Bioresource Technology 97 (2006) 500–505
Eﬃcient conversion of wheat straw wastes into biohydrogen gas by cow dung compost
, Ya-Hui Zhang a, Shu-Fang Zhang a, Hong-Wei Hou a, Bao-Zeng Ren
Department of Chemistry, Zhengzhou University, Zhengzhou 450052, PR China 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 136fold 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 The microbial conversion of agricultural and industrial wastes and residues into hydrogen is attracting increasing interest, this is due to that hydrogen is an excellent 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 hydride and its transmission through natural gas pipelines would be more eﬃcient than the transmission of electricity down power lines (Fan et al., 2004). Earlier studies
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have been done by pure cultures of anaerobic bacteria to study the conversion of carbohydrates (such as glucose and starch) to hydrogen gas, e.g., Enterobacter (Rachman et al., 1997), Aspergillus terreus (Emtiazi et al., 2001) and Clostridium (Taguchi et al., 1994). Recently, 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,
0960-8524/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.02.049
Seed microﬂora Hydrogen-producing microﬂora was taken from cow dung compost in the suburb of Zhengzhou City in this study. however. Experimental methods 2.1.4 g of KH2PO4. Analytical methods The hydrogen gas percentage was calculated by comparing the sample biogas with a standard of pure hydrogen using a gas chromatograph (GC. were selected as target factors in conversion of raw wheat straw wastes into biohydrogen gas by cow dung compost. The concentrations of the volatile fatty acids (VFAs) and the alcohol were analyzed using another GC of the same model with a ﬂame ionization detector (FID) and a 8 feet stainless column packed with 10% PEG-20M and 2% H3PO4 (80/100 mesh). However. it is hard to convert directly raw crop stalk wastes into biohydrogen gas by microbe anaerobic fermentation because of their complex chemical composition. Only in China.
2. Therefore.01 g of CaCl2 Æ 2H2O. The bottles were incubated at 36 ± 1 °C and operated in an orbital shaker with a rotation speed of 90 rpm to provide better contact among substrates. TVS value was deterwheat straw ÀW mined as follows: TVS ¼ W drieddried wheat straw ash Â 100%. (2002) investigated a mesophilic microbial community converting glucose into hydrogen. Fan et al. the oven and the detector were 100 °C. Experimental procedure The batch experiments were performed with 250 ml serum vials as batch reactors ﬁlled to 150 ml comprising the mixture of the composts.4. the mixed solution containing wheat straw wastes and dilute HCl was boiled in a Teﬂon digestor by microwave heating or in a beakers. hemicellulose. For this purpose.1 g of MgSO4 Æ 7H2O. The compost concentration of 80 g/l was maintained in the batch reactors. then neutralized to pH = 7 with either
3. respectively.1. the pretreated wheat straw. Except that some of them were used to make paper or as feedstuﬀ for livestock.3. the accumulative hydrogen yield increased
.Y. 0. Nitrogen was used as the carrier gas at a ﬂow rate of 20 ml/min. is persistent in the environment and remains as an environmental pollutant. 1 depicted the eﬀects of the changes in the acid concentration on hydrogen production yield at the ﬁxed initial pH 6. several environmental factors. the research of the conversion of the biomass containing cellulose. The operational temperatures of the injection port. 0. the value is about 136-fold as compared with that of raw wheat straw wastes. such as wheat straw and corn stalk. 0.2. protein. in the present study. 12. Fang et al. respectively. Before it is used.015 g of MnSO4 Æ 7H2O. 1. 2. initial pH and substrate concentration.01 g of Na2MoO4 Æ 2H2O. 0. fat. The result is encouraging because of its potential commercial and environmental beneﬁts in the future. It is well known that cellulose in nature substrates.-T. the detector and the oven were 220 °C. Maximum hydrogen production yield of 68. 240 °C and a programmed column temperature of 130–175 °C. 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-forming anaerobes. The pH values inside the digesters were measured by a microcomputer pH-vision 6071. the annual yield of wheat straw is 110 million tons (Yang and Wang. e. W 2. Nitrogen was the carrier gas at a ﬂow rate of 20 ml/min. lignin.5 and substrate concentration 15 g/l. 0. The temperatures of the injection port.01 g of NaCl.. Each liter of nutrient stock solution containing 80 g of NH4HCO3. and 3 ml of nutrient stock solution. cellulose. In general. Eﬀects of pretreatment of substrate on hydrogen production Fig. Cellulosic materials can. such as wheat straw. be a valuable and vast renewable resource. Agilent 4890D) equipped with a thermal conductivity detector (TCD) and 6 feet stainless column packed with Porapak Q (80/100 mesh). 0. 2. These vials were gassed with nitrogen gas to remove oxygen and the headspace of the reactors to keep the anaerobic environment. which was slightly modiﬁed from Lay et al. our research interest is to convert wheat straw wastes into hydrogen gas by natural anaerobic microorganism. under the condition of microwave heating. most of them were set on ﬁre or discarded as environmental pollutants. 1999). (1999). into biohydrogen is lacking.1 ml H2/g TVS was observed from the pretreated wheat straw wastes by microorganisms. such as pretreatment conditions. The volume of biogas was determined using glass syringes of 5–50 ml.
