h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /
Industrial
Microbiology
Enhancement
of
pigment
extraction
from
B.
braunii
pretreated
using
CO
2
rapid
depressurization
Edgar
Uquiche
a,b,∗,
Ivette
Antilaf
a,
Sonia
Millao
aaDepartmentofChemicalEngineering,CenterofFoodBiotechnologyandBioseparations,BIOREN,UniversidaddeLaFrontera(UFRO),
Temuco,Chile
bAgriaquacultureNutritionalGenomicCenter,CGNA,TechnologyandProcessesUnit,UFRO,Temuco,Chile
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received6March2015 Accepted25September2015 Availableonline2March2016 AssociateEditor:GiseleMonteirode Souza Keywords: Botryococcusbraunii Celldisruption Chlorophylls Microalgae Supercriticalextraction
a
b
s
t
r
a
c
t
Extraction ofcompoundsfrommicroalgaerequirescelldisruption asa pretreatmentto increaseextractionyield.Botryococcusbrauniiisamicroalgawithasignificantcontentof carotenoidsandotherantioxidantcompounds,suchaschlorophylls.CelldisruptionofB. brauniiusingCO2rapiddepressurizationwasstudiedasapretreatmentfortheextractionof
carotenoidandchlorophyllpigments.Westudiedtheeffectoftemperature(21–49◦C)and pressure(6–13MPa)duringstaticcompressiononpigmentrecoverywithsupercriticalCO2at
40◦C,30MPaandsolventflowof4.7LNPT/min.Withintheexperimentalregion,the extrac-tionyieldofcarotenoidsandchlorophyllsincreasedby2.4-and2.2-foldrespectively.Static compressionconditionsofhighpressureandlowtemperatureincreasedtheextractionof carotenoidsandespeciallychlorophylls.Weselected21◦Cand13MPaasthecelldisruption condition,whichproduced1.91g/kgd.s.ofcarotenoidsand14.03mg/kgd.s.ofchlorophylls. Pretreatedmicroalgagavea10-foldhigherchlorophyllextractionyieldcomparedtothe untreatedsample.Whileforcarotenoidsandtocopherolswere1.25and1.14-foldhigher, respectively.Additionally,antioxidantactivityofpretreatedmicroalga(33.22mmolTE/kg oil)wassignificantlyhigherthanthevaluefortheuntreatedsamples(29.11mmolTE/kgoil) (p≤0.05).Confocalmicroscopyimagesshowedmorphologicaldifferencesbetween micro-colonieswithandwithoutdisruptiontreatment,suggestingthatpartialcelldisruptionby rapiddepressurizationimprovedtheextractionofmicroalgacompounds.
©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Microalgaeareabroadgroupofautotrophicorganismswhich growby photosynthesis and can becultivated as a source
∗ Correspondingauthor.
E-mail:[email protected](E.Uquiche).
ofbioactivecompoundswithhighcommercialvalue.1
Botry-ococcus braunii isaunicellular green microalgaofthe class
Chlorophyceae,characterizedbytheproductionofchlorophyll pigments.B.brauniicellsareheldtogetherbyan extracellu-larmatrixcomposedofacross-linkedaldehydepolymercore
http://dx.doi.org/10.1016/j.bjm.2016.01.020
1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
andarecapableofproducinglargeamountsofhydrocarbons, exopolysaccharidesandcarotenoids.2,3 Thesehydrocarbons
arelargelystoredintheextracellularmatrix.4B.brauniiis
clas-sifiedintothreeracesA,BandL,dependingonthetypesof hydrocarbonsproduced.5Thepresenceofcarotenoidsismore
pronouncedinracesBandL.6Thecarotenoidsfoundinclude
-carotene,lutein,violaxanthin,canthaxanthin,astaxanthin, zeaxanthin.7,8 B. braunii isan interestingmicroalga forthe
extractionofhigh-valuecompoundsforusesinnutraceutical applications.7,9
Oneofthecharacteristicsofmicroalgaeistherigidityof theircellwalls.InB.brauniithewallofeachcellhasan inter-nal fibrillar layer made of polysaccharide and an external trilaminarsheath.4 CellwallofB. braunii iscomposed ofa
cellulose-likepolysaccharide(as-1,4-and/or-1,3-glucan).10
Cell disruptionis thereforenecessary torelease intracellu-larcompoundsandimproveextractionsolventaccess.11The
followingmethodshavebeenusedformicroalgalcell disrup-tion: sonication,11 high-pressure homogenizers,12 chemical
disruption,13enzymaticdegradation,14beadmilling,11,15and
microwaves.16
Studiescomparing methodsofmicroalga celldisruption have been reported inliterature. Differentmethods ofcell disruptiontoidentifythemosteffectivemethodfor extract-inglipidsfrommicroalgae(Botryococcussp.,Chlorellavulgaris,
and Scenedesmus sp.) wasinvestigated.13 Amongthe
meth-odstested(autoclaving,beadmilling,microwaves,sonication, andtreatmentwith10%NaClsolution),themicrowaveoven wasthemostefficientforlipidrecovery.Inotherstudywas investigateddifferentcelldisruptionmethodsforextracting lipidsfrommicroalgae(Chlorellasp.,Nostocsp.andTolypothrix
sp.),includingautoclaving,beadmilling,microwave, sonica-tionandtreatmentwith10%NaClsolution.17Thesonication
