Turning biomass into functional composite materials: rice husk for fully renewable immobilized biocatalysts

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ContentslistsavailableatScienceDirect

EFB

Bioeconomy

Journal

journalhomepage:www.elsevier.com/locate/bioeco

Turning

biomass

into

functional

composite

materials:

Rice

husk

for

fully

renewable

immobilized

biocatalysts

Mariachiara

Spennato

a

_,

_Anamaria

_Todea

a

_,

_Livia

_Corici

a,#

_,

_Fioretta

_Asaro

a

_,

_Nicola

_Cefarin

b

_,

Gilda

Savonitto

c

_,

_Caterina

_Deganutti

a

_,

_Lucia

_Gardossi

a,∗

a Department of Chemical and Pharmaceutical Sciences, University of Trieste, Via L. Giorgieri 1, Trieste 34127, Italy b Elettra-Sincrotrone Trieste SCpA, SS 14, km 163.5, Basovizza, Trieste TS 34149, Italy

c Department of Life Sciences, University of Trieste, Via L. Giorgieri 10, Trieste 34127, Italy

a

r

t

i

c

l

e

i

n

f

o

Keywords:

Biocatalysis for bioeconomy Covalent immobilization of lipases Rice husk

Renewable immobilization carriers Laccase oxidation of cellulose

a

b

s

t

r

a

c

t

Ricehuskisanunderexploited,lowdensityandhighlyrobustcompositematerial,massivelyavailablefromrice processing.Herewereporttwonewproceduresfortheformulationofimmobilizedlipasesapplicableinfats andoilstransformations.Theenzymeswerecovalentlyanchoredonaldehydegroupsintroducedonricehusk bylaccase-catalysedoxidationofthecellulosecomponent.Themethodavoidstheuseoftoxicglutaraldehyde whileallowsfortheapplicationandrecyclingofthebiocatalystsinaqueousmedia.Thesecondmethoduseda ﬂuidizedbedgranulatorforthecoatingoftheparticlesofricehusk(200–400𝜇m)inthepresenceofwater-soluble binders.Theformulationsaremechanicallystableandsuitableforapplicationsindiﬀerenthydrophobicmedia. Bothmethodsallowfortherecoveryandreuseofthericehuskattheendofthelifecycleofthebiocatalysts.

1. Introduction

Whenconsidering thelargest scaleapplicationsofenzymesin in-dustry, they include glucose isomerase for production of high fruc-tosesyrup(107 _ton/y),_{𝛽-galactosidases}_for_lactose_hydrolysis_in_milk (105_ton/y),_and_lipase_for_{transesteriﬁcation}_of_food_oil₍₁₀5_ton/y). Alltheseprocessesareembeddedinbioeconomyvaluechainsand uti-lizeimmobilizedenzymes(DiCosimoetal.,2013).Animmobilized in-solubleenzymaticformulationnotonlyenablestheirreusability,thus loweringproductioncosts,butalsoreduces wastestreamgeneration (Girellietal.,2020).Inthelastyears,wehavetackledthechallenge ofmakingbiocatalysismoresustainableandsuitableforthelarge-scale processesofbioeconomybydevelopingimmobilizedbiocatalyststhat exploitresidualbiomassfromagriculturalproducts.Achievingboththe environmentalandeconomicsustainabilityofimmobilizedenzymesis quitechallenging,sincethe“readytouse” carriers,suchasfossil-based methacrylicresins,arequiteexpensiveandalsoresponsibleof green-housegasemission(Kimetal.,2009).Finally,thestabilityoforganic fossil-basedcarrierswasreportedunsatisfactoryunderchemicaland me-chanicalstress(Hilterhausetal.,2008;Pellisetal.,2015;Koruppetal., 2010).

Considering that for bulk products the allowable cost contribu-tion of immobilized enzymes should be around 0.05 € kgproduct−1

∗_{Corresponding}_author.

E-mailaddress:gardossi@units.it(L.Gardossi).

#_Present_Addresses:_“Coriolan_Dr_ăgulescu_{” Institute}_of_Chemistry,_Mihai_Viteazul_24,₃₀₀₂₂₃_Timi_șoara,_Romania

(Tufvessonetal., 2011),ricehusks(RH)attractedourattentionasa potentialrenewablecarrierforenzymeimmobilization.This lignocel-lulosic material is the second most abundant biomassglobally, pro-duced in around 120 Mt per year, of which only 20 Mt are used (Tucketal.,2012).RHisanextremelyrobustcompositematerialmade ofSiO2,lignin,hemicelluloseandcellulose,(Coricietal.,2016)which provides aversatilechemicalplatformforintroducing functionalities (Raynaud2014).Italsomeetscircularitycriteriabecauseitcanbe re-utilizedattheendofitsproposedindustrialapplication(Contrerasetal., 2012).Finally,asnaturalmaterial,ithasthesubsidiaryadvantagethat itissubjectedtolessstringentlegislativeconstraintsevenafterchemical modiﬁcation(Raynaud2014).Thetubularstructureoftracheidsconfers toRHaverylowdensity(<0.4gmL−1_),_while_SiO

2isresponsiblefor itsremarkablemechanicalrobustness.

