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1 The Algae-in-a-Bottle Experiment: A High-Impact Learning Activity W. Sean Chamberlin, PhD Professor, Earth Sciences, Fullerton College

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TheAlgae-in-a-BottleExperiment:

AHigh-ImpactLearningActivity

W.SeanChamberlin,PhD

Professor,EarthSciences,FullertonCollege

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TableofContents

Summary........................................................................................................................................................................... 4StudentLearningOutcome ......................................................................................................................................... 4LessonPlan....................................................................................................................................................................... 4Whystudyalgae?........................................................................................................................................................................... 5Introducesomebasicfactsaboutmicroalgae. ........................................................................................................................ 5Introducephotosynthesis............................................................................................................................................................ 5Introducechlorophylla................................................................................................................................................................ 6Introducerespiration. .................................................................................................................................................................. 6Emphasizethatphotosynthesisplusrespirationmakealgaegrow. .................................................................................. 7Introducelimitingfactors. ........................................................................................................................................................... 7Askscientificquestions................................................................................................................................................................ 8Discusscarbondioxide................................................................................................................................................................. 8Discussandaddsalts. ................................................................................................................................................................... 9Discussandaddnutrients. .......................................................................................................................................................... 9Discusslightandremovethelabelonthebottle..................................................................................................................10(Optional)Discusslightandnutrientsandmaximumbiomass. .......................................................................................11Don’tforgettomakenotes:thescientificnotebook. ...........................................................................................................11Takepictures!Learngoodscientifichousekeeping. ............................................................................................................11Addalgae.Observe.Taketimezeromeasurements. ...........................................................................................................12Letthealgaeincubate! ...............................................................................................................................................................12Makeobservationsormeasurementsperiodically. .............................................................................................................12Framethosephotos! ...................................................................................................................................................................13Compileresultsandgraphthem. .............................................................................................................................................13Shareandinterprettheresults. ...............................................................................................................................................13

BackgroundandExplanationofApproach..........................................................................................................14TheSlowApproach ..................................................................................................................................................................14TheBasics........................................................................................................................................................................................................ 14TheScientificNotebook ............................................................................................................................................................................ 14Pre-Tests .......................................................................................................................................................................................................... 15DissolvedGases............................................................................................................................................................................................. 15WaterChemistry .......................................................................................................................................................................................... 15BiologicallyImportantNutrients(akafertilizers)......................................................................................................................... 16LimitingFactors-Nutrients: ..................................................................................................................................................................... 16LightIntensity ............................................................................................................................................................................................... 17TheLight-DarkCycle .................................................................................................................................................................................. 17Chlorophylla .................................................................................................................................................................................................. 18Photoadaptation ........................................................................................................................................................................................... 18LightSources.................................................................................................................................................................................................. 19LightintheBottle......................................................................................................................................................................................... 20ScientificQuestions&Predictions........................................................................................................................................................ 20TheSeasonalApproach ............................................................................................................................................................................. 21

TheFastApproach....................................................................................................................................................................21ScientificQuestions&Predictions........................................................................................................................................................ 21PicturesandNotebooks ............................................................................................................................................................................ 22LabelingBottles ............................................................................................................................................................................................ 23ConducttheExperiment ........................................................................................................................................................................... 23KeepTakingPicturesandMakingObservations............................................................................................................................ 23ASimpleObservationActivity................................................................................................................................................................ 24AssessAttitudesandUnderstanding................................................................................................................................................... 24

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MaterialsandOptionalEquipment........................................................................................................................25BottledWater.............................................................................................................................................................................25Saltwater......................................................................................................................................................................................25SourcesofAlgae ........................................................................................................................................................................25SourcesofNutrientMedia .....................................................................................................................................................25LightSources ..............................................................................................................................................................................26BulbFixturesandBulbs ............................................................................................................................................................................ 26FluorescentLights ....................................................................................................................................................................................... 26AquariumLEDFixtures ............................................................................................................................................................................. 26MobileLightingSystems ........................................................................................................................................................................... 26

ScientificEquipment................................................................................................................................................................27HelpfulResources ........................................................................................................................................................27

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Summary

TheAlgae-in-a-BottleExperimentprovidesanengagingandflexiblehigh-impactteachingtoolfor

helpingstudentstoknow,understand,andapplyanumberofconceptsrelatedtothebiologyandecology

ofaquaticplantsandtheirenvironments.Itisalsorelevanttomethodsbeingdevelopedfortheuseof

algaeasanalternativeenergysource,thatis,biofuels.Theprotocolsinthisexperimentcanbeadapted

useasademonstrationactivityinoneortwoclasssessions,orasanatureofscience,inquiry-based

activityoverafewtoseveralweeks.Theeasy-to-obtainandinexpensivematerialsusedinthe

experimentmakeitaccessibletoinstitutionswhereresourcesandspacearelimited,aslongassunlight

orartificiallightareavailabletocarryouttheexperiment.Preliminaryresultswithnon-majorsenrolled

inintroductorygeneraleducationoceanographycoursesinacommunitycollegeindicateincreased

engagementandahighlevelofenthusiasmfortheexperiments,andsuggestabetterknowledgeand

understandingoftheeffectsoflightandnutrientsonphotosynthesis,andgreaterappreciationforthe

natureofscience.WebelievethattheAlgae-in-a-BottleExperimentoffersaneffectivemeansfor

improvingscienceliteracyandforintroducingscientificresearchtodiverselearnersfrommiddleschool

tocollege.

StudentLearningOutcome

Uponsuccessfulcompletionofthisexperiment,studentswillbeabletowritetheequationof

photosynthesisandexplainhowvaryingconditionsoflightandnutrientswillaffectthegrowthratesof

microalgaeandtheproductivityoffoodwebsthatdependonthem.

LessonPlan

Thetopicsandstepsoutlinedbelowrepresentasequenceofdiscussionsandactivitiesthatintroduce

theprocessofphotosynthesisandthefactorsthataffectthegrowthofalgaeinaquaticenvironments.The

lessonplanisapplicabletostudentsenrolledinintroductorybiology,botany,plantphysiology,algology,

marinebiology,environmentalbiology,ecology,andoceanographywhereageneralunderstandingof

photosynthesis,primaryproduction,theglobalcarboncycle,andaquaticfoodwebsisdesired.

Thelevelofcomplexityofeachstepandactivitycanbetailoredaccordingtothegoalsofinstruction,

orbackgroundoftheinstructor.Thesequencemaybesimplified,expanded,accelerated,orsloweddown

dependingonthetimeandresourcesavailabletotheinstructor.Theentiresequencemaybecompleted

asademonstrationactivityinthetimeframeofasingle-class,oritmaybecarriedoutasaninquiry

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activityoveranumberofclasses.Inthelongertimeframe,eachsteporactivitymaybeinterwovenwitha

lectureorotheractivity,assomestepscanbecompletedin10-15minutes.Thelongertimeframe,akind

ofWhatAboutBob?baby-stepsapproach,offersthegreatestopportunitytoachievethehighestlevelsof

contentmasteryandconceptualunderstanding.Forexample,theinstructormaychoosetogivestudents

bottlesofwaterthatalreadycontainalgae,andaskstudentstoobservethechangesincolorthatappear

overthecourseofafewdaysorweeks.Studentsmayeventakethebottleshometomakethese

observations.Alternatively,theinstructormaychoosetocoveronly1-2stepsinthelessonplaneach

class,permittinggreatertimeforintroductionanddiscussionofconcepts,andallowingstudentstocarry

outindividualinvestigationsintheformofaninquiryorresearchactivity.

Themaingoalhereistoprovideinstructorswithafairlysimpleactivity(inpractice)thatisfunand

engagingforstudents,andthatallowsstudentsandinstructorstoexploreandlearnaboutfundamental

processesofglobalimportance.Studentslearnbestbydoing,andit’sinthatspiritthatthisactivityis

presented.

Whystudyalgae?

Tellthemtheyaregoingtocreateahomeforamostmarvelousorganism,onethatservesasthebaseof

thefoodweb,and,indoingso,providesfishandshellfishtomuchoftheworld;onethatregulatescarbon

dioxide,and,assuch,controlsthetemperatureofourplanet,andonethatmayonedayprovidefuel,what

hasbecomeknownasbiofuels,fortransportation.Thisorganismbelongstoagroupoforganismsknown

asthealgae,orbecausetheyaretiny,themicroalgae.Intheocean,werefertomicroalgaeas

phytoplankton,thealgaedrifters.

Introducesomebasicfactsaboutmicroalgae.

Microalgaearesingle-celled,microscopic,photosyntheticorganisms,relatedtoseaweeds,thatinhabit

thelightedregions(i.e.,thephoticzone)ofallaquaticenvironments,freshwaterandsaltwater,including

yourcatordog’swaterdish,yourAuntMary’sbirdbath,andyourkidbrother’sgoldfishbowl.Theclass

willgrowaspeciesofmarinemicroalgae.Distribute8-ozbottledwatertoindividualorteamsofstudents.

Asktheclasstoexaminetheirbottles.Whatwouldbetheidealenvironmentfortheiralgaeinabottle?

Whatareessentialingredientsoftheirnewhome?Writetheirresponsesontheboard.

Introducephotosynthesis.

