TheInfluenceofCarbonDioxideonAlgaeGrowth

Thefirstobjectiveofthisexperimentistoshowthatincreasedatmospheric concentrationsofcarbondioxide,CO2,canstimulatealgaegrowth.Thesecondobjective ofthisexperimentistodemonstratetomiddleschoolstudentsthatdissolvedCO2can movefromoneliquidreservoirthroughtheairtoanotherliquidreservoir. Thisexperimentusesaliquidreservoirinaclosedsystemthatcanbemanipulated tochangetheamountofCO2intheair.Theclosedsystemincorporatesabeakeroffresh wateralgae,Closterium,ingrowthmediasittinginsidea1/2galcontainerpartiallyfilled withawaterreservoir(Figure1A).Oncethesystemissetup, Closteriumalgaeisgrown inthepresenceoflightfromafluorescentlightbulbon12hourslightdarkcycle. Additionally,thesystemcanbesetuptocollectoxygengasreleasedfromthealgaeby addingaglassfunnelandaninvertedtesttubefilledwithgrowthmedia(Figure1B).

500mlbeakerorFlask AlgaeCulture

1/2GalScrewCapContainer Reservoir
Water CarbonatedWater 0.4MKOH

Figure1A:Closedsystemforshowingtheaffectofcarbondioxideonalgaegrowth.

InvertedTesttube forcollectingoxygen 600mlbeaker 1/2GalScrewCapContainer Reservoir AlgaeCulture

Water CarbonatedWater 0.4MKOH

Figure1B:Closedsystemforcollectingreleaseofoxygengasbyalgae. Theexperimentconsistsminimallyofthreegroups1)thenormalcontrolwitha reservoirofdistilledwater,2)theexperimentalgroupwithareservoirofcarbonated water,and3)thenegativecontrolwithareservoirof0.4Mpotassiumhydroxide.The normalcontrolofdistilledwaterrepresentsthenaturalconditionofdissolvedgasesin

water.Theexperimentalgroupisa1:2mixtureofbottledcarbonatedwatertodistilled water,whichisenrichedwithCO2gas.Thenegativecontrolwith0.4Mpotassium hydroxidedepletestheairofanyCO2 andshowsthatsomeCO2isrequiredforalgae growth.Carbondioxidereactswithwatertoformcarbonicacid. CO2(g)+H2O(l)H2CO3 (aq) Potassiumhydroxidereactswithcarbondioxide,CO2,toformK2CO3(s). 2KOH(aq)+CO2(g) K2CO3 (aq)+H2O(l) ThepresenceofKOHeffectivelyremovesCO2fromtheatmosphereoftheclosed system.Thisactivityrequiresanadulttosetuptheexperiment,becauseoftheglassware manipulationsandthebasicpotassiumhydroxide.Inourclasses,wehadthestudents makehypotheses,observethealgaecultures,conductparallelsimulations,collectand analyzedata. Materials (6)gallonRubbermaidplasticcontainerswithlids (3)600mlglassbeakers (3)500mlglassflasks (3)glassfunnels (3)12milliliterglasstesttubes (36)15milliliterglasstesttubes (3)testtuberacks 2.0literdistilledwater 0.5literofcarbonatedwater 1.2liter0.4Molarpotassiumhydroxidesolution 1tube50XAlgaGro:(CarolinaBiologicalSupply#HT153751) 101510PlantFood ClosteriumAlgae:(CarolinaBiologicalSupply#HT152115) Parafilm Plasticwrap Glassmarkingpen Fluorescentlight Electrictimer ExperimentalSetup Aweekpriortosettinguptheexperiment,establishunderartificialornatural lightanactivelygrowingClosteriumalgaeculturein100mlof1XAlgaGrowmedium. Thedaybeforetheexperiment,obtaincarbonatedwater,make0.4Mpotassium hydroxide(26.9gofKOHin1.2Lofdistilledwater),andcleanglassbeakers.Beakers forculturingthealgaewerewashedwithdishsoapandthenrinsedthreetimeswith distilledwatertoinsurethatcontaminateswouldnotinhibitalgaegrowth. AlgaeGrowthSystem Thedayoftheexperimentalsetupforalgaegrowth,labeleachofthreelarge plasticcontainers:normalcontrol,experimentalgroup,andnegativecontrol.Intothe normalcontrolcontainer,transfer600mlofdistilledwater.Intotheexperimentalgroup

