Metabolic flux and transcriptional analysis elucidate higher butanol/acetone ratio feature in ABE extractive fermentation by Clostridium acetobutylicum using cassava substrate

Abstract

In acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum ATCC 824 using corn-based substrate, the solvents are generally produced at a ratio of 3:6:1 (A:B:E, w/w). A higher butanol/acetone ratio of 2.9:1 was found when cassava was used as the substrate of an in-situ extractive fermentation by C. acetobutylicum. This ratio had a 64% increment compared to that on corn-based substrate. The metabolic flux and (key enzymes) genes transcriptional analysis indicated that weakened metabolic fluxes in organic acids (especially butyrate) formation and re-assimilation pathways, which associated with lower buk and ctfAB transcriptional levels, contributed to higher butanol and lower acetone production rate in fermentations on cassava. Moreover, NADH generation was enhanced under the enriched reductive environment of using cassava-based substrate, which converted more carbon flux to butanol synthesis pathway, also leading to a higher ratio of butanol/acetone. To further increase butanol/acetone ratio, tiny amount of electron carrier, neutral red was supplemented into cassava-based substrate at 60 h when butonal production rate reached maximal level. However, neutral red addition enhanced NADH production, followed with strengthening the metabolic fluxes of organic acids formation/re-assimilation pathways, resulted in unchanged in butanol/acetone ratio. A further increase in butanol/acetone ratio could be realized when NADH regeneration was enhanced and the metabolic fluxes in organic acids formation/reutilization routes were controlled at suitably low levels simultaneously.