Reinforcement of bacterial cellulose aerogels with biocompatible polymers

Bacterial cellulose (BC) aerogels, which are fragile, ultra-lightweight, open-porous and transversally isotropic materials, have been reinforced with the biocompatible polymers polylactic acid (PLA), polycaprolactone (PCL), cellulose acetate (CA), and poly(methyl methacrylate) (PMMA), respectively, at varying BC/polymer ratios.

jo u r n al h om ep a g e :w w w e l s e v i e r c o m / l o c a t e / c a r b p o l

polymers

a University of Natural Resources and Life Sciences Vienna, Division of Chemistry of Renewables, Konrad-Lorenz-Straße 24, A-3430 Tulln, Vienna, Austria

b University of Natural Resources and Life Sciences Vienna, Department of Wood Science, Konrad-Lorenz-Straße 24, A-3430 Tulln, Vienna, Austria

c Clermont Université, ENSCCF, Institute of Chemistry of Clermont-Ferrand, BP 10448, 63000, Clermont-Ferrand, France

d CNRS, UMR 6296, ICCF, 24 av des Landais, 63171 Aubière, France

a r t i c l e i n f o

Article history:

Received 11 November 2013

Received in revised form 30 March 2014

Accepted 10 April 2014

Available online 21 April 2014

Keywords:

Bacterial cellulose

Cellulosic aerogels

Cellulose composite materials

Interpenetrating polymer networks

Reinforcement

Supercritical carbon dioxide

a b s t r a c t

Bacterialcellulose(BC)aerogels,whicharefragile,ultra-lightweight,open-porousandtransversally isotropicmaterials,havebeenreinforcedwiththebiocompatiblepolymerspolylacticacid(PLA), poly-caprolactone (PCL),cellulose acetate(CA), andpoly(methyl methacrylate)(PMMA), respectively,at varyingBC/polymerratios.Supercriticalcarbondioxideanti-solventprecipitationandsimultaneous extractionoftheanti-solventusingscCO2 havebeenusedascoretechniquesforincorporatingthe secondarypolymerintotheBCmatrixandtoconverttheformedcompositeorganogelsintoaerogels Uniaxialcompressiontestsrevealedaconsiderableenhancementofthemechanicalpropertiesas com-paredtoBCaerogels.Nitrogensorptionexperimentsat77Kandscanningelectronmicrographsconfirmed thepreservation(orevenenhancement)ofthesurface-area-to-volumeratioformostofthesamples Theformationofanopen-porous,interpenetratingnetworkofthesecondpolymerhasbeen demon-stratedbytreatmentofBC/PMMAhybridaerogelswithEMIMacetate,whichexclusively extracted cellulose,leavingbehindself-supportingorganogels

©2014TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBYlicense

(http://creativecommons.org/licenses/by/3.0/)

1 Introduction

Bacterialcellulose (BC) is anextracellular natural byproduct

ofthemetabolismof variousbacteria(Deinema&Zevenhuizen,

1971),withAcetobacterspp.strainsbeingmostcommonlyused.BC

isproducedbytherespectivebacteriastrainsinresponseto

spe-cificenvironmentalconditions.Acetobacterxylinum,forexample,

producescellulosepelliclesthatkeepthebacteriumfloatingonthe

surfacetomaintainsufficientoxygensupply.Otherbacteria,such

astheplantpathogenAgrobacteriumtumefaciens,usecellulosefor

betterattachmenttoplants,similartothesymbioticRhizobiumspp

Bacterialcellulose,grownundercontrolledconditionson

appro-priatecarbonandnitrogensources,formshighlyporousnetwork

structures, whose voids are filled with the culture medium

Themacroscopicappearance(pellicles,sheets,tubes,etc.)varies

dependingonthetechnologicalapproach(staticvs.agitated,batch

vs.continuouscultivation,rotaryvs.diskfermenters,e.g.).After

∗ Corresponding author Tel.: +43 1 47654 6452.

E-mail address: falk.liebner@boku.ac.at (F Liebner).

1 Current address: Swiss Federal Institute of Technology Zurich, Institute for

Building Materials, Schafmattstraße 6, 8093 Zurich, Switzerland.

removing the culture medium and thorough washing, a taste-less,colorless,andodorlesstranslucentandchewygelisobtained which, todate, ismainlycommercialized asadietaryauxiliary However, applications in skin care (Nanomasque®; Amnuaikit, Chusuit,Raknam,&Boonme,2011)andtopologicalwound heal-ing (Suprasorb®X, Bioprocess®,XCell®,and Biofill®; Petersen & Gatenholm,2011),whichbothtakeadvantageofthehighpurity

ofBC,itspositiveeffectonskintissueregeneration(Sutherland, 1998)anditsgreatwater-retainingandmoisturizingcapabilities, arecurrentlyadvancingstrongly.Beyondthat,good biocompatibil-ityandlowimmunogenicpotential(Heleniusetal.,2006;Klemm, Schumann,Udhardt,&Marsch,2001)renderBCapromising mate-rialforvariousbiomedicalapplications.Thiscomprisestheiruse

asartificialbloodvessels(Klemmetal.,2001), semi-permanent artificialskin(Petersen&Gatenholm,2011),aswellasmatrices forslow-releaseapplications(Haimeretal.,2010),nervesurgery (Klemm et al., 2001), engineering of bone tissue (Zaborowska

etal.,2010)orartificialkneemenisci(Bodin,Concaro,Brittberg,

&Gatenholm,2007)

