Isolation and molecular characterization of Thraustochytrium strain isolated from Antarctic Peninsula and its biotechnological potential in the production of fatty acids

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Environmental

Microbiology

Isolation

and

molecular

characterization

of

Thraustochytrium

strain

isolated

from

Antarctic

Peninsula

and

its

biotechnological

potential

in

the

production

of

fatty

acids

Esteban

Caama ˜no

_,

_Lyliam

_Loperena

_,

_Ivonne

_Hinzpeter

_,

_Paulina

_Pradel

_,

Felipe

Gordillo

_,

_Gino

_Corsini

_,

_Mario

_Tello

_,

_Paris

_Lavín

_,

_Alex

_R.

_González

a_{LaboratoriodeMicrobiologíaAmbientalyExtremófilos,DepartamentodeCienciasBiológicas,UniversidaddelosLagos,Osorno,Chile} b_{InstitutodeIngenieríaQuímica,DepartamentodeBioingeniería,UniversidaddelaRepública,Montevideo,Uruguay}

c_{DepartamentodeGobiernoyEmpresa,UniversidaddelosLagos,Osorno,Chile}

d_{CentrodeInteracciónPlanta-SueloyBiotecnologíadeRecursosNaturales,LaboratoriodeFisiologíayBiologíaMolecularVegetal,}

UniversidaddeLaFrontera,Temuco,Chile

e_{CentrodeBiotecnologíadelosRecursosNaturales,FacultaddeCienciasAgrariasyForestales,UniversidadCatólicadelMaule,Talca,}

Chile

f_{InstitutodeCienciasBiomédicas,FacultaddeCienciasdelaSalud,UniversidadAutónomadeChile,Santiago,Chile} g_{UniversidadCientíficadelSur,Lima,Perú}

h_{CentrodeBiotecnologíaAcuícola,DepartamentodeBiología,FacultaddeQuímicayBiología,UniversidaddeSantiago,Santiago,Chile} i_{LaboratoriodeComplejidadMicrobianayEcologíaFuncional,InstitutoAntofagasta,UniversidaddeAntofagasta,Antofagasta,Chile}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received18September2016 Accepted31January2017 Availableonline10June2017 AssociateEditor:Dra.VaniaMelo

Keywords: Thraustochytrids Antarctic Docosahexaenoicacid Eicosapentaenoicacid Temperature

a

b

s

t

r

a

c

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ThraustochytridsareunicellularprotistsbelongingtotheLabyrinthulomycetesclass,which arecharacterizedbythepresenceofahighlipidcontentthatcouldreplaceconventional fattyacids.Theyshowawidegeographicdistribution,howevertheirdiversityinthe Antarc-ticRegionisratherscarce.Theanalysisbasedonthecompletesequenceof18SrRNAgene showedthatstrain34-2belongstothespeciesThraustochytriumkinnei,with99%identity.The totallipidprofileshowsawiderangeofsaturatedfattyacidswithabundanceofpalmitic acid(16:0),showingarangeof16.1–19.7%.Ontheotherhand,long-chainpolyunsaturated fattyacids,mainlydocosahexaenoicacidandeicosapentaenoicacidarepresentinarange of24–48%and6.1–9.3%,respectively.Allfactorsanalyzedincells(biomass,carbon consump-tionandlipidcontent)changedwithvariationsofculturetemperature(10◦Cand25◦C).The growthinglucoseatatemperatureof10◦Cpresentedthemostfavorableconditionsto pro-duceomega-3fattyacid.Thisresearchprovidestheidentificationandcharacterizationofa

Thraustochytridsstrain,withatotallipidcontentthatpresentspotentialapplicationsinthe productionofnutritionalsupplementsandaswellbiofuels.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mail:Alex.gonzalez@ulagos.cl(A.R.González).

