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 (inline-formulaδ¹⁵N) of soil-emitted NO and its utility in providing mechanistic information on soil NO dynamics. However, interpretation of soil inline-formulaδ¹⁵N-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 inline-formula% inline-formulaO₂), oxic (20 inline-formula% inline-formulaO₂), and hypoxic (0.5 inline-formula% inline-formulaO₂) incubations of an agricultural soil were conducted to quantify the net N isotope effects (inline-formula¹⁵η) for NO production in denitrification, nitrification, and abiotic reactions of nitrite (inline-formula $M10inlinescrollmathmlchem{\mathrm{normal\; NO}}_{\mathrm{normal\; 2}}^{-}$ 25pt16ptsvg-formulamathimg5f68580b6b50ed5200a71c6309703243 bg-18-805-2021-ie00001.svg25pt16ptbg-18-805-2021-ie00001.png ) using a newly developed inline-formulaδ¹⁵N-NO analysis method. A sodium nitrate (inline-formula $M12inlinescrollmathmlchem{\mathrm{normal\; NO}}_{\mathrm{normal\; 3}}^{-}$ 25pt16ptsvg-formulamathimg737339a8d3517116341490f01d8cfecf bg-18-805-2021-ie00002.svg25pt16ptbg-18-805-2021-ie00002.png ) containing mass-independent oxygen-17 excess (quantified by a inline-formulaΔ¹⁷O notation) and three ammonium (inline-formula $M14inlinescrollmathmlchem{\mathrm{normal\; NH}}_{\mathrm{normal\; 4}}^{+}$ 24pt15ptsvg-formulamathimgcb88f58b3b25b473f7c5a29ace587a7f bg-18-805-2021-ie00003.svg24pt15ptbg-18-805-2021-ie00003.png ) fertilizers spanning a inline-formulaδ¹⁵N gradient were used in soil incubations to help illuminate the reaction complexity underlying NO yields and inline-formulaδ¹⁵N dynamics in a heterogeneous soil environment. We found strong evidence for the prominent role of inline-formula $M17inlinescrollmathmlchem{\mathrm{normal\; NO}}_{\mathrm{normal\; 2}}^{-}$ 25pt16ptsvg-formulamathimg3e662158e7d6a0c69e79cdad3103a280 bg-18-805-2021-ie00004.svg25pt16ptbg-18-805-2021-ie00004.png re-oxidation under anoxic conditions in controlling the apparent inline-formula¹⁵η for NO production from inline-formula $M19inlinescrollmathmlchem{\mathrm{normal\; NO}}_{\mathrm{normal\; 3}}^{-}$ 25pt16ptsvg-formulamathimg7248c728767abac31fc80ac33e5f4469 bg-18-805-2021-ie00005.svg25pt16ptbg-18-805-2021-ie00005.png in denitrification (i.e., 49 inline-formula‰ to 60 inline-formula‰). These results highlight the importance of an under-recognized mechanism for the reversible enzyme inline-formula $M22inlinescrollmathmlchem{\mathrm{normal\; NO}}_{\mathrm{normal\; 2}}^{-}$ 25pt16ptsvg-formulamathimg57d92df6ed78c83b450a25437527dccc bg-18-805-2021-ie00006.svg25pt16ptbg-18-805-2021-ie00006.png oxidoreductase to control the N isotope distribution between the denitrification products. Through a inline-formulaΔ¹⁷O-based modeling of co-occurring denitrification and inline-formula $M24inlinescrollmathmlchem{\mathrm{normal\; NO}}_{\mathrm{normal\; 2}}^{-}$ 25pt16ptsvg-formulamathimg9980dde299c5939f6aa6f5b78f3cf120 bg-18-805-2021-ie00007.svg25pt16ptbg-18-805-2021-ie00007.png re-oxidation, the inline-formula¹⁵η for inline-formula $M26inlinescrollmathmlchem{\mathrm{normal\; NO}}_{\mathrm{normal\; 2}}^{-}$ 25pt16ptsvg-formulamathimg5659f1ec0d19e14da6dd21bc92929b68 bg-18-805-2021-ie00008.svg25pt16ptbg-18-805-2021-ie00008.png