Improved isotopic model based on 15 N tracing and Rayleigh-type isotope fractionation for simulating differential sources of N2 O emissions in a clay grassland soil.

RATIONALE: Isotopic signatures of N2 O can help distinguish between two sources (fertiliser N or endogenous soil N) of N2 O emissions. The contribution of each source to N2 O emissions after N-application is difficult to determine. Here, isotopologue signatures of emitted N2 O are used in an improved isotopic model based on Rayleigh-type equations.
METHODS: The effects of a partial (33% of surface area, treatment 1c) or total (100% of surface area, treatment 3c) dispersal of N and C on gaseous emissions from denitrification were measured in a laboratory incubation system (DENIS) allowing simultaneous measurements of NO, N2 O, N2 and CO2 over a 12-day incubation period. To determine the source of N2 O emissions those results were combined with both the isotope ratio mass spectrometry analysis of the isotopocules of emitted N2 O and those from the 15 N-tracing technique.
RESULTS: The spatial dispersal of N and C significantly affected the quantity, but not the timing, of gas fluxes. Cumulative emissions are larger for treatment 3c than treatment 1c. The 15 N-enrichment analysis shows that initially ~70% of the emitted N2 O derived from the applied amendment followed by a constant decrease. The decrease in contribution of the fertiliser N-pool after an initial increase is sooner and larger for treatment 1c. The Rayleigh-type model applied to N2 O isotopocules data (δ15 Nbulk -N2 O values) shows poor agreement with the measurements for the original one-pool model for treatment 1c; the two-pool models gives better results when using a third-order polynomial equation. In contrast, in treatment 3c little difference is observed between the two modelling approaches.
CONCLUSIONS: The importance of N2 O emissions from different N-pools in soil for the interpretation of N2 O isotopocules data was demonstrated using a Rayleigh-type model. Earlier statements concerning exponential increase in native soil nitrate pool activity highlighted in previous studies should be replaced with a polynomial increase with dependency on both N-pool sizes.

INTRODUCTION

Agricultural soils rely on external n class="Cn class="Chemical">hemical">nitrogen (N) inputs and constitute a major source of <n class="Chemical">hemical">span class="Chemical">nitrous oxide (N2O) and nitric oxide (NO) emissions, accounting for around 10% of greenhouse gas (GHG) emissions from human activities1 and contributing to the formation of acid rain, eutrophication and ground level ozone.2 In soil, nitrification and denitrification are the most important microbial processes involved in the production of N2O, requiring high and low oxygen (O2) concentrations for the activation of each process, respectively. Moreover, when denitrification occurs, N applied to soils can be emitted back to the atmosphere as dinitrogen (N2). Many observations have suggested that sequential synthesis of denitrification enzymes is responsible for the delay in N2 appearance relative to N2O.3, 4, 5

Amongst the strategies to identify <n class="Chemical">span class="Chemical">N2O sources in the soil and their variation in hemical">space and time, the study of the natural abundance of stable isotopic signatures of <hemical">span class="Chemical">N2O,6, 7 such as the δ15N and δ18O values and the 15N site preference (SP), have gained attention ever since the early 2000s.8, 9, 10 The N2O produced from denitrification in soils tends to be associated with δ15N signatures with values in the range of −13 to −54‰11, 12 while those derived from nitrification are up to −60‰.11, 13 Moreover, reduction of N2O to N2 from denitrifying bacteria can be determined by isotopic discrimination as a consequence of the difference in reaction rates of the isotopically light (14N, 16O) and heavy (15N, 18O) molecules of N2O.14, 15, 16 Interpretation of N2O isotopomers as indicators of source processes has also been developed.17, 18 This approach is based on the difference in 15N occupation of the peripheral (β) and central N‐positions (α) of the linear molecule that defines the intra‐molecular 15N SP.19, 20 The SP is not dependent on the isotopic signature of the precursor,21 in contrast to average δ15N and δ18O values of N2O. However, Sutka et al22 found that the SP is increased during fungal denitrification and nitrification whereas N2O reduction via denitrification increases the SP by increasing the α‐site 15N‐enrichment in the residual N2O.9, 15 Wu et al23 subsequently quantified the potential bias on SP‐based N2O source partitioning using a closed‐system model.

n class="Chemical">Nitrogen fertiliser application to agricultural land can affect the isotopic signature of <hemical">span class="Chemical">N2O and result in two different pools of emissions: pool 1 from fertiliser addition and pool 2 from the native soil N. In addition to those two pools, spatial heterogeneity of denitrification can have a significant impact on N‐isotope patterns which might only occur in situations where available N and C are added at the same time, e.g. slurry, grazing excreta, urea fertiliser.24, 25, 26, 27 The isotope fractionation during N2O production7, 12 and reduction,15, 16 or when both processes take place simultaneously,26 has been previously reported. Moreover, a comprehensive review of isotope effects and isotope modelling approaches was recently presented by Denk et al.28 Previously, using a Rayleigh equation to describe isotopic fractionation,29 Well and Flessa12 concluded that the isotopic fingerprint of soil‐emitted N2O is a useful parameter to evaluate the contribution of different processes to the N2O flux in soils. However, the spatial extent and specific denitrification rates of hypothesized pools could only be constrained by fitting measured and modelled δ15Nbulk values, which were associated with considerable uncertainties on the volume and denitrification rates of the assumed pools. Modelling the isotope fractionation during production and reduction based on the measured temporal pattern of the δ15Nbulk‐N2O values suggested that there was a multi‐pool (non‐homogenous) distribution of nitrate (NO3 −) in the soil.25 Thus, evaluation of isotopologue signatures for identifying source processes was hampered by the simultaneous occurrence of several factors contributing to the time course of isotopic signatures, which could thus not be fully explained. In this sense, Lewicka‐Szczebak et al26 showed that higher denitrification rates resulted in decreasing net isotope effects during N2O production for 15N using a modelling approach. For N2O reduction, clearly diverse net isotope effects were observed for the two distinct soil pools. In addition, in a laboratory incubation carried out at different saturation levels for a grassland soil, Cardenas et al30 found that added N produced higher denitrification rates than soil N, resulting in less isotopic fractionation.

