XP1菌株强化湿地植物脱氮及其对根际微生物的影响侯庆杰

24820118ResearchofEnvironmentalSciencesVol．24No．8Aug．20112010[e-mail]．*[e-mail]1112*121．2501002．250061XP1、XP13PCR－DGGEXP13．3＞＞3＞＞XP1、1.5∶1.3∶1．XP1、、314%56%56%98%ρTN《》GB3838—2002Ⅲ〔ρTN≤1mgL〕3XP1340%53%67%．PCR－DGGEX522A1001－6929201108－0857－08EnhancedDenitrificationwithWetlandPlantsbyStrainXP1andItsEffectonRhizosphereMicroorganismsHOUQing-jie1PEIHai-yan12HUWen-rong121．SchoolofEnvironmentalScienceandEngineeringShandongUniversityJinan250100China2．ShandongProvincialEngineeringCenterofEnvironmentalScienceandTechnologyJinan250061ChinaAbstractAdenitrifyingbacterialstrainnamedXP1withgooddenitrificationpropertieswasisolatedfromtheconstructedwetlandofNansiLake．TostudytheenhanceddenitrificationofXP1intherhizospheresoilsofthreeplantsArundodonaxPhragmitesaustralisandTyphathebacteriumstrainXP1wasinoculatedintotherhizospheresoilofthethreeplants．ThechangeofXP1intherhizospheresoilmicroorganismofthethreeplantsinthecourseofenhanceddenitrificationwasinvestigatedbypolymerasechainreactionanddenaturinggradientgelelectrophoresisPCR-DGGE．TheresultsshowedthatwithouttheenhanceddenitrificationforsimulatedwetlandstheorderofnitrogenremovalcapacityinthethreeplantwetlandswasArundodonax＞Phragmitesaustralis＞Typhaunderthesameconditions．ThequantitativeorderofthedominantmicroorganismsaroundthethreeplantswasArundodonax＞Phragmitesaustralis＞Typhaalthoughtherewasnoobviousdifferenceinthevarietyofmicroorganismsintherhizospheresoilofthethreeplants．TheratioofindigenousstrainXP1intherhizospheresoilsofthethreeplantswas1.5∶1.3∶1．Comparedtothecontrolgrouptheenhanceddenitrificationofthethreeplantswereincreasedfrom14%56%56%tomorethan98%respectivelywhichmeanttheXP1hadasignificantenhancementindenitrification．WhenρTNofthewetlandwaslessthanthatoftheClassⅢcriterionintheEnvironmentalQualityStandardsforSurfaceWaterofChinaρTN≤1mgLthediversityandquantitiesofthemicroorganismsinthethreeplants'rhizospheresoilsdecreasedthoughtherewasnomarkedchangeinthemicrobialcommunitystructure．ForstrainXP1thequantitiesintherhizospheresoilsofthethreeplantsdecreasedbyuptoDOI10.13198/j.res.2011.08.25.houqj.0012440%53%and67%respectively．KeywordsNansiLakedenitrifyingbacteriastrainenhanceddenitrificationPCR-DGGEmicrobialcommunity1．、、、、2．、3TN10%4．5-67-10．11-12、13-1415-16．1718．DGGE、19201．、21．XP1、XP13DGGE、ρTN、．11.1XP1Paenibacillussp．．pH7.0～9.0pH8.07ρTN30mgL．LB．343mgKNO3357mgK2HPO41.5gMgSO4·7H2O0.1g1L．EDTA50.0gZnSO42.2gCaCl25.5gMnCl2·4H2O5.06gFeSO4·7H2O5.0gNH46Mo7O2·4H2O1.1gCuSO4·7H2O1.57gCoCl2·6H2O1.61g1000mLpH7.016．