Low voltage iron electrocoagulation as a tertiary treatment of municipal wastewater

Delft University of Technology

Low voltage iron electrocoagulation as a tertiary treatment of municipal wastewater

removal of enteric pathogen indicators and antibiotic-resistant bacteria

Bicudo Perez, B.; van Halem, Doris; Trikannad, Shreya Ajith; Ferrero, Giuliana; Medema, Gertjan

DOI

10.1016/j.watres.2020.116500

Publication date

2021

Document Version

Final published version

Published in

Water Research

Citation (APA)

Bicudo Perez, B., van Halem, D., Trikannad, S. A., Ferrero, G., & Medema, G. (2021). Low voltage iron

electrocoagulation as a tertiary treatment of municipal wastewater: removal of enteric pathogen indicators

and antibiotic-resistant bacteria. Water Research, 188, 1-10. [116500].

https://doi.org/10.1016/j.watres.2020.116500

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Water Research 188 (2021) 116500

ContentslistsavailableatScienceDirect

Water

Research

journalhomepage:www.elsevier.com/locate/watres

Low

voltage

iron

electrocoagulation

as

a

tertiary

treatment

of

municipal

wastewater:

removal

of

enteric

pathogen

indicators

and

antibiotic-resistant

bacteria

Bruno

Bicudo

a,∗

_,

_Doris

_van

_Halem

a

_,

_Shreya

_Ajith

_Trikannad

a

_,

_Giuliana

_Ferrero

b

_,

Gertjan

Medema

a,c,d

a Faculty of Civil Engineering and Geosciences, Water Management Department, TU Delft, The Netherlands

b Water Supply, Sanitation and Environmental Engineering Department, IHE Delft Institute for Water Education, The Netherlands c KWR Watercycle Research Institute, The Netherlands

d Michigan State University, Michigan, USA

a

r

t

i

c

l

e

i

n

f

o

Article history: Received 8 July 2020 Revised 15 September 2020 Accepted 5 October 2020 Available online 6 October 2020 Keywords: Antimicrobial resistance electrocoagulation iron pathogens secondary effluent water reclamation

a

b

s

t

r

a

c

t

Inthispaperweanalysethefeasibilityoflowvoltageironelectrocoagulationasameansofmunicipal secondaryeffluenttreatmentwithafocusonremovalofmicrobialindicators,AntibioticResistantBacteria (ARB)andnutrients.Alaboratoryscalebatchunitequippedwithironelectrodeswasusedonsynthetic andrealsecondaryeffluent fromamunicipalwastewatertreatmentplant.Syntheticsecondaryeffluent wasseparatelyassayedwithspikedEscherichiacoliWR1andwithbacteriophageX174,whilereal ef-fluentsampleswerescreenedbeforeandaftertreatmentforE.coli,ExtendedSpectrum Betalactamase-producing E.coli,Enterococci,VancomycinResistantEnterococci,Clostridiumperfringenssporesand so-maticcoliphages.Chargedosage(CD)andchargedosagerate(CDR)wereusedasthemainprocess con-trolparameters.Experimentswithsyntheticsecondaryeffluentshowed>4log10and>5log10removalfor phageX174 and for E.coli WR1,respectively. Inrealeffluents, bacterialindicator removal exceeded 3.5log10,ARBwereremovedbelowdetectionlimit(≥2.5log10),virusremovalreached2.3log10andC. per-fringens spore removalexceeded 2.5log10.Experiments inbothreal and syntheticwastewater showed thatbacterialremovalincreasedwithincreasingCDanddecreasingCDR. Virusremovalincreasedwith increasingCD butwasirresponsivetoCDR. C.perfringenssporeremoval increased withincreasingCD yetreachedaremovalplateau,beingalsoirresponsivetoCDR.Phosphateremovalexceeded99%,while totalnitrogenandchemicaloxygendemandremovalwerebelow15%and58%,respectively.Operational costestimatesweremadeforpowerandironplateconsumption,andwerefoundtobeintherangeof 0.01to0.24€/m3_{forthedifferentassayedconfigurations.Inconclusion,lowvoltageFe-ECisapromising} technologyforpathogenreductionofsecondarymunicipaleffluents,withlog10 removalscomparableto thoseachievedbyconventionaldisinfectionmethodssuchaschlorination,UVorozonation.

© 2020TheAuthor(s).PublishedbyElsevierLtd. ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/)

1. Introduction

At present, one-third oftheworld’s population livesin water-stressed countriesand by 2025 the figure is expected to rise to two-thirds (Elimelech, 2006). Discharge of untreated wastewater into the water sources can degrade waterquality, increasing the risk to humanhealth andecosystemdegradation. Inthiscontext,

∗_{Corresponding author.}

E-mail address: b.bicudoperez@tudelft.nl (B. Bicudo).

waterreuseisoftenrecognisedasasolutionwithgreatpotential inreducing thegapbetweenavailability anddemand.Agriculture iscurrentlythelargestconsumerofreclaimedwater,providingan allyearroundwatersourcewithanestimate 200millionfarmers worldwideusingeitherrawortreatedwastewaterforirrigationof over2000km2 (Qadiretal.,2007,Raschid-sally&Jayakody,2008), constitutingroughly8% ofallirrigated landintheplanet,mostof whichhappensinAsia(Howell,2001).

TheUnited Nationsagenda forSustainableDevelopmentGoals targetsimprovementinwaterquality byreducingpollution, elim-inating the discharge of polluted waters, halving the proportion

https://doi.org/10.1016/j.watres.2020.116500

of untreatedwastewaterand increasing safewaterreuse globally (Anfruns-Estradaetal.,2017).Despitereclamationbeingan attrac-tive concept, municipalwastewaterharbours a wide rangeof en-teric pathogenssuch asvirus, bacteria, protozoa, parasiticworms andeggs,andits(re)usecalls forcarefulmanagementofits asso-ciated healthrisks.Such risksdepend onthetype ofwatertobe recycled,thetypeandconcentrationofpathogens,andin particu-lar, theabilityofsuch pathogensto survivetreatment,aswell as the type ofexposure ofsusceptiblecommunitymembersto such waters.Therequiredlevelofpathogenreductioninreclaimed wa-ter dependson the nature of reuseapplication andpotential for humanexposuretowater.

