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Readily biodegradable COD as an indicative parameter in estimating the efficacy of sewage for biological… Manoharan, Ramanathan 1988

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READILY BIODEGRADABLE COD AS AN INDICATIVE PARAMETER IN ESTIMATING THE EFFICACY OF A SEWAGE FOR BIOLOGICAL EXCESS PHOSPHORUS REMOVAL by RAMANATHAN MANOHARAN B . S c . ( C i v i l Eng.), U n i v e r s i t y of P e r a d e n i y a , 1977 . E n g . ( C i v i l Eng.), U n i v e r s i t y of B r i t i s h Columbia, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of C i v i l E n g i n e e r i n g ) We accept t h i s t h e s i s as conforming t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA December 1988 w Ramanathan Manoharan, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of C i v i l Engineering The University of British Columbia Vancouver, Canada D a t e D e c . U , 1988 DE-6 (2/88) - i i -ABSTRACT The objectives of t h i s research were to develop a r e l i a b l e measurement technique to quantify the readily biodegradable substrates present in a sewage and to investigate their role in b i o l o g i c a l excess phosphorus removal, under acclimated conditions for each substrate added, leading to the characterization of a given sewage with respect to i t s natural e f f i c a c y in the b i o l o g i c a l excess phosphorus removal process. The experimental work during t h i s study involved two laboratory scale systems in which one was used to quantify the readily biodegradable substrates in the feed while the other was used as a b i o l o g i c a l excess phosphorus removal system. The readily biodegradable content of the feed was changed using d i f f e r e n t dosages of sodium acetate, sodium propionate, sodium butyrate and glucose. The e f f e c t s of these compounds (both as s p e c i f i c substrates and as general readily biodegradable substrates) on the various elements of the b i o l o g i c a l excess phosphorus removal mechanism (such as anaerobic phosphorus release, aerobic phosphorus uptake, o v e r a l l phosphorus removal, anaerobic carbon storage and aerobic carbon consumption) were investigated. Results of t h i s study showed that almost a l l the diff e r e n t elements of the b i o l o g i c a l excess phosphorus removal process compared very well among the d i f f e r e n t substrates used when the — i i i — re a d i l y biodegradable substrates (quantified as rea d i l y biodegradable COD) was used as the unit of measurement. Also, the o v e r a l l phosphorus removal e f f i c i e n c y improved with increasing amounts of rea d i l y biodegradable substrates entering the system. A d i r e c t relationship existed between the phosphorus release in the anaerobic zone and the phosphorus uptake in the aerobic zone, according to P uptake (mg/L) = 1.21 + 1.701 x P release (mg/L) with the constant of co r r e l a t i o n being 0.985. The r e s u l t s of t h i s study also showed the importance of carbon storage. A d e f i n i t e l i n k existed between the carbon storage (as PHB or PHV) and phosphorus release under the anaerobic conditions, and between the carbon consumption and phosphorus uptake under aerobic conditions. However, during the glucose addition t r i a l , a second carbon storage compound (glycogen) was found to be stored under aerobic conditions and consumed under anaerobic conditions. An excellent l i n e a r r e l a t i o n s h i p existed between the PHB synthesis and glycogen consumption, with the estimated mean value for the increase in the amount of PHB per unit of glycogen consumed being approximately 0.41, on a weight basis. In terms of s p e c i f i c substrates, for the same dosage as COD, t h e i r effectiveness in b i o l o g i c a l excess phosphorus removal had the following decreasing order: - i v-acetate > propionate > butyrate > glucose It was noticed during the study that whenever there was a reduction in the dosage of the added simple carbon substrates, the steady-state b i o l o g i c a l excess phosphorus removal continued for a period of upto 5 days at the same higher level before decreasing to the new lower l e v e l , r e f l e c t i n g the reduced substrate dosage. But, as a c o r o l l a r y , when the substrate dosage was increased, the excess phosphorus removal increased immediately without the presence of any s i g n i f i c a n t lag period. -v-TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES v i i i LIST OF FIGURES ix ACKNOWLEDGEMENT x i i 1. INTRODUCTION 1 2. LITERATURE REVIEW 8 2.1. B i o l o g i c a l Excess Phosphorus Removal 8 2.2. Concept of Readily Biodegradable Substrates 15 2.3. The Role of Readily Biodegradable Substrates on B i o l o g i c a l Excess Phosphorus Removal 23 3. EXPERIMENTAL METHODS 32 3.1. Experimental Design 32 3.2. Quantification of Readily Biodegradable Substrates 33 3.2.1. Experimental Set-up 33 3.2.2. Measurement Procedure 38 3.2.3. Calculation Procedure 39 3.2.4. Example Calculation 39 3.3. Laboratory Scale Operation of Phosphorus Removal System 40 3.3.1. Wastewater Source 40 3.3.2. Chemical Addition 43 3.3.3. Anaerobic Reactor 44 3.3.4. Anoxic Reactor 44 - v i -Page 3.3.5. A e r o b i c Reactor 45 3.3.6. C l a r i f i e r 45 3.3.7. O p e r a t i o n 46 3.4. A n a l y t i c a l Methods 46 3.4.1. Chemical Oxygen Demand 46 3.4.2. D i s s o l v e d Oxygen 48 3.4.3. Glycogen 48 3.4.4. N i t r o g e n 48 3.4.5. O x i d a t i o n Reduction P o t e n t i a l 49 3.4.6. pH 50 3.4.7. Poly-B-hydroxybutyrate and Poly-B-h y d r o x y v a l e r a t e 50 3.4.8. Phosphorus 52 3.4.9. S o l i d s 54 3.4.10. V o l a t i l e F a t t y A c i d s 54 3.5. C o l d Storage T e s t i n g s , 55 3.6. S t a t i s t i c s 56 4. RESULTS AND DISCUSSION 57 4.1. A c e t a t e A d d i t i o n 58 4.2. P r o p i o n a t e A d d i t i o n 60 4.3. B u t y r a t e A d d i t i o n 63 4.4. Glucose A d d i t i o n 65 4.5. R e a d i l y Biodegradable COD 68 4.6. Phosphorus Release and Uptake 72 4.6.1. Anaerobic Zone 79 Page 4.6.2. Aerobic Zone 86 4.6.3. Anoxic Zone .. 90 4.6.4. Phosphorus Accumulation in Sludge 94 4.7. Carbon Storage and Consumption 95 4.7.1. Anaerobic Zone 100 4.7.2. Aerobic Zone 106 4.7.3. Anoxic Zone 107 4.7.4. Carbon Storge and Consumption as Glycogen 110 4.8. Overall Phosphorus Removal 117 4.9. Overall Nitrogen Removal 125 4.10. Experimental Run with Combined Acetate and Propionate 130 4.11. Lag Response during Dosage Transitions 133 5. CONCLUSIONS AND RECOMMENDATIONS 141 5.1. Conclusions 141 5.1.1. Conclusions D i r e c t l y Related to Objectives 141 5.1.2. Other Conclusions Related to Readily Biodegradable Compounds in General 143 5.1.3. Other Conclusions Related to S p e c i f i c Readily Biodegradable Compounds 145 5.2. Recommendations 148 BIBILIOGRAPHY 150 APPENDICES 157 A l . Development of the Method Used for the Determination of the Readily Biodegradable COD ... 157 A2. Raw Data from the Various Experimental Runs 169 A3. Raw Data from the Cold Storage Testings 192 —v i i i -LIST OF TABLES Page 3.1. Summary of Operating Parameters for the Laboratory Excess Phosphorus Removal System 47 4.1. Results of Acetate Addition Experiments 59 4.2. Results of Propionate Addition Experiments 62 4.3. Results of Butyrate Addition Experiments 64 4.4. Results of Glucose Addition Experiments 66 4.5. Phosphorus Uptake for Acetate Run 75 4.6. Phosphorus Uptake for Propionate Run 76 4.7. Phosphorus Uptake for Butyrate Run 77 4.8. Phosphorus Uptake for Glucose Run 78 4.9. Average Phosphorus Uptake for A l l Runs 80 4.10. Carbon Consumption for Propionate Run 99 4.11. Carbon Consumption for Butyrate Run 99 4.12. Carbon Consumption for Glucose Run 101 4.13. Ov e r a l l Nitrogen Removal for A l l Runs 129 4.14. Phosphorus Uptake, Carbon Consumption and Nitrogen Removal during the Run with Combined Acetate and Propionate 131 4.15. Phosphorus and Carbon Balances for the Butyrate Run during the Dosage Transitions 138 - i x -LIST OF FIGURES Page 2.1. P r o f i l e of oxygen uptake rate with domestic wastewater as feed 17 2.2. P r o f i l e s of oxygen uptake rates under two d i f f e r e n t feeding conditions 17 2.3. P r o f i l e s of oxygen uptake rates with two d i f f e r e n t types of feed 19 2.4. P r o f i l e of oxygen uptake rate with glucose as substrate 19 2.5. Process configuration used for the determination of the readily biodegradable COD of feed 22 2.6. D e n i t r i f i c a t i o n rates used in the batch d e n i t r i f i c a t i o n method 22 2.7. Oxygen uptake rate p r o f i l e used in the batch aerobic method 22 2.8. The modified Ludzack-Ettinger process for b i o l o g i c a l nitrogen removal 24 2.9. The modified UCT process for b i o l o g i c a l nitrogen and phosphorus removal 24 3.1. Schematic layout of the process configuration for the determination of the readily biodegradable COD of the feed 34 3.2. Schematic layout of the laboratory scale experimental set-up of the b i o l o g i c a l excess phosphorus removal system 41 4.1. Total readily biodegradable COD in feed vs. chemical addition to feed, expressed as COD 71 4.2. Total readily biodegradable COD in feed vs. chemical addition to feed, expressed as mg/L 71 4.3. Mass of phosphorus entering and leaving each individual reactor per unit i n f l u e n t flow 73 4.4. The anaerobic phosphorus release with chemical dosage, expressed as COD entering the anaerobic zone 85 4.5. The anaerobic phosphorus release with r e a d i l y biodegradable COD available in the anaerobic zone ... 85 -x-Page 4.6. The aerobic phosphorus uptake with chemical dosage, expressed as COD in the feed 87 4.7. The aerobic phosphorus uptake with readily biodegradable COD in the feed (including the chemical addition) 87 4.8. Relationship between the aerobic phosphorus uptake and the anaerobic phosphorus release 92 4.9. The anoxic phosphorus uptake with chemical dosage, expressed as COD in feed 92 4.10. Relationship between the aerobic sludge percent phosphorus and the chemical dosage, expressed as COD in feed 96 4.11. Relationship between the aerobic sludge percent phosphorus and the readily biodegradable COD in feed (including the chemical addition) 96 4.12. Mass of carbon storage compounds entering and leaving each i n d i v i d u a l reactor per unit influent flow 98 4.13. A s i m p l i f i e d model for anaerobic metabolism of bio-P bacteria 101 4.14. Relationship between carbon storage and phosphorus release in the anaerobic zone for the propionate run 104 4.15. Relationship between carbon storage and phosphorus release in the anaerobic zone 104 4.16. Anaerobic carbon storage vs. chemical dosage, as COD entering the anaerobic zone 105 4.17. Anaerobic carbon storage vs readily biodegradable COD (from the feed and the chemical addition) entering the anaerobic zone 105 4.18. A s i m p l i f i e d model for aerobic metabolism of bio-P bacteria 108 4.19. Relationship between carbon consumption and phosphorus uptake in the aerobic zone 108 4.20. Relationship between carbon storage and phosphorus uptake in the anoxic zone 112 4.21. Relationship between the phosphorus uptake and glycogen consumption in various zones for glucose run 112 -xi-Page 4.22. Relationship between the i n t r a c e l l u l a r PHB and glycogen for glucose run 118 4.23 The o v e r a l l phosphorus removal of the system vs. the chemical addition, expressed as COD in feed 118 4.24. The o v e r a l l phosphorus removal of the system vs. the re a d i l y biodegradable COD in feed (including the chemical addition) 122 4.25. The o v e r a l l phosphorus removal of the system vs. the chemical dosage, expressed as mg/L in feed 122 4.26. Relationship between the o v e r a l l phosphorus removal of the system and the anaerobic phosphorus release ... 124 4.27. Relationship between the o v e r a l l phosphorus removal of the system and the anaerobic carbon storage 124 4.28. Overall nitrogen removal e f f i c i e n c y of the system vs. the chemical dosage, expressed as COD in feed 127 4.29. Overall nitrogen removal e f f i c i e n c y of the system vs. the r e a d i l y biodegradable COD in feed (including the chemical addition) 127 4.30 P r o f i l e of the effluent ortho phosphorus for the acetate run 135 4.31. P r o f i l e of the effluent ortho phosphorus for the propionate run 135 4.32. P r o f i l e of the effluent ortho phosphorus for the butyrate run 136 4.33. P r o f i l e of stored PHB in the various zones of the system for the butyrate run 136 - x i i -ACKNOWLEDGEMENT I wish t o express my s i n c e r e thanks t o Dr. W.K. Oldham, Head of the C i v i l E n g i n e e r i n g Department a t the U n i v e r s i t y of B r i t i s h Columbia, f o r h i s e n t h u s i a s t i c s u p p o r t and a d v i c e g i v e n d u r i n g the e n t i r e p e r i o d of t h i s r e s e a r c h . I a l s o w i s h t o thank Dr. D.S. M a v i n i c , Dr. K . J . H a l l and Dr. T. B e a t t y f o r t h e i r a s s i s t a n c e i n the p r e p a r a t i o n of t h i s t h e s i s . I thank Susan L i p t a k , P a u l a P a r k i n s o n and Romy So f o r t h e i r i n v a l u a b l e a s s i s t a n c e i n the l a b o r a t o r y a n a l y s i s , Guy K i r s c h f o r h i s a s s i s t a n c e i n the c o n s t r u c t i o n of the l a b o r a t o r y e x p e r i m e n t a l system, Kannappar Mukunthan f o r h i s a s s i s t a n c e i n c o l l e c t i n g sewage f o r the stu d y , the Department of C i v i l E n g i n e e r i n g f o r the f i n a n c i a l support from the NSERC g r a n t s of Dr. W.K. Oldham and Yogeswary Manoharan f o r her c o n s t a n t moral support. -1 -CHAPTER ONE INTRODUCTION The impact of increased nutrient loadings on natural water bodies has fast become an important concern with increased levels of urbanization and land e x p l o i t a t i o n , causing eutrophication of water bodies with a l g a l blooms y i e l d i n g numerous physical and aesthetic problems. They also cause foul taste and odour of water, disruption of r e c r e a t i o n a l a c t i v i t i e s and high water treatment costs. Furthermore, f i s h k i l l s could result due to the depletion of dissolved oxygen r e s u l t i n g from the decay of the a l g a l blooms. The three major nutrients required for any b i o l o g i c a l growth are carbon, nitrogen and phosphorus. To c u r t a i l any unwanted growth, the l i m i t i n g nutrient should be f i r s t i d e n t i f i e d and subsequently c o n t r o l l e d . It i s usually not possible to control the aquatic input of carbon to an extent that i t w i l l become the l i m i t i n g nutrient due to i t s natural abundance from either the organic compounds or the bicarbonate ion (Black and Khettry, 1980). The assimilable forms of nitrogen for most aquatic growth are n i t r a t e and ammonia. However, when these forms become no longer a v a i l a b l e , some species (e.g. blue-green algae) can f i x the nitrogen from the atmosphere and add i t to the aquatic system. Thus, c o n t r o l l i n g the input of nitrogen to c u r t a i l any unwanted a l g a l growth in a water body becomes -2-i r r a t i o n a l . This leaves the control of phosphorus as the most p r a c t i c a l means of c u r t a i l i n g the eutrophication of the natural water bodies. Phosphorus has been recognized as the l i m i t i n g nutrient in most of the eutrophication situations (Porter, 1975). The phosphorus available to the organisms in a given ecosystem enters the system primarily v i a anthropogenic point and d i f f u s e sources, since the natural source of phosphorus from the minerals of the earth's crust i s usually scarce due to their low s o l u b i l i t y . The contribution of phosphorus to the ecosystem through non-point sources are d i f f i c u l t to distinguish and for the most part, t h e i r c o n t r o l would require a monumental e f f o r t . In these s i t u a t i o n s wider controls, such as the proper management of f e r t i l i z e r a p p l i c a t i o n to the a g r i c u l t u r a l lands and a control of man's a c t i v i t i e s in terms of extensive urbanization may prove to be more e f f e c t i v e . Point sources, on the other hand, are e a s i l y i d e n t i f i a b l e and controlled, based on s i t e - s p e c i f i c needs. Often, the major point source of phosphorus to receiving water bodies i s the discharge of domestic wastewaters. The primary sources of phosphorus in the domestic wastewater are human waste, synthetic laundry detergents and water treatment chemicals used to protect the water d i s t r i b u t i o n systems from corrosion (U.S.EPA, 1976). Municipal wastewaters are most often treated using the - 3 -activated sludge process. In a t y p i c a l activated sludge treatment process, 1.0-1.5 mg/L of phosphorus i s removed (for the basic metabolic purposes) for every 200 mg/L of COD removal (U.S.EPA, 1976) and the phosphorus makes up approximately 1.5% of the dry weight of biomass (Hoffmann and Marais, 1977). Since the phosphorus removal obtained by t h i s conventional treatment process i s often inadequate to protect the environment, better phosphorus removal i s being required by many government agencies. Two major strategies are a v a i l a b l e ; chemical or b i o l o g i c a l (excess) phosphorus removal. Chemical phosphorus removal i s achieved by p r e c i p i t a t i n g phosphates with c e r t a i n chemicals, followed by coagulation and sedimentation. The most commonly used chemicals for t h i s purpose are lime, alum and f e r r i c c h loride, which provide the cations necessary for the p r e c i p i t a t i o n of phosphates in the wastewater. These chemicals are added either at the i n i t i a l stage or d i r e c t l y into the b i o l o g i c a l process or as a f i n a l stage of treatment. Although extremely low e f f l u e n t phosphorus l e v e l s may be obtained through t h i s process, i t suffers from a number of drawbacks such as the production of a large amount of chemical sludge that requires subsequent d i s p o s a l . High chemical costs also have made th i s method of phosphorus removal p r o h i b i t i v e for many communities. Another method which does not require any chemical addition or produce increased volumes of sludge to dispose of, i s -4 -based on the b i o l o g i c a l excess phosphorus ( b i o - P ) removal. In the b i o - P p r o c e s s , phosphorus i s taken up i n excess of the normal metabolic requirements d e s c r i b e d e a r l i e r . I t i s g e n e r a l l y b e l i e v e d to be s t o r e d by the organisms i n the form of long c h a i n s of i n o r g a n i c polyphosphate, known as v o l u t i n g r a n u l e s ( L e v i n and Shapi r o , 1965). The excess phosphorus removal i n a st a n d a r d a c t i v a t e d sludge process i s s t i m u l a t e d by adding an anaerobic zone upstream of the normal a e r o b i c zone. Under the anaerobic c o n d i t i o n s , phosphorus i s r e l e a s e d from the r e t u r n sludge i n t o the l i q u i d phase and upon e n t e r i n g the a e r o b i c zone, a l l the r e l e a s e d phosphorus as w e l l as i n f l u e n t phosphorus i s taken up by a p r o p e r l y a c c l i m a t e d biomass, r e s u l t i n g i n excess phosphorus removal by the system. The reason f o r t h i s behaviour was f i r s t h y p o t h e s i z e d t o be the consequence of the a n a e r o b i c phosphorus r e l e a s e , with t h a t r e l e a s e being the r e s u l t of the anaerobic s t r e s s (Barnard, 1976). However, f u r t h e r r e s e a r c h has shown t h a t the anaerobic phosphorus r e l e a s e was more c l o s e l y r e l a t e d t o the c o n c e n t r a t i o n and the nature of the carbon-based s u b s t r a t e s a v a i l a b l e t o the micro-organisms i n the anaerobic zone, r a t h e r than to the degree of anaerobic s t r e s s ( S i e b r i t z et a l . , 1982). I t has a l s o been demonstrated t h a t simpler carbon s u b s t r a t e s (such as a c e t a t e , p r o p i o n a t e e t c . ) produce b e t t e r anaerobic phosphorus r e l e a s e , f o l l o w e d by b e t t e r excess phosphorus uptake i n the a e r o b i c zone. -5-It i s now a standard practice to supply these simple carbon substrates to the anaerobic zone either through external additions or by producing these simple carbon substrates in s i t u . This can be done in a primary sludge fermenter placed up-stream of a b i o l o g i c a l excess phosphorus removal process, for sewages with low COD concentrations (Rabinowitz, 1985). However, with strong sewages (such as those in South A f r i c a ) , these simple carbon substrates could apparently be generated either in the anaerobic zone of the main process with longer hydraulic retention times (2-3 hours of nominal hydraulic retention time) or in the sewage c o l l e c t i o n system through fermentation. It i s c l e a r that the amount of external simple carbon substrates needed or the degree of the fermentation necessary to produce a desired degree of phosphorus removal depends on the a v a i l a b i l i t y of these simple substrates in the raw feed i t s e l f . The simple substrates present in the feed sewage depends on the c h a r a c t e r i s t i c s of the c o l l e c t i o n system: i t varies d i r e c t l y with the hydraulic retention time provided by the c o l l e c t i o n system, due to the possible presence of fermentative conditions. It should be noted that the low ve l o c i t y of flow i s also important to the presence of sludge deposits and zero dissolved oxygen necessary for any fermentation a c t i v i t y . The biodegradable portion of a municipal wastewater i s composed of two f r a c t i o n s : a readily biodegradable soluble f r a c t i o n that i s used by the microorganisms at a rapid rate; and - 6 -a slowly biodegradable particulate f r a c t i o n that requires storage and enzymatic breakdown pr i o r to transfer through the c e l l wall (Dold et a l . , 1980). Although many researchers have investigated the ef f e c t s of s p e c i f i c simple substrates in the feed (such as short chain v o l a t i l e f a t t y acids) on b i o l o g i c a l excess phosphorus removal, no s i g n i f i c a n t attempt has been made to quantify the presence of these preferred substrates on a more generalized basis. There were many instances where the phosphorus removal e f f i c i e n c y of a b i o l o g i c a l excess phosphorus removal process was found to be good, although no s i g n i f i c a n t amount of any v o l a t i l e f a t t y acids was found in the feed (Koch, 1984). This indicates that other preferred substrates or compounds that lend themselves to be ea s i l y converted to the preferred substrates in the anaerobic zone of the process were present in the feed. Since i t i s not p r a c t i c a l to identif y every possible preferred substrate present in a sewage, a more generalized parameter i s necessary to estimate the a v a i l a b i l i t y of the t o t a l preferred substrates in a given sewage. This research investigates the p o s s i b i l i t y of using the parameter "readily biodegradable COD" to quantify the presence of simple preferred carbon substrates in a sewage, in order to assess the s u i t a b i l i t y of a given sewage for i t s natural e f f i c a c y in the b i o l o g i c a l excess phosphorus removal process. -7-The o b j e c t i v e s o f t h i s r e s e a r c h may be summarized as f o l l o w s : ( i ) D e v e l o p m e n t o f a r e l i a b l e measurement t e c h n i q u e f o r t h e q u a n t i f i c a t i o n o f t h e r e a d i l y b i o d e g r a d a b l e s u b s t r a t e s ( a s r e a d i l y b i o d e g r a d a b l e . C O D ) p r e s e n t i n a sewage. ( i i ) I n v e s t i g a t i o n o f t h e r o l e o f r e a d i l y b i o d e g r a d a b l e s u b s t r a t e s e n t e r i n g a b i o l o g i c a l e x c e s s p h o s p h o r u s r e m o v a l s y s t e m on t h e v a r i o u s e l e m e n t s o f t h e p r o c e s s ( s u c h a s a n a e r o b i c p h o s p h o r u s r e l e a s e , a e r o b i c p h o s p h o r u s u p t a k e , o v e r a l l p h o s p h o r u s r e m o v a l , a n a e r o b i c c a r b o n s t o r a g e a n d a e r o b i c c a r b o n c o n s u m p t i o n ) . ( i i i ) I n v e s t i g a t i o n o f t h e e f f i c a c y o f u s i n g r e a d i l y b i o d e g r a d a b l e COD a s t h e p a r a m e t e r f o r a g e n e r a l c h a r a c t e r i z a t i o n o f a sewage w i t h r e s p e c t t o t h e b i o l o g i c a l e x c e s s p h o s p h o r u s r e m o v a l p r o c e s s . -8-CHAPTER TWO LITERATURE REVIEW The r e a l i z a t i o n of the importance of having low phosphorus concentrations in sewage treatment plant discharges to control the eutrophication of natural waterbodies has led to extensive research work in t h i s f i e l d . The b i o l o g i c a l excess phosphorus (bio-P) removal technique i s considered to be one of the best methods available for removing phosphorus from domestic wastewater discharges, as discussed in the previous chapter. This chapter provides a review of previous work that has been used in developing the objectives and methods used in t h i s research. 