international journal of hydrogen energy 35 (2010) 4041–4047

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Biohydrogen production from poplar leaves pretreated

by different methods using anaerobic mixed bacteria

Maojin Cui, Zhuliang Yuan, Xiaohua Zhi, Liling Wei, Jianquan Shen*
Beijing National Laboratory for Molecular Sciences (BNLMS), Laboratory of New Materials, Institute of Chemistry, Chinese Academy of
Sciences, Zhongguancun North First Street 2, Beijing 100190, PR China

article info abstract

Article history: Leaves are one of the main by-products of forestry. In this study, batch experiments were
Received 14 September 2009 carried out to convert poplar leaves pretreated by different methods into hydrogen using
Received in revised form anaerobic mixed bacteria at 35  C. The effects of acid (HCl), alkaline (NaOH) and enzymatic
5 February 2010 (Viscozyme L, a mixture of arabanase, cellulase, b-glucanase, hemicellulase and xylanase)
Accepted 6 February 2010 pretreatments on the saccharification of poplar leaves were studied. Furthermore, the
Available online 12 March 2010 effects of acid and enzymatic pretreatment on hydrogen production, together with their
corresponding degradation efficiencies for the total reducing sugar (TRS) and metabolites
Keywords: were compared. A maximum cumulative hydrogen yield of 44.92 mL/g-dry poplar leaves
Poplar leaves was achieved from substrate pretreated with 2% Vicozyme L, which was approximately
Pretreatment 3-fold greater than that in raw substrate and 1.34-fold greater than that from substrate
Anaerobic fermentation pretreated with 4% HCl. The results show that enzymatic pretreatment is an effective
Biohydrogen production method for enhancing the hydrogen yield from poplar leaves.
ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

1. Introduction attracted increasing attention. Many studies on biohydrogen

Fossil fuels such as oil, coal and natural gas are the main
energy sources in current society. However, the inevitable
depletion of fossil fuels, combined with concerns about their
impact on our environment has led to an interest in non-
polluting and alternative energy sources. In comparison with
fossil fuels, hydrogen is a promising alternative fuel of the
future as it is a clean energy carrier with a high energy-yield
(122 kJ/g) [1,2], the highest of all known fuels, and generates
no pollutants producing only water upon combustion.
Cellulosic biomass from agricultural and forest residue is
an abundantly renewable resource [3]. The annual yield of
biomass wastes is more than 0.7 billion tons in China alone [4].
Much cellulosic biomass, such as poplar leaves, however, is
burned or discarded as environmental pollutants. Therefore,
the production of hydrogen from cellulosic biomass has
from cellulosic biomass have been carried out [4–15]. The
conversion of raw cellulosic biomass into hydrogen directly by
microorganisms, however, is very difficult due to the
complicated polymer structure of hemicellulose and cellulose.
To enhance hydrogen yield, therefore, it is necessary to
pretreat the substrate [5]. Nowadays, the methods of pre-
treating cellulosic biomass mainly include steam explosion,
mechanical, thermal, acid, alkaline, ammonia and oxidative
treatment [4–12,16]. Acid hydrolysis is considered to be one of
the most effective methods of solubilizing hemicellulose,
hydrolysis products contain not only soluble sugars but also
a variety of by-products, such as phenol, furan and furfural
compounds, adversely inhibiting the capability of hydrogen-
producing bacteria to produce hydrogen [10,17]. In addition,
acid hydrolysis is commonly carried out at high temperature
and corrodes equipment [18]. In contrast, enzymatic

* Corresponding author. Tel.: þ86 10 62620903; fax: þ86 10 62559373.

