RaghuChandrasekarana,1,LuciaBarraa,b,1,SaraCarilloc,ToninoCarusod, MariaMichelaCorsaroc,FabrizioDalPiazd,GiuliaGrazianie,FedericoCoratoa, DeboraPepea,AlessandroManfredoniaa,IdaOreficea,AlexanderV.Rubanf, ChristopheBruneta,∗
aStazioneZoologicaAntonDohrn,VillaComunale,80121Napoli,Italy
bCNR-IBBR,ViaUniversità133,80055Portici(NA),Italy
cUniversityofNaples“FedericoII”,DipartimentodiScienzeChimiche,ComplessoUniversitarioM.S.Angelo,ViaCintia4,80126Napoli,Italy
dUniversityofSalerno,DipartimentodiChimicaeBiologia,ViaGiovanniPaoloII132,84084Fisciano(SA),Italy
eUniversityofNaples“FedericoII”,DepartmentofAgriculturalandFoodScience,ParcoGussone,80055Portici(NA),Italy
fSchoolofBiologicalandChemicalSciences,QueenMaryUniversityofLondon,MileEndRoad,LondonE14NS,UK
a r t i c l e i n f o
Articlehistory:
Received25June2014
Receivedinrevisedform8October2014 Accepted13October2014
Availableonline27October2014
Keywords:
Biotechnology Bluelight Carotenoids Microalgae Lipids
a b s t r a c t
ThebiochemicalprofileandgrowthofthecoastaldiatomSkeletonemamarinoiwasinvestigatedunder fourdifferentdailybluelightdoses(sinusoidallightpeakingat88,130,250and450molphotons m−2s−1,respectively).Abilityofcellstoregulatethelightenergyinputcausedalterationsingrowthand differentbiosyntheticpathways.Thelightsaturationindexforphotosynthesis(Ek),whichgovernsthe photoacclimativeprocesses,rangedbetween250and300molphotonsm−2s−1.Cellsthatwereadapted tolowlight(<Ek)enhancedtheircarotenoid,lipidandproteincontentsandloweredcarbohydratecontent, andviceversaunderhighlight(≥Ek).Variationsinfattyacid,pigmentandaminoacidcompositionswere aresultoflightadaptation.Ourdatashowthatlightisapotentfactorformanipulatingbiomasssynthesis inmicroalgae,suchasdiatomsformicroalgalbiotechnology.
©2014ElsevierB.V.Allrightsreserved.
1. Introduction
Useofmicroalgaeforbiotechnologicalapplicationscoversmany aspects,suchasfoodadditivesinnutraceuticsandanimal feed-ing,cosmetics,pharmaceuticsandenergetics(Barraetal.,2014;
Cadoretetal.,2012;Chisti,2013;Levitanetal.,2014;Muniretal., 2013).Inaneconomiccontext,microalgaepresentmany advan-tagescomparedtootherphotosyntheticorganisms,suchastheir highgrowthrateandtherequirementoflittlespaceforbiomass production.Indeed,Cadoretetal.(2012)reportedaproductivity of10gm−2day−1formicroalgaecomparedto1–2gm−2day−1for higherplants.Despitetheirgreatpotentialforthebiotechnological applications(Cadoretetal.,2012;Fuetal.,2013),theextensiveuse ofphotosyntheticprotistshastobeenforced.Themainchallenge istoenhancebiomassandbioactivemoleculessynthesis,and,in
∗ Correspondingauthor.Tel.:+390815833243.
E-mailaddress:christophe.brunet@szn.it(C.Brunet).
1 Theseauthorscontributedequallytothiswork.
parallel,toavoidtheincreaseinproductioncosts(Blankenetal., 2013).Tocopewiththesebiotechnologicalexpectations,thereisa needto(i)increasethediversityofalgalspeciesused,(ii)undertake thegeneticengineeringofthemostadequatestrains(Cadoretetal., 2012)and(iii)todeeplyinvestigatealternativewaysforenhancing biomasssynthesis,suchasthe“photosyntheticregulation biotech-nology”,lightmanipulationbeingapowerfultoolforincreasing microalgalproductivity(Barraet al.,2014;Perrineet al.,2012).
