Botryococcus braunii strains compared for biomass productivity, hydrocarbon and carbohydrate content

ContentslistsavailableatScienceDirect

Journal of Biotechnology

jo u r n al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / j b i o t e c

Botryococcus braunii strains compared for biomass productivity, hydrocarbon and carbohydrate content

Joao Diogo Gouveia

, Jesus Ruiz

, Lambertus A.M. van den Broek

, Thamara Hesselink

, Sander Peters

, Dorinde M.M. Kleinegris

, Alison G. Smith

, Douwe van der Veen

, Maria J. Barbosa

, Rene H. Wijffels

aWageningenUniversityandResearch,BioprocessEngineeringGroup,AlgaePARC,Droevendaalsesteeg1,6708PB,TheNetherlands

bWageningenURFood&BiobasedResearch,P.O.Box17,6700AAWageningen,TheNetherlands

cUniversityofNordland,FacultyofBiosciencesandAquaculture,N-8049Bodø,Norway

dPlantResearchInternational,BusinessUnitofBioscience,ClusterAppliedBioinformatics,PlantResearchInternational,Droevendaalsesteeg1,6708PB Wageningen,TheNetherlands

eDepartmentofPlantSciences,UniversityofCambridge,DowningStreet,CambridgeCB23EA,UK

a r t i c l e i n f o

Articlehistory:

Received21November2016 Receivedinrevisedform1March2017 Accepted11March2017

Availableonline20March2017

Keywords:

Botryococcusbraunii Hydrocarbons Carbohydrate Galactose Fucose Microalgae

a b s t r a c t

Botryococcusbrauniicanproducebothlong-chainhydrocarbonsaswellascarbohydratesinlargequan- tities,andisthereforeapromisingindustrialorganismfortheproductionofbiopolymerbuildingblocks.

ManystudiesdescribetheuseofdifferentstrainsofBotryococcusbrauniibutdifferencesinhandlingand cultivationconditionsmakethecomparisonbetweenstrainsdifficult.Inthisstudy,16B.brauniistrains obtainedfromsixculturecollectionswerecomparedfortheirbiomassproductivityandhydrocarbon andcarbohydratecontent.BiomassproductivitywashighestforAC768strainwith1.8gL⁻¹day⁻¹,while hydrocarbonproductionrangedfromnonetoupto42%pergrambiomassdryweight,withShowashow- ingthehighesthydrocarboncontentfollowedbyAC761.Thetotalcarbohydratecontentvariedfrom20%

to76%pergramofthebiomassdryweight,withCCALA777asthehighestproducer.Glucoseandgalactose arethemainmonosaccharidesinmoststrainsandfucosecontentreached463mgL⁻¹inCCALA778.

©2017ElsevierB.V.Allrightsreserved.

1. Introduction

Humanactivitiesgreatlydependonpetroleumasbothanenergy sourceandindustrialrawmaterial(Dale,2007).Petroleumusage inthelongtermisbothunsustainableduetodepletingeconomi- callyrelevantsourcesandbyrapidreleaseofcarbondioxideinthe environment.Onepotentialsourceofbiofuelsandotherbiobased rawmaterialcompoundsaremicroalgae.Thesephotoautotrophic organismsareabletotransforminorganiccarbonintolipidssuchas triacylglycerols(TAGs)atafasterratethanagriculturaloleaginous crops,anddonotcompeteforarableland(WijffelsandBarbosa, 2010).Besideslipids, otherproductsofinterest thatmicroalgae mayproduceinlargequantitiesarehydrocarbonsandcarbohy- drate.Hydrocarbonsarenaturaloccurringcompoundsconsisting entirelyofhydrogenandcarbon,andareoneofthemostimportant energyresources(TimmisandQin,2010).Hydrocarbonsderived

∗Correspondingauthor.

E-mailaddress:[email protected](J.D.Gouveia).

frommicroalgaecanbehydrocrackedandtransformedintoavia- tionturbinefuel(Hillenetal.,1982).Carbohydratehavearange ofindustrialuses,includingasthickeners,stabilisersandgelling agentsinfoodproducts(Donotetal.,2012),aswellasinthephar- maceuticalandcosmeceuticalindustries(Borowitzka,2013;Buono etal.,2012).

Onepromisingproductionhostofbiofuelsandbiobasedmateri- alsisBotryococcusbraunii.Thiseukaryoticmicroalgaspeciecanbe foundacrosstheworldasavarietyofstrainswithdifferentphysio- logicalcharacteristics.SomestrainsofB.brauniicanproducetoup to86%hydrocarbonsoncelldryweightbasis(Brownetal.,1969), whereasotherstrainscanproducetoupto4.5gL⁻¹carbohydrates intothemedium(Fernandesetal.,1989).OneadvantageofB.brau- niiisthatitsecretsextracellularhydrocarbonsandcarbohydrates (Kalachevaetal.,2002;Lupietal.,1994;Volovaetal.,1998;Weiss etal.,2012;Wolf,1983)whichallowsthedevelopmentofstrategies forinsituextractionsuchas“milking”(Moheimanietal.,2013).

B.brauniicanproducehydrocarbonswithdifferentchemical structures.Thesehydrocarbonsplayaroleinthenaturalgrowth cycleofB.braunii(Khatrietal.,2014).Dependingonwhattype

http://dx.doi.org/10.1016/j.jbiotec.2017.03.008 0168-1656/©2017ElsevierB.V.Allrightsreserved.

Table1

OrigenofBotryococcusbrauniistrains.Informationregardingtheindividualstrainsandtheculturecollectionsfromwheretheywerepurchased.*strainsusedforgrowthin bubblecolumnsphotobioreactors.

