Interactive effects of MnO₂, organic matter and pH on abiotic formation of N₂O from hydroxylamine in artificial soil mixtures

Abstract

Abiotic conversion of the reactive nitrification intermediate hydroxylamine (NH₂OH) to nitrous oxide (N₂O) is a possible mechanism of N₂O formation during nitrification. Previous research has demonstrated that manganese dioxide (MnO₂) and organic matter (OM) content of soil as well as soil pH are important control variables of N₂O formation in the soil. But until now, their combined effect on abiotic N₂O formation from NH₂OH has not been quantified. Here, we present results from a full-factorial experiment with artificial soil mixtures at five different levels of pH, MnO₂ and OM, respectively, and quantified the interactive effects of the three variables on the NH₂OH-to-N₂O conversion ratio (R_NH2OH-to-N2O). Furthermore, the effect of OM quality on R_NH2OH-to-N2O was determined by the addition of four different organic materials with different C/N ratios to the artificial soil mixtures. The experiments revealed a strong interactive effect of soil pH, MnO₂ and OM on R_NH2OH-to-N2O. In general, increasing MnO₂ and decreasing pH increased R_NH2OH-to-N2O, while increasing OM content was associated with a decrease in R_NH2OH-to-N2O. Organic matter quality also affected R_NH2OH-to-N2O. However, this effect was not a function of C/N ratio, but was rather related to differences in the dominating functional groups between the different organic materials.

Introduction

Nitrous oxide (N₂O) is a potent greenhouse gas that can be formed by several soil processes, such as microbial nitrification and denitrification. The N₂O production from nitrification, especially from its reactive intermediate hydroxylamine (NH₂OH), has received increasing attention in the recent past, fostered by the development of analytical techniques for the determination of the ¹⁵N site preference in the N₂O molecule that allows for constraining the contribution of different source processes to total N₂O formation^1,2,3,4. Also, increasing knowledge from molecular biological and genetic studies has contributed to elucidating the different N₂O formation mechanisms during nitrification³. Still, the role of NH₂OH in N₂O formation in the soil is insufficiently understood. While there is evidence, e.g., from measurements in wastewater treatment systems that NH₂OH can contribute about 65% of total N₂O formation², the formation of N₂O from NH₂OH in soil and its controlling factors have rarely been studied^5,6.

Hydroxylamine was first identified by Lees (1952)⁷ as an intermediate of the first step of nitrification by ammonia oxidizing bacteria (AOB), in which ammonia is oxidized to nitrite. Understanding the nitrification process in ammonia-oxidizing archaea (AOA), however, is much more fragmentary, but NH₂OH has been identified as an intermediate of ammonia oxidation also in AOA⁸. In most circumstances, NH₂OH is quickly oxidized to nitrite in the periplasm of the AOB, and N₂O may be produced as a side product during this process³. However, also a leakage of NH₂OH from the periplasm across the outer membrane of the AOB into the soil matrix, followed by a chemical reaction with soil constituents yielding N₂O, could be a potential mechanism of N₂O formation during nitrification. This assumption is supported by the fact that AOB can take up NH₂OH from the surrounding medium⁹ as well as by the observation that the medium of AOB cultures contains measurable amounts of NH₂OH. The latter was found for Nitrosomonas europaea under oxic conditions, both for wild-type N. europaea and even more so for NirK and NorB-deficient mutants¹⁰. In accordance with this assumption, a positive relationship between NH₂OH content of the soil and soil N₂O emissions under oxic conditions has been detected in natural forest soil samples¹¹. In addition, abiotic formation of N₂O from NH₂OH has been observed in sterilized soil samples from different ecosystems⁶.

