ActivelyN₂O‑Reducing Oxygen-TolerantMicrobial Consortium Attained by Using a High-Dilution-Rate ChemostatFed with Methanol

Nitrous oxide-reducing bacteria (N₂ORB) are generally considered the only biological sink for the potent greenhouse gas N₂O. Although N₂O consumption activities by diverse heterotrophic N₂ORB have been detected, knowledge gaps remain about the phylogenies, physiologies, and activities of N₂ORB. Here, we successfully enriched a methylotrophic N₂ORB consortium under intermittent oxygen and N₂O supplies. ¹⁵N tracer analysis showed that the N₂O consumption activity of the enriched consortium was higher than its N₂O production activity in the presence of either a single or multiple electron acceptors (i.e., nitrogen oxides). The observed maximum N₂O consumption was 80.7 μmol·g-biomass^–1·h^–1. Quantitative PCR results showed that clade I <i>nosZ</i> bacteria overwhelmed clade II <i>nosZ</i> bacteria at high (0.41 mmol·min^–1) and low (0.08 mmol·min^–1) N₂O loading rates. The dilution rate and N₂O loading rate affected the microbial community composition and activity. A higher N₂O loading rate stimulated active and oxygen-tolerant N₂ORB that boosted N₂O consumption by approximately 50% in the presence of oxygen. Metagenomic analysis unraveled the predominance of a novel methylotrophic N₂ORB, possessing entire denitrifying genes and high-affinity terminal oxidase genes, from the reactor with a high N₂O loading rate. The unique physiological traits of the consortium enriched by methanol shed light on a novel functionaerobic N₂O consumption by N₂ORBand pave the way for innovative N₂O mitigation strategies applying powerful N₂O sinks in engineered systems.