Culturecollection Botryococcus brauniiStrain

Race Location Isolation,date

ofisolation

Reference

Berkeley Showa * RaceB culturingtanks,

Berkley

Byunknown, 1980

Wolfetal.(1985)

ScandinavianCulture CollectionofAlgaeand Protozoa(SCCAP)

SCCAPK-1489 Notknow Belgium,

Nieuwoort

ByG.Hansen, 2008

Noreference

CultureCollectionof AlgaeatGoettingen University(SAG)

SAG30.81 RaceA Peru,Dpto.

Cuzco,Laguna Huaypo

ByE.

Hegewald, 1977

Dayanandaetal.(2007)

CultureCollectionof AutotrophicOrganisms (CCALA)

CCALA-777 * Notknow Portoda

Castanheira (Poc¸odos Basilios) Portugal

BySantos,1975 Fernandesetal.(1989)

CCALA-778 * Notknow SerradaEstrela

(Barragemda ErvadaFome) Portugal

BySantos,1997 Noreference

CCALA-835 RaceA Peru,Dpto.

Cuzco,Laguna Huaypo

ByE.

Hegewald, 1977

Dayanandaetal.(2007)

CultureCollectionof AlgaeandProtozoa (CCAP)

CCAP-807-2 * RaceA Grasmere,

Cumbria, England

ByJaworski, 1984

Hiltonetal.(1989)

UTEXCulture CollectionofAlgae

UTEXBb572 RaceA Madingley,

Cambridge, England

ByM.R.Droop, 1950

ErogluandMelis (2010)

UTEXBbLB572 RaceA Cambridge,

England

ByM.R.Droop, 1950

Erogluetal.(2011)

ALGOBANK-CANE AC755 * RaceA Lingoult-

Morvan, France

ByPierre Metzger,1981

Metzgeretal.(1985a,b)

AC759 * RaceB Ayame,Ivory

Coast

ByPierre Metzger,1984

Metzgeretal.(1988)

AC760 RaceB Kossou,Ivory

Coast

ByPierre Metzger,1984

Metzgeretal.(1988)

AC761 * RaceB Paquemar,

Martinique, France

ByPierre Metzger,1983

Metzgeretal.(1985a,b)

AC765 RaceL Kossou,Ivory

Coast

ByPierre Metzger,1984

Metzgeretal.(1988)

AC767 RaceL Songkla

Nakarin, Thailand

ByPierre Metzger,1985

Metzgeretal.(1987)

AC768 RaceL Yamoussoukro,

IvoryCoast

ByPierre Metzger,1984

Metzgeretal.(1987)

ofhydrocarbonsareproduced,B.brauniiissubclassifiedintofour chemical races, designated A, B and L (Metzger and Largeau, 2005), and S, a recent assignment(Kawachiet al., 2012).Race Astrainssynthesizeodd-numbered alkadienesand trienes(C₂₅ toC₃₁)(Dayanandaetal.,2007; Erogluand Melis,2010;Hilton etal., 1988; Metzger etal., 1986; Metzger etal., 1989), raceB strainssynthesizeaclassofisoprenoidderivedcompoundstermed botryococcenes(C₃₀toC₃₇)andmethylatedsqualenes(C₃₁toC₃₄) (MetzgerandCasadevall,1983;Metzgeretal.,1985b;Metzgeretal., 1987;Nanamura,1988),raceLstrainssynthesizelycopadiene(C40) andraceSstrainssynthesizeC₁₈epoxy-n-alkanesandC₂₀saturated n-alkanes(MetzgerandCasadevall,1987).

Inadditiontohydrocarbons,B.brauniistrainscanproducelarge amountsofcarbohydrateswiththehighestamountssofarreported as4.0–4.5gL⁻¹ (Fernandesetal.,1989).Extensivecarbohydrate productionwasfirst observedby anincrease ofbrothviscosity duringgrowth(Casadevallet al.,1985).Sincethis initialreport, otherstrainswere foundtoproduce carbohydrateswithyields of250mgL⁻¹ for race Aand Bstrains, and 1gL⁻¹ for a race L strain(AllardandCasadevall,1990).Later,1.6gL⁻¹forB.braunii LB572and0.7gL⁻¹ forSAG30.81 (Dayanandaetal.,2007)were

reported.Galactosewasidentifiedasthemainmonomericsugar constituentofallcarbohydratesexamined,withfucoseandrham- noseasaccompanyingmonomers.GlucosewasdetectedintheL strainonly(AllardandCasadevall,1990).

OnedrawbackofusingB.brauniiasanindustrialhostisitsslow growthcompared tootherphotoautotrophicmicroorganisms.B.

brauniibiomassproductivitiesrangebetween0.1and0.2gL⁻¹d⁻¹ (Cabanelas et al.,2015; Eroglu et al., 2011)where other green microalgaesuchasChlorellasp.canachieve0.5gL⁻¹d⁻¹(Hempel et al., 2012). One commonly reported hypothesis for the slow growthisduetothesynthesisofenergeticallyexpensivehydro- carbons(Banerjeeetal.,2002).

Thereisanextensivebodyofworkinlastfewdecadesdescribing differentstrainsofB.brauniianditisclearthatthereisahighdegree ofmorphologicalplasticityandphysiologicaldiversityamountthe genus.ItisprobablyduetothishighdiversityinthegenusthatB.

brauniiisnotaneasyorganismtomaintainandgrowunderlabo- ratoryconditions.Manymethodsofcultivation,differenttypesof growthmediumorcultureconditionshavebeenreportedinthe literaturetostudymoreindepthB.brauniiindividualstrainsas wellasforcomparingstrainsdiversity(AllardandCasadevall,1990;

Table2

HydrocarbonprofileofsevenstrainsofBotryococcusbrauniigrowninshakeflasks.Strainsaregroupedintosubclades3and5asaresultofthephylogeneticplacementresults.

