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

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Pleasecitethisarticleinpressas:FurlanVJM,etal.ProductionofdocosahexaenoicacidbyAurantiochytriumsp.ATCCPRA-276.BrazJMicrobiol.

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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

aUniversidadeFederaldoPampa(UNIPAMPA),Itaqui,RS,Brazil

bInternationalInstituteforAppliedSystemsAnalysis(IIASA),Laxenburg,Austria

cPortugueseInstituteofSeaandAtmosphere(IPMA,I.P./DMRM),Lisbon,Portugal

a r t i c l e i n f o

Articlehistory:

Received3August2015 Accepted13October2016 Availableonlinexxx

AssociateEditor:RosaneFreitas Schwan

Keywords:

Carbonsource Docosahexaenoicacid Nitrogensource

Polyunsaturatedfattyacids Thraustochytrids

a bs t r a c t

The highcostsandenvironmentalconcernsassociatedwithusingmarineresourcesas sourcesofoils richinpolyunsaturatedfattyacidshaveprompted searchesforalterna- tive sources ofsuch oils. Some microorganisms, among them members of the genus Aurantiochytrium,cansynthesizelargeamountsofthesebiocompounds.However,various parametersthataffectthepolyunsaturatedfattyacidsproductionoftheseorganisms,such asthecarbonandnitrogensourcessuppliedduringtheircultivation,requirefurthereluci- dation.Theobjectiveofthisinvestigationwastostudytheeffectofdifferentconcentrations ofcarbonandtotalnitrogenontheproductionofpolyunsaturatedfattyacids,particularly docosahexaenoicacid,byAurantiochytriumsp.ATCCPRA-276.Weperformedbatchsystem experimentsusinganinitialglucoseconcentrationof30g/Landthreedifferentconcentra- 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,weobservedthehigh- estlevelofpolyunsaturatedfattyacidsproduction(3.6g/L),withdocosahexaenoicacidand docosapentaenoicacid␻6concentrationsreaching2.54and0.80g/L,respectively.

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

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atherosclerosis, rheumatoid arthritis, and Alzheimer’s disease,³ aswellasinpreventingbreastandcoloncancer.⁴ Docosapentaenoicacid(DPA,C22:5␻6)isanotherPUFAthatis importantforhumanhealth.DPAhasbeenfoundtohelppre- 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 Theappro- priateconcentrationsofcarbonandnitrogenareessentialfor thraustochytridstobiosynthesizeandaccumulatepolyunsat- 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-276cellsgrownonpotatodex- 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^®Bplusbiore- 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)anddif- 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/Lmonosodiumglu- 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^◦Cfor15mininaCer- toClavCV-EL-18Lautoclave.Thebioreactorwassterilizedin anAJCUniclave77-127Lautoclavefor60min.Theothercom- ponentsofthemediumwereﬁltered-sterilizedusing0.22␮m membranes(Millipore).

Aftersterilization,thedissolvedcomponentswereadded to the bioreactor along with the following metal solutions: 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 following 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 wasdeter- mined each 24h using the spectrophotometric method described byMiller,¹³ usingaUV/Visdualbeamabsorption spectrophotometer(AtiUnicamHeliosAlpha,UK).

Totalnitrogenconcentration

Thetotalnitrogen(deﬁnedandcomplexsources)contentof theculturesupernatantwasdeterminedeach24hfollowing theproceduredescribedbyFurlanetal.¹¹

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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 preparedbyesteri- ficationusingtheacidcatalysismethoddescribedbyCohen et al.¹⁴ A gas chromatography system (Varian, CP 3800) equippedwithanautosampler,injector,andflameionization detector(FID),bothofthelatterat250^◦C,wasusedtoidentify themethylestersinthesamples.Themethylestersweresep- 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 bycomparingtheirretentiontimeswiththoseofchromato- graphicstandards(Sigma–AldrichCo,St.Louis,MO,USA).

Theresultswere analyzedusingananalysisofvariance (ANOVA) and the mean values were compared using the Tukeytest,withthesigniﬁcancelevelsetat5%.Beforeper- 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 initial 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 concentration 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

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Biomass (g/L)

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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)

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Biomass (g/L)

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Glucose (g/L)

0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45

C

0 24 48 72 96 120 144 168 192 Culture time (h)

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Biomass (g/L)

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Glucose (g/L)

0,0 0.05 0.10 0.15 0.20 0.25 0.30

D

0 24 48 72 96 120 144 168 192 Culture time (h)

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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.

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Biomass (g/L)

PUFAs (mg/g) PUFAs (g/L)

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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):

PUFAs (g/L):

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,theaverageglucoseconsump- tionratewas0.17g/Lhandtheglucosetobiomassconversion factor (YBiomass/Glucose) was1.28. Theaverage nitrogencon- 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,theaveragetotalnitro- genconsumptionratewas0.0016g/LhandtheYBiomass/Nitrogen

conversionfactorwas49.15,meaningthat49.15gofbiomass wasproducedpergramofnitrogenconsumed.

Fattyacidproﬁle

Fig.2showstheaveragePUFAsconcentrationsinthebiomass thatwerereachedthroughoutAurantiochytriumsp.ATCCPRA- 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 highest PUFAs concentration (2.85g/L) was obtained at 168h of cultivation in the experiments using an initial nitrogen 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 concentration 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)ofthebiomasswascomposedofPUFAsat168hofcul- 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),account- ingfor3.34%ofthetotalbiomass(0.80g/L).Wealsoobserved

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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

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Total fatty acids, %

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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 glutamate,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,whichalsoresultedalsoinahighercellconcentra- tion(23.9g/L).GanuzaandIzquierdo¹⁸usedSchizochytriumsp.

