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,∗

a_Institute_of_Materials_Science_&_{Nanotechnology,}_Bilkent_University,_06800,_Bilkent,_Ankara,_Turkey b_{UNAM-National}_{Nanotechnology}_Research_Center,_Bilkent_University,_06800,_Bilkent,_Ankara,_Turkey c_Polatlı_Science_and_Literature_Faculty,_Biology_Department,_Gazi_University,_06900,_Polatlı,_Ankara,_Turkey d_Life_Sciences_Application_and_Research_Center,_Gazi_University,_06830,_Golbasi,_Ankara,_Turkey

e_Department_of_Medical_Biology_and_Genetics,_Faculty_of_Medicine,_Gazi_University,_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◦_C_for_three_months_and_they_were_found_as_potentially

storableforkeepingencapsulatedbacterialcellsalive.Overall,theresultssuggestthatelectrospun nanoﬁ-brouswebsaresuitableplatformsforpreservationoflivingbacterialcellsandtheycanbeuseddirectly asastartinginoculumforbioremediationofwatersystems.

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:ttekinay@gazi.edu.tr(T.Tekinay),uyar@unam.bilkent.edu.tr

(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∼1010_cfu/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_–108_cfu/mL_per₁₀_mg,

equivalentwebsamples(withw/vratioof1mg/mL)wereprepared forinitiatingMBbioremovalexperiments.Theinitialbacterialcell viabilities of bacteria/PVA and bacteria/PEO webs were around 107_–108_cfu/mL_and₁₀8_cfu/mL_per₁₀_mg,_{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 viablebacterialcellswereadjustedaround108_cfu/mL_to

compen-satetheinitialcellviabilitiesoffreeandencapsulatedbacteria.In

ordertoevaluatetheroleofdeadcellsinMBremoval,deadcells werealsotestedforMBremovalatthesameconditions.Bacterial cellviabilitieswereadjusted as∼1010 _cfu/mL,_{corresponding}_to

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 upontheR2_values_of_zero,_ﬁrst,_second_and_third_order_plots_of

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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_–108_cfu/mL_for

bacte-ria/PVAand108_cfu/mL_for_bacteria/PEO_webs._Bacteria/PEO_web

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

TheR2_values_of_different_order_plots_for_MB_removal_are_listed

inTableS1.Whilebacteria/PVAwebhasshownthehighest cor-relationwiththezeroordermodel(R2₌_0.9797),_{free-bacteria}_and

bacteria/PEOwebsampleshaveshownthehighestcorrelationwith theﬁrstordermodel(R2_values_of_0.9912_and_0.943,_{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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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◦_C_for₃_months_and_(b)₂₅◦_C_for₁₀_days_(n₌_3,_graphs_show

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.

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