Biogeosciences, 14, 711732, 2017 www.biogeosciences.net/14/711/2017/ doi:10.5194/bg-14-711-2017 Author(s) 2017. CC Attribution 3.0 License.
Dominika Lewicka-Szczebak1, Jrgen Augustin2, Anette Giesemann1, and Reinhard Well1
1Thnen Institute of Climate-Smart Agriculture, Federal Research Institute for Rural Areas, Forestry and Fisheries, Bundesallee 50, 38116 Braunschweig, Germany
2Leibniz Centre for Agricultural Landscape Research, Eberswalder Strae 84, 15374 Mncheberg, Germany
Correspondence to: Dominika Lewicka-Szczebak ([email protected])
Received: 1 July 2016 Discussion started: 30 August 2016
Revised: 19 January 2017 Accepted: 19 January 2017 Published: 14 February 2017
Abstract. Stable isotopic analyses of soil-emitted N2O ( 15Nbulk, 18O and 15Nsp=15N site preference within the
linear N2O molecule) may help to quantify N2O reduction to N2, an important but rarely quantied process in the soil nitrogen cycle. The N2O residual fraction (remaining unreduced N2O, rN2O) can be theoretically calculated from the measured isotopic enrichment of the residual N2O. However, various N2O-producing pathways may also inuence 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, applying two reference methods for quantication of rN2O: helium incubation with direct measurement of N2 ux and the 15N gas ux 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 to the accompanying N2O and N2 source processes and the most critical is the determination of the initial isotopic signature of N2O before reduction ( 0). We show that 0 can be well determined experimentally if stable in time and then successfully applied for determination of rN2O based on 15Nsp values. Much more problematic to deal with are temporal changes of 0 values leading to failure of the approach based on 15Nsp values only. For this case, we propose here a dual N2O isotopocule mapping approach, where calculations are based on the relation between 18O and 15Nsp values. This
Quantifying N2O reduction to N2 based on N2O isotopocules validation with independent methods (helium incubationand 15N gas ux method)
allows for the simultaneous estimation of the N2O-producing pathways contribution and the rN2O value.
1 Introduction
N2O reduction to N2 is the last step of microbial denitrication, i.e. anoxic reduction of nitrate (NO3) to N2 through the following intermediates: NO3 ! NO2 ! NO ! N2O !
N2 (Firestone and Davidson, 1989; Knowles, 1982). Commonly applied analytical techniques enable us to quantitatively analyse only the intermediate product of this process, N2O, but not the nal product, N2. This is due to the high atmospheric N2 background precluding direct measurements of N2 emissions (Bouwman et al., 2013; Saggar et al., 2013). Hence, N2O reduction to N2 is the least well understood N transformation and constitutes a key quantity of the N cycle, as potential signicant loss of reactive N to the atmosphere. N2 and N2O denitrication uxes cause lowering of both plant-available N, and N leaching while N2O reduction to N2 decreases N2O uxes (Butterbach-Bahl et al., 2013).
To overcome the problems with N2 quantication, three methods for N2-ux estimation are applicable (Groffman, 2012; Groffman et al., 2006): direct N2 measurements under a N2-free helium atmosphere (helium incubation method),
15N analyses of gas uxes after addition of 15N-labelled substrate (15N gas ux method), and the reduction inhibition method based on the comparison of N2O uxes with and without acetylene application (acetylene inhibition method).
Published by Copernicus Publications on behalf of the European Geosciences Union.
712 D. Lewicka-Szczebak et al.: Quantifying N2O reduction to N2 based on N2O isotopocules
These methods were widely applied in laboratory studies to determine the contribution of N2O reduction to N2, which is usually expressed as the fraction of the residual unreduced N2O: rN2O = yN2O / (yN2 + yN2O) (y: mole fraction). The
whole scale of possible rN2O 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 15N gas ux method can be applied under eld conditions to determine the rN2O (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 eld studies due to catalytic NO decomposition in presence of C2H2 and O2 (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 eld-based measurements of soil N2 emissions are available and this important component in the soil nitrogen budget is still missing. This constitutes a serious shortcoming in understanding and mitigating the microbial consumption of nitrogen fertilizers (Bouwman et al., 2013; Seitzinger, 2008), and the N2O emission, which signicantly contributes to global warming and stratospheric ozone depletion (IPCC, 2007; Ravishankara et al., 2009).
N2O isotopic fractionation studies could potentially be used for quantication of rN2O under eld conditions (Park et al., 2011; Toyoda et al., 2011; Zou et al., 2014). Its advantage over the 15N gas ux method lies in its easier and non-invasive application, lack of a need for additional fertilization, and much lower costs. This expands the application potential of the isotopic fractionation method and enables its more widespread use. This kind of study uses the isotopic analyses of the residual unreduced N2O, of which three isotopic signatures can be determined: of oxygen ( 18O), bulk
nitrogen ( 15Nbulk), and nitrogen site preference ( 15Nsp),i.e. the difference in 15N between the central and the peripheral N atom of linear N2O molecules (Brenninkmeijer and Rckmann, 1999; Toyoda and Yoshida, 1999). All these three isotopic signatures ( 18O, 15Nbulk and 15Nsp) are al
tered during the N2O reduction process and the magnitude of the observed change depends largely on the N2O 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 N2O, provided that the isotopic signature of the initially produced N2O before reduction ( 0) and the net isotope effect associated with N2O reduction ( red) are known (Lewicka-Szczebak et al., 2014).
150N and 180O values depend largely on the isotopic signatures of the N2O precursors, i.e. of NH+4, NO3, NO2, and
H2O, and on the transformation pathways such as nitrication or denitrication (Perez et al., 2006). 150Nsp values,
however, are independent of the precursors, but differ according to different pathways, e.g. nitrication or denitrication (Sutka et al., 2006), and different microbial communities, e.g. bacterial or fungal denitriers (Rohe et al., 2014; Sutka et al., 2008) involved in the N2O production. Therefore, 0 values may vary between different soils and due to different conditions, e.g. moisture, temperature, fertilization. red values are variable depending on experimental conditions, but these variations are largest for 18redO and 15redNbulk, whereas for 15redNsp, quite stable values in the range from 7.7 to 2.3
with an average of 5.4 1.6 have been found (Lewicka-
Szczebak et al., 2014). Moreover, recently this value has been conrmed under oxic atmosphere (Lewicka-Szczebak et al., 2015); hence, it can be expected that 15Nsp values can be applied as a robust basis to calculate N2O reduction for eld studies.
However, some open questions still remain: (i) are the isotopic fractionation factors for denitrication processes determined in laboratory experiments transferable to eld conditions? (ii) How robustly can the N2O residual fraction be determined? (iii) Is the quantication of the entire nitrogen loss due to denitrication possible? In this study we present a validation of the calculations based on the N2O isotopic fractionation performed in laboratory experiments. Two different reference methods for quantication of N2O reduction were applied: incubation in a N2-free helium atmosphere and the 15N gas ux method. Helium incubations allow for simultaneous determination of the N2O isotopic signature and the rN2O from the same incubation vessel (Lewicka-Szczebak et al., 2015), whereas in 15N gas ux experiments, parallel incubations of 15N-labelled and natural abundance treatments are necessary. Nevertheless, 15N-labelled treatments provide additional information on the coexisting N2O-forming processes (Mller et al., 2014), which might possibly impact the N2O 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, aiming at a better understanding of the complex N2O production and consumption in these soils. The main aims of this study were to (i) check how precisely the N2O residual fraction can be calculated with the isotopic fractionation approach, (ii) identify the sources of possible bias (e.g. coexisting N2O forming processes), and (iii) search for the possibilities to improve the precision and applicability of this calculation approach.
2 Methods
The list with explanations of all abbreviations and specic terms used in the manuscript can be found in the Supplement (Table S1).
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D. Lewicka-Szczebak et al.: Quantifying N2O reduction to N2 based on N2O isotopocules 713
2.1 Experimental set-ups
2.1.1 Experiment 1 helium incubation as reference method (Exp 1)
Two soil types were used: a mineral arable soil with silt loam texture classied as a Haplic Luvisol (Min soil) and an organic grassland soil classied as Histic Gleysol (Org soil).The soils were air dried and sieved at 4 mm mesh size. Afterwards, the soil was rewetted to obtain 70 % water-lled pore space (WFPS) and fertilized with 50 mg N (added as NO3)
per kilogram of soil. Then the soils were thoroughly mixed to obtain a homogenous distribution of water and fertilizer and 250 cm3 of wet soil were repacked into each incubation vessel with bulk densities of 1.4 g cm3 for the Min soil and0.4 g cm3 for the Org soil. Afterwards the water decit to the target WFPS: 70 or 80 % WFPS depending on the treatment, was added on the top of the soil. The incubations were performed using a special gas-tight incubation system allowing for application of a N2-free atmosphere. This system has been described in detail by Eickenscheidt et al. (2014). Here we briey present its general idea.
The incubation vessels were cooled to 2 C, repeatedly evacuated (to 0.047 bar), ushed with He to reduce the N2 background, and afterwards ushed with a continuous stream of He + O2 for at least 60 h. When a stable and low N2 back
ground (below 10 ppm) was reached, temperature was increased to 22 C. The incubation lasted 5 days, while the headspace was constantly ushed with a continuous ow of 20 % O2 in a helium (HeO2) mixture for the rst 3 days and then with pure He for the following 2 days, at a ow rate of ca. 15 cm3 min1. The uxes of N2O and N2 were directly analysed and the samples for N2O isotopocule analyses were collected at least twice a day. The N2O residual fraction was determined based on the direct measurement of N2O and N2 uxes.
The data from two selected samplings of this experiment have already been published, with particular emphasis on the O isotopic fractionation (experiment 2.32.6 in Lewicka-Szczebak et al., 2016).
2.1.2 Experiment 2 15N gas ux as reference method (Exp 2)
The same soils (Min soil and Org soil) as in Exp 1 were used for parallel incubations under either an anoxic (N2) or an oxic (78 % He + 2 % N2+ 20 % O2) atmosphere with con
tinuous gas ow at 10 cm3 min1. The N2 background concentration in the oxic incubation was reduced to increase the sensitivity of the 15N gas ux method (Meyer et al., 2010).
