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.

Iron (Fe²⁺), manganese (Mn²⁺), and ammonium (NH₄⁺) are the three most common contaminants in anaerobic groundwater and are typically removed in rapid sand filters in a series of simultaneous, uncontrolled, and interconnected redox reactions. In this study, we demonstrated separation of these oxidation processes, including reversing the order of NH₄⁺and Mn²⁺oxidation, allowing Mn²⁺to oxidize before NH₄⁺. To achieve this uncommon sequence, the filter was operated with low O₂ concentrations (∼0.02 mmol/L, ∼0.5 mg/L) and a high pH (∼8). Under these conditions, Mn²⁺ oxidation is consuming all available O₂, suppressing the occurrence of NH₄⁺oxidation. In the filter with low O₂ (0.08 mmol/L, ∼3 mg/L) and low pH (∼6.8), the opposite was observed, as Mn²⁺ oxidation was delayed under these conditions, resulting in complete O₂ consumption by NH₄⁺-oxidizing bacteria. Reactive transport modelling and parameter estimation revealed that Mn²⁺ oxidation is one order of magnitude faster in absence of NH₄⁺ oxidation (1.4 × 10⁻² vs 2.5 × 10⁻³ mmol/L), whereas NH₄⁺ oxidation seemed to be accelerated by simultaneous Mn²⁺ oxidation (6.8 × 10⁻³ vs 2.9 × 10⁻² s⁻¹). This interconnection between Mn²⁺ and NH₄⁺ oxidation was further emphasized by the observation of Mn²⁺ release in the presence of NO₂⁻. In conclusion, this study has shown that a shift from conventional aerated groundwater treatment to sequential oxidation in separate filters offers (i) a more controllable system, (ii) the potential to optimize the rates of each oxidation process separately, which would ultimately result in higher flows and less backwashing.

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.

Division of metabolic labour is a defining trait of natural and engineered microbiomes. Denitrification-the stepwise reduction of nitrate and nitrite to nitrogenous gases-is inherently modular, catalysed either by a single microorganism (termed complete denitrifier) or by consortia of partial denitrifiers. Despite the pivotal role of denitrification in biogeochemical cycles and environmental biotechnologies, the ecological factors selecting for complete versus partial denitrifiers remain poorly understood. In this perspective, we critically review over 1500 published metagenome-assembled genomes of denitrifiers from diverse and globally relevant ecosystems. Our findings highlight the widespread occurrence of labour division and the dominance of partial denitrifiers in complex ecosystems, contrasting with the prevalence of complete denitrifiers only in simple laboratory cultures. We challenge current labour division theories centred around catabolic pathways, and discuss their limits in explaining the observed niche partitioning. Instead, we propose that labour division benefits partial denitrifiers by minimising resource allocation to denitrification, enabling broader metabolic adaptability to oligotrophic and dynamic environments. Conversely, stable, nutrient-rich laboratory cultures seem to favour complete denitrifiers, which maximise energy generation through denitrification. To resolve the ecological significance of metabolic trade-offs in denitrifying microbiomes, we advocate for mechanistic studies that integrate mixed-culture enrichments mimicking natural environments, multi-meta-omics, and targeted physiological characterisations. These undertakings will greatly advance our understanding of global nitrogen turnover and nitrogenous greenhouse gases emissions.

Nitrous oxide (N₂O) is a potent greenhouse gas of primarily microbial origin. Oxic and anoxic emissions are commonly ascribed to autotrophic nitrification and heterotrophic denitrification, respectively. Beyond this established dichotomy, we quantitatively show that heterotrophic denitrification can significantly contribute to aerobic nitrogen turnover and N₂O emissions in complex microbiomes exposed to frequent oxic/anoxic transitions. Two planktonic, nitrification-inhibited enrichment cultures were established under continuous organic carbon and nitrate feeding, and cyclic oxygen availability. Over a third of the influent organic substrate was respired with nitrate as electron acceptor at high oxygen concentrations (>6.5 mg/L). N₂O accounted for up to one-quarter of the nitrate reduced under oxic conditions. The enriched microorganisms maintained a constitutive abundance of denitrifying enzymes due to the oxic/anoxic frequencies exceeding their protein turnover—a common scenario in natural and engineered ecosystems. The aerobic denitrification rates are ascribed primarily to the residual activity of anaerobically synthesised enzymes. From an ecological perspective, the selection of organisms capable of sustaining significant denitrifying activity during aeration shows their competitive advantage over other heterotrophs under varying oxygen availabilities. Ultimately, we propose that the contribution of heterotrophic denitrification to aerobic nitrogen turnover and N₂O emissions is currently underestimated in dynamic environments.

