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Acoustic, electrochemical and microscopic
characterization of interaction of Arthrospira platensis
biofilm and heavy metal ions
Nadèje Tekaya, Ibtissèm Gammoudi, Mohamed Braiek, Hakim Tarbague,
Fabien Morote, Vincent Raimbault, Nawfel Sakly, Dominique Rebière, Hatem
Ben Ouada, Florence Lagarde, et al.
To cite this version:
Nadèje Tekaya, Ibtissèm Gammoudi, Mohamed Braiek, Hakim Tarbague, Fabien Morote, et al..
Acoustic, electrochemical and microscopic characterization of interaction of Arthrospira platensis
biofilm and heavy metal ions. Journal of Environmental Chemical Engineering, Elsevier, 2013, 1
(3), pp.609-619. �10.1016/j.jece.2013.07.006�. �hal-00878629�
Acoustic,
electrochemical
and
microscopic
characterization
of
interaction
of
Arthrospira
platensis
biofilm
and
heavy
metal
ions
Nade`je
Tekaya
a,b,*
,
Ibtisse`m
Gammoudi
c,
Mohamed
Braiek
a,b,
Hakim
Tarbague
c,
Fabien
Morote´
d,
Vincent
Raimbault
c,
Nawfel
Sakly
b,
Dominique
Rebie`re
c,
Hatem
Ben
Ouada
e,
Florence
Lagarde
a,
Hafedh
Ben
Ouada
b,
Touria
Cohen-Bouhacina
d,
Corinne
Dejous
c,
Nicole
Jaffrezic
Renault
aa
Univ.Lyon,InstitutdesSciencesAnalytiques,CNRS/ENSUMR5280,Universite´ ClaudeBernard,69100Villeurbanne,France
b
Univ.Monastir-LaboratoiredesInterfacesetMate´riauxAvance´s,Faculte´ desSciencesdeMonastir,Monastir5000,Tunisia
c
Univ.Bordeaux,Laboratoiredel’Inte´grationduMate´riauauSyste`me,CNRSUMR5218,IPB,Univ.Bordeaux1,33405Talence,France
d
Univ.Bordeaux1,LaboratoireOndesetMatie`red’Aquitaines,CNRSUMR5798,351crsLibe´ration,33405Talence,France
e
Univ.Monastir,InstitutNationaldesSciencesetTechnologiesdelaMer,RoutedeKhniss,Monastir5000,Tunisia
Introduction
Environmentalcontaminationwithheavymetalshasincreased throughouttheworldduetothedisposalofhazardouseffluentinto receivingwaters[1,2].Humansandanimalsareplacedhighlyin the food chain and in particular the marine food chain. Non biodegradable,heavymetalsaccumulateinphotosyntheticorgan ismsandtransferpollutantstoconsumers,includinghumans[3,4]. Indeed, substances such as cadmium or mercury have been classifiedas‘‘priorityhazardoussubstances’’inDecisionNo.2455/ 2001/EC[5] and Directive 2008/32/CE[6] for which industries should implement the necessary measures in order to reduce humananthropicactivity.Thisistopreserveecosystems.TheU.S.
EnvironmentalProtectionAgency’sRoadmapforMercury(July5, 2006) promotes the reduction of mercury in processes and products. Theoverallgoalof theGlobal MercuryPartnership of the United Nations Environment Program (Governing Council Decision25/5,Nairobi,Kenya,February16 20,2009)istoreduce andeventuallyeliminatemercuryuseinproductsandprocesses andraisingawarenessofmercury freealternatives.Qualitycontrol ofaquaticecosystemsrequirestoolsofinsitucontinuousdetection of contaminatedenvironments, suchaselectrochemical[7] and electromechanical[8]platformdetection.
Biosensorsthatareemergentmicro technologiesandcharac terizedbytheirsmallsize,rapidresponsewouldallowcontinuous insitutoxicitymonitoring.Recently,thedevelopmentofwhole cellbiosensorshasraisedanincreaseinterest.Microalgaesuchas Chlorellavulgaris,wereusedinvariousstudiestodevelopwhole cell biosensors for the control of toxic pollutants in aquatic environments[9 12].Acousticbiodetectionplatformbasedonthe bacteria Escherichia coli, has been developed for heavy metals detection in liquid medium [8]. For the same purpose, a dried ARTICLE INFO Keywords: Spirulina Heavymetals Admittance Lovewave SEM AFM ABSTRACT
ThisstudyexaminesabiofilmofArthrospiraplatensisanditsinteractionswithcadmiumandmercury, usingelectrochemicaladmittancespectroscopytechniquecombinedwithhighlysensitiveLovewave platformforthereal timedetectioninliquidmedium.Spirulinacellswereimmobilizedviamultilayers ofpolyelectrolyte(PEM)onSi/SiO2surfaceofbothtransducersandcharacterizedusingatomicforce
microscopy(AFM).Scanningelectronmicroscopy(SEM)cellimagesrevealedafirstdefensemechanism againstcadmiumat10 12Manditimmediatelytakesplaceafter4sfrominjection.Thecyanobacteria
biofilmbecomesmoreconductive,duetoanincreaseofpolyphosphatebodies.Anincreaseofdensity inducesadecreaseoffrequency.Responsetimet90%ofthebiofilmtowardCd2+wasbetween6and8min,
whileitdidnotexceedafewsecondstowardHg2+at10 12M.However,theinitialrapidstageofmercury
adsorptiontook40storeachthesaturatedstage.Onceexternalsorptionreachedthesaturatedstage, internalmercuryuptakebegan;cationsweretransportedacrossthecellmembraneintothecytoplasm andabeta HgSprecipitationtookplace,inducingconductivitybiofilmdecrease,andgeneratingan increaseofdensity,andthusafrequencydecrease.SEMimagesrevealedthebeginningcelldamageat 10 06Mofcadmiumandmercury.
