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High temperature biological treatment of Kraft pulping effluent Tai, Judy Yuet Wah 1998

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H I G H T E M P E R A T U R E B I O L O G I C A L T R E A T M E N T O F K R A F T P U L P I N G E F F L U E N T by J U D Y Y U E T W A H T A I B.A.Sc., The University of British Columbia, 1994 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE in THE F A C U L T Y OF GRADUATE STUDIES DEPARTMENT OF CHEMICAL ENGINEERING We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA APRIL 1998 © Judy Yuet Wah Tai, 1998 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. The University of British Columbia Vancouver, Canada Department DE-6 (2/88) A B S T R A C T The pulp and paper process produces hot effluent streams which currently must be cooled prior to biological treatment. Cooling such streams requires capital for construction of cooling towers or heat exchangers, as well as additional money for maintenance and operating costs. This project was to develop and test a high temperature activated sludge (HTAS) technology for the treatment of Kraft pulping effluents. Studies are based on two laboratory scale activated sludge bioreactors. The operating temperature of the laboratory reactors was increased over the temperature range 35°C to 55°C. The response of the reactors was measured in a number of ways. First, conventional treatment parameters such as biochemical oxygen demand (BOD), chemical oxygen demand (COD), solids content, oxygen uptake rate (OUR), toxicity and pH were monitored. Second, changes in the microbial ecosystem were indirectly monitored by measurement of changes in substrate uptake profiles, floe structure, settleability, and kinetics of methanol and formate uptake. Results have proven that the bioreactor was able to achieve BOD, COD and toxicity reductions of 94.8%, 35.8% and 93.8% respectively at 55°C. Effluent volatile suspended solids concentration increased with increasing temperature and averaged 63.3 mg/L at 55°C. Most probable number analyses of substrate use profiles indicated that the population of microorganisms responsible for degrading methanol, formate and resin acids decreased at temperatures over 45°C. The high temperature bioreactor recovered from a rapid drop in temperature within 24 hours. Kinetic constants obtained from ii measuring the substrate uptake of methanol and formate show that biomass has a weaker capacity to degrade methanol at elevated temperatures but an approximately equal formate-degrading activity at both temperatures. ii i TABLE OF CONTENTS A B S T R A C T i i L I S T O F T A B L E S v i i L I S T O F F I G U R E S ix A C K N O W L E D G E M E N T S x i Chapter 1 I N T R O D U C T I O N 1 1.1 Kraft Pulping 2 1.2 Biological Wastewater Treatment 3 1.3 Wastewater Treatment at Western Pulp 6 Chapter 2 B A C K G R O U N D 8 2.1 Cell Structure and Composition of Microorganisms 8 2.2 Thermophilic Microorgansims 9 2.2.1 Microbial Adaptation to High Temperature Environment 12 2.3 Activated Sludge Process 13 2.3.1 Process Description 13 2.3.2 Microbiology 18 Bacteria 19 Protozoa and Rotifers 19 Filamentous Bacteria and Fungi 20 2.4 Kinetics 21 2.5 Effect of Temperature on the Activated Sludge Process 23 Chapter 3 O B J E C T I V E S 27 iv Chapter 4 E X P E R I M E N T A L A P P A R A T U S A N D M E T H O D S O F A N A L Y S I S . . . 2 8 4.1 Experimental Apparatus 28 4.1.1 Bioreactors 29 4.1.2 Clarifier 31 4.1.3 Feed Storage Tank 31 4.1.4 Heating and Cooling System 32 4.2 The Experimental Laboratory-Scale Activated Sludge System 32 4.2.1 Aeration 34 4.2.2 Sludge Wastage 34 4.2.3 Clarifier Sitrring 34 4.2.4 Sludge Recycling 35 4.2.5 Control Units 35 4.3 Source and Treatment of Activated Sludge and Primary Clarified Effluent 35 4.4 Methods of Analysis 37 4.4.1 Biochemical Oxygen Demand 37 4.4.2 Chemical Oxygen Demand 38 4.4.3 Solids 40 4.4.4 Oxygen Uptake Rate 40 4.4.5 Toxicity 41 4.4.6 pH 42 4.5 The Practical Upper Temperature Limit of HTAS 43 4.6 Effect of High Temperature and Substrates on Species Diversity 43 4.6.1 Substrate Utilization Profile 44 4.6.2 OUR 46 4.6.3 Volatile Solids and Settleability Study 46 4.6.4 Microscopic Floe Structure 47 4.7 Response of HTAS Bioreactors to Temperature Shock 47 4.8 Kinetics of Substrate Uptake In Mesophilic and Thermophilic Bioreactors 48 Chapter 5 R E S U L T S A N D D I S C U S S I O N 50 5.1 Start-up and Steady-State Operation at 35°C 50 5.2 The Practical Upper Temperature Limit of HTAS 51 5.2.1 Operational Disruptions 51 5.2.2 Comparison of Mesophilic and Thermophilic AS System 52 BOD Removal 54 COD Removal 56 Solids 58 SOUR 61 Toxicity 61 5.3 Effect of High Temperature and Substrates on Species Diversity 64 5.3.1 Substrates Utilization Results 64 5.3.2 Floe Structure and Settleability 65 5.4 Response of HTAS Bioreactors to Temperature Shock 69 5.5 Kinetics of Substrate Uptake In Mesophilic and Thermophilic Bioreactors.... 71 5.5.1 Comparison within Individual Bioreactors 71 5.5.2 Comparison between Mesophilic and Thermophilic Bioreactors 72 Chapter 6 C O N C L U S I O N S 74 Chapter 7 R E C O M M E N D A T I O N S 77 R E F E R E N C E S 78 A P P E N D I X 82 A 5.1 Steady-State Operation Data 83 A 5.2 The Practical Upper Temperature Limit of HTAS 88 A 5.3 Effect of High Temperature and Substrates on Species Diversity 97 A 5.4 Response of HTAS Bioreactors to Temperature Shock 99 A 5.5 Kinetics of Substrate Uptake In Mesophilic and Thermophilic Bioreactors... 106 A 5.6 COD Calibration Curve 113 vi LIST OF TABLES Table 1.1 Typical chemical composition of woods 1 Table 2.1 Temperature range of different groups of microorganisms 10 Table 2.2 Known upper temperature limits for growth of specific from various microbial groups 11 Table 4.1 Characteristics of the received Western Pulp PCE 36 Table 4.2 Substrates used in the substrates utilization profile study 44 Table 5.1 Steady state performance of R2 at 35°C 50 Table 5.2 Treatment parameters at different temperatures 53 Table 5.3 Results of statistical significance of treatment parameters 53 Table 5.4 Change in Floe structure with increasing operating temperatures 67 Table 5.5 Mesophilic bioreactor performance before and after temperature shock 69 Table 5.6 Thermophilic bioreactor performance before and after temperature shock 70 Table 5.7 Kinetic values for methanol and formic acid at different temperatures 71 Table A 5.1.1 Steadystate BOD removal at 35 °C 83 Table A 5.1.2 Steady state COD removal at 35 °C 84 Table A 5.1.3 Steady state MLVSS and VSS removal at 35 °C 85 Table A 5.1.4 Steady state OURs and SOURs removal at 35 °C 86 Table A 5.1.5 Steady state toxicity removal at 35 °C 87 Table A 5.2.1 BOD removal between 37°C and 55°C 88 Table A 5 2.2 COD removal between 37°C and 55°C 89 Table A 5.2.3 M L V S S and VSS concentration during 37°C and 55°C 90 Table A 5.2.4 OURs and SOURs during 37°C and 55°C 91 vii Table A 5.2.5 Toxicity removal during 37°C and 55°C 93 Table A 5.2.6 Data for statistical significance test between 35°C and 55°C 94 Table A 5.2.7 Data for statistical significance test between 35°C and 45°C 95 Table A 5.2.8 Data for statistical significance test between 45°C and 55°C 96 Table A 5.3.1 Number of bacteria estimated by the M P N method 97 Table A 5.3.2 Sludge settleability as a function of reactor operating temperature 98 Table A 5.4.1 BOD removal for both bioreactors after temperature shock 99 Table A 5.4.2 COD removal for both bioreactors after temperature shock 100 Table A 5.4.3 M L V S S and VSS concentration after temperature shock 101 Table A 5.4.4 OURs and SOURs after temperature shock 103 Table A 5.4.5 Toxicity removal after temperature shock 104 Table A 5.4.6 Data for statistical significance test for mesophilic bioreactor after the shock study ....105 Table A 5.4.7 Data for statistical significance test for thermophilic bioreactor after the shock study 106 Table A 5.5.1 Kinetic constants data for utilization methanol at 35°C I l l Table A 5.5.2 Kinetic constants data for utilization methanol at 45°C I l l Table A 5.5.3 Kinetic constants data for utilization formic acid at 35°C 112 Table A 5.5.4 Kinetic constants data for utilization formic acid at 45°C 112 Table A 5.6 Data for COD calibration curve 113 vii i LIST OF FIGURES Figure 1.1 Kraft pulping process flowsheet 3 Figure 2.1 A typical prokaryotic cell 8 Figure 2.2 A typical eukaryotic cell 9 Figure 2.3 Schematic diagram of an activated sludge process 15 Figure 4.1 Laboratory scale activated sludge system 30 Figure 4.2 Schematic of experimental apparatus 33 Figure 4.3 Actual laboratory-scale AS process set-up 33 Figure 4.4 COD Calibration curve 39 Figure 4.5 Respirometer set-up 49 Figure 5.1 BOD removal efficiency as a function of reactor operating temperature 55 Figure 5.2 COD removal efficiency as a function of reactor operating temperature 57 Figure 5.3 Biomass concentration as a function of reactor operating temperature 59 Figure 5.4 Effluent solids concentration as a function of reactor operating temperature. 60 Figure 5.5 SOUR as a function of reactor operating temperature 62 Figure 5.6 Effluent toxicity removal efficiency as a function of reactor operating temperature 63 Figure 5.7 microscopic picture of floe structure at 38°C 66 Figure 5.8 Microscopic picture of floe structure at 55°C 66 Figure 5.9 Settleability of biomass with increasing operating temperature 68 Figure A 5.4.1 BOD removal efficiency of RI (mesophilic bioreactor) after temperature shock 99 Figure A 5.4.2 BOD removal efficiency of R2 (thermophilic bioreactor) after temperature shock 99 ix Figure A 5.4.3 COD removal efficiency of RI after temperature shock 100 Figure A 5.4.4 COD removal efficiency of R2 after temperature shock 100 Figure A 5.4.5 M L V S S concentration in RI after temperature shock 101 Figure A 5.4 6 M L V S S concentration in R2 after temperature shock 101 Figure A 5.4.7 VSS concentration in RI after temperature shock 102 Figure A 5.4,8 VSS concentration in R2 after temperature shock 102 Figure A 5.4.9 SOUR in RI after temperature shock 103 Figure A 5.4.10 SOUR in R2 after temperature shock 103 Figure A 5.4.11 Toxicity removal efficiency of RI after temperature shock 104 Figure A 5.4.11 Toxicity removal efficiency of R2 after temperature shock 104 Figure A 5.5.1 OUR versus time 107 Figure A 5.5.2 Oxygen consumed versus substrate metabolized 108 Figure A 5.5.3 SUR versus BOD 108 Figure A 5.5.4 Linearized Monod equation 109 x ACKNOWLEDGEMENTS I would like to thank Dr. Sheldon Duff for giving me the opportunity to work on this project. His endless patience and understanding have encouraged me and made this thesis possible. I would also like to thank the industrial support of Western Pulp's Squamish mill and Ms. Jeanne Taylor. The financial support of Science Council of B C through G.R.E.A.T. award is greatly appreciated. The technical support of the Pulp and Paper Centre and the Department of Chemical Engineering are also appreciated. In addition, I would like to thank my colleagues and friends at PPC for making my life at school enjoyable. xi C h a p t e r 1 INTRODUCTION The pulp and paper process utilizes wood residuals as raw material. Wood is mainly composed of fibres. Fibres are made of cellulose, hemicellulose, lignin and extractives. Typical composition of wood is shown on Table 1.1 (Thomas, 1981). Table 1.1 Typical chemical composition of woods. C e l l u l o s e H e m i c e l l u l o s e L i g n i n E x t r a c t i v e s P e r c e n t a g e i n W o o d 45-50 20-25 20-30 0-10 Not all these materials favour the process. The best balance of papermaking properties occurs when most of the lignin is removed while retaining substantial amounts of hemicellulose and all of the cellulose. Thus, the primary purpose of Kraft pulping is the removal of lignin from the rest of the wood contents. Lignins are characterised as very complex, crosslinked, three-dimensional polymers formed from phenolic units (Thomas, 1981). The linkages between different organic groups within the lignin structure are broken during pulping. There are various techniques to carry out pulping. The two major categories are chemical pulping and mechanical pulping. In chemical pulping the lignin in the fibre is degraded and dissolved away with chemicals, leaving behind most of the cellulose and hemicellulose. The yield is low relative to mechanical pulping, usually between 40% and 50% of the wood substance. Mechanical pulping separates the fibres by application of 1 mechanical stress and energy. Since there is no significant chemical composition change, pulp yield is above 90% (Ionides, 1995). 1.1 Kraft Pulping The dominant pulping process in the world is a chemical pulping process, the Kraft process. Kraft is the German word for strength and the pulp produced from the Kraft process is known for its superior strength properties (Smook, 1992). The complex lignin structure is broken down into various organic compounds during the process. Most of the important chemicals used during the Kraft pulping such as sodium and calcium are recovered. A typical Kraft process is shown in Figure 1.1. Wood chips are first fed into the digester and are cooked with a solution of inorganic chemicals. Two products come out after digestion: cooked pulp and black liquor. The cooked pulp is separated from the black liquor in the brown stock washers and goes to the bleaching stages. In the early stages of bleaching most of the residual lignin in the pulp is removed. The later stages employ oxidizing agents to brighten the pulp. Under a typical bleaching sequence of DEDED, the pulp is first treated with an acid agent, usually CIO2, to solubilize and oxidize the remaining lignin. This is the D stage. Then, in the E stage, the chlorinated lignin compounds are extracted using caustic soda, NaOH. The process is then repeated with a second D stage to oxidize and brighten the pulp and another E stage to extract the soluble compounds so that they can be washed out. A final D stage is used to bring the pulp to market brightness. 2 Bleach Plant a Bleached A Pulp Foul Digester Condensates — — -J Foul Condensate Steam I Recovery Boiler Caustic Sewer Chemical Recovery Line Acid Sewer Multiple Effect Evaporator Pulp Line Black liquor —• Miscellaneous Green Liquor Clarifie White White Liquor Causticiser Figure 1.1 Kraft pulping process flowsheet. Black liquor from brown stock washers goes to the recovery cycle to recover chemicals and energy. It is concentrated in the multiple effect evaporators and then goes to the recovery boiler where the sodium and sulphur compounds are recovered. In addition, organic components are burned and energy is recovered in the process. 1.2 Wastewater Treatment An important consideration in modern pulp and paper mills is to treat mill effluents so that their impact on the environment is minimal. Of most concern to the pulp and paper industry from a water pollution perspective are solids, oxygen demand and toxicity (Smook, 1992). Dissolved oxygen is essential to the survival of aquatic life. When natural waters become seriously deficient in dissolved oxygen, their ability to support aquatic life is impaired. A large fraction of naturally-occurring organic waste is biodegradable by organisms in water to produce CO2 and H2O as the ultimate products, according to the following example reaction: C 6 H i 2 0 6 (glucose) + 6 0 2 -> 6 C 0 2 + 6H 2 0 The rate of the biological breakdown and therefore the rate of oxygen consumption from the water varies depending on the nature of the waste and the physical conditions in the environment. Some effluent constituents are degraded rapidly, whereas more complex structures, such as lignin derivatives and organic solids, are slow to degrade. Effluent solids interfere with the feeding habits of fish. Deposition of settleable solid particles can 4 also affect the viability of bottom-dwelling organisms. Several groups of compounds including resin acids, unsaturated fatty acids and chlorinated phenolics in pulp mill effluent have been identified as toxic and can affect the habitat of fish and other aquatic life. The major sources of wastewater in the pulping process are evaporator and digester foul condensates, and bleach plant washer filtrates. Evaporator and digester condensates are highly contaminated with low-molecular-weight organic matter and normally account for up to one third of the BOD load (Springer, 1993). This organic matter is mainly methanol, which represents most (80%) of the BOD, as well as other alcohols, ketones, phenolic substances, sulfur compounds and terpenes (Springer, 1993). The acid and caustic filtrates from bleaching contribute a large portion of toxic substances. Principle toxic chemicals from the caustic filtrates include chlorinated compounds, resin acids and unsaturated fatty acid derivatives (Springer, 1993). Thus, it is important to have proper wastewater treatment before discharging effluents into natural waters. A typical pulp mill wastewater treatment plant consists of primary treatment and secondary treatment. In primary wastewater treatment, screening and sedimentation are employed to remove floating and settleable solids. After primary treatment, the wastewater is further treated in secondary or biological treatment. It is called biological because the process utilizes naturally-occurring microorganisms to convert nonsettleable colloidal particles and dissolved organic material to environmentally-benign materials. Thus, the objectives of biological treatment are to reduce the BOD and the toxicity of the effluent. 