The soils were air dried and sieved at 4 mm mesh size. Afterwards, the soil was rewetted to obtain a WFPS of 70 % and fertilized with 80 mg N (added as NO3) per kilogram of soil. Half of each soil sample was fertilized with Chile saltpeter (NaNO3, Chili Borium Plus, Prills-Natural origin,
supplied by Yara, Dlmen, Germany), i.e. nitrate fertilizer from atmospheric deposition ore with 15N at natural abundance level (NA treatment). This fertilizer was used to enable the determination of O exchange between denitrication intermediates and water based on the 17O anomaly of Chile saltpeter (Lewicka-Szczebak et al., 2016). The other half of the soil was fertilized with 15N-labelled NaNO3 (98 atom %
15N) (15N treatment). Then the soils were thoroughly mixed to obtain a homogenous distribution of water and added fertilizer. A total of 500 cm3 of wet soil was repacked into incubation vessels with bulk densities of 1.4 g cm3 for the Min soil and 0.4 g cm3 for the Org soil. Afterwards the water decit to the target WFPS of 75 % for Min soil and 85 % for Org soil was added on the top of the soils. Glass jars (0.8 dm3J. WECK GmbH u. Co. KG, Wehr, Germany) were used with airtight rubber seals and with two three-way valves installed in their glass cover to enable continuous gas ow and sampling. The sampling vials were connected to vents of the incubation jars (Well et al., 2008) and were exchanged each 24 h. The soils were incubated for 9 days at constant temperature (22 C). During each sampling, gas samples were collected in two 12 cm3 Labco Exetainers (Labco Limited, Ceredigion, UK) and for NA treatment additional samples were collected in one 120 cm3 crimped vials.
2.2 Chromatographic analyses
In Exp 1, online trace gas concentration analysis of N2 was performed with a micro gas chromatograph (Agilent Technologies, 3000 Micro GC), equipped with a thermal conductivity detector (TCD). Concentrations of trace gases were analysed by a GC (Shimadzu, Duisburg, Germany, GC14B) equipped with an electron capture detector (ECD) for N2O and CO2. The measurement precision was better than 20 ppb for N2O and 200 ppb for N2, respectively.
In Exp 2 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 (1) of four standard gas mixtures, was typically 1.5 %.
2.3 Soil analyses
Soil water content was determined by weight loss after 24 h drying in 110 C. Soil nitrates and ammonium were extracted in 0.01 M CaCl2 solution (1 : 10 ratio) by shaking at
room temperature for 1 h, and NO3 and NH+4 concentrations were determined colorimetrically with an automated analyser (Skalar Analytical B.V., Breda, the Netherlands).
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714 D. Lewicka-Szczebak et al.: Quantifying N2O reduction to N2 based on N2O isotopocules
2.4 Isotopic analyses in NA treatments
2.4.1 Isotopic signatures of N2O
Gas samples were analysed using an isotope ratio mass spectrometer (Delta V, Thermo Fisher Scientic, Bremen, Germany) coupled to an automatic preparation system (Precon +
GC Isolink, Thermo Fisher Scientic, Bremen, Germany) where N2O was pre-concentrated, separated, and puried. In the mass spectrometer, N2O isotopocule values were determined by measuring m/z 44, 45, and 46 of the intact N2O+ ions as well as m/z 30 and 31 of NO+ fragment ions. This allows the determination of average 15N ( 15Nbulk), 15N
( 15N of the central N position of the N2O molecule), and 18O (Toyoda and Yoshida, 1999). 15N ( 15N of the peripheral N position of the N2O molecule) was calculated from 15Nbulk = ( 15N + 15N )/2 and 15N site preference
( 15Nsp) from 15Nsp = 15N 15N . The scrambling fac
tor and 17O-correction were taken into account (Rckmann et al., 2003). Pure N2O (Westfalengas; purity > 99.995 %)
was used as internal reference gas. It had been analysed 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 15Nsp values (Mohn et al., 2014).
All isotopic values are expressed as deviation from the
15N / 14N and 18O / 16O ratios of the reference materials (i.e. atmospheric N2 and Vienna Standard Mean Ocean Water (V
SMOW), respectively). The analytical precision determined as standard deviation (1) of the internal standards for measurements of 15Nbulk, 18O, and 15Nsp was typically 0.1,
0.1, and 0.5 , respectively.
2.4.2 Isotopic signatures of NO3
18O and 15N of nitrate in the soil solution were determined using the bacterial denitrication method (Sigman et al., 2001). The analytical precision determined as standard deviation (1) of the international standards was typically0.5 for 18O and 0.2 for 15N.
2.4.3 Soil water analyses
Soil water was extracted with the method described by Kniger et al. (2011) and 18O 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 (1) of the internal standards was below 0.1 . The overall error associated with the soil water extraction method determined as standard deviation (1) of the ve samples replicated was below 0.5 .
2.5 Isotopic analyses in 15N treatments
2.5.1 15NO3 and 15NH4
15N abundances of NO3 (aNO
3 ) and NH+4 (aNH+
4 ) were
measured according to the procedure described in Stange et al. (2007). NO3 was reduced to NO by Vanadium-III-chloride (VCl3) and NH+4 was oxidized to N2 by Hypobromide (NaOBr). NO and N2 were used as measurement gas. Measurements were performed with a quadrupole mass spectrometer (GAM 200, InProcess, Bremen, Germany).
2.5.2 15N2O and 15N2
The gas samples from the 15N treatments of Exp 2 were analysed for m/z 28 (14N14N), 29 (14N15N), and 30 (15N15N) of N2 using a modied GasBench II preparation system coupled to an isotope ratio mass spectrometer (MAT 253, Thermo Fisher Scientic, Bremen, Germany) according to Lewicka-Szczebak et al. (2013a). This system allows a simultaneous determination of isotope ratios 29R (29N2 / 28N2) and 30R (30N2/28N2) representing three separated gas species (N2, N2+N2O, and N2O), all measured as N2 gas after N2O re
duction in a Cu oven.For each of the analysed gas species (N2, N2+N2O, and
N2O) the fraction originating from the 15N-labelled pool (fP) was calculated after Spott et al. (2006) as follows:
fP =
aM abgd aP abgd
, (1)
where aM :15N abundance in total gas mixture is as follows.
aM =
29R + 230R
2(1 + 29R + 30R)
(2)
abgd : 15N is the abundance of non-labelled pool (atmospheric background or experimental matrix), aP : 15N is the abundance of 15N-labelled pool, from which the fP was derived as follows:
aP =
30xM aM abgd
aM abgd
. (3)
The calculation of aP is based on the non-random distribution of N2 and N2O isotopologues (Spott et al., 2006) where 30xM is the fraction of 30N2 in the total gas mixture:
30xM =
30R
1 + 29R + 30R
. (4)
Identical calculations are performed for each separated gas species, providing the values fP_N2, aP_N2, fP_N2O, aP_N2O, fP_N2+N2O, and aP_N2+N2O. Importantly, in our incubations
under articial atmosphere, we have no background N2O, hence the 15N abundance of total N2O (aM_N2O) results from the mass balance of the 15N abundances and sizes of the
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D. Lewicka-Szczebak et al.: Quantifying N2O reduction to N2 based on N2O isotopocules 715
pools contributing to N2O production. Because aP_N2O represents the 15N abundance of the 15N-labelled pool emitting
N2O, the aM_N2O value enables the distinction between N2O originating from labelled 15NO3 pool (fP_N2O) and that from non-labelled natural abundance pools, like NH+4 or organic N (fN_N2O), as follows:
aM_N2O = aP_N2O fP_N2O + 0.003663 fN_N2O, (5)
where 0.003663 is the fraction of 15N in non-labelled N2O and fN_N2O = 1 fP_N2O.
Based on the determined fP_N2 and fP_N2+N2O we can cal
culate rN2O as follows:
rN2O =
yN2O
yN2 + yN2O =
The hybrid fraction, for either N2O or N2, is calculated as follows:
fH =
HL + H
, (11)
and:
fL + fH = 1 (12)
2.6 Co-existence of other N-transformation processes
The mineral N concentrations and 15N abundances allow for a quantication of the following.
i. formation of natural abundance NO3 via gross nitrication (n) based on the dilution of the 15N-labelled NO3 pool, which is obtained from the initial (subscript 0) and nal (subscript t) concentration (c) and 15N abundance (a) in soil nitrate (Davidson et al., 1991):
n = (cNO3_0 cNO3_t)
fP_N2+N2O fP_N2 fP_N2+N2O
, (6)
where y represents the mole fractions. This approach appeared to be more suitable than directly using fP_N2O, because (i) direct isotopic analysis of the N2O was not possible in samples with low N2O concentration and (ii) fP_N2 and fP_N2+N2O were quantied in one sample based on the same
method whereas fP_N2O includes analysis of isotope ratios of the N2O peak and analysis of N2O concentration by gas chromatography in a replicate gas sample, thus resulting in potential bias in fP_N2O due to the difculty of collecting exactly identical replicate gas samples (Lewicka-Szczebak et al., 2013b).
