Microfluidic technology for plankton research

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Mathias Girault, Thomas Beneyton, Yolanda del Amo, Jean-Christophe Baret

To cite this version:

Mathias Girault, Thomas Beneyton, Yolanda del Amo, Jean-Christophe Baret. Microfluidic

tech-nology for plankton research. Current Opinion in Biotechtech-nology, Elsevier, 2019, 55, pp.134-150.

�10.1016/j.copbio.2018.09.010�. �hal-02118999�

Microfluidic

technology

for

plankton

research

Mathias

Girault

,

Thomas

Beneyton

,

Yolanda

del

Amo

and

Jean-Christophe

Baret

Abstract

Planktonproducesnumerouschemicalcompoundsusedin cosmeticsandfunctionalfoods.Theyalsoplayakeyroleinthe carbonbudgetontheEarth.Inacontextofglobalchange,it becomesimportanttounderstandthephysiologicalresponse ofthesemicroorganismstochangingenvironmental

conditions.Theiradaptationsandtheresponsetospecific environmentalconditionsareoftenrestrictedtoafewactive cellsorindividualsinlargepopulations.Usinganalytical capabilitiesatthesubnanoliterscale,microfluidictechnology hasalsodemonstratedahighpotentialinbiologicalassays. Here,wereviewrecentadvancesinmicrofluidictechnologies toovercomethecurrentchallengesinhighcontentanalysis bothatpopulationandthesinglecelllevel.

Addresses

1

CentredeRecherchePaulPascal,Unite´ MixtedeRecherche5031, Universite´ deBordeaux,CentreNationaldelaRechercheScientifique, 33600Pessac,France

2_{Universite´ deBordeaux-OASU,UMRCNRS5805EPOC}

(EnvironnementsetPale´oenvironnementsOce´aniquesetContinentaux), StationMarined’Arcachon,33120Arcachon,France

Correspondingauthor:

Baret,Jean-Christophe(jean-christophe.baret@u-bordeaux.fr)

CurrentOpinioninBiotechnology2019,55:134–150

ThisreviewcomesfromathemedissueonAnalyticalbiotechnology EditedbySauliusKlimasauskasandLinasMazutis

ForacompleteoverviewseetheIssueandtheEditorial Availableonline13thOctober2018

https://doi.org/10.1016/j.copbio.2018.09.010

0958-1669/_ã2018TheAuthors.PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBY-NC-NDlicense( http://creative-commons.org/licenses/by-nc-nd/4.0/).

Introduction

Algaeare locatedat thebase of thetrophicweb. They supportbivalveandfishrecruitmentratesaswellasmany other marine organism growth and hence significantly contributeto thelocal economy in numerous countries [1]. They are also used to produce various high-values chemicalcompoundssuchas biopharmaceuticalsor cos-meticsandthereforehaveahugeeconomicalimpact[2]. Among the algae, the phytoplankton are microscopic organisms which have benefited humankind since the earliestdaysoflifeontheplanet,bycontributingtothe changeoftheatmosphereoftheplanetinthepast.They are still responsible of more than 40% of the inorganic

carbonfixationontheEarth,andplaytheirsignificantrole in climate control [3]. Those microorganisms are an importantcompartmentrulingthefluxesofbothoxygen andcarbonattheworldwidescale.However,evidences ofsubstantialenvironmentalchanges,asillustratedbythe effectofclimatewarmingonsurfaceoceanandonwater column stratification,canlead to drasticchangesof the planktondiversityandcommunitystructureatthesurface of the Ocean [4]. The stratification of the Ocean and subsequent faster inorganic nutrient depletion in the surfacelayersduetophytoplanktonuptakeareexpected tofavorsmallpicoplankton growthandmicrobial domi-nated food webs at the expense of larger microphyto-planktonspeciesandcarbonexporttowardsdeeplayers [5].Therefore,modifications ofbiological carbon pump are expected at the global scale leading to important changes of the carbon export in the Ocean. However, some plankton speciesdevelop alternative strategies to survive to changing environments. For example, the activation of a set of enzymes to access the organic compounds as well as changes in the motility patterns canbeefficientstrategiestocopewithnutrientlimitation orpredation.

Withinasinglepopulation,thesestrategiesarenot acti-vated in each cell suggesting a wide diversity in the physiologicalresponseofthecelllivingunder thesame environments.Inaddition,fromanevolutionviewpoint, adaptationtochangesinenvironmentalconditionsarises fromvariantshavinggenotypicandphenotypicproperties differentthan themeanof thepopulation.Therefore,a singlecellapproachunravelingstatisticallyextraordinary behaviourisrequiredtounderstandhowplankton popu-lationsadapttoenvironmentalchanges.Thediversityof these physiological adaptative responses of cells is not easilydetected withclassical sampling strategies which hide single event in the response of the population. Microfluidic technology has become a key technology to preciselycontrol, manipulate and monitor small vol-umeatthepicoliterandnanoliterscalesinamicrofluidic chipdevice.Thetechnologyenablestheminiaturization andparallelizationofbiochemicalassaystoachievesingle cell level and high-throughput with low cost and time footprint.Thisminiaturizationassociatedallows microor-ganismstudiesinthefieldinconfinedenvironmentssuch asshipsor spacestations[6].

In this review, we present the recent developments in microfluidicsdedicatedtoplanktonresearch.Wefocuson howmicrofluidicplatformsaddressthemainchallengesof

thefield,suchasanalysisatalowdensityoforganismsin environmental samples, difficulties to cultivate plank-tons, pre-concentrate, detectand sort them,and onthe how analytical microfluidic platforms dedicated to the interactionsbetweenplanktonandtheirenvironmentare implemented (swimmingspeed of plankton, toxicity of phytoplankton,plankton-bacteriainteractionsandmodes of nutritionofplankton).

Analytical

microfluidic

platform

Culturing bacteria or eukaryotic plankton in confined microfluidic chambers or water-in-oil (w/o) droplets offers severaladvantages over the traditional culturing methods.Inparticular,smalldropletsactingasnanoliter batchculturesactastrapforhighlymotilecells.Fragile cells can also be studied in a microfluidic device by encapsulating living cells in alginate-based hydrogels [7].On-chipculturesconductedin asmallheight chan-nel(<50mm)reducestheculturechambertoaquasitwo dimensionalspaceand favourstheobservationofliving planktonusingclassicalmicroscope.Inacontinuousflow experiment,microfluidictechnologyenablestheprecise and dynamic control of the cellular environment. Dynamic modifications of nutrient conditions are cre-atedbyasimplepulseofeithernutrient-richornutrient exhausted medium.Forexample,Lukeetal.measured inreal-timethegrowth rate,cellsizeand chlorophylla content of cyanobacteria in response to ammonium pulsesinthemicrofluidicdevice[8].Theyrevealedthat cyanobacteria growing in on-chip showed a dynamic response to the nutrient additions similar to those observed for natural conditions. They concluded that the on-a-chip culture method is suitable for probing physiological changes in a dynamically changing envi-ronment. Culturingplankton in confined environment, such asw/odroplets, alsorevealsthecommunity inter-actions between two species. Especially, overyielding mechanism isshownwhendifferent speciesare encap-sulatedwithinthesamedroplet[9].However,confined culture environments lead to a rapid consumption of nutrients[10].Asaconsequence,cellscultivatedwithin microfluidic devicescould deviate fromanatural toan unrealisticcultureconditionifnoprecautionsaretaken. Thetimescaleforwhichtheonchipgrowthrateon-chip differsfromthosemeasuredwithlargeculturecondition ishighlyvariableintheliterature.Thistimespreadfrom several hours up to 1 month (Figure 1) [11,12]. This highvariabilityismainlyexplainedbythephysiologyof the cells,theconcentrationofnutrientsand thesizeof theincubationchamber(i.e.cultureinacontinuousflow ordiscretew/odroplets).Duringthetimeperiodwhere microfluidic incubation mimics large flask conditions, the on-chip cell culturing is suitable to prospect the optimum of cell growth depending on the nutrient concentrations[13,14].Externalfactorssuch asthe car-bondioxideconcentrationorlightexposurecandirectly