2.1 BIOLOGICAL EXCESS PHOSPHORUS REMOVAL Comprehensive l i t e r a t u r e reviews on the b i o l o g i c a l excess phosphorus removal have been presented by S i e b r i t z et a l . (1983), Comeau (1984) and others. This section w i l l b r i e f l y cover some of the same subject matter, plus provide an update on the more recent research. B i o l o g i c a l excess phosphorus removal was f i r s t reported by Srinath et a l . (1959), followed by Alarcon (1961). However, - 9 -neither Srinath et a l . nor Alarcon offered any explanation for the occurrence of t h i s phenomenon or why i t was observed only in ce r t a i n treatment f a c i l i t i e s . The f i r s t attempt to explain t h i s b i o l o g i c a l excess phosphorus removal phenomenon was made by Feng (1962) using a ser i e s of batch t e s t s . He concluded that the excess phosphorus removal was stimulated by a process temperature of about 2 5 ° C , aeration and appropriate food to micro-organism r a t i o , although these conditions are often found in treatment processes that do not e x h i b i t any excess phosphorus removal. His finding that the phosphorus release could occur with inadequate aeration l a t e r emerged as an important factor in the phosphorus removal process. Levin and Shapiro (1965) were the f i r s t researchers to propose a biochemically based explanation for excess phosphorus removal by r e l a t i n g the role of phosphorus to the aerobic u t i l i z a t i o n of carbohydrates. They also noted that c e r t a i n microorganisms have the a b i l i t y to store phosphorus in long chains of inorganic polyphosphates and concluded that the prospects of achieving a reduction in the dissolved phosphorus content of a sewage, using a modified activated sludge process, were promising. As a resu l t of these p a r t i a l explanations, a number of researchers, such as Shapiro et a l . (1967), Vacker et a l . (1967) , Wells (1969) , Bargman et a l . (1970) and Milbury et a l . (1970, -10-1971) began investigating the b i o l o g i c a l excess phosphorus removal phenomenon. Although many invaluable findings were made during these studies, such as the phosphorus release under anaerobic conditions, phosphorus uptake under aerobic conditions and the minor role that inorganic p r e c i p i t a t i o n of phosphates plays in the ov e r a l l excess phosphorus removal process, no exact indisputable explanations for that removal were given. However, at this stage, the mechanism of the excess phosphorus removal was generally accepted to be b i o l o g i c a l in nature. Barnard (1974) reported about 97% phosphorus removal from a modified activated sludge process (referred to as the "Bardenpho" process) in which an anaerobic-aerobic sequence was employed. Although the author refe r s to the unaerated zones of the process as the anaerobic zones, they are in fact anoxic zones due to the presence of recycled n i t r a t e s in them. Fuhs and Chen (1975) were the f i r s t researchers to s p e c i f i c a l l y investigate the role of t h i s anaerobic-aerobic sequence on the excess phosphorus removal. They reported that the anaerobic/aerobic sequence allowed Acinetobacter (which are capable of storing excess phosphorus) to f l o u r i s h in the process, since the anaerobic conditions promoted the growth of a fac u l t a t i v e anaerobic population of microorganisms that produced carbon sources (such as ethanol, acetate and succinate) necessary for the slow growing Acinetobacter. Although t h i s theory was the f i r s t attempt to explain the excess phosphorus removal in a -11-process having an anaerobic-aerobic sequence on a microbiological basis, i t d i d not explain either the anaerobic phosphorus release or i t s role in the b i o l o g i c a l excess phosphorus removal. According to Davelaar et a l . (1978) and Toerien et a l . (1979), the micro-organisms responsible for the b i o l o g i c a l excess phosphorus removal are ubiquitous and the i r p r o l i f e r a t i o n depends pri m a r i l y on the appropriate environmental conditions. The f i r s t researcher to link the anaerobic phosphorus release as an i n t r i n s i c part of the b i o l o g i c a l excess phosphorus removal mechanism was Barnard (1976). He pointed out that the excess phosphorus removal occurs when the anaerobic phosphorus release i s induced through an anaerobic stress that can be indicated by the oxidation-reduction potential (ORP). He also reported the adverse e f f e c t s of nit r a t e s entering the anaerobic zone on the excess phosphorus removal, through the reduction of the anaerobic stress, and the increase in the ORP. Other researchers (McLaren and Wood, 1976; Ni c h o l l s , 1978), while investigating the detrimental effects of nitrates entering the anaerobic zone, also confirmed the importance of the anaerobic phosphorus release in the b i o l o g i c a l excess phosphorus removal. According to Comeau et a l . (1985), the two es s e n t i a l c h a r a c t e r i s t i c s of bacteria responsible for b i o l o g i c a l excess phosphorus removal (bio-P bacteria) are the a b i l i t y to store polyphosphate a e r o b i c a l l y and the a b i l i t y to store carbon - 1 2 -anaerobically, in such a form as poly-B-hydroxybutyrate (PHB). When simple preferred carbon substrates (such as acetate or propionate) are present in the anaerobic zone, the bio-P bacteria store these substrates as carbon reserves (PHB or poly-B-hydroxyvalerate) by cleaving polyphosphates (to maintain proton motive force) and releasing phosphorus into s o l u t i o n . The concentration of the external carbonaceous substrates i s generally low in the aerobic zone of a completely mixed activated sludge process (Manual of Practice No.8, WPCF, 1977). Therefore, the bio-P bacteria with their i n t e r n a l stored carbon reserves w i l l be able to compete better with other microorganisms upon entering the aerobic zone. Under these conditions, the bio-P bacteria degrade their stored carbon reserves and store polyphosphates by removing phosphorus from s o l u t i o n . Thus, the presence of preferred substrates in the anaerobic zone and the anaerobic-aerobic sequence (with recycle) are important to esta b l i s h a s u f f i c i e n t proportion of bio-P b a c t e r i a . It may take 6 to 8 weeks to develop the microorganisms responsible for the b i o l o g i c a l excess phosphorus removal (Oldham and Stevens, 1984; Manning and Irvine, 1985). However, once these organisms are established, a short period of unfavourable conditions would not wipe them out. In fact, i t has been reported by Manning and Irvine (1985) that i t took only 2 days to reestablish good phosphorus removal e f f i c i e n c y a f t e r a 13 day period of upset. - 1 3 -Since q u a n t i f i c a t i o n of the anaerobic stress required for the phosphorus release and the subsequent excess phosphorus removal using oxidation-reduction potential (ORP) measurements are d i f f i c u l t and unreliable, an alternate parameter was proposed by Rabinowitz and Marais (1980) to quantify the anaerobic stress. The parameter, known as the "anaerobic capacity" or "anaerobic p o t e n t i a l " , was defined as the difference between the d e n i t r i f i c a t i o n capacity of the anaerobic reactor and the mass of ni t r a t e s entering the reactor (both expressed in mg N per l i t r e of feed). The authors concluded that at least 9 mg N/L of anaerobic capacity i s necessary to achieve the anaerobic phosphorus release and the subsequent aerobic phosphorus uptake. S i e b r i t z et a l . (1982, 1983), while investigating t h i s concept of anaerobic capacity, reported that an anaerobic-aerobic process did not produce any phosphorus release, although i t had an anaerobic capacity of 35 mg N/L. They concluded that the anaerobic phosphorus release and the subsequent excess phosphorus uptake are clos e l y linked to the readi l y biodegradable substrates avail a b l e in the anaerobic zone rather than any other parameter. This w i l l be discussed in d e t a i l in section 2.3. At t h i s stage, biochemical models to explain the b i o l o g i c a l excess phosphorus removal phenomenon started to emerge.A good l i t e r a t u r e review with a special emphasis on the biochemical models for the b i o l o g i c a l excess phosphorus removal has been presented by Simm (1988). -14-A s i g n i f i c a n t biochemical explanation for excess phosphorus removal was presented by Hall et a l (1978) and Nicholls and Osborn (1979), indicating two survival mechanisms for the aerobic organisms under anaerobic conditions, as outlined below. (i) the breaking-up of the i n t r a c e l l u l a r polyphosphate chain to provide the energy needed, and ( i i ) formation of PHB, a common carbon reserve compound, for the accumulation of the hydrogen ions and electrons in order to process more substrates. These ideas were further extended by Rensink (1981). He suggested that the lower f a t t y acids present in the l i q u i d phase under anaerobic conditions are stored as PHB. The energy necessary for t h i s storage r e s u l t s from the polyphosphate cleavage, thus creating conditions for the slow-growing Acinetobacter to survive and to better compete with the other micro-organisms in the b i o l o g i c a l excess phosphorus removal systems. According to Comeau et a l . (1987), the carbon storage in the form of poly-B-hydroxyvalerate (PHV) would exceed the carbon storage in the form of PHB when short chain v o l a t i l e fatty acids (or their s a l t forms) containing an odd number of carbon atoms (e.g. propionate) are added to the system. Conversely, PHB would -15-become the dominant form of carbon storage, i f short chain fatty acids (or their s a l t forms) containing an even number of carbon atoms (e.g. acetate, butyrate) are added. Comeau (1984) in his biochemical model for the excess phosphorus removal, proposed that the polyphosphate reserves (in addition to t h e i r role in the storage of carbon) are used to supply energy to maintain the proton motive force of the bio-P bacteria. However, Wentzel et a l . (1986) pointed out that the mechanism proposed by Comeau (1984) for maintaining the proton motive force in the anaerobic zone gives r i s e to charge and proton imbalances across the cytoplasmic membrane of the c e l l . They presented a modified biochemical model based on the ef f e c t s of anaerobic and aerobic phases on the i n t r a c e l l u l a r NADH/NAD and ATP/ADP ra t i o s and their influence on the biochemical regulation of carbon and phosphorus metabolic pathways. This model maintained both the proton motive force and the charge n e u t r a l i t y . 2.2 CONCEPT OF READILY BIODEGRADABLE SUBSTRATES Ekama and Marais (1978), while trying to develop a general model for the dynamic behaviour of the activated sludge process, observed a precipitous drop (or a step change) in the oxygen -16-uptake rate (OUR) at the termination of the feed (Fig. 2.1). The system used was a single reactor, completely mixed activated sludge process with a 12 hour c y c l i c loading of municipal wastewater at 20°C, pH of 7 and a short process sludge age of 2.5 days. At f i r s t , t h i s precipitous drop in the oxygen uptake rate was believed to be a behavioural c h a r a c t e r i s t i c of n i t r i f i c a t i o n . In order to v e r i f y t h i s hypothesis, the authors operated two similar laboratory scale activated sludge units with the f i r s t unit (unit C) being operated under c y c l i c loading of low nitrogen wastewater, while the second unit (unit N) was operated under steady continuous loading of the same low nitrogen wastewater with a c y c l i c loading of saline ammonia superimposed. Under these conditions, the precipitous drop in the oxygen uptake rate was observed only in unit C (Fig. 2.2). The same experiments were repeated with both units being operated under c y c l i c loadings, but with unit C receiving a low nitrogen wastewater and unit N receiving a high nitrogen wastewater. Both units exhibited the precipitous drop in the oxygen uptake rate under these conditions of operation (Fig. 2.3). From the r e s u l t s of these two sets of experiments, i t was concluded that the step change observed in the oxygen uptake rate was in response to the energy requirement for the adsorption of - 1 7 -° START o FEED is o H - O £° z o oo o z • 32 >-X o o o ^ . 0 0 STOP FEED START FEED 4.00 8 , 0 0 nriE 2* 0(VR 6S) 0 0 2 ° ' ° ° 2 4 ' ° ° 2 8 - 0 0 F i g . 2.1. P r o f i l e of oxygen uptake rate with domestic wastewater as feed (after Ekama and Marais, 1978). 8 ^ *> o e o UJ M H < I -0 . to I, z UJ CD >-x O m UNIT C UNIT N A V HI -4 -2 0 2 4 6 TIME (hours) Unit C: C y c l i c loading of low N wastewater Unit N: Continuous loading of low N wastewater + c y c l i c loading of saline ammonia F i g . 2.2. P r o f i l e s of oxygen uptake rates under two d i f f e r e n t feeding conditions (after Ekama-and Marais, 1978). -18-the carbonaceous material and not due to a behavioural c h a r a c t e r i s t i c of n i t r i f i c a t i o n . Therefore, as soon as the feeding ceased, there was a step-wise decrease in the rate at which oxygen was u t i l i z e d . Dold et a l . (1980) c r i t i c i z e d t h i s explanation, since i t led to the conclusion that adsorption was energy demanding, in contrast to the basic thermodynamic p r i n c i p l e which states that the adsorbed state i s associated with a lower energy l e v e l than the unadsorbed state. This doubt was reinforced when a r e l a t i v e l y large precipitous drop was observed when a soluble and e a s i l y biodegradable substrate (glucose) was used as the feed (Fig. 2.4). From these pure substrate experiments, the authors hypothesized that the municipal wastewater i s composed of two fract i o n s , namely (i) a readily assimilable soluble (readily biodegradable) f r a c t i o n that i s used r a p i d l y , and ( i i ) a slowly biodegradable p a r t i c u l a t e f r a c t i o n requiring storage and enzymatic breakdown p r i o r to being transferred through the c e l l membrane. Under t h i s hypothesis, the step change in the oxygen uptake rate was attributed to the cessation of oxygen u t i l i z a t i o n - 1 9 -^ o o £ o -4 UNIT C A UNIT N • -2 0 2 4 6 TIME (hours) Unit C: Cyc l i c loading of low N wastewater Unit N: Cy c l i c loading of high N wastewater F i g . 2.3- P r o f i l e s of oxygen uptake rates with two di f f e r e n t types of feed (after Ekama and Marais, 1978). STOP FEED 8.00 12.00 16.06 TIME (HOURS) 20.00 24.00 F i g . 2.4. P r o f i l e of oxygen uptake rate with glucose as substrate (after Dold et a l . , 1980). -20-for the metabolism of the rea d i l y biodegradable substrates in the feed. Subsequent to t h i s step change in the oxygen uptake rate, the oxygen uptake behaviour remains a consequence of the slowly biodegradable substrates made available before the termination of the feed. At least three d i f f e r e n t methods are reported in the l i t e r a t u r e for the measurement of the readily biodegradable substrates (as COD) in a sewage. They are b r i e f l y outlined below. The continuous aerobic method, developed at the University of Cape Town (UCT) by Ekama and Marais (1984), involves the operation of a short sludge age completely mixed activated sludge system ( F i g . 2.5). The basis of t h i s method of determining the r e a d i l y biodegradable carbonaceous substrates in a sewage consists of measuring the step change in the oxygen uptake rate at the feed termination and i s discussed in d e t a i l in section 3.2. Large scatter in the r e s u l t s by t h i s method was reported by N i c h o l l s et a l . (1985), in contrast to the results achieved by the researchers at the University of Cape Town. The second method, known as the batch d e n i t r i f i c a t i o n  method, operates on the basis that there are three di f f e r e n t and d i s t i n c t d e n i t r i f i c a t i o n rates in the activated sludge process, as reported by Stern and Marais (1974). The i n i t i a l rapid rate of d e n i t r i f i c a t i o n i s related to the a v a i l a b i l i t y of the readily biodegradable carbonaceous substrates and the measurement of t h i s -21-d e n i t r i f i c a t i o n rate i s used to estimate the readily biodegradable substrate content of the feed. According to Nicholls et a l . (1985), the second d e n i t r i f i c a t i o n rate l i n e i s extrapolated back to the Y-axis (Fig. 2.6) and the AN0 3 determined. The readily biodegradable COD i s then calculated by using the following formula: Readily biodegradable COD = 8.6 x ANO^ since each mg of nitrate (as N) r e s u l t s in the oxidation of 8.6 mg of readily biodegradable substrate (as COD) (van Haandel et a l . , 1981). Recovery studies conducted with sodium acetate, using t h i s method, gave a recovery of 10.4 mg/L from the added concentration of 10.5 mg/L (Nicholls et a l . , 1985). In the recently developed batch aerobic method (Nicholls et a l . , 1985), a batch sample of activated sludge i s d i l u t e d with the sewage under investigation such that a high food to microorganism r a t i o i s obtained. The v a r i a t i o n of the oxygen uptake rate with time i s pl o t t e d , as shown in F i g . 2.7. The readily biodegradable COD i s reported to be proportional to the shaded area with i t s concentration given by the following formula (Nicholls et a l . , 1985): Readily biodegradable COD = Area 0.33 where 0.33 i s the general conversion factor for COD to oxygen - 2 2 -A E R A T I O N R E A C T O R S E T T L I N G T A N K E F F L U E N T S L U D G E R E C Y C L E F i g . 2.5. Process configuration used f o r the determination of the readily biodegradable COD of feed (after Ekama and Marais, 1984). A N O , NO, \ 1 s t R a t e ^ v ^ ^ 2 n d R a t e 3 r d R a t e T imt F i g . 2.6. D e n i t r i f i c a t i o n rates used i n the batch d e n i t r i f i c a t i o n method (after Nicholls et a l . , 1985). O x / g e n U t i l i s a t i o n R a t e T i • e F i g . 2.7. Oxygen uptake rate p r o f i l e used i n the batch aerobic method (after Nicholls et a l . , 1985). -23-(Ekama and Marais, 1984). However, preliminary investigations with t h i s method, using sodium acetate as substrate, showed poor recoveries. 2.3 THE ROLE OF READILY BIODEGRADABLE SUBSTRATES IN BIOLOGICAL EXCESS PHOPHORUS REMOVAL The defining of the prerequisites for the b i o l o g i c a l excess phosphorus removal by S i e b r i t z et a l . (1982, 1983), in terms of the readily biodegradable substrates a v a i l a b i l i t y in the anaerobic zone, was a very s i g n i f i c a n t step in the understanding of the nature of the b i o l o g i c a l excess phosphorus removal mechanism. It also produced a major s h i f t in emphasis away from the previously hypothesized anaerobic stress to recognize the importance of the nature of the avai l a b l e carbon substrates in the anaerobic zone. This s i g n i f i c a n t f i n d i n g was made by the authors while investigating the a p p l i c a b i l i t y of the anaerobic capacity hypothesis (proposed by Rabinowitz and Marais, 1980) to both the modified Ludzack - Ettinger (MLE) and the modified University of Cape Town (UCT) processes, with regard to b i o l o g i c a l excess phosphorus removal. The schematic layouts of these processes are shown in Figures 2.8 and 2.9. Three MLE processes and a modified UCT process were set up and fed from the same wastewater source. - 2 4 -ANOXIC AEROBIC REACTOR REACTOR F i g . 2.8. The modified Ludzack - Ettinger process f o r b i o l o g i c a l nitrogen removal (after S i e b r i t z et a l . , 1982). ANAEROBIC ANOXIC AER03IC REACTOR REACTORS REACTOR F i g . 2.9. The modified UCT process for b i o l o g i c a l nitrogen and phosphorus removal (after S i e b r i t z et a l . , 1982). -25-The three MLE units were given unaerated sludge mass fractions of 40, 55 and 70 percent respectively and the recycle ratios were set to achieve the anaerobic capacities ranging from 6 to 35 mg N/L in the anoxic reactors. Over two months of operation, neither phosphorus release nor excess phosphorus removal was observed in any of the MLE units. In contrast, the modified UCT process with a 10% anaerobic sludge mass f r a c t i o n consistently gave good phosphorus release and excess phosphorus removal, c l e a r l y indicating a breakdown in the anaerobic capacity hypothesis for the b i o l o g i c a l excess phosphorus removal. The d i f f e r e n t phosphorus release patterns in the modified MLE and UCT processes were explained in terms of the readily biodegradable substrates (quantified in terms of readily biodegradable COD) surrounding the organisms in the unaerated reactor. The unaerated reactor in the MLE process was anoxic (due to the presence of n i t r a t e s through the recycle) whereas the f i r s t unaerated reactor in the modified UCT process was t r u l y anaerobic (since no n i t r a t e s enter t h i s reactor through the rec y c l e ) . In the MLE process, s u f f i c i e n t n i t r a t e s are recycled to the anoxic reactor to u t i l i z e a l l the available readily biodegradable COD, since each mg of n i t r a t e (as N) results in the oxidation of 8.6 mg of rea d i l y biodegradable substrate, as COD -26-(van Haandel et a l . , 1981). In contrast, in the modified UCT process, the concentration of the readily biodegradable substrate available for anaerobic conditioning of the phosphorus-storing organisms i s maximized, since no n i t r a t e s are recycled to the anaerobic reactor. Therefore, the process configurations such as the modified UCT process, which can ensure zero n i t r a t e discharge to the anaerobic zone, are at a s i g n i f i c a n t advantage with respect to b i o l o g i c a l excess phosphorus removal. From these observations, the authors concluded that the concentration of the readily biodegradable COD surrounding the microorganisms in the anaerobic reactor i s the key parameter in determining whether or not the phosphorus release and excess phosphorus uptake takes place. The results from t h e i r studies also showed that the concentration of the rea d i l y biodegradable substrate surrounding the organisms in the anaerobic zone must exceed approximately 25 mg/L as COD to ensure good phosphorus release and subsequent excess phosphorus uptake. It was also concluded that the b i o l o g i c a l excess phosphorus removal increased with increasing readily biodegradable COD concentration in the anaerobic zone beyond 25 mg/L. Marais et a l . (1983) further summed up the b i o l o g i c a l excess phosphorus removal in terms of r e a d i l y biodegradable substrate, as follows: "Poly-P accumulation serves as an energy reser v o i r , to sustain the organism during the anaerobic stressed state, -27-but p r i n c i p a l l y to gain a positive advantage over non-P accumulating organisms by p a r t i t i o n i n g 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". The r e a d i l y biodegradable substrates (or preferred substrates) necessary for the b i o l o g i c a l excess phosphorus removal, through the p r o l i f e r a t i o n of the appropriate microorganisms, are usually made available to the anaerobic zone. This can be accomplished either through external additions of simple carbon substrates (such as acetate, propionate etc.) or produced in s i t u by employing a primary sludge fermenter or thickener ahead of the process (Oldham and Stevens, 1984; Oldham, 1985; Rabinowitz, 1985). Oldham (1985), while working with a f u l l scale b i o l o g i c a l excess phosphorus removal treatment plant in Kelowna, B r i t i s h Columbia, reported that the a v a i l a b i l i t y of the primary sludge thickener supernatant to the fermentation zone of the process had a marked influence on the phosphorus removal capacity of the system. The o v e r a l l phosphorus removal e f f i c i e n c y quickly dropped to l e v e l s found in conventional activated sludge plants when the thickener supernatant flow was removed. Conversely, the addition of t h i s supernatant to a unit that was marginally achieving good phosphorus removal, quickly restored the excellent phosphorus removal. He also reported that when the thickener supernatant -28-flow was changed from one p a r a l l e l module of the treatment plant to the other, phosphorus removal c a p a b i l i t i e s were quickly lost in the f i r s t module, and even more quickly restored in the other. Once the basic hypothesis for the ro l e of s p e c i f i c substrates in the b i o l o g i c a l excess phosphorus removal process was developed, many research workers started to focus their attention on the effects of these s p e c i f i c substrates on the various aspects of the phosphorus removal mechanism. Fukase et a l . (1982), while conducting a series of laboratory scale batch experiments using acclimated biomass and synthetic feed made up of either acetate or glucose as the BOD source, found that the phosphorus release was concomitant with the disappearance of the substrates from the sol u t i o n . The rel a t i o n s h i p observed between the reduction in organic matter and the increase in soluble phosphate was given as ^acetate:AP0 4 being 1:1 and aglucose:APO^ being 2:1, in terms of molar r a t i o s . They also found that the carbon was stored by the microorganisms under anaerobic conditions as PHB during the addition of acetate, and as glycogen during the addition of glucose. However, the opposite finding was made by Mino et a l . (1987) where glycogen was found to be consumed under anaerobic conditions. During their study of two laboratory scale anaerobic-aerobic prosesses using synthetic sewage (consisting of acetic acid, sodium propionate, glucose and peptone), they found two -29-carbon storage compounds - PHB and glycogen. As reported by many other researchers, PHB was found to be stored under anaerobic conditions (associated with phosphorus release) and consumed under aerobic conditions (associated with phosphorus uptake). However, glycogen showed the opposite trend with anaerobic consumption and aerobic storage. They concluded that the NADH required for the PHB synthesis from acetate, under anaerobic conditions, i s supplied from the consumption of glycogen, through the Embden-Meyerhof-Parnas (EMP) pathway. They also indicated that the anaerobic/aerobic acclimatized sludge was converting the stored PHB to glycogen during the aerobic phase, so as to maintain the required l e v e l of glycogen for the consumption during the anaerobic phase. As discussed by Mino et a l . (1987), the decrease of the i n t r a c e l l u l a r carbohydrate concentration under anaerobic conditions was also reported by Tsuno et a l . (1986) and could be att r i b u t e d to the consumption of glycogen stored in the microbial c e l l s . Somiya et a l . (1988), in the i r study of b i o l o g i c a l excess phosphorus removal, reported the existence of a linear r e l a t i o n s h i p between the increase in the amount of i n t r a c e l l u l a r PHB and the decrease in the amount of i n t r a c e l l u l a r carbohydate, when the e x t r a c e l l u l a r glucose i s depleted. The estimated mean value for the increase in the amount of PHB per unit mass of carbohydrate u t i l i z e d was reported to be approximately 0.2, on a -30-weight basis or approximately 0.3, on TOC ( t o t a l organic carbon) basis. Potgieter and Evans (1983), using batch experiments with unacclimated biomass, investigated the e f f e c t s of various substrates on phosphorus release, by adding equal amounts of substrates as COD, to non-aerated reactors. They reported the following decreasing order of e f f e c t , with the f i r s t named substrate being accompanied by the greatest phosphorus release. acetate > propionate > formate > butyrate > hydroxybutyrate > glucose. Oldham and Koch (1982) in a si m i l a r batch experiment with unacclimated biomass, observed the following decreasing order of phosphorus release among the various substrates tested. sodium acetate > propionic acid > ac e t i c acid > glucose > is o -butyric acid. Jones et a l . (1985), working with continuous laboratory scale excess phosphorus removal systems with acclimated biomass, reported that the degree of phosphorus release was substrate s p e c i f i c and the phosphorus release and the subsequent phosphorus uptake had the following decreasing order of e f f e c t . butyric acid > ethanol > methanol > acetic acid > sodium - 3 1 -acetate They also reported that although s i g n i f i c a n t differences in the phosphorus release were apparent, the difference in the phosphorus uptake or removal did not appear to be s i g n i f i c a n t . While studying the e f f e c t s of short-chain carbon compounds on the k i n e t i c s of the b i o l o g i c a l nutrient removal, Gerber et a l . (1986) reported that the most favourable net phosphate removal from solu t i o n i s obtained by the use of acetate, butyrate, propionate and la c t a t e . Formate was found to stimulate good phosphorus release under anaerobic conditions, but was worst with respect to the o v e r a l l net phosphorus removal. They also concluded that the phenomenon of anaerobic phosphorus release from sludges accomplishing enhanced phosphorus removal was p r i m a r i l y dependent on the nature of the substrate interacting with the b a c t e r i a l mass. - 3 2 -CHAPTER THREE EXPERIMENTAL METHODS This chapter d e t a i l s the experimental methods used in t h i s study. Section 3.1 deals with the experimental design. The measurement of readily biodegradable COD of feed, the laboratory operation of the b i o l o g i c a l excess phosphorus removal system, the sampling and analysis techniques used and the procedure used to study the eff e c t of cold storage (at 4°C) on sewage are a l l outlined in sections 3.2, 3.3, 3.4 and 3.5 respectively. 3.1 EXPERIMENTAL DESIGN The experiments involved two p a r a l l e l laboratory scale systems in which one was used to quantify the re a d i l y biodegradable substrates in the feed (the elements and the development of t h i s system are described in section 3.2 and appendix A1 respectively) while the other was used as a b i o l o g i c a l excess phosphorus removal system (described in section 3.3). The readily biodegradable content of the feed was changed using d i f f e r e n t dosages of sodium acetate, sodium propionate, sodium butyrate and glucose. The ef f e c t s of these compounds (both as s p e c i f i c substrates and as general readily biodegradable substrates) on the various elements of the b i o l o g i c a l excess phosphorus removal mechanism (such as anaerobic phosphorus -33-release, aerobic phosphorus uptake, overa l l phosphorus removal, anaerobic carbon storage and aerobic carbon consumption) were investigated. 3.2 QUANTIFICATION OF READILY BIODEGRADABLE SUBSTRATE The technique used to quantify the readily biodegradable substrate of feed during t h i s study was an adaptation of the procedure used by Ekama and Marais (1984). The adaptations were made both for convenience and to suppress unwanted n i t r i f i c a t i o n and filamentous growth. The development of these modifications are outlined in d e t a i l in Appendix A1. The basis of the method for determining the readily biodegradable substrate consists of measuring the step change in the oxygen uptake rate (OUR) at the feed termination, in a completely mixed activated sludge process operated at a very short sludge age. 3.2.1 EXPERIMENTAL SET-UP A schematic layout of the process i s shown in Fig.3.1. The elements of t h i s experimental set-up are outlined below. Chemical Addition Chemical Container A i r Supply Scraper E f f l u e n t C l a r i f i e r Return Sludge Feed Tank F i g . 3 . 