Knowing rN2O we can estimate the total denitrication [N2+ N2O] ux using the measured [N2O] ux and the de
termined rN2O as follows:
[N2+N2O]ux = [
log(aNO3_0/aNO3_t)
log(cNO3_0/cNO3_t)
. (13)
ii. formation of 15N-labelled NH+4, most probably due to DNRA (dissimilatory nitrate reduction to ammonium)
or due to coupled immobilization-mineralization (Rutting et al., 2011), based on 15N mass balance of nal (subscript t) and initial (subscript 0) ammonium concentration (c) and 15N abundance (a) in nal and initial ammonium and average (of initial and nal value, subscript av) 15N abundance in nitrate:
DNRA =
cNH4_t aNH4_t cNH4_0 aNH4_0 aNO3_av
N2O]ux fP_N2O
rN2O
+ [N2O]ux fN_N2O. (7)
Moreover, from the comparison of the aP_N2 or aP_N2O with aNO
3 values obtained from NO3 analysis of soil extracts, the contribution of hybrid N2 (fH_N2) and N2O (fH_N2O) can be estimated. If aP < aNO
3 this can be due to the combination of two N sources, labelled and non-labelled, to form N2O or N2 (Spott and Stange, 2011). Hence, the fractions of three pools: non-labelled (N), labelled non-hybrid (L) and labelled hybrid (H) contributing to N2 or N2O formation were determined according to Spott and Stange (2011):
N =
a2NO
. (14)
iii. mineralization (m) the amount of natural abundance N which was added to the system, based on N balance, including nal and initial ammonium concentration (cNH4_t, cNH4_0), nitrication (n), non-labelled
N2O ux (fN_N2O [N2O] ux) and DNRA:
m = cNH4_t cNH4_0 + n
+fN_N2O [N2O]ux DNRA. (15)
iv. nitrate immobilization (i) the magnitude of N sink not explained by other processes, including nal and initial nitrate concentration (cNO3_t, cNO3_0), nitrication (n), total N-gas ux [N2O + N2] ux, and DNRA:
i = cNO3_0cNO3_t+nDNRA[N2O+N2] ux. (16)
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+aNO3 (230x29x)+30x
3 (abgd aNO
3 )2
, (8)
L =
a2bgd + abgd(230x29x)+30x(abgd aNO
3 )2
, (9)
29x 2aNO
3 ) + aNO
3 (2
30x+
29x) 2
30x
H =
abgd(230x+
2 . (10)
(abgd aNO
3 )
716 D. Lewicka-Szczebak et al.: Quantifying N2O reduction to N2 based on N2O isotopocules
2.7 N2O isotopic fractionation to quantify N2O reduction
The N2O fractionation approach is based on the changes in N2O isotopic signatures due to partial N2O reduction to N2, which alters the 18O, 15Nbulk, and 15Nsp of the residual
unreduced N2O ( r). All these isotopic signatures depend on the N2O residual fraction (rN2O) according to the following isotopic fractionation equations applying the closed-system
Rayleigh model (Mariotti et al., 1981):
1 + r
1 + 0 =
(rN2O) red. (17) In simplied, approximated form (applied only for graphical interpretations in Sect. 3.4.1), it is as follows:
r 0 + red ln(rN2O). (18)
To be able to determine rN2O from N2O isotopic values of individual samples according to Eq. (17), isotopic fractionation factors associated with N2O reduction ( red) and initial
N2O isotopic signature before reduction ( 0) must be known. We tested various experimental approaches to determine red and 0 values to check which value yields best t between calculated and measured N2O reduction, and thus to identify which of the methods to determine red and 0 is the most suitable one.
2.7.1 Estimating red and 0 values
Mean red and 0 values for the entire experiment
From the statistically signicant logarithmic ts between rN2O and measured r values, we can estimate the isotopic fractionation by N2O production ( 0) and N2O reduction ( red) according to Eq. (18), where the slope represents the red (the isotope effect associated with N2O reduction), and the intercept gives 0 (the initial isotopic signature for the produced N2O unaffected by its reduction) (Fig. 4).
For 18O and 15Nbulk, 0 values are expressed as relative values in relation to the source, i.e. soil water ( 18O(N2O / H2O)) and soil nitrate ( 15Nbulk(N2O / NO3)).
This allows us to reasonably compare different treatments differing in soil water isotopic signatures and properly interpret 15Nbulk values which are related to the isotopic signature of nitrate, getting enriched with incubation time. 150Nsp is independent of the isotopic signature of the source, hence the measured 15Nsp values were directly used for determination of correlations.
Temporarily changing red and 0 values
The interpretations and calculations based on values are difcult when we deal with the simultaneous variations in rN2O and 0 values. Usually, to calculate rN2O a stable 0 is assumed (Lewicka-Szczebak et al., 2015), and to precisely determine temporal changes in 0, we need independent data on
rN2O (Kster et al., 2015). In eld studies, neither rN2O nor 0 can be determined precisely, but rather the possible ranges for each parameter can be given (Zou et al., 2014). In our experiments we have measured rN2O with independent methods, hence we can assess the 0 changes with time, under the assumption that red is stable, or conversely, assess changes in red assuming stable 0 values. The assumption of a stable red value is best justied for 15redNsp, which shows the narrowest range of variations from 7.7 to 2.3 with a mean
of 5 (Lewicka-Szczebak et al., 2014, 2015). Hence, a
xed 15redNsp value of 5 was used to calculate a 150Nsp
value for each sample and thus to estimate its change with time. To calculate the possible temporal change in red values, 0 was assumed constant. The respective 0 value derived from the correlation between ln(rN2O) and r (Mariotti et al., 1981) was used.
Fungal fraction estimated from 0 values
From the calculated 150Nsp values, the fraction of N2O originating from fungal denitrication (fF) can be estimated using the isotopic mass balance. Isotopic end-members for 15Nsp
values were assumed to be 35 for fungal denitrication (Rohe et al., 2014) and 5 for heterotrophic bacterial den
itrication (Sutka et al., 2006; Toyoda et al., 2005). The mixing end-member characterized by higher 15Nsp values can theoretically also originate from nitrication (hydroxylamine oxidation pathway), but only in the oxic treatments. However, in our experimental set-up, due to high nitrate amendment, the absence of ammonia amendment, and high soil moisture, N2O ux from nitrication should be much lower than from denitrication (Zhu et al., 2013). Therefore, the signicant shifts in 150Nsp values observed here are instead discussed as a result of fungal denitrication admixture.
2.7.2 Calibration and validation of rN2O quantication
The precision of the quantication of the N2O reduction based on the N2O isotopic fractionation approach was checked by comparison of the calculated values and the values measured by the reference methods, i.e. direct N2 measurements in He incubation (for Exp 1) and the 15N gas ux
method (for Exp 2). The 0 and red values needed to determine rN2O with Eq. (18) were found from the natural log t between the isotopic signature of residual unreduced N2O and rN2O determined by the independent method, as shown in the previous Sect. 2.7.1.
The calibration of the isotopic fractionation approach was performed by applying 150Nsp and 15redNsp values obtained in the particular experiment to calculate rN2O from the same experiment. The precision of this approach was evaluated by comparing measured and calculated rN2O and determining the standard error of calculated rN2O.
The validation of the isotopic fractionation approach was performed by applying 150Nsp and 15redNsp values determined
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denitrication we used the values of the controlled soil incubation only (from 17.4 to 21.4 ) and disregarded pure culture studies which show a large range of possible values due to various O exchange with 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 N2O is quite stable (Lewicka-
Szczebak et al., 2016).)
150Nsp for fungal denitrication and nitrication based on pure culture studies: for fungal denitrication from30.2 to 39.3 (Maeda et al., 2015; Rohe et al., 2014;
Sutka et al., 2008) and for nitrication from 32.0 to38.7 (Frame and Casciotti, 2010; Heil et al., 2014;
Sutka et al., 2006). As both processes overlap, a common end-member value for N2O production by fungal denitrication of 34.8 is used. (A recent study also indicated a lower 150Nsp value for one individual fungal species, which was disregarded here due to its very low
N2O production: C. funicola showed 150Nsp of 21.9 but less than 100 times lower N2O production with nitrite compared to other species, and no N2O 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 N2O production (> 10 mg N2O
N g1 biomass).)
180O(N2O / H2O) for fungal denitrication and nitrication based on pure culture studies: for fungal denitrication from 40.6 to 51.9 (Maeda et al., 2015; Rohe et al., 2014; Sutka et al., 2008) and for nitrication from35.6 to 55.2 (Frame and Casciotti, 2010; Heil et al., 2014; Sutka et al., 2006). As both processes overlap, a common end-member value for N2O production by fungal denitrication of 43.6 is used. (The relevant values for fungal denitrication are selected after the same criteria as above for 150Nsp.)
Isotopic fractionation factors associated with N2O reduction: values obtained from controlled soil incubations are 15redNsp from 7.7 to 2.3 with a mean
of 5 and of 18redO values from 25 to 5 with
a mean of 15 (Jinuntuya-Nortman et al., 2008;
Lewicka-Szczebak et al., 2014; Menyailo and Hungate, 2006; Ostrom et al., 2007; Well and Flessa, 2009a).Although the range of possible red variations is quite large, it has been shown recently that the mean values and typical 15redNsp/ 18redO ratios are applicable for oxic or anoxic conditions unless N2O reduction is almost complete, i.e. rN2O < 0.1 (Lewicka-Szczebak et al., 2015).
The 15Nsp / 18O slope of the mixing line between the end-member value for N2O production of fungal denitrication or nitrication is distinct from the slope of the reduc-
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D. Lewicka-Szczebak et al.: Quantifying N2O reduction to N2 based on N2O isotopocules 717
in a parallel experiment to calculate rN2O of the validation experiment with the same soil. The validation was performed in three ways (Val1Val3):
i. Val1 used 150Nsp and 15redNsp values obtained from a previous static experiment performed with the same soil (Exp 1EF in Lewicka-Szczebak et al., 2014) to calculate rN2O for Exp 1 and 2 based on the measured 15Nsp values of residual unreduced N2O.
ii. Val2 used 150Nsp and 15redNsp values obtained from Exp 1 to calculate rN2O for Exp 2, and vice versa.
iii. Val3 used the same 150Nsp as Val2, but for 15redNsp the common value of 5 was applied, as recently sug
gested as a mean robust 15redNsp (Lewicka-Szczebak et al., 2014). Here we checked how our results are affected when we use this common value instead of the 15redNsp value determined for the particular soil.