dilute NaOH or HCl solution.g.0278 g of FeCl2. Results and discussion 3. Chemical pretreatment of the substrate The wheat straw wastes used as substrate were obtained from the suburbs of Zhengzhou city. Before the substrate were degraded by microorganisms. / Bioresource Technology 97 (2006) 500–505
to generate hydrogen from sucrose or glucose in batch experiments. As can be seen from Fig. 80 °C and 150 °C.
the hydrogen yields increased from 0. we deduced that an increase in the hydrogen yield possibly was due to an increase in the soluble sugar in the composition of the acid pretreated substrate.9 ml H2/g TVS.
Cumulative hydrogen (ml H2/g TVS)
20 15 10 5 0 0
2 4 6 8 10 Microwave heating time (min)
Fig. heating time (b) on hydrogen production yield.9 ml H2/g TVS was observed in the test using the pretreated substrate (2.0% HCl concentration by ferv.60%. and the cellulose and hemicellulose contents decreased from 22.1 ml H2/g TVS. heating time (min)
Fig.2. while the initial pH of the culture medium was 9. heating. The eﬀect of pretreatment time on hydrogen production yield. e.5% and 21.5% to 15. 3. concentration of substrate. As far as we know.-T. heating 30 min).0.0% HCl). However. heating. 2 showed the effects of micro-wave heating (a) and ferv. Fan et al. (a) 2. / Bioresource Technology 97 (2006) 500–505
Cumulative hydrogen (ml H2/g TVS)
25 20 15 10
Cumulative hydrogen (ml H2/g TVS)
Microwave 20 15 10 5 0 0 1 2 3 4 HCl concentration (%) 5 6 Ferv. Then.5. concentration of substrate. the cumulative hydrogen yield dropped to 22.5. For example. respectively.0–9.0. the hydrogen yield increased with the increase of the heating time. heating time also aﬀected the hydrogen-production yield for the acid pretreated substrate. the results are plotted in Fig. the change curve of hydrogen yield was similar to that by microwave heating.0% to 6. As shown in Fig.01 ml H2/g TVS to 24. the acid pretreated wheat straw was then used for biohydrogen production at different initial pH values from 4.0. the maximal hydrogen yield from the acid pretreated wheat straw by microwave heating was higher than that by ferv.0% HCl concentration by microwave heating and (b) 1. the maximal hydrogen yield was only 0. microwave heating time. e. Maximum hydrogen yield of 22. 2. 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. The eﬀect of chemical pretreatment of wheat straw on hydrogen-production potential. 1.g.7 ml H2/g TVS. heating of 50 min. Although the higher acid concentration was in favor of the hydrolyzation of the substrate. As can be seen from Fig. while the initial pH level rose from 4. Other variables are constant at their respective levels as follows: initial pH. 4 min (or ferv.5 ml H2/g TVS by the cow dung compost in the test using the raw wheat straw.5–2.0 ml H2/g TVS occurred at the microwave heating of 8 min and the ferv.. heating.. Thereafter. Other variables are constant at their respective levels as follows: initial pH. the composition of the wheat straw was analyzed in the test.
from 0. Fig. except that the maximum hydrogen yield
. Compared with the raw wheat straw.0 to 9. heating.g. heating.0%.9 ml H2/g TVS at HCl concentration of 2.. the hydrogen yield gradually declined as HCl concentration increased from 22.0%. the direct conversion of raw wheat straw into hydrogen gas by anaerobic fermentation is very diﬃcult because of its complex polymer structure such as cellulose. but the high ClÀ anion concentration in the batch tests heavily inhibited the growth of hydrogen production bacteria (Wang et al. Eﬀect of initial pH value on hydrogen-production yield To investigate the eﬀect of initial pH on start-up a hydrogen-producing reactor.40% and 12. 15 g/l. respectively. the hydrogen yield slightly decreased with increased initial pH of the culture medium in the range of initial pH 7.0 to 7. Accordingly.
