The aim of this study was to investigate the effects of different doses (10–200 mg/L) of magnetite (FeO·Fe2O3), Fe0, Ni0, and FeCl3 on the efficiency of dark fermentative H2 production from cheese whey using Thermoanaerobacterium thermosaccharolyticum SP-H2. Energy conversion efficiency and hydrogen yield increased significantly by 22.7 % and 34.8 %, respectively, when 10 mg/L of magnetite was added, and hydrogenase activity increased by 23.6 % when 100 mg/L of Fe0 was added compared to the control (p < 0.001). Using scanning electron microscopy and energy dispersive X-ray spectroscopy, it was found that during the early exponential phase of hydrogen evolution, T. thermosaccharolyticum cells accumulated up to 11.2 times more Fe (up to 2.8 wt%) in the presence of optimal concentrations of Fe additives compared to the control. Intracellular iron accumulation and hydrogen yield were positively correlated with acetate concentration (p < 0.001), suggesting an efficient acetate pathway for dark fermentation. During the stationary growth phase, with almost no H2 production, the intracellular iron content decreased by up to 67.9 %. The phenomenon of changes in the amount of metal in cells, depending on the phase of hydrogen production, was demonstrated for the first time. The findings suggest that intracellular metals can be used in cell metabolism to produce H2, and the mechanisms behind this process need further investigation. Overall, this study provides valuable insights into the optimal forms and doses of metals for enhancing biohydrogen production, as well as possible research directions for the potential use of metals by microorganisms during additive-assisted dark fermentation of carbohydrate-rich wastes.