Table Of Content

ScienceoftheTotalEnvironment631–632(2018)1342–1349 ContentslistsavailableatScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv Salinity is a key factor driving the nitrogen cycling in the mangrove sediment HaitaoWanga,b,c,d,JackA.Gilbertd,YongguanZhua,b,XiaoruYanga,b,⁎ aInstituteofUrbanEnvironment,ChineseAcademyofSciences,Xiamen,FujianProvince361024,China bCenterforExcellenceinRegionalAtmosphericEnvironment,InstituteofUrbanEnvironment,ChineseAcademyofSciences,China cSchoolofLifeSciences,XiamenUniversity,Xiamen,FujianProvince361102,China dTheMicrobiomeCenter,DepartmentofSurgery,UniversityofChicago,Chicago,IL60637,USA H I G H L I G H T S G R A P H I C A L A B S T R A C T • Salinityelevationdecreasedboththeac- tivityandabundanceofdenitrifiers. • Thenitrifiers(AOB)weremostabun- dant under the intermediate salinity conditions. • Salinitysignificantlyshapedthenitrify- inganddenitrifyingcommunitystruc- tures. • Thesefindingsmayincreaseconcernre- gardingthreatsofsalinityintrusion. a r t i c l e i n f o a b s t r a c t Articlehistory: Coastalecosystemsarehotspotsfornitrogencycling,andspecificallyfornitrogenremovalfromwaterandsed- Received9January2018 imentthroughthecouplednitrification-denitrificationprocess.Salinityisgloballyimportantinstructuringbac- Receivedinrevisedform5March2018 terialandarchaealcommunities,buttheassociationbetweensalinityandmicrobially-mediatednitrificationand Accepted9March2018 denitrificationremainsunclear.Thedenitrificationactivityandcompositionandstructureofmicrobialnitrifiers Availableonlinexxxx anddenitrifierswerecharacterizedacrossagradientofmanipulatedsalinity(0,10,20and30ppt)inamangrove sediment.SalinitynegativelycorrelatedwithbothdenitrifyingactivityandtheabundanceofnirKandnosZ Editor:JayGan denitrifyinggenes.Ammonia-oxidizingbacteria(AOB),whichdominatednitrification,hadsignificantlygreater Keywords: abundanceatintermediatesalinity(10and20ppt).However,apositivecorrelationbetweenammoniaconcen- Salinity trationandsalinitysuggestedthatnitrifyingactivitymightalsobeinhibitedathighersalinity.Thecommunity Nitrification structureofammonia-oxidizingarchaea(AOA)andbacteria(AOB),aswellasnirK,nirSandnosZdenitrifying Denitrification communities,wereallsignificantlycorrelatedwithsalinity.Thesechangeswerealsoassociatedwithstructural Abundance shiftsinphylogeny.Thesefindingsprovideastrongevidencethatsalinityisakeyfactorthatinfluencesthenitro- Communitystructure gentransformationsincoastalwetlands,indicatingthatsalinityintrusioncausedbyclimatechangemighthavea broaderimpactonthecoastalbiospheres. ©2018ElsevierB.V.Allrightsreserved. 1.Introduction Coastalwetlandssuchasmangrovesdominatethetropicalandsub- tropicalcoastlines,butaregloballydisappearing(Dukeetal.,2007). Theseecosystemsare hotspotsof nutrientcyclingandplaypivotal ⁎ Correspondingauthorat:InstituteofUrbanEnvironment,ChineseAcademyof rolesinnutrienttransformationandavailability(FisherandAcreman, Sciences,Xiamen,FujianProvince361024,China. E-mailaddress:[email protected](X.Yang). 2004).Recenteffortshavebeenfocusedondetermininghowclimate https://doi.org/10.1016/j.scitotenv.2018.03.102 0048-9697/©2018ElsevierB.V.Allrightsreserved. H.Wangetal./ScienceoftheTotalEnvironment631–632(2018)1342–1349 1343 change may influence these cycling processes (Duke et al., 2007; nirSandnosZ)wereestimatedbyreal-timequantitativePCR(qPCR). Gallowayetal.,2008;GruberandGalloway,2008),includingtheconse- Thenitrifyingcommunitycompositionandstructure(AOAandAOB) quencesofsalinityintrusionresultingfromgroundwaterabstraction, wasassessedbyclonelibrary,whilehigh-throughputsequencingwas declining sediment loads and rising sea level, on nitrogen cycling performed for denitrifying communities (nirK, nirS and nosZ). This (Bernhardetal.,2005;Bernhardetal.,2007;Franklinetal.,2017;Liu study aims to reveal the impact of salinity on both nitrifying and etal.,2017;Shengetal.,2015;Zhouetal.,2017).Nitrificationanddeni- denitrifying processes and the potential influences of changes in trificationareresponsibleforammoniaoxidationandnitratereduction, couplednitrification-denitrificationonthenitrogentransformations respectively.Thecouplednitrification-denitrificationprocessdrivesthe andecosystemfunctions. nitrogenremovalinwetlands,thuspreventingeutrophication(Fisher andAcreman,2004;Vymazal,2007). Manyeffortshavefocusedonhownitrifyinganddenitrifyingcom- 2.Materialsandmethods munities respond to the salinity elevation in coastal wetlands (Bernhardetal.,2005;Bernhardetal.,2010;Bernhardetal.,2007; 2.1.Incubationexperiment Martonetal.,2012;Shengetal.,2015;Xieetal.,2014;Zhangetal., 2015),yettheanswerremainsambiguous.Ammoniaoxidation,which Thesurfacesediment(0–50cm)fromanunvegetatedareainthe isthefirststepofnitrificationconductedbytheammonia-oxidizingar- mangrovewetlandlocatedinthemudflatofJiulongRiverestuary(24° chaea(AOA)andammonia-oxidizingbacteria(AOB)showsvaryingre- 27′N;117°54′E)wascollectedaspreviouslydescribed(Wangetal., sponses to salinity in different environments. Moderate salinity 2014).Theoriginalsalinityofthesedimentwasaround15ppt(parts- (10–20ppt)isassociatedwithanincreasedabundanceofAOAandpo- per-thousand).Toaccuratelymanipulatetheincubations,sediment tentialnitrificationrates,whileAOBeithershownoornegativecorrela- wasair-dried,groundandsieved(b2mm)beforeexperimentalsetup. tionwithincreasesalinity(Bernhardetal.,2005;Bernhardetal.,2010; ThepHandsalinityofthetreatedsedimentmeasuredassedimentto Bernhardetal.,2007;Zhangetal.,2015).AstudyintheDouroRiveres- waterratioof1:5(g/mL)were7.06and3pptrespectively.Theconcen- tuaryobservedincreasednitrificationratewithasalinityincreasefrom trationsofammonium(NH+)andnitrateandnitrite(NO−)were12.9 4 x 0to15ppt(Magalhaesetal.,2005).Estuarinenitrifiersappeartogrow and1.94μgg−1drysediment,respectively.Thedriedsedimentwas optimallyat5–10ppt,andareinhibitedifsoilsalinityexceeds10ppt keptforthreemonthsbeforethestartoftheincubationexperiment. (Zhouetal.,2017).Thesestudiessuggestthatoptimumnitrification Analiquotof10gdrysedimentwasplacedin120-mLserumbottles rateornitrifierabundancenormallyoccursinmiddlesalinityrangesal- and then 10 mL distilled water was added to each bottle. All the thoughsomevaryingresultsexisted.Increasingsalinitycanimpactni- serumbottlesweresealedwiththewrapswithtinyholestolettheair trification directly by constraining the fitness of nitrifiers and gothroughbutpreventthewaterrunningoff.Thenthesebottleswere indirectlybyinfluencingtheavailabilityofoxygenwhichcanfurther incubatedat25°Cinadarkandaerobicenvironmentforonemonth impactthesoilrespiration(Zhouetal.,2017).However,ammoniaad- torecoverthemicroorganisms.Afterthat,thedifferentsalinitieswere sorptioninsedimentisalsodecreasedwithanincreaseinsalinity,po- manipulated by adding solutions with different concentrations of tentially contributing to increased ammonia efflux, which further NaCl.Theincubationswerecategorizedinto4groupsrepresenting4sa- stimulatesnitrification(Seitzingeretal.,1991;Rysgaardetal.,1999). linitygradients.Eachgroupreceivedcorresponding5-mLNaClsolu- Denitrification,whichreturnsnitrogenbacktotheatmosphereas tions.Thefinalsalinitygradientswere0ppt,10ppt,20pptand30ppt N OandN , also showsdifferent relationships with salinity. Ithas ascalculatedbytheaddedNaCltotheaddedwater(15mLintotalfor 2 2 beenshowntobenegativelyassociatedwithsedimentsalinityinthe eachbottle).InadditiontoNaCl,300μMKNO wasalsoaddedtoeach 3 range of 0–36 ppt in certain estuaries (Giblin et al., 2010; Santoro incubationtostimulatethenitrogenprocess.Afteraddingthesolutions, etal.,2008),butalsotohavenoassociationwithsalinitiesbetween2 allthebottleswereincubatedat25°Cinadarkandaerobicenviron- and24pptinotherplaces(Fearetal.,2005).Denitrifierabundance mentforanotheronemonth.Sedimentsamplesweretakenat0,7,14, andpotentialhavebeenassociatedwithlowsalinitiesaround5ppt 21and28days.Foreachsampling,twoofthreeincubationsofeachsa- (Franklinetal.,2017;Martonetal.,2012).Salinitycaninfluencedenitri- linitygradientweretakenforDNAextractionandnutrientmeasure- ficationbyalteringtheaccessibilityoforganicsubstrateswhicharees- ment,andfordeterminingdenitrifyingactivity.Theusedincubations sentialforheterotrophicbacteria(Franklinetal.,2017).Theinfluence wereexcludedfromfuturesamplingtoavoidtheunbalanceofsample ofincreasedsalinityonnitrificationcanchangethenitrateavailability, quantities.Intotal,120incubationswereprocessed,including60each whichcanalsoaffectthedenitrification(Giblinetal.,2010).Moreover, (4salinitygradients×5timepoints×3replicates)forDNAextraction dissimilatorynitratereductiontoammonium(DNRA)cancompetefor anddenitrifyingactivitydetermination. nitratewithdenitrificationsinceDNRAisfavoredoverdenitrification withhighstorageofsulfideswhichisalwaysassociatedwithhighsalin- ityconditionssuchascoastalandmarineecosystems(Giblinetal., 2.2.Denitrifyingactivityandnutrientmeasurement 2013;Marchantetal.,2014).Salinityalsoexhibitsasignificantimpact onsoilbacterialandfungalcommunitystructure(Asgharetal.,2012; Thepotentialdenitrifyingactivitywasmeasuredaccordingtothe Beheraetal.,2017;Chenetal.,2017;MohamedandMartiny,2011). C H inhibitionmethod.Ateachtimepoint,threeincubationsofeach 2 2 However,onlyseveralstudieshaveexploredtheassociationbetween salinitygradientweresealedwithrubberstoppersandthenevacuated salinityandnitrifyingordenitrifyingcommunitystructure(Bañeras andflushedwith99.999%heliumthreetimes.C H wasinjectedtoa 2 2 etal.,2012;Bernhardetal., 2007;Franklin etal.,2017;Sahanand finalcompositionof10%(vol/vol).Thesedimentswerethenincubated Muyzer,2008;Xieetal.,2014). at25°Conamagneticstirringapparatusfor12days.Theheadspace Thedifferencesoftheresponsetosalinityinnitrogencyclingpro- gaseswereanalyzedatevery2,4or12hforthefirsttwodays,and cessesandthestructureoftheresponsiblemicroorganismsaretobeex- thenevery24or48hforthenext10dayswithagaschromatographer pected because of the influence of un-measured environmental equippedwithanECDdetectoraspreviouslydescribed(Molstadetal., variables.Itishenceessentialtocontrolenvironmentalsystemsas 2007).Thedenitrifyingactivitywascalculatedbylinearregressionof muchaspossible.Inthisstudy,alaboratoryincubationexperiment accumulatedN Opergramdrysedimentversustime.Theconcentra- 2 wasconductedtoinvestigatetheresponseofmangrovesedimentnitri- tionsofNH+andNO−weredeterminedusing2MKClextractswitha 4 x fyinganddenitrifyingcommunitiestodifferentsalinities(0,10,20and 1:10ratiooffreshsedimenttoKClsolution(g/mL).Theextractswere 30ppt).DenitrifyingactivitywasmeasuredasstimulatedN Oemission, furtheranalyzedbyFIAQC8500continuousflowinjectionanalyzer 2 andtheabundanceofnitrifiers(AOAandAOB)anddenitrifiers(nirK, (LACHAT,USA). 