Enzyme and Microbial Technology 45 (2009) 8087

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Enzyme and Microbial Technology

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Characterization of enzymatic saccharication for acid-pretreated

lignocellulosic materials with different lignin composition
Gia-Luen Guo , Deng-Chieh Hsu, Wen-Hua Chen, Wei-Hsi Chen, Wen-Song Hwang
Cellulosic Ethanol Project, Institute of Nuclear Energy Research, 32546, Taiwan

a r t i c l e

i n f o

Article history:
Received 14 October 2008
Received in revised form 7 May 2009
Accepted 21 May 2009
Keywords:
Lignocellulosic materials
Lignin
Enzymatic digestibility
Cellulase

a b s t r a c t
The enzymatic saccharication of three different feedstocks, rice straw, bagasse and silvergrass, which had
been pretreated with different dilute acid concentrations, was studied to verify how enzymatic saccharication was affected by the lignin composition of the raw materials. There was a quantitatively inverse
correlation between lignin content and enzymatic digestibility after pretreatment with 1%, 2% and 4%
sulfuric acid. The lignin accounted for about 18.821.8% of pretreated rice straw, which was less than
the 23.126.5% of pretreated bagasse and the 21.524.1% of pretreated silvergrass. The maximum glucose
yield achieved, under an enzyme loading 6.5 FPU g1 DM for 72 h, was close to 0.8 g glucose/g glucan from
the enzymatic hydrolysis of the pretreated rice straw; this was twice that from bagasse and silvergrass. A
decrease in initial rate of glucose production was observed in all cases when the raw materials underwent
enzymatic saccharication with 4% sulfuric acid pretreatment. It is suggested that the higher acid concentration led to an inhibition of -glucosidase activity. Fourier transform infrared (FTIR) spectroscopy
further indicated the chemical properties of the rice straw and silvergrass become more hydrophilic after
pretreatment using 2% of sulfuric acid, but the pretreated bagasse tended to become more hydrophobic.
The hydrophilic nature of the pretreated solid residues may increase the inhibitive effects of lignin on the
cellulase and this could become very important for raw materials such as silvergrass that contain more
lignin.
2009 Elsevier Inc. All rights reserved.

1. Introduction
The Institute of Nuclear Energy Research (INER), a governmental
organization of Taiwan, is being supported to develop lignocellulosic ethanol production technology from local raw materials.
Agricultural wastes that are abundant in Taiwan, such as rice straw
and bagasse, were preferentially chosen as feedstocks. In addition,
in order to reduce the production costs of lignocellulosic ethanol,
the development of energy crops as feedstocks is also considered
essential. Thus, short-rotation perennial herbs, one example being
silvergrass, are believed to have potential as a raw material for
ethanol production.
The performance of lignocellulosic biomass-to-ethanol processes greatly depends upon the characteristics of feedstocks and
conversion technology including pretreatment, enzymatic saccharication and fermentation. Particularly, the operational integration
of pretreatment and enzymatic saccharication for the different
feedstocks is a crucial issue that may enhance the production ef-

Corresponding author at: Institute of Nuclear Energy Research, Atomic Energy

Committee, Executive Yuan, No. 1000, Wunhua Rd., Jiaan Village, Longtan Township,
Taoyuan County 32546, Taiwan, ROC. Tel.: +886 3 4711400 5039.
E-mail address: glguo@iner.gov.tw (G.-L. Guo).
0141-0229/$ see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2009.05.012

ciency of the fermentable sugars. From the engineering aspect,

dilute acid pretreatment has been considered to be a promising pretreatment technology for lignocellulosic ethanol production [1,2].
This pretreatment technology retains most of the glucan fraction in
the solid phase and the glucan can be further converted to glucose
by a follow-up enzymatic conversion.
However, the activity during the enzymatic conversion of the
glucan fraction in the pretreated solid residues is closely linked
to the structural properties of the raw material and thus dilute
acid pretreatment may be employed at various severity levels in
order to adjust these properties. Numerous obstacles may act to
reduce the performance of the next step, enzymatic saccharication. For example, cellulase must bind to the cellulose surface
before the hydrolytic reaction can occur and thus the amount of
assessable surface area in the pretreated solid residue can affect
the performance of the enzymes [3,4]. Furthermore, the cellulase
attack reaction is principally initiated on the amorphous portions
of cellulose. Thus, it is widely accepted that the crystallinity of the
pretreated solid residues may negatively affect the efciency of
enzyme contact with the cellulose [5]. In addition, the lignin component of the solid residue is thought to be a key factor affecting
enzymatic saccharication. The lignin polymer is considered to be
an enzyme adsorbent, which will diminish the amount of cellulase available for the hydrolytic reaction [6,7]. Finally, numerous

