Mixed carbon sources fermentation by bacteria is a promising approach for biohydrogen (H-2) production biotechnology. In the present study, growth and H-2 production by purple bacteria Rhodobacter sphaeroides MDC6521 during mixed carbon sources (succinate + acetate, succinate + malate, and malate + acetate) photo-fermentation was investigated. The growth rate of bacteria in mixed carbon sources containing medium was of similar to 0.33 h(-1) which was considerably higher (1.3-1.7-fold) compared with sole carbon substrate containing one. Moreover, the H-2 production during photo-fermentation of succinate and acetate mixture was of similar to 6.5 mmol H(2)g(-1) (dry weight of biomass) and significantly more (similar to 2-3-fold) than that with appropriate sole sources and higher (1.5-fold) than that with succinate and malate mixture. Probably, supplementation of the mixed carbon sources into bacterial culture alters the mode of metabolism, resulting in enhanced H-2 production, thus they can be preferable compared to the sole carbon source. The changed FOF1-ATPase activity of membrane vesicles suggested its important role in the increase of H-2 production efficiency. The results showed that mixed carbon sources provide more H-2 than the sole carbon substrates and succinate with acetate mixture is better than succinate with malate. (C) 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.