The Effect of Natural Buffer on Biohydrogen Production


  • Miftahul Choiron Jember University Indonesia
  • Seishu Tojo Tokyo University of Agriculture and Technology
  • Tadashi Chosa Tokyo University of Agriculture and Technology


Hydrogen is the promising ideal energy carrier with no emission but water on its combustible in the next generation. Hydrogen production using biological methods is greener than other methods using fossil fuel. One of the major factors affecting the operation of biohydrogen production is pH level in bioreactors. Restrain of declining pH is expected to increase hydrogen production. Pretreatment is one key factor in successful biohydrogen fermentation using mixed microbes. This study aims to investigate the natural buffer effect on biohydrogen using hot compressed water pretreatment. This batch fermentation experiment was operated in a 110 mL glass reactor with 3.75 g/L glucose as substrate. Mixed culture was obtained from cow dung compost treated with hot compressed water pretreatment at 150 ºC, 0.5 MPa for 40 minutes. Fine dried eggshell powder and calcinated eggshell were added with 1 g/L, 3 g/L, and 5 g/L concentrations as buffer agents. The result showed that the addition of 1 g/L eggshell obtained the highest hydrogen production rate of 0.92 mol H2/mol glucose. Butyric acid and acetic acid are recognized as an indicator of hydrogen production and the Butyric/Acetic molar ratio over 2.6 as efficient biohydrogen fermentation. The highest B/A ratio in this experiment was 4.62 on 3g/L addition of eggshell powder.


Author Biographies

Miftahul Choiron, Jember University Indonesia

Faculty of Agricultural Technology, Agroindustrial Department, Jember University. Jl. Kalimantan No.37, Jember, Indonesia

Seishu Tojo, Tokyo University of Agriculture and Technology

Institute of Agriculture, Tokyo University of Agriculture and Technology, Japan 183-8538 Tokyo, Fuchu, Harumicho, 3 Chome−8−1

Tadashi Chosa, Tokyo University of Agriculture and Technology

2Institute of Agriculture, Tokyo University of Agriculture and Technology, Japan 183-8538 Tokyo, Fuchu, Harumicho, 3 Chome−8−1


Oreggioni, G.D, Baboo L.G, Savvas A.T, Giuseppe B, Matthew R, Marie E.K, Trisha A.T and Mike K.T., Potential for Energy Production from Farm Wastes Using Anaerobic Digestion in the UK: An Economic Comparison of Different Size Plants. Energies, 10, 2017, 1396.

Ge Xumeng, Fuqing Xu, Yebo Li., Review: Solid-state Anaerobic Digestion of Lignocellulosic Biomass: Recent Progress and Perspectives. Bioresource Technology, 205, 2016, pp. 239-249.

Wang, A.J., Cao, G.L., and Liu, W.Z., Biohydrogen Production from Anaerobic Fermentation. In: Bai FW., Liu CG., Huang H., Tsao G. (eds) Biotechnology in China III: Biofuels and Bioenergy, Advances in Biochemical Engineering Biotechnology, 128. Springer, Berlin, Heidelberg, 2011.

Argun H. and Dao S., Bio-hydrogen Production from Waste Peach Pulp by Dark Fermentation: Effect of Inoculum Addition. International Jour-nal of Hydrogen Energy, 42, 2017, pp. 2569-2574.

Zeng G.H., Wang L and Kang Z.H., Feasibility of Biohydrogen Production from Tofu Wastewater with Glutamine Auxotrophic Mutant of Rhodobacter Sphaeroides, Renewable Energy, 35, 2010, pp. 2910-2913.

Kim M-S and Lee D-Y, Fermentative Hydrogen Production from Tofu-Processing Waste and Anaerobic Digester Sludge Using Microbial Consortium, Bioresource Technology, 101, 2010, S48–S52.

Kim D-H, Lee D-Y, and Kim M-S, Enhanced Biohydrogen Production from Tofu Residue by Acid/Base Pretreatment and Sewage Sludge Addition, International Journal of Hydrogen Energy, 36, 2011, pp. 13922-13927.

Monlau F., Aemig Q., Trably E., Hamelin J., Steyer J-P., and Carrere H., Specific Inhibition of Biohydrogen-producing Clostridium sp. After Dilute-Acid Pretreatment of Sunflower Stalks, International Journal of Hydrogen Energy, 38 2013, pp. 12273-12282.

Levin D.B., Islam R., Cicek N., and Sparling R., Hydrogen Production by Clostridium Thermo-cellum 27405 from Cellulosic Biomass Substrates, International Journal of Hydrogen Energy, 31, 2006, pp. 1496–1503.

Soltan M., Elsamadony M., and Tawfik A., Biological Hydrogen Promotion via Integrated Fermentation of Complex Agro-Industrial Wastes, Applied Energy, 185, 2017, pp. 929–938.

