Hydrogen, Fuel Cell & Energy Storage

Hydrogen, Fuel Cell & Energy Storage

A Critical Review of Continuous Biohydrogen Production

Document Type : Research Paper

Authors
Atefeh Farjadmanesh 1
Khosrow Rostami 1
Fatemeh Boshagh 2
1 Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran
2 School of Chemical Engineering and Materials Science, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea
10.22104/hfe.2025.7307.1336
Abstract
Biohydrogen is a feasible and environmentally sustainable fuel for the world's growing energy demand. As an alternative renewable energy source, hydrogen contains 2.75 times more energy per gram than any other known source. Biohydrogen is produced from various microorganisms and renewable materials through anaerobic dark fermentation and phototrophic bacteria, operating in batch, semi-continuous, and continuous modes. Anaerobic dark fermentation has a higher production rate and yield compared to photo-fermentation. In this review, the dark fermentation of hydrogen is discussed concerning key influential parameters and environmental factors such as temperature, pH, hydraulic retention time, mass transfer coefficient, and recycle ratio. A low initial pH affects metabolic pathways, prolongs the lag phase, and enhances Fe-hydrogenase activity. Mesophilic, thermophilic and ultrathermophilic strains could tolerate maximum operating temperature of 40, 65 or 108°C, respectively. A varity of fermenters are continuously used for biohydrogen production, with a few discussed here. Tower-type fermenters are more feasible than CSTRs as they provide a higher hydraulic retention time for continuous biohydrogen production. Fluidized beds are used for both short- and long-term hydraulic retention time operations. Hydraulic retention time (HRT) typically depends on the bioreactor type, geometry, and feed composition, particularly the carbon source. When wastewater is used, HRT generally ranges from 8 to 14 hours. However, the process requires more detailed data to fully understand and overcome thermodynamic limitations. These limitations could be addressed through genetic modification or metabolic pathway alterations to enhance biohydrogen yield, making large-scale and commercial production more feasible.
Keywords
Biohydrogen
Fermenters
Continuous operation
Dark fermentation
Subjects
[1] Ni M, Leung DYC, Leung MKH, Sumathy K. An overview of hydrogen production from biomass. Fuel Processing Technology. 2006;87:461-72.
[2] Cui M, Yuan Z, Zhi X, Wei L, Shen J. Biohydrogen production from poplar leaves pretreated by different methods using anaerobic mixed bacteria. International Journal of Hydrogen Energy. 2010;35:4041-7.
[3] Boshagh F, Rostami K. A review of application of experimental design techniques related to dark fermentative hydrogen production. Journal of Renewable Energy and Environment. 2020;7(2):27-42.
[4] Ghimire A, Frunzo L, Pirozzi F, Trably E, Escudie R, Lens PNL, et al. A review on dark fermentative biohydrogen production from organic biomass: process parameters and use of byproducts. Applied Energy. 2015;144:73-95.
[5] Das D, Veziroˇglu T. Hydrogen production by biological processes: a survey of literature. International Journal of Hydrogen Energy. 2001;26:13-28.
[6] Tamburic B, Zemichael FW, Maitland GC, Hellgardt K. Parameters affecting the growth and hydrogen production of the green alga Chlamydomonas reinhardtii. International Journal of Hydrogen Energy. 2011;36:7872-6.
[7] Chong M, Sabaratnam V, Shirai Y, Hassan MA. Biohydrogen production from biomass and industrial wastes by dark fermentation. International Journal of Hydrogen Energy. 2009;34:3277-87.
[8] F¨orberg C, H¨aggstr¨om L. Control of cell adhesion and activity during continuous production of acetone and butanol with adsorbed cells. Enzyme and microbial technology. 1985;7(5):230-4.
[9] Budiman P, Wu T. Ultrasonication pretreatment of combined effluents from palm oil, pulp and paper mills for improving photofermentative biohydrogen production. Energy Convers Manag. 2016;119:142-50.
[10] Zhang Y, Liu G, Shen J. Hydrogen Production in batch culture of mixed bacteria with sucrose under different iron concentrations. International Journal of Hydrogen Energy. 2005;30:855-60.
