This study investigates the role of Zero-Valent Iron (ZVI) in anaerobic digestion, emphasizing the critical influence of bicarbonate and feeding regimes. Abiotic tests revealed that higher bicarbonate concentrations enhanced hydrogen production. However, two-cycle experiments with ZVI and bicarbonate showed reduced hydrogen in the second cycle due to ZVI passivation. A similar phenomenon was observed when anaerobic granular sludge (AGS) was introduced into the system. Glucose at 4, 8, and 16 g/L was tested with AGS, with ZVI-containing samples achieving superior methane production, exceeding 95 % methane and CO