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Biochemical models for biological excess phosphorus removal from wastewater Comeau, Yves 1984

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BIOCHEMICAL MODELS FOR BIOLOGICAL EXCESS PHOSPHORUS REMOVAL FROM WASTEWATER by YVES COMEAU B.Ing., Ecole Polytechnique de Montreal, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of C i v i l Engineering) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1984 © Yves Comeau, 1984 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r b y h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f C i v i l Engineering The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main M a l l V a n c o u v e r , C a n a d a V6T 1Y3 D a t e June 15, 1984 DE-6 (3/81) ABSTRACT The objectives of this research were to propose some biochemical models that would explain the basic mechanisms of biological excess phosphorus removal from wastewater, and to provide selected experimental confirmations of the proposed models. The models postulated had to be based on the observations available from the literature as well as to be consistent with principles of bacterial biochemistry and microbiology. The phenomena of phosphate release and uptake were explained by the role of carbon substrates being stored anaerobically as poly-g-hydroxybutyrate (PHB), and by the aerobic utilization of PHB for energy production. It was postulated that under anaerobic conditions (in absence of both free oxygen and nitrate), substrates such as acetate would diffuse in cells and de-energize the membrane. Bacteria that had polyphosphate reserves could re-energize their membrane by neutral phosphate expulsion, thus causing a net ejection of protons. Speculations on the possible role of polyphosphate as an anaerobic energy source were provided. The experimentation involved preliminary technique development, and batch testing under aerobic and anaerobic conditions. The effect of the addition of acetate, nitrate, and 2,4-dinitrophenol (a toxicant) and of various pH's on phosphate release were also studied. The following observations were made from the experimentation. The expected role for PHB was confirmed. Nitrate addition anaerobically resulted in phosphate uptake. Potassium, magnesium, and calcium were found to be co-transported with phosphate. Under anaerobic conditions, with identical - i i -amounts of acetate addition, a higher pH resulted in an increased magnitude of rapid phosphate release. The addition of 2,4-dinitrophenol resulted in a higher degree of anaerobic phosphate release when acetate was added. The postulated models were found to be consistent with most observations from the literature and from this research. From the understanding provided by these models, recommendations were given for efficient approaches to the operation of treatment plants removing phosphorus biologically, and for optimum processes for biological excess phosphorus removal. - i i i -TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i i i LIST OF FIGURES ix ACKNOWLEDGEMENTS . . . x i i i 1. INTRODUCTION 1 2. LITERATURE REVIEW 4 2.1. Introduction 4 2.2. Process Schemes for Biological Excess Phosphorus Removal. . 4 2.2.1. Biological Nitrogen Removal Processes 6 2.2.2. Biological Phosphorus (and Nitrogen) Removal . . . 10 2.3. Requirements for Excess Phosphorus Removal 15 2.3.1. Phosphate Uptake 15 2.3.2. Phosphate Release 18 2.4. Bacterial Storage for Bio-P Removal 25 2.4.1. Polyphosphate Metabolism 25 2.4.2. PHB Metabolism 29 2.5. Bacteria Involved in Bio-P Removal 31 2.6. Mechanistic Model for Bio-P Removal from the Literature . . 34 3. BIOCHEMICAL MODELS - 37 3.1. Introduction 37 3.2. Postulated Model for Aerobic Conditions 37 - Iv -3.3. Postulated Model for Anaerobic Conditions 38 3.3.1. D i f f i c u l t i e s i n Proposing a Biochemical Model for Anaerobic Conditions 38 3.3.2. Postulated Model 41 3.3.3. Speculations on Energy Sources for PHB Storage . . . 46 METHODOLOGY 53 4.1. Batch Testing 53 4.2. UBC P i l o t Plant Activated Sludge 53 4.3. A n a l y t i c a l Techniques 55 4.3.1. Residue 55 4.3.2. Metals 58 4.3.3. Nitrogen 58 4.3.4. Oxidation-Reduction P o t e n t i a l (ORP) 59 4.3.5. pH 59 4.3.6. Phosphorus 59 4.3.7. V o l a t i l e Fatty Acids (VFA's) 61 4.3.8. Poly-3-Hydroxybutyrate (PHB) 61 4.3.9. A l k a l i n e Phosphatase 64 RESULTS AND DISCUSSION 66 5.1. Introduction 66 5.2. A l k a l i n e Phosphatase 66 5.2.1. Introduction 66 5.2.2. Results 67 5.2.3. Discussion 67 5.3. C y t o l o g i c a l Staining 69 Page 5.4. Polyphosphate Chemical Quantification 69 5.4.1. Introduction 69 5.4.2. Results 70 5.4.3. Discussion 70 5.5. Poly-g-Hydroxybutyrate Chemical Quantification 75 5.5.1. Introduction 75 5.5.2. GC Peak Separation 75 5.5.3. Hydroxybutyric Ester Formation and Sludge Sampling . 77 5.6. Pilot Plant Surveys for PHB 84 5.6.1. Introduction 84 5.6.2. First Pilot Plant Survey for PHB 84 5.6.3. Second Pilot Plant Survey for PHB 84 5.6.4. Discussion 89 5.7. Batch Tests with Acetate Addition 89 5.7.1. Introduction 89 5.7.2. Preliminary Experiment 90 Results 90 Discussion 92 5.7.3. Experiment on the Effect of Acetate and Nitrate . . 92 Introduction 92 Results 93 Discussion . 101 5.7.4. Experiment on the Effect of pH and DNP 109 Introduction 109 Results I l l - v i -Page Discussion 114 5.8. Consistency of the Models with Observations 118 5.8.1. Introduction 118 5.8.2. Aerobic Conditions 120 5.8.3. Anaerobic Conditions 121 5.9. Applications Suggested by the Model 124 5.9.1. Carbon Storage 124 5.9.2. Discussion of Nutrient Removal Processes 127 6. CONCLUSIONS AND RECOMMENDATIONS 132 6.1. Conclusions 132 6.1.1. Observations from Batch Tests 132 6.1.2. Model for Bio-P Removal . 133 6.2. Recommendations 134 BIBLIOGRAPHY 137 APPENDICES 145 A. Symbols 145 B. Glossary 147 C. Glycolysis Pathway and TCA Cycle 149 D. Elements of Bioenergetics 151 E. Procaryotic Membrane Transport 155 - v i i -LIST OF TABLES Page 2.1 Effect of various types of substrate on phosphate release 24 2.2 Effect of pH on phosphate release 24 2.3 List of polyphosphate-accumulating bacteria 32 3.1 Possible reactions for transforming acetate to acetyl CoA 48 3.2 Free energy of hydrolysis of energy-rich compounds 49 4.1 Typical Influent and effluent characteristics of the UBC bio-P pilot plant 57 5.1 Alkaline phosphatase level under anaerobic conditions 68 5.2 Polyphosphate salts recovery for various phosphorus determinations 71 5.3 Recovery of polyphosphate from activated sludge samples 72 5.4 Reproducability of phosphorus analysis of activated sludge 73 5.5 GC/MS identification of the peaks adjacent to the HB ester peak 78 5.6 Reproducibility of the GC injection technique 79 5.7 Reproducibility of the PHB determination 81 5.8 Recovery of hydroxybutyrate from sludge samples 82 5.9 Residue concentrations (SS) for the pilot plant survey #2 87 5.10 Determination of the molar ratio of positive charges to phosphate molecules transported 110 - v i i i -LIST OF FIGURES Figure Page 2.1 Conventional activated sludge process 5 2.2 Ludzack and Ettinger process for biological nitrogen removal 9 2.3 Wuhrmann process for biological nitrogen removal 9 2.4 Modified Ludzack and Ettinger (MLE) process for biological nitrogen removal 9 2.5 Phostrip process for biological phosphorus removal 9 2.6 Bardenpho process for biological nitrogen and phosphorus removal 9 2.7 Phoredox process for biological nitrogen and phosphorus removal 9 2.8 Modified Phoredox process for biological nitrogen and phosphorus removal 12 2.9 UCT process for biological nitrogen and phosphorus removal 12 2.10 Modified UCT process for biological nitrogen and phosphorus removal 12 2.11 A/0 process for biological phosphorus removal 12 2.12 Effect of 2,4-dinitrophenol addition on phosphate uptake under aerobic conditions 17 2.13 Effect of alternative aerated and unaerated periods in a batch test on phosphate release and uptake 17 2.14 Effect of CO2 bubbling and acetic acid addition on phosphate release under anaerobic conditions 19 2.15 Effect of various levels of acetate addition on phosphate release 19 2.16 Effect of various levels of acetate addition on (A) phosphate release and (B) denitrification 21 2.17 Effect of acetate addition on phosphate release 23 2.18 Effect of various types of substrates on phosphate release 23 - Ix -Figure Page 2.19 Chemical structure of polyphosphate 26 2.20 Polyphosphate metabolic pathways 26 2.21 Patterns of polyphosphate accumulation in Aerobacter  aerogenes 26 2.22 Poly-g-hydroxybutyrate metabolic pathways 30 3.1 Conceptual model for aerobic metabolism in bio-P bacteria 39 3.2 Postulated model for aerobic metabolism in bio-P bacteria 40 3.3 Postulated model for anaerobic metabolism of bio-P bacteria 45 3.4 Speculations on energy sources for acetic acid storage as PHB 47 3.5 Speculations on the role of polyphosphate, and a source of NADH from the TCA cycle 51 3.6 Speculations on the role of polyphosphate and a source of NADH from glycolysis and amino acids degradation 52 4.1 Batch testing apparatus 54 4.2 Configuration of the UBC pilot plant processes 56 5.1 UBC pilot plant survey no. 1: "A" side 85 5.2 UBC pilot plant survey no. 1: "B" side 85 5.3 UBC pilot plant survey no. 2: "A" side 88 5.4 UBC pilot plant survey no. 2: "B" side 88 5.5 Effect of acetate and air addition on: (A) phosphate release and uptake, and (B) poly-g-hydroxybutyrate 91 5.6 Effect of various levels of acetate and nitrate addition on: the SRP profile 94 5.7 : the nitrate profile 94 - x -Figure Page 5.8 : the ORP profile 95 5.9 : the VFA's profile 95 5.10 : the potassium profile 98 5.11 : the magnesium profile 98 5.12 : the calcium profile 99 5.13 : the sodium profile 99 5.14 : the PHB profile 100 5.15 : the PHB-GC r t 5.89 min. compound profile 100 5.16 : the pH profile 102 5.17 Relationship between the phosphate released and the acetate available for phosphate release 104 5.18 Relationship between the SRP concentration and the rate of SRP decrease from solution after nitrate addition 104 5.19 Relationship between the potassium and SRP concentrations 108 5.20 Relationship between the magnesium and SRP concentrations 108 5.21 Combined effect of acetate addition, and pH adjustment or 2,4-dinitrophenol addition on: the SRP profile 112 5.22 : the potassium profile 112 5.23 : on the magnesium profile 113 5.24 : on the pH profile 113 5.25 Relationship between the potassium and SRP concentrations 116 5.26 Relationship between the magnesium and SRP concentrations 116 5.27 Proposed optimum process for bio-P removal 128 5.28 A proposed efficient process for plant upgrading aiming at bio-P removal 130 - xi -Figure Page 5.29 A proposed efficient process for plant upgrading aiming at bio-P and N removal 131 C-l Outline and regulation of the glycolysis pathway and of the TCA cycle 150 D-l Summary of bacterial bioenergetics 154 E- l Summary of procaryotic membrane transport 157 - x i i -ACKNOWLEDGEMENT S I thank Dr. K. J. Hall for his assistance in the research and prepara-tion of this thesis. I am obliged to Dr. W.K. Oldham for Integrating me in his bio-P team. I thank F.A. Koch and Barry Rabinowitz for providing activated sludge from the UBC pilot plant, and also for discussion and occasional batch test assistance. I am grateful to Dr. R.E.W. Hancock for discussion and providing microbiology information. I also thank Susan Liptak, Paula Parkinson and Susan Jasper for labora-tory guidance, Timothy Ma for the GC/MS work, Troy Vassos and Ashok Gupta for assistance in a batch test, Dr. F.M. Harold (Denver, CO.) for his donation of polyphosphate salts, Carol Lore and Kelly Lamb for typing the manuscript, the Department of C i v i l Engineering for financial support from the NSERC grants #67-8945 of Dr. D.S. Mavinic and #67-8935 of Dr. K.J. Hall, and Claire LauzS for her constant moral support. - x i i i -1. 1. INTRODUCTION Increased levels of urbanization, industrialization and land exploitation have resulted in increased nutrient loadings causing pollution problems in receiving water bodies. In most of these natural aquatic environments, nitrogen or phosphorus are the limiting nutrients. This means that the growth of aquatic plants and algae Is controlled by the limited supply of nitrogen or phosphorus. As a result of human acti v i t i e s , streams or rivers shallow enough to allow light penetration can support the growth of rooted plants. In some cases, the natural flow can become impeded and backflooding may occur. With nocturnal oxygen consumption by plants, the dissolved oxygen concentration may be reduced drastically, affecting fish and other oxygen-dependent organisms. In lakes, ponds and reservoirs, increased nutrient loadings result in aquatic plant growth in shallow water. The most obvious and damaging effect i s observed with sudden algal proliferation, commonly referred to as blooms. Their accumulation on the shoreline or in coves can Interfere with recreational activities in particular. When decomposing, they cause serious nuisance of unsightliness and odor. Problems of taste and odor In water supplies can occur. Since the decomposition process consumes oxygen, the dissolved oxygen level can decrease drastically. To control mass production of algae or aquatic plants, phosphorus is the "key" element to remove. Carbon cannot become a limiting nutrient in most cases due to i t s natural abundance from organic compounds or the bicarbonate ion. Nitrogen is not a true growth restraint either. The assimilable forms 2. of nitrogen for most plants and microorganisms are nitrate and ammonia. When these forms become depleted, blue-green algae can f i x the nitrogen gas from a i r and add nitrogen to the system. Phosphorus, however, has i t s only source from the earth crust and i t s limited solubility makes i t scarce in most natural aquatic systems. Control of nutrient enrichment in water bodies requires a nutrient budget in order to determine important sources and determine the best strategy for nutrient removal. In some instances, the contribution from non-point sources, such as urban runoff or agricultural lands, may be so great that the benefits from nutrient removal may become insignificant. In numerous cases, however, removal of nutrients from point sources such as domestic or industrial wastewaters have proven beneficial (WPCF, 1983). The major sources of phosphorus in domestic wastewater are human waste, synthetic laundry detergents and water treatment chemicals (used to protect the water distribution system against corrosion) (U.S. EPA, 1976). In municipal plants, removal of phosphorus from wastewater i s most widely done by chemical precipitation. Another method that does not require chemical addition and thus, does not result in increased volumes of sludge to dispose of, is based on biological excess phosphorus removal. In this method, phosphate uptake in excess of the normal metabolic requirements i s stimulated by modifying an activated sludge process. The basic modification to an activated sludge plant consists of adding a non-aerated zone upstream of the normal aerated zone. A high soluble reactive phosphate concentration (SRP) i n the non-aerated zone i s observed to be associated with a very low SRP concentration In the aerated zone; less than 0.5 mg P0^-P/£ can be achieved. Although good phosphorus removal by biological excess removal processes 3. i s pos s i b l e , i r r e g u l a r performance at treatment plants st imulated research i n the under ly ing mechanisms. Much e f f o r t was spent i n "black box" studies aiming at the opt imizat ion of the process by changing a parameter and the observat ion of i t s e f f e c t s . With the recogn i t ion of the m i c rob io log i ca l nature of the mechanism, pre l iminary models exp la in ing the biochemical mechanisms were proposed. It was the purpose of th i s research to support and improve such b i o -chemical models. Batch tests were used to provide complementary observa-t i o n s . Concerning the improvement of b iochemical models, the consistency between the proposed models and known p r i n c i p l e s of biochemistry and b ioenerget i cs was p a r t i c u l a r l y emphasized. In th is repor t , a l i t e r a t u r e review on b i o l o g i c a l phosphorus removal leads to the postulated biochemical models. The methodology chapter spec i f i e s the batch tes t ing approach, the cha r a c t e r i s t i c s of the UBC p i l o t p lant s ludge, and the a n a l y t i c a l techniques fol lowed for parameters determinat ion. Resul ts and d i scuss ion are deal t with i n one chapter because of the many subsections invo l ved . The consistency of the model with observat ions, and app l i ca t i ons suggested by the model are given at the end of th i s chapter. F i n a l l y , conclusions and recommendations for future research are proposed. In the Appendices, a l i s t of the symbols used throughout the t ex t , a g lossary , and elements of the g l y c o l y s i s pathway, of the TCA cyc l e , of b a c t e r i a l b ioene rge t i c s , and of c e l l u l a r t ransport are presented. These Appendices are re fer red to throughout the t he s i s . 4. 2. LITERATURE REVIEW 2.1. Introduction Phosphorus Is an essential element for microorganisms because of i t s use in energy transfer and for c e l l components such as the c e l l membranes and the genetic material. In conventional activated sludge treatment receiving primary effluent (see Fig. 2.1), between 1.0 and 1.5 mg/i of total phosphorus (TP) is removed per 200 mg/£ of chemical oxygen demand (COD) removed. This translates Into a reduction in TP of 20 to 30 percent across the plant for a typical raw sewage of 8 mg TP/Jfc (Metcalf and Eddy, 1979). Therefore, this normal metabolic requirement for phosphorus is inadequate to provide an effluent level below 1.0 mg TP/A. With the new approach of biological excess phosphorus removal (bio-P removal), effluent TP levels lower than 0.5 mg P/£. can be achieved. In this literature review, the historical development of the modifications of activated sludge processes to remove nitrogen and/or phosphorus, are f i r s t presented. Then, the requirements to achieve excess phosphorus removal are reviewed. The metabolism of two important reserve materials, namely polyphosphate and poly-3-hydroxybutyrate, are presented thereafter. Since some species of bacteria were identified to be responsible for bio-P removal, another section deals with this aspect. Finally, biochemical models presented in the literature are reviewed. 2.2. Process Schemes for Biological Excess Phosphorus Removal The development of bio-P removal processes is closely related to biological nitrogen removal by nitrification-denitrification. A historical description of these processes w i l l be helpful to understand the role of each reactor and i t s interactions with the other reactors of the process. 5. SLUDGE WASTAGE RAW SEWAGE WASTAGE J (SLUDGE WASTAGE) F i g . 2.1 Conventional a c t i v a t e d sludge process (adapted from Metcalf and Eddy, 1979.) 6. 2.2.1. Biological Nitrogen Removal Processes The purpose of this section is to show how the development of nitrogen removal processes by nitrification-denitrification eventually led to biological excess phosphorus removal. For a more detailed coverage of nitrogen removal, refer to WPCF (1983) or U.S. EPA (1975). Major aspects of nitrogen metabolism in wastewater treatment are deamination, n i t r i f i c a t i o n and denitrification. The reactions and the conditions under which each reaction occurs w i l l be reviewed before discussing activated sludge processes aiming at nitrogen removal. (a) Deamination Ammonia can be produced from organically-bound nitrogen (Painter, 1970): (1) from extra-cellular organic nitrogen-containing compounds by chemical or biochemical degradation (e.g.: from amino acids, urea); (2) from living bacterial c e l l during endogenous respiration; and (3) from dead and lyzed c e l l s . A general equation for deamination can be written as follows: organic nitrogen (+02) + H20 > fatty acid (+C02) + ammonia (2.1) (amino acids, urea) Under aerobic conditions, carbon dioxide is produced by bacterial deamination. Under anaerobic conditions, saturated or unsaturated fatty acids are produced depending on i f a reduction process happened or not (Sawyer and McCarty, 1978). 7. (b) N i t r i f i c a t i o n N i t r i f i c a t i o n i s the name given to the oxidation of ammonia to n i t r i t e then nitrate by autotrophic organisms. These organisms derive their energy from these oxidations and not from the oxidation of organic compounds (Painter, 1970). Autotrophs such as Nitrosomonas form n i t r i t e from ammonia: NH* + 3/2 0 2 » 2H+ + H20 + N0~ (2.2) Autotrophs such as Nitrobacter form nitrate from n i t r i t e : N0~ + 1/2 0 2 » N0~ (2.3) Oxygen is essential for these reactions to occur. With H + production in equation (2.2), the pH and the alkalinity of the solution w i l l both tend to decrease. (c) Nitrogen Removal by Denitrification Nitrogen removal in wastewater treatment is achieved by sludge wastage and denitrification. Denitrification is a process by which nitrate or n i t r i t e is transformed into nitrogen gas (nitrous oxide gas can also be produced). A large number of bacteria and fungi can u t i l i z e nitrate to produce energy by respiration. The reaction of nitrate reduction to nitrogen gas can be represented as: 8. N0~ + 6 H + + e~ > 1/2 N 2 + 3 H20 (2.4) Denitrification is inhibited by the presence of free oxygen. Indeed, even with active nitrate-reducing enzymes, cells w i l l readily use oxygen instead of nitrate as the terminal electron acceptor. In addition, the formation of nitrate-reducing enzymes Is inhibited by free oxygen (Painter, 1970). (d) Nitrogen Removal Processes by Activated Sludge Numerous process configurations have been proposed for nitrogen removal by nitrlfication-denitrification of municipal sewage by activated sludge. A short review of these processes Is given here to show how process configura-tions for biological phosphorus removal were eventually developed. Ludzack and Ettinger (1962) were f i r s t to propose a process configura-tion u t i l i z i n g the biodegradable material In the influent as the main energy source for denitrification (see Fig. 2.2). In this process the non-aerated reactor 1 is in partial communication with the aerated reactor and the return sludge from the settling tank Is discharged to the aerobic reactor. N i t r i f i -cation takes place in the aerobic reactor and n i t r i f i e d mixed liquor is recycled to the non-aerated reactor in an Indeterminate way by the mixing action of the two reactors. As a consequence, the denitrification perform-ance of the process is variable. Wuhrmann (1964) was f i r s t to propose a self-generated energy source for denitrification in a nitrification-denitrification process (see Fig. 2.3). 1 The non-aerated reactor is sometimes called "anoxic" or anaerobic in the literature. Refer to the Glossary (App. B) for a definition of the terminology used in this thesis. 9. Aneiie Aerobic Aerobic Anemic 2.2 Ludzack and Ettinger process Fig. 2.3 Wuhrman process for for biological nitrogen removal biological nitrogen (from Van Haandel et a l . , 1981). removal (from Van Haandel et a l . , 1981). . 2.4 Modified Ludzack and Fig- 2.5 Phostrip process for biological Ettinger (MLE) process phosphorus removal (from WPCF, for biological nitrogen 1983). removal (from Van Haandel, 1981). Primary onoiie (vector Aerobic reactor Secondory onoiie reactor MiMd liquor recycle Reoerotion raoctor White flow Sartlar Primary Aarebic Secondary onoiie raoctor onoiie raoctor raoctor Miied liquor recycle 5f?*£8,'on —3 raoctor Wore flow 1 Seiner Effluent 2.6 Bardenpho process for biological Fig. 2.7 Phoredox process for nitrogen and phosphorus removal biological nitrogen (from Van Haandel, 1981). and phosphorus removal (from Van Haandel, 1981). 