The role of EPS in the foaming and fouling for a MBR operated in intermittent aeration conditions

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Biochemical

Engineering

Journal

j ou rn a l h o m ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / b e j

Regular

article

The

role

of

EPS

in

the

foaming

and

fouling

for

a

MBR

operated

in

intermittent

aeration

conditions

Riccardo

Campo

_,

_Marco

_Capodici

_,

_Gaetano

_Di

_Bella

_,

_Michele

_Torregrossa

b_{DipartimentodiIngegneriaCivile,UniversitàdegliStudidiPalermo,Ambientale,Aerospaziale,deiMateriali,ScuolaPolitecnica,VialedelleScienzeed8,}

90128Palermo,Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received18April2016

Receivedinrevisedform29August2016 Accepted16November2016

Availableonline18November2016 Keywords: IA-MBR Filamentousbacteria Foaming Fouling EPS

a

b

s

t

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a

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ThisworkinvestigatesthecausesoffoamingandfoulinginanIntermittentAerated–Membrane BioRe-actor(IA-MBR)usedforwastewatertreatment.Theexperimentwasdividedintothreeperiodswith differentaeratedregimesexpressedwithdifferenttaeration/tcycleratio(PeriodI:60min/180min,PeriodII: 80min/180min,PeriodIII:30min/90min).

Theadvancedfoamingtestsusedallowthestudyoftheroleofextracellularpolymericsubstances (EPSs)onfoamingandfouling.Ingeneral,inthePeriodsIandII,goodcorrelationsbetweentheEPSs andtheModifiedScumIndexwithoutpurification(MSI0)andtheFoamPowerwereobserved.Theresults shownthatthefilamentousmicroorganisms,whichmainlygrewinthePeriodIII,playalsoakeyfactorin thefoamingandfouling.Inparticular,whenanetproliferationoffilamentousbacteriaoccurred(during periodIIIwithahighernumberofcyclesperday)bothEPSsconcentrationandfilamentousabundance influencethemodifiedscumindexobtainedaftertwopurificationsteps(MSI2).Finally,theprocessing datashowthattheEPSshamperedalsothemembranefiltrationbutimprovethepre-filtrationactionof cakelayer.Ontheotherhand,theco-presenceofEPSsandfilamentousbacteriareducedtheeffectocake layeraspre-filter.

©2016ElsevierB.V.Allrightsreserved.

1. Introduction

Membrane Bioreactors (MBRs) represent nowadays a

well-consolidatedadvanced available technologyin orderto comply

withstringentmandatoryrequirementsintermsofquality

stan-dardsof treated wastewater.Indeed, MBRsystems guarantee a

highereffluentqualityalsointermsofpathogenicbacteriaremoval,

alowerecologicalfootprintandalowersludgeproductionif

com-paredtoaconventionalactivatedsludge(CAS)system.However,

thegreatestdownsideofMBRsisrepresentedbytheoccurrence

offouling andfoaming phenomena[1].Membrane fouling

con-sistsof thedepositionof particlesonthemembrane surfaceor

colloidalparticleswithintheporesofthemembrane.Thetangible

consequence of fouling is representedby an increase of

trans-membranepressure(TMP)yieldingareductionofpermeabilityand

thusadecreasingeffluentflow[1].Fourgroupsoffactorsaffect

membranefouling:membrane materials,mixedliquor features,

influentwastewatercompositionandoperatingconditionssuchas

∗ Correspondingauthor.

E-mailaddress:[email protected](G.DiBella).

sludgeretentiontime (SRT),hydraulicretentiontime(HRT)and

foodtomicroorganismratio(F/M)[2–4].Particularly,the

mem-branefoulingisstronglyinfluencedbymixedliquorfeatures.More

indetail,amongothersparameters,sludgeviscosity,mixedliquor

suspendedsolids(MLSS)concentration,presenceandabundanceof

filamentousbacteria,extracellularpolymericsubstances(EPSs)and

solublemicrobialproducts(SMPs)concentrationsarerecognized

tobethekeyfactorsaffectingthemembranefouling[5].Despite

manyauthorsoutlinedthatmembranefoulingispromotedbyhigh

EPSconcentrations[6,7],thereisstilluncertaintywhetherfouling

resultsmainlyinfluencedbyproteinsorpolysaccharidesfractionof

EPSandSMP.Suchuncertaintyislikelyduetodifferencesin

exper-imentalmethodsavailableforEPScontentassessmentandalsoto

thedifferentconfigurationofMBRtreatmentplants[3,8].

Ontheotherhand,thefoamingrepresents,aswellasfouling,

anotherdownsideofMBRsthatcanleadtoasignificantdecreasing

ofbiologicalperformancesofMBRsandalsotoadecreasein

mem-branefiltrationefficiency[9].Foamingarisesasaviscouslayeron

thesurfaceofwastewatertreatmentplantreactors;itresultsin

afloatingbiomassthatcancauseseriousoperationalproblemsto

thetreatmentprocess.In particular,itispossibletodistinguish

two differentkindof foaming:biologicalfoamingand chemical

http://dx.doi.org/10.1016/j.bej.2016.11.012

foaming.Thebiologicalfoamingtriggerfactorisrecognizedtobe

theunbalancedF/Mratio;whilethechemicalfoamingisattributed

toa loadingshock caused by thepresence of synthetic

surfac-tants[10].Amongthevariouskindofmicroorganismstraceable

intheactivatedsludge,a significantpresenceof Microthrix

par-vicellaandNocardioformshasbeenobservedusingconventional

microscopyduringbiological foamingoccurrence.For such

rea-son,theaforementionedmicroorganismshavebeendefined“foam

formingbacteria”[11].Furthermore,otherstudiesoutlinedalsothe

presenceofotherbacteriaspeciesduringfoamingoccurrence.For

thisreason,bacterialstrainssuchastheEikelboomType0092have

beendefined“notfamousfoam-formingbacteria”[9,11].

