Metagenomics reveals the role of micron zero-valent iron in semi-continuous anaerobic digestion of food waste: The non-negligible impact of organic loading rate

Micron zero-valent iron (mZVI) is a promising additive for enhancing anaerobic digestion, yet its role under varying operational conditions remains unclear. This study investigates how micron zero-valent iron influences semi-continuous anaerobic digestion of food waste under different organic loading rates (OLR). At low OLR (1.05 gVS/L/day), mZVI (5 g Fe/L) severely inhibits hydrolytic enzymes (amylase, protease, lipase) by 30–50 %, inhibits hydrolytic enzymes (amylase, protease, lipase), acetate kinase, coenzyme F420, and ATP synthesis, reducing abundances of hydrolytic/acidogenic microbes (Fermentimonas, Aminobacterium) and leading to a 15–20 % lower methane yield compared to the control. Conversely, under high OLR (3.38–4.22 gVS/L/day), mZVI's inhibitory effects diminish: it enhances ATP production by 60–80 %, boosts methanogens (Methanobacterium, Methanosarcina) abundances by 2–3 folds, upregulates methanogenesis-related genes (acs, frd) by 0.5–1.0 log2 fold change, and maintains a stable methane yield of 373.2 ± 24.1 mL/gVS (vs. system breakdown in the control at 4.22 gVS/L/day). The dual role of mZVI stems from competition for oxidative power: limited substrate metabolism at low OLR restricts its benefits, while robust substrate energy flow at high OLR activates mZVI's reductive capacity to aid methanogenesis. These findings highlight OLR-dependent mechanisms of mZVI in optimizing anaerobic digestion efficiency, providing insights for sustainable organic waste-to-energy strategies.