Metabolomics provides insights into the actual physiology of cells rather than their mere "potential", as provided by genomic and transcriptomic analysis. We investigate the modulation of nitrous oxide (N₂O) accumulation by intracellular metabolites in denitrifying bacteria using metabolomics and genome-based metabolic network modeling. Profiles of metabolites and their rates of production/consumption were obtained for denitrifying batch cultures under four conditions: initial COD:N ratios of 11:1 and 4:1 with and without nitrite spiking (28 mg-N L^-1). Only the nitrite-spiked cultures accumulated N₂O. The NO₂^- spiked cultures with an initial COD:N = 11:1 accumulated 3.3 ± 0.57% of the total nitrogen added as N₂O and large pools of tricarboxylic acid cycle intermediates and amino acids. In comparison, the NO₂^- spiked cultures with COD:N = 4:1 showed significantly higher (p = 0.028) N₂O accumulation (8.5.3 ± 0.9% of the total nitrogen added), which was linked to the depletion of C11-C20 fatty acids. Metabolic modeling analysis shows that at COD:N of 4:1 the denitrifying cells slowly generate electron equivalents as FADH₂ through β-oxidation of saturated fatty acids, while COD:N of 11:1 do it through the TCA cycle. When combined with NO₂^- shock, this prolonged the duration over which insufficient electron equivalents were available to completely reduce NOx to N₂, resulting in increased N₂O accumulation. Results extend the understanding of how organic carbon and nitrite loads modulate N₂O accumulation in denitrification, which may contribute to further design strategies to control greenhouse gas emissions from agricultural soils or wastewater treatment systems.

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