Open Archive Toulouse Archive Ouverte (OATAO) OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible. This is an author-deposited version published in: http://oatao.univ-toulouse.fr/ Eprints ID: 4573 To link to this article: DOI: 10.1016/j.watres.2010.10.016 http://dx.doi.org/10.1016/j.watres.2010.10.016 To cite this version: Boulêtreau, Stéphanie and Charcosset, Jean-Yves and Gamby, Jean and Lyautey, Emilie and Mastrorillo, Sylvain and Azémar, Frédéric and Moulin, Frédéric and Tribollet, Bernard and Garabétian, Frédéric (2011) Rotating disk electrodes to assess river biofilm thickness and elasticity. Water Research, vol. 45 (n° 3). pp. 1347-1357. ISSN 0043-1354 Any correspondence concerning this service should be sent to the repository administrator: [email protected] Rotating disk electrodes to assess river biofilm thickness and elasticity Ste´phanie Bouleˆtreaua,b,*, Jean-Yves Charcosseta,b, Jean Gambyd, Emilie Lyauteya,b, Sylvain Mastrorilloa,b, Fre´de´ric Aze´mara,b, Fre´de´ric Moulinc, Bernard Tribolletd, Fre´de´ric Garabetiane aUniversite´deToulouse,UPS,INP,EcoLab(Laboratoired’e´cologiefonctionnelle),118routedeNarbonne,F-31062Toulouse,France bCNRS,EcoLab,F-31062Toulouse,France cInstitutdeMe´caniquedesFluidesdeToulouse,UMR5502,Alle´eduProfesseurCamilleSoula,31400Toulouse,France dLaboratoireInterfacesetSyste`mesElectrochimiquesLISE,UPR15duCNRS,Universite´PierreetMarieCurie,4placeJussieu, 75252Pariscedex05,France eUniversite´deBordeaux,EPOC-OASU,UMR5805,StationMarined’Arcachon,2rueduProfesseurJolyet,33120Arcachon,France a b s t r a c t Thepresentstudyexaminedtherelevanceofanelectrochemicalmethodbasedonarotating diskelectrode(RDE)toassessriverbiofilmthicknessandelasticity.Aninsitucolonisation experiment in the River Garonne (France) in August 2009 sought to obtain natural river biofilmsexhibitingdifferentiatedarchitecture.Aconstrictedpipeprovidingtwocontrasted flowconditions(about0.1and0.45ms 1ininflowandconstrictedsectionsrespectively)and containing24RDEwasimmersedintheriverfor21days.Biofilmthicknessandelasticity werequantifiedusinganelectrochemicalassayon7and21daysoldRDE-grownbiofilms Keywords: (t7 and t21, respectively). Biofilm thickness was affected by colonisation length and flow Epilithon conditionsandrangedfrom36!15mm(mean!standarddeviation,n¼6)inthefastflow Periphyton sectionatt7to340!140mm(n¼3)intheslowflowsectionatt21.Comparingtheelectro- Biofilmarchitecture chemical signal to stereomicroscopic estimates of biofilms thickness indicated that the Biofilmdeformation methodconsistentlyallowed(i)todetectearlybiofilmcolonisationintheriverand(ii)to Voltammetry measurebiofilmthicknessofuptoafewhundredmm.Biofilmelasticity,i.e.biofilmsqueeze Electrochemistry byhydrodynamicconstraint,wassignificantlyhigherintheslow(1300!480mmrpm1/2, n¼8)thaninthefastflowsections(790!350mmrpm1/2,n¼11).Diatomandbacterial density, and biofilm-covered RDE surface analyses (i) confirmed that microbial accrual resulted in biofilm formation on the RDE surface, and (ii) indicated that thickness and elasticity represent useful integrative parameters of biofilm architecture that could be measuredonnaturalriverassemblagesusingtheproposedelectrochemicalmethod. 1. Introduction solidsubstrata(Lock,1993).Embeddedinamucilagematrixof microbially generated biopolymers (EPS: extracellular polymeric Riverepilithicbiofilmsarecomplexmicrobialconsortiaofalgae, substances), these aggregates have relatively high mechanical bacteriaandothermicro-andmeso-organismsthatdevelopon stability and cell density. River biofilm dynamics influences * Correspondingauthor.Tel.:þ33(0)561557348;fax:þ33(0)561556096. E-mailaddress:[email protected](S.Bouleˆtreau). 0043-1354/$eseefrontmatterª2010ElsevierLtd.Allrightsreserved. doi:10.1016/j.watres.2010.10.016 variousinstreamprocessessuchasprimaryproduction(Wetzel, methodforinsituexperiments.Asflowrateandbiofilmmatu- 1975), river food web (Feminella and Hawkins, 1995), organic ration are proved to influence biofilm architecture (Peterson, matterandnutrientcycling(Pauletal.,1991;Battinetal.,2003a; 1996), we designed an experimental device to produce 7-day Teissieretal.,2007),andaccumulationofcontaminantssuchas and21-day-oldbiofilmsinsituwhilevaryingtheflowrate. pesticides (Dorigo et al., 2007) and toxic metals (Cheng et al., 2008;ThuyDongetal.,2008). Biofilm architecture (e.g. thickness, cohesion) varies with 2. Materials and methods communitymaturationandresistancetocurrentvelocity,both for monospecific biofilms (e.g. Mukherjee et al., 2008) or for 2.1. Experimentaldesign complex river biofilms (Peterson, 1996). Architecture partly conditionsbiofilm functionsaffectingmasstransfer between 2.1.1. Biofilmproductiondevice aggregatesandbulkwater,influencingforexampletherelative An experimental pipe device for biofilm production was uptake of substrates differing in bioavailability (Battin et al., designedandscaledtoprovidetwocontrastedcurrentvelocity 