Effect of soil saturation on denitrification in a grassland soil

. Nitrous oxide (N 2 O) is of major importance as a greenhouse gas and precursor of ozone (O 3 ) destruction in the stratosphere mostly produced in soils. The soil-emitted N 2 O is generally predominantly derived from denitriﬁcation and, to a smaller extent, nitriﬁcation, both processes controlled by environmental factors and their interactions, and are inﬂuenced by agricultural management. Soil water content expressed as water-ﬁlled pore space (WFPS) is a major controlling factor of emissions and its interaction with compaction, has not been studied at the micropore scale. A laboratory incubation was carried out at different saturation levels for a grassland soil and emissions of N 2 O and N 2 were measured as well as the isotopocules of N 2 O. We found that ﬂux variability was larger in the less saturated soils probably due to nutrient distribution heterogeneity created from soil cracks and consequently nutrient hot spots. The results agreed with denitriﬁcation as the main source of ﬂuxes at the highest saturations, but nitriﬁcation could have occurred at the lower saturation, even though moisture was still high (71 % WFSP). The isotopocules data indicated isotopic similarities in the wettest treatments vs. the two drier ones. The results agreed with previous ﬁndings where it is clear there are two N pools with different dynamics: added N producing intense denitriﬁcation vs. soil N resulting in less isotopic fractionation

to relate WFPS with emissions (Schmidt et al., 2000;Dobbie and Smith, 2001;Parton et al., 2001;del Prado et al., 2006;Castellano et al., 2010) but the "optimum" WFPS for N2O emissions varies from soil to soil (Davidson, 1991). Soil structure could be influencing this effect and it has been identified to strongly interact with soil moisture (Ball et al., 1999; van Groenigen et al., 2005) through changes in WFPS. Particularly soil compaction due to livestock treading and the use of heavy machinery affect soil structure and emissions as reported by studies relating bulk density to fluxes (Klefoth et al., 2014b); and degrees of tillage to emissions (Ludwig et al., 2011).
Compaction is known to affect the size of the larger pores (macropores) thereby reducing the soil air volume and therefore increasing the WFPS (for the same moisture content) (van der Weerden et al., 2012). However, little is known about the effect of compaction on the smaller soil pores (micropores) and this could provide valuable information for understanding the simultaneous behaviour of the dynamics of water in the various pore sizes in soil. Such an understanding would lead to the development of better N2O mitigation strategies via dealing with soil compaction issues.
The role of water in soils is closely linked to microbial activity but also relates to the degree of aeration and gas diffusivity in soils (Morley and Baggs, 2010). Water facilitates nutrient supply to microbes and restricts gas diffusion, thereby increasing the residence time of gases in soil, and the chance of further N2O reduction before it can be released to the atmosphere. This is further aided by the restriction of the diffusion of atmospheric O2 (Dobbie and Smith, 2001), increasing the potential for denitrification. In consequence, counteracting effects (high microbial activity vs low diffusion) occur simultaneously making it difficult to predict net processes and corresponding outputs (Davidson, 1991). Detailed understanding of the sources of N2O and the influence of physical factors, i.e. soil structure and its interaction with moisture, is a powerful basis for developing effective mitigation strategies.
Isotopocules of N2O represent the isotopic substitution of the O and/or the two N atoms within the N2O molecule.
The isotopomers of N2O, are those differing in the peripheral (β) and central N-positions (α) of the linear molecule (Toyoda and Yoshida, 1999) with the intramolecular 15 N site preference (SP; the difference between δ 15 N αδ 15 N β ) used to identify production processes at the level of microbial species or enzymes involved (Toyoda et al., 2005;Ostrom, 2011). Moreover, δ 18 O, δ 15 N and SP of emitted N2O depend on the denitrification product ratio (N2O / (N2+N2O)), and hence provide insight into the dynamics of N2O reduction (Well and Flessa, 2009;Lewicka-Szczebak et al., 2014;Lewicka-Szczebak et al., 2015). Koster et al. (2013) for example recently reported δ 15 N bulk values of N2O between -36.8‰ and -31.9‰ under the conditions of their experiment, which are indicative of denitrification according to Perez et al. (2006) and Well and Flessa (2009) who proposed the range -54 to -10‰ relative to the substrate. Baggs (2008) summarised that values between -90 to -40‰ are indicative of nitrification. Determination of these values are normally carried out in pure culture studies or in conditions favouring either production or reduction of N2O (Well and Flessa, 2009). The SP is however considered a better predictor of the N2O source due to its independence from the substrate signature (Ostrom, 2011).
Simultaneous occurrence production and reduction of N2O as in natural conditions presents a challenge for isotopic factors determination due to uncertainty on N2 reduction and the co-existence of different microbial communities producing N2O (Lewicka-Szczebak et al., 2014). Recently, using data from the experiment reported here, where soil was incubated under aerobic atmosphere and the complete denitrification process occurs, Lewicka-Szczebak et al. (2015) determined fractionation factors associated with N2O production and reduction using a modelling approach. The analysis comprised measurements of the N2O and N2 fluxes combined with isotopocule data. Net isotope effects (η values) are variable to a certain extent as they result from a combination of several processes causing isotopic fractionation (Well et al., 2012). The results generally confirmed the range of values of η (net isotope effects) and η 18 O/η 15 N ratios reported by previous studies for N2O reduction for that part of the soil volume were denitrification was enhanced by the N+C amendment. This did not apply for the other part of the soil volume not reached by the N+C amendment, showing that the validity of published net isotope effects for soil conditions with low denitrification activity still needs to be evaluated. Lewicka-Szczebak et al. (2015) observed a clear relationship between 15 N and 18 O isotope effects during N2O production and denitrification rates. For N2O reduction, differential isotope effects were observed for two distinct soil pools characterized by different product ratios N2O / (N2+N2O). For moderate product ratios (from 0.1 to 1.0) the range of isotope effects given by previous studies was confirmed and refined, whereas for very low product ratios (below 0.1) the net isotope effects were much smaller. In this paper, we present the results from the gas emissions measurements from soils collected from a long-term permanent grassland soil to assess the impact of different levels of soil saturation on N2O and N2 and CO2 emissions after compaction. CO2 emissions were measured in addition as an estimate of aerobic respiration and thus of O2 consumption, which indicates denitrification is promoted. The measurements included the soil isotopomer ( 15 Nα, 15 Nβ and site preference) analysis of emitted N2O, which in combination with the bulk 15 N and 18 O was used to distinguish between N2O from bacterial denitrification and other processes (e.g. nitrification and fungal denitrification) (Lewicka-Szczebak, 2017).
We conducted measurements at defined saturation of pores size fractions as a prerequisite to model denitrification as a function of water status (Butterbach Bahl et al., 2013 andMüller andClough, 2014). We have under controlled conditions created a single compaction stress of 200 kPa (typical of soils compacted after grazing) in incremental layers using a uniaxial pneumatic piston to simulate a grazing pressure. We hypothesized that at high water saturation, spatial heterogeneity of N emissions decreases due to more homogeneous distribution of the soil nutrients and/or anaerobic microsites. We also hypothesized that even at high soil moisture a mixture of nitrification and denitrification can occur. We base this on the creation of pockets of aerobicity as well of anaerobicity at high soil moisture, mainly driven by soil respiration after application of N and C (using up O2) and further recovery after nutrients are used becoming limiting (increasing aeration). We also aimed to assess how these effects (spatial heterogeneity and source processes) occur in a relatively narrow range of moisture (70-100%). As far as we know there no other studies going to this level of detail. They mostly rely on the knowledge of the effect of moisture on soil processes, whilst in our study, we combined direct measurements of both N2O and N2 with isotopomers of N2O to verify the source processes. In addition, the packing of the cores in our study was of great precision increasing our potential to achieve reproducibility in the replicates where a mixture of aerobic/anaerobic pores might have occurred. We aimed to understand changes in the ratio N2O/(N2O+N2) at the different moisture levels studied in a controlled manner on soil micro and macropores. The N2 emissions were based on direct measurements from the incubated soils, avoiding methodologies that rely on inhibitors such as acetylene with limitations in diffusion in soil and causing oxidation of NO (Nadeem et al., 2013). Moreover, we used isotopocule values of N2O to evaluate if the contribution of bacterial denitrification to the total N2O flux was affected by moisture status.

