Research Article

Received: 25 January 2013 Revised: 31 March 2013 Accepted: 1 April 2013 Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2013, 27, 1517–1526

Isotopomer and isotopologue signatures of N2O produced inalpine ecosystems on the Qinghai–Tibetan Plateau

Tomomichi Kato1,2*, Sakae Toyoda3, Naohiro Yoshida3,4,5, Yanhong Tang6

and Eitaro Wada11Research Institute for Global Change, Japan Agency for Marine-Earth Science and Technology, Yokohama, Kanagawa236-0001, Japan2Laboratoire des Sciences du Climat et de l’Environnement, IPSL, CEA-CNRS-UVSQ, Orme des Merisiers, 91191 Gif surYvette, France3Department of Environmental Science and Technology, Tokyo Institute of Technology, Yokohama 226-8502, Japan4Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, Yokohama 226-8502, Japan5Earth-Life Science Institute, Tokyo Institute of Technology, Meguro, Tokyo 152-8551, Japan6National Institute for Environmental Studies, Tsukuba, Ibaraki 305-8569, Japan

RATIONALE: Static-chamber flux measurements have suggested that one of the world’s largest grasslands, the Qinghai-Tibetan Plateau (QTP), is a potential source of nitrous oxide (N2O), a major greenhouse gas. However, production andconsumption pathways of N2O have not been identified by in situ field measurements.METHODS: Ratios of N2O isotopomers (14N15N16O and 15N14N16O) and an isotopologue (14N14N18O) with respect to14N14N16O in the atmosphere, static chambers, and soils were measured by gas chromatography and mass spectrometryin the summer of 2005 and the following winter of 2006 at three typical alpine ecosystems: alpine meadow, alpine shrub,and alpine wetland, on the QTP, China.RESULTS: Site preference (SP) values of soil-emitted N2O were estimated as 33.7% and 30.1% for alpine meadow andshrub, respectively, suggesting larger contributions by fungal denitrification, than by bacterial denitrification andnitrifier-denitrification, to N2O production. Statistical analysis of the relationship between SP and d15Nbulk valuesindicated that in alpine meadow, shrub, and wetland sites fungal denitrification contributed 40.7%, 40.0%, and 23.2%to gross N2O production and the produced N2O was reduced by 87.6%, 82.9%, and 92.7%, respectively.CONCLUSIONS: The combined measurements of N2O concentration, flux, and isotopomeric signatures provide a robustestimation of N2O circulation dynamics in alpine ecosystems on the QTP, which would contribute to the development ofecosystem nitrogen cycle model. Copyright © 2013 John Wiley & Sons, Ltd.

(wileyonlinelibrary.com) DOI: 10.1002/rcm.6595

Nitrous oxide (N2O) is a major greenhouse gas influencingglobal climate change, and thus the mechanisms of itsproduction and consumption processes are of great interest.Natural ecosystems are a major source of N2O, which isproduced through soil microbial activities of nitrification anddenitrification. These processes occur naturally and can beenhanced in soils and waters that are enriched in nitrogen-containing species. In nitrification, N2O is produced as a by-product in hydroxylamine oxidation to nitrite, as follows:

NH3 ! NH2OH ! N2O (1)

In denitrification, N2O is produced as an intermediate bybacterial/fungal denitrification:

NO�3 ! NO�

2 ! NO ! N2O ! N2 (2)

* Correspondence to: T. Kato, Laboratoire des Sciences duClimat et de l’Environnement, IPSL, CEA-CNRS-UVSQ,Bât. 712, Orme des Merisiers, 91191 Gif sur Yvette. France.E-mail: tomomichi.kato@lsce.ipsl.fr

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In addition to the above two processes, N2O can beproduced from nitrite through nitrification, which is oftentermed nitrifier-denitrification:

NH3 ! NO�2 ! N2O (3)

Although many field studies have examined the temporaland spatial variations in N2O fluxes between terrestrialecosystems and the atmosphere, the processes of productionand consumption of N2O gas before its release from soillayers have received less attention. Therefore, muchuncertainty remains in our understanding of soil N2O andits potential response to present and future climaticvariations, as well as to anthropogenic disturbances alteringnatural and agricultural ecosystems at unprecedented scales,both in rate and geographical extent.[1]

Stable isotope techniques can provide much informationregarding the attribution of N2O to different microbialprocesses of production and consumption, and physicalprocesses of transportation between soil and theatmosphere.[2–4] Isotopomer analysis has recently been usedto further refine the isotopic fingerprint of N2O. In contrast

