Production of docosahexaenoic acid by Aurantiochytrium sp. ATCC PRA-276

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

Food

Microbiology

Production

of

docosahexaenoic

acid

by

Aurantiochytrium

sp.

ATCC

PRA-276

Valcenir

Júnior

Mendes

Furlan

a,∗

_,

_Victor

_Maus

b

_,

_Irineu

_Batista

c

_,

Narcisa

Maria

Bandarra

c

a_{UniversidadeFederaldoPampa(UNIPAMPA),Itaqui,RS,Brazil}

b_{InternationalInstituteforAppliedSystemsAnalysis(IIASA),Laxenburg,Austria} c_{PortugueseInstituteofSeaandAtmosphere(IPMA,I.P./DMRM),Lisbon,Portugal}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received3August2015

Accepted13October2016

Availableonline20January2017

AssociateEditor:RosaneFreitas

Schwan

Keywords:

Carbonsource

Docosahexaenoicacid

Nitrogensource

Polyunsaturatedfattyacids

Thraustochytrids

a

b

s

t

r

a

c

t

The highcostsandenvironmentalconcernsassociatedwithusingmarineresourcesas

sourcesofoils richinpolyunsaturatedfattyacidshaveprompted searchesfor

alterna-tive sources ofsuch oils. Some microorganisms, among them members of the genus

Aurantiochytrium,cansynthesizelargeamountsofthesebiocompounds.However,various

parametersthataffectthepolyunsaturatedfattyacidsproductionoftheseorganisms,such

asthecarbonandnitrogensourcessuppliedduringtheircultivation,requirefurther

eluci-dation.Theobjectiveofthisinvestigationwastostudytheeffectofdifferentconcentrations

ofcarbonandtotalnitrogenontheproductionofpolyunsaturatedfattyacids,particularly

docosahexaenoicacid,byAurantiochytriumsp.ATCCPRA-276.Weperformedbatchsystem

experimentsusinganinitialglucoseconcentrationof30g/Landthreedifferent

concentra-tionsoftotalnitrogen,including3.0,0.44,and0.22g/L,andfed-batchsystemexperiments

inwhich0.14g/Lofglucoseand0.0014g/Loftotalnitrogenweresuppliedhourly.Toassess

theeffectsofthesedifferenttreatments,wedeterminedthebiomass,glucose,totalnitrogen

andpolyunsaturatedfattyacidsconcentration.Themaximumcellconcentration(23.9g/L)

wasobtainedafter96hofcultivationinthebatchsystemusinginitialconcentrationsof

0.22g/Ltotalnitrogenand30g/Lglucose.Undertheseconditions,weobservedthe

high-estlevelofpolyunsaturatedfattyacidsproduction(3.6g/L),withdocosahexaenoicacidand

docosapentaenoicacid␻6concentrationsreaching2.54and0.80g/L,respectively.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis

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

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

Introduction

The search for nutraceutical products that can prevent

and/ortreatdiseaseshasintensifiedduringthelastdecade.

∗ _{Correspondingauthor.}

E-mail:juniorfurlan@yahoo.com.br(V.J.Furlan).

Among these products, types ␻3 and ␻6 polyunsaturated

fatty acids (PUFAs) havereceived a great dealof attention

duetotheirhealthbenefitsandtheirextensiveapplications

in the food and pharmaceutical industries.1

Docosahe-xaenoic acid (DHA, C22:6 ␻3), for example, is necessary

for the brain development of newborn children and

con-tributes to increasing their intelligence and verbal and

reasoning skills.2 _{Furthermore, DHA is helpful in treating}

http://dx.doi.org/10.1016/j.bjm.2017.01.001

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC

atherosclerosis, rheumatoid arthritis, and Alzheimer’s

disease,3 _{aswellasinpreventingbreastandcoloncancer.}4

Docosapentaenoicacid(DPA,C22:5␻6)isanotherPUFAthatis

importantforhumanhealth.DPAhasbeenfoundtohelp

pre-ventvariousdiseases,suchascardiovasculardisorders(such

as myocardial infarction, thrombosis, and atherosclerosis),

diabetes,asthma,inflammationandrheumatism(including

arthritisandosteoporosis).5

Themaincommercialsourceofthesecompounds,

partic-ularlyDHA,isoilobtainedfrommarinefish.6 _However,the

widespreadconsumptionoftheseoilsislimitedbymarine

chemicalpollution,decliningfishstocks,seasonalvariations

inthecompositionoffishoils,theirpooroxidativestability,

typicalunpleasantodourandtaste,andthehighcostoftheir

extractionand purification processes.7 _{Thisproblems have}

inspiredthedevelopmentofnewmethodsforthelarge-scale

productionofoilsbysafeandhealthysources.1,8

Heterotrophic microorganisms that are members ofthe

Thraustochytridgrouparealternative sourcesofthese oils.

