Photo-fermentative H-2 production by mixed bacteria and pure bacterium Rhodopseudomonas faecalis RLD-53 using single and mixed substrate as carbon source was investigated in batch culture. Experimental results showed that 60 mmol/L acetate was the optimal concentration for mixed bacterial H-2 production and maximum cumulative H-2 volume was 2468 +/- 123 mL H-2/L-culture. It was also found that propionate or butyrate was a key factor for enhancing H-2 production in mixed substrate system. Photo-H-2 production can be greatly promoted when proper concentration of propionate and butyrate were added into acetate medium as mixed substrate and a higher H-2 yield of 2931 +/- 146 mL H-2/L-culture was obtained. In addition, it was worth noting that when the strain RLD-53 was added into mixed bacteria with different concentration ratios, H-2 yield did not yet increase. Interestingly, H-2 production capacity gradually decreased with ratio of strain RLD-53 to mixed bacteria from 8:0 to 4:4, and then gradually increased from 4:4 to 0:8. This implied that the competition relationship between strain RLD-53 and mixed bacteria in substrate utilization strongly influenced their H-2 production. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.