Acetone-butanol-ethanol (ABE) fermentation process is a promising bioenergy option amid rising concerns over the environmental impact of fossil fuel usage. However, the commercialization of the ABE process has been marred by challenges of low product yield and titer, thereby non-competitive process economics. Here, we coupled low-cost reducing agents with a controlled feeding strategy to improve both product titer and yield. Reducing agents promote cofactor dependent butanol production while fed-batch operation enhances glucose consumption, final ABE titer, and partly mitigates product toxicity. We investigated the effects of ascorbic acid, L-cysteine, and dithiothreitol (DTT) on fed-batch ABE production using Clostridium acetobutylicum. Moreover, to study the metabolic modifications triggered by these reducing agents, we performed NADH, ATP, extracellular amino acid secretion, and NADH- dependent butanol dehydrogenase (BDH) assays. L-cysteine and DTT improved ABE solvent titer by 2-fold, producing 24.33 and 22.98 g/L ABE with solvent yields of 0.38 and 0.37 g/g, respectively. NADH, BDH, and ATP levels increased significantly which also reflected in elevated ABE titer and yield. Furthermore, histidine secretion emerged as an important factor in Clostridial acid stress in this study. The results demonstrate that reducing agents and the fed-batch combination enables efficient utilization of glucose and remarkably enhances ABE production.

2020