2003b). In spite of its major interest, the in situ character- conditions within the same pipe, sothat all factors affecting isationofbiofilmarchitectureremainsachallengesincetools biofilmdynamicsotherthanflowcouldbeconsideredsimilar. areveryscarce,inconvenienttouseinthefieldandsomewhat According to the volume continuity equation for an incom- semiquantitative.Amongarchitecturalparameters,thickness pressiblefluid,throughapipeconstriction(fromthesection#1 isthemostintegrativeandinformativewithrespecttovaria- of area A to the section #2 of area A ), (i) the fluid velocity 1 2 tion in key parameters including volume, wet weight, and increasesand(ii)thisincreaseinvelocity(fromv tov )issetto 1 2 number of species. However river biofilm thickness is rarely thedecreaseinsectionareaasfollows:v2=v1¼A1=A2. measured and studies often intentionally use biomass as an The constricted pipe consisted in three main parts: an indirect estimation of thickness (Dodds et al., 1999). Several upstream first cylinder (section #1, slow flow) followed by destructive (scanning electron microscopy, cryoembedding) aconvergingconicalinlet(anglea )andaseconddownstream 1 andnondestructive(lightmicroscopy,scannerwithanimage cylindrical throat (section #2, fast flow) (Fig. 1.). The current acquisitionsystem,alasertriangulationsensor,confocallaser- velocity v was determined by the local river currentvelocity 1 scanning microscopy andtwo-photonexcitationmicroscopy) andfollowedriverflowvariationsduringthewholeexperiment. optical methods are available to measure biofilm thickness The currentvelocity v dependson v value and on the ratio 2 1 (Paramonova et al., 2007). They are ideal tools for biofilm betweendiametersðF2=F1Þ.Diameterdimensionswerechosen monitoringatthemicrometerscalespatialresolution.Investi- (i) to provide a quite easily handling structure, (ii) to ensure gationsonbacterialbiofilmsarealsoorientedtowardsnano- relativelyhomogeneousflowconditionsineachsectionand(iii) scopic spatial arrangement using a combination of confocal toensurearatiov2=v1around4.Inletandthroatdiameterswere laser-scanning microscopy and atomic force microscopy setto20and10cmrespectively.Adivergingrecoverypart(angle (Schmidetal.,2008).Themaindrawbackfortheirapplicationto a )followedbyathirdcylindricalthroat(section#3;diameter 2 riverbiofilmistheincompatibilitybetweentheirobservation F3¼F1) was added to the structure to ensure a straight exit scaleandthecentimetreormetrescaleofbiofilmdevelopment stream. Convergence and divergence angles were chosen inrivers(e.g. onrock substratessuchaspebbles). Anoptical accordingtovaluesminimisingflowdetachmentandheadloss method(BakkeandOlsson,1986),periodicallyappliedforriver inVenturipipes:a ¼20&anda ¼14&.Numerousformulasare 1 2 andestuarinebiofilms(Sekaretal.,2002;Rao,2003)determines foundtoestimatetheentrancelength(l)ofcylindricalductsi.e. e biofilmthicknessastheverticalsampledisplacementrequired the position beyond which flow is fully developed (Anselmet to move the focal plane of the microscope from the water- etal.,2009).Applicationofsuchformulastothepresentflow ebiofilm interface to the biofilmesubstratum interface. It is conditionsyieldsvaluesofle=Febetween20and30leadtotoo limited in that an estimate of the refractive index of the long pipe dimensions to be handled in the river. Entry and transparentfilmisrequiredanditcanonlybeappliedtobiofilm constrictedsectionlengthsweresetto3and4timesthediam- thinnerthan100mm(Paramonovaetal.,2007). eter,thetotallengthbeingtherefore186cm.AtRDElocations, Herbert-Guillou et al. (1999) reported an electrochemical viscousshearstressonthecylinder(andincidentallyonbiofilm) methodbasedontheanalysisofatraceroxidationcurrenton isaround10timeslargerintheconstrictedthanintheentry arotatingdiskelectrode(RDE)wherebiofilmhasdeveloped. section, ensuring relative homogeneous and contrasted local This electrochemical technique was applied to detect very flowsatRDEsurfaces. thin bacterial biofilms developed in sea and tap waters Theconstrictedpipewasmadeof3-mmthickPlexiglas!to (Herbert-Guillou et al., 2000; Gamby et al., 2008). Beside ensure light diffusion. Pipe sections for which diameter was thicknessmeasurement,Herbert-Guillouetal.