Writeontheboardabasicequationforphotosynthesis:CO2+H20!sugars+O2.Discuss.Whatdothe

symbolsinthisequationmean?Simplyput,plantsandalgaeusecarbondioxideandwatertoproduce

sugars,akindofenergymolecule,andintheprocess,produceoxygenasabyproduct.What’smissing?

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Algae,likeplants,needlighttogrow.Addlight:CO2+H20+light!sugars+O2.Whatelsedoplantsneed

togrow?Theyalsoneedfertilizers.Inthealgaebusiness,wecallthembiologicallyimportantnutrients,

orsimply,nutrients.Examplesofcommonlawnandgardenfertilizersmaybeshown.Onthefrontofthe

packagearetheletters,N,P,andK.Whatdothesesymbolsstandfor?Thesearethebasicmacronutrients

requiredbyallplantsandalgae.Addnutrients:CO2+H20+light+nutrients!sugars+O2.(Atthispoint,

moreadvancedcoursesmaydevelopfurtherdetailsofphotosynthesis,suchasthelightanddark

reactions,andthewater-splittingreactions,whichhelpsexplainhowsolarenergyistransformedinto

chemicalenergywhichisthenusedasanenergysourcetomanufacturecellularmaterials,andhelps

explainthattheO2producedbyplantsandalgaecomesfromthesplittingofwater,notthesplittingof

carbondioxide.)

Introducechlorophylla.

Thecentralplayerinphotosynthesisisamoleculecalledchlorophylla.Thismoleculeistheprimarylight-

absorbingmoleculeofallplantsandalgaeandphotosyntheticbacteria.Askstudentsiftheyhaveever

seenchlorophyll?Ifawindowisnear,askthemtolookoutsideatatree.Cantheyseechlorophyll?

Explainthatthegreencolorofplantsandalgaecomesfromchlorophyll.Thismoleculeabsorbssunlight

andtransferstheenergyfromsunlightintoasystemthattransformsthesolarenergyintoaformof

chemicalenergythatcanbeusedbytheplant.Theindividualcellsofalgaearetoosmalltosee,butwhen

thealgaeareabundant,theirchlorophyllisvisible.Askstudentsiftheyhaveevernoticedchangesinthe

coloroftheoceanfromwintertospringtosummertofall.Inwinterandsummer,theoceanmaybeblue

andmostlytransparent.Inthespringandfall,theoceanmaybegreenandopaque.Thedifferenceincolor

andthechangeincolorfrombluetogreencomesfromthepresenceofphytoplankton.Whentheoceanis

blue,therearefewphytoplanktoninthewater.Whentheyoceanisgreen,phytoplanktonareabundant.

Intheexperiment,thecolorinsidethebottleprovidesanindicationoftheamountofalgae.Changesin

thecolorinsidethebottleoveraperiodofdaysindicatehowfastthealgaearegrowing.Itwillbe

importanttoobserveanddocumentthesechanges.

Introducerespiration.

Askstudentswhathappenstoplantswhentheydon’tgetenoughlight?Studentswillgenerallyanswer

thattheplantswilldie,buttheinstructormaycounterbyaskingifplantsdieatnight?Whydon’tplants

dieatnight?Plantsandalgaestoreenergyinthesamemannerashumansdo.Whenlight(orfood)isnot

available,plants(andhumans)usetheirstoredenergy.Plants,algae,andhumansstoreenergyasfatty

acids,whichwemaythinkofasenergymolecules,justlikesugars.Whenplants,algae,andhumansneed

energy,theybreakdownthesefattyacidsinaprocesscalledcellularrespiration.Here’stheequation:

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fattyacids+O2!CO2+H20.Doesthisequationlookfamiliar?Infact,respirationresembles

photosynthesisintheoppositedirection.Organismsuseoxygentobreakdownenergymolecules(sugars

andfats)and,indoingso,producecarbondioxideandwater.Whyisthisimportant?Becauseplantsand

algaeusesomeoftheenergytheyproduce,andtheyuseoxygentobreakdownthatenergy.Simplyput,

plantsandalgaecarryoutrespirationandrequireoxygen.Repeat:plantsandalgaerequireoxygen.

Fortunatelyforus,plantsandalgaeproducemoreenergymoleculesandmoreoxygenthantheyneed.

Otherwise,wemightonlyfindplantsonPlanetEarth!

Emphasizethatphotosynthesisplusrespirationmakealgaegrow.

Photosynthesisturnssolarenergyintochemicalenergy,andrespirationtakesthatchemicalenergyand

usesittobuildallofthethingsthatmakeupaplantoralgalcell,includingthemolecularmachinerythat

letsplantsandalgaephotosynthesize.Photosynthesisandrespirationworktogether.Growthinplants

andalgaeoccursviacellulardivision:onecelldividesintotwocells,twocellsdivideintofourcells,andso

onandsoforth.Becausealgaeareunicellular,wecanmeasurehowfasttheyaregrowingbycountingthe

increaseincellnumbersoveraperiodoftime.Alternatively,wecanmeasurechangesinthe

concentrationofchlorophyll,eithervisuallyaschangesincolor,orquantitatively,usingscientific

instrumentsdesignedtodetectchlorophyll(e.g.,colorimeters,spectrophotometers,fluorometers,etc.)If

wewanttoconductexperimentsonwhatmakesalgaegrow,thentrackingtheirgrowthratewillbe

important.Theinstructormaywanttoholduptwodifferentbottlesofalgaewithdifferent

concentrationsofchlorophylltoemphasizethispoint,orprojectamicroscopicimageofmicroalgae.

(Advancedclassesmaybeintroducedtogrowthcurvesandgrowthequations,ifdesired.)

Introducelimitingfactors.

Innature,somethingalwayslimitshowfastplantsoralgaecangrow.Ifit’snotlight,thenitmightbe

nutrients.Ifit’snotlightandnutrients,thenitmightbewater(forplants)orsomeotherfactor(for

algae).Theideathatsomethingalwayslimitshowfastplantsoralgaecangrowprovestobeanimportant

one,especiallyinagriculture.Ifyouwanttogrowmorecropsfaster,youneedtoidentifythefactorthatis

limitingthegrowthofthecrops.Inthemid-1800s,JustinvonLiebig,auselesschemistrystudent(inthe

eyesofhisprofessors)cameupwiththeideaofalimitingfactor,theonefactorrequiredforthegrowthof

aplantthatisintheleastsupply.Itmightbelight,itmightbenutrients,oritmightbesomethingelse.

ThuswasbornLiebig’sLawoftheMinimum.Anillustrationofawoodenbucketwithdifferentlengthsof

planksservesasausefulanalogy:thelengthoftheshortestplankdeterminestheamountofwaterheld

bythebucket.Analogously,thelimitingfactordeterminesthegrowthrateoftheplantoralgae.This“law”

hasimportantimplicationsforunderstandingthegrowthofalgaeinabottle,andinnature.Inadditionto

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agriculture,Liebig’sLawoftheMinimumhasbeenappliedtounderstandingthebiologyandecologyof

phytoplanktongrowthintheocean.

Askscientificquestions.

TheAlgae-in-a-BottleExperimentmaybeconductedasademonstrationactivity,wherestudentsoptimize

waterlevels,preparesaltwater,addnutrients,findsuitablelight,addalgae,andwatchthealgaegrow,or

itmaybepresentedasaninquiryactivity,wherestudentscomeupwith(oraregiven)scientific

questionstoexplore,formulatehypothesesandpredictions,setupexperimentalconditions,carryoutthe

experiments,andanalyzeandcommunicatetheresults.Ineitherapproach,it’susefultoaskstudentsto

workinpairsorteamstocomeupwithafewscientificquestionsconcerningthegrowthoftheiralgaeat

differentlightlevelsanddifferentnutrientconcentrations.Whathypothesesmighttheytest?Whatdo

theypredictwillhappen?Alternatively,theinstructormaywriteafewquestions,hypotheses,and

predictionsontheboard.Toavoidconfusion,startwithlightasalimitingfactorfirst.Forexample,how

fastwillalgaegrowinhighlight?Howfastwilltheygrowunderlowlight?Howfastwillalgaeathigh

lightgrowwithshortdaysandlongnights,suchasfoundinwinterintemperatezones?Howfastwill

theygrowwithlongdaysandshortnights,resemblingsummerintemperatezones?Thereareanumber

ofvariationshere,butitcanbeusefultoallowstudentstoexplorethepossibilities,justasascientist

might.Similarquestion-generatingcanbecarriedoutwithnutrients,butwithoneimportantextension:

theconcentrationofnutrientsinthebottlewilldeterminethemaximumbiomassofthealgaeinthe

bottle.Lowernutrientconcentrationswillproducelowerbiomass,andhighernutrientconcentrations

willproducehigherbiomass.

Discusscarbondioxide.

Holdupapieceofwood.Whatkindofatomsdoesitcontain?Carbon.Fromwheredoesthecarboninthe

woodcome?Carbondioxide.Howdoplantsobtaincarbondioxide?Fromtheatmosphere.Howdoalgae

obtaincarbondioxide?Fromcarbondioxidedissolvedinthewater.(It’snotnecessarytointroducethe

formsofdissolvedcarbondioxideinseawater,i.e.,thecarbonatebufferingsystem,asthistendsto

overcomplicatethematter.)Howdoescarbondioxidegetintothewater?Acrosstheair-waterinterface.

