BiodegradabilityviaFluorescence

AmyBigelow

SustainableIRESCohort2015

July31,2015

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AbstractIn-depth analysis of thebiodegradability of aDecentralizedWastewater Treatment System (DEWATS)plant in South Africa was performed using in-situ fluorescence spectroscopy. Tryptophan-likefluorescencewasmonitoredthroughoutthesystem’sthreetreatmentstreetswithspatialandtemporalvariations to discover how biodegradability rates changed within individual chambers, treatmentstreets, and the systemas awhole.Auniformbiodegradationwas foundwithin individual chambers,Tryptophan concentrations were found to decrease throughout the system, especially within theanaerobic filters, and the efficiency of the third treatment system, with only four anaerobic baffledreactor chambers, was questioned. Results from this analysis will help to improve DEWATS plantsaroundtheworldandweremadeinanefforttoimprovesanitationforunderdevelopedareas.

Introduction

Throughout the world, wastewater treatment has been performed in a variety of methods.Conventionally, in the USA and other developed areas, it has been done using clarifiers and aerobicprocesses.TheGermanNGOBORDA,however,hasdevelopedatreatmentsystemusingwhatisknownas an anaerobic baffled reactor (ABR) to deliver sustainable sanitation solutions to underdevelopedareas. One such system was built through the partnership of the University of Kwazulu-Natal, theEthekwiniMunicipality,andBORDAintheNewlands-MashuareajustoutsideofDurban,SouthAfrica.

Servingapproximately83houses,theNewlands-Mashuplantconsistsofthreetreatmentstreets.Eachstreetcontainsaninitialsettlerwherescumisseparated,anABR,andanaerobicfilters(AF).TheABRisaseriesofcompartments(baffles)that forcewastewatertoflowdownintoeachchamber,thenupandover each baffle allowing organic matter to be digested anaerobically (microbial treatment) whileparticles settle to the bottom creating a sludge layer (physical treatment) (Tilley et al., 2014). AschematicoftheprimarysettlerandABRcanbeseenbelow.

Figure1.SchematicofAnaerobicBaffledReactor.Source:Tilleyetal.2014.

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Twoof the streets have sevenABR chambers, twoAF, and are then discharged into awetlands areaconsistingofaverticalplantedgravityfilter(VGF)andahorizontalplantedgravityfilter(HGF).ThethirdstreetconsistsofonlyfourABRchambers,twoAFchambers,andeffluentatthispointisfedbackintothemunicipalsystem(seepicturebelow).

Researchwasconductedonthissitetodeterminetheamountofbiodegradabilityoccurringwithineachtreatmentsystemusingin-situfluorescencespectroscopy.Thisisasensitivetechniquethatattemptstomonitortheconcentrationoftryptophan-like(TRP-like)substancesandchromophoricdissolvedorganicmatter (CDOM) in the water. TRP-like substances, in particular, weremonitored as these have beenmorecorrelatedtobiodegradabilityexperiments.ResearchdonebyHudson,Naomi,etal.foundthatinaerobictreatmentsystems,fluorescenceofTRP-likesubstanceshadastrong,positiverelationshiptothebiochemical oxygen demand (BOD) of the wastewater, a direct measurement of the biodegradationoccurring(Hudsonetal.2008).ThismeantthatahigherconcentrationofTRP-likesubstancesresultedinhigherbiodegradationratesoccurring.

AdditionalresearchinthenearbywatersoftheUmgeniRiverandMsundunziRiverinKwazulu-NatalwasconductedbyA.Bakeretal. thatpositivelycorrelated thepresenceofE. coli toTRP-like fluorescencepeaks.ThisresearchfoundthatatlowerTRP-likeconcentrationsorlowpeakintensities,E.coliwasalsofoundinlowerconcentrationsandviceversa(Bakeretal,2015).