Theseoleaginousmicroorganismscanaccumulatemorethan

50%oftheir weightaslipids,with ahigh concentrationof

DHA ofgreater than 25% ofthe total lipids.9 _Furthermore,

thelipidsofthraustochytridscontainaspecificPUFA(DHA)

insteadofamixtureofPUFAs.Therefore,theiroilhasahigher

levelofoxidativestabilitythanthatoffishoil.10 _The

appro-priateconcentrationsofcarbonandnitrogenareessentialfor

thraustochytridstobiosynthesizeandaccumulate

polyunsat-urated fatty acids.Theconcentration ofthe carbon source

affects the synthesis of organic molecules and the

avail-abilityofenergy,whereastheconcentrationofthenitrogen

sourceaffectsthesynthesisofaminoacidsandnucleicacids.

Therefore,understanding theeffectsofthesesubstrateson

cultivatedmicroorganismsiscrucialforoptimizingtheiroil

production.Herein,wepresenttheresultsofastudyofthe

effectofdifferentconcentrationsofcarbonandnitrogenon

thePUFAsproductionofAurantiochytriumsp.ATCCPRA-276.

Materials

and

methods

Microorganism

Aurantiochytriumsp.ATCCPRA-276cellswereobtainedfrom

theAmericanTypeCultureCollection(Manassas,VA,USA).

Preparingtheinoculum

Aurantiochytriumsp.ATCCPRA-276cellsgrownonpotato

dex-troseagarandstoredat4◦Cweretransferredto500mLflasks

containing100mLofculturemediumconsisting(g/L)ofyeast

extract (1.0), peptone (15.0) and glucose (20.0) dissolved in

seawater(1.5% w/v).Theglucosestock solutionwas

steril-izedseparately.Thecellswereincubatedinanorbitalshaker

(Ika,KS260B)rotatingat150rpmat30◦Cwithout lightfor

48h.11

Preparingtheculturemedia

Wecultivated the microorganismina Biostat®Bplus

biore-actor (Sartorius Stedim Biotech., Germany) containing a

5L borosilicate glass vessel and equipped with pressure

flow meters and gas and liquid-flow controllers. We

con-ducted batch and fed-batch experiments. For the batch

systemexperimentsweusedamediumconsistingofKH2PO4

(0.3g/L),MgSO4·7H2O(5.0g/L),NaCl(10.0g/L),NaHCO3(0.1g/L),

CaCl2·2H2O(0.3g/L),KCl(0.28g/L),glucose(30.0g/L)and

dif-ferent total nitrogen (TN) concentrations: 3.0g/L (1.36g/L

(NH4)2SO4,13.63g/Lyeastextractand13.63g/Lmonosodium

glutamate), 0.44g/L (0.2g/L (NH4)2SO4, 2.0g/L yeast extract

and 2.0g/Lmonosodiumglutamate),and 0.22g/L(0.1g/Lde

(NH4)2SO4,1.0g/Lyeastextractand1.0g/Lmonosodium

glu-tamate). For the fed-batch system experiments, 0.14g/Lof

glucoseand0.0014g/Loftotalnitrogenweresuppliedhourly

(6.66×10−4g/Lhof(NH4)2SO4,6.66×10−3g/Lhofyeastextract

and 6.66×10−3g/Lhof monosodiumglutamate). Theyeast

extract,monosodiumglutamateandglucosesolutionswere

separatelysterilizedbytreatmentat121◦Cfor15minina

Cer-toClavCV-EL-18Lautoclave.Thebioreactorwassterilizedin

anAJCUniclave77-127Lautoclavefor60min.Theother

com-ponentsofthemediumwerefiltered-sterilizedusing0.22␮m

membranes(Millipore).

