The optimum values of hydraulic retention time (HRT) and organic loading rate (OLR) of an anaerobic sequencing batch reactor (ASBR) for biohydrogen production from palm oil mill effluent (POME) under thermophilic conditions (60 degrees C) were investigated in order to achieve the maximum process stability. Microbial community structure dynamics in the ASBR was studied by denaturing gradient gel electrophoresis (DGGE) aiming at improved insight into the hydrogen fermentation microorganisms. The optimum values of 2-d HRT with an OLR of 60 gCOD l(-1) d(-1) gave a maximum hydrogen yield of 0.27 l H(2) gCOD(-1) with a volumetric hydrogen production rate of 9.1 l H(2) l(-1) d(-1) (16.9 mmol l(-1) h(-1)). The hydrogen content, total carbohydrate consumption, COD (chemical oxygen demand) removal and suspended solids removal were 55 +/- 3.5%, 92 +/- 3%, 57 +/- 2.5% and 78 +/- 2%, respectively. Acetic acid and butyric acid were the major soluble end-products. The microbial community structure was strongly dependent on the HRT and OLR. DGGE profiling illustrated that Thermoanaerobacterium spp., such as Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium bryantii, were dominant and probably played an important role in hydrogen production under the optimum conditions. The shift in the microbial community from a dominance of T. thermosaccharolyticum to a community where also Caloramator proteoclasticus constituted a major component occurred at suboptimal HRT (1 d) and OLR (80 gCOD l(-1) d(-1)) conditions. The results showed that the hydrogen production performance was closely correlated with the bacterial community structure. This is the first report of a successful ASBR operation achieving a high hydrogen production rate from real wastewater (POME). (C) 2009 Published by Elsevier Ltd on behalf of international Association for Hydrogen Energy.