Inthiscontext,thefeasibilityofIron(0)Electrocoagulation (Fe-EC)formicrobialattenuationinmunicipalsecondaryeffluentswas explored. Previous studies have demonstrated the effect of Fe-EC in the removalof a wide range of microorganismsfrom bac-teria to viruses in different water matrices, mainly for drinking water applications (Ghernaout et al., 2019, Heffron et al., 2019a,

Heffron etal., 2019c,Delaire. 2016,). The applicationofFe-EC for thereclamationofsecondarymunicipaleffluentsprovides interest-ingadvantages,sincetheirhigherconductivitiesreduceelectrolysis cost, plus residual iron is a lesserconcern thanfor drinking wa-ter. Inspite ofthis, only few Fe-EC studies focussedon real sec-ondary effluents (Ding et al., 2017, Anfruns-Estrada et al., 2017,

Llanosetal.,2014)andtothebestofourknowledge,none inves-tigatedtheremovalofAntibioticResistantBacteria(ARB).

The main objectiveof the present research is to evaluate the performance oflow voltageFe-ECduringthetreatmentof munic-ipalsecondaryeffluents(forreclamationpurposes)intheremoval of microbial pathogenindicators (Escherichia coli, enterococci, so-matic coliphages and Clostridium perfringens spores) and Antibi-otic ResistantBacteria (ExtendedSpectrumBeta Lactamase(ESBL) andCarbapenemResistant(CRE)-E.coliandVancomycinResistant Enterococci-VRE). Other parameters of importance such as nutri-ents, COD, turbidity,colour, pHandresidual iron were also anal-ysed (SI Table S4). Charge Dosage (CD) and Charge Dosage Rate (CDR) were selected as the main process-control parameters for theirondosage,directlylinkedtotheelectrolysistimeandcurrent intensity,respectively(Ghernaoutetal.,2019,VanGenuchtenetal., 2017,Delaireetal.,2015,Amroseetal.,2013,Guetal., 2009).CD isdefinedasthetotalelectricchargeperunitvolumeappliedtoa givenwatersample,whileCDRisdefinedasthespeedof applica-tion ofelectriccharge(ChargeDosage perunit time).Inthisway CD[C/L]representsthetotaldoseofcurrent,whileCDR[C/L/min] representsthecurrentdosingspeed.TheinfluenceofCDandCDR in the microbial removal of municipal secondary effluents using Fe-ECiscentraltothisresearch.

2. MaterialsandMethods

2.1. Fieldsamplingandselectionofmicrobialindicators

Prior to the beginning ofthe EC experiments andin orderto collect dataon theexpectedbackgroundlevels onpathogenic in-dicator organisms and ARB in secondary effluents, a six month samplingcampaign(June-Dec2018)wasconductedinamunicipal wastewater treatment plant (WWTP) from the Netherlands. This facilityisoftheactivatedsludgetype,withprimaryandsecondary treatment and no disinfection. Grab samples from raw sewage and secondary effluent were collected approximately every two weeks andscreened fordiverseindicators,namelyE.coli, entero-cocci, Extended-SpectrumBetalactamase-producingE.coli (ESBLE), Carbapenemase-producing-E.coli,(CRE)Vancomycin Resistant En-terococci (VRE),C.perfringens sporesandsomatic coliphages.The premiseunderlyingsuchselectionwastocoverbacterial,viraland protozoan indicators,as well as ARB. The screening was

culture-based,usingselectiveagarmediumforeachoneofthe aforemen-tionedindicators.Thismicrobialtoolkitisdescribedinfurther de-tailinSupportingInformation(SITableS1).

2.2. Laboratorysetup

Parallelexperimentsincylindricalglassbeakers(0.5Lfor syn-theticeffluents and1L forrealeffluents) were conductedin the laboratory, as depicted in Fig. 1. The dual power source was a 30V-3ATENMA72-10500benchDCpowersupply,connectedwith crocodileclip cablestotwo S235steelplates(maximum percent-ages:0.14%carbon,0.10%silicium,0.80%manganese,0.025% phos-phorous,0.015%sulphur,0.010%nitrogen,0.20%copperand0.080% aluminium). Dimensions of the steel plates were 6cm x4cm, of which4cmx4cmweresubmerged(2cmemergingtoconnectthe clipcables),beingpolishedwithcoarseandfinesandpaperbefore each use. The plates were mounted inthe end of a plastic tube withcarvedparallelslotsensuringtheplatesremainedparalleland spacedapproximately1cmasdescribedelsewhere (Heffron etal., 2019b,Anfruns-Estrada etal., 2017,Ndjomgoue-Yossa etal., 2015,

Merzouk et al., 2009). The beakers were mounted on identical LABNICO L23 magnetic stirrers and fitted withPTFE coatedbars forstirringpurposes.Duringeachindividualexperiment,onlytwo process-controlparameters were varied, namely CD and CDR,by controlling the electrolysis time and the supplied amperage, re-spectively. These parameter combinations were selected before-handforeachexperiment. Stirring wasprovidedby themagnetic stirrersandthespeedwassetto100rpmforallexperiments.

For each assay, after the current wasapplied, the iron plates were removed fromthe beaker, andthe latter wascovered with aluminiumfoiltopreventthe entryofdust.Particleswereleft to settleovernight (asreportedbyDelaire etal., 2016,Delaire etal., 2015), after which the supernatant was carefully harvested with theuseofasterile 50mlserologicalpipette.Supernatantwas col-lectedfromthesurfaceuntilonlya1-2cmlayerofwateroverthe sedimentswasleft.Theharvestingprocedurewasconducted care-fully, inorder not to generateripples that could disturb the set-tledparticles.Thecollectedsupernatantwastransitorilydeposited inacleancontainer,andusedimmediatelyformicrobiologicaland physical/chemicalcharacterization.

