Integrated co-cultivation and subsequent esterification: Harnessing Saccharomyces cerevisiae and Clostridium tyrobutyricum for streamlined ester production.

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 °C 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¯, 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¯ and 21.43 ± 1.66 g L¯, respectively. Further enhancement of product concentrations was explored through a fed-batch cultivation strategy yielding up to 45.62 ± 3.82 g L¯ of butyric acid and 18.61 ± 4.11 g L¯ 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¯ (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.