The effects of Fe and Ni nanoparticles (NPs) versus Fe2+ and Ni2+ ions on the efficiency of mesophilic dark hydrogen fermentation from glucose were investigated using the heat shock pretreated anaerobic sludge. The initial glucose concentration and the pH of the reactors were 5 g/L and 5.5, respectively, and the concentrations of the supplemented metals were varied between 0 and 50 mg/L. The hydrogen and biogas yields of the control test were 247 and 391 mL/g VS, respectively. The results showed that the effect of Ni2+ ions on the hydrogen yield was highly significant (P < 0.01), improving the hydrogen yield by 55%. The effects of Fe-0 NPs and Fe2+ ions were significant too (P < 0.05), enhancing the hydrogen yield by 37% and 15%, respectively, whereas the effect of Ni-0 NPs was insignificant (P > 0.05). The analysis of the soluble metabolites revealed that the hydrogen fermentative yield was mainly affected by ethanol and propionate production, while the production of butyrate favored the hydrogen fermentation. (C) Copyright 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.