1 . Schematic layout of the process configuration f o r the determination of the r e a d i l y biodegradable COD of the feed. - 3 5 -(a) FEED The feed, in which the readily biodegradable COD concentration i s to be found, was continuously added to the system at a rate of 12 l i t r e s per day from a constantly s t i r r e d p l a s t i c tank with a l i d . The s t i r r i n g was slow and gentle to keep the p a r t i c u l a t e matter from s e t t l i n g , but to avoid any a i r entrainment into the feed. The feed to the b i o l o g i c a l excess phosphorus removal system used in t h i s study was also pumped from the same container. (b) CHEMICAL ADDITION The chemical substrate additions (at the same concentrations as in the b i o l o g i c a l excess phosphorus removal system) were done using a separate Masterflex pump, with the actual concentrations in the chemical container adjusted according to the flow rates (except in the case of acetate addition where the sodium acetate solution was d i r e c t l y added to the feed tank, at approximately 8 hour i n t e r v a l s ) . The 500 ml chemical feed container was f i l l e d on a d a i l y basis. (c) AEROBIC REACTOR The reactor consisted of a c y l i n d r i c a l p l e x i - g l a s s tank -36-with a l i q u i d volume of 2 l i t r e s , complete with a s t i r r e r for constant mixing. The mixed liquor in the reactor was aerated using a fine bubble purging stone attached to a glass tube. The dissolved oxygen concentration was manually controlled between 1.5 and 2.5 mg/L. A l l l i q u i d streams to and from the reactor entered below the l i q u i d surface in order to avoid any a i r entrainment. Mixed l i q u o r suspended so l i d s were wasted d i r e c t l y from t h i s reactor on a twice d a i l y basis to achieve a syste: s o l i d s retention time (SRT) of 3 days, giving an approximate aerobic SRT of 1.5 days. The loss of sol i d s through the effluent, due to the r e l a t i v e l y high effluent suspended s o l i d s concentrations, was taken into account while wasting from the aerobic reactor. (d) RETENTION TANK FOR RETURN SLUDGE A c y l i n d r i c a l p l e x i - g l a s s tank, where the l i q u i d volume could be varied between 0.5 to 2 l i t r e s , was used as the retention tank for the return sludge. The l i q u i d volume of t h i s reactor during t h i s study was 1 l i t r e for the acetate, propionate and butyrate runs and 0.5 l i t r e during the glucose run. The surface of t h i s reactor was covered with a f l o a t i n g cover to minimize any entrainment of a i r into the reactor contents. The primary purpose of t h i s holding tank was to control filamentous growth in t h i s short sludge age system, as discussed in d e t a i l in Appendix A1. The contents of t h i s reactor were s t i r r e d at a l l -37-times, using a magnetic s t i r r e r . (e) CLARIFIER Mixed liquor from the aerobic reactor was allowed to flow into a c y l i n d r i c a l s e t t l i n g tank (with a centre b a f f l i n g tube) having a l i q u i d volume of 1 l i t r e . The s e t t l e d sludge was continuously recycled back to the aerobic reactor through the sludge retention tank, with a recycle r a t i o of 1:1. A 1 rpm scraper mechanism was i n s t a l l e d to prevent the sludge from adhering to the side walls of the c l a r i f i e r . (f) OPERATION For every run during the study, this system was considered to be in steady state when the d a i l y oxygen uptake rate and the aerobic mixed liquor suspended s o l i d s showed approximately steady values (less than 10 percent v a r i a t i o n ) . More than 5 mg/L of ammonia was always present in the effluent of the system thereby keeping the same oxygen requirement for n i t r i f i c a t i o n , before and a f t e r the termination of the feed. This was ensured by the selection of an appropriate short l i q u i d retention time, as discussed in d e t a i l under the section dealing with the development of t h i s method (Appendix A l ) . -38-3.2.2 MEASUREMENT PROCEDURE The f o l l o w i n g procedure was c a r r i e d out d u r i n g each measurement of the r e a d i l y b i o d e g r a d a b l e COD of the f e e d . ( i ) The oxygen uptake r a t e under c o n t i n u o u s s u b s t r a t e f e e d i n g c o n d i t i o n s was measured by r a i s i n g the d i s s o l v e d oxygen c o n c e n t r a t i o n of the a e r o b i c mixed l i q u o r t o about 6 mg/L, s w i t c h i n g o f f the a i r s u p p l y , and m o n i t o r i n g the r e s u l t a n t decrease of the d i s s o l v e d oxygen c o n c e n t r a t i o n u s i n g an OmniScribe c h a r t r e c o r d e r (by Houston I n s t r u m e n t ) . The s l o p e of t h i s p l o t was then c a l c u l a t e d and the oxygen uptake r a t e expressed as mg/L/h. ( i i ) The above procedure was r e p e a t e d , except t h a t the f e e d ( i n c l u d i n g the s u b s t r a t e a d d i t i o n ) was stopped i n the mi d d l e of the procedure (when the d i s s o l v e d oxygen c o n c e n t r a t i o n was about 3 mg/L) and the decrease i n the c o n c e n t r a t i o n of the d i s s o l v e d oxygen was moni t o r e d c o n t i n u o u s l y u n t i l i t was l e s s than 1 mg/L. The t e r m i n a t i o n of the feed would have caused a change i n the sl o p e of the d i s s o l v e d oxygen vs time p l o t and the new reduced oxygen uptake r a t e was c a l c u l a t e d . The above procedures were repeated t o ensure r e p r o d u c i b i l i t y , and average v a l u e s used to c a l c u l a t e oxygen uptake r a t e w i t h and wi t h o u t feed t e r m i n a t i o n . -39-3.2.3 CALCULATION PROCEDURE T h i s procedure was adapted from the work of Ekama and Marais (1984). The f o l l o w i n g i n f o r m a t i o n i s r e q u i r e d to c a l c u l a t e the c o n c e n t r a t i o n of the r e a d i l y b i o d e g r a d a b l e COD i n the i n f l u e n t f eed. Q = Flow r a t e of the feed (L/h) V = Volume of the a e r o b i c r e a c t o r (L) OUR^ = Average OUR bef o r e the feed t e r m i n a t i o n (mg/L/h) OURa = Average OUR a f t e r the feed t e r m i n a t i o n (mg/L/h) Then, the r e a d i l y biodegradable COD c o n c e n t r a t i o n of the feed i s given by, (OUR. - OUR ) x V R e a d i l y biodegradable COD = * 0.334 x Q where 0.334 = General c o n v e r s i o n f a c t o r f o r COD to oxygen (Marais and Ekama, 1984). 3.2.4 EXAMPLE CALCULATION The f o l l o w i n g data was obtained on the f i r s t day of the 30 mg COD/L a c e t a t e run. Q = 0.49 L/h -40-2 L 39.5 mg/L/h 32.4 mg/L/h Therefore, r e a d i l y biodegradable COD = (39.5 - 32.4) x 2 0.334 x 0.49 = 87 mg/L 3.3 LABORATORY SCALE OPERATION OF PHOSPHORUS REMOVAL SYSTEM A bench top, laboratory scale b i o l o g i c a l excess phosphorus removal system was operated during t h i s study. The schematic diagram of t h i s experimental set-up i s shown in F i g . 3.2, and the d e t a i l s of the various components are outlined below. 3.3.1 WASTEWATER SOURCE The wastewater used during most of t h i s study was obtained from the wastewater storage tanks of the p i l o t sewage treatment plant situated on the University of B r i t i s h Columbia campus in Vancouver, B r i t i s h Columbia. The wastewater source for t h i s treatment plant was a main sewer l i n e servicing the student residences, on-campus housing and the university sports centre. The raw sewage from t h i s l i n e was pumped d a i l y , commencing at 10 a.m. using a submersible macerator pump, into two mechanically mixed p l a s t i c storage tanks (each with a capacity of 9000 l i t r e s ) V = OURb = OUR = F i g . 3.2. Schematic layout of the laboratory scale experimental set-up of the b i o l o g i c a l excess phosphorus removal system. -42-u n t i l the tanks were f u l l . Due to the low a l k a l i n i t y of thi s sewage (80-120 mg/L as CaC0 3), additional a l k a l i n i t y of approximately 100 mg/L as CaC0 3 was added to these tanks in the form of sodium bicarbonate. From these tanks, the sewage was coll e c t e d every two weeks in 25 l i t r e carboys. It was subsequently stored in the laboratory cold room at 4°C, for use in the lab scale experiments of t h i s study. During a portion of t h i s study, the wastewater source was switched from the p i l o t treatment plant at the University of B r i t i s h Columbia campus to the f u l l scale sewage treatment plant in Richmond, B r i t i s h Columbia. This was done during the 15 and 10 mg COD/L of acetate and during the 25 and 20 mg COD/L of propionate runs due to the closure of the p i l o t plant for upgrading, and during the 30 and 75 mg COD/L of glucose runs due to the low COD strength of the p i l o t plant wastewater. The strength of the c o l l e c t e d wastewater was adjusted ( i f necessary) by d i l u t i n g with tap water, such that the t o t a l COD entering the system (including the chemical addition) l i e s within the range of 250-275 mg/L. However, during the glucose runs, the COD of the raw sewage was kept around 200 mg/L (irr e s p e c t i v e of the glucose dosage) to maintain the composition of the feed as mostly sewage, since higher glucose dosages (up to 150 mg COD/L) were required to e f f e c t good excess phosphorus removals. Occasionally, the phosphorus content of the feed was increased by adding t r i b a s i c sodium phosphate (Na-PO-.12H,0) to maintain a - 4 3 -t o t a l phosphorus concentration of about 4 mg/L. The sewage was added to the experimental phosphorus removal system at a rate of 12 l i t r e s per day from a constantly s t i r r e d p l a s t i c tank. To avoid changing the c h a r a c t e r i s t i c s of the wastewater due to excess aeration, the tank was covered with a p l a s t i c l i d and the s t i r r e r speed was kept as low as possible, while s t i l l keeping the p a r t i c u l a t e s of the feed in suspension. The feed for the system used to determine the readily biodegradable COD of the feed was pumped from the same laboratory storage tank. The d a i l y aliquot of feed, having been stored at 4°C, was allowed to acclimate to ambient temperature (approximately 20°C) before being added to the system. 3.3.2 CHEMICAL ADDITION The chemicals added during t h i s study were sodium acetate, sodium propionate, sodium butyrate and glucose. The chemical dosages during the various experimental runs (as mg COD/L entering the anaerobic reactor) were 30, 25, 20, 15, 10 and 5 for acetate; 25, 20, 15, 10 and 5 for propionate; 30, 25, 20, 15 and 10 for butyrate; and 75, 60, 45 and 30 for glucose. During the acetate run, appropriate amounts of sodium acetate solution were mixed with raw sewage and added to the feed tank, at approximately 8 hour i n t e r v a l s . However, th i s practice -44-was changed during the other runs and the chemical substrates were pumped separately from the feed, using a separate chemical pump. The actual concentration in the chemical feed container was adjusted, according to the chemical and feed flow rates, to achieve the desired dosage of the chemical substrate in the anaerobic reactor. The chemical container had a capacity of 500 ml and was f i l l e d on a d a i l y basis. 3.3.3 ANAEROBIC REACTOR This reactor was a c y l i n d r i c a l plexi-glass tank with a l i q u i d volume of 1.5 l i t r e s , and i t provided the anaerobic conditions necessary for the excess phosphorus removal. It was mixed using a s t i r r e r and was f i t t e d with a fl o a t i n g cover to minimize any a i r entrainment due to surface turbulence. This reactor was fed continuously with the various chemical substrates together with the sewage feed. 3.3.4 ANOXIC REACTOR A c y l i n d r i c a l p l e xi-glass tank with a l i q u i d volume of 2.25 l i t r e s was used as the anoxic reactor. The contents of t h i s reactor were constantly mixed with a s t i r r e r and the l i q u i d surface was covered using a fl o a t i n g cover. The primary purpose of t h i s anoxic reactor was to d e n i t r i f y the return sludge from -45-the c l a r i f i e r , thus preventing any NO bleeding into the anaerobic reactor through the anoxic-anaerobic recycle. The necessity and the advantage of preventing the NOx from entering the anaerobic zone, are discussed in d e t a i l in Chapter 4. 3.3.5 AEROBIC REACTOR This reactor consisted of a pl e x i - g l a s s tank with a l i q u i d volume of 4 l i t r e s . The reactor contents were aerated using a fine bubble sparger stone attached to a glass tube and the dissolved oxygen concentration was manually controlled between 1.5 and 2.5 mg/L. The contents of t h i s reactor were completely mixed at a l l times with a s t i r r e r . 3.3.6 CLARIFIER Mixed liquor from the aerobic reactor was allowed to flow into a c y l i n d r i c a l p l e x i - g l a s s c l a r i f i e r (with centre baffling) having a l i q u i d volume of 1 l i t r e . The s e t t l e d sludge was recycled back to the anoxic reactor with a recycle r a t i o of 1:1. A 1 rpm scraper mechanism was i n s t a l l e d to prevent the sludge from adhering to the side walls of the c l a r i f i e r . -46-3.3.7 OPERATION The system was operated at a system sludge retention time (SRT) of 18 days, which was maintained by wasting the mixed liqu o r suspended s o l i d s d i r e c t l y from the aerobic reactor, on a d a i l y basis. Since the effluent of t h i s system contained a r e l a t i v e l y high concentration of suspended s o l i d s , the aerobic wastage was adjusted to compensate for the loss of s o l i d s through the e f f l u e n t . A l l recycle ratios during t h i s study was kept at 1:1. The system was considered to have reached steady state during each run when the mixed liquor suspended s o l i d s and the ef f l u e n t phosphorus concentrations showed r e l a t i v e l y steady values. Table 3.1 presents a summary of operating parameters. 3.4 ANALYTICAL METHODS A l l f i l t r a t i o n s of the samples during t h i s study were done using Whatman No.4 f i l t e r s , except during s o l i d s analysis in which Whatman 934-AH glass f i b r e f i l t e r s were used. The a n a l y t i c a l methods used for the determination of each parameter in the study are now described. 3.4.1 CHEMICAL OXYGEN DEMAND (COD) The samples were preserved with concentrated s u l f u r i c acid - 4 7 -Table 3.1 Summary of Operating Parameters for the Laboratory B i o l o g i c a l Excess Phosphorus Removal System Parameter Values Actual Hydraulic Retention Time (h) Anaerobic 1 .5 Anoxic 1 .5 Aerobic 4.0 Sludge Retention Time (d) System 18 Aerobic approx.10 Feed Flow (L/d) 12 Recycle Ratio Return sludge 1:1 Anoxic to anaerobic 1:1 Aerobic Dissolved Oxygen (mg/L) 1.5-2.5 -48-(pH < 2.0) and analysed in accordance with the Standard Methods (A.P.H.A. et a l . , 1980). 3.4.2 DISSOLVED OXYGEN Dissolved oxygen concentrations in the bio-reactors were measured using Model 54A dissolved oxygen meters (Yellow Spri Instrument Co.). The membranes and the e l e c t r o l y t e s in the probes were replaced once a week on average. The probes were c a l i b r a t e d using the Winkler method, as outlined in the Standard Methods (A.P.H.A. et a l . , 1980). 3.4.3 GLYCOGEN The determination of glycogen in the sludge was by hydrolysis in 30% (wt./vol.) potassium hydroxide, followed by p r e c i p i t a t i o n with ethanol and using the anthrone colorimetric method, as outlined in in the Manual of Methods in General Microbiology (A.S.M. et a l . , 1981). 3.4.4 NITROGEN Two types of nitrogen measurements were made during t h i s research, namely NOx (which includes both n i t r a t e s and n i t r i t e s ) -49-and Total Kjeldahl Nitrogen (TKN). A l l values are presented as mg/L N. (a) N0 X - NITROGEN The analysis was done using a copper-cadmium column in which n i t r a t e s are reduced to n i t r i t e s , followed by colorimetric measurements on a Technicon Autoanalyser I I , in accordance with the Technicon I n d u s t r i a l Method No.100-70W (1973). (b) TOTAL KJELDAHL NITROGEN (TKN) Samples were digested in a Technicon block digestor 40 and analysed according to Technicon Block Digester Instruction Manual (1974). 3.4.5 OXIDATION REDUCTION POTENTIAL (ORP) ORP measurements were made using Broadley James Corporation's combined ORP probes, with peripheral junctions using Ag/AgCl as reference couples, connected to high impedance d i g i t a l panel meters with l i q u i d c r y s t a l displays. Regular maintenance of these electrodes involved a thorough cleaning with a paper towel. A minimum of three hours was allowed after each -50-c l e a n i n g of the probes f o r them to e q u i l i b r a t e with the r e a c t o r c o n t e n t s before t a k i n g any rea d i n g . 3.4.6 pH The pH of the b i o - r e a c t o r s , was measured with a F i s h e r Accumet Model 320 expanded s c a l e pH meter wi t h g l a s s and r e f e r e n c e e l e c t r o d e s combined i n a s i n g l e probe. The meter was s t a n d a r d i z e d a g a i n s t F i s h e r pH 4 and pH 7 b u f f e r s o l u t i o n s as o u t l i n e d i n the Standard Methods (A.P.H.A. et a l . , 1980). 3.4.7 POLY-B-HYDROXYBUTYRATE AND POLY-B-HYDROXYVALERATE The chemical q u a n t i f i c a t i o n of poly-B-hydroxybutyrate (PHB) and poly-B-hydroxyvalerate (PHV) were done u s i n g an adapted procedure of Braunegg et a l . (1978). The method i n v o l v e d d e p o l y m e r i z a t i o n of PHB and PHV by s u l f u r i c a c i d , c o n v e r s i o n i n t o v o l a t i l e methyl e s t e r s with a c i d i f i e d methanol and e x t r a c t i o n i n c h l o r o f o r m f o r i n j e c t i o n on a gas chromotograph. Mixed l i q u o r samples from the a c t i v a t e d sludge process were c e n t r i f u g e d to g i v e approximately 30 mg of suspended s o l i d s and the sludge p e l l e t s f r o z e n f o r s t o r a g e . Once enough sludge p e l l e t s were accumulated, they were then l y o p h i l i z e d u s i n g e i t h e r a V i r t i s 10-234 or a M u l t i - D r y (by FTS Systems Inc.) l y o p h i l i z e r . -51-Two ml of a c i d i f i e d methanol (3% H2S0^) containing benzoic acid as the i n t e r n a l standard and 2 ml of chloroform were added to a measured amount of l y o p h i l i z e d sludge in 15 ml Pyrex test tubes with T e f l o n - l i n e d caps. The samples and the standards (sodium s a l t of D-L 3-hydroxybutyric acid dissolved in a c i d i f i e d methanol) were then heated at 100°C for approxmately 3.5 hours and allowed to cool to room temperature. Two ml of the denser chloroform phases were then transferred to 10 ml Pyrex test tubes containing 0.5 ml of d i s t i l l e d water and vigorously shaken for about 5 minutes. These were then centrifuged for about 10 minutes at 1500 g and the chloroform phases containing the PHB and PHV methyl esters transformed to gas chromatograph v i a l s for 1 ml s p l i t injections into a Hewlett-Packard 5880A gas chromotograph. This additional p u r i f i c a t i o n step was necessary to achieve r e l i a b i l i t y of the method and to avoid the premature degradation of the gas chromotograph column. The gas chromatograph was equipped with a programmable auto sampler (7672A) and a c a p i l l a r y column (15 m long and 0.52 mm in t e r n a l diameter) coated with 1 um of DB-Wax. Experimental conditions for the chromatograph were as follows: Temperature of the in j e c t i o n port Temperature of the detector port Linear v e l o c i t y of the c a r r i e r gas (helium) = 210°C = 220°C = 20 cm/s -52-The following oven temperature p r o f i l e was used: I n i t i a l temperature = - 50°C I n i t i a l time = = 1 min Program rate = = 8°C/min F i n a l temperature • = 160°C F i n a l time = - 5 min A f i n a l period of 4 minutes with an oven temperature of 200°C was also employed to clear any sample residue from the column aft e r the analysis of each sample. A r a t i o of HV to HB of 1.211 was used to c a l i b r a t e the gas chromatograph's HV response, since no standards were available for i t s dir e c t c a l i b r a t i o n . The following correction factors in terms of l y o p h i l i z e d mass of the samples were also applied to the PHB and PHV ca l c u l a t i o n s , since the recoveries obtained by t h i s method were found to be inversely proportional to the l y o p h i l i z e d mass (Comeau et al.,1988). PHB correction factor = 1.000 + 0.00361 x l y o p h i l i z e d mass PHV correction factor = 1.000 + 0.00780 x l y o p h i l i z e d mass 3.4.8 PHOSPHORUS Three types of phosphorus measurements were made during -53-t h i s study, namely ortho-phosphorus, t o t a l phosphorus and percent phosphorus content in sludge. (a) ORTHO-PHOSPHORUS Ortho-phosphorus analysis was done either by using the stannuous chloride technique outlined in the Standard Methods (A.P.H.A. et a l . , 1980) or by the automated ascorbic acid reduction method on a Technicon Auto-analyser II, in accordance with Technicon Industrial Method NO.94-70W (1973). Both methods produced comparable r e s u l t s . (b) TOTAL PHOSPHORUS The samples were f i r s t subjected to acid digestion using a Technicon block digestor 40 and s u l f u r i c acid, prior to analysis by Technicon Auto-analyser I I , according to the Technicon I n d u s t r i a l Method No.327-73W (1974). (c) PERCENT PHOSPHORUS CONTENT IN SLUDGE The phosphorus content in the sludge was determined by subjecting a measured amount of f i n e l y ground oven dried (at 104°C) sludge to acid digestion (as described in the Technicon -54-Block Digester Instruction Manual, 1974) and analysing for the t o t a l phosphorus, as described above in section 3.4.8 (b). 3.4.9 SOLIDS (a) TOTAL SUSPENDED SOLIDS (TSS) This was done by vacuum f i l t e r i n g a known volume of sample through a pre-washed and oven dried Whatman 934-AH glass f i b r e f i l t e r and oven drying for a minimum of 1 hour at 104°C, as outlined in the Standard Methods (A.P.H.A. et a l . , 1985). (b) VOLATILE SUSPENDED SOLIDS (VSS) This was determined by i g n i t i n g the n o n f i l t r a b l e s o l i d s obtained in 3.4.9 (a) at 550°C, as outlined in the Sta;. Methods (A.P.H.A. et a l . , 1985). 3.4.10 VOLATILE FATTY ACIDS (VFA) The v o l a t i l e fatty acid measurements were done using a computer-controlled Hewlett-Packard 5880A gas chromatograph, equipped with a flame ionization detector (FID) and using helium as the c a r r i e r gas. The glass column (0.91 m long with a 6 mm -55-external diameter and 2 mm internal diameter) was packed with 0.3% Carbowax/0.1 % H 3 P 0 4 ° n Supelco Carbopak C (supplied by Supelco Inc.). The column was conditioned according to the procedure outlined in the Supelco B u l l e t i n 751E (1982). Samples to be analysed were f i l t e r e d using Whatman 4 f i l t e r s and 1.0 u l aliquots were injected using microsyringes (Hamilton Model 701N, 10 ul) and a Hewlett-Packard auto-sampler (Model 7672A). Samples were a c i d i f i e d using a 1% solution of phosphoric acid to bring the pH below 3.0, before t h e i r i n j e c t i o n s . Experimental conditions for the chromatograph were as follows: Flow rate of c a r r i e r gas (helium) = 20 ml/min V o l a t i l e fatty acids analysed included acet i c , propionic and butyric acids with quantification done by the external standard methods, using reagent grade standards. 3.5 COLD STORAGE TESTINGS Detector port temperature Isothermal oven temperature Injection port temperature 150°C 200°C 120°C Since the sewage c o l l e c t e d during t h i s study was to be kept at 4°C for approximately 2 weeks, i t was decided to study -56-the e f f e c t s of c o l d s t o r a g e on sewage c h a r a c t e r i s t i c s a t t h e b e g i n n i n g of the e x p e r i m e n t s . A b r i e f s t u d y was u n d e r t a k e n t o l o o k a t t h e v a r i o u s p a r a m e t e r s of t h e sewage and t h e i r v a r i a t i o n o v e r a two week p e r i o d i n c o l d s t o r a g e . Two d i f f e r e n t b a t c h e s of sewage were c o l l e c t e d from t h e p i l o t p l a n t w a s t e w a t e r s t o r a g e t a n k s , as d e s c r i b e d i n s e c t i o n 3.3.1, c o m p l e t e l y m i x e d i n l a r g e c o n t a i n e r s and s t o r e d i n s e v e n s e p a r a t e 500 ml a i r t i g h t p l a s t i c b o t t l e s i n t h e c o l d room a t 4°C. Over a p e r i o d of two weeks, one b o t t l e was t a k e n e v e r y o t h e r day and a n a l y s e d f o r t h e f o l l o w i n g p a r a m e t e r s i n d u p l i c a t e . ( i ) B i o c h e m i c a l oxygen demand (BOD) ( i i ) C h e m i c a l oxygen demand (COD) ( i i i ) N i t r a t e s and n i t r i t e s (NO x) ( i v ) T o t a l K j e l d a h l n i t r o g e n (TKN) (v) T o t a l p h o s p h o r u s (TP) ( v i ) O r t h o - p h o s p h o r u s ( P 0 4 ~ P ) ( v i i ) V o l a t i l e f a t t y a c i d s (VFA) 3.6 STATISTICS The l i n e a r r e g r e s s i o n a n a l y s i s of t h e d a t a was done, u s i n g a Texas I n s t r u m e n t T I - 6 6 programmable c a l c u l a t o r , i n a c c o r d a n c e w i t h t h e TI-66 Sourcebook (T e x a s I n s t r u m e n t s , 1 9 7 7 ) . - 5 7 -CHAPTER FOUR RESULTS AND DISCUSSION The r e s u l t s o f t h e bench s c a l e e x p e r i m e n t s c o n d u c t e d i n t h i s s t u d y a r e d e t a i l e d and d i s c u s s e d i n t h i s c h a p t e r . The i n i t i a l f o u r s e c t i o n s d e s c r i b e t h e f o u r i n d i v i d u a l r u n s s e p a r a t e l y , w i t h d i s c u s s i o n r e l e v a n t t o t h e s e p a r t i c u l a r r u n s . S u b s e q u e n t s e c t i o n s p r e s e n t t h e d i s c u s s i o n u n d e r t h e b r o a d e r a s p e c t s o f t h e b i o l o g i c a l e x c e s s p h o s p h o r u s r e m o v a l t e c h n o l o g y i n t h e a c t i v a t e d s l u d g e p r o c e s s , b y c o m b i n i n g t h e r e s u l t s o b t a i n e d f r o m t h e i n d i v i d u a l e x p e r i m e n t a l r u n s o f t h i s s t u d y . The r e s u l t s o f t h e s t u d y on c o l d s t o r a g e ( a t 4°C) o f sewage showed no s i g n i f i c a n t v a r i a t i o n i n any o f t h e p a r a m e t e r s t e s t e d ( S e c t i o n 3.4) d u r i n g a p e r i o d o f two weeks. The v a r i a t i o n s n o t i c e d were c o m p a r a b l e t o t h o s e e x p e c t e d d u r i n g t h e c h e m i c a l a n a l y s i s t h r o u g h e x p e r i m e n t a l e r r o r s . The raw d a t a o b t a i n e d d u r i n g t h i s s t u d y a r e g i v e n i n A p p e n d i x A3. As d e s c r i b e d i n C h a p t e r 3, t h e r e a d i l y b i o d e g r a d a b l e COD f r a c t i o n o f t h e i n f l u e n t d u r i n g t h e e x p e r i m e n t s was v a r i e d u s i n g d i f f e r e n t d o s a g e s o f sodium a c e t a t e , s o d ium p r o p i o n a t e , s o d ium b u t y r a t e and g l u c o s e . -58-4.1 ACETATE ADDITION Sodium acetate was added continuously to the system at dosages of 30, 25, 20, 15, 10 and 5 mg/L as COD in the anaerobic reactor. The raw data for these experimental runs are given in Appendix A2. The r e s u l t s shown in Table 4.1 indicated that at 5 mg COD/L of acetate addition, the phosphorus removal was only 1.0 mg of P/L of feed and the percent phosphorus in the aerobic sludge was 1.5%. This value corresponds to the expected dry weight percent phosphorus content of organisms not exhibiting any excess b i o l o g i c a l phosphorus removal (Hoffmann and Marais, 1977). More than 100 percent improvement in phosphorus removal, from 1.0 to 2.2 mg of P/L of feed was obtained when the acetate addition was 10 mg COD/L. The percent phosphorus in the aerobic sludge was also higher at 2.2 %, indicating the presence of b i o l o g i c a l excess phosphorus removing organisms. This trend continued with increasing loadings. However, s i g n i f i c a n t improvement in b i o l o g i c a l excess phosphorus removal was not achieved by increasing acetate loading beyond 20 mg COD/L. This i s due to the phosphorus l i m i t i n g conditions of the i n f l u e n t . Thus, in t h i s circumstance, no s i g n i f i c a n t advantage in excess phosphorus removal could be obtained by increasing the acetate loading in the anaerobic reactor beyond 20 mg COD/L. -59-Table 4.1 Results of Acetate Addition Experiments Acetate Dosage in mg COD/L Parameter 30 25 20 15 1 0 5 TOTAL P : Influent 4.5 4.2 4.1 4.4 4. 1 4.4 (mg/L) Ef f l u e n t <0.2 <0.2 0.4 0.8 1.9 3.4 ORTHO-P : Influent 3.2 3.2 3.2 3.2 3.0 3.3 (mg/L) Anaerobic 18.1 16.0 10.5 5.2 3.6 3.4 Anoxic 15.6 13.5 8.2 4.4 3.3 3.5 Aerobic <0.1 <0.1 0.3 0.6 1.7 3.1 E f f l u e n t <0.1 <0.1 0.3 0.8 1.8 3.4 Aerobic sludge % P 5.7 5.5 4.8 4.6 3.0 1 .5 AP (mg/L) 4.3 4.0 3.7 3.6 2.2 1 .0 COD: Influent 219 224 230 234 240 246 Tmg/L) Added 60 50 40 30 20 10 E f f l u e n t 35 40 32 29 32 36 Readily biodegrad-able COD (mg/L) 84 77 71 63 55 48 -60-The ortho-P concentrations in both anaerobic and aerobic reactors showed that the phosphorus was released in the anaerobic zone and subsequently taken up in the aerobic zone. Both the phosphorus release and the uptake increased with increasing acetate dosages. 