* Correspondingauthorat:Univ.Lyon,InstitutdesSciencesAnalytiques,CNRS/ ENSUMR5280,Universite´ ClaudeBernard,69100Villeurbanne,France. Tel.:+33611901065.
E-mailaddress:tekayanadeje@yahoo.fr(N.Tekaya).
biomassof cyanobacteria, Arthrospira platensis,called Spirulina, wasused,inthepresentstudy.
ThechoiceofSpirulinawasbasedontwoprincipalreasons:the first is that its biomass is environmental friendly, easier and harmless when manipulated. The second is that Spirulina has neverbeenusedasabioreceptorforbiosensorsdestinedtodirectly detectpollutants.Mostpreviousworkemployedalgalandbacteria biomasstoextractheavymetalsfromeffluentsolutionsandfor bioremediation. In fact, some microalgae and cyanobacteria species(suchasSpirulina)canbindawiderangeofheavymetals incontaminatedecosystems[13 16].SpirulinaisaGramnegative bacterium,alsoconsideredasbluegreenmicroalgae.Components found in the cell wall of Spirulina, such as peptydoglycan, teichuronicacid,teichoicacid,polysaccharidesandproteins[17]
whichdisplaymainlycarboxylic,hydroxylandphosphategroups
[18,19]maygivealgalwallbindingproperties.ThecellwallofA. platensishaslotsofnegativecarboxylandphosphategroups,which arethedominantbindingsitesoftoxicandmetalliccations[20,21]. Furthermore,it hasbeenfoundthatmicroalgae possessa large surfaceareaandhighbindingaffinity[22],hence,theyareavery effectivebiosorbents.
The resultsinthis studyhelp toprovideaninsight into the different interactions of dried biomass of A. platensis toward metalliccationsusingacombinationofelectrochemical,acoustic andmicroscopictools.Thesethreetechniqueshavebeenchosen forspecificreasons:first,admittancespectroscopyisapowerful tool for thestudy of dynamic electrical propertiesof dielectric materials[23].Second,weappliedacousticwaveplatforminorder toperformrealtimemonitoringoftheinteractionofaheavymetal solutionincontactwithSpirulinabiofilm.Thiswillinducechanges of its viscoelastic parameters. Due to their high sensitivity to surface perturbations and their transverse wave type (Shear horizontallypolarized surface guided waves),sensors based on Love waves areideally suited for (bio)chemicalapplications in gasesandliquids[24].Theacousticwavedelay linewasinsertedin anoscillation loop and associated witha Polydimethylsiloxane (PDMS) chip, resulting in a small platform convenient for fast detection. Therefore, admittance and acoustic characterization wascarriedaftereachinjectionofheavymetals.Third,microscopic tools(AFMandSEM)wereusedascomplementarytechniquesin ordertoprovideinformationoftheobservedSpirulinacells(shape, size, biovolume, etc.). AFM is a powerful imaging tool that mechanically probes a surface with a high resolution to give morphological(ortopographical)detailsofsamplesurface.Itcan alsoprovideinformationaboutthemechanicalsurfaceproperties atthelocalscalesuchasviscoelasticity,chemicalcompositionand morphology evolution [25 30]. The AFM can also be used to capturedynamicaspectsofindividualbiologicalmolecules[31 36]andtheirinteractionswiththeenvironment[37,38]providing new insights into how macromolecules may work on the nanometerscale.AFMimageswereachievedtodetecta change inSpirulinacellsbymeasuringtheelasticmodulusviaforcecurves athighconcentrationsofmetalliccations.SEMwasperformedon Spirulinacellsinordertocharacterizeheavymetalseffectatlow concentrations(10 12M).Thistechnologyallowstheobservation
ofmicrostructuralchangesofbiologicalsamplesintheirnatural state,undercontrolledconditionsoftemperatureandpressure.
Spirulinacellswereimmobilizedontheelectrodesurface(Si/ SiO2)andonLovewavesensorswithasiliconoxidesurface,viaa
polyelectolyte multilayers (PEM) using a layer by layer (LBL) method. The LBL assembly technique consists in the alternate depositionofpolyanionsandpolycationsfromaqueoussolutions to build ultrathin multilayered films on flat substrates [39]. Currentlythesefilmsareintenselystudiedbecauseoftheirmany potential applications [40,41] and recently, PEM were used to immobilizeE.colionSi/SiO2substrate[8].
