Selective extraction of neutral lipids and pigments from Nannochloropsis salina and Nannochloropsis maritima using supercritical CO2 extraction: Effects of process parameters and pre-treatment

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Selective extraction of neutral lipids and pigments from Nannochloropsis salina and Nannochloropsis maritima

using supercritical CO2 extraction: Effects of process parameters and pre-treatment

Adil Mouahid, Kanitta Seengeon, Mathieu Martino, Christelle Crampon, Avery Kramer, Elisabeth Badens

To cite this version:

Adil Mouahid, Kanitta Seengeon, Mathieu Martino, Christelle Crampon, Avery Kramer, et al.. Selec-

tive extraction of neutral lipids and pigments from Nannochloropsis salina and Nannochloropsis mar-

itima using supercritical CO2 extraction: Effects of process parameters and pre-treatment. Journal

of Supercritical Fluids, Elsevier, 2020, 165, pp.104934. �10.1016/j.supflu.2020.104934�. �hal-03232116�

Selective extraction of neutral lipids and pigments from Nannochloropsis salina and Nannochloropsis maritima using supercritical CO

extraction: Effects of process parameters and pre-treatment

AdilMouahid^a,∗,KanittaSeengeon^a,MathieuMartino^a,ChristelleCrampon^a, AveryKramer^b,ElisabethBadens^a

aAixMarseilleUniv,CNRS,CentraleMarseille,M2P2,Marseille,France

bCellana,LLC,590CypressHillsDrive,Encinitas,CA92024,UnitedStates

h i g h l i g h t s

•Theeffectsofprocessparameterson neutrallipids andpigmentsextrac- tionfrom Nannochloropsis sp were investigated.

•Extraction kinetics were compared betweenairflowdriedandringdried biomass.

•RSM was used to investigate the effectsofpressureandtemperature ontherecoveryofneutrallipidsand pigments.

•Sovová’smathematical wasapplied tobetterunderstandthemechanism ofextractionkinetics.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Articlehistory:

Availableonline18June2020

Keywords:

SupercriticalCO2extraction Ringdryer

Pigments Sovová’smodel

Nannochloropsis,pre-treatment.

a b s t r a c t

SupercriticalCO2extractionexperimentswereconductedtoinvestigatetheeffectsofpretreatmentand processparametersonneutrallipids,chlorophyllsandcarotenoidsrecoveryontwospeciesofNan- nochloropsis.ForNannochloropsismaritima,afactorialexperimentaldesignwasperformed(P:[100–300]

bar,T:[313–333]K).Thehighestextractionyieldswereobtainedatthehighestpressuresandtemper- atures.Twodryingmodes,ringdryingandairflowdrying,werecompared.Althoughtotalextraction yieldandextractionkineticswereobservedtobegreaterusingairflowdriedmicroalgae,extractsfrom thisdryingmethodresultedinpartialdegradationofglyceridesinfreefattyacids.Ringdriedextracts maintainedthesameneutrallipidcompositionastheinitialbiomass.Basedontheseresults,ringdried NannochloropsissalinawasextractedusingsupercriticalCO2at333Kandboth300–400bar.Extraction curves

Abbreviations: AFD,Airflowdrying/Airflowdried;Chla,Chlorophylla;DM,Drymatter;EPA,Eicosapentaenoicacid;FD,Freezedrying/FreezeDried;FFA,Freefatty acids;HPTLC,Highperformancethinlayerchromatography;RD,Ringdrying/RingDried;RSM,Responsesurfacemethodology;SC−CO2,SupercriticalCO2;SEM,Scanning electronmicroscopy;TAG,Triacylglycerides;TLC,Thinlayerchromatography.

∗Correspondingauthor.

E-mailaddress:adil.mouahid@univ-amu.fr(A.Mouahid).

weremodelledusingtheSovová’smathematicalmodel.