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to d18O and average d15N (d15Nbulk) values, the differencebetween central and peripheral 15N enrichment (d15Na –d15Nb= 15N site preference: SP) is considered to beindependent of the isotopic signature of the precursor[5] andthus supplies process information even if isotopic signaturesof additional N species are lacking. However, there are stillrelatively few in situ field measurements available forcomprehending the natural abundance of N2O isotopic andisotopomeric values.The Qinghai-Tibetan Plateau (hereafter referred to as

QTP) is one of the largest grasslands (~2.0� 106 km2) inthe world, with high elevations (more than 4000 m abovesea level (a.s.l.)).[6] Alpine grasslands, which arecharacterized by low temperature and plentiful sunlight,might be a net carbon sink because of the lowerdecomposition rate of organic matter and relativelyfavorable photosynthetic conditions compared with highlatitude cold ecosystems, such as tundra or taiga forest.Alpine grassland soils store an estimated 33.52 Pg C inorganic form, accounting for approximately 2.5% of theglobal soil carbon pool.[7] Several field measurements ofN2O flux have been conducted on the QTP. Kato et al.[8]

examined the spatial variation in N2O fluxes and reportedthat some sites acted as sinks and others as sources ofatmospheric N2O. They also showed several relationshipsbetween the N2O flux and environmental factors, includingan exponentially negative relationship with the C/N ratioof surface soil in drier ecosystems and a negative correlationwith soil pH in wetlands. Lin et al.[9] showed that yak dungand urea caused slightly higher emission of N2O in an alpinemeadow. Considering that finding, the QTP may be apotential N2O source because of livestock raising and couldhave a substantial impact on the global N2O budget.Complicated interactions between microbes, nutrients, andphysical and chemical conditions lead to variations in therelative intensity of nitrification, denitrification, and N2Oreduction. Previous studies have mainly examined thecontributions of each production and consumption processby indirect methods, such as the relationship between netecosystem N2O flux and environmental factors. However,not only quantitative but also qualitative approaches,such as stable isotope analysis, are needed for betterunderstanding of the mechanisms of N2O production,reduction, and transportation in field settings of the QTP.The emerging ecosystem modeling approach to clarify

the behavior of green house gases on continental andglobal scales also demands more in situ information aboutthe N2O production and consumption pathways for theparameterization and validation of the model. To reducethe uncertainties in the global N2O budget estimation forbetter future climate projection, therefore, we have toclarify the mechanisms involved in the N2O cycle in thevast QTP alpine ecosystems both qualitatively andquantitatively. Here we conducted the in situ fieldmeasurement of isotopic signature of N2O in three typicalalpine ecosystems in northeastern QTP in the summer of2005 and the following winter of 2006. We sampled thegas in the atmosphere and soil layers, and soil-emittedgas in static closed chambers at the soil surface, andexamined the temporal and vertical changes in the isotopicsignature of N2O. Considering that the net N2O flux iscontrolled by N2O production and reduction occurring in

wileyonlinelibrary.com/journal/rcm Copyright © 2013 John Wil

the aerobic/anaerobic part of soil layers, we raise severalquestions that would provide a better understanding of themechanisms of N2O circulation in the QTP alpine ecosystems:

1. Does the N2O pathway show clear diurnal and seasonalchanges?

2. What microbial types are the most likely end members andhow much is the fractional contribution of each endmember for gross N2O production?

3. How much is the gross N2O production reduced byoxidation in soil?

EXPERIMENTAL

Site description

The isotopic signatures and fluxes of N2O were measured indaytime in summer 2005 and winter 2006 at three typical QTPecosystems: alpine meadow, alpine shrub, and alpine wetland.The sites were located within approximately a 2-km radius ofthe Haibei Alpine Meadow Ecosystem Research Station,Northwest Plateau Institute of Biology, Chinese Academyof Science, China (37�29’–45’N, 101�12’–23’E; 3250 m a.s.l.).The annual average temperature and precipitation for1981–2000 were �1.7�C and 561 mm, respectively. In thealpine meadow, the soil is clay loam of Mat Cry-gelicCambisol. In the alpine shrub, the soil is silty clay loam ofMol-Cryic Cambisol.[10] The wetland is characterized by amixed hummock-hollow terrain with hummocksrepresenting 40%, hollows 55%, and other features 5% ofthe landscape. The study sites are not closed by fence andare grazed by yaks and sheep every winter.

