The Effect of Dark Period Duration on Synechococcus WH8102 Growth Rates. by Marius Ibuye Balola A PROJECT. submitted to. Oregon State University

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1 The Effect of Dark Period Duration on Synechococcus WH8102 Growth Rates by Marius Ibuye Balola A PROJECT submitted to Oregon State University University Honors College in partial fulfillment of the requirements for the degree of Honors Baccalaureate of Science in BioHealth Sciences (Honors Associate) Presented June 4, 2015 Commencement June 2015

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3 AN ABSTRACT OF THE THESIS OF Marius Ibuye Balola for the degree of Honors Baccalaureate of Science in BioHealth Sciences presented on June 4, Title: The Effect of Dark Period Duration on Synechococcus WH8102 Growth Rates. Abstract approved: Kimberly Halsey Synechococcus is one of the most abundant groups of primary producers in the marine environment. They play a crucial role in the food web by being the main source of alimentary energy to many marine organisms that contribute to human nutriments. The ability of Synechococcus to use light as a source of energy through photosynthesis makes them important in the marine environment. Understanding how these photosynthetic organisms respond and adapt to fluctuations in their environment is important to studies of phytoplankton physiology. To date, no studies have been done that measured the effect of dark period duration on the growth rate of Synechococcus. This sparked our curiosity to examine how the duration of a dark period impacts the growth rate of the ecologically important cyanobacteria, Synechococcus sp. WH8102. We found that exposure of Synechococcus WH8102 to a dark period did not enhance growth rate at any light intensity studied thus far. More research is needed to more finely resolve the potential effects of dark period on growth in this organism. These results will provide insights to understand the global success of this organism in marine environments. Key Words: Cyanobacteria, Synechococcus, dark periods, growth rates

4 Corresponding address:

5 Copyright by Marius Ibuye Balola June 4, 2015 All Rights Reserved

6 The Effect of Dark Period Duration on Synechococcus WH8102 Growth Rates by Marius Ibuye Balola A PROJECT submitted to Oregon State University University Honors College in partial fulfillment of the requirements for the degree of Honors Baccalaureate of Science in BioHealth Science (Honors Associate) Presented June 4, 2015 Commencement June 2015

7 Honors Baccalaureate of Science in BioHealth Science project of Marius Ibuye Balola presented on June 4, APPROVED: Kimberly Halsey, Mentor, representing Oregon State University, Department of Microbiology Steve Giovannoni, Committee Member, representing Oregon State University, Department of Microbiology Bethan Jones, Committee Member, representing Oregon State University, Department of Microbiology Toni Doolen, Dean, University Honors College I understand that my project will become part of the permanent collection of Oregon State University, University Honors College. My signature below authorizes release of my project to any reader upon request. Marius Ibuye Balola, Author

8 Introduction Synechococcusisoneofthemostabundantgroupsofprimaryproducersinthe marineenvironment.aswithmanyothercyanobacterialspecies,synechococcussp. playsacrucialroleinthefoodweb.theyconstitutethebaseofaquaticfoodweb, thusprovidingnutrientstomicroorganismsaszooplankton,whichinturnare consumedbylargerorganisms. [1] Thisfoodweb,therefore,makesSynechococcus importantforhumanfoodproductionsincethefishstocksdependontheprimary producers. CyanobacteriaareresponsiblefortheoxygenationofEarth satmosphere. CyanobacteriaareknowntobethefirstoxygenEproducingorganisms. [2] They contributevastlytoecologicaloxygencycle.asacomponentofthislargeand diversegroupofphytoplankton,synechococcuscontribute70to80percentofthe oxygenintheatmosphereandsimultaneously,theyfixaround25megatonsof carbondioxide. [3] Thesebacteriareducecarbondioxideintheoceansthroughtheir photosyntheticprocessesconvertingitintoinorganiccarbon. Synechococcusisaprokaryoticunicellularcyanobacteriumandthuscarriesout oxygenicphotosynthesisandrespirationactivitieswithinitsplasmamembrane.itis mostlyfoundinwellelitsurfaceofmarineenvironmentwherelightisnonelimiting. Duringphotosynthesis,Synechococcususeslightenergytomakeorganiccarbon, whichiseventuallyusedtomakeenergyneededforgrowth. Occasionally,Synechococcuswillusehydrogensulfideaselectrondonor,however wateristhepreferredelectronsource,causingoxygentobecreatedasabyproduct. [4] Throughthisprocesselectronsbecomeavailableforreductionofcarbondioxide intoorganiccarbon.photosyntheticelectrontransportalsodrivestheformationofa protongradientthatisusedtogenerateatp.duringtheseprocesses,different metabolicactivitiescanbeconductedtogetherorseparatelyduringdarkorlight periods.phycobilisomesandchlorophyllareusedtoharvestlighttocarryout photosynthesisduringtheday.inthedark(atnight),thecellswilluseoxygenasa terminalelectronacceptor,andthroughaerobicrespiration,theybreakdowntheir storedcarbohydratetofuelothermetabolicprocesses. [5] Incontrasttoeukaryoticalgae,whichcanspatiallyseparatecarbonfixationand electrontransportwithinchloroplastsandmitochondria,respectively, Synechococcuslacksmembraneboundorganellesandthususesitsplasma membraneforbothactivities.thisdifferenceincellularorganizationledusto hypothesizethatsynechococcuswouldbenefitfromatemporalseparationof photosyntheticcarbonfixationandrespiration. Synechococcushasadaptedtovariousenvironments.Itssuccessfuladaptationis basedonitsabilitytoliveinenvironmentsthatfluctuateinavailabilitiesoflightand nutrients,suchasoligotrophicareasthatarecharacterizedbyextremelylow