Our previous studies reported the chemical oxidative cleavage (NaIO4)ofthecellulosepresentinRHfortheintroductionofcarbonyl functionalities,whichwereexploitedforthebindingofenzymesinthe presenceof glutaraldehydeascrosslinking agent(Corici etal.,2016; Cespuglietal.,2018;Pellisetal.,2017).

Applicationsoflipasesinanaqueousenvironmentrequirethe cova-lentanchoringoftheenzymeonthecarriersforpreventingthe detach-mentoftheproteinanditspartitionin themedium(Biermannetal. 2000,SchmidandVerger1998).Despitethewidenumberofstudies presentintheliterature(Hanefeldetal.,2009),veryfewexamplesof

co-https://doi.org/10.1016/j.bioeco.2021.100008

Received23March2021;Receivedinrevisedform26May2021;Accepted26May2021

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valentbindingonsolidsupportswithsatisfactoryandclearlystated im-mobilizationyieldshavebeenreportedsofarbecauseoftheirstructural andconformationalfeatures(Hilterhaus etal., 2008;Cantoneet al., 2013).Wehavepreviouslyreported(Cespuglietal.,2018)thatCaLB canbeimmobilizedcovalentlyonchemicallyoxidizedRHafterthe in-troductionofahexamethylenediaminespacer(HMDA)andemploying glutaraldehydeasbi-functionalagentabletoformiminebondswiththe spacerandtheaminegroupsoflysineresiduespresentonthesurfaceof thelipase.ThetoxicityofGAiswidelydocumentedalthoughitisstill usedinfoodindustryaccordingtostrictlydefinedofficialregulations. Theadversehealtheffectsonhumansincludesensitizationofskinand respiratoryorgansandclosed-systemsarerecommendedtoprevent haz-ardsfromGAexposureinindustrialplants(TakigawaandEndo2006; ToxicologicalProfile,2015;EFSA,2011)

Thepresentstudyillustratestwosustainableandscalablemethods thatdemonstratehowitispossibletoexploitRHasimmobilization car-rierwhileovercomingtheuseoftoxicreagents,suchasNaIO4and glu-taraldehyde.Theﬁrstapproachexploitslaccaseenzymesforthe oxida-tionofthecellulosecomponentofRHandtheintroductionofaldehyde groupsusableforthedirectcovalentanchoringofproteins.Themethod wasvalidatedwithlipaseBfrom Candida antarctica (CaLB)andlipase fromThermomyces lanuginosus (TLL),appliedinthehydrolysisof triglyc-eridesinaqueousmedia.Thesecondimmobilizationmethodemploysa ﬂuidizedbedgranulatorforthecoatingofRHwithTLLinthepresence ofwater-solublebindersandallowsfortheinexpensiveimmobilization oflipasestobeusedinlow-watermedia.

2. Materials and methods 2.1. Chemicals

LipaseB from Candida antarctica (EC3.1.1.3,CASnumber9001– 62–1,liquidsolution,activity:1372TBU g −1 _(TBU₌_enzymatic_Units calculatedwithtributyrinhydrolysis),proteincontent=84mgmL−1₎ andLaccaseNovozym51,003(EC1.10.3.2.,CASnumber80,498–15– 3)from Myceliophthora thermophila expressedin Aspergillus sp . (activ-ity: 64 U_ABTS mL−1₎ _were _from _Novozymes_(Denmark). _Lipase _from Thermomyces lanuginosus (speciﬁc activity: 3176 TBU g −1_, _liquid so-lution, protein content = 38 mg mL−1₎ _{maltodextrin,} _{Kollidon®25,} 2,2,6,6-tetramethylpiperidin-1-yl)oxy (TEMPO), NaOH, NaIO4, Brad-ford reagent, tributyrin and 2,2′ -Azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)diammoniumsalt(ABTS)werefrom Sigma-Aldrich. Li-pasefrom Ryzopus oryzae (powder,activity:29,496U g −1₎_was_kindly donatedbyAmanoCorporation(Japan).LaccaseC(EC1.10.3.2)from Trametes versicolor (2176 UABTS g −1) was from ASA Spezialenzyme GmbH(Germany).PEG3000wasfromMerck.Arabicgumwasfrom FlukaTM_._{Hydroxyethylcellulose}_was_from_Esperis_S.p.A._(Italy)._The_RH waskindlydonatedbyRiseriaCusaro,Binasco(Italy),milledandsieved aspreviouslyreported(Cespuglietal.,2018).Allenzymaticspeciﬁc ac-tivitiesandproteincontentreportedaboveweredetermined indepen-dently.

2.2. Chemical oxidation of rice husk

ThechemicaloxidationofRHwasperformedaspreviouslydescribed byCespuglietal.(2018)

2.3. Assays for enzymes activity

ActivityoflaccaseswasevaluatedusingtheABTSmethodreported byPiscitellietal.(2005)(seesupplementarymaterialsmethodS1).