http://dx.doi.org/10.1016/j.bjm.2017.01.011

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Themicroorganisms belongingto the Labyrinthulomycetes

class and family Thraustochytriaceae are unicellular protists

whichare present inmarine ecosystems. Theyhave akey

roleontheinitialstageofthemicrobialchainfood,asorganic

matterdegraders.1,2 _{Thesemicroorganismshavebeen}

stud-ied at the morphological, ecological and biotechnological

levels.3,4 _{Due to the presenceof a high lipid content that} couldreplaceconventionalsourcesoffattyacids;the Thraus-tochytriaceae family is of great interest.5–8 _{Particularly, the}

moststudied metabolitesare docosahexaenoic acid (C22:6,

DHA)andeicosapentaenoicacid(C20:5,EPA).9,10_These

essen-tialbiomoleculesoftheomega-3family areinvolvedinthe

physiological development of children and adults,

choles-terolregulation,prostaglandin,thromboxaneandleukotriene

biosynthesis. Furthermore, they have a preventive role on

differentpathologiessuchasarteriosclerosis,asthma, throm-bosis, arthritisand awide range oftumors.11–13 _{Besides, a}

varietyofbiomoleculeswithunknownfunctionsthatcould

haveapotentialbiotechnologicalapplication,suchas

extra-cellular polysaccharides, carotenoids, squalene, enzymes,

osmolytes, unsaturated and saturated fatty acids could be

used as biofuel.14 Taxonomic classification of the

Thraus-tochytriaceaefamilycomprisesthegeneraAplanochytrium, Ulke-nia, Thraustochytrium, Japonochytrium, Aurantiochytrium (also known asSchizochytrium), Botryochytrium, Parietichytriumand

Sicyoidochytrium.15,16 _{Their geographic distribution includes} theNorthSea,India,Indonesia,Japan,Australia,South Amer-icaandtheAntarcticContinent.17,18_{Thelatterplacepresents}

poorstudiesofthemarinemicrobialdiversityandthe

num-berofspeciesvarieswidelybetweentaxons.19–21 _Currently, inthediversitystudiesoftheThraustochytrids from Antarc-ticwaterstwospecieshavebeendescribed:Thraustochytrium antarticum(SoutheasternIndianOcean)andThraustochytrium rossii(SouthwesternPacificOcean).23_{Morerecently,onlyone}

microorganism has been characterized by molecular

phy-logeny;Aplanochytriumstocchinoi(TerraNovaBay).23

Microorganismshavebeenusedasalternativefattyacids

sourceinbacteria,fungi,yeastsand microalgae.24–26 Protist species suchas Crythecodiniumcohnii, Schizochytrium sp.and

Ulkeniasp.havebeencharacterizedbytheirrapidgrowth,high photosyntheticactivityandhighproductionofbiomass.27–29 In vitro culture of these microorganisms with high car-bon/nitrogenratiofavorslipidsaccumulationandadecrease incelldevelopment.29_{However,thisisnotclearlyestablished,} sinceinSchizochytriumstrains,thefattyacidsincreaseis asso-ciatedwithbiomassgrowth.30 _{Besides,invitrostudies with}

carbon and nitrogen sourcescould favorthe biomass

pro-ductionaslipidcontents,becausethesemoleculesarelinked tothedevelopmentofabiologicalmodelsuccessfulonfatty acidsproduction.18,29

Thisstudyprovidesabiologicalandbiotecnologicalfocus, usinginvitrostudiesofastrainbelongingtothe Thraustochytri-aceaefamilyisolatedfromtheAntarcticcoastofKingGeorge Island.Theobjectiveofthisresearchisbasedonthe

morphol-ogyandtaxonomicclassification(18SrRNAgene)studiesof

thecultureparameterssuchasthecarbonsourceandthe tem-peratureonbiomassproductionwithhighcontentofessential

fatty acids.Thisresearchoffersnewinformationabout the

biodiversityofLabyrinthulomycetesclassintheAntarctic con-tinentand their potentialapplicationasa biotechnological toolonLC-PUFAsorbiofuelsproduction.

Materials

and

methods

Isolationandcultureconditions

TwosampleswerecollectedonthecoastofKingGeorgeIsland, specificallyoncoordinatesS62◦1234.8 W58◦5534.3 and

the microorganisms were isolated from the watercolumn

(2.4◦CandpH8.1).Thraustochytridswereobtainedusingthe pinepolenmethod.31_{Then,theywereincubatedatdifferent} temperatures (10◦Cand 25◦C)and observedusinganoptic

microscopeduring10daystoimprovemicroorganismfixation.

InoculeswerecultivatedinliquidmediumusingHondaetal. (1998)58_{modifiedprotocol(0.2%yeastextractand0.2%sodium} glutamate)inartificialseawaterwith5%glucoseor5%starch, atthesametemperatureconditionsduring8days.32_Finally, thesampleswerelyophilized,centrifugedandstoredat−20◦_C

forposterioranalysis.Morphologicalanalysiswasperformed

withanOlympusCX21lightmicroscope(Tokyo,Japan).

