Mixing method and hydraulic retention time (HRT) have been shown to affect the photo-fermentation hydrogen production process. In this study, different bioreactors with diverse mixing methods, batch and continuous modes, and a range of HRTs were investigated to determine the optimum operating conditions. The pH value, cell optical density (OD660), reducing sugar concentration, and cumulative hydrogen yield were monitored during experiments. Experimental results show that magnetic-stirring had negative impacts on hydrogen production due to the OD660 value, substrate conversion efficiency, and cumulative hydrogen yield were all less than half of the static bioreactor. The baffle photo-fermentative bioreactor had the highest hydrogen production rate of 7.37 mmol L-1 h(-1) with the highest cumulative hydrogen yield of 512.29 mmol Consequently, it was selected as the preferred bioreactor for continuous hydrogen production. In addition, we found the digestion stability increased with an increase in HRT using the baffle photo-fermentative bioreactor. However, the highest cumulative hydrogen yield of 589.21 mmol L-1 and satisfactory substrate conversion efficiency of 40.48% occurred at an HRT of 24 h. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.