The three perennial sedges Kobresia humilis, K. pygmaea, andK. tibetica dominated the plant community at the alpinemeadow site.[6] A Rosaceous shrub, Potentilla fruticosa, andthe above three sedges dominated at the alpine shrub site,[11]

and K. tibetica, Carex pamirensis,Hippuris vulgaris, and Blysmussinocompressus dominated at the wetland.[12] In the wetland,the soil surface under plant canopies was largely covered bymoss (Distichium inclinatum [Hedw.], Bruch and Schimp.,Brachythecium spp., and Encalypta spp.), and free water waspartially covered by Potamogeton pectinatus L.[13] Vascularplants start to grow in May and die out in October.[6]

Gas sampling

Gas samples for isotopic analysis were collected from one of thestatic flux chambers after sampling for the flux measurementthrough a capillary tube to avoid a sudden pressure changeinside the chamber. We sampled four times on a single day(10:00, 14:00, 18:00, and 24:00 at Beijing Standard Time [BST])to investigate the diurnal cycle of N2O flux and isotopicsignature. In summer, samples were taken on 21 July at thealpine meadow, 23 July at the alpine shrub, and 25 July 2005at the alpine wetland. In winter, samples were taken on4 March at the alpine meadow, 6 March at the alpine shrub,and 8March 2006 at the alpine wetland. In addition, N2O gaswas collected from the atmosphere at 2.2 m height and fromthe soil layer through a capillary tube and cylindrical soil-gas-phase probes made of stainless steel (GL Science. Inc.,Tokyo, Japan) installed individually at depths of 10, 30, and

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70 cm at 14:00 and 24:00 (BST) in summer and winter at thealpine meadow, 30 cm at 14:00 in summer at the alpine shrub,and at no depth at the alpine wetland. The gas was stored inan evacuated 500-mL container made of glass or stainlesssteel. For soil gas, 10-, 30-, and 70-cm depths were chosenfor sampling; no soil gas was sampled in the wetland.

Flux measurement of N2O

A polyvinyl chloride (PVC) cylinder chamber (200 mm indiameter by 200 mm high) was used to measure the fluxes.Four chambers were used in summer and two were used inwinter. In both seasons, the chambers were set in 5 m� 5 mrectangle grids spaced at 5-m intervals. They were buried atapproximately 70-mm depth vertically at least 24 h beforesampling. At every sampling time, the PVC lids were puton the chamber tops and sealed tightly by vinyl tape. Theair inside the chambers was sampled in 10-mL glass vialsfor N2O gas at 1, 10, 20, 30, and 60 min in summer, and at1, 10, 30, 60, and 90 min in winter. The flux was calculatedas a linear slope of the concentration evolution over the timecourses. The concentrations of N2O in the air samples weremeasured by gas chromatographs (models GC-7A and GC-14B;Shimadzu, Kyoto, Japan) equipped with a (63Ni)-electron-capture detector. From each vial, 2 mL of air was withdrawnusing a gas-tight syringe, and injected into a gas chromatographand purged with pure N2 gas at a flow rate of 40 mL min–1.The samples were directly injected from sample vials into thegas chromatograph with a gas-tight syringe (A-2 type gassyringes; VICI Precision Sampling, Baton Rouge, LA, USA).

On-site measurements of environmental factors

Soil temperature and water temperature (in case of an open-water site) at 5 cm around the chambers were measured bythermometers. The average values of soil-water content at 0and 12 cm depths were determined by time domainreflectometry (TDR) sensors (CS-620; Campbell Sci. Inc.,Logan, UT, USA) only in summer. In wetlands, the soiloxidation–reduction potential (ORP) was measured using aglass electrode. After chamber gas sampling, 5-cm-deep soilcores were collected. Using these cores, the soil air-filled spacewas measured with a soil three-phase meter (model DIK-1130;Daiki, Tokyo, Japan). The water-filled-pore space (WFPS) wasalso determined by dividing the soil-water-filled space, asevaporated soil water from oven-dried soil, by the porosity,which is calculated as a sum of the air-filled space and theevaporated soil water from oven-dried soil. Above-groundplant mass was collected by mowing. Below-ground plantmass and mineral soil were separately collected by sievingthe soil samples collected with a core sampler from 0- to30-cm depths. Each sample was then weighed before andafter oven drying at around 80�C for 48 h. The total carbonand nitrogen contents of plant bodies and of sieved anddecarboxylated soil samples were measured with an NCanalyzer (Sumigraph NC-900; Sumika Chemical AnalysisService Ltd., Osaka, Japan).

