Fermentative H(2) production was operated at different hydraulic retention time (HRT = 4-12 h) using a continuously stirred tank reactor (CSTR) containing suspended H(2)-producing sludge. The H(2) production rate (HPR) increased with a decrease of HRT from 12 to 6 h, giving an optimal HPR of 0.61/h/l at HRT = 6 h for CSTR operation. However, when the HRT was further shortened to 4 h, both HPR and H(2) yield decreased significantly The poor H(2)-producing performance at 4 h HRT is mainly attributed to the marked decrease in biomass content in the reactor due to severe cell washout, as the biomass concentration decreased to a low level of 1.63 g VSS/l (volatile suspended solid, VSS). Bioreactor design strategies were applied to improve cell retention under a high substrate feeding rate. Silicone-immobilized cells (immobilized-cell-seeded anaerobic bioreactor (ICSAB system)) or powered activated carbon carriers (AGSB system) were added into the reactor to either maintain stable biomass concentration in the reactor or enhance biomass content by stimulating sludge granulation. Both ICSAB and agitated granular sludge bed (AGSB) showed improvement in biomass retention while operating at a HRT of 4 h. In particular, the biomass concentration in AGSB system went up to 10.3 VSS/l, leading to a drastic enhancement in H(2) producing-performance (0.971/h/l and 1.54 mol H(2)/mol glucose) Bacterial community analysis by denatured gradient gel electrophoresis (DGGE) indicates a transition in bacterial composition in CSTR under different HRT operation. Moreover, under the same HRT (4 h) the major bacterial populations in AGSB and ICSAB reactors were very different from those observed in CSTR, indicating that the performance of H(2) production seemed to be in close connection with the bacterial community structure. Several Clostridium species known as H(2) producers were also detected in the sludge samples by DGGE and 16S rDNA sequence matching, revealing the effectiveness of the H(2)-producing sludge used in this study. (c) 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.