5 Biological treatment depends on a mixed culture of microorganisms that use organic matter (BOD) in the effluent as food source and convert it into CO2, H2O and cell mass. Normally, the microorganisms can reduce BOD by approximately 95% and render the effluent non-toxic to fish (Environment Canada, 1983). Bacteria are the primary BOD degraders in the treatment system. Other microorganisms include fungi, protozoa, and rotifers which remove dispersed bacteria, thereby reducing solids in the discharged effluent (Winkler, 1981). The process can take place either in the presence of oxygen (aerobic systems), or without oxygen (anaerobic systems). The biological treatment process consists of creating conditions for a healthy population of microorganisms to develop and feed on the organic matter in the effluent. The parameters that are important include: pH, temperature, hydraulic and solids retention time, level of nutrients and, in the case of a aerobic system, dissolved oxygen concentration. There are a variety of common biological treatment technologies employed in the pulp and paper industry. One of the most popular is the activated sludge process. It is commonly used in situations where land is limited. The process principles and microbiology of activated sludge are discussed in detail in section 2.2. 1.3 Wastewater Treatment at Western Pulp Western Pulp Limited Partnership (WP) operates a Kraft mill at Squamish, British Columbia, producing 750 tonnes/day of pulp. The combined mill effluent is treated by a UNOX® pure oxygen activated sludge treatment system before discharge to Howe Sound. The biological reactor normally operates at the accepted optimum temperature 6 range of between 35°C and 40°C. However, operational difficulties are encountered, especially during the summer months. The mill uses four plate heat exchangers to cool the four hottest streams prior to treatment. The two bleach plant effluent streams routinely plug the heat exchangers due to fibre carry-over. Plugged heat-exchangers have to be taken off-line for cleaning, and it is common to have only three heat exchangers operating for part of any one month. When this happens, the reduction in cooling capacity is offset by direct injection of cooling water, however during summer months, the fresh water supply is at a minimum, and the demand is at its peak. A forced mill shutdown is possible due to inadequate supply of water needed to cool the effluent prior to treatment. At the Squamish mill, the water is drawn from a water-shed on the mountain behind the mill. Relying on this water supply for cooling effluents leaves the mill and other users of the water-shed in a vulnerable position, especially in the hottest periods of the year. Moreover, cooling of effluents uses energy and is costly. Finally, using large quantities of fresh water for cooling increases the discharge of effluent. Therefore, i f the treatment process could be operated at a higher temperature, less fresh water would be required and the potential for a forced mill shutdown could be reduced. Considerable savings in energy and maintenance of heat exchangers would also be realized. 7 Chapter 2 B A C K G R O U N D 2.1 Cel l Structure and Composition of Microorganisms Despite the complexity and variety, microorganisms can be grouped into two categories: prokaryotes and eukaryotes. Prokarya includes bacteria whereas the Eukarya includes fungi, protozoa, higher plants and animals. There are major differences in the cell structure between these two categories. The prokaryotes have a simpler cell structure than the eukaryotes. The general structure of a typical prokaryotic cell consists of a cell wall, a cytoplasmic membrane, one or more ribosomes, and a nucleus (Figure 2.1). These cells are approximately 1-5 urn in diameter (Brock et al., 1994). The nuclear region is very different from that of an eukaryotic cell, in that it does not have a membrane and contains only one single molecule of DNA. Cell membrane Cytoplasm Nucleus (no membrane) Rigid cell wall Ribosomes Figure 2.1 A typical prokaryotic cell. 8 An eukaryotic cell is much bigger and more complex than the prokaryotic cell (Figure 2.2). It is about 20 \im in diameter and contains a membrane-enclosed nucleus which has more than one copy of DNA. It also contains different types of distinct cellular structures called organelles which are absent in the prokaryotic cell. For example, one such organelle, the mitochondrion, is the site where energy generation takes place. 2.2 Thermophilic Microorgansims The activities of microorganisms are strongly dependent on the physical and chemical conditions in their environment. Temperature, one of the main physical factors, is very important to the growth and the survival of microorganisms. For a particular group of microorganisms, the minimum temperature is where no growth occurs. The optimum temperature is where they grow most rapidly. Growth is no longer possible when the temperature is above their maximum temperature level. A l l microorganisms respond differently to a given temperature. A given temperature range harmful to one Cell membrane Nuclear membrane Extracellular organelles Ribosomes Figure 2.2 A typical eukaryotic cell. 9 type of microorganism may be beneficial to another type. Based on their temperature range for growth, microorganisms can be generally classified into four major groups: psychrophiles, mesophiles, thermophiles and extreme thermophiles (Table 2.1). Thermophiles are of particular research and industrial importance because of their unique cell structures and heat resistant properties. T a b l e 2.1 Temperature range of different groups of microorganisms (Brock et al.,1994). Groups General temperature range rc) Psychrophiles 10-20 Mesophiles 30-40 Thermophiles 55-65 Extreme thermophiles above 80 According to Brock (1978), thermophiles are organisms which live at elevated temperatures and usually have an optimum growth temperature between 45-80°C. Thermophiles include both prokaryotes and eukaryotes and exhibit a wide range of growth characteristics. As with mesophiles, thermophilic bacteria obtain their nutrients in many different ways. According to Brock et al. (1994), thermophiles cannot grow as fast as mesophiles since thermophiles must devote more energy to cell maintenance due to the higher rate of protein turnover at elevated temperatures. Table 2.2 lists some typical thermophilic microorganisms and the upper temperature limit at which they can survive (Brock, 1978). 10 Table 2.2 Known upper temperature limits for growth of specific species from various microbial groups. Groups Upper temperature limits (°C) Eukaryotes: Microorpanisms-Protozoa 56 Algae 55-60 Fungi 60-62 Prokaryotes: Bacteria-Cyanobacteria 70-74 Photosynthetic bacteria 70-73 Lithotrophic bacteria >100 Heterotrophic bacteria >100 Archaea-Hyperfhermophilic methanogens 110 A l l prokaryotic thermophiles are able to grow optimally at temperatures higher than eukaryotes. The upper temperature limit for most eukaryotes is around 60°C (Brock, 1986), a value much lower than that of prokaryotes. The inability of eukaryotes to grow at high temperatures lies in the cell membrane structure. As mentioned above (Section 2.1), eukaryotes contain membranous intracellular organelles such as mitochondria to carry out cellular functions. The membranes surrounding these organelles have pores which allow selective passage of macromolecules (Brock, 1986). As temperature is increased, membrane leakiness increases and the cell becomes metabolically unstable (Brock, 1978). Comparing the cell membrane structure between mesophiles and thermophiles, the major components of the cell membranes of both bacterial groups are lipids, which are polymers of fatty acids. However, unlike mesophiles, thermophiles contain a large 11 proportion of saturated fatty acids which allow the membranes to remain thermally stable and functional even at high temperatures (McElhaney, 1974; Brock et al., 1994; Bitton, 1994). 2.2.1 Microbial Adaptation to High Temperature Eenvironment The ability to survive and grow at elevated temperatures also depends strongly on the molecular structure of thermophiles. First, enzymes and other proteins in thermophiles are more thermostable than are those of mesophiles (Ljungdahl and Sherod, 1974). Studies have shown that proteins from thermophiles are rather similar to mesophiles in many respects (Ljungdahl and Sherod, 1974; Brock et al., 1994). However, subtle changes in the hydrogen bonds, hydrophobic interactions, ionic bonds and sulfur-sulfur bonds allow the molecules to fold differently and thus confer thermostability (Brock, 1986). Ljungdahl and Sherod (1974) suggested that proteins in thermophiles can structurally stabilize themselves by interacting with each other to form macromolecular complexes, which are more thermostable than individual proteins. In the protein-synthesizing machinery, which includes ribosomes and other constituents, the rRNA from thermophiles has a higher G-C content than mesophiles. The greater G-C content results in a higher protein melting point (Brock, 1986). Studies also show that ribosomes in thermophiles can withstand higher temperatures without melting because of the interaction between R N A and the protein within the ribosome (Ljungdahl and Sherod, 1974; Brock, 1986). 12 The activated sludge process is one of the most common methods of treating industrial wastewater. Even though conventional methods of biological treatment employ mesophiles, thermophiles have been suggested by engineers to be more advantageous, especially in industrial processes. The most common thermophiles found in the activated sludge process are genus Bacillus (Brown et al, 1967). 2.3 Activated Sludge Process Activated sludge (AS) is the most common technology used in secondary wastewater treatment. Ardern and Lockett (Tchobanoglous and Burton, 1991) developed the process to treat municipal wastewater in England in 1914. It was so named because it involved the production of an activated mass of microorganisms capable of stabilizing a waste aerobically (Tchobanoglous and Burton, 1991). The ability to reduce the oxidation time of sewage from days to hours made the AS process attractive. A number of variations of the basic system have been developed over the years and now a wide variety of process options exist. In addition to its application to municipal sewage, activated sludge is now a widely-used process for the treatment of many industrial wastewaters such as pulp mill effluent. 2.3.1 Process Description The principle of the process is that wastewater is brought into contact with a mixed suspended-growth culture of microorganisms in an aerated and agitated tank. The 13 organic materials in the wastewater are first absorbed onto the surface of the microorganisms and are later oxidized through microbial metabolism (Winkler, 1981). The microorganisms utilize the organic material (BOD) in the wastewater as a food and energy source, and convert a portion of it to CO2 and H2O and the rest to new cell material. BOD + O2 + Microorganisms ->C02 + H2O + more Microorganisms A schematic of a typical activated sludge process is shown in Figure 2.3. The aeration tank is where the aerobic oxidation of organic matter takes place. The primary effluent enters and is mixed with bacteria to form the mixed liquor (ML). Aeration is usually provided by mechanical means. Mixed liquor suspended solids (MLSS) is the total amount of suspended solids, including microorganisms, in the mixed liquor. The organic portion of the MLSS is the mixed liquor volatile suspended solids (MLVSS). Typically, M L V S S represents 65-75% of MLSS (Bitton, 1994). A clarifier is used for the settling of the sludge. The products from the clarifier are the treated effluent (supernatant) and the settled sludge. The treated effluent is separated from the flocculent microbial sludge mass by gravity settling in a separate vessel known as a clarifier. Most of the settled sludge is returned to the aeration tank and is called returned activated sludge (RAS). The remainder is removed for disposal in order to prevent an excess of biomass from accumulating as well as to maintain a steady microbial population within the reactor. The 14 sludge removal can also be done directly from the reactor. The wasted sludge is known as waste activated sludge (WAS). Waste Activated Sludge (WAS) Figure 2.3 Schematic diagram of an activated sludge process. 15 In the operation of the activated sludge system, several important parameters must be considered. They are the hydraulic retention time (HRT), solids retention time (SRT), and the food to microorganisms ratio (F/M). HRT is defined as the average time spent by the liquid in the aeration tank and is thus the time of contact between the sludge and the wastewater. In a completely mixed continuous system, this is the volume of the reactor divided by the influent flowrate. HRT = — (1) Q where V = volume of aeration tank (L) Q = influent flowrate (L/hr) HRT is generally a fixed value determined during the design stage. Typically, HRT is 2-12 hours or more to allow the microorganisms to degrade the organic matter completely within the reactor (Barr, 1994). Thus, the majority of degradation happens inside the aeration basin and not the clarifier. Longer HRT, however, increases the volume of the aeration basin which can be a problem when space is limited. Shorter HRT allows greater flow rates and biomass production, but reactors operating in this way tend to be less efficient in removing organic matter. The sludge retention time, also known as mean cell residence time or sludge age, is the average time that a microbial cell stays in the reactor. For a completely mixed process operating at steady state conditions, SRT is usually defined as the mass of organisms in the reactor divided by the mass of organisms removed from the system and, when wastage is directly from the aeration basin, is given by the following expression: 16 S R T = ™ Q w X + Q e X e (2) Where X = M L V S S concentration (mg/L) X e = concentration of microorganisms in effluent (mg/L) Q w = flowrate of liquid in the wastage stream (L/day) Q e = flowrate of clarified effluent leaving the system (L/day) Because the activated sludge process recycles a large proportion of the biomass, the SRT is much greater than the HRT. This helps maintain a large number of microorganisms that can effectively oxidize organic matter in a relatively short time (Bitton, 1994). Usually, maintaining an SRT of about 3 to 15 days results in the production of a high quality effluent and good sludge settling characteristics (Tchobanoglous and Burton, 1991). If the SRT is too short, the cells are washed out faster than they can reproduce, producing an effluent quality similar to that of the influent. For longer SRTs, less food is available to the microorganisms, and more oxygen is consumed. Thus the significance of the SRT as an important operational parameter is because of its control over sludge activity and settleability. Another important operational parameter is the food to microorganism ratio (F/M). It is regarded as the amount of food available to the microorganisms: 17 F / M = - ^ . X x V (3) Where S = substrate concentration (mg/L) In a completely mixed activated sludge process, F/M values of 0.2 to 0.6 ensure an optimum amount of food for the microorganisms (Sundstrom and Klei , 1979). It is controlled by the rate of wasting. Higher wasting rates result in higher the F/M ratios. Since the F/M determines the amount of food available to microorganisms, it controls the growth rate of microorganisms. 2.3.2 Microbiology Activated sludge is comprised of a mixture of microorganisms. The components of the sludge in a particular system will depend on the composition of the wastewater and the operating conditions. Generally, the mixed culture consists of bacteria, protozoa, filamentous bacteria and fungi, together with inert organic and inorganic material. They exist either singly or clumped together. Each of the species play a different but significant role in an activated sludge. In the following sections microorganisms commonly found in activated sludge are described. 