[11] Muloiwa M, Nyende-Byakika S, Dinka M. Comparison of unstructured kinetic bacterial growth models. South African Journal of Chemical Engineering. 2020;33:141-50.
[12] Boshagh F, Rostami K, Moazami N. Immobilization of Enterobacter aerogenes on carbon fiber and activated carbon to study hydrogen production enhancement. Biochemical Engineering Journal. 2019;144:64-72.
[13] Boshagh F, Rostami K, Moazami N. Biohydrogen production by immobilized Enterobacter aerogenes on functionalized multi-walled carbon nanotube. International Journal of Hydrogen Energy. 2019;44:14395-405.
[14] Chen C, Lin C, Lin M. Acid-base enrichment enhances anaerobic hydrogen production process. Applied Microbiology and Biotechnology. 2002;58:224-8.
[15] Kumar N, Das D. Continuous hydrogen production by immobilized Enterobacter cloacae IITBT 08 using lignocellulosic materials as solid matrices. Enzyme and Microbial Technology. 2001;29:280-7.
[16] Stoodley P, Sauer K, Davies D, Costerton J. Biofilms as complex differentiated communities. Annual Review of Microbiology. 2002;56:187-209.
[17] Fang H, Liu H, Zhang T. Characterization of a hydrogen production granular sludge. Biotechnology and Bioengineering. 2002;78:44-52.
[18] Show K, Zhang Z, Tay J, Liang D, Lee D, Ren N, et al. Critical assessment of anaerobic processes for continuous biohydrogen production from organic wastewater. International Journal of Hydrogen Energy. 2010;35:13350-5.
[19] Show K, Zhang Z, Tay J, Liang D, Lee D, Ji W. Production of hydrogen in a granular sludgebased anaerobic continuous stirred tank reactor. International Journal of Hydrogen Energy. 2007;32:4744-53.
[20] Zhang M, Fan Y, Xing Y, Pan C, Zhang G, Lay J. Enhanced biohydrogen production from cornstalk wastes with acidification pretreatment by mixed anaerobic cultures. Biomass and Bioenergy. 2007;31:250-4.
[21] Paillet F, Barrau C, Escudi´e R, Trably E. Inhibition by the ionic strength of hydrogen production from the organic fraction of municipal solid waste. International Journal of Hydrogen Energy. 2020;45:5854-63.
[22] Lay C, Wu J, Hsiao C, Chang J, Chen C, Lin C. Biohydrogen production from soluble condensed molasses fermentation using anaerobic fermentation. International Journal of Hydrogen Energy. 2010;35:13445-51.
[23] Chen C, Lin C. Using sucrose as a substrate in an anaerobic hydrogen-producing reactor. Advances in Environmental Research. 2003;7:695-9.
[24] Davila-Vazquez G, Cota-Navarro C, RosalesColu L, Le´on-Rodr´ıguez A, Razo-Flores E. Continuous biohydrogen production using cheese whey: Improving the hydrogen production rate. International Journal of Hydrogen Energy. 2009;34:4296-304.
[25] Antonopoulou G, Gavala H, Skiadas I, Lyberatos G. Effect of substrate concentration on fermentative hydrogen production from sweet sorghum extract. International Journal of Hydrogen Energy. 2011;36:4843-51.
[26] Antonopoulou G, Gavala H, Skiadas I, Lyberatos G. Influence of pH on fermentative hydrogen production from sweet sorghum extract. International Journal of Hydrogen Energy. 2010;35:1921-8.
[27] Arooj M, Han S, Kim S, Kim D, Shin H. Continuous biohydrogen production in a CSTR using starch as a substrate. International journal of hydrogen energy. 2008;33:3289-94.
[28] Kotsopoulos T, Fotidis I, Tsolakis N, Martzop G. Biohydrogen production from pig slurry in a CSTR reactor system with mixed cultures under hyper-thermophilic temperature (70 C). Biomass Bioenergy. 2009;33:1168-74.
[29] Reungsang A, Sreela-or C, Plangklang P. Nonsterile bio-hydrogen fermentation from food waste in a continuous stirred tank reactor (CSTR): Performance and population analysis. International Journal of Hydrogen Energy. 2013;38:15630-7.
