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Biological hydrogen production from probiotic wastewater as substrate by selectively enriched anaerobic mixed microflora
Renewable Energy 34 (2009) 937–940
Contents lists available at ScienceDirect
Renewable Energy journal homepage: www.elsevier.com/locate/renene
Technical Note
Biological hydrogen production from probiotic wastewater as substrate by selectively enriched anaerobic mixed microflora D. Sivaramakrishna a, D. Sreekanth a, V. Himabindu a, *, Y. Anjaneyulu b a
Centre for Environment, Institute of Science and Technology, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad 500072, Andhra Pradesh, India b TLGVRC, JSU Box 18739, JSU, Jackson, MS 32917-0939, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 August 2007 Accepted 19 April 2008 Available online 9 July 2008
Biohydrogen production from probiotic wastewater using mixed anaerobic consortia is reported in this paper. Batch tests are carried out in a 5.0 L batch reactor under constant mesophillic temperature (37 C). The maximum hydrogen yield 1.8 mol-hydrogen/mol-carbohydrate is obtained at an optimum pH of 5.5 and substrate concentration 5 g/L. The maximum hydrogen production rate is 168 ml/h. The hydrogen content in the biogas is more than 65% and no significant methane is observed throughout the study. In addition to hydrogen, acetate, propionate, butyrate and ethanol are found to be the main by-products in the metabolism of hydrogen fermentation. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Biohydrogen gas Probiotic wastewater Pretreatment Mixed microflora Fermentation Mesophilic
1. Introduction Energy is one of the most important factors to global prosperity, however, as a main energy source used, fossil fuel has been overly consumed and is one of the significant causes of global warming and acid rain. Hence the need for a new energy source attraction to fossil fuel has become a sky rocketing demand. Hydrogen, a clean renewable energy source is regarded as an attractive fossil fuel. Hydrogen is a high-value industrial commodity with a wide range of applications. It is an ideal fuel, producing only water upon combustion. It may be converted into electricity via fuel cells or directly used in internal combustion engines. It can also be used for the synthesis of ammonia, alcohols and aldehydes, as well as for the hydrogenation of edible oil, petroleum, coal and shale oil [1]. Hydrogen is produced in large amounts by chemical and physical methods like steam reforming, partial oxidation of fossil fuels, which make hydrogen production expensive and also huge environmental pollution. However, hydrogen production via biological route provides a cost-effective and environmentally harmless
* Corresponding author. Tel./fax: þ91 040 2315 6133. E-mail addresses: [email protected], [email protected] (V. Himabindu). 0960-1481/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.renene.2008.04.016
alternative carried out under mild operating conditions with a wide variety of renewable resources. Biological hydrogen production may be either by fermentation [2,3] or photosynthesis [4,5]. Fermentative hydrogen production is more advantageous over photosynthetic hydrogen production since it does not rely on the availability of light sources and the transparency of the mixed liquor [6]. Production of hydrogen by fermentation has been studied for a large group of fermentative bacteria, such as Enterobacteria [7,8], Klebsiella [9], Citrobacter [10] and Escherichia coli [11]. However studies of hydrogen production by mixed cultures have attracted research attention only recently [12]. Extensive research is carried out for hydrogen production using various waste materials and wastewater from industrial process such as rice slurry wastewater [13], food and domestic wastewaters [14] and citric acid wastewater [15]. Use of industrial wastewater as substrate facilitates both treatment and renewable extraction of clean gas simultaneously. However, no previous study has been reported on the use of probiotic wastewater as substrate for the production of hydrogen. The aim of the present study, therefore, is to evaluate anaerobic biohydrogen production process using probiotic wastewater which is a live carbohydrate rich feed supplement used for farm animals and humans. Additionally, system operating conditions such as effect of wastewater, pH and substrate concentration are also monitored.
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2. Materials and methods 2.1. Probiotic wastewater Probiotic wastewater (suspended solids (SS) – 640 mg/L; total dissolved solids (TDS) – 1430 mg/L; total solids (TS) – 2164 mg/L; COD – 9480 mg/L and pH 7.4) is obtained from a local fermentation company Uni-sanqiuo, Hyderabad, India is used as substrate. Influent is prepared by using the raw wastewater as the sole carbon source, without adding any nutrients. The pH of the mixed liquor in the reactor was adjusted by using 6 N NaOH and 6 N HCl solutions. Prior to addition in to the reactor, the wastewater is heat treated at 50 C for 10 min to inactivate non-sporogenic bacteria present in the wastewater. No significant change in the feed stock is observed following heat treatment.
