Bioresource Technology 97 (2006) 500–505

Efficient conversion of wheat straw wastes into biohydrogen gasby cow dung compost

Yao-Ting Fan a,*, 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 Chinab 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.

� 2005 Elsevier Ltd. All rights reserved.

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

1. Introduction

The microbial conversion of agricultural and indus-

trial wastes and residues into hydrogen is attracting

increasing interest, this is due to that hydrogen is anexcellent alternative energy candidate for the future

and producing only water instead of greenhouse gases

on burning. In addition, it is also the raw material for

the synthesis of ammonia, alcohols, and aldehydes, as

well as the hydrogenations of petroleum, edible oils,

and coal. Hydrogen can be easily stored as a metal hy-

dride and its transmission through natural gas pipelines

would be more efficient than the transmission of electric-ity down power lines (Fan et al., 2004). Earlier studies

0960-8524/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2005.02.049

* Corresponding author. Tel./fax: +86 371 7766017.

E-mail address: yt.fan@zzu.edu.cn (Y.-T. Fan).

have been done by pure cultures of anaerobic bacteria

to study the conversion of carbohydrates (such as glu-

cose and starch) to hydrogen gas, e.g., Enterobacter

(Rachman et al., 1997), Aspergillus terreus (Emtiazi

et al., 2001) and Clostridium (Taguchi et al., 1994). Re-cently, the considerable attention of research activity on

fermentative hydrogen-production has been focused on

the conversion of biomass reproducible resources to

hydrogen by mixed cultures (Lay et al., 1999; Ginkel

et al., 2001; Fan et al., 2002). For example, Ueno

et al. studied the hydrogen-production from an artificial

medium containing cellulose powder by thermophilic

anaerobic microflora enriched from sludge compost(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);

Lay et al. (1999) studied the mixed bacterial cultures,

taken from a compost pile, a potato field and sludge,

Y.-T. Fan et al. / Bioresource Technology 97 (2006) 500–505 501

to generate hydrogen from sucrose or glucose in batch

experiments. Fang et al. (2002) investigated a mesophilic

microbial community converting glucose into hydrogen.

However, the research of the conversion of the biomass

containing cellulose, such as wheat straw and corn stalk,

into biohydrogen is lacking. In general, it is hard toconvert directly raw crop stalk wastes into biohydrogen

gas by microbe anaerobic fermentation because of their

complex chemical composition, e.g., cellulose, hemi-

cellulose, lignin, protein, fat.

It is well known that cellulose in nature substrates,

such as wheat straw, is persistent in the environment

and remains as an environmental pollutant. Only in

China, the annual yield of wheat straw is 110 milliontons (Yang and Wang, 1999). Except that some of them

were used to make paper or as feedstuff for livestock,

most of them were set on fire or discarded as environ-

mental pollutants. Cellulosic materials can, however,

be a valuable and vast renewable resource.

Therefore, in the present study, our research interest

is to convert wheat straw wastes into hydrogen gas by

natural anaerobic microorganism. For this purpose,several environmental factors, such as pretreatment

conditions, initial pH and substrate concentration,

were selected as target factors in conversion of raw

wheat straw wastes into biohydrogen gas by cow dung

compost. Maximum hydrogen production yield of

68.1 ml H2/g TVS was observed from the pretreated

wheat straw wastes by microorganisms, the value is

about 136-fold as compared with that of raw wheatstraw wastes. The result is encouraging because of its

potential commercial and environmental benefits in the

future.

2. Experimental methods

2.1. Seed microflora

Hydrogen-producing microflora was taken from

cow dung compost in the suburb of Zhengzhou City in

this study. Before it is used, cow dung compost was

placed into a stainless steel pizza pan to a depth of

1 cm and broken up in the infrared oven for 2 h in order

to inhibit the bioactivity of hydrogen consumers and to

harvest high yield hydrogen-producing spore-forminganaerobes.

2.2. Chemical pretreatment of the substrate

The wheat straw wastes used as substrate were ob-

tained from the suburbs of Zhengzhou city. Before the

substrate were degraded by microorganisms, the mixed

solution containing wheat straw wastes and dilute HClwas boiled in a Teflon digestor by microwave heating

or in a beakers, then neutralized to pH = 7 with either

dilute NaOH or HCl solution. TVS value was deter-

mined as follows: TVS ¼ W dried wheat straw�W ashW dried wheat straw

� 100%.