[30] Hastuti Z, Chu C, Rachman M, Purwanto W, Dewi E, Lin C. Effect of concentration on biohydrogen production in a continuous stirred bioreactor using biofilm induced packedcarrier. International Journal of Hydrogen Energy. 2016;41:21649-56.
[31] P´’ortner R, Faschian R. Design and Operation of Fixed-Bed Bioreactors for Immobilized Bacterial Culture. IntechOpen; 2019.
[32] Zhang Z, Show K, Tay J, Liang D, Lee D. Biohydrogen production with anaerobic fluidized bed reactors-A comparison of biofilm-based and granule-based systems. International Journal of Hydrogen Energy. 2008;33:1559-64.
[33] Wu S, Lin C, Chang J. Hydrogen Production with Immobilized Sewage Sludge in ThreePhase FluidizedBed Bioreactors. Biotechnology Progress. 2003;19:828-32.
[34] Wu K, Chang C, Chang J. Simultaneous production of biohydrogen and bioethanol with fluidized-bed and packed-bed bioreactors containing immobilized anaerobic sludge. Process Biochemistry. 2007;42:1165-71.
[35] Lin C, Wu S, Chang J. Fermentative hydrogen production with a draft tube fluidized bed reactor containing silicone-gel-immobilized anaerobic sludge. International Journal of Hydrogen Energy. 2006;31:2200-10.
[36] Lee K, Wu J, Lo Y, Lo Y, Lin P, Chang J. Anaerobic hydrogen production with an efficient carrier-induced granular sludge bed bioreactor. Biotechnology Bioengineering. 2004;87:648-57.
[37] Leite J, Fernandes B, Pozzi E, Barboza M, Zaiat M. Application of an anaerobic packed-bed bioreactor for the production of hydrogen and organic acids. International Journal of Hydrogen Energy. 2008;33:579-86.
[38] Fritsch M, Hartmeier W, Chang J. Enhancing hydrogen production of Clostridium butyricum using a column reactor with square-structured ceramic fittings. International Journal of Hydrogen Energy. 2008;33:6549-57.
[39] Elreedy A, Tawfik A, Enitan A, Kumari S, Bux F. Pathways of 3-biofules (hydrogen, ethanol and methane) production from petrochemical industry wastewater via anaerobic packed bed baffled reactor inoculated with mixed culture bacteria. Energy Conversion Management. 2016;12:119-30.
[40] Si B, Liu Z, Zhang Y, Li J, Xing X, Li B, et al. Effect of reaction mode on biohydrogen production and its microbial diversity. International Journal of Hydrogen Energy. 2015;40:3191-200.
[41] Dounavis A, Ntaikou I, Lyberatos G. Production of biohydrogen from crude glycerol in an upflow column bioreactor. Bioresource Technology. 2015;198:701-8.
[42] Carrillo-Reyes J, Cortes-Carmona M, BarcenasR C. Cell wash-out enrichment increases the stability and performance of biohydrogen producing packed-bed reactors and the community transition along the operation time. Renewable Energy. 2016;97:266-73.
[43] Junior A, Zaiat M, Gupta M, Elbeshbishy E, Hafez H, Nakhla G. Impact of organic loading rate on biohydrogen production in an up-flow anaerobic packed bed reactor (UAnPBR). Bioresource Technology. 2014;164:371-9.
[44] Fuess L, Kiyuna L, Garcia M, Zaiat M. Operational strategies for long-term biohydrogen production from sugarcane stillage in a continuous acidogenic packed-bed reactor. International Journal of Hydrogen Energy. 2016;41:8132-45.
[45] Azbar N, Kapdan IK. State of the Art and Progress in Production of Biohydrogen, first ed., canada: Bentham Science; 2012.
[46] B K, I K. The effect of HRT on biohydrogen production from acid hydrolyzed waste wheat in a continuously operated packed bed reactor. International Journal of Hydrogen Energy. 2018;43(23):10678-85.
[47] Si B, Li J, Li B, Zhu Z, Shen R, Zhang Y, et al. The role of hydraulic retention time on controlling methanogenesis and homoacetogenesis in biohydrogen production using upflow anaerobic sludge blanket (UASB) reactor and packed bed reactor (PBR). International Journal of Hydrogen Energy. 2015;40:11414-21.
