Nitrogen isotopic fractionations during nitric oxide production in an agricultural soil

Abstract. 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.


Briefly, an aliquot of soil KCl extract with 60 nmol NH4 + was pipetted into a 20 mL serum vial containing an 270 acidified glass fiber disk. The solution was made alkaline by adding magnesium oxide (MgO) to volatilize NH3, which was subsequently captured on the acidic disk as NH4 + . After incubation under 37 ֯ C for 10 d, NH4 + was eluted from the disk using deionized water, diluted to 10 µM, oxidized by BrOto NO2 -, and finally measured for δ 15 N as NO2at 20 nmol using the denitrifier method. International NH4 + reference standards IAEA-N1, USGS25, and USGS26 underwent the same preparation procedure as the soil KCl extracts and were used along with the NO3 -275 reference standards to correct for blanks and instrument drift. The precision of the δ 15 N-NH4 + analysis is ±0.5‰ (Yu and Elliott, 2018). δ 15 N of NO collected in the TEA solution was measured following the method described in Yu and Elliott (2017). Briefly, the TEA collection samples were first neutralized with 12 N HCl to pH ~7, and then 10 to 20 nmol of the collected product NO2 -+NO3was converted to N2O using the denitrifier method. In light of the low δ 15 N 280 values of soil-emitted NO and the presence of NO2as the dominant collection product, a low δ 15 N NO2isotopic standard (KNO2, RSIL20, USGS Reston; δ 15 N = -79.6‰) was used together with the international NO3reference standards to calibrate the δ 15 N-NO analysis. Following the identical treatment principle, we prepared the isotopic standards in the same matrix (i.e., 20% TEA) as the collection samples and matched both the molar N amount and injection volume (±5%) between the collection samples and the standards to minimize the blank interferences 285 associated with the bacterial medium and the TEA solution. The precision and accuracy of the δ 15 N-NO analysis, https://doi.org/10.5194/bg-2020-344 Preprint. Discussion started: 20 October 2020 c Author(s) 2020. CC BY 4.0 License. determined by repeated sampling of an analytical NO tank (δ 15 N-NO = -71.4‰) under diverse collection conditions, is ±1.1‰ (Yu and Elliott, 2017).

Results
Sixty-three NO collection samples were obtained from the incubation experiments. The NO collection efficiency 290 calculated based on the measured NO2 -+NO3concentration in the TEA solution and the theoretical concentration based on the measured net NO production rate (Yu and Elliott, 2017) was on average 99.1±3.7%. Out of the sixtythree collection samples, four samples had a NO collection efficiency lower than 95%. These samples were excluded from further data analysis and interpretation. The measured N concentrations, net NO production rates, and isotope data from all the incubation experiments are available in Table S5 to Table S11 in the Supplement. 295
The net NO production was significantly higher in the hypoxic incubation (fNO-hypoxic; 9.0 to 10.4 ng N·g -1 ·h -1 ) than in the oxic incubation (fNO-oxic; 7.1 to 8.5 ng N·g -1 ·h -1 ) (Fig. 3c). The measured δ 15 N-NO values ranged from -16.8±0.3 to -54.9±0.8‰ in the oxic incubation and from -21.3±0.0 to -51.4±0.4‰ in the hypoxic incubation ( Fig.   3f). Pooling all the δ 15 N-NO measurements, we found that δ 15 N values between NH4 + and NO differed from 58.9 to 330 70.7‰ across the three δ 15 N-NH4 + treatments in the oxic incubation and from 50.4 to 69.6‰ in the hypoxic incubation (Fig. 4). In both incubations, the largest difference was observed under the high δ 15 N-NH4 + treatment, while the smallest difference was observed under the low δ 15 N-NH4 + treatment. Under both oxic and hypoxic conditions, there was a significant linear relationship between the measured δ 15 N-NO and δ 15 N-NH4 + values from all three δ 15 N-NH4 + treatments (Fig. 4). The slope of the linear relationship is 0.78±0.03 (± 1 SE) and 0.61±0.05 for the 335 oxic and hypoxic incubations, respectively (Fig. 4).

