Formation and ripening of alginate-like exopolymer gel layers during and after membrane filtration

(1)

Delft University of Technology

Formation and ripening of alginate-like exopolymer gel layers during and after membrane

filtration

Pfaff, N. M.; Kleijn, J. Mieke; van Loosdrecht, Mark C.M.; Kemperman, Antoine J.B.

DOI

10.1016/j.watres.2021.116959

Publication date

2021

Document Version

Final published version

Published in

Water Research

Citation (APA)

Pfaff, N. M., Kleijn, J. M., van Loosdrecht, M. C. M., & Kemperman, A. J. B. (2021). Formation and ripening

of alginate-like exopolymer gel layers during and after membrane filtration. Water Research, 195, 10.

[116959]. https://doi.org/10.1016/j.watres.2021.116959

Important note

To cite this publication, please use the final published version (if applicable).

Please check the document version above.

Copyright

Other than for strictly personal use, it is not permitted to download, forward or distribute the text or part of it, without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license such as Creative Commons. Takedown policy

Please contact us and provide details if you believe this document breaches copyrights. We will remove access to the work immediately and investigate your claim.

(2)

Water Research 195 (2021) 116959

ContentslistsavailableatScienceDirect

Water

Research

journalhomepage:www.elsevier.com/locate/watres

Formation

and

ripening

of

alginate-like

exopolymer

gel

layers

during

and

after

membrane

ﬁltration

N.-M.

Pfaff

a,c,∗

_,

_J.

_Mieke

_Kleijn

b

_,

_Mark

_C.M.

_van

_Loosdrecht

a

_,

_Antoine

_J.B.

_Kemperman

c,d

a TNW Applied Sciences, TU Delft, Van der Maasweg 9, 2629 HZ Delft, The Netherlands

b Physical Chemistry and Soft Matter, Wageningen University, Helix, 124, Stippenweg 4, 6708 WE Wageningen, The Netherlands c Wetsus, European Center of Excellence for Sustainable Water Technology, Oostergoweg 9, 8911 MA Leeuwarden, The Netherlands

d Membrane Science and Technology cluster, Faculty of Science and Technology, Mesa + Institute for Nanotechnology, University of Twente, P.O. Box 217,

7500 AE Enschede, The Netherlands

a

r

t

i

c

l

e

i

n

f

o

Article history:

Received 21 October 2020 Revised 17 February 2021 Accepted 19 February 2021 Available online 23 February 2021

Keywords:

Extracellular polymeric substances Calcium-binding

Hydrogel Donnan potential Bioﬁlm

a

b

s

t

r

a

c

t

ThepropertiesofbiofilmEPSaredeterminedbythemultipleinteractionsbetweenitsconstituentsand thesurroundingenvironment.BecauseofthehighcomplexityofbiofilmEPS,itsconstituents’ character-isationisstillfarfromthorough,andidentificationoftheseinteractionscannotbedoneyet.Therefore, weusegelsofbacterialalginate-likeexopolysaccharides(ALEs)asamodelcomponentforbiofilmEPSin thiswork.ThesegelshavebeenexaminedfortheircohesivepropertiesasafunctionofCaCl2 andKCl

concentration.Hereto,ALEgellayerswereformedonmembranesbydead-endﬁltrationofALEsolutions. AccumulationofthecationsCa2+_and_K+_in_the_gels_could_be_well_predicted_from_a_Donnan_equilibrium

model basedontheﬁxednegativechargesinthe ALE.Thissuggeststhatthere isnospeciﬁcbinding ofCa2+ _to_the _ALE_and _that_on_the_time_scale _of_the_experiments,_the_Ca2+ _ions_can _distribute_freely

overthegeland thesurroundingsolution.TheconcentrationofﬁxednegativechargesintheALEwas estimatedaround1mmol/gVSS(volatilesuspendedsolids,organicmass)fromtheDonnanequilibrium. Moreover,anaccumulation ofH+ _was_predicted._Gels _with_more_CaCl

2 inthesupernatantweremore

compactandboreahigherosmoticpressurethanthosewithlessCaCl2,revealingtheroleofCa2+ions

inthenetworkcrosslinking.Itishypothesisedthatthismechanismlatertransitionsintoarearrangement oftheALEmolecules,whicheventuallyleadstoaﬁbrousnetworkstructurewithlargevoids.

1. Introduction

The integrity of bioﬁlms, also when exposed to harsh clean-ing attempts, has been assigned to their polymeric matrix (Seviour et al., 2019). The matrixis often alsoreferred to as ex-tracellular polymeric substances(EPS). It hasbeen found to con-sist of a complex mixture of biopolymers, such as polysaccha-rides and proteins, complemented by lipids, humic substances and eDNA. The EPS matrix has been described as a physically crosslinkedhydrogel(Seviouretal., 2009),inreferencetoits abil-itytoincorporateupto99%waterwhileprovidingalasting poly-mericnetworkstructure.Incontrasttochemicallycrosslinked net-works, the crosslinks in physically linked networks are provided bynon-covalentinteractions.Theseareparticularlyelectrostatic

in-∗_{Corresponding author: Hauffstr. 18, 34125 Kassel, Germany}

E-mail addresses: n.dietrich@tudelft.nl , n.m.pfaff@gmail.com , natascha.m.pfaff@gmail.com (N.-M. Pfaff), M.C.M.vanLoosdrecht@tudelft.nl (M.C.M. van Loosdrecht).

teractions,hydrophobicinteractions,H-bondingandvan-der-Waals forces,andentanglements. Theyare reversible. Essential parame-tersof hydrogelsare their degree of crosslinking,determined for examplebythenumberofchargesonthepolymers,their interac-tionwithcounter-ions,andtheirhydrophilicity(Ganjietal.,2010). Theinterplayofforcesfollowingfromtheseparametersdetermines thestateofswellingofthehydrogelundersteady-stateconditions (Bajpai,2001)andtheirpotentialforwaterstorage.

