- Email: [email protected]
Effects of acid and alkaline pretreatments on the biohydrogen production from grass by anaerobic dark fermentation
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 1 2 0 e1 1 2 4
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Effects of acid and alkaline pretreatments on the biohydrogen production from grass by anaerobic dark fermentation Maojin Cui, 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:
Batch experiments were carried out to study the effects of acid (HCl) and alkaline (NaOH)
Received 11 November 2010
pretreatments on saccharification of grass and subsequent microbial hydrogen production
Received in revised form
at 35 C and initial pH 7.0. Liquid metabolites and hydrogen production characteristics
25 January 2011
were also studied. Results show that the saccharification efficiency and hydrogen
Accepted 14 February 2011
production from grass pretreated by acid and alkali were higher than those from unpre-
Available online 15 March 2011
treated grass and that acid pretreatment was better than alkaline pretreatment for enhancing the hydrogen yield from grass. A maximum cumulative hydrogen yield of
Keywords:
72.21 mL/g-dry grass was achieved from substrate pretreated with 4% HCl, which was
Grass
16.45-fold greater than that from untreated substrate. Only H2 and CO2 were present in the
Pretreatment
biogas. The main liquid metabolite found after fermentation was acetic acid.
Anaerobic dark fermentation
Copyright ª 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
Biohydrogen
1.
Introduction
Global energy requirements depend heavily upon fossil fuels such as coal, oil and natural gas. There is urgency in the search to replace these sources of energy, as the depletion of limited fossil fuels is inevitable [1]. The extensive use of fossil fuels has also caused an environmental issue where emission of CO2, CO, NOx and SOx during combustion has caused air pollution and global warming. Compared with fossil fuels, hydrogen is a promising candidate as a clean energy carrier in the future because it has high-energy yield (122 kJ/g) and generates no pollutants, producing only water when used as a fuel [2]. Hydrogen production by physical or chemical methods has been well developed. Biological processes, such as dark and photo fermentation, however, are environmentally benign and less energy intensive compared with physical and chemical processes [1]. Dark fermentation has been reported recently as the hydrogen production system having
reserved.
the greatest potential [3]. Some of the basic advantages of dark fermentation over other processes include process simplicity, better process economy resulting from lower energy requirements, higher hydrogen production rates and utilization of low-value wastes as raw materials [3,4]. Biomass residues such as leaves, grass and crop stalks are abundant in the world. It was reported that the annual global yields of biomass residues exceed 220 billion tons [5]. Most of them, however, are discarded or burned as environmental pollutants. They can be a valuable and vast renewable resource if their energy content is stored as hydrogen. Numerous studies have been carried out to produce hydrogen by using biomass residues as substrate such as poplar leaves [6], beer lees [7], rice straw [8,9] and corn stalks [10,11,12,13]. The direct utilization of biomass residues by microorganisms, however, is difficult because of the heterogeneity and crystallinity of such materials [14]. For instance, the maximum hydrogen yields from raw beer lees and poplar leaves were
* Corresponding author. Tel.: þ86 10 62620903; fax: þ86 10 62559373. E-mail address: [email protected] (J. Shen). 0360-3199/$ e see front matter Copyright ª 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2011.02.078
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 1 2 0 e1 1 2 4
3.16 and 15.04 mL/g-S, respectively [6,7]. Thus, biomass has to be pretreated to release its underlying monomeric sugar prior to bacterial fermentation. Physical, chemical and biological substrate pretreatment methods, or their combinations, have proven useful for enhancing hydrogen yields [6,7,8,9,10,11,12,13]. The objective of this research was to evaluate the effects of acid and alkaline pretreatment on hydrogen production from grass using anaerobic mixed bacteria. For this purpose, the total reducing sugar (TRS) and hydrogen production from raw and pretreated substrate were studied. Liquid metabolites and hydrogen production characteristics were also studied. A maximum cumulative hydrogen yield of 72.21 mL/g-dry grass was achieved from pretreated substrate, which was 16.45-fold greater than that from raw substrate. The result is encouraging because of its potential commercial and environmental benefits in the future.
2.
Materials and methods
2.1.
Seed microorganisms
The hydrogen-producing mixed cultures were enriched from cracked cereal and identified to be dominated by Clostridium pasteurianum [15]. The cultures’ acclimation and medium were the same as our previous work described in Ref [6].
2.2.
Raw materials and pretreatment
The grass used in this study was collected in winter from the lawn of our institute, 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. 1.0 g of dry grass 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.
Experimental procedures
Batch experiments were carried out in 120 mL serum vials. The raw and pretreated grass was used as substrate. The total work volume was 80 mL (approximately 1.5% (w/v) solids loading) in each case. 1 L 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. 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.
2.4.
1121
Analytical methods
The hydrogen content was determined by a gas chromatograph (Techcomp. Co., China, 7890II) equipped with a thermal conductivity detector (TCD) and a 2-m stainless steel column packed with Porapak Q (80e100 mesh). The operating temperatures of the injection port, the oven and the detector were set at 80, 60 and 100 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 [16]. The cumulative hydrogen volume was calculated by equation (1) [17]: VH;i ¼ VH;i1 þ CH;i VG;i VG;i1 þ VH;0 CH;i CH;i1
(1)
where VH, i is the cumulative hydrogen volume at the current time interval, VH, i 1 is the cumulative hydrogen volume at the previous time interval, VG, i is the total biogas volume in the current time interval, VG, i -1 is the total biogas volume in the previous time interval, CH, i is the fraction of hydrogen in the headspace of the bottle at the current time interval, CH, i-1 is the fraction of hydrogen in the headspace of the bottle at the previous time interval and VH,0 is the total volume of headspace in the bottle. Detection of the alcohols and volatile fatty acids (VFAs, C2-C5) was measured by a gas chromatograph using a flame ionization detector (FID) and a 2-m glass column packed with Unisole F-200 (30e60 mesh). The temperatures of the injection port, the oven and the detector were set at 200, 165 and 200 C, respectively. The carrier gas was argon at a flow rate of 30 mL/min. The concentration of the total reducing sugar was determined by the phenol-sulfuric acid method using glucose as a standard [18]. The contents of hemicellulose, cellulose and lignin were determined according to Van Soest’s method [19].
