Biohydrogen Production from Cheese Whey Wastewater in a Two-Step Anaerobic Process


Cheese whey-based biohydrogen production was seen in batch experiments via dark fermentation by free and immobilized Enterobacter aerogenes MTCC 2822 followed by photofermentation of VFAs (mainly acetic and butyric acid) in the spent medium by Rhodopseudomonas BHU 01 strain. E. aerogenes free cells grown on cheese whey diluted to 10 g lactose/L, had maximum lactose consumption (∼79%), high production of acetic acid (1,900 mg/L), butyric acid (537.2 mg/L) and H2 yield (2.04 mol/mol lactose; rate,1.09 mmol/L/h). The immobilized cells improved lactose consumption (84%), production of acetic acid (2,100 mg/L), butyric acid (718 mg/L) and also H2 yield (3.50 mol/mol lactose; rate, 1.91 mmol/L/h). E. aerogenes spent medium (10 g lactose/L) when subjected to photofermentation by free Rhodopseudomonas BHU 01 cells, the H2 yield reached 1.63 mol/mol acetic acid (rate, 0.49 mmol/L/h). By contrast, immobilized Rhodopseudomonas cells improved H2 yield to 2.69 mol/mol acetic acid (rate, 1.87 mmol/L/h). The cumulative H2 yield for free and immobilized bacterial cells was 3.40 and 5.88 mol/mol lactose, respectively. Bacterial cells entrapped in alginate, had a sluggish start of H2 production but outperformed the free cells subsequently. Also, the concomitant COD reduction for free cells (29.5%) could be raised to 36.08% by immobilized cells. The data suggest that two-step fermentative H2 production from cheese whey involving immobilized bacterial cells, offers greater substrate to- hydrogen conversion efficiency, and the effective removal of organic load from the wastewater in the long-term.

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  1. 1.

    Zhu, H., Suzuki, T., Tsygankov, A. A., Asada, Y., & Miyake, J. (2011). Hydrogen production from tofu wastewater by Rhodobacter sphaeroides immobilized in agar gels. International Journal of Hydrogen Energy, 24, 305–310.

    Article  Google Scholar 

  2. 2.

    Abboud, M. M., Aljundi, I. H., Khleifat, K. M., & Dmour, S. (2010). Biodegradation kinetics and modeling of whey lactose by bacterial hemoglobin VHb-expressing Escherichia coli strain. Biochemical Engineering Journal, 48, 166–172.

    Article  CAS  Google Scholar 

  3. 3.

    Mohan, S. V., Babu, V. L., & Sarma, P. N. (2007). Anaerobic biohydrogen production from dairy waste water treatment in sequencing batch reactor (AnSBR): effect of organic loading rate. Enzyme and Microbial Technology, 41, 506–515.

    Article  CAS  Google Scholar 

  4. 4.

    Ferchichi, M., Crabbe, E., Gil, G.-H., Hintz, W., & Almadidy, A. (2005). Influence of initial pH on hydrogen production from cheese whey. Journal of Biotechnology, 120, 402–409.

    Article  CAS  Google Scholar 

  5. 5.

    Davila-Vazquez, G., Alatriste-Mondragon, F., De Leon-Rodriguez, A., & Razo-Flores, E. (2008). Fermentative hydrogen production in batch experiments using lactose, cheese whey and glucose: Influence of initial substrate concentration and pH. International Journal of Hydrogen Energy, 33, 4989–4997.

    Article  CAS  Google Scholar 

  6. 6.

    Singh, S. P., Srivastava, S. C., & Pandey, K. D. (1994). Hydrogen production by Rhodopseudomonas at the expense of vegetable starch, sugarcane juice and whey. International Journal of Hydrogen Energy, 19, 437–440.

    Article  CAS  Google Scholar 

  7. 7.

    Antonopoulou, G., Stamatelatou, K., Venetsaneas, N., Kornaros, M., & Lyberatos, G. (2008). Biohydrogen and methane production from cheese whey in a two-stage anaerobic process. Industrial and Engineering Chemistry Research, 47, 5227–5233.

    Article  CAS  Google Scholar 

  8. 8.

    Stamatelatou, K., Antonopoulou, G., Tremouli, A., & Lyberatos, G. (2011). Production of gaseous biofuels and electricity from cheese whey. Industrial and Engineering Chemical Research, 50, 639–644.

    Article  CAS  Google Scholar 

  9. 9.

    Azbar, N., Dokgoz, F. T., Keskin, T., Eltem, R., Korkmaz, K. S., Gezgin, Y., Akbal, Z., Oncel, S., Dalay, M. C., & Gonen, C. (2009). Comparative evaluation of bio-hydrogen production from cheese whey waste water under thermophilic and mesophilic anaerobic conditions. International Journal of Green Energy, 6, 192–200.

