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The effect of operational and environmental parameters on the acid-phase anaerobic digestion of primary.. 1993

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e required stan THE EFFECT OF OPERATIONAL AND ENVIRONMENTAL PARAMETERS ON THE ACID-PHASE ANAEROBIC DIGESTION OF PRIMARY SLUDGE by PANAGIOTIS ELEFSINIOTIS Dipl. Eng. (Civil Engineering), National Technical Univ. of Athens, Greece, 1979 M.A.Sc. (Environmental Engineering), The University of Toronto, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Civil Engineering) We accept this thesis as conforming THE UNIVIV SITY BRITISH COLUMBIA January 1993 Panagiotis Elefsiniotis In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. (Signature) Department of  Civil Engineering The University of British Columbia Vancouver, Canada Date ^January 25, 1993 DE-6 (2/88) ABSTRACT 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 °C. 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 ii ABSTRACT ^ iii 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 ABSTRACT^ iv utilization of all three substrates increased with an increase in HRT, but (with the exception 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. TABLE OF CONTENTS PAGE ABSTRACT^ ii TABLE OF CONTENTS^ v LIST OF TABLES viii LIST OF FIGURES^ x GLOSSARY OF TERMS xii ACKNOWLEDGEMENTS^ xiii 1. INTRODUCTION 1 2. LITERATURE REVIEW^ 3 2.1. AN OVERVIEW OF ANAEROBIC DIGESTION^  3 2.2. WASTEWATER COMPOSITION^ 6 2.2.1. Carbohydrates ^  7 2.2.2. Proteins  10 2.2.3. Lipids ^  11 2.3. PATHWAYS OF VFA FORMATION^ 12 2.3.1. Carbohydrate Metabolism  13 a) Hydrolysis ^  13 b) Fermentation of Sugars^  14 2.3.2. Protein Metabolism  17 a) Hydrolysis ^  17 b) Amino Acid Fermentation^  18 2.3.3. Lipid Metabolism  20 a) Hydrolysis ^  20 b) Anaerobic Degradation of Fatty Acids^ 21 2.4. OTHER PATHWAYS OF ANAEROBIC METABOLISM^ 22 2.4.1. Products Formed by Enterobacteria  23 2.4.2. Products Formed by Lactic Acid Bacteria  24 2.5. FACTORS AFFECTING VFA PRODUCTION^ 25 2.6. APPLICATIONS OF THE ACID-PHASE DIGESTION^ 27 2.6.1. The Biological Phosphorus Removal Process 28 2.7. PROCESS CONFIGURATION^ 30 3. RESEARCH OBJECTIVES^ 32 v TABLE OF CONTENTS^ vi PAGE 4. EXPERIMENTAL METHODS AND ANALYTICAL PROCEDURES^ 34 4.1. WASTEWATER SOURCE^ 34 4.2. EXPERIMENTAL SET-UP AND OPERATION^ 35 4.2.1. General System Configuration^  35 4.2.2. Operation ^  37 4.3. ANALYTICAL PROCEDURES 41 4.3.1. Chemical Oxygen Demand (COD)^ 43 4.3.2. Total Organic Carbon (TOC) 43 4.3.3. Organic Acids^ 43 a) Volatile Fatty Acids (VFAs)^ 43 b) Lactic and Pyruvic Acids 44 c) Formic Acid^ 45 4.3.4. Solids^ 45 a) Total Solids (TS) and Volatile Solids (VS) ^ 45 b) Total Suspended Solids (TSS) and Volatile Suspended Solids (TSS) ) ^  45 4.3.5. Nitrogen^ 46 a) Ammonia Nitrogen (NH 3 -N)^ 46 b) Total Kjeldahl Nitrogen (TKN) 47 4.3.6. Proteins ^ 47 4.3.7. Lipids  48 a) Dry Extraction Method^ 48 b) Wet Extraction Method 48 4.3.8. Carbohydrates ^  49 4.3.9. Phosphorus 50 a) Orthophosphate (PO43) ^  50 b) Total Phosphorus  50 4.3.10. pH and Alkalinity^ 50 4.3.11. Other Soluble Organics 51 4.3.12. Gas Analysis 51 4.4. COLD STORAGE TESTING^ 52 4.5. STATISTICS^ 52 5. RESULTS AND DISCUSSION^ 53 5.1. GENERAL CHARACTERISTICS 53 5.1.1. Feed Composition ^  53 5.1.2. Cold Storage Experiments^ 56 5.1.3. Acclimation and Stability of Operation ^  57 5.2. THE EFFECT OF HRT - STAGE 1 59 5.2.1. HRT as a Control Parameter^ 59 5.2.2. VFA Production^ 61 5.2.3. VFA Speciation  65 TABLE OF CONTENTS^ vii PAGE 5.2.4. Particulate Organic Carbon Solubilization^ 69 5.2.5. Substrate Degradation^  74 5.3. THE EFFECT OF SRT - STAGE 2 78 5.3.1. SRT as a Control Parameter 78 5.3.2. VFA Production and Speciation ^  79 5.3.3. Organic Carbon Solubilization and Substrate Degradation^ 84 5.3.4. Gas Production ^  89 5.4. REPLICATION AND THE EFFECT OF FEED SOURCE - STAGE 3^ 91 5.4.1. Replication Experiments (Run 3A)^ 91 5.4.2. The Effect of Feed Source (Run 3B) 93 5.5. THE EFFECT OF pH - STAGE 4^ 97 5.5.1. pH as a Selective Parameter 97 5.5.2. Buffering Capacity 98 5.5.3. VFA Production and Speciation^  100 5.5.4. Organic Carbon Solubilization and Substrate Degradation^ 106 5.6. GENERAL REVIEW^ 111 5.6.1. VFA Formation  111 5.6.2. Formation of Other Soluble End-Products ^  112 5.6.3. Rate-Controlling Step and Nature of Soluble Compounds ^ 116 5.6.4. Mass Balances: Solids and Phosphorus  120 5.6.5. Substrate Utilization Patterns^  122 5.6.6. Potential Application of Findings  125 6. CONCLUSIONS AND RECOMMENDATIONS^  127 6.1. CONCLUSIONS^ 127 6.2. RECOMMENDATIONS 130 REFERENCES^ 131 APPENDIX A - BIOCHEMICAL PATHWAYS^ 146 APPENDIX B - REACTOR OPERATION (HRT AND pH VALUES)^ 151 APPENDIX C - CHEMICAL PARAMETERS 163 APPENDIX D - VFA DISTRIBUTION^ 200 APPENDIX E - VARIOUS EXPERIMENTAL RESULTS AND CONVERSION FACTORS 206 LIST OF TABLES TABLE^ PAGE 2.1. Organic Composition of Primary Sludge ^  7 4.1. Operating Conditions (Mean Values) 39 4.2. Duration of Experimental Runs and Amount of Biomass (VSS) in the Reactors^ 42 5.1. Influent Sludge Characteristics (Iona Island WWTP)^ 54 5.2. Influent Sludge Characteristics (Lions' Gate WWTP) 55 5.3. Organic Composition of Feed^ 56 5.4. Cold Storage Testing^ 57 5.5. VFA Specific Production Rate as Function of HRT^ 63 5.6. Comparison of Reactor and Effluent VFA Concentrations ^ 65 5.7. Percent VFA Distribution as a Function of HRT^ 67 5.8. Specific Solubilization Rates of COD and TOC as a Function of HRT ^ 70 5.9. Percent VSS and TSS Reduction as a Function of HRT^ 73 5.10. Percent Substrate Degradation as a Function of HRT 76 5.11. VFA Specific Production Rate as a Function of SRT^ 80 5.12. Percent VFA Distribution as a Function of SRT  83 5.13. Specific Solubilization Rates of COD and TOC as a Function of SRT ^ 86 5.14. Percent VSS and TSS Reduction as a Function of SRT^ 86 5.15. Percent Substrate Degradation as a Function of SRT  87 5.16. Comparison of Replication Results at Iona WWRP^ 92 5.17. Percent VFA Distribution (Stage 3) ^ 92 viii LIST OF TABLES^ ix TABLE^ PAGE 5.18. t-Test Results for Runs 1C, 3A (Iona Island WWTP) and Run 3B (Lions' Gate WWTP)^ 93 5.19. Comparison of Results from Different Feed Sources ^  95 5.20. Percent Distribution of C 5 Branched VFAs and Iso-butyric Acid (Stage 3) ^  96 5.21. pH Values in Bioreactors (Stages 1 to 3) ^  99 5.22. VFA Specific Production Rate as a Function of pH^  103 5.23. Percent VFA Distribution as a Function of pH  104 5.24. Specific Solubilization Rates of COD and TOC as a Function of pH^ 106 5.25. Percent Soluble COD in the Form of VFAs as a Function of pH^ 107 5.26. Percent VSS and TSS Reduction as a Function of pH^ 108 5.27. Protein Degradation and its End-Products as a Function of pH^ 110 5.28. Summary of Percent VFA Distribution^  113 5.29. Other Soluble End-Products (Mean Values)  113 5.30. Operational Parameters and Percent Soluble COD in the Form of VFAs from Various Acid-Phase Anaerobic Digestion Studies ^ 119 5.31. Percent Recovery Based on Mass Balance ^  121 LIST OF FIGURES FIGURE^ PAGE 2.1. Pathways of Anaerobic Metabolism ^  5 4.1. Experimental Layout^ 36 5.1. VFA Profile (Runs 1C & 1D)^ 62 5.2. Net VFA Production as a Function of HRT^ 62 5.3. Percent VFA Distribution (Stage 1)  68 5.4. Soluble COD Profile (Runs 1C & 1D)^ 71 5.5. Net COD Solubilization as a Function of HRT^ 71 5.6. Percent Soluble COD in the Form of VFAs (Stage 1)  75 5.7. Carbohydrate Degradation as a Function of HRT^ 75 5.8. Protein Degradation as a Function of HRT 77 5.9. Lipid Degradation as a Function of HRT^ 77 5.10. Net VFA Production as a Function of SRT 81 5.11. Percent VFA Speciation as a Function of SRT^  81 5.12. Net COD Solubilization as a Function of SRT    85 5.13. Percent Soluble COD in the Form of VFAs (Stage 2) ^ 85 5.14. Carbohydrate Degradation as a Function of SRT  88 5.15. Protein Degradation as a Function of SRT^ 88 5.16. Lipid Degradation as a Function of SRT 89 5.17. Reactor pH and VFA Concentration (CMR System) ^  101 5.18. Reactor pH and VFA Concentration (UASB System)  101 x LIST OF FIGURES^ xi FIGURE^ PAGE 5.19. VFA Concentration as a Function of pH^  102 5.20. Percent Substrate Degradation as a Function of pH in the CMR System^  109 5.21. Percent Substrate Degradation as a Function of pH in the UASB System  109 GLOSSARY OF TERMS ATP:^adenosine-5' -triphosphate CH 2O: carbohydrates CMR:^completely mixed reactor CoA: coenzyme A COD:^chemical oxygen demand CoV: coefficient of variation EMP:^Embden-Meyerhof-Parnas [glycolytic pathway] HAc: acetic acid HRT:^hydraulic retention time NAD(H 2):^nicotinamide adenine nucleotide (reduced) NAD(P): nicotinamide adenine nucleotide (phosphate) NH3 -N:^ammonia nitrogen ORP: oxidation-reduction potential PHB:^poly-13-hydroxybutyrate PHV: poly-I3-hydroxyvalerate PO4 -3 :^orthophosphate STD: standard deviation SRT: solids retention time TKN:^total Kjeldahl nitrogen TOC: total organic carbon TP: total phosphorus TS:^total solids TSS: total suspended solids UASB:^upfow anaerobic sludge blanket VFAs: volatile fatty acids VS: volatile solids VSS:^volatile suspended solids WWTP: wastewater treatment plant ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my supervisor, Dr. William K. Oldham, for his guidance, knowledge, insight, enthusiasm, and continuous encouragement 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 constructive criticisms and suggestions in the preparation of the final report. I am also grateful to Susan Harper, Paula Parkinson, Timothy Ma, Zufang Zhou, and Romy So for their invaluable analytical assistance, moral support and co- operation which created a pleasant working atmosphere in the environmental engineering laboratory; to Guy Kirsch for his skilled manufacturing of the experimental 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 sharing their knowledge and contributing to many stimulating discussions concerning the nature of this research. I would also like to thank my many fellow graduate students, among others, David Wareham, Prayoon Fongsatitkul, Patrick Coleman and Ramanathan Manoharan for their comradeship, advice, and encouragement; and Christodoulos Labridis and Athanasios Loukas for their companionship and support. Financial support during this study was provided by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC). CHAPTER 1 INTRODUCTION Throughout recorded history humankind has continually struggled to manage the natural environment in order to improve its well-being. The ability to control many aspects of the environment is the main characteristic that has set people apart from other species on the planet. In the last half of this century, however, environmental quality problems have surfaced at an accelerated pace. The population explosion, greater energy use, increased food production needs, changes in life-style, and numerous technological developments have created many strains on parts of the global ecosystem. As a result, an increased concern for the environment is now being witnessed in many parts of the world. Liquid waste management has long been recognized as a necessary action in order to improve environmental quality. Collection, treatment, disposal, and reuse of the wastewater generated in urban or rural areas has become a priority, wherever the social and economic conditions permit. Anaerobic processes have always been an integral part of the wastewater treatment scheme. In the septic tank, one of the oldest and widest applications in municipal 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 anaerobic 1 CHAPTER I. INTRODUCTION 2 processes suffered largely in the 1950s and 1960s, because aerobic and physical- chemical methods became attractive alternatives with their advantage in operation and impurity removal efficiency. The energy crisis in the early 1970s and the rapid growth of biotechnology have rekindled the interest in anaerobic processes. Today the use of anaerobic microorganisms covers a wide array of applications ranging from the wastewater treatment field to the production of food, medicines and industrial chemicals, to genetic engineering and enzyme technology. In the wastewater treatment realm, anaerobic digestion has been traditionally employed to prepare municipal sludges for ultimate disposal and provide a source of energy. Accordingly, most of the research has been focused on the methane generating phase of the process. Little attention has been paid to the acid-phase digestion, the phase in which complex organic substances, such as carbohydrates, proteins and lipids, are converted anaerobically to volatile fatty acids (VFAs) and other low molecular weight soluble carbon compounds. Improved knowledge of the acid-phase digestion can be useful in a variety of situations, ranging from the operation of the overall digestion process itself, to its effect on subsequent treatment processes. A better understanding of digester dynamics during shock loading or digester operational stability can be obtained by exploring the acidogenic step. In addition, since the main products of this phase are soluble 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 2 LITERATURE REVIEW 2.1. AN OVERVIEW OF ANAEROBIC DIGESTION Anaerobic digestion is a response to controlled conditions of a series of reactions which occur in many circumstances in nature. It is a biological process in which organic matter is ultimately converted to methane and carbon dioxide in the absence of molecular oxygen. The overall process entails direct and indirect symbiotic associations between several distinct groups of microbial populations. A number of investigators have attempted to elucidate the different steps and pathways involved in anaerobic metabolism (Holland et al., 1987). As many as nine recognizable steps, each mediated by a specific group of microorganisms, have been identified (Harper and Pohland, 1986). For the purpose of this research, however, the scheme proposed by Kaspar and Wuhrmann (1978a) seems to be the most appropriate, since it provides a more comprehensive insight on the initial steps involved in the degradation of biopolymers. According to this scheme, the following six 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. 3 CHAPTER 2. LITERATURE REVIEW^ 4 4) 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 methanogenesis is implied by the last three. A summary of the above sequence of reactions is depicted in Figure 2.1. Although bacteria are the main biological agents involved in anaerobic degradation of organic compounds, fermentative ciliate and flagellate protozoa, and several anaerobic fungi may also contribute in some ecosystems (McInerney and Bryant, 1981). Hydrolysis of organic matter is a process accomplished by extracellular enzymes. The reaction rate can be greatly affected by the pH and the operating conditions of the system (Verstraete et al., 1981). Complex carbohydrates such as cellulose and starch are hydrolyzed to simple sugars, proteins to amino acids, and lipids to long-chain fatty acids. Fermentation, in this context, can be defined as a microbial metabolic process in which organic compounds serve both as electron donors and as electron acceptors. Any hydrogen generated during fermentation originates from dehydrogenation of pyruvate. This hydrogen production mechanism is not inhibited by high partial pressures of hydrogen, up to 0.5 atm H2 (Thauer et al., 1977). Sugars and amino acids are the substrates undergoing fermentation and they produce biomass, intermediate degradation products (propionate, butyrate, etc.), and the methane precursors acetate and hydrogen (Figure 2.1). METHANE ACETATE HYDROGEN AMINO ACIDS SUGARS 2 FATTY ACIDS ALCOHOLS CARBOHYDRATES INTERMEDIATE PRODUCTS PROPIONATE, BUTYRATE, VALERATE, ETC. PROTEINS LIPIDS 1 CHAPTER 2. LITERATURE REVIEW^ 5 1) HYDROLYSIS 2) FERMENTATION 3) ANAEROBIC OXIDATION OF FATTY ACIDS 4) ANAEROBIC OXIDATION OF INTERMEDIATE PRODUCTS 5) ACETOCLASTIC METHANOGENESIS 6) 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 6 In anaerobic oxidation, molecular hydrogen serves as the main sink for electrons. The principal pathway of hydrogen formation is via oxidation (transfer of electrons to protons) of reduced pyridine dinucleotides and ferredoxin (Kaspar and Wuhrmann, 1978a). It has been demonstrated that the degradation of long-chain fatty acids under anaerobic conditions occurs by a mechanism called 13-oxidation (Jeris and McCarty, 1965). 2.2. WASTEWATER COMPOSITION Composition, in general, refers to the actual amount of physical, chemical and biological constituents in wastewater. Since the organic chemical constituents are of paramount importance in anaerobic digestion, the following discussion will revolve around these compounds. Municipal wastewater is a principal source of organic matter entering the aquatic environment. The suspended impurities in liquid wastes from residential units, hotels, hospitals, restaurants, offices and commercial buildings are on average 70 percent organic in nature (Fair et al., 1971). Settling of wastewater, through primary sedimentation, provides raw or primary sludge, which is often used as a feed to anaerobic digesters. Primary sludge contains a great number of organic compounds, however, most of them can be grouped in three major classes: carbohydrates, proteins and lipids. Although many current research efforts have been directed towards the identification of specific organic chemicals in raw sludge (eg. aromatic hydrocarbons, chlorinated compounds etc.), the three classes mentioned here CHAPTER 2. LITERATURE REVIEW^ 7 are the key players in anaerobic processes. The composition of primary sludge, measured by various researchers, is shown in Table 2.1. Variation in composition may not only reflect the differences in individual wastes, but also recent changes in life style, as indicated by the high carbohydrate and low lipid content of the sludge used in this study. TABLE 2.1. ORGANIC COMPOSITION OF PRIMARY SLUDGE REFERENCE % VOLATILE SOLIDS % TS Carbo- hydrates Proteins Lipids Total VS Balmat 30 32 25 87 78 Buswell & Neave 18 32 41 91 61 Heukelekian 17 36 45 98 76 Heukelekian & Balmat 30 31 24 85 65 Higgins et al. 66 12 15 93 75 Hunter & Heukelekian 44 19 18 81 81 Maki 62 29 22 113 65 0' Rourke - 22 23 - 80 AVERAGE 38 27 27 92 73 This study 58 21 17 96 75 2.2.1. CARBOHYDRATES Carbohydrates are the most abundant organic compounds in the biosphere. They can be precisely defined as polyhydroxy aldehydes or ketones with the general CHAPTER 2. LITERATURE REVIEW 8 formula (CH 20)n , where ri3. Depending on the number of carbon atoms included, carbohydrates can be classified as monosaccharides (simple sugars containing 3 to 9 carbon atoms), oligosaccharides (mainly disaccharides with 12 carbon atoms) and polysaccharides (Bailey and 011is, 1977). Polysaccharides are extremely large molecules. The majority of carbohydrates in nature exist as such macromolecules with molecular weights ranging from 25,000 to 15 million. They consist, for the most part, of simple and derived sugars linked together by glycosidic bonds. Polysaccharides are insoluble in water and can form colloidal suspensions (Gaudy and Gaudy, 1980). The most important polysaccharides found 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 from 50,000 to over 1 million. The glycosidic linkage occurs between the 1 and 4 carbons of successive glucose units (13-1,4 bonding). Generally, cellulose is not easily biodegradable, 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 (Hunter and Heukelekian, 1965; Higgins et al., 1982). Hemicellulose is a group of heteropolymers with frequent side chains. The common 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 9 Hemicellulose is the next most significant constituent (after cellulose) of polysaccharides in wastewater, ranging from 20 to 25% (Hunter and Heukelekian, 1965). Pectin comprises a family of complex polysaccharides containing mostly methylated poly-D-galacturonic acid, arabinose and galactose (Conn and Stumpf, 1976). Starch has the general formula (C 6H 1005 ) x • It occurs in two forms: amylose and amylopectin. Amylose is a linear polymer of D-glucose units linked together by a-1,4 bonds. The amylose molecule contains 100 to 1,000 glucose units and it is insoluble in water. Amylopectin is a branched polymer of glucose containing both a-1,4 bonds and a-1,6 bonds which initiate side chains. It is much larger than amylose (500 to 5,000 glucose units), is soluble in water and can form gels by absorbing water (Bailey and 011is, 1977). Both pectin and starch can be found in small amounts in wastewater (less than 10% of the total carbohydrates and lignin). Lignin is a complex polymeric aromatic substance of variable structure making up 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 an important structural role in plants and one of the main processes of the pulp and paper industry is to separate these two components. Lignin is considered to be a refractory compound not amenable to biodegradation. It comprises 5 to 15% of the non-nitrogenous, alcohol-insoluble matter in domestic wastewater (Hunter and Heukelekian, 1965) and in primary sludge (Higgins et al., 1982). CHAPTER 2. LITERATURE REVIEW^ 10 2.2.2. PROTEINS Proteins constitute the most complex organic compounds in the biosphere. They all contain carbon, hydrogen, oxygen and nitrogen. Phosphorus and sulphur are present in a few. Proteins are an essential part of all living matter and a major dietary constituent. They are polymers of a-amino acids joined together by peptide bonds. These covalent bonds arise by elimination of the elements of water from the carboxyl group of one amino acid and the a-amino group of the next. The molecular weight of proteins, depending on the number of the polymers, can vary from a few thousand to several million (Gaudy and Gaudy, 1980). Proteins are divided into two major classes on the basis of their conformation: fibrous and globular. Fibrous proteins are physically tough and are insoluble in water or dilute salt solutions. On the other hand, most globular proteins are soluble in aqueous systems and they usually have a mobile or dynamic function in the cell (Lehninger, 1975). The importance of proteins in anaerobic digestion stems from their significant buffering capacity (due to the presence of hydroxy and amino groups) as well as their ability to serve as carbon and energy sources. The nutritional value of the individual amino acids to the microorganisms is an additional asset (Tsao, 1984). Almost all of the 20 known amino acids have been identified in untreated sewage. Alanine, aspartic acid, glutamic acid, leucine and iso-leucine are the most predominant ones (Heukelekian and Balmat, 1959; Kahn and Wayman, 1964). The former investigators have reported that the amino acids accounted for 65 to 80% of the total nitrogenous matter. According to Hunter and Heukelekian (1965), the amino CHAPTER 2. LITERATURE REVIEW 11 acids averaged 55% of the total organic nitrogen. Painter et al. (1961) and Hanson and Lee (1971) have reported that about 35 to 40% of the total organic nitrogen was in the amino acid form. It should be noted, however, that this diversity in amino acid content in raw sewage may be mainly the result of analytical determinations (particulate vs. soluble forms of nitrogen). Higgins et al. (1982) have found that the amino acid content of the primary sludge averaged about 65% of the total organic nitrogen. 2.2.3. LIPIDS Lipids are organic biomolecules which are soluble in non-polar solvents such as chloroform, benzene or ether, and practically insoluble in water. Consequently, lipids are diverse in their chemical structure and biological function (Conn and Stumpf, 1976). Lipids have been classified in several different ways. The most satisfactory classification, based on their common chemical characteristics, includes simple, compound and non-saponifiable lipids (Gaudy and Gaudy, 1980). Fats, oils and waxes are all simple lipids. Fats and oils are esters of various fatty acids and the trihydroxy alcohol glycerol. Waxes are esters of fatty acids and long-chain monohydroxy alcohols. The most common fatty acids contain 16 or 18 carbon atoms and may be saturated such as palmitic and stearic or unsaturated such as oleic, linoleic, linolenic and palmitoleic (Gurr and James, 1971). Compound lipids are also esters of various fatty acids and alcohols. The addition of phosphorus and nitrogen compounds results in the creation of CHAPTER 2. LITERATURE REVIEW^ 12 phospholipids, and the addition of carbohydrates in that of glycolipids. Non-saponifiable lipids do not contain fatty acids and, hence, do not yield soaps (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). The esterified fatty acids are the main lipid component (50 to 70% of total lipids) in raw sewage, followed by the unsaponifiable matter (15 to 25%). Free fatty acids and phospholipids have been found in small amounts (Hunter and Heukelekian, 1965). In primary sludge, however, free fatty acids may contribute between 40 and 60% of the total lipids, as a result of the rapid hydrolysis of the fatty acid esters to free fatty acids (Heukelekian and Mueller, 1958). Saturated fatty acids represent about 70 to 80% of the total fatty acids identified, esterified or free. Palmitic and stearic acids are the most commonly found saturated acids and oleic acid the predominant unsaturated one (Higgins et al., 1982). 2.3. PATHWAYS OF VFA FORMATION The ability to produce volatile fatty acids under anaerobic conditions is a widespread attribute in the microbial world. A large number of bacterial species is capable of utilizing complex organic substrates such as carbohydrates, proteins and lipids to produce VFAs and other soluble carbon compounds via a variety of anaerobic metabolic pathways. In the following discussion, the metabolic pathways CHAPTER 2. LITERATURE REVIEW^ 13 of the three major organic components of the primary sludge will be reviewed. 2.3.1. CARBOHYDRATE METABOLISM a) Hydrolysis Bacteria are unable to take up particulate polysaccharides, because biopolymers as such cannot penetrate the cell membrane. Therefore, microorganisms excrete enzymes that are capable of degrading the complex biopolymers to small transportable molecules. These enzymes can either be set free by the organisms or remain associated with them. Cellulose can be hydrolyzed by a number of anaerobic bacteria. Bacteroides succinogenes was the first one isolated from rumen (Hungate, 1949). Other common cellulose hydrolyzing organisms include Clostridium lochheadii and Clostridium cellobioparum (Hungate, 1957), Butyrivibrio fibrisolvens (Shane et al., 1969), and Clostridium thermocellum (Gottschalk, 1986). All cellulolytic bacteria excrete the enzyme complex called cellulase. It consists, in general, of three major enzyme components (Gong et al., 1979): 1) endoglucanase which cleaves the (3-1,4 glycosidic bonds in the cellulose molecule, 2) exoglucanase which removes cellobiose (a disaccharide unit of cellulose) from non-reducing ends of the molecule, and 3) cellobiase which hydrolyzes cellobiose or cellotriose to two or three molecules of glucose respectively. CHAPTER 2. LITERATURE REVIEW 14 A detailed description of the mode of action of this exoenzyme complex is provided by Cuskey et al. (1982). Cellulose hydrolysis yields the simple sugar glucose. Hemicellulose can be degraded by anaerobic organisms producing the exoenzyme complex hemicellulase. The complexity of this enzyme system far exceeds that of cellulose, as hemicellulose is composed of a greater variety of monomers linked together by different types of bonds. A comprehensive review of hemicellulase excreted by anaerobic microbes is presented by Dekker and Richards (1976). Pectin hydrolysis involves bacteria found in rumen such as Bacteroides succinogenes, Bacteroides ruminicola, and Butyrivibrio fibrisolvens. Three types of enzymes 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, lyase depolymerizes the chains to form the final products of hydrolysis (Tsao, 1984). Starch, as a storage material, is amenable to biodegradation. Among the many anaerobes 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 types of specific enzymes (Fogerty and Kelly, 1979). b) Fermentation of Sugars Glucose, the main simple sugar generated from polysaccharide hydrolysis, is CHAPTER 2. LITERATURE REVIEW 15 commonly used by fermentative microorganisms as an energy source. Most of the products formed in the fermentation of glucose originate from pyruvic acid which is produced via the glycolytic Embden-Meyerhof-Parnas (EMP) pathway. According to this pathway, two molecules of pyruvic acid are produced per molecule of glucose through a series of reactions (Figure Al, Appendix A). In addition, two molecules of adenosine-5' -triphosphate (ATP) are generated and two molecules of nicotinamide adenine dinucleotide (NAD) are reduced in the process. Since no oxygen is involved, this pathway is common in both aerobic and anaerobic metabolism (Gaudy and Gaudy, 1980). Depending on the anaerobic microbial species present, subsequent pyruvic acid fermentation can lead to the production of different VFAs. Acetic acid is produced in a number of fermentations. There are certain bacteria, 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 ferment 1 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 dioxide to acetate (Figure A2, Appendix A). Clostridium aceticum and Acetobacterium woodii ferment hydrogen and carbon dioxide to acetate. It should be noted that carbon monoxide 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^ 16 Acetic acid is also generated by mixed-acid producers. They metabolize pyruvate 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 many anaerobes of the Propionibacterium genus. P. pentosaceum and P. shermanii can degrade pyruvic acid via the succinate-propionate pathway. Lactyl-CoA and acrylyl- CoA are intermediates, while electron-transferring flavoprotein functions as hydrogen carrier. 