Influence of pretreatments for extraction of lipids from yeast by using supercritical carbon dioxide and ethanol as cosolvent

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_{Hegel, Pablo Ezguiel and Camy, Séverine and}

Destrac, Philippe and Condoret, Jean-Stéphane (2011) Influence of

pretreatments for extraction of lipids from yeast by using supercritical

carbon dioxide and ethanol as cosolvent. The Journal of Supercritical

Fluids, vol. 58 (n°1). pp. 68-78. ISSN 0896-8446



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Influence

of

pretreatments

for

extraction

of

lipids

from

yeast

by

using

supercritical

carbon

dioxide

and

ethanol

as

cosolvent

P.E.

Hegel

a,b

_,

_S.

_Camy

a,b

_,

_P.

_Destrac

a,b

_,

_J.S.

_Condoret

a,b,∗

a_{UniversitédeToulouse,INPT,UPS,LaboratoiredeGénieChimique,4,AlléeEmileMonso,F-31030Toulouse,France} b_{CNRS,LaboratoiredeGénieChimique,F-31030Toulouse,France}

a

b

s

t

r

a

c

t

Saccharomycescerevisiaeisoneofthemoststudiedandindustriallyexploitedyeast.Itisanon-oleaginous yeastwhoselipidsaremainlyphospholipids.Inthiswork,theextractionofyeastlipidsbysupercritical carbondioxide(SCCO2)andethanolasaco-solventwasstudied.Inparticularourattentionwasfocused

ontheselectivitytowardtriglycerides,andinasubsequentextractionofthephospholipidspresentinthe yeast.IndeedCO2isanon-polarsolventandisnotanefficientsolventfortheextractionofphospholipids.

However,SCCO2 canbeusedtoextractneutrallipids,astriglycerides,andtheadditionofpolar

co-solventslikeethanol,atdifferentcompositions,allowsamoreefficientextractionoftriglycerides,and alsoanextraction–fractionationofphospholipids.InthisworkSCCO2extractionsofaspecificmembrane

complexofS.cerevisiae,obtainedfromanindustrialprovider,werecarriedoutat20MPaand40◦_C,using

ethanolasaco-solvent(9%,w/w).Itwasshownthatdifferentpretreatmentsarenecessarytoobtain goodextractionyieldsandhaveagreatimpactontheextraction.Thekineticoftheextractionswere successfullymodeledusingSovova’smodel.Fromthefittingofthemainparametersofthemodelitwas possibletocomparetheeffectsofthepretreatmentsovertheyeastmaterial,andtobetterunderstand theextractionprocess.Amongtheseventestedpretreatmentsthemoreappropriatewasfoundtobean acidhydrolysisfollowedbyamethanolmaceration.

1. Introduction

Microorganismshaveoftenbeenconsideredfortheproduction ofoilsandfatsasanalternativetoagriculturalandanimalresources [1].Theoleaginousyeastspeciesmayaccumulatemorethan20% lipidsbymassfractionoftheirbiomass.Themainlipidic compo-sitionofoleaginousspecies(CryptococcusalbidusandRhodotorula glutinis)aretriglycerides(TG),andinminorquantities phospho-lipids(PL)andsterols[2,3].

The extraction of lipids from yeasts with different conven-tionalorganicsolvents,suchaschloroformandmethanol,hexane and/orpetroleumether and theirapplicationeven atindustrial scalehavebeenreported[3].However,theuseoftheseorganic solventsatpilotandindustrialscalehastobesubstitutedinthe nearfuture bynon-flammable, less toxic andmore benign sol-ventsinordertoobtainsustainableprocesses[4,5].Carbondioxide (CO2)isaninert,inexpensive,easilyavailable,odorless,tasteless,

environment-friendly,and GRAS(GenerallyRecognizedAsSafe)

∗ Correspondingauthorat:UniversitédeToulouse,INPT,UPS,Laboratoirede GénieChimique,4,AlléeEmileMonso,F-31030Toulouse,France.

Tel.:+33534323713;fax:+33534323690.

E-mailaddress:[email protected](J.S.Condoret).

solvent.Thesupercritical technologyisa greensustainable pro-cess[6,7]inwhichthesolventpowerandselectivitycanbetuned accordingtotheoperatingconditions.

ExtractionoflipidsfromyeastusingSCCO2hasbeenscarcely

considered in the literature. Supercritical studies have been reported for the extraction of high added-value productsfrom yeast,suchasastaxanthinandsqualene[1,8,9].Morestudiescan befoundforSCCO2 lipidextractionfromalgaematerial[10].On

the otherhand, few Refs.[1,11] can be foundin theliterature aboutthedetailedextractionoflipidsfromyeastswithCO2 and

co-solventsandthekineticanalysisoftheprocess,especially tak-ingintoaccounttheyeastpretreatment,whichmayhaveamarked influenceontheextractionefficiency.Nevertheless,topalliatethis scarceknowledge,itcouldbeofinterest,byanalogy,toconsider worksaboutoilextractionfromseeds.TheuseofSCCO2 and

co-solventsfor theselectiveextraction ofTGhasbeenstudied,for example,fortheextraction ofcanola,soybeanlecithinand sun-floweroil[12–17].CoceroandCalvo[13]havereportedavalueof sunfloweroilsolubilityinCO2around3.5g/kgat20MPaand42◦C,

valuewhichincreasesuptonearly25g/kgwhen10%ofethanol bymassfractionisusedasaco-solvent.Also,theadditionofpolar co-solventsmayenhancethesolubilityofPLwhicharenormally insolubleinpureCO2[13,14].However,DunfordandTemelli[14]

Nomenclature

a0 specificsurfaceareaperunitvolumeofextraction

bed(m−1₎

as specificareabetweentheregionsofintactand

bro-kenmembranes(m−1₎

dp particlediameter(m)

DL axialdispersioncoefficient(m2/s)

D12 binarydiffusioncoefficient(m2/s)

e extractionyield(lipids/(TG-freeyeast)kg/kg) jf flux from broken cells or membranes to solvent

(kg/m3_s)

js fluxfromintactcellsormembranestobrokencells

(kg/m3_s)

kf fluid-phasemasstransfercoefficient(m/s)

ks solid-phasemasstransfercoefficient(m/s)

K partitioncoefficient(TG-freeyeast/solvent,kg/kg) L extractionbedlength(m)

P pressure(MPa) Pe Pecletnumber PL phospholipids

q relativeamountofthepassedsolvent(CO2/TG-free

yeast,kg/kg)

r grindingefficiency(fractionofbrokenmembranes ornon-boundlipids)

t extractiontime(s) T temperature(◦_C)

TG triglycerides

U interstitialfluidvelocity(m/s)

xu concentrationintheuntreatedsolid(lipids/TG-free

yeast,kg/kg)

xt transition concentration (lipids/TG-free yeast,

kg/kg)

x1 concentrationinbrokencells(lipids/TG-freeyeast,

kg/kg)

x10 initialconcentrationinbrokencells(lipids/TG-free

yeast,kg/kg)

x2 concentrationin intactcells (lipids/TG-freeyeast,

kg/kg)

x20 initialconcentrationinintactcells (lipids/TG-free

yeast,kg/kg)

y fluid-phaseconcentration(lipids/solvent,kg/kg) y0 initial fluid-phase concentration (lipids/solvent,

kg/kg)

ys solubility(lipids/solvent,kg/kg)

y*(x1) equilibrium fluid-phase concentration

(lipids/solvent,kg/kg) z axialco-ordinate(m) ε bedvoidfraction f solventdensity(kg/m3)

s soliddensity(TG-freeyeast/solidphase,kg/m3)

 viscosity(Pas)

oilfromcanolaflakeswasnotaffectedbytheadditionofethanol (8%bymassfraction)evenat55.2MPaand70◦_{C.Theseauthors}

reportedtheextractionofanincreasingamountofPLwhencanola flakesofreducedoilcontentwereextractedatthesameoperating conditions.Teberikleretal.[15]reportedselectivitytowards phos-phatidylcholineat20MPaand 60◦_{Cwithalowthermodynamic}

solubilityvalues,around0.15g/kgintheSCCO2,with10%(w/w)of

ethanolasaco-solvent.

