Nitrite isotope characteristics and associated soil N transformations

Abstract

Nitrite (NO₂⁻) is a crucial compound in the N soil cycle. As an intermediate of nearly all N transformations, its isotopic signature may provide precious information on the active pathways and processes. NO₂⁻ analyses have already been applied in ¹⁵N tracing studies, increasing their interpretation perspectives. Natural abundance NO₂⁻ isotope studies in soils were so far not applied and this study aims at testing if such analyses are useful in tracing the soil N cycle. We conducted laboratory soil incubations with parallel natural abundance and ¹⁵N treatments, accompanied by isotopic analyses of soil N compounds (NO₃⁻, NO₂⁻, NH₄⁺). The double ¹⁵N tracing method was used as a reference method for estimations of N transformation processes based on natural abundance nitrite dynamics. We obtained a very good agreement between the results from nitrite isotope model proposed here and the ¹⁵N tracing approach. Natural abundance nitrite isotope studies are a promising tool to our understanding of soil N cycling.

Introduction

Nitrite (NO₂⁻), as an intermediate of nearly all N transformations, is a crucial compound to understand the complexity of the N soil cycle with its many contributing pathways. Moreover, as a very reactive compound it usually occurs at very low concentrations, hence conveying information on currently active N transformations. The Ntrace model used for interpretation of ¹⁵N labelled soil studies has been recently expanded with the NO₂⁻ content and isotopic analyses, which vastly increased its interpretation perspectives¹. Thanks to incorporation of NO₂⁻ dynamics in this model it appeared possible to distinguish and quantify three NO₂⁻ and N₂O production pathways: denitrification, autotrophic nitrification and heterotrophic nitrification. Although ¹⁵N tracing studies can precisely identify various soil N transformations^1,2, they require addition of ¹⁵N -labelled substances, which is associated with additional fertilization, soil disturbance, and potential problems with label distribution homogeneity^3,4. Moreover, due to high costs and fast consumption of the ¹⁵N label, ¹⁵N tracing approach can be applied mostly for short-term and micro-plot studies⁵. Development of reliable methods for identifying N transformations based on natural abundance stable isotopes can overcome these problems and provide an approach allowing studies in undisturbed soil conditions ensuring original N transformation rates that can be traced in larger time and space scale.

Natural abundance NO₂^–isotope studies are so far mostly applied in aquatic studies^6,7,8,9 and appeared particularly informative for the oceanic oxygen deficient zones, where NO₂⁻ can be accumulated^7,9. However, for soil studies the natural abundance NO₂⁻ analyses are so far lacking. Also in soils NO₂⁻ accumulation may happen and the monitoring of NO₂⁻ content in soils can provide important information to understand the N cycle^10,11,12. In particular, NO₂⁻ plays a central role for N₂O formation^12,13. However, even in situations when NO₂⁻ accumulation is not observed, and the interpretation of, typically very low, soil NO₂⁻-contents is ambiguous, the N transformations can potentially be followed by the stable isotopic signature of NO₂⁻, which has neither been tested nor applied so far.

Nitrite can be formed during nitrate reduction (NAR) in the course of denitrification, ammonium oxidation (AOX) in the course of autotrophic nitrification and organic N oxidation (ORG) associated with heterotrophic nitrification, and consumed during nitrite reduction (NIR) to NO or N₂O, and nitrite oxidation (NIOX) to NO₃^−1,7. Each of these sources and sinks are characterised by specific isotopic fractionation^7,9,14, which makes it possible to trace them back to their origins and sinks of NO₂⁻, and consequently, for a better understanding of the N cycling⁷.

This study presents the first attempt to interpret the NO₂⁻ isotopic signatures (δ¹⁵N_NO2− and δ¹⁸O_NO2−) in agricultural soil to decipher soil transformation processes. Three laboratory incubations were performed: under oxic conditions with lower water content (L1), under oxic conditions with higher water content (L2) and under anoxic conditions (L3), to monitor the differences when various N transformation processes are enhanced. The incubations at natural abundance level (NA treatment) and under ¹⁵N enrichment (¹⁵NO₃ treatment and ¹⁵NH₄ treatment) were performed simultaneously. Based on the ¹⁵N treatments the Ntrace model¹ was applied to determine NO₂⁻ sources and sinks. The results of NA treatment were used to construct the soil NO₂⁻ model, which is based on the model used for oceanic studies⁷, including the processes that have contributed to production and consumption of NO₂⁻ in soils. This study provides the first attempt to validate the results of NO₂⁻ isotope modelling with an independent ¹⁵N tracing approach.

Results

Soil NO₂ ⁻ characteristics

The oxic experiment was performed in two moisture treatments: L1 (dryer conditions) and L2 (wetter conditions), with water addition in the middle of experiment which increased the soil moisture from 61 to 68% water-filled pores space (WFPS) for L1a and L1b and from 72 to 81% WFPS for L2a and L2b, respectively. The detailed experimental conditions and information on general soil properties can be found in¹⁵ and in the supplement. NO₂⁻ content varied from 0.6 to 1.4 μmol N kg⁻¹ soil for L1 and from 0.1 to 4.7 for L2, whereas the NO₃⁻ content was three orders higher and quite stable ranging from 1300 to 1700 μmol N kg⁻¹ soil. The δ¹⁵N_NO2− was similar for L1 and L2 with a mean of 3.2 ± 4.2‰ and 3.9 ± 4.2‰, respectively, whereas δ¹⁵N_NO3− was very stable with a mean of 4.5 ± 0.4‰ and 4.7 ± 0.6‰, respectively. There was a negative correlation between δ¹⁵N_NO2− and the NO₂⁻-content (Fig. 1A). Similar values for δ¹⁸O_NO2− were found for both L1 and L2 with a mean of 11.8 ± 2.8‰ and 12.5 ± 5.0‰, respectively.