DNAextractionandamplificationofthe18SrRNAgene

DNA extraction was performed using Mo and Rinkevich33

modified protocol. Cultureswere centrifuged at14,000rpm

for 3min, supernatant was discarded, and buffer

extrac-tion wasaddedonpellet(0.2MTris–HClpH8.0,1.4MNaCl,

0.1MEDTAand1.5%SDS)andsonicatedfor30s.Then,the

solutionwascentrifugedat12,000rpmandthesupernatant

was transferred to a new tube to obtain the DNA using

the phenol:chloroform solution (5:1, pH 4.8). The mixture

was centrifugedat13,000rpmfor5min.Theaquosephase

wasprecipitatedwithethanolat4◦Covernight.Finally, the

resulting pelletwassuspendedonnucleasefreewaterand

quantifiedinInfinity200ProNanoQuant(TECAN)andstored

at−20◦C.

PCRamplificationof18Sgenewascarriedoutusing

com-ercialkitGoTaq® GreenMasterMix(ReactionBufferpH8.5,

400␮M ofdNTPs Promega and 3mMMgCl2), and Genomic

DNA (with an average ratio 260/280 of 1.6) and 1mM of

specificprimers(FA15-AAAGATTAAGCCATGCATGT-3,RA15

-AGCTTTTTAACTGCAACAAC-3;FA2 5

-GTCTGGTGCCAGCAG-CCGCG-3, RA2 5-CCCGTGTTGAGTCAAATTAAG-3; FA3 5

-CTTAAAGGAATTGACGGAAG-3 and RA3 5

-CAATCGGTAGG-TGCGACGGGCGG-3).34_{Thermocyclingprofilewasperformed}

withaninitialdenaturationstepof3mina95◦C,35cyclesof amplification(1min94◦C,1min53◦Cand1min72◦C),and

finalelongationat72◦Cof10min.ThePCRfragmentswere

visualized on 1.2%agarose gel using10␮g/mLofethidium

bromide.

Sequencingandmolecularphylogeneticanalysis

The18SrRNAfragmentswereamplifiedandthensequenced

using automated DNA sequencer ABI-Prism (Pontifical

Fig.1–LightmicroscopyofcellsinAntarcticstrain34-2stainedwithmethylviolet.AVegetativecells(vc),sporangia(s)and ectoplasmicnets(arrows).Scalebar100␮m.BMaturesporangiumwithzoospores(z)andproliferativebody(pb).Scalebar 50␮m.

wascarriedonthehomologsequencesavailableonthedata

baseofgenes(GenBankdatabaseoftheNationalCenterfor

BiotechnologyInformation).Homologuesequences

compari-sonwasdoneusingalgorithmmegablastavailableonNCBI

server.35_{SequencesalignmentswereperformedusingClustal} X2.37_{Phylogeneticanalysis,weredonewithNeighbor-joining}

method36_{and thetreetopologywithMEGA 4.0.2.}38 _Species

relations were evaluated with statistical analysis through

bootstrapprotocol.39_{Togroupthephylogeneticanalysisofthe} familyThraustochytriaceaetheProrocentrumgenuswasusedas outgroup.

