O
pen
A
rchive
T
oulouse
A
rchive
O
uverte
(OATAO)
OATAO is an open access repository that collects the work of some Toulouse
researchers and makes it freely available over the web where possible.
This is an author’s version published in:
http://oatao.univ-toulouse.fr/
20536
To cite this version:
Dominguez-Benetton, Xochitl and Godon, Jean-Jacques and Rousseau, Raphaël and
Erable, Benjamin and Bergel, Alain and Delia-Dupuy, Marie-Line Exploring natural vs.
synthetic minimal media to boost current generation with electrochemically-active
marine bioanodes. (2016) Journal of Environmental Chemical Engineering, 4 (2).
2362-2369. ISSN 2213-3437
Any correspondance concerning this service should be sent to the repository administrator:
tech-oatao@listes-diff.inp-toulouse.fr
Exploring
natural
vs.
synthetic
minimal
media
to
boost
current
generation
with
electrochemically-active
marine
bioanodes
X.
Dominguez-Benetton
a,*
,
J.J.
Godon
b,
R.
Rousseau
a,
B.
Erable
a,
A.
Bergel
a,
M.L.
Délia
aaLaboratoiredeGénieChimique,CNRS—UniversitédeToulouse(INPT),4alléeEmileMonso,BP84234,31432Toulouse,France
bInstitutNationaldelaRechercheAgronomique(INRA),LaboratoiredeBiotechnologiedel'Environnement,AvenuedesEtangs,11100Narbonne,France
Keywords:
Microbialelectrochemicaltechnologies
Microbialfuelcells
Bioanodes
Electrochemically-activebacteria
Marinebiofilms
ABSTRACT
OneofthegreatestchallengesinthedevelopmentofMicrobialElectrochemicalTechnologies(METs)is theachievementofefficientbioanodes,whichnotonlycanoperateforlongtermbutwhichcanalsobe effectivelyandpromptlyregeneratedforthesustainedandsuccessiveproductionofelectricityoutof wasteorganicscontainedinaqueousstreams.Simplestrategiesthatfacilitatetheengineeringofthese systemsarethenpursued.Sustainableelectricitygenerationwashereachievedusing electrochemically-active marine biofilms, which reached up to 6.8Am!2 in the best case. These biofilms showed
deteriorated current generation after successive transfers in fresh natural media. The electricity-generationfunctionalityofthesemarinebiofilmswasrecuperatedaftertheirrelocationintosynthetic minimalmedia(i.e.uptoabout3.8Am!2afteradecaydowntoabout1–2Am!2).Uponthisrelocation,
theoverallelectrochemicalmechanismswerepreserved.Fluctuatingnutrientstressintensifiedtheeffect of minimal media on currentgeneration. The changefrom natural to minimal media showed an importantimpactontheselectionandadaptationofmicrobialcommunities,characterizedbyCE-SSCP profiles;yet,robust bioanodesinwhichsomemicrobial specieswerepreserved wereobtained in syntheticminimalmedia,thesebeingsufficientforareproducibleelectrochemicalfunctionality.The systematiccyclingbetweennaturalmediaandminimalmedia,regardedasperiodicstressconditioning, isthereforeproposedasaconvenientstrategytoboostcurrentgenerationinrobust electrochemically-activemarinebioanodes.
1.Introduction
MicrobialElectrochemicalTechnologies(METs)usemicrobial biofilmswhichdevelopoverthesurfaceofelectrodesandactas catalystsforelectrochemicaloxidationorreductionreactions[1]. Such type ofmicrobial electrocatalysis allowsa broad range of applications when operating in galvanic (power generating) or electrolytic (power consuming) mode, for instance, electricity generation[2],treatmentoflow strength[3] andhighstrength wastewaters [4],water desalination[5],and electrosynthesisof organic chemicals [6], among others. Although some of these systemsarestillinearlydevelopment—particularlyformicrobial electrosynthesis—theypromisesignificantbreakthroughsforthe
valorisation waste streams into a selection of higher value chemicalsandfuels[7–9].
ThecrucialcomponentofsomeMETsisthemicrobialanode. Thesourceofinoculumforitsdevelopmentisconsideredtobeone ofthemostinfluential factorsontheirperformance. Numerous sourceshavebeenalready proposed, includingactivatedsludge [10], various wastewaters and mixtures thereof [11–17], and freshwateroritssediments[18–20].
Particularly, saline environmentsprovide theopportunity to developmicrobialanodesthatoperateathighionicstrength[21– 23].Suchtypeofelectrolytesenhancethetransportofionsinthe bulkliquid[24],whichasaconsequencereducestheohmicdrop [25,26]orionicresistanceoftheelectrolyte.Infact,mediawith highionicconductivityareappropriateforbetterperformanceof any electrochemical system. However, in spite of the evident advantagesforbetterelectrochemicalperformance,the develop-ment of microbial anodes in highly conductive environments (>50mS/cm)isstillonearlystages.Themajorityof electrochemi-cally-active (EA) bacteria revealed so far would be drastically affectedbythechangesinosmoticpressurethatmediawithhigh
* Correspondingauthor.Presentaddress:SeparationandConversionTechnology,
FlemishInstituteforTechnologicalResearch(VITO),Boeretang200,Mol2400,
Belgium.
ionicstrength(seesalinity)wouldentail.Yet,thereexist environ-mentswherehalotolerantmicrobialcommunitiessystematically facethischallenge.Saltwateroritssedimentshavebeenemployed showingmodestperformancesoncurrentgeneration[27–29]. Air-tolerant marinebiofilms have beenemployed inmicrobial fuel cells (MFCs) and microbial electrolysis cells (MECs) showing suitable performance and reproducibility [30,31]. Marine sedi-ments[32–36]andinlesserextentsaltmarshsediments[37–41] have also been tested as inoculum sources in sediment-MFCs. Although the latter inhibit the prevalence of methanogenic bacteriawhicharedetrimentalforelectricitygenerationinMFCs andsomecaseshavefavourablyshowedtogenerateupto85Am!2
[42],thesedimentfractionseemssofaranessentialconstituentfor longtermoperation,which results inconvenientforengineered systems.Handling the solid fraction is simplynot practical for industrialprospectionofMETs.
Likewise,nutritionalconditionsrepresentamedullaraspectin theperformance ofMETs.Directfeedof naturalmedia[43–45], wastewaterscontaining diverse substrate organics [46–48] and othertypesofwastes[49,50],supplementedornotwithparticular nutriments,havebeenextensivelyexplored.
Althoughhighlydesirablefromtheperspectiveofvalorisingor improving waste streams, the utilisation of such media might appearimpracticalfromcertainperspectives.Fromthescientific point of view, their complex composition obscures the under-standingsomefundamentalphenomenaandextracellularelectron transfer mechanisms in microbial communities; they involve unidentifiedsidereactionsoccurringatthebulkphase,adsorption oforganiccompoundsandothereventsmaskingthe electrochem-icalinfluenceofthebiofilm.Theelectrochemicalactivityofthe biofilms may involve direct electron transfer via multi-heme cytochromes, electrontransfer throughnanowires, and indirect transferviasolublemediatorsorelectronshuttles.Simplermedia wouldallowabetterdemonstrationofsuch occurrences;atthe sametime,thesewouldmakeeasiertoaddresstheeffectofcertain operationalparametersandsubsequentlytobetterregulatethem. Fromtheengineering perspective,other concernsarise. Notall typesofusableindustrialwastewaterscontainsignificantorganic loads. For instance, desalination processes waters typically undergothroughastepofreductionofthetotaldissolvedsolids (TDS)content;seawaterdesalination startswith35–45g/LTDS, while brackish water starts with 1.5–15g/L TDS, and they are reducedtobelow0.5g/LTDS.Therefore,completeculturemedia are seldom representative for conducting studies with true applicability.