Holdupabottleandask,ifalgaeusethecarbondioxideinthewater,howwilltheygetmore?Itmight

takesomepromptingtogetstudentstorealizeit,butthecapwillhavetoberemovedfromthebottleto

periodicallyrefreshtheairinthebottle.Thebottlemayneedtobeshaken.Whatelsecanbedonebesides

removingthecap?(Moreprompting.)Createaheadspace.Atthispoint,studentsmaydrinkorpourouta

portionofthewatersothatthewaterlevelisevenwiththeshoulderofthebottle,rightwhereitbeginsto

taper.

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Discussandaddsalts.

Iftheclassisgrowingmarinealgae,whatmusttheydotothewaterinthebottle?Addsalts.(Depending

onthelevelofcomplexitydesired,theinstructormayintroducethepropertiesofwater,thenatureof

dissolving,anionsandcations,themajorconstituentsofseawater,thePrincipleofConstantProportions,

salinity,andmeasuringsalinityusingconductivity,buttheseconceptsarenotessentialformeetingthe

learningoutcomesfortheexperiment.)Whatkindsofsaltsmakeupseawater?Mostly,wefindsodium

chlorideinseawater,butwealsofindnineothermajorelements,themajorconstituents,andmorethan

80traceelements,theminorconstituents.Fortunately,wecanbuysaltsforsaltwateraquariums,and

that’swhatwewilluseforouralgae.(Atthispoint,theinstructormayinstructthestudentstoadda

teaspoonofseasaltstotheirbottle,orletstudentscalculatetheamountneededaccordingtothe

directions,forexample,ahalfcupofsaltspergallonofwater.Theproportionalitycalculationisagood

exerciseforstudents.Studentswillneedtomeasuretheamountoffreshwaterintheirbottle,nowthat

theyhavereduceditsvolumetolessthan8ounces.)

Discussandaddnutrients.

Whatelsedoalgaeneedintheirseawatertogrow?Ifstudentswerepayingattentionearlier,theywill

answernutrients.Buttheinstructormayneedtoreferthembacktotheequationofphotosynthesis

writtenontheboard.Whatwouldhappenifnonutrientswereadded?Whereisaplaceonlandwithlow

nutrients?Deserts.Whereisaplaceintheoceanwithlownutrients?Thetropicalocean.There’svery

littleplantgrowthindesertsbecauseofwaterandlownutrients,andverylittlephytoplanktongrowthin

thetropicaloceanbecauseoflownutrients.Soifnonutrientswereadded,therewouldbelittletono

growthofalgae.Whatiflotsofnutrientswereadded?Domorenutrientsnecessarilymeanhigher

growth?Itcanbeusefultoshowagraphofgrowthrateversusnutrientconcentrationforlimiting

concentrationsofnutrients.Atsomepoint,asaturatingconcentrationofnutrientsisreached,beyond

whichadditionalincreasesintheconcentrationdonotproducefastergrowth.Instructorsmayalsowant

toreferbacktothediscussionoflimitingfactorstoexplainthatathighnutrientconcentrations,nutrients

maynolongerbelimiting,and,asaresult,someotherfactormightlimittheirrateofgrowth.What

happenswhenthenutrientsrunout?Thealgaestopgrowing,andthecolorinsidethebottleremains

constant.(Ultimately,theamountofnutrientsinsidethebottledeterminesthefinalbiomasswithinthe

bottle.Soonesimpleexperimentsimplyinvolvescomparingthecoloroftwobottles—onewithlow

nutrientsandonewithhighnutrients—attheendofagivenperiodoftime.Ifnutrientconcentrationsare

saturating,andthetwobottlesheldatthesamelightintensities,theymaybothgrowatthesamerate,but

thebottlewiththehighernutrientconcentrationwillpermitgreatergrowthandahigherfinal

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concentrationofalgae.Thisisapointthattheinstructormaychoosetoexplainwhentheexperiments

havebeencompletedtoallowstudentstoderivethisconclusionontheirown.)Aswithseasalts,algae

nutrientsareavailableforpurchase,albeitinahighlyconcentratedform.Theinstructorwillwantto

prepareasolutionofnutrientsaheadoftimefromwhichstudentscanpipetteagivenamount.The

amountdispensedtostudentswilldependonthetypeofexperimentthattheinstructororstudentshave

chosentocarryout.Inthesimplestapproach,somestudentswilladdafewdropsandotherswilladd

doubleortriplethenumberofdrops.Intheabsenceofinstrumentationtomeasuretheconcentrationsof

nutrientsinthebottles,there’sabitofguessworkhere.Experimentwithit.That’sscience!

Discusslightandremovethelabelonthebottle.

Fortunately,discussionsoflightinvolvemorefamiliarterritory.Studentsgenerallyunderstandhighlight

andlowlight.Theymaybelessfamiliarwithlight:dark(L:D)cycles,thoughsomepromptingand

questioningaboutlengthofdaysinsummerversuswinter,timeitgetsdarkatnight,timeitgetslightin

themorning,etc.,helpsstudentsgrasptheidea.Keytothisstepisadiscussionofthelightenvironment

insidethebottle.Holdingupabottle,theinstructormightaskwhatpropertiesofthebottleitselfmay

impedethequantityoflightthatthealgaereceiveinsidethebottle.Somestudentswillimmediately

recognizethatthecapblockslight.Adiscussionoftheorientationofthebottlewithrespecttothelight

sourcecanbehelpful.Whichisbetter,illuminationfromaboveorilluminationfromtheside?Whatare

thepotentialadvantagesanddisadvantagesofeitherapproach?Savvystudentswillalsorecognizethat

thebottle’slabelwillblocklight.Theclassshouldremovethelabelsfromtheirbottleatthispoint.Ifthe

instructorhasalreadychosenthelightintensityorlightintensitiesfortheexperiment,thenheorshe

maywanttoaskstudentstoworkinpairsorteamstopredictgrowthratesunderthatlightintensity(or

rangeoflightintensities),assumingthatnutrientsarenotlimiting.Ifnaturalsunlightistobeused,the

instructormightaskhowgrowthrateswillvarythroughtheday,andatnight(!)AdiscussionofL:D

cycles,whichofferanotherexperimentalvariable,maybecarriedoutamongstudentsorasaclass.Inthe

end,itwillbeimportantforstudentstoknowortohavedecidedthelightintensity(low,medium,or

high)andthelightregime(12:12,16:8,8:16)fortheirparticularexperiments.(Adventurousinstructors

maywanttointroduceBeer’sLaw,theexponentialdiminishmentoflightasitpassesthroughaliquid.

Thiscanbeusefulfordiscussionsofself-shadingofalgaewithinthebottle,orasanexplanationfor

differencesingrowthratesthatmayoccurinshallowerordeeperbottles.Opportunitiesalsoexistto

coverelectromagneticradiation,theseasonalcycle,theabsorptionpropertiesofwater,theabsorption

spectraofphotosyntheticpigments,whytheoceanisblue,andsatelliteoceanography,amongothers.)

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(Optional)Discusslightandnutrientsandmaximumbiomass.

Asmentionedearlier,theconcentrationofnutrientswilldeterminethemaximumbiomassofalgaethat

willgrowwithinthebottle.However,thespeedwithwhichthemaximumbiomassisachievedwill

dependonlightintensity.Foragivennutrientconcentration,ahighlightintensitywillpromotefaster

growthandaquickeruseofnutrientsthanalowlightintensity.Asaresult,underhighlight,the

maximumbiomassmaybereachedinamatterofafewdays,whereasunderlowlight,themaximum

biomassmaynotbereachedforseveraldays.Instructorsmaywanttopromptstudentstothinkabout

whatishappeninginsidethebottleasthealgaegrow.Forexample,whatishappeningtothenutrient

concentrationoftheseawaterinthebottle?It’sdecreasing.Whatishappeningtothebiomass?It’s

increasing.Whathappenstothelightintensityinsidethebottleasthebiomassofthealgaeincreases?

Thelightisdiminishedbecausethealgaeareabsorbingit.Wheretimepermits,discussionofthese

interactionscanreallyhelptosolidifystudentsunderstandingoftheconcepts.Studentsmaybeaskedto

createamatrixofpossibleoutcomes,ortheymaybeaskedtocreateconceptmaps.Shortanswerand

essayquestionsonthetopicscoveredtothispointwillhelpinstructorsidentifymisconceptions,andwill

helpensurethatstudentsaremakingthelinkbetweenthetheoreticalconceptsandtheactual

experiment.Dostudentsgraspwhatisgoingonwithinthebottle?Dotheyhavetheconceptual

understandingtosupportandexplainwhateverresultstheyachieve?

Don’tforgettomakenotes:thescientificnotebook.

Dependingonthetypeofcourseandtheexperiencelevelofthestudents,itmaybenecessaryto

introduceappropriatemethodsfordocumentingtheexperimentalmethods,observations,anddataina

scientificnotebook.

Takepictures!Learngoodscientifichousekeeping.