Measurement of isotopomers and an isotopologue of N2O

The relative abundances of isotopomers (14N15N16O and15N14N16O) and an isotopologue (14N14N18O) with respect to14N14N16O in the gas samples were determined in the

Copyright © 2013 JRapid Commun. Mass Spectrom. 2013, 27, 1517–1526

laboratory at Tokyo Institute of Technology (Yokohama, Japan)using an on-line analytical system described elsewhere.[14,15]

Briefly, an aliquot of gas sample containing 1–5 nmol of N2Owas manometrically measured and N2O was cryogenicallyconcentrated and separated fromH2O and CO2 using chemicaladsorbents. After further purification by gas chromatography,the N2O was injected into an isotope-ratio mass spectrometer(MAT 252; Thermo Fisher Scientific K. K., Yokohama, Japan).Site-specific N-isotope analysis in N2O was conducted usingion detectors that had been modified for mass analysis of theN2O fragment ions (NO+), which contain the N atoms in thecenter positions of the N2O molecules. Bulk (average) N- andO-isotope ratios were determined from the molecular ions.[16]

Isotopic calibration was carried out by measuring a syntheticair standard containing 349 nL L–1 of N2O (Japan Fine ProductsCo., Ltd., Kawasaki, Japan), which had been previouslycalibrated against international standards for atmospheric N2

and standard mean ocean water.[16] The notation of theisotopomer or isotopologue ratios is shown below. Themeasurement precision was typically better than 0.1% ford15Nbulk and d18O values, and better than 0.5% for d15Na andd15Nb values:

d15 Ni ¼ 15Risample=

15Rstd � 1Þ � 1000 %ð Þ i ¼ a; b; or bulkð Þ�

(4)

d18O ¼ 18Rsample=18Rstd � 1

� �� 1000 %ð Þ (5)

where 15Ra and 15Rb represent the 15N/14N ratios at the centerand end sites of N atoms, respectively, and 15Rbulk and 18Rdenote the isotope ratios for average 15N/14N and 18O/16O,respectively. The subscripts ’sample’ and ’std’, respectively,indicate the isotope ratios for the sample and the standard,with the standard being atmospheric N2 for N and Viennastandard mean ocean water (V-SMOW) for O. We also definethe 15N site preference (hereafter SP) as an illustrativeparameter of the intramolecular distribution of 15N:

15N site preference SPð Þ ¼ d15Na � d15Nb (6)

The N2O concentration was obtained simultaneously withthe isotopomer and isotopologue ratios from the peak areaof the major ions (masses 44 and 30 in the molecular ionand fragment ion analysis, respectively) measured in thesample and the reference gas (the synthetic air), and thevolume of the processed sample and reference.

Definition of enrichment factors

The relationships between isotope ratios and concentrationsof substrate or accumulated products are expressed by theRayleigh equation:

dS tð Þ ¼ dS t ¼ 0ð Þ þ eln f (7)

where dS(t) and dS(t = 0) are the d values of substrate S attimes t and t = 0, respectively, e is an enrichment factor,and f equals the remaining fraction of substrate S at timet ([S][t]/[S][t= 0]). Equation (7) holds only if e is constantduring the progress of the reaction.[17] In this study, e wasused for analysis as an enrichment factor only when theuptake of flux by soil occurred in the alpine meadow andalpine shrub sites.

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Calculation of the isotopic signature of N2O emitted fromthe soil

WhenN2O is emitted from the soil, the collectedN2O in thefluxchambers is a mixture of atmospheric and soil-derived N2O.The isotopic signatures of the soil-emitted N2O (dsoil values)were calculated from those of the chamber gas samples(dchamber values) and the ambient air samples (dair values)assuming two-component mixing. We conducted samplingonly once during each measurement time; therefore, it wasdifficult to obtain the stable dsoil values from the slope of theregression line for dchamber values versus inverse Cchamber

(Keeling plot). Thus, the dsoil value was calculated using thefollowing mass balance equation:

dchamber�Cchamber ¼ dair�Cair þ dsoil�Csoil (8)

where C is the N2O concentration and Csoil =Cchamber�Cair.The dsoil values obtained from small Csoil values (<10 ppbv)were not used for further data analysis because in such casesdchamber was equal to dair within the precision of the analysisand the error propagated in the calculation of dsoil was large.