TheobjectiveofthisworkisrelatedtotheSCCO2extractionof

TGfromyeastmaterial.Theextractionconditionswereselected accordingtopreviousworksat20MPaand40◦_{C,using9%(w/w)}

ofethanolasaco-solventinordertoobtainsolventpowerand selectivitytowardsTGextraction. Therawmaterialusedin this workwasayeastmembranecomplexofhighlipidiccontent, pro-videdbyamajorEuropeanindustrialyeastprovider.Thematerial wassubmittedtodifferentpretreatmentsinordertoincreaseyield andselectivity.Modelingoftheextractionkineticswasdoneusing theSovova’smathematicalmodelofbrokenandintactcells(BIC), veryoftenusedintheliterature.Whenappliedtofitthe experi-mentalcumulativeextractioncurves,itsapplicationtoassessthe SCCO2+ethanol lipidextraction provedtobeusefulforabetter

understandingofthepretreatmentsefficiency. 2. Materialsandmethods

2.1. Yeasts

Aspecificmaterialtermed“driedmembranecomplexof Sac-charomycescerevisiae”(DMCS)ofhighlipidcontentandprovided byLesaffreGroup® _{hasbeenusedin thiswork.Thiscomplexis}

obtainedbyharvestingautolysedyeasts,thenheating(∼100◦_C)

anddryingbyatomizationat60◦_{C,toobtainadrypowderwitha}

meanparticlediameterof70mmandahumiditycontentofaround 8%.DMCSexhibitshighlipidiccontent,nearly20%bymass frac-tionofdrymaterial.ThemeasuredsoliddensityoftheDMCSis 1130kg/m3_.

2.2. Soxhletextractionandcharacterizationofextractedlipids Lipid content of the yeasts was gravimetrically determined bySoxhletextraction(8h)withsolventsoftechnicalgrade and solvent mixtures of different polarities (hexane, ethanol and chloroform–methanol in different volume ratios). The solvents wererecoveredinarota-evaporatorwhichwasoperatedwitha vacuumpump.ThecrudelipidicextractsobtainedfromSoxhlet extractionscontaincarbohydrates,proteins,andotherunwanted materialswhichwereseparatedbyamethodbasedonthework ofHubbardetal.[18].AccordingtoSoxhletextractionsdonewith differentsolvents,Table1showsthetotallipidiccontentafter sep-arationfromnon-lipidicmaterial.

Phospholipids(PL)presentinthetotallipidicamount(1–3g) obtainedbySoxhletextractionwereseparatedandquantifiedby dispersionincoldacetone(90ml),whichisassumedtoonly dis-solveneutrallipids[19,20].Inthiswork,thefractionoflipidswhich isinsolubleinacetoneisassumedtobethePLfraction.Lipidswhich aresolubleinacetoneareinturnassumedtobeTGandfattyacids. Table2showsthefractionoflipidswhichisinsolubleinacetone.

Fatty acid analysis of extracted lipids was carried out by gaschromatography(Thermo-Finiganchromatograph).The equip-mentcomprisedaflameionizationdetector(FID)andacapillary column(WCOTFused Silica25m×0.25mmIDDF=0.4Coating, CP-sil8CB).AsolutionofTMSH(trimethylsulfoniumhydroxide)in methanol(0.2M)wasusedtooperatelipidtransesterificationfor theanalysisoffattyesterscompositionpresentinTG.Heliumwas usedasthegascarrierataflowrateof2ml/minandatasplitflow equalto1/20.Theinjectoranddetectortemperatureswere220◦_C

and260◦_{C,respectively.Theoventemperatureprogramconsisted}

instartingat50◦_{Cduring6min,thensettingarampof7}◦_C/minup

to210◦_C,arampof15◦_C/minupto250◦_{C,andfinallymaintaining}

250◦_{Cduring15min.}

Fattyacidcompositionofacetonesolublelipidsisreportedin Table3andFig.1showsatypicalgaschromatogram.

2.3. Supercriticalextractions

Supercriticalextractionswerecarriedoutinapilotplantfrom SeparexChimieFine,France(SF300type)representedinFig.2

Table1

Soxhletlipidextractionsbyorganicsolvents.Totallipidcontentinsubstrates(lipids/yeast,w/w).

Hexane Ethanol Chloroform:methanol(2:1) Chloroform:methanol(1:2)

Memb.complex(C1) 0.001 0.22 0.212 0.23

Memb.complex(C2) 0.050 – 0.199 –

Memb.complex(C3) 0.051 – 0.230 –

C1:heatedanddriedbyatomization,C2:acidpretreatmentby4h,C3:acidpretreatmentby8h.

Table2

PhospholipidscontentbymassfractioninyeastobtainedbydispersionincoldacetoneofthetotallipidsextractedbySoxhlet(lipids/yeast,w/w).

Hexane Ethanol Chloroform:methanol(2:1) Chloroform:methanol(1:2)

Memb.complex(C1) – 0.066 0.062 0.069

Memb.complex(C2) 0.0 – 0.014 –

Memb.complex(C3) 0.0 – 0.01 –

C1:heatedanddriedbyatomization,C2:acidpretreatmentby4h,C3:acidpretreatmentby8h.

Table3

FattyacidcompositionofacetonesolublelipidsextractedbySoxhletwith chloro-form:methanol(2:1)assolvent.Massfractionsarenormalizedaccordingtothetotal fattyacidsfoundintheanalysis.

C14 C16:0 C16:1 C18:0 C18:1 Fattyacids%,w/w 1.3 12.5 51.4 3.8 30.5 Totalfattyacids%,w/w 72.1

It comprises two cyclone separatorsin seriesand a maximum CO2 flowrateof6kg/hcanbeoperated.Extractioncylindersof

differentcapacities:54cm3_,104cm3_,and228cm3 _(41.5mmi.d.)

wereavailable.Adetaileddescriptionoftheexperimental proce-dureandtheexperimentalset-upcanbefoundinpreviouspapers [17,21].Inthiswork,aco-solvent(ethanol9%bymassfraction) wasusedtoincreasethesolventpowerofCO2andalsotoallow

easiermechanicalrecoveryoftheextractintheseparators.Briefly, theheatingsystemwassetatthedesiredtemperature(40◦_C);the

extractioncylinderwasfilledwithagivenamountofsubstrates (33g,inthesmallvessel,60ginthemediumcapacityand140g inthebiggestone)andplacedinside theextractor vessel.Once thehighpressureextractorvesselwasclosed,carbondioxidewas pumpedatthedesiredtemperatureintotheextractoruptoreach theoperatingpressure(20MPa);thenagivenamountofethanol waspumpedtothevesseluptoobtainthedesiredfractionbymass percentofethanolasco-solvent.Astaticextractionperiodof15min wasinitiatedtoreachthedesiredprocessconditions,andsolvent andco-solventweresubsequently pumpedat thedesired flow-ratewhichwassetat30g/minfortheseextraction studies.The

extractionpressurewasmaintainedbyabackpressureregulator (BPR).TheoutletlineoftheBPRwasconnectedtothefirst sepa-ratorregulatedat7.0MPabyanotherBPRvalvewhoseoutletline wasconnectedtothelastseparatoroperatedat5.0MPa.

