T o b i a s V a l d e m a r R y b n e r S e c t i o n f o r S u s t a i n a b l e B i o t e c h n o l o g y

A a l b o r g U n i v e r s i t y C o p e n h a g e n

Improvingthebiomassproductivityandphycocyaninconcentrationbymixotrophic

cultivationofArthrospiraplatensis

Master Thesis, June 2016

AalborgUniversityCopenhagen

Copyright©2014.Thisreportand/orappendedmaterialmaynotbepartlyorcompletelypublishedorcopiedwithoutpriorwrittenapprovalfromtheauthors.Neithermaythecontentsbeusedforcommercialpurposeswithoutthiswrittenapproval.

Semester: 3and4Title:ImprovingthebiomassproductivityandphycocyaninconcentrationbymixotrophiccultivationofArthrospiraplatensisProjectperiod:1September,2015–10June,2016 Supervisor:PeterWestermann(AAU)Education:MasterofScienceinSustainableBiotechnologyECTS:60Departmentname:DepartmentofChemistryandBioscienceSectionname:SustainableBiotechnologyNumberofpages:57Appendix:2Student:TobiasRybnerStudynumber:20112770Submitted:10.6.2016

Abstract:ArthrospiraPlatensisisaphototrophicorganismthatnaturallycontainsthepigmentphycocyanin.A.platensisiscultivatedinindustrialautotrophicopenracewaypondsthathaveseveraldrawbacksincludingriskofcontaminationandlowproductivity.Manyofthedisadvantagesassociatedwithopenpondscultivationsystemscanbeimprovedbycultivationinclosedphotobioreactors.Cultivationinclosedsystemsisstillanexpensivemethodmainlyassociatedtoahigh-energyconsumption.Therefore,inordertomakeitmoreprofitabletocultivateinclosedsystems,itisnecessarytoreducetheenergyconsumption.ThiscanbeaccomplishedbyusingLEDlightsourcesforcultivationandbyimprovingtheproductivityofA.platensistoreducecultivationtime.InthisstudytheeffectonproductivityofA.platensiscultivatedmixotrophicwithacetateasorganiccarbonsourceunderdifferentlightconditionswasinvestigated.Theaveragebiomassproductivitywasincreasedwitharound20%forthemixotrophicculturesbutthephycocyanincontentpergrambiomasswasthesameforboththeautotrophicandmixotrophiccultures.Lowerlightintensitiesseemtoimprovethephycocyanincontent.However,cultivatingwithonlyredlightdidnotseemtoeffectphycocyanincontent.Italsoseemslikethereisacorrelationbetweenhighbiomassdensityandlowphycocyanincontent.ThebiomassproductivitywasincreasedwhenA.platensiswascultivatedwithacetateandphycocyanincontentwasincreasedwithlowlightintensities,suggestingthatacetatecouldbeusedasasubstrateforcultivationofA.platensisatlowlightintensitiesinordertoimproveproductionofphycocyaninandreduceenergyconsumption.

Aalborg University Copenhagen A.C. Meyers Vænge 15 2450 København SV, Denmark Secretary: Christina Maxwell Berthou Phone: +45 9940 7651 cmp@staff.aau.dk

AcknowledgementsFirstandforemost,IwouldliketothankmysupervisorPeterWestermannforagoodcooperationandvaluableguidancedoingthemasterthesis.Ihaveenjoyedourcollaborationandtheopportunitytodevelopaprojectwhichliesalittleoutsidethesectionsnormalresearchareas,whichhavebeenextremelyexcitingandeducational.IwillalsoliketothankGitteHinz-BergourheadlabtechnicianforhelpingmeinthelaboratoryandforanalysingmyHPLCsamples.Finally,IwouldliketothanksAalborgUniversitystudyboardofchemistryandenvironmentalengineeringforapprovalofmythesisproject.

Contents1 Introduction.........................................................................................................61.1 Surplusproductionofwindpower...............................................................71.2 GeneralArthrospiraplatensis.......................................................................71.3 MorphologyandtaxonomyforArthrospira.................................................81.4 EcologyofArthrospira................................................................................101.5 Effectoftemperatures...............................................................................101.6 EffectofpH.................................................................................................101.7 Mixotrophiccultivation..............................................................................111.8 Phycocyaninandthephotosynthesis.........................................................121.9 Markedforphycocyanin.............................................................................131.10 Industrialcultivationofphotosyntheticorganism......................................141.11 PhotobioreactorandartificiallightingwithLightemittingdiode(LED).....16

2 Projectdescription.............................................................................................182.1 Problemstatement.....................................................................................182.2 Objective:...................................................................................................182.3 Selectionoforganismandexperiments.....................................................192.4 Hypotheses:................................................................................................19

3 Materialandmethods........................................................................................203.1 Sterilization.................................................................................................203.2 Microorganism...........................................................................................203.3 Culturemedia.............................................................................................203.4 ArtificialLEDlightsources..........................................................................233.5 Highperformanceliquidchromatography(HPLC).....................................243.6 Biomassproduction....................................................................................243.7 Phycocyaninconcentration........................................................................263.8 Acetobacteriumcultivation........................................................................273.9 TestofgrowthkineticforA.platensis........................................................273.10 TestofphycocyaninextractionmethodsonArthrospiraplatensis............283.11 TestgrowthrateonArthrospiraplatensiswhenculturedAuto-,hetero-andmixotrophic............................................................................................................293.12 TestgrowthforArthrospiraplatensisinamediumforAcetobacteriumsuppliedwithacetate.............................................................................................303.13 Mixotrophiccultivationwithdifferentconcentrationofacetate..............303.14 TestoflowilluminationwithintheredandbluelightspectrainabatchPhotobioreactorsetup...........................................................................................313.15 TestoflowilluminationwithintheredlightspectrainabatchPhotobioreactorsetup...........................................................................................32

4 Resultsanddiscussion........................................................................................334.1 Acetobacteriumsp.cultivation...................................................................334.2 TestofgrowthkineticforArthrospiraplatensis.........................................334.3 TestofphycocyaninextractionmethodsonArthrospiraplatensis............344.4 TestgrowthrateonArthrospiraplatensiswhenculturedAuto-,hetero-andmixotrophic............................................................................................................364.5 TestgrowthforArthrospiraplatensisinamediumusedtoAcetobacteriumsuppliedwithaceticacid........................................................................................394.6 Mixotrophiccultivationwithdifferentconcentrationofsodiumacetate..40

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4.7 Photobioreactorcultivationwithlowilluminationintheredandbluelightspectra....................................................................................................................44

5 Conclusion..........................................................................................................496 Perspective.........................................................................................................507 Listofreferences................................................................................................528 Appendices.........................................................................................................578.1 Appendix1.Extractionprotocolforphycocyanin......................................578.2 Appendix2.Experimentaldataandcalculations.......................................57

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ListofFiguresFigure1Photosyntheticlightharvestingcomplexincyanobacteria..........................12Figure2Absorbancespectraofdifferentalgaepigments.........................................13Figure3,A)LightspectracultivationlightL2,L3,L4...............................................24Figure4StandardcurveforA.platensisbiomassVS.OD..........................................25Figure5StandardcurvePhycocyaninconcentrationVS.Abs....................................26Figure6.GrowthcurveforArthrospiraplatensiscultivatedinbasalZarroukmedium.

............................................................................................................................34Figure7Phycocyaninyieldfrom1grambiomass[mgPC/gX]from5different

extractionconditions..........................................................................................34Figure8GrowthcurveforArthrospiraplatensiscultivatedautotrophic,mixotrophic,

heterotrophic.....................................................................................................37Figure9GrowthcurveforArthrospiraplatensistestedinDSMZAcetobacterium....39Figure10GrowthcurveforArthrospiraplatensiscultivatedautotrophicand

mixotrophic........................................................................................................41Figure11GrowthcurveforArthrospiraplatensiscultivatedautotrophicmixotrophic.

............................................................................................................................41Figure12sAcetateconsumptionbyArthrospiraplatensis.........................................43Figure13GrowthcurveforArthrospiraplatensiscultivatedmixotrophicunder

differentillumination.........................................................................................45Figure14Acetateconcentrationinthephotobioreactorsovertime.........................47

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ListofTablesTable1advantagesanddisadvantagesforopenandclosedphotocultivationsystems

............................................................................................................................16Table2Componentfor1litreBasalZarroukmedium...............................................21Table3Componentstoprepare1litreA5micronutrientsolution............................21Table4Componentstomake1litre1Msodiumacetatesolution............................21Table5Componentsusedtoprepare1litre135aAcetobacteriummedium............22Table6Componentusedtoprepare1litretraceelementsolution..........................22Table7Componentusedtoprepare1litrevitaminsolution....................................23Table8LEDlightsourcesandtheirlightintensity.....................................................23Table9GrowthparametersforA.platensiscultivatedinbasalZarroukmedium.....33Table10Resultfromtestof5extractionmethods....................................................36Table11GrowthparametersforArthrospiraplatensiscultivatedautotrophicand

mixotrophic.1.....................................................................................................38Table12Phycocyanincontentinthebiomassatday23.Cultivatedautotrophicand

mixotrophic1.....................................................................................................38Table13GrowthparametersforArthrospiraplatensiscultivatedautotrophicand

mixotrophic2.....................................................................................................43Table14Phycocyanincontentinthebiomassatday20.cultivatedautotrophicand

mixotrophic2.....................................................................................................44Table15Growthparametersforcultivatedmixotrophicwith1g/Lacetateunder

differentillumination.........................................................................................46Table16Resultsfromphycocyaninextractionfrombiomasscultivatedmixotrophic

with1g/Lacetateunderdifferentilluminationatday30andday35...............48Table17ResultsfromphycocyaninextractionfromArthrospiraplatensisbiomass

cultivatedmixotrophicwith1g/Lacetateunderlowilluminationredlightspectra................................................................................................................48

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ListofPicturesPicture1FilamentsofArthrospiraplatensisseenthroughamicroscope....................9Picture2ArthrospiraplatensiscultivatedonsolidZarroukmediumseenthrougha

microscope...........................................................................................................9Picture3Openpondracewaytrackforalgaeproduction.........................................14PictureThreetypesofclosedphotobioreactorsystem.............................................15Picture5.FilamentofArthrospiraplatensisseenthroughamicroscopeafter9days

cultivationinDSMZ135aAcetobacteriummedium..........................................40Picture6CultivationofA.platensiswithdifferentacetateconcentration................42PicturePhotobioreactorRHandsystemusedtoextractbiomass.............................44

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1 Introduction.Thetermmicroalgaehasnoformaltaxonomicuprightbutisoftenusedtoembodyahighlydiversegroupofnon-cohesiveO2evolvingphotosyntheticmicroorganisms,whichareabletoliveunderavarietyofdifferentconditions,suchashighandlowtemperatures,lightintensities,pHandsalinity(Barsantietal.,2008).Thegeneraldefinitionofalgaeisthattheyareconsideredsimplephotosyntheticmicroorganismsbecausetheyarenotorganisedintoorgans,likehigherplants(Wijffelsetal.,2013).Microalgaeareanevolutionarilydiversegroupofunicellular,eukaryoticorganisms,althoughprokaryoticcyanobacteriaareoftenincludedinthesamecontext.Theevolutionaryandphylogeneticvarietyofthesephotosyntheticorganismsalsoresultsinawidevarietyofchemicalcompositionswithintheseorganisms.Thebroaddiversityinthechemicalcompositionfoundinmicroalgaeandcyanobacteriahasincreasedtheinterestforthemandthereforemakesthemveryattractiveforbioprospecting,whilstalsohavingpotentialforindustrialproductionofavarietyofbiologicalcompounds(Borowitzka,2013).Microalgaealreadyplayakeyroleinthecommercialproductionofhighvaluechemicalssuchascarotenoid,longchainpolyunsaturatedfattyacidsandphycobilins(KuddusandRamteke,2012).Theprokaryoticcyanobacteria,whichisoftenreferredtoasblue-greenalgae,andeukaryoticmicroalgaeareallpromisingtocommercialproducersofbiochemical,biofuelsandfoodadditive(Wijffelsetal.,2013).Phototrophicorganismsproducesugarandoxygenfromcarbondioxide,water,nutrientsandsunlightbyphotosynthesis(LeuandBoussiba,2014).AbigadvantageofcultivatingPhototrophicorganismsisthattheycanbecultivatedinmarginalorsalinewateronunproductivedryland.Thismeansthattheydonothavetocompetewithagricultureforarablelandandfreshwater.Thesepropertiesmakethemaverypromisingsourceforasustainableproductionofvariousbiomaterials,biochemicalandbiofuels,especiallyonlargeunexploited,sunnyanddrylandareaswhereotheragriculturalactivitiesarenotsuitablebecauseofotherfactorssuchaslackofcleanwaterforirrigationandfertilesoil(LeuandBoussiba,2014).TheideaofculturingmicroalgaeinthelaboratorywasfirstpresentedbyWarburgin1919whereheculturedChlorellaaspartofhisworkstudyingphotosynthesis.Hediscoveredthatsomemicroalgaecoulddailyincreasetheirbiomasssignificantlyandthatthedrymatterfromthemcouldcontainover50%ofcrudeprotein(RichmondandSoeder,1986).ThefirstattempttoupscaletheculturingofChlorellawasperformedinapilotplantinMassachusettsandTokyoin1950s.JapanbecamethefirstcountrytoproduceChlorellabiomassasahealthfood(RichmondandSoeder,1986;Skjånesetal.,2013).Intheearly1960sthefirstcommercialuseoftheeukaryoticmicroalgaeChlorellainalarge-scaleproductionwasstarted,whichwassoonfollowedbythecyanobacteriaArthrospira(sometimereferredtoasSpirulina)inthe1970s.By1980thecommercial

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productionoffoodsupplementswiththemicroalgaeChlorellawasestablishedinAsia,India,USA,IsraelandAustraliaandhassincebecomeanimportantmarketandaresoldashealthfoodallovertheworld(Wijffelsetal.,2013).Thealgaebiomassisveryrichinproteinandcanbeusedasanimalfeedorfoodsupplementswhilsttheextractsfromthealgaecanbeusedintheproductionofcosmeticsandpharmaceuticals(Skjånesetal.,2013).Theinterestandresearchformicroalgaebiotechnologyhasincreasedinthelastdecadeandisnowconsideredtobeaverypromisingtechnologyforbiofuelsandbiochemicals,whichmeansthattheresearchinalgaecultivationandprocessingisnotonlyfocusingonimprovingproductsbutalsoonnewproductsandapplications(Wijffelsetal.,2013).

