Bioprocess and Biosystems Engineering

, Volume 37, Issue 6, pp 1017–1029 | Cite as

Simultaneous single-cell protein production and COD removal with characterization of residual protein and intermediate metabolites during whey fermentation by K. marxianus

  • J. S. S. Yadav
  • J. Bezawada
  • S. Elharche
  • S. Yan
  • R. D. TyagiEmail author
  • R. Y. Surampalli
Original Paper


Cheese whey fermentation with Kluyveromyces marxianus was carried out at 40 °C and pH 3.5 to examine simultaneous single-cell protein production and chemical oxygen demand (COD) removal, determine the fate of soluble whey protein and characterize intermediate metabolites. After 36 h of batch fermentation, the biomass concentration increased from 2.0 to 6.0 g/L with 55 % COD reduction (including protein), whereas soluble whey protein concentration decreased from 5.6 to 4.1 g/L. It was confirmed through electrophoresis (SDS-PAGE) that the fermented whey protein was different from native whey protein. HPLC and GC–MS analysis revealed a change in composition of organic compounds post-fermentation. High inoculum concentration in batch fermentation resulted in an increase in biomass concentration from 10.3 to 15.9 g/L with 80 % COD reduction (including protein) within 36 h with residual protein concentration of 4.5 g/L. In third batch fermentation, the biomass concentration increased from 7.3 to 12.4 g/L with 71 % of COD removal and residual protein concentration of 4.3 g/L after 22 h. After 22 h, the batch process was shifted to a continuous process with cell recycle, and the steady state was achieved after another 60 h with biomass yield of 0.19 g biomass/g lactose and productivity of 0.26 g/L h. COD removal efficiency was 78–79 % with residual protein concentration of 3.8–4.2 g/L. The aerobic continuous fermentation process with cell recycle could be applied to single-cell protein production with substantial COD removal at low pH and high temperature from cheese whey.


Single-cell protein Cheese whey COD Whey protein Intermediate metabolites Cell recycle 



The authors are sincerely thankful to the Natural Sciences and Engineering Research Council of Canada (Grant A4984, RDCPJ379601-08, and Canada Research Chair) for their financial support.


