Isolation of high-lipid content strains of the marine microalga Tetraselmis suecica for biodiesel production by flow cytometry and single-cell sorting
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! 1! ! Isolation)of)high)lipid/content)strains)of)the)marine)microalgae)Tetraselmis* suecica)for)biodiesel)production)by)flow)cytometry)and)single/cell)sorting) ) María)F.)Montero,)Manuela)Aristizábal,)Guillermo)García)Reina) Centro!de!Biotecnología!Marina! Muelle!de!Taliarte!s/n! Universidad!de!Las!Palmas!de!Gran!Canaria!! 35214!Telde,!Gran!Canaria,!Spain! ! Corresponding!author:!MF!Montero! Email:[email protected]! Tel:!34J928454547! Fax:!34J928454490! ! ) Abstract) Biodiesel!from!algae!is!considered!an!alternative!for!a!third!generation!of!biofuels.! However,! most! microalgae! are! not! lipogenic! during! fast! growth!periods,! but! high! lipid!content!occurs!at!resting!stages.!Microalgae!biomass!production!for!biodiesel! needs!continuous!high!volumetric!and!aerial!yields!and!large!amount!of!neutral!lipid! in!the!biomass.!These!requirements!are!similar!to!demanding!a!marathon!runner!to! be! obese.! We! show! that! by! using! cellJsorting! capabilities! of! flow! cytometers,! in! combination! with! the! lipidJsoluble! fluorescent! dye! Nile! Red,! we! can! isolate! and! select!cells!with!a!high!and!stable!lipid!content.!In!our!study!we!were!able!to!select! Dz dz through! three! sorting! events! obtained!from!wild!populations!of!Tetraselmis*suecica! * Keywords:! Citometry,! FluorescenceJactivated! cell! sorting! (FACS),! Nile! Red! (NR),! Neutral!lipids,!Tetraselmis*suecica* Manuscript &OLFNKHUHWRGRZQORDG0DQXVFULSW0RQWHURBUHYLHZHGGRF &OLFNKHUHWRYLHZOLQNHG5HIHUHQFHV
! 2! Introduction) ) Nowadays,!there!is!growing!interest!on!the!use!of!microalgae!as!a!source!of! biomass! for! alternative! fuel! production! (Williams! 2007;! Chen! et! al! 2009).! Many! studies!aim!at!biodiesel!production!from!microalgae!(e.g.!Pulz!2001;!Dismukes!et!al! 2008),!considered!as!an!alternative!for!a!third!generation!of!biofuels.!!Some!consider! microalgae!to!be!the!only!renewable!biofuel!capable!of!meeting!the!global!demand! for! transport! fuels! (Chisti! 2007).! ! Amongst! other! advantages,! compared! with! traditional! biofuel! production! based! on! higher! plants,! microalgae! cultivation! technology! is! well! developed,! does! not! compete! with! food! production! for! arableJ land!or!water!supply,!is!independent!on!soil!fertility,!and!the!annual!yields!per!unit! area! are! much! greater! than! those! for! higher! plants! (Williams! 2007).! Indeed,! microalgae!are!gaining!increased!scientific!attention!due!to!their!potential!ability!to! synthesize!and!accumulate!large!amounts!of!neutral!lipids!(25J50%!of!dry!weight),! mainly!in!the!form!of!triacylglycerol!(TAG)!(Hu!et!al!2008,!Chen!et!al!2009),!which!is! suitable! for! biodiesel! production.! Nevertheless,! to! achieve! profitable! microalgae! cultures! for! biodiesel,! the! strains! should! combine! two! (apparently)! antagonistic! ǣ Ǥ Dz dzǤ! Here!we!describe!a!method!combining!the!use!of!fluorescenceJactivated!cell! sorting!(FACS)!and!lipidJsoluble!fluorescent!dye!Nile!Red!(NR;!McGinnis!et!al!1997;! Elsey!et!al.!!2007)!to!identify,!isolate,!and!select!cells!with!a!higher!and!stable!lipid! content!from!wild!(and!fast!growing)!strains!of!Tetraselmis*suecica.* T.* suecica!is!considered!to!be!a!high!lipid!productivity!strain!(Rodolfi!et!al,! 2009)! ! with! an! average! lipid! productivity! of! 32! mg! LJ1! dJ1! (Griffiths! &! Harrison,! 2009).!Previous!work!carried!out!in!our!lab!(unpublished!results)!indicates!that!T.** suecica!achieves!a!lipid!productivity!of!27!!mg!LJ1!dJ1!,!!a!TGA!productivity!!of!!9!mg!LJ 1!d!1!!and!a!biomass!production!of!0.20!g!afdw!LJ1!dJ1!!in!cultures!grown!in!1.5!L! photobioreactors.!!This!marine!microalgae!is!between!the!best!biomass!producers! and!robust!enough!to!be!regarded!of!high!potential!for!oil!production!(Chini!Zittelli! 2006;! Rodolfi! 2009).! Moreover,! it! was! observed! that! neutral! lipids! were! present! both!during!the!exponential!and!stationary!phase!(Mendoza!et!al!2009).!Therefore,! we! selected! T.* suecica! as! a! suitable! candidate! to! reach! our! goals,! due! to! its! fast!