1.1 Surplusproductionofwindpower

Astheeffectsofclimatechangearebecomingmoreconcrete,therehasbeenanincreasedfocusontheproblemscausedbyclimatechangeandthedevelopmentofrenewableenergysourcesinordertoliberateusfromfossilenergysources.Thishascreatedagrowinginterestinwindpower,especiallyinDenmark,butalsoinGermanyandseveralothercountries,whichhasledtomassiveinvestmentinwindenergyprojects.Thishasresultedinamorefrequentsurplusproductionofwindpower(Neslen,2015).AsDenmarkandothernorthEuropeancountriesproducealargeramountoftheirelectricitywithwindpower,therewillbedaysofhighwindswhereelectricityproductionfromwindpowerwillexceedtheamountofelectricityrequired.Duringtheseperiods,theelectricityissoldatverylowpricesoritmayforshortperiodsevencostmoneytogetridoftheelectricity(Wittrup,2013).Therefore,thereisaneedforamethodtostoreorutilizethesurplusproductionofelectricitygenerated.Themostwelltestedstoragemethodforelectricityusedtodayischemicalstorageinbatteries.Theenergyinabatteryisofhighquality,howeverthereisgreatlossofenergyassociatedwithstoringelectricityinthisway.Inrelationtotheamountofenergythatwillneedtobestored,itisnotpossibletheenergycouldbestoredinthiswayand,inaddition,batteriesareveryexpensivemakingitaveryuneconomicalwaytostoreelectricity(Energinet.dk,2011).Thus,untilbettertechnologiesforstoringsurplusproductionofelectricitygeneratedbecomesavailableortheprocessbecomescheapertherewillbeafoundationfortransformingtheenergytootherproducts.Forinstance,toconvertthesurplusproductionofelectricityintohydrogenbyelectrolysis(Mogensen,2015).

1.2 GeneralArthrospiraplatensisCyanobacteriabelongtothekingdomofEubacteriaandconstituteoneofthelargestgroupsofprokaryotes.Theyaresomeofthesimplestlifeformsonearthandthecellularstructureisasimpleprokaryote,whichcanperformphotosynthesislikeplantsbutwithouttheplantcellwallsresemblingprimitivebacteria(Stanieretal.,1981).Likeanimalscelltheyalsohavecomplexsugars,suchasglycogen,ontheircell

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membrane(Singhetal.,2005).However,theyaretrulyprokaryoticlackingnuclearmembranes,internalorganellesandhistoneproteinassociatedwithchromosomes.CyanobacteriaareabletoliveautotrophicutilisingCO2astheirsolecarbonsourceusingthereductivepentosephosphatepathwayorCalvincycle(StalandMoezelaar,1997).Theyaremainlyaquaticandlargerthanothertypesofbacteria(Mühling,2000).Sincetheyareabletophotosynthesisandareaquatic,theyareoftenmentionedasblue-greenalgae,whichcanbemisleadingsincetheyarenotanalgae(eukaryote).Also,theyareallunicellular,althoughmanygrowincoloniesorfilaments(Singhetal.,2005).Someofthemaretoxicwhileothersareedible.AmongsttheediblegeneraareNostoc,ArthrospiraandAphanizomenon.Arthrospirahavebeencommerciallycultivatedandsoldasafoodsupplementsduetoitshighproteincontent(60-70%ofitdryweight)(Sánchezetal.,2003).Thecyanobacteriabiomassisrichincarotenoid,chlorophyll,phycocyanin,aminoacid,mineralsandmanyotherbioactivecomponents,whichmakesitidealtouseasafoodadditivealongwithmanyotherapplicationswithinbiology,biotechnology,foodandmedicineindustry(Eriksen,2008;Sánchezetal.,2003).Thecompositionofthenutrientsinthebiomassdependsonthegrowthconditionssuchaslightintensity,temperature,pH,salinityetc.,whichcanhaveamajoraffectonthelipidandpigmentcontentinthecells(StalandMoezelaar,1997).ThecyanobacteriumArthrospiraplatensisisgainingmoreattentiononkeybiotechnologyresearchbecauseofitseconomical,ecologicalandnutritionalimportance.A.platensishasagreatpotentialtoprovidetheindustrywithbiologicalproducedingredientsthatcanbeusedforfoodproductionandrelatednutritionalmaterials,suchascolouringagents,vitamins,γ-linolenicacidandenzymesetc.(Borowitzka,2013).TherearemanydifferentproteinspresentinA.platensisincludingphycobilinproteins,whichareafamilyofbrilliantlycoloured,hydrophilicandstablefluorescentpigmentproteinsthatareclassifiedintothreemaingroups:phycocyanin(C-PC),phycoerythrin(C-PE)andallophycocyanin(C-APC)withdifferentinherentcolourandabsorbanceproperties,whichwillbeexaminedlaterinthereport.Phycocyaninisthemainproteininthephycobilinproteinandcanbeusedformorethanjustanutritiveingredientandnaturaldyeinfoodsandcosmetics.Itcanalsobeusedasapotentialtherapeuticagentinoxidativediseasesandasafluorescentmarkerinbiomedicalresearch(Anteloetal.,2010).

1.3 MorphologyandtaxonomyforArthrospiraArthrospiragenusaremulticellularfilamentouscyanobacteria,whichifseenthroughamicroscopeappearasblue-greenfilaments(picture1)(Ciferri,1983).Thebluecolourcomesfromphycocyaninandthegreencolourisfromchlorophyll,howeverthetwopigmentscoverathirdgroupofpigment,thecarotenoids,whichnormalappearsasared,orangeoryellowcolour(RichmondandSoeder,1986).

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Picture1showthefilamentsofArthrospiraplatensisseenthroughamicroscope(40x).(photobyT.Rybner)

Thefilamentsarecomposedofcylindricalcellsarrangedinunbranchedhelicoidaltrichomesthatareacharacteristicforthegenus.Thehelicalshapeofthetrichomeischaracteristicforthegenus.However,thehelicalstructurecandifferinlength,pitchandthehelixdimensionfromdifferentspeciesandevenwithinthesamespecies(RichmondandSoeder,1986).Theirfilamentsaremotileglidingalongtheiraxisanddonothaveheterocysts.Theyonlyhavethehelicalshapewhengrowninaliquidmedium.Whengrownonasolidmediumthefilamentsshapetakestheformofaflatspiral(Picture2)(Ciferri,1983;Sánchezetal.,2003).

Picture2showsArthrospiraplatensiscultivatedonsolidZarroukmediumseenthroughamicroscope(40x).

(photobyT.Rybner)

Thediameterofthecellsvariesfrom1to3μminthesmallerspeciesandupto12μminthelargerspecies,suchasA.platensisandA.maxima.Thelargerspecieshaveagranularcytoplasmthatusuallycontainsagasvacuolesandaeasilyvisiblesepta(RichmondandSoeder,1986).ThecellwallinArthrospiraiscomposedoffourlayersnumberedfromtheinnermostandoutwardsasLI,LII,LIII,andLIV.ItistheLIIlayerthatiscomposedof

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peptidoglycansubstancesthatgivesthecellwallitsrigiditywhilstthethreeotherlayersareveryweakandprovideonlyalittlestructuralsupport(Ciferri,1983;Sánchezetal.,2003).

1.4 EcologyofArthrospiraArthrospiracanbefoundinmanydifferentenvironmentssuchassoil,marshes,freshwater,brackishwaterandseawater.Arthrospiracolonizemanyextremeenvironmentswhereotherorganismshavedifficultiestolive.AnexampleofthiscanbefoundinsomealkalinelakesinAfricaandMexicowhereA.platensisandA.maximaarethedominantorganisms(VonshakandRichmond,1988).ThiswasextensivelystudiedbyIltisin1967intheAfricanalkalinelakeswithintheChadregionwhereheclassifiedthesedifferentbodiesofwaterintothreecategoriesaccordingtotheirsaltconcentration,whichmainlyconsistedofcarbonateandbicarbonate(Ciferri,1983).Thecategoriesare:

1. Lakeswithsaltconcentrationbelow2.5g/L.2. Mesohalinelakes,saltconcentrationrangingfrom2.5to30g/L.3. Alkalinelakes,saltconcentrationabove30g/L.

Inthefirstcategorywithsaltconcentrationbelow2.5g/LhefoundavariedpopulationofmicroorganismsbelongingtoChlorophyceae,cyanobacteriaanddiatoms.Inthesecondcategorywherethesaltconcentrationwasrangingfrom2.5to30g/Lhefoundthatthecyanobacterialpopulationwaspredominantlycomprisedofmanydifferentspeciessuchas,Synechocystis,Oscilfatoria,ArthrospiraandAnnbaenopsis.Inthethirdcategorywithsaltconcentrationabove30g/LthecyanobacterialpopulationwasalmostmonospecificwithArthrospiratheonlymicroorganismpresent.A.platensishasbeenisolatedfromwaterwithsaltconcentrationreaching270g/L,however,growthseemstobeoptimalwithsaltconcentrationrangingfrom20to70g/L(Ciferri,1983;RichmondandSoeder,1986).

1.5 Effectoftemperatures

Arthrospiraisamesophilemicroorganismthatcantolerateandstillgrowintemperaturesrangingfromuptoaround40˚Canddowntoaround15˚C,althoughthismaydeviatebetweenstrains(RichmondandSoeder,1986).TheoptimaltemperatureforA.platensisgrowthis30˚C,whichhasbeenshownbyOgbondawhoinvestigatedtheeffectoftemperatureandpHonbiomassproductionforArthrospiraspecies(Ogbondaetal.,2007).

1.6 EffectofpH

Cyanobacteriacanbefoundinmanydifferentenvironmentsandconditions.Forexample,Arthrospiraplatensisthrivesinextremealkalineenvironments.However,evenamongcyanobacteria,A.platensis’sabilitytoadapttohighpHvaluesisveryuniquebecauseeventhoughmostcyanobacteriacantoleratealkalineconditions,

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theystillhaveoptimalgrowthinaneutralmedium(pH7)(Ciferri,1983).ThisisnotthecaseforA.platensis,whichfailstogrowatpH7orlowerandtheoptimalgrowthisobtainedaroundpH9to10(Ogbondaetal.,2007;Schlesingeretal.,1996).EvenatpH11.5thegrowthrateforA.platensishasbeenapproximately80%oftheoptimalgrowthrateanditcanthereforeclearlybedefinedasanobligatealkaliphilemicroorganism(Schlesingeretal.,1996).

1.7 Mixotrophiccultivation

ThemaincarbonfixationrouteinArthrospiraisbyphotosynthesis.Whenbothlightandanorganiccarbonsourceareavailableduringthecultivation,Arthrospirahavetheabilitytocombineautotrophicphotosynthesiswithheterotrophicassimilationofanorganiccarbonsourceinacombinedprocessknownasmixotrophic(AndradeandCosta,2007).Thephotosyntheticfixationofcarbondioxide(CO2)isregulatedbythelightintensityandtheheterotrophicassimilationoforganiccarbonisregulatedbytheaccessibilitytotheorganiccarbonsource(Zhangetal.,1999).CulturingArthrospiraunderamixotrophicconditionthenthegrowthwillnotstrictlydependonthephotosynthesis,whichmeansthatlightstopsbeingtheonlylimitinggrowthfactor(AndradeandCosta,2007;ChojnackaandNoworyta,2004).Therefore,mixotrophicculturingbecomesaduallimitationprocessinwhichatoohighconcentrationoftheorganiccarbonsourceortoohighlightintensitiescanbelimitingforthegrowth.However,toolowconcentrationoforganiccarbonortoolowlightintensitiescanalsohaveaninhibitingeffectoncellgrowthandthereforelightintensityandorganiccarbonsubstratebecomethetwomostimportantgrowthfactors(ChojnackaandNoworyta,2004;Zhangetal.,1999).MixotrophiccultivationofArthrospiracouldincreasethebiomassconcentration,whilsttheon-goingimprovementinclosedphotobioreactorsformasscultivationofphototrophicmicroorganismcouldalsocontributetomakethemixotrophicgrowthofcyanobacteriaeconomicalfeasible(Chen,1996;ChenandZhang,1997;Marquezetal.,1995).ExperimentswiththefilamentouscyanobacteriaA.platensisandglucoseasanorganiccarbonsourceshowedthatthebiomassandphotosyntheticpigmentproducedwhengrowingmixotrophicwasincreasedby1.5-2foldwhencomparedtoculturesgrownphotoautotrophic.However,thepigmentconcentrationpergrambiomasswasalmostthesameinthemixotrophicandphotoautotrophiccultures(Marquezetal.,1995;Vonshaketal.,2000).

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Figure1Showsthephotosyntheticlightharvestingcomplexincyanobacteria.Thephotosyntheticelectron

flowandATPsynthesisinthethylakoidmembrane(Johnson,2006).

