Biohydrogen production through water–gas shift (WGS) reaction by a biocatalyst was conducted in batch fermentation. The isolated photosynthetic bacterium Rhodopseudomonas palustris PT was able to utilize carbon monoxide and simultaneously produce hydrogen. Light exposure was provided as an indispensable requirement for the first stage of bacterial growth, but throughout the hydrogen production stage, the energy requirement was met through the WGS reaction. At ambient pressure and temperature, the effect of various sodium acetate concentrations in presence of CO-rich syngas on cell growth, carbon monoxide consumption, and biohydrogen production was also investigated. Maximal efficiency of hydrogen production in response to carbon monoxide consumption was recorded at 86 % and the highest concentration of hydrogen at 33.5 mmol/l was achieved with sodium acetate concentration of 1.5 g/l. The obtained results proved that the local isolate; R. palustris PT, was able to utilize CO-rich syngas and generate biohydrogen via WGS reaction.