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Volatile fatty acid metabolism in thermophilic aerobic digestion of sludge Chu, Angus 1995

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VOLATILE FATTY ACID METABOLISM IN THERMOPHILIC AEROBICDIGESTION OF SLUDGEbyANGUS CHUB.Sc., The University ofBritish Columbia, 1988M.Sc., The University ofBritish Columbia, 1990A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FORTHE DEGREE OFDOCTOROF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Department ofCivil Engineering)We acceptthisthesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAJuly 1995© Angus Chu, 1995In presenting this thesis in partialfulfilment of therequirements for an advanceddegree at the Universityof British Columbia, Iagree that the Library shall make itfreely available for reference andstudy. I further agreethat permission for extensivecopying of this thesis for scholarlypurposes may be grantedby the head of mydepartment or by hisor her representatives. It isunderstood that copying orpublication of this thesis for financialgain shall not be allowedwithout my writtenpermission.(Signature)_________________________Department ofCi/ILeN(7,Ne’,,v6The University of British ColumbiaVancouver, CanadaDateJUJ2?S’DE-6 (2/88)11AbstractThe efficacyofVolatile Fatty Acid (VFA) productionin Thermophilic AerobicDigestion (TAD) ofprimary sludge was investigated.Thisresearchprogramwas carried out inapilot scale, TAD process, located inthe wastewatertreatmentpilotplant site, atthe UniversityofBritish Columbia. Preliminaryresults showedthatthe highestaccumulationofVFA (950mg/L as acetate) had occurred, undermicroaerobic conditions (airflowrateofbetween0-0.17V/V-h), inthe first stage ofthe 150 L, 2-stageprocess. Thetwo other aerationconditionsexamined (transition-air flowrate of0.28 V/V-h and aerobic-airflowrate of0.6 V/V-h)accumulatednegligible amountsofVFA. Therefore, the subsequent researchconcentratedonthe first stage ofthe TAD process, under microaerobic conditions. The two independentvariables examined were air flowrates and solids retentiontimes (SRT). Thethree SRTstestedwere 3, 4.5 and 6 days. The four air flowrates examinedwere assignedthe labelstrueanaerobic, lowflow microaerobic, mediumflowmicroaerobic andhigh flowmicroaerobicconditions. NetVFAproductionwas found to be a functionofboth aeration and SRT. Ingeneral, as SRT and air flowrates decreased, netVFA production increased(specificallyacetateandpropionate). The measured concentration ofany speciesofVFA, atany giventime, was afunctionofboththe relative rates ofits synthesis andbiodegradation. Decreasing orincreasingthe aerationrate and/or SRTresulted in aproportional change in VFA accumulation. Themaximummeasured acetate accumulation rate occurred underthe 4.5 d SRT andthe trueanaerobic condition.111A biochemical model was developed in order to explaintheprocess ofVFAmetabolismin TAD. In thisprocess, under strict anaerobic conditions, bacteriamust achieveoxidation/reductionbalance by diverting the catabolic flowofcarbonto fermentativeendproducts (eg. propionate)thatwill consumeNADH (NicotinamideAdenine Dinucleotide). Thekey issue in fermentation is the recycling ofNADH bythe conversion ofspecific intermediatesto differentfermentationproducts whichregenerateNAD. The oxidationofintermediatesthatrequiredthe netreductionofNADcannotproceed under fermentative conditions.Consequently, these catabolicintermediates addedunder batchtestconditions,using TADsludge, under anaerobic conditions, remained intheirunoxidizedform andpersisted inthemedium. The oxidationofintermediates whichrequiredno netreductionofNADcanand didproceedunder fermentativeconditions. Under strictanaerobic conditions, theVFAprofiles inthepilot scale TAD processwere similarto fermentationtypeprocesses (eg. anevendistributionofVFAbetweenacetate andpropionate).Whenthe bioreactorswere operatedundermicroaerobicconditions (ie. oxygendemandis greaterthanoxygen supply), metabolismresultedinacharacteristic VFA distributionprofilewithacetate asthe predominantVFAproduced (up to 80% ofthetotal VFA). Propionateconstitutedthe second largest fraction at 11%. Under thismicroaerobic condition, theNADHproduced during oxidationofsubstrates couldbe reoxidizedby operationofthe respiratorychain. Therefore, the carbonflowcouldbe uncoupledfrom the necessityto maintainredoxbalance viafermentative means. This separationwouldpresumably allow the organisms inaTAD processto maximizeATP (adenosine triphosphate) productionby increasing the flux ofivintermediatesto acetate. The majority ofthe substrates examinedunderbatchtestconditions,withTAD processbiomass, undermicroaerobic conditions, were oxidizedto an acetateintermediate.VTable ofContentsABSTRACTiiTABLE OF CONTENTSvLIST OF FIGURESviiiLIST OF TABLESxiiiACKNOWLEDGMENTSxiv1. INTRODUCTION12. LITERATURE REVIEW32.1 AutothermalThermophilic AerobicDigestion32.1.1 The Autothermal Process42.1.2 ProcessDevelopment 72.2 Enhanced BiologicalPhosphorusRemovalProcessModel 82.3 AcetateProduction inATAD 112.4 ThermophilicPre-StageProcess (DualDigestion) 132.4.1 Anaerobic Digestion: Substrate SpecificityDuring Phase Separation 152.4.2 Phase Separation 162.5 Thermodynamics and Metabolism 182.5.1 Energy Production (ATP) 212.5.2 Bacterial Energetics 222.5.3NADH 263. METHODS AND MATERIALS 293.1 ThermophilicAerobicDigester 293.1.1 Preliminary OperationalPhase 293.1.2 SecondPhase ofOperation 313.2 Aeration 33vi3.2.1 Preliminary Operational Phase343.2.2 Second Phase ofOperation343.3 VolatileFattyAcids353.4 Ethanol and Propanol Determination363. 5 Pyruvic and LacticAcid Determination363.6 Totaland Inorganic Carbon363.7 Solids373.8 On LineData373.9 BatchExperiment384. RESULTS AND DISCUSSION414.1 BatchExperiments414.1.1 Fermentative and OxidativeVFA Metabolism inTAD414.1.2 SubstrateAdditionExperiments454.1.2.1 Valerate andButyrateAdditionExperiments494.1.2.2 Isobutyrate, Isovalerate and2-MethylbutyrateAdditionExperiments544.1.2.3 Pyruvate andLactateAdditionExperiments604.1.2.4 Ethanol and Propanol AdditionExperiments664.1.2.5 Effects ofDifferentClassesofMacromoleculesonVFAMetabolism 724.1.3 2, 4-Dinitrophenol AdditionExperiment 814.1.4 Response ofBiomassfrom a FermenterProcess to Anaerobic and MicroaerobicConditions 864.1.5 Fermentative TAD Experiments 954.1.6 SalmonArmATAD Performance 994.2 Preliminary Pilot Scale TAD Experiments 1054.3 3 x 3 Pilot ScaleTAD Experiments 1154.3.1 Temperature VariationofPilot Scale TAD experiments 1164.3.2 OnLine ORP Measurementsas StateVariable 1214.3.3 VFAAccumulationinTAD 1234.3.3.1 Variability ofVFAMeasurements 1234.3.3.2 VFAAccumulationinPilot Scale TAD Experiments 1294.3.3.3 Kendall’s Tau-b and Analysis ofVariance ofVFA Data 1374.3.3.4 Response Surface Plots ofVFAProductioninPilot Scale TAD 1404.3.4 TAD Pilot Scale ProcesspH 1434.3.5 Total andVolatile SolidsReductionofthe Pilot Scale TAD Process 146vi’4.3.5.1 Role ofEnzymes in Solids Destruction1524.3.5.2 Solids DestructionVariability Discussion1534.3.6 Salmon ArmATAD1545. OVERVIEWAND SUMMARY1595.1 BiochemicalModelofSubstrateMetabolism in TAD1595.2 AcetateAccumulationPhenomenon in TAD1655.3 ‘C-AcetateLabelExperiment1675.4 Acetate Production in Microorganisms1686. CONCLUSIONS AND RECOMMENDATIONS1746.1 Conclusions1746.2 Recommendations1767. REFERENCES180APPENDIXA: BATCH TEST RESULTS 190APPENDIX B: 3X3 PILOT SCALE TAD RESULTS250APPENDIX C: PRELIMiNARYPILOT SCALE TAD RESULTS 293APPENDIX D: TEST CHEMICALS299vii’ListofFigures2. 1 Heat balance schematic ofathermophilic aerobic digester(adapted from EPA, 1990).62. 2 Biochemicalmodel ofphosphate accumulationthe a) anaerobic and b) aerobic zonesofaBio-P process (from Comeau, 1988).102. 3 Variations inthe concentrationsofVFA duringthe biodegradationofyeast cellsby aerobicthermophilic bacteriaunder oxygen limiting conditions(from Mason, 1986). 122. 4 Principal sequences ofanaerobic digestion (Fox andPohiand, 1994). 172. 5 Overall organizationofelectrontransportand oxidative phosphorylation(adapted fromLehninger, 1982).252. 6 Pathways involved in the fermentationofglucose (adaptedfromBoyd, 1984).283. 1 Schematic ofthe UBCpilot scaleTAD process a) operating in series (1stand 2ndphases)and b) underthe parallelmode ofoperation (A and B sides).304. 1 Comparisonofacetate andpropionate concentrationsbetweenthe anaerobic andmicroaerobic conditions, a) acetate responseand b) propionate response.424. 2 Comparisonofbutyrate and2-methylbutyrate concentrationsbetweenthe anaerobic andmicroaerobic conditions, a) butyrate response andb) 2-methylbutyrate response.434. 3 Comparisonofisovalerate and valerate concentrations betweenthe anaerobicandmicroaerobic conditions. a) isovalerate response andb) valerate response. 444. 4 VFAresponse inbatchTAD experiment to propionate addition, a) microaerobic controlb)microaerobicwithpropionate addition c) anaerobic control d) anaerobic withpropionateaddition. 474. 5 Differenceplots ofthepropionate addition experimentunder a) microaerobic andb)Anaerobic conditions. 504. 6 Difference plots ofthe valerate addition experimentunder a) microaerobic and b) anaerobicconditions. 514. 7 Difference plots ofthe butyrate addition experiment under a) microaerobic and b) anaerobicconditions. 52ix4. 8 n-oxidationofbutyric and valerie acids (adapted from Lehninger, 1982).534. 9 Differenceplotsofthe isobutyrate addition experimentunder a)microaerobic andb)anaerobic conditions.554. 10 Difference plots ofthe isovalerate addition experiment under a)microaerobicand b)anaerobic conditions.564. 11 Difference plots ofthe 2-methylbutyrate additionexperimentundera) microaerobicandb) anaerobic conditions.574. 12 Reactions for the oxidation ofthebranched chainamino acidsby bacteria(adapted fromSokatchetal., 1968).584. 13 Differenceplotsofthepyruvate additionexperimentunder a) microaerobic andb)anaerobic conditions.614. 14 Difference plots ofthe lactate additionexperimentunder a) microaerobic andb) anaerobicconditions.624. 15 Formation ofpropionate and acetatefromDL-lactate viathe acrylatepathway (Gottschalk,1986). 654. 16 Differenceplots ofthe ethanol additionexperimentunder a) microaerobicandb)anaerobic conditions. 674. 17 Differenceplots ofthepropanol addition experimentunder a) microaerobic andb)anaerobic conditions. 684. 18 VFA differenceprofilesunder anaerobic conditionsfor a) linoleic acid andb) glucoseadditionexperiments. 734. 19 VFA differenceprofilesunder anaerobic conditionsforthe a)dextrinandb) peptoneaddition experiments. 744. 20 VFA differenceprofilesundermicroaerobic conditions forthe a) linoleic acid andb)glucose addition experiments. 754. 21 VFAdifferenceprofiles underthe microaerobic conditionforthe a) dextrin and b)peptone addition experiments. 764. 22 Fermentation ofpyruvateto propionate viathe succinate-propionatepathway (Gottschalk,1986). 78x4. 23 FormationofC02,lactate and ethanol from glucoseby the heterofermentativepathway.(Gottschalk, 1986).794. 24 2, 4-Dinitrophenol addition experiment under a) anaerobicand b) microaerobicconditions.834. 25 VFA, pH and ORP profiles inthe 2, 4-dinitrophenol addition experimentundermicroaerobic conditions, a) control conditionand b) 2,4-dinitrophenol addition condition.844. 26 Fermenterprocess sludgeresponseto increasingprimary sludge additionunder anaerobicconditions. a) acetate profiles and b) propionate profiles.874. 27 Fermenterprocess sludge response to increasing primary sludgeadditionundermicroaerobic conditions, a) acetate profiles and b) propionate profiles.884. 28 TAD process sludge responseto increasing primary sludge additionunder anaerobicconditions. a) acetateprofiles andb) propionate profiles.894. 29 TAD process sludgeresponse to increasing primary sludge additionunder microaerobicconditions. a) acetate profiles and b) propionate profiles.904. 30 ComparisonofmaximumrateofVFAproductionbetweenTAD and fermenterprocessbiomassunder a) anaerobicandb) microaerobic conditions.934. 31 Comparison ofmaximumachievableconcentrationofVFA betweenTAD andfermenterprocess biomassunder a) anaerobic and b) microaerobicconditions.944. 32 VFAprofilesoffermentativeTAD biomassresponseto increasing primary sludgeadditionunder a) microaerobic andb) anaerobic conditions.974. 33 VFAprofilesofcontrol side TAD biomass response to increasingprimary sludge additionunder a) microaerobic and b) anaerobic conditions. 984. 34 VFAdifference plots ofSalmonArmATAD sludgeunderanaerobic conditionsto a)primary and b) waste activatedsludge additions. 1014. 35 VFA difference plots ofSalmonArmATAD sludgeunder anaerobic conditionsto a)propionate and b) amixture ofprimary andwaste activated sludge additions. 1024. 36 VFA difference plots ofSalmonArmATAD sludge undermicroaerobic conditionsto a)primary and b) waste activated sludge additions. 103xi4. 37 VFA differenceplots ofSalmonArm ATAD sludge undermicroaerobic conditionsto a)propionate and b) amixture ofprimary and waste activated sludgeadditions. 1044. 38 Thetransition condition in the first stage oftheTAD process (air flowrate of0.28 V/Vh). a) An example ofone cycleofORP andtemperatureprofiles. b) VFAprofilesofthesame cycle.1074. 39 ORP and VFAprofiles inthe first stage ofthe TAD process under a)microaerobic (airflowrate of0 V/V-h) and b) aerobic conditions (0.6 V/V-h).1084. 40 ORP andVFAprofiles inthe first stage ofthe TAD process during a switchfrom a)microaerobicto aerobic conditions and from b) aerobic to microaerobicconditions. 1094. 41 Comparisonofthe net acetate andnettotal VFAproductioninthe firststage oftheTADprocessunder the three aeration conditions.1104. 42 Examples ofon line temperature measurements duringpilot scale TADexperiments 1184. 43 Temperature effects in TAD reactorsfrom changing Turboratorrotational speed. 1194. 44 Examplesofon line ORP measurements duringpilot scale TAD experiments, a) ORPvariationofboth sides overaperiodof12 days and b) an extractofthe sameperiodshowing sharktoothpattern. c) FastFouriertransform ofthis ORP data. 1204. 45 Variationofacetate concentrations overtime inthe pilot scale TAD experiments with a 3d SRT. a) A side andb) B side. 1244. 46 Variationofacetate concentrations over time inthepilot scale TAD experimentswitha4.5 d SRT. a) A side and b) B side. 1254. 47 Variation ofA sidepropionate concentrations overtime for all 10 runs. 1264. 48 Variation ofB sidepropionate concentrations overtime for all 10 runs. 1274. 49 Acetate accumulationinpilot scale TAD experimentsonthe a) A and b) B sides. 1304. 50 Propionate accumulation inpilot scale TAD experimentsonthe a) A andb) B sides. 1314. 51 Isobutyrate accumulation inpilot scale TAD experiments onthe a) A andb) B sides. 1324. 52 Isovalerate accumulationin pilot scale TAD experimentsonthe a) A and b) B sides. 133xli4. 53 VFA accumulationnormalized to their respective control values, a) acetateand b)propionate.1354. 54 VFA accumulationnormalized to their respective control values, a) isobutyrate andb)isovalerate.1364. 55 Response surfaceplotsofdaily VFA accumulationnormalizedto theirrespective control values for a) acetate and b) propionate.1414. 56 Response surface plots ofdaily VFA accumulationnormalizedto theirrespectivecontrolvalues for a) isobutyrate and b) isovalerate.1424. 57 Experimentalreactor (B side) pH values overtime forall 10 runs.1444. 58 AveragepH values ofthe a) A side and b) B side bioreactors.1454. 59 Total solids destructioncapacity inpilot scale TAD experiments onthe a) A (control)andb) B (experimental) sides. 1484. 60 Total volatile solids destructioncapacity inpilot scale TAD experiments onthe a)A(control) andb) B (experimental) sides. 1494. 61 Total andvolatile solids destruction capacitiesnormalizedto their control values, a) Totalsolids and b) volatile solids. 1504. 62 DistrictofSalmonArmATADprocess schematic. 1554. 63 District ofSalmonArmATAD reactortemperaturesduringthe a) March, 1993 visit andb) May, 1994 visit. 1564. 64 SalmonArmATAD VFAprofiles in all three cells during the a) March, 1993 visitand b)May, 1994 visit. 1575. 1 Biochemical model ofacetateproductionin TAD under a) microaerobic andb) anaerobicconditions. 1605. 2 Summary ofcarbonflowfrom substrate additionexperiments under a) microaerobic and b)anaerobic conditions. 1615. 3 Comparisonbetweenoxygen demand and oxygen supply ofany aerobicallymetabolizingculture. 164xliiListofTables3. 1 Comparisonofpilot scale TAD parameters andrecommended values (EPA, 1990; Kelly,1990).313. 2 Combination ofaerationand SRT for eachexperimentalrun.324. 1 Selected acetogenic reactions.694. 2 Durationofmicroaerobiosis duringthe three aerationconditions inthe first stageoftheTAD process.1114. 3 ComparisonofVFA distributionbetween2 thermophilic aerobic digestionprocesses and2fermentationprocesses.1124. 4 Descriptive statisticsofair flowrates and SRTsofpilot scaleTAD experiments. 1164. 5 MatrixofKendall’s tau-b correlationcoefficients forbothacetate andpropionateaccumulationvalues versus time.1384. 6 Analysisofvariancetable for acetate and propionate accumulation.140xivAcknowledgmentsI would like to express my gratitude to all the individualsinvolved in the completionofthis thesis:I would first like to thankmy supervisor, Dr. D.S. Mavinic, Headofthe EnvironmentalEngineering Group at UBC, for his encouragement,understanding andunwavering supportthroughoutthe completionofthis research. I am gratefulto Dr. W. K. Oldham, Dr. W. D.Ramey, Dr. E. Hall and H. G. Kelly for serving onmy supervisolycommittee and fortheirconstructive criticisms andvaluable suggestionsthroughboththe researchphase andthepreparationofthe final report.I would also like to acknowledgethe excellenttechnical supportofthe staffoftheEnvironmentalEngineering Lab. Special thanks goesto the DistrictofSalmonArmswastewatertreatmentplant operators, HartFrese and Lee Robinson, fortheir assistanceandcooperationduringthe finalphase ofthe researchprogram.Financial supportforthe study wasprovidedbybothgrants andinkind contributionsfromtheNatural Sciences andEngineeringResearchCouncil ofCanada(NSERC), DaytonandKnightLtd. and TurboratorTechnologies Inc.11. IntroductionThe widespreadapplicationofsewagetreatmentprocessesto satisfy increasinglystringentlegislation concerning aqueous discharges into surfacewaters has resultedinincreasedsludgeproductionand exacerbatedproblemsofsludge disposal. Throughouttheworldconsiderable attentionis being directedinto thetreatmentandultimatedisposal ofsludgeresulting fromthe purificationofdomesticwastewaters.In 1991, aresearchprogramwasundertaken, inthe Civil Engineering Department,at theUniversity ofBritishColumbia, to investigate specific aspects ofaprocess known asThermophilic Aerobic Digestion (TAD). Thisthesis is specifically concernedwiththephenomenonofVolatile Fatty Acid (VFA) metabolisminTAD process biomass, as well as thefactorsthat affectVFAproduction. The purpose for studying VFAmetabolisminTAD will beintroduced andreviewed inthe following section. This literature reviewbriefly introducesthevarioustopicsofinterestwhich specifically dealswiththe autothermal thermophilic aerobicdigestionprocess.The objectives ofthe researchwere two fold. The firstresearchgoal wasto map themetabolicpathways ofvarious carbon substrates inamixed culture biomass. Abatchtestapparatuswas usedto examine the biochemistry involved in substrate metabolism inTAD,specifically acetate metabolism (Results and Discussion, section 4.1). The second objective wasto maximize VFAproductionby optimizationoftwo independentvariables (ie. Solids Retention2times [SRT] and aeration rates). The effectsofvaryingthesetwo parameterswere studied on apilot scale TAD process, located inthe wastewatertreatmentpilotplant site, atthe UniversityofB.C. (sections 4.2 and4.3).The overviewand summary sectioncovers the proposed biochemicalmodel ofsubstratemetabolism in TAD as well as some aspects ofacetate production inmicroorganisms. The finalsection deals withthe main conclusionsand subsequentrecommendationswhicharose from thisresearchprogram.32. LiteratureReviewThe following literature reviewis only meant as abriefintroductionto the various phasesofthe researchprogram. Amore detailed and comprehensive reviewofsome ofthesubjectsareincluded withinthe results and discussion sectionunder manyofthe subheadings.Since the maintopicofthisthesis deals withthe productionofVFAinTAD, thepotential augmentationofotherprocesses, withthe VFAricheffluent, mustbeaddressed. Thetwo processesthathave thepotentialto complementTAD are the biological phosphorusremovalprocess andthe secondphase ofanaerobic digestion. Abriefcritique ofeach oftheseprocessesis included inthe literature review.2.1 Autothermal ThermophilicAerobicDigestionBacteriaare an abundantnatural resource, butthe only waythey canbe utilized ineffective technologicalprocesses is through a comprehensive understanding oftheirphysiologyandbiochemistry. Temperature is one very important factoraffectingthephysiology andbiochemistryofmicroorganisms. Largely outofconvenience, microbiologists have classifiedbacteriaaccording to their optimum growthtemperatures, thereby introducingthethree artificialcategorieswe seetoday. These categories includepsychrophiles, mesophiles andthennophiles.There is still considerable debate concerningthe temperature cutoffs betweenthese three groups.Ingeneral, psychrophilic organisms have anoptimal growthtemperatureof<100C, mesophilicorganismshave optimal growthtemperaturesranging from 15 to 40°C andthennophilicorganisms have optimum growthtemperaturesof>45°C.4The study ofhightemperature aerobic digestion ofsludge began inthe 1960’s withWoodley (1961) and Kambhu andAndrews (1969).The developmentofthese plantswasinitially limited by the belief, by some, thatpure oxygenwas necessary to achieve the neededelevated operating temperatures. However, FuchsandFuchs (1980) andHoffmanand Crauer(1973) reported developmentalworkwhich used air inlaboratory scaleand inprototypefacilities. The 1980’s sawthe emergenceofmany full scaleplants in Europe, but the fewoperating inNorthAmericawere confinedto western Canada.AutothermalThermophilicAerobicDigestion (ATAD) systemsare aerobic sludgedigestionprocesses that operate undertemperature conditionsinthe thermophilic range (40to80° C) withoutexternally supplied supplemental heat. The processreliesonthe conservationofheatreleased during operationto generate and sustainthe desiredtemperatures. The heatgenerated originates fromboththe bacterialmetabolic activity andthe mixing/aeration system.Thetwo major advantages associatedwiththeATAD process are pasteurizationofthe sludgewhichreducespathogen loads, andhigherbiological reactionrates. Althoughadvances havebeenmade inATAD technology froman engineeringperspective (sinceits original development20 years ago), little isknown concerningthemicrobial ecology andthermodynamicsofthissystem.2.1.1 TheAutothermalProcessAutothermal aerobic digestion is capable ofdegrading complexorganic substances intoendproducts including carbon dioxide andwater. To achievethis outcome,an adequate supply5ofbiodegradable organic matter, oxygen andnutrientsare required. A fractionofthe energyreleasedby microbial degradation is consumed to formnewcellular material. The other fractionis released as heat energy. Typical biological heatproductionvalues reported inthe literaturerange from 14,190 to 14,650 kJ/kg02 (Andrews and Kambhu, 1973; Cooney eta!., 1968). Theoxygenrequirements vary (eg. from 1 to 3 kg02/kgVolatile Suspended Solids [VSS] oxidized)but are often consideredto fall withinthe values ofmesophilic digestionwhich is 1.42 kg02/kgofVSS oxidized. Accordingto the EPAreportonATAD technology, theheatreleased bythedigestionprocess is the majorheat sourceusedto achievethe desired operatingtemperature(Kambhu andAndrews, 1969). However, it is notyetclearas to what fractionbiologicallyproduced heat contributes to the total heat input ofthe system. Figure 2.1 showsthe variousinputs, outputs andheatproductionitemsto be included in aheatbalance.Autothermal conditionsrequire an“adequatelythickened” sludgeto providethe neededsubstrate, a suitably insulated reactor to minimize conductive heat loss, goodmixing and anefficient aerationdevice whichminimizes heat loss due to aeration. Sufficientheat shouldbegenerated and sustained intheprocess suchthatheat exchangers between feed and effluent, towarmthe incoming sludge, are notnormally required, unless thetemperature ofthe incomingfeed sludge is low (eg. 8-9° C).6Enthay of Feed SludgeEnthalpy loss fromsensible and LatentWater Vapor HeatLoss In Gas EffluentEnthalpy loss in SludgeEffluentHeat Balance Schematic of a Thermophilic Aerobic Digester(adapted from EPA. 1990)Enthalpy loss dueto sensible heat Lossto surroundingsInfluent Gas (A’Jr)Rgure 2.1:72.1.2 ProcessDevelopmentMuch ofthe developmental work leading to the ATAD process occurred inGermany andhas been described by Popel (Popel and Ohnmacht, 1972). The early studiesonATAD used aself-aspirating aeration devise manufacturedby DeLaval, Inc.They were marketed intheU.S.forthe treatmentofhigh strength industrial and liquid manurewaste in the 1970s, in apatentedprocesscalledthe LICOM (Liquid Composting) system. Several batchtestswithvariousindustrial andanimal wasteswere reported. Thermophilic temperaturesof50°C to 60°Cwereachieved (DeLaval Separation Company). Other studies on dairy,beefand swine wastesdemonstratedthat autoheating to thermophilictemperatureswaspossible (Hoffmanand Craver,1973; Terwillegerand Craver, 1975). Highpurity oxygen systems were also investigated,atthepilot scale, in the early 1970s, by UnionCarbide (Matsch andDmevich, 1977). The mostextensive U.S. study ofATAD using airwas conducted at Binhampton, NewYork, in 1977 and1978 (JewelI eta!., 1982). Currently, the only knownATAD systems formunicipal sludgestabilizationinNorthAmericaare in Canada, where four systems havebeen installedinBritishColumbiaand one inAlberta(Kelly, 1989; Kelly, 1990).Today the Germans and Swiss are the leaders inthistechnology withover 80 operatingplants betweenthem. Other countries using ATAD includeNorway, Britainand SouthAfrica(EPA, 1990). All reportedATAD facilities inthe ERG (FederalRepublic ofGermany) havebeenreported easy to operate andrequire very littleprocesscontrol andmaintenance. Inmostcases, process control consists ofperforming periodic suspended solids tests andpH analysis,monitoringreactortemperatures and controlling the pumping ofspecific volumes ofsludge to8the ATAD reactors on abatchbasis. The normal control parameterfor sludge volumemanagement is the filling levelsofthetanks. Atpresent, there is nocomprehensive real time, onlinemonitoringmethod forreactorperformance. The two most common on lineparameters aretemperature andpH.Dissolved oxygen levels in several reportedATAD studieshave variedfrom 0.7 mg/L tomorethan 3 mg/L (EPA, 1990). Othermeasurementsin asingle stage ATADsystemhaveshownnegligible dissolvedoxygen concentrations, ranging between 0 and 0.2 mgIL.Thesemeasurements suggestthat oxygenconcentrations canbe alimiting factor foraportionofthebatchcycleafter initial sludge feedhasbeenintroduced.OxidationReduction Potential (ORP) hasbeenproposed as atool to assessreactorconditions(Kelly, 1990). Itwas concludedthat ORP was auseful indicator for determinationofpoor oxygentransfer, odorpotential,thin sludge, over aeration and overfeeding. WhenusedinconjunctionwithpH andtemperaturemeasurements, ORP monitoringpermitsthe identificationofoperationalproblems andthe evaluationofpossible corrective actions.2.2 EnhancedBiologicalPhosphorusRemovalProcessModelThe process ofbiologicalphosphorusremoval (Bio-P) is amodificationofaconventional activatedsludgeprocess. Thisprocess usually consists ofananaerobic zonefollowedby anaerobic zone. The anaerobic zone induces the recycledbiomass to releasephosphorus into solution, while accumulating intracellular substrate reserves, to be used later in9the aerobic zone. Inthe aerobic zone, the biomassrecoversthephosphorusreleased intheanaerobic zone, as well as the initial phosphorus presentin the incomingwastewater,byoxidizing the stored carbonreserves, thusresulting inthe accumulationofphosphorus.Thebiomassofanenhanced Bio-P plant is capable ofaccumulating up to 3% (or greater)phosphorusby dry weight, in comparisonto conventional activated sludge biomasswhichtypically contains1%. Inthemodel proposedby Comeau eta!. (1986) andWentzel etal. (1986), thebacteriaresponsible forphosphate accumulation are believed to derive an advantage over other bacteriaby exchangingpolyphosphate forthe accumulationand storage ofpolyhydroxyalkanoate(PHA)inthe anaerobiczone. This stored carbon, inturn, provides energy to drive the accumulationofphosphate under aerobic conditionswhere carbonavailability is limited. This model explains thebeneficial effectsofsimple acetateandpropionate additionsto the anaerobic zone, sincetheseshort chainfatty acids favorthe accumulationofPHAwhichwould inturn, favorthe aerobicaccumulationofphosphorus (Potgieter and Evans, 1983; Siebritz eta!., 1983; ArvinandKristensen, 1985). A simplified representationofthe model is shownin Figure 2. 2. Therefore,thekeyto efficientperformance lies inthe adequate supply oflowmolecularweightVFA(specifically acetate) inthe anaerobic step.Inpractice, this VFAprerequisitehasbeenachieved inanumber ofways. Thesemethods include increasing the solids retentiontimesinthe primary clarifiersto promotefermentationto produce theneededVFA, and/or fermentingtheprimary sludge in a separatereactorthat feeds the VFArich fermented sludge directly into the anaerobic zone (Comeau eta!.,10bio-PbacteriaHP1availablecarbonsubstratesacetate &(a)proplonate(b)Figure 2.2: Biochemical model of phosphate accumulation(adapted from Comeau, 1988)(a) anaerobiczone (b) aerobic zone02(or NO3)bio-Pbacteria111987; Rabinowitzand Oldham, 1985). Thesemethods allrely onthe process offermentationtoproducethe VFAneeded for efficientphosphorusremoval.2.3 AcetateProduction inATADTheATAD processhas thepotential to provide the acetaterequiredin Bio-P butthisalternative hasnotbeen systematically evaluated. Masonetal. (1987) hadreportedthatacetateformation inATAD greatly exceeded all other carboxylicacids and, at 2000 to 2500mg/L, was5 to 10 timesthe concentrationofotherVFA. Hamer (1987) also conductedATAD experimentsinvolvingthe solubilization andbiodegradationofyeast cells,by thermophilicbacterialpopulationsin a laboratory scale bioreactor, operating in afill anddrawmode(ie. semi-continuous). Themixed culture ofprocessbacteriausedwas obtained fromafull scale,thermophilic aerobic waste sludge pre-treatmentprocess. The resultsshowed an accumulationofup to 6000 mgfL acetate. ThenexthighestVFA concentrationwas propionate at 800 mgIL.Theresults fromthis study are shown inFigure 2. 3.Kelly (1990) also conductedexperiments onVFAproductionat a full scaleATADprocess in SalmonArm, B.C. Sludge treatmentinthe digesterwas found to produce upto10,000 mg/L (personal communication). However, theproportionofacetic acidout ofthetotalisnotknown. Ineachofthese studies, fermentationwas assumedto beresponsible forthemeasuredVFA.12z0I-.I—zUiC-,0C-)Figure 2. 3: Variations in the concentrations of VFA during the biodegradation of yeastcells by aerobic thermophilic bacteria under oxygen limiting conditions (Mason, 1986).