Theactivityoflipases(hydrolysisoftributyrin)wasassayedas pre-viouslydescribedbyCespuglietal.(2018),Martinsetal.(2013b).

2.4. Enzymatic oxidation of RH

Protocolat20°C:a10mMsolutionofTEMPOwasaddedto0.2gof RHpreviouslywashedwith10mLsodiumcitratebuffer0.1MpH5.The laccaseandthephosphatebufferwereaddedtoobtainafinal concen-trationof8or40UmL−1_in₂₅_mL_total._The_protocol_at₇₀_°C_employed sodiumcitratebuffer0.1MpH5usingafinallaccaseconcentrationof 8UmL−1._The_reaction_mixtures_were_magnetically_stirred₍₃₅₀_rpm) andairwasinsufflatedfor2minevery8htoincrease theexposure oftheenzymetooxygen.Attheendofthereaction,themixturewas filtered andwashedwithdistilledwater(15mL,4times).The recy-clabilityofthelaccasewasdemonstratedasreportedinESI(Electronic SupplementaryInformation,methodS2).

2.5. Immobilization of lipases on oxidized RH

The lipases were immobilized according to the protocol of Cespuglietal.(2018) modifiedasfollows:on0.2gofoxidizedRHby adding1mLofsolutioncontaining2000U(TBU)ofeachcommercial lipase,dilutedinphosphatebuffer(0.5MatpH8)andaddingPEG-3000 (0.5mLofa2mgmL-1_solution_in_phosphate_buffer_0.5_M_at_pH₈₎_as stabilizer.

2.6. Determination of carbonyl and carboxylic groups content

Thecontentofcarbonylandcarboxylicgroupsweredeterminedas previouslydescribedbyCespuglietal.(2018).Thedetailsareincluded intheESImaterialasmethodS3andS4

2.7. Physical immobilization of lipase TLL on RH

The immobilization of TLL on 100 g of RH in the presence of polyvinylpyrrolidone (Kollidon®25), maltodextrin and hydroxyethyl cellulosewascarriedoutinafluidbedgranulatorMini-Glattfluidized bed(GlattGMbH,Binzen,Germany)equippedwithaconicalvessel (vol-umeof0.75L),threemetallicfiltersandatimingfilterblowing.The immobilizationwasperformedbyadaptingaprocedurepreviously de-scribedby Trastulloetal. (2015) anddetailsareavailable inESI as methodS5.

2.8. Energy dispersive X-ray spectroscopy (EDS)

Non-metallizedsampleswereinvestigatedwithZeissSupra40 high-resolution Field Emission Gun (FEG) Scanning Electron Microscope (SEM). Imageswereobtained bycollecting secondaryelectronswith theelectronhightension(EHT)equalto10keVthankstoan Everhart-Thornleydetector.ThemicroscopewasequippedwithanOxfordAztec energydispersiveX-rayspectroscopy(EDS)systemandanX-act10mm silicondriftdetector(SDD)forcompositionalanalysis.Thesameenergy of10keVwasexploitedtocollectthesiliconsignalonthesample (Fig-ureS1).

2.9. SEM microscopy

SamplesweremetallizedwiththeS150ASputterCoaterinstrument (EdwardsHighVacuum,Crawley,WestSussex,UK)beforebeing ob-servedwiththeLeicaStereoscan430iscanningelectronmicroscope (Le-icaCambridgeLtd.,Cambridge,UK)integratedwithanSidetector(Li) PENTAFETPLUSTM,withanATWTMwindow(OxfordInstruments, Oxfordshire,England)formicroanalysis.

2.10. Stereoscopic microscopy

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Fig.1.Schematiccomparisonofthechemicaland enzymatic mediated oxidation of cellulose. The NaIO4 causes theoxidativecleavage ofthe

glu-cosewiththeformationoftwoaldehydegroups. Thelaccase/TEMPOsystemoxidizestheprimary hydroxylgrouptoanaldehyde,whichundergoes spontaneousoxidationtocarboxylgroup.

2.11. Synthesis of butyl butyrate catalyzed by immobilized TLL in hexane and toluene

TheimmobilizedTLL(160mg)wereincubatedfor1hat48°Cin hexaneortoluene(6.676mL).Thereactionwasinitiatedbytheaddition of1-butanol(0,618mL)andbutyricacid(0,206mL)at3:1molarratio. Thereactionswerecarriedoutat48°C,inanorbitalshaker(250rpm) for24handmonitoredbyHPLCasreportedinESI(methodS6).Control reactionswithoutenzymewereperformedunderidenticalconditions. Alltheexperimentsweremadeinduplicateandthemeanvalueswere considered.

2.12. Synthesis of butyl butyrate catalyzed by physically immobilized TLL in isooctane

1-butanol(0.25mL)andbutyricacid(0.25mL)(1:1 molarratio) wereaddedto9.5mlofisooctaneand130mgimmobilizedTLL.The reactionwasincubatedat45°Cinanorbitalshaker(250rpm)for21h andmonitoredasreportedinESI(methodS7).Alltheexperimentswere madeinduplicateandthemeanvalueswereconsidered.