Nomenclature

AARD Averageabsoluterelativedeviation(%)

a_s Specificareabetweentheregionsofintactandbro- kencells(m⁻¹)

C_u Solutecontentintheuntreatedsolid(kg_solute/kg

solid)

d_p Microalgaeparticlediameter(m)

e Mathematicalmodelextractionyield(kgextract/kg

insolublesolid)

E Amountextracted(kg)

k_s Solid-phasemasstransfercoefficient(m/s) n Numberofexperimentalpoints

N Solidchargeintheextractor(kg) N_m Chargeofinsolublesolid(kg) P Pressure(bar)

q Relativeamountofthepassedsolvent(kg_solvent/kg

insolublesolid)

Q˙ CO₂flowrate(kg/s)

r Grindingefficiencyorfractionofbrokencells t Extractiontime(s)

T Temperature(K)

x_u Concentrationintheuntreatedsolid(kg_solute/kg

insolublesolid)

y_s Solutesolubility(kg_solute/kg_solvent) Greekletters

Solventtomatrix ratioin thebed(kg _solvent / kg

insolublesolid) ε Bedvoidfraction

Introduction

Microalgallipidsandantioxidantsarepromisingrawmaterials for the nutraceutical, food,energy, cosmetic and pharmaceuti- cal industries [1]. Microalgallipid composition varies between speciesandisalsoaffectedbygrowthconditionsincludingmedium composition,temperature,light intensity,and aeration rate[2].

Amongmicroalgaespecies,Nannochloropsisspeciesisverypromis- ingforseveralindustrialapplicationsandisconsideredasource ofcommerciallyvaluablepigments[3–5].Underidealconditionsit contains25upto45%lipidsthatcanbeusedasfeedstockforbiofuel productionandthenutraceuticalindustryasbotheicosapentaenoic acid(EPA)andotherlongchaintriacylglycerides(TAGs)[1,2,6].For thesereasons,Nannochloropsissp.wereselectedforthisstudy.

Microalgallipidextractionistypically performedusingtoxic andnon-selective(polarandneutrallipidsarebothsolubilized) organicsolventssuchasn-hexane.Ifindustriallyrelevantfractions oftheextracted oilare desired,additional separation stepsare required.Awidelyreported[7–10]alternativetoorganicsolvents issupercriticalCO₂(SC−CO₂).Thereareseveraladvantagesofusing SC−CO2includingitsselectivityofneutrallipids,itsgaseousstate atambientpressuremakingitsimpletoremoveresidualsolvent, anditsrecyclability. AlgalSC−CO₂ extractionefficiency primar- ilydependsonpre-treatmentconditions.Thefirstpretreatment stepforanefficientextractionisdewatering(solardrying,airflow drying,freezedrying,microwave,...)followedbytheincreaseof thecrackingofspecificsurfaceareabymechanicalmethods(bead milling,ultra-sonication,...).Non-mechanicalmethodslikechem- icalcelldisruptionorenzymaticcellhydrolysiscanbeappliedfor helpingthereleaseofsolutesofinterestandtheirfurtherrecovery duringextraction.Extractionefficiencyalsodependsonfreesolute accessibilityandinternaldiffusion(choiceofprocessparameters and/ortheuseofco-solvent)[7,10–14].

AsummaryofrecentstudiesdealingwiththeSC−CO₂extrac- tionofneutrallipidsandvaluablecompoundsfromNannochloropsis species[4,11,15–25]arereportedinTable1.Althoughadetailed and accuratecomparisonbetweenthese studiesis difficultdue totherangeofdifferentNannochloropsisstrains,growingcondi- tions, pre-treatments,and extractionconditions(CO₂ flow rate, particlesizediameter,initialwatercontent,etc.).Someinteresting tendenciescanbehighlighted.Studiesdealingwiththepretreat- menteffectsontheextractionkineticsandefficiency[11,17,25]

showedthatairflowdrying(AFD)wasthemostsuitabledrying modeleadingtohighextractionkineticsofneutrallipidsincompar- isontofreezedrying(FD).Indeed,FDpreservestheintegrityofthe microalgacellmembranesandthusinducesmorelimitationfordif- fusionleadingtoslowerextractionkinetics.Addingdistilledwater todriedN.oculatatomoisturecontentsupto20wt%seemstohave noinfluenceontheoilextractionyields[11].Extractscontained both oiland water.Another pretreatmentrecentlyinvestigated suggeststhatcellwalldamageofmarineNannochloropsissp.caused byanosmoticgradientcanhaveapositiveeffectonSC−CO₂extrac- tionkineticswithethanolasco-solvent[25].Performingoneto threeconsecutivewashingstepswithdistilled watercreatedan osmoticpressureimbalanceacrossthecellwallpriortoFD.The authorsshowedthatthepretreatmentdoubledthetotal extrac- tionyieldwhileincreasingtheconcentrationofacylglyceridesand freefattyacids.Nevertheless,hydrolysisofglycerideswasobserved probablyduetothepretreatmentwashingprocedure.

