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Bioresource Technology 97 (2006) 500–505

Efficient conversion of wheat straw wastes into biohydrogen gas


by cow dung compost
a,*
Yao-Ting Fan , Ya-Hui Zhang a, Shu-Fang Zhang a, Hong-Wei Hou a, Bao-Zeng Ren b

a
Department of Chemistry, Zhengzhou University, Zhengzhou 450052, PR China
b
College of Chemical Engineering, Zhengzhou University, Zhengzhou 450052, PR China

Received 7 August 2004; received in revised form 26 February 2005; accepted 26 February 2005
Available online 17 May 2005

Abstract

Efficient conversion of wheat straw wastes into biohydrogen gas by cow dung compost was reported for the first time. Batch tests
were carried out to analyze influences of several environmental factors on biohydrogen production from wheat straw wastes. The
performance of biohydrogen production using the raw wheat straw and HCl pretreated wheat straw was then compared in batch
fermentation tests. The maximum cumulative hydrogen yield of 68.1 ml H2/g TVS was observed at 126.5 h, the value is about 136-
fold as compared with that of raw wheat straw wastes. The maximum hydrogen production rate of 10.14 ml H2/g TVS h was
obtained by a modified Gompertz equation. The hydrogen content in the biogas was 52.0% and there was no significant methane
observed in this study. In addition, biodegradation characteristics of the substrate were also discussed. The experimental results
showed that the pretreatment of the substrate plays a key role in the conversion of the wheat straw wastes into biohydrogen by
the composts generating hydrogen.
Ó 2005 Elsevier Ltd. All rights reserved.

Keywords: Biohydrogen gas; Wheat straw wastes; Pretreatment; Natural anaerobic microorganisms; Fermentation

1. Introduction have been done by pure cultures of anaerobic bacteria


to study the conversion of carbohydrates (such as glu-
The microbial conversion of agricultural and indus- cose and starch) to hydrogen gas, e.g., Enterobacter
trial wastes and residues into hydrogen is attracting (Rachman et al., 1997), Aspergillus terreus (Emtiazi
increasing interest, this is due to that hydrogen is an et al., 2001) and Clostridium (Taguchi et al., 1994). Re-
excellent alternative energy candidate for the future cently, the considerable attention of research activity on
and producing only water instead of greenhouse gases fermentative hydrogen-production has been focused on
on burning. In addition, it is also the raw material for the conversion of biomass reproducible resources to
the synthesis of ammonia, alcohols, and aldehydes, as hydrogen by mixed cultures (Lay et al., 1999; Ginkel
well as the hydrogenations of petroleum, edible oils, et al., 2001; Fan et al., 2002). For example, Ueno
and coal. Hydrogen can be easily stored as a metal hy- et al. studied the hydrogen-production from an artificial
dride and its transmission through natural gas pipelines medium containing cellulose powder by thermophilic
would be more efficient than the transmission of electric- anaerobic microflora enriched from sludge compost
ity down power lines (Fan et al., 2004). Earlier studies (Ueno et al., 2001). Fan et al. have successfully used a
heat-shocked cow dung compost to convert a simulated
organic wastewater into hydrogen gas (Fan et al., 2003);
*
Corresponding author. Tel./fax: +86 371 7766017. Lay et al. (1999) studied the mixed bacterial cultures,
E-mail address: yt.fan@zzu.edu.cn (Y.-T. Fan). taken from a compost pile, a potato field and sludge,