wasthe mostefficient methodforlipid recovery. However, thesonicationmethodhasbeenindicatedtobeunscalable. Beadmillingandhigh-pressurehomogenizingarescalablefor industrialuse.Celldisruptionbybeadmillisbasedon sub-jectingcellstohighstressproducedbyabrasionduringrapid agitationwithglassorceramicbeads.Thismethodiseffective withdifferenttypesofmicroorganism.18Incelldisruptionby
high-pressurehomogenizer,thecellsuspensionisforcedto passthroughanadjustabledischargevalvewitharestricted orifice.19 Castor and Hong,20 pointed out that mechanical
celldisruptionmethodsarenon-selectiveincellwall disrup-tion;this leadstotheformation ofsmall fragmentsofcell wall,increasingthedownstreampurificationburdenbecause these fragments are difficult to separate from the process stream.18
Gasparetal.21studiedtheeffectofthedecompressionrate
ondisruptionefficiencyintrichomesfromoriganumbracts. Theyobservedthatasthedecompressionrateincreased,the pressuredropacrosstheglandwallalsoincreased,resultingin higherdisruptionefficiency.Thus,disruptionoftheseglands wascausedbyapressure gradientformedacrossthegland wallsduringrapiddepressurization.DuringtheCO2
compres-sionstage,glandswereslightlypermeabletothepassageof CO2byaprocessakintodiffusion.
Studies ofcell disruptionusing CO2 rapid
depressuriza-tiontoimprovetheavailabilityofextractedsoluteshavebeen reportedinliterature.Thismethodisbasedonintroducing
a pressurized subcritical or supercritical gas into the cells followedbyrapiddepressurization,causingcelldisruption.18
During thestageofstatic compression,supercriticalCO2 is
verydiffusibleand canpenetratecells.21Afterthecells are
saturated with CO2, a sudden depressurisation is applied
and a pressure gradient across the cell wall is generated. Theyobservedthatasthedecompressionrateincreased,the pressure drop across the glandwall alsoincreased, result-inginhigherdisruptionefficiency.Thus,disruptionofthese glandswascausedbyapressuregradientformedacrossthe gland wallsduringrapiddepressurization.The cell disrup-tionoccursduetotheexpansionoftheCO2,whichimproves
thespeedandincreasestheextractionyield.22,23Furthermore,
cellsareexposedtominimalshearforces,sonoheatis gen-eratedwhichmightdamageheatsensitivecompounds,such ascarotenoidsandchlorophylls.
Temperature, pressure and exposure time during static compression are the operational variables that may affect the efficiency of cell disruption.24 Juhász et al.23 studied
the effect ofCO2 rapiddepressurization on endoglucanase
recoveryfromEscherichiacoli.Thetemperature(32–45◦C)and pressure (12–25MPa) affected the recovery of the enzyme, whileexposuretime(5–60min)hadnosignificanteffect.Rapid depressurization has been applied to disruption bacterial cells,25–27yeastcells,28,29gojiberryseeds,30rapepollen31and
trichomes.21,32
Extraction withsolvents isthe traditionaltechniquefor lipid extraction from microalgae. Lipids are traditionally extracted using nonpolar solvents, commonly n-hexane.33
However,theUnitedStatesEnvironmentalProtectionAgency listed n-hexaneamong187hazardous airpollutantsinthe 2002 National-Scale Air ToxicsAssessments because of its toxic nature.34 Supercritical fluid extraction has received
increasing attentionas anextractiontechnique, because it can provide high solubility, improved mass-transfer rates, and increased selectivity with small changes in the tem-peratureandpressureoftheextractionoperation.35 Carbon
dioxide (CO2) is probably the most widely used
supercrit-ical fluid, and has emerged as a substitute for n-hexane for the extractionof nonpolar solutesfrom biological sub-strates, duetoits inertness,non-toxicity, non-flammability andnon-explosiveness.36Furthermore,itsrelativelylow
crit-icalpropertiesmakeCO2(Tc=31.1◦C,Pc=7.38MPa)anideal
solventfortheextractionofthermallylabilecomponents.We studiedextractionfromB.brauniiusingCO2supercriticalfluid
at40◦Candpressures of12.5,20 and30MPa.37The
extrac-tionyieldandthefractionofthehydrocarbonsintheextracts bothincreasedwithpressureandat30MPathesecompounds wereobtainedrapidly.Theauthorsreportedthatchlorophylls werenotdetectedintheextracts.Santanaetal.38studiedthe
extractionoflipidsfrom B.braunii forbiodieselproduction. Theseexperimentswereconductedattemperaturesfrom50 to 80◦C and pressures from 20 to 25MPa. Lipid extraction yieldwasfoundtodecreasewithtemperatureandtoincrease with pressure.Carotenoids and chlorophylls are important antioxidants.39,40 Thesepigments can beextracted from B.