The swelling state has been identified as a crucial parame-ter with regards to adhesion, mechanical strength, permeability and degradation behaviour of hydrogels (Davidovich-Pinhas and Bianco-Peled,2010).Thesepropertiesalsohavebeenusedto char-acterisebiofilms.Adhesionandmechanicalstrengthhavebeen cor-related with fouling potential (Li and Elimelech, 2004) and suc-cess of cleaning strategies (Safari et al., 2015). The increase of hydraulic resistance resulting frombiofouling on membranes has beenlabelledasahuge impedimentformembranefiltration sys-tems (Flemming, 2020). It strikes, therefore, that the number of studiesaboutswellingofbiofilmsandEPSisminorsofar.Changes

https://doi.org/10.1016/j.watres.2021.116959

(3)

inswellingbehaviourofEPShavebeendescribedasafunctionof pH forEPSextractedfromactivatedsludgeaswellasforEPS ex-tractedfromdifferentbacterialcultures(Radchenkovaetal.,2018). Tounderstandthemolecularinteractionsandallocatefunctions to molecules orfunctional groups, simpliﬁcation of the EPS ma-trixisnecessary.Apromisingmodelarealginate-likeexopolymers (ALE), which are identiﬁed as crucial structural EPS components (Lin et al., 2010). Like the well-characterised and often used al-ginate extracted frombrown algae, gel-formation withCa2+ _ions

has been observed (Felz et al., 2020a). A full chemical analysis of ALEis still pending (Seviour etal., 2019). Still, it wasused in thisworkasasimpleapproximationforthecomplexityofbioﬁlm EPS.Ca2+ _availability_has_been_identiﬁed_as_an_essential_factor_for

biofilmstability(Körstgensetal.,2001)andhasbeenshownto in-duce crosslinking of ALE. Therefore,the effectofvariations inits concentration wasinvestigatedinthisstudyaswell.Based onthe impact of monovalent ions (Wang and Spencer, 1998) and ionic strength (van den Brink et al., 2009) on the structurally similar Ca-alginategels,KClwaschosenasasecond ioniccomponent. Ca-ALE gel layers were produced on membranes in dead-end filtra-tionmode.Althoughusuallymembranefiltrationoperatesin cross-flow mode,dead-endfiltrationwaschosensincethismethodwas found toproduce sufficientlythick filmsto investigateusingOCT, and to focus on the cohesive forces of the bulk ALE gel. From the swelling behaviour of the obtainedthick ALE layers, conclu-sionsweredrawnonthebindingbehaviourofALEinthe network-formation ofEPS, witha particularfocus onthe interaction with Ca2+_. _The _final _composition _(density, _ions) _and _cake _layer

resis-tances of the gel layers were correlated with the availability of Ca2+_and_K+_and_the_solutions’_ionic_strength._Furthermore,_the_gel

layers’ ripeningwasobserved for12days.The resultswere anal-ysedbasedontheDonnanequilibrium.

2. Materialsandmethods

ALEgel layers cross-linked withCa2+ _were_produced _by

pres-sure driven dead-end ﬁltration inthe presence ofCaCl2 and KCl.

Theirswellingbehaviourwasobservedforuptotwoweeks. Even-tually,theircompositionwasdeterminedandinterpreted.

2.1. ALEextractionandcharacterisation

Thealginate-likeexopolymersusedinthisstudywereextracted from Nereda® sludge, collected from the wastewater treatment plantinGarmerwolde,theNetherlands(describedbyPronketal., 2015).AcombinationofaddedNa2CO3,sonication,andhigh

tem-peraturewasusedforextraction,followingproceduresdeﬁnedby

Felzetal.,2016.

Granuleswerecollectedbydecanting.About150gofwet gran-ules (20 gdryweight)were mixedwith1 Ldemineralisedwater and10gNa2CO3 (VWR,TheNetherlands),resultingina1%(m/v)

carbonatesolution.ThemixturewashomogenisedwithaBranson Sonifier250for5minat70%(of 200W)inpulsedmode. Over-heatingofthesolutionwaspreventedusinganicebath.The mix-ture wasthenheatedto 80˚C andvigorouslystirredfor30 min. After centrifugation (Allegra X-12R Centrifuge, Beckman Coulter, 20 min, 3750 rpm), the supernatant wasacidified with1 M hy-drochloric acid(Merck Millipore, Germany)to a final pH 2- 2.5. The solution was centrifuged again(20 min, 3750 rpm), andthe pelletwasstoredat-80_˚Cuntilfurtheruse.Itishereafterreferred toasALE.

After extraction, theALEwastested forits gel-formingability withCaCl2 (Felzetal., 2016). The acidicpelletwasdissolved and

neutralised with1M NaOH(MerckMillipore,Germany).Dropsof the neutralALE were dripped into a 2.5 % (m/v)CaCl2 solution.

Gelling wasconsidered successfulifgel beads could be observed inthesolution.

ThedryandorganicmassesoftheALEextractweredetermined in triplicate. Samples were weighed into dry porcelain crucibles (msample)andheatedto105°Cfor24h.Afterwards,thedrysample

weight(total suspendedsolids,TSS)wasdetermined.After subse-quentheatingto550°Cfor2hours,theash’smasswasmeasured (mash). Theorganic mass (VSS) wasdeﬁned asthe difference

be-tweenTSSandmash.

2.2. Experiments

ExperimentswereperformedwithtwodifferentALE concentra-tions.Gellayerswith60mg/Land45mMionicstrengthwere ob-served forstructuralchangeswithOCT over ﬁvedays.Duplets of layerswith1mMCaCl2/42mM KCl,3mMCaCl2/36mMKCland

15mMCaCl2wereused.Duetotheirstructuralinhomogeneity,the

lattergelswerenotfurtheranalysed.

Gellayers with120mg/LALEstayedstructurallyuniformover thewholeobservationperiodandwere usedtocalculateion dis-tributions. An overview of their ionic combinations is given in

Table1.Foreachioncombination,three feedsolutions were pro-duced,each splitover twomembranes.While twoofthe gel lay-ers were directly analysed for their composition, quadruplets of each combinationwere observed fortwo weeksconcerning their swellingbehaviour(storageat4°Ctoretardmicrobialgrowth).

Afurthertestontheinﬂuenceofthesupernatantcomposition ontheswellingstatewasdonewiththeunderlinedcombinations inTable 1.Threegel layers were preparedwith3mM CaCl2 and

6 mM KCl. One sample (F) was directly transferred to a storage solutionwith12mMCaCl2and6mMKCl,theothertwo(ablank

andthetestgellayer“L”)wereobservedfortwodaysinthe corre-spondingstoragesolution.Aftertwodays,sampleLwasalso trans-ferred to a storage solution with 12 mM CaCl2 and 6 mM KCl,

andthethicknessesofallgellayerswererecordedforanotherﬁve days.

EspeciallyincaseofthedivalentCa2+ _ion, _a_signiﬁcant

differ-encebetweenthenominalconcentrationc_iandtheeffective activ-itya_iwasexpected. ApplyinganextendedDebye-Hückelequation thatconsidersionsizes(Kielland,1937),theactivitycoeﬃcients

γ

i

weredeterminedasafunctionoftheionicstrengthI,thevalence z_iandthehydratedradiusr_ioftheions.

log

γ

i = −B· z 2 i · √ I 1+ri· C· √ I (1)

Inwaterat25°C,thevaluesforBandCareB=0.51M−0.5 and C₌3.3M−0.5nm−1 (HamerandWu,1972).Thehydratedradiifor therelevantionsweretakenas0.3nmforK+ andCl−,0.6nmfor Ca2+_,_and_0.9_nm _for_H+ ₍_Kielland, ₁₉₃₇_)._The _activity_coeﬃcients

calculatedforthesolutionsofTable1canbefoundinthe support-inginformation(SI),TablesF.