Unequallytimespacedsamples(12–18min)weretakeninall casesduringthetotalextractiontime(120–180min).Thesamples fromthebottomof separatorswerecollected alongthetimein 15cm3_{capacityglassvials.Thenethanolwasrecoveredat40}◦_Cin

arota-evaporatoroperatedwithavacuumpump.

Additionalstudiesweredoneinthe228cm3_{extractionvesselto}

checkifinsufficientresidencetime,inrespecttopossibleexternal masstransferproblems,couldhaveaffectedtheextractionresults.

3. Modelingoftheextractionprocess

Themathematical model adoptedin this workis themodel ofBrokenandIntactCells(BIC),ageneralapproachproposedby Sovová[22].Thismodelisusefulbecauseitaccountsforthe pseudo-linearinitialpartoftheextractioncurves,whichhasoftenbeen experimentallyobserved,especiallyinthecaseofoilseed extrac-tion.It isparticularlysuitedforfittingthiskindofexperimental dataasitalmostindependentlysimulatestwoextractionperiods, thefirstonebeinggovernedbyphaseequilibriumandthesecond onebyinternaldiffusioninsideparticles.

Animportant feature of theBIC model proposedby Sovová [22]istomakeitpossibletoconsiderdifferenttypesof pseudo-equilibriain thefirst extraction period:independentonmatrix (solubilityequilibrium)and/oradsorbedonthematrix(partition

Fig.1. TypicalgaschromatographanalysisoftheyeastmembranecomplexDMCSextractedbySoxhletwithorganicsolvent,afterseparationofphospholipidsbydispersion incoldacetone.

Fig.2.SimplifiedflowsheetofthepilotSF300:B,CO2cylinder;E,extractor;S, separators;P,pump;G,coolingandheatinggroup;D,backpressureregulatorvalves; C,co-solvent;VE,ventingsystem.

coefficient).From anexaminationofextraction curvesobtained inthepresentwork,andespeciallyfromtheorderofmagnitude oftheobservedfluidphaseoutputcompositionduringtheinitial extractionstep,ithasbeendecidedtouse‘type A’and‘typeD’ oftheBICmodel,dependinguponthedifferentpretreatmentsof theDMCS.Indeed,‘typeA’extractioncurvesarerepresentativeof systemswithoutsolute-matrixinteraction,andthevalueofthe initialoutputfluidphasecompositioncorrespondingtothefirst partoftheextractioncurve(alsotermedintheliteratureas‘initial apparentsolubility’)is closetothethermodynamicsolubilityof thesoluteinthesupercriticalsolvent,exceptwhenstrongexternal masstransfereffectsarepresent.

‘Type D’ extraction curves are representative of strong matrix–soluteinteractionsandthepseudo-equilibriumispresent inthesystemfromtheverybeginningoftheextraction.The max-imumcompositionofsoluteinthesolventisproportionaltothe solutecompositioninthebrokencellsandisgivenbyapartition coefficient(K).Inthiscase,theinitialoutputfluidphase composi-tionmaybemuchlowerthanthethermodynamicsolubilityofthe soluteinthesupercriticalsolvent.

Thereadercanbefamiliarizedwiththeparametersinvolvedin themodelinAppendixwhichpresentsthefinalsystemoftime ordi-narydifferentialequations,withthespatialderivativeterm

∂

y/

∂

z representedbyfinitedifferences(1y/1z).Theseequationswere numericallyintegratedusingtheRunge–Kutta–Felhbergmethod andyieldedthecompositionoflipidsinsidethesolidmaterialasa functionoftheextractiontime.

Theexternalmasstransfercoefficients(k_fa0),valueswere

esti-matedaccordingtodifferentcorrelations[23–27].Toestimatethe degreeofaxialdispersionintheextractorvessel,Peclet(Pe) num-bers,basedontheheightofthepackedbed,werecalculatedbythe

correlationofCatchpole[28].Table4summarizesthevaluesofthe estimatedparametersasafunctionofparticlediameter.

Porosity of thesolid wasestimated from the valueof solid densityoftheinertmaterial,sol,andvalueoftheapparent

den-sity,ap,obtainedfromthemassofmaterialtreatedpervolume

ofcylindercapacity.Soliddensitiesoftheinertmaterialwerein turnexperimentallyestimatedbypycnometry.Thevaluesofthe physico-chemicalpropertiesofthesolvent(,andD12)usedin

thisworkaresummarizedinTable5.

Fractionsofbrokencellsornon-boundlipidsandinternalmass transferparameterwereleftasparameterstobefitted.Inthecaseof ‘typeD’oftheBICmodel,athirdparameter,thepartitioncoefficient, K,wasdeterminedfromtheslopeofthefirstpartoftheextraction curveandthefractionofbrokencellsofthematerial.

4. Resultsanddiscussion

4.1. Selectivityandsolubilityoflipidswiththemixture CO2+ethanol

To uncouple matrix effects from the solvent efficiency, a preliminary study of the solvent power and selectivity of the CO2–ethanol mixture in respect to the extractable

com-poundshasbeenproposed.Thiswasdonebydirectlyprocessing samples of lipidic extract obtained from Soxhlet extraction (usingmethanol+chloroformmixtures).Glassbeads(1mm)were impregnated with a given amount of lipidic extract (ca. 3.6g) obtainedfromtheSoxhlet(thisamountcorrespondstothequantity oflipidscontainedin17.7gofyeast).Thesmallerextractionvessel of54cm3_{wasusedinthisstudy,followingtheextractionprocedure}

explainedabove.Thetargetwastoestimatetheselectivityofthe solvent(SCCO2+ethanol9%,w/w)inrespecttolipids.The

extrac-tioncurveisshowninFig.3and69%ofthetotallipidswerefound tobeextractedwhilethenon-extractedpartremainedontheglass beadsinsidetheextractionvessel.Theremainingnon-extracted lipidsontheglassbeadswerecheckedtobeacetoneinsoluble,in agreementwiththepreviousresultsobtainedintheSoxhlet stud-ies,andarethereforeassumedtobePLs.Fattyacidanalysisofthe extractedlipidsshowedacompositionsimilartothatofthelipids extractedbySoxhletandthenfractionatedbycoldacetone,within thisspecificcase80%bymassfractionoftotalestercomposition.It canbeseeninFig.3thatanestimatedspecificsolventmassratio, q,equalto55madeitpossibletoselectivelyextractalmost90%of theTGs.Thisvalueoftheselectivitywasalsoobservedinoilseed extraction.Forexample,CoceroandCalvo[13],atsimilaroperating conditions,obtainedonly0.06gofPL/kgoilextracted.Montanari etal.[16]obtained0.7gofPL/kgof“defatted”soybeanflakesat 23MPaand60◦_C.

Theoutputfluidphasecompositionobservedinthisstudywas 4.5gof lipids/kgCO2. Thisrelativelyhighvalue isnevertheless

significantlylower than theinitialapparentsolubilityobserved Table4

ParametersusedintheBICmodelfortheSCCO2+ethanolextractionoflipidsfromthemembranecomplexofS.cerevisiae.Estimatedbycorrelationsfromliterature.