Materialsandmethods Chemicalandbiological
Two types of polyelectrolytes (PE) were used: polyallyla minehydrochloride(PAH),acationictype,andpolysodium4 styrenesulfonate(PSS),ananionictype.PAHandPSShavethe molecularweightofabout56,000and70,000respectively,and they were purchased from Sigma Aldrich. Solutions of PE (5mg/mL)werepreparedinTBS(TrisBufferedSaline)solution (pH=7.2 at0.15M).
The stock solutions (1g/L) of cadmium (Cd2+) and mercury
(Hg2+)asheavymetals,werepreparedfromCd(NO
3)2(H2O)4and
Hg(NO3)2(H2O) in TBS, purchased from Sigma Aldrich. Stock
solutionswerestoredat48Canddilutionswerepreparedbefore eachseriesofmeasurements.
A. platensis (Compere 1968/3786 strain) or Spirulina, was cultivated under sterile conditions in Zarrouik liquid medium containing:(g/L)NaNO3,2.50;K2HPO4,0.50;NaHCO3,10.00;NaCl,
1.00;MgSO47H2O,0.2;CaCl22H2O,0.02;and FeSO47H2O,0.01.
AllsaltswereofanalyticalgradeandwerepurchasedfromAcros Organics. The medium was adjusted to pH 9.0 using NaOH solution. Cultivation was conducted in 5L Erlenmeyer flasks. Cultures were maintained at 2618C under air bubbling and continuouslyexposedtofluorescentlamps(100
m
molphoton/m2s).After, the biomass was recovered by filtration, washed with physiologicalwaterfortheremovalofnutrientsalts,andthendried at408Cfor48h.Then,itwaslyophilizedandtheresultingpowder wasprotectedfrommoisturebystorageinaclosedvesselat48C.The SpirulinawasdissolvedinHEPESanditwasfiltered(0.8
m
m)before analysis.AsSpirulinaismadeoftransparentcellsstackedend to end andentrappedwithasheathformingaspiralfilament,thesheathwas brokenthroughfiltrationinordertoseparateindividualcells.This stepwasverycrucialforourstudies.Lovewavedelaylines
TheLovewavesensor,resultingfrompreviousstudies[42],is an electromechanicalsensor based on a SAW(surface acoustic wave)delaylinewithmetalizedinterdigitaltransducers(IDT)to generateandreceiveanacousticwaveonapiezoelectricsubstrate (Fig.1a).ItconsistedofadualdelaylinedepositedonATcutquartz substrate (Euler angles: 08, 121.58, and 908) used as the piezoelectric material.IDTs weremade bysputtering 70nm of goldontopofa30nmtitaniumadhesionlayertoachieveagood surfaceadhesion(Fig.1a).EachIDTwascomposedof44splitpairs of electrodes with a 40
m
m periodicity which defined the wavelengthl
.Each electrodewas5m
mwide withan aperture w of 40l
, while the center to center path length between electroacoustic transducers (Lcc) was equal to 209l
. A 4m
mplasma enhanced chemical vapordeposited(PECVD) SiO2 layer
was used as the guiding layer. It generated a guided shear horizontal surface acoustic wave (guided SH SAW), also called Lovewave.Thisconfinementofthewaveenergynearthesurface maximized the sensor sensitivity. It also ensured mechanical isolationofelectrodesfrombiologicalsamples.Thesecharacter isticsledtoa117MHzsynchronousfrequencyf0.
The obtained Love wave delay lines were inserted into an oscillation loop. Physicochemical perturbation onto the sensor surfacewillalterthewavevelocity,whichcanbemeasuredwith highaccuracythroughthefrequencyshiftsoftheoscillationloopof aradiofrequencyamplifier.Thisleadstotheachievementofan oscillator with ultra high stability, which had a considerable impactonthedetectionthresholdoftheLovewavesensor.This oscillator reached a short term stability lower than 1Hz/s at workingfrequencieshigherthan100MHz.Theresonantfrequency 2
oftheoscillatorwasmeasured witha 12 bitfrequencycounter (Agilent53131A).
Themicrofluidicchipswithintegratedmicrochannels(Fig.1c andd)werefabricatedbystandardsoftphotolithographymethods, togetherwith micromachiningforexternal3Dshapes[43].This processimpliesatwostepsprocess:(1)aclassicSU 8resinnegative moldonasiliconsubstrateforthemicrochannels;(2)thisnegative moldwasinsertedinamicromachinedaluminummoldthatdefines theoutsideshapeofthefinalmicrofluidicchips.Thisfeaturewasof greatinterest,asthemicrofluidicchipswerenotirreversiblybonded onthesensorsurface,but heldinplacebymechanicalpressure, hencetheneedforpreciselycalibratedPDMSchips.