18 B a c t e r i a Bacteria are the predominant and most important microorganisms present in the activated sludge system. Their two major attributes are their abilities to degrade organic matter and to form floes. Some bacteria produce polysaccharides and other polymeric material that aid in the flocculation of microbial biomass (Bitton, 1994). One of the most common floc-forming bacteria is Zoogloea ramigera (Grady and Lim, 1980). Sludge floes are clusters of several million bacterial cells. The formation of microbial floes facilites the adsorption and agglomeration of the suspended matter in the wastewater and the separation of the sludge from the treated supernatant. Therefore floc-forming bacteria play a very important role in the process. P r o t o z o a a n d R o t i f e r s Protozoa and rotifers play a significant role in the activated sludge process. The most important function they perform is the removal of dispersed bacteria, thus ensuring that bacteria are not discharged with the treated wastewater (Jerkins et al., 1993). Previous experiments have shown that sludge without protozoa produced effluent containing large numbers of dispersed bacteria (Pike and Curds, 1971). The numbers of dispersed bacteria decreased dramatically shortly after inoculation with a Protozoa culture (Pike and Curds, 1971). Usually, protozoa constitute as much as 5% of the mass of the sludge (Richard, 1989). Ciliates are the dominant protozoa present in the activated sludge system (Jones, 1976). Others include flagellates and amoebae. Rotifers in activated sludge, besides removing dispersed bacteria, contribute to floe formation by 19 producing fecal pellets surrounded by mucus (Bitton, 1994). Philodina sp., and Lecane sp. are common rotifers found in activated sludge. Filamentous Bacter ia and Fung i Not all bacteria in activated sludge are floc-formers. Some of them are filamentous. There are major physiological differences between floc-forming and filamentous bacteria. Filamentous bacteria have a higher surface-to-volume ratio than their floc-forming counterparts, and thus are able to survive under low dissolved oxygen concentrations and low-nutrient conditions (low F/M) (Bitton, 1994). Many types of filamentous microorganisms are bacteria with a small portion of fungi (Richard, 1989). A certain amount of filamentous microorganisms can be beneficial to the activated sludge process because filaments are hypothesized to serve as a " backbone" to floe structure, allowing the formation of larger, stronger floes. They serve to catch and hold small particles during sludge settling, providing a lower turbidity effluent (Richard, 1989). A lack of filamentous organisms can lead to formation of pin-flocs which are small and easily sheared and which leave the effluent turbid (Richard, 1989). When filamentous growth is in excess, bulky, loosely packed, poorly settling floes result. This can cause operational problems such as bulking, in which floes tend to be carried out of the clarifier with the treated effluent. Thus, a bulking sludge causes the discharge of excess organic matter and the loss of sludge inventory. Possible reasons for sludge bulking are low dissolved oxygen, nutrient deficiency, low pH and low food to microorganism (F/M) ratio (Richard, 1989). 20 2.4 Kinetics One of the common approaches to modelling activated sludge process kinetics is the application of the Monod equations (1949) for the evaluation of the substrate limited rate of biological growth: — = uX (4) dt V = ^ T c (5> Ks + S Where S = growth limiting substrate concentration (mg/L) K s = half saturation constant (mg/L) u\ = specific growth rate constant u\m = maximum specific growth rate constant These two equations were originally developed for pure cultures, but have often been used for mixed cultures such as activated sludge. Since it is more important to remove organic matter than to cultivate biomass in wastewater treatment, it is desirable to investigate the rate of substrate removal. Biomass growth and substrate removal can be related by the observed biomass yield coefficient, Y 0 b s - The rate of increase of microorganisms in a culture (dX/dt) is proportional to the rate of substrate uptake (dS/dt) by microbial cells. 21 Y o b s — dX dt (6) dS dt where dX/dt = the rate of increase in microorganisms (mg MLVSS/L/day) dS/dt = the rate of substrate removal (mg BOD/L/day) Y o b s = the growth yield coefficient (mg MLVSS/mg BOD) Combining equations 4, 5 and 6 gives: dS 1 _ Urn S dt X ~ Y o b s K s + S (7) Substituting q for (-dS/dt)(l/X), and q m for u\m/Y0bs> a equation for specific substrate removal rate is obtained By analogy to the specific growth rate, the constant K s is equal to S when q = 1/2 q m (Shuler et al., 1992). At high substrate concentration S » K S , and q = q m , the substrate is removed at a constant rate whereas for S « K S , q = (qm/K s)S, substrate is removed at a decreasing rate (Cech et al., 1984). K s represents the affinity of the microorganisms for the substrate. The values for q m and K s both are influenced by temperature, type of carbon source and other factors. They can be obtained simply by plotting equation 8. Another way is to take the reciprocal of the equation to obtain a linear equation: S (8) q = qm K s + S 22 1 K s l 1 (9) q q m S q m Thus, by plotting 1/q against 1/S, a straight line with a slope of K s / q m and y-intercept at l / q m is obtained. 2.5 Effect of Temperature on the Activated Sludge Process The influence of temperature on activated sludge treatment is complex. It affects the metabolic activities of the microbial population, oxygen transfer rates and settling characteristics (Tchobanoglous and Burton, 1991). Biochemical reactions are catalyzed by active proteins called enzymes. Enzyme activity is a temperature-dependent function, with reaction rate generally increasing with increasing temperature. However, elevated temperatures denature protein and therefore reduce activity. As temperature is increased beyond the optimal growth temperature, the effective reaction rate begins to decline due to enzyme denaturation (Friedman, 1970). Different groups of microorganisms exhibit their optimum metabolism and growth at different temperature ranges. For monocultures growing below their optimum temperature, the change in rate constant with temperature can be characterized by the modified Arrehnius equation: KT = K.2O0 .CT -T20) (10) 23 Where K j = growth rate at temperature T T = temperature (°C) K 2 0 = growth rate at 20°C 6 = temperature coefficient (constant) T20 = temperature at 20°C According to the Van't Hoff-Arrhenius rule, biochemical reaction rates will approximately double with each 10°C increase in temperature. The temperature range over which this rule applies to is very small (Surucu, 1976). In a mixed culture biological system, the Arrhenius and Van't Hoff-Arrhenius rule does not predict the reaction rate very well (Sawyer and McCarty, 1994). In a mixed culture system, temperature changes can lead to a change in the dominant species. Therefore, the reaction rate varies as different species dominate different temperatures. Moreover, according to Friedman (1970) a given species probably possesses several alternative metabolic pathways. Each step of each pathway has its own enzyme characteristics and temperature rate dependence. The ability to adapt to changes in temperature varies dramatically between different microorganisms (Ganczarczyk, 1983). Thus, the reaction rate and the temperature coefficient are not constant and change with temperature (Ganczarczyk, 1983). The metabolic activity of the microorganisms involved is not the only consideration in determining the effect of temperature on wastewater treatment. Oxygen transfer is an another factor affected by temperature. Gehm (1956) investigated the effect of high temperature on an AS process treating Kraft mill effluent and concluded that 24 dissolved oxygen was not found at any time at the higher temperature but sufficient oxygen was delivered to maintain aerobic conditions. However, it has been suggested that the low oxygen saturation values in water at high temperature could be offset by the higher oxygen diffusivity coefficient (Surucu et al., 1976; Jackson et al., 1983). Intense agitation was also suggested to compensate for the reduced oxygen solubility at high temperature (Kalinske, 1974). The effect of elevated operating temperatures on solids settleability has been reported, but the data were inconclusive. Increased suspended solids in the effluent of activated sludge processes has been observed at high operating temperatures. Brown et al. (1967) found that there was a greater amount of bacterial cells in the effluent for an activated sludge process operated at 55°C than the one operated at 30°C. Duke et al. (1981) found an increase in effluent suspended solids when running a pilot plant activated sludge process at 115°F (46°C). Similar results were also reported by Flippin and Eckenfelder (1994). Kalinske (1974) concluded from his studies that the high concentration of suspended solids in the effluent from an activated sludge process was partly due to lack of bioflocculation properties of the thermophilic aerobes. He also found that the absence of protozoa at 55°C was a possible reason for the large number of dispersed bacterial cells, since protozoa feed on these cells as their food and energy source. On the other hand, good solids settling characteristics (Gehm, 1956) and an absence of dispersed bacterial growth at elevated temperatures were reported (Jackson, 1983). Barr (1994) and Rintala and Lepisto (1993) also reported effluent turbidity was not noticeably increased at elevated temperatures. 25 Other treatment parameters, such as BOD and COD removal have also been reported for high temperature activated sludge. Duke et al. (1981) concluded that temperature did not affect the ability to remove soluble BOD to the required levels, but affected the rate of soluble BOD removal. Rintala and Lepisto (1993) concluded that COD removals were comparable to mesophilic system when treating bleached Kraft mill effluents at 55°C. Graczyk (1984) reported that optimum BOD and COD removal were obtained at 55-60°C in a laboratory scale activated sludge system. In a recent study, Barr (1994) stated that BOD removal over a temperature range (41-50°C) was comparable to that at 35°C, and COD removal was slightly, but significantly better than that of the mesophilic reactor. Carpenter et al. (1968) conducted an experiment in which the activated sludge operating temperature was increased from 37°C to 52°C in 10 days to determine the effect of temperature on treating pulp and paper wastes. The results showed that a deterioration in performance was observed at an operating temperature of 52°C. However, biomass may not have been fully acclimated to a 15°C increased in temperature within such a short period of time (10 days). The results may not have shown the actual steady state performance of the reactor. In general, previous research on thermophilic activated sludge process has shown inconclusive results. Microbial activities in activated sludge are complex and dictated by a wide range of physical, chemical and biological factors. Further work is required to evaluate the feasibility of activated sludge operation at elevated temperatures. 26 Chapter 3 O B J E C T I V E S The purpose of this work is to determine the effect of elevated operating temperatures on an activated sludge process treating Kraft mill effluent. The specific research objectives were as follows: 1. Obtain steady state performance data for two bioreactors operating at 35°C. 2. Determine the practical upper temperature limit for operation of the activated sludge process by increasing the bioreactor operating temperature from 35°C to 55°C. Evaluation of bioreactor performance was based on removal of BOD, COD, solids, toxicity, and on microbial viability (OUR). 3. Determine the effect of high temperature operation on microbial species diversity. Response variables include: substrate utilization profiles, floe structure and settleability. 4. Evaluate the stability of the high temperature AS process when subjected to rapid changes in temperature. 5. Obtain and compare kinetic constants of substrate uptake in mesophilic and thermophilic AS process. 27 Chapter 4 E X P E R I M E N T A L A P P A R A T U S A N D M E T H O D S O F A N A L Y S I S In this project two bioreactors were used to determine the viability of a high temperature activated sludge technology (HTAS) for treating Kraft pulping effluents. To monitor performance, influent and effluent from the bioreactors were assayed for biochemical oxygen demand (BOD), chemical demand (COD), total suspended solids (TSS), volatile suspended solids (VSS), and acute toxicity as measured by the Microtox toxicity assay (EC50). In addition, the concentration of sludge within the reactor (MLVSS) was measured, and the viability of the sludge was determined through measurements of oxygen uptake rate (OUR). 4.1 Exper imenta l Apparatus The major laboratory equipment involved in this project included: two bioreactors, (RI & R2), two clarifiers, one feed tank, and two water baths. Other accessories included a refrigerator, two wooden frames, an electronic timer as well as controllers, pumps and tubing. 28 4.1.1 Bioreactors The two 4-litre activated sludge bioreactors were constructed out of 6 mm thick Plexiglas (Figure 4.1). Each bioreactor had an inner tank with diameter 20.5 cm and an outer tank with diameter 25.5 cm. The height of each bioreactor was 24 cm. The inner tank contained the activated sludge and wastewater. To maintain the bioreactor temperature, water was circulated to the outer tank from a temperature-controlled water bath. Lids made with Plexiglas were used to cover each bioreactor to prevent heat loss and evaporation. Six holes were made in each lid to allow tubes to go through and to equalize pressure. The reactor vessel was placed on a large stirring plate (Fisher Scientific, Vancouver, BC). A 7.5-cm long Teflon-coated magnetic stir bar was placed on a thin sheet of stainless steel plate at the bottom of each bioreactor to maintain proper mixing of the reactor contents. The stainless steel plate was used to prevent direct contact between the stir bar and the Plexiglas bottom; otherwise wearing would occur and damage the Plexiglas. To connect the bioreactor to the clarifier and the water bath, three nozzles were made on each bioreactor. The nozzle that connected to the clarifier penetrated the inner tank so that the reactor contents could pass to the clarifier. It was located 10 cm from the base of the inner bioreactor vessel and was 15 mm in diameter. The other two nozzles only penetrated the outer jacket. One nozzle was placed where the water entered the outer jacket, 3 cm up from the base of the bioreactor. The other was placed where the water 29 returned to the water bath 18 cm up from the base of the bioreactor. The diameters of these two nozzles were 7 mm and 10 mm respectively. Figure 4.1 Laboratory scale activated sludge system. 30 4.1.2 Clarifier Two 2-litre Pyrex Erlenmeyer flasks were modified to serve as clarifiers (Figure 4.1). Modifications were made by the Canadian Scientific Glass Blowing Company (Richmond, BC). The base of each flask was removed to allow an opening of 16 cm and the top of the flask was narrowed to about 11 mm in diameter. The modified flask became a clarifier by simply turning it upside down. Two 1 lmm-diameter spouts were constructed on each clarifier. One was a horizontal spout located at the midpoint of the clarifier, connected by a hose to the nozzle exiting from the bioreactor. The other spout was about 13 cm below the top of the clarifier and was angled down 15° from the vertical, and served as the outlet for treated wastewater. A small funnel was placed inside each clarifier. A hose was run through the funnel and out the angled spout. The height at which the funnel was located determined the volume of the liquor in the bioreactor. In this project, the volume of the mixed liquor was fixed at 4 litres. When the mixed liquor content was over 4 litres, excess liquor would be forced over the sides of the funnel and exited as treated effluent. 4.1.3 Feed Storage Tank A rectangular tank was made to contain approximately 60 L of feed wastewater. The tank was made with 1 cm-thick Plexiglas with height 59.5 cm, width 35.5 cm and length 40.5 cm. The tank was kept in an Admiral refrigerator and maintained at 4°C to prevent wastewater degradation. To allow direct access from the bioreactors to the 31 wastewater, a 3 cm hole was drilled into the side of the refrigerator so that a tube could run from the tank, out of the refrigerator, through a pump to the bioreactors. 