Oehlenschlager_2025_Biotechnol.Biofuels.Bioprod_18_98

The rising demand for natural products is accelerating research into sustainable methods for producing bio-based flavourings like ethyl butyrate. In this study, ethyl butyrate was successfully produced through the enzymatic esterification of butyric acid and ethanol, which were derived from the co-cultivation of Clostridium tyrobutyricum and Saccharomyces cerevisiae. Initial monoculture experiments with both strains were performed to investigate compromised fermentation conditions for co-cultivation. Based on these findings, anaerobic co-cultivation conditions were established at 37 degreesC and 150 rpm, with the pH controlled at 6. The effects of varying inoculation times in co-culture were examined, considering the solvent and acid tolerance of both strains. Due to the limited acid tolerance of S. cerevisiae, with significant inhibition at butyric acid concentrations above 10 g L(1), a time-delayed inoculation with C. tyrobutyricum was implemented. In batch experiments, the final concentrations of butyric acid and ethanol were 13.98+/-3.06 g L(1) and 21.43+/-1.66 g L(1), respectively. Further enhancement of product concentrations was explored through a fed-batch cultivation strategy yielding up to 45.62+/-3.82 g L(1) of butyric acid and 18.61+/-4.11 g L(1) of ethanol. Ethyl butyrate was formed from the fermentation products by lipase-catalysed enzymatic esterification in a two-phase system through the addition of an organic phase. The ester concentration in the organic phase reached a maximum of 23.93+/-0.68 g L(1) (esterification yield 25%). This study presents a viable approach to the production of bio-based ethyl butyrate offering a sustainable alternative to traditional chemical synthesis methods.