Screening

JournalofEnvironmentalSciencesVo1.17.No.3,PP440—444,2005lssN100l0742

    ;Cl1—2629/X

    ;ArticleID:1001.0742(2005)03一O44O.05CLCnumber:X131;X7Documentcode:A

    ;Screeningonoil—decomposingmicroorganismsandapplicationinorganicwaste

    ;treatmentmachine

    ;LUYi-tong,CHENXiao-bin,ZHOUPei,LIZhen-hong

    ;(DepartmentofResourcesandEnvironmentalScience,ShanghaiJiaotongUniversity,Shanghai201101,China.E-mail:ytlu@sjtu.edu.cn)

    ;Abstract:AsanoiI.decomposablemixtureoftwobacteriastrains(Bacillussp.andPseudomonassp.),Y3wasisolatedafter50d

    ;domesticationundertheconditionthatoi1wasusedastheIimitedcarbonsource.ThedecomposingratebyY3washigherthanthatby

    ;eachseparateindividuaIstrain.indicatingasynergisticeffectofthetwobacteria.UndertheconditionsthatT=25—40,DH=6—8.HRT

    ;(Hydraulicretentiontime)=36handtheoiIconcentrationat0.1%,Y3yieldedthehighestdecomposingrateof95.7%.Y3wasalso

    ;appliedinanorganicwastetreatmentmachineandacertainrateofactivatedbacteriawasputintothestuffing.Aseriesoftestsincluding

    ;humidity,DH,temperature,C/NrateandoiIpercentageofthestuffingwerecarriedouttochecktheefficacyofoil—decomposition.Results

    ;showedthattheoiIcontentofthestuffingwithinoculumswasonlyhalfofthatofthecentreI.Furthermore.thebacteriawerealsobeneficiaI

    ;tomaintainthestabilityofthemachineoperating.Therefore.thebacteriamixtureaswelIasthemachinesinthisstudycouldbeveryusefuI

    ;forwastetreatment.

    ;Keywords:oil—decomposingmicroorganism;screening;application;organicwastetreatmentmachine;decomposingrate

    ;Introduction

    ;TheBOD(biochemicaloxygendemand)offatsandoils

    ;derivedfromanimal,plantormineral,isabout1.8rag/L,

    ;indicatingthatfatsandoilsmaybeeasilierdecomposedthan

    ;themostotherorganiccompounds.Afterbeingintroduced

    ;intotheenvironment,however,fatsandoilscanform

    ;compactfilms,whichcanpreventthetransferofO2acrossthe

    ;interfaeesbetweendifferentmedia,andcausesharmtothe

    ;aerobicdecompositionbyotherorganisms.Therefore,

    ;treatmentsofsolid/liquidwastecontainingfatsandoilshave

    ;beingincreasinglyconcerned.

    ;Theconventionaltechniquesofseparatingfatsandoils

    ;fromwastewaterdependongravityandfloatationsuchas ;grease—trap.Unfortunately,therearealwayssomerestaurants ;thatproduceagreatdealofwastewatercontainingtOOmuch ;fatandoiltobetreatedbysuchequipments.Furthermore. ;solidwastewithfatsandoilsisdifficulttobetreated ;wastewater.InChina,bigger—scalerestaurantsproducelotsof

    ;kitchen-wastewithahighconcentrationoffatsandoils, ;whichcannotbeusedtofeedlivestockaccordingtothe ;Chinese.Theorganicwastetreatmentmachineisakindof ;novelequipmentrelyingonmicroorganismstodegradeorganic ;waste,whichhasbeendevelopedandappliedofteninmany ;citiesinChina.However.therestillremainsome ;limitations.especiallyduringthetreatmentofkitchen—waste

    ;containinglotsoffatsandoils,theanaerobicconditionand ;gooeyderivedfromaccumulatedfat/oilwillbafflethemachine ;fromwell—balancedrunning.Itiswellknownthatanumber ;ofnaturalmicroorganisms,including28speciesofbacteria, ;4speciesofaetinomyeetes,10speciesofyeasts,can ;decomposefat/oilreadily.Therefore,itisimportanttouse ;microorganismstodecomposeeffectivelyfatsandoilsinthe ;waste.