2.3. Experimental procedure

The batch experiments were performed with 250 mlserum vials as batch reactors filled to 150 ml comprising

the mixture of the composts, the pretreated wheat straw,

and 3 ml of nutrient stock solution. These vials were

gassed with nitrogen gas to remove oxygen and the

headspace of the reactors to keep the anaerobic environ-

ment. The bottles were incubated at 36 ± 1 �C and oper-ated in an orbital shaker with a rotation speed of 90 rpm

to provide better contact among substrates. The com-post concentration of 80 g/l was maintained in the batch

reactors. Each liter of nutrient stock solution containing

80 g of NH4HCO3, 12.4 g of KH2PO4; 0.1 g of

MgSO4 Æ 7H2O; 0.01 g of NaCl; 0.01 g of Na2MoO4 Æ2H2O; 0.01 g of CaCl2 Æ 2H2O; 0.015 g of MnSO4 Æ7H2O; 0.0278 g of FeCl2, which was slightly modified

from Lay et al. (1999). The volume of biogas was deter-

mined using glass syringes of 5–50 ml.

2.4. Analytical methods

The hydrogen gas percentage was calculated by com-

paring the sample biogas with a standard of pure hydro-

gen using a gas chromatograph (GC, Agilent 4890D)

equipped with a thermal conductivity detector (TCD)

and 6 feet stainless column packed with Porapak Q(80/100 mesh). The operational temperatures of the

injection port, the oven and the detector were 100 �C,80 �C and 150 �C, respectively. Nitrogen was used asthe carrier gas at a flow rate of 20 ml/min. The concen-

trations of the volatile fatty acids (VFAs) and the alco-

hol were analyzed using another GC of the same model

with a flame ionization detector (FID) and a 8 feet stain-

less column packed with 10% PEG-20M and 2% H3PO4(80/100 mesh). The temperatures of the injection port,

the detector and the oven were 220 �C, 240 �C and aprogrammed column temperature of 130–175 �C, res-pectively. Nitrogen was the carrier gas at a flow rate

of 20 ml/min. The pH values inside the digesters were

measured by a microcomputer pH-vision 6071.

3. Results and discussion

3.1. Effects of pretreatment of substrate on hydrogen

production

Fig. 1 depicted the effects of the changes in the acid

concentration on hydrogen production yield at the fixed

initial pH 6.5 and substrate concentration 15 g/l. As canbe seen from Fig. 1, under the condition of microwave

heating, the accumulative hydrogen yield increased

10

15

20

25

0 2 4 6 8 10 12Microwave heating time (min)

Cum

ulat

ive

hydr

ogen

(m

l H2/

g T

VS)

0

5

10

15

20

0 10 20 30 40 50 60 70

Ferv. heating time (min)

Cum

ulat

ive

hydr

ogen

(m

l H2/

g T

VS)

(a)

(b)

Fig. 2. The effect of pretreatment time on hydrogen production yield.

Other variables are constant at their respective levels as follows: initial

pH, 6.5; concentration of substrate, 15 g/l; (a) 2.0% HCl concentration

by microwave heating and (b) 1.0% HCl concentration by ferv.

heating.

0

5

10

15

20

25

0 1 2 3 4 5 6HCl concentration (%)

Cum

ulat

ive

hydr

ogen

(m

l H2/

g T

VS) Microwave

Ferv.

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

hydrogen-production potential. Other variables are constant at their

respective levels as follows: initial pH, 6.5; concentration of substrate,

15 g/l; microwave heating time, 4 min (or ferv. heating 30 min).

502 Y.-T. Fan et al. / Bioresource Technology 97 (2006) 500–505

remarkably with the increase of HCl concentration in

the range of 0.5–2.0%. Maximum hydrogen yield of22.9 ml H2/g TVS was observed in the test using the pre-

treated substrate (2.0% HCl). Then, the hydrogen yield

gradually declined as HCl concentration increased from

22.9 ml H2/g TVS at HCl concentration of 2.0% to 6.0

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-

tration in the batch tests heavily inhibited the growth ofhydrogen production bacteria (Wang et al., 1995).

Under the condition of ferv. heating, the change curve

of hydrogen yield was similar to that by microwave

heating, except that the maximum hydrogen production

yield was only 17.9 ml H2/g TVS. However, the maximal

hydrogen yield from the acid pretreated wheat straw by

microwave heating was higher than that by ferv.

heating.In addition, both microwave heating and ferv. heat-

ing time also affected the hydrogen-production yield

for the acid pretreated substrate. Fig. 2 showed the ef-

fects of micro-wave heating (a) and ferv. heating time

(b) on hydrogen production yield. As shown in Fig. 2,

the hydrogen yield increased with the increase of the

heating time, the maximal hydrogen yield of 22.5 ml

H2/g TVS and 18.0 ml H2/g TVS occurred at the micro-wave heating of 8 min and the ferv. heating of 50 min,

respectively. The results showed that the microwave

heating was a better method for hydrogen production

from the acid pretreated substrate as compared with

that by ferv. heating.

