藻类对BPA的降解Word格式.docx

藻类对BPA的降解Word格式.docx

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●MicroalgaepromotedbioaccumulationandbiodegradationofBPA

●BPAincreasedtheFAMEandcarbohydratecontentofmicroalgae

BiodegradationofbisphenolAbythefreshwatermicroalgaeChlamydomonasmexicanaandChlorellavulgaris

Min-KyuJia,AkhilN.Kabraa,JaewonChoib,Jae-HoonHwanga,JungRaeKimc,RedaA.I.Abou-Shanabd,Byong-HunJeona

aDepartmentofEnvironmentalEngineering,YonseiUniversity,Wonju220-710,SouthKorea

bWaterAnalysisandResearchCenter,KoreaInstituteofWaterandEnvironment,KoreaWaterResourcesCorp., 

Daejeon306-711,SouthKorea

cSchoolofChemicalandBiomolecularEngineering,PusanNationalUniversity,Busan,609-735,SouthKorea

dDepartmentofEnvironmentalBiotechnology,CityofScientificResearchandTechnologyApplications,NewBorgElArabCity,Alexandria21934,Egypt

*Correspondingauthor.Tel:

+82337602446;

Fax:

+82337602571

E-mailaddress:

bhjeon@yonsei.ac.kr（BHJ）

Abstract

Theendocrine-disruptingchemical,bisphenolA（BPA）hasattractedmuchattentionduetoitsestrogenicactivityandwidespreadenvironmentaldistribution.ThetoxicityandcellularstressesofBPAtoChlamydomonasmexicanaandChlorellavulgarisanditsbiodegradation/bioaccumulationbybothmicroalgaewereinvestigated.The120-hEC50ofBPAforC.mexicanaandC.vulgariswere44.8and39.8mgL-1,respectively.ThedrycellweightandchlorophyllacontentofbothmicroalgaedecreasedwithincreasingBPAconcentrationhigherthan10mgL-1.GrowthofC.vulgariswassignificantlyinhibitedat50mgL-1BPAcomparedtoC.mexicana.Totalnitrogen（TN）andtotalphosphorous（TP）removalwashigherinC.mexicanathaninC.vulgaris.MicroalgaeperformedthebioaccumulationandbiodegradationofBPAtovaryingextentsatdifferentinitialBPAconcentrations.ThehighestratesofBPAbiodegradation,24and23%byC.mexicanaandC.vulgaris,respectively,wereachievedat1mgL-1BPA.BoththetotalfattyacidandcarbohydratecontentsincreasedwithincreasingBPAconcentration.ThisstudydemonstratedthatC.mexicanawasmoretoleranttoBPAandcouldbeusedfortreatmentofBPAcontaminatedaqueoussystems.

Keywords:

BisphenolA,Biodegradation,Microalgae,Carbohydrates,Fattyacids

1.Introduction

Populationgrowthandurbanizationtogetherwithparallelglobalindustrializationhaveresultedinsignificantcontaminationofwaterstreamswithawidevarietyofendocrine-disruptingchemicals（EDCs）[1].BisphenolA 

（BPA）,whichisemployedfortheproductionofepoxyresinsandpolycarbonate（PC）plastics,isutilizedinvariousfoodanddrinkpackaging,babybottlesanddentalsealants[2].BPAisastrongendocrinedisruptorandalsoleadstocarcinogenesis[3].Despiteitshazardouseffects,BPAhasbeenextensivelyused,increasingitsglobalconsumptionatanannualrateof5.5%during2009-2012[4].IthasbeenreportedthathumansareprimarilyexposedtoBPAbyingestion,inhalationandskincontactontheorderofmicrogramsperkilogramofbodyweightdaily[5].BPAreleasedfrommanufacturingsitesoritsresiduesfromurbanandindustrialwastewaterseverelycontaminatetheenvironment,primarilyitsaquaticsystems.BPAhasbeendetectedatconcentrationsofapproximately150μgL−1inindustrialwastewaters[6],21μgL−1inrivers[7]and17,200μgL−1inlandfillleachates[8].BPAimposesdeleteriouseffectsonaquaticorganisms,evenatconcentrationsoflessthan1μgL−1[9],makingitsdetectionandremovaltonon-toxiclevelaprimaryconcerninwaterqualitymanagement.

Methodstoremoveenvironmentalpollutants,especiallyBPAincludephoto-degradation[10],oxidation[11],photoelectrocatalyticoxidation[12]andbiodegradation[13].Phytoremediationisasolarpower-driven,ecologicallysoundandsustainablereclamationstrategythatusesplantsforcleaningcontaminatedsites[14].Inrecentyears,phytoremediationofcontaminatedwatersbyphotoautotrophicaquaticorganismssuchasalgae,hasbeendemonstratedtobesuccessfulfortheremovalofbothorganicandinorganicpollutants[15,16].Asprimaryproducersandresidingatthebaseofaquaticfoodchains,microalgaeplayanimportantroleinmaintainingthebalanceofaquaticecosystems;

however,theyareknowntoberelativelysensitivetochemicals[17].Microalgaehavebeenreportedtoaccumulatepollutantssuchasheavymetals,hexachlorobenzene,herbicides,insecticidesandphenol[15,18].Thebiodegradationofenvironmentalorganiccontaminantsbyalgaehasalsobeenreported[19,20],indicatingthatalgaehavethepotentialtoremovepollutantsfromwastewaterandcanbeemployedinwastewatertreatmentfacilities.