betestedinamicrofluidicdevice[15,16].Forexample, theeffectoflightintensityonthegrowthof phytoplank-tonismeasuredusingamicrofluidicdevicecomposedof two independent and superimposed microfluidic net-works: one layer for cell incubation and a second top layerwheredarkdyesflowwithdifferentconcentrations toimpactlightintensity(Figure1d).Thelightexposure canalsobedirectlycontrolledbyaliquidcrystaldisplay where each pixel of the screen matches an incubation zone containing plankton cells [17]. These original approaches assessseveral potentiallimiting factorsand are highly suitable to test numerous combinations of environmentalconditionsinasingleassay.Althoughon chipculture ofplankton issuitablefortestingeffectof environmentalparametersonthecell,discriminationof cells within theculture chamber is neededin orderto focusoncellsofinterest.Suchdiscriminationofcellsina microfluidic chamber is performed using either detec-tion orrecognition step.

Detectionandrecognitionofplankton

If detection of focused cells within a culture presents obvious interest, the study of in situ coastal waters for seawater quality control or natural assemblage assay is alsoanimportantissue.Forinstance,detectionofharmful algal bloom and identification of invasive species are crucialtocorrectlymanageanecologicalcrisisandassess theriskofpollution.Overthepastdecades,the intensifi-cation of transport of goods by sea has increased the release of ship ballasts containing phytoplankton cells incoastalenvironments.Thosecells,whetherbelonging toforeignortoxicspeciesactasaninoculumreleasedinto theenvironmentandcanthereforebecomeinvasiveand representathreatforlocalspeciesandeconomy.Shipsare traditionally not equipped to analyse seawater and the crewmembersnotformedtoidentifyspeciestrappedin theballast.Becauseofthesizeof amicrofluidicdevice, on-chip flow cytometers are proposed as a method to detect cellsof interest inseawater suchas ballast. This system should be transportable, affordable and easy to operate.Afirstapproachistoconsiderthateachparticlein theballastispotentiallyacell.Toachievethedetection ofparticlesflowinginthechannel,thesimpledifference inlightintensitywhenaparticle passesinthefrontofa detector isusedfor afastcellcount[18].However,the presence/absenceof objectsisnotsuitablefor a qualita-tivediscriminationbetweencellsandinorganicparticles. Inthiscontext,Wangetal.,developedacompactandlow costmicrofluidicdevicecapabletosimultaneouslydetect threesignals:thechlorophyllfluorescence,theside scat-ter and the resistance pulse sensing of microalgae cells [19]. The chlorophyll a signal discriminates inorganic particles or debris from living autotrophic cells. The resistance pulse sensing (change of electricalresistance betweentwoelectrodes)isusedtodeterminethesizeof themicroalgaecellflowinginthedetectionareaandthe sidescatterisusedforthecellroughness.Combinationof

thesethree signals hasbeen usedto discriminate up to three photosynthetic living cells. Benazzi et al., also developedahighspeedmicrofluidicplatformto discrim-inate three species with a diameter >2mm [20]. This microfluidic cytometer combines the measurement of impedance(asaproxyoftheparticlesize)anddifferent wavelengthfluorescencesignals(pigmentcontentofthe particles)to achievecelldiscriminationataflowrate of 3cms 1. Pigment content is also used to identify five differentspeciesinthestudy ofSchaapet al.[21].The analysis,performedatthespeed of3–5mms 1isbased onthegenerationofdistinctivewaveletsoffluorescence whenthepigmentsofthecellareexcitedbyaseriesof lasers.

Althoughmostofsystemsdiscriminate wellthecellsin smallheight channels,the widerange ofplankton size limitsthe detectionefficiency. Planktonflowingat dif-ferent depths in the microfluidic channel sometimes leadstoadifficult settingofthe optimumfocus plane. Typically,onlycellswith asizelower than 2–3mm are poorlydetectedin thechannel andareoftenbelowthe detection limit of a microfluidic flow cytometers. To optimizethedetectionofvariablecellsizes,Mawetal., proposedtofitthesizeofthemicrofluidicchannelinthe detectionareatothetargetsize[22].Thissystem recog-nizescellsfromsmallbacteria(Escherichiacolior Entero-coccusfaecalis, 1–1.5mm) tomicroalgae (Platymonas sub-cordiformis,20mm).Anothermethodconsistsinfocusing the flowof particles in thechannel in order toreduce theirdispersion [23].Upstream ofthedetectionarea,a seriesofchevron-likeobstaclesisaddedonthetopand bottom of the channel. These obstacles progressively focus the flow of particles towards the center of the

channel.Thisthree-dimensionalhydrodynamicfocusing technique is capable to decrease the detection limit down to 1mm (Synechococcus) but is also suitable for studying larger cells such as Karenia brevis (50mm) in asinglechip.

Anotherapproachtoaccessthetoxicityorpathogenrisks consistsinthedistinctionoflivinganddeadcells. Detec-tionofpigment presence as a proxyof autotrophic cell viabilityisinsufficientincoastalregionwherethecellular debris overestimated the count of living cells. To dis-criminatelivingcellstodeadones,Songetal.,developed alabel-freemethodbased onacapacitance change[24]. Thisrobustmethodreliesonthedifferenceinthe capac-itance change between a living and adead cell (living cellshave ahighercapacitancechangethan thesample suspension).Inadditiontocellviability,aprecise detec-tionofthepresenceofspecificcellscanalsobeneededfor monitoringharmfulinvasivespeciesinballastwatersand coastalenvironments.Microfluidictechnologyassociated with qualitative polymerase chain reactionanalysis is a powerful combination of techniques to accurately esti-matethepresenceoftargetspeciesorpathogen[25].To detect-specific species showing environmental risks in coastalwaters,Mahonetal. developedachipcomposed of electrodesand carbon nanotubes functionalised with oligonucleotides[26].Themethodconsistsin the injec-tionofthesamplecontainingamplifiedDNAinthechip device. The amplified DNA of the sample binds the DNAreversecomplement.Thebinds leadtoa modifi-cation of the impedance measured by the electrodes. Theseimpedancemodificationsindicatethattarget spe-ciesarepresentinasample.Totestthismethod,Mahon etal., successfully detectthree invasive species usinga