Studies reporting the effects of process parameters [4,15,16,18,21,22] showed that when FD was performed, the highest extraction kinetics were obtained for pressures higher than 300bar and temperatures close to 323K. The highest extractedamountofchlorophylla(Chla)and totalcarotenoids withpureSC−CO₂wasobtainedat400barand60^◦C.Thehighest carotenoids/Chl a concentration ratio was obtained at 200bar and333K[16].Theyieldofextractedpigmentsdependsonthe operating conditions (influencing the SC−CO₂ density and the pigmentvaporpressure)andonthenatureoftheotherextracted compoundsthatcanplaytheroleofco-solvents[18].

Whentheeffectsofco-solventswereinvestigated[4,19,22–25], itwaspossibletoobservethatethanolwasthemostappropriate co-solventwhenpigments(chlorophyll,carotenoids,...)weretar- geted.TheyieldofextractiondependedstronglyontheSC−CO2

densityandtheamountofethanolintroduced[4].Whenacetone wasusedasco-solvent,theextractionyieldandlipidicextractcom- positionweren’tsignificantlyenhancedcomparedtotheextraction withpureSC−CO₂at320barand313K[19].Furthermore,nosig- nificantdifferencewasfoundinthefattyacidsprofile.Azeotropic mixture (Hexane/Ethanol) helped to extract a high amount of neutralandpolarlipidsbutalsoEPAat340barand353Kwhen microwavepretreatmentwasappliedtothedriedrawbiomassto enhancethecelldisruption[24].

AreviewofthestudiesreportedinTable1offersinsightsbut also identifies gaps in the consistency of reported information (particlesizediameter,initialwatercontent,descriptionofdry- ingmethod,...).Thesemissingdataareofprimeimportancefor comparisonpurposesandscaleupstudies.Fewstudiesreportpre- treatmentconditionssuchasdryingtreatmentmodeandeffects ofhighwater−COntent.Finally, theextractionof pigmentswas mainlyinvestigatedusingaco-solvent.AdditionalSC−CO₂inves- tigationsareneededtofillthesegapssoindustrialscale-upofthis technologycanberealized.

ThegoalofthisstudyistodeepenthestudiesontheSC−CO₂ extractionofvaluablecompounds(neutrallipids,chlorophylland total carotenoids) from Nannochloropsis species by investigat- ingtheeffectsofoperating parameters(pressure,temperature), pretreatment (drying modeand effects of highwater content) with pure SC−CO2 and ethanol as co-solvent. Two species of

Table1

MainrecentstudiesrelatedtotheSC−CO2extractionofvaluablecompoundsfromNannochloropsisspecies.

ExtractionwithpureSC-CO2

Microalgae pre-treatments/particlesize P(bar) T(K) CO2flowrate Extractiontime

(h)

Major extracted compounds

Bestyield/goal References

Nannochloropsissp. FD,dp=370␮m 400,500,700 313,328 10kg/h 6 C16:0,

C16:1:n-7,EPA

Highestextractionyield:25%at 700barand328K/Investigatethe effectsoftheoperatingconditions onextractionkinetics,yieldand fattyacidcomposition.

[15]

Nannochloropsis gaditana

FD 100,200,300,

400,500

313,323,333 0.012kg/h 3 Chla,

carotenoids

Highestextractionyieldat400bar and333K/Theaimwasto ascertaintheinfluenceofpressure andtemperatureonSC-CO2

extractionofChlaandcarotenoids.

[16]

Nannochloropsissp. Ovendryingat343K Grinding

125,200,300 313,333 0.021, 0.037kg/h

– Neutrallipids Bestextractionyield:33%at 300bar,313Kand0.037kg/h/The aimwastousethebiomassand applybiorefineryapproachforthe productionofoil,highadded-value compoundsandbiohydrogen.