Quantitativereplacementofwaterbyanorganicsolventand subsequentextractionoftheorganicsolventfromtheporousBC matrixwithsupercriticalcarbondioxide(scCO2)hasbeen demon-strated to be the most successful approach for converting BC http://dx.doi.org/10.1016/j.carbpol.2014.04.029

0144-8617/© 2014 The Authors Published by Elsevier Ltd This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/3.0/ ).

mis-ciblewithbothwaterandscCO2,asitisthecasefor,e.g.,ethanol

oracetone.Thisdryingprocedurepreservesthefragilecellulose

networkstructure and thehierarchicalsystemof micro-,meso,

andmacropores(Liebneretal.,2010;Maeda,Nakajima,Hagiwara,

Sawaguchi,&Yano,2006b).Bacterialcelluloseaerogelsfeaturean

outstandinglylowbulkdensityindrystate(≥10mgcm−3),have

lowheattransmissionandthermalexpansioncoefficients,arefully

re-hydratableand sharealloftheabovepropertiesrelevant for

biomedicalapplications.Therefore,BCaerogelsexpandthescope

of BC applications considerably, be it in terms of sensing (e.g

byquantumdots),thermal oracousticinsulation, specific

sorp-tion(from gases orliquids), catalysis, orslow release of active

compounds

However,despitethehightensilemodulusandstrengthof

indi-vidualBCribbons (>10GPaand>17MPa,respectively,Svensson

etal.,2005),theresistanceofBCaerogelsandtheirhydrated

pre-cursorstowardscompressivemechanicalstressisnotsufficiently

highformanyapplicationsthatinvolvemechanicwear.Numerous

reinforcingstrategieshavebeenthereforeinvestigated,including

preparationofall-cellulosecomposites,controllingfibrilproperties

byadding special additivestothenutrient medium,

incorpora-tionofstrength-impartingpolymersduringBCgrowth,chemical

surfacemodification,orcross-linking(Seifert,Hesse,Kabrelian,&

Klemm,2004; Yano,Maeda, Nakajima, Hagiwara,& Sawaguchi,

2008)

Three-dimensionalnetworksofasecondarypolymer

interpen-etratingandreinforcingthatofbacterialcellulosecanbeprepared

bysoaking BC witha solutionof the respectivemonomer and

covalentgraftingontoBC(e.g.BC-g-PMMA,BC-g-PBA,

BC-g-PMMA-co-PBA;Lacerda,Barros-Timmons,Freire,Silvestre,&Neto,2013)

Furthertechniquesareinsitugenerationoftheinterpenetrating

network by loadingand subsequent chain-growth

polymeriza-tionofa suitable monomersuchasmethacrylic acid(Hobzova,

Duskova-Smrckova,Michalek,Karpushkin,&Gatenholm,2012)or

precipitationofthereinforcing polymerfroma compatible

sol-vent, filling thevoids of thecellulosic network, as it hasbeen

described in our previous work for BC/cellulose acetate

com-posites (Liebner,Aigner, Schimper, Potthast, & Rosenau, 2012)

BChybridmaterialscontaininganinorganicpolymerhavebeen

obtainedbyloadingofsilicasolinto(Yanoetal.,2008)or

poly-merizationofsilicateprecursorswithintheBCstructure(Maeda,

Nakajima,Hagiwara,Sawaguchi,&Yano,2006a).Anotherprocess

thataffordsorganic/inorganichybridmaterialsisbiomineralization

ofappropriatelyfunctionalizedcellulosicscaffolds,asittakesplace

in(simulated)bodyfluids(Zimmermann,LeBlanc,Sheets,Fox,&

Gatenholm,2011)

Themajorityof previousstudiesusedtheaboveapproaches

eithertoreinforcethinBCfilmsdirectlyortoobtainmechanically

resistantBCsheetsfrommodifiedbulkBCorganogelsafter

com-paction.However,tomakeuseoftheintriguingnativemorphology

of three-dimensional BC aquogels, the reinforcing approaches

shouldaimatafar-reachingpreservationoftheinherentBC

cel-lulosenetworkarchitecture

ThecurrentstudyinvestigatesthereinforcementofBCaerogels

withinterpenetrating,biocompatibleandpartiallybiodegradable

polymers,suchaspolylacticacid(PLA),polycaprolactone(PCL),

cel-luloseacetate(CA)andpoly(methylmethacrylate) (PMMA).The

three-dimensionalnetwork oftheentangledBCfibershasbeen

studiedasatemplateforthepreparationofporousPLA-,PCL-,

CA-,andPMMAscaffoldsofBC-likemorphologyunderpreservation

orenhancementofthesurface-to-volumeratio.Supercritical

car-bondioxideanti-solventprecipitationandextraction,respectively,

havebeenusedascoretechniquesfordepositingthesecondary

polymerwithintheBCmatrixandtoconverttheformedcomposite

organogelsintoaerogels

2 Materials and methods

PLAwasobtainedfromNatureWorksLLC(PLAPolymer4042D;