E-mail address: jqshen@iccas.ac.cn (J. Shen).
0360-3199/$ – see front matter ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2010.02.035
4042 international journal of hydrogen energy 35 (2010) 4041–4047
hydrolysis is considered to be an effective method for pre-
treating cellulosic materials because it can be carried out
under mild conditions without corroding equipment [18].
In this study, we explored the feasibility of converting
poplar leaves into hydrogen with acid and enzymatic (Visco-
zyme L, a mixture of arabanase, cellulase, b-glucanase, hem-
icellulase and xylanase) pretreatments using anaerobic mixed
microbial cultures, aiming at enhancing hydrogen yield by the
pretreatment of substrate. For this purpose, the changes in the
total reducing sugar (TRS) with different pretreatment
methods and the effects of HCl and Viscozyme L concentra-
tions on hydrogen production and liquid metabolites were
studied by anaerobic fermentation in batch cultivation. The
results show that enzymatic pretreatment is better than acid
pretreatment in enhancing hydrogen yield from poplar leaves.
To the best of our knowledge, this is the first report of
hydrogen production from poplar leaves with pretreatment
using anaerobic mixed bacteria.
2. Materials and methods
2.1. Materials
The poplar leaves used in this study were collected in autumn
from a suburb of Beijing, China. They were dried in sunlight,
and were then comminuted to more than 20-mesh using
a comminutor and dried again in a thermoelectric oven for 3 h
at 105  C. All chemicals were analytical reagent grade except
where otherwise specified.
2.2. Seed microorganisms
The hydrogen-producing mixed cultures were enriched from
cracked cereal and identified to be dominated by Clostridium
pasteurianum [19]. The cultures were acclimated in
a completely stirred tank reactor (CSTR) in a chemostat for
approximately one month. The reactor was operated at 35  C,
an 8 h hydraulic retention time, and stirred by gas circulation
[20]. One liter of culture medium used to ferment contained
NH4HCO3, 3770 mg; K2HPO4, 125 mg; NaHCO3, 2000 mg;
CuSO4$5H2O, 5 mg; MgCl2$6H2O, 100 mg; MnSO4$4H2O, 15 mg;
FeSO4$7H2O, 25 mg; CoCl2$6H2O, 0.125 mg.
2.3. Pretreatment
2.3.1. Acid and alkaline pretreatment
1.0 g of dry poplar leaves were mixed with 20 mL of dilute HCl
(or NaOH) aqueous solution at different concentrations (0.5%,
1%, 2%, 4% and 8% (w/v), respectively) (5% (w/v) solids loading)
and boiled for 30 min in serum vials. The mixture was then
neutralized to pH 7.0 by the addition of dilute NaOH (or HCl)
aqueous solution at different concentrations (0.5%, 1%, 2%, 4%
and 8% (w/v), respectively).
2.3.2. Enzymatic pretreatment
Viscozyme L (VL) was purchased from Novozymes (China)
Biotechnology Co., Ltd. (batch number KTN02161). VL is a mixture
of many enzymes, including arabanase, cellulase, b-glucanase,
hemicellulase and xylanase. The optimal working conditions for
VL are pH 3.3–5.5 and temperature 25–55  C.
1.0 g of dry poplar leaves were mixed with 20 mL of VL
aqueous solution at different concentrations ([VL]), pH, enzy-
molysis time (t) and temperatures (T ), respectively (5% (w/v)
solids loading). Four series of experiments were carried out:
Series 1 investigated the effect of VL concentration (0.25–4%,
v/v) on the saccharification of poplar leaves; Series 2 investi-
gated the effect of pH (3.0–7.0) on the saccharification of
poplar leaves, and a buffer containing 0.1 mol/L citric acid and
0.1 mol/L trisodium citrate was used to adjust the pH; Series 3
investigated the effect of enzymolysis time (1–5 h) on the
saccharification of poplar leaves; Series 4 investigated the
effect of enzymolysis temperature (35–55  C) on the sacchar-
ification of poplar leaves.
2.4. Experimental procedures
Batch experiments were carried out to produce hydrogen by
anaerobic fermentation in 120 mL serum vials using poplar
leaves pretreated with HCl and VL as substrate. The total
working volume was 80 mL (approximately 1.25% (w/v) solids
loading) in each case. (1) Acid pretreatment: approximately
40 mL of mixture after pretreatment (20 mL of dilute HCl used
to pretreat substrate and approximately 20 mL of dilute NaOH
used to adjust pH), 20 mL inoculum, 10 mL nutrient solution
(1 L contained NH4HCO3, 30 160 mg; K2HPO4, 1000 mg;
NaHCO3, 16 000 mg; CuSO4$5H2O, 40 mg; MgCl2$6H2O, 800 mg;
MnSO4$4H2O, 120 mg; FeSO4$7H2O, 200 mg; CoCl2$6H2O, 1 mg),
and approximately 10 mL distilled water; (2) enzymatic
pretreatment: 20 mL of mixture after pretreatment, 20 mL
inoculum and 40 mL nutrient solution (1 L contained
NH4HCO3, 7540 mg; K2HPO4, 250 mg; NaHCO3, 4000 mg;
CuSO4$5H2O, 10 mg; MgCl2$6H2O, 200 mg; MnSO4$4H2O, 30 mg;
FeSO4$7H2O, 50 mg; CoCl2$6H2O, 0.25 mg). The air was
removed from the solution and the headspace by argon gas for
3 min before the vials were capped with rubber septum
stoppers and placed in a reciprocal shaker (120 rpm). The
batch experiments were performed at 35  C in the dark. Each
experimental condition was carried out in triplicate.
2.5. Analytical methods
The hydrogen content was determined by a gas chromato-
graph (Techcomp. Co., China, 7890II) equipped with a thermal
conductivity detector (TCD) and a 2-m stainless steel column
packed with Porapak Q (80–100 mesh). The operating
temperatures of the injection port, the oven and the detector
were set at 70, 50 and 70  C, respectively. Argon was used as
the carrier gas at a flow rate of 30 mL/min. At each time
interval, the total volume of biogas production was measured
by a plunger displacement method using appropriately sized
glass syringes, ranging from 10 to 100 mL [21]. The cumulative
hydrogen volume was calculated by Eq. (1) [22],