G.-L. Guo et al. / Enzyme and Microbial Technology 45 (2009) 8087

studies have indicated that dilute acid pretreatment seems to redistribute the chemical structure of lignin and this might be expected
to have a signicant inhibition on hydrolytic rate [8,9]. In reality,
the enzymatic saccharication of lignocellulosic materials is a very
complicated reaction and this is due both to the heterogeneous
structure of raw material and to the presence of non-glycans such
as lignin.
The study of enzymatic saccharication for lignocellulosic materials has been extensively carried out, and most of this research has
concentrated on evaluating the enzymatic digestibility of specic
feedstocks that have undergone various pretreatment technologies
[1013]. Less attention has been paid to investigating the effects of
the structural features of the various feedstocks on enzymatic saccharication [1416]. Notwithstanding this, even research in this
area has seldom examined the enzymatic digestibility of raw materials pretreated with a range of sulfuric acid concentrations.
Rice straw, bagasse and silvergrass are quite different externally and also in terms of their structure and in terms of their
chemical constituent. Moreover, these raw materials, which are
considered typical biomass materials, are found widely in Asia. The
objective of this study was the characterization of the enzymatic
saccharication of these three feedstocks, which have different
lignin compositions. These feedstocks were studied using pretreatment with various concentrations of sulfuric acid. Fourier transform
infrared (FTIR) spectroscopy was used to characterize the changes
to the chemical structure when these feedstocks were pretreated.
The effect of the structural features of the various pretreated solid
residues on enzymatic digestibility was also examined.
2. Materials and methods

81

ing incubator using 100 rpm and 2% dry matter (DM). Especially for the enzymatic
hydrolysis test of pretreated solid residue with 4% sulfuric acid, the 10N NaOH was
extra used to adjust the pH of reaction mixtures to the value as mentioned above.
The enzyme loading was set at 6.5 FPU g1 DM and used a commercial cellulase,
namely Spezyme CP (from Genencor International, USA) with a lter paper activity
of 60 FPU ml1 . The enzymatic hydrolysis experiments were performed at a constant
temperature of 50 C for 72 h; then samples from enzymatic saccharication were
taken at regular intervals and underwent sugar analysis.
2.4. Analysis
All samples were ltered through a 0.45 m lter and diluted appropriately with
eluent (4 mM H2 SO4 ). The quantitative analysis for the various sugars was performed
at 45 C using an HPLC system (Agilent 1200 series, Agilent Technologies) equipped
with a refractive index detector. The separation involved a polystyrene-sulfonic acid
column (Coregel-87H3 column with a length of 30 cm and an inner diameter 7.8 mm,
Transgenomic Technologies) at 65 C with 4 mM H2 SO4 as the eluent at a ow rate
of 1.0 ml min1 .
The constituent of raw materials and pretreated materials was determined by
a standard analysis procedure of biomass composition, which was modied by the
National Renewable Energy Laboratory (NREL) analytic methods [17]. The sugar content was determined based on monomer content measured after a two-step acid
hydrolysis procedure. The samples were treated with 72% (w/w) H2 SO4 at 30 C for
2 h in rst step. The reaction mixture was then diluted to 4% (w/w) H2 SO4 and autoclaved at 121 C for 1 h. The hydrolysis solution was ltered and analyzed for sugar
content and acetyl content by HPLC as described above and for acid soluble lignin
(ASL) from absorbance at 240 nm. The remaining solid residue was dried overnight
at 105 C and further placed in the mufe furnace at 575 25 C for least 24 6 h.
The weight of ash was recorded and used to calculate the content of acid insoluble
lignin (AIL).
In addition, FTIR analysis was performed on both the original feedstock before
pretreatment and the pretreated solid residue after acid hydrolysis with 2% sulfuric
acid. All solid samples were dried and then pressed into a disc with KBr. The discs
used in this work were thin enough to obey the BeerLambert law. Infrared spectra
were obtained using a BIO-RAD FTS-40 (Bio-Rad Laboratories, US) FTIR spectrometer
with a resolution of 0.5 cm1 in the range of 400 cm1 and 4000 cm1 .

