This work sought to optimize fermentation parameters in a batch process for hydrogen production from molasses by a newly isolated Clostridium butyricum W5. Hydrogen yield and production rate, bacterial biomass and volatile fatty acids, including acetic, lactic and butyric acids, were measured. Key fermentation operation parameters, including concentration of carbon and nitrogen sources, growth temperature and pH, and inoculum size were investigated. The best results in terms of hydrogen yield and productivity were obtained under the conditions of 100 g/L molasses, 1.2 g/L NH4NO3, 39 degrees C at pH 6.5 with initial cell concentration of 9 x 10(4) cell/ml. Maximum hydrogen yield was 1.85 mol hydrogen/mol hexose, corresponding to a hydrogen production rate of 17.38 mmol/h/L Experimental data showed that the acetic butyric acid ratio remained relatively stable with an increase in molasses concentration, while the unfavoured product, lactic acid, portion increased. No solvent (ethanol, butanol and acetone) was detected during the fermentation. Propionic acid was measured at a very low level in the hydrogen fermentation. Statistical analysis showed that hydrogen yield increased exponentially with the increase in cell growth, and that there was no correlation between the hydrogen yield and ratio of acetic acid to butyric acid. (C) 2008, The Society for Biotechnology, Japan. All rights reserved.