DespitethefoaminginCASisattributedtotheabundanceof

theaforementionedstrains,ithastobestressedthatfoamingin

MBRsystemscanalsooccurintheabsenceof“foam-forming”or

“notfamousfoam-forming” microorganisms. In these casesthe

foamabundancewasattributedtotheconcentrationoftheprotein

fractionofEPS[12].However,bothEPSsandfilamentousbacteria

influencethesludgehydrophobicityandthereforeimpacton

foam-ingoccurrence. Although thefoaming occurrence canseriously

affectthecorrectworkingofaMBR,studiesdealingwithfoamingin

MBRsarescarce[9,12,13].Furthermore,foamingoccurrencecanbe

encouragedbytheimplementationofnewmanagementstrategies

suchastheIntermittentAeration(IA).Althoughsuchprocesscan

offerseveraladvantages[14,15],theintermittentaerationimpact

onmicroorganismscanresultinaproliferationoffoam-forming

microorganismssuchasfilamentousbacteria.Indetails,theIA

pro-cessconsistsofalternatingaerobicandanoxicphaseswithinthe

samestirred reactor.The implementationofsuchmanagement

strategyallowsachievingtheammonia oxidationaswellasthe

nitratereductionsavingenergy.Indeed,theairsupplynecessary

tothebioreactorresultdecreasedandalsotheabsenceofreturn

activatedsludgerichinnitratesfromnitrificationtodenitrification

yieldtheIAacosteffectivesolution[15].However,thepresence

oftheanoxicphasecanseriouslyimpactinthestrains

develop-mentfavoringtheproliferationoffoam-formingmicroorganisms

likefilamentousbacteria[14].Bearinginmindtheprevious

obser-vations,and considering that extensivestudies onIntermittent

Aerated-MembraneBioReactor(IA-MBR)arelacking,thepresent

studyaimstoinvestigatethefoulingandfoamingmechanismsinan

IA-MBRsystemtreatingdomesticwastewater.Specificfoamtests

andadetailedfoulingtendencyassessmenthavebeencarriedout

inordertooutlinetherootsofbothphenomenainIA-MBRsystems.

Furthermore,adetailedinvestigationfocusedtohighlightthe

influ-enceexertedbyotherparameters,suchasEPSandSMPfractions

andconcentration,sludgeviscosity,TSSconcentrationand

filamen-tousmicroorganisms’abundancehasbeenreported,improvingthe

knowledgeaboutthesetwodrawbacksofthisnewandinnovative

MBRconfiguration.

2. Materialandmethods

2.1. Experimentalsetup

ThepilotplantwasbuiltatPalermomunicipalwastewater

treat-mentPlantof“AcquadeiCorsari”anditwasthesameasthatused

byCapodicietal.[15].Inparticular,theIAreactor(400L),theMBR

(50L)tankandtheOxygenDepletionReactor(ODR)(28L),mainly

composedthepilotplant.Thefirstonewasprovidedwith

aera-tionandstirringdevices;theMBRreactorwasdownstreamtheIA

reactoranditwascontinuouslyaeratedinordertomitigatethe

membranefouling;theODRreactorwascontinuouslystirredin

ordertoavoidTSSsettling.AlogicalschemeofthePilotplantis

showninFig.1.

Fig.1. PilotPlantLay-out.

Throughoutalltheexperimentalperiod,thefollowingmost

rel-evantoperationalparametersweremeasured:DissolvedOxygen

(D.O.),pH,OxidationReductionPotential (O.R.P.).Theywereall

monitoredon-linebymeansofprobesappropriatelylocatedinthe

differentsectionsofthepilotplant[15].

Thetemperaturewasnot regulatedandwasaffectedbythe

environmentalconditions(roomtemperature):however,the

vari-ationwasrelativelysmall,changingfrom18to24◦Cduringthe

experimentalperiod.

TheMBRcompartmentwasequippedwithtwoultrafiltration

(UF)hollowfiberGEZenonZW10®modules(nominalsurfaceof

singlemembrane unit:0.93m2_{; pore dimension:0.04␮m)in a}

submergedconfiguration.

Thedurationsofsuctionandbackwashweresetto9and1min,

respectively,withatotalfiltrationcycleof10min.Theplantwas

fedcontinuouslyandthepermeatefluxfromeachmembranewas

equalto20–23Lm−2h−1;whilethenetpermeatefluxwasequal

to17–19Lm−2h−1.

Thetransmembranepressure(TMP)wasmeasuredbymeans

of analogical vacuum-meters connected to the upper heads of

the membrane modules. TMP data were acquired with two

data-loggers connected tothevacuum-meters,stored and then

post-processed.

Theexperimentalcampaign,whichlasted120days,wasdivided

intothreeperiodswithdifferentaerationregimes[15].Indetails,

Period I (lasted 57days) had a 180min cycle length (tcycle)

with60minofaeration (taeration)and120minwithoutaeration

(taeration/tcycle=60/180).InperiodII(lasted47days)thecyclelength

wasthesameasthatofperiodIandthetaeration/tcycleratiowas

equalto80/180.Finally,periodIII(lasted16days)was

character-izedwithacyclelengthof90minwithtaeration/tcycle=30/90.During

thenot-aeratedphase,themixedliquorwascontinuouslymixedto

avoidSuspendedsolidssettling.Themainoperationalparameters

arereportedinTable1.

Biochemicaloxygendemand(BOD5),chemicaloxygendemand

(COD), soluble COD, ammonium (NH4-N), nitrate ammonium

(NO3-N),nitriteammonium(NO2-N),totalsuspendedsolids(TSS)

andvolatilesuspendedsolids(VSS)weremeasuredthreetimesper

weekinordertoevaluatethepilotplantperformance,accordingto

StandardMethods[16].

2.2. Foamingtestandfilamentousinvestigations

Proposedfoamtestsandcharacterizationscanbecategorized

as:

1)Qualitytest:Foamingrateandfoampower;

Table1

Mainoperationalparameters.