•z:7a,Ez0IIUiC-)a0C-,UiIUiC-)tIME (HOuRS)4813Experience in SalmonArm, B.C. (Kelly, 1990) had shownthat VFAconcentrations inATAD were related to pH and ORP. By changing the rateofsupplied air andmixingeffectiveness, changes in VFA concentrations did occur. Sincethe SalmonArm facility operatesas a biologicalphosphorus removal plant, the potential foraugmentingthe supplyofVFA totheprocess viathe digesters would be attractive. Sludgestabilizationand VFAproductioncouldultimately be accomplished in one operationand, as such,was examined at SalmonArm. Thispotential could also be extended to applications in other communities thathaveBio-Pprocesses.The initial batchexperimentswere done by diverting the contentsofone ATAD reactor into theanaerobic zone. The results showed only modest increasesin bothphosphorusreleaseanduptakerates forrespective anaerobic and aerobic periods, whencomparedto the control.Althoughthesebatchtests showedthatbiological phosphorus removal was slightlyimproved,further investigation wasnotcarried out. Amore detaileddiscussionis presentedin section 5(ie. Overview and Summary).2.4 ThermophilicPre-StageProcess (DualDigestion)Athermophilic, pre-stage process is an aerobicthermophilicpretreatmentsystem appliedpriorto conventionalmesophilic anaerobic digestion(Zwiefelliofer, 1985). Pre-stage systemsdiffer fromATAD systems in severalrespects. There is normally only one pre-stage reactorpriorto anaerobicdigestion. The residence time inthethermophilic reactor is normally between18 and 24 hours. Pre-stage systems are not autothermal andrequire supplemental heating. Heatexchangers are normally used in these systems. For raw sludge heating,a sludgeto sludge heat14exchanger is included in the thermophilic pre-treatmentstep. This exchanger recovers heatfromthe hot sludge prior to introduction into the mesophilicanaerobic digester. Wastegas fromthereactor is often recirculated into the air injectiondeviceto minimize vent gas heatloss.Althoughpre-stage systems are referredto as aerobic,“aerated” is probably amoreappropriatedescriptor. Air input into the system is typically lessthan the stoichiometric demandfor oxygen,and there is no measurable dissolved oxygenresidual.Because ofthe limited oxygensupply, theconcentration ofsoluble degradable organic compoundssuch as VFA is normally veryhigh(BalerandZwiefelhofer, 1991) and sludge leaving theprocess has notbeen fully stabilized.Aerobicthermophilictreatment ofsewage sludgehasbeen successfully appliedtopretreatmentpriorto amesophilicanaerobicdigestionprocess. Whenthis thermophilic processis incorporated ahead ofmesophilic anaerobic digestion,the benefit ofstimulationofWAproductionbecomes evident. The acetate,inparticular wouldbe beneficial to the directmetabolismofmethanogenicorganisms inthe followingstage. Some authorshave describedTAD as apossible acidification stage (Keller and Berninger, 1984).Bomio etal. (1989)investigatedthispossible beneficialnatureofTAD sludge. Cultivations carried outatvery lowaerationrates (O.IVIV-h), withabench scalebioreactorconfigurationfedwith amixture ofprimary andwaste activated sludges from a full scalewaste watertreatmentplant, showed littleproduction ofVFA.152.4.1 AnaerobicDigestion: Substrate SpecificityDuring PhaseSeparationAnaerobic digestion consists ofa complex sequence ofbiologicalreactions, duringwhichtheproducts ofone group oforganisms are utilized as substratesby another group. Theprincipalreaction sequences canbe classified into threeor fourmajor groups. The firstreactionsequence is the hydrolysis ofcomplex, insoluble organicsubstrates into simplermore solubleintermediates.The second reaction is the fermentation/acidificationofsoluble substrates into moreoxidized intermediates, primarily VFA, by fermentative organisms.In the past, anaerobicconversionofparticulate, biodegradable organic compoundsto methane and carbondioxide wasthoughtto comprise three steps: hydrolysis, acid formationandmethaneproduction. Onthebasis ofthis model,methanogenesis from fatty acidswas consideredtherate limiting step inthedigestionofdissolved organic compounds (Ghoshand Pohiand,1974; Novac and Carison, 1970)andthehydrolysisofinsolubleparticulate organicswasregarded as rate limiting forthe overallprocessofsludge digestion(Ghosheta!., 1975). Kasparand Wuhrmann(1978) were among thefirstto suggest thatthe degradationofacetate,ratherthanmethanogenesis from fatty acids, wasthe rate limitingreactioninthe anaerobic degradationofdissolved organic matter. Thiscarbonflowmodel based onpercentage oftotal flowoftheoretical chemical oxygendemand, suggestedthat 54% ofthetotalmethaneproducedin anaerobic digestionis evolvedfrom acetogenicreactionsthatproducehydrogen and acetate frommorereduced compounds (eg. propionate,otherVFA).16In the third majorreaction, VFA andhydrogen are convertedinto methane andcarbondioxide by two coupled reactions, mediatedby acetogenic andmethanogenic bacteria.Acetogenic organisms convertthe products offermentationinto acetate, formate andhydrogen,which serve as substrateswhichmethanogens convertto methane and CO2.A distinctionbetweenacetogenic and acidificationreactions is notalways clear. Acetate and hydrogenareproducedby bothacidificationand acetogenic reactions,andbothacetate andhydrogenaresubstratesfor methanogenesis. The syntrophic couplingofmany acetogenicreactions tomethanogenicreactions is critical, becausethe conversionofVFA toacetate and hydrogenisonlythermodynamically favorable inthepresence ofmethanogens (Fox andPohland, 1994). Ifthe conversionis energeticallyunfavorable, as it is in the case ofbutyrate,propionate andethanol, the concentrationofproductsmustbe maintained atlow levels in order for acetogenesisto occur. These lowconcentrationscanbe accomplishedbytheutilizationofthe acetate andhydrogenby methanogens as the intermediatesare produced.Inthis case, it is the interspeciestransferofreducingequivalencethat is essential forthe conversionofthese substratesto theircorrespondingproducts (Thiele andZiekus, 1988).2.4.2 Phase SeparationThe goal ofatwophase digestion systemis to enhance the anaerobicbiodegradationbycontrolled separationofthe majorreactions. Figure 2. 4 illustratesthe singlephase as well asthetwo phase digestionprocess. Theprinciple oftwo phase digestion isto separatethehydrolysis and fermentation/acidificationreactions fromthe acetogenic/methanogenic reactions.C02,CH4Single-Phase DigestionComplexSubstrateTwo-Phase Digestion170)Cl)0Acetatec’Ja)Cl)-0AcetateFigure 2. 4 Principal sequences ofanaerobic digestion (Fox andPohland, 1994).18Becauseofthis separation, the syntrophic effects involvingacidogenesis andmethanogenesiswill be altered. From anoperational pointofview, enhancing anaerobicdigestionby phaseseparation is accomplishedby providing optimal environments(eg. SRT, HRT, pH) foreachmajorgroup ofbacteriaand their associatedbiologicalreactions.The two steps commonly cited as rate limiting in theprocessofanaerobic digestionarethe hydrolysisofcomplex substrates andmethanogenesis(Wuhnnan, 1978; Ghosh etal., 1975).Two phase digestionisolateseachpotentialrate limitingstep andthereby allowstheoptimizationofeach. However, the syntrophy betweenacetogenesis andmethanogenesismuststill exist intwo phase separation. In order for acetateto beproducedinthe secondphase, theproductsofacetogenesis (eg. acetate andH2),must be maintainedatextremely lowlevels.These levels are achievedby utilizingthese products as substrateby methanogenic organisms.This syntrophic couple couldpotentially be brokenby introducing aprocess upstreamofanaerobicdigestion that couldproduce acetate as itsmajorby-product(eg. ThermophilicPrestage Process).2.5 ThermodynamicsandMetabolismTo understandthe details ofthe biochemical model which describes substrate metabolisminthe TAD process, some fundamental aspects ofthermodynamics andmetabolismmustbeaddressed. The following sectionsreviewsome ofthe basic centralpathways involved intheoxidationofsubstrate molecules (eg. Glycolysis, Citric Acid Cycle andthe ElectronTransport19Chain) which are integral to understanding the mechanisms ofthemodel. The introductionofthe molecules responsible forenergy production and electrontransport (eg. ATP andNADH)must also be considered inorderto develop acomprehensive understandingofthe basicfundamentals oftheproposedbiochemical model.Metabolism is the sumtotal ofallthe biochemical reactionsthattake placewithin anorganism and like other chemical reactions is governedby the laws ofthermodynamics.Energytransformationfrom one formto another is thebasis ofthe science ofthermodynamics andobservations made by physicistsand chemistshave ledto two fundamental laws, whichmay bestated simply as follows:FirstLaw: Inanyphysical or chemical change thetotal amount ofenergy intheuniverseremains constant.SecondLaw: All spontaneousphysical or chemicalchanges tendto proceed suchthatuseful energy (ie. free energy) undergoesirreversible degradation into arandom, disorderedform; this is called entropy.Biochemical reactions, inwhichreactants are convertedto products, can be describedthermodynamically since there is a change in their energy contents. Gibbs explainedthe changesinthe energy contentofsuch a system withthe following equation (Lehninger, 1982):zG=AH-ThSzGrefers to the change in free energyofthe reacting system, AH is the change initsheatcontent (ie. Enthalpy), T is the absolute temperature atwhichthe process is talcing place andiS20is the change in entropy ofthe universe. In most casesiHisnearly equal toGandthe two areoftenused interchangeably. IfzH ofareaction is positive, it is referredto as anendergonicreaction, whichmeans thatthe forwardreactioncannotproceed unlessenergy is put into thesystem. The reverse reaction will howeverproceed spontaneously. Ifthe zH is negative,this isreferred to as anexergonic reactionandtendsto proceed spontaneously inthe forwarddirectionwiththe release offree energy.The formofenergy that cells can andmust use is free energy, whichcan do work atconstanttemperature and pressure. Heterotrophic cells obtaintheir free energy from energyrichnutrientmolecules. Thousands ofmetabolic reactions occur in the cell; some are exergonicwhile others are endergonic. Exergonicreactions such as the oxidative reactions involvingcarbohydrates, fats and proteins, as well as the hydrolysisreactions involving energyrichmolecules such asATP release free energy. Endergonic reactionsrequire the additionofenergy,for example some ofthe biosynthetic reactionsthat are involved inthe synthesis ofproteins,lipids and carbohydrates. Every chemicalreactionhas a standard free energy change,AG0This standard free energy change is aconstant forany givenreactionand canbe calculatedfromthe equilibriumconstantofthe reactionunder standardtemperature (25° C) andpressure (1atmosphere). For examplethe equilibriumconstant forthe reactionA+B+-*C+D is given by,KeqProductof[Products]/Productof[Reactants]= [C][DJ/[Aj[B]Once the equilibriumconstant (ie. K.eq) ofareactionis determined its standard freeenergy change (ie. AG°) canthenbe calculatedby the following expression,21LG°RTlflKeqR is the gas constant (1.987callmol*degree),T is the absolute temperature and lnKisthe natural log ofthe equilibriumconstant. If, for example the equilibriumconstant is high, alarge amountofproduct has beenproduced, the reactiontendsto go to completion and the freeenergy change is negative. Ifthe equilibriumconstantis low, littleproduct is formed, thereaction does not goto completion, andthe free energy changeispositive. Inthis scenario, theconversion,ofreactants to productsrequiresthat energybe supplied to the system. High energycompounds, such as ATP, areresponsible for supplyingmostofthe energy requiredforcompletionofendergonicreactions (Boyd, 1984).2.5.1 EnergyProduction (ATP)Energy production in any organism is based on oxidationreductionreactions inwhichamolecule donates electrons (ie. thus becoming oxidized) and another moleculeaccepts electrons(ie. thusbecomingreduced). Electrons will flowfrom acompoundwithahigherpotentialenergyto acompound withlowerpotential energy withadeclinein free energy (ie. -zSfI) Someofthe energy is lost as heatbutmuch ofthe free energy released from cellularfuels during theircatabolism is conservedby the coupled synthesis ofphosphorylated compounds such as thenucleoside triphosphates (eg. ATP and GTP). Most ofthe energy suppliedto endergonicreactions (ie. reactionsthatrequire energy input) isprovidedbyATP. Heterotrophs, dependingonthe species ofelectron acceptor, canproduce ATP using differentormodifiedmetabolicpathways.22Fermentation is an oxidation/reduction reaction inwhichthe electrondonors and electronacceptors are organic. It is an incomplete oxidationprocessin whichthe endproducts formedpossess considerable amounts ofextractable free energy.These compounds are usually variousacids and alcohols excreted by the organism intotheenvironment. Molecular oxygen isnotinvolved. Respiration, on the other hand, is aprocess inwhicheither an organic or inorganicelectrondonor is oxidized and the fmal electronacceptoris oxygen. The substrates orproductsfromfermentationcan serve as electron donors inrespiration. Fermentationendproducts canserve as substratesinrespiration since thisprocess resultsin acomplete oxidationandextractionofall biologically available energy fromthe donor molecule.Complete oxidation oforganicmolecules are characterizedby the formation ofCO2andH20.2.5.2 BacterialEnergeticsGlycolysis is an almostuniversal central pathwayofglucose catabolism, not only inanimalsandplants, but also in agreatmany microorganisms.The glycolytic sequenceofreactions differs from one speciesofbacteriato anotheronly inhowits rate is regulated and inthe subsequentmetabolicfate ofthepyruvateproduced.During glycolysis, muchofthe freeenergy produced is conserved inthe formofATP. The conversionofglucose into twomoleculesofpyruvate is shownby the equation:Glucose+2P1+2ADP -÷2 Pyruvate +2 ATPThus, foreachmolecule ofglucose degraded, two moleculesofATP are generatedfromADP and Pi. The conversionofglucose to pyruvate iscatalyzed by 10 enzymes acting in23sequence. Glycolysis is an essentialset ofreactions driven to completionby the large decreasein free energy.When cells catabolize glucose to lactate, this compound containsapproximately 93% ofthe available energy ofthe original glucose molecule.This is because lactic acid isalmostascomplex a molecule as glucose and has undergoneno net oxidation. The free energyreleased oncomplete combustionoforganic molecules isin approximateproportionto the ratioofhydrogenbound carbonatomsto thetotal numberofcarbons. Only by theremoval ofallthehydrogenatoms fromthe carbon atomsoforganic substrates andtheirreplacementwithoxygento yieldCO2, can all theirbiological free energy be realized. Carbohydrates,fatty acids and mostoftheamino acids are ultimately oxidizedto CO2andH2Oviathe citric acidcycle (ie. Krebs cycle).Firsthowever, before these nutrients canenterthe Krebs cycletheircarbonbackbonesmust bedegraded so thatthey yield the acetyl group ofacetyl-CoA, the forminwhichthe Krebscycleacceptsmost ofits fuel input. Underaerobic conditions, thenext stepinthe generationofenergy from glucose is the oxidative decarboxylationofpyruvate to form acetyl-CoA. Theequation forthis is as follows:Pyruvate + C0A+NAD —* Acetyl-CoA+ CO2+NADH +HThe formationofacetyl-CoAfrompyruvate is akey irreversiblestep inmetabolism.Acetyl-CoA andNADH, whichare theproducts ofthe oxidationofpyruvate,inhibittheenzyme complexthatmediatesthisreaction (ie. pyruvatedehydrogenasecomplex). Theseinhibitory effects are reversed by CoA andNAD.24Figure 2. 5 shows the overall organizationofelectrontransport andoxidativephosphorylation. One cytochrome system is considered inthis figure. Ineachturnaround thecitric acid cycle, fourpairs ofhydrogen atoms are removedfrom isocitrate, cL-ketoglutarate,succinate and malate bythe actionofspecific dehydrogenases. Thesehydrogen atoms donatetheir electronstothe electrontransport chain andbecomeHions, which escape into the aqueousmedium. The electrons arethentransported along achainofelectroncarrying moleculesuntilthey reach cytochrome oxidase, whichpromotesthe transferofthe electronsto oxygen, the fmalelectronacceptor in aerobic metabolism. In additionto the fourpairsofelectrons arising fromthe citric acid cycle, others come from the dehydrogenasesthat act uponpyruvate, fatty acidsand amino acids duringtheir degradationto acetyl-CoA andother intermediateproducts. Inaerobic cells virtually all thehydrogen atoms derivedby the action ofdehydrogenases onsubstrate moleculesultimatelydonatetheirelectronsto the respiratory chain,the final commonpathway leadingto the terminal electronacceptor, oxygen. Aseachpairofelectronspassesdownthe respiratory chainfromNADH to oxygen,the coupled synthesisofthreemoleculesofATP fromADP andP1takesplace.In carbohydrate catabolism, there are three energy yielding stages, glycolysis, the citricacid cycle and oxidative phosphorylation. Each is so regulatedby its own setofcontrolsthatitproceeds ataratejustsufficientto satisf’ the minute to minute needs ofthecell for itsproducts.These three stages are coordinatedwith each other so thatthey functiontogether in an economicand selfregulatingmanner, like a smoothly running piece ofmachinery, toproduce ATP and252eNADH dehy&ogenase12e-VUbquInoneElectron transportI 2e-and oddattvephosphorykiiionCyochrome b4,2e..Cytochrome C1Cytochrorne CI2e-VCytochrome oxidase+/O H20Agure 2. 5 Overall organizailon of electron transport andoxidative phosphorylailon.(Lehninger, 1982)Nnho -lPvi’uvoteUiCo2FattysCitric Acid CycleSuccinate0)UiAWADP+P,AlPADP+P,AlP26specific intermediates such aspyruvate and citrate whicharerequiredasprecursors in thebiosynthesis ofother cell components. The integrationofthese 3 stages ismade possible byinterlocking regulatory mechanisms. For example the relativeconcentrations ofATP andADPnot only control the rate ofelectrontransport and oxidative phosphorylation, but alsothe ratesofthe citric acid cycle and glycolysis.2.5.3 NADHAll living cells containnicotinamide adenine dinucleotides [NADH], which serveascofactors inmany metabolic reactions. The reduced form [NADHI is ahighenergy molecule,which suppliesreducing equivalence to many intracellular redoxreactions.The levelofNADHincultures is afunctionofthe numberofcells, the energy balance withinthose cells and thelevel ofmetabolic activity. The centralroleplayed byNADH in oxidative reactionswithinorganisms inevitably implicates it in theregulationofrespiration, sinceNADH is, after all, themainimmediate electron donorto the respiratory chain. As a substrate forrespiration,NADHmustexert sometype ofinfluence on its rate.One important feature offermentativemetabolism is anevenhydrogenbalance. Sincetheir is no external electronacceptor such as oxygen, NADHproducing andNADH consumingreactions haveto balance. Sincethe amountofNADHto berecycledvaries withthe nature ofthe substrate, so mustthe compositions ofthemixture offermentationproducts. Hexoses suchas glucose or fructose, produce two NADHper C6 molecule upon conversionto pyruvate;however, hexitols (eg. sorbitol or manitol) produce threeNADHper C6 molecule. To achieve a27proper fermentationbalance, it is necessary to matchthe NADHproduced withthe NADHconsumed by the excretionofspecific fermentation endproducts.Inpractice, E. coliuses amixture ofethanol, lactate and acetate, all ofwhich consume differentamountsofprotonsperC6 molecule. By varyingtheproportions ofeachoftheseproductsit is possibleto matchthesubstrate to achieve redox balance (Clarke, 1989). Inthepresenceofoxygen, NADH generatedduring glycolysis, the TCA cycle and associatedreactions is reoxidized by operationoftherespiratory chain (Ingeldew and Poole, 1984).During fermentation, neither therespiratorychains linkedto oxygennorthose linkedto alternativeelectronacceptors (eg. nitrates, sulfates)are functional. The TCA cycle andpyruvate dehydrogenase reactionswhichgenerateNADHinlarge amounts are largely inoperativeunderanaerobic conditions (Spencer and Guest, 1985;SmithandNeidhardt, 1983). HoweverNADH producedby glycolysismust bereoxidizedtoNADso thatthe glycolytic sequence canproceed. Thus, thekey issuein fermentation is therecycling ofreducing equivalence by conversion ofsubstrate tospecific fermentation endproducts. Figure 2. 6 shows the necessary sequenceofreactions offermentativeorganismstomaintainredoxbalance (Boyd, 1984).28Glucose2ATP‘ir 2ADP2 glyceraldehyde-3-phosphate 2 lactate*2NADH+212 1 ,3-blphosphogtycerate2 pyruvate4ADP 4A1PFIgure 2. 6 Pathways involved in thefermentation of glucose.(Boyd, 1984)293. Methods and Materials3.1 ThermophilicAerobicDigester3.1.1 Preliminary Operational PhaseThe sludge feed source for allpilot scale and batch experimentsoriginated fromthesewerthat connects the University ofB.C. campus residencesto the sewage collectionsystem.Therawsewagewas pumped into three, 12,000L sewage holding tanks. This sewagediversionwas donetwice daily duringpeak flows (ie. from 10:00to 12:00 and from 17:00 to 19:00) tomaximize the solids contentofthe subsequentprimarysludge. Fromthesetanksthe sewage waspumped at aconstantrate into aprimary clarifier. Theprimarysludgewasthenpumped into aprimarythickener. The thickened sludge wasthenmetered and fed into the firststage oftheTAD process. A2-stage, 150 L, pilot scale TAD (Figure3. la), located inthe WastewaterTreatmentPilotPlant site attheUniversity ofB.C.,wasused inthispreliminary study. Each 75L reactorwas equippedwith a 250 mmfoambreaker. Aeration and mixing weresuppliedwiththe Turboratoraerator designed by Turborator TechnologiesInc.(Guarnaschelli and Elstone,1987). This aerator is aselfaspiratingtype ofaerator-mixer.Heat was suppliedby biologicalactivity and the mechanical mixing energiesfromthe aerator/mixer. The reactors operatedinseries under daily batchfeed conditions. Table3. 1 is acomparisonofoperating conditions andsolids destructionefficienciesachieved inthe pilot scaleTAD units to those recommendedforfull scale practice (EPA, 1990; Kelly, 1990). The pilotscale TAD feedwas approximately 1/330_ii1NcIen.da) Preliminary phase of operation (seriesconfiguration)b) Second phase of operation (parallel configuration)Rgure 3. 1 SchematIc of the VBC pilotscale TAD process a) operatinginserIes (1st and 2nd phases) and b) underthe parallel modeof operation (A and B sides).3 12,000 1 sewageholding tanksPrimary Clarifier1.3-1.8 h HRTRimary SludgeThickener 2.3 h HRTTborator 2j161ThickenedTijbcrotor 1Sludg’ - fedonce/d(25 L/d)751TAD 1aC.II75 1TAD 2Data===-I4.Digested Sludgewasted oncefdayTbOAIThickenedPrimarySludge - fedon timedpumps(l5sec on/52.6mm off)4LI a.od862 I.1jrborator B= DataAside (control) TAD1=I =I=ci________4868 1.BsIde TADDigested Sludgewasted once/day31ofthe recommended concentration. The operating degree day productwas belowtherecommendedrange of400-500.Table 3. 1 Comparisonofpilot scale TAD parametersand recommended values (EPA, 1990;Kelly, 1990).Variables Pilotscale TAD values RecommendedvaluesNumber of stages2 2Temperature range of 1st stage(°C) 37-47 35-50Temperature range of 2nd stage(°C) 54-60 55-65Total SRT (d) 6(3/stage)5-10Total solids in feed(%) 1.7 4-6Total solids destruction(%) 29 40Power Density(W/m3) 3000 100-250Degree-day product 294400-5003.1.2 SecondPhase ofOperationBased onpreliminary results, the first stageofthe 2 stagepilot scale TAD processproducedthe highestmeasuredconcentrations ofVFA (up to 950 mg/L as acetate). The secondphase ofoperation exploredthis stage indetail. Theseries configurationofthereactors waschanged to aparallel modeofoperation (Figure3. 1b). This scheme resultedin acontrol side (Aside) and anexperimental side (B side). Each side receivedan identical primary sludge feed.The feedingregime wasalso changedfromonce perday to approximately once perhour. Thesludge within eachTAD reactorwas allowedto accumulatethroughoutthe day andwaswastedonce per day.32Table 3. 2 Combination ofaeration and SRT for each experimental run.Air flow rate (mL/min.) Aeration SRT (days)3 4.5 6True anaerobic 0 n/d run 10 n/d0 Low run 2 run 1 run 3117 Medium run 5 run 4 n/d164 High run 6 runs 7, 9 run 8n/d- not doneA 3x3 factorial experimentwas conductedunder thisnewmode ofoperation. The twoindependent variables examinedwere aerationand SRT. The range ofairflowswas from 0-165mL/minandthe SRT range was 3-6 days. This 3 level factorial designwas madeup of9individual cells orruns and is shown in Table 3. 2. Within all 9 runs, theA side controlindependent variables weremaintained attheirrespective medians (ie. 4.5 d SRT andmediumaeration), whilethe B side experimentalvariables were adjustedto theirappropriate values. Thisexperimental designfacilitatedthe comparisonofthe test condition (B side) to its correspondingcontrol condition (A side) within eachrun. Thiswould alleviatethe variability ofprimarysludge feed characteristic andexternal environmental conditioneffects onthe measuredresponses, since bothreactors were fedfromthe same sludge source and were operated overthesametime period. As canbe seenfromTable 3. 2 notall the combinations ofSRT andaerationwere done. Underthe true anaerobic condition (run#10) only onerunwas attempted(eg. 4.5 d SRT) and the combinationofmedium aeration and 6 d SRT was not done. Priortoeach individual run, the sludge contents ofboth control and test sides were thoroughly mixed, toensure thatboth sides would startwith an identical sludge composition, andallowedto acclimate33attheir respective set conditions forone SRT. Samples for analysis werecollected for a furthertwo SRTs, after whichthe contents were thoroughly mixed in preparation for subsequentruns. Atenth experiment was conductedwhich compared the mediancontrol conditionforboth aerationand SRT (A side) to afully fermentative TAD. To achievea fully anaerobic environmentthetest side reactor (B side) was purgedwith an.anaerobic gas mixture insteadofair (eg. 90%N2,5% CO2and 5% 112).3.2AerationCleanwater aerationexperiments performed earlier onthe aeratorsindicatedthatsignificant amounts ofoxygenwere entrainedby mixing alone (Boulanger,1994). Theseresultssuggestedthatthe standardoxygentransferratewas 28 mgIL-hat anambientwatertemperatureof150C and rotational turborator speed of964 rpm. Surface entrainmentofoxygen is aninterface phenomenonbetweenthe bulk liquid andthe overlying gas phase.The gas inthe headspace inthepilot scale TAD reactor, under cleanwateraerationconditions,containsapproximately 20%02 by volume. The oxygenpercentage inthe gaswithinthe head space ofanoperatingTAD process is afunctionofthe aeration conditions employed. UnderBoulanger’s(1994) oxygendeficient condition, the oxygencontentintheheadspace was as lowas 4% byvolume. The oxygen satisfied conditionresulted in a 12 -15% oxygen inthe gas withinthe headspace. Consequently, the standard oxygentransferrates, due to surface entrainment, wouldinvariably change depending onthepercentages ofthe head space gases. Temperature andturboratorrotational speed would also effectthe standard oxygentransferrates due strictly tosurface entrainment.343.2.1 Preliminary OperationalPhaseForthe purpose ofthis study, thetested air flowrate forthe aerobic conditionwas 0.6Volumeofair/Volume ofsludge-hour, the rate forthe intermediatetransitioncondition at 0.28V/V-h andthe rate forthe microaerobic condition at 0 V/V-h. The air flow ratesin all 3conditions were measured with an inline rotameter (Cole Parmermodel 044-40C). Theflowrateforthethird condition as measured by the flowmeter was zero. A 0.126 V/V-hmaximumflowrate forthe microaerobic conditionmaybe calculatedby extrapolatingavalueusing standardoxygentransferrate data. Since azero air flowrate correlated to ameasurabletransferrateof28mg/L-h, extrapolationofthetransferrate backto zero resulted in a calculatedmaximum airflowrate of0.126 V/V-h. However, itmustbe rememberedthatthis flowrate is based oncleanwateraerationtests at ambienttemperatures. Underthese conditionsthepercentageofoxygenwithinthehead space ofthe TAD unitswas 20%. Underthe microaerobic conditioninwhichtheoxygenpercentage inthehead space is considerably less, the calculatedflowrate of0.126 V/V-his probably agross overestimate ofthe actual air flowrate due to mixing alone.3.2.2 Second PhaseofOperationSince the TAD sludge was fedwiththickenedprimary sludge once/hour and was allowedto accumulate throughoutthe day withinthe reactor, the retentionvolumeofeachreactorwascontinuously increasinguntil wastagewas achieved (once/day). Forthis reason, the air flowrates cannotbe normalizedto the reactorvolume. Instead, the rates are reportedas flowrates(eg. Volume/Time). Amuchnarrowerrange ofairflowrates were used inthe secondphase ofoperation (0-165 mL/min or approximately 0-0.17 V/V-h). This entire range resulted in a35microaerobic environment within the bioreactors. For the purposesofthis study, themicroaerobic environmentwas dividedup into 3 subconditions.The lowflowmicroaerobiccondition correspondedto an air flowrateof0 mL/min as measuredby an in line rotameter(Cole Parmermodels FM032-15, FMO12-10). This correspondedto a calculated flowrateof0.126 V/V-h. The medium air flowmicroaerobic conditionrepresents ameanairflowrate of117 mL/minandthe highflowmicroaerobic conditioncorresponds to amean air flowrateof164 mL/rnin (Table 3. 2).3.3 VolatileFattyAcidsSamples ofTAD sludge takenatvarious times after feeding were centrifuged inamicrocentrifuge (IEC Micro-MB centrifuge) for 10 minutes. One mL ofsupematantwasputinto asealed glass container with 100.tL of3% phosphoric acidto drop thepHto 3. Thecontainerswerethen storedat4 °C until analysis was done.TheVFAdeterminationwasconductedusing aHewlett-Packard 5880A gas chromatograph, equippedwithaFlameIonizationDetector (FID). Heliumwas used as the carriergas. The packing materialwas 0.3%Carbowax20M10.l%H3PO4on Supelco Carbopak C 2 mm ID. The columnwas conditionedaccording to theprocedures described in Supelco Bulletin 751E (1989). The operatingparameterswere: (a) Injectortemperature of150 OC; (b) Detector Temperature of200OC;(c)Oventemperature of 120 °C for 1 minute andthenrampedup 5 °C/min. to 150 °C for 5 minutes;and d) Heliumflowrate of20 mL/min. Quantitationofresponse peaks weredone bycomparisonto external reagent grade standards.363.4EthanolandPropanolDeterminationEthanol andpropanol analysis was done ina similarmannerto VFA determinationexceptthatthe oventemperature was set at800C in contrastto VFA determinationwhichwascarried out at 120°C.3. 5PyruvicandLacticAcidDeterminationSamplesofTAD sludge weretakenand centrifuged inamicrocentrifugefor 10 minutes(IEC Micro-MB centrifuge). One mL ofsupematantwasput into asealed glass containerwith100Lof0.3 M oxalic acid. The containerswerethenstored at 4° C until analysis couldbeperformed. Pyruvic and lactic acid determinationwereperformedby injecting 1 JLL ofsampleinto anHP 5880A gas chromatograph, equippedwith anFlame IonizationDetector. The columnpackingmaterialwas 4% Carbowax 20Mon Supelco Carbopak B-DA 2mmID. The operatingparameterswere: (a) Injectortemperature of 175°C; (b) Detectortemperature of200° C; (c)Isothermaloventemperature of175° C, and (d) Helium flowrate of24 mL/min.Quantitationofresponse peakswere doneby comparisonwithreagentgrade standards.3.6 TotalandInorganic CarbonSamples ofTAD sludge were takenand centrifuged in a microcentrifuge (IEC Micro-MBcentrifuge) for 10 minutes. Approximately 1 mL ofsupematantwas takenwithadisposableplasticpipette. The liquidwithinthepipettewas forced to the base ofthepipettebefore sealingthe end ofthe pipette by passing itoveran open flame. The samples were thenfrozen and storedat170C until analysis couldbe conducted. The samples werethawed atroomtemperature and37subjectedto the appropriate dilution. A40jiL aliquotwas then injected into a ShimadzuTotalCarbonAnalyzer (Model TOC-500), using a seriesoflow andhigh standards (Shimadzucorporation, 1987). Each sample was analyzed3 times and a standarddeviationand coefficientofvariation were calculated.3.7SolidsTotal andvolatile solidswere determinedby evaporating aknownvolumeofsample inaFisher Isotemp (Model 350) forced draft ovenat104°C and igniting the residue at 550°CinaLindberg muffle furnace (type 51828), respectively.Both analyseswereperformed as outlinedinStandardMethods (A.P.H.A. etal., 1989).3.8 OnLineDataEachreactor containedtwo Oxidation Reduction Potential(ORP) combinationelectrodeprobesusingAg-AgC1 referencehalfcells and one thermocouple temperatureprobe. Two ORPprobeswereusedto ensure the accuracy ofthe values obtained. Duringthe preliminaryoperationalphase (3.1.1), both ORP andtemperaturemeasurementswere logged at 1 minuteintervals on anon-line dataacquisitionpackage (LabtechNotebook/XE).Thetwo ORP valueswere thenaveraged and graphed as a moving average, withanintervalof10 datapoints. Duringthe secondphase ofoperation, both ORP and temperature measurements weretakenevery 10seconds and logged at 5 minute intervals. Each logged value wasthe resultofthe average valueofthe previous 30 measurements (ie. 10 seconds x 30 measurements= 5 minutes).383.9Batch ExperimentA batchtest apparatus was commissioned in orderto examine the biochemistryinvolvedin VFAmetabolismin TAD, specifically acetatemetabolism.The basic principleofthisexperiment wasto test specific substratesand inhibitor compounds fortheireffect onVFAmetabolism. The tested substrateswere:1. Propionate 10. n-Propanol2. Butyrate 11. Peptone3. Isobutyrate 12. Glucose4. 2-Methylbutyrate 13. Dextrin5. Valerate 14. Linoleic acid6. Isovalerate 15. Primary sludge7. Lactate 16. Primary sludge supematant8. Pyruvate 17. Primary sludge washedpellet9. EthanolThe inhibitor agentstestedwere:1. Sodium cyanide2. Sodium fluoride3. 