The net accumulation and potential emission of any nitrogen intermediate is a result of the imbalance between its production and consumption rates. Nitrite, a central metabolite exchanged between AOB, NOB and DEN (Fig. 1a), always accumulated before the N2O peaks (Fig. 2). To understand the NO2− flux balance dynamics, we focused on the DNA, expressed proteins and ex situ activity ratios of NO2−-producing and -consuming guilds. At all levels (genomic, proteomic and kinetic), the DEN guild did not display notable seasonal dynamics (Figs. 4c and Supplementary Figs. 9 and 20). In contrast, the (im)balance between AOB (NO2− producer) and NOB (NO2− consumer) fluctuated during the monitored period. The ratio between the total abundances of AOB and NOB, both at the DNA and protein level, was up to threefold higher during periods of high effluent NO2− concentrations compared with the rest of the year (Fig. 5a,b). At the individual protein level, including MAG and unbinned proteins, the ratios between the expression of the key NH3-consuming enzyme (represented by the beta-subunit of the ammonia monooxygenase, AmoB) and NO2−-producing enzyme (represented by the hydroxylamine oxidoreductase, Hao) of AOB relative to the catalytic subunit of the NO2− oxidoreductase of NOB (NxrA) were also higher at high effluent NO2− concentrations (Fig. 5c and Supplementary Data 1). The ratio between the maximum NH4+ and NO2− oxidation activities was consistently higher during high NO2− concentration periods (Fig. 5d).
a,b, Ratios between the total relative abundance of DNA (a) and protein (b) of AOB and NOB (circles). The symbols represent the mean and the error bars represent the standard deviations of technical duplicates independently analysed by liquid chromatography–tandem mass spectrometry (LC–MS/MS); some error bars are smaller than the symbols. c, Ratios between the relative abundance of NO2−-producing and -consuming enzymes of AOB and NOB, respectively: AmoBAOB/NxrANOB (diamonds) and HaoAOB/NxrANOB (circles). The enzyme abundances include proteins belonging to the MAGs and unbinned fraction. The symbols in b,c represent the mean and the error bars represent the standard deviations of technical duplicates independently analysed by LC–MS/MS; some error bars are smaller than the symbols. The corresponding enzyme conversions are represented on the right. d, Ratio between the maximum ex situ NH4+ and NO2− oxidation rates (rmax) measured at 20 °C (circles). The data in a–d are overlaid on the weekly average NO2− concentration in the effluent (seven parallel lanes pooled together, grey shading, right axis). The correlation coefficients between the WWTP parameters and microbial ratios represented here and their statistical significance are reported in Supplementary Table 7.
Roothans, N., Pabst, M., van Diemen, M. et al. Long-term multi-meta-omics resolves the ecophysiological controls of seasonal N2O emissions during wastewater treatment.
Nat Water (2025).
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Roothans, N., Pabst, M., van Diemen, M. et al. Long-term multi-meta-omics resolves the ecophysiological controls of seasonal N2O emissions during wastewater treatment.
Nat Water (2025). https://doi.org/10.1038/s44221-025-00430-x
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