2.3. Modelgellayerproduction

ALEcakelayerswerepreparedbypressure-drivendead-end fil-trationoffeedsolutionsthroughultrafiltrationmembranes(UP150, polyethersulfone (PES), 150 kDa cut-off, Microdyn Nadir, Wies-baden,Germany).The compositions ofthe various feed solutions appliedarespecifiedinsection2.2.

For a total volume of 2 L feed solution, the frozen ALE was neutralisedwith0.1MNaOH(MerckMillipore,Germany)and dis-solvedin1Ldemineralisedwaterbystirringandheatingto40˚C for1hour.CaCl2•2H2O(VWR,theNetherlands)andKCl(VWR,

Bel-gium)were dissolved in 500 mLdemineralisedwater. Both solu-tions were combined slowlyand understirring, andthe mixture

(4)

N.-M. Pfaff, J.M. Kleijn, M.C.M. van Loosdrecht et al. Water Research 195 (2021) 116959

Table 1

Overview of ionic compositions of gel layers. The numbers indicate the ionic strength of the feed solutions. The underlined combinations were subsequently used for testing the reversibility of swelling.

CaCl 2 [mM] → KCl [mM] ↓ 0 1 3 6 8 12 14 24 24 mM - - 42 mM - - - 15 - - 24 mM 33 mM - - - 6 6 mM - 15 mM 24 mM 30 mM 42 mM - 0 - 3 mM 9 mM 18 mM 24 mM - 42 mM

wasﬁlledupto2Lwithdemineralisedwater.Theionic composi-tionwascheckedwithionchromatography(IC,MetrohmCompact IC761).500mLofstoragesolutionswerepreparedforeach exper-iment,withthecorrespondingcompositionofCaCl2 andKCl.

Themembraneswerecutintocircleswithadiameterof7.5cm and immersed in demineralised water for 1 hour. They were mounted atthebottom of450mLstainlesssteeldead-end ﬁltra-tioncells.Fortheﬁltration,twocellswereconnectedinparallelto a10Lpressurevesselthatcontainedthefeedsolution.

2.3.1. Filtrationandcakelayerresistance

Duringﬁltrations,thefeedpressurewassetto1_{± 0.1}bar.The actualpressure(pa)wasrecordedalongsidethemassoftheﬁltered

solution(mf).Toallowuniformgellayerformation,nostirringwas

appliedinthecells.Allfiltrationswereperformedatroom temper-ature,23.9± 0.7°C.Initially,theclean waterflux wasdetermined forall membranesbyfiltration of750mLofdemineralisedwater at1bar.Then,2LoftheALEfeedsolutionwereaddedtothe pres-surevesselandfilteredthroughthetwomembranes,until600mL were filteredthrougheachcell.Filtrationtookbetween18and20 hours. The membranes with the model gel layers were removed and stored at 4°C in Petri dishes submerged in the correspond-ing storage solutions. Betweenexperiments, the cells andtubing werecleanedwith1%NaOClandrinsedwithdemineralisedwater. Thedensityofwaterwasapproximatedas

ρ

H2O = 1g/mLforthe

courseoftheexperiments,inordertotranslatetheloggeddataof ﬁlteredmassofwatermf attimettotheﬁlteredvolumeofwater

V_f.

Thetransmembranepressure(TMP)wasapproximatedwiththe loggedappliedpressurepa.Thetotalresistanceoftheﬁltration Rf

wascalculatedfrompaandtheﬂuxJ,usingthedynamicviscosity

ofwaterat25°C,0.89mPa•s(Nagashima,1977)for

η

H2O: Rf

(

t

)

=

pa

η

H2O· J

(

t

)

(2)

Subtractionofthemembraneresistance(calculated fromclean waterﬂux)fromthetotalresistanceprovidedthecakelayer resis-tance,Rcl.

Tocharacterisethegellayers,theaveragecakelayerresistance overthelasthourpriortoterminationoftheﬁltrationwas consid-ered. Incombinationwiththe organicmassVSS peractive mem-braneareaAm (38.5cm2),thespeciﬁccakeresistance

α

mwas

cal-culated.

2.4. Observationandanalysisofthemodelgellayers

AGanymedeSD-OCT(ThorLabs,Dachau,Germany)wasusedin combinationwiththeThorImage® Softwaretoobtaininformation oneachsample’sstructureandthickness(h).Becausethegellayers consistedformorethan90% ofwater,therefractiveindexof wa-ter at25°Cof1.33 wasused.Gel layerthicknesseswereobserved overup totwoweeks.Thegellayers werealsovisuallyinspected for accumulation ofbacteria using an Olympus BX40 witha 40x magniﬁcationobjective.

After the observationperiod, excesswater wasremoved from the gel layers by gently tapping it off the perpendicular

mem-brane onto a paper towel.The membranewas puton the paper for10 s.Next,the ALEgel layers were scratchedfromthe mem-branes.TheirTSSandVSSwere determined accordingtothe pro-cedure described in section 2.1. Considering these amounts sta-ble over the observation period, thisdata wasused to calculate thespeciﬁc cake resistance

α

m aswell astheorganic mass

den-sity

ρ

VSS,thelatterwiththeobservedthicknessaschanging

vari-able.The ashremaining afterTSS determinationwasdissolved in 69%HNO3(VWR,France),heatingupinamicrowaveoven(Ethos

EASY Advanced Microwave Digestion System, Milestone, Sorisole, Italy)with1500 mWto 200°Cwithin 15 min,and200°C for an-other15min.TheamountsofCa2+_and_K+_in_the_gel_layers₍_m

ion)

were determined usinginductively coupled plasmaoptical emis-sionspectroscopy (ICP-OES,Perkin Elmer,type Optima 5300DV). The molar concentration was calculated for the different ions in referencetotheoriginalALEgellayervolume.

2.5. Electro-chemicalinterpretation

Forthe interpretation ofthe results, thesystem isconsidered as two compartments: the gel layer (compartment 1) and the supernatant (compartment 2) (Fig. 1). The gel is fully penetra-ble for water and all ions contained in the system (Ca2+_, _K+_,

Cl−, H+, OH−). In addition, the gel layer is considered to con-tainﬁxednegativecharges,includingcarboxylicacidsandsulfated glycosaminoglycans-likepolymers(Felzetal.,2020b).