    ;Theaimofthisworkwastodealwithdomesticationbya ;mixtureoftwobacteriawithahighdecomposingrateoffat/ ;oil,anditsapplicationintheorganicwastetreatment ;machine.Thecharacteristicsofthebacterialcultureandthe ;effectofthiscultureontheperformanceofmachinetreatment ;arealsodescdbed.

    ;Correspondingauthor

    ;lMaterialsandmethods

    ;1.1Samplesforscreening

    ;Fivesludgesampleseollecte(1fromfourrestaurantsand ;aWasteWaterTreatmentPlantinShanghaiCitywereusedin ;thisexperiment.Thesampleswerekeptat25ccinthe ;asepticlaboratorybeforeused.

    ;1.2Organicwastetreatmentmachine

    ;Theorganicwastetreatmentmachine(YFL001)was ;providedbyTaiwanYaoFengCompany.

    ;1.3Screeningonthemicroorganismsfromsamples ;About5gofeachsludgesamplewasputinto250rrd

    ;Erlenmeyerflaskswith45mlsterilizeddistilledwaterand ;glassballs.Theywerethoroughlymixedbyshakingonan ;oscillatorat300r/minfor10min.Then10mldiluentswere ;pipettedinto90mlenrichmentmediumin250mlErlenmeyer ;flasks.Theflaskswerethenincubatedat40ccandshakenat

    ;2(x)r/min.Thecompositionoftheenrichmentmedium(g/L ;distilledwater)was:NaCl,5.0;peptone,5.0;KH2P04, ;0.3;MgSO’70,0.1;K2HPO,0.3;(NHe)!SO,1.0;

    ;peanutoil.10m1.andtheinitialpHwas7.0.Onemlpeanut ;oilwasthenpipettedintotheflaskinwhichoilcouldbe ;decomposedwelleveryday.Tenmlcultureswerepipettedinto ;90mlfreshenrichmentculturewith2%peanutoilevery5d ;toimprovemicrobialabilityofdecomposingfat/oil.Tile ;domesticationprocesslasted50d.Afterenrichment.thefat/ ;oil—decomposingmicroorganismswerescreenedonisolation ;plates(theenrichmentmediumwithoutpeptonebut2%agar) ;withNeutralred,Bromocresolpurple(Yu,1990),orblank ;isolationplates.Thecolonieswerepickedupindividually. ;andinoculatedintoliquidmediuminflaskstoascertaintheir ;abilitiestodecomposefat/oil.Thecompositionoftheliquid ;medium(g/Ldistilledwater)was:NaCl,5.0;peptone, ;10.0;beefextract,5.0;KHPO4,0.3;MgSO4.7H20,0.5; ;l<2HPO4,0.5;CaCl2,0.02;peanutoil,10ml;pH7.0. ;1.4CultureconditioI1sandrat,o.1biodegradabilitest ;ofY3

    ;Vari0ustestsaboutY3werecaed0utt0ascertaintheir ;efctiveness,andtoinvestigatethei1uenceofv0us ;culturec0nditionsincludinginitialpH0fInedium,incubati0n ;

    ;No.3S(reeningonoil—decomposingmicroorganismsandapplicationinorganicwastetreatmentnmch

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    ;temperature,treatmenttime,02concentration,differentkinds ;offat/oilascarbonsourcesanddifferentnitrogensources,on ;fat/oilbiodegradabilityofY3.About2mlofeachinoculum ;wasinoculatedinto50mlmediuminthe250mlEdenmeyer ;flasksandincubatedunderdifferentconditionsaccordingto ;theexperimentaldesign.Controlsetswithoutinoculumwere ;alsoincubatedandanalyzedatthesametime.Mter ;incubation.thesampleswerecollectedforfat/oi1analysis ;usingthemethoddescribedbelow.