Lightintensityanditsfrequencyvariability, aswellasits spec-tralcomposition,stronglyaffectthegrowthandphotosynthesisof microalgae(Dimieretal.,2009;Fuetal.,2013;SchellenbergerCosta etal.,2013a,b).
Amongmicroalgae,diatoms,themostrecentandevolutionary diversifiedgroup(Kooistraetal.,2007),isstillunderexploitedinthe fieldofbiotechnology.However,thesiliceouscellwallofdiatoms isofhugeinterestinthefieldofnanotechnologyfordesigningand producingspecificfrustules(LebeauandRobert,2003;Wangetal., 2013).Furthermore,theycanbehighlysuitableforlarge-scale cul-tivation,since mostofthediatomspeciespresent fastadaptive physiologicalplasticity,andthepresenceoflargecentralvacuole
http://dx.doi.org/10.1016/j.jbiotec.2014.10.016 0168-1656/©2014ElsevierB.V.Allrightsreserved.
R.Chandrasekaranetal./JournalofBiotechnology192(2014)114–122 115
Fig.1.GrowthcurveofS.marinoiunderthefourbluefluencerates;experiments wereperformedduringtheexponentialphaseondays4and5(datarepresent mean±SD;n=3).
allowsthemtobehugelycompetitiveinvariableenvironments (Kooistraet al., 2007).Among thediatom-specific carotenoids, fucoxanthin together with the most common -carotene is of hugeinterestinthefieldofbiotechnology(Pengetal.,2011).The twootherxanthophylls,diadinoxanthinanddiatoxanthin,arenot extensivelystudiedbuttheirpotentialroleasantioxidantshave beenrecentlyreported(Gallinaetal.,2014).Thelipidprofile,both intermsofquantityandquality,makesdiatomssuitable candi-datesforaquaculture(LebeauandRobert,2003).Severalstudies haveshownthatthediatomspolyunsaturatedfattyacids(PUFAs) havepositiveeffectonhumanhealth(HallahanandGarland,2005), whilethemonounsaturatedfattyacids(MUFAs)areconsideredto bethepotentialfeedstockforbiodieselproduction(Levitanetal., 2014).
In this study,we explore therole of lightand the potential ofthe“photosyntheticregulationbiotechnology”tobetterexploit diatomsinthefieldofbiotechnology.Onlybluelightconditionwas selected,beingthemostenergeticandfullyabsorbedwavelengths bymicroalgaeforthephotosyntheticactivity.Furthermore,only bluelight,i.e.withoutredradiation,stronglylimitsphotoprotective processesinthecells(Brunetetal.,2014),potentiallyincreasingthe biochemicalenergyavailableforgrowth.
ThecentricdiatomSkeletonemamarinoiwassubmittedtofour differentbluefluencerates,characterizedbydailylightdoseof2.2, 3.2,6.1and11molm−2day−1,respectively.Ourresults,both on physiologicalpropertyandbiochemicalprofilesofthecells, con-firm that variation of bluefluence ratedoesstrongly influence thebiochemicalcharacteristicsofthecells,reinstatingthatlight manipulationisaconcretewaytomodulatethebiochemicalenergy allocationinmicroalgae,andthusadequateforbiotechnological purposes.
2. Materialsandmethods
2.1. Experimentalstrategyandsampling
ExperimentswereconductedonthecoastalcentricdiatomS.
marinoi(CCMP2092), bycultivatingat20◦C in4.5-Lglassflask withair bubbling,containingnatural sterileseawater amended withf/2nutrients(GuillardandRyther,1962).Alltheexperiments wereperformedintriplicate,lasting2daysduringtheexponential growthphase(Fig.1),onculturespre-acclimatedtoeach exper-imentallightconditionfor2weeksbeforetheexperiments.Four differentsinusoidalbluelightdistributions,peakingat88,130,250 and450molphotonsm−2s−1(dailylightdose:2.2,3.2,6.1and
11molm−2day−1,respectively),havebeenapplied,witha12:12h light:darkphotoperiod.Bluelight(=460±30nm)wasprovided byacustom-builtilluminationsystem(Brunetetal.,2014).Light intensitywasmeasuredinsideeachflaskbyusingalaboratoryPAR 4sensor(QSL2101,BiosphericalInstrumentsInc.,SanDiego,CA, USA).
