Hydrogen Production by Immobilized Cells of Clostridium intestinale Strain URNW Using Alginate Beads


Biological hydrogen (H2) is a promising candidate for production of renewable hydrogen. Using entrapped cells rather than conventional suspended cell cultures for the production of H2 offers several advantages, such as improved production yields related to higher cell density, and enhanced resistance to substrate and end-product inhibition. In this study, H2 production by a novel isolate of Clostridium intestinale (strain URNW) was evaluated using cells entrapped within 2% calcium-alginate beads under strictly anaerobic conditions. Both immobilized cells and suspended cultures were studied in sequential batch-mode anaerobic fermentation over 192 h. The production of H2 in the headspace was examined for four different initial cellobiose concentrations (5, 10, 20, and 40 mM). Although a lag period for initiation of the fermentation process was observed for bacteria entrapped within hydrogel beads, the immobilized cells achieved both higher volumetric production rates (mmol H2/(L culture h)) and molar yields (mol H2/mol glucose equivalent) of H2 compared with suspended cultures. In the current study, the maximum cellobiose consumption rate of 0.40 mM/h, corresponding to 133.3 mg/(L h), was achieved after 72 h of fermentation by immobilized cells, generating a high hydrogen yield of 3.57 mol H2/mol cellobiose, whereas suspended cultures only yielded 1.77 mol H2/mol cellobiose. The results suggest that cells remain viable within the hydrogels and proliferated with a slow rate over the course of fermentation. The stable productivity of immobilized cells over 8 days with four changes of medium depicted that the immobilized cells of the isolated strain can successfully yield higher hydrogen and lower soluble metabolites than suspended cells suggesting a feasible process for future applications for bioH2 production.

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  1. 1.

    Jo, J. H., Lee, D. S., Park, D., & Park, J. M. (2008). Biological hydrogen production by immobilized cells of Clostridium tyrobutyricum JM1 isolated from a food waste treatment process. Bioresource Technology, 99(14), 6666–6672. https://doi.org/10.1016/j.biortech.2007.11.067.

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Gopalakrishnan, B., Khanna, N., & Das, D. (2019). Dark-fermentative biohydrogen production. In Biohydrogen (pp. 79–122). Elsevier. https://doi.org/10.1016/b978-0-444-64203-5.00004-6.

  3. 3.

    Kapdan, I. K., & Kargi, F. (2006). Bio-hydrogen production from waste materials. Enzyme and Microbial Technology, 38(5), 569–582. https://doi.org/10.1016/j.enzmictec.2005.09.015.

    CAS  Article  Google Scholar 

  4. 4.

    Touloupakis, E., Poloniataki, E. G., Ghanotakis, D. F., & Carlozzi, P. (2020). Production of biohydrogen and/or poly-β-hydroxybutyrate by Rhodopseudomonas sp. using various carbon sources as substrate. Applied Biochemistry and Biotechnology. https://doi.org/10.1007/s12010-020-03428-1.

  5. 5.

    Kim, M. S., Kim, D. H., & Lee, J. K. (2011). Biohydrogen. In Comprehensive Biotechnology (Vol. 3, 2nd ed., pp. 115–125). https://doi.org/10.1016/B978-0-08-088504-9.00166-5

  6. 6.

    Zhang, C., Kang, X., Liang, N., & Abdullah, A. (2017). Improvement of biohydrogen production from dark fermentation by cocultures and activated carbon immobilization. Energy and Fuels, 31(11), 12217–12222. https://doi.org/10.1021/acs.energyfuels.7b02035.

    CAS  Article  Google Scholar 

  7. 7.

    Sekoai, P. T., Awosusi, A. A., Yoro, K. O., Singo, M., Oloye, O., Ayeni, A. O., et al. (2018). Microbial cell immobilization in biohydrogen production: a short overview. Critical Reviews in Biotechnology, 38(2), 157–171. https://doi.org/10.1080/07388551.2017.1312274.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Kumar, G., Mudhoo, A., Sivagurunathan, P., Nagarajan, D., Ghimire, A., Lay, C. H., & Chang, J. S. (2016). Recent insights into the cell immobilization technology applied for dark fermentative hydrogen production. Bioresource Technology, 219, 725–737. https://doi.org/10.1016/j.biortech.2016.08.065.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Nemati, M., & Webb, C. (2011). Immobilized cell bioreactors. In Comprehensive Biotechnology (Vol. 2, 2nd ed.). Elsevier B.V. https://doi.org/10.1016/B978-0-08-088504-9.00100-8.

  10. 10.

    Schlieker, M., & Vorlop, K. (2006). A novel immobilization method for entrapment LentiKats®. In Immobilization of enzymes and cells (pp. 333–343). New Jersey: Humana Press.

    Chapter  Google Scholar 

  11. 11.