3.
Results and discussion
3.1. grass
Effect of pretreatment on the saccharification of
Acid and alkaline pretreatments are two effective methods to pretreat cellulosic biomass that mainly convert cellulosic biomass into soluble sugars, which can then be used by microorganisms to produce hydrogen [6,7]. The yields of the total reducing sugar (TRS) resulting from 1.0 g of dry grass after HCl and NaOH pretreatments were analyzed and are shown in Fig. 1. TRS yield from pretreated grass was higher than that from raw grass, indicating that pretreatment is critical for increasing the saccharification of grass. The TRS yield was 56.65 mg/g-S in raw substrate and increased with increasing HCl and NaOH concentrations. The amounts of TRS after 8% HCl and 8% NaOH pretreatment were approximately 6.40-fold and 5.84-fold greater than that in the raw substrate. It was observed that the TRS yield with HCl pretreatment was always higher than that with NaOH pretreatment at equal HCl and NaOH concentrations, in agreement with our previous work [6]. To better investigate the chemical composition of raw and pretreated substrates, the contents of hemicellulose, cellulose and lignin in raw substrate and substrate pretreated with 4%
1122
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 1 2 0 e1 1 2 4
80 UP
HCl
350
0.5%
70
NaOH
1% 300
Cumulative hydrogen (mL)
TRS yield (mg/g-S)
250
200
150
100
50
0
4% 8%
50 40 30 20 10
UP
0.5%
1%
2%
4%
0
8%
0
HCl or NaOH Concentratiton (w/v)
20
40
60
80
100
Time (hour)
Fig. 1 e Effects of HCl and NaOH concentrations on the saccharification of grass. UP means unpretreatment. HCl and 0.5% NaOH were analyzed (Fig. 2). These concentrations were the optimal conditions for acid and alkaline pretreatment for hydrogen production according to Fig. 3, Fig. 4 and Table 1. From Fig. 2, it can be seen that raw grass consisted mainly of hemicellulose (42.97%) and cellulose (30.82%). Compared with raw substrate, the content of hemicellulose in substrate pretreated with 4% HCl decreased dramatically, the content of cellulose decreased slightly, and there was an increase in lignin content. The contents of hemicellulose, cellulose and lignin in substrate pretreated with 0.5% NaOH decreased dramatically compared with their levels in raw substrate. These results indicate that acid pretreatment mainly degraded hemicellulose, which is consistent with our previous work [7], but alkaline pretreatment could efficiently degrade hemicellulose, cellulose and lignin.
3.2.
2%
60
Effect of pretreatment on hydrogen production
Fig. 3 e Cumulative hydrogen volumes from 1.0 g of dry grass pretreated by different HCl concentrations versus corresponding fermentation time. The operation was at 35 C and initial pH 7.0.
hydrogen at an initial pH of 7.0. The effects of HCl and NaOH concentrations on the cumulative hydrogen volumes (CHVs) are presented in Figs. 3 and 4. It can be seen from Fig. 3 that the CHV from raw grass was 4.39 mL and that the CHVs initially increased then decreased as the HCl pretreatment concentration was increased (0.5e8%). The maximum hydrogen volume of 72.21 mL was observed at 4% HCl, which was 16.45fold higher than that from the raw substrate. It can be seen from Fig. 4 that the CHVs initially decreased then increased with increasing NaOH concentration (0.5e4%). A maximum hydrogen volume of 19.25 mL was observed at 0.5% NaOH, which was 4.38-fold higher than that from the raw substrate. 24 h was required to reach the maximum CHV from grass pretreated with 0.5% NaOH but 92 h was required with 4%
1.0 g samples of dry grass pretreated with different HCl and NaOH concentrations were used as substrates to produce UP
20 40
0.5%
UP
1%
4% HCl
2% 4%
Cumulative hydrogen (mL)
Content (% dry weigh)
0.5% NaOH
30
20
15
10
5
10
0 0
Hemicellulose
Cellulose
Lignin
Chemical composition
Fig. 2 e Contents of hemicellulose, cellulose and lignin in grass without pretreatment and pretreated by 4% HCl and 0.5% NaOH. UP means unpretreatment.
0
20
40
60
80
100
120
Time (hour)
Fig. 4 e Cumulative hydrogen volumes from 1.0 g of dry grass pretreated by different NaOH concentrations versus corresponding fermentation time. The operation was at 35 C and initial pH 7.0.