Samples for cell counts, variable fluorescence and electron transportratemeasurements,pigments,particulateorganiccarbon andnitrogen,proteinsandRNAweretakenthreetimesperday:
dawn(time0),midday(time6h)andintheafternoon(time9h).
Samplesfor absorptionspectrum analysis,lipids, carbohydrates andfibersdeterminationweretakenonceduringtheexperiment (middaysamplingonsecondday).
2.2. Cellconcentration
Cellconcentrationwasestimatedontriplicatesub-samples.An aliquotof1mLwasusedtofillaSedgewickRaftercountingcell chamber,andcellcountswereperformedusingaZeissAxioskop2 Plusmicroscope.
2.3. Photochemicalefficiencyandphotosyntheticparameters
Photochemicalefficiencyofphotosystem(PS)IIwasestimated bya Phyto-PAM fluorometer(HeinzWalz,Effeltrich, Germany).
Thevariablefluorescenceanalysiswasperformedon15-min dark-acclimated samples, to measure the maximum photochemical efficiency(Brunetetal.,2014).
Electron transport rate (ETR) versus irradiance curves were determinedbyapplying13increasingredactiniclights(655nm) from1to853molphotonsm−2s−1lasting1mineach.Therelative electrontransportrate(relETR,expressedinmole−1s−1cell−1) wascalculatedasfollows:
relETR=Fv
Fm
·I·0.5·a∗
where I is theincident irradiance (expressed in molphotons m−2s−1),FvandFmarethevariablePSIIfluorescenceyieldand maximalPSIIfluorescenceyield,respectively,forilluminatedcells (measuredattheendofthe1minlastingactiniclight),a*isthe cell-specificabsorptioncoefficient,expressedinm2cell−1(forthe determinationofa*,seeSection2.6).Afactorof0.5wasappliedto correctforthepartitioningofphotonsbetweenPSIandPSII, assum-ingthatexcitationenergyisevenlydistributedbetweenthetwo photosystems.
ETR-IcurveswerefittedwiththeequationofEilersandPeeters to estimate relETRmax (maximal relative rate of linear electron transport),˛(maximumlightuseefficiency),andEk(light satu-rationindexforphotosynthesis).
The non-photochemical quenching (NPQ) was estimated on 15-mindark-acclimatedcellsbyilluminatingthesamplewithan actiniclightsetupat480molphotonsm−2s−1during10min,and themaximumfluorescenceyieldwasestimatedeveryminute.NPQ wasquantifiedbytheStern–Volmerexpression:
NPQ= Fm Fm−1
2.4. Pigments
Pigmentmeasurementwasconductedbyhigh-performance liq-uidchromatography (HPLC), following the proceduredescribed in Brunetetal. (2014).Ten mLaliquotof algalculture was fil-tered(underlowlight)on25mmGF/Fglass-fiberfilter(Whatman, Maidstone,UK)andstoredinliquidnitrogenuntilfurtheranalysis.
Pigmentswereextractedbymechanicalgroundingduring3minin
116 R.Chandrasekaranetal./JournalofBiotechnology192(2014)114–122
2mLofa100%methanolsolution.Pigmentswereseparatedina HewlettPackardseries1100HPLC(HewlettPackard,Wilmington, NC,USA),equippedwithareversed-phasecolumn(2.6m diam-eterC8Kinetexcolumn;50mm×4.6mm; Phenomenex®,USA).
Pigmentsweredetectedspectrophotometricallyat440nmusing aHewlettPackardphotodiodearraydetector,modelDADseries 1100.Determinationandquantificationofpigmentswerecarried outusingpigmentstandardsfromtheD.H.I.Water&Environment (Horsholm,Denmark).