VH;i ¼ VH;i1 þ CH;i VG;i  VG;i1 þ VH;0 CH;i  CH;i1 (1)
where VH, i and VH, i1 are cumulative hydrogen volumes at the
current (i) and previous (i  1) time intervals, VG, i and VG, i1
are the total biogas volumes in the current (i) and previous
(i  1) time intervals, CH, i and CH, i1 are the fraction of
 international journal of hydrogen energy 35 (2010) 4041–4047 4043

hydrogen in the headspace of the bottle at the current (i) and

previous (i  1) time intervals and VH,0 is the total volume of 30 HCl

Content of the total reducing sugar (%)

NaOH
headspace in the bottle. Detection of the alcohols and volatile
fatty acids (VFAs, C2–C5) was measured by a gas chromato- 25

graph using a flame ionization detector (FID) and a 2-m glass

column packed with Unisole F-200 (30–60 mesh). The 20

temperatures of the injection port, the oven and the detector

were set at 200, 165 and 200  C, respectively. The carrier gas 15
was also argon at a flow rate of 30 mL/min. The concentration
of the total reducing sugar was determined by the phenol– 10
sulfuric acid method using glucose as a standard [23].
5
2.6. Model analysis
0
The cumulative hydrogen production during the batch UP 0.5% 1% 2% 4% 8%
experiments followed the modified Gompertz equation HCl and NaOH concentrations (w/v)
[24,25]:
Fig. 1 – Effects of HCl and NaOH concentrations on the
  
Rm e saccharification of poplar leaves. UP means
H ¼ P exp ðl  tÞ þ 1 (2)
P unpretreatment.