1123
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 1 2 0 e1 1 2 4
Table 1 e Effects of HCl and NaOH concentrations on the degradation efficiency of total reducing sugar, H2 yields, CO2 yields and yields of ethanol and VFAs after fermentation. DESa (%) UP HCl (w/v)
NaOH (w/v)
0 0.5% 1% 2% 4% 8% 0.5% 1% 2% 4%
84.61 88.94 92.60 93.45 91.97 86.50 79.93 78.94 73.26 69.06
0.56b 0.26 0.33 0.32 0.14 0.24 1.48 1.35 3.82 2.72
H2 yield (mL/g-S) 4.39 0.20 16.31 0.44 27.71 0.48 37.67 1.43 72.21 0.76 67.79 1.08 19.25 0.58 12.20 0.85 9.66 0.47 16.32 0.38
CO2 yield (mL/g-S) 12.41 36.09 51.69 52.13 66.49 70.21 16.75 17.20 31.14 39.48
0.06 0.46 3.67 1.47 3.75 0.67 0.24 1.40 2.15 1.07
Ethanol (mg/g-S)c 6.70 7.37 6.82 6.73 34.15 53.50 8.21 6.88 6.68 16.49
0.17 0.19 0.14 0.37 0.68 1.17 0.12 0.13 0.23 0.37
Acetic acid (mg/g-S) 101.71 10.05 264.19 11.38 266.93 11.74 250.55 15.60 222.06 4.97 145.71 4.84 240.08 12.37 167.92 2.32 174.45 2.26 105.57 2.88
Propionic acid (mg/g-S) 40.74 91.21 92.15 94.12 33.27 15.98 152.58 148.90 153.63 65.44
0.94 4.39 2.69 4.39 1.12 1.04 8.68 2.38 0.40 1.89
n-Butyric acid (mg/g-S) 10.94 3.84 22.78 1.28 15.12 0.43 10.38 0.32 9.78 0.38 4.55 0.57 30.11 1.76 19.16 0.54 11.05 0.18 5.29 0.22
a DES means degradation efficiency of total reducing sugar. b Mean standard deviation, n ¼ 3. c (mg/g-S) means (mg/g-dry grass).
NaOH, indicating that higher NaOH concentration could delay the completion of fermentation. These results indicate that HCl pretreatment was superior to NaOH pretreatment, consistent with previous studies [5,20]. As shown above, CHV from pretreated substrate was higher than that from raw substrate, suggesting that pretreatment is necessary to enhance hydrogen yield.
NaOH concentration increased from 0.5% to 2% and decreased at 4% NaOH. The yields of n-butyric acid decreased gradually with increasing NaOH concentration (0.5%e4%). Comparing the yields of the four products, the acetic acid concentration was the highest, indicating that dark anaerobic fermentation of grass was mainly an acetic acid type fermentation.
3.4. 3.3.
Kinetic analysis
Effect of pretreatment on metabolites
Gas product analyses showed that only H2 and CO2 were present in the biogas and no other gases (such as CH4) were detected during the course of hydrogen production. The effects of HCl and NaOH concentrations on the degradation efficiency of the total reducing sugar (DES), hydrogen yields, carbon dioxide yields and main liquid metabolites are shown in Table 1. The DES from substrate pretreated by HCl initially increased then decreased with increased HCl concentration (0.5%e8%) and a maximum of 93.45% occurred at 2% HCl. However, the DES from substrate pretreated by NaOH decreased continually with increasing NaOH concentration (0.5%e4%) and a maximum of 79.93% was observed at 0.5% NaOH. Clearly, the DES from substrate pretreated with HCl was higher than that from substrate pretreated with NaOH, indicating that acid pretreatment is more effective for the degradation of TRS than alkaline pretreatment. The hydrogen maximum of 72.21 mL/g-S was observed at 4% HCl and the carbon dioxide maximum of 70.21 mL/g-S was observed at 8% HCl. The liquid product analysis showed that the metabolites found after fermentation were mainly ethanol, acetic acid, propionic acid and n-butyric acid, with little iso-butyric acid, iso-valeric acid and n-valeric acid. The yields of ethanol were low at lower HCl concentrations (0.5%e2%) but high at higher HCl concentrations (4%e8%). The yields of acetic acid changed only slightly when the HCl concentration increased from 0.5% to 4% and decreased at 8% HCl. The yields of n-butyric acid decreased gradually, along with an initial increase in propionic acid, which subsequently decreased when the HCl concentration increased from 0.5% to 8%. The yields of ethanol were low at lower NaOH concentrations (0.5%e2%) and were high at higher NaOH concentration (4%). The yields of acetic acid and propionic acid changed only slightly as the
The hydrogen production potential, maximum hydrogen production rate and lag-phase time were elucidated using the modified Gompertz equation (equation (2)) that has been used to describe the progress of the cumulative hydrogen production from batch experiments [21,22]. Rm e ðl tÞ þ 1 H ¼ P exp P
(2)
where H is the cumulative hydrogen production (mL), P is hydrogen production potential (mL), Rm is the maximum hydrogen production rate (mL/hour), e ¼ 2.71828, l is the lagphase time (hour), and t is the incubation time (hr).The corresponding values of P, Rm and l for each batch were estimated using Origin 7.5, a scientific graphing and data analysis software package. The data in Figs. 3 and 4 were simulated using equation (2) and the hydrogen production characteristics are shown in Table 2. According to the data in Table 2, all R2 values were more than 0.990, indicating that the fitted curves matched well with the experimental points. The specific hydrogen production rate (SHPR) initially increased then decreased with increasing HCl concentration (0.5%e8%), with a maximum of 4.60 mL/h occurring at 4% HCl. The SHPR continuously decreased with increasing NaOH concentration (0.5%e4%). This is possibly because HCl and NaOH pretreatments produced inhibitors, such as furfural, that inhibited the SHPR of hydrogen-producing bacteria [23]. These results suggest that higher HCl and NaOH concentrations were unfavorable to enhancing SHPR. It was noted that the SHPRs at 0.5 and 1% NaOH were very large, indicating that a lower NaOH concentration was favorable to enhancing SHPR. The lag-phase time changed only slightly when the HCl concentration increased from 0.5% to 4%, along with an increase at 8% HCl, however,
1124
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 1 2 0 e1 1 2 4
Table 2 e Hydrogen production characteristics at different HCl and NaOH concentrations.