1.8 Phycocyaninandthephotosynthesis

InCyanobacteriathreemulticomplexlightharvestingsystemsexist:photosystemI,photosystemIIandphycobilisomes,whichareusedtocapturelightenergyfromthevisiblelightspectra(400nmto700nm)andconvertittochemicalenergybythephotosynthesis(Figure1)(Gaoetal.,2016;Tingetal.,2002).Thethreecommontypesofphycobiliproteinshavethemajorbiologicalroleofphotosyntheticlightharvesting:theredphycoerythrinthatabsorbsmoststronglyatwavelengtharound550nm,thebluephycocyaninwhichabsorbsmaximumaround620nmandallophycocyaninthatabsorbsaround650nm,whichislocatedatwavelengthsinthevisiblelightspectrawherechlorophyllshavelowextinctioncoefficients(absorbspoorly)(Figure2)(Madiganetal.,2012).Thephycobilinproteinsarecombinedintogroupscalledphycobilisomes,whichisattachtothethylakoidmembrane.Allophycocyaninarelocatedinthecentreofthephycobilisomescomplexandsurroundedbyeitherphycocyaninorphycoerythrinorbothdependingontheorganism(Figure1)(Johnson,2006;Madiganetal.,2012).Thephycobilisomesareorganisedsophycocyaninabsorbsathigherenergies(shorterwavelength)thenallophycocyanin,whichisplacedclosertothereactioncentrechlorophyllthatabsorbsatlowerenergies(longerwavelengths)thanallophycocyanin.SotheenergyflowisfromphycocyaninèallophycocyaninèchlorophyllofphotosystemII(Eriksen,2008;Madiganetal.,2012).Therefore,phycobilisomesfacilitatetheenergytransferthatallowscyanobacteriatogrowatverylowlightintensities(Madiganetal.,2012).

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Figure2showstheabsorbancespectraofdifferentalgaepigments(WIKI,2014).

1.9 Markedforphycocyanin

PhycocyaninisacommerciallypromisingbiochemicalpresentinA.platensis,however,themajorityofthe3000tonsofdryweightofA.platensisbiomassthatisproducedeveryyeararesoldashealthfoodandanimalfeed,whichhasaloweconomicvalue(around36€perkilo)relativetophycocyanin,whichisconsideredasahighvalueproduct(Borch,2011;Eriksen,2008).DependentofthepurityoftheC-phycocyanin,thesellpriceliesfromaround500US$/kgupto100,000US$/kg,withthecurrentestimatedmarketvalueforphycobiliproteinproductsasgreaterthan60millionUS$(Borowitzka,2013),whilsttheannualmarketforphycocyaninliesbetween5to10millionUS$(Yaakobetal.,2014).ThepurityofC-phycocyaninisdividedintothreeclasses:foodgrade(purityof0.7,definedastheratiobetweentheabsorbanceof620nmto280nm),reactivegrade(purityof3.9)andanalyticalgrade(puritygreaterthan4.0).ThecommercialvalueoffoodgradeC-phycocyaninisaround0.13US$/mgwhilstthereactivegradeisaround1to5US$/mgandtheanalyticalgradecangoashighas15US$/mg(Eriksen,2008;Rito-Palomaresetal.,2001).

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Picture3showsanopenpondracewaytrackforalgaeproductionlocatedinWesternAustralia(Watson,2013).

1.10 IndustrialcultivationofphotosyntheticorganismTherearetwocommonlyusedsystemsforcultivatingphotosyntheticmicroorganism:Theopenracewaypondssystem(Picture3)andclosedphotobioreactorsystem(Picture4)(Harunetal.,2010).ThecommercialcultivationofArthrospiraismainlycarriedoutinopenracewaypoundswherethesolarenergyabsorbedbythecyanobacteriaisusedtofixinorganiccarbon(CO2)(Picture3)(Vonshaketal.,2000).Themaindrawbackoftheopenpoundscultivationsystemisthelowcellconcentrationthatisaround0.4-0.8g/l,whichmeansthatrelativelylargecultivationsystemsarerequiredinordertoproduceanefficientamountofphycocyanin(Chenetal.,2006).Otherdisadvantagesofcultivationinanopenpondssystemistheriskofcontaminationfromothermicroorganism,insufficientstirring/mixingandthelackofcontrolofenvironmentalconditionssuchastemperature,evaporationandlightintensity(ChenandZhang,1997).However,theriskofcontaminationcanbesolvedbycultivationunderextremecultureconditions(highsalinityoralkalinity),whichmeansthatonlyasmallamountoforganismsaresuitedforthiscultivationmethod(BrennanandOwende,2010).However,thesearefactorsthatareundesirablewhenproducingaproduct(phycocyanin)forthechemicalandpharmaceuticalindustriessincethecostforproductrecoveryandpurificationcanbecomecostly,especiallyduetothelowcellconcentrationintheopenpondsystems(ChenandZhang,1997).

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Picture4showsthreetypesofclosedphotobioreactorsystem(A)Columnphotobioreactor(Yeomans,2013).

(B)Horizontaltubularphotobioreactor(Bennekom,2014).(C)Flatplatephotobioreactor(AGICAL,2013).

Someofthedrawbacksforopenpondsystemscanbeavoidedbyculturinginaclosedphotobioreactor,whichprovidesasystemthatcancontrolandevenmanipulatecultivationparametersuchasthenutrientsupplyforgrowth,temperature,pH,dissolvedCO2,lightintensitiesandspecificwavelengthoflightandculturescanbemaintainedaxenic(Eriksen,2008;Harunetal.,2010;Ugwuetal.,2008).Closedphotobioreactorsystemsincludethetubular,flatplateandcolumnphotobioreactors(Picture4).Severalstudieshaveindicatedthatthetypeofphotobioreactorandespeciallycultureconditionsuchas:pHofthemedium,typeofmedia,CO2supply,nitrogenconcentrationandlightintensitysignificantlyaffectthebiomassproductionandphycocyaninaccumulationinArthrospiracultures(Chenetal.,2013;Leemaetal.,2010;Zengetal.,2012).TheseconditioncanbecontrolledinaclosedphotobioreactorandithasbeenshownthattheproductivityofArthrospiraculturesisincreasedwhentheyareculturedinclosedphotobioreactor(Chenetal.,2013).Ishasbeenreportedthatvolumetricbiomassproductivityinclosedphotobioreactorscanbe5to20timesabovewhatcanbeobtaininanopenracewaypondandthatthevolumetricproductivityofphycocyaninisthereforealsoincreasedwhenA.platensisisculturedinaclosedphotobioreactorsystem(Eriksen,2008).ThismayfavourusingaclosedphotobioreactorsystemforcommercialproductionofC-phycocyaninbutthecostofproducingphycocyanininclosedphotobioreactorstillneedstobereducedconsiderablyinordertomakeiteconomicallyfeasibleonanindustrialscale(Xieetal.,2015).Thecostinvolvedforestablishing,runningandmaintainingaclosedphotobioreactorsystemismuchhigherthanthatofanopen

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racewaypondsystem,whichismainlycausedbytheequipmentrequirementandenergyconsumptioninvolvedintheclosedphotobioreactorsystem(Harunetal.,2010).Someoftheadvantagesanddisadvantagesforculturinginopenpondsandclosedphotobioreactorsystemsareoutlinedbelow(Table1).Cultivationsystem Advantages Disadvantages

Tubularphotobioreactor Largesurfaceareaèlightutilization.Noneedforagriculturalland.Goodbiomassproductivity.Suitableforoutdoormassculturing.

Wallgrowthèfouling.Largeareaoflandrequired.Limitationonlengthoftubes.Maintenance.

Flatplatephotobioreactor Highbiomassproductivity.Easytosterilise.LowaccumulationofdissolvedO2.Highphotosyntheticefficiency.Largesurfaceareaèlightutilization.

Difficulttoscaleup.Temperaturecontrol.Wallgrowthèfouling.

Columnphotobioreactor Controlofgrowthconditions.Efficientmixing.Highvolumetricmasstransferrates.Lowcostcomparedtotheotherclosedsystems.Compactandeasytooperate.

Smallilluminationarea.Expensivecomperedtoopenponds.

Openracewayponds Relativelycheaptoconstruct.Easytoclean.Lowenergyinput.Noneedforagriculturalland.Lowenergyinput.Easymaintenance.

Lowbiomassproductivity.Largeareaoflandrequired.Fewphototrophicorganismsaresuited.InsufficientmixingèlightandCO2utilization.Riskofcontamination.

Table1outlinessomeoftheadvantagesanddisadvantagesforopenandclosedcultivationsystemsfor

phototrophicorganism(BrennanandOwende,2010).

1.11 PhotobioreactorandartificiallightingwithLightemittingdiode

(LED).

AmajorobstacletomakeiteconomicalefficientandenvironmentalfriendlytouseartificiallightforcultivatingphototrophicmicroorganismsuchasA.platensisinordertoproducebiofuels,biochemicaletc.isthehighenergyinputassociatedwithtraditionalagriculturallightingsystemsusedforphotosyntheticcultivation(Faunceetal.,2013).Themajorityofclosedphotobioreactorsystemsusedtodayaretubularphotobioreactorswithtubesindifferentsizesandshapesdependingonthereactordesign.Regardlessofthereactordesignused,thebiotechnologicalobjectiveistoensureoptimalcultivationconditions.Thetwoimportant

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parameterstoensuringforoptimalgrowthconditionsforthephototrophicorganismareturbulence(mixing)andillumination(Pulz,2001).Thesetwoparametersareimportanttocontrolinordertoinsureanefficientcultivationprocess.Thecontroloftheilluminationisprobablythemostimportantparameterwhendesigningaclosedphotobioreactorforaphototrophiccultivationprocessthatoperates24hoursperday.Thisisbecausemostoftheenergyusedinaphotobioreactorisforlightningandlightwavelengthmayalsoinfluencethecompositionofthecellsinrelationtopigments,protein,polysaccharidesandlipids(Kocetal.,2013;Rivkin,1989).Therefore,thedesignofaphotobioreactorwillhavetobeeconomical,robust,reliableandhaveaneffectivelightsourceinordertomakeitsuitableforindustrialproduction(BeardallandRaven,2013).Whenchoosingasuitablelightsourceforthephotobioreactoritisimportantthatthespectralcharacteristicsofthelightfluxandthewavelengthrangearerightbecausedifferenttypesofphototrophicmicroorganismmayrequiredifferentlightintensity(photonfluxdensity),durationandwavelengthoflight.Toohighalightintensitymayleadtophotooxidationandphotoinhibition,whilsttoolowlightintensitymaybecomegrowthlimiting(Carvalhoetal.,2011).Asuitablelightsourceforphotobioreactorscouldbelightemittingdiode(LED)becausetheyhaveahighluminousefficiency,lowenergyconsumption,coolemittingtemperature,theoptiontoselectspecificwavelengthandtheyareveryrobust,andhavealonglifetimecomparedtootheragriculturallightsources(Massaetal.,2008).TheLEDtechnologyhasrelativelynarrowwavelengthbandsthatmakesitpossibletoproducespecificwavelength,whichinadditiontomakingitpossibletoilluminatethemicrobeswithlightinthespecificwavelengthsuitedforoptimalgrowthanddesiredproduct,LEDalsohasapositiveeffectontheenergyconsumption,andthusmakingtheprocessmoreenergyefficient.LEDcanreducestressconditionsassociatedtoexcessiveilluminationofthemicrobes,whichoftenappearswithotheragriculturallightsystemsbecausewiththeLEDsystemsthelightintensitycaneasilyberegulated(Kocetal.,2013;YamandHassan,2005).TheLEDtechnologyalsohastheadvantagesofalowheadconductioncomparedtootherlightsystems,whichpreventoverheatingofthegrowthmediumandtherebyreducingtheenergyinputforstabilisingthetemperatureinthephotobioreactor.Finally,LEDaresmallinstructureandeasytoinstall(Kocetal.,2013).AlltheseadvantagesmakeLEDsverysuitableasalightsourceforaphotobioreactor,whilstalsomoreenvironmentallyfriendlythanotheragriculturallightsystems.Therefore,inordertojustify(inasustainableperspective)usingartificiallighttocultivatephotosyntheticorganism,theutilizationoftheenergyinputshouldbeaseffectiveaspossible.ThiscanbeachievedbyusingLEDtechnologythathasahighenergyefficiencyandisabletoprovidelightinthespecificwavelengthandintensitythatisrequiredforthephototrophicorganismtocarryouttheirphotosynthesis(Faunceetal.,2013).

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2 ProjectdescriptionTheincreasinginternationalfocusonsustainabilityandutilizationofbiologicalandrenewableresourceshasresultedinanincreasinginterestinfindingalternativesolutionstothecrudeoilbasedproductionofenergy,fuelsandchemicals.PhycocyaninisahighvalueproductwithmanyapplicationsthatexistnaturallyinA.platensis.PhycocyaninisproducedbycultivationofA.platensisinopenracewaypondsthathavedrawbacksofriskofcontaminationandlowproductivity.ThiscouldbesignificantlyimprovedifA.platensiswascultivatedinclosedphotobioreactorsystems,however,thisisstillaveryexpensivesolution.Therefore,iftheproductivitycouldbeincreasedmorebycultivationA.platensismixotrophicwithacheapandrenewablecarbonsourceitcouldhelpmakeitmoreeconomicalfeasibletocultivateinclosedphotobioreactors.Thus,theideabehindthisprojectisthatthesurplusproductionfromwindturbinescouldbeconvertedtohydrogen(H2)byelectrolysis,andsubsequentlytheH2isusedtoproduceacetatewithhomoacetogenicbacteriabyanaerobicfermentationwithH2plusCO2.TheeffluentcontainingacetatecouldbeusedformixotrophiccultivationofA.platensistoincreaseproductivityofcultivation.Afterthecultivationthephycocyanincanbeextractedfromthebiomassandtherebythesurpluswindpowerproductioncouldbeusedtoincreasetheproductivityofphycocyaninproduction.BasedonthisacetatewasusedasorganiccarbonsourceinthisstudytotestmixotrophiccultivationofA.platensiswithaviewtoextractphycocyanin.