2,4-Dinitrophenol39These inhibitor compounds were usedwithprimary sludge as substrate.Concentratedstocksolutions ofacidic compounds 1-8 were made andadjusted to pH 7 before additionto the batchreactors.Thetested sludges included:1. TAD sludge fromthe pilot scale control side (A side,medium air and 4.5 ci SRT).2. TAD sludge fromthepilotscale experimental side (B side, 0 air and 4.5dSRT).3. Fermenter sludge from a side stream fermentationprocess (PhD candidateAl Gibb’sFGRSGRpilotplant locatedatBC ResearchInc. [FGR-Fixed GrowthReactor,SGR-SuspendedGrowthReactor]).4. SalmonArm full scale ATAD sludge fromthe firstandthird cells oftheprocess.Thebatchtest reactorswere one L Erlenmeyerflasks. Each compoundwastestedunder2 conditions. The anaerobic condition was achievedby filling the Erlenmeyerflaskswithprocess sludgeto within 2 cm fromthe brimofthe flask. The microaerobicconditionwasachieved by filling aone L flaskto the 300 mL gradationwithprocess sludge. Surface aerationinthe microaerobicconditionandmixing inthe anaerobic condition were maintainedby using30 mm stirbars drivenby a stirringplatform. Rotational speedwaskeptbetween 120-150 rpm.TAD sludge experimentswere conductedatthe average 45° C operatingtemperatureofthepilotscale units. Thetemperatures ofthe SalmonArmATAD batch sludge experiments weremaintained attheirrespective full scale reactortemperatures (AppendixA). Thesetemperatureswere controlledby submergingthe flasks andtheir contents into a Haake circulating waterbath40(modelE8) set at specific isothermal temperatures.The fermenter sludge experiments wereconductedat ambientroomtemperature.Eachcompoundtestedhad itsrespective anaerobic andmicroaerobiccontrol, so thattherewere 2 anaerobic flasks and 2 microaerobicflasksforeach compoundtested.Eachexperiment lastedfor approximately50 h. Overthe duration ofeach experiment,grab sampleswere taken toperformVFA and TOC determination.pH and ORP measurements were alsomade.414. Results and Discussion4.1 Batch Experiments4.1.1 Fermentativeand OxidativeVFAMetabolism inTADThe batch experiments with spiked substrates and inhibitors weredoneto investigateVFAmetabolism inTAD. The two batchexperimentalconditionstested were anaerobicandmicroaerobic. For adescriptionofthephysical apparatus, referto Section 3.9. The anaerobicconditionwas selected in orderto investigate the strictfermentativemetabolismofthe TADprocessbiomass. Underthis condition (which lacks oxygenas theterminal electronacceptor),the oxidative metabolismoforganisms inthe process shouldbe completelyinhibited. Themicroaerobic conditionwas chosen in orderto investigatethe shiftinmetabolicactivity whenoxygenwas introducedas aterminal electronacceptor. Figures 4. 1 to 4.3 are results comparingthe baseline accumulationofeachVFA intermsoftheirresponseto bothanaerobicandmicroaerobic conditions. Theprocessbiomass source was fromtheA sidepilotscale TAD unit.The substrate wasthickenedprimary sludge. Underthe anaerobic condition,eachVFAtendedto increase, suggestingthat only fermentative type reactionswereoccurring. It is interesting thatpropionate concentrations increased in the sameproportionto that ofacetate.This observation isconsistentwithresultsthat showfermentationprocessesproduce roughly the same equivalenceofacetateto propionate (Elefsiniotis, 1993; Rabinowitz and Oldham, 1985). Under strictanaerobic conditions, complex organics wouldbe oxidizedto simple organics, resulting intheaccumulationoffermentative endproducts inthe medium. The accumulationofVFAinthea)Batch 3: Acetate response to show replicability ofmeasurementsKII0 10 20 30 4050Time (h)b)Batch 3: Propionate response to show replicabilityofmeasurements200Figure 4. 1 Comparison of acetate andpropionate concentrations betweenthe anaerobic and microaerobic conditions. a)Acetate responseand b) Propionate response.42E‘I4003002001000-JE>150100500microaerobic+run 1 run 2meansanaerobic1 run 2 means0 10 2030 40Time (h)5043a)4030-J!20>100Batch 3: Butyrate responseto show replicability ofmeasurementsb)Batch 3: 2-methylbutyrateresponseto show replicability ofmeasurements20-I10>0microaerobic±run 1Xrun 2 meansanaerobicrun 1 run 2meansFigure 4. 2 Comparison of butyrateand 2-methylbutyrate concentrationsbetween the anaerobic andmicroaerobic conditions. a)Butyrate responseand b) 2-methylbutyrateresponse.Time (h)0 10 2030 40 500 1020 3040 50Time (h)a)5040-J- 30E20>100b)20-J>0044Batch 3: Isovalerate response to show replicabilityofmeasurementsBatch 3: Valerate response toshow replicabdity ofmeasurements50microaerobic+run 1 run 2 ——meansanaerobicrun 1 run 2 -—meansFigure 4. 3 Comparison ofIsovalerate and valerate concentrationsbetweenthe anaerobic and microaerobicconditions, a) Isovalerate responseand b) Valerate response.0 1020 30 40Time (h)x10 2030 4050Time (h)45reactor indicates that these endproducts havebeenmade. However, under microaerobicconditions, the trends ofVFA metabolism are completely different.Duringthe first 15 h,acetate concentrations increasedwhilepropionate concentrationsdecreased. This is similartothe response ofthese two VFA duringthe preliminary operationofthe pilot scale TAD processunderthe transitioncondition (See Section4.2). Overthe next 25 h, netacetate concentrationsdecreaseduntil 40 h into the study, whenmeasurable acetatedisappeared. It is clearfromtheseresults thatthe introductionofaterminal electronacceptor(eg. 02), underthe microaerobiccondition, could accountforthe disappearanceofVFA (ie. oxidationofVFAto CO2and1120).Thesetrends inVFAmetabolismdefine the baselineresponseofTAD sludgebiomassto thesetwo environmental conditions.4.1.2 SubstrateAdditionExperimentsArange ofsubstratesweretested fortheir effecton alteringthebaselineresponseofWAaccumulationunderboththe anaerobic andmicroaerobicconditions. The substratestestedwere:1. Propionate 2. Valerate3. Isovalerate 4. Butyrate5. 2-methylbutyrate 6. Isobutyrate7. Pyruvate8. Lactate9. Ethanol 10. Propanol11. Linoleic acid 12. Glucose13. Dextrin 14. Peptone46The process biomass came fromthe control (A) sidebioreactor. Substrates 1 through 10were spiked andmeasuredwithineach reactorduring the courseofthe batch experiments.Substrates 11 to 14 were spikedto a concentration ofapproximately 1000 mgfL.The individualsubstrates were added atthe beginning ofeach experiment.Figure 4. 4 shows resultsfrom thepropionate addition experiment. Figures4. 4aand 4. 4b show VFAtrend dataforthemicroaerobic control andpropionate spikedreactors,respectively. Thesetrends suggestthatpropionate consumption occurs concurrentlywithacetateproductionabovethatobservedforthecontrol. Netacetate concentrationsstartedto decreasefollowingthe exhaustionofpropionate.Figures 4. 4c and 4. 4d showtheVFAtrend forthe anaerobic control andpropionatespikedreactors, respectively.Underthis condition, propionate concentrations didnotsignificantly change overthe courseofthe experiment. Theresulting acetate concentrationsdidnot significantly deviate fromthatofthe control reactor. The resultsfrom Figure 4. 4 suggestthatunder anaerobic conditions, propionatecannotbe further oxidizedwhileunderthemicroaerobic condition, propionate canbe oxidizedto anacetate intermediate on its way tocomplete oxidationto CO2andH20.The metabolismofpropionate is initiated by its activationto propionyl-C0A.Forthefurthermetabolismofpropionyl-CoA,several differentpathwayshavebeenfoundwhichmay beinvolved. A likely pathway forthe catabolismofpropionate occurs in bothE. coli andAcinetobacter(formerly Moraxella) iwoffi (Gottschalk,1986). These organisms convert47a)1200[VFA](mg/U900 i600f30005?—0 1020 30 40 50Time (hrs)b)0 10 2030 40 50Time (hrs)c)1200[WA]900(mg/i.)600°:ZEE-_—ca:::X x><0 1020 30 4050Time (hrs)d)1200[WA]900(mg/i.)60000 10 2030 40 50Time (hrs)•acetate propionate•isobutyratebutyrateA2-methythutyrate isovalerate)<valerate+TOC[VFA](mg/i.)12009006003000Figure 4. 4 VFA response inbatch TAD experiment to propionateaddition.a) Microaerobiccontrol b) Microaerobic withpropionate additionc) Anaerobiccontrol d) Anaerobic withpropionate addition.48propionyl-CoAto pyruvate by the following reactions,CH,f2H(:112H2O\Cli, CH,CH, Cli CHOHKc=aCoACO-SCoA CO-SCoA CO-SCoACOOHProplonyl-CoA Acrylyl-CoA1ccM-CoA PyruvateAccordingto these reactions,the conversionofpropionyl-CoAto pyruvate liberateshydrogen and electrons, whichare then carried intheformofNADH+H.Thisreduced formrequires reoxidationifthe conversionofpropionate to pyruvateis to continue. Under strictfermentative conditions, the cells are unableto recycle the pooiofNAD , resulting in theaccumulation ofpropionate (Figure 4.4d). The introductionofoxygenresults in afunctionalelectrontransportchainwhichcanpotentially regenerateNAD, thusreplenishingthepooi ofoxidized electron carrier, allowing the conversionto proceed. Onlythe conversionofpropionateto pyruvate is addressed inthis section, sincethe catabolism ofpyruvateto acetate will beaddressed in a subsequent section. Inorderto minimizethenumberoffigures, differenceplotsbetween the substrate additionreactorsand its corresponding control reactorwere constructed.Figures 4. 5 - 4. 10 showthistype ofVFAprofile inresponseto the additionofindividual VFAathighconcentrations atthe start ofeach experiment (ie. substrates 1 through 6 from list).Individual reactors, with substrate added, were comparedto the corresponding control reactorwhichhadno external VFA added. Thetrends seen inthese figures plotthe differencebetweenthe experimental flaskandthe corresponding control flask. Eachpointontheplots is thedifference between a concentration ofmeasured compound inthe substrateadditionreactorand49the corresponding value in the control reactorat each sampling timepoint([zj=[experimental][control]). Atrendthat is localizedto the zero linesuggests no difference in VFA responsebetweenthe control and test conditions.Figure 4. 5 shows the same results as Figure4. 4, exceptthatthe dataare expressed usingthisnewformat. As canbe seen fromFigure 4.5, mostofthe observations from Figure4. 4 alsoshowup inthese figures. Underthe microaerobiccondition (Figure 4. 5a), acetate showsthegreatest deviationfrom its control valueand aspropionate concentrationsapproachthatofthecontrol value, which is zero by 30 h, acetateconcentrations startto fall. Underthe anaerobiccondition (Figure 4. 5b), propionate is notconsumedso the difference betweenthe additionreactorvalue and itscontrol value remains constant overthe length ofthe experiment.4.1.2.1 ValerateandButyrateAddition ExperimentsFigures 4. 6 and4. 7 showthe resultsofthevalerate andbutyrateaddition experiments,respectively. The results are similarto that ofthepropionateadditionexperiment, withtheexceptionofthe valerate additionexperiment. Underthemicroaerobic condition, theconsumptionofvalerate seems to stimulate the production ofacetate andpropionate butto alesser extent. This anomaly is consistentwithknownbiochemicalpropertiesofbacteria. TheseVFA can be degraded by the aerobic f3-oxidationprocess (Lehninger, 1982) knownto occur in anumberofmicroorganisms (eg. Pseudomonads, Acinetobacter, Bacilli andE. coil). The firststep inthe oxidation ofthese VFA is the conversion to their corresponding CoA esters by acyl50a)Batch 4: VFA concentrations relative to controls forpropionate addition under micro-aerobic conditions900b)Batch 4: VFA concentrations relative to controls forpropionate addition under anaerobic conditions900E> 3000 10 2030 40 50• Acetate —C—----— Propionate —f— isoA Butyratebutyrate—&—-—— 2-Methyl • isovalerate—c—---—— ValeratebutyrsteFigure 4. 5 Difference plots of the propionate additionexperiment undera) n,icroaerobic and b) anaerobic conditions.600E>30000 10 20 30 40Time (h)506000Time (h)51-JELl>-JE‘I100-1000 1020 30 40 50• Acetate -C—— Propionate X— iso .& Butyratebutyrate—&--—— 2-Methyt • isovaerate -—— Valeratebutyratea) Batch 6: VFA concentrations relative to controls for valerateaddition under micro-aerobic conditions900700500300100-10010 20Time (h)30 40 50b)Batch 6: VFA concentrations relative to controls for valerateaddition under anaerobic conditions5001300Time (h)Figure 4. 6 Difference plots of the valerate addition experiment undera) microaerobic and b) anaerobic conditions.52-JE>-jE>Time (h)30 4050• Acetate L1— Propionate——X——— so A Butyratebutyrate-—-zt—— 2-Methyl• Isovalerate -—— Valeratebutyratea)Batch 6: VFA concentrationsrelative to controls for butyrateaddition under micro-aerobicconditions900700500300100-10010 20Time (h)30 40 50b)Batch 6: VFA concentrationsrelative to controls for butyrateaddition under anaerobicconditions900700_A -500300iool_________-100010 20Figure 4. 7 Differenceplots of the butyrateaddition experimentundera) microaerobic and b) anaerobicconditions.5.3-oxldatton of Butydc AddCH3— CH,—CH COOH(BulyTic Acid)CoA. ATPocyt-C0A synthetoseCHaCH2 CH2— Co— C0AFADfolly ocyl-C0A dehydrogenciseVCH—CH CH—CO—CoAH203-hydroxyocyl-CoA hydrolyaseCH—CH —CH2—CO—CoAOHL-3-hydroxyacyl-CoA dehydrogenaseADH+H*CH— CH— CH— CHaKetothloIaseCoACHa Co— CoA + CH3— CO— CoA (2 Acet’4 Co.A)p-oxIdatIon of Valetic AcidCH— CH2—CH — COOH(Valeric Acid)I— FAD, NAD, H20, 2CoAacyl-COA synthetaseNADH+HCH3 CO— C0A + CH3— OH2— CO— CoA(Aceiyl CoA + Proplonyl CoA)FIgure 4. 8 f-oxidaiion of butyic and valericacids.(adapted from Lehninger,1982)54C0A synthetase. The C0A esters are thenoxidized inthe3 positionand subsequentlycleavedtoyield acetyl-CoAandthe CoA esterofthe fatty acidshortened by 2 carbon atoms.Thisprocessrequiredthe concerted action offour enzymes,as illustrated in Figure 4. 8. Inthe case ofbutyrate, n-oxidationyieldstwo acetyl-CoA. With valerate, n-oxidationyields acetyl-CoA andpropionyl-CoA in equimolaramounts. Figure 4. 6a showsthat acetateandpropionate are notproduced in equimolarproportions. This is expectedsince propionate, undermicroaerobicconditions, canbe further oxidizedto acetate.For each moleculeofbutyrate or valerateoxidized, there is acorresponding consumptionofone molecule eachofFAD andNAD(Lehninger, 1982). Inorderforp-oxidationto continue, as is the case forpropionate oxidation,theremustbe an operating mechanism to recyclethe oxidized formofthese electroncarriers.Undermicroaerobicconditions, the electrontransport chaincan servethis purpose.4.1.2.2Isobutyrate,Isovalerateand2-MethylbutyrateAdditionExperimentsFigures 4. 9 to 4. 11 showthe differencetrendsinVFAmetabolism forthe isobutyrate,isovalerate and2-methylbutyrate addition experiments. Under theanaerobic condition, eachofthese spikedVFAbehaved similarlyto the other VFA tested.They remained inthemediumandwerenot furtheroxidizedto any intermediateproducts. Undermicroaerobic conditions, thedisappearance ofthese VFA seemed to stimulate atransientacetateproduction. The availableevidence inthe literature suggeststhatbacterial catabolismofthese 3 VFAconverges with thea)Batch 8: VFA concentrationsrelative to controls forIsobutyrate addition undermicro-aerobic conditions15001200b)Batch 8: VFA concentrationsrelative to controls forlsobutyrate addition underanaerobic conditions15001200900XXX_X XX3000 10 2030 40 5060Time (h)• Acetate —{}— Propionate—X—— iso A Butyratebutyrate-—-— 2-Methyl• Isovalerate—0--———— ValeratebutyrateFigure 4. 9 Difference plots ofthe isobutyrate additionexperimentunder a) Microaerobic and b)anaerobic conditions.55.1E>90060030000 10 2030 40Time (h)50 60-IE‘I600-J.£>• Acetate —D Propionate -X—iso A Butyratebutyrate—&--——— 2-Methyl •isovalerate ‘D— ValeratebutyrateFigure 4.10 Difference plots of the isovalerate additionexperiment undera) microaerobic and b) anaerobic conditions.56a)Batch 6: VFA concentrations relativeto controls forisovalerate addition under micro-aerobicconditions900700500300100-100 0 10 20 30 40Time (h)50-jE>b) Batch 6: VFA concentrations relativeto controls forisovalerate addition under anaerobic conditions500 -1-300100 -I-- - --1000 10 20 30 40 50Time (h)57a)Batch 6: VFA concentrations relative to controls for 2-methylbutyrate addition under micro-aerobic conditions900 —700500E300>100I —a —;——-------1000 10 20 30 40 50Time (h)b) Batch 6: VFA concentrations relativeto controls for 2-methylbutyrate addition under anaerobic conditions900700500E300>100-1000 10 20 30 40 50Time (h)• Acetate D Propionate —X--—---— so A Butyratebutyrate2-Methyl • Isovalerate ValeratebutyrateFigure 4.11 Difference plots of the 2-methlybutyrate addition experimentunder a) microaerobic and b) anaerobic conditions.58Vailne Leucine IsoIeucbe1 V2-Ketolsovalerate 2-Ketolsocaproate 2-Keto-3-methylvolerateCoA— CoA—-2HIsobutyiyl-CoA+C02 IsovaleM-CoAI-C02 2-Methylbutyryl-C0A+C022H 2H 2HMethacrytØ-C0A 3-Meth,1crotonyI-CoA llgM-C0AH20Co2ATPH20—3-HydroxysobuiyryI-CoA 3-Methylglutaconyt-C0A 2-Methy-3-hydroxybjtyryI-CoAHO H203-Hydroxylsobutyrate+CoA 3-Hydroxy-3-methylglutaryl÷C0A 2-Methylacetoacetyk.C0AICoA—.N.VMethylmalonic semkildehyde Acetoacetate+acetyl-C0A Proplonyl-C0A+acety)-C0ARopIonaIdehyde+CO2CoAPropionyl-COAFigure 4. 12 Reactions in the oxidation of the branched chain amino acids bybacterk (adapted from Sokatch et al., 1968).59oxidative pathwaysofbranched chain amino acids, valine,leucine and isoleucine (Figure 4. 12).The enzymatic conversions necessary forthe catabolismofbranchedchain aminoacids shownin Figure 4. 12 have beenreported to occur in avarietyoforganisms. However, most studieshave been done using several species ofPseudomonas(Massey etat., 1976).GrowthofP. aeruginosa onvaline results in simultaneousdevelopmentofthe ability ofwhole cellsto oxidize isobutyrate andpropionate (Sokatch etat., 1968). Theenzymes involvedinthe catabolismofleucine were firstdiscovered in speciesofMycobacterium andAlcaligenes(formerly calledAchromobacter) isolated from soil, usinga selective culture mediumwithisovaleric acid as the sole carbon source (Sokatch, 1969). The bacterial oxidationofisoleucineto acetyl-CoAandpropionyl-CoAwas firstdescribed by Conrad eta!. (1974).The synthesis ofthese enzymes was induced by growth on either isoleucine or 2-methylbutyrate.The complete catabolism ofeachbranched chainamino acidrequiresthe cooperationoftwo sequential series ofreactions. The enzymesofthe first series comprisea commonpathwaycatalyzingthe conversionofisoleucine, leucine and valine to theirrespectiveacyl-CoAderivatives. However, the branched chainmetabolites formed subsequentto this commonpathway are catabolisedbythree separate enzyme series,one specific for each amino acid.The first step in the oxidation ofisobutyrate, isovalerate and 2-methylbutyrate aretheconversionto their respective CoA esters. Fromthispoint inthe reactionsequence, theseintermediates can feed directly into the catabolic pathwaysofthebranched chainamino acids.Fromthese sequential series ofreactions(Figure 4. 12),it is clearthat isobutyryl-CoA isoxidizedto propionyl-CoA; isovaleryl-CoAis oxidizedto acetoacetate and acetyl-CoA and 2-60methylbutyryl-CoAare oxidized to acetyl-C0A and propionyl-CoA. The resultsfrom Figures 4.9 to 4. 11 suggests thatthe catabolismoftheseVFA lead only to acetate as anintermediatemetabolite. However, itmust be rememberedthat, under microaerobicconditions, propionylCoAwill be furthermetabolizedto pyruvate andultimately to acetateviaacetyl-CoA.Therefore, itis reasonable to observe no accumulationofpropionate inthe batch reactors. As isthe case for all otherVFAtested, the catabolicpathwaysofthebranched chainamino acidsevolve hydrogen (ie. consumeNAD). Consequently,operationofthese pathways can onlytakeplace whenaterminal electron acceptor, such as oxygen, is available (ie.microaerobiccondition).4.1.2.3PyruvateandLactateAdditionExperimentsFigures4. 13 and 4. 14 showthe pyruvate andlactate addition experiments, respectively.Whatmakes these resultsdifferent fromtheVFAadditionexperiments isthe apparentconsumptionofbothofthese substrates under anaerobicconditions. Underthis condition,pyruvate consumptionwas followedby acetateproduction. Sincethereis no acetate catabolicactivity under anaerobic conditions, acetate accumulatedandpersisted in the medium. Underthemicroaerobic condition, pyruvate disappearancewas followedby only aslight shorttermaccumulationofacetate.The Enterobacteriaceae are ableto synthesize two different enzyme systemsforthebreakdownofpyruvateto acetyl-C0A. Thepyruvatedehycirogenase multienzyme complex is61a) Batch 8: VFA concentrations relative to controls for pyruvateaddition under micro-aerobic conditions1900 T1500 -1100E700>300.‘100NN• — —---.10 20 30 40 50Time (h)60b) Batch 8: VFA concentrations relative to controls for pyruvateaddition under anaerobic conditions15001100• Acetate D Propionate —*—— iso A Butyratebutyrate--—— 2-Methyl• isovalerate -—— Valerate PyruvatebutyrateFigure 4.13 Difference plots of the pyruvate addition experiment undera) microaerobic and b) anaerobicconditions.-IE>700300-100-50010 20 30 40 50 60Time (h)62—I2U>2>a)Batch 2: VFA concentrations relative to controls for lactateaddition under micro-aerobic conditions900700500300100-10010 20Time (h)30 40 50b) Batch 2:VFA concentrations relativeto controls for lactateaddition under anaerobic conditions10 20900700500300100-100• Acetate DPropionate iso A Butyratebutyrate2-Methyl • isovalerate Valerate0 LactatebutyrateFigure 4.14 Difference plots of the lactate addition experiment undera) microaerobic and b) anaerobicconditions.30 40 50Time (h)63involved in the aerobic metabolismofthis compound.Under anaerobic conditions, theseenzymes are no longer synthesized and the enzymestill present is inhibited by NADH. Instead,the synthesis ofpyruvate formate lyase is inducedunder fermentativeconditions (Pecher etal.,1982). The reactionscatalyzed by this enzymeproceedintwo steps, with an acyl-enzymeintermediateand acetyl-CoAand formate asproducts.CH3-CO-COOH+ enzyme-CH3CO-enzyme+ HCOOHCH3CO-enzyme+CoASH-* enzyme+CH3CO-SCoAPyruvate formate lyase is irreversibly andrapidly inactivatedinthepresence ofair sothatitfunctions only infermentative metabolism. Evenunderanaerobic conditions, the enzymeisnotvery stable. Atlow concentrations ofpyruvate, it is convertedto aninactiveformwhichcan bereactivated. Upona shiftto anaerobicconditions, the inactiveform ofpyruvate formatelyase is convertedto the active enzyme by a complicatedreaction seriesinvolving reducedflavodoxin, S-adenosyl methionine and an activatingenzyme (Knappe and Schmitt, 1976;Conradtetal., 1984; Pascal etal., 1981). Themajor advantageofpyruvate formate lyase overthepyruvate dehydrogenase complex is thatthe formationofacetyl-CoA is not accompaniedbythe reductionofNAD. The functional nature ofthis enzyme is consistentwiththe observationsfrom Figure 4. 13, thatpyruvateconsumption, under anaerobic conditions, is accompaniedbyacetate production. The criteriafor balancing reducing equivalencehadbeenmetbyproducingformate insteadofCO2.Under anaerobic conditions,the consumptionoflactate is followed bythe accumulationofbothpropionate and acetate in an approximate2 propionate: 1 acetate molarratio. The most64likely sequence ofreactions forthistypeofconversion is the acrylate pathway. Thispathway isgenerally associatedwith a fewanaerobicmicroorganisms(eg. Clostridiumpropionicum,Megasphaera[formerlyPeptostreptococcus]elsdenii).Figure 4. 15 illustratesthis pathway,whichconverts lactate to propionate and acetateinamolarratio of3:2:1 withCO2and 1120 asby products. This figure shows that L, D orDL-lactatemay serve as substrate; anenzymeispresentwhich interconvertsthe isomers (#1). L-lactateis convertedto L-lactyl-CoAinaCoAtransferasereaction (#2). By reactionsnotyetestablishedin detail, acrylyl-CoAis produced(#3). This intermediate is thenreduced topropionyl-CoA(#4) andpropionate is producedbythe above mentionedC0Atransferase(#2). The hydrogendonor forthe reductionofacrylylC0Ato propionyl-CoAis areduced electrontransferring flavoprotein.These oxidized carriersbecomereducedbythe conversionofD-lactateto acetate, thus achievingredoxbalance withoutthe necessity ofanexternal processto regeneratethepoolofelectroncarriers (eg. respiratorychain). Undermicroaerobicconditions, withafunctioning electrontransport chain, onlytheaccumulationofacetate is seen. Theseresults are consistentwiththepreviousresultsobtainedfortheVFAadditionexperiments undermicroaerobic conditions.Specifically, whenoxygenisintroduced into the bioreactor, propionate (which accumulated inthe lactate additionexperimentunder anaerobicconditions) may eithernotbeproduced or befurther oxidizedto atransientacetate intermediateundermicroaerobicconditions.65OH2CH3—C—CO—CoAI0Figure 4. 15: Formationofpropionate and acetate fromDL-lactate viathe acrylatepathway (Gottschalk, 1986).(2OH(L)2CH3CCOOHIlIi’(D)CH3-C—COOHljH4r2H202CH2=CH—CO—CoA4ETFETFH2CH3 COCOOHfrETFH2CoA FdETF-IC02JFdH2CH3—CO--CoA2CH3—CH2-CO—CoA2ADPCH3—COOHsum: 3 lactate2 CH3—CH2—COOH2 propionate+ acetate+H20664.1.2.4EthanolandPropanolAddition ExperimentsFigures 4. 16 and 4. 17 showthe resultsofthe ethanol andpropanol additionexperiments. Under anaerobicconditions, ethanolconsumptionwas concomitantwiththeproductionofacetate. Underthis same condition,propanol consumption correspondedto theproductionofpropionate. This is a somewhatsurprisingresult since ethanol andpropanolaremorereduced compounds thanacetate andpropionate,respectively. Therefore, theoxidationofthese compoundsto theircorresponding acids necessitatestheproduction ofreduced electroncarriers, and sincethe oxidative chainis notfunctionalunder fermentativeconditions, theremustbe analternative mechanism forrecyclingthe oxidizedpool ofthese electron carriers.Amechanistic process to explainanaerobic consumption ofethanolandproductionofacetate are found inthe anaerobic digestionliterature(HarperandPohiand, 1986). Inorderforcatabolismofglucose to proceed, theNADHproduced duringsubstrate levelphosphorylationofglyceraldehyde-3-phosphatemustberegeneratedtoNAD.This functionis accomplishedby thereductionofprotonsto formhydrogengas, whichis subsequentlyremovedbythehydrogenotrophs(eg. methanogens). Microbial associations,inwhichanH2producing organismcan growonly inthepresence ofanH2consuming organism, are called ‘syntrophicassociations’. The coupling offormationanduse ofhydrogen is called ‘interspecieshydrogentransfer’. Thethermodynamicsofreactionsinwhich some compoundsyield acetate andH2(andCO2inthe casesofpropionate and lactate) are illustratedinTable 4. 1. The free energy changesarepositive forpropionate, butyrate and ethanol. Therefore, the reactions will notproceed from67—IE>• Acetate ———— Prepionate—X——— so A Butyratebutyrate——— 2-Methyl• Isovalerate — Valerate EthanolbutyrateE>a) Batch 8: VFA concentrations relative to controlsfor ethanoladdition under micro-aerobic conditions1000600200-200-600-100010 2060Time (h)b) Batch 8: VFA concentrations relativeto controls for ethanoladdition under anaerobic conditions1000600200-200-600-100010 20 3040 50 60Time (h)Figure 4.16 Difference plots ofthe ethanol addition experiment undera) microaerobic and b)anaerobic conditions.68a) Batch 8: VFA concentrations relativeto controls for propanoladdition under micro-aerobic conditions1000600b) Batch 8: VFA concentrations relativeto controls for propanoladdition under anaerobic conditions1000600E>200-200-600-10001060Time (h)2147 mg/s10 20 30 40 50 60200E-200>-600-1000• Acetate —DPropionate —X— so A Butyratebutyrate—zt—----— 2-Methyl • Isovalerate >--— Valerate IPropanolbutyrateFigure 4.17 Difference plots ofthe propanol addition experiment undera) microaerobic and b) anaerobic conditions.Time (h)69left to rightexceptunder conditions in whichaproductis kept at an extremely lowconcentration. Due to the highaffinity ofthe methanogenicorganismstowardsH2,the partialpressure ofH2, which is aproductin the catabolism ofthese compounds, is maintained aslowas1 atmospheres inthe presence ofthese bacteria.This concentrationis lowenoughto allowtheformationofH2by oxidizingNADH to regenerateNAD as well as allowing thermodynamicallyunfavorable reactions feasible (ie. reactions having a+AG°’)(Wolin, 1976). Whatmustberememberedaboutthe previous results shown is that bothpropionateandbutyrateremainunmetabolized inthemediumunder anaerobicconditions. Theseresults seem to becontradictory tothe ethanol catabolic resultsthat suggest ethanolcatabolism under fermentativeconditions. However, this free energy change is muchmorepositiveforbothpropionate andbutyrate conversionto acetatethan forethanol (ie. +76.1 and +48.1Uversus +9.6ki).Table 4. 1 Selected acetogenic reactions.Acetogenic reactionsAG°’(KJ)aPropionate —* acetateCH32COOH + 2H20—* CH3COOH + CO2+ 3H2 +76.1Butyrate —> acetateCH32COOH+2H20->2CH3COOH + 2H20+48.1Ethanol —* acetateCH32OH+ H20-*CH3COOH + 2H2+9.6Lactate —* acetateCH3CHOHCOOH +2H20-*CH3COOH + CO2+ H20-4.2Hydrogen + carbon dioxide —* acetate4H2+ 2C02-÷CH3COOH + 2H20-107.1Hydrogen + carbon dioxide —* methane4H2+ CO2-* CH4+ H2O-135.6aAdapted from Thauer eta!.(1977) and Gottschalk (1986)70Table 4. 1 lists the free energy changes for eachofthe reactions listed above.Accordingto Harper and Pohiand (1986), thermodynamic calculationsassociated withthese reactionsindicatethatpropionic acid oxidationto acetatebecomesfavorable only athydrogenpartialpressures below 1 atm, while butyric acid oxidationbecomes favorable at 10atmH2orbelow. Incontrast, ethanoloxidationto acetate is not inhibiteduntil thehydrogenpartialpressure approaches 1 atm. Therefore, ifthe hydrogenpartial pressure canbe maintainedbetween 10and 101atm., this wouldtheoretically favorthe oxidation ofethanol toacetate butnotthe oxidationofpropionate orbutyrate. The mechanismby whichthe partial pressure ofhydrogencanbemaintainedwithinthisrange underthe experimental conditionsofthe batchtests isnotwellunderstood. The syntrophicassociationbetweenacetogenicandmethanogenicreactions under TAD conditionsprobably doesnotoccur since methanogensaretypicallynotassociatedwiththe TAD process. There is, however, anothergroup oforganismswhichmayplay arole intheprocess ofhydrogen utilization.Wehave seenthatacetate, so far, is an importantproductin anumberoffermentations.There is agroup oforganisms, however, bywhichacetate is formed as thepredominantnongaseousproduct. Knowledge ofthis very small groUp ofacetogenicorganisms has grown inrecentyearsto include anumberofthermophilic organisms, including Clostridiumthermoautotrophicum, C. thermoaceticum andAcetogenium kivui (Gottschalk, 1986). Thesespeciesofbacteriaare ableto live atthe expense ofacetate formation fromH2andC02,according to the equation illustrated inTable 4. 1. The free energy change is slightly less71negativethanthe free energy change formethanogenic conversionofCO2andH2to methane.According to Harper and Pohiand (1986), the rangeofhydrogenpartialpressures thatencompasses the methanogenic “niche” (ie. H2partial pressurerangeofmethanogenic activity)is between 14and 1Oatm. Presumably, acetogenic conversion ofH2andCO2to acetateoperatesin a higherpartial pressurerange, since the free energy changeofthisreaction is lessnegativethan the free energy change ofthe correspondingmethanogenic conversion. Itmay bepossiblethatthe typesofacetogenic organisms associatedwitha TAD process couldmaintain arange ofhydrogenpartialpressuresthatcould allowethanol conversionto acetate andpropanolconversionto propionate, butinhibitboth oxidation ofpropionate and butyrate.Underthe microaerobic condition, bothethanol andpropanolwere catabolized (Figures4. 16aand 4. 