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Comparison of bio-hydrogen production from hydrolyzed wheat starch by mesophilic and thermophilic 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 5 ( 2 0 1 0 ) 1 3 2 1 4 e1 3 2 1 8
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Comparison of bio-hydrogen production from hydrolyzed wheat starch by mesophilic and thermophilic dark fermentation Ayse Cakır, Serpil Ozmihci, Fikret Kargi* Department of Environmental Engineering, Dokuz Eylul University, Buca, Izmir, Turkey
article info
abstract
Article history:
Hydrogen gas production potentials of acid-hydrolyzed and boiled ground wheat were
Received 12 August 2010
compared in batch dark fermentations under mesophilic (37 C) and thermophilic (55 C)
Received in revised form
conditions. Heat-treated anaerobic sludge was used as the inoculum and the hydrolyzed
6 September 2010
ground wheat was supplemented by other nutrients. The highest cumulative hydrogen gas
Accepted 8 September 2010
production (752 ml) was obtained from the acid-hydrolyzed ground wheat starch at 55 C
Available online 12 October 2010
and the lowest (112 ml) was with the boiled wheat starch within 10 days. The highest rate of hydrogen gas formation (7.42 ml H2 h1) was obtained with the acid-hydrolyzed and the
Keywords:
lowest (1.12 ml H2 h1) with the boiled wheat at 55 C. The highest hydrogen gas yield
Acid hydrolysis
(333 ml H2 g1 total sugar or 2.40 mol H2 mol1 glucose) and final total volatile fatty acid
Bio-hydrogen
(TVFA) concentration (10.08 g L1) were also obtained with the acid-hydrolyzed wheat
Dark fermentation
under thermophilic conditions (55 C). Dark fermentation of acid-hydrolyzed ground wheat
Mesophilic
under thermophilic conditions (55 C) was proven to be more beneficial as compared to
Thermophilic
mesophilic or thermophilic fermentation of boiled (partially hydrolyzed) wheat starch.
Wheat starch
1.
ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.
Introduction
Hydrogen gas is a clean fuel with high energy content (122 kJ g1) and is considered to be one of the major energy carriers in the future. Air pollution and global warming problems caused by fossil fuels can be overcome by utilization of hydrogen with no COx, NOx, SOx emissions. Despite the aforementioned advantages, hydrogen gas is not readily available in nature and is currently produced by energy intensive chemical processes such as steam reforming of natural gas or by water electrolysis [1]. Hydrogen gas production by fermentation of carbohydrate rich raw materials has distinct advantages over other methods due to operation under mild conditions. Major obstacles in bio-hydrogen production are low production rates and yields requiring large fermentation volumes [1e4].
Starch and cellulose containing renewable resources such as biomass and agricultural wastes constitute inexpensive and easily fermentable raw materials for bio-hydrogen production. Hydrolysis (acid or enzymatic) of starch/cellulose to highly concentrated sugar solution is the first step in fermentative hydrogen gas production from waste biomass. Dark fermentation of resulting carbohydrates to volatile fatty acids (VFA), H2 and CO2 by acetogenic-anaerobic bacteria and photo fermentation of VFAs to CO2 and H2 by photo-heterotrophic bacteria (Rhodobacter sp.) are the next steps in fermentative hydrogen gas production [1,2]. Heat-treated anaerobic sludge has been the most widely used culture for dark fermentation of starch and cellulose along with some pure cultures of Clostridia and Enterobacter sp. [5e10]. Bio-hydrogen production from ground wheat starch by
* Corresponding author. Tel.: þ90 232 4127109. E-mail address: [email protected] (F. Kargi). 0360-3199/$ e see front matter ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2010.09.029
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 5 ( 2 0 1 0 ) 1 3 2 1 4 e1 3 2 1 8
dark fermentation has been studied extensively using heattreated anaerobic sludge [11e15]. Partial hydrolysis of ground wheat starch by boiling improved the rate of hydrogen production in dark fermentation [16]. Heat pre-treatment of anaerobic sludge was found to be the most suitable method among the other pre-treatment methods [17]. Starch can be hydrolyzed by several different methods. Acid hydrolysis at high temperature (90e150 C) yields nearly complete breakdown of starch to carbohydrate molecules. Partial hydrolysis of starch by boiling followed by bacterial (enzymatic) hydrolysis is a slow, but inexpensive method commonly used. Hydrolysis method affects the yield and formation rate of hydrogen gas. Dark fermentation of hydrolyzed starch can be realized by using mesophilic (37 C) or thermophilic (55 C) bacteria. Most of the dark fermentation studies were realized at 37 C using mesophilic bacteria [5e15]. Thermophilic dark fermentation has also been used by some investigators for bio-hydrogen production from different substrates [18e24]. In the light of literature reports this study was designed to determine the most suitable hydrolysis method and dark fermentation temperature by using acid-hydrolyzed or boiled ground wheat for dark fermentation at 37 C and 55 C. Hydrogen gas formation rates and the yields were used as comparison criteria for different fermentation conditions.
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the beginning of the experiments to sustain anaerobic conditions. The experimental bottles were placed in constant temperature incubators at 37 C or 55 C after inoculation. A control bottle without inoculation was used for comparison. In another set of experiments, the ground wheat (200 mesh) was boiled for 1.5 h for partial hydrolysis of starch to reducing sugars. The same experimental procedure was used for partially hydrolyzed wheat starch as that of the acidhydrolyzed wheat.