2.2. Inoculum Hydrogen producing microflora for the batch experiments are taken from the pilot scale, UASB reactor treating local slaughterhouse manure treatment plant (Alkabeer, Hyderabad, India). It is stored anaerobically at room temperature for 6 months. Prior to use, the sludge is sieved to remove stone, sand and other coarse material. Thereafter, the seed sludge is heated at 85 C for 1 h and acid treatment at a pH of 3–4 for 24 h before inoculation to inactivate hydrogenotophic methanogens and enrich hydrogen producing bacteria.
Fig. 2. Effect of nutrients.
2.4. Analytical methods The hydrogen gas percentage was calculated by comparing the sample biogas with a standard of pure hydrogen using a Gas Chromatograph (Amil Nucon 5700, Italy) equipped with a thermal conductivity detector (TCD) and 6 feet stainless column packed with porapak Q (80/100 mesh). The operational temperatures of the injection port, the oven and the detector are 100 C, 50 C and 100 C respectively. Nitrogen is used as the carrier gas at a flow rate of 30 ml/min. The concentrations of the volatile fatty acids (VFAs) and the alcohol are analyzed using same GC under following conditions; column: chromosorb 101, carrier gas: nitrogen, flow rate: 30 ml/min, column temperature: 200 C, injector temperature: 220 C, detector temperature 220 C, and detector: FID. The pH values inside the digesters are measured by a microcomputer pHvision 6071.
2.3. Batch reactor The anaerobic batch experiments are performed in a 5 L capacity round bottom flask consisting of wide mouth, suitable inlet and outlet arrangements. The reactor is provided with a three way outlet ports. One is for the gas outlet and other is for transfer of treated biomass and third port is for the removal of sludge. The anaerobic batch reactor is represented in Fig. 1. The gas outlet is connected through rubber tube to the liquid displacement system to measure the gas production. The reactor is placed on a magnetic stirrer for continuous and homogenous mixing. The reactor is covered with an aluminum foil to inhibit the growth of photosynthetic bacteria. Nitrogen gas was sparged in to the digester for 3– 5 min to create strict anaerobic conditions prior to seeding of the active anaerobic sludge. The reactor temperature is maintained at 37 C. The entire system is checked thoroughly for gas leakages.
Fig. 1. Batch reactor.
2.5. Operation Three series of experiments are conducted to investigate the individual effect of operational parameters. In series 1, the effect of raw wastewater on hydrogen production with and without nutrients. In series 2, the effect of pH on hydrogen production and in series 3 the effect of substrate concentration in wastewater are monitored. Each series is repeated three times for reproduction of values. The effluent biogas compositions are simultaneously monitored along with operational parameters and steady-state conditions are reported.
Fig. 3. (a) Effect of pH on total biogas and hydrogen gas. (b) Effect of pH on hydrogen yield and substrate removal.
D. Sivaramakrishna et al. / Renewable Energy 34 (2009) 937–940
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Table 1 VFA and Alcohol production at pH 4.5–7.0 pH 4.5 5.0 5.5 6.0 6.5 7.0
VFA & alcohols (mg/L)
Acetate (%)
Butyrate (%)
Propionate (%)
Ethanol (%)
Methanol (%)
2023 2664 2174 2148 2096 2112
23.4 32.1 36.2 45.3 46.2 47.3
46.1 44.5 41.2 38.4 35.2 33.2
1.3 2.3 3.7 3.7 4.2 6.8
4.2 3.8 3.2 2.8 1.9 –
9.2 6.4 5.8 5.2 4.8 3.2
3. Results and discussion 3.1. Effect of nutrients Representative biogas production curves from batch tests conducted with and without nutrient amendments are shown in Fig. 2. The initial pH of wastewater is maintained at 5.5 for a substrate concentration of 5 g/L carbohydrate. From the graph it is observed that wastewater with nutrients produced a biogas of 232 ml and without nutrients 250 ml. It is concluded from the above results that almost equal amounts of biogas is produced with (or) without addition of nutrients since the probiotic wastewater itself might be rich in nutrients. This indicates that addition of nutrients is not always needed to maximize hydrogen production from the wastewater samples. 