2.3. Syntheticandrealsecondaryeffluents

The formula for the synthetic secondary effluent was based on the specifications from the Organisation for Economic Co-operationandDevelopmentguidelines(OECD,2001)andthen ad-justed based on a preceding water sampling campaign at the WWTP. Based on these samples, readily biodegradable COD was replacedby lessdegradablecompounds asexpectedin secondary effluents: yeast extract was replaced by starch, andpeptone was replacedby microcrystalinecellulose, whichalsoprovided partic-ulates.The nitrogen andphosphoroussources (ureaand dipotas-sium phosphate, respectively) were adjusted following the same principle.Sodiumchloridewasincreasedinordertoprovidea con-ductivityofapproximately1000

μ

S/cm,similartothat ofthegrab samplescollectedfromthemunicipal WWTP.Adoptingthis com-positionasthebaseline,twovariantswereproduced:onemedium with higher nutrients than the baseline (HNM), the other with lower(half)thelevelofnutrients(LNM)(Table1).

Two non-pathogenic organisms were used to spike the syn-theticeffluents,namelyE.coliWR1(NCTC13167)andsomatic col-iphage



X174 (ATCC 13706-B1),bacterial andviralindicators re-spectively.Itisworthnotingthatexperimentswereconducted ei-therwith E.coli orwithcoliphage



X174, butnot both simulta-neouslytopreventE.coli frombeinginfectedbythecoliphage.E.

B. Bicudo, D. van Halem, S.A. Trikannad et al. Water Research 188 (2021) 116500

Figure 1. Bench scale EC laboratory setup.

Table 1

Composition of synthetic effluent ( OECD 2001 ) and synthetic effluent medium with high nutrients (HNM) and low nutrients (LNM).

Concentration (mg/L) Compound OECD HNM LNM Yeast Exctract 22 - - Peptone 32 - - Starch - 8 8 Microcrystaline Cellulose - 5 5 Urea 6 8.6 4.3 Dipotassium Phosphate 28 5.4 2.7 Sodium Chloride 7 60 60

Calcium chloride dihydrate 4 4 4

Magnesium Sulphate Heptahydrate 2 2 2

coli WR1waspropagatedinTYGBbrothfor3hat37°Cto concen-trationsofapproximately2× 108 _{cfu/ml,whilephage}

_

_X174was

propagated following the ISO 10705-2_2000 method, to concen-trationsofapproximately1× 1012 _{pfu/ml.E. coliWR1and}

_

_X174

weredosedintothetestliquidatinitialconcentrationsof approx-imately 1 × 105 _{(cfu/pfu)/L in order to matchthe concentrations}

in therealsecondary effluentdetectedduringthe sampling cam-paign.

Experimentsinvolvingrealsecondaryeffluentswereconducted withunalteredgrab samples(20-30L) collecteddownstream from the secondary settlers of the aforementioned WWTP during the months ofMay-June2019. Allassayson thesesampleswere per-formed immediately upon arrival in the laboratory during the samedayofcollection.Thecompletephysical/chemical character-isticsofthesesamplescanbeobservedinSITableS2.

2.4. OperationalFe-ECparameters

Fe-EC experiments conducted on the spiked synthetic sec-ondaryeffluentsandrealsecondaryeffluentsfollowedthe configu-rationsofCDandCDRdescribedinTable2.Forsyntheticmedium, conditionsapplyforbothkindsofeffluent(HNMandLNM)andfor bothspikedindicators(E.coliWR1andsomaticcoliphage



X174). All Fe-EC experiments withsynthetic effluentwere performedin 0.5L beakers equipped with iron electrodes as described in 2.2. Synthetic medium wasfreshlypreparedevery day beforethe as-says.Experimentsusingrealsecondaryeffluentwereconductedin 1Lbeakers,usingE.coli,ESBL-E.coli,enterococci,VRE,Somatic col-iphages and C. perfringens spores as indicators. The experiments described in this section were conducted on four different days, andhenceunderslightlydifferentrealsecondaryeffluentqualities. Thecharacteristicsoftherealsecondaryeffluentandthe schedul-ing forthedifferent daysandmicrobialgroupscan befound un-derSITablesS2andS3.Thetheoreticallydosedironconcentration Fe_theo wascalculatedaccordingtoFaraday’slaw(SI).

2.5. Analyticalmethods

Microbiologicalscreeningandquantificationwasperformed ei-ther by spread plate method or by membrane filtration accord-ingto APHA-StandardMethods forthe ExaminationofWaterand Wastewater, 23rd Edition, depending on the expected microbial loadof the sample. The specific culture media usedfor each in-dicatorisdetailedinSI TableS1. Screeningofsomatic coliphages wasperformedbypourplatetechniquefollowingISO10705-2.

AnalysisofionssuchasNO₂ −,NO₃ 2 −,NH₄ + ,PO₄ 3 − andCl−in filteredwatersampleswascarriedoutwithMetrohm881basicIC plusand883compactICproIonchromatography.Fortheanalysis oftotalnitrogen,Spectroquant® nitrogencelltestwereused,with digestionat120°Cfor1h,followedbyreadinginaSpectroquant® NOVA60photometer(Merck,Germany).Totaliron(Fe+ 2 ,Fe+ 3 )was measured using Spectroquant® Iron Cell Test (1-50mgFe/L), read inthe aforementioned Spectroquant® NOVA60 (Merck,Germany) photometer. COD analysis wasperformed using HACH-Langetest kits(LCK314,15-150mgO₂ /L)with2hdigestionat148°Cand read-ingina HACHDR3900spectrophotometer. Totalsuspendedsolids analysisofsampleswascarried out accordingtoAPHA- Standard Methods fortheExaminationof WaterandWastewater23rd Edi-tion.TurbiditywasmeasuredusingTurb430IRmultimeter.Colour wasanalysedinbothunfilteredandfilteredsamplesusingUV-VIS spectrophotometerata410nmwavelength.

