处理重金属废水人工湿地中微生物群落结构和酶活性变化

32420114ENVIRONMENTALSCIENCEVol.32No.4Apr2011541004、.PCR-DGGE.4.10±0.72×106～1.61±0.10×1077.21±1.60×104～1.29±0.02×1051.41±0.27×106～3.38±0.11×106.DGGE816SrRNA18SrRNA.0.67±0.2mg/g·24h、40.15±0.14mg/g·24h1.03±0.16mg/g·2h1、31.、、.PCR-DGGEX172A0250-3301201104-1202-082010-06-042010-08-230701K012200771410011862010GXNSFA0130181974～[e-mail]crobialCommunityStructureandEnzymeActivityinConstructedWetlandsReceivingWastewaterContainingHeavyMetalsJINZhen-jiangLIUJieXIAOYuLIJin-chengTIANHai-taoWULeiGuangxiKeyLaboratoryofEnvironmentalEngineeringProtectionandAssessmentGuilinUniversityofTechnologyGuilin541004ChinaAbstractThemicrobialcommunitystructureandenzymeactivitywereinvestigatedinthethree-stagewavysubsurfaceconstructedwetlandW-SFCWreceivingwastewatercontainingheavymetalsCrCuandNi.ColonyformingunitsCFUabundanceofmicrobeswascalculatedbyusingplateculture.ThemicrobialcommunitystructurewasinvestigatedbyusingPCR-DGGEandsequencing.Enzymeactivitywasdetectedbyusingcolorimetry.TheresultsshowedthattheCFUofbacteriarangedfrom4.10±0.72×106to1.61±0.10×107fungirangedfrom7.21±1.60×104to1.29±0.02×105andactinomycetesrangedfrom1.41±0.27×106to3.38±0.11×106.Allof8bandsretrievedfromDGGEgelswereunique16SrRNAor18SrRNAsequencesandrevealedthatthemicrobialcommunitystructureshadchangedspatially.Maximumurease0.67±0.2mg/g·24hinvertase40.15±0.14mg/g·24handalkalinephosphatase1.03±0.16mg/g·2hactivitieswereatfirststagethirdstageandfirststagerespectively.AnegativecorrelationbetweenfungiCFUabundanceandthetotalcontentofCravailableCrcontentandactivationrateofCrwasdetectedanegativecorrelationwasalsofoundbetweentheinvertaseactivityandtotalCuandNicontentsandtheNiactivationrate.KeywordsheavymetalsconstructedwetlandmicrobialcommunityPCR-DGGEenzymeactivitycorrelation1～4.5～7.Yu4Hallberg8Sheoran6.、9--.、DOI:10.13227/j.hjkx.2011.04.0464LeersiahexandraSwartz10.11.1PVCPolyvinylchloride××=2.00m×1.00m×0.70mwavysubsurfaceconstructedwetlandW-SFCW10.20m1.80m30.60m.0.20m3·m2·d－10.36m30.5%0.40m0.10m0.30m.1cm0.50m.1、23.1.mm1Fig.1Schematicdiagramofthree-stageW-SFCW1/mg·L－1Table1Wastewaterqualityofconstructedwetland/mg·L－1CrCuNi10.005.002.501.800.160.151.220～15cm2mm24℃.1.3HNO3+HClO4+HF0.1mol/LHCl∶=5∶1...1.4、NA、MartinGauselg.1.5DNADNAMoBIODNApowersoil.1.6polymerasechainreactionPCRPCR16SrRNA11F338-GC5′-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3′R5185′-ATTACCGCGGCTGCTGG-3′.200bp25μLPCR2×MasterMix12.5μL9.5μLDNAgenomicDNA10ng/μL1μL20μmol/μL1μLPCRtouchdownPCR1294℃5min94℃1min30213265℃1℃55℃72℃55℃1572