[4]

Nannochloropsis oculata

AFDat308K,FDwatercontent 4-5wt%and20wt%dp<160␮m, dp=315-1000␮m

400 333 0.5kg/h – C16:0,

C16:1␻7, C18:1␻9

Extractionyield:12%forAFDand dp<160␮m/Investigatetheeffects ofhighwater-content,drying modeandparticlesizeonextract compositionandextraction kinetics

[11]

Nannochloropsis oculata

AFDat308K,FD

watercontent4-5wt%and20wt%

dp<160␮m,dp=315-1000␮m nitrogenlimitationculture

400 333 0.4,0.5kg/h – C16:0,

C16:1␻7, C18:1␻9, C20:5␻3

Extractionyield:30%forAFD, nitrogenlimitationcultureand dp=160-315␮m/Investigatethe effectsofpre-treatmenton extractionkineticsandmodelling.

[17]

Nannochloropsis gaditana

dp=616␮m,watercontentof 30wt%

317,377,381, 425,464,479, 533,543

309,313,323, 333,337

1.2 Carotenoids,

tocopherol, C16:0,C16:1, C18:1,EPA

Bestextractionconditionsfor carotenoids:533barand337K/ Studyoftheeffectsoftemperature andCO2densityonthecontentof carotenoidsandtocopherols.

[18]

Nannochloropsis salina

Dryingatatmosphericpressure andtemperature(298K)for16h, Watercontentof4.12wt%, dp=500-1000␮m

320 313 1.5kg/h – Palmitoleic

acid,Palmitic acid,Oleicacid, EPA

Extractionyield:6.25%/ Investigatetheextractionoflipids andvaluablecompoundsfrom microalgae.

[19]

Nannochloropsis oculata

Washedwithammoniumformate toremovesalt,driedina dehydratorat318Kduring40h

300 313 9mL/min 2 C16:0,C16:1,

EPA,total carotenoids

Extractionyield:15.6%/ Comparisonoffattyacid composition,totalcarotenoidsand antioxidantswithsub-critical n-butaneextraction.

[20]

Table1(Continued) ExtractionwithpureSC-CO2

Microalgae pre-treatments/particlesize P(bar) T(K) CO2flowrate Extractiontime

(h)

Major extracted compounds

Bestyield/goal References

Nannochloropsis oculata

FD,dp=200␮m 250,450,750 323 1.5kg/h 2 Neutrallipids,

glycolipids, phospholipids

Extractionyield:20%at750and 450bar/Investigatetheselective extractionofneutrallipidsfrom microalgae.

[21]

ExtractionwithSC-CO2+co solvent

Microalgae pre-treatments/particlesize co-solvent P(bar) T(K) CO2flowrate Extractiontime (h)

Major compounds

Yieldandorgoal References

Nannochloropsis gaditana

FD Ethanol5mol% 200,300,400,

500

313,323,333 0.012kg/h 3 Carotenoids

and chlorophylls

Highestyieldofcarotenoidsand chlorophyllsat500barand333K/ Theaimwastoascertainthe influenceofbothpressureand temperaturewhenusingethanol asco-solvent.

[22]

Nannochloropsissp. Ovendryingat343K,grinding Ethanol5, 10wt%

125,200,300 313,333 0.021, 0.037kg/h

– Bestextractionyield:44%at

300bar,313Kand0.021kg/h/The aimwastousethebiomassand applybiorefineryapproachforthe productionofoil,highadded-value compoundsandbiohydrogen.

[4]

Nannochloropsis salina

Ovendryingat310Kfor2days, watercontent16wt%,dp<500␮m

Ethanol5wt% 300 318 0.4kg/h 1.5 C16:0,

C16:1cis␻7, EPA

Extractionyield:30.4%/Increase therecoveryofomega-3andalpha linoleicacidintheextract.

[23]

Nannochloropsis salina

Dryingatatmosphericpressure andtemperature(298K)for16h, waterontentof4.12wt%

dp=500-1000␮m

Acetone 3.2wt%

320 313 1.5kg/h 1.5 Palmitoleic

acid,Palmitic acid,Oleicacid, EPA

Extractionyield:6.38%/Extraction oflipidsandvaluablecompounds frommicroalgae.