2.6. MethodologyforFe-ECcostestimation

Simplifiedoperational costestimatesforeach individual Fe-EC experiment were performedconsidering asinputs the consumed electricpowerandmetallic iron. Asfor2019 average energycost inThe Netherlands for amedium size consumer= 0.0679€/kWh (Eurostat,2019), andthesteelS235 cost ₌0.21_€/kg(MEPS Inter-nationalLtd,2019).

ForagivenFe-ECexperiment,inwhichUistheappliedvoltage (v),Iisthecurrentintensity(mA),tisthetreatmenttime(h)and Visthevolumeofthecell(m3 ),thentheconsumedpowercanbe estimatedas: P



kW.h m3



=U.I.t V (1)

Then,theoperationalcostforeachparticularexperimentis de-termined by theamount ofconsumed power andtheamount of dosediron(describedinSI-Faraday’sequation),multipliedbytheir respectiveunitcosts:

Cost



€/m3



=P



kW.h m3



× 0.0679€/kWh +m_Fe

(

kg

)

× 0.21€/kg (2) 3

Table 2

Operational parameters for synthetic and real secondary effluent assays.

Water type Electrode area (cm 2 ) _{Vol (L) CD (C/L) CDR (C/L/min) Dosed Fe} theo (mg/L)

Synthetic secondary effluent 32 0.5 10 5 - 7.2 - 36 -72 2.9 20 5 - 7.2 - 36 -72 5.8 50 5 - 7.2 - 36 -72 14.5 75 5 - 7.2 - 36 -72 21.8 150 5 - 7.2 - 36 -72 43.9 200 5 - 7.2 - 36 -72 58.1

Real secondary effluent 32 1 50 7.2 14.5

100 7.2 29.0 200 7.2 58.1 400 7.2 116.1 40 1 50 36 14.5 100 36 29.0 200 36 58.1 400 36 116.1 2.7. DataAnalysis

Data seriesofsomaticcoliphageandE.coliattenuationin syn-theticeffluentswasanalysedwiththestatisticaltestANOVA (anal-ysis ofvariance)in orderto determine ifCDR introduced signifi-cantlogarithmicremovalvariationsforthedifferentCDs assayed. In this case, the obtained data was comprised by duplicate mi-crobialsamplinginduplicate assays(n=4). Forrealeffluent sam-ples,obtaineddatawascomprisedby duplicatemicrobialsamples insingleassays(n₌2).Formicrobialindicatorspresentingremoval stagnation, Tukey’s (honest significance) range test was used to verifythesocalled“removalplateau”.Faradaic efficiency determi-nationinrealeffluentexperimentswasdeterminedby theuseof theleastsquarefunctionapproximation.

3. Results

3.1. Microbialindicatorsinrawsewageandsecondaryeffluent Theaverageconcentrationsoftargetmicrobialindicatorsinthe WWTPinfluentwerefoundinthe1× 105 _{-1× 10}8 _{cfu/L(orpfu/L)}

range,whilefortheeffluent,averagevalueswerebetween1× 102

and1_{× 10}5 cfu/L(orpfu/L).Theobserved2-3log10removalis

typ-icalforactivatesludge-basedtreatmentsystems(Hataetal.,2013,

De Luca,etal.,2013,Fu etal.,2010,Tanjietal., 2002,Roseetal., 1996). Concentrations of bacterial indicators (E. coli and entero-cocci)exceededthatofESBLEandVREby2-3log₁₀ inbothinfluent and effluent, indicating that ARB were presentinlower numbers (Fig. 2). Also the removalof ESBLE and VRE by secondary

treat-Figure 2. Microbial indicators E. coli , ESBL- E. coli , enterococci, VRE, C. perfringens spores and somatic coliphages in raw influent and secondary effluent of a conven- tional Dutch WWTP. Number of samples analysed for each indicator is noted on the foot of each bar. Error bars represent standard deviation.

ment wascomparabletothat of E.coli andenterococci.The lev-els of the selectedindicators in the WWTPinfluent andeffluent streamswerein-linewithpublishedliterature(Schmittetal.,2017,

Karonetal., 2016,Harwoodet al., 2005,Lodder & DeRoda Hus-man, 2005, Noble etal., 2004,Hot et al., 2003, Cetinkaya,et al., 2000, Lasobras et al., 1999, Gantzer, et al., 1998); therefore, the WWTPwasselectedassourceofrealwastewaterforfurther labo-ratoryexperiments.OfparticularinterestwerethelevelsofE.coli andsomaticcoliphagesintheeffluent(approximately1× 105 _cfu/L

and1 _{× 10}4_{pfu/L respectively), asthesewere used to determine}

the spike concentration values for E. coli WR1 and somatic col-iphage



X174in thesyntheticeffluentduringFe-EC experiments (section2.3).

3.2. E.coliWR1and



X174removalfromsyntheticeffluent Experiments assessing the removal of E. coli WR1 are shown in Fig.3. Forboth synthetic watertypes, removalof E. coli WR1 increasedgradually withincreasing CD.In theexperiments using LNM(Fig.3a), theeffectofCDRintheremovalappearsnegligible, withnoclearpatternformicrobialattenuation.ANOVAtests con-ductedforallCDsinLNMusingCDRastheindependentvariable, produced p-valuesconsistently below0.05forCDs≥50C/L, mean-ingthatalthoughforagivenCD(_≥50C/L)removalvariationsseem unimportant inoperationalterms (≤0.5log),the influenceof CDR inremovalis statisticallypresent.In theexperiments usingHNM (Fig.3b),similarANOVAtestswereconducted,onceagain produc-ing forCDs≥50C/L, p-valuesbelow0.05, meaningthat the influ-enceofCDRinremovalisstatisticallysignificant,withgreater re-moval values associated to lower CDRs. Obtained E. coli removal using HNM was lower than with LNM, reaching 4.9log₁₀ , hence suggestingthatthepresenceofhighernutrientconcentration neg-ativelyaffectedE.coliremoval.