2.2.
Organisms
2.
Materials and methods
Anaerobic sludge was obtained from acidogenic phase of anaerobic wastewater treatment plant of PAKMAYA Bakers Yeast Co., Izmir, Turkey. After pH adjustment to 5.9, the sludge was boiled for about 1 h in order to eliminate methanogenic bacteria and to select spore forming acidogenic bacteria. The heat-treated sludge was adapted to thermophilic conditions before use by cultivating at 55 C for several growth cycles of 3 days duration. The cultivation media contained glucose (60 g L1), peptone (10 g L1), yeast extract (0.6 g L1), MgSO4$7H2O (0.25 g L1), K2HPO4 (1 g L1), KH2PO4 (1 g L1), 1 L-cysteineeHCl$H2O (0.1 g L ). Argon gas was passed through the cultivation media before incubation and the cultivation flasks were closed with gas-tight rubber stoppers. The cultivated organisms were centrifuged at 8000 rpm (7000g) and resuspended in phosphate buffer to be used for inoculation of the experimental bottles.
2.1.
Experimental system and procedure
2.3.
Four sets of batch dark fermentation experiments were carried out under mesophilic (37 C) and thermophilic (55 C) conditions using either acid-hydrolyzed or boiled ground wheat. Duplicate experiments were carried out using 0.5 L serum bottles (Isolab-Germany Boro 3.3) with 0.25 L fermentation volume. Silicone rubber stoppers and screw caps were used to avoid gas leakage from the bottles. The waste wheat was ground down to 200 mesh size and the pH of the powdered wheat solution (20 g L1 ground wheat) was adjusted to pH ¼ 2.5 using sulfuric acid. The wheat powder (WP) solution was autoclaved at 121 C for 15 min for hydrolysis of the wheat starch to reducing sugar compounds. The yield of starch conversion to soluble total sugar was 95% in acid hydrolysis. The mixture was centrifuged at 8000 rpm (7000g) for 15 min to separate the solids after acid hydrolysis. The supernatant sugar solution was neutralized by adding 10% (w v1) NaOH solution. The initial total sugar (TS) concentration was So ¼ 18.5 0.3 g L1. The serum bottles were inoculated with 10 ml heat-treated anaerobic sludge. The oxidation reduction potential (ORP) was adjusted to nearly 200 mV by addition of L-cysteineeHCl$H2O. Ground wheat contained nearly 97% (w w1) starch, 3.4 mg N g1 PW, 1.72 mg P g1 PW, 1.25 mg Fe g1 PW. Since the nitrogen and phosphorous contents of WP were not sufficient for fermentation, (NH4)2SO4 and KH2PO4 were added to the fermentation media in order to adjust the C/N/P ratio to 100/1/0.3. The fermentation medium was also supplemented with 100 mg L1 MgSO4$7H2O and 25 mg L1 FeSO4$7H2O. Argon gas was passed through the head space of the bottles for 5 min at
Analytical methods
Samples were removed from the liquid phase everyday for analyses of total sugar including starch (TS), and total volatile fatty acids (TVFA). For total sugar (TS) analysis, the samples containing starch and soluble sugar were acidified and boiled for 1.5 h for complete hydrolysis of starch and the resulting total sugar concentration was determined by using the acidephenol method [25]. The samples were also centrifuged at 7000g to remove solids from the liquid media. Soluble sugar and TVFA analyses were carried out in clear supernatants. Soluble sugar content was determined by the acidephenol spectrometric method [25]. TVFA analyses were carried out by using analytical kits (Spectroquant, 1.01763. 0001, Merck, Darmstadt, Germany) and a PC spectrometer (WTW Photolab S12). Hydrogen gas was sampled from the head space of the bottles by using gas-tight glass syringes. Hydrogen gas concentrations in the gas phase were determined by using a gas chromatograph (HP Agilent 6890). The column was Alltech, Hayesep D 80/100 600 1/800 0.08500 . N2 gas was used as carrier with a flow rate of 30 ml min1 and the head pressure was 1.5 atm (22 psig). The amount of total gas produced was determined by water displacement method everyday using sulfuric acid (2%) and NaCl (10%) containing solution. The cumulative hydrogen gas production was determined as explained in our previous publications [11e17]. pH and ORP of the fermentation medium were monitored by using pH and ORP meters with the relevant probes (WTW Sci., Germany). pH of the medium decreased from an initial value of 7.0 to nearly 4.8 due to VFA production which was
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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 5 ( 2 0 1 0 ) 1 3 2 1 4 e1 3 2 1 8
adjusted to 7.0 by addition of 10 M NaOH everyday. ORP values varied between 50 and 150 mV, in general.
3.
