Low-substrate nitrogen drives functional succession toward a cooperative Candidatus Brocadia consortium in anammox...

Mainstream anammox treatment is promising but limited by slow acclimation and unstable performance under low nitrogen. Community succession is often observed, but it is usually explained by kinetic differences among anammox bacteria, which cannot fully account for competitive outcomes. Here, we operated an anammox biofilter under sustained low‑nitrogen stress and combined metagenomics, metatranscriptomics, co‑occurrence networks, and SMETANA to identify ecological adaptation mechanisms. During early acclimation, Ca. Kuenenia reduced the expression of costly biosynthetic pathways, including aromatic amino acid synthesis by 25.6%-38.8%. In contrast, Ca. Brocadia showed broad transcriptional activation, with anammox genes upregulated by ∼18-fold. During long-term operation, the community shifted to a multispecies Ca. Brocadia assemblage characterized by complementary model-inferred auxotrophies, stronger positive associations around the dominant Brocadia species (68.0%), and lower predicted metabolic resource overlap than that observed in the high-nitrogen system (0.66 ± 0.12 vs. 0.77 ± 0.10). Ultimately, Ca. Brocadia replaced Ca. Kuenenia as the dominant functional lineage, increasing from 1.0% to 44.7% in relative abundance and contributing 71.5% of total transcriptional activity. These findings suggest that low-nitrogen stress favors a metabolically complementary and potentially cooperative Ca. Brocadia assemblage rather than a single superior competitor, offering ecological guidance for stabilizing anammox processes in low-strength wastewater.