Figure 1

Relationship between NO₂⁻ isotopic signature δ¹⁵N_NO2− (A) or δ¹⁸O_NO2− (B) and reciprocal NO₂⁻ content (Keeling plot analysis—see “Methods” section). For δ¹⁸O_NO2− (B) the dashed line indicates the δ value of NO₂⁻ in full equilibrium with ambient water in 20 °C of 8.6‰²³ and the arrows indicate the direction of change in δ values of NO₂⁻in course of equilibration with water (points ‘move’ towards full equilibrium). For L3 the first samples taken after 24 h are marked with circles and the second samples taken after 48 h are shown with crosses only. Note the logarithmic scale of the X-axis.

The anoxic experiment L3 was performed to favour denitrification. NO₂⁻ content was much higher when compared to oxic conditions (L1 and L2) reaching 63.9 ± 12.5 μmol N kg⁻¹ soil and 32.4 ± 8.9 μmol kg⁻¹ soil after 24 h and 48 h of incubation, respectively. The average δ¹⁵N_NO2− was − 24.8 ± 3.3‰ without significant differences between the two samplings, whereas δ¹⁸O_NO2− showed significantly lower values of 4.4 ± 0.4‰ after 24 h compared to 6.6 ± 1.0‰ after 48 h (Fig. 1B).

In both ¹⁵N treatments (¹⁵NH₄⁺ and ¹⁵NO₃⁻) in L1 and L2, we observed a sudden drop in ¹⁵N abundance in NO₂⁻ (a¹⁵N_NO2−) from 12.7 to 5.1 at.% after water addition to the soil, whereas ¹⁵N abundance in NO₃⁻ (a¹⁵N_NO3−) showed only slight decrease from 13.2 to 12.1 at.% (means for all treatments, individual values in Fig. S1 and Table S1). This indicates an incorporation of another source of unlabelled NO₂⁻ for the wet part of these experiments. In natural abundance isotopes this change was also reflected in a higher apparent isotope effect ¹⁵η_{NO2−/ NO3−}. For the wet part it was even positive with an average + 1.6‰, whereas for the dry part it was lower with − 4.4‰ (Table 1). Interestingly, this significant isotopic change in NO₂⁻ was not reflected in the N₂O ¹⁵N abundance (a¹⁵N_N2O) in the ¹⁵N treatments (Fig. S1, Table S1).

Table 1 Apparent isotopic fractionation factors for δ¹⁵N of NO₃⁻, NO₂⁻ and N₂O.

Isotope effects between NO₃ ⁻, NO₂ ⁻and N₂O

With the NA dataset for NO₂⁻ presented in this paper and the dataset for N₂O presented in a previous paper for L1 and L2¹⁵, and here for L3 (Table S1), we can investigate the relation between the isotopic characteristics of both N compounds and determine the apparent isotope effects between NO₂⁻ and N₂O, and comparing them with isotope effects between NO₃⁻ and N₂O. Therefore, we need the isotopic signatures of the produced N₂O prior to isotopic fractionation due to N₂O reduction. For L3 the incubations were partially conducted with N₂O reduction inhibition (acetylated treatments) and we only report here the δ_N2O values of the inhibited treatment (Table S1) which represent the produced N₂O isotopic signatures. For L1 and L2 a detailed study of N₂O reduction was performed¹⁵ where the N₂O reduced fraction (r_N2O) was determined with ¹⁵N treatment, and the produced N₂O (δ_{N2O_p}) can be calculated according to the equation:

$${\delta }_{N2O\_p}={\delta }_{N2{O}_{m}}-\mathrm{ln}{r}_{N2O}*{\varepsilon }_{red}$$

based on the measured N₂O (δ_{N2O_m}) and the isotopic fractionation associated with N₂O reduction (ε_red)¹⁵. The determined apparent N isotope effects (¹⁵η) was calculated as:

$${{}^{15}\eta }_{product-substrate}={\delta }^{15}{N}_{product}-{\delta }^{15}{N}_{substrate}$$

Determination of NO₂ ⁻ dominant source

Keeling plots were applied to identify the NO₂⁻ dominant source (see Methods Section for methodical explanation)^15,16,17,18. For the oxic experiment, a significant linear fit between δ¹⁵N_NO2− and reciprocal NO₂⁻ content was found, where an linear equation intercept of − 13.3‰ indicated the isotopic signature of the dominant NO₂⁻ source. It must be denitrification since the applied conditions of quite high soil moisture and nitrate amendment should have favoured denitrification. In a previous study, denitrification was identified as the dominant source for N₂O¹⁵ and also the applied Ntrace model indicated the dominance of denitrification nitrate reduction (NAR) in the NO₂⁻-sources (f_NAR of 0.53 and 0.55 for L1 and L2, respectively, Table 2). Hence, based on the value found from the Keeling plot (Fig. 1A) we can determine the nitrogen isotopic fractionation for denitrification (¹⁵ε_NAR) between δ¹⁵N_NO3− (mean measured value) and δ¹⁵N_NO2− (Keeling plot intercept) for this incubation experiment:

$$^{{{15}}} \varepsilon_{NAR} \left( {{\text{NO}}_{{2}}^{ - } /{\text{ NO}}_{{3}}^{ - } } \right) = - {13}.{3}\textperthousand{-}\left( { + {4}.{5}\textperthousand} \right) = - {17}.{8}\textperthousand $$

This value fits quite well in the literature range^7,14 and is further used in the NO₂⁻ isotope model as ¹⁵ε_NAR.

Table 2 Nitrite stable isotope model to determine sources mixing proportions.

Under anoxic conditions, denitrification should be the only source of NO₂⁻, hence a typical Keeling correlation is not expected. We rather observed the opposite trend in L3 than under oxic conditions, i.e. lower δ¹⁵N_NO2− values with lower NO₂⁻ contents (Fig. 1A). Most probably this reflects the variability of apparent isotope effects, which are typically larger for lower reaction rates^19,20.

