An investigation on biohydrogen production was conducted in a granular sludge-based continuous stirred tank reactor (CSTR). The reactor performance was assessed at five different glucose concentrations of 2.5, 5, 10, 20 and 40 g/L and four hydraulic retention times (HRTs) of 0.25, 0.5, 1 and 2h, resulting in the organic loading rates (OLRs) ranged between 2.5 and 20g-glucose/Lh. Carbon flow was traced by analyzing the composition of gaseous and soluble metabolites as well as the cell yield. Butyrate, acetate and ethanol were found to be the major soluble metabolite products in the biochemical synthesis of hydrogen. Carbon balance analysis showed that more than half of the glucose carbon was converted into unidentified soluble products at an OLR of 2.5g-glucose/Lh. It was found that high hydrogen yields corresponded to a sludge loading rate in between 0.6 and 0.8g-glucose/g-VSSh. Substantial suppression in hydrogen yield was noted as the sludge loading rate fell beyond the optimum range. It is deduced that decreasing the sludge loading rate induced the metabolic shift of biochemical reactions at an OLR of 2.5 g-glucose/L h, which resulted in a substantial reduction in hydrogen yield to 0.36-0.41 mol-H-2/mol-glucose. Optimal operation conditions for peak hydrogen yield (1.84mol-H-2/mol-glucose) and hydrogen production rate (3.26L/Lh) were achieved at an OLR of 20g-glucose/Lh, which corresponded to an HRT of 0.5h and an influent glucose concentration of 10g/L. Influence of HRT and substrate concentration on the reactor performance was interrelated and the adverse impact on hydrogen production was noted as substrate concentration was higher than 20 g/L or HRT was shorter than 0.5 h. The experimental study indicated that a higher OLR derived from appropriate HRTs and substrate concentrations was desirable for hydrogen production in such a granule-based CSTR. (c) 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.