2.5.1. Electroneutrality

Electroneutralitydemands that within each compartment, the chargesarebalanced.

i zi· ci, 2 = 0. (3) i zi · ci, 1 − Z = 0 (4)

i are the ions speciﬁed above, Z isthe concentration of ﬁxed negativechargesinthegel.In theexperiments,theionic compo-sitionin thesupernatant wasconsideredcontrolled and constant duetoitssubstantialvolumetricexcesscomparedtothegellayers. Theconcentrationsassumedare,therefore,thoseshowninTable1, completedwithanegligibleconcentrationof10−4mMforbothH+ andOH− (pH7).

2.5.2. Donnanequilibrium

The Donnan potential describes the electrical potential differ-encebetween two compartmentsdue to theuneven distribution ofionsasaresultoftheexistenceofﬁxedchargesinoneofthem (here inthe ALE). Following fromthe Nernst equation, it can be calculatedasfollows EDonnan=

1−

2 = RT zi · F · ln

(

ai, 2 ai, 1

)

(5)

Asbefore,compartment1representsthegelphaseand2stands forthesolution.

1and

2aretherespectiveelectricalpotentials,

(5)

Fig. 1. Schematic display of the two phases in the system: the ALE gel (1) and the supernatant (2). The ions need to balance the ﬁxed negative charges on the ALE polymers.

a_i,1 andai ,2 theactivities ofioni inthe two compartments,zi is

thevalencyoftheion,Rthegasconstant,Ttheabsolute tempera-ture,andFtheFaradayconstant.

Assuming that there are no speciﬁc interactions, the Donnan potential applies toall ionsinthesystem. EquatingEq.5for dif-ferentionsi₌jandi₌kgives:

aj, 2 aj, 1

1 z_j =

ak, 2 ak, 1}

1 z_k . (6)

Eq. 6 enables calculating the distribution of a non-quantiﬁed ion(likeH+)fromthedetermineddistributionofanotherion(like Ca2+_).

2.5.3. Osmoticpressure

As a consequence of ﬁxed negative charges (as found in the ALE),anuneveniondistributionbetweengelandsupernatantcan beestablished,asdescribedabove.Thiscausesanosmoticpressure differencebetweenthegellayersandthesupernatant.Theosmotic pressure

in each of thecompartments can be calculated from theionicconcentrationsusingthevan’tHoff equation:

= RT· i

ci (7)

Inequilibrium,thegelnetworkwithstandstheosmoticpressure difference between the gel layer and the supernatant. Therefore, thisdifferencecanindicatethestrengthofthenetwork.

3. Results

3.1. Cakelayerresistance

The development of cake layer resistance during ﬁltration is showninFig.2forarepresentativeexperiment(6mMCaCl2/6mM

KCl).ForallexperimentsexecutedwithCaCl2,similargraphswere

obtained.Suchshapeoftheresistancedevelopmentiscommonfor foulingexperiments(Listiarinietal.,2009).Theslightconcave cur-vaturesuggeststhatthecakelayersinthisworkwerecompressed duringformation(RoordaandvanderGraaf,2001).

The speciﬁccakelayer resistance

α

m wascalculatedusingthe

VSS determined directlyafter ﬁltration. The resultsare shown in the bubble chart of Fig. 3. Differences between the tested ionic compositions were small. Only with increasing CaCl2 content, a

slight increasein thespeciﬁc cakelayer resistancewasobserved. AsystematiceffectofKClwasnotfound.

Fig. 2. Development of cake layer resistance throughout ﬁltration, including a trend line for the data above 400 mL. Data is shown for 120 mg/mL ALE, 6 mM CaCl 2 /6 mM KCl. Data points indicate the averaged resistance over 10 min.

3.2. Composition

3.2.1. Swellingbehaviourafterpreparation

Comparing gel layers analysed directly after production with thoseexaminedafter12 daysshowednosigniﬁcant differencesin VSScontent.Nomicrobialgrowthwasobservedonthe12daysold gellayers.TheamountofALEwasconsideredconstantduringthe observationperiod.

Itwasexpectedthataftertheﬁltration,thegellayerswould ad-justtotheremovaloftheappliedpressurebyswellingand even-tuallyreachanequilibriumthickness.Thishydrogel-likebehaviour hasbeenobservedforalginategellayersundersimilarconditions (Davidovich-PinhasandBianco-Peled,2010).Theequilibrationtook a long time, though (see Fig. 4). In a prolonged observation pe-riod,poresappearedinsome gels,asrevealedby OCT,whichwill be discussed insection 3.4.Forall gel layers, an initially fast in-creaseinthicknesswasobserved, followedby a periodofslower

(6)

Fig. 3. Bubble chart of the ﬁnal speciﬁc cake layer resistance [10 15 m/kg] of the ALE gel layers as a function of CaCl

2 and KCl concentrations of the solution. The bubble diameter indicates the speciﬁc cake layer resistance. Complete data, including standard deviations, are in the SI, Table E .

Fig. 4. VSS density ρVSS as a function of time after formation of ALE gel layers with A) only CaCl 2 , B) constant KCl, C) constant CaCl 2 , and D) constant ionic strength. The error bars indicate the standard deviation over 4 samples.

increase. The thicknesswasconvertedinto VSSdensity

ρ

VSS.The

developmentof

ρ

VSS asa functionoftime is showninFig.4 for

thedifferentioniccompositions.

The densityof gel layers withconstant CaCl2 and varyingKCl

concentrations in the supernatant as the only variable showed fairlyidenticalbehaviour(Fig.4C).Incontrast,anincreaseinCaCl2

concentrationresultedinhigherVSSdensities(Fig.4A,4Band4D).

An exception from this observation was found at CaCl2

concen-trations above 8 mM CaCl2: the density and swelling behaviour

didnotdepend onCaCl2 concentrationanymore (Fig. 4A)oronly

slightly(Fig.3B),suggestingsaturation.Slightlysmallerdifferences in VSSdensity were observed between the sampleswith 3 mM, 6mM and8 mM CaCl2 shown inFig.4D(with varying KCl

con-centrations)comparedtoFig.4A(withoutKCl)and4B(withstable

(7)

Table 2

Estimation of osmotic pressure difference [Pa] between the ALE gel layers and the supernatant concentration after 12 days of storage, as calculated by the experimentally determined concentrations of Ca 2+_{and K}+_{. Errors concern the standard} deviation over four samples.