For δ¹⁸O_NO2− values, beside sources mixing, we also deal with isotope exchange of O-atoms between NO₂⁻ and ambient water, hence the Keeling plot method cannot be applied. We observed that δ¹⁸O_NO2− values were modified by the O exchange process, especially under anoxic conditions (L3), where lower NO₂⁻ content and incubation progress shifted δ¹⁸O_NO2− values towards equilibrium with water (NO₂⁻ full eq, Fig. 1B). NO₂⁻ samples taken after 48 h showed more equilibrated δ¹⁸O_NO2− values and lower NO₂⁻ content.

NO₂ ⁻ isotope model

The model is constructed based on the NO₂⁻ isotope model proposed for oceanic studies⁷ and adapted for typical soil N pathways after the Ntrace model, designed for ¹⁵N labelled soil studies applying NO₂⁻ as a key intermediate in soil N transformations¹, assuming steady state conditions. It takes into account three main NO₂⁻ sources (NAR, AOX and ORG) and two main NO₂⁻ sinks (NIR and NIOX), as well as δ¹⁸O_NO2− equilibration with ambient water (Fig. 2), according to the following equations:

$${\delta }^{15}{N}_{NO2-}={\delta }^{15}{N}_{NAR}*{f}_{NAR}+{\delta }^{15}{N}_{AOX}*{f}_{AOX}+{\delta }^{15}{O}_{ORG}*{f}_{ORG}-{{}^{15}\varepsilon }_{NIR}*{f}_{NIR}-{{}^{15}\varepsilon }_{NIOX}*{f}_{NIOX}$$

(1)

$${\delta }^{18}{O}_{NO2-}={(\delta }^{18}{O}_{NAR}*{f}_{NAR}+{\delta }^{18}{O}_{AOX}*{f}_{AOX}+{\delta }^{18}{O}_{ORG}*{f}_{ORG}-{{}^{18}\varepsilon }_{NIR}*{f}_{NIR}-{{}^{18}\varepsilon }_{NIOX}*{f}_{NIOX})*\left(1-x\right)+{\delta }^{18}{O}_{eq}*x$$

(2)

where δ_NO2− is the measured residual NO₂⁻ isotopic signature, δ_NAR/AOX/ORG are the isotopic signatures of source NO₂⁻ calculated with the measured stable isotope values for NO₂⁻ substrates (NO₃⁻, NH₄⁺, Norg, respectively for three sources, Table 2) and the characteristic isotopic fractionation associated with each NO₂⁻ formation pathway (ε_NAR/AOX/ORG, Table 2). ε_NIR/NIOX are the isotopic fractionation factors associated with NO₂⁻ sinks (ε_NIR/NIOX, Table 2). See also Methods Section for detailed description of isotope effects for particular processes; final values used in the model are shown in Table 2. The δ¹⁸O_eq stands for O isotopic signature of NO₂⁻ in complete equilibrium with water, which equals 8.6‰ for the incubation temperature of 20 °C⁷ and δ¹⁸O_H2O of − 5‰. x is the extent of oxygen atom exchange between nitrate and ambient water determined with the ¹⁷O approach²¹ for N₂O originating from denitrification processes under anoxic conditions (L3) and is equal 0.25 (see Methods Section). The exchange for NO₂⁻ cannot be higher than the value determined for N₂O. Since most of the exchange observed for N₂O is associated with the NO₂^—-H₂O isotope exchange²¹ this value was incorporated in the model calculations.

Figure 2

General scheme of the NO₂⁻ stable isotope model. The isotopic signatures of NO₂⁻ sources shown are based on the measured mean isotopic signatures of substrates for L1 and L2 and the isotopic fractionation associated with NAR, AOX and ORG (Table 1). Dashed gray arrows illustrate the mixing of 3 NO₂⁻ sources with mean mixing proportions found in Ntrace study (f_NAR = 0.55, f_AOX = 0.15, f_ORG = 0.30, Table 1) resulting in the produced δ_NO2- (grey open point). This δ value can be modified by NIR (red arrow) and NIOX (blue arrow). The δ¹⁸O_NO2- after reduction or oxidation (red and blue open point, respectively) is further modified by equilibration with ambient water with the extent of 0.25 of the equilibrated oxygen atoms (red and blue filled point, respectively). The ratio of NO₂- reduction and oxidation processes (red-ox ratio, here 4:1, as mean from Ntrace study, Table 1) determines the final δ_NO2- (purple point).

For NAR we were able to determine the isotope effect (¹⁵ε_NAR =  − 17.8) based on the Keeling plot (Fig. 1A) for L1 and L2. This value of − 17.8‰ is on the lower range of previously determined values^{22,23,24,25,26,27,28,29} (see summary in the Methods Section) and was used in the model for L1 and L2. For anoxic experiment L3 the fractionation was larger (Fig. 1A) and we included the typical ¹⁵ε_NAR value of − 30‰ in the model. For ¹⁸ε_NAR the fractionation must be very low to obtain the observed range of δ¹⁸O_NO2−: − 10‰ for L3 and no fractionation for L1 and L2. Similar ranges of ¹⁸ε_NAR values were modelled previously with indication of lower values for smaller reaction rates²¹. This is in accordance with our observations indicating much lower N transformation rates for the oxic experiments L1 and L2 than for the anoxic L3. Also the isotopic fractionation for NIR appears to be lower under anoxic conditions, where NO₂⁻ is accumulating. We obtained best fit between modelled and measured values for L3 when no fractionation associated with NIR was assumed (Table 2). This can be due to observed accumulation of NO₂⁻, indicating that the steady-state model assumption is not valid. In case of NO₂⁻ accumulation the isotopic fractionation associated with its reduction has a very low impact on the final isotopic signature of the residual NO₂⁻.

