In this study two tasks were accomplished. First, a modified simple purification and collection lab system is described for CO2-free bio-hydrogen production. Second, the efficiency of hydrogen production was investigated by mixed dark and photo-fermentative hydrogen producing bacteria Escherichia coil, Clostridium acetobutylicum, Rhodobacter capsulatus and Rhodobacter sphaeroides from sorghum grains crude starch. The crude starch of white sorghum grains showed a higher potency as a carbon source for fermentative hydrogen production than that of red sorghum grains although both varieties are quite applicable for industrial scale hydrogen production. Using mixed R. capsulatus and R. sphaeroides showed a higher efficiency for hydrogen production than using each alone in the combined dark and photo-fermentation due to their varying efficiencies of converting various volatile fatty acids produced by dark fermentation into hydrogen gas. For enhanced hydrogen production, this study suggests using mixed strains of non-oxygenic photosynthetic purple non-sulfur bacteria in the combined dark and photo-fermentation. The high efficiency of hydrogen production from sorghum starch suggests that using this cheap crop in future industrial application of biological H-2 production would increase its economic feasibility in developing countries of arid climate where sorghum is no longer widely used as human food. (C) 2015 Elsevier Ltd. All rights reserved.