(2000)showed smallerthanF1weresurroundedwithanother20-cmdiameter thattheRDEmethodcouldbeusedtoprovidecomplementary Plexiglas!pipetoformasinglecontinuouspipeanddecrease information on biofilm functional properties relative to bio- detachmentoftheexternalflowaroundthepipe.Theadditional filmelasticity. sheath did not affect light penetration: irradiance in both Theobjectivesofthepresentstudywereto(i)adapttheRDE sectionsofthepipe,asmeasuredusingaLI-CORLi100quanta- methodtoestimatenaturalphototrophicbiofilmthicknessand meteratsunlight,exhibitedsimilarvalueswithina10%range. elasticity and particularly, (ii) improve the biofilm elasticity parametercalculation,(iii)assesstherelevanceofthicknessand 2.1.2. Experimentalprocedure biofilm elasticity measurements to differentiate contrasted TwelveRDEwereincorporatedateachdownstreamextremity riverphototrophicbiofilmsand,(iv)provethesuitabilityofthis ofbothsectionsoftheapparatus(Fig.1).TheRDEwerelabelled Fig.1ePhotographandschematicrepresentationoftheexperimentalpipedevice.ThepositionoftheRDEsisindicated onthephotographbyitslabelling.SF:slowflowsection;FF:fastflowsection;7:7days;21:21days.Arrowshowscurrent direction. SF(forslowflow)orFF(forfastflow)accordingtowhichsection collectedintheslowfloworfastflowsectionfollowedby7or theywerelocated.Ineachsection,thesurfaceof6RDEperpipe 21accordingtothesamplingtime,andfollowedbytherepli- side(rightandleft)wasverticallypositionedattheequatorline catenumber;RDESF7#3standsforoneoftheRDEsampledin to prevent particle sedimentation during the colonisation the slow flow section after 7 days of colonisation. Sampled process.TheRDEwerepositionednexttoeachothertoensure RDE were kept in river water at 4 &C in the dark during homogeneous environmental conditions between replicates. transport to the laboratory and measurements were per- They were maintained in order to arise to the pipe internal formedwithin5h.Att the12sampledRDEwerereplacedby 7 surfacewithnyloncableglandallowinganeasyrecovery. stainless-steelcylindersofsimilardiameter. The constrictedpipe was immersed parallel to the water currentatthebottomoftheRiverGaronneatthestudysiteof 2.2. Biofilmarchitecturemeasurements l’Aouach(01&1800000E; 43&2300800N). Thissite isatypicalreach forbiofilmdevelopment(Lyauteyetal.,2005;Bouleˆtreauetal., 2.2.1. Electrochemicalmeasurementtheory 2006).Duringthelow-waterperiod(fromJulytoOctober),the Themethodconsistsofmeasuringthesteady-statediffusion studyriverreachischaracterisedbyashallow(<1.5m),wide currentontheRDEinterfaceatafixedpotentialandatafixed (100m),andunshadedbed.Waterexhibitslowturbidity(<30 rotation speed U without biofilm (t ) and after biofilm devel- 0 NTU) and nutrient concentrations of about 10 mg P L 1 of opment (t and t ). To impose this constant potential, a 3- 7 21 solublereactivephosphorus,1mgNL 1ofbothammonium electrode-system immersed in an electrochemical cell filled andnitrates,and1.5mgCL 1ofdissolvedorganiccarbon.The with a tracer solution and connected to a potentiostat was constrictedpipewasmaintainedontheriverbottominazone used:(i)RDE,theworkingmetallicelectrodeonwhichbiofilm wheretheriverbedwasflatandhomogeneous(boulderrocks), develops;(ii)thereferenceelectrodethatcontrolsthepotential shallow(waterdeptharound50cm)andcurrentvelocitywas of the working electrode and (iii) the counter electrode that slow (around 0.1 m s 1). The experiment was performed on closes the electrical circuit and the overall current goes August 2009 during a low-flow period to exploit the most through.Diffusioncurrentresultsintheoxidationofareduced stable current velocities as possible, and to enable biofilm species at the RDEeelectrolyte interface. Without biofilm, accrualespeciallyinthefastflowsection.Dataondailymean diffusion currentdepends directlyon thediffusion boundary flowweresuppliedbyDIRENMidi-Pyre´ne´es(gaugingstation: layer thickness at the RDEeelectrolyte interface. With RDE Portet-sur-Garonne)andmeancurrentvelocitywasmeasured rotating at a constant rotation speed around its axis, the at the pipe entry using an FLO-MATE portable flowmeter diffusion boundary layer thickness is maintained constant. (Model2000,Marsh-McBirney,USA). Biofilmisconsideredasaninertporouslayerwithrespectto The device was immersed for 21 days, and six RDE per mass transport since it contains more than 95% of water section weresampledafter7 (t7) and 21 (t21) days of coloni- (Characklis,1990).Thebiofilmisalsoconsideredasalayerof sation.ReplicateRDEwerenamedasfollows:SForFFwhen stagnant water on the RDE surface, and the slowconvection existing inside the biofilm is neglected. The diffusion coeffi- surfacearea(sumofwhiteandblackpixels)withImageJ1.37v cient in biofilm was shown to be the same as the diffusion (WayneRasband,NationalInstitutesofHealth,USA). coefficient in water (L’Hostis et al., 1996), this property is Forthicknessestimation,stereomicroscopy(LeicaMZ12.5, extendedforthethickerriverbiofilmsunderinvestigationin 16)magnification)imagesofasideviewofeachcolonisedRDE the present study. This layer adds to the hydrodynamic standinginwaterwerecapturedusingaLeicaDFC320camera boundary layer one,inducing a decreasein diffusion current (Leica Microsystems DI Cambridge). Several focal planes cor- intensity. respondingtovariouscrosssections((x,z)-planesina(x,y,z) coordinate system) were visible on the picture thanks to the 2.2.2. Electrochemicalmeasurementsetting settingofanappropriatedepthoffield.Theprojectedimageof The RDE was made of a 5-mm diameter platinum cylinder thevariousfocal planeswasconvertedtobinaryimageafter (electricalconductor)coatedwithaTeflon!cylinder(electrical biofilmpixelsselection.Themaximalbiofilmheight(maximal insulator). The reference electrode was a saturated calomel z-coordinateofthe(y,z)-plane)oneachabscissaoftheimage electrode (SCE) (REF421, Radiometer Analytical, France). The (x-axis) was measured automatically in pixels using Image J. counterelectrodewasacylindricalgridofplatinumimmersed Conversionfrompixeltommwasperformedusingalinescale into the electrolyte solution that surrounded the working standard. This gives the mean maximal biofilm thickness electrode. A 0.01M potassium ferrocyanide [Fe(CN) ]2 and (meanz )ofthewholecolonisedRDEsurface((x,y)-plane). 