Torquato L.D.M., Pachiega R., Crespi M.S., Nespeca M.G, Oliveira J.E., and Maintinguer S.I., Potential of Biohydrogen Production from Effluents of Citrus Processing Industry Using Anaerobic Bacteria from Sewage Sludge, Waste Management, 59, 2017, pp. 181–193.

Cui M, Yuan Z, Zhi X, Wei L and Shen, J., Biohydrogen Production from Poplar Leaves Pretreated by Different Methods Using Anaerobic Mixed Bacteria. International Journal of Hydrogen Energy, 35, 2010, pp. 4041–4047.

Hernández M.A., Susa M.R., Andres Y., Use of Coffee Mucilage as a New Substrate for Hydrogen Production in Anaerobic Co-digestion with Swine Manure, Bioresource Technology, 168, 2014,pp. 112-118.

Gómez L.M.R., Guerrero C. E. M., Alfaro J.M, Vázquez S.I.S., Olivo A. R., and López A. C. Effect of Carbon/Nitrogen Ratio, Temperature, and Inoculum Source on Hydrogen Production from Dark Codigestion of Fruit Peels and Sewage Sludge, Sustainability, 11(7), 2019, p. 2139.

Moiceanu G., Paraschiv G., Voicu G., Dinca M., Negoita O., Chitoiu M., and Tudor P, Energy Consumption at Size Reduction of Lignocellulose Biomass for Bioenergy, Sustainability, 11(9), 2019,p. 2477.

Sun Y., He J., Yang G., Sun G., and Sage V. A Review of the Enhancement of Bio-Hydrogen Generation by Chemicals Addition. Catalysts, 9, 2019, p. 353.

Lin H, Wang Y., and Zhu M., Evaluation of Spent Mushroom Compost as a Lignocellulosic Substrate for Hydrogen Production by Clostri-dium Thermocellum, International Journal of Hydrogen Energy, 42, 2017, pp. 26687-26694.

Jiang D., Fang Z., Chin S-X., Tian X. and Su T-C. Biohydrogen Production from Hydrolysates of Selected Tropical Biomass Wastes with Clostri-dium Butyricum, Scientific Reports, 6, 2016, p. 27205.

Park J-H, Do-Hyung K, Sang-Hyoun K, Jeong-Jun Y, and Hee-Deung P., Effect of Substrate Concentration on the Competition between Clostridium and Lactobacillus during Biohy-drogen Production, International Journal of Hydrogen Energy, 43(25), 2018, pp. 11460-11469.

Kumar, N., and Das, D., Enhancement of Hydrogen Production by Enterobacter Cloacae IIT-BT 08, Process Biochem, 35, 2000, pp. 589–593.

Mars A.E., Veuskens T., Budde M.A.W., Doeveren P.F.N.M., Lips S.J., Bakker R.R., Vrije T., and Claassen P.A.M., Biohydrogen Pro¬duc¬tion from Untreated and Hydrolyzed Potato Steam Peels by The Extreme Thermo¬philes Caldi-cellulosiruptor Saccharolyticus and Thermo¬toga Neapolitana, International Journal of Hydrogen Energy, 35(15), 2010, pp. 7730-7737.

Pachapur V.L, Kutty P, Pachapur P, Brar S.K, Bihan Y.L, Galvez-Cloutier R and Buelna G. Seed Pretreatment for Increased Hydrogen Production Using Mixed-Culture Systems with Advantages over Pure-Culture Systems, Energies 12(3), 2019, p. 530.

Kuribayashi M., Tojo S., Chosa T., Murayama T., Sasaki K., and Kotaka H., Developing a New Technology for the Two Phase Methane Fermentation Sludge Recirculation Process, Chemical Engineering Transactions, 58, 2017, pp. 475-480.

Cappai G., Gioannis G. D., Muntoni A., Spiga D., Boni M. R., Polettini A., Pomi R., and Rossi A. Biohydrogen Production from Food Waste: Influence of the Inoculum-To-Substrate Ratio, Sustainability, 10(12), 2018,p. 4506.

Alibardi L. and Cossu R., Effects of Carbo¬hydrate, Protein and Lipid Content of Organic Waste on Hydrogen Production and Fermen¬ta¬tion Products, Waste Management, 47, 2016, pp. 69–77.

Wang B, Wei Wan, and Jianlong Wang, Inhibitory Effect of Ethanol, Acetic Acid, Propionic Acid and Butyric Acid on Fermen¬tative Hydrogen Production. International Jour¬nal of Hydrogen Energy 33(23), 2008,pp. 7013-7019.

