发酵型和非发酵型反硝化聚磷菌的鉴定及代谢机理刘晖

　61　9　　　　　　　　　　　　Vol.61　No.9　20109　　CIESC　Journal　　　September　2010刘　晖1,2,孙彦富1,贾晓珊2,周康群1,刘洁萍1(1,510225,2,510275):(DPB),,,。:(1)YU-1、YU-2,YU-1Acinetobacterjunii,,YU-2Escherichiacoli,。(2),YU-11mgPHAPYU-20.2mg。PHAATP,,NADH。ATP、PHANADH。(3),YU-11mgPHAYU-2P0.3mg,12.8%,PHBATP,PHBNADH,PHA。:;;;:X172　　　　　　:A:0438-1157(2010)09-2454-09Identificationoffermentationandnon-fermentationdenitrifyingphosphorusremovingbacteria(DPB)andtheirmetabolicmechanismLIUHui1,2,SUNYanfu1,JIAXiaoshan2,ZHOUKangqun1,LIUJieping1(1DepartmentofEnvironmentalScienceandEngineering,ZhongkaiUniversityofAgricultureandEngineering,Guangzhou510225,Guangdong,China;2SchoolofEnvironmentalScienceandEngineering,SunYat-SenUniversity,Guangzhou510275,Guangdong,China)Abstract:Acomplexsystemofactivatedsludgeandbio-membranesystemwasusedforenrichmentofdenitrifyingphosphorusbacteria(DPB).DPBstrainswereisolatedandidentified.Inordertostudytheirmetabolicmechanism,activitytestofdenitrifyingandaccumulatingphosphoruswascarriedoutonbothfermentedandnon-fermentedbacteria.Theresultshows:(1)YU-1andYU-2wereobtainedbyseparationofDPBwithcombinedactivatedsludgeandbiofilmmethod.TheformerissimilartoAcinetobacterjunii,akindofnon-fermentedDPBandthelattersimilartoEscherichiacoli,akindoffermentedDPB.(2)Duringtheanaerobicstageforevery1mgofPHAgeneratedthephosphorusreleaseis0.2mglargerfornon-fermentedbacteriaYU-1thanthefermentedbacteriaYU-2.Theenergyrequiredforthesynthesisof　　2010-01-05,2010-02-03。:(1973—),,,。:(2008ZX07211-003);(06022869,07003251);(2007-Z-023);(LYM08067)。　　　Receiveddate:2010-01-05.Correspondingauthor:LIUHui,associateprofessor,[e-mail]:supportedbytheNaturalScienceFoundationofGuangdongProvince(06022869,07003251).　　PHAismainlyfromthedegradationofpoly-phosphate,themajorityofcarbonfromtheaceticacid,reducedcoenzymeNADHnotfromtheglycolysisprocessofglycogen.ThemechanismforgenerationofATP,PHAandNADHaredifferentfromthetraditionalmetabolicmechanism.(3)Underanoxicenvironmentforevery1mgofPHAdecomposedthephosphorusaccumulationis0.3mghigherfornon-fermentedbacteriaYU-1thanthefermentedbacteriaYU-2,whichmeanstheaccumulatingefficiencyis12.8%higher.ThereasoncanbethatinthisstagethemostofATPgeneratedfromthedecompositionofPHBisusedforthetransportofphosphateandthesynthesisofpoly-phosphorus,reducedcoenzymeNADHcomefromthebreakdownofPHAforthereductionpowerrequiredforanaerobicprocess.ThemechanismofPHAdecompositionisdifferentfromthemetabolicmechanismoftraditionalphosphorusaccumulatingbacteria.Keywords:fermented;non-fermented;denitrifyingphosphorusremovingbacteria;metabolicmechanism　,,、[1-3]。