3.2. Effect of pH pH is one of the important factors of anaerobic fermentation process. It has a profound effect on hydrogen production. A set of tests are performed at variable pH from 4.5 to 7.0, keeping the other operating conditions (temperature ¼ 37 C, substrate concentration ¼ 5 g/L) constant. It shows that, the initial pH values significantly effects on: (a) total biogas and hydrogen gas; and (b) hydrogen yield and substrate removal. Fig. 3a illustrates that, the hydrogen production increased from 114 ml/h at an initial pH 4.5 to 168 ml/h at an initial pH 5.5. No methane gas is observed at pH 5.5 which may be due to the suppression of methanogenic activity under acidic condition with respect to substrate degradation. This is in coincidence with the results obtained by Fang and Liu [16] who reported the production of methane free biogas at pH 5.5. At pH higher (or) lower than this, the hydrogen production decreased, and it is completely eliminated below pH 4. Kim et al. [17] reported acidogenic conditions to be most suitable for hydrogen generation from anaerobic fermentative consortia. Zhang et al. [18] also reported an optimum pH range of 5.5–6.0 for hydrogen generation by anaerobic fermentation. Suppression of methanogenesis and solvent genesis at this pH indicates that during fermentation some organic acids are also produced as metabolic products [19]. Accumulation of these acids causes a sharp drop of culture pH and subsequent inhibition of bacterial hydrogen production. The poor hydrogen production at pH lower than 5.5 could be due to the increased formation of acidic or alcoholic metabolites, which destroys the cell’s ability to maintain internal pH. It might have resulted in lowering of intracellular level of ATP, thereby inhibiting glucose uptake [20]. Fig. 3b illustrates that substrate degradation increased from 86% to 93% at pH 4.5–5.5, and remained nearly constant (91–93%) for pH ranging from 5.5–7. It can also be inferred that the hydrogen yield reached on optimum at pH 5.5. It shows that the maximum yield of 1.8 0.1 mol H2/mol substrate observed in this study is substantially higher than the yields reported for other mixed cultures. The results showed that the pH control could stimulate the microorganisms to produce hydrogen and would achieve the system having a maximum hydrogen yield, but the activity of
hydrogenases would be inhibited by low (or) high pH values in overall hydrogen fermentation. Overall, pH 5.5 was identified in this study as the suitable pH range for hydrogen production from carbohydrates under the mesophilic condition. Anaerobic hydrogen production is always accompanied with VFA production. The VFA concentration distribution and their fractions have been successfully used as indicators for monitoring hydrogen production [21]. Table 1 lists the distribution of the key VFA and alcohols in the effluents at various pH. It shows that butyrate and acetate are the two main products in all fermentation batches. Increase of pH from 4.5 to 7.0 resulted in the decrease of butyrate but an increase of acetate. With in a pH range of (4.5–7.0) the effluent contained mostly butyrate (46.1–33.2%), followed by acetate (23.4–47.3%). In hydrogen producing process the pathway may shift from VFA producing to alcohol, when the pH is lowered 4.5 or below. However, increase of pH did result in increase of propionate, from 1.3% of the total VFA/alcohols at pH 4.5 to 6.8% at pH 7.0, and the decrease of ethanol, from 9.2% at pH 4.5 to 3.2% at pH 7.0. Methanol is also detected in all batches except pH at 7.0. The above results demonstrate that the distribution of final VFAs and alcohols are influenced by variations in initial pH.
3.3. Effect of substrate concentration The effects of the substrate concentration versus hydrogen yield by the microorganisms are presented in Fig. 4. The hydrogen yield increased remarkably with an increase in the concentration of the substrate, i.e., when the concentration of the substrate increased from 2 to 3.5 and 5 g/L, the hydrogen yield increased from 1.5, 1.6 and 1.8 mol/mol respectively. Thereafter, the hydrogen yield decreased gradually as the concentration of the substrate increased, i.e., while the concentration of the substrate increased from 5, 6.5 to 8 g/L, the hydrogen yield decreased from 1.8, 1.2 to 0.9 mol/mol, respectively. The result showed that the change of the substrate concentration obviously affected the hydrogen yield. Although an increase in the substrate concentration enhanced the hydrogen yield, excessive addition of substrate concentration result in the accumulation of volatile fatty acids and a fall of hydrogen-producing bacteria. In addition, the partial pressure of hydrogen in the batch reactor increased with the increasing substrate concentration resulting in enhanced alcohol production with further inhibition in the hydrogen production [22].
Fig. 4. Effect of substrate concentration on hydrogen yield.