2.5. Particulateorganiccarbonandnitrogen
TenmLaliquotforthedeterminationofparticulateorganic car-bon(POC)andparticulateorganicnitrogen(PON)wasfilteredon pre-combusted (450◦C, 5h) glass-fiberfilters (Whatman, Maid-stone,UK),conservedincellcultureplates(Corning®,CorningInc., NY,USA),andimmediatelystoredat–20◦C.Theanalyseswere per-formedwithaThermoScientificFlashEA1112automaticelemental analyzer(ThermoFisherScientific,MA,USA),followingthe pro-cedurepreviouslydescribedbyBrunetetal.(2014).Filterswere thawedjustpriortoanalysisandallowedtodryat60◦Cthrough adesiccator.Thenfilterswereloadedinsmalltincupsthatwere crimpedclosedandtransferredtotheCHNanalyzer.Asetofempty filterswasprocessedasordinarysamplestoaccomplishtheblank determination.Cyclohexanone2,4-dinitrophenylhydrazone(C,N, H,51.79,20.14,5.07wt%,respectively)wasusedasstandard.
2.6. Absorptionspectrum
Thespectralabsorptionmeasurementswereperformedusing a spectrophotometerHewlettPackard HP-8453Eequippedwith aninvertedLabsphereintegratingsphere(RSA-HP-53Reflectance Spectroscopy Accessory) following the procedure described in Dimieretal.(2009).TenmLaliquotofalgalculturewasfiltered ontoWhatmanGF/Ffiltersandimmediatelyfrozen.Theabsorption (a())wasmeasuredbetween250and800nm,andthusintegrated between400and 700nm.Thisintegratedvaluewasdividedby cellconcentrationfortheestimationofthecell-specificabsorption coefficient,a*,expressedinm2cell−1.
Thephotosyntheticallyusableradiation(PUR)wascalculatedas describedinBrunetetal.(2014).
2.7. Nutrients
Samplesfordeterminingnutrientconcentrationswerecollected in20mLpolyethylenevials,andquicklyfrozenandstoredat−20◦C.
Ammonium,nitrate,nitrite,silicicacidandphosphate concentra-tionsweredeterminedusingaTechniconAutoAnalyzerfollowing classicalmethods(Grasshoffetal.,2009).
Nutrientconcentrationanalyzedfromthemorningsamplingon thefirstandseconddayoftheexperimentwasthereforeusedto estimatetheuptakeofnutrientsreportedbycellnumberincrease duringthe1stand2nddayoftheexperiments:
Nu=N2−N1
C2−C1,
whereNuisthenutrientuptake(nmolcell−1day−1),Cnisthecell concentrationatdaynandNnisthenutrientconcentrationatday n.
2.8. Cellspelletpreparation
The volume sampled (50mL for RNA and protein, 800mL for lipids, carbohydrates and fibers) from each triplicate was centrifugedat4000rpm(3399×g)for20minat4◦C(DR15P cen-trifuge,B.BraunBiotechInternational,Melsungen,Germany);the
supernatantwasdiscarded.ForproteinsandRNAquantification, thepellet hasbeen transferredin a 2mLEppendorf tubes and centrifugedat14,000rpm(20,817×g)for 15minat4◦C(5417R centrifuge,Eppendorf,Hamburg,Germany).Forlipidsand carbo-hydrates,thepelletsobtainedafterthecentrifugation(16tubesof 50mL)waspooledtogetherandcentrifugedagainaspreviously mentionedandweighed.
2.9. RNAanalysis
ThetotalRNAhasbeenextractedfromthepelletfollowingthe proceduredescribedinBarraetal.(2013).ConcentrationofRNA wasmeasuredbyNanodrop(AgilentTechnologies,SantaClara,CA, USA).