remarkably with the increase of HCl concentration in the range of 0. the maximal hydrogen yield of 22. 6. the change trend of the hydrogen yield with initial pH value is similar to that by microwave heating.502
Y. 6.0.0 ml H2/g TVS at HCl concentration of 5. The results showed that the pretreatment of the substrate plays a key role in the conversion of wheat straw wastes into biohydrogen by microorganisms. 15 g/l. the initial pH values signiﬁcantly aﬀect the hydrogen-production yield of the substrate under the condition of the microwave heating. In order to explain the experiment phenomena.5 ml H2/g TVS and 18. we found that the soluble sugar content increased
20 30 40 50 Ferv. 2. Under the condition of the ferv. Under the condition of ferv. 3. hemi-cellulose and lignin. 3. In addition. respectively. both microwave heating and ferv. except that the maximum hydrogen production yield was only 17. 1995).24% to 9.88% for the acid pretreated wheat straw by the microwave heating of 8 min.
the hydrogen yield dropped from 68.. 4.0% HCl concentration by microwave heating 8 min. e. but the activity of hydrogenase would be inhibited by low or high pH values in overall hydrogen fermentation (Fan et al.0% and there was no signiﬁcant methane observed in this study.1 ml H2/g TVS was observed at 126. 3.-T.g.5 h while the pH value decreased from 5.7 ml H2/g TVS to 16. 3.4 ml H2/g TVS at 16 h to 40. 4. 5. (m) 1. 5(c) and (d)). the partial pressure of hydrogen in the batch reactor rose with the increases in substrate concentration.95 to 5. The hydrogen yield increased rapidly from 7. Although an increase in the substrate concentration could enhance the hydrogen yield under the condition of the optimum hydrogen production. acetate. propionate and butyrate reached maximum yields of 1752.. Fig.5 h (Fig. while the concentration of the acid pretreated wheat straw rose from 5 g/l to 25 g/l.0% HCl concentration by ferv. The maximum hydrogen yield of 68. (j) 2. and even inhibited the growth of hydrogen-producing bacteria.
of 10. the optimum pH value of hydrogen-production appeared in the range of 5. 2004). The eﬀect of substrate concentration on hydrogen production yield. 52. The pH value of the medium decreased from 7.7 ml H2/g TVS. Lay et al. The results showed that the change of the substrate concentration obviously aﬀected the hydrogen yield in the test.14 ml H2/g TVS h was by a modiﬁed Gompertz equation (Lay et al. while the concentration of the acid pretreated substrate increased from 25 g/l. The hydrogen content in the biogas was 52.Y. respectively. 3. under the conditions of the microwave heating and ferv. However.7 (Fig. heating.4. 1999). volatile fatty acids (VFAs) and alcohol were selected as main by-products of the composts consuming the substrate. the cumulative hydrogen yield increased from 13. Fan et al. respectively. maximum hydrogen yield of 68..7 with the progress of hydrogen evolution and wheat straw decomposition. During this period.1 ml H2/g TVS. e. Eﬀect of substrate concentration on hydrogen-production yield The eﬀects of the pretreated substrate concentration versus cumulative hydrogen yield by the microorganisms were presented in Fig. 4).g. 234 and 1617 mg/l at 78. 4. 2004.0% HCl concentration by ferv. 30 g/l to 35 g/l. the
cumulative hydrogen yield decreased gradually as the concentration of the pretreated substrate increased. 44. 5(a)). (j) 2. pH value (b). Other variables are constant at their respective levels as follows: concentration of substrate.3 ml H2/g. The maximum hydrogen production rate of 10. the hydrogen evolution began to occur after 4 h of cultivation.
Cumulative hydrogen (ml H2/g TVS)
70 60 50 40 30 20 10 0 5
Microwave Ferv. thus inhibiting hydrogen production (Fan et al. The results showed that the pH control could stimulate the microorganisms to produce hydrogen and would achieve the system having a maximum hydrogen yield. Biodegradation characteristics of the substrate In this paper. (m) 1.0% HCl concentration by microwave heating 8 min.0..3. 5(a). 15 g/l. heating 50 min.1 ml H2/g TVS.3 ml H2/g TVS occurred at initial pH value 8.8 ml H2/g TVS at 31. / Bioresource Technology 97 (2006) 500–505
30 Cumulative hydrogen(ml H2/g TVS) 25 20 15 10 5 0 4 6 Initial pH 8 10 Microwave Ferv.. As shown in Fig. The eﬀect of varied pH value on hydrogen production yield.0–4.1 ml H2/g TVS occurred at the acid pretreated wheat straw of 25 g/l by microorganisms under the conditions of the microwave heating.6 ml H2/g TVS to 68.
Fig. 52. While the partial pressure of hydrogen increased to a certain level in the headspace of reactor. VFAs (c) and alcohol (d) during the conversion of the pretreated wheat straw wastes to biohydrogen by cow dung compost. 5 showed the changes of the accumulative hydrogen yield (a). Thereafter. the cumulative hydrogen yield increased remarkedly with increasing the concentration of the pretreated substrate.8 ml H2/g TVS. Hydrogen production was accompanied with the formation of volatile fatty acids (VFAs) throughout the wheat straw fermentation (Fig..