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Table 2 VFA and Alcohol production at pH 5.5 and various substrate concentrations Substrate conc. 2.0 3.5 5.0 6.5 8.0
VFA & alcohol (mg/L)
Acetate (%)
Butyrate (%)
Propionate (%)
Ethanol (%)
Methanol (%)
983 1684 1923 2116 2726
43.2 41.8 38.2 36.0 33.2
29.4 32.6 34.2 36.3 35.2
1.2 1.5 1.5 1.8 1.9
7.2 8.6 9.2 10.4 11.2
1.9 2.2 2.8 3.1 3.4
Table 2 shows that the total VFA/alcohols concentration increased only with the increase in substrate concentration, from 983 mg/L at 2.0 g/L to 2726 mg/L at 8 g/L. The composition of VFA/ alcohols slightly depended on substrate concentration in wastewater. Increase of substrate concentration resulted in a slight decrease of acetate production (from 43.2 to 33.2%), but increased butyrate (from 29.4 to 35.2%) and ethanol production (7.2–11.2%). It has a little effect of on the contents of propionate (1.2–1.9) and methanol (1.9–3.4). 4. Conclusion The study demonstrated the feasibility of H2 generation from probiotic wastewater as substrate using selectively enriched mixed consortia. The conditions of heat treatment and enrichment pH are the main treatment effects that are significant on the generation. Probiotic wastewater used as the sole carbon source documented its metabolic participation in H2 generation. Fermentation pH 5.5 and substrate concentration 5 g/L are found to be optimum for the overall process efficiency of H2 generation. The described process has a dual benefit of H2 production with simultaneous wastewater treatment in an economical, effective and sustainable way. Acknowledgement The authors wish to thank the Ministry of New and Renewable Energy (MNRE) Government of India (File No. 102/9/2004-NT) for the partial financial support of this study. References [1] Fang HHP, Li C, Zhang T. Acidophilic biohydrogen production from rice slurry. International Journal of Hydrogen Energy 2006;31:683–92. [2] Tao Y, Chen Y, Wu Y, He Y, Zhou Z. High hydrogen yield from a two-step process of dark-and photo-fermentation of sucrose. International Journal of Hydrogen Energy 2007;31:200–6. [3] Ren N, Li J, Li B, Wang Y, Liu S. Biohydrogen production from molasses by anaerobic fermentation with a pilot-scale bioreactor system. International Journal of Hydrogen Energy 2006;31:2147–57. [4] Ghirardi ML, Zhang L, Flynn LJW, Seibert TM, Greenbaum E, et al. Microalgae: a green source of renewable H2. Tibtech 2000;18:506–11.
[5] Chen HC, Newton AJ, Melis A. Role of SulP, a nuclear-encoded chloroplast sulfate perm ease, in sulfate transport and H2 evolution in Chlamydomonas reinhardtii. Photosynthesis Research 2005;84:289–96. [6] Hart D. Hydrogen power: the commercial future of the ultimate fuel. London: Financial Times Energy Publishing; 1997. [7] David B, Levina B, Islamc R, Cicekc N, Sparlingd R. Hydrogen production by Clostridium thermocellum 27405 from cellulosic biomass substrates. International Journal of Hydrogen Energy 2006;31:1496–503. [8] Fabino B, Perego P. Thermodynamics study and optimization of hydrogen production by Enterobacter aerogens. International Journal of Hydrogen Energy 2002;27:149–56. [9] Minnan L, Jinli H, Xiaobin W, Huijuan X, Jinzao C, Chuannan L, et al. Isolation and identification of high hydrogen producing strain Klebsiella oxytoca HP1 from a hot spring. Research in Microbiology 2005;156:76–81. [10] Oh Y-K, Seol E-H, Kim JR, Park S. Fermentative biohydrogen production by a new chemo heterotrophic bacterium Citrobcter sp. Y19. International Journal of Hydrogen Energy 2003;28:1353–9. [11] Chittibabu G, Nath K, Das D. Feasibility studies on the fermentative hydrogen production by recombinant Escherichia coli BL-21. Process Biochemistry 2006; 41(3):682–8. [12] Hawkes FR, Hussy I, Kyazze G, Dinsdale R, Hawkes DL. Continuous dark fermentative hydrogen production by mesophilic microflora: principles and progress. International Journal of Hydrogen Energy 2007;32:172–84. [13] Yu H, Zhu Z, Hu W, Zhang H. Hydrogen production from rice slurry wastewater in an up flow anaerobic reactor by using mixed anaerobic cultures. International Journal of Hydrogen Energy 2002;27:1359–65. [14] Van Ginkel SW, Oh S-E, Logan BE. Biohydrogen gas production from food processing and domestic wastewaters. International Journal of Hydrogen Energy 2004;30:4673–82. [15] Yanga H, Shaoc P, Lub T, Shena J, Wangb D, Xub Z, et al. Continuous biohydrogen production from citric acid wastewater via facultative anaerobic bacteria. International Journal of Hydrogen Energy 2006;31:1306–13. [16] Fang HHP, Liu H. Effect of pH on hydrogen production from glucose by a mixed culture. Bioresource Technology 2002;82:87–93. [17] Kim IS, Hwang MH, Jang NJ, Hyun SH, Lee ST. Effect of low pH on the activity of hydrogen utilizing methanogen in bio-hydrogen process. International Journal of Hydrogen Energy 2004;29:1133–40. [18] Zhang Z-P, Show K-Y, Tay J-H, Liang DT, Lee D-J, Jiang W-J. Effect of hydraulic retention time on biohydrogen production and anaerobic microbial community. Process Biochemistry 