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 Clostridium propionicum and Megasphaera elsdenii which produce propionic acid via the acrylate pathway (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 obligate anaerobes which belong to the four genera: Clostridium, Butyrivibrio, Eubacterium, and Fusobacterium. The reactions involved in the production of butyric acid are presented in Figure A4, Appendix A. In this pathway, the conversion of pyruvate to acetyl-CoA is catalyzed by the pyruvate-ferredoxin oxidoreductase enzyme system. Also, butyryl-CoA is not converted to butyric acid by simple hydrolysis, but via the formation of butyryl phosphate, which yields an additional ATP molecule (Gottschalk, 1986). CHAPTER 2. LITERATURE REVIEW^ 17 2.3.2. PROTEIN METABOLISM a) Hydrolysis The bacteria involved in protein metabolism have the ability to produce proteolytic enzymes which break the biopolymers into their monomeric components (i.e. amino acids) before they can enter the cell membrane and be used either as building blocks or as fermentative substrates. Protein hydrolysis progresses in steps in reverse manner to those in which proteins 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 genus Clostridium (Siebert and Torein, 1969; Hobson and Shaw, 1971). Bacteroides ruminicola has also been found to exhibit similar activity (Hobson and Shaw, 1974). Proteolytic enzymes are divided into two groups, according to their mode of action on a polypeptide chain: endopeptidases and exopeptidases. Pepsin, a typical endopeptidase, has very broad specificity, but preferentially attacks polypeptide chains along their length, whenever residues of aromatic amino acids occur. On the contrary, exopeptidases can only split terminal peptide bonds. They are subdivided into: aminopeptidases, which require a free terminal amino group and are dependent on metal ions for their activity; and carboxypeptidases, which break down peptides with a free terminal carboxy group (Lehninger, 1975). The synergistic action CHAPTER 2. LITERATURE REVIEW^ 18 of both types of enzymes results in the formation of free a-amino acids. b) Amino Acid Fermentation Several single amino acids can serve as energy and carbon sources for strict or facultative anaerobes. Organisms possessing the enzyme dehydrogenase can convert aliphatic amino acids (containing the alkyl group, R) to the corresponding VFAs via reductive deamination. Hydrogen ions act as the hydrogen donor. A general reaction can be written as follows (Doelle, 1975): R-CHNH2COOH + 2H+ ----, R-CH2 COOH + NH3^(2.6) Many microorganisms can specifically ferment individual amino acids to produce VFAs. A few representative cases are outlined below. Clostridium propionicum employs the acrylate pathway (Figure A3, Appendix A) to convert alanine, via lactic acid, to a mixture of propionic and acetic acid (Gottschalk, 1986). Glycine is a preferred substrate for Clostridium histoliticum and Diplococcus glycinophilus, 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 of glutamic acid by obligate anaerobes. Clostridium tetanomorphum employs the methylaspartate pathway, which is rather unusual and is used only by the Clostridium species, for the formation of an acetic and butyric acid mixture with a 3:1 ratio CHAPTER 2. LITERATURE REVIEW 19 (Gottschalk, 1986). The same acids can be also produced (at a 2:1 ratio) via the hydroxyglutarate pathway followed by Acidaminococcus fermentans, Clostridium microsporum and other species (Buckel and Barker, 1974). Not all amino acids can be fermented singly, or at least no organism capable of utilizing certain amino acids has been isolated. The Stickland reaction is an oxidation-reduction reaction between pairs of amino acids. One amino acid acting as the hydrogen donor is oxidized, and a second one acting as the hydrogen acceptor is reduced. This allows the amino acids that cannot be fermented individually to be used as an energy source. This reaction is curried out by many proteolytic clostridia such as C. stickandii, C. sporogenes, and C. histoliticum (Barnard and Akhtar, 1979; Barker, 1981). The coupling of valine (hydrogen donor) and glycine (hydrogen acceptor), for example, results in the formation of iso-butyric acid and acetic acid. It was first demonstrated by Cohen- Bazire et al. (1948): CH3 CHCH 3 CHNH2COOH + 2CH 2NH2COOH + 2H20 CH3 CHCH3 COOH + 2CH3 COOH + 3NH3 + CO2^(2.8) In a similar fashion, iso-leucine or leucine, acting as hydrogen donors can be oxidized to the isomers of the valeric acid. In the case of iso-leucine the predominant isomer is 2-methylbutyric acid, and in that of leucine 3-methylbutyric acid (Elsden and Hilton, 1978). CHAPTER 2. LITERATURE REVIEW^ 20 2.3.3. LIPID METABOLISM a) Hydrolysis The hydrolysis of ester linkages in lipids requires the presence of lipolytic enzymes. Since the environment in which hydrolysis takes place involves a lipid- water interface, this is a heterogeneous enzymatic catalysis. Two common types of lipolytic enzymes include lipases and phospholipases. Lipases catalyze the stepwise and partially reversible hydrolysis of fatty acid ester bonds in simple lipids (triglycerides), with the intermediate formation of di- and monoglycerides and the ultimate release of 3 mol of the corresponding fatty acid and 1 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 the triglyceride), and fatty acid specific (Macrae, 1984). Lipases are widespread in nature. Under anaerobic conditions, they are excreted by microorganisms belonging mainly to the genera Bacillus, Chromobacterium, and Serratia (Finnerty, 1988). Phospholipases are involved in the hydrolysis of phospholipids. Four types of phospholipases are known and are classified according to the ester bond which they hydrolyze (Waite, 1987). Phospholipid metabolism results in the production of CHAPTER 2. LITERATURE REVIEW^ 21 the corresponding fatty acids and a variety of other organic compounds, depending upon the substrate utilized. Phospholipases have been found in many spore-forming anaerobic bacteria such as Clostridium perfrigens and Bacillus cereus (Low, 1981). b) Anaerobic Degradation of Fatty Acids The anaerobic metabolism of long chain fatty acids occurs via a mechanism called (3-oxidation, because the beta carbon (second from the carboxyl carbon) is oxidized. This pathway involves repetition of a sequence of reactions that results in the removal of two carbon atoms as acetyl-CoA with each repetition (Gaudy and Gaudy, 1980). The first step in the (3-oxidation is its activation by one of several enzymes called acyl-CoA synthetases. Since four hydrogen atoms are generated per molecule of acetyl-CoA, the overall reaction can only proceed if much of the hydrogen can be converted to H2 gas. Many anaerobic bacteria form the enzyme hydrogenase, which catalyzes the reversible reaction of hydrogen production from a reduced high-energy electron carrier such as reduced pyridine dinucleotides and ferredoxin (Benemann and Valentine, 1971): 2H+ + 2e - ----, H2^(2.10) The stoichiometry of the (3-oxidation reaction including oxidation of NAD(P)H or ferredoxin is as follows (Jeris and McCarty, 1965; Gujer and Zehnder, 1983): (-CH2 CH2-) + 2H20^CH3COOH + 2H 2^(2.11) CHAPTER 2. LITERATURE REVIEW 22 The 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 pyruvate dehydrogenation (-0.68 V at pH 7) (Wolin, 1976). Based on thermodynamic considerations, partial pressures of H2 gas higher than 0.5 atm may slow down the oxidation 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 various saturated fatty acids except for the activation step. The enzyme catalyzing the activation of fatty acids falls into three distinct categories depending on chain length. There is evidence that unsaturated fatty acids are first hydrogenated and then degraded by the same mechanism (Novak and Carlson, 1970; Hobson et al, 1974). Acetyl-CoA, the main intermediate of (3-oxidation can be converted to either acetic or butyric acid (Harper and Pohland, 1986). Propionic acid may also be formed as an end-product of the metabolism of fatty acids that contain odd numbers of carbon atoms. A three-carbon residue, propionyl-CoA, remains after the removal of the other carbons as acetyl-CoA, and is converted to propionic acid under anaerobic conditions (McInerney and Bryant, 1981). 2.4. OTHER PATHWAYS OF ANAEROBIC METABOLISM A great number of microorganisms can degrade the intermediates of anaerobic metabolism alternatively to form a wide array of end-products. Two groups of anaerobes of particular interest are enterobacteria and lactic acid bacteria. CHAPTER 2. LITERATURE REVIEW^ 23 2.4.1. PRODUCTS FORMED BY ENTEROBACTERIA Enterobacteria are classified into three categories according to the type of fermentation they carry out: mixed acid, butanediol, and propanediol producers. The mixed acid producers belong to the genera Escherichia, Salmonella, and Shigella. A typical member of this group, Escherichia coli, ferments sugars to lactic, acetic, succinic, and formic acids. Smaller amounts of ethanol, carbon dioxide, and hydrogen gas 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, carbon dioxide 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 derived from pyruvic acid. The pathway leading to succinic acid branches off at phosphoenolpyruvate. The amounts of fermentation products formed depend very much on the activity on pyruvic acid of the three enzyme systems involved (Garvie, 1980). Members of the Enterobacteria family, such as Citrobacter freundii, are able to metabolize glycerol (a product of lipid hydrolysis) either to 1,3-propanediol or to glyceraldehyde-3-phosphate. This process is assumed to be independent from the carbohydrate metabolism and occurs only if glycerol is available (Doelle, 1975). CHAPTER 2. LITERATURE REVIEW^ 24 2.4.2. PRODUCTS FORMED BY LACTIC ACID BACTERIA Lactic acid bacteria are morphologically a heterogeneous group and characterized by their main end-product, lactic acid. These microorganisms are highly saccharolytic and lack most anabolic pathways, so they exhibit very specific nutritional requirements. Most lactic acid bacteria are strictly fermentative, but are aerotolerant (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 and Streptococcus species yields only lactic acid: C611 1206^2CH3CHOHCOOH^(2.12) 2) The heterofermentative pathway used mainly by Leuconostoc species yields the following products: C 6H 120 6^CH3CHOHCOOH + CH 3 CH2OH + CO2^(2.13) 3) The bifidum pathway employed by Bifidobacterium species yield lactic acid and acetic acid: 2C6H 1206 ----+ 2CH 3 CHOHCOOH + 3CH 3 COOH^(2.14) Besides glucose, many other saccharides can be utilized by lactic acid bacteria including fructose, galactose, lactose, and pentoses. CHAPTER 2. LITERATURE REVIEW^ 25 2.5. FACTORS AFFECTING VFA PRODUCTION In general, the acid-phase digestion products may be markedly affected by the characteristics of the wastewater, environmental factors such as culture pH, temperature, oxidation-reduction potential (ORP), reactor configuration and available trace 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 a relatively new one in the wastewater treatment field. Traditionally, most of the research in anaerobic sludge digestion has been focused on the methanogenic phase of the process, where VFAs are used as substrate for the methane forming bacteria. Little attention has been paid, therefore, to the optimization of the acidogenic microorganisms coupled with suppression of the methanogenic ones. Most of the valuable contributions on the acid-phase digestion have been obtained from studies using either soluble substrates such as glucose (Ghosh and Pohland, 1974; Uribelarrea and Pareilleux, 1981; Zoetemeyer et al., 1982b; and Cohen 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 and van Andel, 1984); or specific industrial wastewaters generated from sugar refineries (Gil-Pena et al., 1987), and ethanol distilleries (Machado and Sant'Anna, 1987). It is questionable, therefore, whether information available from these sources can be directly applied to the design and operation of anaerobic digesters treating primary sludge from municipal wastewater treatment plants. CHAPTER 2. LITERATURE REVIEW 26 Relatively few studies have been performed using primary sludge from municipal wastewater treatment facilities as a feed. Among them are those by O'Rourke (1968), Chynoweth and Mah (1971), Borchard (1971), Eastman and Ferguson (1981), Rabinowitz (1985), Gupta (1986), and Ghosh (1987). Important findings from a selected number of the above mentioned studies are summarized in the following paragraphs. Andrews and Pearson (1965) have observed that the acidogenic phase is fairly rapid, with an optimum cell residence time of 0.75 days. In addition, the type of volatile acids generated from a given substrate is greatly influenced by variation of the organism residence time. Chynoweth and Mah (1971) have reported a high rate of lipid dissimilation in primary sludge digestion. Acetic, propionic, and butyric acids were the main products. Formic acid was also detected in smaller amounts. On the contrary, Eastman and Ferguson (1981) have found that lipid degradation was minimal in the acid phase, but carbohydrates and proteins were extensively metabolized. The VFA production was significantly affected by pH but not by the influent solids concentration, at least up to 6% VS. According to Zoetemeyer et al. (1982b), the relative production of individual VFAs from glucose depends on the dilution rate and more strongly on the culture pH value, with an optimum pH in the range between 5.0 and 6.0. Rabinowitz (1985) has found that at sludge retention times ranging from 2.5 to 10 days, acetic and propionic acids made up more than 90% of the short chain VFA production, and appeared in the fermenter supernatant in a ratio of CHAPTER 2. LITERATURE REVIEW^ 27 approximately 55:45. Gupta (1986) has reported that the net VFA generation consistently improved with increase in temperature between 10 and 30 °C, while pH control at 7.0 did not make 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 hydraulic retention 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 three reactions in Figure 2.1 need to be encouraged, with the concurrent suppression of the last three ones which are linked to methanogenic activity. Methane formers are very sensitive to environmental factors. Their activity drops drastically at pH below 6 (Zehnder et al., 1981). They are very slow growing organisms as well. The maximum specific growth rate of methanogens can be one order 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 a critical SRT, which appears to be system specific (Ghosh, 1987). Methanogenesis can be suppressed, therefore, by operating the digester at a pH below 6 and an SRT value below the critical one. 2.6. APPLICATIONS OF THE ACID -PHASE DIGESTION Improved knowledge of the acid-phase anaerobic digestion can be useful in a CHAPTER 2. LITERATURE REVIEW^ 28 number of situations, ranging from the operation of the overall digestion process itself 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 organic material. Moreover, since the main products of acidogenic activity are short-chain, soluble organic substrates, they can be used as an energy and carbon source for bacteria carrying out other processes, such as biological phosphorus removal or two- stage biological denitrification. A brief description of the biological phosphorus removal process and the role of VFAs in it is outlined below. 2.6.1. THE BIOLOGICAL PHOSPHORUS REMOVAL PROCESS Biological phosphorus removal has been a viable alternative to chemical precipitation as a means to control nutrient discharges into receiving water bodies. In this process, phosphorus is taken up by certain species of bacteria such as Acinetobacter beyond their need for normal cell maintenance and synthesis (Siebrietz et al., 1983). A continuous flow biological phosphorus removal scheme consists of a bioreactor in which an aerobic zone is preceded by an anaerobic one. The addition of simple soluble carbon substrates such as VFAs in the anaerobic zone results in phosphate release and carbon storage by the biomass (Nicholls and Osborn, 1979; Barnard, 1983; Comeau et al., 1986). Carbon storage occurs mainly in the form of poly-3-hydroxybutyrate (PHB) and poly-P-hydroxyvalerate (PHV) (Comeau et al., CHAPTER 2. LITERATURE REVIEW 29 1988). The fact that phosphorus-removing bacteria can assimilate the acid-phase digestion products in the anaerobic zone provides them with a competitive advantage compared to other heterotrophic microorganisms occurring in activated sludge systems (U.S. EPA, 1987). It has been observed that there is a relationship between the amount of VFAs added and the amount of phosphate released under anaerobic conditions (Fukase et al., 1982; Arvin, 1985). When the anaerobic zone is followed by an aerobic one, the phosphorus removing bacteria take phosphate from solution and store it in polyphosphate pools. The amount of phosphorus uptake in the aerobic zone can be correlated with that released under anaerobic conditions, which in turn is a function of the amount of stored VFAs (in the from of PHB or PHV) available in the aerobic zone (Wentzel, 1984). Hence, the biological phosphorus removal capacity of a plant can be improved by the presence of short chain soluble carbon compounds in the anaerobic zone. Rensick et al. (1984) have reported that acetic acid addition increased phosphorus removal from 45 to 97%. According to Rabinowitz and Oldham (1985), the incorporation of primary sludge digestion into the design of a simplified nutrient removal process resulted in an improvement of more than 100% in phosphorus 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 VFAs required in this zone is 25 to 30 mg/L (as HAc). Since the VFA content of untreated domestic wastewater is usually very low (less than 10 mg/L), an external organic carbon source is needed to trigger the biological phosphorus removal mechanism. CHAPTER 2. LITERATURE REVIEW 30 The soluble carbon concentration in the anaerobic zone of the process can be increased either by the addition of preformed VFAs or by primary sludge digestion with return of the fermented material to the main bioreactor. 2.7. PROCESS CONFIGURATION The basic requirement in anaerobic processes is the maintenance of a sufficient amount of active biomass in the reactor under high organic loading conditions. In order to meet this requirement, many suspended- and attached-growth process configurations have been thoroughly investigated, such as the completely mixed anaerobic digestion (conventional digester), anaerobic contact process (completely mixed reactor with clarifier and solids recycle), anaerobic filter, upflow anaerobic sludge 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-phase digestion of primary sludge: the completely mixed reactor (CMR) with clarifier and solids recycle system; and the upflow anaerobic sludge blanket (UASB) reactor. Completely mixed digesters (with or without solids recycle) have been of fundamental importance in anaerobic treatment, with a wide range of applications in municipal and industrial wastewaters (Speece, 1983; McCarty and Smith, 1986). The upflow anaerobic sludge blanket process is a recent modification of the biolytic tank (Jewell, 1987). In the 1970s, Lettinga and co-workers developed the UASB reactor concept in the Netherlands (Lettinga et al., 1979). It is based on the CHAPTER 2. LITERATURE REVIEW 31 idea that anaerobic sludge has superior settling characteristics, if the physical and chemical conditions for sludge flocculation remain favorable. The sludge blanket (bed) can be considered as a separate fluid phase with its own specific properties. A well-established sludge blanket is fairly stable and can withstand relatively high mixing 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 shape with a 1 to 5 mm diameter (Hulshoff Pol et al., 1983). As such, the system acts as a biofilm in a sense that there is substrate diffusion into the conglomeration of microorganisms and product diffusion out (McCarty and Smith, 1986). Pellets have very good settleability and are readily retained in the reactor without the need of a clarifier (Sam-Soon et al., 1988). Pellet formation in UASB systems, however, depends upon the type of the wastewater used. For example, a high degree of pelletization has been achieved in systems treating carbohydrate wastes (Lettinga et al., 1980; Ross, 1984), but a limited pellet formation has been observed with acetate- propionate mixtures (de Zeeuw and Lettinga, 1980). No pellet formation has been obtained 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 and Vinken, 1980). In addition, several full-scale installations have been successfully employed in Northern Europe treating a variety of industrial wastewaters (Maat and Habets, 1987; Lettinga and Hulshoff Pol, 1991). CHAPTER 3 RESEARCH OBJECTIVES As discussed in the previous chapter (Section 2.5), much of the information available on the acid-phase digestion has been obtained using simple, soluble carbon substrates. In certain cases, where primary sludge was used as feed, the main focus of the research was on methane production optimization (O'Rourke, 1968; Chynoweth and Mah, 1971; Ghosh, 1987), or on phase-separation techniques (Borchard, 1971), or on kinetic modelling (Eastman and Ferguson, 1981). Consequently, information regarding details on the mechanisms and pathways involved in the transformation of the organic substrates and the full spectrum of products formed was rather limited. On the other hand, Rabinowitz (1985) and Gupta (1986) have attempted to investigate the 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 or conventional continuous-flow reactors without solids recycle eliminated the possibility to distinguish between the two retention times (HRT and SRT) of the system. It should be pointed out that HRT and SRT are two different operational parameters and that they may affect any biological process in a distinctly different manner. Moreover, only a few investigations included acid-phase digestion experiments with pH control (Eastman and Ferguson, 1981; Gupta, 1986). Even in these studies, no attempt was made to control the pH at values below 5.5. 32 CHAPTER 3. RESEARCH OBJECTIVES 33 It is, therefore, apparent that there is a need for an in-depth investigation of the acid-phase digestion of primary sludge. Inspired by the above observation, this research 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, reactor configuration, and influent characteristics) on the process. 3) Suggest possible mechanisms and biochemical pathways involved in the conversion 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 of reactor HRT and SRT values. Although CMR systems have long been used in environmental engineering practice for acid-phase digestion of primary sludge, the application of UASB systems, for the same purpose, is a rather novel idea. A better understanding of the nature of the acidogenic phase can be achieved by determining the level and rate of carbohydrate, protein, and lipid utilization, and the 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). Additional valuable measurements included different types of solids and nitrogen forms, as well as pH and ORP. CHAPTER 4 EXPERIMENTAL METHODS AND ANALYTICAL PROCEDURES 4.1. WASTEWATER SOURCE The primary sludge used in this study was obtained from the Iona Island wastewater treatment plant located in Richmond, British Columbia, except for one experimental run, where the sludge was collected from the Lions' Gate treatment plant situated in North Vancouver, British Columbia. Both plants operate as primary treatment facilities, with subsequent anaerobic digestion for sludge stabilization. The sludge was collected, usually once a week, from the underflow lines of the primary clarifiers in 25 L carboys and stored in the laboratory cold room at 4 °C. It was subsequently screened through a 0.6 cm rectangular-shaped mesh and transferred into a 60 L covered plastic container, which was kept at the same temperature (4 °C). The screening removed most of the large fibrous material and nearly 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 adjusted to a value of about 4,000 mg/L before feeding, by diluting with distilled water or by settling and decanting the excess liquid, in order to provide a level of comparable feed 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). 34 CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^35 4.2. EXPERIMENTAL SET-UP AND OPERATION 4.2.1. GENERAL SYSTEM CONFIGURATION This research involved the use of two laboratory-scale, continuous-flow units, having different configurations: a completely mixed reactor (CMR) with clarifier and sludge recycling, and an upflow anaerobic sludge blanket (UASB) reactor, as depicted in Figure 4.1. Both reactors were made of plexiglass (internal diameter: 11.2 cm, total volume: 3.2 L, liquid volume: 3.0 L). The CMR system was equipped with a stainless steel stirrer with blades for mixing of the contents. Visually, complete horizontal and vertical 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 biomass through the effluent line. The bottoms of the clarifier and the UASB reactor were modified to an inverted cone shape (height of cone: 8.0 cm, diameter at bottom: 4.0 cm) in order to provide good settling conditions for the clarifier and a better mixing/diffusion flow pattern in the UASB system respectively. The two reactors were hermetically sealed to avoid any air entrapment. A small, cone-shaped device (height of cone: 6.0 cm, diameter at top: 4.0 cm) was attached internally to the cover of each reactor to act as a gas collection system. The gas 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 and 24.0 cm from the bottom in the CMR reactor; and 8.0, 16.0 and 26.0 cm from the Gas Flow Meter CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^36 COMPLETELY MIXED ^ UPFLOW ANAEROBIC REACTOR (CMR) SLUDGE BLANKET (UASB) Waste^ Waste SR : Sludge Recycle Kr: Pump FEED I^REACTOR I^CLARIFIER ^ REACTOR II FIGURE 4.1. EXPERIMENTAL LAYOUT Gas Flow Meter Q SR ^...- Effluent FEED II CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES 37 bottom in the UASB unit). However, the lowest port in each reactor was exclusively used for wasting purposes. The other ports were periodically used to test the operational 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/AgC1 reference electrode, with a platinum band as the noble metal, and the pH elecrode has a sealed (gel) reference electrode. Each probe was affixed to one end of a piece of rigid plastic tubing which slid inside the sleeve of another plastic tube, with minimum resistance. This latter tube opened up through a ball valve acting as a channel to allow the probe to slide into and out of the reactor. An 0-ring seal inserted between the two cylinder walls prevented liquid being forced by back pressure from the interior of the reactor. The ORP probes were used throughout the entire experimental study, while the pH probes were inserted only during the last two runs (Runs 4A and 4B), as part of the pH control strategy. 4.2.2. OPERATION A total of 11 runs were conducted to investigate the effects of selected operational and environmental parameters on the acid-phase digestion of primary sludge. The research evolved into 4 stages, and in each stage an attempt was made CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^38 to explore the influence of a particular parameter. In Stage 1 (Runs 1A to 1D) the role of Hydraulic Retention Time (HRT) was investigated, while Stage 2 (Runs 2A to 2C) focused on the effect of Solids Retention Time (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 experiments were conducted at an ambient liquid temperature between 18 and 22 °C. A summary of operating conditions is presented in Table 4.1. The SRT of the systems was controlled by wasting the appropriate volume from each reactor on a daily basis. The waste volume was slightly adjusted, when necessary, to compensate for the loss of biomass [measured as Volatile Suspended Solids (VSS)] through the effluent line. For the UASB system, the wastage was based on the sludge blanket volume (eg. to maintain an SRT of 10 days, one tenth of the volume of the blanket was 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 appreciable effect. 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 was calculated daily using a specially graduated stick and converted into the corresponding HRTs. For feeding purposes, the primary sludge was transferred from the 60 L plastic container to two 10 L buckets and, after adjusting the TS content to 4,000 mg/L (as explained in Section 4.1.), it was added to the two feed tanks at the end of each CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^39 experimental day. This was done to minimize possible changes in feed characteristics due to higher temperatures in the early afternoon hours, especially during the summer months. To avoid altering the characteristics of the feed due to excess aeration, the feed tanks were covered with plastic lids and the stirrer speeds were kept as low as possible, while still keeping the particulate matter in suspension. TABLE 4.1. OPERATING CONDITIONS (MEAN VALUES) RUN REACTOR TYPE SRT (d) WASTE (mild) HRT (hr) pH ORP (mV) lA CMR 10 290 9.03 5.25 -326 UASB 10 180 9.25 5.14 -369 1B CMR 10 280 6.14 5.27 -284 UASB 10 250 6.09 5.33 -362 1C CMR 10 290 12.12 5.01 -309 UASB 10 160 12.07 4.96 -385 1D CMR 10 290 14.91 5.10 -325 UASB 10 150 15.28 4.98 -370 2A CMR 15 190 12.20 5.17 -343 UASB 15 110 12.11 5.09 -376 2B CMR 20 140 12.06 5.23 -364 UASB 20 80 11.94 5.09 -391 2C CMR 5 400 12.14 5.63 -354 UASB 5 275 11.89 5.52 -385 3A CMR 10 290 11.84 5.15 -296 UASB 10 170 12.16 4.98 -371 3B CMR 10 290 12.05 5.03 -311 UASB 10 160 12.11 5.05 -367 4A CMR 10 290 12.22 4.42 -278 UASB 10 160 12.13 4.47 -333 4B CMR 10 290 11.97 6.09 -326 UASB 10 160 12.02 6.05 -393 CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES 40 Total solids (TS) were selected as the control parameter, since their analysis involves a simple, direct and readily accessible method. The TS concentration applied in this study was determined by a trial-and-error procedure, as the highest amount of solids contained in the feed without causing frequent operational problems. Two previous attempts, using 10,000 and 7,000 mg/L TS respectively, were proven to be unsuccessful due to a number of regular operational difficulties (ie. blockages of feed lines, pump failures, reactor overflows). The recycle pump (Cole-Parmer Company, Model 7017-21) operated on a cycle of 5 minutes on and 5 minutes off. This combination was found to be adequate to clear 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 was less than 8 hours. A scraper mechanism operating at 1 rpm was installed to prevent the sludge adhering to the clarifier walls. During the pH control experiments (Runs 4A and 4B) a Cole-Parmer Company pH/pump system (Series 7142) was connected to each one of the pH probes which were inserted into the reactors. The pH/pump system was set at a selected pH value and the pump was activated whenever the pH was higher (in the case of acid addition) or lower (in the case of base addition) than the set value. Aqueous solutions of 0.02 N HC1 and 0.02 N NaOH were used to respectively decrease or increase the pH in the reactors. The reactors were seeded with sludge obtained from a fermenter installed at the University of British Columbia pilot plant (a biological phosphorus removal research facility) treating mostly campus wastewater, by adding 1.0 L of seed and 2.0 CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^41 L 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 both systems were drained and cleaned and some of the tubing was replaced. At the same time, the ORP probes were cleaned and their responsiveness tested by immersion in a quinhydrone solution, as described by the A.S.T.M. (1989b), and discarded whenever it was considered necessary. To increase accuracy and reliability, the pH probes 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-state when the volatile acid production showed approximately steady values (ie. less than 10 percent variation in concentration). This was usually achieved in less than two weeks of operation, although sporadic disturbances occurred afterwards in certain cases. To ensure that reasonable steady-state conditions were established, most experiments were operated for about 4 to 5 SRTs, as shown in Table 4.2. 