As far as we know, the direct conversion of raw

wheat straw into hydrogen gas by anaerobic fermenta-

tion is very difficult because of its complex polymerstructure such as cellulose, hemi-cellulose and lignin,

e.g., the maximal hydrogen yield was only 0.5 ml

H2/g TVS by the cow dung compost in the test using

the raw wheat straw. In order to explain the experiment

phenomena, the composition of the wheat straw was

analyzed in the test. Compared with the raw wheat

straw, we found that the soluble sugar content increased

from 0.24% to 9.60%, and the cellulose and hemicel-

lulose contents decreased from 22.5% and 21.5% to

15.40% and 12.88% for the acid pretreated wheat straw

by the microwave heating of 8 min, respectively.

Accordingly, we deduced that an increase in the hydro-gen yield possibly was due to an increase in the soluble

sugar in the composition of the acid pretreated

substrate. The results showed that the pretreatment of

the substrate plays a key role in the conversion

of wheat straw wastes into biohydrogen by micro-

organisms.

3.2. Effect of initial pH value on hydrogen-production

yield

To investigate the effect of initial pH on start-up a

hydrogen-producing reactor, the acid pretreated wheat

straw was then used for biohydrogen production at dif-

ferent initial pH values from 4.0 to 9.0, the results are

plotted in Fig. 3. As can be seen from Fig. 3, the initial

pH values significantly affect the hydrogen-productionyield of the substrate under the condition of the micro-

wave heating, e.g., while the initial pH level rose from

4.0 to 7.0, the hydrogen yields increased from 0.01

ml H2/g TVS to 24.1 ml H2/g TVS, respectively. There-

after, the hydrogen yield slightly decreased with in-

creased initial pH of the culture medium in the range

of initial pH 7.0–9.0. For example, while the initial pH

of the culture medium was 9.0, the cumulative hydrogenyield dropped to 22.7 ml H2/g TVS. Under the condition

of the ferv. heating, the change trend of the hydrogen

yield with initial pH value is similar to that by micro-

wave heating, except that the maximum hydrogen yield

0

5

10

15

20

25

30

4 6 8 10Initial pH

Cum

ulat

ive

hydr

ogen

(ml H

2/g

TV

S)

MicrowaveFerv.

Fig. 3. The effect of varied pH value on hydrogen production yield.

Other variables are constant at their respective levels as follows:

concentration of substrate, 15 g/l; (j) 2.0% HCl concentration by

microwave heating 8 min; (m) 1.0% HCl concentration by ferv. heating

50 min.

Y.-T. Fan et al. / Bioresource Technology 97 (2006) 500–505 503

of 10.3 ml H2/g TVS occurred at initial pH value 8.

The results showed that the pH control could stimulatethe microorganisms to produce hydrogen and would

achieve the system having a maximum hydrogen yield,

but the activity of hydrogenase would be inhibited by

low or high pH values in overall hydrogen fermentation

(Fan et al., 2004; Lay et al., 1999).

3.3. Effect of substrate concentration on

hydrogen-production yield

The effects of the pretreated substrate concentration

versus cumulative hydrogen yield by the microorgan-

isms were presented in Fig. 4. As can be seen from

Fig. 4, the cumulative hydrogen yield increased remar-

kedly with increasing the concentration of the pretreated

substrate, e.g., under the conditions of the microwave

heating and ferv. heating, while the concentration ofthe acid pretreated wheat straw rose from 5 g/l to 25

g/l, 30 g/l, the cumulative hydrogen yield increased from

13.8 ml H2/g TVS, 5.6 ml H2/g TVS to 68.1 ml H2/g

TVS, 52.7 ml H2/g TVS, respectively. Thereafter, the

0

10

20

30

40

50

60

70

80

5 15 25 35 45Substrate concentrtion (g/l)

Cum

ulat

ive

MicrowaveFerv.Crude

hydr

ogen

(m

l H2/

g T

VS)

Fig. 4. The effect of substrate concentration on hydrogen production

yield. Other variables are constant at their respective levels as follows:

initial pH, 7; (j) 2.0% HCl concentration by microwave heating 8 min;

(m) 1.0% HCl concentration by ferv. heating 50 min.

cumulative hydrogen yield decreased gradually as the

concentration of the pretreated substrate increased,

e.g., while the concentration of the acid pretreated sub-

strate increased from 25 g/l, 30 g/l to 35 g/l, the hydro-

gen yield dropped from 68.1 ml H2/g TVS, 52.7 ml

H2/g TVS to 16.3 ml H2/g, 44.1 ml H2/g TVS, respec-tively. However, maximum hydrogen yield of 68.1 ml

H2/g TVS occurred at the acid pretreated wheat straw

of 25 g/l by microorganisms under the conditions of

the microwave heating, the value is about 136-fold as

compared with that of raw wheat straw (Fig. 4).

