Decolorization of acid and basic dyes understanding the meta(4)

Here,wedemonstratedthattheE.colitypestrainDH5αworkedwellonacidandbasicdyesunderaerobicconditions,showingdifferentmechanismsofdecolorization.Bothacidanthraquinone(groupA)andacidazo(groupB)dyeswereremovedfromeffluentsbythecombinedactionofprecipita-tion,celladsorption,andmetabolism.Thesethreeeventshavebeenrarelyconsideredasactingaltogetherinpreviousstudies(Sarataleetal.2011;Giwaetal.2012)whileclaimingthemetabolicactivityofthebacterialbiomassesastheonlyde-colorizingmechanism.Itisimportanttounderlinethatthosephenomenaoccurredduringcellculturegrowthregardlessoftheamountofbiomassandthetypeofaciddyes.Itis

reasonabletoassumethatgroupsAandBdyeswerelikelytoprecipitateinthepresenceofE.colialreadyatt0asdem-onstratedbyA324andA361.Thephenomenoncanbecer-tainlydescribedbyabioticevent,sincecontrolexperimentsintheabsenceofE.colididnotshowanydyeprecipitationinducedbythenutrientmediumorbymechanicalactionsuchascentrifugation.Theeffectofbiomassgrowthresultedinanincreaseofdecolorizationmainlyduetoprecipitation,cellwalladsorption,andintracellularaccumulation,althoughasignificantfractioncouldbealsoassociatedtometabolicevents,relatedtotheactivityofbacterialazoreductases(Liuetal.2009).Similarfindingswerealsodescribedforabasicanthraquinone(groupC)dye,whichhasneverbeenreportedinpreviouspaperstothebestofourknowledge.Eveninthisparticularcase,thepresenceofbacterialbiomassinducedastrongprecipitationeffectatt0,causingareductionofmetab-olismcontribution.Itwasinterestingtonoticethatthediffer-entchemicalnatureofA324andB47anthraquinonedyescausedtheiroppositecontributionbetweenmetabolism(27and45%,respectively)andprecipitationeffect(46and38%,respectively).Thisphenomenonisrelatedtothediffer-entchemicalaffinityofacidorbasicanthraquinonedyestothecellwalloftheGram-negativeE.coli.

Degradationofbasicazodyes(groupD)occurredefficient-lyunderaerobicconditions,confirmingthemetabolicpath-wayofE.coliinvolvingoxygen-resistantazoreductases.Thisresultwasalsoconsistentwithrecentfindings,whichdemon-stratedtheprominentroleofE.coliAzoR-purifiedenzymeunderaerobicconditions(Mercieretal.2013).Theremark-ablyhighrateofdecolorizationforB46wasfoundtobede-pendentontheamountofbiomassandtheconcentrationofdye.Conversely,metaboliccontributiontototaldyereductiononA324,A361,andB47wasevenlowerthan50%,probablybecauseoftheabsenceofredoxmediatorsasrecentlyreported(Cuietal.2015).

Comparativetrialswithsteadyandshakenalivecellscon-firmedthatprecipitationandabsorptionphenomenacontrib-utedinadifferentwaytothetotaldyereductionforA324,A361,andB47,whereasB46underwentenzymaticdegrada-tion.Thisconceptmaybeindirectlyconfirmedbythedecol-orizationofB46,whichresultedmarginallyaffectedbysteadyandshakenbiomass.Inthiscase,itcouldberemarkedthat

ApplMicrobiolBiotechnol

metabolismwastheprinciple,ifnottheunique,modeofdyedegradationactivatedbyE.coli,demonstratedbytheabsenceofanydegradationwithdeadcells.

ExperimentscarriedoutonA324,A361,andB47atvari-ableamountofcolordemonstratedthatE.coliwasabletokeepasimilarbehavioracrossthewholeincubationtimeandregardlessoftheinitialdyeconcentration.Thesefindingsmayhaveimportantimplicationsatindustriallevel:particu-larlyforthetextileindustry,whichexploitsthevastmajorityoftheglobalproductionofdyes.Thevariationofdyeconcen-trationineffluentsisquiteoftenunpredictableandfluctuating,duetodyereactivitywithwater,dyefixation,andvariableexhaustionofthedyeingbath(Ghalyetal.2014).InthecaseofB46,E.colishowedastrongerabilitytometabolizebasicazodyesatthetypicalconcentrationfoundedineffluentsafteradyeingprocesses.Only2hwassufficienttocompletelyremoveB46fromtrialsolution.Massspectrometricanalysisdemonstratedthatmetabolite1wasoriginatedbyreductivecleavage(Pandeyetal.2007)ofB46andfurtherdegradedbyE.coliinaerobicconditions.Also,compound1resultedcompatiblewiththetoxicityprescriptionsofREACH(EC2006).ThecationicnatureofB46wascrucialforitsquanti-tativeuptakeanddegradationbyE.coli,duetothehighaf-finityofthisdyeforthebacterialcellwallandmembranestructure.Thisassumptioncouldbeexplainedtakingintoac-counttheHard-Soft-Acid-basemodel(Ho1975).ThelonepairoftheN′-benzyl-N′-methylmoietyofB46isdelocalizedthroughtheazophenylfragmentintothetriazolering,asshownbytheresonancehybridstructure3,definedasoftcationbecauseofthedistributionofthepositivechargeontomultiplecenters.Itfollowsthatcompoundssuchas3interactquitewellwithlipidsandphospholipidsofthebacterialcellwallandmembrane,sincethenegativelychargedpolarheadsarediffusedsoftanions.Consequently,electrostaticinterac-tionsbetweenthedyeandthecellanionsaresupposedbeingdrivenentropicallytoestablishasoft-cation-soft-anioncouple(theB46cationandthephospholipidanion)andahard-cation-hard-anioncouple(sodiumcationandbromideanion).TheinhibitionofbacterialdecolorizationbyglucoseonB46furtherdemonstratedthatthereductivecleavageoftheazogrouptookplaceunderaerobicconditions.Inaddition,noresidualtoxicitywasfoundintheE.coli-treatedbasicazodyesolutions.Thus,theobserveddifferencesintheacutetoxicitycouldberelatedtoacompleteprocessofdyemetabolism,excludingtheformationofundesirablearomaticamines(EC2006).