3. Results and discussion

3.1. Enzymatic functionalization of RH

Thecovalentbindingofproteinsonanycarrierrequiresthe intro-ductionoffunctionalgroupsabletoformsuitablebondswiththeside chainsofaminoacidspresentontheproteinsurface.Wehave previ-ouslyreportedthefunctionalizationofRHthroughanoxidative cleav-ageofthecellulosiccomponentbymeansofNaIO₄(Coricietal.,2016; Cespugli etal., 2018).To improvethe sustainability andscalability oftheoxidativeprocess,aprotocolwasdevelopedinwhichthetoxic NaIO₄ was replaced bylaccase enzymes,copper-oxidases presentin variousorganismslikebacteria,plants,insectsandfungi(Claus2004). Bigmoleculesandnon-phenoliccompoundsareoxidizedwithdiﬃculty bylaccasesduetothereducedaccessibilityoftheactivesiteandalso becauseoftheir lowredoxpotential (Bourbonnaisetal.1995).This drawbackcanbe overcome withtheuseof a laccase-mediator system (LMS),namelybyaddingasmallmoleculeabletogeneratestable rad-icalspecies(CañasandCamarero2010).Inourworkwehavetaken inspirationfromtheworkofPatelandco-workersthatdescribesthe ox-idationoftheC6hydroxylofglucoseinthecellulosechaincatalyzedby laccaseinthepresenceofTEMPO-radical(Pateletal.,2011)(Fig.1b)

Inthepresentstudy,milledRHparticles(0.2–0.4mm)wereoxidized bylaccasefromTrametes sp.andlaccasefromMyceliophthora thermophile andthecontentof carbonylgroupswas assessedusing the hydroxy-laminehydrochlorideassay(Cespuglietal.,2018;Guigoetal.,2014). DatainFig.2aindicatethatlaccasefromTrametes sp.wasmoreeﬀective inoxidizingtheRHandthattheTEMPOmediatorwasnecessary.The amountoftheresultingcarbonylgroupsdependsonthelaccaseunits, withthehighestconcentrationachievedinthepresenceof40UmL−1 oflaccasefromTrametes sp.

Todecreasetheamountoflaccaseintheoxidationprocess,alarger amountofRHwasusedinthereactionmixturewhilemaintainingthe sameconcentrationofenzyme(Fig.2b).Thetemperaturewasalso in-creasedto70°Candtherecyclabilityofthelaccaseforfurther oxida-tivecycleswasdemonstrated.After48hofenzymatictreatment,the oxidizedRHwasremovedbyfiltrationandsomefreshrice-huskwas addedintheenzymaticsolution.Fig.2bshowsthatwithoutthe addi-tionofTEMPOtheconcentrationofcarbonylgroupsstillincreasedafter 24h,indicatingaresidualpresenceofunreactedTEMPOderivingfrom thefirstcycle.WhenTEMPOwasaddedtotherecycledenzyme,the carbonylgroupsincreasedby166%in24hand177%in48h.Overall, thedatasuggestthattheTEMPOconcentrationcanbereducedinboth cyclesandthattheoxidationoccursmuch fasterin thesecond cycle (terminated in24h),thusdemonstratingtherecyclabilityof the lac-case.Theobservedhigheractivityinthesecondcyclecouldbeascribed toactivationphenomenaoccurringuponexposuretohightemperature ordenaturingfactors,asreportedinourstudyonlaccasefromSteccher- inum ochraceum 1833.Bymeansofmoleculardynamicssimulationswe disclosedminorsuperficialconformationalmodificationsofthelaccase thatimproveitsactivitywithoutcausingsevereunfoldingnorthe dis-ruptionoftheelectrontransferpathway(ETP)(Ferrarioetal.,2015).

Currentdataonthepreservationoftheactivityofthelaccasefrom Trametes sp.during96hofreactionat70°Csuggestthattheenzymatic oxidativefunctionalizationofRHcanbefurtheroptimizedinorderto maximizethenumberofcyclesandminimizeofeconomicimpactof thetreatment.TheTEMPOmediatorseems toplayamajorroleand althoughitsconcentrationcanbereduced,itsenvironmentaland eco-nomicimpactdeservesforfurtherattention.Futurestudieswillbe fo-cusedonthereplacementofTEMPOwithrenewableandinexpensive mediatormolecules,whichhavebeenprovedeﬀectiveinprevious stud-ies(CañasandCamarero2010;Medinaetal.,2013)

Inallcases,thecontentofcarbonylgroupsmeasuredafterthe en-zymaticLMSmethod(Fig.2a)resultedtobeconsiderablylowerthan the1mmol g −1_obtained_with_0.2_M_NaIO

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Fig.2. (a)Concentrationofcarbonylgroupsupondiﬀerentoxidationtreatmentsat20°C.RH1:untreatedrisehusk;RH2:Novozyme51,00320UmL−1_;_RH3:

LaccaseC8UmL−1_;_RH4:_Laccase_C₄₀_U_mL−1_without_TEMPO;_RH5:_Laccase_C₄₀_U_mL−1_with_TEMPO._(b)_Oxidation_at₇₀_°C_and_recycling_of_laccase:_ﬁrst₄₈_h

cycleofoxidation(blackbars);laccasereusedforasecondoxidationcycleonfreshRH(graybars);recycledlaccasebutwithoutadditionofTEMPO.