Extractionanddeterminationoffattyacids

Fattyacids profilewas donewith 50mglyofilizedbiomass,

usingdirecttransesterification.40_{Thefatty acids were}

con-centrate at 1000g at 4◦C and stored at −20◦_{C. They}

werequantifiedbyGasChromatography(GC)(Agilent,7609,

Santa Clara, USA) using as standard Supelco® 37

Compo-nent FAME Mix (Sigma–Aldrich) (Methyl butyrate, Methyl

hexanoate, Methyl octanoate, Methyl decanoate, Methyl

undecanoate, Methyl laurate, Methyl tridecanoate, Methyl

tetradecanoate, Myristoleic Acid Methyl Ester, Methyl

pen-tadecanoate,cis-10-Pentadecenoicacidmethylester,Methyl

palmitate,MethylPalmitoleate,Methylheptadecanoate,

cis-10-Heptadecenoicacid methyl ester,Methyloctadecanoate,

trans-9-Elaidic acid methyl ester, cis-9-Oleic acid methyl

ester, Linolelaidic acid methyl ester, Methyl Linoleate,

Methyl Arachidate, gamma-Linolenic acid methyl ester,

Methylcis-11-eicosenoate,MethylLinolenate,Methyl

hene-icosanoate,cis-11,14-Eicosadienoicacidmethyl,ester,Methyl

docosanoate, cis-8,11,14-Eicosatrienoic acid methyl ester,

MethylErucate,cis-11,14,17-Eicosatrienoicacidmethylester,

Methyl tricosanoate, Methyl cis-5,8,11,14-Eicosatetraenoic,

cis-13-16-Docosadienoicacidmethylester(22:2),Methyl

lig-nocerate, Methylcis-5,8,11,14,17-Eicosapentaenoate, Methyl

Nervonate, cis-4,7,10,13,16,19-Docosahexaenoate). Biomass

quantitative analysis was determined with optical

den-sity and long-chainpolyunsaturated fatty acids (LC-PUFAs;

DHA and EPA) content was carried out using gravimetric

assay.

Neutralfattyacids(triglycerides)in34-2strainwere

visu-alized using Oil red O protocol modified.41 _{The samples}

were stained with 0.3% Oil Red solution for 20min and

visualized under optical microscope (OlympusCX21) using

ISCapture program (http://www.tucsen.com). Images were

edited withImageJprogram (http://imagej.nih.gov/ij/).Data

and graphics were processed using Open Source Software

IGOR Pro 6.36 (https://www.wavemetrics.com) and Canvas

(http://www.canvasgfx.com).

Results

Morphologicandgeneticidentification

The isolated Antarctic strain had spherical cells with an

averageof46±5␮mindiameter(Fig.1A).Morphological

char-acteristics were observed such as; formation of sporangia

andvegetativecellswithfinefilamentsofectoplasmicnets

(plasmamembraneextensions) (Fig. 1).Thestrain34-2has aproliferativebodyassociatedtoenclosedsporeswithinthe cell wallofthe sporangium(Fig.1B).Inordertodetermine

thephylogeneticrelationshipthesearchofthehomologous

sequenceswasperformed(Megablastalgorithm).The

compar-isonof18SrRNAgeneshowsthattheAntarcticstrainexhibits highidentityof99%(overan95%ofalignedlengthof1616bp)

withsequences belongingtoThraustochytriumkinneispecies

(GenBankaccessionnumbers|KF709393.1|and|L34668.1|),

con-firmingaclosetaxonomicclasificationwithThraustochytrids

microorganisms(Fig.2).Thisstrainshowsacommon ances-tor,withthreeunclassifiedThraustochytridaegroupedinclade A,withabootstrapvalueof100%.Inthetreetopology,the

sequence of A. stocchinoi, species isolated from Ross Sea,

appearassociatedtotheAntarcticacladeB.Thephylogenetic analysisrevealedanearlydiversificationwithstrain34-2and therestofthegenera.The18SrRNAsequencesofstrain

34-2 are available in GenBank(accession numbers: LN558422;

Fig.2–Molecularanalysisbasedoncompletealignmentofthe18SrRNAgene.Thephylogeneticaltreewasperformedwith thegenusbelongingtotheThraustochytriaceaefamily,andusingtwospeciesofthegenusProrocentrumasoutgroup.The phylogenywasdevelopedbytheNeighbor–joiningmethod,showingthenumberofGenBankaccessionnumbersforeach sequenceandthebootstrapsvalueobtainedfrom1000replicatesinnodes.Thestraindeterminedinthisstudyisshownin boldtype(CladeA).

Biomassandomega-3fattyacidproduction

Acultureof8daysofstrain34-2wasdoneinorderto deter-minekineticbiomassgeneration(Fig.3).Themicroorganisms wereculturedinfourdifferentconditions,liquidmedia

sup-plemented with 0.5% glucose or starch at 10◦C and 25◦C

(Fig. 3AandB).Both graphicsshow anexponentialgrowth

untilday5.Thehighestgrowthwasregisteredusinga

sup-plementedmediumwithglucoseat25◦C,withcellularvalue of2.2×103_{mg/L.Ontheotherhand,alowgrowthwas}

reg-isteredusingaglucoseculturewithatemperatureof10◦C,

showingamaximumvalueofbiomassat7dayswitharateof

growthof0.7×103_{mg/L.Instarchmedium,maximumvalues}

wereregisteredat25◦Cand10◦Cat6dayswith1.8×103_mg/L

and1×103_{mg/Lrespectively.Theseresultsrevealedthatthis}

microorganismhadahighbiomassproducedindifferent

car-bonsourcesathightemperature.

