Development of a semi-continuous two-stage simultaneous saccharification and fermentation process for enhanced 2,3-butanediol production by Klebsiella oxytoca

Abstract: Klebsiella oxytoca naturally produces a large amount of 2,3-butanediol (2,3-BD), a promising chemical with wide industrial applications, along with various by-products. Previously, we have developed a metabolically engineered K. oxytoca ΔldhA ΔpflB strain to reduce the formation of by-products. To improve 2,3-BD productivity and examine the stability of K. oxytoca ΔldhA ΔpflB strain for industrial application, a semi-continuous two-stage simultaneous saccharification and fermentation (STSSF) process was developed. The STSSF with the K. oxytoca ΔldhA ΔpflB mutant using cassava as a carbon source could produce 108 ± 3·73 g_{(2,3- BD )} l⁻¹ with a yield of 0·45 g_{(2,3- BD )} g_(glucose) ⁻¹ and a productivity of 3·00 g_{(2,3- BD )}l⁻¹ h⁻¹. No apparent changes in the final titre, yield and productivity of 2,3-BD were observed for up to 20 cycles of STSSF. Also, microbial contamination and spontaneous mutation of the host strain with potential detrimental effects on fermentation efficiency did not occur during the whole fermentation period. These results strongly underpin that the K. oxytoca ΔldhA ΔpflB mutant is stable and that the STSSF process is commercially exploitable. Significance and Impact of the Study: There is growing interest in the production of 2,3-butanediol (2,3-BD) from renewable resources by microbial fermentation because of its wide applications to specialty and commodity chemical industries. Klebsiella oxytoca usually produces 2,3-BD as a major end product during the fermentation of carbohydrates. This is the first study to provide a high-efficiency simultaneous saccharification and 2,3-BD fermentation process. Also, this study proves the stability of a metabolically engineered 2,3-BD overproducing K. oxytoca strain for industrial application.