Abiotic NO production
Addition of NO3or NH4 + to the sterilized soil did not result in detectable NO production under either oxic or anoxic condition. Immediate NO release was, however, triggered by NO2addition under anoxic conditions (Fig. 5a). The abiotic NO production rate (fNO-abiotic) reached a steady level of 83±5 ng N·g -1 ·h -1 several minutes after the NO2 -340 addition and then decreased exponentially to < 3 ng N·g -1 ·h -1 over the following 8 days (Fig. 5a). The natural logarithm of fNO-abiotic showed a linear relationship with time (Fig. 5b). The NO produced following the NO2addition had a δ 15 N value of -17.8±0.4‰, giving rise to a δ 15 N offset between NO2and NO of 19.2±0.5‰.

Discussion
Because interpretations of the results from the incubation experiments build upon each other, here we discuss the 345 results from incubation of the sterilized soils (hereafter, abiotic incubation), anoxic incubation, and oxic/hypoxic incubations successively.

Reaction characteristics and N isotopic fractionation during abiotic NO production
The immediate release of NO upon the addition of NO2highlights the chemically unstable nature of NO2and the critical role of chemical NO2reactions in driving soil NO emissions (Venterea et al., 2005;Lim et al., 2018). The 350 strong linearity between ln(fNO-abiotic) and time (Fig. 5b) suggests apparent first-order kinetics for the abiotic NO production from NO2 -(Equations 2 and 3) (McKenney et al., 1990).
In Equations 2 and 3, t is time; kabiotic is the pseudo-first order rate constant for NO2loss; sabiotic is the apparent 355 stoichiometric coefficient for NO production from NO2 -; and [NO2 -]t and [NO2 -]0 are NO2concentration at time t and t=0 in the sterilized soil, respectively. Combining Equations 2 and 3 and then log-transforming both sides yield: Equation (4) According to Equation 4, kabiotic and sabiotic are estimated using the slope and intercept of the linear regression of ln(fNO-abiotic) versus time (Fig. 5b). Given [NO2 -]0 = 8 µg N·g -1 , sabiotic and kabiotic are estimated to be 0.52±0.05 (±SE) 360 and 0.019±0.002 h -1 , respectively, suggesting that NO accounted for 52±5% of the reacted NO2during the abiotic incubation. The estimated kabiotic is within the range (i.e., 0.00055 to 0.73 h -1 ) derived by a recent study based on soil samples spanning a wide range of pH values (3.4 to 7.2) (Lim et al., 2018). Based on the estimated kabiotic, 97% of the added NO2was lost by the end of the abiotic incubation.
Several reaction pathways with distinct stoichiometry have been proposed for abiotic NO production from 365 NO2in soils. Under acidic soil conditions, self-decomposition of HNO2 produces NO and nitric acid (HNO3) with a stoichiometric HNO2-to-NO ratio ranging from 0.5 to 0.66 (i.e., 1 mole of HNO2 produces 0.5 to 0.66 mole of NO) (Van Cleemput and Samater, 1995). Although at pH 5.7, HNO2 constituted <1% of the NO2 -+HNO2 pool in this soil, HNO2 decomposition can occur on acidic clay mineral surfaces, even though bulk soil pH is circumneutral (Venterea et al., 2005). However, given the complete NO2consumption in the abiotic incubation, HNO2 370 decomposition confined to acidic microsites could not account for all observed NO production. Under anoxic conditions, NO2 -/HNO2 can also be stoichiometrically reduced to NO by transition metals (e.g., Fe(II)) and diverse organic molecules (e.g., humic and fulvic acids, lignins, and phenols) in a process termed chemo-denitrification (Zhu-Baker et al., 2015). The produced NO from chemo-denitrification can undergo further reduction to form N2O and N2 (Zhu-Baker et al., 2015). In addition, both NO2and NO in soil solution can be consumed as nitroso donors 375 in abiotic nitrosation reactions, resulting in N incorporation into soil organic matter (Heil et al., 2016;Lim et al., 2018). Therefore, our observation that about half of the reacted NO2was recovered as NO may result from multiple competing NO2sinks, parallel NO-producing pathways, and possibly abiotic NO consumption in the sterilized soil.
