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.

Quantitative PCR (qPCR) is broadly used as the gold standard to quantify microbial community fractions in environmental microbiology and biotechnology. Benchmarking efforts to ensure the comparability of qPCR data for environmental bioprocesses are still scarce. Also, for partial nitritation/anammox (PN/A) systems systematic investigations are still missing, rendering meta-analysis of reported trends and generic insights potentially precarious. We report a baseline investigation of the variability of qPCR-based analyses for microbial communities applied to PN/A systems. Round-robin testing was performed for three PN/A biomass samples in six laboratories, using the respective in-house DNA extraction and qPCR protocols. The concentration of extracted DNA was significantly different between labs, ranged between 2.7 and 328 ng mg⁻¹ wet biomass. The variability among the qPCR abundance data of different labs was very high (1−7 log fold) but differed for different target microbial guilds. DNA extraction caused maximum variation (3–7 log fold), followed by the primers (1–3 log fold). These insights will guide environmental scientists and engineers as well as treatment plant operators in the interpretation of qPCR data.