Rhodopseudomonas palustris P4 can produce H-2 either from CO by water-gas shift reaction or from various sugars by anaerobic fermentation. Fermentative H-2 production by P4 is fast, but its yield is relatively low due to the formation of various organic acids. In order to increase H-2 production yield from glucose, P4 was investigated for the photo-fermentation of acetate which is a major by-product of fermentative H-2 production. Experiments were performed in batch modes using both light-grown and dark-grown cells. When the dark-grown P4 was challenged with light and acetate, H-2 was produced with the consumption of acetate after a lag period of 25 h. H-2 production was inhibited when a nitrogen source, especially ammonium, is present. When the dark-fermentation broth containing acetate was adopted for photo-fermentation with light-grown cells, H-2 production and concomitant acetate consumption occurred without a lag period. The H-2 yield was estimated as 2.4-2.8 mol H-2/mol acetate and the specific H-2 production rate was as 9.8 ml H-2/g cell h. The fact that a single strain can perform both dark- and light-fermentation gives a great advantage in process development. Compared to a one-step dark-fermentation, the combined dark- and light-fermentation can increase the H-2 production yield on glucose by two-fold. (C) 2003 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.