4.1.4 Heating and Cooling System Since the project involved a temperature study, the laboratory scale activated sludge system made use of water as the medium to cool or heat the bioreactors. A 10-litre M G W L A U D A thermostated water bath was used to control the RI bioreactor. The water temperature in RI was maintained at 35°C. A V W R Scientific Model 1162 (VWR, West Chester, PA) automated heating/cooling water bath was connected to the R2 bioreactor to control the temperature. The temperature variation in R2 ranged from 35°C to 55°C. 4.2 The Exper imental Laboratory-Scale Activated Sludge System A schematic diagram is shown in Figure 4.2. The two activated sludge bioreactors were placed side by side and shared a common influent tank. Figure 4.3 shows an actual picture of the setup. A rectangular wooden frame was constructed to support the whole unit. The back part of the frame was sized to fit the stirrer and the bioreactor. The front of the frame served as a holding rack to keep the clarifier at the proper height so as to control the liquid level in the bioreactor. As the operating temperature in R2 was increased above room temperature, two 0.5-cm insulating sheets were wrapped around the bioreactor and the clarifier to reduce heat loss. 32 11 P U M P C L A R I F I E R F E E D T A N K Figure 4.3 Actual laboratory-scale AS process set-up. 33 4.2.1 Aeration The bioreactors were continuously aerated to ensure that the dissolved oxygen level was maintained above a minimum level of 2 mg/L. Air was fed through a stone diffuser from an Optima air pump which was able to deliver 500-5000 mL air/minute. The dissolved oxygen within the bioreactors was measured regularly using a DO meter to ensure it was above the minimum level. 4.2.2 Sludge Wastage The biomass was growing continuously; therefore, wasting of biomass was required to maintain a constant MLVSS. In this project, about 10 mL of M L V S S was removed from each bioreactor each hour. Wasting once per hour was chosen over once per day because removal of a small amount of the bioreactor contents every hour was thought to be less disruptive to the system. 4.2.3 Clarifier Stirring Unlike an industrial clarifier which usually has a small surface area to volume ratio and rakes that remove and mix any biomass that sticks on the wall of the clarifier, the laboratory clarifier had a conical shape and a large surface area to volume ratio. During operation of the laboratory clarifier, a large portion of biomass was found to stick on the walls of the clarifier. To overcome this problem, a pump was activated for 30 34 seconds every 15 minutes at a rate of 40 mL/min to circulate and mix the liquid contents of the clarifier to increase flocculation and improve settling. 4.2.4 Sludge Recycling Biomass recycling was done directly from the bottom of the clarifier by running a tube through a pump and back to the bioreactor. The clarifier temperature was not controlled, meaning temperature changes in the laboratory clarifier would affect biomass viability. Therefore, recycling was continuous to transfer the biomass back to the bioreactor as quickly as possible. The biomass was recycled at 8 mL/min. 4.2.5 Control Units For each activated sludge unit, four Cole Palmer (Chicago, IL) 1-100 R P M peristaltic pumps and four Cole Palmer (Barrington, IL) speed controllers were used to control feed, biomass recycling, biomass wasting and clarifier mixing. A ChronTrol model X T timer (San Diego, CA) was used to control the pumps. 4.3 Source and Treatment of Act ivated Sludge and P r imary Clar i f ied Effluent In order to carry out the studies at conditions similar to Western Pulp's wastewater treatment system, waste activated sludge (WAS) from Western Pulp Limited Partnership's Kraft mill in Squamish BC was used to seed the bioreactors. The WAS was 35 collected from the secondary clarifier and was delivered to the U B C Pulp and Paper Centre the same day. A solids test was performed to determine the concentration of the WAS which was then diluted by distilled water to make up a mixed liquor concentration of 2000 mg/L. Primary clarified effluent (PCE) was used as the influent wastewater for this project. It was collected from the mixing box upstream from the activated sludge reactor at WP. At this point in the process, the effluent had been neutralized and nutrients necessary for biological treatment had been added. PCE was shipped weekly from the mill to U B C and was stored at 4°C to minimize microbial activity before the experiment. The PCE was delivered weekly for several reasons. First, there was limited storage area at UBC. Second, storing of the PCE over a long period of time could result in deterioration. Finally, even though changing the PCE weekly may have caused a variation in influent characteristics, it more closely approximated the conditions at the mill. Table 4.1 contains a summary of mill PCE characteristics over the entire experimental period of 17 months. Table 4 .1 Characteristics of the received Western Pulp PCE. B O D mg/L C O D mg/L Toxicity TU (ECso) pH Low 48.1 501 5.7 (17.4) 6.6 High 603 1729 110 (0.9) 9.6 Average 261±129 1233 ±306 37.1 ±23.2 (3-7) 7.7 ± 0.7 36 4.4 Methods of Analysis Six analytical methods were employed to determine the treatment efficiency of the bioreactors. These methods included BOD, COD, solids, toxicity, OUR and pH. Analyses were conducted according to Standard Methods for the Examination of Water and Wastewater, 17 t h edition. Any deviation from Standard Methods is noted. 4.4.1 Biochemical Oxygen Demand Biochemical oxygen demand (BOD) is the parameter most widely used to characterize organic pollution. BOD is the quantity of oxygen utilized by a mixed population of microorganisms in the aerobic oxidation of organic matter. BOD is often written as BOD5 where the subscript refers to the length of the test in days. Five days is the most commonly used period for the test. In this study, the 5-day BOD test was performed, according to Standard Methods twice weekly on the influent and effluent samples. In each test, seed control samples were run in duplicate, along with one influent dilution and three different effluent dilutions. Both influent and effluent samples were performed in triplicate. Dilution water was prepared one hour before testing. Reagent solutions containing the micronutrients necessary for bacterial growth were added to the dilution waster as specified by Standard Methods. The dilution water was aerated to ensure that it was saturated with dissolved oxygen. Initially, biomass from each bioreactor was used as seed, however, once the 37 temperature study began POLYSEED (Polybac Corp., Bethlehem, PA) was used to provide a more reliable measurement. To carry out the assay, each sample was put into a Wheaton 300-mL BOD bottle. A stir bar was added to each bottle to ensure complete mixing when taking the DO reading. To minimize evaporation of the water through the seal during incubation, each sample was covered with a Wheaton BOD cap. Incubation was done in a dark incubator at 20°C. The DO concentration of all the samples was measured initially and again after 5 days. DO measurements were made originally using a YSI (Yellow Springs Instrument, Yellow Springs, OH) model-50B DO meter and a YSI model-5730 DO probe; however, due to temperature limitations for this equipment, an Orion DO (Analytical Technology, Inc., Boston, MA) meter was substituted part way through the study. The BOD of both influent and effluent samples was calculated according to the formula given in Standard Methods. 4.4.2 Chemical Oxygen Demand The COD test is used to measure the amount of oxygen required for oxidation of organic content using a strong chemical oxidizing agent in an acidic medium. The COD of the wastewater is generally higher than the BOD because more compounds can be chemically oxidized than can be biologically oxidized. The COD test was performed on influent to, and effluent from the reactors according to the Closed Reflux Colorimetric Method specified in Standard Methods. COD was measured three times a week. Distilled water was used as a blank. Influent and 38 effluent samples were run in triplicate while blanks were performed in duplicate. Each sample and the COD reagents were put into a standard 10-mL vial. A Hach model 45600 (Loveland, CO) COD reactor was used to digest the samples at 150°C for 2 hours. After the samples were cooled, their absorbances were measured using a Hach Dr/2000 (Loveland, CO) direct reading spectrophotometer set at 620 nm. Calibration curves were constructed every time a new set of reagents was prepared to ensure that the COD values obtained were correct. COD standards were prepared using potassium acid phthalate ( K H C 8 H 4 O 4 ) and used to generate standard curves. A typical standard curve is shown in Figure 4.4. 600 1 . 1 . 1 Absorbence Figure 4.4 COD calibration curve. 39 4.4.3 Solids The most important physical characteristic of wastewater is its solids content. Suspended solids determination is one of the major parameters used to evaluate the strength of wastewater and to determine the efficiency of the treatment unit. Total suspended solids (TSS) are the solids that are nonfilterable. The volatile suspended solids (VSS) determine the amount of organic matter present. The TSS and VSS of the activated sludge and effluent from the two bioreactors were determined twice per week. A 15-20 mL sample from each bioreactor was collected using a Nichiryo model 5000 syringe pipette. Effluent samples of 100 mL were collected in a graduated cylinder from each clarifier outlet. The samples were filtered using a Whatman glass microfibre filter (No. 934-AH) and were placed in ceramic crucibles. They were first dried at 105°C overnight in a Fisher Isotemp oven and then cooled in a dessicator. The TSS was determined by weighing the samples on an OHAUS API 1 OS analytical balance. The samples were then ignited at 550°C for one hour in a Thermolyne Furnatrol I muffle furnace, and were cooled and weighed to determine the VSS. 4.4.4 Oxygen Uptake Rate Microorganisms in the activated sludge process use oxygen as they consume food. The rate at which they use oxygen, the oxygen uptake rate (OUR), can be taken as a measure of biological activity. High OURs indicate high biological activity; low OURs indicate low biological activity. 40 Mixed liquor was removed from each bioreactor into a beaker and aerated for 15 minutes to raise the DO concentration. The aerated sludge was then poured into a 60-mL respirometer containing a Teflon-coated stir bar. The whole unit sat on top of a stir plate. Water was circulated through the outer jacket of the respirometer at the same temperature as the bioreactor. When the operating temperature of the mixed liquor was below 40°C a YSI model-5730 DO probe was immersed halfway down the respirometer, and the dissolved oxygen was recorded using a YSI model-59 DO meter. An Orion DO meter was used when the operating temperature was above 40°C. The decrease in DO was monitored for 15 minutes or until the DO level inside the respirometer dropped below 2 mg/L. The data was sent directly from the DO meters via a RS-232 interface to a computer. After the test, the mixed liquor was sampled to determined VSS, and the rest was poured back into the bioreactor. 4.4.5 Toxicity Pulp mill effluent contains organic compounds which are toxic to aquatic organisms, so it is important to evaluate the toxicity of the effluent. The Microtox toxicity test was used to determine the toxicity of effluent samples. The test uses light emitting bacteria, Vibrio fisheri. When the bacteria are exposed to toxic compounds, a reduction in light emission can be used as a quantitative measurement of toxicity. Toxicity measurements were taken with the Microtox model-500 Analyzer according to the methodology outlined in the Microtox manual. Influent and effluent 41 were tested three times a week. Influent was diluted to 10% concentration and was assayed using the Basic test. Effluent was assayed using the 100% test without dilution. In the Microtox tests, precision in the amounts of reagents and samples added was important. A slight deviation in the amount added would strongly affect the results. Similarly, a trace of impurities such as unclean pipettes would also affect the results. A Nichiryo model-5000 syringe pipette was used to transfer any solution between 1 and 5 mL. Two different sizes of Eppendorf pipettes were used to transfer solutions between 10 uX-100 p;L and 100 uX-1000 \iL. New pipette tips and curvettes were used for each test. The reading was measured as EC50 which is the effluent concentration that results in a measurable negative effect on 50 percent of the test population. Taylor (1996) concluded from her results that a Microtox reading of >35% will result in a fish toxicity result of >100%. EC50 can be converted to toxicity units (TU) by the formula: TU=100/EC 5 0 4.4.6 pH The pH range suitable for biological treatment is restricted to between 6 and 8.5. The pH of both influent and effluents was monitored daily. A Cole Palmer model-05669-20 pH meter and probe was used to check the pH. If the measurement showed the pH was either too high (>8) or too low (<7), hydrochloric acid or sodium hydroxide would be added to maintain the pH between 7 and 8. 42 4.5 T h e P r a c t i c a l U p p e r T e m p e r a t u r e L i m i t o f H T A S The goal of this experiment was to determine the practical upper temperature limit for operation of activated sludge bioreactors treating pulping effluents. The bioreactors were first allowed to stabilize at 35°C and maintained at this temperature for six weeks to establish steady state conditions. Reactor performance during this period was monitored and used as the baseline data for future comparison with periods when the reactor was operated at a different temperature. At the end of the six weeks the temperature in one reactor (R2) was then increased at a rate of 2°C per week. Once the reactor reached 50°C, it was allowed to acclimate to each new temperature for 2 weeks. The operating temperature of the bioreactors was noted twice a day using a mercury thermometer. At the end of this period, the temperature of the reactor was 55°C. The operating temperature of R2 was then turned down to and remained at 45°C for the rest of the study. 4.6 E f f e c t o f H i g h O p e r a t i n g T e m p e r a t u r e o n Spec ies D i v e r s i t y One of the important effects that may result from increasing the reactor operating temperature is a decrease in the number of species which can exist within the bioreactor. To determine the effect of operating temperature on species diversity, a summer project was developed and carried out by a summer student, Joyce Choi. The temperature range of R2 during the experiment was 32°C to 52°C, over a period of three months. The bioreactor contents were assayed to identify the substrates which could be utilized by the biomass as temperature increased. The substrate uptake profiles were monitored for 15 43 different substrates at each temperature tested (Table 4.1). Other methods to monitor the changes in the microbial community included monitoring OUR, volatile solids concentration, settleability of the biomass, and floe microscopic structure. Table 4.2 Substrates used in the substrates utilization profile study. Substrates Concentration (g/L) Sodium formate 0.5 Benzoic acid 0.05 Palmitic acid 0.5 Linoleic acid 0.5 Guaiacol 0.5 Dehydroabietic acid (DhA) 0.05 Abietic acid (AbA) 0.05 Pimaric acid (PiA) 0.05 Isopimaric acid (IpA) 0.05 12/14-Cl-DhA 0.05 Arabinose 0.5 Galactose 0.5 Glucose 0.5 Methanol 0.5 Ethanol 0.5 4.6.1 Substrate Utilization Profile Two different methods were involved in evaluating the substrate utilization profile of the biomass in R2: (1) microplating, and (2) determination of the Most Probable Number (MPN). Microplating was done by first performing an extraction of biomass from the sludge suspension. Biomass extraction was done by collecting a 70 mL sample of sludge 44 from R2, and centrifuging it in two SS34 centrifuge tubes at 13000 x g. The tubes were decanted leaving behind the pellets which were washed with 0.1M phosphate buffer at pH 7 and then re-centrifuged at 13000 x g. The washing and centrifuging were repeated two times. The washed solids were diluted with saline solution (0.85% NaCl) with 0.01% (v/v) Tween 80 and 0.01% (w/v) sodium pyrophosphate from a 50 g/L standard solution. The mixture was then blended ("grind" setting) using a household blender for 10 seconds. Six dilutions (0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001 g/L) were made from the mixture using saline solution. These diluted biomass samples were used for inoculation of the Microplates. The BIOLOG redox technology developed by BIOLOG Inc. (Hayward, CA) was used in this study. Commercially available BIOLOG MT Microplates which contain 96 wells in each plate with tetrazolium violet inside each well were used to colorimetrically indicate the utilization of the carbon sources. The MT plate was used to develop a characterization plate strictly designed for biomass samples from biological reactors treating bleached Kraft mill effluent (Victorio et al., 1996). The ability to rapidly visualize a metabolic pattern produced by sole-carbon-source utilization by the microbial biomass provides a useful tool to distinguish microbial communities within the wastewater treatment systems (Victorio et al., 1996). For non-volatile substrates, 10 \iL were dispersed into the wells and dried before the biomass suspension (150 jxL) was inoculated. For volatile substrates such as methanol and ethanol, filling was done immediately after the inoculation of biomass suspension. Six Microplates were utilized for each test at each temperature: three replicates for each substrate and three control wells with only biomass inocula. Microplates were incubated at the same temperature as 45 the R2 bioreactor for 10 to 14 days. The plates were examined approximately every 24 hours. Substrate utilization was indicated by a purple colour in the well. Loss of volatile substrates at elevated temperatures represented a source of possible error during the experiment. Attempts were made to minimize the effects of evaporation. For the tests at 50°C and 52°C, volatile substrates together with the biomass inoculum were incubated in sealed test tubes. The M P N technique is based on "The Most Probable Number Technique" (Oblinger,1980). It has been used in different studies to estimate the number of microorganisms in a sample. Gale and Broberg (1994) used the M P N method for enumerating E. coli in drinking water samples. Wrenn and Venosa (1996) applied this technique in enumerating oil-degrading bacteria and stated that the method was accurate and selective. 