reduction to NO and NO reduction to nitrous oxide (inline-formulaN₂O) were constrained to be 15 inline-formula‰ to 22 inline-formula‰ and inline-formula−8 inline-formula‰ to 2 inline-formula‰, respectively. Production of NO in the oxic and hypoxic incubations was contributed by both inline-formula $M33inlinescrollmathmlchem{\mathrm{normal\; NH}}_{\mathrm{normal\; 4}}^{+}$ 24pt15ptsvg-formulamathimgda812f2362704fc8599bd97e87b386fd bg-18-805-2021-ie00009.svg24pt15ptbg-18-805-2021-ie00009.png oxidation and inline-formula $M34inlinescrollmathmlchem{\mathrm{normal\; NO}}_{\mathrm{normal\; 3}}^{-}$ 25pt16ptsvg-formulamathimg983e472baa2b1950fa497a555dcc7b2e bg-18-805-2021-ie00010.svg25pt16ptbg-18-805-2021-ie00010.png consumption, with both processes having a significantly higher NO yield under inline-formulaO₂ stress. Under both oxic and hypoxic conditions, NO production from inline-formula $M36inlinescrollmathmlchem{\mathrm{normal\; NH}}_{\mathrm{normal\; 4}}^{+}$ 24pt15ptsvg-formulamathimg12e0e3705d3dbe24d04fbcadbab425f9 bg-18-805-2021-ie00011.svg24pt15ptbg-18-805-2021-ie00011.png oxidation proceeded with a large inline-formula¹⁵η (i.e., 55 inline-formula‰ to 84 inline-formula‰) possibly due to expression of multiple enzyme-level isotopic fractionations during inline-formula $M40inlinescrollmathmlchem{\mathrm{normal\; NH}}_{\mathrm{normal\; 4}}^{+}$ 24pt15ptsvg-formulamathimgd0b0f5cd174742dc798542982bfda883 bg-18-805-2021-ie00012.svg24pt15ptbg-18-805-2021-ie00012.png oxidation to inline-formula $M41inlinescrollmathmlchem{\mathrm{normal\; NO}}_{\mathrm{normal\; 2}}^{-}$ 25pt16ptsvg-formulamathimgae012be8e049730edc17abc1a5141921 bg-18-805-2021-ie00013.svg25pt16ptbg-18-805-2021-ie00013.png that involves NO as either a metabolic byproduct or an obligatory intermediate for inline-formula $M42inlinescrollmathmlchem{\mathrm{normal\; NO}}_{\mathrm{normal\; 2}}^{-}$ 25pt16ptsvg-formulamathimgef655b7de5138e3536c4788b60d2d1fc bg-18-805-2021-ie00014.svg25pt16ptbg-18-805-2021-ie00014.png production. Adding inline-formula $M43inlinescrollmathmlchem{\mathrm{normal\; NO}}_{\mathrm{normal\; 2}}^{-}$ 25pt16ptsvg-formulamathimgdf58a555dc3e458544843502401ab977 bg-18-805-2021-ie00015.svg25pt16ptbg-18-805-2021-ie00015.png to sterilized soil triggered substantial NO production, with a relatively small inline-formula¹⁵η (19 inline-formula‰). Applying the estimated inline-formula¹⁵η values to a previous inline-formulaδ¹⁵N measurement of in situ soil NOinline-formula_x emission (NOinline-formula $M49inlinescrollmathml{}_x=chem\mathrm{normal\; NO}+chem{\mathrm{normal\; NO}}_{\mathrm{normal\; 2}}$ 69pt13ptsvg-formulamathimg459b3c9cb49280050944b876d58c17d3 bg-18-805-2021-ie00016.svg69pt13ptbg-18-805-2021-ie00016.png ) provided promising evidence for the potential of inline-formulaδ¹⁵N-NO measurements in revealing NO production pathways. Based on the observational and modeling constraints obtained in this study, we suggest that simultaneous inline-formulaδ¹⁵N-NO and inline-formulaδ¹⁵N-inline-formulaN₂O measurements can lead to unprecedented insights into the sources of and processes controlling NO and inline-formulaN₂O emissions from agricultural soils.