Biohydrogen production by sequential operation of dark and photo-fermentation processes is a promising method to produce hydrogen from renewable resources, in a sustainable way. In this study, barley straw hydrolysate (BSEI) dark fermenter effluent (DFE) was used as the biomass feedstock for biohydrogen production through photofermentation. Two different dark fermentation effluents were obtained by performing fermentation with or without addition of yeast extract (YE), using hyperthermophilic dark fermentative bacteria, Caldicellusiruptor saccharolyticus. Photofermentative hydrogen production on BSH DFEs was carried out using purple non-sulfur (PNS) bacterial strains Rhodobacter capsulatus DSM1710 and Rhodobacter capsulatus YO3 (an uptake hydrogenase deleted MT1131 strain). The effects of YE addition during dark fermentation and Fe/Mo supplementation to DFEs on subsequent photofermentation were investigated. Presence of YE in DFEs did not influence the photofermentative hydrogen production. Fe/Mo supplementation of the effluents improved the H-2 production on both DFEs, regardless of the strain used. The highest productivity of 0.58 mmol/(L-c.h) was achieved by R. capsulatus YO3 on Fe/Mo supplemented BSH DFE that had no YE. Biohydrogen production on BSH DFE was achieved at a high rate and yield, showing that it can be used as a feedstock for photofermentative hydrogen production. (C) 2013 Elsevier Ltd. All rights reserved.