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The fascinating diatom frustule—can it play a role for attenuation of UV radiation?

Ellegaard, Marianne; Lenau, Torben Anker; Lundholm, Nina; Maibohm, Christian; Friis, Søren MichaelMørk; Rottwitt, Karsten; Su, Yanyan

Published in:Journal of Applied Phycology

Link to article, DOI:10.1007/s10811-016-0893-5

Publication date:2016

Document VersionPeer reviewed version

Link back to DTU Orbit

Citation (APA):Ellegaard, M., Lenau, T. A., Lundholm, N., Maibohm, C., Friis, S. M. M., Rottwitt, K., & Su, Y. (2016). Thefascinating diatom frustule—can it play a role for attenuation of UV radiation? Journal of Applied Phycology,28(6), 3295–3306. https://doi.org/10.1007/s10811-016-0893-5

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Thefascinatingdiatomfrustule–canitplayaroleforattenuationofUVradiation?

MarianneEllegaard1,TorbenLenau2,NinaLundholm3,ChristianMaibohm4,SørenMichaelMørkFriis4,KarstenRottwitt4,YanyanSu1

1DepartmentofPlantandEnvironmentalSciences,UniversityofCopenhagen,Thorvaldsensvej40,1871Frederiksberg,Denmark.CorrespondingauthorMarianneEllegaardme@plen.ku.dk;phone:+4535320024

2DepartmentofMechanicalEngineering,TechnicalUniversityofDenmark,Produktionstorvet,Building426,2800KongensLyngby,Denmark

3TheNaturalHistoryMuseumofDenmark,UniversityofCopenhagen,Sølvgade83S,DK-1307CopenhagenK,Denmark

4DepartmentofPhotonicsEngineering,TechnicalUniversityofDenmark,ØrstedsPlads343,2800KongensLyngby,Denmark

Keywords:UVprotection,photonics,photobiology,nano-patterning,diatom

Reference: Ellegaard, M., Lenau, T. A., Lundholm, N., Maibohm, C., Friis, S. M. M., Rottwitt, K., & Su, Y. (2016). The fascinating diatom frustule—can it play a role for attenuation of UV radiation? Journal of Applied Phycology. doi:10.1007/s10811-016-0893-5

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Abstract

Diatomsareubiquitousorganismsinaquaticenvironmentsandareestimatedtoberesponsiblefor20-25%ofthetotalglobalprimaryproduction.Auniquefeatureofdiatomsisthesilicawall,calledthefrustule.Thefrustuleischaracterizedbyspeciesspecificintricatenano-patterninginthesamesizerangeaswavelengthsofvisibleandultraviolet(UV)light.Thishaspromptedresearchintothepossibleroleofthefrustuleinmediatinglightforthediatoms´photosynthesisaswellasintopossiblephotonicapplicationsofthediatomfrustule.Oneofthepossiblebiologicalroles,aswellasareaofpotentialapplication,isUVprotection.InthisreviewweexplorethepossibleadaptivevalueofthesilicafrustulewithfocusonresearchontheeffectofUVlightondiatoms.WealsoexplorethepossibleeffectofthefrustulesonUVlight,fromatheoretical,biologicalandanappliedperspective,includingrecentexperimentaldataonUVtransmissionofdiatomfrustules.

Introduction

Thediatomsareuniqueamongalgaeinthatthecelliscompletelyenclosedinacontinuoussilica-basedcover,thefrustule.Thefrustuleisasarulebuiltmoreorlesslikeapetridish,withaninnerandoutervalve,connectedbygirdlebands(Fig.1).Diatomsarealsospecialamongeukaryoticmicroalgaeasduetothefrustuletheyhaveafixedcellshape,andtheylackflagellainthevegetativestages.Diatomsareubiquitousorganismsinaquaticenvironmentsandcanbedominantprimaryproducersinallaquatichabitats,includingice.Theyarethusestimatedtoberesponsiblefor20-25%ofthetotalglobalprimaryproduction(Nelsonetal.1995).Diatomspeciesdiversityisfarfromdiscoveredatpresent.Some10,000specieshavebeenformallydescribed,butitisestimatedthattherealnumberofspeciesis10-20timeshigher(Guiry2012).Traditionally,speciesdescriptionshavebeenbasedondetailsintheintricatenanoscalepatternofholesinthefrustuleandeachspeciesthushasitsowndistinctpatternwhichisreproducedatcelldivision.Whilebenthicdiatomshaveevolvedaspecializedmodeofmovementviaaslitthroughthefrustulecalledtheraphe,plankticdiatomsarenon-motileandarethereforedependentonbuoyancyandwatermovementtostaysuspendedinthephoticzone.Asthefrustuleisgenerallydenserthanbothseawaterandfreshwater,itwouldseemtobeanimpedimenttophotosyntheticorganismsdependentonstayingupinthephoticzone.Possibleadaptiverolesofthediatomfrustulehavethereforebeenexplored,fromthepointofviewthatthefrustulemustgiveadaptiveadvantagesthatoff-setthisseemingdisadvantage.Wewillfocusontheevidenceforaroleinprotectingthediatomcellfromultravioletradiation(UV)damage.Thatthefrustuleinteractswithlightisclearalreadywhenstudyingfrustulesunderthelightmicroscope(Fig.2b).This,andthefactthatsomefrustules,suchasthatshowninFig.2b,lookverysimilartoaverythinsliceofaphotoniccrystalfiberormicro-structuredopticalfiber,haspromptedintensiveresearchintopossiblephotonicapplicationsofdiatomfrustule.InthisreviewwewillfocusonpotentialforUVprotectiveapplications.

Review

Thesuggestedadaptivefunctionsofthediatomfrustule

Thediatomfrustulehasbeensuggestedtobeaby-productofotheradaptationsratherthangivinganadaptiveadvantageperse.Thussomeauthorsstatethatitismetabolicallycheaptoproducerelativetoothertypesofcellcovering(only2%oftheenergybudgetofthecell;Raven1983)althoughotherauthors

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questionthis(FinkelandKotrc2010;Lavoieetal2016).Medlin(2002)putforwardthehypothesisthat,becausesilicametabolismplaysanimportantroleinresistingaging,thesilicafrustuleismainlyaby-productofthis.Otheradaptiveadvantageshavebeensuggested,includingadeliberateincreaseofthedensityofcells,alteringsinkingrateviaspinedevelopment,resistingturgorchangesandresistancetoparasites(reviewedbyRavenandWaite2004).However,thesuggestionswhichhavereceivedmostsupportare:1.defenseagainstpredation,2.proton/pHbufferingand3.modulationoflightwithinthecell.Totestthefirsthypothesis(defenseagainstpredation)Hammetal.(2003)performedastudywheretheytestedthestrengthofdifferentlyshapedfrustules(threespecies),usingglassmicroneedlestopressthecellandmeasuringtheforcenecessarytobreaksinglecells.Theyconcludethatallthreeshapesofcellstestedareremarkablystrongduetotheirarchitectureandthematerialpropertiesofthesilica.Thesecondhypothesis,pHbuffering,hasreceivedsupportfrombothlaboratorytestingoftheefficacyofdiatombiosilicaasapHbuffer(MilliganandMorel2002)andfromtheorieslinkingmajoreventswithintheevolutionaryhistoryofdiatomstochangesinatmosphericCO2levels,andtherebypHinaquaticsystems(e.g.Armbrust2009).Athirdsuiteofhypothesesdealwithdifferentaspectsofcontrollingthelightregimewithinthediatomcell(Fuhrmanetal.2004),byfunctioningasaphotoniccrystal(Fuhrmanetal.2004),byscatteringvisiblelight(RavenandWaite2004)orbyreflecting,absorbingorscatteringlightintheUVrange(below380nm;Davidsonetal.1994;RavenandWaite2004).InthisreviewwefocusonthepossibleroleoffrustulesinmodulatingUVlight,aswellasdiscussingwhichcharacteristicsofthediatomfrustulearecentraltothephotoniceffects.

Photonicapplicationsordiatombiology?