Altogetherthishasfurtherconsequences,microbialconsortia will develop at different levels of community and metabolic complexity[51];simplerculturemediainfluencethedevelopment of microbial consortia with simpler nutritional requirements, whichareknowntobecomemoreeconomicalandeasiertocontrol atprocesslevel.Yet,thissimplermetaboliccomplexitydoesnot necessarily imply a less diverse microbial community.Besides, growthindefinedmediamayresultinincreasedbiofilmformation comparedtorichmedia[52].Furthermore,fromtheperspectiveof microbial electrosynthesis (MES), well controlled and simpler matricesappearpragmaticforasubsequentdownstream process-ingstrategy—ontheperspectivetoobtainhighpurityproductsor easier,fasterandmoreeconomicalrecoverythereof.
Minimalculturemediaarethosewhichonlycontainthecritical nutrientsformicrobialgrowth:acarbonsource,andvarioussalts that provideessential elementssuch as nitrogen, phosphorous, magnesium,sulphurandothertraceelements.Someofthemcan alsobe supplemented by selected agents, usually amino acids, vitaminsorsugars,whenthegrowthofauxotrophsisanticipated. Minimal media meet better the environmental conditions of surface-watersthancompletemedia,reasonwhytheyareoften
used in environmental microbiology togrow wild-type micro-organisms[53].Minimalmediahavebeengenerallyemployedin MFCs with pure cultures of Shewanella oneidensis, Lactococcus lactis,EnterobactercloacaeandBacillussubtilis[54–64]. Neverthe-less,mixedculturebioanodeshavepredominantlyderivedfrom microbialcommunitiesenrichedfromwastewaters,andreportson theirdirectadaptationtominimalmediaarescarce[65].Whatis more, such approach has not been reported for halotolerant bioelectrodes; knowledgeisstill lackingontheperformance of electrochemically-activemixedculturebioanodesunderminimal conditionsincontrasttonaturalorindustrialstreams.
Basedontheoverallconsiderationshereexplained,theaimof thepresentinvestigationwastousehalotolerantcommunitiesto develop EA-biofilms in minimal synthetic media, which were hypothesizedtoprogressintorobust, reproducibleand electro-chemically functional microbial anodes. Microbialanodes were formedfrommarinebiofilmsand theirfunctionaladaptationto minimalculturemediawasassessed.Thepresentstudyprovides significantadvancementoftheexistingstateoftheartforitsdirect implications towards development and maintenance of robust bioelectrodeswithindustrial/engineeredprospection.
2.Materialsandmethods 2.1.Biofilmsamples
Naturalmarinebiofilmswerecollectedfromaplasticfloating bridgeintheportofLaTremblade(N45.4601000,W1.803000;Atlantic
Ocean,France),aspreviouslydescribedbyErableandBergel[66]. The floating bridge is situated in the low water zone and, consequently,itisindirectcontactwithsedimentsforabout6h aday.Thebiofilmwasharvestedusingaplasticscraper;around 50mLofbiomaterialwerestoredinaglassbottlecontaining20mL of fresh seawater, for three days at ambient temperature (17# 4$C).Theseawaterusedfortheexperimentswasfromthe
samelocationandithadaconductivityof49mScm!1.A
biofilm mix was preparedbyadding 1:1 ofnatural seawater and solid biofilm,supplementedwith20mMsodiumacetate,andtakento homogeneityinaceramicmortar.Thebiofilmmixwasmaintained for8hunderorbitalagitationat60rpm,untiluse.
2.2.Electrochemicalinstrumentationandsetup
Experiments were carried out in 700mL (total volume) borosilicate SchottDuran reactors containing500mLof culture mediumsolution,atconstantpolarisationconditions (chronoam-perometry, CA) using a conventional three-electrode system connectedtoa multi-potentiostat(VMP2Bio-LogicSA,software EC-Labv.9.97,Bio-LogicSA).Thereactorswereclosedhermetically, withnogasflow.Theoperationaltemperaturewascontrolledat 30$C throughout all the investigation. The experiments were
conductedinahalf-cellsetuptoevaluatetheperformanceofeach anodeinwell-controlledelectrochemicalconditions.Rectangular slices of 2 types of carbon electrodes were used as working electrodes,feltortissue[67],respectively.Duringpunctualpoints of experimentation, samplesof theelectrodeswere takenwith sharpscissors; therefore,currentdensities(Am!2)arereported
with theproper corrections to theprojected surface area. The carbon materials were initially sterile, as proven by confocal microscopy(datanotshown),andtheywerevigorouslyrinsedin steriledistilledwaterpriortouse.Theelectrodeswereembedded downtohalfoftheliquidvolume.Theelectriccontactwasmade with twirled 90%/10% Platinum/Iridium wires. The auxiliary electrodes were made of Heraeus Vectra 90%/10% Platinum/ Iridium meshes (20cm2 projected surface area) and were
electricpotentialsareherereportedagainsttheSaturatedCalomel standard referenceElectrode(SCE,MaterialsMates,insaturated KClwhichhasapotentialof244mVvs.SHE).Thepotentialofthe anodeswasfixedat+100mV/SCEduringCA.Fromtimetotimethe constantpolarizationwasinterruptedandcyclicvoltammetry(CV) wasinsteadperformedinsituontheactiveanodes,startingfrom open circuitconditions.The potential was scannedthree times between!500and500mV/SCEatscanrateof5mVs!1
;onlydata from the last scan (out of three) are here reported. Control electrodes(seawaterorsyntheticculturemediawithoutinoculum) wereequallytested,showingnoelectriccurrentgeneration. 2.3.Mediaandcultureconditions
FormationoftheEA-biofilmswasachievedbyintroducingthe inoculum (10%v/v)intoanaturalmedium(NM)ordirectlyina syntheticminimalmedium.NMwascomposedoffreshseawater supplementedwithacetate.Foreachindependentexperiment,the microbialanodewasthenrepetitivelytransferredintoasynthetic minimal medium(SMM).Bioanode transfertofreshmediawas carried out once that a peak of current had decayed, as per observations on the response in CA. The SMM consisted of a modifiedStarkeymedium[68],frequentlyusedforculturingstrict anaerobic sulfate-reducing bacteria. The composition of this mediumwas,perliterofdistilledwater:K2HPO40.5g,NH4Cl2g,
oligoelements solution 1mL and NaCl 30g. The media can be supplemented withyeastextract yeastextract(0.2g), inwhich caseitisherereferredasSMM-Y.Theoligoelementssolutionwas composed of, per liter of distilled water: HCl 37% 46mL, MgCl2
%
6H2O 55g, FeSO4(NH4)2SO4%
6H2O 7g, ZnCl2%
2H2O 1g,MnCl2
%
4H2O 1.2g, CuSO4%
5H2O 0.4g, CoSO4%
7H2O 1.3g, BO3H3,1g Mo7O24(NH4)6
%
4H2O, 0.05g NiCl2%
6H2O and Na2SeO3%
5H2O0.01g;thesecompoundsweredissolvedunderstirringat200rpm duringonehour,thenthesolutionwasheatedupuntilcomplete dissolutionofthesolidsalts.Aftercoolingunderagitation,60gof CaCl2
%
2H2Owereadded.The solutionwas stockedindarkflaskunderrefrigeration;itprecipitatesafterseveraldays,soitneedsto bere-homogenizedpriortoutilization.Themediainthereactors weresupplementedwithsodiumacetate.Here,theconcentration ofsodiumacetate(eitherinNMorSMM)ismarkedasasubindex inmM(e.g.SMM20referstoaSMMmediumsupplementedwith
20 sodiumacetate;NM60referstothenaturalmedium
supple-mentedwith60mMsodiumacetate,andsoon).Theconductivity of each mediumwas measuredwithin 48and 52mScm!1,the
temperaturewaskeptconstantat30$Candthesystemshadno
agitation. ThepH wasnaturallymaintained (buffered)between 7.5and8.2duringalltheexperiments.
