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Bacteria encapsulated electrospun nanofibrous webs for remediation of methylene blue dye in water

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ContentslistsavailableatScienceDirect

Colloids

and

Surfaces

B:

Biointerfaces

jou rn a l h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / c o l s u r f b

Bacteria

encapsulated

electrospun

nanofibrous

webs

for

remediation

of

methylene

blue

dye

in

water

Omer

Faruk

Sarioglu

a,b

,

Nalan

Oya

San

Keskin

b,c,d

,

Asli

Celebioglu

a,b

,

Turgay

Tekinay

d,e,∗∗

,

Tamer

Uyar

a,b,∗

aInstituteofMaterialsScience&Nanotechnology,BilkentUniversity,06800,Bilkent,Ankara,Turkey bUNAM-NationalNanotechnologyResearchCenter,BilkentUniversity,06800,Bilkent,Ankara,Turkey cPolatlıScienceandLiteratureFaculty,BiologyDepartment,GaziUniversity,06900,Polatlı,Ankara,Turkey dLifeSciencesApplicationandResearchCenter,GaziUniversity,06830,Golbasi,Ankara,Turkey

eDepartmentofMedicalBiologyandGenetics,FacultyofMedicine,GaziUniversity,06500,Besevler,Ankara,Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received31August2016 Receivedinrevisedform 22December2016 Accepted18January2017 Availableonline19January2017

Keywords: Bacteria Bioremediation Electrospinning Encapsulation Methyleneblue Nanofibers

a

b

s

t

r

a

c

t

Inthisstudy,preparationandapplicationofnovelbiocompositematerialsthatwereproducedby encap-sulationofbacterialcellswithinelectrospunnanofibrouswebsaredescribed.Acommercialstrainof Pseudomonasaeruginosawhichhasmethyleneblue(MB)dyeremediationcapabilitywasselectedfor encapsulation,andpolyvinylalcohol(PVA)andpolyethyleneoxide(PEO)wereselectedasthepolymer matricesfortheelectrospinningofbacteriaencapsulatednanofibrouswebs.Encapsulationofbacterial cellswasmonitoredbyscanningelectronmicroscopy(SEM)andfluorescencemicroscopy,andthe via-bilityofencapsulatedbacteriawascheckedbylive/deadstainingandviablecellcountingassay.Both bacteria/PVAandbacteria/PEOwebshaveshownagreatpotentialforremediationofMB,yet bacte-ria/PEOwebhasshownhigherremovalperformancesthanbacteria/PVAweb,whichwasprobablydue tothedifferencesintheinitialviablebacterialcellsforthosetwosamples.Thebacteriaencapsulated electrospunnanofibrouswebswerestoredat4◦Cforthreemonthsandtheywerefoundaspotentially

storableforkeepingencapsulatedbacterialcellsalive.Overall,theresultssuggestthatelectrospun nanofi-brouswebsaresuitableplatformsforpreservationoflivingbacterialcellsandtheycanbeuseddirectly asastartinginoculumforbioremediationofwatersystems.

©2017ElsevierB.V.Allrightsreserved.

1. Introduction

Therearedifferenttypesofcontaminantsinwastewater efflu-entswhichareutilizedinindustrialprocesses,anddyescomprise agreat portioninthoseindustrial contaminants.Syntheticdyes haveagreatusageinvariousindustries(e.g.textile,leather,paper) anddyeingprocesscanleadtomanyenvironmentalproblems[1]. Methyleneblue(MB)isacommonbasic,cationicdyewithabroad applicationareaintextileindustry,paperindustry,chemistry,

biol-Abbreviations: MB,methyleneblue;PEO,polyethyleneoxide;PVA,polyvinyl alcohol.

∗ Correspondingauthorat:InstituteofMaterialsScience&Nanotechnology, BilkentUniversity,InstituteofMaterialsScience&Nanotechnology,Bilkent,06800, Ankara,Turkey.Tel.:+903122908987;fax:+903122664365.

∗∗ Correspondingauthorat:LifeSciencesApplicationandResearchCenter,Gazi University,06830,Golbasi,Ankara,Turkey.

E-mailaddresses:[email protected](T.Tekinay),[email protected]

(T.Uyar).

ogyandmedicine[1,2–7].Nevertheless,decontaminationofMB fromwatersystemsafteruseisstillamajorchallenge. Conven-tionalwastewatertreatmentmethodscanbeusedforremediation ofMBsuchasphotocatalysis[8],advancedoxidationprocesses[9], reverse osmosis[10] and electrochemicaltreatment[11]. Some ofthesetechniquescanbeusedefficiently forMBremediation, thougheachofthemhastheirownlimitations,henceinnovative approaches have been presented in theliterature for develop-mentofsustainable,environmentallyfriendly,cost-effectiveand efficienttreatmentmethods[1].Bioremediationisanalternative technologyfordecontaminationofwatersystemsbyuseofspecific microorganisms,anditcanprovidegreen,efficient,cost-effective andsustainableremediationofwatercontaminants[12]. Microal-gae,fungiandbacteriacanbeutilizedforbioremediation.These microorganismscanremediatewaterpollutantseitherby biosorp-tionorbioaccumulation.Althoughdeadcellbiomassescanonlybe usedforbiosorption,livingcellscanpossessbothbioaccumulation andbiosorption,hencehigherefficiencyforbioremediationcould beachievedbylivingcellsinsomestudies[12].

http://dx.doi.org/10.1016/j.colsurfb.2017.01.034

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246 O.F.Sariogluetal./ColloidsandSurfacesB:Biointerfaces152(2017)245–251

ThegenusPseudomonascomprisesGram-negative,aerobic, rod-shapedbacteriaandhasabroadmetabolicdiversity[13],havinga potentialtobeusedinbioremediationstudies.Apopular mem-berofthisgenus,Pseudomonasaeruginosahasalreadybeenused effectivelyinbioremediationoforganiccontaminants[1,14–16], implyingthisspeciesasapotentialcandidateforfurther bioreme-diationstudies.