Whendiscussingpotentialphotonicutilizationsofdiatomfrustules,thelightenvironmentthatdiatomsareadaptedtoundernaturalconditionsshouldbetakenintoaccount.Whenlightenterswater,itbecomespartiallypolarizedandisabsorbedmorestronglythaninairinawavelengthdependentway.Red,orangeandyellowarequicklyabsorbedbythewater,onlypenetratingmetersbelowthesurfacewhilebluelightisnotstronglyabsorbedbywaterandcanpenetratemuchdeeper,dependingontheconcentrationofdissolvedmatterandparticles(Depauwetal.2012;seealsoFig.2).AcoupleofmetersbelowthesurfacealmosthalfoftheintegratedlightintensitywillbeintheUVspectralregion,i.e.below400nm,comparedtoonlyabout3%atgroundlevel(JohnsenandSosik2004).DiatomsthusliveinalightenvironmentdominatedbyUVandbluelight,supportingtheideathatthefrustulemayplayaroleinUVlightprotection.Asmoststudiesaimedatcharacterizingthephotonicpropertiesofdiatomfrustulesandelucidatingtheirindustrialpotentialaredoneonrinsedfrustules,theorganicmaterialinthecellcontentistakenintoaccountinneithermeasurementsnorsimulations.Further,mostofthesestudiesareperformedorsimulatedinair,contrarytotheaquaticnaturalhabitatofthediatoms(althoughthereareexceptions,e.g.DeTommasietal.2010;DiCaprioetal.2014;Hsuetal.2012;LeDuffetal.2016).Thereforethecalculatedeffectsoftheseexperimentsandsimulationsapplytothefrustule,andforuseinapplicationsofrinsedfrustules,butcannotgivefullbiologicalanswersastohowthelivingdiatomsbenefitbytheinteractionsbetweenthefrustuleandlight.Thesedifferencesbetweenmeasurementsonrinsed(andoftenseparated)frustulesandthediatominitsnaturalhabitataresummarizedinFig.2.

Anotheraspecttobeconsideredistheorientationofthefrustule.Undernaturalconditions,inaquaticplankticenvironments,lightwillhitthediatomcellfromtheoutsideofthefrustule(theconvexside),butsomeofthelightmayalsobeaffectedbythe(concave)insideoftheoppositevalveasitpassesthroughthe

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cell.Underexperimentalconditionstotesttheeffectofthefrustulenano-patternonincidentlight,thefrustuleswilltypicallyhavebeencleanedoforganicmaterial(cellcontentandanyorganiccovering)andthefrustulewillbeseparatedintoitsparts–thetwovalvesandthegirdlebands(seeFig.1).Typically,measurements(andsimulations)havebeendoneonasinglevalve.Itisnotalwaysclearfromwhichdirectionthelightishittingthevalve,andthequestionishowtheorientationofthevalveaffectsthemeasurements.Dothephotonicpropertiesdifferdependingonwhethertheincidentlighthitsthevalvefromwhatwastheoutsideortheinsideofthecell?Formeasurementsonalayeroffrustules,anotherquestioniswhetheristhereapatterntohowasinglevalvewillnormallyfallwheninadropofwateroranotherliquidmatrix.Basedonthe(sparse)literaturetodateonthis,andonourownunpublisheddata,theanswertoboththesequestionsappearstobeyes.Preliminaryresultsindicatethatinadropofliquidthevalvesfallapproximatelyonethirdmoreoftenwiththeinsideup(Gösslingetal.pers.comm.).Thiscan,however,becontrolledasshownbyWangetal.(2012),wherethevalvesaremadetoallfaceinthesamedirectionbysuspensionandsubsequentlyextractingtheliquid.Wehaveobserveddifferencesinourmeasurementswithregardtointerferencepoints,dependentonthewaythevalvesarefacing(unpublisheddata).ThisisingoodagreementwithfindingsbyRomannetal.(2015),wherenointerferencepoints(seee.g.Maibohmetal.2015a)areobservedwhenthevalveisfacingwiththeoutsidetowardsthemicroscopeintransmissionexperiments.

EffectsofUV-light

UVlightisingeneraldetrimentaltolivingorganisms(althoughnearUVlightmaybephotosyntheticallyactive;XuandGao2010)aswellasmanyman-madematerials;theshorterthewavelength,themoredamaging(withtheeffectfurtherenhancedbyresonantbehavioratspecificwavelengthsfrompossibleabsorptionpeaks).EffectsofincreasedUVlevelsonlivingdiatomshavebeenstudiedinachangedglobalclimatescenariosincehuman-inducedreductionsintheozonelayerofthestratospherewerereporteddecadesago(e.g.Wuetal.2014).Polarregions(receivingincreasingUVlight)aswellasmountainhabitats(naturallyhighUVradiation)havebeenthepreferredhabitatsforUVresearchonalgae.AsmostUVradiationisnotphotosyntheticallyactive,cellstrategieswouldthusbeexpectedtobeeithertoavoidortolerateUVradiation(HolzingerandLütz2006).AUV-inducedeffectisdependentonthelevelofUV,thedurationoftheexposure,thespectralcompositionofthelightintheUVrange,thesensitivityoftheorganism(s)investigated,physiologicalstatus,ambientnutrientconditionsandtemperature(Bumaetal.1996;CullenandLesser1991;LitchmanandNeale2005;Hessenetal.2012).UVradiationharmscellsthroughabsorptionbynucleicacidsandproteinsandhenceinhibitingDNAtranslation,replicationandtranscription(Bumaetal.1995;HolzingerandLütz2006).ButcomparisonsamongstudiesofUVeffectsonalgaearehamperedbydifficultiesincomparingradiationconditionswhichcannotberecalculatedandthuscompared(HolzingerandLütz2006).Thus,infuturestudiesofthepossibleeffectsofthefrustuleonUVlight,itwouldbeadvantageoustostandardizethemethodofregisteringUVintensityandwavelength.

AdecreaseinphotosynthesishasbeenthemostprominentUVeffectinalgae(Waringetal.2006;HolzingerandLütz2006),anddecreasesinbiomassand/orgrowthrateofdiatomsexposedtoUVradiationarecommonlyreported(e.g.Reizopoulouetal.2000;Nahonetal.2010),buttheeffectandtherecoverycapabilityafterUVexposuresapparentlyvaryamongspecies(Bumaetal.1996),asspeciescompositioninexposedcommunitiesarereportedtochangewithtime(Reizopoulouetal.2000).ThisagreeswithvaryingeffectsofUVamongcultureddiatomspeciesbothwithregardtoresponseandsensitivitytoUV(Karentzet

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al.1991;Davidsonetal.1994;Waringetal.2006;Hessenetal.2012;Scholzetal.2014),evenamongspecieswithinagenus(Hargravesetal.1993).ThishasraisedspeculationsastowhydifferencesinUV-effectsvarymoreindiatomsthaninotheralgalgroupswheretheresponsescanoftenbecorrelatedtocellsizeduetosizerelatedeffectsonself-shadingandUV-absorbingcompounds(Waringetal.2006andreferencesherein).Onepossibleexplanationcouldbethatthisdifferenceisduetospeciesspecificdifferencesinthepatternofholesorothercharacteristicsofthefrustule.

ChronicUV-Bexposureondiatomshasoftenbeenrelatedtoincreasesincellvolume(duetoadecouplingbetweencellgrowthandcelldivision)anddecreasesingrowthrates(Karentzetal.1991;Bumaetal.1995;1996;HolzingerandLütz2006andreferencestherein;Nahonetal.2010;Scholzetal.2014).Anincreaseinnumberofchloroplastsandthusincellularpigmentcontenthasbeenobservedinsomediatoms(Bumaetal.1996),whereasothershavefoundareductioninpigmentation(Lohmannetal.1998).Othersagainshowedanincreaseinpotentialphotoprotectivepigmentslikediadinoxanthinanddiatoxanthin(Döhler1995;Leuetal.2006).Inastudyonlong-termacclimation,severaldiatomspeciesacclimatedbychangingphotosynthesisrelatedpigments,whereasonespecies,Amphoracoffeaeformis,lackedthesepigmentchanges(Rechetal.2005).IthasbeensuggestedthatUVabsorbingcompounds,mycosporine-likeaminoacids(MAAs),chemicallyboundtothediatomfrustuleprovidediatomswithUVprotectingproperties(Ingallsetal.2010),andtheyexplainalackofincreaseinMMAsindiatomsinresponsetoUV,incontrasttoflagellates(Davidsonetal.1994),toberelatedtothetightassociationwiththefrustule(Ingallsetal.2010).ThishypothesisissupportedbyhighMAAcontentintwocentricdiatomspeciesrelatedtolackofUVeffectonphotosynthesis,asopposedtolowerMAAconcentrationsintwopennatediatomsaffectedbyUV(Helblingetal.1996).

Nitrate-limitationincreasesthesensitivityofdiatomstoUVlightduetoadecreaseinphotochemicalquantumyield(CullenandLesser1991;Scholzetal.2014).Moreover,ithasbeenreportedthatthesensitivityofdiatomstoUVradiationcouldbeincreasedbyelevatedCO2duetoareductioninrelativeelectrontransportrate(Wuetal.2012).AnadditionalstressfordiatomshasbeenobservedundercertainsalinityconditionscombinedwithUVradiation,andthiscombinedeffectisspecies-dependent(Döhler1984;Rijstenbil2005).