In soil, N₂O can be formed chemically, among a range of possible reactions, according to the following equations¹²:

Owing to its high oxidization potential, manganese dioxide (MnO₂) acts as a strong oxidant in soil that plays an important role not only in the turnover of organic substances^13,14, but also in the N cycle¹⁵, even under anoxic conditions^16,17. Soil organic matter (SOM) plays a crucial role in the storage and release of N as well as in the emission of N₂O from soils. Quick disappearance of nitrite and nitrate within a few hours after addition has been observed in forest soils^18,19,20, whereas NH₂OH disappeared completely in soil several minutes after addition^5,11. Abiotic reactions of SOM and inorganic N may contribute to the quick disappearance, as nitrite and nitrate can react with SOM or dissolved organic carbon (DOC), leading to the formation of organic N, such as nitroso and nitro compounds^21,22, while NH₂OH can also react with carbonyl groups to form oximes^23,24:

The quality of SOM, or more specifically the C/N ratio and the type and abundance of functional groups, influence the bonding of inorganic N to SOM²². Phenolic lignin derivatives, an important constituent of SOM, can covalently bind reactive N compounds and thereby stabilize N in soil^25,26. The N binding form can be affected by the plant species from which the SOM is derived due to the different characteristics of phenolic compounds, e.g. condensed or hydrozable tannin²⁷.

Soil pH is another key factor influencing most nitrogen transformations in soil. High pH can lead to an increase of chemical N₂O production involving nitrite by favoring nitrite accumulation, either directly through increasing nitrite stability, or indirectly by inhibiting biological nitrite oxidation due to a higher concentration of free NH₃ (an inhibitor of nitrite oxidizers) in the soil²⁸. In contrast, high soil N₂O emissions have also been observed in acid forest soils^29,30. In this case, the effect of pH on enzyme activities during denitrification and nitrification was suggested as the main reason³¹. However, also chemical reactions that produce N₂O in the soil, such as the reaction of nitrite with SOM and the reaction of NH₂OH with MnO₂, are subject to a strong pH dependence and can contribute substantially to N₂O emissions under acidic conditions^32,33,34.

The aim of this study was to quantify the interactive effects of the major control factors of abiotic N₂O formation from NH₂OH in soil, i.e. MnO₂ content, pH and OM quantity and quality, by means of experiments with artificial soil mixtures. We hypothesized that the control factors interact with each other in the following way: At higher pH, unprotonated NH₂OH would react more readily with carbonyl groups of OM, leading to oxime formation and making NH₂OH less available for oxidation to N₂O by MnO₂. Lower soil pH would lead to increased protonation of NH₂OH, making NH₂OH more stable against the reaction with carbonyl groups of OM and more prone to the reaction with MnO₂, leading to higher N₂O formation from the same amount of NH₂OH (Fig. 1). To test these hypotheses, we performed two laboratory experiments with artificial soil mixtures, which were produced from pure quartz sand, quartz powder, kaolin clay, MnO₂ powder and different plant-derived organic materials, resembling SOM of different quality, at different mixing ratios. In these experiments, N₂O formation was determined after NH₂OH addition to the different mixtures at different pH levels and related to the different control factors.

Figure 1: Hypothetical model of NH₂OH release by ammonia-oxidizing bacteria to the soil environment and potential abiotic reactions of NH₂OH with MnO₂ and organic matter in the soil at different pH conditions (R₁R₂C=O represents carbonyl groups of SOM).

AMO is ammonia monooxygenase; HAO is hydroxylamine oxidoreductase.

Results and Discussion

R_NH2OH-to-N2O at different pH, MnO₂ and OM contents (%)