brauniiusingsupercriticalCO2,andcanbeusedasan
indica-torofcelldisruptioninthecaseofphotosyntheticmicroalgae speciescontainingchlorophyllandcarotenoids,becausethey arereleasedwhenthecellcollapseoccurs.41
Cell disruptionusing rapiddepressurization can be car-riedoutinsupercriticalextractionequipment,sothatafter pretreatment, temperatureand pressure conditions are set tocarryout the supercriticalextractionofthe lipidic com-pounds.Thisproceduresavestimeandminimizesequipment andlaborrequirementsaswellascontaminationandextract loss.24Also,itcanbescaledup toindustrialscale.20If
pre-treatmentandextractionareconductedinthesameextractor, thesubstratewillbeprotectedfromexposuretooxygenand hightemperature,avoidingoxidationreactions.Microalgacell disruptionusingCO2rapiddepressurizationhasnotyetbeen
reported in literature. In this work, we hypothesized that celldisruptionofB.brauniiusingrapiddepressurizationmay enhance the extractionyieldofpigments, carotenoidsand chlorophylls,byadjustingthetemperatureandpressure con-ditionsunderstaticcompression.Theobjectiveofthiswork wastoimprovetherecoveryofpigmentsfromB.brauniiwith supercriticalCO2,throughapretreatmentofcelldisruption
using CO2 rapiddepressurization. Responsesurface design
wasusedtoevaluatetheeffectofthestaticcompression con-ditionsonpigmentrecoveryfromB.braunii.
Materials
and
methods
Substrate
B.brauniiUTEXLB572wassuppliedbyUniversidadde Antofa-gasta (Antofagasta, Chile). This strain corresponds to race Bandwasculturedunderoutdoorproductioninpilot-scale panel reactors.42 Microalga samples consisted of air-dried
microalgae,whichwerecarefullymilledwithmortarand pes-tleuntilaparticlesizeoflessthanTylermesh40wasobtained. Averageparticlesizewas0.244mm.Themicroalgasamplehad amoisturecontentof7.9±0.2/100gdrysubstrate(d.s.) (deter-minedgravimetricallybydryinginanovenfor10hat102◦C) andanoilcontentof106±2g/kgd.s.(determined gravimet-ricallybyextractionwithtechnicalgradehexaneinSoxhlet apparatusfor10hat70◦C).Sampleswerestoreduntilusein dry,darkconditions,packedintheabsenceofoxygen.
Substratepretreatment
Theinitialsampleofmicroalgasamplesconsistedofair-dried microalgae. Thissample was divided into two fractions: a fractionwithouttreatmentbyrapiddepressurization(as con-trol),andtheotherfractionsubjectedtotreatmentbyrapid depressurization.ThepretreatmentusingCO2rapid
depres-surization began by loading ca. 5g of microalgae sample (substratecharacterized)intoa50cm3extractionvessel
(14-mminternaldiameter).Theextractionvesselwasplacedin anair-convectionovenofaSpe-edSFEunit(Applied Separa-tions,Allentown,PA).Priortopretreatment,airtrappedinthe extractionvesselwaspurgedbymeansofacontrolledflowof CO2.Theextractionvesselwasthenpressurizedwith
high-purity(99.95% pure)CO2 (Linde, Santiago, Chile), and kept
understaticcompression for1h, atdifferentcombinations oftemperature(21–49◦C) andpressure (6–13MPa).TheCO2
wasthenquicklyreleasedbyopeningavalve,whichallowed thepressuretodiminishtonormalatmosphericpressure;the
extractionvesselwasthenkeptatatmosphericpressurefora further10min.
Supercriticalextraction
Itwasthenre-pressurizedandthepretreatedsubstratewas extracted at 40◦C (temperatureof the air convection oven containing the extraction vessel, controlled automatically) and30MPa(extractionpressurecontrolledmanuallywithan air-booster pump), with 4.7L NPT/min of CO2 with a
sin-glesuperficialvelocityof1mm/s.Inallcases,theextraction wascarriedoutfor60min(correspondingtoaspecific con-sumptionofsolventof101.5kgCO2/kgd.s.),afterwhichthe
expansion valve (kept at120◦C) was opened. Of this way, rapid depressurization ofthe microalgae cells and consec-utivesupercriticalCO2extractionwerecarriedoutwiththe
samesubstratechargewithintheextractorvessel.This pro-cedureissimilartothatreportedinothersstudies.31,32The
oilrecoveredwasassessedgravimetricallybydifferenceusing cleanedanddriedglassvials,andtheextractedoilyield(Yoil,
g/kgd.s.)wasmeasured.Eachexperimentalassaywas con-ductedinduplicate.Recoveredoilextractswereusedforlater analysis.
