The synergistic effects and optimization of pH, carbon-to-nitrogen ratio (C/N), and light intensity (I) on the photo-fermentative hydrogen production of Rhodobacter sphaeroides 158 DSM and light conversion efficiency have been investigated under different conditions of pH (6.5-8); C/N (15-35); and light intensity (35-185 W m(-2)). Response surface methodology (RSM) and Box-Behnken experimental design (BBD) were used to identify the optimum values of the three key parameters of pH, C/N, and I, based on the impact on hydrogen production potential (HPP), hydrogen production rate (HPR), and light conversion efficiency eta. With desirability value of 0.91, the optimum values of 7.4, 27.5, and 126 W m(-2) were identified for pH, C/N, and I respectively, with HPP, HPR and eta reaching 960 mL L-1, 41.74 mL L-1 h(-1), and 0.31 respectively. Regression analysis indicated a good fit between experimental and model data. The study showed that both C/N ratio and I have crucial and significant effect on the HPP, HPR and eta, followed by pH, the synergistic effect of pH-I and C/N-I on the light conversion efficiency (eta) was significant while pH -C/N was insignificant. The results and analysis obtained could be very useful for better optimizing the photo-fermentative hydrogen production. (C) 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.