Nitrous oxide reductases (N2OR) are the sole sink of the potent greenhouse gas nitrous oxide (N₂O) in the environment. Having been studied for decades, N2OR have attracted renewed attention following the discovery of a previously unrecognized clade, now termed clade II. This clade exhibits unexpectedly widespread taxonomic distribution and prevalence across diverse environments, prompting research efforts to define and assign distinct clade-specific traits. In this perspective, we aim to critically review and evaluate dichotomous clade-based classifications, addressing oversimplifications and unresolved ambiguities in linking clade identity to physiological traits like substrate affinity, acid tolerance, and aerotolerance. Growing experimental evidence from N₂O-reducing isolates and enrichments suggests a general difference in substrate affinity between the clades. Recent discoveries of N₂O reduction at pH < 5.0 attribute the long-sought acidophilic N₂O reduction exclusively to organisms possessing clade II nosZ, and attempts have also been made to relate clade separation to aerotolerant N₂O reduction. However, it is important to note that such binary characterizations are based on limited observations and lack a solid understanding of the underlying mechanisms, exposing them to bias and oversimplification risks. We emphasize the need for a balanced research effort to establish a robust link between ecophysiology and biochemistry, enabling a more accurate evaluation of clade-based characterizations and, ultimately, a deeper understanding and effective harnessing of N₂O-reducing organisms.

Wastewater treatment plants (WWTPs) are a major source of N₂O, a potent greenhouse gas with 300 times higher global warming potential than CO₂. Several approaches have been proposed for mitigation of N₂O emissions from WWTPs and have shown promising yet only site-specific results. Here, self-sustaining biotrickling filtration, an end-of-the-pipe treatment technology, was tested in situ at a full-scale WWTP under realistic operational conditions. Temporally varying untreated wastewater was used as trickling medium, and no temperature control was applied. The off-gas from the covered WWTP aerated section was conveyed through the pilot-scale reactor, and an average removal efficiency of 57.9 ± 29.1% was achieved during 165 days of operation despite the generally low and largely fluctuating influent N₂O concentrations (ranging between 4.8 and 96.4 ppmv). For the following 60-day period, the continuously operated reactor system removed 43.0 ± 21.2% of the periodically augmented N₂O, exhibiting elimination capacities as high as 5.25 g N₂O m^-3·h^-1. Additionally, the bench-scale experiments performed abreast corroborated the resilience of the system to short-term N₂O starvations. Our results corroborate the feasibility of biotrickling filtration for mitigating N₂O emitted from WWTPs and demonstrate its robustness toward suboptimal field operating conditions and N₂O starvation, as also supported by analyses of the microbial compositions and nosZ gene profiles.

Microorganisms possessing N₂O reductases (NosZ) are the only known environmental sink of N₂O. While oxygen inhibition of NosZ activity is widely known, environments where N₂O reduction occurs are often not devoid of O₂. However, little is known regarding N₂O reduction in microoxic systems. Here, 1.6-L chemostat cultures inoculated with activated sludge samples were sustained for ca. 100 days with low concentration (<2 ppmv) and feed rate (<1.44 µmoles h⁻¹) of N₂O, and the resulting microbial consortia were analyzed via quantitative PCR (qPCR) and metagenomic/metatranscriptomic analyses. Unintended but quantified intrusion of O₂ sustained dissolved oxygen concentration above 4 µM; however, complete N₂O reduction of influent N₂O persisted throughout incubation. Metagenomic investigations indicated that the microbiomes were dominated by an uncultured taxon affiliated to Burkholderiales, and, along with the qPCR results, suggested coexistence of clade I and II N₂O reducers. Contrastingly, metatranscriptomic nosZ pools were dominated by the Dechloromonas-like nosZ subclade, suggesting the importance of the microorganisms possessing this nosZ subclade in reduction of trace N₂O. Further, co-expression of nosZ and ccoNO/cydAB genes found in the metagenome-assembled genomes representing these putative N₂O-reducers implies a survival strategy to maximize utilization of scarcely available electron acceptors in microoxic environmental niches.