处理含酚废水曝气生物流化床中丝状菌种群结构的解析董婧

23820108ResearchofEnvironmentalSciencesVol.23No.8Aug.2010*100012ABFTρ100mg/L400mg/L100%4.SEM、FISHDGGE.ρ.SSUrRNA.16SrDNA18SrDNAPCR－DGGEGeotrichumklebahnii..X703.1A1001－6929201008－1068－08AnalysisoftheStructureofFilamentousMicrobialGroupsfromAeratedBiologicalFluidizedTankTreatingPhenolicWastewaterDONGJingSONGYu-dongZHOUYue-xiYANGJing-huiYANGJianResearchCenterofWaterPollutionControlTechnologiesChineseResearchAcademyofEnvironmentalSciencesBeijing100012ChinaAbstractAnaeratedbiologicalfluidizedtankABFTwasusedtotreatphenolicwastewaterwithfavorabletreatmentefficiency.Astheinfluentphenolconcentrationincreasedfrom100mg/Lto400mg/Lthephenolremovalrateremainedaround100%andstablephenolremovalwasobtainedatthefirstoffourtanks.ThemicrobialcommunitystructureandbiodiversityintheABFTreactorwereexaminedusingmethodssuchasscanningelectronmicroscopySEMfluorescenceinsituhybridizationFISHanddenaturinggradientgelelectrophoresisDGGE.TheresultsofSEMshowedthatwiththeincreaseofinfluentphenolconcentrationfilamentousmicroorganismsgraduallybecamethedominantspeciesinthemembraneoftheABFTstuffing.TheFISHresultstargetingSSUrRNArevealedthatmostofthefilamentousmicroorganismsintheABFTreactorwerefungiwhilethePCR-DGGEresultsforbacterialandfungal16Sand18SrDNAindicatedthatthefilamentousfungimainlybelongedtoGeotrichumklebahniialthoughtherewerealsosomefilamentousbacteriasuchasThiothrixsp.andactinobacterium.Clusteranalysisanddiversityanalysisshowedthatphenolhadstrongselectivityonthemicrobialcommunity.KeywordsphenolaeratedbiologicalfluidizedtankfilamentousmicroorganismGeotric[e-mail].*[e-mail]、、、、、..《》12911.“”68、61.、、、、2..3-45.3.6kg/m3·dDOI10.13198/j.res.2010.08.93.dongj.00382h95%.AeratedBiologicalFluidizedTankABFT、、、、6、、、7-8、、、、、9.10.PCR－DGGEFISH.11.1ABFTABFT1.ABFT46L24L.1130%.8h.1.ρ100200300400mg/L4ρCODCr800mg/L.1ABFTFig.1Flowchartoflab-scaleABFTreactor1.2PHILIPSXL30SERIES.50%70%80%90%100%10min2h.1.3FISH4%4℃PBS3VPBS∶V1∶1.AMANN1250%80%98%3min.0.9mol/LNaCl20mmol/LTris－HCl0.01%SDSX%46℃2h..DAPI5min.OlympusBX－51、.1、wTable1Listanddescriptionof16Sand18SrRNAtargetedoligonucleotideprobesusedinthisstudy5′→3′w/%EUB33816SrRNAGCTGCCTCCCGTAGGAGT20FITC13ARCH91516SrRNAGTGCTCCCCCGCCAATTCCT25FITC14EUK51618SrRNAACCAGACTTGCCCTCC50TAMRA131.4PCR－DGGE1.4.1DNADNAZHOU15.1.4.2DNADNA16.1.4.3DNADNA.1.4.416SrDNAPCR16SrDNAV3DGGEGC-clamp.GC－F3415′－CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG－3′R5345′－ATTACCGCGGCTGCTGG－3′17.96012345pmol/μL.PCR50μL45pmol/μL0.5μL10mmol/LdNTP0.25μL