The effect of substrate concentration on hydrogen production was investigated using a continuous-flow stirred-tank reactor (CSTR). Sucrose was used as a model substrate. The CSTR was started at a sucrose concentration of 30 g COD/L and exhibited stable H-2 production for 271 days at inlet sucrose concentrations of 10-60 g COD/L. Hydrogen production depended on the substrate concentration such that the highest values of 1.09 mol H-2/mol hexose(added), 1.22 mol H-2/mol hexose(consumed), 7.65 L H2L/d, and 3.80 L H-2/g VSS/d were recorded at a sucrose concentration of 30 g COD/L. All bacterial species detected by polymerase chain reaction-denaturing gradient gel electrophoresis analysis were H-producing Clostridium spp. At inlet sucrose concentrations below 20 g COD/L, the H-2 yield per hexose(consumed) decreased along with a significant decrease in the n-butyrate/acetate ratio. At the same range of sucrose concentrations, Clostridium scatologenes (an H-2-consuming acetogen) was found in the sludge. At inlet sucrose concentrations over 35 g COD/L, substrate overload occurred and caused a decrease in the carbohydrate degradation efficiency and H-2 yield per hexose(added). (C) 2005 Elsevier Ltd. All rights reserved.