Crude glycerol (CG) is the main by-product (waste) of biodiesel production (by transesterification of lipids) and by weight it could be as high as 10% (w/w) of the final product. Considering global interest for biodiesel production, there is a need for sustainable management of the waste. In this context, bio-hydrogen production by utilization of CG could be an interesting option. Moreover, hydrogen is considered as the cleanest renewable alternative of fossil fuels. However, CG contains a few impurities, such as methanol, NaCl and soap and their individual and interactive effect on hydrogen production should be investigated. Thus, present study deals with the investigation of the effect of methanol, NaCl and soap on hydrogen production and substrate (glycerol) utilization. Based on the investigation carried out using response surface methodology, it was observed that soap is the most significant factor (p value = 0.0104 < 0.05) to inhibit hydrogen production. Similarly, methanol was also found to have negative effect on hydrogen production in the concentration range (1.3-3.3 g/L) considered. Meanwhile, NaCl (0.15-0.55 g/L) showed positive effect on the process. Overall, presence of different impurities was found to reduce the cumulative H-2 production by 21.56-95.31%. Furthermore, based on present observations a novel integrated strategy for soap removal and process pH management has been hypothesized for improved H2 production by CG bioconversion. (C) 2013 Elsevier Ltd. All rights reserved.