. The objective of the present work is to
investigate the effect of several factors on peracetic acid pretreatment of sugarcane bagasse.

RESULTS: It was found that PAA charge, liquid/solid (l/s) ratio, temperature, time, interactions between PAA
charge and l/s ratio, temperature and time, all had a very significant effect on the enzymatic conversion ratio of
cellulose. The relative optimum condition was obtained as follows: PAA charge 50%, l/s ratio 6:1, temperature
80 ◦ C and time 2 h. More than 80% of the cellulose in bagasse treated under the above conditions was converted to
glucose by cellulase of 20 FPU g−1 cellulose. Compared with H2 SO4 and NaOH pretreatments under the same mild
conditions, PAA pretreatment was the most effective for enhancement of enzymatic digestibility.

CONCLUSION: PAA pretreatment could greatly enhance the enzymatic digestibility of sugarcane bagasse by
removing hemicelluloses and lignin, but removal of lignin was more helpful. This study can serve as a step
to further optimization of PAA pretreatment and understanding the mechanism of enhancement of enzymatic
digestibility.
 2007 Society of Chemical Industry

Keywords: sugarcane bagasse; peracetic acid; pretreatment; enzymatic hydrolysis

INTRODUCTION cellulose digestibility using cellulase enzymes, which

Due to increasing energy consumption and environ-
mental concerns, during recent years there has been
increased interest in using ethanol as a transportation
fuel. Lignocellulosic materials are attractive feedstocks
for ethanol production because they are abundant,
cheap and renewable. One of the major lignocellu-
losic materials to be considered in tropical countries
is sugarcane bagasse, the fibrous residue obtained
after extracting the juice from sugar cane in the sugar
production process.1 In general, sugar factories gener-
ate approximately 270 kg of bagasse (50% moisture)
per metric ton of sugarcane.2 Therefore, it can be
estimated that the yield of sugarcane bagasse (50%
moisture) is approximately 10 million tons per year in
China.
Enzymatic hydrolysis is a promising way to obtain
sugars from lignocellulosic materials, but the low
enzymatic accessibility of the native cellulose is
a key problem for biomass-to-ethanol processes.
Therefore, pretreatment is an essential element in
the bioconversion of lignocellulosic substrates. The
objective of biomass pretreatment is to alter the
structure of the lignocellulosic matrix to increase
can be done by removing lignin, hemicelluloses,
or a combination of the two. Several processes
have been developed for pretreatment of sugarcane
bagasse, including steam explosion,3,4 liquid hot water
process,5 acid hydrolysis,6 alkali pretreatment7 and
wet oxidation.1 All of these processes enhance the
enzymatic digestibility of bagasse to some extent.
However, most of them should be operated at high
temperature resulting in high pressure, which increases
the energy consumption and costs of equipments.
Furthermore, these processes still leave most of
the lignin in the material and limit the complete
bioconversion of cellulose to sugar. Lignin is believed
to be a major hindrance to enzymatic hydrolysis.8 – 12
Low-lignin substrates have improved microbial activity
and enzyme efficiency, eventually lowering the enzyme
requirement.13
Peracetic acid (PAA) is recognized as a powerful
oxidizing agent and is quite selective towards the
lignin structure. It oxidizes the aromatics in lignin,
generating dicarboxylic acid and their lactones.14 The
enzymatic digestibility of PAA-pretreated or PAA pre-
pretreated biomass was effectively enhanced.8,15 – 17

∗
Correspondence to: De-hua Liu, Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China
E-mail: dhliu@tsinghua.edu.cn
(Received 6 June 2007; revised version received 16 July 2007; accepted 17 July 2007)
Published online 15 October 2007; DOI: 10.1002/jctb.1775