Oncestudentshavepreparedtheirseawaterandnutrientsolution,andhaveaclearideaofthelight

protocolthattheywillobserve,it’sagoodtimeforstudentstotakepicturesoftheirbottles(usingtheir

smartphonesorotherdevices),andevenselfieswiththeirbottle.Wehighlyrecommendthatinstructors

simplyinstructstudentstotakepictureswithoutsupplyinganyadditionaldetails.Studentsshouldbe

askedtosharetheirphotoswitheachother,evenpostthemonFacebookorInstagram.Ifpossible,

studentsshouldaskstudentstoemailtheirphotostotheinstructorsothatheorshecanprojectthemon

theclassroomprojectionscreen.Twoorthreephotosfromdifferentstudentsworksideallytomakethe

followingpoints:1)Howwillstudentsbeabletoidentifytheirbottlesamongaclassroomofbottles?2)

Howwilltheybeabletotellfromapictureiftheirbottleshaveturnedgreener?and3)Whymightitbe

importanttoreproduceexactlythewayinwhichthephotographwastaken?Thefirstpointmaybe

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addressedbyassigninguniqueidentifierstoeachstudent.Theinstructormightaskifitmatterswhere

studentsplacetheirlabels.(Thebestapproachistouseapermanentmarkerandlabeltheplasticcapon

thebottle.)Thesecondpointmaybeaddressedbysuggestingthatstudentstaketheirpicturesagainsta

whitebackground.Awhiteboardworksgreatforpicturetaking.Thethirdpointreferstocreatingatime

sequenceofphotographsthatareidenticalineveryrespect:samedistancefromthecamera,same

lighting,sameangle.Reproducingthephotographicconditionsasexactlyaspossibleensuresthatany

changesobservedareduetothegrowthofthealgae,notthewaythepicturewastaken.

Addalgae.Observe.Taketimezeromeasurements.

It’sfinallytimetoaddalgae.Theinstructorwillprovideaflaskorcontainerofstockculturefromwhich

studentsmaypipetteasmallamount(~1ml),ortheinstructormayaddthealgae.Tothedegreepossible,

identicalamountsshouldbeaddedtoeachbottletoensureconsistencyandreproducibilityintheresults.

Followingtheadditionofalgae,studentsshouldcaptheirbottle,andgentlyshakeit.Theymaythentake

pictures(usingthepicture-takingcriteriaestablishedinthepreviousstep).Ifquantitativemeasurements

aretobetaken,thestudentsshouldbeinstructedtotakethesemeasurementsatthistime.Students

shouldmakevisualobservationsoftheiralgae,andwritethoseobservations,alongwiththetimeand

date,intheirnotebooksorontheirworksheet.Theinstructormaywanttoremindstudentstobesure

thattheyhaveproperlylabeledtheirbottles.

Letthealgaeincubate!

Studentscannowplacetheirbottlesinthelocationchosenfortheexperiment(awindowsill,atabletop

withalightsource,aplantgrowthcart,etc.).Theywillwanttonotetheorientationofthebottlewith

respecttothelightsourceassmallperturbationsinthebottle,bottlecaps,labelresidue,mayslightly

changethelightregime.Studentswillwanttomaintainthesameorientationthroughouttheexperiment.

Makeobservationsormeasurementsperiodically.

Followingtheinitialadditionofalgae,thetimetableformakingfurtherobservationsormeasurements

willdependontheclassschedule.Ifstudentsaretakingpictures,observationsmaybecompletedina

shortamountoftimeatthebeginningofclassoveraperiodofaweekorafewweeks.Iftheyaremaking

measurementsofchlorophyll,itmaytakelonger,butwithenoughresourcesandproperorganization,

thesemeasurementsshouldn’ttakelongeither.Anaturalendpointfortheexperimentisthepointwhere

nofurtherchangesincolor(orchlorophyll)areobserved(ormeasured),themaximumbiomasspoint,or

ashortertimeperiod,dependingontheinitialquestionsbeinginvestigated.

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Framethosephotos!

Theavailabilityofappsthatpermituserstocreatecollagesofphotosprovesveryusefulforillustrating

before-and-afterphotosofthealgaeinthebottle.Atimesequenceofseveralphotosmayalsobecreated.

Studentsgenerallyknowtheseappswell,andwillrequirelittledirectionforcreatingacollageoftheir

experiment.

Compileresultsandgraphthem.

InclassroomswhereiPadsorcomputersareavailable,studentsmayenterdataintoaspreadsheetas

theycollectit.Otherwise,theymaycreatetablesandgraphsofdatainacomputerlaboronacomputerat

home.Barchartsillustratingchangesincolor(measuredbyacolorimeterorspectrophotometer)or

chlorophyll(measuredinafluorometer)helpillustratechangesandratesofchangeinalgalgrowth.

Shareandinterprettheresults.

Weencourageinstructorstoallowstudentstoshareandinterprettheirresultswiththerestoftheclass.

Theonlineavailabilityoffreepresentationsoftware(e.g.,GoogleSlides)makesiteasyforstudentsto

prepareafewshortslidesoftheirresults,andmakesiteasyforstudentstoprojecttheirslidesona

classroomprojectionsystemconnectedtotheInternet.Alternatively,studentsmaypreparePowerPoint

presentations,andsubmitthemonaUSBdrive,ortheymaywriteuptheirexperimentsinalaboratory

reportorscientificpaper.Weencourageinstructorstofocusonthepresentationandinterpretationof

theresultsversusaproperaccountingofmethods,thoughthisisimportant.Fartoooftenstudentsfeel

liketheresultsoftheirexperimentsarenotvalid,thattheydidsomethingwrong,orthatsome

experimentalerrorgavethemtheresultsthattheyachieved.Onthecontrary,it’simportanttocomeup

withpossibleexplanationsforanyresultsthestudentsfind.Algaemaygrowornotgrowforavarietyof

reasons,includingtemperaturefluctuations(unaccountedforinourexperiments),bacterial

contamination,differencesinbottlesthatinducedifferencesintheinternallightintensity,accidental

differencesintheamountofsalts,nutrients,oralgaeadded.Studentsshouldbeencouragedtocompare

theirresultswiththoseoftheirteammembers.Ifthetreatmentswereidentical,shouldn’ttheresultsbe

identical?Alivelydiscussionofresultsdemonstratesthatstudentswereengagedintheprocess,thatthey

wereinvestedintheexperiments,andthattheyunderstandtheconceptualunderpinningsofthe

experiments.Thisisthemostrewardingpartoftheexperimentforstudentsandinstructors.

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BackgroundandExplanationofApproach

Theideaforaclassroomexperienceingrowingalgaeemergedfromanundergraduateresearchprojectin

algaebiofuelsatFullertonCollege,alargecommunitycollegeinSouthernCalifornia.Lackingspaceand

resourcesforsophisticatedbioreactors,studentsgrewan“off-the-shelf,”euryhaline(tolerantofawide

rangeofsalinity),marinealgalspecies,Tetraselmis,ina120-gallonaquariumequippedwithLED

aquariumlights.Successfulpropagationofthealgae(i.e.,theydidn’tkillit)ledtotheideaofhavinga

classroomofstudentsgrowthealgaein8-ozwaterbottles.(Smallerwaterbottleswereusedtoreduce

theexpense,but11-or12-ozbottlesshouldworkequallyaswell.)

TheSlowApproach

TheBasics

Inthesimplestversionoftheexperiment,studentsareintroducedtothebasicsofphotosynthesis,

includingthelimitingeffectsoflightandnutrientsonmarinealgalgrowth.Studentsarethengivenan8-

ozbottleofwaterpurchasedatagroceryorsuperstore,andaskedtodiscusswhatarethenecessary

“conditions”toturntheirbottledwaterintoasuitablehomefortheirmarinealgae.Studentsgenerally

understandthatlightisnecessaryforalgalgrowth,buttheyarelesscertainwhatwillhappenunderlow

lightconditions,orunderconditionswithvaryinglight-darkcycles.Similarly,studentsgenerally

understandthatsomekindof“fertilizers”(i.e.,biologicallyimportantelements)areimportanttomake

algaegrow,butlackthesophisticationtopredictwhatmighthappenatdifferentconcentrationsof

nutrients.Nonetheless,studentsareontherighttrackinknowingthattheiralgaewillrequirelightand

nutrients.

TheScientificNotebook

Followingageneralintroductiontotheexperiment,andinpreparationforcarryingouttheexperiment,

it’sagoodideatointroducestudentstotheimportanceofdocumentingeverystepalongtheway.If

scientificnotebooksandscientificnotetakinghavenotbeenintroducedtotheclass,thennowwouldbea

goodtime.Theinstructormayaskstudentstotakenotesindividuallyintheirownnotebooks,orthey

mayprovidecompositionnotebookstotheteamsofstudentswhowillworktogetherontheexperiments.

Introductiontothescientificmethod,posingquestions,formulatinghypotheses,makingpredictions,

carefullydocumentingobservationsandmeasurements,anddatarecordingcanbeintroduced.

Otherwise,iftimeisrestricted,theinstructormaywishtopresentacommonquestionorquestionstothe

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class,andguidestudentsthroughapre-chosensetofhypothesesandpredictionsforagivensetof

experimentalconditions.