2.1 Problemstatement

CantheproductivityofArthrospiraplatensiscultivatedatlowlightintensitiesbeincreasedifacetateisaddedtothegrowthmedium?CanthephycocyaninconcentrationpergramofbiomassbeincreasedifArthrospiraplatensis,undercultivation,isilluminatedonlywithlightintheredlightspectra?

2.2 Objective:

Theoverallobjectiveforthis60-ECTSmasterthesisistoinvestigatetheeffectonbiomassproductionandphycocyaninphycocyaninconcentrationinA.platensiswhencultivatedmixotrophicwithacetateasanorganiccarbonsource.AlsotoinvestigatewhetheritispossibletocultivateA.platensisinthefermentationbrothfromanaerobicacetateproductionbyAcetobacteriumsp.Furthermore,theobjectiveistoupscalethecultivationofS.platensisina2-litrebatchphotobioreactorsetupunderredandblueilluminationwithdifferentlightintensities.

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2.3 Selectionoforganismandexperiments

A.platensiscanbecultivatedmixotrophic,andhasalreadybeentestedwithanumberofdifferentorganiccarbonsources,whichhaveshowntoincreasethebiomassgrowthrate(AndradeandCosta,2007;ChojnackaandNoworyta,2004;Marquezetal.,1993;Vonshaketal.,2000).

2.4 Hypotheses:

1. TheoverallhypothesisisthatA.platensiscanincreaseitsgrowratewhencultivatedmixotrophicwithacetate.

2. FermentationbrothfromanaerobicfermentationofacetatebyAcetobacteriumsp.canbeusedtocultivateSpirulinaplatensismixotrophic.

3. Alsowhencultivatedmixotrophicwithlowlightintensitiesintheredandbluelightspectra,S.platensiswillstillgrowwellandtheconcentrationphycocyaninpergramofbiomasswillstillbehigh,sincephycocyaninabsorbslightintheredspectra.

4. Theprocesscanbecomemoreenergyeffectivebecauseweonlyuselightintherelevantlightspectra’sformaintaininggrowth(redandbluelight)andbyusingLEDtechnologyforillumination.

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3 Materialandmethods

3.1 Sterilization

ThegrowthmediumandglasswareusedforculturingArthrospiraplatensisweresteamsterilizedinanautoclaveat121°Candasteampressureof1barfor15-20min.Tracemetal,micronutrientandvitaminsolutionswerefiltersterilizedthrougha0,2μmcelluloseacetatemembranefilterandaddedtothemediumsafterautoclavingtoavoidprecipitation.

3.2 Microorganism

ThemicroorganismusedinthisstudywasArthrospiraplatensis.ThepurecultureofA.platensiswasprocuredfromExperimentalPhycologyandCultureCollectionofAlgaeattheuniversityofGöttingen(EPSAG),strainnumber21.99.ThestrainwasmaintainedinabasalZarroukmedium,butfromarecipefoundinR.GuptaPh.D.thesis(Gupta,2007;Zarrouk,1966).MaintainingandculturingofstartercultureswascarriedoutunderilluminationofL3andL4LEDlightbolts403lux(Table8).

3.3 Culturemedia

AutotrophiccultivationofA.platensiswascarriedoutinabasalZarrouk(BZ)medium(Table2and3).ThepHofthemediumwasadjustedtoapHvaluearound9to9,5withpotassiumhydroxide(KOH).Cultureswereincubatedinacultureroomat30±3°Candilluminatedwithartificiallightintheredandbluespectra(Table8).ForthemixotrophiccultivationofA.platensistheBZmediumwassuppliedwithandorganiccarbonsourcefromeitheraceticacid99.8-100.5%obtainedfromSigmaAldrichoracetatefroma1Msodiumacetatesolution(Table4).FortheacetateproductionwithhomoacetogenicbacteriabyanaerobicfermentationwithH2plusCO2.Acetobacteriumsp.werecultivatedinDSMZAcetobacteriummedium135aandthecompositionofitisshownbelowintable5,6and7.

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BasalZarroukmedium SolutionI Sodiumbicarbonate(NaHCO3) 18.00gPotassiumphosphatedibasic(K2HPO4) 0.50gDe-ionizedwater(dH2O) 500mlSolutionII Sodiumnitrate(NaNO3) 2.50gPotassiumsulphate(K2SO4) 1.00gSodiumchloride(NaCl) 1.00gMagnesiumsulphate(MgSO4) 0.20gCalciumchloride(CaCl2) 0.04gIronsulphate(FeSO4) 0.01gEthylenediaminetetraacetate(C10H16N2O8) 0.08gDe-ionizedwater(dH2O) 500mlA5micronutrientsolution* 1mlAutoclavesolutionIandIIseparately,assembleaftercoolingandaddsolutionA5insterilesolution.

Table2showsthecomponentfor1litreBasalZarroukmedium(Gupta,2007;Zarrouk,1966).

*PreparationofA5micronutrientsolution Boricacid(H3BO3) 2.860gManganese(II)chloride(MnCl2) 1.810gZincsulphate(ZnSO4) 0.222gSodiummolybdate(Na2MoO4) 0.0177gCopper(II)sulphate(CuSO4) 0.079gDe-ionizedwater(dH2O) 1000mlFiltersterileifsterileconditionisrequired.

Table3showsthecomponentstoprepare1litreA5micronutrientsolutionusedtopreparethebasalZarrouk

medium(Gupta,2007;Zarrouk,1966).

1MSodiumacetatesolution Sodiumacetate(C2H3O2Na) 82gDe-ionizedwater(dH2O) 1000ml

Table4showsthecomponentstomake1litre1Msodiumacetatesolution.

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DSMZ135aAcetobacteriummedium Sodiumchloride(NaCl) 20.00gAmmoniumchloride(NH4Cl) 1.00gMonopotassiumphosphate(KH2PO4) 0.33gDipotassiumphosphate(K2HPO4) 0.45gMagnesiumsulphateheptahydrate(MgSO4x7H2O) 0.10gTraceelementsolution* 20.00mLYeastextract 2.00gSodiumbicarbonate(NaHCO3) 5.00gResazurin 1.00mgVitaminsolution** 10.00mLEthyleneglycol 1.25gL-Cysteine-HCLxH2O 0.50gSodiumsulphidenonahydrate(Na2Sx9H2O) 0.50gDe-ionizedwater(dH2O) 1000.00mLThemediumwaspreparedanaerobicallywithN2andCO2gasmixture.

Table5showsthecomponentsusedtoprepare1litre135aAcetobacteriummedium(DSMZ,2016).

*Preparationoftraceelementsolution Nitrilotriaceticacid 1.50gMgSO4x7H2O 3.00gMnSO4xH2O 0.50gNaCl 1.00gFeSO4x7H2O 0.10gCoSO4x7H2O 0.18gCaCl2x2H2O 0.10gZnSO4x7H2O 0.18gCuSO4x5H2O 0.01gKAI(SO4)2x12H2O 0.02gH3BO3 0.01gNa2MoO4x2H2O 0.01gNiCl2x6H2O 0.03gNa2SeO3x5H2O 0.30mgNa2WO4x2H2O 0.40mgDistilledwater 1000.00mL

Table6Showthecomponentusedtoprepare1litretraceelementsolutionusedinthe135aAcetobacterium

medium(DSMZ,2016).

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**Preparationofvitaminsolution Biotin 2.00mgFolicacid 2.00mgPyridoxine-HCL 10.00mgThiamine-HCLx2H2O 5.00mgRiboflavin 5.00mgNicotinicacid 5.00mgD-Ca-pantothenate 5.00mgVitamineB12 0.10mgp-Aminobenzoicacid 5.00mgLipoicacid 5.00mgDistilledwater 1000.00mL

Table7showsthecomponentusedtoprepare1litrevitaminsolutionusedinthe135aAcetobacterium

medium(DSMZ,2016).

3.4 ArtificialLEDlightsources

TheilluminationforthedifferentexperimentalsetupswasprovidedwithartificialLEDlightintheredandbluespectra,whichisillustratedbelow(Table8).Unit Description Spectra LightintensityE(lux)

L1 Photobioreactorlightingplatform Red,blue 349L1 Photobioreactorlightingplatform red 175

L2 LEDlightpanels Red,blue 839L3 LEDlightbolt Red,blue 403L4 LEDlightbolt Red,blue 403

Table8ShowsthedifferentLEDlightsourcesandtheirlightintensityinlux.

ThewavelengthandlightintensityforL2,L3,andL4wasmeasuredwithaspectralmeter(MetrueSIM-2plus),whichwasprovidedfromStatensByggeInstitute(SBI)(Figure3).ThelightintensityofL1wasmeasuredwithaLuxmeterapp(LuxLightMeter)onanIPhoneSE(Table8).

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Figure3,A)ShowsthelightspectraforL2,B)showsthelightspectraforL3andC)showsthelightspectrafor

L4.

3.5 Highperformanceliquidchromatography(HPLC)

HPLCanalysiswasusedtomonitoranddeterminetheconcentrationofacetateintheBZmediuminordertodeterminetheresidualacetateconcentrationinthemedium.1mLsamplewasextractedsimultaneouswithopticaldensity(OD)measurementforbiomassdeterminationandstoredinthe-20°Cforlateranalysis.ThesamplewasanalysedinaDionexUltimate3000withRI(refractiveindex)detector,eluent4mMsulphuricacid,columnBIORADAminexHPX-87Hat60°C.

3.6 Biomassproduction

ThebiomassconcentrationforA.platensiswasevaluatedbymeasuringtheopticaldensity(OD)ofthesampleatawavelengthof675nm,whichwasfoundbyperformingawavelengthscanofabiomasssampleonaspectrophotometer(modelDR3800™BenchtopSpectrophotometer).AstandardcalibrationcurveofopticaldensityversusA.platensisbiomassconcentrationwasmadebyperformingadilutionseriesintripletsandmeasuringtheopticaldensityofthesampleandplottingtheagentsofthebiomassconcentrationofthesample,whichwasfoundbydryingintriplets3mlofthedilutedbiomasssamplesinanovenat104˚Cuntiltheweightwasstable(Figure4).

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Figure4showsthestandardcurveforA.platensisusedtodeterminethebiomassconcentrationwithoptical

density(OD)measurement.

TheOD675nmvalueswereconvertedtobiomassconcentrationbytheformulaobtainfromthecalibrationcurve:

!"#$%&& ' ( =*+,-. − 0.0229

33.835×109, ; = 0.99

Thespecificgrowthrate(μ=day-1)forA.platensiscultureswasdetermentforthegrowthperiodbytheequation:

<(>%?@A) =ln(E)F

I.e.themaximumslopeofaplotoflnXversustime.WhereXisthebiomassconcentration(gram/litre)andtisthecultivationtime(days)(HillandRobinson,1974;Xieetal.,2015).Theproductivityistheratiobetweenthedifferencesinbiomassconcentrationforaperiodoftime,whichwascalculatedbytheequation:

G =(EH − EI)

FH

WhereP=productivity(gramperLitre/day),Xi=biomassconcentrationattimei(gram/litre),X0=theinitialcellconcentrationandti=isthetimerange(days)betweenXiandX0(AndradeandCosta,2007;Walteretal.,2011).

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Figure5showstheStandardcurvemadetodeterminephycocyaninconcentrationfrommeasuringmaximal

absorbance(Abs).

3.7 Phycocyaninconcentration

TheabsorbanceofPhycocyaninsampleswasmeasuredwithUV/Visspectrophotometer(MecasysOptizenPOPBIO).ThedeterminationofphycocyaninconcentrationwascarriedoutbymakingadilutionseriesofaPCsamplepurchasefromSigmaAldrichandusingittocalibratetheabsorbancecurveat620nmofdilutedsamples(Figure5).Themaximumabsorbance(Abs)ofphycocyaninwasfoundbyconductingawavelengthscan.TheAbsofextractionsampleswerecalculatedbytheequationobtainfromthecalibrationcurve:

Gℎ?K#K?%L"L $' $( =MN&,OI + 0.016

74.721×TUVWXYZT[\]]Z^

, ; = 0,99

WhereAbs620istheabsorbanceat620nmandVsampleisthevolumeofthebiomasssampleandVbufferisthevolumeofthebufferaddedintheextraction.ToevaluatetheyieldofphycocyaninaccumulatedpergramofA.platensisbiomassthefollowingequationwasused:

_̀ abcdcbVeHe(Gf' !"#') =hℎ?K#K?%L"L(' i)N"#$%&&(' i)

Thepurityofthephycocyaninfractionafterextractionwasevaluatedbasedontheratiobetweenabsorbanceofphycocyaninmeasuredat620nmandtheabsorbanceofsomeoftheaminoacidspresentintheproteinsinthesampleat280nm(Walteretal.,2011).

GfX\^Hjb =MN&,OIMN&OkI

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ThephycocyaninsampleswithanAbs620/Abs280ratiohigherthan0.7areconsideredasfoodgradewhileandratiohigherthan4areconsideredasanalyticalgrade(Eriksen,2008).ThemeasurementofAbsorbanceforPhycocyaninwasconductedonaMecasysOptizenPOPBIOspectrometer.

3.8 Acetobacteriumcultivation.

ObjectiveofexperimentTheobjectiveofperformingthisexperimentwastotrytocultivateAcetobacteriumsp.(DSM:2396)inaDSMZ135amediumsuppliedwithhydrogen(H2)andcarbondioxide(CO2)toseeifitwillproduceacetate.TheideaisthatthefermentationbrothcontainingacetatecouldlaterbeusedassubstrateformixotrophiccultivationofA.platensis.ExperimentalsetupThetestwascarriedoutindupletsin20mlvialswith10mlDSMZ135aAcetobacteriummediumunderanaerobicconditions.ThevialswerestartedwithanAcetobacteriumsp.freezeculture(DSM:2396)obtainedfromDSMZculturecollection.Thevialswereinstalledinanincubatorat30°C.Afterstartingthecultures,thevialwasfeedwithhydrogen(H2)twotothreetimesaweek.