6 max ferricyanide[Fe(CN) ]3 solutionwasusedastracerin1MKCl. 6 Ferrocyanideoxidationcurrentintensitywasmeasuredat0V/ 2.2.4. Cellnumeration SCEatwhichnowaterelectrolysisandnooxygenreduction After electrochemical measurements, material on the RDE occur.Measurementswereperformedat20&C. surfacewasremovedwithasterilescalpelandplacedinto1mL In the laboratory, the RDE was mounted on a motor axis of filter-sterilized (0.2 mm pore-size filter) river water and pluggedusingmercurycontactsandwasrotatedbyaDCmotor preserved for storage at 4 &C with the addition of 100 mL of system.Themotorspeedwascontrolledwithaservosystem neutralized formaldehyde to the biofilm suspension. Biofilm and measured using a tachometer. Prior to diffusion current suspension was sonicated in an ultrasonic bath (Elmasonic measurements,theequilibriumpotentialoftheferrocynanide/ S900H,Elma,SouthOrange,NJ)at37kHz(15min)andvortexed ferricyanidecoupleatthesameconcentrationwasmeasured (15min)accordingtoBuesingandGessner(2002).Forbacterial between 0.240 and 0.236 V/SCE in accordance with the counts,500mLaliquotoftheappropriatecellsuspensiondilu- referencepotential( 0.237V/SCE).Diffusioncurrentwasthen tionwasstainedwith200mLDAPI(0.01mgmL 1)andcollected measuredatthepotential0V/SCEforeachRDErotationspeed by filtration on 0.2 mm pore-size black polycarbonate filters between100and1200rpmbystepsof100rpm.Rotationspeed (Nuclepore,Whatman,Maidstone,UK)accordingtoGarabetian waslimitedto1200rpmtopreventbiofilmerosion.Beforet etal.(1999).CountswerecarriedoutonanOlympusBH2RLFA 0 measurements, every RDE were polished using sandpaper microscopeat1250)magnificationandresultswereexpressed (grade1200)andcleanedwithdistilledwater.Aftert andt ascellnumberpercm2.Diatomdensityinbiofilmsuspension 7 21 measurements, each RDE was individually conditioned into was estimated directly (t ) or after 5-fold dilution (t ) using 7 21 riverwateruntilfurtheranalyses. aNageottecountingchamber,bycountingthetotalnumberof Biofilm thickness d (mm) was calculated from diffusion diatomsin30fields(1.25mLeach,0.5mmdepth),usinglight current intensity measurements with ðiðtÞÞand without bio- microscopyat250)magnification(OlympusBH2RLFA). filmðið0ÞÞforeachRDErotationspeed(Uinrpm)asfollows: 2.2.5. Statisticalanalyses d¼nFDC(ShiðtÞ 1 ið0Þ 1i)10;000 (1) Electrochemical parameters (biofilm thickness and elasticity) were deduced by statistical adjustment using Origin 8.1 SR1 with n is the number of electrons, F the Faraday constant (v8.1.1388,OriginLabCorporation,Northampton,USA).Agree- (96485Cmol 1orsAmol 1),Dthediffusioncoefficientinboth mentbetweensimulatedandmeasuredthicknesswasevalu- waterandbiofilmsetto6.8)10 6cm2s 1at20&Caccordingto ated by X2 and R2 application. The non-parametric Deslouisetal.(1980),C*theelectroactivespeciesconcentra- ManneWhitney U-test procedure was used to test for flow tioninthebulksolution(0.00001molcm 3),andStheactive effectsonbiofilmthickness,biofilmelasticity,RDEbiofilmcover, RDEarea(0.196cm2). bacterialanddiatomcellnumbers.Correlationbetweenbiofilm architecture parameters was explored by usingthe Pearson r 2.2.3. Imageacquisitionandanalysis coefficient.Allvaluesaregivenasaverage!standarddeviation ForRDEbiofilmcoverestimations,stereomicroscopy(Olympus (SD). Statistical analyseswereperformed with SPSS15.0 soft- SZX10,24)magnification)imagesofthebareRDE(t0)andthe wareforWindows,andwereconsideredsignificantatp*0.05. wetcolonisedRDE(t ort )surfaceswerecapturedusingan 7 21 Olympus U-TV0.63XC camera (Olympus Corporation, Tokyo, Japan)asTIFFfiles(1600by1200pixels)andimportedinPho- 3. Results toshop CS3 (AdobePhotoshop v 10.0.1).Nostainingwasper- formed.TheimageofthebareRDEsurfacewasusedascontrol. 