A/A/O、A/A/O,、、。(DPB)[4],/,(),,(VFAs),PHA;PHA,(),ATP,,。BCFS、A2N,,,,:(1):DPB,,,,;(2):,;(3):,DOCOD,;(4):30%,,;(5)COD:,COD,COD;(6):50%COD,COD;(7):50%,,,。SBR[5-7],,[8],、,,。,,,,,(DPB),。,,,·2455·　9　　:。,、、,,。1　1.1　1.1.1　活性污泥与生物膜复合系统　1。1m3·d-1,、ORP:(1):,50%COD,HRT5.0h,ORP-100mV;(2):,HRT0.32h;(3):,CODBOD,HRT2.0h,ORP+200mV;(4):ORPCOD,,,HRT0.6h,ORP+50mV;(5):,,HRT3.0h,ORP0～-100mV;(6):DO,,HRT0.3h;(7):,,HRT0.3h。1　Fig.1　Activatedsludgeandbio-membranecombinedsystem1.1.2　同步反硝化聚磷菌的活性试验装置　,,(2)。2　Fig.2　Activitytestequipmentofdenitrifyingphosphorusremovingbacteria　1.2　1.2.1　试验污水　,1。1　Table1　CompositionofsewagewastewaterCompositionConcentration/mg·L-1COD200—350BOD80—120N(NH4Cl)25—30TN30—35P(PO3-4-P)3.5—5.0SS100—160pH7—7.51.2.2　系统运行方案和接种污泥　A2/O。1(MLSS=3500～4000mg·L-1),。3,。1:,,30d。95.4%,90.8%。,,,35d。14.1%89.4%,·2456·　　　　　61　9.7%85.2%。2:,1,。43d,43.7%91.2%,39.9%86.7%。3:,。()。37d。47.5%94.6%,41.86%90.2%。3(NO-3-N93.4%,PO3-4-P89.8%),YU-1、YU-2。1.2.3　培养基和基础培养液　(1),[9]5.0g,KH2PO40.25g,MgSO4·7H2O0.5g,CaCl2·H2O0.2g,(NH4)2SO42.0g,1.0ml,1000ml,pH=7.0。　(2)300mg·L-1,COD300mg·L-1。KH2PO40.025g,PO3-4-P5mg·L-1。1.3　1.3.1　反硝化聚磷菌的分离　1.2.2,,。1.3.2　同步反硝化聚磷菌株显微形态观察　,100×10。1.3.3　同步反硝化聚磷菌的生理生化指标[10-11]　、、、、、。1.3.4　DNA提取、PCR扩增、基因序列比对及进化树构建　,TE(pH=8.0),,SDS/DNA。16SrRNA27f1522r。PCR:94℃4min,94℃1min,55℃1min,72℃2min,30,72℃7min。。16SrDNA,GenBankBLAST。16SrDNA,。MEGA4.0,Neighbour-joining。MEGA4.0SEQBOOTCON-SENSEbootstrap,1000,[12]。1.3.5　反硝化聚磷菌的基质利用试验方法　1.3.1YU-1、YU-22()。OD6500.3(1010·ml-1);,ORP-80mV,2h。KNO3N∶P=2∶1。(ORP-50～-80mV),,,COD、PO3-4-P、NO-3-N、PHA。1.3.6　指标测定方法　PHA,20ml,,2ml,2ml,0.2ml,100℃4h,1ml,()。;COD;;;MLSS:;:;pH、ORP。2　　2.1　YU-1、YU-2YU-1,、、、,30℃,24h1.3mm、48h2.2mm,。、,(0.8～1.2)μm×(1.4～1.8)μm,、。YU-2,、、,,·2457·　9　　:,。30℃,24h1.2mm、48h1.9mm,。,,(0.4～0.7)μm×(1.0～3.0)μm。2.2　YU-1。、、、PHB、、L-、L-、L-。、、L-、L-、L-,,YU-1。YU-2。、、、ONPG、、、PHB、;、、V-P、、、;;、、、、,、。,YU-2Escherichiacoli()。2.3　16SrRNAYU-1、YU-2DNA,16SrDNAPCR,1500bp。(:FJ889443GQ213993)BLASTGenBank,3、4。2.4　YU-1、YU-22.4.1　非发酵型的YU-1菌的释磷和反硝化聚磷特性　PHA(PHB),。,YU-1。5。,(PO3-4-P)5.8mg·L-116.0mg·L-1,PHA14.3mg·L-128.9mg·L-1,ΔPHA=14.6mg·L-1,CH1.5O0.5(PHA)+1.125O2※CO2+0.75H2O,PHACOD1.674,ΔPHA=14.6×1.674=24.4mg·L-1(COD),COD309.2mg·L-1285.5mg·L-1,ΔCOD=23.7mg·L-1,ΔCOD/ΔPHA(COD)=0.969,PHA(96.9%)。。ΔP/ΔPHA=0.7,1mgPHA0.7mgP。3h,16.0mg·L-1·2458·　　　　　61　5　YU-1Fig.5　ReleasingandaccumulatingphosphoruscharacteristicsofYU-1　1.2mg·L-1,ΔP=14.8mg·L-1,92.5%,7.4mg·L-1·h-1,96.8%,PHA28.9mg·L-115.4mg·L-1,PHA4.5mg·L-1·h-1,ΔPHA=13.5mg·L-1,ΔP/ΔPHA