Where H is the cumulative hydrogen production (mL), P is

hydrogen production potential (mL), Rm is the maximum
hydrogen production rate (mL/h), e is 2.72, l is the lag-phase
time (h), and t is the incubation time (h). The corresponding
values of P, Rm and l for each batch were estimated using
Origin 7.5, which is a scientific graphing and data analysis
software.
3. Results and discussion
3.1. Effect of pretreatment on the saccharification
of poplar leaves
of poplar leaves. Therefore, the effects of VL concentration, pH,
enzymolysis time and temperature on the contents of the TRS
from 1.0 g of dry poplar leaves were studied. The results are
shown in Table 1. The TRS content continuously increased
when the VL concentration increased from 0.25% to 1% but the
increment was small when the VL concentration increased from
1% to 4%. Therefore, 1% VL was used to investigate the effect of
pH on the saccharification of poplar leaves. The TRS content
continuously decreased with increasing pH. The optimal pH was
in the range of 3.0–4.0. Therefore, the effect of enzymolysis time
on the saccharification of poplar leaves was studied at pH 4.0.

3.1.1. Effects of HCl and NaOH pretreatment

on the saccharification of poplar leaves Table 1 – Effects of VL concentration, pH, enzymolysis
Acid pretreatment mainly converts hemicellulose in the time and enzymolysis temperature on the
saccharification of poplar leaves.
cellulosic biomass into xylose [26], which is the main
component of hemicellulose [27]. HCl was less active than [VL] (v/v) pH t (h) T ( C) CS (%)
sulfuric acid in the degradation of xylose, which is the feed- 0.25% 4.0 3 50 18.51
stock for producing hydrogen by bacteria [28]. Therefore, HCl 0.5% 20.27
was used to pretreat the substrate. The TRS contents from 1% 25.07
1.0 g of dry poplar leaves after HCl and NaOH pretreatment 2% 25.33
were analyzed and are depicted in Fig. 1. As shown in Fig. 1, 4% 26.03

the TRS content continuously increased with increasing HCl 1% 3.0 3 50 25.75
and NaOH concentrations. When comparing the contents of 4.0 25.07
the TRS from substrate pretreated with 4% and 8% HCl (or 5.0 23.01
6.0 22.51
NaOH), the increment was found to be small. The amounts of
7.0 22.39
TRSs with 4% HCl and 4% NaOH pretreatment were approxi-
mately 2.33-fold and 1.66-fold greater compared with that in 1% 4.0 1 50 22.34
2 23.92
the raw substrate. It was noted that the TRS content with HCl
3 25.07
pretreatment was always higher than that with NaOH
4 25.25
pretreatment at the same HCl and NaOH concentrations. 5 26.00
These results indicate that HCl pretreatment was superior to
1% 4.0 3 35 18.93
NaOH pretreatment, consistent with previous studies [4,5].
40 20.03
45 21.79
3.1.2. Effect of enzymatic pretreatment 50 25.07
on the saccharification of poplar leaves 55 24.63
Enzyme concentration, pH value, enzymolysis time and
‘CS’ means the content of the total reducing sugar.
temperature are the main factors that affect the saccharification
4044 international journal of hydrogen energy 35 (2010) 4041–4047

