Isotopic fractionation of N 2 O to quantify N 2 O reduction to N 2-validation with Helium incubation and 15 N gas flux methods

Stable isotopic analyses of soil-emitted N2O ( 15 N bulk ,  18 O and  15 N sp = 15 N site preference within the linear N2O molecule) may help to quantify N2O reduction to N2, a main unknown magnitude 10 in the soil nitrogen cycling. The N2O residual fraction (rN2O) can be theoretically calculated from the measured isotopic enrichment of the residual N2O. However, various N2O producing pathways may also influence the N2O isotopic signatures, and hence complicate the application of this isotopic fractionation approach. Here this approach was tested based on laboratory soil incubations with two different soil types 15 applying two reference methods for quantification of rN2O: Helium incubation with direct measurement of N2 flux and the 15 N gas flux method. This allowed a comparison of the measured rN2O values with the ones calculated based on isotopic enrichment of residual N2O. The results indicate that the performance of the N2O isotopic fractionation approach is related with the accompanying N2O and N2 source processes and the most critical is the determination of the initial isotopic signature of N2O before 20 reduction (0). We show that 0 can be well experimentally determined if stable in time and successfully applied for determination of rN2O based on  15 N sp values. Much more problematic is to deal with temporal changes of 0 values leading to failure of the approach based on  15 N sp values only. For this case we propose here a dual N2O isotopocule mapping approach, where calculations are based on the relation between  18 O and  15 N sp values. This allows for the simultaneous estimation of the N2O 25 producing pathways contribution and the rN2O value. Biogeosciences Discuss., doi:10.5194/bg-2016-276, 2016 Manuscript under review for journal Biogeosciences Published: 30 August 2016 c © Author(s) 2016. CC-BY 3.0 License.


Introduction
N 2 O reduction to N 2 is the last step of microbial denitrification, i.e., anoxic reduction of nitrate (NO 3 -) to N 2 through the following intermediates: NO 3 - NO 2 - NO  N 2 O  N 2 (Firestone and Davidson, 1989;Knowles, 1982).Commonly applied analytical techniques enable us to quantitatively analyse only the last intermediate of this process, N 2 O, whereas the contribution of N 2 O reduction to N 2 is mostly unknown, since it is challenging to directly measure N 2 emissions due to the high atmospheric background (Bouwman et al., 2013;Saggar et al., 2013).To overcome this problem, three methods for N 2 -flux estimation are applicable (Groffman, 2012;Groffman et al., 2006): direct N 2 -measurements under a N 2 -free helium atmosphere (helium incubation method), 15 N analyses of gas fluxes after addition of 15 N-labelled substrate ( 15 N gas flux method), and the reduction inhibition method based on the comparison of N 2 O fluxes with and without acetylene application (acetylene inhibition method).
These methods were widely applied in laboratory studies to determine the contribution of N 2 O reduction to N 2 , which is usually expressed as the fraction of the residual unreduced N 2 O: r N2O = y N2O /(y N2 +y N2O ) (y: mole fraction).The whole scale of possible r N2O variations, ranging from 0 to 1, had been found in laboratory studies (Lewicka-Szczebak et al., 2015;Mathieu et al., 2006;Morse and Bernhardt, 2013;Senbayram et al., 2012).However, due to technical limitations, only the 15 N gas flux method can be applied in field conditions to determine the N 2 O residual fraction (Aulakh et al., 1991;Baily et al., 2012;Bergsma et al., 2001;Decock and Six, 2013;Kulkarni et al., 2013;Mosier et al., 1986).The acetylene inhibition method is not useful for field studies due to catalytic NO decomposition in presence of C 2 H 2 and O 2 (Bollmann and Conrad, 1997;Felber et al., 2012;Nadeem et al., 2013) and the Helium incubation method requires a sophisticated air-tight incubation system, so far attainable only in laboratory conditions.Hence, no comprehensive data sets from field-based measurements of soil N 2 emissions are available and this important component in soil nitrogen budget is still missing.This constitutes a serious shortcoming in understanding and mitigating the microbial consumption of nitrogen fertilisers (Bouwman et al., 2013;Seitzinger, 2008), and the N 2 O emission, which significantly contributes to global warming and stratospheric ozone depletion (IPCC, 2007;Ravishankara et al., 2009).
N 2 O isotopic fractionation studies could potentially be used for quantification of r N2O in field conditions (Park et al., 2011;Toyoda et al., 2011;Zou et al., 2014).Its advantage over the 15 N gas flux method lies in its easier and non-invasive application, no need of additional fertilization, and much lower costs, thus, the potential for a more widespread use.These studies use isotopic analyses of the residual unreduced N 2 O, of which three isotopic signatures can be determined: of oxygen (δ 18 O), bulk nitrogen (δ 15 N bulk ) and nitrogen site preference (δ 15 N sp ), i.e., the difference in δ 15 N between the central and the peripheral N atom of linear N 2 O molecules (Brenninkmeijer and Röckmann, 1999;Toyoda and Yoshida, 1999).All these three isotopic signatures (δ 18 O, δ 15 N bulk and δ 15 N sp ) are altered during the N 2 O reduction process and the magnitude of the observed change depends largely on the N 2 O residual fraction (Jinuntuya-Nortman et al., 2008;Menyailo and Hungate, 2006;Ostrom et al., 2007;Well and Flessa, 2009a).Hence, principally, this fraction can be calculated from the isotopic enrichment of the residual N 2 O, provided that the isotopic signature of the initially produced N 2 O before reduction (δ 0 ) and the net isotope effect associated with N 2 O reduction (η red ) are known (Lewicka-Szczebak et al., 2014).δ 0 15 N and δ 0 18 O values depend largely on the isotopic signatures of the N 2 O precursors, i.e., of NH 4 + , NO 3 -, NO 2 -, H 2 O, and on the transformation pathways, e.g., nitrification or denitrification (Perez et al., 2006).δ 0 15 N sp values, however, are independent of the precursors, but differ according to different pathways, e.g., nitrification or denitrification (Sutka et al., 2006) and different microbial communities, e.g., bacterial or fungal denitrifiers (Rohe et al., 2014;Sutka et al., 2008) involved in the N 2 O production.Therefore, δ 0 values may vary between different soils and due to different conditions, e.g., moisture, temperature, fertilizing.η red values are variable depending on experimental conditions, but these variations are largest for η red 18 O and η red 15 N bulk , whereas for η red 15 N sp quite stable values in the range from -7.7 to -2.3 ‰ with average of -5.4±1.6 ‰ have been found (Lewicka-Szczebak et al., 2014).Moreover, recently this value has been also confirmed under oxic atmosphere (Lewicka-Szczebak et al., 2015), hence, it can be expected that δ 15 N sp values can be applied as a robust basis to calculate N 2 O reduction also for field studies.
Currently, the most important question is whether the isotopic fractionation factors for denitrification processes determined in laboratory experiments are transferable to field conditions and how robust they are for calculating the N 2 O residual fraction and quantifying the entire nitrogen loss Nevertheless, 15 N-labelled treatments provide additional information on the coexisting N 2 O-forming processes (Müller et al., 2014), which might possibly impact the N 2 O isotopic signatures.Therefore, here we have applied both methods for the same pair of very different soils, a mineral arable and an organic grassland soil, for better understanding of the complex N 2 O production and consumption in these soils.The main aim of this study was to (i) check how precisely the N 2 O residual fraction can be calculated with the isotopic fractionation approach, (ii) identify the sources of possible bias, e.g., the coexisting N 2 O forming processes, and (iii) search for the possibilities to improve the precision and applicability of this calculation approach.

Methods
The list with explanations of all abbreviations and specific terms used in the manuscript can be found in the Supplement (S1).