TheSU 8negativemoldwasobtainedbyspin coatingSU 82100 onasiliconsubstrateusingadequateprocessparameterstoobtaina 200
m
mthicklayer.ThisSU 8negativemoldwastheninsertedin ourmicromachinedaluminuminjectionmold,wherethemixtureof PDMSwasinjectedusingasyringepumpafterdegassing.ThePDMS wasthencuredfor20minat958Candtheresultingchipwiththe microfluidicnetworkpatternwaspeeledofffromthemold.For detectionmeasurements,thesensor wasinsertedinto a custom test cell, maintaining the PDMS chip with proper alignment and pressureon the Love wave device.Thetest cell providesconnectionsanditwasmountedintheretroactionpathof aradiofrequencyamplifier.Fluidsampleswereflowedthroughthe microchannels above the microsensor with a programmable syringe pump with 40
m
L/min flow rate. Liquid samples were confinednearthesensor surfaceand flowedalong theacoustic path,onthemainsensitivepartofthemicrosensorandtheanalysis chamberswere2.6m
Linvolume.Frequency variation was recorded after each deposit fluid. Mean values and error bars were calculated from 3 to 4 experimentswithdifferentdelay lines.
Electrochemicaladmittancespectroscopy
Electricalmeasurementswereperformedinaconventional three electrodeglasscellconsistingof aworking electrodeof
Si/SiO2, a platinum counter electrode, and Ag/AgCl reference
electrode.Theworkingelectrode was aheterostructure of Si/ SiO2 dividedintosquaresof 1cm2; withsilica obtainedform
thermal oxidationof a siliconwafer. Thep type siliconhasa (100)crystalorientation.Thelayerthicknessofthermalsilica was approximately 100nm. Theohmic contactwas obtained with a layerofaluminum depositedonthe backside. Si/SiO2
electrodes were chosenforseveral reasons:the firstonewas due toits negative chargewhich promoted interactionswith the first layer of polycations, inorder to compare with Love wave results achieved on a sensor where itsfinal layer was silica. Thesecond reason isthatthe silicaisa biocompatible materialandSi/SiO2structuresarecompatiblewithmicroelec
tronic technologies.
Theelectrochemicalcell wasdesigned tomaintain a fixed distancebetweentheelectrodes.Itwasmanufacturedwithtwo inlets:oneforthepositioningofthereferenceelectrodeandthe other for heavy metal injections. This prevented further manipulation or movement of the electrodes. Fixing the geometry of the cell also ensured the reproducibility of measurements. The sensitive surface area of the working electrode was measured at 0.3cm2. Theelectrochemical cell
wasplacedinaFaradaycageandinmeasuredindarknesswhile the solutionwas homogenized undermagnetic stirring. Elec trochemicalmeasurements were performedinTBSat 0.15M. Impedance analysis was performed at 258C by varying the frequency from10mHzto100kHzusing a MetrohmAutolab 83139 impedance analyser controlled with FRA (frequency responseanalyser)software.Anexcitationvoltageof10mVwas surimposedtoapotentialof2300mVversusSCE.Thispotential attributestothesiliconaccumulationrangewherethesubstrate behavedlikeametal.
ImpedancedatawasobtainedforthebareandmodifiedSi/SiO2
electrodesandtheyweresuccessfullymodeledusingtheclassical circuit (R and CPE (constant phase element in parallel)) [44]
presented in Fig. 2c Electrical parameters were deduced from modelingbyminimizing
x
2value.Fig.1.(a)SchemeofadualLovewavedelay-line,(b)Spirulinaimmobilizationonapolyelectrolytemultilayer(PEM)coatedwithalayerbylayer(LBL)methodand(candd) hydrodynamicchipwithmicrofluidicnetwork,alignedonadualLovewavedelay-line.
The impedance of the SiO2/electrolyte interface CPE can be
expressedas:
ZCPE
1 ðQj
v
Þnwhere
v
is a circular frequency, Q is a constant and n is the parametervariesfrom0to1.AFMdevice
AFM experiments were performed on the NSI platform of LOMA(Bordeaux1)usingaBioscopeIImountedonanOlympus invertedopticalmicroscopeandoperatedwitha NanosCopeV controller (Veeco Brucker, Santa Barbara, CA). This AFM was equipped with a G scanner (maximum XYZ scan range of 150
m
m150m
m12m
m) [45]. In this work, samples were scannedinairandinthetappingmodeusingPPP NCL 50silicon probes(NANOSENSORSTM)withaspringconstantofabout32N/mandacorrespondingmeasuredresonancefrequencyofabout 165kHz.Incontactmode,triangularSNL 10siliconnitrideprobes (Veeco)withanominalspringconstantof0.58N/mwereusedto probeSpirulinainliquid.Allscansweremeasuredwithscanrates between0.3and1Hz(accordingtothescansizeandthescanning mode).AFM datawereprocessedusingtheNanoscopeversion 7.30(Veeco).Foreachexperiment,siximageswererecordedat the same time: height images (trace and retrace), deflection imagesorsignalerror(traceandretrace)andfrictionimagesin contact(traceandretrace)orphaseimagesintapping(traceand