container,transfer200mloffreshcarbonatedwaterand400mlofdistilledwater.Into thenegativecontrolcontainer,transfer600mlof O.4MKOHsolution.Setupthealgae culturesbylabelingeachofthreeglass600mlbeakersnormalcontrol,experimental group,andnegativecontrol.Filleachbeakerwith400mlofAlgalGromedium.Add1 dropof101510plantfertilizer.Beforetransferringthealgae,stirvigorouslytosuspend thealgaeandthentransfer30mlofactivelygrowingalgaetoeachbeaker.Carefully placethebeakerinsidethelargeplasticcontainer,closethelid,andsealtightlywith parafilm.Placetheculturesnexttoafluorescentlightona12houron/offelectrictimer. Lettheculturesgrowfor7to10days. OxygenCollectionSystem Tocollectoxygengas,thesetupisbasicallythesameexceptaftertransferringthe algaetothebeakerwait10minutesforthealgaetosettletothebottom.Slowlysubmerge aninvertedglassfunnelintoeachalgaecultureuntilthefunnelrestonthebottomofthe beaker.Makesuretheglassstemispartiallysubmergedbeneaththegrowthmedium. Next,fillatesttubecompletelyto thetopwithgrowthmedium,stretchtheparafilmover theopening,andtightlywraptheparafilmaroundthesideofthetesttube.Invertthetube andthereshouldbenoairbubblesinsidethetube,orleakage.Placetheinvertedtube withparafilmovertheglassstem,quicklyandcarefullypuncturetheparafilm,andplace theopeningofthetesttubebeneaththesurfaceofthemedium.Thereshouldbeacolumn ofgrowthmediumwithaminimumamountofair.Marktheamountofairinthetube withawaterproofpen.Closethecontainerandwrapwithparafilm.Repeatfortheother twogroupsandplaceinfrontofalightsource.Also,lettheculturesgrowfor7to10 days. DataCollection Studentsobservedthecolorintensityofthegrowingculturesandrecordedtheir observationsondatasheets.Afteraboutaweekthestudentsconductedtwosimulations ofserialdilutionsoffoodcolorandfiltration.First,topreparethestudentsforlimiting serialdilutions,studentsseriallydilutedoutgreenfoodcolorbypowersof10.The studentsweregivenfour15millilitertesttubesandinstructedtotransfer9millilitersof waterintoeachtesttube.Thestudentswerethengivenatesttubewith10dropsofgreen foodcolordilutedin10millilitersofwater.Thestudentswereinstructedtoremove1 milliliterfromtheconcentratedfoodcolor,transfertothefirstofthefourtesttubes,and mixthoroughly.Thestudentsrepeatedthisprocessbyremoving1milliliterfromthetest tubetheyjustpreparedandtransferringittothenexttesttube.Thisprocesswas continueduntilthefoodcolordisappeared.Thestudentswereabletoseewitheach1:9 dilution,thecolorintensityofthedyedecreaseduntilitdisappeared. Limitingserialdilutionsweredonetodeterminethenumberofalgaegrowingin eachcontainer.Limitingserialdilutionsemployspoweroftendilutionstodeterminethe numberofalgaepermilliliterintheculture.Ataboutthesametimethestudentsare doingtheirserialdilutions,12testtubesarelabeledandfilledwith9millilitersofAlga Gromediumforeachgroup.Removeonemilliliterwithabulbpipettefromthealgae culture,diluteinthefirsttesttube,andmix.Next,removeonemilliliterfromthetesttube containingthealgaethatwasjustdilutedandtransfertothesecondtesttube.Repeatthis processthroughthenext12testtubes.Whenfinished,lightlywrapthetopofthetest