In the present study all three factors, i.e. pH, MnO₂ and OM content, affected R_NH2OH-to-N2O from peat moss significantly (Fig. 2, S1 and S2). The R_NH2OH-to-N2O increased greatly with an increase in MnO₂ content from 0% to 0.1% (Fig. 2). This finding is consistent with Bremner et al.⁵, who studied 19 soils with a wide range of properties and found that the formation of N₂O by decomposition of NH₂OH was highly correlated with oxidized Mn content of the soils. The fact that NH₂OH was used in the past for the selective extraction of Mn oxides from soil samples³⁵ indicates that NH₂OH can efficiently reduce Mn(IV) to Mn(II) or Mn(III) (and in turn is oxidized to N₂O) in natural soil samples. With increasing OM content, R_NH2OH-to-N2O decreased remarkably, especially at high pH (Fig. 2c,d,e). For example, an increase in OM by only 1% at 0.01% MnO₂ led to about 50% and 80% decrease in N₂O emissions at pH 3 and pH 7, respectively (Fig. 2e, S2). This could be caused by the oxime-forming reaction between NH₂OH and carbonyl groups of OM, such as in quinones. The oximes may undergo a tautomeric equilibrium with their corresponding nitrosophenol forms²³. In fact, NH₂OH has been used in a number of previous studies to determine the carbonyl content of humic substances³⁶, indicating a high affinity of NH₂OH to OM that contains carbonyl groups. In the absence of OM and MnO₂, increasing pH led to a slight increase in R_NH2OH-to-N2O due to the self-decomposition of NH₂OH at higher pH, whereas in the presence of OM and absence of MnO₂ nearly no NH₂OH was converted to N₂O (Fig. S2a). In contrast, the effect of increasing pH on R_NH2OH-to-N2O became negative already in the presence of 0.01% MnO₂ (Fig. S2b). This finding suggests that acidic conditions are favorable for the redox reaction between NH₂OH and MnO₂.

Figure 2: Mean NH₂OH-to-N₂O conversion ratios (R_NH2OH-to-N2O) in artificial soil mixtures at different pH as well as MnO₂ and organic matter (OM, peat moss) contents.

The total amount of NH₂OH added was 5 nmol (equivalent to 70 μg N per kg dry material). Different symbols represent R_NH2OH-to-N2O at different OM content (n = 3, SD < 5%, not shown).

Also strong interactive effects of pH and MnO₂, pH and OM, and OM and MnO₂ were observed for the conversion of NH₂OH to N₂O. The largest R_NH2OH-to-N2O found in the present experiment was 81.5% in the absence of SOM at pH 3, and with a MnO₂ content of 0.1% (Fig. 2a), while the lowest R_NH2OH-to-N2O was about 9%, when SOM content was 10% in the presence of 0.1% MnO₂ at pH 7 (Fig. 2e). This suggests that even at the highest MnO₂ level and in all other respects optimal conditions a small fraction of NH₂OH had not been converted to N₂O, but to some other unidentified product.

In the treatments without OM, MnO₂ had only a small effect on R_NH2OH-to-N2O at all pH conditions, while it had a larger effect especially at higher OM content (Fig. 2, S1), suggesting a strong competition between OM and MnO₂ for NH₂OH. The competition was biased by pH, with lower pH favouring the reaction of NH₂OH and MnO₂, while higher pH favoured the reaction of NH₂OH with OM. These findings confirmed our hypothesis that at low pH NH₂OH is more protected against reaction with OM and more available for the oxidation by MnO₂ due to the higher degree of NH₂OH protonation at lower pH.

R_NH2OH-to-N2O as a function of pH, MnO₂ content and OM quality

Organic matter quality had a clear influence on R_NH2OH-to-N2O in this study (Fig. 3, S3, and S4). Most of the OM types were associated with a significantly lower R_NH2OH-to-N2O compared to the mixtures without OM within the pH range of the experiment. In general, the inhibitory effect of the organic materials on the conversion of NH₂OH to N₂O showed a clear pH dependency, but was not a function of C/N ratio (Fig. 3, S3). At acidic conditions (pH 3–4), peat moss and watermilfoil with their relatively large C/N ratio inhibited R_NH2OH-to-N2O the least, while the cyanobacterium material and clover had a stronger inhibitory effect on R_NH2OH-to-N2O despite their smaller C/N ratio (Fig. 3a,b). The differences between peat moss, cyanobacterium and watermilfoil material as OM became smaller at higher pH, and were no longer significant at pH 7 in the presence of 0.01% MnO₂ (Fig. 3e), while clover showed always the smallest R_NH2OH-to-N2O at all pH levels. In the absence of MnO₂, all OM forms showed a R_NH2OH-to-N2O close to zero, except for the watermilfoil material that was associated with a R_NH2OH-to-N2O significantly above zero within the pH range 3–6 (Fig. S4a). A possible explanation could be the fact that, in contrast to the other OM sources, the watermilfoil material contained about 0.03% Mn (Table 1), which could have caused the N₂O emission after NH₂OH addition even without external MnO₂ addition.