Soil used in the study
An agricultural soil, under grassland management since at least 1838 (Barré et al., 2010), was collected from a location adjacent to a long-term ley-arable experiment at Rothamsted Research in Hertfordshire (Highfield, see soil properties in Table 1 and further details in Rothamsted Research, 2006;Gregory et al., 2010). The soil had been under permanent cut mixed-species (predominantly Lolium and Trifolium) vegetation. The soil was sampled as described in Gregory et al. (2010). Briefly it was sampled from the upper 150 mm of the profile, air dried in the laboratory, crumbled and sieved (<4 mm), mixed to make a bulk sample and equilibrated at a pre-determined water content (37 g 100 g -1 ; Gregory et al., 2010) in air-tight containers at 4° C for at least 48 hours.

Preparation of soil blocks
The equilibrated soil was then packed into twelve stainless steel blocks (145 mm diameter; h: 100 mm), each of which contained three cylindrical holes (i.d: 50 mm; h: 100 mm each). The cores were packed to a single compaction stress of 200 kPa in incremental layers using a uniaxial pneumatic piston. The three hole-blocks were used to facilitate the compression of the cores. The 200 kPa stress was analogous to a severe compaction event by a tractor (Gregory et al., 2010) or livestock (Scholefield et al., 1985). The total area of the upper surface of soil in each block was therefore 58.9 cm 2 (3 × 19.6 cm 2 ) and the target volume of soil was set to be 544.28 cm 3 (3 × 181.43 cm 3 ) with the objective of leaving a headspace of approximately 45 cm 3 (3 × 15 cm 3 ) for the subsequent experiment. The precise height of the soil (and hence the volume) was measured using the displacement measurement system of a DN10 Test Frame (Davenport-Nene, Wigston, Leicester, UK) with a precision of 0.001 mm.

Equilibration of soil cores at different saturations
The soil was equilibrated to four different initial saturation conditions or treatments (t0) which were based on the likely distribution of water between macropores and micropores. The first treatment was where both the macro-and micropores (and hence the total soil) was fully saturated; the second treatment was where the macropores were half-saturated and the micropores remained fully saturated; the third treatment was where the macropores were fully unsaturated and the micropores again remained fully saturated; and the fourth treatment was where the macropores were fully unsaturated and the micropores were half-saturated. These four treatments are hereafter referred to as SAT/sat; HALFSAT/sat; UNSAT/sat and UNSAT/halfsat, respectively, where upper-case refers to the saturation condition of the macropores and lower-case refers to the saturation condition of the micropores. In order to set these initial saturation conditions, we referred to the gravimetric soil water release characteristic for the soil, as given in Gregory et al. (2010) (see supplement 1). To achieve target water contents during the incubation, the amount of liquid added with the C/N amendment (15 mL) was considered in the total volume of water added. For the SAT/sat and HALFSAT/sat conditions, two sets of three replicate blocks were placed on two fine-grade sand tension tables connected to a water reservoir. For the UNSAT/sat condition a set of three replicate blocks was placed on a tension plate connected to a water reservoir, and the final set of three replicate blocks were placed in pressure plate chambers connected to high-pressure air. All blocks were saturated on their respective apparatus for 24 h, and were then equilibrated for 7 days at the adjusted target matric potentials which were achieved by either lowering the water level in the reservoir (sand tables and tension plate) or by increasing the air pressure (pressure chambers). At the end of equilibration period, the blocks were removed carefully from the apparatus, wrapped in air-tight film, and maintained at 4 °C until the subsequent incubation.

Incubation
The study was carried out under controlled laboratory conditions, using a specialised laboratory denitrification (DENIS) incubation system (Cardenas et al., 2003). Each block containing three cores was placed in an individual incubation vessel of the automated laboratory system in a randomised block design to avoid effect of vessel. The lids for the vessels containing three holes were lined with the cores in the block to ensure that the solution to be applied later would fall on top of each soil core. Stainless steel bulkheads fitted (size for ¼" tubing) on the lids had a three-layered Teflon coated silicone septum (4 mm thick x 7 mm diameter) for supplying the amendment solution by using a gas tight hypodermic syringe. The bulkheads were covered with a stainless-steel nut and only open when amendment was applied. The incubation experiment lasted 13 days from the time the cores started to be flushed until the end of the incubation. The incubation vessels with the soils were contained in a temperature controlled cabinet and the temperature set at 20°C. The incubation vessels were flushed from the bottom at a rate of 30 ml min -1 with a He/O2 mixture (21% O2, natural atmospheric concentration) for 24 h, or until the system and the soils atmosphere were emitting low background levels of both N2 and N2O (N2 can get down to levels of 280 ppm much smaller than atmospheric values). Subsequently, the He/O2 supply was reduced to 10 ml min -1 and directed across the soil surface and measurements of N2O and N2 carried out at approximately 2 hourly cycles to sample from all the 12 vessels. Emissions of CO2 were simultaneously measured.