Applicationofmicroorganismsforuseinbioremediationcan beperformedwitheitherfreemicroorganismsormicroorganism immobilized bio-hybridmaterials.Immobilized microorganisms can bring advantages than free cells in terms of their poten-tialreusability,lowerspaceandgrowthmediumnecessities,and higherresistancetoenvironmentalextremes[17,18].Electrospun fibrouswebshavebecomeapopularcarriermatrixfor immobi-lizationof specificmicroorganismsfor bioremediation of water systems[11,19–22].Electrospinningcanallowsimple,versatileand cost-effectiveproductionoffibrouswebswithuniqueproperties suchashighsurfaceareaandporosity,makingelectrospunfibrous websaspromisingcandidatesformicrobialintegrationand mem-brane/filterapplications[23].Inrecentyears,anumberofstudies havebeenreportedaboutencapsulationofmicroorganismsinto electrospunfibrousmatrices[23–27].Whileviabilityorbioactivity ofencapsulatedmicroorganismshasbeencheckedinallofthese studies,justonlyveryfewofthemhavereportedenvironmental applications[26–29].

In the present study, bioremediating bacterial cells were successfully encapsulated into polyvinyl alcohol (PVA) and polyethyleneoxide(PEO)polymericmatrices whilekeepingthe bacteriabioactiveandtheviablecellnumbersindesirableamounts. Two water based and biocompatible polymeric matrices were selectedforencapsulationofbacterialcellstoreducetheeffectsof exteriorenvironmentontheviablebacterialnumbers.Thesenewly producedbacteriaencapsulatednanofibrouswebsweretestedfor theirremovalcapacitiesagainstMBdye.Itwasfoundthat, bacte-riaencapsulatedwebshavethepotentialtosuccessfullyremediate MBinwater.Inaddition,thestorabilityofbacteriaencapsulated nanofibrouswebswastestedintermsoftheviablebacterial num-bers.Theresultshaveshownthattheencapsulatedbacteriacanbe storedsafelyforlongtimeperiodswithoutsignificantlossesintheir cellviability.Thesetypesofbio-hybridmaterialscouldbeof inter-estduetoeasyandsafepreservationofbioremediatingbacteriafor potentialwastewatertreatmentapplications.

2. Experimental

2.1. Materials

The chemicals and reagents (polyvinyl alcohol (PVA, Mw ∼125.000,Scientific PolymerProducts,Inc.),polyethyleneoxide (PEO,Mw∼900.000,Sigma-Aldrich),methyleneblue(MB,≥82%, Sigma-Aldrich),Nutrientbroth(Sigma-Aldrich),LB(Luria-Bertani) broth(Sigma-Aldrich)andAgar(Sigma-Aldrich))werepurchased and used without any purification. The deionized water was obtainedfromaMilliporeMilli-QUltrapureWaterSystem.Allthe chemicalswere of highpurity available and were of analytical grade.

2.2. Procurementofthebacterialstrain

Thecommercialbacterial strain utilized in this study (Pseu-domonas aeruginosa ATCC 47085) was purchased from ATCC (AmericanTypeCultureCollection,USA).Thebacterialculturewas enrichedinLBmedium(Luria-Bertani:10g/Ltryptone,5g/Lyeast extract,10g/LNaClin1Lofdistilledwater)andstockcultureswere preparedfromtheinitialbroth.Thestockcultureswerestoredat

4◦Cforshortperiodsandfreshcultureswerepreparedfromthose samplespriortothefurtheruse.

2.3. ElectrospinningofbacteriaencapsulatedPVAandPEO nanofibrouswebs

PVAandPEOnanofiberswereproducedbyusingasinglesolvent system(water),butwithdifferentpolymerconcentrations.While forPVAnanofibers,thepolymerconcentrationwas7.5%(w/v)in theelectrospinningsolution,itwas3.5%(w/v)forPEOnanofibers. Thematerialsusedforpreparationofelectrospunnanofiberswere allsterilizedbyautoclave,andtheinsideofthePlexiglasboxwhere electrospinning wascarriedout wassterilizedby UV-Clightto avoidcontamination.Beforeelectrospinningprocess,2X concen-trationsofpolymermixtures,whichweretwiceasdensedasthe regularconcentrations,werepreparedandthenequalamountsof eitherbacteria-freedistilledwaterorbacteriacontainingdistilled waterweremixedwiththesemixturestoobtain1Xelectrospinning solutions. In order to encapsulate sufficient amounts of bacte-riawithinpolymermatrices,therequiredbacterialamountwas determinedbydrycellbiomass(∼4mgofbacterialbiomassper mLofelectrospinningsolution,correspondingto∼1010cfu/mL).