Similartothesetworesearchanalyses,theresearchdoneattheNewlands-Mashusitewasconductedbased on the premise that higher TRP-like concentrationsmeantmore biodegradation occurring andmore microbial presence. The goal of the research was to answer three main questions: how wasbiodegradation changingwithin each chamber, howdid it change throughout thewhole system, andhowdiditchangebetweenstreets.

Methods

In-situ fluorescence was conducted using a submersible C3 fluorimeter (Cyclops) manufactured byTurnerDesignsthatwasequippedwiththreesensorstodetectthefollowing:chlorophyllinvivoforblueexcitation(465nmex/696nmem),CDOM(325nmex/470nmem),andtryptophan(285nmex/350nm

Figure2.Newlands-MashuDEWATSplantandon-sitelaboratoryfacilities.Source:MonicaPalomo.

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em).Beforeuse,theCyclopswascalibratedforbothTRPandCDOMconcentrationsusingTRPpowderandPonyLakeCDOMpowderrespectivelyinultrapurewater.

Totakemeasurements,theCyclopswassubmergedintothewastewater,slightlyswungtoremoveanyaccumulated air bubbles, and then held as motionless as possible for a specific amount of time.Duplicatemeasurements,whenperformed,simplyconsistedofrepeatingthisprocessasecondorthirdtime.

FourmeasuringorsamplingplanswerecreatedforusewiththeCyclopsincludinginitial,secondary,24-hour, and comparison measurement methods. The initial sampling plan was constructed to detectdifferences in depth and location within an ABR chamber. Three different locations were measuredwithineachABRchamber,inlet,middle,andoutlet,atthreedifferentdepths:0.5m,1.0m,and1.5m(seeABRdiagrambelow). Inlet,middle, andoutletofeachanaerobic filter (AF)weremeasuredaswellbydropping the Cyclops to the rocks then lifting slightly (<0.5m). In addition, the influent into thehorizontalgravityfilter(HGF)wasmeasuredbyfillinga5Lpitcherwithinfluent,coveringthisinablackplasticbag(todiminishadverseeffectofoutsidelightwithfluorescence),thensubmergingtheCyclopsatanangle toaccount forairbubblesandslightly lifting it fromthebottom.TheCyclopswasheldateachlocationforoneminutewithreadingsatonesecondinterval.

Thesecondarysamplingplanconsistedofsubmerging theCyclopsat theoutletonlyofeachchamberwith the same minute, one second interval procedure as for the initial sampling plan. HGFmeasurements were performed in exactly the same manner as in the initial plan. The 24-hour planconsistedofleavingtheCyclopssubmergedinonechamber(eitherABR1orABR7/4)foranentire24hourperiodwithreadingstakeneveryfiveminutes.Lastly,thecomparisonsamplingmethodwasusedtocompareABR1,ABR7/4,andAF2 inStreets1-3.Thiswasdonebysubmerging intotheoutletsofthesechambersandtakingthesameminute,onesecondintervalreadings.

Results

The firstweekofCyclopsmeasurementsweredone toassess thebiodegradabilityatdifferentdepthsanddifferent locationswithineachchamberofstreetone.MeasurementsweretakenonJuly1,2015overthecourseoftheentiredaywithduplicatemeasurementstakenatalllocationsinAF2andABR1.

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Figure3.ProfileandtopviewofanABRchamber.Source:AmyBigelow

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ThefollowingtwographsshowtheTRP-likeconcentrationresultsofABRchambers3and4andcanbetakenasrepresentativedataforallchambersmeasured.

StandarddeviationsforABR3datawerefairly lowrangingfrom0.0108mg/lfromtheoutletat1mto0.701mg/l in themiddleat1mdepth.Standarddeviations forABR4were similarly lowranging from0.009mg/lat the inletat0.5mto0.055mg/lat theoutletat1m.Lowerstandarddeviations inABR4suggestgreaterprecisioninthesemeasurementsthaninABR3.