DNAPCRDNAMarkerOMEGA．、、．SYBRgreenⅠ．UV－2450EppendorfPCREppendorfEppendorfEppendorfpH．1.21.2.1ρNO2－-NN－1－－ρNO3－-NρTNpHpH22．1.2.2、0.5m0.8m．15cm．、10cm．ρTN50mgL．8588XP13①XP1②③XP1．3ρTN．XP1OD6001.5110.1.2.3DGGE1.2.3.13、、34℃．1.2.3.2DNA0.5gOMEGADNADNA．DNA0.8%DNA．1.2.3.3DNA0.8%DNAOMEGA．1.2.3.4DNA16SrDNAV3GC341f5'－CGCCCGCCGCGCGCGGCGGGCGGGGGGGGGGCACGGGGGGCCTACGGGAGGCAGCAG－3'534r5'－ATTACCGCGGCTGCTGG－3'23-24．GCPCRDGGEDNA25．PCR50μLPCRTemplate1μLPrimer110μmolL1μLPrimer210μmolL1μL10×TaqBuffer5μLdNTPMixture2.5mmolL4μLTaq2.5UμL0.5～1μLddH2O50μL．PCR94℃5min．3594℃1min55℃1min72℃2min72℃10min26．1.2.3.5PCRDGGED-codesystemBioradUSADGGE27-28．40μLPCRDGGE8%40%～60%100%70molL40%1×TAE150V60℃6h．10mLSYBRgreenⅠ1×TAE1000045min26．QuantityOneBioradUSA29-31DGGE．22.1XP1XP131．XP1ρTN50mgL72hρTN43mgL48hρTN120hρTN22mgLρTN72hρTN20mgL40h．ρTN3＞＞．32、．33．34．、．3、3．XP1XP13．ρTN48h15mgLρTN45mgL72hρTN1958241XP13Fig．1TheenhancedeffectsofstrainXP1onthedenitrificationofthreewetlandsmgL《》GB3838—2002Ⅲ〔ρTN≤1mgL〕TN98%．ρTN48h10mgLρTN42mgL72hρTN1mgLTN98%．ρTN24～48h48h5mgLρTNⅢρTN．XP1ρTN50mgLXP1ρTN、、314%56%56%98%．3XP1ρTNⅢXP1．2.22.2.1DNAPCRXP116SrDNAV3GC341f534rPCR220bpDNA2.M—marker1—XP12—3—4—5—6—7—8—9—10—．2XP1DNAPCRFig．2AgarosegelelectrophoresisofPCRproductsofstrainXP1andrhizospherebacterium2.2.2DNADGGE2.2.2.1DGGEPCRDGGEDGGE3．DGGEDNA0688XP11～102．3DGGEFig．3DGGEprofilesofdifferentsamplesOperationalTaxonomicUnitOTU35．QuantityOneDGGEGauss．、34XP1Ⅲ55．DGGE1.31433．34Fig．4Comparisonlanesimageofblankgroup5Fig．5ComparisonlanesimageofenhanceddenitrificationinArundodonaxwetland168241DGGETable1ThetotalbandnumbersandopticaldensityofDGGEprofiles23456789104042343637303030251041.661.781.401.531.631.201.401.521.2010.5gOD312．2～102．．abcefgdh．31iXP1DGGE．3iXP15%～7%XP13．3＞＞XP1、1.5∶1.3∶1．1、72hTN50%34%14%．、3637-38．39．．2.1、3．153Ⅲ．TN404142．43．6XP1Fig．6ThedecreaseofstrainXP1opticaldensityinrhizospheremicroorganismsofenhanceddenitrificationwetlandQuantityOneXP110%．ρTNⅢ、XP140%54%67%6．XP1．2688XP12.2.2.2DGGEQuantityOneDGGE7.2～102．7Fig．7Dendrogramwithrhizospheremicroorganisms457①XP13．22．②XP133．③3XP1XP1XP1．3a．XP13＞＞3＞＞XP1、1.5∶1.3∶1．b．XP1ρTN、、314%56%56%98%．XP1．c．3ρTNXP1、40%53%67%XP1．References1．J．200728122856-2860．2SOLANOMLSORIANOPCIRIAMP．ConstructedwetlandsasasustainablesolutionforwastewatertreatmentinsmallvillagesJ．BiosystemEngineering2004871109-118．3．J．2002321265-268．4JANV．RemovalofnutrientsinvarioustypesofconstructedwetlandsJ．SciTotalEnviron200738012348-65．5JOONGKKKYOUNGJPKYO