Mw209.0kgmol−1,6.1%D-isomer).PCL(Mw48.0–90.0kgmol−1,

Mn∼45.0kgmol−1),CA(Mn∼30.0kgmol−1,39.8wt%acetyl)and PMMA(Mw∼350.0kgmol−1)werepurchasedfromSigma-Aldrich (Vienna,Austria).AbsoluteethanolwasobtainedfromFisher Scien-tific(Vienna,Austria).Tetrahydrofuran(HiPerSolvCHROMANORM forHPLC)andacetone(AnalaRNORMAPUR)wereobtainedfrom VWR(Vienna,Austria)

2.1 Preparationofbacterialcellulose BacterialcellulosewaskindlyprovidedbytheResearchCentre forMedical Technologyand Biotechnology(FZMB)Bad Langen-salza,Germany.Thematerialwasproducedbyastaticcultivation

of Gluconacetobacter xylinum AX5 wild type strain on Hestrin-Schrammgrowthmediumfor30daysat30◦C

TheobtainedBClayerwascutinto120mm×20mm×20mm cuboids,heatedthreetimesfor20minin0.1MaqueousNaOHat

90◦C,andfinallyrinsedwithdeionizedwaterfor24h.Afterwards theBCwassubjectedtoasolventexchange,replacingwaterby96% ethanol

2.2 PreparationofBC-basedcompositeaerogels Priortomodification,theBCwascutintosmallercuboids fea-turingedgelengthsofabout10mm.Consideringthetransverse isotropy of BC aerogels (Liebner, Aigner et al., 2012)and with respecttotheevaluationofthemechanicalpropertiesofthe com-posites, thespecimens were markedalong thedirection ofthe

120mmedgesoftheparentBCsamples.Theseedgescorrespond

tooneofthehorizontal(plane)directionsoftheharvestedBCand areperpendiculartotheir(weaker)growthdirection

TherespectiveBCspecimensweretransferredfirstto tetrahy-drofuran(inthecaseofPCLandPLA)oracetone(inthecaseofCA andPMMA),correspondingtothetypeofsolventusedfor dissolu-tionofthereinforcingpolymer,andsubsequentlyintotheloading bathswhichcontainedsolutionsoftherespectivereinforcing poly-meratoverallconcentrationsof10,20,40,80and120mgmL−1 (samplelabelingreferstotheseconcentrations,e.g.:PLA10).All sol-ventexchangeandloadingstepswerecarriedoutintotalvolumes correspondingtotheten-foldvolumeoftherespectiveBCsample Afteraresidencetimeofatleast24hatroomtemperature(PCL,CA, PMMA)and50◦C(PLA),respectively,thesampleswereremoved fromtheloadingbath.Precipitationofthesecondpolymerwithin theBCporenetworkwascarriedoutwitheitherethanol(inthe caseofPLAandPCL)orscCO2(forCAandPMMA).Conversionof compositeorganogelstotherespectiveaerogelswasineithercase accomplishedbyscCO2drying:Theorganogelswereplacedinto

a300mLautoclaveequippedwithaseparatorforcarbondioxide recycling(Separex,France).Dryingwasperformedunderconstant flowofscCO2 (40gmin−1)at10MPaand40◦Cfortwotothree hours.Thesystemwasthenslowlyandisothermallydepressurized

atarateof<0.1MPamin−1

2.3 CharacterizationofBCaerogelsandBCcompositeaerogels Shrinkageoftheorganogelsduringloading/precipitationand subsequentdryingwasdeterminedbymeasuringthedimensions andcalculatingthevolumeofthecuboidsbeforeloadingwiththe respectivepolymersolutionandafterscCO2 drying.Tocalculate densities,theweightoftheaerogelswasdetermined gravimetri-callyafterdrying

(LeicaEMSCD005sputtercoater,layerthickness6nm)was

per-formedonaTecnaiInspectS50instrumentunderhighvacuumat

anaccelerationvoltageof5.00kV

PolarizedlightmicroscopywasperformedonaLeicaDM4000M

microscope.Imageswererecorded witha digitalcamera (Leica

MicrosystemsWetzlarGmbH,Germany)

ThermoporosimetrywasconductedonaMettler-ToledoDSC30

instrument equipped with a liquid nitrogen module calibrated

(bothfortemperatureandenthalpy)withmetallicstandards(In,

Pb,Zn)usingo-xyleneasinterstitialliquid.About10or20mgof

thestudiedmaterialwasplacedintoaDSCpanwhichwasthen

sealedandsubjectedtorepeatedfreezing/thawingcyclesin

com-parisontoanemptyDSCpan.Adetaileddescriptionoftheapplied

temperatureprogramcanbefoundelsewhere(Bruns,Lallemand,

Quinson,&Eyraud,1977;Nedelec,Grolier,&Baba,2006)

