Appl Biochem Biotechnol DOI 10.1007/s12010-014-0961-8
Controlled Continuous Bio-Hydrogen Production Using Different Biogas Release Strategies S. Esquivel-Elizondo & I. Chairez & E. Salgado & J. S. Aranda & G. Baquerizo & E. I. Garcia-Peña
Received: 18 February 2014 / Accepted: 14 May 2014 # Springer Science+Business Media New York 2014
Abstract Dark fermentation for bio-hydrogen (bio-H2) production is an easily operated and environmentally friendly technology. However, low bio-H2 production yield has been reported as its main drawback. Two strategies have been followed in the past to improve this fact: genetic modifications and adjusting the reaction conditions. In this paper, the second one is followed to regulate the bio-H2 release from the reactor. This operating condition alters the metabolic pathways and increased the bio-H2 production twice. Gas release was forced in the continuous culture to study the equilibrium in the mass transfer between the gaseous and liquid phases. This equilibrium depends on the H2, CO2, and volatile fatty acids production. The effect of reducing the bio-H2 partial pressure (bio-H2 pp) to enhance bio-H2 production was evaluated in a 30 L continuous stirred tank reactor. Three bio-H2 release strategies were followed: uncontrolled, intermittent, and constant. In the so called uncontrolled fermentation, without bio-H2 pp control, a bio-H2 molar yield of 1.2 mol/mol glucose was obtained. A sustained low bio-H2 pp of 0.06 atm increased the bio-H2 production rate from 16.1 to 108 mL/L/h with a stable bio-H2 percentage of 55 % (v/v) and a molar yield of 1.9 mol/mol glucose. Biogas release enhanced bio-H2 production because lower bio-H2 pp, CO2 concentration, and reduced volatile fatty acids accumulation prevented the associated inhibitions and bio-H2 consumption. Keywords Controlled continuous culture . Continuous intermittent gas release . Hydrogen partial pressure . Dark fermentation S. Esquivel-Elizondo : I. Chairez : E. I. Garcia-Peña (*) Bioprocesses Department, Unidad Profesional Interdisciplinaria de Biotecnología, Instituto Politécnico Nacional, P.O. Box 07340, Mexico City, Mexico e-mail: [email protected] E. Salgado : J. S. Aranda : G. Baquerizo Bioengineering Department, Unidad Profesional Interdisciplinaria de Biotecnología, Instituto Politécnico Nacional, P.O. Box 07340, Mexico City, Mexico Present Address: S. Esquivel-Elizondo Swette Center for Environmental Biotechnology, The Biodesign Institute at Arizona State University, P.O. Box 875701, Tempe, AZ 85287-5701, USA
Appl Biochem Biotechnol
Introduction Among other available energy sources, hydrogen gas (H2) is a very promising and sustainable option because it has the highest potential amount of energy per gram (122×106 J/kg) [1]. H2 combustion is environmentally ideal because only water vapor and heat energy are produced [2]. In addition, H2 may be used to generate electricity in proton exchange membrane fuel cells (PEMFC) [3, 4]. Bio-H2 production by dark fermentation is an efficient process that requires minimal energy input and no fossil fuels [5]. The bio-H2 yield from dark fermentation is determined by the metabolic pathways and by thermodynamic conditions [6–10]. Bio-H2 production by dark fermentation in batch regimen has been broadly studied [11–13]. These studies showed that low bio-H2 yields are obtained during dark fermentation. In addition, since a continuous regulated supply of bio-H2 is important for a successful use of this energy source. The operation conditions for such a process should be accurately determined to ensure maximum productivity. Moreover, fermentation operated in continuous regimen and inoculated with mixed microflora can deal with non-sterilized organic wastes. This scheme could be reproduced in large scale reactor providing constant production of bio-H2 [14]. Nevertheless, there are some problems in fermentations with mixed microflora: first the bioH2 consumption by homoacetogens microorganisms. These cells generate acetic acid and/or methane. Thus, the general bio-H2 yield is lower and compromises the reaction efficiency. Additionally, it is well known that intermediates accumulation in the liquid and gaseous phases over the fermentative process can negatively influence both the metabolism of anaerobic bacteria and the fermentation pattern. Furthermore, high dissolved bio-H2 and bio-H2 pp may adversely affect the metabolic pathways of bio-H2 production. This effect has been frequently observed during the process; however, it is not completely understood and the extent of inhibition is still a matter of concern [10]. In batch reactions, when bio-H2 pp increases, bio-H2 production decreases and the metabolic pathways shift from butyric and acetic acid formation towards the production of ethanol, propionic, and lactic acids [2, 15]. In addition, hydrogenases enzymes are thermodynamically regulated by the bio-H2 concentration in liquid phase [7, 16]. Bio-H2 production from the oxidation of ferredoxin (Fd) (mediated by hydrogenases) requires a bio-H2 pp lower than 0.3 atm [17]. Recently [18], hypothesized the effect of bio-H2 pp on the synthesis of this compound from the oxidation of NADH to NAD+. They suggested that Gibbs energy of NADH hydrogenase becomes a function of both, bio-H2 pp and NAD+/NADH ratio. Jung et al., [19] showed that bio-H2 synthesis became thermodynamically feasible when bio-H2 pp decreases. Accordingly, higher bio-H2 yield requires fermentation systems designed to remove the accumulated bio-H2. This strategy prevents inhibition events and gas consumption by methanogens. Some approaches have been proposed to reduce bio-H2 pp during dark fermentations. A continuous inflow of inert gases (N2, CO2) is the most commonly reported method for removing bio-H2 [6, 15]. Decreasing bio-H2 pp by inert gas sparging may enhance hydrogenase activity, thereby resulting in thermodynamically favorable conditions for bio-H2 production [20]. This method increases the bio-H2 production beyond 50 % (v/v) in continuous and batch fermentations [6, 12, 15, 20]. Sparging gas in the reactor is very expensive. Moreover, the bio-H2 is diluted after sparging. Thus, gas purification becomes necessary prior to use bio-H2 as energy source. Biogas release as a bio-H2 removal strategy has been less studied than gas sparging. These studies have been done mostly in batch systems [21]. The reported bio-H2 molar yields in those studies ranged from 1.1 to 3.9 mol H2/mol glucose consumed with an average bio-H2 concentration
Appl Biochem Biotechnol
between 50 and 60 % [17, 22, 23]. Few works have been performed at larger scales. Indeed, inhibition phenomena that is more pronounced at larger sizes has been less studied. Even when some aspects of bio-H2 pp effect have been studied, it is necessary to determine its influence over the metabolic inhibition, the biomass growth, and bio-H2 production. The aim of this work was to evaluate different strategies to release biogas from the headspace of the reactor. These strategies were compared with an uncontrolled fermentation with no gas liberation. Concomitantly, the paper also describes how the increment in bio-H2 production by reducing the bio-H2 pp depends on the biogas release scheme (applying intermittent and constant biogas liberation). The effect of the gas release strategy over the bio-H2 production was evaluated by determining the substrate consumption, bio-H2 production, the bio-H2 yield, the bio-H2 production rate, biomass production, and the metabolic pattern. This study can be used to characterize a set of stable environmental conditions to favor the metabolic pathway that leads to the highest bio-H2 molar yield in a continuous operation system.
