Decolorization of acid and basic dyes understanding the meta(3)

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Fig.1aE.coligrowthat25(black)and37°C(white)after16hofincubationat0.3mg/mlofdyeconcentration.bDyedecolorizationreportedinthisfigurewasrelatedtothesametrialinwhichbacterialgrowthhadbeenmonitored

EffectofbacterialbiomassonE.colidecolorizationactivity

DifferentamountsofE.colibiomass(1.5,7.5,15,and30mgofbacterialwetweight)wereinoculatedseparatelyintoeachrepresentativedyesolution,andthecultureswere

Fig.2Decolorizationrateafter16hofE.coliactivityat0.3mg/mlofdyeconcentration:precipitation,adsorption,andmetabolism.Blackportionmetaboliccontribution;whiteportionprecipitationandadsorptiononbacterialcellwall;grayintracellular

accumulation

homogenizedbymechanicalstirring.Analiquot(1ml)wastakenfromeachsampleandcentrifugedattime0(t0)toassesswhetherprecipitationofthedyemayhavebeeninducedbybacterialbiomass.Subsequently,thecultureswereincubatedat37°Cfor48honanorbitalshaker,sampledat2-hintervalfromt0to8h,thenat24and48h.ThedecolorizationresultsobtainedforA324,A361,andB47showedanonproportionalincreaseindyereductionrelatedtothedifferentbiomassamounts.InFig.4,itwasreportedthedecolorizationtrendforexperimentscontainingthemaximumamountofbacterialbiomass.Dyereductionoccurredevenatt0forthosethreedyes(46,17,and38%,respectively)remainingpracticallyconstantfor8h,atimewhichimplicatesatleast24E.coligenerations(Kutsu2007).Finally,thedecolorizationpercent-ageincreasedtoathresholdvalueat48hregardlessofthedye(Fig.4).Theoverallprofileoftheexperimentsappearedtoremainconstant,suggestingthatdecolorizationofA324,A361,andB47hadbeenoccurringwithacombinationofmetabolismandcell-induceddyeprecipitation.Controlexper-imentswerealsopreparedintheabsenceofbacterialbiomass.Aftermechanicalhomogenization,eachsolutionwascentri-fugedwithoutobservinganyprecipitation,leavingthesam-plesonstandingfor24

h.

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Table2Datafromthevisiblespectroscopic(λmax)andmassspectrometric(m/z)analysesofthesupernatantsafterdecolorizationofA324,A361,B47,andB46

DyeLabcodeA324A361B47B46

ab

Referenceλmax(nm)610525575530

massion(m/z)450.44a517.60371.43b321.18b

a

Supernatants1stgeneration615530585n.d.

450.34517.34372.15213.06

2ndgeneration614530587n.d.

450.34517.34372.13213.12

3rdgeneration615528588n.d.

450.33517.40372.15213.12

Anion,analysisunderESI-cmethodCation,analysisunderESI+cmethod

n.dnotdetected

TheexperimentscarriedoutonthedegradationofB46showedthetypicaltrendofametabolicdecolorizingactivity

Fig.3Massspectraofthefirstgenerationsupernatantsfromdecolorizationassay.aA324;bA361;cB47;dB46.Massspectrawerecarriedoutonthesupernatantscollectedafter

centrifugationandwithoutfurtherpurification.ThepresenceofotherpeakswasrelatedtochemicalspeciesoftheLB

mediumafterbacterialtreatment

(Fig.4):thebiomassincreaseinducedaproportionalreduc-tionoftimetoreachtheplateauofbacterialdecolorizationat

ab

cd

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Fig.4EffectofmaximumE.colibiomass(30mg)onthedecolorizationofA324(blacktriangle),B47(whitesquare),A361(blacksquare),andB46(whitetriangle)asafunctionoftime

24h(datanotshown).Thisevidencewassupportiveforaspecificmetabolicactivity,inducedbythepresenceofazoreductaseenzymesproducedbyE.coli(Pandeyetal.2007).

Acomparativeexperimentbetweenaliveanddeadbacte-rialcellsshedlightonthecontributionofbothmetabolismandadsorptiontothedecolorizingactivityofE.coli.SolutionsofA324,A361,B47,andB46wereinoculatedandevaluatedafter16hofincubation.AsreportedinFig.S3,onlyforA361,itwaspossibletoobserveadyereduction(over10%)causedbytheinoculationofdeadcells,whileforA324andB47,thedecolorizationlevelwaslessthan5%.Conversely,therewasnoevidenceofE.coliabsorptionactiv-ityontheB46solution.