    Article  CAS  Google Scholar 

  10. 10.

    Song, W., Cheng, J., Zhou, J., Xie, B., Su, H., & Cen, K. (2010). Cogeneration of hydrogen and methane from protein-mixed food waste water by two-phase anaerobic process. International Journal of Hydrogen Energy, 35, 3141–3146.

    Article  CAS  Google Scholar 

  11. 11.

    Alalayah, W. M., Kalil, M. S., Kadhum, A. A. H., Jahim, J. M., Jaapar, S. Z. S., & Alauj, N. M. (2009). Bio-hydrogen production using a two-stage fermentation process. Pakistan Journal of Biological Sciences, 12, 1462–1467.

    Article  CAS  Google Scholar 

  12. 12.

    Ozmihci, S., & Kargi, F. (2010). Bio-hydrogen production by photo-fermentation of dark fermentation effluent with intermittent feeding and effluent removal. International Journal of Hydrogen Energy, 35, 6674–6680.

    Article  CAS  Google Scholar 

  13. 13.

    Singh, S. P., Srivastava, S. C., & Pandey, K. D. (1990). Photoproduction of hydrogen by a non-sulphur bacterium isolated from root zones of water fern Azolla pinnata. International Journal of Hydrogen Energy, 15, 403–406.

    Article  CAS  Google Scholar 

  14. 14.

    Tanisho, S., & Ishiwata, Y. (1995). Continuous hydrogen production from molasses by fermentation using urethane as a support of flocks. International Journal of Hydrogen Energy, 20, 541–545.

    Article  CAS  Google Scholar 

  15. 15.

    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, 8, 481–484.

    Article  Google Scholar 

  16. 16.

    Rachmann, M. A., Nkashimada, Y., Kakizono, T., & Nishio, N. (1998). Hydrogen production with high yield and high evolution rate by self-flocculated cells of Enterobacter aerogenes in a packed-bed reactor. Applied Microbiology and Biotechnology, 49, 450–454.

    Article  Google Scholar 

  17. 17.

    Kumar, N., & Das, D. (2001). Continuous hydrogen production by immobilized Enterobacter cloacae IIT-BT 08 using lignocellulosic materials as solid matrices. Enzyme and Microbial Technology, 29, 280–287.

    Article  CAS  Google Scholar 

  18. 18.

    Liu, X., Zhu, Y., & Yang, S.-T. (2006). Butyric acid and hydrogen production by Clostridium tyrobutyricum ATCC 25755 and mutants. Enzyme and Microbial Technology, 38, 521–528.

    Article  CAS  Google Scholar 

  19. 19.

    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, 6666–6672.

    Article  CAS  Google Scholar 

  20. 20.

    Wang, Y.-Z., Liao, Q., Zhu, X., Tian, X., & Zhang, C. (2010). Characterstics of hydrogen production and substrate consumption of Rhodopseudomonas palustris CQK 01 in an immobilized-cell photobioreactor. Bioresource Technology, 101, 4034–4041.

    Article  CAS  Google Scholar 

  21. 21.

    Liu, B.-F., Xie, G.-J., Guo, W.-Q., Ding, J., & Ren, N.-Q. (2011). Optimization of photo-hydrogen production by immobilized Rhodopseudomonas faecalis RLD-53. Natural Resources, 2, 1–7.

    Article  Google Scholar 

  22. 22.

    Tian, X., Liao, Q., Liu, W., Wang, Y.-Z., Zhu, X., Li, J., & Wang, H. (2009). Photohydrogen production rate of a PVA-boric acid gel granule containing immobilized photosynthetic bacteria cells. International Journal of Hydrogen Energy, 34, 4708–4717.

    Article  CAS  Google Scholar 

  23. 23.

    Pfennig, N. (1967). Photosynthetic bacteria. Annual Review of Microbiology, 21, 285–324.

    Article  CAS  Google Scholar 

  24. 24.

    Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin–Phenol reagent. Journal of Biological Chemistry, 193, 265–275.

    CAS  Google Scholar 

  25. 25.

    Herbert, D., Phipps, P. J., & Strange, R. E. (1971). In J. R. Norris & D. W. Ribbons (Eds.), Methods in microbiology, vol.5B: Chemical analysis of microbial cells (pp. 209–344). London: Academic.

    Google Scholar 

  26. 26.

    Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28, 350–356.

    Article  CAS  Google Scholar 

  27. 27.

    APHA. (1995). Standard methods for the examination of water and waste water (19th ed.). USA: American Public Health Association.

    Google Scholar 

  28. 28.

