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

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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

a

_,

_Lyliam

_Loperena

b

_,

_Ivonne

_Hinzpeter

c

_,

_Paulina

_Pradel

d

_,

Felipe

Gordillo

e

_,

_Gino

_Corsini

f,g

_,

_Mario

_Tello

h

_,

_Paris

_Lavín

i

_,

_Alex

_R.

_González

a,∗

a_Laboratorio_de_{Microbiología}_Ambiental_y_{Extremóﬁlos,}_Departamento_de_Ciencias_Biológicas,_Universidad_de_los_Lagos,_Osorno,_Chile b_Instituto_de_Ingeniería_Química,_Departamento_de_{Bioingeniería,}_Universidad_de_la_República,_Montevideo,_Uruguay

c_Departamento_de_Gobierno_y_Empresa,_Universidad_de_los_Lagos,_Osorno,_Chile

d_Centro_de_Interacción_Planta-Suelo_y_{Biotecnología}_de_Recursos_Naturales,_Laboratorio_de_Fisiología_y_Biología_Molecular_Vegetal,

UniversidaddeLaFrontera,Temuco,Chile

e_Centro_de_{Biotecnología}_de_los_Recursos_Naturales,_Facultad_de_Ciencias_Agrarias_y_Forestales,_Universidad_Católica_del_Maule,_Talca,

Chile

f_Instituto_de_Ciencias_Biomédicas,_Facultad_de_Ciencias_de_la_Salud,_Universidad_Autónoma_de_Chile,_Santiago,_Chile g_Universidad_Cientíﬁca_del_Sur,_Lima,_Perú

h_Centro_de_{Biotecnología}_Acuícola,_Departamento_de_Biología,_Facultad_de_Química_y_Biología,_Universidad_de_Santiago,_Santiago,_Chile i_Laboratorio_de_Complejidad_Microbiana_y_Ecología_Funcional,_Instituto_Antofagasta,_Universidad_de_Antofagasta,_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

t

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.

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

∗ _{Corresponding}_author_.

E-mail:[email protected](A.R.González).

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

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Introduction

Themicroorganisms belongingto the Labyrinthulomycetes

class and family Thraustochytriaceae are unicellular protists

whichare present inmarine ecosystems. Theyhave akey

roleontheinitialstageofthemicrobialchainfood,asorganic

matterdegraders.1,2 _These_{microorganisms}_have_been

stud-ied at the morphological, ecological and biotechnological

levels.3,4 _Due _to _the _presence_of _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_The_latter_place_presents

poorstudiesofthemarinemicrobialdiversityandthe

num-berofspeciesvarieswidelybetweentaxons.19–21 _Currently,

inthediversitystudiesoftheThraustochytrids from

Antarc-ticwaterstwospecieshavebeendescribed:Thraustochytrium

antarticum(SoutheasternIndianOcean)andThraustochytrium rossii(SouthwesternPaciﬁcOcean).23_More_recently,_only_one

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,_this_is_not_clearly_established,

sinceinSchizochytriumstrains,thefattyacidsincreaseis

asso-ciatedwithbiomassgrowth.30 _Besides,_in_vitro_studies _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,_they_were_incubated_at_different

temperatures (10◦Cand 25◦C)and observedusinganoptic

microscopeduring10daystoimprovemicroorganismﬁxation.

InoculeswerecultivatedinliquidmediumusingHondaetal.

(1998)58_modiﬁed_protocol_(0.2%_yeast_extract_and_0.2%_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_{Thermocycling}_proﬁle_was_performed

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

(3)

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_Sequences_alignments_were_performed_using_Clustal

X2.37_Phylogenetic_analysis,_were_done_with_{Neighbor-joining}

method36_and _the_tree_topology_with_MEGA _4.0.2.38 _Species

relations were evaluated with statistical analysis through

bootstrapprotocol.39_To_group_the_phylogenetic_analysis_of_the

familyThraustochytriaceaetheProrocentrumgenuswasusedas

outgroup.

Extractionanddeterminationoffattyacids

Fattyacids proﬁlewas donewith 50mglyoﬁlizedbiomass,

usingdirecttransesteriﬁcation.40_The_fatty _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;

(4)

(5)

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._On_the_other_hand,_a_low_growth_was

reg-isteredusingaglucoseculturewithatemperatureof10◦C,

showingamaximumvalueofbiomassat7dayswitharateof

growthof0.7×103_mg/L._In_starch_medium,_maximum_values

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

and1×103_mg/L_{respectively.}_These_results_revealed_that_this

microorganismhadahighbiomassproducedindifferent

car-bonsourcesathightemperature.

Theeffectsofthecarbonsourceandtemperaturewere

ana-lyzedonbiomass, carbonintakeand polyunsaturatedfatty

acidcontentfor5daysofculture(Fig.4).Theresultsshowed

thatthereexistsamajorproductionofbiomassat25◦C,with

2×103_mg/L._On _the _other _hand,_low _biomass_was _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/L_for_glucose_and _1.2_×₁₀3_mg/L_for_starch._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 _Identiﬁed_as_fatty_acids_according_standard_retention_time_from

standardmix.

respectively)were obtained,witharatioofomega-3:6fatty

acidsof55.9%(Table1).

Determinationandanalysisoffattyacids

Thefattyacids qualitativestudies weredoneusingOilRed

Ostaining,afat-solublediazodye.Forthispurpose,cultures

A

3

2.5

2

1.5

1

0.5

0

3

2.5

2

1.5

1

0.5

0 0

0 1 2 3 4 5 1 2 3 4 5

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

(6)

2.5 35 4

3

2

1

0 30

EPA

DHA

25

20

15

10

5

0

B

A

C

2

1.5

1

0.5

0

10°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

10°C 25°C

Saturated Unsaturated

25°C 0

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 _the_morphology _of_the

strainsbelongingtothegenusThraustochytriumisolatedfrom

theSoutheasternIndianOceanandtheSouthwesternPaciﬁc

Ocean.Besides,Moroetal.23_described_and_{characterized}_the

(7)

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,_the_molecular_analysis

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_On_the_other_side,

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_The_research_is_focused

mainlyontheproductionofessentialfattyacids,beingDHA28

themoststudiedmetabolite.ThesynthesisofLC-PUFAson

thesemicroorganismsisperformedusingtwodifferent

mech-anisms;oneofthemistheelongation/desaturationpathway

whichhasbeendescribedonThraustochytriumaureum.10,44

Fur-thermore,Naganoetal.45_demonstrated_that_the_content_of

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 _The_high _ratios _of

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

production.49,50 _According_to_this,_the_culture_supplemented

withglucoseat10◦Cevidencedthebestresultswithavalue

of55.9%and5%,respectively(Table1).Ithasbeendescribed

thatproﬁlesoffattyacidsareusedasamethodologyto

estab-lishphylogenetichomologieswithotherstrainsbelongingto

the family Thraustochytriaceae.16,19 _{Consequently,} _the_proﬁle

of strain 34-2 isconsistent with researches carried out by

Leeetal.50_that_cultured₃₆_{Thraustochytrid}_strains_with_0.2%

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 _The_Oil_Red_is_used_mainly_on_the_cytological

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_In_consequence,_our_results_suggest_that_the

ratiocarbon:nitrogenistemperaturedependent;incubatedat

(8)

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_On_the_other_hand,_the_proﬁle_of_fatty_acids

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,_different_areas_with_potential

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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