2.1. Raw materials

3. Results and discussion

The bagasse was kindly supplied by the Taiwan Sugar Corporation (Tainan, Taiwan). Other raw materials, namely the silvergrass (Miscanthus oridulus) and rice
straw, which were used in this study, were collected from a eld near Longtan
(Taoyuan, Taiwan). These raw materials were air-dried and then milled to give a size
less than 0.5 cm. All of the materials were stored in plastic bags at room temperature
until further processed.
2.2. Dilute acid pretreatment
All of the raw materials were dried at 105 C overnight to ensure a low moisture
content prior to treatment. A biomass sample of 10 g on a dry basis was used for
each batch of dilute acid pretreatment experiments. The pretreatments were performed in an autoclave reactor using 60 ml of 1%, 2% or 4% sulfuric acid (w/w). The
samples were held at 130 C for 15 min. After allowing a few minutes for the temperature to drop, the cooled samples were then immediately separated into solid and
liquid fractions by ltration. All of the pretreated solid residues then underwent an
enzymatic hydrolysis test.
2.3. Enzymatic saccharication
The enzymatic saccharication of the pretreated solid residues after dilute acid
pretreatment was performed in 50 mM sodium acetate buffer with pH 5.0 on a shak-

3.1. Raw materials constituent

Table 1 summarizes the constituent of the raw materials and pretreated solid residues. Although glucose and xylose were the main
sugars present in all three feedstocks, the constituent of the rice
straw, other than these sugars, was different to that of bagasse and
silvergrass. The lignin accounted for 13.0% of the rice straw, which
was signicantly lower than that the 18.8% for bagasse and the 18.6%
for silvergrass. The content of acid soluble lignin was only 3.4% of
rice straw, which was lower than 8.1% of bagasse and 11.2% of silvergrass. Furthermore, the ash content of the rice straw was noticeably
higher than the other feedstocks because of the silica-richness of
this raw material. In addition, the glucan and lignin content for
pretreated solid residues was greater than that for raw materials,
as results of xylose release during dilute acid pretreatment. Interestingly, the content of acid soluble lignin was similar for these
pretreated solid residues. This indicated that greater amounts of

Table 1
Chemical constituent of the raw materials and pretreated solid residues.
Constituent

Dry matter (%)

Rice straw

Bagasse

Silvergrass

Original

1%

2%

4%

Original

1%

2%

4%

Original

1%

2%

4%

Glucose
Xylose
Arabinose
Acetyl groups

34.6
21.3
3.8
1.1

45.8
7.5
0.1

47.9
4.2
a

50.7
2.5

40.7
23.6
4.1
2.7

56.9
7.2
0.5

59.0
4.5

61.9
3.2

37.4
23.5
3.5
2.0

53.7
7.5

56.7
4.5

57.6
2.3

Lignin
Acid insoluble lignin
Acid soluble lignin

13.0
9.6
3.4

18.8
13.8
5.0

19.3
15.2
4.4

21.8
17.7
4.1

18.8
10.7
8.1

23.7
19.3
4.4

23.1
18.8
4.3

26.5
22.9
3.6

18.6
7.4
11.2

21.5
16.1
5.3

21.7
16.7
5.0

24.1
19.7
4.4

Ash

14.5

13.1

14.4

13.0

8.2

9.0

8.0

2.5

4.3

3.7

3.5

3.6

Not detected.