Period n◦cycles/day Ox/Cycle[min/min] Anox/Cycle[min/min] F/M[kgBOD5kg−1VSSd−1] COD[gm−3] NH4-N[gm−3] MLSS[kgm−3]

I 8 60/180 120/180 0,08±0.02 380±48 29±5 ∼2.7

II 8 80/180 100/180 0,06±0.01 483±25 23±3 ∼4.3

III 16 30/90 60/90 0,05±0.01 414±34 19±5 ∼3.7

3)Microscopic observation: Filamentous bacteria identification andabundance.

Generally, foam quality is measured on site by meansof a specificapparatus.Todefinethefoam stabilityand “geometric” characteristics(thicknessandfeatures),theFoamingRate (FMR) is used according to Blackall’s method [17]. The FMR value is

measuredbypouring50mLofactivatedsludgeintoa40mm

diam-eter×500mmhighglasscylinderandbubblinggasthroughitvia

a40–90␮mporesizediffuserat200mLmin−1.

Foamcharacteristicsarerecordedintermsofitsvolume,bubble

size,speedofformationandtimetakenforthefoamtocollapseafter

aerationhasceased(Table2).

FoamPower(FP)evaluatesthefoampotential/stabilityofMBR

activatedsludgeinaccordancewiththeconceptsusedinfood

sci-ence.FPisdefinedas“theconsumedsamplevolume(mL)from

foamingby1Lofaeration”[12].100mLofactivatedsludgesample

isplacedinatransparent7cm2_{cross-section(A)verticalcylinder}

andaeratedfor30s(T)atanairflow(QAir)of5Lmin−1.TheFPis

thendeterminedfromthedifferenceintheinterfacelevelsofthe

sampleliquidandfoambeforeandafteraeration(H0):

FP= A·Ho

T·QAir

(1)

Whilesomewhatrudimentary,FMRmeasurementisusefulfor

quicklyassessing the extentof foaming risk (nonefor FMR=0;

overt/onerousforFMR=6–7).Bycontrast,FPisscientifically

rigor-ous,andessentialforanalysisoffoamingpotentialandpredictionof

foamingrisk.Furthermore,FPhasbeencloselycorrelatedwithEPS

concentration[9],andsocanbeindirectlyassociatedtothe

physi-ologyoftheactivatedsludgeandwithotherphenomenadependent

onthem(suchasmembranefoulingrate).However,bothtestsare

simpleandcanbeconducteddirectlyinsituwithsimilarsimple

equipment.

MoreusefulforfoamingassessmentinanMBRplantisthe

Mod-ifiedScumIndex(MSI)[10].TheMSIallowsthequantificationof

thefoamfractionfromEPS(generallymoreabundantbutless

sta-blefoam)comparedwiththatduetothepresenceoffilamentous

bacteria(usuallylowerinmassbutmorehighlystable).

MSIisdeterminedusinga2-laliquotofactivatedsludge,taken

fromtheaerationtank,which isplacedintheflotationcelland

aeratedatanintensityof10LairL−1h−1for15mintoformathick,

stablelayerofscum.Onceasingaerationthescumlayerisseparated

Table3

MSIclassification.

Effectofscumflotationstep Effectofscumpurificationstep

MSI0[%] MSI1[%] MSI2[%] MSI3[%]

Low 0–10 0–10 0–7.5 0–5

Moderate 10–15 10–15 7.5–12.5 5–10

Serious 15–25 15–20 12.5–17.5 10–25

Excessive >25 >20 >17.5 >15

fromthenon-floatingsludgeandthetestrepeatedseveraltimes (dependingonthefilamentousbacteriacontent)ontheresidual unfloatedmaterialuntilallofthefoam-formingmicro-organisms aretransferredintothescum.Witheachsteptheextractedscumis stored.Thisfirstphaseiscalled“flotationstep”.Theportionofscum separatedinthisphasecontainsthefoamproducedbyboththeEPS contributionandthegrowingbiomass.Thedrymassofrecovered scumcanbedeterminedandtheMSI0calculatedas:

MSI= _MassofMass_Suspendedofscum_Solidsrecovered_inthesample×100 (2) Followingtheflotationstep,thefloatingscumisplacedbackin theflotationcellandre-suspendedwithwatertoatotalvolume of 2L.Subsequently, the“purification step”is performedunder controlledaerationconditionsfor15minandtheprocess of re-suspensionanddilution(withpurewater)isrepeated,withsteps of15min,untiltheamountoffoamformedisconstant.These por-tionscontainonlythefoam-formingbiomass.Theequationabove providestheScumIndexforeachpurificationstepinthe step-by-stepprocess(MSI1,MSI2,MSI3,etc)togaugefoamingpropensity (Table3)basedondeterminationsusingsimpleequipment(Fig.2).

Ingeneral,ithasbeendemonstratedthatthedilutionofthe

sep-aratedscumsamplescouldhaveanegativeinfluenceontheoriginal

impactof EPSconcentration.For this reason,it isbelieved that

theMSI0representsmainlytheeffectoffoamingduetoEPS.

Con-versely,purifyingthefoambydilutionandre-suspensionisolates

thecontributionfromfilamentousbacteria.

Concerningmicroscopicinvestigation,theabundanceand

dom-inanceof filamentous micro-organismsby microscopic analysis

can beestimated using the criteria suggested by Jenkinset al.

[18].Thismethodisbasedonobservingmorphologicaland

phys-iologicalcharacteristicsofbacteria,includingbranching,motility,

filamentshapelengthanddiameter,location,growthofattached

Table2

FoamingRateclassification.

FR Effects

0 Reactiontoaerationasforpurewater.Bubblesbreaksurfacebutareunabletofoamorhavenostability.

1 1–3cmoffoamwithfragile,ill-formedbubbles.Insufficientstabilityoffoamfilms.Immediatecollapseonaerationbeinghalted.