1344 H.Wangetal./ScienceoftheTotalEnvironment631–632(2018)1342–1349 2.3.DNAextractionandgenequantification deduced to amino acid to build the neighbor-joining phylogenetic treeswith1000timesbootstrapusingMEGAv7.0(Kumaretal.,2016). TotalDNAwasextractedfrom0.5gfreshsedimentwiththeFastDNA All the sequences of AOA and AOB clones were submitted to SPINKitforsoil(MPBiomedicals,SantaAna,CA,USA)accordingtothe GenBank with the accession numbers MF566141-MF566739 and manufacturer'sinstructions.Thepurityandconcentrationoftheex- MF566740-MF567338,respectively.AllthesequencesofnirK,nirSand tracted DNA were measured using UV–vis spectrophotometer ND- nosZ were submitted to the European Nucleotide Archive of EMBL 1000(NanoDrop,USA).TheextractedDNAwasthenstoredat−20°C withtheaccessionnumberPRJEB21903. formolecularexperiments. Theprimerpairsusedforamplifyingandquantifyingthearchaeal 2.5.Statisticalanalysis andtheβ-proteobacteriaamoAgenes,nirS,nirKandnosZgeneswere describedinpreviousstudies(Wangetal.,2014;Wangetal.,2015). Analysisofvariance(ANOVA)wasemployedtoobservethesignifi- Theconditionsusedforreal-timequantitativePCR(qPCR)ofthese5 canceofthesalinityimpactonthedenitrifyingactivity,nutrientconcen- geneswerealsodescribedinthetwopreviousstudies,withtheexcep- trations,geneabundancesandOTUrelativeabundances.Datafailedto tionthatreactionswereconductedonaLightCycler480IIReal-TimePCR meettheassumptionsofANOVAwerelogtransformedoranalyzedby System(Roche,IN,USA).Toevaluatetherelativeabundanceofeach thenon-parametricKruskal-Wallistest.TheTukeyposthocmultiple gene,theabundancesofbacterialandarchaeal16SrRNAgeneswere comparisons were conducted to compare the differences between alsoquantified.Theprimerpairsandconditionsusedfor16SrRNA eachtwoofthesalinitygradients.Nonmetricmultidimensionalscaling genesweredescribedinapreviousstudy(Wangetal.,2017).Thecon- (nMDS)basedontheBray-Curtisdissimilaritymatrixwasperformedto ditionsofqPCRandprimersforallgenesusedinthisstudyweresum- determinetheinfluenceofsalinityonthenitrifyinganddenitrifying marized in Table S1. Standard curves of qPCR were obtained by communitycompositions.Analysisofsimilarities(ANOSIM)andper- seriallydilutingstandardplasmidscontainingthetargetgeneswith mutationalmultivariateanalysisofvariance(PERMANOVA)wereused knowncopynumbers.NegativecontrolswithoutDNAtemplatewere totestthesignificanceofthesalinityimpactonthemicrobialcommu- includedineachamplification.Inhibitionwaseliminatedbyhighlydi- nitystructures.PairwisePearsoncorrelationswereconductedbetween lutingtheDNAextracts.PCRefficiencyabove90%wasaccepted. denitrifyingactivity,NH+concentration,NO−concentrationandabun- 4 x dancesofnitrifyinganddenitrifyinggenes.AlltheP-valuesweread- justedbyfalsediscoveryrate(FDR)methodformultiplecomparisons 2.4.Clonelibraries,high-throughputsequencingandphylogeneticanalysis andthenullhypothesiswasrejectedwhileP-valueswasb0.05.Allthe statistical analyses were processed in R with vegan, car and Hmisc TheDNAsamplesfromday28wereusedformicrobialcommunity packages. compositionanalysis.ThecommunitycompositionsofAOAandAOB wereanalyzedbycloningwhilethecommunitycompositionsofnirK, 3.Results nirSandnosZweretestedusinghigh-throughputsequencing.ThePCR mixtureandreactionofAOAandAOBweredescribedintheprevious 3.1.Denitrifyingactivityandnutrientconcentrations study(Wangetal.,2015).ThePCRproductsofthe12sampleswere usedtoconstructtheclonelibraries and50clones foreachlibrary TheactivityofdenitrificationwascalculatedastheaverageN O 2 wereselectedforsequencing.Intotal,599cleansequenceswereused emissionperdayduringa12-dayincubationaftersampling(Fig.S1). forclusteringforbothAOAandAOB.Similarityof97%wasusedasthe Theemissionrateinsedimentwithgreatersalinity(20–30ppt)wassig- thresholdforpickingoperationaltaxonomicunit(OTU)usingMothur nificantlylowercomparedtothelowersalinity(0–10ppt).Oneexcep- v1.19(Schlossetal.,2009). tion to this trend was on day 7 and 14 of the incubations, when Fordenitrifyinggenes,eachreverseprimercorrespondingtoacer- emissionsweresignificantlyreducedin10-ppt,whencomparedtothe tain sample was tagged with a six-base barcode. Amplifications of 0-pptcontrol(Fig.1a).Duringthe28-dayincubation,theammonia nirK, nirS and nosZ were processed as previously described (Wang wasdepletedin0-and10-pptsedimentsonday21and28,respectively etal.,2014),butwiththebarcodedprimers.PCRproductswerepurified (Fig.1b).Ammoniaconcentrationswerealsosignificantlylowerthan withtheUniversalDNAPurificationKit(TIANGEN,China).Thepurified thatin30-pptsamplesonday7and14.Althoughammoniaconcentra- productconcentrationswerethenconfirmedusingtheQuantiFluor tionin20-pptsedimentsshowedanincreaseonday14and21,itde- dsDNASystem(Promega,CA,USA).ThebarcodedPCRproductswere creaseddramaticallyonday28.Theseresultssuggesttheinhibitionof equallypooled,whichwerethenprecipitatedbyethanolwithsodium ammoniaconsumptionbyhighsalinity.However,theNO−concentra- x acetateanddissolvedinsterilizedMilli-Qwater.ThefinalDNApool tionexhibitedanoppositepattern.Theconcentrationwassignificantly wassequencedonanIlluminaMiSeqPE300platform.Thesequencing lowerinboththe20-and30-pptsamplesonday14,whilethelowest datawereanalyzedusingQIIMEv1.9.1(Caporasoetal.,2010).Rawse- NO−concentrationoccurredin20-and30-pptsamplesonday21and x quencesweredemultiplexedandlowqualityorambiguousreadswere day28,respectively(Fig.1c).Despitethat,theoverallNO−concentra- x removed.Theframe-shifterrorswerecheckedusingtheHMM-FRAME tionswerenotsignificantlydifferentfromday0today28.Correlation algorithm(ZhangandSun,2011)togetherwithHiddenMarkovModels analysisdemonstratedthattheNH+andNO−concentrationswereneg- 4 x ofdifferentgenesfromtheFunGenedatabase(Fishetal.,2013).Se- ativelyandpositivelyassociatedwiththeaverageN Oemissionrate,re- 2 quenceswithframe-shiterrorswerediscarded.Intotal,therewere spectively(Pb0.05,Fig.S2). 401,963,729,176and508,882cleansequencesfornirK,nirSandnosZ, respectively.FilteredsequenceswerethenclusteredintoOTUsbased 3.2.Theabundancesofnitrifyinganddenitrifyinggenes on 97% similarity using the “pick_otu.py” function with USEARCH method.Thesingletonsandchimericsequenceswereremovedduring Theabundances of amoAgenesforbothAOA(Fig.2a)andAOB theOTUpicking.Afterthat,eachsamplewasrarefiedtoequalsequence (Fig.2b)increasedfromday0today28.ForAOA,thedifferenceonlyoc- depth(thesmallestnumberofthesequencesamongallsamples)for curredonday7thatamoAgeneabundancewassignificantlylowerin downstreamanalyses. highsalinitysamples(20and30ppt)thaninthelowsalinitysamples TherepresentativesequencesofOTUswitharelativeabundance (0 and 10 ppt) (Fig. 2a). Interestingly, the abundance of bacterial above1%wereblastedagainsttheGenBanknucleotidedatabaseand amoAgenewassignificantlyhigherintheintermediatesalinitysamples referencesequenceswerechosenasthehighlysimilarsequencesto (10or20ppt)comparedtothoseinthelow(0ppt)orhigh(30ppt)sa- eachOTU.Thenboththerepresentativeandreferencesequenceswere linity samples (Fig. 2b), suggesting a different response to salinity H.Wangetal./ScienceoftheTotalEnvironment631–632(2018)1342–1349 1345 Fig.1.ChangesintheaverageN2Oemissionrate(a),ammoniaconcentration(b),andnitrateandnitriteconcentration(c)onday0,day7,day14andday28.Thedifferentlettersamong samplesateachtimepointindicatethesignificantdifference(Pb0.05).Thevaluesaregivenasmean±standarddeviation(n=3).*,undetected. impactbetweenAOBandAOA.Moreover,theAOBabundancewassev- eralordersofmagnitudegreaterthanAOAabundance,despitecompa- rable abundance on day 0, suggesting that AOB dominated nitrification.Therelativeabundancecalculatedasthecopynumber ratiooffunctionalgenetocorresponding16SrRNAgenewasinvesti- gated(Fig.S3).TheresultsofrelativeabundancesofAOAandAOB showedasimilartrend,withtheexceptionthatdifferenceofAOArela- tiveabundancealsooccurredonday21whentheabundancewassig- nificantlylowerinhighsalinitysamples(20and30ppt)compared with0-pptsamples(Fig.S3). Salinityhadalimitedinfluenceondenitrifyinggeneabundance. Onlyonday28thelowestandhighestnirKabundancewasdetected in30-pptand0-pptsamples,respectively(Fig.3a).Whiletherewas nosignificantchangeinnirSabundance(Fig.3b),thenosZabundance wassignificantlylowerin30-pptsamplescomparedtotheothersam- plesonday28(Fig.3c).Onday28,thenirK/nirSratiowassignificantly greaterin0-pptsamplescomparedtotheothersamples(Fig.3d),while thenir/nosZratiowasgreatestin30-pptsamples(Fig.3e).Thenir/nosZ ratiowaspositivelyandsignificantlycorrelatedwithdenitrifyingactiv- ityandNO−concentration(Pb0.05,Fig.S2).Therelativeabundancesof x nirK,nirSandnosZallshowedasimilartrendtotheabsoluteabun- dances(Fig.S3). 3.3.Thecommunitycompositionsofnitrifiersanddenitrifiers Intotal,53and26OTUswereobtainedforAOAandAOBcommuni- ties,respectively.nMDSplotsshowedthatbothAOAandAOBcommu- nitiesweregroupedaccordingtodifferentsalinitygradients(Fig.4aand b).BoththeANOSIMandPERMANOVAindicatedthatsalinitysignifi- cantly impacted AOA and AOB community composition (P b 0.01, Table 1). For denitrifying communities, 1607, 7259 and 6148 OTU wereobservedfornirK,nirSandnosZcommunities,respectively.The nirKandnosZcommunitiesofsamplesassociatedwithdifferentsalinity gradientswerewellgroupedwhilethenirScommunitieswerelessdis- tinctlydistributed(Fig.4c,dande).However,thenirK,nirSandnosZ communitycompositionswereallsignificantlyinfluencedbysalinity asdemonstratedbytheANOSIMandPERMANOVAresults(Pb0.05, Table1). 