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The role of carbon storage in biological phosphate removal from wastewater Comeau, Yves 1989

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T H E R O L E O F C A R B O N S T O R A G E I N B I O L O G I C A L P H O S P H A T E R E M O V A L F R O M W A S T E W A T E R by Y V E S C O M E A U B.Ing., Ecole Polytechnique de Montreal, 1980 M . A . S c , The University of British Columbia, 1984 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F P H I L O S O P H I S E D O C T O R (Ph.D.) in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Department of Civil Engineering) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F BRITISH C O L U M B I A May, 1989 ®Yves Comeau, 1989 In presenting this thesis in partial fulfillment 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 Civil Engineering The University of British Columbia 1956 Main Mall Vancouver, B.C., Canada V6T 1Y3 i i A B S T R A C T The objective of this research was to define the importance and role of carbon storage in biological excess phosphorus (bio-P) removal. For that purpose, a sensitive GC technique was developed for poly-/3-hydroxybutyrate (PHB) quantification. This technique also allowed the determination of poly-/3-hydroxyvalerate (PHV). Both PHB and PHV are referred to as poly-£-hydroxyalkanoates (PHA). Characterization of bio-P sludges obtained from an experimental pilot plant was performed in a number of batch experiments. The effect of anaerobic substrate addition was investigated with the following compounds: formate, acetate, propionate, butyrate, iso-butyrate, valerate, iso-valerate, hexanoate, lactate, /3-hydroxybutyrate, succinate, citrate, glucose, glycine and ethanol. In almost all cases, a direct relationship was observed between phosphate (Pi) release/uptake and PHA storage/consumption. These observations supported the concept that anaerobic polyP degradation was used for PHA storage, and that aerobic PHA consumption was used for polyP storage. It was also shown that nitrate, but not nitrite, could be used for Pi uptake and PHA consumption. For the sludge used in this research, acetate and propionate were found to be the two most favorable substrates to induce anaerobic PHA storage. Other substrates also triggered such storage, but at slower rates. The respective proportions of PHB and PHV storage indicated that, in general, substrates composed of an even number of carbons (e.g. acetate, butyrate, ^-hydroxybutyrate) favored PHB formation, - whereas substrates composed of an odd number of carbons (e.g. propionate, valerate, lactate) favored PHV formation. The response of the pilot plant sludge to the anaerobic addition of toxicants (2,4-DNP, high pH, low pH, cyanide, fluoride, C0 2 , H2S) was also tested to help postulate biochemical mechanisms for bio-P removal. Anaerobic Pi release was significantly stimulated by the addition of 2,4-DNP, a high pH or cyanide; in these cases, however, minimal PHA storage was observed. It was proposed that polyP degradation could be regulated by a pH-gradient sensitive enzyme that could be used to expel protons, which, in turn, would assist bio-P bacteria in maintaining a constant proton motive force. Molar ratios calculated between metallic cations and Pi, indicated that potassium, magnesium and calcium were probably co-transported with Pi both for export and import into bio-P bacteria. X l l At lab-scale, four sequencing batch reactors (SBR) were operated in parallel to develop bio-P sludges acclimated to different levels of acetate addition. PHA were also quantified in bio-P sludges taken from an experimental pilot plant at UBC over a five months period, and from the Kelowna full-scale treatment plant on two consecutive days. These continuous systems confirmed the role of PHA in bio-P removal. From the results obtained, a biochemical model was proposed to describe the activity of bio-P bacteria under anaerobic, anoxic and aerobic conditions. A summary of microbial activity in a bio-P biomass was presented. This research indicated that carbon storage as PHA played a central role in explaining bio-P removal mechanisms. It was proposed that to maximize bio-P