Superscriptletteraandbnexttochemicalformulasstandfordifferentmolecularstructures.C?referstounidentifiedcompound.

Chemicalformula Subclade3 Subclade5

Showa AC759 AC760 AC761 K-1489 AC755 CCAP807/2

C23H44320 – – – - – – 73

C25H48348 – – - – – 48 93

C27H52376 – – – – – 58 171

C29H56404 – – – – 97 283 187

C31H60432 – – – – 65 94 –

C33H56452^a – 55 58 – – – –

C33H56452^a 91 – – 109 – – –

C34H58466^b 108 – – 57 – – –

C34H58466^b 599 – 57 509 – – –

C34H58466^b 138 195 334 186 – – –

C34H58466^b 84 270 240 77 – – –

C? 133 – – – – – –

totalhydrocarbons(mgL⁻¹) 1153 520 689 938 162 483 524

Cabanelasetal.,2015;Dayanandaetal.,2007;Erogluetal.,2011;

Heged ˝usetal.,2016;Metzgeretal.,1989;Mouteletal.,inpress).

Whilethishasledtoseveralreportsonhydrocarbonandcarbo- hydrateproductionforvariousB.brauniistrains,itcanbeargued thatitisstilldifficulttocompareandassessphysiologicalchar- acteristics.Thisstudyattemptstoestablishreferenceconditions forfutureinvestigationsofthisinterestingphotosyntheticorgan- ismalongsideitsproduction oflongchainhydrocarbonsand/or carbohydrates.

TheaimwastocomparevariousreadilyavailablestrainsofB.

brauniiunderthesamecultureconditionsforbiomassproductivity andtotalhydrocarbonandcarbohydratecontent.Sixteenstrains weretestedinErlenmeyerflasks,ofwhichsevenpromisingstrains wereadditionallyinvestigatedinbubblecolumns.Comparisonof thesixteenstrainsregardingbiomassproductivity,carbohydrate andhydrocarboncontentisreportedandmajordifferenceshigh- lighted.Twostrainsareputforwardascandidatesforindustrial applications.

2. Materialsandmethods 2.1. Strainsandmedia

Sixteen non-axenic B. braunii strains were obtained from culture collections (Table 1). Upon arrival, each strain was washed with sterile distilled water and revegetated in modi- fied Chu 13 medium (Largeau et al., 1980)without citric acid or vitamins,withthefollowing composition:400mgL⁻¹ KNO₃, 200mgL⁻¹ MgSO4·2H2O, 108mgL⁻¹ CaCl2·2H2O, 104.8mgL⁻¹ K₂HPO₄,20mgL⁻¹ Fe–Na₂EDTA,9.4␮gL⁻¹Na₂O₄Se,2.86mgL⁻¹ H₃BO₃,1.8mgL⁻¹MnSO₄·4H₂O,220␮gL⁻¹ZnSO₄·7H₂O,90␮gL⁻¹ CoSO4·7H2O, 80␮gL⁻¹ CuSO4·5H2O, 60␮gL⁻¹ Na2MoO4·2H2O, 10␮LL⁻¹H2SO4.FinalpHwasadjustedtopH7.2withNaOH.The 16strainswerekeptinanincubationroomwiththeinitialenvi- ronmentparameters:light intensityof60␮mol photonm⁻²s⁻¹, light:darkphotoperiod 18:6h, ambientair CO2,temperature of 23^◦Candmechanicalshakingat60rpm.Growthconditions–prior totheexperiment,the16strainsinoculumwerecultivatedfortwo batchcyclesunderexperimentalconditionstominimizeeffectsof changingenvironmentalparameters.

Shakeflaskcultivation:Forcomparisonofbiomassproductiv- ity,hydrocarbonand carbohydratecontent,the16 strainswere growninInforsHTMultritonincubatorsin250mLconicalflasks andavolumeof150mLmedium.Temperaturewassetat23^◦C, with 2.5% CO2 enriched air and shaking at 90rpm. Illumina- tion was provided by Phillips lamps FL-Tube L 36W/77, with 150␮molphotonm⁻²s⁻¹,andalight:darkphotoperiodof18:6h (photoperiod of 18:6h chosen based on earlier work done as

described in Gouveia,2010).Flasks wereinoculated with algae froma10-dayold,activelygrowingculture,suchthattheinitial absorbanceat680nmwas0.2.TheErlenmeyerflaskswerecapped withaerasealsterilefilm(Alphalabs).Theexperiment wascon- ductedintriplicateandsamplesweretakenatdays0,6,12and18 afterinoculation.Thecultivationperiodof18daysforthisexperi- ment,waschosenbasedonsimilarcultivationtimesfoundinother studies(ErogluandMelis,2010;KojimaandZhang,1999).

Bubblecolumnscultivation:Fordeterminingcultivationviabil- ityatlargerscaleofselectedstrainsfromshakeflaskexperiment, seven strains (marked with a star in Table 1) were cultivated in batch mode in a bubble column with a working volume of 400mL.Thesecustom-madeglasstubes(GlassInstrumentmakerij, WageningenUniversity)are400mminheight,withaninternal andexternaldiameterof40and60mm,respectively.Awaterbath wasusedtopumpwaterthroughanexternalwaterjacket,keep- ingthetemperatureoftheculturesat23^◦C.Thebubblecolumns werekeptverticalbetweentwolargelightpanels(benchpanels:

Mazda5LKNRBECO118I,fluorescenttubes:PhilipsMasterTL- D18W/865)whichprovided150␮molphotonm⁻²s⁻¹ fromtwo sideswithalight:darkcycleof18:6h.Airenrichedwith2%CO2

wascontinuouslyinjectedatthebottomofthereactoratarateof 0.5vvm(volumeairvolumereactor⁻¹ min⁻¹,200mLmin⁻¹).Air flowwascontrolledbyamassflowcontroller(Brooks0254,Brooks instruments),andfiltersterilizedthrougha0.2␮mairfilter(Acro 50,PallCorporation)beforeitpassedthroughaholedplateinthe tubetocreatesmallbubbles.Theenrichedairprovidedinorganic carbonforgrowth,keptthereactormixedandstabilizedpHaround 7.2.Tocompensateforevaporation,MilliQwaterwasaddedtothe aerationtubesthrougha0.2␮mfilter(Minisart,Sartoriusstedim).