Results and discussion
Four sets of duplicate batch experiments were performed for bio-hydrogen production using: (a) boiled ground wheat at 37 C, (b) boiled ground wheat at 55 C, (c) acid-hydrolyzed ground wheat at 37 C, (d) acid-hydrolyzed ground wheat at 55 C in order to determine the most favorable hydrolysis method and fermentation temperature. Boiling was used for partial hydrolysis of wheat starch and acid hydrolysis in autoclave was used for complete hydrolysis before dark fermentation. Boiled wheat powder was subjected to bacterial hydrolysis followed by dark fermentation of resulting carbohydrates. Fig. 1 depicts variation of cumulative hydrogen gas formation (CHF) with time for the four different experiments described above. The highest CHF (752 ml) was obtained with the acid-hydrolyzed wheat starch under thermophilic conditions (55 C) within 10 days. CHF (600 ml) obtained from acidhydrolyzed wheat starch under mesophilic conditions (37 C) was comparable to that obtained at 55 C. CHFs obtained from boiled wheat powder (WP) at 55 C and 37 C were 115 ml and 125 ml, respectively. Hydrogen gas formation from boiled WP at 55 C was faster than that of 37 C. The determining factor for cumulative hydrogen gas volume is the method of hydrolysis rather than fermentation temperature. Sugar solution obtained from acid hydrolysis of wheat starch contained more readily fermentable glucose and maltose for hydrogen gas formation as compared to partially hydrolyzed wheat powder by boiling. Bacterial hydrolysis and fermentation of starch in boiled WP resulted in lower rate and extent of hydrogen gas formation under both mesophilic and thermophilic conditions. Variations of total sugar (starch þ soluble sugar) concentrations with time are depicted in Fig. 2 for the acid-hydrolyzed and boiled WP fermentations under mesophilic and thermophilic conditions. Unlike hydrogen gas formation, total
Fig. 2 e Variation of total sugar concentration with time for A boiled WP fermentation at 55 C, - acid-hydrolyzed WP fermentation at 55 C, : boiled WP fermentation at 37 C, C acid-hydrolyzed WP fermentation at 37 C.
sugar removal from the medium was faster for boiled wheat starch than that obtained from acid hydrolysis. Initial total sugar concentration decreased from 18.5 g L1 to less than 1 g L1 within 120 h (5 days) for mesophilic (37 C) fermentation of boiled WP despite the fact that H2 gas production was low with this fermentation. Polysaccharides derived from partial hydrolysis of boiled starch may have been deposited by the bacteria instead of fermentation to VFA and H2 gas. At the end of 10 days of dark fermentation, more than 85% total sugar was utilized in all cases indicating effective utilization of total sugars derived from starch either by deposition or product formation. Cumulative hydrogen gas formation data depicted in Fig. 1 were correlated with the Gompertz equation and the constants were determined by regression analysis in order to estimate the rate and the extent of hydrogen gas production for different experiments. The Gompertz equation has the following form Rm e ðl tÞ þ 1 HðtÞ ¼ P exp exp P
(1)
where, H is the cumulative hydrogen gas volume at any time t (ml H2), P is the maximum potential hydrogen formation (ml), Rm is the maximum rate of hydrogen formation (ml h1), l is duration of the lag phase, ‘e’ is 2.718 and ‘t’ is time (h). Table 1 presents the Gompertz equation coefficients for different dark fermentation conditions. The highest cumulative hydrogen
Table 1 e Gompertz equation constants for the mesophilic and thermophilic fermentations using acidhydrolyzed and boiled wheat powder. Fig. 1 e Variation of cumulative hydrogen gas formation with time for A boiled WP fermentation at 55 C, - acidhydrolyzed WP fermentation at 55 C, : boiled WP fermentation at 37 C, C acid-hydrolyzed WP fermentation at 37 C.
Substrate Boiled WP-55 C Acid-hydrolyzed WP-55 C Boiled WP-37 C Acid-hydrolyzed WP-37 C
P (ml)
Rm (ml h1)
l (h)
R2
115.7 752.3 125.7 599.9
1.12 7.42 1.38 4.29
24.1 31.6 69.3 44.3
0.99 0.99 0.99 0.99
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 5 ( 2 0 1 0 ) 1 3 2 1 4 e1 3 2 1 8
gas formation (752 ml) was obtained with the acid-hydrolyzed WP fermentation at 55 C (thermophilic fermentation) and the lowest CHF (115 ml) was realized with the boiled WP (partial hydrolysis) fermentation at 37 C (mesophilic fermentation). Clearly, acid hydrolysis of WP yielded readily fermentable glucose and maltose for bio-hydrogen production by dark fermentation. The highest (7.42 ml h1) and the lowest (1.12 ml h1) rates of hydrogen gas formation were also obtained with the acid hydrolyzed and boiled WP fermentations at 55 C, respectively. The lag phases for thermophilic fermentation were much lower than those of the mesophilic fermentation. The results clearly indicated that thermophilic fermentation of acid-hydrolyzed WP was superior to the mesophilic fermentation of acid-hydrolyzed or boiled WP in terms of the rate and extent of hydrogen gas formation. Another important parameter in bio-hydrogen production by dark fermentation is hydrogen gas yield (ml H2 g1 TS or mol H2 mol1 glucose). Fig. 3 depicts hydrogen yields for different fermentation conditions. The yields were calculated by using the following equation: YH2 ¼
VH2 VðSo SÞ
(2)
where YH2 is the hydrogen yield (ml H2 g1 TS), VH2 is the cumulative hydrogen gas volume at the end of dark fermentation (ml H2), V is the fermentation volume (L), So and S are the initial and final total sugar concentrations (g L1). The highest yield (333 ml H2 g1 TS ¼ 2.4 mol H2 mol1 glucose at 30 C and 1 atm) was obtained with the thermophilic (55 C) fermentation of acid-hydrolyzed WP. Boiled WP resulted in the lowest hydrogen yield of 40 ml H2 g1 TS by the mesophilic and thermophilic fermentation. Mesophilic fermentation of acid-hydrolyzed WP resulted in hydrogen gas yield of 220 ml g1 TS (1.6 mol H2 mol1 glucose at 30 C, 1 atm). Fermentation of glucose produced from acid hydrolysis of WP was a much favorable substrate as compared to boiled (partially hydrolyzed) WP resulting in high hydrogen yield and formation rate. Dark fermentation under thermophilic conditions (55 C) was also found to be more advantageous as compared to mesophilic fermentation at 37 C.