4.3. ANALYTICAL PROCEDURES Samples were obtained from three locations, in order to follow the composition changes 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 a CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^42 minimum. Most tests were performed regularly on a biweekly basis. The majority of tests 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 detail below. All sample filtrations were done using Whatman No. 4 filters, with the exception of solids analysis where Whatman 934-AH glass microfiber filters were used. TABLE 4.2. DURATION OF EXPERIMENTAL RUNS AND AMOUNT OF BIOMASS (VSS) IN THE REACTORS RUN SRT (d) DAYS PER RUN # OF SRTs VSS IN REACTORS CMR (g) UASB (g) % DIF. lA 10 86 8.6 42.76 50.21 14.8 1B 10 44 4.4 52.70 66.68 21.0 1C 10 52 5.2 31.23 33.73 7.4 1D 10 47 4.7 23.58 27.27 13.5 2A 15 54 3.6 27.52 33.95 18.9 2B 20 55 2.8 30.01 35.85 15.5 2C 5 28 5.6 14.89 19.22 22.5 3A 10 38 3.8 29.65 32.03 7.4 3B 10 41 4.1 30.81 34.27 10.1 4A 10 38 3.8 28.34 33.81 16.2 4B 10 48 4.8 28.14 29.76 5.4 AVER. 48 4.7 13.9 CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^43 4.3.1. CHEMICAL OXYGEN DEMAND (COD) The filtered samples were preserved by freezing at -7 °C and analyzed in duplicate 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 an automatic Shimadzu Total Organic Carbon Analyzer (Model TOC-500) using a series of low and high standards, as described in the Instruction Manual (Shimadzu Corporation, 1987). The method is based on the principle that the quantity of CO 2 produced during combustion is proportional to the amount of carbon in the sample. 4.3.3. ORGANIC ACIDS a) Volatile Fatty Acids (VFAs) The volatile fatty acid determination was conducted using a computer- controlled Hewlett-Packard 5880A gas chromatograph, equipped with a flame ionization detector (FID). Helium was used as the carrier gas. Volatile fatty acids analyzed include: acetic, propionic, butyric, iso-butyric, valeric, 3-methylbutyric, and 2 -methylbutyri c. The filtered samples were kept frozen at -7 °C in sealed plastic transfer CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^44 pipettes (Canlab No. P5214-1). At the time of analysis, the samples were thawed at room 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 ti aliquots were injected using microsyringes (Hamilton Model 701 N, 10 ill) and a Hewlett-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 conditioned according to the procedure described in the Supelco Bulletin 751E (1989). The operating conditions of the chromatograph were as follows: Injector temperature:^150 °C Detector temperature:^200 °C Isothermal oven temperature:^120 °C Flow rate of helium gas:^20 mL/min Quantification of the response peaks was done by comparison with external standard methods using reagent grade standards. The detection limit of the method is 1 mg/L. At least two aliquots of each sample were injected and the mean values reported. b) Lactic and Pyruvic Acids The same technique used for VFA analysis was applied for lactic and pyruvic acid determination, with the following three modifications: The samples were acidified using 0.03 M oxalic acid, the glass column was packed with 4% Carbowax 20M on Supelco Carbopack B-DA, and the isothermal CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^45 oven temperature was 175 °C (Supelco, 1990). c) Formic Acid Formic acid was analyzed by a colorimetric method outlined by Lang and Lang (1972). The bright yellow and green-yellow fluorescent reaction products from the formic-citric acid reaction change to raspberry red at room temperature, in the same medium. The intensity of the color is proportional to the concentration of the formic acid present. Absorbance measurements were taken at 515 nm on a Bausch & Lomb Spectronic 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 is provided by Kisaalita (1987). 4.3.4. SOLIDS a) Total Solids (TS) and Volatile Solids (VS) Total solids were determined by evaporating a known volume of well-mixed sample in a Fisher Isotemp (Model 350) forced draft oven at 104 °C. Subsequently, by igniting the residue at 550 °C in a Lindberg muffle furnace (Type 51828), the volatile solid content was measured. Both analyses were performed as outlined in Standard 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 determined CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES 46 in accordance with the Standard Methods (A.P.H.A. et al., 1989). A known volume of sample was vacuum filtered through a pre-washed and oven-dried Whatman 934- AH glass microfiber filter and dried at 104 °C for TSS analysis. The VSS were determined by igniting the residue at 550 °C. Since the suspended solids concentration in both reactors was very high, usually more than 10,000 mg/1, the Gooch crucible method was considered impractical (Anderson, 1989). Instead, a known volume of well-mixed sample was transferred into a 50 ml centrifuge tube and spun down at 2500 rpm in an IEC International Centrifuge (Model CS-CC467) for about 15 min. The supernatant was vacuum filtered through a pre-washed and oven-dried glass microfiber filter. The settled 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 °C oven, and finally into the 550 °C furnace, as described above. 4.3.5. NITROGEN a) Ammonia Nitrogen (NH 3-N) Samples for ammonia determination were first filtered and preserved with concentrated sulfuric acid and then stored at 4 °C. Ammonia nitrogen was analyzed in triplicate by the automated phenate method, using a Technicon Autoanalyzer II Continuous Flow System (Industrial Method No. 98-70W). Appropriate dilutions were made prior to determining the intensity of the color complexes formed and then compared with those of a series of standards of known concentrations. CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^47 b) Total Kjeldahl Nitrogen (TKN) Total Kjeldahl nitrogen (TKN) was measured by digesting the samples in a BD-40 Technicon Block Digester with concentrated H 2 SO4 and K2SO4 , to liberate all organically bound nitrogen. Filtered or unfiltered samples were used to determine the soluble or total TKN respectively. Standards and samples were analyzed colorimetrically in triplicate using a Technicon Autoanalyzer II (Industrial Method No. 376-75W), according to Technicon Block Digester Instruction Manual (1974). 4.3.6. PROTEINS The amount of protein in a sample can be estimated by measuring the nitrogen content of the organic matter present. Organic nitrogen includes the nitrogen in amino acids, amines, amides, imides, nitro-derivatives and a number of other compounds. Most of the organic nitrogen that occurs in municipal wastewater, however, is in the form of proteins or their degradation products: polypeptides and amino acids (Sawyer and McCarty, 1979). Therefore, assuming that all organic nitrogen is due to protein and that protein contains on average 16 percent nitrogen (Gaudy and Gaudy, 1980), the protein content can be calculated from the corresponding TKN value by subtracting the inorganic nitrogen concentration (in this case only the NH 3 -N value) and multiplying the difference by 6.25 (100 divided by 16). CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^48 4.3.7. LIPIDS Total lipids were determined by dry extraction (influent and reactor samples) and wet extraction methods (effluent samples). a) Dry Extraction Method Samples were dried overnight at 104 °C, ground in an Oster commercial blender, and weighed on Whatman 941 paper filters (9.0 cm diameter). They were then subjected to continuous extraction for 6 hours in a Soxhlet apparatus using petroleum ether as solvent, in accordance with the procedure described by Triebold and Aurand (1969). Reweighing the samples with a Mettler AC 100-52 balance (after at least 1 day in a vacuum desiccator), allowed calculation of the total lipid content as percent of the TS of the sample. b) Wet Extraction Method According to the Rose-Gottlieb Method (Triebold and Aurand, 1969), a known volume of the sample is treated with ethanol and ammonium hydroxide solution, and then extracted with a 1:1 mixture of ethyl and petroleum ethers. The ethers containing the dissolved lipids are decanted into a weighed flask. The extraction is repeated a second time, after which the solvents are evaporated and the weight of the extracted total lipids determined. CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^49 4.3.8. CARBOHYDRATES Since cellulose is a major component in domestic wastewaters (Gaudy and Gaudy, 1980), a method specifically suited to measure the cellulose content (as the one described below) is considered indispensable for total carbohydrate analysis in this study. The first step in carbohydrate determination involved an acid hydrolysis technique outlined in the A.S.T.M. (1989a). A primary hydrolysis of the samples with 72% H 2SO 4 at 30 °C for 1 hour was followed by a secondary hydrolysis in a preheated autoclave (Barnstead Company, Model C-0704) for 4 hours. The diluted hydrolyzates were then neutralized with a 6 N solution of NaOH. The second step of the analysis included the post-neutralization stages of the ferricyanide method (Handbook of Micromethods, 1974). Adding the appropriate reagents (carbonate-cyanide, ferricyanide, and ferric-iron), a blue complex is formed whose intensity is proportional to the concentration of glucose. The absorbance is then measured at 690 nm and compared with that of a range of glucose standard of known concentrations. For soluble carbohydrate determination, the ferricyanide method was followed entirely. CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^50 4.3.9. PHOSPHORUS a) Orthophosphate (PO 4 -3) Samples for orthophosphate analysis were filtered and preserved as in the case of ammonia (Section 4.3.5.a). Orthophosphate was determined in triplicate by the automatic ascorbic acid reduction method on a Technicon Autoanalyzer II (Industrial Method No. 327-73W). According to this technique, a blue-colored antimony- phosphomolybdate complex is formed when ortho-phosphate reacts with ammonium molybdate and potassium antimonyl tartrate. The peak heights of standards of known concentrations 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). All organically 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 ALKALINITY A Beckman 44 pH meter with automatic temperature compensation was used to determine the pH of the samples. The meter was calibrated daily, prior to measurements, 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 of 4.5 with 0.02 N sulfuric acid. CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^51 4.3.11. OTHER SOLUBLE ORGANICS In order to tentatively identify the nature of other soluble degradation products, a series of samples were analyzed on a Hewlett-Packard 5985B Gas Chromatography - Mass Spectroscopy system. The mass spectrometer was operated in the electron impact mode, using helium as carrier gas, at the following conditions: Ion source temperature: 200 °C Ionizing energy:^70 eV Scan range:^34-350 amu at 1 A/D measurement. The mass spectra were acquired with the data system and the peaks were identified with base peak probability matching using the library EPA-NIH Mass Spectra 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 ANALYSIS Gas samples were extracted periodically from the head space of the reactors using an 1 ml Hamilton syringe and rapidly injected into a Fisher-Hamilton Gas Partitioner (Model 29), using helium as carrier gas and a thermal conductivity detector. The gases were identified by comparing their retention times to standard gases and concentrations were estimated by comparing the peak areas with known CHAPTER 4. EXPERIMENTAL METHODS & ANALYTICAL PROCEDURES^52 standards that were used to determine response factors. 4.4. COLD STORAGE TESTING The effects of cold storage on sludge characteristics were studied at the beginning of the experimental research and at the end of Stage 1. This was considered necessary, since the raw sludge used in this study was to be kept at 4 °C for approximately 1 to 2 weeks. In each testing, over a period of 20 days, samples were taken from the supernatant of one of the 25 L carboys and analyzed for the following parameters in duplicate: chemical oxygen demand (COD), total organic carbon (TOC), and volatile fatty acids (VFA). 4.5 STATISTICS Averages, standard deviations, coefficients of variation and significant difference between the means (t-test) were calculated by the statistics package included in Symphony (release 1.2) of Lotus Development Corporation (Cambridge MA). CHAPTER 5 RESULTS AND DISCUSSION 5.1. GENERAL CHARACTERISTICS A brief description of important general issues concerning the nature of this research is presented below, preceding the detailed analysis of the four experimental stages. 5.1.1. FEED COMPOSITION An understanding of the nature of the wastewater used as feed is essential in the design and operation of any biological treatment process. To promote this understanding, an analysis of important physical and chemical constituents of primary sludge was performed regularly and the data have been included in Appendix C. A summary of the results, along with a basic statistical evaluation, is presented in Table 5.1 (Iona Island WWTP) and Table 5.2 (Lions' Gate WWTP). The total solids concentration of the reactor influent was continually adjusted to 4,000 mg/L, as mentioned earlier (Section 4.1), to provide a uniform feed for the entire 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 both 53 CHAPTER 5. RESULTS AND DISCUSSION 54 the standard deviation (STD) and the coefficient of variation (CoV) are reasonably small. For instance, the CoV of all 4 types of solids is less than 10%, which indicates that the majority of measurements are close to the mean. Comparing the two sources of feed, the most noteworthy difference lies in the VFA and NH 3 -N content. The feed from Lions' Gate plant contains roughly 40% (for both 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 3.3 TS 3260 - 5235 4007 294 7.3 VS 2300 - 4205 2990 279 9.3 TSS 2625 - 5050 3613 315 8.7 VSS 2035 - 3985 2710 263 9.7 CARBOHYDRATES 1140 - 2700 1703 218 12.8 PROTEINS 473 - 834 627 64 10.2 LIPIDS 394 - 693 507 47 9.3 COD 244 - 673 427 73 17.1 TOC 71 - 208 128 25 19.5 VFA (as HAc) 46 - 150 103 19 18.4 NI-13 -N 13 - 35 20 3.8 19.0 TKN 89 - 158 121 11 9.1 PO4-3 (as P) 6 - 17 10 1.8 18.0 TP 14 - 27 19 2.3 12.1 ALKAL. (as CaCO3 ) 141 - 216 184 16 8.7 Note: All values in columns RANGE, MEAN and STD are expressed in mg/L, except pH. CHAPTER 5. RESULTS AND DISCUSSION ^ 55 TABLE 5.2. INFLUENT SLUDGE CHARACTERISTICS (LIONS' GATE WWTP) PARAMETER RANGE MEAN STD CoV(%) pH 5.74 - 6.33 6.00 0.16 2.7 TS 3630 - 4340 4032 204 5.1 VS 2925 - 3585 3285 228 6.9 TSS 3065 - 3870 3539 219 6.2 VSS 2470 - 3310 2956 216 7.3 CARBOHYDRATES 1790 - 2360 2091 182 8.7 PROTEINS 497 - 611 559 36 6.4 LIPIDS 434 - 537 486 32 6.6 COD 359 - 522 445 50 11.2 TOC 91 - 153 128 19 14.8 VFA (as HAc) 29 - 67 44 11 25.0 NH3 -N 5 - 11 8 2.0 25.3 TKN 88 - 109 97 7 7.2 PO43 (as P) 5 - 9 7 1.1 15.7 TP 10 - 16 13 1.8 13.8 ALKAL. (as CaCO 3 ) 153 - 197 172 16 9.3 Note: 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 that carbohydrates are by far the most predominant group in the raw sludge from both facilities (Table 5.3). Proteins and lipids are the other two important classes of organic compounds present. The three groups together account for 95% of the volatile solids content. The sludge from Lions' Gate is richer in carbohydrates but contains less protein and lipid. CHAPTER 5. RESULTS AND DISCUSSION^ 56 The particulate fraction of the organic matter is very high, as indicated by the VSS/VS ratio, which averages about 90%. Lipids and polysaccharides are practically insoluble in water. Analytical determination of soluble carbohydrates and proteins has 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 are normally of globular nature and, therefore, water soluble (Gaudy and Gaudy, 1980). The low soluble protein content of primary sludge indicates that most of the proteinaceous matter is still an integral part of the suspended solids. TABLE 5.3. ORGANIC COMPOSITION OF FEED ORGANIC CLASS IONA ISLAND WWTP LIONS' GATE WWTP RANGE (% of VS) MEAN (% of VS) RANGE (% of VS) MEAN (% of VS) CARBOHYDRATES 49 - 63 56 60 - 67 64 PROTEINS 18 - 25 21 15 - 19 17 LIPIDS 12 - 21 17 12 - 16 15 TOTAL 94 96 5.1.2. COLD STORAGE EXPERIMENTS The results of both tests on stored raw sludge (at 4 °C) showed no significant variation 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 the chemical analysis through experimental errors. Manoharan (1988) has reported that CHAPTER 5. RESULTS AND DISCUSSION^ 57 no 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 and an increase in VFAs occurred, presumably as a result of bacterial activity. To avoid any alterations in feed composition, the maximum storage period was set at 10 days. TABLE 5.4. COLD STORAGE TESTING DAY TEST 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 84 2 426 128 102 440 129 82 4 411 124 96 449 125 79 6 406 122 94 431 130 88 8 415 130 101 443 120 86 10 419 122 95 435 126 80 12 414 117 103 428 125 84 14 408 120 101 402 109 86 16 370 106 113 361 99 93 18 373 102 115 347 97 97 20 298 84 106 5.1.3. ACCLIMATION AND STABILITY OF OPERATION The original heterogeneous population in a bioreactor has to undergo biochemical acclimation and selection of the species best able to grow on the carbon CHAPTER 5. RESULTS AND DISCUSSION^ 58 sources available in order to ensure successful and sustainable operation. In continuous-flow systems, acclimation is a time-dependent process and it can be influenced by the type of seed used, the characteristics of feed, and the chosen operational 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 both the 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 attributed to the synergistic action of a number of factors such as the suitable seed used (taken from an acid-phase digester), the good digestability of the primary sludge, the favorable operating conditions and the small volume of the reactors. It has been reported (Lettinga et al., 1979; de Zeeuw and Lettinga, 1980) that in UASB systems, long acclimation periods (sometimes up to 4 to 8 weeks) may be required, because of the slow formation of sludge blanket. The phenomenon of microbial aggregation and granulation, however, is greatly affected by various nutritional and environmental parameters such as trace metal ions (particularly calcium), temperature, the nature of the inoculum, and feed used (Mahoney et al., 1987; Guiot et al., 1988). Investigating a number of seeding and reactor loading alternatives, Fongsatitkul (1992) has found that, in most cases, the acclimation process was completed within 4 weeks. Using mesophilic granular sludge as seed material, van Lier et al. (1992) have observed that the start-up period in a UASB system was between 1 and 2 weeks. In this study, the good settling properties of the sludge resulted in a sludge blanket formation in about 3 to 6 days with minimal loss CHAPTER 5. RESULTS AND DISCUSSION 59 of 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 and Ferguson (1981) have found that steady-state was achieved within 7 days in 2.5 L completely-mixed acidogenic reactors. Both systems' behavior during the steady-state analysis period was stable. No significant trends were observed in any parameter over the 3 to 9 solids retention times of the experiments. The standard deviation for individual analyses was within 12% of the mean for almost all measured parameters. The higher variability observed in certain parameters in two cases (Runs 1B and 2C) is attributable to bacterial stress imposed by the short HRT and SRT respectively, of those runs. Furthermore, the ORP values measured ranging from -270 to -400 mV (Table 4.1), suggest that good anaerobic conditions were maintained throughout this experimental 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 that the environment is more reductive inside the blanket than it is in the CMR system. 5.2. THE EFFECT OF HRT - STAGE 1 5.2.1. HRT AS A CONTROL PARAMETER The microbial population of most natural environments is usually dominated by a relatively small number of species. A few selective environmental parameters such as pH, ORP, temperature, a toxic factor, the presence or absence of a key CHAPTER 5. RESULTS AND DISCUSSION 60 growth factor, etc., operate to impose a limit on the heterogeneous nature of the bacterial population, and thereby "select" one or more dominant cultures. This phenomenon of species selection has found a variety of applications in environmental engineering and related fields (Ghosh and Pohland, 1971). In closely monitored continuous-flow bioreactors, any or all of the parameters can be controlled and maintained constant. Consequently, it is often possible to select and retain a group of microbial species which would accomplish the desired biochemical conversions at acceptable reaction rates. Survival of individual species, however, in heterogeneous cultures such as those found in wastewater treatment processes, depends upon a variety of less defined factors. Although the population in such processes tends to remain, to some degree, heterogeneous because of mutual microbial interactions (eg. competition, amensalism, parasitism, mutualism, symbiosis, predation, etc.), the relative proportion of each major species changes from one condition to another (Harrison, 1978). An important operational variable which can be easily manipulated is the hydraulic retention time (HRT). It is the average length of time a molecule of liquid remains in the reactor and can be defined as the volume of the reactor divided by the average influent flow rate. HRT governs the amount and type of substrate being used by the cells. Since anaerobic digestion is a two-phase process, HRT can act as a selection parameter for the acidogenic phase only if it encourages the growth of acid formers and concurrently suppresses the growth of methane producers. One of the objectives of this study has been to investigate the effect of the two operational parameters [HRT and SRT (defined in Section 5.3.1.)] independently. For CHAPTER 5. RESULTS AND DISCUSSION 61 this reason, small HRT values are selected in contrast with the SRT. In Stage 1, the HRT varied from 6 to 15 hours, while SRT was kept constant at 10 days. The raw data collected during this stage are tabulated in Appendix C (Tables Cl to C20). 5.2.2. VFA PRODUCTION Short-chain volatile fatty acids (C 2 to C 5 ) are normally the main products of the acidogenic digestion of primary sludge (Chynoweth and Mah, 1971). The high concentrations of total VFAs (expressed as acetic acid for comparison purposes) achieved in both bioreactors clearly support the above observation (Appendix C). For example, the profiles of influent and reactor VFA concentrations, depicted in Figure 5.1 (Runs 1C and 1D), show that a sharp increase in the reactor VFA content occurs in both runs. This suggests that favorable conditions for the growth and maintenance of a healthy population of acid-producing microorganisms have been established during the course of the experiments. The total net VFA production (as acetic acid) at steady-state operation, as a function of HRT, is presented in Figure 5.2. In both systems, VFA concentration increases with HRT to a maximum value at 12 hours. A further increase in HRT results in a drop in concentration by about 80 mg/L. The decline in VFA generation coupled with the higher production rate of gaseous end-products (as explained in Section 5.3.4) provide strong evidence that methane-forming bacteria have been stimulated at an HRT of 15 hours. CHAPTER 5. RESULTS AND DISCUSSION 900 800 700 600 500 400 300 200 100 0 62 800 700 600 500 400 300 200 20 ^ 40 ^ 60 ^ 80 ^ 100 TIME (d) FIGURE 5.1. VFA PROFILE (RUNS 1C & 1 D) SRT = 10 d + REACTOR II (UASB) 6 ^ 9 ^ 12 ^ 15 HRT (hr) FIGURE 5.2. NET VFA PRODUCTION AS A FUNCTION OF HRT CHAPTER 5. RESULTS AND DISCUSSION^ 63 It is obvious that the mean VFA concentration is consistently higher in the UASB reactor. As a result of the good settling properties of the sludge blanket, the amount of active biomass (measured as VSS) lost through the effluent line is considerably lower as compared to the CMR system (Appendix C). Since more bacteria are retained in the upflow reactor (the amount of VSS in the UASB reactor is on the average 14% higher than that in the CMR System - Table 4.2), they can in turn generate a greater amount of products. However, the net VFA specific production 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 be independent of the reactor configuration, at least for the above mentioned conditions. TABLE 5.5. VFA SPECIFIC PRODUCTION RATE AS A FUNCTION OF HRT RUN HRT (hr) CMR (mgVFA/mgVSS*d) UASB (mgVFA/mgVSS*d) 1B 6 0.067 0.061 lA 9 0.083 0.080 1C 12 0.101 0.103 1D 15 0.092 0.089 The specific production rate is largely affected by the change in HRT, reaching its maximum value at 12 hours. The low rate at the shortest HRT is mainly due to the limited time available for substrate assimilation, while the decline noticed at the longest HRT is probably caused by the conversion of soluble VFAs to gaseous products. Although gas generation has been detected for all HRTs (Table E4, CHAPTER 5. RESULTS AND DISCUSSION 64 Appendix E), the sharp increase observed in Run 1D (HRT 15 hours) suggests that methanogenic activity was encouraged in this case. It is possible that the fraction of methane-forming bacteria in the biomass at a 10 day SRT was sufficient to affect net VFA production at the longer HRT. Based on the above results, the optimum HRT for VFA formation (for this type of wastewater) is 12 hours, with an acceptable range of more than 9 and less than 15 hours. The VFA concentrations in the reactor and in the effluent of the CMR system are essentially the same (Table 5.6). It is possible that a dynamic equilibrium exists in the clarifier between the rates of acid formation and volatilization. In general, the rate 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 molecular properties of the specific compound (Loehr et al., 1973). Due to a number of synergistic reasons such as the short retention time of the liquid in the clarifier (2 to 5 hours), quiescent flow conditions, ambient temperature, small surface area (99 cm2), and higher pH values than the corresponding pK A values of the acids, the degree of volatilization is considered to be minimal. Similarly, no appreciable acidogenesis should be taking place, mainly because of the short retention time (6 to 8 hours) of the recyclable biomass. On the contrary, the VFA concentration (Runs 1A and 1B) in the effluent of the UASB system is considerably lower than that in the reactor (sludge blanket), as shown in Table 5.6. The difference in VFA, which is statistically significant according to the t-test performed (Miller et al., 1990), is probably due to flow channelization in the reactor. At shorter HRTs (higher flow rates), the liquid CHAPTER 5. RESULTS AND DISCUSSION^ 65 molecules in the middle of the reactor may move upwards faster (in a jet-like fashion) than those close to the wall and eventually leave the system earlier. This results in an even shorter HRT for part of the reactor's contents diminishing, at the same time, the opportunity for food assimilation by the bacteria, which can ultimately lead to lower VFA concentration in the effluent of the unit. At higher HRTs (12 to 15 hours), the phenomenon of flow channelization appears to be of minor importance, since the VFA values are essentially the same in the sludge blanket and the effluent of the reactor. TABLE 5.6. COMPARISON OF REACTOR AND EFFLUENT VFA CONCENTRATIONS RUN CMR SYSTEM UASB SYSTEM REACTOR (mg/I-) EFFL. (mg/L) REACTOR (mg/L) EFFL. (mg/L) SIGNIF. DIFFER. 1B (6 hr) 407 412 466 370 YES IA (9 hr) 540 530 603 465 YES 1C (12 hr) 632 630 685 665 NO 1D (15 hr) 550 560 610 608 NO 5.2.3. VFA SPECIATION Identification of the individual acids formed during the acid-phase digestion of primary sludge is important, since it may furnish valuable information on the metabolic pathways involved in the process. The VFAs identified include: acetic, propionic, butyric, iso-butyric, valeric, 3-methylbutyric and 2-methylbutyric CHAPTER 5. RESULTS AND DISCUSSION 66 (Appendix D). The above VFAs are normally generated not only during the acidogenic digestion of municipal wastewaters (Perot et al., 1988), but also during the 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 an average 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 to propionic 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 and the 3 isomers of valeric acid account for the remaining 14%. The distribution of the individual acids is essentially the same in both systems. It is interesting to note that the percent VFA distribution, despite some variation in the minor acids from one run to another, is not affected by HRT (at least in the range tested), which is in contrast with the pattern followed by both the net VFA concentration and the production rate. This observation can lead to the speculation that either the majority of acid-producing bacteria is to some extent equally influenced by the variation in HRT, or possible differences in microbial activity counterbalance each other so that the final picture is basically the same. The percent distribution also reveals that there is a shift towards the higher molecular weight VFAs (iso-butyric and the 3 isomers of valeric acid) during the digestion of primary sludge, when compared to the influent VFA distribution. The average values for Stage 1, illustrated in Figure 5.3, show that a relative reduction yn az1 c-) (,) O TABLE 5.7. PERCENT VFA DISTRIBUTION AS A FUNCTION OF HRT VOLATILE^RUN 1B (6 hr)^RUN 1A (9 hr)^RUN 1C (12 hr)^RUN 1D (15 hr) FATTY MEAN ACID^CMR^UASB^CMR^UASB^CMR^UASB^CMR^UASB ACETIC 43.7^45.9^48.0^45.3^47.5^46.0^50.3^43.7^46.3 PROPIONIC^36.8^34.4^33.2^32.5^29.9^30.4^28.1^33.2^32.3 BUTYRIC^7.7^8.0^7.3^7.1^9.9^10.3^7.5^6.9^8.1 ISO-BUTYRIC 3.4 3.1 3.0 2.6 3.2 2.9 4.2 4.6 3.4 VALERIC 3.7 3.9 4.2 6.3 4.4 5.6 5.2 5.7 4.9 3-METHYLBUT. 2.8 2.7 2.9 4.0 3.3 3.1 3.2 4.0 3.2 2-METHYLBUT. 1.9 2.0 1.4 2.2 1.8 1.7 1.5 1.9 1.8 50 INFLUENT REACTOR I (CMR) REACTOR II (UASB) 40 30 20 10 BUTYRIC^VALERIC^2-METHYLBUTYRIC PROPIONIC^ISO-BUTYRIC^3-METHYLBUTYRIC VOLATILE FATTY ACIDS (VFAs) FIGURE 5.3. PERCENT VFA DISTRIBUTION (STAGE 1) 0\ 00 CHAPTER 5. RESULTS AND DISCUSSION^ 69 in propionic and butyric acids occurs with an increase in iso-butyric, valeric, 3- methylbutyric, and 2-methylbutyric acids. On the other hand, acetic acid percent distribution does not seem to be following any trend between the influent and the reactor contents. This shift can be primarily attributed to protein fermentation which results in the production of significant amounts of the above mentioned higher molecular weight VFAs. The rate of protein utilization is considerably higher in the bioreactors 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 conditions prevailing in such treatment units can further contribute to this phenomenon. 