Solid-phase electron donor-dependent redox regulation reprograms metabolism and community assembly in anammox systems

Anaerobic ammonium oxidation (anammox) is a promising nitrogen removal technology but remains constrained by limited operational robustness under environmental perturbations. Here, a solid-phase redox regulation strategy was proposed by coupling hydrogen peroxide (H2O2) with two distinct electron donors, inorganic ferrous sulfide (FeS) and organic lignite, to enhance system stability under continuous-flow conditions. The results show that upon the introduction of redox pairs, both systems achieved high nitrogen removal efficiency, maintaining ammonia and nitrite removal above 96.3% even under a 50% increase in nitrogen loading. Moreover, the specific anammox activity significantly increased by 3.27- (FeS-based system) and 2.87-fold (lignite-based system), respectively. Multi-omics analyses revealed that exogenous redox regulation reinforced electron transport and ATP synthesis, while reprogramming carbon metabolism toward the Wood-Ljungdahl pathway without disrupting the core anammox pathway. Notably, distinct donor-dependent effects were observed. The FeS-based system promoted direct inorganic redox cycling, leading to enhanced energy turnover and the formation of a functionally complementary multi-genus anammox consortium. In contrast, the lignite-based system facilitated co-enrichment of anammox bacteria and denitrifiers, indicating different ecological interaction modes. Both systems activated antioxidant defense mechanisms in response to controlled oxidative perturbation, indicating adaptive redox homeostasis. Overall, this study demonstrates that electron donor properties govern redox-mediated metabolic and community reorganization, providing new insights into the design of robust anammox systems.