3.9 TestofgrowthkineticforA.platensisObjectiveofexperimentThisexperimentwasperformedinordertomakeagrowthcurveforA.platensis.ThegrowthcurvewasusedtodeterminewhenstarterculturesofA.platensisareintheexponentialphaseofgrowthandreadyforinoculationandtodeterminehowoftenIhavetomeasuregrowthinmylaterexperiment.ExperimentalsetupTheexperimentwascarriedoutin250mLbluecapbottlesintripletswith100mlbasalZarroukmediumineachbottle.TheBottleswasinoculatedwith5mLA.platensis(21.99)startercultureprovidedfromEPSAG.Afterinoculationthebottleswereinstalledinanincubatorroom(temperature30±2°C)withagitation(180RPM)andunderconstantartificiallighting(L2-LEDlightpanels839lux).BiomassaccumulationwasmonitoredbyOD675nmmeasurementandplottedinanexcelsheetVStimetoformagraphofthegrowth.1mLbiomasssamplesforOD675nmmeasurementwasextractedevery6hoursinthebeginningbutwaslaterchangedtoevery8andthen12hoursbytheend.TheexperimentranuntilOD675nmmeasurementwasdecreasingforallthreebottles.

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3.10 TestofphycocyaninextractionmethodsonArthrospiraplatensis.ObjectiveofexperimentTheobjectiveforperformingthisexperimentwastofindthemostsuitableprocedureforharvestingandextractingthephycocyaninpigmentfromtheA.platensisbiomassandoptimizeitformyexperiment.Thiswasdoneinordertoevaluatethephycocyaninconcentrationpergramofbiomassandthepurityoftheextractedphycocyanin.Theresultsandexperiencefromtheexperimentwereusedtoconductaprotocolforextractingthephycocyanin,whichwasusedinmylaterwork(appendix1).ExperimentalsetupBasedonaliteraturereview,fiveproceduresweredevelopedandtestedonA.platensisbiomass(Horváthetal.,2013;Lawrenzetal.,2011).Theywerenamedmethod1,2,3,4and5.TheexperimentwasperformedintripletsandtheA.platensisbiomasscamefromthesameculturewhichhadaOD675nm=1.562whichisequaltoacelldensityof45.49g/L.Method1Extractbyfiltration.10mLofA.platensisbiomasssamplewasharvestedandthewaterwasremovedbyvacuumfiltrationthroughaGF/Ffilter.Afterfiltrationthefiltersweretransferredtoa50mLfalcontubeandimmediatelyfrozenat-80°Cuntilfurtherprocessing.Thefilterswerethensubjectedtotwofreeze-thawcyclesof24hourseachat-80°Cand5°C.Afterthefreeze-thawcyclesthefiltersweremechanicallydisruptedbygrindingthefilterswithanicecooledmortarandpestleuntilthefilterwascompletelydisintegratedtoahomogeneousslurry.Duringthegrinding0.5ml0,1MphosphatebufferpH6wasadded.Theslurrywasthentransferredtoa50mLfalcontubeand4mLphosphatebufferwasaddedtothefalcontubes.Inordertoremoveallthefilterparticles,thetubeswerefirstcentrifugedfor30minat10000gand4°C.AfterthecentrifugationthesupernatantcontainingthephycocyaninwasvacuumfilteredthroughacleanGF/Ffilterfollowedbyfiltrationthroughasyringefilterwitha0.45μmcelluloseacetatemembranefilter.Thephycocyaninconcentrationandpurityofthesamplewasthendeterminedwiththemethoddescribedabove.Method2Extractionbycentrifugationandonefreeze-thawcycle.10mLofA.platensisbiomasssamplewasharvestedandcentrifugedina15mLfalcontubefor60minat10000gand4°C.Afterthecentrifugationthesupernatantwasdecantedawayandthecellswerere-suspendedin4mL0,1MphosphatebufferpH6andimmediatelyfrozenat-80°C.thesampleswereexposedtoonefreeze-thawcycleof48hourseach.Afterthefreeze-thawtreatmentthesampleswerecentrifugedfor30minat10000gand4°Candthephycocyanincontainingsupernatantwasextractedandfilteredthroughasyringefilterwitha0.45μmcelluloseacetatemembranefiltertoremoveimpurities.Thephycocyaninconcentrationandpurityofthesamplewasdeterminedwiththemethoddescribedabove.

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Method3Extractionbycentrifugationandtwofreeze-thawcycle.Theprocedurewasthesameasinmethod2butwithtwofreeze-thawcyclesof48hoursinsteadofone.Method4Extractionbycentrifugationandthreefreeze-thawcycle.Sameprocedureasinmethod2butwiththreefreeze-thawcyclesof48hoursinsteadofone.Method5Extractionbycentrifugationandsonication.10mLofA.platensisbiomasssamplewasharvestedandcentrifugedina15mLfalcontubefor60minat10000gand4°C.Afterthecentrifugationthesupernatantwasdecantedawayandthecellswerere-suspendedin4mL0.1MphosphatebufferpH6andsonicatedfor60secondsat50W.Thesampleswereimmediatelyfrozenat-80°Candexposedtoonefreeze-thawcycleof48hours.Thedeterminationofphycocyaninconcentrationandpurityofthesampleswerecarriedoutwiththesameprocedureasdescribedabove.

3.11 TestgrowthrateonArthrospiraplatensiswhenculturedAuto-,hetero-andmixotrophic.

ObjectiveofexperimentTheobjectiveofperformingthisexperimentwastotesttheeffectthataceticacidhasonthegrowthrateofA.platensis.TotestthisA.platensiswascultivatedautotrophic,heterotrophicandmixotrophicwithaceticacidavailableasorganiccarbonsourceforhetero-andmixotrophiccultures.ExperimentalsetupTheexperimentwasperformedintripletsin250mLbluecapbottlesunderdifferentconditions:(A)autotrophiccultivationwith100mLBZmediumineachbottle.(B)mixotrophiccultivation100mLBZmediumwithaconcentrationof2g/Laceticacidineachbottle.(C)heterotrophiccultivation100mLBZmediumwith2g/Laceticacidconcentration.ThepHwasadjustedtoaroundpH9-9,5witha1Mpotassiumhydroxide(KOH)solution.Theystartedwith5mLinoculumwithacelldensityof41.14g/Landgrownasbatchcultures.CultureAandBwasinstalledinanincubatorroom(temperature30±2°C)withagitation(180RPM)andunderconstantartificiallighting(L2-LEDlightpanels839lux).CultureCwaswrappedwithaluminiumfoilandcultivatedwithoutlightintheincubatorroom(temperature30±2°C)withagitation(180RPM).Theywerecultivatedfor25daysandcellconcentrationwasmonitoredeverydaybymeasuringtheOD675nmwiththemethoddescribedearlier.Attheendoftheexperimentthegrowthrateandthephycocyaninconcentrationwasdeterminedinanexcelsheetasdescribedearlier.

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3.12 TestgrowthforArthrospiraplatensisinamediumfor

Acetobacteriumsuppliedwithacetate.ObjectiveofexperimentTheobjectiveofthisexperimentwastotestwhetherA.platensiscangrowinDSMZ135aAcetobacteriummediumsuppliedwithaceticacidindifferentconcentrations(2g/L,3g/Land4g/L).ThiswasconductedtosimulatethefermentationbrothfromanaerobicfermentationwithAcetobacteriumsp.(DSM:2396)andtotestwhetherA.platensiscangrowinitornot.Thelowestconcentrationofaceticacid(2g/L)inthisexperimentwaschosenbecauseA.platensishaspreviouslyshowntogrowwellatthatconcentration.AlsoIhavepreviouslytriedtocultivateAcetobacteriumsp.inDSMZa135mediumandreachanacetateconcentrationof1.9g/L.ExperimentalsetupTheexperimentwascarriedoutasbatchcultivationin250mLbluecapbottlesandperformedintriplets.ThemediumusedintheexperimentwasDSMZAcetobacteriummedium135a,exceptresazurin(usedtoindicateifO2ispresent),Ethyleneglycerol(carbonsourceandIassumethatIwillbeusedupinthefermentationbroth)andSodiumsulphate(usedtohelpkeepthemediumanaerobic).ThecompositionofDSMZ135amediumcanbefoundintable5,6and7.Eachbottlehad100mlDSMZ135amediumwithdifferentaceticacidconcentration:(A)2g/Laceticacid,(B)3g/Laceticacidand(C)4g/Laceticacid.ThepHwasadjustedtopH9with1Mpotassiumhydroxide(KOH).10mLfromthreestartercultureswasusedtostarttheexperimentwithcelldensity:(A)starterculture45.07g/L.(B)starterculture38.25g/L.(C)starterculture26.04g/L.Afterinoculationthebottleswereplacedinanincubatorroom(temperature30±2°C)withagitation(180RPM)andunderconstantartificiallighting(L2-LEDlightpanels839lux).GrowthwasmonitoredbyOD675nmmeasurementasdescribedearlier.

3.13 Mixotrophiccultivationwithdifferentconcentrationofacetate.

ObjectiveofexperimentTheobjectiveofthisexperimentwastoinvestigatewhateffectdifferentconcentrationofacetatewillhaveonthebiomassproduction.Inordertodothis,Iwilltryfourdifferentconcentrationofacetate(1g/L,2g/L,3g/Land4g/L)tofindtheacetateconcentrationbestsuitedformixotrophiccultivationofA.platensis.ExperimentalsetupTheBZmediumwaspreparedasdescribedinthesectionculturemediabutinsteadofusingaceticacidinthemedium,1MsodiumacetatesolutionwasusedinsteadtosupplytheBZmediumwithanorganiccarbonsourceformixotrophiccultivation.Thiswasconductedbasedonexperiencefromtheauto-,heteroand

31

mixotrophiccultivationexperimentwithA.platensiswhereaceticacidwasusedasorganiccarbonsource.Theexperimentwasperformedintripletsandcultivatedasbatchin250mLbluecapbottleswithdifferentacetateconcentrationinthemediumandonewithoutacetate:(A)autotrophicculturewith100mLBZmedium.(B)mixotrophicculture,100mLBZmediumwithanacetateconcentrationof1g/L.(C)mixotrophicculture,100mLBZmediumwithaacetateconcentrationof2g/L.(D)mixotrophicculture,100mLBZmediumwithaacetateconcentrationof3g/L.(E)mixotrophicculture,100mLBZmediumwithaacetateconcentrationof4g/L.Theyallstartedwith10mLinoculumwithacelldensityof52.70g/Landplacedinanincubatorroom(temperature30±2°C)withagitation(230RPM)andunderconstantartificiallighting(L2-LEDlightpanels839lux).GrowthwasmonitoredbyOD675nmmeasurementasdescribedearlier.Attheendoftheexperimentphycocyanincontentfromthebiomasswasdetermined.

3.14 Testoflowilluminationwithintheredandbluelightspectraina

batchPhotobioreactorsetup.

ObjectiveofexperimentTheobjectiveofperformingthisexperimentwastotestA.platensisinBZmediumsupplementedwithacetateatlowlightintensitieswithredandbluelight.Thiswasconductedtoseewhataffectmixotrophiccultivationwillhaveonthephycocyaninconcentrationandthecelldensitywhencultivatedatlowlightintensities.ExperimentalsetupTheexperimentwascarriedoutasbatchcultivationina2litrebluecapbottlewith1500mLBZmediumandanacetateconcentrationof1g/L.Thephotobioreactorwasstartedwith150mLstarterculturewithacelldensityof39.37g/L.Aftertheinoculationthereactorwasplacedinanincubatorroom(temperature30±2°C)underconstantilluminationwithredandbluelightbyL1lightingplatform(349lux).Thephotobioreactorwasmixed/agitatedbypumpingatmosphericairthroughtheaquaticphase.Theevaporationofwaterfromtheaquaticphaseinthephotobioreactorwasadjustedbytheadditionofsterilewater,whichwascorrectedaccordingtomarksplacedonthesideofthereactor.TheadditionofsteriledH2Owascarriedouteveryday.Liquidsampleswerecollectedfromtheculturebrothwithatimeintervalof48hourstodeterminebiomassconcentration,pHandresidualacetateconcentration.Theexperimentranfor35daysandattheendoftheexperimentthephycocyaninconcentrationwasdetermined.

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3.15 Testoflowilluminationwithintheredlightspectrainabatch

Photobioreactorsetup.

ObjectiveofexperimentThisexperimentwascarriedouttotestA.platensisinBZmediumsupplementedwithacetateatlowlightintensitieswithonlyredlight.Thiswasconductedtoseewhataffectmixotrophiccultivationwillhaveonthephycocyaninconcentrationandthecelldensitywhencultivatedmixotrophicwithonlyredlightandlowillumination.ExperimentalsetupTheexperimentwascarriedoutunderthesameconditionasforthebatchphotobioreactorwithredandblueilluminationexceptthatinthisexperimentalsetupthephotobioreactorwasilluminatedonlywithredlightbyL1lightingplatform(175lux).Thephotobioreactorwasstartedwithastarterculturewithcelldensity44.34g/L.

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4 ResultsanddiscussionInthissectiontheexperimentalresultsandexperiencesmadefromtheexperimentalworkwillbecommentedonanddiscussedandwillformthebasisfortheconclusion.Alltheexperimentalresultsandcalculationscanbefoundinappendix2.

