"Applied Science, Faculty of"@en . "Civil Engineering, Department of"@en . "DSpace"@en . "UBCV"@en . "Elefsiniotis, Panagiotis"@en . "2008-09-16T17:23:46Z"@en . "1993"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "This research explored the effect of certain operational and environmental parameters on the acid-phase anaerobic digestion of primary municipal sludge. The operational parameters included the hydraulic retention time (HRT) and the solids retention time (SRT), while pH, reactor configuration and influent characteristics were the environmental factors of interest. Moreover, an attempt was made to identify the most significant metabolic pathways involved in the conversion of the major components of primary sludge (carbohydrates, proteins and lipids) to short-chain volatile fatty acids (VFAs) and other soluble end-products. The experiments were conducted using two continuous-flow three-liter reactors having different configurations: a completely mixed reactor (CMR) with a clarifier and sludge recycle, and an upflow anaerobic sludge blanket (UASB) reactor. Both systems were run at an ambient liquid temperature between 18 and 22 \u00B0C. The research program evolved into the following four stages: In Stage 1 the role of HRT was investigated, while Stage 2 focused on the effect of SRT. The issues of replication and the source of influent sludge were the targets in Stage 3. Finally, in Stage 4 the effect of pH was explored. During the last stage, dilute solutions (0.02N) of HC1 or NaOH were continuously added through an automated pump system to keep the pH at selected values. Favorable conditions for acidogenic digestion were established and maintained resulting, generally, in high VFA and low gas generation rates. The net VFA concentration and the specific production rate increased, in both reactors, with an increase in HRT up to 12 hours, but decreased slightly at longer HRTs. The same pattern was followed not only by the COD concentration but also by the specific solubilization rates of COD and TOC. Variation in SRT had a profound effect on VFA production rate only at the lower (5 day) SRT. At longer SRTs a plateau in acid production appeared to be reached. A decrease in pH from 5.1 to 4.5 did not have an effect on the rate of VFA generation, but an increase to pH 6.1 resulted in significantly lower rates (25 to 30%) of acid production. Acetic acid and propionic acid were the most prevalent VFAs produced and accounted for 45 and 31% (on average) of the total respectively. Butyric acid followed with an average value of 9%. The percent VFA distribution appeared to be independent of HRT, but it was a function of both SRT and pH. Besides VFAs, small amounts of formic acid, ethanol and lactic acid were regularly detected in both systems. Results showed that the steady-state operation of the acid-phase digestion can be replicated and that the seasonal changes in the study (summer-winter) did not affect the process. The use of a different source of influent sludge had an effect on lipid and carbohydrate utilization patterns, which was also reflected in the corresponding VFA production rates. In general, protein degradation percentages were moderate and significantly lower than those obtained for the other two groups of organic compounds. The utilization of all three substrates increased with an increase in HRT, but (with the\r\nexception of proteins) was essentially independent of SRT. The reactor configuration played a role in substrate degradation as well. Although both systems showed a fairly similar behavior in protein utilization, the degradation of carbohydrates and lipids was distinctly and consistently different. Lipids were broken down more efficiently in the CMR system, while higher rates of carbohydrate dissimilation were observed in the UASB reactor."@en . "https://circle.library.ubc.ca/rest/handle/2429/2049?expand=metadata"@en . "13355089 bytes"@en . "application/pdf"@en . "e required stanTHE EFFECT OF OPERATIONAL AND ENVIRONMENTALPARAMETERS ON THE ACID-PHASE ANAEROBIC DIGESTIONOF PRIMARY SLUDGEbyPANAGIOTIS ELEFSINIOTISDipl. Eng. (Civil Engineering), National Technical Univ. of Athens, Greece, 1979M.A.Sc. (Environmental Engineering), The University of Toronto, 1982A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Department of Civil Engineering)We accept this thesis as conformingTHE UNIVIV SITY BRITISH COLUMBIAJanuary 1993Panagiotis ElefsiniotisIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of Civil EngineeringThe University of British ColumbiaVancouver, CanadaDate ^January 25, 1993DE-6 (2/88)ABSTRACTThis research explored the effect of certain operational and environmentalparameters on the acid-phase anaerobic digestion of primary municipal sludge. Theoperational parameters included the hydraulic retention time (HRT) and the solidsretention time (SRT), while pH, reactor configuration and influent characteristicswere the environmental factors of interest. Moreover, an attempt was made toidentify the most significant metabolic pathways involved in the conversion of themajor components of primary sludge (carbohydrates, proteins and lipids) to short-chain volatile fatty acids (VFAs) and other soluble end-products.The experiments were conducted using two continuous-flow three-liter reactorshaving different configurations: a completely mixed reactor (CMR) with a clarifierand sludge recycle, and an upflow anaerobic sludge blanket (UASB) reactor. Bothsystems were run at an ambient liquid temperature between 18 and 22 \u00C2\u00B0C. Theresearch program evolved into the following four stages: In Stage 1 the role of HRTwas investigated, while Stage 2 focused on the effect of SRT. The issues ofreplication and the source of influent sludge were the targets in Stage 3. Finally, inStage 4 the effect of pH was explored. During the last stage, dilute solutions (0.02N)of HC1 or NaOH were continuously added through an automated pump system to keepthe pH at selected values.Favorable conditions for acidogenic digestion were established and maintainedresulting, generally, in high VFA and low gas generation rates. The net VFAconcentration and the specific production rate increased, in both reactors, with aniiABSTRACT^ iiiincrease in HRT up to 12 hours, but decreased slightly at longer HRTs. The samepattern was followed not only by the COD concentration but also by the specificsolubilization rates of COD and TOC.Variation in SRT had a profound effect on VFA production rate only at thelower (5 day) SRT. At longer SRTs a plateau in acid production appeared to bereached.A decrease in pH from 5.1 to 4.5 did not have an effect on the rate of VFAgeneration, but an increase to pH 6.1 resulted in significantly lower rates (25 to 30%)of acid production.Acetic acid and propionic acid were the most prevalent VFAs produced andaccounted for 45 and 31% (on average) of the total respectively. Butyric acidfollowed with an average value of 9%. The percent VFA distribution appeared to beindependent of HRT, but it was a function of both SRT and pH. Besides VFAs, smallamounts of formic acid, ethanol and lactic acid were regularly detected in bothsystems.Results showed that the steady-state operation of the acid-phase digestion canbe replicated and that the seasonal changes in the study (summer-winter) did notaffect the process.The use of a different source of influent sludge had an effect on lipid andcarbohydrate utilization patterns, which was also reflected in the corresponding VFAproduction rates.In general, protein degradation percentages were moderate and significantlylower than those obtained for the other two groups of organic compounds. TheABSTRACT^ ivutilization of all three substrates increased with an increase in HRT, but (with theexception of proteins) was essentially independent of SRT.The reactor configuration played a role in substrate degradation as well.Although both systems showed a fairly similar behavior in protein utilization, thedegradation of carbohydrates and lipids was distinctly and consistently different.Lipids were broken down more efficiently in the CMR system, while higher rates ofcarbohydrate dissimilation were observed in the UASB reactor.TABLE OF CONTENTSPAGEABSTRACT^ iiTABLE OF CONTENTS^ vLIST OF TABLES viiiLIST OF FIGURES^ xGLOSSARY OF TERMS xiiACKNOWLEDGEMENTS^ xiii1. INTRODUCTION 12. LITERATURE REVIEW^ 32.1. AN OVERVIEW OF ANAEROBIC DIGESTION^ 32.2. WASTEWATER COMPOSITION^ 62.2.1. Carbohydrates ^ 72.2.2. Proteins 102.2.3. Lipids ^ 112.3. PATHWAYS OF VFA FORMATION^ 122.3.1. Carbohydrate Metabolism 13a) Hydrolysis ^ 13b) Fermentation of Sugars^ 142.3.2. Protein Metabolism 17a) Hydrolysis ^ 17b) Amino Acid Fermentation^ 182.3.3. Lipid Metabolism 20a) Hydrolysis ^ 20b) Anaerobic Degradation of Fatty Acids^ 212.4. OTHER PATHWAYS OF ANAEROBIC METABOLISM^ 222.4.1. Products Formed by Enterobacteria 232.4.2. Products Formed by Lactic Acid Bacteria 242.5. FACTORS AFFECTING VFA PRODUCTION^ 252.6. APPLICATIONS OF THE ACID-PHASE DIGESTION^ 272.6.1. The Biological Phosphorus Removal Process 282.7. PROCESS CONFIGURATION^ 303. RESEARCH OBJECTIVES^ 32vTABLE OF CONTENTS^ viPAGE4. EXPERIMENTAL METHODS AND ANALYTICAL PROCEDURES^ 344.1. WASTEWATER SOURCE^ 344.2. EXPERIMENTAL SET-UP AND OPERATION^ 354.2.1. General System Configuration^ 354.2.2. Operation ^ 374.3. ANALYTICAL PROCEDURES 414.3.1. Chemical Oxygen Demand (COD)^ 434.3.2. Total Organic Carbon (TOC) 434.3.3. Organic Acids^ 43a) Volatile Fatty Acids (VFAs)^ 43b) Lactic and Pyruvic Acids 44c) Formic Acid^ 454.3.4. Solids^ 45a) Total Solids (TS) and Volatile Solids (VS) ^ 45b) Total Suspended Solids (TSS) and Volatile SuspendedSolids (TSS) ) ^ 454.3.5. Nitrogen^ 46a) Ammonia Nitrogen (NH 3 -N)^ 46b) Total Kjeldahl Nitrogen (TKN) 474.3.6. Proteins ^ 474.3.7. Lipids 48a) Dry Extraction Method^ 48b) Wet Extraction Method 484.3.8. Carbohydrates ^ 494.3.9. Phosphorus 50a) Orthophosphate (PO43) ^ 50b) Total Phosphorus 504.3.10. pH and Alkalinity^ 504.3.11. Other Soluble Organics 514.3.12. Gas Analysis 514.4. COLD STORAGE TESTING^ 524.5. STATISTICS^ 525. RESULTS AND DISCUSSION^ 535.1. GENERAL CHARACTERISTICS 535.1.1. Feed Composition ^ 535.1.2. Cold Storage Experiments^ 565.1.3. Acclimation and Stability of Operation ^ 575.2. THE EFFECT OF HRT - STAGE 1 595.2.1. HRT as a Control Parameter^ 595.2.2. VFA Production^ 615.2.3. VFA Speciation 65TABLE OF CONTENTS^ viiPAGE5.2.4. Particulate Organic Carbon Solubilization^ 695.2.5. Substrate Degradation^ 745.3. THE EFFECT OF SRT - STAGE 2 785.3.1. SRT as a Control Parameter 785.3.2. VFA Production and Speciation ^ 795.3.3. Organic Carbon Solubilization and Substrate Degradation^ 845.3.4. Gas Production ^ 895.4. REPLICATION AND THE EFFECT OF FEED SOURCE - STAGE 3^ 915.4.1. Replication Experiments (Run 3A)^ 915.4.2. The Effect of Feed Source (Run 3B) 935.5. THE EFFECT OF pH - STAGE 4^ 975.5.1. pH as a Selective Parameter 975.5.2. Buffering Capacity 985.5.3. VFA Production and Speciation^ 1005.5.4. Organic Carbon Solubilization and Substrate Degradation^ 1065.6. GENERAL REVIEW^ 1115.6.1. VFA Formation 1115.6.2. Formation of Other Soluble End-Products ^ 1125.6.3. Rate-Controlling Step and Nature of Soluble Compounds ^ 1165.6.4. Mass Balances: Solids and Phosphorus 1205.6.5. Substrate Utilization Patterns^ 1225.6.6. Potential Application of Findings 1256. CONCLUSIONS AND RECOMMENDATIONS^ 1276.1. CONCLUSIONS^ 1276.2. RECOMMENDATIONS 130REFERENCES^ 131APPENDIX A - BIOCHEMICAL PATHWAYS^ 146APPENDIX B - REACTOR OPERATION (HRT AND pH VALUES)^ 151APPENDIX C - CHEMICAL PARAMETERS 163APPENDIX D - VFA DISTRIBUTION^ 200APPENDIX E - VARIOUS EXPERIMENTAL RESULTS ANDCONVERSION FACTORS 206LIST OF TABLESTABLE^ PAGE2.1. Organic Composition of Primary Sludge ^ 74.1. Operating Conditions (Mean Values) 394.2. Duration of Experimental Runs and Amount of Biomass (VSS) in theReactors^ 425.1. Influent Sludge Characteristics (Iona Island WWTP)^ 545.2. Influent Sludge Characteristics (Lions' Gate WWTP) 555.3. Organic Composition of Feed^ 565.4. Cold Storage Testing^ 575.5. VFA Specific Production Rate as Function of HRT^ 635.6. Comparison of Reactor and Effluent VFA Concentrations ^ 655.7. Percent VFA Distribution as a Function of HRT^ 675.8. Specific Solubilization Rates of COD and TOC as a Function of HRT ^ 705.9. Percent VSS and TSS Reduction as a Function of HRT^ 735.10. Percent Substrate Degradation as a Function of HRT 765.11. VFA Specific Production Rate as a Function of SRT^ 805.12. Percent VFA Distribution as a Function of SRT 835.13. Specific Solubilization Rates of COD and TOC as a Function of SRT ^ 865.14. Percent VSS and TSS Reduction as a Function of SRT^ 865.15. Percent Substrate Degradation as a Function of SRT 875.16. Comparison of Replication Results at Iona WWRP^ 925.17. Percent VFA Distribution (Stage 3) ^ 92viiiLIST OF TABLES^ ixTABLE^ PAGE5.18. t-Test Results for Runs 1C, 3A (Iona Island WWTP) and Run 3B(Lions' Gate WWTP)^ 935.19. Comparison of Results from Different Feed Sources ^ 955.20. Percent Distribution of C 5 Branched VFAs and Iso-butyric Acid(Stage 3) ^ 965.21. pH Values in Bioreactors (Stages 1 to 3) ^ 995.22. VFA Specific Production Rate as a Function of pH^ 1035.23. Percent VFA Distribution as a Function of pH 1045.24. Specific Solubilization Rates of COD and TOC as a Function of pH^ 1065.25. Percent Soluble COD in the Form of VFAs as a Function of pH^ 1075.26. Percent VSS and TSS Reduction as a Function of pH^ 1085.27. Protein Degradation and its End-Products as a Function of pH^ 1105.28. Summary of Percent VFA Distribution^ 1135.29. Other Soluble End-Products (Mean Values) 1135.30. Operational Parameters and Percent Soluble COD in the Form of VFAsfrom Various Acid-Phase Anaerobic Digestion Studies ^ 1195.31. Percent Recovery Based on Mass Balance ^ 121LIST OF FIGURESFIGURE^ PAGE2.1. Pathways of Anaerobic Metabolism ^ 54.1. Experimental Layout^ 365.1. VFA Profile (Runs 1C & 1D)^ 625.2. Net VFA Production as a Function of HRT^ 625.3. Percent VFA Distribution (Stage 1) 685.4. Soluble COD Profile (Runs 1C & 1D)^ 715.5. Net COD Solubilization as a Function of HRT^ 715.6. Percent Soluble COD in the Form of VFAs (Stage 1) 755.7. Carbohydrate Degradation as a Function of HRT^ 755.8. Protein Degradation as a Function of HRT 775.9. Lipid Degradation as a Function of HRT^ 775.10. Net VFA Production as a Function of SRT 815.11. Percent VFA Speciation as a Function of SRT^ 815.12. Net COD Solubilization as a Function of SRT 855.13. Percent Soluble COD in the Form of VFAs (Stage 2) ^ 855.14. Carbohydrate Degradation as a Function of SRT 885.15. Protein Degradation as a Function of SRT^ 885.16. Lipid Degradation as a Function of SRT 895.17. Reactor pH and VFA Concentration (CMR System) ^ 1015.18. Reactor pH and VFA Concentration (UASB System) 101xLIST OF FIGURES^ xiFIGURE^ PAGE5.19. VFA Concentration as a Function of pH^ 1025.20. Percent Substrate Degradation as a Function of pH in the CMRSystem^ 1095.21. Percent Substrate Degradation as a Function of pH in the UASBSystem 109GLOSSARY OF TERMSATP:^adenosine-5' -triphosphateCH 2O: carbohydratesCMR:^completely mixed reactorCoA: coenzyme ACOD:^chemical oxygen demandCoV: coefficient of variationEMP:^Embden-Meyerhof-Parnas [glycolytic pathway]HAc: acetic acidHRT:^hydraulic retention timeNAD(H 2):^nicotinamide adenine nucleotide (reduced)NAD(P): nicotinamide adenine nucleotide (phosphate)NH3 -N:^ammonia nitrogenORP: oxidation-reduction potentialPHB:^poly-13-hydroxybutyratePHV: poly-I3-hydroxyvaleratePO4 -3 :^orthophosphateSTD: standard deviationSRT: solids retention timeTKN:^total Kjeldahl nitrogenTOC: total organic carbonTP: total phosphorusTS:^total solidsTSS: total suspended solidsUASB:^upfow anaerobic sludge blanketVFAs: volatile fatty acidsVS: volatile solidsVSS:^volatile suspended solidsWWTP: wastewater treatment plantACKNOWLEDGEMENTSI would like to express my sincere gratitude to my supervisor, Dr. William K.Oldham, for his guidance, knowledge, insight, enthusiasm, and continuousencouragement and support throughout this research.I would like to thank Drs. Kenneth J. Hall, K. Victor Lo, Donald S. Mavinic,and Barry C. McBride for serving in my committee and for their constructivecriticisms and suggestions in the preparation of the final report.I am also grateful to Susan Harper, Paula Parkinson, Timothy Ma, ZufangZhou, and Romy So for their invaluable analytical assistance, moral support and co-operation which created a pleasant working atmosphere in the environmentalengineering laboratory; to Guy Kirsch for his skilled manufacturing of theexperimental apparatus; and to Ian Sellers and the operators at the Iona and Lions'Gate wastewater treatment facilities for their assistance in sludge collection.Thanks are owed to Dr. William D. Ramey and Frederick A. Koch for sharingtheir knowledge and contributing to many stimulating discussions concerning thenature of this research.I would also like to thank my many fellow graduate students, among others,David Wareham, Prayoon Fongsatitkul, Patrick Coleman and Ramanathan Manoharanfor their comradeship, advice, and encouragement; and Christodoulos Labridis andAthanasios Loukas for their companionship and support.Financial support during this study was provided by a grant from the NaturalSciences and Engineering Research Council of Canada (NSERC).CHAPTER 1INTRODUCTIONThroughout recorded history humankind has continually struggled to managethe natural environment in order to improve its well-being. The ability to controlmany aspects of the environment is the main characteristic that has set people apartfrom other species on the planet.In the last half of this century, however, environmental quality problems havesurfaced at an accelerated pace. The population explosion, greater energy use,increased food production needs, changes in life-style, and numerous technologicaldevelopments have created many strains on parts of the global ecosystem. As aresult, an increased concern for the environment is now being witnessed in manyparts of the world.Liquid waste management has long been recognized as a necessary action inorder to improve environmental quality. Collection, treatment, disposal, and reuse ofthe wastewater generated in urban or rural areas has become a priority, wherever thesocial and economic conditions permit.Anaerobic processes have always been an integral part of the wastewatertreatment scheme. In the septic tank, one of the oldest and widest applications inmunicipal sewage treatment, most reactions take place under anaerobic conditions.The first heyday of anaerobic digestion occurred in the period from 1920 to 1935,when it was studied and applied extensively. The popularity of the anaerobic1CHAPTER I. INTRODUCTION 2processes suffered largely in the 1950s and 1960s, because aerobic and physical-chemical methods became attractive alternatives with their advantage in operationand impurity removal efficiency. The energy crisis in the early 1970s and the rapidgrowth of biotechnology have rekindled the interest in anaerobic processes. Todaythe use of anaerobic microorganisms covers a wide array of applications rangingfrom the wastewater treatment field to the production of food, medicines andindustrial chemicals, to genetic engineering and enzyme technology.In the wastewater treatment realm, anaerobic digestion has been traditionallyemployed to prepare municipal sludges for ultimate disposal and provide a source ofenergy. Accordingly, most of the research has been focused on the methanegenerating phase of the process. Little attention has been paid to the acid-phasedigestion, the phase in which complex organic substances, such as carbohydrates,proteins and lipids, are converted anaerobically to volatile fatty acids (VFAs) andother low molecular weight soluble carbon compounds.Improved knowledge of the acid-phase digestion can be useful in a variety ofsituations, ranging from the operation of the overall digestion process itself, to itseffect on subsequent treatment processes. A better understanding of digesterdynamics during shock loading or digester operational stability can be obtained byexploring the acidogenic step. In addition, since the main products of this phase aresoluble organic substrates, they can be used as an energy source for other processes,such as biological phosphorus removal or two-stage biological nitrogen removal.CHAPTER 2LITERATURE REVIEW2.1. AN OVERVIEW OF ANAEROBIC DIGESTIONAnaerobic digestion is a response to controlled conditions of a series ofreactions which occur in many circumstances in nature. It is a biological process inwhich organic matter is ultimately converted to methane and carbon dioxide in theabsence of molecular oxygen. The overall process entails direct and indirectsymbiotic associations between several distinct groups of microbial populations.A number of investigators have attempted to elucidate the different steps andpathways involved in anaerobic metabolism (Holland et al., 1987). As many as ninerecognizable steps, each mediated by a specific group of microorganisms, have beenidentified (Harper and Pohland, 1986). For the purpose of this research, however, thescheme proposed by Kaspar and Wuhrmann (1978a) seems to be the mostappropriate, since it provides a more comprehensive insight on the initial stepsinvolved in the degradation of biopolymers. According to this scheme, the followingsix processes may take place during the anaerobic digestion of a complex substrate:1) Hydrolysis of particulate and soluble biopolymers (carbohydrates, proteins,lipids).2) Fermentation of amino acids and sugars.3) Anaerobic oxidation of long-chain fatty acids and alcohols.3CHAPTER 2. LITERATURE REVIEW^ 44) Anaerobic oxidation of intermediate products such as volatile fatty acids(with the exception of acetate).5) Conversion of acetate to methane.6) Conversion of hydrogen and carbon dioxide to methane.Acid-phase digestion involves the first three reactions, while methanogenesisis implied by the last three. A summary of the above sequence of reactions isdepicted in Figure 2.1. Although bacteria are the main biological agents involved inanaerobic degradation of organic compounds, fermentative ciliate and flagellateprotozoa, and several anaerobic fungi may also contribute in some ecosystems(McInerney and Bryant, 1981).Hydrolysis of organic matter is a process accomplished by extracellularenzymes. The reaction rate can be greatly affected by the pH and the operatingconditions of the system (Verstraete et al., 1981). Complex carbohydrates such ascellulose and starch are hydrolyzed to simple sugars, proteins to amino acids, andlipids to long-chain fatty acids.Fermentation, in this context, can be defined as a microbial metabolic processin which organic compounds serve both as electron donors and as electron acceptors.Any hydrogen generated during fermentation originates from dehydrogenation ofpyruvate. This hydrogen production mechanism is not inhibited by high partialpressures of hydrogen, up to 0.5 atm H2 (Thauer et al., 1977). Sugars and aminoacids are the substrates undergoing fermentation and they produce biomass,intermediate degradation products (propionate, butyrate, etc.), and the methaneprecursors acetate and hydrogen (Figure 2.1).METHANEACETATE HYDROGENAMINO ACIDSSUGARS2FATTY ACIDSALCOHOLSCARBOHYDRATESINTERMEDIATE PRODUCTSPROPIONATE, BUTYRATE,VALERATE, ETC.PROTEINS LIPIDS1CHAPTER 2. LITERATURE REVIEW^ 51) HYDROLYSIS2) FERMENTATION3) ANAEROBIC OXIDATION OF FATTY ACIDS4) ANAEROBIC OXIDATION OF INTERMEDIATE PRODUCTS5) ACETOCLASTIC METHANOGENESIS6) REDUCTIVE METHANOGENESIS (CQ2 + 4H2 ---> CI j + 21-t20)FIGURE 2.1. PATHWAYS OF ANAEROBIC METABOLISM(Adapted from Kaspar and Wuhrmann, 1978a)CHAPTER 2. LITERATURE REVIEW 6In anaerobic oxidation, molecular hydrogen serves as the main sink forelectrons. The principal pathway of hydrogen formation is via oxidation (transfer ofelectrons to protons) of reduced pyridine dinucleotides and ferredoxin (Kaspar andWuhrmann, 1978a). It has been demonstrated that the degradation of long-chain fattyacids under anaerobic conditions occurs by a mechanism called 13-oxidation (Jeris andMcCarty, 1965).2.2. WASTEWATER COMPOSITIONComposition, in general, refers to the actual amount of physical, chemical andbiological constituents in wastewater. Since the organic chemical constituents are ofparamount importance in anaerobic digestion, the following discussion will revolvearound these compounds.Municipal wastewater is a principal source of organic matter entering theaquatic environment. The suspended impurities in liquid wastes from residentialunits, hotels, hospitals, restaurants, offices and commercial buildings are on average70 percent organic in nature (Fair et al., 1971). Settling of wastewater, throughprimary sedimentation, provides raw or primary sludge, which is often used as a feedto anaerobic digesters. Primary sludge contains a great number of organiccompounds, however, most of them can be grouped in three major classes:carbohydrates, proteins and lipids. Although many current research efforts have beendirected towards the identification of specific organic chemicals in raw sludge (eg.aromatic hydrocarbons, chlorinated compounds etc.), the three classes mentioned hereCHAPTER 2. LITERATURE REVIEW^ 7are the key players in anaerobic processes.The composition of primary sludge, measured by various researchers, is shownin Table 2.1. Variation in composition may not only reflect the differences inindividual wastes, but also recent changes in life style, as indicated by the highcarbohydrate and low lipid content of the sludge used in this study.TABLE 2.1. ORGANIC COMPOSITION OF PRIMARY SLUDGEREFERENCE% VOLATILE SOLIDS % TSCarbo-hydratesProteins Lipids Total VSBalmat 30 32 25 87 78Buswell & Neave 18 32 41 91 61Heukelekian 17 36 45 98 76Heukelekian & Balmat 30 31 24 85 65Higgins et al. 66 12 15 93 75Hunter & Heukelekian 44 19 18 81 81Maki 62 29 22 113 650' Rourke - 22 23 - 80AVERAGE 38 27 27 92 73This study 58 21 17 96 752.2.1. CARBOHYDRATESCarbohydrates are the most abundant organic compounds in the biosphere.They can be precisely defined as polyhydroxy aldehydes or ketones with the generalCHAPTER 2. LITERATURE REVIEW 8formula (CH 20)n , where ri3. Depending on the number of carbon atoms included,carbohydrates can be classified as monosaccharides (simple sugars containing 3 to9 carbon atoms), oligosaccharides (mainly disaccharides with 12 carbon atoms) andpolysaccharides (Bailey and 011is, 1977).Polysaccharides are extremely large molecules. The majority of carbohydratesin