MicroalgaeandmarinediatomshavebeenreportedfortheremediationofBPA[13,21],butrelativelyfewstudieshavereportedtheutilizationoffreshwatermicroalgaetoremoveBPA.Thisstudyaimstoinvestigate

（1）thescreeningofBPAtolerantalgalstrains;

（2）microalgalbioaccumulation/biodegradationofBPA;

and（3）theeffectofBPAconcentrationonnutrient（nitrogenandphosphorous）removalandcarbohydrateandfattyacidproductionbymicroalgae.

2.MaterialsandMethods

2-1.Chemicals

Allchemicalsusedinthisstudywereofanalyticalgrade.BPA（purity,>

99.0%）waspurchasedfromSigma-Aldrich（St.Louis,MO,USA）,andmethanolandotherchemicalswereobtainedfromDuksan（Seoul,S.Korea）.

2-2.Algalstrains,cultureconditionsandinoculumpreparation

Fourmicroalgalspecieswereinvestigatedinthisstudy:

ChlamydomonasmexicanaFR751193,ChlorellavulgarisFR751187,MicractiniumreisseriFR751194andScenedesmusobliquusHM103383（Table1）.Themicroalgalstrainswereindividuallyinoculatedin250mLErlenmeyerflaskscontaining100mLBold’sBasalMedium（BBM）at10%concentration（Vinoculum/Vmedia）.Themicroalgalcellswerecultivatedinashakerincubatorat150rpmand27º

Cundercontinuousilluminationofwhitefluorescentlightof45-50μmolphotonm-2s-1fortwoweeks.Themicroalgalsuspensionwasadjustedtoanabsorbanceof1.0atanopticaldensity（OD）of680nmasmeasuredusingaspectrophotometer（HachDR/4000,Loveland,CO,USA）.

2.3.Experimentalprocedure

Forgrowthinhibitiontests,thetoxicityofmethanoltothealgalcellswasinvestigated.Theinitialconcentrationofmethanolinthemediumwas0.03%（v/v）.AlgalcellsattheexponentialphasewereinoculatedintothemediumsupplementedwithdifferentBPAconcentrations.TheeffectiveconcentrationofBPAthatproduceda50%inhibitionofalgalgrowthat120h（120-hEC50）wasobtainedfromthedose-responseregressioncurvebyplottingBPAconcentrationsagainstinhibitionpercentages[22].

InitialexperimentswerecarriedouttoselectBPAtolerantmicroalgalspecieswiththelowestgrowthinhibitionbasedontheirbiomassyieldafter5daysofcultivationat7mgL-1BPAinBBM.Furthermore,BPAremovalandchangesinthebiochemicalcompositionofselectedmicroalgalspeciesat1,5,10,25and50mgL-1BPAweredetermined.Thebatchexperimentswereconductedusing500mLaluminumcrimp-sealedserumbottlescontaining300mLBBMinoculatedwith1.5%oftheinoculum（Vinoculum/VBBM）.Thebottleswereincubatedinashakerincubatorat27°

Cand150rpm,underwhitefluorescentlightillumination（alternatelight/darkperiodsof16h/8h）atanintensityof45-50μmolphotonm−2s−1for10days.

2-4.Measurementofcellgrowthandnutrientremoval

GrowthwasmonitoredbasedonchangesintheOD680concentration.TheOD680valueswereconvertedtodrycellweight（DCW）concentrations（gL-1）,basedonalinearrelationshipbetweenOD680anddrycellweight[23],whichwasobtainedafterextensivedataanalysisandwascalculatedbyEqs.

（1）and

（2）forC.mexicanaandC.vulgaris,respectively,asfollows:

Dryweight（gL-1）=0.3218×

OD680-0.0139（R²

=0.9948）

（1）

Dryweight（gL-1）=0.3065×

OD680-0.0097（R²

=0.9958）

（2）

Forthechlorophyllameasurement,a5mLculturewasharvestedbycentrifugationat4,500gfor10min.Thesupernatantwasdiscardedandthepelletwasre-suspendedin5mLof95%methanol,incubatedat60º

Cfor5minandcentrifugedagainfor10min.Theabsorbanceofthesupernatantat665and652nmwavelengthswasdeterminedwithaHachDR/4000UV-visiblespectrophotometer（Hach,Loveland,CO,USA）,andthechlorophyllaconcentrationoftheextractwascalculatedfollowingtheformuladescribedbyPorraetal.[24]:

Chlorophylla（mgL-1）=16.29×

A665–8.54×

A652（3）

Thespecificgrowthrate（µ

）wascalculatedbyfittingthedrycellweightforthefirst7daysofcultivationtoanexponentialfunction,asshowninEq.4:

（4）

TNandTPfromthesampleweremeasuredusingPersulfateDigestionandAcidPersulfateDigestion,whichareequivalenttoStandardMethods4500-NCand4500P.B.5,forwaterandwastewater,respectively[23].

2.5.DeterminationofresidualBPA

2-5-1.BPAinthemediumandabsorbedbycells

InordertodeterminetheamountofBPAinthemedium,10mLofsamplewascollectedfromthecultureandce