Application of amendment
An amendment solution equivalent to 75 kg N ha -1 and 400 kg C ha -1 was applied as a 5 ml aliquot a solution containing KNO3 and glucose to each of the three cores in each vessel on day 0 of the incubation. Glucose is added to optimise conditions for denitrification to occur (Morley and Baggs, 2010). The aliquot was placed in a stainless-steel container (volume 1.2 l) which had three holes drilled with bulkheads fitted, two to connect stainless steel tubing for flushing the vessel, and the third one to place a septum on a bulkhead to withdraw solution. Flushing was carried out with He for half an hour before the solution was required for application to the soil cores and continued during the application process to avoid atmospheric N2 contamination (a total of one and a half hours). The amendment solution was manually withdrawn from the container with a glass syringe fitted with a three-way valve onto the soil surface; care was taken to minimise contamination from atmospheric N2 entering the system. The syringe content was injected to the soil cores via the inlets on the lids consecutively in each lid (three cores) and all vessels, completing a total of 36 applications that lasted about 45 minutes. Incubation continued for twelve days, and the evolution of N2O, N2 and CO2 was measured continuously. At the end of each incubation experiment, the soils were removed from the incubation vessels for further analysis. The three cores in each incubation vessel were pooled in one sample and subsamples taken and analysed for mineral N, total N and C and moisture status.

Gas measurements
Gas samples were directed to the relevant analysers via an automated injection valve fitted with 2 loops to direct the sample to two gas chromatographs. Emissions of N2O and CO2 were measured by Gas Chromatography (GC), fitted with an Electron Capture Detector (ECD) and separation achieved by a stainless steel packed column (2 m long, 4 mm bore) filled with 'Porapak Q' (80-100 mesh) and using N2 as the carrier gas. The detection limit for N2O was equivalent to 2.3 g N ha -1 d -1 . The N2 was measured by GC with a He Ionisation Detection (HID) and separation achieved by a PLOT column (30 m long 0.53 mm i.d.), with He as the carrier gas. The detection limit was 9.6 g N ha -1 d -1 . The response of the two GCs was assessed by measuring a range of concentrations for N2O, CO2 and N2. Parent standards of the mixtures 10133 ppm N2O + 1015.8 ppm N2; 501 ppm N2O + 253 ppm N2 and 49.5 ppm N2O + 100.6 ppm N2 were diluted by means of Mass Flow controllers with He to give a range of concentrations of: for N2O of up to 750 ppm and for N2 1015 ppm. For CO2, a parent standard of 30,100 ppm was diluted down to 1136 ppm (all standards were in He as the balance gas). Daily calibrations were carried out for N2O and N2 by using the low standard and doing repeated measurements.
The temperature inside the refrigeration cabinet containing the incubation vessels was logged on an hourly basis and checked at the end of the incubation. The gas outflow rates were also measured and recorded daily, and subsequently used to calculate the flux.

Measurement of N2O isotopic signatures
Gas samples for isotopocule analysis were collected in 115 ml serum bottles sealed with grey butyl crimp-cap septa (Part No 611012, Altmann, Holzkirchen, Germany). The bottles were connected by a Teflon tube to the end of the chamber vents and were vented to the atmosphere through a needle, to maintain flow through the experimental system. Dual isotope and isotopocule signatures of N2O, i.e.  18 O of N2O ( 18 O-N2O), average  15 N ( 15 N bulk ) and δ 15 N from the central Nposition (δ 15 N α ) were analysed after cryo-focussing by isotope ratio mass spectrometry as described previously (Well et al., 2008). 15 N site preference (SP) was obtained as SP = 2 * (δ 15 N α - 15 N bulk ). Dual isotope and isotopocule ratios of a sample (Rsample) were expressed as ‰ deviation from 15 N/ 14 N and 18 O/ 16 O ratios of the reference standard materials (Rstd), atmospheric N2 and standard mean ocean water (SMOW), respectively: (1) where X = 15 N bulk , 15 N α , 15 N β , or 18 O

Data analysis and additional measurements undertaken
The areas under the curves for the N2O, CO2 and N2 data were calculated by using GenStat 11 (VSN International Ltd, Hemel Hempstead, Herts, UK). The resulting areas for the different treatments were analysed by applying analysis of variance (ANOVA). The isotopic ( 15 N bulk , 18 O, and site preference (SP) differences between the four treatment for the different sampling dates were analysed by two-way ANOVA. We also used the Student's t test to check for changes in soil water content over the course of the experiments.
Calculation of the relative contribution of the N2O derived from bacterial denitrification (%BDEN) was done according to Lewicka-Szczebak et al. (2015). The isotopic value of initially produced N2O, i.e. prior to its partial reduction (δ0) was determined using a Rayleigh model (Mariotti et al., 1982), were δ0 is calculated using the fractionation factor of N2O reduction (ηN2O-N2) for SP and the fraction of residual N2O (rN2O) which is equal to the N2O/(N2+N2O) product ratio obtained from direct measurements of N2 and N2O flux. An endmember mixing model was then used to calculate the percentage of bacterial N2O in the total N2O flux (%BDEN) from calculated δ0 values and the SP and δ 18 O endmember values of bacterial denitrification and fungal denitrification/nitrification. The range in endmember and ηN2O-N2 values assumed (adopted from Lewicka-Szczebak, 2017) to calculated maximum and minimum estimates of %BDEN is given in Table 4. We also fitted 3 functions through this data (SP vs N2O/(N2+N2O)) including a second-degree polynomial, a linear and logarithmic function.
Because both, endmember values and ηN2O-N2 values are not constant but subject to the given ranges, we calculated here several scenarios using combinations of maximum, minimum and average endmember and ηN2O-N2 values ( At the same time as preparing the main soil blocks, a set of replicate samples was prepared in exactly the same manner, but in smaller cores (i.d: 50 mm; h: 25 mm). On these samples, we analysed soil mineral N, total N and C and moisture at the start of the incubation. The same parameters were measured after incubation by doing destructive sampling from the cores. Mineral N (NO3 -, NO2and NH4 + ) was analysed after extraction with KCl by means of a segmented flow analyser using a colorimetric technique (Searle, 1984). Total C and N in the air-dried soil were determined using a thermal conductivity detector (TCD, Carlo Erba, model NA2000). Soil moisture was determined by gravimetric analysis after drying at 105°C.