nature exist as such macromolecules with molecular weights ranging from 25,000to 15 million. They consist, for the most part, of simple and derived sugars linkedtogether by glycosidic bonds. Polysaccharides are insoluble in water and can formcolloidal suspensions (Gaudy and Gaudy, 1980). The most important polysaccharidesfound in municipal wastewaters include cellulose, hemicellulose, pectin and starch(Hunter and Heukelekian, 1965).Cellulose is the most profuse source of organic carbon on earth. Structurally,it is an non-branched polymer of D-glucose units with a molecular weight span from50,000 to over 1 million. The glycosidic linkage occurs between the 1 and 4 carbonsof successive glucose units (13-1,4 bonding). Generally, cellulose is not easilybiodegradable, since few microorganisms are able to break down the (3-1,4 bonds(Tsao et al., 1978). The major source of cellulose in domestic wastewater is paper.Cellulose is the main constituent of non-nitrogenous, alcohol-insoluble matter (i.e.carbohydrates and lignin) in sewage, accounting for 45 to 60% of the total (Hunterand Heukelekian, 1965; Higgins et al., 1982).Hemicellulose is a group of heteropolymers with frequent side chains. Thecommon monomeric components of hemicellulose include hexoses such as glucose,mannose and galactose; pentoses such as xylose and arabinose; and uronic acids.CHAPTER 2. LITERATURE REVIEW 9Hemicellulose is the next most significant constituent (after cellulose) ofpolysaccharides in wastewater, ranging from 20 to 25% (Hunter and Heukelekian,1965).Pectin comprises a family of complex polysaccharides containing mostlymethylated poly-D-galacturonic acid, arabinose and galactose (Conn and Stumpf,1976).Starch has the general formula (C 6H 1005 ) x \u00E2\u0080\u00A2 It occurs in two forms: amyloseand amylopectin. Amylose is a linear polymer of D-glucose units linked together bya-1,4 bonds. The amylose molecule contains 100 to 1,000 glucose units and it isinsoluble in water. Amylopectin is a branched polymer of glucose containing botha-1,4 bonds and a-1,6 bonds which initiate side chains. It is much larger thanamylose (500 to 5,000 glucose units), is soluble in water and can form gels byabsorbing water (Bailey and 011is, 1977).Both pectin and starch can be found in small amounts in wastewater (less than10% of the total carbohydrates and lignin).Lignin is a complex polymeric aromatic substance of variable structure makingup a substantial portion of the woody parts of plant tissue, where it helps to \"cement\"cellulose fibers together (Lehninger, 1975). Both cellulose and lignin play animportant structural role in plants and one of the main processes of the pulp andpaper industry is to separate these two components. Lignin is considered to be arefractory compound not amenable to biodegradation. It comprises 5 to 15% of thenon-nitrogenous, alcohol-insoluble matter in domestic wastewater (Hunter andHeukelekian, 1965) and in primary sludge (Higgins et al., 1982).CHAPTER 2. LITERATURE REVIEW^ 102.2.2. PROTEINSProteins constitute the most complex organic compounds in the biosphere.They all contain carbon, hydrogen, oxygen and nitrogen. Phosphorus and sulphur arepresent in a few. Proteins are an essential part of all living matter and a majordietary constituent. They are polymers of a-amino acids joined together by peptidebonds. These covalent bonds arise by elimination of the elements of water from thecarboxyl group of one amino acid and the a-amino group of the next. The molecularweight of proteins, depending on the number of the polymers, can vary from a fewthousand to several million (Gaudy and Gaudy, 1980). Proteins are divided into twomajor classes on the basis of their conformation: fibrous and globular. Fibrousproteins are physically tough and are insoluble in water or dilute salt solutions. Onthe other hand, most globular proteins are soluble in aqueous systems and theyusually have a mobile or dynamic function in the cell (Lehninger, 1975).The importance of proteins in anaerobic digestion stems from their significantbuffering capacity (due to the presence of hydroxy and amino groups) as well as theirability to serve as carbon and energy sources. The nutritional value of the individualamino acids to the microorganisms is an additional asset (Tsao, 1984).Almost all of the 20 known amino acids have been identified in untreatedsewage. Alanine, aspartic acid, glutamic acid, leucine and iso-leucine are the mostpredominant ones (Heukelekian and Balmat, 1959; Kahn and Wayman, 1964). Theformer investigators have reported that the amino acids accounted for 65 to 80% ofthe total nitrogenous matter. According to Hunter and Heukelekian (1965), the aminoCHAPTER 2. LITERATURE REVIEW 11acids averaged 55% of the total organic nitrogen. Painter et al. (1961) and Hansonand Lee (1971) have reported that about 35 to 40% of the total organic nitrogen wasin the amino acid form. It should be noted, however, that this diversity in amino acidcontent in raw sewage may be mainly the result of analytical determinations(particulate vs. soluble forms of nitrogen). Higgins et al. (1982) have found that theamino acid content of the primary sludge averaged about 65% of the total organicnitrogen.2.2.3. LIPIDSLipids are organic biomolecules which are soluble in non-polar solvents suchas chloroform, benzene or ether, and practically insoluble in water. Consequently,lipids are diverse in their chemical structure and biological function (Conn andStumpf, 1976). Lipids have been classified in several different ways. The mostsatisfactory classification, based on their common chemical characteristics, includessimple, compound and non-saponifiable lipids (Gaudy and Gaudy, 1980).Fats, oils and waxes are all simple lipids. Fats and oils are esters of variousfatty acids and the trihydroxy alcohol glycerol. Waxes are esters of fatty acids andlong-chain monohydroxy alcohols. The most common fatty acids contain 16 or 18carbon atoms and may be saturated such as palmitic and stearic or unsaturated suchas oleic, linoleic, linolenic and palmitoleic (Gurr and James, 1971).Compound lipids are also esters of various fatty acids and alcohols. Theaddition of phosphorus and nitrogen compounds results in the creation ofCHAPTER 2. LITERATURE REVIEW^ 12phospholipids, and the addition of carbohydrates in that of glycolipids.Non-saponifiable lipids do not contain fatty acids and, hence, do not yieldsoaps (salts of fatty acids) on alkaline hydrolysis. This subclass includes sterols, fat-soluble vitamins and plant pigments.Lipids are contributed to domestic wastewater in butter, lard, margarine,vegetable fats and oils, and other food items (Metcalf and Eddy, 1991). Theesterified fatty acids are the main lipid component (50 to 70% of total lipids) in rawsewage, followed by the unsaponifiable matter (15 to 25%). Free fatty acids andphospholipids have been found in small amounts (Hunter and Heukelekian, 1965). Inprimary sludge, however, free fatty acids may contribute between 40 and 60% of thetotal lipids, as a result of the rapid hydrolysis of the fatty acid esters to free fattyacids (Heukelekian and Mueller, 1958). Saturated fatty acids represent about 70 to80% of the total fatty acids identified, esterified or free. Palmitic and stearic acidsare the most commonly found saturated acids and oleic acid the predominantunsaturated one (Higgins et al., 1982).2.3. PATHWAYS OF VFA FORMATIONThe ability to produce volatile fatty acids under anaerobic conditions is awidespread attribute in the microbial world. A large number of bacterial species iscapable of utilizing complex organic substrates such as carbohydrates, proteins andlipids to produce VFAs and other soluble carbon compounds via a variety ofanaerobic metabolic pathways. In the following discussion, the metabolic pathwaysCHAPTER 2. LITERATURE REVIEW^ 13of the three major organic components of the primary sludge will be reviewed.2.3.1. CARBOHYDRATE METABOLISMa) HydrolysisBacteria are unable to take up particulate polysaccharides, becausebiopolymers as such cannot penetrate the cell membrane. Therefore, microorganismsexcrete enzymes that are capable of degrading the complex biopolymers to smalltransportable molecules. These enzymes can either be set free by the organisms orremain associated with them.Cellulose can be hydrolyzed by a number of anaerobic bacteria. Bacteroidessuccinogenes was the first one isolated from rumen (Hungate, 1949). Other commoncellulose hydrolyzing organisms include Clostridium lochheadii and Clostridiumcellobioparum (Hungate, 1957), Butyrivibrio fibrisolvens (Shane et al., 1969), andClostridium thermocellum (Gottschalk, 1986). All cellulolytic bacteria excrete theenzyme complex called cellulase. It consists, in general, of three major enzymecomponents (Gong et al., 1979):1) endoglucanase which cleaves the (3-1,4 glycosidic bonds in the cellulosemolecule,2) exoglucanase which removes cellobiose (a disaccharide unit of cellulose)from non-reducing ends of the molecule, and3) cellobiase which hydrolyzes cellobiose or cellotriose to two or threemolecules of glucose respectively.CHAPTER 2. LITERATURE REVIEW 14A detailed description of the mode of action of this exoenzyme complex isprovided by Cuskey et al. (1982). Cellulose hydrolysis yields the simple sugarglucose.Hemicellulose can be degraded by anaerobic organisms producing theexoenzyme complex hemicellulase. The complexity of this enzyme system farexceeds that of cellulose, as hemicellulose is composed of a greater variety ofmonomers linked together by different types of bonds. A comprehensive review ofhemicellulase excreted by anaerobic microbes is presented by Dekker and Richards(1976).Pectin hydrolysis involves bacteria found in rumen such as Bacteroidessuccinogenes, Bacteroides ruminicola, and Butyrivibrio fibrisolvens. Three types ofenzymes are associated with the degradation of pectin to galacturonic acid residues.Pectinesterase demethylates pectin to produce poly-D-galacturonic acid and methanol,while hydrolase breaks down the polymer to oligomeric chains. Then, lyasedepolymerizes the chains to form the final products of hydrolysis (Tsao, 1984).Starch, as a storage material, is amenable to biodegradation. Among the manyanaerobes able to hydrolyze starch are Streptococcus bovis, Bacteroides amylophilus,Succinomonas amylolytica, and a number of Lactobacillus species (Tsao, 1984).Complete hydrolysis of starch to glucose requires the synergistic action of four typesof specific enzymes (Fogerty and Kelly, 1979).b) Fermentation of SugarsGlucose, the main simple sugar generated from polysaccharide hydrolysis, isCHAPTER 2. LITERATURE REVIEW 15commonly used by fermentative microorganisms as an energy source. Most of theproducts formed in the fermentation of glucose originate from pyruvic acid which isproduced via the glycolytic Embden-Meyerhof-Parnas (EMP) pathway. According tothis pathway, two molecules of pyruvic acid are produced per molecule of glucosethrough a series of reactions (Figure Al, Appendix A). In addition, two moleculesof adenosine-5' -triphosphate (ATP) are generated and two molecules of nicotinamideadenine dinucleotide (NAD) are reduced in the process. Since no oxygen is involved,this pathway is common in both aerobic and anaerobic metabolism (Gaudy andGaudy, 1980). Depending on the anaerobic microbial species present, subsequentpyruvic acid fermentation can lead to the production of different VFAs.Acetic acid is produced in a number of fermentations. There are certainbacteria, however, which form acetate as the predominant end-product.Representative genera of acetogenic organisms include Clostridia and Acetobacteria.It has been demonstrated, for example, that Clostridium formicoaceticum can ferment1 mol of hexose to 3 mol of acetic acid (Ljungdahl, 1986):C 61-1 120 6 ------+ 3CH 3COOH (2.1)Acetic acid is formed by the EMP pathway and by reduction of carbon dioxideto acetate (Figure A2, Appendix A). Clostridium aceticum and Acetobacterium woodiiferment hydrogen and carbon dioxide to acetate. It should be noted that carbonmonoxide plays an important role as a precursor of the carboxyl group of acetate(Eden and Fuchs, 1982; Cole, 1988):2CO2 + 4H2 ---, CH 3 COOH + 2H20^(2.2)CHAPTER 2. LITERATURE REVIEW^ 16Acetic acid is also generated by mixed-acid producers. They metabolizepyruvate to acetate and other products, as will be discussed later (Section 2.4)Propionic acid is a major end-product of fermentations carried out by manyanaerobes of the Propionibacterium genus. P. pentosaceum and P. shermanii candegrade pyruvic acid via the succinate-propionate pathway. Lactyl-CoA and acrylyl-CoA are intermediates, while electron-transferring flavoprotein functions as hydrogencarrier. The overall reaction is as follows (Doelle, 1975):3CH3 COCOOH + 3H2 -----, 2CH 3 CH2 COOH + CH 3 COOH + CO 2 + H2 O (2.3)Lactic acid is a preferred substrate for certain bacteria such as Clostridiumpropionicum and Megasphaera elsdenii which produce propionic acid via the acrylatepathway (Figure A3, Appendix A). This pathway can be summarized as follows(Papoutsakis and Meyer, 1985):3CH 3 CHOHCOOH -----, 2CH 3 CH2COOH + CH 3COOH + CO 2 +H20 (2.4)Butyric acid can be formed as a main fermentation product by many obligateanaerobes which belong to the four genera: Clostridium, Butyrivibrio, Eubacterium,and Fusobacterium. The reactions involved in the production of butyric acid arepresented in Figure A4, Appendix A. In this pathway, the conversion of pyruvate toacetyl-CoA is catalyzed by the pyruvate-ferredoxin oxidoreductase enzyme system.Also, butyryl-CoA is not converted to butyric acid by simple hydrolysis, but via theformation of butyryl phosphate, which yields an additional ATP molecule(Gottschalk, 1986).CHAPTER 2. LITERATURE REVIEW^ 172.3.2. PROTEIN METABOLISMa) HydrolysisThe bacteria involved in protein metabolism have the ability to produceproteolytic enzymes which break the biopolymers into their monomeric components(i.e. amino acids) before they can enter the cell membrane and be used either asbuilding blocks or as fermentative substrates.Protein hydrolysis progresses in steps in reverse manner to those in whichproteins are synthesized (Sawyer and McCarty, 1978):Protein ---, proteoses -----, peptones -----,polypeptides ---, dipeptides ---, a-amino acids^(2.5)The most common anaerobic proteolytic microorganisms belong to the genusClostridium (Siebert and Torein, 1969; Hobson and Shaw, 1971). Bacteroidesruminicola has also been found to exhibit similar activity (Hobson and Shaw, 1974).Proteolytic enzymes are divided into two groups, according to their mode ofaction on a polypeptide chain: endopeptidases and exopeptidases. Pepsin, a typicalendopeptidase, has very broad specificity, but preferentially attacks polypeptidechains along their length, whenever residues of aromatic amino acids occur.On the contrary, exopeptidases can only split terminal peptide bonds. They aresubdivided into: aminopeptidases, which require a free terminal amino group and aredependent on metal ions for their activity; and carboxypeptidases, which break downpeptides with a free terminal carboxy group (Lehninger, 1975). The synergistic actionCHAPTER 2. LITERATURE REVIEW^ 18of both types of enzymes results in the formation of free a-amino acids.b) Amino Acid FermentationSeveral single amino acids can serve as energy and carbon sources for strictor facultative anaerobes. Organisms possessing the enzyme dehydrogenase canconvert aliphatic amino acids (containing the alkyl group, R) to the correspondingVFAs via reductive deamination. Hydrogen ions act as the hydrogen donor. A generalreaction can be written as follows (Doelle, 1975):R-CHNH2COOH + 2H+ ----, R-CH2 COOH + NH3^(2.6)Many microorganisms can specifically ferment individual amino acids toproduce VFAs. A few representative cases are outlined below. Clostridiumpropionicum employs the acrylate pathway (Figure A3, Appendix A) to convertalanine, via lactic acid, to a mixture of propionic and acetic acid (Gottschalk, 1986).Glycine is a preferred substrate for Clostridium histoliticum and Diplococcusglycinophilus, with acetic acid being the main product according to the equation(Elsden and Hilton, 1978):4CH2NH2COOH + H 2 O ------, 3CH 3COOH + 4NH 3 + 2CO2^(2.7)Two different pathways have been elucidated regarding the fermentation ofglutamic acid by obligate anaerobes. Clostridium tetanomorphum employs themethylaspartate pathway, which is rather unusual and is used only by the Clostridiumspecies, for the formation of an acetic and butyric acid mixture with a 3:1 ratioCHAPTER 2. LITERATURE REVIEW 19(Gottschalk, 1986). The same acids can be also produced (at a 2:1 ratio) via thehydroxyglutarate pathway followed by Acidaminococcus fermentans, Clostridiummicrosporum and other species (Buckel and Barker, 1974).Not all amino acids can be fermented singly, or at least no organism capableof utilizing certain amino acids has been isolated.The Stickland reaction is an oxidation-reduction reaction between pairs ofamino acids. One amino acid acting as the hydrogen donor is oxidized, and a secondone acting as the hydrogen acceptor is reduced. This allows the amino acids thatcannot be fermented individually to be used as an energy source. This reaction iscurried out by many proteolytic clostridia such as C. stickandii, C. sporogenes, andC. histoliticum (Barnard and Akhtar, 1979; Barker, 1981). The coupling of valine(hydrogen donor) and glycine (hydrogen acceptor), for example, results in theformation of iso-butyric acid and acetic acid. It was first demonstrated by Cohen-Bazire et al. (1948):CH3 CHCH 3 CHNH2COOH + 2CH 2NH2COOH + 2H20CH3 CHCH3 COOH + 2CH3 COOH + 3NH3 + CO2^(2.8)In a similar fashion, iso-leucine or leucine, acting as hydrogen donors can beoxidized to the isomers of the valeric acid. In the case of iso-leucine the predominantisomer is 2-methylbutyric acid, and in that of leucine 3-methylbutyric acid (Elsdenand Hilton, 1978).CHAPTER 2. LITERATURE REVIEW^ 202.3.3. LIPID METABOLISMa) HydrolysisThe hydrolysis of ester linkages in lipids requires the presence of lipolyticenzymes. Since the environment in which hydrolysis takes place involves a lipid-water interface, this is a heterogeneous enzymatic catalysis. Two common types oflipolytic enzymes include lipases and phospholipases.Lipases catalyze the stepwise and partially reversible hydrolysis of fatty acidester bonds in simple lipids (triglycerides), with the intermediate formation of di- andmonoglycerides and the ultimate release of 3 mol of the corresponding fatty acid and1 mol of glycerol (Ratledge, 1988):triglyceride I- - - -4 diglyceride + fatty acid ,-----+monoglyceride + fatty acid -----,glycerol + fatty acid^(2.9)Microbial lipases are classified into three groups according to their specificity:non-specific, 1-3 specific (catalyzing reactions at the C 1 and C 3 positions of thetriglyceride), and fatty acid specific (Macrae, 1984). Lipases are widespread innature. Under anaerobic conditions, they are excreted by microorganisms belongingmainly to the genera Bacillus, Chromobacterium, and Serratia (Finnerty, 1988).Phospholipases are involved in the hydrolysis of phospholipids. Four typesof phospholipases are known and are classified according to the ester bond whichthey hydrolyze (Waite, 1987). Phospholipid metabolism results in the production ofCHAPTER 2. LITERATURE REVIEW^ 21the corresponding fatty acids and a variety of other organic compounds, dependingupon the substrate utilized. Phospholipases have been found in many spore-forminganaerobic bacteria such as Clostridium perfrigens and Bacillus cereus (Low, 1981).b) Anaerobic Degradation of Fatty AcidsThe anaerobic metabolism of long chain fatty acids occurs via a mechanismcalled (3-oxidation, because the beta carbon (second from the carboxyl carbon) isoxidized. This pathway involves repetition of a sequence of reactions that results inthe removal of two carbon atoms as acetyl-CoA with each repetition (Gaudy andGaudy, 1980).The first step in the (3-oxidation is its activation by one of several enzymescalled acyl-CoA synthetases. Since four hydrogen atoms are generated per moleculeof acetyl-CoA, the overall reaction can only proceed if much of the hydrogen can beconverted to H2 gas. Many anaerobic bacteria form the enzyme hydrogenase, whichcatalyzes the reversible reaction of hydrogen production from a reduced high-energyelectron carrier such as reduced pyridine dinucleotides and ferredoxin (Benemann andValentine, 1971):2H+ + 2e - ----, H2^(2.10)The stoichiometry of the (3-oxidation reaction including oxidation of NAD(P)Hor ferredoxin is as follows (Jeris and McCarty, 1965; Gujer and Zehnder, 1983):(-CH2 CH2-) + 2H20^CH3COOH + 2H 2^(2.11)CHAPTER 2. LITERATURE REVIEW 22The ATP yield of this reaction is not known. The oxidation of NAD(P)H,however, has a higher redox potential (-0.32 V at pH 7) than that of pyruvatedehydrogenation (-0.68 V at pH 7) (Wolin, 1976). Based on thermodynamicconsiderations, partial pressures of H2 gas higher than 0.5 atm may slow down theoxidation of NAD(P)H as a result of product inhibition (Kaspar and Wuhrmann,1978a).Little variation has been found in the 13-oxidation scheme for the varioussaturated fatty acids except for the activation step. The enzyme catalyzing theactivation of fatty acids falls into three distinct categories depending on chain length.There is evidence that unsaturated fatty acids are first hydrogenated and thendegraded by the same mechanism (Novak and Carlson, 1970; Hobson et al, 1974).Acetyl-CoA, the main intermediate of (3-oxidation can be converted to eitheracetic or butyric acid (Harper and Pohland, 1986). Propionic acid may also be formedas an end-product of the metabolism of fatty acids that contain odd numbers ofcarbon atoms. A three-carbon residue, propionyl-CoA, remains after the removal ofthe other carbons as acetyl-CoA, and is converted to propionic acid under anaerobicconditions (McInerney and Bryant, 1981).2.4. OTHER PATHWAYS OF ANAEROBIC METABOLISMA great number of microorganisms can degrade the intermediates of anaerobicmetabolism alternatively to form a wide array of end-products. Two groups ofanaerobes of particular interest are enterobacteria and lactic acid bacteria.CHAPTER 2. LITERATURE REVIEW^ 232.4.1. PRODUCTS FORMED BY ENTEROBACTERIAEnterobacteria are classified into three categories according to the type offermentation they carry out: mixed acid, butanediol, and propanediol producers. Themixed acid producers belong to the genera Escherichia, Salmonella, and Shigella. Atypical member of this group, Escherichia coli, ferments sugars to lactic, acetic,succinic, and formic acids. Smaller amounts of ethanol, carbon dioxide, and hydrogengas are also formed. On the other hand, species of the genera Enterobacter, Serratia,and Erwinia show a different metabolic activity and are called butanediol producers.Enterobacter aerogenes, for example, forms mainly 2,3-butanediol, ethanol, carbondioxide and hydrogen gas. Acid generation is minimal, except for some formic acid(Wood, 1961).Both groups of bacteria mentioned employ the EMP pathway (Figure A1,Appendix A) for glucose breakdown. All products, except succinic acid are derivedfrom pyruvic acid. The pathway leading to succinic acid branches off atphosphoenolpyruvate. The amounts of fermentation products formed depend verymuch on the activity on pyruvic acid of the three enzyme systems involved (Garvie,1980).Members of the Enterobacteria family, such as Citrobacter freundii, are ableto metabolize glycerol (a product of lipid hydrolysis) either to 1,3-propanediol or toglyceraldehyde-3-phosphate. This process is assumed to be independent from thecarbohydrate metabolism and occurs only if glycerol is available (Doelle, 1975).CHAPTER 2. LITERATURE REVIEW^ 242.4.2. PRODUCTS FORMED BY LACTIC ACID BACTERIALactic acid bacteria are morphologically a heterogeneous group andcharacterized by their main end-product, lactic acid. These microorganisms are highlysaccharolytic and lack most anabolic pathways, so they exhibit very specificnutritional requirements. Most lactic acid bacteria are strictly fermentative, but areaerotolerant (Bergy's Manual, 1974).The three following pathways can be employed for the fermentation of glucose,via pyruvate, to lactic acid and other end-products (Gottschalk, 1986):1) The homofermentative pathway used mostly by Lactobacillus andStreptococcus species yields only lactic acid:C611 1206^2CH3CHOHCOOH^(2.12)2) The heterofermentative pathway used mainly by Leuconostoc species yieldsthe following products:C 6H 120 6^CH3CHOHCOOH + CH 3 CH2OH + CO2^(2.13)3) The bifidum pathway employed by Bifidobacterium species yield lactic acidand acetic acid:2C6H 1206 ----+ 2CH 3 CHOHCOOH + 3CH 3 COOH^(2.14)Besides glucose, many other saccharides can be utilized by lactic acid bacteriaincluding fructose, galactose, lactose, and pentoses.CHAPTER 2. LITERATURE REVIEW^ 252.5. FACTORS AFFECTING VFA PRODUCTIONIn general, the acid-phase digestion products may be markedly affected by thecharacteristics of the wastewater, environmental factors such as culture pH,temperature, oxidation-reduction potential (ORP), reactor configuration and availabletrace minerals, and operational parameters such as hydraulic retention time (HRT)and solids retention time (SRT).The concept of optimizing VFA production by anaerobic digestion is arelatively new one in the wastewater treatment field. Traditionally, most of theresearch in anaerobic sludge digestion has been focused on the methanogenic phaseof the process, where VFAs are used as substrate for the methane forming bacteria.Little attention has been paid, therefore, to the optimization of the acidogenicmicroorganisms coupled with suppression of the methanogenic ones.Most of the valuable contributions on the acid-phase digestion have beenobtained from studies using either soluble substrates such as glucose (Ghosh andPohland, 1974; Uribelarrea and Pareilleux, 1981; Zoetemeyer et al., 1982b; andCohen et al., 1984), a mixture of simple organics (Andrews and Pearson, 1965),lactose (Kisaalita et al., 1987; Hsu and Yang, 1991), a protein, gelatin (Breure andvan Andel, 1984); or specific industrial wastewaters generated from sugar refineries(Gil-Pena et al., 1987), and ethanol distilleries (Machado and Sant'Anna, 1987). Itis questionable, therefore, whether information available from these sources can bedirectly applied to the design and operation of anaerobic digesters treating primarysludge from municipal wastewater treatment