! 3! growth,!high!source!of!biomass,!presence!of!lipids!during!most!steps!of!the!culture! evolution,!and!positiveJresponse!to!cell!staining!with!NR.* ) Material)and)methods) ) T.* suecica! was! obtained! fr Dz dz ȋȌ collection! (strain! BNA! 10_037;! Centro! de! Biotecnología! Marina,! University! of! Las! Palmas,! Canary! Islands,! Spain).! Wild! algal! strain! cultures! were! grown! with! MBA! medium! (Table! 1),! in! a! 1.5! L! polyethylene! terephthalate! (PET)! column! photobioreactor!(Ø!=!9!cm;!height!=!31!cm)!under!greenhouse!sunlight!conditions! (temperature!range!between!20,0!and!30,4!qC;!mean!PAR!doses!of!11870!±!1795!kJ! mJ2!dJ1).!Mixing!was!provided!by!continuous!air!bubbling.!CO2!was!injected!9!times! per!day!in!50!secondsJpulse!intervals.! ! FluorescenceJactivated! cell! sorting! was! carried! out! using! an! EpicsJAltra! cytometer!(BeckmanJCoulter)!equipped!with!a!15!mW!airJcooled!!488!nm!argonJion! laser.!Upon!excitation!by!the!laser,!NR!exhibits!yellow!fluorescence!when!dissolved! in!neutral! lipids,!and! reaches! its!peak!in! emision! intensity!at! 570!nm! (Elsey! et!al!! 2007).! ! NR! fluorescence! and! chlorophyll! autofluorescence! were! detected! by! the! optical! system! in! the! FL3! channel! (575r25! nm! bandpass! filter)! and! FL5! channel! (675!nmr40nm!!bandpass!filter),!respectively.!! ! Sorting!was!performed!at!high!pressure!(31.4J31.5!psi),!using!a!70!Pm!nozzle,! a!drop!drive!frequency!of!about!59!KHz,!and!a!flow!rate!ranging!between!15000!and! 20000!cells!sJ1.!Instrumental!alignments!corrections!were!daily!checked,!based!on! fluorescent! bead! signals! (10! Pm! FlowJ! check! Fluorospheres,! Beckman! Coulter)! represented! in! Forward! Scatter! versus! Side! Scatter! plots! and! fluorescence! histograms.! Next,! the! setting! up! of! the! sort! delay! and! the! sort! recovery! were! controlled! by! counting! with! an! epifluorescence! microscope! the! number! of! sorted! beads!deposited!on!a!slide.!The!recovery!was!consistently!>85%.! ! Once!the!checking!process!was!finished,! the! selection! process!started!with! wild!samples!of!fastJgrowing!T.*suecica.**3!to!4!ml!of!cell!suspensions!(20!x!106!cells! mlJ1)! were! stained! with! 10! µl! per!ml! of!working! solution! of! NR! and! acetone! (0.1! mg/ml!)!concluding!with!3!PM!final!concentration.!The!mixture!was!vortexed!then! incubated! for! 15! minutes! at! 37! °C! in! darkness.! ! Meanwhile,! unstained! cells! were!
