Biohydrogen production processes were investigated using thermophilic bacterial consortia enriched from sludge of the anaerobic digester. A multiple parameter optimization viz. temperature, pH and substrate concentration was performed for maximization of hydrogen production. Heat shock pre-treatment followed by BES (2-bromo ethane sulfonate) treatment was done for the enrichment of hydrogen producing bacteria. Box-Behnken design and response surface methodology were adopted to investigate the mutual interaction among the process parameters. Experimental optimization of process parameters (60 degrees C, pH 6.5 and 10 g/L) gave the maximum hydrogen production and yield of 3985 mL/L and 2.7 mol/mol glucose respectively in the batch system which is higher than the reported value on UASB. These experimental parameters found concurrent with the values obtained from the theoretical model i.e. 58.4 degrees C, pH 6.6, 10.8 g/L and yield of 2.71 mol/mol glucose. At optimized conditions, maximum hydrogen production rate (R-m) of 850 mL/h, gas production potential (P) of 4551 mL/L and lag time (lambda) of 1.98 h were determined using modified Gompertz equation. Using the optimum conditions, hydrogen production from rice spent wash was conducted in which hydrogen yield of 464 mL/g carbohydrate and hydrogen production rate of 168 mL/L h were obtained. PCR-DGGE profile showed that the thermophilic mixed culture was predominated with species closely affiliated to Thermoanaerobacterium sp. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.