The other half of the reacted NO2that could not be accounted for by the measured NO was likely present in the forms of N2O, N2, and/or nitrosated organic compounds in the soil. 380 The observed δ 15 N difference between NO2and NO (i.e., 15 ηNO2/NO(abiotic) = 19.2±0.5‰) likely reflects a combined N isotope effect for all of the competing NO production pathways during the abiotic incubation. While very little isotope data exist for abiotic NO2reactions in the literature, the measured 15 ηNO2/NO(abiotic) in this study is consistent with reported N isotope effects (i.e., 15 to 25‰) for abiotic NO2reduction by Fe(II) at similar NO2consumption rates as this study (0.02 to 0.05 h -1 ) (Buchwald et al., 2016). On the other hand, the measured 385 NO2and H2O (Casciotti et al., 2007;Buchwald and Casciotti, 2010), which effectively erase the isotopic imprints of 465 denitrification on NO2prior to its re-oxidation. The reversibility of NXR and its direct control on O isotopes in NO3  Importantly, there is mounting evidence from the marine N cycle community that NO2re-oxidation plays a critical role in the N isotope partitioning between NO3and NO2 -. At the process scale, NO2re-oxidation cooccurring with dissimilatory NO3reduction can lead to a large δ 15 N difference between NO3and NO2beyond what would be expected to result from NO3reduction alone (Gaye et al., 2013;Dale et al., 2014;Dähnke and Thamdrup, 2015;Peters et al., 2016;Martin and Casciotti, 2017;Buchwald et al., 2018). This large δ 15 N difference is thought to 475 arise from a rare, but intrinsic, inverse kinetic isotope effect associated with NO2re-oxidation (e.g., -13‰) (Casciotti et al., 2009). As such, in a net denitrifying environment, NO2re-oxidation functions as an apparent branching pathway along the sequential reduction of NO3 -, preferentially re-oxidizing 15 NO2back to NO3 -. At the enzyme scale, the bidirectional NXR enzyme has been proposed to catalyze intracellular coupled NO3reduction and NO2oxidation (i.e., bidirectional interconversion of NO3and NO2 -), facilitating expression of an equilibrium N 480 isotope effect between NO3and NO2 -(Reaction 2) (Wunderlich et al., 2013;Kemeny et al., 2016). theoretical quantum calculations (Casciotti, 2009) suggests that this N isotope equilibration favors partitioning of 14 N into NO2with an equilibrium isotope effect ranging from -50 to -60‰ (negative sign is used to denote that this 485 N isotope equilibration partitions 14 N to the left side of Reaction 2). This NXR-catalyzed NO3 -/NO2interconversion was invoked to explain the extremely low δ 15 N-NO2values relative to δ 15 N-NO3 -(up to 90‰) in the surface Antarctic ocean, where aerobic NO2oxidation is inhibited by low nutrient availability (Kemeny et al., 2016).