3.1. Determinationofbiofilmthicknessandelasticity Binaryimagesweregeneratedbyaffectingthewhitecolorto thebarepixelsandtheblackcolortothecolonisedpixels.RDE The reciprocal steady-state current intensity (mA 1) was biofilm cover (surface %) was determined on the platinum plottedagainstthereciprocalsquarerootoftheRDErotation surfaceastheratioofthesurfaceareaofblackpixelstothetotal speed (rpm 0.5) in the Koutecky-Levich coordinates in the Fig. 2. For each EDT, before (t ) or after biofilm colonisation ParametervaluesareresumedintheTable1.Thederivative 0 (t ort ),thecurrentincreasedwiththeRDErotationspeed ofdvs.Umaytendtowardsinfinitywhentherotationspeed 7 21 accordingtotheLevichlaw(Levich,1962).Foragivenrotation tendstowardszero.Thiscanresultinalossofaccuracyond0 speed,thecurrentdecreasedwithbiofilmformation(t7vs.t0 yielding to unrealistic too large d0 for SF21#6, SF21#10 and andt vs.t ).Thisdecreaseinthecurrentintensitymeasured SF21#12parameterfits(asindicatedusingtheinfinitysignin 21 0 betweent andt ort wassignificantandallowedthickness Table 1). Such unrealistic values led us to excludethe corre- 0 7 21 determination using equation (1) for 22 RDE over 24. Con- spondingRDEresults.Thepooragreementbetweenmeasured nectingissueswereattheoriginofthedefectson2RDE(SF7#1 andsimulatedthicknessesathighrotationspeedfortheseRDE andFF21#16).For22RDE(andeventhemostcolonisedones), is likely to suggest that the law is not applicable under high minimalrecordedcurrentintensities(i.e.intensitymeasured rotationspeedsforthickbiofilms.NeverthelessweakX2values attheminimalrotationspeedof100rpm)werehigherthan confirmedgoodfitqualityfor19outof22RDE;thecalculatedd0 several tens of mA suggesting that the measurement was valuesarereliableandrangedfrom16mmafter7daysofcolo- relevant(seeAppendix).Theslopeishigherfor21-thanfor nisationto500mmafter21daysofcolonisation.Electrochemi- 7-day-old biofilms, and for slow than for fast flow grown cally measured biofilm thicknesses were significantly biofilms (Fig. 2). Biofilm thickness measured at each RDE correlatedwithstereomicroscopicestimates(Table2).Electro- rotationspeed(U)wasrepresentedonFig.3.Therelationship chemicalbiofilmthicknessestimateswere1.8-foldlowerthan between thickness and rotation speed can be analysed by stereomicroscopicestimates,rangingfrom70to540mm(Fig.4). consideringthefollowinglaw: 3.2. Insituexperimentalsettings 1 d¼ (2) ðd Þ 1þKU0:5 0 TheRDEsupportingdevicewasdesignedtobeimmersedinto d0 (mm) is biofilm thickness at zero RDE rotation speed and, theriverensuringbothinsituenvironmentalvariability(algal in other words, the theoretical biofilm thickness without andbacterialinoculum,light,temperature,nutrient,etc.)and any particular hydrodynamic constraint. The coefficient K twocontrastedflowconditions.Flowvelocitylevelinthepipe (mm 1rpm 1/2)relatesthedependenceofthicknesswithRDE wascontrolledbynaturaltemporal hydraulic changesinthe rotationspeedandwasusedtoparametisebiofilmelasticity river.Otherthandays5e6e7whenthedailymeanflowpeaked as1=K(mmrpm1/2). at99m3,theriverexperiencedaperiodofquitestableandlow Slowflowt vst Fastflowt vst 7 0 7 0 25 10 SF7#1 FF7#13 SF0#1 FF0#13 SF7#3 FF7#15 20 SF0#3 8 FF0#15 SF7#5 FF7#17 SF0#5 FF0#17 -1mA) 15 SSFF70##77 -1mA) 6 FFFF70##2200 -1ntensity( 10 SSSSFFFF7070####119911 -1 ntensity( 4 FFFFFFFF7070####22223344 i i 5 2 0 0 0.02 0.04 0.06 0.08 0.10 0.12 0.02 0.04 0.06 0.08 0.10 0.12 (electroderotationspeed)-1/2(rpm)-1/2 (electroderotationspeed)-1/2(rpm)-1/2 Slowflowt vst Fastflowt vst 21 0 21 0 25 10 SF21#2 FF21#14 SF0#2 FF0#14 SF21#4 FF21#18 20 SF0#4 8 FF0#18 SF21#6 FF21#19 SF0#6 FF0#19 -1 mA) 15 SSFF021##88 -1 mA) 6 FFFF201##2211 -1ensity( 10 SSSSFFFF0220#11#11##201102 -1ensity( 4 FFFF021##2222 nt nt i i 5 2 0 0 0.02 0.04 0.06 0.08 0.10 0.12 0.02 0.04 0.06 0.08 0.10 0.12 (electroderotationspeed)-1/2(rpm)-1/2 (electroderotationspeed)-1/2(rpm)-1/2 Fig.2eInversecurrentintensityevolutionwiththeelectroderotationspeedmeasuredonelectrodesafterdifferent colonisationtimes(0day,t :closedsymbols;7days,t and21days,t :opensymbols)intwoflowsections(slowflow, 0 7 21 SFandfastflow,FF)withtheferro-/ferricyanidetracer.EachsymbolcorrespondstooneRDE. Slowflowt Fastflowt 7 7 100 50 SF7#3 FF7#13 SF7#5 FF7#15 SF7#7 FF7#17 80 SF7#9 40 FF7#20 SF7#11 FF7#23 FF7#24 thickness (µm) 4600 thickness (µm) 2300 20 10 0 0 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 electroderotationspeed(rpm) electroderotationspeed(rpm) Slowflowt Fastflowt 21 21 400 100 SF21#2 FF21#14 SF21#4 FF21#18 SF21#6 FF21#19 SF21#8 80 FF21#21 300 SF21#10 FF21#22 SF21#12 µm) µm) 60 s ( s ( es 200 es n n k k hic hic 40 t t 100 20 0 0 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 electroderotationspeed(rpm) electroderotationspeed(rpm) Fig.3eThicknessevolutionwiththeelectroderotationspeedmeasuredonelectrodesaftertwocolonisationtimes(7days, closedsymbolsand21days,opensymbols)intwoflowconditions(slowflow,SFandfastflow,FF)withtheferrocyanide tracer.EachsymbolcorrespondstooneRDE. flow(64!10m3s 1)duringtheexperiment,favouringbiofilm 59%onaverageintheslowflowsectionandfrom54to85%on development(datanotshown).Whilemeasurementonday7 average in the fast flow section (Fig. 5c.). Stereomicroscopic highlighted the above mentioned 3-day period of hydraulic thicknesssignificantlyincreasedbetweent andt andsignif- 7 21 disturbance,otherdiscretemeasurementsondays0and21in icantlydecreasedfromtheslowtothefastflowsection(Fig.5d). theslowflowsection(i.e.inletofthepipe)showedquitesimilar