The present study explores the potentiality of different pretreatment to develop a suitable acidogenic culture for biohydrogen production. Experiments were design to enumerate variation in hydrogen production pattern with glucose using" different pretreated inoculum. Among all pretreatment strategies, heat shock pretreatment showed significant improvement of H-2 production. The process was validated using modified Gompertz model (R-2 > 0.99). Substrate conversion efficiency using heat shock pretreated inoculum was high (85.8%) as compared to other acidogenic cultures. Further statistical methodology was considered for optimization of process parameters to maximize the hydrogen production using heat shock pretreated inoculum. Under optimum physico-chemical parameters (6.5 pH, 37 degrees C, 1 g/L YE and 26 C/N ratio), maximum cumulative H-2 production and H-2 yield of 3220 mL/L and 2.6 mol H-2/mol glucose, respectively were observed. Taguchi design of experiment was used to further understand the degree of influence imparted by the above factors on H-2 production. The C/N ratio was found to be most influential parameter among the other factors. The PCR-DGGE analysis showed the dominance of Clostridium sp. in the mixed culture. Maximum gaseous energy recovered using heat shock pretreated acidogenic culture was found to be higher (26.21%) as compared to others. (C) 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.