5.2.4. PARTICULATE ORGANIC CARBON SOLUBILIZATION Particulate organic material must first undergo liquefaction by extracellular enzymes, before being taken up by the bacteria. Since most of the substrate in primary sludge is in the particulate form (about 90% as indicated by the VSS/VS ratio), solubilization of organic matter is a crucial step in anaerobic digestion. Generally, the rate of hydrolysis depends upon the pH, temperature, the type of substrate, the nature of biomass, the size of the particles, and the remaining concentration of the biodegradable suspended matter (Eastman and Ferguson, 1981). Substrate solubilization can be estimated from a number of non-specific parameters such as COD, TOC, TSS and VSS. These parameters were routinely CHAPTER 5. RESULTS AND DISCUSSION^ 70 measured and the results are summarized in Appendix C. As illustrated by the profiles of influent and reactor soluble COD concentrations depicted in Figure 5.4 (Runs 1C and 1D), a distinct increase in COD occurs in both reactors which is the result of substrate conversion from a particulate to 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, expressed as mg of net soluble COD or TOC per mg of VSS per day (Table 5.8). All three maximum values correspond to an HRT of 12 hours, which coincides with the time required for optimum VFA production. In addition, the overall trend is very similar to the one described in the previous section for VFAs. TABLE 5.8. SPECIFIC SOLUBILIZATION RATES OF COD AND TOC AS A FUNCTION OF HRT RUN HRT (hr) COD RATE (mgCOD/mgVSS*d) TOC RATE (mgTOC/mgVSS*d) CMR UASB CMR UASB 1B 6 0.159 0.160 0.054 0.054 lA 9 0.163 0.168 0.057 0.060 1C 12 0.187 0.198 0.070 0.070 1D 15 0.169 0.175 0.064 0.063 The percent soluble COD in the form of VFAs (calculated by converting the VFAs to COD using the appropriate factors shown in Table E5, Appendix E) as a 1.8 1.6 a--.^1.4 DE) oz 7  1.22 i 1.0 z F. U —0^0.8 Z 0 O 0.6 0.4 0.2 1.4 1.3 15126 0.6 0.7 a^1.2---. Er) Z 1.1To'O 21=- . 1.0Q  c 00 z = w b 0.90 z 0O 0.8 9 HRT (hr) CHAPTER 5. RESULTS AND DISCUSSION^ 71 10020 ^ 40 ^ 60 ^ 80 TIME (d) FIGURE 5.4. SOLUBLE COD PROFILE (RUNS 1C & 1 D) FIGURE 5.5. NET COD SOLUBILIZATION AS A FUNCTION OF HRT CHAPTER 5. RESULTS AND DISCUSSION^ 72 function of HRT is presented in Figure 5.6. Although the percent volatile acid COD increases with a change in HRT from 6 to 9 hours, no remarkable variation is observed beyond this point. Similar percentages have been obtained in both reactors for all HRTs. The results suggest that the conversion rate of soluble substrates to VFAs may have reached a plateau. In other words, the rate of metabolism of soluble extracellular intermediate products to VFAs appears to be independent of HRT, above a certain minimum value. A smaller percent volatile acid COD, observed at 6 hours HRT, indicates that the mechanisms for acid generation are influenced by the short HRT more drastically than those involved in hydrolysis or the production of extracellular metabolic intermediates. The extent of organic substrate solubilization can be viewed from a diametrically opposite perspective, namely from the destruction of suspended solids. The percent VSS and TSS reduction was based on a mass balance around the reactors at steady-state conditions. Mass balances were performed in two distinct manners according 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, while the "moving average mass balance" involves averaging the values from multiple balance periods, each equivelent to 1 SRT in length. As it is evident from the example illustrated in Table E6, Appendix E, the two methods yield similar results. Suspended solid solubilization increases with HRT, but the percent change becomes smaller at HRTs higher than 9 hours (Table 5.9). The gradually diminishing sensitivity of the rate of hydrolysis at higher HRTs might be due to the fact that it is actually approaching a maximum value beyond which it probably becomes CHAPTER 5. RESULTS AND DISCUSSION ^ 73 independent of HRT. The relatively high percent VSS reduction obtained provides an additional evidence that the particulate complex substrates in primary sludge are amenable to solubilization. TABLE 5.9. PERCENT VSS AND TSS REDUCTION AS A FUNCTION OF HRT RUN HRT (hr) VSS (%) TSS (%) CMR UASB CMR UASB 1B 6 44.2 43.8 46.1 45.5 lA 9 57.6 63.6 58.8 63.7 1C 12 63.1 70.6 64.2 69.6 1D 15 67.7 72.5 68.3 71.3 The UASB reactor shows an overall better performance (except at the shortest HRT - Run 1B) in hydrolyzing the particulate organic material. This behavior, which is also reflected in the COD solubilization rates (Table 5.8), does not result in higher VFA production rates. This is probably due to the presence of a different mix of microorganisms in the UASB reactor which generate a greater variety of intermediate products during the degradation process. The percent TSS reduction results (Table 5.9) are essentially identical to those obtained from the VSS analysis. Since the VSS account for about 75 to 80% of the TSS in the feed, a substantial fraction of the particulate inorganics (approximately equal 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^ 74 5.2.5. SUBSTRATE DEGRADATION Carbohydrates, proteins and lipids in that order are the three primary sources of organic substrates in primary sludge. Since they basically occur in particulate form, they have to be first hydrolyzed by the action of specific enzymes before undergoing further degradation. The fermentation of carbohydrates is one of the main pathways for the production of VFAs. In domestic wastewaters, carbohydrates are present in the form of polymers, principally as "designated cellulose". The term "designated cellulose" has been proposed by Hobson (1980) to denote that this is a material largely defined by the method of analysis rather than by chemical constitution. Designated cellulose mostly consists of residues of toilet and similar papers and the remains of cooked vegetables in human feces. It can be relatively easily hydrolyzed by cellulases (Ng et al., 1977). The high percentages of total carbohydrate utilization observed, ranging from 43 to 77% (based on a mass balance at steady-state operation), are in agreement with the above statement (Table 5.10). It is apparent that the percent conversion values are a function of HRT, which emphasizes the role of this parameter on the enzymatic hydrolysis of carbohydrates. Protein and lipid utilization patterns follow a trend similar to that of carbohydrates, regarding the influence of HRT (Table 5.10). The conversion percentages, however, are remarkably different. Proteins are degraded at much lower rates than the other two organic classes. In general, proteolytic activity has been found to take place in digesters using a number of feedstocks, but the overall INFLUENT MASS LOADING LOSS IN EFFLUENT AND WASTAGE SYSTEM I (CMR) SYSTEM II (UASB) -- -àA.A..a..^-^.. - CHAPTER 5. RESULTS AND DISCUSSION^ 75 80 70 60 50 40 30 20 10 0^ 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 HRT 30 28 26 24 22 20 18 16 14 12 10 8 6 4 2 0 CHAPTER 5. RESULTS AND DISCUSSION ^ 76 TABLE 5.10. PERCENT SUBSTRATE DEGRADATION AS A FUNCTION OF HRT RUN HRT (hr) CARBOHYDR (%) PROTEINS (%) LIPIDS (%) CMR UASB CMR UASB CMR UASB 1B 6 43.5 43.1 26.4 24.5 63.4 47.5 lA 9 56.1 66.6 35.6 38.5 72.4 53.5 1C 12 60.6 73.4 42.9 45.0 80.9 62.0 1D 15 64.2 76.8 47.7 47.6 83.2 67.3 breakdown percentages are moderate (Summers and Bousfield, 1980; Gujer and Zehnder, 1983). On the other hand, there has been a controversy regarding the extent of lipid degradation during the acid-phase process. Some investigators have reported that lipid dissimilation is minimal during the acid-phase step (Mahr, 1967; Eastman and Ferguson, 1981), while others have observed a significant utilization of lipids (Chynoweth and Mah, 1971; Ghosh, 1987). This subject will be treated in some detail in Section 5.6.5. Concerning the effect of reactor configuration on substrate dissimilation, both systems exhibit a fairly similar behavior in protein reduction rates, but the degradation pattern of carbohydrates and lipids are distinctly and consistently different (Figures 5.7 to 5.9). Lipids are broken down more efficiently in the CMR unit, while higher rates of carbohydrate utilization are observed (except at the shortest HRT - Run 1B) in the UASB reactor. LOSS IN EFFLUENT AND WASTAGE SYSTEM I (CMR) ® SYSTEM II (UASB) -ii g LOSS IN EFFLUENT AND WASTAGE ECEa SYSTEM I (CMR) ZZZ SYSTEM II (UASB) 77CHAPTER 5. RESULTS AND DISCUSSION 10 9 8 7 6 5 4 3 2 1 0 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 HRT RUN 1B (6 hr)^RUN 1A (9 hr)^RUN 1C (12 hr)^RUN 1D (15 hr) FIGURE 5.9. LIPID DEGRADATION AS A FUNCTION OF HRT 13: g CHAPTER 5. RESULTS AND DISCUSSION^ 78 5.3. THE EFFECT OF SRT - STAGE 2 5.3.1. SRT AS A CONTROL PARAMETER Another operational variable which can be used as a selective factor by imposing a stress on bacterial communities is the solids retention time (SRT). It is the average time allowed for a microorganism to remain in the reactor and can be defined as the amount of suspended solids in the reactor divided by the amount of suspended solids leaving the reactor per day. The SRT governs the types of organisms which eventually predominate in the system because it interferes directly with their generation time. The physiology, environmental requirements, and growth kinetics of the acidogenic and the methanogenic groups of microbes may differ greatly from each other. It has been reported (Ghosh and Klass, 1978) that the maximum specific growth rate of the acid-producing bacteria can be up to one order of magnitude higher than that of the methane-producing organisms. This suggests that it is possible to maximize VFA production in an acid-phase digester by operating the system at an SRT below some critical value. The critical SRT (which in many cases coincides with the 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 (Section 2.5) SRT and HRT are almost identical because of the use of batch reactors or conventional continuous-flow systems without solids recycle. The SRT/HRT ratio can be slightly increased to 1.5-2 as a result of withdrawal of digester supernatant (Henze CHAPTER 5. RESULTS AND DISCUSSION 79 and Harremoes, 1983). Nevertheless, SRT and HRT are two different parameters and have different effects on the biological process. For this reason, no clear distinction can be made between the individual influence of the two parameters on the acidogenic phase in these previous investigations. In this study, HRT and SRT were independently controlled through appropriate design and operational strategies. The very nature of the UASB reactor allows for individual manipulation of these two variables, while for the same reason the CMR unit was modified by adding a clarifier with a solids recycling system. The ultimate goal has been to operate at an HRT as low as possible to minimize reactor volume and associated capital costs; and concurrently to maintain a reasonably long SRT to promote growth and proliferation of the acid-generating organisms, process stability and minimal sludge production, without inducing growth of methane-forming bacteria. Based on the results from Stage 1, the optimum HRT for VFA production in both reactors is 12 hours. The SRT in that stage was kept constant at 10 days. In Stage 2, an SRT variation from 5 to 20 days is investigated (resulting in a range of SRT/HRT ratios from 10 to 40). The chemical parameters measured during Stage 1 were also recorded for Stage 2 and the data are presented in Appendix C (Tables C21 to C35). 5.3.2. VFA PRODUCTION AND SPECIATION Volatile fatty acids were the main soluble compounds generated during this set CHAPTER 5. RESULTS AND DISCUSSION 80 of 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 a constant HRT of 12 hours), results in higher VFA concentrations in both reactors. In general, the variation in SRT does not seem to have a profound effect on VFA production, with the exception of Run 2C (5 days SRT). The drastic drop in VFA concentration observed in this case indicates that such a short SRT may impose a strong stress on the metabolic activity of the acidogenic bacteria. The operational stability of either system also suffers, as reflected on the substantially higher standard deviation (STD) values for this run for almost all the chemical parameters analyzed (Tables C31 to C35, Appendix C). Information presented in Table 5.11 shows that the net VFA specific production rate increases with SRT up to 15 days, but a plateau appears to be reached at this value. The influence of SRT on the VFA production rate is rather moderate (as in the previous case), except at the shortest SRT, where the production rate is reduced to almost one half of that calculated at 10 days. Overall, the CMR TABLE 5.11. VFA SPECIFIC PRODUCTION RATE AS A FUNCTION OF SRT RUN SRT (d) CMR (mgVFA/mgVSS*d) UASB (mgVFA/mgVSS*d) 2C 5 0.053 0.056 1C 10 0.101 0.103 2A 15 0.125 0.110 2B 20 0.119 0.109 155 10 20 800 700 0 600 E 500 400 w 300 0 0 200 100 0 70 60 50 40 30 20 10 0 CHAPTER 5. RESULTS AND DISCUSSION ^ 81 SRT (d) FIGURE 5.10. NET VFA PRODUCTION AS A FUNCTION OF SRT 5 ^ 10 ^ 15 ^ 20 SRT (d) FIGURE 5.11. PERCENT VFA SPECIATION AS A FUNCTION OF SRT CHAPTER 5. RESULTS AND DISCUSSION 82 system is slightly more effective in producing VFAs at SRTs of 15 and 20 days (by 14 and 9% respectively) than the other one, but at lower SRTs both systems exhibit similar rates of product formation. (The standard error of the mean for VFA specific production rates ranges from 3 to 5%). The VFA speciation results (Table 5.12) are in agreement with many of the findings mentioned in Stage 1 (Section 5.2.3) such as those concerning the two predominant acids (acetic and propionic) or the influence of reactor configuration (CMR and UASB). It is interesting to note that the VFA distribution is, to some extent, 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 very similar in both systems) is plotted as a function of SRT in Figure 5.11. The most remarkable difference occurs in the case of the 4 "minor" acids (iso-butyric, valeric, 3-methylbutyric and 2-methylbutyric). Their percent distribution increases dramatically with SRT (almost doubles from 5 to 20 days). For both acetic and propionic acid the percent distribution declines slightly with an increase in SRT. In the case of butyric acid, a maximum is reached at 10 days. The overall picture suggests that different pathways for VFA production may predominate at various SRTs. Short SRTs seem to favor the generation of straight C2 to C4 VFAs, but at longer SRTs more branched C4 and C 5 acids are formed. Although the possible presence of slower-growing microorganisms at longer SRTs cannot be excluded, the direct association of the 4 higher molecular weight VFAs with protein fermentation provides strong evidence that this phenomenon is a result of proteinaceous metabolism, as discussed in the following section. TABLE 5.12. PERCENT VFA DISTRIBUTION AS A FUNCTION OF SRT VOLATILE FATTY ACID RUN 2C (5 d) RUN 1C (10 d) RUN 2A (15 d) RUN 2B (20 d) CMR UASB CMR UASB CMR UASB CMR UASB ACETIC 50.5 48.0 47.5 46.9 43.8 45.2 43.8 44.6 PROPIONIC 31.6 32.3 29.9 30.4 29.9 28.2 27.5 26.4 BUTYRIC 6.8 7.6 9.9 10.3 8.1 7.3 7.0 7.1 ISO-BUTYRIC 3.3 2.9 3.2 2.9 3.8 3.7 5.2 5.5 VALERIC 4.0 4.5 4.4 5.6 6.9 8.0 7.9 8.1 3-METHYLBUTYR. 2.4 2.8 3.3 3.1 5.1 5.4 6.0 5.3 2-METHYLBUTYR. 1.4 1.9 1.8 1.7 2.4 2.2 2.6 3.0 ALL 4 MINOR VFAs 11.1 12.1 12.7 12.7 18.2 19.3 21.7 21.9 CHAPTER 5. RESULTS AND DISCUSSION^ 84 5.3.3. ORGANIC CARBON SOLUBILIZATION AND SUBSTRATE DEGRADATION The majority of observations made about the VFA data set are equally applicable to COD and TOC results. For example, the net COD concentration as a function of SRT (Figure 5.12) shows a great degree of similarity with the VFA production (Figure 5.10), dropping sharply at an SRT of 5 days and approaching a plateau 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). A plausible explanation for this phenomenon is that at short SRTs the biochemical pathways followed for VFA production from soluble biopolymers are much more influenced than those involved in hydrolysis. If the same microbial community is responsible for the conversion of particulate organic matter to VFAs, it can be concluded 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 increases drastically with increasing SRT, approaching the 90% level at 20 days (Figure 5.13). It is apparent that longer SRTs favor the conversion of soluble metabolic intermediates 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 the variation 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 4 4 ..2^4 4^ 4^, 4 4 1^■ 4 - '^1 '' 4 4^- ^4^i^4 ►4 4 4 4 4 .^4^ : ^1 4 -^4^ 4^ 4 ► 4 4 ^ ►4 .-... 4^ 4 4^ 4^-1 4 '--^: 4 4 ,-,._^4^ 4 4^ 4^--a-. 4 4^- •► ►4 _►4►4 4 4 -^4^-----^4 '^4  7̂ 4 4 -. 4^ 4   -^4  ^--^4 4 4^ 4 -^4 -^A 4 4^ 4 CHAPTER 5. RESULTS AND DISCUSSION^ 85 1.4 1.2 0) E^1.0 z 0 < c* 0.8cc = F- 0 Z L.L.I 0 Z 0.6 0 0.4 0.2  5 ^ 10 ^ 15 ^ 20 SRT (d) FIGURE 5.12. NET COD SOLUBILIZATION AS A FUNCTION OF SRT 100 90 80 70 60 Lu 50 oCLu 0- 40 30 20 10 RUN 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) 0 CHAPTER 5. RESULTS AND DISCUSSION ^ 86 comparison of VSS and TSS values and the influence of reactor configuration are also valid for Stage 2. TABLE 5.13. SPECIFIC SOLUBILIZATION RATES OF COD AND TOC AS A FUNCTION OF SRT RUN SRT (d) COD RATE (mgCOD/mgVSS*d) TOC RATE (mgTOC/mgVSS*d) CMR UASB CMR UASB 2C 5 0.184 0.192 0.066 0.066 1C 10 0.187 0.198 0.070 0.070 2A 15 0.200 0.193 0.078 0.072 2B 20 0.184 0.181 0.072 0.070 TABLE 5.14. PERCENT VSS AND TSS REDUCTION AS A FUNCTION OF SRT RUN SRT (d) VSS (%) TSS (%) CMR UASB CMR UASB 2C 5 62.8 66.2 64.6 66.9 1C 10 63.1 70.6 64.2 69.6 2A 15 67.1 75.2 68.4 75.4 2B 20 65.6 73.4 66.7 75.3 Table 5.15 shows the percent utilization of carbohydrates, proteins and lipids calculated from the respective mass balances. Considering the suspended solids behavior in the reactors as described above, the three organic classes of interest are not expected to be affected significantly by the variation in SRT. Although, this is CHAPTER 5. RESULTS AND DISCUSSION^ 87 basically true for carbohydrates and lipids, the protein degradation pattern appears to 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 anaerobic metabolism of proteins (Gottschalk, 1986), the increase in protein dissimilation is in agreement with the higher production of these 4 acids at longer SRTs (Table 5.12). TABLE 5.15. PERCENT SUBSTRATE DEGRADATION AS A FUNCTION OF SRT RUN SRT (d) CARBOHYDR (%) PROTEINS (%) LIPIDS (%) CMR UASB CMR UASB CMR UASB 2C 5 59.0 70.4 38.7 37.4 83.1 64.5 1C 10 60.6 73.4 42.9 45.0 80.9 62.0 2A 15 62.5 78.8 51.2 48.7 84.5 69.8 2B 20 61.0 76.5 54.1 55.2 81.4 66.7 Finally, the reactor configuration affects the utilization patterns of the three organic classes in exactly the same way as outlined in Stage 1. The rate of carbohydrate degradation is significantly higher in the UASB reactor (Figure 5.14), lipids are solubilized more effectively in the CMR unit and protein dissimilation rates are similar in both systems (Figures 5.15 and 5.16). INFLUENT MASS LOADING LOSS IN EFFLUENT AND WASTAGE SYSTEM I (CMR) EZZ1 SYSTEM II (UASB) 0 RUN 2C (5 d)^RUN 1C (10 d)^RUN 2A (15 d)^RUN 2B (20 d) 6 5 4 3 2 1 CHAPTER 5. RESULTS AND DISCUSSION 16 14 LOSS IN EFFLUENT AND WASTAGE SYSTEM I (CMR) EZZ SYSTEM II (UASB) 88 rn 2 RUN 2C (5 d)^RUN 1C (10 d)^RUN 2A (15 d)^RUN 2B (20 d) FIGURE 5.14. CARBOHYDRATE DEGRADATION AS A FUNCTION OF SRT FIGURE 5.15. PROTEIN DEGRADATION AS A FUNCTION OF SRT Si^3 g 2 0 RUN 2C (5 d)^RUN 1C (10 d)^RUN 2A (15 d)^RUN 2B (20 d) 4 1 5 LOSS IN EFFLUENT AND WASTAGE SYSTEM I (CMR) EZZ] SYSTEM II (UASB) CHAPTER 5. RESULTS AND DISCUSSION^ 89 FIGURE 5.16. LIPID DEGRADATION AS A FUNCTION OF SRT 5.3.4. GAS PRODUCTION Gas generation is the ultimate goal in the two-phase anaerobic digestion process. 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 pathways followed for substrate metabolism (Appendix A). Ideally, the methane content in the reactor should be negligible. In practice, however, varied amounts of methane have been 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 in the co-existence of heterotrophic methane producers, or the presence of certain fast- CHAPTER 5. RESULTS AND DISCUSSION^ 90 growing autotrophic methanogenic organisms such as Methanobacterium, or both (Novaes, 1986). The relatively low gas production obtained (Table E4, Appendix E) indicates that methanogenesis was successfully suppressed throughout this experimental study. Despite the small volumes of gas generated, certain interesting observations can be made. Gas production appears to be independent of HRT (with the notable exception of 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 substantially encouraged during this run, as compared to any other set of experimental conditions. When the values of the two operational parameters are distinctly different (resulting in SRT/HRT ratio much higher than one), it is possible that the prolonged availability of food may trigger first the mechanism for methanogenesis. Therefore, longer HRTs may stimulate gas production by allowing better contact between the soluble substrates (i.e. VFAs) and the already present methanogens, while shorter HRTs severely limit methanogen activity without significantly affecting acidogenesis. Several analyses of gas composition showed that CO 2 is the predominant gas in 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 70:25:5 ratio 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), no significant changes in gas composition were observed among different runs. CHAPTER 5. RESULTS AND DISCUSSION^ 91 5.4. REPLICATION AND THE EFFECT OF FEED SOURCE - STAGE 3 5.4.1. REPLICATION EXPERIMENTS (RUN 3A) To determine the degree of replication possible in acid-phase digesters, two runs (1C and 3A) were operated under identical conditions, except that the first experiment took place in late spring - early summer (May - June) and the second one in winter (January - February). The operating conditions corresponding to Run 1C (HRT: 12 hours, SRT: 10 days) were selected for the rest of the study. Although longer SRTs resulted in slightly better VFA production rates (Runs 2A and 2B), an SRT of 10 days is considered as a "reasonable" value to ensure high VFA production and at the same time 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 replicate units. The distribution of the individual volatile fatty acids is about the same for both runs as well (Table 5.17). Furthermore, the variation in standard deviation of all measured values is not statistically significant, according to t-test (Table 5.18). On the 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 influent collection (summer - winter) does not seem to play any role in the process. CHAPTER 5. RESULTS AND DISCUSSION ^ 92 TABLE 5.16. COMPARISON OF REPLICATION RESULTS AT IONA ISLAND WWTP ORGANIC PARAMETER CMR SYSTEM UASB SYSTEM RUN IC-S RUN 3A-W RUN 1C-S RUN 3A-W VFA SP. PROD. RATE 0.101 0.104 0.103 0.099 COD SP. SOLUB. RATE 0.187 0.198 0.198 0.197 TOC SP. SOLUB. RATE 0.070 0.070 0.070 0.074 COD IN VFA FORM 71.0 69.5 70.9 68.5 VSS 63.1 65.5 70.6 68.7 TSS 64.2 65.9 69.6 69.9 CARBOHYDRATES 60.6 59.8 73.4 71.8 PROTEINS 42.9 43.5 45.0 43.5 LIPIDS 80.9 79.2 62.0 61.2 Note: 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) VOLATILE FATTY ACID RUN 1C (I.I.) RUN 3A (I.I.) RUN 3B (L.G.) CMR UASB CMR UASB CMR UASB ACETIC 47.5 46.0 47.6 45.4 45.1 46.6 PROPIONIC 29.9 30.4 30.7 32.0 35.3 33.0 BUTYRIC 9.9 10.3 9.4 9.5 8.9 8.6 ISO-BUTYRIC 3.2 2.9 2.8 2.3 4.4 4.6 VALERIC 4.4 5.6 4.9 5.4 4.1 3.8 3-METHYLBUT. 3.3 3.1 3.0 3.7 1.4 2.8 2-METHYLBUT. 1.8 1.7 1.6 1.7 0.8 0.8 Note: I.I.=Iona Island WWTP, L.G.=Lions' Gate WWTP. CHAPTER 5. RESULTS AND DISCUSSION ^ 93 TABLE 5.18. t-TEST RESULTS FOR RUNS 1C, 3A (IONA ISLAND WWTP) AND 3B (LIONS' GATE WWTP) (Level of significance a=0.05) ORGANIC PARAMETER RUNS 1C and 3A  t  <2.074 RUNS 1C & and 3B, It 3A (comb.) <1.960 CMR UASB CMR UASB VFA SP. PROD. RATE 0.548 0.767 3.108 2.388 COD SP. SOLUB. RATE 0.930 0.107 0.741 0.565 TOC 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 0.696 5.4.2. THE EFFECT OF FEED SOURCE (RUN 3B) A common attribute of biological treatment processes is that they are often influenced by the nature of the feed used. Primary sludges from different sources may behave in a different way during the acid-phase digestion step (Chynoweth and Mah, 1971). To investigate the possible dependency of the process on influent characteristics, the two reactors were operated at 12 hours HRT and 10 days SRT (identical conditions with Runs 1C and 3A), using primary sludge from another source which had the composition shown in Table 5.2. CHAPTER 5. RESULTS AND DISCUSSION 94 A 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 comparative purposes. It is interesting to note that all four "general" variables (COD and TOC solubilization rates, and VSS and TSS reduction percentages) are quite similar, but the "specific" parameters (with the exception of proteins) exhibit some trend of variation. For example, the VFA production rates are reduced in Run 3B by about 12% and the percent COD in the form of VFAs by about 20% in both reactors, when compared to Runs 1C and 3A. Moreover, a relatively lower rate of lipid hydrolysis and an accordingly higher rate of carbohydrate breakdown have been observed, which suggests that the lipolytic activity of lipases was to some extent adversely affected but that of carbohydrate-hydrolyzing enzymes was encouraged when the alternative feed was used. Although, as illustrated in Table 5.19, only the variation in VFA production rates and the related COD in the form of VFAs percent values can be classified as significantly different from a strictly statistical point of view (t-test; Miller et al., 1990), the variation in both carbohydrate and lipid utilization patterns are consistent and may be important especially when compared to the negligible percent changes observed during the replication experiments (Table 5.18). The percent VFA distribution shows no appreciable changes regarding the major 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 of 3-methylbutyric and 2-methylbutyric has accordingly decreased in Run 3B. This can be better illustrated by comparing the relative ratios of the two branched C 5 VFAs to iso-butyric acid (Table 5.20). This ratio not only drops dramatically in the case TABLE 5.19. COMPARISON OF RESULTS FROM DIFFERENT FEED SOURCES ORGANIC CMR SYSTEM^ UASB SYSTEM PARAMETER^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^YES COD SP. SOLUB. RATE^0.193^0.195^+0.8^NO^0.198^0.202^+2.3^NO TOC SP. SOLUB. RATE^0.070^0.071^+1.4^NO^0.072^0.069^-4.2^NO COD IN VFA FORM 70.3 55.2 -21.5 YES 69.7 55.7 -20.1 YES VSS 64.3 64.6 +0.5 NO 69.7 70.4 +1.1 NO TSS 65.1 63.2 -2.8 NO 69.8 70.7 +1.4 NO CARBOHYDRATES 60.2 64.1 +6.5 NO 72.6 78.7 +8.5 NO PROTEINS 43.2 44.3 +2.6 NO 44.3 43.5 -1.8 NO LIPIDS 80.1 72.9 -8.9 NO 61.6 57.4 -6.8 NO Note: Specific rates are expressed as mg(Parameter)/mgVSS*d, the rest of the values are (%); Runs 1A and 3B were from Iona Island WWTP, while Run 3B was from Lions' Gate WWTP. cd) (,) (.4 O CHAPTER 5. RESULTS AND DISCUSSION^ 96 of the last run, but also the values calculated are the lowest values obtained in the entire study for either reactor. Since all three acids are directly related to protein metabolism, the above observation suggests a possible difference in protein (i.e. amino acid) composition between the two wastewater sources. The primary sludge from Iona Island WWTP, for example, may contain a smaller amount of valine which can produce iso-butyric acid via the Stickland reaction (Eq. 2.8), and/or higher amounts 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 AND ISO-BUTYRIC ACID (STAGE 3) RUN REACTOR I (CMR) REACTOR II (UASB) Bran. C5 VFAs (%) ISO-BUT. (%) RATIO Bran. C5 VFAs (%) ISO-BUT. (%) RATIO 1C (I.I.) 5.1 3.2 1.59 4.8 2.9 1.66 3A (I.I.) 4.6 2.8 1.64 5.0 2.3 2.17 3B (L.G.) 2.2 4.4 0.50 3.6 4.6 0.78 Note: I.I.=Iona Island WWTP, L.G.=Lions' Gate WWTP Taking into account the spectrum of variations observed using sludge from a different source, it can be concluded that the nature of primary sludge may have an effect upon the hydrolysis of particulate organic matter and furthermore that the bacteria may use different metabolic pathways to utilize certain hydrolysis products. This behavior indicates a higher degree of sensitivity to changes in initial conditions than to changes in operational conditions. CHAPTER 5. RESULTS AND DISCUSSION^ 97 5.5. THE EFFECT OF pH - STAGE 4 5.5.1. pH AS A SELECTIVE PARAMETER The pH of a bioreactor determines the possibility of survival and the rate of reproduction of any microbial species present in this particular environment. In many cases, however, the primary determinants of pH are the organisms themselves (if the pH is not externally adjusted). Microorganisms are able to alter the pH of their environment through various metabolic activities. These changes might be advantageous or not to the microbes that cause them. Some species can create environments in which very few other organisms are able to survive, and if they themselves are not adversely affected by the conditions they create, they may thus eliminate competition. On the other hand, the alterations in pH may encourage the predominance of a competitor. For example, bacteria may produce acidic products that decrease the pH value in an insufficiently buffered environment and allow fungi to predominate (Gaudy and Gaudy, 1980). The ability of microorganisms to alter pH is the basis of important interactions between species. Since pH affects growth rate, changes in pH may cause dramatic shifts in the relative numbers of different species in the population. It has been found that many aspects of microbial metabolism are greatly influenced by the variations in pH over the range within which the organisms can grow (Sakharova and Rabotnova, 1976). These aspects include utilization of carbon and energy sources, efficiency of substrate dissimilation, synthesis of protein and different types of CHAPTER 5. RESULTS AND DISCUSSION 98 storage material, and release of metabolic products from the cell. Moreover, pH variations can affect cell morphology and structure and, therefore, flocculation and adhesion phenomena (Forage et al., 1985). All of the above factors play a crucial role in determining the ability of a given microbial species to compete with others in a heterogeneous environment. 