Fractional composition of N2O production andconsumption pathways estimated by SP and d15Nbulk

Assuming that N2O is produced by two sources and that themixed N2O is also consumed by N2O reduction, x, the fractionof N2O produced by one of two sources, and Fr, the progressof N2O reduction, are estimated by SP and d15Nbulk by MonteCarlo calculation. Details of this calculation are explained inthe Auxiliary Material of the report by Toyoda et al.[15]

In SP–d15N space, the following equation defines a line,which makes it possible to deduce the SP and d15N valuesof the produced N2O, and the relative contributions fromthe two sources, by tracing the observed values backalong the line until it intersects with the mixing line definedby the two sources:

SPsoil � xSPnit þ 1� xð ÞSPdenitf g¼ e SPð Þred=eð15NbulkÞred½d15Nbulk

soil

� xd15Nbulknit þ 1� xð Þd15Nbulk

denit� ��

(9)

where soil, nit, and denit denote soil, nitrification, anddenitrification, respectively, e(SP)red and e(15Nbulk)red arethe fractions of reduction of SP and 15Nbulk, and C is theN2O concentration.We obtained x and Fr, approximate measures of the degree

of N2O reduction, and their uncertainties by Monte Carlocalculation as follows. First, the e and SP values for N2Oproduction and consumption processes were calculated usingrandom numbers that obey a normal distribution, with theaverage and 1s values obtained from published results(’best estimates’ in Table 3, Toyoda et al.[15]). For e(SP)/e(15Nbulk), we used the average values of e(SP)/e(15Nbulk) ineach experimental condition or the literature, instead ofthe independent average of e(SP) and e(15Nbulk) shown inTable 3 of Toyoda et al.[15] The isotopic signature of N2Oproduced by the two sources was then calculated from theobserved isotope ratios in substrates (ammonium and nitrate;if not available, the d15N value of nitrate was calculated fromthe d15N value of ammonium and the enrichment factor for

wileyonlinelibrary.com/journal/rcm Copyright © 2013 John Wil

nitrification) and the randomly determined parameters. Next,the intersection of the fractionation and source-mixing lineswas obtained and x and k were calculated as follows:

x ¼ SPintersect � SPdenitð Þ= SPdenit � SPnitð Þand (10)

k ¼ �SPsoil þ xSPnit þ 1� xð ÞSPdenitf g½ �=e SPð Þred (11)

where SPintersect is the SP value of the calculated intersect, andk is a positive number. If N2O production follows sequentiallywith the reduction, k=�ln(C/C0) and therefore the degree ofN2O reduction, Fr, is calculated as follows:

Fr ¼ 1� C=C0 ¼ 1� exp �kð Þ (12)

In some cases, the intersection could not be obtainedmathematically or was outside the mixing line, and thuswas discarded. The calculation was repeated until 1000results were obtained.

Thismethod can be applied for the combination of two sourcesthat have apparently distant SP values, for example, fourcombinations: fungal denitrification or hydroxylamine oxidation(nitrification) vs. denitrification or nitrifier-denitrification.

RESULTS

Diurnal change in environmental factors, N2O gas flux, andN2O isotopic signature

The soil-surface temperature reached its diurnal maximum at14:00 in summer and 18:00 in winter and reached itsminimum at 24:00 in summer and 6:00 in winter (Fig. 1(a)).The soil water was relatively constant diurnally in the alpinemeadow and shrub, but decreased considerably at 24:00 inthe alpine wetland (Fig. 1(b)).

The N2O gas fluxes fluctuated temporally andoccasionally changed their signs in the range of �9.24 to20.27 mg N2O m–2 h–1 (Fig. 2). The daily averaged fluxes for thesingle chamber that was used for isotopic measurements werepositive, with values of 0.81, 3.01, and 3.35 mg N2O m–2 h–1

for alpine meadow, alpine shrub, and alpine wetland,respectively, in summer. In winter, these values were 1.30,8.29, and 0.26 mg N2Om–2 h–1 for alpine meadow, alpine shrub,and alpine wetland, respectively. The daily averaged fluxes forall four chamber-averaged values were similar to values for thesingle chamber except for the wetland, which had a negativevalue of �1.05 mg N2O m–2 h–1 in summer. The amplitude oftemporal fluctuations was much smaller in winter than insummer for alpine meadow and wetland.

Isotopomers and an isotopologue in soil-emitted N2O (dsoilvalues) showed large diurnal changes in summer and winterfor alpine meadow, and in winter for alpine wetland (Fig. 3).However, they showed relatively stable values in summerand winter for alpine shrub and in summer for alpinewetland (Fig. 3).