2.7.3 Mapping approach to distinguish mixing and fractionation processes
Until now, isotopomer maps, i.e. plots of 15Nsp vs. 15Nbulk or 15Nsp vs. 18O, have been used to differenti
ate between processes (Koba et al., 2009; Zou et al., 2014) or to identify N2O reduction to N2 (Well et al., 2012). Here we present a very rst attempt of simultaneous quantication of fractionation and mixing processes based on the relation between 15Nsp and 18O values, which we call the mapping approach. The graphical illustration of the 15Nsp / 18O
maps is presented in Fig. 1. The approach is based on the different slopes of the mixing line between bacterial denitrication and fungal denitrication or nitrication and the reduction line reecting isotopic enrichment of residual N2O due to its partial reduction. Both lines are dened from the known most relevant literature data on the respective 0 and red values:
150Nsp for bacterial denitrication from pure culture studies: for heterotrophic bacterial denitrication from
7.5 to +3.7 (Sutka et al., 2006; Toyoda et al., 2005)
and for nitrier denitrication from 13.6 to +1.9
(Frame and Casciotti, 2010; Sutka et al., 2006). As both processes overlap, a common mean end-member value for N2O production by bacterial denitrication of
3.9 is used.
180O(N2O / H2O) for bacterial denitrication: for heterotrophic bacterial denitrication from controlled soil incubations from 17.4 to 21.4 (Lewicka-Szczebak et al., 2016; Lewicka-Szczebak et al., 2014) and for nitrier denitrication based on pure culture studies from19.8 to 26.5 (Frame and Casciotti, 2010; Sutka et al., 2006). As both processes overlap, a common end-member value for N2O production by bacterial denitrication of 21 is used. (For heterotrophic bacterial
718 D. Lewicka-Szczebak et al.: Quantifying N2O reduction to N2 based on N2O isotopocules
Figure 1. Scheme of the mapping approach to simultaneously estimate the magnitude of N2O reduction and the admixture of fungal denitrication (or nitrication).
tion line resulting from reduction isotope effects (Fig. 1: reduction line and mixing line, respectively). Isotopic values of the samples analysed are typically located between these two lines, reduction and mixing. From their position on the 15Nsp / 18O map we can estimate the impact of fractionation associated with N2O reduction and admixture of N2O originating from fungal denitrication or nitrication. If we assume bacterial denitrication as the rst source of N2O, then we can deal with two scenarios:
Scenario 1 (Sc1) the N2O emitted due to bacterial denitrication is rst reduced (point move along reduction line up to the intercept with red_mix line) and then mixed with the second end-member (point move along red_mix line to the measured sample point)
Scenario 2 (Sc2) the N2O from two end-members is rst mixed (point move along mixing line up to the intercept with mix_red line) and only afterwards the mixed N2O is reduced (point move along mix_red line to the measured sample point).
While both scenarios yield identical results for the admixture of N2O from fungal denitrication or nitrication, the resulting reduction shift, and hence the calculated rN2O value, is higher when using Sc2.
3 Results
3.1 Exp 1
N2O and N2 uxes and isotopocules of N2O
The detailed results presented as time series are shown in Fig. S1 in the Supplement. 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 rN2O with incubation 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 values, from 21.6 to 59.1 for 18O, from 52.9 to 29.9 for 15Nbulk, and from 0.3 to 19.6 for 15Nsp.
For the Org soil 80 % WFPS treatment, the initial increase in rN2O, 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.
Values of did not show a clear trend over time and ranged from 11.2 to 41.9 for 18O, from 46.4 to 17.4 for
15Nbulk, and from 1.9 to 17.5 for 15Nsp. In the 70 %
WFPS treatment, the gas uxes were below detection limit during the oxic phase.
18O(H2O) of soil water ranged from 6.5 to 5.1 for
Org and Min soil, respectively.
3.2 Exp 2
3.2.1 NA treatment, Exp 2
N2O and N2 uxes and isotopocules of N2O
The detailed results presented as time series are shown in Fig. S2 in the Supplement. For the anoxic treatments we ob-serve a gradual decrease in N2O ux and an increase in N2 ux (calculated with the rN2O values determined in the parallel 15N treatment) with incubation progress. For Min soil, 18O increases from 27.3 to 71.2 , 15Nbulk from 45.6
to 28.2 , and 15Nsp from 5.5 to 34.6 . For Org soil
18O increases from 18.4 to 52.6 , 15Nbulk from 46.2 to +7.5 , and 15Nsp from 4.3 to 31.4 .
Under oxic conditions, we observe much higher standard deviations for both N2O ux and N2O isotopic signatures.
For Min soil no clear trend over time can be described: the N2O ux is decreasing but rises again at the end of the incubation. Similarly, values rst increase and then decrease again, varying between 32.8 and 63.4 for 18O, between
43.2 and 3.0 for 15Nbulk, and between 3.1 and 16.8
for 15Nsp (Fig. S2.2a). For Org soil, values increase until the 5th day, from 17.5 to 46.6 for 18O and from 48.4 to 38.1 for 15Nbulk, and then vary around 46 and 39 ,
respectively. 15Nsp values keep increasing through the entire incubation period from 1.7 to 23.6 (Fig. S2.2b).
18O(H2O) of soil water ranged from 8.5 to 6.1 for
Org and Min soil, respectively.
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D. Lewicka-Szczebak et al.: Quantifying N2O reduction to N2 based on N2O isotopocules 719
3.2.2 15N treatment, Exp 2
N2O and N2 uxes and 15N enrichment of N pools
The detailed results presented as time series are shown in Fig. S3 in the Supplement. The determined rN2O values in the anoxic treatments are decreasing with incubation progress, from 0.58 to 0.02 for Min soil (Fig. S3.1a) and from 0.71 to 0.30 for Org soil (Fig. S3.1b). In the oxic treatments rN2O varies between 0.08 and 0.72. The minimum values are reached about in the middle of the incubation time in both soil types: on the 6th day for Min soil and the 5th day for Org soil incubation.
From all 15N treatments, only the anoxic Org soil treatment provided very consistent 15N atom fractions in all gaseous fractions (aM_N2O, aP_N2O, aP_N2). They ranged from 42 to 46 atom %, which is in close agreement with soil nitrate (aNO3 = 43 atom %) (Fig. S3.1b). For the anoxic
Min soil treatment, aP_N2 and aP_N2O ranged from 49 to 51 atom % and also correspond to aNO3 (51 atom %), but the
15N atom fraction of the emitted N2O (aM_N2O) is significantly lower, decreasing from 49 to 24 atom % with incubation time (Fig. S3.1a). In oxic conditions we deal with even lower 15N atom fractions in total N2O. aM_N2O ranges from 4 to 32 atom % for Min soil (Fig. S3.2a) and from 11 to 37 atom % for Org soil (Fig. S3.2b). Moreover, for oxic treatments lower values of aP_N2 can also be observed, down to 28 atom % for Min soil and 34 atom % for Org soil. For mineral N we observed almost no change in 15N content in the extracted nitrate under anoxic conditions, with maximal change in aNO3 of 0.3 atom %. Under oxic conditions a slight decrease of 1.5 for Min and 3.2 atom % for Org soil occurs.
The non-labelled ammonium pool stays mostly unchanged under oxic treatments, but signicant 15N enrichment is ob-served under anoxic conditions, where aNH4 reaches 8.7 for
Min and 3.5 atom % for Org soil by the end of the incubation (Figs. S3.1a, b).
N transformations
In Table 1, calculated rates of N transformations are shown.
Initial and nal concentrations for nitrate and ammonium were measured, total gaseous N-loss ([N2+ N2O] ux) is cal
culated (Eq. 7), the rates of nitrication (n), DNRA, mineralization (m), and immobilization (i) were estimated according to Eqs. (13)(16). The ux of N2O from non-labelled soil N pools was calculated as fN_N2O [N2O] ux. The nitrica
tion 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 the formation of 15N labelled NH+4 (from 0.02 to 0.10 mg N, for Min soil and Org soil, respectively). Mineralization (m)
appears to be very high for Org soil, both in oxic (1.99 mgN) and anoxic (1.25 mg N) conditions, and lower for Min
soil (0.31 and 0.15 mg N, respectively). Interestingly, in each treatment quite a pronounced additional nitrate sink, most probably due to N immobilization (i), was found, mostly much larger than the total gaseous loss ([N2+N2O] ux) (Ta
ble 1).
N2O and N2 source processes
Based on the non-random distribution of N2O isotopologues obtained in 15N treatments, we can differentiate between the
15N-pool-derived N2O (fP_N2O) and non-labelled N2O fraction (fN_N2O) (Fig. 2). fP_N2O decreases with lowering of total N2O uxes and is higher for anoxic treatments (above0.42 for Min soil and above 0.91 for Org soil) when compared to oxic treatments (from 0.03 to 0.67 and from 0.14 to0.98, respectively). A signicant contribution of non-labelled N2O (fP_N2O < 1) in the anoxic Min soil treatment was thus evident (Fig. 2a), but the lower fP_N2O values are associated with lower N2O uxes at the end of the incubation, and the cumulative ux of non-labelled N2O is only approx. 0.02 of the total denitrication ux [N2O+N2]. This is slightly
higher than for the Org soil anoxic treatment, where the cumulative ux of non-labelled N2O reaches only ca. 0.01 of the total denitrication ux [N2O + N2]. The contribution of
the cumulative non-labelled N2O ux to the total denitrication ux [N2O + N2] is quite signicant for oxic treatments,
with a mean value of 0.18 and 0.29 for Org soil and Min soil, respectively. Within the 15N-pool-derived N2O, the hybrid sub-fraction can be determined (fH_N2O). Hybrid N2O was found only in oxic treatments (Fig. 2). For Min soil, fH_N2O was detected in all measured N2O samples and varied between 0.05 and 0.19. For Org soil, no fH_N2O was found during the rst 2 or 3 days of incubation when the N2O concentration was highest. Afterwards its contribution gradually increased with decreasing N2O concentration, reaching up to 0.25 of the 15N-pool-derived N2O. Similarly, fH_N2 was determined. Very small fH_N2 was detected in anoxic treatments, up to 0.09 for Min soil and up to 0.18 for Org soil, where only ve samples from two vessels indicated possible presence of hybrid N2 (Fig. 3). Signicantly higher fH_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 signicant negative correlation between fH and, both N2O (Fig. 2) and
N2 ux (Fig. 3), whereas no such relation exists for Min soil.