4.2 PROPIONATE ADDITION After the l a s t run with acetate ( i . e . the 5 mg COD/L run), the same system was spiked continuously with sodium propionate at dosages of 25, 20, 15, 10 and 5 mg COD/L, in the anaerobic reactor. These dosages were selected to avoid the phosphorus l i m i t i n g conditions experienced during the acetate runs with higher dosages. Stable steady state conditions were achieved in about 20 days. No s i g n i f i c a n t changes were noticed and the mixed liquor suspended s o l i d s concentration remained v i r t u a l l y unchanged over the whole t r a n s i t i o n period. The experimental run with 10 mg COD/L was repeated since the sewage used during t h i s run was found to contain acetate at a le v e l of 14.7 mg/L as COD. However, during a l l the other experimental runs throughout t h i s study, the raw sewage did not contain any measurable short chain v o l a t i l e fatty acid. -61-Starting from the propionate run, carbon storage (or consumption) in individual reactors was also measured to better understand the b i o l o g i c a l excess phosphorus removal process. S i g n i f i c a n t amounts of poly-B-hydroxyvalerate (PHV) and poly-B-hydroxybutyrate (PHB) were found in mixed liquor suspended solids during the propionate run. PHV was stored during the anaerobic phase and consumed during the aerobic phase. The quantities of t h i s carbon storage and consumption increased with increasing dosages of propionate. The raw data for t h i s series of experiments are l i s t e d in Appendix A2. The results shown in Table 4.2 indicated that the phosphorus removal increased with increasing propionate dosages, showing the same trend observed during the acetate run. At 5 mg COD/L dosage, no s i g n i f i c a n t b i o l o g i c a l excess phosphorus removal was taking place, since the percent phosphorus in the dry aerobic sludge was only 1.4% (Hoffmann and Marais, 1977). Although the b i o l o g i c a l excess phosphorus removal organisms could s t i l l be present, they might not be operating in the "bio-P" mode due to the low concentration of the preferred substrate (propionate, in t h i s case) in the anaerobic zone. The ortho-P concentrations in various zones showed that the phosphorus release in the anaerobic reactor and the phosphorus uptake in the aerobic reactor increased with increasing propionate loading. - 6 2 -Table 4.2 Results of Propionate Addition Experiments Propionate Dosage in mg COD/L Parameter 25 20 15 10 5 1 0* TOTAL P : Influent 4.3 4. 1 4.2 4.0 4. 1 3.9 (mg/L) Effluent 0.3 0.9 1 .6 2.6 3. 1 <0.2 ORTHO-P : Influent 3.2 3.0 3.3 3.1 3.2 2.9 (mg/L) Anaerobic 13.7 10.3 5.3 4. 1 3.3 15.5 Anoxic 11.5 8.7 4.2 3.6 3.0 11.2 Aerobic 0.3 0.9 1 .5 2.5 3.0 <0. 1 Effluent 0.3 0.8 1 .5 2.4 3. 1 <0.1 Aerobic sludge % P 5.5 4.4 3.7 2.2 1 .4 5.2 AP (mg/L) 4.0 3.2 2.6 1 .4 1 .0 3.7 COD: Influent 226 227 234 241 244 239 (mg/L) Added 50 40 30 20 10 20 Effluent 25 25 21 25 24 28 Readily biodegrad-able COD (mg/L) 71 66 59 51 46 71 * The raw sewage during this run had an acetate concentration of 14.7 mg/L as COD. -63-4.3 BUTYRATE ADDITION Once the propionate runs were over, the system was spiked continuously with sodium butyrate at a concentration of 30 mg/L as COD in the anaerobic reactor. This was followed by dosages of 25, 20, 15 and 10 mg COD/L. When the substrate was changed from propionate to butyrate, the mixed liquor suspended s o l i d s of the system decreased as much as ten times (from approximately 2300 mg/L to 250 mg/L in the aerobic and anoxic reactors) within a period of 11 days and gradually increased back to the normal concentration of 2300 mg/L in another 2 weeks. This indicated a s i g n i f i c a n t s h i f t in the population of organisms in the system. However, the system did not exhibit any excess phosphorus removal for a further 5 weeks period ( u n t i l Oct.6,1986). The system started removing phosphorus from the 6th week onwards (from Oct.9,1986) and continued to perform s a t i s f a c t o r i l y thereafter, although no operational conditions were changed. The raw data for t h i s run are given in Appendix A2. The fact that the excess phosphorus removal mechanism was established in a very short period of only 3 days a f t e r Oct.6, with no apparent change in any other parameter, suggested that i t took almost 7 weeks to e s t a b l i s h the correct culture of organisms, and then only 3 days for that culture to multiply to -64-Table 4.3 Results of Butyrate Addition Experiments Butyrate Dosage in mg COD/L Parameter 30 25 20 15 10 TOTAL P : Influent 4. 1 4.2 3.9 4.2 4.0 (mg/L) Ef f l u e n t 0.5 0.9 1.8 2.3 3.1 ORTHO-P : Influent 3.3 3.2 3.0 3.2 3.0 (mg/L) Anaerobic 12.9 10.1 7.4 4.7 3.4 Anoxic 9.8 7.7 5.9 4. 1 3.1 Aerobic 0.5 0.9 1.7 2.3 3.0 Ef f l u e n t 0.5 1 .0 1.7 2.4 2.9 Aerobic sludge % P 4.6 4.3 2.7 2.1 1 .6 AP (mg/L) 3.6 3.3 2.1 1.9 0.9 COD: Influent 220 223 230 234 241 Tmg/L) Added 60 50 40 30 20 Ef f l u e n t 34 38 40 39 34 Readily biodegrad-able COD (mg/L) 69 66 62 59 50 -65-s i g n i f i c a n t l e v e l s . Once t h i s population was s i g n i f i c a n t l y established, the system started to perform successfully. From the day the butyrate addition was started, i t took 76 days for the system to reach the steady state. Since the solids retention time (SRT) of the system was 18 days, the usual accepted adjustment period of 3 SRT's (54 days) was well surpassed in th i s case. This raised an important issue regarding the operational adjustment time required before making the conclusion that a system would not work (or vice versa). As observed in e a r l i e r acetate and propionate runs, the phosphorus removal increased with increasing butyrate dosage.The anaerobic phosphorus release and aerobic phosphorus uptake also followed the same trend. The carbon storage during these runs was primarily as PHB. 4.4 GLUCOSE ADDITION The glucose run was started with the dosage of 60 mg/L as COD in the anaerobic reactor. When the substrate was switched over from butyrate to glucose, the mixed liquor suspended solids (MLSS) of the system thinned out and was re-established at an aerobic MLSS of around 2500 mg/L, indicating a s i g n i f i c a n t population s h i f t in the system. This adjustment period was approximately 5 weeks. -66-Table 4.4 Results of Glucose Addition Experiments Glucose Dosage in mg COD/L Parameter 75 60 45 30 TOTAL P: (mg/L) Influent Effluent 3.9 0.2 3.6 0.7 3.7 1.7 3.7 2.4 ORTHO-P: (mg/L) Influent Anaerobic Anoxic Aerobic Effluent 3.3 13.3 6.9 0.1 0.1 2.7 1 1.2 6.8 0.7 0.6 2.8 6.8 6.0 1.6 1 .6 2.7 4.1 3.1 2.4 2.3 Aerobic sludge % P 4.1 3.4 2.7 1 .7 AP (mg/L) 3.7 2.9 2.0 1 .3 COD: (mg/L) Influent Added Effluent 1 92 1 50 37 189 120 31 199 90 33 214 60 27 Readily able biodegrad-COD (mg/L) 83 70 62 54 -67-Although the s o l i d s reached steady state, no excess phosphorus removal was taking place. The anoxic mixed liquor appeared very gluey with s i g n i f i c a n t gas bubbles (much more than those associated with the formation of nitrogen gas, during d e n i t r i f i c a t i o n , as observed during the other runs), indicating some kind of fermentative condition. D e n i t r i f i c a t i o n in the anoxic reactor was poor and ni t r a t e s were bleeding into the anaerobic reactor. However, the d e n i t r i f i c a t i o n performance of the anoxic reactor improved gradually over a further period of about 6 weeks. Once the n i t r a t e bleeding into the anaerobic reactor was s i g n i f i c a n t l y reduced, the system started removing phosphorus su c c e s s f u l l y . Other dosages used in t h i s run were 45, 30 and 75 mg/L as COD. Unlike the other runs, the f i n a l loading was higher due to the i n i t i a l estimated loading not y i e l d i n g the maximum possible excess phosphorus removal for the given conditions. The raw data for these are given in Appendix A2. As observed in a l l the previous runs, anaerobic phosphorus release, aerobic phosphorus uptake and the o v e r a l l phosphorus removal increased with increasing glucose dosages. Carbon storage and consumption involved two compounds, PHB and glycogen. -68-4.5 READILY BIODEGRADABLE COD The r e a d i l y b i o d e g r a d a b l e COD i n a sewage, i t s s i g n i f i c a n c e i n b i o l o g i c a l excess phosphorus removal p r o c e s s and the methods f o r i t s d e t e r m i n a t i o n were a l l e x p l a i n e d i n d e t a i l i n the p r e v i o u s c h a p t e r s . T h i s s e c t i o n d i s c u s s e s the degree of b i o d e g r a d a b i l i t y ( i n terms of r e a d i l y b i o d e g r a d a b l e COD) of the d i f f e r e n t c h e m i c a l s u b s t r a t e s added t o the e x p e r i m e n t a l system d u r i n g t h i s s tudy. The e f f e c t s of the r e a d i l y b i o d e g r a d a b l e COD on the b i o l o g i c a l excess phosphorus removal p r o c e s s w i l l be d i s c u s s e d under the d i f f e r e n t a s p e c t s of the p r o c e s s , i n s e c t i o n s 4.6 to 4.9. The t e c h n i q u e developed and used i n t h i s s t u d y , f o r the d e t e r m i n a t i o n of the r e a d i l y b i o d e g r a d a b l e COD, was c a p a b l e of measuring o n l y the t o t a l r e a d i l y b i o d e g r a d a b l e COD, which i n c l u d e the r e a d i l y b i o d e g r a d a b l e COD a l r e a d y p r e s e n t i n the raw sewage and the r e a d i l y b i o d e g r a d a b l e COD i n t r o d u c e d by the added c h e m i c a l s u b s t r a t e s . T h e r e f o r e , the r e s u l t s p r e s e n t e d i n t h i s study r e p r e s e n t the t o t a l r e a d i l y b i o d e g r a d a b l e COD and not the r e a d i l y b i o d e g r a d a b l e COD i n t r o d u c e d by the added s i m p l e carbon s u b s t r a t e s a l o n e . The dosages of v a r i o u s e x p e r i m e n t a l runs d e s c r i b e d i n s e c t i o n s 4.1 t o 4.4, were i n mg/L as COD i n the a n a e r o b i c r e a c t o r . To a c h i e v e these dosages, the feed c o n c e n t r a t i o n s were t w i c e as h i g h to account f o r the d i l u t i o n e f f e c t t a k i n g p l a c e i n -69-the anaerobic reactor due to the anoxic-anaerobic recycle (with recycle r a t i o of 1:1). The discussion presented in th i s section describe the chemical dosages with respect to the feed (and not to the anaerobic reactor contents). The plot of r e a d i l y biodegradable COD vs the chemical dosages (as COD) for the various experimental runs (Fig. 4.1) showed that for the same dosage (in mg/L as COD), the readily biodegradable substrates had the following decreasing order of e f f e c t . acetate > propionate > butyrate > glucose As expected, i t seems to suggest that the degree of biodegradability i s some inverse function of the complexity of the added compound. It was also noted that in the more linear ranges of the plot (between the dosages of 20-40 mg/L as COD for acetate, propionate, butyrate and between 60-120 mg/L as COD for glucose), an increase of 1 mg COD/L in chemical dosage increased the readily biodegradable COD in the feed by 0.80, 0.75, 0.45 and 0.27 mg/L for acetate, propionate, butyrate and glucose respectively. However, these values can only be used as approximate estimates, since the d i f f e r e n t batches of sewage used during each run might not have had the same readily biodegradable substrate content. This might also be a reason (besides any -70-l i m i t a t i o n of the method of measuring the r e a d i l y biodegradable COD) for not obtaining complete recovery of the added acetate,a simple e a s i l y degradable compound, as re a d i l y biodegradable COD (as observed by Nicholls et al.,1985). The chemical dosages in terms of either mM/L or mM of carbon/L did not show any s i g n i f i c a n t c o r r e l a t i o n with their degree of biodegradability. Surprisingly, the same dosages of acetate, propionate and butyrate expressed as mg/L of the corresponding v o l a t i l e fatty acid (acetic acid, propionic acid and butyric acid respectively), gave r e l a t i v e l y the same readily biodegradable COD content (Fig. 4.2). Therefore, t h i s i s observed to be a rough guide in estimating the r e a d i l y biodegradable COD of these group of chemicals, commonly encountered in the b i o l o g i c a l excess phosphorus removal process. However, glucose did not follow t h i s trend. Extrapolation of the plot in F i g . 4.2, gave a value of around 40 mg/L of readily biodegradable COD for zero chemical addition, indicating that the approximate average readily biodegradable COD of the raw sewage used in t h i s study was 40 mg/L. Since the average t o t a l COD of the raw sewage used during the period of these experiments was around 225 mg/L, on a percentage basis, 18% of the t o t a l COD in the raw sewage was readily biodegradable. This compares well with the value of 24% with respect to the biodegradable COD (ie about 20% of the t o t a l COD) obtained by Dold et a l . (1980) for South African sewage. - 7 1 -90 0 20 40 60 80 100 120 140 160 Dosoge in m g / L as COD In feed F i g . 4..1. T o t a l r e a d i l y b i o d e g r a d a b l e COD i n f e e d v s . c h e m i c a l a d d i t i o n t o f e e d , e x p r e s s e d as COD. 0 20 40 60 80 100 120 140 Dosoge in m g / L in feed F i g . 4 . . 2 . T o t a l r e a d i l y b i o d e g r a d a b l e COD i n f e e d v s . c h e m i c a l a d d i t i o n t o f e e d , e x p r e s s e d as m g / L . Dosages o f a c e t a t e , p r o p i o n a t e and b u t y r a t e a r e e x p r e s s e d as t h e i r c o r r e s p o n d i n g a c i d s . - 7 2 -The percentage of readily biodegradable COD entering a treatment f a c i l i t y i s s i g n i f i c a n t l y influenced by the type of c o l l e c t i o n system (Paepcke, 1983; Barnard, 1984). Col l e c t i o n systems providing long retention times can provide fermentative conditions, resulting in the breakdown of complex organic molecules to simple compounds causing high r e a d i l y biodegradable content. The c o l l e c t i o n system for the treatment plant, where the sewage used in t h i s study was obtained, had a very short detention time and thus was u n l i k e l y to provide enough fermentative conditions to breakdown the complex compounds. This was also confirmed by the absence of any measurable short chain v o l a t i l e fatty acid in the raw sewage. In addition to the nature of the sewage i t s e l f , t h i s might also be a reason for the s l i g h t l y low percentage of re a d i l y biodegradable substrate content obtained for the sewage used in t h i s study. 4.6 PHOSPHORUS RELEASE AND UPTAKE The prerequisite for b i o l o g i c a l excess phosphorus removal in the activated sludge process i s the presence of an anaerobic zone upstream of the aerobic zone. In general, phosphorus i s released in the anaerobic zone and taken up in the aerobic zone. The quantitative amounts of phosphorus release and uptake could be found by mass balance, using concentrations of phosphorus in various zones of the process. - 7 3 -During this study, phosphorus concentrations in the raw sewage and the effluent were measured both in terms of t o t a l phosphorus and ortho phosphorus. However, only ortho phosphorus was measured in the various reactors, since no simple technique was available for measuring u n f i l t e r e d t o t a l phosphorus without including the phosphorus present in mixed liquor suspended s o l i d s . For this reason, exact phosphorus mass balances in the individual reactors could not be done. However, the extreme range values could be calculated i f an assumption was made on which zone of the bioreactor caused the complex phosphorus ( i . e . , the difference between t o t a l phosphorus and ortho phosphorus in the feed) to be transformed to simpler ortho phosphorus. Tables 4 . 5 to 4 . 8 present the values for the phosphorus uptake in various reactors under different assumptions. The values of phosphorus uptake (with the negative sign indicating phosphorus release), per unit volume of the feed, presented in th i s section were derived in the following way using mass balance ( F i g . 4 . 3 ) . i anox Fig. 4 . 3 . Mass of phosphorus entering and leaving each i n d i v i d u a l reactor per unit i n f l u e n t flow. P indicates the ortho-P concentration. -74-P uptake = Mass of P entering - Mass of P leaving Phosphorus uptake (per unit i n f l u e n t flow) in the anaerobic reactor i s given by (influent ortho-P) + (anoxic ortho-P) - 2x(anaerobic ortho-P) Phosphorus uptake (per unit i n f l u e n t flow) in the anoxic reactor i s given by 2x(anaerobic ortho-P) + (aerobic ortho-P) - 3x(anoxic ortho-P) Phosphorus uptake (per unit i n f l u e n t flow) in the aerobic reactor i s given by 2x(anoxic ortho-P) - 2x(aerobic ortho-P) Depending on the zone in which the complex phosphorus i s transformed into ortho phosphorus, the difference between the t o t a l phosphorus and the ortho phosphorus of the feed i s added to the formula of the corresponding zone. It should also be noted that the d a i l y wastage was not included in c a l c u l a t i n g the phosphorus balance, since i t s contribution was i n s i g n i f i c a n t due to the wastage volume being very small compared to the d a i l y flows. The results presented in Tables 4.5 to 4.8 indicated that the differences between the phosphorus uptake values in the same zone under the d i f f e r e n t assumptions were not large. Therefore, the averages of these extreme values are used in the following -75-Table 4.5 Phosphorus Uptake for Acetate Run Chemical Dosage in Phosphorus Uptake Anaerobic Reactor (mg of phosphorus/L of feed) (mg COD/L) Anaerobic Anoxic Aerobic (a) when the complex phosphorus in the influent i s transformed completely to ortho phosphorus in the anaerobic zone 30 -16.1 -10.5 31.0 25 -14.3 -8.4 26.8 20 -8.7 -3.3 15.8 15 -1.6 -2.2 7.6 10 0.2 -1.0 3.2 5 1.1 -0.6 0.8 (b) when the complex phosphorus in the influent i s transformed completely to ortho phosphorus in the anoxic zone 30 -17.4 -9.2 31.0 25 -15.4 -7.4 26.8 20 -9.6 -2.4 15.8 15 -2.8 -1.0 7.6 10 -0.9 0.1 3.2 5 0 0.5 0.8 (c) when the complex phosphorus in the influent i s transformed completely to ortho phosphorus in the aerobic zone 30 -17.4 -10.5 32.3 25 -15.3 -8.4 27.8 20 -9.6 -3.3 16.7 15 -2.8 -2.2 8.8 10 -0.9 -1.0 4.3 5 0 -0.6 1.9 (d) when the complex phosphorus in the influent i s unchanged and ends up in the sludge 30 -17.4 -10.5 31.0 25 -15.3 -8.4 26.8 20 -9.6 -3.3 15.8 15 -2.8 -2.2 7.6 10 -0.9 -1.0 3.2 5 0 -0.6 0.8 -76-Table 4.6 Phosphorus Uptake f o r P r o p i o n a t e Run Chemical Dosage i n Anaerobic R e a c t o r (mg COD/L) Phosphorus Uptake (mg of phosphorus/L of feed) A n a e r o b i c Anoxic A e r o b i c (a) when the complex phosphorus i n the i n f l u e n t i s t r a n s f o r m e d c o m p l e t e l y t o o r t h o phosphorus i n the a n a e r o b i c zone 25 20 1 5 1 0 5 •11.6 -7.8 -2.2 -0.6 0.5 •6.8 •4.6 •0.5 •0.1 0.6 22.4 15.6 5.4 2.2 0 (b) when the complex phosphorus i n the i n f l u e n t i s t r a n s f o r m e d c o m p l e t e l y t o o r t h o phosphorus i n the a n o x i c zone 25 20-1 5 1 0 5 •12.7 -8.9 -3. 1 -1.5 -0.4 •5.7 •3.5 0.4 0.8 1 .5 22.4 15.6 5.4 2.2 0 (c) when the complex phosphorus i n the i n f l u e n t i s t r a n s f o r m e d c o m p l e t e l y to o r t h o phosphorus i n the a e r o b i c zone 25 20 1 5 10 5 •12, -8, -3, -1 , -0, •6.8 -4.6 •0.5 •0.1 0.6 23.5 16.7 6.3 3. 1 0.9 (d) when the complex phosphorus i n the i n f l u e n t i s unchanged and ends up i n the sludge 25 20 1 5 1 0 5 •12.7 -8.9 -3. 1 -1.5 -0.4 •6.8 •4.6 •0.5 •0.1 0.6 22.4 15.6 5.4 2.2 0 -77-Table 4.7 Phosphorus Uptake for Butyrate Run Chemical Dosage in Anaerobic Reactor (mg COD/L) Phosphorus Uptake (mg of phosphorus/L of feed) Anaerobic Anoxic Aerobic (a) when the complex phosphorus in the influent i s transformed completely to ortho phosphorus in the anaerobic zone 30 25 20 15 10 •12.7 -9.3 -5.9 -2.1 -0.7 •3.1 •2.0 •1 .2 •0.6 0.5 18.6 13.6 8.4 3.6 0.2 (b) when the complex phosphorus in the influent i s transformed completely to ortho phosphorus in the anoxic zone 30 25 20 15 10 •1 1.9 -8.3 -5.0 -1.1 0.3 •2.3 •1 .0 0.3 0.4 1 .5 18, 13, 8, 3, 0, (c) when the complex phosphorus in the influent i s transformed completely to ortho phosphorus in the aerobic zone 30 25 20 15 10 •1 1.9 -8.3 -5.0 -1.1 0.3 •3.1 •2.0 •1 .2 •0.6 0.5 19, 14, 9, 4, 1 , (d) when the complex phosphorus in the influent i s unchanged and ends up in the sludge 30 25 20 15 10 •1 1.9 -8.3 -5.0 -1.1 0.3 •3.1 •2.0 •1 .2 •0.6 0.5 18.6 13.6 8.4 3.6 0.2 -78-Table 4.8 Phosphorus Uptake for Glucose Run Chemical Dosage in Anaerobic Reactor (mg COD/L) Phosphorus Uptake (mg of phosphorus/L of feed) Anaerobic Anoxic Aerobic (a) when the complex phosphorus in the influent i s transformed completely to ortho phosphorus in the anaerobic zone 75 60 45 30 •15.8 •12.0 - 3 . 9 -1 .4 6.0 2.7 •2.8 1 .3 13.6 12.2 8.8 1.4 (b) when the complex phosphorus in the influent i s transformed completely to ortho phosphorus in the anoxic zone 75 60 45 30 -16.4 -12.9 - 4 . 8 - 2 . 4 6.6 3.6 -1 .9 2.3 13.6 12.2 8.8 1 .4 (c) when the complex phosphorus in the influent i s transformed completely to ortho phosphorus in the aerobic zone 75 60 45 30 •16.4 •12.9 - 4 . 8 - 2 . 4 6.0 2.7 -2.8 1 .3 14.2 13.1 9.7 2.4 (d) when the complex phosphorus in the influent i s unchanged and ends up in the sludge 75 60 45 30 -16.4 -12.9 - 4 . 8 - 2 . 4 6.0 2.7 •2.8 1 .3 13.6 12.2 8.8 1.4 -79-discussion. The average phosphorus uptake (or release, i f negative) in various zones for the d i f f e r e n t experimental runs are given in Table 4.9. 4.6.1 ANAEROBIC ZONE The anaerobic zone i s considered to be one in which neither dissolved oxygen nor n i t r a t e (or other forms of oxidized nitrogen) i s present. The presence of an anoxic zone (used for d e n i t r i f i c a t i o n ) in thi s study was to ensure that no N0 X (includes both nitrates and n i t r i t e s ) was introduced to the anaerobic reactor through the recycle. This was achieved during most of the study, except at the higher dosages of the glucose run. Phosphorus release in the anaerobic reactor for various chemical substrate additions, in terms of COD in the anaerobic reactor, i s shown in Fig.4.4. The phosphorus release in the anaerobic reactor, for the same COD dosage, appeared to have the following decreasing order of e f f e c t . acetate, propionate > butyrate > glucose This p e r f e c t l y agreed with the findings of Gerber et a l . (1986), although they used acetic, propionic and butyric acids instead of the i r sodium s a l t s . It i s also in general agreement with other -80-Table 4.9 Average Phosphorus Uptake for A l l Runs Chemical Dosage in Anaerobic Reactor (mg COD/L) Phosphorus Uptake (mg of phosphorus/L of feed) Anaerobic Anoxic Aerobic RUN 1 ACETATE 30 25 20 15 10 5 •16.75 -14.80 -9.15 -2.20 -0.35 0.55 -9.85 -7.90 -2.85 -1 .60 -0.45 -0.05 31 .65 27.30 16.25 8.20 3.75 1 .35 RUN 2 PROPIONATE 25 20 15 10 5 -12.15 -8.35 -2.65 -1 .05 0.05 -6.25 •4.05 •0.05 0.35 1 .05 22.95 16.15 5.85 2.65 0.45 RUN 3 BUTYRATE 30 25 20 15 10 -12.30 -8.80 -5.45 -1 .60 -0.20 -2.70 -1 .50 -0.75 -0.10 1 .00 1 9.00 14.10 8.85 4.10 0.65 RUN 3 : GLUCOSE 75 60 45 30 -16.10 -12.45 -4.35 -1 .90 -6.30 •3.15 •2.35 1 .80 1 3.90 12.65 9.30 1 .90 -81-studies by Potgieter and Evans (1983) and Oldham and Koch (1982), although these studies were conducted using non-acclimatized bio mass in batch experiments. It should be noted that propionic acid, instead of sodium propionate, was used during the study by Oldham and Koch (1982). Although the results reported by Potgieter and Evans (1983) showed the same general trend as those observed during t h i s current study, the anaerobic phosphorus releases with acetate and propionate were much higher than those obtained using butyrate and glucose. The authors reported that acetate, propionate, butyrate and glucose, at a concentration of 110 mg/L as COD, gave phosphorus releases of 58.6, 54.5, 8.2 and 5.0 mg/L respectively. The very low releases observed with butyrate and glucose in their studies could be primarily due to the usage of non-acclimatized biomass in their batch experiments. The much higher phosphorus releases observed with butyrate and glucose in t h i s current study (using acclimatized sludge) indicate the importance of using organisms acclimatized to the substrates under investigation, in studying the e f f e c t s of the various substrates on the b i o l o g i c a l excess phosphorus removal. However, according to Jones et a l . (1985), the phosphorus release due to butyric acid was much greater than the phosphorus release due to acetic acid (for the same COD dosage), somewhat contradicting the observations made in t h i s study. I n s u f f i c i e n t acclimatization period of two weeks allowed between the d i f f e r e n t -82-s u b s t r a t e a d d i t i o n s , d u r i n g the study by Jones et a l . ( l 9 8 5 ) , might be a reason f o r t h i s d i s c r e p a n c y , e s p e c i a l l y i f the a d d i t i o n of a c e t a t e had f o l l o w e d the b u t y r a t e a d d i t i o n . I t took almost f o u r weeks t o complete a s h i f t i n organism p o p u l a t i o n when the c h e m i c a l s u b s t r a t e was s w i t c h e d from p r o p i o n a t e to b u t y r a t e d u r i n g t h i s c u r r e n t study. Phosphorus r e l e a s e i n the a n a e r o b i c r e a c t o r v a r i e d almost l i n e a r l y w i t h the amount of a c e t a t e , p r o p i o n a t e or b u t y r a t e added, f o r dosages above 15 mg COD/L i n which a c t i v e b i o l o g i excess phosphorus removal was t a k i n g p l a c e ( F i g . 4 . 4 ) . The d e v i a t i o n of the 30 mg COD/L a c e t a t e run 'from t h i s t r e n d i s b e l i e v e d t o be due t o the phosphorus l i m i t i n g c o n d i t i o n s i n the feed. Based on thes e l i n e a r ranges, 1.26 mg of phosphorus was r e l e a s e d f o r ev e r y mg of a c e t a t e a v a i l a b l e as COD i n the ana e r o b i c r e a c t o r . T h i s r e p r e s e n t e d a molar r a t i o of 2.60 moles of phosphorus per mole of a c e t i c a c i d . The molar r a t i o v a l u e s f o r p r o p i o n a t e and b u t y r a t e were 3.44 and 3.69 moles of phosphorus per mole of the c o r r e s p o n d i n g a c i d r e s p e c t i v e l y . T h i s i n d i c a t e d t h a t b u t y r a t e r e l e a s e d phosphorus the most and a c e t a t e the l e a s t i n terms of molar r a t i o s . The molar r a t i o of 2.60 moles of phosphorus r e l e a s e d per mole of a c e t i c a c i d , d u r i n g the a c e t a t e run i s r e l a t i v e l y h i g h compared t o the v a l u e of 1.76 o b t a i n e d by R a b i n o w i t z (1985). Fukase et a l . (1982) r e p o r t e d a ph o s p h a t e : a c e t a t e molar r a t i o of 0.9:1, w h i l e A r v i n (1985) and Comeau et a l . (1987) obser v e d a -83-r a t i o of 1.4:1. However, these values were based on batch experiments where they were calcul a t e d by adding d i f f e r e n t concentrations of acetate at the beginning of the experiments and monitoring the corresponding phosphorus releases. These cont r o l l e d conditions are usually not present in continuous flow experiments where acetate i s added together with the sewage feed. The higher molar r a t i o obtained in t h i s current study under these continuous flow conditions might be due to the presence of higher than the added concentrations of acetate, r e s u l t i n g from the fermentation of the raw sewage in the anaerobic reactor. It should also be noted that the above reported r a t i o s were based on substrate u t i l i z a t i o n rather than substrate a v a i l a b i l i t y . S i e b r i t z et a l . (1983) reported a phosphate:acetate molar r a t i o of 2:1 based on substrate a v a i l a b i l i t y . Glucose did not exhibit a l i n e a r r e l a t i o n s h i p of phosphorus release in the anaerobic reactor, with the dosage expressed as COD. The phosphorus release was lower than expected during the 75 mg COD/L run, probably due to n i t r a t e entrainment into the anaerobic reactor. This w i l l be discussed in d e t a i l in section 4.6.3. The anaerobic phosphorus release was f i r s t recognized as an i n t r i n s i c part of the b i o l o g i c a l excess phosphorus removal mechanism by Barnard (1976). He also hypothesized that the anaerobic phosphorus release i s induced through an anaerobic stress that can be indicated by the oxidation reduction potential -84-(ORP) of that zone. The data from this current study shows the i n v a l i d i t y of t h i s anaerobic stress hypothesis. For example, during the experimental runs with 25 mg COD/L of acetate, propionate and butyrate, and 30 mg COD/L of glucose, the average oxidation - reduction potentials of the anaerobic zone were -338, -353, -364 and -353 mV respectively. This indicates that the degree of the anaerobic stress that was present during these runs would be r e l a t i v e l y the same. Should the anaerobic stress hypothesis be true, the phosphorus releases during these runs should have been approximately equal. However, the phosphorus releases were s i g n i f i c a n t l y d i f f e r e n t , with the following decreasing order (with the average phosphorus releases given in brackets, in mg/L). acetate (14.80) > propionate (12.15) > butyrate (8.80) > glucose (1.90) For a l l experimental runs, there was a net release of phosphorus in the anaerobic reactor when the concentration of re a d i l y biodegradable COD in i t was above approximately 25 mg/L. Also, the anaerobic phosphorus release increased with increasing r e a d i l y biodegradable COD present in the same reactor (Fig.4.5). This i s in good agreement with the observations made by S i e b r i t z et a l . (1983) who suggested that a minimum of about 25 mg COD/L of r e a d i l y biodegradable COD concentration in the anaerobic reactor was necessary to stimulate the phosphorus release and that the phosphorus release increased with increasing readily -85-RBD COD in anaerobic zone (mg/L) Fig. 4 . 