3.4.Phylogeneticanalysis ToreducetheeffectofrareOTUsonphylogenetictreeconstruction, wechosetheOTUswitharelativeabundanceN1%.Thedistributionof abundantOTUsfordifferentgenesaredisplayedinFig.S4.According tothephylogenetictreeforAOA(Fig.S5)andAOB(Fig.S6),theabun- dantOTUscouldbegroupedinto4and3clusters,respectively.The Fig.2.ChangesintheamoAgeneabundanceofAOA(a)andAOB(b)onday0,day7,day AOA cluster I, which was associated with Nitrososphaera (Fig. S5), 14 and day 28. The different letters among samples at each time point indicate thesignificantdifference(Pb0.05).Thevaluesaregivenasmean±standarddeviation accountedforthelargestproportion(74.0%)ofAOAsequences.Therel- (n=3). ativeabundanceofAOAclusterIwasslightlygreaterinthe0-and10- 1346 H.Wangetal./ScienceoftheTotalEnvironment631–632(2018)1342–1349 Fig.3.Changesintheabundancesofdenitrifyinggenes,nirK(a),nirS(b)andnosZ(c),aswellasthenirK/nirS(d)andnir/nosZ(e)ratiosonday0,day7,day14andday28.Thedifferent lettersamongsamplesateachtimepointindicatethesignificantdifference(Pb0.05).Thevaluesaregivenasmean±standarddeviation(n=3). pptsamplescomparedtothosein20-and30-pptsamples(Fig.5a).The Thesefindingssuggestthatsalinityintrusionincoastalareasmightin- AOBclusterIIwhichwasaffiliatedwithNitrosomonas(Fig.S6)occupied terruptthebalanceofnutrientcycles,andcouldnegativelycontribute 76.4%ofthetotalAOBmembers.BothAOBclusterIandIIdominated0- toecosystemfunctions. and10-pptsampleswhileonlyclusterIIflourishedin20-and30-ppt SalinityexhibitedalimitedinfluenceonAOAabundanceinthis samples(Fig.5b),whichsuggestedthathighsalinityinhibitedtheclus- study,whichisdifferentfrompreviousstudiesshowingthatAOAwas terIofAOBwhichwasassociatedwithNitrosospira(Fig.S6). themostabundantatintermediate salinity(10–30ppt)(Bernhard Basedonthephylogeneticanalysesofdenitrifyingcommunities,the etal.,2010;Zhangetal.,2015).However,AOBplayedadominantrole nirK(Fig.S7),nirS(Fig.S8)andnosZ(Fig.S9)sequencescanbegrouped innitrificationinthisstudysincetheAOBabundanceoutcompeted into3,10and7clusters,respectively.FornirK,clusterIwasthedomi- AOAabundance.TherelativeimportanceofAOAandAOBfornitrifica- nantclusteraccountingfor58.4%ofthetotalsequences.Thiscluster tion in salineenvironments remains controversial. Consistentwith wassignificantlygreaterinthehighsalinity(20and30ppt)samples thisstudy,theAOBabundancewasgreaterthanAOAinsediments thaninthelowsalinity(0and10ppt)samples(Fig.5c,TukeyHSDPb fromDouroRiverestuarydespiteofthevariedsalinitiesrangingfrom 0.05),suggestingpreferenceofhighsalinityconditionofthiscluster. 1.5 to 26.8 ppt (Magalhães et al., 2009). In some estuaries, it was However,nirKclusterIcannotbeassignedtoaknowntaxon(Fig.S7). foundthatAOApredominatedthenitrificationintheregionswithlow FornirS,onlyclusterVandVIIshowedaslightlydifferentabundance salinitywhileAOBweremoreabundantthanAOAinhighersalinityre- amongdifferentsalinitygradients(Fig.5d).FornosZ,clusterIandclus- gions(MosierandFrancis,2008;Santoroetal.,2008).Incontrast,AOA terIIIwerethedominantclusters,withaproportionof10.8%and20.2%, abundancewasgreaterthanAOBatallthesalinitylevelsinPlumIsland respectively.TheabundanceofclusterIIIwhichmightbeassociated Soundestuary(Bernhardetal.,2010).Thesedivergentresultsindicate withThiobacillus(Fig.S9)wassignificantlylowerin30-pptsamples thatotherfactorsinfluencetheabundanceofAOAandAOB.Itissug- comparedto0-pptsamples(Fig.5e,TukeyHSDPb0.05). gestedthatthedominanceofAOBoverAOAnormallyoccursinman- grovesedimentsastheslightlyacidic or neutral conditionandthe 4.Discussion availableammoniamightfavorthegrowthofAOB(Lietal.,2011;Li andGu,2013;Wangetal.,2015).Moreover,wefoundthatAOBabun- Thedenitrifyingactivity,abundanceofnitrifiersanddenitrifiers,and dancewasgreaterinthe10-or20-pptsedimentsduringthe28-dayin- theircommunitystructureswereinvestigatedinmangrovesediments cubation.Thisfindingsupportsapreviousmeta-analysisshowingthat treatedwithdifferentsalinities.Salinityexhibitedasignificantinfluence nitrificationcanbepromotedwithinacertainrangeofthesalinityand onthenitrogencyclingprocesses.Increaseofsalinityfrom0to30ppt inhibitedwhensalinityexceedsthisrange(Zhouetal.,2017).Forthis decreasedbothdenitrificationactivityandtheabundanceofdenitri- study,theintermediatesalinities(10and20ppt)wereconsideredas fiers,whilethenitrifiersweremostabundantundertheintermediate theoptimalconditionfortheammoniaoxidizers,whichmightalsore- salinityconditions.Thecommunitycompositionofnitrifiersanddeni- sultfromthelong-termadaptionofthemicrobestothelocalhabitats trifierswassignificantlyinfluencedbysalinity.Althoughthisstudyis wherethesedimentsalinitynormallyshiftedbetween10and20ppt basedonthemanipulatedsedimentrecoveredfromthedriedsediment, (Wangetal.,2016).WeobservedthatNH+concentrationwassignifi- 4 therecoveredmicrobialcommunitieswereoriginallyfromthesedi- cantlyhigherinthe20-and30-pptsamples,suggestingthelesscon- mentinthefield,andthuspartlyrepresentingthein-situcondition. sumption of NH+ which might be caused by the reduction of 4 H.Wangetal./ScienceoftheTotalEnvironment631–632(2018)1342–1349 1347 Fig.4.Nonmetricmultidimensionalscaling(nMDS)plotsofAOA(a),AOB(b),nirK(c),nirS(d)andnosZ(e)basedontheBray-Curtisdissimilarities. nitrificationactivity.However,nosignificantcorrelationwasfoundbe- 2005;SahanandMuyzer,2008).Thecontrastingresultsonthedistribu- tweenNH+concentrationandAOBabundance.Thismaybeduetothe tionofNitrosospiramightresultfromtheothercovaryingfactors,such 4 enhancedDNRAactivityasDNRAisfavoredunderthehighersalinity astemperatureandammoniaconcentration;however,salinitywas condition(Giblinetal.,2013;Marchantetal.,2014),whichmightfur- theonlyfactorthatdrovechangesinourstudy.Ourresultsalsoindicate theroffsettheconsumedNH+. thatNitrosomonasmightpossessabroaderadaptiontothesalinity 4 Inadditiontotheabundanceofnitrifiers,salinityexhibitedasignif- levels compared to Nitrosospira. However, the mechanism on how icantimpactonbothAOAandAOBcommunitystructure.Theseresults theytoleratethesalinitystressremainsunknown. supportpreviousstudiesshowingthatsedimentsalinitywasthemain Itissuggestedthatdenitrificationisconsistentlydecreasedbysoil factor controlling the distribution of AOA or/and AOB (Sahan and salinizationacrosscoastalecosystems(Zhouetal.,2017).Insupportof Muyzer,2008;Zhangetal.,2015).ThegenusNitrososphaeradominated this,denitrifyingactivitydeclinedwithincreasedsalinityfrom0to AOAcommunityinallsamples,butwasslightlyrestrictedathighsalin- 30pptinthisstudy.Therearemanymechanismsthatmaycontribute itylevelsinthisstudy.However,asonlyoneNitrososphaeraisolatehas tothisphenomenon.Foralaboratoryincubation,salinityelevation beenidentified(Tournaetal.,2011),littlecanbesaidaboutitspotential mightdirectlyconstrainthefitnessofmicrobesandatthesametimere- interaction.ForAOB,wefoundthatboththehighandlowsalinitiesfa- ducethesoilrespiration(Wongetal.,2008)whichcoulddecreasethe voredNitrosomonaswhileNitrosospiraexistedonlyatlowsalinitylevels. oxygenconsumptionthusinhibitingtheanaerobicdenitrifyingprocess. Twoformerstudiesshowedthatalongasalinitygradient,Nitrosospira Thereductionofnitrificationbysalinityelevationcanreducethenitrate wasenrichedatthehighsalinitysiteswhilethegenusNitrosomonas availability,whichcanalsolimitthedenitrification(Giblinetal.,2010). tendedtoassociatewithlowormiddlesalinitysites(Bernhardetal., Inthisstudy,theenhancedconsumptionofNO−bysalinity,whichin- x creasedduringtheincubation,couldbeduetothepromotedDNRA Table1 under high salinity conditions (Giblin et al.,2013; Marchant et al., TheANOSIMandPERMANOVAresultsshowingtheinfluenceofsalinityonnitrifyingand 2014).Hence, ourresultspotentially indicate thepredominanceof denitrifyingcommunitycompositions. DNRAoverdenitrificationinsedimentsthataresubjecttohighsalin- ANOSIM PERMANOVA ities.Ontheotherhand,theabundanceofdenitrifiers(nirKandnosZ) R Significance R2 Significance wasalsoimpactedbysalinization,butinhibitiononlyoccurredatday 28,suggestingthetoleranceofdenitrifierstosedimentsalinizationat AOA 0.397 0.002** 0.409 0.010** thebeginningoftheincubation.ThedecreaseofthenirK/nirSratio AOB 0.559 0.005** 0.556 0.002** nirK 0.491 0.002** 0.479 0.003** withincreasingsalinityindicatedthattherelativeimportanceofnirK nirS 0.361 0.016* 0.355 0.025* andnirSforreducingthenitritemightshiftaccordingtosalinitylevels nosZ 0.361 0.005** 0.378 0.002** andthatnirS-bearingdenitrifiersmightbemoretoleranttosalinityele- Significancelevelisshownas*,Pb0.05;**,Pb0.01. vationcomparedtothosenirK.Moreover,theenhancednir/nosZratioin 1348 H.Wangetal./ScienceoftheTotalEnvironment631–632(2018)1342–1349 Fig.5.Thedistributionoftheclustersobtainedfromphylogeneticanalysisandtheirrelativeabundances.Thevaluesaregivenasmean±standarddeviation(n=3).Significancelevelis shownas●,Pb0.1;*,Pb0.05;***,Pb0.001. 30-pptsamplesatday28illustratedthatsalinityelevationmightrise Insummary,wefoundtheinhibitingeffectofsalinityelevationon thegeneticpotentialforN Oemissionssincethehighernir/nosZratio thedenitrifyingactivityandabundancesofnirKandnosZ,whilethe 2 canleadtoanincreasedN O/(N O+N )end-productratio(García- nirSabundanceremainedstable.TheabundanceofAOBincreasedatin- 2 2 2 Lledóetal.,2011). termediatesalinitylevelswhileAOAwerelessaffected.However,are- Apreviousstudyfoundthatsalinity,inadditiontoothermajorfac- ductionintheconsumptionofNH+athighsalinitylevelsmightsuggest 4 tors,wasassociatedwiththedynamicsofnirK,nirSandnosZcommunity theinhibitionofnitrificationbysalinization.Therefore,thereductionof compositioninwetlandsediments(Bañerasetal.,2012).Thesalinity couplednitrification-denitrificationprocessmightdecreasethenitro- wasalsofoundtocorrelatewithnosZcommunitystructuresacross genremoval,whichmightfurtherleadtoeutrophicationanddisturb eighttidalwetlands(Franklinetal.,2017).However,astudyinthe thenitrogenbalanceintheenvironment.Whilethereislimitedinfor- SanFranciscoBayestuaryrevealedsignificantcorrelationsbetweensa- mationaboutthesalinityandmicrobialcommunitycomposition,this linityandabundancesofnirKandnirSbutnottheircommunitystruc- studyprovidesstrongevidencethatsalinitycansignificantlyshape tures(MosierandFrancis,2010).Sincethesestudieswereconducted thecommunitystructureofbothnitrifiersanddenitrifiers.Theseresults withsamplesinsitu,theimpactofsalinitywascoupledwithother illustratethatsalinityplaysavitalroleinregulatingthenitrogencycling majorfactorssuchasvegetation,carbonsourceandmetals.Henceour incoastalareas,whichmayincreaseconcernregardingthreatsofsalin- study does provide strong evidence that salinity can significantly ityintrusionassociatedwithclimatechangeandarisingsealevel. shapethecommunitystructuresofdenitrifiersintheabsenceofother factors.AlthoughweobservedthatthedominantnirKclusterIwassig- Acknowledgments nificantlyinfluencedbythesalinity,littleisknownaboutthiscluster. Similarly,theclusterVandVIIofnirSwereslightlyinfluencedbythesa- ThisworkwassupportedbytheStrategicPriorityResearchProgram linity,buttheyarealsounknowntaxa.ThedominantclusterIIIofnosZ ofChineseAcademyofSciences(XDB15020402),theNationalNatural which might be associated with Thiobacillus was decreased by the ScienceFoundationofChina(41430858,41771285,41571130063), highsalinity(30ppt).Mangrovecontainshighlevelofsulfidesgener- andtheInternationalScience&TechnologyCooperationProgramof atedbysulfatereduction(Ferreiraetal.,2007),andsomespeciesbe- China(2011DFB91710).WethankProf.TianlingZheng(deceased)from longing to Thiobacillus can use the sulfur and oxidize sulfide to XiamenUniversityforhishelpwithdesigningandanalyzingthedata. supporttheirautotrophicgrowth(Friedrichetal.,2001).Ithasbeenrec- Theauthorsdeclarenoconflictofinterests. ognizedthatsulfuroxidationcanbecoupledwithnitratereduction (BurginandHamilton,2007);thusreductionofsulfidebyoxidation AppendixA.Supplementarydata mightleadtothedecreaseofsulfur-drivennitratereduction.Therefore, theinhibitionofThiobacillusbysalinizationmightleadtothereduction Supplementarydatatothisarticlecanbefoundonlineathttps://doi. ofdenitrificationasweobservedinthisstudy. org/10.1016/j.scitotenv.2018.03.102. H.Wangetal./ScienceoftheTotalEnvironment631–632(2018)1342–1349 1349 References Magalhaes,C.M.,Joye,S.B.,Moreira,R.M.,Wiebe,W.J.,Bordalo,A.A.,2005.Effectofsalinity andinorganicnitrogenconcentrationsonnitrificationanddenitrificationratesinin- Asghar,H.N.,Setia,R.,Marschner,P.,2012.Communitycompositionandactivityofmi- tertidalsedimentsandrockybiofilmsoftheDouroRiverestuary,Portugal.WaterRes. crobesfromsalinesoilsandnon-salinesoilsrespondsimilarlytochangesinsalinity. 