Figure 3: Mean NH₂OH-to-N₂O conversion ratios (R_NH2OH-to-N2O) in artificial soils at different pH and MnO₂ content, and for organic matter of different origins at a fixed content of 2.5% (w/w).

The total amount of NH₂OH added was 5 nmol. Different symbols represent R_NH2OH-to-N2O for the artificial soil mixtures with the different organic materials (n = 3, SD < 5%, not shown).

Table 1 Element contents (%) and C/N ratios of the organic materials used in this study.

We assumed that R_NH2OH-to-N2O would be a function of the C/N ratio of the different SOM types, as larger C/N ratios would be indicative of a lower degree of N-containing functional groups, i.e. leaving a higher chance for NH₂OH to react with SOM and not to be converted to N₂O. However, as stated above we did not observe any clear relationship between C/N ratio and R_NH2OH-to-N2O, e.g. peat moss had the largest C/N ratio, but did not lead to the lowest R_NH2OH-to-N2O. Instead, clover with a much lower C/N ratio had the largest inhibitory effect on R_{NH2OH-to-N2O.} The addition of 2.5% dry clover powder (C/N ratio = 11.3) to the artificial soil mixture decreased R_NH2OH-to-N2O by 48% at pH 3 (Fig. 3a), which was similar to the effect of 10% peat moss (C/N ratio = 67.2) at the same pH (Fig. 2a). The reason for this observation could lie in the differences in functional groups between the different organic materials used in this study.

A better insight into the effects of C and N functional groups of the different organic materials was obtained from NMR analysis. The peat moss OM had the lowest proportion of ester or amide carbonyl at around 170 ppm of all materials (Fig. 4, Table 2). This is in accordance with the observation that – despite having the largest C/N ratio – peat moss OM had a lower inhibitory effect on R_NH2OH-to-N2O compared to clover and watermilfoil OM (if the background MnO₂ effect was subtracted), i.e. the lack of almost any carbonyl groups in peat moss was clearly visible in its chemical behaviour toward NH₂OH. In addition, peat moss OM exhibited the largest proportion of O-substituted aliphatic compounds, which might have also contributed to the relatively low inhibitory effect on R_NH2OH-to-N2O in comparison to clover and watermilfoil OM. In contrast, cyanobacterium OM had the highest proportion of acid/amide carbonyl of all four organic materials, suggesting the highest inhibitory effect on R_NH2OH-to-N2O due to the competitive reaction of carbonyl groups with NH₂OH. The clover material, however, contained lower amounts of O-substituted aliphatics and di-O-substituted C in comparison to peat moss and watermilfoil OM, which may have increased its affinity for NH₂OH. For the proportion of unsaturated C no clear trend emerged across the different materials, suggesting that the effect of unsaturated C on R_NH2OH-to-N2O is of minor importance.

Figure 4

¹³C- and ¹⁵N-CPMAS-NMR spectra of the different organic materials (cyanobacterium, clover, watermilfoil, peat moss) used in the experiment.

Table 2 Relative proportions of chemical features of the different plant materials derived from ¹³C CPMAS NMR spectra.