10. The f i r s t reactor, which is aerobic, receives the influent flow and sludge return flow from the settling tank. Ni t r i f i c a t i o n of the influent total nitrogen occurs in this aerobic reactor. The contents of the aerobic reactor discharge to the post-denitrification reactor where denitrification takes place. The rate of energy release due to organism death and lysis (self-generated energy source) is low, resulting in a low rate of denitrification. Hence a high degree of denitrification requires a large non-aerated volume fraction, but experience has shown that this may affect the n i t r i f i c a t i o n efficiency of the process since the n i t r i f i e r s being obligate aerobes can only multiply in the aerobic zone of the process (van Haandel et a l . , 1981). Barnard (1975) proposed (1) a complete separation of the non-aerated reactor (also called the pre-denitrification reactor), (2) the introduction of a controlled recycle from the aerobic to the non-aerated reactor, and (3) discharge of the sludge return to the non-aerated reactor (see Fig. 2.4). This process was called the Modified Ludzack-Ettinger (MLE) process. Compared with the original process, the modified one gave improved and more consistent performance. The MLE process cannot reduce the effluent nitrate concentration to near zero because the net concentration of nitrate established in the aerobic reactor is the source for both the recycle and the effluent flows. 2.2.2. Biological Phosphorus (and Nitrogen) Removal The Phostrip process developed by Levin and Shapiro (1965) was the f i r s t biological excess phosphorus removal system to be proposed (Fig. 2.5). It takes advantage of phosphate release under anaerobic conditions In the phosphate stripper, to obtain a relatively small stream (5 to 25% of the influent flow) rich in phosphate. Lime treatment is then applied for 11. phosphate precipitation and ultimate disposal via the lime sludge. This process can be modified to include nitrogen removal by denitrification (WPCF, 1983). Working on nitrogen removal to obtain higher denitrification efficiencies and thus less nitrates in the effluent, Barnard (1975) combined the MLE and Wuhrmann processes. A flash aeration reactor was added before the fi n a l settling tank to strip nitrogen gas bubbles from the floes and to n i t r i f y ammonia released in the post-denitrification reactor. The process was called Bardenpho (for Barnard-Denitrification Phosphorus removal) (see Fig. 2.6). Barnard also observed phosphate release in the post-denitrification zone followed by excess phosphate uptake in the reaeration zone. This observation led him to say that excess biological phosphorus removal is induced i f , at some point In the process configuration, the organism mass is stressed by subjecting i t to an anaerobic state such that phosphate is released by the sludge mass to the bulk liquid. To obtain this condition efficiently, an anaerobic reactor was included ahead of the pre-denitrification reactor to receive the influent and the underflow recycle. This process is known as the Phoredox Process (see Fig. 2.7). A number of research workers investigated Barnard's proposed process and were successful in obtaining phosphorus removal. They were also successful In obtaining phosphorus removal with the Modified Phoredox process (see Fig. 2.8) which is a Phoredox process without the post-denitrification and reaeration reactors. These two reactors were l e f t out since the denitrification in the post denitrification reactor per unit volume was relatively inefficient compared to that achieved in the pre-denitrification reactor. It was also observed regarding the Modified Phoredox process that: 12. F i g . 2.8 Modified Phoredox process f o r b i o l o g i c a l n i t r o g e n and phosphorus removal (from S i e b r i t z et a l . , 1983a). ARAEROSIC ANOXIC ACROIIC REACTOR REACTOR M A C TOR RECYCLE MIXED HOUOR RECYCLE" -3ITE FLOW ' 1ETTL IR ^ E F T L U C R T ANOXIC KACTOR REACTORS MIXCC LIQUOR RECYCLES WASTE PLOW SETTLER Emuctr r F i g . 2.9 UCT process f o r b i o l o g i c a l F i 8 -nit r o g e n and phosphorus removal (from S i e b r i t z et a l . , 1983a). 2.10 Modified UCT process f o r b i o l o g i c a l n i t r o g e n and phosphorus removal (from S i e b r i t z et a l . , 1983a). •(FLUENT F i g . 2.11 A/0 process f o r b i o l o g i c a l phosphorus removal (from WPCF, 1983). 13. (1) the anaerobic reactor was necessary for excess phosphorus removal; (2) nitrate in the sludge recycle adversely affected phosphorus removal; and (3) increasing the volume of the anaerobic reactor increased the excess phosphorus removal capacity. Since 1970, Marais with many researchers at the University of Cape Town contributed to the development of the activated sludge process and particularly to the characterization of the denitrification phenomenon with the Bardenpho and Phoredox processes. They found, in particular, that: (1) for any selected sludge age and minimum temperature, the requirement for efficient n i t r i f i c a t i o n imposes an upper limit on the non-aerated sludge mass fraction (the aerated sludge mass fraction includes the n i t r i f i e r s ) ; and that, (2) the limitation on the non-aerated mass fraction correspondingly limits the concentration of nitrate that can be removed. If the nitrate concentration generated is higher than the denitrification capacity achievable, nitrate w i l l appear in the effluent and, in the Phoredox process, the phosphorus removal w i l l be adversely affected because the return sludge to the anaerobic reactor w i l l carry nitrates (Siebritz et a l . , 1983a). From these findings on the Phoredox process, i t was clear that among other factors that may affect phosphate uptake, a major one was the adverse effect of the presence of nitrates in the sludge recycle discharged directly to the anaerobic reactor. The University of Cape Town (UCT) (see Fig. 2.9) process was then developed to make the anaerobic reactor independent of the effluent nitrate concentration. In the UCT process, the settling tank underflow recycle (s) as well as the mixed liquor recycle (a) are discharged to a denitrification reactor and an additional mixed liquor recycle (r) from the denitrification to the anaerobic reactor i s introduced. The nitrate recycled to the denitrification reactor can be controlled by appropriately 14. adjusting the mixed liquor recycle (a) such that the nitrate concentration i n the outflow of the denitrification reactor remains approximately zero. Thus, optimal anaerobic conditions are created in the anaerobic reactor. From operation with this process, a different type of problem arose that was related to high TKN/COD ra t i o s 2 of the influent wastewater and the limit that should be imposed on the non-aerated sludge mass fraction. In the UCT process, as the influent TKN/COD ratio increases, the (a) recycle ratio needs to be reduced to avoid nitrate discharge to the anaerobic reactor. A reduc-tion in the (a) recycle ratio causes an increase in the actual denitrifica-tion retention time. In some cases, this denitrification retention time may exceed 1 hour. On the other hand, to preserve good settleability in the c l a r i f i e r , an upper limit of 1 hour must be Imposed on the actual d e n i t r i f i -cation retention time. For high TKN/COD sewage between 0.10 to 0.11, the (a) recycle should then be low while a good settleability requires the recycle to be high. To address this problem, another process was proposed, the Modified UCT process (Fig. 2.10), in which the denitrification zone is subdivised into two parts. In the Modified UCT process, the f i r s t denitrification reactor receives the underflow recycle (s), and the denitrified recycle (r) is taken from i t . The second denitrification reactor receives the aerobic recycle (a). This process allows the operation of the recycle ratios to result in an actual 2 Influent TKN/COD ratio: (Total Kjeldahl Nitrogen/Chemical Oxygen Demand ratio). This ratio characterizes the influent sewage and quantifies the denitrification capacity of the process. Usually, the TKN/COD ratio of raw sewage is in the range of 0.06 to 0.08 and for settled sewage 0.09 to 0.11 mg TKN-N/mg COD. For example, a TKN/COD ratio of 0.08 appears to be the upper limit for the Phoredox process while i t is 0.14 for the UCT process for s t i l l being able of complete denitrification of the incoming sewage (Siebritz et a l . , 1983a). 15. denitrification retention time of less than 1 hour while ensuring a nitrate free discharge to the anaerobic reactor. An advantage of this process over the UCT process is that i t does not require the need of a careful control of the aerobic recycle (a) ratio to ensure a nitrate free discharge to the anaerobic reactor. Excellent settleability has also been observed. The maximum TKN/COD ratio allowable, however, is lowered to 0.11 mg TKN-N/mg COD (Siebritz, et a l . , 1983a). The Anaerobic/Oxic (A/0) process has a similar configuration to the Ludzack and Ettinger process except that i t has separate compartments for each zone (see Fig. 2.11). It takes advantage of a short aerobic retention time to prevent n i t r i f i c a t i o n and avoid problems with nitrate recirculation to the anaerobic zone. It is thus a higher rate activated sludge process than the other systems that have been discussed. As a result, a larger amount of sludge must be disposed of than with the other processes presented. This process can include nitrogen removal by the addition of an aerobic sludge recycle (WPCF, 1983). The resulting process would then have a similar configuration to the Modified Phoredox process (Fig. 2.8). 2.3. Requirements for Excess Phosphorus Removal Excess phosphorus removal by the sludge mass requires a high degree of aerobic phosphate uptake preceded by anaerobic phosphate release. The conditions stimulating these phenomena are discussed in this section. 2.3.1. Phosphate Uptake The f i r s t reports about excess phosphorus removal by activated sludge plants were written around 1960 (Marais et a l . , 1983). Phosphorus uptake in excess of the metabolic requirements were associated with sufficiently long 16. aeration time and high enough rates of aeration. Levin and Shapiro (1965) hypothesized that such a phenomenon was biologically mediated. Observing polyphosphate granules, they deducted that excess phosphorus taken up was stored in such reserves. Since the formation of polyphosphate granules requires energy, they also established that the presence of carbonaceous substrates stimulated phosphate uptake and storage. Inhibition of energy production by addition of 2,4-dinitrophenol (DNP) under aerobic conditions showed that the phenomenon was truly biological. Later investigators also verified the effect of DNP, and the results obtained by Rensink et a l . (1981) are shown in Fig. 2.12. Some phosphate removal from solution can be attributed to chemical precipitation by adsorption or precipitation due to biologically mediated chemical changes. However, i t is now evident that the phenomenon of excess phosphorus removal is mostly of a biochemical nature (Marais et a l . , 1983). Shapiro (1967) observed that the prerequisite for excess aerobic phos-phate uptake was anaerobic phosphate release. Such release was possible in the absence of oxygen and at low redox potentials of around -150 mV (E^) 3. It was also observed that the released phosphate was not organically bound to the RNA or DNA of organisms but rather taken from polyphosphate reserves. Wells (1969) showed the reversibility of phosphate uptake and release (see Fig. 2.13). From this last figure, i t appears that the capacity for the activated sludge to take up phosphate decreases with time and that i t is 3 Such a low redox potential Indicates that not only free oxygen (0 2) but also nitrate (NO3) are absent. Thus, these electron acceptors are not available to microorganisms for energy production (see Appendix D). TINE (hours) 2.12 E f f e c t of 2,4-dinitrophenol a d d i t i o n on phosphate uptake under aerobic c o n d i t i o n s . A batch of sludge was kept anaerobic i n i t i a l l y . (from Rensink et a l . , 1981). 2.13 E f f e c t of a l t e r n a t i v e aerated and unaerated periods i n a batch t e s t on phosphate r e l e a s e and uptake (from W e l l s , 1969 taken from Marais et a l . , 1983). 18. proportional to the active mass of microorganisms present (Marais et a l . , 1 9 8 3 ) . Achievement of excess phosphorus removal can be indicated by the ratio of phosphorus removed to COD removed (AP/ACOD) in a treatment plant. Ratios significantly greater than 0 . 0 1 0 are reported by Davelaar et a l . ( 1 9 7 8 ) to be characteristics of bio-P removal. Raper ( 1 9 8 2 ) reviews that ratios of 0 . 0 2 for full-scale plants and up to 0 . 0 4 5 for lab scale plants have been reported. The phosphorus content of the sludge also provides a parameter to indicate excess phosphorus removal. On a dry weight basis, the phosphorus content of conventional activated sludge i s 1 . 5 to 2.0% (WPCF, 1983 and Bundgaard et a l . , 1 9 8 3 ) . From the approximate formula for the organic fraction of activated sludge, C ^ H ^ I M ^ P Q Q 8 3 * t n e Phosphorus content can be calculated to be 2 . 3 percent (Metcalf and Eddy, 1 9 7 9 ) . However, higher percentages based on suspended solids are reported for sludge removing excess phosphorus biologically in full-scale plants: 6 to 7 percent (Rensink et a l . , 1 9 8 1 ) , and 6 . 5 percent (Oldham and Koch, 1 9 8 2 ) . In a lab-scale unit with acetate-fed sludge, Fukase et a l . ( 1 9 8 2 ) reported a maximum of 8 percent phosphorus content of sludge. Of this high phosphorus content, 75 percent was released anaerobically. The phosphorus content of the sludge after phosphate release was calculated to be In the range 1 . 4 to 3 . 6 percent. 2 . 3 . 2 . Phosphate Release Anaerobic phosphate release is caused by the presence of suitable carbon substrates. Fuhs and Chen ( 1 9 7 5 ) observed that addition of acetic acid or carbon dioxide to a culture of bacteria capable of bio-P removal and kept anaerobically, caused rapid phosphate release (see Fig. 2 . 1 4 ) . 19. 40 35 30 c » « flB I ,0 2 a M O c a. 15 10 -5 -.A C0 2 i i O Acetic acid 1 i 1 i If No treatment Days in anaerobic state F i g . 2.14 E f f e c t of CO2 bubbling and a c e t i c a c i d a d d i t i o n on phosphate r e l e a s e under anaerobic c o n d i t i o n s (from Fuhs and Chen, 1975). I o CD CL or to 120 100-80 60-40-20-Time (h) 500 mg/l as Acetic Acid 200 mg/l 100 mg/l 0 mg/l 1^  3 1 4 F i g . 2.15 E f f e c t of v a r i o u s l e v e l s of acetate a d d i t i o n on phosphate r e l e a s e . A batch of sludge taken from the aerobic zone of an acetate-fed l a b - s c a l e p l a n t was kept a n a e r o b i c a l l y f o r the t e s t , (adapted from Fukase et a l . , 1982). 20. During other experimentations, they also observed the presence of poly-8-hydroxybutyrate (PHB), a carbon reserve, in some bacteria. Studies conducted at the University of Capetown (Dold et a l . , 1980) showed the importance of the readily biodegradable fraction of the influent COD4 for denitrication. This concept was extended by Siebritz et a l . (1983a) who showed the requirement of readily biodegradable COD in the anaerobic reactor to obtain phosphate release and excess phosphorus removal. Fukase et a l . (1982), experimenting with an acetate-fed lab-scale unit for bio-P removal, observed that acetate addition to a batch of sludge kept anaerobically, caused phosphate release (see Fig. 2.15). The i n i t i a l rate of release was independent of the amount of acetate added. However, the magnitude of the release appeared to be a direct function of the amount added. Very l i t t l e subsequent release was observed once a maximum magnitude of phosphate release was attained. Expressed as a molar ratio, the phosphate released to acetate added was (1/1.1):1.0. At UBC, Rabinowitz et a l . (1982) obtained results from batch tests with various levels of acetate addition that were similar to those obtained by Fukase and coworkers. In Figures 2.16a and 2.16b, SRP and nitrate concentra-tions are shown. Phosphate release was observed In presence of nitrate, provided sufficient acetate was added. Denitrification activity consumed a l i t t l e less than 40 mg/l of acetate as COD before a net phosphate release was observed. The rate of denitrification increased with the amount of acetate added, up to a maximum level which corresponded to an addition of about 80 mg/l of acetate as COD. .** The counterpart of the readily biodegradable COD fraction, the slowly biodegradable fraction, includes complex substrates or particulates that need to be hydrolyzed before being metabolized. 21. if) if) > I o Q_ CL HI If) 25-20-Legend A 0 fna/L as COD X 3D mfl/L 01 COD O 4D m i / l BI COD • . 80 m a / l " » COD * »0 m | / L o» COD « IPC m i / L a« COD (a) Phosphate r e l e a s e Time (h) 10 in if) > CT) O CD "o •—V-'mn. S - r . p — r Legend 20 mg/L as COD 40 mg/L • s COD 60 mg/L OS COD 80 mg/L as COD 100 mg/L as COD (b) D e n i t r i f i c a t i o n Time (h) F i g . 2.16 E f f e c t of v a r i o u s l e v e l s of acetate a d d i t i o n on phosphate r e l e a s e and d e n i t r i f i c a t i o n . A batch of sludge taken from the aerobic zone of a p i l o t p l a n t was kept anaerobic-a l l y f o r the t e s t (from Rabinowitz et a l . , 1982). 22. The presence of nitrate in the anaerobic zone, coming from either the influent sewage (usually low) or the return sludge Is unfavorable to the efficiency of bio-P removal. In fact, the various process configurations for bio-P plants have gradually developed towards minimizing the amount of nitrate entering the anaerobic zone by provision of a denitrifying zone for the activated sludge recycle and/or the return sludge, as was discussed previously (section 2.3). The effect of various levels of acetate addition on phosphate release was tested by Siebritz et a l . (1983b) on denitrified sludge (see Fig. 2.17). From their results a molar ratio of phosphate released to acetate added of 0.5:1.0 can be calculated. The effect on phosphate release of adding various types of substrates was studied by Potgieter and Evans (1983; see Table 2.1). Equivalent amounts of substrate as COD were fed to non-aerated reactors containing sludge sampled from the aerobic zone of a lab-scale bio-P plant. - Acetate and propionate caused the highest levels of phosphate release. Formate caused about half the level achieved with acetate and f i n a l l y , butyrate, hydroxy-butyrate and glucose caused about 10 times less phosphate release than acetate. Oldham and Koch (1982, see Fig. 2.18) in a similar experiment with sludge from the full-scale plant of Kelowna, B.C., observed the highest degree of phosphate release with acetate and propionate, about 40 percent of this highest level with glucose or acetic acid, and about 20 percent with iso-butyric acid. The effect of pH adjustment on phosphate release from sludge sampled from an aerobic reactor was reported by Potgieter and Evans (1983, see Table 2.2). Maximum phosphate release was observed at pH 4. Phosphate was even taken up from solution at pH values of 8 or 9. At pH values of 3 and 2, bacterial floe disintegration was observed. 23. A C E T A T E ADDED (mgArr / l ) F i g . 2.17 E f f e c t of acetate a d d i t i o n on phosphate r e l e a s e . A batch of sludge taken from the aerobic zone of a l a b - s c a l e plant was d i l u t e d w i t h i t s e f f l u e n t to approx. 1000 mg VSS/1, and kept a n a e r o b i c a l l y f o r the t e s t (from S i e b r i t z et a l . , 1983b). a a o o 2 0 -Aeelote oddition to Control II I I SUBSTRATE ADDITION Doto : August 11/82 Sludge KELOWNA LEGEND • • 7 + x CONTROL I CONTROL II ( A c t i o n oddition) SODIUM ACETATE G L U C O S E PROPIONIC AC I 0 ISO-BUTYRIC ACID ACETIC ACID _L I 2 3 4 5 6 Time ( hours ) F i g . 2.18 E f f e c t of v a r i o u s types of substrates on phosphate r e l e a s e (from Koch and Oldham, 1982). TABLE 2.1 EFFECT OF VARIOUS TYPES OF SUBSTRATE ON PHOSPHATE RELEASE (From Potgieter and Evans, 1983) Substrate Phosphate Release (110 mg/£ as COD) (mg/£) Formate 28.4 Acetate 58.6 Propionate 54.5 Butyrate 8.2 Hydroxybutyrate 5.9 Glucose 5.0 Ribose 0.0 Glycerol 0.0 EDTA 0.0 TABLE 2.2 EFFECT OF pH ON PHOSPHATE RELEASE (From Potgieter and Evans, 1983) pH Phosphate release 3 (mg/O 2 6.0 3 18.3 A 20.6 5 11.0 6 9.8 7 (control) 5.9 8 -1.6 9 -5.0 a Negative values Indicate phosphate uptake. 25. 2.4. Bacterial Storage for Bio-P Removal As seen in the previous section discussing the requirements for bio-P removal, two bacterial reserve materials appear to play a central role, namely polyphosphate and PHB. The metabolism of these compounds is reviewed here. 2.4.1. Polyphosphate Metabolism Polyphosphate reserves in bacteria mainly occur as linear chain varying from only a few units to thousands of units long (see Fig. 2.19). Poly-phosphate forms granules which also contain RNA, proteins, lipi d s and magnesium (Harold, 1966). Such granules can be identified with a light microscope by their property of staining basic dyes metachromatically (with a change of color of the dye; for example, methylene blue turns red-violet). Polyphosphates are referred in the literature as volutin, metachromatic granules or Babes-Ernst granules. The variability of the polyphosphate content of bacteria was observed by Harold (1966) to be one of the most striking aspects of polyphosphate metabolism. In phosphate-starved c e l l s , polyphosphate cannot usually be detected; at the other extreme, i t may accumulate to the point where poly-phosphate is the most abundant cellular phosphorus compound, up to 20 percent of i t s dry weight as polyphosphate. Polyphosphates appear to be only synthesized by the transfer of one phosphate from ATP by the enzyme polyphosphate kinase. The degradation of the polymer can be effected by a reversal of the above reaction depending on the ATP/ADP ratio apparently, but in most cases, i t is accomplished by hydrolysis of the terminal phosphate by action of the enzyme polyphosphatase. Fig. 2.19 Chemical structure of polyphosphate (from Gaudy and Gaudy, 1980). PISTIUS MEMtBANE Fig. 2.20 Polyphosphate metabolic pathways (adapted from Harold, 1966). Fig. 2.21 Patterns of polyphosphate accumulation in Aerobacter aerogenes. (a) Nutrient deprivation: cells were placed in a medium devoid of sulfur at 0 h. (b) "Overplus": cells were placed in medium devoid of phosphate at 0 h; phosphate was restored at A h (adapted from Harold, 1966). 27. These pathways are depicted in Fig. 2.20. Other pathways for the degradation of polyphosphate involve the phos-phorylation of AMP, glucose or fructose. The required enzymes, however, may not be present in a l l bacteria (Harold, 1966). Harold (1966) reports that the accumulation of polyphosphate occurs mainly by two main mechanisms, namely "luxury uptake" and "overplus uptake" under aerobic conditions. "Luxury uptake" occurs when an essential element other than phosphorus is limited, but when sufficient energy is available to transfer and store phosphate into the c e l l . Sulfate starvation is particularly effective in enhancing polyphosphate accumulation by this mechanism (Fig. 2.21a). This phenomenon can be explained by the inhibition of nucleic acid synthesis due to sulfate starvation which causes growth and c e l l division to cease. Other essential elements can have the same effect. Thence, ATP is available for polyphosphate storage. "Overplus uptake" occurs when certain bacteria are temporarily deprived of adequate supplies of phosphate and then re-exposed to an abundance of this element. This reaction i s shown in Fig. 2.21b. The major polyphosphate accumulation can be correlated with the abund-ance of the enzyme polyphosphate kinase as a result of phosphate starvation (Harold, 1966). Three major physiological functions of polyphosphate are suggested: namely a phosphate reserve, an energy source, and a metabolic regulator. It is well established that polyphosphate can supply phosphate for the biosynthesis of nucleic acids and phospholipids (a membrane component). The properties of the polymer are clearly in keeping with a reserve role, since the osmotic equilibrium of the c e l l w i l l undergo much less change than i f 28. Inorganic phosphate were accumulated without polymerization. As the phosphate content of many natural environments is low due to the insolubility of calcium phosphate, the existence of a phosphate reserve material i n microorganisms and the synthesis of the enzymes responsible for its accumula-tion, appears largely reasonable (Harold, 1966). The role of polyphosphate as an energy reserve is supported by a few observations (Kulaev, 1979). F i r s t l y , the high energy of the terminal phosphate bond of the polymer is comparable to the terminal phosphate bond of ATP. Secondly, the reversibility of the polyphosphate kinase enzyme in vitro may also be observed in vivo. Thirdly, the conservation of the phosphate group transfer potential of polyphosphate has been demonstrated for the phosphorylation of glucose in some bacteria. But, many observations conflict with this energy role for polyphosphate. Fi r s t , polyphosphate was observed to not be used under conditions of limited or blocked energy production. Second, the rapid rate of uti l i z a t i o n of poly-phosphate Indicates an absence of regeneration of the polymer, as would occur for ATP for example. Finally, in Aerobacter aerogenes polyphosphate was shown to be hydrolyzed by the enzyme polyphosphatase, losing i t s phosphate bond energy. Thus, i t appears that polyphosphate does not play the same role as ATP in energy transfer. Nevertheless, polyphosphate could play a role of phosphate bond transfer to a sugar or some other compounds. The role of polyphosphate in regulating the cellular level of Inorganic phosphate, ATP, ADP or other high-energy phosphate compounds have been proposed. For instance, to maximize the "phosphate transfer potential" of ATP, the concentration of inorganic phosphate, ADP and H"1" should be kept low. Polyphosphate can serve as a storage compound for this function of a metabolic regulator (Harold, 1966). 