Waste and Biomass Valorization

, Volume 7, Issue 4, pp 753–764 | Cite as

Effect of pH on Continuous Biohydrogen Production from End-of-Life Dairy Products (EoL-DPs) via Dark Fermentation

  • K. P. Stavropoulos
  • A. Kopsahelis
  • C. Zafiri
  • M. KornarosEmail author
Original Paper



To investigate the effect of pH and thus determine the optimal pH value for biohydrogen production from a mixture of End-of-Life Dairy Products (EoL-DPs) through dark fermentation in view of increasing the yield of their subsequent co-digestion with agroindustrial wastes in a two-stage anaerobic digestion system.


A Continuously Stirred Tank Reactor (CSTR) was operated under mesophilic conditions (37 °C) and hydraulic retention time of 6 days in order to enhance biohydrogen production from a typical mixture of EoL-DPs (93 % milk–5 % yoghurt–2 % cheese, w/w). CSTR experiments were performed to investigate the effect of controlled pH (4.0, 4.5, 4.7, 5.0, 5.3, 5.7) on the production of biohydrogen and Volatile Fatty Acids (VFAs).


The maximum hydrogen yield and productivity (0.84 mol H2/mol carbohydrates consumed and 0.76 L L R −1 ·day−1 respectively) was obtained at pH 5.0, whereas the greatest degradation of soluble carbohydrates was observed at pH 4.7. Equally high concentration of total VFAs, 25.1 and 24.6 g/L, was recorded at pH 4.7 and 5.0 respectively. Acetic, butyric and propionic acid were the main volatile fatty acids detected, while lactic acid was identified as a major intermediate metabolite of the studied process which presented an intense accumulation prior to its conversion to butyric and/or acetic acid and hydrogen.


The optimum conversion of the studied EoL-DPs’ mixture to biohydrogen (40.6 mL H2/g VSadded or 24.3 mL H2/g CODadded) was identified at pH 5.0, with hydrogen production being primarily associated with the bioconversion of lactic acid to butyric acid.


End-of-life dairy products Biohydrogen production Dark fermentation Acidogenesis Anaerobic digestion 



Financial support from the European Commission Project LIFE10 ENV/CY/000721 (DAIRIUS) “Sustainable management via energy exploitation of end-of-life dairy products in Cyprus” is gratefully acknowledged.