Anoverflowbottleequippedwith0.2␮mairfilter(Acro50,Pall Corporation)wasconnectedtothetopofthecolumn,toenableair toescapeandpossibleoverflowoftheculturetobekeptfreeof contamination.Thereactorswereinoculatedatbiomassconcen- trationof0.1–0.3gL⁻¹,andsterilemediumwasaddedto400mL.

Twiceaweeksamplesweretakenthroughaportatthesideofthe reactortodeterminepHanddryweight.

2.2. Biomassdryweight

FivemLaliquotsofculturebrothwerefilteredontopre-weighed GF/D glass-fibre membranes(Whatman). The GF/D filters were driedat100^◦Cfor24handweighted,andbiomassamountwas determinedbysubtraction.Fromthedryweight,biomassproduc- tivitywascalculatedusingthefollowingequation:

Productivity=Cx2−Cx1

t2−t1 ,

Fig.1. Phylogenytreeplacementof16Botryococcusbrauniistrains.Strictconsensustreebasedwithoverlaidbootstrapvaluesandconcensusthresholdof50%obtainedby maximum-likelihoodanalysison18-rRNAgenesequencesfrom19Botryococcusculturecollectionstrains,4referencestrainsAJ581910Ayame(B),AJ581911Songkla(L), AJ581912(A)andAY197640Tow(A),31BotstrainsfromKawachietal.,2012andspeciesfromotherchlorophytesthatareusedasanoutgroup.

where Cx1 and Cx2 represent thebiomass concentration at the beginning(t₁)andattheendoftheexperiment(t₂),respectively.A univariateanalysisofvariance(one-wayANOVA)wascarriedout tocomparethebiomassproductivityamongdifferentstrains.As post-hoctesttheTukey’stestwasused.Allstatisticalanalyseswere doneatasignificancelevelof0.05.ThesoftwareusedwasGenstat 64-bitRelease18.1.

2.3. Hydrocarbonextraction

Ourmethodis adaptedfromthemethodologydeveloped by Folchand Dyer (Blighand Dyer, 1959; Folch etal., 1957).One milliliter of culture was transferred to a glass vial and 2.5mL methanoland1.25mLdichloromethanewereaddedandmixedfor 6h.Afterthemixingstep,1.25mLdichloromethanewasaddedand

mixedfor1minfollowedbyadditionof1.25mL0.9%(w/v)NaCland mixedforanotherminute.Hereafter,sampleswerecentrifugedfor 5minat1500×gandthebottomphasewasremovedtoanewglass vialusingaglassPasteurpipetteanddriedundernitrogengas.The residue was resuspended in 300␮L dichloromethane:methanol (v:v)andstoredat−20^◦C.Forthehydrocarbonextractionofthe strainscultivatedinthebubblecolumns,theresiduewasresus- pendedin1000␮Lhexane.Hydrocarbonextractionwasperformed usingthelastdatapointsamplesforboththeErlenmeyerflasksand bubblecolumncultures.

2.4. Hydrocarbonanalysis

Hydrocarbons extracted from the strains cultivated in the Erlenmeyerflasks weremeasuredbygaschromatography com-

Fig.2.Comparisonofphysiologicaltraitsfor16Botryococcusbrauniistrains.(a)Biomassproductivity;(b)totalhydrocarboncontent;(c)totalcarbohydratecontent.Thebars inplotArepresentthestandarderrormean,with3replicatesforallexceptAC760andAC768which1replicatewasused,andAC755,AC761,AC767,CCAP807/2,SAG30.81, CCALA778,UTEXLB572where2wereused.Eachlettersabovebarsingrapha,representstatisticaldifference(P<0.05)byANOVA.

Table3

Monosaccharidecomposition.Monomericsugarcontent(mgL⁻¹)ofthe16strainsofBotryococcusbrauniigroupedbysubcladesfromthephylogeneticplacementresults.

Theentryn.d.standsfornotdetected.

Fucose Rhamnose Arabinose Galactose Glucose Total

Subclade1

AC765 37 3 27 99 118 284

AC768 70 3 12 131 113 328

Subclade2

AC767 24 2 17 144 21 208

Subclade3

Showa 6 3 102 199 163 473

AC759 n.d 5 18 210 82 316

AC760 n.d 4 18 165 99 286

AC761 n.d 3 17 230 93 343

Subclade5

UTEX572 4 3 10 107 158 282

UTEXLB572 7 3 9 86 213 319

AC755 172 3 27 307 151 660

K-1489 80 3 9 321 163 576

CCALA777 279 4 9 1115 374 1781

CCALA778 463 9 21 908 288 1688

CCALA835 29 3 14 88 143 277

SAG30.81 18 9 15 72 123 237

CCAP807/2 136 5 28 134 178 480

bined with mass spectrometry (GC–MS). The instrument used wasa7890A/5975CfromAgilentTechnologies,usingaDB-Petro (100m×0.25mm×0.50␮m)J&WColumnusingheliumasthecar- riergas,splitlessinjector,anoperationtemperatureof280^◦Cand aninjectionvolumeof1␮L.Theovenprogramwassetat40^◦Cfor 0.5min,thenrampedupby30^◦Cperminuteto250^◦Cfollowedby a5^◦Cperminuterampto300^◦Cin37.5min,withatotalruntime of55min.Samplesweredilutedindichloromethane:methanol1:1 (v:v)Squalene(C30H50,M=410gmol⁻¹)wasthereferencestan-

dardused.Theconcentrationofthecalibrationstandardswere50, 100,250and500mgL⁻¹.