29. 2.4.2. PHB Metabolism Poly-B-hydroxybutyrate (PHB) is a polymer of D(-)-B-hydroxybutyrate. Deposits of PHB are readily visible with the light microscope, occurring as dark granules of variable size scattered through the c e l l . Sudan black stains specifically these granules. The major pathways of synthesis and degradation is shown in Fig. 2.22. Other pathways of secondary importance are reviewed by Dawes and Senior (1973). The synthesis of PHB involves the condensation of two acetyl CoA into acetoacetyl CoA which is reduced to 8-hydroxybutyryl CoA with NADH. The 8-hydroxybutyryl CoA is then added to the polymer. Degradation of PHB occurs by hydrolysis into hydroxybutyrate, which is then oxidized by NAD+ to produce acetoacetate. A CoA group addition then forms acetoacetyl CoA which can be degraded to acetyl CoA. The synthesis of PHB is unique among energy storage compounds In not requiring the direct participation of ATP. Reducing power in the form of NADH i s essential, however, and PHB formation may be regarded as a quasi-fermentation process permitting the reoxidation of NADH into NAD+. Such a process is particularly useful under conditions of oxygen limitation, which prevent the re-oxidation of NADH by the electron transport chain 5, or under conditions of nitrogen limitation, which result in intracellular accumulation of NADH, since ATP is not utilized for protein synthesis. Therefore, PHB reserves w i l l accumulate when cells are limited in oxygen or in nitrogen but s t i l l have a carbon source available (Dawes and Senior, 1973). PHB can be seen as the procaryotic reserve material equivalent to fat in See Appendix D. 30. CH,—C^S—CoA acetyl-CoA F i g . 2.22 Poly-g-hydroxybutyrate metabolic pathways (from S t a n i e r et a l . , 1970). 31. higher organisms (Stanier et a l . , 1970). The assimilated carbon stored in cells may accumulate until i t represents as much as 50 percent of the cellular dry weight (Dawes and Senior, 1973). Essentially, PHB synthesis represents a device of accumulating carbon in a form that is "osmotically inert". Indeed, the free carboxyl group of the acid precursors is eliminated through the formation of an ester bond between the subunits of the polymer. PHB degradation w i l l occur when the internal concentration of NAD+ and of CoA w i l l increase while the concentration of acetyl CoA is low. For example, PHB w i l l be degraded in presence of oxygen when the external carbon sources are limited. However, i f both oxygen and a carbon source are present, PHB w i l l not be degraded (Dawes and Senior, 1973). The role of PHB is more one of an energy source than of a carbon source for carbon skeletons when starvation conditions occur (Dawes and Senior, 1973). 2.5. Bacteria Involved in Bio-P Removal In activated sludge, the predominant microorganisms are determined by the characteristics of the influent wastewater, the environmental conditions such as temperature or pH, the process design and the mode of process operation (WPCF, 1977). Osborn and Nicholls (1978) published a l i s t of bacteria capable of accumulating polyphosphate (see Table 2.3). Fuhs and Chen (1975) isolated many groups of bacteria from bio-P treatment plants. They found that members of the Aclnetobacter genus could be responsible for bio-P removal. Isolated bacteria were shown to be obligate aerobes growing on acetate, ethanol and many other substrates but not on glucose, lactose, lactate, formate or propionic acid, in particular. 32. TABLE 2.3 LIST OF POLYPHOSPHATE-ACCUMULATING BACTERIA (From Osborn and Nicholls, 1978) Acetobacter suboxydans Aerobacter aerogenes Azotobacter ag i l i s A. vinelandii Bacillus s u b t i l l i s Bacterium aerogenes B. cloacae B. Friedlanderi Caulobacter vibroides Chlorobium thiosulphatophilum Chromatium Clostridium spec. Corynebacterium diptheria C. xerose Escherichia c o l i Hydrogenomonas spec. Mycobacterium avium M. chalonei M. Phlei M. smagmatis M. thamnaphaeos M. Tuberculosis Rhodopseudomonas palustris Rhodospirillium rubrium Serratia marcascens Thiobacillus thioxydans Nitrobacter Micrococcus denitrifians Staphylococcus aureus Chlamydomoda Mucor racemosus Claviceps purpurea Acinetobacter Zoogloea Ramigera Nitrosococcus Beggiatoa 33. These bacteria could accumulate polyphosphate and PHB. Buchan (1983) investigated the location and nature of polyphosphate storage in various activated sludges from full-scale bio-P plants in South Africa. The predominant organism in the sludges was of the Acinetobacter genus. He supported the idea that these bacteria were responsible for bio-P removal. Brodisch and Joyner (1983) determined the composition of the microflora in one pilot scale and two lab-scale bio-P plants. They found that Acinetobacter species were present only in minor proportions (less than 10 percent), but Aeromonas and Pseudomonas species were dominant (up to 50 percent of the population). They concluded that species other than Acinetobacter were probably also Involved in bio-P removal. Many authors have documented the presence of PHB in sludge involved in bio-P removal: Fuhs and Chen (1975), Nicholls and Osborn (1979), Fukase et a l . (1982) and Buchan (1983). Nicholls and Osborn (1979) did not find glycogen stored in bacteria of two full-scale bio-P plants. Fukase et a l . (1982), however, observed the presence of glycogen instead of the presence of PHB in a lab-scale glucose-fed bio-P plant. From the above observations, i t seems likely that the presence of poly-phosphate and of a carbon reserve such as PHB or glycogen, are the basic requirements for bio-P removal. Since in most plants, large amounts of sugars are seldom found in the influent, bacteria storing glycogen and poly-phosphate w i l l probably not proliferate as much as bacteria storing PHB and polyphosphate. In addition, i t appears that Acinetobacter may not be the only bacteria involved i n bio-P removal. For these reasons, It was decided that, in this thesis, the bacteria responsible for bio P removal should be 34. called by the generic name "bio-P bacteria" instead of a restrictive genus name. It is assumed that most of these bio-P bacteria can store PHB as a carbon storage, but that a l l of them also store polyphosphate. 2.6. Mechanistic Model for Bio-P Removal from the Literature A biochemical model that would satisfactorily explain mechanisms by which bacteria are responsible for bio-P removal under any given set of conditions would be very useful. The f i r s t significant attempt In this direction was done by Nicholls and Osborn (1978). They proposed that poly-phosphate storage assists the bacteria In surviving an "anaerobic stress". In their explanation, polyphosphate would help somehow the bacteria to survive anaerobically. Hall, Nicholls and Osborn (1978) added that PHB plays the role of accumulating hydrogen ions and electrons such that more substrate can be processed under anaerobic conditions. On this basis, they proposed to supplement the amount of VFA's fed to the anaerobic reactor by the addition of fermented primary sludge. Under subsequent aerobic conditions, PHB would provide energy. They also proposed that polyphosphate supplied energy as ATP under anaerobic conditions. Rensink et a l . (1981) extended these ideas by stating that fatty acids added to, or formed in the anaerobic zone are stored as PHB. The energy required for PHB storage would come from polyphosphate forming ATP. By such mechanisms, Acinetobacter, which was thought to be responsible for bio-P removal and which i s a slow-growing bacteria, would survive and proliferate in bio-P plants. Marais et a l . (1983) formulated the following hypothesis: "Poly-P accumulation serves as an energy reservoir, to sustain the organism during the anaerobic stressed state, but princi-pally to gain a positive advantage over non-P accumulating organisms by partitioning off readily biodegradable COD (in the lower fatty acid form) in the anaerobic state for i t s exclusive use subsequently in the aerobic state". Concerning the role of polyphosphate under anaerobic conditions, Marais et a l . (1983) proposed that one ATP i s formed from polyphosphate hydrolysis by the enzyme polyphosphatase. This enzyme was presumed to be activated when the cellular ATP/ADP ratio i s low. On this aspect, Harold (1966) points out that although evidence indicates that the phosphate acceptor in the poly-phosphate breakdown reaction can be other than water, this possibility must remain. According to Marais et a l . (1983), the ATP formed would be used to bring substrate in the c e l l and convert i t in polyphosphate. With acetate as a substrate, two ATP's are required to take up acetate and synthesize acetyl CoA which is then stored as acetoacetate. Thus, two phosphate molecules originating from polyphosphate would diffuse in the bulk liquid for one acetate taken up. With glucose, however, two situations arise: the bacteria with poly-phosphate reserves cannot or can use glucose. In the case of the bacteria with polyphosphate that cannot use glucose (as for Acinetobacter; referring to Fuhs and Chen, 1975) facultative organisms would ferment one glucose into three acetate and increase their energy level by two ATP's for each glucose. The acetate released could then be used as previously explained by bacteria responsible for excess P removal. If glucose can be used by bacteria containing polyphosphate, however, processing glucose to PHB w i l l cause some NADH to accumulate. In a mixed culture medium, however, other bacteria would ferment glucose to acetate which, as previously explained, can be used to form acetoacetate. With the accumulated NADH, acetoacetate could be stored as PHB. Thus, a mixed culture would confer an advantage to bacteria containing polyphosphate (Marais et a l . , 1983). Concerning phosphate uptake, Siebritz et a l . (1983b) point out that the "luxury uptake" and "overplus uptake" mechanisms are inappropriate to explain the behavior observed in activated sludge plants. Indeed, these mechanisms require the limitation of a specific metabolite such as sulfur, nitrogen or phosphate. Such nutrient limitations are highly improbable with sewage as a substrate given the configuration of the bio-P processes. From the hypothesis presented above by Marais et a l . (1983), Siebritz et a l . (1983b) suggested that the presence of PHB supplied enough energy to stimulate polyphosphate storage. NADH: see Appendices C and D. 3. BIOCHEMICAL MODELS 37. 3.1. Introduction In the literature review chapter, requirements for bio-P removal were presented, then synthesized in the model proposed by Marais et a l . (1983). In this chapter, biochemical models of metabolic processes postulated to explain phosphorus removal by bio-P bacteria are presented. F i r s t , aerobic metabolism resulting i n polyphosphate storage is described. Then, anaerobic metabolism resulting in polyphosphate breakdown and PHB storage i s presented. Under anaerobic conditions, i t is postulated that the maintenance of a constant proton motive force is Involved in regulating phosphate expulsion. Another aspect of the model for anaerobic conditions is related to energy production for substrate storage. Speculations are formulated on this last aspect. As previously mentioned, information on the glycolysis pathway, the TCA cycle, bioenergetics and membrane transport i s summarized in Appendices C, D and E for ease of reference and understanding of microbiological concepts. 3.2. Postulated Model for Aerobic Conditions In the anaerobic zone, bio-P bacteria w i l l have accumulated PHB reserves and have decreased their polyphosphate storage. Thus, just before entering the aerobic zone, the bulk liquid solution w i l l show a high soluble phosphate concentration and a low storable carbon substrate 1 concentration. Under aerobic conditions, energy production w i l l require available carbon 1 The storable carbon substrate refers to substrates such as VFA's that can be stored as PHB. 38. substrate to be processed via the tricarboxylic acid (TCA) cycle (refer to Appendix D). Since extracellular sources of substrate are scarce, bacteria that have accumulated PHB w i l l be able to use their reserves for energy production. Dawes and Senior (1973) pointed out that PHB is used mainly for energy production and not as a carbon source for synthesis. Thus, bio-P bacteria w i l l u t i l i z e their PHB reserves to produce energy. With a high cellular ATP/ADP ratio, phosphate uptake and storage w i l l be stimulated as well as the growth and the reproduction of bio-P bacteria. This conceptual mechanism i s depicted in Fig. 3.1. A postulated biochemical model for aerobic conditions is shown in Fig. 3.2. If nitrate i s present in absence of oxygen, some bio-P bacteria could use this alternate electron acceptor for energy production. Therefore, polyphosphate storage and PHB u t i l i z a t i o n are expected to occur both under aerobic conditions or in the presence of nitrate. 3.3. Postulated Model for Anaerobic Conditions 2 3.3.1. D i f f i c u l t i e s in Proposing a Biochemical Model for Anaerobic Conditions The basic problem In proposing a biochemical model for anaerobic conditions resides in two major d i f f i c u l t i e s regarding the actual biochemical processes in which polyphosphate is Involved. The f i r s t d i f f i c u l t y i s to explain why phosphate i s released from bacteria. This behavior is peculiar In i t s e l f since phosphate, being naturally at a low level in natural environments, tends to be accumulated and 2 Anaerobic conditions should be understood as the absence of both free oxygen and of nitrate. The effect of nitrate Is discussed in section 3.2. Extracellularly bound carbon compounds P i F i g . 3.1 Conceptual model f o r aerobic metabolism i n bio-P b a c t e r i a . 40. ( c r e a t i n g a proton motive f o r c e ) F i g . 3.2 P o s t u l a t e d model f o r aerobic metabolism i n bio-P b a c t e r i a . Carbon s u b s t r a t e s , e x t e r n a l i f a v a i l a b l e or i n t e r n a l from PHB,are degraded v i a the TCA c y c l e r e s u l t i n g i n a high i n t e r n a l energy l e v e l . Some energy i s used f o r polyphosphate formation. 41. stored in bacteria for survival rather than expelled from c e l l s . To the writer's knowledge, the microbiological literature provides very l i t t l e information on transport of phosphate for expulsion from bacteria. If bacteria are getting rid of such an essential metabolite as phosphate, they must benefit from such an action under the conditions of a bio-P process. This advantage is proposed to be the "partitioning off" of easily storable carbon. Although the actual coupling mechanism is not established, i t was postulated that the maintenance of a constant proton motive force could explain the phosphate expulsion phenomenon. The second d i f f i c u l t y concerns the role of polyphosphate. Is i t used only as a reserve material or also as an energy source? In section 2.4.1 on polyphosphate metabolism, i t was shown that some mechanisms are available for polyphosphate to serve as an energy source. Nevertheless, reviews on the subject by Harold (1966), Dawes and Senior (1973), and Kulaev (1979) concluded that the major function of polyphosphate was probably not to store energy but rather to serve as a reserve material. As pointed out by Siebritz et a l . (1983b), however, the known mechanisms for polyphosphate storage, "luxury uptake" and "overplus uptake", do not appear to explain satisfac-t o r i l y the mechanisms of bio-P removal. This reason led many authors to suggest that polyphosphate played a role in ATP production. In speculating on energy sources for PHB removal, a role of energy production for polyphos-phate w i l l be presented. 3.3.2. Postulated Model As seen with the model on aerobic metabolism (refer to Fig. 3.2), the presence of PHB reserves is the cornerstone of survival and proliferation of 42. bio-P bacteria in a zone where the competition for soluble carbon is strong. To be stored as PHB, substrates must f i r s t be transported inside the c e l l . Several monocarboxylic acids (with one-COOH group) are transported neutrally across the membrane i f an appropriate pH gradient exists (Harold and Spitz, 1975) . In solution, at a pH greater than 6.5, more than 99 percent of the acetate is in the ionic form. Once in the c e l l , at a pH of about 7.6 (Schuldiner and Padan, 1982), one could expect H"1" accumulation from the dissociation of acetate Into its ionic form. However, since the transforma-tion of acetate into acetyl CoA releases a water molecule and no no net intracellular pH decrease should be observed. Therefore, acetate transport w i l l decrease the pH gradient by one H + for each acetate transported. In fact, acetate, propionate and butyrate as well as other lipophilic acids are used as permeant acids to measure the pH gradient of c e l l membranes (Kaback, 1976) . This pH gradient decrease w i l l reduce the proton motive force which the cells have to maintain at a constant level (Bakker and Mangerich, 1981, Schuldiner and Padan, 1982). Some mechanisms are available for bacteria to counteract the effect of a decreased pH gradient. Under aerobic conditions, H + can be expelled by the electron transport chain. Under anaerobic conditions where no electron acceptor is present, however, this mechanism cannot be functional. Another way to eject H + consists of breaking down ATP at the membrane-bound ATP-ase enzyme (Harold, 1977). With acetate as substrate, however, no ATP can be generated by glycolysis and the reserves of ATP would be rapidly depleted. A third mechanism consists of u t i l i z i n g NADH at the membrane-bound transhydro-genase enzyme to expel H + (Harold, 1977). Once again such a mechanism cannot be functional with acetate as a carbon source. Although acetyl CoA could be 43. fed into the TCA cycle to supply NADH3, the formation of acetyl—CoA from acetate requires an Input of energy which is not available under anaerobic conditions. Finally, the mechanism proposed to expel H* out of the c e l l , i s by co-transport with phosphate. Phosphate is probably expelled across the bacterial membrane in a neutral state (Harold, 1977). This is justified by the fact that the plasma membrane is impermeable to both negative and positive ions. Furthermore, Inward transport of phosphate occurs neutrally, the negative phosphate charge being neutralized by protons, by using the pH gradient as the driving force under aerobic conditions (Harold, 1977) (see Appendix E). Since a favorable pH gradient allows phosphate uptake aerobically, i t appears reasonable that an unfavorable pH gradient, combined with the intra-cellular availability of phosphate, can result in phosphate expulsion i f the ce l l can benefit from such an action. When the expelled phosphate is released in solution, i t w i l l tend to equilibrate with the other phosphate species, namely HgPC^, H2P04~, HPO^-2 and P0^~3 depending on the pH. At an external pH of 6.8, 83 percent of the phosphates w i l l be In the monovalent form and 17 percent in the divalent form. This could result In an average release of 1.17 proton for each phosphate transported. Thus, phosphate extrusion from the c e l l could serve as a way for cells to expel H* and counteract an unfavorable pH gradient. Provided that the intracellular phosphate concentration is high enough, the regulation of the phosphate carrier could respond to a change in either 3 Acetyl-CoA can be fed in the TCA cycle anaerobically by bacteria, but is normally blocked at the ct-ketoglutarate dehydrogenase enzyme by an excess of NADH. If some NADH is used up, the TCA cycle can function at a lower rate just sufficient to resupply the NADH util i z e d (see Appendix C). 44. the pH gradient or the proton motive force. Such a regulation mechanism of a carrier enzyme would not be unique. For example, carrier enzymes have been reported to respond to changes (1) in the pH gradient for inward transport of potassium (Bakker and Mangerich, 1981), (2) in the external pH also for inward movement of potassium (Yamasaki et a l . , 1980), (3) in the charge gradient (Aip) for outward movement of sodium (Sorensen and Rosen, 1982), and (4) in the proton motive force for sugar uptake (Peterkofsky and Gazdar, 1979, and Reider et a l . , 1979). It should be noted that although protons are expelled with phosphate, no is removed from the cytosol. In fact, the H* rejected with a phosphate molecule originates from the dissociation of a water molecule involved in the breakdown of polyphosphate. Even i f phosphate reached equilibrium with i t s monovalent or divalent phosphate species in the cytosol, i t would recombine before being expelled neutrally across the c e l l membrane. In summary, i t i s postulated that the uptake of acetate results i n a transient decrease of the pH gradient across the membrane which is in turn reestablished by neutral expulsion of phosphate molecules from the c e l l . Any substrate (such as acetate), which is taken up by carrying a proton inside the c e l l and is rapidly metabolized, or stored as PHB, is expected to cause a similar transient pH gradient decrease and could result in phosphate expulsion as long as phosphate molecules are available from polyphosphate. Since a phosphate pool Is present in bacteria (Medveczky and Rosenberg, 1971, Rae and Strickland, 1975), phosphate transported neutrally across the membrane could be supplied from this phosphate pool, i t s e l f replenished from the polyphosphate reserves. Thus, the role of polyphosphate Is proposed to be the regulation of the internal inorganic phosphate level. These concepts are summarized in Fig. 3.3. 45. Ac ( p r o t o n m o t i v e f o r c e ) F i g . 3.3 P o s t u l a t e d m o d e l f o r a n a e r o b i c m e t a b o l i s m o f b i o - P b a c t e r i a . A c e t a t e t r a n s p o r t d e c r e a s e s t h e p r o t o n m o t i v e f o r c e w h i c h i s r e - e s t a b l i s h e d b y p h o s p h a t e e x p u l s i o n . S o u r c e s o f e n e r g y f o r P H B f o r m a t i o n a r e s p e c u l a t e d i n F i g s . 3.4, 3.5, a n d 3.6. 46. 