Hypothetically, if expressed at either the process or the enzyme level, the N isotope effect for NO2re-oxidation could propagate into denitrification-produced NO, giving rise to an increased δ 15 N difference between NO3and NO 490 To test whether NO2re-oxidation can explain the observed declines in δ 18 O-NO3and Δ 17 O-NO3values and δ 15 N distribution between NO3 -, NO2 -, and NO, we modified an isotopologue-specific (i.e., 14 N, 15 N, 16 O, 17 O, and 18 O) numerical model previously described by Yu and Elliott (2018) to simulate co-occurring denitrification and NO2re-oxidation in two steps. Without a clear identification of the alternative electron acceptors that coupled with 495 anaerobic NO2oxidation in the studied soil, we followed the reaction scheme proposed by Wunderlich et al. (2013) and Kemeny et al. (2016) (Reaction 1) to parameterize the NXR-catalyzed NO2re-oxidation as the backward reaction of a dynamic equilibrium between NO3and NO2 - (Fig. 6)that is, the NXR-catalyzed NO2re-oxidation (backward reaction) is balanced by an NXR-catalyzed NO3reduction (forward reaction), leading to no net NO2oxidation or NO3reduction in the soil. Importantly, this representation is consistent with the observation that both 500 NO3consumption and NO2accumulation followed a pseudo-zero order kinetics over the anoxic incubation ( Fig. 2a https://doi.org/10.5194/bg-2020-344 Preprint. Discussion started: 20 October 2020 c Author(s) 2020. CC BY 4.0 License. and 2b), which implies no net contribution from the NO3 -/NO2interconversion. Given previous findings that the NXR-catalyzed O exchange between NO3and NO2depends on NO2availability (Wunderlich et al., 2013), the backward NO2re-oxidation was assumed to be first order (with respect to NO2 -), defined by a first order rate constant, kNXR(b). With respect to the O isotope equilibration between H2O and the reacting NO2pool, we considered 505 two extreme case scenarios: (1) no exchange and (2)  and therefore all three O atoms in NO3produced from NO2re-oxidation originate from H2O. Furthermore, we 510 considered both abiotic NO production and denitrification as the source of NO during the anoxic incubation (Fig. 6).
To account for the potential overestimation in kabiotic (see above), we used a reduced kabiotic (0.0027 h -1 ) to model abiotic NO production from NO2 -, while sabiotic and 15 ηNO2/NO(abiotic) were fixed at 0.52 and 19.2‰, respectively. With respect to δ 15 N of denitrification-produced NO, we assumed that NIR-catalyzed NO2reduction to NO and NORcatalyzed NO reduction to N2O were each associated with a kinetic N isotope effect ( 15 ηNIR and 15 ηNOR). The closed-515 system Rayleigh equation was then used to simulate the coupled NO production and reduction in denitrification at With this model of co-occurring denitrification and NO2re-oxidation, we first solved for the rates of denitrifier-catalyzed NO3 -(RNAR), NO2 -(RNIR), and NO (RNOR) reductions and kNXR(b) (4 unknowns) using the 520 measured NO3and NO2concentrations, fNO-anoxic, and Δ 17 O-NO3values (4 measured variables). This first modeling step was robustly constrained by the measured Δ 17 O-NO3 -, which essentially functions as a 15 NO3tracer (Yu and Elliott, 2018) and is therefore particularly sensitive to NO2re-oxidation. In the second modeling step, the measured δ 15 N-NO3 -, δ 15 N-NO2 -, and δ 15 N-NO values (3 measured variables) were used to optimize the kinetic N isotope effects for NAR-catalyzed NO3reduction ( 15 ηNAR), 15 ηNIR, 15 ηNOR, and the equilibrium N isotope effect for NXR-525 catalyzed NO3 -/NO2interconversion ( 15 ηNXR(eq)) (Reaction 2; Fig. 6) (4 unknowns). This modeling system is underdetermined (number of measured variables < number of unknowns) and thus cannot be solved uniquely. Thus, instead of definitively solving for the four unknown isotope effects, we explored their best combination to fit the measured δ 15 N values of NO3 -, NO2 -, and NO. Specifically, to reduce the number of unknowns for model optimization, 15 ηNAR and 15 ηNXR(eq) were treated as known values, and 15 ηNIR and 15 ηNOR were solved by mapping 530 through the entire space of 15 ηNAR and 15 ηNXR(eq) (at a resolution of 1‰), defined by their respective widest range of possible values. We used a range of 5 to 55‰ for 15 ηNAR, consistent with a recent compilation based on soil incubations and denitrifier pure cultures (Denk et al., 2017). Given the existing observational and theoretical constraints (Casciotti, 2009;Brunner et al., 2013), a range of -60 to 0‰ was assigned to 15 ηNXR(eq), which is equivalent to the argument that the impact of NO3 -/NO2interconversion on the N isotope distribution between NO3 -535 and NO2can vary from null to a strong partitioning of 14 N to NO2 -. We further defined the lower 2.5th percentile of the error-weighted residual sum of squares (RSS) between simulated and measured δ 15 N values of NO3 -, NO2 -, and https://doi.org/10.5194/bg-2020-344 Preprint. Discussion started: 20 October 2020 c Author(s) 2020. CC BY 4.0 License. NO as the threshold for selection of the best-fit models. Detailed information regarding model optimization can be found in the Supplement (Text S3.2).
