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12 Pages·2011·0.41 MB·French

by  Boulêtreau, Stéphanie

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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
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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