The kinetics of n class="Chemical">N transformations in soils has been previously explored using an isotopic model based on Rayleigh‐type equations.26 This model was developed to simulate δhemical">15N values of <hemical">span class="Chemical">N2O using process rates and associated fractionation factors, but assumptions had to be made for some of the model parameters due to a lack of available data. The model is able to evaluate the progress in nitrate consumption and the accompanying isotope effect by fitting the δ15N values for the produced N2O where the δ15N values of the residual N2O are calculated based on the known N2O reduction ratio. The latter ratio is calculated from direct measurements of the isotopic signature of the remaining unreduced N2O. The isotopic signature of the instantaneously produced N2O and the fraction of unreduced N2O are calculated, based on direct measurements of N2O and N2 fluxes. A more comprehensive description of the calculation methods and model construction can be found in Lewicka‐Szczebak et al.26 In this context, the aim of the present study was to parameterise the previous two‐pool model via determination of the N2O production and consumption as well as the N2O isotopocule signatures of emitted N2O in a soil treated with a partial and total dispersal of added N and C. The N2O isotopocule data were used to determine the importance of N2O emission from different pools using a Rayleigh‐type model. Controlling the soil volume of pool 1 we assessed the specific denitrification rates of pools 1 and 2 and independently evaluated the contribution of each pool to the total N2O flux using a parallel 15N‐tracing experiment. By applying isotopically labelled N, we were able to gain a deeper insight into the proportion of added N that produced the emitted N2O to estimate the magnitude of pool‐derived fluxes.

EXPERIMENTAL

Set up

A clayey pelostagnogley soil of the Hallsworth series (pH in <n class="Chemical">span class="Chemical">water, 5.6; total N, 0.5%; <hemical">span class="Chemical">ammonium N, 6.1 mg kg−1 dry soil; total oxidized N, 15.1 mg kg−1 dry soil; organic matter, 11.7%; clay, 44%; silt, 40%; sand, 15%; w/w) was collected in November 2013 from a typical grassland in SW England, located at Rothamsted Research, North Wyke, UK (50° 46′ 50″ N, 3° 55′ 8″ W). Spade‐squares (20 × 20 cm to a depth of 15 cm) of soil were taken from 12 locations along a 'W' line across a field of 600 m2 size. After collection, the soil was air dried to ~30% gravimetric moisture content, sieved to <2 mm and stored at 4°C until preparation of the experiment. The experimental design tightly constrained several factors to study the effects of nutrient concentration and fertiliser application area as previously described.27 The soil moisture was adjusted to 85% water filled pore space (WFPS) to promote denitrification conditions, taking the amendment with nutrient solution into account. Before starting the experiment, the soil was preincubated to avoid the pulse of respiration associated with wetting dry soils.31 For this, the required soil was spread to 3–5 cm thickness. Then, while being mixed continuously, the soil was primed by spraying it with water containing 25 kg N ha−1 of potassium nitrate (KNO3), which is a typical yearly rate of N deposition through rainfall in the UK.32, 33 The soil was then left for 3 days at room temperature before being packed into cores and the incubation being started. This was done to promote the growth of denitrifying organisms and prevent a long lag‐phase, therefore reducing the length of the experiment.

The incubation experiment was carried out in a n class="Chemical">specialised gas‐flow‐soil‐core incubation system (DENItrification System (DENIS)3) in which environmental conditions can be tightly controlled. The DENIS simultaneously incubates 12 vessels containing 3 soil cores each (Figure 1). The cores were packed to a bulk density of 0.8 g cm−3 to a height of 75 mm into plastic sleeves of 45 mm diameter. The vessels were purged to exclude atmospheric <span class="Chemical">N2 from the soil and headhemical">space with a He/O2 mixture (80:20) as described by Loick et al.27 The vessels were kept at 20°C during <hemical">span class="Disease">flushing as well as for the 12‐day incubation period after amendment application. The experiment was set up to investigate the effect of a heterogeneous distribution of N and C on gaseous emissions from denitrification, by applying the same amount of N and C to each of the three cores within a vessel (100% of total surface area, treatment 3c) or to one of the three cores (33% of total surface area, treatment 1c) (Figure 1). The treatments were physically separated into different cores to remove subsurface lateral dispersion effects and to control the mass transfer coefficient at the surface (see Loick et al27 for further description).

Figure 1

Schematic showing the N and C application rates and amounts of added N and C with the different treatments. Top values are amounts of N and C in mg added per core; bottom values are amounts of N and C in mg added to the whole vessel and the rate this equates to in kg ha‐1 per vessel: 3c = nutrients applied to all three cores; 1c = nutrients applied to one core; Control = no nutrient application to any core. Each small core contained 95.3 g dry soil

Schematic showing tn class="Chemical">he N and C application rates and amounts of added N and C with the different treatments. Top values are amounts of N and C in <span class="Chemical">mg added per core; bottom values are amounts of N and C in <hemical">span class="Chemical">mg added to the whole vessel and the rate this equates to in kg ha‐1 per vessel: 3c = nutrients applied to all three cores; 1c = nutrients applied to one core; Control = no nutrient application to any core. Each small core contained 95.3 g dry soil

The experiment was carried out with four replicate vessels per treatment (Figure 1): treatment 1c = one of tn class="Chemical">he three cores inside a vessel was amended with hemical">KNO3 and <hemical">span class="Chemical">glucose; treatment 3c = all three of the cores inside a vessel were amended with KNO3 and glucose; Control = only water was applied to each of the three cores. Within each of the treatments 1c and 3c treatments two of the four vessels received 15N‐labelled KNO3 (5 at%). The experiment was carried out twice, resulting in four labelled and four unlabelled replicates per treatment. Considering the total surface area of the vessel (sum of the areas of the three cores in a vessel), N was applied at a rate of 75 kg N ha−1 and C as glucose at 400 kg C ha−1 for treatment 3c where N and C were diluted in 15 mL water and 5 mL of that solution was added to each of the three cores inside one vessel. For treatment 1c, N was applied at a rate of 25 kg N ha−1 and C as glucose at 133.3 kg C ha−1, being applied in solution with 5 mL water to one of the three cores, while the other two cores each received 5 mL water only. The amendment was applied to each of the three cores via a syringe through a sealed port on the lid of the incubation vessel.