2.10. Proteinandaminoacidsanalysis
Thetotalproteinsfromthepelletwasextractedbysonicating thecell pellets for 2.4minin 600L of sterilewater and cen-trifugedat 13,000rpm (17,949×g)for 20min at4◦C. Thenthe supernatantwascollected andthepelletwasre-extractedwith 500Lof0.1NNaOHand0.5%-mercaptoethanol(v/v).The mix-turewaskeptatRTfor1h(withoccasionalshaking)andcentrifuged at13,000rpm(17,949×g)for20minat21◦C.Thesupernatantwas mixedandthepelletwasdiscarded.Proteinswerethenpurified withtrichloroaceticacid(TCA)beforeacidhydrolysisfor amino acidsanalysis,accordingtoBarbarinoandLourenc¸o(2005)andthe crudeextracthasbeenquantifiedwithFolin&Ciocalteureagent (Sigma).
Samplescontaining50gofproteinwereacidhydrolyzedwith 1mLof 6NHCl in vacuum-sealedhydrolysisvials at110◦C for 22h. Norleucinewasadded totheHCl as aninternal standard.
Althoughtryptophanwascompletelylostwithacidhydrolysisand methionineandcysteine+cystinecouldbedestroyedtovarying degreesbythisprocedure,thehydrolysatesweresuitablefor anal-ysisofallotheraminoacids.Thetubeswerecooledafterhydrolysis, openedandplacedinadessicatorcontainingNaOHpelletsunder vacuumuntildry(5–6days).Theresiduewasthendissolvedin a suitable volume of dilution Na–S Rbuffer (pH 2.2; Beckman Instr.),filteredthroughaMilliporemembrane(0.22mporesize) andanalyzedforaminoacidsbyion-exchangechromatographyin aBeckman,model7300instrumentequippedwithanautomatic integrator.
2.11. Lipidandfattyacidsanalysis
Eachwetpelletwassonicatedfor15minat25◦Cin10mLof acetic acid/chloroform(1/9),or acetone/methanol (9/1)or pure methanolin order togetrespectively triglyceride,glycolipid or phospholipidfraction.Theextractedsolutionwaspassedthrough cartridgesSupelcleanTMENVI-Florisil®SPETubes(byAldrich), pre-conditionedwith30mLofchloroform(Popovichetal.,2012).The sampleshavebeensubsequentlyconcentrated underN2 fluxto reachfinalvolumeof1mLandesterifiedwithKOH2Minmethanol accordingto(Grazianietal.,2013).OneLwasinjecteddirectly inaThermoFinniganTRACEgaschromatographequippedwitha fusedsilicacapillarycolumn(FAMEWAXRestek,30m×0.25mm i.d.,0.25mfilmthickness)andanFIDdetector.Thecalibrationhas beenperformedusingastandardPUFA’s(bySupelco)asinternal standard.
Thedegreeofunsaturation(DU)wascalculatedusingthe fol-lowingformulaasreportedinRamosetal.(2009):
DU=(monounsaturatedCn:1,wt%)+2
×(polyunsaturatedCn:2,3,wt%)
R.Chandrasekaranetal./JournalofBiotechnology192(2014)114–122 117
Table1
PhotosyntheticpropertiesofS.marinoiunderthefourbluefluencerates.
88molphotonsm−2s−1 130molphotonsm−2s−1 250molphotonsm−2s−1 450molphotonsm−2s−1
PARdose 2.2 3.2 6.1 11
a* 1.66±0.59 0.90 0.94±0.29 2.26±0.64
PUR 0.51±0.18 0.37 0.87±0.34 4.1±1.2
0.40±0.27 1.24±0.23 0.49±0.13 0.31±0.11
relETRmax 3.16±1.15 2.21±0.08 2.90±1.02
Ek 252±36 241±7 306±13
␣ 0.012±0.003 0.009±0.0006 0.0095±0.003
NPQ 0.64±0.05 0.87±0.03 0.61±0.02
TotalRNA 0.48±0.18 0.30±0.06 0.69±0.22 0.38±0.14
AveragePARdoseexperiencedbythecellsunderdifferentbluelightconditions(molm−2day−1).a*×10−11,absorptioncoefficient(m2cell−1);PUR×10−6,photosynthetically usableradiation(Wcell−1);,growthrate(day−1);relETRmax×10−6(maximalrelativerateoflinearelectrontransport,pmole−1h−1cell−1);˛(maximumlightuseefficiency, pmole−1h−1cell−1(molphotonm−2s−1)−1);Ek(lightsaturationindexforphotosynthesis,molphotonsm−2s−1)andtotalRNAcontent(pgcell−1).Datarepresentmean andSD(n=3exceptfortotalRNA,n=15).