Fig.3. SEMimagesillustratingtheeﬀectofNaIO4(a,c)vsenzymaticoxidation

(b,d)onthemorphologyofRH.CorrosiveeﬀectoftheNaIO4onthesurface(a)

andontheinnercellulosicmatrixoftracheid(d).

unit,whereasinthecaseofLMSoxidationonesinglecarbonylgroup perglucoseunitisformed.Moreover,SEMmicroscopyhighlightedthe corrosiveeﬀectofNaIO4,whichleadstomarkedcovalentmodiﬁcation ofthepolysaccharidebecauseoftheoxidativecleavage,openingthe ac-cesstodeeperlayersofthematerial(Fig.3),bothinsidethetracheid structures(Fig.3c)andontheoutersurface(Fig.3a).

Fig.3indicatesthepoorselectivityoftheNaIO4towardsthevarious componentsof thelignocellulosicmatrixof theRH.Onthecontrary, theenzymatictreatmentwithlaccasecausesnegligiblemorphological modiﬁcationontheexternalsurface,whileleadstoamodestincrease oftheroughnessoftheinternalsurfaceofthetracheids.

MorphologicalstudiesmakinguseofenergydispersiveX-ray spec-troscopy (EDS) were of aid in understanding the robustnessof the outersurfaceofthehuskand,moreprecisely,thelocalizationofSiO2 (Parketal.,2003;Colettaetal.,2013).UsingtheEDS(Fig.4right)the K𝛼1signalofthesilicon(ESIFigureS1)wascollected(greendots)to appreciatethepresenceSiO2onthesurfaceoftheRHandtoconﬁrmits roleinprotectingthericegrainfrommechanicalandchemicalagents.

Theformationofcarboxylicgroupsduringboththechemicaland enzymaticoxidationprocesseswerealsoevaluated(ESIFigureS2)and theyconﬁrmedthattheoxidationofaldehydestocarboxylicgroupsis notlaccasemediated.

3.2. Glutaraldehyde free covalent immobilization of lipases on RH Inthepresentstudy,we aimedatsimplifying theimmobilization protocol,byreducingthesyntheticstepswhileavoidingtheuseof glu-taraldehyde(GA).Wehaveexploredthepossibilitytoformtheimine bonddirectly betweenlysineresidues andthecarbonyl grouponC6 formed via laccaseoxidation,whichappearsmoreaccessiblethanthe di-aldehydegroupsobtainedusingNaIO4.Twomorelipasesweretested inthestudy:lipasefromThermomyces lanuginosus (TLL)andlipasefrom Rhizopus oryzae (ROL),whicharewidelyemployedinthefoodsector for thetransesteriﬁcation of oils andfats (Higashiyama andSumida 2004).Therefore,avoidingtheuse ofglutaraldehyde intheir formu-lationswouldbedesirable(Zeigeretal.,2005).

Thetri-dimensionalmodelsofthelipasesarereportedinESI (Fig-ure S3).TLLandROLundergointerfacialactivation(Ferrarioetal. 2011)whenapproachingahydrophobicphase,whereasCaLBdoesnot undergosigniﬁcantconformationalchangesanditsactivesiteis perma-nentlyaccessible(Ferrarioetal.2011,Bassoetal.,2007).Theanalysis ofthenumberandthepositionoflysineresiduespotentiallyinvolved in thecovalentbindingwiththecarrierindicatesthatCaLBandTLL have9and7lysinesrespectively,whereasROLhas15lysines,oneof themlocatedintheproximityoftheactivesite.Thatfeaturemakesthe immobilizationofROLquitechallenging,sincetheformationofthe co-valentbondcanoccludetheactivesitebutalsopreventthenecessary conformationalchangesconnectedtotheinterfacialactivation.Indeed, previousdatacollectedinourgroupshowedthatthehydrolytic activ-ityiscompletelylostuponcovalentimmobilizationofROLinaqueous buﬀeronmethacrylicepoxyresins(Gardossietal.,2012).

DatareportedinTable1 demonstratethatTLLandCaLBcanbe an-choreddirectlyontheenzymaticallyoxidizedRH,whileavoidingthe useofglutaraldehydeandspacers.Higherloadingwasobtainedinthe caseofRHoxidizedenzymatically,indicatingaregio-preferenceinthe formationofiminebondswiththemoreaccessiblealdehydegroupson C6.

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forma-Fig.4.Left:SEMimageoffragmentsofmilledRH, showingthelinearrippleswithconicalshapeonthe externalsurface.Right:2Dmapthedistributionof SiO2.