17a). The complete consumptionofbothofthese compounds occurredwithin 20h, compared to theiranaerobic catabolicrates whichwere much slower. The catabolismofthesecompounds was followed by anapparent suppressionin acetateproduction,whichresulted intheacetate concentrationsbeing lower in the experimentalreactorthanthe concentrationswere inthe corresponding control reactor (whichbadnoexternal ethanol or propanol added). Theresultsofthese figures suggest that a switchto aerobic ethanolorpropanol catabolism seems toeither stimulatethe catabolic rate ofacetate consumptionor suppresstherate ofacetateanabolism, thusresulting ina quicker disappearanceofacetatefromthemedium.724.1.2.5EffectsofDfferentClassesofMacromolecules on VFA MetabolismIn orderto investigatethe effectsofdifferentclassesofcompounds onVFAmetabolismin TAD, examples ofrepresentative lipids, carbohydratesand proteinswere chosen andtestedunderbatch TAD conditions. The moleculesselectedwere linoleic acid (fatty acid),glucose,dextrin(carbohydrates) andpeptone (protein). Linoleicacid is apolyunsaturatedfatty acid andis amajorconstituentofmany vegetable oils(eg. soybean, peanut, cornand sunfloweroils).Dextrin is ahydrolysisproduct formedwhenstarch is heated at lowtemperaturesfora shortperiodoftime inthepresence oflargeamounts ofacid. Starchconsists ofchainsofD-glucoseresiduesthatare connectedby(x(l-÷4)linkages and atbranchpointsbyc(l—÷6)linkages(Windholz, 1983). Starch is astoragematerial and as such is structuredtobe degraded. It istherefore not surprisingthatpowerful starch decomposingenzymes are producedandexcretedby microorganisms.Two importantenzymes inthis process arec-amylaseandpullulanase. (x-amylase isproducedby bacteriaand it cleaves thec(1—>4) glycosidic linkages inthe starchmolecule atrandom. Pullulanases are calleddebranchingenzymes, becausethey hydrolyzethe cL(1—*6)linkages in starch. Grueningereta!. (1984)prepared crudea-amylase extracts from asubspeciesofBacillusstereothermophi!usisolated from anATADpilotplantinAltenrheim, SG,Switzerland. Sonnleitner andFiechter(1983) over atwo yearperiodisolatedthermophilicmicroorganisms from an aerobic thermophilic, continuouslyoperated sewage sludge treatmentprocess.73a) Batch 7: VFA concentrationsrelative to controls for linoleicacid addition under anaerobic conditions500T300E____80-100LTime (h)b)Batch 7: VFA concentrations relative to controls forglucoseaddition under anaerobic conditions5003001007”O 2040 60 80-100Time (h)• Acetate —&---—— propionate X Iso A Butyratebutvrate—- 2-Methyl• Isovalerate --< ValeratebutyrateFigure 4.18 VFA difference profiles under anaerobicconditions fora) linoleic acid and b)glucose addition experiments.74a)Batch 7: VFA concentrations relative to controls for dextrinaddition under anaerobic conditions500b)Batch 7: VFA concentrations relativeto controls for peptoneaddition under anaerobic conditions50040 6080Time (h)• Acetate — Propionate—X--——- iso A Butyratebutyrate—z---—-— 2-Methyl • isovaierateC’— ValeratebutyrateFigure 4.19 VFA difference profiles underanaerobic conditions for thea)dextrin and b) peptone additionexperiments.-JE>300100-100Time (h)—IE>30075-JE>Time (h)• Acetate -0——— Propionate —X--— so A Butyratebutyrate—r————— 2-Methyl • Isovalerats—<> Valeratebutyrate-IE>a)Batch 7: VFA concentrations relative to controls for linoleicacid addition under micro-aerobic conditions500 -3001000 20 40 60 80-100--Time (h)b) Batch 7: VFA concentrations relative to controls for glucoseaddition under micro-aerobic conditions500300100-10020 40 60 80Figure 4.20 VFA difference profiles under microaerobic conditionsfor thea) linoleic acid and b) glucose addition experiments.76E>a) Batch 7: VFA concentrations relative to controls for dextrinaddition under micro-aerobic conditions500300100-10020 40 60Time (h)8020b)Batch 7: VFA concentrations relative to controlsfor peptoneaddition under micro-aerobic conditions5003300E>100-100Time (h)• Acetate —D Propionate—X——— iso A Butyratebutyrate- 2-Methyl• isovalerate ValeratebutyrateFigure 4.21 VFA difference profiles under themicroaerobic condition for thea) dextrin and b) peptone addition experiments.40 60 8077A representative set ofisolates were characterizedformicrobiological and biochemicalproperties. Morethan 90% ofthe isolates could degrade starchand grew on gummiarabicum asa sole carbon source. Amylasesandproteases were formed in mediacontaining complexcompounds, butwere not found in significantamounts in growthmediacontainingsimplecarbon substrates. Peptone is high in amino acid content and contains anegligiblequantityofproteoses andmore complexnitrogenous constituents.Figures 4. 18 to 4. 21 showthe resultsofthe macromolecule addition experiments.Peptone wasthe only one ofthe fourtested substratesto stimulate only acetateproductionunderfermentative conditions. Glucose anddextrinstimulatedpredominantly propionateproduction.A somewhat surprising resultwasthatlinoleic acid did very little intermsofVFA stimulationineitherthe anaerobic or microaerobic environments. The1-oxidationoflong chain fatty acids didnot seemto readily occur, althoughthis degradationhadbeenproposed in aprevious section(4.1.2.1) asthemodel pathway forthe catabolismofbutyrate and valerate. Possibleexplanationsforthis anomaly lie inthe factthat linoleic acid is amuchlargermoleculethantheotherVFAtested andwouldthereforehave differenttransportcharacteristics across thecytoplasmic membrane. Undermicroaerobic conditions, the otherthreemacromoleculespredominantly stimulatedtheproductionofacetate. Whether or not eachofthese compoundswasbeing actually consumed is notknown since these macromoleculeswere not assayed.The mostlilcely sequenceofreactions forthe anaerobic catabolismofglucose and dextrinto propionate is the succinate-propionatepathway. Thispathway is employed by mostpropionate producing organisms. Succinate is an intermediate but is also produced as anend78CH-CH2_coOH7Figure 4. 22: Fermentationofpyruvate to propionate via the succinate-propionatepathway (Gottchalk, 1986).CH3-CH2—CO-CoACH3—CO-COOHf,..-— biotin—CO[2CH3biotinHOOC—C--CO—CoA(S)I1H8HOOC—C—CO—CoA (R)HOOC-CH2-CO--COOHCH3B12—enzymeJ-NADH + H137f-NADHOOC-CH2-CH2—CO—CoA HOOC—CH2-CHOH—COQHføH2OHOOC—CH=CH—COOI-j6HOOC—CH2—CH2—COOH279[ianoiI1-q- NADJ—NADH+HacetaldehydeCoAjrNADt7I-NADH +Hacetyl—CoAPiCoAj6CH3 C00P03H2acetyl—()H3P04C—C—TPP—ETPP-EH2C=C—TPP—EH2OH2C—cH—TPP—EOHOH5jO=C-HH—C—OHCH2glyceraldehyde—3-- CE)NADNADH + Hlactate[glucose]NADNADH+Hribu1ose—5—CH2OHHO—C—HH-C-OHCH2-O-®xylulose—5—®NADH + HNADFigure 4. 23: FormationofC02,lactate and ethanolfrom glucosebytheheterofermentativepathway. The numbers refertothe sequence ofenzymes involved inthis pathway (Gottchalk, 1986).80product in small or large amounts. The sequence ofreactionsinvolved inthispathway areillustrated inFigure 4. 22 (Gottschalk, 1986).The NADH produced during substrate levelphosphorylationofglyceraldehyde-3-phosphateduring glycolysismustbe regenerated toNAD,in order forthe catabolismofglucose to proceed. This regenerationrequirementcan be metbythe fermentation ofpyruvateto eitherpropionate orbutyrate which consumeNADHto produceNAD. Since butyiic acidwas not detectedinthese experiments, thisresults in thesuccinatepropionate pathway as alikely candidate. Anotherpotentialpathway isthe heterofermentativeprocess whichresults intheproductionoflactate or ethanol and is illustrated in Figure4. 23.Eitherofthe two pathways shown inthese figures (4. 22 and 4. 23) willbalancethe redox.Thelactate accumulationintheheterofermentativepathway canfurtherbe metabolizedto propionateandacetate in anapproximate 2 propionate: 1 acetate molarratio andcould account fortheobserved occurrenceofbothpropionate and acetate. The results fromthe dextrin additionexperiment, under anaerobicconditions (Figure 4. 19a), suggestthatthe heterofermentativesequenceofreactions wasthe modelpathway. The discrepanciesbetweenthe glucose anddextrinadditionresultsmaypossiblybe explained interms oftherelative efficienciesoftheacquisitionofthese substratesby different subpopulations ofbacteria. This type ofkinetic rateargumentcould account forthe discrepancies seenin the VFAprofiles.Although anumberofbacterial species growwith some single amino acids, manypreferto fermentmixturesofamino acids. They carry out coupled oxidation-reductionreactionsbetweenpairs ofamino acids. One amino acid is oxidized and a second one is reduced. Thisallowsthe amino acids that cannot be fermented individually to be used as asourceofenergy.81These reactions are carried outby many proteolytic species ofClostridia (eg.C. sticlaindii, C.sporogenes, C. histotiticum) (Barnard and Akhtar, 1979; Barker, 1981). Forexample,thecoupling ofvaline (hydrogendonor) and glycine (hydrogen acceptor) results in theformationofisobutyric and acetic acids. These reactions were first demonstratedby Cohen-Bazire etal.(1948).CH3CHCHCHNH2COOH+2CH2NHCOOH+2H20CH3CHCHCOOH+2CH3COOH+ 3NH3+CO2Ina similarmanner, isoleucine, acting as ahydrogen donor canbe oxidizedto 2-methylbutyric acid (ElsdenandHilton, 1978).Undermicroaerobicconditions, it is no longer necessaryto maintainredox balancebyshuttling substratesto specific reduced end products. This change allowsthe stimulationofacetate asthepredominantby product. Acetyl-CoA is converted to acetateviaatwo stepreaction. Thispathwayproduces acetic acid and generatesanATP for every acetyl-CoA.Although only asingle ATP is produced,this level is significantwhen comparedwith anetgainoftwoATP/glucose realizedfromthe glycolytic pathway.4.1.3 2, 4-DmitrophenolAdditionExperimentThe purpose ofthis sectionis to investigatethe effectofinhibitorcompounds onsubstrate metabolism in TAD. In aerobic metabolism, the oxidationofNADH andthephosphorylationofADP are coupledreactions. In vivo, theuncoupling ofthese two reactions82canbe achieved by the additionofcertain compounds to the cells. Theseagents makethecytoplasmic membrane permeable to protons. As aconsequence, aApH gradientcannotbeestablishedandATP cannotbe synthesizedby oxidativephosphorylation. Becauseoftheirmode ofaction, uncouplers are also known as ‘protonophores’. One suchcompoundis 2,4-dinitrophenol (Gottschalk, 1986; Lehninger, 1982). Figure 4. 24 shows theresultsoftheadditionofthis compound and its subsequenteffect onVFAmetabolismin the TAD batchexperiment withprimary sludge as substrate. Since fermentativereactions do notutilizeoxidative phosphorylationto produceATP, this agent shouldhave no effect in an anaerobicenvironment. Underthis condition, the expectedresults are seen.However, there is anunexpectedresultunderthemicroaerobic condition, whichistheaccumulationofacetate far in excessofits controlvalues. It is also apparentfromtheseresultsthatthe anabolic activityofacetateconsumptionmaybe suppressed sincethere is no netoxidationofthis intermediate (ie. acetate accumulationinthe medium). Normally, inE. coilcellsgrowing on glucose, approximately 50% ofthe original carbonis released as CO2, withtheremaining 50% being converted into cellularmaterial (Gottschalk, 1986). Inthepresenceof2,4-dinitrophenol, glucose is oxidized almost completely to CO2withvirtually no anabolicintermediates.The results fromFigures 4. 24 and 4. 25 suggestthat, under microaerobic conditions, theprocessbacteriaare able to switchfrom generatingATP viaoxidative meansto employingsubstrate levelphosphorylationreactions. Thisresults intheproductionofacetate, since acetateproduction from acetyl-CoAyieldsATP. The ORP and pH profiles also supportthis12>2>• AcetaRe —C——— Propionate —X— iso A Butyratebutyrate—zt--—-—— 2-Methyl • isovalerate—-— Valeratebutyrate83a)Batch 9: VFA concentrations relative to controls for 2,4-dinitrophenol addition under anaerobic conditions900700500T-10030 40 5060Time (h)b)Batch 9: VFA concentrations relative to controls for 2,4-dinitrophenol addition under micro-aerobic conditions900700500300100-10010 20 30 4050 60Time (h)Figure 4.24 2, 4-dinitrophenol addition experimentunder a) anaerobicand b) microaerobic conditions.84a)Batch Test 9: [VFAJ, TOC, pH,ORP v time (micro-aerobiccontrol w 270 ml A side + 30ml primary sludge)Batch Test 9: [VFAI,TOC, pH, ORP v time (micro-aerobicwb)2,4-dinitrophenol add’n+ 270 ml A side+ 30 mL primarysludge)250Figure 4.25 VFA, pHand ORP profiles in the2, 4-dinitrophenoladditionexperiment under microaerobicconditions. a) controlcondition andb) 2,4-dinitrophenoladdition condition.0-250ORP(mV)_50019pH8740 50 600 10 2030Time (hrs)0-ORP(mV)-2508pH7640 5060•acetate propionate•isobutyrate‘ butyrateA2-methybutyrateisovalerateXvalerate lactate+TOC•pHCORP0 10 2030Time (his)85hypothesis (Figure 4. 25). Under the control microaerobic condition,the ORP remainsrelatively constant atapproximately -250 mV, suggestinga relatively constantbaseline oxygendemand. Inthe 2,4-dinitrophenoladdition reactor, theORP increases until, by the end oftheexperiment, the reactorhas a slightly positive ORP, suggestingthatoxidative metabolismhadbeen suppressed. This may represent some cellular damagefrom long term energeticpoisoning.The large decrease inpHwas consistentwiththe accumulationofacidic endproducts.The catabolicflowofcomplex substrates in theprimary sludgefeed source to apredominant acetate endproduct, generatesNADH, whichmustbe reoxidizedby operationofthe electrontransportchain. Therefore, these results suggestthat, althoughthe oxidativemetabolismmay be suppressed, abasal level ofactivityoftherespiratory chainmustbemaintained to regenerate oxidized electroncarriers(ie. NAD).In additionto uncouplers, therespiratory chain can be impaired byinhibitors ofelectrontransport. Cyanide is one such inhibitor. This compoundblocksthereductionofoxygencatalyzedby theterminalelectroncarrier inthetransport chain. However,this compoundhadno effect onthemetabolismofVFAunderboth anaerobicandmicroaerobic batchtestconditions. Neither didthe anion fluoride. The physiological effectofthis compound is toinhibitglycolysis by blockingthe enzymeenolase to preventtheproductionofphosphoenolpyruvate from2-phosphoglycerate (Lebninger, 1982).864.1.4 Response ofBiomass from a FermenterProcesstoAnaerobic and MicroaerobicConditionsExperimentswith fermenterprocess sludgewere conducted inorderto understandthecomplex interactions atworkin a fermentationprocess. Thissection describesthe behavioroffermenter sludge subjectedto boththe anaerobic and aerobicconditions. The sludge used inthese batchexperiments came from a side streamfermentationprocesslocated atB. C. Research(Al Gibb, PhD thesis, inprogress). This sidestreamfermentationprocess fed its VFArichmixedliquor into anFGR-SGRbiological phosphorus removal process.Figure 4. 26 showsthe acetate and propionateresponsetrendsto increasingamountsofaddedprimary sludge (0-80% by volume) underanaerobic conditions.Within afermentationtype system, propionate concentrations started out approximately equal tothatofacetate. Overthe courseofthe experiment, whichinthis case lastedfor 70 h, propionate concentrations,in allcases, increased at a slightlyhigherratethanthatofacetate. Undermicroaerobic conditions(Figure4. 27) both acetate andpropionate curvesare similarto the responseoftheseVFAunderTAD batchconditions. Specifically, under highprimary sludge feedrates, overtime, propionateconcentrations decrease as acetate concentrations increase. Thesetrend results suggestthattheeffector is the aerationconditions employed. Therefore, it shouldnot behighly process specific.Whenthe increasingprimary sludge addition experimentwas repeatedwith TAD sludge,some interesting observations and similarities appear (Figures 4. 28 and 4. 29). Undermicroaerobic conditions, the maximumtransientacetate concentrations and maximumacetateproductionrates within the batch reactors increasedwith increasing primary sludge addition872.20Batch Test 10: [Acetate]a)2I.60040020000 10 20 30 40 5060 70Time (hrs)b)Batch Test 10: [Propionate]60040020000 10 20 30 40Time {hrs)Proportion primary0% ° 20%40%sludge_______A80%Figure 4.26 Response of fermenter sludgeto primary sludgeaddition under anaerobic conditions. a)Acetate profiles andb) Propionateprofiles.50 60 7088600E0U— 30020020010000 10 20 3040 50 60 70Time (hrs)Proportion primary0% ° 20%40%sludge_______80%Figure 4.27 Response of fermenter sludgeto primary sludgeaddition under microaerobic conditions. a) Acetateprofiles andb) Propionate profiles.a)Batch Test 10: [Acetatel00 10 20 30 4050 60 70Time (hrs)b)Batch Test 10: [Propionate]206002000b)Batch Test 10: [Propionate]50 60 70600400Time (hrs)Figure 4.28 Response of TAD sludge toincreasing primary sludgeaddition under anaerobic conditions. a)acetate profiles andb) propionate profiles.89a)Batch Test 10: [Acetate]4000 10 20 30 40Time (hrs)200200Proportion primary0%‘20% 40%sludge_______—60%A80%90E00a)Batch Test 10: [Acetate]120090060030000 10 20 30 40Time (hrs)50 60 70b)Batch Test 10: [Propionate]300E200100050 60 70• 00/°20°/ • 40%Proportion primary° 0sludge60%A80%Figure 4.29 Response of TAD sludge to increasing primarysludgeaddition under microaerobic conditions. a) acetate profilesandb) propionate profiles.0 10 20 30 40Time (hrs)91rates. These increases in maximum concentrations andproductionrates were apparent at eventhe highestvolume ofprimary sludge added (ie. 80% primaryand 20% TAD sludgesbyvolume). If80% ofthe volume ofa full scale TAD bioreactorwere replacedeachday, thiswould correspondto a 1.25 d SRT forthatreactor. Therefore,theseresults suggestthat solidsretentiontimesthat couldpotentially maximize VFAproductioncouldbe somewhere intherange of 1 d, which is the recommendedvalue forthermophilicprestage systems.Like thepatternfor fermentersludge both acetate andpropionateprofiles forTADincreased inproportionto eachother during anaerobic incubation conditions. BothWAtrendsunderthe microaerobic conditionhad less similarityto theWAtrendsoffermenter sludge.Themost obvious differences arethatpropionate concentrationsdecreased more rapidly and thatacetate concentrations increased more rapidly, resulting inhighertransientconcentrationswhencomparingTAD sludgeto fermentersludgeperformance. However, it is interestingthattheseresults suggestthat fermenter sludge, upon aeration, can behave ina similarmannerto TADsludge. This observationsuggeststhatthe oxidativemetabolismoffacultative anaerobeswithinthe fermentersludgebiomass, uponthe introductionofoxygenastheterminal electron acceptor,is capableofselectively shuttling complex substratesto an acetate endproduct, presumably inorderto maximizeATP production. These results imply thatthese ‘acetate overflow’ systemsare ubiquitous over a large spectrumofmicroorganisms.Figures 4. 30 and 4. 31 showthe maximumnetrate ofVFAproductionand themaximumconcentrations achievedwithbothfermenter andTAD biomass. Eachpointrepresents abatch experimentwithvarying amounts ofaddedprimary sludge (0-80% by92volume). The maximumrate ofacetateproductioninmostcases occurredwithinthe first20 hofeach experiment. Figures 4. 30b and 4. 3lb showthat, in amicroaerobicenvironmentunderhighprimary sludge additionrates, the maximum rateofacetate production andthe maximumtransientacetate concentrationsofTAD sludge was 2.5xhigherthanthe maximumrate andmaximum acetate concentrationsoffermenter sludge.Figure4. 30a shows thatunder anaerobicconditions, fermenter sludge performance was superiorto TAD sludgeperformanceunderlowprimary sludge additionrates.When comparing fermenter sludgeperformance underboth anaerobic andmicroaerobicconditions, some interesting results canbe seen (Figure 4.30). Underfermentative conditions,overthe entire rangeofprimary sludge additionrates, propionate productionwas similartothatofacetate anddecreased as theprimary sludge addedwas increased.The acetateproductionratewas highestwhennoprimary sludge was added (14 mgIL-h). Undermicroaerobicconditions,acetateproduction increased as thevolume ofprimary sludge added increased. Althoughtheproductionratewas only 8 mgfL-hwhenno primary sludge was added, itincreased to 18 mgfLhwiththe highest addedvolume ofprimary sludge. By slightly aerating fermenter sludge, itwaspossibleto improve acetate production during highprimary sludge additionrates.Underambientroomtemperatures, it is difficultto differentiatetherelative effectsofsubstrate (ie.primary sludge) and active biomass (ie. fermenter sludge) under suchhighprimary sludgeadditionrates. UnderTAD conditions, the biomass intheprimary sludge wouldbe consideredpredominantly substrate since incubationtemperaturesofthe batch experiments were 45° C.Therefore, ifproductionrate calculationswerenormalizedto the activebiomass, theresulting93a)Batch 10: Maximum rate of VFA production as a function ofsludge addition under anaerobic conditions20EC0=100.>‘CE15050b)Batch 10: Maximum rate of VFA production as a function ofsludge addition under micro-aerobic conditionsh..CEC0.20.>‘CE50403020100% primary sludge addition100100Bacetate,Uacetate,•proponate propionateTAD fermenter TAD fermenterbiomass biomass biomass biomassFigure 4.30 Comparison of maximum rate of VFA production betweenTAD and fermenter process biomass under a) anaerobic andb) microaerobic conditions.0 20 40 60 80% primary sludge addition0 20 40 60 8094a) Batch 10: Maximum concentration of VFAs as a function ofsludge addition under anaerobic conditions600-JE 400>200E0b)Batch 10: Maximum concentration of VFAs as a function ofsludge addition under micro-aerobic conditions1200•acetate, — acetate,•propionate ——— propionateTAD fermenter TAD fermenterbiomass biomass biomass biomassFigure 4.31 Comparison of maximum achievable concentration of VFAbetween TAD and fermenter process biomass under a) anaerobic andb) microaerobic conditions.0 20 40 60 80% primary sludge addition100-JE>xE90060030000 20 40 60 80% primary sludge addition10095TAD sludge performance would be far superior to fermentersludge.4.1.5 FermentativeTAD ExperimentsThe purpose ofthis sectionis to address the difficultiesthat arise whentryingto compareTAD and fermenterbiomassperformance. The mostobvious confounding factor isthatthesesludges originate from 2 distinctly differentprocesses.Since TAD sludge came from aprocessoperating at45° C and fermenter sludge came from aprocess at ambienttemperatures(approximately200C), the batch experiments on eachcorrespondingbiomasswere thusconducted atdifferenttemperatures. One system is acclimatedto thepresence ofoxygenwhilethe otherprocesswas strictly fermentative. TAD SRTs and Hydraulic RetentionTimes (HRT)are equal while fermentersusually have a much longerSRTthanHRT.Inorderto addressthese discrepancies, oneparallel side ofthepilot scale TAD processwas rununder strictfermentative conditions under athermophilictemperature regime.Toachieve atrue anaerobic environment, one ofthe reactorswaspurgedwithananaerobic gasmixture (ie. 90%N2,5% CO2and 5%H2). Batchexperiments were thenperformedwith sludgedrawn fromthis side as well as the control side. This type ofcomparisonwould eliminatethepreceding confounding independentvariable effects, except for the effectofoxygen.Figure 4. 32 showsthe VFAprofiles ofthe true fermentative TAD sludgeunder bothanaerobic and microaerobicbatchconditions. These results show, thatundermicroaerobicconditions, propionate and acetatetrends were divergentand the decrease inpropionate96concentrations was much slower comparedto aTADprocess acclimated to small amountsofoxygen (Figure 4. 33). These resultshave a striking similarity tothe VFA response offermentersludge undermicroaerobic conditions. The experiment, inwhich25% (by volume) primarysludge was added, yielded amaximum acetateproductionrateof5.5 mgfL-h under anaerobicconditions and 18.0 mgIL-hundermicroaerobic conditions andresulted inahigheracetateconcentration under microaerobic conditionsby the endofthe experiment(Figure 4. 32).Anotherinterestingresult isthat, underthe niicroaerobic condition, thefermentative TAD sludgeexhibited an ORP profilewhichwas consistently less negativethanthatofthe control side TADsludge (Fermentative TAD ORP>-250 mV, control TAD ORP<-250mV). This observation isconsistentwiththe hypothesisthatTAD biomass acclimatedto small amountsofoxygenhave amore efficient02 scavenging systemthan afermentative TAD process, resulting inamorereduced environmentundermicroaerobic conditions. These results suggestthatthe systemsresponsibleforacetate metabolismin TAD, undermicroaerobic conditions, areubiquitousinnature andare notafunctionofthe aerationrate employed, since fermentative TADbiomasscanbehave inasimilarmannertoTAD biomass acclimatedto small amountsof02.97a) Batch Test 11•acetate, 0%primary-— acetate, 25%primary•acetate, 50%primary-— propionate, 0%primaryApropionate,25% primary—— propionate,50% primaryb)1000Batch Test 11•acetate, 0%primary-— acetate, 25%primarypropionate, 0%primaryApropionate,25% primary0 10 20 30 40 50Time (hrs)Figure 4.32 VFA profiles of fermentative TAD biomass response toincreasing primary sludge addition under a) microaerobic andb) anaerobic conditions.1500120090060000-JE00 10 20 30 40 50Time (hrs)E>75050025098a) Batch Test 11500•acetate, 0%primary-— acetate, 25%primary• acetate, 50%250primary-— propionate, 0%primaryApropionate,25% primary0-— propionate,40 5050% primaryb)Batch Test 11500•acetate, 0%primary- acetate, 25%primary250°propionate, 0%primaryApropionate,25% primary00 10 20 30 40 50Time (hrs)Figure 4.33 VFA profiles of control side TAD biomass response toincreasing primary sludge addition under a) microaerobic andb) anaerobic conditions.o 10 20 30Time (hrs)994.1.6 Salmon Arm ATAD PerformanceSince most ofthe experiments were conductedwithpilotscale TAD biomass,confirmationofthese results with full scale ATAD biomasswasnecessary. In 1985, the DistrictofSalmonArm chose abiologicalphosphorusremoval processto replacetheirexistingtricklingfilterprocess. Thisnovel Bio-P system employsfixedand suspendedgrowthreactorsto achievephosphorusremoval (Gibb eta!., 1989). To replace their existing sludge digestionsystem,aerobic mesophilic digesters were retrofittedforATAD operation. This system usesthe sameTurboratorInc. designed aerator/mixer as the UBC pilot scaleTAD process. The digestersoperate in series and are fed withamixtureofprimary andwaste activatedsludges inanapproximate 1:1 ratio. Sincethe majority ofthe batchexperimentswere conductedwithpilotscale TAD sludge, confirmation ofa fewofthese batchexperimentson full scale SalmonArmATAD sludge wasnecessary.Figures 4. 34 to 4. 37 illustratetheresponseofindividual VFA inthe SalmonArmATAD sludge takenfromthe thirdcell relativeto their control values, underbothanaerobic andmicroaerobicbatchtestconditions. The substratestestedwereprimary sludge, waste activatedsludge, amixture ofprimary and waste activated sludges andpropionate. The results are similarto those obtainedusing thepilot scale TAD process biomass. There are, however, afewdiscrepanciesbetweenpilotand full scale VFA trends. Inthepropionate additionexperimentunder anaerobic conditions (Figure 4. 35a), therewas little deviation ofacetate concentrationsfrom its control value until 45 h into the experiment. At this point, acetate concentrations100increased very rapidly until, by the end ofthe experiment,the concentration was approximately800 mg/L higherthanthe corresponding control value. The difference betweenthepropionateconcentrationsofthe additionreactor and the control reactorremained constantoverthe courseofthe experiment, suggesting thatthe addedpropionate wasnotconsumed underanaerobicconditions. Undermicroaerobic conditions (Figure 4. 37a), propionate consumptionoccurredbut was not concomitantwith acetate production. Therewas however atransientincrease inisovalerate concentrationsrelativeto control values.Aninteresting observationwasthatundermicroaerobic conditions, primary sludgewassuperiorto waste activated sludge interms ofits ability to stimulate acetateproduction (Figure 4.36). However, amixture ofprimary andwaste activated sludges seemedto be betteratstimulating acetateproductionthanprimary sludge alone (Figure 4. 37). Thisresultseemscontradictory. Itmustbe rememberedthatthe SalmonArmATAD process is acclimatedtoafeed stream consistingofbothprimary and waste activated sludges. Althoughwaste activatedsludge cannot stimulate acetate production alone, there mustbe somenecessary componentsprovided by this fractionto the digestionprocess.101a)Batch 13: VFA concentrations relative to controlsforprimary addition under anaerobic conditions500300100EU- -100>-300-500b) Batch 13: VFA concentrations relative to controls forsecondary addition under anaerobic conditions400Figure 4.34 VFA difference plots of Salmon Arm ATADsludge underanaerobic conditions toa) primary andb) waste activated sludge additions.30 40Time (h)-JE>702000-200-400-600• Acetate DProptonate X iso A Butyratebutyrate2-Methyl • so Valeratebutyrate ValerateTime (h)102—IE>-JE>• Acetate —Ci—----— Propionate -—X—-— iso AButyratebutyrate——-—- 2-Methyl • lsovaierate 0----Valeratebutyratea) Batch 13: VFA concentrations relative to controls forpropionate addition under anaerobic conditions8006004002000-20010 20 30 40 5060Time (h)b) Batch 13: VFA concentrations relative to controlsfor mixedprimary/secondary addition under anaerobic conditions2000-200Time (h)Figure 4.35 VFA difference plots of Salmon Arm ATADsludge undermicroaerobic conditions to a) propionate and b) a mixture of primary andwaste activated sludge additions-JE>• Acetate —0 Propionate X Iso A Butyratebutyrate—- 2-Methyl • Isovalerate — —---- Valeratebutyrate103—SE>a)Batch 13: VFA concentrations relative to controls for primaryaddition under micro-aerobic conditions8006004002000.200!10 20 30 40 50 60Time (h)b) Batch 13: VFA concentrations relative to controls,secondary sludge addition, micro-aerobic conditions4002000-200-400-60030 40 50 60 70Time (h)Figure 4.36 VFA difference plots of Salmon Arm ATAD sludge undermicroaerobic conditions to a) primary and b) waste activatedsludge additions.104-IE>• Acetate —-—— Propionate —X——— iso- A ButyrateButyrate— 2-Methyl- • iso- ——— Valerate8utyrate Valerate-IE>a)Batch 13: VFA concentrations relative to controls, propionateaddition under micro-aerobic conditions10008006004002000-200Time (h)50 60b) Batch 13: VFA concentrations relativeto controls for mixedprimary I secondary addition under micro-aerobic conditions10008006004002000-20010 20 30 40 50 60Time (h)Figure 4.37 VFA difference plots of Salmon Arm ATAD sludge undermicroaerobic conditions to a) propionate and b) a mixture of primary andwaste activated sludge additions.1054.2 PreliminaryPilotScale TADExperimentsSince therewere no suitable guidelines as to the appropriate aerationratethatwouldstimulate maximumVFAproductionin TAD, an initial broad range ofairflowrateswerechosen. The tested air flowrateswere 0 volume ofair/volume ofsludge-hour,which is defmedas amicroaerobic condition.(ie. calculatedflowrate of0.126 V/V-h), an intermediatetransitioncondition at 0.28 V/V-hand an aerobic condition at 0.6 V/V-h. Eachrunat thespecifiedoperatingconditionlasted fortwo solids retentiontimes (SRTs) or 12 days (SRT equalsHRT).The studies concentratedonthe first stage ofthe two-stage TAD process, sincepreliminaryresults showedthehighestproductionofVFAoccurred inthis stage.Table 3. 1 is acomparisonofoperating conditions and solids destructionefficienciesinthepilot scale TAD and thoserecommendedfor full scale design (EPA, 1990; Kelly, 1990). Thetotal measured solids destructionwasroughly 3/4 ofrecommendeddestructionrates. Theoperating degree day productwasbelowthe recommended range of400 to 500. The 29% totalsolids destructionwas similarto the 30% observedby Koers andMavinic (1977) foraerobicallydigested sludges operating atasimilar degree-dayproduct, butatlowertemperatures.Figure 4. 38 and Figure 4. 39 are examples ofthe daily ORP andtemperatureprofiles inthe first stage ofthepilot scale TAD, representingthe transition, microaerobic and aerobicconditions, respectively. In Figure 4. 38a, the airflowrate of0.28 V/V-hconsistentlyproducedaperiodwithanegative ORP signal ofapproximately -250 mV for about 5 h. Atthe end ofthisperiod, the TAD ‘elbow’ was observed. This elbowcorresponded to thepoint atwhichameasurable concentrationofdissolved oxygenwaspresent. The elbowalso correspondedto the106disappearance ofmeasurable VFA. This graph also showsthat acetate wasthe predominantVFA speciesproduced.Any measured VFA species is afunctionofboth its anabolism and catabolism.Inorderfor acetate to buildup to 45 mg/L, its rate ofsynthesis has to exceed its rateofconsumption.This excess anabolismoccurredinthe first 4.5 hofthe cycle(Figure 4. 38b). Between 4.5 and8h, the catabolic rate exceeded therateofsynthesis. Theresultwas adisappearanceofacetate.Theserelativemetabolic rates respondedrapidly to changes ofaeration. Whenthe aerationratewas increasedfrom 0 to 0.6 V/V-h, at approximately 70 h, measurable acetateconcentrationsrapidly dropped andby 80 h, approached0 (Figure 4. 40a). Therefore, by increasing the airflow, the net observedconcentrationofacetate decreased. This figure also demonstratesthecapacity ofanequilibrated TAD, operatingunderhighly reducedconditionsandaerated farbelowitsmaximum oxygendemand, to immediately adjust itsmetabolic ratesto meettheenvironmentalpressures ofthe increased aerated condition. Figure4. 40b showsthe oppositetrend. Whenthe air flowrate was decreased from 0.6 to 0 V/V-h, at97 h, accumulated acetateconcentrations startedto increase andreachedanequilibriumby approximately 4 days (i.e. 1.3SRTs).Figure4. 38 The transitioncondition in the first stageofthe TAD process (air flowrate of0.28 V/V-h).a) An example ofonecycle ofORP andtemperature profiles. b)VFAproffles ofthe same cycle.107a)20a0ci)Dci)2ci)I-Time (h)b)2LI..>Time (h)—A— Acetate.0—— Propionate —÷-- Isobutyrate108a)>E00-250-300-350lime of feed800700600500400LL300 >200100025.0-4000.05.0 1 d.o1Time (h)2d.0b)(—*— Acetate —.—Propionate —s--Isobutyrate —‘f--- Butyrate>E0r.r0-J0,EU>AcetatePropioflateFigure4. 39 ORP and WA profilesin the first stage ofthe TAD process under a)microaerobic (air flow rateof0 V/V-h)and b) aerobic conditions(0.6 V/V-h).109a)>Ea0-JELI.