[48] Roy S, Vishnuvardhan M, Das D. Continuous thermophilic biohydrogen production in packed bed reactor. Applied Energy. 2014;136:51-8.
[49] Peixoto G, Saavedra N, Varesche M, Zaiat M. Hydrogen production from soft-drink wastewater in an upflow anaerobic packed-bed reactor. International Journal of Hydrogen Energy. 2011;36:8953-66.
[50] Junior A, Wenzel J, Etchebehere C, Zaiat M. Effect of organic loading rate on hydrogen production from sugarcane vinasse in thermophilic acidogenic packed bed reactors. International Journal of Hydrogen Energy. 2014;39:16852-62.
[51] noz P´aez KM, Ruiz-Ordaz N, Garcia-Mena J, Ponce-Noyola M, Ramos-Valdivia A, RoblesGonz´alez I, et al. Comparison of biohydrogen production in fluidized bed bioreactors at room temperature and 35C. International Journal of Hydrogen Energy. 2013;38:12570-9.
[52] Zhang Z, Tay J, Show K, Yan R, Liang D, Lee D, et al. Biohydrogen production in a granular activated carbon anaerobic fluidized bed reactor. International Journal of Hydrogen Energy. 2007;32:185-91.
[53] Mu Y, Yu H. Biological hydrogen in a UASB reactor with granules: Physicochemical characteristics of hydrogen producing granules. Biotechnology. 2006;94:980-7.
[54] Castell´o E, C Y Santos TI, Paolino G, Wenzel J, Borzacconi L, Etchebehere C. Feasibility of biohydrogen production from cheese whey using a UASB reactor: links between microbial community and reactor performance. International Journal of Hydrogen Energy. 2009;34:5674-82.
[55] Liu D, Liu D, Zeng R, Angelidaki I. Hydrogen and methane production from household solid waste in the two-stage fermentation process. Water Research. 2006;40:2230-6.
[56] Chang F, Lin C. Biohydrogen production using an up-flow anaerobic sludge blanket reactor. International Journal of Hydrogen Energy. 2004;29:33-9.
[57] Wang Y, Mu Y, Yu H. Comparative performance of two upflow anaerobic biohydrogen-producing reactors seeded with different sludges. International Journal of Hydrogen Energy. 2007;32:1086-94.
[58] Yang H, Shao P, Lu T, Shen J, Wang D, Xu Z, et al. Continuous biohydrogen production from citric acid wastewater via facultative anaerobic bacteria. International Journal of Hydrogen Energy. 2006;31:1306-13.
[59] Jung K, Kim D, Shin H. Continuous fermentative hydrogen production from coffee drink manufacturing wastewater by applying UASB reactor. International Journal of Hydrogen Energy. 2010;35:13370-8.
[60] Kotsopoulos T, Zeng R, Angelidaki I. Biohydrogen production in granular up-flow anaerobic sludge blanket (UASB) reactors with mixed cultures under hyper-thermophilic temperature (70 ◦C). Biotechnology Bioengineering. 2006;94:296-302.
[61] Carrillo-Reyes J, Celis L, Alatriste-Mondrag´on F, Razo-Flores E. Different start-up strategies to enhance biohydrogen production from cheese whey
in UASB reactors. International Journal of Hydrogen Energy. 2012;37:5591-601.
[62] Chookaew T, Sompong O, Prasertsan P. Biohydrogen production from crude glycerol by immobilized Klebsiella sp. TR17 in a UASB reactor and bacterial quantification under non-sterile conditions. International Journal of Hydrogen Energy. 2014;39:9580-7.
[63] Radjaram B, Saravanane R. Start up study of UASB reactor treating press mud for biohydrogen production. Biomass Bioenergy. 2011;35:2721-8.
[64] Puyol D, Mohedano A, Sanz J, Rodriguez J. Comparison of UASB and EGSB performance on the anaerobic biodegradation of 2, 4-dichlorophenol. Chemosphere. 2009;76:1192-8.
[65] Guo W, Ren N, Wang X, Xiang W, Meng Z, Ding J, et al. Biohydrogen production from ethanoltype fermentation of molasses in an expanded granular sludge bed (EGSB) reactor. International Journal of Hydrogen Energy. 2008;33:4981-8.