3.3.3. Speculations on Energy Sources for PHB Storage To provide a more complete picture of anaerobic metabolic processes i n bio-P b a c t e r i a , speculations, based more on m i c r o b i o l o g i c a l and biochemical p r i n c i p l e s than on experimental evidences, are given here. It i s believed that such explanations can be u s e f u l to help the i n t e r p r e t a t i o n of p e c u l i a r or apparently contradictory observations. When acetate i s metabolized, energy Is required for the production of a c e t y l CoA and for the production of 3-hydroxybutyryl CoA (see Section 2.4.2). Macrae and Wilkinson (1958) found that, under n i t r o g e n - l i m i t i n g conditions, PHB could not be formed from acetate alone. However, acetate combined with pyruvate, glucose or g-hydroxybutyrate, could t r i g g e r PHB synthesis. This observation, as well as known pathways of acetate metabolism, suggest that anaerobic storage of acetate as PHB requires a source of energy contributed by compounds other than acetate. These compounds can be sugars or amino acids which produce energy by the g l y c o l y s i s pathway and/or the TCA c y c l e . It i s speculated that polyphosphate can serve as a source of energy by d i r e c t phosphorylation or ATP formation. Figure 3.4 depicts the proposed sources of energy for storage of acetate as PHB. The formation of PHB i s discussed i n two steps. A) Acetate to Acetyl-CoA A source of energy i s required to process acetate to a c e t y l CoA. Table 3.1 gives a l i s t of known reactions for the transformation of acetate Into a c e t y l CoA. Table 3.2 gives a l i s t of the free energy of hydrolysis of the energy-rich compounds that can be Involved i n this reaction. Most reactions require e i t h e r ATP or a CoA compound to transform acetate to a c e t y l CoA. Unfortunately, under anaerobic conditions, bio-P bacteria have a very limited Bio-P Bacteria PHB Synthesis ENERGY Production Amino Acid hydroxyb 3.4 Speculations on energy sources f o r a c e t i c a c i d storage as PHB. 48. TABLE 3.1 POSSIBLE REACTIONS FOR TRANSFORMING ACETATE TO ACETYL CoA A G°'obs b Reaction 3 (Kcal/mole) (3-1) acetate + ATP + CoA acetyl CoA + ADP + Pi + 0.9 (3-2) acetate + ATP -> acetyl-P + ADP + 3.1 acetyl-P + CoA —> acetyl CoA - 2.2 (3-3) acetate + ATP acetyl AMP + P P i c (-) acetyl AMP acetyl CoA (-) (3-4) acetate + succinyl CoA -> acetyl CoA + succinate - 0.1 (3-5) acetate + acyl CoA acetyl CoA + acylate (-) d a The reference for the reactions and AG°£ S is Thauer et a l . (1977), except for the equation (3-1) which comes from Mlchal (1977). b A negative free energy charge (AG°^g) indicates a reaction that would occur spontaneously. c PPi stands for pyrophosphate. d A(30£s is dependent on the acyl group involved in CoA transfer. TABLE 3.2 FREE ENERGY OF HYDROLYSIS OF ENERGY-RICH COMPOUNDS Energy-rich Compound A^°'obs (Kcal/mole) Acetyl CoA - 8.5 Acetyl Phosphate - 10.7 ATP ADP + Pi) - 7.6 ATP (+ AMP + PPI) - 9.96 PPi (pyrophosphate) - 5.2 Succinyl CoA - 8.4 Polyphosphate ? a - reference: Thauer et a l . (1977). I I - AG°^ g is the free energy charge of hydrolysis at a free Mg concentration of 10 - 3 M, an ionic strength of 0.25, a pH of 7, one atmosphere of pressure, and a concentration of the products and reactants of one molar. 50. supply of both of these energy-rich compounds, but they have reserves of polyphosphate. The free energy of hydrolysis of polyphosphate is not known but, from i t s chemical structure, i t can be supposed to be similar to that of pyrophosphate (-5.2 kcal/mole). With such a low free energy of hydrolysis, i f polyphosphate replaces ATP in eqns. 3-1 and 3-2, i t would seem that there w i l l be a lack of energy for the reaction to occur spontaneously. However, i f the concentration of the product (acetyl-P or acetyl CoA in this case) was much lower than the concentration of the reactant (acetate), the reaction could proceed (Brock, 1979) with polyphosphate as an energy source. It was seen in the postulated model for bio-P bacteria under anaerobic conditions that a supply of phosphate was needed from polyphosphate to expel H"** from the c e l l . Thus, i t seems likely that polyphosphate could be broken down by transferring i t s energy for the production of acetyl CoA from acetate. In turn, the high intracellular inorganic phosphate concentration resulting from such a process could activate the carrier enzyme expelling phosphate. B) Acetyl CoA to PHB Acetoacetyl CoA, formed from two acetyl CoA's, w i l l require NADH to be processed into 3-hydroxybutyryl CoA (see Section 2.4.2). This supply of NADH could come from either acetyl CoA processed via the TCA cycle 1*, or from the glycolysis of sugars, or else from the degradation of amino acids. These possibilities are shown in Figs. 3.5 and 3.6. Future experiments with the combined addition of acetate with glucose and/or amino acids, would provide useful information to test these speculations. ** See the footnote (no. 3) about the TCA cycle functioning anaerobically in section 3.3.2. 7 Acetic Acid F i g . 3.5 Speculations on the r o l e of polyphosphate, and a source of NADH from the TCA c y c l e . F i g . 3.6 Speculations on the r o l e of polyphosphate and a source of NADH from g l y c o l y s i s and amino acids degradation. 53. 4. METHODOLOGY In this chapter are described the procedures used for batch testing, the characteristics of the activated sludge used, and the analytical techniques that served for parameter measurement. 4.1. Batch Testing Batch test reactors were 2.8 l i t r e pyrex erlenmeyers, mixed with magnetic stirrers (see Fig. 4.1). Anaerobic conditions were Insured by a rubber bung with sealed openings for an ORP probe, a Tygon sampling tube and a septum. A syringe was used to sample from a plastic tube. A septum served to insert a nitrogen gas balloon fit t e d on a syringe needle in order to replace the volume of sample withdrawn by an inert gas, or to inject chemical solutions into the reactor. For aerobic conditions, the rubber bung was removed and a Fisher Fritted Glass Air F i l t e r was used, with the flowrate controlled manually via a needle valve linked into the building compressed air supply. The syringe and a plastic tube were used for sampling. For anaerobic conditions, the order of addition of solutions in a reactor were f i r s t , a concentrated buffer solution then, the sludge to f i l l up the reactor, and f i n a l l y other chemicals such as acetate or nitrate (after some sample withdrawal). A l l batch tests were conducted at room temperature (21 ± 1°C). When many reactors were operated simultaneously, the sampling time of each reactor was usually staggered by five minutes. 4.2. UBC Pilot Plant Activated Sludge Activated sludge for batch testing was obtained from the UBC pilot plant 54. Fig. 4.1 Batch testing apparatus. 5 5 . operating on bio-P removal modes. The processes are shown in Fig. 4.2. Domestic sewage from a two thousand-people residential complex provided the influent feed to the plant. Typical influent and effluent characteristics of the plant are given in Table 4.1. The bioreactor configuration consisted of two non-aerated c e l l s , plus one aerated c e l l , and a secondary c l a r i f i e r . For the "A" side, the f i r s t c e l l (the denitrification reactor) received primary supernatant and the sludge recycled from the secondary c l a r i f i e r . Downstream of the denitrifica-tion reactor, another non-aerated c e l l was supplemented by the anaerobic reactor (no fermented sludge was added at the time of the f i r s t pilot plant survey, however; section 5.6.2). For the "B" side, the f i r s t non-aerated c e l l received raw sewage, the sludge recycle and sodium acetate. Acetate addition was equivalent to about 25 mg/£ as COD i n the c e l l where i t was supplemented. Both the influent and the sludge recycle had a flowrate of 3.0 £/min. The hydraulic retention time in the three cells of the bioreactor was about 1.5 h, 1.5 h and 4.6 h. 4.3 Analytical Techniques A l l f i l t r a t i o n s were done through 0.45 um membrane f i l t e r s for metals, nitrogen and phosphorus determinations (A.P.H.A., 1980). Sludge samples were preliminarily centrifuged (5 min, 1800 g) and their supernatant subsequently fi l t e r e d . The centrifuge used was the Model CS of International Equipment Co-. 4.3.1 Residue (a) Total Nonfiltrable Residue (Suspended Solids [SS]) Sodium Acetate PRIMARY CLARIFIER EFFLUENT FERMENTER SUPERNATANT f "A" SIDE Sodium Acetate LEGEND: Z2 n no 0^. but Nitrate present no 0_, and no Nitrate present Aerobic Reactor SECONDARY CLARIFIER EFFLtJENT FERMENTER 'B" SIDE F i g . 4.2 Conf i g u r a t i o n of the UBC p i l o t p l a n t processes. TABLE 4.1 TYPICAL INFLUENT AND EFFLUENT CHARACTERISTICS OF THE UBC BIO-P PILOT PLANT3 Concentration (mg/£) Influent Effluent COD 200-325 40-50 SS 100-175 5.0-15.0 TP 3.5-4.5 0.0-0.4b TKN-N 20-28 1-2 NO3-N - 6-9 (2-4) c a From Koch (1984) for the period of April 1983 to December 1983. b TP effluent given for successfully operating conditions. c The effluent nitrate concentration was reduced by aerobic sludge recycling for denitrification. 58. Nonftitrable residue i s the material retained on a standard glass fibre f i l t e r (Whatman 934AH) and remaining after evaporation and drying to a constant weight at 104°C (A.P.H.A., 1980). (b) Volatile Residue (Volatile Suspended Solids [VSS]) Volatile residue i s determined by igniting the nonfiltrable evaporated residue from Method (a) above at 550°C (A.P.H.A., 1980). 4.3.2 Metals Atomic absorption spectroscopy was used to determine dissolved metal concentrations. Filtered samples were acidified to a pH lower than 2 with concentrated n i t r i c acid (HN03) for preservation. When dilutions were required, a 0.57 percent HN03 solution was used. The determinations followed the procedures described in U.S. EPA (1979). Metals analyzed were: aluminum, cadmium, calcium, iron, magnesium, manganese, potassium and sodium. Radiation buffers at a concentration of 0.1 percent used were lanthanum buffer for calcium and magnesium, sodium buffer for potassium, and potassium buffer for sodium (U.S. EPA, 1979). 4.3.3 Nitrogen (a) Nitrate plus Nitrite-Nitrogen Nitrate plus nitrite-nitrogen was analyzed on the Technlcon Autoanalyzer II according to the Technicon Method 100-70W (1973). In this procedure, nitrate i s reduced to n i t r i t e by a copper-cadmium reductor column. The n i t r i t e ion then reacts with sulfanilamide under acidic conditions to form a diazo compound. This compound then couples with N-l-naphthylethylenediamine dihydrochloride to form a reddish purple azo dye. The detection limit is 0.04 mg NO3-N/JI. 59. 4.3.4 Oxidation-Reduction P o t e n t i a l (ORP) ORP were continuously monitored with a Broadley Jance Corp. combined ORP probe with peripheral junction, using Ag/AgCl as a reference couple. A 1/8 inch band platinum sensor was connected to a high impedance d i g i t a l panel meter with l i q u i d c r y s t a l d isplay (LCD). 4.3.5 pH The pH values were determined with a Fisher Accumet Model 320 expanded scale pH meter with combined glass and reference electrodes i n one probe, standardized against F i s h e r pH 4 and pH 7 buffer solutions (A.P.H.A., 1980). 4.3.6 Phosphorus The words SRP and TP are used i n t h i s thesis when a d i r e c t reference to a concentration i s made. Otherwise, the word "phosphate" i s used to describe SRP uptake from or release into s o l u t i o n . The word "phosphorus" i s used to refer to the o v e r a l l phosphorus removal phenomenon from wastewater by a treatment plant. (a) Soluble Reactive Phosphorus (SRP) Soluble r e a c t i v e phosphorus was determined on 0.45 um membrane-filtered samples using the automatic ascorbic acid reduction method (Technicon Auto-analyzer I I , Method No. 94-70W, 1973) f o r which the detection l i m i t i s 0.2 mg P/£. A l t e r n a t i v e l y , the stannuous chloride method was used (A.P.H.A., 1980). (b) Acid-hydrolyzable Phosphorus (AHP) Acid-hydrolyzable phosphorus was determined on u n f i l t e r e d and f i l t e r e d 60. samples by autoclaving under mild acidic conditions (A.P.H.A., 1980). (c) Total Phosphorus A preliminary digestion step for total phosphorus was performed with the block digestor method using sulfuric acid. Sample analysis was performed according to the Technicon Autoanalyzer II, Method No. 327-73W (1974). The phosphorus standards used for calibration ranged from 0 to 2.67 mg P /J i . (d) Phosphorus Content in Sludge The centrifuged sludge was dried at 104°C. A weighed aliquot of the dried solids was analyzed for total phosphorus as outlined above. The results were expressed as percent mg P per mg SS. (e) Polyphosphate Detection of polyphosphate by cytological staining depends upon the reaction of the polymer with basic dyes such as methylene blue. The poly-phosphate granules are stained dark blue or violet, contrasting with the lighter blue of c e l l s . The procedure used was that of Norris and Swain (1971). The staining solution was prepared by dissolving 0.5 g of methylene blue in 100 ml of d i s t i l l e d water at 50°C, followed by addition of 1 ml of a 1 percent potassium hydroxide solution and by addition of 30 ml of ethanol. The staining procedure consisted of heat-fixing a bacterial smear on a glass slide and applying the stain for 5 to 60 seconds. The prepared smear was then rinsed with water, blot dried and examined under a NIKON, model L-Ke, microscope at 1300 times magnification. Quantification of polyphosphate was done by the acid-hydrolyzable phosphorus technique (A.P.H.A., 1980). An evaluation of this method i s 61. presented in section 5.3. Polyphosphate salts of various chain length (n = # of phosphates) were obtained from Sigma for n = 25 and 65 and from a generous donation by Dr. F.W. Harold (Denver, Colorado) for n = 14, 50, 126, 170, 216 and 1130. 4.3.7 Volatile Fatty Acids (VFA's) Volatile fatty acids were determined by gas chromatography according to the procedure described in the Supelco Bulletin 751E (1982). A computer-controlled gas chromatograph (Model HP 5880, Hewlett-Packard) equipped with a flame ionization detector (FID) was used for a l l analyses. Samples were preserved by f i l t r a t i o n and freezing. Microsyringes (Hamilton Model 75N, #87900, 5 ul) were used to inject 1.0 pi of 0.45 um membrane-filtered samples. The glass column was 0.91 m long with a 4 mm internal diameter, packed with Supelco 60/80 Carbopack C/0.3% Carbowax 20 M/0.1 % H 3P0 4. VFA's analyzed included: acetic, propionic and butyric acids. Just before Injection, the pH was brought below pH 3 by adding phosphoric acid to obtain a 1% acid solution. Experimental conditions for the chromatograph were: injection port temperature = 150°C, detector temperature = 200°C, isothermal oven temperature = 110°C with a flowrate of nitrogen as carrier gas of 20 ml/min. Standards were prepared from reagent grade acetic acid (99.9% pure), propionic acid (96% pure), and butyric acid (98% pure). 4.3.8 Poly-g-Hydroxybutyrate (PHB) The cytological staining method of Norris and Swain (1971) was used to detect PHB in sludge. A bacterial smear was heat-fixed on a glass slide and 62. then flooded with a sudan black stain (0.3 g sudan black B and 100 ml of 70 percent ethanol) for 15 minutes (replenished as i t dried out). The excess of stain was drained off the slide which was blot dried. The slide was then rinsed with xylene, blotted dry again and counterstained with a 9.5 percent aqueous safranin solution for 10 seconds. Finally, the slide was washed with water, blotted dry and examined under 1300 times magnification with a micro-scope. Lipid Inclusions were black, blue, or bluelsh-gray in contrast to the pale red c e l l s . For chemical quantification of PHB, the procedure of Braunegg et a l . (1978) was used. The basis of the method consists of depolymerizing PHB by sulfuric acid and converting hydroxybutyrate to hydroxybutyric acid methyl-ester with acidified methanol. The methylester acids were then extracted in chloroform after water addition, and separated by gas chromatography. The HP chromatograph and syringes used were the same as described for the VFA determinations. Samples were processed immediately after collection. The injection volume was 1.0 ul of extracted sample. The silinized glass column was 1.83 m long by 2 mm internal diameter packed with Chromosorb W AW DMCS 80-100 mesh coated with 5% Carbowax M20 TPA. The column was supplied and packed by Chromatographic Specialties Ltd. The experimental conditions for the chromatograph were: injection port temperature = 150°C, detector temperature = 200°C, oven temperature program: i n i t i a l temperature = 100°C, I n i t i a l time = 1 minute, temperature rate of Increase = 8°C/min, final temperature = 150°C, f i n a l time = 0.25 min, post run temperature = 180°C, post run time = 4 min, temperature equilibrium time = 3 min. The carrier gas (N 2) flowrate was 20 ml/min. The helium and air flowrates were of 30 and 400 ml/min respectively. A l l gases were of U.S. Pharmaceutical (USP) grade. Cell pretreatment consisted of centrifuging down 10 ml of sludge (about 30 mg 63. SS) for 5 minutes at 1800 g (3,000 rpm) in a teflon screw-cap test tube. PHB extraction from samples was then performed as follows: after decanting the liquid phase, the cells were suspended in a mixture of 2 ml acidified methanol (3% H 2S0 4, v/v) and 2 ml chloroform, then heated at 100°C for 3-1/2 hours. After cooling to room temperature, 1 ml of H20 was added, and the sample was vigorously shaken for 10 min. The two phases were allowed to separate, during which c e l l detritus gathered at the interphase. The organic phase (chloroform layer on the bottom of the test tube) which was to be analyzed for PHB, was placed in vials and stored at 4°C. To increase accuracy and reproducibility, benzoic acid was used as an internal standard dissolved in the acidified methanol reagent. Standards were prepared from the sodium salt of D,L-8-hydroxybutyric acid (BDH Chemicals). For hydroxybutyrate (HB) standards, the procedure recommended consisted of adding only 1 ml (versus 2 ml for samples) of chloroform to 2 ml of acidified methanol in which both the hydroxybutyrate salt and benzoate salt were dissolved. The incubation time was 60 minutes (versus 3-1/2 hours for samples) at 100°C. The standards used ranged from 0 to 200 ng HB/u£ chloroform. The detection limit was 0.5 ng HB/u£ chloroform. For gas chromatography/mass spectrometry (GC/MS) of compounds eluted on the PHB chromatogram, a nickel column was packed which size allowed to f i t in the GC/MS oven. This column was used only to get comparable peak separation to that obtained on the GC glass column. Samples were concentrated 6-fold by nitrogen gas blowing before injection. This nickel column was 1.83 m long by 2 mm Internal diameter, packed with Chromosorb W AW DMCS 80/100 mesh coated with 7.25% Carbowax M20. The GC/MS (Hewlett Packard 5985 B Quadripole) was run with the following conditions: helium carrier flow = 32 64. ml/min, oven temperature: 100°C to 180°C at 4°C/min, injection port temperature = 150°C, ion source temperature = 200°C, GC/MS interface temperature = 250°C, electron impact ionization (EI) = 70 electron-volts (ev), electron multiplier voltage for detection = 2000 volts, scan range = 30-450 atomic mass units (AMU), and scan rate = 2.1 seconds. The compound match was done by probability base peak search with the U.S. EPA/NIH (1978) library f i l e . 4.3.9 Alkaline Phosphatase The activity of the enzyme alkaline phosphatase was determined according to the procedure described by Ashley and Hurst (1981). The method is based on the hydrolysis of 1% (w/v) p-nitrophenol phosphate disodium (PNP, Sigma) into p-nitrophenol by the enzyme alkaline phosphatase in a pH 11.4 buffered solution of 0.2 M 2-amino-2-methyl-l-propanol (Sigma). The enzyme alkaline phosphatase was previously extracted from the c e l l by sonication followed by centrifugation. The product, p-nitrophenol, formed a yellow complex under alkaline conditions which was readily measured photometrically at a wave-length of 410 nm. Duplicate 10 ml samples of sludge were sonicated at 60% for 60 seconds (Artek Systems Corp., Sonic 300 dismembrator), centrifuged at 1500 g for 10 minutes and the supernatant filtered through a 1.2 um membrane f i l t e r . One ml of this f i l t r a t e was added to 1.5 ml of buffer and 0.5 ml PNP solution. The resulting solution was incubated at 37°C for 90 minutes. One ml of this solution was added to 2.0 ml of 0.2 M NaOH. After 30 minutes the absorbance was measured at a wavelength of 410 nm on a Pye Unicam UV-spectrophotometer using a one cm c e l l . The absorbance was converted to units of yg PNP hydrolyzed per ml. 65. Standard solutions were prepared with p-nitrophenol (Sigma) solutions undergoing similar treatment. Humic blanks were prepared by replacing the PNP substrate by d i s t i l l e d water in the described procedure for sample treatment. Similarly, substrate PNP blanks were prepared by replacing c e l l sample by d i s t i l l e d water. 66. 5. RESULTS AND DISCUSSION 5.1 Introduction Although the postulated biochemical models were presented before the results and discussion of the experiments, i t should be pointed out that the model was proposed on the basis of the interpretation of both the literature and of the experiments. It was chosen to use the format hypotheses (models), results, and discussion in order to relate results to the postulated models. The experimentation can be divided in three phases. The f i r s t phase dealt with preliminary investigations in which the activity of alkaline phosphatase was determined and cytological staining was performed. Then, in order to get more precise quantification of reserve polymers in sludge, techniques for the determination of polyphosphate and PHB were investigated. Finally, the changes of PHB concentration across the UBC pilot plant was studied, and a series of batch tests provided new information on the bio-P removal mechanisms. The last two sections of this chapter cover a discussion of the consistency of the model with the observations from both the literature and the experimentation, and some applications that can be proposed from an understanding of the model. Due to the large number of distinct parts in the experimentation, the results w i l l be presented and discussed in each section. 5.2 Alkaline Phosphatase 5.2.1 Introduction Ashley and Hurst (1981) showed that the activity of alkaline phosphatase 67. in anaerobic digesters increased prior to the accumulation of VFA's which in turn is commonly used to indicate a state of digester failure to produce methane. Alkaline phosphatase is an enzyme that hydrolyzes the terminal phosphate of a molecule. From this property and the observation that the enzyme activity preceded VFA production, i t was decided to determine i f a relation-ship could be established between the enzyme activity and phosphate release or uptake observed i n bio-P removal, knowing that VFA addition stimulates phosphate release. 5.2.2. Results Sludge taken from an aerobic reactor of the pilot plant (side A) was subjected to anaerobic conditions with acetate added at time zero. The activity of the enzyme alkaline phosphatase was measured in replicate samples and is shown in Table 5.1. At the time of this experiment, the UBC pi l o t -scale bio-P plant was removing phosphorus ef f i c i e n t l y as was indicated by a less than 0.1 mg P/£ effluent total phosphorus concentration. 5.2.3. Discussion The va r i a b i l i t y of the results between duplicate samples led to caution in interpreting the results. The maximum level of activity measured was about 5 ug PNP/ml. Ashley and Hurst (1981) reported that a "normal" level of alkaline phosphatase in anaerobic digesters ranged from 3 to 5 ug PNP/ml and that a high enzyme level was about 16 ug PNP/ml. Therefore, i t appeared that the measured level of alkaline phosphatase was not higher than "normal". TABLE 5.1 ALKALINE PHOSPHATASE LEVEL UNDER ANAEROBIC CONDITIONS Time of Anaerobic Conditions (h) Alk. Phosphatase Level (ug PNP hydrolyzed/ml of sludge) Sample l a Sample 2 a 0 1.3 2.1 0.5 0.0 1.7 1.0 0.0 0.2 1.5 2.3 2.5 2.0 1.5 4.0 3.0 2.7 5.3 a Samples 1 and 2 are replicates. 69. 5.3. Cytologlcal Staining PHB and polyphosphate are bacterial reserve materials that can be stained. Sudan black staining and microscopic observations of PHB-enriched cel l s (after acetate addition under anaerobic condition) and of PHB-depleted cells (after aerated conditions) gave contrasting results. Presence or scarcity of dark dots in cells suggested an anaerobic PHB accumulation when acetate was added, and a PHB util i z a t i o n when the sludge was aerated. Polyphosphate staining by methylene blue did not give contrasting results. Further investigation could have been done for polyphosphate staining but due to the encouraging PHB staining results and also to the repeated literature reports about phosphate being stored as polyphosphate (Fuhs and Chen, 1975; Buchan, 1983; Marais et a l . , 1983) i t was decided to spend efforts on chemical quantification of both polyphosphate and PHB. Indeed, such chemical estimations allowed a more precise quantitative comparison between various sludge samples analyzed while staining provided only a qualitative comparison. 5.4 Polyphosphate Chemical Quantification 5.4.1 Introduction Quantification of polyphosphate can be done by chemical methods or chromatography (the use of chromatography for long polyphosphate chains Is limited; Harold, 1966). Most chemical methods are based on the fractionation of the c e l l constituents followed by hydrolysis of polyphosphate by a strong acid. Harold (1966), and Kulaev (1979) reviewed the methods available for polyphosphate quantification. The techniques described are time-consuming and often require specialized equipment not available in the UBC Environmental Engineering Lab (such as a cold ultracentrifuge for example). 