Biohydrogen (H-2) production with simultaneous wastewater treatment was studied in anaerobic sequencing batch biofilm reactor (AnSBBR) using distillery wastewater as substrate at two operating pH values. Selectively enriched anaerobic mixed consortia sequentially pretreated with repeated heat-shock (100 degrees C; 2 h) and acid (pH -3.0; 24 h) methods, was used as parent inoculum to startup the bioreactor. The reactor was operated at ambient temperature (28 +/- 2 degrees C) with detention time of 24 h in periodic discontinuous batch mode. Experimental data showed the feasibility of hydrogen production along with substrate degradation with distillery wastewater as substrate. The performance of the reactor was found to be dependent on the operating pH. Adopted acidophilic microenvironment (pH 6.0) favored H-2 production (H-2 production rate-26 mmol H-2/day; specific H-2 production-6.98 mol H-2/kgCOD(R)-day) over neutral microenvironment (H-2 production rate-7 mmol H-2/day; specific H-2 production-1.63mol H-2/kgCOD(R)-day). However, COD removal efficiency was found to be effective in operated neutral microenvironment (pH 7-69.68%; pH 6.0-56.25%). The described process documented the dual benefit of renewable energy generation in the form of H-2 with simultaneous wastewater treatment utilizing it as substrate. (C) 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.