Analysisofextracts
Carotenoid concentration (Ccar, g carotenoids/kg oil) was
quantified in an oil sample dissolved in chloroform p.a. (Merck,Darmstadt,Germany).Absorbancewasreadat452nm byspectrophotometryinaGenesys10SUV-Vis spectropho-tometer(ThermoFisherScientificInc.,Madison,WI).32 The
standard fortheanalysisofcarotenoids(-carotene typeI, ≥95% pure)was obtainedfrom Sigma–Aldrich (SaintLouis, MO). The carotenoid extraction yield (Ycar, g/kg d.s.) was
obtainedfromYoil×Ccar.Chlorophyllconcentration(Cchlor,mg
chlorophyllsaandb/kgoil)wasquantifiedinanoilsample dissolvedinethyletherp.a.andabsorbancewasreadat642 and 660nm inthe spectrophotometer.Chlorophyllaand b
contentsweredeterminedusingequationsreportedby Wrol-stadetal.43Thechlorophyllextractionyield(Y
chlor,mg/kgd.s.)
wasobtainedfromYoil×Cchlor.Thetocopherolconcentration
(Ctoc,gtocopherol/kgoil)ofselectedoilsampleswas
quanti-fiedat520nmin␣-tocopherolequivalents.44Thestandardfor
theanalysisoftocopherols␣-tocopherolwasobtainedfrom Sigma–Aldrich.Thetocopherolextractionyield(g/kgd.s.)was estimatedfromYoil×Ctoc.Antioxidantactivityofselectedoil
sampleswasmeasuredusingaTroloxequivalentantioxidant capacityassay.45Antioxidantactivitywasexpressedas
mil-limoleTroloxequivalent/kgoil.Troloxstandardwasobtained fromSigma–Aldrich(SaintLouis,MO).
Confocalmicroscopy
Controlsampleandmicroalgaepretreatedwithrapid depres-surization were observed with confocal microscopy. A microalgasample(100mg)wassuspendedin1mLof phos-phatebuffer(pH7.5at25◦C)inanEppendorftubeandfiltered throughaMilliporefilter(MilliporeCorp.,Bedford,MA).A solu-tion ofwhite calcofluor(Sigma–Aldrich, SaintLouis,MO)in dimethylsulfoxide(MerckKGaA,Darmstadt,Germany) was
Table1–Extractionyieldofcarotenoids(Ycar)andchlorophylls(Ychlor)byCO2at40◦Cand30MPaasafunctionof
temperatureandpressureofstaticcompression.
Run T [◦C] P [MPa] X1 [–] X2 [–] Ycar [gcar/kgd.s.] Ychlor [mgchlor/kgd.s.] 1 25 7.0 −1 −1 1.12 12.19 2 45 7.0 1 −1 0.73 11.53 3 25 12.0 −1 1 1.46 13.78 4 45 12.0 1 1 1.29 9.97 5 21 9.5 −1.41 0 1.72 10.78 6 49 9.5 1.41 0 1.00 7.44 7 35 6.0 0 −1.41 0.78 12.94 8 35 13.0 0 1.41 1.11 16.03 9 35 9.5 0 0 1.02 13.04 10 35 9.5 0 0 1.01 12.68 11 35 9.5 0 0 0.93 11.91 12 35 9.5 0 0 0.99 12.41
prepared in 1:10 ratio v/v. Suspension of microalgae was markedwith100Lofcalcofluorsolutionandincubated at roomtemperaturefor15min.Thesuspensionwascentrifuged inaHitachi centrifugeCT15E(HitachiKokiCo.,Ltd.,Tokyo, Japan). Microalgae samples were viewed using a Confocal LaserMicroscopeFV-1000(OlympusCorp.,Tokyo,Japan). Flu-oViewsoftwareFV-102.0(OlympusCorp.,Tokyo,Japan)was usedforimageacquisition(40×magnification).
Experimentdesign
Central composite rotatable design (CCRD) was used to evaluatetheeffectsoftheindependentvariables:coded tem-perature(X1,Eq.(1),whereTistemperature◦C),andcoded
pressure (X2, Eq. (2), where P is pressure in MPa), both
expressedindimensionlessunits,ontheresponsevariables: extractionyieldofcarotenoids(Ycar)andchlorophylls(Ychlor).