2 Intermittentfilmssufficientlystabletolastfor>5–10s.Usuallygeneratedfromafragilefoamstructureoflimitedheight.Filmsunstableonaerationbeing halted.

3 Foaming(bubblesabout1cmdia.)to3–8cmheight.Infrequentregularfilmformation,withbothfilmandfoamsemi-stableonaerationbeinghalted.Films have10–30sstability.

4 Initially8–15cmoffoam(about1cmdia.bubbles)withstablefilmsbeingformedatregularintervals.Bodyofthefoamandfilmsstablefor3–5minonce aerationceases.

5 Formationofstablefoam5–10cminheightwithin2min;afterwhichcollapseto3–5cmheight,whichisstablewhenaerationishalted.Nofilms. 6 Stablefoam15–30cminheightwithnofilm.Bubblesizeof∼0.5cmduringproduction,increasingto2–3cmdia.within3–5minfromtimethataerationis

halted.

7 Densestablefoam>30cmover2minaeration.Bubblesizeabout0.3cmduringproductionoffoamandmax.1cmdia.within3–5minafteraerationishalted. Foamissufficientlystabletoshownochangeinheight10–15minafteraerationishalted.

Fig.2.MSIprotocol.

epiphyticbacteria,sheath,cellsepta,cellshape,flocsize,sulphur deposits,andNeisserand Gramstaining.Theabundanceof fila-mentousmicro-organisms(Ab)isevaluatedusingthesubjective “scoring”ofthefilamentsaccordingtotheindexproposedby Jenk-insetal.,assigninganumericalvaluetothepresenceoffilaments onascalefrom0to6[18].

Morespecifically,thequantitativeestimationoffoam-forming

micro-organismscanbecalculatedusinga“fastmethod”.A50mL

sampleisplacedontheslideandthesamplestained(Gramand

Neisser)toidentifythenumberofGramandNeisserpositive

fila-ments.Thisisbasedonthenumberofintersectionsofacertaintype

ofpositivefilamentdetectedalongwiththetotalnumberofsuch

positivefilamentsperunitareaofthemicroscopeslide.Themean

numberofintersectionsacrossfivesamples(X_AVG)isnormalised

againstthetotal mass of volatilesuspended solids(VSS)in the

microscopicsample(Vin␮L)andcorrectedforthemagnification

MtoproducetheNormalizedIntersectionsNumber(NIN):

NIN= XAVG·M

VSS·V ·10

6 ₍₃₎

wherethe106_{factorconverts␮ltoL.}

2.3. Foulinganalysis

Themembranefoulinghasbeeninvestigatedbymeanofthe

“ResistancesinSeriesModel”[7].Morespecifically,theprotocol

basedonthephysicalcleaningapproachhasbeenusedtodefinethe

characteristicresistancesderivingfromthemainfouling

mecha-nisms.Indetail,theadoptedcleaningprotocolwascharacterizedby

aseriesofdifferentstepsandactionsreproducedalmostidentically

asbetteroutlinedinthefollowing.

1First,permeatefluxandTMParemeasuredduringnormalplant

operations,beforethecleaningaction.Onthebasisofthese

val-ues, thetotal resistancetofiltrationcan beevaluated(Rtot,1),

accordingtoEq.(4):

Rtot,1=

TMP1

J1·␮

(4)

whereJ1isthepermeatefluxofthefouledmembrane[m3m−2s−1],

TMP1isthetransmembranepressureinthesameconditions[Pa],

␮isthepermeateviscosity[Pas]thatisalmostequaltothatof

wateratworkingtemperature.

2Afterwards,themembraneisextractedfromthebioreactorand

physicallycleanedthrough3operationalsteps.Firstly,the

mem-braneisrinsedwithtapwaterat0.4–0.5barfor15min,gently

rubbingthemembranesurface(orthemembranefibersinthe

case of hollow fiber modules). Thereafter, the membrane is

cleanedinpurewater bymeansofmembraneshaking,either

mechanical(forsmallmembranemodulesusedinbenchscale

plants)ormanual(forbiggermodulesusedinpilotplant

appli-cations). Finally, 5min rinsing with ultrapure water (at low

pressure,<0.2bar)iscarriedout.

3Subsequently,thecleanedmembraneisimmersedincleanwater

andsubjectedtoanormalfiltrationcycle(withthesame

inordertomeasuretheresistancetofiltrationincleanwater

(Rtot,cw),accordingtoEq.(5):

Rtot,cw=TMPcw

J_cw·␮ (5)

whereJcw[m3m−2s−1]andTMPcw[Pa]arethepermeatefluxand

thetransmembranepressureincleanwaterafterphysicalcleaning;

␮isthewaterviscosity[Pas].

4Thecleanedmembraneisthereafterimmersedinthe

bioreac-torandthensubjectedtonormalfiltrationcycles(withthesame

fluxesandeventuallyordinarybackwashingwithanaverageflow

ratenextto900mLmin−1).Itis worthnoting thattheentire

processofphysicalcleaninghadashortduration(about½–1h)

duringwhichtheMLSSconcentrationwasassumedtobealmost

constant.Thus,itispossibletoevaluatethefinaltotalresistance

tofiltration(Rtot,2),accordingtoEq.(6):

Rtot,2=

TMP2

J2·␮

(6)

where J2 is the permeate flux during filtration of the same

mixedliquorasthatofstep1,aftermembranephysicalcleaning

[m3_m−2_s−1_{],TMP2isthetransmembranepressureunderthesame}

conditions[Pa],whereas␮isthepermeateviscosity[Pas].

Thecakecontribution,duetoeitherreversible(RC,rev)or

irre-versible (RC,irr) phenomena, can be computed, after cleaning,

accordingtothesteps2–3oftheaforementionedprotocol.Inthis

context,thetotalresistancecanbeexpressedbasingonthespecific

depositionmechanisms:

Rtot,1=Rm+RPB+RC,rev+RC,irr (7)

whereRPBistheresistancedueto“PoreBloking”.