Gas analyses and data management

The gas emissions were measured every 10 min consecutively in vessels 1 to 12, resulting in bi‐hourly measurements for each vessel. Tn class="Chemical">he fluxes of hemical">N2O, <hemical">span class="Chemical">CO2 and N2 were quantified by gas chromatography using an electron capture detector (ECD) for N2O, and a helium ionization detector (HID) for CO2 and N2, respectively, while the NO concentrations were determined by chemiluminescence, as described by Loick et al.27 The flow rates through the vessel were measured daily and used to correct all gas concentrations and convert them into flux units (kg N or C ha−1 d−1). The CO2 fluxes showed constant emissions of 0.67 kg C ha−1 h−1 before and after the peak in all vessels, which we consider to be a baseline flux. In order to show emissions attributed to amendment application only, the CO2 fluxes in all the treated vessels were adjusted by subtracting this baseline. The initial emission rates for each gas and vessel were determined from the beginning of each peak until the increase in concentrations slowed down, as previously described by Loick et al.27

Analysis of the isotopocules of N2O

Gas samples for isotopocule analysis of the emitted <n class="Chemical">span class="Chemical">N2O were taken 4 h after amendment application and then daily from unlabelled and control treatments. Samples were collected in two 115‐mL septum‐capped serum bottles, which were connected in line to the vent of each vessel. The isotopocule signatures of <hemical">span class="Chemical">N2O, i.e. δ18O (δ18O‐N2O) values, average δ15N (δ15Nbulk‐N2O) values and δ15N values from the central N‐position (δ15Nα), were determined by isotope ratio mass spectrometry.7 The 15N site preference (SP) was obtained as SP = 2 * (δ15Nα – δ15Nbulk‐N2O). The isotopocule ratios of a sample were expressed as ‰ deviation from the 15N/14N and 18O/16O ratios of the reference standard materials, atmospheric N2 and standard mean ocean water, respectively, as described by Bergstermann et al.25

Isotopic analysis of N2O in 15N‐labelled treatments

Gas samples for n class="Chemical">15N analysis were taken just before (0 h) and 4 h after amendment application and then daily for the first week, followed by a final sampling at day 11. The sampling dates were chosen to cover changes in isotopic ratios during the main period of NO and <hemical">span class="Chemical">N2O fluxes, and after the emissions returned to background levels. Samples were taken from the outlet line of each vessel using 12‐mL exetainers (Labco, Lampeter, UK) which had previously been flushed with He and evacuated. The 15N‐enrichment of N2O was determined using a TG2 trace gas analyser (Sercon, Crewe, UK) and an autosampler (Gilson, Dunstable, UK), interfaced to a Sercon 20–22 isotope ratio mass spectrometer. Standard solutions of 6.6 and 2.9 at% ammonium sulfate ((NH4)2SO4) were prepared and used to generate samples of 6.6 and 2.9 at% N2O34 which were used as reference and quality control standards. The 15N content of the N2O was calculated as described by Loick et al27 to determine how much of the measured N2O derived from the NO3 − amendment rather than the native soil N.

Soil analyses

The moisture contents and <n class="Chemical">span class="Chemical">NH4 + and <hemical">span class="Gene">NO3 − concentrations were determined in soil samples taken at the beginning and end of the incubation. At the end of the soil incubation time, each core was divided in half to separate the top section from the bottom section. The WFPS was calculated from the soil moisture contents by drying a subsample (50 g) at 105°C overnight. The soil NH4 +‐N and NO3 −‐N were measured by automated colorimetry from 2 M KCl soil extracts using a SANPLUS analyser (Skalar Analytical B.V., Breda, The Netherlands).35

Model refinement

A comparison of modelled and measured data for the previously used Rayleigh model26 and tn class="Chemical">he Rayleigh model adapted to the hemical">N2O isotopocule data (determined in this study) was applied to account for isotope effects associated with <hemical">span class="Chemical">N2O reduction, taking emissions from two distinct soil pools (NO3 − added with the amendment = pool 1; native soil NO3 − = pool 2) into account. The previously used Rayleigh model26 assumes an exponential increase in the N2O originating from pool 2 after amendment application until nitrate in pool 1 is exhausted. However, this exponential increase was only an assumption and not experimentally confirmed. Hence, we used the 15N‐labelled treatments to determine the equation that best describes the mixing dynamics of the two NO3 − pools. The Rayleigh model was then run with the isotopocule data from the unlabelled treatments, but using the equation determined before using the 15N‐labelled treatments. In this study, the volume reached by the amendment (volume of pool 1) was assumed to be 33% and 100% in treatments 1c and 3c, respectively. For modelling, we applied the equations described in Lewicka‐Szczebak et al.26 Briefly, the isotopic signature of the product, N2O and the isotopic signature of the remaining substrate, NO3 −, was calculated according to Equation 1: where δS is the isotopic signature of the remaining NO3 − (δ15NNO3‐r); δS0 the isotopic signature of the initial NO3 − (δ15NNO3‐i), i.e., fertiliser or soil NO3 ‐l; and ηP‐S the Net Isotope Effect (NIE) between product and substrate.

In this study, we determined the δ<n class="Chemical">span class="Chemical">15N value of the applied fertiliser whereas that of soil <hemical">span class="Gene">NO3 −was adapted from the literature26

δ<span class="Chemical">15Nsoil <hemical">span class="Gene">NO3‐ = 10‰.

f, the fraction of unreduced <n class="Chemical">span class="Gene">NO3 −N, was determined by subtracting the initial <hemical">span class="Gene">NO3 − concentration and the cumulative N loss as denitrification products (N2 + N2O) for each time step of the process: It was assumed that the NO and NO2 − pools were negligible in the overall N balance, as these represent very reactive intermediate products undergoing fast further reduction. ηP‐S represents the Net Isotope Effect (NIE) of N2O production referred to as ηN2O‐NO3. The δ15NN2O‐p (instantaneously produced N2O) value was calculated according to Equation 3: The isotopic signature of the reduced N2O was calculated according to Equation 1, where δS is the isotopic signature of the remaining unreduced N2O (δN2O‐r); δS0 the isotopic signature of the instantaneously produced N2O (δN2O‐p); f the fraction of unreduced N2O, calculated based on direct measurements of the N2O and N2 flux, i.e., the product ratio (N2O/(N2O + N2)); and ηP‐S is the NIE of N2O reduction referred to as ηN2‐N2O.