Development of a stepwise multiple regression model from the artificial soil mixtures and application to natural soils

The multiple regression model obtained from the first experiments was R_NH2OH-to-N2O = 45.9–3.1 SOM + 241.1 MnO₂ − 4.5 pH, R² = 0.62 (P < 0.01), which could explain about 62% variation of R_NH2OH-to-N2O, and the contributions of pH, Mn and SOM content to the model’s performance were all significant (P < 0.01). It could well explain the observations (Fig. 3) for peat moss, watermilfoil and clover OM (R² close to 0.8, P < 0.01, Fig. 5). This demonstrated the general applicability of the model for the OM derived from the different plant and cyanobacterium materials, with different N content, aliphatic C content and C/N ratios. In contrast, the model proved to be not appropriate for the artificial soil mixture without any MnO₂, indicated by the decreased goodness of the simulation.

Figure 5: Results of the application of the artificial soil regression model for the calculation of NH₂OH-to-N₂O conversion ratios (R_NH2OH-to-N2O) to artificial soil mixtures amended with the different organic materials (n = 22).

The three points for which R_NH2OH-to-N2O was determined at pH 3, 4, and 5 without MnO₂ addition were excluded from the simulation.

Finally, R_NH2OH-to-N2O was simulated with the same regression model for the natural soils described in Heil et al.⁶. The results showed that the application of the model to natural soils was promising, no matter if it was applied to fumigated or fresh soils (Fig. 6). The simulated R_NH2OH-to-N2O explained more than 90% of the observed rates, especially for cropland, grassland and deciduous forest soils. However, the model failed at correctly predicting R_NH2OH-to-N2O for the spruce forest soil of Heil et al.⁶, which could be related to the high SOM and relatively low MnO₂ content of the spruce soil as compared to the other soils. This finding suggests that there is a threshold value for the SOM content of 10% above which – and a MnO₂ content of 0.01% below which – the model fails to predict the correct R_NH2OH-to-N2O values.

Figure 6

Results of the application of the artificial soil regression model for the calculation of NH₂OH-to-N₂O conversion ratios (R_NH2OH-to-N2O) to six natural fresh and chloroform-fumigated soils as reported in Heil et al.⁶.

Soil pH, MnO₂ and SOM content were identified as crucial control variables of R_NH2OH-to-N2O, i.e. the conversion ratio of NH₂OH to N₂O in the artificial soil experiments of this study. Organic matter derived from different plant species and a cyanobacterium also affected R_NH2OH-to-N2O due to the differences in composition, type and abundance of functional groups, as more carbonyl C leads to higher reactivity of NH₂OH with organic matter, thereby lowering its availability for the oxidation to N₂O by MnO₂. The multiple regression model of pH, MnO₂ and OM developed here could explain about 60% of the variance of R_NH2OH-to-N2O in the artificial soil mixtures, and proved also to be promising for the prediction of R_NH2OH-to-N2O of chemical N₂O production from NH₂OH in natural soils, when SOM content was below 10% and Mn content was larger than 0.01%. If these findings can be confirmed for other soils from different ecosystems, this improved understanding of the controls of N₂O formation from the reactive nitrification intermediate NH₂OH in soils can have large implications for developing appropriate management options, such as adding organic amendments with suitable chemical characteristics, for mitigating N₂O emissions from agricultural land, the largest anthropogenic source of N₂O to the atmosphere.

Methods

Experimental setup

Two full-factorial artificial soil experiments were conducted. The first experiment comprised three factors (pH, MnO₂ and OM content) and five levels of each factor. The scond experiment comprised also three factors (pH, MnO₂ and OM quality) with five levels of pH and MnO₂, and four different organic materials at the same concentration level (2.5% w/w on a dry weight basis), but of different quality. Each experiment was conducted in triplicate.