Duringthesecondweek,onlytheoutletat0.5mofeachchamberwasmeasuredbutwasmeasuredat9:00,11:00,and13:00onJuly7,2015.ThefollowinggraphsaretheTRP-likeconcentrationsandCDOMconcentrationsforallthreetimesfortheentiretreatmentstreetfromABR1totheHGF.

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Figure4.ABR3TRPconcentrationsplottedbylocationanddepthwithinthechamber

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Figure5.ABR4TRPconcentrationsplottedbylocationanddepthwithinthechamber

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Figure6.9:00samplingattheoutletofeachABRchamber,AFchamber,andHGFat0.5m.

Figure7.11:00samplingattheoutletofeachABRchamber,AFchamber,andHGFat0.5m.

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Inadditiontotheseresults,measurementresultsfora24-hourtimelapseinABR1andABR7(theinletandoutletoftheABRsystem)canbeseeninthefollowinggraphs.Itisimportanttonotewhatdaysoftheweek thesemeasurementswere takenas aweeklypatternmaybeable tobeestablished.ABR1measurementsweretakenfrom16:30July8,2015to15:35July9,2015(aWednesdaytoaThursday).ABR7measurementsweretakenfrom16:00July9,2015to15:10July10,2015(aThursdaytoaFriday).

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Figure8.13:00samplingattheoutletofeachABRchamber,AFchamber,andHGFat0.5m.

Figure9.TRP-likeandCDOMconcentrationsfrom24-hourtimelapseofABR1,Street1.Measuredattheoutletat0.5m.

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020406080100120140160180200

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ThethirdweekofmeasurementsresultedindataforStreet3.ThefollowinggraphsindicatetheresultsforfullStreet3measurementsat9:00,11:00,and13:00withoutreadingsfromtheHGFasStreet3doesnot empty into the gravity filters but rather goes back into themunicipal system. AllmeasurementsweretakenonJuly13,2015at0.5mattheoutletofeachchamber

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Figure10.TRP-likeandCDOMconcentrationsfrom24-hourtimelapseofABR7,Street1.Measuredattheoutletat0.5m.Thereisanoticeable,abruptdecreaseinTRPat9:55am.

Figure11.TRP-likeandCDOMconcentrationsfor9:00samplingofStreet3.Measuredatoutlet,at0.5m.

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Figure12.TRP-likeandCDOMconcentrationsfor11:00samplingofStreet3.Measuredattheoutlet,at0.5m.

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A24-hourtimelapsewasalsotakenoftheinletandoutletoftheABR(ABRchambers1and4).ABR1was measured from 11:50 July 16, 2015 to 8:25 July 17, 2015 (Thursday to Friday) and ABR 4 wasmeasuredfrom10:55July15,2015to9:40July16,2015(WednesdaytoThursday).Theresultscanbeseeninthefollowinggraphs.

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Figure13.TRP-likeandCDOMconcentrationsfor13:00samplingofStreet3.Measuredatoutlet,at0.5m.

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Figure14.TRP-likeandCDOMconcentrationsfor24-hourtimelapseofABR1inStreet3.Measuredatoutlet,at0.5m.

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Lastbutnot least, comparisonmeasurementswere takenofall threestreetson July17,2015at9:00and11:00.Theresultscanbeseeninthefollowinggraph.NoticethatStreet3hasconsistentlyhigherTRPconcentrationsthantheothertwostreetsattheoutletoftheABRandinAF2.

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Figure15.TRP-likeandCDOMconcentrationsfor24-hourtimelapseofABR4,Street3.Measuredatoutlet,at0.5m.Aninstrumentmalfunctioncausedagapindatafrom16:00to0:00atwhichpointnodatawastaken.

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Figure16.TRP-likeconcentrationsfortheinlet(chamber1)andoutlet(chamber4/7)oftheABRinallthreestreets.Measuredatoutlet,at0.5m.