Materials and Methods Inoculum A previously characterized mixed microbial consortium [24], cultured in a 30 L anaerobic digester was used as inoculum. The anaerobic digester was periodically fed with ground vegetable and fruit scraps. The inoculum was heat pre-treated at 80 °C for 35 min in order to reduce H2-consuming microorganisms [3]. Experimental Set-Up A 30 L CSTR (BDE-30 Eili Dicon S. A.) was used in this study. The CSTR was equipped with a pressure control system consisting of a gauge manometer and a valve located at the top of the reactor headspace. Experiments were performed at a 15 L operating volume with a 10 %v/v inoculum (30×103 mg VSS/L) in a mineral medium previously reported [3]. Anaerobic conditions were established just before starting the operation by sparging N2 into the reactor. All the fermentations were started in batch mode operation with an initial glucose concentration of 10×103 mg/L. After the glucose depletion, a complete biogas release was performed and a 10×103 mg/L glucose pulse was applied. Thereafter, bio-H2 accumulation was controlled through biogas release following the control strategies used in the subsequent continuous fermentation. Continuous operation was settled after the glucose pulse and when the microorganisms were in the log growth phase. A hydraulic retention time (HRT) of 31 h was set for continuous fermentations, as determined previously based on experimental kinetic growth parameters for the mixed microbial consortium [25, 26]. Continuous fermentations were operated under some predefined conditions: temperature of 36±1 °C, pH 5.5, mixing speed of 150 rpm, and inlet substrate concentration of 10×103 mg/L [25]. Operation Conditions Three different experimental conditions for the continuous culture were evaluated in this study. These conditions were used to evaluate three different control strategies for the bio-H2 release. The biogas release during all three fermentations was set and controlled with a manometer located in the upper part of the CSTR. The manometer monitors the pressure in the CSTR in
Appl Biochem Biotechnol
units of psig. Release of biogas was considered complete when the manometer reading was 0 atm. The first experiment (named uncontrolled fermentation) was carried out to determine the system performance in terms of bio-H2 productivity without a strict control of biogas overpressure. In this experiment, the system pressure is increased up to its highest but safety operation value of 1.36 atm (with respect to the atmospheric pressure). When the pressure reached this value, biogas was released until the manometer registered 0.0068 atm. Then, the pressure increased again. In the second experiment, namely intermittent biogas release (IBGR) fermentation, biogas was discharged from the headspace to a pressure of 0.136 atm after the maximum pressure value of 0.885 atm was reached. The third fermentation named constant biogas release (CBGR) fermentation keeps the average pressure at or below of 0.116 atm by continuous biogas release. Samples were periodically taken from both the liquid and gaseous phases. Liquid samples were analyzed by duplicate to determine substrate consumption and biomass growth, while gas samples were analyzed to characterize biogas composition. Analytical Methods Bio-H2 in the headspace was periodically measured using a gastight syringe (0.15 mL injection volume) and a gas chromatograph (Gow-Mac Series 580, Bethlehem, PA, USA) as previously reported [3]. Calibration curves were made for different system pressures (0.13, 0.51 and 0.75 and 1.00 atm). Biogas stream was measured with universal flow meter (Agilent Technologies ADM1000, Wilmington, DE, USA). Bio-H2 pp were calculated using Dalton’s Law of partial pressure from bio-H2 concentrations determined by gas analysis and the system gauge pressure. Glucose consumption was measured with the DNS colorimetric method as previously reported in [3]. Biomass was evaluated by quantifying protein with the Bradford method. Biomass concentration was estimated by assuming that protein constituted 25 % of dry biomass weight (experimentally determined). Volatile fatty acids (VFAs) were determined from liquid samples (30 mL injection volume) using an HPLC (Perkin Elmer Series 200 HPLC systems, Shelton, CT, USA) as reported in [26].
Results and Discussion Uncontrolled Fermentation Fermentation Performance This experiment was designed to identify the conditions required to perform continuous operation in a 30 L CSTR reactor. In Fig. 1a, the glucose and biomass concentration and the cumulative bio-H2 production over the whole operation period (295 h) are shown. Figure 1b presents the bio-H2 concentration in the biogas (v/v) and the bio-H2 pp. The reactor was initially operated in batch conditions for 168 h to attain enough biomass to start the continuous culture. During the first 50 h of batch operation, the initial glucose concentration of 10×103 mg/L was almost completely consumed (97 %). The biomass increased from 36 to 135 mg/L due to substrate assimilation. The biomass growth yield during
Appl Biochem Biotechnol
Fig. 1 Uncontrolled fermentation performance: a Glucose consumption (dashed line with white triangle), biomass production (white square), and bio-H2 production (black circle) during fermentation. GR glucose removal percentage. b Bio-H2 partial pressure (white diamond) and bio-H2 concentration (black circle) in biogas. Vertical bars in the graphs separate the process into the first batch culture, the glucose pulse, and four cycles
this period was 0.2 mg biomass/mg glucose consumed. Bio-H2 production initiated after 48 h, once the initial glucose was consumed, reaching a final total volume in the batch culture of 22.9 L (Fig. 1a). After 95 h of batch mode operation, when a bio-H2 pp value of 0.66 atm was reached, the biogas was completely released from the reactor headspace. To start the continuous regimen, a glucose pulse (10×103 mg/L) was added to the system. The bio-H2 pp increased to 0.71 atm as result of a new period of biological activity caused by the new source of nutrients. During this second batch stage, glucose was nearly exhausted (97 %) in 24 h due to the higher biomass concentration (300 mg/L). After 168 h of batch
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operation, the biomass concentration was stable. This condition was used to start the continuous operation. The bio-H2 production increased during the continuous culture, this was correlated with biomass production (around 200 and 300 mg/L) and biogas release. Glucose consumption was kept around 90 %, while cumulative bio-H2 production increased throughout the continuous operation (Fig. 1a). As shown in Fig. 1b, the bio-H2 concentration in the biogas was between 30 and 40 % (v/v) in batch culture. Approximately, 5 h after the glucose pulse (i.e., at 95 h), bio-H2 production rate decreased and the cumulative bio-H2 was practically constant before the continuous culture started. This situation is explained by the inhibition caused to the increase in bio-H2 pp (0.06 to 0.71 atm). Throughout two cycles of the continuous culture, the biogas was accumulated to a gauge pressure of 1.36 atm. Within these cycles, bio-H2 was between 0.12 and 0.63 atm, and bio-H2 concentration in the biogas reached values from 30 to 50 % (v/v). The averaged bio-H2 production rate (bio-H2PR) in these two cycles was 34.2 mL/Lh. This