Aftersterilization,thedissolvedcomponentswereadded

to the bioreactor along with the following metal

solu-tions: MnCl2·4H2O (8.6mg/L), ZnCl2 (0.6mg/L), CoCl2·4H2O

(0.26mg/L), CuSO4·5H2O (0.02mg/L), FeCl3·6H2O (2.9mg/L),

H3BO3 (34.2mg/L), and Na2EDTA (30.0mg/L) and the

fol-lowing vitamin solutions: thiamine (9.5mg/L) and calcium

pantothenate(3.2mg/L),allofwhichhadbeensterilizedusing

0.22␮mmembranefilters(Millipore).Theinoculum(350mL)

wasthenaddedtotheculturemedium(10%v/vrelativetothe

totalvolumeoftheculturemedium).

Cultivation was performed at 23◦C with agitation at

100rpmand thepHofthemediaadjustedto6.0using4N

NaOH.Duringthefirst120hofcultivation,theculturemedium

was aeratedat2.5vvm.Afterthisperiod,airinjectionwas

discontinued.

Biomassconcentration

Thebiomasswasdeterminedevery24husingthemethodof

Min etal.12 _{Analiquot oftheculture mediumwasfiltered}

usingapreviouslyweighedglass-microfiberfilterpaper(GF/C:

1.2␮m, Whatman). Thebiomass retained in the filter was

washedtwiceusingdistilledwateranddriedinanoven

(Mem-mert)at60◦Cfor24h.Thebiomasscontentwascalculatedas

thedifferencebetweentheinitialandfinalweights.

Glucoseconcentration

The glucosecontent ofthe culturesupernatant was

deter-mined each 24h using the spectrophotometric method

described byMiller,13 _{usingaUV/Visdualbeamabsorption}

spectrophotometer(AtiUnicamHeliosAlpha,UK).

Totalnitrogenconcentration

Thetotalnitrogen(definedandcomplexsources)contentof

theculturesupernatantwasdeterminedeach24hfollowing

Fattyacidprofile

Culturesamplescollectedat24hintervalswerecentrifuged

(Kubota,6800)at8742×gfor15minat4◦C,afterwhichthe

biomass was washed with distilled water and centrifuged

again. This process was repeated twice. Thebiomass was

frozenat−20◦_{Canddriedfor48husingafreezedryer(Heto}

PowerDryLL3000).

Between 20 and 100mg ofthe lyophilized biomasswas

weighedand added to50␮LoftheinternalC21:0 standard

solution(10mg/mL)topermitexpressingtheresultsasgof

fattyacids/goflyophilizedbiomass.

Methylestersofthefattyacidswere preparedby

esteri-ficationusingtheacidcatalysismethoddescribedbyCohen

et al.14 _{A gas chromatography system (Varian, CP 3800)}

equippedwithanautosampler,injector,andflameionization

detector(FID),bothofthelatterat250◦C,wasusedtoidentify

themethylestersinthesamples.Themethylesterswere

sep-aratedusingaDB-WAXpolyethyleneglycolcapillarycolumn

(Agilent,30mlong,0.25mminternaldiameter and0.25␮m

thick)usingthefollowingprogram:heatingat180◦C(5min),

graduallyincreasing at4◦C/min to 220◦C (and holding for

25min),andthengraduallyincreasingat20◦C/minto240◦C

(andholdingfor15min).Themethyl esterswereidentified

bycomparingtheirretentiontimeswiththoseof

chromato-graphicstandards(Sigma–AldrichCo,St.Louis,MO,USA).

Theresultswere analyzedusingananalysisofvariance

(ANOVA) and the mean values were compared using the

Tukeytest,withthesignificancelevelsetat5%.Before

per-formingtheANOVA,thenormalityofthedatadistributions

wereevaluatedusingtheKolmogorov–Smirnovtestandthe

homoscedasticityofthedatawasevaluatedusingtheCochran

test.15

Results

Growthkinetics,glucoseandtotalnitrogenconsumption

Fig.1showstheaveragebiomass,glucoseandtotalnitrogen

concentrationsover timeinculturesofAurantiochytriumsp.

ATCCPRA-276growingunderfourdifferentconditions.

As shownin Fig. 1(A), inthe experimentsusing an

ini-tial total nitrogen concentration of 3.0g/L, the maximum

biomassconcentration(9.3g/L)wasreachedat120h,foran

average yieldof0.07g/Lh ofbiomass. Theaverage glucose

consumption rate in these experiments was 0.13g/Lh and

0.43gofbiomasswasproducedpergramofglucoseconsumed

(YBiomass/Glucose).Theaveragetotalnitrogenconsumptionrate

was0.0010g/Lh,resultinginasubstratetobiomassconversion

factor(YBiomass/nitrogen)of65.8.