2006;41:2118–23. [19] Mohan SV, Bhaskar YV, Krishna PM, Rao CN, Babu VL, Sarma PN. Biohydrogen production from chemical wastewater as substrate by selectively enriched anaerobic mixed consortia: influence of fermentation pH and substrate composition. International Journal of Hydrogen Energy 2007;32(13):2286–95. [20] Bowles LK, Ellefson WL. Effects of butanol on Clostridium acetobutylicum. Applied and Environmental Microbiology 1985;50:1165–70. [21] Chang F-Y, Lin C-Y. Biohydrogen production using an up-flow anaerobic sludge blanket reactor. International Journal of Hydrogen Energy 2004;29:33–9. [22] Fan Y-T, Zhang Y-H, Zhang S-F, Hou H-W, Ren B-Z. Efficient conversion of wheat straw wastes into biohydrogen gas by cow dung compost. Bioresource Technology 2006;97:500–5.
Contents lists available at ScienceDirect
Renewable Energy journal homepage: www.elsevier.com/locate/renene
Technical Note
Biological hydrogen production from probiotic wastewater as substrate by selectively enriched anaerobic mixed microflora D. Sivaramakrishna a, D. Sreekanth a, V. Himabindu a, *, Y. Anjaneyulu b a
Centre for Environment, Institute of Science and Technology, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad 500072, Andhra Pradesh, India b TLGVRC, JSU Box 18739, JSU, Jackson, MS 32917-0939, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 August 2007 Accepted 19 April 2008 Available online 9 July 2008
Biohydrogen production from probiotic wastewater using mixed anaerobic consortia is reported in this paper. Batch tests are carried out in a 5.0 L batch reactor under constant mesophillic temperature (37 C). The maximum hydrogen yield 1.8 mol-hydrogen/mol-carbohydrate is obtained at an optimum pH of 5.5 and substrate concentration 5 g/L. The maximum hydrogen production rate is 168 ml/h. The hydrogen content in the biogas is more than 65% and no significant methane is observed throughout the study. In addition to hydrogen, acetate, propionate, butyrate and ethanol are found to be the main by-products in the metabolism of hydrogen fermentation. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Biohydrogen gas Probiotic wastewater Pretreatment Mixed microflora Fermentation Mesophilic
1. Introduction Energy is one of the most important factors to global prosperity, however, as a main energy source used, fossil fuel has been overly consumed and is one of the significant causes of global warming and acid rain. Hence the need for a new energy source attraction to fossil fuel has become a sky rocketing demand. Hydrogen, a clean renewable energy source is regarded as an attractive fossil fuel. Hydrogen is a high-value industrial commodity with a wide range of applications. It is an ideal fuel, producing only water upon combustion. It may be converted into electricity via fuel cells or directly used in internal combustion engines. It can also be used for the synthesis of ammonia, alcohols and aldehydes, as well as for the hydrogenation of edible oil, petroleum, coal and shale oil [1]. Hydrogen is produced in large amounts by chemical and physical methods like steam reforming, partial oxidation of fossil fuels, which make hydrogen production expensive and also huge environmental pollution. However, hydrogen production via biological route provides a cost-effective and environmentally harmless
* Corresponding author. Tel./fax: þ91 040 2315 6133. E-mail addresses: [email protected], [email protected] (V. Himabindu). 0960-1481/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.renene.2008.04.016
alternative carried out under mild operating conditions with a wide variety of renewable resources. Biological hydrogen production may be either by fermentation [2,3] or photosynthesis [4,5]. Fermentative hydrogen production is more advantageous over photosynthetic hydrogen production since it does not rely on the availability of light sources and the transparency of the mixed liquor [6]. Production of hydrogen by fermentation has been studied for a large group of fermentative bacteria, such as Enterobacteria [7,8], Klebsiella [9], Citrobacter [10] and Escherichia coli [11]. However studies of hydrogen production by mixed cultures have attracted research attention only recently [12]. Extensive research is carried out for hydrogen production using various waste materials and wastewater from industrial process such as rice slurry wastewater [13], food and domestic wastewaters [14] and citric acid wastewater [15]. Use of industrial wastewater as substrate facilitates both treatment and renewable extraction of clean gas simultaneously. However, no previous study has been reported on the use of probiotic wastewater as substrate for the production of hydrogen. The aim of the present study, therefore, is to evaluate anaerobic biohydrogen production process using probiotic wastewater which is a live carbohydrate rich feed supplement used for farm animals and humans. Additionally, system operating conditions such as effect of wastewater, pH and substrate concentration are also monitored.