Rapid sand filters (RSF) are an established and widely applied technology for the removal of dissolved iron (Fe²⁺) and ammonium (NH₄⁺) among other contaminants in groundwater treatment. Most often, biological NH₄⁺oxidation is spatially delayed and starts only upon complete Fe²⁺ depletion. However, the mechanism(s) responsible for the inhibition of NH₄⁺oxidation by Fe²⁺ or its oxidation (by)products remains elusive, hindering further process control and optimization. We used batch assays, lab-scale columns, and full-scale filter characterizations to resolve the individual impact of the main Fe²⁺ oxidizing mechanisms and the resulting products on biological NH₄⁺ oxidation. modeling of the obtained datasets allowed to quantitatively assess the hydraulic implications of Fe²⁺ oxidation. Dissolved Fe²⁺ and the reactive oxygen species formed as byproducts during Fe²⁺ oxidation had no direct effect on ammonia oxidation. The Fe³⁺ oxides on the sand grain coating, commonly assumed to be the main cause for inhibited ammonia oxidation, seemed instead to enhance it. modeling allowed to exclude mass transfer limitations induced by accumulation of iron flocs and consequent filter clogging as the cause for delayed ammonia oxidation. We unequivocally identify the inhibition of NH₄⁺oxidizing organisms by the Fe³⁺ flocs generated during Fe²⁺ oxidation as the main cause for the commonly observed spatial delay in ammonia oxidation. The addition of Fe³⁺ flocs inhibited NH₄⁺oxidation both in batch and column tests, and the removal of Fe³⁺ flocs by backwashing completely re-established the NH₄⁺removal capacity, suggesting that the inhibition is reversible. In conclusion, our findings not only identify the iron form that causes the inhibition, albeit the biological mechanism remains to be identified, but also highlight the ecological importance of iron cycling in nitrifying environments.

Rapid sand filters (RSF) are an established and widely applied technology for groundwater treatment. Yet, the underlying interwoven biological and physical-chemical reactions controlling the sequential removal of iron, ammonia and manganese remain poorly understood. To resolve the contribution and interactions between the individual reactions, we studied two full-scale drinking water treatment plant configurations, namely (i) one dual-media (anthracite and quartz sand) filter and (ii) two single-media (quartz sand) filters in series. In situ and ex situ activity tests were combined with mineral coating characterization and metagenome-guided metaproteomics along the depth of each filter. Both plants exhibited comparable performances and process compartmentalization, with most of ammonium and manganese removal occurring only after complete iron depletion. The homogeneity of the media coating and genome-based microbial composition within each compartment highlighted the effect of backwashing, namely the complete vertical mixing of the filter media. In stark contrast to this homogeneity, the removal of the contaminants was strongly stratified within each compartment, and decreased along the filter height. This apparent and longstanding conflict was resolved by quantifying the expressed proteome at different filter heights, revealing a consistent stratification of proteins catalysing ammonia oxidation and protein-based relative abundances of nitrifying genera (up to 2 orders of magnitude difference between top and bottom samples). This implies that microorganisms adapt their protein pool to the available nutrient load at a faster rate than the backwash mixing frequency. Ultimately, these results show the unique and complementary potential of metaproteomics to understand metabolic adaptations and interactions in highly dynamic ecosystems.

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.

The application of partial nitritation-anammox (PN/A) under mainstream conditions can enable substantial cost savings at wastewater treatment plants (WWTPs), but how process conditions and cell physiology affect anammox performance at psychrophilic temperatures below 15 °C remains poorly understood. We tested 14 anammox communities, including 8 from globally-installed PN/A processes, for (i) specific activity at 10–30 °C, (ii) composition of membrane lipids, and (iii) microbial community structure. We observed that membrane composition and cultivation temperature were closely related to the activity of anammox biomasses. The size of ladderane lipids and the content of bacteriohopanoids were key physiological components related to anammox performance at low temperatures. We also indicate that the adaptation of mesophilic cultures to psychrophilic regime necessitates months, but in some cases can take up to 5 years. Interestingly, biomass enriched in the marine genus “Candidatus Scalindua” displayed outstanding potential for nitrogen removal from cold streams. Collectively, our comprehensive study provides essential knowledge of cold adaptation mechanism, will enable more accurate modelling and suggests highly promising target anammox genera for inoculation and set-up of anammox reactors, in particular for mainstream WWTPs.