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Discussion

Results from the first week of sampling compared TRP-like concentrations for varying depths andlocationswithineachchamber.A t-testwasperformedonthedata forABR3andABR4(t-testvaluescanbeseeninthetablebelow)whichdidshowasignificantdifferenceinbothdepthandlocationwitha95% confidence interval, however, based on the graphs seen in the results section above, it wasdetermined that there were no practical differences found within the chamber for either depth orlocation. With no practical differences observed, the chamber could be considered one unit with aconstantbiodegradationratethroughouttheentirechamber.

Table1.ABR3LocationComparisonT-Test

Inlet–Middle Inlet–Outlet Middle–Outlet0.5m 1.049E-19 5.180E-86 4.312E-101.0m 0.918* 2.732E-07 0.459*1.5m 1.924E-15 0.001 9.538E-07

ABR3DepthComparisonT-Test 0.5m–1.0m 0.5-1.5m 1.0–1.5mInlet 0.001 3.430E-13 0.0001Middle 8.254E-17 1.159E-16 0.137*Outlet 2.735E-95 2.810E-92 0.056**P-value<0.05indicatesasignificantdifference.OnlyfourareasinABR3showednosignificantdifference.

Table2.ABR4LocationComparisonT-TestP-values

Inlet–Middle Inlet–Outlet Middle–Outlet0.5m 3.228E-24 0.004 2.722E-271.0m 1.769E-146 1.122E-39 2.318E-421.5m 1.652E-125 3.341E-32 3.621E-46

ABR4DepthComparisonT-Test 0.5m–1.0m 0.5-1.5m 1.0–1.5mInlet 2.395E-117 1.418E-120 8.297E-31Middle 1.581E-09 1.512E-15 3.961E-48Outlet 1.739E-13 7.769E-39 1.472E-14

With a constant biodegradation rate throughout the entire chamber, the subsequentmeasurementsafterweekonewereonlydoneat0.5mattheoutletofeachchamber.TimetrialsdoneonStreet1at9:00, 11:00, and 13:00 at this location in each chamber indicated a general decrease in TRP-likeconcentrationsasthewastewaterwentthroughthetreatmenttrain.Thesteepestdecrease,however,wasseenintheanaerobicfilterchambers.BasedonthecorrelationofTRP-likeconcentrationandE.colipresence found in other research, this would suggest that there was a gradual removal of E. coli orbacteria within the ABR with the largest removal in the AF chambers (Baker et al. 2015). Percentremoval as compared to the concentrations in the previous chamber can be seen in the followinggraphs.

Figure17.TRP-likeconcentrationsfortheABRoutlet(chamber7/4)andAFoutlet(AF2)ofStreets1and3.Measuredatoutlet,at0.5m.

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It is also important to note that the concentrations in ABR 1 (the inlet of the whole system) weretypicallyat3mg/lwithABR7(theoutlet)usuallyat2mg/l,AF2at<1mg/l,andHGFatornear0mg/l.Comparing theseconcentrations to those foundbyBakeretal, theseconcentrationsaremuchhighereven in the AF. They measured TRP-like concentrations ranging from only 0.006-0.196mg/l whichcorrelated to anE. coli concentrationof 146-9,204,500CFU/100ml. Itmustbe added,however, thattheirresearchwasperformedonsewagecontaminatedfreshwaterwhilethisresearchwasonmunicipalwastewaterwhich,aftertreatment,reachedalevelsimilartoorbetterthancontaminatedfreshwater.

