Abstract:[Background] Bio-butanol is a powerful liquid fuel. The products of butanol fermentation (acetone-butanol-ethanol fermentation, ABE fermentation) are the mixture of butanol, acetone and ethanol, weight ratio of the primary product—butanol over the major by-product—acetone (B/A ratio) is about 2.0. [Objective] Butyric acid is an important precursor for butanol synthesis in ABE fermentation. Using butyric acid/glucose as co-substrate would efficiently produce butanol featured with high B/A ratio and improve the quality of ABE fermentation products. [Methods] In a 7-L anaerobic fermentor, the supernatant originated from butyrate fermentation (BFS) with corn starch/waste Pichia pastoris as the raw materials was directly mixed with glucose solution as the ABE fermentation medium, then BFS and concentrated glucose solution were fed during the fermentation as required. [Results] Compared with traditional ABE fermentation using 150 g/L corn, butanol concentration could be maintained at an equivalently high level of 12.7?12.8 g/L, B/A ratio largely increased from 2.0 to 4.4?5.0, butanol yield to total carbon source also increased from 0.32?0.34 to 0.39?0.41 (mole base), butyrate/glucose consumption ratio reached high levels between 37% and 53%. The ABE fermentation benefited from the efficient utilization of butyrate, oligosaccharides, amino acids, etc. in the BFS and the enhanced NADH utilization efficiency. [Conclusion] The proposed fermentation strategy could save raw materials and operating costs of ABE fermentations, largely reduce the residual oligosaccharides concentrations in BFS, increase diversity and flexibility of the fermentation processes based on variations of marketing requirement and products prices, which have reasonably high economic and environmental effects.