A biorefinery concept for the production of fuel ethanol, probiotic yeast, and whey protein from a by-product of the cheese industry


Agroindustrial by-products and residues can be transformed into valuable compounds in biorefineries. Here, we present a new concept: production of fuel ethanol, whey protein, and probiotic yeast from cheese whey. An initial screening under industrially relevant conditions, involving thirty Kluyveromyces marxianus strains, was carried out using spot assays to evaluate their capacity to grow on cheese whey or on whey permeate (100 g lactose/L), under aerobic or anaerobic conditions, in the absence or presence of 5% ethanol, at pH 5.8 or pH 2.5. The four best growing K. marxianus strains were selected and further evaluated in a miniaturized industrial fermentation process using reconstituted whey permeate (100 g lactose/L) with cell recycling (involving sulfuric acid treatment). After five consecutive fermentation cycles, the ethanol yield on sugar reached 90% of the theoretical maximum in the best cases, with 90% cell viability. Cells harvested at this point displayed probiotic properties such as the capacity to survive the passage through the gastrointestinal tract and capacity to modulate the innate immune response of intestinal epithelium, both in vitro. Furthermore, the CIDCA 9121 strain was able to protect against histopathological damage in an animal model of acute colitis. Our findings demonstrate that K. marxianus CIDCA 9121 is capable of efficiently fermenting the lactose present in whey permeate to ethanol and that the remaining yeast biomass has probiotic properties, enabling an integrated process for the obtainment of whey protein (WP), fuel ethanol, and probiotics from cheese whey.

Key points

• K. marxianus–selected strains ferment whey permeate with 90% ethanol yield.

• Industrial fermentation conditions do not affect selected yeast probiotic capacity.

• Whey permeate, fuel ethanol, and probiotic biomass can be obtained in a biorefinery.

Graphical abstract

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MDP is a fellow of the Argentina National Research Council (CONICET); DER, MR, and GLG are members of the Scientific Career of CONICET. AKG and JVM-Jr would also like to acknowledge Dr. Guilherme M. Tavares and Dr. Miriam D. Hubinger for helpful discussions.

Availability of data and material

Raw data of all experiments is registered in hardcopy in the participant laboratories and is available for revision if required

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This study was funded by the FAPESP-CONICET scientific cooperation grants, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de La Plata (UNLP), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, PICT 2014-3137), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant numbers 2015/14109-0 and 2016/50444-0), and Alexander von Humboldt Stiftung for partially funding the research via a Return fellowship to Dr. Romanin. JVM-Jr received a scholarship from FAPESP (grant number 2015/26072-3).

Author information




MDP and JVM-Jr performed experimental work and participated in the study design and manuscript writing. DER performed animal assays and participated in data analysis and manuscript writing. MR, AKG, and GLG participated in study design and conception, funding, and manuscript writing.

Corresponding author

Correspondence to Graciela L. Garrote.

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The experimental protocol of animal assays performed in the article was approved by the Animal Ethics Committee of Faculty of Exact Sciences, National University of La Plata, Argentina (Approval No 011-01-15). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.

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Pendón, M.D., Madeira, J.V., Romanin, D.E. et al. A biorefinery concept for the production of fuel ethanol, probiotic yeast, and whey protein from a by-product of the cheese industry. Appl Microbiol Biotechnol 105, 3859–3871 (2021). https://doi.org/10.1007/s00253-021-11278-y

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  • Kluyveromyces
  • Whey
  • Bioethanol
  • Probiotics
  • Biorefinery