Soil composition
The results after moisture adjustment at the start of the experiment resulted in a range of WFPS of 100 to 71% for the 4 treatments ( Table 2). The results from the end of the incubation also confirmed that there remained significant differences in soil moisture between the high moisture treatments (SAT/sat and HALFSAT/sat) and the two lower moisture treatments (Table 3; one-way ANOVA, p<0.05). Soil in the two wettest states lost statistically significant amounts of water (10% (p=0.006) and 4.4% (p<0.001) for SAT/sat and HALFSAT/sat, respectively) over the course of the 13-day incubation experiment. This was inevitable as there was no way to hold a high (near-saturation) matric potential once the soil was inside the DENIS assembly, and water would have begun to drain by gravitational forces out of the largest macropores (>30 µm). An additional factor was the continuous He/O2 delivery over the soil surface which would have caused some drying. We accepted these as unavoidable features of the experimental set-up, but we assume that the main response of the gaseous emissions occurred under the initial conditions, prior to the loss of water over subsequent days. Soil in the two drier conditions had no significant change in their water content over the experimental period (p= 0.153 and 0.051 for UNSAT/sat and UNSAT/halfsat, respectively). The results of the initial soil composition were, for mineral N: 85.5 mg NO3 --N kg -1 dry soil, 136.2 mg NH4 + -N kg -1 dry soil. The mineral N contents of the soils at the end of the incubation are reported in Table 3 showing that NO3was very small in treatments SAT/sat and HALFSAT/sat (~1 mg N kg -1 dry soil) compared to UNSAT/sat and UNSAT/halfsat (50-100 mg N kg -1 dry soil) at the end of the incubation. Therefore, there was a significant difference in soil NO3between the former, high moisture treatments and the latter drier (UNSAT) treatments which were also significantly different between themselves (p<0.001 for both). The NH4 + content was similar in treatments SAT/sat, HALFSAT/sat and UNSAT/sat (~100 mg N kg -1 dry soil), but slightly lower in treatment UNSAT/halfsat (71.3 mg N kg -1 dry soil), however overall differences were not significant probably due to the large variability on the driest treatment (p>0.05).

Gaseous emissions of N2O, CO2 and N2
All datasets of N2O and N2 emissions showed normal distribution (Fpr.<0.001). The treatments SAT/sat and HALFSAT/sat for all three gases, N2O, CO2 and N2 showed fluxes that were well replicated for all the vessels (see Fig.  1), in contrast for UNSAT/sat and UNSAT/halfsat the emissions between the various replicated vessel in each treatment was not as consistent, leading to a larger within treatment variability in the magnitude and shape of the GHG fluxes measured. The cumulative fluxes also resulted in larger variability for the drier treatments (Table 3).

Nitrous oxide and nitrogen gas.
The general trend was that the N2O concentrations in the headspace increased shortly after the application of the amendment (Fig. 1). The duration of the N2O peak for each replicate soil samples was about three days, except for UNSAT/halfsat in which one of the replicate soils exhibit a peak which lasted for about 5 days. The N2O maximum in the SAT/sat and HALFSAT/sat treatments was of similar magnitude (means of 5.5 and 6.5 kg N ha -1 d -1 , respectively) but not those of UNSAT/sat and UNSAT/halfsat (means of 7.1 and 11.9 kg N ha -1 d -1 , respectively). The N2 concentrations always increased before the soil emitted N2O reached the maximum. The lag between both N2O and N2 peak for all samples was only few hours. Peaks of N2 generally lasted just over four days, except in UNSAT/halfsat where one replicate lasted about 6 days ( Fig. 1). Unlike in the N2O data, there was larger within treatment variability in the replicates for all four treatments. The standard deviations of each mean (Table 3) also indicate the large variability in treatments UNSAT/sat and UNSAT/halfsat for both N2O and N2.
The product ratios, i.e. N2O/(N2O+N2) resulted in a peak just after amendment addition by ca. 0.73 (at 0.49 d), 0.65 (at 0.48 d), 0.99 (at 0.35 d) and 0.88 (at 0.42 d) for SAT/sat, HALFSAT/sat, UNSAT/sat and UNSAT/halfsat, respectively, and then decreases gradually until day 3 where it becomes nearly zero for the 2 wettest treatments, and stays stable for the driest treatments between 0.1-0.2 (see Table 5 where the daily means of these ratios are presented).
The cumulative areas of the N2O and N2 peaks analysed by one-way ANOVA resulted in no significant differences between treatments for both N2O and N2 (Table 3). Due to the large variation in treatments UNSAT/sat and UNSAT/halfsat we carried out a pair wise analysis by using a weighted t-test (Cochran, 1957). This analysis resulted in treatment differences between SAT/sat and HALFSAT/sat, HALFSAT/sat and UNSAT/sat, SAT/sat and UNSAT/sat, but only at the 10% significance level (P <0.1 for both N2O and N2).

Carbon dioxide.
The background CO2 fluxes (before amendment application, i.e. day -1 to day 0) were high at around 30 kg C ha -1 d -1 and variable (not shown). The CO2 concentrations in the headspace increased within a few hours after amendment application.