0960-8524/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2005.02.049
Y.-T. Fan et al. / Bioresource Technology 97 (2006) 500–505 501

to generate hydrogen from sucrose or glucose in batch dilute NaOH or HCl solution. TVS value was deter-
wheat straw W ash
experiments. Fang et al. (2002) investigated a mesophilic mined as follows: TVS ¼ W dried
W dried wheat straw
 100%.
microbial community converting glucose into hydrogen.
However, the research of the conversion of the biomass 2.3. Experimental procedure
containing cellulose, such as wheat straw and corn stalk,
into biohydrogen is lacking. In general, it is hard to The batch experiments were performed with 250 ml
convert directly raw crop stalk wastes into biohydrogen serum vials as batch reactors filled to 150 ml comprising
gas by microbe anaerobic fermentation because of their the mixture of the composts, the pretreated wheat straw,
complex chemical composition, e.g., cellulose, hemi- and 3 ml of nutrient stock solution. These vials were
cellulose, lignin, protein, fat. gassed with nitrogen gas to remove oxygen and the
It is well known that cellulose in nature substrates, headspace of the reactors to keep the anaerobic environ-
such as wheat straw, is persistent in the environment ment. The bottles were incubated at 36 ± 1 °C and oper-
and remains as an environmental pollutant. Only in ated in an orbital shaker with a rotation speed of 90 rpm
China, the annual yield of wheat straw is 110 million to provide better contact among substrates. The com-
tons (Yang and Wang, 1999). Except that some of them post concentration of 80 g/l was maintained in the batch
were used to make paper or as feedstuff for livestock, reactors. Each liter of nutrient stock solution containing
most of them were set on fire or discarded as environ- 80 g of NH4HCO3, 12.4 g of KH2PO4; 0.1 g of
mental pollutants. Cellulosic materials can, however, MgSO4 Æ 7H2O; 0.01 g of NaCl; 0.01 g of Na2MoO4 Æ
be a valuable and vast renewable resource. 2H2O; 0.01 g of CaCl2 Æ 2H2O; 0.015 g of MnSO4 Æ
Therefore, in the present study, our research interest 7H2O; 0.0278 g of FeCl2, which was slightly modified
is to convert wheat straw wastes into hydrogen gas by from Lay et al. (1999). The volume of biogas was deter-
natural anaerobic microorganism. For this purpose, mined using glass syringes of 5–50 ml.
several environmental factors, such as pretreatment
conditions, initial pH and substrate concentration, 2.4. Analytical methods
were selected as target factors in conversion of raw
wheat straw wastes into biohydrogen gas by cow dung The hydrogen gas percentage was calculated by com-
compost. Maximum hydrogen production yield of paring the sample biogas with a standard of pure hydro-
68.1 ml H2/g TVS was observed from the pretreated gen using a gas chromatograph (GC, Agilent 4890D)
wheat straw wastes by microorganisms, the value is equipped with a thermal conductivity detector (TCD)
about 136-fold as compared with that of raw wheat and 6 feet stainless column packed with Porapak Q
straw wastes. The result is encouraging because of its (80/100 mesh). The operational temperatures of the
potential commercial and environmental benefits in the injection port, the oven and the detector were 100 °C,
future. 80 °C and 150 °C, respectively. Nitrogen was used as
the carrier gas at a flow rate of 20 ml/min. The concen-
trations of the volatile fatty acids (VFAs) and the alco-
2. Experimental methods hol were analyzed using another GC of the same model
with a flame ionization detector (FID) and a 8 feet stain-
2.1. Seed microflora less column packed with 10% PEG-20M and 2% H3PO4
(80/100 mesh). The temperatures of the injection port,
Hydrogen-producing microflora was taken from the detector and the oven were 220 °C, 240 °C and a
cow dung compost in the suburb of Zhengzhou City in programmed column temperature of 130–175 °C, res-
this study. Before it is used, cow dung compost was pectively. Nitrogen was the carrier gas at a flow rate
placed into a stainless steel pizza pan to a depth of of 20 ml/min. The pH values inside the digesters were
1 cm and broken up in the infrared oven for 2 h in order measured by a microcomputer pH-vision 6071.
to inhibit the bioactivity of hydrogen consumers and to
harvest high yield hydrogen-producing spore-forming
anaerobes. 3. Results and discussion