Material dpa_{(mm) a0(m}−1_{) Uε(cms}−1_{) kfa0}d_(s−1_{) kfa0}e_(s−1_{) kfa0}f_(s−1_{) kfa0}g_(s−1_{) kfa0(s}−1₎h _Pei

M.C1b _{0.073 38576 0.049 1.30 0.036 0.72 0.062 0.037 166}

M.C2c _{0.309 10078 0.049 0.27 0.038 0.14 0.066 0.037 20}

a_{Meandiameteraftermaterialpretreatments.}

b_{Membramecomplexheatedanddriedbyatomizationandthensubmittedtodifferentpretreatments.}

c _{Membranecomplexsubmittedtoacidpretreatment,thendriedbyconvectionat60}◦_{Candmilledbyknifegrinder.} d _{Tanetal.[23].}

e_{LeeandHolder[24].} f _{Puiggenéetal.[25].} g_{Sovováetal.[26].} h _{CoceroandGarcia[27].}

Table5

PhysicalpropertiesusedintheBICmodelfortheSCCO2extraction.

T(◦_{C) P(MPa) Y*}a_{lipid/CO2(g/kg) f}b_(kgm−3_{) }c_{(Pas) D12}d_(m2_s−1₎

40 20 25 840 7.72×10−5 _5.5×10−9

a_{TGsolubilityfromCoceroandCalvo[13].}

b_{Mixturedensity.CO2DensityfromNationalInstituteforStandardsandTechnology[29].EthanoldensityfromPerry’sHandbook[30].}

c _{MixtureviscosityestimatedaccordingtothemethodofGrunbergandNissan[31].ViscosityofpureCO2estimatedfromJossietal.[31].LiquidethanolfromPerry’s} Handbook[30].

d_{Binarydiffusioncoefficient(D12)fortriglyceridesinCO2estimatedusingtheequationofCatchpoleandKing[32].} byCoceroandCalvo[13]fortheextractionofsunfloweroilwith

SCCO2+ethanol at the same operating conditions (20MPa and

315K),whichwasaround25gofoil/kgCO2.Theoutputfluidphase

compositionincreasedfrom4.5g/kgto9.4g/kgwiththeheight oftheextraction cylinderrangingfrom54cm3 _to228cm3 _(i.d.:

41.5mm)withaCO2flowrateequalto30g/min.Thisincreasewas

morepronouncedatlowerflow-rate(20g/minCO2)whereavalue

of18g/kgwasreached.

Furthermore,similarexperimentswereconductedusing com-mercialsunfloweroil.Thiswasdoneinordertoeasilycomparethe resultswiththoseofCoceroandCalvo[13].Theresultsobtained arein thesame orderofmagnitudethan theonesobtainedfor yeastlipids,i.e.,16gofoil/kgofCO2(228cm3configuration).So,

thisresultcouldbeassociatedtoexternalmasstransferlimitations and/or hydrodynamical problems in oursystem which prevent fromattainingthethermodynamicsolubilityintheCO2 output.

However,the completestudy ofthe hydrodynamicbehavior of supercriticalphase onmasstransferratewasbeyondthescope ofthiswork.Sovováetal.[26]alsoobservedsimilarproblemsin theoilextractionof grapeseedswhenCO2 waspumpedin

up-flowconfiguration(oppositetogravity).Adetailedstudyofthe effectofflowdirectionupontheexternalmasstransfercoefficient canbefoundintheworksofPuiggenéetal.[25]andStüberetal. [33].Theseauthorsobservedastrongdependenceoftheextraction rateuponsolventflow-rateandalsoupongravity-assisted direc-tion,especiallyforparticlesoflargediameterandhighlysoluble compounds.

AnapproachsimilartotheoneofTanetal.[23]wasusedto determinethevolumetricexternalmasstransfercoefficient,kfa0,in

ordertocomparethesevalueswiththoseoftheliterature[23–27]. Table6summarizesthevaluesofkfa0obtainedinthiswork,and

showsthatthesevaluesareoneorderofmagnitudelowerthan kfa0valuesobtainedbyCoceroandGarcia[27]fortheextractionof

sunfloweroilwithSCCO2andethanolatsimilaroperating

condi-tions(k_fa0=0.012s−1andkfa0=0.057s−1forsuperficialvelocities

Fig.3.ExtractionoflipidsfromDMCSwithSCCO2(30g/min,40◦_Cand20MPa) and9%(w/w)ofethanolasco-solvent.(N)experimentaldataofdirectextraction oflipidspreviouslyobtainedbySoxhletwithchloroform:methanolasthesolvent (70%oftheselipidsaresolubleinCO2).

from1.1cm/minto2.5cm/min).Thevaluesobtainedinthisstudy arealsooneorderofmagnitudelowerthanthevaluescalculated bycorrelationsproposedintheliteraturewhichtakeintoaccount theparticlesize(Table4).Also,atthelowestflow-rate,usingthe highestcapacityextractor,theexternalvolumetricmasstransfer coefficient,kfa0,wasshowntoincrease.Axialdispersionwas

esti-mated(from[28])andthePecletnumber(accordingtotheglass beadsdiameter(1mm))wasestimatedaroundPe=5andPe=18, forbedheightsof4.5cmand16cm,respectively.Thisisindicating thataxialdispersionphenomenacanhaveasignificantinfluence ontheextractionefficiency[34].

Fromtheseresults,valuesofkfa0calculatedinthissectionwere

usedtomodelthesupercriticalextractionoftheDMCScomplex subjectedtodifferentpretreatments.Whenfreelipidswerepresent inthesubstrate,‘typeA’ofBICmodelwasselectedtorepresentthe cumulativeextractioncurves.

4.2. SCCO2+ethanolextractionsofdriedyeastmembrane

complex(DMCS)

MostoftheextractionstudiesusingSCCO2withandwithout

co-solventweredonehereinthelowestcapacityextractioncylinder of54cm3_.

Processing of DMCS with pure CO2 (operating conditions:

20MPa,40◦_{Cand30g/min)producedverylowyieldsanditwas}

notpossibletofollowtheextractionkinetics.Around3goflipids by kgof raw material were obtainedafter 150min. Thisvalue wasobtainedwithrecoveryoftheextractbyfinalcleaningofthe extractionplantwithethanol.Indeed,thislowyieldwasexpected regardingresultsoftheSoxhletextraction(Table1)wherehexane extractionproducedonly0.1%bymassfractionoflipids.

Fig.4showstheresultsoftheSCCO2extractionofDMCSusing

9%bymassfractionofethanolinaCO2flowrateequalto32g/min,

at20MPaand40◦_{C.Theeffectofresidencetimeuponinitial}

out-putfluidphasecompositionandrateofinternalmasstransferwas testedbyincreasingtheextractioncylindercapacityfrom54cm3

to104cm3_{.Fromtheinitialslopesofthetwocurves,initial}

out-putfluidphasecompositiony0 werefoundtobe0.36glipids/kg

CO2whatevertheextractorcapacity.ValuesofthePecletnumber

forthedifferentextractorcapacities(Pe=UεL/DL=166forL=0.045

mandPe=298forL=0.077m,axialdispersioncoefficient calcu-latedaccordingto[28]),wereindicatinginthiscasenegligibleaxial dispersioneffects[34].

Becausevery lowinitialoutputfluidphase composition val-uesare characteristic of strongmatrix–solute interaction, ‘type D’modelwasselected.Itsequilibrium adsorptionconstant was estimated from initial slope values and from equation 6 (see Table6

kfa0calculatedfromtheexperimentalresultsaccordingto[23]. Cylinder capacity(cm3₎ Flow-rate (g/min) y0(g/kg) kfa0(s−1) 54 30 4.5 0.0014 228 30 9.4 0.0016 228 20 18 0.0045

0 2 4 6 8 10 12 14 16 200 150 100 50 0 CO2/TG-free Yeast [kg/kg] e, Lipid/TG-free yeast [g/kg]

Fig.4. SCCO2+ethanolextractionofDMCS.Residencetimeeffectoninitialoutput fluidphasecomposition“y0”andinternalmasstransferextractionrate.Symbolsare experimentalcurves:()54cm3_{and(♦)104cm}3_{extractioncylindercapacity.Lines} are‘typeD’ofBICmodel.