Fig. 4 e Final TVFA concentration in dark fermentation of acid-hydrolyzed and boiled wheat powder under mesophilic and thermophilic conditions.
Volatile fatty acids (VFAs) are by products of dark fermentation along with hydrogen gas. The extent of hydrogen gas formation strongly depends on the type and amount of VFAs produced. In dark fermentation, TVFA yield is usually around 60% of total sugar concentration. For the initial total sugar concentration of 18.5 g L1, the expected final TVFA concentration is around 11 g L1 for 60% conversion yield. As depicted in Fig. 4, final TVFA concentrations for dark fermentation of boiled WP were around 2 g L1 for both the thermophilic and mesophilic fermentations indicating low fermentation yields and therefore, low extent of hydrogen gas production. The highest final TVFA concentration (10.08 g L1) was obtained from the dark fermentation of acid-hydrolyzed WP under thermophilic conditions (55 C) which is very close to the theoretical yield. This result is in agreement with the high extent of hydrogen gas production from acid-hydrolyzed WP under thermophilic conditions. Dark fermentation of acidhydrolyzed WP under mesophilic conditions (37 C) yielded a final TVFA concentration of 6.9 g L1 indicating good extent of sugar fermentation yielding high TVFA and H2 gas formation. The results indicated that VFA and H2 gas formations were directly related and high final VFA concentrations yielded high volumes of hydrogen gas formation.
4.
Fig. 3 e Hydrogen yields for dark fermentation of acidhydrolyzed and boiled wheat powder under mesophilic and thermophilic conditions.
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Conclusion
Bio-hydrogen production from dark fermentation of acidhydrolyzed or boiled ground wheat starch was investigated under thermophilic (55 C) and mesophilic (37 C) conditions. The highest rate (7.42 ml h1) and extent (752 ml) of hydrogen gas formation was obtained from the thermophilic fermentation of acid-hydrolyzed wheat powder within 10 days. Hydrogen yield was also the highest (333 ml g1 TS ¼ 2.4 mol H2 mol1 glucose) with the acid-hydrolyzed wheat powder at 55 C. Polysaccharides derived from boiled wheat starch were probably deposited by the bacteria instead of fermentation to VFA and H2 gas. Acid hydrolysis of wheat starch yielded fermentable glucose for production of H2 gas by dark fermentation. High final TVFA concentration obtained
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from dark fermentation of the acid-hydrolyzed wheat starch at 55 C also supported high levels of H2 gas formation.
[13]
Acknowledgement [14]
This study was supported by the Scientific and Technological Research Council of Turkey by a grant number TUB 105M296. [15]
references [16] [1] Kapdan IK, Kargi F. Biohydrogen production from waste materials. Enzyme Microb Technol 2006;38:569e82. [2] Manish S, Banerjee R. Comparison of biohydrogen production processes. Int J Hydrogen Energy 2008;33:279e86. [3] Kotay SM, Das D. Biohydrogen as a renewable energy resource e prospects and potentials. Int J Hydrogen Energy 2008;33:258e63. [4] Meng N, Leung DYC, Leung MKH, Sumathy K. An overview of hydrogen production from biomass. Fuel Process Technol 2006;87:461e72. [5] Yokoi H, Saitsu A, Uchida H, Hirose J, Hayashi S, Takasaki Y. Microbial hydrogen production from sweet potato starch residue. J Biosci Bioeng 2001;91:58e63. [6] Liu G, Shen J. Effects of culture medium and medium conditions on hydrogen production from starch using anaerobic bacteria. J Biosci Bioeng 2004;98:251e6. [7] Liu H, Zhang T, Fang HHP. Thermophilic H2 production from cellulose containing wastewater. Biotechnol Lett 2003;25: 365e9. [8] Lay JJ. Biohydrogen generation by mesophilic anaerobic fermentation of micro-crystalline cellulose. Biotechnol Bioeng 2001;74:281e7. [9] Yokoi H, Maki R, Hirose J, Hayashi S. Microbial production of hydrogen from starch manufacturing wastes. Biomass Bioenergy 2002;22:389e95. [10] Han SK, Shin HS. Biohydrogen production by anaerobic fermentation of food waste. Int J Hydrogen Energy 2004;29: 569e77. [11] Argun H, Kargi F, Kapdan IK, Oztekin R. Biohydrogen production by dark fermentation of wheat powder solution: effects of C/N and C/P ratio on hydrogen yield and formation rate. Int J Hydrogen Energy 2008;33:1813e9. [12] Oztekin R, Kapdan IK, Kargi F, Argun H. Optimization of media composition for hydrogen gas production from
