Data Sheet

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Optimizing oil production from algal cultures, EMD Millipore provides laboratory instruments and products for algal research and productionJim Mulry, Tim Brown, Jason Whalley, Michael Moeller, and Angelica Olcott EMD Millipore Corporation

There are several examples illustrating the

potential of biodiesel fuel derived from algae;

• OnNovember16,2010,theRiverinesailedfrom

PointLomainSanDiego.Itwaspoweredby

a50/50mixofF-76gradeofpetroleumand

hydro-processedalgaedieselrunningaLM2500

gasturbineengine

• OnNovember11,2010,ContinentalAirlinesflew

apassengerjetpoweredbyjetfuelmadefrom

geneticallymodifiedalgae.Whileresearchwill

alwayscontinue,themoveintoproductionis

essentialforprofitabilityandtheoverallgrowth

ofcarbonneutralgreentechnology.

There are 3 key stages to successful production

of biodiesel (lipids) from algae:

1)Selectionofalgalspeciescapableofproducing

significantquantitiesofneutrallipids,which

includeoilscapableofbeingprocessedinto

petroleum.

2)Optimizationofgrowthconditions.

3)Efficientextractionofneutrallipidsfrom

culturesandsubsequentoilproduction.

Asgovernmentsstrivetoincreasethefocusandavailabilityofgreenfuels,fuelsderivedfromorbyliving

organismssuchasalgae,yeast,orbacteria,multiplelabs&start-upcompanieshavesprunguptoprovide

new,greenersourcesoffuels.Oneareaofconsiderablefocusistheproductionofbiodieselfuelfromalgae.

EMD Millipore is a division of Merck KGaA, Darmstadt, Germany

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Algal Species SelectionThegrowthofalgaedependsuponanumber

ofphysicalandchemicalfactors,including

nutrientsource,CO2,andwatersupplywhich

makeforaverysimpleecosystem.Thereforethe

keytosuccessfulproductionofoilfromalgae

isintheselectionofthealgaespeciesthathas

thefastestgrowthcyclebalancedbythemost

lipidproduction.Screeningofmultiplespecies

inmultiplegrowingconditionscanbeatime

intensiveandcostlyexercise.Oneapproachto

indentifythebestproducersisusingthetechnique

offlowcytometry.

The guava® easyCyte™ HT Series Flow CytometersFlowcytometryisanestablishedtechniquefor

thecharacterizationandquantificationofthe

physicalandchemicalpropertiesofmicroscopic

particlesandcellpopulationsinliquidsuspension.

Thistechniqueinvolvesthecreationofathin

fluidstreamwithinwhichcellsarearrangedina

straightline.Thisstreampassesthroughanoptical

windowwithinwhichexcitationsoffluorescence

(bothintrinsicandastheresultofstaining

dyes)areaccomplishedbymeansoflaserlight

atspecificwavelengths.Fluorescencedetectors

collecttheresultingemittedsignals.Usedwidely

forresearchanddiagnosticpurposesinmedicine,

thistechnologyhasalsoshownutilityinthe

fieldofmarineresearchandmorerecentlyfor

optimizationofbiofuel,andothersystemsforlipid

productionbioprocessing.

Comparedtootherflowcytometrysystems,

guava®easyCyte™HTSeriesFlowCytometersare

inexpensive,easytomaintain,anddonotrequire

anexperiencedusertooperate.Inasinglevisit,we

cantrainthenoviceusertopreparesamplesfor

lipidcontentmeasurement,operateandmaintain

theinstrument,andcollectandanalyzedata.These

methodscanbeemployedinthecharacterization

ofalgaespecies,detectionofproductionculture

contaminants,andthemeasurementand

optimizationoflipidproduction.

Cyanobacteria(blue-greenalgae,Division

Cyanophyta)maybeidentified,classified,and

assessedinculturebasedontheirdifferential

expressionoffluorescentproteins(suchas

phycobiliproteins)andchlorophyllcontent,as

thisexpressionvariesbyspecies.Moreover,many

non-algaecontaminantsinbioprocessreactorsdo

notexpresstheseproteins,sothealgaecultures

canbequantifiedseparately.Thefigurebelow

demonstratesastrategytodetectcontamination

ofalgaebloomswithinvasive,non-fluorescent

proteinexpressingbacteriawithinthealgae

bioreactor(Figure1).