In the experiments using an initialnitrogen

concentra-tion of0.44g/L,thehighestbiomassconcentration(17.0g/L)

was obtained at 144h, foran average yield of 0.11g/Lh of

biomass (Fig. 1(B)). The average glucose consumption rate

was0.15g/Lh, resultinginaglucosetobiomassconversion

factor (YBiomass/glucose) of 0.65. The average total nitrogen

consumption rate was 0.0018g/Lh. Each gram of nitrogen

that was consumed was converted into 50.7g of biomass

(YBiomass/Nitrogen).

A

0 24 48 72 96 120 144 168 192 Culture time (h) 1 2 3 4 5 6 7 8 9 10 Biomass (g/L) 0 5 10 15 20 25 30 Glucose (g/L) 2.8 2.82 2.84 2.86 2.88 2.90 2.92 2.94 2.96 2.98 3.0 Total nitrogen (g/L)

B

0 24 48 72 96 120 144 168 192 Culture time (h) 2 4 6 8 10 12 14 16 18 Biomass (g/L) 0 5 10 15 20 25 30 35 Glucose (g/L) 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 Total nitrogen (g/L)

C

0 24 48 72 96 120 144 168 192 Culture time (h) 2 4 6 8 10 12 14 16 18 20 22 24 26 Biomass (g/L) 0 5 10 15 20 25 30 Glucose (g/L) 0,0 0.05 0.10 0.15 0.20 0.25 0.30 Total nitrogen (g/L)

D

0 24 48 72 96 120 144 168 192 Culture time (h) 2 4 6 8 10 12 14 Biomass (g/L) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Glucose (g/L) 0.0 0.05 0.10 0.15 Nitrogen total (g/L)

Biomass (g/L) Glucose (g/L) Total nitrogen (g/L)

Fig.1–Concentrationsofbiomass,glucoseandtotalnitrogenovertimeintheculturemediaofAurantiochytriumsp.ATCC PRA-276.Thegraphsshowthedataobtainedusingdifferenttreatments,asfollows:abatchsystemwithan(A)initial nitrogenconcentrationof3.0g/L,(B)initialnitrogenconcentrationof0.44g/Lor(C)initialnitrogenconcentrationof0.22g/L or(D)afed-batchsystemwith0.14g/Lofglucoseand0.0014g/Lofnitrogensuppliedhourly.

25 20 15 10 Biomass (g/L) PUFAs (mg/g) PUFAs (g/L) 5 0 0 24 48 72 Culture time (h) 96 120 144 168 0 50 100 150 200 4.0 3.0 2.0 1.0 0.0 PUFAs (mg/g): 3.0 (g/L) TN 0.44 (g/L) TN 0.22 (g/L) TN Fed-batch Biomass (g/L): 3.0 (g/L) TN 0.44 (g/L) TN 0.22 (g/L) TN Fed-batch PUFAs (g/L): 3.0 (g/L) TN 0.44 (g/L) TN 0.22 (g/L) TN Fed-batch

Fig.2–PUFAsconcentrationsinthebiomassofAurantiochytriumsp.ATCCPRA-276cultivatedunderdifferentconditions. PUFAs,polyunsaturatedfattyacids;TN,totalnitrogen.

Intheexperimentsusinganinitialnitrogenconcentration

of0.22g/L,thehighestbiomassconcentration(23.9g/L)was

observedat96h(Fig.1(C)),foramaximumyieldof0.23g/Lhof

biomass.Intheseexperiments,theaverageglucose

consump-tionratewas0.17g/Lhandtheglucosetobiomassconversion

factor (YBiomass/Glucose) was1.28. Theaverage nitrogen

con-sumptionratewas0.0022g/Lh,resultinginaYBiomass/Nitrogen

conversionfactorof104.7,meaningthat 104.7gofbiomass

wasproducedpergramofnitrogenconsumed.

When the fed-batch cultivation process was used, the

highestbiomassconcentration(13.2g/L)wasreachedat120h

(Fig.1(D)),foramaximumyieldof0.10g/Lhofbiomass.The

average glucose consumption rate was 0.13g/Lh, resulting

inaglucosetobiomassconversionfactor(YBiomass/Glucose)of

0.64.Usingthis cultivationprocess,theaveragetotal

nitro-genconsumptionratewas0.0016g/LhandtheYBiomass/Nitrogen

conversionfactorwas49.15,meaningthat49.15gofbiomass

wasproducedpergramofnitrogenconsumed.

