Algae Analysis: Biofuels
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Transcript of Algae Analysis: Biofuels
Data Sheet
White Paper
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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Chlorophyll (RED-HLog)
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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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BODIPY (GRN-HLog)
Plot 4: Gated by Chlorophyll A
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High LipidContent Clone
M1 M2
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Plot 4: Gated by Chlorophyll A
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Chlorophyll A (RED-HLog)
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-HLo
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BODIPY Fluorescence
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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.
www.emdmillipore.com/offices
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