UP HCl (w/v)
NaOH (w/v)
0 0.5% 1% 2% 4% 8% 0.5% 1% 2% 4%
P (mL)
Rm (mL/h)
l (h)
R2
4.39 16.31 27.71 37.67 72.21 67.79 19.25 12.2 9.66 16.32
3.53 1.37 2.46 2.96 4.60 3.93 15.35 11.60 2.24 0.31
7.41 7.77 9.31 8.77 8.56 12.82 7.40 7.95 16.12 8.75
1 0.996 0.999 0.999 0.996 0.997 1 1 0.995 0.992
P is hydrogen production potential (mL). Rm is the maximum hydrogen production rate (mL/h). l is the lag-phase time (h). R2 is correlation coefficient.
the lag-phase time initially increased then decreased with increasing NaOH concentration (0.5%e4%). As shown above, the optimal acid and alkaline pretreatment conditions were 4% HCl and 0.5% NaOH, respectively.
4.
Conclusions
In this work, HCl and NaOH were used to pretreat grass to enhance hydrogen yield. It was found that only H2 and CO2 were present in the biogas product, without detectable CH4 during the course of hydrogen production. The saccharification efficiency and hydrogen production from grass pretreated by acid and alkali were higher than those from unpretreated grass, indicating that pretreatment was necessary to enhance the TRS and hydrogen yields. Hydrogen production initially increased then decreased with increasing HCl concentration, however, it initially decreased then increased with increasing NaOH concentration. Acid pretreatment was superior to alkaline pretreatment for enhancing hydrogen production from grass. The main liquid metabolite found after fermentation was acetic acid, indicating that dark anaerobic fermentation of grass was mainly an acetic acid type fermentation.
Acknowledgments The authors would like to thank the Chinese Academy of Sciences for financial support (Item No. KJCX2-YW-H21).
references
[1] Das D, Veziroglu TN. Hydrogen production by biological processes: a survey of literature. Int J Hydrogen Energy 2001; 26:13e28. [2] Kotay SM, Das D. Biohydrogen as a renewable energy resource-prospects and potentials. Int J Hydrogen Energy 2008;33:258e63. [3] Levin DB, Pitt L, Love M. Biohydrogen production: prospects and limitations to practical application. Int J Hydrogen Energy 2004;29:173e85.
[4] Kim DH, Kim SH, Shin HS. Sodium inhibition of fermentative hydrogen production. Int J Hydrogen Energy 2009;34: 3295e304. [5] Zhang M, Fan Y, Xing Y, Pan C, Zhang G, Lay JJ. Enhanced biohydrogen production from cornstalk wastes with acidification pretreatment by mixed anaerobic cultures. Biomass Bioenergy 2007;31:250e4. [6] Cui M, Yuan Z, Zhi X, Wei L, Shen J. Biohydrogen production from poplar leaves pretreated by different methods using anaerobic mixed bacteria. Int J Hydrogen Energy 2010;35: 4041e7. [7] Cui M, Yuan Z, Zhi X, Shen J. Optimization of biohydrogen production from beer lees using anaerobic mixed bacteria. Int J Hydrogen Energy 2009;34:7971e8. [8] Nguyen TAD, Kim KR, Kim MS, Sim SJ. Thermophilic hydrogen fermentation from Korean rice straw by Thermotoga neapolitana. Int J Hydrogen Energy 2010;35: 13392e8. [9] Lo YC, Lu WC, Chen CY, Chang JS. Dark fermentative hydrogen production from enzymatic hydrolysate of xylan and pretreated rice straw by Clostridium butyricum CGS5. Bioresour Technol 2010;101:5885e91. [10] Cao G, Ren N, Wang A, Lee DJ, Guo W, Liu B, et al. Acid hydrolysis of corn stover for biohydrogen production using Thermoanaerobacterium thermosaccharolyticum W16. Int J Hydrogen Energy 2009;34:7182e8. [11] Fan Y, Xing Y, Ma H, Pan C, Hou H. Enhanced cellulosehydrogen production from corn stalk by lesser panda manure. Int J Hydrogen Energy 2008;33:6058e65. [12] Wang Y, Wang H, Feng X, Wang X, Huang J. Biohydrogen production from cornstalk wastes by anaerobic fermentation with activated sludge. Int J Hydrogen Energy 2010;35:3092e9. [13] Ren N, Wang A, Gao L, Xin L, Lee DJ, Su A. Bioaugmented hydrogen production from carboxymethyl cellulose and partially delignified corn stalks using isolated cultures. Int J Hydrogen Energy 2008;33:5250e5. [14] Datar R, Huang J, Maness PC, Mohaghehi A, Czernik S, Chorent E. Hydrogen production from the fermentation of corn stover biomass pretreated with a steam-explosion process. Int J Hydrogen Energy 2007;32:932e9. [15] Liu G, Shen J. Effects of culture and medium conditions on hydrogen production from starch using anaerobic bacteria. J Biosci Bioeng 2003;98:251e6. [16] Owen WF, Stuckey DC, Healy JB, Young LY, McCarty PL. Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Water Res 1979;13:485e93. [17] Logan BE, Oh SE, Kim IS, Ginkel SV. Biological hydrogen production measured in batch anaerobic respirometers. Environ Sci Technol 2002;36:2530e5. [18] Dubios M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956;28:350e6. [19] Van Soest PJ, Robertson JB, Lewis BA. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J Dair Sci 1991;74:3583e97. [20] Pan C, Zhang S, Fan Y, Hou H. Bioconversion of corncob to hydrogen using anaerobic mixed microflora. Int J Hydrogen Energy 2010;35:2663e9. [21] Zwietering MH, Jongenburger I, Rombouts FM, Riet KV. Modeling of the bacterial-growth curve. Appl Environ Microbiol 1990;56:1875e81. [22] Fang HHP, Li C, Zhang T. Acidophilic biohydrogen production from rice slurry. Int J Hydrogen Energy 2006;31:683e92. [23] Laser M, Schulman D, Allen SG, Lichwa J, Antal MJ, Lynd LR. A comparison of liquid hot water and steam pretreatments of sugar cane bagasse for bioconversion to ethanol. Bioresour Technol 2002;81:33e44.