plants.CHAPTER 2. LITERATURE REVIEW 26Relatively few studies have been performed using primary sludge frommunicipal wastewater treatment facilities as a feed. Among them are those byO'Rourke (1968), Chynoweth and Mah (1971), Borchard (1971), Eastman andFerguson (1981), Rabinowitz (1985), Gupta (1986), and Ghosh (1987).Important findings from a selected number of the above mentioned studies aresummarized in the following paragraphs.Andrews and Pearson (1965) have observed that the acidogenic phase is fairlyrapid, with an optimum cell residence time of 0.75 days. In addition, the type ofvolatile acids generated from a given substrate is greatly influenced by variation ofthe organism residence time.Chynoweth and Mah (1971) have reported a high rate of lipid dissimilation inprimary sludge digestion. Acetic, propionic, and butyric acids were the mainproducts. Formic acid was also detected in smaller amounts.On the contrary, Eastman and Ferguson (1981) have found that lipiddegradation was minimal in the acid phase, but carbohydrates and proteins wereextensively metabolized. The VFA production was significantly affected by pH butnot by the influent solids concentration, at least up to 6% VS.According to Zoetemeyer et al. (1982b), the relative production of individualVFAs from glucose depends on the dilution rate and more strongly on the culture pHvalue, with an optimum pH in the range between 5.0 and 6.0.Rabinowitz (1985) has found that at sludge retention times ranging from 2.5to 10 days, acetic and propionic acids made up more than 90% of the short chainVFA production, and appeared in the fermenter supernatant in a ratio ofCHAPTER 2. LITERATURE REVIEW^ 27approximately 55:45.Gupta (1986) has reported that the net VFA generation consistently improvedwith increase in temperature between 10 and 30 \u00C2\u00B0C, while pH control at 7.0 did notmake any significant change in the total acid production.According to Ghosh (1987), the culture pH had a strong effect on carbohydrate,protein and lipid reduction efficiencies. In addition, increase in digester hydraulicretention time increased the degradation of all the three major organic components,while higher temperatures had a more marked effect on protein reduction.In order to optimize the acidogenic phase of anaerobic digestion, the first threereactions in Figure 2.1 need to be encouraged, with the concurrent suppression of thelast three ones which are linked to methanogenic activity.Methane formers are very sensitive to environmental factors. Their activitydrops drastically at pH below 6 (Zehnder et al., 1981). They are very slow growingorganisms as well. The maximum specific growth rate of methanogens can be oneorder of magnitude lower than that of acidogenic bacteria (Ghosh and Klass, 1978).It has been also observed that VFA conversion to methane does not occur below acritical SRT, which appears to be system specific (Ghosh, 1987). Methanogenesis canbe suppressed, therefore, by operating the digester at a pH below 6 and an SRT valuebelow the critical one.2.6. APPLICATIONS OF THE ACID -PHASE DIGESTIONImproved knowledge of the acid-phase anaerobic digestion can be useful in aCHAPTER 2. LITERATURE REVIEW^ 28number of situations, ranging from the operation of the overall digestion processitself to its effect on subsequent treatment processes. The increasing use of the two-phase digestion process creates an opportunity to further explore the acid-phase step.This may result to a better understanding of digester dynamics during shock loading,a greater operational stability of the system, or higher conversion rates of the organicmaterial. Moreover, since the main products of acidogenic activity are short-chain,soluble organic substrates, they can be used as an energy and carbon source forbacteria carrying out other processes, such as biological phosphorus removal or two-stage biological denitrification. A brief description of the biological phosphorusremoval process and the role of VFAs in it is outlined below.2.6.1. THE BIOLOGICAL PHOSPHORUS REMOVAL PROCESSBiological phosphorus removal has been a viable alternative to chemicalprecipitation as a means to control nutrient discharges into receiving water bodies.In this process, phosphorus is taken up by certain species of bacteria such asAcinetobacter beyond their need for normal cell maintenance and synthesis (Siebrietzet al., 1983). A continuous flow biological phosphorus removal scheme consists ofa bioreactor in which an aerobic zone is preceded by an anaerobic one. The additionof simple soluble carbon substrates such as VFAs in the anaerobic zone results inphosphate release and carbon storage by the biomass (Nicholls and Osborn, 1979;Barnard, 1983; Comeau et al., 1986). Carbon storage occurs mainly in the form ofpoly-3-hydroxybutyrate (PHB) and poly-P-hydroxyvalerate (PHV) (Comeau et al.,CHAPTER 2. LITERATURE REVIEW 291988). The fact that phosphorus-removing bacteria can assimilate the acid-phasedigestion products in the anaerobic zone provides them with a competitive advantagecompared to other heterotrophic microorganisms occurring in activated sludgesystems (U.S. EPA, 1987).It has been observed that there is a relationship between the amount of VFAsadded and the amount of phosphate released under anaerobic conditions (Fukase etal., 1982; Arvin, 1985). When the anaerobic zone is followed by an aerobic one, thephosphorus removing bacteria take phosphate from solution and store it inpolyphosphate pools. The amount of phosphorus uptake in the aerobic zone can becorrelated with that released under anaerobic conditions, which in turn is a functionof the amount of stored VFAs (in the from of PHB or PHV) available in the aerobiczone (Wentzel, 1984). Hence, the biological phosphorus removal capacity of a plantcan be improved by the presence of short chain soluble carbon compounds in theanaerobic zone. Rensick et al. (1984) have reported that acetic acid additionincreased phosphorus removal from 45 to 97%. According to Rabinowitz and Oldham(1985), the incorporation of primary sludge digestion into the design of a simplifiednutrient removal process resulted in an improvement of more than 100% inphosphorus removal.In order to induce phosphorus release in the anaerobic zone, Siebritz et al.(1983) found that, in the absence of nitrates, the minimum concentration of VFAsrequired in this zone is 25 to 30 mg/L (as HAc). Since the VFA content of untreateddomestic wastewater is usually very low (less than 10 mg/L), an external organiccarbon source is needed to trigger the biological phosphorus removal mechanism.CHAPTER 2. LITERATURE REVIEW 30The soluble carbon concentration in the anaerobic zone of the process can beincreased either by the addition of preformed VFAs or by primary sludge digestionwith return of the fermented material to the main bioreactor.2.7. PROCESS CONFIGURATIONThe basic requirement in anaerobic processes is the maintenance of a sufficientamount of active biomass in the reactor under high organic loading conditions. Inorder to meet this requirement, many suspended- and attached-growth processconfigurations have been thoroughly investigated, such as the completely mixedanaerobic digestion (conventional digester), anaerobic contact process (completelymixed reactor with clarifier and solids recycle), anaerobic filter, upflow anaerobicsludge blanket (UASB), fluidized bed, and expanded bed (Ross and Smallen, 1981;Metcalf and Eddy, 1991).Two reactor configurations have been selected to investigate the acid-phasedigestion of primary sludge: the completely mixed reactor (CMR) with clarifier andsolids recycle system; and the upflow anaerobic sludge blanket (UASB) reactor.Completely mixed digesters (with or without solids recycle) have been offundamental importance in anaerobic treatment, with a wide range of applications inmunicipal and industrial wastewaters (Speece, 1983; McCarty and Smith, 1986).The upflow anaerobic sludge blanket process is a recent modification of thebiolytic tank (Jewell, 1987). In the 1970s, Lettinga and co-workers developed theUASB reactor concept in the Netherlands (Lettinga et al., 1979). It is based on theCHAPTER 2. LITERATURE REVIEW 31idea that anaerobic sludge has superior settling characteristics, if the physical andchemical conditions for sludge flocculation remain favorable. The sludge blanket(bed) can be considered as a separate fluid phase with its own specific properties. Awell-established sludge blanket is fairly stable and can withstand relatively highmixing forces (Lettinga et al., 1980). The sludge generated in the UASB reactor(which is essentially of a vertical plug-flow type) often is in a very dense well-defined pellet or granular form. These granular particles are nearly spherical in shapewith a 1 to 5 mm diameter (Hulshoff Pol et al., 1983). As such, the system acts asa biofilm in a sense that there is substrate diffusion into the conglomeration ofmicroorganisms and product diffusion out (McCarty and Smith, 1986). Pellets havevery good settleability and are readily retained in the reactor without the need of aclarifier (Sam-Soon et al., 1988). Pellet formation in UASB systems, however,depends upon the type of the wastewater used. For example, a high degree ofpelletization has been achieved in systems treating carbohydrate wastes (Lettinga etal., 1980; Ross, 1984), but a limited pellet formation has been observed with acetate-propionate mixtures (de Zeeuw and Lettinga, 1980). No pellet formation has beenobtained in reactors using olive oil processing wastes (Boari et al., 1984).The UASB concept has been extensively used in laboratory-scale and pilot-scale studies. Its feasibility has been demonstrated in anaerobic treatment of low-strength wastes, acid-phase digestion, and denitrification experiments (Lettinga andVinken, 1980). In addition, several full-scale installations have been successfullyemployed in Northern Europe treating a variety of industrial wastewaters (Maat andHabets, 1987; Lettinga and Hulshoff Pol, 1991).CHAPTER 3RESEARCH OBJECTIVESAs discussed in the previous chapter (Section 2.5), much of the informationavailable on the acid-phase digestion has been obtained using simple, soluble carbonsubstrates. In certain cases, where primary sludge was used as feed, the main focusof the research was on methane production optimization (O'Rourke, 1968; Chynowethand Mah, 1971; Ghosh, 1987), or on phase-separation techniques (Borchard, 1971),or on kinetic modelling (Eastman and Ferguson, 1981). Consequently, informationregarding details on the mechanisms and pathways involved in the transformation ofthe organic substrates and the full spectrum of products formed was rather limited.On the other hand, Rabinowitz (1985) and Gupta (1986) have attempted to investigatethe acid-phase step. Both studies, however, were of an exploratory nature,concentrating on the production of acetic, propionic and butyric acids.In all of the above studies, however, the use of either batch reactors orconventional continuous-flow reactors without solids recycle eliminated thepossibility to distinguish between the two retention times (HRT and SRT) of thesystem. It should be pointed out that HRT and SRT are two different operationalparameters and that they may affect any biological process in a distinctly differentmanner. Moreover, only a few investigations included acid-phase digestionexperiments with pH control (Eastman and Ferguson, 1981; Gupta, 1986). Even inthese studies, no attempt was made to control the pH at values below 5.5.32CHAPTER 3. RESEARCH OBJECTIVES 33It is, therefore, apparent that there is a need for an in-depth investigation ofthe acid-phase digestion of primary sludge. Inspired by the above observation, thisresearch has attempted to explore the following areas:1) Investigate independently the effect of certain operational parameters (i.e.HRT and SRT) on acidogenic digestion.2) Investigate the effect of certain environmental parameters (i.e. pH, reactorconfiguration, and influent characteristics) on the process.3) Suggest possible mechanisms and biochemical pathways involved in theconversion of carbohydrates, proteins and lipids to VFAs and other soluble end-products.To meet the above stated objectives, a series of laboratory-scale, continuous-flow experiments has been employed. Their design allowed for separate control ofreactor HRT and SRT values. Although CMR systems have long been used inenvironmental engineering practice for acid-phase digestion of primary sludge, theapplication of UASB systems, for the same purpose, is a rather novel idea.A better understanding of the nature of the acidogenic phase can be achievedby determining the level and rate of carbohydrate, protein, and lipid utilization, andthe rate and type of product formation (C 1 to C 6 VFAs, straight-chain and branched;alcohols; lactic acid and other soluble end-products; and certain gases). Additionalvaluable measurements included different types of solids and nitrogen forms, as wellas pH and ORP.CHAPTER 4EXPERIMENTAL METHODS AND ANALYTICAL PROCEDURES4.1. WASTEWATER SOURCEThe primary sludge used in this study was obtained from the Iona Islandwastewater treatment plant located in Richmond, British Columbia, except for oneexperimental run, where the sludge was collected from the Lions' Gate treatmentplant situated in North Vancouver, British Columbia. Both plants operate as primarytreatment facilities, with subsequent anaerobic digestion for sludge stabilization.The sludge was collected, usually once a week, from the underflow lines ofthe primary clarifiers in 25 L carboys and stored in the laboratory cold room at 4 \u00C2\u00B0C.It was subsequently screened through a 0.6 cm rectangular-shaped mesh andtransferred into a 60 L covered plastic container, which was kept at the sametemperature (4 \u00C2\u00B0C). The screening removed most of the large fibrous material andnearly all the hair and seeds that may cause major problems in pump lines.The total solids (TS) content of the sludge was determined and then adjustedto a value of about 4,000 mg/L before feeding, by diluting with distilled water or bysettling and decanting the excess liquid, in order to provide a level of comparablefeed throughout the entire study. (The TS content of primary sludge, after screening,ranged from 2,400 to 10,350 mg/L, with an average value of 5,140 mg/L).34CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^354.2. EXPERIMENTAL SET-UP AND OPERATION4.2.1. GENERAL SYSTEM CONFIGURATIONThis research involved the use of two laboratory-scale, continuous-flow units,having different configurations: a completely mixed reactor (CMR) with clarifier andsludge recycling, and an upflow anaerobic sludge blanket (UASB) reactor, asdepicted in Figure 4.1.Both reactors were made of plexiglass (internal diameter: 11.2 cm, totalvolume: 3.2 L, liquid volume: 3.0 L). The CMR system was equipped with a stainlesssteel stirrer with blades for mixing of the contents. Visually, complete horizontal andvertical mixing appeared to be achieved. The use of a clarifier (internal diameter:11.2 cm, liquid volume: 1.0 L) was necessary to avoid a substantial loss of biomassthrough the effluent line. The bottoms of the clarifier and the UASB reactor weremodified to an inverted cone shape (height of cone: 8.0 cm, diameter at bottom: 4.0cm) in order to provide good settling conditions for the clarifier and a bettermixing/diffusion flow pattern in the UASB system respectively.The two reactors were hermetically sealed to avoid any air entrapment. Asmall, cone-shaped device (height of cone: 6.0 cm, diameter at top: 4.0 cm) wasattached internally to the cover of each reactor to act as a gas collection system. Thegas production was monitored by two wet gas flow meters via water trap flasks.Spigots for sampling and wasting were placed at different heights (12.0 and24.0 cm from the bottom in the CMR reactor; and 8.0, 16.0 and 26.0 cm from theGas FlowMeterCHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^36COMPLETELY MIXED^UPFLOW ANAEROBICREACTOR (CMR) SLUDGE BLANKET (UASB)Waste^ WasteSR : Sludge RecycleKr: PumpFEED I^REACTOR I^CLARIFIER^REACTOR IIFIGURE 4.1. EXPERIMENTAL LAYOUTGas Flow Meter QSR^...-EffluentFEED IICHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES 37bottom in the UASB unit). However, the lowest port in each reactor was exclusivelyused for wasting purposes. The other ports were periodically used to test theoperational stability of the systems.At two strategic diametrically opposite points, perpendicular to the feed-wasting-effluent ports, a combination oxidation-reduction potential (ORP) probe(Broadley-James Corporation) and an epoxy-body combination pH probe (Cole-Parmer Company) were inserted into each reactor. The ORP probe uses a Ag/AgC1reference electrode, with a platinum band as the noble metal, and the pH elecrode hasa sealed (gel) reference electrode. Each probe was affixed to one end of a piece ofrigid plastic tubing which slid inside the sleeve of another plastic tube, withminimum resistance. This latter tube opened up through a ball valve acting as achannel to allow the probe to slide into and out of the reactor. An 0-ring sealinserted between the two cylinder walls prevented liquid being forced by backpressure from the interior of the reactor.The ORP probes were used throughout the entire experimental study, while thepH probes were inserted only during the last two runs (Runs 4A and 4B), as part ofthe pH control strategy.4.2.2. OPERATIONA total of 11 runs were conducted to investigate the effects of selectedoperational and environmental parameters on the acid-phase digestion of primarysludge. The research evolved into 4 stages, and in each stage an attempt was madeCHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^38to explore the influence of a particular parameter.In Stage 1 (Runs 1A to 1D) the role of Hydraulic Retention Time (HRT) wasinvestigated, while Stage 2 (Runs 2A to 2C) focused on the effect of Solids RetentionTime (SRT). The question of reproducibility at different conditions (summer/winter)and the source of influent sludge were the targets in Stage 3 (Runs 3A and 3B).Finally, the effect of pH was explored in Stage 4 (Runs 4A and 4B). All experimentswere conducted at an ambient liquid temperature between 18 and 22 \u00C2\u00B0C.A summary of operating conditions is presented in Table 4.1. The SRT of thesystems was controlled by wasting the appropriate volume from each reactor on adaily basis. The waste volume was slightly adjusted, when necessary, to compensatefor the loss of biomass [measured as Volatile Suspended Solids (VSS)] through theeffluent line. For the UASB system, the wastage was based on the sludge blanketvolume (eg. to maintain an SRT of 10 days, one tenth of the volume of the blanketwas wasted). Since the VSS content of the supernatant and the effluent was very low,usually less than 2% of that of the blanket, it was considered to have no appreciableeffect.The HRT in each system was controlled by a Cole-Parmer Company pump(Model 7015-21). The difference in liquid volume in the feed containers wascalculated daily using a specially graduated stick and converted into thecorresponding HRTs.For feeding purposes, the primary sludge was transferred from the 60 L plasticcontainer to two 10 L buckets and, after adjusting the TS content to 4,000 mg/L (asexplained in Section 4.1.), it was added to the two feed tanks at the end of eachCHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^39experimental day. This was done to minimize possible changes in feed characteristicsdue to higher temperatures in the early afternoon hours, especially during the summermonths. To avoid altering the characteristics of the feed due to excess aeration, thefeed tanks were covered with plastic lids and the stirrer speeds were kept as low aspossible, while still keeping the particulate matter in suspension.TABLE 4.1. OPERATING CONDITIONS (MEAN VALUES)RUN REACTORTYPESRT(d)WASTE(mild)HRT(hr)pH ORP(mV)lA CMR 10 290 9.03 5.25 -326UASB 10 180 9.25 5.14 -3691B CMR 10 280 6.14 5.27 -284UASB 10 250 6.09 5.33 -3621C CMR 10 290 12.12 5.01 -309UASB 10 160 12.07 4.96 -3851D CMR 10 290 14.91 5.10 -325UASB 10 150 15.28 4.98 -3702A CMR 15 190 12.20 5.17 -343UASB 15 110 12.11 5.09 -3762B CMR 20 140 12.06 5.23 -364UASB 20 80 11.94 5.09 -3912C CMR 5 400 12.14 5.63 -354UASB 5 275 11.89 5.52 -3853A CMR 10 290 11.84 5.15 -296UASB 10 170 12.16 4.98 -3713B CMR 10 290 12.05 5.03 -311UASB 10 160 12.11 5.05 -3674A CMR 10 290 12.22 4.42 -278UASB 10 160 12.13 4.47 -3334B CMR 10 290 11.97 6.09 -326UASB 10 160 12.02 6.05 -393CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES 40Total solids (TS) were selected as the control parameter, since their analysisinvolves a simple, direct and readily accessible method. The TS concentration appliedin this study was determined by a trial-and-error procedure, as the highest amountof solids contained in the feed without causing frequent operational problems. Twoprevious attempts, using 10,000 and 7,000 mg/L TS respectively, were proven to beunsuccessful due to a number of regular operational difficulties (ie. blockages of feedlines, pump failures, reactor overflows).The recycle pump (Cole-Parmer Company, Model 7017-21) operated on a cycleof 5 minutes on and 5 minutes off. This combination was found to be adequate toclear the recycle line of blockages and to ensure a reliable volumetric throughput.The flow rate was adjusted accordingly so that the retention time in the clarifier wasless than 8 hours. A scraper mechanism operating at 1 rpm was installed to preventthe sludge adhering to the clarifier walls.During the pH control experiments (Runs 4A and 4B) a Cole-Parmer CompanypH/pump system (Series 7142) was connected to each one of the pH probes whichwere inserted into the reactors. The pH/pump system was set at a selected pH valueand the pump was activated whenever the pH was higher (in the case of acidaddition) or lower (in the case of base addition) than the set value. Aqueous solutionsof 0.02 N HC1 and 0.02 N NaOH were used to respectively decrease or increase thepH in the reactors.The reactors were seeded with sludge obtained from a fermenter installed atthe University of British Columbia pilot plant (a biological phosphorus removalresearch facility) treating mostly campus wastewater, by adding 1.0 L of seed and 2.0CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^41L of tap water. Some basic characteristics of the seed are presented in Table El,Appendix E.At the end of Stages 1 and 2, and afterwards, at the end of each run bothsystems were drained and cleaned and some of the tubing was replaced. At the sametime, the ORP probes were cleaned and their responsiveness tested by immersion ina quinhydrone solution, as described by the A.S.T.M. (1989b), and discardedwhenever it was considered necessary. To increase accuracy and reliability, the pHprobes used in Run 4A were also replaced at the end of the run.In every experimental run the systems were considered to be in steady-statewhen the volatile acid production showed approximately steady values (ie. less than10 percent variation in concentration). This was usually achieved in less than twoweeks of operation, although sporadic disturbances occurred afterwards in certaincases. To ensure that reasonable steady-state conditions were established, mostexperiments were operated for about 4 to 5 SRTs, as shown in Table 4.2.4.3. ANALYTICAL PROCEDURESSamples were obtained from three locations, in order to follow the compositionchanges that occurred through the process:1) Influent sample: from the 10 L feed buckets, just before feeding.2) Reactor sample: from the wastage of each reactor.3) Effluent sample: from the effluent line of each system.Sampling, handling and preservation times before analysis were kept to aCHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^42minimum. Most tests were performed regularly on a biweekly basis. The majority oftests were conducted in accordance with Standard Methods (A.P.H.A. et al., 1989).When non-standard testing procedures were performed, they are discussed in detailbelow.All sample filtrations were done using Whatman No. 4 filters, with theexception of solids analysis where Whatman 934-AH glass microfiber filters wereused.TABLE 4.2. DURATION OF EXPERIMENTAL RUNS AND AMOUNTOF BIOMASS (VSS) IN THE REACTORSRUN SRT(d)DAYSPER RUN# OFSRTsVSS IN REACTORSCMR (g) UASB (g) % DIF.lA 10 86 8.6 42.76 50.21 14.81B 10 44 4.4 52.70 66.68 21.01C 10 52 5.2 31.23 33.73 7.41D 10 47 4.7 23.58 27.27 13.52A 15 54 3.6 27.52 33.95 18.92B 20 55 2.8 30.01 35.85 15.52C 5 28 5.6 14.89 19.22 22.53A 10 38 3.8 29.65 32.03 7.43B 10 41 4.1 30.81 34.27 10.14A 10 38 3.8 28.34 33.81 16.24B 10 48 4.8 28.14 29.76 5.4AVER. 48 4.7 13.9CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^434.3.1. CHEMICAL OXYGEN DEMAND (COD)The filtered samples were preserved by freezing at -7 \u00C2\u00B0C and analyzed induplicate using the dichromate reflux procedure outlined in Standard Methods(A.P.H.A. et al., 1989).4.3.2. TOTAL ORGANIC CARBON (TOC)The filtered samples were preserved as above and analyzed in duplicate on anautomatic Shimadzu Total Organic Carbon Analyzer (Model TOC-500) using a seriesof low and high standards, as described in the Instruction Manual (ShimadzuCorporation, 1987). The method is based on the principle that the quantity of CO 2produced during combustion is proportional to the amount of carbon in the sample.4.3.3. ORGANIC ACIDSa) Volatile Fatty Acids (VFAs)The volatile fatty acid determination was conducted using a computer-controlled Hewlett-Packard 5880A gas chromatograph, equipped with a flameionization detector (FID). Helium was used as the carrier gas. Volatile fatty acidsanalyzed include: acetic, propionic, butyric, iso-butyric, valeric, 3-methylbutyric, and2 -methylbutyri c.The filtered samples were kept frozen at -7 \u00C2\u00B0C in sealed plastic transferCHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^44pipettes (Canlab No. P5214-1). At the time of analysis, the samples were thawed atroom temperature and diluted 1:5 (except the influent samples) with distilled water.After being acidified with 5% phosphoric acid to bring the pH below 3, 1.0 tialiquots were injected using microsyringes (Hamilton Model 701 N, 10 ill) and aHewlett-Packard auto-sampler (Model 7672 A). The glass column (length: 91.0 cm,external diameter: 6.0 cm, internal diameter: 2.0 cm) was packed with 0.3%Carbowax 20M/0.1% H3PO4 on Supelco Carbopack C. The column was