Theeffectsofthecarbonsourceandtemperaturewere

ana-lyzedonbiomass, carbonintakeand polyunsaturatedfatty

acidcontentfor5daysofculture(Fig.4).Theresultsshowed thatthereexistsamajorproductionofbiomassat25◦C,with

2×103_{mg/L.On the other hand,low biomasswas present}

inglucose mediumat 10◦C with avalue of0.5×103_mg/L)

(Fig. 4A). The LC-PUFAs content shows the highest values

obtained with glucose as carbon source at 10◦C (Fig. 4B).

The analysis revealed values of 29.8±2.1mg/g DHA and

3.3±0.2mg/gEPA.Otherwise,thelowestvalueswereobtained at25◦Cwith5.6±0.1mg/gDHAand0.7±0.1mg/gEPA.In car-bonintaketheuseofglucoseandstarchat10◦Cpresentedthe highestvalueswith2.4×103mg/Land2.4×103mg/L respec-tively(Fig.4C).Ontheother hand,theresultsat25◦Cwere 1.4×103_{mg/Lforglucoseand 1.2×10}3_{mg/Lforstarch.The}

numbersofanalysisalreadydescribed,showstandard

devia-tionvaluesonlyforvariationsoverorequalto0.1.

Ingeneral,thesevaluesshowthatthegrowthwithglucose at10◦CwasthemostfavorableforDHAandEPAproduction.

Itwas observed thatwith low biomass(0.5×103_{mg/L) the}

highervaluesofDHA andEPA(29.8±2.1and 3.3±0.2mg/g

Table1–FattyacidprofileofAntarcticstrain34-2.The

valuesareshownasapercentageoftotalfattyacid

contentandaratioofomega-3:omega-6andDHA:EPA,

forthetemperatureandcarbonsourceconditionsused

inthisstudy.

Fattyacidsa_{(%) Glucose Starch} 10◦ 25◦ 10◦ 25◦ Saturateda Capricacid(C10:0) 1.9 5.9 1 1 Undecanoicacid(C11:0) 0.6 2.8 0.6 1 Lauricacid(C12:0) 0.8 1.3 0.8 1.2 Tridecanoicacid(C13:0) 0.3 1 0 0 Myristicacid(C14:0) 2.5 2.7 2.4 2.5 Pentadecanoicacid(15:0) 0.8 3.1 0.6 2.9 Palmiticacid(C16:0) 17.9 16.1 18.6 19.7 Stearicacid(C18:0) 4.7 11.3 4.3 10.1 Arachidicacid(C20:0) 1.4 5.1 1.9 4.5 Unsaturateda Elaidicacid(C18:1n9) 1.3 3.4 1.2 3.2 cisOleicacid(C18:1n9c) 1.6 2.8 2 3.2

Linoleicacid(C18:2) 1 0 1.1 1.5

Eicosapentaenoicacid(C20:5) 9.9 6.1 7.4 7.1 Docosahexaenoicacid(C22:6) 46.6 24 48.3 30.7 Unknownretentiontime 9 13.7 9.4 10.7

Total(%) 100 100 100 100

Ratio

DHA/EPA 5 4 6.5 4.2

n-3/n-6 55.9 – 50.6 25.2

a _{Identifiedasfattyacidsaccordingstandardretentiontimefrom}

standardmix.

respectively)were obtained,witharatioofomega-3:6fatty acidsof55.9%(Table1).

Determinationandanalysisoffattyacids

Thefattyacids qualitativestudies weredoneusingOilRed

Ostaining,afat-solublediazodye.Forthispurpose,cultures

A

Days of culture Days of culture

Biomass x 10 3 (mg 1 –1 ) Biomass x 10 3 (mg 1 –1 ) 6 6 7 8 9 10 7 8 9 10

B

Fig.3–Thetemperatureandcarbonsourceeffectonbiomassproduction.(A)Strain34-2supplementedwithglucose5%at 25◦C()and10◦C(䊉).(B)supplementedwithstarch5%at25◦C()and10◦C(䊉).Thisdatawaspresentedasthemeanof triplicate±SD.