TheresistancesRtot,cwandRtot,2canbeexpressedaccordingto

Eqs.(8)and(9):

Rtot,cw=Rm+RPB (8)

Rtot,2=Rm+RPB+RC,rev (9)

Finally,theRISmodelisappliedinthefollowingway,through

Eqs.(10)–(12),inordertocomputethedifferentfouling

mecha-nisms:

RPB=Rtot,cw−Rm (10)

RC,irr=Rtot,1−Rtot,2 (11)

RC,rev=Rtot,2−Rtot,cw (12)

2.4. Viscosity,hydrophobicity,flocdimensionsandextracellular

content

Finally,thefollowingmainparametersthatdefinetheactivated

sludgefeatureshavebeenmeasured:

• Theviscosityofthemixedliquorwasinvestigatedtoevaluatethe

sludgerheologyandtheirroleinfoamingandfouling.Arotational

rheometer(Brookfielddigitalviscometer,modelDV-E)equipped

withconcentriccylindersandanadapterforlowviscositywas

usedtoestimatethemixerliquorviscosityunderconstant

tem-perature(20◦C).

• ForthehydrophobicityofmixedliquortheRosenberg’smethod

wasapplied[19].

• Theflocsizedistributionofthesludgesuspensionwas

deter-minedbyusingaMalvernMastersizer2000instrument(Malvern

InstrumentsLtd., Worcestershire, UK)with a detection range

of0.02–2000␮m.Thecharacteristicdiametersd10,d90anddm

(average)weredetermined.

• The total EPSs (EPST) were evaluated by using the “heating

method”[1,4,20],whichallowstheachievementofthesoluble

fractionofEPST(SMPs)aswellastheboundfractionEPSbound.

Inordertomeasuretheproteinandcarbohydratecontents,the

extractedboundEPSsandSMPswereanalyzedusingLowry’sand

Anthrone’smethods,respectively[21,22].In particular,inthis

workthesumofproteinandcarbohydratecontentwas

consid-eredas:

EPST=[EPSbound,P+EPSbound,C]+[SMPP+SMPC] (13)

wherethesubscriptsymbol“P”or“C”indicatestherelativecontent

ofproteinsorcarbohydratesintheEPSTandSMPs,respectively.

3. Resultsanddiscussion

3.1. Mixedliquorcharacterization(EPS,viscosity,floc

dimensions)

Concerningtheremovalefficiencies,thepilotplantreducedthe

averageinfluentCODof326mg/LtoamedianeffluentCOD

con-centrationof27mg/L(92%removal)inthePeriodI,from421mg/L

to18mg/LinthePeriodII(96%removal),from401mg/Lto14mg/L

inthePeriodIII(97%removal).Ingeneral,eachphase(anoxicand

aerobic)contributedtotheCODremoval,withasimilarspecific

CODremovalinbothphases.

Regardingthe TNremoval, differentbiological performances

wereobservedduringeachperiod.

• InthePeriodI,theaveragenitrificationanddenitrification

effi-ciencieswereabout71%and85%,respectively:theunsatisfying

nitrificationefficiencywaslikelyduetothelowabundanceof

autotrophicbiomassduetolowF/Mratioapplied,thataffected

significantlythenitrificationefficiency.

• InthePeriodII,theaveragenitrificationanddenitrification

effi-ciencieswereabout95%and72%,respectively:inthiscase,the

nitrificationwascompletebecausethephase ofaeration were

higher thanPeriodI (taeration/tcycle=0.44 inPeriod2 and 0.33

inPeriod1);contrarytheefficiencyofnitrificationwasslightly

lower(thanperiod II) becausenowthere aremore nitrateto

denitrified.

• InthePeriodIII,theaveragenitrificationanddenitrification

effi-ciencieswereabout98%and55%,respectively:theunsatisfying

denitrificationefficiencywasstrictlydependentonthenumber

ofdailycycle(withatcycleof90minthereare16cycleperday,

significantlyhigherthan8cycleperdaysofperiod1and2);such

acircumstance(displacementbetweenaeratedandno-aerated

phase)significantlyaffectedthetotalnitrogenremovalefficiency,

thatinperiod3waslowerthan60%,asaveragevalue

Adetaileddiscussionconcerningtheremovalperformanceof

removaloftheplantwasreportedin[15].

On the other hand, someimportant observation concerning

Mixedliquorcharacteristicscanbedonebeforetodiscussfoaming

andfoulingphenomenainIA-MBR.

Duringtheinvestigatedperiodtheaveragemixedliquor

sus-pendedsolidMLSSconcentrationsforthepilot-scaleMBRranged

from5to7gTSS/landtheaveragepercentageofvolatilefraction

wasalmostequalto75%.Theobservedyieldcoefficient(Yobs)was

nearto0.07gVSSgCOD−1.Actually,It’simportanttounderlinethat

themixedliquorsuspendedsolidMLSSconcentrationwaslower

duringtheinitialdays(<7gTSS/l),duetoanexcessivesludge

0

100

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600

700

0

20

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60

80

100

120

0

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0

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EPSt Protein Carbohydrate

I II III

a)

_b)

c)

Fig.3.SpecificEPSbound(a)andSMP(b)distribution,inproteinandcarbohydratefraction,andrelationshipbetweenproteinsandcarbohydrateswithtotalEPSs(c).

reduced,theMLSSvaluesrosereachingafinalvalueof7gTSS/land

thereafternosignificantbiomassgrowthhasbeennoticed[15].