Ca 2+_{[mM] →} K + [mM] _↓ ₁ ₃ ₆ ₈ ₁₂ ₁₄ 24 - - 43.2 ± 25.5 - - - 15 - 0.3 ± 1.4 25.0 ± 18.6 - - - 6 - 18.4 ± 5.4 47.3 ± 7.1 48.9 ± 6.8 59.1 ± 14.7 - 0 29.3 ± 6.8 23.8 ± 4.0 51.4 ± 6.4 67.2 ± 7.0 - 72.7 ± 14.4 Table 3

Osmotic pressure [Pa] in the ALE gel layers transferred to supernatant with higher CaCl 2 concentration. Analysis after 7 days. As a reference, the data for the blank after 12 days is shown as well. Errors concern the standard deviation over four samples, where applicable.

storage time

low blank:3 mM CaCl 2 6 mM KCl

Produced:3 mM CaCl 2 6 mM KCl After 2 days:12 mM CaCl 2 6 mM KCl

Produced:3 mM CaCl 2 6 mM KCl Stored:12 mM CaCl 2 6 mM KCl high blank:12 mM CaCl 2 6 mM KCl 7 d 33.1 46.6 65.8 - 12 d 18.4 ± 5.4 - - 59.1 ± 14.7

KClconcentration).Theinﬂuence ofCaCl2 concentrationappeared

tobemuchmoresigniﬁcant,though. 3.2.2. Iondistribution

Ca2+ _and _K+ _both _accumulated_in _the_gel _layers _(the

concen-trations are summarised in the SI in Tables A and B). While for Ca2+ _the _{concentration} _found _inside _the _gels _initially _was _up _to

25timesthat inthesupernatant,withamaximumof4timesthe supernatantconcentration theaccumulationofK+ inside thegels wasmuchmoremoderate.AsfortheVSSdensities,adecreaseover timewasobserved.

Alltypesofsmallions,providedthattheywerenotirreversibly bound tothegelonthetimescaleoftheexperiments,would dis-tribute overthe two compartmentsaccordingto the Donnan po-tential(cf.section 2.5.1).Whethertheions indeeddistributed ac-cording toa Donnan equilibrium, waschecked by comparingthe Donnan potential values calculated from both the distributions of K+ ions and Ca2+ _ions _(SI, _Tables _H _and _J). _All _values _were

found between −10 mV and −40 mV, in the range where pas-sively established potentials in biological systems were expected (Sperelakis, 2012). The reasonable agreementbetween the values calculatedfromthedistributionsofCa2+_and_K+_suggests_that_both

typesofionscouldfreelymovebetweenthetwocompartmentson thetimescaleoftheexperiments.

WithEq.6theH+activitiesinsidethegellayerswereestimated from the Ca2+ _activities, _considering _the _supernatant _H+ _activity

wasconstantataH ≈ 10−4 mM(pH7).Thecalculationspredicta

slightdecreaseofpHinsidethegellayerstopH6.6-6.8. Based on the Donnan equilibrium and electro-neutrality re-quirement,theconcentrationofﬁxedchargesZintheALEwas es-timated usingEq.4.Forthecalculations, itwasassumedthatthe contribution ofcations other thanCa2+ _and_K+ _to_the

neutralisa-tionofZwasnegligibleandthatCl− (SI,TablesG)wasthe domi-nantanioninthesystem.Anaveragevalueof1.05± 0.20mmol/g VSSwasobtained(datainSI,TablesK).

3.2.3. Osmoticpressure(networkstrength)

Because of their very low molar concentrations, the ALE molecules as well as H+ andOH− hardly contributedto the os-motic pressuredifferenceandonlyCa2+_,_K+_and_Cl− _were

consid-ered. Theresultingvaluesfor

,calculatedwithEq.7,areshown in Table2 andTable 3.After preparationof thegel layers, water ﬂewinsidethegellayerstominimisetheosmoticpressure differ-enceandcausedswelling.Atsomepoint,furtherswellingwas pre-ventedbytheopposingelasticforceoftheALEnetwork.In equilib-rium,theosmoticpressuredifferenceis,therefore,anindicatorfor

thenetworkstrength.TheresultsshowthatwithincreasingCaCl2

concentrationinthesupernatant,thenetworkstrengthslightly in-creased. This indicates that Ca2+ _was _involved _in _crosslinking _of

thegellayers.

3.3. ReversibilityofswellingbyincreasingtheCa2 +concentration To differentiate between the inﬂuence of CaCl2 available

dur-ing ﬁltration and CaCl2 available during the swelling, an

exper-iment with a change in CaCl2 concentration during storage was

performed.Gel layerswere producedwithalow calcium concen-tration (3 mMCaCl2,6mM KCl), andthen transferred tostorage

solutionswithahighcalciumconcentration(12mMCaCl2,6mM

KCl)directly(sampleF)andaftertwo days(sampleL).The devel-opmentoftheirVSSdensityintimeisshowninFig.5,compared toalow blank,preparedandstoredwith3mMCaCl2/6mMKCl.

Alsoshownisahighblank,producedandstoredat12mMCaCl2,

6mMKCl.

Already after 3 minutes, sampleF had a higher density than those stored in the low CaCl2 solution (Fig. 5B). The highblank

wasatthis momentstill much denser. After 30min, thedensity ofsampleFequalledthatofthehighblank.Inthelongerterm,it evenseemedtoreachaslightlyhigherdensitythanthis.

AftertransferringsampleLtoahighcalciumconcentration so-lution,compactionoftheALEgellayerwasobservedwithinafew minutes (Fig. 5C). Thisprocess continued forabout6 h.Then an apparent steady-state wasreachedthat lasted forthe restof the observation period (Fig. 5A). The density achieved by this com-paction stayed below those ofthe highblank andsample F. The swelling was, therefore, described as partly reversible. Similarly, theCa2+_content_per_VSS_in_the_transferred_gel_layers_did_not

com-pletelyreachthatofthehighblank(datanotshown).

Inaccordancewiththedensitydata,theosmoticpressuredata for the reversibility experiment (Table 3) showed a clear hierar-chyinnetworkstrengthincreasingfromthelowblankviatheone transferredto12mMCaCl2after2daystotheonedirectlystored

in12mMCaCl2.Alsointhiscase,whathappenedduringthetwo

daysofstorageinthelowconcentration solution,waspartly irre-versible.

3.4. Additionalripening

The experiments described in section 3.2 were limited to 12 daysbecause that wasfoundto be theperiod overwhich all gel layers kept their macroscopicintegrity, whichwasa requirement to calculate andcompare the densities. When storedfor a more

(8)

Fig. 5. Comparison of the swelling behaviour of gel layers produced with 3 mM CaCl2 and 6 mM KCl and subsequently stored in 12 mM CaCl2/6 mM KCl (high calcium, sample F, + ) and 3 mM CaCl2/6 mM KCl for two days and then transferred to 12 mM CaCl2/6 mM KCl (low calcium, sample L, x). For comparison, blanks produced and stored with either high (circle) or low (diamond) calcium concentration are shown. A) shows the whole range of the experiment, B) zoomed in on the ﬁrst two days and C) zoomed in on the swelling after transfer to the higher calcium concentration after two days. Since those were single experiments, the error bars show the standard deviation over 4 samples only for the high blank.

extendedperiod,orwithlessoptimisedconcentrationsofALEand ions, initially voidswere observed inthe gel layers.Eventually, a ﬁbrous networkdeveloped.Thisdevelopmentisillustrated bythe optical coherencetomography(OCT) picturesinFig.6forgel lay-ersproducedwith60mg/LALEand45mMionicstrength30min afterproduction,after3daysandafter5days.