Experiment 1 -Helium incubation as reference method (Exp1)
Two soil types were used: a mineral arable soil with silt loam texture classified as a Haplic Luvisol (Min soil) and an organic grassland soil classified as Histic Gleysol (Org soil).The soils were air dried and sieved at 4mm mesh size.Afterwards, the soil was rewetted to obtain 70 % water-filled pore space (WFPS) and fertilised with 50 mg N (added as NO 3 ) per kg soil.Then soils were thoroughly mixed to obtain a homogenous distribution of water and fertilizer and 250 cm 3 of wet soil were repacked into each incubation vessel with bulk densities of 1.4 g cm -3 for the Min soil and 0.4 g cm -3 for Org soil.
Afterwards the water deficit to the target WFPS, depending on the treatment 70 or 80 % WFPS, was added on the top of the soil.The incubations were performed using a special gas-tight incubation system allowing for application of N 2 -free atmosphere.This system has been described in detail by Eickenscheidt et al. (2014).Here we present briefly its general idea.
The incubation vessels were cooled to 2 ºC, repeatedly evacuated (to 0. The data from two selected samplings of this experiment have been already published with particular emphasis on the O isotopic fractionation (experiment 2.3-2.6 in (Lewicka-Szczebak et al., 2016)).

Experiment 2 -15 N gas flux as reference method (Exp2)
The same soils (Min and Org soil) as in Exp1 were used for parallel incubations under either an anoxic (N 2 ) or an oxic (78 % He + 2 % N 2 + 20 % O 2 ) atmosphere with continuous gas flow at 10 cm 3 min -1 .
The N 2 background concentration in the oxic incubation was reduced to increase the sensitivity of the 15 N-flux method (Meyer et al., 2010).
The soils were air dried and sieved at 4mm mesh size.Afterwards, the soil was rewetted to added fertilizer.500 cm 3 of wet soil was repacked into incubation vessels with bulk densities of 1.4 g cm -3 for the Min soil and 0.4 g cm -3 for the Org soil.Afterwards the water deficit to the target WFPS of 75 % for Min and 85 % for Org soil was added on the top of the soil.Glass jars (0.8 dm 3 J.WECK GmbH u.Co. KG, Wehr, Germany) were used with airtight rubber seal and with two three-way valves installed in their glass cover to enable continuous gas flow and sampling.The sampling vials were connected to vents of the incubation vials (Well et al., 2008) and were exchanged each 24 h.The soils were incubated for 9 days at constant temperature (22 ºC).During each sampling, gas samples were collected in two 12 cm 3 Labco Exetainers® (Labco Limited, Ceredigion, UK) and for NA treatment additionally in one 120 cm 3 crimped vial.

Chromatographic analyses
In Exp1, online trace gas concentration analysis of N 2 was performed with a micro-GC (Agilent Technologies, 3000 Micro GC), equipped with a thermal conductivity detector (TCD).Concentrations of trace gases were analysed by a GC (Shimadzu, Duisburg, Germany, GC-14B) equipped with an electron capture detector (ECD) for N 2 O and CO 2 .The measurements precision was better than 20 ppb for N 2 O and 200 ppb for N 2 , respectively.
In Exp2 the samples for gas concentration analyses were collected in Labco Exetainer® (Labco Limited, Ceredigion, UK) vials and were analysed using an Agilent 7890A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with an ECD detector.Precision as given by the standard deviation (1σ) of four standard gas mixtures was typically 1.5%.and Yoshida, 1999).δ 15 N β (δ 15 N of the peripheral N position of the N 2 O molecule) was calculated from δ 15 N bulk = (δ 15 N α + δ 15 N β ) / 2 and 15 N site preference (δ 15 N sp ) from δ 15 N sp = δ 15 N αδ 15 N β .The scrambling factor and 17 O-correction were taken into account (Röckmann et al., 2003).Pure N 2 O (Westfalengas; purity > 99.995 %) was used as internal reference gas.It had been analyzed for isotopocule values in the laboratory of the Tokyo Institute of Technology using calibration procedures reported previously (Toyoda and Yoshida, 1999;Westley et al., 2007).Moreover, the standards from a laboratory intercomparison (REF1, REF2) were used for performing two-point calibration for δ 15 N sp values (Mohn et al., 2014).

Soil analyses
All isotopic values are expressed as ‰ deviation from the 15 N/ 14 N and 18 O/ 16 O ratios of the reference materials (i.e., atmospheric N 2 and Vienna Standard Mean Ocean Water (V-SMOW), respectively).The analytical precision determined as standard deviation (1σ) of the internal standards for measurements of δ 15 N bulk , δ 18 O and δ 15 N sp was typically 0.1, 0.1, and 0.5 ‰, respectively.

Isotopic signatures of NO 3
δ 18 O and δ 15 N of nitrate in the soil solution were determined using the bacterial denitrification method (Sigman et al., 2001).The analytical precision determined as standard deviation (1σ) of the international standards was typically 0.5 ‰ for δ 18 O and 0.2 ‰ for δ 15 N.

Soil water analyses
Soil water was extracted with the method described by Königer et al. (2011) and δ 18 O of water samples was measured using a cavity ring down spectrometer Picarro L1115-i (Picarro Inc., Santa Clara, USA).
The analytical precision determined as standard deviation (1σ) of the internal standards was below 0.1 ‰.The overall error associated with the soil water extraction method determined as standard deviation (1σ) of the 5 samples replicates was below 0.5 ‰. 15 N abundances of NO 3 -(a NO3-) and NH 4 + (a NH4+ ) were measured according to the procedure described in Stange et al. (2007).NO 3 -was reduced to NO by Vanadium -IIIchloride (VCl 3 ) and NH 4 + was oxidized to N 2 by Hypobromide (NaOBr).NO and N 2 were used as measurement gas.Measurements were done with a quadrupole mass spectrometer (GAM 200, InProcess, Bremen, Germany).

15 N 2 O and 15 N 2
The gas samples from the 15 N treatments of Exp2 were analysed for m/z 28 ( 14 N 14 N), 29 ( 14 N 15 N) and 30 ( 15 N 15 N) of N 2 using a modified GasBench II preparation system coupled to an isotope ratio mass spectrometer (MAT 253, Thermo Fisher Scientific, Bremen, Germany) according to Lewicka-Szczebak et al. (2013).This system allows a simultaneous determination of isotope ratios 29 R ( 29 N 2 / 28 N 2 ) and 30 R ( 30 N 2 / 28 N 2 ) representing three separated gas species (N 2 , N 2 +N 2 O and N 2 O), all measured as N 2 gas after N 2 O reduction in a Cu oven.
For each of the analysed gas species (N 2 , N 2 +N 2 O and N 2 O) the fraction originating from the 15 Nlabelled pool (f P ) was calculated after Spott et al. (2006) where 0.003663 is the fraction of 15 N in non-labelled N 2 O and f N_N2O = 1f P_N2O .
Based on the determined f P_N2 and f P_N2+N2O we can calculate r N2O as: where y represents the mole fractions.
Moreover, from the comparison of the a P _ N2 or a P _ N2O with a NO3-values obtained from NO 3 analysis of soil extracts, the contribution of hybrid N 2 (f H _ N2 ) and N 2 O (f H_N2O ) can be estimated.If a P < a NO3-this can be due to the combination of two N sources, labelled and non-labelled, to form N 2 O or N 2 (Spott and Stange, 2011).Hence, the fractions of three pools: non-labelled (N), labelled non-hybrid (L) and labelled hybrid (H) contributing to N 2 or N 2 O formation were determined according to Spott and Stange (2011): and the hybrid fraction, for either N 2 O or N 2 , is calculated as:

Co-existence of other N-transformation processes
The mineral N concentrations and 15 N abundances allow for a quantification of: (i) formation of natural abundance NO 3 -via gross nitrification (n) based on the dilution of the 15 Nlabelled NO 3 -pool, which is obtained from the initial (subscript 0) and final (subscript t) concentration (c) and 15 N abundance (a) in soil nitrate (Davidson et al., 1991): nitrate immobilisation -magnitude of N sink not explained by other processes, including final and initial nitrate concentration (c NO3_t , c NO3_0 ), nitrification (n), total N-gas flux [N 2 O+N 2 flux] and

N 2 O isotopic fractionation to quantify N 2 O reduction
The N 2 O fractionation approach is based on the changes in N 2 O isotopic signatures due to partial N 2 O reduction to N 2 , which alters the δ 18 O, δ 15 N bulk and δ 15 N sp of the residual unreduced N 2 O (δ r ).All these isotopic signatures depend on the N 2 O residual fraction (r N2O ) according to the following isotopic fractionation equations applying closed system Rayleigh model (Mariotti et al., 1981): or in simplified, approximated form (applied only for graphical interpretations in Sect.3.4.1): To be able to determine r N2O from N 2 O isotopic values of individual samples according to Eq. ( 17 δ 0 values to check which value yields best fit between calculated and measured N 2 O reduction and thus to identify, which of the methods to determine η red and δ 0 is the most suitable one.