TheEPSsinthemixedliquor,thatwereknownaskeyfactorsin

theformationoffoamingandfoulinginotherplants[12,13,23–27],

areshowninFig.3,intermsofEPSboundandSMPsbothinprotein

andcarbohydratefractions

Thepredominanceofprotein,especiallyintheformofEPSbound,

could be due to the presence of large quantities of

endoen-zymes,whichwereliberatedbylysis,andextracellularproducts

intheflocs,dependingonmicroorganismtype,substrate

proper-ties[28,29]andoperatingconditions.Furthermore,due totheir

hydrophobicity,proteinsgenerallyhavehigheraffinityforsludge

flocsthandopolysaccharides[30].Infact,sincethepolysaccharides

aremore readilybiodegradable than protein, theyare

metabo-lized;contrary,proteins,thataremolecularlymorecomplex,tend

toadheretothesludgeflocsbecomingthemaincomponentsof

EPSbound.Ingeneral,theEPSsformationisfavoredinIAconditions

becausethealternationofaeratedno-aeratedrepresentastress

factorformicroorganisms,whichcouldleadtocellularautolysis

[5].

In particular, until day 36 in the Period I

(taeration/tcycle=60min/180min) high values of EPST were

observed due to acclimation of inoculated biomass (collected

from a CASP) to the new IA condition. Subsequently theEPST

decreased,denotinganacclimatizationofbiomasstotheregime

ofalternationofanoxic/oxicphases.AtthebeginningofPeriodII

(taeration/tcycle=80min/180min),there wasa furtherdecreaseof

EPSTthatwaslikelyduetoadifferentmetabolicbehaviorofthe

biomass[31].However,inthefollowingdaysofPeriodII,anew

biomassadaptationoccurredwithagradualincreaseofEPST.

Finally,thePeriodIII(taeration/tcycle=30min/90min)was

char-acterizedbyafurtherdecreaseofEPSTduetothereductionofthe

anoxictimepercycle,withasubsequentmetabolicinhibition.

Finally,Fig.4ashowsthetrendsofviscosityand

hydrophobic-ityduringthethree periods;thesevariables arestrictly related

tothecontents ofpolysaccharidesandproteins, respectively.In

thiscontext, theconcentrationofpolysaccharides results

corre-latedwelltotheviscosityof themixedliquor(Fig.4b).Onthe

otherhand,inFig.4cthecorrelationbetweenproteincontentand

Fig.4.Distributionofviscosityandhydrophobicityofmixedliquor(a);correlations betweenpolysaccharidesandviscosity(b)andbetweenproteinsand hydrophobic-ity(c).

0 200 400 600 800 0 20 40 60 EPS T [mg · gVSS -1] MSI [%] R² = 0,69 R² = 0,63 R² = 0,97 0 200 400 600 800 1000 0 100 200 300 EPS T [mg · gVSS -1] FP [ml · L-1_]

e)

MSI_0 MSI_1 MSI_2

I II III 0 1 2 3 4 0 20 40 60 80 100 120 FMR [ -] Time [days] 0 50 100 150 200 250 300 0 20 40 60 80 100 120 FP [ml · L -1] Time [days] FP

a)

c)

b)

d)

Fig.5.ModifiedScumIndex(MSI)(a),FoamRate(FMR)(b),FoamPower(FP)(c),andcorrelationsMSI-EPST(d)andFP-EPST(e).

hydrophobicityarereported.Ingeneral,thehydrophobicityis

cou-pledtohighproteinconcentrationinthemixedliquor,andthis

circumstancereducesthesludgeaggregation[32].

3.2. Foamingphenomenonanalysis

TheresultsoftheFoamTests,showninFig.5,arerepresentative

forsamplestakenattheendoftheaerobicphase.

DuringPeriodI,theMSI0(Fig.5a)increaseduntil50%(day39th)

accordingtotheincreaseofEPST.Subsequently,itdecreasedto

val-uesintherangeof10–20%attheendofPeriodI.Similarly,inthe

PeriodII,thevariationofMSI0iscorrelatedtoEPSsvariation:

ini-tiallyMSI0decreased(untilday90th)andthenincreasedtoabout

35%.InperiodIII,theMSI0decreasedtoabout20%.Thus,in

agree-mentwithpreviousstudies[9,12],alsoinanIA-MBRthegeneral

trendofMSI0iscorrelatedwiththevariationofEPSSinthe

biore-actor.Contrarily,afterthecompleteprocessof“purification”the

effectofEPSSwas“masked”.Morespecifically,theMSI2wasless

affectedbytheEPST,andshowedatrendsimilartothatof

filamen-tousmicroorganisms.ThecorrelationbetweenMSI2andthelevel

offilamentousabundanceisdiscussedinthenextparagraph.

Thequalityandstabilityofthefoamlayer,intermsofFMRand

FPtestsrespectively,areshowninFig.5bandc.Bothparameters

increasedcontinuouslyduringthewholemonitoringperiod:this

wasprobablyduetothestabilizingeffectofthefilamentousthat

wereselectedduringtheexperiment(seenextsections).

Ingeneral,usingthepreviousFMRclassification,inthePeriod

IandIIthefoamappearedas“fragileanddecreasingiftheaeration

ceased”.Ontheotherhand,inPeriodIIIthefoamlayerwas3–8cm

inheightwith“infrequentregularfilmformation,having10–30s

sta-bility,andwithbothfilmandfoambeingsemi-stableuponaeration

beinghalted”.

ConcerningFig.5c,theresultsshownthatthetrendofFPstarted

froma value nexttoabout 50mlL−1,then it rapidlyincreased

toover250mlL−1(day36),incorrespondenceofasimultaneous

increaseofEPST(mainlyproteinfraction).Subsequently,thevalue

decreasedtoabout50mlL−1byday92.Finally,attheendofperiod

IIandduringperiodIII,therewasanincreaseto150mlL−1andthis

lattertrendismainlyduetothefilamentousmicroorganisms,as

furtherdiscussed.