Asimpleandwelldocumentedmethodofneutral

lipidstainingisthenemployedtodeterminelipid

content.Thetotalsamplepreparationtimefor

theseassaysislessthan15minutes.Common

dyeswhichenablestainingofneutrallipids

inalgalcellsincludeNileRedandtheBODIPY

(Boron-Dipyrromethene)familyoflipophilicdyes.

Figure 1. A) Clean Culture: homogenous algae cell samples B) Subculture Bloom: separating microbial population from the algae population C) Bacterial Contamination: complete microbial population separated from the algae population

A: Clean Culture C: Bacterial Contamination

B: Subculture Bloom

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Thesedyesrapidlydiffuseintolivingcellswithout

theneedforfixationand/orpermeabilizationand

becomeincorporatedintotheneutrallipidglobules

whichformintracellularlyinoil-producingcells.

DuetoamuchmorerobustsignalfromBODIPYas

opposedtoNileRed(seeFigure2),werecommend

theuseofBODIPY.

Usingthissimplestainingprotocol,theeffects

ofvarioustreatmentsonoilproductionmaybe

assessed.AsshowninFigure3,theassayonthe

culturesbelowshowedvariancesinthenutrient

conditionsresultingindifferentiallipidcontent.

Asimilarstrategycanbeemployedtoevaluate

differentspeciesorstrainsforoilproduction

capability.

Forevaluationofneutrallipidproductionin

greenalgalcells(DivisionChlorphyta),asimple

flow-basedprotocolthathasproveneffectivein

mixed,non-sterilecultures.Thegoalistorestrict

interrogationtochlorophyllA-containingcells

(identifiedonthebasisoffluorescenceintheRed

Channel,seeM1histogrammarkerinleftpanel

below)andtoevaluatethegreenfluorescence

intensityofBODIPY505/515(LifeTechnologies),

alipophilicdyecapableofincorporatingintooil

droplets.AsshowninFigure4(rightpanel),this

AE

Count MFI Geo-Mean x-Median Cells/mL

Negative Control 1000 6.0 5.2 5.4 1.95E+05

Nile Red 1000 213.5 73.1 68.6 1.36E+05

Bodipy 1000 626.2 416.7 437.0 147E+05

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Figure 2. Bodipy reports lipid content in algae.

Figure 3. Lipid content can be a function of cell cycle distribution. Algal cells were stained with BODIPY 505/515 at different times during the cell cycle both to determine neutral lipid per cell by flow cytometry and to image lipid bodies in living cells.

Figure 4. Lipid measurement of chlorophyll A-positive algae. Identification of algal cells containing chlorophyll A; chlorophyll A fluoresces in the red channel (A). Gate applied to select for chlorophyll A-positive cells (B). Histograms showing a wide range of lipid content (as evidenced by BODIPY green fluorescence intensity) for a variety of algal strains (C), with one clone showing as much as 500 times as much lipid content as others.

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Neutral Lipid/BODIPY Signal (RFU)Total Fatty Acid (pg/cell)Fatty Acid Productivity (mg/L)Total Cell Concentration

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High LipidContent Clone

M1 M2

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High LipidContent Clone

M1 M2

A: Not Gated B: Gated by Chlorophyll A C. Histograms showing varying lipid content of different algae clones

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Figure 5. Chlorophyll Mean fluorescence as measured on a Guava® 8HT Flow Cytometer with laser excitation at 488 nm (75mW) and bandpass filtration at 586/23.

staincreatesafluorescentsignatureinoil-rich

cells(redhistogrampeak)withsufficientpeak

separationtoinsurealmostnooverlapwith

unstainedcontrols(pinkhistogrampeak),allowing

easyidentificationofspecies,strains,orcloneswith

highoilproduction.

Theseresultsshowthatguava®easyCyte™flow

cytometerscanbeusedtoevaluateavarietyof

parametersofalgalgrowthandproduction,and

offeranexcellenttoolfordeterminingaspectsof

bioprocesscultureconditions.