! 4! used!as!an!autofluorescence!control!displaying!yellow!and!red!fluorescence!(by!FL3! and! ! FL5! channels,! respectively)! from! bivariate! scatter! ! plots! (Forward! Scatter! versus!FL5!and!FL5!versus!FL3)!and!two!histograms!(FL5!and!FL3)!on!the!computer! screen.!! ! Immediately! after! the! incubation! finished,! the! stained! cells! were! analysed! and!the!sorting!criteria!setJup.!Sorting!windows!were!positioned!to!isolate!the!cells! with!the!highest!relative!yellow!fluorescence!from!histogram!FL3!(between!8!to!25! %!of!the!population!in!10!strains),!corresponding!to!the!extreme!right!tail.!The!first! sorted! cells! were! observed! under! the! microscope,! to! verify! that! they! contained! larger! amounts!of!lipid!droplets!than!the!whole! unJsorted!population.! During!the! sorting!process!the!cells!were!poured!into!Falcon!tubes,!containing!500!Pl!of!MBA!or! seawater,!depending!on!the!treatment!applied,!as!explained!later.!The!cell!cultures! were! transferred! to! glass! conical! tubes! and! placed! in! an! indoor! culture! chamber! with! a! light! intensity! of! 75! Pmol! photons!mJ2! sJ1! in! a! 12Jhour! light/dark! cycle! at! 24qC,!.!Cell!cultures!were!grown!adding!media!MBA!until!the!next!sorting!process.!!!! ! To!decide!the!best!methodology,!several!treatments!were!applied!to!the!wild! strain! to! assess! the! cultivability! of! the! isolated! cells,! based! on! their! viability! and! recovery! during! the! first! 24! hours! after! sorting! (Figure! 1).! The! alternative! treatments!were:!(1)!using!either!a!commercial!sheath!fluid!(Isoton£!II;!Beckman! Coulter)! or! GF/FJfiltered! seawater! as! sheath! fluid! in! the! cytometer;! (2)! resuspending!the!cells!after!extracting!the!supernatant!sheath!fluid!(postJsorting)!in! fresh! MBA! media! or! in! filtered! seawater;! (3)! keeping! isolated! cells! on! seawater! (from! the! sheath! fluid)! or! in! fresh! MBA! during! the! initial! 24! hours! postJsorting! period.!In! all! cases!isolated! cells!were! concentrated! by! soft! centrifugation!(1! min! 300!g)!to!yield!a!1!ml!inoculum!before!transferring!to!glass!tubes.!24!hours!later,!! 500!Pl!MBA!were!added!to!the!the!cells,!and!300!Pl!were!added!the!following!days,! although!trying!to!not!dilute!the!cells!in!excess.!Simultaneously,!the!cells!were!gently! mixed!and!observed!under!the!microscope!for!motility,!growth!and!viability!after!24! hours.!! The!possible!toxic!effect!of!the!Isoton!II!solution!(distributed!as!sheath!fluid! for!many!sorter!equipments)!was!evaluated!by!quantifying!the!cells!viability!under! several! Isoton! dilutions! treatments! (Figure! 2).! 1! ml! of! T.* suecica* culture! (with! a! cellular! density! of! 11x106! cell! mlJ1)! was! dispensed! into! each! of! 9! Falcon! tubes.!