retrace).Formoreclarity,imageswereflattenedandonlythetrace heightimagewasshowninthispaper.Forcecurveswereobtained with the same cantilever in the force calibration mode and collected bymeasuringthecantileverdeflectionasthesample wasmovedtowardthetip[46,47].Ingeneral,inthecaseofanAFM study of a given physico chemical treatment of surface, we performed a comparative study of samples corresponding to different phases of the treatment. In this study and for each treatment a series of three samples: the bare sensor was characterized, the sensor functionalized with PEM and finally thewholebiosensor(thefunctionalizedsensoronwhichSpirulina cells were attached). These experimental force curves were obtainedwiththesamecantilever(stiffnessofabout0.58N/m) andwithaZscanvelocityof1.6
m
m/s.Scanningelectronicmicroscopy(SEM)
The SEM images were captured using a QuantaTM 250
microscope (FEI). A pretreatment of cells was needed before imaging.Cellswerefixed,withcovalentlinksusingglutaraldehyde solution(3%)purchasedfromSigma,speciallypurifiedforuseasan electron microscopy fixative. Spirulina based biosensors were immersedinthissolutionfor45min.Afterthat,dehydrationseries insuccessiveabsoluteethanolsolutionswereappliedfor10min usingincreasedconcentrations(20%,40%,60%,80%,100%ethanol). Immobilizationofcyanobacteriaontransducersurfaces
PEMformationwascarriedoutbythealternativedepositionof previoustwopolyelectrolytes. ThisLBLassemblytechniquewas Fig.2.(a)FrequencyvariationduetothePEcoatingandSpdeposition,(b)reproducibility( :PAH, :PSS);meanvaluesanderrorbarshavebeencalculatedfrom4 experimentswithdifferentdelay-linesystems,(c)equivalentcircuit(RCPEinparallel),(d)overlappingofadmittanceplotsofbareelectrodeandmodifiedonebyPEM depositionandSpirulinacellsimmobilizationobtainedin0.15MTBSbuffer,frequencyrange:10mHz–100kHz,and(e)relativeresistanceevolutionforthreebilayers;mean valuesanderrorbarshavebeencalculatedfrom3experiments.
based on the electrostatic adsorption of oppositely charged polymerchains.Eachpolyelectrolytedepositstepwasfollowed byarinsingstepwithTBS(0.15M).Thisadsorptionprocedurewas repeateduptothreecompletebilayersandahalf(PAH PSS)3 PAH
(Fig.1b).Ahomogeneouspositivelychargedlayeronthelatter was then obtained since the Spirulina is overall negatively charged[48 50].Thenumberofbilayerswasoptimizedtoobtain the maximum shift of frequency when Spirulina cells were deposited on the sensor surface. After each polyelectrolyte coatingstep,theunfixedpolyelectrolytewasremovedbyrinsing. Cyanobacteriacellswere,then,immobilizedbyphysicaladsorp tion(Fig.1b).
Acousticcharacterization
Itis commonly acknowledgedthat the principle of acoustic wavesensordetectionismainlycausedbymechanicaleffectsand in particular mass loading due to the transducer surface perturbationinducedbymaterialdeposition.InFig.2a,itcanbe seen that the signal stabilized within only 2min after each polyelectrolytecoatingstep.Itwasnotedthatthemassandthe thickness of these films grow linearly with the number of deposited bilayers [51 54]. Consequently, as the polymers thicknessincreased, inducingamassloadingeffect,afrequency decrease was recorded after each polyelectrolyte coating step (Fig.2a).
Concerning Spirulina cells immobilization, in our previous study[55],anoptimizationofthesamecyanobacteriabiofunctio nalizationon thesensor surfacehasbeenachieved,usingthree generationsofPDMSchipsalreadymanufactured(employedfora
static, millifluidic and microfluidic setups, respectively). The bioreceptor immobilization response time was greatly reduced to half using the microfluidic set up. The same microfluidic configurationwasemployedinthispresentwork.Furthertothe Spirulinaimmobilization,afrequencyvariationof163kHzwas recorded(Fig.2aandb).
Electrochemicaladmittancespectroscopycharacterization
Complexadmittancespectrawererecordedaftereachdeposi tionofbilayersofpolyelectrolyte(PAH PSS)followedbyimmobi lizationofSpirulina.Anincreaseofthediameterofthesemi circle oftheNyquistplotwasrecordedasthethicknessofthebiofilm increased (Fig. 2d). Resistance evolution for three bilayers is presentedinFig.2ewheremeanvaluesanderrorbarshavebeen calculated from 3 experiments, determined by modeling. The decrease ofthe resistance valueis certainly due theimportant increase of the film conductivity during PEM formation. This parametermaybeexpressedas:
R e
g
Swhere
g
istheconductivity,Sistheelectrodesurface,andeisthe biofilmthickness.TheabsenceofseveralsemicirclesinFig.2dcanbeexplainedby the pseudo stratified polyelectrolyte multilayers structure. Each layerofpolyelectrolyteinterpenetratesanunderlayer[56].Conse quently, therewill be no interface formation duringsuccessive layersofpolyelectrolytedeposition. Ahigherresistancedecrease wasobservedfromthedepositionofthefirstbilayer(Fig.2e).