Fig.5d,esummarizethecorrelationbetweenEPST,MSIandFP

data,confirmingthattheEPSareakeyfactorinthefoaming.In

detail,thecorrelationbetweenMSIandEPSTbecameprogressively

weakergoingfromMSI0toMSI2(Fig.5d).Ontheotherhand,the

absenceofsignificantcorrelationsbetweenMSI2andEPSSconfirms

thatthepurificationstepreducestheeffectofEPSsonfoaming,

infavorofthecontributionoffilamentousbacteria[9,13].In

par-ticular,whenthemixedliquorwaspurifiedfromscumduringthe

foamingtests,themajoramountofEPSwasremovedwiththescum

andsothecorrelationbetweentheextracellularpolymeric

sub-stancesandthesubsequentscumindexesbecamegraduallyless

important.

Finally, Fig.5eshows that alsothe EPSand FP are strongly

correlated. The polymeric and jellynature of EPS, involves the

productionofevermorepersistentbiologicalfoamunderaerobic

conditions,thegreateristheconcentrationofextracellular

poly-mericsubstances.So,FPisaparameterthatsatisfactorilydescribes

the“probability”ofthemixedliquorfoaminginalloperative

condi-tions(suchascausedbyonlyEPS,orcoupledEPSwithfilamentous

bacteria).

3.3. FilamentousbacteriainIA-MBR

Filamentousbacteriaplayanimportantroleinthedevelopment

of sludge features andmembrane permeability. Thus, the

anal-ysisoffilamentous bacteriainthesludge suspension,shownin

Fig.6a,providesusefulinformation.Inthiscontext,themicroscopic

observationsandinvestigationsaccordingtoJenkinsetal.[18]

car-riedoutsinceday44,highlightedthepresenceofthreedominant

speciesof filamentousbacteria:EikelboomType0092, Microthrix

parvicellaand,insmallerquantities,Nocardioforms.

TheEikelboomType0092(“notfamous”bacteria)asoneofthe

dominantspeciesisinagreementwithotherstudies[9,29,33,34].

Fig.6.Trendoffilamentousbacteria(a)(witha1-a3microscopicobservations)andtheircorrelationswithMSI2(b)andFP(c).

favoredbytheoperatingconditionsimposedintheIA-MBRandby

thelackofdissolvedoxygenduringtheanoxicphases,especiallyin

thePeriodIII.

Correlationsbetweenthedevelopmentoftotalfilamentous

bac-terialspeciesandtheScumIndexandthefoampowerinPeriod

IIIarealsoshowninFig.6(bandc).Itisimportanttounderline

that,converselytothecorrelationbetweenMSIandEPSs,thebest

correlationbetweenMSIandNINwasobservedinPeriodIIIwith

MSI2.Thisresultconfirmstheresultsofpreviousexperiments[9]

andpreviousobservations:ingeneral,thedatashowasignificant

correlationafterpurificationsteps.Furthermore,inthePeriodIII,

thepotentialoffoamingisalsocoupledtofilamentous

microorgan-isms,asconfirmedbythecorrelationbetweenFPandNIN(Fig.6c).

In general, in an IA-MBR, the main foam-forming bacteria

growthstronglydependsonthealternationoftheaerobic/anoxic

phases.

• Fewercyclesperdayandhigherdurationoftheanoxicphasefor

singlecycle,determinedagreaterproductionofEPS,whichwas

themaincauseoffoaminginPeriodsIandII.

• Ontheotherhand,ahighernumberofcyclesperday,asinPeriod

III,stimulateaproliferationofdifferentfilamentous

microorgan-ismsthatbecomeakeyfoam-formingfactorintermofstability

andfoampotential.

3.4. Membranefoulinganalysisandrelationwithfoaming

Thevariationofthetotalresistancestofiltrationisreportedin

Fig.7a.Thetotalhydraulicresistancetrendwasstronglyinfluenced

byphysical cleanings,which alloweda partialrecoveryof

per-meabilityandtheestimationofspecificresistances.Noprevailing

foulingmechanism,betweenthereversibleandirreversible

foul-ing,wasobservedfromtheanalysisandcomparisonofthespecific

foulingresistances(Fig.7b).OnlyinthePeriodIII,theirreversible

foulingmechanisms,intermsofRCirrandRPB,weredominant.

On theother hand, Fig.7c underlines theimportant role of

boundand solubleEPSonthemembrane fouling development.

Morespecifically,thehistogramreportsthesingularratiobetween

eachfactor(EPSs,SMPs,RPB,RCake)andthetotalfractionofEPSTor

RT.Inthiscontext,Fig.7cshowsanoppositetrendforthethree

peri-odswithregardtoEPSboundandSMP.FromPeriodItoperiodIII,the

microorganismsmodifytheirmetabolism,decreasingtheircellular

autolysis(soimplyinglowerreleaseofSMP)[5,31]andincreasing

theproductionofexopolymersasstoragesubstrateintheformof

EPSbound.

Particularly, in Period I, which hasa cycle time of 180min

with120minwithoutaeration,biomassissubjectedtoahigher

metabolicstressthanintheremainingtwoperiods.Successfully,

therewasaslightincreaseofboundEPSboundfraction(from77%in

PeriodIto96%inPeriodIII),whichcorrespondstoanequallysmall

decreaseofSMP(from23%inperiodIto4%inperiodIII).

Contem-porarily,anincreaseofirreversiblefouling,bothasirreversiblecake

(Rcake,irr)andasporeblocking(RPB)wasobservedattheexpenseof

adecreaseinthereversiblefouling(Rcake,rev).

Thefoulingfromirreversiblecakeisattributabletothe

produc-tionofEPSbound,whichgivesagelatinousconsistencytothecake

thatcanberemovedwithaphysicalcleaning.

Theconcomitantincreaseofporeblockingisprobablydueto

de-flocculationwhichtypicallyoccursinaMBRsystem,asdiscussedin

thefollowing,andwhichinvolvesflocdisintegration,witha

possi-bleocclusionofthemembraneporesbydispersedmicroorganisms

andsolubleproducts.