InspiteofthereportedeffectsofUVradiation,somestudiesfindnoeffectofUVondiatoms,orlesseffectthanonflagellates(Villafaneetal.1995;Vernet2000;LitchmanandNeale2005;Guihéneufetal.2010;Hessenetal.2012:Wuetal.2012).Sofarnoexplanationhasbeenfoundforthisdifference.WehypothesizethatitisthediatomfrustuleanditscharacteristicswhichreflectorabsorbtheUVandthereforeprotectthediatomcellsagainstUV.Thisissupportede.g.byobservationsofdiatomsnotrespondingtoUVwithachangeinfattyacidcompositionorhavingverylowamountsofMMAsincontrasttomanyothermicroalgaelikedinoflagellates(Jeffreyetal.1999,Guihéneufetal.2010andreferencesherein).AlsoJeffreyetal(1994)underlinedthatthesurvivalofdiatomsexposedtoUVdidnotcorrelatewithabsorptionbye.g.pigments.Potentialindustrialapplicationsofdiatomfrustules

Similartootheralgalgroups,potentialindustrialapplicationsofdiatomsincludebiofuels(e.g.Levithanetal.2014),bioplastics(Hempeletal.2011)andotherusesoftheorganiccellcontents(Bozartetal.2009).Uniqueamongthealgae,however,istheadditionalpotentialforapplicationsofinorganicmaterial:the

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silicafrustule.Geologicaldepositsrichinprehistoricdiatomremains,calleddiatomaceousearth,arealreadyindustriallyexploitedfore.g.filtering(ParkinsonandGordon1999).

Inrecentdecades,diatomfrustuleshavebeenthefocalpointforintenseopticalstudiesduetotheirinherentstronginteractionwithlight(includingUVradiation)andpotentialindustrialusesofspecificcharacteristics.Theideasforpotentialapplicationsforthesilicafrustuleincludephotoniccrystals(Fuhrmanetal.2004,Yamanakeetal.2008;deStefanoetal.2009),chemicalsensors(deStefanoetal.2009),microlenses(deStefanoetal.2007a),enhancedlightharvestinginsolarcells(Ottesen2011),lightfocusingcapabilitiesandapplicationsofthis,suchasopticalswitches(DeTommasietal.2010;Maibohmetal.2015a;Ferraraetal.2014).DuetothedestructiveeffectsofUVlightonlivingorganismsandmanytypesofmaterials,andduetotheunwantedeffectsassociatedwithmanytypesofUVprotectionthereisgreatinterestinfindingnovelmeansofUVprotectionwithoutdeleteriousaspects.UVprotectionisimportante.g.toprotectpolymersfromdeteriorationwhenexposedtosunlightandfortheprotectionofhumanskininsunlotions.ThepotentialadvantageofusingdiatomsasUV-filterswouldbethatitmaybepossibletoselectivelyreflectorabsorbcertainwavelengths.Suchafilterthereforehasthepotentialnottoimpactthecolorofthematerial.Itwouldforinstancebepossibletomakedurabletransparentpolymersandlacquerswithlastingpropertiesinsunlight.ThemosteffectiveUV-filterusedforpolymersiscarbonblackwhichabsorbsnotonlyUVbutalsootherwavelengthsoflightandthereforeappearblack(Coleman2011).ThereareotheradditivestopolymersthatpreventdegradationfromUV-lightincludingHinderedAmineLightStabilizers(HALS)thatworkbytrappingfreeradicalsformedduringphoto-oxidationofthepolymer(Gijsman2011)thusextendingthelifetimeofthepolymer.SunlotionstypicallyprotectstheskinfrombothUVA(400-320nm)andUVB(320-290nm)light.

Parameterscentraltophotonicapplicationsofdiatomfrustules

Forapplicationpurposesitisnotnecessarytoknowthefullinteractionbetweenthelightandthefrustule.Itisenoughtoknowtheeffectswhichareimportanttotheapplicationneeded.

Developmentofsophisticatedmethodsandequipment,whichhaverevealedmaterialcompositionandthreedimensional(3D)structure,havestrengthenedthepotentialforapplicationsofthefrustule.Whenlightinteractswiththefrustule,someeffectscaneasilybeobservedinalightmicroscope(Fig.2b,c).Theintricate,semitransparent,nano-structuredfrustulecauseslighttobetransmittedormultiplereflectedfromthedifferentinterfacesgivingrisetophaseshiftsaswellasfrequencydependentconstructiveanddestructiveinterference(ParkerandTownlee2007;Gordonetal.2009;Maibohmetal.2015a,b).Inadditiontotheseeasilyobservedopticalphenomenamoresophisticatedopticaleffectshavebeentheorized,i.e.wave-guidingandband-gaps(Fuhrmannetal.2004).Ifdiatomfrustulesaretobeusedinindustrialapplicationsweneedtodeterminewhichaspectsarecentralforthedesiredapplication.Althoughthereishugemorphologicalvariationamongtheindividualspeciesofdiatoms,thisreviewdiscusscommontraitsaswellasidentifydifferences(seee.g.Maibohmetal.2015b)whichcanbeexploitedforapplicationpurposes.

Structuralpropertiesofthediatomfrustule

Diatomscanroughlybedividedintotwodifferentgroupsbasedontheirvalvesymmetry,thecentrics(radiallysymmetrical)andthepennates(bilaterallysymmetrical).Forbothforms,thevalveisavery

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complex3Dstructure,asseeninFig.1andingraphenereproductions(Panetal.2014).ThesizefeaturesofnanopatternofthevalvevaryfromspeciestospeciesandareintheorderofwavelengthsofUV-Ctonearinfraredlight.Thisincludesphotosyntheticallyactiveradiation(PAR)(400-700nm),indicatingthatPARlightwillbestronglyinfluencedwhenitinteractswiththefrustule.ThisisseenwhenlightinthePARwavelengthrangeistransmittedthroughthefrustule;theporestructureinteractswiththelightandcreatesawavelengthdependentinterferencepattern(DeTommasietal.2010;Maibohmetal.2015a).Thestructurealsointeractswiththelightinawaythatitredirectslighttowardsthecellinteriorevenwhenlightisstrikingthefrustuleatanotheranglethannormalincidence(Maibohmetal.2015c).

Materialpropertiesofthediatomfrustule

Thefrustulematerialispredominantlysilicaprecipitatedbydiatomsfromsilicicacidwithspecies-specificsizeandpatternofholes.ThespecificsilicastructureofthefrustuleisusualcharacterizedasamorphoushydratedsilicabuttherehavealsobeenindicationsfromX-raydiffractionstudiesthatpartofthefrustulecouldbecomposedofα-quartzwithagrainsizeoffewtenthsofmicrometers(Goswamietal.2012).Inadditiontothis,traceimpuritiessuchasaluminum,magnesium,ironandtitaniumcanbepartofthefrustulematerial(Butcheretal.2005).Thesetraceimpuritiesaretakenupfromthewaterenvironmentandincorporatedintothefrustulebythediatomcell.

Simulationoflightinteractionwiththefrustulestructure

ThetransmissionoflightthroughadiatomvalvecanbesimulatedusingMaxwell’sequations,whichgovernthepropagatingelectromagneticfield.Ageometricalopticsapproachisinsufficientbecause,asdescribedabove,thestructuresofthevalveareonthesamescaleasthewavelengthofvisibleandUVlight;hence,diffractionplaysanimportantrole,andsomeinterferencepatternsarereadilyobservedalreadyunderanormallightmicroscope(Fig.2b,c).Accuratenumericalmodellingofthethree-dimensionallystructuredvalverequiresmanycomputationalresourcessoitisdesirabletosimplifythemodelofthevalveandfocusonfewofitsfeatures.Forsuchsimulations,thevalveisgenerallyassumedtobeaplaneglasssurfacewithapatternofairholes,whichresemblesthelargerholesononesideofthevalveasseeninFig.2b.Somestudieshavealsomodelledtheotherplaneofthevalve,eitherincludingthemoreintricateholestructure(DeTommasietal.2010),orasahomogeneoussurfaceabsentofholes(Yamanakaetal.2008).Intheliterature,twodifferentschemesofsimulationapproacheshavebeenpursued:oneconsidersthetransmissionoflightthroughtheplaneofthevalvewherelightpassesthroughtheholes,andthesilicawallsareconsideredopaqueorpartlytransparent;theedgesoftheholesareconsequentlyresponsiblefordiffractionofthepropagatingbeam.ThetheorybehindthisandanumericalalgorithmtocarryoutthisapproachwasdescribedbyDelenetal.(1998).Theaimofthisapproachistoexplainthelens-lesslightfocusingobservedbyDeStefanoetal.(2007a),whereitwasfoundthatthearrayoflargerholesinthevalveplaysacriticalroleinunderstandingthecomplexdiffractionpatternbehindthevalve;thesmallerholesontheotherplaneofthevalve(inthecribrum)wereignoredinthatmodel.Later,thesameapproach,includingthesmallerholeshowever,wasusedtopredictthat,forvisiblelight,multiplefocuspointsarefoundascloseas100-200µmfromtheplaneofthevalve,butthatnosuchfocusingisfoundforshorterwavelengthsintheUVregime(DeTommasietal.2010).Whilethelatterpredictionremainsunconfirmedbyexperimentalinvestigations,arecentstudyshowedexcellentagreementbetweenexperimentaldataandtheoryandwithoutthesmallerholesinthevisibleregime(Maibohmetal.2015a),thusvalidatingthisnumericalapproach.