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Effects of acid and alkaline pretreatments on the biohydrogen production from grass by anaerobic dark fermentation Maojin Cui, 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:
Batch experiments were carried out to study the effects of acid (HCl) and alkaline (NaOH)
Received 11 November 2010
pretreatments on saccharification of grass and subsequent microbial hydrogen production
Received in revised form
at 35 C and initial pH 7.0. Liquid metabolites and hydrogen production characteristics
25 January 2011
were also studied. Results show that the saccharification efficiency and hydrogen
Accepted 14 February 2011
production from grass pretreated by acid and alkali were higher than those from unpre-
Available online 15 March 2011
treated grass and that acid pretreatment was better than alkaline pretreatment for enhancing the hydrogen yield from grass. A maximum cumulative hydrogen yield of
Keywords:
72.21 mL/g-dry grass was achieved from substrate pretreated with 4% HCl, which was
Grass
16.45-fold greater than that from untreated substrate. Only H2 and CO2 were present in the
Pretreatment
biogas. The main liquid metabolite found after fermentation was acetic acid.
Anaerobic dark fermentation
Copyright ª 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
Biohydrogen
1.
Introduction
Global energy requirements depend heavily upon fossil fuels such as coal, oil and natural gas. There is urgency in the search to replace these sources of energy, as the depletion of limited fossil fuels is inevitable [1]. The extensive use of fossil fuels has also caused an environmental issue where emission of CO2, CO, NOx and SOx during combustion has caused air pollution and global warming. Compared with fossil fuels, hydrogen is a promising candidate as a clean energy carrier in the future because it has high-energy yield (122 kJ/g) and generates no pollutants, producing only water when used as a fuel [2]. Hydrogen production by physical or chemical methods has been well developed. Biological processes, such as dark and photo fermentation, however, are environmentally benign and less energy intensive compared with physical and chemical processes [1]. Dark fermentation has been reported recently as the hydrogen production system having
reserved.
the greatest potential [3]. Some of the basic advantages of dark fermentation over other processes include process simplicity, better process economy resulting from lower energy requirements, higher hydrogen production rates and utilization of low-value wastes as raw materials [3,4]. Biomass residues such as leaves, grass and crop stalks are abundant in the world. It was reported that the annual global yields of biomass residues exceed 220 billion tons [5]. Most of them, however, are discarded or burned as environmental pollutants. They can be a valuable and vast renewable resource if their energy content is stored as hydrogen. Numerous studies have been carried out to produce hydrogen by using biomass residues as substrate such as poplar leaves [6], beer lees [7], rice straw [8,9] and corn stalks [10,11,12,13]. The direct utilization of biomass residues by microorganisms, however, is difficult because of the heterogeneity and crystallinity of such materials [14]. For instance, the maximum hydrogen yields from raw beer lees and poplar leaves were
* Corresponding author. Tel.: þ86 10 62620903; fax: þ86 10 62559373. E-mail address: [email protected] (J. Shen). 0360-3199/$ e see front matter Copyright ª 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2011.02.078
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 1 2 0 e1 1 2 4
3.16 and 15.04 mL/g-S, respectively [6,7]. Thus, biomass has to be pretreated to release its underlying monomeric sugar prior to bacterial fermentation. Physical, chemical and biological substrate pretreatment methods, or their combinations, have proven useful for enhancing hydrogen yields [6,7,8,9,10,11,12,13]. The objective of this research was to evaluate the effects of acid and alkaline pretreatment on hydrogen production from grass using anaerobic mixed bacteria. For this purpose, the total reducing sugar (TRS) and hydrogen production from raw and pretreated substrate were studied. Liquid metabolites and hydrogen production characteristics were also studied. A maximum cumulative hydrogen yield of 72.21 mL/g-dry grass was achieved from pretreated substrate, which was 16.45-fold greater than that from raw substrate. The result is encouraging because of its potential commercial and environmental benefits in the future.
2.
Materials and methods
2.1.
Seed microorganisms
The hydrogen-producing mixed cultures were enriched from cracked cereal and identified to be dominated by Clostridium pasteurianum [15]. The cultures’ acclimation and medium were the same as our previous work described in Ref [6].
2.2.
Raw materials and pretreatment
The grass used in this study was collected in winter from the lawn of our institute, 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. 1.0 g of dry grass 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.
Experimental procedures
Batch experiments were carried out in 120 mL serum vials. The raw and pretreated grass was used as substrate. The total work volume was 80 mL (approximately 1.5% (w/v) solids loading) in each case. 1 L 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. 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.
2.4.
1121
Analytical methods
The hydrogen content was determined by a gas chromatograph (Techcomp. Co., China, 7890II) equipped with a thermal conductivity detector (TCD) and a 2-m stainless steel column packed with Porapak Q (80e100 mesh). The operating temperatures of the injection port, the oven and the detector were set at 80, 60 and 100 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 [16]. The cumulative hydrogen volume was calculated by equation (1) [17]: VH;i ¼ VH;i1 þ CH;i VG;i VG;i1 þ VH;0 CH;i CH;i1
(1)
where VH, i is the cumulative hydrogen volume at the current time interval, VH, i 1 is the cumulative hydrogen volume at the previous time interval, VG, i is the total biogas volume in the current time interval, VG, i -1 is the total biogas volume in the previous time interval, CH, i is the fraction of hydrogen in the headspace of the bottle at the current time interval, CH, i-1 is the fraction of hydrogen in the headspace of the bottle at the previous time interval and VH,0 is the total volume of headspace in the bottle. Detection of the alcohols and volatile fatty acids (VFAs, C2-C5) was measured by a gas chromatograph using a flame ionization detector (FID) and a 2-m glass column packed with Unisole F-200 (30e60 mesh). The temperatures of the injection port, the oven and the detector were set at 200, 165 and 200 C, respectively. The carrier gas was argon at a flow rate of 30 mL/min. The concentration of the total reducing sugar was determined by the phenol-sulfuric acid method using glucose as a standard [18]. The contents of hemicellulose, cellulose and lignin were determined according to Van Soest’s method [19].