conditionedaccording to the procedure described in the Supelco Bulletin 751E (1989).The operating conditions of the chromatograph were as follows:Injector temperature:^150 \u00C2\u00B0CDetector temperature:^200 \u00C2\u00B0CIsothermal oven temperature:^120 \u00C2\u00B0CFlow rate of helium gas:^20 mL/minQuantification of the response peaks was done by comparison with externalstandard methods using reagent grade standards. The detection limit of the methodis 1 mg/L. At least two aliquots of each sample were injected and the mean valuesreported.b) Lactic and Pyruvic AcidsThe same technique used for VFA analysis was applied for lactic and pyruvicacid determination, with the following three modifications:The samples were acidified using 0.03 M oxalic acid, the glass column waspacked with 4% Carbowax 20M on Supelco Carbopack B-DA, and the isothermalCHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^45oven temperature was 175 \u00C2\u00B0C (Supelco, 1990).c) Formic AcidFormic acid was analyzed by a colorimetric method outlined by Lang and Lang(1972). The bright yellow and green-yellow fluorescent reaction products from theformic-citric acid reaction change to raspberry red at room temperature, in the samemedium. The intensity of the color is proportional to the concentration of the formicacid present. Absorbance measurements were taken at 515 nm on a Bausch & LombSpectronic 80 using a 1.0 cm cell. The method has a detection limit of about 5 mg/L.Detail description of the reagents used and the analytical procedure followed isprovided by Kisaalita (1987).4.3.4. SOLIDSa) Total Solids (TS) and Volatile Solids (VS)Total solids were determined by evaporating a known volume of well-mixedsample in a Fisher Isotemp (Model 350) forced draft oven at 104 \u00C2\u00B0C. Subsequently,by igniting the residue at 550 \u00C2\u00B0C in a Lindberg muffle furnace (Type 51828), thevolatile solid content was measured. Both analyses were performed as outlined inStandard Methods (A.P.H.A. et al., 1989).b) Total Suspended Solids (TSS) and Volatile Suspended Solids (VSS)The TSS and VSS contents of influent and effluent samples were determinedCHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES 46in accordance with the Standard Methods (A.P.H.A. et al., 1989). A known volumeof sample was vacuum filtered through a pre-washed and oven-dried Whatman 934-AH glass microfiber filter and dried at 104 \u00C2\u00B0C for TSS analysis. The VSS weredetermined by igniting the residue at 550 \u00C2\u00B0C.Since the suspended solids concentration in both reactors was very high,usually more than 10,000 mg/1, the Gooch crucible method was consideredimpractical (Anderson, 1989). Instead, a known volume of well-mixed sample wastransferred into a 50 ml centrifuge tube and spun down at 2500 rpm in an IECInternational Centrifuge (Model CS-CC467) for about 15 min. The supernatant wasvacuum filtered through a pre-washed and oven-dried glass microfiber filter. Thesettled sludge at the bottom of the tube was scraped out and washed on to the filter.The filter was then placed on its aluminium storage dish and transferred into the 104\u00C2\u00B0C oven, and finally into the 550 \u00C2\u00B0C furnace, as described above.4.3.5. NITROGENa) Ammonia Nitrogen (NH 3-N)Samples for ammonia determination were first filtered and preserved withconcentrated sulfuric acid and then stored at 4 \u00C2\u00B0C. Ammonia nitrogen was analyzedin triplicate by the automated phenate method, using a Technicon Autoanalyzer IIContinuous Flow System (Industrial Method No. 98-70W). Appropriate dilutions weremade prior to determining the intensity of the color complexes formed and thencompared with those of a series of standards of known concentrations.CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^47b) Total Kjeldahl Nitrogen (TKN)Total Kjeldahl nitrogen (TKN) was measured by digesting the samples in aBD-40 Technicon Block Digester with concentrated H 2 SO4 and K2SO4 , to liberateall organically bound nitrogen. Filtered or unfiltered samples were used to determinethe soluble or total TKN respectively. Standards and samples were analyzedcolorimetrically in triplicate using a Technicon Autoanalyzer II (Industrial MethodNo. 376-75W), according to Technicon Block Digester Instruction Manual (1974).4.3.6. PROTEINSThe amount of protein in a sample can be estimated by measuring the nitrogencontent of the organic matter present. Organic nitrogen includes the nitrogen inamino acids, amines, amides, imides, nitro-derivatives and a number of othercompounds. Most of the organic nitrogen that occurs in municipal wastewater,however, is in the form of proteins or their degradation products: polypeptides andamino acids (Sawyer and McCarty, 1979). Therefore, assuming that all organicnitrogen is due to protein and that protein contains on average 16 percent nitrogen(Gaudy and Gaudy, 1980), the protein content can be calculated from thecorresponding TKN value by subtracting the inorganic nitrogen concentration (in thiscase only the NH 3 -N value) and multiplying the difference by 6.25 (100 divided by16).CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^484.3.7. LIPIDSTotal lipids were determined by dry extraction (influent and reactor samples)and wet extraction methods (effluent samples).a) Dry Extraction MethodSamples were dried overnight at 104 \u00C2\u00B0C, ground in an Oster commercialblender, and weighed on Whatman 941 paper filters (9.0 cm diameter). They werethen subjected to continuous extraction for 6 hours in a Soxhlet apparatus usingpetroleum ether as solvent, in accordance with the procedure described by Trieboldand Aurand (1969). Reweighing the samples with a Mettler AC 100-52 balance (afterat least 1 day in a vacuum desiccator), allowed calculation of the total lipid contentas percent of the TS of the sample.b) Wet Extraction MethodAccording to the Rose-Gottlieb Method (Triebold and Aurand, 1969), a knownvolume of the sample is treated with ethanol and ammonium hydroxide solution, andthen extracted with a 1:1 mixture of ethyl and petroleum ethers. The etherscontaining the dissolved lipids are decanted into a weighed flask. The extraction isrepeated a second time, after which the solvents are evaporated and the weight of theextracted total lipids determined.CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^494.3.8. CARBOHYDRATESSince cellulose is a major component in domestic wastewaters (Gaudy andGaudy, 1980), a method specifically suited to measure the cellulose content (as theone described below) is considered indispensable for total carbohydrate analysis inthis study.The first step in carbohydrate determination involved an acid hydrolysistechnique outlined in the A.S.T.M. (1989a). A primary hydrolysis of the samples with72% H 2SO 4 at 30 \u00C2\u00B0C for 1 hour was followed by a secondary hydrolysis in apreheated autoclave (Barnstead Company, Model C-0704) for 4 hours. The dilutedhydrolyzates were then neutralized with a 6 N solution of NaOH.The second step of the analysis included the post-neutralization stages of theferricyanide method (Handbook of Micromethods, 1974). Adding the appropriatereagents (carbonate-cyanide, ferricyanide, and ferric-iron), a blue complex is formedwhose intensity is proportional to the concentration of glucose. The absorbance isthen measured at 690 nm and compared with that of a range of glucose standard ofknown concentrations.For soluble carbohydrate determination, the ferricyanide method was followedentirely.CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^504.3.9. PHOSPHORUSa) Orthophosphate (PO 4 -3)Samples for orthophosphate analysis were filtered and preserved as in the caseof ammonia (Section 4.3.5.a). Orthophosphate was determined in triplicate by theautomatic ascorbic acid reduction method on a Technicon Autoanalyzer II (IndustrialMethod No. 327-73W). According to this technique, a blue-colored antimony-phosphomolybdate complex is formed when ortho-phosphate reacts with ammoniummolybdate and potassium antimonyl tartrate. The peak heights of standards of knownconcentrations are then compared to those of the samples.b) Total Phosphorus (TP)The samples were treated the same way as in TKN analysis (4.3.5.b). Allorganically bound phosphorus, liberated by acid digestion, is oxidized to ortho-phosphate, which can be measured by the ascorbic acid method mentioned above.4.3.10. pH AND ALKALINITYA Beckman 44 pH meter with automatic temperature compensation was usedto determine the pH of the samples. The meter was calibrated daily, prior tomeasurements, using two standard buffer solutions of pH 4.0 and 7.0.Total alkalinity was measured by titrating the samples to an end point pH of4.5 with 0.02 N sulfuric acid.CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^514.3.11. OTHER SOLUBLE ORGANICSIn order to tentatively identify the nature of other soluble degradationproducts, a series of samples were analyzed on a Hewlett-Packard 5985B GasChromatography - Mass Spectroscopy system. The mass spectrometer was operatedin the electron impact mode, using helium as carrier gas, at the following conditions:Ion source temperature: 200 \u00C2\u00B0CIonizing energy:^70 eVScan range:^34-350 amu at 1 A/D measurement.The mass spectra were acquired with the data system and the peaks wereidentified with base peak probability matching using the library EPA-NIH MassSpectra Database as described by Girard (1991).The organic compounds analyzed included: ethanol, butanol, 2-propanol, 1,3-propanediol, 2,3-butanediol, 1,2,3-propanetriol (glycerol), ethanal (acetaldehyde),acetone, and 2,3-butanedione.4.3.12. GAS ANALYSISGas samples were extracted periodically from the head space of the reactorsusing an 1 ml Hamilton syringe and rapidly injected into a Fisher-Hamilton GasPartitioner (Model 29), using helium as carrier gas and a thermal conductivitydetector. The gases were identified by comparing their retention times to standardgases and concentrations were estimated by comparing the peak areas with knownCHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^52standards that were used to determine response factors.4.4. COLD STORAGE TESTINGThe effects of cold storage on sludge characteristics were studied at thebeginning of the experimental research and at the end of Stage 1. This wasconsidered necessary, since the raw sludge used in this study was to be kept at 4 \u00C2\u00B0Cfor approximately 1 to 2 weeks. In each testing, over a period of 20 days, sampleswere taken from the supernatant of one of the 25 L carboys and analyzed for thefollowing parameters in duplicate: chemical oxygen demand (COD), total organiccarbon (TOC), and volatile fatty acids (VFA).4.5 STATISTICSAverages, standard deviations, coefficients of variation and significantdifference between the means (t-test) were calculated by the statistics packageincluded in Symphony (release 1.2) of Lotus Development Corporation (CambridgeMA).CHAPTER 5RESULTS AND DISCUSSION5.1. GENERAL CHARACTERISTICSA brief description of important general issues concerning the nature of thisresearch is presented below, preceding the detailed analysis of the four experimentalstages.5.1.1. FEED COMPOSITIONAn understanding of the nature of the wastewater used as feed is essential inthe design and operation of any biological treatment process. To promote thisunderstanding, an analysis of important physical and chemical constituents of primarysludge was performed regularly and the data have been included in Appendix C. Asummary of the results, along with a basic statistical evaluation, is presented in Table5.1 (Iona Island WWTP) and Table 5.2 (Lions' Gate WWTP).The total solids concentration of the reactor influent was continually adjustedto 4,000 mg/L, as mentioned earlier (Section 4.1), to provide a uniform feed for theentire experimental program.Despite the fact that the range for most parameters appears to be quite wide(especially for the sludge from Iona Island), the statistical evaluation shows that both53CHAPTER 5. RESULTS AND DISCUSSION 54the standard deviation (STD) and the coefficient of variation (CoV) are reasonablysmall. For instance, the CoV of all 4 types of solids is less than 10%, whichindicates that the majority of measurements are close to the mean.Comparing the two sources of feed, the most noteworthy difference lies in theVFA and NH 3 -N content. The feed from Lions' Gate plant contains roughly 40% (forboth parameters) of the amount present in the other source.TABLE 5.1. INFLUENT SLUDGE CHARACTERISTICS (IONA ISLAND WWTP)PARAMETER RANGE MEAN STD CoV(%)pH 5.63 - 6.93 6.09 0.20 3.3TS 3260 - 5235 4007 294 7.3VS 2300 - 4205 2990 279 9.3TSS 2625 - 5050 3613 315 8.7VSS 2035 - 3985 2710 263 9.7CARBOHYDRATES 1140 - 2700 1703 218 12.8PROTEINS 473 - 834 627 64 10.2LIPIDS 394 - 693 507 47 9.3COD 244 - 673 427 73 17.1TOC 71 - 208 128 25 19.5VFA (as HAc) 46 - 150 103 19 18.4NI-13 -N 13 - 35 20 3.8 19.0TKN 89 - 158 121 11 9.1PO4-3 (as P) 6 - 17 10 1.8 18.0TP 14 - 27 19 2.3 12.1ALKAL. (as CaCO3 ) 141 - 216 184 16 8.7Note: All values in columns RANGE, MEAN and STD are expressed in mg/L, except pH.CHAPTER 5. RESULTS AND DISCUSSION^ 55TABLE 5.2. INFLUENT SLUDGE CHARACTERISTICS (LIONS' GATE WWTP)PARAMETER RANGE MEAN STD CoV(%)pH 5.74 - 6.33 6.00 0.16 2.7TS 3630 - 4340 4032 204 5.1VS 2925 - 3585 3285 228 6.9TSS 3065 - 3870 3539 219 6.2VSS 2470 - 3310 2956 216 7.3CARBOHYDRATES 1790 - 2360 2091 182 8.7PROTEINS 497 - 611 559 36 6.4LIPIDS 434 - 537 486 32 6.6COD 359 - 522 445 50 11.2TOC 91 - 153 128 19 14.8VFA (as HAc) 29 - 67 44 11 25.0NH3 -N 5 - 11 8 2.0 25.3TKN 88 - 109 97 7 7.2PO43 (as P) 5 - 9 7 1.1 15.7TP 10 - 16 13 1.8 13.8ALKAL. (as CaCO 3 ) 153 - 197 172 16 9.3Note: All values in columns RANGE, MEAN and STD are expressed in mg/L, except pH.The classification of the organic composition of the influent reveals thatcarbohydrates are by far the most predominant group in the raw sludge from bothfacilities (Table 5.3). Proteins and lipids are the other two important classes oforganic compounds present. The three groups together account for 95% of thevolatile solids content. The sludge from Lions' Gate is richer in carbohydrates butcontains less protein and lipid.CHAPTER 5. RESULTS AND DISCUSSION^ 56The particulate fraction of the organic matter is very high, as indicated by theVSS/VS ratio, which averages about 90%. Lipids and polysaccharides are practicallyinsoluble in water. Analytical determination of soluble carbohydrates and proteinshas shown that only 7 and 16% of the total respectively occurs in soluble form(Tables E2 and E3, Appendix E). Most of proteins in domestic wastewater arenormally of globular nature and, therefore, water soluble (Gaudy and Gaudy, 1980).The low soluble protein content of primary sludge indicates that most of theproteinaceous matter is still an integral part of the suspended solids.TABLE 5.3. ORGANIC COMPOSITION OF FEEDORGANICCLASSIONA ISLAND WWTP LIONS' GATE WWTPRANGE(% of VS)MEAN(% of VS)RANGE(% of VS)MEAN(% of VS)CARBOHYDRATES 49 - 63 56 60 - 67 64PROTEINS 18 - 25 21 15 - 19 17LIPIDS 12 - 21 17 12 - 16 15TOTAL 94 965.1.2. COLD STORAGE EXPERIMENTSThe results of both tests on stored raw sludge (at 4 \u00C2\u00B0C) showed no significantvariation in any of the chemical parameters tested for at least a period of 12 days(Table 5.4). The variations observed are comparable to those expected during thechemical analysis through experimental errors. Manoharan (1988) has reported thatCHAPTER 5. RESULTS AND DISCUSSION^ 57no change in raw sewage characteristics took place within two weeks of cold storage.In this study, however, after 14 to 16 days a gradual decrease in COD and TOC andan increase in VFAs occurred, presumably as a result of bacterial activity. To avoidany alterations in feed composition, the maximum storage period was set at 10 days.TABLE 5.4. COLD STORAGE TESTINGDAYTEST ONE (WINTER) TEST TWO (SUMMER)COD(mg/L)TOC(mg/L)VFA(mg/L)COD(mg/L)TOC(mg/L)VFA(mg/L)0 420 139 98 452 137 842 426 128 102 440 129 824 411 124 96 449 125 796 406 122 94 431 130 888 415 130 101 443 120 8610 419 122 95 435 126 8012 414 117 103 428 125 8414 408 120 101 402 109 8616 370 106 113 361 99 9318 373 102 115 347 97 9720 298 84 1065.1.3. ACCLIMATION AND STABILITY OF OPERATIONThe original heterogeneous population in a bioreactor has to undergobiochemical acclimation and selection of the species best able to grow on the carbonCHAPTER 5. RESULTS AND DISCUSSION^ 58sources available in order to ensure successful and sustainable operation. Incontinuous-flow systems, acclimation is a time-dependent process and it can beinfluenced by the type of seed used, the characteristics of feed, and the chosenoperational and environmental conditions.In this study, acclimation was accomplished in a rather short period of time(6 to 10 days) in both reactors. The phenomenon was considered complete when boththe increase in VFA production and the decline in pH (when it was not controlled)exhibited signs of stability. The short acclimation period observed can be attributedto the synergistic action of a number of factors such as the suitable seed used (takenfrom an acid-phase digester), the good digestability of the primary sludge, thefavorable operating conditions and the small volume of the reactors.It has been reported (Lettinga et al., 1979; de Zeeuw and Lettinga, 1980) thatin UASB systems, long acclimation periods (sometimes up to 4 to 8 weeks) may berequired, because of the slow formation of sludge blanket. The phenomenon ofmicrobial aggregation and granulation, however, is greatly affected by variousnutritional and environmental parameters such as trace metal ions (particularlycalcium), temperature, the nature of the inoculum, and feed used (Mahoney et al.,1987; Guiot et al., 1988). Investigating a number of seeding and reactor loadingalternatives, Fongsatitkul (1992) has found that, in most cases, the acclimationprocess was completed within 4 weeks. Using mesophilic granular sludge as seedmaterial, van Lier et al. (1992) have observed that the start-up period in a UASBsystem was between 1 and 2 weeks. In this study, the good settling properties of thesludge resulted in a sludge blanket formation in about 3 to 6 days with minimal lossCHAPTER 5. RESULTS AND DISCUSSION 59of biomass, which in turn induced steady-state conditions within the next few days.The small volume of the reactor might have also played a critical role. Eastman andFerguson (1981) have found that steady-state was achieved within 7 days in 2.5 Lcompletely-mixed acidogenic reactors.Both systems' behavior during the steady-state analysis period was stable. Nosignificant trends were observed in any parameter over the 3 to 9 solids retentiontimes of the experiments. The standard deviation for individual analyses was within12% of the mean for almost all measured parameters. The higher variability observedin certain parameters in two cases (Runs 1B and 2C) is attributable to bacterial stressimposed by the short HRT and SRT respectively, of those runs.Furthermore, the ORP values measured ranging from -270 to -400 mV (Table4.1), suggest that good anaerobic conditions were maintained throughout thisexperimental investigation. The ORP values were always lower in the UASB reactor.Since the probe was inserted in the sludge blanket of this reactor, it is obvious thatthe environment is more reductive inside the blanket than it is in the CMR system.5.2. THE EFFECT OF HRT - STAGE 15.2.1. HRT AS A CONTROL PARAMETERThe microbial population of most natural environments is usually dominatedby a relatively small number of species. A few selective environmental parameterssuch as pH, ORP, temperature, a toxic factor, the presence or absence of a keyCHAPTER 5. RESULTS AND DISCUSSION 60growth factor, etc., operate to impose a limit on the heterogeneous nature of thebacterial population, and thereby \"select\" one or more dominant cultures. Thisphenomenon of species selection has found a variety of applications in environmentalengineering and related fields (Ghosh and Pohland, 1971).In closely monitored continuous-flow bioreactors, any or all of the parameterscan be controlled and maintained constant. Consequently, it is often possible to selectand retain a group of microbial species which would accomplish the desiredbiochemical conversions at acceptable reaction rates. Survival of individual species,however, in heterogeneous cultures such as those found in wastewater treatmentprocesses, depends upon a variety of less defined factors. Although the populationin such processes tends to remain, to some degree, heterogeneous because of mutualmicrobial interactions (eg. competition, amensalism, parasitism, mutualism,symbiosis, predation, etc.), the relative proportion of each major species changesfrom one condition to another (Harrison, 1978).An important operational variable which can be easily manipulated is thehydraulic retention time (HRT). It is the average length of time a molecule of liquidremains in the reactor and can be defined as the volume of the reactor divided by theaverage influent flow rate. HRT governs the amount and type of substrate being usedby the cells. Since anaerobic digestion is a two-phase process, HRT can act as aselection parameter for the acidogenic phase only if it encourages the growth of acidformers and concurrently suppresses the growth of methane producers.One of the objectives of this study has been to investigate the effect of the twooperational parameters [HRT and SRT (defined in Section 5.3.1.)] independently. ForCHAPTER 5. RESULTS AND DISCUSSION 61this reason, small HRT values are selected in contrast with the SRT. In Stage 1, theHRT varied from 6 to 15 hours, while SRT was kept constant at 10 days. The rawdata collected during this stage are tabulated in Appendix C (Tables Cl to C20).5.2.2. VFA PRODUCTIONShort-chain volatile fatty acids (C 2 to C 5 ) are normally the main products ofthe acidogenic digestion of primary sludge (Chynoweth and Mah, 1971). The highconcentrations of total VFAs (expressed as acetic acid for comparison purposes)achieved in both bioreactors clearly support the above observation (Appendix C). Forexample, the profiles of influent and reactor VFA concentrations, depicted in Figure5.1 (Runs 1C and 1D), show that a sharp increase in the reactor VFA content occursin both runs. This suggests that favorable conditions for the growth and maintenanceof a healthy population of acid-producing microorganisms have been establishedduring the course of the experiments.The total net VFA production (as acetic acid) at steady-state operation, as afunction of HRT, is presented in Figure 5.2. In both systems, VFA concentrationincreases with HRT to a maximum value at 12 hours. A further increase in HRTresults in a drop in concentration by about 80 mg/L. The decline in VFA generationcoupled with the higher production rate of gaseous end-products (as explained inSection 5.3.4) provide strong evidence that methane-forming bacteria have beenstimulated at an HRT of 15 hours.CHAPTER 5. RESULTS AND DISCUSSION90080070060050040030020010006280070060050040030020020^40^60^80^100TIME (d)FIGURE 5.1. VFA PROFILE (RUNS 1C & 1 D)SRT = 10 d+ REACTOR II (UASB)6^9^12^15HRT (hr)FIGURE 5.2. NET VFA PRODUCTION AS A FUNCTION OF HRTCHAPTER 5. RESULTS AND DISCUSSION^ 63It is obvious that the mean VFA concentration is consistently higher in theUASB reactor. As a result of the good settling properties of the sludge blanket, theamount of active biomass (measured as VSS) lost through the effluent line isconsiderably lower as compared to the CMR system (Appendix C). Since morebacteria are retained in the upflow reactor (the amount of VSS in the UASB reactoris on the average 14% higher than that in the CMR System - Table 4.2), they can inturn generate a greater amount of products. However, the net VFA specificproduction rate, expressed as mgVFA/mgVSS*d, is similar in both units (Table 5.5).This is an indication that the ability of biomass to generate VFAs appears to beindependent of the reactor configuration, at least for the above mentioned conditions.TABLE 5.5. VFA SPECIFIC PRODUCTION RATE AS A FUNCTION OF HRTRUN HRT(hr)CMR(mgVFA/mgVSS*d)UASB(mgVFA/mgVSS*d)1B 6 0.067 0.061lA 9 0.083 0.0801C 12 0.101 0.1031D 15 0.092 0.089The specific production rate is largely affected by the change in HRT, reachingits maximum value at 12 hours. The low rate at the shortest HRT is mainly due tothe limited time available for substrate assimilation, while the decline noticed at thelongest HRT is probably caused by the conversion of soluble VFAs to gaseousproducts. Although gas generation has been detected for all HRTs (Table E4,CHAPTER 5. RESULTS AND DISCUSSION 64Appendix E), the sharp increase observed in Run 1D (HRT 15 hours) suggests thatmethanogenic activity was encouraged in this case. It is possible that the fraction ofmethane-forming bacteria in the biomass at a 10 day SRT was sufficient to affectnet VFA production at the longer HRT. Based on the above results, the optimumHRT for VFA formation (for this type of wastewater) is 12 hours, with an acceptablerange of more than 9 and less than 15 hours.The VFA concentrations in the reactor and in the effluent of the CMR systemare essentially the same (Table 5.6). It is possible that a dynamic equilibrium existsin the clarifier between the rates of acid formation and volatilization. In general, therate of desorption of a volatile compound from the liquid phase is a function of pH,temperature, degree of turbulence, viscosity of the liquid, and the molecularproperties of the specific compound (Loehr et al., 1973). Due to a number ofsynergistic reasons such as the short retention time of the liquid in the clarifier (2to 5 hours), quiescent flow conditions, ambient temperature, small surface area (99cm2), and higher pH values than the corresponding pK A values of the acids, thedegree of volatilization is considered to be minimal. Similarly, no appreciableacidogenesis should be taking place, mainly because of the short retention time (6to 8 hours) of the recyclable biomass.On the contrary, the VFA concentration (Runs 1A and 1B) in the effluent ofthe UASB system is considerably lower than that in the reactor (sludge blanket), asshown in Table 5.6. The difference in VFA, which is statistically significantaccording to the t-test performed (Miller et al., 1990), is probably due to flowchannelization in the reactor. At shorter HRTs (higher flow rates), the liquidCHAPTER 5. RESULTS AND DISCUSSION^ 65molecules in the middle of the reactor may move upwards faster (in a jet-likefashion) than those close to the wall and eventually leave the system earlier. Thisresults in an even shorter HRT for part of the reactor's contents diminishing, at thesame time, the opportunity for food assimilation by the bacteria, which canultimately lead to lower VFA concentration in the effluent of the unit. At higherHRTs (12 to 15 hours), the phenomenon of flow channelization appears to be ofminor importance, since the VFA values are essentially the same in the sludgeblanket and the effluent of the reactor.TABLE 5.6. COMPARISON OF REACTOR AND EFFLUENT VFA CONCENTRATIONSRUNCMR SYSTEM UASB SYSTEMREACTOR(mg/I-)EFFL.