Mechanical response profiles towards compressive stress

orthogonallytothe(weaker)growthdirectionofBCwererecorded

onaZwick-Roell MaterialsTestingMachineZ020 Therequired

straintoachieveadeformationspeedof2.4mmmin−1was

mea-suredina500Nloadcell.Yieldstrength(RP0.2)wasdefinedasthe

stressat0.2%plasticdeformation

Nitrogen adsorption/desorption isothermsat 77K havebeen

obtainedonaMicromeriticsASAP2020analyzer.Allsampleswere

degassedinvacuumpriortoanalysis.Specificsurfaceareaswere

calculatedusingtheBrunauer,EmmettandTeller(BET)equation

3 Results and discussion

3.1 Shrinkageandbulkdensity

StaticcultivationofAcetobacterxylinumAX5wildtypestrainon

Hestrin-Schrammmediumaffordsbacterialcelluloseaquogelsthat

canbeconvertedintotherespectiveaerogelsatverylow

shrink-age(1–5%)byscCO2treatment(supercriticalpointofCO2:31.2◦C,

7.38MPa), ifthe interstitialwater is quantitatively replaced by

anappropriateCO2-miscibleorganicsolventpriortothedrying

step(40◦C, 10MPa; Liebneretal.,2010;Maeda, 2006).Ethanol

asa medium-polar solventthat is misciblewithboth H2O and

CO2isfrequentlyusedinscCO2dryingofaerogels.The

morphol-ogyofpolysaccharide-basedgels,suchasofBCaquogels,which

consistofentangledribbon-typecellulosemicrofibrilsand

inter-stitialwater,canbelargelypreservedduringthissolventexchange

(ethanol)andthesubsequentscCO2drying.Thisisreflectedbythe

lowapparentdensityoftheobtainedaerogels(7.8±0.5mgcm−3;

n=5)whichisingoodagreementwithvaluesreportedelsewhere

(8.3±0.7mgcm−3;Liebneretal.,2010)

However, loading of BC gels with the reinforcing polymers

poly(lacticacid) (PLA),polycaprolactone(PCL),cellulose acetate

(CA),and poly(methyl methacrylate) (PMMA)required solvents

otherthanethanolduetosolubilityissues.Whileacetonewasthe

solventforCAandPMMA,tetrahydrofurane(THF)wasusedinthe

caseofPLAandPCL.BCreferencesamples,whichhadbeen

man-ufacturedusingtherespectivesolventsacetoneandTHFinstead

of ethanol and which didnot contain thereinforcing polymer,

revealedthatthetypeoforganicsolventhasaweakimpactonthe

overallshrinkageofthegelsduringprocessingandhenceonthe

apparentdensityandresponseoftheobtainedaerogelstowards

compressivestress.ComparedtoBCaerogelspreparedfromthe

respectivealcogels(7.8±0.5mgcm−3),replacementofthe

inter-stitialethanolbyacetonepriortoscCO2dryingaffordedsomewhat

higherdensitiesof9.4±0.6mgcm−3(referencesamplesCA0and

PMMA0;n=4),similartodatareportedelsewhere;Liebner,Aigner

etal.,2012).Thedensitiesofaerogelsobtainedbysequential

sol-ventexchangefromethanoltoTHFandbacktoethanolpriorto

scCO2drying(referencesamplesPLA0andPCL0)werefoundtobe

inthesamerange(9.6±0.8mgcm−3;n=4)

Theimpactofthetypeofsolventontheapparentdensityofthe aerogelsismostlikelyduetothedifferentstrengthsofinteractions thatoccurbetweentherespectivesolventsandthesurfaceofthe cellulosemicrofibrilsandarestronglyinfluencedbytheabundance

ofOHgroups.AccordingtotheHansenmodelofsolvent–polymer interactions,thecohesiveenergydensity(expressedasHildebrand solubilityparameter)canbecalculatedasthesumofadispersion forcecomponent,apolarcomponentandahydrogenbonding com-ponent.Replacementofethanol (ıSI=26.5MPa1/2)byacetoneor THFdecreasesthetotalHildebrandparametertoıSI=20.0MPa1/2

andıSI=19.4MPa1/2,respectively.Thehydrogenbonding compo-nent,whichis,duetothehighabundanceofOHgroups,supposed

tobeofparticularimportanceforsolvent–polymerinteractions,is evenmoreaffectedanddecreasesfromıH=19.4MPa1/2(ethanol)

to8.0MPa1/2(THF)and7.0MPa1/2(acetone),respectively.A simi-lareffectisassumedtooccurintheinitialphaseofscCO2drying, whenCO2 andsolventformarathernon-polar,expandedliquid phaseinsidetheporesofthegelstobedried.Thisisevidentfrom theextensiveshrinking thathasbeenreportedforthe prepara-tionof aerogelsfromavarietyof biopolymers(starch,alginate, cellulose)