The TRS content continuously increased with the increasing

Table 2 – Hydrogen production characteristics at different
enzymolysis time. The increment, however, was small when HCl concentrations.
the enzymolysis time exceeded 3 h. The effect of enzymolysis
HCl (w/v) P (mL) Rm (mL/h) l (h) R2
temperature on the saccharification of poplar leaves was also
studied using an enzymolysis time of 3 h. The TRS content 0 15.04 1.25 3.60 0.9992
initially increased then decreased with increasing enzymolysis 0.5% 27.56 2.74 11.90 0.9994
1% 28.44 1.73 10.00 0.9993
temperature, and the optimal temperature was 50  C. According
2% 29.14 1.53 9.40 0.9979
to the above results, the optimal conditions for saccharification
4% 33.45 1.18 8.76 0.9937
of poplar leaves were [VL] ¼ 1%, pH ¼ 3.0–4.0, t ¼ 3 h and T ¼ 50  C.
To summarize, the TRS content from pretreated poplar P is hydrogen production potential (mL).
Rm is the maximum hydrogen production rate (mL/h).
leaves was higher than that from raw poplar leaves, indicating
l is the lag-phase time (h).
that pretreatment is very necessary for further saccharifica-
R2 is correlation coefficient.
tion of poplar leaves. The TRS contents after enzymatic
pretreatment were lower than those after HCl pretreatment
but higher than those after NaOH pretreatment. Therefore, production rate continuously decreased with increasing HCl
poplar leaves pretreated by HCl and VL were used as concentration (0.5–4%), possibly because HCl pretreatment
a substrate in the fermentation to produce hydrogen. produced some inhibitors such as furfural that inhibited the
specific hydrogen production rate of hydrogen-producing
3.2. Effect of pretreatment on hydrogen production bacteria [29]. The maximum specific hydrogen production
rate of 2.74 mL/h was observed at 0.5% HCl. The shortest
3.2.1. Effect of HCl pretreatment on hydrogen production duration of 3.60 h occurred with the raw substrate, because
The effects of HCl concentration on the cumulative hydrogen the microorganisms used here were acclimated to raw poplar
volumes are presented in Fig. 2. 1.0 g of dry poplar leaves leaves. The duration continuously decreased with increasing
pretreated with different HCl concentrations were used as the HCl concentration (0.5–4%).
substrate to produce hydrogen at an initial pH of 7.0. It can be
seen from Fig. 2 that the cumulative hydrogen volume from 3.2.2. Effect of enzymatic pretreatment on hydrogen
raw poplar leaves was 15.04 mL but that the cumulative
production
hydrogen volumes continued to increase (27.56–33.45 mL) 1.0 g of dry poplar leaves pretreated with various VL concen-
when the HCl concentration increased (0.5–4%). The trations at optimal pretreatment conditions (pH ¼ 4.0, t ¼ 3 h,
maximum hydrogen volume of 33.45 mL was observed at 4% T ¼ 50  C) were used as substrate to produce hydrogen at an
HCl, which was 2.22-fold higher than that from the raw initial pH of 7.0. The effects of VL concentration on the
substrate. cumulative hydrogen volumes are presented in Fig. 3. As
To further investigate the effect of HCl concentration on shown in Fig. 3, the cumulative hydrogen volumes initially
hydrogen production, the data in Fig. 2 were simulated using increased then decreased with increasing VL concentration,
Eq. (2) and the hydrogen production characteristics are shown suggesting that excessive VL was unfavorable to hydrogen
in Table 2. According to the data in Table 2, all R2 values were production. The maximum hydrogen volume of 44.92 mL was
more than 0.990, indicating that the fitted curves matched observed at 2%, which was 2.99-fold higher than that from the
well with the experimental points. The specific hydrogen

50
UP
35
UP 0.25%
0.5% 0.5%
30 1% 1%
40
2%
2%
Cumulative hydrogen (mL)
Cumulative hydrogen (mL)

4%
25
4%
30
20

15 20

10
10
5

0 0
0 20 40 60 80 0 10 20 30 40 50 60 70
Time (hour) Time (hour)

Fig. 2 – Cumulative hydrogen volumes from 1.0 g of dry Fig. 3 – Cumulative hydrogen volumes from 1.0 g of dry
poplar leaves pretreated by different HCl concentrations poplar leaves pretreated by different VL concentrations
versus corresponding fermentation time. The operation versus corresponding fermentation time. The operation
was at 35 8C and initial pH 7.0. was at 35 8C and initial pH 7.0.
 international journal of hydrogen energy 35 (2010) 4041–4047 4045