2.2. Chemical pretreatment of the substrate 3.1. Effects of pretreatment of substrate on hydrogen
production
The wheat straw wastes used as substrate were ob-
tained from the suburbs of Zhengzhou city. Before the Fig. 1 depicted the effects of the changes in the acid
substrate were degraded by microorganisms, the mixed concentration on hydrogen production yield at the fixed
solution containing wheat straw wastes and dilute HCl initial pH 6.5 and substrate concentration 15 g/l. As can
was boiled in a Teflon digestor by microwave heating be seen from Fig. 1, under the condition of microwave
or in a beakers, then neutralized to pH = 7 with either heating, the accumulative hydrogen yield increased
502 Y.-T. Fan et al. / Bioresource Technology 97 (2006) 500–505

25 25

hydrogen (ml H2/g TVS)


hydrogen (ml H2/g TVS)
Microwave
20 Ferv. 20

Cumulative
Cumulative

15
15
10

5 10
0 2 4 6 8 10 12
0 (a) Microwave heating time (min)
0 1 2 3 4 5 6
HCl concentration (%)

hydrogen (ml H2/g TVS)


20
Fig. 1. The effect of chemical pretreatment of wheat straw on 15

Cumulative
hydrogen-production potential. Other variables are constant at their
respective levels as follows: initial pH, 6.5; concentration of substrate, 10
15 g/l; microwave heating time, 4 min (or ferv. heating 30 min).
5

0
remarkably with the increase of HCl concentration in 0 10 20 30 40 50 60 70
the range of 0.5–2.0%. Maximum hydrogen yield of (b) Ferv. heating time (min)

22.9 ml H2/g TVS was observed in the test using the pre- Fig. 2. The effect of pretreatment time on hydrogen production yield.
treated substrate (2.0% HCl). Then, the hydrogen yield Other variables are constant at their respective levels as follows: initial
gradually declined as HCl concentration increased from pH, 6.5; concentration of substrate, 15 g/l; (a) 2.0% HCl concentration
22.9 ml H2/g TVS at HCl concentration of 2.0% to 6.0 by microwave heating and (b) 1.0% HCl concentration by ferv.
heating.
ml H2/g TVS at HCl concentration of 5.0%. Although
the higher acid concentration was in favor of the hydro-
lyzation of the substrate, but the high Cl anion concen- from 0.24% to 9.60%, and the cellulose and hemicel-
tration in the batch tests heavily inhibited the growth of lulose contents decreased from 22.5% and 21.5% to
hydrogen production bacteria (Wang et al., 1995). 15.40% and 12.88% for the acid pretreated wheat straw
Under the condition of ferv. heating, the change curve by the microwave heating of 8 min, respectively.
of hydrogen yield was similar to that by microwave Accordingly, we deduced that an increase in the hydro-
heating, except that the maximum hydrogen production gen yield possibly was due to an increase in the soluble
yield was only 17.9 ml H2/g TVS. However, the maximal sugar in the composition of the acid pretreated
hydrogen yield from the acid pretreated wheat straw by substrate. The results showed that the pretreatment of
microwave heating was higher than that by ferv. the substrate plays a key role in the conversion
heating. of wheat straw wastes into biohydrogen by micro-
In addition, both microwave heating and ferv. heat- organisms.
ing time also affected the hydrogen-production yield
for the acid pretreated substrate. Fig. 2 showed the ef- 3.2. Effect of initial pH value on hydrogen-production
fects of micro-wave heating (a) and ferv. heating time yield
(b) on hydrogen production yield. As shown in Fig. 2,
the hydrogen yield increased with the increase of the To investigate the effect of initial pH on start-up a
heating time, the maximal hydrogen yield of 22.5 ml hydrogen-producing reactor, the acid pretreated wheat
H2/g TVS and 18.0 ml H2/g TVS occurred at the micro- straw was then used for biohydrogen production at dif-
wave heating of 8 min and the ferv. heating of 50 min, ferent initial pH values from 4.0 to 9.0, the results are
respectively. The results showed that the microwave plotted in Fig. 3. As can be seen from Fig. 3, the initial
heating was a better method for hydrogen production pH values significantly affect the hydrogen-production
from the acid pretreated substrate as compared with yield of the substrate under the condition of the micro-
that by ferv. heating. wave heating, e.g., while the initial pH level rose from
As far as we know, the direct conversion of raw 4.0 to 7.0, the hydrogen yields increased from 0.01
wheat straw into hydrogen gas by anaerobic fermenta- ml H2/g TVS to 24.1 ml H2/g TVS, respectively. There-
tion is very difficult because of its complex polymer after, the hydrogen yield slightly decreased with in-
structure such as cellulose, hemi-cellulose and lignin, creased initial pH of the culture medium in the range
e.g., the maximal hydrogen yield was only 0.5 ml of initial pH 7.0–9.0. For example, while the initial pH
H2/g TVS by the cow dung compost in the test using of the culture medium was 9.0, the cumulative hydrogen
the raw wheat straw. In order to explain the experiment yield dropped to 22.7 ml H2/g TVS. Under the condition
phenomena, the composition of the wheat straw was of the ferv. heating, the change trend of the hydrogen
analyzed in the test. Compared with the raw wheat yield with initial pH value is similar to that by micro-
straw, we found that the soluble sugar content increased wave heating, except that the maximum hydrogen yield
Y.-T. Fan et al. / Bioresource Technology 97 (2006) 500–505 503