NO₂ ⁻ turnover

When δ¹⁸O_NO2- values are not completely equilibrated with soil water, measured δ¹⁸O_NO2− values can be used to estimate the rates of biological NO₂⁻ turnover relative to abiotic exchange⁷. This estimation is based on the abiotic equilibration rate as a function of temperature and pH⁷. Furthermore, we can determine the flux of NO₂⁻ oxygen atoms abiotic exchange as F_eq = k*C_NO2−. The NO₂⁻ flux of biological production (or consumption) can be determined from the δ¹⁸O_NO2- isotope mass balance following the method proposed for oceanic studies⁷ adapted to soil NO₂⁻ transformations:

$${F}_{B}=\frac{{F}_{eq}({ \delta }^{18}{O}_{NO2-}-{\delta }^{18}{O}_{eq})}{{\delta }^{18}{O}_{NAR}*{f}_{NAR}+{\delta }^{18}{O}_{AOX}*{f}_{AOX}+{\delta }^{18}{O}_{ORG}*{f}_{ORG}-{{}^{18}\varepsilon }_{NIR}*{f}_{NIR}-{{}^{18}\varepsilon }_{NIOX}*{f}_{NIOX}-{ \delta }^{18}{O}_{NO2-}}$$

(3)

where δ¹⁸O_NO2- is the measured NO₂⁻ , δ¹⁸O_NAR/AOX/ORG are the calculated NO₂⁻ sources NAR, AOX and ORG, ¹⁸ε_NIR/NIOX are the isotopic fractionation associated with NO₂⁻ sinks NIR and NIOX, f are the respective contributions of NO₂⁻ sources determined by Ntrace model (Table 2), and δ¹⁸O_eq is the value for NO₂⁻ in complete equilibrium with ambient water (of 8.6‰ for this case study). In turnover rate calculations (Table 3) we have neglected NIOX because due to the inverse fractionation of this process for some cases the isotope mass balance did not work due to unrealistic discrepancies between calculated and measured δ¹⁸O_NO2− values. Since ¹⁸ε_NIOX can be very low³⁰ and the NIOX contribution is low for our case study (up to 30%, Table 2), this process has most probably little impact on the final δ¹⁸O_NO2− values. In the NO₂⁻ isotope model, neglecting NIOX would result in higher final modelled δ¹⁸O_NO2 values of 13.3‰ for both treatments, which would fit to the measured values equally well as when the NIOX fractionation is included (Table 2).

Table 3 Nitrite transformation fluxes (due to equilibration (F_eq) and biological turnover (F_B)) and residence time due to biological turnover (Γ_B) or abiotic equilibration (Γ_eq) determined with δ¹⁸O_NO2- values compared to nitrite turnover rate determined with Ntrace model (F_Ntrace).

Our results indicate that the NO₂⁻ flux in L2 is larger than in L1 (Table 3), which is reasonable since L2 was the wetter treatment showing more intensive nitrogen fluxes based N₂O and N₂ fluxes, which were twice as high in L2 compared to L1¹⁵. Similar differences can be observed here for calculated FB values (Table 3), however this is not directly confirmed by the Ntrace results.

Discussion

We found very good congruity between Ntrace and the NA NO₂⁻ model. The modelled δ¹⁸O_NO2− and δ¹⁵N_NO2− values using measured source fractions provided by the Ntrace model differed up to 1.2‰ and 4.0‰, respectively, when compared to true measured values (Table 2). When we solely used the NA NO₂⁻ model to assess the fraction of NO₂⁻-sources contribution based on δ¹⁵N_NO2−, i.e., fitting modelled δ¹⁵N_NO2− values to the true measured values by adjusting the fraction of NO₂⁻-sources contribution, the fitted fractions are in good agreement with fractions provided by the Ntrace model (Table 2). Both results show similar dominance of NAR in NO₂⁻- production (f_NAR of ca. 0.55) but the NA NO₂⁻ model indicates even higher contribution of heterotrophic vs autotrophic nitrification (f_ORG vs. f_AOX).

The Ntrace approach, which is able to identify the contribution of ORG, was actually the first one that paid attention to this process in soils³¹. Here, with the NA NO₂⁻ model we get a confirmation of the potentially high ORG relevance in soil N transformations. Without this process the final δ¹⁵N_NO2− and δ¹⁸O_NO2− values could not be explained. Namely, if only considering two source processes: NAR and AOX, to meet the measured δ¹⁵N_NO2− value we would need domination of NAR (f_NAR > 0.75) and to meet the measured δ¹⁸O_NO2− value we would need an unrealistically high contribution of AOX (f_AOX > 0.70). Hence, the application of both isotope signatures (δ¹⁵N_NO2− and δ¹⁸O_NO2−) simultaneously allows for a proper identification of NO₂⁻ sources.

The presented NO₂⁻ isotope model may not be very typical, since for our case study we had exceptionally high δ¹⁵N_NH4+ values (from 36 to 100‰), hence this worked partially as a naturally low level ¹⁵N tracing allowing for very clear separation of NAR and AOX with δ¹⁵N_NO2− values. In case of similar δ values for substrate NO₃⁻ and NH₄⁺, this separation would be very weak, but still, in combination with δ¹⁸O_NO2− values, may be useful in assessing source contributions. The ¹⁵N enrichment of NH₄⁺ was not purposely induced but was a consequence of the fast ammonium consumption. Ntrace analysis revealed that the dominant ammonium sink is immobilisation, responsible for more than 90% of ammonium consumption. This process is associated with pronounced enrichment of residual ammonium in ¹⁵N^32,33,34, which we observed in this study. The very fast NH₄⁺ immobilisation and its further release due to Norg oxidation to NO₃⁻ were unexpected in this study and cannot be fully explained. The Ntrace model assumes the existence of the labile Norg pool, which is associated with these extremely fast fluxes. In the NA model, the assumed substrate for ORG nitrite production is the measured δ¹⁵N of the bulk organic N pool. This is probably the largest uncertainty in the model, since the labile Norg pool may be isotopically different than the bulk Norg. Similar uncertainties may also be associated with the measured bulk δ¹⁵N_NO3−, since this value may be significantly higher in the intensively denitrifying soil microsites.

