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Sterilization

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SterilizationSteril

    Sterilization

JournaZofEnvironmentalSciencesVo1.16.No.2.PP.348352,2004ISsNi00I一仃742

    ;CNIl2629,X

    ;ArlideID:1001.0742(2004)02.0348.05CLCnumber:X703Documentcode:A

    ;SterilizationofEscherichiacolicellsbytheapplicationofpulsedmagneticfield ;LIMei.QUJiu—hui’.PENGYongzhen

    ;(1.ResearchCenterforEcoEnvironmentalSciences,ChineseAcademyofSciences,Beijing100085,China.Email:jhqu@mail.rcees.ac.cn;2

    ;SchoolofMunicipalandEnvironmentalEngineering,HarbinInstituteofTechnology,Harbin150090,China)

    ;Abstract:Theinactivationofmicroorganismsbypulsedmagneticfieldwasstudied.Itwasimprovedthatthe

    ;applicationofelectromagneticpulsesevidentlycausesaIethaIeffectonE.coilcellssuspendedinphosphatebuffer

    ;solutionNa,HPO/NaH,PO(0.334/0.867mmoI/L).Experimentalresultsindicatedthatthesurvivability(N/N;where

    ;NoandNarethenumberofcellssurvivedpermilliliterbeforeandafterelectromagneticpulsesapplication,

    ;respectively)ofE.coildecreasedwithmagneticfieldintensityBandtreatme

    ttimet.Itwasalsofoundthatthe

    ;mediumtemperatures.thefrequenciesofpulsef.andtheinitiaIbacteriaIcelIconcentrationshavedeterminate

    ;influencesindestructionofE.coilcellsbytheapplicationofmagneticpulses.Theapplicationofanmagnetic

    ;intensityB=160mTatpulsesfrequencyf=62kHzandtreatmenttimet=16hresultinaconsiderabledestruction

    ;IevelsofE.coilcells(/:10).Possiblemechanismsinvolvedinsterilizationofthemagneticfieldtreatment

    ;werediscussed.Inordertoshortenthetreatmenttime.manygroupsofparallelinductivecoi1wereused.The

    ;practicabilitytestshowedthatthetreatmenttimewasshortenedto4hwiththeapplicationofthreegroupsofparalleI

    ;coi1whenthesurvivabilityofE.cogcellswasIessthan0.01%;andthepowerconsumptionwasabout0.2kWh/m.

    ;Keywords:Escherichiacoilbacteria;pulsedmagneticfield;inducedcurrent:celImembrane ;Introduction

    ;vanouschemicaltreatmentshavebeenpredominantly

    ;usedtoinactivateorkillmicrobesinwater.However,the

    ;useofdisinfectantschangesthewatercompositionandcauses

    ;contaminationofthetreatedmediumfromtoxicchemicalby

    ;products.Therefore,physicaltreatmentscanbeusedbecause ;theydonotchangethequalityofwater.Newphysical ;techniquestoinactivatebacteriainwaterarebeingexplored ;aspotentialalternativestothechemicalmethods,including ;theutilizationofpulsedelectricfields,magneticfield, ;intenselightpulsesandsoon.Inparticular,magnetic ;treatmentshaveattractedmuchattentionforover100years, ;andinactivationofmicroorganismsinwaterusingpulsed ;magneticfieldhasbeenstudiedinrecentyears(Guo,1996; ;Lei,1994;Luo,2001).Itsefficiencyisstillacontroversial ;question.Ingeneral,peopleagreeonthefactthat ;electromagneticfieldcancausechangesforcellson ;configuration,formation,andfunction,etc.Evenmagnetic ;fieldoflowintensitycanstronglyinfluencebiologicsystem ;(Bassett,1993).Forexample,magnetictreatmentcauses ;theactivityofbloodplatelet,thebreakdownofcell,the ;inactivationofenzyme(Wang,2000).Pulsedmagneticfield ;hasgreaterbiologiceffectsthanconstantfield(Zhong, ;1998).Possibilitiesofinactivatingthemicroorganismsin ;liquidmediausingapulsedelectromagneticfieldhavebeen ;consideredbymanyresearchersinanefforttodevelopa ;promisingsterilizationmethod.Ithavebeenreportedthatthe ;survivabilityofmicroorganismswhenexposedtoapulsed ;magneticfielddependsonboththemagnitudeofthemagnetic ;fieldintensityandthetreatmenttime(Guo,1996).Despite ;severalstudieswhichhaveexaminedthemagneticfieldeffects ;onthedeathofmicroorganisms,theunderlyingmechanisms ;involvedinthebreakdownofthecellleadingtocelldeathare ;notclear.Inthisstudy,ahomemadeelectromagneticdevice