 2007 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2007/$30.00

XB Zhao, L Wang, DH Liu

Gharpuray found that ball milling followed by Ltd in Japan. The cellulase activity was determined
PAA treatment could decrease the crystallinity and by the method recommended by Ghose,20 and
lignin content and enhance the hydrolysis.18 Teixeira expressed in filer paper units (FPU): 1 FPU
studied PAA pretreatment of woody biomass and was defined as the amount of enzyme capable of
sugarcane bagasse at ambient temperature for a 7- producing 1 µmole of reducing sugars in 1 min.
day period, with PAA concentrations varying from The main monosaccharides in the liquid phase
6% to 60%. The pretreated samples had a greatly were determined by Shimadzu (Tokyo, Japan) high
enhanced enzymatic digestibility.17 He also found performance liquid chromatography (HPLC) using
that alkaline treatments were helpful in reducing an Aminex HPX-87H column and RID-10A detector.
PAA requirements.19 However, these processes are The mobile phase was 0.05 mol L−1 H2 SO4 at a
very slow, leading to a decrease in productivity. flow rate of 0.8 mL min−1 . Standard glucose, xylose
Furthermore, the authors did not discuss the factors and arabinose were purchased from Sigma-Aldrich
affecting PAA pretreatment and their significance. (Shanghai, China).
Therefore, the objective of this present work is to
investigate the factors that affect PAA pretreatment of Pretreatment process
sugarcane bagasse and analyze their significance for The pretreatment was carried out in a 1000 mL glass
enzymatic hydrolysis. flask immersed in a water bath. 30 g of screened
bagasse was packed into the flask and a specific
volume of prepared PAA solution was added. A
EXPERIMENTAL Teflon paddle was used for intermittent stirring to
Materials and analytical methods keep the system as homogeneous as possible. After
Sugarcane bagasse was obtained from Guanxi province pretreatment, the bagasse was washed with water until
in the south of China. It was ground and screened. neutrality and dried at 105 ◦ C for 6 h. The oven-dried
The fraction not passing through a 20-mesh sieve was samples were stored in valve bags for further analysis
used in all pretreatment experiments. The composition and enzymatic hydrolysis. The liquid was collected for
of sugarcane bagasse was determined according to analysis of sugars and recovery of acetic acid.
corresponding Chinese standards. The data are shown
in Table 1. Enzymatic hydrolysis
The chemicals, including anhydrous acetic acid, Before enzymatic hydrolysis, the cellulose content in
30% hydrogen peroxide, potassium permanganate, the treated samples was determined. Then the samples
potassium iodide, sodium thiosulfate and sulfuric acid were digested by cellulase loading of 20 FPU g−1
were analytically pure, obtained from Beijing Beihua cellulose. The enzymatic digestibility tests were
Fine Chemicals Co., Ltd. Peracetic acid was prepared conducted as follows: temperature 50 ± 0.5 ◦ C, pH
by reaction of acetic acid and 30% hydrogen peroxide, 4.8 (0.1 mol L−1 sodium acetate buffer), 130 rpm
with volume ratio 2:1 at room temperature for 72 h. in an air-bath shaker. The digestibility, denoted as
3% (w/w) of sulfuric acid was added as a catalyst. conversion ratio of cellulose (CRC), was defined as
Determination of peracetic acid concentration was the percentage of cellulose converted to glucose after
made in accordance with Chinese standard GB/T 72 h of incubation with cellulase enzyme.
19 108-2003.
The cellulase enzyme used in the experiment was
Cellulase R-10, obtained from Yakuh Honsha Co. RESULTS AND DISCUSSION
Effect of PAA charge and liquid/solid ratio (l/s)
Table 1. Chemical composition of sugarcane bagasse and
PAA charge (based on raw material) and l/s ratio (v/w)
corresponding test methods
were varied from 20–50% and 3:1–7:1, respectively,
Items Values Methods keeping other conditions at 80 ◦ C, reaction time
2 h. Figure 1 shows that the cellulose content in
Moisture content (%, w/w) 3.42–6.07 GB/T 2677.2-1993
the treated materials increases with increasing PAA
Ash (%, w/w) 1.38 GB/T 2677.3-1993
Hot water extractives (%, 5.16 GB/T 2677.4-1993
charge or decreasing l/s ratio. PAA charge and l/s ratio
w/w) reflected the corresponding PAA concentration in the
1% NaOH extractives (%, 34.20 GB/T 2677.5-1993 liquid phase and percentage solids. At a fixed PAA
w/w) charge, increase in l/s ratio (decrease in percentage
Benzene-ethanol extractives 3.17 GB/T 2677.6-1994 solids) resulted in a decrease of PAA concentration
(%, w/w) in the liquid phase (as shown in Fig. 2), which
Cellulose (%, w/w) 44.98 Nitric acid-ethanol reduced the reaction rates of degradation of lignin and
method hemicelluloses. Similarly, at a fixed l/s ratio, increase of
Holocellulose (%, w/w)) 76.76 GB/T 2677.10-1995 PAA charge enhanced the rates of delignification and
Klason lignin (%, w/w) 18.45 GB/T 2677.8-1994 dissolving of hemicelluloses. Therefore, more lignin
Acid-soluble lignin (%, w/w) 1.80 GB/T 747–2003
and hemicelluloses were removed with higher PAA
Total lignin (%, w/w) 20.25 GB/T 2677.8-1994,
GB/T 10337-1989
charge or lower l/s ratio for the same reaction time.
It can be seen that the material contained over 80%