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2. Materials and methods 2.1. Probiotic wastewater Probiotic wastewater (suspended solids (SS) – 640 mg/L; total dissolved solids (TDS) – 1430 mg/L; total solids (TS) – 2164 mg/L; COD – 9480 mg/L and pH 7.4) is obtained from a local fermentation company Uni-sanqiuo, Hyderabad, India is used as substrate. Influent is prepared by using the raw wastewater as the sole carbon source, without adding any nutrients. The pH of the mixed liquor in the reactor was adjusted by using 6 N NaOH and 6 N HCl solutions. Prior to addition in to the reactor, the wastewater is heat treated at 50 C for 10 min to inactivate non-sporogenic bacteria present in the wastewater. No significant change in the feed stock is observed following heat treatment.
2.2. Inoculum Hydrogen producing microflora for the batch experiments are taken from the pilot scale, UASB reactor treating local slaughterhouse manure treatment plant (Alkabeer, Hyderabad, India). It is stored anaerobically at room temperature for 6 months. Prior to use, the sludge is sieved to remove stone, sand and other coarse material. Thereafter, the seed sludge is heated at 85 C for 1 h and acid treatment at a pH of 3–4 for 24 h before inoculation to inactivate hydrogenotophic methanogens and enrich hydrogen producing bacteria.
Fig. 2. Effect of nutrients.
2.4. Analytical methods The hydrogen gas percentage was calculated by comparing the sample biogas with a standard of pure hydrogen using a Gas Chromatograph (Amil Nucon 5700, Italy) equipped with a thermal conductivity detector (TCD) and 6 feet stainless column packed with porapak Q (80/100 mesh). The operational temperatures of the injection port, the oven and the detector are 100 C, 50 C and 100 C respectively. Nitrogen is used as the carrier gas at a flow rate of 30 ml/min. The concentrations of the volatile fatty acids (VFAs) and the alcohol are analyzed using same GC under following conditions; column: chromosorb 101, carrier gas: nitrogen, flow rate: 30 ml/min, column temperature: 200 C, injector temperature: 220 C, detector temperature 220 C, and detector: FID. The pH values inside the digesters are measured by a microcomputer pHvision 6071.
2.3. Batch reactor The anaerobic batch experiments are performed in a 5 L capacity round bottom flask consisting of wide mouth, suitable inlet and outlet arrangements. The reactor is provided with a three way outlet ports. One is for the gas outlet and other is for transfer of treated biomass and third port is for the removal of sludge. The anaerobic batch reactor is represented in Fig. 1. The gas outlet is connected through rubber tube to the liquid displacement system to measure the gas production. The reactor is placed on a magnetic stirrer for continuous and homogenous mixing. The reactor is covered with an aluminum foil to inhibit the growth of photosynthetic bacteria. Nitrogen gas was sparged in to the digester for 3– 5 min to create strict anaerobic conditions prior to seeding of the active anaerobic sludge. The reactor temperature is maintained at 37 C. The entire system is checked thoroughly for gas leakages.
Fig. 1. Batch reactor.