2.4.DNAextraction,CE!SSCPfingerprintingandDNAsequencing Genomic DNA was extracted and purified from the carbon electrodesamplesusingapreviouslydescribedprotocol[69].The total DNA extractedwas purified using a QiAmp DNAmicrokit (Qiagen, Hilden, Germany).DNA amountand purity of extracts wereconfirmedbyspectrophotometry(InfiniteNanoQuantM200, Tecan, Austria). The bacterial communities issued of the EA-biofilms wereanalyzed by thePCR–single strandconformation polymorphism(SSCP)technique.HighlyvariableV3regionsofthe 16S rRNAgene were amplified by PCR from each biofilm DNA sample. Onemicroliterof genomicDNA sampleswasamplified usingtheprimersw49(50-ACGGTCCAGACTCCTACGGG-30,
Escher-ichia coli position F330) and 50-6FAM labelled w104 (50
-TTACCGCGGCTGCTGCTGGCAC-30, E. coli position R533) [70] in
accordance with previously described CE-SSCP amplification methods [71]. CE-SSCP electrophoreses were performed with ABI310(AppliedBiosystems).CE-SSCPprofileswereanalysedusing
GeneScansoftware(AppliedBiosystems)and thepackage ‘Stat-Fingerprints’[72]. Pyrosequencing of the DNAsamples using a 454protocolwasperformedbyResearchandTestingLaboratory (Lubbock,USA).
2.5.Epifluorescencemicroscopy
EA-biofilmcolonisationwasimagedattheendofexperiments by epifluorescence microscopy. The bioanodes were washed carefullywithSMM(withoutcarbonsource)toremoveallexcess materialsand chemicals, except for the attached biofilms. The biofilmsweredyedaccordingtoErableandBergel[73],using0.03% acridineorange(A6014,Sigma)for10min.Afterwards,thesamples wererinsedwithrunningdemineralizedwater.Thesampleswere analyzedwithaCarlZeissAxiotech100microscopeequippedfor epifluorescencewithanHBO50/acmercurylightsourceandthe Zeiss 09 filter(excitorHP450-490,reflector FT 10,barrier filter LP520).Imageswereacquiredwithamonochromedigitalcamera (EvolutionVF)andtreatedwiththeImage-ProPlus5.0softwareto recompose3-Dimages.
3.Resultsanddiscussion
3.1.FunctionalhalotolerantEA-bioanodesprogressivelydeterioratein naturalmarinemedia
Anelectrochemicalreactorstarted upbypolarizingacarbon feltanode(25cm2)at100mV/SCE.Afterreachinga
pseudo-steady-statebehavior (&1h),the reactorwas inoculated with10% v/v marinebiofilmmix.AnEA-biofilmprogressivelydevelopedunder natural seawater supplemented with 60mM sodium acetate (NM60).Every time thecurrent dropped down almostentirely,
the full exhausted medium was drained and replaced by an equivalent volume of fresh NM60. Thissuccessive transfer was
performedthreetimes(Fig.1).Intheprimaryadaptation,current density(j)intensifieduptoabout6.8Am!2duringthe
first10days, followed bya progressivedecay within the subsequent 8days. Afterthisdropincurrentdensity,thebioanodewastransferredto freshNM60.Rightafter,alesssignificantcurrentgenerationpeak
wasattained(upto&1.2Am!2
),withatotaldurationofninedays. Concisely,successive transfersofhalotolerantbioanodesinto freshnaturalmediasupplementedwithasuitablecarbonsource resulted in progressive loss of the generated current. This behaviour was reproducible with 20mM of sodium acetate as
Fig.1.Chronoamperometryofamarinebiofilmdeveloped overcarbontissue
polarizedat100mV/SCE.NM60:Naturalmedium(seawater)supplementedwith
60mMsodiumacetate.ThebioanodewasadaptedtofreshNM60;threesuccessive
transfersfreshNM60tookplace.Numberspointingwitharrowsindicatemoments
carbonsource,withaninferiorperformanceforcurrent genera-tion.Thisbehaviourisexplainedbydepletionofvitalnutriments otherthan the carbon source,for the EA-biofilm tosustain its activity.
3.2.Theuseofminimalmediare-establishescurrentgeneration capacitiesofEA-biofilmsthatformerlydeterioratedinnaturalmedia
Anindependentelectrochemicalreactorstartedupinthesame conditionsthanformerlydescribedforthesystemcharacterizedin Fig.1. Thesole differencewas that a third transfer tonatural mediumwasavoided,inordertopreventtotaldecayincurrent generation.Instead,theexhaustedNM60wasdrainedandreplaced
byasyntheticminimalmediasupplementedwith60mMsodium acetate(namelySMM60).Intheprimaryadaptationand
consecu-tivetransferstoNM60(Fig.2,days0–26)theoverallbehaviourwas
consistentwiththeoneobservedduringthetotaloperationofthe firstreactor(Fig.1):afirstcurrentpeakwithimportantcurrent densitywas obtained,followedwithsubsequentpeakswherein current generation appears significantly deteriorated.However, rightafter transferring the bioanode intoSMM60, not onlythe
current density doubled themagnitude of the precedent peak producedinNM60butalso,afterasecondtransfertoSMM60,the
currentdensityattainedequalledtothemaximallevelachieved withtheinitialNM60(Fig.2,days26–38).Furthermore,suchlevel
was maintained even after a third transfer to SMM60 (day
38onwards).Replicateexperimentsshowed thesametrend.To sum up, successive transfers of halotolerant bioanodes to a syntheticminimal media supplementedwith a suitable carbon source, after progressive loss of current generation in natural media,resultedinrecuperationoftheoriginalfunctionality.This behaviourwasalsoreproduciblewith20mMofacetate,withan inferiormaximalcurrentdensityatthefirstpeak(1–2Am!2).
