TheValueofInoculumManagementinMicroalgae
ProductionValerieHarmon
HarmonConsulting,Inc.Co-Authors:J.McGowen,P.Pienkos,T.Lane,K.Poorey,E.Knoshaug,E.
Wolfrum
• Cultureswillinitiateinalaboratoryinsmallvolumesandarethentransferredincrementallyuntilfullscaleisachieved.• Typicalscaleupwouldassume10– 20%inoculumbyvolumeasthecultureistransferredfromonescaletoanother.• Initialscaleupistypicallyabatchproductionmodewhichmeansthealgaeismovedoutofonesystemcompletelyintothenextculturestage• Asscaleincreases,inoculumsystemscanmovetobatch,semi-continuousorcontinuouscultivationsystems
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Necessaryforachievingscale Anexampleofaproductionschemeforbatchcultureofalgae(LeeandTamaru,1993).
Inoculum:Whyisitimportant?
Whathappenswhenscaleisachieved?
• Isinoculumnecessaryformaintainingscale?• Algaecultivationsystemsdofail!Thisistrueofpondsandofclosedsystems!
• Wearejustbeginningourstudyofculturesystemfailuresasanindustry,whichwillenableustocontrolandmanageoursystemsforimprovedreliabilityandproductivity.
• Howwillsystemrecoverybemanagedinatimeandcosteffectivemannerwhenlargescaleproductionsystemsfail?• Thereisnotjustonewaytoapproachinoculumgeneration
• Batchproduction• SemiContinuousproduction• Continuousproduction
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Anexampleofalgalgrowthdynamics
AlgalGrowthDynamicsDuringScaleUp(FAOFisheriesTechnicalPaper361,1996).EditedbyLavens andSorgeloos
Lag“phase,duringwhichlittleincreaseincelldensityoccurs,isrelativelylongwhenanalgalcultureistransferredfromaplatetoliquidculture.Culturesinoculatedwithexponentiallygrowingalgaehaveshortlagphases,whichcanseriouslyreducethetimerequiredforupscaling.Thelagingrowthisattributedtothephysiologicaladaptationofthecellmetabolismtogrowth,suchastheincreaseofthelevelsofenzymesandmetabolitesinvolvedincelldivisionandcarbonfixation.”
“Inpractice,culturecrashescanbecausedbyavarietyofreasons,includingthedepletionofanutrient,oxygendeficiency,overheating,pHdisturbance,orcontamination.Thekeytothesuccessofalgalproductionismaintainingallculturesintheexponentialphaseofgrowth.”
“Themostimportantfactorsgoverningcontaminantgrowthdynamicsinalgalmassculturesarethegrowthrateofthecontaminant,therateofdilutionofthebioreactorcontainingtheculture,andthesizeoftheinitialinoculum”ofthecontaminant
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ModeledContaminantGrowthDynamics Conclusion
ForeheadandO’Kelly,2013SmallDoses,BigTroubles:ModelingGrowthDynamicsofOrganismsAffectingMicroalgalProductionCulturesinClosedPBRs
RealLifeExamplesATP3TestBedDataonPondReliabilityandPersonalExperience
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AlgaeTestbedPublicPrivatePartnership
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EstablishCollaborativeOpenTestbedsatfivegeographicallydistinctproductionfacilitiesinordertoquantifyseasonaland
environmentalvariabilityinalgaeproduction
• SoutheastCoastal:FL• WesternCoastal:CA• PacificTropicalCoastal:HI
• SoutheastInland:GA• SouthwestDesertInland:AZ
AlgaeTestbedPublicPrivatePartnership
GenerateHigh-ImpactDatafromLongTermCultivationTrials
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• UnifiedFieldStudies(UFS)• All5sitesexecutingsameproductionmethods,systems,includingseedproductionsystems,andstrains
• ScientificDataManagementSystemandHarmonizedSOPsforanalyticalandUFSproductionmethods• Dataintegrity• StrongQCcapabilityofdata
• DatapubliclyavailableviaNationalRenewableEnergyLabwebsite
UnifiedFieldStudyExperimentalDesign