  1. 1.
    Council Directive 1999/31/EC of 26 April 1999 on the landfill of wasteGoogle Scholar
  2. 2.
    Perle, M., Kimchie, S., Shelef, G.: Some biochemical aspects of the anaerobic degradation of dairy wastewater. Water Res. 29, 1549–1554 (1995)CrossRefGoogle Scholar
  3. 3.
    Girotto, F., Alibardi, L., Cossu, R.: Food waste generation and industrial uses: a review. Waste Manag 45, 32–41 (2015)CrossRefGoogle Scholar
  4. 4.
    Cantrell, K.B., Ducey, T., Ro, K.S., Hunt, P.G.: Livestock waste-to-bioenergy generation opportunities. Bioresour. Technol. 99, 7941–7953 (2008)CrossRefGoogle Scholar
  5. 5.
    Ince, O.: Performance of a two-phase anaerobic digestion system when treating dairy wastewater. Water Res. 32, 2707–2713 (1998)CrossRefGoogle Scholar
  6. 6.
    Jeyaseelan, S., Matsuo, T.: Effects of phase separation in anaerobic digestion on different substrates. Water Sci. Technol. 31, 153–162 (1995)CrossRefGoogle Scholar
  7. 7.
    De Gioannis, G., Friargiu, M., Massi, E., Muntoni, A., Polettini, A., Pomi, R., Spiga, D.: Biohydrogen production from dark fermentation of cheese whey: influence of pH. Int. J. Hydrogen Energy 39, 20930–20941 (2014)CrossRefGoogle Scholar
  8. 8.
    Alonso, S., Herrero, M., Rendueles, M., Díaz, M.: Residual yoghurt whey for lactic acid production. Biomass Bioenergy 34, 931–938 (2010)CrossRefGoogle Scholar
  9. 9.
    Wan, C., Li, Y., Shahbazi, A., Xiu, S.: Succinic acid production from cheese whey using Actinobacillus succinogenes 130 Z. Appl. Biochem. Biotechnol. 145, 111–119 (2008)CrossRefGoogle Scholar
  10. 10.
    Löser, C., Urit, T., Förster, S., Stukert, A., Bley, T.: Formation of ethyl acetate by Kluyveromyces marxianus on whey during aerobic batch and chemostat cultivation at iron limitation. Appl. Microbiol. Biotechnol. 96, 685–696 (2012)CrossRefGoogle Scholar
  11. 11.
    Ntaikou, I., Antonopoulou, G., Lyberatos, G.: Biohydrogen production from biomass and wastes via dark fermentation: a review. Waste Biomass Valori. 1, 21–39 (2010)CrossRefGoogle Scholar
  12. 12.
    Boyles, D.: Bioenergy Technology-Thermodynamics and Costs. Wiley, New York (1984)Google Scholar
  13. 13.
    Ghimire, Α., Frunzo, L., Pirozzi, F., Trably, E., Escudie, R., Lens, P.N.L., Esposito, G.: A review on dark fermentative biohydrogen production from organic biomass: process parameters and use of by-products. Appl. Energy 144, 73–95 (2015)CrossRefGoogle Scholar
  14. 14.
    Kotay, S.M., Das, D.: Biohydrogen as a renewable energy resource—prospects and potentials. Int. J. Hydrogen Energy 33, 258–263 (2008)CrossRefGoogle Scholar
  15. 15.
    Dareioti, Μ.Α., Vavouraki, Α.Ι., Kornaros, Μ.: Effect of pH on the anaerobic acidogenesis of agroindustrial wastewaters for maximization of bio-hydrogen production: a lab-scale evaluation using batch tests. Bioresour. Technol. 162, 218–227 (2014)CrossRefGoogle Scholar
  16. 16.
    Rai, P.K., Singh, S.P., Asthana, R.K.: Biohydrogen production from sugarcane bagasse by integrating dark- and photo-fermentation. Bioresour. Technol. 152, 140–146 (2014)CrossRefGoogle Scholar
  17. 17.
    Chandrasekhar, K., Lee, Y.J., Lee, D.W.: Biohydrogen production: strategies to improve process efficiency through microbial routes. Int. J. Mol. Sci. 16, 8266–8293 (2015)CrossRefGoogle Scholar
  18. 18.
    Moreno, R., Escapa, A., Cara, J., Carracedo, B., Gómez, X.: A two-stage process for hydrogen production from cheese whey: integration of dark fermentation and biocatalyzed electrolysis. Int. J. Hydrogen Energy 40, 168–175 (2015)CrossRefGoogle Scholar
  19. 19.
    Dhar, B.R., Elbeshbishy, E., Hafez, H., Lee, H.S.: Hydrogen production from sugar beet juice using an integrated biohydrogen process of dark fermentation and microbial electrolysis cell. Bioresour. Technol. 198, 223–230 (2015)CrossRefGoogle Scholar
  20. 20.
    Gómez, X., Fernández, C., Fierro, J., Sánchez, M.E., Escapa, A., Morán, A.: Hydrogen production: two stage processes for waste degradation. Bioresour. Technol. 102, 8621–8627 (2011)CrossRefGoogle Scholar
  21. 21.
    Giordano, A., Cantu, C., Spagni, A.: Monitoring the biochemical hydrogen and methane potential of the two-stage dark-fermentative process. Bioresour. Technol. 102, 4474–4479 (2011)CrossRefGoogle Scholar
  22. 22.
    Cavinato, C., Bolzonella, D., Fatone, F., Cecchi, F., Pavan, P.: Optimization of two-phase thermophilic anaerobic digestion of biowaste for hydrogen and methane production through reject water recirculation. Bioresour. Technol. 102, 8605–8611 (2011)CrossRefGoogle Scholar