70. Thus, alternative methods were investigated. As mentioned In section 2.4.1, the phosphorus content of the sludge can provide an estimation of the polyphosphate content of the sludge. Another method for the same purpose was used by Potgieter and Evans (1983). They determined the concentration of polyphosphate by the ART measurement (A.P.H.A., 1980). This method is reported to measure condensed phosphates such as pyro-, t r i - , poly-, and hexameta-phosphate. In this research, recovery tests were conducted to verify If poly-phosphates of longer chains could be hydrolyzed by the AHP test. The recovery experiment was done f i r s t with d i s t i l l e d water and then with samples of activated sludge. The reproducibility of the AHP test was also determined in the latter experiment. 5.4.2. Results Duplicate samples were analyzed for the recovery of polyphosphate salts dissolved in d i s t i l l e d water by the SRP, AHP, and TP tests (see Table 5.2). The recovery of a long chain polyphosphate salt (n = 216) from filtered and unfiltered sludge samples was determined with the AHP and TP tests. For this experiment, 0.5 ml of a 26.0 mg P/£ solution of polyphosphate was added to diluted samples of unfiltered or of filtered sludge. The diluted samples were of 1.2 ml and 1.6 ml of unfiltered or fil t e r e d sludge respectively in 50 ml. Results are shown in Table 5.3. Reproducibility tests for SRP, AHP and TP were done on unfiltered and filtered sludge samples (see Table 5.4). 5.4.3. Discussion Duplicate samples were analyzed for the recovery test of polyphosphate 71. TABLE 5.2 POLYPHOSPHATE SALTS RECOVERY FOR VARIOUS PHOSPHORUS DETERMINATIONS % Recovery Poly P Chain Length 3  • SRP AHP TP n % % % 14 6.5 100 96.2 100 97.5 28 - 106 -92 -50 5.4 102 97.5 102 98.9 65 - 106 -104 -126 1.0 106 94.5 87 97.1 170 0.8 105 100.5 106 99.9 216 0.9 104 100.4 92 99.2 1130 0.4 99 98.7 101 99.8 X — 100.8 98.4 S - 5.8 1.8 a The polyphosphate associated cations in the salts, were sodium for n = 14 to 170, and potassium for n = 216 and 1130. b The polyphosphate solutions prepared contained about 30 mg P/£. To fa c i l i t a t e salt dissolution, lithium chloride was added such that a Li/Pi molar ratio equaled about 1.25 (Van Wazer, 1958), and heat was applied. TABLE 5.3 RECOVERY OF POLYPHOSPHATE FROM ACTIVATED SLUDGE SAMPLES Percent Recovery3 (%) Replicate Sample AHP TP f i l t . u n f i l t . f i l t . u n f i l t . 1 100.6 51.9 98.1 104.3 2 100.5 71.1 102.7 95.8 3 98.2 85.6 101.9 83.5 4 97.3 5 97.7 6 102.7 X 99.7 69.9 100.0 94.5 S 1.4 24.1 2.6 11.1 a 0.5 ml of a 26.0 mg P/£ solution of polyphosphate (chain length = 216) was added to a diluted sample of sludge or of sludge f i l t r a t e , namely 1.2 and 1.6 ml in 50 ml respectively. 73. TABLE 5.4 REPRODUCIBILITY OF PHOSPHORUS ANALYSIS OF ACTIVATED SLUDGE3 Replicate Sample Phosphorus Concentration (mg P/£) SRP AHP TP f l i t . u n f i l t . f i l t . u n f i l t f i l t . u n f i l t . 1 19.2 - 19.0 67.1 18.3 103.4 2 18.5 - 18.2 67.5 18.4 111.4 3 18.3 - 18.7 69.6 18.3 106.0 4 18.7 - 18.2 64.9 18.2 107.1 5 19.8 - 19.9 67.2 19.8 105.2 X 19.1 18.8 67.3 18.6 106.6 S 0.6 0.7 1.7 0.7 3.0 a MLSS = 3665 mg/Jl 74. salts by the SRP, AHP and TP tests (see Table 5.2). For shorter polyphos-phate chains (n = 14 and 50), some SRP (about 5 percent) was detected. For longer chains (n = 126 to 1130), less than one percent was measured as SRP indicating very limited hydrolysis in d i s t i l l e d water of the longer chain salts. For both the AHP and TP tests, the recovery was very close to 100 percent. Recovery of polyphosphate (n = 216) from filtered sludge samples gave excellent recovery for the AHP and TP tests (Table 5.3). The unfiltered samples showed good recovery for the TP test (95%) but not as good for the AHP test (70%). The latter result is probably explained by the fact that some adsorption on c e l l detritus occurred even with the high level of solids dilution used. Polyphosphate chains as long as the salt used (n = 216) are reported to occur in microorganisms. For example, Kulaev (1979) reports that 16 percent of the polyphosphate in a yeast averaged a chain length of 260. Thus, the above test performed with a long polyphosphate salt can be considered r e a l i s t i c . Therefore, i t appears that the AHP test does not degrade completely long polyphosphate chains. Table 5.4 shows good results of SRP, AHP and TP reproducibility with unfiltered and fil t e r e d sludge. The values obtained provide another estima-tion of the validity of the AHP test. From the section 2.3.1, the percent phosphorus in sludge depleted from polyphosphate reserves is reported to average about 1.5 percent. Supposing that AHP provides an estimation of the polyphosphate content of sludge, then the percent phosphorus of polyphosphate-depleted sludge can be calculated by the amount of unfiltered phosphorus compounds (TP minus AHP) divided by the SS concentration. This calculation gave 1.1 percent [.(88.0-48.5) *3,665 ]. This value is s i g n i f i -cantly lower than the 1.5 percent value reported by other workers (see section 2.3.1). It was suspected that the AHP test of an unfiltered sludge sample hydrolyzes not only the polyphosphate fraction but also other phosphate-containing compounds such as ATP, other nucleotides and phospho-l i p i d s . From the above considerations, although the hydrolysis of polyphos-phate dissolved in d i s t i l l e d water by the AHP test gave good recovery and reproducibility, i t appeared that the AHP test did not provide an accurate estimation of the polyphosphate content of sludge samples. 5.5. Poly-3-Hydroxybutyrate Chemical Quantification 5.5.1. Introduction The key role of PHB as a carbon reserve material for bio-P removal was pointed out in the literature review (Chapter 2) and served as the basis of the postulated biochemical models (Chapter 3). Cytologlcal staining (section 5.3) supported the concept of anaerobic PHB formation and aerobic PHB degra-dation. To obtain quantitative estimations of PHB, i t was decided to use the GC technique of Braunegg et a l . (1978). The analytical technique of GC determination was described in the section 4.3.8. Some problems with the analysis arose which resulted in a series of investigations to improve the accuracy of the PHB determination. Points investigated included the separa-tion of the GC peaks, the formation of the HB ester peak, and the sampling of sludge. 5.5.2. GC Peak Separation When a sample was processed for PHB analysis, other peaks than the desired HB ester peak appeared on the chromatogram. To obtain an accurate PHB estimation, the HB ester peak should be distinct from any adjacent peak and the peak baseline should be f l a t . Similar conditions should also be f u l f i l l e d with the internal standard (ISTD, benzoic acid ester) which corrects for variations in the volume of sample injected on the GC column. The Injection technique used was the solvent-flush method. It consists of rinsing the syringe of the hydroxybutyrate methylester that could adhere on the syringe wall by flushing a volume of solvent (chloroform) after the sample. Thus, an injected volume of 1.0 p£ of extracted sample was followed by 0.5 u£ of air which separated a flushing solvent volume of 1.0 u£ from the sample. This technique allowed to Insure that most of the HB ester was injected on the GC column. However, i t created problems with the integration of the HB ester peak area by the computer. Indeed, sometimes the HB ester was on the shoulder of a large solvent peak which caused the ester peak area to be underestimated. To correct this problem, a lower volume (about 0.5 u£) of flushing solvent was injected. Alternatively, i t could have been possible to change the column characteristics (such as column length, size, packing) or the GC operating conditions (such as temperature program, gas flow rates). The retention time of the HB ester peak for standards was 4.59 min, but for samples i t was 4.69 min. On some occasions, a small "neighbour" peak appeared in sludge samples at 4.46 min which caused a decrease in the integrated value of the 4.69 min HB ester peak. When the 4.69 min peak was large enough (over 40 GC units), however, the HB ester integration included the area of the preceding "neighbour" peak and thus increased the HB ester peak area value. Such an interference did not happen often, but future work with the PHB determination should provide a good isolation of the HB ester peak for every case. The presence of peaks adjacent to the HB ester peak when an extracted sludge sample was injected led to an Investigation of possible decomposition products of the HB ester. It was observed that most of the adjacent peaks were present in a f a i r l y constant amount in sludge samples. However, some peaks showed variation In their area. Thus, a peak at 5.75 min GC retention time ( r t ) appeared only after prolonged periods of anaerobiosis in batch reactors (25 h) or in the fermenter supernatant of the UBC pilot plant. In addition, the peak at 5.89 min GC r showed a lower concentration in absence of oxygen or nitrate. Upon nitrate addition, however, Its concentration increased suddenly (refer to section The identification of the peaks adjacent to the HB ester was done by GC/MS analysis (see Table 5.5). From the sole name and structure of these compounds i t was d i f f i c u l t to determine whether any of these peaks was derived from chemical changes of PHB under the conditions of reaction and extraction, or they were impurities. Since none of these peaks appeared to change in concentration at the same time as the HB peak, i t appeared that they could well be derived from other cellular constituents than PHB. It should be noted that the preparation of calibration curves from HB standards did not result in the formation of adjacent peaks. The reproducibility of the GC injection technique was tested (see Table 5.6). The coefficient of variation being less than 5 percent, the injection technique was considered satisfactory. 5.5.3. Hydroxybutyric Ester Formation and Sludge Sampling Braunegg et a l . (1978) recommend to use 1 ml of chloroform incubated for 1 h at 100°C to form the HB ester from HB salts for the preparation of standards. For samples, however, 2 ml of chloroform and 3-1/2 h at 100°C incubation time is suggested. In this research, standards were prepared as recommended, and also as for the treatment of samples. No significant 78. TABLE 5.5 GC/MS IDENTIFICATION OF THE PEAKS ADJACENT TO THE HB ESTER PEAK Retention Time on Glass Column (min.) Percent Spectral F i t a GC Area Name of Compound Structure Sample 1 Sample 2 (%) (GC units) 3.61 98 20 Butanoic acid, 3-methyl-methylester CH, • 1 I |3 n -c-c-c-c-c* ^0-CH 3 4.15 97 10 Butanoic acid, ethenyl ester C C C C-C*° 1 1 l 1 O—CH=CH2 4.60 — 4 Furfural ^ ) - C H 0 4.77 85 96 0-200 Butanoic acid, 3-methyl, methylester (HB ester peak) OH ' 1 1 '^0 f i l l 0 C H3 5.75 92 0-50 Pentanolc acid, 3-hydroxy, methylester OH • 1 ' 1 *0 -C-C-C-C-C* 1 1 i 1 0 L H 3 5.89 87 98 60-140 Pentanolc acid, 4-oxo, methylester _c-c-c-c-cCo- C H 6.20 81 86 25 Butanedioic acid, dimethylester H.C-0^ r_lr_c*»° 3 0 ^ L , , ^0-CH3 6.61 — 1200 Benzoic acid methylester (Internal Standard) ^ - c - ° / ^ ^ O - C l ^ a The percent spectral f i t denotes agreement of the mass spectrum of an unknown compound with the mass spectrum of a standard in the U.S. EPA/NIH (1978) library f i l e . 79. TABLE 5.6 REPRODUCIBILITY OF THE GC INJECTION TECHNIQUE Number Average Standard Coefficient Sample of PHB Cone. Deviation of Variation Identification Replicates GC Units S S/AVG n GC Units % I) WITH STANDARDS STD 51.8-A 7 16.1 0.71 4.4 STD 51.8-B 12 12.4 0.62 5.0 STD 103.6 5 30.0 1.12 3.7 II) WITH SAMPLES 1 4 27 3.5 1.3 2 6 26.3 4.6 1.3 80. differences were observed between the various sets of standards prepared. For each set, the linearity of the calibration curves was very good (correlation coefficient higher than 99.4% with five standards ranging from 0 to 200 ng HB/uJl CHC*3). h As shown in Table 5.7, the reproducibility of the PHB determination was tested in several occasions with triplicate sludge samples. The samples for which the coefficient of variation was larger than 10 percent were identified as samples in which the volume of flushing solvent was large enough to cause an underestimation of the HB ester peak. Aside from these samples, the coefficient of variation remained lower than 6.5 percent. Such results were considered satisfactory for the investigatory purpose of the experiments. Some PHB recovery tests were performed with sludge samples. In such tests a given amount of HB salt was added to a sample which was then analyzed for PHB. In Table 5.8 is reported an experiment where various amounts of HB salt, dissolved in the acidified methanol reagent, were added to the sludge pellet. The results showed a f a i r l y poor recovery (60 to 80 percent). Adsorption of the added HB salt on sludge may explain such results. It was also noticed that some water remained trapped in the sludge pellet after centrifugation and decantation. To test the effect of water on the PHB determination, 1.0 ml of water was added to a sludge pellet and 100 ng HB/u£ of HB salt, dissolved in the acidified methanol reagent, was added. The recovery gave 73 percent. Addition of the same amount of HB salt dissolved in the added water (1.0 ml), however, resulted in only 57 percent recovery. Furthermore, when 1.5 ml of water with the HB salt dissolved in i t was added, the recovered fraction gave a low value of 51 percent. With 1.0 ml of water added, the difference between 73 and 57 percent HB recovery could be due to the fact that the HB ester Is more d i f f i c u l t to form i f the HB salt TABLE 5.7 REPRODUCIBILITY OF THE PHB DETERMINATION1 Sample Identification Mean PHB Concentration Standard Deviation Coefficient of Variation X S C.V. ng HB/\ii CHC1 3 ng HB/\iZ CHCI3 % 1 13.1 0.15 1.2 2 17.5 1.1 6.3 3 18.3 3.6 20.0 4 21.7 3.0 14.0 5 57.4 1.2 2.1 6 60.1 1.6 2.6 7 68.6 3.6 5.2 8 84.0 2.6 3.1 9 108.0 11.0 10.0 a A l l with tr i p l i c a t e sludge samples. TABLE 5.8 RECOVERY OF HYDROXYBUTYRATE FROM SLUDGE SAMPLES Amount HB Added HB Measured Recovery ng HB/uJl CHCI3 ng HB/uA CHCI3 % 0.0 (50.0) a -20.7 66.5 80 51.8 101.8 100a 103.6 115.9 64 207.2 203.2 74 b a The PHB content of the sludge (50.0) was estimated from the 51.8 ng HB/uJi CHCI3 addition. b 74% = (203.2 - 50.0) * 207.2 83. is dissolved in water instead of in the acidified methanol reagent where i t can already be partially methylated. The poor recovery obtained were probably due to HB adsorption on sludge residues or the test tube walls. In order to Improve the accuracy of their PHB determinations, Apostolides and Potgieter (1981) used freeze-dried sludge samples. Thus, water could not interfere with the HB ester formation and also, a precise amount of sludge could be weighed. In addition to freeze-drying of sludge, the PHB determination could be Improved by the u t i l i z a t i o n of a boiling bath on a mixing table Instead of a vigorous shaking before incubation in an oven at 100°C. In that way, no sludge solids would adhere on the test tube wall, and the test tube contents would be continuously mixed. PHB is degraded aerobically by microorganisms. Since the sampling from an anaerobic reactor resulted in some air of the syringe getting in contact with the sludge sample, test tubes of sludge for the PHB test were rapidly cooled down by immersion in a -35°C bath (60 percent ethanol with dry ice). Although the necessity of this step was not established, i t was believed to be useful to prevent PHB degradation while processing the sample. For future determination of the PHB content in sludge, i t is recommended that some preliminary work be done to optimize the accuracy and reproduc-i b i l i t y of the test. Such effort should be f i r s t directed towards obtaining good HB ester and benzoic acid ester peak separation on a f l a t baseline, then towards maximizing the yield of HB ester from the reaction and extraction steps, and f i n a l l y towards an efficient sampling technique that would prevent any PHB change and provide an accurate amount of sludge solids to analyze. 84. 5.6 Pilot Plant Surveys for PHB 5.6.1 Introduction Preliminary batch tests with activated sludge from the UBC pilot plant showed that PHB was accumulated under anaerobic conditions when acetate was added. Could the same observation be made by sampling the various zones of the bioreactor of the pilot plant? Two pilot plant surveys for PHB were conducted to answer this question. The results of each experiment are presented prior to a discussion. 5.6.2 Fir s t Pilot Plant Survey for PHB For the f i r s t PHB survey (Oct. 3, 1983), samples were not taken for solids determination. Therefore, PHB results can only be compared for the aerobic and anaerobic reactors where the MLSS concentration is usually similar. The results from the A side are presented in Fig. 5.1. PHB, phosphorus (TP or SRP) and nitrate results are given. The phosphorus removal efficiency was high as shown by a 0.5 mg P/Jl TP effluent concentration. It was in the second anaerobic c e l l , where sodium acetate was added, that the highest concentrations were observed for both SRP (19.7 mg PO^-P/A) and PHB (10.0 mg HB/£). The SRP was surprisingly low in the f i r s t anaerobic c e l l (1.4 mg PO^-P/£) where the PHB content of the sludge mass was also low (3.1 mg HB/Ji). The results from the B side are presented in Fig. 5.2. The TP removal efficiency was poor, the effluent TP being 2.7 mg P/£. Both the SRP and the PHB profiles across the bioreactor showed l i t t l e deviation from an average value. 5.6.3 Second Pi l o t Plant Survey for PHB In a second PHB survey, (October 17, 1983), solids determination were Acetate Added Raw Sewage PHB TP 1.3 4.5 CLARIFIER PHB 3.1 PHB 10.0 PHB 3.7 SRP 1.4 SRP 19.7 SRP 0.5 N03 0.05 N03 0.35 N03 6.7 l 8 t ANAEROBIC 2 n d ANAEROBIC AEROBIC RETURN SLUDGE T Effluent TP 0.5 ( f l i t . ) N03 6.2 SLUDGE WASTAGE F i g . 5.1 UBC p i l o t plant survey n o . l . "A" si d e . Primary Supernatant CLARIFIER PHB TP 1.2 4.5 PHB 1.2 PHB 1.7 PHB 2.6 SRP 3.4 SRP 3.4 SRP 3.7 N03 0.35 N03 0.51 N03 3.8 l 8 t ANAEROBIC 2 n d ANAEROBIC AEROBIC Effluent TP 2.7(filt.) N03 3.4 RETURN SLUDGE F ~ SLUDGE WASTAGE F i g . 5.2 UBC p i l o t plant survey n o . l . *'B" s i d e . A l l u n i t s i n mg/1. 86. made for each sample (see Table 5.9). The VSS/SS ratio determined on sludge from the aerobic c e l l was assumed constant throughout a given bioreactor. This assumption was based on the fact that the same sludge was continuously recycled in a given bioreactor. The PHB concentration was expressed as umole HB/g VSS in order to f a c i l i t a t e the comparison between samples from different reactors and waste streams. Results for the A side, are presented i n Fig. 5.3. The phosphorus removal efficiency was not very good as shown by a TP effluent concentration of 2.0 mg P/A. The SRP profile across the plant showed that l i t t l e phosphate was released anaerobically (maximum SRP = 6.4 mg PO^-P/Jt). The PHB profile, however, showed a maximum value of 75.0 umole HB/g VSS in the second anaerobic zone. This value was much higher than what was found in the f i r s t anaerobic zone (24.0 umole HB/g VSS) or in the aerobic zone (20.0 umole HB/g VSS). This high PHB concentration in the second anaerobic zone suggested that a significant amount of soluble substrate was present according to the postulated models. But, since the feed line of sodium acetate was going to the f i r s t reactor, i t would mean that a significant portion of the soluble substrate was not taken up in the f i r s t anaerobic reactor. Further experimentation is recommended to c l a r i f y such an unexpected result. In side B (Fig. 5.4) there was excellent phosphorus removal as shown by an effluent TP concentration of 0.3 mg P/fc. Fermenter supernatant addition to the second anaerobic c e l l resulted in a high SRP value (21.7 mg PO^-P/A), whereas the SRP of the f i r s t anaerobic c e l l was low (1.4 mg PO^-P/Jl). Similarly, PHB concentration was maximum in the second anaerobic c e l l . From the second anaerobic to the aerobic c e l l , a concurrent decrease of SRP (21.7 to 0.22 mg P04-P/£) and PHB (48.0 to 20.0 umole HB/g VSS) was observed. The low PHB value of the return sludge (6.1 umole HB/£) suggested that the PHB TABLE 5.9 RESIDUE CONCENTRATION (SS) FOR THE PILOT PLANT SURVEY NO.2 SS A Side influent 56 fermenter supernatant 3200 1st anaerobic 2888 2nd anaerobic 3100 aerobic 3430 return sludge 6900 effluent 12 B Side influent 128 1st anaerobic 2100 2nd anaerobic 2150 aerobic 2160 return sludge 5960 effluent 25 a The VSS/SS ratio and percent P in sludge were determined on samples from the aerobic c e l l . For the A side, the VSS/SS ratio gave 78.7%, and the percent P in sludge 3.4%. For the B side, the results were 83.8%, and 1.6% respectively. Acetate Added Raw Sewage PHB 2.0 (180) TP 4.6 TKN 26.8 1 PHB 4.3 (24.0 SRP 4.3 N0 3 0.18 l B t ANAEROBIC I PHB 13.9 (75.01 SRP 6.4 N0 3 0.27 2 n d ANAEROBIC PHB 3.7 (20.0) SRP 2.3 N0 3 7.2 AEROBIC RETURN SLUDGE PHB 2.6 (5.1) F i g . 5.3 UBC p i l o t plant survey no.2. "A" sid e . Fermenter Supernatant Primary VFA's: Acet., Prop., Butyr. 58. 49. 2.5 Supernatant PHB 9.1 (39.) PHB 12.0 (48.! PHB 5.7 (20.) TP 4.6 SRP 1.4 SRP 21.7 SRP 0.22 N0 3 0.1 NO <0.03 NO 9.0 TKN 26.8 j l8 tANAEROBIC 2nd ANAEROBIC AEROBIC RETURN SLUDGE PHB 3.3 (6.1) T SLUDGE WASTAGE T SLUDGE WASTACE TP 2 . 0 ( f i l t . ) N0 3 6.0 TKN 0 . 7 ( f i l t . ) CLARIFIER Effluent TP 0.3 ( f i l t . ) N0 3 8.9 TKN 1.0 ( f i l t . ) F i g . 5.4 UBC p i l o t plant survey no.2. "B" si d e . A l l u n i t s i n mg/1, and also given in(uinole HB/g VSS) for PHB. 89. reserves were decreased before the return sludge was recirculated. It can also be seen that carbon reserves in the form of PHB were built up in the f i r s t anaerobic reactor. 5.6.4 Discussion High anaerobic phosphate release was associated with low effluent phosphorus concentrations. Conversely, poor anaerobic phosphate release was associated with high effluent phosphorus concentration. Identical trends were also observed for PHB concentrations, except in one case. More pilot plant surveys for PHB would be desirable. However, the role of PHB would probably be determined more directly with batch tests. Pilot plant surveys would be more useful at a later stage when more precise relationships between PHB and phosphorus removal would have been established. 5.7 Batch Tests with Acetate Addition 5.7.1 Introduction In order to test the validity of the postulated biochemical models presented in Chapter 3, many batch tests were run. Most of these were conducted under anaerobic conditions. Since hydrogen ions (H +) are proposed to play a key role in the transport of substrate and phosphate, the reactor content was buffered at a pH of about 7.2 with a 10.0 mM tris(hydroxymethyl)amlnomethane (TRIS) buffer. The objective was to characterize, by batch testing, the effect on bio-P bacteria of the addition of acetate, nitrate, oxygen (by aeration), 2,4-dinitrophenol (DNP; a toxicant), or changes of the pH. For these purposes, the concentration of many parameters was determined, namely: SRP, N03, ORP, PHB, VFA, SS, VSS, pH and many metals. In the f i r s t set of experiments, anaerobic conditions were not always 90. efficiently maintained throughout the test. This problem was later solved by maintaining a positive pressure of nitrogen gas inside the reactor with a balloon. The ORP was monitored to show that no air was entrained in the reactor. Another operational problem which affected the earlier analytical results was the hold up of rinsing d i s t i l l e d water in the f i l t e r assembly which diluted the f i l t e r e d sample. This problem was corrected by pre-rinsing the f i l t e r assembly with an aliquot of sample, and by f i l t e r i n g larger sample volumes (40-50 m£). The concentration results were expressed on a molar basis per gram of VSS. Using a molar basis facilitated comparison between parameters, particu-la r l y for the discussion of transport processes. In addition, a unit weight basis allowed a comparison of results from reactors that contained different residue concentrations in a given experiment. VSS rather than SS were chosen to express the weight because VSS represents better the organic matter content and thus, the bacterial mass. The VSS and VSS/SS ratio were reported for the experiments allowing back-calculation of any parameters in mg/Jl units i f desired. 