Results from the first modeling step are summarized in Table 1 and the best-fit models were plotted in Fig.  540 2 to compare with the measured data. Because the NXR-catalyzed NO3 -/NO2interconversion was assumed to result in no change in NO3and NO2concentrations, RNAR (0.158 µg N·g -1 ·h -1 ), RNIR (0.112 µg N·g -1 ·h -1 ), and RNOR (0.039 µg N·g -1 ·h -1 ) can be well-described by zero-order kinetics and are not sensitive to model scenarios for O exchange between NO2and H2O (Table 1). Moreover, the observed NO2accumulation and fNO-anoxic dynamics can be wellreproduced using the modeled denitrification rates and the downward adjustment of kabiotic ( Fig. 2b and 2c). kNXR(b) 545 was estimated to be 0.64 h -1 and 0.25 h -1 under the "no exchange" and "complete exchange" scenarios, respectively (Table 1) (Granger and Wankel, 2016). Therefore, although kNXR(b) cannot be definitively quantified in this study due to the unknown degree of O exchange between NO2and H2O, these simulation results provide confidence 560 in our hypothesis that the observed decreases in δ 18 O-NO3and Δ 17 O-NO3values were driven by the reversible action of the NXR enzyme. It is important to note that the estimated kNXR(b) is fairly large even under the "complete exchange" scenario. Based on the NO2concentration measured at the end of the anoxic incubation (6.9 µg N·g -1 ), a kNXR(b) of 0.25 h -1 would require a NO2re-oxidation rate (1.7 µg N·g -1 ·h -1 ) that is one order of magnitude higher than the estimated RNAR and RNIR. However, the inferred maximum NO2re-oxidation rate under either model 565 scenario (1.7 to 4.4 µg N·g -1 ·h -1 ) is still within the reported range for aerobic NO2oxidation in agricultural soils (e.g., up to 6-7 µg N·g -1 ·h -1 ) (Taylor et al., 2019), indicative of high NOB activity even under anoxic conditions (Koch et al., 2015).
