Microorganisms encoding for the N2O reductase (NosZ) are the only known biological sink of the potent greenhouse gas N2O and are central to global N2O mitigation efforts. Clade II NosZ populations are of particular biotechnological interest as they usually feature high N2O affinities and often lack other denitrification genes. We focus on the yet-unresolved ecological constraints selecting for different N2O-reducers strains and controlling the assembly of N2O-respiring communities. Two planktonic N2O-respiring mixed cultures were enriched at low dilution rates under limiting and excess dissolved N2O availability to assess the impact of substrate affinity and N2O cytotoxicity, respectively. Genome-resolved metaproteomics was used to infer the metabolism of the enriched populations. Under N2O limitation, clade II N2O-reducers fully outcompeted clade I affiliates, a scenario previously only theorized based on pure-cultures. All enriched N2O-reducers encoded and expressed the sole clade II NosZ, while also possessing other denitrification genes. Two Azonexus and Thauera genera affiliates dominated the culture, and we hypothesize their coexistence to be explained by the genome-inferred metabolic exchange of cobalamin intermediates. Under excess N2O, clade I and II populations coexisted; yet, proteomic evidence suggests that clade II affiliates respired most of the N2O, de facto outcompeting clade I affiliates. The single dominant N2O-reducer (genus Azonexus) notably expressed most cobalamin biosynthesis marker genes, likely to contrast the continuous cobalamin inactivation by dissolved cytotoxic N2O concentrations (400 μM). Ultimately, our results strongly suggest the solids dilution rate to play a pivotal role in controlling the selection among NosZ clades, albeit the conditions selecting for genomes possessing the sole nosZ remain elusive. We furthermore highlight the potential significance of N2O-cobalamin interactions in shaping the composition of N2O-respiring microbiomes.

Nitrous oxide (N2O) is the third most important greenhouse gas and originates primarily from natural and engineered microbiomes. Effective emission mitigations are currently hindered by the largely unresolved ecophysiological controls of coexisting N2O-converting metabolisms in complex communities. To address this, we used biological wastewater treatment as a model ecosystem and combined long-term metagenome-resolved metaproteomics with ex situ kinetic and full-scale operational characterization over nearly 2 years. By leveraging the evidence independently obtained at multiple ecophysiological levels, from individual genetic potential to actual metabolism and emergent community phenotype, the cascade of environmental and operational triggers driving seasonal N2O emissions has ultimately been resolved. We identified nitrifier denitrification as the dominant N2O-producing pathway and dissolved O2 as the prime operational parameter, paving the way to the design and fostering of robust emission control strategies. This work exemplifies the untapped potential of multi-meta-omics in the mechanistic understanding and ecological engineering of microbiomes towards reducing anthropogenic impacts and advancing sustainable biotechnological developments.

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.

Microbial communities drive the nitrogen cycle, a fundamental process sustaining life on Earth. However, human activities have disrupted this balance, leading to excessive emissions of nitrous oxide (N2O), a potent greenhouse gas with nearly 300 times the global warming potential of carbon dioxide and a significant contributor to ozone layer depletion. Despite the urgency to reduce emissions, they are projected to increase by 50% in the next 50 years. Our ability to mitigate these emissions is limited by our incomplete understanding of the microbial complexity driving them. To develop effective mitigation strategies, we must determine how microbial communities regulate nitrogen transformations across diverse environments, from natural ecosystems such as soils and oceans to managed and engineered systems like agricultural soils and wastewater treatment plants (WWTPs).

By integrating genomic, proteomic, and metabolic insights, this thesis explores the complexity of microbial nitrogen cycling and N2O 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.

Nitrous oxide (N2O) is a potent greenhouse gas of primarily microbial origin. Aerobic and anoxic emissions are commonly ascribed to nitrification and denitrification, respectively. Beyond this established dichotomy, we quantitatively prove that heterotrophic denitrification can significantly contribute to aerobic nitrogen turnover and N2O emissions in complex microbiomes exposed to frequent oxic/anoxic transitions. Planktonic, nitrification-inhibited denitrifying enrichments respired over a third of the influent organic substrate with nitrate at high oxygen concentrations. N2O accounted for up to one quarter of the aerobically respired nitrate. The constitutive detection of all denitrification enzymes in both anoxic and oxic periods highlight the selective advantage offered by metabolic preparedness in dynamic environments. We posit that aerobic denitrification and associated N2O formation is currently underestimated in dynamic microbial ecosystems.