2.5. Operation Three series of experiments are conducted to investigate the individual effect of operational parameters. In series 1, the effect of raw wastewater on hydrogen production with and without nutrients. In series 2, the effect of pH on hydrogen production and in series 3 the effect of substrate concentration in wastewater are monitored. Each series is repeated three times for reproduction of values. The effluent biogas compositions are simultaneously monitored along with operational parameters and steady-state conditions are reported.
Fig. 3. (a) Effect of pH on total biogas and hydrogen gas. (b) Effect of pH on hydrogen yield and substrate removal.
D. Sivaramakrishna et al. / Renewable Energy 34 (2009) 937–940
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Table 1 VFA and Alcohol production at pH 4.5–7.0 pH 4.5 5.0 5.5 6.0 6.5 7.0
VFA & alcohols (mg/L)
Acetate (%)
Butyrate (%)
Propionate (%)
Ethanol (%)
Methanol (%)
2023 2664 2174 2148 2096 2112
23.4 32.1 36.2 45.3 46.2 47.3
46.1 44.5 41.2 38.4 35.2 33.2
1.3 2.3 3.7 3.7 4.2 6.8
4.2 3.8 3.2 2.8 1.9 –
9.2 6.4 5.8 5.2 4.8 3.2
3. Results and discussion 3.1. Effect of nutrients Representative biogas production curves from batch tests conducted with and without nutrient amendments are shown in Fig. 2. The initial pH of wastewater is maintained at 5.5 for a substrate concentration of 5 g/L carbohydrate. From the graph it is observed that wastewater with nutrients produced a biogas of 232 ml and without nutrients 250 ml. It is concluded from the above results that almost equal amounts of biogas is produced with (or) without addition of nutrients since the probiotic wastewater itself might be rich in nutrients. This indicates that addition of nutrients is not always needed to maximize hydrogen production from the wastewater samples. 3.2. Effect of pH pH is one of the important factors of anaerobic fermentation process. It has a profound effect on hydrogen production. A set of tests are performed at variable pH from 4.5 to 7.0, keeping the other operating conditions (temperature ¼ 37 C, substrate concentration ¼ 5 g/L) constant. It shows that, the initial pH values significantly effects on: (a) total biogas and hydrogen gas; and (b) hydrogen yield and substrate removal. Fig. 3a illustrates that, the hydrogen production increased from 114 ml/h at an initial pH 4.5 to 168 ml/h at an initial pH 5.5. No methane gas is observed at pH 5.5 which may be due to the suppression of methanogenic activity under acidic condition with respect to substrate degradation. This is in coincidence with the results obtained by Fang and Liu [16] who reported the production of methane free biogas at pH 5.5. At pH higher (or) lower than this, the hydrogen production decreased, and it is completely eliminated below pH 4. Kim et al. [17] reported acidogenic conditions to be most suitable for hydrogen generation from anaerobic fermentative consortia. Zhang et al. [18] also reported an optimum pH range of 5.5–6.0 for hydrogen generation by anaerobic fermentation. Suppression of methanogenesis and solvent genesis at this pH indicates that during fermentation some organic acids are also produced as metabolic products [19]. Accumulation of these acids causes a sharp drop of culture pH and subsequent inhibition of bacterial hydrogen production. The poor hydrogen production at pH lower than 5.5 could be due to the increased formation of acidic or alcoholic metabolites, which destroys the cell’s ability to maintain internal pH. It might have resulted in lowering of intracellular level of ATP, thereby inhibiting glucose uptake [20]. Fig. 3b illustrates that substrate degradation increased from 86% to 93% at pH 4.5–5.5, and remained nearly constant (91–93%) for pH ranging from 5.5–7. It can also be inferred that the hydrogen yield reached on optimum at pH 5.5. It shows that the maximum yield of 1.8 0.1 mol H2/mol substrate observed in this study is substantially higher than the yields reported for other mixed cultures. The results showed that the pH control could stimulate the microorganisms to produce hydrogen and would achieve the system having a maximum hydrogen yield, but the activity of
hydrogenases would be inhibited by low (or) high pH values in overall hydrogen fermentation. Overall, pH 5.5 was identified in this study as the suitable pH range for hydrogen production from carbohydrates under the mesophilic condition. Anaerobic hydrogen production is always accompanied with VFA production. The VFA concentration distribution and their fractions have been successfully used as indicators for monitoring hydrogen production [21]. Table 1 lists the distribution of the key VFA and alcohols in the effluents at various pH. It shows that butyrate and acetate are the two main products in all fermentation batches. Increase of pH from 4.5 to 7.0 resulted in the decrease of butyrate but an increase of acetate. With in a pH range of (4.5–7.0) the effluent contained mostly butyrate (46.1–33.2%), followed by acetate (23.4–47.3%). In hydrogen producing process the pathway may shift from VFA producing to alcohol, when the pH is lowered 4.5 or below. However, increase of pH did result in increase of propionate, from 1.3% of the total VFA/alcohols at pH 4.5 to 6.8% at pH 7.0, and the decrease of ethanol, from 9.2% at pH 4.5 to 3.2% at pH 7.0. Methanol is also detected in all batches except pH at 7.0. The above results demonstrate that the distribution of final VFAs and alcohols are influenced by variations in initial pH.
