In this study, some key factors, for example, diluted ratio of effluents, the ratio of dark-photo bacteria, light intensity and light/dark cycle influencing hydrogen production by combining Clostridium butyricum and immobilized Rhodopseudomonas faecalis RLD-53 in batch culture, were investigated. Experimental results showed the photo-hydrogen yield decreased when increasing diluted ratio from 1:0.5 to 1:3, and it reached the maximum value of 4368 ml-H(2)/I-effluents at the ratio of 1:0.5. When the ratio of dark-photo bacteria was at 1:2, the hydrogen yield reached highest value of 4.946 mol-H(2)/mol-glucose and cumulative hydrogen volume was 5357 ml-H(2)/I-culture during the combination process. When the light intensity was at 10.25 W/m(2), the hydrogen volume of photo-fermentation and the combination process reached maximum value of 4260 ml-H(2)/I-effluents and 5892 ml-H(2)/I-culture, respectively. During the combination process, maximum total hydrogen yield was 5.374 mol-H(2)/mol-glucose. Meanwhile, hydrogen production under light/dark cycle was evaluated. (C) 2010 Elsevier Ltd. All rights reserved.