Hydrocarbonanalysisforthestrainscultivatedin thebubble columnswascarriedoutusingGC-FID.Theinstrumentusedwas anAgilentTechnologiesHP6890seriesequippedwithautosam- pler,ausingRestekRxi-5ms(30m×0.25mm×0.25␮m)column.

Heliumwasusedasthecarriergas,andahydrogen/air mixture detection,gassplitlessinjectorsat350^◦Coventemperatureand injectionvolumeof1␮L.Theovenprogramwas50^◦Cfor1min, then15^◦Cperminute to180^◦C,then7^◦Cperminuteto230^◦C,

Fig.3. GrowthcurvesofBotryococcusbrauniistrainsinbubblecolumnsphotobioreactors.Cultivationinbatchmodeintherangeof25–30days.n=1.

then30^◦Cperminuteto350^◦Candholdfor15minwithatotal runningtimeof35min.Samplesweredilutedinhexane,andsev- eraldilutionsofstandardsusingsqualenewereused.Thedilutions were1,0.1,0.01,0.005and0.001%(v/v).

2.5. Carbohydrateextraction

10mLofculturefromthelasttimepointtaken,wasfreezedried usingZirbustechnologySublimator2×3×3andthepelletweight wasdetermined.Apre-hydrolysisstepwasperformedasfollows:

72%(w/w)H2SO4wasaddedtothefreezedriedsample(w/v)and hydrolyzedfor1hourat30^◦C,andstirredwithglasspipetteevery 15min.Afterthepre-hydrolysiswaterwasaddedin11.6v/v(final concentration1MH2SO4)tothemixtureandplacedat100^◦Cfor 3handshakenevery30min.Hereafterthesolutionwasallowed tocoolonicefor15minandsubsequentlycentrifugedfor15min at3000×g.Before analysissampleswerediluted in water and 2.5␮LmL⁻¹ 0.1%(w/v)bromophenolblueinethanolwasadded tothesamples.ForneutralizationthepH,solidbariumcarbonate powderwasaddeduntilamagentacolourwasseen.Subsequently thesolutionwasfilteredthrougha0.45␮mporesizePTFEfilter.

2.6. Carbohydrateanalysis

Monomericsugarcomposition wasdetermined byHighPer- formance Anion Exchange Chromatography (HPAEC) using an ICS-3000 Ion Chromatography HPLC system equipped with a CarboPacPA-1column(2mm×250mm)incombination witha CarboPacPA guard column(2mm×25mm)and a pulsed elec- trochemical detector in pulsed amperometric detection mode (Dionex).Aflowrateof0.3mLmin⁻¹wasusedandthecolumnwas equilibratedwith16mMNaOH.Thefollowinggradientwasused:

0–26min,16mMNaOH;26–33min,16–100mMNaOH;33–78min 0–1Msodiumacetatein100mMNaOH;78–83min1Msodium acetate in100mM NaOH.l-Rhamnose,l-fucose,d-mannose,l- arabinose,d-glucose,d-xylose,d-galactose,sucrose,d-glucuronic acidandd-galacturonicacid(Sigma–Aldrich)wereusedasstan- dardsforidentification.Forthebubblecolumngrowth,thetotal sugarcontent wasmeasuredusingthe DuboisMethod(DuBois etal.,1956).

2.7. 18SrRNAanalysis

The genomicDNA was extracted aftera grinding procedure usinglysingmatrixEtubes(6914–500,MPBiomedicalsEurope) usingaDNeasyPlantMinikit(QiagenGmbH,Germany)accord- ingtothespecificationsofthemanufacturer. PCRamplification offragmentswasdonefollowingKawachietal.(2012).Forward and reversed primer combination consisted of CV1×CV2 and CV3×CV4primersaccordingtoSenousyetal.(2004).

Aftermultiplication,thePCRtemplatesweresequencedwith CV1,CV2,CV3andCV4sequenceprimers.UsingthePREGAP4inter- faceoftheStadenpackage2004(Stadenetal.,2003), rawtrace datawasprocessedintoassemblyreadysequencesandsequences were basecalled by the PHRED base caller (Ewingand Green, 1998).DNAsequence analysisandmaximumdivergenceof18S rRNAsequenceswereperformedaccordingtothemethodusedby Kawachietal.(2012).

3. Results

3.1. 18SrRNAanalysis

Toassesstherelationshipsbetweenour16culturecollection strainsweused18SrRNAsequenceanalysisfollowingthemethod that wasusedtocharacterize 31BOT strainsbyKawachi etal.

(2012), who strongly linkedthe B.braunii chemical races with theirphylogeneticplacement.OurstrainsfitwellwithKawachi’s results(Fig.1).StrainsUTEX572,UTEXLB572,AC755,CCALA835, CCALA777, CCALA778,K-1489, CCAP807/2 andSAG30.81 are in subclade5whichreferstoraceA.StrainsAC759,AC760,AC761 andShowaareinsubclade3whichreferstoraceB, andstrains AC765,AC767andAC768insubclade1and2whichreferstorace L.InFig.2,theassignedsubclades1,2,3and5areusedtogroup thestrainsandthereforeitisalsoincludedinTables2and3.