24-Hour time lapse trialsonStreet1and3were inconclusiveas theywerenot indicativeofanydailycyclical pattern. It was assumed that TRP-like concentrationswould follow the same daily pattern asflowrates,a largepeak inthemorning,asmallerpeak intheevening,andthendecreasingovernight.ThiswasnotseenthroughCyclopssampling,however.DifferencesinTRP-likeconcentrationswereseenbetweendifferentchambersbetweenthedifferentstreets.BothABR1chambersfromStreets1and3only showed a gradual decrease in concentrations while ABR 7/4 showed sudden changes inconcentrations. It is unclear if thiswas a function of differences in daily loads, chambers, instrumentmalfunction,oranotherunknownfactor.Furtherresearch,possiblyleavingtheCyclopsinonechamberforanentireweek,wouldbeneededtofullyunderstandthepatternsinTRP-likeconcentrations.

DifferenttreatmentstreetsshoweddifferentTRP-likeconcentrationsaswell.Intimetrialsdoneat9:00and11:00ofall threestreets,acleardifferencewasfound inStreet3.All threestreetsstartedatthesameconcentrationattheABRinlet(ABR1).BythetimethewaterexitedtheABRatABR7/4,however,concentrationswerenoticeablyhigherinStreet3thantheothertwostreets.Similarly,acomparisonofonlyStreets1and3ABRandAFoutletsshowthatStreet3hadconsistentlyhigherTRPconcentrationsthanStreet1.ThiswouldalsoimplythatStreet3hashigherconcentrationsofE.colithanStreet1.ThisdatacouldbeindicatingthatStreet3,withonlyfourABRchambers,islessefficientatremovingbacteriathan the other two streets. Higher concentrations may have been a function of a lower flow ratethrough Street 3 however. Flow patterns and their effect on TRP concentrations would need to beanalyzedfurthertoconfirmthis.

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Figure18.PercentremovalofTRP-likeconcentrationsfromthepreviouschamberwithinStreet1.

Figure19.PercentremovalofTRP-likeconcentrationsbytreatmentsectioninStreet1:ABR,AF,andHF.

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Conclusion

AltogetherthebiodegradabilityratesviaTRP-like fluorescencewereabletobeestimatedusing in-situfluorescenceinstrumentation.IndividualchamberswithintheDEWATStreatmentstreetswerefoundtohave a uniform biodegradation regardless of depth or location within a chamber. Biodegradationdecreasedthroughoutthetreatmentstreetwiththelargestdecreaseoccurringintheanaerobicfilters.Further research is needed to establishmicrobial treatment patterns as 24-hour time lapses did notrevealanydailycyclicalpatterns.Inadditiontothis,basedontheseresultsitisrecommendednottousefourABRchambersinsteadofsevenasStreet3withonlyfourchambersseemedtobelessefficientforbiodegradation.

All results from theCyclops, however, need tobeused in conjunctionwith standardbiodegradabilitytestssuchasuseofarespirometer,5-dayBiochemicalOxygenDemandtests,andE.colienumeration.Usedalongsidethesetraditionaltests, in-situfluorescenceusingTRP-likepeakintensitiesgivesaquicksnapshot of overall biodegradability. Thismethod could then be extremely useful in evaluating otherDEWATSplantsinanefforttoprovideasolutionforsustainablesanitationaroundtheworld.

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References

Baker,A.,Cumberland,S.A.,Bradley,C.,Buckley,C.,andBridgeman,J.,Towhatextentcanportablefluorescencespectroscopybeusedinthereal-timeassessmentofmicrobialwaterquality?.ScienceDirect.2015,inpress.BORDA,Germany.July30,2015.http://www.borda-net.org/.

Hudson,N.,Baker,A.,Ward,D.,Reynolds,D.,Brunsdon,C.,Carliell-Marquet,C.,Browning,andS.CanfluorescencebeusedasasurrogatefortheBiochemicalOxygen(BOD)testinwaterqualityassessment?AnexamplefromSouthWestEngland.ScienceDirect.2008,391:149-158.Tilley, E., Ulrich, L., Lüthi, C., Reymond, P., and Zurbrügg, C. Compendium of Sanitation Systems andTechnologies.SwissFederalInstituteofAquaticScienceandTechnology(Eawag).2014,2ndEd.