The usedOCT systemhada resolutioninthe rangeof10 μm, meaningthatporescouldonlybedetectedassoonastheyreached thissizeinbothdimensions.Itseemslikelythatthesepores devel-opedonamicro-scalebeforestructuralchangescouldbeobserved withtheOCT.Structural rearrangementsintheALEnetworkwere considered themajorcausefortheobservedslowswellingofthe gel layers described insections 3.1-3.3. Increasingthe resolution, forexample,bytakingSEMpicturesonstabilisedsamples,may re-vealsuchchangesonthemicrostructurelevel.

4. Discussion

Thegellayers’compositionandappearanceresultfromthe bal-ances betweenelectrostatic interactions inthe gel, osmotic pres-suredifferencesbetweenthegelandtheoutsidesolution,andthe ability ofthe gel networkto withstandswelling by waterintake. Thesemechanismsarediscussedbelow.

4.1. Iondistribution

While the high speciﬁcity and chelating nature of the Ca2+

crosslinksinalginateare wellknown,thepresentresultsindicate nospeciﬁc(irreversible)bindingofCa2+_to_ALE._Therefore,_it_is

ex-pectedthatCa2+ _can_be _easily_removed, _for_example,_by _dilution,

orreplacedbyotherions.Basedonthisstudy’sresults,other diva-lentcationsareprobablyalsoabletoinducethecrosslinking. For-mationofstablegelswithcomparableelasticpropertieshasindeed beenconfirmedwithawholerangeofdivalentcations(Felzetal., 2020a).Toidentifythe(non)specificityofthoseinteractions, com-positionanalysislikedoneinthisstudycanbeused.Thedifference withalginate in binding maybe explainedby thefinding that a significant amountof thechargedgroups inALEis sulfaterather thancarboxylate(Felzetal.,2020b).

Basedonelectro-neutralityandtheDonnanpotential,the num-ber ofnegativechargesin theALEwascalculated as1.05 ± 0.20 mmol/g VSS. One Ca2+ _cation _could _neutralise _two _negative

chargesintheory,sothisvalueiswell inlinewiththe measured Ca2+ _{concentrations}_in_the_ALE_gel_layers _of₂₀₀ _-₇₀₀

_μ

_mol _per

g VSS(SI, Fig.A). Itshould benoted, however,that followingthe Donnan potential also Cl− distributed over gel layer and

(9)

Fig. 6. Ripening of ALE gel layers observed by OCT over 5 days. The images show an ALE gel made from a solution of 60 mg/L ALE and 45 mM ionic strength. Note the changed composition in comparison with the measurements described earlier, which accelerated the ripening process.

natant.Asaconsequence,notevery Ca2+ _ion_inside_the_gel_layers

contributedtothecrosslinking.

The pKa values of the carboxylate groups of mannuronic and

guluronic acidwere found at3.38 and3.65(Draget etal., 1994). These groups are all negatively charged at pH 7 and thus con-tributedtoZintheALEgellayers.Sulfatesarestrongacids,so neg-ativelychargedindependentlyofpH.Therefore,thecurrent exper-imentsandcalculationsdidnotidentifywhatgroupsofALEwere involved incrosslinking. Onthebasis offurther chemical charac-terisation of ALE, experiments in which the pH is varied can be usefulfordeeperinsight.

4.1.1. CorrelationbetweenCa2+ _content,_density_and_strength_of_the

ALEgellayers

With increasing CaCl2 concentration in the supernatant, the

ALE gel layers asprepared in thiswork were found denser until a plateau was reached around 8 mM Ca2+ ₍_Fig. ₄_). _Such

corre-lation has beenstudied intensively for alginategels ( Davidovich-Pinhas and Bianco-Peled, 2010) and has been explained with a higher number of crosslinks with increasing Ca2+ _{concentration.}

AlsoforbacterialEPS,Ca2+_availability_has_been_directly_linked_to

denserﬁlms(GoodeandAllen,2011;Körstgensetal.,2001). Thiswork shows,inaddition,acorrelation betweenCa2+

con-tent anddensityandthe networkstrength(cf. section3.2.3).The supernatant’sionic compositiondeﬁnedthe ionconcentration in-side the gels(given the ﬁxed amount of charges ofthe ALE per g VSS)andthus the osmoticpressure difference (section 2.5). At the same time, the network strength determined the maximum amount of water that could be taken up to reduce the osmotic pressure difference. Apparently,over time thismaximumamount increased,associatedwithaslowbutirreversibleweakeningofthe network structure(as showninTable3,further discussionofthe reversibilityinsection4.3).Themechanismsbehindthiseffectare probablyrelatedtotheslowstructuralrearrangementresultingin voidformation(Fig.6)andrequirefurtherinvestigation.

4.2. HydraulicresistanceofALEgellayers

A positive correlation between EPS density and hydraulic re-sistance ofbioﬁlms has been postulated before (Desmond et al., 2018; Jafari et al., 2018). Also, the Ca2+ _{concentration} _in _ALE

films has been correlated to flux decline in membrane filtration (Herzbergetal., 2009). Thisgoesalong withthefinding that the

speciﬁc cake layer resistance of the ALE gel layers in this work slightly increased with supernatant CaCl2 concentration (Fig. 3).

In this context, it attractedattention that while the VSSdensity reached a maximum around 110 mg/cm3_, _the _speciﬁc _resistance

stillslightlyincreased(e.g.between8mMCaCl2and14mMCaCl2, Fig.3).Thedifferencesinrelationtothestandarddeviations(cf.SI, TableE)weresosmall,however,thatnofurtherconclusionscould bedrawnfromthesedata.

4.3. Compressibility,relaxationandripening

Compressibility,asobservedfortheALEgellayersinthiswork, isafeatureofbiofilmsthathasbeeninvestigatedforseveralyears (Jafari et al., 2018), andthat has alsobeen usedasan indication fortheviscoelastic behaviour ofbiofilms(Safari etal., 2015). The relaxationof biofilmsaftercompression has beendescribedwith thehelpofMaxwellsprings(Jonesetal.,2011;Safarietal.,2015), asan approximately exponentialprocess. In ourexperiments, re-laxation wasexpected as soon as the filtration pressure was re-leased.Duetoexperimentalrestrictions,thechangesinthe thick-nessoftheALEgellayersinthisstudywereonlyrecordedfromca. 3minutesafterpressurerelease.Therefore,aquantitativeanalysis of thecollected data appeared unreliable. For accurate collection andanalysisof relaxationdata toextract characteristic viscoelas-ticdata,systemswithcontrollablecompressionandinstantaneous observationofthestressandstrainareneeded. Suchexperiments willalsobeofinteresttodistinguishbetweencross-linkedgel lay-ersandnon-cross-linked cakelayers.Also, theinfluenceof differ-entgelproductionprocedurescouldbeinterestinginthisregard.