X1= T−35
10 (1)
X2= P−
9.5
2.5 (2)
The design was based on a two-factor factorial design (n=2),withtwolevels(codedvalues−1and+1.Thefactors
andtheirlevelsareshowninTable1.TheCCRDmatrixhad 4(2n)cubepoints(runs1–4)and4(2n)starpoints(runs5–8),
atanaxialdistancetothe centerof1.41 (˛=2n/4),and four
replications of the center points (runs 9–12) to determine experimental error(Table1). Experiments were carried out inarandomizedordertominimizetheeffectofunexpected variability inthe observedresponseduetoextraneous fac-tors.Asecond-ordermodel(Eq.(3))wasusedtodescribethe responsevariableYasafunctionofcodedtemperature(X1)
andcodedpressure(X2),
Y=A0+A1X1+A2X2+A12X1X2+A11X12+A22X22 (3)
whereA0isaconstant;A1andA2arelinearcoefficients;A12
isacross-productcoefficient;andA11andA22arequadratic
coefficients.Three-dimensionalsurfaceresponseplotswere generated by varying the two variables within the experi-mental range. The model fit was evaluated byanalysis of variance (ANOVA). Thecoefficientsofthe responsesurface equationwereestimatedusingDesignExpertDesign-Expert Software,version6.0.1(Stat-Ease,Inc.,Minneapolis,MN).The statistical significancewasbasedon thetotal errorcriteria withaconfidencelevelof95%.
Table2–Analysisofvarianceofregressioncoefficientsandstatisticalindicatorsofappropriatenessofthesecondorder modelselected.
Regressioncoefficient Ycar Ychlor
Estimate p-Value Estimate p-Value
A1 −0.1972 0.0003 −1.147 0.0080 A2 +0.1708 0.0009 +0.543 0.1274 A2 1 +0.1892 0.0008 −1.682 0.0019 A2 2 +1.018 0.0234 F-value 29.89* 13.75* r2 0.918 0.887 Adjustedr2 0.888 0.821 Lackoffit 8.00ns 5.30ns Signal-to-noiseratio 16.63 13.63 ∗ Significantatp≤0.001;ns:non-significant(p>0.05).
Results
and
discussion
The experimental results of the extraction yields of carotenoids(Ycar)andchlorophylls(Ychlor)asafunctionofthe
temperatureandpressureappliedduringstaticcompression areshowninTable1.Experimentaldataistheaverageoftwo measurements.Wenotethatthepressuredropoccurred in thefirst 10s.For celldisruption ofrapepollencollected by bees,wasreportedthatthepressure(treatmentat45MPa)was quicklyreleasedwithin1min.31Fortreatmentof
microorgan-ismwithcompressedCO2,wasreportedthatarapidrelease
oftheCO2 pressure(1.5–5MPa)waswithin4s.26 Oil
extrac-tion yield (Yoil) ranged from 84.43 to 103.01g/kg d.s. (full
datanotshown).Carotenoidextractionyield(Ycar,g/kgd.s.)
rangedfrom0.73to1.72g/kgd.s.,2.4-folddifferences. Chloro-phyllextractionyield(Ychlor,mg/kgd.s.)rangedfrom7.44to
16.03mg/kgd.s.,2.2-folddifferences.
Table2summarizesthestatisticalindicatorsobtainedfrom
theanalysisofvarianceappliedtothe second-ordermodel selected (Eqs. (4) and (5)). The modelwas considered ade-quatebecauseofthesignificanceofthemodel(p≤0.001),the non-significance ofthe lack offit (p>0.05)relative topure error,thehighsignal-to-noiseratio(>4)andhighcoefficientof determination(r2).Forinstance,thiscoefficientindicatesthat
themodelexplains91.8%ofthevariabilityinthecarotenoid extractionyieldYcar.The informationprovidedbythe
sta-tisticalindicatorswas complementedbyagoodcorrelation between predicted and experimental responses withinthe experimentalrangeinvestigated,forbothresponses,sincethe plotshowsaclosefitoftheexperimentalwiththepredicted values(datanonshown).
Extractionyieldsofcarotenoidsandchlorophylls
Analysisofvariancewasusedtoevaluatethesignificanceof themodel’sregressioncoefficients(Table2).Alargeregression coefficientandasmallp-valuewouldindicateamore signif-icanteffectontheresponsevariable.Significantcoefficients (p>0.05)wereusedtowritethesecondordermodels.The vari-ablewiththelargesteffectonthecarotenoidextractionyield wasthelineartermofthetemperature(p=0.0003),followed bythequadratictermofthetemperature(p=0.0008)andthe lineartermofthepressure(p=0.0009).Therewasno signifi-canteffectofthequadratictermofthepressure(p=0.5212), noroftheinteractiontermbetweentemperatureandpressure (p=0.2708).Thusthesecond-ordermodel(Eq.(4))establishes a statistically significant relationship between carotenoid recoveryandthetemperatureandpressureconditionswhen carryingoutrapiddepressurizationintheselected experimen-tal range(6≤P≤13MPa;21≤T≤49◦C). Coded variables are importantbecausetheygiveadirect,quantitativeindication oftheeffectoftheindependentvariablesonanydependent variable as a function of the selected experimental range. ThetermA2(=+0.1782),whichmultipliesthelineartermX2,
indicates that the carotenoidextraction yield increases by 0.1782g/kgd.s.whenthecompressionpressureincreasesby 2.5[=0.5(12–7)]MPawhileoperatingat35◦C(X1=0).