Correspondingtotherathermarginal,solvent-dependent dif-ferencesinshrinkage,thespatialdimensionsofBCspecimenwere largelypreservedthroughoutloadingandprecipitationofPLA,CA and PMMA, andduring scCO2 drying ofthe BC/PLA,BC/CAand BC/PMMAhybridorganogels,inparticularforthosevariantswith lowpolymerconcentrationintheloadingbaths.Strongershrinkage wasobservedonlyatconcentrationlevelsof80and120mgmL−1, correspondingtoapolymer-to-BCmassratioof≥7.9,withthe high-estvalueobservedforPMMA120(23.7%,Fig.1)

Reinforcement of cellulose aerogels with polycaprolactone (PCL)throughethanolanti-solventprecipitationfromTHFand sub-sequentscCO2 drying,turnedouttobealessfeasibleapproach

assubstantialcollapsingofthespecimenoccurredduringscCO2 drying This effect, which was observed for all PCL/BC hybrid organogelsexceedingaPCL/BCmassratioof2.0(cf.Fig.1 is trig-geredbythecomparativelystrongexpansionofPCLunderscCO2 conditions,causedbythelowglasstransitiontemperature(TG)of PCL(−60◦C)andthegoodsolubilityofCO2inPCL,whichexistsin

arubberystateundertheconditionsemployed.Whilefast depres-surizationofCO2-expandedneatPCLaffordsstablefoams(Xuetal., 2004), slowdepressurizationratesof lessthan 0.1MPamin−1 –

astypicallyusedtopreservethefragilecellulosenetwork struc-tureofrespectiveorganogels(Liebneretal.,2010)–causesthe expandedPCLrubbertocollapse.Asaresult,compactBChybrid materialswithdensitiesabouttwiceashighasobservedfor all otherhybridaerogelswereobtainedwithcelluloseribbonsstuck togetherbyPCL.Interestingly,collapsingofthenetworkstructure wasmuch more pronounced along thegrowthdirection of BC, indirectlyconfirmingtheiranisotropicmorphologyandresponse towardsmechanicalstress(seebelow).Asimilarshrinkageeffect wasnotobservedforPLA(TG=55–60◦C),PMMA(TG=90–105◦C; Buck,Diem,Schreyer,&Szigeti,1975;Dixitetal.,2009)andCA (TG=140–190◦C;Vallejos,Peresin,&Rojas,2012)whoseglass tran-sitiontemperaturesaredistinctlyabovetheemployedoperation temperatureofthescCO2unit(40◦C)

WiththeexceptionofBC/PCLhybridaerogels,thebulk densi-tiesoftheobtainedsetsofreinforcedBCaerogelsrangedfrom16

to170mgcm−3,dependingontherespectiveconcentrationofthe loadingbathaswellastheextentofshrinkage(Fig.1).Theamount

ofsecondarypolymercontainedinacertainvolumeoftheloading bathwasalsofoundintheobtainedaerogel(Fig.1A,inset), indicat-ingtheabsenceofspecificinteractionsbetweenBCandtheloaded substances which could have caused an enrichment effect In

Fig 1. Bulk density of reinforced BC aerogels vs mass ratio of the secondary polymer ( p ) in the aerogel (A; inset:  p in the aerogel vs concentration of the secondary polymer

in the loading bath (c p )) Overall shrinkage of gels during loading, solvent exchange and scCO 2 extraction vs loading bath concentration (B).

particular at higher loadings, BC composites with CA

exhib-itedlowershrinkage ratesanddensities comparedtotheother

organogels

3.2 MorphologyofBChybridaerogels

The ‘biological spinnerets’ lined up in BC producing

bacte-riareleasecelluloseasanextracellularsubstanceintheformof

elementaryfibrilswhichaggregatetoribbons.Asthecellulose

syn-thesizingsitesareduplicatedduringcelldivision(Brown,Willison,

&Richardson,1976),motheranddaughtercellareconnectedtoone

andthesamecelluloseribbonwhichcausesformationofahighly

interconnectedandentangledthree-dimensionalnetworkof

cellu-lose(Fig.2A).Nitrogensorptionexperimentsat77K,theresultsof

thermoporosimetrymeasurements(Fig.2B)andSEMmicrographs

revealaverybroadporesizedistributionwithvoiddiameters

ran-gingfromthesingledigitnano-tomicrometerrange

Themorphologyoftheaerogelschangesgraduallywith

increas-ing content of loaded polymer once a second component is

introducedintothisnetwork(Fig.3).Theabove-described

aggluti-nativeeffectofPCLcanalreadybeseenatthelowestconcentration

level At higher loadings the formation of distinctly separated

regions,stronglydeviatingintheirmorphologyareclearlyvisible

intherespectiveSEMimages.Whilesomeareasappearvery

sim-ilartotheoriginalBCnetwork,inothersthereinforcedfibersare

agglomeratedtoclustersformingmulti-layeredstructures,

pene-tratingthecompositeperpendiculartothegrowthdirection.These

structuresareassociatedwiththecollapseofthecuboidsathigher

loadings

PLAisprecipitatedinformofsmall,individualsphereswith

par-ticlesizesof about0.5–2.0␮m(PLA10 andPLA20) or ellipsoids

(PLA40)atlowerPLAconcentrations intheloadingbath,

corre-spondingtoPLA/BCratiosof≤3.5.Athigherratios(PLA80and120;