3.2. Shakeflaskcultivation

Thebiomassproductivityofthe16strainswasdeterminedby assessingthetotal biomassincrease overthecultivationperiod between time of inoculation and end of experiment (day 18).

Thebiomassproductivitiesintheshakeflasksvariedupto2-fold betweenstrains and showedstatistically significantdifferences (P<0.05)in biomassproductivitybetweenstrains(Fig.2a).The

Table4

Volumetricproductivity(PV)andmaximumspecificgrowthrate(max)ofBotry- ococcusbrauniigrowninbubblecolumns.PVexpressedingramsperlitreperday andmaxistheamountbiomassgrowthperday.

Strain PV(gL⁻¹d⁻¹) max(d⁻¹)

AC755 0.06 0.05

AC759 0.09 0.07

AC761 0.15 0.11

CCALA777 0.08 0.06

CCALA778 0.12 0.17

CCAP807/2 0.14 0.11

Showa 0.14 0.17

three highest biomass producers were AC768, CCALA778 and AC765with0.18,0.15and0.14gL⁻¹day⁻¹,respectively.With0.08 and 0.07gL⁻¹day⁻¹,strainsAC759 and CCAP807/2 respectively showedthelowestbiomassproductivity.

Thetotalhydrocarboncontentinboththeculturemediumand biomass wasdeterminedat theend ofexperiment. Long-chain hydrocarbonsweredetectedinsevenstrains(Fig.2b).Ofthese, thehighesthydrocarbonproducingstrainswereShowaandAC761, whichyieldedahydrocarbonpercentageofaround40%pergram biomassdry weight.Strain K-1489was thelowestproducer of thehydrocarbonproducingstrains,followedbyAC755.Thestrains Showa,AC759,AC760andAC761producedC33toC37molecules, whereasK-1984,AC755andCCAP807/2producedC₂₅toC₃₁chain hydrocarbons(Table2).Nocorrelationwasfoundbetweenbiomass productivityandhydrocarboncontent.

Totalcarbohydratecontentintheculturemediumandbiomass, basedontheirmonomericsugarcomposition,weredeterminedat theendofthegrowthexperiment.Totalcarbohydratecontentvar- iedbetween9%and74%pergrambiomassdryweight(Fig.2c).Two Aracestrainsshowedhighestamountoftotalcarbohydrateswith 74%perbiomassdryweightforCCALA777whichisthehighest measured,followedbyCCALA778with52%.Themonomericcar- bohydratecompositionwasexaminedandthemainconstituent monosugarsforthe16strainsaregalactose,glucose,fucose,and arabinose(Table3).Rhamnosewasalsodetectedinlowbutconsis- tentamountsinallstrainswithvaluesrangingfrom2to9mgL⁻¹. Themainsugarmonomerwasgalactosefor10 strains,whereas glucosewasthemainmonomerfortheremainingsixstrainsstud- ied.Fucoseisfoundtovarysignificantlybetweenstrains,ranging fromnodetectioninAC759,AC760and AC761to463mgL⁻¹ in CCALA778.OtherB.brauniistrainsshowinghighamountsoffucose areAC755,CCALA777andCCAP807/2with172mgL⁻¹,279mgL⁻¹ and136mgL⁻¹respectively.OfthefourraceBstrains,onlyShowa showedasmallamountoffucosedetectedwith6mgL⁻¹.Arabi- nosevariesformoststrainsbetween9mgL⁻¹and28mgL⁻¹,but forShowaitispresentinhighamountswith102mgL⁻¹.Galac- toseisthelargestportionofthetotalcarbohydrateproduceinthe strainsCCALA777andCCALA778with1115mgL⁻¹and908mgL⁻¹ respectively.Similarlytohydrocarboncontent,nocorrelationwas foundbetweenbiomassproductivityandtotalcarbohydrate.

3.3. Bubblecolumnscultivation

Basedonhighestbiomassproductivity,hydrocarbonsandcar- bohydratecontentfromtheshakeflaskexperiment,sevenstrains werescaled-uptobubblecolumnstoassessthecultivationvia- bility.Fig.3showsthebiomassevolutionforthesestrainsgrown forupto30daysinthebubblecolumns.InTable4thevolumet- ricproductivities(PV)andmaximumspecificgrowthrates(max) aregiven.Growthcurves,volumetricproductivitiesandmaximum specificgrowthrateshighlightShowa,CCALA778,CCAP807/2and AC761asthebestperformingstrainsinthebubblecolumns,with productivitiesrangingbetween0.12and0.15gL⁻¹day⁻¹.Theother

Table5

Percentageofhydrocarbonandtotalcarbohydratecontentperbiomassdryweightof Botryococcusbrauniibiomassculturedinbubblecolumns.Themeasurementswere carriedoutonsamplesfromthelastdayofgrowthforeachstrain.

Strain %hydrocarbon %carbohydrates

AC755 16 25

AC759 21 10

AC761 45 10

CCALA777 10 55

CCALA778 0 9

CCAP807/2 7 5

Showa 25 4

strainsshowslowergrowthwithlowerbiomassproductivity.The hydrocarbonandcarbohydratecontentareshowninTable5.Long chainhydrocarbonsweredetectedinallsevenstrainswithexcep- tionofCCALA778.AC761showsthehighesthydrocarboncontent with45%pergramofbiomassdryweightfollowedbyShowawith 25%.Thelowestamountofhydrocarbonwasfoundinthestrain CCAP807/2with7%ofitsbiomassdryweight.Carbohydratecon- tentinthesevenstrainsvariedbetween4%and55%forShowaand CCALA777respectively.

