Influence of fermentation pH and substrate composition (composite chemical wastewater as primary carbon source) on molecular H-2 production was studied in batch experiments using sequentially pretreated [heat-shock (100 degrees C; 2 h) and acid (pH. 3; 24 h)] anaerobic mixed consortia as inoculum. Sequentially coupled repeated pretreatments showed positive influence on the overall H2 generation. Effective H2 production was evidenced at fermentation pH 6 (1.25 mmol H-2/g COD) compared to 5 (0.71 mmol H-2/g COD) and 7 (0.27 mmol H-2/g COD). Fermentation pH of 6.0 was found to be optimum for effective H2 generation with the pretreated inoculum. The feed consisting of only glucose as primary substrate showed low H-2 yield, while feed with chemical wastewater admixed either with glucose or sewage wastewater as co-substrates demonstrated high H-2 yield. Addition of co-substrate (glucose or sewage wastewater) along with chemical wastewater showed enhanced H-2 yield. Glucose concentration exceeding 2 g/l resulted in reduced H-2 yield. Higher VIA concentrations were recorded in experiments carried out at fermentation pH 5 than 7. At fermentation pH 6 VFA composition showed the presence of acetate, butyrate, and propionate, with relatively lower concentration of ethanol. Acid-forming pathway with acetic acid as a major metabolite dominated the metabolic flow during the H-2 production. (C) 2007 Inter-national Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.