2.5 35 4 3 2 1 0 30 EPA DHA 25 20 15 10 5 0

B

A

C

Glucose Glucose Glucose

Biomass x 10 3 (mg 1 –1) Carbon intake x 10 3 (mg 1 –1 ) Lipids/Biomass (mg g –1 )

Starch Starch Starch

10°C 10°C 10°C 10°C 10°C

25°C 25°C 25°C 25°C 25°C 25°C

Fig.4–Thetemperatureandcarbonsourceeffectonbiomass,omega-3fattyacidcontent(EPAyDHA)andcarbonintake. (A)Quantityofbiomasssupplementedduring5daysofculturewithglucose(blackbars)andstarch(whitebars).(B) RelationshipofDHAandEPAinrelationtobiomass.(C)Carbonsourceconsumptionduring5daysofculture.Thisdatawas presentedasthemeanoftriplicate±SD.

wereselectedwithhigh(glucoseat10◦C)andlower(glucose 25◦C) lipid/biomass proportion according to Fig. 4B.These

results showed the presence of intracellular lipid vesicles

withredcolor(SupplementaryMaterial).Theculture

supple-mentedwithglucoseat10◦Cshowedanintensestainingon

intracellularvesiclesin5and8daysofculture.Ontheother

hand,thetotalcompositionoflipidsofstrain34-2showed

64%ofsaturatedfattyacids(9/14)(Table1).Inthisgroupof

100 80 60 40 20 10°C

Glucose

Total fatty acid content (%)

Starch

Fig.5–Summaryofsaturatedandunsaturatedfattyacid contentofAntarcticstrain34-2.Thevalueshowsthe percentageofTable1.

saturatedfattyacidspalmitic(C16:0)andestearic(C18:0)acids standout.Inunsaturatedfattyacids,theeicosapentanoicacid

(C20:59)and docohexanoic acid (C22:6)showed thehighest

values.

The results indicate that the production of these

biomoleculeschangedwithcarbonsourceandtemperature.

Particularly, some lipids are affected according to carbon

sourceat25◦C.Thechange forglucoseand starch

respec-tivelywasasfollows;capricacid5.9%and1%,linoleicacid0%

and1.5%,undecanoicacid2.8%and1%,palmiticacid16.1%

and 19.7%anddocosahexaenoicacid24% and30.7%.Other

fatty acids varied with temperature changes (10 and 25◦C

respectively)asfollows:estearicacid4.7–11.3%,arachidicacid 1.4–5.1%,bothcultureswithglucose.

In general,themostimportantparameter infatty acids

profilewastheeffectoftemperature.For example,the

cul-turesupplementedwithglucoseshowedthehighestchanges

(Fig.5).Theresultsevidencedadifferenceof37%ofsaturated

fatty acids and 40% ofunsaturated fatty acids. Otherwise,

microorganismssupplementedwithstarchpresentedavalue

of9%anddecrease23%,ofunsaturatedandsaturatedfatty

acidsrespectively.

Discussion

TheThraustochytridsareaeukaryotegroupshowingawide

geographicdistribution,withscarceinformationonthe diver-sityintheseaoftheAntarcticContinent.Nowadays,reports

ofthesemicroorganismsontheAntarcticRegionarelimited

onlytotworesearches.Oneofthemwaspublishedby

Bah-nweg and Sparrow,22 _{and described themorphology ofthe}

strainsbelongingtothegenusThraustochytriumisolatedfrom

theSoutheasternIndianOceanandtheSouthwesternPacific

Ocean.Besides,Moroetal.23_{describedandcharacterizedthe} species Aplanochytriumtocchinoi collected inthe TerraNova

Bay(SouthernOcean,Antarctic),basedontheassayof

Trans-missionElectronMicroscopy(TEM)andmolecularphylogeny.

Themorphologicalstudiesdoneonthestrain34-2revealed

the presenceofstructures described forthe genus

Thraus-tochytrium, withpiriforms sporangiato ovalated,formation

ofzoosporesgroupsbeforeliberationandthepresenceofa

proliferationbody(Fig.1).42,43_{Besides,themolecularanalysis}

basedonthecompletesequenceofthe18SrRNAgeneshowed

thatstrain34-2belongstothespeciesT.kinneiwithidentityof

99%.Thephylogeneticanalysisevidencesacommon

ances-torwith thraustochytrids isolated of the seashore ofChile

(ValparaísoandPuertoMontt)andAustralia(Southwestof Tas-mania),strainslocatedonthecladeA,particularlyT.kinnei |KF709393.1|,Thraustochytriidae sp.|DQ459552.2| and Thraus-tochytriidaesp.|JN675277.1|,respectively(Fig.2).Therefore,this

research provides basicinformation on the first molecular

identificationofthisspeciesontheAntarcticRegion. Accord-inglywiththeirsitesofisolation,wethinkthatdistribution

ofclade A is a consequence ofdynamics of the Southern

Hemispherecurrents,becauseThraustochytridscouldliveor

betransportedonhydrochory,suchassediment, senescent

macroalgaeandfallenmangroveleaves.10,19_{Ontheotherside,}

the topology oftrees show the evidence that A. stocchinoi,

describedabove,andthestrain34-2,whicharelocated geo-graphicallyontheoccidentalregionoftheAntarcticcontinent, presentedanearlydivergence,showingthefirstphylogenetic relationoftwogeneraofthefamilyThraustochytriaceaeisolated fromthecoastsoftheAntarcticRegion(Fig.2,cladeAandB).

