We demonstrate a two-step process used to increase the hydrogen yield from sucrose and to relieve the pollution threat caused by resultant fatty acids in dark-fermentation. In batch tests of dark-fermentation using microflora, the maximum hydrogen production rate was > 360 mL H-2/Lh and the maximum hydrogen yield was 3.67 mol H-2/mol sucrose. The fatty acids produced in dark-fermentation were mainly butyrate and acetate with a small amount of propionate, valerate, n-butyl alcohol, and caproate. Photo-fermentation with Rhodobacter sphaeroides SH2C was then employed to convert these small molecular fatty acids into hydrogen. The total hydrogen yield from sucrose increased from the maximum of 3.67 mol H-2/mol sucrose in dark-fermentation to 6.63 mol H-2/mol sucrose by using the two-step process. No butyrate, acetate, propionate, or valerate was detected in the final fermentation effluent after photo-fermentation. Our results suggest that a two-step process of combining dark- and photo-fermentation may increase hydrogen production capacity from biomass and prevent the environmental problems associated with untreated fermentation effluents. (c) 2006 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.