Butyl butyrate production by Clostridium beijerinckii spo0A mutant through simultaneous ABE fermentation, condensation, and extraction

A Clostridium beijerinckii spo0A mutant was constructed and characterized to investigate the phenotype of Spo0A disruption as a master regulator of the solventogenic switch. We found that the spo0A mutant accumulates both butyrate and butanol at high concentrations in a pH-controlled fermentation. Typically, the wild-type C. beijerinckii does not accumulate butyrate at a concentration greater than 2 g/L. Owing to the unique phenotype, we examined the spo0A mutant for the production of butyl butyrate that is formed by the condensation of butyrate and butanol during the ABE fermentation in the presence of lipase. Butyl butyrate is a value-added chemical that has numerous uses in the flavor and fragrance industry, Also, it is a compatible biofuel for Jet fuel, and a blending stock for biodiesel. To demonstrate butyl butyrate production by the spo0A mutant, we performed a small-scale batch fermentation in a biphasic medium containing 30 ml of P2YE medium and 30 ml of hexadecane using the lipase CalB. The spo0A mutant exhibited acid crash due to butyrate accumulation, and produced only 98 mg/L butyl butyrate in the hexadecane layer. To alleviate butyrate toxicity, the biphasic medium was supplemented with CaCO₃ buffer and butanol as the co-substrate for initiation of the condensation. Through this optimization, the butyl butyrate titer increased to 2.73 g/L. When continuous agitation was carried out, butyl butyrate production was further increased up to 3.98 g/L. In this study, we successfully demonstrated the use of an engineered strain of C. beijerinckii for product diversification of the ABE fermentation.