4.1 Acetobacteriumsp.cultivationTheAcetobacteriumsp.startedfromafreezecultureandwassubsequentlycultivatedforabout1month.ThepurposeofthiswastousethemasastartercultureforaceticacidproductioninalargervolumewheretheeffluentfromthefermentationcouldbeusedtotestwhetheritwassuitableasasubstrateforamixotrophiccultivationofA.platensis.Duetomanyotherexperimentalsetupsandaboundedamountoftime,itwasdecidedtofirsttestA.platensisinthe135amediumenrichedwithaceticacidtoseeifthemediawouldbeinhibitorytoA.platensisbeforeafermentationofacetateinalargervolumewouldbecarriedout.Therefore,anHPLCanalysisfromthestarterculturewascarriedouttodeterminetheacetateconcentrationinthestartercultures.TheHPLCanalysisshowsthattheAcetobacteriumhaveproducedacetate,andthattheacetateconcentrationwas1.9g/L.Thisacetateconcentration(1.9≈2g/L)wasusedtodesignotherexperimentalsetupsformixotrophiccultivationofA.platensis.

4.2 TestofgrowthkineticforArthrospiraplatensisInfigure6,wecanseethatthegrowthcurveisdividedintwobyareddottedline.ThisistoindicatewhenanextraLEDlightsourcewasaddedtotheexperimentalsetupandtoindicatetwodifferentgrowthtrends,oneuptoday16andoneafterday16.Theextralightsourcewasaddedtotheexperimentalsetupbecauseafter15daysofcultivationthegrowthcurveresembledazeroorderreaction,whichcouldindicatethatsomethingwasinhibitingtheprocess.Thegrowthrateincreasedsignificantlyafterday16andtheextralightsourcewasappliedtotheexperimentalsetup,whichshowsthatlightwasthelimitingfactorandthattheL2-LEDlightpaneldoesnotprovideenoughenergy(839lux)tosupportA.platensisfullphotosyntheticpotential(table.9).

Sample Xmax1 Xmax2 P1 P2 μmax1 μmax2

[g/L] [g/L] [gL-1/d] [gL-1/d] [d-1] [d-1]A.platensis 18.47±1.63 69.88±4.91 1.01±0.09 7.91±1.00 2.87±0.08 3.80±0.08

Table9showsthegrowthparametersforA.platensiscultivatedinabasalZarroukmedium.

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Figure6.showsthebiomassproductionofArthrospiraplatensiscultivatedinbasalZarroukmedium.The

dottedredlineindicateday16whereandextralightsourcewasadded.

Figure7showsthephycocyaninyieldfrom1grambiomass[mgPC/gX]from5differentextractionconditions.

4.3 TestofphycocyaninextractionmethodsonArthrospiraplatensis.DuringthetestingofdifferentextractionmethodsofphycocyaninfromArthrospiraplatensis,severalobstaclesemergealongthewayandwillbediscussedinthissection.Thiswascarriedouttofindthemostsuitedmethodforextracting

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phycocyaninfromA.platensisthatwouldbeusedtodeterminethephycocyanincontentinthebiomassthroughoutthiswork.Infigure7itcanbeseenthatmethodM5(sonicationwithonefreeze-thawcycle)gavethehighestphycocyaninyieldpergrambiomass(1.402±0.096[mg/g])ofallthefiveextractionmethodstested,andwithastrongstatisticalevident(T-Test:0.01<p<0.05)whentestedagainstmethodM3(twofreeze-thawcycles)andM4(threefreeze-thawcycles)whicharetheonesclosesttoM5(Figure7).ExtractionmethodM5hadthebestresultswithinthe3parameters(concentration,yieldandpurity)usedtoevaluatethe5extractionmethodsandwascalculatedbasedontheAbs(620nmand280nm)measurementoftheextractedphycocyaninsamples,whichareshownintable10.Thereisnostatisticalevidence(T-Test:p>0.10)thatcouldsupportthatMethodM3ismoreefficientthanM4becausetheirresultsareveryclosetoeachotherandmethodM3hasaverylargestandarddeviation(figure7).However,theresultsobtainfromM3andM4couldindicatethatthethirdfreeze-thawcycledoesn’thaveanysignificanteffectontheextractionofphycocyaninandthereforeareredundantfortheextractionprocess.Thelargestandarddeviationsobservedforthecentrifugedsamplesisproperlycausedbydifficultyofgettingthebiomasssamplesseparatedintotwoperfectphases(solidandliquid)bycentrifugationandthereforecouldeasilylosesomeofthebiomasswhentheliquidphaseisdecantedaway.ExtractionmethodM2hadthelowestphycocyaninyieldofthecentrifugedsamplesandalsoalargestandarddeviation.However,itcanbeseenfromtheresultsthatthereisapositiveeffectontheamountofextractedphycocyaninifthebiomasssamplesareexposedtotwofreeze-thawcyclesinsteadofonefreeze-thawcycleandwithgoodstatisticevidence(T-Test:0.01<p<0.05)(table10).However,thereisnostatisticalevidenceindicatingwhethertwoorthreefreeze-thawcycleshasthebesteffectontheamountofextractedphycocyanin(T-Test;p>0.10).Itiscleartoseefromfigure7andtheresultsshownintable10thatextractionofphycocyaninbyfiltrating(methodM1)thebiomasssamplesisnotasefficientascentrifugingthesamples.Thismaybecausedbythewaythefiltratedsamplesarestoredunderthefreeze-thawcycles.WhenthebiomasssampleswerefilteredthroughaGF/Ffilterpapertheyweresubjectedtofreeze-thawcycleswithoutaphosphatebuffer,whichcouldhavecausedadegradationofthephycocyaninpigmentduringthethawperiodinthefreeze-thawcycle.AsecondfactorthathasanegativeeffectontheconcentrationofphycocyaninextractedinmethodM1isduetothelossofbiomassinthemortarwhentransferringtheslurry(biomassandfilterpaper)toacentrifugetubeafterthemechanicaldisruptionoftheGF/Ffilterpapersinthemortar,therebyalsocontributingtoaloweryield.

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Sample Variable Phycocyanin Yield Purity

[mg/mL] [mgPC/gX] [A620nm/A280nm]

M1 Filtration 0.003±0.000 0.058±0.004 0.18±0.03M2 Onefreeze-thawcycle 0.019±0.010 0.428±0.217 4.44±0.83M3 Twofreeze-thawcycles 0.053±0.005 1.171±0.103 7.57±1.57M4 Threefreeze-thawcycles 0.052±0.001 1.141±0.031 7.33±3.36M5 Sonication,onefreeze-thaw

cycle0.064±0.004 1.402±0.096 8.94±4.82

Table10showstheresultfromthetestof5extractionmethodsfroma10mlbiomasssampleswithacell

densityof45.49g/L.

Basedontheresultsofthetestofextractionmethodsitcanbeseenthatsonicationfollowedbyonefreeze-thawcycleisthemosteffectivemethodtodisruptthecellandextractthephycocyaninpigmentfromthebiomass(table10).TheseresultscorrespondwellwiththeobservationsfromLawrenzandHorvathastudieswherebothsuggestedthatonefreeze-thawcyclefollowedbysonicationwasthemostefficientextractionmethodforphycocyaninextractionfromfilamentouscyanobacteria(Horváthetal.,2013;Lawrenzetal.,2011).TheexperienceandresultsobtaineddoingthistestofextractionmethodswereusedtoconstructaprotocolforextractionphycocyaninpigmentsfromtheA.platensisbiomass(appendix1).TheextractionprotocolwasusedtoextractphycocyaninpigmentfromtheA.platensisbiomassthroughoutthisproject.

4.4 TestgrowthrateonArthrospiraplatensiswhenculturedAuto-,hetero-andmixotrophic.

ThegrowthcurveofArthrospiraplatensiscultivatedunderthreeconditions:(A)autotrophic,(B)mixotrophicand(C)heterotrophicareshowninFigure8.Fromthegrowthcurve(Figure8)itcanbeseenthatthemixotrophic(B)cultureshadalackphaseinthebeginningofthecultivation(day0today4),whichismostlikelyduetotheaceticacidinthemediumandmetabolicadjustmenttothenewsubstrate.Theheterotrophic(C)culturesshowednegativeornogrowthandthesmallvariationsonthegrowthcurve(Figure8)aremostprobablycausedbycelldebrisfloatingaroundinthegrowthmedium.TheresultsfortheheterotrophicandmixotrophicculturescorrespondwellwithRichmondandSoederwhowritethatA.platensisisanobligatephotoautotrophandcannotgrowinthedarkwithorganiccarbonsources(heterotroph)butcanutilizeorganiccarbonsourceswhencultivatedwithlight(mixotrophic)(RichmondandSoeder,1986).TheresultsarealsoinagreementwithHaxthausenwhoshowedthatwhenChrorellasorokinianawascultivatedheterotrophicinthedarkwithacetateasanorganiccarbonsource,thegrowthwasnegativeorequaltozero(Haxthausen,2015).TheresultsdonotfitwithMarquezresults,whichshowedthatA.platensiscanbecultivatedheterotrophicinthedarkinanutrientmediumenrichedwithglucoseasorganiccarbonsource

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(Marquezetal.,1993).ThiscouldindicatethatA.platensisisabletouseglucose(sugar)ascarbonsourcetosupportgrowthwhencultivatedheterotrophicinthedarkbutnotacetate(carboxylicacid)whencultivatedwithoutlight.AsA.platensiswasabletogrowwithacetateasacarbonsourcewhencultivatedmixotrophicwithlightbutnotheterotrophicwithoutlightcouldindicatethatA.platensiscanmetabolisesugarsintoenergybutnotacetateintoenergy,andinsteadstoreitascellularcarboninsteadofmetabolisingittoCO2.Thisisonlyapresumedconclusionandwillrequirefurtherresearchinordertoprovideananswer.Ascanbeseenfromthegrowthcurve(Figure8)noneoftheculturesreachedastablegrowthtrend(exponentialphase),whichisproperlycausedbythelowillumination(839lux)thatalsowasdiscussedintestforgrowthkineticofA.platensis.Therefore,thespecificgrowthratewascalculatedbylinearregressionofthedataplots(Figure8).

Figure8showsbiomassproductionofArthrospiraplatensiscultivatedA)autotrophic,B)mixotrophic(2g/L

aceticacid)andC)heterotrophic(2g/Laceticacid).

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Sample Xmax P μ

[g/L] [gL-1/d] [d-1]A 49.23±2.84 2.08±0.12 1.91±0.29B 61.59±4.16 2.61±0.16 2.78±0.32

Table11showsthegrowthparametersforArthrospiraplatensiscultivatedA)autotrophicandB)mixotrophic.

Xmax=maximumbiomassconcentration,μ=specificgrowthrate,P=productivity.

Themixotrophic(B)cultureshadahigherspecificgrowthratethantheautotrophic(A)culturesandwithgoodstatisticalevidentfortheresults(T-Test;p=0,03)(table11).TheseresultsareinagreementwiththosefromVonshakwhoalsofoundthatmixotrophiccultivationofA.platensiswith2g/Lglucoseinthegrowthmediumhadahighergrowratethanthosecultivatedautotrophic.Vonshakalsosuggestedthatthemixotrophicculturesrequiredlesslightforgrowth,althoughmixotrophicculturesdonotutilizelightenergymoreeffectivelyatlowlightlevelsbutareabletousemorelightenergythantheautotrophiccultures(Vonshaketal.,2000).Ascanbeseenfromthep-valuesonthegrowthcurve(Figure8)thereisnostatisticalevidenceforthebiomassconcentrationbetweentheautotrophicandmixotrophicculturesbeforeday21.After23daysofcultivationthebiomassconcentrationforthemixotrophic(B)cultureswas61.59±4.16g/Landtheautotrophic(A)cultures49.23g/L(T-Test;p=0,02)(table11).Theaverageacetateconsumptionrateforthemixotrophiccultureswas0.021g/L.Themixotrophic(B)cultureshadaproductivityof2.61±0.16[gL-1/d],whichisapproximately20%higherthantheautotrophic(A)culturesandwithstrongstatisticalevidencefortheresult(T-Test;p=0,01)(table11).Intable12theresultsofphycocyaninextractionfromtheautotrophicandmixotrophicculturesareshown.Fromtheresultsshownintable12itdoesnotseemthatmixotrophiccultivationwithacetatehasaneffectonthephycocyaninconcentrationpergramofbiomassandthepurityofthephycocyaninisalsoveryhighforbothsamples(table12).However,themixotrophic(B)sampleshavearelativelyhighstandarddeviation,althoughthisisprobablycausedbylossofbiomassduringtheextractionprocess.

Sample Phycocyanin Yield Purity

[mg/mL] [mgPc/gX] [A620nm/A280nm]

A 0.039±0.002 0.801±0.006 7.89±1.22B 0.049±0.009 0.798±0.199 10.53±4.73

Table12Showsthephycocyanincontentinthebiomassatday23.A)autotrophiccultivatedinbasalZarrouk

medium,B)mixotrophiccultivatedinbasalZarroukmediumwith2g/Laceticacid.

Fromtheresultsitdoesnotseemasthoughmixotrophiccultivationwithacetatehasaneffectonphycocyanincontentpergramofbiomass.However,itdoeshaveasignificanteffectonbiomassproductivity.

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Basedontheresultsitcannotbesaidwhethermixotrophiccultivationwillhaveanyeffectonthemaximumbiomassconcentrationastheexperimentonlyranfor23daysandthereforeitisnotknownwhetherthemixotrophiccultureswouldgetahigherbiomassconcentrationthantheautotrophicculturesovertime.However,basedontheexperimentitisconcludethatA.platensiswhencultivatedmixotrophicwithaceticaciditcanachieveahighbiomassconcentrationfasterthanbyautotrophiccultivation.TheseresultsindicatethepotentialofaceticacidasanorganicsubstrateformixotrophiccultivationofS.platensistoincreasebiomassproductivity.

4.5 TestgrowthforArthrospiraplatensisinamediumusedto

Acetobacteriumsuppliedwithaceticacid.

Figure9ShowsArthrospiraplatensistestedinDSMZAcetobacteriummedium135amixedwithaceticacid.The

figureshowsthebiomassconcentrationin3differentaceticacidconcentration.