Interestingobservations inthisstudywere the long continua-tion of the swelling (Fig. 4), and the eventual development of a fibre-like ALE network (Fig. 6). Fibrous structures have been ob-served in biofilm matrices (Romero et al., 2010) and are usually attributedto amyloids. The present studysuggests that ALE also forms such fibres, in accordance with previous work (Lin et al., 2018). The observation that the swelling process wasonly partly reversible(section3.3)indicates thata slowmolecular rearrange-mentstartedwiththeinitialformationofmicroscopicvoidsinthe structure (cf. Jafari et al., 2018) right when the pressure was re-leased. Because the network hadstarted to be weakened by the voids(cf.Table3,section4.1.1),neitherthedensitynortheosmotic pressuredifferenceofgellayersdirectlystoredinthehigherCaCl2

(10)

Fi-N.-M. Pfaff, J.M. Kleijn, M.C.M. van Loosdrecht et al. Water Research 195 (2021) 116959

brousstructuresandadecreaseindensityoverlongertimescales (between 7 and12 days) were especially found inthe gel layers that werepreparedwithandstoredatthelowerCaCl2

concentra-tions (1and3mM).Apparently, ﬁbreformationwaspreventedor delayedbyCa2+_crosslinks_in_the_network.

AccordingtoDesmondetal.(Desmondetal.,2018b), compres-sioncausedby filtrationpressure isreversibleforstructurally ho-mogeneous biofilms and irreversible for films with a heteroge-neousstructure.Thisworksupportsthisfinding,showingthat net-works withvoidswere weaker than morehomogenous networks withoutvoids.

4.4. Outlook

This work provides valuable insight into the interaction be-tween ALE and Ca2+ _in _the _presence _of _K+ _and _Cl−_. _As _a _next

step, theinﬂuence ofCa2+ _content _of_ALE_gels_on _their

mechan-icalpropertiessuchasmodulus,strengthandadhesionwillbe in-vestigated.Whileacloserlookontheswellingbehaviourcanbea start,e.g.byobservationintheﬁltrationcellduringﬁltration, pos-siblyalsounderapplicationofdifferentpressures,determiningthe gels’viscoelasticpropertiesbyrheologicalmeasurementswill pro-vide quantitativedata.Thiswillmakeitpossibletolinkthe com-positiontomechanicalpropertiesofthegellayers andwillbethe subjectofaforthcomingpaper.

The systemof ALE andspecific ions still represents a simpli-fied modelfor theEPS matrixof biofilms.The modelcan be ex-tended by addingotherkinds ofmolecules,such aspeptides, hu-mic acidsandeDNA,toeventuallygetclosetorealbiofilms’ com-plexity. Evaluating the interactions within the ALE network, also beyondelectrostaticones,withaddedcompounds,andbasedona better chemicalcharacterisationofALEandEPScanleadthepath towards understanding the cohesive forcesof biofilms.Thisis an importantsteptowardstailoredcleaningstrategies.

5. Conclusions

– ThedensityandnetworkstrengthofALEgellayersdependon crosslinkingwithamultivalentcationlikeCa2+_.

– Ca-ALEgellayersproducedonamembranebydead-end ﬁltra-tionswell,afterpressurerelease,forupto12days.Thisprocess isaccompaniedbyaweakeningofthestructureandispartially irreversible.Oneofthemechanismsbehinditisaslow molec-ularrearrangementofALE, culminating inthedevelopmentof aﬁbrousstructure.

– Theaccumulationofcations,includingCa2+_,_over_a_Ca-ALE_gel

andits supernatant, canbe describedasaresultof aDonnan potentialinducedbytheﬁxedchargesontheextracellular poly-mers. Thisindicatesthat nospeciﬁcbindingisinvolvedinthe physicalcrosslinkingofALEgellayersbyCa2+_.

DeclarationofCompetingInterest

Theauthorsdeclarethattheyhavenoknowncompeting ﬁnan-cialinterestsorpersonalrelationshipsthatcouldhaveappearedto inﬂuencetheworkreportedinthispaper.

Acknowledgement

Thisworkwasperformedinthecooperationframeworkof Wet-sus, European Centre Of Excellence For Sustainable Water Tech-nology (www.wetsus.nl). Wetsus is funded by the Dutch Min-istry of Economic Affairs and Ministry of Infrastructure and En-vironment, the European Union Regional Development Fund, the Province of Fryslân, andtheNorthernNetherlands Provinces.The authors wouldlike to thankthe members ofthe research theme “Bioﬁlms” forfruitfuldiscussionsandﬁnancialsupport.

Supplementarymaterials

Supplementary material associated with this article can be found,intheonlineversion,atdoi:10.1016/j.watres.2021.116959.

References

Bajpai, S.K. , 2001. Swelling studies on hydrogel networks - a review. J. Sci. Ind. Res. (India). 60, 451–462 .

Davidovich-Pinhas, M., Bianco-Peled, H., 2010. A quantitative analysis of alginate swelling. Carbohydr. Polym. 79, 1020–1027. doi: 10.1016/J.CARBPOL.2009.10.036 . Desmond, P., Morgenroth, E., Derlon, N., 2018. Physical structure determines com-

pression of membrane bioﬁlms during Gravity Driven Membrane (GDM) ultra- ﬁltration. Water Res. 143, 539–549. doi: 10.1016/j.watres.2018.07.008 .

Draget, K.I., Skjåk Bræk, G., Smidsrød, O., 1994. Alginic acid gels: the effect of alginate chemical composition and molecular weight. Carbohydr. Polym. 25, 31–38. doi: 10.1016/0144-8617(94)90159-7 .

Felz, S., Al-Zuhairy, S., Aarstad, O.A., van Loosdrecht, M.C.M., Lin, Y.M., 2016. Ex- traction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge. J. Vis. Exp. e54534. doi: 10.3791/54534 .

Felz, S., Kleikamp, H., Zlopasa, J., van Loosdrecht, M.C.M., Lin, Y., 2020a. Impact of metal ions on structural EPS hydrogels from aerobic granular sludge. Bioﬁlm 2. doi: 10.1016/j.bioﬂm.2019.10 0 011 .

Felz, S., Neu, T.R., van Loosdrecht, M.C.M., Lin, Y., 2020b. Aerobic granular sludge contains Hyaluronic acid-like and sulfated glycosaminoglycans-like polymers. Water Res 169. doi: 10.1016/j.watres.2019.115291 .

Flemming, H.-C., 2020. Biofouling and me: My Stockholm syndrome with bioﬁlms. Water Res. 173, 115576. doi: 10.1016/j.watres.2020.115576 .