Statistical analysis

Data were analysed to determine normality (Kolmogorov–Smirnov test) and equality of variance (Levene test) conditions. To fulfil these assumptions, tn class="Chemical">he data were log‐transformed before analysis, if needed. Statistical analysis was performed using GenStat 16th edition (VSN International Ltd, Hemel Hempstead, UK). Cumulative emissions were calculated after linear interpolation of the area between sampling points. Differences in total emissions between treatments for each gas measured were assessed by analysis of variance (ANOVA) at p <0.01.

RESULTS

Fluxes and cumulative gas emissions

The fluxes and cumulative emissions of n class="Chemical">NO, hemical">N2O, <hemical">span class="Chemical">N2 as kg N ha−1 and CO2 are shown in Figure 2 and Table 1, respectively. The NO emissions from treatments 1c and 3c increased immediately after amendment application with a peak lasting just over 2 days and a maximum on day 1 (Figure 2) The mean cumulative NO emissions from treatment 3c (same shape) was about 2.3 times greater over the time of the incubation than that from treatment 1c (Table 1). Emissions of NO from the Control treatment were negligible.

Figure 2

Average fluxes of NO, N2O, N2 and CO2 for the different treatments (n = 8). In treatment 1c one of the three cores inside a vessel was amended with KNO3 and glucose (the other two received water); in treatment 3c, all three of the cores inside a vessel were amended with KNO3 and glucose (each core received the same N and C rate as treatment 1c); in the Control treatment, only water was applied to each of the three cores

Table 1

Cumulative emissions of NO, N2O, N2 as kg N ha−1 and CO2 as kg C ha−1. Values were determined in the period between the start and end of the emission peak: NO day 0–4, N2O day 0–10, N2 day 4.5 to 9.5, CO2 day 0–10 after amendment application. Different letters indicate a significant difference between treatments for each measured gas (n = 8 for 1c and 3c, n = 4 for control; p <0.05). Standard errors of the mean are included

Gas	1c	3c	Control
NO	0.0079 ± 0.0005B	0.0183 ± 0.0021A	0.0018 ± 0.0003C
N2O	6.73 ± 1.37B	19.49 ± 5.04A	1.14 ± 0.13C
N2	2.88 ± 0.56B	5.91 ± 2.25A	3.02 ± 0.93B
CO2	192.23 ± 3.65B	313.66 ± 10.07A	122.41 ± 6.73C
Total N	9.46 ± 1.01B	26.12 ± 6.59A	4.28 ± 0.89B

Average fluxes of NO, <n class="Chemical">span class="Chemical">N2O, <hemical">span class="Chemical">N2 and CO2 for the different treatments (n = 8). In treatment 1c one of the three cores inside a vessel was amended with KNO3 and glucose (the other two received water); in treatment 3c, all three of the cores inside a vessel were amended with KNO3 and glucose (each core received the same N and C rate as treatment 1c); in the Control treatment, only water was applied to each of the three cores

Cumulative emissions of NO, <n class="Chemical">span class="Chemical">N2O, <hemical">span class="Chemical">N2 as kg N ha−1 and CO2 as kg C ha−1. Values were determined in the period between the start and end of the emission peak: NO day 0–4, N2O day 0–10, N2 day 4.5 to 9.5, CO2 day 0–10 after amendment application. Different letters indicate a significant difference between treatments for each measured gas (n = 8 for 1c and 3c, n = 4 for control; p <0.05). Standard errors of the mean are included

Similarly to the observed n class="Chemical">NO emissions, the hemical">N2O emissions increased immediately after amendment application (Figure 2). The emissions from treatment 3c peaked 3.5 days after the amendment was applied, before decreasing again. The maximum <hemical">span class="Chemical">N2O emission was larger for treatment 3c than for treatment 1c. In treatment 1c, however, there was a plateau in N2O emissions from about day 2 to day 4 before showing the same decrease as treatment 3c. The cumulative emissions of N2O (Table 1) were 2.9 times greater from treatment 3c than from treatment1c. The Control treatment only showed very small N2O emissions from 1 to 2.5 days after water addition.

The <n class="Chemical">span class="Chemical">N2 fluxes increased after amendment application in treatments 1c and 3c and <hemical">span class="Chemical">water addition in the Control treatment (Figure 2). Slightly higher N2 fluxes were measured in treatment 3c than in treatment 1c and the Control treatment, showing a peak after 2 days in treatment 3c (Figure 2). In contrast to the NO and N2O emissions, the N2 cumulative emissions were similar for treatment 1c and the Control treatment, whereas significant higher N2 cumulative emissions were measured in treatment 3c (Table 1).

The total denitrification was calculated as tn class="Chemical">he sum of all the N emitted (Table 1) and was significantly higher in treatment 3c than in treatment 1c (2.8‐fold) and the Control (6.1‐fold) treatment.

The <n class="Chemical">span class="Chemical">CO2 fluxes showed similar trends to the <hemical">span class="Chemical">N2O fluxes. In treatments 1c and 3c, the CO2 emissions increased immediately after amendment application (Figure 2) and peaked after about 3 days in both treatments. The cumulative emissions of CO2 (Table 1) were 1.6 and 2.6 times greater from treatment 3c than from treatment 1c and the Control treatment, respectively. CO2 emissions above background levels were negligible for the Control treatment.

Soil mineral N

The results of tn class="Chemical">he soil analysis at the end of the incubation are given in Table 2. The NO3 − concentrations were significantly different between the top and the bottom half of the cores for the amended treatments but no significant difference was detected within the Control treatment. The results, if considering the whole vessel, did, however, show that there was a significant difference in the <hemical">span class="Gene">NO3 − concentrations between treatments 1c and 3c in the top layer (p <0.05). Both amended treatments showed significantly higher NO3 − concentrations than those in the Control treatment.