Estimating η red and δ 0 values
Mean η red and δ 0 values for the entire experiment From the statistically significant logarithmic fits between r N2O and measured δ r values we can estimate the isotopic fractionation by N 2 O production (δ 0 ) and N 2 O reduction (η red ) according to Eq. ( 18), where the slope represents the η red , the isotope effect associated with N 2 O reduction, and the intercept gives δ 0 , the initial isotopic signature for the produced N 2 O unaffected by its reduction (Fig. 4)

Temporarily changing η red and δ 0 values
The interpretations and calculations based on δ values are difficult when we deal with the simultaneous variations in r N2O and δ 0 values.Usually, to calculate r N2O a stable δ 0 is assumed (Lewicka-Szczebak et al., 2015) and to precisely determine temporal changes in δ 0 , we need independent data on r N2O (Köster et al., 2015).In field studies both r N2O and δ 0 cannot be determined precisely, but rather the possible ranges for each parameter can be given (Zou et al., 2014) values, δ 0 was assumed constant and the respective value derived from the correlation between ln(r N2O ) and δ r (Mariotti et al., 1981) were used.

Fungal fraction estimated from δ 0 values
From the calculated δ 0 15 N sp values, the fraction of N 2 O originating from fungal denitrification (f F ) can be estimated using the isotopic mass balance.Isotopic endmembers for δ 15 N sp values were assumed to be 35 ‰ for fungal denitrification (Rohe et al., 2014) and -5 ‰ for heterotrophic bacterial denitrification (Sutka et al., 2006;Toyoda et al., 2005).The mixing endmember characterized by higher δ 15 N sp values can theoretically also originate from nitrification (hydroxylamine oxidation pathway), but only in the oxic treatments.However, in our experimental set-up, due to high nitrate amendment, no ammonia amendment and high soil moisture, N 2 O flux from nitrification should be much lower than from denitrification (Zhu et al., 2013).Therefore, the significant shifts in δ 0 15 N sp values observed here are rather discussed as a result of fungal denitrification admixture.

Calibration and validation
The precision of the quantification of the N 2 O reduction based on the N 2 O isotopic fractionation approach was checked by comparison of the calculated values and the values measured by the reference methods.The δ 0 and η red values needed to determine r N2O with Eq. ( 18) were found from the ln fit between the isotopic signature of residual unreduced N 2 O and r N2O determined by the independent method, as shown in the previous section 2.7.1.
The calibration of the isotopic fractionation approach was performed by applying δ 0 15 N sp and η red 15 N sp values obtained in the particular experiment to calculate r N2O from the same experiment.The precision of this approach was evaluated by comparing measured and calculated r N2O and determining the standard error of calculated r N2O .
The validation of the isotopic fractionation approach was performed by applying

Mapping approach to distinguish mixing and fractionation processes
Until now, isotopomer "maps", i.e. plots of SP vs δ 15 N bulk or SP vs δ 18 O, have been use to differentiate between processes (Koba et al. (2009), Zou et al. (2014)) or to identify N 2 O reduction to N 2 (Well et al., 2012).Here we present a very first attempt of simultaneous quantification of fractionation and mixing processes based on the relation between δ 15 N sp and δ 18 O values, which we call 'mapping approach'.The graphical illustration of the δ 15 N sp /δ 18 O "maps" is presented in Fig. 1.The approach is based on the different slopes of the mixing line between bacterial denitrification and fungal denitrification or nitrification and the reduction line reflecting isotopic enrichment of residual N 2 O due to its partial reduction.Both lines are defined from the known most relevant literature data on the respective δ 0 and η red values: δ 0 15 N sp from pure culture studies for bacterial denitrification: for heterotrophic bacterial denitrification from -7.5 to +3.7 ‰ (Sutka et al., 2006;Toyoda et al., 2005) and for nitrifier denitrification from -13.6 to +1.9 ‰ (Frame and Casciotti, 2010;Sutka et al., 2006).As both processes overlap, a common mean endmember value for N 2 O production by bacterial denitrification of -3.9 ‰ is used.
-  ambient water depending on the bacterial strain, whereas soil incubations indicated that this exchange is high (Kool et al., 2007;Snider et al., 2013) and the isotope effect between water and formed N 2 O quite stable (Lewicka-Szczebak et al., 2016).
δ 0 15 N sp for fungal denitrification and nitrification based on pure culture studies: for fungal denitrification from 30.2 to 39.3 ‰ (Maeda et al., 2015;Rohe et al., 2014;Sutka et al., 2008) and for nitrification from 32.0 to 38.7 ‰ (Frame and Casciotti, 2010;Heil et al., 2014;Sutka et al., 2006).As both processes overlap, a common endmember value for N 2 O production by fungal denitrification of 34.8 ‰ is used.A recent study indicated also a lower δ 0 15 N sp value for one individual fungal species, which was disregarded here due to its very low N 2 O production: C. funicola showed δ 0 15 N sp of 21.9 ‰ but less than 100 times lower N 2 O production with nitrite compared to other species, and no N 2 O production with nitrate (Rohe et al., 2014).Similarly, from the study of Maeda et al. (2015) we accepted only the values of strains with higher N 2 O production (> 10mg N 2 O-N/g biomass).
δ 0 18 O(N 2 O/H 2 O) for fungal denitrification and nitrification based on pure culture studies: for fungal denitrification from 40.6 to 51.9 ‰ (Maeda et al., 2015;Rohe et al., 2014;Sutka et al., 2008) and for nitrification from 35.6 to 55.2 ‰ (Frame and Casciotti, 2010;Heil et al., 2014;Sutka et al., 2006).As both processes overlap, a common endmember value for N 2 O production by fungal denitrification of 43.6 ‰ is used.The relevant values for fungal denitrification are selected after the same criteria as above for δ 0 15 N sp .
- While both scenario yield identical results for the admixture of N 2 O from fungal denitrification / nitrification, the resulting reduction shift, and hence the calculated r N2O value, is smaller when using scenario 2. time, from 1 down to 0.25 for 80 % WFPS and down to 0.63 for 70 % WFPS.This is associated with a simultaneous increase in δ values, from 21.6 to 59.1 ‰ for δ 18 O, from -52.9 to -29.9 ‰ for δ 15 N bulk , and from 0.3 to 19.6 ‰ for δ 15 N sp .For the Org soil 80 % WFPS treatment, the initial increase in r N2O , from 0.08 to 0.49 during the oxic phase, is followed by a slight drop (from 0.60 to 0.39) during the anoxic phase.δ values did not show a clear trend over time and ranged from 11.2 to 41.9 ‰ for δ 18 O, from -46.4 to -17.4 ‰ for δ 15 N bulk and from -1.9 to 17.5 ‰ for δ 15 N sp .In the 70 % WFPS treatment, the gas fluxes were below detection limit during the oxic phase.

3
δ 18 O(H 2 O) of soil water ranged from -6.5 to -5.1 ‰ for Org and Min soil, respectively.