9 concentrationsofnutrients.suchenvironmentsaretypicalforopenseaspaces wherethesecellsarespecializedforobtainingnecessarynutrientsandtracemetals. [6] Inadditiontotheresponsesofcyanobacteriatonutrientdepletion,previousstudies haveexaminedhowotherfactorssuchaslightfluctuation,daylength,and temperatureaffectthegrowthrateofcyanobacteria.daylengthandtemperature wereobservedtohaveseparateand/orinteractiveimpactsoncyanobacterial growthrate.ingeneral,theresultsshowedthatcyanobacteriagrewmostefficiently withshortdaylengthsandwarmtemperatures. [7] Wemeasuredgrowthrateresponsesunderdifferentlightintensitiesandlight/dark periods.wehypothesizedthatforagivenlightintensitythegrowthratewouldbe fasterwhenadarkperiodwasprovided.ourhypothesisalsostatedthatthere wouldbeanoptimaldarkperioddurationwheretheenhancementofgrowthrate wouldbeobserved.thelongetermgoalistounderstandhowthesephotosynthetic organismsrespondandadapttofluctuationsintheirenvironment.inthiscase,light periodwasthevariablewechosetostudytolearnhowthesecellsoptimizetheir growthinmarineecosystems.theresultsofthisprojectwillgivecluestothe physiologicalprocessesusedbythisabundantandecologicallyimportant cyanobacterium. MaterialsandMethods Culture.Conditions. Synechococcus WH8102 cultures were grown in artificial seawater, f/2 + Si media, at a constant temperature of 20 ºC and lit by fluorescent tubes. Nine glass flasks were inoculated with Synechococcus WH8102 to an initial concentration of about 10 4 cells per ml. Among these nine flasks, three were exposed to ~ μmol photons m -2 s -1, and six were exposed to lower light intensities that were achieved by screening the fluorescent tubes with neutral density screening. Thus, three flasks were exposed to ~ μmol photons m -2 s -1 and three flasks were exposed to ~5-10 μmol photons m -2 s -1. The intensity of light was measured using a PAR (photosynthetically active radiation) sensor. The experiment was conducted in three parts under a 24 hour cycle each. It should be noted that the light intensities remained the same throughout the experiment (10, 50, and 200 μmol photons m -2 s -1 ). Inthefirstexperiment,allnineflasksweregownwithnodarkperiod(24hourlight period).inthesecondexperiment,flaskswereexposedto12hoursoflightand12 hoursofdarktime,andthethirdexperimentwasplannedsuchthatcultureswould beexposedtolightfor18hoursand6hoursofdarkperiod.afourthexperiment wasplannedsuchthatcultureswouldbeexposedto6hoursoflightand18hoursof darkperiod.notethattimeconstraintsdidnotallowcompletionofthethirdand fourthexperiments.