Table 2

Soil characteristics at the end of the experiment. Total amounts measured for nitrate (NO3 −) and ammonium (NH4 +). ‘1c’ = average values for 12 cores (4 amended with 75 kg N ha−1, 8 unamended) from vessels of treatment 1c; ‘3c’ = average values for 12 cores (12 amended with 75 kg N ha−1) of treatment 3c; ‘control’ = average of 12 cores from the control treatment only receiving water. WFPS values are an average over all three treatments (average values for 36 cores). Different letters indicate a significant difference between treatments for each layer (top or bottom); * indicates significant difference between the top and bottom layer within a single grouping. (n = 10 for ‘1c’ and ‘3c’, n = 4 for ‘control’), p < 0.05). Standard errors are included. NO3 −‐N (mg g−1 dry soil) values were 4.6 10−2 ± 2.0 10−4 and 9.8 10−3 ± 4.0 10−4 before and after priming, respectively, before amendment application. NH4 +‐N (mg g−1 dry soil) amount was 6.0 10−3 ± 9.0 10−6 before amendment application

Parameter	Layer	1c	3c	Control
NO3 − (mg N g−1 dry soil)	Top	1.44 ± 0.06B*	1.68 ± 0.05A*	1.23 ± 0.13B
	Bottom	1.28 ± 0.04A*	1.36 ± 0.04A*	1.13 ± 0.03B
NH4 + (mg N g−1 dry soil)	Top	0.055 ± 0.002B*	0.050 ± 0.001C*	0.060 ± 0.001A*
	Bottom	0.069 ± 0.004A*	0.066 ± 0.003A*	0.076 ± 0.005A*
WFPS (%)	Top	83.2 ± 0.50*
	Bottom	76.0 ± 0.56*

Soil characteristics at the end of tn class="Chemical">he experiment. Total amounts measured for hemical">nitrate (<hemical">span class="Gene">NO3 −) and ammonium (NH4 +). ‘1c’ = average values for 12 cores (4 amended with 75 kg N ha−1, 8 unamended) from vessels of treatment 1c; ‘3c’ = average values for 12 cores (12 amended with 75 kg N ha−1) of treatment 3c; ‘control’ = average of 12 cores from the control treatment only receiving water. WFPS values are an average over all three treatments (average values for 36 cores). Different letters indicate a significant difference between treatments for each layer (top or bottom); * indicates significant difference between the top and bottom layer within a single grouping. (n = 10 for ‘1c’ and ‘3c’, n = 4 for ‘control’), p < 0.05). Standard errors are included. NO3 −‐N (mg g−1 dry soil) values were 4.6 10−2 ± 2.0 10−4 and 9.8 10−3 ± 4.0 10−4 before and after priming, respectively, before amendment application. NH4 +‐N (mg g−1 dry soil) amount was 6.0 10−3 ± 9.0 10−6 before amendment application

Regardless of the treatment, tn class="Chemical">he hemical">NH4 + concentrations were lower than the <hemical">span class="Gene">NO3 − concentrations at the end of the incubation, with significantly higher values in the bottom layer of the core. Both soil NH4 + and NO3 − increased in all treatments compared with the initial soil conditions (6.1 and 15 mg N kg dry soil−1). The NH4 + concentrations were only significantly different between treatments in the top layer, in decreasing order: Control >1c > 3c. The soil moisture content was significantly different between the top (83.2 ± 0.50) and the bottom (76.0 ± 0.56) half of the cores at the end of the incubation in all treatments.

15N‐enrichment of N2O in the 15N‐labelled treatment

The <n class="Chemical">span class="Chemical">15N‐enrichment of the emitted <hemical">span class="Chemical">N2O is shown in Figure 3. Regardless of the N treatment, up to day 4 around 70% of the emitted N2O was derived from the applied amendment, with a constant decrease thereafter (Figure 3). After 4 days, when N2O emissions decrease while the N2 fluxes increase (Figure 4), which indicates that N2O reduction dominates over N2O production, the enrichment in 15N of the N2O decreases. This decrease is faster in treatment 1c than in treatment 3c, reaching a final contribution of fertiliser N to N2O emissions of around 20% and 50%, respectively, by day 11.

Figure 3

Contribution of applied fertiliser‐N to N2O emissions as determined from 15N‐enrichment of the emitted N2O from those 1c and 3c treatments that had received 15N‐labelled KNO3 with their amendment

Figure 4

Comparison of δ15N bulk and δ18O values of soil‐emitted N2O from those 1c and 3c treatments that had received unlabelled KNO3 with their amendment as well as the Control treatment

Contribution of applied fertiliser‐N to <n class="Chemical">span class="Chemical">N2O emissions as determined from <hemical">span class="Chemical">15N‐enrichment of the emitted N2O from those 1c and 3c treatments that had received 15N‐labelled KNO3 with their amendment

Comparison of δn class="Chemical">15N bulk and δ18O values of soil‐emitted <hemical">span class="Chemical">N2O from those 1c and 3c treatments that had received unlabelled KNO3 with their amendment as well as the Control treatment

Isotopic signature of N2O in the non‐labelled treatments

δ15Nbulk values of N2O

The δ<n class="Chemical">span class="Chemical">15Nbulk‐<hemical">span class="Chemical">N2O values were not significantly different between the N‐amended treatments during the first 4 days, and increased from an initial value of about −23.4‰ in both treatments to −1.1‰ and − 5.5‰ in treatments 1c and 3c, respectively (Table 3). After 4 days, the δ15Nbulk‐N2O values remained relatively constant in treatment 3c, in the range of −1.2 to 1.7‰, until the end of the incubation. In contrast, in treatment 1c the δ15Nbulk‐N2O values increased until day 6 (10.4‰) and declined by day 9 (−4.2‰), peaking again on day 11 (51.8‰). Immediately after water addition, the δ15Nbulk‐N2O value of the Control treatment was −23.8‰ and it peaked on day 6 (10.4‰) to decrease afterwards until −20.7‰ on day 11 (Table 3).