! 5! Triplicate! samples! of! 3,! 4! and! 6! ml! Isoton! were! added! to! get! 1:4,! 1:5! and! 1:7! dilutions.!!The!dilution!1:4!corresponds!to!the!usual!one!obtained!in!the!EpicsJAltra! cytometer!during!the!sorting!process!at!cell!densities!of!20!x106!cells!mlJ1.!After!30! minutes!incubation!(the!average!time!that!cells!are!immersed!in!Isoton),!the!tube! cells!were!concentrated!by!low!centrifugation!(1!min!at!300!g),!and!the!Isoton!was! extracted.!Then!the!pellet!was!resuspended!in!2!ml!of!filtered!seawater.!!Finally,!a! FDA! (Fluorescein! DiAcetate)! viability! cell! control! was! performed! to! all! tubes! (as! indicated!in!Coder,!1997).!!We!used!a!fresh!solution!(1:10!in!water),!prepared!from!a! stock!solution!of!FDA!(1mg!mlJ1!in!acetone,!kept!at!4!°C).!!20!Pl!of!this!solution!were! added!to!1!ml!of!each!of!the!9!Falcon!tubes!containing!the!resuspended!cells!and! incubated!for!15!min!at!37!°C.!The!samples!were!immediately!analyzed!in!a!Quanta! flow!cytometer!(BeckmanJ!Coulter)!at!525r25!nm!(excitation!at!480nm)!to!quantify! the!viable!cells.!! ! Results)and)discussion) ) The!best!experimental!conditions!for!sorting!and!successful!growing!of!high! lipidic! strains! were! achieved! with! the! following! protocol:! (1)! using! seawater! as! sheath! fluid,!which!avoids!the!Isoton!toxic! effect!on! cells,!and!makes!unnecessary! the!centrifugation!step!to!extract!the!Isoton!after!sorting!(Figure!1),!(2)!isolating!and! sorting!of!ten!percent!of!the!population!with!the!highest!FL3!signal!(Figure!3),!and! (3)!maintaining!sorted!cells!initially!in!seawater,!by!adding!300J500!Pl!MBA!media! after! 24!hours.!Microscopic! inspection! of! sorted! single!cells!following!these!three! steps! showed! that! most! of! the! sorted! cells! were! motile! and! not! visibly! damaged.! These!cells!were!satisfactorily!used!for!reJgrowth!of!new!algal!strains.!We!obtained! an! average! cell! recovery! of! about! 65%! with! respect! to! the! viable! cells! (80%)! significantly! higher! than! the! 20! to! 30%! reported! by! Sensen! et! al! (1993)! for! phytoplankton! flagellates.!Other!microorganisms!as!bacteria!gave!variable!results,! in!the!range!of!22!to!85%!(ResinaJPelfort!et!al!2001).!! We!found!that,!under!our!experimental!conditions,!the!optimal!concentration! of!NR!both!for!staining!the!cells!and!keeping!them!active!was!3!PM,!at!cell!densities! about!15J20!x106!cells!mlJ1.!This!concentration!is!five!times!lower!than!that!used!by! other!authors!!(15!PM)!who!reported!toxic!effects!of!the!dye!at!cell!densities!of!105!J
! 6! 106! cells! mlJ1!!(de! la! Jara! et! al! 2003,! Mendoza! et! al! 2009),! but! similar! to! the! concentration!used! by!others,!like! Lopes!da!Silva!et! al!(2009).!We!found!a!strong! linear! correlation! (r2=0.81,! n=18)! between! the! fluorescence! signal! FL3! of! cells! stained!with!NR!and!the!triacylglicerol!content!of!the!same!cells,!measured!with!a! commercial! enzymatic! colorimetric! test! (Triglycerid! LJType,! Wako! Chemicals! GmbH),! using! triolein! as! standard.! A! similarly! strong! correlation! (r2=0.87,! n=18)! between!FL3!and!neutral!lipids!estimated!by!gravimetry!was!reported!by!Mendoza! et!al!(2009)!for!the!same!strain!of!T.*suecica*(BNAJ10037).!Other!authors!have!also! reported! highly! significant! correlations! between! the! NR! fluorescence! and! total! microalgal!lipid!content!(Chen!et!al!2009;!Cooksey!et!al!1987;!de!la!Jara!et!al!2003;! da! Silva! et! al! 2009);! thus,! supporting! the! view! that! the! dye! is! a! useful! proxy! for! quantification! of! lipids! in! microalgae! in! order! to! achieve! sorting! of! hyperlipidic! strains.! Our! experiments! analyzing! the! toxicity! effects! of! the! Isoton! on! isolated! ! T.