The NO₂⁻ isotopic signature time series in the ¹⁵N treatments (Fig. S1), strongly indicates the appearance of new unlabelled NO₂⁻ for L1 and L2 after water addition in the course of the incubation. Ntrace clearly indicated an increase in ORG contribution after water addition (from 0.11 to 0.49 and from 0.07 to 0.33 for L1 and L2, respectively, Table S3) and the NA NO₂⁻ model confirms this finding (from 0.11 to 0.52 and from 0.21 to 0.31 for L1 and L2, respectively, Table S3). This suggests that NA NO₂⁻ analyses can be used to trace the dynamic changes in soil N transformations.

The ¹⁵N treatment indicated that N₂O ¹⁵N enrichment (a¹⁵N_N2O) follows rather a¹⁵N_NO3− than a¹⁵N_NO2− (Fig. S1). This indicates that mostly NAR NO₂⁻ is further reduced and emitted as N₂O and suggests that the ORG NO₂⁻ forms an isolated NO₂⁻ pool, as also suggested earlier¹, which is probably not further reduced to N₂O in significant magnitude. The calculated a_{P_N2O} value representing the ¹⁵N enrichment of the ¹⁵N -pool derived N₂O is higher than a¹⁵N_N2O due to the contribution of non-labelled N₂O to the total N₂O flux, so that a¹⁵N_N2O = f_{P_N2O} * a_{P_N2O} + (1- f_{P_N2O} ) * a_NA , where a_NA is the ¹⁵N abundance of the natural abundance samples (0.367 at.%). However, a_{P_N2O} values always have a higher ¹⁵N abundance than found for any soil N-pool (Table S1). This indicates that in denitrification soil microsites, where the ¹⁵N-pool derived N₂O is produced, we deal with higher a¹⁵N_NO2− and a¹⁵N_NO3− values than the mean analysed values. This confirms the N₂O emission originating from various isolated soil N-pools. The fraction of the ¹⁵N -pool N₂O is dominating – from 0.7 to 1.0—with higher values for higher soil moisture (Table S1). This is in contrast to NO₂⁻ which gets ¹⁵N depleted after water addition (Fig. S1) and f_NAR is only around 0.5.

Regarding the NA isotope effects, we can see that for pure denitrification processes under anoxic conditions in L3, we deal with very low ¹⁵η_NO2−NO3 values (Table 1) indicating a pronounced isotope effect between NO₃⁻ and NO₂⁻, whereas for L1 and L2, where NO₂⁻ is formed not only due to NAR but also AOX and ORG (Table S2), this effect is much smaller (as indicated by ¹⁵η_NO2−NO3 closer to 0, Table 1). Interestingly, in the wetter parts of the experiments (L1b, L2b), when increased contribution of ORG NO₂⁻ occurs, even an inverse effect is observed, i.e. NO₂⁻ is ¹⁵N enriched compared to NO₃⁻. As a result, for L1 and L2 very similar isotope effects for N₂O production with both substrates are observed (¹⁵η_N2O-NO3 and ¹⁵η_N2O-NO2 are not significantly different). This is in contrast with L3, where ¹⁵η_N2O-NO2 is much lower compared to ¹⁵η_N2O-NO3. For oxic conditions, clearly the highest isotope effect for N₂O production with both substrates is noted for L2b, for which the ¹⁵η_N2O-NO3 is nearest to the values typical for denitrification, as found in L3. Indeed, for L2b the denitrification pool derived fraction (f_{P_N2O}, Table S1) is the highest. Also the previous study¹⁵ indicated that the N₂O fraction produced due to denitrification, including both bacterial and fungal denitrification, is near 1 for this part of the experiment (L2b)¹⁵. However, the ¹⁵η_N2O-NO2 values are much lower for L2b than for L3 which is probably caused by admixture of other nitrite sources present for L2b but absent for L3.

Despite the fact that NO₂⁻⁻ turnover rates determined with δ¹⁸O_NO2− (F_B) differed somewhat from the F_Ntrace results (Table 3), the congruence can be considered to be adequate because very plausible ranges for NO₂⁻ turnover rates were observed. Both methods provide a similar range of values, however, the Ntrace model does not reflect the significant difference in turnover rates between L1 and L2 (Table 3). This may be due to lower sensitivity of the Ntrace approach in precise NO₂⁻ fluxes determination, since these are determined as a result of complex modelling of all N pools and the final result is an average of the best fit fluxes for both treatments (¹⁵NH₄⁺ and ¹⁵NO₃⁻). This turnover rate estimation provides a unique opportunity to predicting process rates based on natural abundance isotopic measurements.

Summing up, the natural abundance signatures of NO₂⁻ can be applied for identification of NO₂⁻ sources by applying the NA isotope model, which allows to estimate the contribution of the main pathways: NAR, AOX and ORG. This study showed that these are in a very good agreement with the results provided by the Ntrace model. Moreover, analysis of δ¹⁸O_NO2− values allows for estimation of NO₂⁻ turnover rates. The natural abundance signatures of NO₂⁻ may potentially be used in linking the soil N transformations with gaseous emissions in the form of N₂O. However, this connection still cannot be fully understood and needs further studies.

