Metabolic analysis of butanol production from acetate in Clostridium saccharoperbutylacetonicum N1-4 using ¹³C tracer experiments

During acetone-butanol-ethanol (ABE) fermentation by clostridia, acetate is reutilised for butanol production. In this study, we investigated the characteristics of ABE production from acetate and analysed the metabolism of exogenously added acetate by Clostridium saccharoperbutylacetonicum N1-4. Supplementation of 4 g L^-1 exogenous acetate, to media containing glucose, increased not only concentrations of butanol (48.3%) and acetone (90.5%), but also the ratio of acetone to butanol (27.1%), which suggested that acetate addition altered the metabolic flux. Acetate could not be metabolised in the absence of glucose, thus glycolysis appeared to be necessary for acetate utilisation. In order to clarify the metabolism of exogenous acetate, ¹³C tracer experiments were performed by supplementing [1,2-¹³C₂] acetate in a culture broth. Based on the results of gas chromatography-mass spectroscopy analysis, we first confirmed both butanol and acetone formation from acetate. Further, the acetate-to-butanol efficiency will significantly decrease when more acetate than 2-4 g L^-1 is added to the fermentation, while acetate-to-acetone efficiency may remain high (up to a ratio of 2 mol acetate per 1 mol glucose fed). Moreover, the culture supplemented with acetate exhibited an increase in conversion efficiency of glucose to butanol and acetone, from 0.196% to 19.5% and from 0 to 7.64%, respectively, even during acidogenesis. Thus, we first revealed quantitatively that acetate addition induced solvent production during the early growth phase, and increased metabolic flux to acetone and butanol production from both acetate and glucose.