Butanol is a promising biofuel and valuable platform chemical that can be produced through fermentative conversion of glycerol. The initial fermentation conditions for butanol production from pure glycerol by Clostridium pasteurianum DSM 525 were optimized via a central composite design. The effect of inoculum age, initial cell density, initial pH of medium and temperature were quantified and a quadratic model was able to predict butanol yield as a function of all four investigated factors. The model was confirmed through additional experiments and via analysis of variance (ANOVA). Subsequently, numerical optimization was used to maximize the butanol yield within the experimental range. Based on these results, batch fermentations in a 7 L bioreactor were performed using pure and crude (residue from biodiesel production) glycerol as substrates at optimized conditions. A butanol yield of 0.34 mole(butanol) mole(glycerol)(1) was obtained indicating the suitability of this feedstock for fermentative butanol production. (C) 2016 Elsevier Ltd. All rights reserved.