Nguyen Thi Hong Hue, Pham Duc Ngoc, Tran My Hanh, Ngo Anh Tien, Bui Thi Viet Ha

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Abstract: Hydrogen is considered as an ideal substitute to fossil fuels in the energy and non-polluting characteristics. Biological hydrogen production using microorganisms is a promising method to the world's energy industry. The anaerobic, mesophilic, Gram-positive strain Clostridium beijerinckii CB3 (C. beijerinckii CB3) isolated from cattle feces in North of Vietnam has been studied to optimize the biohydrogen production in anaerobic condition. In this study, the effects of culture conditions on hydrogen production by C. beijerinckii CB3 were investigated in batch culture using serum bottles. Various medium components (carbon and nitrogen sources, inorganic salts) and environmental factors (initial pH, temperature of incubation), time and orbital shaker of culture were optimized for hydrogen production by C. beijerinckii CB3. The optimal parameters for the best growth and biohydrogen production in batch tests were incubation time 48 h, 37oC, pH 8.5, and orbital shaker 200 rpm. The maximum cell growth of 1.6 in OD600 and biohydrogen production of 881.25 mL/L were obtained, respectively, in the medium containing 10 g/L of glucose, 10 g/L of yeast extract or 10 g/L of peptone, 480 mL/L of NaHCO3, and 32 mL/L of K2HPO4. These results indicated that C. beijerinckii CB3 is a potential candidate for fermentative biohydrogen production.

Keywords: Biohydrogen production, C. beijerinckii, culture condition, growth, anaerobic condition.


[1] Das D, Veziroglu NT, Hydrogen production by biological processes: a survey of literature, Int J Hydrogen Energy 26 (2001) 13.
[2] Levin DB, Pitt L, Love M., Biohydrogen production: prospects and limitations to practical application, Int J Hydrogen Energy 29 (2004) 173-85.
[3] Kotay SM, Das D., Microbial hydrogen production with Bacillus coagulans IIT-BT S1 isolated from anaerobic sewage sludge, Bioresour Technol 98 (2007) 1183.
[4] O-Thong Sompong, Prasertsan Poonsuk, Karakashev Dimitar, Angelidaki Irini, Thermophilic fermentative hydrogen production by the newly isolated Thermoanaerobacterium thermosaccharolyticum PSU-2, Int J Hydrogen Energy 33 (2008), 124.
[5] Kamalaskar, Leena B, et al., High biohydrogen yielding Clostridium sp. DMHC-10 isolated from sludge of distillery waste treatment plant, International journal of hydrogen energy 35(19) (2010) 10639.
[6] Wang, CC, et al., Producing hydrogen from wastewater sludge by Clostridium bifermentans, Journal of Biotechnology 102(1) (2003) 83.
[7] An, D., Li, Q., Wang, X., Yang, H., & Guo, L, Characterization on hydrogen production performance of a newly isolated Clostridium beijerinckii YA001 using xylose”, International Journal of Hydrogen Energy 39(35) (2014) 19928.
[8] Pan CM, Fan YT, Zhao P, Hou HW, Fermentative hydrogen production by the newly isolated Clostridium beijerinckii Fanp3, Int J Hydrogen Energy 33(20) (2008) 5383.
[9] S. Van Ginkel and S. Sung, Biohydrogen production as a function of pH and substrate concentration, Environ Sci Technol 35 (2011) 4726.
[10] H. Argun, F. Kargi, I. K. Kapdan and O. Oztekin, Biohydrogen production by dark fermentation of wheat powder solution: Effects of C/N and C/P ratio on hydrogen yield and formation rate, Int. J. Hydrogen Energy 33 (2008) 1913.
[11] M. L. Chong, N. A. A. Rahman, P. L. Yee, S. A. Aziz, R. A. Rahim, Y. Shirai and M. A. Hassan, Effects of pH, glucose and iron sulfate concentration on the yield of biohydrogen by Clostridium butyricum EB6, Int J Hydrogen Energy 34 (2009) 8859.
[12] H. Yokoi, A. Saitsu, H. Uchida, J. Hirose, S. Hayashi and Y. Takasaki, Microbial hydrogen production from sweet potato starch residue, J Biosci Bioeng 91 (2011) 58.
[13] H. H. P. Fang, C. Li and T. Zhang, Acidophilic bio-hydrogen production from rice slurry, Int J Hydrogen Energy 31(2005) 683.
[14] Kapdan, Ilgi Karapinar and Kargi, Fikret, Bio-hydrogen production from waste materials, Enzyme and microbial technology 38(5) (2005) 569.
[15] Gray, Clarke T and Gest, Howard, Biological Formation of Molecular Hydrogen: A “hydrogen valve” facilitates regulation of anaerobic energy metabolism in many microorganisms, Science 148(3667) (1965) 186.
[16] Mangayil R et al, Fermentative Hydrogen production from different sugars by Citrobacter sp. CMC-1 in batch culture, International Journal of Hydrogen Energy,Volume 36, Issue 23 (2011) 15187.
[17] Zhao, Xin, et al., Hydrogen production by the newly isolated Clostridium beijerinckii RZF-1108, Bioresource technology 102(18) (2011) 8432.
[18] Dang Thi Yen, Vuong Thi Nga, Lai Thuy Hien, Nguyen Thi Thu Huyen, "Effect of environmental parameters on hydrogen production of Strain Tr2 isolated in Vietnam under microaerobic condition, Journal of science and technology 51c(5) (2016) 587. (Vietnamese).
[19] Alalayah, Walid M, et al., Effect of environmental parameters on hydrogen production using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564), American Journal of Environmental Sciences 5(1) (2009) 80.
[20] Cai, Jinling, et al., Fermentative hydrogen production by a new mesophilic bacterium Clostridium sp. 6A-5 isolated from the sludge of a sugar mill, Renewable energy 59 (2013) 202.
[21] Chong, Mei-Ling, et al., Effects of pH, glucose and iron sulfate concentration on the yield of biohydrogen by Clostridium butyricum EB6, International journal of hydrogen energy 34(21) (2009) 8859.
[22] Ferchichi et al., Influence of culture parameters on biological hydrogen production by Clostridium saccharoperbutylacetonicum ATCC 27021, World Journal of Microbiology and Biotechnology 21 (2005) 855.
[23] Taguchi et al., Continuous hydrogen production by Clostridium sp. strain no. 2 from cellulose hydrolysate in an aqueous two-phase system, Journal of Fermentation and Bioengineering 82(1) (1996) 80.
[24] Lee DJ, Show KY, Su A, Dark fermentation on biohydrogen production: pure culture, Bioresous technol 102 (18) (2011) 8393.
[25] Eriksen et al, Hydrogen production in anaerobic and microaerobic Thermotoga neapolitana, Biotechnol Lett 30(1) (2008) 103.
[26] Narayanaswamy S., Plant cell and tissue culture, Tata McGraw-Hill Education, New Delhi, 1994.
Evvyernie, Dwierra, et al, Identification and characterization of Clostridium paraputrificum M-21, a chitinolytic, mesophilic and hydrogen-producing bacterium, Journal of bioscience and bioengineering 89(6) (2000) 596.