1116 J Chem Technol Biotechnol 82:1115–1121 (2007)

DOI: 10.1002/jctb
 Pretreatment of sugarcane bagasse for enzymatic hydrolysis

(w/w) of cellulose when treated with 50% PAA at a 100

l/s ratio of 3–5:1 at 80 ◦ C for 2 h. However, further
increase of PAA charge at a l/s ratio of 3 conversely

Degree of delignification (%)

80
led to lower cellulose content, compared with those
at l/s ratios of 4 and 5. This may be because the
decreasing l/s ratio accelerated recondensation and 60
deposition of dissolved lignin on the cellulosic fibers,
l/s ratio=3
a phenomenon known to occur in acidic organosolv 40 l/s ratio=4
delignification process.21 It can also be confirmed from l/s ratio=5
Fig. 3 that for a PAA charge over 50% the degree of l/s ratio=6
delignification at a l/s ratio of 3 was lower than those 20 l/s ratio=7
at l/s ratios of 4 and 5, probably due to the effect
of solubility limitations with less liquid being present. 0
On the other hand, some cellulose was also dissolved 0 10 20 30 40 50 60 70
under conditions of high PAA charge and low l/s ratio. PAA Charge
It was found that 8% of the cellulose was dissolved Figure 3. Effect of PAA charge and l/s ratio on degree of
in the liquid phase with a PAA charge of 60% and l/s delignification.
ratio 3:1.
Since PAA was prepared by reaction of acetic acid played the major role in hydrolysis of the
acid and hydrogen peroxide, with sulfuric acid as glycan. Table 2 shows concentrations of the main
a catalyst, the acids in the system could cause monosaccharides (xylose, glucose and arabinose) in
degradation of glycans, especially hemicelluloses. The the liquid phase under different conditions. These
pK a values of acetic acid and PAA at 25 ◦ C were monosaccharides were mainly from the hydrolysis
4.76 and 8.20, respectively.22 Therefore, sulfuric of hemicelluloses. It can be observed that all the
concentrations of monosaccharide increased with PAA
100 charge or concentration of sulfuric acid, but decreased
with l/s ratio. However, when the PAA charge was
l/s ratio=3
90 l/s ratio=4
increased to 60% at a l/s ratio of 3, the xylose
l/s ratio=5 concentration decreased. This may be explained
Cellulose content (%)

80
l/s ratio=6 by decreased solubility of the lignin–hemicellulose
l/s ratio=7 complex polymers owing to reduced liquid volume
and significant oxidation of xylose by PAA when its
70
charge was too high. Formic acid was detected in
the HPLC analysis, which could also be formed by
60
oxidation of xylose. There are also other oxidized
products, which should be analyzed further in future
50
work. Nevertheless, almost no solid cellulose was
lost when the l/s ratio was more than 4:1. This
40
0 10 20 30 40 50 60 indicates that PAA could selectively react towards
PAA charge (%) lignin without significant degradation of cellulose,
which contributes to the fractionation of cellulose from
Figure 1. Effect of PAA charge and l/s ratio on cellulose content in other components.
the treated materials.
The treated materials were hydrolyzed by cellulase
of 20 FPU g−1 cellulose at 50 ◦ C for several days. The
200 time profiles are shown in Fig. 4. It is clear that
20 % PAA the untreated bagasse was hard to digest. Almost
30 % PAA no further sugar was formed after 4 h. However,
40 % PAA
the enzymatic digestibility of treated bagasse was
in the liquid phase (g/L)