[66] Cisneros-P´erez C, Carrillo-Reyes J, Celis L, Alatriste-Mondrag´on F, Etchebehere C, RazoFlores E. Inoculum pretreatment promotes differences in hydrogen production performance in EGSB reactors. International Journal of Hydrogen Energy. 2015;40:6329-39.
[67] Li C, Fang HH. Fermentative hydrogen production from wastewater and solid wastes by mixed cultures. Critical reviews in environmental science and technology. 2007;37(1):1-39.
[68] Guo X, Trably E, Latrille E, Carr`are H, Steyer J. Hydrogen production from agricultural waste by dark fermentation: a review. International Journal of Hydrogen Energy. 2010;35:10660-73.
[69] Balachandar G, Khanna N, Das D. Biohydrogen, first ed., India: Elsevier; 2013.
[70] Wang J, Wan W. Factors influencing fermentative hydrogen production: A review. International Journal of Hydrogen Energy. 2009;34:799-811.
[71] Levin D, Chahine R. Challenges for renewable hydrogen production from biomass. International Journal of Hydrogen Energy. 2010;35:4962-9.
[72] Lin C, Lay C. Carbon/nitrogen-ratio effect on fermentative hydrogen production by mixed microflora. International Journal of Hydrogen Energy. 2004;29:41-5.
[73] Ren N, Gong M. Acclimation strategy of a biohydrogen producing population in acontinuousflow reactor with carbohydrate fermentation. Engineering Life Science. 2006;4:403-9.
[74] Mu Y, Yu H, Wang Y. The role of pH in the fermentative H2 production from an acidogenic granule-based reactor. Chemosphere. 2006;64:350-8.
[75] Kim M, Cha J, Kim D. Biohydrogen, first ed., Republic of Korea: Elsevier; 2013.
[76] Zagrodnik R, Laniecki M. The role of pH control on biohydrogen production by single stage hybrid dark- and photo-fermentation. Bioresource Technology. 2015;194:187-95.
[77] Stavropoulos K, Kopsahelis A, Zafiri C, Kornaro M. Effect of pH on Continuous Biohydrogen Production from End-of-Life Dairy Products (EoLDPs) via Dark Fermentation. Waste and Biomass Valorization volume. 2016;7.
[78] Alexandropoulou M, Antonopoulou G, Trably E, Carrere H, Lyberatos G. Continuous biohydrogen production from a food industry waste: Influence of operational parameters and microbial community analysis. Cleaner Production. 2018;174:1054-63.
[79] Zhao Q, Yu H. Fermentative H2 production in an upflow anaerobic sludge blanket reactor at various pH values. Bioresource Technology. 2008;99:1353-8.
[80] Yu H, Zhu Z, Hu W, Zhang H. Hydrogen production from rice winery wastewater in an upfow anaerobic reactor by using mixed anaerobic cultures. International Journal of Hydrogen Energy. 2002;27:1359-65.
[81] Fang H, Liu H. Effect of pH on hydrogen production from glucose by a mixed culture. Bioresource Technology. 2002;82:87-93.
[82] Li Y, Zhu J, Wu X, Miller C, Wang L. The Effect of pH on Continuous Biohydrogen Production from Swine Wastewater Supplemented with Glucose. Applied Biochemistry and Biotechnology. 2010;162:1286-96.
[83] Sepehri S, Rostami K, Azin M. A Study on the Role of Clostridium Saccharoperbutylacetonicum N1-4 (ATCC 13564) in Producing Fermentative Hydrogen. International Journal of Chemical Reactor Engineering. 2018;17:1-12.
[84] Boshagh F, Rostami K, Niel EV. Application of kinetic models in dark fermentative hydrogen productioneA critical review. International Journal of Hydrogen Energy. 2022;47:21952-68.
[85] Bundhoo M, Mohee R. Inhibition of dark fermentative bio-hydrogen. International Journal of Hydrogen Energy. 2016;41:6713-33.
[86] Liu Y, Liu J, He H, Yang S, Wang Y, Hu J, et al. A Review of Enhancement of Biohydrogen Productions by Chemical Addition Using a Supervised Machine Learning Method. Energies. 2021;41:1-16.