>Time (h)—— Acetate —— Propionate—— Isobutyrate—‘4—-Butyrateb)>E-1l0-J3)Ea)C)Time (h)Figure 4. 40 ORP and WAprofilesin the first stage ofthe TAD processduring a switchfrom a) microaerobic to aerobicconditions and from b) aerobic to microaerobicconditions. The vertical linesrepresentthe points at whichthe TAD process had receivedfeed sludge.1101600Total VFA1400-I0)Acetate,1000800ci4-ci)C)400Total VFA200AcetateAcetate Total VFAAerobic TransitionMicroaerobicAeration conditionFigure 4. 41 Comparisonofthe net acetateand nettotalWAproduction inthefirststage oftheTAD processunder thethree aeration conditions.Included arethemean, standarddeviation andrange.111The first4.5 h ofFigure 4. 38b shows some interesting results.As acetate concentrationsincreased, propionate concentrations decreased and after5 h, propionate had disappearedcompletely. Thisresult indicatedthatacetate and propionate, whicharetwo fermentative endproducts, appearto behave in opposite trends underthermophilic sludgedigesting conditionswhich was similarto the behaviorofthese VFA underbatchtestconditions inthe microaerobicenvironment. Figure 4. 39ashows the optimal rate ofaerationintermsofmaximumnetVFAproduction. With anegative ORP signal that lastedthe entire 24 h cycle, acetateconcentrationsexceeded 700 mg/L. Figure 4. 39b showsthe VFAprofilesunderthe aerobic condition.Itisclear fromthis graphthattherate ofacetate catabolism had exceededtherateofacetatesynthesis. The result wasno accumulationofVFA.Table 4. 2 showsthe durationofmicroaerobiosis observedunderthethreeaerationconditions. The largestdetectable range, ofbetween 6 and 14 hofmicroaerobiosis(ORP valuesofbelow-250 mV), wasunder thetransitioncondition (air flowrate: 0.28 V/V-h). This largerange mightbe dueto dailyvariability in sludge feed characteristics.Table 4. 2 Durationofmicroaerobiosisduringthe three aerationconditionsinthe first stageofthe TAD process. Each feeding cycle lasted for24 h.Variables Microaerobic Transition AerobicAir flow (V/V-h) 0 0.28 0.60Minimum duration (h) 24 6 0.5Maximum duration (h) 24 14 3Figure 4. 41 shows the capacityofthe three aeration conditionsto produce acetate andtotal VFA, respectively. This figure shows the mean, range and standard deviation ofthe net112productionfor each ofthe 3 conditions. It is clearthatthe lowestaerationrate of0 V/V-h gavethe highest acetate andtotal VFA concentrations.Table 4. 3 comparesthe VFA distributionpatternbetween aTADprocess, anATADprocess and two fermentativeprocesses (Oldham et aX., 1992). Whenthe TAD process iscomparedto afermentative process, some striking differencesappear. Infermentativeprocesses, acetatecontributes an averageof48% ofthe total VFA inthePenticton, B.C.treatmentplant and43% inthe Kelowna, B.C. plant. However,the University ofB.C. pilotplant and the SalmonArmATAD processesthe contributionswere 81% and70%, respectively.Table 4. 3 ComparisonofVFA distributionbetween 2 thermophilic aerobicdigestionprocessesand 2 fermentationprocesses.Variables Salmon Arm, UBC pilot Penticton, B.C. Kelowna,B.C.B.C. ATM) TAD Fermenter FermenterAcetate (%) 70 81 48 43Propionate (%) 14 11 42 43Others (%) 16 8 10 14Hamer (1987) conducted aerobicthermophilic experiments whichmeasuredthevariousVFA, as afunctionoftime, in semicontinuous operatingconditions in a laboratory scalebioreactor. Thethermophilic organisms were from afull-scale operatingATAD process. Thesludge feed source was asuspension ofyeastcells. Theirresultswere similarto the resultsofthis study, intermsofoveralltrends andrelative proportionsofeach VFA. Hamer (1987)explainedthose effects by suggestingthat accumulationofcarboxylic acids, predominantlyacetic acid, is the result ofthe fermentative metabolismofthe thermophilicprocess culture. Thismodel suggests that near exhaustionofthe preferred soluble substrate is followedby utilization113ofthe lowmolecular mass carboxylic acids producedearlier (eg. acetate, propionate). However,it does notexplain why areadily usable substratelike acetate canaccumulate to concentrationsof6 g/L, whenthe mixedmicrobial consortiummediatingthe biodegradationprocesswouldbeexpectedto simultaneously use theproduced fermentationproducts (Willdnsonand Hamer,1979). This discrepancy was attributedtothe possibilityofsome sortofinhibitionofcarboxylicacid biodegradation.The resultsfromthispreliminarypilot scale study suggestthatthere was simultaneousutilizationofVFA andthatthemeasured concentrationofVFA, at any giventime, is a functionofboththe relative ratesofits synthesis andbiodegradation.Throughoutthe study, VFAconcentrations increasedwhenthe catabolic rate ofVFAconsumption was expectedto be lowerthanthe anabolicrateofVFAproduction. InFigure4. 38, the netacetate concentration builtupto amaximumwithin 4.5 h andthenhad disappearedby 8 h. The ORP duringthisperiodremained at around -250 mV. Itmay beconvenienttothinkthat this environmentrepresents afermentative condition; however,itmustbe rememberedthat, throughoutthis cycle, the reactorswere aerated at 0.28 V/V-h. Therefore, the oxygendemandbetween 0 and 8 hactually exceededthe oxygensupply. Thisrange also happenedto bethe acetate detectionrange.It is clearfromthe datathatthe measurable presenceofeasily oxidizable substratescontributedto thehigh oxygen demand (eg. acetate) andthatthe disappearance ofthesecompounds correlatedto the ORP elbow, whichwas thetransitionpointbetweenperiodsofhighoxygenrequirement and decreasing oxygenrequirement. Figure4. 40 showsthepatternofacetatebehaviorwhenthe airflowrate is increased or decreased.Whenflowrateswere114increased, the netacetate concentrationfell rapidly. Whenflowrates decreased, the netacetateconcentration increased. Since the response timeofchanges inmeasuredacetate to increase ordecrease inair flowwas negligible, the inhibitionofacetate degradationwas inpartafunctionofaeration. This correlationwas expected, since the most importantmoleculerequired in acetatebiodegradation, or its oxidation, is molecular oxygen. Therefore, it isclearthatthemeasurableaccumulation ofacetate occurreddespite aerobicrespiration.1154.3 3x3PilotScale TADExperimentsFrom thepreliminary study onTAD, it was discovered thatmicroaerobicconditionsproducedthe highest concentrations ofVFA out ofthe three conditions examined (ie.microaerobic, transitionand aerobic conditions). A 3x3 pilotTAD experimentnarrowedtherange ofair flowrates suchthatall flowratestestedwere encompassed withinthe microaerobicrange. The meanflowrateswere 0 mL/min, whichwas definedas a lowflowmicroaerobiccondition, 115 mL/rninwhichwas defined as amedium flowmicroaerobicconditionand 165mL/minwhich correspondedto ahighflowmicroaerobiccondition. SRT wasthe otherindependentvariable tested. The SRT values examinedwere 3, 4.5, and 6 days.To testmainand interactioneffectsofthese two variables, atotalof9 cells orrunswouldhaveto be done. Atotal of9 runswere donewiththehighair flowand 4.5 d SRT combinationrepeated. Thisresultedin only 8 ofthepossible 9 combinations completed. As seenin Table 3.2, due to an error in scheduling, the medium air flowand 6 d SRT combinationwasnot done. Inadditionto these 9 runs, atenthrunwas done in atrue fermentative environmentata4.5 d SRTto examinethemetabolic behaviorofsubstratesunder anaerobic conditions. This anaerobiosiswas achieved bypurgingthetestreactor (B side) withananaerobic gasmixture whichcontained90% nitrogen, 5% carbon dioxide and 5% hydrogen. Since SRTs and air flowratesvaried overthe course ofeachrun, Table 4. 4 showsthe calculated SRTs and aerationrates. Includedarethe mean, standard deviationandrange forthese independently setvariables. In all runs, theAside control independentvariableswere maintained attheirrespective medians (ie. 4.5 d SRTand mediumaeration) while the B side experimental variableswere adjusted to their appropriate116values. Since all control side (A side) runs had the sameSRT and aerationrates,cumulativevalues forall runs were calculated forthis reactor.This experimental design facilitatedthecomparisonofthetestcondition (B side) to the correspondingcontrol condition (A side) withinthe contextofasinglerun.Table 4. 4 Descriptive statistics ofair flowrates and SRTsofpilotscale TAD experiments.B side SRT (d) Aeration(expt.)(mL/min)Run # Mean Standard Range MeanStandard Range(target) deviation (target) deviation1 4.6 (4.5) 0.3 4.1-5.1 0(0) 0 02 3.3 (3.0) 0.4 2.6-4.0 0(0) 0 03 5.6 (6.0) 0.6 4.5-6.7 0(0) 0 04 4.6 (4.5) 0.2 4.3-4.9 110(117) 22 65-1455 3.4 (3.0) 0.1 3.2-3.6 123 (117) 7114-1326 6.4 (6.0) 0.2 6.1-6.7 160(164) 3 156-1647 4.5 (4.5) 0.7 3.1-5.2 165(164) 0 1658 5.5 (6.0) 0.3 5.0-6.0 165(164)1 164-1669 4.6 (4.5) 0.4 3.9-5.3 164(164) 3 160-16810 4.4(4.5) 0.2 4.1-4.7 n/a (n/a) n/a n/aA side 4.5 (4.5) 0.3 3.3-5.7 105 (117) 1924-186(cont.)4.3.1 TemperatureVariation ofPilot ScaleTAD experimentsFromthepreliminarypilot scale study thefirst stageofthe TAD process was showntoproducethehighestmeasured concentrationsofVFA. The temperature range ofthe first stagewas370to 47° C. The secondphase ofoperationattempted to reproducesimilartemperaturerangesunder adifferentprimary sludge feedingregime (once/hour insteadofonce/day). Figure1174. 42 shows examplesofthe on line temperature variations ofbothA and B sides during runs 1and 8. As seenfromthese results, the temperature rangewas approximately 43 to 49° C. Inaddition, there seemedto be a daily cyclic temperature response.The temperatures insidethereactors seemed to be higher during the late morning andafternoonperiods (eg. 10:00-13:00)relative to the night and early morning periods(22:00-03:00). This observationwould beconsistentwithambienttemperature fluctuations overthe courseofaday. The ambienttemperatures outsidethe reactormay have had enoughofaneffectto produceatemperaturecycle withinthe reactors, since during the day, the ambient externaltemperaturewas invariably afewdegreeswarmerthan atnight.One feature ofthesepilot scale units, whichmakethem differentfromotherpilot andbench scale processes, wasthatthere was no needfor any externalheating deviceto maintainreactortemperatures. Reactortemperatures weremaintainedby biological heatproduction,mechanical mixing heat input and an adequately insulatedreactor. Sincethere was noexternalheating controls, isothermal operationcouldnotbe maintainedand somefluctuationsdid occur.However, therewas anindirectway ofcontrolling reactortemperatures. This was achievedbyvaryingthe only controllableheat input, whichwasthe amountofmechanicalmixing energy (ie.changing TurboratorRPMs). Whenreactortemperatureswere increasing,it couldbe reduced byslightly decreasing the RPM ofthe turborator, orvice versa. Figure 4. 43 illustratesthis effect.Whenthe A sidetemperature startedto decrease (Figure 4. 43a), the RPM ofthe Turboratormixer/aeratorwas increasedto 850 from 830 RPM atapproximately 10:30 am. This increase inrotational speedresulted in an increase inreactortemperature. Therefore, a change ofonly 17118a) Temperature Variations, Sides A and B, Run 14846A Side1::40 I I II I I I I I I I I I I I I Ic,l c’1 c’1C’) C’) C C’) C’)C’)0 C)2 2 2 22 2C’) U) N 0) —o o 0 0 0 —Time (days:hours:minutes)b) Temperature Variations, Sides Aand B, Run 848B Side46tASid,44E4)424.0 II I I I I I I I I(‘1(‘4U) U) It) U)U) U)0)1 0)9. 9 9 99 9.o c’4CD CO 0o o 0 00Time (days:hours:minutes)Figure 4.42 Examples of on line temperature measurementsduring pilotscale TAD experimentsa)47.45z1..E43I-41b)5755535149.47I-.434139119Temperature Variations, Sides A and B, Run 7Temperature Variations, Sides A and B, Run 3Figure 4.43 Temperature effects in TAD reactorsfrom changing Turboratorrotational speed.c)00Nc)0C’)0Time (days:hours:mm)NC’)0U,0N00N00Time (days:hours:mm)N0C.,’120a)-320-4100-42000:09:22 04:09:22 08:09:22Time (days:hours:min)12:09:22b)ORP Variations, Sides A and B-320-410-42000:21:22Time (days:hours:min)c)ORP FFTs, Sides A and B4)0’“Li0;Figure 4.44 Examples of on line ORP measurements during pilot scale TADexperiments, a) ORP variation of both sides over a period of 12 days andb) an extract of the same period showing shark toothpattern.c) Fast Fourier transform of this ORPORP Variations, sides A and BiIiI;I0Depression of signal00:09:22 01:09:22Primary Peak4-0 U)If) If) C’) U) U) U)I.., -r- c’10C’) U)Frequency (1/h)CD121RPMresulted in ameasurable temperature increase withinthe reactor. Figure 4. 43b shows theeffectofa Turboratormalfunctionwhichresulted inanincrease ofrotational speed from850 to1030 RPM. The increase inreactortemperature wasdramatic. Approximately 10 h afterthemalfunction, the rotational speed was decreased backto 850 RPMwhichresulted inagradualdecrease intemperature. Thirty six hours afterthe firstmalfunctionhadbeenrectifiedanotheridenticalmalfunctionoccurred, whichresulted in a similartemperature increase. Sinceisothermaltemperatures couldnot be maintained,this couldpotentially have contributed tothevariability in manyofthe measuredparameters.4.3.2 On Line ORP Measurements as StateVariableORP hasbeenusedinthepastto measurethe state ofdifferentprocesses rangingfromanaerobic digestion(Blanc andMolof, 1973; Dirasian eta!.,1963) to biological nutrientremovalprocesses (Kochetal., 1988). Inan attemptto evaluatethe suitability ofORP as astate variableinTAD, on line dataacquisitionofthisvariablewas implemented.Anexample ofthe ORPtrends ofone ofthe runs is shown in Figure 4. 44. Since all runs wereconductedundermicroaerobic aeration conditions, therewas no obvious detectable differencein on line ORPprofilesbetweenthe differentaerationrateswithinthis microaerobic range.Theprofiles ofall 9runsremained consistentlynegative at approximately -400 mV. Thistype ofprofile suggeststhat a highly reduced environmentexistedwithin each reactor. There seemedto be adailytransient ORP perturbation, which correspondedprecisely to the timeofwastage from each122bioreactor. This transienteffectwas short lived and theORP signal returned quickly backto itsbasal response. This couldhave possibly been causedby forcing air into theheadspace ofthebioreactorby wasting afixed volume ofsludge fromthe contentsofthe reactor.Whenthe time scale is expanded, acyclic nature becomes obvious (Figure4. 44b) andassuch was analyzedusing Fourier analysis. The Fouriermodel decomposes atime series intoafmite sumoftrigonometric components (ie. sine and cosine wavesofdifferentfrequencies).Fouriertransforms aretime consumingto computebecausethey involve numeroustrigonometricfunctions. Cooley andTukey (1965) developed afast algorithmforcomputingthetransform ona discrete seriesthatmakes the spectral analysis oflengthy seriespractical.Avariantofthis fastFourier transform algorithmwas used in Excel for Windows (Version4.0). Figure 4. 44c showstherelative amplitude plotted againstthe inverseoffrequency. Thisplot shows oneprimarypeakat 52.6 minutes (frequency 1.12 h’), whichisthe exactperiodbetweenreactor feedings.Whenthereactorswerefed substrate (ie. primary sludge), this seemedtodepress or decrease theORP signal slightly in amore reduced direction (approximately 20 mV).Over the next 52.6minutesthe signals increased gradually until the nextfeedcycle was initiated. This approximateonce/hourfeeding regime created a ‘Sharktooth’ patternresponse intheORP profile. Kelly eta!. (1993) also investigatedthe suitability ofORP measurements asa state variableatthe DistrictofSalmonarmATAD facility, whichhas asimilarfeeding regimeto the UBC pilot scale TADprocess. Stable operationwas found between-50 and -250 mV. They reportedthatthe hourlysludge feedingswere characterized, onthe ORP charts,by abrupt negativedepressions andaslowrise to a lessnegativenorm (ie. ‘sharktoothpattern’).123It is somewhat strange that ORP, althoughinsensitive to changes in aerationratesbetweenruns, was extremely sensitive to the additionofsubstrate within each individual run.These results suggestthatabsolute ORP values arenota suitably stringent statevariable formonitoringreactor conditionsbetweenruns, althoughrelative ORP values are quitesensitive atdetecting slight changes inreactor conditions within anarrowtime window. The resultsfromthis sectionandthose fromthepreliminary pilot scale experiments showthatORP can onlydiscriminatebetweengross changes in aeration (ie. microaerobic, transitionandaerobicconditions).4.3.3 VFAAccumulationin TADThe two independentvariables examinedfortheireffecton VFA accumulationinTADwere aeration and SRT. The following sectionsillustrate and discussthe effects ofthesevariables onVFA accumulationand speciation. The first section introducesthe variablenatureofmeasuring individual VFA speciesovertime andthe factorsthataffecttheirvariability. Thesecond sectiondiscussesthevariables intermsoftheireffectonthe overall accumulationofVFA in TAD. The final sectiondiscussesthe statistical significanceofthe effectofaerationandSRT onVFA accumulation.4.3.3.1 VariabilityofVFAMeasurementsFigures 4. 45 and 4. 46 showthe effectofaeration and SRT onthe variability ofacetate124a)A side [Acetate] control; 4.5 d SRT1000T2800• Medair21600AMed air, 5400_____0200 IMedair,60o 5 10 15Time (days)b)B side [Acetatel with 3 d SRT10002800•Loair,2600AMed air, 5. 400• Hiair,60200o 5 10 15Time (days)Figure 4.45 Variation of acetate concentrations over time in the pilot scaleTAD experiments with a 3 d SRT. a) A side and b) B side(legend key: Symbol; aeration rate; run125a)A side [Acetate] control; 4.5 d SRT10002 800I Med air, 10600i4.0O• Medair, 1AMed air, 4CS. 200Med air, 70 II IMed air, 90 5 10 15________________Time (days)b)B side [Acetate] with 4.5 d SRT1000-—— 0 air, 102800•Loair,1600AMed air, 4400• Hiair,7CS.200Hi air, 90 5 10 15Time (days)Figure 4.46 Variation of acetate concentrations over time inthe pilot scaleTAD experiments with a 4.5 d SRT. a) A side and b) Bside(legend key: Symbol; aeration rate; run #).126a)A side [Propionate] control; 4.5 d SRT6050I___401Medair,230AMed air, 5C.2OMed air, 610______________0 5 10 15Time (days)b)A side [Propionate] control; 4.5 d SRT60__________________50-— Med air, 1030___AMed air, 4Medair, 1Med air, 7-— Med air, 90 5 10 15Time (days)c)A side [Propionatel control; 4.5 d SRT60150_________________!401IMedair,33OCMedair,8j201L I0I0 5 10 15Time (days)Figure 4.47 Variation of A side propionate concentrations over time forall 10 runs (legend key: Symbol; aeration rate; run#).127a) B side[Propionatel with 3 d SRT6002Loair,2400AMed air, 5C200______• Hiair,600 =--0 5 10 15Time (days)b) B side [Propionatel with 4.5 d SRT600________________2— 0 air, 10400•Loair,1—C_______AMed air, 4200• Hiair,70‘r ——-—i-—v vr —A i—— Hi air, 90 5 10 15Time (days)c)B side [Propionate] with 6 d SRT6004001Loair,3C• Hiair,82000• • • • . • • * *0 5 10 15Time (days)Figure 4.48 Variation of B side propionate concentrations over time forall 10 runs (legend key: Symbol; aeration rate; run #).128concentrations betweenthe control (A) side and the experimental (B) side bioreactors. Thevariability overtime oftheA side acetate concentrations inthe reactorwas presumably dueprimarily to variations inuncontrollable environmental conditions (eg. sludge feedcharacteristics, external ambienttemperatures). Even thoughthe SRT and aerationrate intheAside reactorremained constantthroughout the 10 runs (ie. 4.5 d SRT andmedium airflowrate),the acetate concentrationsmeasured inthisreactorvaried considerably. Forexample, inrun6,which correspondedto the highflow/3 d SRT combination, the control (A) sidereactorbeganwithan acetate concentrationof175 mg/L and steadily increasedthroughoutthe experimenttoend at a concentrationof600 mg/L. Whatevereffectorthatresultedinthe increase inacetateconcentrations inthe control side reactor also affectedthe acetateconcentrations ontheexperimental side inthe same manner.A similarbutopposite observationcanbe made forrun 7. The effectorwhichcausedadecrease in acetate concentrationsintheA side reactor from ahighof700 mgfL, atthebeginning, to 200 mg/L atthe terminationofthe experiment, also caused asimilardecrease onthe B side reactor’sacetate concentrations. Figures 4. 47 and 4. 48 showthepropionateprofilesforboththe control and experimental sides ofthe TAD pilot scale reactors forall 10 runs.Underthe control condition(ie. 4.5 d SRT andmediumair flowrate) the propionateconcentrations remained quite low (<60 mg/L). Underthetest conditions (B side), the lowflow/3 d SRT combination andtrue anaerobic aerationconditions stimulatedpropionateaccumulationto well above 200 mg/L within thereactor. By the end ofthe experiment, underthetrue fermentative condition, propionate concentrationswithinthe reactorhad reached 577129mg/L, whichwasproportionalto the concentrationofacetate(635 mgfL, Figure 4. 46!,) atthatpoint. The resultsfrom these figures suggest that, asaerationrates decrease, propionateaccumulationincrease. By thetime atrue fermentativeenvironment is established, propionateaccumulationequals thatofacetate. This is consistentwith other fermentative studiesdealingwiththe acidphase ofanaerobic digestionprocesses.Ofthetotal VFA produced,Elefsiniotis(1993) foundthat46.3% was acetate and 31% waspropionate.4.3.3.2 VFAAccumulation inPilotScale TADExperimentsFigures 4. 49 to 4. 52 showthe effectofthetwo independentvariablesonVFAaccumulationin the control (A side) and experimental reactors(B side). The accumulationofVFAwas calculatedusing amassbalance approach,which is expressed bythe followingequation,VFA accumulation = (Mass in effluent± Netchange inmasswithin system)IVolume ofeffluentSincethe SRTchangedfromrunto run, the mass ofVFA accumulated necessitatednormalizationto avolume componentto alleviate the effectofSRT and to facilitate comparison.This was donebydividing themass accumulatedby thevolumeofeffluent. The netchange inmasswithinthe bioreactormust also be addressed due tothe short SRTs used. The equationforcalculating VFA accumulation is as follows,cVe+(Ce—C- i)VrVFAaccumulation=Ve130a)A Side Acetate Accumulation1000800600g 4002000p.r4.5MedMedSRT (d)MedAerationMedb)B Side Acetate Accumulation1000C800600g 400 14-4-Ir0r 60“ £‘LoSRT (d)MedAeration HiFigure 4.49 Acetate accumulation in pilot scale TAD experimentson the a) A and b) B sides.131a)A Side Propionate Accumulation500•— 400300a2002110000o—MedMedSRT (d)MedAeration Medb)B Side Propionate Accumulation500-- 400300wE200100I0 -4.50LoSRT (d)MedAeration HiFigure 4.50 Propionate accumulation in pilot scale TAD experimentson the a) A and b) B sides.132a)A Side Isobutyrate Accumulation602 50wE404-—30>.o. 20.E 10dz—o0-104.5MedMedSRT (d)MedAeration Medb)B Side Isobutyrate Accumulation602 50wE404-—I___—_____..bC30>.o[----------20oE 10o00-1064.50LoSRT (d)MedAeration HiFigure 4.51 lsobutyrate accumulation rates in pilot scale TAD experimentson the a) A and b) B sides.133a)A Side sovalerate Accumulation140—. 90C40-10r4.5MedMedSRT(d)MedAeration Medb)B Side Isovalerate Accumulation140-I0 —904086oLoSRT (d)MedAeration HiFigure 4.52 Isovalerate accumulation in pilot scale TAD experimentson the a) A arid b) B sides.134where Ce = concentrationofVFA in effluent (mg/L)Ve = daily volumeofeffluent (L)(Ce-Cei) = change in concentration ofVFA inreactor (mg/L)Vr= volumeofreactor attimeofwastage (L)n = numberofdays inrun (d)The A side (control) VFAaccumulationshowed some interestingresults(Figures 4. 49a,4. 50a, 4. 51a, 4. 52a). Althoughthe control side variableswere keptconstant,thevariability ofVFAaccumulationbetweenrunswas large. This impliesthatthere are, asyet, unknowneffectors whichcan cause largedeviations intermsofVFAaccumulationfromone runto thenext. These runs also emphasizedthe need for a control condition,in orderto normalizetestresults against.Figures 4. 53 and 4. 54 showthe experimentalreactor (B side) VFA accumulationnormalizedto acontrol value(A side). This was done bysubtractingtheA sideVFAaccumulationvalue from thatofthe experimental(B) side. Thiswouldpotentially alleviatethevariability inresponse dueto uncontrollableeffectors, since the effect shouldbe similarforbothreactors. Whenthistypeofnormalizing was applied, some main and interactiveeffects ofSRTand aeration appeared. Intermsofacetateaccumulation, there was a generaltrendofincreasingaccumulation as SRT decreasedfrom 6 to 3 d (withthe exceptionofthehighflowaerationcondition) and as aerationdecreasedfromhighto lowairflow. Increasingthe amountofsubstrate (ie. decreasing SRT) seemsto have a similar effectto that ofdecreasing the aerationrate. The maximum measured acetateaccumulation rate wasunderthe 4.5 d andtrue anaerobic135a) Difference, B-A, Acetate Accumulation6005000400I300o 200.g100—04-0-100—-Jr0-20064.50LoSRT (d)MedAerationHib)Differences, B-A, Propionate Accumulation500400a,g300°200 I2•5100‘P00-100604.50LoSRT (d)MedAerationHiFigure 4.53 VFA accumulation normalized to their respective control values.a) Acetate and b)propionate.136a) Differences,B-A, Isobutyrate Accumulation6050a,—øE 4030 r1080-1060Lo SRT(d)MedAeration Hib) Differences, B-A, Isovalerate Accumulation8060I--a,40220Cl-4060LoSRT (d)MedAerationHiFigure 4.54 VFA accumulation normalized to their respective control values.a) Isobutyrate and b) isovalerate.137combination.One important observationwhichmust be consideredfromthese graphs dealswithduplication error. Whenbothcontrol and experimentalbioreactorswere operatedunderidenticalconditions (4.Sd SRT, mediumair flow) an interestingresult appeared. Withthe exceptionofpropionate, the experimentalreactorproduced lessVFAthan its corresponding controlreactor(approximately200 mgfL less acetate). Thiswas unexpected, sincebothreactorswererunningunder identical conditions. Presumably theeffectwas due to minordifferences intemperature,aerationor steady state conditionswithinthe bioreactor,butnothingmoredefmitive canbestatedatthistime.Thepropionate accumulationresponse was also differentthanthatofacetate. Insteadofagradual increase inresponse, thepropionate responsewasdramatic. The accumulationvalueofthe majority ofthe experiments were low, until aerationhadreached areducedenoughcondition. Underthese conditions (ie. lowflowand true anaerobic)thepropionate accumulationvalues were anorderofmagnitudehigherwhencompared tothe other combinations. Theseresults suggestedthat afermentativeprofile couldbe achievedandthat abiochemical switchtopropionateproductionwas triggeredwhena sufficiently reduced environmentwas reached.4.3.3.3 Kendall’s Tau-bandAnalysisofVarianceofVFA DataThe appropriate applicationofaparticular statisticalprocedure depends onhowwell asetofassumptions forthatprocedure are met. One importantrequirementofthe datato beanalyzed withanANOVAmodel is thatthe dataare independently distributed. The138independence assumption is oftenviolatedby datathat are collected over aperiodoftime.EachsetofVFA datafor any particular experimental run are composedofdaily measurementstakenover, typically, two SRTs. As canbe seenfrom Figures 4. 45 and 4. 46, some ofthe acetateconcentrationswithinthe reactors neverreached steady state (Runs 6,7 and 9), thus violatingtheassumption ofindependence. The method chosen to rectify thisviolationofindependentdistribution is to remove all datafrom experimental runs whichviolatethis assumptionand toperform aone-wayANOVA onthe remaining data sets.In orderto assess dependence, correlationcoefficientsmust be calculated. Correlationcomputes various measuresofthe strengthofassociationbetweenvariables (ie. daily VFAproductionrates and days). The coefficient ofcorrelationranges from -1 to +1. Thus, the closerthe coefficientis to +1 or -1, the strongerthe linearrelationship betweenthe variables (eg. VFATable 4. 5 MatrixofKendall’stau-b correlationcoefficients forbothacetate andpropionateaccumulationvalues versus time.Kendall’s tau-b correlation coefficients Operating conditionsRun # Propionate B side Acetate B side Aeration SRT (d)Experimental minus Experimental minus(B) side Control (B) side Control(A) side (A) side1 0.778 0.833 0.833 0.556 Low 4.52 0.444 0.389 0.500 0.333 Low 33 0.111 0.056 0.500 -0.111 Low 64 -0.167 0.000 -0.111 -0.222 Medium 4.55 0.222 -0.111 -0.278 -0.389 Medium 36 0.661 0.333 0.667 0.167 High 37 -0.111 0.000 0.389 0.111 High 4.58 0.648 0.222 0.278 0.167 High 69 -0.056 0.056 0.000 0.056 High 4.510 0.167 -0.222 -0.444 -0.667 True 4.5anaerobic139productionandtime). Onthe other hand,as the coefficient approaches 0, the linearrelationshipbetweenthe variables becomesweaker, regardless ofwhether it is positive or negative.Table 4. 5 shows thematrix ofKendall’s tau-b correlationcoefficientsforall 10experimentalruns betweenbothdaily acetate and propionate accumulationvalues versustime.Includedinthistable are the calculated coefficientsforthe daily accumulation valuesofeachrunfromthe B side experimentalreactor and the coefficientsforthe normalized data.Thisnormalization was done by subtracting the control(A side) reactor’s daily accumulationvaluefromthatofthe experimental (B side) reactor. Inthemajority ofruns, this form oftransformation (ie. normalization)resulted inaweaker correlation coefficient (ie. coefficientscloserto zero). All normalized experimental runs whichhada calculatedcoefficientabove0.4wereremoved (run 10 and 1 forthe acetate andrun1 forthepropionate datasets) and a one-wayANOVAwas appliedto theremaining datasets (Table4. 6). Forbothacetate andpropionate,the statistical decisionisto rejectthe null hypothesis, concludingthattherewas a significantdifferenceinthe average daily accumulationvalues ofthesetwo VFA overthe experimentalrunsanalyzed. Whenruns 2 and 7 wereremovedfrom the acetatedata setandruns 2, 3 and 10 wereremovedfromthepropionate dataset and ANOVA wasperformedonthe remainingruns, thestatistical decisionwas to acceptthe null hypothesis,concluding that for acetate (runs 3, 4, 5, 6,8, 9) andpropionate (runs 4, 5, 6, 7, 8, 9) there wasno difference amongthe average dailyaccumulationvalues ofthesetwo VFA. The conclusion,astestedby aone-wayANOVAmodel,wasthatVFA accumulation in runs 2 and 7 for acetateand runs 2, 3 and 10 forpropionate werestatistically differentfrom the remainingruns.140Table 4. 6 Analysisofvariance table for acetate and propionate accumulation.Compound tested Runs deleted from data set FstatisticFcritical1valuecz=O.05Acetate 1,10 5.261 2.1568.83x1O’1,2,7,10 2.218 2.408 0.067Propionate 1 41.188 2.069 6.95x101,2,3,10 1.303 2.408 0.2784.3.3.4 ResponseSurfacePlotsofVFAProduction inPilotScale TADThe equation calculated intheprevious sectionresulted in one accumulationvalue foreachVFAper experiment. Auseful methodto display these datais to plot a ‘responsesurface’for accumulationvalues forindividual VFA, since SRT and aerationratesvaried slightly overthe courseofeach run. Inthis situation, every daily VFA accumulationvalue wouldhave itsownprecise calculated SRT and aerationrate values. The response surface canthenbebasedonmore than 90 insteadofonly 9 discretepoints. Figures 4. 55 and 4. 56 are response surfaceplotsofindividual daily VFAaccumulationvalues versus SRT and aeration. Eachpointrepresentsindividual daily accumulationvalues fromthe B side normalizedto the control condition(Aside). Thisnormalizationwas done by calculating anaccumulationvalue foreach day underthecontrol conditionand subtractingthis fromthe accumulation value ofeach day calculated fortheexperimental condition. Distanceweighted, least squares (DWLS) smoothing was usedto fitthesurface responseto these data. DWLS smoothing fits a surfacethrougha set ofpointsby leastsquares. This methodproduces alocally weightedthree dimensional surface using analgorithmdevelopedby MeLain (1974) and was used in Systat for Windows (Version 5.0). The discrete141a)‘3b)0Figure 4.55 Response surfaceplots ofdaily VFA accumulationnormalized to theirrespective control values for a) acetate andb) propionate.1200440500400‘3‘‘ 300200142a)200l03iO500.3-b)3jFigure 4.55 Response surfaceplots ofdaily VFA accumulation normalizedto theirrespectivecontrol values fora) isobutyrate andb) isovalerate.iSO143daily accumulationvalues forall runs as well as thefitted surface response are includedin eachgraph.It is clear from Figures 4. 55 and 4. 56 that, as SRT andaeration changed, there wasnoconsistentresponse in VFA accumulation. The responseofacetate and isovalerate seemsto bemore sensitiveto SRT effects athigheraerationratesthanpropionate and isobutyrate(ie. Figure4. 55b and 4. 56b). Unlikethe acetateresponseathighair flows, propionate andisobutyrateseemedto be insensitiveto changes in SRT. Thesetwo VFAare, however, sensitiveto SRT atlow airflowrates.4.3.4 TAD Pilot ScaleProcess pHIngeneral, theprocesspHofaTAD system doesnotrequire control. Thethermophilicoperatingtemperatures suppress nitrificationintheprocess (EPA, 1990). Atypical TAD reactorwithafeed sludgepHof6.5 maintains anearneutralpH inthe firstreactor and oftengreaterthanapH of7.2. Figure 4. 