Usingthesameexperimentalconditions,wedecidedtover-ifywhetherthedecolorizationperformancesofE.coliwereaffectedbytheoxygenationofthecultures(steadyorshaken).Thedyeremovalratedecreasedinarangebetween10and20%forA324,A361,andB47,whenthecultureswerenotunderagitation(Fig.S3).Ontheotherhand,theexperimentofB46didnotdisplayanysignificantdifferencerelatedtothedecolorizingperformancesofE.colicellsinsteadyorshakencondition.EffectofdyeconcentrationonE.colidecolorizationactivity

Culturesampleswerepreparedat0.06-,0.15-,0.3-,0.4-,and0.6-mg/mldyeconcentration,andthesolutionswereinoculat-edwithE.colibiomass.Aliquots(1ml)weresampledat2,6,8,and24h.AsreportedinFig.5,theanalysisofthesuperna-tantsbyvisiblespectrophotometryindicatedthatthehighestpercentagesofcolorremovalfordyesA324,A361,andB47wereobtainedat0.6mg/ml.Decolorizationalsoshowedsim-ilarpatternsfromthelowesttothehighestdyeconcentrationregardlessoftheinitialamountofcolor,highlightinganal-mostlineartrend.ThelargestdifferenceofdecolorizationwasobservedfordyeA324fromthelowesttothehighestconcen-tration,whereasinthecaseofA361andB47,thisphenome-nonwasreducedofabout50%.

ConcerningcompoundB46,thehighestpercentagesofde-colorizationwereobtainedfrom0.06-to0.3-mg/ml

Fig.5Decolorizationatthelowestandthehighestconcentrationsofdye.A324(triangle);A361(square);B47(circle).Whiteindicatorswererelatedtomaximumconcentrationofdye(continuouslines),blackindicatorstotheminimumconcentrationofdye(dashedlines)

concentration(Fig.6a),andsurprisingly,thedecolorizationatt4andt24wassomehowsimilar.Thisresultindicatedthatastheconcentrationofdyeincreased,itwouldnotbeneces-sarytoprolongtheexperimentbeyondthe4h:oratleasttoextenditnecessarilytot24orlonger.Amarkeddifferencewasobservedforthe0.4-and0.6-mg/mlconcentrationsinthesameintervaloftime.Atthemaximumconcentrationtested,wecouldassumethattherelativehighconcentrationofB46hadinducedE.colitoadaptitsmembranetransportsystems.Thedecolorizationafter4hwaslowerthan10%oftheinitialdyeconcentrationasindicatedbythespeedplot:themaxi-mumspeedofdyereductioncouldbeobservedjustafter2hatlowerconcentrations(Fig.6b).

InhibitionofE.coliazoreductaseactivity

Thedecolorizationofazodyesbybacterialstrainsistypicallyinitiatedbyazoreductaseenzymesinanaerobiccondition(Pandeyetal.2007).Onthecontrary,aspreviouslyshown,thereductionofB46wasduetotheaerobicmetabolismofE.coli.Therefore,wedecidedtoverifythekineticofdyedegradationinrelationtotheazoreductaseactivity.Itisknownthatthereductionofazodyesislinkedtothepresenceandavailabilityofcosubstratesascarbonandnitrogensources,andthedegradationrateofbacterialcellsiscorrelatedtothechemicalstructuresofthesemolecules.

Theassayfortheazoreductaseinhibitionwascarriedoutfor6hinaerobicconditionat0.15mg/mlofB46.Foreachreplicate,aspecificamountofglucose(carbonsource),am-moniumchloride,andammoniumsulfate(nitrogensources)wasadded,at1,0.5,and0.5%(w/v),respectively,whichhadalreadydemonstratedtobeabletodecreasedyedecolorization(Modietal.2010).TherewasadecreaseofB46dyereductionupto4%inthecaseofglucosesupplementationandmoder-atescoresfornitrogensubstrates(Fig.S4).Thus,theseresults

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Fig.6Rate(a)andspeed(b)decolorizationwith15-mgwetweightofE.coliatdifferentdyeconcentrationsofB46.Dyeconcentration(mg/ml):triangle0.06;square0.15;plus0.30;circle0.40;cross0.60

showedthatthedecolorizationofB46hadbeenoccurredinaerobicconditionsandincludedazoreductaseactivitytoo.Toxicityassay

TheassessmentofthetoxicityoftheresidualdyemixturestreatedwithE.coliwasaveryimportantaspectforbioreme-diationapplications,todemonstratethatthesolutionsthusobtainedwerenottoxic,aswellasvisuallyclear.Acutetox-icitytestswerecarriedoutontherepresentativedyestreatedwithE.coliafter16hofincubation.AsshowninTableS1,thetoxicityratesofB46-treatedsolutionswereabsentafter6h.ExcellentscoreswereachievedbyA324andA361after12h(after6hca.2and4%,respectively),whiletheworstperfor-manceswereobtainedforB47(after12hca.5%).Accordingtotheseresults,itisreasonabletohypothesizethatthediffer-enceshereobservedintheacutetoxicitybetweenthedifferentdyesmightbemainlyrelatedtoputativelydifferentmecha-nismsadoptedbythisbacteriafordecolorization.

Discussion

Ingeneral,theadaptabilityofE.colitoxenobioticspeciessuchasdyeswasprovedbythecellgrowthandbytheadjust-mentoftheinitialpHofthedyesolutionstonearlyneutralvalues.Thisfindingmeantthatthecellswereinclinedtomaketheirsurroundingenvironmentfavorabletosurvive,althoughthedecolorizationperformancewasfoundnotalwaysstrictlycorrelatedtothebacterialgrowth.

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