(mg/L)REACTOR(mg/L)EFFL.(mg/L)SIGNIF.DIFFER.1B (6 hr) 407 412 466 370 YESIA (9 hr) 540 530 603 465 YES1C (12 hr) 632 630 685 665 NO1D (15 hr) 550 560 610 608 NO5.2.3. VFA SPECIATIONIdentification of the individual acids formed during the acid-phase digestionof primary sludge is important, since it may furnish valuable information on themetabolic pathways involved in the process. The VFAs identified include: acetic,propionic, butyric, iso-butyric, valeric, 3-methylbutyric and 2-methylbutyricCHAPTER 5. RESULTS AND DISCUSSION 66(Appendix D). The above VFAs are normally generated not only during theacidogenic digestion of municipal wastewaters (Perot et al., 1988), but also duringthe digestion of a variety of agricultural industrial wastes (Lettinga et al., 1979; Gil-Pena et al., 1986; Machado and Sant'Anna, 1987). In addition, caproic (hexanoic)acid was seldom detected and never exceeded the 3 mg/L level.Acetic acid and propionic acid are by far the major VFAs produced, with anaverage value of about 46 and 32% respectively of the total (Table 5.7). In general,these two acids have been found to be the most prevalent VFAs (at an acetic topropionic acid ratio of about 1.3 to 1.5) in continuous-flow acid-phase digesters(Rabinowitz and Oldham, 1985). Butyric acid follows with 8%, while iso-butyric andthe 3 isomers of valeric acid account for the remaining 14%. The distribution of theindividual acids is essentially the same in both systems.It is interesting to note that the percent VFA distribution, despite somevariation in the minor acids from one run to another, is not affected by HRT (at leastin the range tested), which is in contrast with the pattern followed by both the netVFA concentration and the production rate. This observation can lead to thespeculation that either the majority of acid-producing bacteria is to some extentequally influenced by the variation in HRT, or possible differences in microbialactivity counterbalance each other so that the final picture is basically the same.The percent distribution also reveals that there is a shift towards the highermolecular weight VFAs (iso-butyric and the 3 isomers of valeric acid) during thedigestion of primary sludge, when compared to the influent VFA distribution. Theaverage values for Stage 1, illustrated in Figure 5.3, show that a relative reductionynaz1c-)(,)OTABLE 5.7. PERCENT VFA DISTRIBUTION AS A FUNCTION OF HRTVOLATILE^RUN 1B (6 hr)^RUN 1A (9 hr)^RUN 1C (12 hr)^RUN 1D (15 hr)FATTY MEANACID^CMR^UASB^CMR^UASB^CMR^UASB^CMR^UASBACETIC 43.7^45.9^48.0^45.3^47.5^46.0^50.3^43.7^46.3PROPIONIC^36.8^34.4^33.2^32.5^29.9^30.4^28.1^33.2^32.3BUTYRIC^7.7^8.0^7.3^7.1^9.9^10.3^7.5^6.9^8.1ISO-BUTYRIC 3.4 3.1 3.0 2.6 3.2 2.9 4.2 4.6 3.4VALERIC 3.7 3.9 4.2 6.3 4.4 5.6 5.2 5.7 4.93-METHYLBUT. 2.8 2.7 2.9 4.0 3.3 3.1 3.2 4.0 3.22-METHYLBUT. 1.9 2.0 1.4 2.2 1.8 1.7 1.5 1.9 1.850INFLUENTREACTOR I (CMR)REACTOR II (UASB)40302010BUTYRIC^VALERIC^2-METHYLBUTYRICPROPIONIC^ISO-BUTYRIC^3-METHYLBUTYRICVOLATILE FATTY ACIDS (VFAs)FIGURE 5.3. PERCENT VFA DISTRIBUTION (STAGE 1)0\00CHAPTER 5. RESULTS AND DISCUSSION^ 69in propionic and butyric acids occurs with an increase in iso-butyric, valeric, 3-methylbutyric, and 2-methylbutyric acids. On the other hand, acetic acid percentdistribution does not seem to be following any trend between the influent and thereactor contents. This shift can be primarily attributed to protein fermentation whichresults in the production of significant amounts of the above mentioned highermolecular weight VFAs. The rate of protein utilization is considerably higher in thebioreactors than in the environment of a sewer system or a primary clarifier,principally because of higher concentrations of biomass and longer SRTs. Moreover,greater availability of soluble extracellular proteins (enhanced by cell lysis and by-products of other biochemical reactions) and favorable environmental conditionsprevailing in such treatment units can further contribute to this phenomenon.5.2.4. PARTICULATE ORGANIC CARBON SOLUBILIZATIONParticulate organic material must first undergo liquefaction by extracellularenzymes, before being taken up by the bacteria. Since most of the substrate inprimary sludge is in the particulate form (about 90% as indicated by the VSS/VSratio), solubilization of organic matter is a crucial step in anaerobic digestion.Generally, the rate of hydrolysis depends upon the pH, temperature, the type ofsubstrate, the nature of biomass, the size of the particles, and the remainingconcentration of the biodegradable suspended matter (Eastman and Ferguson, 1981).Substrate solubilization can be estimated from a number of non-specificparameters such as COD, TOC, TSS and VSS. These parameters were routinelyCHAPTER 5. RESULTS AND DISCUSSION^ 70measured and the results are summarized in Appendix C.As illustrated by the profiles of influent and reactor soluble CODconcentrations depicted in Figure 5.4 (Runs 1C and 1D), a distinct increase in CODoccurs in both reactors which is the result of substrate conversion from a particulateto a soluble state.Variation in HRT has a profound effect not only on the net COD concentration(Figure 5.5), but also on the specific solubilization rates of COD and TOC, expressedas mg of net soluble COD or TOC per mg of VSS per day (Table 5.8). All threemaximum values correspond to an HRT of 12 hours, which coincides with the timerequired for optimum VFA production. In addition, the overall trend is very similarto the one described in the previous section for VFAs.TABLE 5.8. SPECIFIC SOLUBILIZATION RATES OF COD AND TOCAS A FUNCTION OF HRTRUN HRT(hr)COD RATE(mgCOD/mgVSS*d)TOC RATE(mgTOC/mgVSS*d)CMR UASB CMR UASB1B 6 0.159 0.160 0.054 0.054lA 9 0.163 0.168 0.057 0.0601C 12 0.187 0.198 0.070 0.0701D 15 0.169 0.175 0.064 0.063The percent soluble COD in the form of VFAs (calculated by converting theVFAs to COD using the appropriate factors shown in Table E5, Appendix E) as a1.81.6a--.^1.4DE)oz 7 1.22i 1.0z F.U \u00E2\u0080\u00940^0.8Z0O 0.60.40.21.41.3151260.60.7a^1.2---.Er)Z 1.1To'O 21=- . 1.0Q c 00z =w b 0.90z0O 0.89HRT (hr)CHAPTER 5. RESULTS AND DISCUSSION^ 7110020^40^60^80TIME (d)FIGURE 5.4. SOLUBLE COD PROFILE (RUNS 1C & 1 D)FIGURE 5.5. NET COD SOLUBILIZATION AS A FUNCTION OF HRTCHAPTER 5. RESULTS AND DISCUSSION^ 72function of HRT is presented in Figure 5.6. Although the percent volatile acid CODincreases with a change in HRT from 6 to 9 hours, no remarkable variation isobserved beyond this point. Similar percentages have been obtained in both reactorsfor all HRTs. The results suggest that the conversion rate of soluble substrates toVFAs may have reached a plateau. In other words, the rate of metabolism of solubleextracellular intermediate products to VFAs appears to be independent of HRT, abovea certain minimum value. A smaller percent volatile acid COD, observed at 6 hoursHRT, indicates that the mechanisms for acid generation are influenced by the shortHRT more drastically than those involved in hydrolysis or the production ofextracellular metabolic intermediates.The extent of organic substrate solubilization can be viewed from adiametrically opposite perspective, namely from the destruction of suspended solids.The percent VSS and TSS reduction was based on a mass balance around the reactorsat steady-state conditions. Mass balances were performed in two distinct mannersaccording to the method described by Koers (1979). The \"overall mass balance\"refers to a summation period including the entire steady-state length of a run, whilethe \"moving average mass balance\" involves averaging the values from multiplebalance periods, each equivelent to 1 SRT in length. As it is evident from theexample illustrated in Table E6, Appendix E, the two methods yield similar results.Suspended solid solubilization increases with HRT, but the percent changebecomes smaller at HRTs higher than 9 hours (Table 5.9). The gradually diminishingsensitivity of the rate of hydrolysis at higher HRTs might be due to the fact that itis actually approaching a maximum value beyond which it probably becomesCHAPTER 5. RESULTS AND DISCUSSION^ 73independent of HRT. The relatively high percent VSS reduction obtained providesan additional evidence that the particulate complex substrates in primary sludge areamenable to solubilization.TABLE 5.9. PERCENT VSS AND TSS REDUCTION AS A FUNCTION OF HRTRUNHRT(hr)VSS (%) TSS (%)CMR UASB CMR UASB1B 6 44.2 43.8 46.1 45.5lA 9 57.6 63.6 58.8 63.71C 12 63.1 70.6 64.2 69.61D 15 67.7 72.5 68.3 71.3The UASB reactor shows an overall better performance (except at the shortestHRT - Run 1B) in hydrolyzing the particulate organic material. This behavior, whichis also reflected in the COD solubilization rates (Table 5.8), does not result in higherVFA production rates. This is probably due to the presence of a different mix ofmicroorganisms in the UASB reactor which generate a greater variety of intermediateproducts during the degradation process.The percent TSS reduction results (Table 5.9) are essentially identical to thoseobtained from the VSS analysis. Since the VSS account for about 75 to 80% of theTSS in the feed, a substantial fraction of the particulate inorganics (approximatelyequal to that of the corresponding VSS) undergoes solubilization during digestion.This can be attributed to metabolic requirements and the low pH values in the reactors.CHAPTER 5. RESULTS AND DISCUSSION^ 745.2.5. SUBSTRATE DEGRADATIONCarbohydrates, proteins and lipids in that order are the three primary sourcesof organic substrates in primary sludge. Since they basically occur in particulateform, they have to be first hydrolyzed by the action of specific enzymes beforeundergoing further degradation.The fermentation of carbohydrates is one of the main pathways for theproduction of VFAs. In domestic wastewaters, carbohydrates are present in the formof polymers, principally as \"designated cellulose\". The term \"designated cellulose\"has been proposed by Hobson (1980) to denote that this is a material largely definedby the method of analysis rather than by chemical constitution. Designated cellulosemostly consists of residues of toilet and similar papers and the remains of cookedvegetables in human feces. It can be relatively easily hydrolyzed by cellulases (Nget al., 1977). The high percentages of total carbohydrate utilization observed, rangingfrom 43 to 77% (based on a mass balance at steady-state operation), are in agreementwith the above statement (Table 5.10). It is apparent that the percent conversionvalues are a function of HRT, which emphasizes the role of this parameter on theenzymatic hydrolysis of carbohydrates.Protein and lipid utilization patterns follow a trend similar to that ofcarbohydrates, regarding the influence of HRT (Table 5.10). The conversionpercentages, however, are remarkably different. Proteins are degraded at much lowerrates than the other two organic classes. In general, proteolytic activity has beenfound to take place in digesters using a number of feedstocks, but the overallINFLUENT MASS LOADINGLOSS IN EFFLUENT AND WASTAGESYSTEM I (CMR)SYSTEM II (UASB)-- -\u00C3\u00A0A.A..a..^-^.. -CHAPTER 5. RESULTS AND DISCUSSION^ 7580706050403020100^RUN 1 (6 hr)^RUN 1A(9 hr)^RUN 1C (12 hr)^RUN 10 (15 hr)FIGURE 5.6. PERCENT SOLUBLE COD IN THE FORM OF VFAs (STAGE 1)RUN 1 (6 hr)^RUN 1A(9 hr)^RUN 1C (12 hr)^RUN 1D (15 hr)FIGURE 5.7. CARBOHYDRATE DEGRADATION AS A FUNCTION OF HRT302826242220181614121086420CHAPTER 5. RESULTS AND DISCUSSION^ 76TABLE 5.10. PERCENT SUBSTRATE DEGRADATION AS A FUNCTION OF HRTRUNHRT(hr)CARBOHYDR (%) PROTEINS (%) LIPIDS (%)CMR UASB CMR UASB CMR UASB1B 6 43.5 43.1 26.4 24.5 63.4 47.5lA 9 56.1 66.6 35.6 38.5 72.4 53.51C 12 60.6 73.4 42.9 45.0 80.9 62.01D 15 64.2 76.8 47.7 47.6 83.2 67.3breakdown percentages are moderate (Summers and Bousfield, 1980; Gujer andZehnder, 1983).On the other hand, there has been a controversy regarding the extent of lipiddegradation during the acid-phase process. Some investigators have reported thatlipid dissimilation is minimal during the acid-phase step (Mahr, 1967; Eastman andFerguson, 1981), while others have observed a significant utilization of lipids(Chynoweth and Mah, 1971; Ghosh, 1987). This subject will be treated in some detailin Section 5.6.5.Concerning the effect of reactor configuration on substrate dissimilation, bothsystems exhibit a fairly similar behavior in protein reduction rates, but thedegradation pattern of carbohydrates and lipids are distinctly and consistentlydifferent (Figures 5.7 to 5.9). Lipids are broken down more efficiently in the CMRunit, while higher rates of carbohydrate utilization are observed (except at theshortest HRT - Run 1B) in the UASB reactor.LOSS IN EFFLUENT AND WASTAGESYSTEM I (CMR)\u00C2\u00AE SYSTEM II (UASB)-iigLOSS IN EFFLUENT AND WASTAGEECEa SYSTEM I (CMR)ZZZ SYSTEM II (UASB)77CHAPTER 5. RESULTS AND DISCUSSION109876543210 RUN 1B (6 hr)^RUN 1A (9 hr)^RUN 1C (12 hr)^RUN 1D (15 hr)FIGURE 5.8. PROTEIN DEGRADATION AS A FUNCTION OF HRTRUN 1B (6 hr)^RUN 1A (9 hr)^RUN 1C (12 hr)^RUN 1D (15 hr)FIGURE 5.9. LIPID DEGRADATION AS A FUNCTION OF HRT13:gCHAPTER 5. RESULTS AND DISCUSSION^ 785.3. THE EFFECT OF SRT - STAGE 25.3.1. SRT AS A CONTROL PARAMETERAnother operational variable which can be used as a selective factor byimposing a stress on bacterial communities is the solids retention time (SRT). It isthe average time allowed for a microorganism to remain in the reactor and can bedefined as the amount of suspended solids in the reactor divided by the amount ofsuspended solids leaving the reactor per day. The SRT governs the types oforganisms which eventually predominate in the system because it interferes directlywith their generation time.The physiology, environmental requirements, and growth kinetics of theacidogenic and the methanogenic groups of microbes may differ greatly from eachother. It has been reported (Ghosh and Klass, 1978) that the maximum specificgrowth rate of the acid-producing bacteria can be up to one order of magnitudehigher than that of the methane-producing organisms. This suggests that it is possibleto maximize VFA production in an acid-phase digester by operating the system at anSRT below some critical value. The critical SRT (which in many cases coincides withthe HRT of the system) can range from several hours to several days (Ghosh, 1987).In most acid-phase anaerobic digestion studies found in the literature (Section2.5) SRT and HRT are almost identical because of the use of batch reactors orconventional continuous-flow systems without solids recycle. The SRT/HRT ratio canbe slightly increased to 1.5-2 as a result of withdrawal of digester supernatant (HenzeCHAPTER 5. RESULTS AND DISCUSSION 79and Harremoes, 1983). Nevertheless, SRT and HRT are two different parameters andhave different effects on the biological process. For this reason, no clear distinctioncan be made between the individual influence of the two parameters on theacidogenic phase in these previous investigations.In this study, HRT and SRT were independently controlled through appropriatedesign and operational strategies. The very nature of the UASB reactor allows forindividual manipulation of these two variables, while for the same reason the CMRunit was modified by adding a clarifier with a solids recycling system. The ultimategoal has been to operate at an HRT as low as possible to minimize reactor volumeand associated capital costs; and concurrently to maintain a reasonably long SRT topromote growth and proliferation of the acid-generating organisms, process stabilityand minimal sludge production, without inducing growth of methane-formingbacteria.Based on the results from Stage 1, the optimum HRT for VFA production inboth reactors is 12 hours. The SRT in that stage was kept constant at 10 days. InStage 2, an SRT variation from 5 to 20 days is investigated (resulting in a range ofSRT/HRT ratios from 10 to 40). The chemical parameters measured during Stage 1were also recorded for Stage 2 and the data are presented in Appendix C (Tables C21to C35).5.3.2. VFA PRODUCTION AND SPECIATIONVolatile fatty acids were the main soluble compounds generated during this setCHAPTER 5. RESULTS AND DISCUSSION 80of experiments as well. The net VFA concentration plotted as a function of SRT(Figure 5.10), shows that an increase in the SRT of the system, up to 20 days (at aconstant HRT of 12 hours), results in higher VFA concentrations in both reactors. Ingeneral, the variation in SRT does not seem to have a profound effect on VFAproduction, with the exception of Run 2C (5 days SRT). The drastic drop in VFAconcentration observed in this case indicates that such a short SRT may impose astrong stress on the metabolic activity of the acidogenic bacteria. The operationalstability of either system also suffers, as reflected on the substantially higherstandard deviation (STD) values for this run for almost all the chemical parametersanalyzed (Tables C31 to C35, Appendix C).Information presented in Table 5.11 shows that the net VFA specificproduction rate increases with SRT up to 15 days, but a plateau appears to bereached at this value. The influence of SRT on the VFA production rate is rathermoderate (as in the previous case), except at the shortest SRT, where the productionrate is reduced to almost one half of that calculated at 10 days. Overall, the CMRTABLE 5.11. VFA SPECIFIC PRODUCTION RATE AS A FUNCTION OF SRTRUN SRT(d)CMR(mgVFA/mgVSS*d)UASB(mgVFA/mgVSS*d)2C 5 0.053 0.0561C 10 0.101 0.1032A 15 0.125 0.1102B 20 0.119 0.109155 10 208007000 600E 500400w 30000 2001000706050403020100CHAPTER 5. RESULTS AND DISCUSSION^81SRT (d)FIGURE 5.10. NET VFA PRODUCTION AS A FUNCTION OF SRT5^10^15^20SRT (d)FIGURE 5.11. PERCENT VFA SPECIATION AS A FUNCTION OF SRTCHAPTER 5. RESULTS AND DISCUSSION 82system is slightly more effective in producing VFAs at SRTs of 15 and 20 days (by14 and 9% respectively) than the other one, but at lower SRTs both systems exhibitsimilar rates of product formation. (The standard error of the mean for VFA specificproduction rates ranges from 3 to 5%).The VFA speciation results (Table 5.12) are in agreement with many of thefindings mentioned in Stage 1 (Section 5.2.3) such as those concerning the twopredominant acids (acetic and propionic) or the influence of reactor configuration(CMR and UASB). It is interesting to note that the VFA distribution is, to someextent, affected by the variation in SRT, but it appears to be independent of HRT.For better illustration purposes, the average value for each run (since they are verysimilar in both systems) is plotted as a function of SRT in Figure 5.11. The mostremarkable difference occurs in the case of the 4 \"minor\" acids (iso-butyric, valeric,3-methylbutyric and 2-methylbutyric). Their percent distribution increasesdramatically with SRT (almost doubles from 5 to 20 days). For both acetic andpropionic acid the percent distribution declines slightly with an increase in SRT. Inthe case of butyric acid, a maximum is reached at 10 days. The overall picturesuggests that different pathways for VFA production may predominate at variousSRTs. Short SRTs seem to favor the generation of straight C2 to C4 VFAs, but atlonger SRTs more branched C4 and C 5 acids are formed. Although the possiblepresence of slower-growing microorganisms at longer SRTs cannot be excluded, thedirect association of the 4 higher molecular weight VFAs with protein fermentationprovides strong evidence that this phenomenon is a result of proteinaceousmetabolism, as discussed in the following section.TABLE 5.12. PERCENT VFA DISTRIBUTION AS A FUNCTION OF SRTVOLATILEFATTYACIDRUN 2C (5 d) RUN 1C (10 d) RUN 2A (15 d) RUN 2B (20 d)CMR UASB CMR UASB CMR UASB CMR UASBACETIC 50.5 48.0 47.5 46.9 43.8 45.2 43.8 44.6PROPIONIC 31.6 32.3 29.9 30.4 29.9 28.2 27.5 26.4BUTYRIC 6.8 7.6 9.9 10.3 8.1 7.3 7.0 7.1ISO-BUTYRIC 3.3 2.9 3.2 2.9 3.8 3.7 5.2 5.5VALERIC 4.0 4.5 4.4 5.6 6.9 8.0 7.9 8.13-METHYLBUTYR. 2.4 2.8 3.3 3.1 5.1 5.4 6.0 5.32-METHYLBUTYR. 1.4 1.9 1.8 1.7 2.4 2.2 2.6 3.0ALL 4 MINORVFAs 11.1 12.1 12.7 12.7 18.2 19.3 21.7 21.9CHAPTER 5. RESULTS AND DISCUSSION^ 845.3.3. ORGANIC CARBON SOLUBILIZATION AND SUBSTRATEDEGRADATIONThe majority of observations made about the VFA data set are equallyapplicable to COD and TOC results. For example, the net COD concentration as afunction of SRT (Figure 5.12) shows a great degree of similarity with the VFAproduction (Figure 5.10), dropping sharply at an SRT of 5 days and approaching aplateau at longer SRTs. However, the COD and TOC specific solubilization rates(Table 5.13) appear to be independent of SRT (i.e. no decrease at 5 days SRT). Aplausible explanation for this phenomenon is that at short SRTs the biochemicalpathways followed for VFA production from soluble biopolymers are much moreinfluenced than those involved in hydrolysis. If the same microbial community isresponsible for the conversion of particulate organic matter to VFAs, it can beconcluded that SRTs below a certain value pose a limit on acidogenic activity,therefore intermediate soluble products accumulate.On the contrary, the percent soluble COD in the form of VFAs increasesdrastically with increasing SRT, approaching the 90% level at 20 days (Figure 5.13).It is apparent that longer SRTs favor the conversion of soluble metabolicintermediates to end-products.The percent VSS and TSS reduction results, based on a mass balance at steady-state conditions, are tabulated in Table 5.14. From the data, it is evident that thevariation in SRT plays a rather minimal role in the degradation of particulate matter,at least in the range investigated. All observations made in Stage 1 about the^4 ^-Vg^4^4 44 ..2^44^ 4^, 44 1^\u00E2\u0096\u00A04 - '^1 '' 44^- ^4^i^4\u00E2\u0096\u00BA4 44 44 .^4^ :^1 4-^4^ 4^ 4\u00E2\u0096\u00BA 4 4 ^ \u00E2\u0096\u00BA4.-... 4^ 44^ 4^-1 4'--^:44 ,-,._^4^ 44^ 4^--a-. 4 4^- \u00E2\u0080\u00A2\u00E2\u0096\u00BA \u00E2\u0096\u00BA4 _\u00E2\u0096\u00BA4\u00E2\u0096\u00BA44 4 -^4^-----^4'^4 7^ 4 4-. 