5 . The anaerobic phosphorus, release with r e a d i l y biodegradable COD available i n the anaerobic zone. - 8 6 -biodegradable COD present in the anaerobic reactor. The close agreement of the phosphorus release for various substrate additions, with respect to readily biodegradable COD in the anaerobic zone, emphasizes the importance of the a v a i l a b i l i t y of r e a d i l y biodegradable substrate in the anaerobic reactor. This i s a major s h i f t in emphasis away from the i n i t i a l hypothesis that a minimum degree of anaerobic stress i s the necessary precondition for the anaerobic phosphorus release and the subsequent o v e r a l l excess phosphorus removal (as also reported by S i e b r i t z et a l . , 1983). 4.6.2 AEROBIC ZONE Under aerobic conditions, the conditioned biomass removes phosphorus from solution. The uptake of phosphorus in the aerobic zone for various experimental runs are shown in Figs.4.6 and 4.7. The r e s u l t s indicate that the phosphorus uptake in the aerobic reactor increased with the increasing chemical dosage. For the same COD dosage, the aerobic phosphorus uptake had the following decreasing order of e f f e c t . acetate > propionate > butyrate > glucose An apparent d i r e c t relationship was observed between the mass of phosphorus released under anaerobic conditions and the -87 -46 56 65 76 86 RBD COD in feed (mg/L) F i g . I . 7 . The aerobic phosphorus uptake with r e a d i l y biodegradable COD i n the feed (including the chemical a d d i t i o n ) . -88-mass of phosphorus taken up under subsequent aerobic conditions for most portions of the study, except for the runs with higher glucose dosages. Less than expected phosphorus uptake observed during the experimental runs with higher glucose dosages w i l l be addressed in the next section. Increased aerobic phosphorus uptake was observed with increased anaerobic phosphorus release and the r e l a t i o n s h i p was nearly linear (Fig.4.8). For a l l the data points obtained for the aerobic phosphorus uptake and the anaerobic phosphorus release during t h i s study, the l i n e a r regression analysis gave the following 2 r e l a t i o n s h i p between them, with R being 0.830 (where R i s the c o r r e l a t i o n c o e f f i c i e n t ) . P uptake (mg/L) = 1.94 + 1.402 x P release (mg/L) The data points obtained during the higher dosages of glucose (75 and 60 mg COD/L runs), however, should hot be given importance, since these runs had other problems to be discussed in section 4.6.3. Neglecting these two data points gave a s i g n i f i c a n t improvement in the co r r e l a t i o n between the phosphorus uptake in the aerobic zone and the phosphorus release in the anaerobic 2 zone, with R being 0.970 (where R i s the c o r r e l a t i o n c o e f f i c i e n t ) . The rel a t i o n s h i p obtained was as follows: P uptake (mg/L) = 1.21 + 1.701 x P release (mg/L) - 8 9 -However, Wentzell et a l . (1984) developed the following as the r e l a t i o n s h i p between the phosphorus uptake and release in their study of the b i o l o g i c a l excess phosphorus removal. P uptake (mg/L) = 3.14 + 1.145 x P release (mg/L) In the equations discussed above, the phosphorus uptake for zero phosphorus release gives the basic metabolic requirement of the t o t a l organism mass in the system. The higher basic metabolic phosphorus requirement of 3.14 mg/L obtained by Wentzell et a l . (1984), compared to 1.21 mg/L obtained in t h i s current study, i s p r i m a r i l y a t t r i b u t e d to the presence of higher mass of organisms, r e s u l t i n g from the higher COD concentration of the feed. In a t y p i c a l activated sludge process, 1-1.5 mg/L of phosphorus i s removed for the basic metabolism of the c e l l s , for every 200 mg/L of COD removed (U.S. EPA, 1976). Using t h i s as a guideline, the basic metabolic phosphorus requirements of the systems during the study by Wentzell et a l . (feed COD was 500 mg/L) and t h i s current study (feed COD was 225 mg/L, on average) would be 2.50-3.75 mg/L and 1.13-1.69 mg/L respectively. The corresponding values obtained from the equations discussed above, f a l l within these ranges. The lower c o e f f i c i e n t term (of 1.145) associated with the phosphorus release in the equation by Wentzell et a l . (1984), compared to that (1.701) of the current study, seems to suggest that t h e i r system was preferred substrate l i m i t i n g , since t h e i r feed phosphorus concentrations were r e l a t i v e l y high at 15-20 mg/L. -90-4.6.3 ANOXIC ZONE Anoxic zone i s one i n which the e l e c t r o n a c c e p t o r i s a v a i l a b l e i n the form of NO and not as d i s s o l v e d oxygen. S i n c e a A s u b p o p u l a t i o n of the b i o l o g i c a l excess phosphorus removal b a c t e r i a a re c a p a b l e of d e n i t r i f i c a t i o n , phosphorus uptake can take p l a c e i n the presence of NO i n s t e a d of d i s s o l v e d oxygen (McLaren et a l . 1976, Osborn et a l . 1978, Simpkins e t a l . 1978, Iwema et a l . 1984, Comeau et a l . 1985). The replacement of the d i s s o l v e d oxygen w i t h NO as the t e r m i n a l e l e c t r o n a c c e p t o r i s f a c i l i t a t e d by the f o r m a t i o n of the enzyme, n i t r a t a s e , whose p r o d u c t i o n i s g e n e r a l l y i n h i b i t e d by the presence of d i s s o l v e d oxygen. Other b i o l o g i c a l excess phosphorus removing b a c t e r i a t h a t cannot use NO as e l e c t r o n a c c e p t o r , a r e ex p e c t e d t o r e l e a s e A phosphorus, p r o v i d e d t h a t s u f f i c i e n t r e a d i l y b i o d e g r a d a b l e s u s t r a t e s a re a v a i l a b l e . The " b i o - P " d e n i t r i f i e r s , i n the absence of NO , behave l i k e those not ca p a b l e of u s i n g NO as the A X e l e c t r o n a c c e p t o r . The observed phosphorus c o n c e n t r a t i o n i n the a n o x i c zone i s , t h e r e f o r e , the net r e s u l t of the opposing phosphorus r e l e a s e and uptake r e a c t i o n s , and depends on the r e l a t i v e magnitudes of these two r e a c t i o n s . These two opposing r e a c t i o n s can occur s i m u l t a n e o u s l y i n the presence of both the p r e f e r r e d s u b s t r a t e s (such as a c e t a t e or p r o p i o n a t e ) and e l e c t r o n a c c e p t o r s (NO or - 9 1 -Studies by Gerber et a l . (1987) showed that the phosphorus release reaction predominates when a preferred substrate, such as acetate or propionate, i s present in the anoxic (or aerobic) zone in s i g n i f i c a n t concentration. The release reaction continues u n t i l the preferred substrate i s consumed, whereupon the phosphorus uptake commences. The observation of phosphorus release in the presence of a preferred substrate, even under aerobic conditions, may be explained in terms of pH gradient across the c e l l membrane. For example, when acetate i s transported across the c e l l membrane, a pH gradient s h i f t i s established due to a proton accompanying each acetate molecule to f a c i l i t a t e i t s transport across the c e l l membrane as an electrochemically neutral form (HAc) (Comeau, 1984). The pH gradient across the c e l l membrane i s quickly reestablished by the degradation of the stored polyphosphate and thereby releasing phosphorus into s o l u t i o n . At low chemical dosages, most of the readily biodegradable substrates get u t i l i z e d in the anaerobic reactor and very l i t t l e or none enter the anoxic reactor. Thus, the phosphorus uptake reaction becomes dominant over the phosphorus release reaction causing net anoxic phosphorus uptake (provided s u f f i c i e n t N0 x i s present). At higher dosages, some bleeding of the added simple carbon substrates into the anoxic reactor may occur, causing the phosphorus release reaction to become dominant, as reported by Gerber et a l . (1987). This r e s u l t s in a net phosphorus release in the anoxic reactor under these conditions. Fig.4.9 shows that - 9 2 --1 1 3 6 7 9 11 13 16 17 Phosphorus Rdoase (mg of P/ L of f««d) Fig.4.8. Relationship between the aerobic phosphorus uptake and the anaerobic phosphorus release. 0 2 0 40 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 Fig.U.9 . Dosage in feed (mg COD/L) The anoxic phosphorus uptake with chemical dosage, expressed as COD i n feed. -93-t h i s behaviour was observed during most of this study, except with the higher dosages of glucose. During 75 and 60 mg COD/L glucose runs (or 150 and 120 mg COD/L with respect to the feed), the occurrence of fermentative conditions in the anoxic reactor was suspected due to low oxidation reduction potentials, sticky appearance of the mixed liquor and the formation of substantial gas bubbles. According to Wilderer et a l . (1987), fermentative conditions in such a system would enrich the biocommunity for bacteria which reduce n i t r a t e only to n i t r i t e , and such a population s h i f t (towards f a c u l t a t i v e anaerobes) occurs at the expense of d e n i t r i f i e r s . A similar observation was also made by Manoharan et al.(1988), while using glucose as the carbon source for d e n i t r i f i c a t i o n in b i o l o g i c a l treatment of l a n d f i l l leachates. Although no d i f f e r e n t i a t i o n between n i t r i t e s and nit r a t e s were made during t h i s current study, the NOx concentrations in the anoxic reactor were higher than normal for the 75, 60 and 30 mg COD/L dosages of glucose. Since the population s h i f t was at the expense of the normal d e n i t r i f i e r s , the "bio-P" d e n i t r i f i e r s might have been active in the anoxic zone during these runs, causing anoxic phosphorus uptake, as shown in Fig.4.9. It should also be noted that even i f some glucose bled into the anoxic zone at the higher dosages, the studies by Gerber et a l . (1987) showed that glucose does not give r i s e to any -94-phosphorus release unless s t r i c t anaerobiosis p r e v a i l s . The reason for thi s may be that unless s t r i c t anaerobiosis p r e v a i l s , glucose cannot be fermented to form the preferred substrates (such as acetate or propionate) to e f f e c t any phosphorus release, as discussed e a r l i e r . 4.6.4 PHOSPHORUS ACCUMULATION IN SLUDGE As discussed in the preceding sections, phosphorus i s released in the anaerobic zone and taken up in the aerobic zone. The phosphorus taken up from the solution during the aerobic phase i s stored in the sludge and ph y s i c a l l y removed from the system through wasting. Therefore, the dry weight percent phosphorus content of the aerobic sludge i s a good indication of the extent of b i o l o g i c a l excess phosphorus removal. Since the dry weight percent phosphorus content of the organisms in a t y p i c a l activated sludge system, not exhibiting b i o l o g i c a l excess phosphorus removal, i s about 1.5% (Hoffmann and Marais, 1977), any increased phosphorus content above 1.5% i s a di r e c t indication of the degree of excess phosphorus removal. The res u l t s from th i s study showed that the percent phosphorus in the aerobic sludge increased with increasing chemical dosages (Fig.4.10). The percent phosphorus in the aerobic sludge had the following decreasing order of e f f e c t , among the various added compounds, for the same dosage expressed as COD. -95-acetate > propionate > butyrate > glucose This was expected, since for the same dosage, expressed as COD, the aerobic phosphorus uptake had the same decreasing order of e f f e c t , as discussed in section 4.6.2. The percent phosphorus in the aerobic sludge was above 1.5% for a l l the added chemical substrates, when the readily biodegradable COD in the feed exceeded approximately 54 mg/L (corresponds to the anaerobic zone value of 27 mg/L, due to the 1:1 r e c y c l e ) . This confirms the findings of S i e b r i t z et al.(l982) that at least 25 mg/L of readily biodegradable COD in the anaerobic zone i s necessary for any b i o l o g i c a l excess phosphorus removal to occur. The percent phosphorus values were more comparable among the various chemicals when the dosages were expressed in terms of readi l y biodegradable COD (Fig.4.11), rather than as t o t a l COD. 4.7 CARBON STORAGE AND CONSUMPTION Bacteria responsible for the b i o l o g i c a l excess phosphorus removal (hereafter referred to as bio-P bacteria) are those capable of storing both polyphosphate under aerobic conditions and carbon under anaerobic conditions (Comeau et al.,1985). The key to the p r o l i f e r a t i o n of the bio-P bacteria i s l i k e l y the a b i l i t y to store carbon in a stressed environment, where aerobic -96-c a •a D a o 0 z a. ti 0 a. c CL 5 -3 -2 -1 a Acetate + Propionate O Butyrate A Glucose 20 - r -40 I 60 I 60 —I— 100 —I— 120 140 160 F i g . 4 - 1 0 6 Dosage in mg/L as COD in feed R e l a t i o n s h i p between t h e a e r o b i c s l u d g e p e r c e n t p h o s p h o r u s a n d t h e c h e m i c a l d o s a g e , e x p r e s s e d as COD i n f e e d . o o 3 a o i . o o o c a. D 0 JC a. c e u i . o a 4 -2 - a /"cetate + Propionate O Butyrate A Glucooo 40 —r-60 I 60 70 I 80 90 F i g . 4 . 1 1 RBD COD in feed (mg/L) R e l a t i o n s h i p between the a e r o b i c s l u d g e p e r c e n t p h o s p h o r u s and t h e r e a d i l y b i o d e g r a d a b l e COD i n f e e d ( i n c l u d i n g the c h e m i c a l a d d i t i o n ) . -97-metabolism i s not possible. Substrate l i m i t i n g conditions often occur in the aerobic zone of a completely mixed activated sludge process. Therefore the organisms, such as the bio-P bacteria, that have i n t r a c e l l u l a r l y stored carbon have a d e f i n i t e advantage over those that rely on the membrane transport of the substrates. It i s for t h i s reason that an anaerobic, aerobic zone sequence i s a prerequiste for the b i o l o g i c a l excess phosphorus removal process, as described in Section 4.6. No carbon storage analysis was done during the acetate runs. Carbon storage (and consumption) was made up of both poly-B-hydroxyvalerate (PHV) and poly-B-hydroxybutyrate (PHB) during the propionate runs. PHB was the most s i g n i f i c a n t storage compound during the butyrate runs, although some PHV was also found. The glucose runs involved two carbon storage compounds, PHB and glycogen. The quantitative values of carbon consumption (with negative sign indicating carbon storage) presented in t h i s section were derived in the following way, using a mass balance c a l c u l a t i o n (Fig. 4 .12). Feed, with i t s low s o l i d s , was assumed to have no carbon storage compounds (C^ n^ = 0) upon entering the system. Daily wastage from the system was not taken into account, since i t s contribution was i n s i g n i f i c a n t as discussed in Section 4 . 6 . - 9 8 -<k anox Fig.4-.12. Mass of carbon storage compounds entering and leaving each individual reactor per unit influent flow. C indicates the concentration of the carbon storage compounds. *It should be noted that the return sludge has twice the aerobic carbon concentration. C consumption = Mass of C entering - Mass of C leaving Carbon consumption (per unit influent flow) in the anaerobic reactor is given by (anoxic carbon) - 2x(anaerobic carbon) Carbon consumption (per unit influent flow) in the anoxic reactor is given by 2x(anaerobic carbon) + 2x(aerobic carbon) - 3x(anoxic carbon) Carbon consumption (per unit influent flow) in the aerobic reactor is given by 2x(anoxic carbon) - 2x(aerobic carbon) The various carbon balances for the different experimental runs are presented in Tables 4.10 to 4.12. -99-T'able 4.10 Carbon Consumption f o r P r o p i o n a t e Run P r o p i o n a t e Dosage i n Carbon Consumption Anaerobic Reactor (mg /L of feed) (mg COD/L) A n a e r o b i c Anoxic A e r o b i c . PHB PHV PHB PHV PHB PHV 25 -15.5 -11.7 -9.7 -3.1 25.2 14.8 20 -10.5 -4.5 -2.7 -10.5 13.2 15.0 15 -8.5 -8.6 0.9 12.4 7.6 -3.8 1 0 -6.2 -9.8 3.2 5.2 3.0 4.6 .5 -3.4 -9.8 2.8 9.2 0.6 0.6 Table 4.11 Carbon Consumption f o r B u t y r a t e Run B u t y r a t e Dosage i n Carbon Consumption An a e r o b i c Reactor (mg/L of feed) (mg COD/L) A n a e r o b i c Anoxic A e r o b i c PHB PHV PHB PHV PHB PHV 30 -27.7 -7.0 -7.1 4.6 34.8 2.4 25 -19.3 -4.0 -4.7 1 .6 24.0 2.4 20 -11.3 -1.8 -2.5 -0.8 13.8 2.6 1 5 -6.0 -1.4 -0.2 -0.4 6.2 1 .8 10 -4.5 -1.6 0.5 0.8 4.0 0.8 -1 00-4.7.1 ANAEROBIC ZONE In the anaerobic zone of the b i o l o g i c a l excess phosphorus removal process, polyphosphate reserves of the bio-P bacteria are postulated to be broken down and hydrolyzed in order to f a c i l i t a t e transport and i n t r a c e l l u l a r storage of the available simple carbonaceous substrates. Since these substrates, such as acetate and propionate, have to be transported through the membrane of the organisms in an electrochemically neutral f o r -(as acetic a c i d and propionic a c i d ) , degradation ot polyphosphates re-establishes the pH gradient across the c e l l membrane, thereby f a c i l i t a t i n g more carbon storage (Comeau et al.,1985). It i s also suggested that the polyphosphate breakdown serves as an energy source for the carbon storage (Kulaev,1975; Mino et al.,1984). Phosphate r e s u l t i n g from the polyphosphate breakdown i s released into the solution (as discussed in section 4.6.1) since i t i s unusable by the c e l l under such conditions. These are i l l u s t r a t e d in terms of a simple biochemical model in Fig.4.13. According to Comeau et al.(1987), the proportion of carbon storage as PHV would exceed the storage as PHB when short chain fatty acids containing an odd number of carbons (e.g. propionate) are added to the system, whereas more PHB than PHV would be accumulated with the addition of short chain fatty acids containing an even number of carbon atoms (e.g. acetate, butyrate). The authors used batch experiments with pure substrate -101-Table 4.12 Carbon Consumption f o r Glucose Run Glucose dosage i n Carbon Consumption Anaerobic R e a c t o r (mg /L of feed) (mg COD/L) Ana e r o b i c . Anoxic A e r o b i c PHB G l y c . PHB G l y c . PHB G l y c 75 -43.0 108 12.8 -24 30.2 -84 60 -31.9 68 5.5 -10 26. 4 -58 45 -12.2 29 -3.2 5 15.4 -34 30 -7.6 1 3 3.0 1 4.6 -14 Fig.4.13. A sim p l i f i e d model for anaerobic metabolism of bio-P bacteria. Adapted from Comeau et al.(1985). -1 02-additions in reaching these conclusions. This current study confirmed these findings, except at lower propionate dosages. To store 1 mole of PHV, 1 mole of propionate and 1 mole of acetate should be present. The acetate component can be obtained either by breaking down some of the propionate molecules or from the fermentation of the raw sewage. Therefore, at lower propionate dosages, i t i s possible that the number of acetate molecules may outnumber those of propionate. This si t u a t i o n can result in the formation of PHB by some acetate molecules among themselves, thereby r e s u l t i n g in more PHB than PHV during the experimental runs with lower propionate dosages, as observed in t h i s study. Carbon storage and consumption as PHV during the propionate run and as PHB in both butyrate and glucose runs are in very good agreement with the above described biochemical model. Since PHV storage was due to the addition of propionate and followed the standard trend of increasing with increasing phosphorus release, i t was assumed to represent the carbon storage, with respect to the bio-P bacteria, for the propionate run. PHB, as the carbon storage compound during propionate addition, d i d not follow the expected trend during most of the run, with carbon storage decreasing with increasing phosphorus release (Fig.4.14). The presence of PHB could have been due to non-bio-P bacteria (since i t i s a common storage compound in many bacteria) and could have resulted from acetate derived from the -103-fermentation of the feed, as discussed e a r l i e r . However, great importance should not be given to these values, since the carbon analysis during the propionate addition was done only once for each steady state run, compared to three times during the butyrate and glucose runs. As discussed in Section 4.6.1, for the same amount of anaerobic phosphorus release, the addition of chemical substrate necessary (as COD) i s least for propionate and most for glucose ( i f the acetate runs are not considered). This explains the observation that the anaerobic carbon storage was found to be the most during glucose addition and least during propionate addition, for the same amount of anaerobic phosphorus release (Fig.4.15). From the e a r l i e r discussions, i t may be concluded that the role of the anaerobic zone in a b i o l o g i c a l excess phosphorus removal process emerges as the one in which the carbon storage by the bio-P bacteria should be maximized. Fig.4.16 c l e a r l y shows that t h i s could be ensured by the presence of simple carbonaceous substrates in the anaerobic zone. The close agreement among the carbon storage values during various runs in Fig.4.17 shows the importance of quantifying the amount of readily biodegradable COD entering the anaerobic zone, including both the feed and the chemical addition, for any meaningful comparison purposes. (It should be noted that the -104-Phosphorus Release (mg of P /L of feed) .4..14.. Relationship between carbon storage and phosphorus release the anaerobic zone f or the propionate run. 45 AO -35 -30 -25 -~ 20 -15 -10 -+ Propionate O Butyrate Glucose -1 T 3 - r -5 -T-9 11 I 13 I 15 17 Fig.4-15. Phosphor L B release (mg of P /L of feed) Relationship between carbon storage and phosphorus release i n the anaerobic zone. Carbon storage i s as PHV for propionate addition and as PHB for butyrate and glucose additions. - 1 0 5 -45 40 -35 -30 25 -20 -15 10 -5 -+ Propionate O Butyrate Glucose I 20 I 40 I 60 80 4.16. 45 40 Dosage in anaerobic zone (mg COD/L) Anaerobic carbon storage vs. chemical dosage, as COD entering the anaerobic zone. Carbon storage i s as PHV for propionate addition and as PHB for butyrate and glucose additions. 35 -30 25 -20 15 -10 + Propionate O Butyrate A Glucose —T— 24 I 26 I 28 —T— 32 - r -34 - T -36 —I-38 I 40 I 42 22 4.17. 30 RBD COO In anaerobic reactor (mg/L) Anaerobic carbon storage vs. r e a d i l y biodegradable COD (from the feed and the chemical addition) entering the anaerobic zone. Carbon storage i s as PHV for propionate addition and as PHB for butyrate and glucose additions. -106-addition of 1 mg of glucose as COD, contributes only 0.27 mg as re a d i l y biodegradable COD, as discussed in section 4.5). In other words, i t means that the readi l y biodegradable COD entering the anaerobic zone i s an excellent indicating parameter for the anaerobic carbon storage capacity of the system. 4.7.2 AEROBIC ZONE In a b i o l o g i c a l excess phosphorus removal process, the bio-P bacteria have s i g n i f i c a n t carbon reserves and low polyphosphate, when they enter the aerobic zone. It i s very common for the exogenous carbonaceous substrates to be at a low concentration in the aerobic zone of a completely mixed activated sludge process. In fact, an examination of 100 sets of annual performance data from a variety of municipal activated sludge wastewater plants showed that the effluent q u a l i t y , in terms of BODg, to be 50 mg/L or less 95% of the time; equal to 20 mg/L or less 50% of the time; and equal to 10 mg/L or less 19% of the time (W.P.C.F. et a l . , 1977). These values are approximately the same for the f i l t e r e d mixed liq u o r of the aerobic zone in a t y p i c a l completely mixed activated sludge process, indicating a low concentration of available external carbonaceous substrates in the aerobic zone. Under these conditions, the bio-P bacteria degrade t h e i r carbon reserves and store polyphosphate by removing soluble phosphate from the solut i o n . Therefore, phosphorus uptake in the aerobic zone i s accompanied by stored carbon consumption - 1 0 7 -(as i l l u s t r a t e d in Fig.4.18 by a s i m p l i f i e d biochemical model). The results of t h i s study, shown in Fig.4.19, agreed well with t h i s theory. The stored carbon in the c e l l s can be used either for their growth or to take up phosphorus from solution. The results of t h i s study showed that for the same amount of carbon consumption, the associated phosphorus uptake had the following decreasing order of e f f e c t . propionate > butyrate > glucose This means that during propionate addition, a higher proportion of the stored carbon was used for the phosphorus uptake, rather than for growth, indicating the effectiveness of propionate in the b i o l o g i c a l excess phosphorus removal over butyrate and glucose. 4.7.3 ANOXIC ZONE As described in section 4.6.3, when s u f f i c i e n t NO i s present in the absence of any preferred substrate (such as acetate or propionate), the bio-P d e n i t r i f i e r s could use NOx as the electron acceptor, consume the stored carbon and take up phosphorus. This i s believed to be the case during the experimental runs with low propionate and butyrate dosages, since - 1 0 8 -Fig.4-.18. A simplified model for aerobic metabolism of bio-P bacteria. Adapted from Comeau et al.(l985). 40 Phosphorus uptake (mg of P /L of feed) Fig. 4.19. Relationship between carbon consumption and phosphorus uptake i n the aerobic zone. Carbon storage i s as PHV for propionate addition and as PHB for butyrate and glucose additions. -109-the p o s s i b i l i t y of these s u b s t r a t e s b l e e d i n g i n t o the a n o x i c zone i s remote. When a p r e f e r r e d s u b s t r a t e i s pr e s e n t under the a n o x i c (or a e r o b i c ) c o n d i t i o n s , the p r e f e r r e d s u b s t r a t e i s u t i l i z e d and s t o r e d as carbon r e s e r v e s by the organisms; t h i s i s a c c o m p l i s h e d by b r e a k i n g down p o l y p h o s p h a t e s and r e l e a s i n g phosphorus, s i m i l a r to those observed i n the a n a e r o b i c zone. A l t h o u g h these two opposing r e a c t i o n s can tak e p l a c e s i m u l t a n e o u s l y i n the presence of both p r e f e r r e d s u b s t r a t e s and e l e c t r o n a c c e p t o r s , the r e l e a s e r e a c t i o n was r e p o r t e d t o predominate u n t i l the p r e f e r r e d s u b s t r a t e s d i s a p p e a r (Gerber e t a l . , 1987). T h i s might be the case d u r i n g the runs w i t h h i g h e r dosages of p r o p i o n a t e and b u t y r a t e , as d i s c u s s e d i n S e c t i o n 4.6.3. However, the presence of g l u c o s e i n p l a c e of the above d i s c u s s e d p r e f e r r e d s u b s t r a t e s does not g i v e r i s e t o phosphorus r e l e a s e ( i n c o n j u n c t i o n w i t h carbon s t o r a g e ) u n l e s s s t r i c t a n a e r o b i o s i s p r e v a i l s (Gerber e t a l . , 1987). T h i s e x p l a i n s the behaviour observed d u r i n g the g l u c o s e a d d i t i o n (Table 4.12) where s t o r e d carbon was consumed d u r i n g a l l e x p e r i m e n t a l r u n s , except the 45 mg COD/L run. D u r i n g t h i s r u n , w i t h the absence of any NO x and low ORP (-176 mV) i n the a n o x i c zone, the p o s s i b i l t y of complete a n a e r o b i o s i s e x i s t e d , t h e r e b y promoting phosphorus r e l e a s e and carbon s t o r a g e . Whatever the case might be, the r e s u l t s of t h i s c u r r e n t study c l e a r l y showed t h a t i n the a n o x i c zone, phosphorus r e l e a s e was a s s o c i a t e d w i t h carbon s t o r a g e ( s i m i l a r t o the a n a e r o b i c -1 10-zone) and the phosphorus uptake was associated with the carbon consumption (similar to the aerobic zone) (Fig.4.20). 