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hydrolyzed wheat starch by dark fermentation. Int J Hydrogen Energy 2008;33:4083e90. Argun H, Kargi F, Kapdan IK, Oztekin R. Batch dark fermentation of powdered wheat starch to hydrogen gas: effects of the initial substrate and biomass concentrations. Int J Hydrogen Energy 2008;33:6109e15. Argun H, Kargi F, Kapdan IK. Microbial culture selection for bio-hydrogen production from waste ground wheat by dark fermentation. Int J Hydrogen Energy 2009;34: 2195e200. Kargi F, Pamukoglu MY. Dark fermentation of ground wheat starch for bio-hydrogen production by fed-batch operation. Int J Hydrogen Energy 2009;34:2940e6. Argun H. PhD thesis. Izmir, Turkey: Dokuz Eylul University; January 2010. Argun H, Kargi F. Effects of sludge pre-treatment method on bio-hydrogen production by dark fermentation of waste ground wheat. Int J Hydrogen Energy 2009;34: 8543e8. Azbar N, Dokgoz FTC, Keskin T. Continuous fermentative hydrogen production from cheese whey wastewater under thermophilic anaerobic conditions. Int J Hydrogen Energy 2009;34:7441e7. Azbar N, Dokgoz FT, Keskin T. Comparative evaluation of biohydrogen production from cheese whey wastewater under thermophilic and mesophilic anaerobic conditions. Int J Green Energy 2009;6:192e200. Gavala HN, Skiadas IV, Ahring BK, Lyberatos G. Thermophilic anaerobic fermentation of olive pulp for hydrogen and methane production: modelling of the anaerobic digestion process. Water Sci Technol 2006;53:271e9. Kongjan P, O-Thong S, Kotay M, Min B, Angelidaki I. Biohydrogen production from what straw hydrolysate by dark fermentation using extreme thermophilic mixed culture. Biotechnol Bioeng 2009;105:899e908. Zang T, Liu H, Fang HHP. Bio-hydrogen production from starch in wastewater under thermophilic condition. J Environ Manage 2003;69:149e56. Kongjan P, Angelidaki I. Extreme thermophilic hydrogen production from wheat straw hydrolysate using mixed culture fermentation: effect of reactor configuration. Bioresour Technol 2010;101:7789e96. Yokoyama H, Ohmori H, Waki M, Ogino A, Tanaka Y. Continuous hydrogen production from glucose by using extreme thermophilic anaerobic microflora. J Biosci Bioeng 2009;107:64e6. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956;8:350e66.
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Comparison of bio-hydrogen production from hydrolyzed wheat starch by mesophilic and thermophilic dark fermentation Ayse Cakır, Serpil Ozmihci, Fikret Kargi* Department of Environmental Engineering, Dokuz Eylul University, Buca, Izmir, Turkey
article info
abstract
Article history:
Hydrogen gas production potentials of acid-hydrolyzed and boiled ground wheat were
Received 12 August 2010
compared in batch dark fermentations under mesophilic (37 C) and thermophilic (55 C)
Received in revised form
conditions. Heat-treated anaerobic sludge was used as the inoculum and the hydrolyzed
6 September 2010
ground wheat was supplemented by other nutrients. The highest cumulative hydrogen gas
Accepted 8 September 2010
production (752 ml) was obtained from the acid-hydrolyzed ground wheat starch at 55 C
Available online 12 October 2010
and the lowest (112 ml) was with the boiled wheat starch within 10 days. The highest rate of hydrogen gas formation (7.42 ml H2 h1) was obtained with the acid-hydrolyzed and the
Keywords:
lowest (1.12 ml H2 h1) with the boiled wheat at 55 C. The highest hydrogen gas yield
Acid hydrolysis
(333 ml H2 g1 total sugar or 2.40 mol H2 mol1 glucose) and final total volatile fatty acid
Bio-hydrogen
(TVFA) concentration (10.08 g L1) were also obtained with the acid-hydrolyzed wheat
Dark fermentation
under thermophilic conditions (55 C). Dark fermentation of acid-hydrolyzed ground wheat
Mesophilic
under thermophilic conditions (55 C) was proven to be more beneficial as compared to
Thermophilic
mesophilic or thermophilic fermentation of boiled (partially hydrolyzed) wheat starch.
Wheat starch
1.
ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.
Introduction
Hydrogen gas is a clean fuel with high energy content (122 kJ g1) and is considered to be one of the major energy carriers in the future. Air pollution and global warming problems caused by fossil fuels can be overcome by utilization of hydrogen with no COx, NOx, SOx emissions. Despite the aforementioned advantages, hydrogen gas is not readily available in nature and is currently produced by energy intensive chemical processes such as steam reforming of natural gas or by water electrolysis [1]. Hydrogen gas production by fermentation of carbohydrate rich raw materials has distinct advantages over other methods due to operation under mild conditions. Major obstacles in bio-hydrogen production are low production rates and yields requiring large fermentation volumes [1e4].