Ycar=0.9705−0.1972X1+0.1708X2+0.1892X21 (4)
The variable with the largest effect on the chlorophyll extractionyieldwas thequadratictermofthetemperature (p=0.0019),followed bythe linearterm ofthe temperature (p=0.0080)andthequadratictermofthepressure(p=0.0234). Therewasnosignificanteffectofthelineartermofthe pres-sure(p=0.1274)oroftheinteractiontermbetween tempera-tureandpressure(p=0.0670).Thus,astatisticallysignificant relationshipbetweenchlorophyllrecoveryandthe tempera-tureandpressureconditionshasbeenestablishedwiththe followingsecond-ordermodel(Eq.(5)).Thelineartermofthe pressure(A2=+0.543)wasnotremovedfromEq.(5)to
main-tainthehierarchyofthemodel.Tobettervisualizetheeffectof thetemperatureandpressureontherecoveryofcarotenoids
0.68 0.97 1.27 1.56 1.86 Y1 Y2 21 28 35 42 49 6.0 7.8 9.5 11.3 13.0 T: Temperature P: Pressure
A
B
15.45 13.47 11.49 9.51 7.53 13.0 11.3 9.5 7.8 6.0 21 28 35 42 49 T: Temperature P: PressureFig.1–Surfaceplotofextractionyieldat40◦Cand30MPa
withCO2,for:(A)carotenoids(Ycar,gcar/kgd.s.)and(B)
chlorophylls(Ychlor,mgchlor/kgd.s.),asafunctionof
temperature(T,◦C)andpressure(P,MPa)oftherapid
andchlorophyllsintheexperimentalrange,surfaceresponse graphsweregeneratedusingthesecond-ordermodels
Ychlor=12.502−1.147X1+0.543X2−1.682X21+1.018X22 (5)
Effectsoftemperatureandpressure
Carotenoidrecoverydecreasedwithincreasingtemperature inthelowertemperature range(<40◦C).There wasa nega-tivelineareffect(A1=−0.1972)(Eq.(4))oftemperature.Thus,
weobservedthatcarotenoidrecoverydecreasesfrom1.86to 1.16g/kgd.s.whentemperatureincreasesfrom21to40◦Cat 13MPa.Intheuppertemperaturerange,over40◦C,the posi-tivenon-lineareffectoftemperature(A2
1=+0.1892)becomes
important and carotenoid recovery increases slightly with temperature.Similar behavior forYcarwas observedinthe
lowerpressurerange.
Tochlorophyllsrecovery,thenegativelineareffectof tem-perature (A1=−1.147) (Eq. (5)) was observed in the upper
temperature range (over ∼35◦C). When the temperature
increasedfrom35 to49◦Cat13MPa,Ychlor decreasedfrom
15.26to10.36mg/kgd.s.Thenegativenon-lineareffectof tem-perature(A2
1=−1.682)becomesimportantfortemperatures
below35◦C.This was reflected in the plateau for temper-atures between 30 and 35◦C, where there was a minimal changeinYchlor and15.41and 15.26mg/kgd.s.respectively
were obtained. Similar behavior for Ychlor for temperature
changeswasobservedinthelower pressurerange. Accord-ingtoanalysisofvariance,thequadratictermoftemperature contributed28%and37%toexplainingthebehaviorofYcarand
Ychlor,respectively.Thequadratictermofpressurecontributed
13%toexplainingthebehaviorofYchlor.
Carotenoidrecoveryincreasedwithpressure(A2=+0.1708)
(Eq.(4))towhatevertemperatureduringstaticcompression. TherewasapositiveeffectofpressureonYcar.Forinstance
Ycar increasedfrom 1.38 to1.86g/kgd.s. (1.3-fold increase)
whenthepressureincreasedfrom6to13MPaat21◦C.Similar behavior was observedfor Ychlor forpressure increases, in
theupperpressurerangeover9.5MPa.Therewasapositive effect of pressure on Ychlor. For instance Ychlor increased
from 10.81 to13.57mg/kg d.s. (1.3-fold increase)when the pressureincreasedfrom9.5to13MPaat21◦C.Forpressures below9.5MPa,thenon-lineareffectofpressure(A22=+1.018)
becomesimportant,andweobservedslightchangesinYchlor
with pressure. Supercritical extractions were performed underconstanttemperatureandpressureconditions, there-forethesolventpowerofCO2shouldnotchangewithinthe
extractionassays. However,microalgaextractsare complex mixtures of several interacting compounds. Probably the effectofquadratictermswouldbeexplainedbytheexistence ofsolute–soluteinteractions (anti-solvencyandco-solvency effects) and/or solute–matrix interactions that affect the solubility behavior of a compound of the mixture in the supercriticalphaseanditsrecoveryyield46(Fig.1).