    ;wasdevelopedtoinvestigatetheeffectofapulsedmagnetic ;fieldonthesurvivabilityofEscherichiacolisuspendedin ;bufferedphosphatesolutionbothbyfollowingthechangesof ;magneticfieldintensityandtreatmenttime.Theinfluencesof ;pulsefrequency,mediumtemperature,initialbacterialcell ;concentrationsinkillingE.colicellswereinvestigatedalso. ;1Materialsandmethods

    ;1.1Biologicalandchemical

    ;ThepurecultureofthebacteriaEscherichiacolusedin ;thisstudywaskindlyprovidedbytheinstituteof ;microbiology,ChineseAcademyofSciences.TheE.col ;cellsweregrowninEscherichiaLBbrothinanincubatorfor ;24hat37?.Thecellswereharvestedfromthebrothby

    ;centrifugationbeforereachingtheirstationarygrowthphase. ;Centrifugationwascarriedoutat10000r/minfor5minat

    ;4?.I,hecellswerewashedandresuspendedinphosphate ;buffersolution.TheconcentrationNa2HPO4/Nail2PO4was ;(0.334/0.867mmol/L).ThepHofthebufferwas7.4.The ;dilutionofcellsinthesuspensionwaschosentoproduce ;approximately10or10.cells/mlbeforetreatment. ;ThesurvivabilityoftheE.colcellsfollowingthe ;treatmentwasmeasuredbymeansofcolonycountinKinan ;Fonndati.nitem:TheNationalNaturalScienceFoundationofChina(N..50238050)andtheNationalKe

    yTechn.loslesR&DPr0舢一1OhFive.

    ;YearPJ

    ;(2001BA610A05D);*Correspondingauthor

    ;

    ;No.2SterilizationofEscherichiacolicellsbytheapplicationofpulsedmagneticfield349

    ;agardish.Thetreatedsamplewasseriallydilutedwith ;deionizedandsterilizedwater,and1mlofthedilutionswas ;platedonagarmediumandincubatedforabout48h.The ;dilutionsfortheviablecountwerecarriedoutinsuchaway ;thatthenumberofcoloniesontheagarplatewas30300.

    ;Eachreadingwascalculatedfromanaverageofeightplates, ;andforeachexperimentalconditionmeasurementswere ;repeated34times.

    ;Inadditiontothecolonycountingasameasureof ;survivabilityofmicroorganisms,scanningelectronmicroscopy ;(SEM)observations,chromatographicandatomicabsorption ;spetrophotometryanalysistodetectthepresenceofanionsand ;cationswerecarriedout.Forelectronmicroscopythe ;microbial(:ellswerefixedfor2hat4?with2.5%

    ;glutaraldehydein(0.334/0.867mmol/L)phosphatebuffer ;followedbyfiltration(0.2mmmilliporepolycarbonatefilter ;paper).Thefilterpaperwaswashedwithphosphatebuffer ;anddehydratedingradedacetone.Furtherdryingwasdone ;usingacriticalpointdryer.Thefiltercontainingthemicrobes ;wasmountedoncopperstubsandcoatedwithgold.The ;scanningelectronmicrographsweretakenusingaHitachi ;modelS570(SEM).Beforeandaftertreatmentsthe