Isotopocules of N2O
The  15 N bulk of the soil emitted N2O in our study differed significantly among the four treatments and between the seven sampling dates (p<0.001 for both); there was also a significant treatment*sampling date interaction (p<0.001). The maximum  15 N bulk generally occurred on day 3, except for SAT/sat on day 4 ( Table 6).
The maximum  18 O-N2O values were also found on day 3, except for SAT/sat which peaked at day 2 (Table 6).
Overall, the  18 O-N2O values varied significantly between treatment and sampling dates (p<0.001 for both), but there was no significant treatment*time interaction (p>0.05).
The site preference (SP) for the SAT/sat treatment had an initial maximum value on day 2 (6.3‰) which decreased thereafter in the period from day 3 to 5 to a mean SP values of the emitted N2O of 2.0‰ on day 5, subsequently rising to 8.4‰ on day 12 of the experiment ( Table 6). The HALFSAT/sat treatment had the highest initial SP values on day 2 and 3 (both 6.4‰), decreasing again to a value of 2.0‰, but now on day 4 followed by subsequent higher SP values of up to 9.2‰ on day 7 ( Table 6). The two driest treatments (UNSAT/sat and UNSAT/halfsat) both had an initial maximum on day 3 (11.9‰ and 5.9‰, respectively), and in UNSAT/sat the SP value then decreased to day 7 (3.9‰), but in UNSAT/halfsat treatment after a marginal decrease on day 4 (5.4‰) it then increased throughout the experiment reaching 11.8‰ on day 12 (Table 6). The lowest SP values were generally on day 1 in all treatments. Overall, for all parameters, there was more similarity between the more saturated treatments SAT/sat and HALFSAT/sat, and between the two more dry and aerobic treatments UNSAT/sat and UNSAT/halfsat. The N2O / (N2O + N2) ratios vs SP for all treatments in the first two days (when N2O was increasing and the N2O / (N2O + N2) ratio was decreasing) shows a significant negative response of the SP when the ratio increased (Fig. 3). This behaviour suggests that when the emitted gaseous N is dominated by N2O (ratio close to 1) the SP values will be slightly negative with an intercept of -2‰ (Fig. 3), i.e. within the SP range of bacterial denitrification. With decreasing N2O / (N2O + N2) ratio the SP values of soil emitted N2O were increasing to values up to 8‰. This is in juxtaposition with the situation when the N emissions are dominated by N2 or N2O is low, where the SP values of soil emitted N2O were much higher (Fig. 3), pointing to an overall product ratio related to an 'isotopic shift' of 10 to 12.5‰. We fitted 3 functions through this data including a second-degree polynomial, a linear and logarithmic function. The fitted logarithmic function It has been reported that the combination of the isotopic signatures of N2O potentially identifies the contribution of processes other than bacterial denitrification (Köster et al., 2015;Wu Di et al., 2016;Deppe et al., 2017). The question arises to which extent the relationships between the δ 18 O and δ 15 N bulk and between δ 18 O and SP within the individual treatments denitrification dynamics. We checked this to evaluate the robustness of isotope effects during N2O reduction as a prerequisite to calculate the percentage of bacterial denitrification in N2O production. In our data, maximum δ 18 O and SP values, were generally observed at or near the peak of N2 emissions on days 2-3, independent of the moisture treatment (Table 6 and Fig. 3). δ 15 N bulk values of all treatments were mostly negative when N2O fluxes started to increase (day 1, Fig. 1, Table 6), except for UNSAT/halfsat in which the lowest value was before amendment application, reaching their highest values between days 3 and 4 for when N2O fluxes were back to the low initial values, and then decreased during the remaining period. δ 18 O values increased about 10 -20‰ after day 1 reaching maximum values on days 2 or 3 in all treatments, while SP increased in parallel, at least by 3‰ (SAT/sat) and up to 12‰(UNSAT/sat). While δ 18 O exhibited a steady decreasing trend after day 3, SP behaved opposite to δ 15 N bulk with decreasing values while δ 15 N bulk was rising again after days 4 or 5.
We further explored the data by looking at the relationships between the  18 O and  15 N bulk for all the treatments.
The  18 O vs  15 N bulk for all treatments is presented separating the data in three periods (see Fig. 4).: '-1', with  18 O vs  15 N bulk values 1 day prior to the moisture adjustment (and N and C application); '1-2', with values in the first 2 days after the addition of water, N and C were added and N2O emissions were generally increasing in all treatments; and, '3-12', the period in days after moisture adjustment and N and C addition when N2O emissions generally decreased back to baseline soil emissions. There was a strong and significant relationship (P<0.001 and 0.05, respectively) between  18 O vs  15 N bulk for the high moisture treatments (R 2 = 0.973 and 0.923 for SAT/sat and HALFSAT/sat, respectively) at the beginning of the incubation ('1-2') when the N2O emissions are still increasing, in contrast to those of the lower soil moisture treatments that were lower and not significant (R 2 = 0.294 and 0.622, for UNSAT/sat and UNSAT/halfsat, respectively). The relationships between  18 O vs  15 N bulk of emitted N2O for the '3-12' period were significant for SAT/sat and HALFSAT/sat with R 2 values between 0.549 and 0.896 and P values <0.05 and 0.001, respectively (Fig. 4).
Regressions were also significant for this period for the driest treatments (P<0.001). Interestingly, with decreasing soil moisture content (Fig. 4a to 4d) the regression lines of '1-2' and '3-12' day period got closer together in the graphs.
Overall, the  15 N bulk isotopic distances between the two lines was larger for a given δ 18 O-N2O value for SAT/sat and HALFSAT/sat (ca. 20‰) when compared to the UNSAT/sat and UNSAT/halfsat treatments (ca. 13‰) (Fig. 4). So, it seems the  15 N bulk / δ 18 O-N2O signatures are more similar for the drier soils than the two wettest treatments. In addition, then only δ 15 N continue increasing due to fractionation of the NO3during exhaustion of pool 1 in the wet soil (days 3,4,5), finally as pool 1 is depleted and more and more comes from pool 2, the product ratio increases somewhat, and δ 15 N decreases somewhat since pool 2 is less fractionated and, also δ 18 O decreases due to slightly increasing product ratio.
Note that the turning points of δ 18 O and product ratio (Table 3 and 4) for the wetter soils almost coincide.
Similarly to Fig. 4,  18 O vs the SP (Fig. 5) was analysed for the different phases of the experiment. Generally, the slopes (Table 7) for days 1-2 for the three wettest treatments were similar (~0.2-0.3) following the range of known reduction slopes and, also had high and significant (P<0.05) regression coefficients (R 2 = 0.65, 0.90 and 0.87 for SAT/sat, HALFSAT/Sat and UNSAT/sat, respectively). The slopes on days 3-5 were variable but slightly similar on days 7-12 (between 41 and 0.68) for the same three treatments. They were only significant for the 2 driest treatments (P<0.05). On days 7-12 SAT/sat and UNSAT/sat gave significant correlations (P<0.001 and 0.05, respectively).  (Table 6). However, during day 3 to 12 the %BDEN ranged from 78-100% in SAT/sat and 79-100% HALFSAT/Sat, which was generally higher than that estimated at 54-86% for UNSAT/halfsat treatment. The %BDEN of the UNSAT/halfsat in that period was intermediate between SAT/sat and UNSAT/sat with range of range 60-100% (Table 6). The final values were similar to those on day -1, except for the UNSAT/sat treatment.