Besidesitswidedistribution, thesemicroorganisms

pro-duce metabolites with biotechnological applications such

asenzymes and extracellularpolysaccharides, carotenoids,

polyunsaturatedfattyacids(asessentialfattyacids),squalene

andsaturatedandmonounsaturatedfattyacidswith

poten-tialontheproductionofbiodiesel.14_{Theresearchisfocused} mainlyontheproductionofessentialfattyacids,beingDHA28

themoststudiedmetabolite.ThesynthesisofLC-PUFAson

thesemicroorganismsisperformedusingtwodifferent

mech-anisms;oneofthemistheelongation/desaturationpathway

whichhasbeendescribedonThraustochytriumaureum.10,44

Fur-thermore,Naganoetal.45_{demonstratedthatthecontentof}

unsaturatedfattyacidsprecursor(C18andC20)was

consis-tentwiththeidentificationof5-elongaseand4-desaturase

genesonthestrainsofthegenusThraustochytrium.

Accord-ingly, the results of fatty acids profile of the strain 34-2

evidencesimilarvaluesofstearicacid,linoleicacidandDHA,

whichsuggeststhatthismicroorganismcouldusethe

elon-gase/desaturasepathwayforLC-PUFAsproduction(Table1). Inconsistencewith thisit isobservedthat theincrease in

temperatureproducedanincreaseofstearicacid (C18:0)in

bothcultures,whichdecreasedthetotalcontentof

unsatu-ratedfattyacids(Table1andFig.5).Theseresultssuggestthat at25◦Ctheenzyme9-fattyaciddesaturase,inchargeofthe transformationofstearicacidinoleicacid,decreasedits activ-ity,whichcouldberelatedtotheresponseofadaptationtothe changeoftemperatureinthismicroorganism.Conversely,the

increaseinthesynthesisofDHAatlowtemperaturescould

beanadaptativeresponsewhichcould favorthe storingof

thispolyunsaturatedfatty acid.Inconsistence, Jainet al.46 describedthatDHAcouldplayaroleasenergysourceofready

energy during starvation. On the other hand,it is already

knownthatlowtemperaturehasimportantconsequencesfor

thesynthesisofunsaturatedfattyacids,increasing availabil-ityofoxygen,neededfortheactivityofdesaturasesenzymes anddiminishingthefluidityofthemembrane.47,48

Ingeneralterms,ourresultsshowthatindependentofthe

carbon source, the culturesincubated at high temperature

present a majorproductionof biomass,whilethe cultures

incubatedatlowertemperaturesynthesizehighcontentsof

DHA and minor biomass(Figs. 3 and 4Aand B).However,

thecontentofLC-PUFAshasahighconcentrationonallthe

analyzedconditions,particularlyDHAwitharange24–48.3%

(Table 1). These values are in agreement with researches

thatpointthatsynthesisofDHArepresentsmorethan25%

of the total content offatty acids produced by the strains

ofThraustochytrids.3,10 _{Thehigh ratios of}

omega-3:omega-6 and DHA/EPA are important parameters in omega-3 oil

production.49,50 _{Accordingtothis,theculturesupplemented} withglucoseat10◦Cevidencedthebestresultswithavalue of55.9%and5%,respectively(Table1).Ithasbeendescribed thatprofilesoffattyacidsareusedasamethodologyto estab-lishphylogenetichomologieswithotherstrainsbelongingto the family Thraustochytriaceae.16,19 _{Consequently, theprofile}

of strain 34-2 isconsistent with researches carried out by

Leeetal.50_{thatcultured36Thraustochytridstrainswith0.2%}

ofglucose and separated them into eightgroupsbased on

theirfattyacidsand18SrDNAgene,evidencedamean

per-centageof35.6%DHA,9.2%EPAand17.5%palmiticacidfor

thestrainoftheThraustochytriumgenera.Ontheotherside, thequalitativeanalysisoffattyacidsshowedthatusingthe

methodology ofOil RedO, the presenceof vesiclesof red

color was observed(Supplementary Material). Thestaining

intensityevidencedaconsistencywithquantitativeanalysis betweenculturesofglucose at10◦Cand25◦C(Table1and

