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Characteristics of the microbial communities in the integrated vertical-flow constructed wetlands

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Characteristics of the microbial communities in the integrated vertical-flow constructed wetlands

    Characteristics of the microbial communities in the integrated vertical-flow

    constructed wetlands

    Availableonlineat,^,\I\,vv.sciencedirect.com

    ScienceDirect

    JournalofEnvffonmentalSciences21(2009)12611267

    JoInRNALoF

    ENVIRONMENTAL

    SCIENCES

    !:!2

    CN11-2629,X

    www.1esc.ac.on

    Characteristicsofthemicrobialcommunitiesintheintegratedvertical-flow constructedwetlands

    ZHOUQiaohong,HEFeng,ZhangLiping,WANGYanfen,WUZhenbin

    StateKeyLaboratoryofFreshwaterEcologyandBiotechnology,InstituteofHydrobiology,ChineseAcademyofSciences,

    Wuhan430072,China.E-mail:hefeng@ihb.ac.cPl

    Received07November2008;revised15December2008;accepted12January2009 Abstract

    Microorganismsplayanimportantroleinremovingpollutantsfromconstructedwetlands.Weinvestigatedthemicrobia1

    characteristicsinanovelintegratedvertical

    flowconstructedwetland(IVCW),whichhasbeeninoperationin,vuhan.Chinasince 1998.Weusedphospholipidfattyacid(PLFA1andamoAgenetoanalyzethestructureanddiversityofthemicrobialcommunity

    withintheIVCW.PLFAresultssuggestedthattheamountofbacterialPLFAwassignificantlyhigherthanthatoffungalPLFA.but

    thetotalmicrobialbiomassrepresentedbyPLFAindexwaslowinthesystem.Microbialspatialdistributionshowedsignificantly

    higherbacterial(bothGandG

    )andfungalbiomassinthesurfacethaninthesubsurfacelayers.Theratiosofmonounsaturatedto

    branchedPLFAdemonstratedthatananaerobiclayersandwichedbytwoaerobiclayersexistedintheIVCW.consistentwitl1theredox

    potentialresults.AnalysisoftheamoArevealedepresenceofNitrosomonas

    likesequencesinthesurfacesubstrateofthedownflow

    chamberandapparentdiversitiesofammonia

    oxidizingbacteriainthesystem.Theseresultssuggestthatmicroorganisms,despitetheir relativelylowbiomass.haveinhabitedtheIVCVandtheresultswil1offersomevaluableinformationonmicrobetosystemdesigners

    andmanagers.

    Keywords:ammonia

    oxidizingbacteria;constructedwetland;microbialcommunity;PLFA

    DOI:10.10l6/S1001O742(08)624134

    Introduction

    W_etlandslocatebetweenterrestrialandaquaticsystems

    andoftenpossessthecharacteristiesofbothsystems.

    Theyperformmanyimportantbiogeochemicalfunctions

    inwatershedsduetotheiruniquelocation(Wangeta1.,

    2o06,.Theyhavebeenfoundtobeusefulinremediat

    ingenvironmentcontaminatedwithbiochemicaloxygen

    demand(BOD),totalsuspendedsolid(TSS),nitrogen

    andphosphorusaswellasvariousorganicpollutantsor

    metaloxides,orevenpathogens(Thurstoneta1.,2001).

However,asuncontrolledecosystems,naturalwetlands

    sufferfromhydrologicalshortcircuiting.poorcontrol overchemicalandbiologicalprocesses(Mitsch,1994), andarevulnerabletoenvironmentalchanges.Tobetter understandtheirfunctionsandexploretheirapplications, constructedwetlandwasintroducedasamodelsystem

     andhasbeenusedworld.wide.Theseconstructedwet

    landshavebeenprovedvaluableinnotonlyremoving environmentalcontaminants.butalsoprovidinghabitatfor wildlifeandattractivedestinationsfortourists.Thewet

    landremediationprocessesincludebiologicalprocesses Correspondingauthor.Email:hefeng@thb.ac.cn