Based on the modeled denitrification rates and kNXR(b), the best-fit 15 ηNXR(b) was confined to a narrow range from -40 to -35‰ ( Fig. 7a and 7b) and was not sensitive to model scenarios for O equilibration between NO2and 570 H2O (Fig. 8b). While the best-fit 15 ηNAR and 15 ηNXR(b) were positively correlated, especially under the "complete exchange" scenario ( Fig. 7a and 7b), the best-fit 15 ηNAR spanned a wide range (5 to 45‰) and was significantly lower under the "no exchange" scenario (RSS-weighted mean: 19‰) relative to the "complete exchange" scenario (RSS-weighted mean: 30‰) (Fig. 8a). On the other hand, the best-fit 15 ηNIR (15 to 22‰) and 15 ηNOR (-8 to 2‰) did https://doi.org/10.5194/bg-2020-344 Preprint. Discussion started: 20 October 2020 c Author(s) 2020. CC BY 4.0 License. source is mostly likely related to NO3consumption. This is based on the observation of high NO3concentrations in both oxic and hypoxic incubations, as well as the estimated low ROrgN/NH4 (Table 2), which indicates a low availability of labile organic Nanother potential substrate for NO production (Stange et al., 2013) in this agricultural soil. Therefore, based on the assumption that NH4 + oxidation and NO3consumption were the two primary NO sources during the oxic and hypoxic incubations, a two-source isotope mixing model was used to relate 690 the measured δ 15 N-NO values to the concurrently measured δ 15 N-NH4 + and δ 15 N-NO3values: Equation (5) where 15 ηNH4/NO and 15 ηNO3/NO are the net isotope effects for NO production from NH4 + oxidation and NO3consumption, respectively. Rearranging Equation (5) yields Equation (6) Equation (8) Equation (6) essentially dictates that the δ 15 N-NO values can be modeled from the δ 15 N-NH4 + and δ 15 N-NO3values using a hypothetical isotope effect for NO production from the combined soil NH4 + and NO3pool ( 15 ηcomb; the last term in Equation (6)) that is a mixing of 15 ηNH4/NO and 15 ηNO3/NO controlled by fNH4 (Equation 7). Thus, assuming fNH4 700 and 15 ηcomb were constant in each incubation experiment, fNH4 and 15 ηcomb can be solved using the measured δ 15 N-NO, δ 15 N-NH4 + , and δ 15 N-NO3values from all three δ 15 N-NH4 + treatments (Equation 8). fNH4 was estimated to be 0.72 under the oxic incubation (Table 2), indicating that 72% of the measured net NO production was sourced from NH4 + oxidation, with the remainder being ascribed to NO3consumption. Under the hypoxic condition, the share of NH4 + oxidation decreased to 58% (Table 2). 15 ηcomb was estimated to be 56‰ under the oxic condition and 51‰ under the 705 hypoxic condition (Table 2). Combining the δ 15 N-based NO source partitioning with the estimated RNH4/NO3 and RNO3comp, we further estimated NO yield in NH4 + oxidation and NO3consumption, respectively, and where the results are illustrated according to the classic "hole-in-the-pipe" (HIP) concept (Fig 9) (Davidson and Verchot, 2000). NO yield was 1.3% in NH4 + oxidation and 3.2% in NO3consumption in the oxic incubation ( Fig. 9; Table   2). Under the hypoxic condition, NO yield was increased to 5.2% in NH4 + oxidation and 6.1% in NO3consumption 710 ( Fig. 9; Table 2).
Most previous laboratory and field studies suggest that soil NO emissions are predominately driven by nitrification, whereas NO produced from denitrification is further reduced to N2O before it escapes to the soil surface (Kester et al., 1997;Skiba et al., 1997). The minor role of denitrification is largely deduced from the supposition that denitrification is activated only under wet soil conditions (Davidson and Verchot, 2000). However, 715 based on our δ 15 N-based NO source partitioning, about 30% of the net NO production was contributed by NO3consumption under oxic condition, highlighting the potential importance of denitrification in driving soil NO emissions under conditions not typically conducive to its occurrence. There is growing evidence that extensive anoxic microsites can develop in otherwise well-aerated soils due to micro-scale variability of O2 demand and soil texture-dependent gas diffusion limitations (Keiluweit et al. 2018). Although we would not predict high rates of 720 heterotrophic respiration in this agricultural soil with low organic carbon, it is possible that rapid O2 consumption by nitrification may outpace O2 supply through diffusion in soil microsites, fostering development of anoxic niches in https://doi.org/10.5194/bg-2020-344 Preprint. Discussion started: 20 October 2020 c Author(s) 2020. CC BY 4.0 License. close association with nitrification hot spots (Kremen et al., 2005). Based on 15 N labeling and direct 15 NO measurements using a gas chromatograph-quadrupole mass spectrometer, Russow et al. (2009) demonstrated that nitrification contributed about 70% of net NO production in a well-aerated, NH4 + -fertilized silt loam, in strong 725 agreement with our results based on natural abundance δ 15 N measurements. An even lower contribution to NO production, e.g., 26 to 44%, has been reported for nitrification in organic, N-enrich forest soils incubated under oxic conditions (Stange et al., 2013). The persistence of denitrifying microsites in the studied soil is further corroborated by the nearly doubled net NO production from NO3consumption in the hypoxic incubation (Fig. 9). Importantly, the actual NO yield in denitrification might be much higher than those estimated for gross NO3consumption during 730 the oxic and hypoxic incubations (i.e., 3.2% and 6.1%), as denitrification occurring in anoxic niches might only comprise a small fraction of the estimated RNO3comp.