  23. 23.
    Luo, G., Xie, L., Zhou, Q., Angelidaki, I.: Enhancement of bioenergy production from organic wastes by two-stage anaerobic hydrogen and methane production process. Bioresour. Technol. 102, 8700–8706 (2011)CrossRefGoogle Scholar
  24. 24.
    Schievano, A., Tenca, A., Lonati, S., Manzini, E., Adani, F.: Can two-stage instead of one-stage anaerobic digestion really increase energy recovery from biomass? Appl. Energy 124, 335–342 (2014)CrossRefGoogle Scholar
  25. 25.
    Hawkes, F.R., Dinsdal, R., Hawkes, D.L., Hussy, I.: Sustainable fermentative hydrogen production: challenges for process optimisation. Int. J. Hydrogen Energy 27, 1339–1347 (2002)CrossRefGoogle Scholar
  26. 26.
    Wang, J., Wan, W.: Factors influencing fermentative hydrogen production: a review. Int. J. Hydrogen Energy 34, 799–811 (2009)CrossRefGoogle Scholar
  27. 27.
    Kapdan, I.K., Kargi, F.: Bio-hydrogen production from waste materials. Enzyme Microb. Technol. 38, 569–582 (2006)CrossRefGoogle Scholar
  28. 28.
    Das, D., Veziroglu, T.N.: Advances in biological hydrogen production processes. Int. J. Hydrogen Energy 33, 6046–6057 (2008)CrossRefGoogle Scholar
  29. 29.
    Ren, N., Wang, B., Huang, J.-C.: Ethanol-type fermentation from carbohydrate in high rate acidogenic reactor. Biotechnol. Bioeng. 54, 428–433 (1997)CrossRefGoogle Scholar
  30. 30.
    Chen, C.C., Lin, C.Y., Lin, M.C.: Acid-base enrichment enhances anaerobic hydrogen production process. Appl. Microbiol. Biotechnol. 58, 224–228 (2002)CrossRefGoogle Scholar
  31. 31.
    Luo, G., Xie, L., Zou, Z., Zhou, Q., Wang, J.-Y.: Fermentative hydrogen production from cassava stillage by mixed anaerobic microflora: effects of temperature and pH. Appl. Energy 87, 3710–3717 (2010)CrossRefGoogle Scholar
  32. 32.
    Wu, X., Yao, W., Zhu, J.: Effect of pH on continuous biohydrogen production from liquid swine manure with glucose supplement using an anaerobic sequencing batch reactor. Int. J. Hydrogen Energy 35, 6592–6599 (2010)CrossRefGoogle Scholar
  33. 33.
    Ferchichi, M., Crabbe, E., Gil, G.-H., Hintz, W., Almadidy, A.: Influence of initial pH on hydrogen production from cheese whey. J. Biotechnol. 120, 402–409 (2005)CrossRefGoogle Scholar
  34. 34.
    Fang, H.H.P., Liu, H.: Effect of pH on hydrogen production from glucose by a mixed culture. Bioresour. Technol. 82, 87–93 (2002)CrossRefGoogle Scholar
  35. 35.
    Khanal, S.K., Chen, W.H., Li, L., Sung, S.: Biological hydrogen production: effects of pH and intermediate products. Int. J. Hydrogen Energy 29, 1123–1131 (2004)Google Scholar
  36. 36.
    APHA, AWWA, WEF: Standard Methods for the Examination of Water and Wastewater, 19th edn. American Public Health Association, Washington, DC (1995)Google Scholar
  37. 37.
    Joseffson, B.: Rapid spectrophotometric determination of total carbohydrates. In: Grasshoff, K., Ehrhardt, M., Kremling, K. (eds.) Methods of Seawater Analysis, pp. 340–342. Verlag Chemie GmbH, Weinheim (1983)Google Scholar
  38. 38.
    Mohd Yasin, N.H., Rahman, N.A., Man, H.C., Mohd Yusoff, M.Z., Hassan, M.A.: Microbial characterization of hydrogen-producing bacteria in fermented food waste at different pH values. Int. J. Hydrogen Energy 36, 9571–9580 (2011)CrossRefGoogle Scholar
  39. 39.
    Van Ginkel, S., Sung, S., Lay, J.-J.: Biohydrogen production as a function of pH and substrate concentration. Environ. Sci. Technol. 35, 4726–4730 (2001)CrossRefGoogle Scholar
  40. 40.
    Davila-Vazquez, G., Alatriste-Mondragón, F., de León-Rodríguez, A., Razo-Flores, E.: Fermentative hydrogen production in batch experiments using lactose, cheese whey and glucose: influence of initial substrate concentration and pH. Int. J. Hydrogen Energy 33, 4989–4997 (2008)CrossRefGoogle Scholar
  41. 41.
    Azbar, N., Çetinkaya Dokgöz, F.T., Keskin, T., Korkmaz, K.S., Syed, H.M.: Continuous fermentative hydrogen production from cheese whey wastewater under thermophilic anaerobic conditions. Int. J. Hydrogen Energy 34, 7441–7447 (2009)CrossRefGoogle Scholar
  42. 42.
    Yang, P., Zhang, R., McGarvey, J.A., Benemann, J.R.: Biohydrogen production from cheese processing wastewater by anaerobic fermentation using mixed microbial communities. Int. J. Hydrogen Energy 32, 4761–4771 (2007)CrossRefGoogle Scholar