4.7.4 CARBON STORAGE AND CONSUMPTION AS GLYCOGEN One of the reasons for selecting glucose as the added chemical substrate for the f i n a l run was to investigate whether the glucose would be stored and consumed primarily as glycogen or whether i t would f i r s t be reduced to acetate (or propionate) and follow the pathway of PHB storage and consumption, as observed with e a r l i e r runs. Fukase et al . ( l 9 8 2 ) , while using synthetic wastewater with glucose and peptone as the only BOD sources, found that glycogen was stored under anaerobic conditions during batch experiments using acclimated biomass. The maximum glycogen storage was achieved at the time when the soluble glucose disappeared from the solution. However, soon a f t e r the aerobic conditions were i n i t i a t e d , the glycogen concentration started to increase again, but at a slower rate than during the anaerobic conditions in the presence of soluble glucose. No explanation was given for t h i s behaviour. Mino et a l . (1987), in their studies using batch experiments with synthetic feed (containing acetic a c i d , sodium propionate, glucose and peptone), found that the i n t r a c e l l u l a r -111-carbohydrates are consumed under anaerobic conditions and stored under aerobic conditions. The authors a t t r i b u t e the consumption of glycogen to the decrease in the i n t r a c e l l u l a r carbohydrate concentration under anaerobic conditions. The contradictory behaviour observed during these two studies may be due to the difference in the composition of the feed. The presence of compounds (such as acetate) other than glucose i s believed to have played an important ro l e , as w i l l be discussed l a t e r . Since acetate could be formed in the anaerobic zone due to fermentation, when using sewage as feed, the behaviour reported by Mino et a l . (1987) appears to be more l o g i c a l to expect under the conditions of t h i s current study. In f a c t , i t was confirmed by the glucose runs of t h i s study. The re s u l t s of the glucose runs in t h i s study showed that glycogen was consumed in the anaerobic zone while phosphorus was released, and was stored in the aerobic zone while phosphorus was taken up (Fig . 4.21). The amounts of glycogen storage and consumption increased with increasing glucose dosages (Table 4.12). Substantial amounts of PHB were also found to be synthesized in the anaerobic zone and consumed in the aerobic zone. The PHB followed the standard pattern observed with the other runs, as discussed in the previous sections. -1 1 2 -14 - 7 - 6 - 3 - 1 1 3 6 7 Phosphorus uptake (mg of P/L of feed) F i g . 4-.20. R e l a t i o n s h i p between c a r b o n s t o r a g e and p h o s p h o r u s uptake i n t h e a n o x i c z o n e . C a r b o n s t o r a g e i s a s PHV f o r p r o p i o n a t e a d d i t i o n and as PHB f o r b u t y r a t e and g l u c o s e a d d i t i o n s . 20 15 -- 2 0 H 1 1 1 1 1 1 1 T 1 r— - 1 0 0 - 6 0 - 2 0 20 60 100 Glycogen consumption (mg/L of feed) F i g . 4 . . 2 1 . R e l a t i o n s h i p between the p h o s p h o r u s u p t a k e and g l y c o g e n consumption i n v a r i o u s zones f o r g l u c o s e r u n . -113-An a l m o s t l i n e a r r e l a t i o n s h i p e x i s t e d between t h e amounts of i n t r a c e l l u l a r PHB s t o r e d and t h e amounts of i n t r a c e l l u l a r g l y c o g e n d e p l e t e d ( F i g . 4 . 2 2 ) . The e s t i m a t e d mean v a l u e f o r t h e i n c r e a s e i n t h e amount of PHB p e r u n i t mass of g l y c o g e n consumed i s a p p r o x i m a t e l y 0.41 on a weight b a s i s . T h i s compares v e r y w e l l w i t h a s i m i l a r v a l u e of 0.2, o b t a i n e d by Somiya e t a l . d 9 8 8 ) , based on the t o t a l c a r b o h y d r a t e c o n s u m p t i o n r a t h e r t h a n t h e g l y c o g e n c o n s u m p t i o n . T h i s v a l u e would c o n v e r t t o ab o u t 0.48 i n terms of g l y c o g e n c o n s u m p t i o n , b a s e d on t h e f i n d i n g s o f F u k a s e e t a l . ( 1 9 8 2 ) t h a t a p p r o x i m a t e l y 42% of t h e t o t a l s u g a r s s t o r e d i n the s l u d g e was as g l y c o g e n . The above d i s c u s s e d b e h a v i o u r c o u l d be e x p l a i n e d u s i n g m i c r o b i a l pathways as f o l l o w s : Assuming t h a t a c e t a t e would be formed by t h e f e r m e n t a t i v e o r g a n i s m s i n t h e a n a e r o b i c zone, by f e r m e n t i n g e i t h e r t h e raw sewage or t h e added g l u c o s e , i t c o u l d be f i r s t c o n v e r t e d t o a c e t y l - C o A , as shown ( i n a b b r e v i a t e d form) below: ATP ADP CH^ COOH ^ — > Acetyl-CoA ( + ? ± ) The ATP r e q u i r e d f o r t h i s c o n v e r s i o n c o u l d be s u p p l i e d f r o m t h e h y d r o l y s i s of t h e a c c u m u l a t e d p o l y p h o s p h a t e , and the A c e t y l - C o A i s t h e n c o n v e r t e d t o PHB. NADH NAD+ Acetyl-CoA ^ — _ (C H,0 ) -114-A c c o r d i n g t o Matsuo (1985) and Comeau (198 4 ) , t h e NADH r e q u i r e d f o r the s y n t h e s i s o f PHB i s p r o d u c e d by o x i d i z i n g some of the a c e t y l - C o A t o c a r b o n d i o x i d e t h r o u g h t h e t r i c a r b o x y l i c a c i d (TCA) c y c l e . But Mino e t a l . (1987) i n d i c a t e d t h a t the TCA c y c l e , i n g e n e r a l , d o e s not work under a n a e r o b i c c o n d i t i o n s s i n c e s u c c i n a t e d e h y d r o g e n a s e , one of t h e enzymes i n the TCA c y c l e , c a n n o t m a i n t a i n i t s a c t i v i t y u nder a n a e r o b i c c o n d i t i o n s . I n s t e a d , t h e NADH n e c e s s a r y f o r t h e s y n t h e s i s o f PHB from a c e t y l - C o A i s s u p p l i e d t h r o u g h t h e c o n s u m p t i o n of t h e s t o r e d g l y c o g e n . G l y c o g e n c an be c o n v e r t e d t o p y r u v i c a c i d t h r o u g h t h e Embden-Meyerhof-Parnas (EMP) pathway. T h r o u g h f u r t h e r o x i d a t i o n of p y r u v i c a c i d , i t i s c o n v e r t e d t o a c e t y l - C o A g e n e r a t i n g c a r b o n d i o x i d e . ADP ATP ( C 6 H 1 0 0 5 ) n > < A c e t y l - C o A ( + CO,, ) N A D + NADH These p r o p o s e d pathways e x p l a i n t h e p h o s p h o r u s r e l e a s e , PHB s t o r a g e and g l y c o g e n c o n s u m p t i o n o b s e r v e d i n t h e a n a e r o b i c zone of the system d u r i n g t h e g l u c o s e r u n . In t h e a e r o b i c zone o f t h e b i o l o g i c a l e x c e s s p h o s p h o r u s r e m o v a l p r o c e s s , PHB was consumed and g l y c o g e n was s y n t h e s i z e d ( F i g . 4 . 2 2 ) . A c c o r d i n g t o Min o e t a l . ( 1 9 8 7 ) , t h i s i n d i c a t e d t h a t t h e PHB s t o r e d under a n a e r o b i c c o n d i t i o n s was u s e d a s t h e c a r b o n s o u r c e not o n l y f o r t h e m e t a b o l i s m o f t h e c e l l s , but a l s o f o r t h e g l y c o g e n s y n t h e s i s u n d e r a e r o b i c c o n d i t i o n s . They a l s o a r g u e d - 1 1 5 -that " i t is essential for the anaerobic/aerobic acclimatized sludge to convert the stored PHB to glycogen during the aerobic phase so as to maintain the required level of glycogen for the consumption during the subsequent anaerobic phase". This does not provide a sound argument., since i t implies that the biomass anticipate the anaerobic/aerobic sequence. A possible explanation is provided below. The PHB consumption and glycogen synthesis under aerobic conditions might have been two independent events in two different groups of organisms. However, the existence of an excellent correlation between the PHB consumption and glycogen storage during the entire glucose run (Fig.4.22), suggests the p o s s i b i l i t y of a single group of organisms being involved. Under aerobic conditions, energy is produced by processing the PHB via the TCA cycle, generating carbon dioxide. Malate, one of the intermediate products of the TCA cycle, can be transformed by either the "malic enzyme" or malate dehydrogenase to phosphoenol pyruvate, which on further biochemical tranformation yields glucose 6-phosphate and f i n a l l y glycogen, as shown (in abbreviated form) below: PHB A c e t y l - C o A — M a l a t e NADH, C02 G l y c o g e n G l u c o s e 6 - p h o s p h a t e P h o s p h o e n o l p y r u v a t e -116-However, i f a second group of organisms (such as chemoautotrophs) a r e a l s o i n v o l v e d , g l y c o g e n can be s y n t h e s i z e d u s i n g the carbon d i o x i d e ( r e l e a s e d d u r i n g the TCA c y c l e of the f i r s t group of organisms) through the C a l v i n c y c l e , as i n d i c a t e d below: PHB (CALVIN CYCLE) Glycogen -* Glucose 6-phosphate ^ — As o b s e r v e d w i t h the oth e r r u n s , the a n o x i c zone behaved s i m i l a r l y t o e i t h e r the a n a e r o b i c or the a e r o b i c zone, as d i s c u s s e d i n s e c t i o n 4.6.3. In the a n o x i c zone, phosphorus r e l e a s e was accompanied by PHB s t o r a g e and glycogen consumption w h i l e phosphorus uptake was a s s o c i a t e d w i t h PHB consumption and glycogen s t o r a g e ( F i g s . 4 . 2 0 and 4.21). No g l y c o g e n a n a l y s i s was done f o r a c e t a t e , p r o p i o n a t e or b u t y r a t e r u n s . P r e l i m i n a r y i n v e s t i g a t i o n s by N i c h o l l s and Osborn (1979) i n d i c a t e d t h a t no glycogen a c c u m u l a t i o n was p r e s e n t i n two t y p i c a l waste t r e a t m e n t p l a n t s a c h i e v i n g excess phosphorus removal. However, the presence of o t h e r secondary carbon s t o r a g e compounds (such as c a r b o h y d r a t e s ) and t h e i r r o l e i n the b i o l o g i c a l e x c e s s phosphorus removal p r o c e s s should be f u r t h e r i n v e s t i g a t e d . -1 17-4.8 OVERALL PHOSPHORUS REMOVAL Although the phosphorus uptake in the aerobic reactor (section 4.6.2) gives an indication of the degree of expected phosphorus removal by the system, i t is discussed separately in t h i s section with respect to the various aspects of the b i o l o g i c a l excess phosphorus removal process. The difference between the t o t a l phosphorus concentrations of the feed and the ef f l u e n t i s considered as the phosphorus removal of the system and expressed as mg of phosphorus per l i t r e of feed. The v a r i a t i o n of the t o t a l phosphorus in the feed was between 3.9 and 4.5 mg/L during the acetate, propionate and butyrate runs while the v a r i a t i o n during the glucose run was between 3.6 and 3.9 mg/L. The range of the t o t a l phosphorus concentrations of the feed are also presented in the various plots in order to id e n t i f y the runs where phosphorus l i m i t i n g conditions were experienced. The o v e r a l l phosphorus removal, with respect to the added chemical substrates as COD in feed (Fig.4.23), showed that the o v e r a l l removal increased with increasing dosage of the added substrate. For the same chemical substrate dosage, expressed as COD, the o v e r a l l phosphorus removal had the following decreasing order of e f f e c t among the various chemical substrates added. acetate > propionate > butyrate > glucose However, according to Jones et a l . (1985), the differences -118-40 Fig.4.23-Dosoge in mg/L as COD In feed The overall phosphorus removal of the system vs. the chemical addition, expressed as COD i n feed. -119-in the o v e r a l l phosphorus removal with sodium acetate and butyric a c i d was not s i g n i f i c a n t , contrary to the observations made in t h i s current study with sodium acetate and sodium butyrate. This dif f e r e n c e might be the result of the i n s u f f i c i e n t a c c l i m a t i z a t i o n period of 2 weeks provided between the d i f f e r e n t substrate runs during the study by Jones et a l . , (1985), i f the acetate run had succeeded the butyrate run. Gerber et a l . (1986) reported that the net phosphorus uptake was v i r t u a l l y i d e n t i c a l for acetate, propionate and butyrate. This study was conducted using batch experiments with non-acclimated biomass. It should be noted that the net phosphorus removal in t h i s study was r e l a t i v e l y low compared to the performance of continuous flow systems under comparable conditions. These results indicate that s i g n i f i c a n t differences e x i s t between the behaviours of s u f f i c i e n t l y acclimated and non-acclimated biomasses. A more meaningful and r e l i a b l e comparison of the effectiveness of various substrates, with regard to the b i o l o g i c a l excess phosphorus removal, can only be arrived at, by using a s u f f i c i e n t l y acclimated biomass for the substrate under in v e s t i g a t i o n , and for the operating conditions. The r e s u l t s from t h i s current study, under continuous flow conditions with an acclimated biomass, suggest that the effectiveness of a s p e c i f i c substrate on the b i o l o g i c a l excess -120-phosphorus removal i s some d i r e c t function of the s i m p l i c i t y of that substrate molecule. The o v e r a l l phosphorus removal of the system for the various chemical substrate additions in terms of readily biodegradable COD in the feed (Fig.4.24) were in close agreement, indicating the use of t h i s parameter to optimize the system. Once the requirement of the r e a d i l y biodegradable COD that should enter the anaerobic zone to e f f e c t the desired degree of excess phosphorus removal i s established, the amount of external simple carbon substrate additions necessary (to supplement the readily biodegradable COD a v a i l a b l e in the raw feed i t s e l f ) can be calculated using the r e s u l t s obtained in Section 4.5. However, for the same readily biodegradable COD content in feed, the addition of short chain v o l a t i l e fatty acid s a l t s outperformed glucose in b i o l o g i c a l excess phosphorus removal. Any o v e r a l l phosphorus removal in excess of 1.0-1.5 mg/L for every 200 mg/L of COD removed, can be c l a s s i f i e d as excess phosphorus removal, since i t i s generally accepted to be the normal metabolic requirement for the c e l l growth in such activated sludge systems (U.S. EPA, 1976). On t h i s basis, excess phosphorus removal was taking place whenever the readily biodegradable COD in the feed (including the chemical addition) exceeded approximately 50 mg/L (Fig.4.24). This represents a read i l y biodegradable COD of approximately 25 mg/L entering the anaerobic reactor, due to the 1:1 anoxic/anaerobic recycle. -121-T h e r e f o r e , i t c o u l d be concluded t h a t excess phosphorus removal takes p l a c e i f the r e a d i l y b i o d e g r a d a b l e COD a v a i l a b l e i n the an a e r o b i c zone exceeds a p p r o x i m a t e l y 25 mg/L. T h i s i s i n e x c e l l e n t agreement w i t h the e a r l i e r f i n d i n g s of S i e b r i t z e t a l . ( l 9 8 2 ) . A l t h o u g h v a r i o u s c o r r e l a t i o n s between the o v e r a l l phosphorus removal and dosages i n d i f f e r e n t forms (mg/L as a c i d , mM/L, mM of carbon/L e t c . ) were i n v e s t i g a t e d , e x p r e s s i n g dosages i n terms of r e a d i l y b i o d e g r a d a b l e COD proved t o be the b e s t f o r any comparison purposes among the d i f f e r e n t s u b s t r a t e s . But f o r a c e t a t e , p r o p i o n a t e and b u t y r a t e a d d i t i o n s , the o v e r a l l phosphorus removal was r e l a t i v e l y the same f o r the same dosages expressed i n mg/L as t h e i r c o r r e s p o n d i n g v o l a t i l e f a t t y a c i d s ( F i g . 4 . 2 5 ) . T h i s was not s u r p r i s i n g s i n c e these compounds, when pres e n t i n the same mg/L as a c i d c o n c e n t r a t i o n s , p r o v i d e d a l m o s t equal r e a d i l y b i o d e g r a d a b l e COD c o n c e n t r a t i o n s , as d e s c r i b e d e a r l i e r i n s e c t i o n 4.5 ( F i g . 4 . 2 ) . The o v e r a l l phosphorus removal of the system i n c r e a s e d w i t h i n c r e a s i n g a n a e r o b i c phosphorus r e l e a s e ( F i g . 4 . 2 6 ) . T h i s supported an e a r l i e r o b s e r v a t i o n by Barnard (1976) who, i n a d d i t i o n t o b e i n g the f i r s t t o l i n k the an a e r o b i c phosphorus r e l e a s e t o the excess phosphorus removal, a l s o r e p o r t e d t h a t the degree of phosphorus r e l e a s e d u r i n g the a n a e r o b i c phase determined the o v e r a l l phosphorus removal of the system. -122-5 40 60 60 70 80 90 RBD COD in feed (mg/L) Fig .4 . 24 . . The o v e r a l l phosphorus r e m o v a l o f t h e system v s . t h e r e a d i l y b i o d e g r a d a b l e COD i n f e e d ( i n c l u d i n g the c h e m i c a l a d d i t i o n ) . 5 - 1 1 0.5 H 1 1 1 1 1 1 1 1 | 1 1 1 1 1 0 20 40 60 80 100 120 140 Dosage in mg/L In feed F i g . 4 . 2 5 . The o v e r a l l phosphorus r e m o v a l o f the system v s . the c h e m i c a l d o s a g e , e x p r e s s e d as mg/L i n f e e d . Dosages o f a c e t a t e , p r o p i o n a t e and b u t y r a t e a r e e x p r e s s e d as t h e i r c o r r e s p o n d i n g a c i d s . -123-The o v e r a l l phosphorus removal of the system varied d i r e c t l y with the anaerobic carbon storage (Fig.4.27), confirming the recent theory that the anaerobic carbon storage i s of paramount importance to b i o l o g i c a l excess phosphorus removal (Comeau et al.,1984; Arvin and Kristensen,1985); however the anaerobic phosphorus release and the anaerobic carbon storage are cl o s e l y linked (section 4.7.1). The results also indicated that the carbon storage was least with propionate (no carbon analysis was done during the acetate run) and most with glucose, for the same o v e r a l l phosphorus removal of the system. The large dosages used and the additional production of NADH necessary for the carbon storage as PHB, through glycogen consumption (section 4.7.4), might be the reasons for t h i s higher carbon storage during the glucose run. Higher dosages of butyrate, compared to those of propionate were required to provide the same degree of phosphorus removal. This i s believed to be the primary cause for the higher carbon storages obtained with butyrate than with propionate for the same degree of phosphorus removal. Since there were some s i g n i f i c a n t differences during the glucose run, the following should be considered while attempting to compare the results from t h i s run with the other three runs. (i) During the i n i t i a l runs with acetate, propionate and butyrate, the t o t a l COD of the feed (including the added chemical substrate) varied between 254 and 281 mg/L whereas during the glucose run, i t varied between 274 and 342 mg/L due to the higher - 1 2 4 -4 .5 - 7 0.5 - j 1 1 1 1 1 1 1 1 1 0 10 2 0 3 0 40 5 0 Anoorobk: carbon storage (mg/L of feed) F i g . 4 . 2 7 . R e l a t i o n s h i p between the o v e r a l l p h o s p h o r u s r e m o v a l o f the system and t h e a n a e r o b i c c a r b o n s t o r a g e . C a r b o n s t o r a g e i s as PHV f o r p r o p i o n a t e a d d i t i o n and as PHB f o r b u t y r a t e and g l u c o s e a d d i t i o n s . -125-glucose dosages necessary to induce good excess phosphorus removal. As a re s u l t , more COD was removed by the system during the glucose run, creating more c e l l mass (as indicated by the mixed liquor suspended s o l i d s concentrations data) and thereby requiring more phosphorus for the normal metabolic purposes, compared to the other runs. ( i i ) The t o t a l phosphorus concentration in the feed during the glucose run was r e l a t i v e l y low (varied between 3.6 and 3.9 mg/L) compared to the runs with acetate, propionate and butyrate (varied between 3.9 and 4.5 mg/L). ( i i i ) Glucose runs (especially the 60 and 75 mg COD/L runs) were suspected to have had a s i g n i f i c a n t l y d i f f e r e n t type of organisms (f a c u l t a t i v e anaerobes, as dicussed in section 4.6.3) compared to the acetate, propionate and butyrate runs. 4.9 OVERALL NITROGEN REMOVAL Although the process configuration used in t h i s study was not s p e c i f i c a l l y designed to optimize nitrogen removal, the o v e r a l l nitrogen removal of the system increased with increasing dosages of chemical substrates, with the exception of the glucose runs (Fig.4.28). Overall nitrogen removal percentages presented in t h i s section were calculated as follows: -126-% Nitrogen removal = (feed TKN)-(effluent TKN)-(effluent NO jxlOO (feed TKN) x The feed NOx was not considered since i t was neg l i g i b l e throughout the study. The two most probable reasons for the increase in the nitrogen removal e f f i c i e n c y with increasing chemical dosages are as follows: ( i ) The simple chemical substrates used in t h i s study, in addition to being the desired substrates for the b i o l o g i c a l excess phosphorus removal process, are also preferred substrates for b i o l o g i c a l nitrogen removal. The d e n i t r i f i c a t i o n rates using these substrates are much higher than those achieved with more complex organic substrates present in the sewage. According to Gerber et a l . (1986), the d e n i t r i f i c a t i o n rates (in mg N/g MLSS.h) for acetate, propionate, butyrate and glucose (at a concentration of 200 mg COD/L) are 2.51, 1.68, 2.13 and 0.92 respectively, compared to 0.64 for se t t l e d sewage (diluted to a COD l e v e l of 440 mg/L). Fig.4.29 shows a general increasing pattern in the o v e r a l l nitrogen removal of the system with increasing r e a d i l y biodegradable COD content (which i s a di r e c t function of the s i m p l i c i t y of the substrates present) in the feed, except for the glucose run. ( i i ) The decrease in the influent TKN/COD r a t i o by the incremental increase of the added chemical substrates might have - 1 2 7 -o > o E C c s o 0 z F i g . o > o E £ c « o L. 72 70 68 66 64 62 60 58 56 54 52 50 48 46 4.28. 72 70 68 66 64 62 60 58 56 54 52 50 48 46 • Acetate + Propionate O Butyrate A GIUCODO I 20 40 60 —T— 80 —1— 100 I r 120 - i r 140 160 Dosage in mg/L as COD O v e r a l l n i t r o g e n r e m o v a l e f f i c i e n c y o f t h e system v s . t h e c h e m i c a l d o s a g e , e x p r e s s e d as COD i n f e e d . • Acetate + Propionate 6 Butyrate 40 I 50 I 60 —T— 70 — r -80 90 Fig .4-29 . RBD COD In feed (mg/L) O v e r a l l n i t r o g e n r e m o v a l e f f i c i e n c y o f t h e system v s . t h e r e a d i l y b i o d e g r a d a b l e COD i n f e e d ( i n c l u d i n g the c h e m i c a l a d d i t i o n ) . -128-provided improved influent c h a r a c t e r i s t i c s for b i o l o g i c a l nitrogen removal (Table 4.13). Although this explanation appears v a l i d for most parts of the acetate and butyrate runs, i t completely f a l l s apart for the propionate run, due to the v a r i a t i o n of the TKN concentrations in the d i f f e r e n t batches of feed used. The r e l a t i v e l y lower o v e r a l l nitrogen removal during the acetate run, compared to the propionate and butyrate runs, i s believed to be due to the r e l a t i v e l y high TKN/COD ratios of the feed during that run (Table 4.13). The o v e r a l l nitrogen removal pattern during the glucose run was unique among the various runs of thi s study. The removal was quite low at higher dosage runs (75 and 60 mg COD/L in the anaerobic zone), while the runs with lower dosages (45 and 30 mg COD/L i n anaerobic zone) followed the standard pattern established by the other runs. This was believed to be due to the higher glucose concentrations, favouring a s h i f t in organism population to f a c u l t a t i v e anaerobes at the expense of true d e n i t r i f i e r s , as described in section 4.6.3. The s h i f t was accompanied by symptoms of fermentative conditions, such as low ORP, formation of substantial gas bubbles and gluey appearance. -129-Table 4.13 Ov e r a l l Nitrogen Removal for A l l Runs Chemical Dosage in Percent Nitrogen Removal Ratio Anaerobic Reactor of the System of (mg COD/L) (%) TKN/COD RUN 1 : ACETATE 30 57.9 0.0971 25 56.5 0.1007 20 52.9 0.1015 15 52.2 0.1102 10 51.4 0.1131 5 50.9 0.1121 RUN 2 : PROPIONATE 25 66.9 0.0942 20 68.7 0.0970 15 64.1 0.0928 10 62.4 0.0927 5 58.3 0.0898 RUN 3 : BUTYRATE 30 66.9 0.0864 25 66.7 0.0802 20 65.7 0.0885 15 64.3 0.0837 10 61.5 0.0897 RUN 3 : GLUCOSE 75 51.3 0.0564 60 59.9 0.0735 45 63.2 0.0761 30 62.0 0.0785 -1 30-4.10 EXPERIMENTAL RUN WITH COMBINED ACETATE AND PROPIONATE T h i s p a r t i c u l a r combined run ( a c e t a t e p l u s p r o p i o n a t e ) was a c c i d e n t a l , r a t h e r than i n t e n t i o n a l . A f t e r the t e r m i n a t i o n of the p r o p i o n a t e run w i t h 15 mg/L (as COD, i n the a n a e r o b i c z o n e ) , the dosage was reduced to 10 mg/L (as COD, i n the a n a e r o b i c zone) and a new batch of feed was s t a r t e d . The e f f l u e n t o r t h o phosphorus c o n c e n t r a t i o n dropped t o l e s s than 0.1 mg/L from the p r e v i o u s steady s t a t e v a l u e of about 1.4 mg/L, w i t h i n two days a f t e r the feed change was made. A l t h o u g h t h e r e was a decrease i n the added p r o p i o n a t e dosage, t h i s sudden drop i n the e f f l u e n t phosphorus c o n c e n t r a t i o n was accompanied by an i n c r e a s e i n the r e a d i l y b i o d e g r a d a b l e COD c o n c e n t r a t i o n of the f e e d (up from 59 t o 71 mg/L). The v a r i o u s parameters f o r t h i s run are g i v e n i n T a b l e s 4.2 and 4.14. None of these r e s u l t s would have been b e l i e v a b l e i f not f o r the measurement of the r e a d i l y b i o d e g r a d a b l e COD i n the f e e d . A l l the phosphorus r e l e a s e s , carbon s t o r a g e s , o v e r a l l removals e t c . compared w e l l w i t h those observed d u r i n g the o t h e r runs of t h i s s t u d y , o n l y i n terms of the r e a d i l y b i o d e g r a d a b l e COD. A l t h o u g h i t was known t h a t the r e a d i l y b i o d e g r a d a b l e s u b s t r a t e c o n t e n t i n t h i s p a r t i c u l a r b a t c h of f e e d was u n u s u a l l y h i g h , c a u s i n g the e x c e l l e n t excess phosphorus removal, the e x a c t compound or compounds c a u s i n g t h i s was not i d e n t i f i e d u n t i l two weeks l a t e r . -131-Table 4.14 Phosphorus Uptake, Carbon Consumption and Nitrogen Removal during the Run with Combined Acetate and Propionate Parameter Values Phosphorus Uptake (mg of P/L of feed) Anaerobic -16.40 Anoxic -3.00 Aerobic 22.70 Carbon Consumption (mg/L of feed) as PHV: Anaerobic -9.1 Anoxic 3.9 Aerobic 5.2 as PHB: Anaerobic -12.2 Anoxic -2.6 Aerobic 14.8 Percent nitrogen removal 73.8 -1 32-The raw feed samples for v o l a t i l e fatty acid measurements were usually preserved and analysed approximately every two weeks during t h i s study. The v o l a t i l e f a t t y acid results for the feed, during t h i s run, showed the presence of unusually high l e v e l s of acetate. The concentration of the acetate in the raw feed was 14.7 mg/L as COD compared to almost none during the rest of t h i s study. Knowing t h i s information, an estimate of the re a d i l y biodegradable COD in the feed was made as follows, based on the resu l t s obtained in section 4.5. Since 1 mg COD/L of acetate provides 0.80 mg/L of readily biodegradable COD, the contribution from the 14.7 mg COD/L of acetate present in the feed Since 1 mg COD/L of propionate provides 0.75 mg/L of readily biodegradable COD,the contribution from the 20 mg COD/L of propionate added to the feed Since approximately 18% of the t o t a l COD in the raw sewage (= 239 mg/L for this batch of feed) i s readily biodegradable,contribution from the feed = 14.7x0.80 = 11.8 mg/L = 20x0.75 = 15.0 mg/L = 239x0.18 = 4 3.0 mg/L Therefore, t o t a l readily This compared very well mg/L, showing the v a l i d i t y of the biodegradable COD = 69.8 mg/L with the measured value of 71 results obtained in section 4.5 - 1 3 3 -in estimating the r e a d i l y biodegradable COD of the sewage used in t h i s study. This method of estimation i s possible only i f most of the readily biodegradability of the feed re s u l t s from the short chain v o l a t i l e f a t t y acids, a common occurrence where the c o l l e c t i o n systems provide long detention times. The source of the extra biodegradability would have never been found, i f i t had resulted from any simple compound other than the v o l a t i l e f a t t y acids. This c l e a r l y i l l u s t r a t e s the importance and the necessity of measuring the readily biodegradable COD of the sewages to characterize them, in order to optimize the b i o l o g i c a l excess phosphorus removal process. 