150 50 % PAA
PAA concentration

60 % PAA greatly enhanced and maintained a relatively high

saccharification rate for the first 12 h, but no obvious
100 difference was found between samples treated with
PAA charges of 50% and 60%. This shows that
increase of PAA charge beyond 50% gave no
50 enhancement of enzymatic digestibility.
Figures 5 and 6 show the conversion ratio of
cellulose (CRC) after the treated materials were
0 digested for 72 h. A PAA charge of 0 means that
3 4 5 6 7
l/s ratio (v/w)
the bagasse did not undergo pretreatment. The CRC
increased with PAA charge because more lignin and
Figure 2. PAA concentrations in the liquid phase at several l/s ratios. hemecelluloses were removed with higher PAA charge.

J Chem Technol Biotechnol 82:1115–1121 (2007) 1117

DOI: 10.1002/jctb
XB Zhao, L Wang, DH Liu

Table 2. Concentrations of several monosaccharides in the liquid 100

phase at different l/s ratios with different PAA charges and
concentrations of sulfuric acid l/s ratio=3

Conversion ratio of cellulose (%)

80 l/s ratio=4
PAA Concentration Concentration (g L−1 ) l/s ratio=5
charge l/s ratio of sulfuric l/s ratio=6
(%) (v/w) acid (%,w/w) Glucose Xylose Arabinose 60 l/s ratio=7

20 3:1 1.11 0.86 4.19 0.86

30 3:1 1.67 1.10 8.01 1.70 40
40 3:1 2.22 1.26 27.10 3.34
50 3:1 2.78 1.89 36.54 5.24
60 3:1 3.33 2.11 31.39 5.53 20
20 4:1 0.83 0.28 2.63 0.96
30 4:1 1.25 0.31 2.92 1.17
0
40 4:1 1.67 0.56 9.78 2.75 0 10 20 30 40 50 60
50 4:1 2.08 0.82 18.51 2.78 PAA charge (%)
60 4:1 2.50 1.10 32.14 4.69
20 5:1 0.67 0.15 1.16 1.87 Figure 5. Effect of PAA charge on CRC.
30 5:1 1.00 0.26 2.75 2.32
40 5:1 1.33 0.40 8.39 3.26
50 5:1 1.67 0.64 15.07 3.60 100
20% PAA 30%PAA
60 5:1 2.00 0.83 26.27 3.84 40% PAA 50%PAA

Conversion ratio of cellulose (%)

20 6:1 0.56 0.38 2.31 2.30 60%PAA
80
30 6:1 0.83 0.48 3.69 3.92
40 6:1 1.11 0.57 5.12 3.63
50 6:1 1.39 0.75 7.81 3.72 60
60 6:1 1.67 0.85 9.73 3.67
20 7:1 0.48 0.25 1.73 0.29
30 7:1 0.71 0.28 1.99 0.56 40
40 7:1 0.95 0.31 2.89 1.39
50 7:1 1.19 0.55 5.79 1.69
20
60 7:1 1.43 0.73 6.69 1.92

0
90 3 4 5 6 7
control 20% PAA l/s ratio
80 30% PAA 40% PAA
Conversion ratio of cellulose (%)

50% PAA 60% PAA Figure 6. Effect of l/s ratio on CRC.