  1. 1.
    González Siso MI (1996) The biotechnological utilization of cheese whey: a review. Bioresour Technol 57:1–11CrossRefGoogle Scholar
  2. 2.
    Panesar PS, Kennedy JF (2012) Biotechnological approaches for the value addition of whey. Crit Rev Biotechnol 32:327–348CrossRefGoogle Scholar
  3. 3.
    Madureira AR, Pereira CI, Gomes AMP, Pintado ME, Xavier Malcata F (2007) Bovine whey proteins—overview on their main biological properties. Food Res Int 40:1197–1211CrossRefGoogle Scholar
  4. 4.
    Ghaly AE, Mahmoud NS, Rushton DG, Arab F (2007) Potential environmental and health impacts of high land application of cheese whey. Am J Agric Biol Sci 2:106–117CrossRefGoogle Scholar
  5. 5.
    Agustriyanto R, Fatmawati A (2009) Model of continuous cheese whey fermentation by Candida pseudotropicalis. World Acad Sci Eng Technol 33:281–285Google Scholar
  6. 6.
    Smithers GW (2008) Whey and whey proteins—From ‘gutter-to-gold’. Int Dairy J 18:695–704CrossRefGoogle Scholar
  7. 7.
    Ghaly AE, Kamal MA (2004) Submerged yeast fermentation of acid cheese whey for protein production and pollution potential reduction. Water Res 38:631–644CrossRefGoogle Scholar
  8. 8.
    Mawson AJ (1994) Bioconversions for whey utilization and waste abatement. Bioresour Technol 47:195–203CrossRefGoogle Scholar
  9. 9.
    Mansour MH, Ghaly AE, Benhassan RM, Nassar MA (1993) Modeling batch production of single cell protein from cheese whey. Appl Biochem Biotechnol 43:1–14CrossRefGoogle Scholar
  10. 10.
    Grba S, Stehlik-Tomas V, Stanzer D, Vahčić N, Škrlin A (2002) Selection of yeast strain Kluyveromyces marxianus for alcohol and biomass production on whey. Chem Biochem Eng Q 16:13–16Google Scholar
  11. 11.
    Fonseca GG, Heinzle E, Wittmann C, Gombert AK (2008) The yeast Kluyveromyces marxianus and its biotechnological potential. Appl Microbiol Biotechnol 79:339–354CrossRefGoogle Scholar
  12. 12.
    Çinar Ö, Hasar H, Kinaci C (2006) Modeling of submerged membrane bioreactor treating cheese whey wastewater by artificial neural network. J Biotechnol 123:204–209CrossRefGoogle Scholar
  13. 13.
    Cristiani-Urbina E, Netzahuatl-Munoz AR, Manriquez-Rojas FJ, Juarez-Ramrez C, Ruiz-Ordaz N, Galndez-Mayer J (2000) Batch and fed-batch cultures for the treatment of whey with mixed yeast cultures. Process Biochem 35:649–657CrossRefGoogle Scholar
  14. 14.
    Chatzipaschali AA, Stamatis AG (2012) Biotechnological utilization with a focus on anaerobic treatment of cheese whey: current status and prospects. Energies 5:3492–3525CrossRefGoogle Scholar
  15. 15.
    Spălăţelu C (2012) Biotechnological valorisation of whey. Innov Romanian Food Biotechnol 10:1–8Google Scholar
  16. 16.
    Pacheco FP, Galindo AB (2010) Microbial safety of raw milk cheeses traditionally made at a pH below 4.7 and with other hurdles limiting pathogens growth. In: Mendez-Vilas A (ed) Current research, technology and education topics in applied microbiology and microbial biotechnology, Formatex, pp 789–1620Google Scholar
  17. 17.
    Fernández A, Menéndez V, Riera FA (2012) α-Lactalbumin solubilisation from a precipitated whey protein concentrates fraction: pH and calcium concentration effects. Int J Food Sci Technol 47:467–474CrossRefGoogle Scholar
  18. 18.
    Mollea C, Marmo L, Bosco F (2013) Valorisation of cheese whey, a by-product from the dairy industry. ISBN 978-953-51-0911-2Google Scholar
  19. 19.
    Paul D, Mukhopadhyay R, Chatterjee B, Guha A (2002) Nutritional profile of food yeast Kluyveromyces fragilis biomass grown on whey. Appl Biochem Biotechnol 97:209–218CrossRefGoogle Scholar
  20. 20.
    Schultz N, Chang L, Hauck A, Reuss M, Syldatk C (2006) Microbial production of single-cell protein from deproteinized whey concentrates. Appl Microbiol Biotechnol 69:515–520CrossRefGoogle Scholar
  21. 21.
    Michael L, Shuler FK (2002) Bioprocess engineering: basic concepts. Prentice Hall: Upper Saddle River, New YorkGoogle Scholar
  22. 22.
    Parrondo J, García LA, Díaz M (2009) Nutrient balance and metabolic analysis in a Kluyveromyces marxianus fermentation with lactose-added whey. Braz J Chem Eng 26:445–456CrossRefGoogle Scholar
  23. 23.