15 N treatment, Exp2
N 2 O and N 2 fluxes and 15 N enrichment of N pools The detailed results of the experiment presented as time series are shown in the supplement Fig. S3.
From 15 N treatments we can conclude that only for anoxic Org soil treatment very consistent 15 N atom fractions in all gaseous fractions were obtained (a M_N2O , a P_N2O , a P_N2 ), from 42 to 46 at%, which are in close agreement with soil nitrate (a NO3 =43 at%) (Fig. S3.1(b)).For anoxic Min soil treatment, a P_N2 and a P_N2O (from 49 to 51 at%), also correspond to a NO3 (51 at%), but the 15 N atom fraction of emitted N 2 O (a M_N2O ) is significantly lower, decreasing from 49 to 24 at% with incubation time (Fig. S3.1(a)).In oxic conditions we deal with even lower 15 N atom fractions in total N 2 O, for Min soil a M_N2O from 4 to 32 at% (Fig. S3.2(a)) and for Org soil a M_N2O from 11 to 37 at% (Fig. S3.2(b)).Moreover, for oxic treatments also lower values of a P_N2 can be observed, down to 28 at% for Min soil and 34 at% for Org soil.For mineral N we observed almost no change in the extracted nitrate under anoxic conditions, with maximal change in a NO3 of 0.3 at%, and slight decrease under oxic conditions of 1.5 at% for Min and 3.2 at% for Org soil.The non-labelled ammonium pool stays mostly unchanged under oxic treatments, but significant 15 N enrichment is observed under anoxic conditions, where a NH4 reaches 8.7 at% for Min and 3.5 at% for Org soil by the end of the incubation (Fig. S3.1(a), S3.1(b)).

N transformations
In Table 1, calculated rates of N transformations are shown.Initial and final concentrations for nitrate and ammonium were measured, total gaseous N-loss ([N 2 +N 2 O] flux) is calculated (Eq.( 7)), the rates of nitrification (n), DNRA, mineralisation (m), immobilisation (i) were estimated according to Eqs.
(13) -( 16).The flux of N 2 O from non-labelled soil N pools was calculated as f N_N2O × [N 2 O] flux.The nitrification rate (n) was highest for the Org soil in oxic conditions (1.93 mg N per kg soil and 24 h).
But even in anoxic treatments, a low n rate was detected (up to 0.06 mg N).In the anoxic treatments DNRA was also active, which resulted in formation of 15 N labelled NH 4 + (from 0.02 to 0.10 mg N, for Min and Org soil, respectively).Mineralisation (m) appears to be very high for Org soil, both in oxic (1.99 mg N) and anoxic (1.25 mg N) conditions, and lower for Min soil (0.31 and 0.15 mg N, respectively).Interestingly, in each treatment a quite pronounced additional nitrate sink, most probably due to N immobilisation (i), was found, mostly much larger than the total gaseous loss ([N 2 +N 2 O] flux) (Table 1).

N 2 O and N 2 source processes
Based on the non-random distribution of N 2 O isotopologues obtained in 15 N treatments, we can differentiate between the 15 N-pool derived N 2 O (f P_N2O ) and non-labelled N 2 O fraction (f N_N2O ) (Fig. 2).
f P_N2O decreases with lowering of total N 2 O fluxes and is higher for anoxic treatments (above 0.42 for Min and above 0.91 for Org soil) when compared to oxic treatments (from 0.03 to 0.67 and from 0.14 to 0.98, respectively).A significant contribution of non-labelled N 2 O (f P_N2O < 1) in the anoxic Min soil treatment was thus evident (Fig. 2 The contribution of the cumulative non-labelled N 2 O flux to the total denitrification flux [N 2 O+N 2 ] is quite significant for oxic treatments, with a mean value of 0.18 and 0.29 for Org and Min soil, respectively.Within the 15 N-pool derived N 2 O, the hybrid sub-fraction can be determined (f H_N2O ).
Hybrid N 2 O was found only in oxic treatments (Fig. 2).For Min soil, f H_N2O was detected in all measured N 2 O samples and varied between 0.05 and 0.19.For Org soil, no f H_N2O was found during the first two or three days of incubation when the N 2 O concentration was highest, and afterwards its contribution gradually increased with decreasing N 2 O concentration, reaching up to 0.25 of the 15 N-pool derived N 2 O. Similarly, f H_N2 was determined.Very small f H_N2 was detected in anoxic treatments, up to 0.09 for Min soil and up to 0.18 for Org soil, where only five samples from two vessels indicated possible presence of hybrid N 2 (Fig. 3).Significantly higher f H_N2 were observed for oxic conditions, up to 0.90 for Min soil and up to 0.68 for Org soil.For Org soil, there is significant negative correlation between f H and N gas flux, both for N 2 O (Fig. 1) and for N 2 (Fig. 2), whereas no such relation exists for Min soil.Much wider ranges of η red values were found for η red 18 O (from -22.7 to -9.9 ‰) and η red N bulk (from -6.6 to -2.0 ‰).In contrast to quite variable η red values, the determined δ 0 values are very robust, with δ 0 18 O about +36 and δ 0 15 N bulk about -45 ‰.
These relations look very different for Org soil.Firstly, there is no significant correlation between δ r and r N2O for Exp1, whereas all correlations are significant for Exp2 (Fig. 4(b), Table 2).The η red values determined for Exp2 for Org soil (Table 2) are much more negative than for Min soil and also compared to the known literature range of fractionation factors (Jinuntuya-Nortman et al., 2008;Lewicka-Szczebak et al., 2015;Well and Flessa, 2009a).

Temporarily changing η red and δ 0 values
Theoretical δ 0 15 N sp values were calculated for individual samples assuming stable η red values (as described in Sect.2.7.1) and the variations of calculated δ 0 15 N sp with incubation time for both soils are presented in Fig. 5.An increase in δ 0 15 N sp value with time is observed for both soils, but is much larger and clearly unidirectional for Org soil.Since r N2O simultaneously decreases during the incubation, the δ 0 15 N sp value obtained from the correlation between δ 15 N sp and r N2O (Table 2,  Fungal fraction estimated from δ 0 values For Org soil, the time course of δ 0 15 N sp values (Fig. 5) indicated a very pronounced increase in the fraction of N 2 O originating from fungal denitrification (f F ) during the incubation time of Exp2 ( 9days), giving f F values from 10 % at the beginning up to 75 % at the end.For Min soil in Exp2, f F was smaller and varied from 7 to 49 %.

Calibration and validation
From the correlation tested above (Table 2) we found that only for Min soil δ 0 and η red values can be robustly determined from δ 15 N sp values.Hence, we show here the calibration and validation based on these values only.The calibration shows a quite good agreement between the measured and the calculated r N2O with a significant fit to the 1:1 line (Fig. 6).The mean absolute difference between measured and calculated r N2O was 0.08 for Exp1 and 0.04 for Exp2.The mean relative error in the determination of the reduced N 2 O fraction (1-r N2O ) representing the N 2 flux was 36 % for Exp1 and 8 % for Exp2.For Exp1 we have tested if a better fit could be obtained when fractionation factors for oxic and anoxic treatment are determined and applied separately.In Fig. 6, points calculated with mean values for oxic and anoxic treatment (Exp1 mean) as well as calculations for either oxic or anoxic treatments are shown.The fit to a 1:1 line is similar for the calculation using the mean values (Exp1 mean: R 2 =0.83) and the respective oxic and anoxic treatments considered individually (Exp1 oxic: R 2 =0.86 and Exp1 anoxic: R 2 =0.79).In our Min soil, η red values were thus not affected by incubation conditions.
For Val1, i.e. using the δ 0 15 N sp and η red 15 N sp values obtained from a previous static experiment performed with the same soil, no significant correlation with the 1:1 line was obtained (Fig. 7 (red triangles)).For Exp1 the mean absolute difference between the measured and the calculated r N2O reaches 0.41 and the relative error in determining N 2 flux is as high as 234 %, whereas for Exp2 these As shown in Fig. 5, at the beginning we deal with larger dominance of bacterial over fungal N 2 O, which results in lower δ 0 15 N sp than assumed in the calculations, and consequently in an overestimation of the r N2O .
For Val3, i.e. using a common value of -5 ‰ for η red 15 N sp , the fit is very similar as for Val2 (not shown).
For Exp1 the mean absolute difference between measured and calculated r N2O was 0.14 (relative error 60 %), which was slightly higher compared to the 0.10 difference (relative error 54 %) for Val2.For Exp2 this difference was only 0.05 (relative error 9%), hence even lower than 0.07 (relative error 13 %) obtained for Val2.
Summarising the results of these three validation scenarios, we can conclude that actual  0 values must apparently be known to obtain reliable estimates of r N2O , whereas it seems possible to use a general value for η red 15 N sp .