• FiveSites:Southwestdesertinland, Southwestcoastal,Southeastcoastal,Southeastinland,andPacifictropicalcoastal.• FourSeasons:Spring (March– May),Summer (June–August),Fall (September– November),Winter(December– February)• TwoYears: 2014and2015• ThreeStrains:Nannochloropsisoceanica (CellanastrainKA32),Chlorellavulgaris(ASUstrainLRB_AZ_1201),Desmodesmuscf intermedius(CellanastrainC046)• ExperimentalTreatments:Harveststrategies:frequency(Onceperweek(1x)vsThreetimesperweek(3x)) and dilutionrates(0.11,0.214and0.3perday)
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UFSInoculumProductionMethodsforSystemInitiation
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Initialculturedensity~0.1-0.2g/LAveragegrowthrate~0.3g/L/dayGrowthperiodis9-10daysFinalculturedensity~3.5g/L
1to1transfer
Columntopanel
Initialculturedensity~0.2g/LAveragegrowthrate~0.15-0.2g/L/dayGrowthperiodis9-10daysFinalculturedensity~2.0g/L
12-2’x2’panelsyieldprovidebiomassforaminimumtargetof0.05g/L(AFDW)initialmini-pondconcentration(siteshave14available)
UFSInoculumforSystemRecoveryPost-FailureHealthyPond
FailedPondL
SameTreatment
Drain,clean,calibrateAdvantageofnogrowout,rightbacktoharvestingimmediately!Butisthisqualityinoculum?
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PondReliabilityMetrics
• Reliability:Theabilityofanasset(inthiscaseapond)toperformitsintendedfunctionwithoutfailureforaspecifiedperiodoftimeunderspecifiedconditions.• Metrics• MeanTimeBetweenFailure(MTBF)inDays
• Aharmonizedmetricasperthe SocietyforMaintenanceandReliabilityProfessionals• Averagelengthofoperatingtimebetweenfailures;error-freeperformancetime• Thismetriccanonlybecalculatedifapondfails
• MeanTimeToReset(MTTR)inDays,2.26days• Annual#ofPondResets
• 365/(MTBF+MTTR)
• AnnualResetEffortinDays• Annual#ofresetsxMTTR
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MeanTimeBetweenFailures Effort
BatchSeedInoculumvsSemi-ContinuousOpenPondInoculum
19.98
12.79
0
5
10
15
20
25
Seed Pond
Days
InoculumSource:MeanTimeBetweenFailures
21.00
38.5847.37
87.16
0
20
40
60
80
100
120
Seed Pond
Days
InoculumSource:AnnualResetEffort
Annual#ofRepairsAverageAnnualRepairEffort(#Repairs*MTTR)Average
ReliabilityImprovementsRelatedtoInoculumandProductionStrategies– ANumbersGame• SeedInoculumassumedtostartwith0ciliatesandprotozoaperml,butonday3itisassumedthat5ciliatesand1protozoaentertheculture
• PondInoculumassumedtostartwith150ciliatespermland50protozoapermlonday0(pondinoculum)• Ciliatepestsassumedtoincreasetheirpopulationsbyafactorof4daily• Otherprotozoanpestsassumedtoincreasetheirpopulationsbyafactorof2.67daily
0
5E+10
1E+11
1.5E+11
2E+11
2.5E+11
0 2 4 6 8 10 12 14 16
#/ml
Days
TheoreticalContaminantGrowthRatesSeedInoculum:VariousHarvestStrategies
Ciliates:3x,0.214 Protozoa:3x,0.214
Ciliates:1x,0.11 Protozoa:1x,0.11
0
2E+11
4E+11
6E+11
8E+11
1E+12
1.2E+12
1.4E+12
1.6E+12
1.8E+12
0 2 4 6 8 10 12 14 16
#/ml
Days
TheoreticalContaminantGrowthRatesPondInoculum:VariousHarvestStrategies
Ciliates:3x,0.214 Protozoa:3x,0.214
Ciliates:1x,0.11 Protozoa:1x,0.11
BatchInoculumProductioninOpenPonds:LifeLessonsfromAustralia
• Noinoculuminitially,restartedfailedpondsfromanexistingponds• Notachievingbiomassproductiongoals
• Scalefromoneculturesystemtothenext• Lab– tominiponds(700Lvolume)
• Labtooutdoortransition• (14)minipondsto(4)50m2 ponds• (4)50m2 pondsto(2)400m2 ponds• (2)400m2 pondsto(1)4000m2 ponds
• Inoculumnotneeded– toharvestbasinandbiomassnotlost!!!!!• Wheninoculumwasneededonepondperweekcouldbeinoculated• Thisinoculumsystemutilizedexistingcapabilityandstaffing,socostnothingtoimplementotherthannutrientswhichproducedeitherinoculumorharvestablebiomass• Wellworthit!Producedqualityinoculum!• Existingstaffhadaroutinethatprovidedtheinoculumweneededwhenweneededit!• Weachievedourbiomassproductiongoals!