Starch and cellulose containing renewable resources such as biomass and agricultural wastes constitute inexpensive and easily fermentable raw materials for bio-hydrogen production. Hydrolysis (acid or enzymatic) of starch/cellulose to highly concentrated sugar solution is the first step in fermentative hydrogen gas production from waste biomass. Dark fermentation of resulting carbohydrates to volatile fatty acids (VFA), H2 and CO2 by acetogenic-anaerobic bacteria and photo fermentation of VFAs to CO2 and H2 by photo-heterotrophic bacteria (Rhodobacter sp.) are the next steps in fermentative hydrogen gas production [1,2]. Heat-treated anaerobic sludge has been the most widely used culture for dark fermentation of starch and cellulose along with some pure cultures of Clostridia and Enterobacter sp. [5e10]. Bio-hydrogen production from ground wheat starch by
* Corresponding author. Tel.: þ90 232 4127109. E-mail address: [email protected] (F. Kargi). 0360-3199/$ e see front matter ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2010.09.029
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 5 ( 2 0 1 0 ) 1 3 2 1 4 e1 3 2 1 8
dark fermentation has been studied extensively using heattreated anaerobic sludge [11e15]. Partial hydrolysis of ground wheat starch by boiling improved the rate of hydrogen production in dark fermentation [16]. Heat pre-treatment of anaerobic sludge was found to be the most suitable method among the other pre-treatment methods [17]. Starch can be hydrolyzed by several different methods. Acid hydrolysis at high temperature (90e150 C) yields nearly complete breakdown of starch to carbohydrate molecules. Partial hydrolysis of starch by boiling followed by bacterial (enzymatic) hydrolysis is a slow, but inexpensive method commonly used. Hydrolysis method affects the yield and formation rate of hydrogen gas. Dark fermentation of hydrolyzed starch can be realized by using mesophilic (37 C) or thermophilic (55 C) bacteria. Most of the dark fermentation studies were realized at 37 C using mesophilic bacteria [5e15]. Thermophilic dark fermentation has also been used by some investigators for bio-hydrogen production from different substrates [18e24]. In the light of literature reports this study was designed to determine the most suitable hydrolysis method and dark fermentation temperature by using acid-hydrolyzed or boiled ground wheat for dark fermentation at 37 C and 55 C. Hydrogen gas formation rates and the yields were used as comparison criteria for different fermentation conditions.
13215
the beginning of the experiments to sustain anaerobic conditions. The experimental bottles were placed in constant temperature incubators at 37 C or 55 C after inoculation. A control bottle without inoculation was used for comparison. In another set of experiments, the ground wheat (200 mesh) was boiled for 1.5 h for partial hydrolysis of starch to reducing sugars. The same experimental procedure was used for partially hydrolyzed wheat starch as that of the acidhydrolyzed wheat.
2.2.
Organisms
2.
Materials and methods
Anaerobic sludge was obtained from acidogenic phase of anaerobic wastewater treatment plant of PAKMAYA Bakers Yeast Co., Izmir, Turkey. After pH adjustment to 5.9, the sludge was boiled for about 1 h in order to eliminate methanogenic bacteria and to select spore forming acidogenic bacteria. The heat-treated sludge was adapted to thermophilic conditions before use by cultivating at 55 C for several growth cycles of 3 days duration. The cultivation media contained glucose (60 g L1), peptone (10 g L1), yeast extract (0.6 g L1), MgSO4$7H2O (0.25 g L1), K2HPO4 (1 g L1), KH2PO4 (1 g L1), 1 L-cysteineeHCl$H2O (0.1 g L ). Argon gas was passed through the cultivation media before incubation and the cultivation flasks were closed with gas-tight rubber stoppers. The cultivated organisms were centrifuged at 8000 rpm (7000g) and resuspended in phosphate buffer to be used for inoculation of the experimental bottles.
2.1.
Experimental system and procedure
2.3.
Four sets of batch dark fermentation experiments were carried out under mesophilic (37 C) and thermophilic (55 C) conditions using either acid-hydrolyzed or boiled ground wheat. Duplicate experiments were carried out using 0.5 L serum bottles (Isolab-Germany Boro 3.3) with 0.25 L fermentation volume. Silicone rubber stoppers and screw caps were used to avoid gas leakage from the bottles. The waste wheat was ground down to 200 mesh size and the pH of the powdered wheat solution (20 g L1 ground wheat) was adjusted to pH ¼ 2.5 using sulfuric acid. The wheat powder (WP) solution was autoclaved at 121 C for 15 min for hydrolysis of the wheat starch to reducing sugar compounds. The yield of starch conversion to soluble total sugar was 95% in acid hydrolysis. The mixture was centrifuged at 8000 rpm (7000g) for 15 min to separate the solids after acid hydrolysis. The supernatant sugar solution was neutralized by adding 10% (w v1) NaOH solution. The initial total sugar (TS) concentration was So ¼ 18.5 0.3 g L1. The serum bottles were inoculated with 10 ml heat-treated anaerobic sludge. The oxidation reduction potential (ORP) was adjusted to nearly 200 mV by addition of L-cysteineeHCl$H2O. Ground wheat contained nearly 97% (w w1) starch, 3.4 mg N g1 PW, 1.72 mg P g1 PW, 1.25 mg Fe g1 PW. Since the nitrogen and phosphorous contents of WP were not sufficient for fermentation, (NH4)2SO4 and KH2PO4 were added to the fermentation media in order to adjust the C/N/P ratio to 100/1/0.3. The fermentation medium was also supplemented with 100 mg L1 MgSO4$7H2O and 25 mg L1 FeSO4$7H2O. Argon gas was passed through the head space of the bottles for 5 min at