3.3 N2O isotopic fractionation to quantify N2O reduction
3.3.1 Estimating red and 0 values
For Min soil we obtained very consistent correlations between rN2O and measured r values for all treatments except the oxic Exp 2. The N2O uxes for oxic conditions showed large variations within the repetitions and between the treatments (compare Figs. S2.2a and S3.2a) which indi-
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720 D. Lewicka-Szczebak et al.: Quantifying N2O reduction to N2 based on N2O isotopocules
Figure 2. Contribution of 15N-pool-derived N2O in the total N2O ux (fP_N2O diamonds) and the fraction of hybrid N2O within the
15N-pool-derived N2O (fH_N2O triangles) in relation to the total N2O ux for Min (a) and Org (b) soil in oxic (blue data points) and anoxic (black lled data points) conditions. No hybrid N2O was detectable under anoxic conditions. Logarithmic correlation is shown where statistically signicant (fP Min soil: R2 = 0.80, p < 0.001; fP Org soil: R
2 =0.88, p < 0.001; fH Org soil: R2 = 0.59; p = 0.013). Fluxes
lower than 0.01 (detection limit) are shown jointly as < 0.01.
Table 1. Rates of N transformation processes as calculated from 15N-pool dilution for Exp 2 15N treatment. Measured data used for the calculation are provided in the Supplement (Table S2).
N-transformations: calculated rates (mg N kg1 dry soil per 24 h)
Treatment Nitrication Unlabelled N2O ux DNRA Mineralization Total N-gas ux Immobilization fN_N2O [N2O] [N2+ N2O]
Min Soil
oxic 0.30 0.01 b.d. 0.31 0.02 2.18 anoxic 0.05 0.04 0.02 0.15 1.67 2.51
Org Soil
oxic 1.93 0.07 b.d. 1.99 0.34 6.29 anoxic 0.06 0.13 0.10 1.25 10.42 9.53
b.d. 15N below detection limit.
cates that NA and 15N treatments are not directly comparable. Therefore, the results of the oxic incubation (blue diamonds, Fig. 4a) show no correlation between 15Nsp and rN2O. The other three ts indicate an absolutely consistent value for 150Nsp from 4.0 to 4.5 and also quite a consistent value for 15redNsp from 8.6 to 6.7 (Fig. 4a).
Much wider ranges of red values were found for 18redO (from
22.7 to 9.9 ) and redNbulk (from 6.6 to 2.0 ). In
contrast to quite variable red values, the determined 0 values are very robust, with 180O about +36 and 150Nbulk about 45 (Table 2).
These relations look very different for Org soil. Firstly, there is no signicant correlation between r and rN2O for
Exp 1, whereas all correlations are signicant for Exp 2
(Fig. 4b, Table 2). The red values determined for Exp 2 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 red and 0 values
Theoretical 150Nsp values were calculated for individual samples assuming stable red values (as described in Sect. 2.7.1) and the variations of calculated 150Nsp with incubation time for both soils are presented in Fig. 5. An increase in 150Nsp value with time is observed for both soils, but is much larger and clearly unidirectional for Org soil. Since rN2O simultaneously decreases during the incubation, the
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D. Lewicka-Szczebak et al.: Quantifying N2O reduction to N2 based on N2O isotopocules 721
Figure 3. Contribution of hybrid N2 in the total 15N-pool-derived N2 in relation to the N2 ux for Min (a) and Org (b) soil under oxic (blue triangles) and anoxic (black triangles) conditions. Logarithmic correlation is shown where statistically signicant (fH Org soil oxic:
R2 = 0.79; p < 0.001). Fluxes lower than 0.01 (detection limit) are shown jointly as < 0.01.
Figure 4. Examples of the relation between 15Nsp and rN2O: Min soil (a) and Org soil (b). The equation for natural log correlations are given where signicant, n.a. where not signicant.
150Nsp value obtained from the correlation between 15Nsp and rN2O (Table 2, Fig. 4b) is much below the actual one (Fig. 5b). For Min soil this increasing trend is not so large and constant, and hence the correlation between 15Nsp and rN2O (Table 2, Fig. 4a) provides the 150Nsp value which represents the mean of actual variations quite well (Fig. 5a).
It could also be assumed that 0 values are constant during the experiment and the variable values can be calculated. Under this assumption the values through both soils and experiments are extremely variable for 15Nbulk from 59 to +30 , for 15Nsp from 24 to +15 , and for 18O from 143 to +48 .
Fungal fraction estimated from 0 values
For Org soil, the time course of 150Nsp values (Fig. 5) indicated a very pronounced increase in the fraction of N2O originating from fungal denitrication (fF) during the incubation time of Exp 2 (9 days), giving fF values from 10 % at the beginning up to 75 % at the end. For Min soil in Exp 2, fF was smaller and varied from 7 to 49 %.
3.3.2 Calibration and validation of rN2O quantication
From the correlation tested above (Table 2) we found that only for Min soil can 0 and red values be robustly determined from 15Nsp values. Hence, we show here the cali-
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722 D. Lewicka-Szczebak et al.: Quantifying N2O reduction to N2 based on N2O isotopocules
Figure 5. Calculated 15
0 Nsp values for individual samples (assuming common stable
15redNsp value of 5 ) with the respective fraction
of fungal N2O (fF) (calculated with end-member 15
0 Nsp values: 5 for bacterial and 35 for fungal denitrication). The individual
15
0 Nsp values are compared with the general
150 Nsp value calculated from the overall correlation between 15Nsp and rN2O (Table 2). Min
soil (a) and Org soil (b).
Table 2. Fractionation factors of N2O reduction ( red) and isotopic signatures of initial unreduced N2O ( 0) determined from the regression function = red ln (rN2O) + 0 (Eq. 14). Statistical signicance given for = 0.05 with p < 0.05, p < 0.01, and p < 0.001 from
Pearson correlation coefcients.
18O(N2O / H2O) 15Nbulk(N2O / NO
3 ) 15Nsp rN2O range
red 0 red 0 red 0
Min soil, Exp 1
anoxic 15.5 +35.7 6.6 48.7 8.6 +4.4 0.190.75
oxic 22.7 +37.0 5.7 42.0 6.8 +4.5 0.271.00 Min soil, Exp 2
anoxic 9.9 +35.5 2.0 45.2 6.7 +4.0 0.010.59
oxic n/a n/a n/a n/a n/a n/a 0.040.71
Org soil, Exp 1
anoxic n/a n/a n/a n/a n/a n/a 0.300.84 oxic n/a n/a n/a n/a n/a n/a 0.050.56
Org soil, Exp 2
anoxic 38.4 +20.6 32.9 60.9 30.8 3.4 0.090.82
oxic 25.4 +24.6 6.8 47.1 20.8 3.3 0.100.88
n/a not applicable no statistically signicant correlation.
bration and validation based on these values only. The calibration shows quite a good agreement between the measured and the calculated rN2O with a signicant t to the 1 : 1 line
(Fig. 6). The mean absolute difference between measured and calculated rN2O was 0.08 for Exp 1 and 0.04 for Exp 2.
The mean relative error in the determination of the reduced N2O fraction (1 rN2O) representing the N2 ux was 36 %
for Exp 1 and 8 % for Exp 2. For Exp 1 we have tested if
a better t 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 (Exp 1 mean), as well as calculations for either oxic or anoxic treatments, are shown. The t to a 1 : 1 line is similar for the calculation using the mean values
(Exp 1 mean: R2 = 0.83) and the respective oxic and anoxic
treatments considered individually (Exp 1 oxic: R2 = 0.86
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D. Lewicka-Szczebak et al.: Quantifying N2O reduction to N2 based on N2O isotopocules 723
Figure 6. Calibration of the N2O isotopic fractionation approach using Min soil data. rN2O calculated based on Eq. (17) and measured with independent methods are compared. For Exp 1 the values calculated based separately either on an oxic (blue triangles) or an anoxic treatment (lled black triangles), or based on the mean values (reversed blue triangles), are shown. For Exp 2 only anoxic treatment samples are shown, since for oxic treatment the relevant reference data are missing (see discussion in Sect. 3.4.1). Goodness of t to the 1 : 1 line is expressed as R
2 and the statistical significance is determined for = 0.05 with p < 0.05, p < 0.01, and
p < 0.001 from Pearson correlation coefcients.
and Exp 1 anoxic: R2 = 0.79). This indicates that for this
soil red values were not affected by incubation conditions.
For Val1, i.e. using the 150Nsp and 15redNsp values obtained from a previous static experiment performed with the same soil, the calculated and measured values showed a correlation but the observed slope was signicantly lower than 1 (Fig. 7, red triangles). For Exp 1 the mean absolute difference between the measured and the calculated rN2O reaches0.41 and the relative error in determining N2 ux is as high as 234 %, whereas for Exp 2 these values are much lower with0.09 and 16 %, respectively. Signicantly lower errors determined for Exp 2 are due to many data points of extremely low rN2O values.
For Val2, i.e. using 150Nsp and 15redNsp values from Exp 1, the t to the 1 : 1 line was denitely much better than for
Val1, which is shown by the signicant correlation between measured and calculated rN2O (Fig. 7, black triangles). The absolute mean difference between the measured and the calculated rN2O was 0.10 and 0.07 for Exp 1 and Exp 2, and the relative error in determining the N2 ux reached 54 and 13 %, respectively. Nevertheless, for Exp 2 the maximal difference of 0.40 is very high. The four samples showing the highest deviation are the very rst samples of the incubation, which most probably show slightly different microbial activ-
Figure 7. Validation of the N2O isotopic fractionation approach using Min soil data. rN2O calculated based on Eq. (17) and measured with independent methods are compared. For Exp 1 (triangles) and
Exp 2 (diamonds) the values calculated based on previous static experiment (Val1 red points) and on this study (Val2 black points) are shown. Goodness of t to the 1 : 1 line is expressed as R
2 and the
statistical signicance is determined for = 0.05 with p < 0.05,
p < 0.01, and p < 0.001 from Pearson correlation coefcients.
ity compared to the further part of the incubation. As shown in Fig. 5, at the beginning we deal with larger dominance of bacterial over fungal N2O, which results in lower 150Nsp than assumed in the calculations, and consequently in an overestimation of the rN2O.