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G.-L. Guo et al. / Enzyme and Microbial Technology 45 (2009) 8087

Fig. 1. Time-course for glucose yield during enzymatic saccharication of (A) rice
straw; (B) bagasse; (C) silvergrass pretreated with various concentrations of sulfuric
acid () 1%; () 2%; () 4%.

acid soluble lignin were released for bagasse and silvergrass after
pretreatment. The lignin component of raw materials ought to play
a signicant role in the enzymatic saccharication performance
[18]. It implicated that the diversity of lignin composition in pretreated solid residues may affect enzymatic digestibility in addition
to pretreatment conditions employed.
3.2. Enzymatic digestibility
Sugar yields are often used as an indicator to measure the performance of enzymatic saccharication when lignocellulosic material
is used. Fig. 1 shows the variation of glucose yield from the enzymatic saccharication of rice straw, bagasse and silvergrass using
the different pretreatment severity conditions. The glucose yield
was determined by the ratio of the amount of glucose released during enzymatic saccharication relative to the potential maximum
for glucose, which was estimated from a glucan content of each
pretreated solid residues. The severity conditions for the dilute acid
pretreatment were 1%, 2% and 4% sulfuric acid (w/w) at 130 C with
a 15 min of reaction time.

The glucose released from the degradation of cellobiose was

progressively increased with the increase in hydrolytic time. The
highest glucose yield obtained at 72 h was different for rice straw,
bagasse and silvergrass. Fig. 1 indicates that the glucose yield from
enzymatic conversion of the pretreated rice straw was enhanced
when the sulfuric acid concentration was raised from 1% to 2%
and the maximum glucose yield achieved was close to 0.8 g glucose/g glucan; interestingly pretreated bagasse gave the maximum
glucose yields from enzymatic saccharication that were only half
of that from pretreated rice straw. However, the glucose yield was
found to reduce 30% for both of rice straw and bagasse when the
pretreatment was increased in severity to 4% sulfuric acid. This
implies that raw materials pretreated with such a high sulfuric acid
concentration may generate inhibitory effect that affect enzymatic
digestibility, which would result in a reduction in the sugar yield
from enzymatic saccharication. In addition, the glucose yield was
enhanced 1.5 times for silvergrass when sulfuric acid concentration
was increased from 12% to 4% during pretreatment. This result was
quite different with that for rice straw and bagasse.
Moreover, cellobiose is an important intermediate during enzymatic saccharication. An analysis of cellobiose conversion may
give a better insight in the enzymatic saccharication for various
raw materials. Fig. 2 shows the variation in cellobiose concentration
during enzymatic saccharication of pretreated rice straw, bagasse
and silvergrass. In general, the cellobiose concentration achieved
a maximum value at around 48 h, and after that the cellobiose
concentration decreased as hydrolytic time increased. The highest
cellobiose concentration accumulated from enzymatic saccharication of pretreated rice straw, as well as the maximum glucose
yield, was raised with the increase of sulfuric acid concentration
from 1% to 2% during pretreatment. Pretreated bagasse also gave
the highest cellobiose concentration from enzymatic saccharication that was only half of that from pretreated rice straw at the
same enzyme loading. Although the highest cellobiose concentration was observed for bagasse with pretreatment at 2% and 4% of
sulfuric acid, the corresponding glucose yield was not enhanced
under the same pretreatment severities. This indicates that the rate
of glucose production from degradation of cellobiose was diminished and this would obviously result in a decrease of glucose yield
at the end of the 72 h of hydrolytic reaction. Moreover, the highest cellobiose concentration from enzymatic saccharication was
observed for silvergrass with pretreatment at 4% of sulfuric acid.
The results were similar to that for glucose yield during enzymatic
saccharication. This indicates the enzymatic digestibility of silvergrass was obviously improved with dilute acid pretreatment at such
a high severity condition.
3.3. Effect of acid concentration during pretreatment
The initial rate in the release of sugar from enzymatic saccharication of the pretreated solid residues is shown in Fig. 3(A). The
initial rate of sugar release was determined by the amounts of sugar
release within 1 h at the beginning of enzymatic hydrolysis. Overall, the initial rate for sugar release from the pretreated rice straw
was higher than from bagasse and silvergrass. Furthermore, the rate
of cellobiose released from pretreated rice straw and bagasse was
enhanced when the sulfuric acid concentration was raised from 1%
to 2%, but the rate of glucose production was similar after the same
pretreatment. This contrasts with silvergrass where the rates for
1% and 2% sulfuric acid pretreatment were similar. The maximum
rate of cellobiose released was 2.5 g/l/h, 1.3 g/l/h and 1.1 g/l/h, during the enzymatic saccharication of pretreated rice straw, bagasse
and silvergrass, respectively. This was using 2% of sulfuric acid and
the corresponding rates for glucose production were about 1.6 g/l/h
for the pretreated rice straw, but only 0.9 g/l/h for other two feedstocks. Since the outcomes for the initial rates of sugar release were

G.-L. Guo et al. / Enzyme and Microbial Technology 45 (2009) 8087

83

Fig. 3. Effect of pretreatment in terms of increasing acid concentration on (A) initial

sugar-release rates from the enzymatic saccharication of the various raw materials;
(B) xylose and glucose yield from conversion of xylan and glucan by acid-catalyzed
reaction.