Table 3

Measured isotopic ratios of emitted N2O, as δ18O, δ15Nbulk and site preference (SP), in those 1c and 3c treatments that received unlabelled KNO3 with their amendment as well as the control treatment over the time of the incubation

Days after treatment	δ18O values (‰)			δ15Nbulk values (‰)			SP (‰)
	1c	3c	Control	1c	3c	Control	1c	3c	Control
0	25.6	24.0	39.7	−23.4	−23.3	−23.8	−1.6	−4.9	22.4
2	21.4	21.7	18.9	−18.0	−16.9	−26.0	−6.0	−5.7	−4.1
4	37.3	38.9	30.1	−1.1	−5.5	−8.1	−6.3	−5.5	−3.7
6	43.3	41.7	31.1	10.4	−1.2	10.4	3.6	1.8	3.9
9	39.6	42.4	31.9	−4.2	1.0	−19.8	7.0	3.1	6.4
11	42.1	42.1	37.9	51.8	1.7	−20.7	9.4	4.3	22.9

Measured isotopic ratios of emitted n class="Chemical">N2O, as δ18O, δ<hemical">span class="Chemical">15Nbulk and site preference (SP), in those 1c and 3c treatments that received unlabelled KNO3 with their amendment as well as the control treatment over the time of the incubation

15N site preference of N2O

The <n class="Chemical">span class="Chemical">15N site preference of <hemical">span class="Chemical">N2O (SP‐N2O) of both N‐amended treatments decreased slightly for the first 4 days and gradually increased thereafter until the end of the incubation, showing only small differences between them (Table 3). Overall, the SP N2O values increased from an initial value in the range of −1.6 and −4.9‰ to a maximum of approximately 9.4‰ and 4.3‰ in treatments 1c and 3c, respectively (day 11 after application). The SP N2O from the Control treatment increased after the application of water up to 22.5‰ and declined to −4.1‰ by day 2, increasing gradually until the end of the incubation to reach a final value of 22.9‰ (Table 3). The δ15Nα and δ15Nβ values followed a similar trend to the δ15Nbulk values with small differences between the isotope ratios, and generally δ15Nα > δ15Nβ (data not shown).

δ18O values of N2O

Similar to the <n class="Chemical">span class="Chemical">N2O <hemical">span class="Chemical">SP, the δ18O values of N2O showed small differences in the temporal pattern between treatments 1c and 3c (Table 3). Overall, the δ18O values of the N2O in both N‐amended treatments increased continuously from an average 29.4‰ to 40.4‰ at the end of the incubation. In the Control treatment, the δ18O values of N2O increased after water application to 39.7‰, followed by a decline to 18.9‰ by day 2. Afterwards, the value gradually increased until the end of the incubation to about 37.6‰ (Table 3).

An X/Y plot of δ18O‐n class="Chemical">N2O values against δ<hemical">span class="Chemical">15Nbulk‐N2O values is presented in Figure 4. Regardless of the treatment, both isotope ratios increased at a ratio of approximately 1:3 during the incubation. A similar behaviour was observed in both N‐amended treatments, which indicated that the ratio of the simultaneous increase in the δ18O‐N2O and δ15Nbulk‐N2O values did not differ between treatments (Figure 4). Moreover, the δ18O‐N2O and δ15Nbulk‐N2O values grouped into two separate clusters depending on whether they were measured from samples taken before or after the N2O peak. As expected, a different trajectory in the δ15Nbulk‐N2O and δ18O‐N2O values was observed in the Control treatment over the experimental period.

The X/Y plot of δ18O‐<n class="Chemical">span class="Chemical">N2O values against <hemical">span class="Chemical">SP in Figure 5 shows the “map” for the values of δ18O and SP from all unlabelled treatments. Reduction lines (vectors) represent minimum and maximum routes of isotopocule values with increasing N2O reduction to N2 based on the reported range in the ratio between the isotope fractionation factors of N2O reduction for SP and the δ18O values.18 Most of the values measured after amendment application, but before the N2O peak, are below the lower reduction line, but within the area indicating bacterial denitrification. During the N2O peak the samples show increased δ18O values followed by an increased SP after the peak.

Figure 5

SP vs δ18O values from all vessels that had received unlabelled amendment, grouped for four time periods depending on the appearance of the peak in N2O emissions (circles = pre‐amendment; triangles = after amendment application, but before the N2O peak (days 0–3); crosses = during the N2O peak (day 4); squares = post N2O peak (days 5‐12), all with associated trendlines (see legend)). The solid black lines are reduction lines after Lewicka‐Szczebak et al18 representing minimum and maximum routes of isotopocule values with increasing N2O reduction to N2. Endmember areas for fungal denitrification, nitrification and bacterial denitrification are from Lewicka‐Szczebak et al18

n class="Cn class="Chemical">hemical">SP vs δ18O values from all vessels that had received unlabelled amendment, grouped for four time periods depending on the appearance of the peak in <n class="Chemical">hemical">span class="Chemical">N2O emissions (circles = pre‐amendment; triangles = after amendment application, but before the N2O peak (days 0–3); crosses = during the N2O peak (day 4); squares = post N2O peak (days 5‐12), all with associated trendlines (see legend)). The solid black lines are reduction lines after Lewicka‐Szczebak et al18 representing minimum and maximum routes of isotopocule values with increasing N2O reduction to N2. Endmember areas for fungal denitrification, nitrification and bacterial denitrification are from Lewicka‐Szczebak et al18

Modelling 15N‐enrichment of N2O

Measurements of n class="Chemical">15N‐enrichment using the <hemical">span class="Chemical">15N‐labelled treatments 1c and 3c (Figure 3) derived in the polynomial Equations 4 and 5, respectively, were: where f(X) is the contribution of fertiliser N to N2O in % and x is the time after amendment (d).

The Rayleigh model fit adapted to <n class="Chemical">span class="Chemical">15N data for the unlabelled treatments 1c and 3c was evaluated in all vessels, assuming one‐pool and two‐pool emissions. Only two vessels per treatment (n = 4) showed a <hemical">span class="Disease">good polynomial fit (R2 >0.89) of the modelled data to the measured data and an average of them is shown in Figure 6. The equations and R2 values of all the vessels for each N pool are shown in Table S1 (supporting information). The Rayleigh model applied to the δ15Nbulk‐N2O data showed poor agreement with the measurements using the original model for treatment 1c, with the two‐pool model giving better results when using the polynomial equation determined above (Figure 6). In contrast, for treatment 3c little difference was observed between the modelling approaches (Figure 6).