The electrospinning solutions were loaded in 1mLsyringe fit-tedwith a metallic needle of 0.6mminner diameterand they werelocatedhorizontallyonasyringepump(modelKDS-101,KD Scientific,USA).Oneoftheelectrodesofhigh-voltagepower sup-ply(MatsusadaPrecision,AUSeries)wasclampedtothemetallic needle and the other one was clamped to the grounded alu-minumcollector which was covered withan aluminum foilto depositthePVAandPEOelectrospunnanofibers.The electrospin-ningparameterswereappliedas:feedrateofsolutions=1mL/h, applied voltage=10–15kV, tip-to-collector distance=10–12cm. The electrospinning apparatus was enclosed in a Plexiglas box and electrospinning wascarried out at24◦C±1 and ∼20% rel-ativehumidity.Thecollectedbacteriaencapsulatednanofibrous webswerestoredinarefrigerator(+4◦C)forquickorlonger-term use.Onlyinoneexperiment,thebacteriaencapsulatednanofibrous webswerestoredatroomtemperatureaswell.Bacteria-free (pris-tine)nanofiberswerestoredatroomtemperature.

2.4. Viabilityandstoragetests

In order to evaluate whether bacterial cells were properly encapsulatedwithin PVA and PEOnanofibers,thePseudomonas aeruginosaATCC47085cellswerestainedwithfluorescentstains (LIVE/DEADBacLightTMkit)beforemixingwiththe2X concen-trationsofpolymermixtures.Afterpreparationofelectrospinning solutions,PVAandPEOnanofiberswerecollectedonglassslides toobserveunderafluorescencemicroscope.Microscopic evalua-tionofLIVE/DEAD-stainedbacterialcellswasmadebythegeneral assessment:brightgreenfluorescenceemittingcellscorrespondto livingcellsandbrightredfluorescenceemittingcellscorrespondto deadones.PhotographsweretakenbyusingaLeicaoptical micro-scope(Leica,DMI4000B)whichhasanattachedfluorescenceunit. In addition to fluorescence microscopy, the viability of PseudomonasaeruginosaATCC 47085cellsin either electrospin-ning solutions or encapsulated within nanofibrous webs was determinedviaviablecellcounting(VCC)assay.Tofindthe encap-sulationefficiency,equivalentpiecesofthenanofibrousmaterial wasweighedwhichcontaintheencapsulatedbacterialcells. Dis-tilledwaterwasaddedtothesepiecesandtheydissolvedrapidly inwater,serial10-folddilutionsweremadeandthenthebacterial solutionswerespreadonLBagarplates.Afterovernightincubation at30◦C,thenumberofcolony-formingunits(CFU)wascounted.All testsweremadeintriplicate.

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Fig.1. (a)SchematicrepresentationofelectrospinningprocessforbacteriaencapsulatedPVAandPEOwebs,andphotographsofPVAandPEOwebs,(b)representative imagesforbacteriaencapsulatedwebsincludingaSEMmicrographandaschematicrepresentationofabacterialcellinsidePVA/PEOfibers.

Forstoragetest,equivalentpieceswerepreparedforsame sam-plesandtheirviabilitieswerecheckedregularlyfordefinedtime periods.Bacteriaencapsulatednanofibrouswebsweretestedfor storabilityat4◦Cfor3monthsandat25◦Cfor10daysunderdry conditions.

2.5. Methyleneblue(MB)bioremovalexperiments

LBbrothwasutilizedasthebacterialgrowthmediumforMB bioremovalexperiments.ThepHlevelswereconstantand neu-tral(pH7.0).BacteriaencapsulatedPVAandPEOwebswereadded directlytoMBcontainingLBbrothforinitiatingbacterialgrowth. TheeffectofPVAandPEOinthegrowthmediaofdissolvedwebs onbacterialgrowthwasevaluatedbyOD600measurements.After

achievingthebacterialcellviabilitiesof each bacteria encapsu-latedPVAandPEOwebsintherangeof107–108cfu/mLper10mg,

equivalentwebsamples(withw/vratioof1mg/mL)wereprepared forinitiatingMBbioremovalexperiments.Theinitialbacterialcell viabilities of bacteria/PVA and bacteria/PEO webs were around 107–108cfu/mLand108cfu/mLper10mg,respectively.Samples

werecollectedperiodicallytoanalyzeremainingMB concentra-tionsbyaspectrophotometer,andthespecificabsorbanceofthe dyewasmeasuredat660nm.Thesampleswerecentrifugedprior tomeasurementsfor5minat10,000rpm,andthesupernatant frac-tionswereutilizedforspectrophotometricmeasurementstoavoid opticaldensity interferencefrombacterial cells.Threedifferent (10,15,25mg/L)initialMBconcentrationsweretestedfor evalu-ationoftheremovalcapabilitiesofbacteria/PVAandbacteria/PEO webs.Thesampleswereincubatedfor48hat125rpmand30◦C. Freebacteriaandbacteria-freewebsamplesweretestedfortheir MBremovalcapabilitiesaswell,aspositiveandnegativecontrols, respectively.Thebacterialcellviabilitiesofinitialinoculaforfree viablebacterialcellswereadjustedaround108cfu/mLto

compen-satetheinitialcellviabilitiesoffreeandencapsulatedbacteria.In

ordertoevaluatetheroleofdeadcellsinMBremoval,deadcells werealsotestedforMBremovalatthesameconditions.Bacterial cellviabilitieswereadjusted as∼1010 cfu/mL,correspondingto

thetotalviablebacterialnumberwithinelectrospinningsolutions beforestartingtheprocess,beforekillingbacterialcellsat70◦Cfor 3h.Alltestsweredoneintriplicate.

