Adding butyrate significantly enhanced butanol production from glycerol with Clostridium pasteurianum CH4, which predominantly produces butyrate (instead of butanol) when grown on glucose. Hence, the butyrate produced from assimilating glucose can be used to stimulate butanol production from glycerol under dual-substrate cultivation with glucose and glycerol. This proposed butanol production process was conducted by employing sequential or simultaneous addition of the two substrates. The latter approach exhibited better carbon source utilization and butanol production efficiencies. Under the optimal glucose to glycerol ratio (20 g L-1 to 60 g L-1), the simultaneous dual-substrate strategy obtained maximum butanol titer, productivity and yield of 13.3 g L-1, 0.28 g L-1 h(-1), and 0.38 mol butanol/mol glycerol, respectively. Moreover, bagasse and crude glycerol as dual-substrates were also converted into butanol efficiently with a maximum butanol concentration, productivity and yield of 11.8 g L-1, 0.14 g L-1 h(-1), and 0.33 mol butanol/mol glycerol, respectively. (C) 2012 Elsevier Ltd. All rights reserved.