4^ 4 -^4 ^--^44 4^ 4 -^4-^A 4 4^ 4CHAPTER 5. RESULTS AND DISCUSSION^ 851.41.20)E^1.0z0< c* 0.8cc =F- 0ZL.L.I0Z 0.600.40.2 5^10^15^20SRT (d)FIGURE 5.12. NET COD SOLUBILIZATION AS A FUNCTION OF SRT10090807060Lu 50oCLu0- 40302010RUN 2C (5 d)^RUN 1C (10 d)^RUN 2A (15 d)^RUN 2B (20 d)FIGURE 5.13. PERCENT SOLUBLE COD IN THE FORM OF VFAs (STAGE 2)0CHAPTER 5. RESULTS AND DISCUSSION^ 86comparison of VSS and TSS values and the influence of reactor configuration arealso valid for Stage 2.TABLE 5.13. SPECIFIC SOLUBILIZATION RATES OF COD AND TOCAS A FUNCTION OF SRTRUN SRT(d)COD RATE(mgCOD/mgVSS*d)TOC RATE(mgTOC/mgVSS*d)CMR UASB CMR UASB2C 5 0.184 0.192 0.066 0.0661C 10 0.187 0.198 0.070 0.0702A 15 0.200 0.193 0.078 0.0722B 20 0.184 0.181 0.072 0.070TABLE 5.14. PERCENT VSS AND TSS REDUCTION AS A FUNCTION OF SRTRUNSRT(d)VSS (%) TSS (%)CMR UASB CMR UASB2C 5 62.8 66.2 64.6 66.91C 10 63.1 70.6 64.2 69.62A 15 67.1 75.2 68.4 75.42B 20 65.6 73.4 66.7 75.3Table 5.15 shows the percent utilization of carbohydrates, proteins and lipidscalculated from the respective mass balances. Considering the suspended solidsbehavior in the reactors as described above, the three organic classes of interest arenot expected to be affected significantly by the variation in SRT. Although, this isCHAPTER 5. RESULTS AND DISCUSSION^ 87basically true for carbohydrates and lipids, the protein degradation pattern appearsto be SRT dependent. Longer SRTs result in consistently higher protein dissimilation.Most of the protein content in primary sludge is cell protein (Section 5.1.1) and,therefore, not readily available for fermentation. In the bioreactors, however,continuous metabolic activity and cell lysis may increase the soluble protein level,especially at longer SRTs. Since the production of the 4 \"minor\" VFAs (iso-butyric,valeric, 3-methylbutyric and 2-methylbutyric) is mostly associated with the anaerobicmetabolism of proteins (Gottschalk, 1986), the increase in protein dissimilation is inagreement with the higher production of these 4 acids at longer SRTs (Table 5.12).TABLE 5.15. PERCENT SUBSTRATE DEGRADATION AS A FUNCTION OF SRTRUNSRT(d)CARBOHYDR (%) PROTEINS (%) LIPIDS (%)CMR UASB CMR UASB CMR UASB2C 5 59.0 70.4 38.7 37.4 83.1 64.51C 10 60.6 73.4 42.9 45.0 80.9 62.02A 15 62.5 78.8 51.2 48.7 84.5 69.82B 20 61.0 76.5 54.1 55.2 81.4 66.7Finally, the reactor configuration affects the utilization patterns of the threeorganic classes in exactly the same way as outlined in Stage 1. The rate ofcarbohydrate degradation is significantly higher in the UASB reactor (Figure 5.14),lipids are solubilized more effectively in the CMR unit and protein dissimilationrates are similar in both systems (Figures 5.15 and 5.16).INFLUENT MASS LOADINGLOSS IN EFFLUENT AND WASTAGESYSTEM I (CMR)EZZ1 SYSTEM II (UASB)0RUN 2C (5 d)^RUN 1C (10 d)^RUN 2A (15 d)^RUN 2B (20 d)654321CHAPTER 5. RESULTS AND DISCUSSION1614 LOSS IN EFFLUENT AND WASTAGESYSTEM I (CMR)EZZ SYSTEM II (UASB)88rn2RUN 2C (5 d)^RUN 1C (10 d)^RUN 2A (15 d)^RUN 2B (20 d)FIGURE 5.14. CARBOHYDRATE DEGRADATION AS A FUNCTION OF SRTFIGURE 5.15. PROTEIN DEGRADATION AS A FUNCTION OF SRTSi^3g 20RUN 2C (5 d)^RUN 1C (10 d)^RUN 2A (15 d)^RUN 2B (20 d)415LOSS IN EFFLUENT AND WASTAGESYSTEM I (CMR)EZZ] SYSTEM II (UASB)CHAPTER 5. RESULTS AND DISCUSSION^ 89FIGURE 5.16. LIPID DEGRADATION AS A FUNCTION OF SRT5.3.4. GAS PRODUCTIONGas generation is the ultimate goal in the two-phase anaerobic digestionprocess. The acid-phase step is generally characterized by a very low gas production,mostly in the form of CO2 , N2 and H2 , which are by-products of many pathwaysfollowed for substrate metabolism (Appendix A). Ideally, the methane content in thereactor should be negligible. In practice, however, varied amounts of methane havebeen detected in acid-phase digesters (Eastman and Ferguson, 1981; Ghosh, 1987).This may be due to either incomplete separation of the two phases which results inthe co-existence of heterotrophic methane producers, or the presence of certain fast-CHAPTER 5. RESULTS AND DISCUSSION^ 90growing autotrophic methanogenic organisms such as Methanobacterium, or both(Novaes, 1986).The relatively low gas production obtained (Table E4, Appendix E) indicatesthat methanogenesis was successfully suppressed throughout this experimental study.Despite the small volumes of gas generated, certain interesting observations can bemade. Gas production appears to be independent of HRT (with the notable exceptionof Run 1D) but increases rather proportionally with increasing SRT in both systems.However, the two- to three-fold increase in Run 1D (HRT: 15 hours, SRT: 10 days)shows that the activity of methane-forming bacteria has been substantiallyencouraged during this run, as compared to any other set of experimental conditions.When the values of the two operational parameters are distinctly different (resultingin SRT/HRT ratio much higher than one), it is possible that the prolongedavailability of food may trigger first the mechanism for methanogenesis. Therefore,longer HRTs may stimulate gas production by allowing better contact between thesoluble substrates (i.e. VFAs) and the already present methanogens, while shorterHRTs severely limit methanogen activity without significantly affecting acidogenesis.Several analyses of gas composition showed that CO 2 is the predominant gasin this phase. On average, the CH4 :CO2 :N2 percentage was 32:62:6 (by volume),which is in agreement with the range reported in the literature for the acid-phase step(Ghosh et al., 1975; Fongsatitkul, 1992). This ratio is very different from the 70:25:5ratio found in most well-operating two-phase sludge digesters (Metcalf and Eddy,1991). Based on a rather small number of samples analyzed (2 to 4 per run), nosignificant changes in gas composition were observed among different runs.CHAPTER 5. RESULTS AND DISCUSSION^ 915.4. REPLICATION AND THE EFFECT OF FEED SOURCE - STAGE 35.4.1. REPLICATION EXPERIMENTS (RUN 3A)To determine the degree of replication possible in acid-phase digesters, tworuns (1C and 3A) were operated under identical conditions, except that the firstexperiment took place in late spring - early summer (May - June) and the second onein winter (January - February).The operating conditions corresponding to Run 1C (HRT: 12 hours, SRT: 10days) were selected for the rest of the study. Although longer SRTs resulted inslightly better VFA production rates (Runs 2A and 2B), an SRT of 10 days isconsidered as a \"reasonable\" value to ensure high VFA production and at the sametime to minimize the length of the experiments.Analysis of the reactor contents during the steady-state period (Table 5.16)shows that the variation in all parameters measured is minimal between the replicateunits. The distribution of the individual volatile fatty acids is about the same for bothruns as well (Table 5.17). Furthermore, the variation in standard deviation of allmeasured values is not statistically significant, according to t-test (Table 5.18). Onthe basis of these data, it is concluded that the steady-state operation of the acid-phase digestion can be replicated and that the seasonal variation of influentcollection (summer - winter) does not seem to play any role in the process.CHAPTER 5. RESULTS AND DISCUSSION^ 92TABLE 5.16. COMPARISON OF REPLICATION RESULTS AT IONA ISLAND WWTPORGANICPARAMETERCMR SYSTEM UASB SYSTEMRUN IC-S RUN 3A-W RUN 1C-S RUN 3A-WVFA SP. PROD. RATE 0.101 0.104 0.103 0.099COD SP. SOLUB. RATE 0.187 0.198 0.198 0.197TOC SP. SOLUB. RATE 0.070 0.070 0.070 0.074COD IN VFA FORM 71.0 69.5 70.9 68.5VSS 63.1 65.5 70.6 68.7TSS 64.2 65.9 69.6 69.9CARBOHYDRATES 60.6 59.8 73.4 71.8PROTEINS 42.9 43.5 45.0 43.5LIPIDS 80.9 79.2 62.0 61.2Note: Specific rates are expressed as mg(Parameter)/mgVSS*d, the rest of the values are (%);S=Summer, W=Winter.TABLE 5.17. PERCENT VFA DISTRIBUTION (STAGE 3)VOLATILEFATTYACIDRUN 1C (I.I.) RUN 3A (I.I.) RUN 3B (L.G.)CMR UASB CMR UASB CMR UASBACETIC 47.5 46.0 47.6 45.4 45.1 46.6PROPIONIC 29.9 30.4 30.7 32.0 35.3 33.0BUTYRIC 9.9 10.3 9.4 9.5 8.9 8.6ISO-BUTYRIC 3.2 2.9 2.8 2.3 4.4 4.6VALERIC 4.4 5.6 4.9 5.4 4.1 3.83-METHYLBUT. 3.3 3.1 3.0 3.7 1.4 2.82-METHYLBUT. 1.8 1.7 1.6 1.7 0.8 0.8Note: I.I.=Iona Island WWTP, L.G.=Lions' Gate WWTP.CHAPTER 5. RESULTS AND DISCUSSION^ 93TABLE 5.18. t-TEST RESULTS FOR RUNS 1C, 3A (IONA ISLAND WWTP)AND 3B (LIONS' GATE WWTP)(Level of significance a=0.05)ORGANICPARAMETERRUNS 1C and 3A t <2.074RUNS 1C &and 3B, It3A (comb.)<1.960CMR UASB CMR UASBVFA SP. PROD. RATE 0.548 0.767 3.108 2.388COD SP. SOLUB. RATE 0.930 0.107 0.741 0.565TOC SP. SOLUB. RATE 0.000 0.966 0.250 1.415% COD IN VFA FORM 0.951 1.417 4.964 5.043% VSS REDUCTION 0.920 1.621 0.832 1.814% TSS REDUCTION 0.441 1.459 0.567 1.411% CH20 DEGRAD. 0.781 1.030 1.879 1.694% PROTEIN DEGRAD. 0.829 1.064 1.288 1.125% LIPID DEGRAD. 1.465 0.546 1.880 0.6965.4.2. THE EFFECT OF FEED SOURCE (RUN 3B)A common attribute of biological treatment processes is that they are ofteninfluenced by the nature of the feed used. Primary sludges from different sourcesmay behave in a different way during the acid-phase digestion step (Chynoweth andMah, 1971).To investigate the possible dependency of the process on influentcharacteristics, the two reactors were operated at 12 hours HRT and 10 days SRT(identical conditions with Runs 1C and 3A), using primary sludge from anothersource which had the composition shown in Table 5.2.CHAPTER 5. RESULTS AND DISCUSSION 94A summary of the important variables from Run 3B is presented in Table 5.19,along with the combined average values from Runs 1C and 3A for comparativepurposes. It is interesting to note that all four \"general\" variables (COD and TOCsolubilization rates, and VSS and TSS reduction percentages) are quite similar, butthe \"specific\" parameters (with the exception of proteins) exhibit some trend ofvariation. For example, the VFA production rates are reduced in Run 3B by about12% and the percent COD in the form of VFAs by about 20% in both reactors, whencompared to Runs 1C and 3A. Moreover, a relatively lower rate of lipid hydrolysisand an accordingly higher rate of carbohydrate breakdown have been observed, whichsuggests that the lipolytic activity of lipases was to some extent adversely affectedbut that of carbohydrate-hydrolyzing enzymes was encouraged when the alternativefeed was used. Although, as illustrated in Table 5.19, only the variation in VFAproduction rates and the related COD in the form of VFAs percent values can beclassified as significantly different from a strictly statistical point of view (t-test;Miller et al., 1990), the variation in both carbohydrate and lipid utilization patternsare consistent and may be important especially when compared to the negligiblepercent changes observed during the replication experiments (Table 5.18).The percent VFA distribution shows no appreciable changes regarding themajor acids (Table 5.17). A closer examination of the minor products, however,reveals that the production of iso-butyric acid has remarkably increased and that of3-methylbutyric and 2-methylbutyric has accordingly decreased in Run 3B. This canbe better illustrated by comparing the relative ratios of the two branched C 5 VFAsto iso-butyric acid (Table 5.20). This ratio not only drops dramatically in the caseTABLE 5.19. COMPARISON OF RESULTS FROM DIFFERENT FEED SOURCESORGANICCMR SYSTEM^ UASB SYSTEMPARAMETER^RUNS^RUN^(%)^SIGN.^RUNS^RUN^(%)^SIGN.1C & 3A^3B DIFF.^DIFF.^1C & 3A^3B DIFF.^DIFF.VFA SP. PROD. RATE^0.103^0.089^-13.6^YES^0.101^0.090^-10.9^YESCOD SP. SOLUB. RATE^0.193^0.195^+0.8^NO^0.198^0.202^+2.3^NOTOC SP. SOLUB. RATE^0.070^0.071^+1.4^NO^0.072^0.069^-4.2^NOCOD IN VFA FORM 70.3 55.2 -21.5 YES 69.7 55.7 -20.1 YESVSS 64.3 64.6 +0.5 NO 69.7 70.4 +1.1 NOTSS 65.1 63.2 -2.8 NO 69.8 70.7 +1.4 NOCARBOHYDRATES 60.2 64.1 +6.5 NO 72.6 78.7 +8.5 NOPROTEINS 43.2 44.3 +2.6 NO 44.3 43.5 -1.8 NOLIPIDS 80.1 72.9 -8.9 NO 61.6 57.4 -6.8 NONote: Specific rates are expressed as mg(Parameter)/mgVSS*d, the rest of the values are (%); Runs 1A and 3B were from Iona IslandWWTP, while Run 3B was from Lions' Gate WWTP.cd)(,)(.4OCHAPTER 5. RESULTS AND DISCUSSION^ 96of the last run, but also the values calculated are the lowest values obtained in theentire study for either reactor. Since all three acids are directly related to proteinmetabolism, the above observation suggests a possible difference in protein (i.e.amino acid) composition between the two wastewater sources. The primary sludgefrom Iona Island WWTP, for example, may contain a smaller amount of valine whichcan produce iso-butyric acid via the Stickland reaction (Eq. 2.8), and/or higheramounts of leucine and iso-leucine which be oxidized, in a similar way, to 3-methylbyturic and 2-methylbyturic acids (Section 2.3.2.b).TABLE 5.20. PERCENT DISTRIBUTION OF C5 BRANCHED VFAs ANDISO-BUTYRIC ACID (STAGE 3)RUNREACTOR I (CMR) REACTOR II (UASB)Bran. C5VFAs (%)ISO-BUT.(%)RATIO Bran. C5VFAs (%)ISO-BUT.(%)RATIO1C (I.I.) 5.1 3.2 1.59 4.8 2.9 1.663A (I.I.) 4.6 2.8 1.64 5.0 2.3 2.173B (L.G.) 2.2 4.4 0.50 3.6 4.6 0.78Note: I.I.=Iona Island WWTP, L.G.=Lions' Gate WWTPTaking into account the spectrum of variations observed using sludge from adifferent source, it can be concluded that the nature of primary sludge may have aneffect upon the hydrolysis of particulate organic matter and furthermore that thebacteria may use different metabolic pathways to utilize certain hydrolysis products.This behavior indicates a higher degree of sensitivity to changes in initial conditionsthan to changes in operational conditions.CHAPTER 5. RESULTS AND DISCUSSION^ 975.5. THE EFFECT OF pH - STAGE 45.5.1. pH AS A SELECTIVE PARAMETERThe pH of a bioreactor determines the possibility of survival and the rate ofreproduction of any microbial species present in this particular environment. In manycases, however, the primary determinants of pH are the organisms themselves (if thepH is not externally adjusted). Microorganisms are able to alter the pH of theirenvironment through various metabolic activities. These changes might beadvantageous or not to the microbes that cause them. Some species can createenvironments in which very few other organisms are able to survive, and if theythemselves are not adversely affected by the conditions they create, they may thuseliminate competition. On the other hand, the alterations in pH may encourage thepredominance of a competitor. For example, bacteria may produce acidic productsthat decrease the pH value in an insufficiently buffered environment and allow fungito predominate (Gaudy and Gaudy, 1980).The ability of microorganisms to alter pH is the basis of important interactionsbetween species. Since pH affects growth rate, changes in pH may cause dramaticshifts in the relative numbers of different species in the population. It has been foundthat many aspects of microbial metabolism are greatly influenced by the variationsin pH over the range within which the organisms can grow (Sakharova andRabotnova, 1976). These aspects include utilization of carbon and energy sources,efficiency of substrate dissimilation, synthesis of protein and different types ofCHAPTER 5. RESULTS AND DISCUSSION 98storage material, and release of metabolic products from the cell. Moreover, pHvariations can affect cell morphology and structure and, therefore, flocculation andadhesion phenomena (Forage et al., 1985). All of the above factors play a crucial rolein determining the ability of a given microbial species to compete with others in aheterogeneous environment.5.5.2. BUFFERING CAPACITYThe buffering capacity of a biological system is manifested by the degree ofits resistance to pH changes. In acid-phase digestion, many inorganic and organicbuffer systems such as carbonate/bicarbonate, phosphate, borate, silicate, citrate, andproteins may be active in the pH range of interest. The resistance to acidification isa function of the total buffering capacity of the system (Powell and Archer, 1989).Throughout the uncontrolled pH experiments (Stages 1 to 3), the pH values in eitherreactor, after an initial drop during acclimation, were exceedingly stable during thesteady-state operation (Appendix B). The coefficient of variation (CoV) was between2 and 5% for all runs (Table 5.21). This is mainly attributed to the presence ofproteins and VFAs, since the buffering capacity of the Vancouver water supply isvery low (total alkalinity is usually between 100 and 150 mg/L as CaCO 3 ).Proteins and their hydrolysis products (amino acids) act as both hydrogendonors and hydrogen acceptors since they possess the ionizable amino group (-NH 2)and carboxyl group (-COOH). Although the peptide bonds of proteins tie up the a-amino acid and carboxyl groups, there are both amino and carboxyl groups as wellCHAPTER 5. RESULTS AND DISCUSSION^ 99as other ionizable groups (eg. imidazolyl, sulphide etc.) in the side chains of manyacids (Gaudy and Gaudy, 1980). Thus, proteins in solution can buffer against changesin pH. Individual amino acids exert their maximum buffering potential at differentpH values depending on the number of amino and carboxyl groups they possess. Forexample, two of the most prevalent amino acids in domestic wastewaters, asparticacid (pKAi = 3.86) and glutamic acid (pK A , = 4.07) are most effective in acidicenvironments (CRC Handbook of Chemistry and Physics, 1981).TABLE 5.21. pH VALUES IN BIOREACTORS (STAGES 1 TO 3)RUNREACTOR I (CMR) REACTOR II (UASB)MEAN STD CoV (%) MEAN STD CoV (%)lA 5.23 0.17 3.3 5.25 0.15 2.91B 5.27 0.11 2.1 5.33 0.13 2.41C 5.01 0.25 5.0 4.96 0.23 4.61D 5.06 0.14 2.8 5.10 0.13 2.52A 5.17 0.14 2.7 5.03 0.13 2.62B 5.23 0.10 1.9 5.09 0.11 2.22C 5.63 0.18 3.2 5.52 0.11 2.03A 5.15 0.17 3.3 4.98 0.13 2.63B 5.03 0.16 3.2 5.05 0.14 2.8Volatile fatty acids can also act as buffers in a pH range close to their pKAvalues. The two major products of acidogenic digestion, acetic acid (pK A = 4.76) andpropionic acid (pKA = 4.87), attain their highest buffering capacity at pH of aboutCHAPTER 5. RESULTS AND DISCUSSION^ 1005 (Sawyer and McCarty, 1979).The utilization of proteins and amino acids during the digestion process iscounterbalanced by the generation of VFAs, and as a result, at steady-stateconditions, the pH of the system remains fairly constant. The \"equilibrium\" pH rangeattained, however, may be a function of the relative concentrations of the reactantsand the products involved. VFA concentrations below 400 mg/L resulted in anincrease in pH in either reactor, but no appreciable variation in pH (less than 0.3units) occurred at concentrations ranging from 400 to 750 mg/L (Figures 5.17 and5.18).To further investigate the role of pH in the process, two additional experimentswere conducted at controlled conditions. Dilute solutions (0.02N) of hydrochloricacid and sodium hydroxide were used to maintain the pH at selected values. Bothchemicals were specifically chosen because of their low-level interference with themetabolic pathways involved. Sodium is generally tolerated by most microorganisms,particularly in the presence of potassium. Furthermore, chloride ions are notinhibitory to bacteria at the concentrations attained (Forage et al., 1985).5.5.3. VFA PRODUCTION AND SPECIATIONThe total net VFA production (as acetic acid) at steady-state conditions isdepicted in Figure 5.19. VFA concentration does not appear to be affected, in eitherreactor, by a drop in pH from 5.1 to 4.5 (Runs 1C and 4A), but in Run 4B anincrease in pH to about 6.1 results in significantly lower (25 to 30%) total acidi.t 600rnEZ 50005-2 400L^300.%0z00 200>1005.65.4 5.5 5.74.9^5.0^5.1^5.2^5.3REACTOR pHFIGURE 5.17. REACTOR pH AND VFA CONCENTRATION (CMR SYSTEM)6005004003002005.4 5.65.54.9^5.0^5.1^5.2^5.3REACTOR pHFIGURE 5.18. REACTOR pH AND VFA CONCENTRATION (UASB SYSTEM)800700100CHAPTER 5. RESULTS AND DISCUSSION80070010100CHAPTER 5. RESULTS AND DISCUSSION^ 102i 600- ^OOOOO ^00 I,-O 0zcr6zz coa6co1VMSVRXR8V2S8Rt .-818R.8.9328SRRSR442SS612FAR148818ui ui Tf ui ui ui ui Tf ui ui mf ui ui ui ui ui ui ui ui ui ui ui ui Tf ui ui Ti Tf Tr Ti up Ti Tf Tr Tf ui ui^up ui ui^mf ui53 V g tg F: $2 $2^tg 5? 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REACTOR I (CMR) CHARACTERISTICS FROM RUN IADATE DAY P A R AMET ERS (mg/L)TS VS TSS VSS PROTEINS^LIPIDS^CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO412/21/89 9 19595 15295 14490 10800 2445 2250 7855 543 1109 380 440 49 80 9.412/24/89 12 22850 17950 16915 13975 2525 1900 10750 594 1379 476 454 50 92 10.812/29/89 17 29855 22655 20040 16470 2120 1475 12785 649 1334 524 396 57 111 10.301/02/90 21 20140 14980 12000 10145 1940 1680 7980 466 1374 438 353 42 82 9.201/05/90 24 27445 20270 18615 15360 1800 1715 12035 518 1305 501 343 55 110 8.401/09/90 28 23600 17705 17525 14445 1725 1600 10835 552 1149 443 325 49 99 7.701/17/90 36 24905 19090 16855 12910 1630 1510 10345 332 1247 460 313 52 91 10.001/20/90 39 24435 18820 15250 11175 1950 1665 8160 527 1173 357 341 29 84 8.201/23/90 42 26730 19755 16640 12680 1755 1910 10000 616 1127 385 323 43 81 9.601/27/90 46 26400 19405 17180 13205 2125 1675 10235 648 1397 464 399 59 97 10.301/30/90 49 25270 19995 18030 14910 1750 2020 11510 576 1575 552 347 67 106 11.002/02/90 52 31635 24770 21465 17670 1605 2300 13975 424 1481 546 300 43 114 8.602/06/90 56 30025 22880 19605 16040 2020 1615 13325 602 1259 400 376 52 110 8.802/09/90 59 23085 17075 15485 13040 1635 2090 10180 492 1373 401 306 44 85 11.202/13/90 63 19140 15700 14965 12450 1730 1665 9015 511 1454 536 305 28 82 11.702/16/90 66 29840 22870 20520 15740 1625 1890 12580 569 1293 412 299 39 100 10.302/20/90 70 27575 20435 18120 14915 1690 2180 11535 509 1542 515 315 45 91 12.002/24/90 74 26120 20405 17585 14675 2150 1925 11850 507 1213 371 379 35 93 9.802/27/90 77 29295 22735 20840 17255 1835 1715 13649 582 1193 360 325 32 108 8.803/02/90 80 28035 21900 18520 15405 1580 1540 11165 551 1292 355 282 29 98 10.603/06/90 84 26035 20575 19050 16160 1720 1675 13285 577 1112 382 322 47 107 10.203/08/90 86 24670 18515 16940 14125 1925 1855 10870 536 1178 400 347 39 91 12.3MEAN 25758 19717 17574 14252 1876 1811 11087 540 1298 439 345 45 96 10.0STD 3365 2568 2225 1986 257 234 1761 71 134 65 45 10 11 1.2APPENDICESgODccW OW:Ef2 EHMRIVAEIAMFREAMF\u00C2\u00A7M\u00C2\u00A7M\u00C2\u00A7gilWaiRfMriagV11.12 WEiiPliM .- ^ CV INTI Y- CA ..- ... RI 11 1 C71 ^11 !^g CA & g -- 24\"91Pill\"\"H18% 11-3giNg^RcliNg^NNNM^N/7(7; iii \">-ccC3c6V.t&X888.7WTV8828,58Z8z60I-2WIL00^gggEMMgpgglgggiggRW\"\"\"4\u00C2\u00A7C;AiM\u00C2\u00A71.- 1- 1- CD C) CD CD CD CD CD^CP^CD CD CP OD CP CP CD ER CD167TABLE C4. REACTOR II (UASB) CHARACTERISTICS FROM RUN 1ADATE DAY P AR AMETERS (mg/I)TS VS TSS VSS PROTEINS UPI DS^CARBOHYDR-TOT. VFA COD TOC TKN NH3-N TP PO412/21/89 9 40530 32530 30485 22260 7170 8420 13470 487 1445 494 1208 61 281 6.812/24/89 12 53840 42855 40190 30269 9175 8950 18420 601 1546 544 1507 39 371 8.312/29/89 17 52775 42945 41715 31005 8920 9545 17570 652 1376 474 1476 48 380 7.401/02/90 21 55270 44660 42065 32100 7085 8680 21030 537 1561 591 1187 53 394 9.201/05/90 24 53885 44445 45630 34395 7825 11340 20945 526 1729 614 1308 56 375 8.601/09/90 28 51840 41770 39370 30415 7240 8155 20245 659 1269 445 1211 53 352 6.301/17/90 36 48690 38464 35540 27955 8060 9380 14960 492 1438 470 1323 34 311 6.701/20/90 39 47305 37325 32755 26065 6600 9415 13370 595 1664 584 1110 54 288 7.801/23/90 42 50455 39880 36695 29420 7170 8020 19520 733 1425 439 1209 62 324 5.301/27/90 46 50355 41070 39090 28650 6035 9235 18475 692 1552 529 1023 57 302 8.601/30/90 49 54530 44245 42785 31170 8000 10385 16035 560 1672 599 1337 57 384 8.802/02/90 52 45975 35860 33645 26410 5715 9990 18450 580 1732 593 964 50 293 8.202/06/90 56 51950 39410 34285 26110 5530 8425 15440 677 1321 482 949 64 287 7.102/09/90 59 49525 39455 37490 29320 8465 8260 18695 564 1399 479 1389 34 322 7.402/13/90 63 42220 33100 29995 23375 8220 7490 13260 532 1456 496 1360 45 276 6.602/16/90 66 44010 34510 31595 23105 9075 7765 13805 701 1657 524 1512 60 294 6.902/20/90 70 40875 32075 30810 22975 6910 9145 13060 662 1644 579 1152 46 302 7.702/24/90 74 51860 40865 39540 30070 6380 10215 17905 566 1191 419 1056 35 386 8.602/27/90 77 53515 40255 36575 28685 5975 8860 19280 584 1300 464 991 35 371 9.003/02/90 80 49900 41010 37075 27480 7565 7965 17525 626 1342 468 1262 51 350 8.803/06/90 84 46590 36340 30235 23620 6005 9650 15335 644 1520 512 1022 61 311 10.103/08/90 86 52295 40765 39080 28760 7730 9325 16650 589 1559 541 1279 42 358 9.5MEAN 49463 39265 36666 27892 7311 9028 16975 603 1491 515 1220 50 332 7.9STD 4341 3743 4413 3231 1073 928 2525 67 151 57 170 10 39 1.2APPENDICES^169k_N'-q17\u00E2\u0080\u00A2-1fNNqCgigNVN'-ltqqq0WIMr:oirscoduicor:mcoorrscompsoioded.-....._v-010000(01 oNctclqq01411. -0( 0.-0?Nltict6Wo.-mmoiddmmo.-o06oWmoWmmv-.-.....^.-....-^.-.-.-.-tzt SVZSX4RSzESS28148ZM;412V6\u00C2\u00B0)gr:ga- ScsSSS88SwiSSRggE42c74::-E.:LI_.8 S F-lilaMif ggniif \u00C2\u00A7grifMERMAValgEMEME!114WiRAng\u00C2\u00A7MAIMI1 Wsmi.1cr<8a3 F-E22mvals1v4wRosPEGgiaguPssvV-VNA2ggXgniRIWMORZMU\"rg5n EREEEE M E WMFEE?F.>0F-WAMEEFAHNEMMAtle\u00C2\u00A7\u00C2\u00A7,M11\u00C2\u00A71EnIMIRIMUMinpVIIIPM1WWW1_\"\"^ ____^__\"^ _^\"^N\"11M11111111M111110>Q0\u00C2\u00B0)V.^:KARARV*TVSS2SPRIEz6\u00C2\u00B01-2cowUlg:kggggggg:M.FMgEgV4P\"\"\"giraliigl\u00C2\u00A7.-.-.-000000000^000^000igiTABLE C6. INFLUENT CHARACTERISTICS FROM RUN 1BDATE DAY P AR A METERS(mg/L)TS VS TSS VSS PROTEINS^LIPIDS^CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP03/20/90 7 3810 2980 3285 2710 619 568 1715 82 244 71 115 15.8 16.8 7.903/23/90 10 3985 2850 3570 2630 635 498 1520 112 347 104 122 20.4 17.1 8.603/27/90 14 3815 2860 3695 2650 639 519 1670 120 534 171 122 19.3 16.8 9.003/30/90 17 4625 3300 4465 2970 627 564 1975 83 301 92 114 132 17.6 7.704/03/90 21 3600 2740 3300 2500 506 425 1660 119 321 103 96 14.8 14.0 8.504/06/90 24 4325 3145 4065 2905 589 441 2015 126 389 125 110 15.4 15.1 9.904/09/90 27 4110 3100 3830 2890 776 528 1715 110 375 110 142 17.6 26.7 12.104/12/90 30 3485 2545 3185 2415 647 442 1400 136 448 139 124 20.1 22.7 11.704/16/90 34 3820 2825 3535 2550 694 497 1510 91 401 128 130 18.5 19.5 10.304/19/90 37 4035 3010 3710 2755 610 500 1855 99 374 121 112 14.3 17.4 9204/23/90 41 3780 2720 3440 2435 598 424 1605 118 353 109 119 22.9 17.6 8.204/26/90 44 3555 2625 3265 2404 569 419 1620 132 426 130 106 15.0 19.9 11.5MEAN 3912 2892 3612 2651 626 485 1688 111 376 117 117 17.3 18.4 9.6STD 315 214 358 190 63 52 177 17 71 24 11 2.9 3.3 1.5TABLE C7. REACTOR I (CMR) CHARACTERISTICS FROM RUN 1BDATE DAY PARAMETERS (mg/1.)TS VS TSS VSS PROTEINS UPIDS CARBOHYDR TOT. VFA COD TOC 1104 NH3-N TP PO403/20/90 7 24675 18070 17425 13640 2000 2420 9820 359 942 317 349 29 126 6203/23/90 10 27605 20785 19995 16445 1905 3040 11175 605 991 364 357 52 147 10.703/27/90 14 28955 21995 22620 18825 2200 2635 13850 502 1207 425 396 44 155 9.803/30/90 17 25285 19300 20150 16270 1795 2225 11735 312 1203 392 317 29 130 7204/03/90 21 32970 24695 23260 19355 2080 2370 13960 371 1090 357 366 33 172 8.104/06/90 24 31385 23220 23285 18365 2000 2230 14100 315 1128 386 357 37 174 8.004/09/90 27 24910 18490 18990 15810 1615 2685 11445 341 977 319 295 37 139 9.304/12/90 30 25345 19445 20425 16140 1715 2740 11210 452 1045 338 322 47 148 12.004/16/90 34 30095 23420 24420 20375 2160 2900 14695 378 1161 380 377 32 166 7.804/19/90 37 32685 24730 24975 19730 2050 2464 14375 435 1098 331 360 32 176 8.704/23/90 41 26220 20120 19820 16650 1725 2215 12340 418 990 308 320 44 127 8.004/26/90 44 32900 24855 23660 19215 1860 2640 14435 397 1174 344 328 31 145 6.7MEAN -1 28586 21594 21585 17568 1925 2547 12762 407 1084 355 345 37 150 8.5STD 3184 2432 2304 1937 179 260 1584 80 89 34 28 7 18 1.6TABLE C8. EFFLUENT I CHARACTERISTICS FROM RUN 1BDATE DAY PARAMETERS (mg/L)TS VS TSS VSS PROTEINS UPIDS CARBOHYDR TOT. VFA COD TOC TIN NH3-N TP PO403/20/90 7 2622 1872 1070 764 332 100 473 355 1067 388 88 35 112 5.803/23/90 10 2804 1964 1184 920 419 112 558 578 950 303 107 40 14.4 9.403/27/90 14 3680 2738 1878 1420 624 148 836 504 1161 403 139 39 19.3 9.103/30/90 17 3488 2602 1796 1308 447 136 768 316 1228 416 97 25 16.6 7.704/03/90 21 3654 2740 1816 1396 398 153 838 350 1112 364 99 35 17.0 7.004/06/90 24 3186 2262 1318 960 371 102 628 352 1072 361 98 39 13.9 6.204/09/90 27 2548 1820 1144 850 391 93 549 378 1032 356 92 29 14.4 8204/12/90 30 3006 2204 1362 1032 559 105 612 463 1076 369 143 53 16.3 10.404/16/90 34 3688 2618 1700 1222 410 130 730 362 1195 428 96 30 15.8 7.504/19/90 37 2710 1986 1334 958 359 101 618 430 1086 311 92 34 14.2 6.804/23/90 41 3064 2206 1302 1000 415 116 627 447 1103 357 109 43 17.8 8.704/26/90 44 3362 2488 1648 1184 368 138 754 408 1198 372 88 29 16.4 6.1MEAN 3151 2292 1463 1085 424 120 666 412 1107 369 104 36 15.6 7.7STD 405 324 275 207 81 20 112 73 75 36 18 7 2.0 1.4TABLE C9. REACTOR II (UASB) CHARACTERISTICS FROM RUN 18DATE DAY P AR A METERS (mg/L)TS VS TSS VSS PROTEINS^UPIDS^CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO403/20/90 7 47375 35915 36620 26760 5820 8305 14985 426 1046 361 969 38 306 8.903/23/90 10 50825 39040 38650 29905 8025 8760 17865 569 1174 428 1328 44 338 7.403/27/90 14 42200 33220 32795 25715 6390 8360 13765 509 1408 472 1063 41 306 7.003/30/90 17 51980 40865 42180 30655 6375 9705 16975 405 1221 356 1369 29 353 6.204/03/90 21 41755 31410 30485 23170 5560 7780 13080 451 1328 468 922 33 274 7.904/06/90 24 45665 36665 38835 29005 7010 8355 16745 506 1525 467 1152 31 310 5.304/09/90 27 48380 36700 38325 28280 7120 9510 15095 319 1260 429 1169 29 293 5.104/12/90 30 41890 32475 35290 25645 6095 8795 13785 545 1454 493 1026 51 275 7.604/16/90 34 43445 33335 33965 24375 6760 7915 13285 488 1412 429 1128 47 298 8.004/19/90 37 40985 31350 33675 26140 7525 7120 15890 406 1188 371 1247 43 325 9.404/23/90 41 39855 30145 31380 23410 6350 8030 13280 501 1204 388 1046 30 335 6.004/26/90 44 46540 35355 35685 27000 8170 8365 16315 472 1390 449 1341 34 356 6.8MEAN 45075 34706 35657 26672 6933 8417 15089 466 1301 428 1147 37 314 7.1STD 3824 3141 3289 2325 896 685 1586 66 135 47 143 7 26 1.3TABLE C10. EFFLUENT II CHARACTERISTICS FROM RUN 1BDATE DAY P A R AMET ERS (mg/L)TS VS TSS VSS PROTEINS^UPIDS^CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO403/20/90 7 2038 1426 742 628 300 64 460 349 1045 344 83 35 11.7 7.50323/90 10 2492 1770 1062 758 247 75 502 460 1087 360 80 40 12.2 6.903/27/90 14 3006 2172 1264 942 253 77 629 419 1240 390 80 40 14.1 7.103/30/90 17 3636 2706 1696 1352 351 101 888 313 975 331 86 29 15.4 5.804/03/90 21 3240 2380 1434 1110 428 93 739 338 1215 410 97 28 13.9 5.404/06/90 24 2984 2140 1134 804 350 78 563 350 1287 382 91 35 12.2 6.004/09/90 27 3026 2200 1342 998 356 89 675 282 1085 338 87 30 12.6 5.304/12/90 30 3262 2346 1314 1006 267 93 709 448 1301 417 89 46 13.3 6.704/16/90 34 2646 1892 1100 816 358 63 604 334 1148 348 105 48 14.0 8.204/19/90 37 2726 2004 1202 944 416 70 696 352 1010 358 112 45 13.8 8.304/23/90 41 3428 2452 1388 1040 374 82 765 411 976 300 92 32 11.7 4.904/26/90 44 2804 2070 1206 948 310 68 652 388 1193 359 87 38 11.0 6.2MEAN 2941 2130 1240 946 334 79 657 370 1130 361 91 37 13.0 6.5STD 418 323 223 178 57 12 112 52 112 32 9 6 1.2 1.1APPENDICESoicoqqmw^q,\u00E2\u0080\u00A2,i-:Wo1-.1.