    ;concentrationsofanions,chlorine(C1),phosphate(po34),

    ;nitrate(NO;),andsulfate(SO:)ionsweremeasuredusing

    ;aDionex4500ionchromatograph(IC);theconcentrationsof ;cations,natrium(Na),kalium(K)weremeasuredusinga ;HitachimodelZ6100atomicabsorptionspectrophotometry. ;1.2Electrical

    ;Thecircuitconfigurationusedtogeneratedirectcurrent ;pulsewasbasedonthecompletedischargeofacapacitor ;producinganexponentiallydecayingvoltagewaveform.The

    ;capacitorwaschargedusingaDCpowersupply.Itsworking ;principleisshowninFig.1.Thefrequencyofthepulse ;applicationwasvariablebetween400and6000Hz.Four ;highlevelswereusedforthetests.Themagneticfield ;treatmentsetupisshowninFig.2.Themagneticfieldused ;toinactivateorkillmicroorganismswasgeneratedby ;inductivecoilgrouplinkedtothedirectcurrentpulse ;generator,whichwerewrappedaroundtheouterwallofthe ;pipe.Themagneticfieldintensitywasvariedbytheoutput ;powerofthedirectcurrentpulsegenerator.Thelengthof ;eachinductivecoilwas10cm.Thetotallengthofthe ;pipelinewas1.0m,whichwassterilizedbeforetreatment. ;Thetestsamplewasaddedtoaglasscuvetteinatotally ;asepticcondition.Ametricpumpwasusedtomakethe ;samplerecycleinthepipeline.Theflowvelocityof0.2m/s ;wasusedforalJmeas11rements.

    ;2Resultsanddiscussion

    ;2.1Principalonpulsedelectromagneticfieldtreatment ;ofmicrobialcells

    ;Biologiceffectsofelectromagneticfieldincludethe ;thermalandnonthermaleffects.Thethermaleffectsreferto ;thebiologicfunctionchangesduetotemperaturerisingwhen ;cellsabsorbtheenergyofelectromagneticwaves.The ;nonthermaleffectsinvolvethestrongbiologicresponses ;withoutobvioustemperaturerisingincludingchangeson ;physiology,biochemistry,andfunction,whicharerelatedto ;thefrequencyandpowerofelectromagneticfield.The ;nonthermaleffectsoftenhappeninthestatefarawayfrom ;balance.Forthenonthermaleffects,theresponsesofcellsto ;electromagneticwavesarenonlinear,i.e.,theinducements ;fromoutersmallenergycangiverisetothereleaseof ;intracellulargreatenergy.Usingelectromagneticpulseto ;inactivatemicroorganismsdependsonthenonthermaleffects ;primarily(Zhou,2000).

    ;3

    ;!!!!!il

    ;Fig.1Diagramofdirectcurrentpulsegenerator

    ;Fig.2Sketchmapofmagneticfieldtreatmentset-up ;1influencecontainer;2.metricpump;3dischargevalve;4iductivecoi ;group;5.directcurrentpulsegenerator

    ;Cellscanbepenetratedbymagneticfieldbecausetheir ;magneticconductivityissimilarwiththatofvacuum.Asa ;result,theinducedcurrentwillbeproducedincellsfor ;pulsedmagneticfieldbecauseofitsgreatvariablerateof

    ;magneticfieldduetoshortpulsedelay.Studiesshowthatthe ;biologiceffectsofpulsedmagneticfieldlieonthecategory ;andsizeofcells.Cellswithdifferentcategoryandsizehave ;differentendurablecapabilitytopulsedmagneticfield ;intensity.