3.3.Preservationofelectrontransfermechanismsisidentifiedbycyclic voltammetryresponse
CVswererecordedatspecificmomentsduringtheCA experi-mentspresentedinFig.1andFig.2.Fig.3presentsthederivatives of current density with respect to potential (
D
j/D
E), for the oxidationturnoffasobtainedinCV.Withthepurposeofimprovedvisualization,onlythosevaluesbeyondopencircuitvoltage(OCV) areshown. Forthesame purpose, thepotential is presentedin logarithmicscaleandnormalized.First,itbecomesclearthatthe mechanisms of electron transferoccurring for theoxidation of acetateduringthefirstpeaksofcurrentgenerationobtainedwith NM60,aspresentedinFig.1andFig.2respectively,areequivalent
andjustdifferinintensity(Fig.3,&and*).WhentheEA-biofilm was consecutively transferred toNM60 solely, not onlycurrent
generationfailed,butalsoitwasevidentthattheelectrochemical responseofthesystemdiverged(Fig.3,&)whencomparedthe one obtainedin opposed circumstanceswhere the biofilmwas transferringcurrenttowardsthecarbonelectrode (Fig.3,&,*, and').InthecaseoftheEA-biofilmadaptedtoSMM60(Fig.2,day
26onwards),itisappreciatedthattheelectrontransfer mecha-nisms(Fig.3,')remaincomparabletothoseformerlyobtained withtheprimarycolonizersinNM60(Fig.3,*),implyingonlya
succession and not a modification of the functionality.This is, current density was impacted,but not the formof thekinetic response.Tosumup,asobservedfromthetransformationofthe CVresponse(registeredatdifferentcharacteristicstagesoftheCA curves for different electrodes), it was confirmed that the halotolerantEA-bioanodesprogressivelyexhaustedtheircurrent generation functionality over the solid-state electrode when progressively exposed to natural marine media supplemented with a suitable carbon source, as opposed to the positive preservationandupsurgeofsuchcapacitywhenatotal deteriora-tionis preventedbyprompt relocationinfreshminimal media supplementedwiththesamesubstrate.
Fig.2.Chronoamperometryofamarinebiofilmdevelopedovercarbontissue
polarizedat100mV/SCE.NM60:Naturalmedium(seawater)supplementedwith
60mMsodiumacetate.SMM60:Syntheticminimalmediumsupplementedwith
60mMsodiumacetate.ThebioanodewasfirstadaptedtoNM60withtwosuccessive
transferstofreshNM60;subsequentlyitwasrepetitivelytransferredtoSMM60.
Numberspointingwitharrowsindicatemomentswhencyclicvoltammograms
wererecorded.
Fig.3. Derivativeofcurrentdensitywithrespecttopotential(Dj/DE),forthe
oxidationturnoffasobtainedinCV,beyondOCV.TheCVswereperformedat
punctualmoments,asshownonFig.1(F1)andFig.2(F2).CV1orCV2correspondto
thepointsindicatedwitharrowsinthecorrespondingFig.1andFig.2.
Fig.4.CE-SSCPprofilesshowingthechangeofthemicrobialcommunitystructures.
Microbialcommunityexposedtonaturalmedium(F2nat)divergestothatobserved
fortheinoculum(control)andtheonefromexposedtosyntheticmedium(F2
3.4.Preservedelectrochemicalfunctionalityisnottheoutcomeoffully preservedmicrobialdiversity
CE-SSCPprofilespresentedinFig.4correspondtothesystem whoseelectrochemicalresponseisshowninFig.2andFig.3(F2). First,itcanbeappreciatedthatonlyafractionfromtheoriginal biofilmmixsuccessfullycolonizedthecarbonfeltelectrodeandis correlatedtotheelectrochemicalactivityobserved.Itcanalsobe appreciated that although the electrochemical functionality is preserved and even recuperatedat original levels(Fig.2)after transferof thebioanodetothesyntheticminimalmedium, this preservationandrestorationisnotassociatedtotheprevalenceof thesamemicrobialcommunity.Someofthepredominantstrains become extinct whereas others—poorly present at the primary stagewithnaturalmedium—boostafterbeingtransferredtothe minimalmedium.
Certainly, thepresence/absence andconcentrationof certain nutrimentssuchasnitrogenorcertainoligoelementscanfavour thegrowthand performanceofcertainmicrobes.However,it is here believed that the induced stress given by the change in nutrientmakeselectrontransfertowardsthecarbonelectrodethe predominantmechanismfor survival.Giventheconservation of theformoftheCVresponse,asobservedinFig.3,electrontransfer couldbeattributedtoapredominantelectrochemicalmechanism forthismicrobialpopulationbyeitherdirectelectrontransferorby entrapment of self-produced mediators in the extrapolymeric matrix.
3.5.Mildnutrientstressatthethinlinebetweeninactivationand intensificationbooststheeffectofminimalmedia
Bacterial stress can be defined as any deleterious physical, chemical or biological factor that induces modifications in the physiologyofbacteria[74].Adequatenutrientlevelswillpromote metabolicprogressionatamaximumrate,whichischaracteristic foreverymicrobe.Hence,variationsintheselevelsrepresentan environmental stress factor. In fact, in their natural habitat, conditionsthatallowformaximalmicrobialmetabolicratesare fewandfarbetween.Mostmarinebiofilmsarenaturallyadaptedto live in a constant state of stress, due to nutrient scarcity and abundance (sometimes upto the point of toxicity) [75]. Even, fluctuating or seasonalconditionsinvariably placethem in this “feastandstarvation”existence,withthe“hungerbetweenmeals” the morehabitual state [76,77].With this inmind, partof the present investigation consisted on simulating transition states betweenfeastandfamine,whichentailexposuretoan environ-ment inwhich bacteria arelimited for one or more classes of essential nutrients [78]; the in-between hunger state, would emphasize on bacterial scavenging for substrates to further stimulate nutrient acquisition. Of course, such strategy was combined with the standpoint of re-establishing the current generationfunctionalitydeterioratedinnaturalmedia,withthe useofminimalmedia,aspreviouslyexplained.
Anindependentelectrochemicalreactorstartedupinsimilar conditionsthanformerlydescribedforthesystemspresentedin Fig.1andFig.2,withadifferenttypeofcarbontissueassupport material.Theinitialconcentrationofacetatewas20mM(NM20)—
whichisfarfromlimiting.
Aftercurrentdecayofthefirstpeak,theexhaustedmediumwas removed (decanted)andconsecutivelyreplacedbyfreshnatural mediumwithincreasingconcentrationsofacetate:NM40,NM60
andNM80,respectively.Successivetransferstothesefreshnatural
medianegativelyimpactedoncurrentgeneration,evenwhenthe concentration of acetate was progressively increased. This is consistentwiththerationalethattherearedominatingimportant nutrimentaldeficienciesinnaturalseawater,asideofthecarbon
sourceneededbytheEA-bacteriatoproliferate.Itshouldbenoted that this behavior was observed independently of the type of carbonsupportused.
Acetateconcentrationwasperiodicallyfollowedforeachpeak ofcurrentproduction.Almostallacetatewasdepletedbythelapse ofeachtransfertofreshmedium.Still,currentdensitypeaksdid notintensifyaccordingly(Fig.5).Thismaybeexplainedbyeitherof two possibilities: a) the planktonic counterpart was largely consuming the substrate through a fermentative route; b) the biofilmatthesurfacebecomesmoreandmoredenselypopulated bynon-electrochemicallyactivemicroorganisms.
