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 (NO) 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). Only the nitrite-spiked cultures accumulated NO. The NO spiked cultures with an initial COD:N = 11:1 accumulated 3.3 ± 0.57% of the total nitrogen added as NO and large pools of tricarboxylic acid cycle intermediates and amino acids. In comparison, the NO spiked cultures with COD:N = 4:1 showed significantly higher (p = 0.028) NO 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 FADH through β-oxidation of saturated fatty acids, while COD:N of 11:1 do it through the TCA cycle. When combined with NO shock, this prolonged the duration over which insufficient electron equivalents were available to completely reduce NOx to N, resulting in increased NO accumulation. Results extend the understanding of how organic carbon and nitrite loads modulate NO 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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