Appendix).Thevolumetricexternalmasstransfercoefficient, esti-matedaccording to Tanet al. [23], thevalue kfa0=1.3s−1 and

theoneobtainedfromLeeandHolder[24],k_fa0=0.036s−1,were

testedinthemodel.However,theobtainedfinal fitted parame-tersweresimilar.Actually,kfa0valuesgreaterthan0.02s−1proved

tocorrespondtolowexternalmasstransferresistance.Model fit-ting,inthis case, wasdoneby adjustingthefractionof broken cells,r,andtheinternalvolumetricmasstransfer,ksasandfitting

processshowedthattheseparametershavea weak interdepen-dence.Indeed, parameter radjusts thepoint at theend of the firstpartofextractioncurve,whereinitialapparentsolubilityy* orexternalmasstransfer,controlstheextraction.Parameterksas,

inturn,allowsadjustingthesecondpartoftheextractioncurve, whereinternalmasstransfergovernstheprocess.Agood agree-mentbetweenthemodelandexperimentaldatacanbeobserved inFig.4.

Thevaluefoundfortheparameterksas=1.3×10−6_s−1_islowin

respecttovaluesnormallyfoundforSCCO2extractionofoilseeds

[35],which areatleasttwo ordersof magnitudegreater.Fig.5 showsSEM(ScanningElectroMicroscope)imagesofDMCS.Itcan beseenanon-porousmaterial;actually,theplasmamembranesare stronglycorrugatedinawaythatthelipidsmaynotbeexposedto thesolvent(Fig.5bandc).Thewallsofthemembraneseemtobe verycompact(Fig.5d)explainingverylowvaluesofksasadjusted

with‘typeD’oftheBICmodel.

Resultsobtainedfromtheextractionofnonpre-treatedDMCS obviouslyindicatedtheneedforpretreatmentinordertoincrease thematerialporosity,andalsotoweakentheinteractionbetween lipidsandbiopolymerswhicharepresentinthemembranes.

Inthiscase,fattyacidanalysisoftheextractedlipidsrevealed 66%oftotalfattyesters.Indeed,relativecompositionoffattyacids wasverysimilartotheoneobtainedin theanalysisofSoxhlet extractedlipids. Also, this resultcouldbe explainedby the co-extraction of sterolsand glycolipids (solublein acetone) which constitutetheinertpartofthemembranecomplexandwhichare solubilizedwhenethanolisusedasaco-solvent[36].

4.3. InfluenceofpretreatmentsofDMCS

The differentpretreatments applied tothe yeast membrane complexwhichweretestedinthisworkaresummarizedinTable7. Detaileddescriptionsofthepretreatmentsaregivenbelowinthis section.Fig.6showscumulativeextractioncurvesasafunctionof specificmasssolventratio,q,fortheextractionofDMCSsubjected todifferentpretreatments.The54cm3_{cylinderextractorwasused}

intheseextractionexperimentsat20MPaand 40◦_Cwith9%of

ethanolasaco-solventandataCO2flow-rateof30g/min.Fig.6also

includesthebestfitlinesobtainedwiththeBICmodelaccordingto theparametersreportedinTable8.

Pretreatment1:Thermalpretreatmentsarenormallyappliedin theindustryofedibleoilsinordertocoagulateproteins,todecrease oilaffinityforthesolidsurfaceandtoagglomeratetheoilintolarger droplets [37].The materialDMCSwascookedfor 30–40minat 90◦_Cand120◦_{Cinaventilatedoven.Theextractioncurveforthis}

Fig.5. SEMimagesoforiginalyeastmembranecomplexDMCS.(a)RegulardistributionofDMCSmaterialwithcorrugatedmembranes.(bandc)Spatialdistributionofthe membranes.(d)Surfaceofthemembranes.

Table7

PretreatmentsoftheDMCSmaterialstudiedinthisworkfortheSCCO2+ethanol extractionoftriglycerides.

No.ofpretreatment Description

1 Cookingduring30minat90◦_Cor120◦_C

2 Disruptionbyrapiddecompressionafterexposureto SCCO2

3 Extra-dryingofthematerialat60◦_{Covernightand} millingprocess.

4 Macerationinethanolovernight,dryingin rota-evaporatorat60◦_Candmilling.

5 Harvesting,acidpretreatmentduring4h,filtering, heating(>100◦_{C)anddryingbyatomization(whole} pre-treatmentdonebytheprovider).

6 DMCSre-suspendedinanacidicsolutionduring4h (pre-treatmentdonebytheprovider).Thenthe materialwasfilteredanddriedinanairovenat 60◦_{C,overnight.Driedmaterialwasmilledinaknife} grinderandsize-classifiedwithameanparticle diameterof300mm.

7 Idem6.Then,macerationwithmethanolduring1h.

Fig. 6.Extraction of DMCSin 54cm3 _{cylinder extractor volume with SCCO2} (30g/min40◦_{Cand20MPa)and9%(w/w)ofethanol,andsubjectedtodifferent} pre-treatments.Symbolsareexperimentaldata:( )untreatedDMCS,(d)DMCSafter pretreatment2,(♦)DMCSafterpretreatment1.1,(N)DMCSafterpretreatment3, ()DMCSafterpretreatment4.Linesare‘typeD’modelfittingwithparameters reportedinTables6and8.

pre-treatedmaterial(Fig.6)showedthatsolublelipidsstillhave astronginteractionwiththematrixfromthestartofthe extrac-tionprocess.Ityieldedsimilarvalues ofy0 andksas parameters

(Table8).Theoutputfluidphasecompositioninthefirstextraction periodis0.36goflipids/kgofCO2and,asexplainedbefore,isstill

oneorderofmagnitudelowerthantheinitialcompositionobtained intheextractionofpureyeastlipidsasreportedinTable6atthe sameoperatingconditionsandforthelowercapacityextractor.It

istwoordersofmagnitudelowerthanthethermodynamic solu-bility(25goil/kgCO2).Thevalueofpartitioncoefficient,K=0.0019

(calculatedfromtheslopeofthecumulativeextractioncurvesand Eq.(6)asgiveninAppendix),evidencesastrongmatrix-oil interac-tionwhencomparedtothevaluecorrespondingtothenon-bound soluteextraction, usingextractimpregnatedglassbeads,where K=0.026(K=y0/x1,0,werex1,0istheamountofoildividedbythe

massofinertmaterial).However,thefractionofbrokencellsor non-boundlipids,r,wasfoundtoincreasefrom3.8%inthe orig-inalmaterialto5.9%withthethermalpretreatmentat120◦_C,as

showninTable8.Also,bothtemperaturesofthermalconditioning, 90◦_Cand120◦_{C,leadtosimilarvaluesofthevolumetricinternal}

masstransfercoefficientsksas.However,thesevaluesremainin

thesameorderofmagnitudethanthoseobservedintheextraction oftheuntreatedmaterial,indicatingpersistenceofinternalmass transferlimitations.

ChromatographicanalysesoftheextractedlipidsfromtheDMCS afterthispretreatment,reportedafattyacidcompositionvery sim-ilartotheoriginalmaterialwithatotalfractionofesterof61%by massfraction.