Byday21(Fig.5),currentdensitywaslowandstableatabout 0.25Am!2.Atsuchmoment,50%ofthevolumeofthemediumwas
removed (containing about 10mM of residual acetate), and replacedby freshseawater. This stepwas executed in order to address if an immediate dilution of the planktonic population wouldimproveelectricitygeneration.Currentdensity remained withoutprogressforthefollowing fourdays.Subsequently, the anode was re-transferred to fresh NM20, since these were the
conditionsinwhichtheEA-biofilmperformedbestduringalmost one month of experimentation. A slight improvement was observed with this action but, still, current density remained below0.5Am!2anddidnotresumetoitsinitialhighest.
Itwasanticipatedfrompreviousexperimentsthattransferring theEA-bioelectrodetoSMMwouldre-establishcurrent genera-tion. Still, in order todetermine if greater nutrimentalvariety (provided by yeast extract) would favorably impact current production as opposed todirect passageto non-supplemented SMM.ThemediumwasdecantedandfullyreplacedbySMM-Y20,
whereYreferstoSMMsupplementedwith0.4g/Lofyeastextract. AsobservedinFig.5 (days38–49)such enrichmentperformed almostinvariablewithrespecttoprecedingNM20,confirmingthat
nutrient-richerconditionsdonotspeciallyfavorcurrent produc-tion for this system. Additionally, yeast extract could have introducedadditionalelectronacceptors,thusdeviatingelectron transferfromtheanodetotheirelectron-mediatingstructures.
Afteralmost 49days of operation, the bioanode was finally transferredtoSMM20. Intheimmediate coupleof dayscurrent
densitystartedtovisiblyincreaseupto0.7Am!2.Whatismore,in
two consecutive transfers to SMM20 the system not only
re-establishedcurrent generation uptoabout 2.8Am!2and 3.4A
Fig.5.CAofmarinebiofilmdevelopedovercarbontissue.Thebioanodewasfirst
adaptedtoNM20withsuccessiveadditionsofacetateatdifferentconcentrations
(NM40,NM60andNM80).Subsequently,50%oftheexhaustedmediawasremoved
(&NM10remained, presumably)and theremnant was diluted1:1with fresh
seawater.Lateron,thefullexhaustedmediawasreplacedwithagainNM20.Further
thebioanodewastransferredtoSMM-Y20.Sincenocurrentimprovementwas
detectedafterthesestressfactors(&day48),thebioanodewasfinallysuccessively
m!2,respectively,butitsurpassedbymorethanthreetimesthe
current density initially obtained in NM20. An independent
replicatereactorshowedacomparabletrend.
An epifluorescence microscopy image is presented (Fig. 6), showingrepresentativebiofilmcolonizationoverthecarbontissue fibersatday67.Biofilmaggregationwas foundtobesignificant aroundthecarbon-fibers,withanestimatedthicknessofabout20– 30
m
m,whichistypicalinbioelectrochemicalsystems[79].Overthetypicallyusedcarbonfelt,thistypeofmarinebiofilmshave shown80–90
m
mthickness[80]innaturalmedia.3.6.Nutrimentalstressfactorsbyconditionsfluctuatingfromnatural toenrichedtosyntheticminimalmediumpreserveandboostcurrent generationfunctionality
CE-SSCPprofilespresentedinFig.7correspondtothesystem presentedinFig.5,attwodifferentstages(1,Stressnat,and4, Stresssynth).Ingeneral,thesamebehaviourshowninFig.4was observed.Onlyafractionfromtheoriginalbiofilmmixsuccessfully colonizedthecarbonfeltelectrode.Similarly,theelectrochemical functionalitywaspreservedandrecuperated,butinthiscasethe additional stressdue to fluctuating froma natural medium, to excessandfurther todeprivedminimalconditions,significantly improved current density generation. At least 7 peaks are preserved from the initial population developed in natural medium.SuchpeakswerealsopresentintheEA-biofilmsubject ofFig.4atalowerextent.Inbothcasessuchpeakswereenhanced when the bioelectrode was transferred to minimal media, as comparedtotheirsignificanceinnaturalmedia.Itispossiblethat despite of the overall changing microbial community a set of microorganisms which are truly electrochemically active over solid-statecarbonelectrodesaremuchfavouredbythenutrient
minimalconditionandevenfurtherwhensuchisprovidedafter nutrient-fluctuatingstressconditions.
3.7.Implicationsoftheseresults
The results here disclosed reveal a simple, yet innovative, strategy to boost current generation in independent microbial anodes for electrochemical systems. Development of microbial biofilms for effective electro-bioprocesses must be robust and capabletoworkwithminimalrequirements;besides,theyshould displayhighfunctionalityandreproducibilitywhenoperatingin normalcapacityandbeabletoendureperiodicstressconditions. Onlysuchtypesofbiofilmscouldbecomenotonlyindustrialized butpolyvalentfordifferenttypesofBESandexpandtheiruseto newareasandmarkets.TheEA-biofilmsherestudiedpresented thesecharacteristics whensubject tothedeveloped procedure. However,this firstlong-termresearchofhalotolerantmicrobial anodescouldbestillimproved,playingwithseasonalvariationson the original inoculum or shifting batch to continuous mode operation. Improving theoverall current generatingcapacity of theserobustandversatilebioanodesiscritical.Futureworkmay considercontinuous operationand identification ofthe param-eters that will improve current densities, such as nature and concentrationofthecarbonsource,electrodematerial,and start-up electrode potential, osmotic and hydrodynamic conditions, amongothers.Furtherelucidationofmechanisticimplicationsof howEA-biofilmscopewithnutrimentalstresswouldalsoadvance thisscientificfield.
Finally,itisrelevanttostatethatreplicateexperimentsreferred inthismanuscriptshowedthesametrendthantheindependent experiments hereshown, even when different types of carbon support materials and different substrate concentrations were employed.Variabilityinthemagnitudesofcurrent densitywas encountered.Still,suchvariabilitydidnothaveanimpactinthe mainfindings:
1. EA-marineanodesprogressivelydeteriorateinnaturalmarine media supplemented with acetate, not in terms of biomass growthbutintheirfunctionalitytogeneratesustainedcurrent. 2. Theuseofaminimalsyntheticfeedsupplementedwithacetate, re-establishescurrentgenerationcapacitiesofthemarine EA-marineanodesthatformerlydeterioratedinnaturalmedia. 3.The overallelectrontransfer mechanismsfor oxidation
reac-tions arepreservedwhen passing fromnatural toa suitable syntheticmedia,asevidencedfromtheCVresponses. 4.Progressively-inducedmildnutrientstresswhiletheEA-biofilm
deteriorates in natural media not only restores current generationwhentransferringtominimalmedia,butalsoboosts itsinfluenceleadingtounprecedentedcurrentdensitiesforan individualbioanode.
4. Conclusions
Electrochemicallyactivebiofilmsweredevelopedinsynthetic minimalmediashowingeffectiveperformanceintermsofcurrent density(e.g.upto3.8Am!2to6.8Am!2),whencomparedtotheir
deteriorated performance in natural media (e.g. wherein the current densitydropped below 0.1Am!2
).Even,when nutrient stresswasinduced,theuseofsyntheticminimalmedianotonly restoredbutboostedcurrentdensity.