(a) (b) (c) (d) (e) (f) Light blocking layer Light Light-dark cycle control layer Light intensity control layer Microalgae culture layer Algae & culture media inlet

Black dye Black dye DI water DI water Pneumatic valve Waste outlet

Algae colony & culture media flow Single colony trapping site Waste 77 _µm 85 µm r : 40 µm Mineral oil Algae culture ho ha Microscope PDMS chip

Current Opinion in Biotechnology

Droplettrappingusinghydrostaticpressure.(a)Schematicoftheexperimentalsetupofthehydrostaticpressureheadfortrappingthedroplets,(b) time-stampedimagesshowingtheformationofsinglealgaecultureplug(i–iv)anddropletarrays(v–x),and(c)themicrochipaftertrapping 30droplets.Reproduced,withpermissionfrom11.Thehigh-throughputmicrofluidicmicroalgalphotobioreactorarray.(d)Theplatformwas composedoffourlayers–alightblockinglayer,amicrofluidiclight–darkcyclecontrollayer,amicrofluidiclightintensitycontrollayer,anda microalgaeculturelayer.(e)Enlargedviewofasingleculturecompartmenthavingfivesingle-colonytrappingsites.(f)Asingle-colonytrapping sitecomposedoffourmicropillars.Reproduced,withpermissionfrom16.

single device. Recently, amicrofluidicPCR systemhas alsobeenusedtoidentifybacterialassemblagesfixedon theplankton[27].Bothdetectionandrecognitionsystem makespossible thestudyofcellof interestinthe incu-bationchamber.However,apre-concentrationofcellsof interestinthechipareoftenneededforsampleswithlow cellabundance.

Pre-concentrationofcellsinachip

Excepted during largephytoplanktonbloomsor in spe-cificcultureconditions,cellconcentrationinasampleis commonly lower than one cell per nL (i.e. <109 cells L 1). The low cell abundancesfound in natural condi-tionsleadtoalownumberofeventsofinterestduringan on-chip assay. For this reason, numerous methods are developedinordertopre-concentratethesampledirectly on-chip.SamplefiltrationsthroughdifferentsmallPDMS meshes aredescribedin theliterature.Filtrationcanbe achievedbyaddingaseriesofsmallpillarsdisposedina checked patternin alargechannel.Byplayingwiththe dispositionofeachpillarandtheflowvelocity,Singhetal., progressivelyreroutedlargercellstoacollectionchannel locatedinthecentreofthechip[28].Thissmartmethod allowstheseparationofcellsasafunctionoftheirsizesin a continuous flow device. Concentration of cells in a sample isalsoachievedby usingasimple filtersystem. Forexample,Zhuetal.proposedamethodbasedonthe gap between a deep channel and a shallow one [29]. Microbial cells larger in size than the weir gap (1– 4mm) are hence concentrated in the deep channel whereas theliquidandsmallerparticlesareflushedinto theshallowchannel.Aseriesofsmallporesinthechannel isalsoproposedasasolutiontoincreasetheconcentration of cells in a sample. Shape of the filter can be tuned accordingtothemorphologyofthecells ofinterest.For example, Hønsvall et al. designed a series of trilobite shape-like filters to limit their clogging by chain-like microplankton species (such as Chaetoceros spp.) [30]. ThefiltrationmethoddevelopedbyHønsvalletal.,allows an efficient plankton separation based on their sizes. However,thepresence oflargeextracellularmatrix pro-ducedbysomeplanktonspecies(e.g.Thalassiosira weiss-flogii)canclogthefiltersinlongdurationexperiments.To limittheprogressivecloggingofmicrofluidicdeviceswith time, several studies avoid the use of obstacles in the channels.Inabsenceofafiltertypemodule,the concen-trationofplanktonisperformedusingeitherAC electro-kineticsor passivehydrodynamicforces(i.e.theinertial focussing method) [31]. The inertial focussing method mainly consists in a microfluidic device with a long channel.Alongchannelisneeded toreachthe equilib-riumbetweenthesheargradientliftforcewhichpushes particles awayfromchannel centrelineand the surface-effect lift force which moves particles away from the channel.Thestableequilibriumpositionwithinthecross sectionofthemicrochanneldependsontheparticlesize. Asaconsequence,thelongchannelisdisposedeitherin

straight, serpentine or spiral to maximise the particle separationandminimizetheplaceoccupiedonthechip. For example,Wang and Dandy useda single long ser-pentine channel (4mm) to get a flow of concentrated cyanobacteria culturein a collectionchannel[32]. This approachsuitableforharvestingasinglestrainofcellscan beoptimizedtosortandaccumulateparticlesdepending on their sizes. By using inertial focussing method and Dean force,Miller etal., createdaseriesof threespiral channels with different diameters in order to progres-sively cut-off the 100, 50 and 30mmsize-fractions in a sample[33].Toachievethediscriminationof particles, theyconnected,inacascadingway,thediscardoutletofa largespiralchanneltoaninletofasmallerspiralchannel (Figure 2a). This process continuously re-injected the sampleattheinletofaspiralchannelandimprovedthe separation of particles as a function of their sizes. Although the inertial focussing method is popular in microfluidicapplications,largeplanktoncapabletoswim canmoveinthechannelcrosssection.Asaconsequence, the equilibrium between the shear gradient and the surface-effect lift forces isnot reached.In thiscontext, Kumanoetal.managed tohydrodynamicallytraphighly motile cells [34]. The trap consists in a main channel splittingintotwochannels(alargechannelsimilarinsize asthemainchannelandafunneltypechannel).Thefirst cellflowinginthemainchannelistrappedinthefunnel type channeland blockedtheflow in it.Consequently, thesubsequentcellsarere-routedintothelargechannel untilthenext funneltype channel.Byusingaseriesof largeandfunneltypechannels,thecellsareprogressively concentratedinthechip.Anotherstrategytoincreasethe concentrationofhighlymotilecellsinasampleistouse traps which mimic the lobster pots or fish weirs [35]. Bouchillon etal., creatednumerousheart-shaped cham-berswherethemotileplanktoncaneasilyenterinsidethe chamber but hardly escape (Figure 2b). This trapping methodmaybeoptimizedbymeasuringtheinteractions betweenswimmingcellsandsurfaces(i.e.thescattering angleafteracontactwiththewallofthechannel)[36].To summarize,pre-concentrationofplanktonina microflui-dic chip is performed as a function of the cell size or swimmingspeedandbehaviour.Although pre-concentra-tion methods are dedicated to increase the events of interest in a sample, they can not be accurate enough to isolate a specific cell in a population. To achieve a higherdegreeofdiscriminationofcell,asortingsystemis required.

Sortingplanktoninamicrofluidicdevice

Sortingcellsordropletsofinterestinamicrofluidicdevice isaneffervescentresearchfieldwherenumerousmethods have been developed and are continuously improved throughtheyearsandapplications.Thesesorting meth-ods are discriminated into three main approaches: the passive, theactive andthehybrid.