removal, it is important to maximize anaerobic PHA storage by maximizing the addition of certain simple substrates and minimizing the addition of electron acceptors, such as nitrate or oxygen, into the anaerobic zone of a bio-P process. i v TABLE OF CONTENTS page ABSTRACT ii T A B L E OF CONTENTS iv LIST OF TABLES vii LIST OF FIGURES viii ACKNOWLEDGEMENTS xi LIST OF ABBREVIATIONS xii 1. INTRODUCTION 1 1.1. Eutrophication and Phosphorus Removal 1 1.2. Bio-P Removal 2 2. LITERATURE REVIEW 3 2.1. Historical Developments and Bio-P Processes 3 2.1.1 Historical developments 3 a) Importance of an anaerobic zone upstream of the aerobic zone 3 b) Electron acceptors c) Simple substrates 4 5 7 11 11 11 13 15 15 17 18 2.1.2 Bio-P processes 2.2. Microbial Aspects of Bio-P Removal 2.2.1. Microbial Metabolism a) PolyP metabolism b) PHB and PHV c) Glycogen d) Bacterial bioenergetics e) Bacterial membrane transport 2.2.2. Microbiology 3. R E S E A R C H OBJECTIVES AND APPROACH 21 4. MATERIALS A N D METHODS 23 23" 23 25 . 28 28 28 32 32 32 34 34 4.1. Experimental Setup 4.1.1. Batch experiments 4.1.2. SBR 4.2. Characteristics of Treatment Plants Studied 4.2.1. UBC Pilot plant 4.2.2. Kelowna full-scale plant 4.3. Analytical Methods 4.3.1. Standard analytical techniques 4.3.2. Radioactivity 4:3.3. V F A 4.3.4. Total carbohydrates and glycogen V 4.3.5. PHA 35 a) PHA extraction and quantification 35 b) G C / M S 36 4.4. Statistical Techniques 36 5. RESULTS 37 5.1. PHA Determination in Activated Sludge 37 5.1.1. PHA quantification 37 5.1.2. Reproducibility, recovery and sensitivity 37 5.2. Laboratory Batch Experiments 40 5.2.1. ANAEROBIC addition of acetate . 44 a) PHB and PHV storage 44 b) Reproducibility 47 c) Excess acetate 47 d) Metallic cations 54 e) Radioactive acetate 57 5.2.2. AEROBIC presence or addition of acetate and/or propionate 60 5.2.3. Propionate with or without acetate 65 5.2.4. Other substrates 69 5.2.5. Wastewater characterization by its ability to induce PHA storage: Penticton wastewater 75 5.2.6. Metallic cations 79 5.2.7. Nitrate and nitrite 82 5.2.8. Toxicants 85 a) 2,4-DNP 85 b) pH 94 c) Cyanide, Fluoride 100 d) C 0 2 , H 2 S 101 5.3. Studies on Continuous bio-P Wastewater Treatment Plants 101 5.3.1. Sequencing Batch Reactors (SBR) 101 5.3.2. PHA at the UBC Pilot Plant 105 5.3.3. Glycogen at the UBC Pilot Plant 112 5.3.4. PHA at the Kelowna, B.C. Full-scale Plant 113 6. DISCUSSION 118 6.1. Biochemical Model 118 6.1.1. Anaerobic model 118 a. Postulated model 118 b. Anaerobic energy balance for bio-P bacteria 122 6.1.2. Aerobic model 123 a. Postulated model 123 b. Aerobic energy balance for bio-P bacteria 124 .6.1.3. Anoxic model 125 6.2. Phosphate and PHA 125 6.2.1 Synthesis of results 125 a) Methods 125 i) PHA quantification 125 ii) PolyP estimation 125 iii) Wastewater characterization by its fermentability 126 b) Anaerobic substrate addition 126 v i c) Sequencing batch reactors 127 d) UBC pilot plant and Kelowna full-scale plant 129 e) Metallic cations 129 f) Oxidized nitrogen 130 g) Toxicants 130 6.2.2. Molar ratios 133 a) Summary of values 133 b) Acetate 137 c) Other substrates 141 d) Toxicants 147 e) Metallic cations 148 6.2.3. Rates of reaction 151 a) Substrate uptake 151 b) Pi release and uptake 152 i. Pi release 152 ii. Pi uptake 154 6.3. Difficulties of the proposed model to explain some observations 156 6.3.1. Aerobic or anoxic Pi release upon substrate addition 156 a. Proposed explanation for aerobic Pi release: polyP degradation regulated by the pH gradient 156 b. Anoxic Pi release 158 c. Implications of aerobic substrate addition for full-scale treatment 158 d. Implications of aerobic substrate addition for pure culture work 160 6.3.2. Glycogen storage 160 6.3.3. Biochemical mechanisms for the PhoStrip process 161 6.3.4. Minimal PHA storage with propionate or succinate addition 163 6.4. Microbial Activity 163 6.4.1. Classification and activity of microorganisms 164 6.4.2. Energy for bio-P bacteria 166 6.4.3. General considerations for full-scale design 167 7. CONCLUSIONS AND RECOMMENDATIONS 168 7.1 Conclusions 168 7.2 Recommendations for Future Research 170 REFERENCES 172 APPENDICES 185 APPENDIX 1 Chemical structure of compounds 