Figure 6

Comparison of modelled and measured data for the previously used Rayleigh model (model A) and the Rayleigh model adapted according to 15N data (model B) for the two treatments 1c (left) and 3c (right) assuming one‐pool emission (only from fertiliser) and two‐pool emission (from fertiliser and soil nitrate). Equations relate to the adapted two‐pool model B (top equation) and the one‐pool model (bottom equation)

Comparison of modelled and measured data for the previously used Rayleigh model (model A) and tn class="Chemical">he Rayleigh model adapted according to <span class="Chemical">15N data (model B) for the two treatments 1c (left) and 3c (right) assuming one‐pool emission (only from fertiliser) and two‐pool emission (from fertiliser and soil <hemical">span class="Chemical">nitrate). Equations relate to the adapted two‐pool model B (top equation) and the one‐pool model (bottom equation)

DISCUSSION

Soil data and gaseous emissions

Our findings are in agreement with those of Wang et al36 and Loick et al27 who found that the emissions of n class="Chemical">NO, hemical">N2O and <hemical">span class="Chemical">CO2 are related to the amounts of applied NO3 − and C, NO3 − and C thereby being the limiting factors for denitrification activity, rather than the soil area and volume and associated microbial population that receives the amendment. Although the total emissions were not similar, the peaks of N2O, NO and CO2 fluxes were concurrent in treatments 1c and 3c. Moreover, the amendment solution was spread over all three cores in treatment 3c which could have potentially supported a three times larger microbial community with the nutrients than treatment 1c. Loick et al27 found a delay in the N2O emission peak when only one of three cores inside a vessel was amended with the full amount of nutrients, compared with an equal distribution of the treatment into three cores (so each core received 1/3 of the nutrients). In our case, in treatments 1c and 3c all individual cores (one in 1c and three in 3c) received the same amount of nutrients and the response time was similar, showing that denitrifiers transformed the NO3 − added to N2O for the same time period in both treatments, regardless of the soil area/volume amended. Although the cumulative emissions of N2 were higher in treatment 3c, the fluxes were lower than the N2O fluxes in all treatments. It has been demonstrated that many denitrifiers lack one or more of the denitrification enzymes involved in all reduction steps from NO3 − to N2,37 particularly N2O reductase (NosZ) the enzyme reducing N2O to N2. In addition, the last step in denitrification is also the least energetically favourable.38 Therefore, denitrifiers would preferentially reduce NO3 − to N2O rather than N2O to N2. We hypothesised that these reasons explain the accumulation of N2O over N2. 27, 39

Isotope analysis of N2O from 15N‐labelled treatments

The <n class="Chemical">span class="Chemical">15N signature of <hemical">span class="Chemical">N2O was used to determine the contribution of the native soil NO3 − or the NO3 − added with the amendment to the N2O emissions (Figure 3). While in treatment 3c N2O emissions were mainly from the added NO3 − (pool 1) throughout the whole experimental period, in treatment 1c, a low 15N enrichment of the measured N2O was observed after 5 days, indicating that after this time most of the emitted N2O was from the native soil NO3 − (pool 2). This can be explained due to NO3 − limitation in the soil treated in treatment 1c after the N2O peak. Because only one‐third of the soil/microbial community received nutrient amendment, N2O emissions were low in treatment 1c and those from the non‐amended cores are likely to mask the effect of the amendment on N2O production.27 Moreover, after 11 days, N2O production in treatment 3c still came from the NO3 − added.

Analysis of isotopocules of N2O

δ15Nbulk‐N2O values

The increase in δ<n class="Chemical">span class="Chemical">15Nbulk‐<hemical">span class="Chemical">N2O values until day 4 in both treatments 1c and 3c is probably a consequence of the 15N‐enrichment during ongoing NO3 − reduction of the added NO3 −.25 From day 4 onwards the δ15Nbulk‐N2O values increased in treatment 1c, indicating enrichment in 15N from a different pool of NO3 −. The 15N‐enrichment of N2O in the 15N‐labelled treatment 3c showed that some of the N2O (30 to 50%) came from soil‐derived NO3 −. This suggests that pool 1 dominated initially (while the unlabelled treatment showed an increase in δ15Nbulk‐N2O values) whereas, when the relative contribution of soil‐NO3 − increased (which can be seen by lowering of N2O emission from fertiliser), the δ15Nbulk values did not increase further, due to the increasing contribution from pool 2 masking any increases in δ15Nbulk values from pool 1. In treatment 1c, however, changes in the 15N‐enrichment of the N2O could be related to the influence of two N‐pools; one core receiving amendment (soil N + added N) and two cores with only soil N with different denitrification dynamics where the fraction of N2O varied over time. The observed dynamics are in line with earlier observations during incubation of NO3 −/glucose‐amended soil cores25, 26 where the initial increase in δ15Nbulk‐N2O values had been explained by the fast exhaustion of NO3 − and the consequential 15N‐enrichment of residual NO3 − from pool 1 during the earlier phase, followed by declining N2O fluxes from pool 1 after its exhaustion. The lowering of δ15Nbulk values was explained as being from the growing contribution of pool 2 to N2O fluxes, since pool 2 was previously less fractionated than pool 1 due to its lower denitrification rate in the absence of glucose. The final increase in δ15Nbulk values was explained by N2O fluxes from pool 2 since its NO3 − was also progressively reduced and thus fractionated. The latter was verified by modelling of the δ15N‐N2O values and it is further discussed in section 4.4.

The 15N site preference

The <n class="Chemical">span class="Chemical">SP of the <hemical">span class="Chemical">N2O is the result of several mechanisms responsible for N2O production such as nitrification, bacterial and fungal denitrification.15, 40, 41, 42 The range of SP values in this study is in agreement with those from previous studies under denitrifying conditions.18, 25, 43 Moreover, it is known that reduction of N2O to N2 causes 15N accumulation on the central N‐position of the N2O because of the cleavage of NO bonds during this process.15, 40 In fact, we observed a N2 peak after 5 days, in both treatments 1c and 3c, with higher SP values indicating the reduction of N2O to N2.

