Complex multi-pathways of N₂O production across macroscopic and microscopic spatial scales in a full-scale IFAS-CANON...

Completely autotrophic nitrogen removal over nitrite (CANON) is widely implemented for treating high-ammonium wastewater. However, nitrous oxide (N₂O) emissions may offset its climate benefits, and pathway-level evidence from full-scale systems remains scarce. Here, N₂O production, transformation, and underlying pathways were investigated in a full-scale integrated fixed-film activated sludge (IFAS)-CANON system treating sludge digestion supernatant using in situ monitoring, batch tests, biofilm microelectrode profiling, and stable isotope analysis. The N₂O emission factor of the full-scale system reached 1.59 ± 0.50% of the total nitrogen removed. Despite similar microbial community structures across different locations, N₂O production and associated pathways varied spatially. Ammonia-oxidizing bacteria (AOB)-enriched activated sludge and the biofilm outer layer were identified as the main N₂O-producing zones. Driven by the complex wastewater matrix, both biotic and abiotic pathways contributed to N₂O production, with biotic processes dominating. Biotic pathways mainly included AOB denitrification and hydroxylamine (NH₂OH) oxidation, and heterotrophic denitrification also contributed due to limited biodegradable carbon and low denitrifying community diversity. For abiotic N₂O production, NH₂OH oxidation by HNO₂ was a potentially important pathway. Nitrite accumulation was the key driver across all critical pathways. Correspondingly, in the full-scale system, N₂O production was significantly higher at locations where NO₂⁻-N concentrations exceeded 25 mg/L than at downstream locations. These findings provide process-level mechanistic insights and identify key targets for reducing N₂O emissions in full-scale IFAS-CANON systems.