5.5.2. BUFFERING CAPACITY The buffering capacity of a biological system is manifested by the degree of its resistance to pH changes. In acid-phase digestion, many inorganic and organic buffer systems such as carbonate/bicarbonate, phosphate, borate, silicate, citrate, and proteins may be active in the pH range of interest. The resistance to acidification is a 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 either reactor, after an initial drop during acclimation, were exceedingly stable during the steady-state operation (Appendix B). The coefficient of variation (CoV) was between 2 and 5% for all runs (Table 5.21). This is mainly attributed to the presence of proteins and VFAs, since the buffering capacity of the Vancouver water supply is very low (total alkalinity is usually between 100 and 150 mg/L as CaCO 3 ). Proteins and their hydrolysis products (amino acids) act as both hydrogen donors 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 well CHAPTER 5. RESULTS AND DISCUSSION^ 99 as other ionizable groups (eg. imidazolyl, sulphide etc.) in the side chains of many acids (Gaudy and Gaudy, 1980). Thus, proteins in solution can buffer against changes in pH. Individual amino acids exert their maximum buffering potential at different pH values depending on the number of amino and carboxyl groups they possess. For example, two of the most prevalent amino acids in domestic wastewaters, aspartic acid (pKAi = 3.86) and glutamic acid (pK A , = 4.07) are most effective in acidic environments (CRC Handbook of Chemistry and Physics, 1981). TABLE 5.21. pH VALUES IN BIOREACTORS (STAGES 1 TO 3) RUN REACTOR I (CMR) REACTOR II (UASB) MEAN STD CoV (%) MEAN STD CoV (%) lA 5.23 0.17 3.3 5.25 0.15 2.9 1B 5.27 0.11 2.1 5.33 0.13 2.4 1C 5.01 0.25 5.0 4.96 0.23 4.6 1D 5.06 0.14 2.8 5.10 0.13 2.5 2A 5.17 0.14 2.7 5.03 0.13 2.6 2B 5.23 0.10 1.9 5.09 0.11 2.2 2C 5.63 0.18 3.2 5.52 0.11 2.0 3A 5.15 0.17 3.3 4.98 0.13 2.6 3B 5.03 0.16 3.2 5.05 0.14 2.8 Volatile fatty acids can also act as buffers in a pH range close to their pKA values. The two major products of acidogenic digestion, acetic acid (pK A = 4.76) and propionic acid (pKA = 4.87), attain their highest buffering capacity at pH of about CHAPTER 5. RESULTS AND DISCUSSION^ 100 5 (Sawyer and McCarty, 1979). The utilization of proteins and amino acids during the digestion process is counterbalanced by the generation of VFAs, and as a result, at steady-state conditions, the pH of the system remains fairly constant. The "equilibrium" pH range attained, however, may be a function of the relative concentrations of the reactants and the products involved. VFA concentrations below 400 mg/L resulted in an increase in pH in either reactor, but no appreciable variation in pH (less than 0.3 units) occurred at concentrations ranging from 400 to 750 mg/L (Figures 5.17 and 5.18). To further investigate the role of pH in the process, two additional experiments were conducted at controlled conditions. Dilute solutions (0.02N) of hydrochloric acid and sodium hydroxide were used to maintain the pH at selected values. Both chemicals were specifically chosen because of their low-level interference with the metabolic pathways involved. Sodium is generally tolerated by most microorganisms, particularly in the presence of potassium. Furthermore, chloride ions are not inhibitory to bacteria at the concentrations attained (Forage et al., 1985). 5.5.3. VFA PRODUCTION AND SPECIATION The total net VFA production (as acetic acid) at steady-state conditions is depicted in Figure 5.19. VFA concentration does not appear to be affected, in either reactor, by a drop in pH from 5.1 to 4.5 (Runs 1C and 4A), but in Run 4B an increase in pH to about 6.1 results in significantly lower (25 to 30%) total acid i. t 600 rn E Z 500 0 5- 2 400 L^300.%0 z 0 0 200 > 100 5.65.4 5.5 5.74.9^5.0^5.1^5.2^5.3 REACTOR pH FIGURE 5.17. REACTOR pH AND VFA CONCENTRATION (CMR SYSTEM) 600 500 400 300 200 5.4 5.65.54.9^5.0^5.1^5.2^5.3 REACTOR pH FIGURE 5.18. REACTOR pH AND VFA CONCENTRATION (UASB SYSTEM) 800 700 100 CHAPTER 5. RESULTS AND DISCUSSION 800 700 101 0 0 CHAPTER 5. RESULTS AND DISCUSSION ^ 102 i 600 < I cr) E 500 Z 0 Fr-<ccI-- 400zw C.) Z 0 0 300 700 200 6.14.5^4.7^4.9^5.1^5.3 ^ 5.5 ^ 5.7 ^ 5.9 pH RANGE FIGURE 5.19. NET VFA PRODUCTION AS A FUNCTION OF pH production, suggesting a sensitive response of the process at the above pH range. Since the reactor and effluent VFA concentrations of the CMR system are actually the same (Appendix C), no volatilization losses occurred at any pH value. As in the Stage 1 results, the VFA concentration is higher in the UASB reactor, but the specific production rate is similar in both systems (Table 5.22). This is a further indication that the ability of bacteria to produce VFAs is independent of the reactor configuration, at least within the HRT and pH ranges investigated. Both the VFA production rate and concentration follow comparable trends at different pH values. The optimum pH range for the acidogenic phase can be influenced by the characteristics of the feed and operating conditions. The results from this study show CHAPTER 5. RESULTS AND DISCUSSION^ 103 that the runs (without pH control) associated with high VFA production were largely operated at pH values between 5.0 and 5.3, which can be considered as an "optimum" range. The same pH range has been obtained in a UASB reactor treating a synthetic sludge (Fongsatitkul, 1992). It has been frequently reported that acid-phase bioreactors were successfully operated at pH between 5.0 and 6.0 (Eastman and Ferguson, 1981; Zoetemeyer et al., 1982b; Breure et al., 1986; Kisaalita et al., 1989). In some cases, however, the optimum pH was significantly higher. Joubert and Britz (1986) and Perot et al. (1988) have found that the maximum concentration of acid- phase products was achieved at pH 6.5 and 6.8 respectively. The higher optimum pH observed in the last two studies may be due to the composition of the feed (sucrose in the former study and a mixture of primary and waste activated sludge in the latter) or the type of inoculum used. TABLE 5.22. VFA SPECIFIC PRODUCTION RATE AS A FUNCTION OF pH RUN pH RANGE CMR (mgVFA/mgVSS*d) UASB (mgVFA/mgVSS*d) 4A 4.3 - 4.6 0.097 0.100 1C 4.9 - 5.2 0.101 0.103 4B 5.9 - 6.2 0.076 0.078 That the extracellular pH has a strong effect on the pathways of metabolism and products generated by microorganisms is a well known phenomenon in biological processes. However, the mechanism by which metabolic reactions are regulated is not CHAPTER 5. RESULTS AND DISCUSSION^ 104 well understood. Often the response to pH in terms of product formation seems to be a logical adaptation by the microbial species (Forage et al., 1985). As shown in Table 5.23, alterations in pH can also influence the percent VFA distribution. A relative increase in propionic acid is observed at pH 4.3-4.6 (Run 4A) and in butyric acid at pH 5.9-6.2 (Run 4B), as compared to the uncontrolled pH of 5.0-5.3 (Run 1C). In contrast, the range of pH studied (4.3-6.2) does not seem to have any appreciable effect on the percent distribution of the other VFA present. Moreover, acetic acid yield has been found by others to be independent of pH values from 4.5 to 7.0 (Hsu and Yang, 1991). TABLE 5.23. PERCENT VFA DISTRIBUTION AS A FUNCTION OF pH VOLATILE FATTY ACID RUN 4A (pH=4.3-4.6) RUN 1C (pH=4.9-5.2) RUN 4B (pH=5.9-6.2) CMR UASB CMR UASB CMR UASB ACETIC 45.6 43.0 47.5 46.0 43.4 42.4 PROPIONIC 38.0 39.3 29.9 30.4 20.6 22.4 BUTYRIC 5.5 6.1 9.9 10.3 21.0 19.8 ISO-BUTYRIC 3.9 3.2 3.2 2.9 4.6 3.8 VALERIC 3.7 4.1 4.4 5.6 4.8 5.2 3-METHYLBUT. 2.2 3.0 3.3 3.1 3.7 4.3 2-METHYLBUT. 1.1 1.3 1.8 1.7 1.9 2.1 There is evidence in the literature that propionic acid production is encouraged by a drop in pH (down to 4.5). Eastman and Ferguson (1981) have found that CHAPTER 5. RESULTS AND DISCUSSION 105 propionic acid distribution increased steadily with the decrease in pH from 7.0 to 5.0. In another study, the highest propionic acid concentration was obtained at the lowest pH value over the range 4.5 to 8.0 (Zoetemeyer et al., 1982b). Although the optimum growth rate of most propionic-acid bacteria occurs at pH 6.0 or higher, the product yield increases significantly with decreasing pH from 6.0 to 4.5 (Hsu and Yang, 1991). In all of the above investigations the pH was externally controlled and, therefore, changes in productivity occurred as a result of pH manipulation. Since the production of propionic acid is both growth and non-growth associated, the optimum pH for cell growth is not necessarily the optimum value for propionic acid generation (Papoutsakis and Meyer, 1985). A diametrically opposite result to the above pattern has been observed in the case of butyric acid (Table 5.23). The percent distribution increases dramatically (about 3 to 4 times) with increasing pH, especially between the Runs 4A and 4B. It has been reported that pH values of 6.0 or higher favor butyric acid production in acidogenic sewage sludge (primary and secondary) digestion (Joubert and Britz, 1986). On the other hand, the acid-phase degradation of less complex organic substrates (sucrose and lactose) resulted in either no change in the percent butyric acid distribution at pH between 4.5 and 6.5 (Zoetemeyer et al., 1982b) or even a drop in butyric acid with increasing pH (Kisaalita et al., 1987). It has not been conclusively demonstrated whether butyric acid generation is growth associated or not (Morris, 1985; Kisaalita et al., 1989). It appears that substrate composition plays, among others, a critical role in butyric acid production at different pH values. In conclusion, the variation in product distribution as a function of pH in CHAPTER 5. RESULTS AND DISCUSSION^ 106 steady-state, continuous-flow systems may be caused by alterations in the metabolism of the same bacterial population or by changes of the population itself, or both. 5.5.4. ORGANIC CARBON SOLUBILIZATION AND SUBSTRATE DEGRADATION Organic carbon solubilization data, expressed as the corresponding COD and TOC rates, are shown in Table 5.24. It is apparent that a drop in pH from 5.1 to 4.5 (Runs 1C and 4A) did not induce any appreciable changes in the overall hydrolysis pattern of organic substrate. In contrast, an increase in pH from 5.1 to 6.1 (Runs 1C and 4B) resulted in remarkably higher solubilization rates. In fact, these are the highest values obtained for the entire study, in either reactor. The observed increase in substrate solubilization can be attributed to the higher rate of carbohydrate hydrolysis occurring at this pH range in both reactors (Figures 5.20 and 5.21). TABLE 5.24. SPECIFIC SOLUBILIZATION RATES OF COD AND TOC AS A FUNCTION OF pH RUN pH RANGE COD RATE (mgCOD/mgVSS*d) TOC RATE (mgTOC/mgVSS*d) CMR UASB CMR UASB 4A 4.3 - 4.6 0.194 0.184 0.073 0.068 1C 4.9 - 5.2 0.187 0.198 0.070 0.070 4B 5.9 - 6.2 0.219 0.239 0.078 0.084 CHAPTER 5. RESULTS AND DISCUSSION 107 The percentage of COD due to VFAs, as a function of pH, is depicted in Table 5.25. Although no significant changes occur by lowering the pH to 4.5, a drastic drop in the percentage can be observed at pH of about 6.1. VFA production has been reduced at this pH range (Figure 5.19), but hydrolysis of organic matter (measured as soluble COD) is still increasing. This indicates that an increase in pH to about 6.1 adversely affects the acid-generating pathways, but on the contrary, it further stimulates the overall hydrolytic activity, which may result in higher concentration of soluble metabolic intermediate products. These observations are further supported by the percent VSS and TSS reduction data (Table 5.26). The same trends, as above, are followed by both parameters with respect to pH alteration. TABLE 5.25. PERCENT SOLUBLE COD IN THE FORM OF VFAs AS A FUNCTION OF pH RUN pH RANGE COD IN VFA FORM (%) REACTOR I (CMR) REACTOR II(UASB) 4A 4.3 - 4.6 67.1 74.7 1C 4.9 - 5.2 71.0 70.9 4B 5.9 - 6.2 52.5 51.6 Analysis of the degradation behavior of the individual organic classes, however, reveals that certain important deviations from the previously described scheme take place, which cannot be detected in the "generic" parameters. Results presented in Figure 5.20 (CMR unit) and Figure 5.21 (UASB unit) show that none CHAPTER 5. RESULTS AND DISCUSSION ^ 108 TABLE 5.26. PERCENT VSS AND TSS REDUCTION AS A FUNCTION OF pH RUN pH RANGE VSS (%) TSS (%) CMR UASB CMR UASB 4A 4.3 - 4.6 62.5 67.7 64.1 69.3 1C 4.9 - 5.2 63.1 70.6 64.2 69.6 4B 5.9 - 6.2 71.3 76.0 72.9 76.1 of the utilization patterns of the three organic classes is comparable to that followed by VSS (i.e. no significant change between pH 4.5 and 5.1, a moderate increase at pH 6.1). Lipid solubilization deviates from the above trend only at pH 6.1 (Run 4B), where a moderate drop is observed instead of an increase. This might be the result of reduced activity of the lipolytic enzymes at this pH value. The protein degradation picture is quite different. The highest reduction percentage obtained in the entire study is observed at pH 4.5. As pH increases, a significant decrease in the protein hydrolysis rate occurs. It is interesting to note that the high protein dissimilation rate observed at pH 4.3-4.6 (Run 4A) did not induce an increase in any of the end-products associated with protein fermentation. The production of the corresponding VFAs (iso-butyric and the 3 isomers of valeric acid) and ammonia is about the same or even lower in Run 4A compared to the other two runs at higher pH values (Table 5.27). Although many proteolytic organisms usually prefer a neutral pH environment, proteolytic enzymes may exhibit their maximum activity at different pH values ranging from 2 to 10 (Bailey and 011is, 1977). Since  ,L LIPIDS • VSS + CARBOHYDRATES PROTEINS CHAPTER 5. RESULTS AND DISCUSSION^ 109 4.5 ^4.7^4.9 ^ 5.1^5.3 ^ 5.5 ^ 5.7 ^ 5.9 ^ 6.1 pH RANGE FIGURE 5.20. PERCENT SUBSTRATE DEGRADATION AS A FUNCTION OF pH IN THE CMR SYSTEM 4.5 ^ 4.7 ^ 4.9 ^ 5.1^5.3 ^ 5.5 ^ 5.7 ^ 5.9 ^ 6.1 pH RANGE 90 80 70 60 cc a.^50 40 30 90 80 70 60 cc 50 40 30 FIGURE 5.21. PERCENT SUBSTRATE DEGRADATION AS A FUNCTION OF pH IN THE UASB SYSTEM CHAPTER 5. RESULTS AND DISCUSSION ^ 110 enzymatic activity is strongly pH dependent, it is possible that an environment with an extracellular pH of about 4.5 may activate further the proteolytic enzymes without, at the same time, promoting acidogensis. TABLE 5.27. PROTEIN DEGRADATION AND ITS END-PRODUCTS AS A FUNCTION OF pH RUN 4A RUN 1C RUN 4B PARAMETER (pH=4.3-4.6) (pH=4.9-5.2) (pH=5 9-6.2) CMR UASB CMR UASB CMR UASB RELATED VFAs 10.9 11.6 12.7 13.3 15.0 15.4 (% of total) NH3-N (mg/L) 30.3 29.8 29.3 35.7 30.5 28.5 PROTEIN 64.8 60.1 42.9 45.0 39.3 36.8 DEGRAD. (%) On the other hand, carbohydrate degradation increases steadily with increasing pH, in the range investigated (4.3-6.2). A similar trend has been observed by Eastman and Ferguson (1981) in continuous flow reactors treating primary sludge at pH between 5.2 and 6.5. It has been reported that hydrolysis of complex carbohydrates reaches a maximum rate at a pH value between 6.0 to 6.5 (Breure et al., 1986), which suggest that the activity of the enzymes involved increases with pH up to the optimum rate. Regarding the effect of reactor configuration on the utilization of the three organic classes, no variation from the previously described pattern was observed. CHAPTER 5. RESULTS AND DISCUSSION^ 111 5.6. GENERAL REVIEW 5.6.1. VFA FORMATION A summary of the distribution of VFAs generated in the anaerobic digestion of primary sludge is presented in Table 5.28. Acetic acid is the most prevalent product, as it is formed directly from the fermentation of carbohydrates and proteins, as well as the anaerobic oxidation of lipids via a number of metabolic pathways (Sections 2.3.1 to 2.3.3). Propionic acid is formed primarily from carbohydrates, but it can be also produced in the digestion of the other two organic classes (McCarty et al., 1963). On the other hand, butyric acid is mainly generated in the digestion of proteins and lipids. It may also be formed in the fermentation of carbohydrates from pyruvate via an alternative pathway (Figure A4, Appendix A). In mixed-culture fermentations, however, this pathway is considered as a rather minor one (Gottschalk, 1986). Taking into account the relative content of the three organic components in the feed, this can provide an explanation for the remarkable difference in the formation of propionic and butyric acids (Table 5.28). The enhanced production of butyric acid at the pH range of 5.9-6.2 (which coincides with the pH for maximum carbohydrate hydrolysis) may be partially due to activation of the above mentioned biochemical pathway. Iso-butyric acid and the three isomers of valeric acid are largely associated with the fermentation of proteins. They can be formed via reductive deamination of single amino acids (Eq. 2.6) or by an oxidation-reduction reaction between pairs of CHAPTER 5. RESULTS AND DISCUSSION 112 amino acids known as the Stickland reaction (Eq. 2.8). It has been found that the production of all four acids during the digestion of non-proteinaceous substrates is minimal (Cohen et al., 1984). It can be also observed that the percent VFA distribution is remarkably similar in both reactors (Table 5.28). Although there is a significant difference in the degradation rates of carbohydrates and lipids between the two systems, the generation of the same VFAs (i.e. acetic, propionic and butyric acids) from the metabolism of these two substrates results in an overall similar picture with respect to product distribution. 5.6.2. FORMATION OF OTHER SOLUBLE END -PRODUCTS In acid-phase digestion, besides VFAs, a variety of simple soluble C 1 to C4 end-products may be generated such as organic acids (formic and lactic), alcohols (ethanol, butanol, 2-propanol, 1,3-propanediol, 2,3-butanediol and glycerol), ketones (acetone and 2,3-butanedione) and aldehydes (acetaldehyde) (Doelle, 1975; Gottschalk, 1986). Chemical analyses for the above compounds plus pyruvic acid showed that only formic acid, lactic acid and ethanol were regularly detected in both reactors, at relatively low concentrations (Table 5.29). The rest of the chemicals either were not detected at all (2-propanol, 1,3-propanediol, acetone, 2,3-butanedione and acetaldehyde), or found sporadically at very low levels, usually less than 1 mg/L (butanol, 2,3-butanediol and glycerol). Moreover, pyruvic acid, the key intermediate metabolite in carbohydrate fermentation, was never detected. In general, pyruvic acid CHAPTER 5. RESULTS AND DISCUSSION^ 113 TABLE 5.28. SUMMARY OF PERCENT VFA DISTRIBUTION VOLATILE FATTY ACID REACTOR I (CMR) REACTOR II (UASB) RANGE MEAN RANGE MEAN ACETIC 43.4 - 50.5 46.3 42.4 - 48.0 45.1 PROPIONIC 20.6 - 38.0 31.0 22.4 - 39.3 31.3 BUTYRIC 5.5 - 21.0 9.0 6.1 - 19.8 8.9 ISO-BUTYRIC 2.8 - 5.2 3.8 2.3 - 5.5 3.6 VALERIC 3.7 - 7.9 4.9 3.8 - 8.1 5.5 3-METHYLBUT. 1.4 - 6.0 3.3 2.6 - 5.4 3.7 2-METHYLBUT. 0.8 - 2.6 1.7 0.8 - 3.0 1.9 TABLE 5.29. OTHER SOLUBLE END-PRODUCTS (MEAN VALUES) RUN REACTOR I (CMR) REACTOR II (UASB) Formic Acid Lactic Acid Ethanol Formic Acid Lactic Acid Ethanol lA N.A. N.A. 6.5 N.A. N.A. 7.7 1B N.A. N.A. 4.0 N.A. N.A. 10.1 1C 15.2 1.5 6.4 6.3 <1.0 9.0 1D 23.2 <1.0 3.8 7.7 <1.0 6.7 2A 30.6 <1.0 9.2 14.5 1.6 10.5 2B 33.5 1.3 7.7 14.8 <1.0 13.4 2C 15.1 2.5 8.5 7.4 2.0 10.1 3A 21.0 <1.0 5.9 11.1 1.4 9.8 3B 16.5 <1.0 4.0 9.2 <1.0 12.7 4A 14.8 1.7 1.1 6.0 <1.0 2.8 4B 28.5 7.8 17.7 14.1 11.7 28.4 Note: N.A. =Not Analyzed; All values are expressed in mg/L. CHAPTER 5. RESULTS AND DISCUSSION 114 is very rarely excreted by anaerobic microorganisms because it plays a crucial role in bacterial energy balance. The pathway leading from pyruvic acid to the end- products of fermentation may accomplish a dual purpose: oxidation of the already reduced NADH 2 , and production of additional ATP by substrate level phosphorylation (Gaudy and Gaudy, 1980). The first result is essential for the completion of the biochemical cycle, while the second is highly desirable since anaerobic growth is directly proportional to the amount of ATP generated (Gottschalk, 1986). It should be noted that all of the above compounds (except glycerol) are fermentation end-products of carbohydrate metabolism. Glycerol is mainly formed during lipid hydrolysis (Eq. 2.9). These results are in agreement with several studies which have found that generation of soluble non-VFA end-products is minimal in continuous flow systems using a heterogeneous particulate feed (Chynoweth and Mah, 1971; Eastman and Ferguson, 1981; Ghosh, 1987). Significant amounts of lactic acid or ethanol have been obtained in batch experiments (Uribelarrea and Pareilleux, 1981) and in studies using simple soluble substrates such as glucose (Zoetemeyer et al., 1982a), sucrose (Joubert and Britz, 1986) or lactose (Kisaalita et al., 1990). There is no apparent trend in the production of formic acid, lactic acid and ethanol associated with the variations in the operational parameters (HRT and SRT) and the feed source, except a slight increase in formic acid at longer SRTs (Table 5.29, Runs 2A and 2B). Changes in pH, however, affect to a great extent all three compounds. At pH 4.3-4.6 (Run 4A) ethanol production is limited, but at pH 5.9-6.2 (Run 4B) the amounts of both lactic acid and ethanol are remarkably higher. Formic acid concentration increases with pH as well (Run 4B). CHAPTER 5. RESULTS AND DISCUSSION 115 The pH of the environment may have a strong effect on many pathways of anaerobic metabolism. For example, a number of saccharolytic clostridia which normally ferment carbohydrates to butyric acid are able to change their metabolism when the pH is lowered to about 4.0, favoring the production of acetone, and concurrently to convert the butyric acid produced to butanol (Doelle, 1975). It should be noted that the reactor regime appears to play a role in the production of formic acid which is 2 to 3 times higher in the CMR system. On the contrary, ethanol is generated at an almost double rate in the UASB reactor. In carbohydrate fermentation, formic acid and ethanol are formed by decarboxylation of pyruvic acid. As mentioned earlier, the major objective of metabolic reactions that convert pyruvic acid to various products is the reoxidation of the electron carrier (NADH 2) that has already been reduced. Certain microorganisms can utilize a mechanism which removes two hydrogen atoms along with the C 1 fragment without reducing the electron carrier. In this case, the products of the C 1 -C 2 split of pyruvic acid are formic acid and acetyl-CoA. The latter compound may be then reduced to ethanol by accepting four hydrogen atoms, which results in the simultaneous oxidation of two molecules of NADH 2 (Gaudy and Gaudy, 1980). In acidic pH conditions, bacteria able to synthesize the enzyme formic dehydrogenase can degrade formic acid to H2 and CO 2 . Depending on the strain and the pH, variable amounts of formic acid may be converted, with the remainder appearing as a fermentation product. The substantially lower formic acid concentration in the UASB reactor (Table 5.29) may be due to either greater CHAPTER 5. RESULTS AND DISCUSSION^ 116 degradation rates or lower formation rates of the acid in this particular environment, or both. Although lactic acid is an important product in many fermentations (Section 2.4.2), it is usually present at only very low concentrations in anaerobic digestion effluents (Ueki et al., 1978; Uribelarrea and Pareilleux, 1981). It has been demonstrated that lactic acid can be converted to propionic acid and other products by anaerobic bacteria employing the acrylate pathway (Figure A3, Appendix A) or the succinate pathway (Nakamura and Takahashi, 1971). Formation and subsequent conversion of lactic acid occurs as a normal process in digesters. The interaction between lactate-generating and lactate-utilizing bacteria plays an important role in the fermentation of carbohydrates during sludge digestion (Ueki et al., 1980). Since the growth of lactate-utilizing bacteria is suppressed by high glucose concentrations, lactic acid accumulation may occur in digesters treating glucose-rich wastewaters (de la Torre and Goma, 1981; Zoetemeyer et al., 1982a). The relatively low soluble carbohydrate (i.e. glucose) levels measured in this study (Table E2, Appendix E) did not interfere with the metabolism of lactate-utilizers resulting in very small amounts of lactic acid in both reactors (Table 5.29). 5.6.3. RATE-CONTROLLING STEP AND NATURE OF SOLUBLE COMPOUNDS As mentioned in Section 2.1, the anaerobic digestion of complex substrates is a multi-step process. In general, when a process consists of a sequence of reactions, CHAPTER 5. RESULTS AND DISCUSSION 117 one step is usually much slower than the others. The last slow step in the sequence is called the rate-controlling, rate-limiting or rate-determining step (Hill, 1977). In anaerobic digestion, the rate-controlling step is related to the nature of the substrate, process configuration, loading rate, and temperature (Speece, 1983). For instance, the rate of hydrolysis of particulate organics may impose a limit on the overall digestion process if raw cellulosics (including lignin) or grease and lipids of certain industrial origin are involved (Pavlostathis and Giraldo-Gomez, 1991). The particulate organic matter in raw municipal sludge can be relatively easily solubilized (Ghosh et al., 1975). The high VSS reduction percentages obtained in this research are in agreement with the above statement. Detection of rather significant amounts of soluble substrate (carbohydrates and proteins) in the effluent of both systems (Tables E2 and E3, Appendix E) diminishes the possibility that the hydrolysis of particulate substrate is the rate-controlling step of the process. (It should be also noted that no variations in the soluble carbohydrate and protein concentrations were observed between the reactor and the effluent of each system). Based on the analytical results and theoretical considerations (as discussed in the following paragraphs), the conversion of soluble metabolic intermediates to VFAs and other end-products of acidogenic digestion appears to play the most critical role in determining the rate-limiting step in the case of high hydraulic loading experiments (HRTs much less than 1 day). The soluble compounds identified in the effluent of acid-phase digestion systems can be classified into three categories: soluble substrates, extracellular intermediate metabolites, and end-products of the phase. Although the main end- CHAPTER 5. RESULTS AND DISCUSSION 118 products of acidogenic digestion of primary sludge are short-chain VFAs, a number of other soluble compounds can be generated in smaller amounts (Section 5.6.2). A summary of the percent soluble COD in the form of VFAs, according to various researchers, is presented in Table 5.30. The results show a great deal of variation, with mean values ranging from 40 to 90%. Only Zoetemeyer et al. (1982b) have reported significant amounts of other products such as ethanol, lactic acid and formic acid. It should be noted that this was the only study using a simple soluble substrate (glucose) instead of primary sludge. From the results illustrated in Table 5.30, it does not appear to be any obvious and clear relationship between the percent soluble COD in the form of VFAs and the prevailing operating conditions (SRT, SRT/HRT ratio, temperature). Although in the continuous-flow studies included in this summary higher VFA percentages can be associated with a low SRT/HRT ratio, other factors such as the characteristics of the feed and the type of seed used may play a more critical role in this phenomenon. It has not been possible from the information available to identify the nature of the soluble non-VFA compounds contributing to COD in most of the studies included in Table 5.30. In this work, the soluble COD in the form of VFAs averages 66% of the total, in both reactors. Taking into account the soluble carbohydrate and protein fraction in the effluent (Tables E2 and E3, Appendix E) and using the appropriate conversion factors (Table E5, Appendix E), the average values increase to 79 and 74% for the CMR and the UASB units respectively. The minor end-products (Table 5.29) account for an additional 1 to 3% of the soluble COD in both systems. Therefore, the remaining soluble COD (approximately 20% in the CMR unit and 25% in the UASB CHAPTER 5. RESULTS AND DISCUSSION^ 119 unit) can be principally attributed to the metabolic intermediates of the process. It is possible that in high hydraulic loading rate reactors the limited time available for food assimilation may not allow for the completion of the corresponding pathways resulting in accumulation of intermediate products. TABLE 5.30. OPERATIONAL PARAMETERS AND PERCENT SOLUBLE COD IN THE FORM OF VFAs FROM VARIOUS ACID-PHASE ANAEROBIC DIGESTION STUDIES REFERENCE SRT (d) SRT/ HRT TEMP. (0 C) % COD as VFAs RANGE MEAN Gupta et al. (1985) - (B) 3 - 9 1 10 - 30 31 - 48 41 Perot et al. (1988) - (B) 8 - 10 1 35 - 55 36 - 59 46 This study 5 - 20 10 - 40 20 41 - 91 66 Fongsatitkul (1992) 20 - 40 5 - 10 35 56 - 75 68 Zoetemeyer et al. (1982b) 0.2 - 0.4 1 30 59 - 88 74 Ghosh et al. (1975) 0.5 - 1.2 1 36 59 - 91 77 Eastman & Ferguson (1981) 1 - 3 1 35 83 - 95 90 Note: Studies marked with a (B) are batch studies, the rest are continuous-flow experiments. The type and amount of the intermediates in acidogenic metabolism depend largely on substrate composition and concentration, pH, temperature, and operational parameters (Hobson et al., 1974). A few specific examples are given below. In carbohydrate fermentation, succinic acid is an important extracellular intermediate. It is formed by certain fermentative species in the rumen and in sludge, but it cannot be converted to propionic acid by the same species (Scheifinger and Wolin, 1973). CHAPTER 5. RESULTS AND DISCUSSION^ 120 Bacterial deamination under anaerobic conditions can also proceed without reduction (independently of reductive deamination described in Eq. 2.6) to form the corresponding unsaturated acids (Sawyer and McCarty, 1978): R-CH 2CHNH 2COOH^R-CH=CHCOOH + NH 3^(5.1) Aromatic amino acids are fermented by certain bacteria belonging to the genus Clostridium to phenylacetic acid, phenylpropionic acid, benzoic acid, indolylacetic acid, phenol, p-cresol and other products (Elsden et al., 1976). Subsequent conversion of these compounds to short-chain aliphatic acids is very slow, whenever possible (Hobson et al., 1974). Degradation of glycolipids and phospholipids can yield galactose (a soluble C6 aldose), in addition to glycerol (McInerney and Smith, 1981). The whole array of C8 to C14 monocarboxylic acids is normally produced as intermediates during the anaerobic 13-oxidation of long-chain fatty acids. Their solubility is mainly a function of the chain length and at 20 °C ranges from 680 mg/L for octanoic acid (C 8) to 20 mg/L for tetradecanoic acid (C 14) (Streitwieser and Heathcock, 1981). Substantial quantities of these acids have been found in anaerobic digestion systems (Novak and Carlson, 1970). 