AbstractTo investigate the pathways of nitrogen (N) removal and N₂O emission in a one-stage autotrophic N removal process during the non-aeration phase, biofilm from an intermittent aeration sequencing batch biofilm reactor (SBBR) and organic carbon-free synthetic wastewater were applied to two groups of lab-scale batch experiments in anaerobic conditions using a ¹⁵N isotopic tracer and specific inhibitors, respectively. Then, the microbial composition of the biofilm was analysed using high-throughput sequencing. The results of the ¹⁵N isotopic experiments showed that anaerobic ammonium oxidation (Anammox) was the main pathway of N transformation under anaerobic conditions and was responsible for 83–92% of N₂ production within 24 h. Furthermore, experiments using specific inhibitors revealed that when nitrite was the main N source under anaerobic conditions, N₂O emissions from heterotrophic denitrification (HD) and ammonia-oxidizing bacteria (AOB) denitrification were 64% and 36%, respectively. Finally, analysing the microbial composition demonstrated that Proteobacteria, Planctomycetes, and Nitrospirae were the dominant microbes, corresponding to 21%, 13%, and 7% of the microbial community, respectively, and were probably responsible for HD, Anammox, and AOB denitrification, respectively.