Fattyacidprofile

Fig.2showstheaveragePUFAsconcentrationsinthebiomass

thatwerereachedthroughoutAurantiochytriumsp.ATCC

PRA-276cultivationinthedifferentexperiments.

EvaluatingthePUFAsconcentrationsinthebiomass(g/L)

showed that there were significant differences among the

valuesatdifferenttimesthroughoutcultivationinallofthe

experiments.ThehighestPUFAsconcentration(3.6g/L)was

observedat96hofcultivationintheexperimentsusingan

initialnitrogen concentrationof 0.22g/L. Thesecond

high-est PUFAs concentration (2.85g/L) was obtained at 168h

of cultivation in the experiments using an initial

nitro-gen concentrationof 0.44g/L. Inthe fed-batch culture, the

maximal PUFAs concentration of 1.89g/L was reached at

144h of cultivation. In the experiments using an initial

nitrogen concentration of 3g/L, the maximal PUFAs

con-centration of 0.84g/L was reached at 120h of cultivation

(Fig.2).

Fig.3showsthefattyacidprofileatthetimepointwhenthe

highestPUFAsyieldwasobtainedineachexperiment(g/L).

Intheexperimentsusinganinitialnitrogenconcentration

of3.0g/L,9%(w/w)ofthebiomasswascomposedofPUFAsat

120hofcultivation,ofwhich20%wasDPA␻6(Fig.3),which

represented1.8%ofthebiomass(0.17g/L).AsshowninFig.3,

DHAaccountedfor61.3%ofthePUFAspresent,i.e.,5.5%ofthe

biomass(0.51g/L).

Using an initial nitrogen concentration of 0.44g/L, 18%

(w/w)ofthebiomasswascomposedofPUFAsat168hof

cul-tivation,ofwhich22%wasDPA␻6,i.e.,3.9%ofthebiomass

(0.63g/L). We alsoobserved that DHA accounted for 69.2%

ofthePUFAs,whichwas12.5%ofthetotalbiomass(1.97g/L)

(Fig.3).

Intheexperimentsusinganinitialnitrogenconcentration

of0.22g/L,15%(w/w)ofthebiomasswascomposedofPUFAsat

96hofcultivation,ofwhich22.3%wasDPA␻6(Fig.3),

C14:0 C15:0 C16:0 C16:1ω9 C18:1ω9 C16:2ω4 C20:4ω6 C20:5ω3 C22:5ω6 (DPA) C22:5ω3

C22:6ω3 (DHA) Other fatty acids

3.0 (g/L)

0.44 (g/L)

0.22 (g/L)

Fed-batch

0 10 20 30

Total fatty acids, %

40 50 60 70 80 90

Fig.3–Fattyacidprofilesofthebiomassof

Aurantiochytriumsp.ATCCPRA-276whenbatch-cultured for120husinganinitialnitrogenconcentrationof3.0g/L totalnitrogen,at168hwhenbatch-culturedusinganinitial nitrogenconcentrationof0.44g/L,at96hwhen

batch-culturedusinganinitialnitrogenconcentrationof 0.22g/L,andat144hwhenculturedusingthefed-batch process.

thatDHAcomprised70.5%ofthePUFAs(Fig.3),i.e.,10.62%of

thebiomass(2.54g/L).

After144hofcultivationusingthefed-batchsystem,14.9%

(w/w)ofthebiomasswascomposedofPUFAs,ofwhich23.1%

wasDPA␻6(Fig.3),i.e.,3.43%ofthetotalbiomass(0.44g/L).

Inaddition,DHAaccountedfor70.5%ofthePUFAs(Fig.3),i.e.,

10.5%ofthetotalbiomass(1.33g/L).

Discussion

Intheexperimentsusinganinitialnitrogenconcentrationof

3g/L,thecellsconsumedtheleastamountofTN(0.0010g/Lh),

resultinginthelowestcellyield(0.07g/Lh).Thisresultcould

beduetothelowC/N substrateratiointheculture(4). An

excess of nitrogen may have inhibited the growth of the

evaluatedstrain. Chenet al.16 _{conductedastudy inwhich}

theyoptimizedthenitrogensourcesforAurantiochytriumsp.