4.11 LAG RESPONSE DURING DOSAGE TRANSITIONS The experimental runs, with each chemical substrate during t h i s study, were started with the highest dosage and subsequently reduced in steps, except for the glucose run. With t h i s sequence, an interesting phenomenon was observed during the acetate, propionate and butyrate runs. Whenever there was a reduction in the dosage of the added chemical substrate, there was a lag period for the ef f l u e n t phosphorus concentration to increase to a le v e l corresponding to that of the reduced dosage. In other words, the b i o l o g i c a l excess phosphorus removal continued to occur for a short period of time at the l e v e l corresponding to the higher dosage before decreasing to a lower l e v e l , -1 34-corresponding to the new reduced dosage. This lag period was longer (4 to 5 days) during the t r a n s i t i o n s between the higher dosages and shorter (1 to 2 days) during the t r a n s i t i o n s between the smaller dosages (Figures.4.30, 4.31 and 4.32). In order to investigate and explain t h i s lag response, carbon storage analysis was done during the t r a n s i t i o n phases of the butyrate runs, in addition to the steady state periods. Similar to the situation under steady state conditions, carbon was stored in the anaerobic zone and consumed in the aerobic zone (as PHB) during the t r a n s i t i o n periods. This storage and consumption was accompanied by phosphorus release and phosphorus uptake respectively. The p r o f i l e s of the PHB concentrations in the anaerobic, anoxic and aerobic zones during the enti r e butyrate run are shown in Fig.4.33. As soon as the chemical dosage was reduced, the PHB concentrations in a l l reactors started to decrease towards the new steady state concentrations. However, i t was noticed that u n t i l the aerobic PHB concentrations were reduced to the new lower steady state values, the effluent continued to maintain the same low phosphorus concentration, r e s u l t i n g in higher excess phosphorus removal corresponding to the higher dosage. Once the aerobic PHB concentrations reached the new steady state values, the effluent phosphorus concentration started to increase towards the new higher steady state values, r e s u l t i n g in the deterioration of the excess phosphorus removal (Fig.4.30). -1 3 5 -4 Days Fig .4-.31. P r o f i l e o f the e f f l u e n t o r t h o p h o s p h o r u s f o r the p r o p i o n a t e r u n . Arrows i n d i c a t e the days on w h i c h the dosage was changed . * The raw sewage d u r i n g t h i s r u n had an a c e t a t e c o n c e n t r a t i o n o f 14-.7 mg/L as COD. -136-3.5 E £ a « £ a 0 £ -i \ at £ 5> E c • u c o o O I ft. 3 -2.5 -2 -1.5 -0.5 120 Days Fig.4.32. P r o f i l e o f t h e e f f l u e n t o r t h o p h o s p h o r u s f o r the b u t y r a t e r u n . Arrows i n d i c a t e t h e days on w h i c h the dosage was c h a n g e d . Anaerobic sone 120 Days Fig.4.33. P r o f i l e o f s t o r e d PHB i n the v a r i o u s zones o f t h e system f o r t h e b u t y r a t e r u n . Arrows i n d i c a t e t h e days on w h i c h t h e dosage was c h a n g e d . -137-At t h i s point, the reason for t h i s behaviour was believed to be as follows: Although the PHB synthesis during the anaerobic phase started to decrease immediately due to the dosage reduction, the organisms had the luxury of using the extra PHB already stored in them. The a c c e s s i b i l i t y of these extra internal carbon reserves could have helped to maintain the same uptake of phosphorus through higher carbon consumption, leading to the continuation of the higher degree of excess phosphorus removal. Once t h i s extra available PHB reserve i s depleted, the carbon consumption started to decrease towards the lower steady state values, r e s u l t i n g in reduced phosphorus uptake and causing the lower excess phosphorus removal by the system. However, the phosphorus and carbon balances during the t r a n s i t i o n periods did not support t h i s hypothesis. Both the phosphorus uptake and the carbon consumption started to decrease immediately a f t e r the reduction in the dosage (Table 4.15). Therefore, another mechanism must exist through which the organisms were able to maintain the same removal, despite the lower phosphorus uptake and carbon consumption. Further research i s necessary to f u l l y explain and understand t h i s behaviour. Nevertheless, one of the reasons for the absence of thi s behaviour, during the glucose run, might be due to the over-shadowing of t h i s e f f e c t by those of the population s h i f t of the -1 38-Table 4.15 Phosphorus and Carbon Balances for the Butyrate Run during the Dosage Transitions Number of Days Phosphorus Uptake Carbon Consumption after the (mg of P/L of feed) (mg of PHB/L of feed) Dosage Change Anaero Anox Aero Anaero Anox Aero 30 mg COD/L steady state -12.30 -2.70 19.00 -27.7 -7.1 34.8 1 -10.30 -2.70 17.00 -22.8 -7.2 30.0 3 -8.90 -2.90 15.80 -20.5 -6.7 27.2 5 -8.80 -2.10 14.60 -19.4 -5.8 25.2 25 mg COD/L steady state -8.80 -1.50 14.10 -19.3 -4.7 24.0 1 -7.20 -1.50 12.50 -16.1 -3.9 20.0 3 -6.40 -1.10 11.30 -13.7 -3.7 17.4 5 -5.80 -0.60 9.50 -11.4 -3.4 14.8 20 mg COD/L steady state -5.45 -0.75 8.85 -11.3 -2.5 13.8 1 -4.55 -0.25 7.45 -8.8 -2.2 11.0 3 -3.35 -0.05 5.85 -6.8 -1.2 8.0 5 -2.15 -0.25 4.65 -6.3 0.3 6.0 15 mg COD/L steady state -1.60 -0.10 4.10 -6.0 -0.2 6.2 1 -1.10 0.70 2.50 -4.8 -1.0 5.8 3 -0.70 1.40 1.10 -4.6 -0.2 4.8 10 mg COD/L steady state -0.20 1.00 0.65 -4.5 0.5 4.0 -139-organisms, as discussed e a r l i e r in section 4.6.3. On the contrary, when the dosage of the simple substrates was increased, the excess phosphorus removal increased immediately without the presence of any s i g n i f i c a n t lag period. This was observed during the 10 mg COD/L propionate run in which the sewage used had unusually high l e v e l s of acetate. As discussed in section 4.9, when the concentration of the simple substrates entering the system increased from 15 mg COD/L of propionate to 10 mg COD/L of propionate plus 14.7 mg COD/L of acetate, the effluent ortho phosphorus dropped from 1.4 mg/L to less than 0.1 mg/L within two days, ind i c a t i n g an immediate improvement in the excess phosphorus removal. The benefits of the continued higher excess phosphorus removal for a short duration a f t e r a reduction in dosage of the simple carbon substrates and the immediate improvement of the excess phosphorus removal e f f i c i e n c y after an increase in dosage could be quite substantial. Some of the possible advantages of th i s behaviour are l i s t e d below: (i ) The b i o l o g i c a l excess phosphorus removal process shows a considerable short-term s t a b i l i t y ; i . e . the preferred substrate and phosphorus loadings need not balance on a real-time basis. ( i i ) The addition of extra simple carbon substrates in the b i o l o g i c a l excess phosphorus removal treatment plants may be -1 40-optimized using c y c l i c loading patterns with higher and lower dosages resu l t i n g in reduced chemical consumption. However, further research is necessary to investigate as to how long the system could be stressed in t h i s fashion without any i l l - e f f e c t s to the process. ( i i i ) There i s an emerging trend in the design of the b i o l o g i c a l excess phosphorus removal systems to incorporate primary sludge fermenters ahead of the anaerobic zones to generate the necessary simple carbon substrates (Rabinowitz and Oldham, 1985). A better design of the ove r a l l process may be accomplished by providing one fermenter set-up for two or more process t r a i n s , with each process t r a i n receiving the fermenter e f f l u e n t in turns. The type of behaviour dicussed in t h i s section was also observed in the f u l l - s c a l e b i o l o g i c a l excess phosphorus removal treatment plant in Kelowna, B r i t i s h Columbia. When the primary sludge thickener supernatant (containing simple preferred carbon substrates) flow was changed from one module to the other, phosphorus removal in the f i r s t module did not show any sign of deterioration for another 3 days, whereas the phosphorus removal in the module to which the thickener supernatant was made available improved almost immediately (Oldham, 1985). It was also reported that only 2 days were required to re-establish a good phosphorus removal, after 13 days of upset in a b i o l o g i c a l excess phosphorus removal system (Manning and Irvine, 1985). - 1 4 1 -CHAPTER FIVE CONCLUSIONS AND RECOMMENDATIONS 5.1 CONCLUSIONS Sig n i f i c a n t progress has been made in understanding many aspects of the b i o l o g i c a l excess phosphorus removal process during the course of t h i s research. The results obtained in t h i s study have contributed towards a better understanding of the mechanism of the excess phosphorus removal process and have helped in assessing the s u i t a b i l i t y of a given sewage for b i o l o g i c a l excess phosphorus removal. The conclusions of t h i s research may be summarized under three d i f f e r e n t catagories as follows: 5.1.1 CONCLUSIONS DIRECTLY RELATED TO OBJECTIVES (1) The technique proposed (in Section 3.2) for the q u a n t i f i c a t i o n of readily biodegradable substrates a v a i l a b l e in a sewage, was proved to be e f f e c t i v e and r e l i a b l e . However, t h i s technique cannot be used as a f i e l d t e s t , since i t requires the operation of a short sludge age, completely-mixed, activated sludge system under continuous flow conditions. -1 42-(2) Almost a l l the d i f f e r e n t elements of the b i o l o g i c a l excess phosphorus removal process (such as anaerobic phosphorus release, aerobic phosphorus uptake, o v e r a l l phosphorus removal, carbon storage and carbon consumption) compared well among the d i f f e r e n t carbon substrates used in t h i s study when th e i r dosages were expressed in terms of re a d i l y biodegradable COD, rather than t o t a l COD, moles,moles of carbon etc.. In other words, r e l a t i v e l y the same behaviour of the excess phosphorus removal system was observed, with d i f f e r e n t carbon substrate additions, when the readily biodegradable substrates (as readily biodegradable COD) entering the system were used as the unit of measurement. Also, a l l the above mentioned elements of the b i o l o g i c a l excess phosphorus removal mechanism increased with increasing readily biodegradable substrates entering the system. Therefore, i t may be concluded that the e f f i c a c y of a given sewage for b i o l o g i c a l excess phosphorus removal w i l l vary d i r e c t l y with the concentration of the readily biodegradable COD of the sewage; t h i s indicates the v a l i d i t y of using the readily biodegradable COD as a major parameter for a general characterization of sewage with respect to the b i o l o g i c a l excess phosphorus removal. This conclusion was reinforced when the sewage used during one of the runs in t h i s study contained abnormally high levels of acetate. The increase in the concentration of the readily biodegradable COD of the feed was the f i r s t i n d i c a t i o n of t h i s -1 43-s i t u a t i o n and explained the observed dr a s t i c increase in the phosphorus removal e f f i c i e n c y . Therefore, the measurement of the readily biodegradable COD entering the system eith e r through the feed or through the fermenter e f f l u e n t , i f present, could be used to e f f e c t i v e l y optimize the b i o l o g i c a l excess phosphorus removal process, e s p e c i a l l y with regard to the requirements of the external supplementary simple carbon sources necessary to provide the desired degree of phosphorus removal. 5.1.2 OTHER CONCLUSIONS RELATED TO READILY BIODEGRADABLE COMPOUNDS IN GENERAL (1) A d i r e c t linear relationship existed between the phosphorus uptake in the aerobic zone and the phosphorus release in the anaerobic zone. In other words, higher anaerobic phosphorus release promoted higher aerobic phosphorus uptake and thereby resu l t i n g in good overal l phosphorus removal by the system. The rela t i o n s h i p between the aerobic phosphorus uptake and the 2 anaerobic phosphorus release was found to be as follows, with R being 0.970 (where R i s the co r r e l a t i o n c o e f f i c i e n t ) . P uptake (mg/L) = 1.21 + 1.701 x P release (mg/L) This equation of best f i t i s s c i e n t i f i c a l l y defensible, with the f i r s t term representing the basic metabolic requirement of the t o t a l biomass in the system, while the c o e f f i c i e n t of the second -1 44-term i s determined by the degree to which influent phosphorus or preferred substrate i s l i m i t i n g the process, as discussed in Section 4.6.2. (2) The presence of readily biodegradable substrates in the feed entering the anaerobic zone of a b i o l o g i c a l excess phosphorus removal system ensured good anaerobic phosphorus release, subsequent aerobic phosphorus uptake, and o v e r a l l excess phosphorus removal. The results of t h i s study showed that a minimum of 25 mg/L of r e a d i l y biodegradable COD should enter the anaerobic zone for any s i g n i f i c a n t b i o l o g i c a l excess phosphorus removal to take place, as discussed in Section 4.8. (3) A d e f i n i t e d i r e c t l i n k existed between the carbon storage and the phosphorus release during the anaerobic phase of the process. This observation supports the kinetic model for the b i o l o g i c a l excess phosphorus removal mechanism proposed by Comeau et al.(1985), as discussed in Section 4.7.1. The release of phosphorus occurs to f a c i l i t a t e the transport of the simple carbon substrates (such as acetate, propionate etc.) across the c e l l wall to be stored as PHB or PHV by maintaining a proton motive force in the c e l l . A similar link was also found to exist between the carbon consumption and the uptake of phosphorus during the aerobic phase (Section 4.7.2). (4) Whenever there was a reduction in the dosage of the added simple carbon substrates, there was a lag period before a -1 45-deterioration in phosphorus removal occurred. In other words, the steady-state b i o l o g i c a l excess phosphorus removal continued for a short period of time at the same higher l e v e l before decreasing to the new lower l e v e l , r e f l e c t i n g the reduced substrate dosage. This lag period was longer (4 to 5 days) during the tr a n s i t i o n s between higher dosages and shorter (1 to 2 days) during the tr a n s i t i o n s between smaller dosages. As a c o r o l l a r y , when the dosage of the simple carbon substrates was increased, the excess phosphorus removal increased immediately without the presence of any s i g n i f i c a n t lag period, as discussed in Section 4.11. This behaviour could successfully be used in optimizing b i o l o g i c a l excess phosphorus removal systems. 5.1.3 OTHER CONCLUSIONS RELATED TO SPECIFIC READILY BIODEGRADABLE COMPOUNDS (1) The degree of biodegradability was appeared to be some inverse function of the complexity of the added substrate molecule. For the same dosage as COD, the various substrates had the following decreasing order of effectiveness in terms of their degree of biodegradability. acetate > propionate > butyrate > glucose Oxygen uptake rate results showed that 1 mg of acetate (as COD) provided approximately 0.80 mg of readily biodegradable COD. The -1 46-corresponding values for propionate, butyrate and glucose were 0.75, 0.45 and 0.27 respectively. (2) D i f f e r e n t simple carbon substrates exhibited a range of effectiveness in phosphorus release during the anaerobic phase of the b i o l o g i c a l excess phosphorus removal process when COD was used as the unit of measurement. The anaerobic phosphorus release appeared to be a d i r e c t function of the s i m p l i c i t y of the molecule of the added substrate. For the same COD dosage, the various substrates had the following decreasing order of effect with respect to anaerobic phosphorus release. acetate, propionate > butyrate > glucose (3) Carbon was stored i n t r a c e l l u l a r l y during the anaerobic phase and consumed during the aerobic phase. The primary carbon storage compound was poly-B-hydroxybutyrate (PHB) when the added simple carbon substrate was butyrate and poly-B-hydroxyvalerate (PHV) for the addition of propionate. The addition of glucose as the simple carbon substrate involved two carbon storage compounds, PHB and glycogen. Contrary to what happened with PHB, glycogen was consumed in the anaerobic zone and synthesized in the aerobic zone, during the glucose runs, supporting the observations made by Mino et al . ( 198.7). It i s speculated that glycogen consumption during the anaerobic phase provided the NADH required for the PHB synthesis, -1 47-through Embden-Meyerhof-Parnas (EMP) pathway and oxidation, as dicussed in Section 4.7.4. An excellent l i n e a r relationship existed between the PHB synthesis and the glycogen consumption, with the estimated mean value for the increase in the amount of PHB per unit of glycogen consumed being approximately 0.41, on a weight basis. (4) For the same amount of chemical substrate dosage (expressed as COD) the o v e r a l l phosphorus removal had the following decreasing order of ef f e c t among the various substrates added; th i s indicates the degree of effectiveness of these substrates for b i o l o g i c a l excess phosphorus removal. acetate > propionate > butyrate > glucose (5) The presence of glucose in the b i o l o g i c a l excess phosphorus removal system promoted fermentative conditions in the anoxic zone. These conditions probably enriched a biocommunity of bacteria ( f a c u l t a t i v e anaerobes) at the expense of "non-bio-P d e n i t r i f i e r s " . Thus, the "bio-P d e n i t r i f i e r s " might have been active in the anoxic zone, causing higher than expected anoxic phosphorus uptake, as dicussed in Section 4.6.3. This observation indicates that a sub-population of the bio-P bacteria are also d e n i t r i f i e r s . -1 48-5.2 RECOMMENDATIONS Further research work i s recommended in the following areas. (1) Development of a simpler and preferably a chemical technique for the measurement of the readi l y biodegradable COD of the feed, since the method used in this study required the operation of a short sludge age continuous activated sludge system. (2) The role of secondary carbon storage compounds (other than PHB and PHV) such as glycogen found during the glucose runs of th i s study should be investigated. Attention should be given to to the presence of carbohydrates in feed, their storage in c e l l mass and th e i r d i r e c t or indire c t contributions to the b i o l o g i c a l excess phosphorus removal mechanism. (3) A series of batch tests could be performed to determine the fate of the added simple carbon substrates in the b i o l o g i c a l excess phosphorus removal process, using radioactive isotope l a b e l i n g techniques. This would be of part i c u l a r interest, when more than one storage compound i s involved in the phosphorus removal mechanism. (4) The behaviour of the continued higher excess phosphorus removal for a short period aft e r a reduction in the dosage of simple carbon substrates and the immediate improvement of the -1 49-phosphorus removal after an increase in the dosage should be further investigated. A better understanding of t h i s behaviour could be very important, since i t could successfully be incorporated into the designs of the b i o l o g i c a l excess phosphorus removal systems to optimize them. -1 50-BIBILIOGRAPHY Alarcon, G.O. (1961), "Removal of Phosphorus from Sewage", Masters Essay, John Hopkins University, Baltimore, Md. A.P.H.A. 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(1982), "A Parametric Model for B i o l o g i c a l Excess Phosphorus Removal", Presented at the IAWPRC Post-conference Seminar on Phosphate Removal, Pretoria, A p r i l , 1982. S i e b r i t z , I., Ekama, G.A. and Marais, G.v.R. (1983), " B i o l o g i c a l Excess Phosphorus Removal in the Activated Sludge Process", Research Report W 47, Department of C i v i l Engineering, University of Cape Town. Simm, R.A. (1988), " F e a s i b i l i t y Study of the Use of a Rotating B i o l o g i c a l Contactor (RBC) for B i o l o g i c a l Dephosphotation", M.A.Sc. Thesis, Department of C i v i l Engineering, University of B r i t i s h Columbia. Simpkins, M.J. and McLaren, A.R. (1978), "Consistent B i o l o g i c a l Phosphate and Nitrate Removal in an Activated Sludge Plant", Prog. Wat. Tech., 10, 433-442. -155-Somiya, I., Tsuno, H. and Matsumota, M. (1988), "Phosphorus Release - Storage Reaction and Organic Substrate Behaviour in B i o l o g i c a l Phosphorus Removal", Wat. Res., 22, 1, 49-58. Srinath, E.G., Sastry, C A . and P i l l a i , S.C (1959), "Rapid Removal of Phosphorus from Sewage by Activated Sludge", Water and Waste Treatment, 11, 410. Stern, L.B. and Marais, G.v.R. (1974), "Sewage as Electron Donor in B i o l o g i c a l D e n i t r i f i c a t i o n " , Research Report W 7, Department of C i v i l Engineering, University of Cape Town. Supelco (1982), "Separating Aqueous Carboxylic Acids (C2-C5) at ppm Concentrations", Supelco Inc., B u l l . 751 E, Bellafonte, PA. Technicon I n d u s t r i a l Method No. 94-70W (1973), "Orthophosphate in Water and Wastewater (Range: 0-10 mg/L)", Technicon Ind. Systems, Tarrytown, N.Y. Technicon I n d u s t r i a l Method No. 100-70W (1973), "Nitrate and N i t r i t e in Water and Wastewater (Range: 0-2 mg/L)", Technicon Ind. Systems, Tarrytown, N.Y. Technicon Block Digester Manual (1974), "Operation Manual for the Technicon Block Digester, Models BD-20 and BD-40", Technicon Pub. 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(1967), "Phosphate Removal through Municipal Wastewater Treatment at San Antonio, Texas", Jour. Wat. P o l l . Cont. Fed., 39, 5, 750-771. -156-Wells, W.N. (1969), "Differences in Phosphate Uptake Rates Exhibited by Activated Sludges", Jour. Wat. P o l l . Cont. Fed., 41, 5, 765-771. Wentzell, M.C., Dold, P.L., Ekama, G.A. and Marais, G.v.R. (1984), "Kinetics of B i o l o g i c a l Phosphorus Release", Proc. 12th Conf. of the IAWPRC, Post-conference Seminar on Enhanced B i o l o g i c a l Phosphorus Removal from Wastewater, Paris, Sept., 1984. Wentzell, M.C., Lotter, L.H., Loewenthal, R.E. and Marais, G.v.R. (1986), "Metabolic Behaviour of Acinetobacter spp. in Enhanced B i o l o g i c a l Phosphorus Removal - A Biochemical Model", Water S.A., 12, 4, 209-224. Wilderer, P.A., Jones, W.L. and Dau, U. (1987), "Competition in D e n i t r i f i c a t i o n Systems Affecting Reduction Rates and Accumulation of N i t r i t e " , Wat. Res., 21, 2, 239-245. W.P.C.F. (1977), "Wastewater Treatment Plant Design", Manual of Practice No.8, Water Pollution Control Federation, Washington, D.C. - 1 5 7 -APPENDIX A1 DEVELOPMENT OF THE METHOD USED FOR THE DETERMINATION OF THE READILY BIODEGRADABLE COD -1 58-A1.1 INTRODUCTION This gives a b r i e f outline of the development of the method used for the determination of the readily biodegradable COD of the feed. Two i d e n t i c a l laboratory scale systems, as shown by the schematic diagram in F i g . A l . l ( a ) , were operated a f t e r having decided to use the step change in the oxygen uptake rate, at the termination of the feed, as the basis for the readily biodegradable COD measurement. A1.2 SYSTEM START-UP The two continuous flow completely mixed actvated sludge systems were operated at a sludge age of 6 days. The sludge used in the start-up of these units was obtained from the aerobic zone of the p i l o t scale b i o l o g i c a l excess phosphorus removal treatment plant situated at the University of B r i t i s h Columbia campus. The p i l o t plant was operating at a sludge age of 20 days at the time the sludge for these units were taken. Steady state was considered to be achieved when the d a i l y oxygen uptake rate and the concentration of the aerobic mixed liquor suspended so l i d s showed approximately steady values. -159-A1.3 INITIAL OPERATION During the period of steady state operation, the degree of n i t r i f i c a t i o n in both systems was high. The aerobic nit r a t e l e v e l s were around 20-25 mg/L with influent TKN values varying between 20 and 30 mg/L. No s i g n i f i c a n t concentration of ammonia was found in the effluents of the systems. Due to the high degree of n i t r i f i c a t i o n , the oxygen uptake rate drop, at the feed termination, not only included the cessation of oxygen requirement for the metabolism of the readily biodegradable COD of the feed, but also included the cessation of oxygen requirement for n i t r i f i c a t i o n . This resulted in higher oxygen uptake rate drops than those expected due to the readily biodegradable substrates alone, in the feed. A1.4 SUPPRESSION OF UNWANTED NITRIFICATION The estimates of the r e a d i l y biodegradable COD concentration of the feed using these systems were higher than expected, due to the presence of high degree of n i t r i f i c a t i o n . Since t h i s situation was unacceptable for the determination of the readily biodegradable COD of the feed, various steps were taken to suppress the n i t r i f i c a t i o n . These are b r i e f l y outlined in the following sections. -1 60-A1.4.1 REDUCTION OF SLUDGE AGE The sludge age was reduced in steps from 6 days to 2.5 days with no apparent change in the degree of n i t r i f i c a t i o n . Even at the short sludge age of 2.5 days, the n i t r a t e levels in the aerobic reactor averaged around 20 mg/L, indicating a high degree of n i t r i f i c a t i o n . Since the systems were i n i t i a l l y started-up using a good n i t r i f y i n g population of micro-organisms (from the p i l o t plant at the University of B r i t i s h Columbia), i t was decided at th i s stage to r e s t a r t the systems using a mixed liquor with low n i t r i f y i n g population, obtained from the f u l l scale sewage teatment plant in Squamish, B r i t i s h Columbia. A1.4.2 USE OF LOW NITRIFYING MIXED LIQUOR The two systems were restarted, at a sludge age of 3 days, using the aerobic mixed liquor from the f u l l scale treatment plant in Squamish, B r i t i s h Columbia. The aerobic n i t r a t e concentration of the plant, at the time the sludge was taken, was around 5 mg/L. However, the nitr a t e l e v e l s of the aerobic reactors of the laboratory systems started to increase steadily and within 30 days, reached a steady concentration of approximately 20 mg/L. -161-Following t h i s unsuccessful attempt to control the degree of n i t r i f i c a t i o n in the systems, i t was decided to use a n i t r i f i c a t i o n i n h i b i t o r . A1.4.3 ADDITION OF NITRIFICATION INHIBITOR 2 chloro - 6 ( t r i c h l o r o methyl) pyridine was used as the inhib i t o r to control n i t r i f i c a t i o n . This i s widely used in the standard BOD t e s t , at a concentration of 10 mg/L, to i n h i b i t n i t r i f i c a t i o n . The n i t r i f i c a t i o n i n h i b i t o r was added to one of the two systems in four d i f f e r e n t ways and the re s u l t s are summarized in Table A1.1. During the period of t h i s addition, system II (without the addition of i n h i b i t o r ) consistently had high degree of n i t r i f i c a t i o n compared to the system I (with the addition of i n h i b i t o r ) . As expected, i t was also noted that the oxygen uptake rate of the system decreased with increased addition of the i n h i b i t o r . A1.4.4 REDUCTION OF HYDRAULIC RETENTION TIME (HRT) The results presented in Table A1.1 show that the ef f e c t i v e control of n i t r i f i c a t i o n could only be achieved through a continuous addition of the i n h i b i t o r , at concentrations above -1 62-T a b l e A l . 