The effects of hydraulic retention time (HRT) and organic loading rate (OLR) on hydrogen production were investigated with glucose medium containing 2% NaCl. Halophilic hydrogen producing bacterium (HHPB) Clostridium bifermentans 3AT-ma, which can survive under high salt conditions, was used. Sponge media were used as 20% of working volume. The OLR and HRT were varied in 10-60 g-glucose/L-reactor/day and 24-6 h. With OLR of 20 g-glucose/L/day, shorter HRT resulted in higher hydrogen producing rate and yield. When the OLR was increased from 20 to 60 g-glucose/L-reactor/day at HRT 6 h, the hydrogen production rate increased, while the hydrogen production yield decreased due to the increase and accumulation of volatile fatty acids. Biohydrogen production was possible from the salinity substrate using HHPB, and the maximum hydrogen production yield was 1.1 mol-H-2/mol-glucose with optimal conditions of OLR of 20 g-glucose/L/day and HRT of 12 h. (C) 2012 Elsevier Ltd. All rights reserved.