    Yokoi, H., Tokushige, T., Hirose, J., Hayashi, S., & Takasaki, Y. (1998). H2 production from starch by a mixed culture of Clostridium butyricum and Enterobacter aerogenes. Biotechnology Letters, 20, 143–147.

    Article  CAS  Google Scholar 

  29. 29.

    Papageorgiou, G. C., Kalosaka, K., Sotiropoulou, G., Barbotin, J. N., Thomasett, B., & Thomas, T. (1988). Entrapment of active ion-permeable cyanobacteria (Anacystis nidulans) in calcium alginate. Applied Microbiology and Biotechnology, 29, 565–571.

    Article  CAS  Google Scholar 

  30. 30.

    Fabiano, B., & Perego, P. (2002). Thermodynamic study and optimization of hydrogen production by Enterobacter aerogenes. International Journal of Hydrogen Energy, 27, 149–156.

    Article  CAS  Google Scholar 

  31. 31.

    Oh, Y. K., Seol, E. H., Kim, J. R., & Park, S. (2003). Fermentative biohydrogen production by a new chemoheterotrophic bacterium Citrobacter sp. Y19. International Journal of Hydrogen Energy, 28, 1353–1359.

    Article  CAS  Google Scholar 

  32. 32.

    Ferchichi, M., Crabbe, E., Hintz, W., Gill, G.-H., & Almadidy, A. (2005). Influence of culture parameters on biological hydrogen production by Clostridium saccharoperbutylacetonium ATCC 27021. World Journal of Microbiology and Biotechnology, 21, 855–862.

    Article  CAS  Google Scholar 

  33. 33.

    Calli, B., Schoenmaekers, K., Vanbroekhoven, K., & Diels, L. (2008). Dark fermentative H2 production from xylose and lactose—effects of on-line pH control. International Journal of Hydrogen Energy, 33, 522–530.

    Article  CAS  Google Scholar 

  34. 34.

    Fang, H. H. P., Zhu, H., & Zhang, T. (2006). Phototrophic hydrogen production from glucose by pure and co-cultures of Clostridium butyricum and Rhodobacter sphaeroides. International Journal of Hydrogen Energy, 31, 2223–2230.

    Article  CAS  Google Scholar 

  35. 35.

    Liu, B.-F., Ren, N.-Q., Tang, J., Ding, J., Liu, W.-Z., Xu, J.-F., Cao, G.-L., Guo, W.-Q., & Xie, G.-J. (2010). Biohydrogen production by mixed culture of photo- and dark fermentation bacteria. International Journal of Hydrogen Energy, 35, 2858–2862.

    Article  CAS  Google Scholar 

  36. 36.

    Oh, Y.-K., Seol, E.-H., Kim, M.-S., & Park, S. (2004). Photoproduction of hydrogen from acetate by a chemoheterotrophic bacterium Rhodopseudomonas palustris P4. International Journal of Hydrogen Energy, 29, 1115–1121.

    CAS  Google Scholar 

  37. 37.

    Barbosa, M. J., Rocha, J. M. S., Tramper, J., & Wijffels, R. H. (2001). Acetate as a carbon source for hydrogen production by photosynthetic bacteria. Journal of Biotchnology, 85, 25–33.

    Article  CAS  Google Scholar 

  38. 38.

    Chen, C. Y., Lee, C. M., & Chang, J. S. (2006). Feasibility study on bioreactor strategies for enhanced photohydrogen from Rhodopseudomonas palustris WP 3-5 using optical-fibre assisted illumination systems. International Journal of Hydrogen Energy, 31, 2345–2355.

    Article  CAS  Google Scholar 

  39. 39.

    Ren, N.-Q., Liu, B.-F., Zheng, G.-X., Xing, D.-F., Zhao, X., Guo, W.-Q., & Ding, J. (2009). Strategy for enhancing photo-hydrogen production yield by repeated fed-batch cultures. International Journal of Hydrogen Energy, 34, 7579–7584.

    Article  CAS  Google Scholar 

  40. 40.

    Seifert, K., Waligorska, M., & Laniecki, M. (2010). Hydrogen generation in photobiological process from dairy waste water. International Journal of Hydrogen Energy, 35, 9624–9629.

    Article  CAS  Google Scholar 

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This work was supported by grant 103/131/2008-NT from Ministry of New and Renewable Energy, Govt. of India and Hydrogen Energy Centre of this University.

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Correspondence to S. P. Singh.

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Rai, P.K., Singh, S.P. & Asthana, R.K. Biohydrogen Production from Cheese Whey Wastewater in a Two-Step Anaerobic Process. Appl Biochem Biotechnol 167, 1540–1549 (2012).

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  • Enterobacter
  • Rhodopseudomonas
  • Whey
  • H2 production