bio-H2PR was higher than bio-H2PR obtained in the batch operation (16.1 mL/Lh). Two intermittent biogas liberations were carried out during the last continuous fermentation cycles (at 236 and 265 h of culture). These depressurizations resulted in sustained low bio-H2 pp (90 %). The butyrate production was the highest in the IBGR fermentation. In this fermentation, biogas release reduced the overpressure from 0.885 to 0.136 atm (associated to bio-H2 pp in the range of 0.08 to 0.47 atm), and thus, butyrate production was primarily enhanced, since butyric acid fermentation (−257 kJ) becomes thermodynamically more feasible than acetic acid fermentation (−184.2 kJ) [31]. The higher butyrate production is justified by both VFA and bio-H2 removal. Biogas release contributed to prevent the accumulation of these products, thereby enhancing their production. According to the Le Chatelier’s principle, decreasing VFA (acetic and butyric acids) concentrations favor reaction rates that generate them. Acetate concentration was highest (210 mg/L) in the CBGR fermentation. Enhanced acetate production is apparently promoted by the constant low bio-H2 pp. During the CBGR fermentation, the average bio-H2 concentration in the liberated gas was 54.4 %, while the bio-H2 pp
60.4 7.0 9.2
N2 sparging
Control
Internal biogas sparging (200 mL/min)
N2 sparging (200 mL/min)
CO2 sparging (200 mL/min)
CSTR (2.3 L) (wheat starch coproduct)
CSTR (2.9 L)
Sucrose
CSTR (30 L) glucose
Batch culture (294 mL) glucose
22.9 7
N2 sparging
CSTR (2.5 L) glucose
30–50 55 60
Uncontrolled fermentation
62
Continuous gas release (respirometric method)
IBGR fermentation CBGR fermentation
30
Intermittent pressure release (Owen method)
63.2
Bio-H2 percentage (%)
Conditions
System/substrate
Table 1 Comparison results in different systems with controlled partial pressure conditions
111.9 240.7
70.2
0.238
0.182
Cumulative bio-H2 production (L)
1.1 1.9
1.2
1.15
0.91
0.82
0.75
1.9
1.43
Yield
59.0 108.0
36.0 (mL/Lh)
5.91
3.43
3.19
3.2
4.5 3.13 (mL/min gbiomass)
Bio-H2PR (L/g VSS/d)
This study
[24]
[20]
[12]
[6]
Reference
Appl Biochem Biotechnol
0.08–0.5 (0.32)c
Continuous
240.7 (237.3)a
0.07–0.56
0.56 atm) inhibits bacterial metabolism [21]. Homoacetogenesis is thermodynamically feasible at the usual experimental partial pressure of 0.39 atm and acetic acid concentration of 10×10−3 mol [18]. In the present work, the acetic acid concentration was higher than 10×10−3 mol and the bio-H2 pp was lowered to 0.06 atm, then the conditions in the CSTR made homoacetogenic reaction thermodynamically unfeasible, which thus enhanced bio-H2 production. Positive effects of the continuous flow were verified since constant dilution of the VFA concentration was attained, thereby reducing VFA-related inhibitions for bio-H2 production. On the contrary, batch process for bio-H2 production allows the accumulation of metabolic intermediates in the liquid and gas phases. The study [15] reported an extremely high bio-H2 in the system, which caused a shift in metabolic products from organic acids to solvents. Both accumulation of metabolic intermediates and high bio-H2 pp negatively affected bio-H2 production by different inhibitory effects. The VFAs can be stimulatory, inhibitory, or even toxic to the fermentative bacteria depending on their concentration [32]. At high VFAs levels, their no dissociated forms can permeate the bacterial cell membrane, then reducing the pH inside the bacterial cell [33]. Therefore, additional energy must be used to avoid unfavorable conditions, reducing the bacterial growth rate due to less available energy [32]. The overall results showed increments in the cumulative bio-H2 production, the bio-H2PR and the bio-H2MY caused by the reduction in the bio-H2 pp obtained via controlled biogas
Appl Biochem Biotechnol
release. In the CBGR fermentation, the bio-H2 pp was maintained at an average of 0.06 atm, which made the NADH reaction possible. This could be the main cause of the increased bioH2 production in the CBGR experiment. Mandal et al., [17] established a maximum bio-H2 pp as a function of the different electron donors and acceptors in the system with the relation Bio−H2 ppmax ≤e2 FðEH2 −ExÞ=RT , where EH2 =−414 mV. The authors demonstrated that the bio-H2 pp must be lower than 0.3 atm to produce bio-H2 using ferredoxin (Efd =−400 mV). Therefore, the bio-H2 pp maintained in the CBGR also allowed Bio-H2 production through the ferredoxin reaction. Global Mass and Energy Balance The stoichiometric butyrate fermentation from glucose is presented in the following equation. In this equation, ammonium is the nitrogen source of and the formula C5H7O2N represents biomass in mixed cultures [34–36]. In dark fermentation, 10–20 % of the substrate is stoichiometric converted to bio-H2. Fermentation with constant biogas release achieved 16 % glucose conversion to bio-H2, where 12 mol of H2/mol of glucose would be 100 % conversion. The 16 % conversion rate represents approximately 50 % of the maximum possible conversion through acetic acid fermentation. Considering that 15 % of the electrons in the substrate are recovered as bio-H2. The general model of the reaction described above is C 6 H 12 O6 þ HCO−3 þ 0:1N H 4 →CH 3 CH 2 CH 2 COO− þ 0:1C 5 H 7 O2 N þ H 2 O þ 3CO2 þ 2H 2 According to the global mass and energy balance equation, 1 mol of glucose produces 87 g of butyrate and 63.3 L of H2 at 0.76 atm (atmospheric pressure of Mexico City) and 20 °C. CBGR fermentation was mainly a butyric fermentation. The productivity is close to the theoretical value of 60 L of H2/mol of glucose compared to 63.3 theoretical L (Table 4). The average bio-H2 concentration was higher than the theoretical value achieved in the butyric acid fermentation. This is possible because the stoichiometric balance of butyric fermentation only takes into account the bio-H2 production from formate, while the ferredoxin-hydrogenase reaction was also possible with the constant biogas release at low pressure. Conclusions This paper has addressed the effect of bio-H2 pp on the continuous culture considering the gas release strategy. The effect of bio-H2 pp in various ranges was evaluated in a 30 L CSTR. The sustained low pH2 (0.06 atm), obtained by constant biogas release, produced the bio-H2PR increase from 36 to 108 mL/L h. The same condition induced higher biomass growth and glucose removal. Constant biogas release allowed both butyric and acetic acid fermentations with an enhanced bio-H2 production. The reduced CO2 concentrations limited the bio-H2 Table 4 Compared productivities of theoretical butyric acid fermentation and constant gas release fermentation
Theoretical butyric acid fermentation
Constant gas release (0.136 atm) fermentation
Butyrate (g)
87
80.1
Acetate (g)
–
3.8
63.3 40.2
60 54.4
2
1.9
H2 (L) H2 (%) H2 molar yield
Appl Biochem Biotechnol
consumption by homoacetogens. A maximum H2 yield of 1.9 was obtained and the bio-H2 was not diluted in the liberated gas. The continuous culture assured a constant bio-H2 production and avoids VFA accumulation. This fact prevented the modification in bacterial metabolism. Moreover, this condition also limited the bacteria inhibition. Acknowledgments This work was supported through funding provided by the CONACYT grant 60976 and Instituto Politécnico Nacional, grant SIP 20140405. The authors are grateful to Conacyt for the fellowship awarded.
