Effective Removal Of E. coli And Coliform Bacteria By The TivaWater Biosand Filter

Introduction ThedevelopersoftheTivaWaterBiosandfilter(BSF)areprovidingalowcostfilterfortheremoval of E. coliandcoliformbacteriaforhouseholduseindevelopingcountries.ThisBSFwasfirsttestedforuseforareasaroundKampala,UgandaandwasdesignedinKnoxville,Tennessee.IntheKampala,Ugandaarea,TivaWaterprovidessustainableemploymentforarearesidentsastheysell,distributeandservicetheseBSFs. Historically,BSFshaveprovidedanexcellentpoint-of-use(POU)treatmenttechnologywhichisefficientinremovalofpathogensthatcausesignificantintestinaldiseaseinthesedevelopingareas,bothruralandurban.BSFsprovideanaffordableandeconomic,lowmaintenancefilterfortap,well/boreholes,andsurfacewatersources. The following is documentation of the methods and testing results that TivaWater hasproducedsincethedevelopmentofthisfilter.TherearecurrentlythreecompletedexperimentswithinthedocumentregardingtheefficiencyandeffectivenessofthefiltrationoftheTivaWaterfilters.Thefirstexperimentalreviewcontainsthelong-termanalysisofcoliformandE. coli removal bytheColilertIDEXXreagentmethod.Designedtotestthelifespanofthefilters,thismethodprovesthecapabilityofTivaWaterfiltersinremovingcoliformsandE. colibybothpresence/absenceandquantificationof the targetedorganisms.Thesecondexperiment isanattempt toanalyze theinteractionofcoppermetalwithintheTivaWaterfilters.Byunderstandingthemobilityofcopper(II)sulfatethroughthefilter,itispossibletodiscernthecapabilitiesofthefilterinremovingcopper,arsenic, lead,and/orotherpossiblecontaminantmetals similar in ionsizewhichoftenpollutesourcewaters.ThelastreviewconcernsanexperimentwhichimplementsdetectionmethodsofmicrospherespassedthroughfourdifferenttypesofTivaWaterfilters.Theexperimentattemptstoprovewhichsandtypesandmassesofsandaremoreeffectiveinremovingparticles,aswellasgagingwhatsizesofparticlesthosefiltersarecapableofremovingfromsourcewaters.

2008IntroducedtoconcreteBisosandFilters(BSF)duringfieldvisittoUganda.

ExtensivetechnicalandfieldresearchconfirmedthatBSFisthemostpracticalfiltermethodforpotentialbusinessmodel.

IncollaborationwithNealCaldwell,designedmodifiedBSFplasticversionthatincludedpre-filter,waterstorageandtap.

PurchasedIDEXXColilertsystemandperformedextensivetestingforE. coli and coliformwithfunctionalmodel.

Conductedmultiplestudiescomparingflowratesandsandconfiguration.

FlowratesandwaterqualitycomparesfavorablytoHydraidBSFproduct.

FieldmarketresearchfocusedonproductimplementationinUganda.

PatentApplicationdatedJune14,2010andplasticmoldordered.

FirstfiltersmanufacturedatDalenProductsplant.

Testingon6PrototypesatDalenLabwithColilertSysteminitiated.

ServiceteaminKampala,Ugandaestablishedandfieldsandproductioninitiated.

Delivered50filterstoUgandaforhomeinstallationandadditionalwaterqualitytesting.

3,200filtersdeliveredbycontainertoKampala,Uganda.

Installedfiltersat5testsitesinUganda,includingsourcewaterfromsurface,tapandwells.

AwardedwaterqualitycertificationfromUgandaNationalBureauofStandards.

TestingforcopperreductionconductedatUniversityofTennessee(UT)CenterforEnvironmentalBiotechnology(seeattacheddatareports).

4,200filtersdeliveredbycontainertoKampala,Uganda.

FieldtestingexpandedtoincludemanysitesinUganda.

ExperimentswithvarioustypesandmassesofsandconductedatTivaWaterlab.

PhaseIofUTconductstestsforefficiencyofbiolayerusingfluorescentdye,seeattached data reports

PhaseIIofUTtestssimulatingparticlesizesofCryptosporidium,VibriocholeraandRotavirusforreductionincompetingfiltermasses.(seeattacheddatareports)

TivaWater Development Time-line:

2009

2010

2011

2012

2013

Top

Outer Bucket

Inner Bucket

FilterRetainingRing

Filter

SandSeparator Cup

OutletTube

Faucet

Sand

HistoryofColiformandE.coliRemovalMethod TestingwasperformedbytheColilertIDEXXreagentmethod,usingapatentedDefinedSubstrateTechnology(DST).ThismethodoftestingusestwonutrientindicatorsONPGandMUG,whichcanbemetabolizedbythecoliformenzymes.Coliform,usingβ-galactosidasetomovefromacolorlesstoayellowproduct,andE. coli,usingβ-glucoronidasetometabolizeMUGtocreatefluorescence.Non-coliformbacteriaaresuppressedbytheColilert’sspecificallyformulatedmatrix.Threelevelsofcontrolwereutilizedperthemanufacturer’sdirectionsfornewlotsofreagentmaterial.DocumentationofQCfrequencyandperformancecanbesubmittedupon request. During the testing process all QCwere found to be in-range and passed.QuantificationofcoliformandE. colilevelsareinterpretedbytheMostProbableNumber(MPN)techniquewhichprovidesthedensityoftargetorganismsper100mlsample.

DataTable1.PercentandLogRemovalofColiformandE. colithroughFilters1-5andComparisontoHydraidProduct

Filter# No.Dates FlowRate(ml/min)

Coliform E. coli Coliform E. coli

1(sand>145) 65 22 99.82 99.55 2.962 2.8442(sand>100) 14 35 99.93 99.55 3.272 2.8093(un-amended)

65 9 99.94 99.38 3.381 2.862

4(sand>145) 14 41 99.62 99.45 2.732 2.5395(sand>145) 14 41 99.92 99.55 3.228 2.809Hydraid 13 192 89.63 98.26 1.358 2.143

Average%Removal AverageLogRemoval

Discussion Table 1 shows efficiency levels for removal of total coliforms and E. coli bacteria byboth percent and logarithmic removal of a range of TivaWater filters. Logarithmic removal isthestandardsystemforexaminingfiltrationperformanceinwatertesting.Filters1and3weredesignatedlong-termfiltersinordertodeterminetheirlifespanunderconstantconditions.TheHydraidfilteruseditsowndesignatedmediamixtureofrocksandsandandnotthesamesandastheTivaWaterfilters.Alldatapointsreadinglessthan1MPNwereconvertedto0.5sincezerosdonotcomputeinthelogarithmicscale.ThefinalaveragesarelimitedbythemaximumE. coli measurementbyColilertinundilutedsourcewaterofaMPNgreaterthan2419.6/100ml.Thus,ifE. coliconcentrationinthesourcewaterwasgreaterthan2419.6,thanthepercentremovalwouldbeunderestimated.

Filter AverageConcentrationCuSO4Added(mg/L)

AverageConcentrationCuSO4Finished(mg/L)

Average%Removal

AverageCuSO4 Remaining(mg)inSand

1 137.5 0.413 99.69 137.12 52.87 0.327 97.42 52.53 28.9 0.521 98.34 28.44 4.68 0.602 88.48 4.1

Copper(II)SulfateInfluenceandRemovalMethod Thecopperexperiment isasimulationofmetalcontaminationbychargingTivaWaterfilterswith copper(II) sulfate (CuSO4)fromCuVer 5mlCopper Reagent Powder Pillows. Thesimulationprovidesinsightonthemobilityofheavymetalssuchasarsenicandleadbecauseof theirsimilaritieswith theCopper ion.AstocksolutionofCuSO4was first formulatedataconcentration of 3.9g/100ml. Four standard concentrations of 4, 1, 0.4, and 0.1mg/L wereequivalentto3.32,0.86,0.33,and0.08mg/Lrespectively.Eachrunconsistedofaddingknownconcentrationsofthestocksolutiontoeachofthefilters.Filter1received15ml,2-5ml,3-3ml,and4acontrolreceivingnoamountofsolution.Eachinputofstocksolutionwasaddedto5Lofwaterandtheconcentrationofthemixbeforefiltrationrecorded.Afterthefiltrationtimeperiodof24hours,thevolumeofoutputwasrecordedaswellastheconcentrationofCuSO4 remaining.

DataTable2.PercentRemovalofCopper(II)Sulfateintriplicateexperiments.

Discussion Theamountofcopper(II)sulfateremovedbythesandfilterswasdeterminedintriplicateexperiments.Copper(II)sulfatewasaddedtotapwaterbecausetheuseofcreekwaterledtointerferenceinthecopperreagentpowderpillowmeasurements.Inputwaterconsistedof5LitersofwaterwithvaryingamountsofCuSO4.Theaverageoutputoffinishedwaterwas4.78L..Waterretentionwhichremainsinthesandlayerofthefilteraccountsforthedifferencebetweeninputwaterandfinishedwater.Filter4receivednoCuSO4indicatingtraceamountsofcopperwaspresentinthetapwater.TheamountofCuSO4remaininginthesandisshowninTable2.ItisunknownastohowmuchCuSO4canberetainedinthesandbeforebreakthrough.