Somaticcoliphage



X174attenuationduringLNMexperiments (Fig. 4a) and HNMexperiments (Fig. 4b) displayed in qualitative termsaverysimilarbehaviour; removalbeing<1log₁₀ inthe10– 75C/L rangewithoutanysignificant variation, andlevellingoff at 150-200C/L, reaching a plateau of 3.0-4.0log₁₀ removal. ANOVA testsconducted forall CDs in both watermatrices usingCDR as the independent variable produced for all cases p-values above 0.05, meaning that for somatic coliphages the influence of CDR inremovalisstatisticallyinsignificantthroughoutthewholerange ofdosed charge, irrespective of theconsidered water matrix.For theaforementionedplateaus inremovalobservable at150C/Land 200C/L, Tukey tests were performed taking all 16 bars compris-ing both plateaus, as independent variables. No combination of 2 bars yielded a Tukey-p value <0.05, meaning that all 16 bars constitutethesameplateau.Hence,theconcentration ofnutrients

B. Bicudo, D. van Halem, S.A. Trikannad et al. Water Research 188 (2021) 116500

Figure 3. E. coli WR1 removal during Fe-EC experiments in LNM (a) and HNM (b) with increasing CD (10, 20, 50, 75, 150 and 200C/L) and CDRs (5, 7.2, 36, 72C/L/min). Each bar represents the average of four values (duplicate microbial screening in duplicate assays), error bars represent standard deviation.

was not found to affect the response of coliphages to the iron dosing speed (CDR), nor to the value of the maximum removal (plateau).

3.3. Microbialandphysicalchemicalattenuationinrealeffluents Followingtheexperimentsusingspikedsyntheticsecondary ef-fluent, realsecondary effluentfromtheWWTPwasassayed. Two CDRs (7.2 and36C/L/min)incombinationwithfourdifferentCDs (50, 100,200and400C/L)wereassayed. Forall experimentshere described, a singleassay wasconductedin whichduplicate sam-ples were screenedformicrobialindicators.The resultsforE.coli andESBL-E. coli aredepictedinFig. 5.Theremovalwasfoundto increasewithincreasingCD,forbothassayedCDRs.E.coliremoval spanned from0.5log₁₀ atCD50C/L, toa maximumof3.7log₁₀ at 400C/L. ThisisconsiderablylowerthantheresultsobtainedforE. coli insyntheticeffluent,sinceremovalsof5.4log₁₀ wereachieved using half of the iron dose (200C/L). CDR showed a significant effect on E. coli attenuation, with removal rates of 3.7log₁₀ and 2.4log₁₀ at 7.2and 36C/L/min, respectively (ANOVA p-values be-tween same CDs and either CDR<0.05 for 50, 100 and 200C/L). A similarbehaviour wasobserved duringFe-EC experimentswith ESBL-E.coli,withincreasingremovalfollowingincreasesinCD,and favoured by the lower CDR of 7.2C/L/min over 36C/L/min. Mini-mum removal of 0.2log10 wasobserved for 50C/L, while a

max-imum surpassing 2.6log₁₀ was estimated for 400C/L. It is note-worthythatexperimentswithCD200and400C/Linvolving ESBL-E. coli achieved removal rates high enough to hinder detection throughculturebasedmethods,meaningthatthefilteredsamples were below the limit of detection (LOD). For these experiments,

the minimum removal efficiency for ESBL-E. coli was calculated on amathematical basis, considering the methodappreciation of 1cfuandthefilteredvolumeineachcase.Fromacomparison per-spective,obtainedESBL-E.coliremovalunder7.2C/L/minwaslower than that of sensitive E. coli (ANOVA p-values<0.05) while for 36C/L/minremovalwasequalfor50C/L(ANOVAp-value≈0.2)but lowerfor100C/L(ANOVAp-value_<0.05).Thissuggeststhat sensi-tive E.coli isaconservativeindicator forESBL-E.coli,since sensi-tiveE.coliisremovedequallyorlessthanESBL-E.coli.Thefactthat ESBL-E.coliwasremovedbelowLODforCD200and400C/Lwhile sensitiveE.coliwasnot,shouldnotbemisreadasESBL-Ecolibeing betterremoved,sinceconcentrationsofESBL-E.coli were3orders ofmagnitudelowerthansensitiveE.coli intherealsecondary ef-fluentsamplesasdepictedinFig.2.

For enterococci and VRE (Fig. 6) removal also increased with CDforbothCDRs. Enterococciremovalspannedfrom0.4log₁₀ for CDof 50C/L, toa maximum of 3.6log₁₀ at 400C/L, with attenua-tionofenterococcibeingverysimilartothatofE.coli(Fig.5).CDR also showed a major effect on enterococci attenuation, with re-movalrates at7.2C/L/minbeing up to0.9log₁₀ higherthan those at 36.0C/L/min (ANOVA p-values between same CDs and either CDR<0.05 for all CDs). Regarding VRE, attenuation levels were in the same range as those of ESBL-E. coli, with a minimum of 0.3log₁₀ achievedat50C/L(36.0C/L/min)andamaximum exceed-ing2.5log₁₀ at200-400C/L(7.2C/L/min),respectively.VREremoval for200and400C/Lat7.2C/L/min, and400C/Lat36C/L/minwere alsoestimated minimumvaluessince the resultingconcentration fortheseexperimentswasbelowLOD.EnterococciandVREbehave inaverysimilarway,withVREbeingremovedinalmostidentical ratiostothatofsensitiveenterococci,atleastforthesampleswith

Figure 4. Somatic coliphage X174 removal during Fe-EC experiments in LNM (a) and HNM (b) with increasing CD (10, 20, 50, 75, 150 and 200C/L) and CDR (5, 7.2, 36, 72C/L/min). Each bar represents the average of four values (duplicate microbial screening in duplicate assays), error bars represent standard deviation.

