Wastewater treatment plants (WWTPs) exhibit marked seasonality in N₂O emissions. This study aimed at investigating whether the temperature response of the wastewater nitrifying community contributes to this seasonality. NH₄⁺ oxidation and N₂O production rates were determined with indigenous activated sludge in the laboratory at the water temperatures measured in situ at the time of sampling (14.5–27.5 °C) under nonlimiting O₂ availability (>5 mg L^–1 throughout all incubations). The N₂O yield, which ranged between 0.004 and 0.028 mol N₂O–N/mol NH₄⁺, exhibited a significant negative correlation (ρ = −0.53, p = 0.0015) with temperature. Interestingly, N₂O–N yield was also positively correlated with mixed-liquor suspended solid (MLSS) concentration (ρ = 0.41, p = 0.017), a parameter upheld in winter to sustain nitrification rates. This biomass effect was substantiated by subsequent experiments in which N₂O yields of activated sludge (MLSS: 2343 ± 39 and 3760 ± 93 mg L^–1) were significantly higher (1.6- to 1.9-fold) than their 2-fold dilutions, regardless of temperature. Higher NH₂OH levels detected in denser activated sludge during nitrification (peak concentration of 0.25 ± 0.13 μM versus 0.09 ± 0.01 μM of the 2-fold dilution) suggested NH₂OH accumulation as a possible mechanistic explanation. These observations suggest that the higher design MLSS for winter performance may contribute to an increase in N₂O emissions from nitrogen removal WWTPs.

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.