Cell disruptionstudiesusing CO2 rapiddepressurization
havebeen reportedinliterature.Thecell disruptionof Ral-stoniaeutrophatoextractpoly(-hydroxybutyrate)(PHB)was studied.27AmultipurposeSFE-SFCsystemwasusedforthe
celldisruption,anditsefficiencywasmeasuredasPHB recov-ery.Extractionyieldincreasedwithincreasingpressurefrom 15to20MPa,butdecreasedat30MPa.Theauthorsattribute thenegativeeffectofhighpressuretothefactthatdisruption ofthecytoplasmmembraneoccursfasterthandisruptionof thecellwallat30MPa,resultingincellwallshrinkage,which hinderstheextractionofintracellularcompounds.Ina simi-larstudyontherecoveryofPHBfromR.eutrophacells,itwas reportedthatPHBrecoverydecreasedwithincreasingpressure from25to35MPaatconstanttemperature.47Wasstudiedthe
celldisruptionofE.coliusingCO2rapiddepressurizationfor
endoglucanaseenzymerecovery.23Thepressureduringstatic
compression(10–25MPa)hadasignificantpositiveeffecton enzymerecovery.Higherpressureresultedinbetterenzyme recovery. Therefore, the positive effect of pressure during static compressiononcell disruptionhasbeen observedat pressuresbelow20MPa.Withrespecttothecompression tem-perature, Hejazi et al.27 reported that biopolymer recovery
decreasedwhenthecompressiontemperaturerosefrom40 to70◦C.Theauthorsattributedthiseffecttothesupercritical CO2beingclosertoitsliquidstateatlowertemperatures:when
rapiddepressurizationoccurs,theCO2reachesitsgasstate
withmaximumvolumechange,whichinturncausesmore extensivecell walldisruption. Also,Khosravi-Daranietal.47
reportedthatPHBrecoverydecreasedwithincreasing temper-aturefrom30to40◦Catconstantpressure.Thusanegative effectoftemperatureduringstaticcompressiononcell dis-ruptionusingrapiddepressurizationhasbeenobserved.
Table3–ComparisonofsupercriticalCO2extraction(40◦Cand30MPa)betweenmicroalgaeuntreatedandpretreated
withrapiddecompression(21◦Cand13MPa).
Characteristics Untreated Pretreated Foldincrease
Oilextractionyield(g/kgd.s.) 76.97±1.51a 82.37±0.90b 1.07
Concentration(mg/kgoil) Carotenoids 17.02±0.93a 19.75±0.78b 1.16 Chlorophylls 19.36±1.68a 181.26±3.62b 9.36 Tocopherols 8509±55a 9059±39b 1.06 Extractionyield Carotenoids(g/kgd.s.) 1.53±0.15a 1.91±0.09b 1.25 Tocopherols(g/kgd.s.) 0.91±0.01a 1.04±0.01b 1.14 Chlorophylls(mg/kgd.s.) 1.38±0.35a 14.03±0.91b 10.2
Antioxidantactivity(mmolTE/kgoil) 29.11±0.51a 33.22±0.31b 1.14
Accordingtoourresults,theextractionofcarotenoidswas best at 21◦C and 13MPa with Ycar at 1.87g/kg d.s., while
theextraction ofchlorophyll was bestat32◦Cand 13MPa, with Ychlor at 15.45mg/kg d.s. In other words higher
pig-mentextractionwasobtainedintheexperimentalrangeof highpressure and lowtemperature, whichagreeswiththe observationsofpreviousauthors.23,27,47Thisrangeis
charac-terizedbyhigherCO2 density (about810–880kg/m3).When
the CO2 isdenser,its solvent power increasesand in
con-sequence sodoes the permeation capability ofCO2 within
thecell.Furthermore,whenthe CO2 density increases,the
net amount of gas absorbed into the cells increases and thisincreasestheforce causedbyrapiddepressurization.47
AmajorchangeinthevolumeoccurswhentheCO2returns
to the gaseous state due to rapid depressurization.27 This
suddenvolumechangeresultsinmoreeffectivecell disrup-tionandimprovestheavailabilityofpigmentsbysupercritical extraction.