Figure 5. E. coli and ESBL- E. coli removal during real secondary effluent Fe-EC ex- periments. Note: Bars marked with an asterisk ( ∗_{) indicate a minimum estimated}

removal, due to effluent concentration below LOD. Each bar represents the aver- age of two values (duplicate microbial screening, single assay). Error bars indicate standard deviation.

concentrationsaboveLOD(ANOVAp-values>0.05forbothCDRs). This suggeststhatremovalofenterococci canbe usedasa proxy forremovalofVRE,duetotheobservedsimilaritiesintheir atten-uationpatterns.

C. perfringens sporesshowedthemost complexbehaviour un-der Fe-EC(Fig.7). FromaCDperspective,removalincreasedwith CDyetappeared tostagnateunder200and400C/Lreachingover

Figure 6. Enterococci and VRE removal during real secondary effluent Fe-EC ex- periments. Note: Bars marked with an asterisk ( ∗_{) indicate a minimum estimated}

removal due to effluent concentration below LOD. Each bar represents the average of two values (duplicate microbial screening, single assay). Error bars indicate stan- dard deviation.

2log₁₀ .ANOVAtestbetweenthe7.2and36C/L/minseriesrevealed no considerable statistical difference between them (indicating thatCDRplaysnomajorroleinC.perfringenssporeremoval),while Tukeytestindicated twostatisticallydifferentgroups, namelythe 50C/Lsamples,andthe100,200 and400C/Lsamples(confirming the existence ofthe removalstagnation). Thisbehaviour was not observedforanyotherindicatororganisminthisstudy.

B. Bicudo, D. van Halem, S.A. Trikannad et al. Water Research 188 (2021) 116500 Table 3

Simplified operation cost calculation for experiments conducted using real secondary effluent and associated microbial removal.

Settings Costs Microbial log removal

CD (C/L) CDR (C/L/min) Intensity (A) Voltage. (v) Electric ( €/m 3 ) Metal Iron ( €/m 3 ) Operation

( €/m 3 ) _{E.coli ESBL- E.coli Enterococci VRE}

C. perfringens Spores Somatic Coliphages 50 7.2 0.12 7.5 0.007 0.003 0.010 0.6 0.8 0.6 0.5 1.0 0.2 100 7.5 0.014 0.006 0.020 1.0 2.5 1.1 1.3 2.2 0.5 200 7.6 0.029 0.012 0.041 2.9 > 2.6 2.5 > 2.5 2.0 1.0 400 7.7 0.058 0.024 0.082 3.7 > 2.6 3.6 > 2.5 2.2 > 2.3 50 36.0 0.60 27 0.025 0.003 0.028 0.0 0.2 0.4 0.3 0.4 0.4 100 27 0.051 0.006 0.057 0.4 1.9 0.8 0.8 1.8 0.6 200 27 0.102 0.012 0.114 1.3 > 1.9 2.0 2.0 2.8 0.9 400 28 0.211 0.024 0.236 2.4 > 1.9 2.6 > 2.1 2.8 2.1

Figure 7. C. perfringens spores and somatic coliphage removal during real secondary effluent Fe-EC experiments. Note: Bars marked with an asterisk ( ∗_{) indicate a min-}

imum estimated removal due to effluent concentration below LOD. Each bar repre- sents the average of two values (duplicate microbial screening, single assay). Error bars indicate standard deviation.

Forsomaticcoliphages,removalbelow1log₁₀ wasobservedfor the threelower CDsassayed(50, 100 and200C/L),yetincreasing to over 2log₁₀ for 400C/Lunderboth CDRs.CDR seems toplay a lesssignificantroleforthisindicatorcomparedtobacteria,withno major differencebetween7.2and36C/L/min intermsofachieved removal(ANOVAp-values>0.05).

3.4. Nutrientremovalinsyntheticandrealeffluents

Togetherwiththeremovalofmicrobialindicators,PO₄ 3 −,COD, and TN removal wasalso measured for each ofthe samples un-der each configuration ofCD andCDR ineither syntheticor real effluent experiments. For synthetic HNM and LNM experiments, PO₄ 3 − removalincreasedrapidlywithincreasing CDtovalues be-lowLOD(>98%removal),whileTNremovaldisplayedadecreasing trendwithincreasingCDforallCDRs(SIFigureS1andS2).Inreal secondary effluents(Fig.8), removalofPO₄ 3 − washigh,dropping from1.50mg/L tovaluesbelowdetectionwithassociated removal ratesabove99%.CODremovalreached30-40%forthehigherCDs, inparticularforCDRof36C/L/min,whileTNachievedamaximum removalof15.4%forCD200C/LandCDR36C/L/min.Theinfluence ofCDRonphosphateremovalwasnotconclusive, sinceveryhigh removalvalueswerequicklyreached,irrespectiveofCDR. Interest-ingly,CODandTNexhibithigherremovalforhigherCDR,opposite ofwhatisobservedformostmicrobialindicators.

3.5. Costestimation

The operational cost of the Fe-EC process for the real sec-ondaryeffluentexperiments,andtheirachievedmicrobialremoval for each indicator are shown in Table 3. The calculations indi-catelowercostsatlowerCDRs,duetoareducedpower

consump-tionforallCDs.Operationalcosts from0.01to0.08_€/m3_were

ob-tained fortheexperiments using realsecondary effluentunder a CDRof7.2C/L/min,whileforCDRof36C/L/min,estimationsrange from 0.03 to 0.24_€/m3 . It is worth noting that lower CDRs pro-ducebetter outcomesintermsofmicrobialremoval, andalso re-sultin lower operationalcosts due tolower required operational voltage.From theseestimates,althoughthe iron electrodecost is the same for both scenarios, the power cost is the most impor-tant factor in the total operating cost, with an impact of about 70%forCDRof7.2C/L/minand90% forCDRof36C/L/min.Forthe experimental conditions explored in this research, the combina-tionthatproducedthebestoutcomefromamicrobialperspective wasCD400C/LandCDR7.2C/L/min,involvinganassociatedcostof 0.082_€/m3_.

