Abstract Hydrogen gas production by photo-fermentation of dark fermentation effluent of acid hydrolyzed wheat starch was investigated at different hydraulic residence times (HRT = 1–10 days). Pure Rhodobacter sphaeroides (NRRL B-1727) culture was used in continuous photo-fermentation by periodic feeding and effluent removal. The highest daily hydrogen gas production (85 ml d −1) was obtained at HRT = 4 days (96 h) while the highest hydrogen yield (1200 ml H 2 g −1 TVFA) was realized at HRT = 196 h. Specific and volumetric hydrogen formation rates were also the highest at HRT = 96 h. Steady-state biomass concentrations and biomass yields increased with increasing HRT. TVFA loading rates of 0.32 g L −1 d −1 and 0.51 g L −1 d −1 resulted in the highest hydrogen yield and formation rate, respectively. Hydrogen gas yield obtained in this study compares favorably with the relevant literature reports probably due to operation by periodic feeding and effluent removal.