Pre-Tests

Instructorsmayalsowishtoadministerpre-testsdevelopedforthealgae-in-a-bottleexperiment.(See

Appendix.)Studentattitudestowardsscienceandstudentknowledgeofphotosynthesisandalgal(or

plant)growthmaybeassessedtogaugelevelsofenthusiasmandlevelsofknowledgepriortothe

activity.Followingtheexperiment,asetofpost-testscanbeadministeredtodetermineimprovementsin

attitudesandunderstanding,andasatoolforimprovingthewayinwhichtheexperimentiscarriedout.

DissolvedGases

Followingtheintroduction,theinstructormayprovideeachstudentwithabottleofwater.Thefirst

discussionwithstudents,bottleinhand,mayconcernthepresenceofdissolvedgases,andspecificallythe

deliverofcarbondioxide,toalgaeinthebottle.Theinstructormaywanttointroducetheconceptof

respiration,andevengasexchangeacrossanair-waterinterface,ifdesired.Anystudentwhohasowneda

goldfishshouldbefamiliarwiththeimportanceofaerationforoxygenation.Algae,too,needoxygen,

exceptthisisnotusuallyaproblembecausetheyproducetheirown,apointthatcanbereinforcedby

referringtoasimpleequationforphotosynthesis.However,algaealsoneeddissolvedcarbondioxideto

grow.Studentsmaybeaskedhowtheiralgaewillobtaincarbondioxideinabottlethatisfilledtothe

brimandcapped.Anumberofsolutionscanbeexplored—removingthecap,addinganairpump,shaking

thebottleregularly—allofwhichservethepurpose.Themostelegant(andeasiesttoimplement)

solutionistosimplypourwateroutofthebottle.Werecommendreducingthewaterleveltothe

shoulderofthebottle.Heretheinstructormayintroducetheconceptofheadspaceinabottletoallowfor

anappropriateexchangeofgases.Ademonstrationofheadspaceinasodabottleforpreventing

explosionscanbehelpful.Foradvancedclasses,theinverserelationshipbetweenthesaturation

concentrationofagasandwatertemperaturemayalsobeexplored.

WaterChemistry

Furtherdiscussioncanbeelicitedregardingtherequirementsformarine-versus-freshwateralgae.

Invariably,someonementionssaltwater,andadiscussionensuesregardingthechemicalcompositionof

saltwater,andhowtoreproducethatformulainabottleofwater.Anumberofimportantwater

chemistryconcepts,suchasthenatureofdissolving,cationsandanions,thePrincipleofConstant

Proportions,etc,canbeintroducedhere,buttheconversationcanbeshortenedbytellingstudentsabout

“instantocean”saltsmanufacturedforthesaltwaterandreefaquariumhobbyist.Onevariantofthe

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experimentasksstudentstocalculatetheamountofsaltsthatshouldbeaddedtobottleofwaterbased

onthemanufacturer’sdirectionsfortheamountofsaltstoproduceagallonofsaltwater.Studentsmust

grapplewithunitconversionsandratiostocomeupwiththecorrectamountofsalts.Theyalsowillneed

tomeasurethevolumeofwaterintheirbottleaftertheyhaveremovedsomewatertoallowforgas

exchange(asmentionedabove).Instructorsmayextendthispartoftheexperimenttoincludelessonsin

measurement,methodsprotocols,andrecordkeepinginascientificnotebook.Oncestudentshave

preparedtheirsaltwatersolution,theymaycheckthesalinityinsidetheirbottleusingarefractometeror

salinometer(ifavailable).Otherwise,theinstructormaysimplyprovideananswer,allowstudentstoadd

apre-determinedamountofsalt(usuallyateaspoon),andcontinuewiththeexperiment.

BiologicallyImportantNutrients(akafertilizers)

Onceanunderstandingofidealwaterconditionsisestablished,andstudentshavepreparedtheir

seawatersolution,thenextstepistointroducetheneedforbiologicallyimportantnutrients,andthe

conceptofalimitingfactor.Anexpandedversionoftheequationofphotosynthesismaybepresented,or

anintroductionto“fertilizers”andtheimportanceofnitrogen(N),phosphorus(P),andpotassium(K)

maybepresented.TheinstructormayintroducestudentstoJustinvonLiebig,aworthlesschemistry

student,accordingtohisprofessors,buttheoriginatoroftheideathatoneelement(orfactor)aboveall

limitsthegrowthofplants(ormicroalgae),thatis,Liebig’sLawoftheMinimum.Formarinealgae,

nitrogenintheformofnitrateisgenerallylimitingintheocean,butthisfactmayovercomplicatestudent

understandingatthispoint.Wegenerallyaskwhatmighthappenifalgaeweresimplyintroducedtotheir

bottlesatthisstage.Somepromptingofanswersusinghouseholdplantsasexamplesmaybeappropriate.

Wegenerallyaskhowmanystudentshavegrownplants,orhelpedtofertilizealawn.Fordemonstration,

weshowdifferentbagsoffertilizerwiththeirdifferingratiosofN,P,andK.Where’sthesoilinthe

aquaticalgaehome?What’sthepurposeofsoil?Whydowefertilizeplants?Whathappensifwedon’t?

ThestandardnutrientenrichmentmediumforgrowingaquaticalgaeisGuillard’sf/2,thoughother

mixtureshavebeguntoreplacethisone.Afulldescriptionofthesemediaisbeyondthescopeofthistext,

butlinkstousefulresourcesonthistopiccanbefoundintheAppendix.Forthepurposesofthe

experimentshere,wepurchaseliquidf/2fromanaquaculturevendor.

LimitingFactors-Nutrients:

Thecriticaldecisionhereishowmuchnutrientenrichmentmediatoadd.Theinstructormaywishto

showstudentsagraphoftherelationshipbetweennutrientconcentrationandgrowthrate.When

concentrationsarelimiting(thelinearpartofthegraph),algalgrowthwillberegulatedbythenutrient

concentration.Whereconcentrationsaresaturating(thehorizontalpartofthegraph),nutrient

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concentrationswillbesaturatingandsomeotherfactorwilllimitthegrowthofthealgae,ortheywill

growattheirmaximalrate.Asanextensiontotheexperiment,studentsmaybeaskedtohypothesize

whatwillhappenunderconditionsoflimitingandsaturatingnutrients.Whichbottlewillturngreen

faster?Theymayalsobeaskedtoconsidertheendresult(i.e.,thefinalconcentrationofalgaeintheir

bottle,i.e.,themaximumbiomass)undereachoftheseconditions.Whichbottlewillturngreenest?Iftime

islimited,orthecomplexityofthistopicisonethatinstructorsdonotwishtointroduce,studentsmaybe

instructedtoaddapre-setamountofsaturatingnutrients,ortosimplygiveanyamountatry.Inour

experience,studentstendtowanttoaddplentifulnutrients,sotheinstructormaywanttogivestudentsa

morenarrowchoice,2,4,or6drops.Tothoseeagertoaddlotsofnutrients,theinstructormightaskif

it’spossibletoaddtoomuch?(Yes.)Whathappenswhenyouoverfertilizealawn?(Itdies.)What

happenstothesalinityifyouaddtoomuchnutrientmedia?(Thesalinityislowered.)

LightIntensity

Oncenutrientshavebeenadded,it’stimetoconsiderthelightintensityunderwhichthealgaewillbe

grown.Studentsshouldbeaskedtopredictratesofgrowthversusdifferentlightintensities.The

instructormayaskiftherecanbetoomuchlight,ortoolittle.Agraphofgrowthrateversuslight

intensitymaybeshowntostudentsfollowingdiscussionoftheirpredictions.Weretheirprediction

accurate?Theinstructorwillwanttopointoutkeyconceptssuchasthecompensationlightintensity,the

minimumlightintensityneededtomeetthemetabolicdemandsofthealgae(orplant)itself.Belowthis

lightintensity,thealgaewill“starve.”Thesaturationlightintensity,thelightintensityatwhichgrowth

rateismaximal,shouldremindstudentsofthesaturatingnutrientconcentration.Abovethislight

intensity,thegrowthrateofthealgaewillnolongerincrease;thegrowthrateismaximal.However,light

intensitycanbecometoohigh,andtheconceptofphotoinhibitionmaybeintroduced.Studentsmaybe

askedtonameafewcommonplantsthatpreferthesun,andonesthatprefertheshade.Whathappensto

shadeplantswhenyouplacetheminbrightlight?(Theywitheranddie.)Algaereducetheirgrowthrate

atextremelyhighlightintensities.

TheLight-DarkCycle

Inadditiontolightintensity,thelight-dark(L:D)cycleplaysanimportantroleinthegrowthofthealgae.

It’sinstructivetoprobestudentunderstandingoftheL:Dcycleandhowitchangesthroughtheseasonal

cycle.Whenarenightsthelongestanddaystheshortest?Whenaredaysthelongestandnightsthe

shortest?Howlongisdaylightduringthesummer?Howaboutduringthewinter?CommonL:Dcyclesare

16:8hours(summer),12:12hours(spring/fall),and8:16hours(winter).Iftheclassisusingnatural

light,studentscanlookuptheL:DcycleontheUSNavalObservatorywebsite

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(http://aa.usno.navy.mil/data/docs/Dur_OneYear.php).Withartificiallight,theL:Dcyclecanbe

controlledwithasimpletimer(suchasthosefoundinahardwarestore).Studentsmaybeaskedhowthe

L:Dcyclemightaffecttheirexperiments.WillthealgaegrowfasterunderalongerL:Dcycle,orwillthey

simplygrowforalongerperiodoftime?Why?HowwilltheL:Dcycleaffectthetimeneededtoreachthe

maximumbiomassinthebottle?