In this study, the decrease in <n class="Chemical">span class="Chemical">15N SP values of <hemical">span class="Chemical">N2O before the N2O peak followed by an increase suggests that the site‐specific 15N fractionation factor of the reduction of NO3 − to N2O was not constant in treatments 1c and 3c. At the end of the experiment, the maximum SP value was reached, coinciding with minimum fluxes of N2O and the lowest N2O/(N2 + N2O) ratio, suggesting an increase in the extent of the N2O reduction.25 Regardless of the amounts of N and total area amended, the variation in the SP N2O between treatments was relatively small. This agrees with earlier studies12, 25, 43 that explained the decline in SP values as resulting from the initiation of anaerobic conditions after inducing this process by flushing with N2 or with a decreasing contribution from fungal denitrification. It is possible that some N2O emission resulted from nitrification although the soil moisture was adjusted to favour denitrification.7

The δ18O signatures

The values of δ18O‐<n class="Chemical">span class="Chemical">N2O are determined by <hemical">span class="Gene">NO3 −, O2 and soil H2O incorporation and reduction effects during the production of N2O resulting in 18O‐depleted or ‐enriched N2O, respectively, since the 18O–N bond is more stable and 16O is removed more easily from NO3 −.41, 43 It is known that oxygen can be incorporated from H2O to N2O during denitrification to constitute more than 60% of the O in the N2O produced‐.44, 45 During the first four days of the incubation, the δ18O‐N2O values increased indicating an independence of the δ18O‐N2O values from the δ18O‐NO3 values during the production of N2O that can be attributed to a lower O‐exchange with water.12 Our results are in agreement with those reported by Meijide et al43 and Bergstermann et al25 showing stabilisation of δ18O‐N2O values after the N2O peak. However, in contrast to Meijide et al43 we did not observe an increase in δ18O‐N2O values linked to an increase of N2 fluxes.

In this study, different patterns of δn class="Chemical">15Nbulk vs δ18O values (Figure 4 showing two clusters before and after the <hemical">span class="Chemical">N2O peak as well as differently sloped lines for the different treatments) suggested the temporal change in denitrification between the different pools before and after the N2O peak. Before the N2O peak, N2O originated from non‐fractionated NO3 − in pool 1 (NO3 − added from fertiliser) whereas after the N2O peak the main flux might have come from pool 2 (mixture from fertiliser and native NO3 −), which also contained less fractionated NO3 − initially.43 Moreover, the patterns of SP vs δ18O values gave further indications on processes contributing to N2O fluxes:18, 46 pre‐peak values cluster mainly in the bacterial endmember area indicating little contribution from other sources and minor reduction in agreement with flux data, whereas post‐peak values (>day 4) cluster around the reduction line, indicating bacterial production with varying reduction to N2, where the latter is also confirmed by flux data (Figure 3). Interestingly, the peak values form a distinct cluster below the reduction line with SP values below zero per mil, indicative of bacterial production with minor reduction, but the δ18O values are increased by 15 to 20‰ compared with the pre‐flux values. Those data can thus not be explained with the “mapping approach” suggested by Lewicka‐Szczebak et al18 which assumes that the δ18O value of bacterial N2O prior to its reduction is relatively constant due to almost complete O‐exchange with water, implying that a positive shift in the δ18O value must be due to N2O reduction and associated with increasing SP values. Because the δ15Nbulk values exhibited a similar upshift until day 4, we assume that this effect is due to an increase in the δ18O and δ15N values of the NO3 − precursor resulting from fractionation during intense denitrification in this phase of the experiment (day 4). This would also mean, however, that O‐exchange with water during N2O production was incomplete, which has been reported earlier for a dynamic incubation similar to our study.45

Isotopocules model

The Rayleigh model25, 26 was applied to account for tn class="Chemical">he importance of hemical">N2O emissions from the one‐pool and two‐pools using the δ<hemical">span class="Chemical">15Nbulk values of N2O. Until now, this model has been used to simulate the δ15N values of N2O using process rates and associated fractionation factors, but assumptions had to be made for some of the model parameters due to lack of available data.25 In this study, we carried out two incubation experiments in order to parameterise the model. The range of δ15Nbulk values agrees with other studies that identified denitrification as the main N2O‐producing process under similar conditions.43 Data from 15N‐labelling showed an initial increase in the contribution of pool 1 followed by a decrease (Figure 3), which was sooner and larger in treatment 1c. The comparison of the previously used Rayleigh model25, 26 and the Rayleigh model adapted in this study according to δ15Nbulk analysis of N2O showed that a two‐pool model was better for interpreting treatment 1c, whereas for treatment 3c little difference between the modelling approaches was observed. This supports the idea that the amendment was mixed with parts of the soil pool, forming one uniform pool initially dominating N2O emissions in treatment 3c. In this treatment the δ15Nbulk levels stabilise after day 6, which indicates that a second pool contributes to emissions. Previous studies25, 26 assumed that during the N2O emission peak, a small but increasing contribution from pool 2 also occurs and its contribution was fitted assuming an exponential increase of pool 2 emission until reaching the emission observed after the extinction of pool 1. Using two different amendment areas, we found that a third‐order polynomial equation based on empirical δ15Nbulk data improved the fit of the model, especially for treatment 1c.

Although we intended to control the magnitude of pool 1 (33% or 100% of amendment area) in this study, tn class="Chemical">he Rayleigh model fit adapted to the <span class="Chemical">15N‐labelling data showed a good third‐order polynomial fit for only two vessels per treatment. Thus, a better parameterising of the model should be addressed for examination of fractionation factors for various product ratios and reaction rates of pool 2 by future studies.

CONCLUSIONS

Determining n class="Chemical">N2O emissions from different N‐pools in soil is important for the interpretation of <hemical">span class="Chemical">N2O isotopocule data. This study shows the potential for understanding the source of N2O emissions from different N pools using an improved model for the interpretation of N2O isotopocule data. It was indicated that the assumptions regarding the exponential increase in pool 2 activity accepted in previous studies25, 26 should be replaced with a polynomial increase with dependence on both pool sizes. Our results show the value of parameterising models under controlled laboratory conditions using experimental data but further work is required to apply the findings to other soil types and improve the refinement of model parameters.

Table S1. Rayleigh model adapted equations according to <span class="Chemical">15N data (model B) for the 1C and 3C treatments assuming 1‐pool emission (only from fertiliser) and 2‐pool emission (mixture from fertiliser and soil <hemical">span class="Chemical">nitrate). Only vessels with R2 value >0.89 (in bold and underlined)

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