Nitrogen isotopic fractionations during nitric oxide production in an agricultural soil

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 ( 15N) of soil-emitted NO and its utility in providing mechanistic information on soil NO dynamics. However, interpretation of soil 15N-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 (15-) for NO production in denitrification, nitrification, and abiotic reactions of nitrite (NO-2 ) using a newly developed 15N-NO analysis method. A sodium nitrate (NO-3 ) containing mass-independent oxygen-17 excess (quantified by a 117O notation) and three ammonium (NHC 4 ) fertilizers spanning a 15N gradient were used in soil incubations to help illuminate the reaction complexity underlying NO yields and 15N dynamics in a heterogeneous soil environment. We found strong evidence for the prominent role of NO-2 re-oxidation under anoxic conditions in controlling the apparent 15- for NO production from NO-3 in denitrification (i.e., 49% to 60 %). These results highlight the importance of an under-recognized mechanism for the reversible enzyme NO-2 oxidoreductase to control the N isotope distribution between the denitrification products. Through a 117O-based modeling of co-occurring denitrification and NO-2 re-oxidation, the 15- for NO-2 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 NHC 4 oxidation and NO-3 consumption, with both processes having a significantly higher NO yield under O2 stress. Under both oxic and hypoxic conditions, NO production from NHC 4 oxidation proceeded with a large 15- (i.e., 55% to 84 %) possibly due to expression of multiple enzyme-level isotopic fractionations during NHC 4 oxidation to NO-2 that involves NO as either a metabolic byproduct or an obligatory intermediate for NO-2 production. Adding NO-2 to sterilized soil triggered substantial NO production, with a relatively small 15- (19 %). Applying the estimated 15- values to a previous 15N measurement of in situ soil NOx emission (NOx D NOCNO2) provided promising evidence for the potential of 15N-NO measurements in revealing NO production pathways. Based on the observational and modeling constraints obtained in this study, we suggest that simultaneous 15N-NO and 15N-N2O measurements can lead to unprecedented insights into the sources of and processes controlling NO and N2O emissions from agricultural soils.