-\u00C2\u00A7RMEgiEMEF\u00C2\u00A7E6E8 WPIWEIr8 c4r4r, rangNE,M11-.tg^gN^gTWIIIIIIINN^gIPEIE6 g1\u00C2\u00A711\u00C2\u00A7Flip ^gm;r-Q ^mmX^g4^Pizi;Rgoevt--Ruigx8;V4D13iEw4c1IMMWM!Biking:46;iqc,.1Lugwc62<6Q<8an RE4l'tct.-irlvq01040(1.-111(101\"-N0.-Mt-n:0D)ocficooiei.-.-.-If) 0' m .- 4 cq CO N P. CO al P\u00E2\u0096\u00A0 el m.1 , m 6 oi 0 0.- ei .- ci N .- CO Oi1132.13.744X2R$S8Star\u00C2\u00B0EIVRSPPgaSI8288w N VIN I-w-ET 8>MHZ-051MM*MflEgE\u00C2\u00A7MEW.REEMMVIIIEGitrs22mRqSVSSSRteMe$SRRiciRm,,,,,NR0R,Nm ,c,17-Assysnsv.:Pmmuim>.FN\u00E2\u0080\u0094 Eil/AEEEFIEAMPMIAMEFAZIRAEgr.ilnIPMMEriNi^.\u00E2\u0096\u00A0...^C\u00C2\u00B0^NNN22 4 8 y 8 8 A ; A :t g V^sgmuixissIgNs4NRAgNs-4 sa iica039vtl-Rxmi-vvv4mca26w1-giUMMMgg-27:1-\u00E2\u0080\u009C7;W.:1-.:2E888S888g8S8g8TABLE C16. INFLUENT CHARACTERISTICS FROM RUN 1DDATE DAY P AR A METERS(mg/L)TS VS TSS VSS PROTEINS^UPIDS^CARBOHYDT TOT. VFA COD TOC TKN NH3-N TP PO406/28/90 8 4285 3325 3795 3165 774 514 1940 79 467 150 142 18.2 23.1 12.507/02/90 12 3905 3140 3560 2870 647 476 2005 105 615 197 133 29.6 22.3 15.607/05/90 15 4195 3265 3985 3005 686 429 1955 89 352 97 129 19.3 18.9 9.307/09/90 19 4170 3090 3940 2840 667 442 1825 117 367 115 129 22.3 17.5 8.307/12/90 22 3995 2720 3590 2590 632 459 1680 102 396 130 128 26.4 18.8 8.007/16/90 26 3440 2670 3045 2515 524 431 1580 119 307 94 100 15.9 17.2 9.407/19/90 29 4130 3430 3920 3200 782 454 2065 123 385 129 146 21.1 20.7 11.807/23/90 33 4170 3335 3945 3025 641 480 2090 133 474 154 127 24.4 21.7 12.907/26/90 36 3825 2865 3540 2635 683 467 1650 82 453 128 135 25.5 18.0 10.007/30/90 40 4185 3130 3875 2880 688 502 1740 100 402 117 127 17.3 23.0 13.808/02/90 43 4090 3055 3805 2815 690 468 1855 121 386 118 127 16.2 18.4 11.108/06/90 47 4020 2960 3740 2755 721 482 1695 129 458 146 141 25.7 20.9 13.7MEAN 4034 3082 3728 2858 678 467 1840 108 422 131 130 21.8 20.0 11.4STD 219 232 254 207 65 25 164 17 76 27 11 4.3 2.1 2.3TABLE C17. REACTOR I (CMR) CHARACTERISTICS FROM RUN 1DDATE DAY P AR AMETERS(mg/1.)TS VS TSS VSS PROTEINS^UPIDS CARBOHYDC TOT. VFA COD TOC TKN NH3-N TP PO406/28/90 8 15905 12120 11060 8890 1790 1070 6280 457 1127 400 349 62 101 8.707/02/90 12 13575 10075 8705 6815 1605 825 4900 548 1311 442 326 69 83 11.607/05/90 15 12880 9880 9560 7565 1420 935 5425 611 1161 389 300 72 90 12.507/09/90 19 13700 9970 8535 6750 1470 690 4900 504 1455 507 302 67 80 9.307/12/90 22 10990 8385 8755 6390 1285 885 4710 502 1290 405 263 58 79 8.007/16/90 26 15195 11125 10210 8515 1735 670 6240 584 1132 359 340 63 102 8.807/19/90 29 12170 9310 9225 7200 1610 770 5030 617 1200 441 336 78 89 10.907/23/90 33 14195 10785 11485 9355 1570 1005 6815 580 1466 544 319 68 114 12.607/26/90 36 17880 13720 12815 9885 1820 840 7245 615 1286 467 344 53 120 9.407/30/90 40 15015 11090 11380 8670 1595 695 6280 509 1093 416 303 47 87 7.508/02/90 43 11670 8830 8745 6955 1380 640 5240 523 1070 410 270 49 77 11.008/06/90 47 14405 10545 9730 7340 1505 715 5255 555 1174 460 296 55 84 11.4MEAN 13965 10486 10017 7861 1565 812 5693 550 1230 437 312 62 92 10.1STD 1841 1395 1323 1102 157 134 806 50 127 50 27 9 13 1.7TABLE C18. EFFLUENT I CHARACTERISTICS FROM RUN 1DDATE DAY P AR AMETERS (mg/1)TS VS TSS VSS PROTEINS^UPIDS CARBOHYDC TOT. VFA COD TOC TKN NH3-N TP PO406128/90 8 2554 1974 578 456 250 30 331 464 1097 383 107 67 14.1 8.907/02/90 12 2408 1868 584 448 229 36 320 574 1350 421 110 74 16.6 12.407/05/90 15 3176 2376 808 654 344 43 431 618 1199 406 125 70 19.0 12.307/09/90 19 2686 1968 536 392 301 41 278 524 1369 481 109 61 12.8 8.707/12/90 22 2298 1814 490 388 279 28 283 490 1270 399 98 53 13.1 9.107/16/90 26 3274 2390 778 634 339 36 422 591 1136 353 125 71 15.5 8.207/19/90 29 2874 2192 734 564 315 25 399 627 1223 430 128 78 17.2 11.007/23/90 33 2778 2112 626 476 258 24 346 592 1443 493 114 73 16.6 12.107/26/90 38 2550 1980 522 380 236 37 269 621 1291 447 96 58 14.0 9.707/30/90 40 2582 1944 518 393 224 40 274 512 1149 397 90 54 11.8 8.808/02/90 43 2818 2116 688 532 301 29 365 567 1211 389 108 60 16.7 11.008/06/90 47 2490 2000 596 454 252 42 312 542 1169 390 105 65 15.5 11.8MEAN 2707 2061 622 481 277 34 336 560 1242 416 110 65 15.2 10.3STD 282 175 102 91 40 6 55 52 100 39 12 8 2.0 1.5TABLE C19. REACTOR II (UASB) CHARACTERISTICS FROM RUN 1DDATE DAY PARAMETERS(mg/L)TS VS TSS VSS PROTEINS UPIDS CARBOHYDI TOT. VFA COD TOC TKN NH3-N TP PO406/28/90 8 35470 27570 28855 22270 7945 5870 11705 546 1410 453 1352 81 328 11.207/02/90 12 38305 31010 31425 24035 8380 5550 12860 527 1498 527 141 1 70 340 9.107/05/90 15 32145 24415 23335 16570 6225 4265 9655 700 1335 485 1056 60 271 8.007/09/90 19 31285 23605 23340 17435 6085 4160 10160 615 1400 481 1039 66 280 9.407/12/90 22 28645 21720 20815 15470 5500 3315 8650 504 1586 531 946 66 263 7.307/16/90 26 28080 20840 22650 16265 5660 4220 8290 592 1484 509 964 58 288 7.907/19/90 29 30885 23555 22710 16020 7080 3765 8655 693 1309 443 1201 68 295 9.807/23/90 33 28700 21660 22060 16830 7225 3980 9300 703 1600 572 1223 67 302 7.607/26/90 36 36710 28885 28900 20925 8315 4925 11110 641 1317 457 1383 52 317 10.707/30/90 40 31845 24410 25985 18740 6590 3970 10730 538 1418 512 1104 50 311 8.408/02/90 43 30930 25140 23950 17160 7740 4745 8625 619 1295 466 1294 56 293 10.308/06/90 47 28170 21950 23195 16465 6885 4330 8935 645 1222 399 1170 68 277 7.9MEAN 31764 24563 24768 18182 6969 4425 9890 610 1406 486 1178 63 297 9.0STD 3272 3011 3142 2638 948 703 1382 67 113 45 153 8 22 1.3TABLE C20. EFFLUENT II CHARACTERISTICS FROM RUN 1DDATE DAY P AR AMETERS (mg/L)TS VS TSS VSS PROTEINS^UPIDS CARBOHYD( TOT. VFA COD TOC TKN NH3-N TP PO406/28/90 8 2688 1962 320 246 150 14.5 133 552 1260 432 103 79 14.4 11.807/02/90 12 2328 1746 258 190 128 14.0 103 564 1563 510 91 70 11.7 10.007/05/90 15 2362 1784 280 206 126 13.6 115 678 1310 451 83 63 11.1 8.807/09/90 19 2770 2050 364 282 168 14.5 152 645 1252 452 96 70 11.6 9.007/12/90 22 2286 1692 252 186 118 13.7 104 480 1492 482 81 62 10.4 8.107/16/90 26 2602 1886 276 202 127 12.8 111 544 1574 522 79 58 9.8 7.407/19/90 29 2130 1598 228 174 100 11.4 97 714 1182 387 80 64 10.7 8.607/23/90 33 2890 2134 348 258 172 15.9 138 697 1510 576 91 64 12.9 9207/26/90 36 2710 2006 304 224 154 14.1 121 651 1335 434 79 54 14.3 10.507/30/90 40 2452 1802 282 202 124 15.3 108 526 1526 544 66 46 11.8 8.108/02/90 43 2128 1638 256 190 112 16.8 97 597 1244 445 77 59 13.5 11.308/06/90 47 2364 1744 294 232 150 13.7 128 653 1217 430 92 68 12.3 9.7MEAN 2476 1837 289 216 136 14.2 117 608 1372 472 85 63 12.0 9.4STD 241 163 38 31 22 1.3 17 72 142 53 10 8 1.4 1.3TABLE C21. INFLUENT CHARACTERISTICS FROM RUN 2ADATE DAY P AR AMETERS trg/L)TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOO TKN NH3-N IP PO409/06/90 8 3925 3045 3610 2840 645 540 1615 106 434 128 119 15.4 15.7 6.809/10/90 12 3975 2980 3590 2725 592 564 1630 132 489 140 113 18.3 17.9 8.309/13/90 15 3800 2940 3365 2745 546 542 1680 89 425 118 109 21.8 14.8 10.409/17/90 19 3960 3035 3640 2840 596 574 1710 103 537 162 123 27.2 14.7 10.009/20/90 22 4380 3380 4055 3180 597 569 2020 116 560 164 124 28.9 18.4 11.809/24/90 26 4605 3555 4330 3405 679 580 2155 127 587 191 135 26.0 17.0 10.009/27/90 29 4320 3400 3920 3065 613 447 2135 134 590 201 123 25.3 15.1 9.510/01/90 33 4295 3130 3945 2920 623 553 1780 113 402 119 125 25.1 19.3 10.510/04/90 36 3605 2635 3440 2450 611 509 1340 136 373 104 127 28.8 16.9 8.310/09/90 41 4675 3680 4420 3340 754 579 2065 128 426 121 146 25.1 23.8 14.410/11/90 43 4105 3040 3870 2845 716 464 1645 150 530 136 128 13.4 19.3 11.510/15/90 47 3955 2965 3745 2755 630 555 1690 93 447 113 127 26.2 17.0 10.710/18/90 50 4045 3035 3800 2790 630 572 1525 120 331 94 129 27.7 17.0 9.310/22/90 54 4130 3010 3835 2805 663 591 1530 116 319 87 127 20.6 20.2 12.8MEAN 4127 3131 3826 2908 635 546 1751 119 461 134 125 23.6 17.7 10.3STD 289 267 292 248 52 42 240 17 87 33 8 4.8 2.4 1.9TABLE C22. REACTOR I (CMR) CHARACTERISTICS FROM RUN 2ADATE DAY P AR AMETERS(mg/L)TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO409/06/90 8 15275 11610 9505 7715 2600 1160 5755 550 1180 387 477 61 86 7.409/10/90 12 13565 10255 8365 6900 2335 1285 5035 659 1455 502 428 55 77 7.909/13/90 15 16440 12440 11190 8335 2670 1090 6170 695 1308 485 479 52 94 8.809/17/90 19 20685 15420 13535 10780 2920 1835 7380 687 1583 590 545 78 121 9.709/20/90 22 17915 13615 12050 9365 2665 1640 6490 703 1531 527 493 67 107 8.009/24/90 26 17285 12775 11080 8550 3000 1375 6335 720 1483 532 549 69 93 10.309/27/90 29 17440 13550 12405 10080 2795 1590 7130 684 1389 457 511 64 129 7.610/01/90 33 22130 16440 14345 11350 3160 1695 8205 743 1274 451 571 65 137 10.910/04/90 36 16480 12505 11670 9405 2610 1440 6700 654 1502 546 484 66 101 9.310/09/90 41 14425 10715 9980 7995 2395 1290 5875 676 1352 440 449 65 78 8.110/11/90 43 20815 16080 13475 10410 2935 1790 7215 726 1450 550 514 44 116 7.710/15/90 47 18935 13865 11760 9255 3120 1595 6605 802 1286 496 563 64 100 11.210/18/90 50 16980 11985 10030 8265 2560 1410 5805 677 1191 441 479 69 86 9.310/22/90 54 21205 15640 12955 10005 2875 1710 7015 711 1302 509 519 59 122 8.4MEAN 17827 13350 11596 9172 2760 1493 6551 692 1378 494 504 63 103 8.9STD 2527 1893 1645 1226 245 226 783 54 122 52 41 8 18 12TABLE C23. EFFLUENT I CHARACTERISTICS FROM RUN 2ADATE DAY P AR A METERS(mg/L)TS VS TSS VSS PROTEINS^UPIDS^CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO409/06/90 8 2698 2052 648 494 182 28 368 518 1229 395 100 71 12.1 6.909/10/90 12 3144 2376 972 788 231 44 523 609 1469 518 94 57 15.4 8.309/13/90 15 3552 2728 990 812 162 43 537 656 1404 454 87 61 16.1 7.809/17/90 19 3904 3014 1056 840 260 47 544 689 1542 549 114 72 17.8 9.509/20/90 22 3502 2566 874 700 219 41 475 707 1488 500 102 67 15.4 8.209/24/90 26 3352 2470 878 668 243 36 461 760 1592 543 112 74 16.7 10.609/27/90 29 3742 2732 846 668 295 32 450 724 1287 440 110 62 13.2 7.110/01/90 33 3036 2318 740 592 318 34 404 707 1277 445 115 64 16.0 10.310/04/90 36 2856 2186 680 524 243 35 378 631 1469 540 110 71 13.6 8.010/09/90 41 3890 2980 1008 776 211 42 510 715 1460 480 103 69 15.9 8.610/11/90 43 3784 2774 987 780 207 47 496 694 1337 483 82 49 14.7 7.210/15/90 47 3288 2400 788 606 248 41 391 781 1318 501 115 75 16.3 10.710/18/90 50 3098 2312 708 544 178 29 390 675 1173 454 103 75 13.1 8.410/22/90 54 3534 2608 962 780 215 43 512 702 1403 514 103 69 14.2 6.6MEAN 3384 2537 867 684 229 39 460 683 1389 487 104 67 15.0 8.4STD 368 276 130 112 42 6 61 63 118 44 10 7 1.6 1.3TABLE C24. REACTOR II (UASB) CHARACTERISTICS FROM RUN 2ADATE DAY PARAMETERS (mg/1.)TS VS TSS VSS PROTEINS UPIDS CARBOHYDR TOT. VFA COD TOC T)04 NH3-N TP PO409/06/90 8 41140 30545 27400 19575 7850 7190 9335 542 1349 476 1330 74 324 8.209/10/90 12 43540 32640 29835 22705 9145 9015 9725 619 1599 587 1534 71 366 10.409/13/90 15 38635 27220 22605 16565 7580 5765 8670 697 1572 564 1271 58 307 9.309/17/90 19 47325 36405 33825 24705 10425 9665 10805 749 1735 585 1733 65 380 10.509/20/90 22 47740 36165 31375 23455 10650 8925 11005 854 1692 590 1768 64 368 10.509/24/90 26 48755 37030 32390 24095 10205 8040 12510 820 1707 607 1697 64 387 7.809/27/90 29 44920 33220 27685 20000 8955 7400 9400 687 1541 511 1489 57 339 8.710/01/90 33 40945 29975 25535 19340 7615 6595 9760 785 1435 478 1279 61 326 9.910/04/90 36 46440 33810 28990 20555 8650 6285 10655 721 1441 497 1448 62 343 7.310/09/90 41 49705 36300 32845 23495 10130 8340 11420 796 1536 546 1691 70 374 9.010/11/90 43 39850 28885 25245 18880 7845 7670 8205 745 1682 602 1301 46 313 11.710/15/90 47 44865 33970 27600 19720 10025 6835 10025 867 1427 512 1668 64 338 10.310/18/90 50 49245 37175 32590 23150 10920 6495 12665 698 1374 477 1819 72 382 8.810/22/90 54 43870 32495 28870 20820 8690 7700 10160 793 1636 581 1452 62 364 11.6MEAN 44784 33274 29056 21219 9192 7566 10310 741 1552 544 1534 64 351 9.6STD 3489 3078 3185 2303 1151 1098 1257 86 125 48 187 7 26 1.3TABLE C25. EFFLUENT H CHARACTERISTICS FROM RUN 2ADATE DAY P AR AMETERS(mg/L)TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO409/06/90 8 2800 2126 358 300 148 22 173 548 1368 471 92 68 10.8 7.909/10/90 12 3036 2262 372 284 132 26 162 633 1516 522 87 66 11.3 8.109/13/90 15 2670 1984 320 272 154 27 151 685 1534 553 85 60 12.4 9.509/17/90 19 3438 2682 574 480 214 23 256 729 1563 556 91 56 14.6 9.109/20/90 22 3662 2736 612 504 210 18 263 800 1663 593 98 64 14.5 8.309/24/90 26 2928 2220 356 280 139 19 166 797 1594 564 78 56 9.7 6.709/27/90 29 3116 2306 358 276 147 28 155 662 1469 461 74 50 10.9 8.310/01/90 33 3120 2404 380 316 175 31 172 739 1410 469 80 52 11.5 8.010/04/90 36 2538 1890 296 244 148 19 145 744 1469 464 88 64 9.9 6.910/09/90 41 3472 2642 400 328 183 32 177 731 1412 515 86 57 10.8 7.410/11/90 43 3294 2454 358 302 139 22 173 787 1604 584 63 41 13.6 10.210/15/90 47 3542 2710 494 400 178 27 220 846 1416 530 89 61 12.8 7.910/18/90 50 3418 2598 440 364 132 27 196 680 1319 462 92 71 10.6 6.310/22/90 54 3016 2360 394 314 143 29 181 765 1617 588 82 59 12.5 8.0MEAN 3146 2384 408 333 160 25 185 725 1497 524 85 59 11.9 8.0STD 328 261 89 75 26 4 35 75 100 49 8 8 1.5 1.0TABLE C26. INFLUENT CHARACTERISTICS FROM RUN 28DATE DAY P A R A METERS (mg/L)TS VS TSS VSS PROTEINS^UPIDS^CARBOHYDR TOT. VFA COD TOO TKN NH3-N TP PO410/29/90 6 3845 2965 3425 2580 603 515 1615 74 330 94 115 182 192 11.011/01/90 9 3810 2790 3515 2565 612 484 1580 80 322 88 122 24.0 19.4 10.511/05/90 13 4035 2740 3560 2600 610 525 1415 98 421 139 123 25.8 19.8 9.311/08/90 16 3710 2680 2930 2145 563 479 1530 108 453 126 109 19.4 20.4 11.511/12/90 20 3995 2815 3370 2460 664 528 1395 117 401 109 121 14.3 15.6 7.511/15/90 23 4575 3470 4065 3035 557 549 1950 46 265 73 103 14.1 18.8 7.811/19/90 27 3635 2620 3130 2210 494 418 1605 76 344 97 95 16.4 18.9 10.711/22/90 30 3710 2440 3110 2035 566 444 1315 75 272 75 108 17.3 18.9 11.111/26/90 34 3510 2330 3150 2160 516 491 1180 65 393 108 98 15.0 18.6 9.311/29/90 37 4150 2890 3560 2545 652 608 1540 55 280 80 122 17.5 22.8 12.612/03/90 41 4195 2890 3510 2565 648 583 1485 87 375 103 119 15.2 18.5 10.412/06/90 44 4025 3015 3505 2655 629 535 1630 69 334 99 115 14.8 16.1 8.012/10/90 48 3730 2855 3280 2490 583 488 1625 83 359 104 112 18.7 18.3 9.112/13/90 51 3800 2925 3345 2575 618 532 1570 91 401 116 120 21.0 17.5 8.912/17/90 55 3695 2700 3250 2380 563 503 1545 98 419 121 111 21.3 16.6 7.5MEAN 3895 2808 3380 2467 592 512 1532 81 358 102 113 18.2 18.6 9.5STD 263 254 259 243 48 47 166 19 56 18 9 3.4 1.7 1.7TABLE C27. REACTOR I (CMR) CHARACTERISTICS FROM RUN 2BDATE DAY P AR AMETERS (mg/L)TS VS TSS VSS PROTEINS^LIPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO410/29/90 6 18950 14335 12100 9415 3255 1610 7190 530 1411 502 587 66 155 10.711/01/90 9 23595 18200 15515 11005 3940 1965 7780 587 1126 420 686 55 198 7.411/05/90 13 20780 15425 13685 9780 3380 1770 7400 667 1299 450 610 69 169 9.611/08/90 16 22565 16935 13590 9840 4015 2125 7665 721 1217 440 693 50 184 10.111/12/90 20 17475 12880 11875 8590 3090 1550 6390 703 1111 411 536 42 157 8.211/15/90 23 22230 16890 14280 10925 3880 2220 7825 762 967 345 659 39 200 6.211/19/90 27 24975 18900 15225 11525 4445 2335 8340 556 1310 474 763 52 209 6.111/22/90 30 24535 19015 15620 12170 4515 2475 8670 649 1137 434 787 64 224 10.011/26/90 34 22440 16660 14180 10580 3660 2000 7685 714 1215 422 642 56 188 8.411/29/90 37 17990 14075 12210 9325 3470 1880 6420 646 1338 487 620 65 164 8.012/03/90 41 17890 13345 11480 8870 3150 1920 6240 738 1368 480 552 48 157 7.212/06/90 44 19375 14750 12520 9055 3235 2215 6250 745 1400 505 585 67 162 9.812/10/90 48 22650 16975 14065 10340 3960 2375 7085 652 1330 467 695 62 190 6.712/13/90 51 19630 14600 12170 9220 3280 2310 5995 698 1468 548 584 59 158 7.712/17/90 55 21855 16680 13420 9845 3875 1920 7530 743 1386 532 670 50 166 9.0MEAN 21129 15978 13462 10032 3677 2045 7231 674 1272 461 645 56 179 8.3STD 2369 1879 1317 1000 441 270 789 69 134 50 70 9 21 1.4TABLE C28. EFFLUENT I CHARACTERISTICS FROM RUN 2BDATE DAY PA R AMETER S (mg/L)TS VS TSS VSS PROTEINS UPI DS CARBOHYDR TOT. VFA COD TOC TIN NH3-N TP PO410/29/90 6 3526 2604 988 748 234 66 496 560 1318 496 110 73 18.3 10.211/01/90 9 2996 2232 756 542 200 42 391 628 1127 455 93 61 14.3 7.311/05/90 13 3832 2882 1046 806 251 68 518 715 1228 461 102 62 17.9 8.311/08/90 16 3408 2488 910 684 177 56 474 673 1146 425 87 59 17.4 9.011/12/90 20 3112 2210 818 618 166 50 422 694 1096 435 75 49 14.8 7.811/15/90 23 3428 2578 964 702 134 44 481 718 1008 369 61 40 15.6 7.011/19/90 27 2840 2016 686 520 230 36 382 571 1225 461 85 49 13.0 6211/22/90 30 3018 2120 710 554 199 39 397 638 1208 444 96 64 14.9 8.311/26/90 34 3060 2296 758 600 147 47 412 739 1156 431 83 59 14.7 7.411/29/90 37 3536 2564 962 724 177 52 495 672 1242 481 97 69 17.8 8.212/03/90 41 2970 2208 736 552 172 35 395 724 1273 487 76 48 14.3 7.312/06/90 44 3028 2180 710 538 159 41 388 748 1324 500 96 71 16.2 9.012/10/90 48 3616 2662 920 720 229 57 483 686 1266 485 105 68 15.8 6.412/13/90 51 2902 2110 776 604 213 48 430 751 1441 550 93 59 14.1 6.812/17/90 55 3134 2204 732 572 174 43 394 783 1451 511 84 56 162 8.3MEAN 3227 2357 831 632 191 48 437 687 1234 466 90 59 15.7 7.8STD 292 244 116 88 34 10 47 63 117 42 12 9 1.5 1.0TABLE C29. REACTOR II (UASB) CHARACTERISTICS FROM RUN 2BDATE DAY P AR AMETERS(mg/L)TS VS TSS VSS PROTEINS UPIDS CARBOHYDR TOT. VFA COD TOC 11Q4 NH3-N TP PO410/29/90 6 48135 34480 31915 24495 9825 10885 11875 585 1492 495 1636 64 502 8.911/01/90 9 51065 37095 33740 25540 11940 11205 12815 663 1236 455 1964 54 526 7.311/05/90 13 40270 28080 24685 18800 8705 8600 9440 696 1362 489 1444 51 394 10.111/08/90 16 47055 34600 30945 23005 10190 10080 11250 709 1611 613 1695 65 468 6.711/12/90 20 44435 32535 27520 20485 9385 9285 9825 780 1472 561 1550 48 437 9.011/15/90 23 50290 37550 32455 24715 10975 11195 11205 821 1300 461 1801 45 484 7.811/19/90 27 51895 39145 33370 25300 11625 10100 13075 600 1178 388 1922 62 510 6.111/22/90 30 42550 30070 26970 21490 8900 9675 10630 673 1374 519 1493 69 432 7.511/26/90 34 44960 31965 27495 22020 8985 9860 11140 752 1453 580 1492 54 457 9.011/29/90 37 40945 28600 24975 19755 8395 8945 9660 704 1210 426 1391 48 404 10.212/03/90 41 38725 27815 23285 18995 8510 8860 9185 737 1451 539 1433 71 422 7.612/06/90 44 41310 30010 25325 20180 9060 9945 10260 776 1444 536 1518 67 441 11.012/10/90 48 46920 34420 29080 23700 9765 10190 12105 699 1517 544 1615 52 463 7.312/13/90 51 50360 36400 30865 25030 10425 11075 12410 768 1678 559 1716 48 507 6.512/17/90 55 46315 32595 27660 22595 9040 9205 10770 805 1592 529 1507 60 445 8.6MEAN 45682 33024 28686 22407 9715 9940 11043 718 1425 513 1612 57 459 8.2STD 4096 3493 3257 2282 1075 835 1192 67 143 59 170 8 39 1.4TABLE C30. EFFLUENT II CHARACTERISTICS FROM RUN 2BDATE DAY P AR AMETERS(mg/L)TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TIN NH3-N TP PO410/29/90 6 3298 2384 506 392 154 44 232 575 1411 504 92 67 14.1 7.911/01/90 9 3576 2642 608 484 132 48 281 686 1287 421 71 50 13.9 5.411/05/90 13 2792 2150 430 348 168 36 211 702 1403 483 79 52 13.6 9.511/08/90 16 2800 2072 378 308 120 33 189 732 1507 551 86 68 11.0 6211/12/90 20 3440 2494 526 432 114 50 249 773 1333 523 84 46 14.5 7.411/15/90 23 2720 2054 368 276 171 28 173 803 1212 445 83 56 11.1 6.711/19/90 27 2664 1958 366 266 125 29 165 601 1155 415 87 67 10.4 5.511/22/90 30 3066 2208 420 344 143 34 206 623 1421 524 88 65 11.2 6.011/26/90 34 2956 2276 456 360 96 37 216 729 1327 509 72 56 15.4 9.411/29/90 37 3578 2612 524 408 154 42 233 681 1251 475 70 45 12.7 6.812/03/90 41 3212 2404 414 336 114 44 200 756 1509 530 89 70 12.2 7.412/06/90 44 2838 2176 422 324 160 46 192 784 1459 510 94 68 15.5 10.112/10/90 4e 2904 2077 460 350 149 36 211 710 1392 486 82 58 11.2 6.512/13/90 51 3040 2204 504 414 141 42 247 727 1590 575 87 44 11.9 5.112117/90 55 3132 2294 504 398 130 37 246 759 1597 539 79 59 13.9 7.3MEAN 3068 2267 459 363 138 39 217 709 1390 499 80 58 12.8 7.1STD 290 198 67 57 21 6 30 65 127 44 9 9 1.6 1.5TABLE C31. INFLUENT CHARACTERISTICS FROM RUN 2CDATE DAY P A R AMETERS(mg/L)TS VS TSS VSS PROTEINS^LIPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO412/20/90 3 3810 2630 3490 2200 595 454 1535 87 339 91 119 24.2 13.7 6.412/24/90 7 4150 2890 3600 2485 564 514 1525 105 390 106 109 18.8 16.6 7.712/27/90 10 4195 3055 3685 2655 648 457 1795 117 412 121 125 21.4 18.5 9.312/31/90 14 3970 2845 3660 2420 550 500 1620 108 491 141 112 23.9 15.9 7.101/03/91 17 4365 3200 4005 2775 605 506 1885 122 417 119 119 22.2 20.1 10.601/06/91 20 4200 3285 3905 2865 592 487 2045 96 428 126 113 18.6 16.4 9.401/09/91 23 4075 3155 3830 2835 565 438 1990 120 463 144 110 19.6 18.7 10.101/12/91 26 4225 3310 3945 2805 619 506 1970 101 475 141 124 25.0 20.3 9.701/14/91 28 3980 2740 3560 2420 556 452 1585 120 369 100 107 17.9 15.9 6.9MEAN 4108 3012 3742 2607 588 479 1772 108 420 121 115 21.3 17.3 8.6STD 157 232 174 221 31 27 197 12 47 18 6 2.5 2.1 1.5TABLE C32. REACTOR I (CMR) CHARACTERISTICS FROM RUN 2CDATE DAY ATS VS TSS VSS PROTEINS LIPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO412/20/90 3 13515 10130 8805 6910 2760 885 4855 345 1090 365 501 60 76 9.512/24/90 7 11015 8005 6935 5375 2105 654 4015 279 919 300 378 41 63 7.412/27/90 10 11150 8030 6955 5125 2000 601 3880 247 884 295 366 46 56 7.412/31/90 14 9615 6960 5050 3920 1415 526 2760 274 798 245 269 42 46 8.901/03/91 17 9640 7135 5775 4535 1610 609 3380 194 780 257 295 37 52 6.301/06/91 20 9145 6615 5340 4170 1550 517 3015 173 842 251 295 47 41 5.501/09/91 23 10435 7630 6190 4710 1805 622 3550 228 905 306 320 32 55 9.601/12/91 26 9775 6995 5860 4565 2040 651 3255 252 835 266 361 35 57 8.201/14/91 28 11510 8570 7025 5360 1845 603 3990 198 954 320 345 50 67 7.8MEAN 10644 7786 6437 4963 1903 630 3633 243 890 289 348 43 57 7.8STD 1269 1019 1076 834 375 101 595 50 89 37 65 8 10 1.3TABLE C33. EFFLUENT I CHARACTERISTICS FROM RUN 2CDATE DAY P AR AMETERS (mg/L)TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO412/20/90 3 3616 2622 1102 810 299 50 586 327 1100 365 113 65 15.8 8.312/24/90 7 2978 2096 796 552 229 34 436 247 955 302 77 41 11.6 6.312/27/90 10 3548 2524 1084 780 216 45 581 260 845 256 82 47 16.3 9.012/31/90 14 3000 2190 844 586 207 39 455 261 901 297 83 50 13.6 8.201/03/91 17 3516 2604 1040 744 301 51 565 203 808 249 89 41 13.3 6.601/06/91 20 3274 2358 962 668 270 40 517 169 927 300 99 56 12.1 5.801/09/91 23 3062 2120 868 612 231 38 487 217 880 277 79 42 15.5 10.201/12/91 26 3556 2588 1076 706 263 47 544 248 972 318 80 38 13.5 7.001/14/91 28 3012 2132 838 652 236 37 492 195 874 280 91 53 12.4 7.3MEAN 3285 2359 957 679 250 42 518 236 918 294 88 48 13.8 7.6STD 259 215 115 83 33 6 51 44 81 33 11 8 1.6 1.3TABLE C34. REACTOR II (UASB) CHARACTERISTICS FROM RUN 2CDATE DAY P AR AMETERS(mg/L)TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO412/20/90 3 33700 24670 22925 16990 5840 3985 10135 355 1263 402 993 58 221 10.112/24/90 7 27865 20705 18660 13570 4820 3200 8725 334 1045 361 812 41 187 8.712/27/90 10 27270 19360 16730 12175 4375 2830 7840 286 940 305 746 46 172 6.612/31/90 14 29105 21020 18510 13220 5105 2685 8800 290 992 315 852 35 184 7.301/03/91 17 24505 17640 15375 11515 4255 2775 7335 253 891 284 722 41 163 5.201/06/91 20 29510 21635 20565 14745 5010 3265 9640 259 1058 346 854 53 192 9.001/09/91 23 26155 19605 19595 13900 4525 3170 8595 247 985 299 768 44 175 5.001/12/91 26 29710 21320 19870 14815 5105 3335 9080 308 1114 351 851 34 204 6.301/14/91 28 26885 20005 17955 12660 4560 2930 8385 1022 320 778 48 169 7.8MEAN 28301 20662 18909 13732 4844 3131 8726 288 1034 331 820 44 185 7.3STD 2484 1824 2072 1550 460 373 803 36 102 35 76 7 17 1.6TABLE C35. EFFLUENT II CHARACTERISTICS FROM RUN 2CDATE DAY P AR AMETERS(mg/L)TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO412/20/90 3 3144 2326 488 342 176 32 161 342 1175 380 87 59 13.4 9.612/24/90 7 2948 2078 380 256 155 26 137 310 939 298 74 49 10.8 8.212/27/90 10 2442 1710 296 196 160 20 106 274 1040 327 71 45 9.7 7.012/31/90 14 3086 2132 490 338 136 35 160 263 974 319 65 43 11.3 7.401/03/91 17 2824 1954 432 304 131 31 146 237 912 290 88 47 9.5 5.901/06/91 20 2448 1762 388 260 180 28 125 235 1101 373 84 55 11.5 8.201/09/91 23 2690 1950 302 202 149 33 102 240 942 298 71 48 9.4 7.101/12/91 26 2492 1768 344 212 133 34 119 298 1066 348 81 40 8.9 6.001/14/91 28 3100 2204 440 326 174 25 151 225 963 312 84 56 10.5 6.6MEAN 2797 1987 396 271 155 29 134 269 1012 327 74 49 10.6 7.3STD 273 203 69 56 18 5 21 38 83 31 9 6 1.3 1.1TABLE C36. INFLUENT CHARACTERISTICS FROM RUN 3ADATE DAY P A R AMETER S (mg/L)TS VS TSS VSS PROTEINS^UPI DS CARBOHYDR TOT. VFA COD TOC TKN NH3-N 1P PO401/21/91 7 4050 2990 3475 2715 627 514 1715 83 420 122 116 15.3 17.4 9.901/24/91 10 3380 2340 2850 2115 541 483 1300 92 324 108 110 23.9 15.9 9.201/28/91 14 3885 2565 3390 2420 546 530 1350 109 439 118 106 18.5 18.3 12.001/31/91 17 3790 2710 3150 2305 639 520 1415 115 396 110 121 19.2 16.7 10.502/04/91 21 4610 3320 4170 2845 622 559 2005 87 506 152 121 21.4 19.4 10.902/07/91 24 4085 2925 3490 2650 684 465 1580 89 355 104 125 15.3 16.7 8.102/11/91 28 4055 2860 3615 2555 667 491 1500 114 412 129 124 16.8 17.8 10.702/14/91 31 3795 2755 3305 2525 590 516 1495 121 477 137 118 23.2 14.4 6.702/18/91 35 4130 3170 3645 2885 699 533 1785 74 511 160 131 18.7 18.2 10.802/21/91 38 4020 3095 3510 2780 673 520 1790 87 423 132 124 16.0 19.7 11.4MEAN 3980 2873 3460 2580 629 513 1594 97 426 127 119 18.8 17.5 10.0STD 297 278 326 234 53 26 213 15 57 18 7 3.0 1.5 1.5TABLE C37. REACTOR I (CMR) CHARACTERISTICS FROM RUN 3ADATE DAY PARAMETERS (mg/L)TS VS TSS VSS PROTEINS UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO401/21/91 7 16320 11710 10525 7660 1645 865 5935 548 1188 391 307 44 89 5.801/24/91 10 19160 13915 12160 9340 1840 970 7050 594 1331 416 349 55 108 7.801/28/91 14 22560 15765 14635 10905 2120 1090 8245 633 1435 485 389 50 134 9.201/31/91 17 21705 15440 15335 11835 2065 1255 8650 677 1493 505 398 