186 APPENDIX 2 Molecular weights and COD equivalence for substrates 187 APPENDIX 3 Batch experiments - Treatment plants characterization data 188 APPENDIX 4 UBC pilot plant PHB/PHV data 193 v i i LIST OF TABLES Table 2.1 Genera of bacteria isolated from bio-P sludges 19 Table 4.1 Average UBC pilot plant process and sludge characteristics for the period studied (February 9 to July 6, 1987) ?..., 30 Table 4.2 Average Kelowna full-scale plant process and sludge characteristics for July 15 and 16, 1987 30 Table 4.3 Standard analytical techniques 33 Table 5.1 Overview of batch experiments 41 Table 5.2 Effects of 0.55 mM acetate addition on anaerobic Pi release, PHA storage, aerobic Pi uptake, and PHA consumption 46 Table 5.3 Effects of 0.55 mM acetate and/or propionate addition on Pi and PHA 65 . Table 5.4 Effects of the addition of 0.55 mM of various substrates. 71 Table 5.5 Pi release/uptake and PHA storage/consumption for batch experiment of October 28,1986 73 Table 5.6 C O D and acetate concentration of the four Penticton wastewater samples 77 Table 5.7 Effects of 1.0 mM 2,4-DNP addition 88 Table 5.8 Effects of 0.5 and 5.0 mM 2,4-DNP addition 89 Table 5.9 Effects of cyanide (5.0 mM) and fluoride (10 mM) 100 Table 5.10 SBR average influent, reactor and effluent characteristics for weeks 9 to 13 103 Table 5.11 U B C pilot plant average PHA content and % (PHV/PHA) 110 Table 5.12 UBC bio-P pilot plant carbohydrates and glycogen content 113 Table 6.1 Proposed energy balance for anaerobic PHB storage with acetate as substrate 123 Table 6.2 Proposed energy balance for aerobic PHB consumption 124 Tables 6.3-A, -B, -C Summary of molar ratios for batch experiments 134 Table 6.4 Molar ratios of Pi released over substrate taken up 145 Table 6.5 Molar ratios of cations and Pi 149 Table 6.6 Classification of microbial activity at the UBC bio-P pilot plant 165 Table A - l Chemical structure of compounds 186 Table A-2 Molecular weight and COD equivalence for substrates 187 Table A-3 Batch experiments - Treatment plant characterization data 190  4 UBC pilot plant PHB/PHV data 3 v i i i LIST OF FIGURES Figure 2.1 Simplified model for A. anaerobic conditions and Fl aerobic or anoxic conditions 6 Figure 2.2 Configuration of processes for biological nitrogen and phosphorus removal 8 Figure 2.3 Polyphosphate metabolism 12 Figure 2.4 Poly-/S-hydroxybutyrate metabolic pathways 12 Figure 2.5 Chemical structure of PHB and PHV 14 Figure 2.6 Overview of bacterial bioenergetics 16 Figure 2.7 Overview of bacterial membrane transport 16 Figure 4.1 Batch experiment apparatus 24 Figure 4.2 Lab-scale SBR apparatus 26 Figure 4.3 Schematic description of the automated operation of the SBR by two four-channels microprocessor controllers 27 Figure 4.4 Use of ORP to define the time required for complete denitrification in an SBR 27 Figure 4.5 UBC pilot plant process configuration 29 Figure 4.6 Schematic of the Kelowna bio-P wastewater treatment plant 29 Figure 4.7 Process configuration of the Kelowna full-scale bio-P treatment plant 31 Figure 5.1 Chromatogram of methyl esters derivatives extracted from a bio-P activated sludge sample 38 Figure 5.2 Correction factors for A, PHB and B. PHV as a function of the weight of lyophilized sludge extracted 39 Figure 5.3 Effects of anaerobic acetate addition to denitrified bio-P sludge, and of aeration 45 Figure 5.4 Assessment of experimental reproducibility for anaerobic acetate addition 48 Figure 5.5 Limitation of anaerobic acetate uptake by internal polyP reserves availability, and prolonged aerobic Pi release with increasing amounts of acetate added 49 Figure 5.6 Relationship between net Pi release and acetate consumed 50 Figure 5.7 Aeration time required to reach a minimum Pi concentration as a function of the amount of acetate consumed 50 Figure 5.8 Normal acetate addition to bio-P sludge obtained from four SBR acclimated to 0,15, 30 or 45 mg COD/1 acetate addition 52 Figure 5.9 Excess acetate addition (200 mg COD/1) to bio-P sludge obtained from four SBR acclimated to 0,15, 30 or 45 mg COD/1 acetate addition 53 Figure 5.10 Molar ratio of anaerobic Pi release over PHA storage for each SBR with riormaland excess acetate addition 55 Figure 5.11 Effect of metallic cations (potassium, magnesium, calcium I or sodium) on Pi release/uptake, and PHA storage/consumption , 56 Figure 5.12 Radioactive 14C-acetate addition 58 Figure 5.13 Fate of radioactive 14C-acetate into dissolved, particulates and C 0 2 fractions 59 Figure 5.14 Aerobic PHA storage in the 60 and 100 mgCOD/l reactors to which an excess of acetate was added anaerobically 61 Figure 5.15 Slow aerobic polyP degradation over a 10-day period . 