39,1783–1794. SoilBiol.Biochem.47,175–178. Magalhães,C.M.,Machado,A.,Bordalo,A.A.,2009.Temporalvariabilityintheabundance Bañeras,L.,Ruiz-Rueda,O.,López-Flores,R.,Quintana,X.,Hallin,S.,2012.Theroleofplant ofammonia-oxidizingbacteriavs.archaeainsandysedimentsoftheDouroRiveres- typeandsalinityintheselectionforthedenitrifyingcommunitystructureintherhi- tuary,Portugal.Aquat.Microb.Ecol.56,13–23. zosphereofwetlandvegetation.Int.Microbiol.15,89–99. Marchant,H.K.,Lavik,G.,Holtappels,M.,Kuypers,M.M.,2014.Thefateofnitrateininter- Behera,P.,Mahapatra,S.,Mohapatra,M.,Kim,J.Y.,Adhya,T.K.,Raina,V.,etal.,2017.Salin- tidalpermeablesediments.PLoSOne9,e104517. ityandmacrophytedrivethebiogeographyofthesedimentarybacterialcommuni- Marton,J.M.,Herbert,E.R.,Craft,C.B.,2012.Effectsofsalinityondenitrificationandgreen- tiesinabrackishwatertropicalcoastallagoon.Sci.TotalEnviron.595,472–485. housegasproductionfromlaboratory-incubatedtidalforestsoils.Wetlands32, Bernhard,A.E.,Donn,T.,Giblin,A.E.,Stahl,D.A.,2005.Lossofdiversityofammonia- 347–357. oxidizingbacteriacorrelateswithincreasingsalinityinanestuarysystem.Environ. Mohamed,D.J.,Martiny,J.B.,2011.Patternsoffungaldiversityandcompositionalonga Microbiol.7,1289–1297. salinitygradient.ISMEJ.5,379–388. Bernhard,A.E.,Tucker,J.,Giblin,A.E.,Stahl,D.A.,2007.Functionallydistinctcommunities Molstad,L.,Dörsch,P.,Bakken,L.R.,2007.Robotizedincubationsystemformonitoring of ammonia-oxidizing bacteria along an estuarine salinity gradient. Environ. gases(O2,NO,N2ON2)indenitrifyingcultures.J.Microbiol.Methods71,202–211. Microbiol.9,1439–1447. Mosier,A.C.,Francis,C.A.,2008.Relativeabundanceanddiversityofammonia-oxidizing Bernhard,A.E.,Landry,Z.C.,Blevins,A.,delaTorre,J.R.,Giblin,A.E.,Stahl,D.A.,2010.Abun- archaeaandbacteriaintheSanFranciscoBayestuary.Environ.Microbiol.10, danceofammonia-oxidizingarchaeaandbacteriaalonganestuarinesalinitygradi- 3002–3016. ent in relation to potential nitrification rates. Appl. Environ. Microbiol. 76, Mosier,A.C.,Francis,C.A.,2010.DenitrifierabundanceandactivityacrosstheSan 1285–1289. FranciscoBayestuary.Environ.Microbiol.Rep.2,667–676. Burgin,A.J.,Hamilton,S.K.,2007.Haveweoveremphasizedtheroleofdenitrificationin Rysgaard,S.,Thastum,P.,Dalsgaard,T.,Christensen,P.B.,Sloth,N.P.,1999.Effectsofsalin- aquaticecosystems?Areviewofnitrateremovalpathways.Front.Ecol.Environ.5, ityonNH4+adsorptioncapacity,nitrification,anddenitrificationinDanishestuarine 89–96. sediments.Estuar.Coasts22,21–30. Caporaso,J.G.,Kuczynski,J.,Stombaugh,J.,Bittinger,K.,Bushman,F.D.,Costello,E.K.,etal., Sahan,E.,Muyzer,G.,2008.Diversityandspatio-temporaldistributionofammonia- 2010.QIIMEallowsanalysisofhigh-throughputcommunitysequencingdata.Nat. oxidizingArchaeaandBacteriainsedimentsoftheWesterscheldeestuary.FEMS Methods7,335–336. Microbiol.Ecol.64,175–186. Chen,L.,Li,C.,Feng,Q.,Wei,Y.,Zheng,H.,Zhao,Y.,etal.,2017.Shiftsinsoilmicrobialmet- Santoro,A.E.,Francis,C.A.,deSieyes,N.R.,Boehm,A.B.,2008.Shiftsintherelativeabun- abolicactivitiesandcommunitystructuresalongasalinitygradientofirrigation danceofammonia-oxidizingbacteriaandarchaeaacrossphysicochemicalgradients waterinatypicalaridregionofChina.Sci.TotalEnviron.598,64–70. inasubterraneanestuary.Environ.Microbiol.10,1068–1079. Duke,N.C.,Meynecke,J.-O.,Dittmann,S.,Ellison,A.M.,Anger,K.,Berger,U.,etal.,2007.A Schloss,P.D.,Westcott,S.L.,Ryabin,T.,Hall,J.R.,Hartmann,M.,Hollister,E.B.,etal.,2009. worldwithoutmangroves?Science317,41–42. Introducingmothur:open-source,platform-independent,community-supported Fear,J.M.,Thompson,S.P.,Gallo,T.E.,Paerl,H.W.,2005.Denitrificationratesmeasured softwarefordescribingandcomparingmicrobialcommunities.Appl.Environ. alongasalinitygradientintheeutrophicNeuseRiverEstuary,NorthCarolina,USA. Microbiol.75,7537–7541. Estuar.Coasts28,608–619. Seitzinger,S.P.,Gardner,W.S.,Spratt,A.K.,1991.Theeffectofsalinityonammoniumsorp- Ferreira,T.O.,Otero,X.L.,Vidal-Torrado,P.,Macías,F.,2007.Effectsofbioturbationbyroot tioninaquaticsediments:implicationsforbenthicnutrientrecycling.Estuaries14, and crab activity on iron and sulfur biogeochemistry in mangrove substrate. 167–174. Geoderma142,36–46. Sheng,Q.,Wang,L.,Wu,J.,2015.Vegetationalterstheeffectsofsalinityongreenhouse Fish,J.A.,Chai,B.,Wang,Q.,Sun,Y.,Brown,C.T.,Tiedje,J.M.,etal.,2013.FunGene:the gasemissionsandcarbonsequestrationinanewlycreatedwetland.Ecol.Eng.84, functionalgenepipelineandrepository.Front.Microbiol.4,291. 542–550. Fisher,J.,Acreman,M.,2004.Wetlandnutrientremoval:areviewoftheevidence.Hydrol. Tourna,M.,Stieglmeier,M.,Spang,A.,Könneke,M.,Schintlmeister,A.,Urich,T.,etal., EarthSyst.Sci.8,673–685. 2011.Nitrososphaeraviennensis,anammoniaoxidizingarchaeonfromsoil.P.Natl. Franklin,R.B.,Morrissey,E.M.,Morina,J.C.,2017.Changesinabundanceandcommunity Acad.Sci.USA108,8420–8425. structureofnitrate-reducingbacteriaalongasalinitygradientintidalwetlands. Vymazal,J.,2007.Removalofnutrientsinvarioustypesofconstructedwetlands.Sci.Total Pedobiologia60,21–26. Environ.380,48–65. Friedrich,C.G.,Rother,D.,Bardischewsky,F.,Quentmeier,A.,Fischer,J.,2001.Oxidationof Wang,H.T.,Su,J.Q.,Zheng,T.L.,Yang,X.R.,2014.Impactsofvegetation,tidalprocess,and reducedinorganicsulfurcompoundsbybacteria:emergenceofacommonmecha- depthontheactivities,abundances,andcommunitycompositionsofdenitrifiersin nism?Appl.Environ.Microbiol.67,2873–2882. mangrovesediment.Appl.Microbiol.Biotechnol.98,9375–9387. Galloway,J.N.,Townsend,A.R.,Erisman,J.W.,Bekunda,M.,Cai,Z.,Freney,J.R.,etal.,2008. Wang,H.T.,Su,J.Q.,Zheng,T.L.,Yang,X.R.,2015.Insightsintotheroleofplanton Transformationofthenitrogencycle:recenttrends,questions,andpotentialsolu- ammonia-oxidizingbacteriaandarchaeainthemangroveecosystem.J.SoilsSedi- tions.Science320,889–892. ments15,1212–1223. García-Lledó,A.,Vilar-Sanz,A.,Trias,R.,Hallin,S.,Bañeras,L.,2011.Geneticpotentialfor Wang,H.,Liao,G.,D'Souza,M.,Yu,X.,Yang,J.,Yang,X.,etal.,2016.Temporalandspatial NW2aOteermReisss.i4o5n,s5f6r2o1m–5t6h3e2s.edimentofafreewatersurfaceconstructedwetland. Cvahriniaat.ioEnnsviorofng.rSecein.Phooluluste.Rgaess.flInutx.e2s3,fr1o8m73a–1ti8d8a5l.mangrovewetlandinSoutheast Giblin,A.E.,Weston,N.B.,Banta,G.T.,Tucker,J.,Hopkinson,C.S.,2010.Theeffectsofsalin- Wang,H.T.,Marshall,C.W.,Cheng,M.Y.,Xu,H.J.,Li,H.,Yang,X.R.,etal.,2017.Changesin ityonnitrogenlossesfromanoligohalineestuarinesediment.Estuar.Coasts33, landusedrivenbyurbanizationimpactnitrogencyclingandthemicrobialcommu- 1054–1068. nitycompositioninsoils.Sci.Rep.7,44049. Giblin,A.E.,Tobias,C.R.,Song,B.,Weston,N.,Banta,G.T.,Rivera-Monroy,V.H.,2013.The Wong,V.N.L.,Dalal,R.C.,Greene,R.S.B.,2008.Salinityandsodicityeffectsonrespiration importanceofdissimilatorynitratereductiontoammonium(DNRA)inthenitrogen andmicrobialbiomassofsoil.Biol.Fertil.Soils44,943–953. cycleofcoastalecosystems.Oceanography26,124–131. Xie,W.,Zhang,C.,Zhou,X.,Wang,P.,2014.Salinity-dominatedchangeincommunity Gruber,N.,Galloway,J.N.,2008.AnEarth-systemperspectiveoftheglobalnitrogencycle. structureandecologicalfunctionofArchaeafromthelowerPearlRivertocoastal Nature451,293–296. SouthChinaSea.Appl.Microbiol.Biotechnol.98,7971–7982. Kumar,S.,Stecher,G.,Tamura,K.,2016.MEGA7:molecularevolutionarygeneticsanalysis Zhang, Y., Sun, Y., 2011. HMM-FRAME: accurate protein domain classification for version7.0forbiggerdatasets.Mol.Biol.Evol.33,1870–1874. metagenomicsequencescontainingframeshifterrors.BMCBioinformatics12,198. Li,M.,Gu,J.-D.,2013.Communitystructureandtranscriptresponsesofanammoxbacte- Zhang,Y.,Chen,L.,Dai,T.,Tian,J.,Wen,D.,2015.Theinfluenceofsalinityontheabun- ria,AOA,andAOBinmangrovesedimentmicrocosmsamendedwithammoniumand dance,transcriptionalactivity,anddiversityofAOAandAOBinanestuarinesedi- nitrite.Appl.Microbiol.Biotechnol.97,9859–9874. ment:amicrocosmstudy.Appl.Microbiol.Biotechnol.99,9825–9833. Li,M.,Cao,H.,Hong,Y.,Gu,J.-D.,2011.Spatialdistributionandabundancesofammonia- Zhou,M.,Butterbach-Bahl,K.,Vereecken,H.,Bruggemann,N.,2017.Ameta-analysisof oxidizingarchaea(AOA)andammonia-oxidizingbacteria(AOB)inmangrovesedi- soilsalinizationeffectsonnitrogenpools,cyclesandfluxesincoastalecosystems. ments.Appl.Microbiol.Biotechnol.89,1243–1254. Glob.Chang.Biol.23,1338–1352. Liu,X.,Ruecker,A.,Song,B.,Xing,J.,Conner,W.H.,Chow,A.T.,2017.Effectsofsalinityand wet–drytreatmentsonCandNdynamicsincoastal-forestedwetlandsoils:implica- tionsofsealevelrise.SoilBiol.Biochem.112,56–67.

ebook img

Salinity is a key factor driving the nitrogen cycling in the mangrove sediment PDF

2018·1.7 MB·English
by  Haitao Wang
#additional_collections #folkscanomy #folkscanomy_academic #mendeley_climate_change_library
Save to my drive
Quick download
Download
Upgrade Premium
Most books are stored in the elastic cloud where traffic is expensive. For this reason, we have a limit on daily download.