3.
Results and discussion
3.1. grass
Effect of pretreatment on the saccharification of
Acid and alkaline pretreatments are two effective methods to pretreat cellulosic biomass that mainly convert cellulosic biomass into soluble sugars, which can then be used by microorganisms to produce hydrogen [6,7]. The yields of the total reducing sugar (TRS) resulting from 1.0 g of dry grass after HCl and NaOH pretreatments were analyzed and are shown in Fig. 1. TRS yield from pretreated grass was higher than that from raw grass, indicating that pretreatment is critical for increasing the saccharification of grass. The TRS yield was 56.65 mg/g-S in raw substrate and increased with increasing HCl and NaOH concentrations. The amounts of TRS after 8% HCl and 8% NaOH pretreatment were approximately 6.40-fold and 5.84-fold greater than that in the raw substrate. It was observed that the TRS yield with HCl pretreatment was always higher than that with NaOH pretreatment at equal HCl and NaOH concentrations, in agreement with our previous work [6]. To better investigate the chemical composition of raw and pretreated substrates, the contents of hemicellulose, cellulose and lignin in raw substrate and substrate pretreated with 4%
1122
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 1 2 0 e1 1 2 4
80 UP
HCl
350
0.5%
70
NaOH
1% 300
Cumulative hydrogen (mL)
TRS yield (mg/g-S)
250
200
150
100
50
0
4% 8%
50 40 30 20 10
UP
0.5%
1%
2%
4%
0
8%
0
HCl or NaOH Concentratiton (w/v)
20
40
60
80
100
Time (hour)
Fig. 1 e Effects of HCl and NaOH concentrations on the saccharification of grass. UP means unpretreatment. HCl and 0.5% NaOH were analyzed (Fig. 2). These concentrations were the optimal conditions for acid and alkaline pretreatment for hydrogen production according to Fig. 3, Fig. 4 and Table 1. From Fig. 2, it can be seen that raw grass consisted mainly of hemicellulose (42.97%) and cellulose (30.82%). Compared with raw substrate, the content of hemicellulose in substrate pretreated with 4% HCl decreased dramatically, the content of cellulose decreased slightly, and there was an increase in lignin content. The contents of hemicellulose, cellulose and lignin in substrate pretreated with 0.5% NaOH decreased dramatically compared with their levels in raw substrate. These results indicate that acid pretreatment mainly degraded hemicellulose, which is consistent with our previous work [7], but alkaline pretreatment could efficiently degrade hemicellulose, cellulose and lignin.
3.2.
2%
60
Effect of pretreatment on hydrogen production
Fig. 3 e Cumulative hydrogen volumes from 1.0 g of dry grass pretreated by different HCl concentrations versus corresponding fermentation time. The operation was at 35 C and initial pH 7.0.
hydrogen at an initial pH of 7.0. The effects of HCl and NaOH concentrations on the cumulative hydrogen volumes (CHVs) are presented in Figs. 3 and 4. It can be seen from Fig. 3 that the CHV from raw grass was 4.39 mL and that the CHVs initially increased then decreased as the HCl pretreatment concentration was increased (0.5e8%). The maximum hydrogen volume of 72.21 mL was observed at 4% HCl, which was 16.45fold higher than that from the raw substrate. It can be seen from Fig. 4 that the CHVs initially decreased then increased with increasing NaOH concentration (0.5e4%). A maximum hydrogen volume of 19.25 mL was observed at 0.5% NaOH, which was 4.38-fold higher than that from the raw substrate. 24 h was required to reach the maximum CHV from grass pretreated with 0.5% NaOH but 92 h was required with 4%
1.0 g samples of dry grass pretreated with different HCl and NaOH concentrations were used as substrates to produce UP
20 40
0.5%
UP
1%
4% HCl
2% 4%
Cumulative hydrogen (mL)
Content (% dry weigh)
0.5% NaOH
30
20
15
10
5
10
0 0
Hemicellulose
Cellulose
Lignin
Chemical composition
Fig. 2 e Contents of hemicellulose, cellulose and lignin in grass without pretreatment and pretreated by 4% HCl and 0.5% NaOH. UP means unpretreatment.
0
20
40
60
80
100
120
Time (hour)
Fig. 4 e Cumulative hydrogen volumes from 1.0 g of dry grass pretreated by different NaOH concentrations versus corresponding fermentation time. The operation was at 35 C and initial pH 7.0.