10 Cell.Density.Measurements. Throughouttheexperimentsthecelldensityofeachflaskwasmeasuredbyflow cytometrytocalculatethegrowthrateofsynechococcusinresponsetodifferent lightintensityanddurationofdarkperiod.onemilliliterofeachcultureflaskwas transferredtoatubeforcellcountsbyflowcytometry.forsamplesthatwerehighly concentrated,1/10or1/100dilutionwasappliedaccordingly.transferredsamples weremixedwith1μl50%glyceraldehydeandincubatedatroomtemperaturefor 15minutestofixthecellsbeforerunningthesamplesintheflowcytometer. Thecelldensity(cells/mL)wasobtainedusingthefollowingformula: (#events/time)x(flowrate) whereeventsarecellsandtheflowratethroughthecytometerwas1s/μl.the numberofeventswasobtainedfromadelineatedregionknowntocapturethecell sizeandorangefluorescencesignatureofsynechococcus. Theaverageandstandarddeviationofthecell density was taken from the triplicate cultures grown at each light intensity, and results were plotted using Excel. Results SynechococcusWH8102wasgrownintriplicateateachofthreelightintensities(10, 50,200μE)ataconstanttemperature(20ºC). During the first experiment (Figure 1), cultures were grown under zero dark periods (constant light). Thisexperiment lasted 20 days for cultures at 50 and 200 µe and 28 days under 10 µe.we observed the typical three phases of a growth curve for a bacterial batch culture(lag, exponential, stationary). However, variability intheapparentgrowth ratewas observed in cells growing at 10 µe.from these growth curves, I calculated growth rates (Table1)of cells grown at each light intensity. The fastest growthratewas observed for cells growing at 50 µe. Cells growing at 200 µe has a growth rate thatwas abouthalf ofcellsgrownat50µe. Cellsgrowingat10µEgrew the slowest. Light intensity Growth Rate (division/day) 200 µe µe µe 0.15 Table1:Synechococcusgrowthrate(division/time)atthreedifferentlight intensitieswithzerodarkperiods

11 1.00E+07 CellDensity(Cell#/mL) 1.00E E E E µE 50µE 10µE 1.00E Time(Days) Figure 1: Synechococcus WH8102 growth at three different light intensities with zero dark periods (constant light). Inthesecondexperiment(Figure2),culturesweregrownwitha12Ehourdarkand 12Ehourlightperiods.Thisexperimentlasted24daysforculturesgrowingat200 µeand26daysforculturesgrowingat10and50µe.weobservedatypical bacteriumcellcurvewith200µeandcurveswithvariabilityat10and50µe. ThegrowthratescalculatedfromFigure2areshowninTable2.At12Ehourdark periodthegrowthratesat200and50µewerealmostthesame.negligiblegrowth ratewasobservedat10µeduetoinsufficientlighttosustaingrowth. LightIntensity Growth Rate (division/day) 200 µe µe µe 3.0 E-05 (negligible) Table 2: Synechococcus.WH8102growth rates (division/day) atthreedifferentlight intensitieswitha12ehourdarkperiod

12 1.00E+07 Celldensity(cell#/mL) 1.00E E E uE 50uE 10uE 1.00E TIme(days) Figure2:Synechococcus.WH8102growthatthreedifferentlightintensitieswitha 12Ehourdarkperiod Figure3providesasummaryofthegrowthratesobtainedfromthe24hlightand 12hlight/12hdarkexperiments.With12hoursofdarkness,thegrowthrate becamemaximalbetween10and50µe.with12hoursofdarknessthegrowthrate was0.41division/day(200µe),0.44division/day(50ue),andnegligiblegrowth rateat10uecomparedto1.32division/day(200µe),0.58(50µe),and0.15(10µe) atzerodarkperiods. Growthrateswerehigherunderzerodarkperiodsincomparisonto12Ehourdark periods.growthratechangedbetweendifferentlightintensities,butthegrowth ratesfor12ehourdarkperiodperiodsdidnotchangebetween200and50µe.

13 SynechococcusGrowthRate UnderDifferentDarkPeriods Growthrate(division/day) µE 50µE 10µE Lightintensiity Zerodark period 12hoursdark darkperiod Figure3:Summarytableofdifferentgrowthratesatdifferentlightintensitiesunderzero and12ehourdarkperiods. Discussion Thisresearchprojectmeasuredtheeffectofdarkperioddurationonthegrowth rateofsynechococcuswh8102.exposureofthecellstoadarkperiodhada significanteffectongrowthrate.thecombinationoflightintensityanddarkperiod gaveadditionalinformationabouthowsynechococcusoptimizegrowthunder differentlightconditions.thisstudysuggeststhatsynechococcusgrowsoptimally whenprovidewithamoderatelightlevelandconstantlight. Thefastestgrowthratewasunder50µElightintensitywithnodarkperiod.This resultsuggeststhatsynechococcuswh8102hasincreasedabilitytoconvert inorganiccarbontoorganiccarbonatthisgivencondition(constantlightat50µe). Atthisoptimalcondition,carbonwasaccumulatedanduseddirectlyinrespiration tomakeenergy. Growthratewasinhibitedatlightintensitieshigherandlowerthan50µE.It appearedthattherewasaminimumlightintensitythatislessorequalto50µeand