Fig. 2. Time-course for cellobiose concentration during enzymatic saccharication

of (A) rice straw; (B) bagasse; (C) silvergrass pretreated with various concentrations
of sulfuric acid () 1%; () 2%; () 4%.

comparable with that for sugar yield relative to each other, this suggests that enzymatic digestibility was mainly determined by initial
rate of sugar released.
The hydrolytic reaction initially occurs due to cellulase attack
on the cellulose surface. Thus, initial rate of sugar released was
probably determined by the number of enzyme attacking sites
for susceptible glucan in the pretreated solid residues. Fig. 3(B)
indicates that the yield in terms of the release of xylose by xylan
hydrolysis using rice straw was slightly increased from 0.71 to 0.75 g
xylose/g xylan when the sulfuric acid concentration was increased
from 1% to 2% during pretreatment. For the dilute acid pretreatment
of bagasse and silvergrass, the yield from the conversion of xylan
to xylose was generally in the range of 0.600.67 g xylose/g xylan
under the same pretreatment conditions. The removal of xylan may
result in greater exposure of the cellulose structure and thus an
increase in the fraction of susceptible glucan. This may diminish
unproductive interactions between the enzyme and the inert fraction of the pretreated solid residues. The enhancement in the initial
rate of cellobiose released for rice straw and bagasse suggests that
this may be due to the increased number of enzyme attacking sites
in solid residues when the sulfuric acid concentration is increased

from 1% to 2% during pretreatment. However, the effect of sulfuric

acid concentration was not apparent for the initial rate of glucose
production from enzymatic saccharication at the same pretreatment conditions. Besides, such an increase in the initial rate of
sugar release was not observed for silvergrass under similar circumstances.
Fig. 3(B) also shows the yield from the conversion of xylan to
xylose was greatly enhanced to about 0.9 g xylose/g xylan and above
when the sulfuric acid concentration was further increased from
2% to 4% during pretreatment. This indicates that the xylan was
almost all released by pretreatment at high severity condition. The
amounts of glucose released were also increased during the pretreatment at 4% of sulfuric acid. However, a decline in initial rate of
sugar released, especially for glucose production, was found for all
raw materials when they were pretreated with such a high sulfuric acid concentration. The phenomenon seemed to be common to
the three feedstocks despite the differences in their chemical constituent. The glucose released during pretreatment was proposed
from the acid hydrolysis of susceptible glucan. Therefore, the residual slow-reacting glucan may result in the increase of crystallinity
in pretreated solid residues and then for the reduction of initial
rate of cellobiose release from enzymatic saccharication. However, the level in the reduction of initial rate of glucose production
seemed to be particularly apparent when sulfuric acid concentration was at 4% during pretreatment, especially for bagasse and
silvergrass, which has been showed more lignin content in the constituent of pretreated solid residues. Since the enzyme responsible
for converting cellobiose to glucose during enzymatic saccharication is -glucosidase, a decrease in initial rate of glucose production
from enzymatic saccharication may be due to the fact that the glucosidase activity present was inhibited by the lignin content of
pretreated solid residue.
It is well accepted that -glucosidase, which does not have a
cellulose-binding domain, shows high adsorptability onto lignin

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G.-L. Guo et al. / Enzyme and Microbial Technology 45 (2009) 8087

Fig. 4. Relationship between lignin content in pretreated rice straw (, ); bagasse
(, ); silvergrass (, ) and initial sugar-release rates for cellobiose (open symbols)
and glucose (close symbols) during enzymatic saccharication.