Preview Salinity is a key factor driving the nitrogen cycling in the mangrove sediment

ScienceoftheTotalEnvironment631–632(2018)1342–1349 ContentslistsavailableatScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv Salinity is a key factor driving the nitrogen cycling in the mangrove sediment HaitaoWanga,b,c,d,JackA.Gilbertd,YongguanZhua,b,XiaoruYanga,b,⁎ aInstituteofUrbanEnvironment,ChineseAcademyofSciences,Xiamen,FujianProvince361024,China bCenterforExcellenceinRegionalAtmosphericEnvironment,InstituteofUrbanEnvironment,ChineseAcademyofSciences,China cSchoolofLifeSciences,XiamenUniversity,Xiamen,FujianProvince361102,China dTheMicrobiomeCenter,DepartmentofSurgery,UniversityofChicago,Chicago,IL60637,USA H I G H L I G H T S G R A P H I C A L A B S T R A C T • Salinityelevationdecreasedboththeac- tivityandabundanceofdenitrifiers. • Thenitrifiers(AOB)weremostabun- dant under the intermediate salinity conditions. • Salinitysignificantlyshapedthenitrify- inganddenitrifyingcommunitystruc- tures. • Thesefindingsmayincreaseconcernre- gardingthreatsofsalinityintrusion. a r t i c l e i n f o a b s t r a c t Articlehistory: Coastalecosystemsarehotspotsfornitrogencycling,andspecificallyfornitrogenremovalfromwaterandsed- Received9January2018 imentthroughthecouplednitrification-denitrificationprocess.Salinityisgloballyimportantinstructuringbac- Receivedinrevisedform5March2018 terialandarchaealcommunities,buttheassociationbetweensalinityandmicrobially-mediatednitrificationand Accepted9March2018 denitrificationremainsunclear.Thedenitrificationactivityandcompositionandstructureofmicrobialnitrifiers Availableonlinexxxx anddenitrifierswerecharacterizedacrossagradientofmanipulatedsalinity(0,10,20and30ppt)inamangrove sediment.SalinitynegativelycorrelatedwithbothdenitrifyingactivityandtheabundanceofnirKandnosZ Editor:JayGan denitrifyinggenes.Ammonia-oxidizingbacteria(AOB),whichdominatednitrification,hadsignificantlygreater Keywords: abundanceatintermediatesalinity(10and20ppt).However,apositivecorrelationbetweenammoniaconcen- Salinity trationandsalinitysuggestedthatnitrifyingactivitymightalsobeinhibitedathighersalinity.Thecommunity Nitrification structureofammonia-oxidizingarchaea(AOA)andbacteria(AOB),aswellasnirK,nirSandnosZdenitrifying Denitrification communities,wereallsignificantlycorrelatedwithsalinity.Thesechangeswerealsoassociatedwithstructural Abundance shiftsinphylogeny.Thesefindingsprovideastrongevidencethatsalinityisakeyfactorthatinfluencesthenitro- Communitystructure gentransformationsincoastalwetlands,indicatingthatsalinityintrusioncausedbyclimatechangemighthavea broaderimpactonthecoastalbiospheres. ©2018ElsevierB.V.Allrightsreserved. 1.Introduction Coastalwetlandssuchasmangrovesdominatethetropicalandsub- tropicalcoastlines,butaregloballydisappearing(Dukeetal.,2007). Theseecosystemsare hotspotsof nutrientcyclingandplaypivotal ⁎ Correspondingauthorat:InstituteofUrbanEnvironment,ChineseAcademyof rolesinnutrienttransformationandavailability(FisherandAcreman, Sciences,Xiamen,FujianProvince361024,China. E-mailaddress:[email protected](X.Yang). 2004).Recenteffortshavebeenfocusedondetermininghowclimate https://doi.org/10.1016/j.scitotenv.2018.03.102 0048-9697/©2018ElsevierB.V.Allrightsreserved. H.Wangetal./ScienceoftheTotalEnvironment631–632(2018)1342–1349 1343 change may influence these cycling processes (Duke et al., 2007; nirSandnosZ)wereestimatedbyreal-timequantitativePCR(qPCR). Gallowayetal.,2008;GruberandGalloway,2008),includingtheconse- Thenitrifyingcommunitycompositionandstructure(AOAandAOB) quencesofsalinityintrusionresultingfromgroundwaterabstraction, wasassessedbyclonelibrary,whilehigh-throughputsequencingwas declining sediment loads and rising sea level, on nitrogen cycling performed for denitrifying communities (nirK, nirS and nosZ). This (Bernhardetal.,2005;Bernhardetal.,2007;Franklinetal.,2017;Liu study aims to reveal the impact of salinity on both nitrifying and etal.,2017;Shengetal.,2015;Zhouetal.,2017).Nitrificationanddeni- denitrifying processes and the potential influences of changes in trificationareresponsibleforammoniaoxidationandnitratereduction, couplednitrification-denitrificationonthenitrogentransformations respectively.Thecouplednitrification-denitrificationprocessdrivesthe andecosystemfunctions. nitrogenremovalinwetlands,thuspreventingeutrophication(Fisher andAcreman,2004;Vymazal,2007). Manyeffortshavefocusedonhownitrifyinganddenitrifyingcom- 2.Materialsandmethods munities respond to the salinity elevation in coastal wetlands (Bernhardetal.,2005;Bernhardetal.,2010;Bernhardetal.,2007; 2.1.Incubationexperiment Martonetal.,2012;Shengetal.,2015;Xieetal.,2014;Zhangetal., 2015),yettheanswerremainsambiguous.Ammoniaoxidation,which Thesurfacesediment(0–50cm)fromanunvegetatedareainthe isthefirststepofnitrificationconductedbytheammonia-oxidizingar- mangrovewetlandlocatedinthemudflatofJiulongRiverestuary(24° chaea(AOA)andammonia-oxidizingbacteria(AOB)showsvaryingre- 27′N;117°54′E)wascollectedaspreviouslydescribed(Wangetal., sponses to salinity in different environments. Moderate salinity 2014).Theoriginalsalinityofthesedimentwasaround15ppt(parts- (10–20ppt)isassociatedwithanincreasedabundanceofAOAandpo- per-thousand).Toaccuratelymanipulatetheincubations,sediment tentialnitrificationrates,whileAOBeithershownoornegativecorrela- wasair-dried,groundandsieved(b2mm)beforeexperimentalsetup. tionwithincreasesalinity(Bernhardetal.,2005;Bernhardetal.,2010; ThepHandsalinityofthetreatedsedimentmeasuredassedimentto Bernhardetal.,2007;Zhangetal.,2015).AstudyintheDouroRiveres- waterratioof1:5(g/mL)were7.06and3pptrespectively.Theconcen- tuaryobservedincreasednitrificationratewithasalinityincreasefrom trationsofammonium(NH+)andnitrateandnitrite(NO−)were12.9 4 x 0to15ppt(Magalhaesetal.,2005).Estuarinenitrifiersappeartogrow and1.94μgg−1drysediment,respectively.Thedriedsedimentwas optimallyat5–10ppt,andareinhibitedifsoilsalinityexceeds10ppt keptforthreemonthsbeforethestartoftheincubationexperiment. (Zhouetal.,2017).Thesestudiessuggestthatoptimumnitrification Analiquotof10gdrysedimentwasplacedin120-mLserumbottles rateornitrifierabundancenormallyoccursinmiddlesalinityrangesal- and then 10 mL distilled water was added to each bottle. All the thoughsomevaryingresultsexisted.Increasingsalinitycanimpactni- serumbottlesweresealedwiththewrapswithtinyholestolettheair trification directly by constraining the fitness of nitrifiers and gothroughbutpreventthewaterrunningoff.Thenthesebottleswere indirectlybyinfluencingtheavailabilityofoxygenwhichcanfurther incubatedat25°Cinadarkandaerobicenvironmentforonemonth impactthesoilrespiration(Zhouetal.,2017).However,ammoniaad- torecoverthemicroorganisms.Afterthat,thedifferentsalinitieswere sorptioninsedimentisalsodecreasedwithanincreaseinsalinity,po- manipulated by adding solutions with different concentrations of tentially contributing to increased ammonia efflux, which further NaCl.Theincubationswerecategorizedinto4groupsrepresenting4sa- stimulatesnitrification(Seitzingeretal.,1991;Rysgaardetal.,1999). linitygradients.Eachgroupreceivedcorresponding5-mLNaClsolu- Denitrification,whichreturnsnitrogenbacktotheatmosphereas tions.Thefinalsalinitygradientswere0ppt,10ppt,20pptand30ppt N OandN , also showsdifferent relationships with salinity. Ithas ascalculatedbytheaddedNaCltotheaddedwater(15mLintotalfor 2 2 beenshowntobenegativelyassociatedwithsedimentsalinityinthe eachbottle).InadditiontoNaCl,300μMKNO wasalsoaddedtoeach 3 range of 0–36 ppt in certain estuaries (Giblin et al., 2010; Santoro incubationtostimulatethenitrogenprocess.Afteraddingthesolutions, etal.,2008),butalsotohavenoassociationwithsalinitiesbetween2 allthebottleswereincubatedat25°Cinadarkandaerobicenviron- and24pptinotherplaces(Fearetal.,2005).Denitrifierabundance mentforanotheronemonth.Sedimentsamplesweretakenat0,7,14, andpotentialhavebeenassociatedwithlowsalinitiesaround5ppt 21and28days.Foreachsampling,twoofthreeincubationsofeachsa- (Franklinetal.,2017;Martonetal.,2012).Salinitycaninfluencedenitri- linitygradientweretakenforDNAextractionandnutrientmeasure- ficationbyalteringtheaccessibilityoforganicsubstrateswhicharees- ment,andfordeterminingdenitrifyingactivity.Theusedincubations sentialforheterotrophicbacteria(Franklinetal.,2017).Theinfluence wereexcludedfromfuturesamplingtoavoidtheunbalanceofsample ofincreasedsalinityonnitrificationcanchangethenitrateavailability, quantities.Intotal,120incubationswereprocessed,including60each whichcanalsoaffectthedenitrification(Giblinetal.,2010).Moreover, (4salinitygradients×5timepoints×3replicates)forDNAextraction dissimilatorynitratereductiontoammonium(DNRA)cancompetefor anddenitrifyingactivitydetermination. nitratewithdenitrificationsinceDNRAisfavoredoverdenitrification withhighstorageofsulfideswhichisalwaysassociatedwithhighsalin- ityconditionssuchascoastalandmarineecosystems(Giblinetal., 2.2.Denitrifyingactivityandnutrientmeasurement 2013;Marchantetal.,2014).Salinityalsoexhibitsasignificantimpact onsoilbacterialandfungalcommunitystructure(Asgharetal.,2012; Thepotentialdenitrifyingactivitywasmeasuredaccordingtothe Beheraetal.,2017;Chenetal.,2017;MohamedandMartiny,2011). C H inhibitionmethod.Ateachtimepoint,threeincubationsofeach 2 2 However,onlyseveralstudieshaveexploredtheassociationbetween salinitygradientweresealedwithrubberstoppersandthenevacuated salinityandnitrifyingordenitrifyingcommunitystructure(Bañeras andflushedwith99.999%heliumthreetimes.C H wasinjectedtoa 2 2 etal.,2012;Bernhardetal., 2007;Franklin etal.,2017;Sahanand finalcompositionof10%(vol/vol).Thesedimentswerethenincubated Muyzer,2008;Xieetal.,2014). at25°Conamagneticstirringapparatusfor12days.Theheadspace Thedifferencesoftheresponsetosalinityinnitrogencyclingpro- gaseswereanalyzedatevery2,4or12hforthefirsttwodays,and cessesandthestructureoftheresponsiblemicroorganismsaretobeex- thenevery24or48hforthenext10dayswithagaschromatographer pected because of the influence of un-measured environmental equippedwithanECDdetectoraspreviouslydescribed(Molstadetal., variables.Itishenceessentialtocontrolenvironmentalsystemsas 2007).Thedenitrifyingactivitywascalculatedbylinearregressionof muchaspossible.Inthisstudy,alaboratoryincubationexperiment accumulatedN Opergramdrysedimentversustime.Theconcentra- 2 wasconductedtoinvestigatetheresponseofmangrovesedimentnitri- tionsofNH+andNO−weredeterminedusing2MKClextractswitha 4 x fyinganddenitrifyingcommunitiestodifferentsalinities(0,10,20and 1:10ratiooffreshsedimenttoKClsolution(g/mL).Theextractswere 30ppt).DenitrifyingactivitywasmeasuredasstimulatedN Oemission, furtheranalyzedbyFIAQC8500continuousflowinjectionanalyzer 2 andtheabundanceofnitrifiers(AOAandAOB)anddenitrifiers(nirK, (LACHAT,USA). 