Passivesortingsystems

Among passives methods, the inertial technology described above canbeoptimized to sort morphologies of interest using both Dean and passive hydrodynamic forces.Schaapetal.,createdamicrofluidicsystemcapable to discriminate three plankton species with different morphologies(ahigh-aspect-ratio cylindrical Monoraphi-dium griffithii, a small spherical Chlorella vulgaris and a prolate spheroidalCyanotheceaeruginosa) [37].This high throughout methodcan sortplankton with 77% separa-tionefficiency(Figure3a–d).Because ofthehigh poly-dispersityinthecelldimensions,thisvalueislowerthan sorting efficiency of monodisperse microspheres tested under the same conditions. However, this method is suitableasapre-sortingsystemandonlyneedfewdevices (apumpandachipdesignedaccordingtothemorphology of the cells of interest). Later,Syed et al., proposed an inertial sorting method to purify the Tetraselmis suecica culturecontaminatedbythediatomPhaeodactylum tricor-nutum[38].Theyusedaspecies-dedicatedspiral-channel to separate these two species with a very high sorting efficiency(upto95%)withoutaffectingthecellviability. Byusing alongstraitchannelwhere sampleandbuffer flowsinalaminarway,Godinoetal.,reportedan impres-siveseparationofeukaryoticplanktonfrombacteria(up to99%)[39].Thissmartprincipleusedthedifferenceof equilibriumbetweenlargerplanktoncells(locatedinthe centreofthechannel)andsmallerbacteria(closetowall of the channel). As aconsequence, the large cells pro-gressively left the flows of the contaminated culture sample and move in the direction of the centre of the channel where an uncontaminated buffer flows. This passivemethodisonlysuitablefor largesizedifference

betweenspeciessuch as microalgae and small bacteria. Anotherreportedpassivesortingmethodusedthe swim-mingbehaviouroftheplanktonitselftoseparatethemore activeonesfromtheothers.ThestudyofKimetal.used the combination of swimming speed and phototaxis to quicklyisolatestrainswithimprovedphotosynthetic effi-ciencies [40]. In a context of directed evolution, this method is able to quickly sort the cells with higher efficient photosynthetic activity in a mixture of 10.000mutants.Withinapopulationofcellswithsimilar morphologies, this method enables the observation of intra-speciesvariability ofaparticularresponsetostress conditions(i.e.thelightintensity)andthesortingofthe fittestcellscapabletoincreasetheirlipidcontent. Accord-ingtothepassivesortingtechnologylistedinthisreview, themainadvantagesof these approachesarethat there areeasy touse andcost-effective.However,thesorting parameters that is the morphology and the swimming speedofthecellsareratherlimited.Theresolutionofthe sortingsystem limitsthe screeningof cells withsimilar morphologiesandisnotsuitableforworkingatthesingle cell level. Moreover, passive sorting systems are not triggered,whichisleadingtosomecontaminationduring theflowsetting.Inthiscontext,anactivesortingmethod capabletomanipulatecellsordropletsatasingleobject levelisrequired.

Activesortingsystems

Despite the numerous types of active sorting systems reported in the literature, the most extensive used system is the one based on dielectrophoresis force to deflect droplets or cells of interest into a collection channel.Whenthesortingconditionisfulfilled,apulse

(a) (b) 500μm 300μm 200μm Focused Outlet Unfocused Outlet Pumped Flow Recirculating Flow Cascade Flow Pump Reservoir FINAL OUTPUT α>~100 μm α>~50 μm α>~30 μm

Current Opinion in Biotechnology

(a)3spiralrectangularchannels(500,300and200mmhigh)arecascadedtofractionateamixofparticleswithawiderangeofdiameter(100, 50and30m).Reproduced,withpermissionfrom33.(b)Tiledmicrographimagesofaportionofa“tertiaryseries”samplerusedforlabtesting. ConcentrationsofCyclidiumsp.becomegreatly-enrichedtowardsthe5thgalleryintheseries,insomecases,completelyfillingtheterminal gallerywithprotists(inset).Reproduced,withpermissionfrom35.

ofhighDCorACvoltage(typicallymorethan500V)is applied on two electrodeslocated at each side of a Y-junction pullsofdroplets intoacollectionstream.The main differencebetween the described systems is the sorting criteria. Popular systems in algal research use fluorescent labelor takeadvantageof the auto-fluores-cent pigment content of cells for screening cells of interest. In principle, fluorescence-actived droplet sys-tems mimic the fluorescence-activated cell sorting (FACS)technologywithalowersortingspeed.However, in contrast to continuous flow systems including flow cytometry technology,planktonencapsulatedin water-in-oil droplets offer the advantage of maintaining the separationinsmalldiscretemicroenvironmentsafterthe screeningprocess.Asthewater-in-oildropletsofinterest are separatedfrom eachotherinthemicrofluidic chan-nel, accurate throughput sorting method is performed. Bestetal.usedthisrobustmethodinordertosortallthe dropletscontainingcellsfromtheemptyonesataspeed of 300Hz and also sorted droplets containing cells depending on their chlorophyll a content at a speed up to 160Hz [41]. The sorting technology used a

photomultiplier tube (PMT) to measure the

fluorescence and a pulse of 700V to sort droplets of interestwithoutanynotableeffectonthecellmotilityor viability(Figure4).Theabsenceofdestructiveeffectof high voltagepulseoncellsisimportanttocontrolsince water-in-oil droplet was reported asan efficient media forcellelectroporation[42].Interestingly,several phys-iologicalparameterssuchascellviabilityaswellaslipid contentcanbedirectlyprobedusingthedieletrophoresis forces [43]. The advantages of these physiological parametersare that boththe sortingcriteriaand object deflection in the channel are performed at the same time. For example, by adjusting the frequency of the ACvoltagepulsesDengetal.,sortedcellsdependingon theirlipidcontent[43].Finally,recentprogressonhigh speedimageprocessingsystem enablesthesortingcell ordropletsofinterestdependingonthemorphologyand the number of the encapsulated objects. The sorting speedoftheimagesortingsystems(10Hz)isslowerthan thefluorescencebasedactivesortingsystem[12]. How-ever, a wide range of sorting criteria can be used to separate the droplets (morphology, fluorescence, num-berofencapsulatedobjects).Thismethodissuitablefor numerous applications including cell identification, Figure3 (a) (b) (c) (d) (e) Inlet 2 S1 S2 Inlet 1 Droplet generation region Magnetophoretic separation region Magnetophoretic separation region Focusing region Focusing region Inlet 3 camera pump Spiral microchannel

Chlorella Monoraphidium Cyanothece

25 μm 350 μm Position (μm) 0 350 (Oil phase) Permanent magnet Magnetized nickel microstructure (Sample containing MNPs and cells) (Oil phase) Focusing flo w Fo cu_sin g flow 10 mm 12 mm 50 μm 600 μm Microchannel x y

Current Opinion in Biotechnology

(a)Schematicviewoftheexperimentalsetupofpassivesortingsystem.(b)Micrographsofthethreedifferentspeciesofalgae.(c)Examplesof imagescapturedattheendofthespiralchannel(d)Normalizeddistributionsofthealgaeacrossthechannelcrosssection.Reproduced,with permissionfrom37.(e)Schematicviewofahybridsortingsystem;Dropletsaregeneratedandfocusedtothesidewallofthemicrochannelbyoil phaseflowfrominlet3.Then,singlecellencapsulateddropletareseparatedbymagneticfield.Thescalebaris200_mm.Reproduced,with permissionfrom45.