Chlorophylla

Atsomepoint,instructorswillwanttomentionthepresenceofchlorophylla(hereafter,simplycalled

chlorophyll).Chlorophyllisthemajorlightabsorbingpigmentofallphotosyntheticorganisms(including

cyanobacteria,algae,andhigherplants)thatproduceoxygen.(Thislatterdistinctionmayormaynotbe

importanttointroduce:somebacteriacarryoutformsofnon-oxygenicphotosynthesisthatdonotuse

chlorophyll,andwe’llleaveitatthat).Thegreenthatstudentsobserveintheleavesofatree,oranalgae-

filledpond,comesfromchlorophyll.Whileit’snotnecessarytointroducebiochemicalmechanismsby

whichplantscaptureandprocesslightenergy(i.e.,theZ-scheme),itisimportanttopointoutthat

chlorophylltakesenergyfromthesun(orartificiallights)andturnsitintochemicalenergy(i.e.,ATPand

NADPH)thattheplantcanusetogrow.Theterm,chlorophyllantenna,iscommonlyusedinthe

literature,butweprefertothinkofchlorophyllasakindofcatcher’smittthat’susedtocapturebaseballs

(i.e.,photons)oflight.Ultimately,studentswillusechangesintheconcentrationofchlorophyllasan

approximationforgrowthrate.Usingphotography(smartphonesortabletsworkgreat),colorchart

comparison(e.g.,Fore-ulescale),acolorimeter(homebuiltorpurchased),orafluorometer(e.g.,Turner

DesignsAquafluor),studentswilltrackchangesinthegrowthofthealgaeintheirbottles.Studentsmay

beaskedtopredictwhatwillhappentochlorophyllconcentrationsintheirbottlesasthealgaegrow.

Photoadaptation

Dependingontimeandthelevelofcomplexitythattheinstructorwishestointroduce,theconceptof

photoadaptationmaybeintroduced.Thephysiologyofalgae(andplants)permitsthemtotunetheir

photosyntheticmachinerytoavailablelightconditions(nutrientconditions,too,butthat’sanother

topic!).Whilewemaynormallyconsiderthattheamountofchlorophyllperalgalcellisconstant,in

reality,thisratiochangeswithlightconditions.Whenlightintensityislow,individualalgalcellsproduce

greateramountsofchlorophyll.Why?Morecatchers’mitts(i.e.,morechlorophyllantennae)enablethe

algalcelltocapturemorephotonsoflight.(Technicallyspeaking,morechlorophyllincreasesthe

probabilityoflightcapture.Algaemayincreasethesizeoftheirantennae,ortheymaymakemore

antennae,dependingonthespecies.)Thus,underlowlightconditions,individualalgalcellswillbecome

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greener.Attheotherendofthespectrum,underhighlightconditions,algaewillreducetheir

concentrationsofchlorophyllpercell(toavoidphotoinhibitionandtoconserveenergy).Assuch,under

highlightintensities,individualalgalcellswillappearlessgreen.Theinstructormaywanttoaskstudents

toexaminetheleavesofshadeandsunplantsandcomparetheircolor.Typically,shadeplantswill

appearadarkergreenbecauseoftheirhighchlorophyllpercellratio,ascomparedtosunplants,witha

lightergreencolor,thatis,alowerchlorophyllpercellratio.It’spossibleatthispointtointroduce

studentstootherphotosyntheticpigments,suchotherformsofchlorophyll(e.g.,chlorophyllb,

chlorophyllc)andaccessorypigments(e.g.,fucoxanthins)thathelpcapturelight(especially,wavelengths

oflightthatchlorophylladoesnotcapture),orphotoprotectivepigments(e.g.,carotenoids)thatprotect

thephotosyntheticmachinerymuchinthesamewaythatsunglassesprotectoureyes.Theseother

pigmentsbecomevisibleinclimateswheretheleaveschangecolor:absorptionofchlorophyllduringfall

makesthesepigmentsapparentastheyellows,oranges,andredsoffallleaves.Differentspeciesofplants

(andalgae)makedifferentkindsofaccessoryandprotectivepigments,and,assuch,areresponsiblefor

impartingvisiblecolordifferencesinthekindsofplantsthatweseeinnature.Additionaltopicsthatmay

beintroducedhereincludethecharacteristicsofvisiblelight(i.e.,colorsandtheirwavelengths),whywe

seecolor(pigmentsinmaterialsabsorbdifferentcolorsandthecolorweseeisthecolornotabsorbed),

theabsorptionpropertiesofdifferentalgalpigments(chlorophyllprimarilyabsorbsanarrowrangeof

bluelight,~430nm,andotherchlorophyllsandotherpigmentsdifferinthecolorstheyabsorb),algal

andplanttaxonomy(which,tosomeextent,isbasedonthepresenceofparticularpigments),andthe

evolutionofalgaeandhigherplants(thepresenceofchlorophyllbinhighplantssuggeststhatthey

evolvedfromchlorophytes,agroupingofalgaethatcontainchlorophyllb).

LightSources

Thedecisionoflightintensitiesatwhichtogrowthealgaeinabottledependsmainlyontheavailability

ofartificiallightsandspace.Thoughsomelightsourcesarebettersuitedtoalgalgrowththanothers,just

aboutanylightsourcecanbeused,evenatungstenlightbulb.Highandlowlightintensitiesmaybe

simulatedbyplacingthebottlesofalgaeclosertoorfurtherfromthelightsource,respectively.

Alternatively,householdwindowscreenmaybecutandwrappedaroundindividualbottlestoreduce

lightintensity.Ifspaceandalightsourcearelimited,thebottlesofalgaemaybesimplyplaceina

windowsillforstudentstoobserveoverthecourseofafewdaysorweeks.Thereareanumberof

differentoptionshere.SeetheAppendixforpossiblelightsourcesandwheretobuythem.

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LightintheBottle

Followingadiscussionoflight,studentsshouldbeaskedtolookattheirbottles,anddetermineif

anythingaboutthebottlemightblockthepathoflightfromthelightsourceintothebottle.Some

studentsmaysuggestthatthecapmayblocklight,acorrectassertion.Butit’stypicaltogetsmilesand

laughterwhenoneormorestudentssuggestthattheyremovethelabelsfromtheirbottles.(The

importanceofthisstepcannotbeoverstated:thedegreetowhichstudentsunderstandtheroleoflightin

algalgrowthwillbeexpressedintheirconfidencethatremovingthelabelisano-brainer.)Atthispoint,

weletthemremovethelabelanddisposeofitproperly.Theinstructorshouldnowaskhowthebottles

shouldbeorientedinrelationtothelightsource.Shouldthebottlesbelitfromthetoportheside?Would

itbebettertolaythebottlesontheirsideforalightsourceabovethebottles?Iftimepermits,a

discussionofBeer’sLawcanbeintroducedhere,theexponentialdecreaseinlightintensityaslight

passesthroughwater.Thoughit’snotnecessarytointroduceabsorptionandscattering,theinstructor

canprovidetheseprocessesasanexplanationforthereductioninlightintensitywithdepthinacolumn

ofwater.Agraphofthisrelationshipcanbeuseful,especiallyasBeer’sLawhelpsstudentstounderstand

howconcentrationsofchlorophyllcanbedeterminedusinganinstrumentthatmeasuresreductionsin

lightasitpassesthroughatesttubeofchlorophyllsolution,i.e.,acolorimeterorspectrophotometer.

Beer’sLawmayalsobeappliedtounderstandingwhytheoceanisblue,asbluelightisthecolorleast

absorbedbyseawater.Beer’sLawhasanumberofapplicationsinchemistry,andtheinterested

instructormayfinditusefultoexplorethisliterature,andintroduceadditionalconcepts,dependingon

theemphasisandcomplexityofthecourse.Thekeypointhereisthatlightintensitywillbereducedfrom

thelightedsideofthebottletotheoppositesideofthebottleasaresultofabsorptionandscatteringby

water(astrongabsorberandscatterer),bythealgaesuspendedinthewater(specifically,chlorophyll

andotherpigments),and,possibly,theplasticofthebottle

ScientificQuestions&Predictions

Afterstudentshaveremovedtheirlabelsanddecidedontheorientationandplacementoftheirbottlesin

thelightpath,theinstructormayaskstudentstopredictthegrowthrateforthelightintensitythatthey

choseorthat’savailabletothem.Studentsmaybeaskedhowfastwilltheirbottlesturngreen?Howlong

willittakebeforethebottlesstopturninggreen?Whatwillcausegrowthtostopinthebottle,if

anything?Willalgaegrowninhighlightreachtheirpeak“greenness”fasterorslowerthanalgaegrown

inlowlight?Whatroledonutrientsplayinallofthis?Themannerinwhichthesequestionsareexplored

andthetimedevotedtotheirdiscussionwilldependonthedegreetowhichthisexperimentservesasan

inquiry-basedactivityorademonstrationactivity.Thealgaeinabottleexperimentisadaptabletoa

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numberofpedagogicalapproacheswithsimplificationsorexpandeddiscussionspossibleatanyofthe

stepsleadinguptointroductionofthealgaeintothebottle.