Theresultsofthepresentstudyhaveimportantimplica-tionsfromanindustrialpointofview,sincemarginstoimple-mentsuchabioremediationtechnologywouldbeflexibletoachievethedecolorizationoftextileandleatherdyeingwaste-watersinshorttimespells.Moreover,interestingrecentresultshaveopenedtheopportunitytoselectE.coliDH5αpopula-tions,bybacterialbiomassrecycle,inordertoincreasetheir

decolorizationabilities(Corsi,unpublishedobservations).Sofar,thisapproachhasbeenappliedtothenewclassofBnaturalized^dyes(Pellegrinietal.2015),watersolubleandeco-friendly,synthesizedfromchromophoresofcommercialdispersecolorants.Overall,theseperspectiveswillrepresentfurtherattractiveimprovementsforE.colidyedecolorizationintextileandleatherindustries,beyondthegeneralclassesofacidandbasicdyesherestudied.

AcknowledgmentsWegratefullyacknowledgeTintoriaCometaSrl(Prato,Italy)forprovidingthecommercialdyesandBEnteCassadiRisparmiodiFirenze^forfinanciallysupportingthisresearch.EthicalstatementM.Cerboneschistatesthatthereisnoconflictsofinterestbetweentheauthorsofthepresentmanuscript.Thisarticledoesnotcontainanystudiescarriedoutonhumanoranimalsubjects.

References

AliH(2010)Biodegradationofsyntheticdyes–areview.WaterAirSoil

Pollut213:251–273.doi:10.1007/s11270-010-0382-4

AliN,HameedA,AhmedS,KhanAG(2008)Decolorizationofstruc-turallydifferenttextiledyesbyAspergillusnigerSA1.WorldJMicrobiolBiotechnol24:1067–1072.doi:10.1007/s11274-007-9577-2

AlinsafiA,EvenouF,AbdulkarimEM,PonsMN,ZahraaO,

BenhammouA,YaacoubiA,NejmeddineA(2007)Treatmentoftextileindustrywastewaterbysupportedphotocatalysis.DyesPigments74:439–445.doi:10.1016/j.dyepig.2006.02.024

AmoozegarMA,HajighasemiM,HamediJ,AsadS,VentosaA(2011)

Azodyedecolorizationbyhalophilicandhalotolerantmicroorgan-isms.AnnMicrobiol61:217–230.doi:10.1007/s13213-010-0144-yAvlonitisSA,PouliosI,SotiriouD,PappasM,MoutesidisK(2008)

Simulatedcottondyeeffluentstreatmentandreusebynanofiltration.Desalination221:259–267.doi:10.1016/j.desal.2007.01.082

AyedL,AchourS,KhelifiE,CherefA,BakhroufA(2010)Useofactive

consortiaofconstructedternarybacterialculturesviamixturedesignforCongoReddecolorizationenhancement.ChemEngJ162:495–502.doi:10.1016/j.cej.2010.05.050

BerberidouC,AvlonitisS,PouliosI(2009)Dyestuffeffluenttreatment

byintegratedsequentialphotocatalyticoxidationandmembranefil-tration.Desalination249:1099–1106.doi:10.1016/j.desal.2009.06.045

BrockTCM,VanWijngaardenRPA(2012)Acutetoxicitytestswith

Daphniamagna,Americamysisbahia,ChironomusripariusandGammaruspulexandimplicationsofnewEUrequirementsfortheaquaticeffectassessmentofinsecticides.EnvironSciPollutResInt19:3610–3618.doi:10.1007/s11356-012-0930-0

ChanGF,GanHM,RashidNAA(2012)Genomesequenceof

Enterococcussp.StrainC1,anAzoDyedecolorizer.JBacteriol194:5716–5717.doi:10.1128/JB.01372-12

ChangJS,LinCY(2001)Decolorizationkineticsofrecombinant

Escherichiacolistrainharboringazodyedecolorizationdetermi-nantsforRhodococcussp.BiotechnolLett23:631–636.doi:10.1023/A:1010306114286

ChangJS,KuoTS,ChaoYP,HoJY,LinPJ(2000)Azodyedecoloriza-tionwithamutantEscherichiacolistrain.BiotechnolLett22:807–812.doi:10.1023/A:1005624707777