* suecica!!clearly!indicate!that!cell!viability!is!reduced!from!79%!(in!the!cell!control,! from!the!photobioreactor)! to!34%,!25! %,!and! 15%!at!dilutions! 1:4,!1:5! ,!and! 1:7,! respectively!(Figure!2).!!These!results!agree!with!the!observed!absence!or!low!cell! motility!under! microscopy! and! consequently!worst!cell!recovery.!Presumably,! the! toxic!effect!is!associated!with!some!Isoton!components,!that!have!been!referred!in! the! literature! to! affect! the! recovery! of! flagellate! microalgae! (Sensen! et! al! 1993),! bacterial! cultures,! and! bacterioplankton! (Comas! ! and! VivesJRego,! 1997;! Resina! Pelfort! et! al.,! 2001).! Sorting!requires!a!conductive! medium! that!should!be!chosen! according!to!the!sample!being!analysed.!In!the!case!of!seawater!microalgae,!the!use! of! plain!seawater,!instead! of! the!Isoton!sheathJfluid,!during!cell!sorting! increased! cell!viability.!The!addition!of!MBA!media,!24!hours!after!isolation,!accelerated!the! recovery!of!strains!with!high!lipid!content.!! ! The! described! methodology! was! used! to! obtain! two! generations! of! hyperlipidic!cells!from!a!wild!strain!of!cells!(Figure!3).!The!first!sorting!of!8%!of!the! wild! population! (15J20! x106! cells! mlJ1)! exhibited! a! mean! fluorescence! FL3! of! 0.5! (29.8! HPCV).!After!26! days!from! the! first!sorting!event,!the!algae!yielded!(for!1.2! x106! cells!mlJ1)!a! mean!FL3! of! 1.0!(17.3!HPCV).!A! subsequent!second! sorting!(38! days! after)! yielded! a! mean! FL3! of! 2.3! (10.3! HPCV).! ! The! first! isolation! must! be! obtained! from! high! cellular! densities,! being! very! selective! (less! than! 10%! of! the!
! 7! population;!see!the!arrow!indication!in!the!cytogram).!Following!these!conditions,! the! mean! lipid! signal! quadruplicates! at! the! third! generation! of! our! experiments! (from!0.5!to!2.3).) We!measured!an!average!growth!rate!(P)!of!0.39!r!0.07!for!our!isolated!T.* suecica* cells,! corresponding! to! a! mean! doubling! time! of! 43! h.! ! This! value! is! only! slightly!higher!than!the!average!doubling!time!(about!38!h)!estimated!by!Griffiths!&! Harrison!(2009)!for!T.*suecica!in!a!recent!review.!!Considering!our!estimated!mean! doubling!times,!the!FL3!signal!for!the!second!and!third!generations!is!obtained!after! 14! and! 21! cell! divisions,! respectively.! Therefore,! the! mean! lipid! signal! (2.3)! is! obtained!after!more!than!30!cell!divisions!from!the!wild!population,!which!supports! the! idea! of! a! stable! signal! in! the! population.! Our! results! achieved! after! three! generations! of! T.* suecica! provide! a! promising! step! in! isolating! new! strains! of! microalgae! yielding! large! amounts! of! desirable! neutral! lipids.! However,! unlike! T.* suecica,!the!thick!and!rigid!cell!walls!of!many!of!these!algae!hampers!NR!to!enter!the! cell!and!stain!the!neutral!lipids.!Recently,!Chen!et!al!(2009)!have!improved!the!NR! method! gaining! in! effectiveness! and! efficiency.! ! This! improvement! may! open! the! possibility! to! isolate! and! culture! other! oleaginous! green! microalgae! (like! Nannochloropsis!or!Chlorella*)!with!great!potential!on!biodiesel!production,!due!to! their!high!tryacylglycerol!content,!while!combining!the!NR!technique!with!FACS!!as! we!have!described!in!this!study.!! !! Acknowledgements* Dz ±ico! Nacional! de! × ±dz ȋȌ Ȃ!Dz × Ódz ȋȌǡ ȋ J YPF).!!We!would!like!to!thank!Dr!J.L.!GómezJPinchetti!for!his!thoughtful!comments! and!advice!during!the!development!of!this!study.! !
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