The production of biohydrogen and 5-aminolevulinic acid (5-ALA) from sugar beet molasses was investigated within a biorefinery framework. A purple non-sulfur photosynthetic bacterium, Rhodobacter sphaeroides O.U.001, was used for this purpose. The suitability of the molasses for biohydrogen and 5-ALA production was assessed in certain aspects and then five different culture media with various sugar contents (3 g/L, 7 g/L, 14 g/L, 21 g/L and 28 g/L) were prepared. Results have shown that molasses is a promising substrate for the production of biohydrogen and 5-ALA in a biorefinery concept and increasing sugar content results in enhanced product accumulation. Specifically, the highest amount of biohydrogen and 5-ALA was observed in 28 g/L sugar-containing medium (1.01 L H-2/L culture, 23,337 mu M). In conclusion, this paper presents the new findings about the enhanced accumulation of biohydrogen and 5-ALA within a biorefinery context. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.