[19]

Nannochloropsis salina

Classicalmicrowaveovendrying Watercontentof6wt%

Hexane/ ethanol4,8 and12wt%

200,270and 340

313,333,353 6kg/h 1 EPA,C16:1,

C18:1,C16:0

Bestextractionyield:31.37%at 340bar,353Katsolventtosolid ratioof12wt%/Investigationof theSC-CO2extractionoflipids fromalgalbiomassusing azeotropicco-solvents.

[24]

Nannochloropsissp. WashingstepwithMiliQwaterto removesalts,FD,Grinding

Ethanol15%v/v 230and380 308,333 2mL/min 6 Freefattyacids, neutrallipids, phospholipids

Extractionyield:23.1%at230bar, 333K/Createosmoticgradientto damagealgaecellsforabetter extractionyield

[25]

Nannochloropsis were considered: Nannochloropsis maritima (N.

maritima)and Nannochloropsis salina (N.salina).To ourknowl- edge,noSC−CO₂extractionexperimentshavebeenreportedonN.

maritima.Theeffectsofextractionprocessparametersusingpure SC−CO2(AFDpretreatment)onN.maritimausingResponseSur- faceMethodology(RSM)wasinvestigated.Tocompletethestudies reporting effects of drying mode onmicroalgae [10,11,17],the effectsofanewdryingmodewasinvestigatedwithringdrying (RD)asaviablepretreatmentoption.Aringdryerisanindustrial- scale,pneumaticsystemthatwasdevelopedtoincreaseversatility of flashdrying technology. RDis described indetail in thefol- lowing references[26–28].It is usedin a numberof industries andoperatesbyexposingaslurrytosuperheatedairinacircular formfactorthatfacilitatescontrolleddryingconditionsandpre- ventsoverheatingofthefinalproduct.Dryingtimeissignificantly shorterthanAFD(afewsecondsversusseveralhours)andthuslim- itstheriskoflipiddegradation.Toourknowledge,thisdryingmode hasnotbeenreportedforSC−CO2extractionintheliterature.The SC−CO₂extractionofvaluablecompoundsfromRDN.salinawas investigatedwithpureCO₂andwithethanolasco-solvent.Anew methodtodisruptcellwallsbyimpregnatingdriedmicroalgaewith ethanolwasperformed.Theeffectofahigh-watercontentofthe biomassontheextractionkineticsandextractedcompoundswas alsoinvestigated.Experimentalextractioncurvesweremodelled usingSovová’sbrokenandintactcell(BIC)mathematicalmodelto betterunderstandthemechanismofextractionkinetics.

Materialsandmethods

Rawmaterialsandchemicals

NannochloropsissampleswereprovidedbyCellanaLLC(Hawaii, USA).Thealgaeculturesweregrowninoutdoorconditions,har- vested,and thickenedto22%solidswitha Westfaliadiskstack centrifuge.N.maritimasampleswereprovidedbothasringdried (RD)materialandaswetpastematerial.N.salinasampleswere providedasRDmaterial.Theringdryerusedinthisstudyoperated at353Kandtheaverageretentiontimewaslessthan30s.Thewet pastewasairflowdried(AFD)inacirculatingairoven(classical plantdryer)SFP6type(R.E.U.SFrance)whichwassettotheopti- mumconditionsof308Kfor20haccordingtoapreviousstudy[10].

Thefinalwatercontentoftheresultingsampleswasabout13wt%

andabout3.4wt%forAFDandRDbiomassrespectively.Thedried biomasswasgroundintopowderandsizedintohomogenousparti- clesizerangeswithavibratorysieveshakeratameanparticlesize of300–500␮m.Algaepowderwasstoredat277Kpriortoexperi- mentstoavoiddegradationofthecompounds.SC−CO2extraction experimentswerecarriedoutwithcarbondioxideof99.7%purity suppliedbyAirLiquideMéditerranée(France).Absoluteethanol withapurityof99.8%,usedasco-solvent,wassuppliedbyFisher (France).