The use of minimal culture media should carefully be consideredfor industrializedMETs, especiallyin whatconcerns microbial electrolysis for hydrogen generation and microbial electrosynthesisforproductorganics,asthesewouldpermitmore straightforwarddownstreamprocessing.
Fig.6.3-DStructureofanhalotolerantanodicbiofilmcolonizationovercarbon
tissuefibres,after67daysofpolarizationat100mv/SCE,asshowninFig.4.Biofilm
aggregation(green)isperceivedaroundthecarbontissuefibers(yellow).
Fig. 7. CE-SSCP profiles showing the changes on the microbial community
structure. The microbialcommunities exposedto natural media (Stress nat)
divergefromthoseobservedfortheoriginalinoculumsource(control)andtheone
Changefromnaturaltominimalmediahasanimportantimpact on the selection and adaptation of microbial communities; however, robust bioanodes containing some preserved species wereobtainedinsyntheticminimalmediaandshowed reproduc-ibleelectrochemicalfunctionality.
Acknowledgment
This work was partof the “DéfiH12” project funded by the FrenchNationalResearchAgency(ANR-09-BioE-010).
References
[1]X.Dominguez-Benetton,S.Sevda,K.Vanbroekhoven,D.Pant,Theaccurateuse ofimpedanceanalysisforthestudyofmicrobialelectrochemicalsystems, Chem.Soc.Rev.41(2012)7228–7246.
[2]B.E.Logan,J.M.Regan,Microbialfuelcells–challengesandapplications, En-viron.Sci.Technol.40(2006)5172–5180.
[3]B.Erable,L.Etcheverry,A.Bergel,Frommicrobialfuelcell(MFC)tomicrobial electrochemicalsnorkel(MES):maximizingchemicaloxygendemand(COD) removalfromwastewater,Biofouling27(2011)319–326.
[4]S. Sevda, X. Dominguez-Benetton, K. Vanbroekhoven, H. De Wever,T.R. Sreekrishnan,D.Pant,Highstrengthwastewatertreatmentaccompaniedby powergenerationusingaircathodemicrobialfuelcell,Appl.Energy105(2013) 194–206.
[5]X.Cao,X.Huang,P.Liang,K.Xiao,Y.Zhou,X.Zhang,B.E.Logan,Anewmethod forwaterdesalinationusingmicrobialdesalinationcells,Environ.Sci.Technol. 43(2009)7148–7152.
[6]M.Sharma,N.Aryal,P.M.Sarma,K.Vanbroekhoven,B.Lal,X. Dominguez-Benetton,D.Pant,Bioelectrocatalyzedreductionofaceticandbutyricacidsvia directelectron transferusinga mixedculture ofsulfate-reducers drives electrosynthesisofalcoholsandacetone,Chem.Commun.49(2013)6495– 6497.
[7]J.M.Flynn,D.E.Ross,K.A.Hunt,D.R.Bond,J.A.Gralnick,Enablingunbalanced fermentations byusing engineeredelectrode-Interfaced bacteria, MBio1 (2010)e00190–10.
[8]K.Rabaey,P.Girguis,L.K.Nielsen,Metabolicandpracticalconsiderationson microbialelectrosynthesis,Curr.Opin.Biotechnol.22(2011)371–377. [9]K.Rabaey,R.A.Rozendal,Microbialelectrosynthesis—revisitingtheelectrical
routeformicrobialproduction,Nat.Rev.Microbiol.8(2010)706–716. [10]Y.F.Choo,JiyoungLee,InSeopChang,ByungHongKim,Bacterialcommunities
inmicrobialfuel cellsenriched withhigh concentrationsofglucose and glutamate,J.Microbiol.Biotechnol.16(2006)1481–1484.
[11]S. Sevda, X. Dominguez-Benetton, K. Vanbroekhoven, H. De Wever,T.R. Sreekrishnan,D.Pant,Highstrengthwastewatertreatmentaccompaniedby powergenerationusingaircathodemicrobialfuelcell,Appl.Energy105(2013) 194–206.
[12]P.D.Kiely,R.Cusick,D.F.Call,P.A.Selembo,J.M.Regan,B.E.Logan,Anode microbialcommunitiesproducedbychangingfrommicrobialfuel cellto microbialelectrolysiscelloperationusingtwodifferentwastewaters, Bio-resour.Technol.102(2011)388–394.
[13]Z.Du,H.Li,T.Gu,Astateoftheartreviewonmicrobialfuelcells:apromising technologyfor wastewatertreatmentand bioenergy,Biotechnol.Adv.25 (2007)464–482.
[14]B.Min,J.Kim,S.Oh,J.M.Regan,B.E.Logan,Electricitygenerationfromswine wastewaterusingmicrobialfuelcells,WaterRes.39(2005)4961–4968. [15]B.H.Kim,H.S.Park,H.J.Kim,G.T.Kim,I.S.Chang,J.Lee,N.T.Phung,Enrichment
ofmicrobialcommunitygeneratingelectricityusingafuel-cell-type elec-trochemicalcell,Appl.Microbiol.Biotechnol.63(2004)672–681.
[16]H.Liu,B.E.Logan,Electricitygenerationusinganair-Cathodesinglechamber microbialfuelcellinthepresenceandabsenceofaprotonexchange mem-brane,Environ.Sci.Technol.38(2004)4040–4046.
[17]L.T.Angenent,K.Karim,M.H.Al-Dahhan,R.Domiguez-Espinosa,Productionof bioenergyand biochemicalsfrom industrial andagricultural wastewater, TrendsBiotechnol.22(2004)477–485.
[18]B.C.Jong,B.H.Kim,I.S.Chang,P.W.Y.Liew,Y.F.Choo,G.S.Kang,Enrichment performance,andmicrobialdiversityofathermophilicmediatorlessmicrobial fuelcell,Environ.Sci.Technol.40(2006)6449–6454.
[19]N.T.Phung,J.Lee,K.H.Kang,I.S.Chang,G.M.Gadd,B.H.Kim,Analysisof microbial diversity in oligotrophic microbial fuel cells using 16S rDNA sequences,FEMSMicrobiol.Lett.233(2004)77–82.
[20]D.E.Holmes,D.R.Bond,R.A.O'Neil,C.E.Reimers,L.R.Tender,D.R.Lovley, Microbialcommunitiesassociatedwithelectrodesharvestingelectricityfrom avarietyofaquaticsediments,Microb.Ecol.48(2004)178–190.
[21]R. Rousseau,X.Dominguez-Benetton,M.L.Délia,A.Bergel,Microbial bio-anodeswithhigh salinitytoleranceformicrobialfuelcells andmicrobial electrolysiscells,Electrochem.Commun.33(2013)1–4.
[22]M.Helder,D.P.B.T.Strik,H.V.M.Hamelers,A.J.Kuhn,C.Blok,C.J.N.Buisman, Concurrentbio-electricityandbiomassproductioninthreePlant-Microbial Fuel CellsusingSpartinaanglica, Arundinellaanomala andArundo donax, Bioresour.Technol.101(2010)3541–3547.
[23]R.A.Timmers,D.P.B.t.B.Strik,H.V.M.Hamelers,C.J.N.Buisman,Long-term performanceof a plant microbialfuel cell with Spartina anglica,Appl. Microbiol.Biotechnol.86(2010)973–981.