5U/μLTaq0.5μL1μL10×PCRbuffer5μL.PCRPCR94℃5min94℃1min65℃30s72℃35s0.5℃55℃3572℃30s.PCR1%.2ABFTFig.2PhenolremovalofbyABFTduringdifferentruns1.4.518SrDNAPCR18SrDNA.NS15′－GTAGTCATATGCTTGTCTC－3′GCFUNG5′－CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCCATTCCCCGTTACCCGTTG－3′18.PCR16SrDNA.PCR94℃5min94℃1min56℃30s72℃60s3572℃5min.PCR1%.1.4.6DGGEDGGEDcodeBio－Radw8%16SrDNA40%～65%18SrDNA10%～35%.1×TAE200V65℃4h.1×SYBRGold30minUVP..GCPCR.PCR.GenBank.1.5DGGEBio－RadQuantityoneDGGE.Shannon－WienerQuantityoneDGGE19Shannon－WienerH.H=－Σni/Nlgni/NniN.QuantityoneUPGMA20.22.1ABFTABFT2.24.ρρABFTρ.90%.07018ABFT.ρ400mg/L2.17kg/kg·d0.5kg/kg·d.ABFT.2.2ABFT3bρ400mg/L..ρ..ABFT.3、ρFig.3AppearanceofthebiomasscarrierSEMimagesofseedsludgeandbiofilmsamplesatdifferentinfluentphenolconcentrationsinthe1stchamber2.3ABFTFISHFISHEUB338〔4a〕ARCH915〔4c〕EUK516〔4e〕EUK516〔4g〕.4bdfhDAPI.4.2～3μm.EUB338ARCH915EUK5164gh.ABFT.2.4ABFTPCR－DGGEPCR－DGGE52ρ400mg/L.1701231—2—4ρ400mg/LDAPIFig.4FISHimagesoffilamentousmicroorganismsattachedonbiomasscarriersinthe1stchamberattheinfluentphenolconcentrationof400mg/LwithdifferentprobesandstainedwithDAPI.Thiothrixsp.78.ρ1011.5..ρρ1005ABFTSρM－S－16SrDNAPCR－DGGEFig.5DGGEprofileofbacterial16SrDNAgenesofseedsludgebiofilmsattachedonbiomasscarriersM-andsuspendedsludgeS-inthe1stchamber2DGGEGenBankTable2TheclosestbacteriaofmainDGGEbandsDGGE/%GenBank1Lactococcussp.cloneA1100GQ390367.12betaproteobacteriumcloneMEsu06b11H698FJ828501.13Bdellovibriosp.cloneNaukuchia-394FJ405270.14Bdellovibriosp.cloneNaukuchia-396FJ405270.15UnculturedbacteriumisolateDGGEgelbandB2395EU316207.16gammaproteobacteriumcloneNdGal10188FJ752852.17ThiothrixflexilisstrainEJ2M-B98NR_024757.18ThiothrixflexilisstrainEJ2M-B100NR_024757.19UnculturedactinobacteriumcloneDOK_CONFYM_clone70784DQ828920.110FirmicutescloneQEDN2BB0996CU926241.111Nitrospirasp.cloneB884EU850362.127018mg/L78ρ200mg/L、101ρ300mg/L、、34.ρ400mg/L.PCR.PCR－DGGE63..ρ400mg/LGeotrichumklebahniiCandidasp..4...6ABFTρM－S－18SrDNAPCR－DGGEFig.6DGGEprofileoffungal18SrDNAofbiofilmsattachedonbiomasscarriersM-andsuspendedsludgeS-inthe1stchamber3DGGEGenBankTable3TheclosestfungiofmainDGGEbandsDGGE/%GenBank1Geotrichumklebahnii97AB000641.12Geotrichumklebahnii96AB000641.13Geotrichumklebahnii96AB000641.14Geotrichumklebahnii96AB000641.15Dipodascusmacrosporus95AB000640.16Candidasp.BG01－7－24－009A－1－195AY242231.17UnculturedfungusisolateDGGEgelband37100FJ236972.18UnculturedfungusisolateDGGEgelband1192FJ608616.1Phanerochaetechrysosporium、CandidatropicalisStrep