5.7.2 Preliminary Experiment Results In a preliminary experiment, various concentrations of acetate were added to aerated sludge kept under anaerobic conditions. This, caused a rapid SRP and PHB increase. After 7 hours, the air was turned on which caused phosphate uptake and PHB ut i l i z a t i o n (see Fig. 5.5). After four to six hours of aeration, SRP started again to increase, whereas PHB did not show a parallel accumulation. The VSS concentration averaged 2370 mg/& and the VSS/SS ratio 80.5 percent. 91. to 10 > I o CL CD J , D_ Cr: ID 00 CO > CD X JD O E CO X 5.5 1 — r ~ 40 20 30 40 50 Time (h) (a) —r-60 70 40 -30 -20 -10 -Air Ac«tat€ 0.5 mM Ac 1 0 ~r 5 15 20 10 Time (h) (b) Effect of acetate and air addition on: (a) phosphate release and uptake, and (b) poly-B-hydroxybutyrate. A batch of sludge taken from the aerobic zone of the UBC pilot plant was subjected to anaerobic conditions prior to the test. A pH 7.2 TR1S b u f f e r solution was present before sodium acetate addition. Air was turned on later. 92. Discussion From these observations, i t appeared that under anaerobic conditions, acetate is stored as PHB while phosphate is expelled from c e l l s . Under subsequent aerobic conditions, phosphate uptake is accompanied by PHB consumption, as was also observed by Fukase, et a l . (1982). The following gradual phosphate release could be either due to fermentation or lyzed products being released in solution, polyphosphate ut i l i z a t i o n for maintenance energy, or due to a buffer effect. This aspect w i l l be reviewed in the discussion of the following experiment. 5.7.3 Experiment on the Effect of Acetate and Nitrate Introduction Based on previous batch tests and the improvements gradually implemen-ted, a comprehensive experiment was conducted where the concentration of many parameters was measured. These parameters were, on unfiltered samples: PHB, pH, ORP, residue (SS, VSS), and on filtered samples: SRP, N03, VFA, and many metals (potassium, magnesium, calcium, sodium, aluminum, cadmium, Iron and manganese). ORP was monitored to show that anaerobic conditions were maintained throughout the experiment. For PHB, a cooling bath (-35°C; 60 percent ethanol with dry ice) was used to stop any metabolism that could have resulted In PHB u t i l i z a t i o n after samples were taken from the reactor. In this experiment, acetate was added in three concentrations (0.0 mM, 0.5 mM, and 1.0 mM acetate added) and four hours later, 10 mg NO^ -N/X, was added to each reactor. The sludge was obtained from an aerobic reactor of the pilot plant. A pH 7.2, 10.0 mM TRIS buffer solution was present in each reactor. 93. The purpose of this comprehensive experiment was to provide information to support a mechanism for phosphate exchange with acetate. Due to the large amount of information from this experiment, the observation from each graph w i l l be outlined prior to a discussion of their relationships. Results The profiles for SRP and nitrate are shown in Figs. 5.6 and 5.7, respectively. The profiles for ORP and VFA's are shown in Figs. 5.8 and 5.9. The following observations can be made from these four graphs: a) Observations before nitrate spiking Before acetate addition, the SRP concentration was practically zero. Nitrate concentration and ORP values were both gradually decreasing (Figs. 5.6, 5.7, 5.8). With acetate addition, the SRP concentration increased rapidly until acetate disappeared from solution (Figs. 5.6, 5.9). Subsequent increase in SRP occurred at a slower rate. The rate of disappearance of acetate from solution was about 0.36 mmole Ac«g VSS" 1^" 1. The denitrification rate increased significantly with higher levels of acetate addition (Fig. 5.7). The time of complete nitrate disappearance from solution corresponded to a characteristic "knee" (as Indicated by the asterisks) on the ORP plot (Figs. 5.7, 5.8) (see also Koch and Oldham, 1984). In the "control" reactor, phosphate release happened only when nitrate was completely denitrified. Following the "knee", the ORP profiles showed a gradual decrease down to 94. CO to > D> I o CL JD O E J , to CO CO > J3> o E o 5.7 0.5 0.4-0.3-0.2-0.1 0.0 Acetate Nitrate 1.0 mM 0"°-°' 7 0.5 mM Ac ^ 0.0 mM Ac / -r-3 4 5 Time (h) 8 5.6 E f f e c t of va r i o u s l e v e l s of acetate and n i t r a t e a d d i t i o n on: th SRP p r o f i l e . Batches of sludge taken from the aerobic zone of the UBC p i l o t plant were kept i n an a i r - f r e e atmosphere w i t h a pH 7.2 TR1S b u f f e r (10.0 mM) s o l u t i o n f o r the t e s t . Sodium acetate, and l a t e r sodium n i t r a t e (10 mg NOJ-N/l) were added. 0.35 Legend i 0.0 mM Ac x 0.5 mM Ac a 1.0 mM Ac Acetate Nitrate 3 4 5 Time (h) E f f e c t of v a r i o u s l e v e l s of acetate and n i t r a t e a d d i t i o n on: the n i t r a t e p r o f i l e ; see a l s o the footnote of F i g . 5.6. 95. 100 -300 -f—-i 1 1 1 1 1 1 r—i p—i 1 1 1 1  0 1 2 3 4 5 6 7 8 . Time (h) F i g . 5.8 E f f e c t of va r i o u s l e v e l s of acetate and n i t r a t e a d d i t i o n on: the ORP p r o f i l e ; see a l s o the footnote of F i g . 5.6. VSS) 0.4 -> < 0.3-o E J , 0.2 -Acetal 0.1-0.0 A c e t a t e 1.0 m M A c , 0 .5 m M A c I 3 0 1 2 Time (h) Legend LZ2 Acetate • i Propionate ED Butyrate •It ACTOR A - 0 0 mM Ac B O S mM Ac C ' 1.0 mM Ac Accfa I* 7 Hitrole 1 I 1 1-10 20 - 0.4 0.3 - 0.2 > > o E E. 1 — i — r -30 40 50 60 L0.0 Time (h) F i g . 5.9 E f f e c t of various l e v e l s of acetate and n i t r a t e a d d i t i o n on: the VFA's p r o f i l e ; see a l s o the footnote of F i g . 5.6. a common level for the three reactors at a value of about -250 mV (E. _„) Ag-AgCJT (Fig. 5.8). "Bumps" in the ORP profile of two reactors (control and 1.0 nM acetate addition) may have been caused by traces of air introduced in the reactor through a perforated septum while sampling at a rate high enough to create a negative pressure in the atmosphere above the liquid. b) Observations after nitrate addition SRP concentration decreased rapidly after nitrate addition. The rate of SRP disappearance from solution was higher in the reactors that had more SRP present in solution (discussed later). Interestingly, the amount of SRP taken up was about the same for the two reactors that had acetate added: 0.175 mmole P04~P/(10 mg N0~-N«£ - 1). With the disappearance of nitrate, the SRP profile resumed i t s increase (Fig. 5.6). A careful observation of the nitrate curves suggested a two-step denitrification process. The i n i t i a l fast rate was similar to the d e n i t r i f i -cation rate triggered by acetate addition: about 0.26 mmole NO^ -N* g VSS - 1«h - 1. For the 0.0 mM ("control") and 0.5 mM acetate addition reactors, a second lower rate of denitrification was observed which was lower for the "control" reactor (0.086 mmole NO^ -N'g VSS - 1*h - 1 for 0.0 mM, and 0.114 mole NO^ -N'g VSS - 1^" 1 for 0.5 nM). The fact that two rates of denitrification were observed, suggested that bacteria had access to two types of carbon compound. The ORP values Increased by about 100 mV with 10 mg/A of nitrate addition. Again a "knee" Indicated by the asterisks correlated well with the time of nitrate disappearance from solution (Figs. 5.7, 5.8). VFA production by fermentation occurred after about 20 hours of anaerobic conditions. After 54 hours, as high as 0.36 mmole acld/g VSS (64 mg acid/A) was produced of which 72% was acetic acid (Fig. 5.9). The fermented substrate probably originated from lyzed c e l l s . In addition to the four preceding graphs, concentration profiles were also established for potassium (Fig. 5.10), magnesium (Fig. 5.11), calcium (Fig. 5.12), sodium (Fig. 5.13), PHB (Fig. 5.14) and GC Area of the r 5.89 min peak (Fig. 5.15). Other metallic cations that were present in negligible concentrations included manganese, cadmium, iron and aluminum. The concen-tration profiles for potassium, magnesium and calcium showed a similar pattern to the one obtained for SRP (Fig. 5.6). Thus, release or uptake of potassium, magnesium and calcium appeared to be correlated to phosphate transport. Sodium, however, did not seem to be correlated to phosphate changes. The sudden "jumps" on the sodium graph indicated by larger lines were caused by sodium acetate or sodium nitrate additions (Fig. 5.13). PHB profiles showed that acetate addition resulted in higher levels of PHB being stored. Nitrate addition did not seem to be associated with a PHB decrease according to the 0.0 mM and 0.5 mM acetate plots for PHB (Fig. 5.14). For the reactor that had 1.0 mM acetate added, the irregular shape of the curve did not allow a direct correlation between denitrification activity and PHB concentration decrease. A careful comparison of the various peaks on the chromatogram of PHB extracts revealed a peak ( r t = 5.89 min; normalized to an internal standard) that showed a similar profile for a l l three reactors 1. The compound showed 1 In Section 5.5.2, i t was reported that the peaks of a GC r of 5.75 min. and 5.89 min. could show significant conentration variation. In this experiment, only the peak at a GC r t of 5.89 min. varied in concentration. 98. CO CO > O E E. ' l / l (/l 0.30 0.25-0.20 0.15-E 0.10-0.05-0.00 Nitrate Acetate or* I 1.0 m M Ac 0 . 5 m M A c v.—x-~tr 0 . 0 m M A c I 3 4 5 Time (h) T 6 F i g . 5.10 E f f e c t of v a r i o u s l e v e l s of acetate and n i t r a t e a d d i t i o n on: the potassium p r o f i l e ; see a l s o the footnote of F i g . 5.6. CO CO > CD O 3 CD D 0.30 0.25-0.20-0.15 -E 0.10-0.05-0.00-A c e t a t e 1.0 m M A c J ..JX- x — = r . - - D " 0 .5 m M A c x-Ni t r a t e I f'~-~^~f-- a-j--a-f-0 .0 m M A c -6-N " S D -D-T " 3 4 5 Time (h) F i g . 5.11 E f f e c t of v a r i o u s l e v e l s of acetate and n i t r a t e a d d i t i o n on: the magnesium p r o f i l e ; see als o the footnote of F i g . 5.6. 99. 0.30 Ul Ul 0.25-> o 0.20-o CD • mol 0.15--c 0.10-c • alci 0.05-o Legend * 0 .0 m m o l a r Ac x 0.5 m m o l a r A c o 1.0 m m o l a r Ac A c e t a t e * * N i t r a t e -x • -jy-o.oo H—i—r— o 1 2 3 4 5 6 7 8 Time (h) F i g . 5.12 E f f e c t of v a r i o u s l e v e l s of acetate and n i t r a t e a d d i t i o n on: the calcium p r o f i l e ; see a l s o the footnote of F i g . 5.6. Ul Ul > f QJ O 3 T5 O Ul 3.0 2.5 2.0 1.5-1.0 Acetate X-) 1.0 m M Ac 0 .5 m M Ac 0 .0 m M Ac Ni t r a t e I 2 3 4 5 6 7 8 Time (h) F i g . 5.13 E f f e c t of v a r i o u s l e v e l s of acetate and n i t r a t e a d d i t i o n on: the sodium p r o f i l e ; see a l s o the footnote of F i g . 5.6. 100. CO CO > CD CD I D_ O E =1 CD I D_ Time (h) F i g . 5, 14 E f f e c t of v a r i o u s l e v e l s of acetate and n i t r a t e a d d i t i o n on: the PHB p r o f i l e ; see a l s o the footnote of F i g . 5.6. 160-. 140-•"g 120 Z5 o 1 0 0 & 80-60-E en 00 t-ri O O 40 20-l 0 Acetate I V Legend Nitrate A 0.0 mM Ac x Q.5 mM Ac D 1.0 mM Ac 3 4 5 Time (h) F i g . 5.15 E f f e c t of v a r i o u s l e v e l s of acetate and n i t r a t e a d d i t i o n on: the PHB-GC r t 5.89 min compound p r o f i l e ; see als o the footnote of F i g . 5.6. 101. higher levels when nitrate was present than when none was remaining in solution. Indeed, nitrate addition resulted in a sudden increase in the concentration of this compound. This peak was identified by the GC/MS library as pentanoic acid, 4-oxo, methylester (Table 5.5). Profiles of pH are shown in Fig. 5.16. Even with a pH 7.2 buffer added, the pH was lower than expected, averaging 6.7. The pH decrease observed between the last two sets of results could be due to the release of fermentation products into solution (Fig. 5.9). Volatile nonfiltrable residue (VSS) concentration in the reactors averaged 2750 mg VSS/A, and the VSS/SS ratio averaged 82.5 percent. Discussion In this section, relationships are derived from the above observations. The discussion is separated in three aspects: A. phosphate and nitrate, B. PHB, and C. metallic cations. A. Phosphate and Nitrate At the point of acetate addition, some nitrate was s t i l l present In the reactors. Since some of the added acetate was undoubtedly utilized for denitrifIcation, i t was hard to t e l l how much phosphate release resulted from acetate addition. The acetate utilized for denitrification was estimated from the nitrate concentration in the reactor and the following formula: 8 N0~ + 5 CH3C00" + 13 H + > 4 N 2 + 10 C0 2 + 14 H20 (5-1) Since 8 moles of nitrate use up 5 moles of acetate, 0.625 umole Ac" /pmole NO3-N w i l l be required for denitrification. For the 0.5 mM acetate reactor, an estimated 3.6 mg N0I-N/£ w i l l consume 0.44 mmole/I of acetate. 102. 8 Nitrate Legend A 0.0 mM A c x 0.5 mM A c D 1.0 mM A c Time (h) F i g . 5.16 E f f e c t of v a r i o u s l e v e l s of acetate and n i t r a t e a d d i t i o n on: the pH p r o f i l e ; see a l s o the footnote of F i g . 5.6. 103. Thus, i t can be estimated that 0.06 mM (0.50 minus 0.44) of acetate resulted in 0.066 mmole/g VSS of phosphate released. Similarly, for the 1.0 mM acetate reactor, 0.64 mM of acetate resulted in 0.28 mmole/g VSS of phosphate released. Fig. 5.17 shows a graph of these results. Obviously, more points are needed to obtain a valid relationship but i t i s Interesting to note that by removing the effect of nitrate on acetate uti l i z a t i o n , very l i t t l e acetate addition may result in phosphate release. The molar ratio of phosphate released to acetate utilized was approximately 1.0. It would be advisable in later experiments to remove a l l nitrate prior to the experimentation in order to verify this ratio. Fukase et a l . (1982), working with an acetate-acclimated bio-P process, found that the molar ratio of phosphate released to acetate utilized was (1/1.1):1.0. On the basis that acetate uptake causes one H + to be transported Inside the c e l l (see section 3.3.2), the above results suggested that one expelled with about one phosphate molecule could be used to re-establish the pH gradient. The other two cationic charges on the phosphate molecule could be neutralized by H"1" or metallic cations such as K +, Hg*~*~ or Ca"*-1" or a combination of these. From batch tests with acetate addition reported by Siebritz et a l . (1983b) (Fig. 2.17), i t can be calculated that the molar ratio of phosphate released to acetate added i s 0.5:1.0. This result appears to be significantly different from the result of this research, as well as from the one of Fukase et a l . (1982). Although Siebritz et a l . (1983b) reported that they worked with sludge denitrified by twenty minutes of anaerobic conditions, i t should be noted that in this research the denitrification without substrate addition took about 2.5 hours (see Fig. 5.7). Any nitrate remaining in solution would cause some acetate to be utilized for denitrification instead of phosphate release. 104. 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 Acetate (mmole Ac/g VSS) F i g . 5.17 R e l a t i o n s h i p between the phosphate released and the acetate a v a i l a b l e f o r phosphate r e l e a s e ; see a l s o the footnote of F i g . 5.6. 0.30 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 SRP (mmole P04-P/g VSS) F i g . 5.18 R e l a t i o n s h i p between the SRP concentration and the r a t e of SRP decrease from s o l u t i o n a f t e r n i t r a t e a d d i t i o n ; see a l s o the footnote of F i g . 5.6. 105. Following acetate disappearance, a slower rate of phosphate release was observed for a l l three reactors. As explained in the preliminary experiment, this slow release may have been caused by fermentation or lyzed products, the presence of the buffer or phosphate u t i l i z a t i o n for maintenance energy. It would seem unlikely that fermentation or lyzed products caused a release after a few hours as was observed in the experiment. It should be noted that the results reported by Rabinowitz et a l . (1982, see Fig. 2.16) with sludge from the UBC pilot plant did not show a similar slow release. The presence of a buffer suggested an explanation to the slow release observed. The buffer used was TRIS which is considered non-metabolizable by bacteria. However, due to the metabolic diversity of an activated sludge population, i t may be possible that some bacteria have the a b i l i t y to degrade TRIS into products causing phosphate release. A speculative explanation for the observed phenomenon, may be related to the fact that the presence of a buffer prevented bacteria from establishing a desired pH gradient by neutralizing IT" expelled with phosphate molecules. Thus, the bacteria would continuously transport phosphate until the depletion of the polyphosphate reserves when a buffer Is present. Finally, the slow release observed may be explained by the role of polyT phosphate as an energy source under prolonged anaerobic conditions, or prolonged aerobic conditions (see Fig. 5.5). Thus, polyphosphate degradation for energy purposes would result In phosphate release in solution. For future experiments, due to both the d i f f i c u l t y of obtaining a given pH by simple buffer addition without later pH adjustment, and the unknown effect of buffers on the complex population present in activated sludge, i t is suggested to either simply monitor the pH variations In solution, or to control the pH manually or automatically by addition of a base such as NaOH, or an acid, such as HC1. 106. With the same amount of nitrate added to each reactor, at 5.25 h, the rate of phosphate uptake appeared to be dependent on the phosphate concentration as shown in Fig. 5.18. Such a relationship i s characteristic of carrier-mediated active transport, since i t shows a saturation effect (Brock, 1979). In this case, the maximum rate of phosphate uptake in the given conditions of the experiment can be extrapolated to be about 0.28 mmole P04-P»g VSS - 1»h - 1. Two reactors, 0.5 mM and 1.0 mM acetate, had sufficient phosphate in solution for nitrate addition to not cause complete phosphate uptake. It was significant that in both cases, 10 mg NO^ -N/A added caused a phosphate uptake of 0.18 mmole PO^-P/g VSS, which is equivalent to 0.68 mmole P04-P taken up per mmole NO^ -N added. The fact that nitrate addition resulted in phosphate uptake may be important for optimizing biological phosphorus and nitrogen removal processes. For example, downstream of the anaerobic zone, where phosphate is released, a non-aerated zone could cause denitrification of a n i t r i f i e d recycle where some phosphate would also be taken up. Thus, both excess phosphate uptake and nitrogen removal could occur in the same reactor. B. PHB Acetate addition resulted in PHB formation. In the reactor where 0.5 mM of acetate was added, 69 percent 2 of the acetate added was stored as PHB. In the other reactor (1.0 mM acetate added), 17 percent 3 of the acetate was 2 Acetate stored as PHB = (PHB concentration as HB) x (2 acetate for each HB) * (acetate concentration added). 69% = (0.0075 mmole PHB-HB/g VSS) x (2 Acetate/HB) * (0.022 mmole Acetate/g VSS). 3 17% = (0.0200) x (2) T (0.233) 107. stored as PHB. Such a difference (between 69 percent and 17 percent) can be explained by the inexact estimation of the nitrate concentration, by the fact that only acetate was considered to be used for denitrification, and by the analytical d i f f i c u l t i e s in the determination of PHB in sludge samples. Fukase et a l . (1982) found that 44 percent by weight of acetate was stored as PHB in a lab-scale continuous-flow activated sludge process. C. Metallic Cations A comparison of the curves obtained for SRP, potassium, magnesium, and calcium indicated strong simi l a r i t i e s . Graphs of the concentration of potassium against SRP (Fig. 5.19) and magnesium against SRP (Fig. 5.20) are presented. The data points on the graph are connected according to the chronology of the experiment. For the graph of potassium against SRP, most points fitted on a general line of slope 1 K+/2.9 P i . However, the "control" set of data did not f i t on this general line. For this set, the potassium concentration increased even when no phosphate was released. But when nitrate disappeared, potassium was released with phosphate in a ratio close to the slope of the general line. Then nitrate addition resulted in phosphate and potassium uptake. At the moment where the added nitrate disappeared, a sudden increase in potassium concentration was observed, while phosphate was neither taken up or released. This last sudden potassium increase was also noticed for the other reactors in which 0.5 mM and 1.0 mM acetate was added. This sudden increase in potassium concentration can be explained by the fact that upon nitrate depletion, no more Is expelled via the electron transport chain (see Appendix D). Therefore, i t may be speculated that potassium became involved in maintaining a constant proton motive force and was released in solution for that purpose. 108. Ul Ul > CD 0.25 0.20--g 0.15-0.10-O E E D *</) 00 _D O Fig. 0.05-0.00 0.0 Legend * 0.0 mM Ac » 0.5 mM Ac o 1.0 mM Ac_ * n i t r a t e add, 0.1 0.2 0.3 SRP (mmole P04-P/g VSS) 0.4 5.19 Relationship between the potassium and SRP concentrations; see also the footnote of F i g . 5.6. Ul Ul > CD O E E '(/) <D C CD O 0.10 0.08-0.06-0.04 -0.02 Legend a 0.0 mM Ac » 0.5 mM Ac ° 1.0 mM Ac * n i t r a t e add slope = 1/3.5 Fig. 0.00 * 1 r 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 SRP (mmole P04-P/g VSS) 5.20 Relationship between the magnesium and SRP concentrations; see also the footnote of F i g . 5.6. 109. In Fig. 5.20, a graph of magnesium versus SRP shows that nearly a l l data points f i t well on a broken line. The fact that two slopes f i t the data much better than just one remains unexplained. Under the conditions that were studied, at the phosphate concentration of 0.21 mmole PO^P/g VSS (17.5 mg/Jl) the ratio of Mg^rPi transported changed from 1:4.9 to 1:3.5. From Figs. 5.12 and 5.6, i t can be estimated that the molar ratio of calcium to phosphate transported was about 0.06:1. From the previous results, i t can be calculated (Table 5.10) that about 0.94 moles of positive charge are associated with each mole of transported phosphate (or from 0.87 to 1.04 mole of positive charge per mole of PO^-P considering Mg + +:Pi slopes of 1:4.9 and 1:3.5). This ratio i s very similar to the ratio of associated positive charges i n polyphosphate granules. Indeed, in a polyphosphate chain, one negative charge is neutralized for each phosphate (see Fig. 2.19). This could suggest that the c e l l expels as many positive charges in the form of metallic cations as there was associated with the polyphosphate granules. Electron microscopy studies by Buchan (1981) showed that polyphosphate granules were associated mainly with calcium but also with potassium and magnesium. The metallic cations expelled may not be the same as those stabilizing the structure of the polyphosphate chain, however. Since potassium is the most abundant intracellular monovalent cation (Helmer et a l . , 1982) and magnesium the most abundant divalent cation (Sorensen and Rosen, 1982), i t is logical that these cations could be expelled instead of calcium. 5.7.4 Experiment on the Effect of pH and DNP Introduction Potgieter and Evans (1983) reported that low pH resulted in a higher 110. TABLE 5.10 DETERMINATION OF THE MOLAR RATIO OF POSITIVE CHARGES TO PHOSPHATE MOLECULES TRANSPORTED Metal/Phosphate Charge Moles of Positive Molar Ratio Charge Potassium 1/2.9 +1 = 0.345 Magnesium 1/4.2 +2 = 0.476 Calcium 0.06/1 +2 = 0.120 0.94 (moles of positive charges per mole of phosphate transported) 111. phosphate release than higher pH values (see section 2.3.2). In their experiment, aerobic sludge maintained anaerobically was adjusted to different pH's without substrate addition. To supplement these observations, i t was decided to determine the effects on phosphate release of pH adjustments combined with acetate addition. To change the pH, various buffer solutions were used. During the same experiment, the combined effect of DNP and acetate addition under anaerobic conditions was also studied. The literature showed that the inhibitory effect of DNP had only been tested on phosphate uptake under aerobic condition (see section 2.3.2). The same amount of acetate (1.0 mM) was added to each buffered reactor. The expected pH with 10.0 mM of buffer were pH 7.2 (control) with TRIS buffer, pH 5.9 with potassium hydrogen phthalate (KH phthalate), and pH 8.5 with TRIS. The observed pH values were somewhat different from the expected values and averaged 6.8 (control), 6.7 (KH phthalate) and 7.8 (TRIS) over the time of the experiment. In-reactor pH adjustment would probably be required to achieve a constant pH. The reactor with toxicant (DNP) had the same buffer conditions as the control. The expected pH was 7.2 with TRIS, but a pH 6.7 was observed. DNP was added 10 minutes prior to acetate addition. Results The concentration profile of four parameters directly related to the proton motive force were determined for each reactor, namely SRP (Fig. 5.21), potassium (Fig. 5.22), magnesium (Fig. 5.23) and pH (Fig. 5.24). VSS concentrations averaged 2890 mg VSS/Jl (±50) with a VSS/SS ratio of 80.7 percent for the reactors without DNP. The reactor with DNP had a VSS concen-tration of 2615 mg VSS/A with a VSS/SS ratio of 81.2 percent. 