Mapping approach to distinguish mixing and fractionation processes
As a qualitative indicator of mixing and fractionation processes we analysed relations between pairs of isotopic signatures to determine the slopes for the measured δ values.The same was done for the δ 0 values calculated using the measured r N2O values (Eq.( 17)).All the calculated slopes are presented in Table 3, and graphical illustrations are shown in the supplement (Fig. S4).The δ 15 N sp /δ 18 O slopes for Org soil are generally higher (from 0.65 to 0.76) than for Min soil (from 0.30 to 0.64) (Table 3).But we can also notice that for both soils, the slopes in Exp1 are lower than in Exp2 The slopes between δ 18 O/δ 15 N bulk observed in our study range mostly from 1.94 to 3.25 (Table 3).Only for Org soil in anoxic conditions (in both Exp1 and 2) this slope is largely lower from 0.61 to 0.84.
With the mapping approach we used dual isotope values, i.e. δ 15 N sp and δ 18 O, to calculate r N2O and the fraction of N 2 O originating from fungal denitrification or nitrification (f F ) as described in Sect.
2.7.3.This was done for both soils but with Exp2 data only (Fig. 8).Both scenarios provide identical results for f F values, whereas r N2O values are always higher for Sc2 ("first reduction, then mixing") when compared to Sc1 ("first mixing, then reduction") with maximal difference up to 0.39 between them.Figure 8 shows the comparison between calculated and measured r N2O values.For most results the measured value is within the range of values obtained from both scenarios.For Org soil, Sc2 results show better agreement with the measured values, but rather the opposite is observed for the Min soil.
The oxic treatment for Min soil shows the worst agreement with the measured values, i.e., the calculated values indicate pronounced underestimation of r N2O .The calculated f F values exhibit a continuous increase with incubation time for all treatments except the oxic treatment of Min soil.

N 2 O and N 2 source processes
In this study quite a high contribution of non-labelled N 2 O was documented (Fig. 1, Fig. 3).
However, in the conditions favouring denitrification with high soil moisture (WFPS 75 %) the typical N 2 O yield from nitrification is much lower compared to the N 2 O yield from denitrification (Butterbach-Bahl et al., 2013;Well et al., 2008).Therefore, in these experimental conditions the contribution of nitrification to N 2 O fluxes should be rather negligible.Most surprising is the significant contribution of non-labelled N 2 O (f P_N2O < 1) in the anoxic Min soil treatment associated with lower N 2 O fluxes at the end of incubation (Fig. 1(a)).Moreover, for both soils in the anoxic treatment the cumulative nonlabelled N 2 O flux in mg N is higher than the initial NH 4 + pool plus the NH 4 + possibly added due to DNRA (Table S1).This indicates that oxidation of organic N must be active in these treatments.
Recently, it has been shown that this process can be even the dominant N 2 O producing pathway (Müller Biogeosciences Discuss., doi:10.5194/bg-2016-276, 2016 Manuscript under review for journal Biogeosciences Published: 30 August 2016 c Author(s) 2016.CC-BY 3.0 License.
et al., 2014); however, it is questionable if this can be active also under anoxic conditions.Nitrifier denitrification or eventually also some abiotic N 2 O production would be the most probable processes to produce non-labelled N 2 O in anoxic treatments, but since the substrate is NH 4 + , it must have been preceded by ammonification of organic N.
A higher contribution of non-labelled N 2 O was noted for anoxic treatments (Fig. 1).This flux can be well explained by nitrification, because it represents, respectively, 2 and 3 % of the nitrification rate (Table 1), which is at the upper end of the known range for the nitrification product ratio (Well et al., 2008).Nitrification was quite significant in oxic treatments and the observed increase in NO 3 exceeded largely the NH 4 + available at the beginning of the incubation (Table S1).This indicated that a pronounced amount of organic N must have been mineralised first or was partially oxidised to NO 3 through the heterotrophic nitrification pathway (Zhang et al., 2015).
To our best knowledge, this is one of the very few studies that document a significant hybrid N 2 and N 2 O production in natural soils without addition of any nucleophiles, i.e.compounds used as the second source of N in codenitrification (Laughlin and Stevens, 2002;Long et al., 2013;Selbie et al., 2015).All these previous studies identified codenitrification as the major N 2 -producing process, with contribution of hybrid N 2 in the total soil N 2 release from 0.32 to 0.95 (Laughlin and Stevens, 2002;Long et al., 2013;Selbie et al., 2015).In our study this contribution is lower, namely 0.18 and 0.05 of the cumulative soil N 2 flux, respectively for Min and Org soil.No hybrid N 2 O was found previously (Laughlin and Stevens, 2002;Selbie et al., 2015), whereas in our study a slight contribution was detected representing 0.027 and 0.009 of the cumulative N 2 O flux for Min and Org soil, respectively.
Interestingly, we observe higher f H values for oxic treatments.This may indicate the fungal origin for hybrid N 2 and N 2 O, since it has been shown that fungal denitrification may be activated in presence of oxygen (Spott et al., 2011;Zhou et al., 2001).Similarly, Long et al. (2013)  substrates may get exhausted.This reinforces the previous observations of enhanced codenitrification for higher ratio between potential nucleophiles and NO 2 -or NO and with decreasing availability of organic substrates (Spott et al., 2011).
A precondition for the proper quantification of various process rates based on the 15 N tracing technique is the homogeneity of 15 N tracer in soil.Recently, a formation of two independent NO 3 -pools in the soil was described for an experimental study (Deppe et al., submitted), where one pool containing the undiluted 15 N tracer solution and thus high 15 N enrichment was mostly the source for N 2 O, whereas the rest of soil NO 3 -representing the other pool was largely diluted by nitrification input and, therefore, the total soil NO 3 -(a NO3 ) showed lower 15 N enrichment than the 15 N-pool derived N 2 O (a P_N2O ).This strong discrepancy between pool enrichments could be explained by the large amount of ammonia applied in that experiment and subsequent fast nitrification in aerobic domains of the soil matrix.For our data, a P values are never significantly higher than a NO3 , and for anoxic treatments agree perfectly (Fig. S3.1(a), S3.1(b)), which indicates that the non-homogeneity problem does not apply here.The reason for better homogeneity achieved in our experiments is probably the much higher soil moisture applied, resulting in more anoxic conditions inhibiting nitrification, and absence of ammonia amendment.Hence, as we can assume homogenous 15 N distribution, our results on f P and f H should be adequate.strong disagreement with previous knowledge on possible η values (Jinuntuya-Nortman et al., 2008;Lewicka-Szczebak et al., 2014;Ostrom et al., 2007;Well and Flessa, 2009a).In the further interpretation of data we therefore suppose that δ 0 values were variable and η values constant.While we cannot rule out that η values varied to some extent, it is not possible to verify that using the current data set.For Org soil, much higher absolute values of η red were found (Table 2) being in contrast to all previous studies (Jinuntuya-Nortman et al., 2008;Lewicka-Szczebak et al., 2015;Well and Flessa, 2009a).Hence, it has to be questioned if this observation is not an experimental artefact.Actually, the Org soil anoxic treatment was the only case where 15 N-pool derived N 2 O was dominant (Fig. S3.1(b)), hence the isotopic signatures should not be altered due to different N 2 O producing pathways but mostly governed by the r N2O .But for Org soil we observe a constant and very significant increase in the contribution of N 2 O from fungal denitrification during the incubation (Fig. 5).

Calibration and validation
The successful calibration shows that δ 0 15 N sp and η red values were stable enough within incubation experiments for calculating r N2O using the isotope fractionation approach.
The results of the calibration were very similar if we treated the oxic and anoxic conditions separately and if we used a mean η red and δ 0 15 N sp value of the oxic and anoxic phase of Exp.1 to all the results (Fig.