References 1. Park, W., Hyun, S. H., Oh, S. E., Logan, B. E., & Kim, I. S. (2005). Removal of headspace CO2 increases biological hydrogen production. Environmental Science and Technology, 39, 4416–4420. 2. Nath, K., & Das, D. (2004). Improvement of fermentative hydrogen production: various approaches. Applied Microbiology and Biotechnology, 65, 520–529. 3. García-Peña, E., Guerrero-Barajas, C., Ramirez, D., & Arriaga-Hurtado, L. (2009). Semi-continuous biohydrogen production as an approach to generate electricity. Bioresource Technology, 100, 6369–6377. 4. Hallenbeck, P., & Ghosh, D. (2010). Improvements in fermentative biological hydrogen production through metabolic engineering. Journal of Environmental Management, 95, 360–364. 5. Ueno, Y., Haruta, S., Ishii, M., & Igarashi, Y. (2001). Microbial community in anaerobic hydrogenproducing microflora enriched from sludge compost. Applied Microbiology and Biotechnology, 57, 555–562. 6. Mizuno, O., Dinsdale, R., Hawkes, F., Hawkes, D., & Noike, T. (2000). Enhancement of hydrogen production from glucose by nitrogen gas sparging. Bioresource Technology, 73, 59–65. 7. Lee, H., Salerno, M., & Rittmann, B. (2008). Thermodynamic evaluation on H2 production in glucose fermentation. Environmental Science and Technology, 42, 2401–2407. 8. Prakasham, R., Sathish, T., & Brahmaiah, P. (2010). Biohydrogen production process optimization using anaerobic mixed consortia: a prelude study for use of agroindustrial material hydrolysate as substrate. Bioresource Technology, 101, 5708–5711. 9. Hung, C., Chang, Y., & Chang, Y. (2011). Roles of microorganisms other than Clostridium and Enterobacter in anaerobic fermentative biohydrogen production systems—a review. Bioresource Technology, 102, 8437–8444. 10. Oh, Y., Raj, S., Jung, G., & Park, S. (2011). Current status of the metabolic engineering of microorganisms for biohydrogen production. Bioresource Technology, 102, 8357–8367. 11. Fang, H., Zhang, T., & Liu, H. (2002). Microbial diversity of mesophilic hydrogen production sludge. Applied Microbiology and Biotechnology, 58, 112–118. 12. Hussy, I., Hawkes, F. R., Dinsdale, R., & Hawkes, D. L. (2003). Continuous fermentation hydrogen production from a wheat starch coproduct by mixed microflora. Biotechnology and Bioengineering, 84, 619–626. 13. Van-Ginkel, S., Oh, S., & Logan, B. (2005). Biohydrogen gas production from food processing and domestic wastewaters. International Journal of Hydrogen Energy, 30, 1535–1542. 14. Hawkes, F., Dinsdale, R., Hawkes, D., & Hussy, I. (2002). Sustainable fermentative hydrogen production: challenges for process optimization. International Journal of Hydrogen Energy, 27, 1339–1347. 15. Valdez-Vazquez, I., Rios-Leal, E., Carmona-Martinez, A., Muñoz-Paez, K., & Poggi Varaldo, H. (2006). Improvement of biohydrogen production from solid wastes by intermittent venting and gas flushing of batch reactors headspace. Environmental Science and Technology, 40, 3409–3415. 16. Angenent, L. T., Karim, K., Al-Dahhan, M., Wrenn, B., & Dominguez-Espinosa, R. (2004). Production of bioenergy and biochemicals from industrial and agricultural wastewater. Trends in Biotechnology, 22, 477–485. 17. Mandal, B., Nath, K., & Das, D. (2006). Improvement of biohydrogen production under decreased partial pressure of H2 by Enterobacter cloacae. Biotechnology Letters, 28, 831–835. 18. Bastidas-Oyanedel, J. R., Mohd-Zaki, Z., Zeng, R. J., Bernet, N., Pratt, S., Steyer, J. P., & Batstonebet, D. J. (2012). Gas controlled hydrogen fermentation. Bioresource Technology, 110, 503–509. 19. Jung, K. W., Kim, D. H., Kim, S. H., & Shin, H. S. (2011). Bioreactor design for continuous dark fermentative hydrogen production. Bioresource Technology, 102, 8612–8620.
Appl Biochem Biotechnol 20. Kim, D., Han, S., Kim, S., & Shin, H. (2006). Effect of gas sparging on continuous fermentative hydrogen production. International Journal of Hydrogen Energy, 31, 2158–2169. 21. Kraemer, J., & Bagley, D. (2008). Optimisation and design of nitrogen-sparged fermentative hydrogen production bioreactors. International Journal of Hydrogen Energy, 33, 6558–6565. 22. Logan, B., Oh, S., Kim, I., & Van-Ginkel, S. V. (2002). Biological hydrogen production measured in batch anaerobic respirometers. Environmental Science and Technology, 36, 2530–2535. 23. Oh, S., Zue, Y., Zhang, H., Guiltinan, M., Logan, B., & Regan, J. (2009). Hydrogen production by Clostridium acetobutylicum ATCC 824 and megaplasmid-deficient mutant M5 evaluated using a large headspace volume technique. International Journal of Hydrogen Energy, 34, 9347–9353. 24. Garcia-Peña, E. I., Parameswaran, P., Kang, D., Canul-Chan, M., & Krajmalnik-Brown, R. (2011). Anaerobic digestion and co-digestion processes of vegetable and fruit residues: process and microbial ecology. Bioresource Technology, 102, 9447–9455. 25. Canul, M., Salgado, E., Aranda, J., & Garcia, E. I. (2010). Hydrogen and electricity production from the organic fraction of solid waste. Proceedings of the Third International Symposium on Energy from Biomass and Waste. Italy. 26. Garcia-Peña, E. I., Canul-Chan, M., Chairez, I., Salgado-Manjarez, E., & Aranda-Barradas, J. (2013). Biohydrogen production based on the evaluation of kinetic parameters of a mixed microbial culture using glucose and fruit–vegetable waste as feedstocks. Applied Biochemistry and Biotechnology, 171, 279–293. 27. Hallenbeck, P. C., & Ghosh, D. (2009). Advances in fermentative biohydrogen production: the way forward? Trends in Biotechnology, 27, 287–297. 28. Arriaga, S., Rosas, I., Alatriste-Mondragón, F., & Razo-Flores, E. (2011). Continuous production of hydrogen from oat straw hydrolysate in a biotrickling filter. International Journal of Hydrogen Energy, 36, 3442–3449. 29. Kongjan, P., O-Thong, S., Kotay, M., Min, B., & Angelidaki, I. (2010). Biohydrogen production from wheat straw hydrolysates by dark fermentation using extreme thermophilic mixed culture. Biotechnology and Bioengineering, 105, 899–908. 30. Arooj, M. F., Han, S. K., Kim, S. H., & Dong-Hoon, K. (2008). Continuous biohydrogen production in a CSTR using starch as a substrate. International Journal of Hydrogen Energy, 33, 3289–3294. 31. Lee, D., Li, Y., & Noike, T. (2009). Continuous H2 production by anaerobic mixed microflora in membrane bioreactor. Bioresource Technology, 100, 690–695. 32. Zheng, X. J., & Yu, H. Q. (2005). Inhibitory effects of butyrate on biological hydrogen production with mixed anaerobic cultures. Journal of Environmental Management, 74, 65–70. 33. Gottschalk, G. (1986). Bacterial metabolism (2nd ed.). New York: Springer. 34. Rittmann, B. E., & McCarty, P. L. (2001). Environmental biotechnology: principles and applications. New York: McGraw-Hill. 35. Cai, G., Jin, B., Saint, C., & Monis, P. (2010). Metabolic flux analysis of hydrogen production network by Clostridium butyricum W5: effect of pH and glucose concentration. International Journal of Hydrogen Energy, 35, 6681–6690. 36. Das, D. (2009). Advances in biohydrogen production processes: an approach towards commercialization. International Journal of Hydrogen Energy, 34, 7349–7357.