1123
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 1 2 0 e1 1 2 4
Table 1 e Effects of HCl and NaOH concentrations on the degradation efficiency of total reducing sugar, H2 yields, CO2 yields and yields of ethanol and VFAs after fermentation. DESa (%) UP HCl (w/v)
NaOH (w/v)
0 0.5% 1% 2% 4% 8% 0.5% 1% 2% 4%
84.61 88.94 92.60 93.45 91.97 86.50 79.93 78.94 73.26 69.06
0.56b 0.26 0.33 0.32 0.14 0.24 1.48 1.35 3.82 2.72
H2 yield (mL/g-S) 4.39 0.20 16.31 0.44 27.71 0.48 37.67 1.43 72.21 0.76 67.79 1.08 19.25 0.58 12.20 0.85 9.66 0.47 16.32 0.38
CO2 yield (mL/g-S) 12.41 36.09 51.69 52.13 66.49 70.21 16.75 17.20 31.14 39.48
0.06 0.46 3.67 1.47 3.75 0.67 0.24 1.40 2.15 1.07
Ethanol (mg/g-S)c 6.70 7.37 6.82 6.73 34.15 53.50 8.21 6.88 6.68 16.49
0.17 0.19 0.14 0.37 0.68 1.17 0.12 0.13 0.23 0.37
Acetic acid (mg/g-S) 101.71 10.05 264.19 11.38 266.93 11.74 250.55 15.60 222.06 4.97 145.71 4.84 240.08 12.37 167.92 2.32 174.45 2.26 105.57 2.88
Propionic acid (mg/g-S) 40.74 91.21 92.15 94.12 33.27 15.98 152.58 148.90 153.63 65.44
0.94 4.39 2.69 4.39 1.12 1.04 8.68 2.38 0.40 1.89
n-Butyric acid (mg/g-S) 10.94 3.84 22.78 1.28 15.12 0.43 10.38 0.32 9.78 0.38 4.55 0.57 30.11 1.76 19.16 0.54 11.05 0.18 5.29 0.22
a DES means degradation efficiency of total reducing sugar. b Mean standard deviation, n ¼ 3. c (mg/g-S) means (mg/g-dry grass).
NaOH, indicating that higher NaOH concentration could delay the completion of fermentation. These results indicate that HCl pretreatment was superior to NaOH pretreatment, consistent with previous studies [5,20]. As shown above, CHV from pretreated substrate was higher than that from raw substrate, suggesting that pretreatment is necessary to enhance hydrogen yield.
NaOH concentration increased from 0.5% to 2% and decreased at 4% NaOH. The yields of n-butyric acid decreased gradually with increasing NaOH concentration (0.5%e4%). Comparing the yields of the four products, the acetic acid concentration was the highest, indicating that dark anaerobic fermentation of grass was mainly an acetic acid type fermentation.
3.4. 3.3.
Kinetic analysis
Effect of pretreatment on metabolites
Gas product analyses showed that only H2 and CO2 were present in the biogas and no other gases (such as CH4) were detected during the course of hydrogen production. The effects of HCl and NaOH concentrations on the degradation efficiency of the total reducing sugar (DES), hydrogen yields, carbon dioxide yields and main liquid metabolites are shown in Table 1. The DES from substrate pretreated by HCl initially increased then decreased with increased HCl concentration (0.5%e8%) and a maximum of 93.45% occurred at 2% HCl. However, the DES from substrate pretreated by NaOH decreased continually with increasing NaOH concentration (0.5%e4%) and a maximum of 79.93% was observed at 0.5% NaOH. Clearly, the DES from substrate pretreated with HCl was higher than that from substrate pretreated with NaOH, indicating that acid pretreatment is more effective for the degradation of TRS than alkaline pretreatment. The hydrogen maximum of 72.21 mL/g-S was observed at 4% HCl and the carbon dioxide maximum of 70.21 mL/g-S was observed at 8% HCl. The liquid product analysis showed that the metabolites found after fermentation were mainly ethanol, acetic acid, propionic acid and n-butyric acid, with little iso-butyric acid, iso-valeric acid and n-valeric acid. The yields of ethanol were low at lower HCl concentrations (0.5%e2%) but high at higher HCl concentrations (4%e8%). The yields of acetic acid changed only slightly when the HCl concentration increased from 0.5% to 4% and decreased at 8% HCl. The yields of n-butyric acid decreased gradually, along with an initial increase in propionic acid, which subsequently decreased when the HCl concentration increased from 0.5% to 8%. The yields of ethanol were low at lower NaOH concentrations (0.5%e2%) and were high at higher NaOH concentration (4%). The yields of acetic acid and propionic acid changed only slightly as the
The hydrogen production potential, maximum hydrogen production rate and lag-phase time were elucidated using the modified Gompertz equation (equation (2)) that has been used to describe the progress of the cumulative hydrogen production from batch experiments [21,22]. Rm e ðl tÞ þ 1 H ¼ P exp P
(2)
where H is the cumulative hydrogen production (mL), P is hydrogen production potential (mL), Rm is the maximum hydrogen production rate (mL/hour), e ¼ 2.71828, l is the lagphase time (hour), and t is the incubation time (hr).The corresponding values of P, Rm and l for each batch were estimated using Origin 7.5, a scientific graphing and data analysis software package. The data in Figs. 3 and 4 were simulated using equation (2) and the hydrogen production characteristics are shown in Table 2. According to the data in Table 2, all R2 values were more than 0.990, indicating that the fitted curves matched well with the experimental points. The specific hydrogen production rate (SHPR) initially increased then decreased with increasing HCl concentration (0.5%e8%), with a maximum of 4.60 mL/h occurring at 4% HCl. The SHPR continuously decreased with increasing NaOH concentration (0.5%e4%). This is possibly because HCl and NaOH pretreatments produced inhibitors, such as furfural, that inhibited the SHPR of hydrogen-producing bacteria [23]. These results suggest that higher HCl and NaOH concentrations were unfavorable to enhancing SHPR. It was noted that the SHPRs at 0.5 and 1% NaOH were very large, indicating that a lower NaOH concentration was favorable to enhancing SHPR. The lag-phase time changed only slightly when the HCl concentration increased from 0.5% to 4%, along with an increase at 8% HCl, however,
1124
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 1 2 0 e1 1 2 4
Table 2 e Hydrogen production characteristics at different HCl and NaOH concentrations.
