Clostridium butyricum and Rhodopseudomonas faecalis RLD-53 were employed to produce hydrogen in mixed culture with glucose as sole substrate. Due to the great difference on growth rate and acid-resistant capacity between photo-fermentative bacteria and dark-fermentative bacteria, directly mixed culture of the two kinds of bacteria in different ratio was studied in this work. Hydrogen yield, volatile acids, pH and biomass in different periods were evaluated. Acetic acid and butyric acid produced by C. butyricum were dominant terminal fermentation products, and they were effective substrates for photo-fermentative bacteria. The cooperation was formed in a way like food chain. But compared to the production rate of volatile acids produced by C. butyricum, the utilization rate by photo-fermentative bacteria was far slower. The results demonstrated that the growth of photo-fermentative bacteria was limited when pH decreased sharply. The best ratio of C. butyricum to R. faecalis RLD-53 was 1:600. The maximum yield of hydrogen reached 122.4 ml-H(2)/vessel and hydrogen production rate was 0.5 ml-H(2)/ml-culture/day. (C) 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.