Table1

FormulationsoflipasescovalentlyimmobilizedonRH.Inallcases10.000UoflipasepergofRHwereemployed.Theactivityisexpressed astheaverageofthreemeasurements.LMS=laccasefromTrametessp.+Tempomediator.Lastthreeentrieswerepublishedinref. (Cespuglietal.,2018)andarereportedforcomparison.

Lipase Oxidation method Other functionalization Protein loaded (%) hydrolytic activity (Ug -1 dry ) immobilization yield (%)

CALB LMS no 65 590 ± 2 5.9 TLL LMS no 72 974 ± 65 9.74 ROL LMS no 53 328 ± 42 3.28 CALB NaIO 4 no 33 290 ± 45 2.90 TLL NaIO 4 no 56 643 ± 67 6.43 ROL NaIO 4 no 32 171 ± 14 1.71 CALB LMS GA 55 155 ± 5 1.55 CALB NaIO 4 GA 65 < 50 < 0,5 CaLB LMS HMDA + GA § ₁₇ _{56 ± 25} _0.56

CaLB NaIO 4 HMDA + GA § 72 316 3.16

CaLB n.a. Epoxy (on methacrylic resin) § ₉₅ ₇₀₉ _7.09

§_previously_published.

tionofunspecificcovalentbondsandcrosslinks.Whencomparedtoour previouslypublisheddataofimmobilizationofCaLBonRHchemically oxidizedandfunctionalizedwithdiaminospacerswithGA,thespecific activitywiththesimplifiedprotocolisalmostdoubled.The immobiliza-tionyieldiscomparabletothatobservedforCaLBimmobilizedonepoxy methacryliccarrier(Cespuglietal.,2018)

The immobilization yields are in line with previous experimen-taldata obtained in thecovalentimmobilizationof different lipases on epoxy methacrylic resins using aqueous bufferasimmobilization medium, which have been always below 10% (Pellis et al., 2016; Gardossietal.,2012).Thedifficultiesencounteredinthecovalent im-mobilizationoflipaseshavebeendiscussedpreviouslyandtheyare as-cribablebothtotheirconformationalandsuperficialstructuralfeatures (Cantoneetal.,2012;Ferrarioetal.,2011).Conversely,veryfewstudies reporttheactualimmobilizationyieldaccompaniedbytheevaluation oftheproteinleachingfromthesupport,asaproofoftherobust an-chorageoftheenzymeonthecarrier.Atthebestofourknowledge,no covalentlyimmobilizedlipaseiscommercializedforindustrial applica-tions,althoughthehydrolyticapplicationsoftheseenzymesrequestthis typeofformulationforenablinganefficientrecyclingofthebiocatalyst andthereductionofthecosts.Asamatteroffact,ananalysisof Tufves-sonandco-workers(Tufvessonetal.,2011)indicatedthatinthecaseof biocatalystsimmobilizedbyadsorption63%ofthematerialcostis as-cribabletothecarrierandonly37%totheenzyme.Takingintoaccount thatthecostofcarriersforcovalentimmobilizationisabout2–6times higher,itisevidentthattheeconomicviabilityofthesebiocatalystsis mainlylinkedtothepriceofthecarrierandthepreservationofthe en-zymeactivityacrossmultiplereactioncycles(i.e.productivity). There-fore,thereareseveralpotentialadvantagesderivingfromthecovalent immobilizationoflipasesonoxidizedricehusk.Firstlythecostofthe carrierisreduced;secondlytheenzymecanbeseparatedbythe hydrol-ysisproductandrecycled,thusfurtherdecreasingtheeconomicimpact ofthebiocatalyst;finally,there-useofthenon-fossilandbiodegradable

carrierattheendofthebiocatalystlifecyclewouldintegrate environ-mentalandeconomicsustainability.

3.3. Stability and recyclability of covalently immobilized lipases in aqueous media

Thestabilityof theimmobilizedlipases wasevaluated inthe hy-drolysisoftributyrin,undervigorousmechanicalstirring.Theresultsin Fig.5indicateanexcellentstabilityofCaLBonricehuskandTLLonrice huskafter10reactioncycles,withabout80%and70%ofrecovered ac-tivity,whereasROLretainsonly15%ofactivity,whichwasascribedto theleachingofROLfromRH,asexperimentallyobserved(ESI,Figure S4).Notably,allbiocatalystsreportedinTable1 weretestedfor pro-teinleachingandonlyROLformulationsshowedthedetachmentofthe proteinfromthecarrier.

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Fig.5. OperationalstabilityofCaLBonricehusk(blackbar)TLLonricehusk(graybar)andROLonricehusk(whitebar)inmultiplehydrolyticcyclesexpressed asthepercentageofretainedactivityaftereachcycle.Measurementswerecarriedoutinduplicateandreportedastheaveragevalue.

3.4. Physical immobilization of TLL in a ﬂuidized bed

Thesecondpartofthestudywasaimedbydevelopingscalable pro-ceduresfor theimmobilizationoflipases tobeapplied in low-water hydrophobicmedia,whileensuringenvironmentalandeconomic sus-tainability.Generally,theapplicationofimmobilizedenzymesin hy-drophobicmediadoesnotrequirethecovalentbindingoftheproteinto thecarrier,sincetheenzymehasaloweraﬃnityforthehydrophobic phase(Pellisetal.,2015).

