氮沉降

See discussions, stats, and author profiles for this publication at: https:www.researchgate.netpublication229325081Low δ 18 O Values of Nitrate Produced fromNitrification in Temperate Forest SoilsArticle in Environmental Science Technology · July 2012DOI: 10.1021es300510r · Source: PubMedCITATIONS15READS1037 authors, including:Some of the authors of this publication are also working on these related projects:nitrogen deposition View projectYunting FangInstitute of Applied Ecology, Chinese Academ91 PUBLICATIONS 1,617 CITATIONS SEE PROFILEKeisuke KobaKyoto University124 PUBLICATIONS 2,059 CITATIONS SEE PROFILEXueYan LiuTianjin University31 PUBLICATIONS 307 CITATIONS SEE PROFILEMuneoki YohUniversity of Tennessee73 PUBLICATIONS 809 CITATIONS SEE PROFILEAll content following this page was uploaded by XueYan Liu on 19 January 2014.The user has requested enhancement of the downloaded file. All intext references underlined in blue are added to the original documentand are linked to publications on ResearchGate, letting you access and read them immediately.Lowδ18OValuesofNitrateProducedfromNitrificationinTemperateForestSoilsYuntingFang,†,‡KeisukeKoba,†,AkikoMakabe,†FeifeiZhu,†,ShaoyanFan,†XueyanLiu,†andMuneokiYoh††GraduateSchoolofAgriculture,TokyoUniversityofAgricultureandTechnology,Saiwaicho358,Fuchu,Tokyo1838509,Japan‡StateKeyLaboratoryofForestandSoilEcology,InstituteofAppliedEcology,ChineseAcademyofSciences,Shenyang110164,ChinaKeyLaboratoryofVegetationRestorationandManagementofDegradedEcosystems,SouthChinaBotanicalGarden,ChineseAcademyofSciences,Guangzhou510650,ChinaSSupportingInformationABSTRACT:Analysesofδ18Oofnitrate(NO3−)havebeenwidelyusedinpartitioningNO3−sources.Howevertheδ18OvalueofNO3−producedfromnitrification(microbialNO3−)iscommonlyestimatedusingtheδ18Oofenvironmentalwaterandmolecularoxygenina2:1ratio.Hereourlaboratoryincubationofninetemperateforestsoilsacrossa1500melevationgradientdemonstratesthatmicrobialNO3−haslowerδ18Ovaluesthanthepredictedusingthe2:1ratio(by5.2−9.5‰atlowelevationsites),incontrasttopreviousreportsshowinghigherδ18Ovalues(upto+15‰)thantheirpredictedvalues.Elevatedδ18OvaluesofmicrobialNO3−wereobservedathighelevationsiteswheresoilwasmoreacidic,perhapsduetoacceleratedOexchangebetweennitrite,anintermediateproductofnitrification,andwater.Lowerδ18OofmicrobialNO3−thanthepredictedandfrompreviousobservationssuggeststhatthecontributionofanthropogenicNinputs,suchasfertilizerandatmosphericdeposition,toagivenecosystemandtheprogressofdenitrificationinnitrogenremovalaregreaterthanweknow.Morethanhalfoftheδ18OofstreamNO3−lowerthanthepredictedvaluealongtheelevationgradientalsoindicatetheimproprietyusingthe2:1ratiofordifferentiatingNO3−sources.INTRODUCTIONTheglobalnitrogen(N)cyclehasbeendisruptedbytheNreleaseassociatedwithhumanactivitiesincludingfossilfuelcombustion,agriculturalfertilization,andNfixingplantcultivation.1−3Consequently,itisimportanttoconstrainNsourcesandsinksintheenvironment.Analysisofstableisotoperatios(δ15Nandδ18O)ofnitrate(NO3−)hasbeenwidelyusedtodifferentiatesourcesofNO3−ingroundwater(e.g.,ref4)andsurfacewater(e.g.,refs5−8),andtoconstraintheoceanicNbudget.9However,theδ18OvaluesofNO3−producedfrommicrobiologicallymediatednitrification(microbialNO3−)wereoftennotmeasured,andinsteadwerecommonlyestimatedusingbiochemicalstoichiometry,thatis,reflectingtheisotopiccompositionoftheenvironmentalwater(H2O)andatmosphericmolecularoxygen(O2)ina2:1ratio,inmostofthesestudies(e.g.,refs5,10,and11).SuchestimatesarebaseduponknowledgeofthefractionalcontributionsofOsourcesduringchemolithoautotrophicnitrification.12−14Withδ18Ovaluesforwatersinthenormalrangeof−25‰(relativetoViennaStandardMeanOceanWater(VSMOW))to+4‰,andwiththeδ18OvalueforsoilO2ofabout+23.5‰(atmosphericO2),theδ18OvalueofsoilmicrobialNO3−producedfrominsitunitrificationispredictedtobebetween−10‰and+10‰.15Afewstudieshavemeasuredδ18OvaluesofsoilmicrobialNO3−,butactuallyfoundthatthemeasuredvaluesareevenmorepositive(upto+15‰)thanthevaluespredictedusingbiochemicalstoichiometry(inthe2:1ratio).Moreover,themeasuredvaluesareoftenhigherthantheδ18OvaluesofNO3−ingroundorsurfacewaters(seeref16).Theutilizationofsuchasimple2:1ratioaboveforcalculationoftheδ18Oinvolvesanassumptionthatexchangeandfractionationofoxygenisotopesduringnitrificationareminimal.Thisassumptioncontraststotherecentworksshowingthatlargekinetic18OisotopeeffectsareassociatedwiththeincorporationofO2andH2Oinlaboratorypureculturestudiesusingmarinenitrifyingbacteria.17,18Inmostcases,kineticisotopicfractionationengenderspreferentialreactionorincorporationofmoleculescontaininglightisotopes,whichwilllowertheδ18OvalueofNO3−producedReceived:February10,2012Revised:June17,2012Accepted:July19,2012Published:July19,2012Articlepubs.acs.orgest©2012AmericanChemicalSociety8723dx.doi.org10.1021es300510r|Environ.Sci.Technol.2012,46,8723−8730throughnitrificationfromthepredictedvalueusingthe2:1ratio.Inaddition,Oexchangebetweennitrite(NO2−),anintermediateproductofnitrification,andH2Oislikelytooccurduringnitrification,whichmighteliminatetheisotopiccompositionofincorporatedO2.16−19Arecentstudyusing18Olabeledwaterincubationsshowsvariabledegrees(37−88%)ofOexchangeinagriculturalandforestsoils.16Takingthesefactorsintoaccount,forexample,theδ18OvaluesofNO3−producedduringnitrificationinseawaterwithaδ18Oof0‰arecalculatedas−8.3‰to−0.7‰,beingmuchlowerthanthosepredictedbythe2:1ratio(+7.8‰to+12‰)andmuchlowerthanthoseofdeepoceanNO3−(+1.5‰to+2.5‰;18).Therefore,itisimportanttoascertainhowmuchlowerorhighertheδ18OvaluesofmicrobialNO3−arethanthepredictedvaluesinthefieldsettings.ItisalsoimportanttoelucidatetheextenttowhichOexchangeaffectsδ18OvaluesofmicrobialNO3−.ThisinformationiscrucialforevaluatingthecontributionofanthropogenicNinputsandNprocessessuchasnitrificationanddenitrification.Inthisstudy,weconductedalaboratoryincubationofarangeoftemperatesoilsalonganelevationgradientincentralJapanandusedthedenitrifiermethodforanalyzingNO3−isotopes.Wefoundmuchlowerδ18OvaluesofNO3−producedfromnitrificationintheinvestigatedsoilsthanthepredictedusingthe2:1ratioasmentionedabove.Weexamineddifferentscenariosofisotopiceffectsofoxygenatomincorporationandexchangewithanattempttointerpretthemeasuredδ18Ovalues.Inaddition,welinkedtheδ18OvaluesofsoilmicrobialNO3−totheδ18OvaluesofstreamwaterNO3−andofatmosphericallydepositedNO3−toseehowmuchthecontributionofatmosphericallydepositedNO3−couldbeunderestimatedifthe2:1ratiowasused.EXPERIMENTALSECTIONCollectionandProcessingofSoilandStreamwaterSamples.Alonganelevationgradientoverabout1500mfrom223to1688mabovethesealevelintheheadwaterareaoftheTamaRiverincentralJapan,nineforestsiteswereselectedonSeptember29,2011forsoilcollection,withthreesitesinlow,middle,andhighelevations,respectively(SupportingInformationFigureS1,Table1).Alongthisgradient,apreviousstudyshowsthatstreamNO3−concentrationvariedwitharangeofnearly100fold.20Therefore,theselectedforestsareexpectedtoencompassalargerangeofNstatus.Oftheninesites,onesite(SiteE,Table1)wasadeciduousbroadleafforest,andeightsiteswereconiferousforestwithJapanesecedar(Cryptomeriajaponica)dominantinthelowandmiddleelevations(sitesA−D,andF)andJapaneseLarch(Larixkaempferi)inthehighelevations(sitesG−H).The0−5cmmineralsoilsfromthemiddlepartofslopeateachsitewerecollected.Samplingwasperformedaweekafterastrongrainevent(220mm,datafromJapanMeteorologicalAgency,http:www.jma.go.jpjmaindexe.html).Consequently,soilshadmoderatemoisture(0.41−1.20gH2Og−1soildw.,Table1),whichwasexpectedtofacilitateNO3−productionandreducetheoccurrenceofdenitrificationinthelaboratoryincubation.Threecompositesoils(eachfrom3−5samplingpoints)ineachforestsitewereobtainedafterremovaloftheorganiclayer.Soilsampleswerekeptincoolingbags.Atthesametime,foursoilcoresfromeachsiteweretakentodeterminethesoilbulkdensityandthefractionofsoil<2mm.Inthelaboratory,soilsweresievedimmediatelybypassingthrough2mmsievesforlateranalyses.Twodayslater,10gofthesievedsoilswereextractedwith50mLdeionizedwater.Aftershakingfor1handcentrifugingfor10min,thesupernatantwasfilteredusingglassfilters(GFF;WhatmanInt.Ltd.,Maidstone,UK)thathadbeenpreviouslymuffledat450°Cfor4htoreduceNblankbeforeuse.ThesoilextractwasdeterminedforionconcentrationandtheNandOisotopesofNO3−.TheconcentrationsofNH4+andNO3−insoilextractweredeterminedusingionchromatography(DionexDX120;DionexCorp.).Subsampleswereovendriedtoconstantweightat60°Candgroundusingaballmill,andweremeasuredforconcentrationsofC,N,and15NnaturalabundanceusingEAIRMS(EA1112coupledwithDeltaXP;ThermoFisherScientificK.K.,Yokohama,Japan).Subsampleswerealsoovendriedtoconstantweightat105°Ctoobtaininitialwatercontents.Streamwaterclosetoeachsoilsamplingsite(usuallywithin200m)wassampledonthedaysoilswerecollected.Tocovertheseasonalityandtohavemorerepresentativesites,25streamwatersamplingsitesalongthesameelevationgradientincludingtheninesitesforsoilsamplingwereset(SupportingInformationFigureS1).InadditiontothesamplinginSeptember2011,streamwaterwasalsocollectedinMayandSeptemberin2009andinMarchandSeptemberin2010usingprerinsedpolypropylenesyringes.Thewaterwasthenfilteredinthefieldthrough0.45μmfiltersinto50mLbottlesprerinsedTable1.SoilProperties(0−5cmMineralSoil)andStreamNO3−ConcentrationfromNineTemperateForestsalongtheElevationGradientinCentralJapansiteselevation(m,a.s.l)bulksoildensity(gcm−3)<2mmsoildensity(gcm−3)gravimetricsoilmoisture(gH2Og−1soildw.)soilpH(H2O)soilC(%)soilN(%)soilCNasoilNO3−(mgNkg−1)bnetnitrification(mgNkg−1day−1)streamNO3−(mgNL−1)A2230.93(0.05)0.37(0.00)0.75(0.10)5.9(0.0)8.9(0.9)0.65(0.04)13.6(0.7)9.0(1.8)1.2(0.1)0.8B2490.92(0.05)0.47(0.07)0.41(0.01)5.4(0.1)5.9(1.0)0.39(0.05)14.9(0.9)6.8(0.6)1.3(0.2)1.4C2990.86(0.06)0.45(0.03)0.81(0.00)5.5(0.2)12.4(1.1)0.79(0.06)15.8(0.4)11.7(2.0)1.8(0.1)1.2D7220.67(0.08)0.34(0.05)0.84(0.13)5.7(0.3)13.8(2.5)1.02(0.15)13.4(0.6)7.7(2.1)1.4(0.3)0.3E7580.36(0.03)0.29(0.02)1.00(0.15)5.1(0.2)16.1(2.9)1.10(0.18)14.6(1.2)4.2(0.8)1.5(0.3)0.5F9000.60(0.07)0.28(0.02)0.70(0.10)5.4(0.1)11.3(1.6)0.90(0.15)12.6(0.3)3.7(1.0)1.3(0.2)0.2G12710.44(0.03)0.40(0.02)0.90(0.02)4.7(0.1)15.9(0.5)1.02(0.04)15.5(0.5)3.0(0.4)1.4(0.2)0.1H12890.42(0.03)0.38(0.03)1.20(0.08)4.4(0.0)18.0(1.0)1.09(0.06)16.6(1.4)6.0(0.5)2.9(0.2)0.2I16680.49(0.05)0.32(0.02)0.78(0.05)4.4(0.1)17.2(1.7)1.24(0.10)13.8(0.7)3.3(0.3)1.7(0.1)0.1Pc0.0070.2270.0260.002<0.001<0.0010.7630.0200.2510.003aOnaweightbasis.bBeforeincubation.cPvaluesoftheregressionsagainstelevationacrossthenineforestsites.Shownaremeansandstandarderrorsofthemeansinparentheses;n=4forsoildensityand3forothervariables.EnvironmentalScienceTechnologyArticledx.doi.org10.1021es300510r|Environ.Sci.Technol.2012,46,8723−87308724threetimeswiththefiltrate.Thesampleswerekeptincoolingbagsduringthesampling.TheconcentrationsofNH4+andNO3−instreamwaterweredeterminedusingthesamemethoddescribedaboveforsoilextract.SoilIncubation.Twodaysaftersampling,about10goffreshlysievedsoilfromeachcompositesamplewasputintoaglassjar(120mL)andincubatedataconstanttemperateof25°Cforaweek.Duringthelaboratoryincubation,thejarswereopenforabout30minevery1−2daystopreventtheoccurrenceofdenitrification.Wedidnotaddmorewatertokeepthesoilmoistureconstantduringtheincubationortoadjusttoagivenmoisturecontent(e.g.,60%ofthewaterholdingcapacity)sothattheoxygensourcefromsoil−waterwasexpectednotbegreatlyaltered.Toexaminetheinfluenceofwaterlossduringtheincubation,soilmoisturewasmeasuredimmediatelyafterincubation.Resultsshowthatonly1.9±0.1%ofthesoil−waterwaslostduringincubation.Consequently,suchatinyamountofwaterlosshadaminorinfluenceon18Oofsoil−water.Incubatedsoilswereextractedandionconcentrationsinsoilextractweredeterminedinthesamewayasdescribedabove.ThenitrificationratewascalculatedasthedifferenceinNO3−concentrationbetweenthosemeasuredbeforeandafterincubation.NitrateIsotopeAnalyses.Wechosethedenitrifiermethod21,22todeterminetheδ15N(versusairN2)andδ18O(versusVSMOW)ofNO3−insoil−waterextractandstreamwaterbecausepreconcentrationandpurificationofNO3−arenotnecessarywhenusingthismethod.TheN2OproducedbydenitrifiersfromNO3−wasintroducedintoanisotoperatiomassspectrometer(DeltaXP;ThermoFisherScientificK.K.,Yokohama,Japan)coupledwithagaschromatograph(HP6890;HewlettPackardCo.,PaloAlto,CA)equippedwithaPoraplotcolumn(25m×0.32mm)andGCinterfaceIII(ThermoFisherScientificK.K.,Yokohama,Japan).TheNandOisotopiccompositionwasmeasuredforN2O.Weranseveralstandards(USGS32,34,35andIAEANO3−)toobtainthecalibrationcurvefordrift,oxygenisotopicexchange,andblank.23Theaveragestandarddeviationsforreplicateanalysisofanindividualsamplewere±0.2‰forδ15NinNO3−and±0.5‰forδ18O.Theδ15Nandδ18OvaluesforNO3−newlyproducedfromnitrificationwerecalculatedusingthefollowingequation.δδδ=×−×−−−−−−−−EEE[(NOconcentration)(NOconcentration)](NOconcentrationNOconcentration)iiiNO3newlyproducedNO3afterincubation3afterincubationNO3beforeincubation3beforeincubation3afterincubation3beforeincubation(1)Therein,iEis15Nor18O.AsthedenitrifierusedinthedenitrifiermethodcanconvertNO2−toN2OandNO2−isreadytoexchangeOatomswithwater,thepresenceofNO2−cancauseinterferenceonisotopeanalysisofNO3−.22However,noNO2−wasfoundinsoilextracteitherbeforeorafterincubationinourstudy.ModelExerciseofIsotopicEffectsonδ18OofNO3−ProducedfromNitrification.InanattempttoseehowandtowhatextentofkineticisotopiceffectsandOexchangepotentiallyaffectδ18OvaluesofNO3−producedfromnitrification,weexaminedthreescenarios.Asdescribedinpreviousreports(e.g.,refs16−18),nitrificationoccursasatwostepprocess;oxidizingammonia(NH3)toNO2−byammoniaoxidizingbacteria(AOB)andammoniaoxidizingarchaea(AOA)asthefirststepandoxidizingNO2−toNO3−byNO2−oxidizingbacteria(NOB)asthesecondstep.O2isincorporatedduringtheoxidationofNH3tohydroxylamine(NH2OH),whereasH2OisincorporatedduringtheoxidationofbothNH2OHtoNO2−andNO2−toNO3−.12−14Duringthenitrificationprocess,fivekineticisotopeeffects(18εk=(18k16k−1)×1000)cantakeplace:(1)selectionofNH2OH(18εk,NH2OH),(2)selectionofNO2−(18εk,NO2),(3)incorporationofoxygenfromO2(18εk,O2),and(4)and(5)incorporationofoxygenfromH2OduringNH2OHoxidationandNO2−oxidation(18εk,H2O,1and18εk,H2O,2,respectively,seeref17).Inadditiontokineticisotopeeffectsduringoxygenatomincorporation,theexchangeofoxygenatomsbetweenNO2−andwatercatalyzedbyammoniaoxidizingbacteria(xAOB)andnitriteoxidizingbacteria(xNOB)islikelytoincreasethedependenceofδ18ONO3‑onδ18OH2Oandloweritsdependenceonδ18OO2.ThefirsttwokineticisotopeeffectscouldbeofminorimportanceasthereactionsofNH2OHandNO2−oxidationarenotratelimiting.17AndxNOBisshowntobeminimalinarecentstudy(<3%,18).ConsideringkineticisotopeeffectsduringOincorporation,theOexchangeinthefirststepofnitrification(xAOB),theequilibriumisotopeeffectsduringtheOexchange(18εeq),δ18ONO3‑canbedescribedbyeq2.16δδεδεδεδε=+++−++++−xxO23{[12(O)12(O)](1)(O)}13(O)kkk18NO318O2,O21818H2O,H2O,118AOB18H2Oeq18AOB18H2O,H2O,218(2)Rearrangingeq2togrouptermscontainingoxygensources,kineticisotopeeffectsandeffectsofOexchangeinordergiveseq3.δδδεεεδδεεε=+++++−+−−−xO13O23O13()13(OO2)kkkkk18NO318O218H2O18,O2,H2O,118,H2O,21818H2O18O218eq,O218,H2O,118AOB(3)IgnoringanykineticisotopeeffectsandOexchange(i.e.,the2:1ratio,Scenario1),δ18ONO3−canbecalculatedbyeq4.