The effect of the iron concentration on hydrogen yield was investigated in batch tests by heat-shocked mixed cultures growing on a sucrose mineral medium at 35 ° C. The hydrogen production yield increased obviously with increasing iron concentration from 0 to 1600 mg FeSO4 l(-1), and then slightly decreased when the iron concentration increased from 1600 to 5000 mg FeSO4 l(-1). The maximum amount of hydrogen production (2.73 mol/mol sucrose) was obtained at the iron concentration of 1600 mg FeSO4 l(-1). The hydrogen production yield and the butyric acid/acetic acid ratio followed a similar trend, suggesting that the formation of butyrate seems to favor hydrogen production. © 2004 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.