growthrateassaysaswellasbiologicalassay.Becauseof the high content image processing and microfluidic technology

Hybridsortingsystems

An hybrid sorting method can be defined as a system capabletosortnumerouseventsataspecifictime.This system cannot fullyworkat asingle cell/dropletlevel evenifthesortingtechnologycanbeoptimizedtoreach thislevel.Hybridsystemsincludesomeparameterssuch as density, compressibility, or lipid content as sorting criteria. Acoustophoretic force is for example used to manipulate water-in-oil droplets depending on size, density and compressibility of objects flowing in the microfluidic channel. A typical example of droplet manipulation is the sorting of droplets containing numerous plankton inside [44]. The difference ofcell concentration per droplet leads to a difference in the averageddensityand/orcompressibilityforeachdroplet. The acoustophoretic force applied to each droplet deflects them to the pressure node when they pass throughanacousticstanding wavefield.Thislabel-free sorting method enables the collection ofdroplets con-tainingeukaryoticplanktoncellsorbacteriawithahigh growing rate (i.e. droplets containing high number of cells).A similar approach namedmagnetophoretic sys-tem allows the on-chip sorting of droplets containing cellsfromemptyones in thechip. Thismethod usesa combination ofmagnet nanoparticles and apermanent

magnet.Inprinciple,the magnetophoretic system con-sists in the addition of magnet nanoparticles to the sample. When a cell is encapsulated in a water-in-oil droplet, its volumeis not occupied by magnetic nano-particles. The difference of number of nanoparticles between the cells containing and the empty droplets createsdifferentmagneticforceswhenthedropletsflow closeto apermanent magnet(Figure3e).This sorting systemcansortdropletscontainingasinglediatomfrom thoseemptydropletswith>94%purityataflowrateof 300mL/h [45].Thedifferenceinlipidcontentcan also beusedassortingcriteriasince,ACvoltagedeflectscell depending on lipid concentrations [46]. By using the dielectrophoresis force and continuous flow device, Hadady et al., sort the high lipid content cells to the populationwith anefficiency of 75%[47]. Hybrid sys-tems are suitable for applications mostly dedicated to celldensityanalysisordirected evolutionexperiments. However, biological assays and investigation of intra-species variability of responses to stress factors often needahigherdegreeofobjectmanipulation.Forthese reasons,a more controllable screening method such as activesortingsystemissometimesrequiredtoachievea higherdegreeofpurification.

Microfluidic

platform

for

the

measurement

of

cell-environment

interactions

Thesecondpartofthereviewfocusesontheinteractions betweenplankton andtheirenvironments.The studied

(a) (b) _{Positive channel} (c)

Negative channel Before sorting

Current Opinion in Biotechnology

SortingC.reinhardtiilow-chlorophyllcell-containingdropletsfromemptydroplets.(a)Imagestakenofdropletsbeforesorting;(b)91%droplets collectedinthe“positive”channelcontaincellswhereasonly6%ofnegativedropletsareoccupied,false“negatives”.Scalebar=100mm.(c) Imagesrecordedduringsortingshowthatcell-containingdropletsaredeflectedintothe“positive”channel(toppanel),whereasemptydroplets flowintothe“negative”channel(bottompanel)41.

interactionsfound intheliteraturerevolvetothe swim-ming speed of plankton, the diagnostic of the water quality, themicrobial loops and the modes of nutrition of plankton as function of nutrient concentration and light.

Theswimmingspeedofmotileplanktonspecies

Nycthemeral migrationofplankton in theseawater col-umn, light attenuation, nutrient concentrations and hydrodynamics areimportantfactorsleadingtothe het-erogeneous distributionfound withinheterotrophic and autotrophic organisms in the ocean. Such patchiness distributionatsmallerscaleremainsscarcelyinvestigated in literature. In particular, the behaviour of eukaryotic plankton andbacteriain changingor turbulent environ-mentisstillnotwellunderstood.Phytoplankton dynam-icsinflowcanbeaffectedbymultipleprocessesrelatedto cellmorphologyandmotility.Amongtheforces control-ling thedistributionatthelower scale,shearstress and small vortices are suggested to play key roles in this spatial heterogeneity of cell distribution. By trapping cellsinalowvolume,themicrofluidictechnologieshelp theobservationofmotileplanktonandprovideasuitable tooltoperformatestbenchoftheeffectofshearstresson livingcells.Study ofthepennatediatomsforming colo-nies(Bacillariaparadoxa)conductedinmicrofluidictends to confirm the light-dark cycleof a high gliding speed during the light period and a non-motile and aligned forms during the dark period [48]. Interestingly, the period of vibration of the diatom colonies does not depend on the water flow speed. However, when the flow speed is high (1.1mms 1), the amplitude of the vibration is higher than in still waters suggesting that diatomsmovefasterinflowingwater.Themotilityspeed anddirectioninaflow-controlledenvironmentindicated thatdiatomcellsautomaticallyadjusttheirgliding direc-tionstothedirectionoftheflow.Inlessthan10min,the directionofglidingisalsorapidlyadjustedtothedirection of theflow offeringthe smallestresistance. This align-ment of cells has also been observed for freshwater phytoplankton [49]. The alignment of cell is reported todependonthespeciesandlinktothevalueoftheshear rate(Figure5)[50].Typically,valuesofshear>1s 1are reportedtohaveaneffectonthedistributionofflagellate speciesinachannel.Thevariabilityinthedistributionof speciesremainednotwellunderstoodpurely,becausethe cellhydrodynamicprofile(i.e.morphology,locationofthe flagella) cannot entirely explain their accumulations eitherinthelowshearorhighshearregion.The modifi-cationoftheflagellatebeatpatterndependingontheflow speedandcellpreferenceprobablyplaysakeyroleinthe cellorientations.Byusingamicrofluidicdeviceandhigh speed camera, Chengala et al., found that Dunaliella primolectacanmigrateacrossthestreamwithoutrotation. This-specificswimleadstoacollectivemigrationanda 2-D thindispersionlayer[51].

Themotilityofplanktonisalsoinvestigateddepending on the presence of obstacles. Wang et al., proposed to studytheroleofconfinement andbendingchannelina microfluidicdevice[52].Theyshowedthatthespeedof cellsdependsonthesizeofthemicrofluidicchannel.In particular,theyshowed thatthechannel cross-sectional area toprotozoancross-sectionalarea had astatistically significant effect on the swimming speed (i.e. a small channelcrosssectionleadstoalowswimmingspeedof thecell).TheswimmingspeedofEuplotesvannus mea-sured on chip (70mms 1) is significantly lower than previously reported in literature (430mms 1). This is unexpected and addresses the question related to the appropriatechannel sizeformeasuring thecellmotility [52]. The effects of obstacles on cells have also been usedtocharacterizethebioluminescenceofsome plank-ton. Latz et al., investigated the latency between the impactofdinoflagellatesonobstaclesand theemission ofabioluminescence signal [53].Theminimum biolu-minescence response latency is ofinterest asit repre-sentsthemostrapidactivityofsignallingpathways.The resultsrevealedthat cellcantriggerabioluminescence flash in only 15ms after a choc with an obstacle. The minimallatencybetweentwoflashesis4mssuggesting that reactivation involves a subsystem of the entire mechanosensory signalling pathway. The reasons of theemissionofbioluminescencesignalarestillnotfully understoodbutareprobablyduetothehighshearstress closed to the obstacle which mimics a predation [53]. This relation between the predator and the prey is particularly complex at the scale of the cell. By using a microscale particle analysis in a microfluidic device, Gemmell et al., showed without the so called ‘wall effect’, that zooplanktoncan dissipate the vortices sig-naturein4ms(newhatchedN1)to>1sfortheadultone [54].These timevalues arein thesameorderof biolu-minescentlatencyandseemtoconfirmthat biolumines-cenceisasuitableescapetechniquefordinoflagellates. According to therapidresponse ofplanktontoastress such as predation,the swimming speedis also used to probe environmental conditions or pollutant effect on the cells.