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Conclusions:InoculumBestPractices
• Aretherewaystomanageourinoculumthatwillhaveasignificanteffectonmicroalgaeproduction?• Quality
• Freeofcontamination,oratleastascleanaspossible
• Fastgrowing,notpaststage2• Nutrientreplete
• Providelittletonotransferstress• Qualityoftheinoculumwilldeterminethelongevityoftheproductionsystem• Testbeddatacanprovidedirectionintermsofhowbesttomanageourinoculumforlargescaleproduction• Inoculumresearchison-goingincurrentATP3research
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Acknowledgements
FloridaAlgaeStevenSchlosserChrisWithstandleyMaryRiddleNancyPhamHo(FIT)
CellanaMartinSabarskyJohannaAntonMarcelaSaraccoDavidAntonKeao Bishop-Yuan
Valicor RenewablesKiranKadamBrianGoodall
ASUGaryDirksJohnMcGowenThomasDempsterPeteLammersMiltSommerfeldWilliamBrandtJessicaChengSarahArrowsmithSarahKempkesDavidCardelloTheresaRosovMariaBautistaMaryCuevasRichardMalloyHenriGerkenPierreWenselLindaBoedekerJamieRockSarahMasonTravisJohnsonSydneyLines
UTEXSchonnaManningJerryBrand
NRELPhilPienkosLieveLaurensEricKnoshaugEdWolfrumRyanDavisChristopherKinchinStefanieVanWychenSandiaNationalLabsRonPateToddLaneKunalPooreyPatriciaGharagozlooThomasReichardtJeriTimlinJessicaDrewryPamelaLane
CalPolyTrygLundquistBradenCroweEricNicolai
CommercialAlgaeProfessionalsAlbertVitaleJeremyWeir
ASUUndergradsShaylinMcgheeDanielBreyfogleLeviBunkerThomasChengattuDelaneyDeHertoghMark FordRileyGreerDilon HurleyKevinLawsonShelbyLuiCarlosLunaMiguelMartinezJoePalaskiCaseyPetersonJeremyPoetAlexandraPrassasSherrySaethao
JayTenisonJessicaVaughnWyatt WesternMykalYbarra
GTUndergradsFariha HassanJerryDuncanFrazierWoodruffShusuke DoiHao FuPatriciaPenalver-ArguesoAllison DunbarAllisonCarrSichoonParkPriyaPradeepTerrySnell
CatherineAchukwuChristineYi
CPUndergradsAydee MelgarGulce OzturkKaitlynJonesMichaelAntoineTrung KTranJakeBenderHeatherFreedDanielMcBroomMicheleHendricksonGerardNguyenDevenDilibertoJackSunderlandDanAverbujAnnMarieSequeiraLaurenMiller
MicheleHendricksonEmilyWangJackSunderlandAnnMarieSequeiraSoroush AboutalebiLaurenMillerSamanthaLuiMicheleHendricksonGabriellaCamposWillBrilesLettyThottathil
GeorgiaTechYongshengChenStevenVanGinkelThomasIgouYingiang SunZixuanHu
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Thankyouforyourtimeandattention!