This study evaluated the possibility of using sugarcane vinasse for biological hydrogen production under non-sterile conditions via sequential two-stage fermentative and phototrophic processes, which simultaneously allows bioenergy production and chemical oxygen demand (COD) removal. The fermentative process was performed in an anaerobic fluidized bed reactor (AFBR) fed with a mixture of sucrose and sugarcane vinasse (5:10 g COD L-1). The phototrophic process was performed using a microbial consortium (Rhodopseudomonas related microorganisms) as inoculum and batch reactors fed with different concentrations of the AFBR effluent (10, 20, 40, 70 and 80%, v/v). The hydrogen yield (HY) (0.34 mol H-2 g(-1) CODinfluent) observed in the fermentative step was justified by the establishment of microbial community composed predominantly by Lactobacillus related microorganisms. The highest HY observed in the phototrophic stage (5.5 mmol H-2 g(-1) CODremoved) was observed for the lowest AFBR concentration. Increasing the AFBR effluent concentration as the substrate for the phototrophic reactors had a negative impact on cell growth, hydrogen production and COD removal mostly due to the occurrence of sulfate reduction. Hydrogen production was not observed in batch reactors fed with 70 and 80% of AFBR effluent; however, under those conditions, the sulfate removal rate was 86.5 and 86.9%, respectively. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.