Vertical profile of N2O isotopomers and isotopologue

At the alpine meadow site, the N2O concentration in the soilgas was similar to that in the atmosphere at 0.1 and 0.3 mbelow the surface, but it increased to 2273 ppb at 0.7 m in

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summer (Fig. 4). The ratios of the isotopomers or theisotopologue of N2O in the shallower soil layers were alsosimilar to those in the atmosphere and showed distinct valuesat 0.7 m in summer (d15Nbulk =�7.9%, d15Na=7.3%, d15Nb=�23.2%, d18O= 47.7%, SP=30.5%). In contrast, suchincreases or decreases in concentration and d values at thedeep soil layer were not observed in winter.At the alpine shrub site, the N2O concentration showed a

monotonic increase from the atmosphere (333 ppb) to 0.3 mbelow the soil surface (5483 ppb; sampling was conductedonly in summer). On the other hand, the isotopic signatureshowed a corresponding increase (SP, from 19.3 to 32.1%)or decrease (d15Nbulk values, from 7.9 to �17.6%; d15Na

values from 17.6 to �1.5%; d15Nb values from �1.7 to�33.6%), although d18O in the soil gas at 0.1 and 0.3 m

Copyright © 2013 JRapid Commun. Mass Spectrom. 2013, 27, 1517–1526

showed nearly the same values (38.6% and 39.3%,respectively), which were slightly lower than the value forthe atmosphere (41.9%).

DISCUSSION

Diurnal change in N2O flux and the isotopomers andisotopologue

The observed N2O fluxes showed diurnal variations inseveral cases (Fig. 2). At the alpine shrub site, the N2O fluxwas higher at 14:00 and 18:00 than at 10:00 and 24:00 in thewinter, corresponding with the variation in soil-surfacetemperature (Fig. 1). This might suggest that the microbial

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Figure 3. Diurnal variations in isotopes and isotopomers from soil-emitted N2O gas at the alpinemeadow, alpine shrub, and alpine wetland sites in summer 2005 (filled circles ●) and winter 2006(open squares □). Error bars show propagated error calculated from 1s of the isotopomer ratioand concentration analysis.

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activity that produces N2O depends on temperature. In othercases, however, the N2O fluxes were not correlated withsurface temperature or soil moisture.The ratios of the N2O isotopomers or isotopologue also

showed temporal variations with larger amplitudes especiallyat the alpine meadow site and alpine shrub site (Fig. 3).

wileyonlinelibrary.com/journal/rcm Copyright © 2013 John Wil

However, attention should be paid to the uncertainty of thedata because the amplitude of flux is basically small, as shownin Fig. 2 and mentioned in previous reports of studies at thesame sites.[8] Therefore, the calculated d values for soil-emitted N2O have large uncertainty as represented by thelarge error bars in their diurnal courses in Fig. 3. At the alpine

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Figure 4. Vertical profile of the N2O concentration andisotopic signature in the atmosphere (2.2 m) and the soillayers (�0.1, –0.3, and �0.7 m) at the alpine meadow andalpine shrub sites in summer 2005 (filled circles●) and winter2006 (open squares □).

N2O isotopomers in alpine ecosystems

152

shrub site, the d values were almost constant both in thesummer and winter, probably indicating that the productionand consumption pathways changed little. Thus, we consider

Copyright © 2013 JRapid Commun. Mass Spectrom. 2013, 27, 1517–1526

that no diurnal changes in N2O isotopic signatures could bedetected in soil-emitted N2O in this chamber-based approachpartly due to the very low N2O flux.

Vertical profile of the N2O concentration, isotopomers, andisotopologue

Direct measurement of N2O in the soil gas revealed that thesummer vertical profile had significantly high N2Oconcentrations and high or low isotopic signatures in thedeeper soil layers, 0.7 m and deeper in alpine meadow and0.3 m and deeper in alpine shrub (Fig. 4). This suggests thatN2O is intensively produced in the deeper layers rather thanin the shallower layers, and that the produced N2O can betransported to the shallower layers or atmosphere bydiffusion. Because the negative flux (i.e., atmosphere to soils)was observed on several occasions (Fig. 2), consumption ofN2O also could have occurred in the shallow layers. The lackof gradient in the concentration and isotopic signature in thewinter vertical profile of N2O at the alpine meadow siteindicates that there was no significant microbial activity inN2O production.

In order to discuss the production and consumptionprocesses of N2O, we estimated the isotopic signature of thesoil N2O source using the relationship between d valuesand the inversed concentration (i.e., Keeling plot; Fig. 5). Insummer, the d15Nbulk and SP values in the atmosphere,soil-emitted gas (chambers), and soil layers show significantcorrelation with the inverse concentration (determinantcoefficient [r2] >0.80), except for SP at the alpine wetlandsite. This suggests that the observed N2O concentrationand isotopic signatures can be explained by mixing of twocomponents (two end-members): atmospheric N2O andsoil-produced N2O. The isotopic signature of the latter wasestimated from the y intercept of the regression line, andvalues of �12.7, –17.6, and �2.4% for d15Nbulk, and 33.7,30.1, and 23.1% for SP, were obtained at the alpine meadow,shrub, and wetland sites, respectively.