Controlled Continuous Bio-Hydrogen Production Using Different Biogas Release Strategies S. Esquivel-Elizondo & I. Chairez & E. Salgado & J. S. Aranda & G. Baquerizo & E. I. Garcia-Peña
Received: 18 February 2014 / Accepted: 14 May 2014 # Springer Science+Business Media New York 2014
Abstract Dark fermentation for bio-hydrogen (bio-H2) production is an easily operated and environmentally friendly technology. However, low bio-H2 production yield has been reported as its main drawback. Two strategies have been followed in the past to improve this fact: genetic modifications and adjusting the reaction conditions. In this paper, the second one is followed to regulate the bio-H2 release from the reactor. This operating condition alters the metabolic pathways and increased the bio-H2 production twice. Gas release was forced in the continuous culture to study the equilibrium in the mass transfer between the gaseous and liquid phases. This equilibrium depends on the H2, CO2, and volatile fatty acids production. The effect of reducing the bio-H2 partial pressure (bio-H2 pp) to enhance bio-H2 production was evaluated in a 30 L continuous stirred tank reactor. Three bio-H2 release strategies were followed: uncontrolled, intermittent, and constant. In the so called uncontrolled fermentation, without bio-H2 pp control, a bio-H2 molar yield of 1.2 mol/mol glucose was obtained. A sustained low bio-H2 pp of 0.06 atm increased the bio-H2 production rate from 16.1 to 108 mL/L/h with a stable bio-H2 percentage of 55 % (v/v) and a molar yield of 1.9 mol/mol glucose. Biogas release enhanced bio-H2 production because lower bio-H2 pp, CO2 concentration, and reduced volatile fatty acids accumulation prevented the associated inhibitions and bio-H2 consumption. Keywords Controlled continuous culture . Continuous intermittent gas release . Hydrogen partial pressure . Dark fermentation S. Esquivel-Elizondo : I. Chairez : E. I. Garcia-Peña (*) Bioprocesses Department, Unidad Profesional Interdisciplinaria de Biotecnología, Instituto Politécnico Nacional, P.O. Box 07340, Mexico City, Mexico e-mail: [email protected] E. Salgado : J. S. Aranda : G. Baquerizo Bioengineering Department, Unidad Profesional Interdisciplinaria de Biotecnología, Instituto Politécnico Nacional, P.O. Box 07340, Mexico City, Mexico Present Address: S. Esquivel-Elizondo Swette Center for Environmental Biotechnology, The Biodesign Institute at Arizona State University, P.O. Box 875701, Tempe, AZ 85287-5701, USA
Appl Biochem Biotechnol
Introduction Among other available energy sources, hydrogen gas (H2) is a very promising and sustainable option because it has the highest potential amount of energy per gram (122×106 J/kg) [1]. H2 combustion is environmentally ideal because only water vapor and heat energy are produced [2]. In addition, H2 may be used to generate electricity in proton exchange membrane fuel cells (PEMFC) [3, 4]. Bio-H2 production by dark fermentation is an efficient process that requires minimal energy input and no fossil fuels [5]. The bio-H2 yield from dark fermentation is determined by the metabolic pathways and by thermodynamic conditions [6–10]. Bio-H2 production by dark fermentation in batch regimen has been broadly studied [11–13]. These studies showed that low bio-H2 yields are obtained during dark fermentation. In addition, since a continuous regulated supply of bio-H2 is important for a successful use of this energy source. The operation conditions for such a process should be accurately determined to ensure maximum productivity. Moreover, fermentation operated in continuous regimen and inoculated with mixed microflora can deal with non-sterilized organic wastes. This scheme could be reproduced in large scale reactor providing constant production of bio-H2 [14]. Nevertheless, there are some problems in fermentations with mixed microflora: first the bioH2 consumption by homoacetogens microorganisms. These cells generate acetic acid and/or methane. Thus, the general bio-H2 yield is lower and compromises the reaction efficiency. Additionally, it is well known that intermediates accumulation in the liquid and gaseous phases over the fermentative process can negatively influence both the metabolism of anaerobic bacteria and the fermentation pattern. Furthermore, high dissolved bio-H2 and bio-H2 pp may adversely affect the metabolic pathways of bio-H2 production. This effect has been frequently observed during the process; however, it is not completely understood and the extent of inhibition is still a matter of concern [10]. In batch reactions, when bio-H2 pp increases, bio-H2 production decreases and the metabolic pathways shift from butyric and acetic acid formation towards the production of ethanol, propionic, and lactic acids [2, 15]. In addition, hydrogenases enzymes are thermodynamically regulated by the bio-H2 concentration in liquid phase [7, 16]. Bio-H2 production from the oxidation of ferredoxin (Fd) (mediated by hydrogenases) requires a bio-H2 pp lower than 0.3 atm [17]. Recently [18], hypothesized the effect of bio-H2 pp on the synthesis of this compound from the oxidation of NADH to NAD+. They suggested that Gibbs energy of NADH hydrogenase becomes a function of both, bio-H2 pp and NAD+/NADH ratio. Jung et al., [19] showed that bio-H2 synthesis became thermodynamically feasible when bio-H2 pp decreases. Accordingly, higher bio-H2 yield requires fermentation systems designed to remove the accumulated bio-H2. This strategy prevents inhibition events and gas consumption by methanogens. Some approaches have been proposed to reduce bio-H2 pp during dark fermentations. A continuous inflow of inert gases (N2, CO2) is the most commonly reported method for removing bio-H2 [6, 15]. Decreasing bio-H2 pp by inert gas sparging may enhance hydrogenase activity, thereby resulting in thermodynamically favorable conditions for bio-H2 production [20]. This method increases the bio-H2 production beyond 50 % (v/v) in continuous and batch fermentations [6, 12, 15, 20]. Sparging gas in the reactor is very expensive. Moreover, the bio-H2 is diluted after sparging. Thus, gas purification becomes necessary prior to use bio-H2 as energy source. Biogas release as a bio-H2 removal strategy has been less studied than gas sparging. These studies have been done mostly in batch systems [21]. The reported bio-H2 molar yields in those studies ranged from 1.1 to 3.9 mol H2/mol glucose consumed with an average bio-H2 concentration
Appl Biochem Biotechnol
between 50 and 60 % [17, 22, 23]. Few works have been performed at larger scales. Indeed, inhibition phenomena that is more pronounced at larger sizes has been less studied. Even when some aspects of bio-H2 pp effect have been studied, it is necessary to determine its influence over the metabolic inhibition, the biomass growth, and bio-H2 production. The aim of this work was to evaluate different strategies to release biogas from the headspace of the reactor. These strategies were compared with an uncontrolled fermentation with no gas liberation. Concomitantly, the paper also describes how the increment in bio-H2 production by reducing the bio-H2 pp depends on the biogas release scheme (applying intermittent and constant biogas liberation). The effect of the gas release strategy over the bio-H2 production was evaluated by determining the substrate consumption, bio-H2 production, the bio-H2 yield, the bio-H2 production rate, biomass production, and the metabolic pattern. This study can be used to characterize a set of stable environmental conditions to favor the metabolic pathway that leads to the highest bio-H2 molar yield in a continuous operation system.
