Efficient growth of Kluyveromyces marxianus biomass used as a biocatalyst in the sustainable production of ethyl acetate

Background Whey is just turning from a waste of milk processing to a renewable raw material in biotechnology for producing single-cell protein, bio-ethanol, or ethyl acetate as an economic alternative. Conversion of whey-borne sugar into ethyl acetate requires yeast biomass as a biocatalyst. A high cell concentration results in a quick ester synthesis, but biomass growth means consumption of sugar at the expense of ester production. Efficient and cost-saving biomass production is thus a practical requirement. Whey is poor in nitrogen and has therefore to be supplemented with a bioavailable N source. Methods Several aerobic growth tests were performed with Kluyveromyces marxianus DSM 5422 as a potent producer of ethyl acetate in whey-borne media supplemented with various N sources. Preliminary tests were done in shake flasks while detailed studies were performed in a stirred bioreactor. Results Ammonium sulfate resulted in strong acidification due to remaining sulfate, but costly pH control increases the salt load, being inhibitory to yeasts and causing environmental impacts. Ammonium carbonate lessened acidification, but its supplement increased the initial pH to 7.5 and delayed growth. Urea as an alternative N source was easily assimilated by the studied yeast and avoided strong acidification (much less base was required for pH control). Urea was assimilated intracellularly rather than hydrolyzed extracellularly by urease. Conversion of urea to ammonium and usage of formed ammonium for biomass production occurred with a similar rate so that the amount of excreted ammonium was small. Ammonium hydroxide as another N source was successfully added by the pH controller during the growth of K. marxianus DSM 5422, but the medium had to be supplemented with some ammonium sulfate to avoid sulfur limitation and to initiate acidification. Non-limited growth resulted in 82 mg N per g of biomass, but N-limited growth diminished the N content. Conclusions K. marxianus could be efficiently produced by supplementing the whey with nitrogen. Urea and ammonia were the favored N sources due to the proton neutrality at assimilation which lessened the salt load and reduced the supply of alkali for pH control or made this even needless.