1344 H.Wangetal./ScienceoftheTotalEnvironment631–632(2018)1342–1349 2.3.DNAextractionandgenequantification deduced to amino acid to build the neighbor-joining phylogenetic treeswith1000timesbootstrapusingMEGAv7.0(Kumaretal.,2016). TotalDNAwasextractedfrom0.5gfreshsedimentwiththeFastDNA All the sequences of AOA and AOB clones were submitted to SPINKitforsoil(MPBiomedicals,SantaAna,CA,USA)accordingtothe GenBank with the accession numbers MF566141-MF566739 and manufacturer'sinstructions.Thepurityandconcentrationoftheex- MF566740-MF567338,respectively.AllthesequencesofnirK,nirSand tracted DNA were measured using UV–vis spectrophotometer ND- nosZ were submitted to the European Nucleotide Archive of EMBL 1000(NanoDrop,USA).TheextractedDNAwasthenstoredat−20°C withtheaccessionnumberPRJEB21903. formolecularexperiments. Theprimerpairsusedforamplifyingandquantifyingthearchaeal 2.5.Statisticalanalysis andtheβ-proteobacteriaamoAgenes,nirS,nirKandnosZgeneswere describedinpreviousstudies(Wangetal.,2014;Wangetal.,2015). Analysisofvariance(ANOVA)wasemployedtoobservethesignifi- Theconditionsusedforreal-timequantitativePCR(qPCR)ofthese5 canceofthesalinityimpactonthedenitrifyingactivity,nutrientconcen- geneswerealsodescribedinthetwopreviousstudies,withtheexcep- trations,geneabundancesandOTUrelativeabundances.Datafailedto tionthatreactionswereconductedonaLightCycler480IIReal-TimePCR meettheassumptionsofANOVAwerelogtransformedoranalyzedby System(Roche,IN,USA).Toevaluatetherelativeabundanceofeach thenon-parametricKruskal-Wallistest.TheTukeyposthocmultiple gene,theabundancesofbacterialandarchaeal16SrRNAgeneswere comparisons were conducted to compare the differences between alsoquantified.Theprimerpairsandconditionsusedfor16SrRNA eachtwoofthesalinitygradients.Nonmetricmultidimensionalscaling genesweredescribedinapreviousstudy(Wangetal.,2017).Thecon- (nMDS)basedontheBray-Curtisdissimilaritymatrixwasperformedto ditionsofqPCRandprimersforallgenesusedinthisstudyweresum- determinetheinfluenceofsalinityonthenitrifyinganddenitrifying marized in Table S1. Standard curves of qPCR were obtained by communitycompositions.Analysisofsimilarities(ANOSIM)andper- seriallydilutingstandardplasmidscontainingthetargetgeneswith mutationalmultivariateanalysisofvariance(PERMANOVA)wereused knowncopynumbers.NegativecontrolswithoutDNAtemplatewere totestthesignificanceofthesalinityimpactonthemicrobialcommu- includedineachamplification.Inhibitionwaseliminatedbyhighlydi- nitystructures.PairwisePearsoncorrelationswereconductedbetween lutingtheDNAextracts.PCRefficiencyabove90%wasaccepted. denitrifyingactivity,NH+concentration,NO−concentrationandabun- 4 x dancesofnitrifyinganddenitrifyinggenes.AlltheP-valuesweread- justedbyfalsediscoveryrate(FDR)methodformultiplecomparisons 2.4.Clonelibraries,high-throughputsequencingandphylogeneticanalysis andthenullhypothesiswasrejectedwhileP-valueswasb0.05.Allthe statistical analyses were processed in R with vegan, car and Hmisc TheDNAsamplesfromday28wereusedformicrobialcommunity packages. compositionanalysis.ThecommunitycompositionsofAOAandAOB wereanalyzedbycloningwhilethecommunitycompositionsofnirK, 3.Results nirSandnosZweretestedusinghigh-throughputsequencing.ThePCR mixtureandreactionofAOAandAOBweredescribedintheprevious 3.1.Denitrifyingactivityandnutrientconcentrations study(Wangetal.,2015).ThePCRproductsofthe12sampleswere usedtoconstructtheclonelibraries and50clones foreachlibrary TheactivityofdenitrificationwascalculatedastheaverageN O 2 wereselectedforsequencing.Intotal,599cleansequenceswereused emissionperdayduringa12-dayincubationaftersampling(Fig.S1). forclusteringforbothAOAandAOB.Similarityof97%wasusedasthe Theemissionrateinsedimentwithgreatersalinity(20–30ppt)wassig- thresholdforpickingoperationaltaxonomicunit(OTU)usingMothur nificantlylowercomparedtothelowersalinity(0–10ppt).Oneexcep- v1.19(Schlossetal.,2009). tion to this trend was on day 7 and 14 of the incubations, when Fordenitrifyinggenes,eachreverseprimercorrespondingtoacer- emissionsweresignificantlyreducedin10-ppt,whencomparedtothe tain sample was tagged with a six-base barcode. Amplifications of 0-pptcontrol(Fig.1a).Duringthe28-dayincubation,theammonia nirK, nirS and nosZ were processed as previously described (Wang wasdepletedin0-and10-pptsedimentsonday21and28,respectively etal.,2014),butwiththebarcodedprimers.PCRproductswerepurified (Fig.1b).Ammoniaconcentrationswerealsosignificantlylowerthan withtheUniversalDNAPurificationKit(TIANGEN,China).Thepurified thatin30-pptsamplesonday7and14.Althoughammoniaconcentra- productconcentrationswerethenconfirmedusingtheQuantiFluor tionin20-pptsedimentsshowedanincreaseonday14and21,itde- dsDNASystem(Promega,CA,USA).ThebarcodedPCRproductswere creaseddramaticallyonday28.Theseresultssuggesttheinhibitionof equallypooled,whichwerethenprecipitatedbyethanolwithsodium ammoniaconsumptionbyhighsalinity.However,theNO−concentra- x acetateanddissolvedinsterilizedMilli-Qwater.ThefinalDNApool tionexhibitedanoppositepattern.Theconcentrationwassignificantly wassequencedonanIlluminaMiSeqPE300platform.Thesequencing lowerinboththe20-and30-pptsamplesonday14,whilethelowest datawereanalyzedusingQIIMEv1.9.1(Caporasoetal.,2010).Rawse- NO−concentrationoccurredin20-and30-pptsamplesonday21and x quencesweredemultiplexedandlowqualityorambiguousreadswere day28,respectively(Fig.1c).Despitethat,theoverallNO−concentra- x removed.Theframe-shifterrorswerecheckedusingtheHMM-FRAME tionswerenotsignificantlydifferentfromday0today28.Correlation algorithm(ZhangandSun,2011)togetherwithHiddenMarkovModels analysisdemonstratedthattheNH+andNO−concentrationswereneg- 4 x ofdifferentgenesfromtheFunGenedatabase(Fishetal.,2013).Se- ativelyandpositivelyassociatedwiththeaverageN Oemissionrate,re- 2 quenceswithframe-shiterrorswerediscarded.Intotal,therewere spectively(Pb0.05,Fig.S2). 401,963,729,176and508,882cleansequencesfornirK,nirSandnosZ, respectively.FilteredsequenceswerethenclusteredintoOTUsbased 3.2.Theabundancesofnitrifyinganddenitrifyinggenes on 97% similarity using the “pick_otu.py” function with USEARCH method.Thesingletonsandchimericsequenceswereremovedduring Theabundances of amoAgenesforbothAOA(Fig.2a)andAOB theOTUpicking.Afterthat,eachsamplewasrarefiedtoequalsequence (Fig.2b)increasedfromday0today28.ForAOA,thedifferenceonlyoc- depth(thesmallestnumberofthesequencesamongallsamples)for curredonday7thatamoAgeneabundancewassignificantlylowerin downstreamanalyses. highsalinitysamples(20and30ppt)thaninthelowsalinitysamples TherepresentativesequencesofOTUswitharelativeabundance (0 and 10 ppt) (Fig. 2a). Interestingly, the abundance of bacterial above1%wereblastedagainsttheGenBanknucleotidedatabaseand amoAgenewassignificantlyhigherintheintermediatesalinitysamples referencesequenceswerechosenasthehighlysimilarsequencesto (10or20ppt)comparedtothoseinthelow(0ppt)orhigh(30ppt)sa- eachOTU.Thenboththerepresentativeandreferencesequenceswere linity samples (Fig. 2b), suggesting a different response to salinity H.Wangetal./ScienceoftheTotalEnvironment631–632(2018)1342–1349 1345 Fig.1.ChangesintheaverageN2Oemissionrate(a),ammoniaconcentration(b),andnitrateandnitriteconcentration(c)onday0,day7,day14andday28.Thedifferentlettersamong samplesateachtimepointindicatethesignificantdifference(Pb0.05).Thevaluesaregivenasmean±standarddeviation(n=3).*,undetected. impactbetweenAOBandAOA.Moreover,theAOBabundancewassev- eralordersofmagnitudegreaterthanAOAabundance,despitecompa- rable abundance on day 0, suggesting that AOB dominated nitrification.Therelativeabundancecalculatedasthecopynumber ratiooffunctionalgenetocorresponding16SrRNAgenewasinvesti- gated(Fig.S3).TheresultsofrelativeabundancesofAOAandAOB showedasimilartrend,withtheexceptionthatdifferenceofAOArela- tiveabundancealsooccurredonday21whentheabundancewassig- nificantlylowerinhighsalinitysamples(20and30ppt)compared with0-pptsamples(Fig.S3). Salinityhadalimitedinfluenceondenitrifyinggeneabundance. Onlyonday28thelowestandhighestnirKabundancewasdetected in30-pptand0-pptsamples,respectively(Fig.3a).Whiletherewas nosignificantchangeinnirSabundance(Fig.3b),thenosZabundance wassignificantlylowerin30-pptsamplescomparedtotheothersam- plesonday28(Fig.3c).Onday28,thenirK/nirSratiowassignificantly greaterin0-pptsamplescomparedtotheothersamples(Fig.3d),while thenir/nosZratiowasgreatestin30-pptsamples(Fig.3e).Thenir/nosZ ratiowaspositivelyandsignificantlycorrelatedwithdenitrifyingactiv- ityandNO−concentration(Pb0.05,Fig.S2).Therelativeabundancesof x nirK,nirSandnosZallshowedasimilartrendtotheabsoluteabun- dances(Fig.S3). 3.3.Thecommunitycompositionsofnitrifiersanddenitrifiers Intotal,53and26OTUswereobtainedforAOAandAOBcommuni- ties,respectively.nMDSplotsshowedthatbothAOAandAOBcommu- nitiesweregroupedaccordingtodifferentsalinitygradients(Fig.4aand b).BoththeANOSIMandPERMANOVAindicatedthatsalinitysignifi- cantly impacted AOA and AOB community composition (P b 0.01, Table 1). For denitrifying communities, 1607, 7259 and 6148 OTU wereobservedfornirK,nirSandnosZcommunities,respectively.The nirKandnosZcommunitiesofsamplesassociatedwithdifferentsalinity gradientswerewellgroupedwhilethenirScommunitieswerelessdis- tinctlydistributed(Fig.4c,dande).However,thenirK,nirSandnosZ communitycompositionswereallsignificantlyinfluencedbysalinity asdemonstratedbytheANOSIMandPERMANOVAresults(Pb0.05, Table1). 