H-2 is regarded as a promising energy resource because of its high energy content (122 kJ/g), and since capable of replacing fossil energy sources. To enhance the hydrogen production, two pure bacterial strains were jointly applied as mixed cultures in a single fermenter, thus combining the starch hydrolysis process and H-2 production process. Strain A1 was responsible for starch hydrolysis by producing amylase and strain B1 was in charge of hydrogen production. Since the starch pre-treatment conduces to starch hydrolysis and the metal ions are the important elements of hydrogenase, the hydrogen production using corn starch was moreover assessed when either pre-treating the starch feed, or by adding Fe2+ or Mg2+, possibly in association with L-cysteine. The cumulative H-2 production increased as average of triplicate experiments from 838 to 1186 mL when the corn starch was pre-treated by boiling. The H-2 production rate of pre-treated corn starch was much higher than that of untreated corn starch. The H-2 yield of pre-treated corn starch reached 1.19 mol H-2/mol glucose, 40% higher than using untreated corn starch (0.85 mol H-2/mol glucose). By adding trace elements (Fe2+ and Mg2+) and L-cysteine, the H-2 yields were higher than for the control, except when adding Mg2+. The enhancement of the H-2 production by the sole addition of Fe2+ and L-cysteine was significant (by 105% and 60%, respectively). The sole addition of Fe2+ to the system had the highest effect with a maximum cumulative H-2 production of 1928 mL and H-2 yield of 1.94 mol H2/mol glucose. (C) 2013 Elsevier Ltd. All rights reserved.