Figure9showsthattheArthrospiraplatensiswereunabletogrowthinDMSZ135amedium.ThiscouldindicatethatsomethingintheDMSZmediumhasaninhibitoryeffectonA.platensis,sincepreviousexperimentswithaceticacidinthebasalZarroukmediumhaveshownthataddingaceticacidtothegrowthmediumhasapositiveeffectongrowthforA.platensis(figure8).Therefore,itmustbesomethingintheDMSZmediumthatA.platensiscan’ttolerateoranabsenceinthemediumsinceA.platensiscan’tgrow.Cysteine-HClcouldbetheinhibitingfactorintheDSMZmediumasitisarelativelystrongreductant,however,thiswouldrequirefurtherinvestigationstodeterminewhetherthisistrue.Afterinoculationthecellconcentrationdropped.Thenafteronly2daysofincubationthecellsconcentrationwasalmostzeroforallbottles,whichshowsthatallthecellsweredead(figure9).Thiscanalsobeseenfrompicture5whichshowswhatbelievedtobeadeadA.platensiscellthepictureisfromoneofcultureAbottlesafter9daysofincubationinDSMZ135amedium.

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BasedonthisexperimentitcanbeconcludedthatA.platensiscannotgrowintheDSMZAcetobacterium(135a)medium.InordertousethecombinationtoproduceacetatewithAcetobacteriumst.andusetheeffluenttoincreasethebiomassproductivityofA.platensisthemediumusedforproducingacetatemustbemodifiedoranewmediumshouldbeusedbeforetheeffluentcanbeusedtocultivateA.platensis.

Picture5.ShowafilamentofArthrospiraplatensisseenthroughamicroscope(40x)frombottleA(2g/L

acetate)after9dayscultivationinDSMZ135aAcetobacteriummedium.(photobyT.Rybner).

4.6 Mixotrophiccultivationwithdifferentconcentrationofsodium

acetate.

Theobjectiveofthisexperimentwastotestfourconcentrationsofacetate(1,2,3,4[g/L])inZarrouk’sgrowthmediuminordertofindtheconcentrationbestsuitedformixotrophiccultivationofA.platensismeasuredbygrowthrate,productivityandmaximalbiomassconcentration.AsodiumacetatesolutionwasusedinsteadofaceticacidasitmakesiteasiertocontrolpHwhenpreparingthegrowthmedium.Duetoanerrormadeinthelaboratory,theacetateconcentrationbecameonlyonetenthofwhatwasplannedsoinsteadof1g/Litbecame0.1g/Lacetateandsoon.Therefore,thisexperimentcannotbeusedtodeterminewhichacetateconcentrationwasbestsuitedformixotrophiccultivationofA.platensis.Insteadtheexperimentwillbeusedtolookatphycocyanincontentinthebiomass,autotrophicandmixotrophiccultivationandtheacetateconsumptionrateforthemixotrophiccultures.

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Figure10showsthegrowthcurveforArthrospiraplatensiscultivatedautotrophic(A)andmixotrophic(Band

C).Theverticalredlineindicateday8.TheP-valuesforbiomassconcentrationatday8and23areshown

downintherightcorner.

Figure11showsthegrowthcurveforArthrospiraplatensiscultivatedautotrophic(A)andmixotrophic(Dand

E).Theverticalredlineindicateday8.Thep-valuesforbiomassconcentrationatday8and25areshowndown

intherightcorner.

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Theautotrophiccultures(A)hadthelowestgrowthrate,productivityandachievedthelowestbiomassconcentration,althoughthedifferencetothemixotrophiccultures(B,C,D,E)areverysmallandtheresultsareallveryclose(table13).Thisisprobablybecausethemixotrophiccultureshadtoolittleacetateandthereforetheacetateconcentrationinthegrowthmediumwasnotenoughtomakeasignificantdifferenceongrowth.Therewasastopinthegrowthforallculturesonaroundday8,whichisindicatedbyareddottedlineonthegrowthcurvesinfigure10and11.Thisisprobablycausedbydifficultytakinganentirelyhomogeneoussamplefromtheculturebottlesandthatthesamplesweretakenwithonlyonedayapartinsteadoftwodays.CulturesA,BandChadmaximumbiomassconcentrationsaroundday23andDandEaroundday25(figure10and11).Thebiomassconcentrationsintheculturesdifferconsiderably,whichisalsoreflectedinerrorbarsandp-valuesinfigure10and11.Thisisprobablyduetodifferenceingrowthratebetweentripletcultures.Thedifferenceingrowthratebetweensamplescouldbeduetoanunequaldistributionoflightbetweensamplesbecauseofhowtheywereplacedintheshakerduringincubation(picture6).Therefore,thebottlesweremovedaroundduringcultivationtotrytocompensatefortheunequallightdistributionintheshaker.

Picture6showshowthebottleswereplacedintheshakerduringcultivationofA.platensiswithdifferentacetateconcentration.

Astherewasnoclearexponentialgrowthphaseprobablyduetolowillumination(839lux)(figure10and11).Thespecificgrowthratewascalculatedbylinearregressionofthedatasetsfromday0to20fortheautotrophiccultures(A)andfromday0to17forthemixotrophiccultures(B,C,D,E)(table13).

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Sample Xmax P μ

[g/L] [gL-1/d] [d-1]A 60.12±2.91 2.80±0.12 3.04±0.16B 64.55±1.31 3.18±0.23 3.36±0.25C 61.63±3.13 2.98±0.23 3.15±0.31D 64.18±1.12 3.07±0.25 3.05±0.18E 64.22±2.70 3.02±0.10 3.09±0.09

Table13showsthegrowthparametersforArthrospiraplatensiscultivatedautotrophicandmixotrophic.Xmax

=maximumbiomassconcentration,μ=specificgrowthrate,P=productivity

Theacetateconsumptionrateforthemixotrophicculturesisshowninfigure12andwasdeterminedbylinearregressionofacetateconcentrationVStime.Theacetateconsumptionrateisalmostthesameforallcultures,respectivelyB=0.017,C=0.017,D=0.018,E=0.019day-1.TheseresultscouldindicatethattheconsumptionrateofacetateforA.platensiscultivatedmixotrophicdependsontheilluminationandnottheacetateconcentrationinthemedium.However,thiswillrequirefurtherinvestigation.

Figure12showtherateofacetateconsumptionbyArthrospiraplatensis

Phycocyaninyieldismoreorlessthesameforallcultures,andpurityofthephycocyaninsamplesisalsohighforallcultures(table14).ThephycocyaninyieldwasmuchlowerforA.platensisinthiscultivationthaninpreviouscultivations.Thebiomasssampleswerebyamistakeonlysonicatedfor30secondsat30Winsteadof60secondsby50W,astheyshouldhavebeenthereforetheyweresonicatedagainfor30secondsat50Wwhichmayhaveinfluencedtheresultsand,thereforetheresultscannotbeusedtocomparewithpreviousexperiments.Thedifferenceinphycocyaninconcentrationpergrambiomassandthepurityofthesamplesismorelikelyduetofactorassociatedtotheextractionprocessthanthecultivationconditions.

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Sample Phycocyanin Yield Purity

[mg/mL] [mgPc/gX] [A620nm/A280nm]

A 0.020±0.001 0.342±0.022 5.96±0.94B 0.021±0.001 0.344±0.028 6.84±2.27C 0.022±0.001 0.359±0.037 6.02±1.75D 0.023±0.001 0.386±0.009 10.37±5.54E 0.019±0.001 0.318±0.008 8.55±0.22

Table14Showsthephycocyanincontentinthebiomassatday20.

Theresultsofthisexperimentcouldindicatethatacetateaddedtothegrowthmediumincreasesthegrowthrate,productivityandmaximalbiomassconcentrationofA.platensis.However,thereisnostatisticalevidenceofthisstatementfromtheseresults(T-Test:p>0.10).Theresultscouldalsoindicatethatthereisacorrelationbetweenacetateconsumptionandlightintensity,althoughitisonlycircumstantialevidenceandwillrequirefurtherinvestigation.

4.7 Photobioreactorcultivationwithlowilluminationintheredand

bluelightspectra.

ForthephotobioreactorexperimentthecultivationofA.platensiswasperformedintwoexperimentalsetupswithdifferentilluminationlevels,respectively175luxatonlyredlightand349luxbyacombinationofbothredandbluelight.Inthisdiscussion,theywillbereferredtoasRLforreactoratlowillumination(175lux)andRHforreactoratthehighillumination(349lux).

Picture7showsthephotobioreactorRHandsystemusedtoextractbiomasssamplesduringcultivation.

RH=Reactorhighillumination(349lux)redandbluelightspectra.(photobyT.Rybner)

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Figure13showsthegrowthcurveforArthrospiraplatensiscultivatedmixotrophicwith1g/Lacetateunder

differentillumination.RH=Reactorhighillumination(349lux)redandbluelightspectra,RL=Reactorlow

illumination(175lux)redlightspectra.

Fromthegrowthcurve(figure13)ofthetwomixotrophiccultivationsofA.platensisthatisusedtoillustratetheirgrowthpatternsitcanbeseenthatgrowthwasalmostidenticalforbothprocesses.Fromthegrowthcurveitcanalsobeseenthatbyday33anduntiltheendoftheexperimentRHincreasedsuddenly(figure13).ItisdifficulttosaywhatthesuddengrowthinRHwascausedby;however,onereasoncouldbethewaybiomasssampleswereextractedduringcultivation.Duringcultivationthebiomasssamplesweretakenasepticallywitha60mLsyringethroughatubeinthelidofthephotobioreactor(Picture7).Attheendoftheexperimentthelidwasremovedandthebiomasssampleweretakendirectlyfromthephotobioreactor.Extractionofsamplesthroughthetubemayhavecausedproblemsofgettingahomogenoussampleasthebiomassdensitybecamehighercausingthebiomass(cells)toclomptogetherandtherebymakingbiggerclompsofbiomassthatwereunabletobeextractedthroughthetube.Alargeclumpofbiomassinthesampletakenattheendoftheexperimentcanthereforebethereasonforthesuddenincreaseinthebiomassconcentration.GenerallyithasbeendifficultthroughoutthisstudyofA.platensistodeterminetheexactbiomassconcentrationbecauseA.platensistendtoclumptogethermakingitdifficulttogetacompletelyhomogenoussampletaken.TheproblemofA.platensisclumpingtogetheralsomeansthatwhenthebiomassconcentrationisdeterminedbyODmeasurementsinaspectrophotometer,themeasurementsdeviatealotandalsocontributestotheuncertaintyofthebiomassconcentration.ThiscouldperhapsofbeensolvedbydisruptingthecellsfirstbysonicationbeforetheODmeasurementtoensureahomogeneoussample.

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Theseproblemsalsoemphasizethenecessityofhavinganeffectivecirculationinaphotobioreactor,whichwillalsoensureabetterdistributionofnutrientsandlightenergy(photons)tothephototrophicmicroorganismcultivated.TheimportanceofcirculationandhomogeneityinthephotobioreactorisalsooneofthethingsthatPulzhighlightsinhisreviewofproductionsystemsforphototrophicmicroorganismsasaveryimportantfactortoensureoptimalcultivationconditionsinaphotobioreactor(Pulz,2001).

Sample Xmax P μ

[g/L] [gL-1/d] [d-1]RH 33.75 0.89 0.62RL 24.00 0.59 0.56

Table15showsthegrowthparametersforArthrospiraplatensiscultivatedmixotrophicwith1g/Lacetate

underdifferentillumination.RH=Reactorhighillumination(349lux)redandbluelightspectra,RL=Reactorlow

illumination(175lux)redlightspectra.Xmax=maximumbiomassconcentration,μ=specificgrowthrate,P=productivity.

RHachievedthehighestbiomassconcentrationof33.75g/LwhereasRLonlyreached24.00g/L(table15).ThehighestspecificgrowthrateandproductivitywasalsoobservedinRH(table15).TheseresultsareperhapsalittlemisleadingsinceRHandRLwerealmostidenticaluntilday33asseenfrombiomassconcentrationmeasurements(figure13).However,theactualbiomassconcentrationinRHandRLmayhavebeenhigherforthesamereasonasjustdiscussedaboveabouthomogeneityofthebiomasssamplesfromthephotobioreactor.Asthephotobioreactorexperimentswerenotperformedindoubletsortriplets,thereisnostatisticalevidencetosaywhethertheresultsaresignificant.ComparedwiththeothercultivationexperimentsofA.platensisperformedinthisstudy,theRHandRLexperimentsachievedonlyapproximately50%ofthebiomassconcentrationthatwasachievedinthepreviousautotrophicandmixotrophiccultivationofA.platensisatalightintensityof839lux(table9,11and13).Duringtheexperiment,thepHincreasedinbothRHandRLandattheendoftheexperiment(day35)thepHvalueinRHwaspH10.3andpH10.4inRL.Infigure14,theacetateconcentrationinRHandRLareshownovertime.Theacetateconsumptionratewasdeterminedbylinearregression,whichisillustratedbytheredandbluedottedlineinfigure14.TheacetateconsumptionrateisalmostthesameforA.platensiscultivatedinRHredandbluelight(349lux)andRLonlyredlight(175lux).However,theacetateconsumptionratewasslightlyloweratRL(175lux)thanatRH(349lux),respectivelyaround0.004day-1inRLand0.005day-1inRH(figure14).

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Figure14showstheacetateconcentrationinthephotobioreactorovertime.RH=Reactorhighillumination(349

lux)redandbluelightspectra,RL=Reactorlowillumination(175lux)redlightspectra.