Methods

Laboratory incubations

Oxic incubations: L1 and L2 experiment

Silt loam soil Albic Luvisol from arable cropland of Merklingsen experimental station located near Soest (North Rhine-Westphalia, Germany, 51° 34′ 15.5″ N, 8° 00′ 06.8″ E) was used in the incubations (0.87 silt, 0.11 clay, 0.02 sand). The soil density of intact cores was 1.3 g cm⁻³, pH value 6.8, total C content 0.0130, total N content 0.0016, organic matter content 0.0214, initial NO₃⁻ content 864 μmol N kg⁻¹ dry soil and initial NH₄⁺ content 50 μmol N kg⁻¹ dry soil . The soil, upper 30 cm soil layer, was collected on the 18.01.2018 and the incubation was conducted from 19.02.2018 to 05.03.2018. The soil was air dried and sieved at 4 mm mesh size. Afterwards, the soil was rewetted to achieve a water content equivalent to 60% water-filled pore space (WFPS) and fertilised with 20 mg N per kg soil, added as NaNO₃ (10 mg N) and NH₄Cl (10 mg N). Three treatments were prepared: natural abundance (NA), labelled with ¹⁵N nitrate (¹⁵NO₃) and labelled with ¹⁵N ammonium (¹⁵NH₄). For the ¹⁵NO₃ treatment, NaNO₃ solution with 72 atom % ¹⁵N was added and for the ¹⁵NH₄ treatment, NH₄Cl solution with 63 atom % ¹⁵N was added. Then soils were thoroughly mixed to obtain homogenous distribution of water and fertilizer and an equivalent of 1.69 kg dry soil was repacked into each incubation column with bulk density of 1.3 g cm⁻³.

For each treatment 14 soil columns were prepared, and half of them received additional water injected on the top of the column (100 mL water added) to prepare two moisture treatments: L1 (61% WFPS) and L2 (72% WFPS). The incubation lasted 12 days. In the meantime, on the 6th day of incubation, water addition on the top of each column was repeated (80 mL water added) to increase the soil moisture in both treatments to ca. 68% WFPS in L1 and ca. 81% WFPS in L2. The strategy of adding water on the top of the column to achieve target water content was necessary to allow mixing and compaction at a suitable (low) water content of the soil and thus to optimise homogeneity of water and fertilizer distribution³. The incubation temperature was 20 °C. The columns were continuously flushed with a gas mixture with reduced N₂ content to increase the measurements sensitivity (2% N₂ and 21% O₂ in He³⁵) with a flow of 9 mL min⁻¹. Gas samples were collected daily into two 12 mL septum-capped Exetainer vials (Labco Limited, Ceredigion, UK) connected to the vents of the incubation columns. Soil samples were collected 5 times during the incubation by sacrificing one incubation column per sampling event, which was then divided into three subsamples (replicate samples of mixed soil).

Anoxic incubations: L3 experiment

The same soil was used for the static incubations performed under an anoxic atmosphere (N₂) in closed, gas-tight vessels, where denitrification products accumulated in the headspace. The incubation was conducted from 13.07.2020 to 15.07.2020. The soil was air dried and sieved at 4 mm mesh size. Afterwards, the soil was rewetted to achieve a water content equivalent to 70% water-filled pore space (WFPS) and fertilised with 100 mg N per kg soil, added as NaNO₃ using natural Chile saltpetre (NaNO₃, Chili Borium Plus, Prills-Natural origin, supplied by Yara, Dülmen, Germany, δ¹⁸O = 56‰, Δ¹⁷O = 21.8‰) to prepare 12 incubation soil samples of NA treatment and Na¹⁵NO₃ to prepare 6 incubation soil samples of ¹⁵NO₃⁻ treatment. The soil was thoroughly mixed to obtain a homogenous distribution of water and fertilizer and an equivalent of 85 g of dry soil was repacked into each incubation jar at bulk densities of 1.3 g cm⁻³. The 0.5 dm³ Mason jars were used with airtight rubber seals and with two three-way valves installed in their cover to enable sampling and flushing. The jars were flushed with N₂ at approximately 500 cm³ min⁻¹ (STP: 273.15 K, 100 kPa) for 10 min to create anoxic conditions. In 6 NA vessels and three ¹⁵NO₃⁻ vessels 50 dm³ of headspace N₂ was replaced with 50 dm³ of acetylene to inhibit N₂O reduction to N₂. Half of the incubation vessels of each treatment was incubated for 45 h and the other half was finished after 21 h for destructive sampling for soil mineral N analyses. The incubation temperature was 20 °C. Four gas samples were collected in 10 to 12 h-intervals by transferring 30 cm³ of headspace gases into two pre-evacuated 12 cm³ Exetainer vials (Labco Limited, Ceredigion, UK). The excess 3 cm³ of headspace gas in each vial ensured that no ambient air entered the vials. The removed sample volume was immediately replaced by pure N₂ gas.

Soil analyses

All soil samples were homogenized. Soil water content was determined by weight loss after 24 h drying at 110 °C. Soil pH was determined in 0.01 mol CaCl₂ solution (ratio 1:5). Nitrate and ammonium concentrations were determined by extraction in 2 M KCl in 1:4 ratio by 1 h shaking. Nitrite concentration was determined in alkaline extraction solution of 2 M KCl with addition of 2 M KOH (25 mL per L) in 1:1 ratio for 1 min of intensive shaking³⁶. The amount of added KOH was adjusted to keep the alkaline conditions in extracts (pH over 8). After shaking, the samples were centrifuged for 5 min and filtered. The extracts for NO₂⁻ measurements were stored at − 4 °C and analyzed within 5 days. NO₃⁻, NH₄⁺ and NO₂⁻ concentrations were determined colorimetrically with an automated analyser (Skalar Analytical B.V., Breda, the Netherlands).