Effect of soil moisture
The observed decrease in total N emissions with decreasing initial soil moisture reflects the effect of soil moisture as reported in previous studies (Well et al., 2006). The differences when comparing the cumulative fluxes however, were only marginally (p<0.1) significant (Table 3) mostly due to large variability within replicates in the drier treatments (see Fig. 1b). Davidson et al. (1991) provided a WFPS threshold for determination of source process, with a value of 60% WFPS as the borderline between nitrification and denitrification as source processes for N2O production. The WFPS in all treatments in our study was larger than 70%, above this 60% threshold, and referred to as the "optimum water content" for N2O by Scheer et al. (2009), so we can be confident that denitrification was likely to have been the main source process in our experiment. In addition, Bateman et al. (2004) observed the largest N2O fluxes at 70% WFPS on a silty loam soil, lower than the 80% value for the largest fluxes from the clay soil in our study (Fig. 2) suggesting that this optimum value could change with soil type. Further, the maximum total measured N lost (N2O+N2) in our study occurred at about 95% WFPS (Fig. 2), but not many studies report N2 fluxes for comparison and we are still missing measurements of nitric oxide (NO)  and ammonia (NH3) to account for the total N losses. It is however possible that the N2O+N2 fluxes in the SAT/sat treatment were underestimated due to low diffusivity in the water filled pores (Well et al., 2001). Gases would have been trapped (particularly in the higher saturation treatments) due to low diffusion and thus possibly masked differences in N2 and N2O production since this fraction of gases was not detected (Harter et al. 2016). It is worth mentioning that there was some drying during the incubation. The flow of the gas is very slow (10 ml/min) simulating a low wind speed so normally this would dry the soil in field conditions too. It would represent a rainfall event where the initial moisture differs between treatments but some drying occurs due to the wind flow. We believe however, that the effect of drying will be more relevant (and significant relative to the initial moisture) later in the incubation.
The smaller standard errors in both N2O and N2 data for the larger soil moisture levels (Table 3 and Fig. 1) could suggest that at high moisture contents nutrient distribution (N and C) on the top of the core is more homogeneous making replicate cores to behave similarly. At the lower soil moisture for both N2O and N2, it is possible that some cracks appear on the soil surface causing downwards nutrient movement, resulting in heterogeneity in nutrient distribution on the surface and increasing variability between replicates, reflected in the larger standard errors of the fluxes. Laudone et al. (2011) studied, using a biophysical model, the positioning of the hot-spot zones away from the critical percolation path (described as 'where air first breaks through the structure as water is removed at increasing tensions') and found it slowed the increase and decline in emission of CO2, N2O and N2. They found that hot-spot zones further away from the critical percolation path would reach the anaerobic conditions required for denitrification in shorter time, the products of the denitrification reactions take longer to migrate from the hot-spot zones to the critical percolation path and to reach the surface of the system. The model and its parameters can be used for modelling the effect of soil compaction and saturation on the emission of N2O. They suggest that having determined biophysical parameters influencing N2O production, it remains to determine whether soil structure, or simply saturation, is the determining factor when the biological parameters are constrained. Furthermore, Clough et al. (2013) indicate that microbial scale models need to be included on larger models linking microbial processes and nutrient cycling, in order to consider spatial and temporal variation. Kulkarni et al. (2008) refers to "hot spots" and "hot moments" of denitrification as scale dependant and highlight the limitations for extrapolating fluxes to larger scales due to these inherent variabilities. In addition, in order to understand heterogeneity of added amendment, we assumed (for modelling purposes) multiple pools after N and glucose amendment. In Bergstermann et al.
(2011) for example we presumed they occupied 10% of the core volume (pool 1), because this resulted in a good fit for measured and modelled N2 and N2O fluxes as well as δ 15 N bulk values. In the current study, we could assume that in the wettest treatment this (proportional) volume was smaller i.e. similar to the pore volume displaced by the added 5 ml of amendment since pores were almost completely filled with water. Furthermore, that it would have been the largest in the driest treatment where the amendment solution was able to infiltrate the partly saturated pore space and thereby increasing the water content in the infiltrated volume. With regards to leaching, it was minimal (< 0.5 mL water in the core) and so significant leaching of amendment can thus be excluded. Other techniques such as X ray and MRI could help determine the distribution of added nutrients in the soil matrix. Well et al. (2003) found that under saturated conditions there was good agreement between laboratory and field measurements of denitrification, and attributed deviations, under unsaturated conditions, to spatial variability of anaerobic microsites and redox potential. Dealing with spatial variability when measuring N2O fluxes in the field remains a challenge, but the uncertainty could be potentially reduced if water distribution is known. Our laboratory study suggests that soil N2O and N2 emission for higher moisture levels would be less variable than for drier soils and suggests that for the former a smaller number of spatially defined samples will be needed to get an accurate field estimate. This applied to a lesser extent to the CO2 fluxes.

Relationship with soil parameters to determine processes
Our results, for the two highest water contents (SAT/sat and HALFSAT/sat), indicated that N2O only contributed 20% of the total N emissions, as compared to 40-50% at the lowest water contents (UNSAT/sat and UNSAT/halfsat, Table 3). This was due to reduction to N2 at the high moisture level, confirmed by the larger N2 fluxes, favoured by low gas diffusion which increased the N2O residence time and the chance of further transformation (Klefoth et al., 2014a).
We should also consider the potential underestimation of the fluxes in the highest saturation treatment due to restricted diffusion in the water filled pores (Well et al., 2001). A total of 99% of the soil NO3was consumed in the two high water treatments, whereas in the drier UNSAT/sat and UNSAT/halfsat treatments there still was 35% and 70% of the initial amount of NO3left in the soil, at the end of the incubation, respectively (Table 3). The total amount of gas lost compared to the NO3consumed was almost 3 times for the wetter treatments, and less than twice for the 2 drier ones. This agrees with denitrification as the dominant process source for N2O with larger consumption of NO3at the higher moisture and larger N2 to N2O ratios (5.7, 4.7 for SAT/sat and HALFSAT/sat, respectively), whereas at the lower moisture, ratios were lower (1.5 and 1.0 for UNSAT/sat and UNSAT/halfsat, respectively) (Davidson, 1991). This also indicates that with WFPS above the 60% threshold for N2O production from denitrification, there was an increasing proportion of anaerobic microsites with increase in saturation controlling NO3consumption and N2/N2O ratios in an almost linear manner. With WFPS values between 71-100 % and N2/N2O between 1.0 and 5.7, a regression can be estimated: Y=0.1723 X -11.82 (R 2 =0.8585), where Y is N2/N2O and X is %WFPS. In summary, we propose that heterogeneous distribution of anaerobic microsites could have been the limiting factor for complete depletion of NO3and conversion to N2O in the two drier treatments. In addition, in the UNSAT/halfsat treatment there was a decrease in soil NH4 + at the end of the incubation (almost 50%; Table 3) suggesting nitrification could have been occurring at this water content which also agrees with the increase in NO3 -, even though WFPS was relatively high (>71%) ( Table 3). It is important to note that as we did not assess gross nitrification, the observed net nitrification based on lowering in NH4 + could underestimate gross nitrification since there might have been substantial N mineralisation during the incubation. However, under conditions favouring denitrification at high soil moisture the typical N2O produced from nitrification is much lower compared to that from denitrification (Lewicka-Szczebak et al., 2017) with the maximum reported values for the N2O yield of nitrification of 1-3 % (e.g. Deppe et al., 2017). If this is the case, nitrification fluxes could not have exceeded 1 kg N with NH4 + loss of < 30 kg * 3% ~1 kg N. This would have represented for the driest treatment, if conditions were suitable only for one day, that nitrification-derived N2O would have been 6% of the total N2O produced. Loss of NH3 was not probable at such low pH (5.6). The corresponding rate of NO3production using the initial and final soil contents and assuming other processes were less important in magnitude, would have been < 1 mg NO3 --N kg dry soil -1 d -1 which is a reasonable rate (Hatch et al., 2002). The other three treatments lost similar amounts of soil NH4 + during the incubation (23-26%) which could have been due to some degree of nitrification at the start of the incubation before O2 was depleted in the soil microsites or due to NH4 + immobilisation (Table 3) (Geisseler et al., 2010).
A mass N balance, considering the initial and final soil NO3 -, NH4 + , added NO3and the emitted N (as N2O and N2) results in unaccounted N-loss of 177.2, 177.6, 130.6 and 110.8 mg N kg -1 for SAT/sat, HALFSAT/sat, UNSAT/sat and UNSAT/halfsat, respectively, that could have been emitted as other N gases (such as NO), and some, immobilised in the microbial biomass. NO fluxes reported by Loick et al. (2016) for example, result in a ratio N2O/NO of 0.4. In summary, unaccounted-for N loss is two to three times the total measured gas loss (Table 3). In addition, in the SAT/sat treatment there was probably an underestimation of the produced N2 and N2O due to restricted diffusion at the high WFPS (e.g. Well et al., 2001). Well et al. (2003) found that under saturated conditions there was good agreement between laboratory and field measurements of denitrification, and attributed deviations, under unsaturated conditions, to spatial variability of anaerobic microsites and redox potential. Dealing with spatial variability when measuring N2O fluxes in the field remains a challenge, but the uncertainty could be potentially reduced if water distribution is known. Our laboratory study suggests that soil N2O and N2 emission for higher moisture levels would be less variable than for drier soils and suggests that for the former a smaller number of spatially defined samples will be needed to get an accurate field estimate. This applied to a lesser extent to the CO2 fluxes.