Materials and Methods Inoculum A previously characterized mixed microbial consortium [24], cultured in a 30 L anaerobic digester was used as inoculum. The anaerobic digester was periodically fed with ground vegetable and fruit scraps. The inoculum was heat pre-treated at 80 °C for 35 min in order to reduce H2-consuming microorganisms [3]. Experimental Set-Up A 30 L CSTR (BDE-30 Eili Dicon S. A.) was used in this study. The CSTR was equipped with a pressure control system consisting of a gauge manometer and a valve located at the top of the reactor headspace. Experiments were performed at a 15 L operating volume with a 10 %v/v inoculum (30×103 mg VSS/L) in a mineral medium previously reported [3]. Anaerobic conditions were established just before starting the operation by sparging N2 into the reactor. All the fermentations were started in batch mode operation with an initial glucose concentration of 10×103 mg/L. After the glucose depletion, a complete biogas release was performed and a 10×103 mg/L glucose pulse was applied. Thereafter, bio-H2 accumulation was controlled through biogas release following the control strategies used in the subsequent continuous fermentation. Continuous operation was settled after the glucose pulse and when the microorganisms were in the log growth phase. A hydraulic retention time (HRT) of 31 h was set for continuous fermentations, as determined previously based on experimental kinetic growth parameters for the mixed microbial consortium [25, 26]. Continuous fermentations were operated under some predefined conditions: temperature of 36±1 °C, pH 5.5, mixing speed of 150 rpm, and inlet substrate concentration of 10×103 mg/L [25]. Operation Conditions Three different experimental conditions for the continuous culture were evaluated in this study. These conditions were used to evaluate three different control strategies for the bio-H2 release. The biogas release during all three fermentations was set and controlled with a manometer located in the upper part of the CSTR. The manometer monitors the pressure in the CSTR in
Appl Biochem Biotechnol
units of psig. Release of biogas was considered complete when the manometer reading was 0 atm. The first experiment (named uncontrolled fermentation) was carried out to determine the system performance in terms of bio-H2 productivity without a strict control of biogas overpressure. In this experiment, the system pressure is increased up to its highest but safety operation value of 1.36 atm (with respect to the atmospheric pressure). When the pressure reached this value, biogas was released until the manometer registered 0.0068 atm. Then, the pressure increased again. In the second experiment, namely intermittent biogas release (IBGR) fermentation, biogas was discharged from the headspace to a pressure of 0.136 atm after the maximum pressure value of 0.885 atm was reached. The third fermentation named constant biogas release (CBGR) fermentation keeps the average pressure at or below of 0.116 atm by continuous biogas release. Samples were periodically taken from both the liquid and gaseous phases. Liquid samples were analyzed by duplicate to determine substrate consumption and biomass growth, while gas samples were analyzed to characterize biogas composition. Analytical Methods Bio-H2 in the headspace was periodically measured using a gastight syringe (0.15 mL injection volume) and a gas chromatograph (Gow-Mac Series 580, Bethlehem, PA, USA) as previously reported [3]. Calibration curves were made for different system pressures (0.13, 0.51 and 0.75 and 1.00 atm). Biogas stream was measured with universal flow meter (Agilent Technologies ADM1000, Wilmington, DE, USA). Bio-H2 pp were calculated using Dalton’s Law of partial pressure from bio-H2 concentrations determined by gas analysis and the system gauge pressure. Glucose consumption was measured with the DNS colorimetric method as previously reported in [3]. Biomass was evaluated by quantifying protein with the Bradford method. Biomass concentration was estimated by assuming that protein constituted 25 % of dry biomass weight (experimentally determined). Volatile fatty acids (VFAs) were determined from liquid samples (30 mL injection volume) using an HPLC (Perkin Elmer Series 200 HPLC systems, Shelton, CT, USA) as reported in [26].
Results and Discussion Uncontrolled Fermentation Fermentation Performance This experiment was designed to identify the conditions required to perform continuous operation in a 30 L CSTR reactor. In Fig. 1a, the glucose and biomass concentration and the cumulative bio-H2 production over the whole operation period (295 h) are shown. Figure 1b presents the bio-H2 concentration in the biogas (v/v) and the bio-H2 pp. The reactor was initially operated in batch conditions for 168 h to attain enough biomass to start the continuous culture. During the first 50 h of batch operation, the initial glucose concentration of 10×103 mg/L was almost completely consumed (97 %). The biomass increased from 36 to 135 mg/L due to substrate assimilation. The biomass growth yield during
Appl Biochem Biotechnol
Fig. 1 Uncontrolled fermentation performance: a Glucose consumption (dashed line with white triangle), biomass production (white square), and bio-H2 production (black circle) during fermentation. GR glucose removal percentage. b Bio-H2 partial pressure (white diamond) and bio-H2 concentration (black circle) in biogas. Vertical bars in the graphs separate the process into the first batch culture, the glucose pulse, and four cycles
this period was 0.2 mg biomass/mg glucose consumed. Bio-H2 production initiated after 48 h, once the initial glucose was consumed, reaching a final total volume in the batch culture of 22.9 L (Fig. 1a). After 95 h of batch mode operation, when a bio-H2 pp value of 0.66 atm was reached, the biogas was completely released from the reactor headspace. To start the continuous regimen, a glucose pulse (10×103 mg/L) was added to the system. The bio-H2 pp increased to 0.71 atm as result of a new period of biological activity caused by the new source of nutrients. During this second batch stage, glucose was nearly exhausted (97 %) in 24 h due to the higher biomass concentration (300 mg/L). After 168 h of batch
Appl Biochem Biotechnol
operation, the biomass concentration was stable. This condition was used to start the continuous operation. The bio-H2 production increased during the continuous culture, this was correlated with biomass production (around 200 and 300 mg/L) and biogas release. Glucose consumption was kept around 90 %, while cumulative bio-H2 production increased throughout the continuous operation (Fig. 1a). As shown in Fig. 1b, the bio-H2 concentration in the biogas was between 30 and 40 % (v/v) in batch culture. Approximately, 5 h after the glucose pulse (i.e., at 95 h), bio-H2 production rate decreased and the cumulative bio-H2 was practically constant before the continuous culture started. This situation is explained by the inhibition caused to the increase in bio-H2 pp (0.06 to 0.71 atm). Throughout two cycles of the continuous culture, the biogas was accumulated to a gauge pressure of 1.36 atm. Within these cycles, bio-H2 was between 0.12 and 0.63 atm, and bio-H2 concentration in the biogas reached values from 30 to 50 % (v/v). The averaged bio-H2 production rate (bio-H2PR) in these two cycles was 34.2 mL/Lh. This bio-H2PR