For Val3, i.e. using a common value of 5 for 15redNsp,
the t is very similar as for Val2 (not shown). For Exp 1 the mean absolute difference between measured and calculated rN2O was 0.14 (relative error 60 %), which was slightly higher compared to the 0.10 difference (relative error 54 %)
for Val2. For Exp 2 this difference was only 0.05 (relative error 9%), hence even lower than 0.07 (relative error 13 %) obtained for Val2.
Summarizing the results of these three validation scenarios, we can conclude that actual 0 values must apparently be known to obtain reliable estimates of rN2O, whereas it seems possible to use a general value for 15redNsp.
3.3.3 Mapping approach to distinguish mixing and fractionation processes
As qualitative indicators of mixing and fractionation processes, we analysed relations between pairs of isotopic signatures to determine the slopes for the measured values.The same was done for the 0 values calculated using the measured rN2O values (Eq. 17). All the calculated slopes are presented in Table 3, and graphical illustrations are shown in
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724 D. Lewicka-Szczebak et al.: Quantifying N2O reduction to N2 based on N2O isotopocules
Figure 8. The calculated contribution of N2O originating from fungal denitrication or nitrication (fF, upper graph, diamonds) and the calculated residual N2O fraction (rN2O) with two scenarios (triangles) compared to the measured values (crosses). Filled black symbols represent anoxic incubation and open blue symbols represent oxic incubation. Min soil (a) and Org soil (b).
the Supplement (Fig. S4). The 15Nsp / 18O 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 Exp 1 are lower than in Exp 2. The slopes between 18O / 15Nbulk observed in our study range mostly from 1.94 to 3.25 (Table 3). Only for Org soil in anoxic conditions (in both Exp 1 and Exp 2) is this slope substantially lower, from 0.61 to 0.84.
With the mapping approach we used dual isotope values,i.e. 15Nsp and 18O, to calculate rN2O and the fraction of
N2O originating from fungal denitrication or nitrication (fF) as described in Sect. 2.7.3. This was done for both soils but with Exp 2 data only (Fig. 8). Both scenarios provide identical results for fF values, whereas rN2O values are always higher for Sc2 (rst reduction, then mixing) when compared to Sc1 (rst mixing, then reduction) with maximal difference up to 0.39 between them. Figure 8 shows the comparison between calculated and measured rN2O 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 rN2O. The calculated fF values exhibit a continuous increase with incubation time for all treatments except the oxic treatment of Min soil.
4 Discussion
4.1 N2O and N2 source processes
In this study quite a high contribution of non-labelled N2O was documented (Figs. 2, 3). Non-labelled N2O may originate from nitrication or nitrier denitrication (Wrage et al., 2001). However, in the conditions favouring denitrication with high soil moisture (WFPS 75 %) the typical N2O yield from nitrication is much lower compared to the N2O yield from denitrication (Butterbach-Bahl et al., 2013; Well et al., 2008). Therefore, in these experimental conditions the contribution of nitrication to N2O uxes should be rather negligible. Most surprising is the signicant contribution of non-labelled N2O (fP_N2O < 1) in the anoxic Min soil treatment associated with lower N2O uxes at the end of incubation (Fig. 2a). Moreover, for both soils in the anoxic treatment the cumulative non-labelled N2O ux in milligrams of
N is higher than the initial NH+4 pool plus the NH+4 possibly added due to DNRA (Table S2). This indicates that oxidation of organic N must be active in these treatments. Recently, it has been shown that this process can even be the dominant N2O-producing pathway (Mller et al., 2014); however, it is questionable if this can also be active under anoxic conditions. Nitrier denitrication or eventually also some abiotic N2O production would be the most probable processes to produce non-labelled N2O in anoxic treatments, but since the substrate is NH+4, it must have been preceded by ammonication of organic N.
A higher contribution of non-labelled N2O was noted for oxic treatments (Fig. 2). This ux can be well explained by nitrication, because it represents up to 3 % of the nitrication rate (Table 1), which is at the upper end of the
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D. Lewicka-Szczebak et al.: Quantifying N2O reduction to N2 based on N2O isotopocules 725
Table 3. Relations between isotopic signatures of emitted N2O: 15Nsp / 18O, 15Nsp / 15Nbulk, 18O / 15Nbulk, and mean rN2O of the corresponding data sets. The slopes for linear t are given. Statistical signicance given for = 0.05 with p < 0.05, p < 0.01, and
p < 0.001 from Pearson correlation coefcients. The graphical presentation of the correlations is shown in the Supplement (Fig. S4).
15Nsp / 18O 15Nsp / 15Nbulk 18O / 15Nbulk rN2O mean
Slope Slope Slope
Min soil, Exp 1
anoxic 0.47 1.01 2.21 0.46 oxic 0.30 0.59 1.94 0.77
Min soil, Exp 2
anoxic 0.64 2.16 3.25 0.14 oxic n/a n/a n/a 0.39
Org soil, Exp 1
anoxic 0.65 0.55 0.84 0.59 oxic n/a n/a n/a 0.34
Org soil, Exp 2
anoxic 0.76 0.82 0.61 0.48 oxic 0.73 2.07 3.07 0.44
Min soil, all data
calculated 0 n/a n/a 0.56
Org soil, all data
calculated 0 0.68 0.74 1.04
n/a not applicable no statistically signicant correlation.
and Org soil, respectively. Interestingly, we observe higher fH values for oxic treatments. This may indicate the fungal origin for hybrid N2 and N2O, since it has been shown that fungal denitrication may be activated in presence of oxygen (Spott et al., 2011; Zhou et al., 2001). Similarly, Long et al. (2013) identied fungal codenitrication as the major N2-producing process. In our study, higher fH values were generally observed for lower N2 and N2O uxes (especially for Org soil, Figs. 2b, 3b). Most probably, towards the end of the incubation, when N2 and N2O uxes decrease, the concentration of intermediate products NO2 and NO also decrease and the organic substrates may get exhausted. This reinforces the previous observations of enhanced codenitrication for a higher ratio between potential nucleophiles and NO2 or NO and with decreasing availability of organic substrates (Spott et al., 2011). But we cannot exclude the possibility that hybrid N2 also originated from other processes, i.e. abiotic codenitrication or anammox (Spott et al., 2011).
A precondition for the proper quantication of various process rates based on the 15N tracing technique is the homogeneity of 15N tracer in soil. Recently, a formation of two independent NO3 pools in the soil was described for an experimental study (Deppe et al., 2017). One pool contained the
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known range for the nitrication product ratio (Well et al., 2008). Nitrication was quite signicant in oxic treatments and NO3 production from nitrication largely exceeded the
NH+4 available at the beginning of the incubation (Table S2). This indicated that a pronounced amount of organic N must have been mineralized rst or was partially oxidized to NO3 through the heterotrophic nitrication pathway (Zhang et al., 2015).
To our best knowledge, this is one of the very few studies that document a signicant hybrid N2 and N2O production in natural soils without the addition of any nucleophiles, i.e. compounds used as the second source of N in codenitrication (Laughlin and Stevens, 2002; Long et al., 2013; Selbie et al., 2015). All these previous studies identied codenitrication as the major N2-producing process, with contribution of hybrid N2 in the total soil N2 release from 0.32 to0.95 (Laughlin and Stevens, 2002; Long et al., 2013; Selbie et al., 2015). In our study this contribution is lower, namely0.18 and 0.05 of the cumulative soil N2 ux for Min soil and Org soil, respectively. No hybrid N2O was found previously (Laughlin and Stevens, 2002; Selbie et al., 2015), whereas in our study a slight contribution was detected representing0.027 and 0.009 of the cumulative N2O ux for Min soil
viously determined value of 2.7 . While that previous
value was within the 150Nsp range of bacterial denitrication (7.5 to 1.3 , Toyoda et al., 2005), the clearly higher
actual values indicate that the previous method must have strongly inuenced the microbial denitrifying communities, most probably favouring bacterial over fungal denitrication.Much wider ranges of red values were found for 18redO (from
22.7 to 9.9 ) and redNbulk (from 6.6 to 2.0 , Ta
ble 2), which is also consistent with the previous ndings, indicating that these values depend on enzymatic and diffusive isotope effects and as result can vary in quite a wide range (Lewicka-Szczebak et al., 2014). The red determined in Exp 1 are similar to the previous results (18 for 18redO and 7 for 15redNbulk, Lewicka-Szczebak et al., 2014),
whereas in Exp 2 the absolute values are much smaller, suggesting a different fractionation pattern there. Most probably this difference is an effect of a different range of rN2O in both experiments (Table 2). In Exp 2 we partially deal with extremely low rN2O values, which results in smaller overall isotope effects, as also shown before (Lewicka-Szczebak et al., 2015). But 150Nbulk values are very robust since the actual 150Nbulk (45 , Table 2) corresponds very well to the one
previously determined (46 ) using the acetylene method.
Conversely, 180O is much higher (+36 , Table 2) compared
to the value of 19 obtained previously (Lewicka-Szczebak et al., 2014). This may indicate a signicant admixture of fungal denitrication characterized by higher 180O but similar 150Nbulk values (Lewicka-Szczebak et al., 2016; Rohe et al., 2014).