To determine isotopic signatures of mineral nitrogen in NA treatments, microbial analytical methods were applied. For nitrate, the bacterial denitrification method with Pseudomonas aureofaciens was applied^37,38. For nitrite, the bacterial denitrification method for selective nitrite reduction with Stenotrophomonas nitritireducens was applied⁶, also for ¹⁵N -enriched samples from ¹⁵N treatments. For ammonium, a chemical conversion to nitrite with hypobromite oxidation³⁹ followed by bacterial conversion of nitrite after pH adjustment was applied⁴⁰. δ¹⁵N of the organic N was analysed in the flushed and dried soil sample after mineral N extractions by EA combustion coupled to Delta Plus mass spectrometer (Thermo Finnigan, Bremen, Germany).

In ¹⁵N treatments, ¹⁵N abundances of NO₃⁻ (a_NO3−) and NH₄⁺ (a_NH4+) were measured as described in Eschenbach, et al.⁴¹. NO₃⁻ was reduced to NO by Vanadium-III chloride (VCl₃) and NH₄⁺ was oxidized to N₂ by hypobromite (NaOBr). NO and N₂ were used as measurement gas. Measurements were performed on isotope ratio mass spectrometer (Delta Plus, Thermo Finnigan, Bremen, Germany).

Soil water was extracted with the method described by Königer, et al.⁴² and the δ¹⁸O of water samples (with respect to VSMOW) was measured using cavity ringdown spectrometer Picarro L1115-i (Picarro Inc., Santa Clara, USA). The measurement repeatability (1σ) of the internal standards (three calibrated waters with known δ¹⁸O: − 19.67‰, − 8.60‰, + 1.37‰) was below 0.1‰. The overall error associated with the soil water extraction method determined as standard deviation (1σ) of the 5 samples replicates was below 0.5‰.

All isotopic values are expressed as ‰ deviation from the ¹⁵N/¹⁴N and ¹⁸O/¹⁶O ratios of the reference materials (i.e. atmospheric N₂ and Vienna Standard Mean Ocean Water (VSMOW), respectively).

Gas analyses

The samples for gas concentration analyses were collected in Exetainer vials (Labco Limited, Ceredigion, UK) and were analysed using an Agilent 7890A gas chromatograph (GC) (Agilent Technologies, Santa Clara, CA, USA) equipped with an electron capture detector (ECD). Measurement repeatability as given by the relative standard deviation (1σ) of four standard gas mixtures was typically 1.5%.

The gas samples collected from ¹⁵N treatments were analyzed for a¹⁵N_N2O (¹⁵N abundance in the emitted N₂O), a_{P_N2O} (¹⁵N abundance in the ¹⁵N-pool derived N₂O) and f_{P_N2O} (¹⁵N-pool derived fraction of N₂O)¹⁵ with a modified GasBench II preparation system coupled to MAT 253 isotope ratio mass spectrometer (Thermo Scientific, Bremen, Germany) according to Lewicka-Szczebak et al.⁴³. In this set-up, N₂O is converted to N₂ during in-line reduction, and stable isotope ratios ²⁹R (²⁹N₂/²⁸N₂) and ³⁰R (³⁰N₂/²⁹N₂), of N₂ are determined.

The gas samples of the NA treatment were analysed for N₂O isotopocules (δ¹⁵N_N2O, δ¹⁸O_N2O, δ¹⁵N^SP_N2O) using a Delta V isotope ratio mass spectrometer (Thermo Scientific, Bremen, Germany), coupled to an automatic preparation system with Precon + Trace GC Isolink (Thermo Scientific, Bremen, Germany), where N₂O was pre-concentrated, separated and purified, and m/z 44, 45, and 46 of the intact N₂O⁺ ions as well as m/z 30 and 31 of NO⁺ fragment ions were determined. The results were evaluated accordingly^44,45,46 which allows the determination of average δ¹⁵N, δ¹⁵N^α (δ¹⁵N of the central N position of the N₂O molecule), and δ¹⁸O. δ¹⁵N^β (δ¹⁵N of the peripheral N position of the N₂O molecule) was calculated as δ¹⁵N = ( δ¹⁵N^α + δ¹⁵N^β)/2 and ¹⁵N site preference (δ¹⁵N^SP) as δ¹⁵N^SP = δ¹⁵N^α—δ¹⁵N^β.

Determination of Δ¹⁷O excess in N₂O and NO₃ ⁻ and estimation of O-atoms exchange (x)

N₂O samples collected in the L3 NA treatment and N₂O produced from soil NO₃⁻ by the bacterial denitrifier method were analysed for Δ¹⁷O after microwave equilibration in a sapphire tube and separation of N₂ and O₂ on a mole sieve column⁴⁷. The ¹⁷O excess, Δ¹⁷O, is defined as⁴⁸:

$$ \Delta^{17} {\text{O}} = \frac{{{1} + \delta^{{{17}}} {\text{O}}}}{{{(1} + \delta^{{{18}}} {\text{O)}}^{{{0}{\text{.5279}}}} }} - 1 $$

(4)

The measurement repeatability (1σ) of the international standards (USGS34, USGS35) was typically 0.5‰ for Δ¹⁷O.

The extent of isotope exchange (x) was determined based on the comparison of Δ¹⁷O in soil nitrate and produced N₂O. It requires the application of nitrate characterised by high Δ¹⁷O. Therefore, for this determination, soils in L3 were amended with natural NaNO₃ Chile saltpetre showing high Δ¹⁷O (of 21.8‰) and the Δ¹⁷O of the N₂O product was measured. Δ¹⁷O of soil water was assumed to be 0‰.

The magnitude of oxygen isotope exchange (x) was calculated as:

$$ x = 1 - \frac{{\Delta^{17} {\text{O}}({\text{N}}_{{2}} {\text{O}})}}{{\Delta^{17} {\text{O}}({\text{NO}}_{3}^{-} )}} $$

(5)

The accuracy of x determination was better than 1%.