3.4.Phylogeneticanalysis ToreducetheeffectofrareOTUsonphylogenetictreeconstruction, wechosetheOTUswitharelativeabundanceN1%.Thedistributionof abundantOTUsfordifferentgenesaredisplayedinFig.S4.According tothephylogenetictreeforAOA(Fig.S5)andAOB(Fig.S6),theabun- dantOTUscouldbegroupedinto4and3clusters,respectively.The Fig.2.ChangesintheamoAgeneabundanceofAOA(a)andAOB(b)onday0,day7,day AOA cluster I, which was associated with Nitrososphaera (Fig. S5), 14 and day 28. The different letters among samples at each time point indicate thesignificantdifference(Pb0.05).Thevaluesaregivenasmean±standarddeviation accountedforthelargestproportion(74.0%)ofAOAsequences.Therel- (n=3). ativeabundanceofAOAclusterIwasslightlygreaterinthe0-and10- 1346 H.Wangetal./ScienceoftheTotalEnvironment631–632(2018)1342–1349 Fig.3.Changesintheabundancesofdenitrifyinggenes,nirK(a),nirS(b)andnosZ(c),aswellasthenirK/nirS(d)andnir/nosZ(e)ratiosonday0,day7,day14andday28.Thedifferent lettersamongsamplesateachtimepointindicatethesignificantdifference(Pb0.05).Thevaluesaregivenasmean±standarddeviation(n=3). pptsamplescomparedtothosein20-and30-pptsamples(Fig.5a).The Thesefindingssuggestthatsalinityintrusionincoastalareasmightin- AOBclusterIIwhichwasaffiliatedwithNitrosomonas(Fig.S6)occupied terruptthebalanceofnutrientcycles,andcouldnegativelycontribute 76.4%ofthetotalAOBmembers.BothAOBclusterIandIIdominated0- toecosystemfunctions. and10-pptsampleswhileonlyclusterIIflourishedin20-and30-ppt SalinityexhibitedalimitedinfluenceonAOAabundanceinthis samples(Fig.5b),whichsuggestedthathighsalinityinhibitedtheclus- study,whichisdifferentfrompreviousstudiesshowingthatAOAwas terIofAOBwhichwasassociatedwithNitrosospira(Fig.S6). themostabundantatintermediate salinity(10–30ppt)(Bernhard Basedonthephylogeneticanalysesofdenitrifyingcommunities,the etal.,2010;Zhangetal.,2015).However,AOBplayedadominantrole nirK(Fig.S7),nirS(Fig.S8)andnosZ(Fig.S9)sequencescanbegrouped innitrificationinthisstudysincetheAOBabundanceoutcompeted into3,10and7clusters,respectively.FornirK,clusterIwasthedomi- AOAabundance.TherelativeimportanceofAOAandAOBfornitrifica- nantclusteraccountingfor58.4%ofthetotalsequences.Thiscluster tion in salineenvironments remains controversial. Consistentwith wassignificantlygreaterinthehighsalinity(20and30ppt)samples thisstudy,theAOBabundancewasgreaterthanAOAinsediments thaninthelowsalinity(0and10ppt)samples(Fig.5c,TukeyHSDPb fromDouroRiverestuarydespiteofthevariedsalinitiesrangingfrom 0.05),suggestingpreferenceofhighsalinityconditionofthiscluster. 1.5 to 26.8 ppt (Magalhães et al., 2009). In some estuaries, it was However,nirKclusterIcannotbeassignedtoaknowntaxon(Fig.S7). foundthatAOApredominatedthenitrificationintheregionswithlow FornirS,onlyclusterVandVIIshowedaslightlydifferentabundance salinitywhileAOBweremoreabundantthanAOAinhighersalinityre- amongdifferentsalinitygradients(Fig.5d).FornosZ,clusterIandclus- gions(MosierandFrancis,2008;Santoroetal.,2008).Incontrast,AOA terIIIwerethedominantclusters,withaproportionof10.8%and20.2%, abundancewasgreaterthanAOBatallthesalinitylevelsinPlumIsland respectively.TheabundanceofclusterIIIwhichmightbeassociated Soundestuary(Bernhardetal.,2010).Thesedivergentresultsindicate withThiobacillus(Fig.S9)wassignificantlylowerin30-pptsamples thatotherfactorsinfluencetheabundanceofAOAandAOB.Itissug- comparedto0-pptsamples(Fig.5e,TukeyHSDPb0.05). gestedthatthedominanceofAOBoverAOAnormallyoccursinman- grovesedimentsastheslightlyacidic or neutral conditionandthe 4.Discussion availableammoniamightfavorthegrowthofAOB(Lietal.,2011;Li andGu,2013;Wangetal.,2015).Moreover,wefoundthatAOBabun- Thedenitrifyingactivity,abundanceofnitrifiersanddenitrifiers,and dancewasgreaterinthe10-or20-pptsedimentsduringthe28-dayin- theircommunitystructureswereinvestigatedinmangrovesediments cubation.Thisfindingsupportsapreviousmeta-analysisshowingthat treatedwithdifferentsalinities.Salinityexhibitedasignificantinfluence nitrificationcanbepromotedwithinacertainrangeofthesalinityand onthenitrogencyclingprocesses.Increaseofsalinityfrom0to30ppt inhibitedwhensalinityexceedsthisrange(Zhouetal.,2017).Forthis decreasedbothdenitrificationactivityandtheabundanceofdenitri- study,theintermediatesalinities(10and20ppt)wereconsideredas fiers,whilethenitrifiersweremostabundantundertheintermediate theoptimalconditionfortheammoniaoxidizers,whichmightalsore- salinityconditions.Thecommunitycompositionofnitrifiersanddeni- sultfromthelong-termadaptionofthemicrobestothelocalhabitats trifierswassignificantlyinfluencedbysalinity.Althoughthisstudyis wherethesedimentsalinitynormallyshiftedbetween10and20ppt basedonthemanipulatedsedimentrecoveredfromthedriedsediment, (Wangetal.,2016).WeobservedthatNH+concentrationwassignifi- 4 therecoveredmicrobialcommunitieswereoriginallyfromthesedi- cantlyhigherinthe20-and30-pptsamples,suggestingthelesscon- mentinthefield,andthuspartlyrepresentingthein-situcondition. sumption of NH+ which might be caused by the reduction of 4 H.Wangetal./ScienceoftheTotalEnvironment631–632(2018)1342–1349 1347 Fig.4.Nonmetricmultidimensionalscaling(nMDS)plotsofAOA(a),AOB(b),nirK(c),nirS(d)andnosZ(e)basedontheBray-Curtisdissimilarities. nitrificationactivity.However,nosignificantcorrelationwasfoundbe- 2005;SahanandMuyzer,2008).Thecontrastingresultsonthedistribu- tweenNH+concentrationandAOBabundance.Thismaybeduetothe tionofNitrosospiramightresultfromtheothercovaryingfactors,such 4 enhancedDNRAactivityasDNRAisfavoredunderthehighersalinity astemperatureandammoniaconcentration;however,salinitywas condition(Giblinetal.,2013;Marchantetal.,2014),whichmightfur- theonlyfactorthatdrovechangesinourstudy.Ourresultsalsoindicate theroffsettheconsumedNH+. thatNitrosomonasmightpossessabroaderadaptiontothesalinity 4 Inadditiontotheabundanceofnitrifiers,salinityexhibitedasignif- levels compared to Nitrosospira. However, the mechanism on how icantimpactonbothAOAandAOBcommunitystructure.Theseresults theytoleratethesalinitystressremainsunknown. supportpreviousstudiesshowingthatsedimentsalinitywasthemain Itissuggestedthatdenitrificationisconsistentlydecreasedbysoil factor controlling the distribution of AOA or/and AOB (Sahan and salinizationacrosscoastalecosystems(Zhouetal.,2017).Insupportof Muyzer,2008;Zhangetal.,2015).ThegenusNitrososphaeradominated this,denitrifyingactivitydeclinedwithincreasedsalinityfrom0to AOAcommunityinallsamples,butwasslightlyrestrictedathighsalin- 30pptinthisstudy.Therearemanymechanismsthatmaycontribute itylevelsinthisstudy.However,asonlyoneNitrososphaeraisolatehas tothisphenomenon.Foralaboratoryincubation,salinityelevation beenidentified(Tournaetal.,2011),littlecanbesaidaboutitspotential mightdirectlyconstrainthefitnessofmicrobesandatthesametimere- interaction.ForAOB,wefoundthatboththehighandlowsalinitiesfa- ducethesoilrespiration(Wongetal.,2008)whichcoulddecreasethe voredNitrosomonaswhileNitrosospiraexistedonlyatlowsalinitylevels. oxygenconsumptionthusinhibitingtheanaerobicdenitrifyingprocess. Twoformerstudiesshowedthatalongasalinitygradient,Nitrosospira Thereductionofnitrificationbysalinityelevationcanreducethenitrate wasenrichedatthehighsalinitysiteswhilethegenusNitrosomonas availability,whichcanalsolimitthedenitrification(Giblinetal.,2010). tendedtoassociatewithlowormiddlesalinitysites(Bernhardetal., Inthisstudy,theenhancedconsumptionofNO−bysalinity,whichin- x creasedduringtheincubation,couldbeduetothepromotedDNRA Table1 under high salinity conditions (Giblin et al.,2013; Marchant et al., TheANOSIMandPERMANOVAresultsshowingtheinfluenceofsalinityonnitrifyingand 2014).Hence, ourresultspotentially indicate thepredominanceof denitrifyingcommunitycompositions. DNRAoverdenitrificationinsedimentsthataresubjecttohighsalin- ANOSIM PERMANOVA ities.Ontheotherhand,theabundanceofdenitrifiers(nirKandnosZ) R Significance R2 Significance wasalsoimpactedbysalinization,butinhibitiononlyoccurredatday 28,suggestingthetoleranceofdenitrifierstosedimentsalinizationat AOA 0.397 0.002** 0.409 0.010** thebeginningoftheincubation.ThedecreaseofthenirK/nirSratio AOB 0.559 0.005** 0.556 0.002** nirK 0.491 0.002** 0.479 0.003** withincreasingsalinityindicatedthattherelativeimportanceofnirK nirS 0.361 0.016* 0.355 0.025* andnirSforreducingthenitritemightshiftaccordingtosalinitylevels nosZ 0.361 0.005** 0.378 0.002** andthatnirS-bearingdenitrifiersmightbemoretoleranttosalinityele- Significancelevelisshownas*,Pb0.05;**,Pb0.01. vationcomparedtothosenirK.Moreover,theenhancednir/nosZratioin 1348 H.Wangetal./ScienceoftheTotalEnvironment631–632(2018)1342–1349 Fig.5.Thedistributionoftheclustersobtainedfromphylogeneticanalysisandtheirrelativeabundances.Thevaluesaregivenasmean±standarddeviation(n=3).Significancelevelis shownas●,Pb0.1;*,Pb0.05;***,Pb0.001. 30-pptsamplesatday28illustratedthatsalinityelevationmightrise Insummary,wefoundtheinhibitingeffectofsalinityelevationon thegeneticpotentialforN Oemissionssincethehighernir/nosZratio thedenitrifyingactivityandabundancesofnirKandnosZ,whilethe 2 canleadtoanincreasedN O/(N O+N )end-productratio(García- nirSabundanceremainedstable.TheabundanceofAOBincreasedatin- 2 2 2 Lledóetal.,2011). termediatesalinitylevelswhileAOAwerelessaffected.However,are- Apreviousstudyfoundthatsalinity,inadditiontoothermajorfac- ductionintheconsumptionofNH+athighsalinitylevelsmightsuggest 4 tors,wasassociatedwiththedynamicsofnirK,nirSandnosZcommunity theinhibitionofnitrificationbysalinization.Therefore,thereductionof compositioninwetlandsediments(Bañerasetal.,2012).Thesalinity couplednitrification-denitrificationprocessmightdecreasethenitro- wasalsofoundtocorrelatewithnosZcommunitystructuresacross genremoval,whichmightfurtherleadtoeutrophicationanddisturb eighttidalwetlands(Franklinetal.,2017).However,astudyinthe thenitrogenbalanceintheenvironment.Whilethereislimitedinfor- SanFranciscoBayestuaryrevealedsignificantcorrelationsbetweensa- mationaboutthesalinityandmicrobialcommunitycomposition,this linityandabundancesofnirKandnirSbutnottheircommunitystruc- studyprovidesstrongevidencethatsalinitycansignificantlyshape tures(MosierandFrancis,2010).Sincethesestudieswereconducted thecommunitystructureofbothnitrifiersanddenitrifiers.Theseresults withsamplesinsitu,theimpactofsalinitywascoupledwithother illustratethatsalinityplaysavitalroleinregulatingthenitrogencycling majorfactorssuchasvegetation,carbonsourceandmetals.Henceour incoastalareas,whichmayincreaseconcernregardingthreatsofsalin- study does provide strong evidence that salinity can significantly ityintrusionassociatedwithclimatechangeandarisingsealevel. shapethecommunitystructuresofdenitrifiersintheabsenceofother factors.AlthoughweobservedthatthedominantnirKclusterIwassig- Acknowledgments nificantlyinfluencedbythesalinity,littleisknownaboutthiscluster. Similarly,theclusterVandVIIofnirSwereslightlyinfluencedbythesa- ThisworkwassupportedbytheStrategicPriorityResearchProgram linity,buttheyarealsounknowntaxa.ThedominantclusterIIIofnosZ ofChineseAcademyofSciences(XDB15020402),theNationalNatural which might be associated with Thiobacillus was decreased by the ScienceFoundationofChina(41430858,41771285,41571130063), highsalinity(30ppt).Mangrovecontainshighlevelofsulfidesgener- andtheInternationalScience&TechnologyCooperationProgramof atedbysulfatereduction(Ferreiraetal.,2007),andsomespeciesbe- China(2011DFB91710).WethankProf.TianlingZheng(deceased)from longing to Thiobacillus can use the sulfur and oxidize sulfide to XiamenUniversityforhishelpwithdesigningandanalyzingthedata. supporttheirautotrophicgrowth(Friedrichetal.,2001).Ithasbeenrec- Theauthorsdeclarenoconflictofinterests. ognizedthatsulfuroxidationcanbecoupledwithnitratereduction (BurginandHamilton,2007);thusreductionofsulfidebyoxidation AppendixA.Supplementarydata mightleadtothedecreaseofsulfur-drivennitratereduction.Therefore, theinhibitionofThiobacillusbysalinizationmightleadtothereduction Supplementarydatatothisarticlecanbefoundonlineathttps://doi. ofdenitrificationasweobservedinthisstudy. org/10.1016/j.scitotenv.2018.03.102. H.Wangetal./ScienceoftheTotalEnvironment631–632(2018)1342–1349 1349 References Magalhaes,C.M.,Joye,S.B.,Moreira,R.M.,Wiebe,W.J.,Bordalo,A.A.,2005.Effectofsalinity andinorganicnitrogenconcentrationsonnitrificationanddenitrificationratesinin- Asghar,H.N.,Setia,R.,Marschner,P.,2012.Communitycompositionandactivityofmi- tertidalsedimentsandrockybiofilmsoftheDouroRiverestuary,Portugal.WaterRes. crobesfromsalinesoilsandnon-salinesoilsrespondsimilarlytochangesinsalinity. 