    ;Themechanismsforpulsedmagneticfieldbiologic ;effectsoncellsareexplainedasfollows:Forpulsedmagnetic ;field,atransientmagneticfluxmustbeproducedincells ;resultedfromtheinstantaneousappearanceanddisappearance ;ofmagneticfield.TheinducedelectromotiveforceEdueto ;thechangesofmagneticfluxisgivenas:

    ;E=d/dt=SdB/dt,(1)

    ;thecorrespondinginducedcurrentIisasfollows: ;

    ;350LIMeietalVo1l6

    ;,:E/(SR)=RdB/dt,(2)

    ;whereSisthecellsectionalareapenetratedverticallybythe ;magneticfield;Risthecellcircuitresistance;dB/dtisthe ;averagevariationalratio.Theinducedcurrentsize, ;direction.andforn1areprimaryfactorstodeterminethe ;biologiceffectsofmagneticfieldoncells.Thehigherthe ;inducedcurrentis.themoreobviousthebiologiceffectsare. ;Theforcedensityoftheinteractionbetweentheinduced ;currentandmagneticfieldcandestroynormalphysiologic ;functionsofcells.Largecellsareeasiertobeinactivated ;thansmallcellsbecausetheforcedensityisproportionalto ;thediameterofcells.Otherwise,theinducedcurrentgives ;risetoanelectricpotentialdifferencebetweentwosidesof ;cellmembranebecauseofitselectricresistance.Asthen, ;thispotentialdifferencechangesthetransmembranepotential, ;whichbringdestructiontocellmembrane.Ontheother ;hand,themovementofelectricionsespeciallyelectronsand ;ionswithlittlequalitiesislimitedwithinarestrictedarea. ;Theycannottransferthroughmembranenormally,and ;normalphysiologicfunctionsarenotavailableforcells.For ;biomacromoleculessuchasenzymetheirconformationsare ;twistedortransformed.Asaresult.thenormalphysiologic ;functionsofcellaredestroyed.

    ;2.2LethaleffectonE.colicells

    ;2.2.1Effectofelectricfield

    ;TheviabilityofE.coliasafunctionoffieldatroom ;temperaturewithadifferenttreatmenttimeisshowninFig. ;3.Ateachleveloftreatmenttimeafiniterateofcelldeath ;increasedexponentiallyasthefieldintensityincreased.Asa

    ;functionoffield,survivabilitydeclinedrapidlyatlow ;strengthsandmoregraduallyathigherstrengths.Withafield ;intensityaslowas160mT.itwaspossibletoreducethe ;viablecellnumbersbyabore85%.Withfurtherincreasein ;fieldintensity,largernumberofcellswerekilled;however, ;therategraduallydeclined.

    ;ThesurvivabilityofE.colasafunctionoffield

    ;intensityatdifferentfrequenciesfisshowninFig.4.Atall ;fourtestfrequenciesthedeathraterapidlyincreasedwitha ;fieldintensityupto120T:whereasatslightlyhigherfield ;intensities(>160T),thedeathrateremainedrelatively ;constant.Itwaspossibletoreducetheviablecellcountto ;10mlat2xmTwithapplicationof62kHzfrequency.

    ;Thetreatmenttimeappliedwaskeptconstant(t=6h)for ;allthesetests.However,actualexposuretimetomagnetic ;fieldwasdifferentasthepulsenumbervariedwiththe ;frequency.Atelevatedpulsefrequencies,thepulsenumber ;increasedforeachdischargingofthecapacitor.Hence,the ;~equencyeffectscausehigherdeathrateduetoanincreased ;exposuretime(Hulsheger,1981).Withthepresentsetupit ;wasthereforenotpossibletotesttheeffectsof~equencyon ;thelethalityofmicroorganismsabove70kHz.

    ;2.2.2Effectoftreatmenttime

    ;SurvivabilityasafunctionoftandTatfieldintensitv ;

    ;

    ;l

    ;Magneticfieldintensity,BImT

    ;Fig.3SurvivabilityofE.colicellsasafunctionofmagneticfieldintensity

    ;Bwithdifferentreatmenttimes

    ;?t=6h;?t=12h;?t:16h;T:25%;pH:7.3;,=56kHz

    ;

    ;{

    ;‘E

    ;Magneticfieldintensity,BImT

    ;Fig.4SurvivabilityofE.colicellsasafunctionofmagneticfieldintensity

    ;Batdifferentpulsefrequencies

    ;?,=46kHz;?,=50kHz;?f=56kHz;Of=62kHz;T=25%;pH=

    ;73

    ;ofl60mI’isshowninFig.5.Asafunctionoftreatment

    ;timethecellsurvivabilityratefeUrapidlyatfirstandthen ;tendedtodecrease,reachingarelativelyconstantrateatthe ;longertreatmenttimes.AsthefieldintensityWashigher.the ;survivabilityofE.colwasreducedby80%abovewith