Fig.3.(aandb)ThreedimensionalAFMheightimagesobtainedincontactmodeandinliquid(0.15MTBSsolution)showingimmobilizedSpirulinacells;(candd)SEM images(WD,workingdistance;HFW:highfieldview(distancex,yscanned);detLFD,typeofuseddetectorisETDEverhartThornley(calledsecondaryelectrondetector– topographiccontrast);mag:magnificationvaluerelativelytoapolaroidformat;HV:acceleratingvoltage).
Microscopiccharacterization
AFMexperimentsperformedondifferentareasoftheelectrode inliquid and contact mode revealedthe coexistenceof several populations:singlecellswhoseaveragesizewasbetween0.4and 0.9
m
m;whileothercellswereclusteredingroupsoftwoorthree (Fig.3a and b).Here,it wasdifficulttoestimateindividualcell dimensions. We can, however, give an order of magnitude of smallercluster sizeswhich wereof theorderof 1.5 3.5m
min length,0.6 1.5m
minwidthand300 400nmintheheightandthe correspondingbiovolumewasabout0.4 2.2m
m3.Othermanipulationswereperformedonthesamesampleinairandintapping mode;differentdimensionswereobservedgivingslightlylower cells volumes at about 0.1 1
m
m3. Once back in the liquid,Spirulinacellsregainedtheirshapesandtheirvolumesandthey remainedstableafterseveralscansofthesurface.
SEMimagesinFig.3canddpresentedalsoseveralpopulations ofcellswhoseaveragesizewasbetween0.4and0.9
m
m. HeavymetalinjectionThefollowingLovewavemeasurementprotocolwasapplied: eachLovewaveplatformcomprisedoftwodelaylines,onebeing usedasthedetectionlinewithimmobilizedcellsviaPEM,while theotherwasusedasacontrolline(basedonPEMintheabsenceof Spirulinacells).Thistechniquealloweddifferentialmeasurement andlimitedexternalperturbationsfore.g.temperaturevariations. Moreover,differentkinetics(responsetimeandinitialresponse)
couldleadtofurtherobservationsrelatedtotheSpirulinabiofilm interactionsforvariousheavymetals.Afteracompletestabiliza tioninTBSbufferfollowingSpirulinaimmobilization,increasing concentrationsofCd2+andHg2+insolutionwereinjectedinthe
rangefrom10 12Mto10 02M.
Admittancevariationwasplottedfordifferentconcentrations ofmercuryandcadmiuminsolution,incontactwiththeSpirulina cellsmodifiedelectrode.Increasingconcentrationsofheavymetals insolution,wereinjectedintherangefrom10 14Mto10 06M.As
a control, PEM response was studied toward metallic cation applyingadmittancespectroscopy.
Resultsanddiscussion:interactionwithheavymetalions Cadmium/biofilminteractions
The typical real time response obtained through acoustic measurements with Cd2+ injection is presented in Fig. 4a and
steady state frequency shifts are summarized in Fig. 5a. No frequencyvariationwasobservedonPEMbasedcontrolline(inthe absenceofSpirulinacells).Itcanbeseen,inFig.4b,thatthereal time frequencyresponsedecreases atthefirstinjection ofCd2+
(10 12M) andtheinduction time(time beforedetection ofthe initialresponse)wasinstantaneousandabout4s.Theresponse time
t
90% of the biofilm toward Cd2+ was 8min. A saturationphenomenonfrom10 09Mwasobserved(Fig.4a).
Fig.4.Responseofacousticandelectrochemicalbiosensorforsuccessiveincreasingconcentrationsofcadmiumobtainedin0.15MTBSbuffer:(a)typicalreal-timeresponse oftheSpirulina-basedacousticbiosensor(flow40mLmin 1
),(b)zoomoftheresponsefurthertheinjectionof10 12
M(Cd2+
),and(c)evolutionofcomplexadmittance spectraoftheSi/SiO2/biofilmstructure,frequencyrange:10mHz–100kHz.
Fig.5.Parametersevolutionfordifferentconcentrationsofcadmium:(a)steady-statefrequencyshiftsand(b)relativeresistanceevolution;meanvaluesanderrorbarshave beencalculatedfrom3experimentswithdifferentelectrodes.
Admittancevalueevolutionwasobservedinmediumandhigh frequencies,withSpirulinabasedbiofilmatthefirstinjectionof cadmiumwherethefinalconcentrationinthesolutionwasabout 10 14M(Fig.4c). In fact, theincreaseof imaginary admittance
(Yim)and theshift of thereal admittance position(Yreal) were
recorded. After modeling Nyquist plots using the equivalent circuit,adecreaseoftheresistanceofthefilmwasobservedaswell asanincreaseoftheCPE(equivalenttocapacitance).Therelative variation of resistance R versus concentration of cadmium concentrationincontactispresentedinFig.5b.Arapidsaturation phenomenonwasobtainedatthesecondinjection10 12M(Cd2+) (Fig.4c).Thisisinagreementwiththerealtimeresponserecorded withLovewaveplatformwherethefrequencydecreasesatthefirst injection of cadmium(10 12M) followed by a rapid saturation
phenomena (Figs. 4a and 5a). SEM image presented in Fig. 6a revealstheeffectofcadmiumcationsat10 14MonSpirulinacells.