Thelackofdataforthe450molphotonsm−2s−1conditionisduetothebreakdownofthePAMfluorometerduringtheexperiment.
2.12. Carbohydrateanalysis
Thepelletsobtainedforcarbohydrateanalysisweresonicated for5minin5mLoff/2culturemediumandcentrifuged.The super-natantandthecellulardebrispelletwerestoredseparatelyforthe analysis.Totalcarbohydratescontent,calculatedonboth super-natantandpelletofeachsample,wasdeterminedbymicrophenol assayasreportedinKobata(1972).
2.13. Fiberanalysis
TotalfibercontentofS.marinoi,composedbysolubleand insol-uble fraction, wasdeterminedby theAOAC 985.29gravimetric method(Grazianietal.,2013).
2.14. Statisticalanalysis
Student’s t-test and Spearman’s rank correlation were per-formedusingSystat7software.
3. Resultsanddiscussion
3.1. Lightmodulationofphotosyntheticpropertyandgrowth
Cellsgrownunder88,130and250molphotonm−2s−1had similargrowthcapacity(Fig.1)inagreementwithalowrangeof variabilityofthelinearelectrontransportrate(relETRmax,from2.2 to3.1×10−6pmole−1h−1cell−1,Table1).Thesimilar photosyn-theticandgrowthratesmeasuredunderthedifferentillumination conditionsareprobablyrelatedtobiochemicalandphysiological adjustments withinthecells. Indeed,thelight saturationindex forphotosynthesis(Ek,Table1),≈250–300molphotonsm−2s−1, revealed that cells grown in 88 and 130mol photon m−2s−1 conditions were potentially light limited, while under the 250 and450molphotonm−2s−1 cellswerelightsaturatedor over-saturated(450molphotonm−2s−1).Thepronouncedlowlight acclimationstateofthecellsunder88molphotonm−2s−1 was confirmedbythehighestvalueofthemaximumlightuseefficiency parameter(˛,Table1),andbytheincreaseincell-specific absorp-tioncoefficient(a*,Table1).Ontheopposite,underthehighest lightcondition,450molphotonm−2s−1,cellsgrewfaster reach-ingahighermaximumcellnumber(11×105cellsmL−1)compared tothethreeotherconditions(∼7×105cellsmL−1;Fig.1).Indeed, thelightenhancementwasonlylittlecompensatedbyadjustments inlightabsorption,asshownbythehighestphotosyntheticusable radiation(PUR,Table1).
Noneofthenutrientswaslimitingduringthe2daysof exper-iment (Table 2), while the increase in cell concentration was
sustained by the uptake of NO3−, PO43− and SiO32− (Table 2, Fig.2).Bycontrast,NO2−andNH4+increasedbetweenthe2days of experiment, probably in relationwiththe rapid and contin-uous recycling of these nutrients in the medium (Barra et al.,
Fig.2. Relationshipbetweengrowthrate(;day−1)andnutrientuptakepercell (nmolcell−1day−1):(a)NO3−,(b)PO43−and(c)SiO32−(datarepresentmean±SD;
n=3).
118 R.Chandrasekaranetal./JournalofBiotechnology192(2014)114–122
Table2
Growthrateandnutrientconcentrationsunderthefourbluefluenceratesduringthetwodaysofexperiments.
88molphotonsm−2s−1 130molphotonsm−2s−1 250molphotonsm−2s−1 450molphotonsm−2s−1 Cellcon.