112. 1.2 ^2 to q> 0.8 O Q_ _QJ O E E, 0.6 0.4 CL 0.2 m 0.0 F i g . 5.21 Acetate (DNP) Legend A TRIS - pH 6.8 x -TR1S+DNP - pH 6.7 D KHpntholote - pH 6.7 * TRIS -pH7.8 0 5 10 15 20 25 30 35 40 Time (h) Combined e f f e c t of acetate a d d i t i o n , and pH adjustment or 2,4-dinitrophenol a d d i t i o n on: the SRP p r o f i l e . Batches of sludge were taken from the aerobic zone of the UBC p i l o t p l a n t and kept a n a e r o b i c a l l y f o r the t e s t with a 10.0 mM bu f f e r s o l u t i o n . 0.5 0.4 -CO > CD „ O 0.3 E CO CO o 0_ 0.2 -0.1-0.0 Acetate (DNP) Legend A TRIS - pH 6 8 x TRlS-hDNP - pH 6.7 o TRIS - pH 7.8 10 15 20 25 Time (h) 30 35 40 F i g . 5.22 Combined e f f e c t of acetate a d d i t i o n , and pH adjustment or 2,4-dinitrophenol a d d i t i o n on: the potassium p r o f i l e ; see a l s o the footnote of F i g . 5.21. 113. Ul Ul > CD O E E, E c CD O 0.5 0 .4-0 .3-0.2 -0.1-0.0 Legend * TRIS - pH 6.8 x -TRIS+DNP - pH 6.7 o KHphtholatc - pH 6.7 * -TRIS - pH 7.8 Acetate (DNP) 10 1^ 15 20 25 Time (h) 30 35 40 F i g . 5.23 Combined e f f e c t of acetate a d d i t i o n , and pH adjustment or 2,4-dinitrophenol a d d i t i o n on: the magnesium p r o f i l e ; see a l s o the footnote of F i g . 5.21. CL 9.0 8.5 8.0 7.5 -7.0-6.5 6.0 Acetate (DNP) r- b u f f e r add. (pK 7.8) a a — a — —. Legend A TRIS - pH 6.8 TRlS-rONP - pn 6 7 KHpbtnolot* - pH 6.7 TRlS - pH 7 8 • : 3 - a -b u f f e r add. (pH 6.7-KKP) 10 15 20 25 30 Time (h) 35 40 F i g . 5.24 Combined e f f e c t of acetate a d d i t i o n , and pH adjustment or 2,4-dinitrophenol a d d i t i o n on: the pH p r o f i l e ; see als o the footnote of F i g . 5.21. 114. Discussion Following the rapid phosphate release caused by acetate addition, a slower, but f a i r l y rapid rate of phosphate release was observed in a l l reac-tors. A similar slow release was discussed with the interpretation of the previous experiment. For the reactor with TRIS buffer (pH 6.8), a plateau was observed in the SRP profile between 3 and 5 hours. It is suspected that the injection of the acetate solution in the reactor was also accompanied by the injection of some ai r that was present in the syringe barrel. The consequent phosphate uptake combined with phosphate released, resulted in a plateau in the SRP profile. A higher pH (7.8) resulted in a greater magnitude of rapid phosphate release (about 0.41 mole PO^-P/g VSS), than observed at a lower pH (6.7) (about 0.20 mmole PO^-P/g VSS). These results are i n agreement with the postulated model for anaerobic conditions of section 3.3. Indeed, a lower pH in solution would make i t easier for the bacteria to take up acetate since more external are avail-able for transport, while s t i l l maintaining a desired pH gradient. Thus, less phosphate should be released at a lower pH, as was observed. Potgieter and Evans (1983) have reported that a lower pH caused more phosphate to be released when no substrate was added (see Table 2.2). Their results also showed that no phosphate release or uptake occurred at a pH of about 7.8. Above this value, phosphate uptake was reported. These results are opposed to those of our observations and are d i f f i c u l t to explain on the basis of phosphate expulsion playing a role of a hydrogen ion carrier to maintain a given pH gradient. More experiments should be conducted to c l a r i f y the effect of pH on phosphate and cations transport. The presence of dinitrophenol at a pH of 6.7 resulted in a high level of 115. rapid phosphate release (about 0.50 mmole PO^-P/g VSS). Within 8 hours of anaerobic conditions, the SRP concentration in the reactor with DNP was about as high as after 35 hours in the other reactors (0.78 mmole PO^-P/g VSS). Having started with the same sludge, this released phosphate probably indicated a complete exhaustion of the internal polyphosphate reserves of bio-P bacteria. The effect of DNP i s to cancel the pH gradient across the inner membrane of a bacterial c e l l by "channelling" hydrogen ions across the membrane (Lehninger, 1982 and see Appendix D). Since the role of phosphate i s postu-lated to be one of a hydrogen ion carrier re-establishing the pH gradient, as expected, the addition of DNP combined with acetate resulted In a higher level of phosphate release than the addition of acetate alone. B. pH From Fig. 5.24, i t can be seen that with acetate addition to the reactors, the pH slightly Increased except for the reactor that had DNP. In this latter reactor, i t decreased for about one hour before going up. The sudden pH jumps for the low and high pH reactors are due to injections of an additional 10.0 nM solution of buffer. Since buffers are present, i t is d i f f i c u l t to discuss the biochemical significance of the slight pH Increase and decrease observed. C. Metallic Cations As in the previous experiment, the general aspect of the concentration profiles for potassium and magnesium are very similar to the profile for SRP (Figs. 5.21, 5.22, 5.23). Figs. 5.25 and 5.26 show the correlation between potassium and SRP, and magnesium and SRP, respectively. Slopes calculated by 116. 0.5 SRP (mmole P04-P/g VSS) F i g . 5.25 R e l a t i o n s h i p between the potassium and SRP concentrations; see a l s o the footnote of F i g . 5.21. Ul Ul > CD o E E E 'on <D C CD D 0.30 0.25-0.20-0.15 0.00 slope = 1/2.9 / L/f ft >^ / 7 3 . 6 ^ b u f f e r a d d i t i o n V Legend * TRIS - pH 6.8 1/4.0 yTy^ •^T/4.1 * TRIS+DNP - pH 6.7 = KHphthalote - pH 6.7 • TRIS - pH 7.8 F i g . 0 0.2 0.4 0.6 0.8 1 SRP (mmole P04-P/g VSS) 5.26 R e l a t i o n s h i p between the magnesium and SRP concentrations; see a l s o the footnote of F i g . 5.21. 117. least-squares method are provided on the graph. Since KH phthalate was used as buffer in the low pH reactor, potassium concentrations were not measured in this reactor. With most data points of Fig. 5.25 f i t t i n g on a straight line, the ratio of released potassium to phosphate (K +:Pi) is calculated to be 1:3.1. In the previous experiment, i t was very similar, being 1:2.9. A constant K +:Pi ratio for a l l reactors would support the idea that the ratio of potassium to phosphate released (or taken up as seen in the previous experiment where nitrate was added) is not dependent on small changes in the external pH or on the addition of DNP when a given amount of a substrate is added (1.0 nM in this case). Four lines are observed to f i t the data points on the graph of Mg"1-*" versus SRP (Fig. 5.26). The results from both the control (pH 6.8) and the reactor with DNP f i t well on the same line of slope 1:3.6 (Mg + +:Pi). In the reactor with a high pH (7.8) a slope f a i r l y close to the one of the control is observed (1:4.1). An extrapolation of the line indicates that no magnesium accompanied phosphate release prior to the release of about 0.07 mmole PO^-P/g VSS i n solution. The major differences between the pH 6.8 and pH 7.8 reactors were the pH and the concentration of chloride ion (from HC1) used to adjust the pH of the buffer. The pH 6.8 reactor had much more chloride ions than the pH 7.8 reactor. In this latter reactor, some buffer was added during the experiment but no change was observed in the best-fit line. The gap between the lines of the control (pH 6.8) and the pH 7.8 reactors can be explained by the much higher concentration of chloride ions in the control reactor. A higher chloride concentration gradient across the bacterial membrane appeared to cause some Mg"*-1" to be released in solution. 118. Such a mechanism would allow cells to re-establish the charge gradient across their membrane. This mechanism appears to be peculiar in that potassium, and not magnesium has been described to play the role of maintaining a constant proton motive force (Yamasaki et a l . , 1980; Bakker and Mangerich, 1981, and Schuldiner and Padan, 1982). For the low pH reactor (6.7) with KH phthalate, two lines f i t the data points. Until the addition of a buffer solution, the slope (Mg"'"*':?!) was similar to the slope of the control and pH 7.8 reactors. With buffer addition, a sudden Mg"***" release was observed (equivalent to 0.045 mM Mg"*-1") and the slope became steeper, indicating that more Mg"*"*" were transported with each Pi expelled into solution. Since there was practically no pH difference between the two reactors (pH's of 6.7 and 6.8), such a Mg + + release could probably be related to the type of buffer added. The chemical structure of phthalate is a benzene ring with two carboxylic groups. Such a structure can give chelating properties to phthalate. Nicas and Hancock (1980) reported that Pseudomonas aeruginosa can show a high content of Mg"*-*" in their c e l l envelope. Thus, the addition of two 10.0 mM aliquots of KH phthalate to an activated sludge sample could well have resulted in step increases in the soluble Mg"1 "^ concentration due to the chelation of magnesium from the c e l l wall of bacteria by phthalate molecules. 5.8. Consistency of the Models with Observations 5.8.1. Introduction An ideal biochemical model should explain a l l observations and allow someone to forecast the behavior of bio-P bacteria under any given set of conditions. 119. In the discussion of the results from this research, the observations were satisfactorily explained by the postulated biochemical models presented in Chapter 3. Where re evant, information from the literature was discussed at the same time. However, from these discussions, two discrepancies between the results and the Information published in the literature appeared. The f i r s t one i s related to the molar ratio of acetate taken up to phosphate released (APi/AAc) under anaerobic conditions. This ratio gave roughly 1.0:1.0 in this research, and (1/1.1):1.0 according to Fukase et a l . (1982). Siebritz et a l . (1983b), however, reported a ratio of 0.5:1.0. It Is believed that both the presence of nitrates and the pH value w i l l affect the (APi/AAc) molar ratio. Further investigation to c l a r i f y this aspect Is suggested. The other discrepancy is related to the effect of the external pH on phosphate release under anaerobic conditions. In this research, a higher pH with acetate addition resulted in a higher level of phosphate release as was expected according to the postulated model (sections 3.3 and Potgieter and Evans (1983) reported that a lower pH resulted in a higher level of phosphate release (see Table 2.2) which is opposed to the prediction of the proposed model. These authors did not add substrate, as was done In this research. Further experimentation is also recommended to c l a r i f y the effect of pH on phosphate release. In this section, the information not highlighted in the discussion of the results, and the information from the literature that has not been discussed, is reviewed and used to verify the consistency of the biochemical models with the observations. 120. 5.8.2. Aerobic Conditions The postulated model for aerobic conditions was summarized in Figs. 3.1 and 3.2. 1. In presence of oxygen and availability of stored PHB, , phosphate was taken up from solution (see Fig. 5.5a, and Marais et a l . , 1983). Since bio-P bacteria have a high level of energy from PHB degradation, they can accumulate phosphate and store It as polyphosphate. 2. In the presence of nitrate, but no oxygen, phosphate was taken up from solution in a molar ratio of phosphate to nitrate of 0.68:1.0 (see Fig. 5.6). Thus, the role of nitrate was apparently similar to oxygen which is to serve as an electron acceptor for the electron transport chain (see Appendix D). It appears that at least a fraction of the bio-P bacteria can u t i l i z e nitrate for this purpose. This aspect w i l l be further discussed in Section 3. PHB was depleted under aerobic conditions (Fig. 5.5b). The basic advantage of bio-P bacteria is to have access to an intracellular reserve of carbon for energy production. As long as the c e l l u t i l i z e s energy for polyphosphate accumulation, reproduction, or c e l l maintenance, the PHB reserves are expected to decrease in size. 4. In presence of 2,4-dinitrophenol (DNP), phosphate uptake from solution is prevented (see Fig. 2.12). Phosphate is taken up from solution by two transport systems, one coupled to ATP for energy, and the other coupled to the proton motive force (Russell and Rosenberg, 1979). DNP is a molecule that "channels" hydrogen ions across the membrane causing depletion of the pH gradient (see Appendix D). Thus, i t is expected that without a pH gradient, cells cannot produce ATP and w i l l not be able to take up phosphate by any of the two phosphate transport systems. 121. 5.8.3. Anaerobic Conditions Anaerobic conditions should be understood as the absence of free oxygen and nitrate In solution. The postulated model was shown in Fig. 3.3 and speculations on energy sources were presented in Figs. 3.4, 3.5 and 3.6. 1. Phosphate was released in solution when acetate was added (see Fig. 5.6). According to the model, acetate uptake caused the pH gradient across the membrane to be transiently decreased. To re-establish the pH gradient, bio-P bacteria expelled phosphate as a neutral molecule. Outside the membrane, phosphate molecules dissociating in their monovalent and divalent species released H+ to re-establish the pH gradient. 2. With nitrate added to a continuous-flow non-aerated reactor, either phosphate release or uptake was observed. Similarly, air entrained from too vigorous mixing was also reported to cause either a net phosphate release or uptake in the reactor (Oldham, 1984). These two observations can be explained by the fact that both phosphate release, performed by bio-P bacteria storing carbon, and phosphate storage, performed by bio-P bacteria u t i l i z i n g the available electron acceptors (nitrate or oxygen), occurred at the same time. The net result was probably dependent on the total amount of nitrate added or oxygen entrained. 3. Studying the effect of glucose, Fukase et a l . (1982) observed that sludge capable of bio-P removal and acclimated to a glucose feed, did not store PHB but rather glycogen under anaerobic conditions. Although in Fig. 3.6, i t is speculated that glucose may be stored in bio-P bacteria as PHB, i t is quite possible that bacteria could also store glycogen instead of PHB i f the feed is glucose-rich. For most sewages, however, i t is expected that sugars would be fermented. Indeed, 12 2. fermentation provides a source of energy as ATP. Since VFA's are a common waste product of fermentation, the released VFA's could then be stored as PHB i n bio-P bacteria, thus causing phosphate release. The level of phosphate release is reported to be about half as high with glucose as i t is with acetate, for identical levels of COD addition (same number of carbon atoms), (Fig. 2.18 and Table 2.1). The lower degree of phosphate release by glucose may be explained by the fact that only a fraction of the carbon atoms of glucose are found as VFA's which are in turn are stored in bio-P bacteria. A certain fraction could also be lost as C0 2, cellular material, or utilized by non bio-P bacteria. 4. Phosphate release is not as high with acetic acid as i t is with sodium acetate for the same COD concentration (see Fig. 2.18). Both acetate and acetic acid should be found in solution i n the anionic form at a normal pH of activated sludge (pH of about 6.5). Also, both compounds w i l l be transported intracellularly as a neutral molecule with H+. But with more H + added with the acid than with the salt, i t is possible that less protons of the proton motive force w i l l be carried in with the acid than with the salt. Therefore, less phosphate would have to be expelled with the acid than with the salt, as Is reported. Thus, for equivalent COD additions, the same amount of PHB could be expected to be found in bio-P bacteria although less phosphate would be expected to be released with acetic acid than with sodium acetate. PHB measurements under such conditions would be desirable to verify the proposed explanation. 5. Carbon dioxide bubbled in an anaerobic reactor containing a culture of Acinetobacter bacteria capable of bio-P removal, caused immediate phos-phate release (see Fig. 2.14). Bubbling carbon dioxide in solution decreases the pH of a solution. Fuhs and Chen (1975) thought that a 123. microsomal pH lowering while the pH of the bulk liquid remained near neutrality was responsible for phosphate release. An alternative explanation can be proposed from the model. It i s known that C0 2 is transported across c e l l membranes by passive diffusion (Thauer et a l . , 1977 and see Appendix E). Thus, C0 2 bubbling can result in CO2 accumulation inside bio-P bacteria. Once inside, the C02 could be hydrated into R^CO^ and then reach equilibrium with the other carbonate species. In reaching the equilibrium, some H+ would be released intracellularly, thus decreasing the pH gradient across the membrane. In a similar mechanism as outlined with the biochemical model for anaerobic conditions, phosphate molecules would be expelled from bio-P bacteria in order to re-establish the pH gradient. 6. Propionate and acetate added in the same COD ratio resulted in the same magnitude of phosphate release (Fig. 2.18 and Table 2.1). From the model presented in Fig. 3.3, i t would be expected that the same molar, and not the same COD, ratio of acetate and propionate would cause identical phosphate release. Indeed, for each acetate or propionate molecule transported inward, one proton is carried along which requires the same number of phosphate molecules to be released in both cases to re-establish the pH gradient. From the observed result, for equlmolar additions, the ratio of phosphate released by propionate as compared to acetate should be 1.755. Since the metabolic processing of propionate to acetyl CoA and PHB ** Hydration of C0 2 i s the rate-limiting step in the carbonate system equilibrium (Stumm and Morgan, 1970). 5 Th.O.D. (propionate) * Th.O.D. (acetate) = 1.75. The ratio of Theoretical Oxygen Demand (Th.O.D.) i s used to approximate the COD ratio. 124. probably requires the release of one C0 2 molecule, i t is possible that this C(>2 Is responsible for the increased phosphate expulsion. As previously explained; the surplus of C0 2 could cause a transient intracellular accumulation, causing expulsion of phosphate. The cumulative effect of propionate transport, and C0 2 production could be responsible for the high phosphate release observed with propionate. 5.9. Applications Suggested by the Model Applications are given which aim at improving the efficiency of bio-P removal processes. They were derived from an understanding of the postulated models for aerobic and anaerobic conditions. The ideas are grouped into carbon storage, and a discussion of nutrient removal. The objective of this section i s not to give an exhaustive l i s t of possible applications for bio-P removal, but rather to show how biochemical models that satisfactorily explain the bio-P processes, can be applied to better design and operation of bio-P treatment plants. 5.9.1. Carbon Storage The role of the anaerobic zone (in which there is no free oxygen and no nitrate) is proposed to be the maximization of PHB storage In bio-P bacteria. For this reason, i t is called the "carbon storage" zone. This role is justified by the proposition that the storage of carbon as PHB provides the bio-P bacteria with the advantage of being able to sequester anaerobically a significant portion of the available substrate. This advantage w i l l be manifested in a greater a b i l i t y of the sludge to remove 125. phosphate from solution under subsequent aerobic conditions. Factors Involved in maximizing the amount of carbon stored as PHB include (1) maximizing the amount of readily storable compounds such as VFA's, (2) the absence of electron acceptors such as oxygen or nitrate, and (3) maximizing the polyphosphate reserves in bio-P bacteria. With these considerations, one can easily assess the pertinence of a proposed modifica-tion to a bio-P process at either the design or the upgrading stage. The available amount of substrate that can be stored as PHB influences directly the amount of PHB that can be accumulated in the carbon storage zone. Thus, the fermentation of primary sludge can be optimized to maximize the yield of VFA's produced to be added i n the carbon storage zone. Wastewater fermentation, which was reported to occur in long sewers (Barnard, 1983), can contribute to the amount of VFA's added to the carbon storage zone. Likewise, fermentation in a non-aerated reactor of the process with a sufficiently long hydraulic retention time can also produce VFA's, as was proposed by Rensink et a l . (1981). In addition, combining municipal influent and an industrial wastewater that is rich i n VFA's, or in other compounds that can be degraded easily into VFA's, can contribute to maximize the amount of PHB stored. Food processing wastewater i s an example of such an industrial effluent. Finally, a direct addition of VFA salts such as sodium acetate, can also supplement the VFA's available for carbon storage. Minimizing the amount of oxygen or nitrate present upstream of, or in the carbon storage zone i s particularly Important. Indeed, in presence of such electron acceptors, storable carbon substrates would be preferentially oxidized for energy production instead of being stored for subsequent aerobic phosphate uptake. 126. To minimize the amount of oxygen in the wastewater entering the bio-reactor, air-entraining equipment should be avoided. Thus, the use of screw pumps, hydraulic jumps, aerated grit chambers or any device entraining a i r should be prevented. Air entrainment in the carbon storage zone can also occur by a too vigorous mixing of the anaerobic reactor (Oldham, 1984). Sludge recirculation may also entrain oxygen i f the dissolved oxygen level of the aerobic reactor i s too high (Barnard, 1983). Similarly, oxygen entrain-ment from the sludge recycle may happen i f a screw pump or a turbulent open-channel i s used to return the sludge from the secondary c l a r i f i e r . Minimizing nitrate entrainment in the carbon storage zone is of prime importance. Thus, denitrification of aerobic sludge should not be performed in the carbon storage zone as is done In the Modified Ludzack and Ettinger, and the Bardenpho processes (see Figs. 2.4 and 2.6). In addition, the return sludge should be denitrified before i t is recirculated to the carbon storage zone. This pre-denitrification step is not done in the Phostrip, the Phoredox and the Modified Phoredox processes (see Figs. 2.5, 2.7 and 2.8). The UCT and the Modified UCT processes, however, have provisions for nitrate removal from the return sludge (see Figs. 2.9 and 2.10). In the case of the Modified UCT process, the return sludge and the aerated sludge are even denitrified in different reactors. ORP measurements can serve as a useful parameter to insure the absence of oxygen and nitrate recirculation into the carbon storage zone (Koch and Oldham, 1984). Finally, maximizing the polyphosphate reserves in bio-P bacteria means that the TP/COD ratio of the influent wastewater should be high enough to allow bio-P bacteria to accumulate sufficient polyphosphate reserves to accumulate the available storable carbon as PHB. Besides Influent wastewater 127. characteristics, the design and operation of the plant should provide the opportunity for bio-P bacteria to maximize their polyphosphate content in the aerobic zone. For this purpose, i t is li k e l y that the most important parameters are the dissolved oxygen level, the degree of n i t r i f i c a t i o n , the hydraulic retention time, the sludge retention and the temperature of the aerobic zone. A l l these parameters should affect the degree of polyphosphate storage in bio-P bacteria as well as the effluent TP level achieved (less than 0.5 mg PO^-P/A can be attained; Oldham et a l . , 1984). 