5.6.4. MASS BALANCES: SOLIDS AND PHOSPHORUS Mass balance is a useful method to evaluate the performance of a biological treatment process with respect to a specific parameter. In addition to the mass CHAPTER 5. RESULTS AND DISCUSSION^ 121 balances discussed previously, the overall behavior of another set of parameters (TS, VS, TP, PO 4 -3 -P) is outlined here. The TS and VS measurements account for the dissolved and suspended solid content in the systems, thus any missing solids can be presumed to be lost in the form of gases (CH 4 , CO 2). Total phosphorus (TP) is a "conservative" component in this process, since the biological mechanism for phosphorus removal is not feasible under anaerobic conditions. Both the TS and VS mass balances (exclusive of losses in gaseous end- products) show high recovery percentages, with a range from 90 to 98% and an average value of 94% (Table 5.31), except for Run 1D where the average solids recovery is 84%. During this run, however, the gas production was considerably higher than the rest of the experiments (Table E4, Appendix E). TABLE 5.31. PERCENT RECOVERY BASED ON MASS BALANCE PARAMETER CMR SYSTEM UASB SYSTEM RANGE MEAN RANGE MEAN TS (exc. Run 1D) 90.9 - 96.1 93.6 93.2 - 98.0 94.9 TS (Run 1D) 83.8 84.5 VS (exc. Run 1D) 90.6 - 95.2 92.9 92.9 - 97.5 94.7 VS (Run 1D) 83.3 83.0 TP 96.5 - 105.0 101.9 100.6 - 109.0 104.7 ORTHO-P 75.7 - 90.2 83.4 67.8 - 84.8 75.7 The TP mass balance shows a slight increase in phosphorus through the process, averaging about 3%. The order of magnitude of this error is typical in CHAPTER 5. RESULTS AND DISCUSSION^ 122 analytical measurements. Other researchers have reported that similar errors may range from 5 to 9% (Jenkins and Mavinic, 1989; Wareham, 1992). The orthophosphate mass balance reveals that a considerable reduction in soluble PO 43- occurs in the process (17% in the CMR unit and 24% in the UASB unit). Orthophosphates, which are incorporated into poly-phosphates and ATP, are used by the cells to meet their energy requirements. The lower percent recovery in the UASB system may be attributed to both the higher amount of biomass retained in this reactor (Table 4.2) and differential phosphate precipitation, most likely in the form of calcium phosphate or ammonium magnesium phosphate (Snoeyink and Jenkins, 1980). Precipitation of orthophosphate in UASB reactors has been also mentioned by Fongsatitkul (1992). It is possible that the slow flow pattern in the UASB system creates certain "pockets" where the solubility-product constants of the corresponding phosphate salts are exceeded, resulting in their precipitation. It should be noted, however, that the solubilities of ammonium magnesium phosphate and calcium phosphate in water at 20 °C and neutral pH are 200 mg/L and 20 mg/I, respectively (CRC Handbook of Chemistry and Physics, 1981). Since the solubility of both salts increases dramatically with decreasing pH, it can be assumed that at the pH range of this study (4.3 to 6.2) precipitation of phosphate salts was rather limited. 5.6.5. SUBSTRATE UTILIZATION PATTERNS The degree of dissimilation of various substrates determines the types of bacteria resident in the particular environment under investigation. At any given CHAPTER 5. RESULTS AND DISCUSSION^ 123 moment, the population of the microorganisms depends on the operational and environmental parameters applied. As stated earlier (Section 5.2.5), the overall lipid degradation patterns observed by various researchers have been rather controversial. The conversion percentages have been either minimal or over the 60% level. It should be noted, however, that it is difficult to interpret the results of different studies on lipid degradation, as various methods of extraction of "total lipids" or "fats" have been employed by different investigators. The total lipid fraction of sludge may contain in addition to glycerides, phospholipids, free fatty acids, waxes, hydrocarbon grease and oils (Hobson et al., 1974). Although the composition of lipids in the feed may have played a role in the above mentioned controversy, since certain fractions are more easily biodegraded than others (eg. saponifiable vs. non-saponifiable lipids), it has been demonstrated that inhibition of lipid degradation is mainly a function of SRT and the enrichment of sludge used (Chynoweth and Mah, 1971). Long SRTs permit development of organisms with long generation times, while short SRTs can eliminate such organisms. Novak and Carlson (1970) have found that rapid dissimilation of linoleic and oleic acids occurred at SRTs of 4 days or longer, but essentially no fatty acid conversion took place at an SRT shorter than 4 days. Furthermore, protein- and carbohydrate-enriched sludges suppress the degradation of lipids present (Mahr, 1969). These observations along with the "polarized" ranges of lipid conversion percentages reported suggest that lipolytic bacteria may not function at all under certain conditions, but if a viable population is established, then they can degrade the CHAPTER 5. RESULTS AND DISCUSSION^ 124 substrate available at very high rates. High percentages of carbohydrate degradation were also observed in this study. It has been found that the enzymatic hydrolysis of cellulose (the main carbohydrate component in primary sludge) can be enhanced by the presence of "cellulosome" particles (Lamed et al., 1983). The quaternary structure of cellulosome particles (a macromolecular complex of molecular weight of about 1.2 million produced by certain anaerobic Clostridia species) plays a dual role by binding to the surface of cellulose substrate and, at the same time, by bringing individual cellulolytic enzymes to within close proximity of one another. Protein conversion percentages in this study are generally moderate and significantly lower than those obtained for the other two substrate fractions. Studies have illustrated that proteins can be singly fermented at high rates to VFAs (Breure and van Andel, 1984; Breure et al., 1985). In municipal sludges, however, proteins are present simultaneously with lipids and carbohydrates. Often substantial degradation of proteins cannot be achieved in anaerobic wastewater treatment (Gujer and Zehnder, 1983). It has been found that, in pure cultures, easily fermentable carbohydrates can repress the synthesis of exopeptidases, a group of enzymes involved in protein hydrolysis (Glenn, 1976; Pansare et al., 1985). There is also evidence that the degradation of a protein, gelatin, was progressively retarded with increasing concentrations of carbohydrates present in the medium as a second substrate (Breure et al., 1986). It is possible that the high carbohydrate content in the primary sludge may have reduced, to some extent, the amount of the proteolytic enzymes synthesized, which resulted in lower conversion rates. CHAPTER 5. RESULTS AND DISCUSSION^ 125 The reactor configuration appears to influence the degradation patterns of both lipids and carbohydrates. It is believed that the observed behavior is due to the impact of the physical environment on the molecular structures of lipids and carbohydrates. In the CMR system, lipids have been dissimilated at a higher rate throughout the experimental study. Vigorous mixing conditions result in better dispersion of the lipids and may accelerate the rate of reaction of the heterogeneous catalysis by increasing contact between substrate and enzyme at the lipid-water interface (Heukelekian and Mueller, 1958). On the contrary, the slow diffusion flow regime in the UASB reactor enhances the hydrolysis of carbohydrates (mainly cellulose) which have a considerably more complex physical structure than that of lipids. Since cellulolytic enzymes need a longer time to diffuse and penetrate the macromolecular structure of the substrate (Tsao, 1984), the microenvironment provided in this type of reactor favors their activity. In conclusion, the heterogeneous population of microorganisms engaged in the acid-phase digestion process is in a dynamic equilibrium state. Various selective pressures may encourage growth of certain bacterial species. Such changes may result in a shift in the population which is reflected in the different rates of substrate conversion and product formation. 5.6.6. POTENTIAL APPLICATION OF FINDINGS The results obtained in this research have contributed towards a better understanding of some basic mechanisms of the acid-phase anaerobic digestion of CHAPTER 5. RESULTS AND DISCUSSION^ 126 primary sludge. In the realm of biological process design, the findings of this study may be useful in a number of cases. Some general ideas are presented below. Since soluble COD was found to be, on the average, significantly higher (over 50%) than the amount of VFAs generated, in biological phosphorus removal applications soluble COD may be useful as a replacement test for VFA to determine the usefulness of digester supernatant for phosphorus removal. Furthermore, the acid-phase digestion process exhibited a remarkable degree of operational stability. It was observed that the amount of VFAs produced in most runs under "normal" operating conditions is high enough (in the range of 550 to 650 mg/L as acetic acid) to support subsequent biological P removal processes (Section 2.6.1). For example, assuming that the TSS content of raw sewage is 250 mg/L, 60% of TSS is removed during primary clarification and the TSS concentration in primary sludge is 3,600 mg/L (Table 5.1), then a 600 mg/L VFA production by acidogenic digestion transcribes into a concentration relative to influent sewage flow of about 25 mg/L. However, the amount of VFAs produced under an environmental stress (eg. short HRTs or SRTs; Runs 1B and 2C) may not be high enough to initiate the phosphorus removal mechanism. A 200 mg/L VFA concentration, for instance, transcribes into an influent concentration of 8 mg/L. This remark clearly illustrates the importance of the acid-phase digestion in biological nutrient removal processes. The overall consistency of the lipid and carbohydrate utilization patterns suggests that a CMR system can be used to treat more effectively wastes with a high lipid content. On the other hand, for carbohydrate-rich wastes a higher degree of organic matter degradation can be achieved in UASB reactors. CHAPTER 6 CONCLUSIONS AND RECOMMENDATIONS 6.1. CONCLUSIONS The operational and environmental parameters investigated had a distinct effect on the acid-phase anaerobic digestion process. Based on the results of this research, the following conclusions can be made: 1) A favorable environment for acidogenic digestion has been established and maintained resulting, in most cases, in high VFA production. At the same time, the methanogenic phase has been successfully suppressed, as indicated by the high TS and VS percent recovery and the low gas generation rates. 2) The net VFA concentration and specific production rate (expressed as mgVFA/mgVSS*d) increase, in either reactor, with an increase in HRT (up to 12 hours). The decrease observed at an HRT of 15 hours can be attributed to the onset of methanogenesis. 3) Variation in HRT has a profound effect not only on the net COD concentration, but also on the COD and TOC specific solubilization rates, expressed as mgCOD(or mgTOC)/mgVSS*d. All three maximum values correspond to an HRT of 12 hours, which coincides with the time required for optimum VFA production. 4) The net VFA concentration, VFA specific production rate and net COD concentration as a function of SRT follow the same pattern, showing a sharp decrease 127 CHAPTER 6. CONCLUSIONS AND RECOMMENDATIONS 128 at an SRT of 5 days and approaching a plateau at SRTs of 10 days or longer. On the contrary, the COD and TOC solubilization rates are not affected by the changes in SRT. 5) Both systems exhibit similar VFA production rates at SRTs up to 10 days, regardless of the HRT. At longer SRTs, however, the CMR unit becomes slightly more effective (by about 12 %) than the UASB reactor. 6) Although a decrease in pH from 5.1 to 4.5 does not have an effect on the specific rate of VFA generation, an increase to pH 6.1 results in significantly lower rates (25 to 30 %) of acid production. 7) Acetic acid and propionic acid are the most prevalent VFAs produced averaging about 45 and 31% of the total respectively. Butyric acid follows with an average value of 9%. The percent VFA distribution appears to be independent of HRT, but it is a function of both SRT and pH. The relative distributions of iso- butyric and the three isomers of the valeric acid increase dramatically with SRT. Moreover, a low pH range (4.3-4.6) encourages the production of propionic acid (39% of total VFAs), while at a pH range of 5.9-6.2 more butyric acid is formed (20% of total VFAs). 8) The percent solubilization of organic matter (measured by the VSS content) increases with HRT, but it is not influenced by the variation in SRT. A moderately higher VSS reduction can be also observed at pH 5.9-6.2, as compared to the other two pH ranges. In all experimental runs TSS solubilization follows a trend identical to that of the VSS. 9) The steady-state operation of the acid-phase anaerobic digestion can be CHAPTER 6. CONCLUSIONS AND RECOMMENDATIONS^ 129 replicated and the seasonal variation of influent collection (summer-winter) does not seem to play any significant role in the process. 10) The use of a different source of influent sludge has an effect on lipid and carbohydrate utilization patterns, which is also reflected in the corresponding VFA production rates. 11) Besides VFAs, relatively small amounts of formic acid, ethanol, and lactic acid were regularly detected in both reactors. No other end-products were found at any appreciable concentration. 12) Lipids and carbohydrates are generally utilized at higher rates (expressed as g/d) than proteins, regardless of the prevailing experimental conditions. The degradation of all three organic components of primary sludge increases with an increase in HRT, but only in the case of proteins is a similar behavior observed for increases in SRT. Carbohydrate and lipid dissimilation is essentially independent of SRT. In a similar fashion, variation in pH significantly affects the protein degradation pattern, but only has a small effect upon the other two organic classes. 13) The reactor configuration has an effect on substrate dissimilation as well. Although both systems exhibit a fairly similar behavior in protein degradation, the utilization patterns of carbohydrates and lipids are distinctly different. Lipids are broken down more effectively in the CMR unit, while higher rates of carbohydrate dissimilation have been obtained in the UASB reactor. Furthermore, in the case of easily hydrolyzable wastes, UASB systems may be used to treat wastewaters that have a reasonably high particulate content (in the range of 3,000 to 4,000 mg/L TSS). CHAPTER 6. CONCLUSIONS AND RECOMMENDATIONS^ 130 6.2 RECOMMENDATIONS Consideration for further research should be focused on the following areas: 1) The role of temperature on the process can be investigated. 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"Microbiology of Methane Bacteria", Proceedings of the 2nd International Symposium on Anaerobic Digestion, Travemtinde, Germany, pp. 45-68. REFERENCES^ 145 Zoetemeyer, R.J., Borderding, P.H., van den Heuvel, J.C., Cohen, A. and C. Boelhouwer, (1982a), "Pilot-Scale Anaerobic Acidification of Wastewater Containing Sucrose and Lactate", Biomass, 2, pp. 201-211. Zoetemeyer, R.J., van den Heuvel, J.C. and A. Cohen, (1982b). "pH Influence on Acidogenic Dissimilation of Glucose in an Anaerobic Digester", Water Research, 16, pp. 303-311. APPENDIX A BIOCHEMICAL PATHWAYS FIGURE^ PAGE A1. The Embden-Meyerhof-Parnas (EMP) Pathway for Glucose Catabolism ^ 147 A2. The Pathway for Autotrophic Formation of Acetic acid^ 148 A3. The Acrylate Pathway for Acetic Acid and Propionic Acid Formation^ 149 A4. The Pathway of Butyric Acid Formation from Pyruvic acid  150 146 147APPENDICES Glucose --- ATP ------•- ADP Glucose 6-phosphate Fructose 6-phosphate ---- ATP ------..- ADP V Fructose 1,6-diphosphate Dihydroxyacetone phosphate ...^ Glyceraldehyde 3-phosphate Pi (2) NAD (2) NADH2 (2) 1,3-diphosphoglyceric acid --- (2) ADP ----ii- (2) ATP (2) 3-phosphoglyceric acid (2) 2-phosphoglyceric acid ---,-- (2) Hp (2) Phosphoenolpyruvic acid ---̂  (2) ADP \ ^.- (2) ATP (2) Pyruvic acid FIGURE A1. THE EMBDEN-MEYERHOF-PARNAS (EMP) PATHWAY FOR GLUCOSE CATABOLISM (Adapted from Gaudy and Gaudy, 1980) (V H 2 CO2 .-',^ V Formic acid Acetyl-CoA HSCoA [CO-Ni-E] -4,^4.- Acetyl CoA V CO [Methyl-CoE]^[CoE] Acetyl-CoA ... V (2) Pyruvic acid HSCoA^HSCoA Cell material A ATP ^ Tetrahydrofolic acid ---..- ADP+ Pi Pi -----..- HSCoA v Acetylphosphate --- ADP ---..- ATP v Acetic acid 148 APPENDICES Glucose Formyltetrahydrofolic acid^Methyltetrahydrofolic acid FIGURE A2. THE PATHWAY FOR AUTOTROPHIC FORMATION OF ACETIC ACID (Adapted from Ljungdahl, 1986) Note: CO-Ni-E is the complex between carbon monoxide and its nickel-containing dehydrogenase; CoE is the corrinoid protein. CoA CO2 Acetyl-CoA ADP+ Pi CoA -^ATP 149APPENDICES CoA (2) L-Lactic acid D-Lactic acid Pyruvic acid (2) p-Hydrohypropionyl-CoA (2) H2O (2) p -Propenyl-CoA ETF ETFH2 ETFH2 ETF (2) Propionyl-CoA CoA Acetic acid (2) Prop onic acid FIGURE A3. THE ACRYLATE PATHWAY FOR ACETIC ACID AND PROPIONIC ACID FORMATION (Adapted from Gottschalk, 1986) Note: ETF is the electron-transferring flavoprotein; Fd is the ferredoxin electron carrier. CoA transferace completes the cycle in propionic acid production. APPENDICES^ 150 (2) Pyruvic acid (2) HSCoA - (2) CO2 + (2) FI2 I (2) Acetyl-CoA \---- HSCoA Acetoacetyl-CoA 7--- NADH2 ----i- NAD 13 -Hydroxybutyryl-CoA `------ H2o I Crotonyl-CoA --- NADH2 ---• NAD I Butyryl-CoA Butyric acid Acetic acid Acetyl-CoA FIGURE A4. THE PATHWAY FOR BUTYRIC ACID FORMATION FROM PYRUVIC ACID (Adapted from Gaudy and Gaudy, 1980) 151 APPENDIX B REACTOR OPERATION (HRT AND pH VALUES) TABLE^ PAGE Bl. Operating Conditions of Run 1A^  152 B2. Operating Conditions of Run 1B  153 B3. Operating Conditions of Run 1C  154 B4. Operating Conditions of Run 1D^ 155 B5. Operating Conditions of Run 2A 156 B6. Operating Conditions of Run 2B 157 B7. Operating Conditions of Run 2C 158 B8. Operating Conditions of Run 3A^ 159 B9. Operating Conditions of Run 3B 160 B10. Operating Conditions of Run 4A  161 B 11. 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APPENDICES ^ 158 TABLE B7. OPERATING CONDITIONS OF RUN 2C DATE DAY HRT (hr) pH VALUES Reactor I Reactor II Influent Reactor I^Reactor II^Effluent I Effluent II 12/18/90 1 12.55 12.55 6.54 5.20 5.28 5.34 521 12/19/90 2 11.69 11.87 6.50 5.28 5.31 5.43 524 12/20/90 3 12.00 12.00 6.52 5.41 5.39 5.50 5.26 12/21M0 4 10.88 11.00 5.99 5.42 5.35 5.49 5.41 12/22/90 5 12.11 13.27 6.02 5.50 5.50 5.48 5.48 12/23/90 6 12.17 11.85 6.64 5.51 5.60 5.55 5.57 12/24/90 7 11.33 11.41 6.52 5.78 5.55 5.82 5.56 12/25/90 8 12.86 12.50 6.35 5.64 5.49 5.75 5.55 12/27/90 10 12.47 12.45 6.22 5.84 5.71 5.95 5.66 12/28/90 11 12.33 11.12 6.10 5.90 5.73 5.90 5.77 12/29/90 12 12.15 12.00 5.99 5.61 5.54 5.68 5.63 12/30/90 13 11.94 12.14 6.38 5.88 5.48 5.86 5.42 12/31/90 14 12.57 12.35 6.49 5.66 5.59 5.75 5.70 01/01/91 15 12.51 11.41 6.51 5.74 5.61 5.83 5.66 01/02/91 16 11.68 12.05 6.24 5.87 5.60 5.84 5.51 01/03/91 17 11.91 12.69 6.27 5.79 5.44 5.77 5.40 01/04/91 18 11.89 11.62 6.11 5.69 5.54 5.73 5.44 01/05/91 19 12.04 11.65 6.04 5.51 5.66 5.57 5.49 01/06/91 20 12.40 11.08 6.15 5.43 5.63 5.46 5.73 01/07/91 21 12.86 11.13 5.90 5.50 5.50 5.59 5.45 01/08/91 22 12.61 11.59 6.27 5.67 5.44 5.78 5.32 01/09/91 23 11.80 11.81 6.11 5.88 5.63 5.83 5.40 01/10/91 24 12.52 12.03 6.01 5.78 5.47 5.70 5.67 01/11/91 25 12.21 11.70 6.35 5.70 5.55 5.77 5.61 01/12/91 26 11.73 12.06 6.13 5.61 5.60 5.53 5.50 01/13/91 27 12.34 11.66 6.13 5.55 5.49 5.58 5.47 01/14/91 28 12.18 12.14 6.18 5.60 5.44 5.62 5.38 MEAN 12.14 11.89 6.25 5.63 5.52 5.67 5.50 STD 0.44 0.53 0.20 0.18 0.11 0.16 0.15 ^ C D  C D  C3 CD CD C D  C ) C D  CD  CD  C 3 CD  C D CD  C 3 CD  C D CD^ CD  C3  CD  C a CD  C D CD  C3  C D CD  C D CD  C3  C , CD  C D CD ^ N .Q .Q ., Q Q .N . ,, ,N .N ,N ,, ^ g . . . . . . . . - . ....--,„- --x-- _-: ^ 1 . 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N r' N N N -' N "r 'N -' N N •- •- L N -L N h 3 N N "N "- 4 tg W @ k iD 3 R t3 g t8 N if iM 8 8 1u 1 S W V 8 8 R ig g IT M S 1 1 4 8 1 9 8 k O n ik in i8  ^ hN d o.. . 1 c" ") P0 9 " ) 0 91 91 9)0 9, 0 0 9 " I PP ( n 9I P P P P o P P o P P P P 9 " /) P P P P 9 7 m 0 P9 ) 0 9 " ) P 9 ) IL ,8 k; t3 ;1 8S 53 =t -N ki ti k9 8M iU RS ig IN SW g8 8S 2C 9N - 41 218 Wei k;8 Q;I R.: 189 0 P a P o m P s n c h m P P P P m 9 , 91 05 7) Pm P , m P m 9 )0 9 )0 1 9 )P cn m sA P P 9 IP P m 9 IP P o m m cm . :1 8 ° ) N 8 '. 2 2 N 8 W 8 M A N 8 .5 8 . 8 8 . 2 8 F A R R e 2 T 2 1 V 9 2 W 8 8 3 2 8 .7.1 .8 ° N o p m p o m p p o o m m o p m p o m p s n p p p o m p m p s n m p s n m m o p p p p p p c n m p o p p sy, i. lg iI s w ii .8 8 .7 .8 u p s 8 ti g g 8 N 8 c - A 2 th s 8 E g g i. 2 8 9 Q 8 s g s z 3 g : 4 T 8 8 ° P 0 O P P 0 0 0 91 9 "n o m P o P 9 1 9 " " " P 5 n P m P P P m P 9 1 9 " ) 91 P P P M P P P P P P P M P P .:I R .8 . :I N E R 9 , 8W 8" :1 gP 3 .8 8 % 8 ;1 C 1 8 W 8 8 8 S 8 .(1 8 8 8 . 2 8 8 3 W 3 8 2 S E a R g iV .R W R o m P P P P P m 9 , 0 5 A 0 0 5 4 9 )9 1 0 9 )P m sn P m m 9 , 09 )9 , m 9) m m 9) 9, 09 )9 , Pc nc 8m 09 10 19 )0 )5 09 )9 , 11 -1 88 8W Wz 18 11 8. 8k 88 Sg bi R1 92 QD 3: 1N %1 -1 Cl it @R th tt ig iS WI CI CI WI RD IM 5 I g m m 4 i m 1el = mR F 12 2 0 3 6 ON APPENDIX C CHEMICAL PARAMETERS TABLE^ PAGE Cl. Influent Characteristics from Run lA^  165 C2. Reactor I (CMR) Characteristics from Run lA^ 166 C3. Effluent I Characteristics from Run lA 167 C4. Reactor II (UASB) Characteristics from Run lA 168 C5. Effluent II Characteristics from Run lA^ 169 C6. Influent Characteristics from Run 1B  170 C7. Reactor I (CMR) Characteristics from Run 1B  171 C8. Effluent I Characteristics from Run 1B 171 C9. Reactor II (UASB) Characteristics from Run 1B^ 172 C10. Effluent II Characteristics from Run 1B 172 C 11. Influent Characteristics from Run 1C^  173 C12. Reactor I (CMR) Characteristics from Run 1C^  174 C13. Effluent I Characteristics from Run 1C 174 C14. Reactor II (UASB) Characteristics from Run 1C  175 C15. Effluent II Characteristics from Run 1C^ 175 C16. Influent Characteristics from Run 1D 176 C17. Reactor I (CMR) Characteristics from Run 1D 177 C18. Effluent I Characteristics from Run 1D^ 177 C19. Reactor II (UASB) Characteristics from Run 1D^ 178 C20. Effluent II Characteristics from Run 1D  178 C21. Influent Characteristics from Run 2A 179 C22. Reactor I (CMR) Characteristics from Run 2A 180 C23. Effluent I Characteristics from Run 2A^ 180 C24. Reactor II (UASB) Characteristics from Run 2A^ 181 C25. Effluent II Characteristics from Run 2A  181 C26. Influent Characteristics from Run 2B 182 C27. Reactor I (CMR) Characteristics from Run 2B  183 C28. Effluent I Characteristics from Run 2B^ 183 C29. Reactor II (UASB) Characteristics from Run 2B^ 184 C30. Effluent II Characteristics from Run 2B 184 C31. Influent Characteristics from Run 2C^  185 C32. Reactor I (CMR) Characteristics from Run 2C^ 186 C33. Effluent I Characteristics from Run 2C 186 C34. Reactor II (UASB) Characteristics from Run 2C 187 C35. Effluent II Characteristics from Run 2C 187 163 APPENDICES^ 164 TABLE PAGE C36. Influent Characteristics from Run 3A^ 188 C37. Reactor I (CMR) Characteristics from Run 3A^ 189 C38. Effluent I Characteristics from Run 3A 189 C39. Reactor II (UASB) Characteristics from Run 3A 190 C40. Effluent II Characteristics from Run 3A 190 C41. Influent Characteristics from Run 3B^  191 C42. Reactor I (CMR) Characteristics from Run 3B^  192 C43. Effluent I Characteristics from Run 3B 192 C44. Reactor II (UASB) Characteristics from Run 3B 193 C45. Effluent II Characteristics from Run 3B^ 193 C46. Influent Characteristics from Run 4A 194 C47. Reactor I (CMR) Characteristics from Run 4A 195 C48. Effluent I Characteristics from Run 4A 195 C49. Reactor II (UASB) Characteristics from Run 4A^ 196 C50. Effluent II Characteristics from Run 4A^ 196 C51. Influent Characteristics from Run 4B 197 C52. Reactor I (CMR) Characteristics from Run 4B 198 C53. Effluent I Characteristics from Run 4B^ 198 C54. Reactor II (UASB) Characteristics from Run 4B^ 199 C55. Effluent II Characteristics from Run 4B 199 APPEND ICES^ 165 LL0tr85 1-.Why.c.gt48G.= vE .- w g ilm q w e lq u Ilq lq q 0 1 1 0 1 4 ,0 1 g 1 0 r d .-6 1 1 ,6 w w 6 0 0 — rs o m W o .-o rs :w .-6 0 c 4 .-^ r ^ .-.-.-^ .-.-.-.- y q q q N N (P v m . - m m q *.(0010/ 11 o iw n w S w w n :fO rZ o ie n tio irsr■ w w w o R w c u .- .- Z w .- w c4 CI Pt 0  0  0  9  Pt 0 7! cu c! CT .7 0 V C4 0 CI M g 0 C 4 1 , 71 (uVaCIIXI W W R O ri Mie (7,188032( 0"cp "R S S O S 8 V 8 X X M F ite WI Cq goE8co-wo 012<1,1- g 3 1 ; 0 4 4 0 Z 8 V 8 8 W S W a r 0 1 3 8 1 ! R R R . . . . .  - - - - MMAMEDAMRIRVili S 8 S 2 S t;P 8 5 ;8 2 M eiraraS S M S S R I m0m-.,- ER4 01§1F1§§§§1§§§fFEEM .-..-.-0.1N F a ia n n igM M E M A gn igl M lg lE H IM M Ig 2 ilg N>2co>W 8sRmssomgggpqgs& iR m m oJcV cM N N N A JN 4N N N R IM R W 4 mm^ m cv; 1;11151;11;1128101/1;111 eA .iqsl S3 m 53 m N rt2migivRpl?Wci 53 ru g ., ;;^ ;4 ;4 g 5i Z4 ZG 3 Cq g Z* 14 " 4 2 1 2111 E111 " 41 V 5 :g^&V Ili ;; V g;^41^ .r^ el X V g1? ›-a0 c5V.tFIXRARV$4VSS28RA8SEiw2 w e w1--0 28,518 M g R M M M M --,Fft § n l i l l E P E 8 .-....-c000000005555b751586g TABLE C2. REACTOR I (CMR) CHARACTERISTICS FROM RUN IA DATE DAY P A R AMET ERS (mg/L) TS VS TSS VSS PROTEINS^LIPIDS^CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 12/21/89 9 19595 15295 14490 10800 2445 2250 7855 543 1109 380 440 49 80 9.4 12/24/89 12 22850 17950 16915 13975 2525 1900 10750 594 1379 476 454 50 92 10.8 12/29/89 17 29855 22655 20040 16470 2120 1475 12785 649 1334 524 396 57 111 10.3 01/02/90 21 20140 14980 12000 10145 1940 1680 7980 466 1374 438 353 42 82 9.2 01/05/90 24 27445 20270 18615 15360 1800 1715 12035 518 1305 501 343 55 110 8.4 01/09/90 28 23600 17705 17525 14445 1725 1600 10835 552 1149 443 325 49 99 7.7 01/17/90 36 24905 19090 16855 12910 1630 1510 10345 332 1247 460 313 52 91 10.0 01/20/90 39 24435 18820 15250 11175 1950 1665 8160 527 1173 357 341 29 84 8.2 01/23/90 42 26730 19755 16640 12680 1755 1910 10000 616 1127 385 323 43 81 9.6 01/27/90 46 26400 19405 17180 13205 2125 1675 10235 648 1397 464 399 59 97 10.3 01/30/90 49 25270 19995 18030 14910 1750 2020 11510 576 1575 552 347 67 106 11.0 02/02/90 52 31635 24770 21465 17670 1605 2300 13975 424 1481 546 300 43 114 8.6 02/06/90 56 30025 22880 19605 16040 2020 1615 13325 602 1259 400 376 52 110 8.8 02/09/90 59 23085 17075 15485 13040 1635 2090 10180 492 1373 401 306 44 85 11.2 02/13/90 63 19140 15700 14965 12450 1730 1665 9015 511 1454 536 305 28 82 11.7 02/16/90 66 29840 22870 20520 15740 1625 1890 12580 569 1293 412 299 39 100 10.3 02/20/90 70 27575 20435 18120 14915 1690 2180 11535 509 1542 515 315 45 91 12.0 02/24/90 74 26120 20405 17585 14675 2150 1925 11850 507 1213 371 379 35 93 9.8 02/27/90 77 29295 22735 20840 17255 1835 1715 13649 582 1193 360 325 32 108 8.8 03/02/90 80 28035 21900 18520 15405 1580 1540 11165 551 1292 355 282 29 98 10.6 03/06/90 84 26035 20575 19050 16160 1720 1675 13285 577 1112 382 322 47 107 10.2 03/08/90 86 24670 18515 16940 14125 1925 1855 10870 536 1178 400 347 39 91 12.3 MEAN 25758 19717 17574 14252 1876 1811 11087 540 1298 439 345 45 96 10.0 STD 3365 2568 2225 1986 257 234 1761 71 134 65 45 10 11 1.2 APPEN D IC ES gODccW OW:E<licmwQEa= m o O tq w !N c lo , ..- 01 0 0 ( N m q v : " 01 r :o o m r .r .m c c ia m o ir :r ...- c o o m o ie o .- 0 0 6 .- ...^ .-^ .........- CO h. CA CO N. AD CI CD  OD WI N : NY CA CO N : AD  CD  CA CO CD  N— ,it: wi sr r. U) A) ... W) 4 CV 0 1 6  CO  N: so v. M AO e4 CO  1 6 . CO '- 1 c4 zI- ? 8 V 8 2 ( 1 7 8 8 8 8 2 . 7 1 7 8 8 $ 8 8 8 V ; Z : = 1 IR 52 RI a IR ER I:2 IR 6 .  P? :2 V :t PR PR ;t !2 S! a 53 PR 8 I - 8 u) ii„8; C:I- ii if ii 1 2 Z ^ill ii Ei ii il 2 g f.; fi ii li ii ti E il& § A g fi ? F F :7 i ii 'A ii : E i? €i !? k iE F '41 g 2 ii A t.3 ii ii li E ii :1 E :i li ii g ii 1 2 ii 2 i igiiEi 7.: ESliccco2mRm ii ii ^?, ^1 li ii § ; i f,i li €i E li t E 1 ^: PR ER a P2 P2 f5 IR a SR g ;.: 6 IR IR A! se f5 E. s SR 12 IR IMUggligEAMMEIER 2 E;SR :2 co>f2 EHMRIVAEIAMFREAMF §M§M§gilWaiRfMria gV11.12 WE iiPliM .- ^ CV INTI Y- CA ..- ... RI 11 1 C71 ^11 !^ g CA & g -- 24 " 9 1 P i l l " " H 1 8 % 11-3 giNg^ RcliNg^ NNNM ^ N/7(7; iii " >-ccC3 c6V . t & X 8 8 8 . 7 W T V 8 8 2 8 , 5 8 Z 8 z6 0I- 2W IL0 0̂ g g g E M M g p g g l g g g i g g R W " " " 4 § C ; A i M § 1.- 1- 1- CD C) CD CD CD CD CD^ CP^ CD CD CP OD CP CP CD ER CD 167 TABLE C4. REACTOR II (UASB) CHARACTERISTICS FROM RUN 1A DATE DAY P AR AMETERS (mg/I) TS VS TSS VSS PROTEINS UPI DS^CARBOHYDR-TOT. VFA COD TOC TKN NH3-N TP PO4 12/21/89 9 40530 32530 30485 22260 7170 8420 13470 487 1445 494 1208 61 281 6.8 12/24/89 12 53840 42855 40190 30269 9175 8950 18420 601 1546 544 1507 39 371 8.3 12/29/89 17 52775 42945 41715 31005 8920 9545 17570 652 1376 474 1476 48 380 7.4 01/02/90 21 55270 44660 42065 32100 7085 8680 21030 537 1561 591 1187 53 394 9.2 01/05/90 24 53885 44445 45630 34395 7825 11340 20945 526 1729 614 1308 56 375 8.6 01/09/90 28 51840 41770 39370 30415 7240 8155 20245 659 1269 445 1211 53 352 6.3 01/17/90 36 48690 38464 35540 27955 8060 9380 14960 492 1438 470 1323 34 311 6.7 01/20/90 39 47305 37325 32755 26065 6600 9415 13370 595 1664 584 1110 54 288 7.8 01/23/90 42 50455 39880 36695 29420 7170 8020 19520 733 1425 439 1209 62 324 5.3 01/27/90 46 50355 41070 39090 28650 6035 9235 18475 692 1552 529 1023 57 302 8.6 01/30/90 49 54530 44245 42785 31170 8000 10385 16035 560 1672 599 1337 57 384 8.8 02/02/90 52 45975 35860 33645 26410 5715 9990 18450 580 1732 593 964 50 293 8.2 02/06/90 56 51950 39410 34285 26110 5530 8425 15440 677 1321 482 949 64 287 7.1 02/09/90 59 49525 39455 37490 29320 8465 8260 18695 564 1399 479 1389 34 322 7.4 02/13/90 63 42220 33100 29995 23375 8220 7490 13260 532 1456 496 1360 45 276 6.6 02/16/90 66 44010 34510 31595 23105 9075 7765 13805 701 1657 524 1512 60 294 6.9 02/20/90 70 40875 32075 30810 22975 6910 9145 13060 662 1644 579 1152 46 302 7.7 02/24/90 74 51860 40865 39540 30070 6380 10215 17905 566 1191 419 1056 35 386 8.6 02/27/90 77 53515 40255 36575 28685 5975 8860 19280 584 1300 464 991 35 371 9.0 03/02/90 80 49900 41010 37075 27480 7565 7965 17525 626 1342 468 1262 51 350 8.8 03/06/90 84 46590 36340 30235 23620 6005 9650 15335 644 1520 512 1022 61 311 10.1 03/08/90 86 52295 40765 39080 28760 7730 9325 16650 589 1559 541 1279 42 358 9.5 MEAN 49463 39265 36666 27892 7311 9028 16975 603 1491 515 1220 50 332 7.9 STD 4341 3743 4413 3231 1073 928 2525 67 151 57 170 10 39 1.2 APPEN D IC ES^ 169 k _ N '- q 1 7 • - 1 f N N q C g ig N V N '- lt q q q 0 W I M r:o irs c o d u ic o r:m c o o rrs c o m p s o io d e d .- ....._ v - 0 1 0 0 0 0 (0 1 o N c tc lq q 0 1 4 11. - 0 ( 0 .- 0 ? N ltic t 6 W o .- m m o id d m m o .- o 0 6 o W m o W m m v -.- . . . . . ^ .-....-^ .-.-.-.- tzt  SVZSX 4R SzESS28148ZM ;412V6°) g r:g a - ScsSSS88SwiSSRggE42c74:: - E . :LI_ .8 S F- lila M if g g n iif § g rif M E R M A V a lgE M E M E ! 114W iR A ng§M A IM I1 W s mi.1cr<8a3 F-E22m vals1 v4 w R osP E G giagu P ssv V -V N A 2 ggX gn iR IW M O R ZM U " rg5n E R E E E E  M  E  W M F E E? F.>0F- W A M E E FA H N E M M A tle §§,M11§1EnIMIRIMUM inpVIIIPM 1W W W 1 _""^ ____^ __"^ _^"^ N " 11M11111111M111110 >Q0 °)V .