Faradaicefficienciesduringrealsecondaryeffluentassayswere calculatedusingleastsquaremethod,withreportingvaluesof113% forCDRof7.2C/L/minand114%for36.0C/L/min(SIFigureS3).

4. Discussion

4.1. Effectofwatermatrixonremovalefficiencies

Thepresentstudyconfirmsthe influenceofwatermatrix, i.e., syntheticversusrealsecondaryeffluent,forbacteriaandvirus in-dicatorremoval by EC, aswell as nutrient removal .In real sec-ondaryeffluents,E.coliremovalwas1-2log₁₀ lower thanthat ob-served forE. coli insyntheticeffluents, evenwhen the dosageof ironwasdoubled.Similarobservationsweremaderegardingphage



X174,withremovalalsodropping by1-2log₁₀ inrealsecondary effluents.Althoughtheresponse obtainedwithsyntheticandreal secondary effluent wassimilar in qualitative terms, removal ob-tainedforE.coliandphage



X174stilldiffersbyordersof magni-tude.

It was hypothesised that the complexity of the water ma-trixfromrealsecondary effluents,andits higherconcentration of organic matter, iron-scavenging anions and complexation agents (such as phosphates, citrates, carbonates and sulphates) are re-sponsibleforsubstantiallyreducedcoagulantgenerationor micro-bial removals, coinciding with the observations of Heffron etal., (2019a),VanGenuchtenetal.,(2017),andGhernaoutetal.,(2019). ThesecompoundsarerecognizedasresponsibleforhinderingFe+ 2 oxidation into insoluble Fe+ 3 , hence reducing coagulant precipi-tationandsubsequentsweep flocculation.C.perfringens spore re-movalwasonlyassessedinrealsecondaryeffluents,showing sim-ilarcharacteristicsboth inquantitativeandqualitativetermswith previous Fe-EC research conductedin realsewage and secondary effluentsbyAnfruns-Estradaetal.,(2017).Inthementionedstudy they determined the maximum removal of C. perfringens spores to 2.79log10, with removal stagnating as the dosage of current

progressed. This behaviour was also observed in the present re-search, particularlyat lower CDRs underlining that increasing CD

Figure 8. Removal of phosphate, COD and TN for real secondary effluent experiments. Each data point represents the average of four values (duplicate chemical screening in duplicate assays). Error bars indicate standard deviation.

will not enhancesporesremoval beyondacertain plateau, what-ever the CDR. In terms of PO₄ 3 − COD and TN removal, the ob-tained results are well within range of previous research, where high PO₄ 3- _{removalranging from50% to98%, 26% to 85%for}

or-ganicsandlessthan30%forTNwasobserved(Zaleschietal.,2013,

Ikematsu, et al., 2006, Inan & Alaydin, 2014, Lacasa et al., 2011,

Malinovicetal.,2016).

4.2. Microbialattenuationmechanisms

Removal of all bacterial indicators was in general terms very similar, irrespective of their resistance to antibiotics or their gram classification,withremovalbeingstronglydependentonthe amount and speed of iron dosage. Similar conclusions are also valid forsomatic coliphages(although appearinglesssensitive to the speed of dosage), but do not fully apply for C. perfringens spores.ThisevidencesadifferingresponsetotheFe-ECprocessfor each type ofmicroorganism. The aimofthis research wasnotto look intothe mechanisms ofremoval foreach one, yetthe body of literature recognizes three pathwaysfor microbial attenuation, namely: a) entrapment or adsorption in the metallic hydroxide flocs, and removal by sedimentation; b) inactivation due to for-mation of ReactiveOxygen Species (ROS)or disinfectants;andc) inactivationduetoelectricalcurrent.

According to most researchers, entrapment seems to be the dominantremovalmechanismforbacteria(Ghernaoutetal.,2019,

Delaire etal., 2016,Delaire etal., 2015), mainly duetothe affin-ity of their surface functional groups such as teichoic acids and phospholipids, with the EC precipitates. These functional groups are foundinsimilar amountsingram positiveandgram negative bacterialcellwalls(Delaireetal.,2016,Borrok,etal., 2005).Virus removal has been attributed to both iron hydroxide floc entrap-ment(Heffronetal.,2019a,Tanneru&Chellam,2012),and inacti-vation byeitherROSorchlorine-basedoxidantsformedby reduc-tionintheanode(Heffronetal.,2019).FormationofCl₂ wasruled out asa mechanismofdisinfectioninourexperiments,since Cl− concentrationwasmeasuredbyICP-MSbeforeandafterthe appli-cation of current, and no variationswere detected in any ofthe samples,alsoinlinewiththefindingsofDelaireetal.,(2015)and

Diaoetal.,(2004).Inactivationduetotheeffectofelectriccurrent has been the least reported biocidal pathway, with the research fromJeongetal.(2006)givingitalargerrelevanceathighcurrent

densities, from33 to100mA/cm2 .It is noteworthy that through-outtheexperimentsconductedinthispublication,currentdensity neverexceeded 20mA/cm2 ,for whichthispathwayof removalis notregardedasdominant.