[19]. Moreover, the deposition of lignin droplets has been observed

on the surface of pretreated solid residues after dilute acid pretreatment under high severity conditions [20]. The redistributed
lignin is thought to deposit on the cell wall matrix. Therefore,
take into account our work and other research ndings as mentioned above, the pretreatment with such a high acid concentration
may promote the formation of redistributed lignin deposit and
then hinder the interaction of cellulase with the cellulose structure. It was suggested to enhance the possibility in the adsorption
of -glucosidase onto lignin component. In addition, the constituent of pretreated solid residues (Table 1) also implicated that
the acid soluble lignin was released and suggested to generate
some inhibitors during dilute acid pretreatment. Thus, the presence of inhibitors may also affect cellulase activity in enzymatic
saccharication.

Fig. 5. The variation in cellobiose released rate (A), and glucose-release rate (B) during the process of enzymatic saccharication of the various raw materials pretreated
with 2% of sulfuric acid (relative value for initial of sugar-release rate was set to 1.0).

3.4. Effect of lignin content in the raw materials

Above results show that enzymatic digestibility was not
enhanced for bagasse and silvergrass when the sulfuric acid concentration was increased from 1% to 2% during pretreatment. Since
the lignin matrix of lignocellulosic materials may impose various
structural restrictions on cellulase and because unproductive binding may retard cellulase attack, it is suggested that the effect of
lignin on enzymatic digestibility became more crucial when a pretreated material has greater lignin content. This study found that
the lignin content of pretreated materials was negatively correlated
with the initial rate of sugar release from enzymatic saccharication
(Fig. 4); for initial rate of glucose production, it was about 1.45 g/l/h
for lignin content at 18.8% of pretreated rice straw, and shown an
about 3.4-fold enhancement for that at 26.5% of pretreated bagasse.
Moreover, a quantitative inverse relationship was also observed
between lignin content and the glucose yield at 72 h of hydrolytic
time.
This study revealed that the enzymatic digestibility of rice
straw was superior to that of bagasse and silvergrass due to lower
lignin content in pretreated solid residues. In fact, the enzymatic
digestibility of rice straw has been investigated in several studies. It
was shown that the glucose yield from enzymatic saccharication
of rice straw was commonly above 70% under various pretreatment technologies in use [2123]. The bagasse was shown diverse
results in enzymatic digestibility, which seems to be determined
by the degree of delignication in pretreated solid residues. The
sugar yield above 70% was only observed when the removal of
lignin in bagasse was greater than 60% by alkaline/peracetic acid
or alkaline/oxidative pretreatment [2426]. For dilute sulfuric acid
and wet oxidation pretreatment, the pretreated bagasse gave the

sugar yield below 50% from enzymatic saccharication [27,28]. In

addition, little research was focused on the enzymatic saccharication of silvergrass or Miscanthus sp. perennial herb [29,30]. It
was indicated that the glucose yield of enzymatic saccharication
was greatly improved when Miscanthus sp. was delignied by pretreatment [30]. Thus, above results suggest the lignin is the most
critical resistance factor against the enzymatic saccharication for
the feedstock with higher content of lignin, such as bagasse and
silvergrass. These agree with the results from enzymatic saccharication in this work.
Furthermore, the decrease in the rate of sugar released can
be used to describe the trend in terms of enzyme deactivation
during enzyme saccharication of the pretreated solid residues.
Fig. 5 illustrates the variations in the rate of cellobiose released and
glucose production during enzyme saccharication of rice straw,
bagasse and silvergrass after pretreatment with 2% sulfuric acid.
The decrease in sugar-release rate from the enzymatic saccharication was exponential. Interestingly, the decay in cellobiose-release
rate was predominantly depended on the lignin content of lignocellulosic materials. Pretreated bagasse and silvergrass have a
similar level of decrease in cellobiose-release rate and in both cases
the amount of cellobiose release were almost half that from pretreated rice straw. However, while the rate of glucose release were
both reduced by 50% for these two feedstocks, the amount of glucose released from the pretreated rice straw was a 1.5-fold and
2.1-fold higher for bagasse and silvergrass, respectively. This suggests that deactivation of enzyme responsible for the release of
glucose may be highly important to the enzymatic saccharication of pretreated silvergrass. Therefore, we suggest that differences

G.-L. Guo et al. / Enzyme and Microbial Technology 45 (2009) 8087

85

Table 2
The relative absorbance of the various bands due to the presence of the different chemical groups in the original feedstocks and the pretreated solid residues.
Group