4.6.2 OUR (see Section 4.4.4) 4.6.3 Volatile Solids and Settleability Study Determination of volatile solids (VS) was conducted according to the Standard Methods. The settleability test was done by collecting enough mixed liquor from the reactor to yield 1 g of VS, and diluting the sample to a VS concentration of 1000 mg/L. The well-mixed suspension was allowed to settle in a 500-mL graduated cylinder for 30 46 minutes at the same temperature as the bioreactor. The settleabilty was measured as the height of the settled solid in millimetres. 4.6.4 Microscopic Floe Structure Sludge samples at different elevated temperatures were examined under a light microscope using 5X and 10X objectives. Floe structure was observed regularly as temperature increased. 4.7 Temperature Shock Study The temperature shock study was used to investigate the response of thermophilic activated sludge bioreactors to rapid changes in temperature associated with shutdown and start-up of mills. A series of experiments was performed to evaluate the ability of the high temperature bioreactor to start up quickly and produce an acceptable effluent quality within a short reheating time. During this study, the normal assay schedule was augmented with periods of rapid sampling during reactor reheating. The temperature of both RI and R2 were reduced to 10°C in three stages within 30 hours. The temperature of R I , was first lowered to 28°C, then to 20°C after 10 hours and finally to 10°C after another 10 hours. Similarly, the temperature of R2, was first lowered to 33°C, then to 22°C after 10 hours, and finally to 10°C after another 10 hours. 47 Both bioreactors were maintained at 10°C for two days without feed or air, but reactor stirring was maintained during this time. Feed and air were stopped once the temperatures were under 30°C. This particular experimental methodology was chosen to mimic the situation observed at Western Pulp during mill shutdown. To start up, the temperatures of both bioreactors were raised to their previous operating values of 35°C and 45°C within 30 hours in the same manner as the temperature was lowered. The feed and air were started when the temperatures reached 30°C. After 30 hours, assays were performed to evaluate the change in performance of the bioreactors due to the temperature shock. The sampling began immediately after the 30 hours of heating, followed by sampling at 1, 6,10, 15, 24, 36, 48 and 63 hours after startup. 4.8 K i n e t i c s o f S u b s t r a t e U p t a k e i n M e s o p h i l i c a n d T h e r m o p h i l i c B i o r e a c t o r s Kraft pulping effluents contain hundreds of different constituents. It is possible that temperature will exert a differential effect on the metabolism of some of these constituents by the microbial culture. In this phase of the work, the ability of microorganisms in the mesophilic and thermophilic bioreactors to metabolize two of the major constituents of Kraft pulping effluents was evaluated and compared. Microbial kinetics were measured by using a respirometic method developed by Cech and Chuboda (1984). Compounds tested were methanol and formic acid. A schematic of the respirometer used is shown in Figure 4.5. The general measuring technique is the same as described in Section 4.4.4. After the base oxygen 48 uptake rate was recorded, a known volume of a single substrate was added to the mixed liquor. The change in OUR as well as the amount of O2 consumed were measured. The concentration of methanol added to Rland R2 ranged from 0.1 to 1.9 mg/L and 0.06 to 1.0 mg/L, respectively. The concentration of formic acid added was 0.4 to 0.6 mg/L for RI and 0.5 to 0.9 mg/L for R2. The amount of substrate did not exceed this range because, at higher substrate concentrations, dissolved oxygen dropped below the minimum level (1 mg/L) before the substrate was completely oxidized. On the other hand, efforts were made to obtain data at lower substrate concentrations. However, at very low substrate concentrations oxidation was completed before any significant change in OUR could be recorded. Moreover, the baseline OUR was unstable during the measurement and peaks were difficult to identify. DO P R O B E F E E D A IR STONE W A T E R OUT W A T E R IN STIR B A R W A T E R J A C K E T Figure 4.5 Respirometer set-up. 49 Chapter 5 R E S U L T S A N D D I S C U S S I O N 5.1 Star t-Up and Steady-State Operat ion at 35°C To start the bioreactors, a total solids test was done on the waste activated sludge received from Western Pulp to determine the concentration. It was found to be 50.7 g/L. The two bioreactors were filled with the desired amount of sludge and distilled water to achieve an MLSS of 2000 mg/L. Feed was introduced and reactor performance was monitored. M L V S S in reactor 1 (RI) began to decline after start-up. After one week of operation M L V S S in RI decreased to below 1000 mg/L and failed to treat influent to the required standard. RI was stopped and started again. On the other hand, R2 performed efficiently and remained at 35°C for six weeks. Its steady state performance was measured and results are summarized in Table 5.1. Table 5.1 Steady state performance of R2 at 35°C. Temp B O D C O D Toxici ty S O U R V S S M L V S S Removal Removal Removal mg0 2 / °C % % % ( E C 5 0 ) mgMLVSS-min mg/1 mg/1 35 95.4 ±4.3 47.6 ±9.6 94.2 ±5.1 12.5 ±7.9 34.3 ± 32.0 2522±1180 (74.4) 50 5.2 The Pract ical Upper Temperature L i m i t of H T A S Following a period of steady-state operation at 35°C, the effect of an increase in operating temperature on performance of R2 was studied. The goal of this part of the experiment was to determine the practical upper temperature limit for operation of activated sludge bioreactors treating Kraft pulping effluents. 5.2.1 Operational Disruptions The temperature ramping phase of this study took place between June, 1996 and January, 1997. During this part of the study, there was an unforeseen mill closure at the end of July, which lasted for two weeks. When WP shut down to facilitate installation of a new bleaching stage, no advance notification was given and thus no PCE was supplied. As a result, an immediate switch to PCE from the Harmac Pacific mill in Nanaimo, BC was made in order to allow the laboratory activated sludge systems to continue operating. However, the PCE from Harmac had different characteristics from the WP PCE. The average BOD, COD and Toxicity were 127.3 mg/L, 1015.5 mg/L and 41.0 (TU), respectively. The biomass in R2 had to acclimate to the new environment which probably affected its performance. Since temperature was meant to be the sole variable in this study, changes in influent characteristics affected the results. Thus, the data obtained during that particular period were not included in the calculation of performance data, nor in the discussion. Western Pulp resumed operation after two weeks of closure and regular shipments of PCE resumed. It took another two weeks for the bioreactors to recover from 51 disruption. Therefore, in total, the period from July 26th to August 23rd was not considered. Other operational problems also occurred unexpectedly. On August 21st and 24th 1996, power outages at the PPC interrupted oxygen supply, stirring and cooling in the bioreactors. These power outages affected the treatment performance and normal progress on the experiment was delayed for two weeks each time while the bioreactors restabilized. Also the water bath controlling the high temperature reactor (R2) ran out of water on September 21, 1996 which caused the temperature of the reactor to decrease to room temperature between September 21st and 22nd. Finally, due to installation of new power outlets, the laboratory scale AS system was disconnected from its power supply for 10 hours on October 15, 1996. A l l these unexpected operational disruptions have undoubtedly upset the bioreactor to a certain extent. However, from the results obtained, only the switch in influent and power outages in August caused a noticeable difference in treatment performance. 5.2.2 Comparison of Mesophilic and Thermophilic AS System Table 5.2 summarizes the treatment performance at three different temperatures. The bioreactor was operated long enough at 35°C and 55°C to obtain steady state values, whereas the bioreactor operated at 45°C for only two weeks and results obtained at this temperature cannot be considered to be steady state values. 52 Table 5.2 Treatment parameters at different temperatures. Temp °C B O D Removal % C O D Removal % Toxici ty Removal %(EC 5 0 ) S O U R mg02/ mgMLVSS-min V S S mg/1 M L V S S mg/1 35 95.4 ±4.3 47.6 ±9.6 94.2 ±5.1 (74.4) 12.4 ±7.9 34.3 ± 32.0 2522±1180 45 96.0 ±1.3 45.3 ± 19.5 94.5 ± 1.7 (64.1) 14.1 ±7.3 28.9 ±18.9 2108 ±433 55 94.8 ± 2.3 35.8 ±8.8 93.8 ±3.0 (61.3) 12.4 ±4.3 63.3 ±36.1 2210 ±549 The results were statistically compared using a Smith-Satterthwaite t-test (Devore, 1991) to determine whether the results at different operating temperatures were significantly different. The level of significance for all calculations was 0.05. Table 5.3 presents the conclusions from the statistical analysis. A " N O " indicates there was no significant difference in the treatment performance at the two temperatures compared whereas a " Y E S " means there was a significant different in treatment performance. Table 5.3 Results of statistical significance of treatment parameters. Columns represent comparison of means for data from two different temperatures. 35°C/45°C 35°C/55°C 45°C/55°C B O D NO NO NO C O D NO Y E S NO M L V S S NO NO NO V S S NO Y E S Y E S S O U R NO NO NO Toxici ty NO NO NO 53 Of the parameters measured, COD removal (lower at elevated temperatures), and effluent VSS (higher at elevated temperatures) were significantly affected by elevated operating temperature (55°C). B O D Removal The percentage removal of BOD by R2 ranged from 39.3% to 99.2% over the temperature range 37°C to 54°C (Figure 5.1). Even though the influent BOD concentration varied from 48.1 mg/L to 603.0 mg/L, R2 consistently reduced the effluent BOD concentration to below 60 mg/L and maintained an average percentage removal above 90% over the entire test period. As the reactor temperature increased, BOD removal performance did not change significantly. The bioreactor remained at 55°C for two and a half months in order to study its steady state performance. During this two and a half months, the bioreactor showed consistent BOD removal and the overall BOD removal was 94.8% at 55°C. There are a few points for which BOD removal was much lower than the average value. These periods are due to the switch to the Harmac effluent (point A), and to subsequent power outages (point B and C), discussed in Section 5.2.1. The percentage removal started to decline from August 7th 1996 and reached a minimum of 39.3% on August 23rd. The removal rate went back to 97.9% by August 29. 54 Figure 5.1 BOD removal efficiency as a function of reactor operating temperature. A = switch to Harmac influent (July 26, 1996); B = power outages (August 21 & 23, 1996); C = water bath malfunction (September 21-22,' 1996); D = power outages (October 15, 1996); E, F = low influent COD (July 9, 1996 & Jan 7, 1997). 55 C O D Removal Figure 5.2 shows the COD removal efficiency of reactor R2 over the temperature range from 37°C to 55°C. The percentage COD removal ranged from 19% to 66%. As was the case for BOD removal, an increase in reactor operating temperature did not dramatically affect COD removal. COD removal declined slowly from its steady state value 49.6% with increasing temperature, to 38.4% at 55°C (Table 5.3). According to the statistical test, there was a significant difference in the treatment performance between 35°C and 55°C. One reason for the decrease in the COD removal in the effluent is due to the COD contribution of the increased concentration of suspended solids. At temperatures over 45°C, turbidity was observed in the effluent. There were a few data points showing low COD removal. On July 9th, 1996 (point E) the low COD removal (19%) was likely attributable to an influent COD which was significantly lower than the normal value 1218 mg/L (Table 4.1). A similar situation on Jan 7fh 1997 (point F) (influent COD 681.0 mg/L) resulted in a decline in the percentage removal to 15%. It seems that whenever the influent COD was much lower than the normal range, low removal efficiency resulted. On the other hand, the disruptions associated with switching effluents and power outages had no obvious effects on COD removal. 56 Figure 5.2 COD removal as a function of reactor operating temperature. A = switch to Harmac influent (July 26, 1996); B = power outages (August 21 & 23, 1996); C = water bath malfunction (September 21-22, 1996); D = power outages (October 15, 1996); E, F = low influent COD (July 9, 1996 & Jan 7, 1997). 57 Solids An attempt was made to maintain an MLVSS concentration close to 2000 mg/L through adjustment of the wastage rate. The mixed liquor volatile suspended solids (MLVSS) concentration during the temperature ramping phase of the increasing operating temperature varied between 1160 mg/L and 3840 mg/L. The volatile suspended solids (VSS) of the effluent varied from 11 mg/L to 128 mg/L (Figure 5.3 and Figure 5.4). There was a general trend of increasing VSS concentration in the effluent as temperature was increased, especially once the temperature was over 50°C. At operating temperatures over 50°C, poor biomass settleability in the clarifier became noticeable and turbidity of the effluent was observed. The VSS at 55°C was significantly higher than what was observed at lower temperatures. The issue of solids settleability at elevated temperatures is difficult to resolve. A decline in sludge settleability and elevated effluent solids levels have been observed in some previous studies (Duke et al., 1980; Flippin and Echkenfelder, 1994; Kalinske, 1974) but not in others (Barr et al., 1996; Rintala and Lepisto, 1993). The present work supports the contention that solids settleability is impacted by high temperature operation, but must be qualified by the other operational disruptions which occurred during the study. The unexpected influent change-over happened when the reactor was operating at 48°C. Although the reactor seemed to recover after receiving a new and dramatically different influent for two weeks, the possibility cannot be eliminated that this change had a more lasting impact. As a result, the issue of sludge settleability at elevated temperatures must be considered unresolved. 58 5000 60 0 -I 1 1 1 1 1 1 1 1 L 0 <N <N ' ^ „ © Time (Date) Figure 5.3 Biomass concentration as a function of reactor operating temperature. A = switch to Harmac influent (July 26, 1996); B = power outages (August 21 & 23, 1996); C = water bath malfunction (September 21-22, 1996); D - power outages (October 15, 1996); E, F = low influent COD (July 9, 1996 & Jan 7,1997). 59 Time (Date) Figure 5.4 Effluent solids concentration as a function of operating reactor temperature. A = switch to Harmac influent (July 26, 1996); B = power outages (August 21 & 23, 1996); C - water bath malfunction (September 21-22, 1996); D = power outages (October 15, 1996); E, F = low influent COD (July 9, 1996 & Jan 7,1997). 