For Org soil, much higher absolute values of 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 whether this observation is not an experimental arte-fact. Actually, the Org soil anoxic treatment was the only case where 15N-pool-derived N2O was dominant (Fig. S3.1b), hence the isotopic signatures should not be altered due to different N2O-producing pathways but mostly governed by the rN2O. But for Org soil, based on the NA treatment, we ob-serve a constant and very signicant increase in the contribution of N2O from fungal denitrication during the incubation (Fig. 5). Future studies should clarify whether such a rapid microbial shift is possible. Fungal denitrication adds N2O characterized by higher 15Nsp values and presumably also higher 18O values (Lewicka-Szczebak et al., 2016; Rohe et al., 2014). As a result the red values determined from correlation slopes are biased because the production of 18O-
and15N -enriched N2O increased in time parallel to a decrease in rN2O. In 15N treatments this increase in N2O added from fungal denitrication cannot be distinguished from bacterial denitrication because both originate from the same
15N nitrate pool.
The Org soil data thus demonstrate that a high and variable in-time contribution of fungal denitrication complicates the
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726 D. Lewicka-Szczebak et al.: Quantifying N2O reduction to N2 based on N2O isotopocules
undiluted 15N tracer solution and thus high 15N enrichment was mostly the source for N2O. The rest of soil NO3 representing the other pool was largely diluted by nitrication input and, therefore, the total soil NO3 (aNO3) showed lower
15N enrichment than the 15N-pool-derived N2O (aP_N2O) (Table 4). This strong discrepancy between pool enrichments could be explained by the large amount of ammonia applied in that experiment and subsequent fast nitrication in aerobic domains of the soil matrix. For our data, aP values are not signicantly higher than aNO3, and for anoxic treatments agree perfectly (Fig. S3.1a, 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 nitrication, and the absence of ammonia amendment. Hence, as we can assume homogenous 15N distribution, our results for fP and fH should be adequate.
4.2 N2O isotopic fractionation to quantify N2O reduction
4.2.1 Estimating red and 0 values
With respect to robust estimation of N2O reduction, a rst question arises: to what extent 0 values and values were variable or constant during incubations. When assuming constant 0 values during the experiment, calculated values were highly variable. The large ranges obtained are clearly in strong disagreement with previous knowledge on possible 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 0 values were variable and values constant. While we cannot rule out that values varied to some extent, it is not possible to verify that using the current data set.
Another question is whether the assumption of isotopic fractionation pattern of closed systems holds. Logarithmic ts provided best correlations with the measured data, whereas linear correlations that would be indicative for open system dynamics (Decock and Six, 2013) yielded worse ts (data not shown). This indicates that the N2O reduction follows the pattern of a closed system according to Rayleigh distillation equation (Eq. 13), as suggested previously (Kster et al., 2013; Lewicka-Szczebak et al., 2015;Lewicka-Szczebak et al., 2014).
To what extent are the observed red and 0 values in agreement with previous data and how could differences be explained? For Min soil we can compare the red and 0 values obtained here to the previous experiment, carried out with the same soil (Exp 1E, 1F, Lewicka-Szczebak et al., 2014) but using the acetylene inhibition technique. The actual 15redNsp values from 8.6 to 6.7 (Fig. 4a) are quite
close to that previous result of 6.0 , whereas 150Nsp val
ues from 4.0 to 4.5 are signicantly higher than the pre-
red and 150Nsp using the acetylene inhibition technique included several experimental limitations that might have affected results. Specically, this approach was based on separate parallel experiments with and without N2O reduction, acetylene amendment required an anoxic atmosphere, and the duration of incubation had to be shorter than 48 h. These limitations most probably inuence the microbial denitrifying community and do not provide the true 150Nsp values.
Whereas nding the true 150Nsp values is rather challenging, fewer problems seem to be related to the 15redNsp values.
For them similar values were found in all the experiments, where He incubations, 15N gas ux or acetylene inhibition methods were applied. The determined values were also similar to the mean literature 15redNsp value of 5 (Lewicka-
Szczebak et al., 2014). Therefore, applying this common literature value for the calculations (Val3) also provided a very good agreement between measured and calculated rN2O values. Hence, this reinforces the previous conclusion that the 15redNsp value of 5 can be commonly applied for rN2O
calculation (Lewicka-Szczebak et al., 2014), but major caution should be paid to the proper determination of 150Nsp values, which may cause much larger bias of the calculated rN2O.
4.2.3 Mapping approach to distinguish mixing and fractionation processes
The emitted N2O is analysed for three isotopocule signatures and the relations between them ( 15Nsp / 18O, 15Nsp / 15Nbulk, 18O / 15Nbulk) can be informative.
Namely, the observed correlation may result from the mixing of two different sources or from characteristic fractionation during N2O 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 eld 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 rN2O (> 0.1)
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D. Lewicka-Szczebak et al.: Quantifying N2O reduction to N2 based on N2O isotopocules 727
Table 4. Results from a laboratory incubation experiment to distinguish between N2O emitted from nitrication and denitrication in a sandy loam soil (Deppe et al., 2017) in comparison with the results of this study (Min and Org soil). Results of Deppe et al. (2017) show large differences between average 15N enrichment of NO
3 in the bulk soil, as analysed in extracted NO
3 , and 15N enrichment of NO
3 in
denitrifying microsites producing N2O, as calculated from the non-equilibrium approach after Spott et al. (2006) and Bergsma et al. (2001).
Deppe et al. Min soil, Org soil, Min soil, Org soil, (2017) oxic oxic anoxic anoxic
aNO3 of added fertilizer 12.5 51.1 43.2 51.1 43.2 aNO3 at nal sampling 2.24 0.02 49.6 0.1 39.9 0.2 50.8 0.2 43.0 0.2
aP_N2O at nal sampling 13.0 0.9 47.7 0.5 37.2 1.0 51.2 0.1 45.9 0.3
aP_N2 at nal sampling n.d. 49.3 1.5 38.7 1.0 49.8 0.4 43.3 1.3
application of the N2O isotopic fractionation approach for quantication of N2O reduction. This is because a highly variable contribution implies that changes in the measured 15Nsp values can either result from variations in 150Nsp or rN2O. Only when the contribution of fungal denitrication is stable, robust rN2O values can be derived from 15Nsp data.
Although the Min soil exhibited a smaller range in fF, the contribution of fungal denitrication was apparently also not constant. Simultaneous application of the other isotopic signatures, i.e. 15Nbulk and/or 18O, as discussed further in Sect. 4.2.3, may help solving this problem.
4.2.2 Calibration and validation of rN2O quantication
The successful calibration shows that 150Nsp and red values were stable enough within Min soil incubation experiments for calculating rN2O 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 red and 150Nsp value of the oxic and anoxic phase of Exp 1 to all the results (Fig. 6). This indicates that the fractionation factors determined experimentally under anoxic conditions may also be applied for isotopic modelling for oxic conditions, e.g. for parallel eld studies in regard to denitrication processes. But importantly, our experiments were performed under high soil moisture and the majority of cumulative N2O ux also in oxic treatments originated from denitrication (Sect. 3.3), which explains the similar 150Nsp values obtained for oxic and anoxic conditions.
For lower soil moisture, differences in 150Nsp values should be expected due to the possible signicant admixture of nitrication processes under oxic conditions.
The results of validation show very different agreement between measured and calculated rN2O values depending on the experimental approach used for determination of red and 150Nsp values (Fig. 7). When the experiments performed in this study were used (Val2) the agreement was quite good.
These experiments are characterized by simultaneous N2O production and reduction and a longer duration of the experiment of 5 to 9 days. However, when we used values found in a previous experiment using the acetylene inhibition technique (Val1), the agreement is much worse. Estimation of
728 D. Lewicka-Szczebak et al.: Quantifying N2O reduction to N2 based on N2O isotopocules
the 15Nsp / 18O slopes characteristic of N2O reduction are quite consistent with previous ndings (Lewicka-Szczebak et al., 2015), i.e. they 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 signicantly differ from the slopes resulting from mixing of bacterial and fungal denitrication, characterized by higher values of about 0.63 and up to 0.85 (Lewicka-Szczebak et al., 2016).
In theory, the slopes for calculated 0 values are not inuenced by N2O reduction and hence should be mostly caused by the variability of mixing processes, whereas the slopes of the measured values reect both mixing and fractionation due to N2O reduction. For Min soil, there is no correlation between calculated values of 150Nsp and 180O (Table 3), which indicates that the correlation observed for measured values was a result of fractionation processes during N2O reduction. In contrast, for Org soil all the correlations for calculated 0 values are still very strong and show similar slopes as the correlations for measured values (Table 3). This indicates a very signicant impact of the mixing of various N2O-producing pathways.
The 15Nsp / 18O 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. 4.2.1 about a higher contribution of fungal N2O in Org soil. But we can also notice that the slopes in Exp 1 are lower than in Exp 2. Most probably less stable microbial activity is present under the longer incubation in Exp 2 (9 days) compared to short phases analysed in Exp 1 (3 days). As observed from the calculated 0 values (Fig. 5) the estimated contribution of fungal N2O most probably increases with incubation time. Hence, the higher slopes for Exp 2 probably result from the admixture of fungal denitrication and the lower slopes for Exp 1 better represent the typical bacterial reduction slopes. The 15Nsp / 18O slopes may thus be helpful in indicating the admixture of various N2O sources.
Interestingly, there is no correlation between isotopic values in oxic Exp 2 for Min soil. A single process or the combination of several processes, which cause large variations in 15Nsp but not in 18O, seems to be present there. This might be due to admixture of N2O from different microbial pathways and possibly also due to O exchange with water.In this treatment we also observe the lowest N2O uxes and also the lowest fP_N2O values, which suggest the largest input from nitrication. The 15Nsp values for hydroxylamine oxidation during nitrication are much larger (ca. 33 ) than for bacterial denitrication or nitrier denitrication (ca. 5 )
(Sutka et al., 2006), whereas 18O 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 15Nsp and 18O (Table 3).