Measurementofwaterqualityusingplankton

Motilityasasurvivalstrategyisanimportantmechanism ofthecellandafactorofitsresilienceinanenvironment. As a consequence, both the swimming pattern and swimming speedare both a functionofthe physiology stateofplanktonaswellasanindicatorofenvironmental conditions. Inthis context,motilityofthemarine phy-toplankton is used as a sensor for assessing pollutant toxicity.Bytrappingtwomarine phytoplanktonspecies in a series of smallincubation chambers, Zheng et al., tracked both the swimming pattern and speedof cells undereightdifferentpollutantconcentrations[55].The differentconcentrationsofpollutantsaresimultaneously generatedbythediffusionofasolutioncontainingahigh

concentration of pollutant into a sample with a low concentrationof pollutant.The successive divisions of the channel allow totest of eight different concentra-tions flowing in different incubation chambers. The curvilinear, average path and straight line velocities are used to quickly characterizethe swimming pattern and speedof plankton understress. Thehalf maximal effective concentration -which defined the response halfway between the baseline and maximum- is esti-matedinonlytwohours.Later,Fengetal.,improvedthe systembyparallelizingtheoperationandincreasingthe number ofspecies tested at the sametime [56]. How-ever, an important dataset of plankton behaviour

recorded under different controlled culture conditions (temperature, cell density and light conditions) is required. Once the calibration done, the results indi-catedthatthemicrofluidicsystemissuitableforarapid diagnosticofthewaterqualitywhentheidentificationof thepollutantis notneeded.

Reactiveoxygenspecies(e.g.H2O2)areimportant mole-cules in celllife and control numerous physiology pro-cesseswithincells.Commonlyobservedinbalancewith antioxidant molecules when the cell is healthy, the increase of reactive oxygen species expresses a stress andcanleadtothecelldeath.Thegenerationofreactive (a) (e) (g) (f) (h) (b) (c) (d) Heterosigma Heterosigma Amphidinium Amphidinium Dunaliella Dunaliella Chlamydomonas Chlamydomonas Heterosigma Amphidinium Dunaliella Chlamydomonas 0 0 0 0 2 4 6 8 10 0.5 1.0 1.5 2.0 25 50 75 100 125 150 175 0.025 0.050 swimming speed, v_s (μm s–1₎ probability density

mean square displacement, MSD (

× 10 5 μ m 2) time (s) flow velocity ( μ m s –1) cross-channel position, x (μm) measurements parabolic fit 0 0 –200 –100 100 200 300 600 1 cm y x z x y 425 μm 750 μ m

Current Opinion in Biotechnology

(a–d)Phasecontrastmicroscopyofthefourspeciesofphytoplankton.(e)Probabilitydensityfunctionofmeasuredswimmingspeedsforeach species.(f)Timeseriesofthemeansquaredisplacementforeachspecies.Thesolidlinerepresentsalinearfittothediffusiveregime,fromwhich theeffectivetranslationaldiffusivity,D,wasobtained.(g)Schematicoftheserpentinemicrofluidicdevice(left,planview;right,isometricview,not toscale)showingtheimagingplane(lightblue)andtheflowprofile(red)atthechannelmid-depth.(h)Flowvelocityprofilesmeasuredatthe mid-depthplaneusingcellsastracers(bluedots),fittedwithaparabolicprofile(redline).Errorbarscorrespondtothestandarderrorofthemean[50].

oxygenspeciesislinkedtothepresenceofabioticfactors (e.g.temperature,UVirradiation)aswellastoxicants(e.g. metalandinorganicnanoparticles).Themeasurementof reactive oxygen species is commonly performed using fluorescentdyes.Thefluorescentdyescanbecombined withdielectrophoresisinordertostackcellsina2-Dfilm [57]. By taking advantage of microfluidic technology, Koman etal., developed anon-invasivemethodto con-tinuouslymeasureon-chiptheconcentrationof extracel-lular H2O2 [58]. The results demonstrated a positive relationship between the kinetics of H2O2 generation and Cd2+concentration.However,thebalancebetween reactiveoxygenspeciesandantioxidantisre-established in1hourandsuggestedanefficientresponseofalgaetoa Cd2+stress.Thecumulativeeffectofadouble consecu-tiveexpositionofthepollutantCd2+onthealgaeleadsto ahigherandfastergenerationrateofH2O2.Theseresults obtainedbythecontinuousmonitoringofH2O2 genera-tion highlighted the advantage of on chip devices by discriminating the different phases of cellular response to the pollutant such as the detection of inflammatory response.

Finally, fluorescence of microalgae is also used as an indicator ofthetoxicityof several chemicalcompounds suchasherbicideorpesticide[59].Lefevreetal., devel-oped a portable fluorescent biosensor for detecting the herbicide Diuron with a very high sensitivity (7.5nM) compared to the portable amperometric biosensor sys-tems and transportable commercial fluorescence equip-ment [60]. However, the biosensor is dedicated to the detectionof onlyoneherbicide.

Measurementofphycotoxins

Due in part to the eutrophication of the coastal areas, harmfulalgalbloomingbecomesacommonphenomenon at a worldwide scale. The bloom of planktonic species capabletoproducetoxinsconstitutesathreatforaquatic ecosystems, humanhealthand activities.The measure-ment of toxin concentrationsin theseawateris particu-larlyimportantforpreservingthehealthofecosystemand managingtheecologicalriskforhumanhealth. Miniatur-izationandcost-affordablesystemsofferedbythe micro-fluidictechnologyarepromising.However,the develop-ment of functionalmicrofluidicsystemsincludingafull characterizationofknowntoxinsisstillindevelopment. The numerous series of complexreactions requiredfor every toxinassay iscomplicated to integrateinasingle microfluidicsystem.