Fig.4B).Becausethismethodologyisbasedontriglycerides

present on thesemicroorganisms, which constituteamong

70–98%ofthetotallipids,which45% presentDHAontheir

structure.51,52 _{TheOilRedisusedmainlyonthecytological} andhistologicalanalysis;howeverthereisnoevidencethat pointtheiruseinThraustochytrids.41,50

Consequently,theresultsshowthatgrowthonglucoseat

10◦Cwasthemostfavorableconditionfortheproductionof omega-3fattyacidobservedatthe5thday.Inparallel,itwas observedthatthemajorchangeswerepresentwiththe varia-tionofthetemperature,affectingbiomass,contentofDHAand carbonintake(Figs.3–5andTable1).Particularlyitshouldbe highlightedthatat10◦Cthereexistsmoreconsumptionofthe

carbonsource,productionofDHA,EPAandminorbiomass.On

thecontrary,theculturesat25◦Cpresentresultsoppositeto thosedescribedbefore.Thisshowsthatathightemperatures thecarbonsourceisnotcompletelyusedup,whichtranslate intoaminorproductionofomega-3fattyacids,however,the increaseofthebiomasssuggeststhatthesourceofnitrogen isbeingused(Figs.3and4C).Studiespointoutthatahigh proportionofcarbon/nitrogenincultureincreasesthe

synthe-sisofDHAonmicroorganismsthatsynthetizebigamountsof

fattyacids.10,53_{Inconsequence,ourresultssuggestthatthe} ratiocarbon:nitrogenistemperaturedependent;incubatedat 25◦Cfavorsthebiomassformation,whileat10◦Cthe high

consumptionofthecarbonsourceisconsistentwiththe con-tentofDHAandEPA,diminishingtheculturebiomass(Fig.4). Finally,isimportanttoconsiderthequalityofthelipids

produced by strain 34-2, with the essential fatty acids as

theDHA,EPAandthelinoleicacid(Table1).Thisconfers a greatinterestintheclinicalarea,particularlyontreatments whereinflammationisakeyphysiologicalresponseonseveral pathologies.54,55_{Ontheotherhand,theprofileoffattyacids} presentsapotentialfortheproductionofbiofuels, particu-larlyonculturessupplementedwithglucoseatatemperature

of25◦C(Table1andFig.5).Thisisduetothefactthat

pro-duction of biofuels requires high quantities offatty acids,

mainlyC16and C18thatallowamajoroxidativeand

ther-malstability.4,28,56,57 _{However,differentareaswithpotential} inbiotechnologyarestillpoorlyunderstoodsuchas;presence ofviruses, extracellularpolysaccharides,osmolytesystems,

their role in organic matter decomposition, the biological

associationwithmarineinvertebrates,potentialin bioreme-diation,compatiblesoluteandheavymetalsresistance.

Inconclusion,thisresearchprovidesanidentificationof thediversityinAntarcticThraustochytrids.Thephylogenetic analysisallowstheclassificationofthestrain34-2witha99% ofidentitywiththespeciesT.kinnei.Thetotallipidcontentof thisstrain suggeststhat itofferspotentialbiotechnological

applicationssuchas productionofbiofuelsand nutritional

supplements.Theseresultsallowustofocus onmolecular

studiestodeterminethe effectsofthetemperature onthe

expressionofthegenesthatcodifyforenzymespresenton

theLC-PUFAsbiosyntheticpathwayandotherbioactive

com-poundswhichcouldactatlowerorroomtemperatures.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

TheauthorswishtothankMauricioQuirozforhisvaluable

assistancein microscopy work and Barbara Burmeister for

proof-readingthe manuscript.Thiswork wassupportedby

DIULA09/14(AG), PROJECT79112042 from PROGRAMA

PAI-CONICYT(FG),GrantBasalesUsa1555USACH-MECESUPtoMT,

FundResearchSupport30/2014DPIUniversidadAutónomade

Chileto(GC)andCONICYTDoctoralFellowshipN◦21130177

andChileanAntarcticInstituteGrantsDG07-16(PP).

Appendix

A.

Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2017.01.011.

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