BR-MP4-A1andobtainedamaximumbiomassconcentration

(9.27g/L)bysupplying2.4g/LofTN(asmonosodium

gluta-mate,yeastextract,and tryptone),whichresultedinaC/N

ratio (5). In the experiments in which we used an initial

nitrogenconcentrationof3g/L,asimilarmaximumbiomass

concentrationof9.3g/Lwasreached(Fig.1(A)).

Using an initial TN concentration of 0.44g/L to

culti-vate Aurantiochytrium sp.BR-MP4-A1, Li et al.17 _{obtained a}

maximum biomass concentration of 14.5g/L, whereas we

observeda maximum biomass concentrationof 17.0g/L in

culturesofAurantiochytriumsp.ATCCPRA-276(Fig.1(B)).This

differenceisduetothesubstratetobiomassconversionrate

(YBiomass/Glucose)being0.53inthepreviousstudy,whereasin

thisstudy,eachgramofglucoseconsumedwasconcertedto

0.65g/Lofbiomass.Thedifferenceintheconversionratecan

beattributedtotheuseofdifferentspeciesinthestudies.

Cultivation using aninitial TNconcentration of0.22g/L

(Fig. 1(C)) resulted in higher substrate consumption rates

(0.17g/Lhglucoseand0.0022g/LhTN)andhighercellbiomass

productivity(0.23g/Lh)comparedwiththoseobtainedusing

the other TN concentrations tested in this study. In

addi-tion, the glucose to biomass conversion factor (1.28) was

higher undertheseconditionsthan underthe other tested

conditions,whichalsoresultedalsoinahighercell

concentra-tion(23.9g/L).GanuzaandIzquierdo18_{usedSchizochytriumsp.}

G13/2Stostudytheeffectofsubstratelevelsonlipid

accumu-lation.Theseauthorsfoundthatusinginitialconcentrations

of0.30g/LofTNand40g/Lofglucoseresultedinabiomass

yieldof15.7g/L.

Intheexperimentsusingfed-batchsystem(Fig.1(D)),the

concentration of TN decreased over time because its

con-sumptionratewashigher(0.0016g/LhofTN)thanitssupply

rate(0.0014g/LhofTN).Theconcentrationofglucoseinthe

culturemediumdecreaseduntil144hofcultivationand

sub-sequentlyincreased.Thisphenomenoncanbeexplainedby

theglucosesupplybeinggreaterafter144hthanthatrequired

forcelldevelopmentandmaintenance.

Thefattyacidprofilesobservedinourexperiments(Fig.3)

aresimilartothosepreviouslyobtainedbyZhuetal.19 and

Furlan et al.20 _{using Schizochytrium limacinum OUC88 and}

Thraustochytrium sp. ATCC 26185, respectively. Zhu et al.19

foundC14:0(3.8–9.6%),C15:0 (2.1–10.1%),C16:0 (32.6–43.3%),

DPA ␻6 (7.1–8.2%)and DHA (29.8–36.5%) as the mainfatty

acids. Furlan et al.20 _{found C14:0 (1.5–9%), C15:0 (21–35%),}

C16:0 (5–33%), DPA ␻6 (7–9%) and DHA (20–31.5%) as the

main fatty acids. These results are similar to our results:

C14:0(1.8–16%),C15:0(5–16%),C16:0(9–32.5%)andthe

polyun-saturated, including DPA ␻6 (6.5–14%) and DHA (20–43%).

Therefore,thefattyacidsproducedbymembersofthe

Thraus-tochytriidaefamilyarelikelytobemainlyC14:0,C15:0,C16:0,

DPA␻6andDHA.

Lietal.17_{studiedthecompositionoftheAurantiochytrium}

sp.BR-MP4-A1biomassandconcludedthatDPA␻6andDHA

comprised6.6%and28.9%,respectively,ofthetotalfattyacids.

TheseauthorsalsoobservedthattheDPA␻6(18%)andDHA

(78%)levelswerehigherthanthoseoftheotherPUFAsthat

werequantitated,similartotheresultsobtainedinthisstudy

(Fig.3).

WealsoobservedareductioninPUFAsproduction

rela-tivetothatofthetotalfattyacidswhentheTNconcentration

decreased.Incontrast,C16:1␻9andC18:1␻9productionwas

increasedbecausethesefattyacidsaretheprecursorsusedfor

PUFAssynthesis.Therefore,thesmallerthefractionsofC16:1

␻9andC18:1␻9,thehigherthefractionofPUFAs(Fig.3).