Mapping approach to distinguish mixing and fractionation processes
The emitted N 2 O is analysed for three isotopocule signatures and the relations between them (δ 15 N sp /δ 18 O, δ 15 N sp /δ 15 N bulk , δ 18 O/δ 15 N bulk ) can be informative.Namely, the observed correlation may result from the mixing of two different sources or from characteristic fractionation during N 2 O reduction, or from the combination of both processes.If the slopes of the regression lines for these both cases were different, mixing and fractionation processes could be distinguished.Such slopes were often used for interpretations of field data (Opdyke et al., 2009;Ostrom et al., 2010;Park et al., 2011;Toyoda et al., 2011;Wolf et al., 2015) but recently this approach was questioned because of very variable isotopic fractionation noted during reduction for O and N isotopes (Lewicka-Szczebak et al., 2014;Wolf et al., 2015).A recent study showed, that for moderate r N2O (>0.1) the δ 15 N sp /δ 18 O slopes characteristic for N 2 O reduction are quite consistent with previous findings (Lewicka-Szczebak et al., 2015), i.e., vary from ca. 0.2 to ca. 0.4 (Jinuntuya-Nortman et al., 2008;Well and Flessa, 2009a).Hence, in such cases, the reduction slopes may significantly differ from the slopes resulting from mixing of bacterial and fungal denitrification, characterised by higher values of about 0.63 and up to 0.85 (Lewicka-Szczebak et al., 2016).
In theory, the slopes for calculated δ 0 values are not influenced by N 2 O reduction and hence should be mostly be caused by the variability of mixing processes, whereas the slopes of the measured δ values reflect both mixing and fractionation due to N 2 O reduction.For Min soil, there is no correlation between calculated values of δ 0 15 N sp and δ 0 18 O (Table 3), which indicates that the correlation observed for measured δ values was a result of fractionation processes during N 2 O reduction.In contrast, for Org soil all the correlations for calculated δ 0 values are still very strong and show similar slope as the correlations for measured δ values (Table 3).This indicates a very significant impact of the mixing of various N 2 O producing pathways.
The δ 15 N sp /δ 18 O slopes for Org soil are generally higher (from 0.65 to 0.76) than for Min soil (from 0.30 to 0.64) (Table 3).This supports the hypothesis from the previous Sect.Interestingly, there is no correlation between isotopic values in oxic Exp2 for Min soil.A process or the combination of several that cause large variations in δ 15 N sp but not parallel in δ 18 O seems to be present there.This might be due to admixture of different microbial pathways and maybe to some extent also due to O-exchange with water.In this treatment we observe the lowest N 2 O fluxes and also the lowest f P_N2O values, which suggests the largest input from nitrification.The δ 15 N sp values for hydroxylamine oxidation during nitrification are much larger (ca.33 ‰) than for bacterial denitrification or nitrifier denitrification (ca.-5 ‰) (Sutka et al., 2006), whereas δ 18 O may be in the same range for both processes (Snider et al., 2013;Snider et al., 2011).This could be an explanation for the missing correlation between δ 15 N sp and δ 18 O (Table 3).
The graphical interpretations including δ 15 N bulk values are more difficult since the isotopic signature of the N precursor must be known, but can be also informative and were often used (Kato et al., 2013;Snider et al., 2015;Toyoda et al., 2011;Toyoda et al., 2015;Wolf et al., 2015;Zou et al., 2014).The slopes between δ 18 O and δ 15 N bulk observed in our study range mostly from 1.94 to 3.25 (Table 3), which corresponds quite well to the previously reported results from N 2 O reduction experiments with the range from 1.9 to 2.6 (Jinuntuya-Nortman et al., 2008;Well and Flessa, 2009a)).
Only for Org soil in anoxic conditions (in both Exp1 and 2) this slope is largely lower from 0.61 to 0.84.
These values are more similar to δ 18 O/δ 15 N bulk slopes for the calculated δ 0 values, 0.56 for Min soil and 1.04 for Org soil (Table 3), which is significantly lower than typical reduction slopes, thus most probably be rather due to mixing of various N 2 O sources.However, the calculated δ 0 values cannot be explained with mixing of bacterial and fungal denitrification only (Fig. S4.3(b)).For the relation of δ 15 N sp /δ 15 N bulk (Fig. S4.2) the reduction and mixing slopes cannot be separated so clearly but, similarly as for δ 18 O/δ 15 N bulk , the calculated δ 0 values are not all situated between the mixing endmember of bacterial and fungal denitrification.This is due to some data points showing very low δ 0 15 N bulk The attainable precision of the method, determined as mean absolute difference between the measured and the calculated N 2 O residual fraction (r N2O ), is about ±0.10, but for individual measurements this absolute difference varied widely from 0.00 up to 0.39.The precision in N 2 flux quantification depends strongly on the r N2O of a particular sample and varied in a very wide range from 0.01 up to 2.41 for Exp1 and 0.00 up to 0.93 for Exp2, with a mean relative difference between measured and calculated N 2 flux of 0.46 and 0.13, respectively.The highest relative errors in the calculated N 2 flux (>1) occur for the very low fluxes only (r N2O > 0.9).
However, for soils of more complex N dynamics, as shown for the Org soil in this study, the determination of N 2 O reduction is more uncertain.The method successfully used for Min soil was not applicable due to failed determination of proper δ 0 15 N sp values, which were significantly changing with incubation progress.Here we suggest an alternative method based on the relation between δ 15 N sp and δ 18 O values ('mapping approach').This allows for the estimation of both the fraction of fungal N 2 O and the plausible range of residual N 2 O.

