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Abstract

Nitric oxide (NO) and nitrous oxide (N2O) are formed during N-cycling in complex microbial communities in response to fluctuating molecular oxygen (O2) and nitrite (NO2−) concentrations. Until now, the formation of NO and N2O in microbial communities has been measured with low spatial and temporal resolution, which hampered elucidation of the turnover pathways and their regulation. In this study, we combined microsensor measurements with metabolic modeling to investigate the functional response of a complex biofilm with nitrifying and denitrifying activity to variations in O2 and NO2−. In steady state, NO and N2O formation was detected if ammonium (NH4+) was present under oxic conditions and if NO2− was present under anoxic conditions. Thus, NO and N2O are produced by ammonia-oxidizing bacteria (AOB) under oxic conditions and by heterotrophic denitrifiers under anoxic conditions. NO and N2O formation by AOB occurred at fully oxic conditions if NO2− concentrations were high. Modeling showed that steady-state NO concentrations are controlled by the affinity of NO-consuming processes to NO. Transient accumulation of NO and N2O occurred upon O2 removal from, or NO2− addition to, the medium only if NH4+ was present under oxic conditions or if NO2− was already present under anoxic conditions. This showed that AOB and heterotrophic denitrifiers need to be metabolically active to respond with instantaneous NO and N2O production upon perturbations. Transiently accumulated NO and N2O decreased rapidly after their formation, indicating a direct effect of NO on the metabolism. By fitting model results to measurements, the kinetic relationships in the model were extended with dynamic parameters to predict transient NO release from perturbed ecosystems.