δδδ=+−O13O23O18NO318O218H2O(4)WhennoOexchange(i.e.,xAOB=0,Scenario2)occurs,thenδ18ONO3‑canbecalculatedusingeq5.δδδεεε=++++−O13O23O13()kkk18NO318O218H2O18,O2,H2O,118,H2O,218(5)WhencompleteOexchange(i.e.,xAOB=1,Scenario3)occurs,thenδ18ONO3‑canbecalculatedusingeq6.δδεε=++−OO1323k18NO318H2O18,H2O,218eq(6)EnvironmentalScienceTechnologyArticledx.doi.org10.1021es300510r|Environ.Sci.Technol.2012,46,8723−87308725Inthepresentstudy,theδ18Ovaluesofsoil−waterhadnotbeendetermined.Themonthlymeansofδ18OvaluesofprecipitationinsummerandautumninTokyowereobservedto−6‰to−8‰(WaterIsotopeSystemforDataAnalysis,Visualization,andElectronicRetrieval,http:wwwnaweb.iaea.orgnapcihIHSresourcesisohis.html).Usingthisrangeforsoil−water,theδ18OvaluesofmicrobialNO3−canbepredictedtobe+2.5‰to+3.8‰bythe2:1ratio(Scenario1),ifweassumethatsoilO2hasaconstantδ18Ovalusof+23.5‰.24Usingmarinenitrifyingbacteria,thecombinedisotopicfractionationforoxygenatomincorporation(18εk,O2+18εk,H2O,1)wasestimatedas−37.6‰to−17.9‰duringNH3oxidation,thefirststepofnitrification,17andtheisotopicfractionationforoxygenatomincorporation(18εk,H2O,2)wasestimatedas−18.2‰to−12.8‰duringNO2−oxidation,thesecondstepofnitrification.18The18εeqwasshownto+14.4‰(nearnaturalpHand21°C,25).Usingthesereportedfractionationfactors,thenδ18ONO3‑werecalculatedas−16.1‰to−6.4‰and−4.5‰to−0.7‰,respectively,forScenarios2and3(Figure2).RESULTSANDDISCUSSIONSoilGeneralPropertiesandNetNitrificationRates.ThesoilsforlaboratoryincubationhadalargerangeofCandNconcentrationsandrelativelyconstantC:Nratios(Table1).Aselevationincreased,soilCandNconcentrationsincreased,whereassoilbulkdensityandsoilpHdecreased(Table1).Soil−waterextractableNO3−concentrationandstreamwaterNO3−concentrationdecreasedsignificantlywithincreasingelevation(Table1),suggestingthatNcyclingwasmoreopeninlowelevationsitesthanhighelevationsites.ThenetratesofsoilNO3−production(1−3mgNkg−1day−1,Table1)showednodifferencealongtheelevation.NitrogenandOxygenIsotopicCompositionofMicrobialNO3−.Theδ15NvaluesofNO3−insoil−waterextractbeforeincubationvariedfrom−8.5‰to−3.5‰,withanaverageof−5.0‰(Figure1a).Theδ15NvaluesofNO3−newlyproducedfromnitrificationduringthelaboratoryincubation(i.e.,microbialNO3−)wassimilartothoseofsoilextractatthreelowelevationsites,butdivergedatmiddleandhighelevationsites(Figure1b).Asaresult,δ15NvaluesofNO3−newlyproducedincreasedsignificantlywithelevation(Figure1b).Wefoundthat15NabundanceofnewlyproducedNO3−largelyreliedonthatofbulksoilN,withδ15Nvaluesbeingabout5‰lowerregardlessofelevation(Figure2b).Theδ18OvaluesofNO3−newlyproducedfromnitrificationrangedfrom−9.3‰to+2.9‰,withanaverageof−4.0±0.4‰(Figure2b).ThisrangecloselyresembledtherangeforthesoilextractNO3−beforeincubation,whichwasbetween−8.7‰and−0.9‰,withanaverageof−4.1±0.4‰(Figure2a).Consequently,theδ18OvaluesofNO3−newlyproducedfromnitrificationwerecorrelatedsignificantlywiththoseofsoilextractNO3−(R2=0.65,P<0.0001).Becausethesoilsusedforwaterextractwerecollectedaweekafteraheavyrainandkeptincoolerfortwodaysaftersampling,itcanbeexpectedthat