1 . Summary of the i n h i b i t o r e f f e c t s on the p r o c e s s used i n the measurement of r e a d i l y b i o d e g r a d a b l e COD CONCENTRATION OF INHIBITOR DURATION OF ADDITION RESULTS I. 10 mg/1 with respect Instantaneous No change to reactor volume I I . 100 mg/1 with respect Instantaneous Nitrate level dropped from 22 mg/1 to reactor volume to 8 mg/1 the next day; gradually increased to around 20 mg/1 i n 12 days I I I . 100 mg/1 with respect 2 days Nitrate level reduced from 21 mg/1 to influent to less than 0.5 mg/1 75 mg/1 with respect 2 days Less than 0.5 mg/1 to influent 50 mg/1 with respect 2 days Less than 0.5 mg/1 to influent 25 mg/1 with respect 2 days Less than 0.5 mg/1 to influent 10 mg/1 with respect 20 days Slowly increased to 16 mg/1 i n 20 to influent days and was s t i l l increasing; decided to increase the duration of i n h i b i t o r addition - 1 6 3 -Table A l . l . (continued) CONCENTRATION Cf INHIBITOR DURATION OF ADDITION RESULTS IV. 100 mg/1 with respect 4- days Nitrate l e v e l decreased to 0.8 mg/1 to influent 75 mg/1 with respect 4 days Less than 0.5 mg/1 to influent 50 mg/1 with respect k days Less than 0.5 mg/1 to influent 25 mg/1 with respect to influent 20 days Within 10 days nitrat e l e v e l increased to 3 mg/1 and stayed around 3 mg/1 10 mg/1 with respect to influent 18 days Slowly increased to 12 mg/1 and was showing an upward trend -164-10 mg/L. Since the addition of i n h i b i t o r during the periods of oxygen uptake rate determinations was not desired, i t was decided to decrease the HRT of the systems. The nominal HRT of both systems were reduced from 6 hours, in steps of 2 hours. (a) HRT of 4 hours During the 15 days of operation at 4 hours of nominal HRT, the average concentration of ni t r a t e s in system I was less than 10 mg/L, compared to 15-20 mg/L in system I I . This caused the estimates of the readily biodegradable COD using system II to be consistently higher than those obtained using system I. (b) HRT of 2 hours During the 30 days of operation at this nominal HRT, the n i t r a t e concentration in system I was less than 5 mg/L, compared to 5-10 mg/L in system I I . However, both systems gave comparable estimates of the readily biodegradable COD of the feed. At t h i s point, i t was decided to operate one scaled down system, as shown by the schematic diagram in Fig.A1.1(b), in order to reduce the high requirement of feed. -1 65-A1.5 SLUDGE BULKING After about 4 months of successful operation, a severe bulking problem occurred and the system began to collapse due to the s o l i d s being l o s t through the e f f l u e n t . Microscopic examination of the mixed liquor revealed the presence of s i g n i f i c a n t filamentous growth. The following remedies were taken to control the sludge bulking and none of them worked. (1) The dissolved oxygen concentration of the aerobic reactor was increased above 4 mg/L from the normal l e v e l of 1.5 -2.5 mg/L. (2) The dissolved oxygen concentration of the aerobic reactor was decreased below 1 mg/L. (3) The entire system was restarted with a f r a c t i o n of i t s s e t t l e d mixed l i q u o r . (4) The supply of a i r was terminated for d i f f e r e n t lengths of time to create temporary anaerobic environments. These were ca r r i e d out with and without feeding. (5) The organic loading to the system was increased to create a high food to micro-organism r a t i o . -166-(6) The organic loading to the system was decreased to create a low food to micro-organism r a t i o . (7) Phosphorus concentration in the feed was increased to eliminate any possible nutrient deficiency. (8) The system was fed using a 12 hour c y c l i c loading. (9) The system was treated with hydrogen peroxide at concentrations of 100, 150, 200 and 250 mg/L, with respect to the feed, over 24 hour periods. (10) The system was subjected to anaerobic conditions p r i o r to the hydrogen peroxide treatment. (11) After treating the system with 300 mg/L of hydrogen peroxide for 48 hours, the system was restarted with less than 100 mg/L of mixed liquor suspended s o l i d s . When every possible remedy found in the l i t e r a t u r e for eliminating the sludge bulking proved to be i n e f f e c t i v e , the system was redesigned to incorporate a permanent anaerobic retention tank for the return sludge, as shown in Fig.A1.1(c). During the optimization period, the influent flow rate was reduced to 0.5 l i t r e s per hour, without any n i t r i f i c a t i o n problem due to the increased HRT. -1 6 7 -Waste Inf. AEROBIC a) (b) eff. 1 1/h V Vol = 6 1J \ 1:1 Recycle • Vol = 1 1 Waste Inf. / AEROBIC Y \ / eff. 1 1/h Vvol = 1 1J \ 1:1 Recycle ' Vol = 1 1 (c) 1:1 Recycle F i g . A l . l . D i f f e r e n t stages i n the development of the process used i n the measurement of r e a d i l y biodegradable COD - 1 6 8 -Th i s system performed s u c c e s s f u l l y throughout the study, with n e i t h e r high degree of n i t r i f i c a t i o n nor sludge b u l k i n g ever being p r e s e n t . An e f f l u e n t ammonia c o n c e n t r a t i o n of a t l e a s t 5 mg/L was always maintained to e l i m i n a t e any decrease i n the oxygen u t i l i z a t i o n at the t e r m i n a t i o n of the f e e d due to the c e s s a t i o n of n i t r i f i c a t i o n . -1 6 9 -APPENDIX A2 RAW DATA FROM THE VARIOUS EXPERIMENTAL RUNS - 1 7 0 -RAW DATA OF PHOSPHORUS FROM THE ACETATE RUNS Dosage in Day # Total P Ortho - P Aero. anaerobic a f t e r (mg/L) (mg/L) Slud. zone as steady %P mg COD/L state inf eff inf anae anox aero e f f 1 4.6 <0.2 3.2 18.2 16.2 <0. 1 <0.1 5.8 4 4.4 <0.2 3.0 18.6 16.8 <0. 1 <0.1 5.9 7 4.6 <0.2 3.1 17.8 14.3 <0. 1 <0.1 5.5 1 1 4.6 <0.2 3.4 17.9 15.1 <0.1 <0.1 5.6 14 4.5 <0.2 3.3 18.0 15.4 <0. 1 <0.1 5.9 1 4.3 <0.2 3.2 16.4 13.4 <0. 1 <0.1 5.4 4 4.2 <0.2 3.4 15.8 15.9 <0.1 <0.1 5.6 8 4.2 <0.2 3.3 16.6 14.4 <0.1 <0.1 5.1 11 4.4 <0.2 3.0 14.4 11.6 <0. 1 <0.1 5.8 15 3.9 <0.2 3.1 16.6 12.4 <0.1 <0.1 5.5 1 4.2 0.-4 5 4.0 0,2 8 4.2 0.4 12 4.0 0.4 15 4.1 0.5 19 4.2 0.3 3.2 11.6 8.4 3.3 1 1 .4 9.2 3.3 8 .4 7 .9 2 .9 10.6 8.8 3.1 10.4 8.4 3.4 10.6 6.4 0.2 0.3 4 .6 0.2 0.2 4.7 0.4 0.4 5.0 0.4 0.3 4.8 o . a 0.3 4 .7 0.2 0 .3 5. 1 1 4.4 0.8 5 4.3 0.6 8 4.3 0.7 12 4.6 0.8 15 4.2 1.0 19 4.3 0.8 3.1 6.8 4.2 3.3 3.4 2 .9 3.4 7.1 7.1 3.4 5.4 5.8 2 .9 5.6 3.2 3 .3 2 .8 3.2 0.4 0.8 4 .5 0.5 0.7 4 .8 0.8 0.8 4.6 0.7 0.7 4 .5 0.6 0.8 4.7 0.8 0.8 4.3 1 4.1 1.7 2 .9 3 .9 3.8 1.7 1.7 3.2 5 4.0 1.8 3.1 3 .9 4 .0 1 .6 1 .8 2.9 8 3.9 1.9 3.1 3.8 2 .7 1.8 1.8 3.0 12 4.1 1.8 3.2 3.4 3.2 1 .6 1 .8 2.7 15 4.2 2.1 2 .9 2 .9 2 .9 1.8 1.9 3.1 1 4.3 3.4 5 4.5 3.4 8 4.2 3.3 12 4.4 3.5 15 4.5 3.5 3.2 3 .5 3.2 3 .6 3.2 3.6 3.4 3.3 3.4 3.6 3.6 3.6 2 .8 3 .5 3.8 3.4 3.4 1 .6 3.3 3.4 1 .3 2.8 3.3 1 .7 2.9 3.3 1 .7 3.1 3.4 1 .4 -171-RAW DATA OF NITROGEN FROM THE ACETATE RUNS Dosage in Day # TKN anaerobic a f t e r (mg/L) zone as steady mg COD/L state inf eff NO (mg?L) inf anae anox aero e f f 30 1 27.5 4 26.9 7 26.4 11 27.1 14 27.8 1.8 0.1 1.8 0.1 1.3 <0.1 2.4 <0.1 2.9 <0.1 <0.1 0.2 <0.1 0.2 <0.1 0.1 <0.1 <0.1 <0.1 <0.1 10.4 10.3 9.8 9.4 8.8 8 .8 9.1 8 .9 9.8 9 .8 25 1 27.8 4 26.6 8 27.6 11 27.2 15 28.7 2.4 0.1 2.2 0.2 1.8 <0.1 2.8 <0.1 2.4 0.3 <0.1 0 .2 <0.1 0.3 <0.1 0.1 <0.1 <0.1 0.1 0.2 10.3 10.6 10.0 9 .8 10.6 10.6 9.4 9.2 8.6 8 .2 1 26.9 2.6 0.2 <0. 1 0.2 9.8 9 .6 5 27.3 3.1 <0.1 <0. 1 0.3 8.6 8 .4 8 28.2 2.3 <0.1 <0. 1 0.3 10.2 9.8 12 26.8 3.2 0.1 <0. 1 0.2 10.6 10.4 15 27.4 3.4 <0.1 <0.1 0.1 10.9 10.6 19 27.6 2.1 <0.1 <0.1 <0. 1 11.2 11.6 1 29.4 3.6 0.2 <0. 1 <0.1 9.8 9 .6 5 29.8 3.8 0.2 <0. 1 <0.1 1 1 .8 1 1 .6 8 28.4 4.8 0.1 <0. 1 0.1 8.8 8.8 12 28.0 5.1 <0. 1 <0. 1 0.1 8.4 8 .4 15 30.2 3.3 <0. 1 <0. 1 0.3 10.6 10.4 19 29.0 3.5 0.1 <0. 1 0.2 10.7 10.7 1 29.6 4.4 0.2 <0. 1 0.2 11.6 1 1 .4 5 30. 1 3.9 0.2 <0. 1 <0.1 1 1 .6 11.6 8 29.4 3.3 0.1 <0. 1 <0.1 10.3 10.5 12 28.6 4.8 0.1 <0. 1 0.2 8.8 8 .6 15 29.4 4.5 <0.1 <0. 1 0.3 8.6 8 .6 1 28.4 3.4 <0.1 <0. 1 0.2 10.4 10.2 5 29. 1 3.8 0.2 <0. 1 0.2 10.8 10.8 8 28.1 3.3 <0. 1 <0. 1 <0. 1 11.6 1 1 .4 12 29.2 2.9 <0.1 <0.1 <0.1 9.6 9.8 15 28.8 4.2 0.2 <0. 1 <0.1 10.6 10.6 -1 72-RAW DATA OF SOLIDS FROM THE ACETATE RUNS Dosage in Day # TSS VSS anaerobic a f t e r (mg/L) (mg/L) zone as steady mg COD/L state anae anox aero eff anae anox aero eff 1 1220 2380 4 1260 2380 7 1310 2340 1 1 1240 2360 1 4 1200 2380 2340 20 850 2340 29 880 2320 21 890 2290 10 840 2300 16 800 1920 1960 14 1880 1990 18 1940 1930 18 1960 1960 -1900 1820 1 1 1 1280 2340 4 1240 2310 8 1 1 30 2320 1 1 1280 2360 1 5 1220 2380 2320 1 1 820 2300 15 870 2340 27 880 2290 39 900 2330 34 810 1960 1720 6 1920 1920 1 4 1880 1890 23 1870 1880 30 1840 1760 25 1 1210 2320 5 1200 2350 8 1 1 20 2280 1 2 1320 2340 15 1 340 2300 19 1290 2340 1 1210 2340 5 1 1 30 2280 8 1 190 2300 1 2 1260 2300 1 5 1200 2320 19 1 180 2370 2340 39 920 2340 35 860 2250 1 1 840 2360 10 890 2280 25 880 2320 19 910 2320 20 740 2240 29 810 2290 14 800 2300 23 800 2320 26 770 2310 30 730 1820 1810 26 1860 1860 22 1740 1830 6 1840 1820 -1730 1700 18 1710 1800 1 4 1820 1760 14 1630 1740 22 1780 1690 10 1810 1820 16 1830 1870 20 1800 1740 23 1 1200 231 0 5 1 180 2290 8 1220 2340 1 2 1230 2360 1 5 1 1 90 2300 2280 29 840 2240 20 850 2300 16 860 2320 15 920 2290 9 900 1800 1890 22 1770 1820 1 4 1 780 1880 9 1820 1790 10 1850 1820 -1 1210 2310 5 1230 2390 8 1 190 2330 12 1230 2320 15 1230 2300 2250 20 930 2270 15 830 2300 16 850 2290 22 840 2280 17 820 1840 1800 18 1870 1830 10 1830 1790 9 1800 1820 1 4 1740 1760 1 1 -1 73-RAW DATA OF COD AND ORP FROM THE ACETATE RUNS Dosage in Day # Influent Effluent Total ORP anaerobic after COD COD RBD COD (mV) zone as steady (mg/L) (mg/L) of feed mg COD/L state (mg COD/L) anae anox 30 1 219 32 87 -342 -168 4 227 35 79 -346 -167 7 203 30 83 -341 -156 1 1 221 41 86 -348 -164 14 227 38 85 -350 -142 25 1 228 43 74 -341 -158 4 231 37 78 -338 -156 8 219 31 80 -340 -160 1 1 226 49 79 -340 -1 72 15 218 41 73 -329 -1 54 20 1 242 32 73 -321 -128 5 218 27 71 -319 -120 8 235 34 73 -315 -1 45 1 2 224 36 69 -319 -149 15 230 32 70 -320 -149 19 231 31 69 -322 -1 54 1 5 1 227 27 63 -31 1 -1 40 5 238 33 59 -332 -143 8 244 31 64 -328 -1 39 1 2 220 22 68 -310 -1 38 1 5 236 29 60 -316 -141 19 240 31 66 -321 -151 10 1 251 34 57 -326 -146 5 238 29 59 -314 -1 46 8 235 33 54 -298 -123 1 2 228 37 51 -300 -1 46 15 249 28 54 -294 -142 5 1 241 29 46 -289 -143 5 239 39 49 -220 -139 8 256 43 48 -288 -147 1 2 249 33 47 -297 -1 52 15 246 38 49 -281 -1 42 -174-RAW DATA OF PHOSPHORUS FROM THE PROPIONATE RUNS Dosage in Day # Total P Ortho - P Aero. anaerobic a f t e r (mg/L) (mg/L) Slud. zone as steady %P mg COD/L state inf eff inf anae anox aero eff 25 1 4.3 0.3 3.2 14.1 11.5 0.3 0.3 5.4 5 4.3 0.4 3.2 13.6 11.2 0.3 0.3 5.6 8 4.4 0.3 3.4 13.9 10.5 0.3 0.3 5.4 12 4.3 0.2 3.0 13.8 1 1 .8 0.2 0.2 5.5 1 5 4.2 0.2 3.0 13.2 12.4 0.2 0.2 5.5 20 1 4.0 0.9 3.1 10.4 8.4 0.9 0.9 4.5 4 4.2 0.8 2.9 10.2 8.6 0.8 0.7 4.3 8 4.1 1.0 2.9 9.8 9.2 0.9 0.9 4.3 1 1 4.1 1.0 3.0 11.2 8.4 0.9 0.8 4.4 15 4.3 0.9 3.1 9.8 8.8 0.9 0.9 4.4 15 1 4.1 1 .6 3.3 5.4 4.2 1 .5 1 .5 3.6 5 4.1 1 .4 3.4 4.9 4.6 1 .3 1.4 3.7 8 4.2 1 .6 3.2 5.6 4.1 1 .7 1 .6 3.6 12 4.4 1 .8 3.2 5.6 3.9 1 .5 1 .5 3.8 1 5 4.2 1 .5 3.4 4.8 4.0 1 .4 1 .4 3.6 * 10 1 3.9 <0.2 2.9 15.4 11.2 <0.1 <0. 1 5.3 5 3.9 <0.2 2.9 15.8 10.8 <0.1 <0.1 5.4 8 4.0 <0.2 3.0 16.2 1 1 .8 <0.1 <0. 1 5.0 1 2 3.9 <0.2 2.9 15.0 1 1 .4 <0.1 <0. 1 5.2 1 5 3.8 <0.2 2.8 14.9 10.9 <0.1 <0. 1 5.2 10 1 4.0 2.6 3.2 3.3 3.3 2.5 2.4 2.3 4 4.1 2.7 3.1 4.5 3.8 2.5 2.6 2.1 8 4.0 2.5 3.1 3.8 3.2 2.5 2.5 2.1 1 1 3.9 2.4 2.9 4.6 3.9 2.4 2.3 2.2 1 5 4.2 2.6 3.2 4.1 4.0 2.4 2.4 2.1 5 1 4.0 3.1 2.9 2.9 2.7 3.1 3.1 1 .5 5 3.9 3.0 3.0 3.6 2.9 2.9 3.0 1 .4 8 4.1 3.2 3.2 3.7 3.4 3.0 3.0 1 .5 12 4.2 3.2 3.4 3.3 3.1 3.0 3.1 1 .3 15 4.1 3.1 3.3 3.0 2.8 3.1 3.2 1 .3 The raw sewage during t h i s run had an acetate concentration of 14.7 mg/L as COD. -175-RAW DATA OF NITROGEN FROM THE PROPIONATE RUNS Dosage in Day # TKN NOv anaerobic after (mg/L) (mg?L) zone as steady mg COD/L state inf eff inf anae anox aero eff 25 1 26.2 2.1 <0. 1 <0.1 0.1 7.2 7.0 5 26.0 1 .8 <0.1 <0.1 0.1 6.8 6.8 8 25.8 1 .4 <0. 1 <0.1 <0.1 6.6 6.4 12 25.7 1 .8 <0.1 <0.1 <0.1 6.8 6.6 1 5 26.2 2.0 <0. 1 <0.1 0.1 7.0 7.0 20 1 25.8 1 .7 <0.1 <0.1 0.2 6.8 6.8 4 25.8 2.1 <0.1 <0.1 0.1 6.0 5.9 8 26.0 1.6 <0. 1 <0.1 <0.1 6.6 6.4 1 1 26.2 1.5 <0.1 <0.1 0.1 6.5 6.5 15 25.7 1.5 <0. 1 <0.1 <0.1 6.8 6.2 15 1 24.7 1.8 <0. 1 <0.1 0.2 7.1 7.1 5 24.6 1.8 <0. 1 <0.1 0.2 6.9 7.0 8 24.3 1 .6 <0.1 <0. 1 <0.1 7.3 7.2 1 2 24.0 1 .4 <0.1 <0.1 0.1 7.3 6.9 15 24.8 2.0 <0.1 <0.1 <0.1 7.5 7.5 * 10 1 27.4 2.1 <0.1 <0.1 <0.1 5.2 5.1 5 27.0 2.0 <0. 1 <0.1 <0.1 5.1 5.1 8 27.2 2.1 <0.1 <0.1 <0.1 5.0 5.0 12 26.8 1 .8 <0. 1 <0.1 <0.1 5.8 5.6 1 5 27.2 1 .6 <0. 1 <0. 1 <0.1 5.2 5.2 10 1 23.9 1 .7 <0.1 <0. 1 0.1 7.2 7.1 4 24. 1 1.7 <0. 1 0.1 0.2 7.9 7.9 8 24.0 1 .6 <0. 1 <0. 1 0.2 7.6 7.4 1 1 24.5 1.6 <0. 1 <0.1 <0.1 7.6 7.2 15 24.7 1 .7 <0.1 0.1 0.1 7.2 7.2 5 1 22.3 1 .9 <0.1 <0.1 0.1 8.0 7.9 5 22.8 2.1 <0. 1 0.1 0.1 6.8 6.8 8 22.8 1 .8 <0. 1 0.1 <0. 1 7.8 7.8 12 23. 1 1 .8 <0.1 <0.1 0.2 7.8 7.6 15 22.9 1.9 <0. 1 <0.1 0.1 8.1 7.9 * The raw sewage during t h i s run had an acetate concentration of 14.7 mg/L as COD. -176-RAW DATA OF SOLIDS FROM THE PROPIONATE RUNS Dosage in Day # TSS VSS anaerobic a f t e r (mg/L) (mg/L) zone as steady mg COD/L state anae anox aero eff anae anox aero e f f 1 1220 2290 2310 22 840 1830 1840 16 5 1230 2290 2300 24 850 1820 1830 12 8 1220 2280 2270 28 880 1830 1830 18 12 1240 2200 2320 18 840 1800 1810 13 15 1240 2290 2310 20 830 1810 1790 14 1 1200 2280 2300 24 820 1820 1800 20 4 1210 2300 2300 22 800 1800 1810 24 8 1210 2280 2290 27 830 1900 1880 22 1 1 1 220 2300 2280 16 810 1840 1820 -1 5 1210 2290 2270 20 820 1800 1800 15 1 1200 2260 2290 26 860 1800 1790 20 5 1 200 2240 2280 28 840 1860 1840 26 8 1 180 2240 2300 26 820 1840 1850 19 12 1200 2260 2280 25 820 1800 1810 18 15 1 190 2230 2310 20 830. 1820 1810 14 1 1230 2320 2360 18 820 1860 1880 6 5 1240 2320 2340 26 840 1880 1890 -8 1260 2340 2340 12 860 1880 1900 8 1 2 1240 2350 2360 20 860 1860 1840 1 1 15 1230 2340 2340 15 810 1840 1840 1 1 1 1 180 2250 2270 28 810 1810 1820 22 4 1 190 2240 2260 20 800 1800 1800 16 8 1 190 2230 2270 16 790 1800 1 790 1 1 1 1 1 170 2230 2270 10 810 1810 1790 8 15 1 190 2240 2280 22 790 1790 1800 16 1 1200 2250 2270 30 800 1800 1790 21 5 1 160 2250 2260 22 780 1780 1 790 18 8 1 180 2220 2240 20 780 1770 1780 16 12 1 170 2230 2260 18 800 1800 1790 12 15 1 180 2260 2290 26 780 1800 1790 14 The raw sewage during this run had an acetate concentration of 14.7 mg/L as COD. -177-RAW DATA OF COD AND ORP FROM THE PROPIONATE RUNS Dosage in Day # Influent E f f l u e n t Total ORP anaerobic after COD COD RBD COD (mV) zone as steady (mg/L) (mg/L) of feed mg COD/L state (mg COD/L) anae anox 25 1 220 24 68 -354 -180 5 225 26 72 -354 -182 8 228 27 73 -352 -180 12 230 27 72 -350 -178 1 5 227 20 72 -354 -180 20 1 230 23 67 -344 -180 4 229 28 67 -342 -178 8 224 24 66 -342 -174 1 1 225 24 64 -340 -178 15 229 24 66 -328 -170 1 5 1 230 22 58 -325 -160 5 232 21 59 -320 -162 8 229 21 58 -322 -168 12 239 22 61 -320 -170 1 5 238 20 61 -310 -160 * 10 1 238 27 70 -360 -180 5 235 29 70 -355 -186 8 240 24 72 -350 -182 1 2 242 30 73 -358 -188 15 240 28 70 -360 -180 10 1 234 24 49 -310 -150 4 240 22 51 -308 -148 8 243 22 52 -306 -148 1 1 245 30 53 -310 -152 15 242 26 52 -310 -150 5 1 240 24 45 -300 -148 5 244 23 46 -290 -146 8 242 23 46 -290 -150 12 248 26 47 -285 -150 15 245 24 46 -290 -149 The raw sewage during t h i s run had an acetate concentration of 14.7 mg/L as COD. -1 78-' RAW DATA OF CARBON STORAGE FROM THE PROPIONATE RUNS Dosage in anaerobic zone as mg COD/L Day # after steady state anae PHB (mg/L) anox aero anae PHV (mg/L) anox aero 25 8 11.6 11.5 4.1 15.2 14.9 2.3 20 8 7.2 9.9 2.4 9.6 8.7 2.1 15 12 6.3 4.0 5.9 7.8 7.1 3.3 * 10 5 11.0 9.8 2.4 6.7 4.3 1.7 10 8 8.1 6.4 4.1 5.7 5.2 3.7 5 8 6.8 3.8 3.5 3.5 3.6 3.3 The raw sewage during t h i s run had an acetate concentration of 14.7 mg/L as COD. - 1 7 9 -RAW DATA OF PHOSPHORUS FROM THE BUTYRATE RUNS Dosage in Day # Total P Ortho - P Aero. anaerobic after (mg/L) (mg/L) Slud. zone as steady %P mg COD/L state inf e f f inf anae anox aero eff 1 4 . 2 0 . 5 3 . 3 1 2 . 6 9 . 8 0 . 5 0 . 5 4 . 3 5 4 . 2 0 . 6 3 . 4 1 3 . 2 10 .0 0 . 5 0 . 4 4 . 6 8 4 . 0 0 . 5 3 . 3 1 2 . 8 9 . 6 0 . 5 0 . 6 4 . 7 12 4.1 0 . 6 3 . 3 1 3 . 0 9 . 8 0 . 6 0 . 5 4 . 8 15 4 . 0 0 . 5 3 . 4 1 2 . 6 1 0 . 0 0 . 5 0 . 6 4 . 8 19 4 . 2 0 . 6 3 . 2 1 3 . 0 9 . 4 0 . 6 0 . 6 4 . 4 1 4 . 2 0 . 9 3 . 2 1 0 . 4 8.1 0 . 9 1 .1 4.1 4 4 . 2 1 . 0 3 . 3 8 . 8 7 . 9 0 . 8 1.0 4 . 3 8 4 . 0 1.0 3.1 1 0 . 3 7 . 4 0 . 8 0 . 9 3 . 9 1 1 4 . 3 0 . 9 3.1 9 . 9 7 . 8 0 . 9 1 . 1 4 . 6 1 5 4 . 4 0 . 8 3 . 2 1 1 . 3 7 . 5 0 . 9 0 . 9 4 . 4 1 3 . 7 1 .8 3.1 7 . 0 5 . 7 1.7 1.8 2 . 7 5 3 . 8 1 .9 2 . 9 7 . 4 5 . 9 1.6 1.7 2 . 7 8 4.1 1 . 9 3 . 0 7 . 5 6 . 4 1.6 1.6 3 . 0 1 2 3 . 9 1 . 7 2 . 8 8 . 0 6 . 7 1 .8 1 . 8 2 . 6 15 4 . 0 1 .8 3.1 7.1 5 . 0 1.8 1.7 2 . 4 1 4.1 2 . 2 3.1 5 . 0 4 . 6 2 . 4 2 . 4 1.9 4 4 . 2 2 . 3 3 . 2 5.1 3 . 9 2 . 4 2 . 4 2 . 0 8 4 . 0 2 . 3 3 . 4 4 . 3 3 . 8 2 . 3 2 . 3 2 . 4 1 1 4 . 4 2 . 4 3 . 2 4 . 5 4 . 3 2 . 3 2 . 4 2 . 2 15 4 . 3 2 . 4 3 . 0 4 . 7 4 . 0 2 . 3 2 . 5 1 .8 1 4.1 3.1 2 . 8 3 . 3 3 . 0 3 . 2 3 . 0 1.5 5 3 . 8 3 . 4 3 . 0 3 . 4 3.1 2 . 9 2 . 9 1.5 8 3 . 8 3 . 0 3 . 0 3 . 4 3 . 2 2 . 9 3 . 0 1 .7 12 4 . 3 2 . 8 2 . 9 3 . 0 2 . 6 2 . 8 2 . 8 1 .6 15 3 . 9 3 . 2 3 . 2 3 . 7 3 . 4 3 . 0 2 . 9 1 .8 -180-RAW DATA OF NITROGEN FROM THE BUTYRATE RUNS Dosage in Day # TKN anaerobic after (mg/L) zone as steady mg COD/L state inf eff NO (mg?L) inf anae anox aero eff 30 1 24.8 2.2 <0. 1 <0.1 0.1 6.0 5.8 5 23.2 1 .8 <0.1 <0.1 <0.1 5.8 6.0 8 24.9 2.1 <0. 1 <0.1 <0.1 6.1 6.2 12 24.0 2.2 0.2 <0.1 0.1 6.0 5.8 15 23.8 2.0 <0.1 <0.1 <0.1 5.9 6.2 19 24.6 2.3 0.2 <0.1 0.1 6.6 5.2 25 1 22.4 1 .8 <0.1 <0.1 0.1 6.0 6.1 4 23.0 2.1 <0.1 <0.1 0.1 6.4 6.4 8 21.1 1 .1 0.1 <0.1 <0.1 5.8 5.6 1 1 21 .2 1 .1 <0.1 <0.1 <0.1 6.0 5.9 15 21 .9 0.8 <0. 1 <0.1 <0.1 5.3 5.3 20 1 23.8 2.0 <0.1 <0.1 <0. 1 6.9 6.8 5 24.2 2.2 <0.1 <0.1 <0. 1 7.0 7.0 8 25.0 1 .3 <0.1 <0.1 0.1 5.7 5.8 12 23.1 1 .6 <0.1 <0. 1 <0.1 6.0 5 .9 15 23.6 1 .6 <0.1 <0.1 <0.1 6.0 6.1 15 1 22.0 1 .1 <0.1 <0.1 <0.1 6.8 6.8 4 22.8 1.0 <0.1 <0.1 0.2 7.6 7.4 8 21 .3 0.8 <0.1 <0.1 <0.1 7.4 7.4 1 1 23.2 1 .1 <0.1 <0.1 <0. 1 6.0 6.1 15 21 .1 1 .7 <0.1 <0.1 0.1 6.5 6.5 10 1 24.1 2.1 <0.1 <0. 1 <0.1 7.8 8.0 5 23.4 2.3 0.2 <0.1 <0. 1 6.9 6.9 8 23.8 3.1 <0.1 <0.1 0.2 6.8 6.8 12 22.1 1.6 <0.1 <0.1 <0.1 6.9 7.0 15 23.5 1 .1 0.1 <0.1 <0.1 6.2 6.2 -181-RAW DATA OF SOLIDS FROM THE BUTYRATE RUNS Dosage in Day # TSS VSS anaerobic after (mg/L) (mg/L) zone as steady mg COD/L state anae anox aero eff anae anox aero eff 1 1230 2290 2290 18 890 1800 1820 14 5 1230 2300 2320 23 900 1 790 1820 19 8 1 300 2320 2320 31 900 1830 1850 24 12 1220 2340 2330 19 920 1880 1830 13 15 1260 2290 2320 12 890 1820 1800 8 19 1210 2300 2310 23 930 1800 1840 12 1 1 190 2260 2280 28 930 1 720 1730 21 4 1230 2400 2390 31 980 1770 1770 24 8 1 230 2370 2390 21 920 1690 1680 16 1 1 1270 2290 2270 32 990 1 780 1810 22 15 1 180 2210 2190 14 880 1680 1620 9 1 1 150 2230 2200 23 870 1 620 1600 18 5 1 170 2230 2230 19 890 1690 1710 15 8 1090 2180 2220 31 920 1580 1600 27 12 1 140 2190 2210 1 5 860 1610 1640 10 15 1020 2080 2060 22 790 1490 1490 1 4 1 1000 21 10 2090 30 760 1 600 1580 22 4 1210 2220 2180 27 870 1660 1610 20 8 1 160 2200 2190 19 820 1690 1720 1 1 1 1 1 040 2090 2100 9 790 1 540 1590 -15 1060 2130 2210 1 1 800 1590 1630 7 1 1050 2100 2170 18 830 1610 1630 10 5 990 2070 21 30 31 710 1570 1610 23 8 1010 2090 2060 30 830 1560 1550 21 12 1 120 2210 2260 19 860 1690 1700 12 15 980 1990 2040 28 690 1480 1520 20 -1 82-RAW DATA OF COD AND ORP FROM THE BUTYRATE RUNS Dosage in Day # Influent Effluent Total ORP anaerobic a f t e r COD COD RBD COD (mV) zone as steady (mg/L) (mg/L) of feed mg COD/L state (mg COD/L) anae anox 30 1 222 29 71 -362 -190 5 218 31 71 -358 -200 8 220 37 69 -350 -194 12 216 36 68 -370 -198 15 225 39 70 -370 -204 19 217 32 67 -364 -196 25 1 226 40 63 -365 -163 4 231 43 66 -360 -175 8 219 29 70 -366 -165 11 225 34 62 -370 -159 15 212 43 71 -360 -159 20 1 236 45 59 -362 -145 5 242 51 64 -355 -189 8 227 22 68 -358 -135 12 219 41 53 -360 -148 15 224 39 65 -351 -1 47 15 1 251 38 63 -370 -165 4 230 36 59 -345 -155 8 227 41 59 -367 -153 1 1 231 33 51 -339 -149 15 229 49 64 -342 -162 10 1 241 38 48 -344 -134 5 255 25 53 -352 -143 8 251 42 51 -338 -131 12 223 46 56 -339 -129 15 236 21 44 -329 -139 -183-RAW DATA OF CARBON STORAGE FROM THE BUTYRATE RUNS Dosage in anaerobic zone as mg COD/L Day # aft e r steady state anae PHB (mg/L) anox aero anae PHV (mg/L) anox aero 30 1 8 19 26.8 24.3 25.2 22.4 24.2 22.7 5.8 6.3 4.9 7.9 3.8 5.9 6.2 3.2 4.9 3.6 4.0 3.1 25 1 8 15 17.4 18.4 17.6 15.9 16.6 16.3 5.1 3.9 4.0 3.6 3.0 2.9 2.3 1.9 2.9 0.9 1 .3 1 .5 20 1 12 1 5 8.9 10.9 12.3 11.1 9.4 9.9 3.3 3.9 2.5 1.9 1 .6 3.0 2.1 2.6 3.0 1 .6 0.9 1 .3 15 1 8 15 4.9 6.2 5.9 6.1 5.4 4.8 3.0 2.1 1.8 0.9 1 .9 1.0 -1 .6 0.9 1.0 0.2 0.3 0.5 10 1 8 15 4.3 4.9 3.8 4.3 4.1 3.8 1.9 2.3 2.0 0.8 0.6 2.1 0.9 0.7 0.7 0.3 0.5 0.3 -184-RAW DATA OF CARBON STORAGE AND PHOSPHORUS FROM THE BUTYRATE RUNS (Transient State) Dosage in anaerobic zone as mg COD/L Day # after dosage change anae PHB (mg/L) anox aero anae Ortho -(mg/L) anox P aero 30 1 3 5 21 .5 19.3 18.1 20.2 18.1 16.8 5.2 4.5 4.2 11.4 10.4 10.2 8.8 8.2 7.9 0.5 0.5 0.8 25 1 3 5 15.0 12.9 1 1 .0 13.9 12.1 10.6 3.9 3.4 3.2 8.9 8.1 7.8 6.9 6.3 6.1 0.9 0.9 1 .6 20 1 3 5 8.5 6.6 5.8 8.2 6.4 5.3 2.7 2.4 2.3 6.6 5.7 4.9 5.2 4.6 4.2 1 .7 1 .9 2.1 15 1 3 4.9 4.5 5.0 4.4 2.1 2.0 4.2 -3.8 3.6 3.2 2.6 2.9 10 -185-RAW DATA OF PHOSPHORUS FROM THE GLUCOSE RUNS Dosage in Day # Total P Ortho - P Aero. anaerobic a f t e r (mg/L) (mg/L) Slud. zone as steady %P mg COD/L state inf eff inf anae anox aero e ff 1 3.6 0.7 2.8 11.4 6.7 0.6 0.6 3.5 5 3.4 0.6 2.4 10.9 6.6 0.6 0.8 3.3 8 3.6 0.7 2.6 10.8 6.8 0.7 0.6 3.5 19 3.5 0.7 2.7 11.3 7.1 0.7 0.5 3.6 23 3.7 0.8 2.8 11.6 6.6 0.7 0.7 3.1 1 3.9 1.8 3.0 6.8 6.0 1.7 1 .7 2.8 4 3.6 1.5 2.8 7.4 6.2 1 .6 1.5 2.7 8 3.7 1.7 2.7 6.2 5.9 1 .5 1 .6 2.7 1 1 3.8 1.8 2.8 6.8 6.1 1.7 1 .7 2.6 15 3.7 1.6 2.8 7.0 6.0 1.6 1 .6 2.9 1 3.7 2.4 2.6 3.8 3.0 2.4 2.3 1.7 5 3.7 2.3 2.8 4.2 3.4 2.4 2.6 1 .7 8 3.9 2.5 2.6 4.4 3.4 2.4 2.4 1.8 12 3.7 2.5 2.9 4.2 3.0 2.3 2.0 1.6 15 3.5 2.3 2.8 3.9 2.9 2.3 2.3 1.8 1 4.0 0.2 3.3 13.2 7.1 <0. 1 <0. 1 4.1 6 3.9 0.1 3.3 13.6 6.9 0.1 0.1 4.2 8 4.1 0.3 3.4 13.8 7.2 0.2 0.2 4.0 12 3.8 0.1 3.2 12.9 6.6 <0.1 <0. 1 4.1 15 3.9 0.2 3.1 12.9 6.8 0.1 0.2 3.9 -186-RAW DATA OF NITROGEN FROM THE GLUCOSE RUNS Dosage in Day # TKN anaerobic after (mg/L) zone as steady mg COD/L state inf eff NO (mg?L) inf anae anox aero eff 1 23.2 5 22. 1 8 24. 1 19 22.3 23 21.9 2.1 <0.1 2.9 <0.1 1.8 <0.1 2.0 <0.1 2.5 <0.1 <0.1 0.1 <0.1 0.2 <0.1 0.2 <0.1 0.1 <0.1 0.3 6.2 6.1 7.0 6.8 7.4 7.4 6.8 6.9 7.0 6.9 45 1 22.6 4 21.3 8 21.7 11 20.8 15 23.4 2.4 <0.1 1.8 <0.1 3.4 0.1 1.4 <0.1 2.8 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 6.2 6.2 6.4 6.3 6.0 6.2 5.9 5.9 6.1 6.2 1 20.9 1.8 <0. 1 <0. 1 0.1 6.4 6.2 5 22.3 1.7 <0. 1 <0. 1 <0. 1 6.9 6.6 8 21.4 1.7 <0. 1 <0. 1 0.2 5.4 5.2 12 21.8 1.6 <0. 1 <0. 1 0.2 6.8 6.9 15 20.9 2.0 <0.. 1 <0. 1 0. 1 6.6 6.6 1 19.8 1.2 <0. 1 <0. 1 0.2 8.3 8.4 6 18.9 1.3 <0. 1 <0. 1 0.3 9.8 9.8 8 20.0 1.3 <0.1 <0. 1 0.3 7.4 7.1 12 19.4 1.4 0.2 <0. 1 0.2 7.2 7.3 15 18.6 1 .2 <0. 1 <0. 1 0.2 8.1 8.1 -187-RAW DATA OF SOLIDS FROM THE GLUCOSE RUNS Dosage in Day # anaerobic a f t e r zone as steady TSS (mg/L) VSS (mg/L) itate anae anox aero eff anae anox aero eff 1 1410 2540 2560 24 1030 2070 2080 18 5 1380 2480 2490 23 1075 1920 1990 18 8 1430 2590 2580 10 11 20 2100 2140 6 19 1290 2520 2550 8 980 2040 2180 4 23 1330 2540 2500 20 1020 1990 2060 12 60 1 1290 2320 4 1300 2400 8 1340 2310 1 1 1280 2310 15 1260 2300 2320 4 960 2360 18 980 2290 10 1000 2360 21 990 2340 20 1020 1850 1860 -1900 1920 18 1870 1800 8 1820 1790 16 1860 1900 16 1 1240 2400 5 1260 2300 8 1 190 2340 12 1240 231 0 15 1290 2430 1 1620 2800 6 1660 2800 8 1610 2810 12 1590 2740 1 5 1620 2790 241 0 10 940 2290 14 960 2310 23 820 2340 22 940 2410 15 990 2810 20 1 190 2790 10 1220 2840 5 1200 2820 21 1 180 2740 20 1260 1790 1770 8 1740 1780 1 1 1810 1820 20 1810 1810 16 1820 1830 10 2250 2300 16 2190 2210 8 2320 2330 -2310 2310 14 2240 2260 13 -1 88-RAW DATA OF COD AND ORP FROM THE GLUCOSE RUNS Dosage in Day # Influent Ef f l u e n t Total ORP anaerobic a f t e r COD COD RBD COD (mV) zone as steady (mg/L) (mg/L) of feed mg COD/L state (mg COD/L) anae anox 60 1 190 36 70 -398 -136 5 198 39 74 -390 -131 8 176 26 66 -393 -121 19 210 32 77 -382 -129 23 172 23 64 -390 -1 36 45 1 190 26 61 -386 -179 4 201 29 66 -362 -171 8 176 36 58 -344 -169 1 1 232 43 67 -366 -188 15 198 32 59 -362 -1 72 30 1 222 29 58 -360 -1 42 5 206 28 52 -350 -1 44 8 232 31 58 -358 -148 12 204 23 47 -356 -151 15 208 26 53 -342 -1 47 75 1 174 29 77 -401 -129 6 180 36 79 -412 -131 8 204 39 87 -408 -128 12 194 38 84 -390 -110 15 208 42 88 -406 -1 17 -189-RAW DATA OF CARBON STORAGE FROM THE GLUCOSE ; RUNS Dosage in anaerobic zone as mg COD/L Day # after steady state anae PHB (mg/L) anox aero anae Glycogen (mg/L) anox aero 60 1 8 23 23.6 25.2 25.3 16.3 18.7 17.4 5.0 4.3 3.6 183 1 79 180 440 429 421 442 461 475 45 1 8 15 10.9 1 1 .7 11.1 9.7 10.3 2.2 2.9 3.1 169 180 175 380 362 394 389 402 396 30 1 8 15 6.4 5.8 6.0 4.5 3.9 5.4 2.9 2.5 1 .6 1 64 169 151 329 342 335 341 363 322 75 1 6 15 32.4 31 .8 29.9 19.8 20.5 19.0 4.9 4.9 4.2 159 162 172 -450 422 437 498 463 472 -1 90-RAW DATA OF THE EFFLUENT ORTHO-P PROFILE (section 4.11) ACETATE Days Ortho-P 1 0. 1 4 0.1 7 0. 1 1 1 0. 1 1 4 0. 1 15 0. 1 16 0. 1 17 0. 1 18 0. 1 19 0. 1 20 0. 1 21 0.1 22 0. 1 23 0. 1 24 0.1 25 0. 1 28 0. 1 32 0. 1 35 0.1 39 0. 1 40 0. 1 41 0. 1 42 0. 1 43 0.1 44 0. 1 45 0. 1 46 0. 1 47 0. 1 48 0.3 49 0.3 53 0.2 56 0.4 60 0.3 63 0.3 67 0.3 68 0.3 69 0.2 70 0.3 71 0.3 72 0.4 73 0.3 74 0.7 PROPIONATE Days Ortho-P 1 0.3 5 0.3 8 0.3 12 0.2 15 0.2 16 0.2 17 0.3 18 0.2 19 0.2 20 0.3 21 0.4 22 0.6 23 0.5 24 0.8 25 1 .0 26 0.9 29 0.7 33 0.9 36 0.8 40 0.9 41 0.9 42 0.8 43 0.9 44 0.9 45 1 .1 46 1 .2 47 1 .2 48 1 .4 49 1 .5 50 1 .5 54 1 .4 57 1 .6 61 1 .5 64 1 .4 65 1 .2 66 0.1 67 0.1 68 0.1 69 0.1 70 0.1 71 0.1 75 0.1 BUTYRATE Days Ortho-P 1 0.5 5 0.4 8 0.6 12 0.5 15 0.6 19 0.6 21 0.5 22 0.6 23 0.5 24 0.7 25 0.8 26 0.9 27 0.9 28 0.8 29 1.0 30 0.9 31 0.9 32 0.8 33 1 .1 36 1 .0 40 0.9 43 1 .0 47 0.9 49. 0.9 50 0.8 51 0.9 52 1 .4 53 1 .6 54 1 .8 55 1 .7 56 1 .8 57 1 .8 61 1.7 64 1 .6 68 1 .8 71 1 .7 73 1 .7 74 1 .8 75 1.9 76 2.1 77 2.1 78 2.3 (continued) -191-RAW DATA OF THE EFFLUENT ORTHO-P PROFILE (section 4.11) ACETATE PROPIONATE BUTYRATE Days Ortho-P Days Ortho-P Days Ortho 75 0.8 78 0.1 79 2.4 76 0.7 82 0.1 80 2.4 77 0.8 85 0.1 81 2.3 81 0.9 86 0.2 82 2.4 84 0.8 87 0.3 85 2.4 88 0.7 88 0.3 89 2.3 91 0.8 89 0.3 92 2.4 95 0.7 90 0.4 96 2.5 98 0.8 91 0.7 98 2.6 102 0.8 92 0.9 99 2.9 103 0.8 • 93 1.3 100 3.0 104 0.8 94 2.0 101 3.0 105 0.9 95 2.5 102 2.9 106 0.8 96 2.4 103 3.0 107 0.8 99 2.6 104 3.0 108 1.0 102 2.5 105 3. 1 109 1.3 106 2.3 106 3.0 1 10 1.7 110 2.4 110 2.9 1 1 1 1 .6 1 1 1 2.5 113 3.0 1 12 1.7 1 12 2.6 1 17 2.8 1 13 1.7 113 2.8 120 2.9 116 1.8 1 14 3.1 119 1.7 115 3.0 123 1.8 1 16 3.2 -127 1.8 1 1 7 3.1 131 1.8 118 3.1 134 1.9 1 19 3.2 135 1 .8 120 3.1 136 1.8 124 3.0 137 1 .8 127 3.0 138 2.1 131 3.1 139 3.1 134 3.2 140 3.0 141 3.5 142 3.3 145 3.3 148 3.4 152 3.4 155 3.3 159 3.3 162 3.4 -1 9 2 -APPENDIX A3 RAW DATA FROM THE COLD STORAGE TESTINGS -1 93-RAW DATA FROM THE COLD STORAGE TESTING I ( A l l concentrations are in mg/L) Day # BOD COD NO TKN Total P PO.-P Total VFA X ft 110 263 <0.1 28.3 4.4 3.3 <3 119 268 <0.1 29.4 4.1 3.3 <3 104 251 <0.1 27.6 4.3 3.1 <3 112 257 <0.1 27.1 4.3 3.1 <3 98 255 <0.1 28.1 4.4 3.4 <3 107 255 <0.1 28.2 4.5 3.4 <3 106 261 <0.1 26.9 4.2 3.4 <3 106 249 <0.1 27.3 4.1 3.4 <3 110 246 0.1 27.3 4.2 3.3 <3 104 252 <0.1 27.9 4.2 3.2 <3 10 117 237 <0.1 29.4 4.4 3.0 <3 122 241 <0.1 28.2 4.1 3.0 <3 12 103 252 <0.1 26.3 4.2 3.3 <3 99 239 0.1 26.9 4.0 3.3 <3 14 102 248 <0.1 28.8 4.3 3.2 <3 104 239 <0.1 27.1 4.0 3.1 <3 -1 94-RAW DATA FROM THE COLD STORAGE TESTING II ( A l l concentrations are in mg/L) Day # BOD COD NOx TKN Total P P0 4~P Total VFA 89 232 <0.1 24.3 4.1 3.2 <3 87 236 0.1 24.6 4.0 3.2 <3 92 228 <0.1 23.1 4.2 3.2 <3 83 219 <0.1 22.9 4 .3 3.1 <3 97 227 <0.1 24.0 4.2 3.1 <3 98 230 <0.1 24.0 4.2 3.1 <3 81 234 0.1 24.6 4.1 3.3 <3 80 234 0.1 25.1 4.0 3.4 <3 86 225 <0.1 23.4 4.0 3.3 <3 81 219 <0.1 24.0 3.9 3.3 <3 10 90 217 <0.1 23 .5 4.2 3.4 <3 94 224 <0.1 23.8 4 .3 3.4 <3 12 83 230 <0.1 22.4 4.2 3.1 <3 85 231 <0.1 22.9 4.1 3.1 <3 14 79 224 <0.1 23.1 4.0 3 .3 <3 88 218 <0.1 23.6 4.0 3.2 <3 

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