Application of the Keeling plot

The original idea for Keeling plot application applies for mixing of the background low level (atmospheric CO₂) and one dominant source responsible for the significant increase of the CO₂ concentration¹⁶. In such a case, plotting the δ values against the reciprocal CO₂ concentration reveals the isotopic signature of the dominant as intercept of the linear fit¹⁶. Afterwards, the application of Keeling approach to isotopic studies has expanded to the other environments and substances, including nitrates source identification^17,18,49. In these studies the requirement of only two sources is not necessarily fulfilled, but the occurrence of a clear linear relation between isotopic signature and reciprocal concentration of the studied substance indicates that there is a dominant source which can be isotopically characterised⁴⁹. This is clearly the case for our nitrite samples, where we find a very significant linear relation (Fig. 1A). Nitrite contents in soils are typically very low and only rarely accumulate, mostly as a result of intensified nitrification or denitrification processes^{11,12,49,50,51}. Hence, with Keeling plot we can isotopically identify the dominant NO₂⁻ source and identify the pathway responsible for this accumulation.

Isotope fractionation factors for the nitrite model

The isotope fractionation factors are always expressed as:

$$ \varepsilon_{{{\text{product}}/{\text{substrate}}}} = \, \delta_{{{\text{product}}}} - \, \delta_{{{\text{substrate}}}} $$

Hence, negative ε values inform about normal isotope effect resulting in product depletion in heavy isotopes.

Nitrite sources

NAR is associated with quite high isotopic fractionation of N and O, resulting in significant depletion in ¹⁵N and ¹⁸O in the product NO₂⁻. The nitrate reductase enzymatic experiments showed a mean ¹⁵ε_NAR of − 26.6 ± 0.2‰, similar to ¹⁸ε_DEN with a mean of − 24.9 ± 0.3‰²⁵. In pure culture bacterial studies much larger variations of ¹⁵ε_DEN were observed, i.e. ranging from − 30.5 to − 5.4‰^22,24,26 and it has been suggested that the range from − 15 to − 10‰ is most representative for typical cellular nitrate reduction rates for bacterial strains²⁷. The strongest fractionation was found for pure culture fungal studies with a mean ¹⁵ε_NAR of − 37.8 ± 6.6‰²⁰. Similar values were found for ¹⁸ε_DEN in pure culture studies: ranging between − 30 and − 25‰ for bacterial denitrification²³ and between − 30 and − 10‰ for fungal denitrification²⁰ . In the sediment denitrification experiments ¹⁵ε_DEN ranged from − 24.4 to − 18.9‰ and ¹⁸ε_DEN from − 21.9 to − 15.8‰^8,29. A slightly lower ¹⁵ε_DEN of − 29.4 ± 2.4‰ was determined for soil studies²⁸.

Nitrite produced from AOX is depleted in ¹⁵N compared to its ammonium substrate. Bacterial ammonia oxidation show a mean ¹⁵ε_AOX of − 25.8 ± 9.8‰⁵², similar to archaeal ammonia oxidation with a mean ¹⁵ε_AOX of − 22 ± 5‰⁵³. δ¹⁸O_NO2- from AOX depends on δ¹⁸O_O2 (+ 23.5‰), δ¹⁸O_H2O (− 5‰) and δ¹⁸O_N2Oeq (8.6‰) according to the equation^7,54:

$${\delta }^{18}{O}_{AOX}=0.5*\left({\delta }^{18}{O}_{O2}+{\delta }^{18}{O}_{H2O}+20\right)*0.92+\left({\delta }^{18}{O}_{H2O}+{\delta }^{18}{O}_{NO2-eq}\right)*0.08$$

(6)

Nitrite produced from ORG show much lower ¹⁵N enrichment with a mean ¹⁵ε_ORG of about − 2‰ as measured for marine sediments fractionation⁵⁵. δ¹⁸O_NO2− from ORG was assumed to be the same as for AOX according to Eq. (3) (+ 18.4‰).

Nitrite sinks

Two major nitrite sinks—reduction and oxidation—show opposite isotopic fractionation. Nitrite reduction is associated with normal isotope effect resulting in enrichment in ¹⁵N and ¹⁸O of the nitrite pool, whereas nitrite oxidation is characterised by inverse isotope effect, where heavy isotopes are preferentially transferred to the oxidised product leaving nitrite pool depleted in ¹⁵N and ¹⁸O⁷. For NIR different fractionation may be associated with various nitrite reductases involved, showing a ¹⁵ε_NIR of − 22 ± 2‰ and an ¹⁸ε_NIR of − 2 ± 2‰ for Cu-NIR and − 8 ± 2‰ and − 6 ± 2‰ respectively for Fe-NIR⁵⁶. In batch experiments with environmental bacterial communities a ¹⁵ε_DNIR ranging from − 15 to − 10‰ was observed when nitrite was investigated as an intermediate product but much lower when nitrite was a substrate²⁹. Here we probably also observe this for L3—where nitrite is accumulating we get the best fit with the measured values when no fractionation associated with NIR is assumed (Table 2).

For nitrite oxidation the inverse isotope effects with a ¹⁵ε_NOX of + 12.8⁵⁷ and an ¹⁸ε_NOX of + 5‰³⁰ were found.

Nitrite equilibration with water

The oxygen isotope signature of NO₂⁻ is additionally modified by the abiotic equilibrium exchange with ambient water²³. The magnitude of this exchange is governed by the equilibrium isotope effect between NO₂⁻ and water (ε_eq) which is a function of temperature^7,23 and the extend of O atoms exchange. ε_eq for the incubation temperature of 20 °C equals 13.63, δ¹⁸O_H2O is − 5‰, consequently, the δ¹⁸O of nitrite in complete equilibrium with water is 8.6‰. The extend of O atoms exchange was determined with the ¹⁷O approach²¹ for N₂O originating for denitrification processes in anoxic experiment L3 and equalled 0.25.