Analytical methods
Samples were removed from the liquid phase everyday for analyses of total sugar including starch (TS), and total volatile fatty acids (TVFA). For total sugar (TS) analysis, the samples containing starch and soluble sugar were acidified and boiled for 1.5 h for complete hydrolysis of starch and the resulting total sugar concentration was determined by using the acidephenol method [25]. The samples were also centrifuged at 7000g to remove solids from the liquid media. Soluble sugar and TVFA analyses were carried out in clear supernatants. Soluble sugar content was determined by the acidephenol spectrometric method [25]. TVFA analyses were carried out by using analytical kits (Spectroquant, 1.01763. 0001, Merck, Darmstadt, Germany) and a PC spectrometer (WTW Photolab S12). Hydrogen gas was sampled from the head space of the bottles by using gas-tight glass syringes. Hydrogen gas concentrations in the gas phase were determined by using a gas chromatograph (HP Agilent 6890). The column was Alltech, Hayesep D 80/100 600 1/800 0.08500 . N2 gas was used as carrier with a flow rate of 30 ml min1 and the head pressure was 1.5 atm (22 psig). The amount of total gas produced was determined by water displacement method everyday using sulfuric acid (2%) and NaCl (10%) containing solution. The cumulative hydrogen gas production was determined as explained in our previous publications [11e17]. pH and ORP of the fermentation medium were monitored by using pH and ORP meters with the relevant probes (WTW Sci., Germany). pH of the medium decreased from an initial value of 7.0 to nearly 4.8 due to VFA production which was
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adjusted to 7.0 by addition of 10 M NaOH everyday. ORP values varied between 50 and 150 mV, in general.
3.
Results and discussion
Four sets of duplicate batch experiments were performed for bio-hydrogen production using: (a) boiled ground wheat at 37 C, (b) boiled ground wheat at 55 C, (c) acid-hydrolyzed ground wheat at 37 C, (d) acid-hydrolyzed ground wheat at 55 C in order to determine the most favorable hydrolysis method and fermentation temperature. Boiling was used for partial hydrolysis of wheat starch and acid hydrolysis in autoclave was used for complete hydrolysis before dark fermentation. Boiled wheat powder was subjected to bacterial hydrolysis followed by dark fermentation of resulting carbohydrates. Fig. 1 depicts variation of cumulative hydrogen gas formation (CHF) with time for the four different experiments described above. The highest CHF (752 ml) was obtained with the acid-hydrolyzed wheat starch under thermophilic conditions (55 C) within 10 days. CHF (600 ml) obtained from acidhydrolyzed wheat starch under mesophilic conditions (37 C) was comparable to that obtained at 55 C. CHFs obtained from boiled wheat powder (WP) at 55 C and 37 C were 115 ml and 125 ml, respectively. Hydrogen gas formation from boiled WP at 55 C was faster than that of 37 C. The determining factor for cumulative hydrogen gas volume is the method of hydrolysis rather than fermentation temperature. Sugar solution obtained from acid hydrolysis of wheat starch contained more readily fermentable glucose and maltose for hydrogen gas formation as compared to partially hydrolyzed wheat powder by boiling. Bacterial hydrolysis and fermentation of starch in boiled WP resulted in lower rate and extent of hydrogen gas formation under both mesophilic and thermophilic conditions. Variations of total sugar (starch þ soluble sugar) concentrations with time are depicted in Fig. 2 for the acid-hydrolyzed and boiled WP fermentations under mesophilic and thermophilic conditions. Unlike hydrogen gas formation, total
Fig. 2 e Variation of total sugar concentration with time for A boiled WP fermentation at 55 C, - acid-hydrolyzed WP fermentation at 55 C, : boiled WP fermentation at 37 C, C acid-hydrolyzed WP fermentation at 37 C.
sugar removal from the medium was faster for boiled wheat starch than that obtained from acid hydrolysis. Initial total sugar concentration decreased from 18.5 g L1 to less than 1 g L1 within 120 h (5 days) for mesophilic (37 C) fermentation of boiled WP despite the fact that H2 gas production was low with this fermentation. Polysaccharides derived from partial hydrolysis of boiled starch may have been deposited by the bacteria instead of fermentation to VFA and H2 gas. At the end of 10 days of dark fermentation, more than 85% total sugar was utilized in all cases indicating effective utilization of total sugars derived from starch either by deposition or product formation. Cumulative hydrogen gas formation data depicted in Fig. 1 were correlated with the Gompertz equation and the constants were determined by regression analysis in order to estimate the rate and the extent of hydrogen gas production for different experiments. The Gompertz equation has the following form Rm e ðl tÞ þ 1 HðtÞ ¼ P exp exp P
(1)
where, H is the cumulative hydrogen gas volume at any time t (ml H2), P is the maximum potential hydrogen formation (ml), Rm is the maximum rate of hydrogen formation (ml h1), l is duration of the lag phase, ‘e’ is 2.718 and ‘t’ is time (h). Table 1 presents the Gompertz equation coefficients for different dark fermentation conditions. The highest cumulative hydrogen
Table 1 e Gompertz equation constants for the mesophilic and thermophilic fermentations using acidhydrolyzed and boiled wheat powder. Fig. 1 e Variation of cumulative hydrogen gas formation with time for A boiled WP fermentation at 55 C, - acidhydrolyzed WP fermentation at 55 C, : boiled WP fermentation at 37 C, C acid-hydrolyzed WP fermentation at 37 C.
