Decolorization of acid and basic dyes understanding the meta(2)

ApplMicrobiolBiotechnolTable1Commercialdyes

Typeofdye

GroupAacidanthraquinone

LabcodeA277A260A40A25A43A324A361A118A97A357A151B47B46B41B51

NameAcidBlue277AcidBlue260AcidBlue40AcidBlue25AcidViolet43AcidBlue324AcidRed361AcidRed118AcidRed97AcidRed357AcidYellow151BasicBlue47BasicRed46BasicBlue41BasicRed51

CAS3925797-81-362168-86-96424-85-76408-78-24430-18-688264-80-661931-22-412217-35-510169-02-561951-36-812715-61-612217-43-512221-69-112270-13-212270-25-6

λmax(nm)605590620605580610525505495490435575530605520

GroupBacidazo

GroupCbasicanthraquinoneGroupDbasicazo

Decolorizationassays:experimentalandanalyticalprocedures

BatchdecolorizationassayswerecarriedoutbyinoculatingE.coliin50-mlFalcontubes(BDBiosciences),containing20mlofLBmediumamendedseparatelywitheachofthedyesreportedinTable1,atknownconcentrations.Thedecol-orizationperformancesweresystematicallyanalyzedasafunctionofthefollowingparameters:staticorshaken(150rpm)cultures,incubationtemperature(25and37°C),initialdyeconcentration,and15-mgwetweightbiomass.Theeffectofglucose,ammoniumchloride,andammoniumsulfateonE.colidecolorizingactivitywasalsoinvestigated,byaddingseparatelythesenutrients(500,250,and250mg/l,respectively)tothedecolorizingmedium.Negativecontrolswithuninoculateddyemediumandheat-killedE.colicells(10minat100°C)werealsoperformed.Treatmentsandcon-trolswererunintriplicate,inthreeindependentexperiments.IncubationofE.coliondecolorizingmediumwaskeptuptoamaximumof48h.Aliquotswereperiodicallyandasep-ticallysampled,centrifuged(5000g,5minatroomtempera-ture)tocollectthebiomassasapellet,andfilteredon0.2-μmsterilecelluloseacetateNalgene?filter(VWR,Milan,Italy).AUV–Visspectrophotometer(Infinite200Pro,Tecan,Seetrasse,Switzerland)wasusedtomeasuretheλmaxofthesupernatantsofeachdye.Thepercentageofdyereduction(DR%)wascalculatedaccordingtothefollowing:

.ih

DR?eA0?A1TA0?100

whereDR=decolorizationaspercentage;A0andA1,initialandfinalabsorbanceattheλmaxofeachdyeinthevisibleregion.ThepHofthesupernatantswasalsomeasured,using

aBasic20-CrisonpHmeter(Carpi,Italy).Bacterialgrowthwasmeasuredascellbiomass.

Experimentaldataweresubjectedtostatisticalanalysistodeterminemeansandthecorrespondingstandarddeviations.Significantdifferencesbetweenmeansofexperimentsweredeterminedbyanalysisofvariance(ANOVA)withasignifi-cancelevelof0.05.Massspectrometry

A50.0-μlaliquotofeachdyesolutionsubjectedtodecol-orizationwasdilutedwith2mlofHPLCgrademethanol(FisherScientific,Leicestershire,UK)andthenfilteredonTitan2?SyringeFilter(ThermoFisherScientific,AustinTX,USA).ThefilteredsamplewasanalyzedusingaThermoScientificLCQ-Fleetmassspectrometerunderelectrosprayionization(ESI)(ThermoScientific,AustinTX,USA)bydirectinfusionwitha500KLHamiltonmicrosyringeandusinga10-μl/minflow.Datawereac-quiredoverhundredscans,afterstabilizationoftheioniccurrentfor5minattheabovementionedsampleflow.Themassspectrometerparametersusedwereasfollows:sheetgasflow=10,auxiliarygasflow=0,sweepgasflow=0,sprayvoltage=5.00kV,capillarytubetempera-ture=220°C,capillaryvoltage=12.0V,andtubelens=77.8V.Toxicityassay

Theshort-termteststodetermineacutetoxicitywerecarriedoutusingthewaterfleaDaphniamagna,astandardizedtestorganismfortoxicityassaysandproposedasarepresentative

invertebrateintheEUGuidanceDocumentonAquaticEco-toxicologicalandamongEUcriteriaforclassificationofdan-geroussubstances(Verma2008;Crosbyetal.1966;BrockandVanWijngaarden2012).Youngorganisms(6–24-hlife)wereusedfortoxicitybioassayandwerenotfedduringthetestperiod.Foreachsampleandeachexperimentalcondition,25organismsweretestedintriplicate,alongwithnegative(water)andpositive(untreateddye)controls.Thenumberofimmobileindividualswasevaluatedafter48-hincubationat21±1°C,witha16-hlight/8-hdarkphotoperiod.D.magnaindividualsareofficiallyacceptedasdeadiftheydonotshowanymobilitywithin30sofobservation.