Theguava®easyCyte™HTSeriesinstrumentsare

easytouse.Trainingcantakeplaceinthealgae

laboratoryandnospecialschoolingisrequired

tooperatethesystem.Operatorsonlyneedtobe

skilledatusingapipetteforsamplepreparation

andhavebasiccomputerskills.Withthesetwoskills

anyonecanbefullytrainedtosetupalgalassaysin

amatterofhours.EMDMilliporeprovidestalented

FieldApplicationSpecialiststoassistlaboratories

learningtooperateaguava®easyCyte™HTseries

instrument.

Theguava®easyCyte™HTflowcytometrysystems

allemploythepatentedcapillarycellcounting

systemforreproducibleresultsandtrustedanalysis.

Apumpisusedtoaspiratethesampleforanalysis.

Thesamplevolumeaspiratedismeasured.The

sampleispulledthroughacapillarybyaprecision

pump.Thisprocessassistsinaligningthecellsfor

countingandmeasurementasthecellspassthe

pointofinterrogationwhereeachcellisindividually

countedandmeasuredbythelaser.

Theguava®easyCyte™HTseriesmakesupto

eightseparatemeasurementsoneachindividual

cellasitpassesthelaser.Forwardlightscatter

providesinformationusedtodeterminerelative

sizeandvolumeofthecell.Sidescatterisemployed

toprovideinformationconcerningtheinternal

structureofthecellincludingnucleustocytoplasm

ratio,granularity,andthenuclearshapeinside

thecell.Guava®easyCyte™HTseriesinstruments

offerthecapacitytoutilizeuptosixdifferentcolor

wavelengthscombinedwithapplicablefluorescent

stainsforindividualintracellularanalysis.Each

guava®easyCyte™HTseriesinstrumentis

customizedwithasmanyastwolaserstoprovide

thebestanalysisforthealgaelaboratory.

Theguava®easyCyte™HTseriesinstrumentsare

employedinresearchingwhichspeciesofalgae

yourbusinesswillselectforgrowing.Express

ProsoftwarecanbeutilizedtoforchlorophyllA

analysis.Measurementsareconductedinthered

channeltomakeintracellularanalysistodetermine

thepresenceandsizeofChlorophyllA.Chlorophyll

Ahelpstoproducethelipidsdesiredforoil

production.

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Figure 6. Sybr Green 1 Dye used to detect algae viability. Algae can be stained at cell concentrations of 1,000 to 5 X 105 cells/mL and detected on the guava® easyCyte™ 5/5HT.

Figure 7. InCyte™ software can be applied to different metrics to algae samples within the same plot, offering a level of detail beyond simple detection of mean fluorescence. Here we offer the breadth of analysis for Chlorophyll A detection in a group of samples.

TheExpressProsoftwareemployedintheguava®

easyCyte™HTseriessystemsiseasytouseforlipid

analysis.Acommercialproductmostutilizedin

lipidanalysisisthestainBodipy.Bodipyisaspecific

markerthatstainslipidsinthegreenchannel.

ExpressProsoftwareisthenusedtoanalyzethelist

modeanalysisofeachcellmeasuredtodetermine

themeancelldensityandthemeanlipidcontent.

Thesearecellcharacteristicsusefulforspecies

selection.

Cellgrowth,cellviability,andcellproliferationare

importantcharacteristicsutilizedindetermining

speciesselection.Theguava®HTseriesalsohave

onboardassaysandreagentkitsavailableto

determiningcellviability,cellgrowth,andcell

proliferation.

ThenewpowerfulInCyte™softwareallowsyouto

comparemultiple96wellplatesofalgaesamplesat

onetime.Uptosixcharacteristicsimportantinyour

speciesselectioncanbeassignedtoeachsample.

Itispossibletorunall300knownspeciesofalgae

knowntoproducethelipidsusedinoilproduction.

Aheatmapcanbegeneratedidentifyingthose

samplesthathaveallsixofthecharacteristicsthat

theoperatorhasdeterminedimportantforspecies

selection.

