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<p>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 (<span class="inline-formula"><i>δ</i>¹⁵N</span>) of soil-emitted NO and its utility in providing mechanistic information on soil NO dynamics. However, interpretation of soil <span class="inline-formula"><i>δ</i>¹⁵N</span>-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 <span class="inline-formula">%</span> <span class="inline-formula">O₂</span>), oxic (20 <span class="inline-formula">%</span> <span class="inline-formula">O₂</span>), and hypoxic (0.5 <span class="inline-formula">%</span> <span class="inline-formula">O₂</span>) incubations of an agricultural soil were conducted to quantify the net N isotope effects (<span class="inline-formula">¹⁵<i>η</i></span>) for NO production in denitrification, nitrification, and abiotic reactions of nitrite (<span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M10" display="inline" overflow="scroll" dspmath="mathml"><mrow class="chem"><msubsup><mi mathvariant="normal">NO</mi><mn mathvariant="normal">2</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="25pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="5f68580b6b50ed5200a71c6309703243"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-18-805-2021-ie00001.svg" width="25pt" height="16pt" src="bg-18-805-2021-ie00001.png"/></svg:svg></span></span>) using a newly developed <span class="inline-formula"><i>δ</i>¹⁵N</span>-NO analysis method. A sodium nitrate (<span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M12" display="inline" overflow="scroll" dspmath="mathml"><mrow class="chem"><msubsup><mi mathvariant="normal">NO</mi><mn mathvariant="normal">3</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="25pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="737339a8d3517116341490f01d8cfecf"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-18-805-2021-ie00002.svg" width="25pt" height="16pt" src="bg-18-805-2021-ie00002.png"/></svg:svg></span></span>) containing mass-independent oxygen-17 excess (quantified by a <span class="inline-formula">Δ¹⁷O</span> notation) and three ammonium (<span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M14" display="inline" overflow="scroll" dspmath="mathml"><mrow class="chem"><msubsup><mi mathvariant="normal">NH</mi><mn mathvariant="normal">4</mn><mo>+</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="24pt" height="15pt" class="svg-formula" dspmath="mathimg" md5hash="cb88f58b3b25b473f7c5a29ace587a7f"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-18-805-2021-ie00003.svg" width="24pt" height="15pt" src="bg-18-805-2021-ie00003.png"/></svg:svg></span></span>) fertilizers spanning a <span class="inline-formula"><i>δ</i>¹⁵N</span> gradient were used in soil incubations to help illuminate the reaction complexity underlying NO yields and <span class="inline-formula"><i>δ</i>¹⁵N</span> dynamics in a heterogeneous soil environment. We found strong evidence for the prominent role of <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M17" display="inline" overflow="scroll" dspmath="mathml"><mrow class="chem"><msubsup><mi mathvariant="normal">NO</mi><mn mathvariant="normal">2</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="25pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="3e662158e7d6a0c69e79cdad3103a280"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-18-805-2021-ie00004.svg" width="25pt" height="16pt" src="bg-18-805-2021-ie00004.png"/></svg:svg></span></span> re-oxidation under anoxic conditions in controlling the apparent <span class="inline-formula">¹⁵<i>η</i></span> for NO production from <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M19" display="inline" overflow="scroll" dspmath="mathml"><mrow class="chem"><msubsup><mi mathvariant="normal">NO</mi><mn mathvariant="normal">3</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="25pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="7248c728767abac31fc80ac33e5f4469"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-18-805-2021-ie00005.svg" width="25pt" height="16pt" src="bg-18-805-2021-ie00005.png"/></svg:svg></span></span> in denitrification (i.e., 49 <span class="inline-formula">‰</span> to 60 <span class="inline-formula">‰</span>). These results highlight the importance of an under-recognized mechanism for the reversible enzyme <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M22" display="inline" overflow="scroll" dspmath="mathml"><mrow class="chem"><msubsup><mi mathvariant="normal">NO</mi><mn mathvariant="normal">2</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="25pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="57d92df6ed78c83b450a25437527dccc"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-18-805-2021-ie00006.svg" width="25pt" height="16pt" src="bg-18-805-2021-ie00006.png"/></svg:svg></span></span> oxidoreductase to control the N isotope distribution between the denitrification products. Through a <span class="inline-formula">Δ¹⁷O</span>-based modeling of co-occurring denitrification and <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M24" display="inline" overflow="scroll" dspmath="mathml"><mrow class="chem"><msubsup><mi mathvariant="normal">NO</mi><mn mathvariant="normal">2</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="25pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="9980dde299c5939f6aa6f5b78f3cf120"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-18-805-2021-ie00007.svg" width="25pt" height="16pt" src="bg-18-805-2021-ie00007.png"/></svg:svg></span></span> re-oxidation, the <span class="inline-formula">¹⁵<i>η</i></span> for <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M26" display="inline" overflow="scroll" dspmath="mathml"><mrow class="chem"><msubsup><mi mathvariant="normal">NO</mi><mn mathvariant="normal">2</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="25pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="5659f1ec0d19e14da6dd21bc92929b68"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-18-805-2021-ie00008.svg" width="25pt" height="16pt" src="bg-18-805-2021-ie00008.png"/></svg:svg></span></span> reduction to NO and NO reduction to nitrous oxide (<span