Isotopocule trends.
Trends of isotopocule values of emitted N2O coincided with those of N2 and N2O fluxes. The results from the isotopocule data (Table 6 and Fig. 3) also indicated that generally there were more isotopic similarities between the two wettest treatments when compared to the two contrasting drier soil moisture treatments.
Isotopocule values of emitted N2O reflect multiple processes where all signatures are affected by the admixture of several microbial processes, the extent of N2O reduction to N2 as well as the variability of the associated isotope effects . Moreover, for δ 18 O and δ 15 N bulk the precursor signatures are variable (Decock and Six, 2013), for δ 18 O the O exchange with water can be also variable (Lewicka-Szczebak et al., 2017). Since the number of influencing factors clearly exceeds the number of isotopocule values, unequivocal results can only be obtained if certain processes can be excluded or be determined independently, Lewicka-Szczebak, 2017).
The two latter conditions were fulfilled in this study, i.e. N2O fluxes were high and several orders of magnitude above possible nitrification fluxes, since the N2Oto-NO3ratio yield of nitrification products rarely exceeds 1% (Well et al., 2008;Zhu et al., 2012). Moreover, N2 fluxes and thus N2O reduction rates were exactly quantified.
The estimated values of % BDEN indicate that in the period immediately after amendment application all moisture treatments were similar, reflecting that the microbial response to N and C added was the same and denitrification dominated. This was the same for the rest of the period for the wetter treatments. In the drier treatments, proportions decreased afterwards and were similar to values before amendment application, possibly due to recovery of more aerobic conditions that could have encouraged other processes to contribute. As N2 was still produced in the driest treatment, (but in smaller amounts), this indicated ongoing denitrifying conditions and thus large contributions to the total N2O flux from nitrification were not probable, but some occurred as suggested by NH4 + consumption.
The trends observed reflect the dynamics resulting from the simultaneous application of NO3and labile C (glucose) on the soil surface as described in previous studies (Meijide et al., 2010;Bergstermann et al., 2011) where the same soil was used, resulting in two locally distinct NO3pools with differing denitrification dynamics. In the soil volume reached by the NO3 -/glucose amendment, denitrification was initially intense with high N2 and N2O fluxes and rapid isotopic enrichment of the NO3 --N. When the NO3and/or glucose of this first pool were exhausted, N2 and N2O fluxes were much lower and dominated by the initial NO3pool that was not reached by the glucose/NO3amendment and that is less fractionated due to its lower exhaustion by denitrification, causing decreasing trends in δ 15 N bulk of emitted N2O. This is also reflected in Fig 4 where N2O fluxes from both pools exhibited correlations (and mostly significant) between δ 15 N bulk and δ 18 O due to varying N2O reduction, but δ 15 N bulk values in days 1 and 2 -i.e. the phase when Pool 1 dominated -were distinct from the previous and later phase.
The fit of 15 N bulk / 18 O data to two distinct and distant regression lines can be attributed to two facts: Firstly, in the wet treatment (Fig 4a, b) Pool 1 was probably completely exhausted and there was little NO3formation from nitrification (indicated by final NO3values close to 0, Table 3) whereas the drier treatment exhibited substantial NO3formation and high residual NO3 -. Hence, there was probably still some N2O from Pool 1 after day 2 in the dry treatment but not in the wetter ones. Secondly, the product ratios after day 2 of the drier treatments were higher (0.13 to 0.44) compared to the wetter treatments (0.001 to 0.09). Thus the isotope effect of N2O reduction was smaller in the drier treatments, leading to a smaller upshift of δ 15 N bulk and thus more negative values after day 2, i.e. with values closer to days 1 +2.
This finding further confirms that δ 15 N/δ 18 O patterns are useful to identify the presence of several N pools, e.g.
typically occurring after application of liquid organic fertilizers which has been previously demonstrated using isotopocule patterns (Koster et al., 2015).
Interestingly, the highest  15 N bulk and δ 18 O values of the emitted N2O were found in the soils of the HALFSAT/sat treatment, although it may have been expected that the highest isotope values from the N2O would be found in the wettest soil (SAT/sat) because N2O reduction to N2 is favoured under water-saturated conditions due to extended residence time of produced N2O (Well et al., 2012). However, N2O/(N2+N2O) ratios of the SAT/sat and SAT/halfsat treatments were not different (Table 5). Bol et al. (2004) also found that some estuarine soils under flooded conditions (akin to our SAT/sat) showed some strong simultaneous depletions (rather than enrichments) of the emitted N2O  15 N bulk and δ 18 O values. These authors suggested that this observation may have resulted from a flux contribution of an 'isotopically' unidentified N2O production pathway. Another explanation could be complete consumption of some of the produced N2O in isolated microniches in the SAT/sat treatment due to inhibited diffusivity in the fully saturated pores space. N2 formation in these isolated domains would not affect the isotopocule values of emitted N2O and this would thus result in lower apparent isotope effects of N2O reduction in water saturated environments as suggested by Well et al. (2012).
The SP, believed to be a better predictor of the N2O source as it is independent of the substrate isotopic signature (Ostrom, 2011), has been suggested as it can be used to estimate N2O reduction to N2 in cases when bacterial denitrification can be assumed to dominate N2O fluxes (Koster et al., 2013;Lewicka-Szczebak et al., 2015). There was a strong correlation between the SP and N2O / (N2O+N2) ratios on the first 2 days of the incubation for all treatments up until the N2O reached its maximum (Fig. 3) which reflects the accumulation of δ 15 N at the alpha position during ongoing N2O reduction to N2. Later on in the experiment, beyond day 3, this was not observed probably because in that period the product ratio remained almost unchanged and very low (Table 6). Similar observations have been reported by Meijide et al. (2010) and Bergstermann et al. (2011), as they also found a decrease in SP during the peak flux period in total N2+N2O emissions, but only when the soil had been kept wet prior to the start of the experiment . These results confirm from 2 independent studies (Lewicka-Szczebak et al., 2014) that there is a relationship between the product ratios and isotopic signatures of the N2O emitted. The δ 18 O vs SP regressions indicate more similarity between the three wettest treatments as well as high regression coefficients, suggesting this SP/δ 18 O ratio could also be used to help identify patterns for emissions and their sources.