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Algae CultivationThesuccessfulgrowthofalgaerequireswater,

light,carbon,nitrogen,andphosphorus.Forthe

mostoptimalgrowthconditionsitisadvisableto

useareliablesourceforhighqualitynutrientsto

enhancealgalgrowth.

Millicell® HY FlasksGrowingalargenumberofcellsforspecies

selectionrequiresahighthroughputflaskto

enhanceyourproductivitybysavingspaceand

time.Eachhighyieldflaskprovidesaconsistent

qualitygrowthenvironmentacrossalllayers

withthesamevolumeofmediaineachlayer.

Recoveringcellsisaseasyasgrowingthem.

Witheasyaccesstopipettingorpouring,Millicell®

HYflasksdeliverreproducible,highyieldsof

healthycells.YourEMDMilliporeApplications

Specialistcanassistyouinselectingthebestflasks

foralgaeanalysis.

Monitoring DevicesOnceyourculturehasbeeninitiateditiscritical

tobemonitoringthestatusofyouralgalculture.

Keyaspectstomonitorarealgalcounts,size

andvolumeplusdeterminingifthecultureis

contaminatedbymicrobialpopulations.

EMDMilliporeoffersseveralkindsofsampling

devices.Thesedevicescomeindifferentshapes,

sizes,andmeetmanydifferentspecifications.

CheckwithyourEMDMilliporerepresentativeto

determinewhichsamplingdeviceisrequiredfor

youraquaticbioreactor.

Scepter™ Handheld Cell CountersForprocessmonitoringyoumaywanttoevaluate

theScepter™HandheldCellCounters.TheScepter™

handheldautomatedcellcounterprovidesa

fastandconvenientmethodforcountingcells

andparticles.Thesystememploystimetested

impedancecountingderivedfromtheCoulter

principle.Itiseasytouseandtakesonly30

secondstocountalgalcells.

The Scepter™ cell counter’s screen displays:

• Cellconcentration

• Averagecellsize

• Averagecellvolume

• Ahistogramofsizeorvolumedistribution

3 4

2

Average cell volume (pL)

Average cell diameter (µm)

Cell concentration (cells/mL)

Histogram displayed as function of cell diameter or cell volume

1

RELATED PRODUCTS

Effective monitoring of bacterial contamination of culture

Commonissuethathasanegativeimpacton

algaecultureiscontaminationfrombacteria.A

bacterialcontaminationcanresultinlossofyour

algaeculturewhichhastobediscardedandanew

cultureinitiated–losingtimeandmoney.

7

Milliflex® Quantum Rapid Detection System TheMilliflex®Quantumrapiddetectionsystem

isaneasytouse,nondestructive,fluorescent

staining-basedlaboratoryinstrumentforfaster

microbialdetection.TheMilliflex®Quantumwill

detectunwantedmicrobialcontaminationinwater

faster,improvingyourprocesscontrolsforaclosed

aquaticsystem.Ideallysuitedfortestingwater,

theMilliflex®Quantumisatruebreakthroughfor

aquaticgrowthsystems.Theoperatorcaneasily

recoverandcollectanymicroorganismsdetected

foridentificationusingexistingIDtechnology.It

iseasytouse.Withasimpleprotocol,hardware,

andworkflow,itonlytakesafewhoursto

familiarizeyourselfwiththesystemandbegin

usingit.Validationisstreamlinedandresults

arecomparablewiththetraditionalmethods.

Samplepreparationandincubationconditions

remainidenticaltotraditionalmicrobiology

methods.However,theMilliflex®Quantumismuch

fasterthantraditionalmethods.Microbiological

resultsareproducedinafractionofthetime

oftraditionalmethodsallowingyoutorespond

tocontaminationearlierintheprocessand

enhancingyourproductivitybysavingyoutime

andmoney.

ExtractionEMDMilliporeoffersacompletelineofchemicals.

Algalproductionusescarbonasanutrient.