  43. 43.
    Venetsaneas, N., Antonopoulou, G., Stamatelatou, K., Kornaros, M., Lyberatos, G.: Using cheese whey for hydrogen and methane generation in a two-stage continuous process with alternative pH controlling approaches. Bioresour. Technol. 100, 3713–3717 (2009)CrossRefGoogle Scholar
  44. 44.
    Teli, A., Ficara, E., Malpei, F.: Bio-hydrogen production from cheese whey by dark fermentation. Chem. Eng. Trans. 37, 613–618 (2014)Google Scholar
  45. 45.
    Lateef, S.A., Beneragama, N., Yamashiro, T., Iwasaki, M., Ying, C., Umetsu, K.: Biohydrogen production from co-digestion of cow manure and waste milk under thermophilic temperature. Bioresour. Technol. 110, 251–257 (2012)CrossRefGoogle Scholar
  46. 46.
    Thauer, R.K., Jungermann, K., Decker, K.: Energy conservation in chemotrophic anaerobic bacteria. Bacteriol. Rev. 41, 100–180 (1977)Google Scholar
  47. 47.
    Kim, S.H., Shin, H.S.: Effects of base-pretreatment on continuous enriched culture for hydrogen production from food waste. Int. J. Hydrogen Energy 33, 5266–5274 (2008)CrossRefGoogle Scholar
  48. 48.
    Noike, T., Takabatake, H., Mizuno, O., Ohba, M.: Inhibition of hydrogen fermentation of organic wastes by lactic acid bacteria. Int. J. Hydrogen Energy 27, 1367–1371 (2002)CrossRefGoogle Scholar
  49. 49.
    Wang, G., Mu, Y., Yu, H.Q.: Response surface analysis to evaluate the influence of pH, temperature and substrate concentration on the acidogenesis of sucrose-rich wastewater. Biochem. Eng. J. 23, 175–184 (2005)CrossRefGoogle Scholar
  50. 50.
    Ding, J., Liu, B.F., Ren, N.Q., Xing, D.F., Guo, W.Q., Xu, G.F., Xie, G.J.: Hydrogen production from glucose by co-culture of Clostridium butyricum and immobilized Rhodopseudomonas faecalis RLD-53. Int. J. Hydrogen Energy 34, 3647–3652 (2009)CrossRefGoogle Scholar
  51. 51.
    Zhang, Y.-J., Jiang, J.-G., Wang, J.-M.: Effect of pH value on VFA concentration and composition during anaerobic fermentation of kitchen waste. China Environ. Sci. 33, 680–684 (2013)Google Scholar
  52. 52.
    Zheng, M., Zheng, M., Wu, Y., Ma, Y., Wang, K.: Effect of pH on types of acidogenic fermentation of fruit and vegetable wastes. Biotechnol. Bioprocess E 20, 298–303 (2015)CrossRefGoogle Scholar
  53. 53.
    Stiles, M.E., Holzapfel, W.H.: Lactic acid bacteria of foods and their current taxonomy. Int. J. Food Microbiol. 36, 1–29 (1997)CrossRefGoogle Scholar
  54. 54.
    Parawira, W., Murto, M., Read, J.S., Mattiasson, B.: Volatile fatty acid production during anaerobic mesophilic digestion of solid potato waste. J. Chem. Technol. Biotechnol. 79, 673–677 (2004)CrossRefGoogle Scholar
  55. 55.
    Akao, S., Tsuno, H., Horie, T., Mori, S.: Effects of pH and temperature on products and bacterial community in l-lactate batch fermentation of garbage under unsterile condition. Water Res. 41, 2636–2642 (2007)CrossRefGoogle Scholar
  56. 56.
    Castelló, E., García y Santos, C., Iglesias, T., 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. Int. J. Hydrogen Energy 34, 5674–5682 (2009)CrossRefGoogle Scholar
  57. 57.
    Antonopoulou, G., Gavala, H.N., Skiadas, I.V., Angelopoulos, K., Lyberatos, G.: Biofuels generation from sweet sorghum: fermentative hydrogen production and anaerobic digestion of the remaining biomass. Bioresour. Technol. 99, 110–119 (2008)CrossRefGoogle Scholar
  58. 58.
    Wang, X., Zhao, Y.-C.: A bench scale study of fermentative hydrogen and methane production from food waste in integrated two-stage process. Int. J. Hydrogen Energy 34, 245–254 (2009)CrossRefGoogle Scholar
  59. 59.
    Infantes, D., González del Campo, A., Villaseñor, J., Fernández, F.J.: Influence of pH, temperature and volatile fatty acids on hydrogen production by acidogenic fermentation. Int. J. Hydrogen Energy 36, 15595–15601 (2011)CrossRefGoogle Scholar
  60. 60.
    Zoetemeyer, R.J., van den Heuvel, J.C., Cohen, A.: pH influence on acidogenic dissimilation of glucose in an anaerobic digestor. Water Res. 16, 303–311 (1982)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • K. P. Stavropoulos
    • 1
  • A. Kopsahelis
    • 1
    • 2
  • C. Zafiri
    • 2
  • M. Kornaros
    • 1
    Email author
  1. 1.Laboratory of Biochemical Engineering and Environmental Technology (LBEET), Department of Chemical EngineeringUniversity of PatrasPatrasGreece
  2. 2.Green Technologies LtdPatrasGreece

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