复合垂直流湿地氨氧化菌种群结构及活性的空间分布黄德锋

,＊(,　200092):,(AOB),PCR-DGGE.,,、、DO,.,0.79mg·(kg·h)-1(NO-3-N,).,,,0.17mg·(kg·h)-1.,(1.92)(1.65).,,Nitrosomonassp.,.Unculturedbetaproteobacterium、Comamonassp.Nitrosomonasoligotropha.,,.:DGGE;;;:X172　:A　:0250-3301(2008)08-2160-06:2007-08-24;:2007-10-20:(05dz12006):(1978～),,,,E-mail:[e-mail]＊,E-mail:[e-mail]ndActivityofAmmonia-OxidizingBacteriaCommunityintheIntegratedVertical-FlowConstructedWetlandsHUANGDe-feng,LITian(StateKeyLaboratoryofPollutionControlandResourceReuse,TongjiUniversity,Shanghai200092,China)Abstract:Theactivityofammonia-oxidizingbacteria(AOB)indifferentlayersofIntegratedvertical-flowconstructedwetlands(IVCW)treatingeutrophicscenicwaterwasmeasured,andthediversityandspatialdistributionofAOBcommunitystructureinIVCWwasinvestigatedusingPCR-DGGE.Theresultsindicatedthatbecauseoftheintegratedinfluenceofcompetitionofplantrhizodeposition,heterotrophicbacteria,DOandammoniaconcentration,thereweresignificantspatialdifferencesintheactivityanddiversityofAOBalongtheflowdirectionofwetland.TheactivityofAOBwas0.79mg·(kg·h)-1(inNO-3-N,thesamebelow)nearthesurfaceofdown-flowsysteminIVCW.Fromthesurfaceofdown-flowsystemtoup-flowsystem,theactivityofAOBdecreasedgradually,andslightlyincreasedto0.17mg·(kg·h)-1nearthesurfaceofup-flowsystem.ThespatialvariationofdiversityofAOBshowedthesimilarchangetrendwiththeactivityinIVCWandthediversityindexindown-flowsystem(1.92)werehigherthanthoseinup-flowsystem(1.65).MostofAOBbelongtooligotrophicbacteriuminIVCW,andthepopulationofammonia-oxidizingbacteriashowedahigherpercentageofNitrosomonas-likesequencesfromthewetlandsamples.Unculturedbetaproteobacterium,Comamonassp.,Nitrosomonasoligotrophawerealsodetected.ThevariationoftheAOBcommunitydemonstratedspatialpatterninIVCW,whichmightberelatedtodifferentwetlandenvironment.Keywords:DGGE;constructedwetland;scenicwater;ammonia-oxidizingbacteria　　,,、、,.,,.,(AOB),,.,DO、pH、、,[1～4].,,.,,,[5].,PCR-DGGE29820088　　　　　　ENVIRONMENTALSCIENCEVol.29,No.8Aug.,2008DOI:10.13227/j.hjkx.2008.08.013,、、,,.1　1.1　(0.5m×0.5m×0.6m)(0.5m×0.5m×0.5m)2,,3～5mm,.,.,8～10m2.:,(5)1.1　Table1　InflowwaterqualityoftheconstructedwetlandpHNTUDOmg·L-1Chlamg·m-3CODmg·L-1TNmg·L-1NH+4-Nmg·L-1TPmg·L-18.129.42.3928.936.84.652.250.38(SD)0.153.930.435.234.240.670.290.021.2　1a,.0.113m3(m2·d),,,20075,S1(5～10cm)、S2(20～25cm)、S3(35～40cm)、S4(35～40cm)、S5(20～25cm)、S6(5～10cm),,(1～6),.1.3　,100g,250mL,100mLNH+4(25mgL),,(25℃,150～160rmin)24h,,NO-3-N,NO-3-N[(1kg)(1h)NO-3-N(mg)][6].1.4　