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TheothercommonapproachfoundintheliteratureisthepropagationoflightintheplaneofthevalveasfirstpresentedbyFuhrmannetal.(2004);itisshownthatthevalvemayactasawaveguideinwhichtheopticalpropertiesaredeterminedbytheperiodicstructureofthelargeairholesinthedirectionofpropagation.Suchastructuremayleadtophotonicbandgaps(PBG;seelater)dependingontheparametersofthespecificstructureandtherefractiveindexcontrastbetweenthevalvematerialandthematerialsintheholes(airorwater).ThestudybyFuhrmannetal.(2004)andalaterstudybyKieuetal.(2014)shownoPBGforaccuratemodelsofdiatomfrustules.Ontheotherhand,ithasbeenshownthatiftherefractiveindexofthefrustuleisincreasedto2.4(whichmayberealizedbymaterialsubstitutionasmentionedbelow)afullPBGisfoundatawavelengthof2.2µm(DeStefanoetal.2009).Thissuggeststhatthefrustulemayfinduseinindustryasanewopticalchemicalsensor(DeStefanoetal.2009).

BesidesthediscoveryofPBGs,thestudyofphotonicbandsgivesmoreinformationabouttheinteractionoflightwiththediatomfrustule:inanotherstudy,strongabsorptionwasfoundintheblueregionofvisiblelightandpartoftheUVAregionbytransmittinglightthroughafrustule(Yamanakaetal.2008).Simulationsofthephotonicbandsofanaccuratemodelofthefrustuleshowedthatastronginteractionbetweenthefrustuleandlightofwavelengthsintheshorterpartofthevisiblespectrum.

Photoluminescence

Poroussilica,oneofthemainconstituentfrustulematerials,isknowntoemitphotoluminescence(PL)inthevisiblespectralregionwhenirradiatedwithUV-light(Cullisetal.1997).ThediatomfrustulescanalsoexhibitPLwithoneormorespectralpeakswhichcanbemoreorlesspronounced(Butcheretal.2003,2005;deStefanoetal.2007b;Mazumderetal.2010;Vijietal.2014).Thusresearchershaveforexcitationusedwavelengthsof325nm,withemissionat539.4nm(deStefanoetal.2009)and300,370and380–allwithemissionat440nm(Mazumderetal.2010;Vijietal.2014).ExperimentsshowgreatdifferencesinPLactivityamongdiatomspeciesandevenamongdiatomsfromsametheculture,fromnoPLactivitytointensitiesmeasurablewiththenakedeye(Butcher2005;Maibohmetal.2015c).Ourownexperimentswithcultivateddiatoms,fromthesameculture,showlowPLactivityorsometimesnoPLatall;underliningthattheuptakeofmineralsfromthesurroundingsandotherinfluencingparameterstothePLactivityareofimportanceontheindividualcelllevel(Maibohmetal.2015c).Forapplicationpurposesthelocalenvironmentofthecleanedfrustuleshasahugeinfluence.Anoxygen-containingatmosphereworksasafastquencherofPLactivitywhileacontrolledatmosphere,asinaultrahighvacuumsystem,thefrustulePLactivityhasbeenshowntoactasagassensorbybindingvarioustypesofgassesandtherebyeitherquenchorenhancethePLactivity(deStefanoetal.2007b).ThepositionandshapeofthePLspectrumisdependentontheUVexcitationwavelength,andthepresenceofmultiplepeaks,i.e.multiplede-excitationpathways,isattributedtonon-uniformcrystallitesize,trappingstatesandimpuritiesinthefrustules(Goswamietal.2012).ThePLspectrumisdominatedbypeaksfromtheamorphoussilica,butitisalsoaffectedbythesmallamountsoftraceimpuritiesinthefrustule.Uptakeoftraceelementswillchangetheabsorptionandtransmissioncharacteristicsofthefrustuleagain,thedegreedependingontheelementandtheconcentrationofit.Thenaturalamountoftraceimpuritiesvaries,dependingonthehabitat,andthetraceimpuritiescaneitherbedepositedinthefrustulebythecelloradsorbedtothefrustuledirectlyfromtheenvironment(deJongeetal.2010).ThePLcanalsobeaffectedbyadsorptionofmaterialwhichquenches,enhancesoraltersthePLspectra(deStefanoetal.2007b;Galeetal.2009;Vijietal.2014).ChangesinPLspectrahavebeenusedasanoveltypeofgassensorwherebindingabsorptionofmolecules

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onthefrustulesleadstoameasureablechange(deStefanoetal.2007b).Metaldopedfrustulescanalsobestimulatedbyanelectriccurrentandsubsequentlyemitelectroluminescence(Jeffryesetal.2008).

Photonicbandgaps,wave-guidingandBraggscattering

Thesethreephotonicphenomenaareofcentralrelevanceandinterestforstudyingdiatomfrustules,particularlywithregardtothepossibilityforusingdiatomfrustulestoselectivelyscreenoutspecificwavelengthsoflight.

Photoniccrystalsareperiodicstructuresthatphotonsofcertainwavelengthscannotpropagatethrough.Thenon-propagatingwavelengthorusualthebandwidthofnon-propagatingwavelengthsconstitutestheso-calledphotonicbandgap(PBG).FormationofaPBGinastructurecanbeviewedasthesynergeticinterplaybetweenthemacroscopicBraggscatteringresonance(whichstemsfromtheperiodicstructureofalternatingmaterialandcreatesstopgapswhentheperiodofthestructurecoincideswithanintegernumberofhalfthewavelengthofthelight)andthemicroscopicscatteringresonance,whichstemsfromthesingleunitcellofthestructure,calledtheBrillouinzoneandisrelatedtohowthestructureisordered.Inthecaseofthediatomfrustule,themacroscopicscatteringwouldbefromtheholes(consistingofwateror,whencleaned,air)alternatingwiththesilicawalls.Themicroscopicscatteringwouldbefromhowtheholesarearrangedinthefrustule,forinstanceahexagonalstructure.IfboththemacroscopicandmicroscopicresonanceoccursatthesamewavelengthformationofaPBGisgreatlyenhanced.Thewavelengthorratherthebandwidthoverwhichlightisnottransmitteddependsalsoonthedifferenceinrefractiveindexbetweenthetwomaterialsofthemicrostructure.Ingeneral,thelargerthedifferenceinrefractiveindex,theeasieritistoobtainaPBG.ThecenterwavelengthandthebandwidthofthePBGarethereforedeterminedbythemacroscopicperiodicity(i.e.thedistancebetweentheholesandthesizeoftheholes),microscopicsymmetry(howtheholesarearrangedcomparedtoeachother),materialcompositionandpolarizationofthelight.Thepropagationdirectionofthelightintheabovesectionispresumedtobeintheplaneofthealternatingstructuresandthereforelightneedstobecoupledto(orredirectedto)thestructureinthisplane.Thecomplexityofthephotoniccrystal,andtherebytherelationbetweenthebeforementionedparametersincreaseswiththeneedofmultiplepropagationdirectionswhichincludesaPBG.WavelengthsincludedinthePBGandinthepropagationdirectionofthePBGisreflectedfromthestructureandmanyapplicationsbenefitfromthestrongwavelength-dependentpropagationoflight.Manyoftheseapplicationsdonotevenrequirethattheperiodicallymodulatedmaterialexhibitallpropertiesofphotoniccrystalsbutonlythat,lightisreflecteddifferentlydependingonitswavelength,i.e.themacroscopicBraggscattering.Thiseffectcanbeseeninthecolorsofcertainbutterflieswherethescatteringiswavelengthdependentandthereforethespectralresponsealsodependsontheobserver.