Pretreatment2:AdisruptionmethodusingpressurizedCO2(SCF

disruption)wasdoneaccordingtotheproceduredescribedbyLin etal.[38].Briefly,thematerialwasplacedintotheextractorand CO2 wasfed uptothe experimentalpressureand temperature

(20MPaand40◦_{C).Thesystemwasmaintainedstaticduring1h}

andthenthepressurewassuddenlyreleasedandthe decompres-sionsteplastedaround5s.Twosuddendecompressionsweredone toattemptopeningcorrugated membranes oftheDMCS. How-ever,SCFdisruptionofthecomplexbarelyimprovedtheresults in respecttotheoriginal material(seein Table8thevalues of themodelparameters).Indeed,thefractionofbrokencellsor non-boundlipidsincreasedfrom3.9%to5.1%.Finally,theseresultsare insameorderofmagnitudethanthoseobtainedbythethermal pretreatmentat90◦_{C.Itisimportanttomentionthat,inthiswork,}

theSCFdisruptionwasappliedtoadriedmaterial,whilethe pro-ceduredescribedintheliteraturewasappliedtoawetmaterial.In addition,Linetal.[38]originallyproposedthisproceduretobreak intactyeastcellswhiletheyeastmembranecomplexusedinthis workwasindeedverydifferentfromintactcellsofS.cerevisiaefrom amorphologicalpointofview.Thismayexplainthepoorresults observedwiththispretreatment.Lipidcompositionofextracted lipidswasalsofoundsimilartoourpreviousresults(Table3).

Pretreatment3:Incompletedryingofthematerialcouldinduce problemsintheextractionbecausepresenceofwatermayreduce thesolventpowerofCO2 [12].However,ethanolasaco-solvent

couldhelpdehydratingthematerialbecauseofitsnaturalaffinity withwater.First,toevaluatethehumiditycontentofthe origi-nalmaterial,DMCSwassubmittedtoovernightextradryingina convectionovenat60◦_{C,anditsinitialhumiditycontentwasthus}

estimatedtobe8%bymassfraction.Theextra-driedmaterialwas thenre-milledbyaknifegrindertoavoidmaterialagglomeration

Table8

ParametersusedintheBICmodelfortheSCCO2+ethanolextractionoflipidsfromthemembranecomplexofS.cerevisiaesubmittedtodifferentpretreatments(pretreatments arenumeratedaccordingtoTable7).Parametersksasandrareobtainedfromnumericalfittingoftheexperimentalcumulativeextractioncurvesobtainedinextractorcapacity of54cm3_.

No. Material dp(mm) ε s(kgm−3_{) ksas(s}−1_{) r y0(g/kg)}

0 DMCS 0.073 0.54 1130 1.3×10−6 _{0.039 0.36} 1 DMCS-Cooking90◦_{C 0.073 0.54 1130 2.6×10}−6 _{0.051 0.36} 1.1 DMCS-Cooking120◦_{C 0.073 0.54 1130 2.6×10}−6 _{0.058 0.36} 2 DMCS-SCFdisruption 0.073 0.54 1130 2.4×10−6 _{0.051 0.36} 3 DMCS-drying-milling 0.070 0.53 1100 3.2×10−6 _{0.110 0.65} 4 DMCS-EtOHovernight 0.071 0.63 1050 3.0×10−6 _{0.140 2.00} 5 acidpretreatment1 0.074 0.63 908 2.4×10−6 _{0.110 0.13} 6 acidpretreatment2 0.309 0.48 990 1.6×10−5 _{0.310 3.5}

(nochangeoftheaverageparticlediameterandofthematerial propertieswereobserved).Thisnewmaterialformwasextracted bySCCO2+ethanolatthesameoperatingconditionsthanprevious

one.Onthecumulativeextractioncurveobtainedinthisspecific case(seeFig.6),avaluey0=0.65glipid/kgCO2oftheoutputfluid

phasecompositioncanbeobserved,withparametersr=0.11and ksas=3.2×10−6s−1 accordingto‘typeD’oftheBICmodel.Here

again,nosignificantchangeswereobservedinthefattyacid com-positionofthelipidextractedinrespecttothepreviousresults.

Pretreatment4:Polarsolvents,e.g.,alcohols,arelikelytoallow disrupting higher degrees of interaction of lipids with other biopolymermolecules containinghydrogen bonds(COOH,NH3,

andpolarfunctionalgroupsofproteins)thannonpolarsolvents suchashexaneorchloroform,aspointedoutbyourprevious Soxh-letextractions.Inthiswork,thecomplexDMCSwaswettedwith ethanol(30%bymassfractionofalcohol),letincontactwithethanol overnight,andthenethanolwasevaporatedinarota-evaporator at60◦_{C.Thematerialwasthenmilledusingaknifegrinderbefore}

extraction.ResultsofthispretreatmentareseeninFig.6,andreveal animportantimpactsinceaninitialoutputfluidphasecomposition of2goflipids/kgofCO2isobserved.Thisincrementindicatesthe

occurenceoffree-oil.So,‘typeA’oftheBICmodelwasusedinthis casetofittheexperimentaldata,usingthevalueofthe thermo-dynamicsolubilityys=25goflipids/kgofCO2,asgivenbyCocero

andCalvo[13].Thus,alowvalueoftheexternalvolumetricmass transfercoefficient(kfa0=0.0014s−1,obtainedinSection4.1)was

usedinordertofitthefirstpartoftheexperimentalcumulative extractioncurve.Indeed,themodelwasnotabletofitthe exper-imentalinitialoutputfluidphase compositionwhenk_fa0 values

greaterthankfa0=0.0025s−1wereusedtomodeltheexperimental

data.

The model gave fitted parameters r=0.14 and ksas=3.0×10−6_s−1_{. The low value of the internal volumetric}

mass transfer coefficient,in comparison with thevalues found for theextraction of vegetableoils fromoleaginous seeds[35], stillindicatesstronglimitationoftheinternaldiffusioninsidethe inertmatrix.Conversely,thefractionofnon-boundlipidsinthe membranewassignificantlyincreasedafterthispretreatmentand

ethanol actuallyweakenedtheinteraction betweenbiopolymer moleculesand lipids. Fatty acidanalysisof thelipidsextracted revealed, after transesterification, a total amount of ester of 85%(w/w)inlipidsextractedinthefirstpartofthecumulative extractioncurve.Inthesecondperiodoftheextractionthetotal amountofesterdecreasesto70%(w/w).Thereisaclearincrement inthetotalamountoflipidsinthefirstpartoftheextractionin comparison withpreviouspretreatments and this couldbethe effect of themaceration in ethanol. On the otherhand, in the secondstageoftheextraction,wheretheinternalmasstransfer ratecontroltheprocess,thetotalamountoflipidsdiminishesat almostthesamelevelasinpreviouspretreatments.

Pretreatment 5: Another common method used to facilitate extractionofyeastlipidsisacidoralkalihydrolysis.Thistechnique breaksthebondsconnectinglipidstootherbiopolymers,causing denaturationofproteins,andinactivationoflipases,butalso pos-sibledegradationofthelipids.Acidhydrolysisisoneofthemost employedtechniquesforyeastcellconditioningatlaboratoryscale andcanbescaledupforpilot-plantoperation[3].