Production and consumption processes of N2O in alpinesoils

The estimated values of SP and d15Nbulk of N2O produced inthe alpine soils are plotted in Fig. 6 together with literaturedata. Our data have conspicuously high SP values, especiallyfor alpine meadow and shrub soils (33.7 and 30.1%,respectively). These values are close to the SP values reportedfor N2O produced by nitrification (oxidation of NH2OH,about 33%, Sutka et al.[18–20]) or fungal denitrification (37%,Sutka et al.[21]), rather than by bacterial denitrification ornitrifier-denitrification (reduction of nitrite, about 0%, Sutkaet al.[18–20] and Toyoda et al.[14]). Therefore, assuming thatnitrification does not occur in deep soils that are notcultivated nor fertilized and that N2O reduction is also notoccurring, the N2O in the alpine meadow and shrub soilswould be produced predominantly by fungal denitrification.In the case of alpine wetland soil, the estimated SP value(23.1%) could be explained by the combination of fungaldenitrification and bacterial denitrification because the latterprocess can be activated under the anaerobic conditioninduced by waterlogging and because nitrification is likelyto be suppressed under anaerobic conditions.

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3

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Figure 5. Relationship of d15Nbulk and SP values against the reciprocal of the N2Oconcentration in the atmosphere (2.2 m; filled circles ●) and the soil gas emitted from thesurface (chamber, open squares □) and the soil layer (�0.1, –0.3, and �0.7 m; filleddiamonds ◆) at the alpine meadow, alpine shrub, and alpine wetland sites in summer 2005.

T. Kato et al.

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However, we cannot exclude the possibility of othermechanisms of N2O production because high SP values arealso observed when N2O is partly reduced by denitrifying

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Figure 6. Correlation diagram of site preference (SP) andd15Nbulk values at the three sites in summer 2005 and at varioussites from the literature. Literature values are averages ofmultiple measurements at each site. Crop (field) consists ofvalues from Perez et al.,[28] Park et al.,[29] and Toyoda et al.;[15]

crop (incubation) consists of values from Well et al.;[30,31]

grassland (field) consists of values from Yamulki et al.,[32]

Opdyke et al.,[25] and Ostrom et al.;[33] grassland (incubation)consists of values from Bol et al.[34] and Cardenas et al.;[35] forest(field) consists of values from Well et al.[36] and Park et al.;[29]

and forest (incubation) consists of values from Perez et al.[37]

wileyonlinelibrary.com/journal/rcm Copyright © 2013 John Wil

bacteria.[22] Hence, we further applied the computationalanalysis of production and reduction of N2O based onisotopic data as presented by Toyoda et al.[15]

Assuming that fungal denitrification and bacterialdenitrification are the major two end-members of N2Osources in the alpine soils, the degree of N2O reduction (Fr)and the relative fraction of fungal denitrification to the grossN2O production (x) were estimated from SP and d15Nbulk

values of soil N2O (see the previous subsection). In addition,the d15N value of nitrate in the soil (�27 to approximately�11%, based on the d15N value of organic nitrogen measuredfor soils between 0 and 0.1-m depth and the reportedenrichment factor for conversion of ammonium into nitrate)was estimated using the procedure explained in theExperimental section. Other input parameters required forthe calculation were taken from Toyoda et al.[15]: the SP valuesof N2O produced by fungal and bacterial denitrificationswere 37.0� 2.6% and �0.40� 5.50%, respectively; the15Nbulk-enrichment factor e(15Nbulk) for N2O produced byfungal or bacterial denitrification was �26� 11%; thee(15Nbulk) for N2O reduction was �9.8� 6.0%; and the ratioof e(SP) to e(15Nbulk) for N2O reduction was 0.86� 0.36. Theaveraged value of x obtained from 1000 iterations of MonteCarlo calculations was 40.7%, 40.0%, and 23.2% for alpinemeadow, alpine shrub, and alpine wetland soils, respectively(Table 1). The averaged Fr values were 87.6%, 82.9%, and92.7% for alpine meadow, alpine shrub, and alpine wetlandsoils, respectively (Table 1).