DNA:,,200mL,10min,120rmin30min,8000rmin10min,,,-70℃,DNA.DNA:3S()DNA,DNA0.8%.1.5　PCRPCR,1PCRβ-ProteobacteriaCTO189fCTO654r[7],2PCR16SrDNAV3GC-338f518r[8].50μLPCR:DNA100ng;10×PCRbuffer5μL;200μmol/LdNTP(10mmolL)15μL;20pmol;Taq2.5U;50μL.:94℃10min;94℃45s,56℃45s(1)60℃45s(2),72℃45s,30;72℃10min.Mycycler(Bio-Rad,USA)PCR,12PCR465bp250bp,1.5%.1.6　DGGE2PCRBIO-RADDcodesystem.8%,35%～60%.:1×TAE60℃、200V10min,150V4h.,EB.Bio-RadQuantityOne4.6.3.PCR,GCF338R518,.1.5%,.NCBI,,MEGA3.1.21618:1.7　PCR-DGGEShannon-Weaver(H′),:H′=-∑si=1niNlnniN(1),s,nii,N,.H′.Bio-RadQuantityOne4.6.3,,.2　2.1　1.1,(5～10cm),0.79mg·(kg·h)-1,,,,0.13mg·(kg·h)-1,,,0.17mg·(kg·h)-1.,,,　　　1　Fig.1　ActivityofAOBindifferentlayersofIVCW　2.,,DO,.,,DO,,.,,.2　DO、pHmg·L-1Table2　ChangesofDO,pHandpollutantsconcentrationinIVCWmg·L-1123456DO1.841.660.970.320.180.230.270.26pH8.047.797.657.517.337.277.166.98COD35.221.615.413.29.89.68.27.0TN5.404.633.742.901.961.701.100.75NH+4-N1.621.250.880.570.520.360.240.21TP0.390.320.260.230.130.090.060.042.2　DGGE2.,,,,,.,(2),、.3～5,,.(#),,,,.,,[9].,(&),(Δ)2162　　　　　　29,,,,.2　AOBDGGEFig.2　DGGEfingerprintsofAOBcommunityinIVCW　2.3　.Ibekwe[10],,,,、.Shannon-Weaver,3.,pH、DO,,.:,,(1.92)(1.65),S2(2.02),,1.52,.,、、[7,11].,,,.,,.,,.,,,.,、DO(2),.3　Fig.3　DiversityindexofAOBcommunityinIVCW　23,(S1),DO,S1S2.,DO,,,DO,,.,DO,[12,13].,,.,、,[14].2.4　DGGE(1～6,2),,,4.4,Nitrosomonassp.,,Nitrosomonassp.,.[15],βNitrosomonassp..,21638:,.,,.UnculturedbetaproteobacteriumCh6S、UnculturedbetaproteobacteriumBa1.0UnculturedbetaproteobacteriumBa0.25-2[16],Unculturedammonia-oxidizingbacteriumcloneLKD3、BetaproteobacteriumHTCC349UnculturedbacteriumDW-NsoE9-9、,Nitrosomonasoligotropha.Nitrosomonasoligotropha,[17].4　Fig.4　Phylogenetictreeofammonia-oxidizingbacteriainIVCW　　　24,,.4,(S1),Nitrosomonassp.,,,Comamonassp.Nitrosomonasoligotropha,,、DOpH,.,.,,.3　(1)、DO,.(2),.(3),.、,.(4),,,Nitrosomonassp.,.,.:[1]　SchrammA,BeerDD,GiesekeA,etal.Microenvironmentsand2164　　　　　　29distributionofnitrifyingbacteriainamembrane-boundbiofilm[J].EnvironmentalMicrobiology,2000,2(6):680-686.[2]　CébronA,CociM,GarnierJ,etal.Denaturinggradientgelelectrophoreticanalysisofammonia-oxidizingbacterialcommunitystructureinthelowerSeineRiver:ImpactofPariswastewatereffluents[J].AppliedandEnvironmentalMicrobiology,2004,70(11):6726-6737.[3]　ParkHD,NogueraDR.Evaluatingtheeffectofdissolvedoxygenonammonia-oxidizingbacterialcommunitiesinactivatedsludge[J].WaterResearch,2004,38(14-15):3275-3286.[4]　PrinˇciˇcA,MahneI,MeguarF,etal.EffectsofpHandoxygenandammoniumconcentrationsonthecommunitystructureof