Meneses L.R., Otor O.F., Bonturi N., Orupõld K., and Kikas T., Bioenergy Yields from Sequential Bioethanol and Biomethane Production: An Optimized Process Flow. Sustainability, 12(1), 2020, p. 272.

Hosseini S.E, Wahid M.A, Jamil M.M, Azli A.A.M., and Misbah M.F, A Review on Biomass-based Hydrogen Production for Renewable Energy Supply, International Journal Energy Research, 39, 2015, pp. 1597–1615.

Ai B, Li Z, Chi X, Meng J, Jha A.K, Liu C, and Shi E., Effect of pH and Buffer on Butyric Acid Production and Microbial Community Characteristics in Bioconversion of Rice Straw with Undefined Mixed Culture, Biotechnology and Bioprocess Engineering, 19, 2014, pp. 676-686.

Dolińska B., Jelińska M, Musioł B.S., and Ryszka F., Use of Eggshells as a Raw Material for Production of Calcium Preparations. Czech Journal of Food Science, 34(4), 2016, pp. 313–317.

Damayanti A, Sarto, Siti Syamsiah, and W.B. Sediawan , The Influence of Chicken Eggshell Powder as a Buffer on Biohydrogen Production from Rotten Orange (Citrus Nobilis Var. Microcarpa) with Immobilized Mixed Culture, AIP Conference Proceedings 1855, 2017, 070006.

Choi J., and Ahn Y. Biohydrogen Fermentation from Sucrose and Piggery Waste with High Levels of Bicarbonate Alkalinity, Energies, 8, 2015, pp. 1716-1729.

Choiron M, Seishu Tojo, and Tadashi Chosa, Biohydrogen Production Improvement using Hot Compressed Water Pretreatment on Sake Brewery Waste, International Journal of Hydrogen Energy, 45(35), 2020, pp. 17220-17232.

Meneses L.R., Otor O.F., Bonturi N., Orupõld K., and Kikas T., Bioenergy Yields from Sequential Bioethanol and Biomethane Production: An Optimi¬zed Process Flow, Sustainability, 12(1), 2020, p. 272.

Zwietering, M.H., Jongenburger, I., Rombouts, F.M., and Riet, K., Modeling of the Bacterial Growth Curve, Applied Environment Micro-biology, 56, 1990, pp. 1875–1881.

Basak B., Fatima A., Jeon B-H, Ganguly A., Chatterjee P.K., and Dey A., Process Kinetic Studies of Biohydrogen Production by Co-Fermentation of Fruit-Vegetable Wastes and Cottage Cheese Whey, Energy for Sustainable Development, 47, 2018, pp. 39–52.

De Gioannis, G., Friargiu, M., Massi, E., Muntoni, A., Polettini, A., Pomi, R., and Spiga D., Biohydrogen Production from Dark Fermen¬ta¬tion of Cheese Whey: Influence of pH. Inter¬national Journal of Hydrogen Energy, 39(36), 2014, pp. 20930 - 20941.

Ghimire, A., Sposito, Frunzo L., Trably E., Escudié R., Pirozzi F., and Esposito G., Effects of Operational Parameters on Dark Fermentative Hydrogen Production From Biodegradable Complex Waste Biomass, Waste Management, 50, 2016, pp. 55-64.

Pecorini, I., Baldi, F., and Iannelli, R., Bioche¬mical Hydrogen Potential Tests Using Different Inocula, Sustainability, 11, 2019, p. 622.

Gou C., Guo J., Lian J., Guo Y., Jiang Z., Yue L.,and Yang J., Characteristics and Kinetics of Biohydrogen Production with Ni2+ using Hydrogen-producing Bacteria, International Journal of Hydrogen Energy, 40(1), 2015, pp. 161-167.

Wu S-Y., Chu C-Y., and Shen Y-C., Effect of Calcium Ions on Biohydrogen Production Performance in A Fluidized Bed Bioreactor with Activated Carbon-Immobilized Cells, International Journal of Hydrogen Energy, 37, 2012, pp. 15496-15502.

Yuan Z., Yang H., Zhi X., and Shen J., Increased Performance of Continuous Stirred Tank Reactor with Calcium Supplementation, International Journal of Hydrogen Energy, 35 ,2010, pp. 2622–2626.

Messet J, Calusinska M, Hamilton C, Hiligsmann S Joris B, Wilmotte A, and Thonart, Fermentative Hydrogen Production from Glucose And Starch Using Pure Strains And Artificial Co-Cultures of Clostridium spp. Biotechnology for Biofuels, 2012, pp. 5:35. Retrieved from

Bujang K, Sujang S, and Adeni DSA. Effects of Calcium Carbonate in Fermentation of L-Lactic Acid from Hydrolyzed Sago Starch, Annual Report of ICBiotech 2003.