class="inline-formula">N₂O</span>) were constrained to be 15 <span class="inline-formula">‰</span> to 22 <span class="inline-formula">‰</span> and <span class="inline-formula">−</span>8 <span class="inline-formula">‰</span> to 2 <span class="inline-formula">‰</span>, respectively. Production of NO in the oxic and hypoxic incubations was contributed by both <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M33" display="inline" overflow="scroll" dspmath="mathml"><mrow class="chem"><msubsup><mi mathvariant="normal">NH</mi><mn mathvariant="normal">4</mn><mo>+</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="24pt" height="15pt" class="svg-formula" dspmath="mathimg" md5hash="da812f2362704fc8599bd97e87b386fd"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-18-805-2021-ie00009.svg" width="24pt" height="15pt" src="bg-18-805-2021-ie00009.png"/></svg:svg></span></span> oxidation and <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M34" display="inline" overflow="scroll" dspmath="mathml"><mrow class="chem"><msubsup><mi mathvariant="normal">NO</mi><mn mathvariant="normal">3</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="25pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="983e472baa2b1950fa497a555dcc7b2e"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-18-805-2021-ie00010.svg" width="25pt" height="16pt" src="bg-18-805-2021-ie00010.png"/></svg:svg></span></span> consumption, with both processes having a significantly higher NO yield under <span class="inline-formula">O₂</span> stress. Under both oxic and hypoxic conditions, NO production from <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M36" display="inline" overflow="scroll" dspmath="mathml"><mrow class="chem"><msubsup><mi mathvariant="normal">NH</mi><mn mathvariant="normal">4</mn><mo>+</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="24pt" height="15pt" class="svg-formula" dspmath="mathimg" md5hash="12e0e3705d3dbe24d04fbcadbab425f9"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-18-805-2021-ie00011.svg" width="24pt" height="15pt" src="bg-18-805-2021-ie00011.png"/></svg:svg></span></span> oxidation proceeded with a large <span class="inline-formula">¹⁵<i>η</i></span> (i.e., 55 <span class="inline-formula">‰</span> to 84 <span class="inline-formula">‰</span>) possibly due to expression of multiple enzyme-level isotopic fractionations during <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M40" display="inline" overflow="scroll" dspmath="mathml"><mrow class="chem"><msubsup><mi mathvariant="normal">NH</mi><mn mathvariant="normal">4</mn><mo>+</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="24pt" height="15pt" class="svg-formula" dspmath="mathimg" md5hash="d0b0f5cd174742dc798542982bfda883"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-18-805-2021-ie00012.svg" width="24pt" height="15pt" src="bg-18-805-2021-ie00012.png"/></svg:svg></span></span> oxidation to <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M41" display="inline" overflow="scroll" dspmath="mathml"><mrow class="chem"><msubsup><mi mathvariant="normal">NO</mi><mn mathvariant="normal">2</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="25pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="ae012be8e049730edc17abc1a5141921"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-18-805-2021-ie00013.svg" width="25pt" height="16pt" src="bg-18-805-2021-ie00013.png"/></svg:svg></span></span> that involves NO as either a metabolic byproduct or an obligatory intermediate for <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M42" display="inline" overflow="scroll" dspmath="mathml"><mrow class="chem"><msubsup><mi mathvariant="normal">NO</mi><mn mathvariant="normal">2</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="25pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="ef655b7de5138e3536c4788b60d2d1fc"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-18-805-2021-ie00014.svg" width="25pt" height="16pt" src="bg-18-805-2021-ie00014.png"/></svg:svg></span></span> production. Adding <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M43" display="inline" overflow="scroll" dspmath="mathml"><mrow class="chem"><msubsup><mi mathvariant="normal">NO</mi><mn mathvariant="normal">2</mn><mo>-</mo></msubsup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="25pt" height="16pt" class="svg-formula" dspmath="mathimg" md5hash="df58a555dc3e458544843502401ab977"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-18-805-2021-ie00015.svg" width="25pt" height="16pt" src="bg-18-805-2021-ie00015.png"/></svg:svg></span></span> to sterilized soil triggered substantial NO production, with a relatively small <span class="inline-formula">¹⁵<i>η</i></span> (19 <span class="inline-formula">‰</span>). Applying the estimated <span class="inline-formula">¹⁵<i>η</i></span> values to a previous <span class="inline-formula"><i>δ</i>¹⁵N</span> measurement of in situ soil NO<span class="inline-formula">_<i>x</i></span> emission (NO<span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M49" display="inline" overflow="scroll" dspmath="mathml"><mrow><msub><mi/><mi>x</mi></msub><mo>=</mo><mrow class="chem"><mi mathvariant="normal">NO</mi></mrow><mo>+</mo><mrow class="chem"><msub><mi mathvariant="normal">NO</mi><mn mathvariant="normal">2</mn></msub></mrow></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="69pt" height="13pt" class="svg-formula" dspmath="mathimg" md5hash="459b3c9cb49280050944b876d58c17d3"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-18-805-2021-ie00016.svg" width="69pt" height="13pt" src="bg-18-805-2021-ie00016.png"/></svg:svg></span></span>) provided promising evidence for the potential of <span class="inline-formula"><i>δ</i>¹⁵N</span>-NO measurements in revealing NO production pathways. Based on the observational and modeling constraints obtained in this study, we suggest that simultaneous <span class="inline-formula"><i>δ</i>¹⁵N</span>-NO and <span class="inline-formula"><i>δ</i>¹⁵N</span>-<span class="inline-formula">N₂O</span> measurements can lead to unprecedented insights into the sources of and processes controlling NO and <span class="inline-formula">N₂O</span> emissions from agricultural soils.</p>