57 showspHprofilesofthe experimental side over all 10 runs. Thecontrol (A) sidepHwasrelatively constant with anaverage pHof7.2 (Figure 4. 58). However,the pHofthe experimental (B) side seemedto decreasewithdecreasing aerationrates and SRTs.The average pH ofthe lowflowandtrue anaerobicconditions wastypically much lowerthanmedium orhighflowconditions. Underthe lowflowand 3 d SRT combination, thepH dropped144a)z7.55.5b)=7.55.5c)80B side pH with 3 d SRTB side pH with 4.5 d SRTTime (days)B side pH with 6 d SRTTime (days)Loair,3• Hiair,8Figure 4.57 Experimental reactor (B side) pH values over time for all 10runs (legend key: Symbol; aeration rate; run #).•Loair,2AMed air, 5• Hiair,60 5 10 15Time (days)—D-———0 air, 10AMed air, 4• Hiair,7—0——--Hi air, 95 10 1560 5 10 15145a) A sideAverage pH8Meda) B side average pHLoSRT (d)MedAeration HiAeration MedFigure 4.58 Average pH of a) A side and b) B sidebioreactors146to as lowas 5.5 by the end ofthe experiment(run 2). These results suggestthatthere is apointatwhichavailable oxygenbecomes so limitingthatproductionofacidic endproductsresultsin asubstantial depression ofthe processpH.4.3.5 TotalandVolatile Solids ReductionofthePilot Scale TAD ProcessOne importantpurposeofthermophilic aerobicdigestion, like other sludge digestionprocesses, is the reductionofthe totalmass ofsludge andtheproductionofastabilizedfinalproduct suitable for disposal orreuse. Digestionprocessesreduce sludgemass mainlybyconverting carbonaceous substratesto gaseous endproductsthatescape into the atmosphere.Thepercent solids reductionachievableby digestionvaries, depending onthetype offeedsludge and operating conditions employedinthetreatmentprocess. For example,volatile solidsdestructioncanbe influencedby processresidencetime, operatingtemperature, concentrationoffeed sludge and airflowrates. Thetwo parametersinvestigatedinthis study were SRT andaeration. One ofthemostimportant directlymeasurableparametersinassessing digesterperformance or efficiency is thereductionintotalandvolatile sludgemass over aspecifiedperiodoftime. The processperformanceofthepilot scale TAD digesters was calculatedutilizing amass balance approach. This method canbe expressedby the following equation,Change inmass=massentering (primarysludge) - mass in effluent (TAD sludge)± net change withinsystemThe determinationofdaily solids concentrations in bothinfluentandeffluent streamsallowedthe use ofthis mass balancetechnique.All solids added or removed fromthe system,147during any particularrun, were used to calculate a singlevalue forpercent solidsreductionforthat run. The equation used forthese calculationswas as follows (Knezevic, 1993),CiVi—CeVe-zCrVr% solids destroyed=100LCIVii=1where Ci = concentrationofsolids in influent(mg/L)Vi = daily volume ofinfluent sludge (L)Ce = concentrationofsolids in effluent (mgfL)Ve = daily volumeofeffluent(L)Cr concentrationofsolids inreactor (mgIL)Vr =volumeofreactor(L)n =numberofdaysinrun(d)Figures 4. 59 and 4. 60 summarizethe total andvolatile solids destructionratesinboththe control (A side) andexperimental (B side) reactorsunder all combinationsofSRT andaerationtested. The control side destructionrateswere relatively constantwiththe exceptionofthe considerablyhigher rate forrunnumber 2. The average A side volatile solids destructionwas 20.8%, whichwas similartothat observedby Boulanger (1994) inthe first stage ofaTADoperatingata 3 d SRT underboth anoxygendeprived and oxygenexcess environment. Intheexperimental reactor (B side), the solids destructionseemed to change as aerationand SRTincreased.148a) A side TS destruction30I?o2010___11‘4.5MedMedSRT (d)AerationMedb) A side TVS destruction30CIo20--, _-I10MedMed:RT (d)MedAerationMedFigure 4.59 Total solids destruction capacity in pilot scale TADexperiments: A side (control) a) TS and b) TVS.149a)B side TS destruction°220%Lo3SRT(d)MedAeration Hib)B side TVS destruction300.‘ 60L7Lo/SRT(d)MedAerationFigure 4.60 Total solids destruction capacity in pilot scale TADexperiments: B side (experimental) a) TS and b) TVS.Hi150a)b)Difference plot of TS destruction, B-A% difference5.0-5.-10--l5Difference plot of TVS destruction, B-A% difference50-5-10-156SRT (d)6SRT (d)Figure 4.61 Total and volatile solids destruction capacities relativetotheir control values (ie, B-A): a) TS and b) TVS.AerationHiAerationHi151In orderto properly compare thedatabetween the runs conductedonthe B sideexperimental reactor, the destructionresultswere normalizedto the A sidecontrol values. Thiswas done by subtracting theA side destructionrates fromthe B side destructionrates for eachrun combination. This normalizationresulted in difference plots forbothtotal andvolatile solidsdestructionrates (Figure 4. 61).As can be seen fromthese plots, the 3d SRT under all aerationratesresulted in a lowerpercentdestructionthan its correspondingcontrolcondition. Similarly,underhighflowrates andall SRTs, thepercentdestructionwas considerablylowerthanthecorresponding controls. The only combinationthatresulted in abetter solids destructionthanthecontrol conditionwasthe lowflow/6 d SRT combination.One interesting observation is thatthedestructionrate resulting fromatrue fermentativecondition (0 flow/4.5 d SRT) was similarto the destructionrate resulting fromhighair flows.Mason(1986) observed similarresultsunder oxygenlimited and oxygen sufficient conditions.Laboratory scale bioreactors fedwitha suspensionofyeastcellswereusedinthat study.Aerobic thennophilic bacteriawere obtained fromanoperating municipalthermophilicaerobicsludge digester. Comparisonbetweenthe extentofsolubilization/biodegradationunder oxygensufficient conditionsshowedthat, formostresidencetimes studies, considerably greater solidsremovalwaspossibleunderthe oxygenlimited setofconditions. Therewasno definite trendinthe variationofsolubilization/biodegradationwithresidence time.1524.3.5.1 RoleofEnzymes in SolidsDestructionIn all digestionprocesses, to achieve solids destruction,one group oforganismsutilizeother organisms as substrate (Mason, 1986).In orderforthisprocessto occur, thesubstratebacteriafirsthave to lose viability and subsequentlylyse. In TAD processes, the substrateorganisms are first subjected to atemperature shock andthento the effectsofautolysisorto theactionoflytic enzymesproduced by the thermophilicorganisms. Only after lysis hasoccurred,is itpossible fortheprocess culture to utilizethe lyticproducts as substrate. Ina study ofbacteriaisolated from athermophilic aerobic sludgetreatmentprocess,Sonnleitner andFiechter(1983) characterized athermophilic populationofbacteriaofwhich over 90% were able todegrade starch. Extracellular enzymeproduction, particularlyproteasesandpolysaccharasesarewellknownfeaturesofspecies ofBacillus (Norris etat., 1981).Giventheroleofextracellular enzymeproductioninthe solubilizationandbiodegradationprocess, whichisthe first step inthe destructionofsolids,the factorsregulatingtheirproductionmustbe considered. The resultsfromthis study indicate thatthe highest solidsdestructions occurredwithinarange ofaeration; operationbeloworabovethisrangeresultedina decrease in destructioncapacity. These results suggestthat optimal extracellular enzymeproductionmay occurwithinthisrange ofairflowrates. This beliefis supportedbytheobservationthatamylaseproductionis inducedby oxygenlimited conditionsin a shake flaskculture ofthermophiuic Bacilli derived from a TAD process (Grueninger etat., 1984).1534.3.5.2 SolidsDestruction VariabilityDiscussionThere aretwo major sources ofvariability whichmust be considered whenthawingconclusions fromthe solids data. The firstsource ofvariability was inthe changingsludge feedsource characteristics and composition.TheA side bioreactorsolids destruction(Figures 4. 59aand 4. 60a) varied considerably from a lowof17 to ahighof29%, eventhoughthe independentvariableswere maintained attheirrespectivemeans (eg. 4.5 d SRT andmedium airflow).Althoughtherewas alarge variabilityinthe A sidebioreactor fromone runtothe next, thevariability from onereactorto the other(A and B sides) was much smaller. Whenbothreactorswere operatedunderthe same conditionsofSRT and aeration, theA side bioreactor’stotal solidsdestructionwas21%, comparedto arateof17.4% inthe B sidereactor.The secondmajor source ofvariability only becomesevidentwhenone oftheexperimentalruns wasreplicated.Runs 7 and 9 are replicatesofthe highair flowrate and4.5dSRT combination. Although solids destructioninboththeAand B side bioreactorswere lowerinrun 7 when compared to run 9 (eg. 3-5% lower), the differencebetweenthe reactorsforeitherruns 7 or 9 remained fairly constant.This suggeststhat althoughthe measured values aredifferentbetweenruns, the overall effect is similar.These results indicate thatcomparisonofdatasetsbetweenruns are inappropriateunderthesepilot scale experiments. In ordertodecipherthe effectofSRT and aerationon solids destructioncapacity, the comparisonmustbemadewithineach individual run.1544.3.6 Salmon Arm ATADIn 1985, the DistrictofSalmonArm chose fortheirplantexpansionabiologicalphosphorus removal facility and ahightemperatureaerobic digestionprocess. Aspartofthesolids train, the original aerobic digesterwasretrofitted for autothermalthermophilicaerobicdigestion. This was done by sectioningthe existingrectangulardigesterinto 3 unequally sizedcompartments, whichoperate in series and are fedamixtureofprimary and waste activatedsludges semicontinuouslythroughoutthe dayby sludgepumps operated ontimers. The aerationdevice chosenwas aBritish Columbiadeveloped aerator/mixer(Turborator).The schematic ofthe SalmonArmATAD process is shownin Figure 4. 62. Since thisprocessutilizedthe sameaerationdevice as the UBC pilot scale TAD process, valuable full scaleVFA informationcouldbe obtained.ObtainingVFA datafromanoperating full scale ATAD process wasthoughtnecessaryto augmentthe existingdatafromthepilot scale process. Samplesweretaken overtwo visitstothe sewage treatmentplant(ie. once in March, 1993 and asecondvisit inMay, 1994). Figure4.63 showsthe daily operating temperatureprofiles ofallthree cells during eachofthe visits. Therange oftemperatures, ofthe first and second cells, were invariablymuchgreaterthanthetemperature range ofthethird cell, duringbothperiods. Figure 4. 64 showsthe VFAprofile ofthe SalmonArmATAD process acrossthe 3 cells.During the 1994 visit, the propionate concentrationinthe first cell was almostashighasthat ofacetate. This somewhatunexpected resultwas probably due to the uncharacteristically155LEGENDMRDENSfl PROBESCREENED RW SEWMEWASTE BIOLOGICAL SOLIDSPUIAPFOUL AIRCRUDE SLUDGELOW PRESSURE MRFOAM BREM(ERTURBORATORDIGESTED SLUDGESEflED SEWETiMERTEMPERATURE PROBEO.R.P. PROBEDISTRICT OF SALMONARM — PROCESSSCHEMATICFigure4. 62District ofSalmon Arm ATAD process schematic.156a)Salmon Arm ATAD Temperatures80201I—0I •‘II IICC) c’CY)O—)O) C’J—)O) OO) C’O)Date (dd-mm-yy)1 St cell - 2 nd 3rd cell ambientcell sludgetemp.b)Salmon Arm ATAD Temperaturesi20 -‘ 100I‘--tOO) C%JO)<OC)<O)O-)O)Date (dd-mm-yy)1 St cell-- 2 rid 3 rd cellambientcellsludgetemp.Figure 4.63 District of Salmon Arm ATAD reactortemperatures duringthe a) March, 1993 visit and b) May, 1 994 visit.15720001800160021400- 12001000800>6004002000Salmon Arm ATAD VFA Profile140012001000800600>4002000•Acetate-— Propionate• Isobutyrate-— Butyrate2-Methylbutyrate-— Isovalerate• Valerate1 stcell2ndcellATAD cell3rdcellSalmon Arm ATAD VFA Profile1 Stcell•Acetate- Propionate• Isobutyrate-0-—Butyrate- 2-Methylbutyrate-— Isovalerate• Valerate2ndcellATAD cell3rdcellFigure 4.64 Salmon Arm ATAD VFA profile in all three cells during thea) March, 1993 visit and b) May, 1994 visit.158high solids concentration in the first cell of9.4 %. During thistimeperiod, the sewage treatmentplant operators were attempting to decrease the sludge blanket intheirprimary clarifiersbyincreasing the pumping rate ofprimary sludge into the firstcell.This effectively increasedthesolid content inthe first cell beyondthe capacity ofthe Turboratoraerator/mixer to maintainadequately mixed conditions. Visual inspection ofthe first cell confirmedthatthe contents, eventhoughthe Turboratorwas operatingnormally, were static. The inabilityofthe Turboratortocompletelymix sludge atthese concentrationswouldresult inthe majority ofthe bioreactorbecoming anaerobic. Consequently, this fermentative environmentwouldresultintheWAprofile seen. Another indicationthat anaerobic conditions existed inthe first cellwasthe lowerpH ofthis cell, whencomparedto the others. ApH of5.62 would be consistentwiththeaccumulation oflarge amounts offermentativeacidic endproducts. TheWAprofiles inthesecond andthird cells weremore inline withtypicalWAprofilesofTAD processes, sincetheconcentrationofthe sludgehaddecreased sufficientlythatthe aerator could maintaincompletelymixedconditions. Theseprofiles showthat acetate wasthepredominantVFAproduced inall 3stages ofthe SalmonArmATAD facility andthathigherVFA concentrationsunder full scaleoperation(up to 2,000 mg/L) couldbe achievedwhencompared to thepilot scale experiments.1595. Overviewand Summary5.1 BiochemicalModelofSubstrateMetabolism inTADTo understandthe biochemistry involved in substratemetabolismin TAD, one firstmustunderstand some basic fundamentalsofbacterial energetics. An exampleofE. coli utilizingglucose is considered. This organism isafacultative anaerobe and as such cangrowbothaerobically,and anaerobically by utilizing sugars, suchas glucose, as a sole carbonandenergysource (Gottschalk, 1986). The first stepin glucose energy metabolism, irrespectiveofthepresence or absence ofoxygen, is thetransport into thecell and its catabolism into pyruvate. Itis inthe furthermetabolismofpyruvatethat differencesbetween aerobic and anaerobicconditions occur.Inan aerobic environment, NADH generated during glycolysis, theTCA cycle and otherassociatedreactions, is reoxidized bythe operationoftherespiratory chain(IngeldewandPoole,1984)(Figure 5. la). Inthe absence ofoxygen, the respiratorychain is nonfunctional. TheTCAcycle andpyruvate dehydrogenasereactionsthat generatelarge amounts ofNADH areinoperative. However, NADHproducedby glycolysismustbe reoxidizedtoNADso that theglycolytic sequence canproceed (Figure 5. lb). Therefore, thekey issue in fermentation istherecycling ofNADH by conversionofpyruvate to differentfermentationproducts which cangenerate the oxidized form ofNADH. Since theratio ofNADH toNADvarieswiththe natureofthe substrate, so mustthe composition ofthemixture offermentative endproducts. Thus, toachieve aproper fermentationbalance, it is necessaryto matchtheNADH produced with160Figure 5.1 Biochemical modelof acetate prouclion in TAD undera) microaerobicandb) anaerobicconditionsa) mlcroaeroblcElectronTransportChainACETATEb) anaerobicElectronTransportChain(inactIve)Other fermentative end-producleg lactate, ethanol,WAs161a) mlcroaeroblc conditionsIsobutyrate Fropionate6HGlucose,Dextrin2H42H4(+2HNADHi-H4 NAD’ElectronTransportChain‘/2O+2H H20b) anaerobic conditionsPyruvatePFLAcetyl-C0ARgure 5.2 Summary of carbon flow from substrateaddition experiments undera) microaeroblc andb) anaerobic conditionsLactateFroplonyl-CoAAcetyl-C0A2-Methyl Butyrate,Valerate4HPropionyl-C0A+ Acetyl-C0A2H1/2Be lsovarateAETME3 LactateHHZ2Froplonyl!C0AAcetyl-C0APyruvateFROPIONATEACETATE162theNADH consumedby excretionofspecificproducts. By varying the proportionsoftheseproducts, it is possibleto matchtheNADHproduced from the oxidationofvarioussubstrates,thus achieving redox balance.Inthe TAD batch studies, under fermentative conditions,the process bacteriamustsimilarly achieve redox balance by divertingthe catabolicflowofcarbonto fermentative endproductsthatwill consumeNADH(Clarke, 1989). Figure 5. 2b shows the fluxofcarbon as wellas the redoxbalance formost ofthe substrates tested.The oxidationofsubstrates that resultinthenetaccumulationofNADcannotproceedunderfermentative conditions. Consequently, thesubstratesadded remained intheirunoxidizedform andpersisted inthemedium (eg. 2-methylbutyrate, propionate, valerate,butyrate isovalerte, isobutyrate). The oxidationofsubstrateswhich canmaintainredox balance canand did proceedunderanaerobic conditions.Usingthe acrylatepathway (Gottschalk, 1986), lactate canbe convertedto propionate andacetateby the following equation,3 lactate —* 2 propionate + acetate+CO2+H20Thehydrogenevolvedduring oxidationoflactateto propionate was consumedbytheproductionofacetate from lactate, thusrecyclingthe reducedNADH to regenerate the oxidizedformin order to maintainthe netredox balance. The oxidationofglucose and dextrin couldoperateviaa similarprincipalto lactate oxidation. NADHproduced during glycolysiscould bereoxidized by coupling this oxidationreactionto the reductive conversionofpyruvatetopropionate.163In the TAD batch studies, undermicroaerobicconditions, theNADHproducedduringoxidation ofsubstrates could be reoxidizedby operationofthe respiratory chain (Figure5. 2a).Therefore, the flowofcarbonmay be uncoupledfromthe necessityto maintainredox balanceviafermentative means. This uncouplingwouldpresumably allowthe organismsin a TADprocessto achieve the global objectiveofmaximizingATP generationby increasingthe fluxofsubstrates to acetate (Majewski andDomach, 1990).Figure 5. 2aillustratesthenetflowofcarbonfrom mostofthe substrates examined.This figure showsthatthe majority oftheoxidized substrates evolvedhydrogen, inthe form ofreduced electroncarrier, and acetate.These reduced electroncarriers canthenbereoxidizedby operationofthe electrontransportchain, allowingthe catabolismofthe substrates addedtoproceed in an oxidative direction.Since theterminal electron acceptor, oxygen, waslimitedin amicroaerobicenvironment, therewouldbe alimited rate oftransportofelectrons downtherespiratory chain, whichwould,inturn, limitthe rate ofoxidationofNADHtoNAD. This limitationwouldresult intheaccumulationofNADH.Themodel suggeststhatthe organismscanmeetpartoftheNADHrequired foroxidativephosphorylationby operationofsubstrate level redoxreactionsandtheremainderby operationofthe TCA cycle to generateNADH. Inresponseto this condition, thebacteriain aTADprocess couldpreferentially shuttle acetyl-CoAthrough an acetyl-phosphate intermediatetoacetate, transferringthe highenergy phosphate bondto ADP generatingATP. Thesereactionsgenerate energy withoutthe reduction ofNAD as wouldbe the situationifacetyl-C0A is feddirectly into the TCA cycle. Thus, by employing substrate level redoxreactionsand a limited1640.1Oxygen Demand(mol/I-h)0.1Rgure 5.3 Comparison between oxygendemand and oxygen supplyof anyaerobically metabolizing cultureOxygen Supply Capacity(mol/l-h)165flowofcarbonthroughthe TCA cycle,bacteriain a TAD process couldmatchtheNADHproducedto meetthe limited capacity oftherespiratory chainto transportelectrons, while atthesametime, maximizing ATP productionby channeling excess acetyl-CoAto acetate. Byincorporatingthis strategy these organisms canmaximizeATP production inthe02 limitingenvironmentofaTAD process.5.2AcetateAccumulationPhenomenonin TADMicrobial cultures are often oxygenlimited. Oxygenlimitations can occurbecauseoflimits inthe capacityofthe respiratory systemsoftheprocessmicrobes or because oftheoxygenation limits ofthe external aeration devices.Whenoxygenbecomes limiting, thesubstrate is onlypartially oxidized, whichleadsto byproduct evolution. This secretionofmetabolic byproducts is coupledto the generationofenergywhilemaintaining cellularredoxbalance.Figure 5. 3 graphically showsthebalance betweenoxygendemandand oxygen supply ofan aerobically metabolizing biological system. Ifthe aerationequipmentcantransfermore02into solutionthanthebiomass can utilize, as is the case fortypical activatedsludge systemswhichoperate in adissolved oxygen concentrationrange of1-3 mgfL, aerobic conditionswillprevail. However, ifthe oxygendemand ofthe systemis higherthanthe aeration capacitytomeetthis requirement, microaerobic conditionswill prevail (Figure 5. 3), whichwill beaccompaniedby byproduct secretion.166Mason (1986) investigated some effects ofdissolvedoxygenconcentrationson VFAproduction in a laboratory scale bioreactor operatedin acontinuous flowmode. Aerobicthermophilic organismswere obtainedfrom an operating waste sludge aerobic thermophilicdigester. Bakersyeastwas used asthe sole biodegradableparticulate carbon source. Nocarboxylic acidwas detectedunder oxygen sufficientconditions. The amountofeachVFAproducedunderoxygen limiting conditionsvariedwithresidence time, although, clearly,acetatewasproduced in thehighest concentrations; at2500mg/L, it was 5 to 10 timeshigherthanthenexthighestVFAmeasured (eg. propionate). Inabatchstudy usingthe sameprocess microbesand feedyeastcells, considerable amounts ofVFAwereproduced and subsequentlyutilized(Hamer, 1987). These results showed an accumulationofup to 6000 mgfL acetate and 800 mg/Lpropionate (Figure 2. 3). Boulanger (1994) andChueta!. (1994) also reported similareffects.Theproductionofhighconcentrations ofVFA was evident onlywhenthe 02 demandwashigherthanthe ability ofthe aerationequipmentto meetthis demand, resultingin amicroaerobicenvironment. Similarly, whenKelly (1990) conducted experiments onVFAproduction in anautothermalthermophilic aerobic digester at SalmonArm, B. C., Canada, the firststage digesterproduced upto 10,000 mg/L (personal communication).VFAproductionhas also been demonstrated in anumber ofaerobic thermophilicpretreatmentprocesses. Thesepretreatment systems also providethermophilic digestionand arenormally incorporatedin the process aheadofconventional anaerobicdigestion. BaierandZwiefelhofer (1991) reportedVFA concentrations as highas 6081 mgfL after aerobicthermophilicpretreatment; 3315 mg/L was acetate. Zwiefelhofer (1985) reportedmuchlower167VFAconcentrations from afouryear full scale demonstrationproject in Switzerland. AverageVFA concentrations inthe effluent oftheAEROTHERMplantwere 658 mg/L acetate,75 mgfLpropionate and 26 mg/L butyrate. As can be seen fromthemajority ofthese studies,acetatewascited as the mostpredominantVFAproduced inATAD. The results fromthisthesisare similarto those from the references thatimplicate acetateas thepredominantVFAproduced.5.3‘4C-AcëtateLabelExperimentHäner etal. (1994) examinedthe nature andfate ofthe organicfractionreleased duringaerobic thermophilicbiodegradation ofmicrobialcells inalaboratory scale treatmentsystem.Theprocess culture was obtained from a full scale, aerobicthermophilic, sludgepretreatmentplant in Switzerland. To simulate feed sludge, acultureofKlebsiellapneumoniae wasusedasthe feed stock. Inorderto assess the dynamic patternoftheproductionandutilizationofbiodegradable intermediates, cultureswere spikedwith14C-radiolabelledacetate. By 50 hintothe experiment, the relativeradioactivity associatedwiththe acetatefractionwas approachingzero, whiletheVFA concentrationwas approximately500 mg/L. Sincebacteriacannotdistinguishbetweenlabeled andunlabelled acetate, theauthors suggestedthatthe productionofacetatemusthave occurredconcomitantwith its utilization, whichwas a departure frompreviousmodels ofacetatemetabolismthat suggested oxidative metabolismwas somehowinhibited intheATAD process (Hamer, 1987). This mostrecentmodel containedthe firstpieceofdirectevidence thatrecognizes acetateproductioncan beconcomitantwith its oxidation, whichis animportant assertionmade inthisthesis. Althoughamajor fractionofthe addedradiolabel was168converted to carbon dioxide, some was alsoincorporated into biomass and a significantfractionwas either released orexcreted into theculture supematantin the formofunidentified,radiolabelled, dissolved organic carbon.5.4 AcetateProduction inMicroorganismsAerobicproductionofacetic acidinEscherichiacolihasbeenreportedby anumberofinvestigators. (Browneta!., 1985;Curless eta!., 1989; Ishikawaand Shado, 1983;Pan eta!.,1987; Meyeretal., 1984). The productionofacetatebyE. colitypically occurs atrapid growthrates and commences afteracritical growthrate has beenexceeded. Acetateproductionisasymptomofachange incellularphysiological state.The accumulationofacetate arises fromanoverflowphenomenonwhere acetyl-CoA is divertedfromtheTCA cycleto firstacetylphosphateandthento acetate,whichresults intheproductionofone substrate levelATPpermolecule ofacetate.Haneta!. (1991) studied acetic acid formation inEcoli fermentation ina continuousculture system. Theybelievedthat the limitedTCA cycle was responsible forthe limited energymetabolismwhichresulted inacetic acid formation.Reicheltand Doelle (1971) looked attheinfluence ofdissolvedoxygenconcentrations,onthe enzymephosphofructokinase, inE. coliK12 (ie. Pasteureffect). Acetic acid productiondid notoccur above an oxygenpartialpressureof25.6 nunHg, whichwas referred toas an excess oxygenstate. Belowthispartial pressure,169acetateproduction increasedprogressively with anadditional sharp increase belowapartialpressure of5.9 mm Hg.MacDonaldandNeway (1990) lookedattheeffectsofmediaquality ontheexpression ofhumanInterleukin-2 (IL-2) athighcell densitiesoffermentercultures ofE. coilK12. Thedissolved oxygenconcentrationsweremaintainedat40% ofair saturationand celldensities at an0D680of60 (ie. 2.5% solids) throughoutthe experiments.InductionofIL-2 accumulationathighculture optical densitieswas also accompaniedby increased acetate accumulationintheculture mediumofup to 300 mM (ie. 18 gIL). Theirresults suggestedthatthe accumulationofdiffusable inhibitors, such as acetate, during inductionmay be asignificantfactor limitingIL2expression in high density cultures.Recently, stoichiometrically based methodshavebeendevelopedforthe study ofoptimalmetabolic behavior (Fell and Small, 1986; Savinell andPalsson, 1992). In an attemptto developarationale foracetate secretion, Majewski andDomach(1990) examinedacetateoverflowmetabolism in E. coli fromthe capacitated flownetworkviewpoint. Thisviewpointfocuses ontherouting ofmetabolicflows (ie. fluxes) throughaconnectednetworkwhere loadsexist.Capacitationrefersto the existenceofconstraints suchthateach reactionprocesswithinthenetworkhas afinite capacity. Inotherwords, the total flowthroughthe networkhas anupperlimitdefined bythe limiting reactionprocess.Theterm load suggeststhat some metabolites aredrainedfromthe cycle atarate dependent on growthrate. Itwas foundthat, whenfluxconstraints are imposed at either the level ofNADHturnoveror the activity ofakey TCA cycleenzyme (eg. x-ketoglutyrate dehydrogenase), switchingto acetate overflowwaspredicted. In170their analysis, maximizationofATP and GTP productionwas considered to be a globalobjectiveofE. coilmetabolism.In a study by Varmaetal. (1993), a flux balanceapproachwas used to determineoptimalmetabolicperformance ofE. coli undervariableoxygenlimitations. Thismethoduses linearoptimizationto find optimal metabolic fluxpatternswith respectto cell growth. Fromcomputations, increasedoxygenlimitations werefound to resultinthe secretionofacetate,formate and ethanol. The computedoptimalgrowthunderincreasing02 limitationrevealed4critical growthrates at whichchanges inthe byproductsecretionpatterns were concomitantwithchanges inmetabolic pathwayutilization. The redoxpotential was identified as alikelytriggerthat ledto shifts in metabolicflows. Doelle eta!. (1982)suggested thatthe accumulationofNADH switches carbonflowtowards acetic acid. Theirmodel suggestedthatahighglucosefeed concentration represses enzymes inthe TCAcycle and electrontransport system. Thisconditionresulted inahighaccumulated intracellularNADH concentration, whichultimatelyswitched carbonflowinthe directionofacetateproduction.Thismodel is consistentwiththebiochemical model ofsubstrate metabolismin TAD. Underbatchtestconditions, inthemicroaerobic environment, the limited flowthroughthe electrontransportsystem, wouldresultinahigh concentrationofaccumulated intracellularNADH. Theresultsfromthe batchexperiments showthatunderthis condition, carbonflowwas predominantlyintheproductionofacetate.Harrison andPirt (1967) investigatedthe influence ofdifferentdissolvedoxygenconcentrations onthe metabolismandrespirationofKiebsiellaaerogenes by means ofa171continuous flowculture technique.Different dissolved oxygentensionswere obtained byvarying the partial pressureofoxygen inthe gas phase. Inthe excessoxygen state, acetic acidwas formed. Thetransition fromexcessoxygen to limiting oxygenconcentrationswasconsistentlymarkedby alarge increase involatileacidproduction. Withinthe limitedoxygenstate, which encompassesarange ofoxygen tensions,thehighestpercentageofvolatile acids asacetic acid occurred athigher oxygensupplies. With decreasing oxygensupply, formic acidproductionincreaseduntil it equaledthe concentrationofacetic acid. Thus, to maximizeaceticacid productionand to minimizeother fermentativeendproducts, there existedanoptimalaerationrate.Britten (1954) observed thatacetate accumulatedinthemediumofwell aerated culturesofE. coil growingonglucose. Hadjipetrowetal. (1964) observed similarresultsforAerobacteraerogenes. The lattergroup showedthat, following glucose exhaustion, oxidationoftheaccumulated acetate occurred. Coultate and Sundaram(1975) werethefirstto confirmsimilarobservationswithatherniophilic system (Bacillusstearothermophilus). They observedthat agreaterproportionofglucose carbonwas leftas acetate, at growthtemperaturesafewdegreeshigherthanthe optimal growth temperature.Their suggested reasoningwasthatthe oxidative(eg. Tricarboxylicacid [TCA] cycle andthe electrontransport chain) and non-oxidative (eg.glycolysis) phasesofglucosemetabolismare less coordinated atthesetemperatures.It is possiblethatthe accumulationofacetatein aTAD process is at leastpartially due toan inefficientcoordinationor uncouplingbetweentheoxidative andnon-oxidative biochemicalsystems (ie. acetate overflowphenomenon), ratherthansimply due to pure fermentation. The172anomalies in the TAD results, that donot correlateto a fermentative model, maybe explainedwithin the contextofthe biochemicalmodelpresented inthis thesis. Thisalternative processwould account forthe relatively high levelsofacetate in the VFA samples. Ingeneral,thefermentationofsludge generates an acetateto total VFAratio of40% (Rabinowitzand Oldham,1985; Fongsatitkul, 1991). InbothTADpublications by Hamer (1987) andMasonetal.