Results

PreliminaryscreeningonE.colidyedecolorizationabilityInitially,ageneralscreeningabouttheE.colidecoloriza-tionabilitieswascarriedoutforthewholeselectionofdyes,toevaluatethebacterialactivityonawiderangeofcolorants.ThesolutionswereinoculatedwithanovernightstartercultureofE.coliinshakentubesatconstanttem-perature(25and37°C).ThepHofeachsamplewasmon-itoredatthebeginningandattheendoftheexperiment.E.colimodifiedtheinitialpHofeachsampletoavalueoscillatingbetween6and7regardlessoftheincubationtemperature.Thisbehaviorwascoherentwiththebacterialgrowth,whichwasrecordedpositiveforeachsampleandgenerallyhigherat37°C,asexpected(Fig.1a).Therefore,itwasnotsurprisingthatthevisiblespectrophotometricanalysisofthesupernatantsshowedasensibledecreaseindyeconcentration(Fig.1b).

Thedecolorizationwasstatisticallysimilarat37and25°C,exceptfordyeA40.Conversely,astrongerdecolor-izationwasobtainedforcompoundsA357,B41,B46,andB51at37°C.Also,itwasinterestingtonotethatwhenthebacterialbiomassofeachexperimentwasseparatedfromthetreateddyesolutionsandvisuallyexamined,thebacte-rialpelletsretainedsomeoftheoriginalcolorinthecaseofdyesofgroupsA,B,andC.Ontheotherhand,nocolora-tionwasobservedfordyesofgroupD(datanotshown),sincetheoriginalcolorhadcompletelyvanishedfromboththesupernatantandthepellet.Itisknownthatsomebacte-riamaydegradeazodyes,hererepresentedbygroupsBandD,throughspecificazoreductaseenzymesandduringan-aerobicgrowth(Liuetal.2009).Inourcase,thiseventmayhavehappenedinaerobicgrowthconditions.However,thedatadidnotallowtounderstandwhichmechanismswereinvolvedinthedecolorizationofdyes.Therefore,A324,A361,B47,andB46wereselectedasrepresentativedyesofeachgroup,inordertocarryoutfurtherinvestigationtoclarifythemodeofactionofE.coli.

ApplMicrobiolBiotechnol

AnalysisofdecolorizationbehaviorofE.coli

Thedecolorizationofdyesmaytakeplaceaccordingtoverydifferentmechanisms,suchasadsorptionanddegra-dation(Sarataleetal.2011).Inthefirstcase,dyesareadsorbedontothebacterialcellsurface,whilewhendyesaregoingtobedegradedbybacterialenzymes,theyarefirstcompartmentalizedinsidethebacterialcells.Therep-resentativecolorantsweretreatedwithE.colifor16hat37°C.Afterward,thebiomasseswereseparatedfromthesupernatants(hereafternamedBfirstgenerationsuperna-tants^forthetotaldecolorization),andtheresultingbacte-rialpelletswerewashedwithwater.Thisstepwasrepeateduntiltheabsorbanceofthesupernatantwashingwaterwascompletelyclearatnakedeye.Thesesupernatants(hereaf-ternamedBsecondgenerationsupernatants^)correspondedtothewashingofthedyeprobablyprecipitatedandadsorbedonthebacterialcellsurface.Subsequently,eachpelletwassubjectedtothermallysisinwaterat100°Cfor10minandseparatedfromthesupernatant(hereafternamedBthirdgenerationsupernatants^)whichcontainedtheintracellulardyeportion(Fig.2).Thespectrophotomet-ricanalysisinthevisibleregionofthesupernatantsthusobtainedfromA324,A361,andB47confirmedthattheirλmaxwasconsistentwiththatoftheuntreateddye(Table2).Theseresultswerealsoconsistentwiththedataderivedfromthemassspectrometricanalysisofthesupernatants(Fig.3).TheE.colipelletsderivedfromthedecolorizationonA324andA361releasedthecolorbothduringthewash-ingandthelysisprocedure,suggestingthepresenceofthedyesbothonthebacterialsurfaceandinsidethebacterialcellwall,respectively.ThesamealsooccurredforB47,althoughtoalesserextent.However,dataobtainedfromsecondgenerationsupernatantscouldnotexcludeapoten-tialdyeprecipitationinducedbybacterialadditionandcellgrowth.VisiblespectraofB46supernatantsdidnotexhibitanyλmaxconsistentwiththisdye.Thedifferencesbetweentheabsorbanceofthefirstgenerationsupernatantsandthosecombinedfromthesecondandthirdgenerationsu-pernatantswerecorrelatedtothemetaboliccontributionofE.colitotheoveralldecolorization.Metabolismwas27,36,and45%forA324,A361,andB47,respectively.InthecaseofB46,thispercentagereached98%,clearlyindicat-ingthatmetabolismwastheonlymechanisminvolvedindecolorization.Inaddition,massspectrometricanalysisconfirmed(Table2andFig.3)thepresenceofN′-benzyl-N′-methylbenzene-1,4-diamine1,havingamolecularweightof212(Fig.S1)whichwouldoriginatefromthereductivecleavageoftheazogroupofB46,promotedbyE.coli(Fig.3dandFig.S1).Thistransformationmayhavebeendrivenalsobythecationicnatureofthisdye,asrep-resentedbyitsresonancehybridstructure3(Fig.S2)dur-ingitsinteractionwiththedegradingbacteria.