    ;1.5Applicationinorganicwastetreatmentmachine ;andtheanalysis

    ;Activatedinoculumswereputatacertainrateintothe ;machineoperatedunderusualconditions.Atthesametime, ;contro1machinesalsoranunderthesameconditions ;comparatively.Themachineisabletotreat1.5kgorganic ;wasteperday.Therefore,leachingintoeachmachineevery ;dayputlto1.5kgdriedkitchenwaste.Thestuffingineach ;machinewasanalyzedevery7d.andasetofdataoffat/oil

    ;percentage,pH,temperature,C/Nratio,humidity, ;organism.totalN.ete.wereobtained.

    ;1.6Estimarionofresidualfat/oil

    ;Themethodforestimatingresidualfat/oilinliquid ;culturewasdifferentfromthatforsolidstuffing.Residualfat/ ;oilinliquidculturewasestimatedusingGB50096.whichis ;thestandardmethodreleasedbyChinesegovemment.The ;residuefat/oilinstumngwasextractedbySoxhletExtraction ;for24hwithpetroleumether(30—60(?),andtheremained

    ;petroleumetherwasevaporatedbyarolling—evaporation

    ;equipment.Thedriedfractionwasputintoanair—ovenand

    ;driedtoaconstantweightatl05?beforeextraction.The

    ;amountofdecomposedfat/oilwasestimatedbythe1ossof ;weightattributedtotheinoculums.

    ;1.7Stuffingsamplingandanalysis

    ;Thestuffingsampleswerecollectedevetw7dfrom5 ;differentsitesinthemachines.ThepHofthesampleswere ;determinedataratioofl:2.5(soll:water).Humiditywas ;measuredbydryingsamplestoaconstantweightinanair- ;ovenatl05?for2h.Temperaturewasmeasuredinthe

    ;stuffingat5differentsiteseveryday.Volatileorganicswere ;determinedinmuffleat550?.Ammoniumandorganic

    ;nitrogen(TKN)weremeasuredwithdrycombustionusingan ;automatedNanalyzer(Gerhardt).

    ;1.8Identificationofmicroorganisms

    ;Onthebasisofthebiochemicalandmorphological ;characteristics.isolateswereidentifiedaccordingtothe ;descriptionsinthe”Berggy’sManualofSystematic

    ;Bateriology”.

    ;2Resultsanddiscussion

    ;2.1Screeningonfat/soil-decomposingmicroorganisms ;Sevencolonies,Y1一Y7.werefoundontheplatein

    ;thefirstisolation.ItwasclearthroughflaskteststhatY3had ;thehighestabilitytodecomposefat/oil.whichcanincrease ;theODvalueat540nmoftheliquidcultureto5.046(taking ;theculturewithoutinoculumsasblankone)andhadn0any ;separationbetweenwaterandoilafter48hincubation. ;However,whenY3wasseparatedfurtherintotwokindsof ;bacteria,Y3.1andY3.2.theabilitytodecomposefat/oi1 ;decreasedobviously.Y3.1(akindofbacteria)couldnot ;su1wiveformorethanonemonthalone,whilehadahigher ;abilitvofdecomposingfat/oilthanY3.2.Therefore,itcould ;beconcludedthatthereexistedaassociationbetweenboth ;kindsofbacteriafortheirsurvivinganddecomposeoffat/oil.

    ;Threekindsofindicators.Neutralred,Bromocresol ;purpleandCuSO4wereusedduringtheisolation.Neutralred ;andBromocresolpurplecouldbeservedforindicating ;whetherornotpHreducingwascausedbyfattyacids ;oroductionfromthedecomposeoffat/oil.WhileCuSO4 ;indicationisbasedonthedepositedoppositereaction ;causedbyCuSOa1dfattyacidsproducedbydecomposingof ;fat/oil.