Band

Relative adsorption
Rice straw

Bagasse

Silvergrass
Original

Pretreated

OH

3300

1.233

1.285

1.043

1.143

1.040

0.901

Phenolic OH
Phenolic OH
1ry OH
2nd OH

1375
1325
1044
1220

1.000
0.991
1.150

0.952
0.968
1.197

1.007
1.003
1.107

0.937
0.966
0.819
0.976

1.016
1.000
1.134

1.015
1.040
1.163
1.105

1.047

1.039

1.039

0.925

1.050

1.081

0.842
0.980
0.975
1.016

0.697
0.880
0.880
0.977

0.871
0.904
0.996
1.014

0.797
0.869
0.899
0.937

0.764
0.802
0.976
1.024

0.820
0.843
0.951
1.000

0.953

0.859

0.946

0.875

0.892

0.904

Mean value
Methoxy-OCH3

2835
2950
1460
1425

Mean value

Original

Pretreated

Original

Pretreated

EtherO

1120

1.166

1.285

1.107

1.106

1.134

1.196

EstersO CO

1150
1250

1.125
1.063

1.242
1.143

1.079
1.049

1.074
0.977

1.086
1.054

1.204
1.082

1.118

1.224

1.078

1.053

1.091

1.160

1.125
0.933

0.898
0.713
0.821

0.987
1.008

0.849
0.742
0.741

1.007
0.992

0.860
0.820
0.860

1.030

0.811

0.998

0.777

1.000

0.847

1.000
1.000
0.855

0.767
1.000
0.679

0.985
1.000
0.903

0.842
1.000
0.787

0.984
1.000
0.866

0.820
1.000
0.885

0.952

0.815

0.963

0.876

0.950

0.902

Mean value
Ketone C O
Aldehyde C O
Carboxyl acid HOC O

1650
1720
1710

Meanvalue
CH vibration of aromatic ring

Mean value

1600
1500
834

in chemical structure of the pretreated solid may affect the level

of decay in glucose production rate in addition to the lignin content.
3.5. Characterization of the chemical structure of the pretreated
solid residues
FTIR analysis was used to investigate the effect of the dilute
acid pretreatment on the chemical structure of lignin in rice straw,
bagasse and silvergrass. Table 2 shows how the relative absorbance
was dened as the ratio of the intensity of different lignin bands
and the intensity of CH vibration of aromatic ring at 1500 cm1
[31]. The absorbance by hydroxyl groups occurs in as a number of
different bands. In addition to the broad band at 3300 cm1 due
to hydroxyl groups, phenolic hydroxyl group bands are observable
at 1375 cm1 and 1325 cm1 . Absorbance by hydroxyl groups also
occurs in bands at 1050 cm1 and 1100 cm1 due to the primary
hydroxyl group and secondary hydroxyl group present in lignin,
respectively. The mean value for the relative absorbance of phenolic hydroxyl groups and primary hydroxyl groups was reduced
for pretreated rice straw and bagasse but increased for pretreated
silvergrass. Thus, the effect of dilute acid pretreatment on chemical properties of raw materials varied between the feedstocks. In
principle, the interactions that occur during the adsorption of cellulase onto lignin may involve the formation of hydrogen bonding,
non-specic hydrophobic interactions and electrostatic interactions [19]. The phenolic hydroxyl groups have been demonstrated to
be critical to the inhibitory effect of lignin on cellulase [32]. Therefore, pretreated silvergrass, which has a greater value for the relative
absorbance of phenolic hydroxyl groups, ought to have a higher
adsorption of cellulase onto lignin by means of hydrogen bond-