Diatomwallshaverepeatednanostructures(Figs1,2b)intherelevantsizerangeofPARandUVlightandhavebeenshowntohavecertaincharacteristicsofphotoniccrystals.Theorderedstructureoftheholeshasindicatedthepossibilityofphotonicbandgapswhenlightispropagatinginthevalveplane.Thishasbeencalculatedforbothvalvesandgirdlebandsbuthasneverbeenshownexperimentally(Fuhrmannetal.2004;Yamanakaetal.2008).IftheaimistopreventtheshortwaveUV-light(280-380nm)fromenteringthecell,diatomwallswithaperiodicityofholesoftheorderof200-250nmmaybeexpectedtodothiswhileallowingvisiblelighttopassthrough.However,calculationsshowthattherefractiveindexcontrastbetweensilica(1.45)andeitherwater(1.33)orair(1)togetherwithperiodicity,symmetryand

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polarizationisnotenoughtoformafullPBGinthefrustule.Furthermore,therefractiveindexcontrastbetweenwaterandthefrustuleinnaturalenvironmentofdiatomsisalsotoolowforefficientBraggscatteringbuttheperiodicstructureisstillshowntohaveastrongabsorptioninthebluespectralregion(Fuhrmannetal.2004;Yamanakaetal.2008).Whenacleanedfrustuleisilluminatedinair,therefractiveindexcontrastislargeenoughforefficientBraggscatteringandatleastpartialphotonicbandgapsoccur.Thisisseenintransmissionmeasurementsandcalculationswherespecificwavelengthsareattenuatedmorethanothers(Fuhrmannetal.2004;Yamanakaetal.2008;Kieuetal.2014).

Inexperimentalmeasurementswheretheincidentlightstrikesthefrustulenormaltothesurface,onlyaweakcouplingoflighttothevalveplaneoccursandtheindexcontrastoftheperiodicstructuregivingrisetoasmalleffect.Furthermore,onlythedirecttransmittedandscatteredoutofplanecontributionofthelightfromthefrustuleisobservedandnotthelightguidedinthevalveplane,makingitdifficulttoprobethetruephotonicbandgapstructureofthefrustule.Iftheangleofincidenceisvariedfromnormal,awavelengthdependentredirectionofthelighttowardsthecellinteriorisobserved.Thisindicatesastronginteractionbetweenthelightandthefrustuleindicatingastrongeffectoftheperiodicstructure(Romannetal.2015,Maibohmetal.2015c).Tofurtherincreasetherefractiveindexcontrastandtherebyenhancetheinteractioneffectbetweenlightandthefrustule,silicacanbesubstitutedinthegrowthmediumwithhighlevelsofthetracesubstances(Townleyetal.2007;Jeffryesetal,2008;Langetal.2013).Duetothefactthateventhetraceamountofimpuritiesmayinfluencephotoniccharacteristicsofthefrustule,cultivationconditionsmustbecontrolledtoensurereproducibleproductsforindustrialapplications.Oritcanbedoneby3Dstructuralpreservingchemicalsubstitutionafterremovaloforganicmaterialinthecleaningprocess(Sandhageetal.2002;Baoetal.2009;vanEyndeetal.2013).Tofurtherquantifyandunderstandthelight-frustuleinteraction,itsimplicationonbiologicalfunctionsofthediatomandthepossibleindustrialapplications,moreadvancedphotonicstudieshavetobeconducted.

TestsoftheeffectofthefrustuleonUVlight

Wehavefoundthatfourofthestudiesaimedatdeterminingpotentialindustrialapplicationsofdiatomfrustuleshaveanalyzedormodelledtheeffectofthefrustulesilicaandnano-patternonUVlight,experimentallyinvestigatingatotalofthreecentricspeciesaswellasperformingsimulations.InastudyontheopticalpropertiesofthecentricdiatomArachnoidiscussp.,Ferreraetal.(2014)simulatedthefocusingeffectofafrustuleinaironUV-lightof300nmandfoundthatthefocusingspotsatthiswavelengthstartedca.900µmfromthevalve,incontrasttofocusingspotsofPARlight(640nm),whichstartedatca.200µm.TheyalsomeasuredtransmittanceintensityprofileatUVBandfoundthespotintensitylowanddistancefarfromthevalve.Inapreviousstudy(onCoscinodiscuswailesii;DeTomassietal.2010)theyshowedbysimulationthatinwaterorcytoplasm,thefocusingspotsforPARwouldbeclosertothevalve.DeTomassietal.(2010)furthertestedtheeffectofavalveofCoscinodiscuswailesiionlightat280nm,andshowedthat,incontrasttolightatPARwavelengthsandabove(532nm,633nm&1000nm),thevalveformednofocusingspotsatthetestedUVwavelength.Goswamietal.(2010)studiedUV-luminescencepropertiesofCyclotellameneghiniana.InaUV-visiblespectroscopyanalysisofasinglevalvetheyfoundpeakabsorbanceat274nm.DeStefanoetal(2007b)simulatedUVshieldingpropertiesofpennateandcentricdiatomsbydigitalizingscanningelectronmicrographsoftwodifferentfrustules(fromThallassiosirarotulaorCoscinodiscuswailesiiandCocconeisscutellum).Davidsonetal.(1994)studiedtheeffectofUV-BirradiationongrowthandsurvivalofsixdifferentAntarcticdiatoms(Nitzschialecointei,N.curta,Proboscisalata,

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Thallasiosiratumida,Odentellaweisflogii,Chaetocerossimplex).Theirfocuswasthusonthebiologyofthediatoms(seediscussionabove)ratherthanonpotentialindustrialapplications;however,oneoftheparameterswhichtheyanalyzedwasabsorptionspectraoftherinsedvalves.Theyfoundthatgenerallyforallsixspeciesabsorbancefellalongthespectrum250-800nm.Theycalculatedthat,oftotalUV-Babsorbance,ca.20%wasduetothefrustule.Thiscould,however,beduestrictlytothematerialpropertiesofthesiliceousfrustuleandnottotheporestructure.MostofthesestudiesweredoneoncleanedfrustulesinairwheretheindexcontrastisgreaterthatinwaterandthereforethereispotentialforstrongerBragg-scattering.Therefore,asdiscussedpreviously,itcanbedifficulttoextrapolatetheseresultstoUV-protectionforlivediatomsinawateryenvironment.

TestsofUVandPARspectraofsinglefrustulesandlayersoffrustulesandwholecells

Experimentsaimedattesting,forpotentialapplications,methodsforcoatingasurfacewithbothrinsedfrustulesandcellsdriedwithcellcontentwerecarriedouttoexploreifthetransparencywasdifferentforUVandvisiblewavelengthswhendiatomswerearbitrarilydriedontoaglassplate(Fig.3).

LighttransmissionwasmeasuredfortwospeciesofCoscinodiscus:C.graniiandC.concinnus.Bothdriedintactdiatomswithcellcontents(dead,butwithremnantsofcellcontents)anddiatomvalvesrinsedoforganicmaterialwereused.ThediatomsweredrippedontoaUV-transparentquartzsilicamicroscopeglassplateanddriedatroomtemperature.Theprocesswasrepeatedseveraltimestoincreasetheconcentration.Inspiteofthis,wedidnotalwaysachieveahighdegreeofcoverageontheglassplate.TransparencyspectraweremeasuredusingaShimadzuUV-2600spectrometerwithbuild-inlightsourcesandintegratingsphere.MicroscopepicturesweretakenusinganOlympusGX41lightmicroscope.Allmeasurementswererepeatedatleasttwice.

AllsampleshadthehighesttransmissionforthelongestwavelengthsandthelowestintheshortwaveUV-spectrumasshowninFig.4.ForrinseddriedfrustulesofC.concinnusthetransmittancewasreducedfromca.67%toca.60%inthevisiblepartofthespectrumfrom700-400nm.IntheUV-Bregion(from320-280nm)thereductionwasfromca.57%toca.53%,i.e.theslopeofthetransmittancereductionwassteeperintheUV-Bregion.AsimilartendencywasseenforC.granii,buttheconcentrationoffrustuleswaslowandthereforethesignalwaslesspronounced.Forthedrieddiatomswithcellcontents,thereductionintransparencyissteeperandstartsat500nm.ForC.granii,thereductionwithintheUV-AandUV-Bspectrum(400-280nm)isfrom14to3%.ForC.concinnus,thereductionintheUV-spectrumisfrom24to9%.Thetransmissionvariedforthedifferentsamplesdependingontheconcentrationofthediatoms.Fortherinseddiatomfrustulestheconcentrationvariedbetween45and85%andforthedriedlivingdiatomsitwas85-95%asshownintable1andillustratedinFig.3.