60 S O U R The specific oxygen uptake rate (SOUR) varied from 8.8 mg 0 2 / g MLVSS-h to 42.9 mg 0 2 / g MLVSS-h over the course of the study (Figure 5.5). The degree of variability in the SOUR data makes it difficult to draw any conclusions The SOUR was the highest 42.9 mg /g MLVSS-h immediately after the reactor was switched back to WP PCE, after receiving Harmac PCE for two weeks. However, no definite conclusion can be drawn since no noticeable change observed in the control reactor (RI). At 55°C, the average SOUR wasl2.17 mg 0 2 / g MLVSS-h. Toxicity The toxicity test was not performed until the Microtox Analyzer was returned from repair and upgrade in mid-July, 1996. The percent toxicity removal was calculated based on T U (Section 4.4.5) and varied from 78.4 % to 97.0 % (Figure 5.6). An increase in operating temperature had no significant effect on toxicity removal. Interestingly, the operational disruptions described previously did not seem to affect the toxicity removal. 61 Figure 5.5 SOUR as a function of reactor perating temperature A = switch to Harmac influent (July 26, 1996); B = power outages (August 21 & 23, 1996); C = water bath malfunction (September 21-22, 1996); D = power outages (October 15, 1996); E, F = low influent COD (July 9, 1996 & Jan 7,1997). 62 F i g u r e 5.6 Effluent toxicity removal as a function of reactor operating temperature. A = switch to Harmac influent (July 26, 1996); B = power outages (August 21 & 23, 1996); C = water bath malfunction (September 21-22, 1996); D = power outages (October 15, 1996); E, F = low influent COD (July 9, 1996& Jan 7, 1997). 63 5.3 Effect of High Operating Temperature on Species Diversity The effect of temperature on species diversity in the MLVSS was studied by monitoring the substrate utilization profiles, OUR, floe structure and biomass settleability as temperature increased from 35°C to 52°C. 5.3.1 Substrates Utilization Results Table A 5.3.1 summarized the results obtained from the BIOLOG test and M P N method. The number of resin acid degrading bacteria appeared to decrease from 107 to 104 cells/g MLVSS when the reactor temperature was increased from 42°C to 45°C. This decrease in M P N indicates that the microbes capable of degrading resin acids (DhA, AbA, PA, IpA, 12/14-Cl-DhA) are mesophiles. The decrease in resin acid degraders did not result in a decrease in the reactor's ability to remove toxicity from the effluent. This may be a result of significant under-utilization of resin acid degrading capacity under normal operation conditions. The bacteria responsible for fatty acid degradation appeared to be less affected by the increase in temperature. The M P N for palmitic acid did not vary much (2.87 x 10 cell/g M L V S S at 35°C and 1.00 x 106 cell/g MLVSS at 52°C) as temperature increased, indicating that population of palmitic acid-degrading microbes were not impacted by the change in operating temperature and survived at elevated temperature. Similarly, the M P N for linoleic acid also appeared not to be affected by the increasing temperature (from 8.4x10s cell/g MLVSS at 48°C to 2.0xl0 4 cell/g MLVSS at 50°C). 64 A l l sugars tested were extensively utilized throughout the study. The MPN's for arabinose (3.1xl0 9 cells/g MLVSS) and glucose (2.8xl0 8 cells/g MLVSS) remained high even at high temperatures (52°C) whereas galactose utilisers seemed to reduce by two orders of magnitude; from 1.5xl07 cells/g MLVSS at 48°C to 6.2 x lO 5 cells/g M L V S S at 52°C. In general, the results indicated that sugars degrading bacteria generally can survive at elevated temperature. Bacteria responsible for metabolism of methanol, ethanol and sodium formate appeared to decrease in number with increasing temperature. MPNs for these three substrates at 39°C were 1.4xl08, 8.0x107, and l.OxlO7 cells/g MLVSS for methanol, ethanol and sodium formate, respectively. However, when the temperature was increased beyond 40°C, MPN's for these substrates fell substantially. At 42°C, they were 1.0x10s, 8.0xl0 5, and 6.2xl0 5 cells/g MLVSS respectively, and continued to decrease as temperature increased. Since these compounds are responsible for the bulk of BOD in Kraft pulping effluents, a decrease in the number of bacteria which are capable of degrading them could result in an impaired ability to handle shock loads of BOD. However, an increase in operating temperature had no significant effect on steady state BOD removal performance (section 5.2.2). 5.3.2 Floe Structure and Settleability As temperature increased, the shape of the floes changed. Figure 5.7 and 5.8 illustrate typical floe structure at 35°C and 52°C. The floes changed from a rounded, compact shape to a longer and more extended structure, which contained a lot of void 65 Figure 5.7 Microscopic picture of floe structure at 38°C. space. Table 5.4 surnmarizes detailed observation of floe structure. Such changes in floe structure appeared to coincide with an increase in the number of filamentous microorganisms at elevated temperatures. Table 5 .4 Change in floe structure with increasing operating temperatures. Temperature Shape Structure Dimensions' Remarks C O (um) , 38 Irregular Rounded Compact 140 Small floes Free-swimming ciliates 42 Irregular Rounded Less compact 200 45 Irregular Relatively open 250 No ciliates 50 Irregular Slender More open 160 Filamentous 52 Irregular Long Extended Open 250 Large floes Filamentous * Floe dimensions were approximate values. This change in floe structure can affect biomass settleability. Settleability tests (Figure 5.9) indicated that the settleability of biomass from R2 decreased at elevated temperature. While these settleability data are based on a single data point at each temperature, the poor settleability and turbid effluent observed at elevated reactor temperatures (Section 5.2.2) support the contention that settleability was reduced at high temperature. 67 80 j 70 -60 -s 50 -B •*-> J= 40 -op 30 -20 -10 -0 -30 35 40 45 Temperature (°Q 50 55 Figure 5 .9 Settleability of biomass with increasing operating temperature. By studying the substrate utilization profiles, floe structure and settleability, it can be seen that, as temperatures increases, the population structure changed. Such a decrease in species diversity is commonly associated with a decrease in ecosystem stability and resistance to perturbations. 68 5.4 Response of H T A S Bioreactors to Temperature Shock The temperature shock study was designed to investigate the response of thermophilic and mesophilic activated sludge bioreactors to rapid changes in temperature associated with shut downs and start up of mills. This study was started on January 10, 1997. However, data was not collected frequently enough after the start-up to closely observe the recovery of the bioreactor. A second trial was performed beginning July 31, 1997. Table 5.5 and 5.6 summarize the treatment performance of the mesophilic bioreactor and the thermophilic bioreactor. The results represent the average values for two weeks leading up to the shock and 63 hours after the shock. Recovery profiles for individual parameters are given in Appendix 5.4. Table 5.5 Mesophilic bioreactor performance before and after temperature shock. Mesophil ic Bioreactor Parameters Before After Significance BOD Removal (%) 96.0 ± 0.4 96.1 ±0.6 No COD Removal (%) 49.6 ± 1.1 42.5 ±4.8 Yes VSS (mg/L) 22.4 ± 8.7 6.3 ±4.3 Yes M L V S S (mg/L) 4439 ± 254 3956 ± 690 No SOUR (mg 0 2 /g MLVSS-h) 5.1 ±2.0 6.9 ±1.4 No Toxicity Removal (%) 95.6 ±0.7 95.5 ± 1.0 No The mesophilic bioreactor showed a significant decline in COD removal. On the other hand, the bioreactor showed an improvement in reducing effluent VSS. After the 69 shock the supernatant was free from noticeable turbidity. Moreover, improved biomass settleability after the temperature shock was observed. Other treatment parameters showed no significant difference before and after the temperature shock. Table 5.6 summarizes the treatment performance of the thermophilic bioreactor before and after the temperature shock. BOD removal did not recover to the level observed prior to the shock. Similarly, COD and toxicity removal were significantly lower after the shock. Other treatment parameters showed no significant difference before and after the shock. Table 5.6 Thermophilic bioreactor performance before and after temperature shock. T lermuphilic Bioreactor Parameters Before After Significance BOD Removal (%) 93.3 ±2.1 85.9 ±2.0 Yes COD Removal (%) 44.8 + 2.5 38.4 ±3.8 Yes VSS (mg/L) 41.6 ±27.3 42.4 ± 22.6 No MLVSS (mg/L) 3063 ±781 3295±1395 No SOUR (mg0 2/gMLVSS-h) 9.3 ±7.7 9.2 ±2.0 No Toxicity Removal (%) 94.6 ± 0.6 92.1 ±2.7 Yes The results obtained from both bioreactors were compared with their own steady state values; however, it is difficult to make comparison between these two bioreactors due to difference in MLVSS concentration. The mesophilic bioreactor had a higher M L V S S concentration (4439 mg/L) than the thermophilic bioreactor (3063 mg/L) before the shock, as a result it probably was more capable of withstanding the drastic temperature upset. Since the temperature is the prime variable in this study, the difference 70 in MLVSS concentration makes it difficult to draw unqualified conclusions when comparing the performance of the two bioreactors. 5.5 Kinet ics of Substrate Uptake i n H i g h and N o r m a l Temperature A S Process Kinetics of metabolism of formate and methanol were determined for M L V S S from reactor RI (35°C) and R2 (45°C) (Table 5.7). Table 5.7 Kinetic values for methanol and formic acid at different temperatures. T=35°C T=45°C Methanol Formic A c i d Methanol Formic A c i d qmax (mg BOD/g MLVSS-min) 0.7245 0.5056 0.4585 0.4717 K s (mg BOD/L) 0.0821 0.1014 0.0527 0.0443 Y i e l d (mg MLVSS/mg BOD) 0.2476 0.0682 0.0931 0.0159 5.5.1 Comparison within Individual Bioreactors For the mesophilic biomass, the maximum substrate uptake rate, q m a x , is higher for methanol than for formic acid. This indicates that the biomass is more active in metabolizing methanol than formic acid. The half saturation constant Ks; however, was slightly higher for formic acid than for methanol. The yield of biomass from methanol was higher than from formic acid, indicating that, methanol is a more easily utilized substrate. 71 The biomass from the reactor operating at elevated temperature exhibited similar activity in metabolizing both substrates as indicated by similar values for both substrates. The K value was slightly higher for methanol than formic acid. This means that the biomass would reach to its maximum substrate uptake rate faster for formic acid than for methanol. As was the case at lower temperatures, the yield of biomass on methanol was higher than on formic acid. The results in the thermophilic bioreactor showed a different pattern from the mesophilic bioreactor in metabolizing the two substrates. Such a difference indicated that the microbial population shifted as the temperature of the treatment system changed from the mesophilic range to the thermophilic range. 5.5.2 Comparison between Mesophilic and Thermophilic Bioreactors The maximum substrate uptake rate for methanol by mesophiles was higher than for the thermophilic biomass. This agreed with the M P N result (Section 5.3.1) which indicated that the number of bacteria responsible for degrading methanol decreased with temperature. Bioreactors operated at elevated temperature would probably be more sensitive to changes in methanol loading and may consequently fail to treat the influent adequately i f there was a sudden increase in methanol concentration in the influent. The yield of biomass at mesophilic temperatures was higher than at thermophilic temperatures. This result does not agree with the results from Al-Awadhi et al (1988) who concluded that the growth rate of the Bacillus sp. on methanol was higher at 55°C 72 than at 35°C. However, that experiment used single species in a batch culture, which is difficult to compare to a mixed continuous culture. The qmax of the mesophilic biomass was similar to that of the thermophilic biomass in the test using formic acid, indicating that the microorganisms at both temperatures performed similarly in degrading formic acid. The half saturation constant and the biomass yield were higher in the mesophilic bioreactor. In general, from the kinetics of substrate uptake, the thermophilic bioreactor has a lower maximum substrate uptake rate. This could be a potential disadvantage especially in the case of a black liquor spill or other incident which would result in elevated BOD loads entering the treatment plant. The thermophilic bioreactor would be less capable than the mesophilic bioreactor of removing the increased amount of BOD, and a decline in treatment performance could result. The biomass yield for both sets of biomass is extremely low with the Y value in the thermophilic reactor being lower than those for its mesophilic counterpart. This can be explained by the fact that both methanol and formic acid are single carbon compounds, and are not readily utilized for cell synthesis. Moreover, formic acid, and to a lesser degree methanol, are highly oxidized. From an operational perspective, such low biomass yields result in reduced sludge production. Sludge handling represents a significant cost at many pulp and paper operations. A previous study has reported that one of the advantages of thermophilic AS operation is the reduced amount of sludge (Surucu, 1976). 73 Chapter 6 CONCLUSIONS The effect of elevated operating temperatures on an activated sludge process treating Kraft mill effluent was studied and the following conclusions were made: 1. There were no significant difference in BOD removal, M L V S S , SOUR and toxicity removal when the operating temperature was increased from 35°C to 55°C. 2. COD removal declined with increasing temperature. Increasing effluent VSS and turbidity were observed at elevated temperature (>45°C). Biomass settleability declined with increasing temperature but operational disruptions may have impacted on the treatment performance. 3. The upper operating temperature limit for the laboratory-scale AS process was below 55°C as some decline in treatment performance was observed at 55°C. 4. The number of microorganisms which degraded resin acids, methanol, ethanol and sodium formate utilization decreased at temperatures above 42-45°C. Microbes which metabolized other substrates such as fatty acids and sugars utilization were relatively stable as temperature increased. 74 5. The floes changed from a small, compact shape at mesophilic temperatures to a large, open structure at elevated temperatures. Increased filamentous growth was observed with increasing temperature and hence hindered sludge settling. 6. There was no significant treatment difference in the mesophilic bioreactor in terms of BOD removal, M L V S S , SOUR and toxicity removal in the first 63 hours after a shutdown / start up and associated temperature change. However, COD removal declined by 14% after the shock. The effluent VSS concentration and settleability were significantly improved after the shock. 7. The thermophilic reactor showed no significant treatment difference in M L V S S , effluent VSS, SOUR and toxicity removal in the first 63 hours after the shock. BOD and COD removal were reduced by 8% and 14%, respectively after the shock 8. The maximum substrate removal rate q m a x for methanol was higher than for formic acid in the mesophilic bioreactor but the q m a x value for the two substrates were similar in the thermophilic bioreactor. The yield of biomass from methanol is higher than from formic acid for both bioreactors. 9. The maximum substrates removal rate q m a x for methanol was higher in the mesophilic bioreactor than the thermophilic bioreactor. The q m a x values of formic acid were similar in both bioreactors. The yield of biomass from both substrates were higher in the mesophilic bioreactor than its thermophilic counterpart. 75 10. The thermophilic bioreactor would be less capable of removing an increased amount of BOD such as would be associated with a black liquor spill. On the other hand, the thermophilic bioreactor would produce less sludge and hence reduce sludge handling costs. 