TheSeasonalApproach

Onewaytoconnecttheexperimentswithecosystemprocessesistotakeaseasonalapproachwhereby

teamsofstudents“duplicate”conditionsoflightandnutrientsthatarepresentduringeachofthefour

seasonsintemperatezonewaters.Typically,lightintensityislowestinwinter,butdissolvednutrient

concentrationsarehighest.Conversely,lightintensityreachesitsmaximuminsummer,butnutrients

maybeundetectableinsurfacewaters.Springandfall,timeswhenalgaebloomsarecommoninlakes

andintheocean,providesufficientlightandplentifulnutrients.L:Dcyclesandwatertemperaturesdiffer,

too.Thefollowingcombinationsmaybeexploredforexploringtheseasonalcycleofproductivityin

aquaticecosystems:

• Winter:lowlighthighnutrients,8:16L:Dcycle,coldesttemperatures

• Spring:mediumlight,highnutrients,12:12L:Dcycle,warmertemperatures

• Summer:highlight,lownutrients,16:8L:Dcycle,warmesttemperatures

• Fall:mediumlight,mediumnutrients,12:12L:Dcycle,coldertemperatures

Studentsshouldbeaskedtopredictgrowthratesandtimetoreachmaximumbiomassforeachofthe

“seasons.”Inpresentingtheirresults,studentsmaybeaskedtocomparewhathappenedintheirbottles

tothetypicalseasonalresponseofalgae“inthewild.”Weretheresultsasexpected?Ifnot,why?

TheFastApproach

Foregoingmostofthedetaileddiscussionoutlinedabove,allofthestepstothispointcaneasilybe

accomplishedinasingleclasssessionprovidedtheinstructorhasthematerialsreadyandhasprepareda

placeforthebottlestobeplacedaheadoftime.Studentscaneasilyremovesomeofthewaterinthe

bottle,addateaspoonofseasalts,addonetoseveralsquirtsofnutrients,andremovethelabelonthe

bottleinthespaceoflessthananhour.However,instructorswillwanttogathertheclassandslowdown

thepacetoaddressacouplequestionsofmethodologythatwillmaximizetheeducationalbenefitsofthe

experiment,andthatwillensurethemostreliableresults.

ScientificQuestions&Predictions

First,instructorswillwanttoaskstudentswhatisthestartingpointfortheexperiment.Isitthebottleof

preparedwaterwithoutalgae,orisitthebottleofwaterplusthealgae?Whatarethedifferences

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betweenthebottlewithoutalgaeandthebottlewithalgae?Howcanweobserveandmeasurethose

differences?Themostappropriatestartingpointforobservingormeasuringthegrowthofalgae,thetime

zero,asitwere,couldbetheobservationormeasurementthatoccursoncetheinitialaliquotofalgaehas

beenadded.However,ifquantitativemeasuresaregoingtobeused,suchasfluorometry,itwillbe

importanttoobtainablank.Whatdoestheinstrumentreadwithoutanyalgae?Wethinkitcanbeuseful

andimportanttointroducetheconceptoftheinstrumentorobservationalblank.Studentsshouldbe

madeawareofthestateofthebottleanditswaterpriortointroductionofthealgae(theblank)aswellas

thetimezero,thestateofthebottleoncethealgaehavebeenintroduced.Quantitativedeterminationof

theconcentrationofalgaeinthebottlerequiresablank,themeasurementobtainedpriortointroduction

ofthealgaetoaccountforinstrumenteffectsonthemeasurement.Thedegreetowhichthisis

emphasizeddependsonthemethodsthatwillbeusedtodocumentchangesinalgalgrowth.But,inany

case,wethinkit’simportanttomakeobservationsormeasurementspriortointroductionofthealgaeto

theirnewhomes.

PicturesandNotebooks

Second,studentsmaybeaskedhowtheyaregoingtodocumentthestartoftheexperiment.Willthey

takepictures?Willtheymakeobservationsonthecolorinsidethebottleoncethealgaehavebeen

introduced?Willtheypickacoloronacolorchart?Regardlessofthemethodschosen,westrongly

recommendthatstudentstakepictures,butthere’salessonhere.Doesitmakeadifferencehowtheytake

thepicture?Attheoutset,weletstudentstakepictureswithoutanydiscussionofhowtheydoit.

Followingthepicturetaking,includingtimeforstudentstotakeselfieswiththeirbottlesofalgae,weask

studentstosharetheirphotoswitheachother.Dotheynoticeanything?Instructorsmayaskafew

studentstoemailtheirphotostotheinstructor’semailaddresssothattheinstructorcanprojectthemon

aclassroomLCDprojector,ifavailable.Typically,studentsplacetheirbottlesontheirdesksoratableand

takepicturesfromanyangleandanydistancewithoutanythoughtofwhethertheycanseecolorinside

theirbottles,ortakethesameshotatafuturedate,oncethealgaehavegrown.Atthispoint,instructors

shouldemphasizetheimportanceofreproducibilityinmakingobservationsormeasurements.Ask

studentstocommentoneachother’sphotoswithregardtothevisibilityandcolorofthecontentsinside

thebottle.Cantheyimproveonthepicture?Doesitmatter?Moststudentswillrealizethattheirpictures

arenotideal.Adiscussionoftheidealphotoshouldfollow.Intheend,instructorswillwanttoemphasize

thatitisimportanttotakethepicturewiththesamecamerainthesameposition(withrespecttothe

bottle),anditwillbeimportantthatthebottleisinaplacewiththesamelightingandagainsta

backgroundwherethecolorinsidethebottlecanbeeasilydiscerned.Wehavefoundthatplacingthe

bottlesontheledgeofawhiteboardworksverywell,aslongasstudentstaketheirpicturessothatthe

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entirebottleiscaptured,sothatthebottlefillstheframeofthecamera,andsothatthecameraisheld

perpendiculartothebottle.

LabelingBottles

Third,andthisisapointthatinstructorscanwaittointroduce,howwillstudentsbeabletoidentifytheir

bottleofalgae?Howwilltheytellonebottlefromtheother?Thiscanbeafunlessonifstudentsareleftto

theirowndevicesattheoutset.Iftheinstructorhasaskedforafewphotosviaemail,heorshecanshow

thephotoandasktheclasstowhomitbelongs.Inmostcases,studentswillhaveneglectedtolabelthe

bottlesoridentifyownershipofthebottleinawaythat’svisibleintheirpictures.Oncestudentsrealize

thisshortcoming,theygetit!Wefindthatit’susefultodiscussandcomeupwithasetofidentifiers

uniquetotheclass(ifdifferentsectionsarecarryingouttheexperiment),theteam(ifstudentsare

workinginteams),andtheindividual(thepersonresponsibleforaparticularbottle).Wealsofindthat

it’susefultoaskstudentswheretheyshouldplacetheirlabels.Ifstudentshavelearnedthelessonsof

lightpenetration,theywillsuggestthatthebottlecapistheappropriateplacebecauseplacingalabelor

writingonthebottlewillreducelightinsidethebottle.Atthispoint,weaskstudentstoproceedwith

labelingoftheirbottles,andtoretakepicturesusinganow-well-definedprotocol.

ConducttheExperiment

Finally,it’stimetoaddalgae.Werecommendaconsistentandpre-determinednumberofdropsforall

students;typically1-3dropsareideal,moreifthestockculturedensityislow.Obviously,thestarting

concentrationwillinfluencethenumberofdaysuntilpeakalgaldensityisreached.Instructorsmaywish

toruntheexperimentaheadoftimewithanumberofdifferentstartingconcentrationstofindthe

concentrationthatbestfitsthetimetabledevotedtotheexperiment.Theimportantconsiderationisto

allowachangetobeobservableordetectablefortheconditionsoflightandnutrientsunderwhichthe

experimentisconducted.Bottleswithhighlightintensitiesandhighnutrientconcentrationswillshow

visiblechangesinashorterperiodoftimethanbottlesgrownatlimitingnutrientconcentrationsunder

lowlight.Intruth,withoutquantitativemeasurementsoflightintensitiesandnutrientconcentrations,it’s

goingtobedifficulttopredictwithanycertaintythedailyrateofchangeingrowth.Butthat’sthefunof

it!Instructorsmaywanttoletsomestudentsstartwithmoredrops.It’sanexperiment.Aslongasallof

thevariablesarecarefullydocumented,thenthere’sknowledgetobegainedinanyapproach.