ThesepreliminaryresultsindicatethatthevalvesaremoretransparenttolightinthevisiblespectrumthanintheUVspectrum,orinotherwordsthatthevalvesfiltersomeoftheUVlight.Thistendencybecomesmorepronouncedtheshorterthewavelength.Forthecleanedvalves,thefilteringeffectintheUVspectrumisseeminglyrathersmall(below10%).However,thiseffectislikelytobecomelargerifacompletecoverageisachieved.Inthemeasuredsamplesonlyabout45-95%ofthesurfaceareawascovered.Toestimatethesizeoftheexpectedspectraltransmissionforacompletecoveragethedatawasnormalizedtothedegreeofcoverageonthequartzmicroscopeglassbymultiplyingwiththefoundcoveragepercentage.TheresultingcurvesinFig.5showthattransparencyishigherforC.concinnus

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comparedtoC.graniiforcleanedfrustulesandfordiatomsdriedwithcontent.C.concinnusalsohasarelativelylowertransparenceintheUV-area.ThetransparencylevelforC.graniiwas30-40%(lowestfortheshortestwavelengths)whichwassimilartothetransmissionobservedthroughasinglevalvedrop-castedonquartz(Fig.6).ThelightbeamwasinthiscasemaskedwithapinholetoonlyallowlightthroughasinglefrustuleasshowninFig.1a.

Formanypotentialindustrialapplicationsitwillbenecessarytoincorporatethediatomsintosomekindofamatrix,e.g.paintorcreme.Thisraisesquestionsofhowtoachievethis,howhighadegreeofcoverageisnecessaryforthedifferentapplicationsandwhetherthelayermustbeuniformand/orflat.

Forthediatomsdriedintactwithcellcontent,amuchmorepronouncedfilteringcanbeobserved.ForC.graniionly3%ofshortwaveUV-Bat280nmisallowedthroughthesampleincontrasttothe28%transmittanceforred700nmlight.Inadditiontothepresenceofcellcontents,anotherdifferencebetweenthetwotreatmentsisthatinthecaseofthediatomsdriedintactwithcellcontents,thefrustuleisintactwiththetwovalvesstilltogether.Thismeansthatthelightwillalwaysstrikethesideofthevalvefacingtotheoutsideofthecell,andasmentionedpreviously,thereappeartobedifferencesinthephotonicpropertiesdependingonwhichsideofthevalveisfacingtowardsthelight.

ThetendencyinthesefindingsisinconcordancewiththefindingsbyNoyesandcolleagues(2008).TheymeasuredlighttransparencyinthediatomC.wailesiiandfoundapproximately80%forredlight(633nm),30%forgreenlight(543nm)and20%forbluelight(472nm).Theyexplainthevaryingdegreeoftransparencywithdiffractionintheholestructureandpointoutthatitisthelargeholesontheinsideofthevalvethatareresponsibleforthediffraction.Similarly,asstatedpreviously,Goswamietal.(2010)foundpeakabsorbanceofthefrustuleofCyclotellameneghinianaat274nmandDavidsonetal.(1994)foundthatthefrustulesofsixdifferentspeciesaccountedforca.20%ofthetotalUVabsorbance.

Summary/Conclusions

Ouroverallconclusionisthat,whileonlyafewspecieshavebeentestedsofar,theresearchintotheroleofdiatomfrustulesinUVprotectionshowgreatpotential,bothwithregardtodiatombiologyandpotentialapplications.

- LessthantendiatomspecieshavebeentestedforeffectsofthefrustuleonUVlight.- Studiesshowthatthefrustuleaccountsforupto20%reflectance/absorbanceintheUVspectrum- Lightfocusingdistancedependsonthewavelength,withlongerdistancesforUVlight.Thiscould,

however,beveryspeciesdependent,becauseofthedifferencesinperiodicstructuresbetweenspecies.

- Therearetwogeneralapproachestomodelingphotoniceffectsofdiatomfrustules.Oneconsidersthetransmissionoflightthroughtheplaneofthevalvewithlightpassingthroughtheholes,andthesilicawallsmoreorlessopaque.Theotherconsidersthepropagationoflightintheplaneofthevalveandfocussesontheperiodicstructureofthelargerholes.

- PhotoluminescenceeffectsareseenwhenilluminatingfrustuleswithUVlight.Thesearestrongestundervacuumconditions.

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- Theabilitytocompletelyexcludelightofcertainwavelengths(PBGs)dependsontherefractiveindexbetweenthefrustuleandthematrixaswellastheperiodicityofthenanostructure.SofarcompletePGBshavenotbeenexperimentallyshownindiatoms.

- LivingdiatomshaveamorevariableresponsetoUVlightthanothergroupsofalgae–thiscouldbeduetodifferencesinUVpropertiesofthefrustule

- ImportantelementstoconsiderforUV-mediatingeffectsare:theperiodicityofthelargeholes(areolae),therefractivecontrastbetweenthefrustuleandthemedium,theorientationofthevalves,thedegreeofcoverageofalayeroffrustules.Thesmallholes(inthecribrum;seefigure1)andcentralareaappeartobelessimportant.

Acknowledgements

ThisworkwasfundedbyTheDanishResearchCouncil(projectALPHA12-127569).SandraWalbyhelpedwithlaboratoryworkandJacobSnebjørnBrøggerKristensendidpreliminarystudiesontransmissionthroughaspin-coatedlayeroffrustules.TomasBenzonhelpedwithgraphicsinfigure2.Threeanonymouseviewersarethankedfortheircommentsthathelpedimprovethemanuscript.

References

1. ArmbrustEV(2009)Thelifeofdiatomsintheworld´soceans.Nature459:185-1922. BaoZ,ErnstEM.YooS,SandhageKH(2009)SynthesesofPorousSelf-SupportingMetal-Nanoparticle

Assemblieswith3DMorphologiesInheritedfromBiosilicaTemplates(DiatomFrustules).AdvMater21:474-478

3. BozarthA,MaierU-G,ZaunerS(2009)Diatomsinbiotechnology:moderntoolsandapplications.ApplMicrobiolBiotechnol82:195–201

4. BumaAGJ,vanHannenEJ,VeldhuisMJW,RozaL,GieskesWWC(1995)MonitoringUV-BinducedDNAdamageinindividualdiatomcellsbyimmunofluorescentthyminedimerdetection.JPhycol31:314-321

5. BumaAGJ,ZemmelinkHJ,SjollemaK,GieskesWWC(1996)UVBradiationmodifiesproteinandphotosyntheticpigmentcontent,volumeandultrastructureofmarinediatoms.MarEcolProgSer142:47-54

6. ButcherKSA,FerrisJMPhillipsMR.(2003)Photoluminescenceandcathodoluminescencestudiesofdiatoms—nature'sownnano-poroussilicastructures.In:CashionJ,FinlaysonT,PaganinD,SmithA,TroupG(Eds.)Proceedingsofthe27thAandNZCondensedMatterandMaterialsMeeting,4–7February,CharlesSturtUniversity,WaggaWagga,NSW,p.51

7. ButcherKSA,FerrisJM,PhillipsMR,Wintrebert-FouquetM,JongWahJW,JovanovicN,VyvermanW,ChepurnovVA(2005)Aluminescencestudyofporousdiatoms.MaterSciEngC25:658-663

8. ColemanEA(2011)Polymeradditives.In:KutzM(ed)AppliedPlasticsEngineeringHandbook,WilliamAndrew,pp.419-428.doi:10.1016/b978-1-4377-3514-7.10021-2

9. CullenJJ,LesserMP(1991)Inhibitionofphotosynthesisbyultraviolet-radiationasafunctionofdoseanddosagerate–resultsforamarinediatom.MarBiol111:183-190.

10. CullisAG,CanhamLT,CalcottPDJ(1997)Thestructuralandluminescencepropertiesofporoussilicon.JApplPhysics82:909

11. DavidsonAT,BramichD,MarchantHJ,McMinnA(1994)EffectsofUV-BirradiationongrowthandsurvivalofAntarcticmarinediatoms.MarBiol199:507-515

12. DelenN,HookerB(1998)Free-spacebeampropagationbetweenarbitrarilyorientedplanesbasedonfulldiffractiontheory:afastFouriertransformapproach.JOptSocAmA15:857-867

14

13. DepauwFA,RogatoA,d'AlcalaMR,FalciatoreA(2012)Exploringthemolecularbasisofresponsestolightinmarinediatoms.JExpBot63:1575-1591

14. DöhlerG(1995)ImpactofUV-AandUV-BirradianceonthepatternsofpigmentsandN-15-ammoniumassimilationofthetropicalmarinediatomBellerocheayucatanensis.BotMar38:513-518.