LipaseTLLwas selectedforthestudyanditwas immobilizedon milled RH (200–400 μm) in a ﬂuidized bed in the presence of an aqueoussolutionofdiﬀerentbindersapprovedforpharmaceuticalsand foodapplications:Kollidon25(polyvinylpyrrolidone=PVP)(Reddyand Sharma,2020),hydroxyethylcellulose(HEC)(DiGiuseppe2018)and maltodextrin(MLDX)(Velásquez-Cocketal.,2018).Auniformlayerof enzymecoatedtheRHandtheimmobilizedbiocatalystsappearas dis-tinctparticles,withoutanyexampleofagglomerates(Fig.6).

3.5. Synthesis of butyl butyrate catalyzed by TLL physically immobilized on rice husk particles

Theefficiencyof theTLLformulations obtainedthroughphysical immobilizationwasevaluatedinthesynthesisofbutylbutyrate start-ingfrombutyricacidand1-butanol(Martinsetal.,2013a).Thisshort chainfruityester,presentinpineappleflavor(Martinsetal.,2013),is usedinperfumesandfragrances,waxes,washing,cleaning,cosmetics andpersonalcareproducts(Xinetal.,2016).Threeorganicsolvents weretested:toluene(logP2.5),hexane(logP3.5)andisooctane(log P4.6).Figs.7illustratesthetime-courseofthereactionsinhexaneand toluene,indicatingstrikingdifferencesofperformanceofthe biocata-lysts.Inhexane,PVP-TLLleadstocompleteconversioninlessthan5h, whereastheprofilesoftheothertworeactionssuggestsomelag-time beforethestartingofthereactions,possiblyascribable tosomemass transferlimitation.Conversions>93%wereachievedinallcasesandit

wasnoticedthattheabsenceofagglomeratesconfershighmechanical stabilitytotheformulations,preventingdisaggregationandproduction ofﬁnesuponstirring.

Allreactionscarriedoutintoluenewereconsiderablyslower: PVP-TLLwasthebestperformingformulationwith > 80%conversionafter 8hofreaction,whileMLDX-TLLstoppedworkingafter10%of conver-sion.

Controlreactionswereperformedintheabsenceofthebiocatalyst, usinglipasefreeparticlesofRHtreatedintheﬂuidizedbedonlywiththe binders.Moreover,blankexperimentsdemonstratedtheabsenceofany aspeciﬁcadsorptionofthesubstrateonthebiocatalystwhenincubated intoluene(ESIFigureS5).

Whenthe synthesiswas conductedin isooctane(Batistella etal., 2012),thesubstrateswereemployedinequimolaramounts,resulting inalowerconcentrationof1-butanolascomparedtotheprevious syn-thesis.Inthiswaywewantedtoverifythepossibilitytoachieve quan-titativeconversionevenwithoutusinganexcessofthepolaralcohol, whichisknowntoaﬀectnegativelythestabilityofenzymes.

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intrin-Fig.6. TLLphysicallyimmobilizedonricehuskparticlesand pre-pared using a fluidized bed reactor. Images obtained by means of stereoscopicmicroscopyat differentmagnificationvalues: PVP (a)−10x(b)−60xandHEC(a)−10x(b)−60x.

Fig.7. ReactionproﬁlesofthesynthesisofbutylbutyratesynthesiscatalyzedbyTLLphysicallyimmobilizedonricehuskparticlesinhexane(a)andtoluene(b).

sicactivityofthelipase,whereas thepartitionphenomenaappearto predominate.

3.6. Stability of physically immobilized TLL and reusability of rice husk PVP-TLLandHEC-TLLweretestedunderhydrolysisconditionsand vigorous stirringin order toevaluatethemechanical stabilityof the formulationsbutalsotomonitorthedetachmentoftheenzymefrom theRH.Thedeterminedhydrolyticactivities(TBU)were4459Ug-1_for HEC-TLLand6609Ug-1_for_PVP-TLL._Compared_to_the_previously im-mobilizedTLLontosilicagranules,thehydrolyticactivitiesobtainedin thisstudywereatleast2timeshigher(Ferreretal.,2002).Microscope

images(ESIFigureS6)demonstratethattheintegrityoftheRHisfully preserved after10 cyclesof reactionundervigorousmechanical stir-ring.Itmustbenoted,thatafter4yearsofstorageat4°CPVP-TLLand HEC-TLLpreserved96%and82%ofhydrolyticactivityrespectively.No microbialcontaminationwasobserved,indicatingthelong-term stabil-ityofthetwoformulations.

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Fig.8. SynthesisofbutylbutyratecatalyzedbythethreediﬀerentformulationsofimmobilizedTLLinisooctane(a)andisooctanewithadditionof0.1%(v/v)of H2O(b).

beingmadebyasinglerobustfragmentofcompositematerial,which doesnotundergodisaggregationortheformationofﬁnesunderphysical stress.