The results showed that the change of the substrate

concentration obviously affected the hydrogen yield in

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

excessive substrate concentration would result in the

accumulation of volatile fatty acids (VFAs) and a fall

of pH value in the reactor, and even inhibited the

growth of hydrogen-producing bacteria. In addition,

the partial pressure of hydrogen in the batch reactor

rose with the increases in substrate concentration. Whilethe partial pressure of hydrogen increased to a certain

level in the headspace of reactor, the microorganisms

would switch to alcohol production, thus inhibiting

hydrogen production (Fan et al., 2004).

3.4. Biodegradation characteristics of the substrate

In this paper, volatile fatty acids (VFAs) and alcoholwere selected as main by-products of the composts con-

suming the substrate. Fig. 5 showed the changes of the

accumulative hydrogen yield (a), pH value (b), VFAs

(c) and alcohol (d) during the conversion of the pre-

treated wheat straw wastes to biohydrogen by cow dung

compost.

As shown in Fig. 5(a), the hydrogen evolution began

to occur after 4 h of cultivation. The hydrogen yieldincreased 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

68.1 ml H2/g TVS was observed at 126.5 h (Fig. 5(a)).

The maximum hydrogen production rate of 10.14

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

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

was 52.0% and there was no significant methane ob-served in this study.

The pH value of the medium decreased from 7.0 to

4.7 with the progress of hydrogen evolution and wheat

straw decomposition, the optimum pH value of hydro-

gen-production appeared in the range of 5.0–4.7 (Fig.

5(b)). Hydrogen production was accompanied with the

formation of volatile fatty acids (VFAs) throughout

the wheat straw fermentation (Fig. 5(c) and (d)). Duringthis period, acetate, propionate and butyrate reached

maximum yields of 1752, 234 and 1617 mg/l at 78.5 h,

0

20

40

60

80

Com

mul

ativ

eH

2 (m

l H2/g

TV

S)

4

5

6

7

8

pH

0

400

800

1200

1600

2000

Vol

atile

aci

ds (

mg/

l)

AcetatePropionateButyrate

0

200

400

600

0 50 100 150

Time (hours)

Alc

ohol

s (m

g/l)

EthanolPropanolButanol

(a)

(b)

(c)

(d)

Fig. 5. Developments of cumulative hydrogen yield, pH value, VFAs

and alcohols in the batch reactor during the conversion of the substrate

to biohydrogen under the pretreated condition of microwave heating.

504 Y.-T. Fan et al. / Bioresource Technology 97 (2006) 500–505

respectively. The ethanol began to produce after 4 h cul-tivation and increased up to 482 mg/l at 126.5 h. When

the reaction reached the quasi-steady state, the produc-

ers of volatile fatty acids and ethanol plateaued. Hydro-

gen production stopped when the available substrate

was consumed up, and the ethanol, acetate and butyrate

as significant by-products were left in the batch reactor,

during which acetate and butyrate accounted for 70–

80% of total VFAs, but amounts of propionate werevery low in total VFAs. This result is similarly those

in biohydrogen fermentation from glucose, in which

VFAs mainly consists of acetate and butyrate (Fan

et al., 2002).

These phenomena were expected because hydrogen

production appears to be usually accompanied with

the formation of VFAs and alcohol while both of them

are the main by-products in the metabolism of hydrogenfermentation. This result also implied the competition

among the acetate, butyrate and propionate producers.

However, under the optimum pH condition of hydrogen

production, the acetate and butyrate producers were ac-

tive and competed with the propionate producer. In

order to convert the wheat straw into biohydrogen by

the microorganisms, the activity of the propionate pro-

ducer must be suppressed.

4. Conclusion

The acid pretreatment of the substrate plays a key

role in efficient conversion of the wheat straw wastes

into biohydrogen gas by the cow dung composts. The

pretreated HCl concentration of 2.0% was optimalunder the microwave heating time of 8 min. The maxi-

mum hydrogen yield of 68.1 ml H2/g TVS was observed

at the fixed initial pH 7.0 and substrate concentration

25 g/l, the value was about 136-fold higher than the

maximum value obtained for 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 was52.0% and there was no significant methane observed

in this study.

The hydrogen production was usually accompanied

with the formation of VFAs and alcohol while both of

them were the main by-products in the metabolism of

hydrogen fermentation, during which acetate and buty-

rate accounted for about 76–80% of VFAs.

Acknowledgements

This work was supported by the China National Key

Basic Research Special Funds (No. 2003CB214500), the

National Natural Science Foundation of China (Nos.

20171040 and 20471053) and the Energy and Technol-

ogy Program from Zhengzhou University.

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