Anammox Community Following Synergistic Inhibition by Low Temperature and BPA (Metagenomic)
Anaerobic ammonium oxidation (anammox) offers an energy-efficient route for nitrogen removal, yet its tolerance to organic micropollutants can be disproportionately eroded by cooling. How temperature decline propagates through the physical, regulatory, and ecological layers of this cooperative consortium to amplify xenobiotic toxicity remains unresolved. Here, we operated three continuous-flow anammox biofilm reactors at 35 C, 25 C, and 15 C and challenged them with stepwise bisphenol A (BPA) concentrations (0.2-20 mg/L) to determine how cooling reshapes BPA tolerance across structural, regulatory, and ecological scales. Cooling sharply narrowed the tolerance boundary. The half-maximal inhibitory concentration (IC50) decreased from 25.05 mg/L at 35 C to 5.89 mg/L at 25 C, and at 15 C, a trace dose of 0.2 mg/L caused near-complete functional loss. Confocal imaging revealed cold-driven EPS densification and depth-dependent viability loss consistent with intensified diffusion limitation. Quorum sensing signals, transcription of core anammox genes (hzsB, hzo), and activities of hydrazine synthase and hydrazine oxidoreductase declined in a coordinated cascade, linking physical constraints to regulatory shutdown. Metagenome-resolved analyses showed that core nitrogen cycle genes persisted under cooling, but low temperature constrained enrichment of heterotrophic partners encoding xenobiotic degradation and cofactor biosynthesis pathways, severing the metabolic cross-feeding that buffers anammox bacteria against BPA. Together, these findings reveal a cross-scale vulnerability cascade in which cooling does not simply slow metabolism but dismantles the cooperative partnerships that underpin xenobiotic tolerance in anammox biofilms.