tubeswithplasticwraptopreventdustandsporesfromenteringtheculture.Placethe culturesinfrontofthelightsourcefor710days.Theculturescanbeobservedevery otherdaytoseeifthealgaearegrowinginthecultures. Next,thestudentsfilteredpreparedculturesofalgaetoseehowdifferentamounts ofgrowingalgaewillproducedifferentcolorintensitiesonfilterpaper.Studentswere givencircularfilterpaperandshownhowtofolditintoacone.Groupsweregiven10 millilitersofundilutedalgaeculture,10milliliters1:3dilutedalgaeculture,10milliliters 1:10dilutedalgaeculture,and10milliliters1:100dilutedalgaeculture.Thestudents thenlabeltheirfilterpaperandfilterthealgaethroughtheirfilterpapercones. Theamountofoxygengasreleasedbyeachculturewasdeterminedbyremoving thelidfromthecontainer.Beforeremovingtheinvertedtesttube,amarkerforwritingon glassisusedtomarkthechangeingasvolumeonthetesttube.Thetesttubeisthen removedfromtheinvertedfunnel,allowinganyremaininggrowthmediumtobepoured out.Thevolumeofthegasisrepresentedbyfindingthevolumeofwaterthatisadded backintothetesttubeatthepremarkedlines. . Results Thestudentsobservedthattheexperimentalgroupwithcarbonatedwatergrew betterastheculturesweregreenerandproducedmoreoxygen.Thenegativecontrolof 0.4MKOHhadlittledetectablealgaegrowthoroxygengasproduction.Theserial 9 dilutedculturesindicatedthattheexperimentalgrouphad1x10 algaepermilliliterof 7 5 growthmedium,compareto1x10 algaepermilliliterforthenormalcontroland1x10 algaepermilliliterforthenegativecontrol.Theamountofoxygengascollectedalso correlatedwithalgaegrowthwith1.6milliliterfortheexperimentalgroup,comparedto 0.6milliliterforthenormalcontrol andnooxygenforthenegativecontrol.Eachgroup wasfilteredandthequalitativelysupportedtheabovefindings. Table1:SummaryofExperimentalResults GrowingAlgaeColorIntensity AlgaeConcentration(Cells/ml) OxygenGas(ml) FilterAlgaeColorIntensity Normal Green 7 1x10 0.6 +++ Experimental DarkGreen 9 1x10 1.6 +++++ Negative LightGreen 5 1x10 0 +

Conclusion Thisexperimentalsystemshowsthepositiveinfluencecarbondioxidehasonalgae growth.Itshowsthatcarbondioxidedissolvedinwatercandiffuseoutofwaterintothe atmosphereoftheclosedsystemandintothegrowthmediumofthealgaeculture.The increasedamountofcarbondioxidehadapositivegrowthaffectontheclosteriumalgae hasevidencedbyTable1.Itproduced100timesmorealgaepermilliliterthandidthe normalcontrol.Inaddition,thesystemshowstheessentialrequirementforcarbon dioxideforalgaegrowth.Thepotassiumhydroxidesolutionremovescarbondioxide fromtheairbyformingK2CO3(s),whichinhibitsalgaephotosynthesis.Thissystem

allowsstudentstoseethatCO2isessentialforphotosynthesisandhaspositiveinfluence onalgaegrowth.

TheaboveprocedurewasdevelopedwithsupportfromBobChen,Ph.D.ofOSEI2,NE COSEE, Dan Repeta, Ph.D Marine Chemistry & Geochemistry Department, Woods HoleOceanographicInstitution,andCurtisOlsenDepartmentofEnvironmental, Coastal,andOceanSciences(ECOS)ofUMassBoston.Theprocedurewasdeveloped byPamelaDrouin,MichelleFalconandDavidJ.Welty,Ph.D.oftheFairhavenPublic SchoolsandCherylBelknap,CurriculumDirectorOceanStudiesCurriculumatthe AtlantisCharterSchoolinFallRiver.

Copyright2005DavidWelty(Fairhaven,MassachusettsPublicSchools).DevelopedforTheCenterfor OceanSciencesEducationExcellenceNewEngland(COSEENE).Thismaterialmayonlybeusedfor personaloreducationaluses.Commercialorforprofitusesofthismaterialareprohibitedwithoutthe writtenpermissionofDavidWelty.ThismaterialisbasedonworksupportedbytheNationalScience FoundationunderGrantNo.0215456.Anyopinions,findingsandconclusionsorrecommendations expressedinthismaterialarethoseoftheauthor(s)anddonotnecessarilyreflecttheviewsoftheNational ScienceFoundation.