Biofilm thickness significantly increased with time, flow velocity values around 0.11 m s 1 that correspond to meansrangingfrom100to340mminslowflowandfrom36 atheoreticalReynoldsnumberof23,000(Table3).Accordingto to72mminfastflow(Fig.5e).Biofilmthicknesswassignifi- thedevicedimensions,flowvelocityandReynoldsnumberin cantlyaffectedbyflowconditionsatbothsamplingtimes. thefastflowsectioncanbecalculatedfromtheformerdatato Significant (or quasi significant) changes in biofilm elas- bearound0.46ms 1and46,000,respectively. ticity values ð1=KÞ occurred between t and t and between 7 21 flowconditions(Fig.5f.).Meanð1=KÞvaluesweresignificantly higher in the slow (1300 mm rpm1/2) than in the fast flow 3.3. Biofilmfeatures section(790mmrpm1/2)(ManneWhitneyU-test,p¼0.032). Electrochemicalthicknessmeasurementsweresignificantly Diatom accrual contributed to biofilm formation on the RDE. correlatedwithRDEbiofilmcover,diatomandbacterialdensi- Diatomdensityincreasedduringcolonisationwith27)103and ties (Table 2). In addition, significant correlation was also 102)103individualspercm2intheslowflowsectionandwith observed between biofilm elasticity and other parameters 8)103and33)103individualspercm2inthefastflowsection exceptbacterialdensity. on average at t and t respectively (Fig. 5a). Consistently 7 21 bacterial densities increased during colonisation reaching 32)106and27)106cellspercm2onaverageatt intheslow 21 and fast flow sections, respectively (Fig. 5b.). Comparing the 4. Discussion two sections, diatoms densities were significantly different, whereasbacterialdensitieswerenot.Asexpected,RDEbiofilm Ecologistsagreetoconsiderthicknessincreaseasthedriving cover significantly increased between t and t from 36 to forceofbiofilmstructuralandfunctionalproperties(Sabater 7 21 Table1eResultsofparameterfits(minimisationChi- square):parametervalues(average±squaredeviation) andfitquality(c2/degreeoffreedom;R2)foreachRDE. c d0(mm) K(mm 1rpm 1/2) do2f R2 Slowflowt7 SF7#3 87!2 0.00084!0.00001 0.13 0.9978 SF7#5 65!1 0.00116!0.00001 0.04 0.9987 SF7#7 193!13 0.00076!0.00002 1.52 0.9926 SF7#9 49!1 0.00103!0.00001 0.03 0.9986 SF7#11 90!1 0.00106!0.00001 0.04 0.9993 Fastflowt7 FF7#13 44!1 0.00222!0.00002 0.01 0.9995 FF7#15 30!1 0.00241!0.00003 0.02 0.9978 FF7#17 16!0 0.00124!0.00002 0.00 0.9970 FF7#20 39!0 0.00203!0.00001 0.01 0.9995 FF7#23 27!0 0.00205!0.00003 0.02 0.9973 FF7#24 58!1 0.00260!0.00002 0.02 0.9991 Fig.4eRelationshipbetweenelectrochemicaland Slowflowt21 stereomicroscopicmeasurementsofbiofilmthickness. SF21#2 501!108 0.00077!0.00003 5.81 0.9869 SF21#4 252!18 0.00071!0.00002 1.70 0.9939 SF21#6 þNa 0.00090!0.00004 58 0.9328 However,intheirpreviousexperiments,electrochemicalesti- SF21#8 277!13 0.00042!0.00001 1.43 0.9971 SF21#10 þNa 0.00053!0.00018 1222 0.7460 matesofbiofilmthicknesswerevalidatedbymeansofconfocal SF21#12 þNa 0.00044!0.00002 265 0.9304 laser-scanning microscopy (L’Hostis, 1996). In the present study,stereomicroscopywasusedsincethewholecolonised Fastflowt21 RDEsurfacecanbeexamined,andmicrobialcountscanthen FF21#14 114!3 0.00084!0.00001 0.26 0.9973 further be done on fresh material since it does not require FF21#18 86!4 0.00094!0.00003 0.78 0.9861 FF21#19 48!2 0.00076!0.00003 0.40 0.9780 any previous processing such as staining, cryoembedding FF21#21 69!1 0.00097!0.00001 0.09 0.9976 or cryosectioning. Stereomicroscopic measurements cannot FF21#22 40!1 0.00099!0.00002 0.07 0.9949 provideabsolutethicknessvalues,butgavetheupperlimitof a þNIndicatesanunrealistictoolargethicknessvalue. biofilmthicknessrangeforeachRDE.Nevertheless,theagree- mentbetweenelectrochemicalmeasurementsandstereomi- croscopicestimatesofbiofilmthickness,2-foldhigherthanthe electrochemicalone,confirmedtherelevance oftheelectro- andAdmiraal,2005),but,studiesonriverbiofilmssufferfrom chemical approach to usefully measure thicknesses ranging a lack of available tools to characterise biofilm architecture. fromafewmmtoseveralhundredsofmm.Theelectrochemical The present study intended to assess the ability of an elec- method is suitable forstudying biofilms containing notonly trochemical method based on rotating disk electrode to prokaryotic but also eukaryotic microorganisms such as measure and evaluate two features of biofilm architecture: microphytobenthicalgae,andparticularlydiatoms.Stackingof thicknessandelasticity. diatomcells,typicallyseveral10mminsize,wouldgiveabiofilm Previously,theelectrochemicalmethodmeasuredonlyvery clusterofhundredsofmminthicknesses.Ourmeasurements thin bacterial biofilms, between 0.9 and 3.5-mm thick in tap are thus consistent with the expected thicknesses for such water(Gambyetal.,2008),andupto10-mmthickinseawater biofilms. (Herbert-Guillouetal.,1999).Theuseof1MKClintheelectro- The second parameter measurable by electrochemistry is chemicalassaycouldbeexpectedtocausethicknessunderes- biofilm elasticity. Initially Herbert-Guillou