    ;2.2ChangesinpHofliquidmediumwithdifferent ;initialPH

    ;Itisimportanttomaintainaninitia1pHbeforethe ;incubationanddegradationoffat/oilcultures.Resultsshow ;thattheculturegrewthebestatthepHrangingfromneutral ;toalkalinewithafat/oildegradationratehigherthan85%. ;pooratpH>9orpH<5withafat/oildegradationratelower ;than50%(Fig.1).Itisalsoobservedthatnomatterwhat ;theinitia1pHwas.pHvaluesaroundpH7.5couldbe ;observedafter24hincubation(Fig.2).Itwaswellknown ;thatthemicrobialdegradationoffat/oilonlyoccursattheoil—

    ;waterinterface(Brock,l991),whichmeansthatadequate ;mixinganddispersionbenefitfat/oildegradation.AlkalipH ;conditionsresultinabetteremulsificationinliquidmedium ;thanacidicpHconditions.thereforethefat/oilinkitchen ;wastecouldbeeasilierandbetterdegradedby

    ;microorganisms.However.itisalsonecessarytoconsiderthe ;microbialactivityaswellasemulsificationbecausemicrobial ;growthcanberestrainedatpH>9.Inaddition.no ;significantdecreaseofpHvaluecausedbyfattyacid ;accumulationintheliquidmediumwasobserved. ;10

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    ;2.3Effectsoftemperatureandshakingspeedonthe ;decomposingrate

    ;葛-uI害A唇8o0

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    ;442LUYi—tongetalVo1.17

    ;Theculturedecomposedfat/oileffectivelyat

    ;temperaturesrangingfrom25to45cc(Fig.3),thehighest ;decomposingratereached87.2%at30c《二.Itshouldbe

    ;notedthatbothincubationandmixingofwaterandfat/oil ;couldbeoptimizedwithinthetemperaturerangeasZobbel ;(Zobbel,1969).Theshakingspeedandtemperaturecan ;affecttwofactorsinfluencingdecomposingrateoffat/oil,first ;isthedissolvedO.theanotheristheadequatemixingof ;waterandfat/oil.Therefore.therearethreedifferentculture ;conditionsthatcanaffectthemixingkitchenwasteinmachine ;directlyorindirectly(Table1).Theadequatemixingcanbe ;regardedasthepreconditionofmicrobialdecomposition. ;Therewouldnotbeanymicrobialdecompositionoffat/oil ;withoutadequatemixinganddispersingoffat/oil,waterand ;microorganisms.Itwasobservedthatthefat/oil ;decompositionwasastrict02一demandingbiochemical

    ;reaction,assimilartopreviousreports(Zhang,1996).There ;wasasaturatedtendencywiththeincreaseofshakingspeed. ;100

    ;80

    ;60

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    ;Fig.3Effectsoftemperatureondecomposingrate ;Table1Effectsofshakingspeedondecomposingrate ;2.4Effectsoftreatingtimeonthedecomposingrate ;Thedegradedamountoffat/oilincreasedwithtreating ;time,whenfat/oilconcentrationwas1%(Fig.4).However, ;aftera36htreatmentthistendencywasnolongerobvious, ;indicatingtheinhibitionofthelipasebyfattyacids(Smith, ;1996).Consideringtheeconomicfactor.33to36hfor ;treatmentwasproposed.Thetreatmenttimeshouldbe ;adjustedaccordingtothevariousfat/oilconcentrationofthe ;waste.

    ;Fig.4EffectsofHRTondecomposingrate

    ;2.5Effectsofcarbonsourcesondecomposingrate ;Thedifferentfats/oilswereaddedascarbonresourceon ;thebasisoftheliquidmediumtoexaminetheeffectscaused ;byindividua1.Itshowsthatthedecomposingrateoffat/oil ;fromplantswashigherthanthefromanimals.Usually,fats ;fromanimalshaveahighermeltingpointthanthatfrom ;plants,leadingtoformconglomerations,havingresistanceto ;thedecompositionintheliquidmedium.Theantimicrobial ;activitiesagainstsomebacteriabycertainfattyacidesters ;reportedbyKwakuetaf.(Kwaku.1999)werenotobserved

    ;inthisstudy.Thewastefat/oilfromkitchenwastewasa ;mixtureofplant—andanimal—originalfat/oil.sothe

    ;degradabilityofwastewashigherthanthelatterbutlower ;thanthattheforifler.Atiera48htreatment.the ;decomposingratereached74%(Fig.5).