Indeed,therearesignificantchangesontheexternalsurfaceand remarkableirregularitiesappearedontheoutercellwallsuchason thesecondwall.Itwasfoundthattheexternalsurfaceofsome bacteria develop a protective layer of excreted polymer, as a defenseagainstcadmiumbyrestrictingtheionpermeabilityinto the cell [57 59]. This phenomenon can also explain that the
response time for the substrate of alkaline phosphatase in Spirulina has been increased after exposure to cadmium and mercury[60].
AccordingtoRangsayatornetal.,transmissionelectronmicro graphs of Spirulina sections revealed the effects induced by cadmium:disintegrationanddisorganizationofthylakoidmem branes,presenceoflargeintrathylakoidalspace,reductionofgas vacuolesandanincreaseofpolyphosphatebodies(PPBs).[13].Ruiz et al. demonstrated using X ray microanalysis of the electron dense vacuoles or polyphosphate bodies of Chlamydomonas reinhardtii [61] that there were large amounts of phosphorus, magnesium,calciumandzinc.TheincreaseofPPBs,inpresenceof cadmiumcouldexplaintheobserveddecreaseofresistance;the subsequentreductionofgasvacuolescouldleadtoanincreaseof densityandofthedielectricconstant,andthusconnectedtoan increase of capacitance of the Spirulina film from1.3410 8F
withoutcadmiumto1.3710 8Ffor[Cd2+]equalto10 6M.The
observeddecreaseofthefrequency,4safterinjection(3100Hzas a mean value (Fig. 5a)) could be attributed tothe increase of density. SEMexperimentsperformed on Spirulinaexposedtoa concentrationof10 09Mofcadmium,showsthebeginningofcell lysisandadecreaseoftheirdiameter(Fig.7b).
Fig.6.SEMexperimentsonSpirulinacellsaftercadmiumexposure:(a)Cd2+
(10 14
M)and(b)Cd2+
(10 09
M).
Fig.7.Responseofacousticandelectrochemicalbiosensorforsuccessiveincreasingconcentrationsofmercuryobtainedin0.15MTBSbuffer:(a)typicalreal-timeresponseof theSpirulina-basedacousticbiosensor(flow40mLmin 1
),(b)zoomoftheresponsefurthertheinjectionof10 12
M(Hg2+
),(c)evolutionofcomplexadmittancespectraof theSi/SiO2/biofilmstructure,frequencyrange:10mHz–100kHzand(d)enlargedcurves.
Mercury/biofilminteractions
AcontrolmeasurementwasperformedwiththePEMwithout Spirulinatoshowthatnovariationofadmittancecouldbeobserved whenaddingHg2+.Inpresenceofcyanobacteriacellsandfollowing
successive injections of mercury, a decrease of the imaginary admittancewasrecorded(Fig.7candd).AftermodelingtheNyquist plotsusingtheequivalentcircuit,anincreaseoftheresistanceofthe filmwasobservedaswellasadecreaseoftheCPE(equivalenttoa capacitance). The relative increase of the resistance (
D
R/R) is presentedinFig.8b.Itvariesbetween15and18,incomparisonwith thecontrolmeasurementrecordedwithPEM,wheretherelative variationofresistance(D
R/R)didnotexceed0.17,calculatedfrom themodeledspectrafor10 06MofHg2+.Atthesecondinjectionofmercury(10 12M),asmallvariation
oftheresistance value wasrecorded(Fig.8b) and a saturation phenomenonwasobservedjust after.Theseobservations were, also,inagreementwiththefrequencyshiftsrecordedinrealtime at10 12Mand10 09MofmercuryusingtheLovewaveplatform
(Fig.7aandb):detectionofmercuryatthefirst(10 12M)andat
the second (10 09M) injections, followed by a saturation
phenomena.Inductiontimeofmercurywasabout40safterthe firstinjection(10 12M)andtheresponsetime
t
90%ofthebiofilm
didnotexceedfewsecond(Fig.7b).
Cogne et al.[62] studied Zn,Mg, Fe, Mn, Cu, K, uptake by Spirulinaingeneral,andtheyreportedthatmicroorganism metal interaction consisted in physical adsorption and chemical
absorption. The partial reversibility of theadmittance noticed afterarinsingstepcantheneliminatecationsphysicallyfixedon thecellwall(Fig.7c).
SEM images were achieved in the same conditions and performedonSpirulinacellsexposedtothesameconcentration (10 14M)ofmercury.Therewasaclearmodificationofthetotal
cellmorphologyexpressedbydepressionsandholes(Fig.9a). Generally,theuptakeofmetalionsbymicroorganismshasbeen reportedintwostages[63,64]:aninitial,rapidstageandalater, slowerstage.Inthefirststage,whichiscalledtherapidphase,the metalionsarefixedontothesurfaceofmicroorganisms.Thisvery fastuptakeinthecellenvelopewasconfirmedin[57].Accordingto WangandChen[65],thereisstoichiometricinteractionbetween functionalgroupsofcellwallcomposition,includingphosphate, carboxyl,amineaswellasphosphodiester.Inthesecondstage,a longeruptakeperiodinsidethecelltakesplace[57].Themetalions aretransportedacrossthecellmembraneintothecytoplasm[63]. Also,accordingtoWangandChen[65],thereisaphysicochemical inorganicdeposition viaadsorptionorinorganicprecipitationin thissecondstage.