(1987),the acetateconstituted 80-90% ofthetotal VFA. This raisesthe possibilitythat, inadditiontofermentation, there are otherprocessesthatcould contributeto the productionofVFA (eg.acetate overflowphenomenon). This effectcould also accountforthe sequentialutilizationofVFA and isconsistentwithknownbiochemicalproperties ofpure cultures ofbacteria.Acetyl-CoA is converted to acetateviaan acetylphosphate intermediate. The successivereactions are catalyzedby 2 enzymes(Rose etal., 1954; Fox and Rosemann,1986). Thispathwayproduces acetic acidandgeneratesanATP forevery acetyl-CoA. Althoughonlyasingle ATP is obtained, this level is significantwhen comparedwithanet gainof2 ATP/glucoserealizedfromthe glycolytic pathway. Whenaeratedculturesof E. coil growonglucose,themajority ofthe acetyl-CoA is converted to acetateviathispathway, and only aminority ismetabolized viathe TCA cycleto giveNADH (nicotinamideadenine dinucleotide) andCO2.Thus, acetate accumulatesinthemedium in largeamounts, even whenoxygenis present,although aerobic cells growing onglucoseproduce negligibleamountsofother fermentationendproducts. Whenthe glucose supplyis exhausted, there is abrieflag period, afterwhichtheaccumulated acetate is takenback into thecells andrespired(Kornberg, 1966; Maloy andNunn,1982). These observations are consistentwiththe results obtained fromthe batch experiments173conducted with TAD sludge. Specifically, that acetateaccumulation was atransientphenomenon, and could be subsequently oxidizedwhenthe more complex substrateswereexhausted.Jenson and Michelson(1992) investigated carbonand energy metabolismofatp mutantsofEscherichia coli. The membrane boundH+ATPaseplays akey role in free energytransductionofbiological systems. Theauthorsreporthowthe carbon and energymetabolismchanges in response to deletionofthe ATP operonthatencodesthis enzyme. Comparedwiththewildtype strain, the atp mutant strainproducedtwiceasmuchacetate as aby-product andexhibited increased flowthroughthe glycolyticpathway and TCA cycle. This leadsto anincrease in substrate levelphosphorylation.They suggestedthatthe atp mutants use uncoupledrespirationinorderto profit from increasedsubstrate levelphosphorylation.It is possible thatthemixedconsortiumoforganisms inthe TADprocessbehavesinasimilar mannerto atp mutants.These TAD organisms may support a similarprocess, to profitfrom increasedsubstrate levelphosphorylation.Thiswouldundoubtedly be advantageous(intermsofacompetitive edge) underthe oxygen limitingenvironmentofmany TADprocesses.The resultsofthisthesis suggestthatthere maybe anumber ofprocessesinvolved intheproductionofVFA. A degreeoffermentationis probably occurringdue to the highly reducednature ofpart ofthe TAD process (eg.ORP values ofbelow -250 mV). In addition, there isprobably simultaneousaerobic oxidationofVFA, which is mostlikely limitedbythe aerationrate, even underthe mostreducedofconditions.1746. Conclusions and Recommendations6.1 ConclusionsDuring the courseofthisresearch, adetailedexaminationofVFAmetabolismin TADwasundertaken atbothbenchandpilotscale. Abiochemicalmodel was developedinan attemptto explainthisphenomenon. Based onresults ofthe researchprogram,the followingconclusions can be drawn:1. Apilot scale TAD processwas studiedunder arange ofaerationratesand SRTs. VFAaccumulation was found to be afunctionofboth SRT and aeration. In general, asSRT and airflowrates decreased, VFAaccumulationincreased(specificallyacetate andpropionate). Themaximum accumulationofacetateoccurredunderthe 4.Sd/true anaerobic combination.2. Undermicroaerobic conditions,themajorbyproductofTAD was acetate,whichaccountedforbetween70 to 80% ofthetotalVFAproduced. This characteristicVFApatternis differentthanthe typical profiles offermentationprocesses.3. Under strictanaerobic conditions, theVFAprofiles in thepilotscale TAD processweresimilarto fermentationtypeprocesses (ie.aneven distributionofVFA between acetate andpropionate). This anaerobic condition alsostimulatedthehighestproductionofpropionate.4. ORP profileswere ableto delineatebetween gross changes in aeration (eg. betweenmicroaerobic, transition andaerobicconditions).5. Fourieranalysisperformed onthe ORPdata, ofthe pilot scale TAD process, indicated acyclic nature withaperiod of52.6 minutes.The approximate once/hour feeding regime createda ‘shark tooth’ patternresponse inthe ORP profile. Althoughinsensitive to changes inaeration175rates between runs, the ORP signals wereextremely sensitive to theadditionofsubstrates(ie.primary sludge) within each run. These resultssuggestthat absoluteORP values arenotasuitably stringent state variable formonitoringand comparingreactor conditionsbetween runs,undermicroaerobic conditions,althoughrelative ORP values arequite sensitiveat detectingslight changes inreactor conditionswithin anarrowtime window.6. Inthe TAD batch studies, theoxidationofsubstratesthat requirethe netreductionofNADcannotproceed underanaerobicconditions. Consequently, the substratesadded, underbatchtestconditions, remained intheirunoxidizedformandpersistedinthemedium (eg.2-methylbutyrate, propionate, valerate,butyrate, isovalerate, isobutyrate).Thepathwaysforoxidation ofsubstratesto endproducts,which canmaintainredox balance,under anaerobicconditions, canand doesproceed(eg. lactate, pyruvate, glucose, dextrin).7. Inthe TAD batchstudies, undermicroaerobicconditions, themajority ofthe substratesexaminedwere oxidizedtoan acetate intermediate. Theseresults suggestthattheNADHproduced during oxidationofsubstrates canbereoxidized by operationofthe respiratorychain.Therefore, the flowofcarbonmay be uncoupled from the necessityofmaintainingredoxbalance viafermentativemeans.8. Intermsoffermenter sludgethe highest productionrate oftotal VFAoccurred underanaerobic batchtest conditions,atambientroomtemperatures, whenno primary sludgefeedwasadded. However,by slightly aerating fermenterbiomass it waspossible to improve acetateproductionunderhigh substrateadditionrates (ie. primary sludge),whencomparedto strictfermentative conditions.1766.2Recommendations1) Traditionalmicrobiological methodologyhas focused onmonospecies culturesgrowing ondefinedmediaunder stringently controlledconditions. Bull and Quale (1982)have argued thatsuchapproaches have resulted inarestrictedperspective ofmicrobial behaviorin complexmixed culture environments. TAD, whichis aprocess comprisedofmixedcultures, is obviouslymore complex in many respects,thanare monoculture systems. Itis forthisreasonthatthegenerationofdefmitive evidence forthe existenceandoperationofspecificbiochemicalpathways is anunrealistic goal whenworkingwith such acomplex system. Alternatively,monospecies ofrepresentative isolatesfrom TAD processes shouldbe studiedunderstringentlycontrolled conditions, inorderto investigatethe biochemistry involvedinthephenomenonofacetate overflow.2) Throughoutthe batchexperiments,there wasno directevidencethattheadded substrateswere metabolized to anacetate intermediate.Inorderto prove the carbonthat originatedfromthe added substrate accumulatesinthe acetatefraction, radiolabelled‘4Cexperiments shouldbeconducted. Inadditionto therandom labelingofsubstrates, radiolabel ofspecific carbonswithina substrate shouldbe done. These experimentsshould give insight into thebiochemicalpathwayspossibly involved inthe conversionofsubstrate to acetate. For example, the labelingofthe first, second orthird carbonsinpropionate andthe distributionpatternoflabel inthebyproducts (eg. acetate,C02)should elucidatethe specific pathwaysutilizedby the organisms inthe catabolism ofpropionate.1773) From an operational perspective, enhancinganaerobic digestionby phase separation isaccomplished byproviding more optimalenvironmentsfor eachmajorgroup ofbacteriaandtheirassociatedbiochemical reactions.Thetwo steps commonly citedas rate limiting intheprocess ofanaerobic digestionare:1) the hydrolysisofcomplex substratesand2)methanogenesis (Kaspar and Wuhrman,1978). Inthe secondphaseoftwo phase digestion,VFA andhydrogenare convertedto methaneand carbondioxideby two coupledreactionsmediated by acetogenic andmethanogenicbacteria. Acetogenicbacteriaconverttheproductsoffermentation into acetate, formate andhydrogen,which act as substratesformethanogenicconversionto methane and carbondioxide. The syntrophic couplingofmany acetogenicreactionsto methanogenic reactionsis oftencritical, sincethe conversionofVFA to acetate andhydrogenis onlythermodynamically favorableinthepresence ofmethanogens; theseorganismsthenutilize these products as substrateand canthereforemaintainthem atlowconcentrations.Consequently, the second phase reactormustprovide abalanceofoptimal environments forbothgroupsoforganisms.Themethane derivedfrommethanogenesis comes from two main sources.Approximately 70% isproducedfromacetate andthe remaining 30% fromCO2andH2(MetcalfandEddy, 1991). Since methanogenicorganisms can only utilize a very narrowrangeofsubstrates, aprocessthatcouldpotentiallypretreatthe sludge andprovide a substrate (eg.acetate) amenable formethanogenesiswouldbe invaluable. Such aprocess isreferredto, intheliterature, as athermophilicprestageor dual digestion system (EPA, 1991). Zwiefelhofer (1985)claimsthatpretreatmentofsludge, usingthermophilic aerobic digestionpriorto mesophilic178anaerobic digestion, throughsolubilizationofparticulateorganic matter, allows short sludgeages in the anaerobic stage (10 d); insoluble, complexcompounds are thus transformed intosoluble byproducts ofbacterial metabolism (eg. VFA)orreadily degradable intermediateproductsofless complex structure, that serve assubstratesthat are more amenabletoanaerobicdigestionthanuntreatedraw sludge.According to the biochemical modelproposed foracetate metabolism in TAD, thepredominantbyproductis acetate. The acetate richeffluentfromathermophilic prestageprocesshasthepotential ofuncouplingthe syntrophicrelationshipthat existsbetweenmethanogenicandacetogenic organisms by providing methanogenswithareadilyutilizable substrate, thusrelievingthe necessityto optimizethe environmentalconditionswithinthe reactor forbothofthese groups oforganisms. The feasibility ofathermophilicprestageprocessto provide acetate,inorderto uncouple the syntrophicrelationship betweenacetogenic andmethanogenicreactionsinthe secondphase ofanaerobicdigestion, requiresmore study. Specifically,ifthis syntrophicrelationcan be uncoupled, canmethanogenicreactionsbe further optimized?4) The effectof2,4-dinitrophenol onVFAmetabolisminTAD fedwithprimary sludgehasbeenexamined. These experiments have shownsome interestingresults. Inadditionto primarysludge, other substrates shouldbetested intermsof2,4-dinitrophenol effects onmetabolism (eg.propionate, butyrate, glucose, etc.).5) Thebiochemical model ofacetateproduction, undermicroaerobic conditions, inTAD sludgeis notrestrictedto thermophilictemperatures or organisms.Themajority ofevidencethatdealswiththis acetate ‘overflow’ anomaly comes fromresearchdone onmesophilic organisms. The179batch experiments conducted, aspartofthis thesis, withpilot scale fermenter sludgeatmesophilictemperatures, under both anaerobic andmicroaerobic conditions, indicate that underhighprimary sludge additionrates, VFAproduction rateswere improvedunder the lattercondition. It would be avaluablefollowup tothisthesis to study the effectofslightly aeratingpilot scale fermenter sludge onVFAproductionratesand composition, andtheir subsequenteffects onthe biologicalphosphorusremovalprocess.Thiscould be done attheUBC Bio-Ppilotplantfacility, whichcontainsbothcontrol (traditionalfermentation) andexperimental(aeratedfermentation) sides.1807. ReferencesAndrewsJ.F. andKambhuK. (1973) Thermophilicaerobic digestionoforganic solid wastes.EPAI67O-2-73-061. August. NTIS PB 73-222396.A.P.H.A. (1989) StandardMethodsforthe Examination ofwaterand Wastewater. 17 th.Edition. American Public Health Association, Washington,D.C.Appleton AR. and VenosaA.D. 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(1985) Aerobicthermopbilic/anaerobicmesophilictwo stagesewage sludgetreatment: Practical experiencesin Switzerland.Conservation andRecycling 8/1, 2,285-301.190AppendixA: BatchTestResultsBatch Test 1: [VFA]v time (0% primaryfeed)Batch Test 1: [VFA]v time (10% primaryfeed)Batch Test I: [VFA]v time (20% primary feed)Batch Test 1: [VFA]v time (30% primary feed)Batch Test 2: {VFA],TOC, pHvtime (micro-aerobiccontrol w300 mL A sludge)Batch Test 2: [VFA],TOC, pH vtime(micro-aerobicw lacticacid and 300 mL Aside sludge)BatchTest 2: [WA],TOC, pH v time(anaerobic w lacticacidand 1 L A side sludge)Batch Test 2: [WA],TOC, pH v time(anaerobic control w1 LA side sludge)BatchTest 4: [VFA],TOC vtime (micro-aerobiccontrol w300 mL A side sludge)BatchTest 4: [WA],TOC vtime (micro-aerobicwpropionateand 300 mL A sidesludge)BatchTest 4: [WA],TOC v time (anaerobicwpropionate and1 L A side sludge)Batch Test 4: [WA],TOC vtime (anaerobiccontrol w IL Aside sludge)Batch Test 5:[WA], TOC,pH v time (micro-aerobicwlocyanide, 300 mLA side and 30mL primary sludge)Batch Test 5: [WA],TOC, pH v time (micro-aerobicw cyanide,300 mL A side and30 mL primarysludge)Batch Test 5: [WA],TOC, pH v time(anaerobicw/o cyanide,1 L A side and100 mL primary sludge)Figure Al.!Figure Al.2Figure A1.3Figure Al.4Figure A2. 1Figure A2.2Figure A3. 1Figure A3.2Figure A4. 1Figure A4.2Figure A4.3Figure A4.4Figure A5. 1Figure AS 2Figure A5.3198198198199199199200200200201201201202202202191Figure A5.4 BatchTest 5: [VFA], TOC, pHv time (anaerobic w cyanide,1 L A side and 100 mLprimary sludge)203Figure A6. 1 Batch Test6: [VFA], TOC, pH,ORP v time (anaerobic controlw 1 L A side sludge)204Figure A6.2 BatchTest 6: [WA], TOC, pH,ORP v time (anaerobic wvalerateadd’n + 1 L A side sludge)204Figure A6.3 BatchTest 6: [WA], TOC,pH, ORP v time (anaerobicwiso-valerate add’n +1 L A side sludge)205Figure A6.4 BatchTest 6: [WA], TOC,pH, ORP v time (anaerobicw butyrateadd’n + 1 L A sidesludge)205Figure A6.5 BatchTest 6: [WA], TOC,pH, ORP v time (anaerobicwmethylbutyrate add’n+ 1 L A side sludge)206Figure A6.6 Batch Test6: [WA], TOC, pH,ORP v time (micro-aerobiccontrolw 300 mL Aside sludge)206FigureA6.7 BatchTest 6: [WA], TOC,pH, ORP v time (micro-aerobicwvalerate add’n+ 300 mL A side sludge)207Figure A6.8 BatchTest 6: [WA], TOC,pH, ORP v time (micro-aerobicwiso-valerate adcl’n+ 300 mL A side sludge)207Figure A6.9 BatchTest 6: [WAJ, TOC,pH, ORP v time (micro-aerobicwbutyrate add’n+ 300 mL A side sludge)208FigureA6.10 BatchTest 6: [WA],TOC, pH, ORP v time(micro-aerobicwmethylbutyrate add’n+ 300 mL A side sludge)208Figure A7. 1 BatchTest 7: [WA],TOC, pH, ORP vtime (anaerobic controlw 1 L A side sludge)209FigureA7.2 BatchTest7: [WA], TOC, pH,ORP v time (anaerobicw linoleicacid add’n + 1 L Aside sludge)209Figure A7.3 BatchTest 7: [WA], TOC,pH, ORP v time(anaerobic w glucoseadd’n+lLAsidesludge)210Figure A7.4 BatchTest 7: [WA], TOC,pH, ORP v time (anaerobicwdextrinadd’n + 1 L A sidesludge)210Figure A7.5Figure A7.6Figure A7.7Figure A7.8Figure A7.9Figure A7.10Figure A8.1Figure A8.2FigureA8.3Figure A8.4Figure A8.5Figure A8.6Figure A8.7FigureA8.8Figure A8.9192BatchTest 7: [WA],TOC, pH, ORP v time (anaerobicw peptoneacld’n +1 L A sidesludge)211Batch Test 7:[WA), TOC, pH, ORPv time (micro-aerobiccontrol w 300mL A side sludge)211Batch Test 7:[WA], TOC, pH, ORPv time (micro-aerobic wlinoleic acid add’n+ 300 mL A side sludge)212Batch Test 7: [WA],TOC, pH, ORP vtime(micro-aerobicwglucose add’n +300 mL A side sludge)212Batch Test 7: [WA],TOC, pH, ORPv time (micro-aerobic wdextrin add’n+ 300 mL A side sludge)213Batch Test 7: [WA],TOC, pH, ORPvtime (micro-aerobicwpeptone add’n+ 300 niL A sidesludge)213Batch Test 8: [WA],TOC, pH, ORPv time (anaerobic controlw1 L A side sludge)214Batch Test 8:[WA], TOC, pH,ORP v time (anaerobic wethanoladd’n + 1 L A side sludge)214Batch Test 8: [WA],TOC, pH, ORPvtime (anaerobicw propanoladd’n + 1 L A sidesludge)215BatchTest 8:[WA), TOC, pH,ORP v time (anaerobicwisobutyrateadd’n + 1 L A sidesludge)215BatchTest 8: [WA],TOC, pH, ORPv time (anaerobic wpyruvateadd’n + 1 L Aside sludge)216Batch Test 8:[WA), TOC, pH,ORP v time (micro-aerobiccontrolw 300 mL A sidesludge)216BatchTest 8: [WA),TOC, pH, ORPv time (micro-aerobicwethanol add’n+ 300 mL A side sludge)217BatchTest 8: [WA),TOC, pH, ORP v time(micro-aerobicwpropanol add’n + 300mL A side sludge)217Batch Test 8: [WA],TOC, pH, ORPvtime (micro-aerobicwisobutyrate add’n+ 300 niL A side sludge)218193Figure A8.10 Batch Test8: [VFA], TOC, pH, ORPv time (micro-aerobic wpyruvate add’n +300 mL A side sludge)218Figure A9. 1 Batch Test9: [VFAI, TOC, pH, ORPv time (anaerobic controlw 700 mL A side and300 mL primary sludge)219Figure A9.2 BatchTest9: [VFA], TOC, pH, ORPv time (anaerobic w 2.4-dinitrophenol add’n + 700mL A side + 300 mL primarysludge) 219FigureA9.3 Batch Test9: [VFA], TOC, pH, ORPv time (anaerobic wNaFadd’n + 700 mL A side+ 300 mL primary sludge)220Figure A9.4 Batch Test9: [VFA], TOC, pH,ORP v time (anaerobicwpelletadd’n + 700 mL Aside + 300 mL primary sludge)220Figure A9.5 Batch Test9: [WA], TOC, pH,ORP v time (anaerobicw supematantadd’n + 700 mL A side+ 300 mL primary sludge)221Figure A9.6 Batch Test9: [WA], TOC, pH, ORPv time (micro-aerobiccontrol w 270 mL A sideand 30 mL primary sludge)221FigureA9.7 Batch Test9: [WA], TOC, pH, ORPv time (micro-aerobicw2.4-dinitrophenol add’n+ 270 mL A side + 30 mLprimary sludge) 222Figure A9.8 BatchTest 9: [yEA], TOC,pH, ORP v time (micro-aerobicwNaFadd’n + 270 mL A side+ 30 mL primary sludge)222Figure A9.9 Batch Test9: [VEAl, TOC, pH,ORP v time (micro-aerobicwpellet add’n + 270 mLA side + 30 mL primarysludge)223Figure A9.10 BatchTest9: [WA], TOC, pH,ORP v time (micro-aerobicwsupernatant add’n+ 270 mL A side + 30 mLprimary sludge)223Figure AlO. 1 Batch Test10: [VFA], TOC,pH, ORP v time (anaerobiccontrolw 1 L A side +0mLprimary sludge)224Figure A10.2 Batch Test10: [WA], TOC, pH,ORP v time (anaerobic w800 mLA side + 200 mL primarysludge)224FigureA10.3 Batch Test10: [WA], TOC,pH, ORP vtime (anaerobicw 600 mLA side + 400 mL primarysludge)225Figure A10.4 Batch Test10: [WA], TOC, pH,ORP v time (anaerobic w400 mLA side + 600 mL primarysludge)225194Figure A10.5 BatchTest 10: [WA], TOC,pH, ORP v time (anaerobicw 200 mLA side + 800 mLprimary sludge)226Figure A10.6 BatchTest 10: [WA], TOC,pH, ORP vtime (micro-aerobicw300 mL A side+0 mLprimary sludge)226Figure A10.7 BatchTest 10: [WA], TOC,pH, ORP v time (micro-aerobicw240 mL fermenter+ 60 mL primary sludge)227Figure A10.8 Batch Test10: [WA], TOC, pH,ORP vtime (micro-aerobicw180 mL fermenter+ 120 mL primary sludge)227Figure A10.9 Batch Test10: [WA], TOC, pH,ORP v time (micro-aerobicw120 mL fermenter+ 180 mL primary sludge)228Figure AlO.10Batch Test10: [WA], TOC, pH,ORP v time (micro-aerobicw60 mL fermenter+240 mL primarysludge)228Figure AlO.11 Batch Test10: [WA], TOC,pH, ORP v time (anaerobiccontrolw 1 L fermenter+ 0 mL primary sludge)229Figure AlO.12BatchTest 10: [WA], TOC,pH, ORP v time (anaerobicw 800 mLfermenter+ 200 mLprimary sludge)229Figure AlO.13BatchTest10: [WA], TOC, pH,ORP v time (anaerobicw 600 mLfermenter + 400 mLprimary sludge)230Figure AlO.14BatchTest 10: [WA], TOC,pH, ORP vtime (anaerobicw400 mLfermenter+600 mL primary sludge)230Figure AlO.l5BatchTest 10: [WA],TOC, pH, ORP vtime (anaerobic w200 mLfermenter+ 800mL primary sludge)231Figure AlO.l6BatchTest 10: [WA], TOC,pH, ORP v time (micro-aerobiccontrolw 300mL fermenter+ 0 mL primary sludge)231Figure AlO.l7BatchTest 10: [WA], TOC,pH, ORP v time(micro-aerobicw240 niL fermenter+ 60 niL primary sludge)232Figure AlO.18Batch Test10: [WA], TOC,pH, ORP v time(micro-aerobicw180 mL fermenter+ 120 mL primary sludge)232Figure 10.19 BatchTest 10: [WA], TOC,pH, ORP v time (micro-aerobicw120 niL fermenter +180 niL primary sludge)233195Figure 10.20 BatchTest 10: [VFA], TOC,pH, ORP v time (micro-aerobicw60 mL fermenter +240 mL primary sludge)233Figure Al1.1 BatchTest 11: [VFA], TOC,pH, ORP v time (anaerobiccontrol wILA)234Figure Al1.2 BatchTest 11: [VFA], TOC,pH, ORP v time (anaerobicA side w25% primarysludge)234Figure Al1.3 BatchTest 11: [VFA],TOC, pH, ORP vtime (anaerobiccontrolw1 LB side)235Figure Al1.4 BatchTest 11: [VFA], TOC,pH, ORP v time (anaerobicB sidew25% primary sludge)235Figure Al1.5 BatchTest 11: [VFA], TOC,pH, ORP vtime (MicroaerobicA sidew25%primary sludge)236Figure Al1.6 Batch Test11: [VFA], TOC, pH,ORP vtime (micro-aerobiccontrol w 1 L Aside sludge)236Figure Al1.7 BatchTest11: [VFA], TOC,pH, ORP v time (micro-aerobicA sidew 50%primary sludge)237Figure Al1.8 BatchTest 11: {VFA], TOC,pH, ORP v time (micro-aerobiccontrol with 1 LB side sludge)237Figure Al1.9 Batch Test11: [WA], TOC,pH, ORPv time (micro-aerobicB side w 50% primarysludge)238Figure All.l0Batch Test11: [WA], TOC,pH, ORP v time (micro-aerobicB sidew 25%primary sludge)238FigureA12. 1 SalmonArm 1st stageATAD: [WA], TOC,pH, ORP v time(anaerobic control)239FigureA12.2 SalmonArm 1st stage ATAD:[WA], TOC, pH,ORP v time(anaerobic w 50%primary sludge)239Figure Al2.3 SalmonArm 1st stage ATAD:[WA], TOC,pH, ORP v time(anaerobic w 50%secondary sludge)240Figure A12.4 SalmonArm 1st stage ATAD:[WA], TOC, pH,ORP vtime(anaerobic wpropionate add’n)240196Figure A12.5 SalmonArm 1st stage ATAD: [VFA],TOC, pH, ORP v time(anaerobic w mixedsecondary and primary sludge)241Figure A12.6 SalmonArm 1st stage ATAD: [VFA],TOC, pH, ORP v time(micro-aerobic control)241Figure A12.7 SalmonArm 1st stage ATAD:[VFA], TOC, pH, ORP v time (micro-aerobicw 50% primarysludge)242Figure A12.8 SalmonArm 1st stage ATAD: [VFA],TOe, pH, ORP v time (micro-aerobic w 50% secondarysludge)242Figure A12.9 SalmonArm 1st stage ATAD:[VFA], TOC, pH, ORP vtime(micro-aerobicw propionate add’n)243Figure A12.10SalmonArm 1st stage ATAD: [WA],TOC, pH, ORP vtime (micro-aerobic w mixed secondaryand primary sludge)243Figure Al3.1 SalmonArm 3rd stage ATAD: [WA],TOC, pH, ORP v time(anaerobic control)244Figure A13.2 SalmonArm 3rd stage ATAD: [WA],TOC, pH, ORP v time(anaerobic w 50% primarysludge)244Figure A13.3 SalmonArm 3rd stage ATAD:[WA], TOC, pH, ORPvtime(anaerobic w 50% secondarysludge)245Figure A13.4 SalmonArm 3rd stage ATAD:[WA], TOC, pH,, ORPv time(anaerobic w propionateacld’n)245FigureA13.5 SalmonArm 3rd stage ATAD:[WA], TOC, pH,ORP v time(anaerobic w mixed secondaryand primary sludge)246Figure A13.6 SalmonArm 3rd stage ATAD:[WA], TOC, pH, ORPv time (microaerobic control)246FigureA13.7 SalmonArm 3rd stage ATAD:[WA], TOC, pH, ORPvtime (microaerobicw 50%primary sludge)247Figure A13.8 SalmonArm 3rd stage ATAD:[WA], TOC, pH,ORP v time (microaerobic w 50% secondarysludge)247Figure A13.9 SalmonArm 3rd stage ATAD:[WA], TOC, pH, ORP vtime (microaerobic w propionateadd’n)248197Figure A13.10SalmonArm 3rd stage ATAD:{VFAI, TOC, pH, ORP v time (microaerobic w mixed secondaryand primary sludge)248Table Al Concentrationofsludges for all batchexperiments249198Figure Al.1: Batch Test 1: [VFA] v time(0% primary feed)6005004003002001000Figure Al.2: Batch Test 1: [VFA] v time (10%primary feed)600500[VFAJ400(mg/I..)30020010:Time (hrs)Figure Al.3: Batch Test 1: [VFA]v time (20% primary feed)20010:Time (hrs)[WA](mg/L)0 1020 30 40Time (hrs)50600500[VFA]400(mg/I)300•acetate propionate•isobutyrate butyrateA2-methyl-butyrate isovalerate(valerate199Figure Al.4: Batch Test1: [VFA] v time (30% primary feed)1000800::200Time hrs)Figure A2.1: Batch Test 2:[VFAI, TOC, pH v time (micro-aerobic control w 300 mL Aside sludge)1500 -81200[VFA](mg/I..)900pH600300____ _________0XX60 10 2030 4050Time (hrs)Figure A2.2: BatchTest 2: [VFAJ, TOC,, pHv time (microaerobic w lactic acidand 300 mL A side sludge)1500i:o:L .Time (Ins)•acetate propionate•isobutyrate<butyrateA2-methylbutyrate isovalerateXvalerate lactate+TOC•pH2001500Figure A3.1: Batch Test 2: EVFA],TOC, pH v time(anaerobic w lactic acid and 1 L A sidesludge)Figure A3.2: Batch Test 2: [VFA],TOC, pH v time(anaerobic control w 1 L A sidesludge)Figure A4.1: Batch Test 4: [VFA],TOC v time (micro-aerobic control 300 ml A sidesludge)1200[VFA](mgJI)60030:0 1020 30 4050Time (hrs)15001200[WA](mg/i.)90060030008pH0 10 20 3040 50Time (hrs)15001200[WA](mg/I..)900600300pH-70 1020 30 40 50Time (hrs)•acetate propionate•isobutyrate‘ butyrateA2-methylbutyrate isovalerate)<valerate ‘actate + TOC•pH201[VFA](mg/I)150012009006003000Figure A4.2: BatchTest 4: [VFA], TOC v time (micro-aerobic w propionate and300 mL A sludge)50Figure A4.3: BatchTest 4: [VFA], TOC v time(anaerobic w propionateand 1 1 A side sludge)0 1020 30 4050Figure A4.4: BatchTest 4: LVFAI, TOC vtime(anaerobic control w1 L A side sludge)600____________________30__0 1020 3040 50•acetate propioriate•isobutyrate‘ butyrateA2-methylbutyrate isovalerateXvalerate lactate+TOC•pH0 1020 30 40Time (hrs)[VFA](mg/I)15001200900::e-0Time (hrs)15001200[VFA](mg/I)900Time (hrs)202Figure A5.1: Batch Test 5:[VFA], TOC, pH v time (micro-aerobic w/o cyanide, w 300mLA side and 30 mL primarysludge)15008[VFA]1200(mg/I.)900pH01020304050Time (hrs)Figure A5.2: Batch Test5: [VFA], TOC, pH v time (micro-aerobicw cyanide, 300 mL Aside and 30 mL primary sludge)150081200 -t[VFA](mg/I.)900pH760030:___-Z260 1020 30 4050Time (hrs)Figure A5.3: Batch Test 5: [VFA],TOC, pH v time(anaerobicw/o cyanide, 1 1 Aside and 100 mL primarysludge)1500[VFA]1200(mg/I.)900.pH.7600 +30:- -- ——4)506Time (hrs)•acetate propionate•isobutyratebutyrateA2-methylbutyrate isovalerate)<valerate*(lactate+TOC•pH203Figure A5.4: Batch Test 5:[VFAJ, TOC, pH v time(anaerobic w cyanide, 1 L A side and100 mL primary sludge)15001200[WA](mg/I..)900pH600 ....—....z*, I30:O1O203O5OTime (hrs)•acetateDpropionate•isobutyratebutyrateA2-methylbutyrate isovalerateXvalerate lactate+TOC•pHa204-250 —250ORP(mV)-5000-250 — 500ORP(mV)-500Figure A61: Batch Test 6: [VFAI,TOC, pH, ORP v time(anaerobic control w 1 L A sidesludge)Figure A6.2: BatchTest 6: [VFA], TOC, pH, ORP vtime(anaerobicw valerateadd’n + 1 1 A side sludge)50•acetate propionate•isobutyratebutyrateA2-methylbutyrate isovalerate><valerate lactate + TOC•pH0ORP0 500pH80 1020 30 4050Time (hrs)9pH80 1020 3040Time (hrs)72050-250ORP(mV)-500-250 —500ORP(mV)-500Figure A6.3: Batch Test6: [VFA], TOC, pH, ORP vtime(anaerobic w iso-valerateadd’n + 1 L A side sludge)Figure A6.4: BatchTest 6: [VFAI, TOC, pH,ORP v time(anaerobic wbutyrate add’n + 1 1 Aside sludge)50•acetateDpropionate•isobutyrate0butyrateA2-methylbutyrate isovaferate><valerate lactate±TOC•pHCORP10009pH80 1020 30 4050Time (hrs)7010009pH80 1020 3040Time (hrs)7206Figure A6.5: Batch Test 6:[VFA], TOC. pH, ORP v time(anaerobic w methylbutyrate‘n + 1 1 A side sludge)FigureA66: Batch Test6: [VFA], TOC, pH, ORPv time(micro-aerobic control w300 ml A side sludge)90 10009-250 —500ORP(mV)-500pH80 1020 30 4050Time (hrs)701000-250— 500ORP(mV)-500pH87500 1020 3040Time (hrs)•acetate propionate•isobutyratebutyrateA2-methylbutyrateAisovalerate)<valerate lactate +TOC•pHCORP207Figure A6.7: BatchTest 6: [VFAI, TOC, pH, ORP vtime(micro-aerobic w valerateadd’n + 300 ml A side sludge)Figure A6.8: BatchTest 6: [VFAI, TOC, pH, ORPv time(micro-aerobic w iso-valerateadd’n + 300 ml Aside sludge)90 10009-250 —500ORP(mV)-500pH80 1020 3040 50Time (hrs)701000-250— 500ORP(mV)-500pH87500 1020 3040Time (hrs)•acetate propionate•isobutyratebutyrateA2-methylbutyrate isovalerate><valerate lactate+TOC•pHCORP208Figure A6.9: Batch Test 6: [VFAI, TOC,pH, ORP v time(micro-aerobic w butyrate addn÷ 300 mL A side sludge)Figure A6.10: Batch Test6: [VFAI, TOC, pH, ORP v time(micro-aerobic w methylbutyrateadd’n + 300 mL A sidesludge)-I-o woo-250ORP(mV)-5009pH870 10 2030 40 50Time (hrs)9pH875°0-250 — 500ORP(my)-500•acetate propionate•isobutyrate0butyrateA2-methylbutyrate ‘ isovalerate>(valerate lactate+TOC•pHCORP0 10 2030 40Time (hrs)209Figure A7.1: BatchTest 7: [VFA], TOC. pH, ORP vtime(anaerobic control w 1 LA side sludge)Figure A7.2: BatchTest 7: [VFA], TOC, pH,ORP v time(anaerobic w linoleicacid add’n + 1 L A sidesludge)01000-250 — 500ORP(mV)-5008pH760 10 2030 40 50 6070 80Time (hrs)0 5008pH760 10 2030 40 5060 70 80-250 —250ORP(mY)-500•acetate propionate•isobutyratebutyrateA2-methylbutyrate isov&erate)<valerate ‘actateTOC•pHCORPTime (hrs)210Figure A7.3: Batch Test 7: [VFA], TOC, pH,ORP v time(anaerobic w glucose add’n+ 1 1 A side sludge)Figure A7.4: Batch Test 7:[VFA], TOC, pH, ORP v time(anaerobic w dextrin add’n +1 L A side sludge)80 1000-250ORP(mV)-5008pH70 10 2030 40 50 60 7080Time (hrs)601000pH-250—5007ORP(mV)-50060 1020 30 40 5060 70 80Time(hrs)•acetate propionate•isobutyratebutyrateA2-methylbutyrate isovalerateXvalerate lactate÷TOC•pHCORP211butyrateFigure A7.5: Batch Test 7: [VFA], TOC, pH, ORP vtime(anaerobic w peptone add’n + 1 1 A side sludge)40Time (hrs)Figure A7.6: Batch Test 7: [VFAJ,TOC, pH, ORP v time(micro-aerobic control w 300 mL A sidesludge)o1000-250 — 5004°Time (hrs)•acetate propionate•isobutyrateA2-methylbutyrate isovalerate><valerate lactate+TOC212Figure A7.8: Batch Test7: [VFA], TOC, pH, ORP v time(micro-aerobic w glucose add’n+ 300 ml A side sludge)8Figure A7.7: Batch Test 7: LVFAI,TOC, pH,, ORP v time(micro-aerobic w linoleic acid add’n+ 300 ml A sidesludge)80-250ORP(mV)-500pH70 10 20 3040 50 60 70 80Time (Ins)601000pH-250 —5007ORP(mV)-50060 10 20 3040 50 6070 80Time (hrs)•acetate propionate•isobutyrateGbutyrateA2-methylbutyrate isovalerate><valerate$(lactate+TOC•pHCORP213Figure A7.9: Batch Test 7: [VFAJ, TOC, pH, ORP v time(micro-aerobic w dextrin add’n + 300 ml A side sludge)Figure A7.10: Batch Test 7: [VFA], TOC, pH, ORP v time(micro-aerobicw peptone add’n + 300 ml A sidesludge)0 1000-250 —500ORP(mV)-5008pH760 10 20 30 40 50 60 7080Time (hrs)01000pH8-250— 500ORP(mV)-500Time (hrs)BacetateDpropionate•isobutyratebutyrateA2-methylbutyrate isovalerateXvalerate lactate+TOC•pH - ORP0 10 20 3040 50 60 70 80214Figure A8.1: Batch Test 8: [VFA], TOC, pH, ORP v time(anaerobic control w 1 L A side sludge)8Figure A8.2: Batch TestS: [VFA], TOC, pH, ORPv time(anaerobic w ethanol add’n + 1 1 A side sludge)0 1000-250 — 500ORP(mV)-500pH760 10 20 30 40 5060Time (hrs)0 20008p11-250 —10007ORP(mV)-500640 50 60•acetate propionate•sobutyrate•ethanol0butyrateA2-methylbutyrate ‘ isovalerateXvalerate lactate TOC•pHCORP0 10 20 30Time (hrs)215ORP(mV)0 2000Figure A8.3: Batch Test 8: [VFA], TOC,, pH, ORP v time(anaerobic w propanol add’n + 1 L A side sludge)8pH76-250ORP(mV)-5000 10 20 30 4050 60Time (hrs)Figure A8.4: Batch Test 8: CVFA], TOC, pH,ORP v time(anaerobic w isobutyrate add’n + 1 L Aside sludge)02000 8[VEAl,TOC(mgit)-2500007p-500 00102030405060Time (hrs)•acetate propionate•isobutyrate•propanol‘ butyrateA2-methylbutyrate isovalerate)<valerate lactate+TOC•pH0ORP216-250 -4000ORP(mV)-500-250 — 500ORP(mV)-500Figure A8.5: Batch Test 8: [VFA], TOC, pH, ORPv time(anaerobic w pyruvate add’n + 1 L A side sludge)Figure A8.6: Batch Test 8: [VFAI, TOC, pH,ORP v time(micro-aerobic control w 300 ml A side sludge)0 10 2030 40 50 60•acetate propionate•isobutyrate pyruvatebutyrateA2-methylbutyrate isovalerateXvalerate lactate ÷ TOC•pHCORP0 20008pH70 10 20 30 40 5060lime (hrs)601000 8pH7Time (hrs)6217Figure A8.7: Batch Test 8: [VFA],TOC, pH, ORP v time(micro-aerobicw ethanol add’n +300 ml A side sludge)0-250ORP(my)-5008pH760 10 2030 40 50 60Time (hrs)Figure A8.8: Batch Test 8:[VFA], TOC, pH, ORP v time(micro-aerobicw propanol add’n+ 300 ml A side sludge)9pH80-250 — 500ORP(mV)-5000 10 20 3040 50 60Time (hrs)•acetate propionate•isobutyrate•ethanol•propanol‘ butyrateA2-methylbutyrate isovalerate<valerate lactate+TOC•pH0ORP7218-250 -4000ORP(mV)-5000-250—1000ORP(mV)-500Figure A8.9: Batch Test B: [VFAI, TOC, pH, ORP v time(micro-aerobicw isobutyrate add’n + 300 mL A side sludge)Figure A8.10: Batch Test 8: [VFA],TOC, pH, ORP v time(micro-aerobic w pyruvate add’n + 300mL A side sludge)40 50 60•acetate propionate•isobutyrate•pyruvatebutyrateA2-methylbutyrate isovalerate)<valerate lactate+TOC•pHCORP0 20009pH80 10 20 30 40 50 60Time (hrs)79pH80 10 20 30Time (hrs)7219Figure A9.1: Batch Test 9: [VFAI, TOC, pH, ORPv time(anaerobic control w 700 mL A side and 300 mL primarysludge)Figure A9.2: Batch Test 9: [VFAJ, TOC, pH, ORPv time(anaerobic w 2,4-dinitrophenol add’n + 700ml A side +300 mL primary sludge)o 1000-250 —500ORP(mV)-500pH70 10 20 3040 50 60Time (hrs)0 5008(mg/ipH250—250C .7I...-.. .....ORP0606Time (hrs)•acetateDpropionate•isobutyrate0butyrateA2-methylbutyrateLisovalerate)<valerate lactate±TOC•pH0ORP220Figure A9.3: Batch Test 9: [VFA], TOC, pH, ORP v time(anaerobic w NaF add’n + 700 mL A side+ 300 ml primarysludge)[VFA],TOC(mg/1) •.Figure A9.4: Batch Test 9: [VFA],TOC, pH, ORP v time(anaerobic w pellet add’n + 700 mLA side + 300 mlprimary sludge)0 1000-250ORP(mV)8pH-7-500 0 60 10 20 30 4050 60Time (hrs)0-250 —ORP(mV)-500pH0 10 20 3040 5060Time (hrs)•acetate propionate•isobutyrate0butyrateA 2-methylbutyrate isovalerateXvalerate lactate + TOC•pHCORP221Figure A9.5: Batch Test 9: [VFAJ, TOC, pH, ORP v time(anaerobic w supematant add’n + 700 mL A side + 300mL primary sludge)Figure A9.6: Batch Test 9: [VFAJ, TOC, pH, ORP v time(micro-aerobic control w 270 ml A side + 30 mL primarysludge)0 500-250 —250ORP(mV)-5008pH760 10 20 30 40 5060Time (hrs)pH80-250—250ORP(mV)-500Time (hrs)•acetateUpropionate•isobutyrate0butyrateA2-methylbutyrate isovalerate)<valerate lactate+TOC•pHCORP0 10 20 3040 50 60222Figure A9.7: Batch Test 9: [VFAL TOC, pH, ORP v time(micro-aerobic w 2,4-dinitrophenoladd’n + 270 ml A side+ 30 mL primary sludge)8Figure A9.8: Batch Test 9: [VFA], TOC, pH, ORP v time(micro-aerobicw NaF add’n + 270 ml A side ÷ 30 mLprimary sludge)250 20000ORP(mV)-250pH760 10 20 30 40 50 60Time (hrs)pH80-250 — 500ORP(mV).500Time (lirs)acetate propionate•isobutyratebutyrateA2-methylbutyrate isovalerateXvalerate ‘actate+TOC•pH0ORP0 10 20 30 40 5060223Figure A9.9: Batch Test 9: [VFAJ, TOC, pH, ORP v time(micro-aerobic w pellet add’n + 270 mL A side + 30 mLprimary sludge)0 500-250 —250ORP(mV)-5009pH870 10 20 30 40 50 60Time (hrs)-1-Figure A9.10: Batch Test 9: LVFAI, TOC, pH, ORP v time(micro-aerobic w supernatant add’n + 270 mL A side + 30mL primary sludge)pH0-250—250ORP(mV)-500Time (hrs)•acetate propionate•isobutyrate‘ butyrateA2-methylbutyrate isovalerateXvalerate lactate+TOC•pCORP0 10 20 3040 50 60224Figure A10.1: Batch Test 10: [VFAJ, TOC, pH, ORP v time(anaerobic control w 11 A side and 0 mL primary sludge)Figure A10.2: Batch Test 10: [VFA], TOC, pH, ORP v time(anaerobic w 800 ml A side + 200 mL primary sludge)0500-250 —250ORP(mV)-500pH70 10 20 30 40 50 60 70Time (hrs)0 500-250 —2508pH7ORP(mV)-500 650 60 70•acetate propionate•isobutyratebutyrateA2-methylbutyrate isovalerate)<valerateXlactate+TOC•pHCORP0 10 20 30 40Time (hrs)2250 1000Figure A10.3: Batch Test 10: [VFA], TOC, pH, ORP v time(anaerobic w 600 mL A side + 400 ml primary sludge)[VFA],TOC(mg/I.)