KeepTakingPicturesandMakingObservations

Followingadditionofalgae,studentswillwanttotakeanotherroundofpictures.Inclassroomswhere

quantitativemeasurementsarebeingcarriedout,thefirstsetofmeasurementsshouldbetakenatthis

24

pointtoestablishtheinitialconcentrationofalgaeineachbottle.Verylikely,despitecarefulattentionto

waterbottlevolumesandtheamountofnutrientsandalgaeadded,therewillbeslightdifferencesamong

students.Anystudentswhosemeasurementsdifferwidelyfromotherstudentsshouldbequeriedasto

theprocedurestheyfollowed.Wetypicallyrequirestudentstoworkinteamsof3or4andcarryout

identicaltreatments.Thus,agiventeamofstudentsperformstheexperimentintriplicateor

quadruplicate,andresultswithineachteamshouldbesimilar.Dependingonthedegreetowhichtheir

initialmeasurementsdifferfromtheexpected,andtimeavailability,instructorsmaychoosetomakethe

studentorstudentstostartover,ortheymayallowtheteamtoproceedwiththeirexperimentsasis.

ASimpleObservationActivity

Forsomeinstructors,thealgae-in-a-bottleexperimentmayconsistsimplyofanactivitywherestudents

prepareabottleofalgae,andwatchitgrow,eitherintheclassroomorathome.Forothers,classsessions

followingthetimezeromeasurementswillrequiretimeforcontinuingtodocumentgrowthusing

photographandothermeasures.Datamaybecompiledfortheclassasawholeastheexperiment

proceeds,orattheendoftheexperiment.Instructorsmayaskstudentstocreateapicturecollageoftheir

bottles,prepareatableofobservations,createbarorlinechartsofanyquantitativemeasures,and/or

writeuptheresultsoftheirexperimentusingalaboratoryreportformat.Wheresufficientquantitative

measuresareavailable,studentsmaycalculatetherateofchangeinchlorophyllconcentrationby

subtractingpreviousvaluesfromsubsequentones,orbysimplysubtractingthetimezeroconcentration

fromthefinalone.Resultscanbepresentedorallyinfrontoftheclass,ortheinstructormaychooseto

compileandpresentselectresultstoillustratekeyfindings.Studentsmaybeaskedtoexplainany

differencesintheirresultsfromtheoutcomestheyexpected,orhowtheymightimproveupontheir

experiments.Therearenumerouspossibilitiesforexploringanddiscussingthephysical,chemical,and

biologicalprocessesthatoccurintheiralgaehomes.

AssessAttitudesandUnderstanding

Followingtheexperiments,instructorsmaywanttoassesschangesinattitudestowardstheexperiments

(i.e.,theaffectivedomain)andchangesinconceptualunderstanding(i.e.,theeffectivedomain)usingthe

post-testsprovidedintheAppendix.Theresultsofthepre-postassessmentscanprovidevaluable

informationontheeffectivenessofeachstepoftheactivity,andpointoutwaysthatdeliveryofthe

experiment,i.e.,thelessonplan,canbemodifiedandimproved.Aboveall,wehopethatimplementation

oftheexperimentreapsbenefitsforhelpingstudentstounderstandthenatureofscience,andfor

bringingtostudentsadeeperconceptualunderstandingofthefactorsthatgovernthegrowthofplants

andalgaeinthenaturalworld.

25

MaterialsandOptionalEquipment

TheAlgae-in-a-BottleExperimentreliesprimarilyoneasy-to-find,inexpensive-to-purchasematerials

availableinlocalgroceryorboxstores,oronline.Instructorswishingtoadoptmorerigorous,research-

basedprotocolsmay,too,findmaterialseasilyavailable,albeitatgreaterexpense.

BottledWater

Bottlesof8-ounce,springwaterworkbestandaretheleastexpensive.

Saltwater

Mortonorotherbrandsofseasalts.

Professionalgradeseasalts:$75,Oceanic81050NaturalSeaSaltMix,makes200gallons

http://www.amazon.com/Oceanic-81050-Natural-200-Gallon-Bucket/dp/B000256EWG

InstantOcean,Coralife,andothersmaybesuitable.

SourcesofAlgae

Liquidmicroalgaecultures:$10-$30,FloridaAquaFarms

http://florida-aqua-farms.com/shop/liquid-microalgae-cultures/

Research-grademicroalgaecultures,$175,NationalCenterforMarineAlgaeandMicrobiota

https://ncma.bigelow.org

SourcesofNutrientMedia

MiracleGro,$9,Amazon

http://www.amazon.com/Miracle-Gro-1001233-Purpose-Plant-Food/dp/B000P6QYJK

MicroalgaeGrowMassPack,$20+,FloridaAquaFarms

http://florida-aqua-farms.com/product-category/store/micro-macro-nutrients/

Researchgradef/2media,$85,NationalCenterforMarineAlgaeandMicrobiota

https://ncma.bigelow.org

26

LightSources

Aninfinitevarietyoflightfixturesarepossible,rangingfromsimplebulbandfluorescentlightfixturesto

LEDaquariumfixtures.Thetypeyouchoosewilldependonbudgetandspaceconsiderations.Forbest

growth,choosebulbsdesignedspecificallyforplants(thatis,bulbsthatemitasmuchbluelightas

possible,thecolormostabsorbedbychlorophylla)

BulbFixturesandBulbs

Bulbfixturescangeneratelotsofheat,buttheyareinexpensive,andanumberofbulbsdesigned

specificallyforplants,includingcompactfluorescentbulbsandLEDbulbs,arenowavailable.Type

“growbulb”inAmazonorclickbelow.

http://www.amazon.com/s/ref=nb_sb_noss_1?url=search-alias%3Daps&field-

keywords=grow+bulb

FluorescentLights

Fluorescentfixturesarecooler,andoffertheadvantagethatyoucanstandthemonedgeandplace

alotofbottlesinfrontofthem.Choosefullspectrumorplantgrowbulbsforthebestresults.Type

“growbulbsfluorescent”inAmazonorclickbelow:

http://www.amazon.com/s/ref=nb_sb_noss?url=search-alias%3Daps&field-

keywords=grow+bulbs+fluorescent&rh=i%3Aaps%2Ck%3Agrow+bulbs+fluorescent

AquariumLEDFixtures

Designedfortrue(hardorstony)corals,whichcontainalgalsymbionts,aquariumLEDfixtures

offerthemostadvancedlightingsystemsforgrowingalgae.Theyarenotinexpensive,buttheir

wavelengthsofemissionareidealforalgae,andmostLEDfixturespermityoutovarythelight

intensity.WeusetheTaoTronics165WDimmableLEDAquariumLights,buttheyarenot

availableatthetimeofthiswriting.Type“LEDreefaquariumlighting”inAmazon.Wesuggestthat

youreadthereviewsandmakeyourowndecisionbasedonyourbudgetandspace

considerations.

http://www.amazon.com/s/ref=nb_sb_noss_1?url=search-alias%3Daps&field-

keywords=led+reef+aquarium+lighting&rh=i%3Aaps%2Ck%3Aled+reef+aquarium+lighting

MobileLightingSystems

Amobilesystemisidealforuseinmulti-useclassrooms(e.g.,mostcollegeclassrooms)wherethe

experimentalsetupcannotbeleftinplace.Mobilelightcartscanbewheeledinandoutofa

27

classroom,andstoredsafelyinaclosetorotherlocationwhereelectricaloutletsareavailable.

Pricesrangefromthefewtoseveralhundredsofdollars.Weusemobile,4-shelf,plantgrowcarts

withfluorescentlamps,purchasedfor$782.50atCarolinaBiologicalSupply.Type“plantgrow

cart”inGoogleforotheroptions.

ScientificEquipment

Measuringcups,teaspoons,andtablespoons,availableatyourlocalgroceryorboxstore

Plastictransferpipettes,$4.39,packof100

http://www.amazon.com/Plastic-Transfer-Pipettes-Gradulated-

Pack/dp/B005IQTSE0/ref=sr_1_1?ie=UTF8&qid=1428780519&sr=8-1&keywords=pipettes%27

Refractometerformeasuringsalinity,$49.99,MarineDepot

http://www.marinedepot.com/ps_viewitem.aspx?idproduct=MD2101&child=MD2101&utm_sour

ce=adwordsfroogle&utm_medium=cse&utm_campaign=adwordsfroogle&utm_content=MD2101&

gclid=CjwKEAjwjKOpBRChjsTyicbFy3QSJADP1gTNYxtayxnYdL87VtARFk9DOuffxNO8mH_wVOCO

elk-wBoCLGfw_wcB

EducationalColorimeterKit,formeasuringcolorchanges,$145.00,IORodeo

http://www.iorodeo.com

AquaFluorHandheldfluorometerformeasuringchlorophyll,~$3000,TurnerDesigns

http://www.amazon.com/Plastic-Transfer-Pipettes-Gradulated-

Pack/dp/B005IQTSE0/ref=sr_1_1?ie=UTF8&qid=1428780519&sr=8-1&keywords=pipettes%27

HelpfulResources

CulturingMethodsandInformation,NationalCenterforMarineAlgaeandMicrobiota

https://ncma.bigelow.org/culturing-methods

HowtoGrowAlgae,Wiki-How

http://www.wikihow.com/Grow-Algae

MeasuringSalinitywithaRefractometer

http://reefkeeping.com/issues/2006-12/rhf/

TheBasicsofPhotosynthesis,SimpleEnglishWikipedia

28

http://simple.wikipedia.org/wiki/Photosynthesis

TheBasicsofChlorophyllMeasurement

http://www.ysi.com/media/pdfs/T606-The-Basics-of-Chlorophyll-Measurement.pdf