was higher than bio-H2PR obtained in the batch operation (16.1 mL/Lh). Two intermittent biogas liberations were carried out during the last continuous fermentation cycles (at 236 and 265 h of culture). These depressurizations resulted in sustained low bio-H2 pp (90 %). The butyrate production was the highest in the IBGR fermentation. In this fermentation, biogas release reduced the overpressure from 0.885 to 0.136 atm (associated to bio-H2 pp in the range of 0.08 to 0.47 atm), and thus, butyrate production was primarily enhanced, since butyric acid fermentation (−257 kJ) becomes thermodynamically more feasible than acetic acid fermentation (−184.2 kJ) [31]. The higher butyrate production is justified by both VFA and bio-H2 removal. Biogas release contributed to prevent the accumulation of these products, thereby enhancing their production. According to the Le Chatelier’s principle, decreasing VFA (acetic and butyric acids) concentrations favor reaction rates that generate them. Acetate concentration was highest (210 mg/L) in the CBGR fermentation. Enhanced acetate production is apparently promoted by the constant low bio-H2 pp. During the CBGR fermentation, the average bio-H2 concentration in the liberated gas was 54.4 %, while the bio-H2 pp
60.4 7.0 9.2
N2 sparging
Control
Internal biogas sparging (200 mL/min)
N2 sparging (200 mL/min)
CO2 sparging (200 mL/min)
CSTR (2.3 L) (wheat starch coproduct)
CSTR (2.9 L)
Sucrose
CSTR (30 L) glucose
Batch culture (294 mL) glucose
22.9 7
N2 sparging
CSTR (2.5 L) glucose
30–50 55 60
Uncontrolled fermentation
62
Continuous gas release (respirometric method)
IBGR fermentation CBGR fermentation
30
Intermittent pressure release (Owen method)
63.2
Bio-H2 percentage (%)
Conditions
System/substrate
Table 1 Comparison results in different systems with controlled partial pressure conditions
111.9 240.7
70.2
0.238
0.182
Cumulative bio-H2 production (L)
1.1 1.9
1.2
1.15
0.91
0.82
0.75
1.9
1.43
Yield
59.0 108.0
36.0 (mL/Lh)
5.91
3.43
3.19
3.2
4.5 3.13 (mL/min gbiomass)
Bio-H2PR (L/g VSS/d)
This study
[24]
[20]
[12]
[6]
Reference
Appl Biochem Biotechnol
0.08–0.5 (0.32)c
Continuous
240.7 (237.3)a
0.07–0.56
0.56 atm) inhibits bacterial metabolism [21]. Homoacetogenesis is thermodynamically feasible at the usual experimental partial pressure of 0.39 atm and acetic acid concentration of 10×10−3 mol [18]. In the present work, the acetic acid concentration was higher than 10×10−3 mol and the bio-H2 pp was lowered to 0.06 atm, then the conditions in the CSTR made homoacetogenic reaction thermodynamically unfeasible, which thus enhanced bio-H2 production. Positive effects of the continuous flow were verified since constant dilution of the VFA concentration was attained, thereby reducing VFA-related inhibitions for bio-H2 production. On the contrary, batch process for bio-H2 production allows the accumulation of metabolic intermediates in the liquid and gas phases. The study [15] reported an extremely high bio-H2 in the system, which caused a shift in metabolic products from organic acids to solvents. Both accumulation of metabolic intermediates and high bio-H2 pp negatively affected bio-H2 production by different inhibitory effects. The VFAs can be stimulatory, inhibitory, or even toxic to the fermentative bacteria depending on their concentration [32]. At high VFAs levels, their no dissociated forms can permeate the bacterial cell membrane, then reducing the pH inside the bacterial cell [33]. Therefore, additional energy must be used to avoid unfavorable conditions, reducing the bacterial growth rate due to less available energy [32]. The overall results showed increments in the cumulative bio-H2 production, the bio-H2PR and the bio-H2MY caused by the reduction in the bio-H2 pp obtained via controlled biogas
Appl Biochem Biotechnol
release. In the CBGR fermentation, the bio-H2 pp was maintained at an average of 0.06 atm, which made the NADH reaction possible. This could be the main cause of the increased bioH2 production in the CBGR experiment. Mandal et al., [17] established a maximum bio-H2 pp as a function of the different electron donors and acceptors in the system with the relation Bio−H2 ppmax ≤e2 FðEH2 −ExÞ=RT , where EH2 =−414 mV. The authors demonstrated that the bio-H2 pp must be lower than 0.3 atm to produce bio-H2 using ferredoxin (Efd =−400 mV). Therefore, the bio-H2 pp maintained in the CBGR also allowed Bio-H2 production through the ferredoxin reaction. Global Mass and Energy Balance The stoichiometric butyrate fermentation from glucose is presented in the following equation. In this equation, ammonium is the nitrogen source of and the formula C5H7O2N represents biomass in mixed cultures [34–36]. In dark fermentation, 10–20 % of the substrate is stoichiometric converted to bio-H2. Fermentation with constant biogas release achieved 16 % glucose conversion to bio-H2, where 12 mol of H2/mol of glucose would be 100 % conversion. The 16 % conversion rate represents approximately 50 % of the maximum possible conversion through acetic acid fermentation. Considering that 15 % of the electrons in the substrate are recovered as bio-H2. The general model of the reaction described above is C 6 H 12 O6 þ HCO−3 þ 0:1N H 4 →CH 3 CH 2 CH 2 COO− þ 0:1C 5 H 7 O2 N þ H 2 O þ 3CO2 þ 2H 2 According to the global mass and energy balance equation, 1 mol of glucose produces 87 g of butyrate and 63.3 L of H2 at 0.76 atm (atmospheric pressure of Mexico City) and 20 °C. CBGR fermentation was mainly a butyric fermentation. The productivity is close to the theoretical value of 60 L of H2/mol of glucose compared to 63.3 theoretical L (Table 4). The average bio-H2 concentration was higher than the theoretical value achieved in the butyric acid fermentation. This is possible because the stoichiometric balance of butyric fermentation only takes into account the bio-H2 production from formate, while the ferredoxin-hydrogenase reaction was also possible with the constant biogas release at low pressure. Conclusions This paper has addressed the effect of bio-H2 pp on the continuous culture considering the gas release strategy. The effect of bio-H2 pp in various ranges was evaluated in a 30 L CSTR. The sustained low pH2 (0.06 atm), obtained by constant biogas release, produced the bio-H2PR increase from 36 to 108 mL/L h. The same condition induced higher biomass growth and glucose removal. Constant biogas release allowed both butyric and acetic acid fermentations with an enhanced bio-H2 production. The reduced CO2 concentrations limited the bio-H2 Table 4 Compared productivities of theoretical butyric acid fermentation and constant gas release fermentation
Theoretical butyric acid fermentation
Constant gas release (0.136 atm) fermentation
Butyrate (g)
87
80.1
Acetate (g)
–
3.8
63.3 40.2
60 54.4
2
1.9
H2 (L) H2 (%) H2 molar yield
Appl Biochem Biotechnol
consumption by homoacetogens. A maximum H2 yield of 1.9 was obtained and the bio-H2 was not diluted in the liberated gas. The continuous culture assured a constant bio-H2 production and avoids VFA accumulation. This fact prevented the modification in bacterial metabolism. Moreover, this condition also limited the bacteria inhibition. Acknowledgments This work was supported through funding provided by the CONACYT grant 60976 and Instituto Politécnico Nacional, grant SIP 20140405. The authors are grateful to Conacyt for the fellowship awarded.