Regardingthefoulingrate(Fig.8a)expressedasincreaseinthe

totalresistanceovertime,itisgreaterinthefirstperiodbecause

thelowconcentrationofTSSinmixedliquoravoidedthe

Fig.7.Totalresistancefouling(a),specificresistances(b)andinfluenceofSMPsandEPSboundonfoulingmechanism(c).

developmentof a cakelayer,mainlymade ofirreversiblecake,

impliedaconsiderabledecreaseoffoulingratefortheremaining

daysofexperimentation.Furthermore,thefoulingrateisclosely

relatedtotheproteincontentofthemixedliquorexpressedbythe

ratioProteins/Polysaccharides(PN/PS),asshowninFig.8b.Dueto

theirhydrophobicnature(Fig.4c),proteinstendtoadheretothe

surfaceoftheflocs,effectivelyconstitutingEPSbound,and

contribut-ingtotheirreversiblecakedeposition.

3.5. Analysisoftheflocdimensions

The resultsof themorphology observations are reported in

Fig.9a,wheredmtrendsareshownforeachphaseaswellasthe

d10andthed90data.

Ingeneral,thedataconfirmthatintheMBRthebiological

aggre-gatesare smallerthanflocs ina ConventionalActivated Sludge

Plant.Thisisduetotheloworganicloadingrateconditions,and

tothehighturbulence inmembrane bioreactors [35–37].More

Fig.9. Flocdimensionsvariations(a)andcorrelationbetweenspecificcakeresistancetofiltrationandthemeanflocdimensions(b).

specifically,throughoutthethreeperiods,therewasaslightsludge

de-flocculation.Inparticular,inPeriodI,de-flocculationoccurred

untilday36thwithapartialre-flocculationoccurredattheendof

thisperiodduetotheincreaseofEPSsinthebioreactor(seeFig.4a).

ThePeriodIIwascharacterizedbyaslightde-flocculation,whilein

periodIIIanewre-flocculationwasnoted,partiallydueto

filamen-tousmicroorganisms,whichactasbridgesfortheformationofnew

sludgeflocs.

Consequently,thespecificcakelayerresistance(␣)was

influ-enced by de-flocculation (Fig.9b). In particular, thesuperficial

cakedepositionincreasedwhen theflocsize decreased. In this

circumstance, the permeability of the resulting cake layer was

stronglyaffectedbythepresenceoffilamentousmicroorganisms

orexcessiveflochydrophobicityandsludgeviscosity.

3.6. Considerationontherelationshipbetweenfoamingand

fouling

InFig.10,allcorrelationbetweenthemainoperational

param-eter(EPS,andNIN)andspecificfoulingandfoamingmeasurement

(MSI,Resistances),areshown.

In general, the high production of extracellular polymeric

substances (Period I and II) involves the formation of a very

hydrophobicsludgethatimpliestheformationofcompactcake

layer,improvingitspre-filtrationaction(Fig.10a).On theother

hand,the filamentous bacteria (Fig. 10b)promote thebridging

betweenthecellularaggregatesand theformationofaswollen

cakewithareductionofthepre-filtereffect(PeriodIII).

Finally,therewassimilarcorrelationbetweenMSI0andFPwith

theRt(Fig.10candd).Thisindicatesthatthephenomenaof

foam-ingandfoulingarenotindependentbutinterconnectedbymeans

ofEPSs.Indeed,anincreaseoffoamingquantity(intermsofMSI0)

andpotential(intermsofFP)inevitablyinvolvesaworseningof

thepermeabilityofthemembrane.Thisresultisapparentlyin

dis-agreementwiththeviewtakenbytheauthorsCosenzaetal.[13],

thataffirmedthatmembranefoulingtendtodecreaseiffoaming

increase.Likely,thedifferenceisduetothehigherpresenceof

over-allEPSboundintheIA-MBR,whichcontributetotheproduction

ofahigherquantityoffoam.Furthermore,inthecurrentwork,the

foamingintheIA-MBRsystemwasmainlyduetotwodifferent

causes:firsttoEPSs(PeriodIandII)andsubsequently(PeriodIII)

tofilamentousbacteriaandEPSstogether.Contrarily,inthe

experi-enceofCosenzaandcoauthors,thefoamingwasmainlycorrelated

toEPSscontribution(withaverylowfilamentousabundance).In

anycase,itisimportanttorememberthatthefoam“persistence”is

relatedtothegeometricfeaturesofBioreactor,whicharedifferent

inbothexperimentations.

4. Conclusions

ThefoamformationoccurredspontaneouslyintheIA-MBR,with

highvaluesof MSI(>30%)and FP(>150mLL−1).In general,the

resultsshowthatextracellularpolymericsubstancesplaykeyroles

infoamandfouling.Furthermore,ifthehighconcentrationofEPS

iscoupledtothepresenceoffilamentousbacteria(asitoccurswith

shortdurationoftheaeration-noaerationcycles)thefoaming

phe-nomenonworsens.Fortheanalyzedcasestudy,inPeriodIandII

(bothwithacycledurationof180min)thefoamwasduetoEPSs

production,asshownbytheMSI0trend.Intheseperiods,thereare

feweraerationcyclesperdaybuttheanoxicphaseforeachcycleis

longer.Ontheotherhand,thehighernumberofcyclesperdayin

PeriodIII(durationofthecycleof90min),despitetheshorter

dura-tionoftheanoxicphaseforeachcycle,promotedtheproliferation

offilamentousbacteriathatbecameakeyfoam-formingfactor,as

confirmedbyFP-NINcorrelation.

However,thisstudyprovidesanewoutlookabouttheroleofEPS

inthefoamingforMBRsandalsoanewinsightintothemembrane

foulingmechanism.

Acknowledgements

ThisresearchwasfundedbytheNationalResearchProjectPRIN

2009(ProjectTitle:ExperimentalanalysisonMBRsystemstostudy

andtorealizeamodeloffoulingincludingthephysicaland

micro-biologicalfeaturesofsludgeandtheoperationalconditionsthat

influencethisphenomenonCUPB71J11000610001);Italian

Min-istryofEducation,UniversityandResearch.

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