et al. (2000) found timation due to EPS constriction (Frank and Belfort, 1997). direct variation of bacterial biofilm thickness with electrode Table2eCorrelationvalues(Pearsonrcoefficient)betweenbiofilmphysiognomyparameters. Parameter d0 1/K Bacterial Diatom RDEbiofilm Stereomicroscopic density density cover thickness d0 1.000 0.615** 0.480* 0.764*** 0.680*** 0.833*** 1/K 1.000 0.428 0.696*** 0.700*** 0.781*** Bacterialdensity 1.000 0.533** 0.561** 0.646*** Diatomdensity 1.000 0.714*** 0.755*** RDEbiofilmcover 1.000 0.822*** Stereomicroscopicthickness 1.000 Starsindicatethesignificancelevel(*p*0.05;**p*0.01;***p*0.001). speedrotation,dependingonbiofilmdevelopmentconditions. Table3eTheoreticalhydrauliccharacteristicsintheslow Therefore, they calculated biofilm deformation as the differ- andfastflowsectionsatt (firstday),t (7colonisation 0 7 days)andt (21colonisationdays)estimatedfrom ence between electrochemical thickness at 100 rpm and 21 measurementsattheinletofthepipeandpipe thicknessatagivenrotationspeed,andrepresentedthislatter dimensions. as a function of electrode rotation speed. This simple rela- Parameter t t t tionship was not observed in the present study, probably 0 7 21 because the studied biofilms contained algae and inorganic Slowflow v(ms 1) 0.11 0.30 0.12 particles. Adapted from Foret (2006) that demonstrated the Re 22,000 60,000 24,000 Fastflow v(ms 1) 0.44 1.20 0.48 dependenceofelectrochemicalthicknesswithkU 0:5 inwater Re 44,000 120,000 48,000 circuit biofilms, an original parameterisation of biofilm elas- ticity resulting from the assessment of an empirical 220000xx110033 a 5500xx110066 b ** pp==00..001100 tt77 tt77 tt2211 tt2211 4400xx110066 -2-2m)m) 115500xx110033 -2-2cm)cm) NNSS pp==00..442233 diatom density (ind. cdiatom density (ind. c 1155000000xxxx111100003333 **** pp==00..000044 ** pp==00..003377 bacterial density (cells bacterial density (cells 232311000000xxxxxx111111000000666666 **** pp==00..000044 NNSS NNpp==SS00 pp..77==440099..005555 **** pp==00..000044 00 00 SSllooww ffllooww FFaasstt ffllooww SSllooww ffllooww FFaasstt ffllooww 110000 c NNSS pp==00..005555 tttt77 660000 d **** pp==00..000066 tttt77 2211 2211 %)%) 8800 **** pp==00..000044 ** pp==00..003377 µm)µm) 550000 over (surface over (surface 6600 **** pp==00..000044 pic thickness (pic thickness ( 343400000000 **** pp==00..000066 **** pp==00..000044 m cm c 4400 scosco E biofilE biofil omicroomicro 220000 **** pp==00..000044 DD ee RR 2200 erer stst 110000 00 00 SSllooww ffllooww FFaasstt ffllooww SSllooww ffllooww FFaasstt ffllooww 550000 e tt 22000000 f **** pp==00..000066 tt ** pp==00..001111 tt77 tt77 2211 2211 440000 ss (µm)ss (µm) 330000 ** pp==00..002255 1/21/2µm rpm)µm rpm) 11550000 NNSS pp==00..005533 NNSS pp==00..005533 ckneckne ** pp==00..002255 city (city ( 11000000 ofilm thiofilm thi 220000 ** pp==00..002288 m elastim elasti **** pp==00..000066 bibi 110000 biofilbiofil 550000 00 00 SSllooww ffllooww FFaasstt ffllooww SSllooww ffllooww FFaasstt ffllooww Fig.5eEffectsofflowconditions(slowflowvs.fastflow)andcolonisationtime(t ,blackverticalbarvs.t ,greyverticalbar) 7 21 ondiatomdensity(a),bacterialdensity(b),biofilm(electrochemical)thickness(c),elasticity(d),biofilmcover(e),and stereomicroscopicthickness(f). relationshipbetweenbiofilmthicknessandRDErotationspeed 46,000) discriminated between optimal (Re near 22,000) and U 0:5wasproposedhere.Resultingelasticityvalues,displaying suboptimal biofilm growth conditions (Re > 40,000; Godillot awiderangeofmagnitudefromabout400to2400mmrpm1/2, et al., 2001). Consistently, higher diatom densities and bio- express the magnitude of biofilm thickness variation due to film thicknesses were found in the optimal flow section as increasing rotation speed and quantify the extent to which compared to the other section. To our knowledge, only one biofilm can be reduced by hydrodynamics constraint. The study has quantified the effect of hydrodynamics on the valuescannotbecomparedtoexistingdata,however. thickness of stream microbial biofilms (Battin et al., 2003b): Theinsituexperimentwasdesignedtocomparecorebio- thicknesses deduced from confocal laser-scanning micros- logical parameters to electrochemical parameters on natural copy images of cryosections of biofilm were significantly river biofilms. As time is one of the main drivers of biofilm higherforbiofilmscultivatedonceramiccouponsintheslow structuring, biofilms were sampled at two stages of biofilm flowcondition(0.065ms 1;Re¼1869)thaninthefastflow accrual pattern, colonisation and maturation. Successional condition (0.23 m s 1; Re ¼ 7559). The relationship between changes driven by changes in benthic microalgal species biofilmthicknessandReynoldsnumberintheformerandin strategies result in temporal changes in biofilm structure the present study were consistent with Godillot et al. (2001) (McCormick and Stevenson, 1991; Biggs et al., 