Comparisonbetweenuntreatedandpretreatedmicroalgae
Table3shows acomparisonofsupercriticalCO2 extraction
(40◦Cand30MPa)usinguntreatedmicroalgaeandmicroalgae pretreatedusingrapiddepressurization.Forrapid depressur-izationpretreatment,weselected21◦Cand13MPaconditions tofavorrecoveryofcarotenoidsandchlorophylls.Thestatic time was kept constant at 60min. The extraction yields forcarotenoidand chlorophyll reportedin Table3 confirm the predictive power of the selected second-order models (Eqs. (4) and (5)). Table 3shows that there was significant difference (p≤0.05) incompound recoveryand antioxidant activity betweenoilsextracted withsupercriticalCO2 from
untreatedandpretreatedsamplesusingrapid depressuriza-tion.Weincludedthe measurementofantioxidant activity andquantifiedtotaltocopherols,whichareimportant antiox-idants.Thehighestdifferencewasobservedintheextraction ofchlorophylls,1.38and14.03mg/kgd.s.,foruntreatedand pretreated samples respectively. Chlorophyll pigments are produced and stored intracellularly in the chloroplasts.48
Mendesetal.37reportedthatinsupercriticalextracts(at40◦C
and 12.5–30MPa) obtainedfrom freeze-dried samples ofB. braunii,chlorophyllswerenotdetected.Therefore,thehigher extractionofchlorophyllsfrompretreatedsampleswithrapid depressurizationwouldbeaverygoodindicatorofcell disrup-tion.Tosupporttheseresults,observationsweremadeunder confocallasermicroscopy.
ConfocalmicroscopyimagesinFig.2showtheeffectofCO2
rapiddepressurization pretreatment on the morphology of microalgaecells.Fig.2Ashowsamicro-colonyofB.brauniinot treatedwithrapiddepressurizationascontrolsample.Intact cellwallscanstillbedistinguishedinthemicro-colony.Thisis clearwhenweobservethewallssurroundingthecellsstained blue(emission450nm),duetothereactionofcalcofluorwith thecellulosecomponentsofthecellwall.Fig.2Bshowsmajor destruction ofcellwalls inthe micro-colony towhichCO2
rapiddepressurization wasapplied:largecell wallintegrity waslost.Cellunitsarenotclearlydifferentiated,sincesome cellshavefusedtogether.Inaddition,redareasappeardue toanincreaseinthereleaseofchlorophyllpigments (emis-sion650–750nm),which wouldpermithigher extractionof
A
B
Intac cell wall
Partial cell disruption Release of chlorophylls
Fig.2–Confocalmicroscopyimagesofmicro-colonyofB.
braunii:(A)nottreatedwithCO2rapiddepressurization,as
controlsampleand(B)treatedwithrapiddepressurization
(bar=20m).
chlorophyllpigmentswithsupercriticalCO2ascomparedto
thecontrolsample(withouttreatmentwithrapid depressur-ization).Therewasthereforeanobservabledifferenceincell wall integrity between the untreated and pretreated sam-ples, whichresultedinincreasedextractionofcarotenoids, chlorophyllsandtocopherols,andinhigherantioxidant activ-ity in pretreatedsamples. These observations suggest that pretreatment using CO2 rapid depressurization improved
pigment extraction due to partial cell disruption of the microalgae.
Conclusions
ThisworkinvestigatedcelldisruptionusingCO2rapid
depres-surizationasasubstratepretreatmentforpigmentextraction from the microalga B. braunii. According to the response surfaceswe suggest aregion oflowtemperature and high pressureforthestaticcompressioncondition,whichfavors the extraction of carotenoids and especially chlorophylls. The combination of high pressure (13MPa) and low tem-perature(21◦C)duringstatic compression wasselected for the pretreatment ofmicroalgaewith purposesof compari-sonwithuntreatedmicroalgae.Oilfrompretreatedmicroalgae presentedbetterantioxidantactivityduetothehigher con-centrationofcarotenoids,chlorophyllsandtocopherols.From confocalmicroscopyimages,itwasobservedthatpartialcell disruptionofmicroalgaeimprovedpigmentextraction.CO2
rapiddepressurizationisasimpleandefficientmethodthat canbeemployedtorecoverimportantintracellular compo-nents,suchasthe pigmentsfrom B.braunii.Moreover,this pretreatmentisperformedatmoderatetemperatureswhich wouldprotecttheseheatsensitivecompounds.Thisisthefirst reportoncelldisruptionusingCO2rapiddepressurizationof
amicroalga.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
ThisresearchwasfundedbytheUniversidaddeLaFrontera (project DI11-0055) and Innova-CORFO (project 09CTEI-6860).OuracknowledgmentstoAgriaquacultureNutritional GenomicCenter,CGNA(R10C1001).
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