30 cumulative hydrogen yield decreased gradually as the


hydrogen(ml H2/g TVS) 25 concentration of the pretreated substrate increased,
e.g., while the concentration of the acid pretreated sub-
Cumulative

20
Microwave strate increased from 25 g/l, 30 g/l to 35 g/l, the hydro-
15 Ferv.
gen yield dropped from 68.1 ml H2/g TVS, 52.7 ml
10
H2/g TVS to 16.3 ml H2/g, 44.1 ml H2/g TVS, respec-
5 tively. However, maximum hydrogen yield of 68.1 ml
0 H2/g TVS occurred at the acid pretreated wheat straw
4 6 8 10
Initial pH
of 25 g/l by microorganisms under the conditions of
the microwave heating, the value is about 136-fold as
Fig. 3. The effect of varied pH value on hydrogen production yield. compared with that of raw wheat straw (Fig. 4).
Other variables are constant at their respective levels as follows:
The results showed that the change of the substrate
concentration of substrate, 15 g/l; (j) 2.0% HCl concentration by
microwave heating 8 min; (m) 1.0% HCl concentration by ferv. heating concentration obviously affected the hydrogen yield in
50 min. the test. Although an increase in the substrate concen-
tration could enhance the hydrogen yield under the con-
dition of the optimum hydrogen production, but the
of 10.3 ml H2/g TVS occurred at initial pH value 8. excessive substrate concentration would result in the
The results showed that the pH control could stimulate accumulation of volatile fatty acids (VFAs) and a fall
the microorganisms to produce hydrogen and would of pH value in the reactor, and even inhibited the
achieve the system having a maximum hydrogen yield, growth of hydrogen-producing bacteria. In addition,
but the activity of hydrogenase would be inhibited by the partial pressure of hydrogen in the batch reactor
low or high pH values in overall hydrogen fermentation rose with the increases in substrate concentration. While
(Fan et al., 2004; Lay et al., 1999). the partial pressure of hydrogen increased to a certain
level in the headspace of reactor, the microorganisms
3.3. Effect of substrate concentration on would switch to alcohol production, thus inhibiting
hydrogen-production yield hydrogen production (Fan et al., 2004).