70

60
50
40
30
20
10
0
0 10 20 30
Time (h)
Figure 4. Time profiles of enzymatic hydrolysis of bagasse treated
with different PAA charge at a l/s ratio of 6:1.
For PAA charges greater than 50%, the CRC increased
only slightly or decreased. Thus, 50% of PAA was
considered the optimum for the pretreatment process.
It can be seen from Fig. 6 that the CRC was initially
enhanced with increase of the l/s ratio. In fact, for
the same PAA charge (based on raw material), a
higher PAA concentration in the liquid was obtained
at a lower l/s ratio. It thus appears that a lower
l/s ratio should more effectively enhance enzymatic
saccharification. In fact, experimental results proved
that a too small l/s ratio reduced the CRC: a l/s
ratio of 6:1 gave the highest reducing sugar yield,
with 80% of the original cellulose being transformed
to glucose. When the l/s ratio was increased to 7:1,
the CRC drastically reduced, probably due to the
decrease of PAA concentration in the liquid, which
reduced the degree of delignification. In a chemical
pulping process, cellulose fibers cannot be significantly
40 50 60 separated unless the degree of delignification is over
80%. It was found that the bagasse was softened when
treated at a l/s ratio of 7:1, however, its structure was
still compact. In comparison, at a l/s ratio of 6:1, most
of the bagasse became pulp whose specific surface
was greatly increased and the CRC was significantly
enhanced.
Thus, the effects of l/s ratio on CRS can be
summarized as follows. First, the l/s ratio affected
the PAA concentration in the liquid, consequently
affecting the removal of lignin and hemicelluloses;
however, when the l/s ratio was too high, it reduced
the PAA concentration in the liquid phase, resulting
in a decrease in CRC. Second, sufficient liquid is
required to dissolve lignin, hemecelluloses and their
degradation products; too small a l/s ratio decreases

1118 J Chem Technol Biotechnol 82:1115–1121 (2007)

DOI: 10.1002/jctb
 Pretreatment of sugarcane bagasse for enzymatic hydrolysis

the solubility because less liquid is present, and this
accelerates recondensation and deposition of dissolved
lignin, leading to a decrease in CRC.
Analysis of variance (ANOVA) was then used to
further study the effects of PAA charge and l/s ratio on
CRC. The results are shown in Table 3, which shows
that PAA charge, l/s ratio and their interaction all had
very significant effects on CRC.
Effect of reaction temperature and time
Temperature and time are two important factors
affecting reaction rate. Experiments were done
to investigate their effects on pretreatment, by
fixing the PAA charge at 50%. The effects of
temperature and time on cellulose content are shown
in Fig. 7. Two l/s ratios, 4 and 6, were selected for
comparison. Prolonging the reaction time naturally
increases cellulose content by removing more lignin,
hemicelluloses and other substances. It is also clear
that the cellulose content increased markedly at a l/s
ratio of 6 when the reaction temperature was increased.
However, at a l/s ratio of 4:1, no improvement was
obtained when bagasse was treated at temperatures
above 70 ◦ C for 2 h.
Increasing temperature could dramatically enhance
the xylose concentration in the liquid phase at a
l/s ratio of 4:1 (see Table 4). This is because the
higher temperature leads to greater degradation of
hemicelluloses. However, the concentrations of the
three monosaccharides were not increased significantly
at a l/s ratio of 6. Degradation of glycans was mainly
Table 3. Analysis of variance for effects of PAA charge and l/s ratio
on CRC
Source SS DF SS/DF
caused by acid hydrolysis, which is a temperature-
dependent process. In the conventional acid hydrolysis
of lignocellulose, high temperatures and pressures are
usually employed, which leads to the formation of
furfural from xylose. No furfural was detected in the
liquid phase here, probably due to the mild conditions
of the PAA pretreatment.
Figure 8 shows the effect of temperature and
time on CRC after 72 h. CRC increased when
the temperature was changed from 60 ◦ C to 80 ◦ C.
However, further increase of temperature did not
notably enhance CRC. Similarly, temperature had
a more significant influence on CRC at a l/s ratio
of 6 than at 4. It can also be seen that a large
increase in CRC was observed when increasing the
pretreatment time from 1 h to 2 h, but according
to these experiments, longer time did not give
more glucose. There are several fundamental factors
that affect enzymatic digestibility of lignocellulose,
including lignin content, crystallinity, surface area,
particle size, etc.11 These factors are always related
to each other. Details on the mechanisms of PAA
pretreatment for the enhancement of enzymatic
digestibility will be discussed in more detail in future
studies.
The results of ANOVA shown in Table 5 indicate
that temperature, time and their interaction all had
very significant effects on CRC.
The optimum conditions for PAA pretreatment of
bagasse were obtained as follows: PAA charge 50%,
l/s ratio 6:1, temperature 80 ◦ C, time 2 h. PAA charge,
l/s ratio, temperature, time and interactions between
PAA charge and l/s ratio, temperature and time, all
had very significant effects on CRC. Treated under
these ‘optimum’ conditions, the bagasse could be
F Proba- Signi- easily digested by cellulase. Over 80% of the original
Value bility ficance

