activated sludge metagenome Raw sequence reads

A membrane aerated biofilm reactor (MABR) was constructed and operated to investigate autotrophic nitrogen removal under low dissolved oxygen (DO) conditions without external organic carbon addition. After 120 days of continuous operation, the ammonia removal efficiency (ARE) approached 100%, and the total nitrogen removal efficiency (TNRE) stabilized at approximately 75%. Batch cycle tests indicated that a stable autotrophic nitrogen conversion pathway was gradually established in the system. Selective inhibition experiments using 1-octyne, combined with nitrogen mass balance analysis, showed that during the stable stage, the nitrite (NO2--N) required for anaerobic ammonium oxidation (Anammox) was mainly supplied by the complete ammonia oxidation (Comammox) pathway. The contribution of conventional ammonia-oxidizing bacteria (AOB) was relatively minor. Microbial community analysis revealed that Comammox Nitrospira and Candidatus Brocadia gradually became enriched in the biofilm during reactor operation, while the relative abundance of some conventional nitrifiers and heterotrophic bacteria decreased. Metagenomic analysis further detected genes related to ammonia oxidation and nitrite oxidation in Nitrospira-associated genomes, and key metabolic genes for Anammox in Brocadia-associated genomes. The reactor performance, inhibition tests, and multi-omics results collectively indicate that an autotrophic nitrogen removal pathway was established in the MABR system. In this pathway, Comammox Nitrospira produces NO2--N, which is subsequently utilized by anaerobic ammonium-oxidizing bacteria (AnAOB) for nitrogen removal. This study provides new evidence for the cooperative roles of nitrogen-cycling microorganisms in MABR systems under low DO conditions. The results also offer insights for optimizing low-carbon nitrogen removal processes in mainstream wastewater treatment.