    Presentaddress:DepartmentofBiologyandChemistry.CityUniver- sityofHongKong.

    suchasmicrobialmetabolicactivities,andplantuptakeas wellasphysicochemicalprocessessuchassedimentation, adsorptionandprecipitation(KadlecmadKnight,1996) atthewater-sediment.rootsedimentandplant.waterin

    terfaces.Forexample,nitrificationanddenitrification reactionsarethedominantnitrogenremovalmechanisms inconstructedwetlands,andthechemolithoautotrophic

    ammoniaoxidizingbacteria(AOB)areresponsibleforthe ratelimitingstepofthenitrificationreaction,andtherefore contributesubstantiallytothegloba1cyclingofnitrogen. Duetoitsimportanceinremovingcontaminantsfrom theconstructedwetlands,themicrobialcommunitywithin thesewetlandshasreceivedgreatattentionrecently,es

    peciallywithrespecttopathogenremoval(Perkinsand Hunter,2000;Thurstoneta1.,2001),metabolismprocess ofpollutants(Giraudeta1.,2001:W_eavereta1.,2004)

    andimpactonthemicrobialcommunities(Ibekwceta1.. 2003:HallbergandJohnson,2005).Ofallthesestudies, theinvestigationofmicrobialcommunitieshasbecomea focusinstudyingdecontmninationintheecologicalsystem ofconstructedwetlands.

    Theintegratedverticalflowconstructedwetlands

    fIVCW1areanewtypeofconstructedwetlandwith subsurfaceflowTheonelocatedinhanChinaisone

    exampleandithasbeeninoperationfor7years.Asa 1262ZHOUQiaohongeta1.

    subprojectoftheinternationalscientificfruitofINC0

    DCproject(No.ERBIC18CI'960059)supportedbythe4th FrameworkProgrammeoftheEuropeanCommission,the IVCWwasconstructedtoenhancethepotentialremoval abilityofasinglevertica1constructedwetland.Thesystem integratedtwokindsofwatercurrentpatterns.includingof verticaldownflowandverticalupflow.Theuniquesystem designwouldresultindifierentsubstratephysicochemical andbiologicalcharacteristicswithinthewetland.Inthis article,themicrobesinthisIVCWwerecharacterised tooffersomevaluableinformationtosystemdesigners andmanagers.Specifically,amoAgenesequenceand phospholipidsfattyacidfPLFAprofileswereanNyzedto studythegeneticdiversitiesandspatialdistributionofthe microbesintheIVCWsystem.

    1Materialsandmethods

    1.1Experimentalwetlandsystem

    TheIVCWwasinstalledneartheEastLakeinWuhun. China.Thevenicalcrosssectionviewofthewetlandis showninFig.1.Briefly.thesystemhasasurfaceareaof

    162m,whichwasdividedequallyintotwochambers, withthefirstbeingadownflowchamberandthesecond upflowchamber.Eachchambercomposedoftwolayers wicfldrentparticlesizes:200mmdepthofcoarsegravel (816mmindiameter)onthebottominbothchambers, 550mm(inthedownflowchamber),and450mltl(inthe

    upflowchamber)depthofsandr04mmindiameter)

    onthetop.Al1waterpipesweremadefrompolyviny1 chloride(100mmdiameter).A5%.gradientventpipewas placedOllthebottomtodraineasily.Forevendistribution ofwater,twoinfluentpipeswithholes(5mmdiameter) ontheundersidewereplacedacrossthesurfaceofthe sandlayer.Onthebottomofbothchambers,collecting pipessentwatercomingfromthedownflowchamber

    tOtheupflowchamber.Theeflluentpipesweresituated onthesurfaceoftheupflowchamber.Thesystemhas

    beenoperatingsinceitwasbuiltinApril.1998.Zizania caducifloraandAcoruscalatIIIASwereoriginallyplanted inthedownflowandupflowchambers,respectively,but ZcaduciflorawasreplacedbyCannaindicainMarchof 2001,becauseZ.caduciomwitherednthewi:iterand