Theremovalcapacities(Qeq)offreebacteriacells,andbacteria

encapsulatedwebswerecalculatedbyEq.(1):

Qeq(mg/g)= (C0−−−Cf).V/M (1)

C0istheinitialMBconcentration(mg/L),Cf isthefinalMB

con-centration(mg/L),Visthesolutionvolume(L)andMisthetotal bacterialcellbiomass(g)atequilibrium[30].

2.6. Scanningelectronmicroscopy(SEM)

Millimeter-lengthPVAandPEOwebswerepreparedforSEM analysis toevaluatemorphologies of bacteria-free and bacteria encapsulatedwebs.Sampleswerecoatedwith5nmAu-Pdprior toSEMimaging(Quanta200FEGSEM,FEIInstruments,USA).The averagefiberdiameter(AFD)wasdeterminedfromtheSEMimages, andaround100fiberswereanalyzed.

2.7. Reactionkineticsstudies

TheorderofreactionsforMBremovalprocesswasevaluated upontheR2valuesofzero,first,secondandthirdorderplotsof

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248 O.F.Sariogluetal./ColloidsandSurfacesB:Biointerfaces152(2017)245–251

Fig.2.SEMmicrographsof(a)pristinePVA(b)pristinePEO(c)bacteria/PVA(d)bacteria/PEOwebsandfluorescencemicroscopyimagesof(e)bacteria/PVAand(f)bacteria/PEO webs.

3. Resultsanddiscussion

3.1. Encapsulationofbacteriawithinnanofibrouswebsand evaluationofbacterialcellviability

Theelectrospinningprocessforbacteriaencapsulated nanofi-brouswebsissummarizedinFig.1schematically. Althoughthe appliedvoltageforelectrospinningprocessishighlydetrimental forbacterialcells,itwasneededtoproducePVAandPEOnanofibers atthesepolymer concentrations.Therefore, in ordertoachieve thebacterialcellviabilitiesindesiredamountsfortheelectrospun nanofibers,bacterialamountsintheelectrospinningsolutionswere highlycondensed,sothatevenafterelectrospinningandcell viabil-itylosses,thereweresufficientamountsofviablebacteriawithin electrospunnanofibers.Theencapsulationprotocolisnotanovel practiceandthereareseveralpapersrelatedwithencapsulation ofbacterialcellswithinelectrospunfibrouswebsfor bioremedi-ation[31–33].Asmentionedinthesepapers,thesurvivingcells cansuccessfullypreservetheirremovalcapabilitiesandenzymatic activitiesagainsttargetcontaminants.Inaddition,the experimen-talmethodusedinthisstudywasutilizedafterseveraloptimization steps(the appliedvoltage was determined after optimization), whichwastheleastharmfulprotocolforbacterialcellsamong

dif-ferentexperimentalconditions.Weagreethattheprocessisstill detrimentalforbacteria,yetconsiderable(andsufficientfor start-inganewculture)amountsoflivingcellscouldbeobtainedafter theprocess.Someadditionalpreservationstepscanalsobe uti-lized,forinstanceaddingglyceroltotheelectrospinningsolution toincreasethebacterialcellviabilitiesduringelectrospinning pro-cess[34],yetsinceourstudyisaproofofconceptstudy,wedidnot includethem.Furthermore,useofbiocompatibleand biodegrad-ablematerials(e.g.polyvinylalcohol,polyethyleneoxide,alginate) forencapsulationcanenhance thesurvivabilityof encapsulated cellsduringstorage.Themorphologiesofbacteria-freeand bacte-riaencapsulatedelectrospunPVAandPEOnanofibrouswebswere evaluatedbySEMimaging.TheaveragediametersofpristinePVA andPEOnanofibersweremeasuredas420±35and230±20nm, respectively.Whilebead-freenanofiberswereobtainedforpristine PVAnanofibrousweb(Fig.2a),beadedstructureswereobtained forpristinePEOnanofibrouswebat3.5%(w/v)polymer concen-tration(Fig.2b).Itwasfoundthatbacterialcellsweresuccessfully encapsulatedwithinPVAandPEOnanofibrouswebs.Encapsulation ofbacterialcellscausedlocalwideningoffibersincertainregions andball-likestructureswereobservedintheseareas(Fig.2cand d).Inaddition,itwasnoticedthattheball-likestructuresdueto bacterialencapsulationarequitedifferent(biggerandthicker)and

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Table1

Removalcapacitiesoffree-bacteria,bacteria/PVAwebandbacteria/PEOweb sam-plesatequilibriumattheendoftheremovalprocess.T=30◦C,agitationrate: 125rpm,incubationtime:48h(n=3,resultsshowmeans±S.E.M).

