Endogenous reactive iron sulfide triggers oxidative toxicity and activity decay in intermittently starved anammox consortia.

Iron is an essential element for anammox metabolism, but its role in activity decay is poorly understood. This study elucidates a "Trojan Horse" mechanism that endogenous iron becomes a catalyst for self-inflicted oxidative stress under redox oscillations. Three anammox consortia under full-load (RF), persistent light-load (RP), and intermittent starvation-refeeding (RI) regimes were systematically compared by integrating C-N-S-Fe cycling, biochemical and multi-omics analyses. Results showed that intermittent starvation initiated extensive cell lysis and the subsequent release of organic matter, iron and sulfur-containing compounds. These products fueled heterotrophic growth and established a strongly reducing environment that promoted sulfide production. Meanwhile, the Fe(II) fraction in sludge increased from less than 32% to more than 90%, facilitating the formation of an FeS layer containing 16.1% of surface iron. Upon refeeding, this FeS layer and associated Fe(II) induced a threefold intracellular ROS surge and severe lipid peroxidation through Fenton-like reactions, which directly resulted in 63% decline in specific anammox activity compared with RP consortia. This work identifies the endogenous iron-sulfur cycle as a new and critical control point for process stability, which provides a scientific basis for developing innovative engineering strategies.