Alpine meadow and shrub showed moderate contributionsby fungal denitrification of 40.7% and 40.0%, respectively, tototal N2O production. Recent studies[23,24] have also shown

ey & Sons, Ltd. Rapid Commun. Mass Spectrom. 2013, 27, 1517–1526

Table 1. Fractional contributions to the N2O productionpathway of two sources (fungal denitrification andbacterial denitrification) and N2O reduction at the Haibeistation site in the summer of 2005

x (%; mean�standarddeviation; share of

fungal denitrificationbetween the

two productionpathways)

Fr (%; mean� standard

deviation; fractionof N2O reduction)

Alpine meadow 40.7� 22.6 87.6� 14.8Alpine shrub 40.0� 21.3 82.9� 18.5Alpine wetland 23.2� 17.2 92.7� 10.0

N2O isotopomers in alpine ecosystems

that fungal denitrification was significant in N2O productionin alpine ecosystems. On the other hand, this statisticalanalysis suggests that bacterial denitrification contributedlargely, by 59.3% and 60.0%, in alpine meadow and shrub,respectively, similar to the relatively high contributions bybacterial denitrification reported from N2O isotopomericmeasurement studies, such as in temperate agricultural soilsin Michigan, USA (61–92%[25]), and in temperate coniferousforests in Japan (96.8%[26]). Finally, we can conclude that theN2O emitted from the soil surface was conveyed by diffusionfrom deeper soil layers, in which the N2O produced by bothfungal and bacterial denitrifications was greatly reduced.The alpine wetland had a lower contribution of fungal

denitrification (23.2%) than the predominant contribution of81% found in peat incubation in tropical wetland soil withrespiration-inhibiting antibiotics in Indonesia.[27] It can beconcluded that the N2O gas was produced predominantly bybacterial denitrification, and also that the produced N2O gaswas strongly eliminated in the soil layers by N2O reduction.

152

CONCLUSIONS

This study provided the isotopic signature of N2O produced inthe soils of three alpine ecosystems in the QTP, which addednew insights into the production mechanisms of N2O in thesehardly explored environments. The vertical profile of N2Oconcentration and isotopomer or isotopologue ratios suggestedthat N2O is produced in the deeper soil layers (about 0.7 mbelow the surface), where significant contributions by fungaldenitrification to gross N2O production were expected basedon the high SP values in alpine meadow and alpine shrub sites(33.7% and 30.1%, respectively). Our results also showed thestraight vertical profile of N2O concentration and isotopomeror isotopologue ratios in an alpine meadow in winter, andindicated that there was much less or no activity of N2Oproduction because of the cold conditions.The relative contributions of N2O production pathways

and N2O reduction were estimated statistically by the MonteCarlo method, assuming that there were two dominant N2Oproduction pathways: fungal and bacterial denitrification.The N2O production in the soil appeared to be moderatelycontrolled by fungal denitrification (23.2–40.7%) and stronglycontrolled by bacterial denitrification in the three alpineecosystems (59.3–76.8%). Under the above assumption, theproduced N2O gas was estimated to be strongly reduced,

Copyright © 2013 JRapid Commun. Mass Spectrom. 2013, 27, 1517–1526

ranging from 87.6 to 92.7%. This supported our observationsof the high N2O concentration in deeper soil and smaller N2Oflux at the soil surface. Thus, the combined measurement ofN2O concentration,N2O flux, andN2O isotopomeric signaturescould provide a robust estimation of N2O-circulation dynamicsin alpine ecosystems on the QTP. However, the N2Oisotopomeric measurements have high uncertainty due to thelack of iterative evidence of temporal isotopic change,especially in the diurnal cycle, and the lack of isotopicmeasurement on N2O substrates. Further investigations areneeded to both qualitatively and quantitatively clarify theinteraction between production pathways and substrates, andthe reduction contribution.

AcknowledgementsThis study was supported by JSPS Grant-in-Aid for ScientificResearch (Kiban-A, No. 19201006) from the Ministry ofEducation, Science, Culture, Sports and Technology of Japan.The authors thankAssociate Prof. DrK. Yamada andDrA. Fujii,and other members in Prof. N. Yoshida’s laboratory in theDepartment of Environmental Chemistry and Engineering,Tokyo Institute of Technology, for providing help andinstruments for isotopomer measurement of N2O gas. Theauthors also thank Dr Y. Yashiro, River Basin ResearchCenter, Gifu University, and Prof. H. Toda, Faculty ofScience, Shinshu University, for providing the instrumentsand advice for measuring N2O gas concentrations.

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