Dark fermentative biohydrogen production (DFBHP) has potential for utilization of rice starch wastewater (RSWW) as substrate. The hydrogen production of Enterobacter aerogenes MTCC 2822 and Clostridium acetobutylicum MTCC 11274, in pure culture and co-culture modes, was evaluated. The experiments were performed in a 2L bioreactor, for a batch time of 120h. The co-culture system resulted in highest cumulative hydrogen (1.13L H2/L media) and highest yield (1.67mol H2/mol glucose). Two parameters were optimized through response surface methodology (RSM)-substrate concentration (3.0-5.0g/L) and initial pH (5.5-7.5), in a three-level factorial design. A total of 11 runs were performed in duplicate, which revealed that 4.0g/L substrate concentration and 6.5 initial pH were optimal in producing hydrogen. The metabolites produced were acetic, butyric, propionic, lactic and isobutyric acids. The volumetric H2 productions, without and with pH adjustments, were 1.24L H2/L media and 1.45L H2/L media, respectively.