^:K A R A R V *T V SS2SPR IE z6 °1- 2co w U lg:kggggggg:M .FM gE g V 4P " " "giraliigl§ . - . - . - 0 0 0 0 0 0 0 0 0 ^ 0 0 0 ^ 0 0 0 igi TABLE C6. INFLUENT CHARACTERISTICS FROM RUN 1B DATE DAY P AR A METERS(mg/L) TS VS TSS VSS PROTEINS^LIPIDS^CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP 03/20/90 7 3810 2980 3285 2710 619 568 1715 82 244 71 115 15.8 16.8 7.9 03/23/90 10 3985 2850 3570 2630 635 498 1520 112 347 104 122 20.4 17.1 8.6 03/27/90 14 3815 2860 3695 2650 639 519 1670 120 534 171 122 19.3 16.8 9.0 03/30/90 17 4625 3300 4465 2970 627 564 1975 83 301 92 114 132 17.6 7.7 04/03/90 21 3600 2740 3300 2500 506 425 1660 119 321 103 96 14.8 14.0 8.5 04/06/90 24 4325 3145 4065 2905 589 441 2015 126 389 125 110 15.4 15.1 9.9 04/09/90 27 4110 3100 3830 2890 776 528 1715 110 375 110 142 17.6 26.7 12.1 04/12/90 30 3485 2545 3185 2415 647 442 1400 136 448 139 124 20.1 22.7 11.7 04/16/90 34 3820 2825 3535 2550 694 497 1510 91 401 128 130 18.5 19.5 10.3 04/19/90 37 4035 3010 3710 2755 610 500 1855 99 374 121 112 14.3 17.4 92 04/23/90 41 3780 2720 3440 2435 598 424 1605 118 353 109 119 22.9 17.6 8.2 04/26/90 44 3555 2625 3265 2404 569 419 1620 132 426 130 106 15.0 19.9 11.5 MEAN 3912 2892 3612 2651 626 485 1688 111 376 117 117 17.3 18.4 9.6 STD 315 214 358 190 63 52 177 17 71 24 11 2.9 3.3 1.5 TABLE C7. REACTOR I (CMR) CHARACTERISTICS FROM RUN 1B DATE DAY PARAMETERS (mg/1.) TS VS TSS VSS PROTEINS UPIDS CARBOHYDR TOT. VFA COD TOC 1104 NH3-N TP PO4 03/20/90 7 24675 18070 17425 13640 2000 2420 9820 359 942 317 349 29 126 62 03/23/90 10 27605 20785 19995 16445 1905 3040 11175 605 991 364 357 52 147 10.7 03/27/90 14 28955 21995 22620 18825 2200 2635 13850 502 1207 425 396 44 155 9.8 03/30/90 17 25285 19300 20150 16270 1795 2225 11735 312 1203 392 317 29 130 72 04/03/90 21 32970 24695 23260 19355 2080 2370 13960 371 1090 357 366 33 172 8.1 04/06/90 24 31385 23220 23285 18365 2000 2230 14100 315 1128 386 357 37 174 8.0 04/09/90 27 24910 18490 18990 15810 1615 2685 11445 341 977 319 295 37 139 9.3 04/12/90 30 25345 19445 20425 16140 1715 2740 11210 452 1045 338 322 47 148 12.0 04/16/90 34 30095 23420 24420 20375 2160 2900 14695 378 1161 380 377 32 166 7.8 04/19/90 37 32685 24730 24975 19730 2050 2464 14375 435 1098 331 360 32 176 8.7 04/23/90 41 26220 20120 19820 16650 1725 2215 12340 418 990 308 320 44 127 8.0 04/26/90 44 32900 24855 23660 19215 1860 2640 14435 397 1174 344 328 31 145 6.7 MEAN -1 28586 21594 21585 17568 1925 2547 12762 407 1084 355 345 37 150 8.5 STD 3184 2432 2304 1937 179 260 1584 80 89 34 28 7 18 1.6 TABLE C8. EFFLUENT I CHARACTERISTICS FROM RUN 1B DATE DAY PARAMETERS (mg/L) TS VS TSS VSS PROTEINS UPIDS CARBOHYDR TOT. VFA COD TOC TIN NH3-N TP PO4 03/20/90 7 2622 1872 1070 764 332 100 473 355 1067 388 88 35 112 5.8 03/23/90 10 2804 1964 1184 920 419 112 558 578 950 303 107 40 14.4 9.4 03/27/90 14 3680 2738 1878 1420 624 148 836 504 1161 403 139 39 19.3 9.1 03/30/90 17 3488 2602 1796 1308 447 136 768 316 1228 416 97 25 16.6 7.7 04/03/90 21 3654 2740 1816 1396 398 153 838 350 1112 364 99 35 17.0 7.0 04/06/90 24 3186 2262 1318 960 371 102 628 352 1072 361 98 39 13.9 6.2 04/09/90 27 2548 1820 1144 850 391 93 549 378 1032 356 92 29 14.4 82 04/12/90 30 3006 2204 1362 1032 559 105 612 463 1076 369 143 53 16.3 10.4 04/16/90 34 3688 2618 1700 1222 410 130 730 362 1195 428 96 30 15.8 7.5 04/19/90 37 2710 1986 1334 958 359 101 618 430 1086 311 92 34 14.2 6.8 04/23/90 41 3064 2206 1302 1000 415 116 627 447 1103 357 109 43 17.8 8.7 04/26/90 44 3362 2488 1648 1184 368 138 754 408 1198 372 88 29 16.4 6.1 MEAN 3151 2292 1463 1085 424 120 666 412 1107 369 104 36 15.6 7.7 STD 405 324 275 207 81 20 112 73 75 36 18 7 2.0 1.4 TABLE C9. REACTOR II (UASB) CHARACTERISTICS FROM RUN 18 DATE DAY P AR A METERS (mg/L) TS VS TSS VSS PROTEINS^UPIDS^CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 03/20/90 7 47375 35915 36620 26760 5820 8305 14985 426 1046 361 969 38 306 8.9 03/23/90 10 50825 39040 38650 29905 8025 8760 17865 569 1174 428 1328 44 338 7.4 03/27/90 14 42200 33220 32795 25715 6390 8360 13765 509 1408 472 1063 41 306 7.0 03/30/90 17 51980 40865 42180 30655 6375 9705 16975 405 1221 356 1369 29 353 6.2 04/03/90 21 41755 31410 30485 23170 5560 7780 13080 451 1328 468 922 33 274 7.9 04/06/90 24 45665 36665 38835 29005 7010 8355 16745 506 1525 467 1152 31 310 5.3 04/09/90 27 48380 36700 38325 28280 7120 9510 15095 319 1260 429 1169 29 293 5.1 04/12/90 30 41890 32475 35290 25645 6095 8795 13785 545 1454 493 1026 51 275 7.6 04/16/90 34 43445 33335 33965 24375 6760 7915 13285 488 1412 429 1128 47 298 8.0 04/19/90 37 40985 31350 33675 26140 7525 7120 15890 406 1188 371 1247 43 325 9.4 04/23/90 41 39855 30145 31380 23410 6350 8030 13280 501 1204 388 1046 30 335 6.0 04/26/90 44 46540 35355 35685 27000 8170 8365 16315 472 1390 449 1341 34 356 6.8 MEAN 45075 34706 35657 26672 6933 8417 15089 466 1301 428 1147 37 314 7.1 STD 3824 3141 3289 2325 896 685 1586 66 135 47 143 7 26 1.3 TABLE C10. EFFLUENT II CHARACTERISTICS FROM RUN 1B DATE DAY P A R AMET ERS (mg/L) TS VS TSS VSS PROTEINS^UPIDS^CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 03/20/90 7 2038 1426 742 628 300 64 460 349 1045 344 83 35 11.7 7.5 0323/90 10 2492 1770 1062 758 247 75 502 460 1087 360 80 40 12.2 6.9 03/27/90 14 3006 2172 1264 942 253 77 629 419 1240 390 80 40 14.1 7.1 03/30/90 17 3636 2706 1696 1352 351 101 888 313 975 331 86 29 15.4 5.8 04/03/90 21 3240 2380 1434 1110 428 93 739 338 1215 410 97 28 13.9 5.4 04/06/90 24 2984 2140 1134 804 350 78 563 350 1287 382 91 35 12.2 6.0 04/09/90 27 3026 2200 1342 998 356 89 675 282 1085 338 87 30 12.6 5.3 04/12/90 30 3262 2346 1314 1006 267 93 709 448 1301 417 89 46 13.3 6.7 04/16/90 34 2646 1892 1100 816 358 63 604 334 1148 348 105 48 14.0 8.2 04/19/90 37 2726 2004 1202 944 416 70 696 352 1010 358 112 45 13.8 8.3 04/23/90 41 3428 2452 1388 1040 374 82 765 411 976 300 92 32 11.7 4.9 04/26/90 44 2804 2070 1206 948 310 68 652 388 1193 359 87 38 11.0 6.2 MEAN 2941 2130 1240 946 334 79 657 370 1130 361 91 37 13.0 6.5 STD 418 323 223 178 57 12 112 52 112 32 9 6 1.2 1.1 APPEN D IC ESo ico q q m w < tv: 011- m co , ^ P- 0 0 0 M  t: w w o W a id • - d m . - com m e o m N ro m m A iN m o N m e l oicduir: p W m N a :M o ia m N . - . - . - N N . . . . c . ^ .- NN 'tqN cgocl i g i t l q RVRRTm-R 7E F A X ,F aR R R I' gR r4 R8t37FIRV=5*-RXRV W 4 gigS82R R SSR :=R   R lAin aglarib M I W <  M tR S I S V 8 S F A S R M IL>^ q ,• , i-: W o1-. 1.- §RMEgiEMEF§E 6<ao_ n MiA2E§RUISEU 8 IIEHMEni2PE 2 RV :igA R V 4 V a c--) 2 /6 M M M k W 8 t327—tc -=Z"V 888 8 8 8 S 8 8 S S 8 S S 0zigw8ggggm,wZ6w81- 173 TABLE C12. REACTOR I (CMR) CHARACTERISTICS FROM RUN IC DATE DAY P AR A METERS (mg/I.1_ TS VS TSS VSS PROTEINS^UPIDS^CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 05/03/90 6 14315 11235 9570 7825 1455 755 5445 498 1390 463 301 69 68 11.2 05/07/90 10 16095 13190 10445 8265 1690 965 5975 537 1368 469 330 59 66 11.8 05/10/90 13 18540 14375 13095 10950 1910 1140 7615 699 1502 502 344 38 89 7.4 05/14/90 17 20630 16680 14020 11605 2150 1280 7815 684 1567 558 390 46 101 8.5 05/17/90 20 17300 13745 12645 10150 1745 1075 6750 577 1305 467 318 39 94 7.1 05/21/90 24 16465 12820 11580 9030 1695 950 6075 609 1608 562 313 42 82 9.3 05/24/90 27 21560 17205 15085 12300 1920 1230 8590 621 1177 383 359 52 103 9.8 05/28/90 31 22380 18000 16335 13360 2205 1385 9140 676 1543 540 399 46 110 7.4 05/31/90 34 20255 15690 13120 10405 1880 1120 7230 594 1310 467 366 66 87 10.3 06/04/90 38 18570 14735 12935 11010 1740 1255 7435 657 1561 482 332 54 89 10.7 06/07/90 41 17680 14360 11415 9675 1690 880 6725 719 1405 468 314 44 75 8.6 06/11/90 45 21425 17280 14055 11200 1955 1210 7670 582 1283 494 374 61 93 9.6 06/14/90 48 16730 13310 11550 9880 1810 1130 6365 704 1429 523 349 60 76 12.0 06/18/90 52 18975 15705 12540 10065 1945 1285 7350 694 1586 588 361 50 91 8.2 MEAN 18637 14881 12742 10410 1842 1119 7156 632 1431 498 347 52 87 9.4 STD 2287 1893 1732 1441 189 171 982 66 129 51 29 10 12 1.6 TABLE C13. EFFLUENT I CHARACTERISTICS FROM RUN 1C DATE DAY P AR AMETERS (mg/L) TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 05/03/90 6 2350 1664 516 418 268 37 268 490 1315 455 109 67 16.6 11.3 05/07/90 10 2452 1782 656 510 322 39 317 559 1421 476 115 64 15.7 9.6 05/10/90 13 3510 2598 1004 818 334 49 481 672 1541 511 99 46 18.5 8.0 05/14/90 17 3146 2234 716 566 318 52 356 690 1552 518 88 37 14.8 8.4 05/17/90 20 3370 2426 928 706 348 27 457 576 1223 469 101 45 18.1 7.3 05/21/90 24 3104 2298 670 514 374 53 326 648 1507 554 101 41 14.4 8.8 05/24/90 27 2628 1938 628 490 261 51 312 620 1177 391 91 49 14.9 9.7 05/28/90 31 2862 2046 622 506 255 51 303 699 1496 528 94 54 12.7 7.6 05/31/90 34 2990 2138 558 454 235 44 282 580 1324 476 106 68 15.4 10.8 06/04/90 38 3098 2292 744 562 242 34 349 623 1582 523 96 57 16.7 9.3 06/07/90 41 3086 2284 552 412 284 31 264 682 1304 489 94 49 13.9 8.1 06/11/90 45 2696 1934 582 474 236 42 298 594 1338 501 98 61 16.2 10.3 06/14/90 48 2972 2200 696 528 332 51 323 709 1296 461 115 62 17.5 11.6 06/18/90 52 3244 2320 680 544 254 48 336 681 1562 586 97 56 17.2 8.0 MEAN 2965 2154 682 536 290 44 334 630 1403 496 100 54 15.9 9.2 STD 323 246 133 105 45 8 61 62 131 46 8 9 1.6 1.3 APPEND ICES ^ 175 LLa468Fa - c)2 z4. m;WOP-2g<6m= '4:"-cicoNaq'tqw1ww1 4101cgwci .-NcoMrmoo.-cdt-Ca).- ...-^ ._.. ^ ._ -^ o _ EA§REMP/10,W 884VMst1282SZ8Se zgAMILAIR11 13 - goE8 111-3MW4H§VER 2 IHUMMFIgiEF „.^ ^, ^Nts^ e < V01 2PvIWIP VS§R8SSPRIEW mm WIIIIIMPOW 2 "Pr"IIIIPP p 0 , , S ^ 8 6 0 , - 2a.8 ;11111111I111111 > E 8 W P I W E I r 8  c4 r4 r, ran g N E,M 1 1- .t g ^ g N ^ g TWIIIIIII N N ^ g IPEIE 6 g1§11§Fli p ^gm;r-Q ^mmX^g4^Pizi; R g oevt--Ruigx8;V4D13iE w4c1 I M M W M ! Biking:46;i q c, .1Lugwc62<6Q<8an RE4 l'tc t. - ir lv q 01040(1.-111(1 01"-N 0 .- M t - n : 0 D ) o c f ic o o ie i.- .- .- If) 0' m  .- 4  cq  C O  N  P. CO a l P■ e l m .1 , m  6 oi 0 0.- ei .- ci  N  .-  CO  O i 1132.13.744X2R$S8Star° EIVRSPPgaSI8288w N VIN I-w- ET 8> MHZ-051MM* MflEgE§MEW. REEMMVIIIEG itrs22m R q S V S S S R t e M e $ S R RiciRm,,,,,N R0 R,N m ,c, 17-Assysnsv.:Pmmui m>.FN— Eil/AEEEFIEAMP MIAMEFAZIRAE gr.ilnIPMME r i N i ^ .■ ...^ C °^ N N N 22 4 8 y 8 8 A ; A :t g V^sg m u ixissIg N s4 N R A g N s-4 sa ii ca 039vtl-Rxmi-vvv 4mca26 w1-g i U M M M g g -27:1-“7;W.:1-.:2 E888S888g8S8g8 TABLE C16. INFLUENT CHARACTERISTICS FROM RUN 1D DATE DAY P AR A METERS(mg/L) TS VS TSS VSS PROTEINS^UPIDS^CARBOHYDT TOT. VFA COD TOC TKN NH3-N TP PO4 06/28/90 8 4285 3325 3795 3165 774 514 1940 79 467 150 142 18.2 23.1 12.5 07/02/90 12 3905 3140 3560 2870 647 476 2005 105 615 197 133 29.6 22.3 15.6 07/05/90 15 4195 3265 3985 3005 686 429 1955 89 352 97 129 19.3 18.9 9.3 07/09/90 19 4170 3090 3940 2840 667 442 1825 117 367 115 129 22.3 17.5 8.3 07/12/90 22 3995 2720 3590 2590 632 459 1680 102 396 130 128 26.4 18.8 8.0 07/16/90 26 3440 2670 3045 2515 524 431 1580 119 307 94 100 15.9 17.2 9.4 07/19/90 29 4130 3430 3920 3200 782 454 2065 123 385 129 146 21.1 20.7 11.8 07/23/90 33 4170 3335 3945 3025 641 480 2090 133 474 154 127 24.4 21.7 12.9 07/26/90 36 3825 2865 3540 2635 683 467 1650 82 453 128 135 25.5 18.0 10.0 07/30/90 40 4185 3130 3875 2880 688 502 1740 100 402 117 127 17.3 23.0 13.8 08/02/90 43 4090 3055 3805 2815 690 468 1855 121 386 118 127 16.2 18.4 11.1 08/06/90 47 4020 2960 3740 2755 721 482 1695 129 458 146 141 25.7 20.9 13.7 MEAN 4034 3082 3728 2858 678 467 1840 108 422 131 130 21.8 20.0 11.4 STD 219 232 254 207 65 25 164 17 76 27 11 4.3 2.1 2.3 TABLE C17. REACTOR I (CMR) CHARACTERISTICS FROM RUN 1D DATE DAY P AR AMETERS(mg/1.) TS VS TSS VSS PROTEINS^UPIDS CARBOHYDC TOT. VFA COD TOC TKN NH3-N TP PO4 06/28/90 8 15905 12120 11060 8890 1790 1070 6280 457 1127 400 349 62 101 8.7 07/02/90 12 13575 10075 8705 6815 1605 825 4900 548 1311 442 326 69 83 11.6 07/05/90 15 12880 9880 9560 7565 1420 935 5425 611 1161 389 300 72 90 12.5 07/09/90 19 13700 9970 8535 6750 1470 690 4900 504 1455 507 302 67 80 9.3 07/12/90 22 10990 8385 8755 6390 1285 885 4710 502 1290 405 263 58 79 8.0 07/16/90 26 15195 11125 10210 8515 1735 670 6240 584 1132 359 340 63 102 8.8 07/19/90 29 12170 9310 9225 7200 1610 770 5030 617 1200 441 336 78 89 10.9 07/23/90 33 14195 10785 11485 9355 1570 1005 6815 580 1466 544 319 68 114 12.6 07/26/90 36 17880 13720 12815 9885 1820 840 7245 615 1286 467 344 53 120 9.4 07/30/90 40 15015 11090 11380 8670 1595 695 6280 509 1093 416 303 47 87 7.5 08/02/90 43 11670 8830 8745 6955 1380 640 5240 523 1070 410 270 49 77 11.0 08/06/90 47 14405 10545 9730 7340 1505 715 5255 555 1174 460 296 55 84 11.4 MEAN 13965 10486 10017 7861 1565 812 5693 550 1230 437 312 62 92 10.1 STD 1841 1395 1323 1102 157 134 806 50 127 50 27 9 13 1.7 TABLE C18. EFFLUENT I CHARACTERISTICS FROM RUN 1D DATE DAY P AR AMETERS (mg/1) TS VS TSS VSS PROTEINS^UPIDS CARBOHYDC TOT. VFA COD TOC TKN NH3-N TP PO4 06128/90 8 2554 1974 578 456 250 30 331 464 1097 383 107 67 14.1 8.9 07/02/90 12 2408 1868 584 448 229 36 320 574 1350 421 110 74 16.6 12.4 07/05/90 15 3176 2376 808 654 344 43 431 618 1199 406 125 70 19.0 12.3 07/09/90 19 2686 1968 536 392 301 41 278 524 1369 481 109 61 12.8 8.7 07/12/90 22 2298 1814 490 388 279 28 283 490 1270 399 98 53 13.1 9.1 07/16/90 26 3274 2390 778 634 339 36 422 591 1136 353 125 71 15.5 8.2 07/19/90 29 2874 2192 734 564 315 25 399 627 1223 430 128 78 17.2 11.0 07/23/90 33 2778 2112 626 476 258 24 346 592 1443 493 114 73 16.6 12.1 07/26/90 38 2550 1980 522 380 236 37 269 621 1291 447 96 58 14.0 9.7 07/30/90 40 2582 1944 518 393 224 40 274 512 1149 397 90 54 11.8 8.8 08/02/90 43 2818 2116 688 532 301 29 365 567 1211 389 108 60 16.7 11.0 08/06/90 47 2490 2000 596 454 252 42 312 542 1169 390 105 65 15.5 11.8 MEAN 2707 2061 622 481 277 34 336 560 1242 416 110 65 15.2 10.3 STD 282 175 102 91 40 6 55 52 100 39 12 8 2.0 1.5 TABLE C19. REACTOR II (UASB) CHARACTERISTICS FROM RUN 1D DATE DAY PARAMETERS(mg/L) TS VS TSS VSS PROTEINS UPIDS CARBOHYDI TOT. VFA COD TOC  TKN NH3-N TP PO4 06/28/90 8 35470 27570 28855 22270 7945 5870 11705 546 1410 453 1352 81 328 11.2 07/02/90 12 38305 31010 31425 24035 8380 5550 12860 527 1498 527 141 1 70 340 9.1 07/05/90 15 32145 24415 23335 16570 6225 4265 9655 700 1335 485 1056 60 271 8.0 07/09/90 19 31285 23605 23340 17435 6085 4160 10160 615 1400 481 1039 66 280 9.4 07/12/90 22 28645 21720 20815 15470 5500 3315 8650 504 1586 531 946 66 263 7.3 07/16/90 26 28080 20840 22650 16265 5660 4220 8290 592 1484 509 964 58 288 7.9 07/19/90 29 30885 23555 22710 16020 7080 3765 8655 693 1309 443 1201 68 295 9.8 07/23/90 33 28700 21660 22060 16830 7225 3980 9300 703 1600 572 1223 67 302 7.6 07/26/90 36 36710 28885 28900 20925 8315 4925 11110 641 1317 457 1383 52 317 10.7 07/30/90 40 31845 24410 25985 18740 6590 3970 10730 538 1418 512 1104 50 311 8.4 08/02/90 43 30930 25140 23950 17160 7740 4745 8625 619 1295 466 1294 56 293 10.3 08/06/90 47 28170 21950 23195 16465 6885 4330 8935 645 1222 399 1170 68 277 7.9 MEAN 31764 24563 24768 18182 6969 4425 9890 610 1406 486 1178 63 297 9.0 STD 3272 3011 3142 2638 948 703 1382 67 113 45 153 8 22 1.3 TABLE C20. EFFLUENT II CHARACTERISTICS FROM RUN 1D DATE DAY P AR AMETERS (mg/L) TS VS TSS VSS PROTEINS^UPIDS CARBOHYD( TOT. VFA COD TOC TKN NH3-N TP PO4 06/28/90 8 2688 1962 320 246 150 14.5 133 552 1260 432 103 79 14.4 11.8 07/02/90 12 2328 1746 258 190 128 14.0 103 564 1563 510 91 70 11.7 10.0 07/05/90 15 2362 1784 280 206 126 13.6 115 678 1310 451 83 63 11.1 8.8 07/09/90 19 2770 2050 364 282 168 14.5 152 645 1252 452 96 70 11.6 9.0 07/12/90 22 2286 1692 252 186 118 13.7 104 480 1492 482 81 62 10.4 8.1 07/16/90 26 2602 1886 276 202 127 12.8 111 544 1574 522 79 58 9.8 7.4 07/19/90 29 2130 1598 228 174 100 11.4 97 714 1182 387 80 64 10.7 8.6 07/23/90 33 2890 2134 348 258 172 15.9 138 697 1510 576 91 64 12.9 92 07/26/90 36 2710 2006 304 224 154 14.1 121 651 1335 434 79 54 14.3 10.5 07/30/90 40 2452 1802 282 202 124 15.3 108 526 1526 544 66 46 11.8 8.1 08/02/90 43 2128 1638 256 190 112 16.8 97 597 1244 445 77 59 13.5 11.3 08/06/90 47 2364 1744 294 232 150 13.7 128 653 1217 430 92 68 12.3 9.7 MEAN 2476 1837 289 216 136 14.2 117 608 1372 472 85 63 12.0 9.4 STD 241 163 38 31 22 1.3 17 72 142 53 10 8 1.4 1.3 TABLE C21. INFLUENT CHARACTERISTICS FROM RUN 2A DATE DAY P AR AMETERS trg/L) TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOO  TKN NH3-N IP PO4 09/06/90 8 3925 3045 3610 2840 645 540 1615 106 434 128 119 15.4 15.7 6.8 09/10/90 12 3975 2980 3590 2725 592 564 1630 132 489 140 113 18.3 17.9 8.3 09/13/90 15 3800 2940 3365 2745 546 542 1680 89 425 118 109 21.8 14.8 10.4 09/17/90 19 3960 3035 3640 2840 596 574 1710 103 537 162 123 27.2 14.7 10.0 09/20/90 22 4380 3380 4055 3180 597 569 2020 116 560 164 124 28.9 18.4 11.8 09/24/90 26 4605 3555 4330 3405 679 580 2155 127 587 191 135 26.0 17.0 10.0 09/27/90 29 4320 3400 3920 3065 613 447 2135 134 590 201 123 25.3 15.1 9.5 10/01/90 33 4295 3130 3945 2920 623 553 1780 113 402 119 125 25.1 19.3 10.5 10/04/90 36 3605 2635 3440 2450 611 509 1340 136 373 104 127 28.8 16.9 8.3 10/09/90 41 4675 3680 4420 3340 754 579 2065 128 426 121 146 25.1 23.8 14.4 10/11/90 43 4105 3040 3870 2845 716 464 1645 150 530 136 128 13.4 19.3 11.5 10/15/90 47 3955 2965 3745 2755 630 555 1690 93 447 113 127 26.2 17.0 10.7 10/18/90 50 4045 3035 3800 2790 630 572 1525 120 331 94 129 27.7 17.0 9.3 10/22/90 54 4130 3010 3835 2805 663 591 1530 116 319 87 127 20.6 20.2 12.8 MEAN 4127 3131 3826 2908 635 546 1751 119 461 134 125 23.6 17.7 10.3 STD 289 267 292 248 52 42 240 17 87 33 8 4.8 2.4 1.9 TABLE C22. REACTOR I (CMR) CHARACTERISTICS FROM RUN 2A DATE DAY P AR AMETERS(mg/L) TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 09/06/90 8 15275 11610 9505 7715 2600 1160 5755 550 1180 387 477 61 86 7.4 09/10/90 12 13565 10255 8365 6900 2335 1285 5035 659 1455 502 428 55 77 7.9 09/13/90 15 16440 12440 11190 8335 2670 1090 6170 695 1308 485 479 52 94 8.8 09/17/90 19 20685 15420 13535 10780 2920 1835 7380 687 1583 590 545 78 121 9.7 09/20/90 22 17915 13615 12050 9365 2665 1640 6490 703 1531 527 493 67 107 8.0 09/24/90 26 17285 12775 11080 8550 3000 1375 6335 720 1483 532 549 69 93 10.3 09/27/90 29 17440 13550 12405 10080 2795 1590 7130 684 1389 457 511 64 129 7.6 10/01/90 33 22130 16440 14345 11350 3160 1695 8205 743 1274 451 571 65 137 10.9 10/04/90 36 16480 12505 11670 9405 2610 1440 6700 654 1502 546 484 66 101 9.3 10/09/90 41 14425 10715 9980 7995 2395 1290 5875 676 1352 440 449 65 78 8.1 10/11/90 43 20815 16080 13475 10410 2935 1790 7215 726 1450 550 514 44 116 7.7 10/15/90 47 18935 13865 11760 9255 3120 1595 6605 802 1286 496 563 64 100 11.2 10/18/90 50 16980 11985 10030 8265 2560 1410 5805 677 1191 441 479 69 86 9.3 10/22/90 54 21205 15640 12955 10005 2875 1710 7015 711 1302 509 519 59 122 8.4 MEAN 17827 13350 11596 9172 2760 1493 6551 692 1378 494 504 63 103 8.9 STD 2527 1893 1645 1226 245 226 783 54 122 52 41 8 18 12 TABLE C23. EFFLUENT I CHARACTERISTICS FROM RUN 2A DATE DAY P AR A METERS(mg/L) TS VS TSS VSS PROTEINS^UPIDS^CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 09/06/90 8 2698 2052 648 494 182 28 368 518 1229 395 100 71 12.1 6.9 09/10/90 12 3144 2376 972 788 231 44 523 609 1469 518 94 57 15.4 8.3 09/13/90 15 3552 2728 990 812 162 43 537 656 1404 454 87 61 16.1 7.8 09/17/90 19 3904 3014 1056 840 260 47 544 689 1542 549 114 72 17.8 9.5 09/20/90 22 3502 2566 874 700 219 41 475 707 1488 500 102 67 15.4 8.2 09/24/90 26 3352 2470 878 668 243 36 461 760 1592 543 112 74 16.7 10.6 09/27/90 29 3742 2732 846 668 295 32 450 724 1287 440 110 62 13.2 7.1 10/01/90 33 3036 2318 740 592 318 34 404 707 1277 445 115 64 16.0 10.3 10/04/90 36 2856 2186 680 524 243 35 378 631 1469 540 110 71 13.6 8.0 10/09/90 41 3890 2980 1008 776 211 42 510 715 1460 480 103 69 15.9 8.6 10/11/90 43 3784 2774 987 780 207 47 496 694 1337 483 82 49 14.7 7.2 10/15/90 47 3288 2400 788 606 248 41 391 781 1318 501 115 75 16.3 10.7 10/18/90 50 3098 2312 708 544 178 29 390 675 1173 454 103 75 13.1 8.4 10/22/90 54 3534 2608 962 780 215 43 512 702 1403 514 103 69 14.2 6.6 MEAN 3384 2537 867 684 229 39 460 683 1389 487 104 67 15.0 8.4 STD 368 276 130 112 42 6 61 63 118 44 10 7 1.6 1.3 TABLE C24. REACTOR II (UASB) CHARACTERISTICS FROM RUN 2A DATE DAY PARAMETERS (mg/1.) TS VS TSS VSS PROTEINS UPIDS CARBOHYDR TOT. VFA COD TOC T)04 NH3-N TP PO4 09/06/90 8 41140 30545 27400 19575 7850 7190 9335 542 1349 476 1330 74 324 8.2 09/10/90 12 43540 32640 29835 22705 9145 9015 9725 619 1599 587 1534 71 366 10.4 09/13/90 15 38635 27220 22605 16565 7580 5765 8670 697 1572 564 1271 58 307 9.3 09/17/90 19 47325 36405 33825 24705 10425 9665 10805 749 1735 585 1733 65 380 10.5 09/20/90 22 47740 36165 31375 23455 10650 8925 11005 854 1692 590 1768 64 368 10.5 09/24/90 26 48755 37030 32390 24095 10205 8040 12510 820 1707 607 1697 64 387 7.8 09/27/90 29 44920 33220 27685 20000 8955 7400 9400 687 1541 511 1489 57 339 8.7 10/01/90 33 40945 29975 25535 19340 7615 6595 9760 785 1435 478 1279 61 326 9.9 10/04/90 36 46440 33810 28990 20555 8650 6285 10655 721 1441 497 1448 62 343 7.3 10/09/90 41 49705 36300 32845 23495 10130 8340 11420 796 1536 546 1691 70 374 9.0 10/11/90 43 39850 28885 25245 18880 7845 7670 8205 745 1682 602 1301 46 313 11.7 10/15/90 47 44865 33970 27600 19720 10025 6835 10025 867 1427 512 1668 64 338 10.3 10/18/90 50 49245 37175 32590 23150 10920 6495 12665 698 1374 477 1819 72 382 8.8 10/22/90 54 43870 32495 28870 20820 8690 7700 10160 793 1636 581 1452 62 364 11.6 MEAN 44784 33274 29056 21219 9192 7566 10310 741 1552 544 1534 64 351 9.6 STD 3489 3078 3185 2303 1151 1098 1257 86 125 48 187 7 26 1.3 TABLE C25. EFFLUENT H CHARACTERISTICS FROM RUN 2A DATE DAY P AR AMETERS(mg/L) TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 09/06/90 8 2800 2126 358 300 148 22 173 548 1368 471 92 68 10.8 7.9 09/10/90 12 3036 2262 372 284 132 26 162 633 1516 522 87 66 11.3 8.1 09/13/90 15 2670 1984 320 272 154 27 151 685 1534 553 85 60 12.4 9.5 09/17/90 19 3438 2682 574 480 214 23 256 729 1563 556 91 56 14.6 9.1 09/20/90 22 3662 2736 612 504 210 18 263 800 1663 593 98 64 14.5 8.3 09/24/90 26 2928 2220 356 280 139 19 166 797 1594 564 78 56 9.7 6.7 09/27/90 29 3116 2306 358 276 147 28 155 662 1469 461 74 50 10.9 8.3 10/01/90 33 3120 2404 380 316 175 31 172 739 1410 469 80 52 11.5 8.0 10/04/90 36 2538 1890 296 244 148 19 145 744 1469 464 88 64 9.9 6.9 10/09/90 41 3472 2642 400 328 183 32 177 731 1412 515 86 57 10.8 7.4 10/11/90 43 3294 2454 358 302 139 22 173 787 1604 584 63 41 13.6 10.2 10/15/90 47 3542 2710 494 400 178 27 220 846 1416 530 89 61 12.8 7.9 10/18/90 50 3418 2598 440 364 132 27 196 680 1319 462 92 71 10.6 6.3 10/22/90 54 3016 2360 394 314 143 29 181 765 1617 588 82 59 12.5 8.0 MEAN 3146 2384 408 333 160 25 185 725 1497 524 85 59 11.9 8.0 STD 328 261 89 75 26 4 35 75 100 49 8 8 1.5 1.0 TABLE C26. INFLUENT CHARACTERISTICS FROM RUN 28 DATE DAY P A R A METERS (mg/L) TS VS TSS VSS PROTEINS^UPIDS^CARBOHYDR TOT. VFA COD TOO TKN NH3-N TP PO4 10/29/90 6 3845 2965 3425 2580 603 515 1615 74 330 94 115 182 192 11.0 11/01/90 9 3810 2790 3515 2565 612 484 1580 80 322 88 122 24.0 19.4 10.5 11/05/90 13 4035 2740 3560 2600 610 525 1415 98 421 139 123 25.8 19.8 9.3 11/08/90 16 3710 2680 2930 2145 563 479 1530 108 453 126 109 19.4 20.4 11.5 11/12/90 20 3995 2815 3370 2460 664 528 1395 117 401 109 121 14.3 15.6 7.5 11/15/90 23 4575 3470 4065 3035 557 549 1950 46 265 73 103 14.1 18.8 7.8 11/19/90 27 3635 2620 3130 2210 494 418 1605 76 344 97 95 16.4 18.9 10.7 11/22/90 30 3710 2440 3110 2035 566 444 1315 75 272 75 108 17.3 18.9 11.1 11/26/90 34 3510 2330 3150 2160 516 491 1180 65 393 108 98 15.0 18.6 9.3 11/29/90 37 4150 2890 3560 2545 652 608 1540 55 280 80 122 17.5 22.8 12.6 12/03/90 41 4195 2890 3510 2565 648 583 1485 87 375 103 119 15.2 18.5 10.4 12/06/90 44 4025 3015 3505 2655 629 535 1630 69 334 99 115 14.8 16.1 8.0 12/10/90 48 3730 2855 3280 2490 583 488 1625 83 359 104 112 18.7 18.3 9.1 12/13/90 51 3800 2925 3345 2575 618 532 1570 91 401 116 120 21.0 17.5 8.9 12/17/90 55 3695 2700 3250 2380 563 503 1545 98 419 121 111 21.3 16.6 7.5 MEAN 3895 2808 3380 2467 592 512 1532 81 358 102 113 18.2 18.6 9.5 STD 263 254 259 243 48 47 166 19 56 18 9 3.4 1.7 1.7 TABLE C27. REACTOR I (CMR) CHARACTERISTICS FROM RUN 2B DATE DAY P AR AMETERS (mg/L) TS VS TSS VSS PROTEINS^LIPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 10/29/90 6 18950 14335 12100 9415 3255 1610 7190 530 1411 502 587 66 155 10.7 11/01/90 9 23595 18200 15515 11005 3940 1965 7780 587 1126 420 686 55 198 7.4 11/05/90 13 20780 15425 13685 9780 3380 1770 7400 667 1299 450 610 69 169 9.6 11/08/90 16 22565 16935 13590 9840 4015 2125 7665 721 1217 440 693 50 184 10.1 11/12/90 20 17475 12880 11875 8590 3090 1550 6390 703 1111 411 536 42 157 8.2 11/15/90 23 22230 16890 14280 10925 3880 2220 7825 762 967 345 659 39 200 6.2 11/19/90 27 24975 18900 15225 11525 4445 2335 8340 556 1310 474 763 52 209 6.1 11/22/90 30 24535 19015 15620 12170 4515 2475 8670 649 1137 434 787 64 224 10.0 11/26/90 34 22440 16660 14180 10580 3660 2000 7685 714 1215 422 642 56 188 8.4 11/29/90 37 17990 14075 12210 9325 3470 1880 6420 646 1338 487 620 65 164 8.0 12/03/90 41 17890 13345 11480 8870 3150 1920 6240 738 1368 480 552 48 157 7.2 12/06/90 44 19375 14750 12520 9055 3235 2215 6250 745 1400 505 585 67 162 9.8 12/10/90 48 22650 16975 14065 10340 3960 2375 7085 652 1330 467 695 62 190 6.7 12/13/90 51 19630 14600 12170 9220 3280 2310 5995 698 1468 548 584 59 158 7.7 12/17/90 55 21855 16680 13420 9845 3875 1920 7530 743 1386 532 670 50 166 9.0 MEAN 21129 15978 13462 10032 3677 2045 7231 674 1272 461 645 56 179 8.3 STD 2369 1879 1317 1000 441 270 789 69 134 50 70 9 21 1.4 TABLE C28. EFFLUENT I CHARACTERISTICS FROM RUN 2B DATE DAY PA R AMETER S (mg/L) TS VS TSS VSS PROTEINS UPI DS CARBOHYDR TOT. VFA COD TOC TIN NH3-N TP PO4 10/29/90 6 3526 2604 988 748 234 66 496 560 1318 496 110 73 18.3 10.2 11/01/90 9 2996 2232 756 542 200 42 391 628 1127 455 93 61 14.3 7.3 11/05/90 13 3832 2882 1046 806 251 68 518 715 1228 461 102 62 17.9 8.3 11/08/90 16 3408 2488 910 684 177 56 474 673 1146 425 87 59 17.4 9.0 11/12/90 20 3112 2210 818 618 166 50 422 694 1096 435 75 49 14.8 7.8 11/15/90 23 3428 2578 964 702 134 44 481 718 1008 369 61 40 15.6 7.0 11/19/90 27 2840 2016 686 520 230 36 382 571 1225 461 85 49 13.0 62 11/22/90 30 3018 2120 710 554 199 39 397 638 1208 444 96 64 14.9 8.3 11/26/90 34 3060 2296 758 600 147 47 412 739 1156 431 83 59 14.7 7.4 11/29/90 37 3536 2564 962 724 177 52 495 672 1242 481 97 69 17.8 8.2 12/03/90 41 2970 2208 736 552 172 35 395 724 1273 487 76 48 14.3 7.3 12/06/90 44 3028 2180 710 538 159 41 388 748 1324 500 96 71 16.2 9.0 12/10/90 48 3616 2662 920 720 229 57 483 686 1266 485 105 68 15.8 6.4 12/13/90 51 2902 2110 776 604 213 48 430 751 1441 550 93 59 14.1 6.8 12/17/90 55 3134 2204 732 572 174 43 394 783 1451 511 84 56 162 8.3 MEAN 3227 2357 831 632 191 48 437 687 1234 466 90 59 15.7 7.8 STD 292 244 116 88 34 10 47 63 117 42 12 9 1.5 1.0 TABLE C29. REACTOR II (UASB) CHARACTERISTICS FROM RUN 2B DATE DAY P AR AMETERS(mg/L) TS VS TSS VSS PROTEINS UPIDS CARBOHYDR TOT. VFA COD TOC 11Q4 NH3-N TP PO4 10/29/90 6 48135 34480 31915 24495 9825 10885 11875 585 1492 495 1636 64 502 8.9 11/01/90 9 51065 37095 33740 25540 11940 11205 12815 663 1236 455 1964 54 526 7.3 11/05/90 13 40270 28080 24685 18800 8705 8600 9440 696 1362 489 1444 51 394 10.1 11/08/90 16 47055 34600 30945 23005 10190 10080 11250 709 1611 613 1695 65 468 6.7 11/12/90 20 44435 32535 27520 20485 9385 9285 9825 780 1472 561 1550 48 437 9.0 11/15/90 23 50290 37550 32455 24715 10975 11195 11205 821 1300 461 1801 45 484 7.8 11/19/90 27 51895 39145 33370 25300 11625 10100 13075 600 1178 388 1922 62 510 6.1 11/22/90 30 42550 30070 26970 21490 8900 9675 10630 673 1374 519 1493 69 432 7.5 11/26/90 34 44960 31965 27495 22020 8985 9860 11140 752 1453 580 1492 54 457 9.0 11/29/90 37 40945 28600 24975 19755 8395 8945 9660 704 1210 426 1391 48 404 10.2 12/03/90 41 38725 27815 23285 18995 8510 8860 9185 737 1451 539 1433 71 422 7.6 12/06/90 44 41310 30010 25325 20180 9060 9945 10260 776 1444 536 1518 67 441 11.0 12/10/90 48 46920 34420 29080 23700 9765 10190 12105 699 1517 544 1615 52 463 7.3 12/13/90 51 50360 36400 30865 25030 10425 11075 12410 768 1678 559 1716 48 507 6.5 12/17/90 55 46315 32595 27660 22595 9040 9205 10770 805 1592 529 1507 60 445 8.6 MEAN 45682 33024 28686 22407 9715 9940 11043 718 1425 513 1612 57 459 8.2 STD 4096 3493 3257 2282 1075 835 1192 67 143 59 170 8 39 1.4 TABLE C30. EFFLUENT II CHARACTERISTICS FROM RUN 2B DATE DAY P AR AMETERS(mg/L) TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TIN NH3-N TP PO4 10/29/90 6 3298 2384 506 392 154 44 232 575 1411 504 92 67 14.1 7.9 11/01/90 9 3576 2642 608 484 132 48 281 686 1287 421 71 50 13.9 5.4 11/05/90 13 2792 2150 430 348 168 36 211 702 1403 483 79 52 13.6 9.5 11/08/90 16 2800 2072 378 308 120 33 189 732 1507 551 86 68 11.0 62 11/12/90 20 3440 2494 526 432 114 50 249 773 1333 523 84 46 14.5 7.4 11/15/90 23 2720 2054 368 276 171 28 173 803 1212 445 83 56 11.1 6.7 11/19/90 27 2664 1958 366 266 125 29 165 601 1155 415 87 67 10.4 5.5 11/22/90 30 3066 2208 420 344 143 34 206 623 1421 524 88 65 11.2 6.0 11/26/90 34 2956 2276 456 360 96 37 216 729 1327 509 72 56 15.4 9.4 11/29/90 37 3578 2612 524 408 154 42 233 681 1251 475 70 45 12.7 6.8 12/03/90 41 3212 2404 414 336 114 44 200 756 1509 530 89 70 12.2 7.4 12/06/90 44 2838 2176 422 324 160 46 192 784 1459 510 94 68 15.5 10.1 12/10/90 4e 2904 2077 460 350 149 36 211 710 1392 486 82 58 11.2 6.5 12/13/90 51 3040 2204 504 414 141 42 247 727 1590 575 87 44 11.9 5.1 12117/90 55 3132 2294 504 398 130 37 246 759 1597 539 79 59 13.9 7.3 MEAN 3068 2267 459 363 138 39 217 709 1390 499 80 58 12.8 7.1 STD 290 198 67 57 21 6 30 65 127 44 9 9 1.6 1.5 TABLE C31. INFLUENT CHARACTERISTICS FROM RUN 2C DATE DAY P A R AMETERS(mg/L) TS VS TSS VSS PROTEINS^LIPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 12/20/90 3 3810 2630 3490 2200 595 454 1535 87 339 91 119 24.2 13.7 6.4 12/24/90 7 4150 2890 3600 2485 564 514 1525 105 390 106 109 18.8 16.6 7.7 12/27/90 10 4195 3055 3685 2655 648 457 1795 117 412 121 125 21.4 18.5 9.3 12/31/90 14 3970 2845 3660 2420 550 500 1620 108 491 141 112 23.9 15.9 7.1 01/03/91 17 4365 3200 4005 2775 605 506 1885 122 417 119 119 22.2 20.1 10.6 01/06/91 20 4200 3285 3905 2865 592 487 2045 96 428 126 113 18.6 16.4 9.4 01/09/91 23 4075 3155 3830 2835 565 438 1990 120 463 144 110 19.6 18.7 10.1 01/12/91 26 4225 3310 3945 2805 619 506 1970 101 475 141 124 25.0 20.3 9.7 01/14/91 28 3980 2740 3560 2420 556 452 1585 120 369 100 107 17.9 15.9 6.9 MEAN 4108 3012 3742 2607 588 479 1772 108 420 121 115 21.3 17.3 8.6 STD 157 232 174 221 31 27 197 12 47 18 6 2.5 2.1 1.5 TABLE C32. REACTOR I (CMR) CHARACTERISTICS FROM RUN 2C DATE DAY A TS VS TSS VSS PROTEINS LIPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 12/20/90 3 13515 10130 8805 6910 2760 885 4855 345 1090 365 501 60 76 9.5 12/24/90 7 11015 8005 6935 5375 2105 654 4015 279 919 300 378 41 63 7.4 12/27/90 10 11150 8030 6955 5125 2000 601 3880 247 884 295 366 46 56 7.4 12/31/90 14 9615 6960 5050 3920 1415 526 2760 274 798 245 269 42 46 8.9 01/03/91 17 9640 7135 5775 4535 1610 609 3380 194 780 257 295 37 52 6.3 01/06/91 20 9145 6615 5340 4170 1550 517 3015 173 842 