AmongthemajorfattyacidsthatarePUFAs,thecontent

ofDPA␻6(20–23.1%)variedlittle,whereasthecontentofDHA

(61.3–70.5%)variedgreatly. Highconcentrationsofavailable

totalnitrogenintheculturemediumfacilitatethesynthesis

ofother fattyacids inadditiontoDHA andDPA ␻6,which

formasignificantfractionofthePUFAspresent.Forexample,

Table1–TotalfattyacidcontentoftheAurantiochytriumsp.ATCCPRA-276biomasswiththehighestPUFAs

concentrationundereachexperimentalcondition.

Totalnitrogen 3.0g/L 0.44g/L 0.22g/L Fed-batchb

C/N 4 27 54 100

Culturetime(h) Totalfattyacids(mg/g)a

120 129.15±0.15

168 448.47±0.05

96 455.62±0.09

144 526.20±0.20

a _{Meanvalues±standarddeviation.}

b _{0.14g/Lofglucoseand0.0014g/Loftotalnitrogensuppliedeachhour.}

2.06%,respectively,ofthefattyacidsobservedinthebiomass

obtainedinculturesgrownusinganinitialnitrogen

concentra-tionof3g/LTNandC16:2␻comprised1.95%ofthefattyacids

observedinculturesgrownusinganinitialnitrogen

concen-trationof0.44g/L(Fig.3).

The main commercial sources of PUFAs are species of

fatty fish,suchasherring,mackerel, salmonand sardines.

TheAurantiochytriumstrainusedinthis studyaccumulated

higherconcentrationsofPUFAs(28–70%)thanthosereported

in sardineoil (31.1%) by Morais.21 _{The DHA concentration}

(20–43%)producedbythisoleaginousmicroorganismwas

2-to4-fold higherthan that foundinthe sardineoil(11%).21

CultivatedAurantiochytriumsp.ATCCPRA-276isapromising

alternativesourceofoilrichinPUFAs.Usingfishforthe

large-scaleproductionofsuchoilislimitedbythechangesinthe

lipidcompositionsandcontentsandthefattyacidprofilesof

fish,whichareaffectedbytheseasonsandtheirspecies,sex,

size,reproductivestatus,catchlocation,dietandnutritional

status.22_{Moreover,fishoilexhibitsagreatdiversityoffatty}

acidswithdifferentchainlengthsanddegreesof

unsatura-tionandthus,requiresexpensiveextractionandpurification

processes.8

Cultivatingan oleaginous microorganism in the

labora-toryundercontrolledenvironmentalconditionsreducesthe

risk ofcontamination and can increase fatty acid

produc-tionatalowcost. Inthisstudy,weobservedthatthetotal

fattyacidcontentofthebiomass(%,w/w)increasedasthe

TNconcentrationwasdecreased.Thisphenomenonoccurred

becauselipids generallyaccumulateinoleaginous

microor-ganismswhenthemediumcontainsanexcessofthecarbon

sourceandalimitedamountofnitrogen(highC/Nratio).In

thepresenceoflow-levelnitrogen,thesynthesisofproteins

andnucleicacids islimitedbythe enhancedconversionof

carbontooil.23,24 _{Thisprocesswasobservedinthe}

experi-mentsusingafed-batchsystem,inwhichC/Nratiowashigh,

eventuallyleadingtotheaccumulationofahighleveloftotal

fattyacids(526.20mg/g)inthebiomass(Table1).However,

fed-batchculturesexhibitedlowerPUFAsproduction(1.89g/L)and

consequentlylowerDPA␻6(0.44g/L)andDHA(1.33g/L)

pro-ductionthanthoseofthebatchculturesbecausetheyieldsof

thesefattyacidsaredependentontheaccumulationofPUFAs

inthetotallipidsaswellasontheaccumulationofoilsinthe

biomass.Additionally,thefattyacidyieldwasalsorelatedto

thecellconcentrationatagiventime.

For example, in the experiments using an initial TN

concentrationof0.22g/L,therewasahighersubstrate

con-sumptionrate,increasedbiomassproductivityandahigher

C/N ratio (54), which resulted in higher yields of DPA ␻6

(0.80g/L)andDHA(2.54g/L).