Samplename Initialconcentration(C0) Qeq(mg/g)

Free-bacteria 10mg/L 25.04±0.58 15mg/L 66.75±6.59 25mg/L 74.02±8.61 Bacteria/PVAweb 10mg/L 27.79±3.18 15mg/L 45±1.38 25mg/L 89.22±1.17 Bacteria/PEOwe 10mg/L 23.57±3.24 15mg/L 60.04±1.46 25mg/L 89.47±3.94

canbeeasilydifferentiatedfromtheordinarybeadsofpristinePEO

nanofibers(Fig.2d).Fluorescencemicroscopyimageshaveshown

that,thecellviabilitieswerepreservedfortheencapsulated bacte-riainthePVAandPEOnanofibermatrices(Fig.2eandf).

The bacterial cell viabilities were also checked by applying VCCassayonequivalentsamplesofbacteria/PVAandbacteria/PEO webs.Asmentionedpreviously,beforeusinginMBremoval exper-iments, thecell viabilities were determined for each 10mg of bacteriaencapsulatedwebsasaround107–108cfu/mLfor

bacte-ria/PVAand108cfu/mLforbacteria/PEOwebs.Bacteria/PEOweb

samples had always higher cell viabilities, possibly due tothe higherw/vratioofbacterialcellbiomassafterdehydrationfor bac-teria/PEOwebs.Afterensuringthatthewebsampleshavesufficient amountsofviablebacterialcells,biodegradationexperimentswere initiatedwithequivalentsofthosewebsamples.

3.2. MBdyeremovalcapabilitiesofbacteria/PVAand bacteria/PEOwebs

Electrospun bacteria/PVAand bacteria/PEO webs arereadily water-solublebiocompositeswhichcouldbehandycarriermatrix forbacterialstorageandcanbealternativetolyophilizedbacteria forenvironmentalremediationapproachesinwater.Theeffectof PVAandPEOpolymericsolutionsonbacterialgrowthwas evalu-atedandnoapparentdifferenceswerefoundforbacteriawhich weregrowninpolymericsolutions(Fig.S1),hencethesewebscan beusedsafelyforstartingbacterialinocula.Here,MBremoval capa-bilitiesofbacteria/PVAandbacteria/PEOwebsweretestedatthree differentinitialMBconcentrations(10,15,25mg/L).Whileboth webshaveshownlowerremovalyieldsat10mg/LofinitialMB (52.5%forbacteria/PVAweband44.4%forbacteria/PEOweb),their removalyieldsincreasedat15mg/L(57%forbacteria/PVAweband 76%forbacteria/PEOweb)and25mg/L(68%forbacteria/PVAweb and69%forbacteria/PEOweb)ofinitialMB,suggestingtheremoval processisbiologicalratherthanadsorptionbased,andmightbe enhancedbygeneticswitchingatadefinedconcentrationrange (Fig.3a).EventhoughPVAandPEOnanofibrouswebswerequickly dissolvedinMBaqueoussolution,averynegligibledecreasewas observedinMBconcentrationwiththeadditionofpristinePVA andPEOnanofibers,implyingtheremovalperformancesof bac-teria/PVA and bacteria/PEO webs were primarilybased onthe bacterialexistence(Fig.3b).Inaddition,deadbacterialcellshave shownaverynegligibledecreaseintheinitialMBconcentration after48hincubation,suggesting MBdyewasprimarily remedi-atedbyviablebacterialcells(Fig.S2).Removalcapacities(Qeq)of

free-bacteriacellsandbacteriaencapsulatedwebswerecalculated foreachconcentrationandarepresentedinTable1.TheQeqvalues

offree-bacteria,bacteria/PVAwebandbacteria/PEOwebsamples aresimilarat10mg/L.At15mg/LofinitialMBconcentration, free-bacteriasamplehasahigherQeqvaluethanbacteriaencapsulated

webs,andbacteria/PEOweb hasahigherQeq valuethan

bacte-ria/PVAweb.At25mg/LofinitialMBconcentration,theQeqvalues

ofbacteria/PVAandbacteria/PEOwebsareveryclosetoeachother andbothofthemarehigherthanthefree-bacteriasample.Since bacteria/PEOwebsampleshadhigheramountsofviablebacteriafor theinitialinoculum,theirremovalperformanceswerehigherthan thatofbacteria/PVAwebsamplesingeneral,suggestingthe bacte-riaencapsulatedwebsamplescanbeimprovedformoreefficient MBremovalbyincreasingtheencapsulatedbacterialcell viabili-ties.Inaddition,byusingamorecapablebacterialstrainforMB removal,higherremovalperformanceseveninshortertime peri-odscanbeobtained,aspresentedpreviously[1,35].Notaregular trendwasobservedwhencomparingtheremovalperformancesof free-bacterialcellsandbacteriaencapsulatedwebsamples.While thehighest MBremovalwasobserved byfree-bacterial cellsat 15mg/L,thehighestMBremovalwasobservedbybacteria/PEO websampleat25mg/L,andthehighestMBremovalwasobserved bybacteria/PVAwebsample at10mg/L.Theseresultsmight be occurredduetothedifferencesinencapsulationefficiencyfor dif-ferentbacteriaencapsulatedwebsamples,leadingdifferencesin initialinoculaandhence maximalgrowthofthebacterialcells. Sinceencapsulationefficiencycanbeinfluencedbatchtobatchdue toslightenvironmentalchanges(e.g.humidity),somefluctuations inbacterialnumberswereobservedforequivalentsamples.