76 Chapter 7 R E C O M M E N D A T I O N S Repeat the temperature shock study to examine the response of the mesophilic and thermophilic AS process. M L V S S and other reactor conditions except temperature in both reactors should be kept close enough to allow direct comparison. Investigate sludge settleability regularly to evaluate the sludge volume index with increasing operating temperature. Evaluate the kinetics of resin acid uptake for both bioreactors, since resin acid is one of the major constituents responsible for effluent toxicity and operating temperature will affect metabolism of the compound. This will serve to confirm and extend the result of the biolog tests done in this report. Perform quantitative shock loading tests on each bioreactor to examine the response of the AS at different to sudden increases/decreases in effluent concentration. This study will help in evaluating the response of the bioreactors to upset conditions such as black liquor spills in the mill. Evaluate the effect of temperature on dewaterability of the waste sludge from each bioreactor. 77 R E F E R E N C E S Al-Awadhi, N., Egli, T., Ffamer G., Growth Characteristics of a Thermotolerant Methylotrophic Bacillus sp. (NCIB 12522) in Batch Culture, Applied Microbiology and Biotechnology, 29:485-493, 1988. American Public Health Association, Standard Methods for the Examination of Water and Wastewater, 17th Edition, Washington, DC, 1989. Barr, T., Effects of High Operating Temperatures, Hydraulic Retention Time and Solids Residence Time on Acitvated Sludge Treatment of Kraft Pulping Effluent, M.A.Sc. Thesis, University of British Columbia, 1994. Bitton, G. ' , Wastewater Microbiology, Wiley-Liss, Inc., 1994. Brock, T. D., Madigan, M. T., Martinko, J. M., Parker, J., Biology of Microorganisms, Prentice Hall, Inc., New Jersey. 1994. Brock, T. D., Thermophiles: General, Molecular, and Applied Microbiology, John Wiley & Sons, New York, 1986. Brock, T. D., Thermophilic Microorganisms and Life at High Temperatures, Springer, New York, 1978. Brown, L. R, Tischer, R. G., Ladner, C. M., Bustwick, C. D., The Effect of Elevated Temperatures on the Treatment of Normal Domestic Sewage, Water Resources Inst., 1967. Carpenter, W. L., Vamvakias, J. G., Gellman, I., Temperature Relationships in Aerobic Treatment and Disposal of Pulp and Paper Wastes, Journal of Water Pollution Control Federation, Vol . 40, No. 5, p.733-740, 1968. Cech, J. S., Chudoba, J., Grau, P., Determination of Kinetic Constants of Activated Sludge Microorganisms, Water Science Technology Vol . 17, pp259-272, 1984. Devore, J. L., Probability and Statistics for Engineering and the Sciences, 3rd Ed., Brooks/Cole Publishing Company, Pacific Grove, California, 1991. Duke, M. L., Templeton, M. E., Eckenfelder, Jr., W. W., Stowe, J. C , High-Temperature Effects on the Activated Sludge Process Treating Industrial Wastewaters, Proceedings of the 35th Industrial Waste Conference, p.817-825, 1981. 78 Environment Canada, Environmental Protection Service, The Basic Technology of the Pulp and Paper Industry and Its Environmental Protection Practices - Training Manual, EPS 6-EP-83-1, 1983. Flippin, T. H., Eckenfelder, Jr., W. W., Effects of Elevated Temperature on the Activated Sludge Process, 1994 Environmental Conference, 1994. Friedman, A. A., Temperature Effects on Growth Rate and Yield for Activated Sludge, PhD Thesis, University of California, UMI Dissertation Services, Michigan, 1970. Gale, P., Broberg, PJ., Use of a Commercial Gene Probe Assay Kit for Rapid MPN Enumeration of Escherichia Coli in Drinking Water, Letters in Applied Microbiology, Vol . 18 (6) pp. 346-348, June, 1994. Ganczarczyk, J. J., Activated Sludge Process: Theory and Practice, Marcel Dekker, New York, 1983. Gehm, H. W., Activated Sludge at High Temperatures and High pH Values, Biological Treatment of Sewage and Industrial Wastes, Vol 1, p.352-355, 1956. Graczyk, M., Purification of Pulp Industry Effluents- A Modification of the Activated Sludge Method in a Thermophilic System, Gas Woda Technika Sanitarna, Vol . 58, No. 6, pp. 142-147, 1984. Grady, C. P. L., Lim, H. C , Biological Wastewater Treatment: Theory and Applications, Marcel Dekker, New York, 1980. Ionides, G., Mechanical Pulping, Technical course notes of Chemical Engineering 470, Department of Chemical Engineering, University of British Columbia, 1995. Jackson, M. L., Thermophilic Treatment of a High-Biochemical Oxygen Demand Wastewater: Laboratory, Pilot-plant and Design, Proceedings of the 37th Industrial Waste Conference, p.753-763, 1983. Jerkins, D., Richard, M . G., Daigger, G. T., Manual on the Causes and Control of Activated Sludge Bulking and Foaming, 2nd edition, Lewis Publishers, Inc., Michigan, 1993. Jones, G. L. Microbiology and Activated Sludge, Process Biochem., Jan/Feb., p.3-5 and 24, 1976. Kalinske, A . A., Thermophilic Bio-Oxidation of High-Strength Organic Wastewaters and Sludges GVC/AIChE Joint Meeting with Jahrestreffen, p. 17-20, September 1974. 79 Ljungdahl, L. G., Sherod, D., Proteins from Thermophilic Microoganisms, Ed. Milton R. Heinrich, Extreme Environments, Academic Press, Inc., New York, 1974. McElhaney, R. N., The Biological Significance of Alterations in the Fatty Acid Composition of Microbial Membrane Lipids in Response to Changes in Environmental Temperature, Ed. Milton R. Heinrich, Extreme Environments, Academic Press, Inc., New York, 1974. Oblinger, J. L., Kobuzer, J. A., Compendium of Methods for the Microbiological Examination of Foods, Speck Marvin L. (ed.), American Public Health Association, Washington, DC 1980. Pike, E. B., Curds, C. R. The Microbial Ecology of the Activated Sludge Process, in Microbial Aspects of Pollution, eds. Sykes, G. and Skinner, F.A., Academic Press, p. 123-147, 1971. Richard, M., Activated Sludge Microbiology, The Water Pollution Control Federation, Virginia, 1989. Rinatala, J . , Lepisto, R., Thermophilic Anaerobic-Aerobic and Aerobic Treatment of Kraft Bleaching Effluents, Water Science and Technology, Vol. 28, No. 2, ppl 1-16, 1993. Sawyer, C. N., McCarty, P. L., Chemistry for Environmental Engineering McGraw-Hill Publishing Company, New York, 1978. Shuler, M. L., Kargi, F., Bioprocess Engineering Basic Concepts, Prentice Hall, Inc., New Jersey, 1992. Smook, G. J., Handbook for Pulp & Paper Technologist, Angus Wilde Publications, 1992. Springer, A . M., Industrial Environmental Control Pulp and Paper Industry, 2nd Edition, Tappi Press, Atlanta, GA, 1993. Streebin, L. E., Comparison Between Thermophilic and Mesophilic Aerobic Biological Treatment of Synthetic Organic Waste, UMI Dissertation Services, Michigan, 1968. Sundstrom, D. W., Kel i , H. E., Wastewater Treatment, Prentice-Hall, Inc., Englewood Cliffs, NJ, 1979. Surucu, G. A., Chian, E. S. K. Engelbrecht, R. S., Aerobic Thermophilic Treatment of High Strength Wastewaters, Journal of Water Pollution Control Federation, Vol . 48, No. 4, p.669-679, April 1976. 80 Thomas, R. J. , Wood Anatomy and Ultrastructure, in Wood: Its Structure and Properties, Wangaard, F. F., Ed., Clark C Heritage Memorial Series on Wood Vol . 1 pp.110-146. 1981. Taylor, J. , Activate Sludge Treatment of Kraft Pulp Mill Effluent, Pulp & Paper Canada, 97:11, 1996. Tchobanoglous, G., Burton, F. L., Wastewater Engineering: Treatment, Disposal and Reuse, McGraw-Hill Inc., New York, 1991. Victorio, L., Gilbride, K. A., Allen, D. G., Liss, S. N., Phenotypic Fingerprinting of Microbial Communities in Wastewater Treatment Systems, Water Research, Vol . 30, No. 5, pp. 1077-1086, 1996. Winkler, M . A., Biological Treatment of Waste-water, Ellis Horwood Ltd., Connecticut, 1981. Wrenn, B. A., Venosa, A. D., Selective Enumeration of Aromatic and Aliphatic Hydrocarbon Degrading Bacteria by a Most-Probable-Number Procedure, Canadian Journal of Microbiology, Vol. 42, pp. 252-258, 1996. 81 APPENDIX 82 A 5.1 Steady-State Operat ion Data Table A 5.1.1 Steady state BOD removal at 35°C. Date iliGE;:-;; Effluent % Removal Date P C E Effluent % Removal 4/29/96 127.7 3.2 97.5 10/18/96 48.1 1.0 97.9 4/30/96 297.1 2.8 99.1 10/23/96 88.0 2.0 97.8 5/3/96 247.8 13.2 94.7 10/25/96 94.5 3.6 96.2 5/9/96 180.9 0.5 99.7 10/31/96 102.0 6.2 94.0 5/16/96 141.0 5.1 96.4 11/6/96 325.9 2.4 99.3 5/22/96 215.9 9.1 95.8 11/8/96 224.7 1.8 99.2 5/24/96 211.9 4.1 98.1 11/14/96 131.5 1.9 98.5 5/29/96 276.8 14.7 94.7 11/20/96 224.6 1.7 99.8 6/6/96 394.3 31.6 92.0 11/22/96 348.5 0.9 99.7 6/14/96 232.3 5.6 97.6 11/27/96 378.6 29.3 92.3 6/19/96 131.6 5.8 95.6 11/29/96 426.1 4.6 98.9 6/21/96 263.0 35.0 86.7 12/4/96 395.5 18.6 95.3 6/28/96 176.1 3.9 97.8 12/6/96 422.8 21.3 95.0 7/2/96 118.8 3.6 97.0 12/11/96 407.4 17.1 95.8 7/5/96 135.0 23.5 82.6 12/15/96 379.0 10.6 97.2 7/10/96 241.9 18.2 92.5 1/7/97 175.2 8.4 95.2 7/12/96 303.4 24.7 91.9 1/9/97 187.6 12.0 93.6 7/18/96 234.5 52.7 77.5 1/18/97 188.3 10.1 94.6 7/19/96 190.0 36.4 80.8 1/22/97 204.7 8.4 95.9 7/25/96 246.8 14.7 94.1 1/30/97 277.5 6.4 97.7 7/31/96 131.3 4.7 96.5 2/5/97 236.1 5.8 97.5 8/7/96 138.9 7.3 94.8 2/7/97 221.9 8.8 96.0 8/9/96 111.9 2.5 97.8 2/19/97 511.9 15.2 97.0 8/14/96 115.9 2.2 98.1 2/26/97 358.4 11.5 96.8 8/23/96 67.3 1.6 97.6 2/28/97 410.4 11.7 97.1 8/29/96 215.1 2.4 98.9 3/5/97 374.7 11.9 96.8 8/30/96 264.0 1.8 99.3 3/7/97 400.4 9.0 97.8 9/4/96 107.0 2.5 97.6 3/12/97 379.0 16.1 95.7 9/11/96 430.4 9.0 97.9 3/14/97 331.0 7.8 97.6 9/13/96 244.4 18.0 92.6 3/18/97 390.6 11.8 97.0 9/18/96 154.6 15.0 90.3 3/26/97 415.6 9.4 97.7 9/20/96 152.3 11.4 92.5 5/15/97 465.4 8.6 98.2 9/27/96 120.8 2.2 98.2 5/29/97 245.0 6.1 97.5 9/28/96 603.0 24.0 96.0 6/18/97 432.2 20.7 95.2 10/3/96 170.1 28.3 83.4 7/23/97 476.8 17.6 96.3 10/4/96 114.3 10.9 90.5 7/31/97 455.3 20.2 95.6 10/16/96 63.7 1.4 97.8 Mean STDEV 95.4 ±4.3 83 Table A 5.1.2 Steady state COD removal at 35°C. Date P C E Effluent % Removal Date P C E Effluent % Removal 6/3/96 1300.9 670.3 48.5 10 30 96 1310.4 823.8 37.1 6/8/96 1728.9 808.5 53.2 11/5/96 1611.7 753.9 53.2 6/11/96 1662.1 1035.1 37.7 11/13/96 1444.9 679.4 53.0 6/19/96 831.3 515.9 37.9 11/20/96 1475.4 749.4 49.2 6/21/96 1042.6 531.2 49.0 12/9/96 1473.6 700.1 52.5 6/25/96 1026.4 528.5 48.5 12/20/96 1455.7 682.1 53.1 7/3/96 867.4 410.2 52.7 1/7/97 681.0 328.8 51.7 7/9/96 500.8 428.3 14.5 1/18/97 811.9 403.2 50.3 7/11/96 961.3 341.6 64.5 1/31/97 704.3 255.3 63.8 7/16/96 1039.0 537.6 48.3 2/4/97 889.0 327.0 63.2 7/18/96 1020.9 557.4 45.4 2/19/97 1597.4 631.0 60.5 7/23/96 941.5 642.3 31.8 2/25/97 1427.0 729.6 48.9 7/26/96 1022.7 524.9 48.7 3/6/97 1425.2 677.6 52.5 7/31/96 1031.8 544.8 47.2 3/14/97 1419.8 735.0 48.2 8/8/96 999.3 506.0 49.4 4/10/97 1433.7 738.2 48.5 8/14/96 1069.7 513.2 52.0 4/24/97 1516.8 788.1 48.0 8/22/96 1057.1 536.7 49.2 5/5/97 1669.8 868.6 48.0 8/27/96 988.4 494.2 50.0 5/20/97 1589.4 755.3 52.5 8/31/96 1088.7 515.1 52.7 6/16/97 1498.2 737.0 50.8 9/11/96 1301.2 595.7 54.2 6/30/97 1508.2 790.6 47.6 9/17/96 952.3 407.1 57.3 7/16/97 1594.7 801.3 49.8 9/25/96 1259.5 592.6 53.0 7/19/98 1600.2 787.32 50.8 10/2/96 1333.0 645.3 51.6 7/26/98 1552.3 781.00 49.7 10/16/96 1045.0 569.1 45.5 7/31/97 1489.6 772.7 48.1 10/22/96 1150.8 513.5 55.4 Mean STDEV 47.6 ±9.6 84 Table A 5.1.3 Steady state MLVSS and VSS concentration at 35°C Date M L V S S (mg/L) V S S (mg/L) Date M L V S S (mg/L) V S S (mg/L) 4/22/96 1605 8.0 10/16/96 2050 3.7 4/25/96 1540 5.0 10/23/96 2867 63.0 4/29/96 1575 11.0 10/28/96 2833 32.0 5/1/96 1940 7.0 11/4/96 2115 30.0 5/6/96 2765 7.0 11/14/96 4767 107.0 5/9/96 2390 3.0 11/20/96 4880 47.0 5/13/96 2535 11.0 11/25/96 5350 16.0 5/17/96 2205 1.0 12/3/96 3787 86.0 5/23/96 3505 11.0 12/9/96 1665 60.0 5/28/96 3705 28.0 12/12/96 2747 30.0 6/4/96 1295 23.0 12/17/96 3880 42.0 6/6/96 1045 41.0 1/2/97 3527 31.0 6/10/96 1235 16.0 1/7/97 3512 22.0 6/19/96 1105 31.0 1/17/97 3493 15.0 6/22/96 895 145.0 1/30/97 3000 10.0 6/25/96 860 110.0 2/4/97 2913 31.0 6/28/96 955 45.0 2/8/97 3287 13.0 7/2/96 865 107.0 2/24/97 3175 54.0 7/8/96 750 79.0 2/28/97 3014 10.5 7/11/96 800 51.0 3/4/97 2984 8.5 7/15/96 890 60.0 3/12/97 2854 9.0 7/17/96 745 71.0 3/20/97 2710 11.2 7/19/96 200 45.0 4/3/97 2612 7.3 7/23/96 2000 50.7 4/25/97 2547 4.0 8/28/96 1865 15.0 5/27/97 2488 14.0 9/3/96 2755 3.5 6/4/97 2841 13.0 9/11/96 3420 22.0 6/6/97 2969 15.0 9/17/96 1385 42.0 6/17/97 3214 19.0 9/25/96 1405 69.0 7/17/97 4600 27.0 10/2/96 1960 116.0 7/31/97 4153 23.0 Mean STDEV 2522 ±1180 34.3 ±32.0 85 Table A 5.1.4 Steady state OURs and SOURs at 35°C Date M L V S S (mg-L) O U R (mg0 2.L-min) S O U R (mgOr' gMLVSS-h) Date M L V S S (mg/L) O U R (mg0 2/L-min) S O U R (mg0 2/ gMLVSS-h) 5/1/96 1940 0.4 11.5 10/23/96 2867 0.5 9.8 5/6/96 2765 0.4 9.1 10/28/96 2833 0.7 14.3 5/9/96 2390 0.3 7.7 11/4/96 2115 0.6 17.5 5/13/96 2535 0.4 9.6 11/8/96 2115 0.8 21.6 5/17/96 2205 0.3 7.2 11/14/96 4767 0.7 9.4 5/21/96 3375 0.6 9.9 11/20/96 4880 0.6 6.8 5/23/96 3505 0.4 6.1 11/25/96 4823 0.7 8.7 5/28/96 3705 0.9 15.3 11/28/96 2987 0.4 7.8 5/30/96 3835 0.4 6.2 12/9/96 1665 0.5 18.3 6/4/96 1295 0.3 12.6 12/17/96 3880 0.6 9.8 6/6/96 1045 0.3 17.2 1/2/97 3527 0.4 6.9 6/10/96 1235 0.5 25.9 1/17/97 3493 0.3 6.0 6/13/96 1235 0.3 13.9 1/30/97 3000 0.3 6.3 6/19/96 1105 0.4 21.0 2/4/97 2913 0.2 4.5 6/22/96 895 0.4 28.4 2/24/97 3175 1.3 24.3 6/25/96 860 0.3 18.1 2/28/97 3014 0.8 15.5 6/28/96 955 0.3 19.0 3/4/97 2984 0.7 14.7 7/2/96 865 0.2 11.9 3/12/97 2854 0.7 15.0 7/8/96 750 0.3 21.7 3/20/97 2710 0.7 15.4 7/11/96 800 0.3 22.0 4/3/97 2612 0.3 7.5 7/15/96 890 0.4 27.5 4/25/97 2547 0.2 5.6 7/19/96 200 0.1 42.8 5/27/97 2488 0.2 4.1 7/23/96 2000 0.8 23.1 6/4/98 2841 0.2 3.7 7/31/96 1360 0.4 18.3 6/6/98 2969 0.3 5.2 8/8/96 1110 0.3 14.3 6/17/97 3214 0.3 4.8 8/13/96 1120 0.3 14.6 7/17/97 4120 0.2 2.3 8/19/96 2093 0.4 10.7 7/18/97 4368 0.3 4.5 8/24/96 1715 0.3 11.5 7/20/97 4268 0.4 5.1 9/3/96 2755 0.2 4.8 7/25/97 4243 0.5 6.4 10/16/96 2050 0.3 9.8 7/31/97 4153 0.5 7.2 Mean STDEV 12.4 ±7.9 86 Table A 5.1.5 Steady state toxicity removal at 35°C. P C E Effluent Removal P C E Effluent Removal Date E C 5 0 T U E C 5 0 T U % Date E C 5 0 T U E C 5 0 T U % 5/3/96 5.7 17.4 69.0 1.4 91.7 12/4/96 2.9 35.0 98 4 1.0 97.1 7/11/96 4.1 24.4 61.5 1.6 93.3 12/9/96 2.9 34.6 49.0 2.0 94.1 7/17/96 2.2 45.9 21.0 4.8 89.6 12/13/96 3.9 25.5 50.9 2.0 92.3 7/22/96 6.5 15.4 22.8 4.4 71.7 12/17/96 2.1 46.7 100.0 1.0 97.9 8/7/96 2.4 41.0 100.0 1.0 97.6 1/8/97 10.1 9.9 100.0 1.0 89.9 8/16/96 4.8 20.7 84.1 1.2 94.3 1/29/97 4.3 23.4 100.0 1.0 95.7 8/23/96 17.4 5.7 100.0 1.0 82.6 2/10/97 3.7 27.2 90.3 1.1 95.9 9/11/96 3.7 26.7 31.1 3.2 88.0 2/25/97 2.5 40.3 42.7 2.3 94.2 9/26/96 3.4 29.5 100.0 1.0 96.6 3/4/97 3.9 25.5 100.0 1.0 96.1 10/2/96 2.0 50.2 51.7 1.9 96.1 3/17/97 2.0 50.2 100.0 1.0 98.0 10/4/96 3.0 33.8 63.9 1.6 95.4 3/25/97 7.2 13.8 100.0 1.0 92.8 10/9/96 3.0 33.6 58.0 1.7 94.9 3/31/97 2.7 36.7 77.0 1.3 96.5 10/16/96 1.0 105.2 86.1 1.2 98.9 4/22/97 3.8 26.4 100.0 1.0 96.2 10/23/96 1.2 84.4 95.6 1.0 98.8 5/15/97 1.1 95.1 67.6 1.5 98.4 10/30/96 3.7 27.4 81.8 1.2 95.5 5/22/97 3.1 32.5 100.0 1.0 96.9 11/1/96 3.7 27.0 68.5 1.5 94.6 6/17/97 0.9 110.2 45.8 2.2 98.0 11/13/96 3.3 29.9 72.8 1.4 95.4 7/17/97 3.7 26.9 68.2 1.5 94.5 11/15/96 4.7 21.3 91.9 1.1 94.9 7/20/97 3.3 30.8 75.4 1.3 95.7 11/20/96 4.6 22.0 29.1 3.4 84.4 7/25/97 2.8 35.5 69.0 1.4 95.9 11/25/96 1.4 71.0 66.7 1.5 97.9 7/28/97 2.4 41.1 61.7 1.6 96.1 11/28/96 1.5 66.0 100.0 1.0 98.5 Mean 74.4 94.2 STDEV ±24.3 ±5.1 87 A 5.2 The Pract ical Upper Temperature L i m i t of H T A S Table A 5.2.1 BOD removal between 37°C to 55°C. Date P C E Effluent Removal Temp Date P C E Effluent Removal Temp % (°C) % <°C) 6/6/96 394.3 25.9 93 A 37 9/11/96 430.4 22.8 94.7 53 6/14/96 232.3 14.7 93.7 39 9/13/96 244.4 46.1 81.1 54 6/19/96 131.6 11.1 91.6 41 9/18/96 154.6 26.3 83.0 54 6/21/96 263.0 17.3 93.4 41 9/27/96 120.8 8.6 92.9 54 6/28/96 176.1 9.8 94.4 43 9/28/96 603.0 48.5 92.0 54 7/2/96 118.8 2.8 97.6 43 10/3/96 170.1 11.9 93.0 54 i Average values at T=45°C A .verage values at T=55°C 115196 135.0 4.6 96.6 45 10/16/96 63.7 3.6 94.3 55 7/10/96 241.9 6.4 97.4 45 10/18/96 48.1 4.8 90.0 55 7/12/96 303.4 11.4 96.2 45 10/23/96 88.0 4.4 95.0 55 7/18/96 234.5 11.0 95.3 45 10/25/96 94.5 4.1 95.7 55 7/19/96 190.0 10.7 94.4 45 11/6/96 325.9 4.0 98.8 55 Mean 96.0 11/8/96 224.7 3.7 98.3 55 STDEV ± 1.3 11/14/96 131.5 7.7 94.2 55 11/20/96 224.6 195.5 92.8 55 7/25/96 246.8 15.6 93.7 48 11/22/96 348.5 6.1 98.3 55 7/31/96 131.3 13.1 90.0 50 11/27/96 378.6 24.7 93.5 55 8/7/96 138.9 33.7 75.7 50 11/29/96 426.1 33.4 92.2 55 8/9/96 111.9 29.6 73.6 50 12/4/96 395.5 20.4 94.8 55 8/14/96 115.9 55.3 52.2 50 12/6/96 422.8 14.6 96.5 55 8/23/96 67.3 40.8 39.3 51 12/11/96 407.4 18.8 95.4 55 8/29/96 215.1 4.5 97.9 51 12/15/96 379.0 23.6 93.8 55 8/30/96 264.0 2.0 99.2 52 1/7/97 175.2 9.0 94.8 55 9/4/96 107.0 8.9 91.7 52 1/9/97 187.6 12.3 93.4 55 Mean 94.8 STDEV ±2.3 88 Table A 5.2.2 COD removal during 37°C to 55°C. Date P C E Effluent % Removal Temp (°C) 6/3/96 1300.9 616.1 52.6 37 6/8/96 1728.9 888.8 48.6 37 6/11/96 1662.1 1042.3 37.3 39 6/19/96 831.3 504.1 39.4 41 6/21/96 1042.6 532.1 49.0 41 6/25/96 1026.4 524.0 48.9 41 7/3/96 867.4 410.2 52.7 43 Average values at T=45°C 7/9/96 500.8 405.7 19.0 45 7/11/96 961.3 324.4 66.3 45 7/16/96 1039.0 543.9 47.7 45 7/18/96 1020.9 526.7 48.4 45 Mean 45.3 STDEV + 19.5 7/23/96 941.5 505.0 46.4 48 7/26/96 1022.7 559.2 45.3 48 7/31/96 1031.8 544.8 47.2 50 8/8/96 999.3 534.8 46.5 50 8/14/96 1069.7 567.4 47.0 50 8/22/96 1057.1 573.7 45.7 50 8/27/96 988.4 507.8 48.6 51 8/31/96 1088.7 592.1 45.6 52 9/11/96 1301.2 712.6 45.2 53 9/17/96 952.3 575.3 