Developmentofsolventsforextractionwill

alsorequirechemicals.Shouldyourextraction

methodbefiltration,EMDMilliporeoffersthe

bestqualityfilters.Ifcentrifugationisyourchoice,

EMDMilliporehascentrifugationfilters.Should

flocculationbeyourmethod,EMDMilliporeoffers

acompletelineofneutralsolvents,polarsolvents,

andmixturesincludingchloroform,acetone,and

methanolincorporatedinchemicalextraction

methods.

Water FiltrationEMDMilliporeofferstheverybestinwater

filtrationproducts.EMDMilliporemakesscreen

filtersusedinsomefiltrationandflocculation

separationtechnologies.Thefilterscanbeusedto

helpseparatethealgaebiomassfromthewater.

Millex® FiltersIfyouareanalyzingalgaefrommarinesamples

inaguava®flowcytometer,ithelpstofilterout

largerparticles.Algaeusedinproductionoflipids

foroilstendtobelessthan10micronsinsize.

UseofaMillex®filterisrecommendedforsample

preparation.Idealforaqueoussolutions,Millex®

filterunitsofferunsurpassedquality,consistency

andreliabilitytoacceleratethefiltrationprocess.

Durapore(PVDF)filterscombinefastflowwith

lowproteinbinding.Nylonfiltersprovidebroad

chemicalcompatibilityforusewithaqueous

organicsolutionssuchasalgallipds.Millipore

Express®Plus(PES)filtershavethefastestflow

ratesandhigherthroughput.HydrophobicPTFE

filtershaveexcellentsolventresistanceandare

idealforHPLCsamplepreparation.

ProductionOnceoilisharvestedfromalgae,itmust

beprocessedandrefinedintoproduct.

Chromatographyisutilizedtodeterminethe

weightandgradeoftheoilproduct.EMDMillipore

hasproductsthatcanaidintheproductionand

refiningofalgaloil.

RELATED PRODUCTS

QuikScale™ Chromatography ColumnsChromatographyisusedtoweighoilmadefromalgallipids.Thisrobust,

scalablefamilyofchromatographycolumnshelpstomaximizeyields,boost

productivity,andshortenthetimetomarket.QuikScale™columnsachieve

ultra-high-throughputanddelivergreaterproductpurityatfasterlinear

velocities.Aninnovativeflowdistributorassuresuniformmediautilization

assuringreproducibleandreliableseparations.

Samplicity™ Filtration SystemHighpressureliquidchromatographyisusedtoweighthealgaloilproduct

forrefining.Toimproveyourproductivity,investintheacquisitionofa

newandexcitingSamplicity™filtrationsystem.TheSamplicity™filtration

systemisaninnovativenewtechnologythatprovidesaconvenient,high-

throughputalternativetothetraditionalsyringe-tipfilterswhenpreparing

samplesforchromatographyanalysis.Theeasy-to-useSamplicity™system

isthefirstvacuumdrivensystemdesignedtofilteruptoeightsamples

atoncedirectlyintoHPLCvials.Justattachthevacuumpump,loadthe

sampleswithapipettor,andflipthelevertorecoverparticulate-free

samplesinjustseconds.UtilizationofaSamplicity™systemcanaccelerate

yoursampleproductionbyasmuchas35%.PleasecontactyourEMD

Milliporerepresentativeforadditionalinformation.

RELATED PRODUCTS SummaryWhetheryouareintheresearchorproductionofalgaeto

oil,itpaystoconsiderEMDMilliporeasareliablesource

oflaboratoryinstrumentsandtests.EMDhasproducts

thatareapplicableforeverythingfromalgaespecies

selectiontoqualitycontrolofrefining.Foradditional

information,pleasecontactyourlocalEMDMillipore

representative,orvisit:www.millipore.com.

EMDMillipore,theMlogo,easyCyte,InCyte,Scepter,Samplicity,andQuikScalearetrademarksofMerckKGaA,Darmstadt,Germany.guava,Millicell,Milliflex,Millex,andMilliporeExpressareregisteredtrademarksofMerckKGaA,Darmstadt,Germany.LitNo.AN5563EN00LS-SBU-12-067497/2012PrintedintheUSA.©2012EMDMilliporeCorporation,Billerica,MAUSA.Allrightsreserved.

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