The combination of dark- and photo-fermentative bacteria is quite a new approach in hydrogen (H-2) production biotechnology, which offers potential efficient production of H-2 from renewable resources. This study is focused on the attempt to obtain high H-2 yield by pure and mixed cultures of dark-fermentative (Escherichia coli) and photo-fermentative (Rhodobacter sphaeroides) bacteria using distillers grains (DG), the ethanol fermentation waste. During the growth in diluted DG media H-2 production by pure and mixed cultures started at 24 h growth. Moreover, the mixed culture produced significantly more H2 from DG: 2-folds diluted media provided the similar to 1.5-3-folds higher H-2 yield in comparison with pure culture. These effects were obtained for continuous H-2 production at 72-96 h growth. DG dilution and neutralization to pH 7.0 were required. The results indicated that DG can be used as effective and valuable source of organic substances in H-2 production by bacteria. This study can provide novel approach for an inexpensive energy generation, as well as to resolve the problem of waste utilization. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.