PAAcharge 12412.3 4 3103.08 1063.03 0 Very Table 4. Concentrations of several monosaccharides in the liquid
l/s ratio 5323.7 4 1330.92 455.94 0 Very phase at different temperatures for different reaction times
Interaction 2435.3 16 152.21 52.14 0 Very
Error 73 25 2.92 Concentration (g L−1 )
Total 20244.3 49 Temperature Time l/s ratioa
(◦ C) (h) (v/w) Glucose Xylose Arabinose

65 1 4:1 0.50 2.84 0.80

100 70 1 4:1 0.54 5.12 0.84
l/s ratio=6:1, for 1h l/s ratio=6:1, for 2h 80 1 4:1 0.56 19.66 2.77
l/s ratio=4:1, for 1h l/s ratio=4:1, for 2h
90 90 1 4:1 0.59 31.37 2.50
65 2 4:1 0.53 6.28 1.37
Cellulose content (%)

70 2 4:1 0.63 14.49 2.73

80 80 2 4:1 0.82 18.51 2.78
90 2 4:1 1.17 37.76 3.91
60 1 6:1 0.19 2.11 0.48
70 70 1 6:1 0.24 2.84 0.79
80 1 6:1 0.35 3.45 1.14
90 1 6:1 0.44 4.74 1.47
60
60 2 6:1 0.34 4.51 1.46
70 2 6:1 0.44 5.73 1.67
50 80 2 6:1 0.75 7.81 1.78
60 65 70 75 80 85 90 90 2 6:1 0.86 9.44 1.88
Temperature (°C)
aThe concentrations of sulfuric acid in the liquid phase was 2.08%
Figure 7. Effect of temperature and time on cellulose content. (w/w) at a l/s ratio of 4:1 and 1.39% (w/w) at 6:1.

J Chem Technol Biotechnol 82:1115–1121 (2007) 1119

DOI: 10.1002/jctb
XB Zhao, L Wang, DH Liu

100 hemicelluloses was dissolved. NaOH pretreatment

l/s ratio=4:1, for 1h dissolved nearly half of the raw materials with
l/s ratio=4:1, for 2h
significant removal of hemicelluloses; however, the
Conversion ratio of cellulose (%)

80 l/s ratio=6:1, for 1h

l/s ratio=6:1, for 2h residue still had relatively high lignin content. PAA
pretreatment gave the highest degree of delignification
60 with the least degradation of hemicelluloses. When
treated bagasse was digested by cellulase, the PAA
treated sample gave the highest saccharification rate
40
(Fig. 9) and CRC (Table 6). The H2 SO4 -treated
sample gave a CRC of less than 10%. Thus,
20 H2 SO4 pretreatment under mild conditions did not
effectively to enhance enzymatic digestibility, probably
0
because the temperature was not high enough to
60 65 70 75 80 85 90 remove significant hemicelluloses to increase enzyme
Temperature (°C) accessibility. Using a higher temperature and pressure
would hopefully alter the structure of the materials
Figure 8. Effect of temperature and time on CRC.
and help to increase CRC. NaOH pretreatment
could remove most hemicelluloses, but the CRC
Table 5. Analysis of variance for effects of temperature and time on
was lower than that for the PAA pretreatment. The
CRC
experimental results further indicated that in PAA
F– Proba- Signi- pretreatment, the enzymatic digestibility was increased
Source SS DF SS/DF value bility ficance due to removal of hemicelluloses and lignin, but
removal of lignin was more helpful than removal of
PAA charge 3470.16 3 1156.72 851.27 0 Very
l/s ratio 1836.55 1 1836.55 1351.59 0 Very
hemicelluloses. However, the optimum conditions for
Interaction 108.78 3 36.26 26.68 0.0002 Very H2 SO4 and NaOH pretreatments would be at higher
Error 10.87 8 1.36 temperatures, so a further economic comparison
Total 5426.36 15 taking into account the different optimum conditions
for these three processes should be done in the future.