Ganuza and Izquierdo18 _{observedgreater DPA␻6(3.85%}

w/w) and DHA(15.4% w/w)accumulation bySchizochytrium

sp. G13/2S cells grown using initial nitrogen and glucose

concentrationsof0.30g/Land40g/L,respectively,than was

observedinourexperiments(3.34%w/wDPA␻6,and10.62%

w/wDHA)usinganinitialnitrogenconcentrationof0.22g/L.

However, their DPA ␻6 (0.6g/L) and DHA (2.42g/L)

produc-tionrateswerelowerthanours,whichcanattributedtothe

highermaximumbiomassconcentration(23.9g/L)obtainedin

ourexperimentscomparedwiththatreportedbyGanuzaand

Izquierdo18_(15.7g/L).

UsingasimilarculturemediumwithaninitialTN

concen-trationof0.44g/LtogrowSchizochytriumsp.ATCC20889,Jiang

etal.8_{observedthatDHAaccountedfor26%ofthetotalfatty}

acids after120hofcultivation,whichisverysimilartothe

25.5%DHAlevelinthetotalfattyacidsobservedinourstudy.

Inourstudy,theaccumulatedbiomassconsistedof

approxi-mately12.5%(w/w)DHA,whichishigherthanthevalue(8.8%)

reportedbyJiangetal.8

Burjaetal.24 _{evaluatedtheeffectofdifferent}

concentra-tionsofnitrogenonfattyacidproductionbyThraustochytrium

sp. ONC-T18. Using aninitial TNconcentrationof 0.75g/L,

these authors obtained 0.04g/L of DHA, corresponding to

0.53%(w/w)ofthebiomass,andreportedalowbiomass

con-centration (7.5g/L) and alow levelofDHA inthe biomass.

Burjaetal.24_{alsoobservedthatusingahigherinitialTN}

con-centration(1.23g/L),1.56g/LofDHAwasobtained,whichis

approximately3timestheDHAconcentration(0.51g/LDHA)

observedinourexperimentsusinganinitialTNconcentration

of3.0g/L.

Conclusions

Herein,wepresentedthe resultsofastudy oftheeffectof

differentconcentrationsofcarbonandnitrogenonPUFAs

pro-ductionbyAurantiochytriumsp.ThePUFAsproductionofthis

microorganism dependson the accumulationoftotal fatty

acidsandontheconcentrationofthebiomass.Therefore,the

culturemediumshouldfacilitatethegrowthofthis

microor-ganismandprovideanadequatenitrogensupplywithrespect

totheC/Nratiobecauseitaccumulates oilswhenthetotal

nitrogensupplyislimited.

Themainpolyunsaturatedfattyacidsfoundinthe

and DHA(61.3–70.5%). Themaximum cellconcentration of

23.9g/L (with45.5% of its weight consisting offatty acids)

wasobservedat96hofcultivation usinginitial

concentra-tionsof30g/Lofglucoseand0.22g/Loftotalnitrogen.Under

these conditions, the highest PUFAs concentration (3.6g/L)

wasreached,withtheDHAandDPA␻6concentrationsbeing

2.54and0.80g/L,respectively.

Theresultsofthisstudyshowedthatthegrowthof

Auran-tiochytriumsp.ATCCPRA-276anditsaccumulationofPUFAs,

particularly DHA, are dependent on the concentrations of

thecarbonandnitrogensubstrates.Theresultsalso

demon-stratedthatthecultivationperiodisanimportantvariablefor

PUFAsproductionbyAurantiochytriumsp.ATCCPRA-276.

Aurantiochytriumsp.ATCCPRA-276iscapableofproducing

highlevelsofPUFAs.Therefore,developingnewtechniques

forcultivatingthismicroorganismcouldreducethecostand

increasetheproductionofoilsforuseinfoodandmedicines.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

This study was supported by the Coordenac¸ão de

Aperfeic¸oamento de Pessoal de Nível Superior of Brazil

(CAPES)anddevelopedatthePortugueseInstituteofSeaand

Atmosphere(IPMA)inLisbon,PT,withtheaidofascholarship

grant awarded to the first author by the Doctoral in the

CountrywithInternshipAbroadProgramme(PDEE)(grantno.

6906/10-9).TheauthorsalsothanktheALGAENEProjectand

DepsiextractaBiologicalTechnologies,Lda.fortheirsupport.

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