    Ugalde UO, Castrillo JI (2002) Single cell proteins from fungi and yeasts. In: Dilip KA, George GK (eds) Applied mycology and biotechnology. Elsevier, AmsterdamGoogle Scholar
  24. 24.
    American Public Health A, Eaton AD, American Water Works A, Water Environment F (2005) Standard methods for the examination of water and wastewater, APHA-AWWA-WEF, Washington, DCGoogle Scholar
  25. 25.
    Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  26. 26.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  27. 27.
    López CVG, del Carmen Cerón García M, Fernández FGA, Bustos CS, Chisti Y, Sevilla JMF (2010) Protein measurements of microalgal and cyanobacterial biomass. Bioresour Technol 101:7587–7591Google Scholar
  28. 28.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  29. 29.
    Dionex the determination of carbohydrates, alcohols, and glycols in fermentation broths. pp 1–11Google Scholar
  30. 30.
    Dragone G, Mussatto SI, Oliveira JM, Teixeira JA (2009) Characterisation of volatile compounds in an alcoholic beverage produced by whey fermentation. Food Chem 112:929–935CrossRefGoogle Scholar
  31. 31.
    Ghaly AE, Kamal M, Correia LR (2005) Kinetic modelling of continuous submerged fermentation of cheese whey for single cell protein production. Bioresour Technol 96:1143–1152CrossRefGoogle Scholar
  32. 32.
    Lukondeh T, Ashbolt NJ, Rogers PL (2005) Fed-batch fermentation for production of Kluyveromyces marxianusFII 510700 cultivated on a lactose-based medium. J Ind Microbiol Biotechnol 32:284–288CrossRefGoogle Scholar
  33. 33.
    Verduyn C (1991) Physiology of yeasts in relation to biomass yields. Antonie Van Leeuwenhoek 60:325–353CrossRefGoogle Scholar
  34. 34.
    AAFCO (2010) Association of American feed control official incorporatedGoogle Scholar
  35. 35.
    Didelot S, Bordenave-Juchereau S, Rosenfeld E, Piot JM, Sannier F (2006) Peptides released from acid goat whey by a yeast–lactobacillus association isolated from cheese microflora. J Dairy Res 73:163–170CrossRefGoogle Scholar
  36. 36.
    Hamme V, Sannier F, Piot JM, Didelot S, Bordenave-Juchereau S (2009) Crude goat whey fermentation by Kluyveromyces marxianus and Lactobacillus rhamnosus: contribution to proteolysis and ACE inhibitory activity. J Dairy Res 76:152–157CrossRefGoogle Scholar
  37. 37.
    Ramírez-Zavala B, Mercado-Flores Y, Hernández-Rodríguez C, Villa-Tanaca L (2004) Purification and characterization of a serine carboxypeptidase from Kluyveromyces marxianus. Int J Food Microbiol 91:245–252CrossRefGoogle Scholar
  38. 38.
    Mahajan SS, Goddik L, Qian MC (2004) Aroma compounds in sweet whey powder. J Dairy Sci 87:4057–4063CrossRefGoogle Scholar
  39. 39.
    Karagül-Yüceer Y, Drake MA, Cadwallader KR (2003) Aroma-active components of liquid cheddar whey. J Food Sci 68:1215CrossRefGoogle Scholar
  40. 40.
    Boender LGM, De Hulster EAF, Van Maris AJA, Daran-Lapujade PAS, Pronk JT (2009) Quantitative physiology of Saccharomyces cerevisiae at near-zero specific growth rates. Appl Environ Microbiol 75:5607–5614CrossRefGoogle Scholar
  41. 41.
    Fonseca GG, Gombert AK, Heinzle E, Wittmann C (2007) Physiology of the yeast Kluyveromyces marxianus during batch and chemostat cultures with glucose as the sole carbon source. FEMS Yeast Res 7:422–435CrossRefGoogle Scholar
  42. 42.
    Ben-Hassan RM, Ghaly AE (1995) Continuous production of single cell protein from cheese whey lactose using Kluyveromyces fragilis. Am Soc Agric Eng 38:1121–1127CrossRefGoogle Scholar
  43. 43.
    Anvari M, Khayati G (2011) Submerged yeast fermentation of cheese whey for protein production and nutritional profile analysis. Adv J Food Sci Technol 3:122–126Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • J. S. S. Yadav
    • 1
  • J. Bezawada
    • 1
  • S. Elharche
    • 2
  • S. Yan
    • 1
  • R. D. Tyagi
    • 1
    Email author
  • R. Y. Surampalli
    • 3
  1. 1.Centre Eau, Terre et Environnement, Institut National de la Recherche ScientifiqueUniversité du QuébecQuebecCanada
  2. 2.Institut Supérieur des Hautes Études en Développement DurableRabatMorocco
  3. 3.USEPAKansas CityUSA

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