PLA/BC:8.2and12.5)asecond,interpenetratingporousstructure

isformedwithintheBCaerogel.The establishmentofthis

sec-ondarynetworkisalsoapparentfromtheresponseofthesesamples

towardscompressivestress,asbothEmodulusandyieldstrength

increasesignificantly(Fig.6,Table1)

In contrast to thepolyesters PLA and PCL, cellulose acetate

and poly(methyl methacrylate) areevenly precipitatedin close

proximitytothesurfaceoftheBCfibrils.Uptoapolymer/BCratio

ofabout4(concentrationlevel≤40mgmL−1)theBCnetworkacts

asatemplatethatgovernsthemorphologyofthesecondary

poly-mernetwork andsupportstheformationofhighlyopen-porous

compositematerialswithporecharacteristicssimilartothoseof

pureBCaerogels.Atpolymer/BCratios≥5themorphologyofthe

obtainedaerogelsisincreasinglydominatedbythepropertiesofthe

pervadingpolymerandresemblestheopenporousmicrostructure

ofpureCAorPMMAfilmsobtainedbyvariousscCO2 processes (Reverchon&Cardea, 2004;Reverchon,Cardea,&Rappo,2006; SoaresdaSilva,Viveiros,Coelho,Aguiar-Ricardo,&Casimiro,2012) Theformationofanopen-porous,interpenetratingnetworkof thesecondpolymerwasconfirmedbytreatingselectedBC/PMMA hybrid aerogels with the cellulose solvent 1-ethyl-3-methyl-imidazoliumacetate(EMIMacetate).EvenatahighPMMA/BCratio

ofabout8,representingoneoftheleastfavorablecasewithregard

toeasinessofcellulosedissolutioncellulosewasextractedbythe ionicliquidat50◦C,leavingbehindorganogelswhichwerelargely transparentpriortodryingandwhosemorphologiescorresponded

tothoseoftherespectivecomposites(Fig.4).ATR-IRanalysisof theextractedsubstanceconfirmedtheextractionofpurecellulose (morethan90%oftheamountofcelluloseoriginallypresentinthe compositeaerogel)duringthisprocess(FigureS1,supplementary data)

3.3 Responsetowardscompressivestress Theanisotropicresponseofbacterialcellulosetowards mechan-ical stress is an issue that must be considered in both characterizationandutilizationofBC-basedmaterials.Static cul-tivationofBC-producingbacteriafor3–4weekstypicallyaffords

a bacterial cellulose fleece of a few centimeters in thickness However,asallBCproducingbacteriastrainsareaerobic microor-ganisms, the release of cellulosic elementary fibrils and their aggregationtoribbonsoccursonlyincloseproximitytothephase boundarybetweenculturemediumandair.Itisknownthatthe densityofBCfilmsthusvariesintherangeofmicrometersfromtop

tobottom(Bodin,Bäckdahletal.,2007).Overtime,gravitypulls thethickeningcellulose fleece deeperintothe culturemedium whichisassumedtohappeninmicrosteps.Asaresult,a trans-verselyisotropicbacterialcellulosenetwork isproduced, which features a higher density and degreeof entanglements parallel

totheliquid/airphaseboundarythaningrowthdirection.Even thoughthelesspronouncedentanglementanddensityofBC ribb-onsingrowthdirectionisnotdirectlyevidentfromrespectiveSEM

orESEMpictures,itcanbeindirectlyvisualizedbyscanning elec-tronmicroscopyoffreeze-driedmaterial(Fig.5A)orpolarization microscopy offrozen BCsamples (Fig.5B).During freezingthe expandingwaterpushesthecellulosenetworkalongitsweaker directionapart,formingalternatinglayersofcompactedandless compactedcelluloseribbons.Dependingonthefreezingconditions thedistancebetweenthose compactedcelluloselayersisinthe lowermicrometerrange(3–10␮m)whichisinaccordancewith theliterature(Bodin,Bäckdahletal.,2007)

The significantly lower stiffness and strength of BC sheets

in thedirection ofgrowth is alsoevident from theanisotropic

Fig 2.SEM picture of an unmodified BC aerogel at 10.000× magnification (A) and its void size distribution as analyzed by thermoporosimetry using o-xylene as confined solvent (B; inset: Thermogram of a deep-frozen BC aerogel soaked with o-xylene).

response of BCaerogels towardscompressive stress.The latter

wereobtainedbyscCO2drying(40◦C,10MPa,3h)ofrespective

BCsamplesafterreplacingtheinterstitialwaterbyethanol.While

aYoung’smodulusofE=0.057±0.007MPaandyieldstrengthof

RP,0.2=4.65±0.48kPa wasobserved alongthe growthdirection,

therespectivevaluesfortheothertwospatialdirectionswere

sig-nificantlyhigher(A:E=0.149±0.023MPa,RP,0.2=7.05±0.55kPa;

B: E=0.140±0.036MPa, RP,0.2=7.84±1.06kPa) Because of this

anisotropy,mechanicaltestingofallpreparedBCcomposite

aero-gelswasperformedbyapplyingtherespectivecompressivestress

along one of its stronger directions, which can be

unambigu-ouslyidentifiedbythelongedgesofthesuppliedBCsamples(ca

120×20×20mm),whichdonotrepresentthegrowthdirection

The mechanical response profiles of BC/PLA, BC/CA and

BC/PMMA composite aerogels towards compressive stress are

largelysimilartothoseofaerogelspreparedfrompureBC,atleast

uptoapolymer/BCratioofabout4(Fig.6A–C;forfullresponse

curvesofthoseBCaerogelsthatwerereinforcedwiththe high-est amounts of PLA (A), PMMA (B) and CA (C)see Figure S2) Theyarecharacterizedbyanadjustmentphase(≤3–5%strain),in whichsample irregularitiesareevenedout,followedbya com-parativelynarrowrangeoflinearelasticdeformation(<10%).The most eye-catching feature however is the pronounced plateau region(15–40%),causedbyplasticdeformationthroughcell col-lapsingandeventuallyfollowedbyanexponentialincreaseofstress overstrainduetomaterialdensification.Similartoaerogelsfrom regeneratedcellulose(Liebner,Dunareanu etal.,2012)–andin contrasttobrittlefoamsandsilicaaerogels–allcompositeaerogels deformedinaductilewayonthemicroscale

WhilethestiffnessoftheBC/CAandBC/PMMAaerogels(Fig.6B andC)significantlyincreasedwitheachascendingconcentration level,thesameeffectwasobservedforBC/PLAonlyforthetwo highestconcentrations(Fig.6A).Themostregularreinforcingeffect was observed for theBC/CA composites, where the Emodulus

Table 1

Density and mechanical properties (n = 3) under uniaxial compression (rCA samples for comparison: Aerogels obtained by cellulose coagulation from Ca(SCN) 2 ·8H 2 O solution,

CA loading from acetone and subsequent scCO 2 anti-solvent precipitation and drying).

Fig 3. Scanning electron images of reinforced BC aerogels at 10.000× magnification Numbers on the left side are referring to the concentration of the loading bath in mg

mL−1.

increasedlinearlyuptoaCA/BCratioof8(CA80;Fig.6D)which

isingoodagreementwithoneofourpreviousstudies(Liebner,

Aigneretal.,2012)

Thequotient of Young’s modulus and bulk density (specific

modulusE␳ isaconvenientparametertocomparethestiffnessof

materialsofvaryingdensity.ForpureBCaerogelsitwascalculated

tobe19(ethanol),21(THF)and25MPacm3g−1(acetone;Table1)

whichisremarkablyhighcomparedtootherporousmaterials.For

apolyurethanefoamofadensityof90mgcm−3,whichis

compara-bletocompositeswithapolymer/BCratioof8,aspecificmodulus

of7.8MPacm3g−1hasbeenpreviouslyreported(Patel,Shepherd,

&Hukins,2008)whilethatoftherespectiveBC/PMMAcomposite

aerogelwas122MPacm3g−1

ComparedtopureBCaerogels,thehighestgaininspecific mod-uluswasachievedforPMMA80(4.8-fold)andPMMA120(5.5-fold) ForBC/PLAaerogelsthespecificmodulusfellbelowthatofthepure

BCaerogel(increaseindensitywithoutareinforcingeffect)and exceededitonlyaftertheinterpenetratingnetworkhadbeenfully developedwithintheBCnetwork(2.8-foldforPLA120)

Reinforcementwithcelluloseacetatewastheonlyvariantthat affordedproductswithE␳valuesincreasingnearlylinearly, start-ingalready fromthe lowestloading(CA10) Thisindicatesthat

atcontentsofthesecondaryconstituent,comparabletothemass

ofthelowdensityBCstructureitself,CAhasthehighest suppor-tingfunctionamongtheappliedpolymers,promotingadhesionof

BCfibriljunctionsalreadyatlowconcentrations(Table1).Atthe

Fig 4.BC/PMMA80 organogel during extraction of BC with an ionic liquid, containing regions of varying amounts of residual BC (opaque) SEM pictures: morphology of a BC/PMMA80 aerogel (A) and of an aerogel as obtained from (A) after extraction of BC by EMIM acetate.

Fig 5.SEM picture of freeze-dried BC (A) and cross-polarization micrograph of a frozen BC sample (B) The white arrows indicate the direction of BC growth.

Fig 6. Mechanical response profiles towards compressive stress for BC aerogels reinforced with PLA (A), PMMA (B) and CA (C) Grey areas indicate standard deviations (D) Correlation between Young’s modulus and bulk density of BC/CA composites (triangles: CA0-80; circles: values from Liebner, Aigner, Schimper, Potthast, & Rosenau, 2012 ) and silica aerogels (diamonds: values from Alaoui, Woignier, Scherer, & Phalippou, 2008 ).