68 137 10.302/04/91 21 19555 13620 12170 9230 1800 910 7240 576 1595 534 326 38 111 7.102/07/91 24 16485 11880 10945 8490 1625 860 6325 609 1392 473 304 44 98 6.402/11/91 28 23745 17260 14700 10820 2200 1105 8205 648 1306 433 410 58 121 5.402/14/91 31 22945 16220 13505 10475 2065 950 8240 567 1423 490 386 56 126 5.702/18/91 35 19955 14885 12040 8760 1780 920 6345 531 1372 479 328 44 95 8.702/21/91 38 22650 15835 14485 11305 1930 1285 8475 581 1246 421 358 49 132 9.0MEAN 20508 14653 13050 9882 1907 1021 7471 596 1378 463 356 51 115 7.5STD 2518 1747 1616 1303 190 147 964 43 113 43 37 8 16 1.6TABLE C38. EFFLUENT I CHARACTERISTICS FROM RUN 3ADATE DAY P AR AMETERS(mg/L)TS VS TSS VSS PROTEINS^LIPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO401/21/91 7 2576 1880 516 412 298 56 281 524 1153 393 99 51 13.8 9.201/24/91 10 2936 2176 596 468 323 70 309 559 1284 434 107 56 12.6 7.501/28/91 14 2656 1944 464 328 250 53 211 638 1400 490 88 48 14.1 10.301/31/91 17 3388 2438 716 556 235 76 374 655 1322 462 103 65 15.9 9.802/04/91 21 2836 2014 494 392 211 54 272 581 1639 579 80 47 12.2 7.802/07/91 24 3302 2410 754 608 268 80 397 569 1286 453 92 49 13.5 6.802/11/91 28 3110 2178 696 510 307 64 357 628 1301 493 102 53 12.4 7.502114/91 31 2692 1992 568 406 329 56 274 536 1527 547 113 60 10.2 5.902/18/91 35 2584 1804 492 352 268 50 244 508 1317 485 93 50 11.8 9.202/21/91 38 2792 2080 530 380 293 55 268 602 1238 446 104 57 14.7 10.8MEAN 2887 2092 583 441 278 61 299 580 1347 478 98 54 13.1 8.5STD 277 200 99 87 37 10 57 48 135 51 9 8 1.5 1.5TABLE C39. REACTOR II (UASB) CHARACTERISTICS FROM RUN 3ADATE DAY PAR AMETERS (mg/L)TS VS TSS VSS PROTEINS UPIDS^CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO401/21/91 7 30935 23100 20620 15770 7130 5750 8860 550 1279 426 1201 80 226 6.801/24/91 10 33260 23970 21435 17330 7265 5935 9315 612 1394 467 1217 55 257 9.701/28/91 14 36380 26755 24775 19605 8050 6560 10300 603 1604 550 1353 65 280 8.701/31/91 17 39715 29315 24790 18765 8750 6075 10240 662 1556 522 1448 48 265 7.602/04/91 21 40660 30345 27310 21920 9015 7135 11495 715 1697 569 1489 47 297 10.402/07/91 24 36975 27880 25120 19055 8025 6310 10450 629 1410 508 1341 57 261 8.302/11/91 28 42805 31030 28505 22285 9120 7020 11475 598 1326 467 1504 45 308 6.302/14/91 31 34450 24660 22465 17155 7480 5935 9335 686 1433 541 1239 42 242 7.002/18/91 35 33500 24375 21630 16520 7245 5680 9190 607 1590 591 1219 80 245 11.102/21/91 38 40745 29150 24820 20010 8085 6220 10805 572 1547 574 1350 56 281 9.0MEAN 36923 27058 24147 18842 8017 6262 10147 623 1484 522 1336 53 266 8.5STD 3689 2738 2453 2081 707 477 898 48 128 51 110 7 24 1.5TABLE C40. EFFLUENT II CHARACTERISTICS FROM RUN 3ADATE DAY PARAMETERS (mg/l)TS VS TSS VSS PROTEINS UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO401/21/91 7 2284 1606 258 184 137 19.3 108 544 1223 397 80 58 9.1 7201/24/91 10 2862 1844 382 288 150 28.1 188 557 1322 417 80 56 12.4 9.301/28/91 14 2198 1538 294 236 104 24.8 129 626 1428 485 83 67 9.6 7.001/31/91 17 3026 2224 356 268 134 22.6 163 692 1511 507 70 49 10.7 7.802/04/91 21 2590 1890 388 288 112 27.0 183 686 1579 518 64 46 12.1 9.202/07/91 24 3170 2346 406 308 110 17.7 198 677 1524 539 67 49 10.2 7.002/11/91 28 3056 2170 334 260 117 19.8 156 628 1262 434 81 42 8.8 6.102/14/91 31 2830 2008 418 322 160 16.9 205 691 1485 533 64 39 9.0 5.802/18/91 35 2564 1794 400 302 122 17.4 191 584 1553 551 73 53 13.4 10.202/21/91 38 3005 2060 348 276 138 202 162 619 1390 502 72 50 10.3 6.8MEAN 2759 1948 358 273 128 21.4 186 630 1428 488 71 51 10.6 7.6STD 318 250 49 38 17 3.8 29 53 118 51 7 8 1.5 1.4APPENDICESillaaril!IIIII5MII191TABLE 042. REACTOR I (CMR) CHARACTERISTICS FROM RUN 3BDATE DAY PAR AMETERS (mg/L)TS VS TSS VSS PROTEINS UPIDS^CARBOHYDR TOT. VFA COD TOC TIN NH3-N TP PO402/28/91 6 16620 12935 10330 8560 1515 945 6620 388 1311 444 294 52 77 7.003/04/91 10 17815 14480 12490 10245 1720 1090 7825 462 1249 424 314 38 93 5.203/07/91 13 19185 15610 11775 9545 1800 965 7500 477 1337 480 331 43 82 5.103/11/91 17 22255 17725 14330 11335 1995 1205 8855 542 1588 536 349 30 103 5.903/14/91 20 20500 16020 13715 10535 1950 1100 8260 512 1394 493 339 27 97 5.603/18/91 24 18525 14835 11500 8840 1560 1025 6870 583 1742 601 289 40 76 4.203/21/91 27 22310 18130 15110 12120 2080 1270 9415 592 1615 534 366 34 114 6.903/25/91 31 19640 15975 13425 10235 1865 1140 7930 488 1485 503 339 40 91 4.403/28/91 34 17060 13870 12100 9310 1735 995 7095 451 1559 518 327 50 81 7.304/01/91 38 22815 18055 14720 11315 2030 1165 8610 483 1273 435 362 37 105 5.004/04/91 41 19310 16340 14335 10950 1765 1090 8490 515 1326 447 330 48 96 4.3MEAN 19640 15816 13075 10272 1820 1090 7952 499 1444 492 331 40 92 5.5STD 2033 1630 1463 1070 176 97 835 56 156 51 23 8 12 1.1TABLE C43. EFFLUENT I CHARACTERISTICS FROM RUN 3BDATE DAY P AR AMETERS (mg/L)TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC T1041 NH3-N TP PO402/28/91 6 2798 2054 624 500 252 68 337 401 1169 409 97 56 9.7 6.403/04/91 10 3246 2542 844 692 208 85 459 447 1232 412 84 51 10.8 4.803/07/91 13 3446 2726 930 764 281 96 499 478 1303 448 88 43 11.3 5.203/11/91 17 3012 2320 628 492 230 67 335 566 1474 506 71 35 9.0 5.603/14/91 20 2886 2228 682 546 196 74 366 514 1286 455 60 28 9.7 5.203/18/91 24 3020 2416 718 572 244 79 390 594 1687 548 84 45 9.5 4.403/21/91 27 2654 2134 588 474 187 73 319 597 1390 446 78 48 9.4 6.203/25/91 31 3152 2458 680 560 261 93 358 481 1466 481 77 35 9.1 4.703/28/91 34 3316 2680 756 608 275 93 404 463 1527 499 97 53 12.0 7.004/01/91 38 2932 2342 744 612 229 98 403 478 1191 422 76 40 11.0 4.904/04/91 41 2732 2090 640 520 204 72 342 526 1328 438 87 54 8.5 4.3MEAN 3018 2363 712 576 233 82 383 504 1368 460 82 44 10.0 5.3STD 239 217 98 85 31 11 53 59 151 42 10 9 1.1 0.8TABLE C44. REACTOR II (UASB) CHARACTERISTICS FROM RUN 313DATE DAY P AR AMETERS(mg/L)TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO402/28/91 6 36870 28780 25370 21015 6865 6020 10685 474 1429 465 1140 41 231 6.603/04/91 10 40990 32215 27645 23465 8460 7255 11160 489 1447 486 1406 52 260 5.803/07/91 13 35885 29040 25730 22005 7330 6635 11145 537 1559 527 1211 38 252 4.103/11/91 17 42165 32690 29895 24795 8340 7570 11560 544 1500 511 1375 41 278 4.603/14/91 20 34240 26935 22815 19405 6715 5650 10390 639 1607 546 1108 33 211 5.903/18/91 24 39125 30595 27860 23125 7255 6930 11350 608 1863 602 1190 30 246 5.603/21/91 27 35795 27470 23610 20085 6665 5845 10030 527 1801 588 1116 50 220 3.803/25/91 31 42310 32295 26070 22040 8045 7035 10800 601 1618 526 1323 36 265 6.703/28/91 34 34380 26405 22920 19175 6685 5470 9705 626 1715 549 1098 28 209 7.104/01/91 38 37485 29720 25635 21280 7625 6345 11020 521 1546 491 1254 34 243 4.004/04/91 41 34370 26000 22545 19200 7250 5695 9970 565 1500 482 1201 41 215 4.6MEAN 37601 29286 25463 21417 7385 6405 10710 557 1599 525 1220 39 239 5.3STD 2949 2323 2257 1789 627 689 584 53 135 42 103 7 22 1.1TABLE C45. EFFLUENT II CHARACTERISTICS FROM RUN 3BDATE DAY P AR AMETERS(mg/L)TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC MN NH3-N TP PO402/28/91 6 2758 2206 338 284 132 33 150 452 1265 418 63 42 8.9 5.803/04/91 10 2414 1934 270 228 154 29 115 481 1325 431 78 53 7.4 5.203/07/91 13 3000 2470 352 284 115 35 141 524 1450 491 63 45 6.0 3.603/11/91 17 3198 2638 404 340 97 44 188 521 1316 436 56 41 7.9 4.303/14/91 20 3194 2502 352 290 128 36 155 539 1414 496 57 36 8.6 5.303/18/91 24 3300 2716 460 392 93 46 217 596 1661 590 49 34 9.0 4.703/21/91 27 3018 2374 322 266 98 32 138 534 1576 548 69 54 6.8 4.003/25/91 31 2808 2292 338 276 124 34 146 610 1422 510 52 32 8.7 6.103/28/91 34 3112 2568 404 328 129 41 179 628 1642 559 51 30 10.1 6.304/01/91 38 2444 2006 356 286 148 31 159 512 1344 446 64 41 6.8 3.704/04/91 41 2956 2440 440 370 113 45 208 570 1418 487 65 47 8.7 42MEAN 2927 2377 367 304 121 37 163 542 1439 492 61 41 8.1 4.8STD 282 237 53 46 19 6 30 52 127 54 8 8 1.2 0.9TABLE C48. INFLUENT CHARACTERISTICS FROM RUN 4ADATE DAY P AR AMETER S (mg/L)TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC -^TKN NH3-N TP PO404/15/91 7 4295 2990 3860 2695 562 430 1870 87 571 134 106 16.1 19.3 8.304/18/91 10 4150 3230 3735 3025 610 538 1915 106 375 116 116 18.7 20.4 12.404/22/91 14 3510 2845 3210 2685 543 446 1665 126 353 102 110 22.7 20.6 9.604/25/91 17 4130 3325 3720 3110 684 562 1870 133 398 123 134 25.0 18.3 9.104/29/91 21 3675 2895 3330 2705 599 492 1685 101 419 129 116 20.3 17.2 9.405/02/91 24 4380 3570 4050 3360 720 591 2055 122 460 156 132 16.6 17.0 8.305/06/91 28 4070 3015 3745 2765 644 509 1715 135 475 128 119 15.6 20.0 12.105/09/91 31 3895 2975 3480 2750 625 518 1610 104 387 102 120 20.2 22.4 13.405/13/91 35 3955 2885 3465 2635 667 538 1520 120 418 119 129 22.0 23.7 13.505/16/91 38 4200 3110 3845 2850 711 531 1665 109 452 127 129 15.5 18.2 10.4MEAN 4026 3084 3644 2858 637 516 1757 114 431 124 121 19.3 19.7 10.7STD 258 218 249 222 57 47 155 15 59 15 9 3.1 2.1 1.9TABLE C47. REACTOR I (CMR) CHARACTERISTICS FROM RUN 4ADATE DAY P AR AMETERS(mg/L)TS VS TSS VSS PROTEINS^LIPIDS CARBOHYDR TOT. VFA COD TOC TIN NH3-N TP PO404/15/91 7 13975 11005 10275 8445 1310 980 6440 494 1215 427 241 31 118 6.204/18/91 10 17950 14250 12680 10735 1545 1055 8280 547 1153 411 293 45 141 5.504/22/91 14 15150 11910 10375 8740 1335 865 6705 523 1394 469 268 54 122 5.704/25/91 17 13170 10265 8995 7680 1200 900 5845 572 1318 449 254 62 106 7.704/29/91 21 19560 15560 13570 11495 1910 1285 8290 594 1515 516 361 56 164 10.805/02/91 24 19635 14000 12025 10055 1730 1250 7650 608 1591 563 319 42 146 11.905/06/91 28 16560 11975 10945 9185 1295 1125 6860 686 1425 500 261 54 128 9.305/09/91 31 14465 11125 9990 8285 1210 970 6480 614 1197 419 254 60 130 7.605/13/91 35 16850 12820 10415 8880 1750 895 6825 530 1411 484 334 54 114 7.205/16/91 38 20550 15580 12930 10960 1935 1235 8145 593 1373 473 349 39 155 8.4MEAN 16787 12849 11220 9446 1522 1056 7152 576 1359 471 293 50 132 8.0STD 2458 1811 1415 1220 274 151 828 53 133 45 42 9 18 2.0TABLE C48. EFFLUENT I CHARACTERISTICS FROM RUN 4ADATE DAY P AR AMETERS(mg/1..)TS VS TSS VSS PROTEINS^LIPIDS CARBOHYDR TOT. VFA COD TOC TIN NH3-N TP PO404/15/91 7 2708 2018 630 572 144 43 402 514 1198 406 59 36 12.2 7.404/18/91 10 2580 1942 584 496 136 36 389 551 1096 388 67 45 10.6 5.504/22/91 14 3136 2384 846 720 162 55 493 538 1367 445 81 56 12.8 4.804/25/91 17 3620 2712 1026 754 197 64 558 562 1269 411 92 61 15.8 7.904/29/91 21 3500 2520 1010 680 170 58 557 624 1458 489 86 59 17.0 10.205/02/91 24 2840 2176 738 534 137 44 456 602 1572 520 66 44 17.2 12.005/06/91 28 2852 2164 660 520 133 40 410 686 1327 452 73 51 14.5 9.205/09/91 31 3470 2658 852 742 184 52 495 664 1194 417 92 62 15.5 8.105/13/91 35 3386 2574 914 748 170 61 516 533 1334 464 83 55 16.0 7.905/16/91 38 2952 2316 714 626 147 47 443 597 1265 437 67 44 13.8 8.2MEAN 3104 2346 797 639 158 50 472 587 1308 443 77 51 14.5 8.1STD 350 255 148 97 21 9 59 55 130 38 11 8 2.1 2.0TABLE C49. REACTOR II (UASB) CHARACTERISTICS FROM RUN 4ADATE DAY PARAMETERS(mg/L)TS VS TSS VSS PROTEINS UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO404/15/91 7 36500 26490 24520 21485 6005 6370 11585 618 1297 439 999 38 330 6.304/18/91 10 36905 27370 25780 22635 6420 6655 12520 574 1331 442 1081 54 347 7.004/22/91 14 32430 23415 22095 19890 5060 5820 10735 648 1544 547 868 58 309 5.904/25/91 17 40440 30390 26635 23215 6815 6945 12095 684 1681 570 1141 50 365 8.004/29/91 21 37610 27975 24560 21025 6105 6335 11490 701 1360 475 1022 45 327 11.605/02/91 24 34405 25150 24325 22005 5210 6720 11835 764 1560 557 888 54 337 11.205/06/91 28 31875 22715 20555 18660 5105 5730 10125 748 1457 505 861 44 303 9.705/09/91 31 34830 24655 22455 20035 5645 6190 10345 670 1399 470 964 61 310 8.105/13/91 35 40210 30015 25510 22510 6200 6900 11755 691 1687 595 1033 41 347 6.805/16/91 38 32955 24085 22630 19855 5450 6480 10555 729 1491 524 918 46 314 8.8MEAN 35816 26226 23907 21132 5802 6415 11304 683 1481 512 977 49 329 8.3STD 2893 2541 1810 1410 569 394 768 55 131 52 90 7 19 1.9TABLE C50. EFFLUENT II CHARACTERISTICS FROM RUN 4ADATE DAY P AR AMETERS(mg/l)TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO404/15/91 7 2490 1918 448 332 81 19 232 616 1279 438 51 38 9.8 5.804/18/91 10 2788 2070 420 306 98 14 207 562 1205 420 71 55 10.1 6.204/22/91 14 2408 1874 366 266 88 13 198 629 1577 563 75 61 9.5 6.304/25/91 17 3194 2412 454 346 122 18 235 680 1622 578 68 49 11.9 8.004/29/91 21 3322 2496 580 424 119 20 295 675 1321 462 67 48 15.2 10.305/02/91 24 3222 2486 468 332 108 22 214 722 1464 515 75 57 14.3 10.405/06/91 28 3538 2732 594 444 129 16 310 745 1477 506 65 44 12.9 7.905/09/91 31 2988 2208 574 418 92 21 284 698 1286 475 75 61 13.4 8.005/13/91 35 2700 2074 442 344 79 14 247 735 1665 578 52 40 10.8 6.405/16/91 38 3270 2616 552 438 87 17 308 707 1520 544 85 52 12.5 7.0MEAN 2992 2289 490 365 100 17 253 677 1442 508 86 50 12.0 7.6STD 360 285 75 58 17 3 41 55 152 55 8 8 1.9 1.6TABLE C51. INFLUENT CHARACTERISTICS FROM RUN 4BDATE DAY P AR AM ET ER S (mg/L)TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO405/23/91 6 3705 2725 3295 2525 657 472 1405 78 590 137 128 23.0 19.4 9.705/27/91 10 4435 3380 4035 2900 669 481 2120 102 673 201 132 25.1 25.0 14.505/30/91 13 3880 2910 3415 2500 575 509 1620 103 551 177 123 30.5 21.7 11.106/03/91 17 3795 2940 3415 2595 524 417 1760 61 316 87 105 21.0 18.1 9.306/06/91 20 3990 2805 3550 2445 619 524 1365 79 361 106 117 18.4 16.7 8.806/10/91 24 4150 3050 3610 2650 532 555 1600 112 365 132 101 15.8 20.2 12.106/13/91 27 4205 3085 3850 2620 517 463 1850 145 527 166 103 20.1 21.6 13.606/17191 31 4085 3100 3595 2715 703 479 1895 109 503 157 139 26.1 22.8 16.106/20/91 34 3960 2920 3685 2545 538 518 1620 117 414 129 104 18.3 20.7 13.906/24/91 38 4175 3050 3590 2720 666 542 1640 106 432 120 127 20.5 20.1 11.806/27/91 41 4260 3230 3750 2850 679 552 1805 88 450 133 131 22.2 24.4 16.707/01/91 45 3985 2785 3665 2465 541 470 1555 105 504 162 104 17.8 18.3 10.307/04/91 48 3875 2970 3405 2545 580 475 1630 111 465 127 118 25.6 19.0 11.0MEAN 4038 2996 3605 2621 600 498 1682 101 473 141 118 21.9 20.6 12.2STD 196 175 194 136 65 38 196 20 96 29 13 3.9 2.4 2.5TABLE C52. REACTOR I (CMR) CHARACTERISTICS FROM RUN 46DATE DAY P AR AMETERS (mg/L)TS VS TSS VSS PROTONS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO405/23/91 6 16230 11830 10155 7795 1765 1180 5140 420 1642 542 351 66 130 6.905/27/91 10 20730 15080 13320 9925 2100 1565 6795 472 1730 605 376 40 165 8.205/30/91 13 16680 12915 11410 8550 1815 1385 5750 460 1651 567 349 59 149 10.206/03/91 17 20145 14895 12125 8955 2210 1625 5950 431 1456 469 417 64 145 8.606/06/91 20 22225 16165 13065 9740 2460 1815 6530 483 1396 459 438 44 169 11.606/10/91 24 17720 12830 11055 8575 1730 1280 6005 495 1360 477 322 46 153 10.506/13/91 27 23050 17150 14390 10355 2515 1630 7305 513 1515 508 448 45 181 6.106/17/91 31 21060 15390 12170 9270 1960 1390 6440 408 1328 445 374 61 150 8.306/20/91 34 23255 17485 14375 10740 2455 1840 7515 412 1446 487 446 53 173 10.706/24/91 38 19175 14390 13700 9910 1835 1375 7140 429 1390 455 342 49 160 8.106/27/91 41 21230 16040 14175 10615 2320 1620 6470 472 1614 538 412 41 167 7.607/01/91 45 18235 13885 12010 8330 1900 1265 5875 426 1377 457 357 53 141 11.507/04/91 48 20610 14570 11550 9195 2035 1645 7020 459 1482 508 386 60 148 10.8MEAN 20027 14817 12577 9381 2085 1509 6457 452 1491 501 386 52 156 9.2STD 2195 1614 1313 886 270 202 667 32 124 47 41 9 14 1.7TABLE C53. EFFLUENT I CHARACTERISTICS FROM RUN 48DATE DAY P AR AMETERS (mg/4TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO405/23/91 6 2428 1866 296 236 257 60 166 430 1608 531 111 70 10.9 7.605/27/91 10 2524 1880 372 292 250 71 200 451 1762 625 78 38 12.9 8.805/30/91 13 3268 2454 486 408 305 82 271 489 1635 562 110 61 16.4 10.706/03/91 17 2664 1958 360 284 267 64 198 428 1447 492 108 65 13.5 9.006/06/91 20 2754 2108 408 328 290 76 228 501 1380 456 92 46 16.8 12.706/10/91 24 3078 2372 316 244 299 58 171 467 1465 508 93 45 15.4 12.006/13/91 27 3312 2520 440 360 328 71 255 502 1598 530 103 50 13.9 8.906/17/91 31 3120 2436 448 366 288 78 258 411 1412 464 110 64 13.3 8.206/20/91 34 2584 1938 368 286 240 62 201 401 1392 477 85 47 15.8 11.806/24/91 38 2732 2010 414 344 259 70 244 414 1406 495 92 50 13.8 9.006/27/91 41 2810 2146 368 358 300 63 262 490 1691 602 88 40 15.3 10.307/01/91 45 3102 2340 422 292 232 80 186 418 1330 434 82 45 16.7 12.907/04/91 48 2638 1980 378 296 303 64 209 439 1498 506 108 60 15.9 11.5MEAN 2847 2154 390 315 278 69 219 449 1510 514 97 52 14.7 10.3STD 282 229 51 49 28 8 35 35 130 54 11 10 2 2TABLE C54. REACTOR II (UASB) CHARACTERISTICS FROM RUN 4BDATE DAY PAR AMETERS (mg/L)TS VS TSS VSS PROTEINS UPIDS^CARBOHYDR TOT. VFA COD TOC TIN NH3-N TP PO405/23/91 6 36750 26235 23450 16625 7275 7140 6940 415 1812 578 1207 43 370 7.705/27/91 10 32475 24180 21120 15090 6850 6775 6175 471 1903 658 1131 35 344 7.605/30/91 13 42015 30910 29805 21920 8200 7890 9520 512 1624 560 1366 54 482 8.806/03/91 17 42165 29775 27265 19450 7935 7610 8655 484 1849 651 1328 59 433 9.006/06/91 20 39275 28560 25335 18535 7355 7325 8055 459 1527 526 1229 52 412 10.306/10/91 24 42630 31695 29660 21045 8315 8030 9010 524 1453 486 1378 47 469 10.506/13/91 27 40005 30085 27450 19230 8160 7775 8285 547 1508 506 1363 57 428 8.706/17/91 31 35075 25380 24570 17515 6765 7035 7985 469 1718 549 1143 61 390 8.506/20/91 34 36715 25770 23715 17205 7055 6840 7415 518 1482 493 1182 53 383 11.206/24/91 38 41200 28355 27640 19350 7880 7795 8900 467 1618 540 1302 41 431 10.406/27/91 41 42015 30215 29005 20195 8405 8015 8870 455 1489 476 1387 42 450 7.807/01/91 45 36800 26650 24140 17440 6930 7490 6985 520 1825 641 1159 50 388 8.607/04/91 48 37675 27435 24855 18155 7405 6760 8365 488 1569 515 1245 60 402 9.6MEAN 38830 28096 26001 18597 7579 7422 8089 487 1644 552 1263 50 414 9.1STD 3061 2262 2573 1800 570 454 931 35 152 61 91 8 38 1.1TABLE C55. EFFLUENT II CHARACTERISTICS FROM RUN 4BDATE DAY P AR AMETERS (mg/L)TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO405/23/91 6 2268 1716 150 114 162 46 49 412 1653 534 74 48 9.4 7.405/27/91 10 2656 1984 168 134 160 51 56 457 1832 614 62 36 9.8 7.405/30/91 13 2846 2248 196 160 194 57 67 503 1506 487 80 49 11.0 8.206/03/91 17 2968 2256 164 128 204 44 55 514 1794 566 93 60 10.5 8.206/06/91 20 3134 2410 222 186 189 67 76 454 1548 524 82 51 13.1 9.906/10/91 24 2340 1806 144 106 153 38 48 530 1461 501 75 50 12.3 10.406/13/91 27 2914 2264 172 134 210 43 57 537 1568 500 93 59 10.2 7.806/17/91 31 3240 2452 200 154 217 58 64 464 1627 566 93 59 9.8 7.006/20/91 34 2664 1938 142 108 157 39 50 541 1508 490 74 48 13.0 11.106/24/91 38 2996 2274 148 120 175 54 49 464 1586 541 70 42 11.7 9.606/27/91 41 3130 2432 204 162 220 58 72 443 1382 469 82 47 10.9 8.007/01/91 45 2602 1888 146 110 158 41 47 532 1798 603 77 52 9.5 7.407/04/91 48 2714 2086 162 134 169 53 57 498 1644 524 89 62 11.0 8.6MEAN 2806 2135 171 135 182 50 57 488 1608 532 80 51 10.9 8.5STD 288 238 26 24 24 8 9 40 131 43 9 7 1.2 1.3APPENDIX DVFA DISTRIBUTIONTABLE^ PAGEDl. Influent VFA Distribution^ 201D2. Reactor I (CMR) VFA Distribution^ 202D3. Effluent I (CMR) VFA Distribution 203D4. Reactor II (UASB) VFA Distribution 204D5. Effluent II (UASB) VFA Distribution^ 205200APPENDICES^201TABLE D1. INFLUENT VFA DISTRIBUTIONRUN PARAMETERS(mg/L)Acetic Propionic Butyric 1w-butyric Valeric 3-methyl-butyric2-methyl-butyricTotal VFAs(as FiAc)1AMEAN 52 44 12 0.4 02 1.0 1.1 98STD 11 11 4 221BMEAN 57 50 15 1.3 1.4 0.7 0.9 111STD 8 10 4 181CMEAN 53 46 19 1.3 1.6 0.9 1.3 106SID 13 12 6 271DMEAN 58 45 16 0.9 1.6 1.6 0.7 108STD 9 9 4 182AMEAN 62 52 18 1.3 1.5 1.6 0.9 119STD 9 10 5 172BMEAN 45 34 10 0.7 0.8 1.3 0.5 81STD 10 8 4 192CMEAN 58 45 14 0.7 1.3 1.5 1.0 108STD 9 8 4 123AMEAN 51 41 18 02 1.4 0.6 0.1 97STD 8 8 3 1635MEAN 22 20 7 0.5 0.3 0.1 0.1 44STD 6 6 2 114AMEAN 62 44 18 1.9 2.2 1.9 1.2 114STD 7 7 2 1548MEAN 51 41 20 2.1 1.9 1.2 0.7 101STD 9 8 4 21APPENDICES^202TABLE D2. REACTOR I (CMR) WA DISTRIBUTIONRUN PARAMETERS (mg/L)Acetic Propionic Butyric Iso-butyric Valerie 3-methyl-butyric2-methyl-butyricTotal VFAs(as HAc)1AMEAN 298 206 45 19 26 18 9 540STD 49 28 8 3 4 4 3 721BMEAN 206 174 36 16 18 14 9 407STD 56 36 9 4 4 4 3 841CMEAN 347 218 72 23 32 25 13 632STD 40 32 8 5 6 5 4 681DMEAN 318 177 48 26 33 20 9 550SID 30 21 7 4 5 4 2 522AMEAN 357 245 66 31 56 41 20 692STD 39 33 7 5 8 8 5 562BMEAN 351 220 56 42 63 48 21 674STD 38 24 6 6 7 7 3 712CMEAN 140 87 19 9 11 7 4 243STD 40 14 3 2 3 1 1 533AMEAN 327 212 65 20 34 21 11 596STD 14 29 8 3 4 3 2 463BMEAN 259 202 50 26 25 8 5 499STD 36 21 8 5 5 1 2 594AMEAN 300 251 37 26 25 15 8 576STD 26 25 7 5 5 3 2 55413MEAN 234 111 113 25 26 20 10 452STD 19 16 15 4 4 4 2 34APPENDICES^ 203TABLE D3. EFFLUENT I (CMR) VFA DISTRIBUTIONRUN PARAMETERS(mg/L)Acetic Propionic Butyric Iso-butyric Valeric 3-methyl-butyric2-methyl-butyricTotal VFAs(as HAc)1AMEAN 287 205 48 a) 24 18 7 530STD 44 33 8 3 4 3 2 731BMEAN 214 173 34 15 19 12 10 412STD 47 32 8 3 3 3 3 761CMEAN 341 226 69 24 35 22 13 630STD 29 37 8 5 6 3 3 641DMEAN 317 185 50 26 35 19 13 560STD 30 22 8 4 5 4 2 642AMEAN 346 246 63 33 59 40 20 683STD 41 28 11 6 8 8 4 662BMEAN 355 227 57 44 63 49 20 687STD 32 29 7 6 7 8 3 652CMEAN 132 86 20 9 12 7 5 236STD 30 14 5 2 2 2 1 473AMEAN 321 202 65 20 31 19 12 580STD 20 23 7 5 5 3 2 5038MEAN 262 202 54 26 22 11 5 504STD 29 31 8 5 5 2 1 624AMEAN 309 255 45 22 21 14 8 587STD 26 26 8 3 3 3 1 581 413MEAN 231 109 117 24 25 20 11 449STD 20 14 19 5 5 4 2 36APPENDICES^204TABLE D4. REACTOR fl (UASB) WA DISTRIBUTIONRUN PARAMETERS(mgIL)Acetic Propionic Butyric Iso-butyric Valeric 3-methyl-butyric2-methyl-butyricTotal VFAs(as HAc)1AMEAN 318 229 50 19 44 28 15 603STD 34 35 8 4 8 6 4 6818MEAN 248 185 43 16 21 15 11 466STD 40 30 6 3 4 3 3 691CMEAN 366 242 83 23 44 24 14 685STD 29 30 8 3 7 4 3 541DMEAN 312 237 49 33 41 28 14 610STD 34 28 7 6 6 5 2 702AMEAN 394 246 63 32 70 47 19 741STD 49 33 7 5 11 7 4 9026MEAN 379 225 60 48 68 44 26 718STD 26 26 7 7 10 7 5 692CMEAN 159 106 25 10 15 9 7 288STD 19 17 6 3 3 2 2 383AMEAN 328 231 69 16 40 27 13 623STD 22 23 8 3 5 5 2 5138MEAN 299 212 56 30 24 17 5 557STD 24 24 8 5 5 2 1 554AMEAN 339 311 49 26 33 24 10 683STD 29 27 7 5 7 3 2 584BMEAN 247 130 116 22 30 25 12 487STD 17 19 18 5 5 4 2 36APPENDICES^205TABLE D5. EFFLUENT H (UASB) VFA DISTRIBUTIONRUN PARAMETERS(mg/L)Acetic Propionic Butyric Iso-butyric Valeric 3-methyl-butyric2-methyl-butyricTotal VFAs(as HAc)1AMEAN 242 180 42 12 38 22 7 465STD 26 31 8 3 7 5 2 571BMEAN 199 144 33 14 17 11 9 370STD 31 25 7 3 3 2 2 551c 'MEAN 362 233 76 24 40 21 15 665STD 30 31 9 5 7 5 3 531DMEAN 324 215 55 30 40 29 15 608STD 38 28 7 , 6 6 6 3 752AMEAN 392 236 60 31 72 43 19 725STD 41 31 7 5 10 7 2 7833MEAN 373 219 60 50 70 46 23 709STD 28 29 6 7 9 7 4 672CMEAN 150 98 23 9 13 8 6 269STD 18 16 6 3 2 2 2 403AMEAN 326 233 76 19 42 25 15 630STD 30 21 9 3 6 5 3 563BMEAN 283 208 56 30 29 17 6 542STD 30 30 9 6 6 3 2 544AMEAN 350 294 49 26 30 19 12 677STD 33 24 6 5 6 4 2 5848MEAN 254 121 114 25 29 25 13 488STD 12 15 19 6 5 5 4 42APPENDIX EVARIOUS EXPERIMENTAL RESULTS AND CONVERSION FACTORSTABLE^ PAGEEl. Seed Characteristics ^ 207E2. Soluble Carbohydrates 207E3. Soluble Proteins 207E4. Gas Production (Mean Values) ^ 208E5. Conversion Factors^ 208E6. Mass Balance Calculation Example (Run 1B, CMR System)^ 208206207APPENDICESTABLE El. SEED CHARACTERISTICSPARAMETERS MEAN VALUES(mg/L)STD(mg/L)pH 6.2 0.3TS 6800 608VS 5030 612TSS 5890 709VSS 4850 556COD (soluble) 830 109TOC 240 36VFAs (as HAc) 35 8NH3-N 28 4TKN (soluble) 44 6TABLE E2. SOLUBLE CARBOHYDRATESINFLUENT EFFLUENTRUN Reactor I (CMR) Reactor II (UASB)Mean^Percent Mean^STD Mean^STD(mg/L)^of Total (mg/L)^(mg/L) (mg/L)^(mg/L)lA 125^7.2 52^14 35^71B 96 5.7 78 19 68 111C 135^8.0 47^9 36^81D 131 7.1 53 13 29 42A 116^6.6 44^11 27^52B 112 7.3 50 8 30 52C 113^6.4 78^14 28^63A 107 6.7 66 12 29 43B 199^9.5 64^7 37^44A 130 7.4 69 10 33 64B 135^8.0 118^16 39^6MEAN 127 7.3TABLE E3. SOLUBLE PROTEINSINFLUENT EFFLUENTRUN Reactor I (CMR) Reactor II (UASB)Mean^Percent Mean^STD Mean^STD(mg/L)^of Total (mg/L)^(mg/L) (mg/L)^(mg/L)lA 116^18.3 83^11 68^81B 89 14.2 89 13 76 101C 108^16.9 66^6 47^71D 87 12.8 71 9 45 52A 109^17.2 47^6 31^42B 82 13.8 24 4 21 32C 92^15.7 63^8 54^83A 107 17.0 69 9 46 43B 107^19.1 56^5 52^74A 94 14.8 67 8 37 44B 103^17.1 70^7 60^8MEAN 99^16.1A. CONVERSION FACTORS FOR VFAsPARAMETER Mol. Weight mgVFA/mgHAcACETIC 80.05 1.000PROPIONIC 74.08 0.817BUTYRIC 88.10 0.682VALERIC 102.13 0.588APPENDICES^ 208TABLE E4. GAS PRODUCTION (MEAN VALUES)RUNSRT(days)HRT(hr)Reactor I Reactor IIMean(mUd)STD(mt./4Mean(mUd)STD(mUd)1A 10 9 26 5 24 61B 10 6 33 7 31 71C 10 12 24 4 34 61D 10 15 80 10 92 112A 15 12 47 5 39 52B 20 12 45 7 52 92C 5 12 21 3 25 53A 10 12 33 5 37 638 10 12 38 5 49 74A 10 12 21 4 31 548 10 12 25 5 28 4TABLE E5. CONVERSION FACTORSB. MISCELLANEOUS CONVERSION FACTORSPARAMETER CONVERSION FACTOR BASISACETIC ACID COD 1.067 mg/mg Acid Acetic AcidPROPIONIC ACID COD 1.514 mg/mg Acid Propionic AcidBUTYRIC ACID COD 1.818 mg/mg Acid Butyric AcidVALERIC ACID COD 2.039 mg/mg Acid Veloric AcidNITROGENOUS COD 9.58 mg/mg Org. N (C4 H6.1 012 N)xor 1.533 mg/mg Protein Org.N=16% ProteinCARBOHYDRATE COD 1.067 mg/mg GlucoseFORMIC ACID COD 0.348 mg/mg Formic AcidETHANOL COD 2.087 mglmg EthanolLACTIC ACID COD 1.066 mg/mg Lactic Acid TABLE E6. MASS BALANCE CALCULATION EXAMPLE (RUN 113. CMR SYSTEM)A. MONING AVERAGE MASS BALANCEDAY Int. Flow Waste Et Flow Inf. VSS Waste VSS Elf. VSS Rate In Rate Out Reduction(Loll (110 UM 01044 Ong/14 (n0114 (aid) OA^CM7 11.97 0.28 11.69 2710 13640 764 32.44 12.7510 11.07 0.28 10.79 2630 16445 920 29.11 14.5314 11.33 0.28 11.05 2650 18825 1420 30.02 20.96^47.317 12.26 0.28 11.98 2970 16270 1308 36.41 20.23 41.721 11.46 0.28 11.18 2500 19355 1396 28.65 21.03^34.624 11.50 0.28 11.22 2905 18365 960 33.41 15.91 41.927 11.94 0.28 11.66 2890 15810 850 34.51 14.34^46.930 11.95 0.28 11.67 2415 16140 1032 28.86 16.56 51.634 12.11 0.28 11.83 2550 20375 1222 30.88 20.16^45.837 11.63 0.28 11.35 2755 19370 958 32.04 16.30 42.241 11.72 0.28 11.44 2435 16650 1000 28.54 16.10^42.544 11.68 0.28 11.40 2405 19215 1184 28.09 18.88 42.2B. OVERALL MASS BALANCEMEAN VALI^11.73^028^11.451^2651^17568^10851^31.08^17.34^442MOVING AVERAGE MASS BALANCE: S VSS REDUCTION = 43.7OVERALL MASS BALANCE: % VSS REDUCTION = 44.2"@en . "Thesis/Dissertation"@en . "1993-05"@en . "10.14288/1.0050447"@en . "eng"@en . "Civil Engineering"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "The effect of operational and environmental parameters on the acid-phase anaerobic digestion of primary sludge"@en . "Text"@en . "http://hdl.handle.net/2429/2049"@en .