39.6 54 9/25/96 1259.5 770.6 38.8 54 10/2/96 1333.0 709.0 46.8 54 Average values at T=55°C 10/16/96 1045.0 683.0 34.6 55 10/22/96 1150.8 668.7 41.9 55 10/30/96 1310.4 760.1 42.0 55 11/5/96 1611.7 898.2 44.3 55 11/13/96 1444.9 944.9 34.6 55 11/20/96 1475.4 987.0 33.1 55 12/9/96 1473.6 876.7 40.5 55 12/20/96 1455.7 925.1 36.4 55 1/7/97 681.0 579.0 15.0 55 Mean 35.8 STDEV ±8.8 89 Table A 5.2.3 M L V S S and VSS concentration during 37 to 55°C. Date M L V S S V S S Temp Date M L V S S V S S Temp (mg/L) (mg/L) (T ) (mg/L) (mg/L) (°C) 6/4/96 3840 10.0 37 8/24/96 1440 32.0 50 6/6/96 3810 25.0 37 8/28/96 1933 21.0 50 6/10/96 2960 63.0 39 9/3/96 2220 44.0 50 6/19/96 1210 102.0 41 9/11/96 1753 96.7 51 6/22/96 1380 64.0 41 9/17/96 1627 28.0 52 6/25/96 1430 74.0 41 9/25/96 1633 73.0 53 6/28/96 1640 30.0 43 10/2/96 2410 65.0 54 7/2/96 1460 17.5 43 Average values at T=55°C Average values at T=45°C 10/16/96 1660 23.0 55 7/8/96 1515 11.0 45 10/23/96 1160 78.9 55 7/11/96 1835 14.7 45 10/28/96 1392 105.4 55 7/15/96 2280 21.5 45 11/4/96 1733 35.0 55 7/17/96 2620 54.4 45 11/14/96 2746 26.0 55 7/19/96 2290 43.0 45 11/20/96 2510 91.2 55 Mean 2108 28.9 11/25/96 2790 89.4 55 STDEV 433 ± 18.9 11/28/96 2930 124.4 55 12/3/96 2123 24.0 55 7/23/96 2313 24.0 48 12/9/96 2373 108.9 55 7/26/96 2060 47.0 48 12/12/96 2800 50.6 55 7/31/96 1635 28.0 48 12/17/96 2220 28.0 55 8/8/96 1167 48.0 48 1/2/97 2250 68.0 55 8/13/96 1330 128.3 48 1/7/97 2246 34 55 8/19/96 1310 35.0 50 Mean 2210 63.3 STDEV 549 + 36.1 90 Table A 5.2.4 OURs and SOURs during 37°C to 55°C. Date M L V S S O U R S O U R Temp (mg/L) (mgO:-' L-min) (mgOVgMLVSS-h) ("C) 6 4 96 3840 0.6 9.8 37 6/5/96 3810 1.3 20.1 37 6/6/96 2960 0.5 9.2 39 6/19/96 1210 0.3 17.1 41 6/21/96 1380 0.4 19.2 41 6/25/96 1430 0.3 10.7 41 6/28/96 1640 0.4 15.5 43 7/2/96 1460 0.3 10.7 43 Average values at T=45°C 7/8/96 1515 0.2 8.8 45 7/11/96 1835 0.4 13.5 45 7/15/96 2280 1.0 26.6 45 7/17/96 2620 0.5 12.2 45 7/19/96 2290 0.4 9.2 45 Mean 14.1 STDEV + 7.3 7/23/96 2313 0.7 19.2 48 7/26/96 2060 0.7 21.6 48 7/31/96 1635 0.3 12.0 50 8/8/96 1167 0.8 42.9 50 8/13/96 1330 0.6 27.8 50 8/19/96 1310 0.8 35.6 50 8/24/96 1440 0.3 12.8 51 8/28/96 1933 0.3 9.5 51 9/3/96 2220 0.3 8.9 52 9/11/96 1753 0.8 27.8 53 9/17/96 1627 0.8 29.5 54 9/25/96 1633 0.8 27.7 54 10/2/96 2410 0.6 13.9 54 Average values at T=55°C 10/16/96 1660 0.4 14.0 55 10/23/96 1160 0.3 15.5 55 10/30/96 1392 0.4 19.2 55 11/4/96 1733 0.4 12.3 55 11/14/96 2746 0.6 12.6 55 11/20/96 2510 0.3 7.8 55 11/25/96 2790 0.4 9.2 55 91 11/28/96 2930 0.4 8.8 55 12/9/96 2373 0.7 17.0 55 12/12/96 2800 0.5 11.1 55 12/17/96 2220 0.6 16.9 55 1/2/97 2250 0.2 4.8 55 1/7/97 0.2258 0.2 6.0 55 Mean 12.4 STDEV ±4.3 92 Table A 5.2.5 Toxicity removal during 37°C to 55°C. P C E Effluent Date E C 5 0 T U E C 5 0 T U % Removal Temp (°C) Average values at T=45°C 7/11/96 4.1 24.4 95.4 1.0 95.7 45 7/17/96 2.2 45.9 32.8 3.0 93.4 45 Mean 64.1 94.5 STDEV ±44.3 ± 1.7 7/22/96 6.5 15.4 43.4 2.3 85.1 48 8/7/96 2.4 41.0 44.2 2.3 94.5 50 8/16/96 4.8 20.7 42.0 2.4 88.5 50 9/11/96 3.7 26.7 17.4 5.7 78.4 53 9/26/96 3.4 29.5 49.6 2.0 93.2 54 10/2/96 2.0 50.2 64.3 1.6 96.9 54 10/4/96 3.0 33.8 100.0 1.0 97.0 54 10/9/96 3.0 33.6 33.8 3.0 91.2 54 Average values atT= =55°C 10/17/96 1.0 105.2 55.4 1.8 98.3 55 10/23/96 1.2 84.4 70.4 1.4 98.3 55 10/31/96 3.7 27.4 51.1 2.0 92.9 55 11/1/96 3.7 27.0 52.1 1.9 92.9 55 11/13/96 3.3 29.9 67.7 1.5 95.1 55 11/15/96 4.7 21.3 41.1 2.4 88.6 55 11/20/96 4.6 22.0 37.8 2.6 88.0 55 11/25/96 1.4 71.0 44.5 2.2 96.8 55 11/28/96 1.5 66.0 49.2 2.0 96.9 55 12/4/96 2.9 35.0 55.9 1.8 94.9 55 12/9/96 2.9 34.6 79.3 1.3 96.4 55 12/11/96 3.8 26.1 63.7 1.6 94.0 55 12/13/96 3.9 25.5 75.2 1.3 94.8 55 12/17/96 2.1 46.7 40.7 2.5 94.7 55 12/24/96 2.7 37.2 100.0 1.0 97.3 55 1/8/97 10.1 9.9 100.0 1.0 89.9 55 Mean 61.3 93.8 STDEV ±20.7 ±3.0 93 Statistical Significance Test Nomenclature X Sample mean S Standard deviation N Number of sample v Degree of freedom f Test statistic value t o.o5,v Critical value for the t-distribution with 95% confidence interval Table A 5.2.6 Data for statistical significance test between 35°C and 55°C. illililli:?!'- S N S 2/N V t' t 0.05.V B O D T=35°C 95.4 4.3 68.0 0.3 47.8 0.8 1.7 T-55°C 94.8 2.3 17.0 0.3 C O D T=35°C 47.6 9.6 43.0 2.1 12.4 3.6 1.8 T=55°C 35.8 8.8 9.0 8.5 M L V S S T=35°C 2522 1180 63.0 22101 43.7 1.5 1.7 T=55°C 2210 549 14.0 21529 111' T=35°C 34.3 32.0 63.0 16.3 17.8 2.8 1.7 T=55°C 63.3 36.1 14.0 93.1 S O U R T=35°C 12.4 7.9 54.0 1.2 30.1 0.004 1.7 T=55°C 12.4 4.3 12.0 1.5 Toxici ty T=35°C 94.2 5.1 35.0 0.7 42.5 0.4 1.7 T=55°C 93.8 3.0 15.0 0.6 94 Table A 5.2.7 Data for statistical test between 35°C and 45°C. X S N S2/N v t' t 0.05.V BOD T=35°C 95.4 4.3 68.0 0.3 12.5 0.7 1.9 T=45°C 96.0 1.3 5.0 0.3 C O D T=35°C 47.6 9.6 43.0 2.1 3.1 0.2 2.4 T=45°C 45.3 19.6 4.0 95.1 M L V S S T=35°C 2522 1180 63.0 22101 9.9 1.7 1.8 T=55°C 2108 433.0 5.0 37497.8 VSS T=35°C 34.3 32.0 63.0 19.9 6.4 0.6 1.9 T=45°C 28.9 18.9 5.0 73.6 SOUR T=35°C 12.4 7.9 54.0 1.2 4.9 0.5 1.9 T=45°C 14.1 7.3 5.0 10.7 Toxicity T=35°C 94.2 5.1 35.0 0.7 2.4 0.2 2.4 T=45°C 94.5 1.7 2.0 1.4 95 Table A 5.2.8 Data for statistical test between 45°C and 55°C. X S N S2/N V t' t 0.05.V BOD T=45°C 96.0 1.2 5.0 0.3 13.9 1.5 1.8 T=55°C 94.8 2.3 17.0 0.3 C O D T=45°C 45.3 19.6 4.0 95.6 3.5 0.9 2.1 T=55°C 35.8 8.8 9.0 8.5 M L V S S T=45°C 2108 433 5.0 374988 9.0 0.4 1.8 T=55°C 2210 549 14.0 21529 VSS T=45°C 28.9 18.9 5.0 71.3 13.9 2.7 1.8 T=55°C 63.3 36.1 14.0 93.1 SOUR T=45°C 14.1 7.3 5.0 10.7 5.2 0.5 2.0 T=55°C 12.4 4.3 12.0 1.5 Toxicity T=45°C 94.5 1.7 2.0 1.4 2.1 0.5 2.9 T=55°C 93.8 3.0 15.0 0.6 96 A 5.3 Effect of H i g h Operat ing Temperature on Species Diversi ty Table 5.3.1 Number of bacteria estimated by M P N method. t°c D h A A b A P i A IpA 12/14-Cl-DhA 35 2.0 x 103 2.0 x 103 2.0 x IO3 6.2 x IO6 1.5 x 10b 38 2.9 x 106 2.9 x 106 6.2 x 106 1.6 x 107 2.0 x 103 39 1.4 x 107 8.0 x 107 1.4 x 107 2.9 x 107 8.0 x 10* 42 4.3 x 106 1.5 x 106 4.3 x 106 1.3 x 10s 1.0 x 106 45 2.0 x 104 2.0 x 104 2.0 x IO4 2.0 x IO4 2.0 x IO4 48 2.0 x 104 2.0 x IO4 2.0 x IO4 2.0 x IO4 2.0 x IO4 50 2.0 x 104 2.0 x 104 2.0 x 104 2.0 x IO4 2.0 x IO4 52 2.0 x 104 2.0 x 104 2.0 x 104 2.0 x IO4 2.0 x IO4 Benzoic A c i d Sodium Formate Palmitic A c i d Linole ic A c i d Guaiaco l 35 2.0 x 103 2.9 x 106 2.8 x 107 2.6 x 106 1.0 x 107 38 2.0 x 103 6.2 x 106 3.1 x 10s 2.9 x IO4 4.3 x 106 39 6.2 x 107 1.0 x 107 5.0 x 107 1.4 x 107 2.9 x IO6 42 2.0 x 104 6.2 x 10* 1.4 x 107 4.3 x IO7 2.0 x IO4 45 2.0 x 106 2.7 x 104 2.6 x IO7 1.0 x 107 2.0 x IO4 48 2.0 x 104 2.0 x 104 1.0 x 107 8.4 x 105 2.0 x IO4 50 2.0 x 104 2.0 x IO4 5.0 x IO7 2.0 x IO4 2.0 x 104 52 2.0 x 104 2.0 x IO4 1.0 x IO6 2.0 x IO4 2.0 x IO4 Arabinose Galactose Glucose Methanol E thanol 35 1.6 x 10s 2.9 x 107 1.6 x 108 2.9 x IO5 1.0 x 107 38 5.0 x 10b 2.9 x 107 1.6 x IO8 2.9 x IO6 5.0 x 107 39 5.0 x 107 1.6 x 108 1.4 x 108 1.4 x 108 8.0 x 107 42 5.0 x 10s 4.3 x 10b 1.4 x 107 1.0 x \Ob 8.0 x 105 45 1.4 x 106 8.0 x 10s 5.0 x 105 2.0 x IO4 2.0 x IO4 48 2.50 x 108 1.5 x 107 2.4 x 108 2.0 x IO4 2.0 x IO4 50 2.9 x 106 2.6 x 105 2.9 x IO6 2.0 x IO4 2.0 x IO4 52 3.1 x 109 6.2 x 105 2.9 x 10s 2.0 x IO4 2.0 x IO4 97 Table A 5.3.2 Sludge settleability as a function of reactor operating temperature. Temp (°C) Height (mm) 35 53 38 32 39 36 42 48 45 60 48 60 50 70 52 60 98 A 5.4 Response of H T A S Bioreactors to Temperature Shock Table A 5.4.1 BOD removal for both bioreactors after temperature shock. Time (h) /PGEtl T=35°C % Removal T=55°C % Removal 0.0 455.3 20.2 95.6 41.0 91.0 1.0 418.5 18.9 95.5 57.4 86.3 4.0 418.5 17.5 95.8 58.2 86.1 14.0 343.5 14.4 95.8 47.0 86.3 18.5 343.5 12.6 96.3 57.4 83.3 25.0 343.5 15.3 95.5 49.0 85.7 40.5 472.5 15.3 96.8 48.9 89.6 63.0 535.5 17.2 96.8 85.4 84.1 > 1 Q O CO 100 98 96 94 92 90 -r± o — — © * *\ n s T=35C i - -Steady state i i 20 40 Time (h) 60 80 Figure A 5.4.1 BOD removal efficiency of RI (mesophilic bioreactor) after temperature shock. > © s 04 O O M 20 40 Time (h) 60 80 Figure A 5.4.2 BOD removal efficiency of R2 (thermophilic bioreator) after temperature shock. 99 Table A 5.4.2 COD removal for both bioreactors after temperature shock. Time (h) P C E T=35°C % Removal T=55°C % Remova l 1.0 1649.8 1017.2 38.3 1010.4 38.8 4.0 1649.8 962.7 41.6 1039.1 37.0 14.0 1559.8 995.4 36.2 872.7 44.1 18.5 1559.8 898.6 42.4 1007.7 35.4 25.0 1559.8 809.9 48.1 1009.1 35.3 40.5 1360.7 797.6 41.4 773.1 43.2 63.0 1562.5 790.8 49.4 1014.5 35.1 > i Q O U 10 20 30 40 Time (h) 50 60 70 Figure A 5.4.3 COD removal efficiency of RI after temperature shock. 50 0 10 20 30 40 50 60 70 Time (h) Figure A 5.4.4 COD removal efficiency of R2 after temperature shock. 100 Table A 5.4.3 MLVSS and VSS concentration after temperature shock. Time (h) T=35°C T=55°C M L V S S (mg/L) V S S (mg/L) M L V S S (mg/L) V S S (mg/L) 0 4153 23 1860 90 1.0 4030 10 3120 45 4.0 3960 3 2930 86 14.0 2560 2 4340 32 18.5 3980 6 1870 39 25.0 3920 14 1280 47 40.5 4520 5 4867 11 63.0 4720 4 4660 37 10 20 30 40 Time (h) 50 60 70 Figure A 5.4.5 MLVSS concentration in RI after temperature shock. 6000 5000 l| 4000 $ 3000 j 2000 1000 0 0 10 20 30 40 50 60 70 Time (h) Figure A 5.4.6 MLVSS concentration in R2 after temperature shock. 101 Figure A 5.4.8 VSS concentration in RI after temperature shock. 102 Table A 5.4.4 OURs and SOURs after temperature shock. T=35°C T=55°C T ime M L V S S O U R S O U R M L V S S O U R S O U R (h) (mg/L) (mgO :.' (mg0 2/ (mg/L) (mg0 2/ (mg0 2 ' L-min) gMLVSS-h) L-min) gMLVSS-h) 1.0 4030 0.470 7.002 3980 0.496 7.482 4.0 3960 0.449 6.804 3120 0.515 9.913 14.0 2560 0.418 9.808 2930 0.451 9.240 18.5 3980 0.413 6.227 4340 0.377 5.213 25.0 3920 0.439 6.722 1870 0.342 10.966 40.5 4520 0.427 5.667 1280 0.238 11.160 63.0 4720 0.461 5.862 4867 0.682 8.404 S 12 «» 0 -I 1 1 1 0 20 40 60 80 Time (h) Figure A 5.4.9 SOUR in RI after temperature shock. 0 10 20 30 40 50 60 70 Time (h) Figure A 5.4.10 SOUR in R2 after temperature shock. 103 Table A 5.4.5 Toxicity removal after temperature shock. Time (h) P C E T=35°C T=55°C 111! ! ! ! ! ! : : T U E C 5 0 T U % Removal E C 5 0 T U % Removal 0 2.4 41.1 61.7 1.6 96.1 48.8 2.0 95.0 1.0 3.6 28.0 78.2 1.3 95.4 26.3 3.8 86.4 4.0 3.6 28.0 77.2 1.3 95.4 42.8 2.3 91.7 14.0 3.6 28.0 70.7 1.4 94.9 44.8 2.2 92.0 18.5 3.6 28.0 100.0 1.0 96.4 67.3 1.5 94.7 25.0 3.6 28.0 55.9 1.8 93.6 50.3 2.0 92.9 40.5 3.6 28.0 100.0 1.0 96.4 60.4 1.7 94.1 63.0 3.6 28.0 100.0 1.0 96.4 52.3 1.9 93.2 100 80 H 1 1 1 1 1 1 0 10 20 30 40 50 60 70 Time (h) Figure A 5.4.11 Toxicity removal efficiency of RI after temperature shock. 100 T ¥ 95 *-> o 8 90 -•S 8 5 " o H 80 -0 Figure A 5.4.12 Toxicity removal efficiency of RI after temperature shock. -T=45C •Steady state i 1 1 1 1 1 10 20 30 40 50 60 70 Time (h) 104 Statistical Significance Test Table A 5.4.6 Data for statistical significance test for mesophilic bioreactor after shock study. X S N S 2 / N v t' t 0.05.V B O D Before 96.0 0.4 4 0.04 7.8 0.3 1.9 After 96.1 0.5 6 0.1 C O D Before 49.6 1.1 4.0 0.3 5.8 3.5 1.9 After 42.5 4.8 6.0 3.8 M L V S S Before 4439 253.8 4 152412 6.0 1.0 1.9 After 3956 690.2 6 79396 V S S Before 22.4 8.7 4.0 18.9 4.0 3.4 2.1 After 6.3 4.3 6.0 3.0 S O U R Before 5.1 2.0 4.0 1.0 4.9 1.6 2.0 After 6.9 1.4 6.0 0.3 Toxici ty Before 95.6 0.7 4.0 0.1 8.0 0.2 1.9 After 95.5 1.0 6.0 0.2 105 Table A 5.4.7 Data for statistical significance test for thermophilic bioreactor after the shock study. X S \ S 2 /N v t' t 0.05.V BOD Before 93.3 2.2 4 1.2 6.2 6.7 1.9 After 85.9 2.0 6 0.7 C O D Before 44.8 2.5 4 1.6 8.0 3.2 1.9 After 38.4 3.8 6 2.4 M L V S S Before 3063 780.8 4 152412 7.9 0.3 1.9 After 3295 1395.2 6 324430 VSS Before 41.6 27.3 4 186.3 5.7 0.0 1.9 After 42.4 22.6 6 85.1 SOUR Before 9.3 7.7 4 14.8 3.3 0.03 2.4 After 9.2 2.0 6 0.7 Toxicity Before 94.6 0.6 4 0.1 5.7 2.2 1.9 After 92.1 2.7 6 1.3 106 A 5.5 Kinetics of Substrate Uptake i n Mesophi l ic and Thermophi l ic A S System Methodology A typical results from the respirometric kinetic assay are shown in Figure A5.5.1. This particular result was obtained by using formic acid at 1.2 mg/L as the substrate. 1.5 i -1 J Time (min) Figure A 5.5.1 OUR versus time. The raised portion represents the amount of oxygen required to metabolize the added substrate (formic acid). By plotting the area of the raised portion against different amount of added substrate, a graph of the oxygen utilized versus substrate added is obtained (FigureA5.5.2). The slope of this line represents the oxygen requirement o the microorganisms. The linear relationship confirms that the oxygen requirement is constant for a given biomass and substrate. It can be seen that the change in OUR due to the addition of substrate is related to the amount of substrate added. This relationship follows Monod kinetics (equation 8) and is shown in Figure A 5.5.3. 107 1.6 y=0.9841x+0.0416 R2=0.9945 0.0 0.2 0.4 0.6 0.8 1.0 1.2 BOD(II^L) 1.4 1.6 Figure A 5.5.2 Oxygen consumed versus substrate metabolized. 1.2 £ 1 . 0 | 0.8 O « 0.6 W) § 0 . 4 £ 0.2 0.0 y=0.1139Ln(x) + 0.9647 R2 = 0.7691 0.0 0.2 0.4 0.6 0.8 1.0 BOD (mg/L) 1.2 1.4 1.6 Figure A 5.5.3 SUR versus BOD. 108 By plotting a double reciprocal plot of the Monod equation, the kinetic parameter K and qm ax were obtained according to equation 9 (Figure A 5.5.4). Figure A 5.5.4 Linearized Monod equation. 109 Sample Calculations 1. Preparation of methanol stock solution Density of methanol = 0.791 g/mL Amount of methanol = 0.05 mL Amount of water = 99.95 mL Concentration of the stock solution . _ 0.05mL = 0J9lg/mLx lOOmL = 3.96xlO"4g/mL 2. Amount of substrate added Methanol CH 3 OH + l.5 0 2 ^ C 0 2 + H 2 0 l mole of methanol exerts 1.5 moles of BODu (ultimate BOD). BOD5 is approximately 80% of BODu . Thus l mole of methanol exerts 1.2 moles of BOD5. A 5 mL of stock solution has = 5mLx3.96xlCT4g/mLxl.2BOD5 = 2.38xlO" 4gBOD 5 Concentration of substrate in 300 mL respirator: 2 .38xl0" 4 gBOD 5 300mL - 7.93x10"7g/mL or 0.79 mg/L 110 Table A 5.5.1 Kinetic constants data for utilizing methanol at 35°C. s AOUR o 2 1/S 1/<l consumed (mg/L) (mg02/L-min) (ms 'U (mgBOD/gM L VSS-•min) 1.8984 1.0592 1.4513 1.4077 0.5268 0.7104 1.1865 0.9708 0.8317 1.2902 0.8428 0.7751 0.9492 0.9642 0.7089 1.2815 1.0535 0.7803 0.7119 1.1890 0.5039 1.5802 1.4047 0.6328 0.4746 0.8627 0.3611 1.1466 2.1070 0.8721 0.3797 0.8514 0.2850 1.1315 2.6338 0.8838 0.2848 1.0340 0.1529 1.3743 3.5117 0.7276 0.2373 0.6232 0.1795 0.8283 4.2141 1.2073 0.1898 0.7056 0.1420 0.9378 5.2676 1.0663 0.1424 0.6728 0.0840 0.8942 7.0235 1.1183 0.1187 0.7093 0.1308 0.9427 8.4282 1.0607 0.0949 0.5684 0.0562 0.7554 10.5352 1.3238 Qmax K Y (mgBOD/gMLVSS- min) (mgBOD/L) (mgMLVSS/mgBOD) 0.7245 0.0821 0.2476 Table A 5.5.2 Kinetic constants for utilizing methanol at 45°C. s A O U R o 2 llllllSiiii||iiiiiiii|iiiii 1/S 1/q consumed (mg-L) (mg02/L-min) (ms'I-) (mgBOD/gMLVSS- min) 0.9492 0.6636 0.8442 0.7317 1.0535 1.3667 0.7119 0.5989 0.5811 0.6603 1.4047 1.5144 0.4746 0.5424 0.3689 0.5981 2.1070 1.6720 0.2373 0.4931 0.1716 0.5437 4.2141 1.8392 0.1424 0.6428 0.0788 0.7088 7.0235 1.4108 0.1187 0.4421 0.0692 0.4875 8.4282 2.0514 0.0949 0.3882 0.0664 0.4281 10.5352 2.3362 Qmax K Y (mgBOD/gMLVSS-min) (mgBOD/L) (mgMLVSS/mgBOD) 0.4585 0.0527 0.0931 111 Table A 5.5.3 Kinetic constants for utilizing formic acid at 35°C. s AOUR o2 q 1/S 1/q consumed (mg/L) (mg02/L-min) (mg/L) (mgBOD/gMLVSS-•min) 1.0184 0.5934 1.0125 0.6368 0.9819 1.5703 0.8487 0.5725 0.8158 0.6144 1.1783 1.6277 0.7638 0.7518 0.7247 0.8068 1.3092 1.2395 0.6790 0.6089 0.6909 0.6535 1.4728 1.5302 0.5941 0.6314 0.6598 0.6776 1.6833 1.4757 0.5092 0.5613 0.5150 0.6024 1.9638 1.6602 0.4243 0.6278 0.4468 0.6737 2.3566 1.4843 0.3395 0.5724 0.3668 0.6143 2.9457 1.6280 0.2546 0.4946 0.2704 0.5308 3.9276 1.8839 0.1697 0.4402 0.4724 5.8914 2.1167 Qmax K Y (mgBOD/gMLVSS-•min) (mgBOD/L) (mgMLVSS/mgBOD) 0.5056 0.1014 0.0682 Table A 5.5.4 Kinetic constants for utilizing formic acid at 45°C. s (mgL) A O U R (mgOa/L-min) o2 consumed (mg/L) q (mgBOD/gMLVSS-min) 1/S 1/q 1.3579 0.9085 1.3189 0.9232 0.7364 1.0832 1.1882 1.0256 1.2119 1.0422 0.8416 0.9595 1.0184 0.9090 1.0587 0.9237 0.9819 1.0826 0.8487 0.8893 0.9268 0.9037 1.1783 1.1066 0.6790 1.0021 0.7248 1.0183 1.4728 0.9820 0.5092 0.8887 0.5680 0.9031 1.9638 1.1073 0.3395 0.8589 0.3927 0.8728 2.9457 1.1458 0.2546 0.8285 0.2879 0.8419 3.9276 1.1878 0.1697 0.6838 0.1934 0.6948 5.8914 1.4392 0.0849 0.6673 0.0803 0.6780 11.7828 1.4749 qmax (mgBOD/gMLVSS-min) K (mgBOD/L) Y (mgMLVSS/mgBOD) 0.4717 0.0443 0.0159 112 A 5.6 C O D Calibration Curve Table A 5.6 summarizes the data for the COD calibration curve. The relationship between absorbence and COD was subjected to a linear fit (r2 = 0.9999) calculated by Microsoft Excel 97 and the equation for the curve is, COD (mg/L) = 2656 x absorbence + 25.5 Table A 5.6 Data for COD calibration curve. Absorbence* C O D mg L 0.18 500 0.10 300 0.07 200 0.03 100 0.01 50 *Data for absorbence are average of three replicate. 113 

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