Preparation of the artificial soil mixtures

The artificial soil mixtures consisted of 15% (expressed as percentage of dry weight) fine quartz sand (50% of the particles 0.05–0.2 mm), representing the sand fraction, 65% quartz powder (0.002–0.063 mm), representing the silt fraction, and 20% kaolin clay (≤0.002 mm), representing the clay fraction, mimicking the soil texture of the agricultural Terrestrial Environmental Observatories (TERENO) field site Selhausen³⁷. Freeze-dried, finely ground and sieved (<0.75 mm) peat moss (Sphagnum magellanicum, collected from Dürres Maar, Eifel, Germany) was amended as SOM to the artificial soil mixtures at levels of 0%, 1%, 2.5%, 5%, 10% dry weight, while the relative amount of sand, clay and silt was reduced according to the amount of peat moss added. The water holding capacity (WHC) was determined for each of the artificial soil mixtures. The WHC increased with increasing organic matter (OM) content, and amounted to 29%, 44%, 55%, 76%, and 132% for the five OM contents, respectively. Each of those artificial soil mixtures was amended with MnO₂ (Merck, Darmstadt, Germany) at five different levels (0%, 0.01%, 0.025%, 0.05%, 0.1% Mn), then the ingredients were thoroughly homogenized.

Preparation of artificial soil mixtures with different OM qualities

Organic materials with different C/N ratios (Table 1) were derived from two different plant species, i.e. watermilfoil (Myriophyllum spec.) and clover (Trifolium repens), and from a cyanobacterium (Spirulina platensis). Watermilfoil and clover had been collected previously on the campus of Forschungszentrum Jülich (2004 and 2014, respectively), while the cyanobacterium material had been purchased in 2006 (Concept Vitalprodukte, Schwerte, Nordrhein-Westfalen, Germany). The finely ground, freeze-dried and sieved (<0.75 mm) organic material was amended to the inorganic quartz-kaolin mixture as described above at a rate of 2.5% dry weight, while the relative amount of sand, clay and silt was reduced accordingly. Also for this experiment, each of the artificial soil mixtures was amended with MnO₂ at five different levels (0%, 0.01%, 0.025%, 0.05%, 0.1% Mn), and again mixed thoroughly to obtain a homogeneous composition.

Addition of NH₂OH to the artificial soil mixtures and analysis of the N₂O formed

One gram of each artificial soil mixture was weighed into individual 22-mL gas chromatograph (GC) vials. Subsequently, NH₂OH in different buffer solutions was added to each vial to obtain a soil water content of 50% WHC, which required addition of varying volumes of buffer solution to the different soil mixtures depending on the OM content, and adaptation of the NH₂OH concentration of each of the buffer solutions accordingly. The total amount of NH₂OH added to each of the soil mixtures was always 5 nmol (equivalent to 70 μg N per kg dry material). The pH buffer solutions at pH 3, 4, 5 and 6 were prepared with citric acid (0.1 M) and sodium citrate (0.1 M) according to Gomori³⁸, whereas the buffer at pH 7 was prepared with tris(hydroxymethyl)aminomethane and maleate (Tris-maleate buffer). The vials were closed immediately after NH₂OH addition. After 10 hours of incubation, the N₂O concentration in the headspace of the vials was measured with a GC equipped with an electron capture detector (Clarus 580, PerkinElmer, Rodgau, Germany). Details of the GC setup and analytical conditions have been described previously¹¹.

Calculation of the NH₂OH-to-N₂O conversion ratio

The NH₂OH-to-N₂O conversion ratio (R_NH2OH-to-N2O, moles N₂O-N per mole NH₂OH-N, %) was determined according to the following equation:

where c₀ is the background N₂O mixing ratio in the headspace of the control with the same amount of water instead of NH₂OH solution (nL L⁻¹); c₁ is the N₂O mixing ratio in the headspace of the sample with NH₂OH addition (nL L⁻¹); the factor 2 represents the molar N ratio of N₂O and NH₂OH; V is the volume of the vial headspace (0.022 L); V_m is the molar volume of N₂O at standard pressure and room temperature (24.465 L mol⁻¹); n is the amount of NH₂OH added to the sample vials (5 nmol).

Determination of the basic properties of the organic materials

Three replicates of each organic material were analyzed to determine its basic properties. The C and N content of the different organic materials was analyzed by weighing 200–300 μg dry material into tin capsules, followed by combustion at 1080 °C in an elemental analyzer (EuroEA, EuroVector, Milan, Italy) interfaced to an isotope-ratio mass spectrometer (Isoprime, Isoprime Ltd, Stockport, United Kingdom). The C and N content was determined through peak integration of m/z 44 (CO₂) and 28 (N₂), respectively, and calibrated against elemental standards.

