Nitrogen-removal collapse and recovery: Integrative meta-omics insights into seasonal microbiome dynamics and cold-adaptive mechanisms in a full-scale activated sludge system.

The underlying mechanisms by which seasonal cooling impairs nitrogen removal in ambient-temperature bioreactors remain largely unresolved. Here, we monitored a full-scale livestock wastewater activated sludge (LWAS) system throughout the summer-winter transition, integrating meta-omics analyses to link process performance with microbial community dynamics and intracellular functional adaptation. Nitrogen-removal rate fell from averaged 92.7 g N/m3/d in summer to zero in autumn, then partially recovered in winter. Concurrently, the microbial Shannon diversity of nitrogen-metabolizing communities declined in autumn and rebounded in winter. Seasonal cooling increased influent immigration and compressed the niche space of nitrogen-metabolizing guilds. Denitrification remained functionally robust, with Pseudomonas and Corynebacterium supplanted summer-dominant Thauera and Thiobacillus. In contrast, ammonia-oxidizing bacteria (AOB) were strongly inhibited: both species abundance and ammonia monooxygenase activity declined in autumn. During winter, Nitrosomonas eutropha_B LWAS_194 dominated and maintained ammonium oxidation at 4°C by up-regulating genes for post-translational modification, energy generation, cryoprotectant production, membrane fluidity, and antioxidant defense. These findings highlight the cold-induced inhibition of AOB as the key limitation for nitrogen removal. Strategies including AOB-specific carriers, bioaugmentation with cold-adapted AOBs and thermal insulation offer practical solutions. This multiscale microbiome-engineering framework is broadly applicable to wastewater and ecological systems experiencing seasonal cold stress.