Tables:
Table 1: Rates of nitrification, mineralisation and DNRA as calculated from 15 N-pool dilution for Exp2 15 N treatment.Source measured data used for the calculation are provided in the supplement (Table S1). N Biogeosciences Discuss., doi:10.5194/bg-2016-276,2016 Manuscript under review for journal Biogeosciences Published: 30 August 2016 c Author(s) 2016.CC-BY 3.0 License.due to denitrification.In this study we present a validation of the calculations based on the N 2 O isotopic fractionation performed in laboratory experiments applying two different reference methods for quantification of N 2 O reduction: incubation in N 2 -free Helium atmosphere and the 15 N gas flux method.Helium incubations allow for simultaneous determination of the N 2 O isotopic signature and the N 2 O residual fraction from the same incubation vessel (Lewicka-Szczebak et al., 2015), whereas in 15 N gas flux experiments, parallel incubations of 15 N-labelled and natural abundance treatments are necessary.
047 bar), flushed with He to reduce the N 2 background and afterwards flushed with a continuous stream of He+O 2 for at least 60 hours.When a stable and low N 2 background (below 10ppm) was reached, temperature was increased to 22 ºC.The incubation lasted 5 days, while the headspace was constantly flushed with a continuous flow of 20 % O 2 in Helium (He/O 2 ) mixture for the first 3 days and then with pure He for the following 2 days, at a flow rate of ca. 15 cm 3 min -1 .The fluxes of N 2 O and N 2 were directly analyzed and the samples for N 2 O isotopocule analyses were collected at least twice a day.The N 2 O residual fraction was determined based on the direct measurement of N 2 O and N 2 fluxes.
obtain a WFPS of 70 % and fertilised with 80 mg N (added as NO 3 -) per kg soil.Half of each soil was fertilized with Chile saltpeter (NaNO 3 , Chili Borium Plus, Prills-Natural origin, supplied by Yara, Dülmen, Germany), i.e., nitrate fertilizer from atmospheric deposition ore with δ 15 N at natural abundance level (NA treatment).This fertilizer was used to enable determining O exchange between denitrification intermediates and with water based on the 17 O anomaly of Chile saltpeter (Lewicka-Szczebak et al., 2016).The other half of the soil was fertilized with 15 N-labelled NaNO 3 (98 at% 15 N) ( 15 N treatment).Then soils were thoroughly mixed to obtain a homogenous distribution of water and Biogeosciences Discuss., doi:10.5194/bg-2016-276,2016 Manuscript under review for journal Biogeosciences Published: 30 August 2016 c Author(s) 2016.CC-BY 3.0 License.
Knowing r N2O we can estimate the total denitrification [N 2 +N 2 O] flux using the measured [N 2 O] flux and the determined r N2O as: Biogeosciences Discuss., doi:10.5194/bg-2016-276,2016 Manuscript under review for journal Biogeosciences Published: 30 August 2016 c Author(s) 2016.CC-BY 3.0 License.
13) Biogeosciences Discuss., doi:10.5194/bg-2016-276,2016 Manuscript under review for journal Biogeosciences Published: 30 August 2016 c Author(s) 2016.CC-BY 3.0 License.(ii) formation of 15 N-labelled NH 4 + , most probably due to DNRA (dissimilatory nitrate reduction to ammonium) or due to coupled immobilisation-mineralisation (Rutting et al., 2011), based on 15 N mass balance of final (subscript t) and initial (subscript 0) ammonium concentration (c) and 15 N abundance (a) in final and initial ammonium and average (subscript av) 15 N abundance in nitrate : of natural abundance N which was added to the system, based on N balance, including final and initial ammonium concentration (c NH4_t , c NH4_0 ), nitrification (n), nonlabelled N 2 O flux (f N_N2O *[N 2 O flux]) and DNRA: ), isotopic fractionation factors associated with N 2 O reduction (η red ) and initial N 2 O isotopic signature before reduction (δ 0 ) must be known.We tested various experimental approaches to determine η red and Biogeosciences Discuss., doi:10.5194/bg-2016-276,2016 Manuscript under review for journal Biogeosciences Published: 30 August 2016 c Author(s) 2016.CC-BY 3.0 License.
δ 18 O and δ 15 N bulk , δ 0 values are expressed as relative values in relation to the source, i.e., soil water (δ 18 O(N 2 O/H 2 O)) and soil nitrate (δ 15 N bulk (NO 3 /N 2 O)).This allows us to reasonably compare different treatments differing in soil water isotopic signatures and properly interpret δ 15 N bulk values which are related to the isotopic signature of nitrate, getting enriched with incubation time.δ 0 15 N sp is independent of the isotopic signature of the source, hence the measured δ 15 N sp values were directly used for determination of correlations.
δ 0 15 N sp and η red 15 N sp values determined in a parallel experiment to calculate r N2O of the validation experiment with the same soil.The validation was performed in three ways (Val1 -Val3): Biogeosciences Discuss., doi:10.5194/bg-2016-276,2016 Manuscript under review for journal Biogeosciences Published: 30 August 2016 c Author(s) 2016.CC-BY 3.0 License.(i) Val1 used δ 0 15 N sp and η red 15 N sp values obtained from a previous static experiment performed with the same soil (Exp 1E-F in Lewicka-Szczebak et al. (2014)) to calculate r N2O for Exp1 and 2 based on the measured δ 15 N sp values of residual unreduced N 2 O. (ii) Val2 used δ 0 15 N sp and η red 15 N sp values obtained from Exp1 to calculate r N2O for Exp2, and vice versa.(iii) Val3 used the same δ 0 15 N sp as Val2, but for η red 15 N sp the common value of -5 ‰ was applied, as recently suggested as a mean robust η red 15 N sp (Lewicka-Szczebak et al., 2014).Here we checked how our results are affected when we use for the calculations this common value instead of the η red 15 N sp value determined for the particular soil.

26. 5 ‰
(Frame and Casciotti, 2010;Sutka et al., 2006).As both processes overlap, a common endmember value for N 2 O production by bacterial denitrification of 21 ‰ is used.For heterotrophic bacterial denitrification we used values of the controlled soil incubation because pure culture studies show a large range of possible values due to various O-exchange with fractionation factors associated with N 2 O reduction: values obtained from controlled soil incubations are η red 15 N sp from -7.7 to -2.3 ‰ with a mean of -5 ‰ and of η red 18 O values from -25 to -5 ‰ with a mean of -15 ‰ (Jinuntuya-Nortmanet al., 2008;Lewicka-Szczebak et al., 2014;Menyailo and Hungate, 2006;Ostrom et al., 2007;Well and Flessa, 2009a).Although the range of possible η red variations is quite large, it has been shown recently that the mean values Biogeosciences Discuss., doi:10.5194/bg-2016-276,2016 Manuscript under review for journal Biogeosciences Published: 30 August 2016 c Author(s) 2016.CC-BY 3.0 License.and typical η red 15 N sp / η red 18 O ratios are applicable for oxic or anoxic conditions unless N 2 O reduction is almost complete, i.e. r N2O < 0.1 (Lewicka-Szczebak et al., 2015).The δ 15 N sp /δ 18 O slope of the mixing line between the endmember value for N 2 O production of fungal denitrification / nitrification and heterotrophic bacterial denitrification / nitrifier denitrification is distinct from the respective slope of the reduction line resulting from reduction isotope effects (Fig. 1: reduction line and mixing line, respectively).Isotopic values of the samples analyzed are typically located between these two, reduction and mixing, lines.From their location we can estimate the impact of fractionation associated with N 2 O reduction and admixture of N 2 O originating from fungal denitrification / nitrification.If we assume bacterial denitrification as the first source of N 2 O, then we can deal with two scenarios: (i) Scenario 1 (Sc1): the N 2 O emitted due to bacterial denitrification is first reduced (point move along reduction line up to the intercept with red_mix line) and then mixed with the second endmember (point move along red_mix line to the measured sample point) (ii) Scenario 2 (Sc2): the N 2 O from two endmembers is first mixed (point move along mixing line up to the intercept with mix_red line) and only afterwards the mixed N 2 O is reduced (point move along mix_red line to the measured sample point).
The detailed results of the experiment presented as time series are shown in the supplement Fig.S1.In general, the switch from oxic to anoxic conditions resulted in an increase of gaseous N-losses.For both treatments of the Min soil (70 and 80 % WFPS), we observed a gradual decrease in r N2O with incubation Biogeosciences Discuss., doi:10.5194/bg-2016-276,2016 Manuscript under review for journal Biogeosciences Published: 30 August 2016 c Author(s) 2016.CC-BY 3.0 License.
and isotopocules of N 2 O of the natural abundance (NA) treatment, Exp2The detailed results of the experiment presented as time series are shown in the supplement Fig.S2.For the anoxic treatments we observe a gradual decrease in N 2 O flux and an increase in N 2 flux with incubation progress.Consequently, r N2O is decreasing, from 0.58 to 0.02 for Min soil (Fig.S2.1(a)) and from 0.71 to 0.30 for Org soil (Fig.S2.1(b)).This decrease in r N2O is clearly associated with N 2 O enrichment in heavy isotopes.For Min soil, δ 18 O increases from 27.3 to 71.2 ‰, δ 15 N bulk from -45.6 to -28.2 ‰, and δ 15 N sp from 5.5 to 34.6 ‰ and for Org soil δ 18 O increases from 18.4 to 52.6 ‰, δ 15 N bulk from -46.2 to +7.5 ‰, and δ 15 N sp from 4.3 to 31.4 ‰.Under oxic conditions, we observe much higher standard deviations for both N 2 O flux and N 2 O isotopic signatures.For Min soil no clear trend over time can be described: the N 2 O flux is decreasing but rises again at the end of the incubation and r N2O reaches a minimum of 0.08 on the 6 th incubation day, and otherwise varies between 0.23 and 0.63.Similarly, δ values first increase and then decrease again varying between 32.8 and 63.4 ‰ for δ 18 O, between -43.2 and -3.0 ‰ for δ 15 N bulk and between 3.1 and 16.8 ‰ for δ 15 N sp (Fig.S2.2(a)).For Org soil, r N2O decreases from 0.72 on the 2 nd incubation day to 0.28 on the 5 th incubation day and stays stable afterwards.Similarly, δ values increase until the 5 th day, from 17.5 to 46.6 ‰ for δ 18 O and from -48.4 to -38.1 ‰ for δ 15 N bulk , and then vary around 46 and -39 ‰, respectively.δ 15 N sp values keep increasing through the entire incubation period from 1.7 to 23.6 ‰ (Fig. S2.2(b)).Biogeosciences Discuss., doi:10.5194/bg-2016-276,2016 Manuscript under review for journal Biogeosciences Published: 30 August 2016 c Author(s) 2016.CC-BY 3.0 License.δ 18 O(H 2 O) of soil water ranged from -8.5 to -6.1 ‰ for Org and Min soil, respectively.
(a), but the lower f P_N2O values are associated with lower N 2 O fluxes at the end of incubation, and the cumulative flux of non-labelled N 2 O is only approx.0.02 of the total denitrification flux [N 2 O+N 2 ].This is slightly higher than for Org soil anoxic treatment, where the cumulative flux of non-labelled N 2 O reaches only ca.0.01 of the total denitrification flux [N 2 O+N 2 ].