    didnotbourgeonwellinthenextspring.Thesourcewater wasfromEastLake,Wuhanandthehydraulicloadingwas 420mm/d,withdiscontinuousfilling.Duringsampling Vb1.21

    periods,theinfluentandetttuentwaterqualityislistedin 1ble1.Conductivity.pH.andTDSweremeasuredusinga TherinoOrion5starportablemetersitu.whilechemica/ oxygendemand(CODcr),totalnitrogen(TN),andt0tal phosphorusfTP)weremeasuredwithin24hinlaboratory

    accordingtoEditorialboardofenvironmentprotection bureauofChinar1997).

    Table1InfluentandeffluentwaterqualityintheIVCW Datainbracketrepresentstandarddeviationofthemear1. 1.2Substratesamples

    Thesubstratesampleswerecollectedusingcylindrical corerbetweenSeptemberandOctoberin2004.Eachcore samplewasdividedintofoursubsamplesaccordingtothe depth.Thesubsamplesat05,1520.30-35and45-50

    cmdepthsweredesignatedSltoS4forthedown.flow chamberandS5t0S8fortheup.flowchamber.Andeach samplewasequallycollectedfromfivesymmetricalsites. Thesulfuricacidpotassiumdichromatemethodwasused tomeasuretheorganicmattercontent.

    1.3Phospholipidextractionandanalysis

    Phospholipidswereextractedfromeachsubsampleand analyzedusingGC/MS.Briefly,5goffleshsubstrate withoutanyroot,witheredbranchesorleaveswasextract

    edwithachloroforill:methanol:pH7.4phosphatebuffer (1:2:0.8,vN/v),andthenthetotallipidsextractedwere furtherfractionatedintoneu~allipids.glycollipidsand

    phospholipidsonasilicabondedphasecolumn(SPESi,

    500mg/6mL.Agilent).Thepolarlipidsweretranses

    terifiedwithmildalkalitorecoverthePLFAasmethyl estersinhexane.ThePLFAswereseparated.idenfified andquantifiedbyaHewlett.Packard6890NGC/5873IMS withanonpolarcapillarycolumn(HP5MS,30mx0.25

    mmx0.25"m).Heliumwasusedasthecarriergas.The temperatureofthejnjectorwassetat250.C.Samples (1L)wereinjectedinthesplitlessmode.Theinitial

    columntemperaturewasmaintainedat80.Cfor2min. DownflowchamberUpflowchamber

    Fig.1VerticalcrosssectionviewoftheIVCWsystem.

    No.9Characteristicsofthemicrobialcommunitiesintheintegratedvertical

    flowconstructedwetlands1263

    thenrampedtol50.Cat50.C/min.raisedto195.Cat 2.5.C/min.whichremainedconstantfor3minbefore beingincreasedto240.C.Massspectraweredetermined byelectronimpactat70eV.PLFAswerequalifiedand quantifiedaccordingtothemethoddescribedbyMac

    naughtoneta1.f1999).Fattyacidnomenclatureusedin isstudycomplywiththatdescribedbyMacnaughtonet Z.r19991andPonderandTadros(2002).

    1.4AnalysisofammoniaoxidizingbacteriausingamoA gene

    TheprimerpairsspecificforamoAgene,amoA1F (5'GGGG1TTC11ACTCCTGGT.3'1andamoA2Rf5'

    CCCCTCKGSAAAGCCTTCTTC3'1(Hoshinoeta1..