. Other variables are constant at their respective levels as follows: initial pH. 1999). 5(b)). In addition.1 ml H2/g TVS. the value is about 136-fold as compared with that of raw wheat straw (Fig. but the excessive substrate concentration would result in the accumulation of volatile fatty acids (VFAs) and a fall of pH value in the reactor. As can be seen from Fig.0 to 4. 7. Crude
15 25 35 Substrate concentrtion (g/l)
Fig. heating 50 min. 30 g/l.5 h. the microorganisms would switch to alcohol production.
S. but amounts of propionate were very low in total VFAs.
Fig. Biodegradation 12... 87–93. 358–363... Feasibility of biological hydrogen production from organic fraction of municipal solid waste. Rachman..J. 33 (11).0% was optimal under the microwave heating time of 8 min. 2003CB214500). 259–263. The ethanol began to produce after 4 h cultivation and increased up to 482 mg/l at 126.. Bordbar. Lay.P. G. Bioeng. A. J. Water Res.W. Noike.. the acetate and butyrate producers were active and competed with the propionate producer. 83 (4). Y.. However. the activity of the propionate producer must be suppressed. Naghavi. Kakizono.1 ml H2/g TVS was observed at the ﬁxed initial pH 7. Zhang.J. 35 (24). G. Fang.. during which acetate and butyrate accounted for 70– 80% of total VFAs.. J. When the reaction reached the quasi-steady state. 370–374.. 1997.T. 152–155 (in Chinese). In order to convert the wheat straw into biohydrogen by the microorganisms.. 5. Technol. and the ethanol.L. the producers of volatile fatty acids and ethanol plateaued. This result also implied the competition among the acetate. Y. 91. H.V. Y. Y. the National Natural Science Foundation of China (Nos. Biodegradation of lignocellulosic waste by Aspergillous terreus. Hydrogen production stopped when the available substrate was consumed up. Ferment.H. 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 hydrogen fermentation. Lee. Environ.T.J.. M. Nakashimada.A. Bioresour. Lay.5 h.. 1999.14 ml H2/g TVS h was obtained by a modiﬁed Gompertz equation.T. 22 (4). Environ.W. This result is similarly those in biohydrogen fermentation from glucose. Nishio.. 2002. Li. S. H. 2001. H. H.. Technol. H.0% and there was no signiﬁcant methane observed in this study. China Environ. N.. the value was about 136-fold higher than the maximum value obtained for raw wheat straw wastes.J.0 and substrate concentration 25 g/l.W. pH value. Hou. 2002). Study on biohydrogen production by anaerobic biological fermentation of organic wasters. 2579–2586.
respectively.. Fan.. Furutani. Lay. Lay. 189–193. Ch. Technol. Fan. Lu. Sci. Optimization of initial substrate and pH levels for germination of sporing hydrogen-producing anaerobes in cow dung compost. Sci. T. Hou. N. Bioresour. Enhanced hydrogen production in altered mixed acid fermentation of glucose by enterobacter aerogenes.. The maximum hydrogen yield of 68. Liu. 20171040 and 20471053) and the Energy and Technology Program from Zhengzhou University. Liao. Ginkel.504
Commulative H2 (ml H2 /g TVS)
80 60 40 20 0
Y. Y. 24 (3). T. 2003.. 4726–4730. Lu.. during which acetate and butyrate accounted for about 76–80% of VFAs. / Bioresource Technology 97 (2006) 500–505
4. acetate and butyrate as signiﬁcant by-products were left in the batch reactor.W.Ch. Zhang.-T... Sci.. Hou.Sh. The maximum hydrogen production rate of 10. J. Li. under the optimum pH condition of hydrogen production. Y. 2001. Ch.. Fan et al. 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. T.J.. H. Sung..J.
.J. in which VFAs mainly consists of acetate and butyrate (Fan et al. The pretreated HCl concentration of 2. J. 82. The hydrogen content in the biogas was 52. J. 2004.L. Fan. 2002.
Volatile acids (mg/l)
6 5 4
2000 1600 1200 800 400 0
600 400 200 0 0
Acetate Propionate Butyrate
Ethanol Propanol Butanol
50 100 Time (hours)
Acknowledgements This work was supported by the China National Key Basic Research Special Funds (No. X. Studies on biohydrogen production by biohydrogen fermentation of natural anaerobic microorganism. VFAs and alcohols in the batch reactor during the conversion of the substrate to biohydrogen under the pretreated condition of microwave heating. Developments of cumulative hydrogen yield. H.... Biohydrogen production as a function of pH and substrate concentration.. butyrate and propionate producers. 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. Eﬀect of pH on hydrogen production from glucose by a mixed culture.
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