Table 2

Influence of aerogel composition on specific surface area and

surface-area-to-volume ratio.

highestloadingleveltheobtainedcompositeaerogels(CA120)

fea-tureda3.2-foldmultiplicationof specificmoduluscomparedto

pureBC

Comparedtosilicaaerogelswhicharealreadycommercialized

forhigh-performancethermalinsulation(Nanogel®,Spaceloft®),

theE-moduleofBC/CAandBC/PMMAcomposite aerogelsisnot

onlysignificantlyhigheratcomparabledensity,butalsoresponds

muchstrongertochangesindensity(Fig.6D).Thespecificmoduliof

lowdensitysilicaaerogelsareclearlyoutmatchedbytherespective

BC/CAcompositeaerogels.WhileE␳ofBC/CAcompositeaerogels

equaled50MPa cm3g−1 ata bulkdensityof84.2mgcm−3,that

ofa comparablesilicaaerogelwasabout4MPacm3g−1 (Alaoui,

Woignier,Scherer,&Phalippou,2008)

Theapplicabilityoftheinvestigatedreinforcementstrategyfor

BC aerogels was also tested for aerogels obtained by

coagula-tionofcellulosefromsolutionstate,basicallyfollowingamethod

describedelsewhere(Hoepfner,Ratke,&Milow,2008).Inbrief,a

smallamountofcottonlinterswasdissolvedincalciumthiocyanate

octahydrateat140◦Caffordinga1wt%cellulosesolution

Coagula-tionofcellulose(‘regeneration’)wasthenaccomplishedbyaddition

ofethanol.Followingexhaustivesaltleechingwithwaterand

sol-ventexchangetoacetone,thereinforcingprocesswascarriedout

asdescribedforBCsamplesCA40andCA80.Accordingly,the

sam-pleswerelabeledrCA40andrCA80.SEManalyzesrevealedthat,

apartfromthehigherdensityofthecellulosemesh,theresulting

compositesfeaturedopenporousmorphologiessimilartotheirBC

counterparts.ComparedtotheBC/CAaerogels,thespecificmoduli

E␳oftheCA-reinforcedaerogelsfromregeneratedcellulosewere

foundtobesignificantlyhigher.WhileE␳ wastwiceashighfor

rCA40,thenextloadinglevel (rCA80)afforded materialswhose

specificmoduliexceededthatoftherespectiveCA80samplesby

afactorofthree(Table1)

3.4 Porecharacteristics

Aspreviouslydiscussed,bacterialcelluloseisamaterialof

hier-archicalarchitecturecomprisingmicro-,meso-andmacropores

Accordingtothermoporosimetrymeasurements(datanotshown)

thepeaksizedistributionpeaksintherangeofsmallmacropores

(ca.80–100nm).Nitrogensorptionexperimentsat77Kconfirmed

thattheformationofasecondarypolymernetworkaffords

materi-alsoflowerspecificsurfacearea(SSA),comparedtothespecific

surface area of BC aerogels (77m2g−1, see Table 2; calculated

fromthedesorptionbranchof theisotherm) Asthisis

primar-ilyduethehigherdensityofthecompositeaerogels,thesurface

areawasrelatedtosamplevolume(surface-area-to-volumeratio;

SAV), rather thantomass It wasevidentthat, withthe

excep-tionofPMMA20andPMMA40,allBCcompositeaerogelsfeatured

significantly higher SAV values (1.1–6.0×106m2m−3)than the

BCaerogels(0.7×106m2m−3).ForBC/PLA,BC/CA,andBC/PMMA

aerogelsthehighestSAVvalueswereobtainedforthe120mgmL−1

loading concentration level In general, the far-reaching

main-tenance or even enhancement of the surface-area-to-volume

ratio confirms the preservation of the aerogel’s open-porous

morphologythroughouttheanti-solventprecipitationanddrying procedures

4 Conclusions

LoadingofPLA,CA,andPMMAfromsolutionsinTHFor ace-toneontobacterialcellulose,followedbyanti-solventprecipitation

oftherespectivepolymerinsidethehighlyporousBCorganogels andsubsequentscCO2 dryinghavebeendemonstratedtoafford

BCcompositeaerogelsofsignificantlyenhancedmechanical resis-tancetowardscompressivestressatfar-reachingpreservationof theopen-porousBCmorphology

TheuseofBC(oraerogelsfromregeneratedcellulose)as tem-poraryscaffold forthecreationofporousPMMA aerogels,with morphologiesresembling theguiding host network,as demon-stratedinthiswork,isaninterestingapproachwhichwillbefurther followedinfuturestudies

Acknowledgements

The financial support by the Austrian Science Fund (FWF: I848-N17),theFrenchL’AgenceNationaledelaRecherché (ANR-11-IS08-0002),andtheAustrianAgencyforInternationalCooperation

in Education and Research (OeAD: FR10/2010) is thankfully acknowledged

Appendix A Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.carbpol.2014.04.029

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