62 Figure 5.16 Aerobic Pi release and PHA storage with aerobic acetate and/or propionate addition 64 Figure 5.17 PHA storage with anaerobic acetate and/or propionate addition 67 Figure'5.18 Amounts of net PHB and PHV storage for a total of-30 mg COD/1,of acetate and/or propionate addition ,68 Figure 5.19 PHA storage with anaerobic acetate and/or propionate addition 70 Figure 5.20 PHA storage with anaerobic short chain fatty acids (SCFA) addition 74 Figure 5.21 Net PHB/PHV anaerobic storage corresponding to the addition of 30 mg C O D / l of various substrates 76 Figure 5.22 Characterization of a wastewater fermentability by its ability to induce anaerobic Pi release and PHA storage by a bio-P sludge 78 Figure 5.23 Metallic cations concentration profiles in an experiment with : various levels of anaerobic acetate addition 80 Figure 5.24 Molar ratios of metallic cations and Pi transport for anaerobic and aerobic conditions for acetate addition 81 Figure 5.25 Reduction in the magnitude of anaerobic Pi release due to acetate addition due to increasing amounts of nitrate addition !• 83 Figure 5.26 Quantification of the effect.of nitrate addition on anaerobic Pi release 84 Figure 5.27 Comparison of oxygen, nitrate and nitrite as electron acceptors for Pi uptake and PHA consumption 86 Figure 5.28 Effect of 2,4-DNP, high pH (9.0) and low pH (4.0) on anaerobic Pi release 87 Figure 5.29 Effect of the addition of a neutralized 2,4-DNP solution on anaerobic Pi release and PHA storage 91 Figure 5.30 Effect of 2,4-DNP addition on metallic cations 92 Figure 5.31 Molar ratios of metallic cations and Pi transport for anaerobic and aerobic conditions for 2,4-DNP addition 93 Figure 5.32 Anaerobic Pi release and limitation of acetate uptake at high pH (8.3 to 9.0) : 95 Figure 5.33 Anaerobic Pi release at high pH (9.0) but not at low pH (4.0) 97 Figure 5.34 Effect of high and low pH on metallic cations 98 Figure 5.35 Molar ratios of metallic cations and Pi transport at high and low pH 99 Figure 5.36 Anaerobic Pi release with H 2 S and C 0 2 addition 102 Figure 5.37 Typical concentration profiles of A. Pi, B. nitrate, C. PHB and D. PHV over an 8-hour cycle in the four SBR 104 Figure 5.38 Correlations between the amount of acetate added and A. Pi release, B. %P content and C PHA concentration for the SBR ' 106 Figure 5.39 Correlation between the amounts of Pi release and PHA stored for the SBR . -. . • 107 Figure 5.40 MLSS concentration at the UBC pilot plant in sides "A" and "B" between January and September 1987 107 Figure 5.41 Average, over the monitoring period, of A. Pi release and uptake and B. PHA storage and consumption in each reactor of the UBC pilot plant 109 Figure 5.42 Relationship and molar ratios for A Pi uptake versus Pi release, B. Pi release versus PHA storage, and C Pi uptake versus PHA consumed for data obtained at the UBC pilot plant 111 Figure 5.43 Pi, nitrate concentration, or PHA content for A. the North side and R the South side of the Kelowna full-scale bio-P treatment plant 114 Figure 5.44 Mass balance for A. Pi and B. PHA data collected at the Kelowna full-scale bio-P treatment plant 116 Figure 5.45 Correlation between Pi and PHA for data obtained at the Kelowna full-scale bio-P treatment plant. 