15. DöhlerG(1984)EffectofUV-BRadiationontheMarineDiatomsLauderiaannulataandThalassiosirarotulaGrowninDifferentSalinities.MarBiol83:247-253

16. vanEyndeE,TytgatT,SmitsM,VerbruggenSW,HauchecorneB,LenaertsS(2013)Biotemplateddiatomsilica–titaniamaterialsforairpurification.PhotochemPhotobiolSci12:690-695

17. FerraraMA,DardanoP,DeStefanoL,ReaI,CoppolaG,RendinaI,CongestriA,AntonucciA,DeStefanoM,DeTommasiE(2014)OpticalPropertiesofDiatomNanostructuredBiosilicainArachnoidiscussp:Micro-OpticsfromMotherNature.PLOSONE9DOI:0103750

18. FinkelZV,KotrcB(2010)Silicausethroughtime:Macroevolutionarychangeinthemorphologyofthediatomfrustule.GeomicrobiolJ27:596-608

19. FuhrmannT,LandwehrS,ElRharbi-KuckiM,SumperM(2004)Diatomsaslivingphotoniccrystals.ApplPhysB78:257–260

20. GaleDK,GutuT,JiaoJ,ChangCH,RorrerGL(2009)PhotoluminescenceDetectionofBiomoleculesbyAntibody-FunctionalizedDiatomBiosilica.AdvFunctMater19:926–933

21. GijsmanP(2011).PolymerStabilization.InKutzM(ed)AppliedPlasticsEngineeringHandbook,WilliamAndrew,pp.375–399.doi:10.1016/b978-1-4377-3514-7.10021-2

22. GuihéneufF,FouquerayM,MimouniV,UlmannL,JacquetteB,TremblinG(2010)EffectofUV-stressonthefattyacidandlipidclasscompositionintwomarinemicroalgaePavlovalutheri(Pavlovophyceae)andOdontellaaurita(Bacillariophyceae).JApplPhycol22:629-638

23. GordonR,LosicD,TiffanyMA,NagySS,SterrenburgFA(2009)TheGlassMenagerie:diatomsfornovelapplicationsinnanotechnology.TrendsBiotechnol27:116-27

24. GoswamiB,ChoudhuryA,BuragohainAK(2012)Luminescencepropertiesofananoporousfreshwaterdiatom.Luminescence27:16-19

25. GuiryMD(2012)Howmanyspeciesofalgaearethere?JPhycol48:1057–106326. HammCE,MerkelR,SpringerO,JurkojkP,MaierC,PrechtelK,SmetacekV(2003)Architectureand

materialpropertiesofdiatomshellsprovideeffectivemechanicalprotection.Nature421:841-84327. HargravesPE,ZhangJ,WangR,ShimizY(1993)GrowthcharacteristicsofthediatomsPseudonitzschia

pungensandP.fraudulentaexposedtoultravioletradiation.Hydrobiol269:207-21228. HelblingEW,ChalkerBE,DunlapWC,Holm-HansenO,VillafañeVE(1996)Photoacclimationof

Antarcticmarinediatomstosolarultravioletradiation.JExpMarBiolEcol204:85–10129. HempelF,BozarthAS,LindenkampN,KlinglA,ZaunerS,LinneU,SteinbüchelA,MaierUG(2011)

Microalgaeasbioreactorsforbioplasticproduction.MicrobCellFact10:8130. HessenDO,FrigstadH,FaerovigPJ,WojewodzicMW,LeuE(2012)UVradiationanditseffectsonP-

uptakeinarcticdiatoms.JExpMarBiolEcol411:45-5131. HolzingerA,LützC(2006)AlgaeandUVradiation:Effectsonultrastructureandrelatedmetabolic

functions.Micron37:190-20732. IngallsAE,WhiteheadK,BridouxMC(2010)Tintedwindows:ThepresenceoftheUVabsorbing

compoundscalledmycosporine-likeaminoacidsembeddedinthefrustulesofmarinediatoms.GeochimCosmochimActa74:104–115

33. JeffryesC,GutuT,JiaoJ,RorrerGL(2008)MetabolicInsertionofNanostructuredTiO2intothePatternedBiosilicaoftheDiatomPinnulariasp.byaTwo-StageBioreactorCultivationProcess,ACSnano2:2103-2112

34. deJongeMD,HolznerC,BainesSB,TwiningBS,IgnatyevK,DiazJ,HowardDL,LegniniD,MiceliA,McNultyI,JacobsenCJ,VogtS(2010)Quantitative3DelementalmicrotomographyofCyclotellameneghinianaat400-nmresolution.PNAS107:15676–15680

15

35. Johnsen,S.,Sosik,H.(2004)Sheddinglightonthelightintheocean.OceanusMagazine43:36. KarentzD,CleaverJE,MitchellDL(1991)Cellsurvivalcharacteristicsandmolecularresponsesof

Antarcticphytoplanktontoultraviolet-Bradiation.JPhycol27:326-34137. KieuK,LiC,FangY,CohoonG,HerreraOD,HildebrandM,SandhageKH,NorwoodRA(2014)

Structure-basedopticalfilteringbythesilicamicroshellofthecentricmarinediatomCoscinodiscuswailesii.OptExpress22:15992-15999

38. LeuE,WangbergSA,WulffA,Falk-PetersenS,OrbaekJB,HessenDO(2006)EffectsofchangesinambientPARandUVradiationonthenutritionalqualityofanArcticdiatom(Thalassiosiraantareticavar.borealis).JExpMarBiolEcol337:65-81

39. LangY,delMonteF,RodriguezBJ,DockeryP,FinnDP,PanditA(2013)IntegrationofTiO2intothediatomThalassiosiraweissflogiiduringfrustulesynthesis.SciRep3,articlenumber3205.

40. LavoieM,RavenJA,LevassaurM(2016)Energycostandputativebenefitsofcellularmechanismsmodulatingbuoyancyinaflagellatemarinephytoplankton.JPhycol52:239-251.

41. LevithanO,DinamarcaJ,HochmanG,FalkowskiPG(2014)Diatoms:afossilfuelofthefuture.TrendsBiotechnol32:117-124

42. LitchmanE,NealePJ(2005).UVeffectsonphotosynthesis,growthandacclimationofanestuarinediatomandcryptomonad.MarEcolProgSer300:53-62.

43. LohmannM,DöhlerG,HuckenbeckN,VerdiniS(1998)EffectsofUVradiationofdifferentwavebandsonpigmentation,15N-ammoniumuptake,aminoacidpoolsandadenylatecontentsofmarinediatoms.MarBiol130:501-507

44. MazumderN,GogoiR,KalitaRD,AhmedGA,BuragohainAK(2010)Luminescencestudiesoffreshwaterdiatomfrustules.IndianJPhys84:665-669

45. MaibohmC,FriisSMM,EllegaardM,RottwittK(2015a)InterferencepatternsandextinctionratioofthediatomCoscinodiscusgranii.OptExpress23:DOI:10.1364/OE.23.009543

46. MaibohmC,FriisSMM,SuY,RottwittK(2015b).Comparingopticalpropertiesofdifferentspeciesofdiatoms.ProcofSPIEVol.936093600B-1

47. MaibohmC,NielsenJH,RottwittK(2015c)Lightinteractionwithnano-structureddiatomfrustule,fromUV-AtoNIR.MSRadvanceshttp://dx.doi.org/10.1557/adv.2015.15

48. MedlinLK(2002)Whysilicaorbetteryetwhynotsilica?Speculationsastowhythediatomsutilizesilicaastheircellwallmaterial.DiatomRes17:453-459

49. MilliganAJ,MorelFMM(2002)Aprotonbufferingroleforsilicaindiatoms.Science297:1848-185050. NahonS,CharlesF,LantoineF,VétionG,EscoubeyrouK,DesmaladesM,PruskiAM(2010)Ultraviolet

radiationnegativelyaffectsgrowthandfoodqualityofthepelagicdiatomSkeletonemacostatum.JExpMarBiolEcol383:164-170