The effects of the pretreated substrate concentration 3.4. Biodegradation characteristics of the substrate
versus cumulative hydrogen yield by the microorgan-
isms were presented in Fig. 4. As can be seen from In this paper, volatile fatty acids (VFAs) and alcohol
Fig. 4, the cumulative hydrogen yield increased remar- were selected as main by-products of the composts con-
kedly with increasing the concentration of the pretreated suming the substrate. Fig. 5 showed the changes of the
substrate, e.g., under the conditions of the microwave accumulative hydrogen yield (a), pH value (b), VFAs
heating and ferv. heating, while the concentration of (c) and alcohol (d) during the conversion of the pre-
the acid pretreated wheat straw rose from 5 g/l to 25 treated wheat straw wastes to biohydrogen by cow dung
g/l, 30 g/l, the cumulative hydrogen yield increased from compost.
13.8 ml H2/g TVS, 5.6 ml H2/g TVS to 68.1 ml H2/g As shown in Fig. 5(a), the hydrogen evolution began
TVS, 52.7 ml H2/g TVS, respectively. Thereafter, the to occur after 4 h of cultivation. The hydrogen yield
increased rapidly from 7.4 ml H2/g TVS at 16 h to
40.8 ml H2/g TVS at 31.5 h while the pH value decreased
from 5.95 to 5.0. The maximum hydrogen yield of
80
Microwave
68.1 ml H2/g TVS was observed at 126.5 h (Fig. 5(a)).
70 Ferv. The maximum hydrogen production rate of 10.14
hydrogen (ml H2/g TVS)

60 Crude ml H2/g TVS h was by a modified Gompertz equation


(Lay et al., 1999). The hydrogen content in the biogas
Cumulative

50
40 was 52.0% and there was no significant methane ob-
30
served in this study.
The pH value of the medium decreased from 7.0 to
20
4.7 with the progress of hydrogen evolution and wheat
10
straw decomposition, the optimum pH value of hydro-
0
5 15 25 35 45 gen-production appeared in the range of 5.0–4.7 (Fig.
Substrate concentrtion (g/l) 5(b)). Hydrogen production was accompanied with the
formation of volatile fatty acids (VFAs) throughout
Fig. 4. The effect of substrate concentration on hydrogen production
yield. Other variables are constant at their respective levels as follows: the wheat straw fermentation (Fig. 5(c) and (d)). During
initial pH, 7; (j) 2.0% HCl concentration by microwave heating 8 min; this period, acetate, propionate and butyrate reached
(m) 1.0% HCl concentration by ferv. heating 50 min. maximum yields of 1752, 234 and 1617 mg/l at 78.5 h,
504 Y.-T. Fan et al. / Bioresource Technology 97 (2006) 500–505

4. Conclusion

H2 (ml H2 /g TVS)
80
(a)
Commulative 60
40 The acid pretreatment of the substrate plays a key
20
role in efficient conversion of the wheat straw wastes
into biohydrogen gas by the cow dung composts. The
0
pretreated HCl concentration of 2.0% was optimal
8 under the microwave heating time of 8 min. The maxi-
(b)
7 mum hydrogen yield of 68.1 ml H2/g TVS was observed
at the fixed initial pH 7.0 and substrate concentration
pH

6
5
25 g/l, the value was about 136-fold higher than the
maximum value obtained for raw wheat straw wastes.
4
The maximum hydrogen production rate of 10.14
2000 ml H2/g TVS h was obtained by a modified Gompertz
Volatile acids (mg/l)

(c)
1600 equation. The hydrogen content in the biogas was
1200 Acetate 52.0% and there was no significant methane observed
Propionate
800 Butyrate in this study.
400 The hydrogen production was usually accompanied
0 with the formation of VFAs and alcohol while both of
600 them were the main by-products in the metabolism of
Alcohols (mg/l)

(d) hydrogen fermentation, during which acetate and buty-


400 Ethanol rate accounted for about 76–80% of VFAs.
Propanol
200 Butanol

0 Acknowledgements
0 50 100 150
Time (hours)
This work was supported by the China National Key
Fig. 5. Developments of cumulative hydrogen yield, pH value, VFAs
Basic Research Special Funds (No. 2003CB214500), the
and alcohols in the batch reactor during the conversion of the substrate
to biohydrogen under the pretreated condition of microwave heating. National Natural Science Foundation of China (Nos.
20171040 and 20471053) and the Energy and Technol-
ogy Program from Zhengzhou University.

respectively. The ethanol began to produce after 4 h cul-


tivation and increased up to 482 mg/l at 126.5 h. When References
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