    2001,wereusedtoamplifythe16SrDNAfragment fromsampleS1.Amplificationswereperformedin25uL reactionmixture,whichcontained0.5?Lof10mlTIOl/L

    dNTPs,0.5of10mo1/Lprimers,1UTaqpolymerase andluLtemplateDNA.PCRprofileconsistedofinitial denaturationfor5minat94.C,followedby35cyclesof denaturationat94.Cfor30s.annealingat50.Cfor30 sandextensionat72.Cfor60s.Thefinalextensionat 72.Clastedfor10min.ThePCRproductswereassessed usingagarosege1f1.0%1electrophoresis. Afterelectrophoresis,thePCRproductswereexcised fromthegel,purifiedandconcentratedusingtheGlass.

    milkDNApurificationkit(BiostarInternationa1.Canada). ThepurifiedDNAwasligatedtoPMD18Tvectorand

    theplasmidscontainingthePCRfragmentswerethen usedtotransformEscherichiacoliDH5acompetentcells. Screenedpositivecloneswereculturedat37.Cfor2hor moreandthenweresenttobesequenced.

    1.5Dataanalysis

    ThePLFAanalysiswasperformedintriplicatesand thedatawereanalyzedbyanalysisofvariance(AND, andprincipalcomponentanalysis(PCA).Allnucleotide sequencesweresubmittedtoGenbank(No.FJ603083. FJ603096)andalignedbythecomputerprogramClustal X.ThehomologoussequencesNitrosornonasoligotropha (AF272406),N/trosomonasaestuarii(AF272400),Nitro

    somonasmarina(AF272405),Nitrosomonascryotolerans (AF272402),Nitrosomonascommunis(AF272399),Ni

    trosomonaseuropaea(AF037107),Nitrosospirabriensis (AY1238211wereobtainedfromNCBIandaneithbor

    joiningphylogeneticUPGMAtreewasconstructedusing Mega2.1.

    2Results

    2.1PLFAtypes

    ThePLFAprofilesfromtheIVCWarepresentedinFig. 2.ThePLFAsextractedfromal1thesubsatesamples

    at0-50cmdepthcontalnedsaturated,monounsaturated (MUFA),branched,cyclopropyl,polyunsaturated(PUFA) andhydroxylfattyacids.StraightchainPLFAswere

    relativeabundantwithinallthesamplescollected,with 16:0havingthehighestconcentration(540.0pmol/gsoil), followedbybranchedPLFAsf319.0pmo~gsoil).And

cyclopropanePLFAs(cy17:0andcyl9:0),andfinally

    hydroxylPLFAs(20H10:0,2OH12:0,3OH12:0,2 0H14:0.3.OH14:0and2OH16:0)havethelower concentrations.76.1and57.1pmol/gsoil.respectively.

    Figure3showstheresultsoftheratiosofMUFAto

    r_]r-]

    .~I--11"]r"nr-nI"qf_1nnnr-]r_]lln几厂]r_]lInr_]000000 寸中

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    00

    0.J00S

    

    0

    量茎

    型堕

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    PLFAtype

    Fig.2Concentrationsofdifferenttypesofphospholipidsfattyacid(PLFA)intheIVCW

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    0

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    O5O5O5O5O5O5O如加"2

    0gdLl0J1L1uL10u

    0_z一王0Ic

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    SlS2S3S4S5S6S7S8

    Samplingsite

    Fig.3RatiosofMUFAtobranchedPLFAintheIVCW.S1toS4 representthesamplesat0-5,15-20,30-35and45-50cmdepthinthe downflowchamber,andS5toS8representthesamplesat0-5.15-20. 30-35and4550cmdepthsintheupflowchamber,respectively. branchedfattyacids.Andtheresultsindicatedthatthe ratiosofallsamplesinthesubsurfacesiteswereless than1.

    2.2MicrobialPLFAcontents

    Themicrobialcommunitiescanbeclassifiedintodif- ferentgroupsaccordingtotheirPLFAcomposition.In thisstudy.themostbacterialPLFAswererepresentedas thesumofthePLFAsffomGrampositivebacteriafG

    (il5:0,a15:0,6:0,l6:lm7c,i17:0)andGramnegative

    bacteria(G)(cy17:0,18:lto9candcy19:0)(Tscherkoet a1..2004).Figure4showsthePLFAconcentrationsoftotal

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