Filter# %BeadRemoval Coliform E. coliSilica10lb. 80.14 72.22 85.98Silica20lb. 99.16 87.74 96.18USAS10lb. 96.19 93.19 98.80USAS20lb. 98.86 98.48 95.49

%ColilertRemoval

FluorescentMicrosphereRemovalMethod Mixes of Constellation Microspheres from Life Technologies of assorted colors andsizeswereadded tovariousTivaWaterfilters inorder todetermine the rangeof removalofsizedparticlesbyfiltration.Thesizesofthemicrospheresrangedfrom0.02micrometersto4micrometersandthecolorsrangedfromblueacrossthespectrumtored.TheAccuriC6FlowCytometerwasusedtoidentifypopulationsofbeadswithininputandoutputsamplesaswellasthesizesofthebeadspresentinbeforeandafterfiltrationsamples.Atemplateofknownbeadsizepopulationsfromapreviousexperimentwasoverlaidacrossastandardofthebeadstopredictthesizesofthebeads.Allfiltersinvolvedwiththeexperimentwereuniqueinthattwosetsofsandtypesandsandmassesweretested.TheefficiencyofcoliformandE. coli removal bythecolilerttestonfivedifferentrunswasusedtoverifythefunctionalityofthebiolayerinTivaWaterfilters.

DataTable3.PercentRemovalofFluorescentMicrospheresamongVaryingFilterSandsandMasses

Discussion Inall experiments, threepopulationsofmicrospheresof twodistinct sizescontained95%ofthemeasuredbeads.Populations1and2were1micrometerbutdifferedincolorwith1and2beinggreen.Population3was2micrometersandlabeledaredcolor.Thesensitivityofthecytometerwaslimitedtoquantifyingparticlesequalorgreaterthan1micrometer.Thepercentbeadremovalwas80%to99%withahigherpercentageremovalinthe20lbfiltersthanthe10lbsandfilterswhichwasconsistentwiththehigherpercentageremovalofE. coli andcoliforms.

Conclusion ColiformandE. coliremovalexperimentsindicatethatfilterswith20lbsofsandconsistentlyremovedover99.62%ofcoliformsand99.37%ofE. coliformorethan2years.Theminimumlogremovalofcoliformswas2.73and themaximum log removalofcoliforms3.38. Similarly, theminimum E. coli logremovalwas2.53andthemaximumE. coli logremovalwas2.86. TheseminimumlogremovalvaluesexceededtheaverageHydraidlogremovalvaluesbylog1.17and0.72forcoliformsandE. coli,respectively. Copper(II)sulfateremovalexperimentsindicatethat20lbsandfiltersremovedbetween97.42and99.69%of theaddedcopper(II)sulfate.Copperwasusedasametalsurrogate forarsenicandleadbecauseuseofarsenicwasprohibitedduetoitstoxicityanddisposalissues.It iscurrentlyunknownwhetherpollutionofheavymetals isaproblem in thecityofKampala,Uganda.

Thefluorescentmicrospherebeadexperimentsindicatethatbeadsinthesizerangeof1to2micrometersareremovedmoreefficientlyinthe20lb.sandfilterscomparedtothe10lb.sandfiltersandrangedfrom80%inthe101b.silicasandfiltertogreaterthan99%inthe20lbsilicasandfilters.Particlesthatare1micrometerinsizearesmallerthanmostbacterialandprotozoanpathogenssuchasVibrio cholerae(2-3microns)andCryptosporidium(5-6microns).

Insummary,thecombinedcolilerttestingandmicrospheretestingsuggeststhatthefilterswilleffectively removebacterialandprotozoanpathogens.While the lifespanof theTivaWaterfilterisundetermined,theColilertexperimentconcludesthatefficacyisnotdiminishedaftertwoyearsofconstantfiltration.Moretestingwillbeneededtoverifyremovalofmetals.

SamuelA.BlountUniversityofTennesseeB.S.EnvironmentalScienceMinorinSustainability

AliceLayton,Ph.D.TheUniversityofTennesseeResearchAssociateProfessorCenterforEnvironmentalBiotechnology

BenCurryTheUniversityofTennesseeGraduateResearchAssistantPh.D.Student

TheTivaWaterFilterExperimentswereperformedunderthesupervisionofDr.AliceC.Layton,ResearchAssociateProfessoroftheUniversityofTennessee’sCenterforEnvironmentalBiotechnologyLab.Mr.BenCurry,GraduateResearchAssistanceandPh.D.studentoftheUniveristyofTennessee,alsoprovidedas-sistanceinanalyzingexperimentaldata.TheexperimentswerecarriedoutbyMr.SamuelBlount,UndergraduateResearchAssistantattheUniversityofTennesseeandTivaWaterResearcher.Dr.Laytonhasreviewedthefinalreport.