4. Discussion

16strainsofB.brauniiwerecomparedunderidenticalgrowth conditions.Theresultsshowsimilarstrainvariationasobserved intheotherstudieswithdifferentgrowthconditions.Anditalso showsdifferencesbetweendifferentstudieswhencomparingthe samestrains.

Intheshakeflaskcultivation,biomassproductivityvariessta- tisticallysignificantly(P<0.05)amongstrainsintheshakeflask cultures,andalignwithreportsfromidenticalstrainsinsimilar experimentalstudies(Erogluetal.,2011).Thestrainwiththehigh- estproductivity(0.18gL⁻¹day⁻¹)AC768,producednodetectable hydrocarbonsandhadarelativelylowlevelofcarbohydrates(10%, w/w).However,thisinversecorrelationisnotobservedforallthe strains.ForexampleAC767andSAG30.81alsoshowlowcarbohy- dratesandnohydrocarbonproduction,whilebiomassproductivity remainedata lowrange with0.10and 0.07gL⁻¹day⁻¹ respec- tively.On theotherhandstrainsthatproducehighamountsof hydrocarbons such as Showa showed much higher productiv- ity(0.11gL⁻¹day⁻¹).Similarresultsinthebubblecolumnswere obtained, which suggeststhat thelow biomass productivityof B.brauniicomparedtootheralgalspeciesisspecificandisindepen- dentofB.brauniiproducinghydrocarbonsorcarbohydrates.Low biomassproductivityofB.brauniiatthisstagehastobeaccepted whenutilizedinlargescaleproductionfortheextractionofhydro- carbonsandcarbohydratesuntilastrainwithhighproductivitycan befoundthatalsocanproducehighamountsofproductofinterest.

Cleardifferencesarealsovisiblebetweenstrainswhencompar- inghydrocarboncontentunderthesameconditions.Sevenstrains outofsixteenproducedhydrocarbonsrangingfrom6%pergramof biomassdryweightinK-1489to42%intheShowastrain,andthese valuesarealsofoundin literature(MetzgerandLargeau,2005).

Fourstrains(Showa,AC759,AC760andAC761)produceC₃₃–C₃₇ hydrocarbonsandtheremainingthreestrainsforwhichhydrocar- bonsweredetected(K-1489,AC755,CCAP807/2)produceC₂₅–C₃₁ hydrocarbons,inaccordancewithpreviousreports(Metzgeretal., 1985a;Metzgeretal.,1988).ForK-1489strain,thisisthefirsttime thatthehydrocarboncontentisreported.ThestrainSAG30.81is reportedtocontainupto30%ofhydrocarbons(Dayanandaetal., 2007)yetinourstudynohydrocarbonsweredetected.Also,fora majorityofotherstrainstested,nohydrocarbonsweredetected.

Mediacompositionbetweenthevariouspreviousstudiesandthis workdo notvary significantly,therefore theabsenceofhydro-

carbonsinthesestrainsareprobablyduetootherfactors.Other cultivationconditions, suchas lengthof cultivation period and lightregimemightplayarole.Forexample,thegrowthconditions forAC765,AC767andAC768strainswerecultivatedincylindri- caltubes,aeratedwith1%CO2undercontinuousilluminationas described(Metzgeretal.,1988;Metzgeretal.,1985b).ForSAG30.81 andUTEXLB572,lightconditionswerevariedwithdifferentinten- sitiesincontinuousandphoto-periodilluminationparametersas describedbyDayanandaetal.(2007).ForCCALA777,thelightinten- sitywasof250␮molphotonm⁻²s⁻¹ incontinuousillumination, with1%CO2aerationincylindricaltubesasdescribedbyFernandes etal.(1991).Thesedifferencesinthegrowthconditionscouldhave changedthehydrocarbonsynthesisrateandexplainwhyitwas notdetectedinsomestrainsofB.brauniiinourstudy.Anotherpos- sibleexplanationforthesedifferentresultsmightbeduetothe differentmethodsofhydrocarbonextractionused.Inthisstudywe useddichloromethane:methanolassolventcomparedtohexane extractionappliedbytheotherstudiespreviouslymentioned.But thisreasondoesnotstandsowellaswehaveobtainedhydrocar- bonsintherangeof6%to42%forK-1489andShowarespectively aswellashydrocarbonchainsbelowandhigherthanC30.

Differencesintotalcarbohydratecontentarealsoevidentrang- ingfrom4%ofbiomassdryweightintheShowastrainto74%inthe CCALA777strain.ThehighviscosityonlyappearsintheCCALA777 andCCALA778.Thisisnotsurprisingasthereareonlyafewreports intheliteratureontheabilityofB.brauniitoproducelargeamounts ofcarbohydrates(AllardandCasadevall,1990;Dayanandaetal., 2007;Fernandeset al.,1991;Fernandesetal.,1989;Lupietal., 1994).Thehighestamountoftotalcarbohydratesextractedfrom theculturebrothinCCALA777andCCALA778was1.78gL⁻¹ and 1.68gL⁻¹ respectively.Similarresultsaredescribedinliterature for theCCALA777 strain,which is also knownby its identifier Acoi58(Fernandesetal.,1989).Itiswellknowntheapplication ofcarbohydratesinthepharmaceuticalindustry(Moscovici,2015) duetothediversebiologicalactiveagentspresent.Carbohydrates fromCCALA778contain27%offucosefromthetotalcarbohydrates, whichtranslatesto140goffucoseperKgofbiomassdryweight.