G13/2Stostudytheeffectofsubstratelevelsonlipidaccumu- 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 culturemediumdecreaseduntil144hofcultivationandsub- 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%)andthepolyun- saturated, including DPA ␻6 (6.5–14%) and DHA (20–43%).

Therefore,thefattyacidsproducedbymembersoftheThraus- tochytriidaefamilyarelikelytobemainlyC14:0,C15:0,C16:0, DPA␻6andDHA.

Lietal.¹⁷studiedthecompositionoftheAurantiochytrium sp.BR-MP4-A1biomassandconcludedthatDPA␻6andDHA comprised6.6%and28.9%,respectively,ofthetotalfattyacids.

TheseauthorsalsoobservedthattheDPA␻6(18%)andDHA (78%)levelswerehigherthanthoseoftheotherPUFAsthat werequantitated,similartotheresultsobtainedinthisstudy (Fig.3).

WealsoobservedareductioninPUFAsproductionrela- 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, C16:2␻4,C20:5␻3andC22:5␻3comprised5.19%,4.84%and

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Table1–TotalfattyacidcontentoftheAurantiochytriumsp.ATCCPRA-276biomasswiththehighestPUFAs concentrationundereachexperimentalcondition.

Totalnitrogen 3.0g/L 0.44g/L 0.22g/L Fed-batch^b

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 obtainedinculturesgrownusinganinitialnitrogenconcentra- tionof3g/LTNandC16:2␻comprised1.95%ofthefattyacids observedinculturesgrownusinganinitialnitrogenconcen- 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%)producedbythisoleaginousmicroorganismwas2- to4-fold higherthan that foundinthe sardineoil(11%).²¹ CultivatedAurantiochytriumsp.ATCCPRA-276isapromising alternativesourceofoilrichinPUFAs.Usingfishforthelarge- scaleproductionofsuchoilislimitedbythechangesinthe lipidcompositionsandcontentsandthefattyacidprofilesof fish,whichareaffectedbytheseasonsandtheirspecies,sex, size,reproductivestatus,catchlocation,dietandnutritional status.²²Moreover,fishoilexhibitsagreatdiversityoffatty acidswithdifferentchainlengthsanddegreesofunsatura- 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 generallyaccumulateinoleaginousmicroor- 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,therewasahighersubstratecon- 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).

UsingasimilarculturemediumwithaninitialTNconcen- trationof0.44g/LtogrowSchizochytriumsp.ATCC20889,Jiang etal.⁸observedthatDHAaccountedfor26%ofthetotalfatty acids after120hofcultivation,whichisverysimilartothe 25.5%DHAlevelinthetotalfattyacidsobservedinourstudy.

Inourstudy,theaccumulatedbiomassconsistedofapproxi- mately12.5%(w/w)DHA,whichishigherthanthevalue(8.8%) reportedbyJiangetal.⁸

Burjaetal.²⁴ evaluatedtheeffectofdifferentconcentra- 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,andreportedalowbiomasscon- centration (7.5g/L) and alow levelofDHA inthe biomass.

Burjaetal.²⁴alsoobservedthatusingahigherinitialTNcon- centration(1.23g/L),1.56g/LofDHAwasobtained,whichis approximately3timestheDHAconcentration(0.51g/LDHA) observedinourexperimentsusinganinitialTNconcentration of3.0g/L.

Conclusions

Herein,wepresentedthe resultsofastudy oftheeffectof differentconcentrationsofcarbonandnitrogenonPUFAspro- ductionbyAurantiochytriumsp.ThePUFAsproductionofthis microorganism dependson the accumulationoftotal fatty acidsandontheconcentrationofthebiomass.Therefore,the culturemediumshouldfacilitatethegrowthofthismicroor- ganismandprovideanadequatenitrogensupplywithrespect totheC/Nratiobecauseitaccumulates oilswhenthetotal nitrogensupplyislimited.

ThemainpolyunsaturatedfattyacidsfoundintheAuran- tiochytriumsp.ATCCPRA-276biomasswereDPA␻6(20–23.1%)

329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375

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and DHA(61.3–70.5%). Themaximum cellconcentration of 23.9g/L (with45.5% of its weight consisting offatty acids) wasobservedat96hofcultivation usinginitialconcentra- tionsof30g/Lofglucoseand0.22g/Loftotalnitrogen.Under these conditions, the highest PUFAs concentration (3.6g/L) wasreached,withtheDHAandDPA␻6concentrationsbeing 2.54and0.80g/L,respectively.

TheresultsofthisstudyshowedthatthegrowthofAuran- tiochytriumsp.ATCCPRA-276anditsaccumulationofPUFAs, particularly DHA, are dependent on the concentrations of thecarbonandnitrogensubstrates.Theresultsalsodemon- stratedthatthecultivationperiodisanimportantvariablefor PUFAsproductionbyAurantiochytriumsp.ATCCPRA-276.

Aurantiochytriumsp.ATCCPRA-276iscapableofproducing highlevelsofPUFAs.Therefore,developingnewtechniques forcultivatingthismicroorganismcouldreducethecostand increasetheproductionofoilsforuseinfoodandmedicines.

Conﬂicts of interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

This study was supported by the Coordenação de Aperfeiçoamento 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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