Inthisstudytheharvestedmembranecomplexwasfirst submit-tedtoacidtreatment,i.e.,boiledwith1NHClsolutionfor4hand subsequentlyheatedanddriedbyatomization(operationsdoneby ourproviderandpartner,theLesaffreCompany).Thispretreatment changedsignificantlythephysicalpropertiesofthematerial,such assoliddensityandporosity,andhadanimportantinfluenceonthe cumulativeextractioncurve.The‘typeD’BICmodelwasusedand yieldedafractionoffree-lipidsr=0.11withaninternalmass trans-fercoefficientsksas=2.4×10−6s−1.However,inthiscasethefinal

extractionyield,ataspecificsolventmassratioof250(CO2

/TG-freeyeast,kg/kg),wassimilartotheoneobtainedafterthethermal pretreatmentat120◦_{C(pretreatment1).Theobservedvalueofthe}

initialoutputfluidphasecompositionwasy0=0.13goflipid/kgof

CO2.ItcorrespondstoapartitioncoefficientKequalto0.0007(kg

TG-freeyeast/kgCO2)fortheadsorptionequilibrium,avalueeven

lowerthattheonefoundfortheextractionofuntreatedDMCS. Pretreatment6:Similartopretreatment5,theacidhydrolysis oftheMCSwasherecarriedoutduring4h.Thenthematerialwas washedandfiltered.ThewetMCS(80%,w/w,ofwater content)

Fig.7. SEMimagesofasectionofDMCSsubjectedtoacidhydrolysisanddriedat60◦_{Covernight.(a)Irregulardistributionofmembraneparticles.(b)Generalshapeofthe} particles.(c)Zoomofthenarrowphasethemembranesshowingaporousmaterial.(d)SurfaceoftheyeastmembranecomplexDMCSafterpretreatment6.

wasthendriedat60◦_{Cin aconvectionovenovernight.Finally,}

thematerialwasmilledusingaknifegrinderuptoafinalaverage particlediameterof300mm.

Fig.7showsSEMimagesofthecomplexMCSsubmittedtothis pretreatment6.BycomparisonwithFig.5,themorphological dif-ferencesofyeastcomplexesMCSdependingonthepretreatment andthedryingprocessareobvious.Inthislastcase,thesizeand particleshapesweremoreirregular(Fig.7a).Mostimportant,the material seemsto beporous,potentially allowinga much bet-terpenetrationforthesolventandbettercontactwiththelipids (Fig.7c).Wallsofthemembraneappearthinnerfavoringashorter pathfortheinternaldiffusionfromintacttobrokenmembranes (Fig.7d).

Fromtheexaminationoftheextractioncurve(Fig.8)obtained withthelowerextractorcapacity (54cm3_{)avalueoftheinitial}

outputfluidphasecompositiony0=3.2goflipids/kgofCO2,canbe

deducedandthisvalueisoneorderofmagnitudegreaterthanthe valuefoundintheextractionofuntreatedDMCS.Thispretreatment allowedtheextractionofonethirdofthelipidsinthefirstpartof theprocess,correspondingtoaspecificsolventmassratioofq=30. CPGanalysesoftheextractedlipidsrevealedconstantfattyacid compositionallalongthecumulativeextractioncurvewithatotal esterfractionof79%(w/w)aftertransesterification.Normalized fattyacidcompositionshowedsimilarresultstotheonesreported inTable3.

Theextractioncylinderof228cm3_{wasemployedtoassessthe}

effectofahigherresidencetimeontheextractionyield.Asitcan beobservedinFig.8,theinitialoutputfluidphasecompositionwas increasedfrom3.2to14goflipids/kgofCO2,whentheDMCS

mate-rialprocessedwasincrementedform30gto140g,respectively. Asinthecaseofpretreatment4,becauseanimportantinitial outputcomposition of lipidsin the solventindicated the pres-enceoffree-oil,‘typeA’oftheBICmodelwasusedinthis case tofitthecumulativeextractioncurves.Thevaluekfa0=0.0014s−1

obtainedinSection4.1wasusedtomodeltheexperimentaldata. Asexplainedbefore,thismodelwasnotabletofitthefirstpart ofthecumulativeextractioncurvewhenkfa0valuesgreaterthan

kfa0=0.0025s−1wereused.

Itisimportanttonotethatthelowkfa0valueusedinthiswork

whenfree-oilwaspresentinthesubstratecouldalsoartificially accountforaninadequatedescriptionofflowpattern[22]. How-ever,thestudyofthehydrodynamicsinsidetheextractioncylinder wasbeyondthescopeofthisworkandadditionalspecific experi-mentswouldbenecessarytoassessthishypothesis.

Withthispre-treatment,thefree-oilornon-boundlipidswere clearlyincremented(r=0.32),showinganimportantimprovement

0 50 100 150 200 250 200 150 100 50 0 CO2/TG-free yeast Kg/Kg e Li p id s/ T G -f re e y e a st g /K g

Fig.8.ExtractionfromDMCSwithSCCO2(30g/min,40◦_{Cand200bar)and9%(w/w)} ofethanol.Materialdriedat60◦_{Cinaconvectionovenby12h.Symbolsare} exper-imentaldata:()extractioncylinderof54cm3_{(N)extractioncylinderof228cm}3_. Lines:BICmodel‘typeA’fittingaccordingtomodelparameters(randks)reported inTable8forpretreatmentno.6andkfa0reportedinTable6.

intheextractioninrespecttotheoriginalmaterialandprevious pretreatments.Thevolumetricinternalmasstransfercoefficient wasalsoincrementedtoksas=1.6×10−5_s−1_{,avaluemorein}

agree-ment with values of internal mass transfer coefficients usually obtainedintheSCCO2extractionofoilseeds[35].Theincrement

ofprocessedquantity,from30gto140g,barelymodifiedthefitted parameters,thefractionofbrokencellsrbeingequalto0.3,andthe valueofthevolumetricinternalmasstransferparameter;ksasto

1.3×10−5_s−1_.

Pretreatment7:AcidhydrolysisofDMCSfollowedbymethanol macerationwasperformed.ItisseeninTable8thatthis pretreat-mentresultsinanimportantincrementoffree-oilornon-bound lipid ratio (r=0.47), influencing strongly the first part of the extraction. However,theinfluenceofalcohol macerationinthe pretreatmentwasnotreflectedinthevalueoffinalyieldbecause internalmasstransferwasstilllimitingtheextraction,asshown bythevalueofthecoefficientksasobtainedaccordingto‘typeA’of

theBICmodel.Thisvaluewaslowerthantheoneobtainedafterthe acidpretreatmentduring4h(seepretreatmentno.7inTable8).

Methanolanddryingpretreatmentsseemedtoopenthe corru-gatedmembranes,favoringthelipidextraction.Polarityofethanol alsohelpstoweakeninteractionoflipidswithbiopolymers[3] increasinginitialoutputfluidphasecompositionobservedinthe cumulative extraction curves. On the other hand, the cooking process at hightemperatures barely improves resultsshowing thathightemperatureprovokesproteinstocoagulate,leadingto moredenseparticles;however,lipidsarenotfreeorcondensed indropletsasitusuallyhappenedinoilseedextractionprocesses [37].

BICmodelparametersreportedinTable8areindicatingsevere internal mass transfer limitations for pretreatments using high temperatures,likecookingprocessattemperatureabove100◦_C

anddryingbyatomization,whereksasvaluesaround10−6s−1were

found.

Theincreaseofparameterrcouldbetheresultofthemilling pretreatment and of a chemical attack over theyeast material structure. Forexample,acidhydrolysishasbeenusedasa pre-treatmentforlignocellulosicmaterialintheethanolbioprocesses [39].Whenlignocellulosicmaterialissubmittedtoacidhydrolysis, hemicelluloseistotallyorpartiallyhydrolyzedintooligomericand monomericsugars[39].Acidhydrolysiscouldhavethesameeffect ontheyeastmembranecomplexproducingagreaterfractionof non-boundlipidsinthematerialafterthepretreatment.