Inaddition,Lefebvreetal.[66]studiedthebiotransformationof Hg(II)bycyanobacteria.Theyrevealedthatmeta cinnabar(beta HgS)constitutedthemajorbiotransformedandcellularlyassoci atedmercurypool.Inthepresenceofmercury,therewasarapid synthesisofbeta HgSandHg(0),however,theproductionratefor thelatter decreasedquickly. Consequently,cyanobacteriacould acttoconvertsubstantialamountsofHg(II)intobeta HgS.Kaoud Fig.8.Parametersevolutionfordifferentconcentrationsofmercury:(a)steady-statefrequencyshiftsand(b)relativeresistanceevolution;meanvaluesanderrorbarshave beencalculatedfrom3experimentswithdifferentelectrodes.
Fig.9.SEMexperimentsonSpirulinacellsaftermercuryexposure:(a)Hg2+
(10 14
M),(b)Hg2+
(10 09
M). 8
etal.[67]suggestthatSpirulinaplatensiscouldchelateHgions producingastablecomplex.
Basedonthesestudies,wecanconcludethatmercurycations canpenetratedirectlyintoSpirulinacell.Theinitialrapidphase of mercury uptake took 40s to reach the saturation stage (inductiontime).Onceexternalsorptionreachedthesaturation stage, internal uptake began [58], cations were transported acrossthe cellmembraneinto thecytoplasm anda beta HgS synthesistookplace[65].Thischemicalcompound,precipitated asablackpowder,isinsolubleinwater,stableandhadacrystal structure.Therefore,mercurysulfideprecipitationmaydecrease thebiofilmconductivity,whichisinagreementwiththestrong increaseoftheresistancevalueobservedinpresenceofmercury cations(Fig.8b).Theobserveddecreaseof capacitanceofthe Spirulina film (from 1.4110 8F without mercury to
1.2510 8F for [Hg2+] equal to 10 6M) could also be in
agreement with the formation of mercury sulphide whose dielectricconstant isabout25, andislower than thatof the Spirulinacellequalto40[68].Theformationofmercurysulfide precipitationgeneratesanincreaseofdensity,andsoadecrease offrequencyinrealtime(Fig.7a)withameanvalueofabout 2700Hzaccumulatedaftertwosuccessiveinjectionsofmercury (10 12Mand10 09M)(Fig.8a).
BiofilmexposedtohighconcentrationsofCd2+andHg2+
Above10 03Mofcadmiumandmercury,completelysisofthe
SpirulinacellswasobservedbytheAFMcharacterizationinliquid medium (contact mode). Indeed, it can be seen from the comparisonbetweenFig.10aandb,adisappearanceofSpirulina cells,resultingintracks,surroundedbythecellmaterial,wasalso inagreementwiththeforcecurvespresentedinFig.10c.Similarly, comparingtheforcecurvesperformedoncells(beforeinjectionof Cd2+) and those performed on the prints of Spirulina cells,
confirmedthoseresults.Infact,followingtheinjectionofavery highconcentrationofCd2+(>10 3M),themodulusidentifiedon
theprintswasthesameasthoseidentifiedontheinitialsubstrate (PEM) accrediting Spirulina cells degradation. This observed burstingofthecellsisin agreementwithtransmissionelectron micrographs observations of a Spirulina section reported in Rangsayatornetal.[13],revealingSpirulinacelllysisinducedby cadmiumexposure.
Conclusion
ImmobilizationofSpirulinacellswascarriedoutsuccessfully via multilayers of polyelectrolytes. Analysis of the acoustic Fig.10.AFMobservation(contactmodeinliquidmedium)oftheSpirulinaimmobilizedonthesensorsurface:(a)beforeheavymetalsinjection,(b)afterinjectionofhigh concentrationofCd2+
(10 3
M)and(c)comparisonofforcescurves(approach)observedonPELandSpirulinacellbeforeandafterinjectionofahighconcentrationof cadmium.
response,associatedtoadmittancespectroscopyandcharacteri zationofsurfaceusingAFMandSEM,allowedtoproposeandto verify some physico chemical mechanisms involved in the interaction between lyophilized biomass of Spirulina cells and heavymetals.Biofilminteractionmechanisms,analyzedtoward cadmiumandmercuryweretotallydifferentandadetectionlimit wasdeterminedtobe10 14Mforeach metal.Thatbioreceptor
response is sensitive enough to provide preliminary in situ environmentalanalysis.Thisbiosensorisnotspecificforasingle metal,butitprovidesaglobalresponseofthepresenceofheavy metals in trace.Such rapid and non destructive measurements couldbeappliedwithothermicro organismsforachievingtoxicity tests.
Acknowledgments
ThisworkwasfinanciallysupportedbytheCMCU,projectNo. 10G1103.PDMSchipsweredesignedintheframeofANRproject BIOALERT.AuthorswouldliketothankMichaelLeefortheEnglish revision.
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