Ganji, F., Vasheghani-Farahani, S., Vasheghani-Farahani, E., 2010. Theoretical de- scription of hydrogel swelling: a review. Irymer Journalanian Pol. 19, 375–398. doi: 10.10 07/s12303-0 09-0 0 04-6 .

Goode, C. , Allen, D.G. , 2011. Effect of calcium on moving-bed bioﬁlm reactor bioﬁlms. Water Environ. Res. 83, 220–232 10.2175/106143010 ×12780288628255 .

Hamer, W.J., Wu, Y.-C., 1972. Osmotic coeﬃcients and mean activity coeﬃcients of uni-univalent electrolytes in water at 25 °C. J. Phys. Chem. Ref. Data 1, 1047– 1100. doi: 10.1063/1.3253108 .

Herzberg, M., Kang, S., Elimelech, M., 2009. Role of Extracellular Polymeric Sub- stances (EPS) in biofouling of reverse osmosis membranes. Environ. Sci. Technol. 43, 4393–4398. doi: 10.1021/es90 0 087j .

Jafari, M., Desmond, P., van Loosdrecht, M.C.M., Derlon, N., Morgenroth, E., Piciore- anu, C., 2018. Effect of bioﬁlm structural deformation on hydraulic resistance during ultraﬁltration: a numerical and experimental study. Water Res 145, 375– 387. doi: 10.1016/j.watres.2018.08.036 .

Jones, W.L., Sutton, M.P., McKittrick, L., Stewart, P.S., 2011. Chemical and antimicro- bial treatments change the viscoelastic properties of bacterial bioﬁlms. Biofoul- ing 27, 207–215. doi: 10.1080/08927014.2011.554977 .

Kielland, J., 1937. Individual activity coeﬃcients of ions in aqueous solutions. J. Am. Chem. Soc. 59, 1675–1678. doi: 10.1021/ja01288a032 .

Körstgens, V., Flemming, H.-C., Wingender, J., Borchard, W., 2001. Inﬂuence of calcium ions on the mechanical properties of a model bioﬁlm of mucoid Pseu- domonas aeruginosa. Water Sci. Technol. 43, 49–57. doi: 10.1371/journal.pone. 0091935 .

Li, Q., Elimelech, M., 2004. Organic fouling and chemical cleaning of nanoﬁltration membranes: measurements and mechanisms. Environ. Sci. Technol. 38, 4683– 4693. doi: 10.1021/es0354162 .

Lin, Y.M., de Kreuk, M., van Loosdrecht, M.C.M., Adin, A., 2010. Characterization of alginate-like exopolysaccharides isolated from aerobic granular sludge in pilot- plant. Water Res 44, 3355–3364. doi: 10.1016/j.watres.2010.03.019 .

Lin, Y.M., Reino, C., Carrera, J., Pérez, J., van Loosdrecht, M.C.M., 2018. Glycosylated amyloid-like proteins in the structural extracellular polymers of aerobic granular sludge enriched with ammonium-oxidizing bacteria. Microbiologyopen 7. doi: 10.1002/mbo3.616 .

Listiarini, K., Sun, D.D., Leckie, J.O., 2009. Organic fouling of nanoﬁltration membranes: Evaluating the effects of humic acid, calcium, alum coagulant and their combinations on the speciﬁc cake resistance. J. Memb. Sci. 332, 56–62. doi: 10.1016/j.memsci.2009.01.037 .

Nagashima, A., 1977. Viscosity of water substance—new international formulation and its background. J. Phys. Chem. Ref. Data 6, 1133–1166. doi: 10.1063/1.555562 . Pronk, M., de Kreuk, M.K., de Bruin, B., Kamminga, P., Kleerebezem, R., van Loos- drecht, M.C.M., 2015. Full scale performance of the aerobic granular sludge process for sewage treatment. Water Res 84, 207–217. doi: 10.1016/j.watres.2015.07. 011 .

Radchenkova, N., Boyadzhieva, I., Atanasova, N., Poli, A., Finore, I., Di Donato, P., Nicolaus, B., Panchev, I., Kuncheva, M., Kambourova, M., 2018. Extracellu- lar polymer substance synthesized by a halophilic bacterium Chromohalobac- ter canadensis 28. Appl. Microbiol. Biotechnol. 102, 4 937–4 94 9. doi: 10.1007/ s00253- 018- 8901- 0 .

Romero, D., Aguilar, C., Losick, R., Kolter, R., 2010. Amyloid ﬁbers provide structural integrity to Bacillus subtilis bioﬁlms. Proc. Natl. Acad. Sci. 107, 2230–2234. doi: 10.1073/pnas.0910560107 .

Roorda, J.H., van Loosdrecht, M.C.M., 2001. New parameter for monitoring fouling during ultraﬁltration of WWTP eﬄuent. Water Sci. Technol. 43, 241–248. doi: 10. 2166/wst.2001.0631 .

(11)

Safari, A., Tukovic, Z., Walter, M., Casey, E., Ivankovic, A., 2015. Mechanical properties of a mature bioﬁlm from a wastewater system: from microscale to macroscale level. Biofouling 31, 651–664. doi: 10.1080/08927014.2015.1075981 .

Seviour, T., Derlon, N., Dueholm, M.S., Flemming, H.C., Girbal-Neuhauser, E., Horn, H., Kjelleberg, S., van Loosdrecht, M.C.M., Lotti, T., Malpei, M.F., Nerenberg, R., Neu, T.R., Paul, E., Yu, H., Lin, Y., 2019. Extracellular polymeric substances of bioﬁlms: Suffering from an identity crisis. Water Res. 151, 1–7. doi: 10.1016/j. watres.2018.11.020 .

Seviour, T., Pijuan, M., Nicholson, T., Keller, J., Yuan, Z., 2009. Understanding the properties of aerobic sludge granules as hydrogels. Biotechnol. Bioeng. 102, 1483–1493. doi: 10.1002/bit.22164 .

Sperelakis, N., 2012. Chapter 10 - Gibbs-donnan equilibrium potentials. In: Cell Physiology Source Book. Elsevier BV, pp. 147–151. doi: 10.1016/ B978- 0- 12- 387738- 3.0 0 010-X .

van den Brink, P., Zwijnenburg, A., Smith, G., Temmink, H., van Loosdrecht, M., 2009. Effect of free calcium concentration and ionic strength on alginate fouling in cross-ﬂow membrane ﬁltration. J. Memb. Sci. 345, 207–216. doi: 10.1016/ j.memsci.2009.08.046 .

Wang, X., Spencer, H.G., 1998. Calcium alginate gels: formation and stability in the presence of an inert electrolyte. Polymer (Guildf) 39, 2759–2764. doi: 10.1016/ S0 032-3861(97)0 0597-1 .