[24]S.B.Velasquez-Orta,I.M.Head,T.P.Curtis,K.Scott,Factorsaffectingcurrent production inmicrobialfuel cellsusing differentindustrialwastewaters, Bioresour.Technol.102(2011)5105–5112.
[25]B.Erable,N.M.DuÛeanu,M.M.Ghangrekar,C.Dumas,K.Scott,Applicationof electro-activebiofilms,Biofouling26(2010)57–71.
[26]R.A.Rozendal,H.V.M.Hamelers,K.Rabaey,J.Keller,C.J.N.Buisman,Towards practicalimplementationofbioelectrochemicalwastewatertreatment,Trends Biotechnol.26(2008)450–459.
[27] C.E.Reimers,H.A.StecherIII,J.C.Westall,Y.Alleau,K.A.Howell,L.Soule,H.K. White,P.R.Girguis,Substratedegradationkinetics,microbialdiversity,and currentefficiencyofmicrobialfuelcellssuppliedwithmarineplankton,Appl. Environ.Microbiol.73(2007)7029–7040.
[28]D.R.Bond,D.E.Holmes,L.M.Tender,D.R.Lovley,Electrode-reducing micro-organismsthatharvestenergyfrommarinesediments,Science295(2002) 483–485.
[29]L.M.Tender,C.E.Reimers,H.A.StecherIII,D.E.Holmes,D.R.Bond,D.A.Lowy,K. Pilobello,S.J.Fertig,D.R.Lovley,Harnessingmicrobiallygeneratedpoweron theseafloor,Nat.Biotechnol.20(2002)821–825.
[30]L.D.Munoz,B.Erable,L.Etcheverry,J.Riess,R.Bassguy,A.Bergel,Combining phosphatespeciesandstainlesssteelcathodetoenhancehydrogenevolution inmicrobialelectrolysiscell(MEC),Electrochem.Commun.12(2010)183–186. [31] B.Erable,A.Bergel,Firstair-toleranteffectivestainlesssteelmicrobialanode obtainedfromanaturalmarinebiofilm,Bioresour.Technol.100(2009)3302– 3307.
[32]I. Vandecandelaere,O. Nercessian, M.Faimali,E. Segaert, A.Mollica, W. Achouak,P.DeVos,P.Vandamme,Bacterialdiversityofthecultivablefraction ofamarineelectroactivebiofilm,Bioelectrochemistry78(2010)62–66. [33]D.R.Lovley,Themicrobeelectric:conversionoforganicmattertoelectricity,
Curr.Opin.Biotechnol.19(2008)564–571.
[34]C.Dumas,A.Mollica,D.Feron,R.Basseguy,L.Etcheverry,A.Bergel,Checking graphiteandstainlessanodeswithanexperimentalmodelofmarine mi-crobialfuelcell,Bioresour.Technol.99(2008)8887–8894.
[35]Z.He,H.Shao,L.T.Angenent,Increasedpowerproductionfromasediment microbialfuelcellwitharotatingcathode,Biosens.Bioelectron.22(2007) 3252–3255.
[36]D.R.Lovley,Bugjuice:harvestingelectricitywithmicroorganisms,Nat.Rev. Microbiol.4(2006)497–508.
[37] R.Rousseau,X.Dominguez-Benetton,M.L.Délia,A.Bergel,Microbial bio-anodeswithhighsalinitytoleranceformicrobialfuelcellsandmicrobial electrolysiscells,Electrochem.Commun.33(2013)1–4.
[38]D.E.Holmes,D.R.Bond,R.A.O'Neil,C.E.Reimers, L.R.Tender,D.R.Lovley, Microbialcommunitiesassociatedwithelectrodesharvestingelectricityfrom avarietyofaquaticsediments,Microb.Ecol.48(2004)178–190.
[39]I. Vandecandelaere,O. Nercessian, M.Faimali,E. Segaert, A.Mollica, W. Achouak,P.DeVos,P.Vandamme,Bacterialdiversityofthecultivablefraction ofamarineelectroactivebiofilm,Bioelectrochemistry78(2010)62–66. [40]X. Dominguez-Benetton, S. Navarro-Avila, C. Carrera-Figueiras,
Electro-chemicalevaluationofTi/TiO2-polyanilineanodes formicrobialfuel cells
usinghypersalinemicrobialconsortiaforsynthetic-wastewatertreatment,J. NewMater.Electrochem.Syst.13(2010)1–6.
[41]B.O.Ortega-Morales,M.J.Chan-Bacab,S.d.C.DelaRosa-Garcia,J.C. Camacho-Chab,Valuable processes andproducts from marineintertidal microbial communities,Curr.Opin.Biotechnol.21(2010)346–352.
[42]R.Rousseau,X.Dominguez-Benetton,M.L.Délia,A.Bergel,Microbial bio-anodeswithhighsalinitytoleranceformicrobialfuelcellsandmicrobial electrolysiscells,Electrochem.Commun.33(2013)1–4.
[43]D.Y.Huang,S.G.Zhou,Q.Chen,B.Zhao,Y.Yuan,L.Zhuang,Enhancedanaerobic degradationoforganicpollutantsinasoilmicrobialfuelcell,Chem.Eng.J.172 (2011)647–653.
[44]A.Esteve-Nuñez,A.Reija, F.Moreno-Garzon,B.Barroeta, A.Aguilera,J.P. Busalmen,J.Martin,I.Ortiz-Bernad,Harvestingbioelectricityfromnatural environments:microbialfuelcellfuelledbyriceplants,NewBiotechnol.25 (2009)S280.
[45]L.M.Tender,S.A.Gray,E.Groveman,D.A.Lowy,P.Kauffman,J.Melhado,R.C. Tyce,D.Flynn,R.Petrecca,J.Dobarro,Thefirstdemonstrationofamicrobial fuelcellasaviablepowersupply:poweringameteorologicalbuoy,J.Power Sour.179(2008)571–575.
[46]B.Tartakovsky,P.Mehta,J.S.Bourque,S.R.Guiot,Electrolysis-enhanced an-aerobicdigestionofwastewater,Biores.Technol.102(2011)5685–5691. [47] Y.Ahn,B.E.Logan, Effectivenessofdomesticwastewatertreatmentusing
microbialfuelcellsatambientandmesophilictemperatures,Biores.Technol. 101(2010)469–475.
[48]S.A.Patil,F.Harnisch,B.Kapadnis,U.Schröder,Electroactivemixedculture biofilmsinmicrobialbioelectrochemicalsystems:theroleoftemperaturefor biofilmformationandperformance,Biosens.Bioelectron.26(2010)803–808. [49]S.Freguia,E.H.The,N.Boon,K.M.Leung,J.Keller,K.Rabaey,Microbialfuelcells
operatingonmixedfattyacids,Biores.Technol.101(2010)1233–1238. [50]B. Cercado-Quezada, M.L. Délia, A. Bergel, Testing various food-industry
wastesforelectricityproductioninmicrobialfuelcell,Biores.Technol.101 (2010)2748–2754.
[51]H.W.Paerl,J.L.Pinckey,Amini-reviewofmicrobialconsortia:theirrolesin aquaticproductionandbiogeochemicalcycling,Microbial.Ecol.31(1996) 225–247.