    ;PeanutLardOliveTallowSaladWaste

    ;oiloiloiloil

    ;Speciesoffat/oil

    ;Fig.5Effectofdifferentfat/oilascarbonsourcesonthedecomposingrate

    ;2.6Effectsofnitrogensourcesonthedecomposingrate ;Theeffectsofdifferentkindsoforganicandinorganic ;nitrogenresourcesonthedecomposingrateoffat/oilinthe ;liquidmediumwerestudied.Thedecomposingratecould ;reachover80%withorganicnitrogenresources.while ;decreasingtobelow50%withinorganicnitrogenresources. ;suchas(NH4)2SO4andNH4C1(Fig.6).Fromthispoint, ;theinoculumsaresuitabletotreatthewastecontaininglotsof ;naturalorganicnitrogensourcesfromrestaurant. ;NH4CIPeptoneYeastextract

    ;Beefexutxact(Ntt4)2SO4Powerofbean

    ;Speciesofnitrogensources

    ;Fig.6Effectsofdifferentnitrogensourcesonthedecomposingrate ;2.7Variationinfat/oilc0ncentratiOnofthestuffingin ;theorganicwastetreatmentmachine

    ;Accordingtothevariationinfat/oilconcentrationofthe ;stuffing,theinformationaboutthesituationofmicrobial ;degradationintheorganicwastetreatmentmachinecouldbe ;obtained.Comparedwiththemachinewithoutinoculum.a ;loweraccumulationoffat/oilwasobservedinthemachine ;withinoculum.Thefat/oilconcentrationofthestuffinginthe ;inoculatedmachinedidnotvaryobviouslyduringthewhole ;???柏加0

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    ;No.3Screeni”gon0il-dec0posi”g1nicroorganisnlsandapplicati0nin0ganicwastetreatmentII1arhine

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    ;n?atment.However,therewasanobviousincreaseoffat/oil ;inthecontrolmachinewithoutinoculums.Ontheday48, ;thefat/oilcontentintheinoculatedmachinewasonlyahalf ;ofthatinthecontrolmachine(Fig.7).Theinitialfat/oil

;concentrationwasnotzerobecausecertainamountofwood—

    ;scrapscontainingfat/oilwasaddedintotheorganicwaste ;treatmentmachineasstuffingtoadjusttheoperation ;conditions,includingC/Nratio,dissolvedO2,etc.inthe ;machiBe.

    ;.

    ;墨15

    ;i,0

    ;霎l7l42l28354046

    ;t,d

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    ;?_Inoculumedmachine—Contralmachine

    ;Fig.7Curveoffat/oilconcentrationofthestuffinginthemachine ;2.8Variationintemperatureofthestuffinginthe ;organicwastetreatmentmachine

    ;Temperatureisalwaysoneofthemostimportantfactors ;toinfluencethetreatment

    ;temperaturecanreduceinsect

    ;efficiencyofwaste.High

    ;eggsandpathogensassociated

    ;withorganicwaste.aswellastheinoculums,anddamageto ;themachineatsomedegree.Therefore.itisveryimportant ;tomaintainoptimizedtemperature.above55—65?.inthe

    ;machine.Fig.8showsthatthetemperatureintheinoculated ;machinecouldbemaintained6?higherthanthatinthe

    ;controlone.Itcanbeconcludedthattheinoculumcouldnot ;onlyimprovethedegradationoffat/oilbutalsomaintainthe ;stabilityatarelativelyhightemperatureinthemachine. ;65

    ;60

    ;55

    ;50

    ;45

    ;40

    ;35

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    ;—

    ;?_Inoculumedmathine—Contralmachine

    ;Fig.8Curveoftemperatureinthewastetreatmentmachine ;2.9Variationinhumidityofthestuffingintheorganic ;wastetreatmentmachine

    ;Itisessentialtomaintainacertainhumidityofthe ;stuffinginthemachineforinoculums.Itiswellknownthat

    ;abilityofmicroorganismsobtainingnutritionwillattenuate ;whenthehumiditydecreasetobelow30%.Whenthe ;humidityincreasestoover65%.almostalloftheporesin ;thestuffingcanbefilledbywater,preventingO2from ;transport(Smith,1996),leadingt?