39,1783–1794. SoilBiol.Biochem.47,175–178. Magalhães,C.M.,Machado,A.,Bordalo,A.A.,2009.Temporalvariabilityintheabundance Bañeras,L.,Ruiz-Rueda,O.,López-Flores,R.,Quintana,X.,Hallin,S.,2012.Theroleofplant ofammonia-oxidizingbacteriavs.archaeainsandysedimentsoftheDouroRiveres- typeandsalinityintheselectionforthedenitrifyingcommunitystructureintherhi- tuary,Portugal.Aquat.Microb.Ecol.56,13–23. zosphereofwetlandvegetation.Int.Microbiol.15,89–99. Marchant,H.K.,Lavik,G.,Holtappels,M.,Kuypers,M.M.,2014.Thefateofnitrateininter- Behera,P.,Mahapatra,S.,Mohapatra,M.,Kim,J.Y.,Adhya,T.K.,Raina,V.,etal.,2017.Salin- tidalpermeablesediments.PLoSOne9,e104517. ityandmacrophytedrivethebiogeographyofthesedimentarybacterialcommuni- Marton,J.M.,Herbert,E.R.,Craft,C.B.,2012.Effectsofsalinityondenitrificationandgreen- tiesinabrackishwatertropicalcoastallagoon.Sci.TotalEnviron.595,472–485. housegasproductionfromlaboratory-incubatedtidalforestsoils.Wetlands32, Bernhard,A.E.,Donn,T.,Giblin,A.E.,Stahl,D.A.,2005.Lossofdiversityofammonia- 347–357. oxidizingbacteriacorrelateswithincreasingsalinityinanestuarysystem.Environ. Mohamed,D.J.,Martiny,J.B.,2011.Patternsoffungaldiversityandcompositionalonga Microbiol.7,1289–1297. salinitygradient.ISMEJ.5,379–388. Bernhard,A.E.,Tucker,J.,Giblin,A.E.,Stahl,D.A.,2007.Functionallydistinctcommunities Molstad,L.,Dörsch,P.,Bakken,L.R.,2007.Robotizedincubationsystemformonitoring of ammonia-oxidizing bacteria along an estuarine salinity gradient. Environ. gases(O2,NO,N2ON2)indenitrifyingcultures.J.Microbiol.Methods71,202–211. Microbiol.9,1439–1447. Mosier,A.C.,Francis,C.A.,2008.Relativeabundanceanddiversityofammonia-oxidizing Bernhard,A.E.,Landry,Z.C.,Blevins,A.,delaTorre,J.R.,Giblin,A.E.,Stahl,D.A.,2010.Abun- archaeaandbacteriaintheSanFranciscoBayestuary.Environ.Microbiol.10, danceofammonia-oxidizingarchaeaandbacteriaalonganestuarinesalinitygradi- 3002–3016. ent in relation to potential nitrification rates. Appl. Environ. Microbiol. 76, Mosier,A.C.,Francis,C.A.,2010.DenitrifierabundanceandactivityacrosstheSan 1285–1289. FranciscoBayestuary.Environ.Microbiol.Rep.2,667–676. Burgin,A.J.,Hamilton,S.K.,2007.Haveweoveremphasizedtheroleofdenitrificationin Rysgaard,S.,Thastum,P.,Dalsgaard,T.,Christensen,P.B.,Sloth,N.P.,1999.Effectsofsalin- aquaticecosystems?Areviewofnitrateremovalpathways.Front.Ecol.Environ.5, ityonNH4+adsorptioncapacity,nitrification,anddenitrificationinDanishestuarine 89–96. sediments.Estuar.Coasts22,21–30. Caporaso,J.G.,Kuczynski,J.,Stombaugh,J.,Bittinger,K.,Bushman,F.D.,Costello,E.K.,etal., Sahan,E.,Muyzer,G.,2008.Diversityandspatio-temporaldistributionofammonia- 2010.QIIMEallowsanalysisofhigh-throughputcommunitysequencingdata.Nat. oxidizingArchaeaandBacteriainsedimentsoftheWesterscheldeestuary.FEMS Methods7,335–336. Microbiol.Ecol.64,175–186. Chen,L.,Li,C.,Feng,Q.,Wei,Y.,Zheng,H.,Zhao,Y.,etal.,2017.Shiftsinsoilmicrobialmet- Santoro,A.E.,Francis,C.A.,deSieyes,N.R.,Boehm,A.B.,2008.Shiftsintherelativeabun- abolicactivitiesandcommunitystructuresalongasalinitygradientofirrigation danceofammonia-oxidizingbacteriaandarchaeaacrossphysicochemicalgradients waterinatypicalaridregionofChina.Sci.TotalEnviron.598,64–70. inasubterraneanestuary.Environ.Microbiol.10,1068–1079. Duke,N.C.,Meynecke,J.-O.,Dittmann,S.,Ellison,A.M.,Anger,K.,Berger,U.,etal.,2007.A Schloss,P.D.,Westcott,S.L.,Ryabin,T.,Hall,J.R.,Hartmann,M.,Hollister,E.B.,etal.,2009. worldwithoutmangroves?Science317,41–42. Introducingmothur:open-source,platform-independent,community-supported Fear,J.M.,Thompson,S.P.,Gallo,T.E.,Paerl,H.W.,2005.Denitrificationratesmeasured softwarefordescribingandcomparingmicrobialcommunities.Appl.Environ. alongasalinitygradientintheeutrophicNeuseRiverEstuary,NorthCarolina,USA. Microbiol.75,7537–7541. Estuar.Coasts28,608–619. Seitzinger,S.P.,Gardner,W.S.,Spratt,A.K.,1991.Theeffectofsalinityonammoniumsorp- Ferreira,T.O.,Otero,X.L.,Vidal-Torrado,P.,Macías,F.,2007.Effectsofbioturbationbyroot tioninaquaticsediments:implicationsforbenthicnutrientrecycling.Estuaries14, and crab activity on iron and sulfur biogeochemistry in mangrove substrate. 167–174. Geoderma142,36–46. Sheng,Q.,Wang,L.,Wu,J.,2015.Vegetationalterstheeffectsofsalinityongreenhouse Fish,J.A.,Chai,B.,Wang,Q.,Sun,Y.,Brown,C.T.,Tiedje,J.M.,etal.,2013.FunGene:the gasemissionsandcarbonsequestrationinanewlycreatedwetland.Ecol.Eng.84, functionalgenepipelineandrepository.Front.Microbiol.4,291. 542–550. Fisher,J.,Acreman,M.,2004.Wetlandnutrientremoval:areviewoftheevidence.Hydrol. Tourna,M.,Stieglmeier,M.,Spang,A.,Könneke,M.,Schintlmeister,A.,Urich,T.,etal., EarthSyst.Sci.8,673–685. 2011.Nitrososphaeraviennensis,anammoniaoxidizingarchaeonfromsoil.P.Natl. Franklin,R.B.,Morrissey,E.M.,Morina,J.C.,2017.Changesinabundanceandcommunity Acad.Sci.USA108,8420–8425. structureofnitrate-reducingbacteriaalongasalinitygradientintidalwetlands. Vymazal,J.,2007.Removalofnutrientsinvarioustypesofconstructedwetlands.Sci.Total Pedobiologia60,21–26. Environ.380,48–65. Friedrich,C.G.,Rother,D.,Bardischewsky,F.,Quentmeier,A.,Fischer,J.,2001.Oxidationof Wang,H.T.,Su,J.Q.,Zheng,T.L.,Yang,X.R.,2014.Impactsofvegetation,tidalprocess,and reducedinorganicsulfurcompoundsbybacteria:emergenceofacommonmecha- depthontheactivities,abundances,andcommunitycompositionsofdenitrifiersin nism?Appl.Environ.Microbiol.67,2873–2882. mangrovesediment.Appl.Microbiol.Biotechnol.98,9375–9387. Galloway,J.N.,Townsend,A.R.,Erisman,J.W.,Bekunda,M.,Cai,Z.,Freney,J.R.,etal.,2008. Wang,H.T.,Su,J.Q.,Zheng,T.L.,Yang,X.R.,2015.Insightsintotheroleofplanton Transformationofthenitrogencycle:recenttrends,questions,andpotentialsolu- ammonia-oxidizingbacteriaandarchaeainthemangroveecosystem.J.SoilsSedi- tions.Science320,889–892. ments15,1212–1223. García-Lledó,A.,Vilar-Sanz,A.,Trias,R.,Hallin,S.,Bañeras,L.,2011.Geneticpotentialfor Wang,H.,Liao,G.,D'Souza,M.,Yu,X.,Yang,J.,Yang,X.,etal.,2016.Temporalandspatial NW2aOteermReisss.i4o5n,s5f6r2o1m–5t6h3e2s.edimentofafreewatersurfaceconstructedwetland. Cvahriniaat.ioEnnsviorofng.rSecein.Phooluluste.Rgaess.flInutx.e2s3,fr1o8m73a–1ti8d8a5l.mangrovewetlandinSoutheast Giblin,A.E.,Weston,N.B.,Banta,G.T.,Tucker,J.,Hopkinson,C.S.,2010.Theeffectsofsalin- Wang,H.T.,Marshall,C.W.,Cheng,M.Y.,Xu,H.J.,Li,H.,Yang,X.R.,etal.,2017.Changesin ityonnitrogenlossesfromanoligohalineestuarinesediment.Estuar.Coasts33, landusedrivenbyurbanizationimpactnitrogencyclingandthemicrobialcommu- 1054–1068. nitycompositioninsoils.Sci.Rep.7,44049. Giblin,A.E.,Tobias,C.R.,Song,B.,Weston,N.,Banta,G.T.,Rivera-Monroy,V.H.,2013.The Wong,V.N.L.,Dalal,R.C.,Greene,R.S.B.,2008.Salinityandsodicityeffectsonrespiration importanceofdissimilatorynitratereductiontoammonium(DNRA)inthenitrogen andmicrobialbiomassofsoil.Biol.Fertil.Soils44,943–953. cycleofcoastalecosystems.Oceanography26,124–131. Xie,W.,Zhang,C.,Zhou,X.,Wang,P.,2014.Salinity-dominatedchangeincommunity Gruber,N.,Galloway,J.N.,2008.AnEarth-systemperspectiveoftheglobalnitrogencycle. structureandecologicalfunctionofArchaeafromthelowerPearlRivertocoastal Nature451,293–296. SouthChinaSea.Appl.Microbiol.Biotechnol.98,7971–7982. Kumar,S.,Stecher,G.,Tamura,K.,2016.MEGA7:molecularevolutionarygeneticsanalysis Zhang, Y., Sun, Y., 2011. HMM-FRAME: accurate protein domain classification for version7.0forbiggerdatasets.Mol.Biol.Evol.33,1870–1874. metagenomicsequencescontainingframeshifterrors.BMCBioinformatics12,198. Li,M.,Gu,J.-D.,2013.Communitystructureandtranscriptresponsesofanammoxbacte- Zhang,Y.,Chen,L.,Dai,T.,Tian,J.,Wen,D.,2015.Theinfluenceofsalinityontheabun- ria,AOA,andAOBinmangrovesedimentmicrocosmsamendedwithammoniumand dance,transcriptionalactivity,anddiversityofAOAandAOBinanestuarinesedi- nitrite.Appl.Microbiol.Biotechnol.97,9859–9874. ment:amicrocosmstudy.Appl.Microbiol.Biotechnol.99,9825–9833. Li,M.,Cao,H.,Hong,Y.,Gu,J.-D.,2011.Spatialdistributionandabundancesofammonia- Zhou,M.,Butterbach-Bahl,K.,Vereecken,H.,Bruggemann,N.,2017.Ameta-analysisof oxidizingarchaea(AOA)andammonia-oxidizingbacteria(AOB)inmangrovesedi- soilsalinizationeffectsonnitrogenpools,cyclesandfluxesincoastalecosystems. ments.Appl.Microbiol.Biotechnol.89,1243–1254. Glob.Chang.Biol.23,1338–1352. Liu,X.,Ruecker,A.,Song,B.,Xing,J.,Conner,W.H.,Chow,A.T.,2017.Effectsofsalinityand wet–drytreatmentsonCandNdynamicsincoastal-forestedwetlandsoils:implica- tionsofsealevelrise.SoilBiol.Biochem.112,56–67.

See more

The list of books you might like

Upgrade Premium
Most books are stored in the elastic cloud where traffic is expensive. For this reason, we have a limit on daily download.
DMCA & Copyright: Dear all, most of the website is community built, users are uploading hundred of books everyday, which makes really hard for us to identify copyrighted material, please contact us if you want any material removed.
Copyright @ PDFdrive.to, 2022 | We love our users