cellulose was hydrolyzed to glucose when digested at

50 ◦ C for 72 h by a cellulase loading of 20 FPU g−1
80
cellulose.
70
Conversion ratio of cellulose (%)

COMPARISON OF PAA PRETREATMENT WITH 60

H2 SO4 AND NAOH PRETREATMENTS UNDER 50

THE SAME CONDITIONS
The PAA solution contains H2 SO4 , which is the 40
major acid causing degradation of hemicelluloses. 30 control
Removal of hemicelluloses can help the enhancement sulfric acid pretreatment
20 sodium hydroxide pretreatment
of enzymatic digestibility. In order to further study
PAA pretreatment
the effectiveness of PAA pretreatment, we compared 10
it with H2 SO4 and NaOH pretreatments under the
same conditions (chemical loading 50%, l/s ratio 6:1, 0
0 10 20 30 40 50 60
80 ◦ C for 2 h). The results are shown in Table 6
Time (h)
and Fig. 9. It can be seen that H2 SO4 treatment
dissolved only 21.3% of the raw materials and Figure 9. Time profiles of enzymatic hydrolysis of bagasse pretreated
3% of the original lignin, but about 64% of the by different methods.

Table 6. Comparison of PAA pretreatment with H2 SO4 and NaOH pretreatments

Control (Untreated bagasse) H2 SO4 treated bagasse NaOH treated bagasse PAA treated bagasse

Dissolved (%) 0 21.3 46.7 34.8

Cellulose content (%) 44.98 57.43 76.10 70.34
Holocellulose content (%) 76.76 71.90 83.82 90.04
Hemicellulose removeda (%) 0.00 64.17 87.05 59.58
Total lignin content (%) 20.25 24.93 15.28 5.60
Lignin removed (%) 0 3.02 59.74 81.97
CRC after 72 h (%) 6.16 9.18 47.93 82.07
a Hemicellulose content was determined by difference of holocellulose and cellulose.

