Phototrophic hydrogen production was conducted using individual substrates, acetate and butyrate, which are the main products of dark fermentation. Effects of initial pH (ranging 5.0-10.0) and individual substrate concentrations (acetate ranging from 800 to 4100mg/l, and butyrate ranging from 1000 to 5 100 mg/l) on phototrophic hydrogen production were evaluated. The maximum hydrogen yields were 2.5 mol-H-2/mol-acetate at an initial pH of 8.0 treating 800 mg/l of acetate, 3.7 mol-H-2/molbutyrate at an initial pH of 9.0 treating 1000 mg/l of butyrate. Analyses of DGGE (denaturing gradient gel electrophoresis) profiles of 16S rDNA fragments and FISH (fluorescent in situ hybridization) images show that both phototrophic hydrogen-producing sludges comprised only one predominant species resembling Rhodobacter capsulatus with over 80% relative abundance. (c) 2005 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.