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 5100mg/l) on phototrophic hydrogen production were evaluated. The maximum hydrogen yields were 2.5mol-H 2 /mol-acetate at an initial pH of 8.0 treating 800mg/l of acetate, 3.7mol-H 2 /mol-butyrate at an initial pH of 9.0 treating 1000mg/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.