1998; Wellnitz showingamaximumbiofilmbiomassforReabout22,000.As and Brader, 2003). Successional processes were also reported forbiofilmelasticityinthepresentstudy,biofilmsproducedin for river biofilm bacterial communities (Jackson et al., 2001; theslowflowsectionexhibitedhigherelasticityvaluesthan Lyauteyetal.,2005;Learetal.,2008).Inthestudiedsectionof biofilms produced in the fast flow section. Most of the theRiverGaronne,biofilmbacterialrichnessprovedtoincrease microorganismsthatformedriverbiofilmbiovolumearefitted from0to7days,anddecreasefrom7to21days(Lyauteyetal., with cellular structures maintaining cellular shape (e.g. 2005), justifying the selected sampling times. The biofilm bacterial cell walls, and diatom siliceous frustules). Biofilm support material is known to influence biofilm community elasticity most probably resulted rather from intercellular composition(CattaneoandAmireault,1992)andbiofilmscol- spacereductionthanfromcellsizeconstriction.Indeed,bio- onising RDE platinum may have exhibited distinctive taxo- film elasticity as defined in the present study might thus nomic assemblages as compared to biofilms colonising river refertovoids(poresandchannels)withinbiofilmand/orthe pebbles.Anin-depthcomparisonofbiofilmstructure,biomass loosenessofcelladhesioninbiofilm.Biofilmelasticitycould and composition between platinum and natural substrata is thusfit withthesinuosityindexof Battinet al. (2003b). The stilltobeperformed,sincenodataonassemblagecomposition multiplicationofporesorvoidswithinbiofilmcontributesto wasrecordedinthepresentstudy.Abundancesofbacteriaand enlarge biofilm surface area within biofilm and therefore diatoms were monitored, showing evidence of a microbial facilitates biofilm e water interactions and advective solute accrualonimmersedRDEsurfaces.Recovereddensitieswere transport(DeBeeretal.,1996).Suchmechanical propertyis comparabletothosepreviouslyobservedintheRiverGaronne well studied in biofilm models used to design and evaluate biofilmsfordiatoms,namely105e107individualspercm2(Eulin, performance ofbiofilmreactors (e.g.Picioreanuet al., 1998). 1997)andbacteria,about107e108cellpercm2(Lyauteyetal., Biofilm elasticity as defined in the present study could be 2010). Temporal evolution of microbial densities of RDE bio- considered as an integrative parameter of biofilmewater films fitted with measured thickness enhancement. Interest- interaction ability, in analogy with biofilm surface enlarge- ingly,RDEbiofilmcoverincreasedwithmicrobialdensitiesand ment in studies of bacterial biofilms of industrial environ- thickness suggesting that phototrophic river biofilms extend ments. For example, the reduction of biofilmewater bothhorizontallyandverticallyinaccordancewiththetypical interactions forming a barrier for advective solute transport model ofbiofilmdevelopmentfromisolatedcolumnforming could be an adaptative response of biofilm submitted to clusterstoconnectedmushrooms(Costertonetal.,1987).The chemical stress. Indeed communities exposed to cadmium proposed electrochemical assay was recommended to detect were primarily dominated by short stalked and ad-pressed and survey fouling of man-made devices in marine and diatom species whereas control communities were domi- drinkingwaters(Herbert-Guillouetal.,1999;Gambyetal.,2008). nated by filamentous diatom species (Feurtet-Mazel et al., It could also be used to evaluate the early dynamics of river 2003).Riverbiofilmarchitecturewasalsoaffectedbychronic biofilme.g.thekineticsintheveryearlystageofcolonisationin copper exposure through the growth of the chain-forming time course experiments or the patchiness of early accrual diatomMelosiravarianschangingfromlongfilamentstoshort zonesinmicroscaleexperiments. tufts(Barranguetetal.,2002).Suchaqualitativeobservation Anothermaindriverofbiofilmstructuringisflow.TheRDE mightbequantifiedbymeasuringbiofilmelasticityusingthe supportingdevicewasimaginedonthepatternofoneVenturi proposed electrochemical method. Further studies, address- pipe immersed into the river ensuring both in situ environ- ing the relationship between biofilm architecture and the mental variability (algal and bacterial inoculum, light, proposed measure of elasticity, might then allow to test temperature, nutrient, etc.) and two contrasted flow condi- whether biofilm physiognomic properties would reflect bio- tions. As intended, generated current velocities, 0.11 and filmfitnessatthecommunityscale. 0.46ms 1,wereinthevelocityrangethatfavourssuchbiofilm development (Horner and Welch, 1981). Despite disturbed hydraulicconditionsfora3-dayperiod,stableandlowdaily 5. Conclusion mean flows occurred during most of the experiment espe- ciallyduringthewholematurationperiod.Duringstableand The present study showed the suitability of an electro- low-flow periods, typical Reynolds numbers (23,000 and chemical method based on rotating disk electrode to assess