[52]P. Landini,Cross-talkmechanisms inbiofilmformationandresponses to environmentalandphysiologicalstressinEscherichiacoli,Res.Microbiol.160 (2009)259–266.
[53]M.Madigan,J.Martinko(Eds.),PrenticeHall,BrockBiologyofMicroorganisms, 2003.
[54]A.A.Carmona-Martinez,F.Harnisch,L.A.Fitzgerald,J.C.Biffinger,B.R. Ring-eisen,U.Schröder,Cyclicvoltammetricanalysisoftheelectrontransferof ShewanellaoneidensisMR-1and nanofilamentandcytochrome knock-out mutants,Bioelectrochemistry81(2011)74–80.
[55]S.R.Higgins,D.Foerster,A.Cheung,C.Lau,O.Bretschger,S.D.Minteer,K. Nealson, P. Atanassoy, M.J. Cooney, Fabrication ofmacroporous chitosan scaffoldsdopedwithcarbonnanotubesandtheircharacterizationinmicrobial fuelcelloperation,EnzymeMicrob.Technol.48(2011)458–465.
[56]M.A.Rosenbaum,H.Y.Bar,Q.K.Beg,D.Segrè,J.Booth,M.A.Cotta,L.T.Angenent, Shewanellaoneidensisinalactate-fedpure-cultureandaglucose-fed co-culturewithLactococcuslactiswithanelectrodeaselectronacceptor,Biores. Technol.102(2011)2623–2628.
[57]F.Qian,Z.He,M.P.Thelen,Y.Li,Amicrofluidicmicrobialfuelcellfabricatedby softlithography,Biores.Technol.102(2011)5836–5840.
[58]N.Uria,D.Sanchez,R.Mas,F.X.Muñoz,J.Mas,Effectofthecathode/anoderatio andthechoiceofcathodecatalystontheperformanceofmicrobialfuelcell transducersforthedeterminationofmicrobialactivity,Sens.Actuat.B:Chem. 170(2012)86–94.
[59]A.Kloke,S.Rubenwolf,C.Bücking,J.Grescher,S.Krezenmacher,R.Zengerle,F. Von Stetten, Aversatile miniature bioreactorandits application to bio-electrochemistrystudies,Biosens.Bioelectron.25(2010)2559–2565. [60]A.V.Samrot,P.Senthilkumar,K.Pavankumar,G.C.Akilandeswari,N.
Raja-lakshmi,K.S.Dhathathreyan,ElectricitygenerationbyEnterobactercloacae SU-1inmediatorlessmicrobialfuelcell,Int.J.HydrogenEnergy35(2010)7723– 7729.
[61]S.Freguia,M.Masuda, S.Tsujimura,K.Kano,Lactococcuslactiscatalyses electricitygenerationatmicrobialfuelcellanodesviaexcretionofasoluble quinone,Bioelectrochemistry76(2009)14–18.
[62]V.R.Nimje,C.Y.Chen,C.C.Chen,J.S.Jean,A.S.Reddy,C.W.Fan,K.Y.Pan,H.T.Liu, J.L.Chen,StableandhighenergygenerationbyastrainofBacillussubtilisina microbialfuelcell,J.PowerSour.190(2009)258–263.
[63]A.K.Manohar,F.Mansfeld,Theinternalresistanceofamicrobialfuelcelland itsdependenceoncelldesignandoperatingconditions,Electrochim.Acta54 (2009)1664–1670.
[64]A.K.Manohar,O.Bretschger,K.H.Nealson,F.Mansfeld,Thepolarization be-havioroftheanodeinamicrobialfuelcell,Electrochim.Acta53(2008)3508– 3513.
[65]S.A.Patil,F.Harnisch,B.Kapadnis,U.Schröder,Electroactivemixedculture biofilmsinmicrobialbioelectrochemicalsystems:theroleoftemperaturefor biofilmformationandperformance,Biosens.Bioelectron.26(2010)803–808.
[66]B.Erable,A.Bergel,Firstair-toleranteffectivestainlesssteelmicrobialanode obtainedfromanaturalmarinebiofilm,Biores.Technol.100(2009)3302– 3307.
[67]D.Pocaznoi,B.Erable,L.Etcheverry,M.L.Delia,A.Bergel,Formingmicrobial anodesunderdelayedpolarisationmodifiestheelectrontransfernetworkand decreasesthepolarisationtimerequired,Biores.Technol.114(2012)334–341. [68]R.L.Starkey,Astudyofsporeformationandothermorphological
character-isticsofVibriodesulfuricans,Arch.Mikrobiol.9(1938)268–304.
[69]J.J.Godon,E.Zumstein,P.Dabert,F.Habouzit,R.Moletta,Molecularmicrobial diversityofananaerobicdigestorasdeterminedbysmall-subunitrDNA se-quenceanalysis,Appl.Environ.Microbiol.63(1997)2802–2813.
[70]C.Delbès,R.Moletta,J.J.Godon,Monitoringofactivitydynamicsofan an-aerobicdigester bacterialcommunity using 16S rRNA polymerasechain reaction–single-strand conformation polymorphism analysis, Environ. Microbiol.2(2000)506–515.
[71]N.Wery,V.Bru-Adan,C.Minervini,J.P.Delgenes,L.Garrelly,J.J.Godon, Dy-namicsofLegionellaspp.andbacterialpopulationsduringtheproliferationof L.pneumophilainacoolingtowerfacility,Appl.Environ.Microbiol.74(2006) 3030–3037.
[72]R.J.Michelland,S.Dejean,S.Combes,L.Lamothe,L.Cauquil,StatFingerprints:a friendlygraphicalinterfaceprogramforprocessingandanalysisofmicrobial fingerprintprofiles,Mol.Ecol.Resour.9(2009)1359–1363.
[73]B.Erable,A.Bergel,Firstair-toleranteffectivestainlesssteelmicrobialanode obtainedfromanaturalmarinebiofilm,Biores.Technol.100(2009)3302– 3307.
[74]L.A.Rodriguez-Romo,A.E.Yousef,InactivationofSalmonellaentericaserovar EnteritidisonshelleggsbyozoneandUVradiation,J.FoodProtect68(2005) 711–717.
[75]A.G.Moat,J.W.Foster,M.P.Spector(Eds.),Microbialphysiology,Ed.,WileyLiss Inc.,2002.
[76]T.Ferenci,Hungrybacteria–definitionandpropertiesofanutritionalstate, Environ.Microbiol.3(2001)605–611.
[77]A.L.Koch,TheadaptiveresponsesofEscherichiacolitoafeastandfamine existence,Adv.Microb.Physiol.6(1971)147–217.
[78]T.Ferenci,Hungrybacteria–definitionandpropertiesofanutritionalstate, Environ.Microbiol.3(2001)605–611.
[79]X.Dominguez-Benetton,S.Sevda,K.Vanbroekhoven,D.Pant,Theaccurateuse ofimpedanceanalysisforthestudyofmicrobialelectrochemicalsystems, Chem.Soc.Rev.41(2012)7228–7246.
[80]B.Erable,A.Bergel,Firstair-toleranteffectivestainlesssteelmicrobialanode obtainedfromanaturalmarinebiofilm,Biores.Technol.100(2009)3302– 3307.