3.3. Evaluationoforderofreactions

TheR2valuesofdifferentorderplotsforMBremovalarelisted

inTableS1.Whilebacteria/PVAwebhasshownthehighest cor-relationwiththezeroordermodel(R2=0.9797),free-bacteriaand

bacteria/PEOwebsampleshaveshownthehighestcorrelationwith thefirstordermodel(R2valuesof0.9912and0.943,respectively).

Theseresultsconformwiththeresultsfromtheliterature,since enzyme-catalyzedreactionsoftenfallunderthezeroordermodel

[36]andfirstorderreactionscanfittotheenzyme-drivenreactions forbiologicalremovalofwatercontaminants[21].

3.4. Storabilityandapplicabilityofbacteriaencapsulatedwebs Thebacteriaencapsulatedwebsamplesweretestedfor storabil-ityatdifferenttimeperiodsviaVCCassay,intermsofcellviability preservation.Different levelsof cellviabilitieswereachievedin two different experiments(at 4◦C and 25◦C)and bacteria/PVA websamplehadhigherinitialcellviabilitythanbacteria/PEOweb sampleforthestoragetestat25◦C(Fig.4b),unlikefromthe pre-viousexperiments,probablyduetoaninfluentialbatchtobatch variation.Nevertheless,thesedifferencesdidnothavesignificant impactsontheassessmentofstoragetest,sinceeachsamplehas beenevaluatedindividuallyandthecomparisonsweremadeon theirinitialtofinalcellviabilityratios.Inaddition,althoughthere aresomevariationsinthecellviabilitymeasurementsofbacteria encapsulatedweb samplesforthestoragetestat4◦C,theseare restrictedincertainlevels,henceitwassupposedthatthepartial dissimilaritiesinthecellviabilitynumbersforequivalentsamples mightbethereasonforthesevariations.Itwasfoundthat,while bacteriaencapsulatedwebsamplescanbesafelystoredat4◦Cfor monthswithoutsignificantlossesintheinitialcellviabilities,the cellviabilitiesrapidlydecreaseat25◦Canddonotallowlong-term storageatthistemperature(Fig.4,TableS2).Therefore,itwas con-cludedthat,thebacteriaencapsulatedwebsamplescanbestored safelyforlongtimeperiods,yetitneedscoolertemperaturesfor cellviabilitypreservation.

Inbrief,thisstudyfocusesondesign anddevelopmentofan alternativesystemwithdistinctfeatures,ratherthantheremoval efficiencyforMBremediation.Totheauthors’knowledge,thisis thefirststudywhichpresentsremediationofMBbybacteria encap-sulatedelectrospunfibrouswebs.Thebacteriaencapsulatedwebs

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250 O.F.Sariogluetal./ColloidsandSurfacesB:Biointerfaces152(2017)245–251

Fig.3. (a)MBremovalprofilesoffree-bacteria,bacteria/PVAwebandbacteria/PEOwebsamplesatinitialconcentrationsof10,15and25mg/L.(b)Concentrationvs.time graphoffree-bacteria,bacteria/PVAweb,bacteria/PEOweb,pristinePVAwebandpristinePEOwebsamplesat15mg/LofinitialMB(n=3,graphsshowmeans±S.E.M).

Fig.4.Viablecellcounting(VCC)resultsofbacteria/PVAandbacteria/PEOwebsamplesforstorageat(a)4◦Cfor3monthsand(b)25Cfor10days(n=3,graphsshow

means±S.E.M).

havelowerspaceandweightcomparetofree-bacteriain liquid media,whichprovideseaseofapplicationandlower transporta-tioncosts,asinlyophilizedbacteria.Inaddition,thesewebscanbe storedatcoolertemperatureswithoutsignificantlossesinthecell viability.Byoptimizationofenvironmentalparametersandusinga morecapablebacterialstrainintermsofremovalefficiencyagainst MBdye,moreefficientbiocompositescanbeproducedfor remedia-tionofMB.Inthissense,thefindingsherearepromisingforfurther developmentsinthisfield.

4. Conclusions

Inthisstudy,wehavedevelopedfunctionalbiocomposite mate-rialsthat wereproduced byencapsulationofa MBremediating PseudomonasaeruginosastrainwithinelectrospunPVAand PEO nanofibrouswebs.Thebacterialcellviabilitieswerecheckedby viable cell counting (VCC) assay and fluorescence microscopy imaging.Sufficientamountsofviablebacterialcellscouldbe encap-sulatedwithinelectrospunPVAandPEOnanofibrouswebs,and thesewebsweretestedforMBremovalinwater.TheresultsofMB removalexperimentsrevealedthatMBremovalcapabilitiesof bac-teriaencapsulatedwebswerebasedonthebacterialpresence,and similarremovalperformanceswereobservedforfree-bacteria.It wasinferredthatMBremovalwasachievedbybiologicalremoval ratherthanadsorption,andtheremovalperformancescanbe opti-mized by increasing the initialcell viability numbers or using amore capablebacterialstrain.Inaddition, storagetestresults showedthatbacteriaencapsulatedwebscanbestoredsafelyfor longtimeperiodsat4◦C,whilepreservingtheinitialcell viabil-itynumbers.Thistypeofstoragecanbealternativetolyophilized bacteriaandbringsomeadvantagesoverstorageinculturemedia;

suchasthewebsamplesdonotneedanyminimalgrowthmedium anditrequiresverysmallspacesforstorage.Inconclusion, bacte-riaencapsulatedelectrospunnanofibrouswebscanbeeffectively usedforremediationofMBinwaterwithstorableandimprovable properties.