251 295 47 41 5.5 01/09/91 23 10435 7630 6190 4710 1805 622 3550 228 905 306 320 32 55 9.6 01/12/91 26 9775 6995 5860 4565 2040 651 3255 252 835 266 361 35 57 8.2 01/14/91 28 11510 8570 7025 5360 1845 603 3990 198 954 320 345 50 67 7.8 MEAN 10644 7786 6437 4963 1903 630 3633 243 890 289 348 43 57 7.8 STD 1269 1019 1076 834 375 101 595 50 89 37 65 8 10 1.3 TABLE C33. EFFLUENT I CHARACTERISTICS FROM RUN 2C DATE DAY P AR AMETERS (mg/L) TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 12/20/90 3 3616 2622 1102 810 299 50 586 327 1100 365 113 65 15.8 8.3 12/24/90 7 2978 2096 796 552 229 34 436 247 955 302 77 41 11.6 6.3 12/27/90 10 3548 2524 1084 780 216 45 581 260 845 256 82 47 16.3 9.0 12/31/90 14 3000 2190 844 586 207 39 455 261 901 297 83 50 13.6 8.2 01/03/91 17 3516 2604 1040 744 301 51 565 203 808 249 89 41 13.3 6.6 01/06/91 20 3274 2358 962 668 270 40 517 169 927 300 99 56 12.1 5.8 01/09/91 23 3062 2120 868 612 231 38 487 217 880 277 79 42 15.5 10.2 01/12/91 26 3556 2588 1076 706 263 47 544 248 972 318 80 38 13.5 7.0 01/14/91 28 3012 2132 838 652 236 37 492 195 874 280 91 53 12.4 7.3 MEAN 3285 2359 957 679 250 42 518 236 918 294 88 48 13.8 7.6 STD 259 215 115 83 33 6 51 44 81 33 11 8 1.6 1.3 TABLE C34. REACTOR II (UASB) CHARACTERISTICS FROM RUN 2C DATE DAY P AR AMETERS(mg/L) TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 12/20/90 3 33700 24670 22925 16990 5840 3985 10135 355 1263 402 993 58 221 10.1 12/24/90 7 27865 20705 18660 13570 4820 3200 8725 334 1045 361 812 41 187 8.7 12/27/90 10 27270 19360 16730 12175 4375 2830 7840 286 940 305 746 46 172 6.6 12/31/90 14 29105 21020 18510 13220 5105 2685 8800 290 992 315 852 35 184 7.3 01/03/91 17 24505 17640 15375 11515 4255 2775 7335 253 891 284 722 41 163 5.2 01/06/91 20 29510 21635 20565 14745 5010 3265 9640 259 1058 346 854 53 192 9.0 01/09/91 23 26155 19605 19595 13900 4525 3170 8595 247 985 299 768 44 175 5.0 01/12/91 26 29710 21320 19870 14815 5105 3335 9080 308 1114 351 851 34 204 6.3 01/14/91 28 26885 20005 17955 12660 4560 2930 8385 1022 320 778 48 169 7.8 MEAN 28301 20662 18909 13732 4844 3131 8726 288 1034 331 820 44 185 7.3 STD 2484 1824 2072 1550 460 373 803 36 102 35 76 7 17 1.6 TABLE C35. EFFLUENT II CHARACTERISTICS FROM RUN 2C DATE DAY P AR AMETERS(mg/L) TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 12/20/90 3 3144 2326 488 342 176 32 161 342 1175 380 87 59 13.4 9.6 12/24/90 7 2948 2078 380 256 155 26 137 310 939 298 74 49 10.8 8.2 12/27/90 10 2442 1710 296 196 160 20 106 274 1040 327 71 45 9.7 7.0 12/31/90 14 3086 2132 490 338 136 35 160 263 974 319 65 43 11.3 7.4 01/03/91 17 2824 1954 432 304 131 31 146 237 912 290 88 47 9.5 5.9 01/06/91 20 2448 1762 388 260 180 28 125 235 1101 373 84 55 11.5 8.2 01/09/91 23 2690 1950 302 202 149 33 102 240 942 298 71 48 9.4 7.1 01/12/91 26 2492 1768 344 212 133 34 119 298 1066 348 81 40 8.9 6.0 01/14/91 28 3100 2204 440 326 174 25 151 225 963 312 84 56 10.5 6.6 MEAN 2797 1987 396 271 155 29 134 269 1012 327 74 49 10.6 7.3 STD 273 203 69 56 18 5 21 38 83 31 9 6 1.3 1.1 TABLE C36. INFLUENT CHARACTERISTICS FROM RUN 3A DATE DAY P A R AMETER S (mg/L) TS VS TSS VSS PROTEINS^UPI DS CARBOHYDR TOT. VFA COD TOC TKN NH3-N 1P PO4 01/21/91 7 4050 2990 3475 2715 627 514 1715 83 420 122 116 15.3 17.4 9.9 01/24/91 10 3380 2340 2850 2115 541 483 1300 92 324 108 110 23.9 15.9 9.2 01/28/91 14 3885 2565 3390 2420 546 530 1350 109 439 118 106 18.5 18.3 12.0 01/31/91 17 3790 2710 3150 2305 639 520 1415 115 396 110 121 19.2 16.7 10.5 02/04/91 21 4610 3320 4170 2845 622 559 2005 87 506 152 121 21.4 19.4 10.9 02/07/91 24 4085 2925 3490 2650 684 465 1580 89 355 104 125 15.3 16.7 8.1 02/11/91 28 4055 2860 3615 2555 667 491 1500 114 412 129 124 16.8 17.8 10.7 02/14/91 31 3795 2755 3305 2525 590 516 1495 121 477 137 118 23.2 14.4 6.7 02/18/91 35 4130 3170 3645 2885 699 533 1785 74 511 160 131 18.7 18.2 10.8 02/21/91 38 4020 3095 3510 2780 673 520 1790 87 423 132 124 16.0 19.7 11.4 MEAN 3980 2873 3460 2580 629 513 1594 97 426 127 119 18.8 17.5 10.0 STD 297 278 326 234 53 26 213 15 57 18 7 3.0 1.5 1.5 TABLE C37. REACTOR I (CMR) CHARACTERISTICS FROM RUN 3A DATE DAY PARAMETERS (mg/L) TS VS TSS VSS PROTEINS UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 01/21/91 7 16320 11710 10525 7660 1645 865 5935 548 1188 391 307 44 89 5.8 01/24/91 10 19160 13915 12160 9340 1840 970 7050 594 1331 416 349 55 108 7.8 01/28/91 14 22560 15765 14635 10905 2120 1090 8245 633 1435 485 389 50 134 9.2 01/31/91 17 21705 15440 15335 11835 2065 1255 8650 677 1493 505 398 68 137 10.3 02/04/91 21 19555 13620 12170 9230 1800 910 7240 576 1595 534 326 38 111 7.1 02/07/91 24 16485 11880 10945 8490 1625 860 6325 609 1392 473 304 44 98 6.4 02/11/91 28 23745 17260 14700 10820 2200 1105 8205 648 1306 433 410 58 121 5.4 02/14/91 31 22945 16220 13505 10475 2065 950 8240 567 1423 490 386 56 126 5.7 02/18/91 35 19955 14885 12040 8760 1780 920 6345 531 1372 479 328 44 95 8.7 02/21/91 38 22650 15835 14485 11305 1930 1285 8475 581 1246 421 358 49 132 9.0 MEAN 20508 14653 13050 9882 1907 1021 7471 596 1378 463 356 51 115 7.5 STD 2518 1747 1616 1303 190 147 964 43 113 43 37 8 16 1.6 TABLE C38. EFFLUENT I CHARACTERISTICS FROM RUN 3A DATE DAY P AR AMETERS(mg/L) TS VS TSS VSS PROTEINS^LIPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 01/21/91 7 2576 1880 516 412 298 56 281 524 1153 393 99 51 13.8 9.2 01/24/91 10 2936 2176 596 468 323 70 309 559 1284 434 107 56 12.6 7.5 01/28/91 14 2656 1944 464 328 250 53 211 638 1400 490 88 48 14.1 10.3 01/31/91 17 3388 2438 716 556 235 76 374 655 1322 462 103 65 15.9 9.8 02/04/91 21 2836 2014 494 392 211 54 272 581 1639 579 80 47 12.2 7.8 02/07/91 24 3302 2410 754 608 268 80 397 569 1286 453 92 49 13.5 6.8 02/11/91 28 3110 2178 696 510 307 64 357 628 1301 493 102 53 12.4 7.5 02114/91 31 2692 1992 568 406 329 56 274 536 1527 547 113 60 10.2 5.9 02/18/91 35 2584 1804 492 352 268 50 244 508 1317 485 93 50 11.8 9.2 02/21/91 38 2792 2080 530 380 293 55 268 602 1238 446 104 57 14.7 10.8 MEAN 2887 2092 583 441 278 61 299 580 1347 478 98 54 13.1 8.5 STD 277 200 99 87 37 10 57 48 135 51 9 8 1.5 1.5 TABLE C39. REACTOR II (UASB) CHARACTERISTICS FROM RUN 3A DATE DAY PAR AMETERS (mg/L) TS VS TSS VSS PROTEINS UPIDS^CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 01/21/91 7 30935 23100 20620 15770 7130 5750 8860 550 1279 426 1201 80 226 6.8 01/24/91 10 33260 23970 21435 17330 7265 5935 9315 612 1394 467 1217 55 257 9.7 01/28/91 14 36380 26755 24775 19605 8050 6560 10300 603 1604 550 1353 65 280 8.7 01/31/91 17 39715 29315 24790 18765 8750 6075 10240 662 1556 522 1448 48 265 7.6 02/04/91 21 40660 30345 27310 21920 9015 7135 11495 715 1697 569 1489 47 297 10.4 02/07/91 24 36975 27880 25120 19055 8025 6310 10450 629 1410 508 1341 57 261 8.3 02/11/91 28 42805 31030 28505 22285 9120 7020 11475 598 1326 467 1504 45 308 6.3 02/14/91 31 34450 24660 22465 17155 7480 5935 9335 686 1433 541 1239 42 242 7.0 02/18/91 35 33500 24375 21630 16520 7245 5680 9190 607 1590 591 1219 80 245 11.1 02/21/91 38 40745 29150 24820 20010 8085 6220 10805 572 1547 574 1350 56 281 9.0 MEAN 36923 27058 24147 18842 8017 6262 10147 623 1484 522 1336 53 266 8.5 STD 3689 2738 2453 2081 707 477 898 48 128 51 110 7 24 1.5 TABLE C40. EFFLUENT II CHARACTERISTICS FROM RUN 3A DATE DAY PARAMETERS (mg/l) TS VS TSS VSS PROTEINS UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 01/21/91 7 2284 1606 258 184 137 19.3 108 544 1223 397 80 58 9.1 72 01/24/91 10 2862 1844 382 288 150 28.1 188 557 1322 417 80 56 12.4 9.3 01/28/91 14 2198 1538 294 236 104 24.8 129 626 1428 485 83 67 9.6 7.0 01/31/91 17 3026 2224 356 268 134 22.6 163 692 1511 507 70 49 10.7 7.8 02/04/91 21 2590 1890 388 288 112 27.0 183 686 1579 518 64 46 12.1 9.2 02/07/91 24 3170 2346 406 308 110 17.7 198 677 1524 539 67 49 10.2 7.0 02/11/91 28 3056 2170 334 260 117 19.8 156 628 1262 434 81 42 8.8 6.1 02/14/91 31 2830 2008 418 322 160 16.9 205 691 1485 533 64 39 9.0 5.8 02/18/91 35 2564 1794 400 302 122 17.4 191 584 1553 551 73 53 13.4 10.2 02/21/91 38 3005 2060 348 276 138 202 162 619 1390 502 72 50 10.3 6.8 MEAN 2759 1948 358 273 128 21.4 186 630 1428 488 71 51 10.6 7.6 STD 318 250 49 38 17 3.8 29 53 118 51 7 8 1.5 1.4 APPEN D IC ES illaaril! IIIII5MII 191 TABLE 042. REACTOR I (CMR) CHARACTERISTICS FROM RUN 3B DATE DAY PAR AMETERS (mg/L) TS VS TSS VSS PROTEINS UPIDS^CARBOHYDR TOT. VFA COD TOC TIN NH3-N TP PO4 02/28/91 6 16620 12935 10330 8560 1515 945 6620 388 1311 444 294 52 77 7.0 03/04/91 10 17815 14480 12490 10245 1720 1090 7825 462 1249 424 314 38 93 5.2 03/07/91 13 19185 15610 11775 9545 1800 965 7500 477 1337 480 331 43 82 5.1 03/11/91 17 22255 17725 14330 11335 1995 1205 8855 542 1588 536 349 30 103 5.9 03/14/91 20 20500 16020 13715 10535 1950 1100 8260 512 1394 493 339 27 97 5.6 03/18/91 24 18525 14835 11500 8840 1560 1025 6870 583 1742 601 289 40 76 4.2 03/21/91 27 22310 18130 15110 12120 2080 1270 9415 592 1615 534 366 34 114 6.9 03/25/91 31 19640 15975 13425 10235 1865 1140 7930 488 1485 503 339 40 91 4.4 03/28/91 34 17060 13870 12100 9310 1735 995 7095 451 1559 518 327 50 81 7.3 04/01/91 38 22815 18055 14720 11315 2030 1165 8610 483 1273 435 362 37 105 5.0 04/04/91 41 19310 16340 14335 10950 1765 1090 8490 515 1326 447 330 48 96 4.3 MEAN 19640 15816 13075 10272 1820 1090 7952 499 1444 492 331 40 92 5.5 STD 2033 1630 1463 1070 176 97 835 56 156 51 23 8 12 1.1 TABLE C43. EFFLUENT I CHARACTERISTICS FROM RUN 3B DATE DAY P AR AMETERS (mg/L) TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC T1041 NH3-N TP PO4 02/28/91 6 2798 2054 624 500 252 68 337 401 1169 409 97 56 9.7 6.4 03/04/91 10 3246 2542 844 692 208 85 459 447 1232 412 84 51 10.8 4.8 03/07/91 13 3446 2726 930 764 281 96 499 478 1303 448 88 43 11.3 5.2 03/11/91 17 3012 2320 628 492 230 67 335 566 1474 506 71 35 9.0 5.6 03/14/91 20 2886 2228 682 546 196 74 366 514 1286 455 60 28 9.7 5.2 03/18/91 24 3020 2416 718 572 244 79 390 594 1687 548 84 45 9.5 4.4 03/21/91 27 2654 2134 588 474 187 73 319 597 1390 446 78 48 9.4 6.2 03/25/91 31 3152 2458 680 560 261 93 358 481 1466 481 77 35 9.1 4.7 03/28/91 34 3316 2680 756 608 275 93 404 463 1527 499 97 53 12.0 7.0 04/01/91 38 2932 2342 744 612 229 98 403 478 1191 422 76 40 11.0 4.9 04/04/91 41 2732 2090 640 520 204 72 342 526 1328 438 87 54 8.5 4.3 MEAN 3018 2363 712 576 233 82 383 504 1368 460 82 44 10.0 5.3 STD 239 217 98 85 31 11 53 59 151 42 10 9 1.1 0.8 TABLE C44. REACTOR II (UASB) CHARACTERISTICS FROM RUN 313 DATE DAY P AR AMETERS(mg/L) TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 02/28/91 6 36870 28780 25370 21015 6865 6020 10685 474 1429 465 1140 41 231 6.6 03/04/91 10 40990 32215 27645 23465 8460 7255 11160 489 1447 486 1406 52 260 5.8 03/07/91 13 35885 29040 25730 22005 7330 6635 11145 537 1559 527 1211 38 252 4.1 03/11/91 17 42165 32690 29895 24795 8340 7570 11560 544 1500 511 1375 41 278 4.6 03/14/91 20 34240 26935 22815 19405 6715 5650 10390 639 1607 546 1108 33 211 5.9 03/18/91 24 39125 30595 27860 23125 7255 6930 11350 608 1863 602 1190 30 246 5.6 03/21/91 27 35795 27470 23610 20085 6665 5845 10030 527 1801 588 1116 50 220 3.8 03/25/91 31 42310 32295 26070 22040 8045 7035 10800 601 1618 526 1323 36 265 6.7 03/28/91 34 34380 26405 22920 19175 6685 5470 9705 626 1715 549 1098 28 209 7.1 04/01/91 38 37485 29720 25635 21280 7625 6345 11020 521 1546 491 1254 34 243 4.0 04/04/91 41 34370 26000 22545 19200 7250 5695 9970 565 1500 482 1201 41 215 4.6 MEAN 37601 29286 25463 21417 7385 6405 10710 557 1599 525 1220 39 239 5.3 STD 2949 2323 2257 1789 627 689 584 53 135 42 103 7 22 1.1 TABLE C45. EFFLUENT II CHARACTERISTICS FROM RUN 3B DATE DAY P AR AMETERS(mg/L) TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC MN NH3-N TP PO4 02/28/91 6 2758 2206 338 284 132 33 150 452 1265 418 63 42 8.9 5.8 03/04/91 10 2414 1934 270 228 154 29 115 481 1325 431 78 53 7.4 5.2 03/07/91 13 3000 2470 352 284 115 35 141 524 1450 491 63 45 6.0 3.6 03/11/91 17 3198 2638 404 340 97 44 188 521 1316 436 56 41 7.9 4.3 03/14/91 20 3194 2502 352 290 128 36 155 539 1414 496 57 36 8.6 5.3 03/18/91 24 3300 2716 460 392 93 46 217 596 1661 590 49 34 9.0 4.7 03/21/91 27 3018 2374 322 266 98 32 138 534 1576 548 69 54 6.8 4.0 03/25/91 31 2808 2292 338 276 124 34 146 610 1422 510 52 32 8.7 6.1 03/28/91 34 3112 2568 404 328 129 41 179 628 1642 559 51 30 10.1 6.3 04/01/91 38 2444 2006 356 286 148 31 159 512 1344 446 64 41 6.8 3.7 04/04/91 41 2956 2440 440 370 113 45 208 570 1418 487 65 47 8.7 42 MEAN 2927 2377 367 304 121 37 163 542 1439 492 61 41 8.1 4.8 STD 282 237 53 46 19 6 30 52 127 54 8 8 1.2 0.9 TABLE C48. INFLUENT CHARACTERISTICS FROM RUN 4A DATE DAY P AR AMETER S (mg/L) TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC -^TKN NH3-N TP PO4 04/15/91 7 4295 2990 3860 2695 562 430 1870 87 571 134 106 16.1 19.3 8.3 04/18/91 10 4150 3230 3735 3025 610 538 1915 106 375 116 116 18.7 20.4 12.4 04/22/91 14 3510 2845 3210 2685 543 446 1665 126 353 102 110 22.7 20.6 9.6 04/25/91 17 4130 3325 3720 3110 684 562 1870 133 398 123 134 25.0 18.3 9.1 04/29/91 21 3675 2895 3330 2705 599 492 1685 101 419 129 116 20.3 17.2 9.4 05/02/91 24 4380 3570 4050 3360 720 591 2055 122 460 156 132 16.6 17.0 8.3 05/06/91 28 4070 3015 3745 2765 644 509 1715 135 475 128 119 15.6 20.0 12.1 05/09/91 31 3895 2975 3480 2750 625 518 1610 104 387 102 120 20.2 22.4 13.4 05/13/91 35 3955 2885 3465 2635 667 538 1520 120 418 119 129 22.0 23.7 13.5 05/16/91 38 4200 3110 3845 2850 711 531 1665 109 452 127 129 15.5 18.2 10.4 MEAN 4026 3084 3644 2858 637 516 1757 114 431 124 121 19.3 19.7 10.7 STD 258 218 249 222 57 47 155 15 59 15 9 3.1 2.1 1.9 TABLE C47. REACTOR I (CMR) CHARACTERISTICS FROM RUN 4A DATE DAY P AR AMETERS(mg/L) TS VS TSS VSS PROTEINS^LIPIDS CARBOHYDR TOT. VFA COD TOC TIN NH3-N TP PO4 04/15/91 7 13975 11005 10275 8445 1310 980 6440 494 1215 427 241 31 118 6.2 04/18/91 10 17950 14250 12680 10735 1545 1055 8280 547 1153 411 293 45 141 5.5 04/22/91 14 15150 11910 10375 8740 1335 865 6705 523 1394 469 268 54 122 5.7 04/25/91 17 13170 10265 8995 7680 1200 900 5845 572 1318 449 254 62 106 7.7 04/29/91 21 19560 15560 13570 11495 1910 1285 8290 594 1515 516 361 56 164 10.8 05/02/91 24 19635 14000 12025 10055 1730 1250 7650 608 1591 563 319 42 146 11.9 05/06/91 28 16560 11975 10945 9185 1295 1125 6860 686 1425 500 261 54 128 9.3 05/09/91 31 14465 11125 9990 8285 1210 970 6480 614 1197 419 254 60 130 7.6 05/13/91 35 16850 12820 10415 8880 1750 895 6825 530 1411 484 334 54 114 7.2 05/16/91 38 20550 15580 12930 10960 1935 1235 8145 593 1373 473 349 39 155 8.4 MEAN 16787 12849 11220 9446 1522 1056 7152 576 1359 471 293 50 132 8.0 STD 2458 1811 1415 1220 274 151 828 53 133 45 42 9 18 2.0 TABLE C48. EFFLUENT I CHARACTERISTICS FROM RUN 4A DATE DAY P AR AMETERS(mg/1..) TS VS TSS VSS PROTEINS^LIPIDS CARBOHYDR TOT. VFA COD TOC TIN NH3-N TP PO4 04/15/91 7 2708 2018 630 572 144 43 402 514 1198 406 59 36 12.2 7.4 04/18/91 10 2580 1942 584 496 136 36 389 551 1096 388 67 45 10.6 5.5 04/22/91 14 3136 2384 846 720 162 55 493 538 1367 445 81 56 12.8 4.8 04/25/91 17 3620 2712 1026 754 197 64 558 562 1269 411 92 61 15.8 7.9 04/29/91 21 3500 2520 1010 680 170 58 557 624 1458 489 86 59 17.0 10.2 05/02/91 24 2840 2176 738 534 137 44 456 602 1572 520 66 44 17.2 12.0 05/06/91 28 2852 2164 660 520 133 40 410 686 1327 452 73 51 14.5 9.2 05/09/91 31 3470 2658 852 742 184 52 495 664 1194 417 92 62 15.5 8.1 05/13/91 35 3386 2574 914 748 170 61 516 533 1334 464 83 55 16.0 7.9 05/16/91 38 2952 2316 714 626 147 47 443 597 1265 437 67 44 13.8 8.2 MEAN 3104 2346 797 639 158 50 472 587 1308 443 77 51 14.5 8.1 STD 350 255 148 97 21 9 59 55 130 38 11 8 2.1 2.0 TABLE C49. REACTOR II (UASB) CHARACTERISTICS FROM RUN 4A DATE DAY PARAMETERS(mg/L) TS VS TSS VSS PROTEINS UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 04/15/91 7 36500 26490 24520 21485 6005 6370 11585 618 1297 439 999 38 330 6.3 04/18/91 10 36905 27370 25780 22635 6420 6655 12520 574 1331 442 1081 54 347 7.0 04/22/91 14 32430 23415 22095 19890 5060 5820 10735 648 1544 547 868 58 309 5.9 04/25/91 17 40440 30390 26635 23215 6815 6945 12095 684 1681 570 1141 50 365 8.0 04/29/91 21 37610 27975 24560 21025 6105 6335 11490 701 1360 475 1022 45 327 11.6 05/02/91 24 34405 25150 24325 22005 5210 6720 11835 764 1560 557 888 54 337 11.2 05/06/91 28 31875 22715 20555 18660 5105 5730 10125 748 1457 505 861 44 303 9.7 05/09/91 31 34830 24655 22455 20035 5645 6190 10345 670 1399 470 964 61 310 8.1 05/13/91 35 40210 30015 25510 22510 6200 6900 11755 691 1687 595 1033 41 347 6.8 05/16/91 38 32955 24085 22630 19855 5450 6480 10555 729 1491 524 918 46 314 8.8 MEAN 35816 26226 23907 21132 5802 6415 11304 683 1481 512 977 49 329 8.3 STD 2893 2541 1810 1410 569 394 768 55 131 52 90 7 19 1.9 TABLE C50. EFFLUENT II CHARACTERISTICS FROM RUN 4A DATE DAY P AR AMETERS(mg/l) TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 04/15/91 7 2490 1918 448 332 81 19 232 616 1279 438 51 38 9.8 5.8 04/18/91 10 2788 2070 420 306 98 14 207 562 1205 420 71 55 10.1 6.2 04/22/91 14 2408 1874 366 266 88 13 198 629 1577 563 75 61 9.5 6.3 04/25/91 17 3194 2412 454 346 122 18 235 680 1622 578 68 49 11.9 8.0 04/29/91 21 3322 2496 580 424 119 20 295 675 1321 462 67 48 15.2 10.3 05/02/91 24 3222 2486 468 332 108 22 214 722 1464 515 75 57 14.3 10.4 05/06/91 28 3538 2732 594 444 129 16 310 745 1477 506 65 44 12.9 7.9 05/09/91 31 2988 2208 574 418 92 21 284 698 1286 475 75 61 13.4 8.0 05/13/91 35 2700 2074 442 344 79 14 247 735 1665 578 52 40 10.8 6.4 05/16/91 38 3270 2616 552 438 87 17 308 707 1520 544 85 52 12.5 7.0 MEAN 2992 2289 490 365 100 17 253 677 1442 508 86 50 12.0 7.6 STD 360 285 75 58 17 3 41 55 152 55 8 8 1.9 1.6 TABLE C51. INFLUENT CHARACTERISTICS FROM RUN 4B DATE DAY P AR AM ET ER S (mg/L) TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 05/23/91 6 3705 2725 3295 2525 657 472 1405 78 590 137 128 23.0 19.4 9.7 05/27/91 10 4435 3380 4035 2900 669 481 2120 102 673 201 132 25.1 25.0 14.5 05/30/91 13 3880 2910 3415 2500 575 509 1620 103 551 177 123 30.5 21.7 11.1 06/03/91 17 3795 2940 3415 2595 524 417 1760 61 316 87 105 21.0 18.1 9.3 06/06/91 20 3990 2805 3550 2445 619 524 1365 79 361 106 117 18.4 16.7 8.8 06/10/91 24 4150 3050 3610 2650 532 555 1600 112 365 132 101 15.8 20.2 12.1 06/13/91 27 4205 3085 3850 2620 517 463 1850 145 527 166 103 20.1 21.6 13.6 06/17191 31 4085 3100 3595 2715 703 479 1895 109 503 157 139 26.1 22.8 16.1 06/20/91 34 3960 2920 3685 2545 538 518 1620 117 414 129 104 18.3 20.7 13.9 06/24/91 38 4175 3050 3590 2720 666 542 1640 106 432 120 127 20.5 20.1 11.8 06/27/91 41 4260 3230 3750 2850 679 552 1805 88 450 133 131 22.2 24.4 16.7 07/01/91 45 3985 2785 3665 2465 541 470 1555 105 504 162 104 17.8 18.3 10.3 07/04/91 48 3875 2970 3405 2545 580 475 1630 111 465 127 118 25.6 19.0 11.0 MEAN 4038 2996 3605 2621 600 498 1682 101 473 141 118 21.9 20.6 12.2 STD 196 175 194 136 65 38 196 20 96 29 13 3.9 2.4 2.5 TABLE C52. REACTOR I (CMR) CHARACTERISTICS FROM RUN 46 DATE DAY P AR AMETERS (mg/L) TS VS TSS VSS PROTONS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 05/23/91 6 16230 11830 10155 7795 1765 1180 5140 420 1642 542 351 66 130 6.9 05/27/91 10 20730 15080 13320 9925 2100 1565 6795 472 1730 605 376 40 165 8.2 05/30/91 13 16680 12915 11410 8550 1815 1385 5750 460 1651 567 349 59 149 10.2 06/03/91 17 20145 14895 12125 8955 2210 1625 5950 431 1456 469 417 64 145 8.6 06/06/91 20 22225 16165 13065 9740 2460 1815 6530 483 1396 459 438 44 169 11.6 06/10/91 24 17720 12830 11055 8575 1730 1280 6005 495 1360 477 322 46 153 10.5 06/13/91 27 23050 17150 14390 10355 2515 1630 7305 513 1515 508 448 45 181 6.1 06/17/91 31 21060 15390 12170 9270 1960 1390 6440 408 1328 445 374 61 150 8.3 06/20/91 34 23255 17485 14375 10740 2455 1840 7515 412 1446 487 446 53 173 10.7 06/24/91 38 19175 14390 13700 9910 1835 1375 7140 429 1390 455 342 49 160 8.1 06/27/91 41 21230 16040 14175 10615 2320 1620 6470 472 1614 538 412 41 167 7.6 07/01/91 45 18235 13885 12010 8330 1900 1265 5875 426 1377 457 357 53 141 11.5 07/04/91 48 20610 14570 11550 9195 2035 1645 7020 459 1482 508 386 60 148 10.8 MEAN 20027 14817 12577 9381 2085 1509 6457 452 1491 501 386 52 156 9.2 STD 2195 1614 1313 886 270 202 667 32 124 47 41 9 14 1.7 TABLE C53. EFFLUENT I CHARACTERISTICS FROM RUN 48 DATE DAY P AR AMETERS (mg/4 TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 05/23/91 6 2428 1866 296 236 257 60 166 430 1608 531 111 70 10.9 7.6 05/27/91 10 2524 1880 372 292 250 71 200 451 1762 625 78 38 12.9 8.8 05/30/91 13 3268 2454 486 408 305 82 271 489 1635 562 110 61 16.4 10.7 06/03/91 17 2664 1958 360 284 267 64 198 428 1447 492 108 65 13.5 9.0 06/06/91 20 2754 2108 408 328 290 76 228 501 1380 456 92 46 16.8 12.7 06/10/91 24 3078 2372 316 244 299 58 171 467 1465 508 93 45 15.4 12.0 06/13/91 27 3312 2520 440 360 328 71 255 502 1598 530 103 50 13.9 8.9 06/17/91 31 3120 2436 448 366 288 78 258 411 1412 464 110 64 13.3 8.2 06/20/91 34 2584 1938 368 286 240 62 201 401 1392 477 85 47 15.8 11.8 06/24/91 38 2732 2010 414 344 259 70 244 414 1406 495 92 50 13.8 9.0 06/27/91 41 2810 2146 368 358 300 63 262 490 1691 602 88 40 15.3 10.3 07/01/91 45 3102 2340 422 292 232 80 186 418 1330 434 82 45 16.7 12.9 07/04/91 48 2638 1980 378 296 303 64 209 439 1498 506 108 60 15.9 11.5 MEAN 2847 2154 390 315 278 69 219 449 1510 514 97 52 14.7 10.3 STD 282 229 51 49 28 8 35 35 130 54 11 10 2 2 TABLE C54. REACTOR II (UASB) CHARACTERISTICS FROM RUN 4B DATE DAY PAR AMETERS (mg/L) TS VS TSS VSS PROTEINS UPIDS^CARBOHYDR TOT. VFA COD TOC TIN NH3-N TP PO4 05/23/91 6 36750 26235 23450 16625 7275 7140 6940 415 1812 578 1207 43 370 7.7 05/27/91 10 32475 24180 21120 15090 6850 6775 6175 471 1903 658 1131 35 344 7.6 05/30/91 13 42015 30910 29805 21920 8200 7890 9520 512 1624 560 1366 54 482 8.8 06/03/91 17 42165 29775 27265 19450 7935 7610 8655 484 1849 651 1328 59 433 9.0 06/06/91 20 39275 28560 25335 18535 7355 7325 8055 459 1527 526 1229 52 412 10.3 06/10/91 24 42630 31695 29660 21045 8315 8030 9010 524 1453 486 1378 47 469 10.5 06/13/91 27 40005 30085 27450 19230 8160 7775 8285 547 1508 506 1363 57 428 8.7 06/17/91 31 35075 25380 24570 17515 6765 7035 7985 469 1718 549 1143 61 390 8.5 06/20/91 34 36715 25770 23715 17205 7055 6840 7415 518 1482 493 1182 53 383 11.2 06/24/91 38 41200 28355 27640 19350 7880 7795 8900 467 1618 540 1302 41 431 10.4 06/27/91 41 42015 30215 29005 20195 8405 8015 8870 455 1489 476 1387 42 450 7.8 07/01/91 45 36800 26650 24140 17440 6930 7490 6985 520 1825 641 1159 50 388 8.6 07/04/91 48 37675 27435 24855 18155 7405 6760 8365 488 1569 515 1245 60 402 9.6 MEAN 38830 28096 26001 18597 7579 7422 8089 487 1644 552 1263 50 414 9.1 STD 3061 2262 2573 1800 570 454 931 35 152 61 91 8 38 1.1 TABLE C55. EFFLUENT II CHARACTERISTICS FROM RUN 4B DATE DAY P AR AMETERS (mg/L) TS VS TSS VSS PROTEINS^UPIDS CARBOHYDR TOT. VFA COD TOC TKN NH3-N TP PO4 05/23/91 6 2268 1716 150 114 162 46 49 412 1653 534 74 48 9.4 7.4 05/27/91 10 2656 1984 168 134 160 51 56 457 1832 614 62 36 9.8 7.4 05/30/91 13 2846 2248 196 160 194 57 67 503 1506 487 80 49 11.0 8.2 06/03/91 17 2968 2256 164 128 204 44 55 514 1794 566 93 60 10.5 8.2 06/06/91 20 3134 2410 222 186 189 67 76 454 1548 524 82 51 13.1 9.9 06/10/91 24 2340 1806 144 106 153 38 48 530 1461 501 75 50 12.3 10.4 06/13/91 27 2914 2264 172 134 210 43 57 537 1568 500 93 59 10.2 7.8 06/17/91 31 3240 2452 200 154 217 58 64 464 1627 566 93 59 9.8 7.0 06/20/91 34 2664 1938 142 108 157 39 50 541 1508 490 74 48 13.0 11.1 06/24/91 38 2996 2274 148 120 175 54 49 464 1586 541 70 42 11.7 9.6 06/27/91 41 3130 2432 204 162 220 58 72 443 1382 469 82 47 10.9 8.0 07/01/91 45 2602 1888 146 110 158 41 47 532 1798 603 77 52 9.5 7.4 07/04/91 48 2714 2086 162 134 169 53 57 498 1644 524 89 62 11.0 8.6 MEAN 2806 2135 171 135 182 50 57 488 1608 532 80 51 10.9 8.5 STD 288 238 26 24 24 8 9 40 131 43 9 7 1.2 1.3 APPENDIX D VFA DISTRIBUTION TABLE^ PAGE Dl. Influent VFA Distribution^ 201 D2. Reactor I (CMR) VFA Distribution^ 202 D3. Effluent I (CMR) VFA Distribution 203 D4. Reactor II (UASB) VFA Distribution 204 D5. Effluent II (UASB) VFA Distribution^ 205 200 APPENDICES ^ 201 TABLE D1. INFLUENT VFA DISTRIBUTION RUN PARAMETERS(mg/L) Acetic Propionic Butyric 1w-butyric Valeric 3-methyl- butyric 2-methyl- butyric Total VFAs (as FiAc) 1A MEAN 52 44 12 0.4 02 1.0 1.1 98 STD 11 11 4 22 1B MEAN 57 50 15 1.3 1.4 0.7 0.9 111 STD 8 10 4 18 1C MEAN 53 46 19 1.3 1.6 0.9 1.3 106 SID 13 12 6 27 1D MEAN 58 45 16 0.9 1.6 1.6 0.7 108 STD 9 9 4 18 2A MEAN 62 52 18 1.3 1.5 1.6 0.9 119 STD 9 10 5 17 2B MEAN 45 34 10 0.7 0.8 1.3 0.5 81 STD 10 8 4 19 2C MEAN 58 45 14 0.7 1.3 1.5 1.0 108 STD 9 8 4 12 3A MEAN 51 41 18 02 1.4 0.6 0.1 97 STD 8 8 3 16 35 MEAN 22 20 7 0.5 0.3 0.1 0.1 44 STD 6 6 2 11 4A MEAN 62 44 18 1.9 2.2 1.9 1.2 114 STD 7 7 2 15 48 MEAN 51 41 20 2.1 1.9 1.2 0.7 101 STD 9 8 4 21 APPENDICES ^ 202 TABLE D2. REACTOR I (CMR) WA DISTRIBUTION RUN PARAMETERS (mg/L) Acetic Propionic Butyric Iso-butyric Valerie 3-methyl- butyric 2-methyl- butyric Total VFAs (as HAc) 1A MEAN 298 206 45 19 26 18 9 540 STD 49 28 8 3 4 4 3 72 1B MEAN 206 174 36 16 18 14 9 407 STD 56 36 9 4 4 4 3 84 1C MEAN 347 218 72 23 32 25 13 632 STD 40 32 8 5 6 5 4 68 1D MEAN 318 177 48 26 33 20 9 550 SID 30 21 7 4 5 4 2 52 2A MEAN 357 245 66 31 56 41 20 692 STD 39 33 7 5 8 8 5 56 2B MEAN 351 220 56 42 63 48 21 674 STD 38 24 6 6 7 7 3 71 2C MEAN 140 87 19 9 11 7 4 243 STD 40 14 3 2 3 1 1 53 3A MEAN 327 212 65 20 34 21 11 596 STD 14 29 8 3 4 3 2 46 3B MEAN 259 202 50 26 25 8 5 499 STD 36 21 8 5 5 1 2 59 4A MEAN 300 251 37 26 25 15 8 576 STD 26 25 7 5 5 3 2 55 413 MEAN 234 111 113 25 26 20 10 452 STD 19 16 15 4 4 4 2 34 APPENDICES^ 203 TABLE D3. EFFLUENT I (CMR) VFA DISTRIBUTION RUN PARAMETERS(mg/L) Acetic Propionic Butyric Iso-butyric Valeric 3-methyl- butyric 2-methyl- butyric Total VFAs (as HAc) 1A MEAN 287 205 48 a) 24 18 7 530 STD 44 33 8 3 4 3 2 73 1B MEAN 214 173 34 15 19 12 10 412 STD 47 32 8 3 3 3 3 76 1C MEAN 341 226 69 24 35 22 13 630 STD 29 37 8 5 6 3 3 64 1D MEAN 317 185 50 26 35 19 13 560 STD 30 22 8 4 5 4 2 64 2A MEAN 346 246 63 33 59 40 20 683 STD 41 28 11 6 8 8 4 66 2B MEAN 355 227 57 44 63 49 20 687 STD 32 29 7 6 7 8 3 65 2C MEAN 132 86 20 9 12 7 5 236 STD 30 14 5 2 2 2 1 47 3A MEAN 321 202 65 20 31 19 12 580 STD 20 23 7 5 5 3 2 50 38 MEAN 262 202 54 26 22 11 5 504 STD 29 31 8 5 5 2 1 62 4A MEAN 309 255 45 22 21 14 8 587 STD 26 26 8 3 3 3 1 58 1 413 MEAN 231 109 117 24 25 20 11 449 STD 20 14 19 5 5 4 2 36 APPENDICES ^ 204 TABLE D4. REACTOR fl (UASB) WA DISTRIBUTION RUN PARAMETERS(mgIL) Acetic Propionic Butyric Iso-butyric Valeric 3-methyl- butyric 2-methyl- butyric Total VFAs (as HAc) 1A MEAN 318 229 50 19 44 28 15 603 STD 34 35 8 4 8 6 4 68 18 MEAN 248 185 43 16 21 15 11 466 STD 40 30 6 3 4 3 3 69 1C MEAN 366 242 83 23 44 24 14 685 STD 29 30 8 3 7 4 3 54 1D MEAN 312 237 49 33 41 28 14 610 STD 34 28 7 6 6 5 2 70 2A MEAN 394 246 63 32 70 47 19 741 STD 49 33 7 5 11 7 4 90 26 MEAN 379 225 60 48 68 44 26 718 STD 26 26 7 7 10 7 5 69 2C MEAN 159 106 25 10 15 9 7 288 STD 19 17 6 3 3 2 2 38 3A MEAN 328 231 69 16 40 27 13 623 STD 22 23 8 3 5 5 2 51 38 MEAN 299 212 56 30 24 17 5 557 STD 24 24 8 5 5 2 1 55 4A MEAN 339 311 49 26 33 24 10 683 STD 29 27 7 5 7 3 2 58 4B MEAN 247 130 116 22 30 25 12 487 STD 17 19 18 5 5 4 2 36 APPENDICES ^ 205 TABLE D5. EFFLUENT H (UASB) VFA DISTRIBUTION RUN PARAMETERS(mg/L) Acetic Propionic Butyric Iso-butyric Valeric 3-methyl- butyric 2-methyl- butyric Total VFAs (as HAc) 1A MEAN 242 180 42 12 38 22 7 465 STD 26 31 8 3 7 5 2 57 1B MEAN 199 144 33 14 17 11 9 370 STD 31 25 7 3 3 2 2 55 1c ' MEAN 362 233 76 24 40 21 15 665 STD 30 31 9 5 7 5 3 53 1D MEAN 324 215 55 30 40 29 15 608 STD 38 28 7 , 6 6 6 3 75 2A MEAN 392 236 60 31 72 43 19 725 STD 41 31 7 5 10 7 2 78 33 MEAN 373 219 60 50 70 46 23 709 STD 28 29 6 7 9 7 4 67 2C MEAN 150 98 23 9 13 8 6 269 STD 18 16 6 3 2 2 2 40 3A MEAN 326 233 76 19 42 25 15 630 STD 30 21 9 3 6 5 3 56 3B MEAN 283 208 56 30 29 17 6 542 STD 30 30 9 6 6 3 2 54 4A MEAN 350 294 49 26 30 19 12 677 STD 33 24 6 5 6 4 2 58 48 MEAN 254 121 114 25 29 25 13 488 STD 12 15 19 6 5 5 4 42 APPENDIX E VARIOUS EXPERIMENTAL RESULTS AND CONVERSION FACTORS TABLE^ PAGE El. Seed Characteristics ^  207 E2. Soluble Carbohydrates 207 E3. Soluble Proteins 207 E4. Gas Production (Mean Values) ^ 208 E5. Conversion Factors^ 208 E6. Mass Balance Calculation Example (Run 1B, CMR System)^ 208 206 207APPENDICES TABLE El. SEED CHARACTERISTICS PARAMETERS MEAN VALUES (mg/L) STD (mg/L) pH 6.2 0.3 TS 6800 608 VS 5030 612 TSS 5890 709 VSS 4850 556 COD (soluble) 830 109 TOC 240 36 VFAs (as HAc) 35 8 NH3-N 28 4 TKN (soluble) 44 6 TABLE E2. SOLUBLE CARBOHYDRATES INFLUENT EFFLUENT RUN 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 35^7 1B 96 5.7 78 19 68 11 1C 135^8.0 47^9 36^8 1D 131 7.1 53 13 29 4 2A 116^6.6 44^11 27^5 2B 112 7.3 50 8 30 5 2C 113^6.4 78^14 28^6 3A 107 6.7 66 12 29 4 3B 199^9.5 64^7 37^4 4A 130 7.4 69 10 33 6 4B 135^8.0 118^16 39^6 MEAN 127 7.3 TABLE E3. SOLUBLE PROTEINS INFLUENT EFFLUENT RUN 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 68^8 1B 89 14.2 89 13 76 10 1C 108^16.9 66^6 47^7 1D 87 12.8 71 9 45 5 2A 109^17.2 47^6 31^4 2B 82 13.8 24 4 21 3 2C 92^15.7 63^8 54^8 3A 107 17.0 69 9 46 4 3B 107^19.1 56^5 52^7 4A 94 14.8 67 8 37 4 4B 103^17.1 70^7 60^8 MEAN 99^16.1 A. CONVERSION FACTORS FOR VFAs PARAMETER Mol. Weight mgVFA/mgHAc ACETIC 80.05 1.000 PROPIONIC 74.08 0.817 BUTYRIC 88.10 0.682 VALERIC 102.13 0.588 APPENDICES ^ 208 TABLE E4. GAS PRODUCTION (MEAN VALUES) RUN SRT (days) HRT (hr) Reactor I Reactor II Mean (mUd) STD (mt./4 Mean (mUd) STD (mUd) 1A 10 9 26 5 24 6 1B 10 6 33 7 31 7 1C 10 12 24 4 34 6 1D 10 15 80 10 92 11 2A 15 12 47 5 39 5 2B 20 12 45 7 52 9 2C 5 12 21 3 25 5 3A 10 12 33 5 37 6 38 10 12 38 5 49 7 4A 10 12 21 4 31 5 48 10 12 25 5 28 4 TABLE E5. CONVERSION FACTORS B. MISCELLANEOUS CONVERSION FACTORS PARAMETER CONVERSION FACTOR BASIS ACETIC ACID COD 1.067 mg/mg Acid Acetic Acid PROPIONIC ACID COD 1.514 mg/mg Acid Propionic Acid BUTYRIC ACID COD 1.818 mg/mg Acid Butyric Acid VALERIC ACID COD 2.039 mg/mg Acid Veloric Acid NITROGENOUS COD 9.58 mg/mg Org. N (C4 H6.1 012 N)x or 1.533 mg/mg Protein Org.N=16% Protein CARBOHYDRATE COD 1.067 mg/mg Glucose FORMIC ACID COD 0.348 mg/mg Formic Acid ETHANOL COD 2.087 mglmg Ethanol LACTIC ACID COD 1.066 mg/mg Lactic Acid TABLE E6. MASS BALANCE CALCULATION EXAMPLE (RUN 113. CMR SYSTEM) A. MONING AVERAGE MASS BALANCE DAY 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^CM 7 11.97 0.28 11.69 2710 13640 764 32.44 12.75 10 11.07 0.28 10.79 2630 16445 920 29.11 14.53 14 11.33 0.28 11.05 2650 18825 1420 30.02 20.96^47.3 17 12.26 0.28 11.98 2970 16270 1308 36.41 20.23 41.7 21 11.46 0.28 11.18 2500 19355 1396 28.65 21.03^34.6 24 11.50 0.28 11.22 2905 18365 960 33.41 15.91 41.9 27 11.94 0.28 11.66 2890 15810 850 34.51 14.34^46.9 30 11.95 0.28 11.67 2415 16140 1032 28.86 16.56 51.6 34 12.11 0.28 11.83 2550 20375 1222 30.88 20.16^45.8 37 11.63 0.28 11.35 2755 19370 958 32.04 16.30 42.2 41 11.72 0.28 11.44 2435 16650 1000 28.54 16.10^42.5 44 11.68 0.28 11.40 2405 19215 1184 28.09 18.88 42.2 B. OVERALL MASS BALANCE MEAN VALI^11.73^028^11.451^2651^17568^10851^31.08^17.34^442 MOVING AVERAGE MASS BALANCE: S VSS REDUCTION = 43.7 OVERALL MASS BALANCE: % VSS REDUCTION = 44.2

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