ing. This result implies that the inuence of unproductive binding

between lignin and cellulase maybe more signicant for the enzymatic saccharication of pretreated silvergrass. These results are
useful because they explain why pretreated silvergrass shows a
higher decrease in the glucose released rate, which has been mentioned earlier.
Moreover, other adsorption peaks occur at around 1425 cm1 ,
1460 cm1 , 2835 cm1 and 2950 cm1 and have been proposed
to be due to the methoxy groups (OCH3 ) that are present in
lignin [33]. These features are recognized as common functional
groups that are associated with the structure of lignin, such as
coniferyl alcohol and sinapyl alcohol [34]. A reduction in the relative
absorbance of these bands was observed for rice straw and bagasse
after dilute acid pretreatment. This suggests that hydrolysis of these
methoxy groups to phenolic hydroxyl groups may have occurred.
However, the dilute acid pretreatment caused the mean value for
the relative absorbance of the methoxy group bands to increase for
silvergrass. This may be attributed to acid-catalyzed degradation of
the lignin with the aliphatic chain of phenyl propane units being
affected. Although the methoxy group itself does not seem to have
an inhibitive effect on cellulase [32], an increase in the number of
methoxy groups to generate coniferyl lignin may act as a barrier to
limit the cellulose conversion [35].
In addition, the CO bond absorption peaks differed between the
various raw materials. The ether bonds (COC) can be detected
at 1120 cm1 and were present in all original feedstocks; however, two other peaks between 1100 cm1 and 1300 cm1 due to
ester bonds (O COC) were only found in raw bagasse and silvergrass. These peaks have been proposed as linkages associated with
the presence of lignin and lignincarbohydrate complexes. Interestingly, the relative absorbance of ether bonds and ester bonds

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G.-L. Guo et al. / Enzyme and Microbial Technology 45 (2009) 8087

were enhanced after dilute acid pretreatment of rice straw and

silvergrass. This may be attributed to the dehydration of alcohols
and it is suggested that this may result in the condensation of the
lignin, which would obviously be unfavorable to enzymatic saccharication because of the presence of lignin droplet precursors
[30].
The ketone group absorption peak involving intramolecular
hydrogen bonds was found at around 1650 cm1 in these raw materials. The FTIR analysis of bagasse and silvergrass further showed an
aldehyde group absorption peak was clearly present at 1720 cm1
[36]. This absorbance has been suggested to be due to acetyl groups
in the lignin or hemicellulose structure. The relative absorbance
of these two kinds of C O groups was reduced in the pretreated
solid residues. This reduction in the ketone and aldehyde groups
may be due to degradation of the aliphatic chain of phenyl propane
units in the lignin molecules. Finally, it has been conrmed that
adsorption peaks at 1710 cm1 are due to carboxyl acid groups
and were present in all pretreated solid residues. Overall, xylan
hydrolysis may also cause a decline in the absorbance of aldehyde
group.
When the above results are considered as a whole, the distributions of adsorption peaks for pretreated solid residues were
quite dissimilar between the different feedstocks and this suggests
that their structural features were also quite different. The results
indicated that the chemical properties of bagasse became more
hydrophobic after 2% dilute acid pretreatment, while those of pretreated rice straw and silvergrass became more hydrophilic. The
latter effect was due to an increase in hydroxyl groups, ether bonds
and ester bonds. The hydrophilic nature of the pretreated silvergrass may tend to increase the inhibitory effect of the higher lignin
content of this feedstock on cellulase and as a result the decay in
the glucose production rate for silvergrass was much more obvious
than for rice straw and bagasse.
4. Conclusions
The study demonstrated that enzymatic digestibility was significantly affected by the lignin content of the pretreated materials
and by the pretreatment employed in terms of various concentration of dilute acid. Using raw material pretreated with 1%, 2%
and 4% dilute sulfuric acid, the results show that a lower lignin
content in the pretreated materials resulted in a higher sugar
yield as well as an improved sugar released rate during enzymatic
saccharication. However, the decrease in initial rate of glucose
production indicated that an inhibition effect on -glucosidase
activity was present when the pretreatment severity was increased
to 4% sulfuric acid. From the viewpoint of enhanced enzymatic
digestibility, rice straw was the best raw material in terms of
enzymatic saccharication performance, perhaps due to the lower
lignin content when dilute acid pretreatment in used. The chemical structure of lignin may also affect the enzymatic digestibility
of the raw materials with greater lignin content, such as bagasse
and silvergrass. The silvergrass, after 2% sulfuric acid pretreatment, seemed to become more hydrophilic and this may have
increased further the inhibitory effect of lignin. Therefore, an analysis of the characteristics of feedstocks would seem to be essential
in order to clarify the connection between the functional groups
present on lignin and the inhibitory effect of lignin on cellulase.
Acknowledgements
The authors thank Dr. Gene Tang and Dr. Lee-Chung Men for their
helpful discussions. Mr. Sheng-Shieh Chen and Chen-Chien Lin are
also appreciated for their technical assistance.

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