Nitrogen metabolism and microbial responses in anammox under dual stress of N,N-dimethylformamide

N,N-dimethylformamide (DMF) is a common toxic industrial solvent. It often enters with low C/N ratio and high NH₄⁺-N wastewater, posing a dual stress of organic matter and toxicity to Anammox process. However, the long-term impact mechanisms and microbial response patterns remain unclear. The effects of gradient DMF stress on the nitrogen removal performance, nitrogen transformation pathways, cellular physiological stress, microbial community succession, and functional gene expression of the Anammox system were investigated, and the system's recovery potential were evaluated. The results showed that low concentrations of DMF, acting as organic matter, enhanced the nitrogen removal performance of the Anammox system. However, DMF was not readily biodegradable by the associated heterotrophic bacteria, and its accumulation exerted a significant toxic effect on Anammox bacteria with a half-inhibition concentration (IC₅₀) of 10.26 mg/L. Low concentrations of DMF slightly stimulated exopolymer secretion and antioxidant enzyme activity; high concentrations of DMF triggered a reactive oxygen species burst, inhibited superoxide dismutase activity, disrupted cell membrane integrity, and reduced ATP content, leading to the collapse of energy metabolism. As the DMF concentration increased, the relative abundance of Candidatus_Brocadia and Candidatus_Jettenia first increased and then decreased, while the relative abundance of Candidatus_Kuenenia decreased continuously. The core genes of AnAOB (hzsA/B/C, hdh) and the autotrophic carbon fixation pathway were downregulated, whereas heterotrophic denitrification genes were upregulated, shifting the nitrogen removal pathway from autotrophic processes to heterotrophic denitrification. The denitrification performance, community structure, and functional genes of the Anammox system partially recovered after the cessation of DMF addition.