5.9.2. Discussion of Nutrient Removal Processes Of the available processes aiming at biological nitrogen and phosphorus removal, the Modified UCT process appears to best f u l f i l l the previously formulated requirements for the carbon storage zone. Indeed, oxygen and nitrate of the sludge recycle stream are removed in a non-aerated reactor before the sludge is recirculated to the carbon storage zone. In addition, aerated sludge is not recirculated into the carbon storage zone. It is proposed that the optimum process for combined bio-P and N removal would be a Modified UCT process where primary fermented liquor would be added to the carbon storage zone. The other suggestions previously formulated regarding the carbon storage zone (see Section 5.9.1) should also be considered for an optimum process. If only phosphorus and not nitrogen is desired to be removed from a given wastewater, the optimum process is proposed to be as shown in Fig. 5.27. In this process, fermented primary sludge is added to the carbon storage zone and the sludge recycle is denitrified prior to addition to the carbon storage zone. No aerobic recycle i s provided. It i s expected that the sludge retention time would be significantly lower than for processes RETURN CARBON SLUDGE STORAGE DENITRIFICATION AEROBIC REACTOR REACTOR REACTOR (OPTIONAL DENITRIFIED ACETATE SLUDGE SLUDGE ADDITION) RECYCLE ' WASTAGE RAW SEWAGE INFLUENT FERMENTER F i g . 5.27 Proposed optimum process f o r bio-P removal. 129. requiring n i t r i f i c a t i o n . A modification of such a process could be easily applied for upgrading existing plants. It would consist of reversing the order of the two non-aerated zones as shown in Fig. 5.28. Such a process would provide similar advantages to these of the proposed optimum process for bio-P removal, but would eliminate the need of a denitrified sludge recycle to the carbon storage zone. Removing this recycle could translate Into significant savings due to lower pumping costs. A disadvantage of this process, however, is related to the u t i l i z a t i o n of storable substrate compounds present In the primary supernatant for denitrification rather than for carbon storage as PHB. A second disadvantage would be that simultaneous phosphate uptake and denitrification would not be allowed to occur in a non-aerated reactor located downstream of the carbon storage reactor. This last process could be modified for bio-P and N removal by adding an aerobic sludge denitrification zone downstream of the carbon storage zone (see Fig. 5.29). RETURN SLUDGE DENITRI- CARBON FICATION STORAGE AEROBIC REACTOR REACTOR REACTOR RAW SEWAGE INFLUENT PRIMARY CLARIFIER (OPTIONAL ACETATE ADDITION) SLUDGE WASTAGE FERMENTER SECONDARY CLARIFIER EFFLUENT >( SLUDGE WASTAGE) F i g . 5.28 A proposed e f f i c i e n t process f o r p l a n t upgrading aiming at bio-P removal. RETURN SLUDGE DENITRI-FICATION REACTOR MIXED CARBON LIQUOR STORAGE DENITRIFICATION REACTOR REACTOR AEROBIC REACTOR (OPTIONAL ACETATE ADDITION) MIXED LIQUOR RECYCLE (a) SLUDGE WASTAGE A RAW SEWAGE INFLUENT PRIMARY CLARIFIER SECONDARY CLARIFIER EFFLUENT > >( SLUDGE WASTAGE) FERMENTER F i g . 5.29 A proposed e f f i c i e n t process f o r plant upgrading aiming at bio-P and N removal. 132. 6. CONCLUSIONS AND RECOMMENDATIONS 6.1. Conclusions This research on bio-P removal contributed new observations and verified others from the literature. Biochemical models explaining the behavior of bio-P bacteria were also postulated. The major experimental observations and the essence of the models are summarized here. 6.1.1. Observations from Batch Tests From batch testing under aerobic conditions, PHB reserves were observed to decrease while phosphate was removed from solution. Under anaerobic conditions, the following observations were made with acetate addition: (1) phosphate was released un t i l no more acetate was available for uptake from solution. Acetate limiting conditions were not reached, however; (2) potassium, magnesium and calcium appeared to be co-transported with phosphate across bacterial membranes; (3) the molar ratio of the sum of metallic cations (potassium, magnesium, calcium) to phosphate expelled was about 0.94; (4) PHB accumulated as shown by both cytological staining and GC measurements; (5) with DNP added, more phosphate was released than without DNP addition; and (6) with a higher pH (7.8), more phosphate was released than at a lower pH (6.8). It was also observed that under anaerobic conditions, nitrate addition caused 133. phosphate uptake from solution. 6.1.2. Model for Bio-P Removal Bacteria responsible for biological excess phosphorus removal were called bio-P bacteria. It was supposed that they have the particular a b i l i t y to store both polyphosphate and PHB (or glycogen) as reserve materials. Aerobic u t i l i z a t i o n of PHB in bacteria serves for energy production. Thus, aerobic phosphate uptake from solution and storage in bio-P bacteria as polyphosphate is presumably triggered by a high intracellular level of ATP made possible by the avail a b i l i t y of PHB. Otherwise, the strong competition for substrate in the aerobic zone of activated sludge plants would not allow bacteria that "wasted" their energy In polyphosphate storage to proliferate. Nitrate addition in absence of free oxygen can also result in phosphate uptake. PHB reserves are formed under anaerobic conditions due to the presence of polyphosphates In bio-P bacteria. The actual role and mechanism by which polyphosphate i s involved i s s t i l l controversial, but i t i s postulated that i t serves for proton expulsion from the c e l l . Indeed, acetate transport in the c e l l removes a hydrogen ion from outside the c e l l membrane and thus decreases both the pH gradient and the proton motive force. Since the proton motive force must remain constant, bio-P bacteria expelling neutral phosphate molecules are proposed to re-equilibrate the transiently decreased pH gradient across the membrane. The role of polyphosphate i n that mechanism is thus to supply phosphate molecules that can be expelled. For PHB formation from substrates such as acetate, i t was speculated that polyphosphate could play a direct role of an energy source by the phosphorylation of transported carbon substrates. On the other hand, the 134. carrier enzyme expelling phosphate could be activated by the presence of high intracellular inorganic phosphate concentration resulting from polyphosphate degradation. The postulated model was found to be conceptually consistent with most observations. The effect of external pH and the molar ratio of acetate taken up to phosphate released anaerobically, however, remain to be c l a r i f i e d . From the understanding provided by this postulated model, a number of applications for bio-P plants were suggested. They included recommendations concerning carbon storage and a discussion of optimum bio-P processes. This postulated biochemical model for bio-P removal provides a valuable tool for designing experiments to test i t s validity and for assessing the pertinence of a design or an operational modification on bio-P plants. 6.2. Recommendations The following recommendations should confirm various aspects of the proposed biochemical models for bio-P removal. 1. A significant portion of this research dealt with reviewing the literature and providing selected experimental observations In order to propose a satisfactory biochemical model that would explain the behavior of bio-P bacteria. A particular effort was made to apply principles of bacterial biochemistry and microbiology to develop a valuable model. More batch tests are required to verify and improve i t s validity. Unless pure culture studies are undertaken, some aspects of the model should always remain speculative. 2. For future batch tests, improved analytical techniques for measuring PHB and polyphosphate would be desirable. Pre-denitrification of the sludge prior to batch testing would allow the effects of the addition of a 135. chemical to be better characterized. Thus, more precise relationships between parameters, such as polyphosphate, PHB, nitrate and SRP could be defined. 3. Batch tests to be conducted under anaerobic conditions should focus on defining the role of polyphosphate and characterizing the transport and storage of substrate into PHB. The relationships between polyphosphate uti l i z a t i o n and PHB formation should be established for various types of substrates such as VFA's, sugars, amino acids, alcohols, various combination of these substrates, and eventually raw sewage. The effect of pH on these relationships should also be established. To c l a r i f y the role of polyphosphate, tests should be conducted with different sludges varying in their polyphosphate content. To describe correctly how substrates are transported across the c e l l membrane, transport mechanisms could be studied by using various types of toxicants such as DNP, valinomycin and cyanide. 4. Batch tests conducted under aerobic conditions should concentrate on explaining the role of PHB as related to phosphate uptake. Comparing the use of various electron acceptors such as oxygen, nitrate and n i t r i t e , should help to define the rate of PHB u t i l i z a t i o n , the rate of phosphate uptake, and the degree to which phosphate uptake can be achieved (down to less than 0.5 mg PO^-P/A, for example). These experiments should also be repeated with sludges containing various levels of PHB, and in presence of soluble substrates added during aeration. 5. As previously mentioned, pure culture studies would be the only reliable method to verify the actual biochemical mechanisms. Postulated bio-chemical models based on observations made on bio-P activated sludge can prove very useful to understand and predict the behavior of bio-P bacteria, but no definite conclusions can be drawn on actual cellular mechanisms without pure culture studies. Peculiar a b i l i t i e s of the bio-P bacteria that deserve to be elucidated, include the mechanism of phosphate expulsion from the c e l l , and the co-transport of phosphate and metallic cations. The proposed role of phosphate to re-establish a transient decrease in the pH gradient by expulsion of H + also deserves a major consideration. In addition, the transport mechanisms of Mg when a large amount of Ci~ i s added could be investigated. The source of energy for substrate storage should be determined and the speculated role of polyphosphate for that purpose be considered. A d i f f i c u l t y in working with pure cultures resides in the loss of microbial species interactions that might have an Important role for blo-P removal. For example, sugar fermentation may not occur in a given culture. Another problem Is related to the various metabolic capacities, such as denitri-fication or glycogen storage of various types of bio-P bacteria. This means that many species would probably have to be isolated and studied. This latter kind of work would be better tackled by microbiologists than environmental engineers since a special training in microbiology techniques is required. Therefore, i t appears that a collaborative effort between microbiologists and environmental engineers would seem most appropriate to verify the validity of the postulated models for biological excess phosphorus removal. 137. BIBLIOGRAPHY Amer. Public Health Assn. (A.P.H.A.). Standard Methods: for the Examination of Water and Wastewater. Washington, D.C.: A.P.H.A., 15th ed., 1980. Apostolides, Z., and D.J.J. Potgieter. "Determination of PHB In Activated Sludge by a Gas Chromatographic Method." European J . Appl. Microbiol. Biotechnol., 13 (1981), 62-63. Ashley, N.V., and Tamara J. Hurst. "Acid and Alkaline Phosphatase Activity in Anaerobic Digested Sludge: A Biochemical Predictor of Digester Failure." Water Res., 15 (1981), 633-638. 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APPENDIX A SYMBOLS A/O process : Anaerobic/Oxic process Ac Acetate ADP : adenosine diphosphate AHP : acid-hydrolyzable phosphorus ATP : adenosine triphosphate bio-P : (related to) biological excess phosphorus (removal) COD chemical oxygen demand C.V. : coefficient of variation DNP : 2,4-dinitrophenol DO : dissolved oxygen FAD+ : flavin adenine dinucleotide FADH2 : dihydrogen flavin adenine dinucleotide GC : gas chromatograph, or gas chromatography GC/MS : GC-mass spectrometer, or GC-mass spectrometry GC r GC-peak retention time GDP : guanosine diphosphate GTP : guanosine triphosphate H + hydrogen ion; proton HB : hydroxybutyrate KH phthalate : potassium hydrogen phthalate (a buffer) MLE : Modified Ludzack Ettinger process a Other abbreviations used include the SI units and the chemical elements of the periodic table. N : nitrogen P : phosphorus P l : phosphate PHB : poly-g-hydroxybutyrate pmf : proton motive force (Au +) H PN : p-nitrophenol PNP : p-nitrophenol phosphate PolyP : polyphosphate PPi : pyrophosphate S : standard deviation SRP : soluble reactive phosphorus SS : suspended solids (total nonfiltrable residue) STD : standard (for calibration curves) TCA : tricarboxylic acid cycle (Krebs cycle) TKN : total Kjeldahl nitrogen TP : total phosphorus TRIS : t r i s (hydroxymethyl) aminoraethane (a buffer) UBC : University of British Columbia UCT : University of Capetown VFA : volatile fatty acid VSS : volatile suspended solids (non-filtrable volatile residue) X : average ApH : pH gradient (of the pmf) : proton motive force (pmf) : charge gradient (of the pmf) 147. APPENDIX B GLOSSARY8 aerobic reactor : anaerobic reactor: anoxic reactor: bio-P bacteria: bio-P process: bio-P removal: bio-P and N removal: bioreactor: carbon storage reactor: denitrification reactor: the aerated reactor in a treatment plant; (see carbon storage reactor); (see denitrification reactor); bacteria responsible for bio-P removal; they are proposed to be capable of both polyphosphate and PHB storage; a treatment plant process aiming at bio-P removal; biological excess phosphorus removal by a modified activated sludge treatment plant; combined biological nitrogen and bio-P removal: the basin(s) that include the various reactors of the activated sludge process (excluding the secondary c l a r i f i e r ) ; a non-aerated reactor in which phosphate is released from the sludge mass, and storable carbon substrates stored as PHB; a non-aerated reactor in which nitrates are removed by denitrification; a The purpose of this GLOSSARY i s to provide a definition to engineering terms that were used in this thesis for the description of bio-P removal phenomena. b In the thesis the words "reactor" and "zone" are used interchangeably. 148. the bacterial process by which ammonia is converted to nitrate; the overall phosphorus removal from a treatment process; microbial phosphate release from the sludge mass to the solution; microbial phosphate uptake from the solution by the sludge mass; carbon compounds which when added to the carbon storage reactor can be stored as PHB (e.g.: VFA's). 149. APPENDIX C Glycolysis Pathway3 and TCA Cycle: Outline and Regulation In Fig. C-l is shown an outline of the glycolysis pathway and of the TCA cycle. The degradation of fatty acids or amino acids into acetyl CoA i s represented. Some elements of regulation of these metabolic processes i s also provided. This information is shown here as a complementary reference to the simplified pathways described in the thesis. For more information on these processes, refer to Brock (1979), Lehninger (1982), or Stryer (1981). a The glycolysis pathway i s also known as the Embden-Meyerhoff pathway (MP). 150. Gli L-ATP J*ADP *ADGlucose 6-phosphate Fructose 6-phosphate -ATP •ADP Fructose 1,6-diphosphate f Dihydroxyacetone phosphate Glyceraldehyde 3-phosphate J Glyceraldehyde 3-phosphate ^NAD* + P, ^NADH 1,3-Diphosphoglycerate ^ADP S»ATP 3-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate ADP ^ATP Pyruvate + 1 NAD ^ T-NADH-4 Acetyl CoA Inhibited by ATP. acetyl CoA. and NADH Inhibited by ATP Oxalo-ses tate /^NADH A NAD+ Citrate Malate \ c/'s-Aconitate Fumarate t » FADK \- FAD** Succinate \ Isocitrate NAD + y NADH ^ 7 Stimulated by ADP a-Ketoglutarate Succinyl P » J » Inhibited by succinyl CoA NAD + N A D H NADH F i g . C - l O u t l i n e and r e g u l a t i o n of the g l y c o l y s i s pathway and of the TCA c y c l e (adapted from S t r y e r , 1981). 151. APPENDIX D Bioenergetics: The Proton-Motive Force The f i e l d of bioenergetics deals with energy transformation i n organisms. A major aspect of bioenergetics for bacteria i s their need to establish a proton motive force (pmf). The pmf is a force resulting from two gradients across the bacterial membrane. The f i r s t component results from a charge gradient such that there is a net negative charge inside the c e l l membrane as compared to the outside (interior negative). The second component is a pH gradient such that more H+ are present outside than inside the membrane (interior alkaline). As derived from the Nernst equation, the equation for the proton-motive is written as: Au^f = Aip - ( 2.3 RT F -) ApH (D-l) where, A uo+ = proton motive force Ai|/ = charge gradient ApH = pH gradient R = gas constant T = temperature, °K F = Faraday constant and 2.3 RT/F 60 mV As an example, with a charge gradient of 140 mV (interior negative) and a pH gradient of 1.4 (interior alkaline), the resulting pmf is 220 mV 152. (Harold, 1977). The major roles of the pmf are: (1) to produce ATP by the membrane-bound ATP-ase enzyme, (2) to transport substrates (see Appendix-E), and (3) to provide energy for cellular movement (Harold, 1977, Harold, 1978). To establish a proton-motive force, non-photosynthetic bacteria can translocate protons by three major mechanisms. The f i r s t one is of major importance and makes use of the electron transport chain to expel H* from the c e l l when substrates and an electron acceptor (mainly oxygen or nitrate) are present. This process i s called respiration. Substrates should f i r s t be processed via the glycolysis or TCA cycle to produce NADH that are used at the electron transport chain. In absence of electron acceptors, the accumu-lation of NADH w i l l Inhibit further NADH production from metabolic pathways. In absence of electron acceptors, ATP breakdown at the ATP-ase site can also be used to translocate protons. This mechanism is a reversal of the one used to produce ATP from the proton motive force. The third mechanism makes use of the enzyme NADH-transhydrogenase to break down NADH into NAD+ in order to translocate Y& (Harold, 1977). The various components of the pmf can be neutralized individually or simultaneously by the addition of toxicants. The charge gradient can be cancelled by potassium transport across the membrane by valinomycin, for example. To neutralize only the pH gradient, acetate or other weak acids can be used (Kaback, 1976). Indeed, such acids form a neutral molecule before diffusing through the membrane. Once in the c e l l , the acids dissociate and remain in the ionic form, thus trapped inside the c e l l . Since H + was removed from outside and released inside, the pH gradient w i l l have decreased. 153. However, since the IT*" was transported neutrally with acetate, the charge gradient remains unaffected. Another compound that affects only the pH gradient is nigericin, an antibiotic which causes one H"1" to be transported inside the c e l l in exchange for one which Is transported out of the c e l l . To affect both the charge gradient and the pH gradient, 2,4-dinitrophenol (DNP) can be used. This toxicant shuttles H* across the membrane such that the H+ gradient is dissipated. Since H"1" influences both the pH gradient and the charge gradient both of these w i l l be affected by DNP. DNP is also called an "uncoupler" because i t uncouples ATP formation at the ATP-ase site from respiration (which uses oxygen or nitrate at the electron transport chain) by dissipating the proton motive force. Although the charge gradient or the pH gradient may be affected by external conditions, bacteria w i l l tend to maintain a constant proton motive force (Bakker and Mangerich, 1981). For example, i f the external pH is suddenly decreased, the high H+ concentration outside the c e l l w i l l cause the pH gradient to increase. To maintain a constant pmf, the charge gradient can be decreased by cation expulsion. Potassium can be used for that purpose (Bakker and Mangerich, 1981). The mechanisms explained above are summarized in Fig. D-l. 154. transhydrogenase ( r e v e r s i b l e i f the ETC i s not f u n c t i o n i n g ) F i g . D-1 Summary of b a c t e r i a l b i o e n e r g e t i c s . 155. APPENDIX E  Procaryotlc Membrane Transport The purpose of this discussion is to summarize the membrane transport mechanisms available to procaryotlc microorganism1 (see Fig. E - l ) . The structure of a bacterial membrane w i l l f i r s t be briefly described. Bacteria have a plasma membrane that plays the role of a permeability barrier. A c e l l wall that surrounds the plasma membrane confers ri g i d i t y and shape to the bacteria. For some bacteria, the gram-negatives, an additional outer membrane serves as a barrier to large molecules (Brock, 1979). There are three kinds of transport: passive diffusion, facilitated diffusion, and active (energized) transport. Passive diffusion is a transport mechanism by which neutral molecules tend to distribute themselves in such a way that the concentration on both sides of the membrane is the same. Water, carbon dioxide and oxygen are transported by passive diffusion across the plasma membrane. In facilitated diffusion, the molecule combines with a membrane carrier and is transported inside the c e l l with i t s concentration gradient. Facilitated diffusion without specificity occurs at the outer membrane of gram-negative bacteria via porins. Facilitated diffusion with specificity occurs with glycerol at the plasma membrane. There are three categories of active transport: ATP-dependent, group translocation, and coupled to the proton motive force. In active transport a 1 Refer to Harold (1977), Harold (1978), Saier (1979), and Silver and Perry (1982) for more information on membrane transport. 156. specific carrier i s required for each solute. The solute i s then accumulated such that i t s internal concentration can exceed i t s external concentration. In ATP-dependent transport, the hydrolysis of ATP drives the internal accumulation of solutes such as glutamine and negatively charged amino acids. In group translocation transport, the solute i s modified during i t s transport. For example, the phosphotransferase system can be. used to phosphorylate several types of sugars such as glucose, fructose, and lactose. A proton motive force can be generated in three ways in non-photo-synthetic bacteria (see Appendix D). The proton motive force can be used to transport cations, anions, and neutral molecules. Cations, such as K + can accumulate In the c e l l i n response to the charge gradient (interior negative). Anion transport can occur with protons such that the molecule i s neutral when i t effectively crosses the membrane. Phosphate, sulfate, and negatively charged amino acids are mostly transported by this mechanism. For neutral molecules, such as sugars or amino acids, the carrier proteins effectively transfer a positive molecule where protons are bound to the carrier for i t s activation. The proton motive force can also be used In transport for the expulsion of cations such as potassium or calcium by an antiport mechanism with protons. 157. uncharged amino acids sugars Antiport Mechanism Neutral Molecules Coupled to the Proton Motive Force III ACTIVE TRANSPORT Anions peptidoglycan layer (25 nm) GRAM-Positive Cells .periplasmic space • plasma membrane peptidoglycan layer (3 nm) GRAM-Negative Cells outer membrane Membrane Structures H?0, 2' CO. PASSIVE TRANSPORT II FACILITATED DIFFUSION .1 without specificity H H2P04, HSO, , Cations .2 Group ranslocation' .1 A T P -Dependent small molecules ,2 with specificity porins glycerol negatively charged amino acids K pyruvate Sugar-Pi phospho^ enol-pyruvat« glutamine ADP + Pi ATP sugars (glucose, fructose mannose, lactose) glutamine (negatively charged amino acids) Fig. E-l Summary of procaryotic membrane transport. 


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