Acknowledgements

The Scientific and Technological Research Councilof Turkey (TUBITAK, project #114Y264) is acknowledged for fundingthe research.Dr.UyaracknowledgesTheTurkishAcademyofSciences - Outstanding Young Scientists Award Program (TUBA-GEBIP) for partialfundingof theresearch. A.Celebioglu acknowledges TUBITAKproject#113Y348forapostdoctoralfellowship.O.F. Sar-iogluacknowledgesTUBITAK BIDEB(2211-C)for NationalPh.D. Scholarship.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.colsurfb.2017.01. 034.

References

[1]N.O.San,A.Celebioglu,Y.Tümtas¸,T.Uyar,T.Tekinay,Reusablebacteria immobilizedelectrospunnanofibrouswebsfordecolorizationofmethylene bluedyeinwastewatertreatment,RSCAdv.4(2014)32249–32255.

[2]N.S.Maurya,A.K.Mittal,P.Cornel,E.Rother,Biosorptionofdyesusingdead macrofungi:effectofdyestructure:ionicstrengthandpH,Bioresour. Technol.97(2006)512–521.

(7)

[3]A.Saeed,M.Iqbal,S.I.Zafar,ImmobilizationofTrichodermaviridefor enhancedmethylenebluebiosorption:batchandcolumnstudies,J.Hazard. Mater.168(2009)406–415.

[4]K.Vijayaraghavan,S.W.Won,J.Mao,Y.S.Yun,Chemicalmodificationof Corynebacteriumglutamicumtoimprovemethylenebluebiosorption,Chem. Eng.J.145(2008)1–6.

[5]V.J.P.Vilar,C.M.S.Botelho,R.A.R.Boaventura,Methyleneblueadsorptionby algalbiomassbasedmaterials:biosorbentscharacterizationandprocess behavior,J.Hazard.Mater.147(2007)120–132.

[6]X.Wang,X.Chen,K.Yoon,D.Fang,B.S.Hsiao,B.Chu,Highfluxfiltration mediumbasedonnanofibroussubstratewithhydrophilicnanocomposite coating,Environ.Sci.Technol.39(2005)7684–7691.

[7]N.Zaghbani,A.Hafiane,M.Dhahbi,Separationofmethylenebluefrom aqueoussolutionbymicellarenhancedultrafiltration,Sep.Purif.Technol.55 (2007)117–124.

[8]R.Wang,J.Guo,D.Chen,Y.E.Miao,J.Pan,W.W.Tjiu,T.Liu,Tubebrushlike ZnO/SiO2hybridtoconstructaflexiblemembranewithenhanced

photocatalyticpropertiesandrecyclingability,J.Mater.Chem.21(2011) 19375–19380.

[9]Y.Zhan,H.Li,Y.Chen,Copperhydroxyphosphateascatalystforthewet hydrogenperoxideoxidationofazodyes,J.Hazard.Mater.180(2010) 481–485.

[10]S.K.Nataraj,K.M.Hosamani,T.M.Aminabhavi,Distillerywastewater treatmentbythemembrane-basednanofiltrationandreverseosmosis processes,WaterRes.40(2006)2349–2356.

[11]E.Rosales,M.Pazos,M.A.Sanromán,Comparativeefficienciesofthe decolourisationofleatherdyesbyenzymaticandelectrochemicaltreatments, Desalination278(2011)312–317.

[12]A.Malik,Metalbioremediationthroughgrowingcells,Environ.Int.30(2004) 261–278.

[13]M.T.Madigan,J.M.Martinko,BrockBiologyofMicroorganisms,eleventhed., PrenticeHall,NewJersey,2006.

[14]A.K.Karamalidis,A.C.Evangelou,E.Karabika,A.I.Koukkou,C.Drainas,E.A. Voudrias,Laboratoryscalebioremediationofpetroleum-contaminatedsoilby indigenousmicroorganismsandaddedPseudomonasaeruginosastrainSpet, Bioresour.Technol.101(2010)6545–6552.

[15]Y.Wang,J.Song,W.Zhao,X.He,J.Chen,M.Xiao,Insitudegradationofphenol andpromotionofplantgrowthincontaminatedenvironmentsbyasingle Pseudomonasaeruginosastrain,J.Hazard.Mater.192(2011)354–360.

[16]R.Pasumarthi,S.Chandrasekaran,S.Mutnuri,Biodegradationofcrudeoilby PseudomonasaeruginosaandEscherichiafergusoniiisolatedfromtheGoan coast,Mar.Pollut.Bull.76(2013)276–282.

[17]E.Eroglu,V.Agarwal,M.Bradshaw,X.Chen,S.M.Smith,C.L.Raston,K.S.Iyera, Nitrateremovalfromliquideffluentsusingmicroalgaeimmobilizedon chitosannanofibermats,GreenChem.14(2012)2682–2685.

[18]L.Hall-Stoodley,J.W.Costerton,P.Stoodley,Bacterialbiofilms:fromthe Naturalenvironmenttoinfectiousdiseases,Nat.Rev.Microbiol.2(2004) 95–108.