IntermsofCDRinpromotingeitheroftheaforementioned mit-igation mechanism, Heffronetal., (2019b) correlated ferrousiron oxidationrateandbacteriophageremoval,concludingthatfast ox-idationofFe+ 2 leadstoashorterexposuretimeandhencepoorer contactbetween the phagesand the reactive iron species, yield-ing a less important disinfection. In other words, implying that ROSgenerated duringferrous ironoxidation are a major contrib-utorinthedisinfectionduringFe-EC,withtheeffectofthese be-ingstrongerforslowlyoccurringoxidations.However,theselected overnightsettlingforallexperimentslikelyinfluencesoxygen dif-fusion into the effluent promoting the slow oxidation of ferrous iron, hencepotentially impacting in theremoval (besides fromit having a reduced practicality on a municipal scale). The present researchobservedforbothsyntheticandrealeffluentsan increas-ing removalefficiencyforbacterial andviralindicatorsunder de-creasingCDRs(lessprominentforviruses),evenwhentheamount ofdosed iron (and coagulation products)was identical.Although thisstudydidnotlookintothemechanisticaspects ofthe micro-bialremovalvia Fe-EC,the observeddependencyofmicrobial re-movalonCDRsuggeststhattheproductionofROScouldinfactbe a contributingfactor duringFe-EC. Theaerobic oxidation of Fe+ 2 releasedduringtheanodeoxidationproducesacascadeofreactive specieswhichincludes superoxideion(•O₂ −),hydrogen peroxide (H₂ O₂ )andhydroxylradical(•OH−)(VanGenuchten&Peña,2017,

Hug&Leupin,2003,Rush,etal., 1990),allofwhichareknownto havedisinfectantproperties(Tanneru&Chellam,2012,Jeongetal., 2006, ElenaPulido 2005). Thisimpliesthat microbial removalby Fe-EC could be in fact a combinationof physical separation and chemical inactivation, and not just an adsorption-sedimentation phenomenon.Itcould alsoexplainwhyspores(dormantbacterial structures,highlyresistanttochemicalattack)aresignificantlyless affectedthanbacterialindicatorsbyvaryingCDorCDR.

4.3. Fe-ECasadisinfectiontechnology

When compared against typical wastewaterdisinfection tech-nologiesin termsofpathogen removal, Fe-ECperforms ina sim-ilar way to that of conventional alternatives such as

chlorina-B. Bicudo, D. van Halem, S.A. Trikannad et al. Water Research 188 (2021) 116500

tion,ozonationorUVlight.Typicalremovalvaluesforchlorination rangefrom2-6log₁₀ forbacteria,0-4log₁₀ forvirusesand0-3log₁₀ forprotozoa.Disinfectionvaluesforozonationinclude2-6log₁₀ for bacteria,3-6log₁₀ forvirusesand_>2log₁₀ forprotozoa.ForUV, ex-pected performance is 2-4log₁₀ for bacteria, 1-3log₁₀ for viruses and2-3log₁₀ forprotozoa(Collivignarellietal.,2018,USEPA,2012,

Bitton, 2005, USEPA, 2003, Rose et al., 1996, WHO, 1989). This means that Fe-EC can in fact be regarded as an unconventional chemical-addition free disinfection technology, based on compa-rableperformance tootherclassically accepteddisinfection meth-ods. Abou-Elela et al. (2012) provides reference O&M cost val-ues for municipal secondary effluent disinfection using chlorine (32mg/L, 15 minutes contact time), ozone (15mg/L, 15 minutes contact time)andUV irradiation (164mWs/cm2 ,15 minutes con-tact time). Cost estimates are 0.024€/m3 _{for chlorine, 0.030€/m}3

for ozoneand0.044€/m3 _{forUV irradiation. Forthe lowest}

stud-iedCDRofthisresearch(7.2C/L/min),theobtainedoperationcosts (0.01to0.082€/m3 _{)fallwithincomparablerangetothoseobtained}

by Abou-Elelaetal.(2012),althougha propercomparisonshould bemadeonabasisofequalmicrobialinactivation.

5. Conclusions

• Lowvoltage Fe-ECisa promisingtechnology formicrobial re-moval in secondary municipal effluents, with log₁₀ removal comparable to those achieved by conventional disinfection methodssuchaschlorination,UVorozonation.

• For real secondary effluents, achieved bacterial removal ex-ceeded 3.5log₁₀ , ARB removal reached or exceeded 2.5log₁₀ , spores were removedbetween 2-3log₁₀ andvirus elimination reached orexceeded 2.3log₁₀ . In synthetic secondary effluent, bacterialandviruslog₁₀ removalwasconsistently higherwith 1-2ordersofmagnitude.

• Microbial removal was found to increase with CD, while de-creasing CDRs showed a higher mitigation of bacteria,yet no significanteffectonvirusesorspores. Thelattershoweda dif-ferent removal trend, withelimination reaching a plateau for medium-highCDs,thisbeingslightlyhigherforhigherCDRs. • Sensitive E. coli and Enterococci appear as conservative

indi-cators for ESBL-E. coli and VRE respectively, although it must benotedthatARBdeterminationwaslimitedbyrelativelylow concentrationsinthesecondaryeffluent.

• Iron plates and electric consumption were the main compo-nents contributing to the costs, with the latter having the largestimpact(70-90%)fortheassayedconditions.Forthemost favourablemicrobialremovalsetofconditions(CD400C/L,CDR 7.2C/L/min) theestimatedunit costoftheprocessis0.08_€/m3 , within comparable range to other conventional disinfection technologiesaschlorine,UVorozone.

• Besidesfrommicroberemoval, Fe-ECoffersadditionalbenefits overtraditionaldisinfectionmethods,suchasnutrientandCOD removal.

DeclarationofCompetingInterest

Theauthorsdeclarethattheyhavenoknowncompeting finan-cialinterestsorpersonalrelationshipsthatcouldhaveappearedto influencetheworkreportedinthispaper.

Acknowledgments

The authors wouldlike to expresstheir gratitude to NWO for theirsponsorshipoftheLOTUSHR project(https://lotushr.org)from which thisresearch ispartof.Wewouldliketoacknowledgethe staff from TU Delft Waterlab, especially Armand Middeldorp and Patricia vanden Bos, and alsothe microbiology technician Anita

vander Veen(KWR) for her traininginsomatic coliphage detec-tion.Our gratitude goes aswell to Dr.Case vanGenuchten, who helpedussetupourfirstFe-ECunitsandprovideduswith guid-ance during ourtrials. Lastly, we would like to acknowledge the operators ofthe WWTP,Paul WeijandAbdel elIdrissifortaking thetimetoprovideuswithfresheffluentsamples.

Supplementarymaterial

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