[87] Li Y, Chen H, Zhao T, Xiong X, Yao X, Han W, et al. Effects of different metal ions on the hydrogen production capacity of Biohydrogenbac terium R3. Solar Energy Journal. 2013;34:1280-7.
[88] Zhang J, Zhang Y, Quan X, Chen S. Effects of ferric ions on hydrogen production efficiency of anaerobic fermentation in UASB reactor. Environmental Science & Policy. 2013;34:2290-4.
[89] Lee D, Li Y, Oh Y, Kim M, Noike T. Effect of iron concentration on continuous H2 production using membrane bioreactor. International Journal of Hydrogen Energy. 2009;34:1244-52.
[90] Lee Y, Miyahara T, Noike T. Effect of iron concentration on hydrogen fermentation. Bioresource Technology. 2001;80:227-31.
[91] Dabrock B, Bahl H, Gottschalk G. Parameters affecting solvent production by Clostridium pasteurianum. Applied and Environmental Microbiology. 1992;58:1233-9.
[92] Peguin S, Soucaille P. Modulation of carbon and electron flow in Clostridium acetobutylicum by iron limitation and methyl viologen addition. Applied and Environmental Microbiology. 1995;61:403-5.
[93] Zhang Y, Shen J. Effect of temperature and iron concentration on the growth and hydrogen production of mixed bacteria. International Journal of Hydrogen Energy. 2006;31:441-6.
[94] Hussy I, Hawkes F, Dinsdale R, Hawkes D. Continuous fermentative hydrogen production from a wheat starch co-product by mixed microflora. Biotechnology and Bioengineering. 2003;84:619-26.
[95] Chen C, Lin C, Chang J. Kinetics of hydrogen production with continuous anaerobic cultures utilizing sucrose as the limiting substrate. Applied Microbiology and Biotechnology. 2001;57:56-64.
[96] Mizuno O, Dinsdale R, Hawkes F, Hawkes D, Noik T. Enhancement of hydrogen production from glucose by nitrogen gas sparging. Bioresource Technology. 2000;73:59-65.
[97] Ueno Y, Otsuka S, Morimoto M. Hydrogen production from ndustrial wastewater by anaerobic microflora in chemostat culture. Journal of Fermentation and Bioengineering. 1996;82:194-7.
[98] Yokoi H, Maki R, Hirose J, Hayashi S. Microbial production of hydrogen from starchmanufacturing wastes. Biomass Bioenergy. 2002;22:389-95.
[99] Yokoi H, Saitsu A, Uchida H, Hirose J, Hayashi S, Takasaki Y. Microbial hydrogen production from sweet potato starch residue. Journal of Bioscience and Bioengineering. 2001;91:58-63.
[100] Lay J. Modelling and optimization of anaerobic digested sludge converting starch to hydrogen. Biotechnology and Bioengineering. 2000;68:269-78.
[101] Beckers L, Masset J, Hamilton C, Delvigne F, Toye D, Crine M, et al. Investigation of the links between mass transfer conditions, dissolved hydrogen concentration and biohydrogen production by the pure strain Clostridium butyricum. Biochemical Engineering Journal. 2015;98:18-28.
[102] Jung K, Kim D, Kim S, Shin H. Bioreactor design for continuous dark fermentative hydrogen production. Bioresource Technology. 2011;102:8612-20.
[103] Chisti M, Moo-Young M. Airlift reactors: characteristics, applications and design considerations. Chemical Engineering Communications. 1987;60:195-242.
[104] Najafpour G, KU I, Younesi H, Mohamed A, Kamaruddin A. Performance of biological hydrogen production process from synthesis gas, mass transfer in batch and continuous bioreactors. International Journal of Engineering. 2004;17:105-20.
[105] Lima D, Zaiat M. The influence of the degree of back-mixing on hydrogen production in an anaerobic fixed-bed reactor. International Journal of Hydrogen Energy. 2012;37:9630-5.
[106] Levenspiel O. Chemical reaction engineering. 3rd ed. Wiley; 1999.
[107] Zhang Z, Show K, Tay J, Liang D, Lee D. Enhanced continuous biohydrogen production by immobilized anaerobic microflora. Energy & Fuels. 2008;22:87-92.
Hydrogen, Fuel Cell & Energy Storage
Volume 13, Issue 2
Spring 2026
Pages 83-104