3.3. Effect of substrate concentration The effects of the substrate concentration versus hydrogen yield by the microorganisms are presented in Fig. 4. The hydrogen yield increased remarkably with an increase in the concentration of the substrate, i.e., when the concentration of the substrate increased from 2 to 3.5 and 5 g/L, the hydrogen yield increased from 1.5, 1.6 and 1.8 mol/mol respectively. Thereafter, the hydrogen yield decreased gradually as the concentration of the substrate increased, i.e., while the concentration of the substrate increased from 5, 6.5 to 8 g/L, the hydrogen yield decreased from 1.8, 1.2 to 0.9 mol/mol, respectively. The result showed that the change of the substrate concentration obviously affected the hydrogen yield. Although an increase in the substrate concentration enhanced the hydrogen yield, excessive addition of substrate concentration result in the accumulation of volatile fatty acids and a fall of hydrogen-producing bacteria. In addition, the partial pressure of hydrogen in the batch reactor increased with the increasing substrate concentration resulting in enhanced alcohol production with further inhibition in the hydrogen production [22].
Fig. 4. Effect of substrate concentration on hydrogen yield.
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Table 2 VFA and Alcohol production at pH 5.5 and various substrate concentrations Substrate conc. 2.0 3.5 5.0 6.5 8.0
VFA & alcohol (mg/L)
Acetate (%)
Butyrate (%)
Propionate (%)
Ethanol (%)
Methanol (%)
983 1684 1923 2116 2726
43.2 41.8 38.2 36.0 33.2
29.4 32.6 34.2 36.3 35.2
1.2 1.5 1.5 1.8 1.9
7.2 8.6 9.2 10.4 11.2
1.9 2.2 2.8 3.1 3.4
Table 2 shows that the total VFA/alcohols concentration increased only with the increase in substrate concentration, from 983 mg/L at 2.0 g/L to 2726 mg/L at 8 g/L. The composition of VFA/ alcohols slightly depended on substrate concentration in wastewater. Increase of substrate concentration resulted in a slight decrease of acetate production (from 43.2 to 33.2%), but increased butyrate (from 29.4 to 35.2%) and ethanol production (7.2–11.2%). It has a little effect of on the contents of propionate (1.2–1.9) and methanol (1.9–3.4). 4. Conclusion The study demonstrated the feasibility of H2 generation from probiotic wastewater as substrate using selectively enriched mixed consortia. The conditions of heat treatment and enrichment pH are the main treatment effects that are significant on the generation. Probiotic wastewater used as the sole carbon source documented its metabolic participation in H2 generation. Fermentation pH 5.5 and substrate concentration 5 g/L are found to be optimum for the overall process efficiency of H2 generation. The described process has a dual benefit of H2 production with simultaneous wastewater treatment in an economical, effective and sustainable way. Acknowledgement The authors wish to thank the Ministry of New and Renewable Energy (MNRE) Government of India (File No. 102/9/2004-NT) for the partial financial support of this study. References [1] Fang HHP, Li C, Zhang T. Acidophilic biohydrogen production from rice slurry. International Journal of Hydrogen Energy 2006;31:683–92. [2] Tao Y, Chen Y, Wu Y, He Y, Zhou Z. High hydrogen yield from a two-step process of dark-and photo-fermentation of sucrose. International Journal of Hydrogen Energy 2007;31:200–6. [3] Ren N, Li J, Li B, Wang Y, Liu S. Biohydrogen production from molasses by anaerobic fermentation with a pilot-scale bioreactor system. International Journal of Hydrogen Energy 2006;31:2147–57. [4] Ghirardi ML, Zhang L, Flynn LJW, Seibert TM, Greenbaum E, et al. Microalgae: a green source of renewable H2. Tibtech 2000;18:506–11.
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