References 1. Park, W., Hyun, S. H., Oh, S. E., Logan, B. E., & Kim, I. S. (2005). Removal of headspace CO2 increases biological hydrogen production. Environmental Science and Technology, 39, 4416–4420. 2. Nath, K., & Das, D. (2004). Improvement of fermentative hydrogen production: various approaches. Applied Microbiology and Biotechnology, 65, 520–529. 3. García-Peña, E., Guerrero-Barajas, C., Ramirez, D., & Arriaga-Hurtado, L. (2009). Semi-continuous biohydrogen production as an approach to generate electricity. Bioresource Technology, 100, 6369–6377. 4. Hallenbeck, P., & Ghosh, D. (2010). Improvements in fermentative biological hydrogen production through metabolic engineering. Journal of Environmental Management, 95, 360–364. 5. Ueno, Y., Haruta, S., Ishii, M., & Igarashi, Y. (2001). Microbial community in anaerobic hydrogenproducing microflora enriched from sludge compost. Applied Microbiology and Biotechnology, 57, 555–562. 6. Mizuno, O., Dinsdale, R., Hawkes, F., Hawkes, D., & Noike, T. (2000). Enhancement of hydrogen production from glucose by nitrogen gas sparging. Bioresource Technology, 73, 59–65. 7. Lee, H., Salerno, M., & Rittmann, B. (2008). Thermodynamic evaluation on H2 production in glucose fermentation. Environmental Science and Technology, 42, 2401–2407. 8. Prakasham, R., Sathish, T., & Brahmaiah, P. (2010). Biohydrogen production process optimization using anaerobic mixed consortia: a prelude study for use of agroindustrial material hydrolysate as substrate. Bioresource Technology, 101, 5708–5711. 9. Hung, C., Chang, Y., & Chang, Y. (2011). Roles of microorganisms other than Clostridium and Enterobacter in anaerobic fermentative biohydrogen production systems—a review. Bioresource Technology, 102, 8437–8444. 10. Oh, Y., Raj, S., Jung, G., & Park, S. (2011). Current status of the metabolic engineering of microorganisms for biohydrogen production. Bioresource Technology, 102, 8357–8367. 11. Fang, H., Zhang, T., & Liu, H. (2002). Microbial diversity of mesophilic hydrogen production sludge. Applied Microbiology and Biotechnology, 58, 112–118. 12. Hussy, I., Hawkes, F. R., Dinsdale, R., & Hawkes, D. L. (2003). Continuous fermentation hydrogen production from a wheat starch coproduct by mixed microflora. Biotechnology and Bioengineering, 84, 619–626. 13. Van-Ginkel, S., Oh, S., & Logan, B. (2005). Biohydrogen gas production from food processing and domestic wastewaters. International Journal of Hydrogen Energy, 30, 1535–1542. 14. Hawkes, F., Dinsdale, R., Hawkes, D., & Hussy, I. (2002). Sustainable fermentative hydrogen production: challenges for process optimization. International Journal of Hydrogen Energy, 27, 1339–1347. 15. Valdez-Vazquez, I., Rios-Leal, E., Carmona-Martinez, A., Muñoz-Paez, K., & Poggi Varaldo, H. (2006). Improvement of biohydrogen production from solid wastes by intermittent venting and gas flushing of batch reactors headspace. Environmental Science and Technology, 40, 3409–3415. 16. Angenent, L. T., Karim, K., Al-Dahhan, M., Wrenn, B., & Dominguez-Espinosa, R. (2004). Production of bioenergy and biochemicals from industrial and agricultural wastewater. Trends in Biotechnology, 22, 477–485. 17. Mandal, B., Nath, K., & Das, D. (2006). Improvement of biohydrogen production under decreased partial pressure of H2 by Enterobacter cloacae. Biotechnology Letters, 28, 831–835. 18. Bastidas-Oyanedel, J. R., Mohd-Zaki, Z., Zeng, R. J., Bernet, N., Pratt, S., Steyer, J. P., & Batstonebet, D. J. (2012). Gas controlled hydrogen fermentation. Bioresource Technology, 110, 503–509. 19. Jung, K. W., Kim, D. H., Kim, S. H., & Shin, H. S. (2011). Bioreactor design for continuous dark fermentative hydrogen production. Bioresource Technology, 102, 8612–8620.
Appl Biochem Biotechnol 20. Kim, D., Han, S., Kim, S., & Shin, H. (2006). Effect of gas sparging on continuous fermentative hydrogen production. International Journal of Hydrogen Energy, 31, 2158–2169. 21. Kraemer, J., & Bagley, D. (2008). Optimisation and design of nitrogen-sparged fermentative hydrogen production bioreactors. International Journal of Hydrogen Energy, 33, 6558–6565. 22. Logan, B., Oh, S., Kim, I., & Van-Ginkel, S. V. (2002). Biological hydrogen production measured in batch anaerobic respirometers. Environmental Science and Technology, 36, 2530–2535. 23. Oh, S., Zue, Y., Zhang, H., Guiltinan, M., Logan, B., & Regan, J. (2009). Hydrogen production by Clostridium acetobutylicum ATCC 824 and megaplasmid-deficient mutant M5 evaluated using a large headspace volume technique. International Journal of Hydrogen Energy, 34, 9347–9353. 24. Garcia-Peña, E. I., Parameswaran, P., Kang, D., Canul-Chan, M., & Krajmalnik-Brown, R. (2011). Anaerobic digestion and co-digestion processes of vegetable and fruit residues: process and microbial ecology. Bioresource Technology, 102, 9447–9455. 25. Canul, M., Salgado, E., Aranda, J., & Garcia, E. I. (2010). Hydrogen and electricity production from the organic fraction of solid waste. Proceedings of the Third International Symposium on Energy from Biomass and Waste. Italy. 26. Garcia-Peña, E. I., Canul-Chan, M., Chairez, I., Salgado-Manjarez, E., & Aranda-Barradas, J. (2013). Biohydrogen production based on the evaluation of kinetic parameters of a mixed microbial culture using glucose and fruit–vegetable waste as feedstocks. Applied Biochemistry and Biotechnology, 171, 279–293. 27. Hallenbeck, P. C., & Ghosh, D. (2009). Advances in fermentative biohydrogen production: the way forward? Trends in Biotechnology, 27, 287–297. 28. Arriaga, S., Rosas, I., Alatriste-Mondragón, F., & Razo-Flores, E. (2011). Continuous production of hydrogen from oat straw hydrolysate in a biotrickling filter. International Journal of Hydrogen Energy, 36, 3442–3449. 29. Kongjan, P., O-Thong, S., Kotay, M., Min, B., & Angelidaki, I. (2010). Biohydrogen production from wheat straw hydrolysates by dark fermentation using extreme thermophilic mixed culture. Biotechnology and Bioengineering, 105, 899–908. 30. Arooj, M. F., Han, S. K., Kim, S. H., & Dong-Hoon, K. (2008). Continuous biohydrogen production in a CSTR using starch as a substrate. International Journal of Hydrogen Energy, 33, 3289–3294. 31. Lee, D., Li, Y., & Noike, T. (2009). Continuous H2 production by anaerobic mixed microflora in membrane bioreactor. Bioresource Technology, 100, 690–695. 32. Zheng, X. J., & Yu, H. Q. (2005). Inhibitory effects of butyrate on biological hydrogen production with mixed anaerobic cultures. Journal of Environmental Management, 74, 65–70. 33. Gottschalk, G. (1986). Bacterial metabolism (2nd ed.). New York: Springer. 34. Rittmann, B. E., & McCarty, P. L. (2001). Environmental biotechnology: principles and applications. New York: McGraw-Hill. 35. Cai, G., Jin, B., Saint, C., & Monis, P. (2010). Metabolic flux analysis of hydrogen production network by Clostridium butyricum W5: effect of pH and glucose concentration. International Journal of Hydrogen Energy, 35, 6681–6690. 36. Das, D. (2009). Advances in biohydrogen production processes: an approach towards commercialization. International Journal of Hydrogen Energy, 34, 7349–7357.