Substrate Boiled WP-55 C Acid-hydrolyzed WP-55 C Boiled WP-37 C Acid-hydrolyzed WP-37 C
P (ml)
Rm (ml h1)
l (h)
R2
115.7 752.3 125.7 599.9
1.12 7.42 1.38 4.29
24.1 31.6 69.3 44.3
0.99 0.99 0.99 0.99
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gas formation (752 ml) was obtained with the acid-hydrolyzed WP fermentation at 55 C (thermophilic fermentation) and the lowest CHF (115 ml) was realized with the boiled WP (partial hydrolysis) fermentation at 37 C (mesophilic fermentation). Clearly, acid hydrolysis of WP yielded readily fermentable glucose and maltose for bio-hydrogen production by dark fermentation. The highest (7.42 ml h1) and the lowest (1.12 ml h1) rates of hydrogen gas formation were also obtained with the acid hydrolyzed and boiled WP fermentations at 55 C, respectively. The lag phases for thermophilic fermentation were much lower than those of the mesophilic fermentation. The results clearly indicated that thermophilic fermentation of acid-hydrolyzed WP was superior to the mesophilic fermentation of acid-hydrolyzed or boiled WP in terms of the rate and extent of hydrogen gas formation. Another important parameter in bio-hydrogen production by dark fermentation is hydrogen gas yield (ml H2 g1 TS or mol H2 mol1 glucose). Fig. 3 depicts hydrogen yields for different fermentation conditions. The yields were calculated by using the following equation: YH2 ¼
VH2 VðSo SÞ
(2)
where YH2 is the hydrogen yield (ml H2 g1 TS), VH2 is the cumulative hydrogen gas volume at the end of dark fermentation (ml H2), V is the fermentation volume (L), So and S are the initial and final total sugar concentrations (g L1). The highest yield (333 ml H2 g1 TS ¼ 2.4 mol H2 mol1 glucose at 30 C and 1 atm) was obtained with the thermophilic (55 C) fermentation of acid-hydrolyzed WP. Boiled WP resulted in the lowest hydrogen yield of 40 ml H2 g1 TS by the mesophilic and thermophilic fermentation. Mesophilic fermentation of acid-hydrolyzed WP resulted in hydrogen gas yield of 220 ml g1 TS (1.6 mol H2 mol1 glucose at 30 C, 1 atm). Fermentation of glucose produced from acid hydrolysis of WP was a much favorable substrate as compared to boiled (partially hydrolyzed) WP resulting in high hydrogen yield and formation rate. Dark fermentation under thermophilic conditions (55 C) was also found to be more advantageous as compared to mesophilic fermentation at 37 C.
Fig. 4 e Final TVFA concentration in dark fermentation of acid-hydrolyzed and boiled wheat powder under mesophilic and thermophilic conditions.
Volatile fatty acids (VFAs) are by products of dark fermentation along with hydrogen gas. The extent of hydrogen gas formation strongly depends on the type and amount of VFAs produced. In dark fermentation, TVFA yield is usually around 60% of total sugar concentration. For the initial total sugar concentration of 18.5 g L1, the expected final TVFA concentration is around 11 g L1 for 60% conversion yield. As depicted in Fig. 4, final TVFA concentrations for dark fermentation of boiled WP were around 2 g L1 for both the thermophilic and mesophilic fermentations indicating low fermentation yields and therefore, low extent of hydrogen gas production. The highest final TVFA concentration (10.08 g L1) was obtained from the dark fermentation of acid-hydrolyzed WP under thermophilic conditions (55 C) which is very close to the theoretical yield. This result is in agreement with the high extent of hydrogen gas production from acid-hydrolyzed WP under thermophilic conditions. Dark fermentation of acidhydrolyzed WP under mesophilic conditions (37 C) yielded a final TVFA concentration of 6.9 g L1 indicating good extent of sugar fermentation yielding high TVFA and H2 gas formation. The results indicated that VFA and H2 gas formations were directly related and high final VFA concentrations yielded high volumes of hydrogen gas formation.
4.
Fig. 3 e Hydrogen yields for dark fermentation of acidhydrolyzed and boiled wheat powder under mesophilic and thermophilic conditions.
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Conclusion
Bio-hydrogen production from dark fermentation of acidhydrolyzed or boiled ground wheat starch was investigated under thermophilic (55 C) and mesophilic (37 C) conditions. The highest rate (7.42 ml h1) and extent (752 ml) of hydrogen gas formation was obtained from the thermophilic fermentation of acid-hydrolyzed wheat powder within 10 days. Hydrogen yield was also the highest (333 ml g1 TS ¼ 2.4 mol H2 mol1 glucose) with the acid-hydrolyzed wheat powder at 55 C. Polysaccharides derived from boiled wheat starch were probably deposited by the bacteria instead of fermentation to VFA and H2 gas. Acid hydrolysis of wheat starch yielded fermentable glucose for production of H2 gas by dark fermentation. High final TVFA concentration obtained
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from dark fermentation of the acid-hydrolyzed wheat starch at 55 C also supported high levels of H2 gas formation.
[13]
Acknowledgement [14]
This study was supported by the Scientific and Technological Research Council of Turkey by a grant number TUB 105M296. [15]
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