1120 J Chem Technol Biotechnol 82:1115–1121 (2007)

DOI: 10.1002/jctb

CONCLUSION
PAA pretreatment can effectively enhance the enzy-
matic digestibility of sugarcane bagasse under mild
conditions. It was shown that PAA charge, l/s ratio,
temperature, time and interactions between PAA
charge and l/s ratio, temperature and time, all had very
significant effects on the enzymatic conversion ratio of
cellulose. Optimum conditions for PAA pretreatment
of bagasse were obtained as follows: PAA charge of
50%, l/s ratio 6:1, temperature 80 ◦ C, time 2 h. Over
80% of the cellulose in bagasse treated under the
above conditions was converted to glucose by cellulase
of 20 FPU g−1 cellulose at 50 ◦ C for 72 h. Compared
with H2 SO4 and NaOH pretreatments under the same
conditions, PAA pretreatment obtained the highest
CRC. It was found that the enhancement of CRC by
PAA pretreatment was due to the removal of hemicel-
luloses and lignin, with removal of lignin being more
helpful. This study thus can serve as a step towards
further optimization of PAA pretreatment and under-
standing the mechanism of enhancement of enzymatic
digestibility.
REFERENCES
1 Martı́n C, Klinke HB and Thomsen AB, Wet oxidation as a pre-
treatment method for enhancing the enzymatic convertibility
of sugarcane bagasse. Enzyme Microb Technol 40:426–432
(2007).
2 Xu F, Sun JX, Liu CF and Sun RC, Comparative study of
alkali- and acidic organic solvent-soluble hemicellulosic
polysaccharides from sugarcane bagasse. Carbohyd Res
341:253–261 (2006).
3 Kaar WE, Gutierrez CV and Kinoshita CM, Steam explosion of
sugarcane bagasse as a pretreatment for conversion to ethanol.
Biomass Bioenergy 14:277–287 (1998).
4 Martin C, Galbe M, Nilvebrant NO and Jonsson LJ, Com-
parison of the fermentability of enzymatic hydrolyzates of
sugarcane bagasse pretreated by steam explosion using differ-
ent impregnating agents. Appl Biochem Biotechnol 98:699–716
(2002).
5 VanWalsum GP, Allen SG, Spencer MJ, et al, Conversion of
lignocellulosics pretreated with liquid hot water to ethanol.
Appl Biochem Biotechnol 57-8:157–170 (1996).
6 Silva SS, Matos ZR and Carvalho W, Effects of sulfuric acid
loading and residence time on the composition of sugarcane
bagasse hydrolysate and its use as a source of xylose for xylitol
bioproduction. Biotechnol Progr 21:1449–1452 (2005).
J Chem Technol Biotechnol 82:1115–1121 (2007)
DOI: 10.1002/jctb
Pretreatment of sugarcane bagasse for enzymatic hydrolysis
7 Playne MJ, Increased digestibility of bagasse by pretreat-
ment with aklalis and steam explosion. Biotechnol Bioeng
26:426–433 (1984).
8 Mohamed AF, Hossam MS and Ahmed IE, Effect of peracetic
acid, sodium hydroxide and phosphoric acid on cellulosic
materials as pretreatment for enzymatic hydrolysis. Enzyme
Microb Technol 5:421–424 (1983).
9 Schwald W, Brownell HH and Saddler J, Enzymatic hydrolysis
of steam treated aspen wood: Influence of partial hemicellu-
lose and lignin removal prior to pretreatment. J Wood Chem
Technol 8:543–560 (1988).
10 Mooney CA, Mansfield SD, Touhy MG and Saddler JN, The
effect of initial pore volume and lignin content on
the enzymatic hydrolysis of softwood. Bioresource. Technol
64:113–119 (1998).
11 Chang VS and Holtzapple MT, Fundamental factors affect-
ing biomass enzymatic reactivity. Appl Biochem Biotechnol
84–86:5–37 (2000).
12 Kim TH and Lee YY, Pretreatment and fractionation of corn
stover by ammonia recycle percolation process. Bioresource
Technol 96:2007–2013 (2005).
13 Kim TH, Kim JS, Sunwoo C and Lee YY, Pretreatment of corn
stover by aqueous ammonia. Bioresource Technol 90:39–47
(2003).
14 Teixeira LC, Linden JC and Schroeder HA, Simultaneous sac-
charification and cofermentation of peracetic acid–pretreated
biomass. Appl Biochem Biotechnol 84–86:111–127 (2000).
15 Taniguchi M, Tanaka M, Matsuno R and Kamikubo T, Evalu-
ation of chemical pretreatment for enzymatic solubilization of
rice straw. Eur J Appl Microbiol Biotechnol 14:35–39 (1982).
16 Ando S, Kakimoto T, Itoh K, Arai I, Kiyoto K and Hanai S,
Increased digestibility of cedar by pretreatment with peracetic
acid and steam explosion. Biotechnol Bioeng 34:802–804
(1988).
17 Teixeira LC, Linden JC and Schroeder HA, Optimizing per-
acetic acid pretreatment conditions for improved simultane-
ous saccharification and co-fermentation (SSCF) of sugar
cane bagasse to ethanol fuel. Renew Energy 16:1070–1073
(1999).
18 Gharpuray MM, Lee YH and Fan LT, Structural modification
of lignocellulosics by pretreatments to enhance enzymatic
hydrolysis. Biotechnol Bioeng 25:157–172 (1983).
19 Teixeira LC, Linden JC and Schroeder HA, Alkaline and per-
acetic acid pretreatments of biomass for ethanol production.
Appl Biochem Biotechnol 77–79:19–34 (1999).
20 Ghose TK, Measurement of cellulase activities. Pure Appl Chem
59:257–268 (1987).
21 Nada Abd-Alla MA, Ibrahem AA, Fahmy Y and Abou-
Yousef HE, Peroxyacetic acid pulping of bagasse. I. Two-
stage pulping. Cell Chem Technol 36:123–136 (2002).
22 Dean JA, Lange’s Handbook of Chemistry, 15th edn, Section 8.
McGraw-Hill, New York (1999).
1121