    Gungormusler-Yilmaz, M., Cicek, N., Levin, D. B., & Azbar, N. (2016). Cell immobilization for microbial production of 1,3-propanediol. Critical Reviews in Biotechnology, 36(3), 482–494. https://doi.org/10.3109/07388551.2014.992386.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Prakash, J., Gupta, R. K., Xx, P., & Kalia, V. C. (2018). Bioprocessing of biodiesel industry effluent by immobilized bacteria to produce value-added products. Applied Biochemistry and Biotechnology, 185(1), 179–190. https://doi.org/10.1007/s12010-017-2637-7.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Kosseva, M. R. (2011). Immobilization of microbial cells in food fermentation processes. Food and Bioprocess Technology, 4(6), 1089–1118. https://doi.org/10.1007/s11947-010-0435-0.

    Article  Google Scholar 

  14. 14.

    Szczsna-Antczak, M., & Galas, E. (2001). Bacillus subtilis cells immobilised in PVA-cryogels. Biomolecular Engineering, 17(2), 55–63. https://doi.org/10.1016/S1389-0344(00)00065-4.

    Article  Google Scholar 

  15. 15.

    Ghorbani, F., Younesi, H., Esmaeili Sari, A., & Najafpour, G. (2011). Cane molasses fermentation for continuous ethanol production in an immobilized cells reactor by Saccharomyces cerevisiae. Renewable Energy, 36(2), 503–509. https://doi.org/10.1016/j.renene.2010.07.016.

    CAS  Article  Google Scholar 

  16. 16.

    Tamayol, A., Akbari, M., Annabi, N., Paul, A., Khademhosseini, A., & Juncker, D. (2013). Fiber-based tissue engineering: Progress, challenges, and opportunities. Biotechnology Advances, 31(5), 669–687. https://doi.org/10.1016/j.biotechadv.2012.11.007.

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Tamayol, A., Akbari, M., Laforte, V., Annabi, N., Khademohosseini, A., & Juncker, D. (2013). Continuous manufacturing of robust living fibers that withstand common textile processing for tissue engineering applications. Freiburg: MicroTAS.

    Google Scholar 

  18. 18.

    Guisan, J. (Ed.). (2006). Encapsulation of cells in alginate gels. In Immobilization of Enzymes and Cells (pp. 323–332). New Jersey: Humana Press.

  19. 19.

    Annabi, N., Tamayol, A., Uquillas, J. A., Akbari, M., Bertassoni, L. E., Cha, C., et al. (2014). 25th anniversary article: Rational design and applications of hydrogels in regenerative medicine. Advanced Materials, 26(1), 85–124. https://doi.org/10.1002/adma.201303233.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Lee, K. S., Wu, J. F., Lo, Y. S., Lo, Y. C., Lin, P. J., & Chang, J. S. (2004). Anaerobic hydrogen production with an efficient carrier-induced granular sludge bed bioreactor. Biotechnology and Bioengineering, 87(5), 648–657. https://doi.org/10.1002/bit.20174.

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Yokoi, H., Tokushige, T., Hirose, J., Hayashi, S., & Takasaki, Y. (1997). Hydrogen production by immobilized cells of aciduric Enterobacter aerogenes strain HO-39. Journal of Fermentation and Bioengineering, 83(5), 481–484. https://doi.org/10.1016/S0922-338X(97)83006-1.

    CAS  Article  Google Scholar 

  22. 22.

    Zhao, L., Cao, G. L., Wang, A. J., Guo, W. Q., Liu, B. F., Ren, H. Y., et al. (2012). Enhanced bio-hydrogen production by immobilized Clostridium sp. T2 on a new biological carrier. International Journal of Hydrogen Energy, 37(1), 162–166. https://doi.org/10.1016/j.ijhydene.2011.09.103.

    CAS  Article  Google Scholar 

  23. 23.

    Wu, S. Y., Lin, C. N., Chang, J. S., Lee, K. S., & Lin, P. J. (2002). Microbial hydrogen production with immobilized sewage sludge. Biotechnology Progress, 18(5), 921–926. https://doi.org/10.1021/bp0200548.

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Plangklang, P., Reungsang, A., & Pattra, S. (2012). Enhanced bio-hydrogen production from sugarcane juice by immobilized Clostridium butyricum on sugarcane bagasse. International Journal of Hydrogen Energy, 37(20), 15525–15532. https://doi.org/10.1016/j.ijhydene.2012.02.186.

    CAS  Article  Google Scholar 

  25. 25.

    Ramachandran, U., Wrana, N., Cicek, N., Sparling, R., & Levin, D. B. (2011). Isolation and characterization of a hydrogen- and ethanol-producing Clostridium sp. strain URNW. Canadian Journal of Microbiology, 57(3), 236–243. https://doi.org/10.1139/W11-005.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Thinesh, P. (2015). Characterization of cell growth , substrate utilization , end-product synthesis and gene expression patterns in cellulose degrading cocultures of Clostridium termitidis CT1112 and Clostridium intestinale URNW. MSc Thesis, University of Manitoba, MB, Canada.

  27. 27.

    Levin, D. B., Pitt, L., & Love, M. (2004). Biohydrogen production: Prospects and limitations to practical application. International Journal of Hydrogen Energy, 29(2), 173–185. https://doi.org/10.1016/S0360-3199(03)00094-6.

    CAS  Article  Google Scholar 

  28. 28.