UP HCl (w/v)
NaOH (w/v)
0 0.5% 1% 2% 4% 8% 0.5% 1% 2% 4%
P (mL)
Rm (mL/h)
l (h)
R2
4.39 16.31 27.71 37.67 72.21 67.79 19.25 12.2 9.66 16.32
3.53 1.37 2.46 2.96 4.60 3.93 15.35 11.60 2.24 0.31
7.41 7.77 9.31 8.77 8.56 12.82 7.40 7.95 16.12 8.75
1 0.996 0.999 0.999 0.996 0.997 1 1 0.995 0.992
P is hydrogen production potential (mL). Rm is the maximum hydrogen production rate (mL/h). l is the lag-phase time (h). R2 is correlation coefficient.
the lag-phase time initially increased then decreased with increasing NaOH concentration (0.5%e4%). As shown above, the optimal acid and alkaline pretreatment conditions were 4% HCl and 0.5% NaOH, respectively.
4.
Conclusions
In this work, HCl and NaOH were used to pretreat grass to enhance hydrogen yield. It was found that only H2 and CO2 were present in the biogas product, without detectable CH4 during the course of hydrogen production. The saccharification efficiency and hydrogen production from grass pretreated by acid and alkali were higher than those from unpretreated grass, indicating that pretreatment was necessary to enhance the TRS and hydrogen yields. Hydrogen production initially increased then decreased with increasing HCl concentration, however, it initially decreased then increased with increasing NaOH concentration. Acid pretreatment was superior to alkaline pretreatment for enhancing hydrogen production from grass. The main liquid metabolite found after fermentation was acetic acid, indicating that dark anaerobic fermentation of grass was mainly an acetic acid type fermentation.
Acknowledgments The authors would like to thank the Chinese Academy of Sciences for financial support (Item No. KJCX2-YW-H21).
references
[1] Das D, Veziroglu TN. Hydrogen production by biological processes: a survey of literature. Int J Hydrogen Energy 2001; 26:13e28. [2] Kotay SM, Das D. Biohydrogen as a renewable energy resource-prospects and potentials. Int J Hydrogen Energy 2008;33:258e63. [3] Levin DB, Pitt L, Love M. Biohydrogen production: prospects and limitations to practical application. Int J Hydrogen Energy 2004;29:173e85.
[4] Kim DH, Kim SH, Shin HS. Sodium inhibition of fermentative hydrogen production. Int J Hydrogen Energy 2009;34: 3295e304. [5] Zhang M, Fan Y, Xing Y, Pan C, Zhang G, Lay JJ. Enhanced biohydrogen production from cornstalk wastes with acidification pretreatment by mixed anaerobic cultures. Biomass Bioenergy 2007;31:250e4. [6] Cui M, Yuan Z, Zhi X, Wei L, Shen J. Biohydrogen production from poplar leaves pretreated by different methods using anaerobic mixed bacteria. Int J Hydrogen Energy 2010;35: 4041e7. [7] Cui M, Yuan Z, Zhi X, Shen J. Optimization of biohydrogen production from beer lees using anaerobic mixed bacteria. Int J Hydrogen Energy 2009;34:7971e8. [8] Nguyen TAD, Kim KR, Kim MS, Sim SJ. Thermophilic hydrogen fermentation from Korean rice straw by Thermotoga neapolitana. Int J Hydrogen Energy 2010;35: 13392e8. [9] Lo YC, Lu WC, Chen CY, Chang JS. Dark fermentative hydrogen production from enzymatic hydrolysate of xylan and pretreated rice straw by Clostridium butyricum CGS5. Bioresour Technol 2010;101:5885e91. [10] Cao G, Ren N, Wang A, Lee DJ, Guo W, Liu B, et al. Acid hydrolysis of corn stover for biohydrogen production using Thermoanaerobacterium thermosaccharolyticum W16. Int J Hydrogen Energy 2009;34:7182e8. [11] Fan Y, Xing Y, Ma H, Pan C, Hou H. Enhanced cellulosehydrogen production from corn stalk by lesser panda manure. Int J Hydrogen Energy 2008;33:6058e65. [12] Wang Y, Wang H, Feng X, Wang X, Huang J. Biohydrogen production from cornstalk wastes by anaerobic fermentation with activated sludge. Int J Hydrogen Energy 2010;35:3092e9. [13] Ren N, Wang A, Gao L, Xin L, Lee DJ, Su A. Bioaugmented hydrogen production from carboxymethyl cellulose and partially delignified corn stalks using isolated cultures. Int J Hydrogen Energy 2008;33:5250e5. [14] Datar R, Huang J, Maness PC, Mohaghehi A, Czernik S, Chorent E. Hydrogen production from the fermentation of corn stover biomass pretreated with a steam-explosion process. Int J Hydrogen Energy 2007;32:932e9. [15] Liu G, Shen J. Effects of culture and medium conditions on hydrogen production from starch using anaerobic bacteria. J Biosci Bioeng 2003;98:251e6. [16] Owen WF, Stuckey DC, Healy JB, Young LY, McCarty PL. Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Water Res 1979;13:485e93. [17] Logan BE, Oh SE, Kim IS, Ginkel SV. Biological hydrogen production measured in batch anaerobic respirometers. Environ Sci Technol 2002;36:2530e5. [18] Dubios M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956;28:350e6. [19] Van Soest PJ, Robertson JB, Lewis BA. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J Dair Sci 1991;74:3583e97. [20] Pan C, Zhang S, Fan Y, Hou H. Bioconversion of corncob to hydrogen using anaerobic mixed microflora. Int J Hydrogen Energy 2010;35:2663e9. [21] Zwietering MH, Jongenburger I, Rombouts FM, Riet KV. Modeling of the bacterial-growth curve. Appl Environ Microbiol 1990;56:1875e81. [22] Fang HHP, Li C, Zhang T. Acidophilic biohydrogen production from rice slurry. Int J Hydrogen Energy 2006;31:683e92. [23] Laser M, Schulman D, Allen SG, Lichwa J, Antal MJ, Lynd LR. A comparison of liquid hot water and steam pretreatments of sugar cane bagasse for bioconversion to ethanol. Bioresour Technol 2002;81:33e44.