However,severalstudiesproposedsomeadvanced micro-fluidic systems suitable for toxin measurement. For example, Wu et al., simply created a microfluidic chip capable to trap single cells and control their lyses by ultrasonication[61].Althoughthesystemispurposedfor thiscontent,nobiologicalassayisdescribedinthisstudy. Inthesameyear,Zhangetal.,developedamoreadvanced

system for measuring three major cyanotoxins [62]. Theyusedconventionalenzymelinkedimmunosorbent assay (ELISA) to detectand measuremicrocystin, saxi-toxin and cylindrospermopsin(Figure6).Inaddition to the good limit of detection (0.02ngml 1), the main advantageofthemicrofluidicsistheshorttimetoanalyse asample(15minfor saxitoxin,and25minfor microcys-tinsandcylindrospermopsinanalysis)comparedto classi-calELISAmethod(60minforsaxitoxinanalysis,115min formicrocystinanalysisand75minfor cylindrospermop-sinanalysis).Thisrobustsystemconsumes3 ordersless reagents and is suitable for on-field measurements. Recent progress in microfluidic fabricationenables the detectionoftoxindirectlyinfoodusinganhybridPDMS/ paperchip deviceand aptamerfunctionalizedwith gra-pheneoxide[63].Thedetectionofplanktontoxinsuchas okadaic acid or brevetoxin can be performed in only 5minutes with a similar limit of detection as ELISA detectionkits.

Bacteria-planktoninteractions

Amongthebacteria-planktoninteractions,theswimming responseofcelltochemicalcompounds(i.e.chemotaxis) iscommonlyobservedinnaturalpopulation.Forexample motilebacteriaorientatetheirswimmingdirectionalong the gradient of organic material. The chemotactic response to a concentration gradient is well known to modifythedynamicofbothphytoplanktonandbacteria populationsinculture.Forexample,themotileresponse of bacteria to some-specific amino acids, carbohydrates andpreyswasshownincultures[64].Thesechemotactic responsesto somelocalconditionssuggest thepresence ofaphycospherewheretheheterotrophicbacteria inter-actswithalgaeproductsbybeingattractedand/orgrowing inthisregion.Theconceptofphycospherewhere phyto-planktonattractmotilebacteriaisdemonstratedoneyear later byBelland Mitchell,[65].Thisconceptof phyco-sphere suggeststhat astrongheterogeneousspatial dis-tributionof microalgae andbacteria existsatverysmall scales.Themanipulationofsmallvolumesandsinglecell analysismakethemicrofluidicsasuitabletoolfor moni-toring interactionbetweencells attondhemicron-scale. By using simple microfluidic devices and cell tracking system, Seymour et al., measured the chemotactic responsetothenutrientpulses[66].Thestudyevidenced arapid aggregation of bacteria (downto <10sfor Pseu-doalteromonas haloplanktis) in thecenter of the channel where nutrientconcentration is higher[67].The aggre-gationof bacteriain thecenterof thechannelpersisted during 15–20minsuggestingaconsumptionofnutrients in thisarea.Interestingly,theextracellularcellproducts includingtheexudatesreleasedbytoxicplanktonalsoact as astrongchemoattractantforbacteria(Figure7)[68]. The velocity response of bacteria to the extracellular productsishighlyvariableandspeciesdependant.This variability in notonlylinkedto theswimmingspeedof bacteria but also depend on their chemoreceptor

sensitivity.Forexample, Min˜oet al.,reportedthat only fastswimmercellsof thebacteriaconsumer,Salpingoeca rosetta, are able to use the pH gradient to orient their swimmingdirection[69].AstheS.rosettacanalsoswima lowerspeed,fastswimmersprobablycharacterizecellsin anactive research phase. Accordingto thefact thatthe highconcentrationofbacteriacaninducelocalacidicpH, theattractionofS.rosettatotheregionwithlowpHvalues suggestedthatthisspeciesusedpHasanindicatorofthe presenceof preys.

It should be noted that small pH changes may favour distinct groupsofbacteria atthe community level and potentially createashift inthemicrobial compositions [70].Indeed,thealkalineconditionsinmarine environ-mentsarecommonlyconsideredasstressconditionsfor bacteria which need special physiological strategies to

copewith it[71].TheacidicpHgeneratedbybacteria alsolocallyregulates theform ofcarbonpresentinthe seawater.Asphytoplanktonpreferentialstorage of inor-ganiccarbonistheformofHCO3 ,aresearchstrategyto find the regions of high concentration of HCO3 is expectedforthesecells.Theregionofneutral or alka-line pH containing a low concentration of bacteria is probably more suitable in terms of algae fitness espe-ciallysincebacteriaarecommonlyfoundtocompetethe planktonfornutrientacquisition.By usingmicrofluidic channels filledwith ion permeableagarose membrane, Choietal.,exploredthechemotaxisofmicroplanktonas a function of HCO3- concentration [72]. The results obtained confirm that algae actively migrated to an optimum zone where HCO3 is abundant (26mM of HCO3 ). Thisactive researchactivity is dependanton circadian rhythm with a peak of activity in the dark (a) (b) Secondary antibody Protein A coated Microspheres 1. Protein A coated Microspheres in antibody HRP labeled antigen antigen substrate Valve 1 Valve 2 2. Secondary

antibody in 3. Wash solution

5. Wash solution 6. Antigen & HRP

labeled antigen in

7. Wash solution 8. substrate solution

4. Antibody in

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(a)Schematicdiagramofthecompetitiveimmunoassayintheimmune-reactioncolumns.(b)Illustrationofthechipoperationstocompletethe immunoassay.Thecentercircleareaofthechipisgraphicalshown:eachprocessofthereagentloadingwascontrolledbytwovalves(Valve 1andValve2).Statusofthevalvesisclarifiedbydifferentcolours:redforaction;grayforinaction.Reproduced,withpermissionfrom62.

period and a minimum during the light period. This peak of research activity for HCO3 in the dark is explained by the need of Chlamydomonas reinhardtii to compensatethelossofCO2duringitsrespirationphase. In additiontothatreason,themaximumofchemotaxis forHCO3 islinkedtothemaximumofchemotaxisfor ammonium.Asaconsequence,thecellduringthedark phase actively prospects the high carbon and nutrient sourcesandwaitsforthedaylighttoinitiatethe photo-synthesis and ammonium uptake/assimilation.In some extends, these results obtained in microfluidics may chemically explained the vertical migration observed inthe seawatercolumn.

Finally, bothbacteria andplankton can beattractedby inorganicandorganiccompounds.Theattractivenessofa chemical compound is function of the species and can varyatthesinglecelllevel.Theswimmingresponseof cell to chemical compounds is one method to find the optimumofgrowthforcell.However,marinecellsneeda setofsensitivechemoreceptorsandanactivepropulsion methodtoreachthesuitablearea.Thesemechanismsof detection and motility are not ubiquitous among all groups and species. In this context, the development of methods probing the nutritional mode and growth optimum oflow motilityareneeded.

Modesofnutritionandpreferenceforplanktongrowth Researches of modes of nutrition phytoplankton and growthoptimum conditionsare of particularinterestfor bothfundamentalknowledgeonplanktonbehaviourand thebiotechnologyapplications.Themodesofnutritionof planktonaremainlyautotrophy,mixotrophyand hetero-trophy.Theswitchbetweendifferentmodesofnutrition withinonesinglespeciesisoftenlinkedtothenutrientor light availability in the seawater. For example, low

concentrationsoforthophosphateleadtoanactivationof asetofenzymesbysomeplanktonicspecies.Amongthe enzymesactivatedduringorthophosphatestress,the alka-linephosphataseisreportedtobeagoodindicatorofthe physiologicalstateandhencethemodeofnutritionofthe cell.To identifythe modeof nutritionandevaluatethe phosphatestress,alkalinephosphataseassaysatthesingle celllevelwasdevelopedinmicrofluidicdevice(Figure8) [73].Theresultsindicatedthatcellcultureshaveasimilar timingin theactivationofalkalinephosphatase(e.g.the switch in nutrition mode between the autotrophy and osmotrophy is activated within a coupled of day at the communitylevel).However,atasinglecelllevel,ahigh variabilityinthekineticsofalkalinephosphataseactivity hasbeenmeasuredunderthesameenvironmental condi-tions and suggestedthateachcellhad differentresponsesto astressinorthophosphatenutrient.