AllardandCasadevall(1990)alsoshowsimilarfucosecontentina raceAandLstrainwith32%oftotalcarbohydratecontent.Fucose isconsideredofhighindustrialvalue(WijesingheandJeon,2012) and it haspotentialmedicinal applications,namelyanti-cancer properties(Liaoetal.,2013).ThecarbohydratesofCCALA777and CCALA778strains, shouldbeanalyzed indepthforitspotential applicationsin industryand aswellasmorestudies toinvesti- gatethemechanismsbehindthehighcontentofcarbohydratesand fucose.Othermonomericcompositionofthetotalcarbohydrates shows galactose and glucose asthe main monomers acrossall strains.ForCCALA777andCCALA778galactoseisbyfarthelargest fractionofthecarbohydratesmoietiesandispresumablythereason whyviscosityincreases(AllardandCasadevall,1990;DíazBayona andGarcés,2014;Fernandesetal.,1989).Recentworkshowsthat galactosecanbeusedfortheproductionofbioethanolviafermen- tation(Leeetal.,2011;Parketal.,2014),thereforewiththehigh amountsofgalactoseproducedbyCCALA777andCCALA778,these strainscanbeconsideraspotentialcandidatesasrawmaterialfor theproductionofbioethanol.Futureresearchininsituextraction ofthecarbohydratesfromthesetwostrainsinacontinuousculti- vationsystemwouldfurtherincreasethefeasibilityforcommercial scaleapplication.

Onepointtobearinmindwhencomparingthestrainsisthe factthatwehavenon-axenic(bacteriapresent)strains.Microalgae andbacteriainteractionsareofmajorinterestasithasbeenshown thatbacteriacanhaveinfluenceonthecultivationofmicroalgae (Cole,1982).Therefore weconsider thatattempts in makingB.

brauniiaxenicisofadvantageasitcanimprovecultureproduc- tivitiesandinthiscaseincreasetheaccuracyofthecomparison

betweenstrains.Alsoisofinteresttostudythepossiblesymbiosis existingwithB.brauniiandbacteriaasarecentstudyusingB.brau- niiBa10strainshowsenhancementofbiomassproductivitieswith thepresenceofbacteria(Tanabeetal.,2015).Inthiscaseforexam- ple,CCALA778strainproduceshighamountsofcarbohydratesand iftheculturecontainshighamountsofbacteria,intheorycouldbe thatthesearedegradingthesugarsforcarbonsourceandultimately decreasingtheamountofproductaccumulatedandavailablefor harvest.

Fromthebubblecolumncultivation,wecanonlyspeculateon theresultsasthebatchmodecultivationwasrunonlyonceforthe selectedstrains.StrainsCCALA778,AC761,ShowaandCCAP807/2 seemviableforscaleupcultivationasvolumetricproductivities showvaluesinsimilarrangeorhigherthantheshakeflaskculti- vation.Therearedifferencesobservedinbiomassproductivities, suchasforAC755andShowa,andalsochangesinthecarbohydrate andhydrocarboncontent.Thesedifferencesmaybeexplainedby physicalpropertiesofthecultivationsystemsused.Biomasspro- ductivitiescanincreaseinsystemssuchasbubblecolumnreactors, forexamplebecauseofimprovedlightdistributionandavailabil- itypercell(KojimaandZhang,1999;Ugwuetal.,2005),orbetter gasmasstransferrates(Posten,2009).Ontheotherhand,biomass productivitiescandecreaseinthesesamesystemsbecauseofshear stressinducedbygaspurgecausingdamagetocells(Barbosaetal., 2003)andincaseofB.braunii,possiblydamagetothecolonies.

FurtherworkwithstrainssuchasCCALA778andAC761shouldbe doneatlargerscaletooptimizeandcharacterizetheirpotentialas biofuelandbioasedrawmaterials.

Twoaspectsforfutureresearchandcomparisonofstrainsare (1)thecharacterizationoftheextracellularandintracellularhydro- carbons.Becauseinsitu(“milking”)extractionisa viableoption asshownbyMoheimanietal.(2013),awidecomparisonwould informalsowhichstrainsarebestforhydrocarbonproductionfacil- ity.(2)thecharacterizationofcolonyformationandstructureasit couldaffecttheextractabilityofproducts,growthratesrelatedto lightabsorptionandpossiblydownstreamprocesses.

Whatalsohasbeenshownandisagainevident,isthevariabil- ityofthephysiologicalcharacteristicsofB.brauniistrainsrelative todifferentliteraturestudies.Thereforetherewouldbeabenefit ofimprovingthespeciesreferenceortaxonomycataloguewhen dealingwiththecosmopolitanB.braunii,forexample,asimilar approachtakenbyDarienkoetal.(2015),whodescribedtheclosely relatedspeciesCoccomyxausingintegrativetaxonomyandDNA barcoding.Thistypeof approachis suitablefor microalgaethat havehighmorphologicalandphysiologicalsimilarities.Arecent publicationbyHeged ˝usetal.(2016)followssimilarapproachwith B.brauniiAraces.

5. Conclusion

Thisstudypresentsthephysiologicaldiversityofcommercially availableB.brauniistrainsgrowninsimilarconditionswithrespect tobiomassproductivity,andhydrocarbonandcarbohydratecon- tent.ThephysiologicalcharacteristicsofB.brauniifromthisstudy complementedwiththeliteraturecanbeasdiverseasthenum- berofstrainscompared.Thisvariabilitykeepsscientistschallenged inthecharacterizationandunderstandingofB.brauniiandatthe sametimepositivethattheexplorationofthisspeciecaninthe futureyieldastrainthatwillhavetheminimumrequiredcharac- teristicsforindustrialapplication.CCALA778strainsshowpotential ascarbohydrateproducerswithfeasibleapplicationsindifferent industries.For example,galactoseinfermentation processesfor theproductionofbioethanolandfucosefortheapplicationincan- certreatment.ForhydrocarbonsproductionthestrainAC761could beexploited.Theslowgrowthof B.brauniiisnot correlatedto