51. NelsonDM,TreguerP,BrzezinskiMA,LeynaertA,QueguinerB(1995)Productionanddissolutionofbiogenicsilicainocean–revisedglobalestimates,comparisonwithregionaldataandrelationshiptobiogenicsedimentation.GlobalBiogeochemCyc9:359–372

52. NoyesJ,SumperM,VukusicP(2008)Lightmanipulationinamarinediatom.JMatRes23:3229-323553. OttesenP.(2011)Processingandcharacterizationofdiatomsforlightharvestingmaterialsonsolar

cells.Mastersthesis,NorwegianUniversityofScienceandTechnology54. PanZ,LerchSJL,XuL,LiX,ChuangY-J,HoweJY,MahurinSM,DaiS,HildebrandM(2014)

Electronicallytransparentgraphenereplicasofdiatoms:anewtechniquefortheinvestigationoffrustulemorphology.SciRep4:6117

55. ParkerAR,TownleyHE(2007)Biomimeticsofphotonicnanostructures.NatureNanotechnol2:347-5356. ParkinsonJ,GordonR(1999)Beyondmicromachining:thepotentialofdiatoms.TrendsBiotechnol17:

190-19657. RavenJA(1983)Thetransportandfunctionofsiliconinplants.BiolRev58:179–207

16

58. RavenJA,WaiteAM(2004)Theevolutionofsilicificationindiatoms:inescapablesinkingandsinkingasescape?NewPhytol162:45-61

59. RechM,MougetJL,Morant-ManceauA,RosaP,TremblinG(2005)Long-termacclimationtoUVradiation:effectsongrowth,photosynthesisandcarbonicanhydraseactivityinmarinediatoms.BotMar48:407-420

60. ReizopoulouS,SantasP,DanielidisD,HaderDP,SantasR(2000)UVeffectsoninvertebrateanddiatomassemblagesofGreece.JPhotochemPhotobiolB56:172-180

61. RijstenbilJW(2005)UV-andsalinity-inducedoxidativeeffectsinthemarinediatomCylindrothecaclosteriumduringsimulatedemersion.MarBiol147:1063–1073

62. RomannJ,ValmaletteJ-C,RøysetA,EinarsrudM-A(2015)Opticalpropertiesofsinglediatomfrustulerevealedbyconfocalmicrospectroscopy.OptLett40:740-3

63. SandhageKH,DickersonMB,HusemanPM,CarannaMA,CliftonJD,BullTA,HeibelTJ,OvertonWR,SchoenwaelderMEA(2002)Novel,BioclasticRoutetoSelf-Assembled,3D,ChemicallyTailoredMeso/Nanostructures:Shape-PreservingReactiveConversionofBiosilica(Diatom)Microshells.AdvMat14:429–433

64. ScholzB,RuaA,LiebezeitG(2014)EffectsofUVradiationonfivemarinemicrophytobenthicWaddenseadiatomsisolatedfromtheSolthorntidalflat(LowerSaxony,southernNorthSea)-PartI:growthandantioxidativedefencestrategies.EurJPhycol49:68-82

65. DeStefanoL,ReaI,RendinaI,DeStefanoM,MorettiL(2007a)LenslesslightfocusingwiththecentricmarinediatomCoscinodiscuswailesii.OptExpr15:18082-18088

66. DeStefanoL,DeStefanoM,MaddalenaP,MorettiL,ReaI,MocellaV,RendinaI(2007b)Playingwithlightindiatoms:smallwaterorganismswithanaturalphotoniccrystalstructure.Proc.SPIE6593PhotonicMaterials,Devices,andApplicationsII:6593-59313

67. DeStefanoL,MaddalenaP,MorettiL,ReaI,RendinaE,DeTommasiM,DeStefanoM(2009)Nano-biosilicafrommarinediatoms:Abrandnewmaterialforphotonicapplications.SuperlatticeMicrost46:84-89

68. DeTommasiE,ReaI,MocellaV,MorettiL,DeStefanoM,RendinaI,DeStefanoL(2010)Multi-wavelengthstudyoflighttransmittedthroughasinglemarinecentricdiatom.OptExpress18:12203

69. TownleyHE,WoonKL,PayneFP,White-CooperH,ParkerAP(2007)ModificationofthephysicalandopticalpropertiesofthefrustuleofthediatomCoscinodiscuswailesiibynickelsulfate.JNanotechnol18:295101

70. VernetM(2000)EffectsofUVradiationonthephysiologyandecologyofmarinephytoplankton.In:DeMoraSJ,DemersS,VernetM(eds)TheEffectsofUVRadiationintheMarineEnvironment,CambridgeUniversityPress,Cambridge,pp.237–278

71. VijiS,AnbazhagiM,PonpandianN,MangalarajD,JeyanthiS,SanthanamP,Devi,AS,ViswanathanC(2014)Diatom-BasedLabel-FreeopticalBiosensorforBiomolecules.ApplBiochemBiotechnol174:1166-1173

72. VillafañeVE,HelblingEW,Holm-HansenO,ChalkerBE(1995)AcclimatizationofAntarcticnaturalphytoplanktonassemblageswhenexposedtosolarultravioletradiation.JPlanktonRes17:2295-2306

73. WangY,PanJ,CaiJ,ZhangD(2012)FloatingassemblyofdiatomsCoscinodiscussp.microshells.BiochemBiophysResCommun420:1-5

74. WaringJ,UnderwoodGJC,BakerNR(2006)ImpactofelevatedUV-Bradiationonphotosyntheticelectrontransport,primaryproductivityandcarbonallocationinestuarineepipelicdiatoms.PlantCellEnviron29:521-534

75. WuXJ,GaoG,GiordanoM,GaoKS(2012)GrowthandphotosynthesisofadiatomgrownunderelevatedCO2inthepresenceofsolarUVradiation.FundamApplLimnol180:279-290

76. WuY,CampbellDA,GaoK(2014)FasterrecoveryofadiatomfromUVdamageunderoceanacidification.JPhotochemPhotobiolB140:249-254.

17

77. XuJ,GaoK(2010)UseofUV-AEnergyforPhotosynthesisintheRedMacroalgaGracilarialemaneiformis.PhotochemPhotobiol86:580–585

78. YamanakaS,YanoR,UsamiH,HayashidaN,OhguchiM,TakedaH,YoshinoK(2008)Opticalpropertiesofdiatomsilicafrustulewithspecialreferencetobluelight.JApplPhys103:074701

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Figure1:ScanningelectronimagesofCoscinodiscusgranii,showingavalveseenfromtheoutsideofthecellandgirdlebandspartlydetached.Insert:Scalebar500nm;smallsectionofadiatomvalveshowingthe3Dstructure.

Figure2:Overviewoverrelevantfeaturesthatdifferbetweenthediatominitsnaturalhabitatandarinseddiatomvalveinatypicalexperimentalphotonicset-up(a).b:Adiatomvalverinsedoforganicmaterialandseenunderlightmicroscope.c:Alivediatominwater(lightmicrograph).d:Aschematicofspectralchangesinlightthroughawatercolumn

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Figure3:LightmicroscopepicturesofdrieddiatomsonaUV-transparentquartzsilicamicroscopeglassplate.Scalebar0.2mm.Coverage(seetable1)isestimatedwithinthewhitesquare.a:Coscinodiscusconcinnus,cleanedfrustulesb:C.granii,cleanedfrustules.c:C.concinnus,driedcellswithcellcontent.d:C.granii,driedcellswithcellcontent.

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Figure4:Transmittancespectrafora:Coscinodiscusconcinnus,cleanedfrustulesb:C.granii,cleanedfrustules.c:C.concinnus,driedcellswithcellcontent.d:C.granii,driedcellswithcellcontent.ThereferencespectrumistheUV-transparentquartzsilicamicroscopeglassplatewithnothingontopofit.

Figure5:TransmittancespectrafromFigure4normalizedtodegreeofcoverageonthequartzmicroscopeglass.a:Coscinodiscusconcinnus,cleanedfrustulesb:C.granii,cleanedfrustules.c:C.concinnus,driedcellswithcellcontent.d:C.granii,driedcellswithcellcontent.

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Figure6:TransmissionthroughasinglevalveofC.Graniinormalizedtothebackground.

Table1:AveragediatomdiameterandcoveragepercentagefordiatomsonquartzglassshowninFig.3.

Approximatediameterofdiatom

valve(µm)

Standarddeviationdiameterofdiatom

%coverage

1.Cconcinnuscleaned

49 6.09 Ca85

2.Cgraniicleaned

62 2.61 Ca45

3.Cconcinusdriedwithcellcontent

43 12.18 Ca85

4.Cgraniidriedwithcellcontent

146 1.71 Ca95