Interestingly, while RNH4/NO3 was significantly lower in the hypoxic incubation, the net NO production from NH4 + oxidation was similar between the two incubation experiments, indicating a higher NO yield in nitrification when O2 availability became limited (Fig. 9). However, mechanisms underlying the differential NO yield in 735 nitrification are difficult to elucidate owing to the high complexity of biochemical pathways of NO production by AOB and AOA. In AOB, the prevailing view of NH3 oxidation is that it occurs via a two-step enzymatic process, involving hydroxylamine (NH2OH) as an obligatory intermediate (Fig. 10). The first step is catalyzed by NH3 monooxygenase (AMO), which uses copper and O2 to hydroxylate NH3 to NH2OH. Next, a multiheme enzyme,  (Hooper et al., 2005;Beeckman et al., 2018). However, there is recent strong evidence that HAO generally catalyzes the three-electron oxidation of NH2OH to NO under both aerobic and anaerobic conditions; the HAO-produced NO is further oxidized to NO2by an unknown enzyme 745 (Caranto et al., 2017). In this way, NO would not be a byproduct of incomplete NH2OH oxidation, but rather required as an obligatory intermediate for NO2production (Fig. 10). It was further proposed that AOB-encoded copper-containing NIR may catalyze the final one-electron oxidation of NO to NO2by operating in reverse (Lancaster et al., 2018). Under this 'NH2OH/NO obligate intermediate' model, high intracellular NO concentrations arise when the rate of NO production outpaces the rate of its oxidation to NO2 -, leading to NO leakage from cells. 750 Consequently, under O2 stress, decreases in the rate of NO oxidation to NO2might be expected, and this may explain the observed increase in nitrification NO yield in the hypoxic incubation. Additionally, some AOB strains can produce NO in a process termed 'nitrifier-denitrification', in which NO is produced through NIR-catalyzed NO2reduction and can be further reduced to N2O by AOB-encoded NOR (Wrage-Mönning et al., 2018) (Fig. 10).
Compared to AOB, the NH3 oxidation pathway in AOA remains unclear (Beeckman et al., 2018). The current model 755 is that NH3 is first oxidized by an archaeal AMO to NH2OH and subsequently converted to NO2by an unknown HAO counterpart (Kozlowski et al., 2016). NO seems to be mandatory for archaeal NH2OH oxidation and has been proposed to act as a co-substrate for the NO2production (Kozlowski et al., 2016). Consequently, NO is usually produced and immediately consumed with tighter control in AOA than in AOB (Kozlowski et al., 2016). https://doi.org/10.5194/bg-2020-344 Preprint. Discussion started: 20 October 2020 c Author(s) 2020. CC BY 4.0 License.
To shed further light on the inner workings of net NO production from NH4 + , we turn to constraining 760 Tables   Table 1. Mean and 95% confidence interval of modeled denitrification rates and NO2re-oxidation rate constant under the 'no exchange' and 'complete exchange' scenarios.