-250—500ORP(mV)-500 0ORP(mV)-500Figure A1O.4: Batch Test 10: LVFAJ, TOC, pH, ORP v time(anaerobic w 400 mL A side + 600 ml primary sludge)Time (hrs)•acetate propionate•isobutyratebutyrateA2-methylbutyrate isovalerateXvalerate lactate+TOC•pHCORP8pH70 10 20 30 40 50 60 70Time (hrs)601000-250 — 500pH60 10 20 30 40 50 60702260 1000Figure A10.5: Batch Test 10: [VFAJ, TOC, pH, ORP v time(anaerobic w 200 ml A side + 800 ml primary sludge)LVFA),TOC(mg/i..)-250 —500ORP(mV)-500 00-250—250ORP(mV)-500Figure Al0.6: Batch Test 10: [VFAI, TOC, pH, ORP v time(micro-aerobic control w 300 ml A side + 0 ml primarysludge)50 60 70•acetate propionate•isobutyrateObutyrateA2-methylbutyrate isovalerateXvalerateXlactate+TOC•pHCORP6.5pH5.50 10 20 30 40 50 60 70Time (hrs)4.5‘.5pH7.50 10 20 30 40lime (hrs)6.52272500ORP(mV)-2500-250 — 500ORP(mV)-500Figure A10.7: Batch Test 10: LVFA], TOC, pH, ORP v time(micro-aerobic w 240 ml A side + 60 ml primary sludge)0 10 20 30 40 50 60 70pHFigure A10.8: Batch Test 10: [VFA], TOC, pH, ORP v time(micro-aerobic w 180 ml A side + 120 ml primary sludge)•acetate propionate•isobutyratebutyrateA2-methylbutyrateAisovalerate)<v&erate lactate+TOC•pHCORP87Time (hrs)6pH70 10 20 30 40 50 60 70Time (hrs)228o iooo[VFAJ,TOC(mg/I)-250 —500ORP(mV)-500 0Figure A10.9: Batch Test 10: [VFAI, TOC, pH, ORP v time(micro-aerobic w 120 ml A side + 180 mL primary sludge)9pH870 10 20 30 40 50 60 70Time (hrs)-1-Figure A10.10: Batch Test 10: [VFAJ, TOC, pH, ORPv time(micro-aerobic w 60 mlA side + 240 ml primarysludge)pH702000[VFA],TOC(mg/i.)-250 —1000ORP(mV)-500 0•acetate propionate•isobutyratebutyrateA2-methylbutyrate isovalerate)<valerate*(lactate+TOC•pH0ORP0 10 20 30 40 50 6070Time (hrs)229Figure Al0.11: Batch Test 10: [VFA], TOC, pH, ORP v time(anaerobic control w 11 fermenter and 0 mL primary sludge)Figure A10.12: Batch Test 10: [VFAL, TOC, pH, ORP v time(anaerobicw 800 mlfermenter ÷ 200 ml primary sludge)70 500-250 —250ORP(mV)-5007.5pH6.55.50 10 20 30 40 50 60 70Time (hrs)0 500-250 —250ORP(mV)-500pH6550 60 700 10 20 30 40Time {hrs)•acetate propionate•isobutyrate0butyrateA2-methylbutyrate isovalerateXvalerate lactate+TOC•pHC’ORP230Figure A10.13: Batch Test 10: [VFAI, TOC, pH, ORP v time(anaerobic w 600 mL fermenter + 400 ml primary sludge)7Figure A10.14: Batch Test 10: EVFA], TOC, pH, ORP v time(anaerobic w400 mlfermenter + 600 mL primary sludge)0 500-250—250ORP(mV)-500pH650 10 20 30 40 50 60 70Time (hrs)7pH60-250 — 500ORP(mV)-500•acetate propionate•isobutyrateObutyrateA2-methylbutyrate isovalerate><valerate lactate+TOC•pHC’ORP0 10 20 30 40 50 6070Time (hrs)5231Figure A10.15: Batch Test 10: [VFA], TOC, pH, ORP v time(anaerobic w 200 mL fermenter + 800 mL primary sludge)6.5pH5.5Figure A10.16: Batch Test 10: [VFAI, TOC, pH, ORP v time(micro-aerobic control w 300 mLfermenter + 0 mL primarysludge)0 1000-250ORP(mV)-5000 10 20 30 40 50 60 70Time (hrs)4.50 8.5pH-250---250 7;5ORP(mV)-500 6.550 60 70•acetate propionate•isobutyrate0butyrateA2-methylbutyrate . isovalerateXvalerate lactate+TOC•pHCORP0 10 20 30 40Time (hrs)232Figure A10.17: Batch Test 10: [VFA], TOC, pH, ORP v time(micro-aerobic w 240 mL fermenter + 60 mL primarysludge)Figure A10.18: Batch Test 10: [VFAJ, TOC, pH, ORP v time(micro-aerobic w 180 mL fermenter + 120 mL primarysludge)250 5000ORP(mV)-2508.5pH7.56.50 10 20 30 40 50 60 70Time (hrs)0 8.5pH-250—2507.5ORP(mV)-500 6.550 60 70•acetate propionate•isobutyratebutyrateA2-methylbutyrate isovalerateXvalerate lactate TOC•pHCORP0 10 20 30 40Time (hrs)233Figure A10.19: Batch Test 10: [VFA], TOC, pH, ORP v time(micro-aerobic w 120 ml fermenter + 180 ml primarysludge)8Figure A10.20: Batch Test 10: [VFA]I TOC, pH, ORP v time(micro-aerobic w 60 mlfermenter + 240 mL primary sludge)0 500-250 —250ORP(mV)-500pH760 10 20 30 40 50 60 70Time (hrs)pH70-250—250ORP(mV)-500Time (hrs)•acetate propionate•isobutyrateGbutyrateA2-methylbutyrate isovalerate<valerate lactate±TOC•pHCORP0 10 20 30 40 5060 70234Figure Al1.1: Batch Test 11: [VFA], TOC, pH, ORP v time(anaerobic control w 1 L A side sludge)Figure All.2: Batch Test 11: [VFA], TOC, pH, ORP v time(anaerobic A side w 25% primary sludge)01000-250 —500ORP(mV)-500pH70 10 20 30 40 50Time (hrs)0 1000-250 — 500ORP(mV)-500pH70 10 20 30 4050Time (hrs)•acetate propionate•isobutyrate0butyrateA2-methylbutyrate isovalerateXvalerate lactate+TOC•pHCORP235Figure Al1.3: Batch Test 11: [VFAI, TOC, pH, ORP v time(anaerobic control w 1 L B side sludge)pH0 10 2030 40 50Figure Al1.4: Batch Test 11: [VFAJ, TOC, pH, ORP v time(anaerobic B side w 25% primary sludge)0 20006.5[VFA],TOC(mg/I)-2501000________5.5ORP(mV)4.5-500 0Time (hrs)01000-250 — 500ORP(mV)-500pH60 10 2030 40 50Time (hrs)•acetate propionate•isobutyratebutyrateA2-methylbutyrate isovalerateXvalerate lactate±TOC•pHCORP236o iooo-250 —500ORP(mV)-5000-250ORP(mV)-500Figure Al1.5: Batch Test 11: LVFA], TOC, pH, ORP v time(micro-aerobic A side w 25% primary sludge)Figure Al1.6: Batch Test 11: LVFA], TOC, pH, ORP v time(micro-aerobic control w 11 A side sludge)•acetate propionate•isobutyratebutyrateA2-methylbutyrate isovaferateXvalerate4(lactate+TOC•pH0ORP8.50 10 20 30 40 50Time (hrs)0 10 20 30 40 50Time (hrs)2372500ORP(mV)-25002000Figure Al1.7: Batch Test 11: [VFAI, TOC, pH, ORP v time(micro-aerobic A side w 50% primary sludge)Figure Al1.8: Batch Test 11: [VFAI, TOC, pH,ORP v time(micro-aerobic control w 1 L B side sludge)[VFALTOC(mg/I)-250—1000ORP(mV)-500 0•acetate propionate•isobutyratebutyrateA2-methylbutyrate isovalerate><valerate lactate±TOC•p1-ICORPpH10 200 40 5030Time (hrs)0 10 20Time (hrs)30 40 50238-250-—i 000ORP(my)-500-250 —1000ORP(mV)-500Figure Al1.9: Batch Test 11: [VFA], TOC, pH, ORP v time(micro-aerobic B side w 50% primary sludge)FigureAll10: Batch Test 11: LVFA],TOC, pH, ORP v time(micro-aerobic B sidew 25% primary sludge)•acetate propionate•isobutyratebutyrateA2-methylbutyratetisovalerate)<valerate*(lactate+TOC•pH0ORP0 2000pH0 10 20 30 4050Time (hrs)02000pH0 10 2030 40 SOTime (hrs)239•acetate propionate•isobutyratebutyrateA 2-methylbutyrate sovalerateXvalerateXlactatetTOC•pH ORPFigure Al2.1: Salmon Arm1St stage ATAD: [VFAI, TOC,pH, ORP v time (anaerobiccontrol)0.250ORP(mV)-500pH50 10 20 30 4050 60 70 80Time (hrs)0 2000Figure A12.2: Salmon Arm1st stage ATAD: [VFAI, TOC,pH, ORP v time(anaerobic w 50% primary sludge)6-250 1000ORP(mV)-500pH5450 60 70800 10 20 3040Time (hrs)240Figure A12.3: Salmon Arm 1st stage ATAD: [VFA], TOC,pH, ORP v time (anaerobic w 50% secondary sludge)Figure Al2.4: Salmon Arm 1St stage ATAD: [VFA], TOC,pH, ORP v time (anaerobic w propionate add’n)60 2000-250—1000ORP(mV)-5006pH540 10 20 30 40 50 60 70 80Time (lirs)02000-250 —1000ORP(mV)-500pH5450 60 70 800 10 20 3040Time (hrs)•acetate propionate•isobutyratebutyrateA2-methylbutyrate isovalerateXvalerateXlactate±TOC•pHCORPxzCoa.a.-_____________0QI0:0I—.:I—/+?•r/‘.+‘/‘,1if.:.+:9.o‘—...v.-40E.I—•>‘II+\_________\__1.2•.i+02-Xou-a.808800000C..Ic.j0o.0000000c.Jo.su242Figure A12.7: SalmonArm 1st stage ATAD: (VFAI, TOC,pH, ORP v time (micro-aerobic w50% primary sludge)Figure A12.8: SalmonArm 1st stage ATAD:[VFA], TOC,pH, ORP v time (micro-aerobicw 50% secondarysludge)250 2000(WA],TOC(mg/i)0 1000ORP(mV)-250 06.5pH5.54.50 10 20 3040 50 60 70 80Time (hrs)0-250ORP(mV)-5006.5pH54.550 60 7080Iacetate propionate+isobutyrateObutyrateA2-methylbutyrate isovalerateXvalerate lactate+TOC•pHCORP0 10 2030 40lime (hrs)243Figure Al2.9: SalmonArm 1St stage ATAD: [VFA],TOC,pH, ORP v time(micro-aerobic w propionateadd’n)Figure Al2.10: SalmonArm 1St stage ATAD:LVFA], TOC,pH, ORP v time(micro-aerobicw mixedsecondary andprimary sludge)8o 2000-250 —40007.5pHORP(mV)-5006.50 10 2030 40Time (hrs)5.550 60 7080-I-0 2000-250 —1000ORP(mV)-500pH760 1020 30 4050 60 7080Time (hrs)•acetate propionate•isobutyratebutyrateA 2-methylbutyrate isovalerate><valerate lactate± TOC•pHC’ORP244Figure A13.l: Salmon Arm 3rd stageATAD: [VFA], TOC,pH, ORP v time (anaerobic control)FigureAl3.2: SalmonArm 3rd stage ATAD: [VFA],TOC,pH, ORP v time (anaerobicw 50% primary sludge)-250 10000 10 2030 40 5060Time (hrs)•acetate propionate•isobutyrateObutyrateA2-methylbutyrate isovalerate><valerate<lactate±TOC•pHCORP0-250ORP(mV)-5008pH760 10 20 3040 50 60Time (hrs)0 2000[VFA],TOC(mg,t)e..•..--......ORP(mV)7pH65-500 0245Figure A13.3: Salmon Arm3rd stage ATAD: [VFA], TOC,pH, ORP v time (anaerobicw 50% secondary sludge)Figure Al3.4:Salmon Atm 3rd stageATAD: [VFAL TOC,pH, ORP v time (anaerobicw propionate add’n)0 20007pH6-2501000ORP(mV)-5000 1020 30 4050 60Time (hrs)5020007.5pH-250—10006.5ORP(mV)-5005.540 5060•acetate0propionate•isobutyratebutyrateA2-methylbutyrateisovalerateXvalerate lactate±TOC•pHCORP0 1020 30Time (hrs)246Figure A13.5: Salmon Arm 3rdstage ATAD: [VFAI, TOC,pH, ORP v time (anaerobicw mixed secondary and primarysludge)Figure A13.6: Salmon Arm3rd stage ATAD: [VFA],TOC,pH, ORP v time(micro-aerobic control)ORP(mV)-500 OX0 1020 3040 5060Time (hrs)70-250ORP(mV)-500pH650 10 2030 40 5060Time (hrs)O2000LVFA],tToc(1•••(mg/1) .........+-25010007.5pH6.55.5•acetate propionate•isobutyratebutyrateA 2-methylbutyrate isovalerateXvalerate lactate+TOC•pHCORP247Figure A13.7: SalmonArm 3rd stage ATAD: IVFAI,TOC,pH, ORP v time (micro-aerobicw 50% primary sludge)Figure A13.8: SalmonArm 3rd stage ATAD: CVFAI,TOC,pH, ORP v time(micro-aerobicw 50%secondary sludge)/-..—0 1020 3040 5060Time (hrs)•acetate propionate•isobutyratebutyrateA2-methylbutyrateisovalerateXvalerate lactate+TOC•pHCORP250 20000ORP(mV)-2505pH550 1020 3040 5060Time (hrs)020007pH6[VFA],TOC(mg/Ij-2501000OO248Figure A13.9:Salmon Arm 3rd stageATAD: [VFA], TOC,pH. ORP v time (micro-aerobicw propionate add’n)7.5Figure A13.1O:Salmon Arm 3rd stageATAD: [VFA],TOC,pH, ORP v time(micro-aerobic wmixed secondary andprimary sludge)-f0 2000-250ORP(my)-500pH6.55.50 1020 3040 5060Time (hrs)8pH760 2000LVFA3,TOC(mg/1)-250—4000ORP(mV)-5000Time (hrs)•acetate propionate•isobutyratebutyrateA2-methylbutyrateisovalerateXvalerate lactate±TOC•pHCORP0 1020 3040 5060249Table Al: Concentration ofsludges for all batch experimentsBatch# ProcessConcentrationFeed sludge ConcentrationIncubationsludge (%)source (%)temperaturesource(°C)3 A side TAD1.51 Primary2.37454 A side TAD1.63 n/an/a 456 A side TAD1.97 n/an/a 458 A side TAD1.95 n/an/a452 A side TAD1.71 n/an/a457 A side TAD1.89 n/an/a 459 A sideTAD 1.86Primary1.81 45l°FntSide stream1.82 Primary2.1620fermenter1OTADA side TAD1.96 Primary2.16 45ll(Aside)A side TAD1.63 Primary1.12451l(B side)B side TAD1.66 Primary1.12 4513 Salmon2.1 Salmon Arm5.07 (prim.) 59Arm 3rd cellprimary and3.85 (sec.)ATADsecondary250Appendix B: 3x3 Pilot ScaleTAD ResultsFigureB1.1Figure B1.2Figure B1.3FigureB1.4Figure B1.5Figure B1.6FigureB1.7FigureB1.8FigureB1.9FigureB1.10FigureB1.11FigureB1.12FigureB2.1FigureB2.2FigureB2.3FigureB2.4FigureB2.5FigureB2.6FigureB2.7FigureB2.8256256256257257257258258258259259259260260260261261261262262A side [Acetate] with 4.5d SRTA side [Acetate] with4.5 d SRTA side [Acetate] with4.5 d SRTA side [Propionate]with 4.5 d SRTA side [Propionate]with 4.5 d SRTA side [Propionate] with4.5 d SRTA side [Isobutyrate]with4.5 d SRTA side [Isobutyrate]with 4.5 d SRTA side [Isobutyrate]with 4.5 d SRTA side [Isovalerate]with 4.5 d SRTA side [Isovalerate]with 4.5 d SRTA side [Isovalerate] with4.5 d SRTB side [Acetate] with 3d SRTB side [Acetate]with 4.5 d SRTB side [Acetate]with 6 d SRTB side [Propionate]with 3 d SRTB side [Propionate]with4.5 d SRTB side [Propionate]with 6 d SRTB side [Isobutyrate]with 3 d SRTB side [Isobutyrate]with4.5 d SRT251FigureB2,9Figure B2.10Figure B2.11Figure B2.12FigureB2.13FigureB2.14Figure B2.15FigureB3.iFigureB3.2Figure B3.3FigureB3.4FigureB3.5FigureB3.6FigureB3.7FigureB3.8FigureB3.9FigureB3.10Figure B3.11FigureB3.12FigureB4. 1FigureB4.2FigureB4.3262263263263264264264265265265266266266267267267268268268269269269B side [Isobutyratel with 6 dSRTB side [Isovalerate] with 3 d SRTB side [Isovalerate] with 4.5 dSRTB side [Isovalerate] with 6 d SRTB side [Butyrate] with 3 d SRTB side [Butyrate] with 4.5 d SRTB side [Butyrate] with 6 d SRTPrimary [Acetate] with B side 3 d SRTPrimary [Acetate] with B side 4.5d SRTPrimary [Acetate] with Bside 6 d SRTPrimary [Propionate] withBside 3 d SRTPrimary [Propionate] withBside 4.5 d SRTPrimary [Propionate] with B side6 d SRTPrimary [Isobutyrate] withB side3 d SRTPrimary [Isobutyrate] withBside 4.5 d SRTPrimary [Isobutyrate] withB side6 d SRTPrimary [Butyrate] with B side3 d SRTPrimary [Butyrate] with B side4.5 d SRTPrimary [Butyrate] withBside 6 d SRTA side TOC with4.5 d SRTA side TOC with 4.5 d SRTA side TOC with 4.5 d SRT252Figure B5. 1FigureB5.2Figure B5.3Figure B6. 1FigureB6.2Figure B6.3Figure B7.1FigureB7.2FigureB7.3Figure B8. 1FigureB8.2FigureB8.3FigureB9. 1FigureB9.2FigureB9.3FigureB10. 1Figure B10.2FigureB10.3FigureB11.1FigureB11.2Figure B11.3FigureB12. 1270270270271271271272272272273273273274274274275275275276276276277B side TOC with 3 d SRTB side TOC with 4.5 d SRTB side TOC with 6 d SRTPrimary TOC with B side 3 d SRTPrimary TOC with B side 4.5 d SRTPrimary TOC with B side 6 d SRTA side pH with 4.5 d SRTA side pH with4.5 d SRTA side pHwith4.5 d SRTB side pHwith 3 d SRTB side pHwith 4.5 d SRTB side pHwith 6 d SRTA side solids levels withB side3 d SRTA side solids levels withB side4.5 d SRTA side solids levels withB side 6 d SRTB side solids levels with3 d SRTB side solids levels with 4.5 dSRTB side solids levelswith 6 d SRTPrimary solids levelswith B side 3 d SRTPrimary solids levels with Bside 4.5 d SRTPrimary solids levels withBside 6 d SRTA side air flowwith 4.5 d SRT253Figure B12.3Figure B13.1Figure B13.2Figure B13.3FigureB14. 1FigureB14.2FigureB14.3FigureB15.1FigureB15.2Figure B15.3FigureB16.1FigureB16.2FigureB16.3FigureB17.1FigureB17.2FigureB17.3Figure B18.1FigureB18.2FigureB18.3FigureB19.1277277278278278279279279280280280281281281282282282283283283284FigureB12.2 A side air flow with 4.5 d SRTA side air flow with 4.5 d SRTB side air flowwith 3 d SRTB side air flow with 4.5 d SRTB side air flow with 6 d SRTA side mixerRPMwith 4.5 d SRTA side mixerRPM with 4.5 d SRTA side mixerRPMwith 4.5 d SRTB side mixerRPM with 3 d SRTB side mixerRPMwith 4.5 d SRTB side mixer RPM with 6 d SRTA side actual SRT fornominal B side 3 d SRTA side actual SRT fornominal B side 4.5 d SRTA side actual SRT fornominal B side 6 d SRTB side actual SRT for nominal B side 3 d SRTB side actual SRT fornominal B side 4.5 d SRTB side actual SRT for nominal B side 6 d SRTTemperature Variations, Sides A and B, run 1ORP Variations, Sides A and B, run 1ORP FFTs, Sides A and B, run 1Temperature Variations, Sides A and B, run 2Figure B19.2 ORP Variations, Sides A and B, run 2 284254Figure B19.3 ORP FFTs, Sides A and B, run 2284Figure B20. I Temperature Variations, Sides A and B, run 3285FigureB20.2 ORP Variations, Sides A and B, run 3 285Figure B20.3 ORP FFTs, Sides A and B, run 3285FigureB21.1 Temperature Variations, Sides A and B, run 4 286FigureB21.2 ORP Variations, Sides Aand B, run 4 286FigureB21.3 ORP FFTs, Sides A and B, run 4 286Figure B22. 1 Temperature Variations, Sides A and B, run 5 287FigureB22.2 ORP Variations, Sides Aand B, run 5 287FigureB22.3 ORP FFTs, Sides A and B, run 5 287Figure B23. 1 Temperature Variations, Sides A and B, run 6 288FigureB23.2 ORP Variations, Sides A and B, run 6 288FigureB23.3 ORP FFTs, Sides A and B, run 6 288Figure B24. 1 Temperature Variations, Sides A and B, run 7 289FigureB24.2 ORP Variations, Sides A and B, run 7 289FigureB24.3 ORP FFTs, Sides A and B, run 7 289FigureB25. 1 Temperature Variations, Sides A and B, run 8 290FigureB25.2 ORP Variations, Sides A and B, run 8 290FigureB25.3 ORP FFTs, Sides A and B, run 8 290FigureB26. 1 Temperature Variations, Sides A and B, run 9 291FigureB26.2 ORP Variations, Sides A and B, run 9 291FigureB26.3 ORP FFTs, Sides A and B, run 9 291255FigureB27.I Temperature Variations,Sides A and B, run 10292FigureB27.2 ORP Variations, Sides Aand B, run 10292FigureB27.3 ORP FFTs, Sides Aand B,run 10292256Figure Bi.1: A side [Acetatej with 4.5 d SRT800T700 -600•. 500 +• 400AMed air, 5300_____200 IMedair,2Med air, 6100_______________________________0 5 10 15Time (days)Figure Bi.2: A side [Acetate] with 4.5 d SRT800Med air, 10700600______• Med air, 1E 500400AMed air, 4300• Med air, 7200Med air, 9— 1000 5 10 15Time (days)Figure Bi.3: A side [Acetate] with 4.5 d SRT800700I600I________E 500 -IMedair,3___Med air, 8300_________________U200t1000 5 10 15Time (days)257Figure B1.4: A side [Propionate] with4.5 d SRT602 50!40Medair,2AMed air, 5Med air, 60 5 1015Time (days)Figure Bi .5: A side [Propionate]with 4.5 d SRT60Med air, 102 50.. 40Medair,130AMed air, 4Med air, 710Medair,90 510 15Time (days)Figure 81.6: A side [Propionate]with 4.5 d SRT6050________!4oMedair,331___j201Medair 80100I0 510 15Time (days)258Figure Bi.7: A side [Isobutyratelwith 4.5 d SRT502- 40E• Medair,230AMed air, 5, 20 +Med air, 6101______________0 510 15Time (days)Figure BI.8: A side [bobutyrate]with 4.5 d SRT50T,4O1\_• Med air, 1— 30AMed air, 4, 20I °-Medair,10• Med air, 7.10°Med air,900 510 15Time (days)Figure Bi.9: A side[Isobutyrate] with 4.5 d SRT5040________EMed air, 33020Med air, 8.00 510 15Time (days)2590 5 1015Figure Bi.11: A side [Isovalerate] with 4.5 dSRT10080E6040200___0 5 1015Time (days)5 10Time (days)Figure Bi.10: A side [Isovalerate] with 4.5 d SRT1008060. 40(U0.! 20-:• Med air, 2AMed air, 5• Med air, 6Time (days)Med air, 10Med air, 1AMed air, 4• Med air, 7—a-——- Med air, 9Figure B1.12: A side [Isovalerate] with4.5 d SRT100T: 80E60. 40(U>0200Medair,3Med air, 8015260Figure B2.1: B side [Acetate]with 3 d SRT10008001•Loair,2600AMed air, 5400• Hiair,602000 5 1015Time (days)Figure B2.2: B side [Acetate] with4.5 d SRT10000 air, 10800•Loair,1600AMed air, 44 400• Hiair,70200Hi air, 90 510 15Time (days)Figure B2.3: B side[Acetate] with 6 d SRT1000T2800! 600k°Loair,3400• Hiair,80I2OO00 510 15Time (days)261Figure B2.4: B side [Propionatejwith 3 d SRT600140:0 5 1015Time (days)Figure B2.5: B side [Propionate]with 4.5 d SRT6004000 510 15Time (days)Figure B2.6: B side [Propionate]with 6 d SRT600TLoair,3• Hiair,8E00.20.200•Loair,2AMed air, 5• Hiair,6EI-’00.20.2000—°——— 0 air, 10•Loair,1AMed air, 4• Hiair,7—°-—— Hi air, 9E4002000-0 510 15Time (days)2626040.o 20IFigure B2.7: B side [Isobutyrate] with3 d SRT80T602:A•••Loair,2AMed air, 5• Hiair,60 510 15Time (days)Figure B28: B side [Isobutyrate]with 4.5 d SRT800—a———0 air, 10•Loair,1AMed air, 4• Hiair,7—°—-— Hi air,90 510 15Time (days)Figure B2.9: B side [Isobutyrate]with 6 d SRT801260-a 20U,Loair,3• Hiair,80 510 15Time (days)263Figure B2.10: B side [Isovalerate] with 3 d SRT1502. iooLoair,2AMed air, 5a.€ f’______• Hiair,63. ,00 5 10 15Time (days)Figure 2.11: B side [Isovalerate] with 4.5 d SRT150——- 0 air, 102•Loair,1.!.100AMed air, 4a0 I• Hiair,73.0°Hi air, 90 5 1015Time (days)Figure B2.12: B side [Isovalerate]with 6 d SRT1502.!iooIILoair,3I___I• Hiair,85000—e------• • • • • • • • I0 5 1015Time (days)264Figure B2.13: B side [Butyrate] with 3 d SRT150_jioo• Loair,2AMedair,5• Hiair,6Time (days)Figure B2.14: B side [Butyrate] with 4.5 d SRT150°-—Oair,10iooLoair,1AMedair,450 Hiair,7Hi air, 900 5 1015Time (days)Figure B2.15: B side [Butyrate] with 6d SRT150J100— ILo air, 3• Hiair,80• • • • • • . . . •‘;—0 510 15Time (days)265Figure 83.1: Primary [Acetate] with Bside 3 d SRT2002•Loair,2AMed air, 5.100(V• Hiair,6U0 5 1015Time (days)Figure B3.2: Primary [Acetate] with B side4.5 d SRT20020air,10Loair,1AMed air, 4100• Hiair,7UHi air, 90 510 15Time (days)Figure B3.3: Primary [Acetate] with Bside 6 d SRT200Loair,3:T 100• Hiair,8U____________________010 510 15Time (days)2665000500010101515Figure B3.4: Primary [Propionate] with B side3 d SRT150-J!100C020.•Loair,2AMed air, 5• Hiair,65Time (days)Figure B3.5: Primary LPropionate] with B side4.5 d SRT150.g100C00.2a-—D———0 air, 10•Loair,1AMed air, 4• Hiair,7—0-———Hi air, 95Time (days)E-IC0a.2a-Figure B3.6: Primary [Propionate] withB side 6 d SRT150100500°Loair,3• Hiair,80 5 1015Time (days)267-IEa0EIFigure B3.7: Primary [Isobutyrate] with B side 3 dSRT6420•Loair,2AMed air, 5• Hiair,60 5 10Time (days)15Figure B3.8: Primary [Isobutyrate] with B side 4.5d SRT6420————0 air, 10•Loair,1AMed air, 4• Hiair,7----——°-——— Hi air, 90 5 10Time (days)15Figure B3.9: Primary [Isobutyrate]with B side 6 d SRT6.0000 5 1015Time (days)268Figure B3.10: Primary [Butyrate] with B side 3 d SRT2015___Loair,210A.Medair,5• Hiair,6-00 5 10 15Time (days)Figure B3.11: Primary [Butyrate] with B side 4.5 d SRT200 air, 1015Loair,110AMedair,4• Hiair,7Hi air, 90 5 10 15Time (days)Figure B3.12: Primary [Butyrate] with B side 6 d SRT20,15• Hiair,8[&Loair,300 5 10 15Time (days)269Figure B4.1: A side TOC with 4.5 d SRT1000800600AMed air, 5Med air 2g 400Med air, 6200I I0 5 10 15Time (days)Figure B4.2: A side TOC with 4.5 d SRT1000—°——Med air, 108003• Med air, 1p600__AMed air, 4g400• Med air, 7b200Med air, 90 5 1015Time (days)Figure B4.3: A side TOC with 4.5 d SRT10008002600 Medair,3g 400• Med air, 8b20000 5 10 15Time (days)270Figure B5.1: B side TOC with 3 d SRT10008003 •Loair,2600AMed air, 5g 400• Hiair,6b200 --________________01 II0 5 10 15Time (days)Figure B5.2: B side TOC with 4.5 d SRT12000 air, 1010002 •Loair,1800AMedium air, 4200 I600__• Hiair,7b400°Hi air, 90 5 10 15Time (days)Figure B5.3: B side TOC with 6 d SRT10001800126001Loair,3Hiair,84oo1______bI20010 I0 5 10 15Time (days)271Figure B6.3: Primary TOC with B side 6 dSRT500400300200I-1000Figure B6.1: Primary TOC with B side 3 d SRT500T400300200 ib100•Loair,2AMed air, 5• Hiair,60 5 10 15Time (days)EC,0IFigure B6.2: Primary TOC with B side 4.5 d SRT0air,10400•Loair,1300AMedium air, 4200• Hiair,7100Hi air, 90I I0 10 15Time (days)5Lo air, 3• Hiair,80 5 1015Time (days)272Figure B7.1: A side pH with 4.5 d SRT8A74f54j4 Med air, 2AMedair,5Med air, 660 5 10 15Time (days)Figure B7.2: A side pH with 4.5 d SRT8Medair 10AMedair,.4Med air, 7Med air, 96I I0 5 10 15lime (days)Figure 87.3: A side pH with 4.5 d SRT9-Med air, 3/\•0 15Time (days)B.272273Figure B8.1: B side pH with 3 dSRT7.5 -•Loair,255AMed air, 5• Hiair,60 510 15Time (days)Figure B8.2: B side pH with 4.5 dSRT0 air, 10AMed air, 4• Hiair,7°Hi air, 95.5 II0 510 15Time (days)Figure 88.3: B side pH with6 d SRT8Lo air, 30. I• Hi air, 86-l0 510 15Time (days)274Figure B9.1: A side solids levels with B side 3 d SRT2.:..:------ Med air,TVSii. II0 5 10 15“° Hi air, TVSTime (days)Figure B9.2: A side solids levels with B side4.5 d SRT0 air, TS•Lo air, TS2.25AMed air, TSE• Hi air (1), TS1 75Hi air (2) TS1 250 D 0 air TVS•/--—• -I--—Lo air, TVS— Med air, TVS0.75I0 510 15Hiair(1),TVSTime (days)Hi air (2), TVSFigure B9.3: A side solids levelswith B side 6 d SRT2.5-JE°Loair,TS2• Hiair,TS.-.•ELoair,TVSC0 i..Hiair, TVSCl)1I0 510 15Time (days)275Figure B1O.1: B side solidslevels with 3 d SRT2.52AMed air,TS:-- Med air, TVS1I0 510 15- Hi air, TVSTime (days)Figure B1O.2: B side solidslevels with 4.5 d SRT0 air, TSLo air, TS2.5A Med air,E •Hiair(1),TS2Hi air,TS1 51_.— : —.Me air IS0 510 15Hi air (1), TVSTime (days)Hi air (2), TVSFigure B1O.3: B side solidslevels with 6 d SRT2.5T-I21____1.5 +D -G.D\\,,/aD—Lo air, TVSHiair,TVS1I0 510 15Time (days)276Figure Bi 1 .1: Primary solids levels with Bside 3 d SRT3.5T—2Loair,TSAMed air, TS2.512• Hiair,TS1 5- -- --- Lo air TVSMed air, WS1 I I0 510 15Hi air, TVSTime (days)Figure 811.2: Primary solids levels withB side 4.5 d SRT0 air. TS•Lo air, TSAMed air, TS• Hi air (1), TS2.5..:.—0——Hi air (2), TS2;....‘h.. :.. ...-Oair,TVSø:AMe illS0 510 15Hiair(1),TVSTime (days)•--0--- Hi air (2), TVSFigure Bi1.3: Primary solids levelswith B side 6 d SRT3.5 --JE3 Loair,TSo2.5.., Hiair, TSGLoairTVS1.5 ..Hi air, TVS0______________1II0 510 15Time (days)277Figure B12.1: A side air flowwith 4.5 d SRT140120• Medair,2AMedair,5100 Medair,680 II0 510 15Time (days)Figure Bi2.2: A side airflowwith 4.5 d SRT150Med air, 10130Med air, 1AMed air, 4Med air, 7110Med air, 990 I0 510 15Time (days)Figure B12.3: A side airflowwith 4.5 d SRT140 -120-’E• • • jMed air, 3I100Med air, 880I0 510 15Time (days)278Figure Bi3.1: B side air flow with 3 d SRT180160AMedair,5140Hi air, 61200 5 10 15Time (days)Figure Bi3.2: B side airflow with 4.5 d SRT170AMed air, 4150Hi air, 7130110 I° Hi air, 90 5 10 15Time (days)Figure B13.3: B side air flow with 6 d SRT200180___160[Hiair,8140 I I0 5 10 15Time (days)279Figure B14.1: A side mixerRPM with 4.5 d SRT900880 i• Med air, 2860AMed air, 5840 +• Med air, 6800I820 10 510 15Time (days)Figure B14.2: A sidemixer RPM with 4.5d SRT900bMed air, 10880• Med air, 1860AMed air, 4800I840• Med air, 7820°Med air, 90 510 15Time (days)Figure 814.3: A sidemixer RPM with 4.5 dSRT900 -880--860Med air, 30840 -i-• Med air, 8820 +800 I0 510 15Time (days)280Figure B15.1: B side mixer RPM with 3 d SRT900880860840820800•Loair,2A-Med air, 5• Hiair,60 5 10Time (days)15Figure B15.2: B side mixer RPM with 4.5 d SRT860840820800780760 -0—D——0 air, 10•Loair,1AMed air, 4• Hiair,7—°-—-— Hi air, 95 10Time (days)15Figure B15.3: B side mixer RPM with6 d SRT10501000950900850800750Loair,3• Hiair,80 510 15Time (days)281Figure B16.1: A side actual SRT for nominal B side 3 d SRT76• Med air, 2:Medair,5• Med air, 63___________________2I I0 5 10 15Time (days)Figure B16.2: A side actual SRT for nominal B side 4.5 dSRT76 Med air 10:::::::3• Medair,72 IIMedair,90 5 10 15________________Time (days)Figure Bi6.3: A side actual SRT for nominal B side 6d SRT7:::::::Time (days)282Figure 817.1: B side actual SRT fornominal B side 3 d SRT76Loair,2‘-5___AMedair,5Hiair,6Time (days)Figure B17.2: B side actual SRT fornonunal B side 4.5 dSRTTE743Hiair,72IIHiair90 510 15_______________Time (days)Figure 817.3: B side actual SRTfor nominal B side 6 d SRTI Loair,34Hiair,32I I0 510 15Time (days)0I..IU)04).c90283Figure 818.1: Temperature Variations, Sides A and B, Run 148 -46444240/NASide0 c’1cO 0Time (days)00Figure B18.2: ORP Variations, Sides A and B, Run 1%%wj000(0Time (days)0U)Figure B18.3: ORP FFTs, Sides Aand B, Run 10 U) U) U)() U) U) U)CC40 (‘1C’) IflFrequency (1/h)284C-,II00E0I->00Time (days)Figure B19.1: Temperature Variations, Sides A and B, Run 24644424038Side0 c’J U) co 0Time (days)0U)cJFigure Bi92: ORP Variations, Sides A and B, Run 200UA0 (0 0Figure B19.3: ORP FFTs, Sides A and B, Run 20_____0I.!_ 0(5C490 1i—A0 U) U) U) c•) U) U) U)‘:c•• F-0 c•.J c) U)(0Frequency (1/h)285I5048B20.1: Temperature Variations, Sides A and B, Run 34644420 CD 0Time (days)00B20.2: ORP Variations, Sides A and B, Run 30I.- -00 c’J CD 0Time (days)Figure B20.3: ORP FFTs, Sides A and B, Run 30. ..0SeSide.0 Lfl U) U) C’) It) U)It)r c’i0 C’)U)CoFrequency (1/h)286Figure B21 .1: Temperature Variations,Sides A and B, Run 4SideA side444240I I I I I I I I II I Ioc.i 1DTime (days)Figure B21.2: ORP Variations, Sides Aand B, Run 400C0Eo01—A00I I I I II I I I I I I I I IC-I.6 66 6 6ao 0 00 0 — —Time (days)Figure 821.3: ORP FFIs,Sides A and B, Run 4JB0JASid0 If)If) If) C)If) If) U)C- C’JC-‘3-If)Frequency (1/h)28746I..440E4).—42c..l CDFigure B22.3: ORP FFTs, Sides Aand B, Run 548Figure B22.1: Temperature Variations, Sides A and B, Run 5AVNr400Time (days)00C?Figure B22.2: ORP Variations, Sides A and B,Run 5000InSIC’4Time (days)CD0______________________C’41JAS0 In Ct)Ct) C In In InCD0C’) InFrequency (1/h)288Figure 823.1: Temperature Variations, Sides A and B, Run 6—.4240I I I I I I IoCDTime (days)Figure 823.2: ORP Variations, Sides A and B,Run 600a.0%&.A0124 i I I II I‘0c%JCDTime (days)Figure 823.3: ORP FFTs, Sides Aand B, Run 60C4C)¶r‘I0ccIIASide-Frequency (1/h)289ç0.-Se48Figure B24.1: Temperature Variations, Sides A and B, Run 7546/‘BSide-\M7Z1!444)0.E!4240 -0 C’1 CDTime (days)Figure B24.2: ORP Variations, Sides A and B, Run 700C>18000LfloLL(‘1 CDTime (days)C’JFigure B24.3: ORP FFTs, Sides A and B, Run 7A.0 L) 1.0 C’) tO1.0 tO CDr- r-.0 (‘4 C’)U)Frequency (1/h)290Figure B25.1: Temperature Variations0Sides A and B, Run 848Se46ASide4)444)E421I I I I I I IIoCD2Time (days)Figure B25.2: ORP Variations,Sides A and B, Run 800CB00o i I I II2Time (days)Figure B25.3: ORP FFTs,Sides A and B, Run 804)0- I0—0 CDCD CD C’) CDIt) It) CDF-.0C’J C’)It)Frequency (1/h)48G464)S“4)0.E4)42400004)(V4)(V4)(V(V291Figure B26.1: Temperature Variations, Sides A and B, Run 9Time (days)Figure B26.2: ORP Variations, Sides A and B, Run 9a0 0 00 — —CO 00BsL+rL400000“ASde’*’Time (days)Figure B26.3: ORP FFTs, Sides A and B, Run94)=0.E4)4)00U) U) C’) U) U) U)r C C’-0C’) U)Frequency (1/h)292Figure B27.1: Temperature Variations, Sides A and B, Run10484610.40I Io C4 (0 0 C4Time (days)Figure B27.2: ORP Variations, Sides A and B, Run 1000..B0or’A S1è0o I I I I I I I I I ICoTime (days)Figure 27.3: ORP FFIs, Sides A and B, Run 100CD.—ULBSe0Side 1o0 (0 CD CDC’) (0 (0 (0c’J .a (‘1C’) U)Frequency (1/h)293Appendix C: PreliminaryPilot Scale TAD ResultsFigureCl.1: Temperatureprofiles over 1 SRT underthetransition condition(0.28 V/V-h)294Figure C1.2: ORP profilesover 1 SRT under thetransition condition(0.28 V/V-h)294Figure C2.1: Temperature profilesover 1 SRT underthe aerobiccondition(0.6 V/V-h)295Figure C2.2: ORP profilesover 1 SRT undertheaerobic condition (0.6 V/V-h)295Figure C3.1: Temperatureprofiles over 1 SRTunderthe microaerobic condition(0 V/V-h)296Figure C3.2: ORP profilesover 1 SRT underthemicroaerobic condition(0 V/V-h)296Figure C4.1: [VFA] in stage1 ofTAD andtotal solidsin primary sludgeundertheaerobic condition (0.6V/V-h)297Figure C4.2: [VFAI in stage1 ofTAD andtotal solidsin primary sludge underthetransition condition(0.6 V/V-h)297Figure C4.3: [WA] in stage1 ofTAD andtotal solidsin primary sludgeunderthemicroaerobic condition(0.6 V/V-h)297Figure CS.1: [VFAI inthe primary sludgeunderaerobic conditions(0.6 V/V-h) 298Figure C5.2: [WA] in theprimary sludge underthe transition condition(0.28 V/V-h)298Figure CS.3: [WA] inthe primary sludgeunder the microaerobiccondition(0 V/V-h)298294656O55.5oa)Ea)-4O35Figure C1.1: Temperatureprofiles over 1 SRT underthe transitioncondition(0.28 V/V-h)12nd stage- -.....-........::.....--:‘/— /7 —/7///////1 St stage//I I I•o 20 40 6080 100 120 140160Time (h)200>0-200-300FigureCl.2: ORP profilesover 1 SRT underthetransition condition(0.28 V/V-h)E....,:.i4Z.E7jj4:.j. I•II I I0 20 4060 80 100120 140 160Time (h)— ORP I --- ORPIA - ORP2 ORP 2A129565G60554-’CucD4540‘-35300___7-.EzEE1.-1‘J-....‘7 ..--...-- -..1J -—•-I I I II I I0 2040 6080 100 120140 160Time (h)— ORP I ----ORP IA--ORP 2ORP 2AFigure 2.1: Temperatureprofiles over 1 SRT undertheaerobic condition(0.6 V/V-h).2nd stage:E: 2.. Z;;7?ZlststageI I II I I I I0 2040 60 80100Time (h)120 140 160Figure 2.2: ORPprofiles over1 SRTunderthe aerobiccondition (0.6 V/V-h).200100//I1’1’ I/71//‘IILi-200-30014296Figure C3. 1: Temperatureprofiles over 1 SRT underthemicroaerobiccondition (0 V/V-h).656055.5045‘40352nd stage/ /7zz...:7Zz ;2.tz..1st stage- - -.. -I I II II I ITime (h)Figure C3.2:microaerobic200ORP profiles over1 SRT underthecondition (0 V/V-h).100-200—ORP 1---ORP IA- ORP 2 ORP2A30I -l0 2040 60 80100 120 140160100/1L .7i1I/II] .4........._...44Jz,*..--300-400. -.I I I0 20 4060 80100 120 140160Time (h)297251.922011.8.l.7<101(I)LI>5.0I-0L—Acetate — Propionate— Total solidsFigure C4.2: [VFA]in stage 1 ofTAD andtotalsolids inprimary sludgeunderthetransition condition(0.28V/V-h).21.91.81.71.61.51.41 I I I1.31 2 3 4 5 67 8 9101112Time (d)Acetate— PropionatePrimarytotal solidsFigure C4.3: [VFA] instage 1 ofTAD andtotalsolids inprimary sludge underthe microaerobiccondition(0 V/V-h).1000800600400200 -ot0 2 46 8 10Time (h)1— Acetate— PropionateIsobutyrate--Primarytotalsolids— 2-methylbutyrate-IsovalerateFigure C4. 1: [VFA] in stage1 ofTAD andtotal solids inprimary sludge under theaerobic condition (0.6 V/V-h)./\...I 1.50 2 46 8 10 12Time (d)-J0)U>200150100500—0)Li>120100-J0)6040200120100-J6040200298Figure C5. 1: [VFA] in theprimary sludge under aerobicconditions (0.6 V/V-h).1 2 3 4 5 67 8 9 10 11 12ii..’—Acetate —1ropionateIsobutyrate--Butyrate2-methylbutyrate--IsovalerateFigure C5.2: [VFAI inthe primary sludgeunderthetransitioncondition(0.28 V/V-h).1 2 3 45 6 7 8 910(I.— AcetateIsobutyrate--Gutyrate2-methylbutyrate-‘-Isovalerate-J0)EU>Figure C5.3: [VFA] intheprimarysludgeunderthemieroaerobiccondition(0 V/V-h).120100806040200-••-1 2 3 4 5 67 8 9 10 11r;... ii.12—Acetate-÷ropionate--Isobutyrate--- Butyrate2-methylbutyrate--Isovalerate299Appendix D: TestChemicalsTableDl: Chemical structure ofcompounds.Compound StructureAcetic acid CH3COOHEthanol CH2OHPropanol CH3OHPropionic acid CM3CH2COOHLactic acid CHCHOHCOOHPyruvic acid CH3COCOOHButyric acid (CH2)COOHIsobutyric acid (CH3CHCOOHValeric acid CH(CHCOOHIsovaleric acid (CH)2CHCHCOOHGlucose CHO(CHOH)4CHOHDextrin (C6H1005)NxH2OLinoleic acid C18302-methylbutyric acid CH3CHCOOHToxicants:2,4-dinitrophenol (N02)C6H30HFluoride FSodium cyanide NaCN

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