The production of biohydrogen through dark fermentation of palm oil mill effluent (POME) was evaluated in two-stages of biohydrogen in an anaerobic sequencing batch reactor (ASBR) system using enriched mixed culture for the first time. This study attempts to examine the effect of HRT and its interaction behavior with the solid retention time (SRT), and the sugar consumption. The effluent after discharged from the thermophilic reactor contained 7.61 g/L TC and 22.87 g/L TSS was fed to the secondary mesophilic reactor system. Results indicated that the overall sugar consumption reached 88.62% at the optimum HRT of 12 h with the SRT set to 20 h. The optimum hydrogen yield and HPR in the thermophilic stage were 2.99 mol H-2/mol-sugar and 8.54 mmol H-2/L.h respectively, while for the mesophilic stage were 1.19 mol H-2/mol-sugar and 1.47 mmol H-2/L.h respectively. The overall HPR showed an improvement and increase from 8.54 mmol H-2/L.h to 10.34 mmol H-2/L.h. Microbial community analysis of mixed culture in the two-stage thermophilic (55.0 degrees C) and mesophilic (37.0 degrees C) ASBR reactor was dominated by Thermoanaerobacterium sp. based on the PCR-DGGE technique. (C) 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.