Thisvariabilityinresponseisalsoparticularlyimportant for research in biofuel production. Especially, since nutritionmodeandnutritivestressleadstovariablelipid content ofmicroalgae cell. Nutrient concentration(i.e. thenitrogenstarvation) iscommonlyreportedtoplaya keyroleintheaccumulationoflipidinthecell[74].The highstorageoflipidinalgaecanbepromotedbyasingle stressinnitrate.However,combinationoftwodifferent stresses (such as nitrate starvation-high temperature, nitrate starvation-highsalinity as wellashigh tempera-ture-highsalinity)isalsoreportedtostimulatethelipid production [75]. Similarly to the alkaline phosphatase activityresults,theresponseoftheplanktontoastressis highly variablein a populationof a samespecies [76]. These high variations in lipid production led to the development of specific detection system suitable for the identification of the cells with the highest lipid content. Although the characterization of the lipid Figure7

(a)

(b)

300 μm

Current Opinion in Biotechnology

(a)SwimmingtrajectoriesofSilicibactersp.cellsacrossthemicrofluidicchannelfollowinginjectionoff/2growthmediumasachemoattractant (control).Eachwhitepathisthetrajectoryofasinglebacterium.(b)SwimmingtrajectoriesofSilicibactersp.cellswithinabandofSynechococcus elongatusextracellularproducts,demonstratingstrongaccumulationinthebandofchemoattractant.Reproduced,withpermissionfrom68.

contentofalgaecanbemeasuredusingaRaman micro-spectroscopy, measurement of the fluorescence-based maximum quantum yield (Fv/Fm), a proxy of nitrate starvation, inculture of diatoms have also been devel-oped[77,78].Later,Guoetal.,developedamicrofluidic devicecapabletosimultaneouslydetectthefluorescence andtoimagecellsatafrequencyof10kHz[79].They demonstrate the combination of the fluorescence of boron-dipyrromethene (BODIPY)labelled cell with an opticalparameter(opacity ofcells)toefficientlydetect thelipidcontentatthesinglecelllevel.Thedetectionof optimum of lipid production under different nutrient condition was recently integrated into complex micro-fluidicanalysisplatforms(Figure9)[80].Withone of themostadvancedmicrofluidicplatforms,thecultureof cells under different nutrient conditions, the cellular

staining of lipids and the measurement of the lipid content at a single cell level was recently achieved [80]. This recent development of microfluidic tools linked to the biotechnological applications opens the doortothehigherdegreeofcellquantificationandwill probably be useful to unravel the complexity of the planktonecology.

Conclusions

and

perspectives

Withintheactualcontextofaglobalchange, understand-ingtheplanktonbehaviourandinteractionsofeachcell with its changing environment is a challenge for the future in Oceanography. The intra-specific response of cellsgrowingunder thesame conditionsandthe detec-tionofactivecellswithinthepopulationoracommunity arehiddeninmeasurementsatthecommunity level.In

(a) _(b) (c) 20 μm 20 μm 20 μm 20 μm 20 μm

T=0 T=30s T=2min T=3min T=1min

T=10min T=9min T=8min T=7min T=6min T=5min T=1min

Current Opinion in Biotechnology

Photomicrographsoflabeledcells.Greenlabelsshowenzyme-labeledfluorescentcompoundslocatedatthealkalinephosphatasesites.Redlabels arechlorophyllapigments.(a)Typicalfluorescenceandbright-fieldimagesoffourdropletscontainingsinglelivinglabeledcells.(b)Differencein labelingbetweenthreedeadcellswithnumerouslabelsatthesurface(upperimage)andalivingcellwithasinglelabellocatedatthebottomofthe flagellarapparatus(bottomimage).(c)Exampleofthekineticsofasinglelivingcelllabeledindroplet.Reproduced,withpermissionfrom73.

thiscontext,theriseofthehigh-contentcellanalysiswill inexorablyleadtothedevelopmentofsuitablesingle-cell technology in plankton research. Microfluidics is a key technology since the manipulation of small volumes at thesubnanoliterscalecombinesbothsinglecell cultiva-tion and high content biological assays. The advanced microfluidicsystemsdescribed inthis review revealthe versatilityofmicrofluidicplatformsforplanktonresearch. The confinementof asingle celltrappedin small chan-nelsorinw/odropletsenablesafinecontrolofcellsand testvariousenvironmentalscenariiinreal-time.Stability of the w/o emulsion over time and reduced analytical volumes are themain advantagesof microfluidic. Para-doxically,thissmallanalyticalvolumeisalsothecurrent weaknessofmicrofluidicsystems.Thecellconcentration ofsomeplankton(suchasdiatomaswellasdinoflagellate) canbeverylowinnaturalsamplesbutseveralprogresses

in the pre-concentration of samples or sorting systems presented in this review alreadyallow high throughput purificationofcells.Integratingaseriesofmodulessuch ascellconcentration,biologicalassay,incubation,sorting and analysis is the next challenge for the technology. Whencontroled,suchanintegratedapproachwillenable the automation of complex biological workflows and decreasethepotential errorsdone bytheexperimenter. Integrationisthereforeoneofthenextchallengestobe tackledinthenear future.

Conflict

of

interest

statement

Acknowledgements

WeacknowledgefinancialsupportbytheERC(FP7/2007-2013/ERCGrant Agreement306385SofI),bytheRegionAquitaine,andbytheFrench’ Figure9

Culture chamber

Droplet synchronizaton

Nile red droplet generation Droplet merging Incubation chamber Rinsing channel Observation chamber

Merged droplet showing low growth or oil production

Fresh oil Stained oil

Droplet containing microalgae

Droplet containing Nile red staining solution

Merged droplet (microalgae + Nile red) Merged droplet showing high_{growth and oil production}

Microalgae droplet generation Electrodes Culturing region On-chip staining region Rinsing/analysis region

Current Opinion in Biotechnology

Illustrationofthedropletmicrofluidicsbasedmicroalgaescreeningplatformforanalyzingmicroalgalgrowthandoilproduction.Theplatformis composedofthreefunctionalparts(i)thedropletgeneration/culturingregionforcultureandgrowthmonitoring,(ii)theonchipstainingregionfortagging Nileredfluorescentdyetooilbodiesofmicroalgae,and(iii)therinsing/analysisregionforoilquantification.Reproduced,withpermissionfrom80.

BordeauxInitiativeofExcellence(IDEXBordeaux)[ReferenceAgence NationaledelaRecherche(ANR)-10-IDEX-03-02].JCBacknowledgesthe supportoftheInstitutUniversitairedeFrance.Weacknowledgesupportby theUniversityofBordeauxforapost-docfellowship.

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