    Taherzadeh, M., & Karimi, K. (2007). Acid-based hydrolysis processes for ethanol from lignocellulosic materials: A review. Bioresources, 2(3), 472–499.

    CAS  Google Scholar 

  29. 29.

    Carrillo-Reyes, J., Albarrán-Contreras, B. A., & Buitrón, G. (2019). Influence of added nutrients and substrate concentration in biohydrogen production from winery wastewaters coupled to methane production. Applied Biochemistry and Biotechnology, 187(1), 140–151. https://doi.org/10.1007/s12010-018-2812-5.

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Lee, H. S., & Rittmann, B. E. (2009). Evaluation of metabolism using stoichiometry in fermentative biohydrogen. Biotechnology and Bioengineering, 102(3), 749–758. https://doi.org/10.1002/bit.22107.

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Ho, K. L., Chen, Y. Y., & Lee, D. J. (2010). Biohydrogen production from cellobiose in phenol and cresol-containing medium using Clostridium sp. R1. International Journal of Hydrogen Energy, 35(19), 10239–10244. https://doi.org/10.1016/j.ijhydene.2010.07.155.

    CAS  Article  Google Scholar 

  32. 32.

    Ho, K. L., & Lee, D. J. (2011). Harvesting biohydrogen from cellobiose from sulfide or nitrite-containing wastewaters using Clostridium sp. R1. Bioresource Technology, 102(18), 8547–8549. https://doi.org/10.1016/j.biortech.2011.04.031.

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Ho, K. L., Lee, D. J., Su, A., & Chang, J. S. (2012). Biohydrogen from lignocellulosic feedstock via one-step process. International Journal of Hydrogen Energy, 37(20), 15569–15574. https://doi.org/10.1016/j.ijhydene.2012.01.137.

    CAS  Article  Google Scholar 

  34. 34.

    Ye, X., Zhang, X., Morgenroth, E., & Finneran, K. T. (2012). Anthrahydroquinone-2,6-disulfonate increases the rate of hydrogen production during Clostridium beijerinckii fermentation with glucose, xylose, and cellobiose. International Journal of Hydrogen Energy, 37(16), 11701–11709. https://doi.org/10.1016/j.ijhydene.2012.05.018.

    CAS  Article  Google Scholar 

  35. 35.

    Rai, P. K., Singh, S. P., & Asthana, R. K. (2012). Biohydrogen production from cheese whey wastewater in a two-step anaerobic process. Applied Biochemistry and Biotechnology, 167(6), 1540–1549. https://doi.org/10.1007/s12010-011-9488-4.

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Penniston, J., & Gueguim Kana, E. B. (2018). Impact of medium pH regulation on biohydrogen production in dark fermentation process using suspended and immobilized microbial cells. Biotechnology and Biotechnological Equipment, 32(1), 204–212. https://doi.org/10.1080/13102818.2017.1408430.

    CAS  Article  Google Scholar 

  37. 37.

    Canbay, E., Kose, A., & Oncel, S. S. (2018). Photobiological hydrogen production via immobilization: understanding the nature of the immobilization and investigation on various conventional photobioreactors. 3 Biotech, 8(5), 1–8. https://doi.org/10.1007/s13205-018-1266-3.

    Article  Google Scholar 

  38. 38.

    Randolph, K., & Studer, S. (2017). Hydrogen production cost from fermentation, DOE Hydrogen and Fuel Cells Program Record 16016, Department of Energy, USA.

  39. 39.

    Kayfeci, M., Keçebaş, A., & Bayat, M. (2019). Hydrogen production. Solar hydrogen production: Processes, systems and technologies. https://doi.org/10.1016/B978-0-12-814853-2.00003-5.

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AT gratefully acknowledges the financial support of NSERC through PDF program.


This work was funded by The Scientific And Technological Research Council Of Turkey (TUBITAK, BIDEB 2214), and Natural Sciences and Engineering Research Council of Canada (NSERC), through the Strategic Programs grant (STPGP 306944-04), by Genome Canada, through the Applied Genomics Research in Bioproducts or Crops (ABC) program for the grant titled, “Microbial Genomics for Biofuels and Co-Products from Biorefining Processes.”

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MG and AT designed and investigated the study. MG validated the experiments. MG prepared the original draft. DBL administrated the project and provided funding. AT and DBL reviewed and edited the manuscript. MG, AT, and DBL read the manuscript. MG, AT, and DBL approved the submission of the manuscript to the journal.

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Correspondence to Mine Güngörmüşler.

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• C. intestinale URNW entrapped in sodium alginate successfully produced biohydrogen.

Higher molar yields in mol H2/mol hexose eq. were achieved with immobilized cells.

The immobilized cells showed stability during sequential batch-mode over 192 h.

Four different initial cellobiose concentrations were tested for both cultures.

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Güngörmüşler, M., Tamayol, A. & Levin, D.B. Hydrogen Production by Immobilized Cells of Clostridium intestinale Strain URNW Using Alginate Beads. Appl Biochem Biotechnol 193, 1558–1573 (2021). https://doi.org/10.1007/s12010-021-03503-1

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  • Biohydrogen
  • Clostridium intestinale URNW
  • Immobilization
  • Hydrogel