Experimentalsetup

Theprocessdiagramoftheexperimentalsetupisgivenin Fig.1Extractionswereperformedusinglaboratoryscaleequip- mentprovidedbySEPAREX(Champigneulles,France).TheCO₂high pressurepump(3)usedforthisstudywasahigh-pressureliquid CO₂ pumpJascoPU-4386(JascoFrance).Ahigh-pressuresolvent pump(10), GILSON307Pumpwithstandardheadpumpof5SC type(GilsonInc.,USA),wasconnectedtotheextractionpilotin ordertocleanthepipesandthemicrometricvalve(V3)attheend ofeachexperimentorforco-solventextractions.Allexperiments werecarriedoutina10cm³ extractionvessel(6)filledtoavol-

umeof80%,whichcorrespondedtoasamplemassof5gforeach experiment.

Considering the small charges of biomass in the extraction vessel,theamountofextractedneutrallipidsEwascalculatedcon- sideringthemasslossesoftheextractionautoclave.Weightlosses wererecordedpriortoextractionand30minafterextractionto allowliberationofresidualCO₂.Extractsampleswererecovered in glassvesselswhich weresubsequentlytoppedwithnitrogen and storedat 255K untilcompositional analysis.Theyield was expressedasthe massratioof thesample massloss(E)tothe massintroducedinitiallyintheextractionautoclave(N).Thisyield wasassimilatedtoneutrallipidsyieldsinceitiswellknownthat SC−CO₂solubilizesnonpolar-lipids.

Extractionexperiments withpureCO₂ were conductedwith valvesV4andV5closed.TheCO2flowandpressurewerecontrolled withmicrometricvalveV3.TheCO₂flowratewasmeasuredthanks toaflowmeterlocatedattheendoftheextractionline(8).When theextractionexperimentswerecompleted,pipesandmicromet- ricvalvewerewashedwithabsoluteethanolbyclosingvalveV2 andopeningvalveV4.Theapparatuswasthendriedwithaflow ofgaseousCO2duringafewminutes(ValvesV2,V3andV5open, valveV4closed).

Forexperimentswithco-solvent,absoluteethanolwasadded atsystempressuretoSC−CO2viaauniontee(1/8)priortothe extractionvessel(bluelinein

Fig.1).Onlyaqualitativeevaluationoftheextractionismade byanalyzingtheextractcomposition.Co-solventexperimentswere conductedfor3handtheextractswereanalyzedforcomposition asdescribedbelow.

ExperimentsperformedonN.Maritima

ExtractionexperimentsperformedondriedN.maritimawere conducted without co-solvent at a constant CO2 flow rate of 0.15kg/h.Theeffectsofpressureandtemperatureontheyieldof extractedneutrallipids,onChlaandtotalcarotenoidsconcentra- tionintheextractsataCO2/microalgaemassratioof180kg/kg were investigated using Response Surface Methodology (RSM).

Threelevelswereconsideredforthepressureandthetempera- ture(-1,0,1respectivelyfor pressuresof 100,200and300bar andtemperaturesof313,323and333K).Thedegreeofsignifi- canceofeach factor(p-value)wasgivenbyNemrodWsoftware and wasdiscussed.A classicalplancomposedof9experiments wasconsidered.EachresponseY(neutrallipidsyield,chlorophylla concentrationandtotalcarotenoidsconcentration)wasmodelled usingasecond-orderpolynomialmodelgiveninEq.1.

Y =b0+b1P+b2T+b11P²+b22T²+b12.T.P (1) Whereb₀,b₁,b₂,b₁₁,b₂₂,b₁₂arethecoefficientoftheresponsesur- faceequationcalculatedbyNemrodWsoftware(LPRAI,Marseille, France).

Theeffectofdryingmodeonextractionkineticswasinvesti- gatedat100barand313K.

AllexperimentalconditionsweresummarizedinTable2.

ExperimentsperformedonN.Salina

SC−CO₂extractionexperimentsonRDN.salinawereperformed atpressuresof300and400bar,atemperatureof333KandaCO₂ flow rateof 0.15kg/hwithpureCO2,ethanol asco-solventand withbiomassathighwatercontent.Theoperatingconditionswere chosenbybuildingonthepreviousreportedstudy[22].Allexper- imentsperformedonRDN.salinawerereportedinTable2.

Twokindsofexperimentswereperformedusingethanolasco- solvent.Thefirstexperimentconsistedinimpregnatingtheground andsievedmicroalgaewithabsoluteethanolfor24h.Thebiomass