The graphical interpretations including 15Nbulk values are more difcult 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, 2015; Wolf et al., 2015; Zou et al., 2014). The slopes between 18O and 15Nbulk observed in our study range mostly from 1.94 to 3.25 (Table 3), which corresponds quite well to the previously reported results from N2O reduction experiments where values in the range from 1.9 to 2.6 were reported (Jinuntuya-Nortman et al., 2008; Well and Flessa, 2009a). Only for Org soil in anoxic conditions (in both Exp 1 and 2) is this slope largely lower and it ranges from 0.61 to0.84. These values are more similar to 18O / 15Nbulk slopes
for the calculated 0 values (0.56 for Min soil and 1.04 for Org soil (Table 3)) and are signicantly lower than typical reduction slopes. Thus, most probably, they are instead due to the mixing of various N2O sources. However, the calculated 0 values cannot be explained with mixing of bacterial and fungal denitrication only (Fig. S4.3b).
For the relation of 15Nsp / 15Nbulk (Fig. S4.2) the reduc
tion and mixing slopes cannot be separated so clearly. The calculated 0 values are not all situated between the mixing end-member of bacterial and fungal denitrication. This observation is similar to that for 18O / 15Nbulk and is due to
some data points showing very low 150Nbulk(N
2O/NO3) values down to ca. 70 . This value exceeds the known range of
the 15N fractionation factors due to the NO3 / N2O steps of denitrication, i.e. based on pure culture studies, from 37
to 10 for bacterial and from 46 to 31 for fungal
denitrication (Toyoda et al., 2015) (as displayed on graphs in Fig. S4) and, based on controlled soil studies, from 55
to 24 (Lewicka-Szczebak et al., 2014; Well and Flessa,
2009b). This additional N2O input may originate from nitrier denitrication, as already suggested based on the 15N
treatments results (Sect. 3.3). Frame and Casciotti (2010) determined that fractionation factors for nitrier denitrication are "15NbulkNH4/N2O = 56.9 , "18ON2O/O2 = 8.4 , and
"15Nsp = 10.7 . When recalculated for values presented
in our study, 180ON2O/H2O will range from 22 to 25 (taking the variations in 18OH2O into account). Unfortunately, the 150Nbulk value for this process could not be assessed in our study, since the 15NNH4 was not measured. In case the 15NNH4 is lower than 0 , the very low 150Nbulk(N
2O/NO3) values may be well explained with nitrier denitrication.
Although the interpretation of the relations between particular isotopic signatures is not completely clear yet, it seems to have potential to differentiate between mixing and fractionation processes. Note that by using the literature ranges of isotopic end-member values, they must be recalculated according to respective substrate isotopic signatures for the particular study; hence 15NNH4, 15NNO3, and 18OH2O should be known. Only the 150Nsp can be directly adopted. Progress in interpretations could be made if all three isotopic signatures would be evaluated jointly in a modelling approach. In order to produce robust results, precise information on 0 values for all possible N2O source processes must be available for the particular soil. Unfortunately, the complete modelling
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D. Lewicka-Szczebak et al.: Quantifying N2O reduction to N2 based on N2O isotopocules 729
is not possible for the data presented here as information on the NH+4 isotopic signature and the 150Nbulk value for possible nitrication processes is lacking.
The mapping approach had been used before based on 15Nsp and 15Nbulk to estimate the fraction of bacterial N2O (Zou et al, 2014). Because N2 uxes were not measured in that study, scenarios with different assumptions for N2O reduction were applied to show the possible range of the bacterial fraction. Here, we evaluated the mapping approach for the rst time using independent estimates of N2O reduction.
Most informative are the relations between 15Nsp and 18O,
because 150Nbulk was poorly known, whereas the estimation of 180O is quite robust due to the large O exchange with water and constant fractionation during O exchange, as shown previously (Lewicka-Szczebak et al., 2016). Therefore we proposed here a method based on 15Nsp and 18O values
to simultaneously calculate the N2O residual fraction (rN2O) and the contribution of the mixing end-members as described in Sect. 2.7.3. From Fig. 8 we can assume that the method works quite well in the case of a signicant admixture of fungal N2O and allows the quantication of its fraction (fF).
For the three treatments where a good agreement between measured and calculated rN2O is observed, we deal with a signicant contribution of fungal N2O (Sect. 4.2.1). The fF values calculated here from the mapping approach are very consistent with the values found based on estimated 150Nsp only (Fig. 5), i.e. without considering 18O values. In the oxic
Min soil treatment we probably deal with a signicant contribution of N2O originating from nitrication or nitrier denitrication, as supposed previously from the 15N treatment (Sect. 4.1) and from the isotopic relations discussed above.The oxic Min soil treatment thus results in rather poor agreement of the mapping approach results. The combination of these processes seems to be too complex to precisely quantify their contribution in N2O production based on three isotopocule signatures only.
Importantly, for Org soil where fF values are very high and variable with time (see also Sect. 4.2.1), the mapping approach was the only method to get any estimation of both fF and rN2O. The other approach, presented in Sect. 2.7.2 and successfully applied for Min soil, failed for Org soil due to the inability to assess a stable 150Nsp. Hence, for the case of varying contribution of fungal N2O, the mapping approach presented here may be the only way of assessing the range of possible fF and rN2O values. However, the precision of the results obtained from the mapping approach is a complex issue depending on the size of end-member areas and variability of values. We did not aim to determine the resulting uncertainty in the present paper. The following paper will address the precision problem in detail (Buchen et al., 2017).
5 Conclusions
We have shown that the N2O isotopic fractionation approach based on 15Nsp values is suitable to identify and quantify N2O reduction under particular conditions, most importantly, quite stable N2O production pathways. It has been conrmed that the range of 15redNsp values dened in previous studies is applicable for the calculations. The calculated N2O residual fraction is much more sensitive to the range of possible 150Nsp values than to 15redNsp values. Therefore, 150Nsp values must be determined with considerable caution. The method can be used in eld studies, but to obtain robust results, in situ measurement of isotopocule uxes should be complemented by laboratory determinations of 150Nsp values. For this aim, the He incubation technique or the 15N gas
ux method can be applied as reference methods, but not the acetylene inhibition method, since it most probably affects the microbial community, which results in biased 150Nsp values. Anoxic incubations may be applied and the determined 150Nsp values are representative for N2O originating from denitrication, even under the oxic atmosphere, which means they are also representative in eld studies.
The attainable precision of the method, determined as mean absolute difference between the measured and the calculated N2O residual fraction (rN2O), is about 0.10, but
for individual measurements this absolute difference varied widely from 0.00 up to 0.39. The relative error of N2 ux quantication depends strongly on the rN2O of a particular sample and varied in a very wide range from 0.01 up to 2.41 for Exp 1 and from 0.00 up to 0.93 for Exp 2, with a mean relative difference between measured and calculated N2 ux of 0.46 and 0.13, respectively. The highest relative errors in the calculated N2 ux (> 1) occur for the very low uxes only (rN2O > 0.9).
However, for soils of more complex N dynamics, as shown for the Org soil in this study, the determination of N2O reduction is more uncertain. The method successfully used for Min soil was not applicable due to failed determination of proper 150Nsp values, which were signicantly changing with incubation progress. Here we suggest an alternative method based on the relation between 15Nsp and 18O values (mapping
approach). This allows for the estimation of both the fraction of fungal N2O and the plausible range of residual N2O.
6 Data availability
The data used in this paper can be found in the Supplement.
The Supplement related to this article is available online at http://dx.doi.org/10.5194/bg-14-711-2017-supplement
Web End =doi:10.5194/bg-14-711-2017-supplement .
Competing interests. The authors declare that they have no conict of interest.
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730 D. Lewicka-Szczebak et al.: Quantifying N2O reduction to N2 based on N2O isotopocules
Acknowledgements. This study was supported by German Research Foundation (DFG: We/1904-4, LE 3367/1-1). Many thanks are due to Martina Heuer for help in N2O isotopic analyses,
Bertram Gusovius for assistance by helium incubations, Stefan Burkart for assistance by microcosm incubations, Kerstin Gilke for help in chromatographic analyses, Roland Fu for advice in statistical evaluation, and Caroline Buchen for supplying soil for laboratory incubations.
Edited by: Y. KuzyakovReviewed by: three anonymous referees
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Abstract
Stable isotopic analyses of soil-emitted N<sub>2</sub>O (δ<sup>15</sup>N<sup>bulk</sup>, δ<sup>18</sup>O and δ<sup>15</sup>N<sup>sp</sup> = <sup>15</sup>N site preference within the linear N<sub>2</sub>O molecule) may help to quantify N<sub>2</sub>O reduction to N<sub>2</sub>, an important but rarely quantified process in the soil nitrogen cycle. The N<sub>2</sub>O residual fraction (remaining unreduced N<sub>2</sub>O, r<sub>N<sub>2</sub>O</sub>) can be theoretically calculated from the measured isotopic enrichment of the residual N<sub>2</sub>O. However, various N<sub>2</sub>O-producing pathways may also influence the N<sub>2</sub>O 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, applying two reference methods for quantification of r<sub>N<sub>2</sub>O</sub>: helium incubation with direct measurement of N<sub>2</sub> flux and the <sup>15</sup>N gas flux method. This allowed a comparison of the measured r<sub>N<sub>2</sub>O</sub> values with the ones calculated based on isotopic enrichment of residual N<sub>2</sub>O. The results indicate that the performance of the N<sub>2</sub>O isotopic fractionation approach is related to the accompanying N<sub>2</sub>O and N<sub>2</sub> source processes and the most critical is the determination of the initial isotopic signature of N<sub>2</sub>O before reduction (δ<sub>0</sub>). We show that δ<sub>0</sub> can be well determined experimentally if stable in time and then successfully applied for determination of r<sub>N<sub>2</sub>O</sub> based on δ<sup>15</sup>N<sup>sp</sup> values. Much more problematic to deal with are temporal changes of δ<sub>0</sub> values leading to failure of the approach based on δ<sup>15</sup>N<sup>sp</sup> values only. For this case, we propose here a dual N<sub>2</sub>O isotopocule mapping approach, where calculations are based on the relation between δ<sup>18</sup>O and δ<sup>15</sup>N<sup>sp</sup> values. This allows for the simultaneous estimation of the N<sub>2</sub>O-producing pathways' contribution and the r<sub>N<sub>2</sub>O</sub> value.
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