Link to modelling approaches.
Since isotopocule data could be compared to N2 and N2O fluxes, the variability of isotope effects of N2O production and reduction to N2 by denitrification could be determined from this data set  and this included modelling the two pool dynamics discussed above. It was demonstrated that net isotope effects of N2O reduction (ηN2O-N2) determined for both NO3pools differed. Pool 1 representing amended soil and resulting in high fluxes but moderate product ratio, exhibited ηN2O-N2 values and the characteristic η 18 O/η 15 N ratios similar to those previously reported, whereas for Pool 2 (amendment-free soil) characterized by lower fluxes and very low product ratio, the net isotope effects were much smaller and the η 18 O/η 15 N ratios, previously accepted as typical for N2O reduction processes (i.e., higher than 2), were not valid. The question arises, if the poor coincidence of Pool 2 isotopologue fluxes with previous N2O reduction studies reflects the variability of isotope effects of N2O reduction or if the contribution of other processes like fungal denitrification could explain this (Lewicka-Szczabak et al., 2017). The latter explanation is evaluated in section 4.3 Liu et al. (2016) noted that on the catchment scale potential N2O emission rates were related to hydroxylamine and NO3 -, but not NH4 + content in soil. Zou et al. (2014) found high SP (15.0 to 20.1‰) values at WFPS of 73 to 89% suggesting that fungal denitrification and bacterial nitrification contributed to N2O production to a degree equivalent to that of bacterial denitrification.
To verify the contribution of fungal denitrification and/or hydroxylamine oxidation we can first look at the ηSPN2O-NO3 values calculated in the previous modelling study applied on the same dataset, (Table 1, the final modelling Step, Lewicka-Szczebak et al., 2015). For Pool 1 there are no significant differences between the values of various treatments, SP0 ranges from (-1.8±4.9) to (+0.1±2.5). Pool 1 emission was mostly active in days 1-2, hence these values confirm the bacterial dominance in the emission at the beginning of incubation, which originates mainly from the amendment addition and represent similar pathway for all treatments. However, for the Pool 2 emission we could observe a significant difference when compared the two wet treatments (SAT/sat and HALFSAT/sat: (-5.6±7.0)) with the UNSAT/sat treatment (+3.8±5.8). This represents the emission from unamended soil which was dominating after the third day of the incubation and indicates higher nitrification contribution for the drier treatment.

Contribution of bacterial denitrification.
An endmember mixing approach has been previously used to estimate the fraction of bacterial N2O (%BDEN), but without independent estimates of N2O reduction (Zou et al., 2014), but due to the unknown isotopic shift by N2O reduction, the ranges of minimum and maximum estimates were large, showing that limited information is obtained without N2 flux measurement.
In an incubation study with two arable soils, Koster et al. (2013) used N2O/(N2+N2O) ratios and isotopocule values of gaseous fluxes to calculate SP of N2O production (referred to as SP0), which is equivalent to SP0 using the Rayleigh model and published values of ηN2O-N2. The endmember mixing approach based on SP0 was then used to estimate fungal denitrification and/or hydroxylamine oxidation giving indications for a substantial contribution in a clay soil, but not in a loamy soil. Here we presented for the first time an extensive data set with large range in product ratios and moisture to calculate the contribution of bacterial denitrification (%BDEN) of emitted N2O from SP0. The uncertainty of this approach arises from three factors, (i) from the range of SP0 endmember values for bacterial denitrification of -11 to 0 per mil and 30 to 37 for hydroxylamine oxidation/fungal denitrification, (ii) from the range of net isotope effect values of N2O reduction (ηN2O-N2) for SP which vary from -2 to -8 per mil , and iii) system condition (open vs. closed) taken to estimate the net isotope effect (Wu et al., 2016).
The observation that %BDEN of emitted N2O was generally high (63-100%) in the wettest treatment (SAT/sat) was not unexpected. However interestingly %BDEN in the HALFSAT/sat treatment was very similar (71-98%), pointing to the role of the wetter areas of the soil microaggregates contributing to high %BDEN values. The slightly lower values, i.e. down 60% in UNSAT/sat %BDEN range of 60-100%, suggest that the majority of N2O derived from bacterial denitrification still results from the wetter microaggregates of the soils, despite the fact that the macropores are now more aerobic. Only, when the micropores become partially wet, as in the UNSAT/halfsat treatment, do the more aerobic soil conditions allow a higher contribution of nitrification/fungal denitrification ranging from 0 -46% (1 -% BDEN, Table 6) on days 3-12 (Zhu et al., 2013). Differences in the contribution of nitrification/fungal denitrification between the flux phases when different NO3pools were presumably dominating are only indicated in the driest treatment, since 1-%BDEN was higher after day 2 (14 to 46%) compared to days 1+2 (0 to 33 %). This larger share of nitrification/fungal denitrification can be attributed to the increasing contribution from Pool 2 to the total flux as indicated by the modeling of higher SP0 for Pool 2 (see previous section and Lewicka-Szczebak et al. (2015). In addition, indication for elevated contribution of processes other than bacterial denitrification were only evident in the drier treatments during phases before and after N2, N2O fluxes were strongly enhanced by glucose amendment. The data supply no clue whether the other processes were suppressed during the anoxia induced by glucose decomposition or just masked by the vast glucose-induced bacterial N2O fluxes.

Figure 5
Site Preference vs δ 18 O in all treatments for three periods (day -1, days 1-2 and days 3-12) in the experiment: a. SAT/sat treatment; b. HALFSAT/sat; c. UNSAT/sat; d. UNSAT/halfsat. Equations of fitted functions and correlation coefficients are in Table 7 for 1-2, 3-5 and 7-12 (5-12 for c.). Endmember areas for nitrification, N; bacterial denitrification, D; fungal denitrification, FD and nitrifier denitrification, ND and corresponding vectors or reduction lines (black solid lines) are from Lewicka-Szczebak et al., (2017), and 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 NO reduction for SP and δ 18 O (Lewicka-Szczebak et al., 2017). 5c .