ConcerningthereusabilityofRH,dataconﬁrmthattheenzyme de-tachesfromRH(> 90%)afteronecycleinaqueoussolution(FigureS6) butthecarrierremainsintact(FigureS7).Therefore,attheendofthe biocatalystlifecycle,theRHcanbereutilizedeitherasimmobilization carrierorforsecondaryuses(Contrerasetal.,2012).

Inordertoperformaverypreliminaryanalysisoftheeconomic sus-tainabilityofnewvaluechainsforthevalorizationofrice,we consid-eredtheItaliancontext.Ricemillssellonlyafractionofricehuskfor amaximumpriceof90€ ton−1₍_CCIAA,₂₀₂₁_)._That_rice_husk_is_used byflorists,farmersforanimallitterandpoultryfarms.Someoftherice huskisemployedbymillsalsofortheinternalproductionofenergyand steamrequiredforparboiledriceproduction.However,theashcontent ofburntricehuskisveryhigh(about20%byweight)comparedtoother biomasssuchaspoplar(1.0%)anddealingwiththelargeamountof re-mainingashisextremelydifficult.Therefore,ricehuskscanbe sustain-ablyusedasafuelforenergyrecoveryonlywhentheirashisusedasa resource.Whenburntat700°C,ricehuskyieldsashcontains97%silica inamorphousform,whichhasmanyapplicationssuchasin construc-tionalworks,intheproductionofporcelainorasamendantinpaddy fields.(SekifujiandTateda2019).

Notably,thecostsofcommercialorganiccarriers,arearound50€ kg−1 _for_adsorption _{immobilization,}_which _means_that _they_have _no chemicalfunctionalitiesontheirsurface,buttheysimplyinteractwith theproteinbyweaknon-covalentinteractions(Tufvessonetal.,2011). Carrierspricereaches100–300€ kg−1_when_they_are_{functionalized}_for covalentimmobilization,andtheseﬁguresmakeevidentthatthereis potentialfornewusesofricehuskandthatricemillswouldﬁndmore advantageoustoselllargeramountsofricehusktoindustriesableto transformitinaddedvalueproducts,which,attheendoftheirlife cy-cle,couldbetransformedinenergyoreventuallyreturntothesoil.The latterusewouldbeparticularlyfeasibleforbiocatalystsappliedinthe foodsector,wherenontoxicsubstratesareprocessed.

Conclusions

DataherereporteddemonstratethatRHisaversatilenatural com-positematerialthatcanbeusedbothforthecovalentandphysical im-mobilizationoflipases.Thecovalentimmobilizationwasperformedby anchoringtheproteindirectlyonthefunctionalizedcarrier,withoutthe needofspacersorglutaraldehyde,leadingtostableformulationsofTLL andCaLB,retaining>70%ofactivityafter10cyclesofhydrolysis.These biocatalysts,becauseoftheirstabilityandrobustness,areapplicablein variousreactionmediaandundermechanicalstress.The

functionaliza-tionofRHwasachievedbymeansoflaccasesinthepresenceofTEMPO mediatorandthelaccasewasreusedfordiﬀerentoxidativecycles.The nextoptimizationstepswillbefocusedonthereplacementofTEMPO withrenewablemediatormolecules.

TheinexpensiveandeasilyscalableimmobilizationoflipaseTLLwas achieved usingaﬂuid-bedgranulatorandwater-solublebinders. Be-causeofthelowtoxicityofthematerialsandthepossibilityofreusing theRHattheendofthebiocatalystlifecycle,thismethodappears ap-propriateforindustrialapplicationsinlow-aqueousmediarequesting low-toxic,inexpensiveandsustainablesolutions.

Overall,themethodsherepresentedintendtoprovideacontribution tothedevelopmentofanewwaveofrenewableindustrialcarriersfor enzymeimmobilization,abletoreplacepetrol-basedmaterialsandto overcometheirnaturalcapitalcost(RaynaudJ.,2014).

Authors contribution

Conceptualization:LuciaGardossi,MariachiaraSpennato Methodology:MariachiaraSpennato,LiviaCorici

Validation:MariachiaraSpennato,AnamariaTodea,FiorettaAsaro Investigation: Mariachiara Spennato, Nicola Cefarin, Gilda Savonitto,CaterinaDeganutti

Writing -Original Draft Preparation:Mariachiara Spennato, Ana-mariaTodea,LuciaGardossi,LiviaCorici,CaterinaDeganutti

Writing-Review&Editing:MariachiaraSpennato,AnamariaTodea, LuciaGardossi,FiorettaAsaro

SupervisionLuciaGardossi FundingacquisitionLuciaGardossi Declaration of Competing Interest

Theauthorsdeclarethattheyhavenoknowncompetingﬁnancial interestsorpersonalrelationshipsthatcouldhaveappearedtoinﬂuence theworkreportedinthispaper.

Acknowledgment

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Supplementary materials

Supplementarymaterialassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bioeco.2021.100008.

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