    ;applyingtreatmenttimeof10h,whenthemediumwas ;250C.Comparedtothis.atT=600C.itwaspossibleto ;obtainamuchhigherreductioninsurvivabilityat ;correspondingtreatmenttimes.Itwaspossibletoreducethe ;viablecellcountto10mlat600Cwithapplicationof16h ;treatmenttime.Cellswerealsokilledduetothetemperature ;effectalonewhileincreasingthemediumtemperature.As ;such,numberofviablecellspermilliliterbeforetreatmentat ;highertemperatures(N0)waslowerthaninitialnumberof ;viablecellspermilliliter(N)atroomtemperature.When ;thetemperatureofthemediumwasraisedto800C.almostall ;thecellswerekilledbythethermaleffectevenpriortothe ;applicationoftheelectricfield.Fig.5showsthatthekilling ;ofE.colicellsduetopulsedfieldintensityapplicationwas ;temperaturedependent.Thetemperatureofthemediumin ;whichcellsaresuspendedhasasignificantinfluencein ;determiningthemembranefluidityproperties.Atlow ;temperatures.thephospholipidsarecloselypackedinarigid ;gelstructure,whileathightemperaturestheyarelessordered ;

    ;No.2Sterilizati0n0fEscherichiacolicellsbytheapplicationofpulsedmagneticfield351

    ;andthemembranehasa”liquid—crystalline”structure

    ;(Jayaram,1991).Thephasetransitionfromgeltoliquid ;crystallineisthermaldependentandhencecanaffectthe ;physicalstabilityofthecellmembrane.Ariseintemperature ;isknowntoincreasethelateraldiffusionrateoflipidsbyat ;leasttwoordersofmagnitudeaslipidschangefromgeltothe ;liquidcrystallinephase.AsseenfromFig.5,alarger ;reductioninsurvivabilityofE.coliwasobservedwhenthe ;liquidtemperaturewashigher(60~C)atcomparable

    ;magnitudeoftreatmenttimethanatlowertemperatures. ;Basedonthis,itisproposedthattemperaturerelatedphase ;transitionofthephospholipidmoleculesfromgeltoliquid

    ;crystallinephaseandtheassociatedreductioninbilayer ;thicknessmaymakethecellmoresusceptibleforfieldeffects ;atarelativelyhightemperature.

    ;

    ;

    ;l

    ;Tre~cnttime,h

    ;Fig.5Sur~’ivabi[ityofE.coilceilsasafunctionof~eatmenttimetat

    ;differenttemperatures

    ;?T=25~C.N.=N0=1.6×10;?T:30~C,N:4.6×10,N0=

    ;4.2×10;?T:45~C,N=5.1×10,N0=4.8×106;?T=60~C,N

;=72×10,N0=43×106;B=160mT;_=62kHz;pH=7.3

    ;Asafunctionoftreatmenttime,whilethefieldand ;temperaturewereconstant,thedegreeofdeathdeclined ;rapidlyatfirstandthenmoreslowly,asshowninFig.6.It ;wasobservedthatanincreaseinthekillingrateofE.coli ;withtheincreaseintheinitialbacterialcellconcentrations. ;Thefollowingisapossibleexplanationfortherelationship ;betweenthenumberoflivecellspresentpriortopulse ;applicationandthesurvivingcellsbasedontheappearanceof ;inducedcurrentduetothevariationofmagneticflux(Kaler, ;1983).Theinducedcurrentisadominantfactorforthe ;breakdownofcellsinvolvedintheapplicationofmagnetic ;field,whichgivesrisetoaninteractionalforcewithmagnetic ;field.Thisforceactingonthecellsurfacecausesirreversible ;breakdownofmembrane,whosemagnitudedepends

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