The elemental composition of the organic materials was analyzed by using inductively coupled plasma optical emission spectrometry (ICP-OES) in the central analytical laboratory (ZEA-3) of Forschungszentrum Jülich. Briefly, 100 mg of sample material were mixed with 3 mL HNO₃ and 2 mL H₂O₂, heated in the microwave at 800 W for 30 min. The mixtures were subsequently filled up to 14 mL and diluted 10-fold with deionized water followed by the ICP-OES measurement.

For the determination of characteristic molecule structures and functional groups of the different organic materials used in the experiments, ¹³C and ¹⁵N cross-polarisation magic-angle spinning (CPMAS) nuclear magnetic resonance (NMR) spectra were obtained. ¹³C CPMAS spectra were obtained on a 7.05 T Varian INOVA^TM Unity (Varian Inc., Palo Alto, CA, USA) at a ¹³C resonance frequency of 75.4 MHz. ¹⁵N CPMAS spectra were obtained on a 14.09 T Varian NMR system (Varian Inc., Palo Alto, CA, USA) at a ¹⁵N resonance frequency of 60.8 MHz. Samples were packed into 6 mm diameter cylindrical zirconia rotors with Vespel^® drive tips and spun at 8000 ± 3 Hz in an HX Apex probe. The spectra were collected with a sweep width of 25 kHz and an acquisition time of 20 ms. In preliminary experiments, the optimal contact time and recycle delay for the cross-polarization experiment were determined. A contact time of 1 ms and a 5 s recycle delay time were used for ¹³C, whereas a contact time of 1 ms and a 1 s recycle delay time were used for ¹⁵N. During cross-polarization the ¹H radio frequency (RF) field strength was set to 47 kHz for ¹³C and to 33.7 kHz for ¹⁵N, respectively. The ¹³C and ¹⁵N RF field strength was set to 41 and 41.7 kHz, respectively. An ascending ramp of 15 and 12.2 kHz on the ¹H-RF field was used for ¹³C and ¹⁵N during contact time to account for inhomogeneities of the Hartmann-Hahn condition, respectively³⁹. Proton decoupling was done using a spinal sequence with a ¹H field strength of 50 and 35.6 kHz, a phase of 4.5° and 5.5°, and a pulse length of 12 and 9.5 μs, respectively.

The free induction decays (FID) were recorded with VnmrJ (Version 1.1 RevisionD, Varian Inc., Palo Alto, CA, USA) and processed with Mestre-C (Version 4.9.9.9, Mestrelab Research, Santiago de Compostela, Spain). All FIDs were fourier-transformed with an exponential filter function with a line broadening (LB) of 20 to 50 Hz. Baseline correction was done using the manual baseline correction function of Mestre-C.

The ¹³C chemical shifts are reported relative to tetramethylsilane (=0 ppm) using adamantane as an external reference. The relative intensities of the regions were determined using the integration routine of the MestRe-C software. The ¹⁵N chemical shifts are reported relative to ammonium nitrate (NH₄⁺ = 0 ppm).

Data analysis

The homogeneity of variance was tested with the Bartlett test. One-way analysis of variance (one-way ANOVA) of the main controlling factors in the two experiments was performed, followed by a Tukey Honest Significant Difference (HSD) test. A stepwise multiple regression model for the NH₂OH-to-N₂O conversion ratio was developed on the basis of the co-variables pH, MnO₂ and SOM content by using the data from the first experiment. In this case, significance was tested with the F test. Linear regression was performed for simulated and measured R_NH2OH-to-N2O in artificial and natural soils described in Heil et al.⁶ and tested for significance. All analyses were performed with the R software package (version 3.1.0, R Development Core Team, 2013)⁴⁰.