Basedontheresultsofacetateconsumptionrate,maximalbiomassconcentrationandlightintensityfromthemixotrophicandheterotrophiccultivationexperimentsconductedinthisstudy,itcouldindicatethattherateA.platensisutilizeacetatedependsinthelightintensity.However,thisisjustanobservationbasedonacetateconcentrationinthemediumandthelightintensityusedduringthecultivationofA.platensisandwillthereforerequirefurtherinvestigationinordertodeterminewhetherthisisthecase.Ascanbeseenfromtable16,theyieldofphycocyanininmgpergramofbiomassisgoodforbothRHandRLcomparedtopreviousphycocyaninyield,yetthepurityofthephycocyaninsamplesisnotasgoodasthepreviousphycocyaninsamples(table12and14).FromtheexperimentalresultsofRHandRLitseemsunlikelythatthereareanypositiveeffectsonthephycocyanincontentinA.platensiswhencultivatedwithonlyredlightastheRLsamplesislowerthanRHsamplesforbothday30andday35(table16).AccordingtoT-TestperformedonthephycocyaninyieldresultsforRHandRLfromday30andday35thereisverystrongstatisticalevidenceagainstthisobservationatday30(T-Test:p<0.01)andstrongstatisticalevidenceagainstthisobservationatday35(T-Test:0.01<p<0.05).However,basedontheresultsfromthisexperimentitcannotbesaidwhetherthisiscausedbytheredlightorthedifferenceinlightintensity.AlthoughresearchconductedbyDanesiwithA.platensisshowedthatlowerlightintensitiesincreasedthepigmentcontent(Danesietal.,2004).Thus,bymakingtheassumptionthatthepigmentcontentshouldbehigheratlowerlightintensitiesandthatredlightshould

48

favourphycocyaninIdonotthinkthatthereisasignificanteffectonthephycocyanincontent,cultivatingA.platensiswithonlyredlight,however,thiswillrequirefurtherexperimenttoconfirm.

Sample Phycocyanin Yield Purity

[mg/mL] [mgPC/gX] [A620nm/A280nm]

RH(day30) 0.022±0.000 1.093±0.021 3.25±2.50RL(day30) 0.016±0.001 0.822±0.032 2.61±0.60RH(day35) 0.030±0.002 0.945±0.065 3.22±0.75RL(day35) 0.017±0.000 0.705±0.004 2.68±0.60

Table16ShowstheresultsfromphycocyaninextractionfromArthrospiraplatensisbiomasscultivated

mixotrophicwith1g/Lacetateunderdifferentilluminationatday30andday35.RH=Reactorhighillumination

(349lux)redandbluelightspectra,RL=Reactorlowillumination(175lux)redlightspectra.

ThebiomasssamplesweretakenfromRLtodeterminethephycocyanincontentinthebiomassatday20,25,30and35.ThiswasconductedtoseeifthereisachangeinphycocyanincontentinA.platensisduringcultivation(table17).Ascanbeseenfromtable17,thereisnosignificantchangeinthephycocyanincontentpergrambiomass,whichisaround0.8mgphycocyaninpergrambiomassforallsamples.Thedifferencebetweenthesamplesisverysmallandisprobablycausedbyvaryingefficiencyoftheextractionprocess(table17).ThisisalsosupportedbyaT-TestperformedonthephycocyaninyieldofA.platensisfromRL,whichshowedthatthephycocyaninyieldwasnotsignificantly(T-Test:p>0.10)influencedovertimeexceptforday35(T-Test:p<0.01).TheseresultsindicatethatthephycocyanincontentisconstantoronlychangelittleinA.platensisduringcultivation.However,thisobservationconflictswithXiewhoreportedthatbyfed-batchcultivationofA.platensisthephycocyanincontentincreasedovertimebutafterawhiledecreasedagain,whichwasattributedtothelowercellviabilityassociatedwiththeextendedcultivationtime(Xieetal.,2015).ThiscouldexplainwhyphycocyaninyieldishigherforbothRHandRLatday30thanatday35,eventhoughthebiomassconcentrationishigheratday35forbothcultures.Whetherthisisthecaseorwhetheritisduetootherfactors,suchaslossofbiomassdoingextractionisnotknown.

Sample Phycocyanin Yield Purity

[mg/mL] [mgPC/gX] [A620nm/A280nm]

RL(day20) 0.012±0.000 0.806±0.017 1.89±0.53RL(day25) 0.016±0.001 0.835±0.036 2.48±0.48RL(day30) 0.016±0.001 0.822±0.032 2.61±0.60RL(day35) 0.017±0.000 0.705±0.004 2.68±0.60

Table17ShowstheresultsfromphycocyaninextractionfromArthrospiraplatensisbiomasscultivated

mixotrophicwith1g/Lacetateunderlowillumination(175lux)redlightspectra.

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5 ConclusionFromthisstudyofmixotrophiccultivationofA.platensisusingacetateasorganiccarbonsourceitseemsunlikelythatacetatehasaneffectonthephycocyanincontentinthebiomass.TheresultsinthestudyalsoindicatethatthephycocyanincontentinA.platensisisconstantoronlychangeslittleduringcultivation,yetdoeschangewithdifferentlightintensities.Furthermore,thisstudyshowsthatthereisnoorlittleeffectonthephycocyanincontentwhencultivatingA.platensiswithonlyredlight,however,itwouldseemthatthereisacorrelationbetweenahighcelldensityandlowerphycocyanincontent.Although,thisitisnotpossibletodeterminefromthisstudy,astherearemanyuncertainfactorsthatmaybeaffectingtheresults.Thebestresultsforextractionofphycocyaninwereachievedbydisruptingthecellwithsonicationfollowedbyonefreeze-thawcycle,whichisinagreementwithwhatsimilarstudieshaveshown.ResultsfromthemixotrophiccultivationofA.platensiswithacetateinthegrowthmediumshowedthatwhencultivatedatlightintensitiesof839luxorlowerA.platensisdidn’treachastablegrowthpattern,indicatingthatlightintensitiesarestillakeylimitingfactorforgrowthofA.platensis.ThestudyalsoindicatedthatA.platensis’sabilitytoutilizeacetateasorganiccarbonsourceisdependentonlightenergy,sincewhencultivatedatlowerlightintensitiestheacetateconsumptionratedecreased.AnotherfactorcontributingtothisobservationwasthatA.platensiswasn’tabletogrowheterotrophicwithacetate.However,A.platensisinsimilarstudieswithglucoseandamylaseinthegrowthmediumhavebeenshowntogrowheterotrophic.ItwasnotpossibletocultivateA.platensisinanAcetobacteriummediumenrichedwithacetate.Ifthishadbeenpossible,itcouldbeusedforconvertingsurplusoutputelectricityfromwindturbinestohighvalueproductssuchasphycocyaniniftheelectricitywasconvertedtohydrogen,whichcouldbeusedtofeedAcetobacteriumtoproduceacetateandusetheeffluentformixotrophiccultivationofA.platensis.Inthisway,surplusproductionfromwindturbinescouldbeusedtoproduceahighvalueproductlikephycocyanin.However,basedonthisstudyanewmediaforAcetobacteriumwouldhavetobeusedinordertofulfilthisidea.FromthisstudyitcannotbesaidwhethermixotrophiccultivationwithacetatecanincreasethemaximumbiomassconcentrationofA.platensis.However,mixotrophiccultivationofA.platensiswithacetatecanincreaseproductivityandinthiswaycontributetomakeitmoreprofitabletoproducephycocyaninbyreducingcultivationtime.TheeconomyofphycocyaninproductionwithA.platensiscouldfurtherbeimprovedbysellingthesolidfraction(celldebris)thatisleftafterthephycocyaninisextracted.Thesolidfractioncontainingcelldebrisisveryrichonproteinandthereforecouldbesoldasfoodsupplements,animalfeed,substrateforbiogasplantsorusedonan

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onsitebiogasplanttoproduceelectricity,whichcouldlowerenergycostfortheproductionfacility.Finally,theresultsfromthisstudyindicateacetatepotentialasanorganiccarbonsourceformixotrophiccultivationofA.platensisandthatacetateissuitableasasubstrateforproductionofhighvalueproductssuchasphycocyanin.

6 PerspectiveForfutureworkwithA.platensisitcouldbeinterestingtolookatfed-batchandcontinuescultivationsincethisisanareawhereitmightbepossibletoincreasetheproductivityandmaximalbiomassconcentrationevenmore(Xieetal.,2015).Subsequently,itcouldalsobeinterestingtoupscaletheprocessandlookmoreintophotobioreactordesignssinceshape,circulation,andilluminationseemtohaveahugeimpactonallgrowthparametersinvolvedinasuccessfulcultivationandthattheparameterswillproperlychancewhenupscalingtoahighervolume.Therefore,thisisanareawhereitreallyispossibletomakeimprovementsthatcouldreallymakeadifferenceandmakephycocyaninproductionmoreeconomicalfeasible(Carvalhoetal.,2011;Pulz,2001).Basedontheexperienceobtaineddoingthisstudyitcouldalsobeinterestingtolookmoreintoextractionmethodsasitultimatelycomesdowntohowmuchphycocyanincanbeextractedfromthebiomass,whichcouldmakeiteasierinfutureworktodeterminetheactualphycocyanincontentinthebiomass.Amajorchallengeintheextractionofphycocyanininthisstudyhasbeentoisolatethebiomasswithoutloss.Thiscouldperhapsbeimprovedbyusingamembraneinsteadofcentrifugationtoseparatethebiomassfromtheliquidwhereithasbeendifficultbycentrifugationtoestablishaclearphaseseparationbetweentheliquidandbiomassthathasresultedinalossofbiomass.Ifamembranecouldbeusedtoseparatetheliquidfromthebiomassandsubsequentlywashingthebiomassoffthemembranewiththephosphatebufferusedintheextractionprocessofphycocyanin,thelossofbiomasswouldbeminimizedensuringahomogeneitybetweensamples.Itcouldalsobeinterestingtofurtheroptimizetheefficiencyofthesonicationbytestingdifferentsonicationtimeandtheintensity(watt)ofthesonicationinordertofindtheoptimalcombination.Inconnectionwiththis,itwouldalsoberelevanttofindabettermethodtooextractmorehomogenousbiomasssamplesandmeasuringthebiomassconcentrationwithopticaldensity(OD).Abettercirculation/stirringinthegrowthmediummightensuremorehomogeneityandextractionofalargervolumeofbiomassthatcouldhelpachieveamoreaccuratesample.TheproblemsassociatedwithODmeasurementscouldperhapsbereducedifthebiomasssamplesweresonicatedbeforeODmeasurementsisperformedinordertoinsuremorestableresults.

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BasedontheresultsfrommixotrophiccultivationofA.platensiswithacetateinthegrowthmedium,itcouldberelevanttolookfurtherintohowA.platensisutilizeacetateunderdifferentlightconditionsastheresultsfromthisstudyindicatethattherewasaconnectionbetweenacetateconsumptionandlightintensities.Inaddition,furtherworkinthiscontextcouldalsolookathowacetateisincorporatedintheTCA-cycleandhowA.platensisutilizedifferentcarbonsourcesbecauseitwasnotpossiblefromthisstudytoexplainwhyA.platensiscouldnotbecultivatedheterotrophicwithacetateasorganiccarbonsourcebutinsimilarstudieshaveshowntogrowthheterotrophicwithglucose(Marquezetal.,1993).ItcouldalsobeinterestingtofurtherinvestigatehowthesurplusproductionfromwindturbinescouldbeconvertedtoacetatethatcouldsupportamixotrophiccultivationofA.platensis.ItwillbenecessarytoinvestigatewhyA.platensisdidnotgrowintheAcetobacteriummediumandhowthisinhibitioncouldbemanaged.Finally,itwouldalsobeinterestingtolookfurtherintotheeffectofdifferentlightspectresandlightintensitiesandtheeffectthatthishasonthepigmentcontentinA.platensis.Inordertoseehowdifferentwavelengthsandintensitiesoflightwouldaffectthepigmentcontentofthepigmentsinvolvedinlightharvestingandnotonlyonthephycocyanincontent.

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8 Appendices

8.1 Appendix1.ExtractionprotocolforphycocyaninExtractionofphycocyaninwithcentrifugationandsonication:

1. extract5-10mLbiomassandtransferto15mlfalcontubesandcentrifugethemfor60minat10,000g(RCF)at4°Candremovethesupernatant(usea1000µLpipettoremovethefloatingcellsfromthesupernatantandpourtherestofthesupernatantaway)

2. Resuspendthebiomassin5mLphosphatebufferpH63. Sonicatethebiomassin60secondat50watt4. Freezeat-80°Cuntilfurthertreatment(min2hours)5. Performonefreeze-thawcycleof48hoursat5°C6. Centrifugeagainfor60minat10,000g(RCF)at4°Candremovethe

supernatantcontainingthephycocyanin7. Removeremainingimpuritiesbyfiltratingthesupernatantthroughaclean

syringefilter(poresize0.45µmcelluloseacetatemembrane)8. Thephycocyaninconcentrationinthesupernatantcannowbedetermined

usingspectrophotometerandtheformulabelowSpectrophotometricquantification:

1. Absorbance(Abs)fromthepurifiedextractsismeasuredat620nminaphotospectrometerin1cmquartzglasscuvettesagainstthephosphatebufferasblank.

2. Purityoftheextractionsampleisdeterminedfromtheratiobetweentheabsorbancemeasuredat620nmand280nm(purity=Abs620nm/Abs280nm)

3. ThephycocyaninconcentrationcanbecalculatedwiththefollowingequationwhichwasfoundbymakingacalibrationcurvefromapurchasedphycocyaninsamplefromSigmaAldrich

4. Gℎ?K#K?%L"L $' $( = l[UmnopI.IA,-q.-OA

× rstuvwx

ryz{{x|, ; = 0,99

8.2 Appendix2.ExperimentaldataandcalculationsAsthisisaneducationwithfocusonsustainabilitytheexcelsheetwasnotprintedonpaperbutinsteadavailableelectronicallyonaUSB-Driveattachedtothereportordownloadedfromthelinkbelow:https://www.dropbox.com/sh/64a0r67itv92q6v/AADiavI6-ICPK7FvnBbMFZkua?dl=0