1stday/2ndday 443,989±205,449 214,214±31,979 439,509±73,147 854,291±282,535
678,394±336,142 738,348±99,598 715,593±122,325 1,072,880±160,228
Cellcon.increase 234,405±225,457 524,134±112,078 276,083±145,257 218,588±176,819
NH4+
1stday/2ndday 0.60±0.37 0.92±0.21 0.84±0.78 1.81±0.80
0.93±0.32 1.17±0.70 0.97±1.08 1.05±0.08
NO2−
1stday/2ndday 4.89±1.13 4.21±0.49 4.93±1.06 6.68±0.05
5.94±0.56 7.12±0.67 8.20±1.29 15.31±1.23
NO3−
1stday/2ndday 369.16±9.5 858.93±46.35 694.16±142.58 594.70±44.01
300.54±28 756.56±33.23 629.32±60 515.60±42.56
PO43−
1stday/2ndday 10.13±0.91 24.13±0.29 19.54±2.38 14.25±0.72
2.08±0.04 13.73±2.97 11.10±4.34 1.74±0.45
[SiO3]2−
1stday/2ndday 68.03±19.89 124.29±17.43 148.69±58.31 64.08±0.76
38.75±23.79 63.86±27.86 56.58±40 5.38±5.50
Cellconcentration(Cellcon.;cellsmL−1)andnutrientconcentration(nmolmL−1day−1).
2014). Growth rate was significantly correlated to the uptake of NO3− and PO43−,(p<0.05 for NO3− and p<0.01 for PO43−; Fig.2), revealing theirrelevant role indriving biomass synthe-sisand thatlight intensitydidnot affectdirectlytheuptake of thesenutrients.Bycontrast,thehigherSiO32−uptakewas mea-suredunderhighlightcomparedtothelowlightconditionsand itwasnotrelatedtothegrowthrate(p>0.05,Fig.2).Thisagrees withapotentialincreaseinphotorespirationrateinducedbyhigh light (Brunetet al., 2011; Schnitzler Parker et al.,2004), being this processa wayforcells todissipatetheexcess biochemical energy, andto thefact thatsilicate metabolism ismore linked torespiration and cell cycle than photosynthesis (Norici et al.,
2011).
TheRNA content percell didnot showsignificant variation amongthelightconditions(p>0.05,n=15),rangingfrom0.30to 0.70pgcell−1 (Table1).TheRNAcontentpercelldisplayed cir-cadian oscillation in thecells (datanot shown), that might be relatedtovariationsinthecellularcontentofproteins,lipids, car-bohydratesorpigments(Fábregasetal.,2002).Inallconditions, theRNAcontent percellincreasedat middaycompared tothe morningandafternoonsamples,whilesomestudies(e.g.Berdalet etal.,1992)showedenhancedRNAcontentatthebeginningof thelightperiod.Thisdiscrepancyisduetothelightdistribution, providedinasinusoidalwayinourstudyandinaquadraticway fortheotherstudies,asrecentlydemonstrated(Orefice,personal communication).
3.2. Lightmodulationofcarotenoidsandchlorophylls
Themainphotosyntheticpigments,chlorophylla(Chla)and fucoxanthin(Fuco),weresignificantlycorrelated(p<0.001,n=36) presentingasignificantdecreaseunderthehighlightconditions (p<0.05, n=9; Fig. 3a and b). Under low light (E<Ek, 88 and 130molphotonm−2s−1),thetotalcarotenoidcontentandChl apercellwassimilar(Fig.3a,Table3),despitethedifferencein dailylightdose(2.2and3.2molm−2day−1).Fuco/Chlawas simi-laramongthedifferentlightconditions(p>0.05,n=9),exceptfor 450molphotonm−2s−1whereitsignificantlydecreased(p<0.01, n=9).Thus,S.marinoitendstomodifytheantennastructureby decreasingFucocomparedtotheChlamoleculesinthereaction centerunderthelattercondition(Dimieretal.,2009),whileunder lowlightcondition,S.marinoitendstomodifyallthereactioncenter andantennastructuretomaintainsimilarthepigmentratio.
OntheoppositeofFucoandChla,chlorophyllc(Chlc)content percellexponentiallydecreasedfromthelowesttothehighestlight dose(Fig.3a),inagreementwiththeresultsobtainedon
OntheoppositeofFucoandChla,chlorophyllc(Chlc)content percellexponentiallydecreasedfromthelowesttothehighestlight dose(Fig.3a),inagreementwiththeresultsobtainedon