Hydrogen producing bacteria IODB-O3 was isolated from sludge and identified as Clostridium sp. by 16S rDNA gene analysis. In this study, biohydrogen production process was developed using low-cost agro-waste. Maximum H-2 was produced at 37 degrees C and pH 8.5. Maximum H-2 yield was obtained 2.54 +/- 0.2 mol-H-2/mol-reducing sugar from wheat straw pre-hydrolysate (WSPH) and 2.61 +/- 0.1 mol/H-2/mol-reducing sugar from pre-treated wheat straw enzymatic-hydrolysate (WSEH). The cumulative H-2 production (ml/L), 3680 +/- 105 and 3270 +/- 100, H-2 production rate (ml/L/h), 153 +/- 5 and 136 +/- 5, and specific H-2 production (ml/g/h), 511 +/- 5 and 681 +/- 10 with WSPH and WSEH were obtained, respectively. Biomass pre-treatment via steam-explosion generates ample amount of WSPH which remains unutilized for bioethanol production due to non-availability of efficient C-5-fermenting microorganisms. This study shows that Clostridium sp. IODB-O3 is capable of utilizing WSPH efficiently for biohydrogen production. This would lead to reduced economic constrain on the overall cellulosic ethanol process and also establish a sustainable biohydrogen production process. (C) 2014 Elsevier Ltd. All rights reserved.