Generally,resultsaresuggestingthatheatingathigh tempera-ture(>100◦_{C)anddryingprocessbyatomizationofmaterialDMCS}

isnotappropriateasa pretreatmentforSCCO2 extraction(with

orwithoutethanol asa co-solvent). Thiscouldbetheresultof incompleteeliminationofwater whichproved tohave prejudi-cialeffects in this case [12],becauseagglomeration of proteins encapsulatesthelipids.Ontheotherhand,thedryingprocessat 60◦_{Cproducesahighporositymaterialwithgreatermass}

trans-ferrates.Acidhydrolysisoralcoholicmacerationseemsnecessary inordertoobtainfreelipidstoextract.Ahighselectivityand sol-ventpowerwereobtainedwhenworkingattheselectedoperating conditions(40◦_{Cand20MPa—9%ofethanol)andappropriate}

pre-treatments,butwithinternalmasstransferlimitation,similarto theoneencountered inSCCO2 extractionof vegetableoils[35].

Thefattyacidanalysisofthelipidsobtainedfromtheextraction withpureCO2(20MPaand40◦C)ofthecomplexDMCS

submit-tedtopretreatment 6showed a total amountof 95% (w/w)of fattyesters.However,only2.5%ofthetotalextractablelipidswere obtained(5goflipids/200goftotallipids).So,furtherincrements throughtriglyceridesselectivitycouldbeobtainedbyextraction andfractionationwithpureCO2ofthelipidspreviouslyobtained

byCO2+ethanol,whereinitialselectivityisnear90%inrespectto

5. Conclusions

Extractionoflipidsfrommembrane complexofS.cerevisiae, using SCCO2+ethanol as a co-solvent, has been carried out at

20MPaand40◦_{C.Conventionalorganicsolventextractionsusing}

aSoxhletapparatuswerealsoperformed.Extractionofthe mem-branecomplexwithchloroform/methanolsolventsyieldedalipid contentof21–23%bymassfractionofmaterial,witha30%ofthese lipidsbeinginsolubleinacetone.Whenthelipidsobtainedby Soxh-letextractionweresubjectedtoSCCO2+ethanol(9%)aselective

extractionoftheTGwasobtained,asexpectedaccordingtothelow solubilityofPLattheseoperatingconditions.TheSCCO2extraction

revealedstronginteractionbetweenthelipidstoextractandthe rawmaterial.Besides,itwasobservedthattheperformanceofthe SCCO2 extractionofthemembranecomplexdependedmarkedly

onthepretreatmentofthematerialanddryingprocessdetermined thesuccessoftheTGextraction.Anacidhydrolysisfollowedbya dryingprocessatlowtemperature(60◦_{C)wasconsideredasthe}

bestpretreatmentandyieldeda30%ofnon-boundlipidfraction andinternalmasstransfercoefficientsinthesameorderof mag-nitudethanthoseobtainedintheSCCO2extractionofvegetable

oils.

Finally,SCCO2extractionofTGfromyeastscanbeperformed

selectivelywitha9%ofethanolastheco-solventat20MPaand 40◦_{C and gave a residual material rich in PL, which could be}

extractedafterthisfirststagebyincreasingthepressureorbyan increaseoftheethanolcomposition.

Acknowledgments

TheauthorswouldliketothanktheFrenchAgenceNationalede laRecherche(ANR)foritsfinancialsupporttothisresearchthrough theLIPICAEROproject,andoneofthepartners,theLesaffreGroup, forprovidingthemembranecomplexofS.cerevisiae,usedinthis work.

AppendixA.

Inthissectionitispresentedthegeneralmathematical model-ingapproachusedinthisworkwherethespatialterm(

∂

y/

∂

z)has beenrepresentedbyfinitedifferences(1y/1z),sothereadercan befamiliarizedwithparametersandtermsgiveninthearticle.The generalmodeliswellpresentedbySovová[22]andmostofthe expressionsgiveninthisAppendixarebasedonthisarticle,where thereaderisreferredforfurtherdetailsandderivations.

Thedifferentialmassbalanceequationsinthesolventphase(1) andsolidparticles(2and3)accordingtoSovová’smodelare: fε



∂y ∂t+U ∂y ∂z



=kfa0f(y∗−y) (1) rs(1−ε)∂x1 ∂t =ksas(x2−x1)−kfa0f(y ∗_{−y) (2)} (1−r)s(1−ε)∂x2 ∂t =−ksas(x2−x1) (3) Theinitialandboundaryconditionsare:

y|t=0=ys;x1|t=0=x1,0;x2|t=0=x2,0;y|z=0=0 (4)

InEqs.(1)–(3):y,x1andx2arethecompositionofoilinthesolvent

phase,inthebrokencellsandintheintactcells,respectively;tisthe extractiontime;z,istheaxialcoordinate,U,istheinterstitialfluid velocity;r,isthefractionofbrokencellsornon-boundlipids;f

andsarethedensitiesofSCCO2andsolidparticles,respectively;

εisthevoidvolumefractionofthebed,a0isthespecificparticle

externalsurface(a0=6(1−ε)/dp,wheredpistheaverageparticle

diameter),asisthespecificareabetweentheregionsofintactand

brokencells,kf istheexternalmasstransfercoefficient,ksisthe

internalmasstransfercoefficient,whichgloballyaccountsforthe internaldiffusionalresistanceusingthelineardrivingforcemodel approach,andy*isthefluidphasecompositionatequilibriumwith thesolidphase.IntheBICmodeltheexpressionofy*dependsupon atransitioncomposition,xt.y*iseitherequaltothethermodynamic

solubilityysorproportionaltotheoilcompositioninthebroken

cellsbecauseofanadsorptionequilibrium,accordingtoEq.(5): y∗_=y

s for x1>xt (5a)

y∗_=Kx

1 for x1<xt (5b)

‘TypeA’extractioncurvesarecharacteristicofsystemswithout solute–matrixinteraction.Inthismodelthetransition composi-tionisxt=0,andtheequilibriumfluidphasecompositionisthe

thermodynamicsolubility,Eq.(5a).‘TypeD’extractioncurvesare characteristicofastrongmatrix–soluteinteractionandthe pseudo-equilibriumpresentinthesystemisgovernedbyEq.(5b)fromthe beginningoftheextraction.Themaximumcompositionofsolutein thesolventisproportionaltothesolutecompositioninthebroken cellsbythepartitioncoefficientK.Inthiscase,theinitialoutput fluidphasecompositiony0couldbemuchlowerthanthe

thermo-dynamicsolubilityofthesoluteinthesupercriticalsolvent.The valueofKinthiscasecanbedeterminedfromtheslopeofthefirst partoftheextractioncurveandthefractionofbrokencellsfrom thephysicalpropertiesofthematerialbyEq.(6):

y0=Kx1,0, x1,0= rxu r+ Kwith = fε s(1−ε), xu−xt≤ rKxt (6)

InEq.(6),y0 istheinitialoutputfluidphasecomposition,x1,0

istheinitialcompositionofoilinthebrokencells,xuistheinitial

compositionofsoluteinthesolidmaterial.

TheextractorisdividedinNequalparts(N=100inthiswork) andafterthedifferentiationofthespatialterm,Eqs.(1)–(3)take thefollowingform:

fε



_dy dt +U yi−yi−1 1z



=kfa0f(y∗−yi) rs(1−ε) dx1,i dt =ksas(x2,i−x1,i)−kfa0f(y ∗_−y i) (1−r)s(1−ε)dx2,i dt =−ksas(x2,i−x1,i) Initialconditionsare:

yi|t=0=ys; x1,i|t=0=x1,0; x2,i|t=0=x2,0

Thissetofthreeordinarydifferentialequationsissolvedforeach ipartofN=100inwhichtheextractorhasbeendivided.

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