the course of hydrogen production, suggesting that there

Table 3 – Hydrogen production characteristics at different
VL concentrations. were no methanogens in the anaerobic mixed bacteria used
here. The effects of HCl and VL concentrations on the sugar
VL (v/v) P (mL) Rm (mL/h) l (h) R2
degradation efficiency, H2 contents, H2 yields, and liquid
0 15.04 1.25 3.60 0.9992 metabolites are shown in Table 5. The sugar degradation
0.25% 27.32 1.85 8.04 0.9922 efficiency exceeded 92%, indicating that most of the sugar
0.5% 33.13 2.14 7.71 0.9938
was consumed by the bacteria, and the changes were small
1% 36.90 2.17 7.88 0.9957
with increasing HCl and VL concentrations. Hydrogen
2% 44.92 1.97 7.98 0.9922
4% 35.47 1.32 7.13 0.9863 contents in the biogas varied from 28.80% to 32.51% with
a change in HCl concentration from 0.5% to 4% and the
divergence was also small. The maximum of 32.51% was
observed at 1% HCl. Hydrogen contents in the biogas varied
raw substrate and better than that from pretreated substrate
from 26.92% to 46.08% with a change in VL concentration
with 4% HCl, indicating that enzymatic pretreatment was
from 0.25% to 4% and the maximum of 46.08% was
better than HCl pretreatment.
observed at 0.5% VL.
To further investigate the effect of VL concentration on
The liquid product analysis showed that the metabolites
hydrogen production, the data in Fig. 3 were simulated using
found after fermentation were ethanol, acetic acid, propionic
Eq. (2) and the hydrogen production characteristics are shown
acid and butyric acid. It should be noted that high yields of
in Table 3. According to the data in Table 3, all R2 values were
ethanol and VFAs were obtained from the raw poplar leaves
more than 0.986, indicating that the fitted curves matched
but a relatively low cumulative hydrogen yield (15.04 mL/g-dry
well with the experimental points. The specific hydrogen
poplar leaves) was produced. This is possibly because some of
production rate initially increased and then decreased with
the hemicellulose and cellulose in raw poplar leaves was
increasing VL concentration, suggesting that excessive VL was
consumed in the production of ethanol and VFAs [26]. When
unfavorable to enhance the specific hydrogen production rate.
the HCl concentration increased from 0.5% to 4%, the yields of
The maximum specific hydrogen production rate of 2.17 mL/h
acetic acid and propionic acid decreased gradually, while
was observed at 1%. The shortest duration of 3.60 h occurred
ethanol and n-butyric acid initially increased then decreased.
with the raw substrate, possibly due to the same reason given
A maximum of four liquid metabolites appeared at 2% VL. The
above. The changes in duration with poplar leaves pretreated
possible metabolic pathway of soluble sugar (mainly glucose)
with different VL concentrations were small with increasing
to produce hydrogen at the maximum hydrogen yield (2% VL
VL concentration (0.25–4%).
pretreatment) is shown as Eq. (3):
To summarize, the cumulative hydrogen volume from
poplar leaves with pretreatment was higher than that from
raw poplar leaves, indicating that pretreatment is necessary
C6H12O6 / 1.315H2 þ 1.873CO2 þ 0.086CH3CH2OH
for increasing hydrogen production. The result was consistent
þ 0.552CH3COOH þ 0.125CH3CH2COOH
with previous studies, which are summarized in Table 4. As
þ 0.074CH3CH2CH2COOH (3)
shown in Table 4, the maximum hydrogen yields from pre-
treated substrates were much higher than hydrogen yields
As shown in Eq. (3), the metabolic pathway was mainly
from raw substrates. The TRS content from the pretreated
acetic acid fermentation, and the conversion efficiency of H
substrate with 4% HCl was higher than that from the pre-
element in glucose into H2 was 22%.
treated substrate with 2% VL but the former cumulative
The mechanism of hydrogen production from glucose and
hydrogen volume was lower than the latter. This is possibly
xylose by bacterial fermentation has been reported previously
because HCl pretreatment produced some inhibitors [29],
[30–32]. Theoretically the metabolic pathways of the four
inhibiting the hydrogen-producing bacteria from producing
liquid products (ethanol, acetic acid, propionic acid and n-
hydrogen using sugar.
butyric acid) from xylose and glucose are as follows:

C5H10O5 / 1.67CH3CH2OH þ 1.67CO2 (4)

3.3. Effect of pretreatment on metabolites

Gas product analyses showed that only H2 and CO2 were

C5H10O5 þ 1.67H2O / 1.67CH3COOH þ 3.33H2 þ 1.67CO2 (5)
present in the biogas, without any detectable CH4 during

