Two-stage anaerobic digestion process has been frequently applied to the sequential production of hydrogen and methane from various organic substrates/wastes. In this study, a cost-effective byproduct of food industry, molasses, was used as a sole carbon source for the two-stage biogas-producing process. The two-stage process consisted of two reactor parts named as the first-stage hydrogenic reactor (HR) operated at pH 5.5 and 35 degrees C and the second-stage methanogenic reactor (MR) at pH 7.0 and 35 degrees C. Microbial community analysis revealed that Clostridium butyricum was the major hydrogen-producing bacteria and methanogens consisted of hydrotrophic bacteria like Methanobacterium beijingense and acetotrophic bacteria like Methanothrix soehngenii. In the first-stage process, hydrogen could be efficiently produced from diluted molasses with the highest production rate of 2.8 (+/- 0.22) L-H(2)/L-reactor/d at the optimum HRT of 6 h. In the second-stage process, methane could be also produced from residual sugars and VFAs with a production rate of 1.48 (+/- 0.09) L-CH(4)/L-reactor/d at the optimum HRT of 6 d, at which overall COD removal efficiency of the two-stage process was determined to be 79.8%. Finally, economic assessment supported that cost-effective molasses was a potent carbon source for the sequential production of hydrogen and methane by two-stage anaerobic digestion process. (C) 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.