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. Nitric oxide (NO) emissions from agricultural soils play a critical role in atmospheric chemistry and represent an important pathway for loss of reactive nitrogen (N) to the environment. With recent methodological advances, there is growing interest in the natural abundance N isotopic composition (δ15N) of soil-emitted NO and its utility in providing mechanistic information on soil NO dynamics. However, interpretation of soil δ15N-NO measurements has been impeded by the lack of constraints on the isotopic fractionations associated with NO production and consumption in relevant microbial and chemical reactions. In this study, anoxic (0 % O2), oxic (20 % O2), and hypoxic (0.5 % O2) incubations of an agricultural soil were conducted to quantify the net N isotope effects (15η) for NO production in denitrification, nitrification, and abiotic reactions of nitrite (NO2−) using a newly developed δ15N-NO analysis method. A sodium nitrate (NO3−) containing mass-independent oxygen-17 excess (quantified by a Δ17O notation) and three ammonium (NH4+) fertilizers spanning a δ15N gradient were used in soil incubations to help illuminate the reaction complexity underlying NO yields and δ15N dynamics in a heterogeneous soil environment. We found strong evidence for the prominent role of NO2− oxidation under anoxic conditions in controlling the apparent 15η for NO production from NO3− in denitrification (i.e., 49 to 60 ‰). These results highlight the importance of an under-recognized mechanism for the reversible enzyme NO2− oxidoreductase to control the N isotope distribution between the denitrification products. Through a Δ17O-based modeling of co-occurring denitrification and NO2− re-oxidation, the 15η for NO2− reduction to NO and NO reduction to nitrous oxide (N2O) were constrained to be 15 to 22 ‰ and −8 to 2 ‰, respectively. Production of NO in the oxic and hypoxic incubations was contributed by both NH4+ oxidation and NO3− consumption, with both processes having a significantly higher NO yield under O2 stress. Under both oxic and hypoxic conditions, NO production from NH4+ oxidation proceeded with a large 15η (i.e., 55 to 84 ‰) possibly due to expression of multiple enzyme-level isotopic fractionations during NH4+ oxidation to NO2− that involves NO as either a metabolic byproduct or an obligatory intermediate for NO2− production. Adding NO2− to sterilized soil triggered substantial NO production, with a relatively small 15η (19 ‰). Applying the estimated 15η values to a previous δ15N measurement of in situ soil NOx emission (NOx = NO + NO2) provided promising evidence for the potential of δ15N-NO measurements in revealing NO production pathways. Based on the observational and modeling constraints obtained in this study, we suggest that simultaneous δ15N-NO and δ15N-N2O measurements can lead to unprecedented insights into the sources of and processes controlling NO and N2O emissions from agricultural soils.

Nitrogen isotopic fractionations during nitric oxide production in an agricultural soil