Table 4 – Biohydrogen production from various cellulosic biomasses with different pretreatment methods.
Feedstock Pretreatment method H2 yield from Maximum H2 yield from Reference
raw material pretreated substrate

Poplar leaves Viscozyme L 15.04 mL/g 44.92 mL/g This study

Beer lees HCl 3.16 mL/g 53.03 mL/g [26]
Cornstalk HCl 3.16 mL/g-TVS 149.69 mL/g-TVS [4]
Wheat straw HCl-Microwave 0.5 mL/g-TVS 68.1 mL/g-TVS [6]
Corn stalk Biopretreatment 20 mL/g-TS 176 mL/g-TS [11]
4046 international journal of hydrogen energy 35 (2010) 4041–4047

Table 5 – Effects of HCl and VL concentrations on the degradation efficiency of total reducing sugar, H2 contents, H2 yields
and yields of ethanol and VFAs after fermentation.
DESa H2 contentb H2 yield Ethanol Acetic acid Propionic acid n-Butyric acid
(%) (%) (mL/g-S) (mg/g-S)c (mg/g-S) (mg/g-S) (mg/g-S)

UP 0 93.28 26.89 15.04 4.20 26.10 9.28 6.06

HCl (w/v) 0.5% 94.98 30.09 27.56 2.27 17.31 5.76 2.21
1% 95.81 32.51 28.44 4.02 16.01 5.16 2.81
2% 94.68 28.80 29.14 5.49 15.96 2.64 1.38
4% 92.44 30.06 33.45 4.87 9.15 2.17 0.94

VL (v/v) 0.25% 95.48 39.25 27.32 3.41 29.72 5.45 2.93

0.5% 96.03 46.08 33.13 4.31 36.01 5.30 3.00
1% 96.03 40.37 36.90 4.30 34.80 6.73 4.40
2% 95.76 41.25 44.92 5.31 44.68 12.42 8.78
4% 95.82 26.92 35.47 4.18 35.80 9.63 5.01

a DES means degradation efficiency of total reducing sugar.

b H2 content means percentage of the total cumulative hydrogen produced to total biogas.
c (mg/g-S) means (mg/g-dry poplar leaves).

pretreatment. Therefore, enzymatic pretreatment is an ideal

C5H10O5 þ 1.67H2 / 1.67CH3CH2COOH þ 1.67H2O (6)
method for increasing the hydrogen yield from poplar leaves
by anaerobic fermentation.

C5H10O5 / 0.83CH3(CH2)2COOH þ 1.67H2 þ 1.67CO2 (7)

Acknowledgments
C6H12O6 / 2CH3CH2OH þ 2CO2 (8)
The authors would like to thank the Chinese Academy of
Sciences for financial support (Item No. KJCX2-YW-H21).
C6H12O6þ2H2O / 2CH3COOH þ 4H2 þ 2CO2 (9)
references

C6H12O6 þ 2H2 / 2CH3CH2COOH þ 2H2O (10)

C6H12O6 / CH3(CH2)2COOH þ 2H2 þ 2CO2 (11)
According to Eqs. (4)–(11), the production of ethanol has no
influence on the production of hydrogen whereas the production
of acetic and n-butyric acid favors the production of hydrogen. In
contrast, the production of propionic acid resulted in less
hydrogen production. According to Eq. (3), it was mainly acetic
acid fermentation, so it favored the production of hydrogen.
4. Conclusions
In this work, three different methods, acid (HCl), alkaline
(NaOH) and enzymatic treatment, were used to pretreat
poplar leaves. The TRS content in pretreated substrate was
higher than that in raw substrate, indicating that pretreat-
ment is required for further saccharification of substrate. The
TRS contents after enzymatic pretreatment were lower than
those after HCl pretreatment but higher than those after
NaOH pretreatment. Nevertheless, the maximum cumulative
hydrogen yield (44.92 mL/g-dry poplar leaves) from substrate
pretreated by 2% VL was higher than that from substrate
pretreated by 4% HCl (33.45 mL/g-dry poplar leaves), indi-
cating that enzymatic pretreatment was better than HCl
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