℃5min.PCR1.2%.18SrRNA13NS15′-GTAGTCATATGCTTGTCTC-3′Fungi-GC5′-CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCCATTCCCCGTTACCCGTTG-3′400bpPCR95℃15min91℃1min57℃1min72℃2min3568℃10min.PCR1.2%.1.7denaturegradientgelelectrophoresisDGGEBio-Rad475Model475GradientDeliverySystem.35%～60%8%.200V60℃4min90V60℃9h15%～30%200V60℃4min100V7h.3min10min23minBio-RadGelDoc-2000QuantityOneBio-Rad、14.1.8DGGE67DNA1.5mL4℃DNA.PCRPCR.PCRDNAEZgenecyclepurekitBiomigapEASY-T3cloningkitTransGen.Invitrogen.1.9NCBIhttp//blast.ncbi.nlm.nih.gov/Blast.cgi2Mega4.1.21000.GenBankHQ009744～HQ009747HQ009748～HQ009751.1.101535-24hgmg-24hgmg2hgmg.1.11MicrosoftExcel2000SPSS13.0.22.122323.、23.233.、.21Table2Changeofheavymetalsindifferentstagesofconstructedwetland/mg·kg－1/mg·kg－1/%CrCuNiCrCuNiCrCuNiS1169.10±98.02a47.78±10.71b56.79±0.03b3.64±2.43a6.56±5.31ab3.15±0.57a2.09±0.23b17.37±0.85a5.54±1.00aS2202.30±36.85a57.00±1.56a78.36±0.66a5.85±1.74a8.46±0.14a3.96±1.32a2.86±0.34a14.83±0.65a5.04±1.64aS354.94±4.61b29.83±0.32c29.24±0.41c0.23±0.05b6.97±0.48b2.22±0.05b0.42±0.05c23.37±1.37a7.61±0.29a1S1、S2S31、23p0.05402142.22.2.12.1～34.10±0.72×1061.61±0.10×1071.26±0.16×10727.97±0.24×1047.21±1.60×10431.29±0.02×1053.38±0.11×1062.47±0.51×1061.41±0.27×106F/B1.97±0.29×10－22Fig.2ChangeofmicrobialCFUindifferentstagesofconstructedwetlandS1-1S1-2、S2-1S2-2S3-1S3-2S1、S2S32318SrRNADGGEFig.3DGGEprofileof18SrRNA4.52±1.29×10－31.03±0.14×10－2.2.2.2318SrRNADGGE416SrRNADGGE56.5DGGES1S26DGGES2S3.3.DGGE1212.3DGGEF1、EG2H3E、F、GH18SrRNA4DGGE2D3A、BCA、B、CD16SrRNA.GenGank16SrRNA18SrRNA783.416SrRNADGGEFig.4DGGEprofileof16SrRNA5021325DGGEFig.5ClusteranalysisofbacteriaDGGEprofile6DGGEFig.6ClusteranalysisoffungiDGGEprofile3Table3ChangeofbandnumberfromDGGEprofileandShannonindexindifferentstagesofconstructedwetlandS141.51±0.50b3.38±0.02a32.00±0.00a3.64±0.05aS244.00±0.00a3.33±0.05a29.50±0.50b3.72±0.05aS345.00±1.00a3.29±0.08a30.00±0.00b3.86±0.11a7DGGEDNA16SrRNAFig.7Phylogenetictreebasedonpartialsequencesof16SrRNAretrievedfromDGGEbandsshowninbold8DGGEDNA18SrRNAFig.8Phylogenetictreebasedonpartialsequencesof18SrRNAretrievedfromDGGEbandsshowninbold2.2.34.340.15±0.14mg/g·24h130.67±0.25mg/g·24h4/mg·g·24h－1Table4Changeofsoilenzymeactivityindifferentstagesofconstructedwetland/mg·g·24h－1S1S2S332.49±3.53b23.64±3.74c40.15±0.14a0.67±0.25a0.40±0.04b0.58±0.12a1.03±0.16a0.52±0.04b0.05±0.00c0.58±0.12mg/g·24h11.03±0.16mg/g·24h30.05±0.00mg/g·24h.33.1、、、1617.3.、R2－0.733*、60214－0.867*－0.733**p0.05.、18.19、20.Kuperman21Cr、CuNi7658.7mg·kg－13446.6mg·kg－1121.0mg·kg