14 greaterthan10µethatresultsinthemaximalgrowthrateobservedat50and200 µe., However,thepresenceofdarkperiodcausedamaximalgrowthratethatisabout halfofthatobservedinconstantlight.interestingly,whenprovided12ehourdark period,lightintensitiesgreaterthat50µeresultedinsimilargrowthrates.adark perioddoesnotappeartoenhancegrowthratesatagivenlightintensity.more experimentsareneededtodeterminethecombinedaffectsoflightintensityand darkperiod,inparticulartoassessifshorterdarkperiodswouldleadtoenhanced growthrates,particularlyathigher(200µe)light. Therearevariableresultsobservedbetweenspecieswhenexposedtodifferent periodsofdarkness.inastudyoffourdifferenteukaryoticalgae,twospecies decreasedtheirgrowthratesandtwospecieshadgrowthratesthatwere unchangedwhenexposedtoa12hdarkperiodcomparedtoconstantlight [8] At50 µeand12hdarkness,twospeciesdecreasedgrowthratesandtwoincreased growthratescomparedtoconstantlight. [8] SimilartoourresultsforSynechococcus.WH8102,thegrowthrateoftheArctic cyanobacteriumschizothirix.calcicola,.didnotrespondpositivelytoadarkperiod exceptwhencombinedwithwarmerincubationtemperatures. [7] Conclusion SynechococcusWH8102 grewoptimallywhenprovidedamoderatelightleveland constantlight.however,thepresenceofadarkperiodcausedamaximalgrowth ratethatisabouthalfofthatobservedinconstantlight.interestingly,when provideda12ehourdarkperiod,lightintensitygreaterthat50µeresultedinsimilar growthrates.attheresolutionofthedarkperiodstestedthusfar,adarkperiod doesnotappeartoenhancegrowthratesatagivenlightintensity.more experimentsareneededtodeterminethecombinedaffectsoflightintensityand darkperiod. Futureworkwillincludemeasurementsofchlorophyllcontent,carbonstorage,and theirchangeswiththeday/nightcycle.

15 Acknowledgements ThisresearchwasconductedatOregon State University: Department of Microbiology --Kimberly Halsey s laboratory under supervision of Dr. Kimberly Halsey with aid of Dr. Bethan Jones SpecialappreciationtoFordFamilyFoundationandOregonStateUniversity: HonorsCollegeandNationalScienceFoundationscholarship

16 References 1. Lidndney,Rebecca;Scott,Michon;(2010). WhatArePhytoplankton.NASA. Earth..Observatory.. 2. Nadis,Steve(2003)."TheCellsThatRuletheSeas".Scientific.American289 (6): PisciottaJM,ZouY,BaskakovIV;Zou;Baskakov(2010).Yang,ChingEHong, ed."lightedependentelectrogenicactivityofcyanobacteria".plos.one5(5) 4. CohenY,JorgensenBB,RevsbechNP,PoplawskiR;Jørgensen;Revsbech; Poplawski(1986)."Adaptationtohydrogensulfideofoxygenicand anoxygenicphotosynthesisamongcyanobacteria".applied.and. Environmental.Microbiology.51(2): Díez,Beatriz;Ininbergs,Karolina(2013). EcologicalImportanceof Cyanobacteria.Cyanobacteria:.An.Economic.Perspective.41E63 6. Palenik,B.;Brahamsha,B.;Larimer,F.W.;LandM.;Hauser,L.;Chain,P.; Lamerdin,J.;Regala,W.;Allen,E.E.;McCarren,J.;Paulsen,I.;Dufresne,A.; Partensky,F.;Webb,E.A;Waterbury,J.(2003). TheGenomeofaMotile MarineSynechococcus.Nature.424,1037E Tang,Evonne;Vicent,Warwick(2010). Effectofdaylengthandtemperature onthegrowthandphotosynthesisofanarticcyanobacterium,schizothrix. calcicola(oscilatoriaceae).european.journal.of.phycology.35(3): Litchman,Elena(2000). Growthratesofphytoplanktonunderfluctuating light.freshwater.biology.44,223e235..