[19]O.F.Sarioglu,O.Yasa,A.Celebioglu,T.Uyar,T.Tekinay,Efficientammonium removalfromaquaticenvironmentsbyAcinetobactercalcoaceticusSTB1 immobilizedonanelectrospuncelluloseacetatenanofibrousweb,Green Chem.15(2013)2566–2572.

[20]N.O.San-Keskin,A.Celebioglu,T.Uyar,T.Tekinay,Microalgaeimmobilizedby nanofibrouswebforremovalofreactivedyesfromwastewater,Ind.Eng. Chem.Res.54(2015)5802–5809.

[21]N.O.San-Keskin,A.Celebioglu,O.F.Sarioglu,A.D.Ozkan,T.Uyar,T.Tekinay, Removalofareactivedyeandhexavalentchromiumbyareusablebacteria attachedelectrospunnanofibrousweb,RSCAdv.5(2015)86867–86874.

[22]O.F.Sarioglu,A.Celebioglu,T.Tekinay,T.Uyar,Evaluationofcontacttimeand fibermorphologyonbacterialimmobilizationfordevelopmentofnovel surfactantdegradingnanofibrouswebs,RSCAdv.5(2015)102750–102758.

[23]W.Salalha,J.Kuhn,Y.Dror,E.Zussman,Encapsulationofbacteriaandviruses inelectrospunnanofibres,Nanotechnology17(2006)4675–4681.

[24]W.Y.Fung,K.H.Yuen,M.T.Liong,Agrowaste-basednanofibersasaprobiotic encapsulant:fabricationandcharacterization,J.Agric.FoodChem.59(2011) 8140–8147.

[25]Y.Liu,M.H.Rafailovich,R.Malal,D.Cohn,D.Chidambaram,Engineeringof bio-hybridmaterialsbyelectrospinningpolymer-microbefibers,Proc.Natl. Acad.Sci.U.S.A.106(2009)14201–14206.

[26]S.Klein,R.Avrahami,E.Zussman,M.Beliavski,S.Tarre,M.Green, EncapsulationofPseudomonassp:ADPcellsinelectrospunmicrotubesfor atrazinebioremediation,J.Ind.Microbiol.Biotechnol.39(2012)1605–1613.

[27]H.W.Tong,B.R.Mutlu,L.P.Wackett,A.Aksan,Manufacturingofbioreactive nanofibersforbioremediation,Biotechnol.Bioeng.111(2014)1483–1493.

[28]N.Jiang,G.L.Ying,S.Y.Liu,L.Shen,J.Hu,L.J.Dai,X.Y.Yang,G.Tian,B.L.Su, Aminoacid-basedbiohybridsfornano-shellizationofindividualdesulfurizing bacteria,Chem.Commun.50(2014)15407–15410.

[29]X.Y.Yang,G.Tian,N.Jiang,B.L.Su,Immobilizationtechnology:asustainable solutionforbiofuelcelldesign,EnergyEnviron.Sci.5(2012)5540–5563.

[30]C.J.Buchko,L.C.Chen,Y.Shen,D.C.Martin,Processingandmicrostructural characterizationofporousbiocompatibleproteinpolymerthinfilms,Polymer 40(1999)7397–7407.

[31]H.W.Tong,B.R.Mutlu,L.P.Wackett,A.Aksan,Silica/PVAbiocatalytic nanofibers,Mater.Lett.111(2013)234–237.

[32]S.Klein,J.Kuhn,R.Avrahami,S.Tarre,M.Beliavski,M.Green,E.Zussman, Encapsulationofbacterialcellsinelectrospunmicrotubes,

Biomacromolecules10(2009)1751–1756.

[33]S.Klein,R.Avrahami,E.Zussman,M.Beliavski,S.Tarre,M.Green, EncapsulationofPseudomonassp.ADPcellsinelectrospunmicrotubesfor atrazinebioremediation,J.Ind.Microbiol.Biotechnol.39(2012)1605–1613.

[34]W.Salalha,J.Kuhn,Y.Dror,E.Zussman,Encapsulationofbacteriaandviruses inelectrospunnanofibres,Nanotechnology17(2006)4675–4681.

[35]N.A.El-Sersy,BioremediationofmethylenebluebyBacillusthuringiensis4G1: applicationofstatisticaldesignsandsurfaceplotsforoptimization, Biotechnology6(2007)34–39.

[36]I.Tinoco,K.Sauer,J.C.Wang,PhysicalChemistry–PrinciplesandApplications inBiologicalSciences,thirded.,PrenticeHall,NewJersey,1996.

Şekil

Fig. 1. (a) Schematic representation of electrospinning process for bacteria encapsulated PVA and PEO webs, and photographs of PVA and PEO webs, (b) representative images for bacteria encapsulated webs including a SEM micrograph and a schematic representat
Fig. 2. SEM micrographs of (a) pristine PVA (b) pristine PEO (c) bacteria/PVA (d) bacteria/PEO webs and fluorescence microscopy images of (e) bacteria/PVA and (f) bacteria/PEO webs.
Fig. 4. Viable cell counting (VCC) results of bacteria/PVA and bacteria/PEO web samples for storage at (a) 4 ◦ C for 3 months and (b) 25 ◦ C for 10 days (n = 3, graphs show means ± S.E.M).

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