For
Min soil we obtained very consistent correlations for all treatments except the oxic Exp2.The N 2 O fluxes for oxic conditions showed large variations within the repetitions and between the treatments (compare Fig.S2.2(a) and S3.2(a)) which indicates that NA and 15 N treatment are not directly comparable.Therefore, the results of the oxic incubation (blue diamonds, Fig.4(a)) show no correlation between δ 15 N sp and r N2O .The other three fits indicate an absolutely consistent value for δ 0 15 N sp from 4.0 to 4.5 ‰ and also a quite consistent value for η red 15 N sp from -8.6 to -6.7 ‰ (Fig.4(a)).
Fig. 4(b)) is much below the actual one (Fig.5(b)).For Min soil this increasing trend is not so large and constant, and hence the correlation between δ 15 N sp and r N2O (Table2,Fig.4(a)) provides the δ 0 15 N sp value which represents the mean of actual variations quite well (Fig. 5(a)).Biogeosciences Discuss., doi:10.5194/bg-2016-276,2016 Manuscript under review for journal Biogeosciences Published: 30 August 2016 c Author(s) 2016.CC-BY 3.0 License.It could also be assumed that δ 0 values are constant during the experiment and the variable η values can be calculated.Under this assumption the η values through both soils and experiments are extremely variable for η 15 N bulk from -59 to +30 ‰, for η 15 N sp from -24 to +15 ‰, and for η 18 O from -143 to +48 ‰.
Biogeosciences Discuss., doi:10.5194/bg-2016-276,2016 Manuscript under review for journal Biogeosciences Published: 30 August 2016 c Author(s) 2016.CC-BY 3.0 License.values are much lower with 0.09 and 16 %, respectively.Significantly lower errors determined for Exp2 are due to many data points of extremely low r N2O values.For Val2, i.e. when δ 0 15 N sp and η red 15 N sp values obtained from Exp1 were used, the fit to the 1:1 line is definitely much better than for Val1, which is shown by the significant correlation between measured and calculated r N2O (Fig. 7 (black triangles)).The absolute mean difference between the measured and the calculated r N2O was 0.10 and 0.07 for Exp1 and 2, and the relative error in determining the N 2 flux reached 54 % and 13 %, respectively.Nevertheless, for Exp2 the maximal difference of 0.40 is very high.The four samples showing the highest deviation are the very first samples of the incubation, which most probably show slightly different microbial activity compared to the further part of the incubation.
identified fungal codenitrification as the major N 2 -producing process.In our study, higher f H values were generally observed for lower N 2 and N 2 O fluxes (especially for Org soil, Fig. 1(b), 2(b)).But we cannot exclude the possibility that hybrid N 2 also originated from other processes, i.e. abiotic codenitrification or annamox (Spott et al., 2011).Most probably, towards the end of the incubation, when N 2 and N 2 O fluxes decrease, also the concentration of intermediate products NO 2 -and NO decrease and the organic Biogeosciences Discuss., doi:10.5194/bg-2016-276,2016 Manuscript under review for journal Biogeosciences Published: 30 August 2016 c Author(s) 2016.CC-BY 3.0 License.
With respect to robust estimation of N 2 O reduction, a first question arises, to which extent δ 0 values and η values were variable or constant during incubations.When assuming constant values of δ 0 values during the experiment, calculated η values were highly variable.The large ranges obtained are clearly in BiogeosciencesDiscuss., doi:10.5194/bg-2016Discuss., doi:10.5194/bg--276, 2016     Manuscript under review for journal Biogeosciences Published: 30 August 2016 c Author(s) 2016.CC-BY 3.0 License.
4.2.1 about a higher contribution of fungal N 2 O in Org soil.But we can also notice that the slopes in Exp1 are lower than in Exp2.Most probably less stable microbial activity is present under the longer incubation in Exp2 (9 days) compared to short phases analysed in Exp1 (3 days).As observed from the calculated δ 0 values Biogeosciences Discuss., doi:10.5194/bg-2016-276,2016 Manuscript under review for journal Biogeosciences Published: 30 August 2016 c Author(s) 2016.CC-BY 3.0 License.(Fig. 5) the estimated contribution of fungal N 2 O most probably increases with incubation time.Hence, the higher slopes for Exp2 probably result from the admixture of fungal denitrification and the lower slopes for Exp1 represent more the typical bacterial reduction slopes.The δ 15 N sp /δ 18 O slopes may thus be helpful in indicating the admixture of various N 2 O sources.

(
N2O/NO3-) down to ca. -70 ‰.This exceeds the known range of the 15 N fractionation range due to denitrification, Biogeosciences Discuss., doi:10.5194/bg-2016-276,2016 Manuscript under review for journal Biogeosciences Published: 30 August 2016 c Author(s) 2016.CC-BY 3.0 License.reference methods, but not the acetylene inhibition method, since it most probably affects the microbial community, which results in biased δ 0 15 N sp values.Anoxic incubations may be applied and the determined δ 0 15 N sp values are representative for N 2 O originating from denitrification, also for oxic conditions, which means, also in field studies.

Table 2 :Figure 1 :Figure 2 :
Figure 1: Scheme of the mapping approach to simultaneously estimate the magnitude of N 2 O reduction and the admixture of fungal denitrification (or nitrification).

Figure 6 :
Figure 6: Calibration of the N 2 O isotopic fractionation approach.r N2O calculated based on Eq. (13) and measured with independent methods are compared.For Exp1 the values calculated based separately either on an oxic (blue triangles) or an anoxic treatment (filled black triangles) 5

Figure 7 :Figure 8 :
Figure 7: Validation of the N 2 O isotopic fractionation approach.r N2O calculated based on Eq. (13) and measured with independent methods are compared.For Exp1 (triangles) and Exp2 (diamonds) the values calculated based on previous static experiment (Val1 -red points) and on

2.4 Isotopic analyses in NA treatments 2.4.1 Isotopic signatures of N 2 O
. In our experiments we have measured r N2O with independent methods, hence we can assess the δ 0 changes with time, under the assumption that η red is stable, or conversely, assess changes in η red assuming stable Szczebak et al., 2014).Hence, a fixed η red 15 N sp value of -5 ‰ was used to calculate a δ 0 15 N sp value for each sample and thus to estimate its change with time.To calculate the possible temporal change in η red Biogeosciences Discuss., doi:10.5194/bg-2016-276,2016 Manuscript under review for journal Biogeosciences Published: 30 August 2016 c Author(s) 2016.CC-BY 3.0 License.
(Lewicka- Szczebak et al., 2016;Rohe et al., 2014) rapid microbial shift is possible.Moreover, fungal denitrification adds 15 N-pool derived N 2 O characterised by higher δ 15 N sp values and presumably also higher δ 18 O values(Lewicka- Szczebak et al., 2016;Rohe et al., 2014).As a result the η red values determined from correlation slopes are biased because the production of 18 O and 15 N α enriched N 2 O increased in time parallel to a decrease in r N2O .The Org soil data thus demonstrate that a high and variable in time contribution of fungal denitrification complicates the application of the N 2 O isotopic fractionation approach for quantification of N 2 O reduction.This is because a highly variable contribution implies that changes in the measured δ 15 N sp values can either result from variations in δ 0 15 N sp or r N2O .Only when the contribution of fungal denitrification is stable, robust r N2O values can be derived from δ 15 N sp data.Although the Min soil exhibited a smaller range in f F , the contribution of fungal denitrification was apparently also not constant.Simultaneous application of the other isotopic signatures, i.e. δ 15 N bulk and/or δ 18 O, as discussed in further Sect.4.2.3, may help solving this problem.