117 Figure 6.1 Biochemical model of bio-P bacteria for A anaerobic and R aerobic (or anoxic) conditions 119 Figure 6.2 Proposed simplified pathway of carbon storage as PHB and PHV 128 Figure 6.3 Proposed model of the effect of 2,4-DNP on bio-P bacteria 132 Figure 6.4 Proposed model of the effect of a high pH on bio-P bacteria 132 Figure 6.5 Molar ratio of PHA storage over Pi release calculated from many experiments with acetate addition 138 Figure 6.6 Absence of a general relationship between PHA storage and Pi release for a variety of A. substrates and B. toxicants 144 Figures 6.7 Rate and maximum rate of anaerobic Pi release 153 Figure 6.8 Rate of aerobic Pi uptake versus PHA content of the sludge 156 Figure A - l Process configuration at the time of sludge sampling for the batch experiments 189 x i ACKNOWLEDGEMENTS I gratefully acknowledge the following persons for their guidance and support during my studies and research at The University of British Columbia: Dr. Kenneth J. Hall, Associate Professor of Civil Engineering and Assistant-Director of the Westwater Research Centre, and Dr. William K. Oldham, Professor and Head of the Civil Engineering Department and coordinator-founder of the bio-P research group, both of whom supervised my work and encouraged me throughout my research, Dr. Robert E.W. Hancock, Professor of Microbiology for enlightening discussions on microbial metabolism, Dr. N. Ross Bulley former Professor of Bio-Resources Engineering at UBC now Head of the Agricultural Engineering Department at the University of Manitoba, and Dr. Richard M.R. Branion, Professor of Chemical Engineering, for serving on my committee and their recommendations, Frederick A. Koch,, research associate, Barry Rabinowitz, former graduate student, and Craig C. Peddie, research engineer, for operating the UBC bio-P pilot plant that supplied the activated sludge biomass for batch experiments and for pilot plant monitoring of carbon storage, Dave G. Wareham, Robert A. Simm, Ashok Gupta, R. Manoharan, B. Neal Carley, Richard D. Bitcon, fellow graduate students, Allan Bronsro and Robert Hicks, summer students, for assistance with the batch experiments; Gerard J.F.M. Vlekke, a Dutch student sent by the International Association for the Exchange of Students for Technical Experience (IAESTE) for experimental work with the sequencing batch reactors using nitrate and air; Gerry M. Stevens, plant superintendant, and Mark L. Watt, laboratory technician for their cooperation with the characterization of the sludge taken at the Kelowna full-scale plant, Dr. William D. Ramey, Instructor of Microbiology, and fellow graduate students Oussama Turk, Patrick F. Coleman, Larry R. Chow, Ramanathan Manoharan, Kirk M. Morrison, Nelson Lee and many others for contributing to a stimulating and pleasant work environment, Paula D. Parkinson and Susan C. Liptak, Environmental Engineering Laboratory Technicians for their invaluable analytical guidance, Timothy J. Ma, gas chromatograph/mass spectrometrist (GC/MS), for the GC/MS work in this thesis, and Guy Kirsch, Civil Engineering Workshop Technician, for his skilled manufacturing of the sequencing batch reactors, Claire Lauze, my spouse, whose encouragement and optimism inspired me throughout my work, and The Natural Sciences and Engineering Research Council of Canada (NSERC) for financial assistance. x i i LIST OF ABBREVIATIONS Anoxic/Oxic process adenosine triphosphate Barnard Denitrification Phosphorus removal process (related to ) biological phosphate removal (e.g. bacteria, process) biochemical oxygen demand chemical oxygen demand 2,4-dinitrophenol dissolved oxygen gas chromatograph(y) GC/mass spectrometer (-metry) D,L-3-hydroxybutyrate sodium salt hydraulic retention time mixed liquor suspended solids mixed liquor volatile suspended solids modified U C T process n number of replications O R P oxidation-reduction potential P phosphorus P i phosphate P H A poly-y9-hydroxyalkanoates P H B "• poly-0-hydroxybutyrate (made of C-4 subunits) P H V poly-^-hydroxyvalerate (made of C-5 subunits) pmf proton motive force polyP polyphosphate r correlation coefficient s standard deviation SBR sequencing batch reactor S C F A short chain fatty acids (volatile and non volatile F A ) S R T sludge retention time SS suspended solids (non-filtrable) T K N total Kjeldahl nitrogen T P total phosphorus U B C University of British Columbia U C T process University of Cape Town (S.A.) process V F A volatile fatty acids VSS volatile suspended solids (non-filtrable) v/v volumetric proportion (volume by volume) Note: in this thesis the terms "anaerobic" will refer to the absence of both free oxygen and oxidized nitrogen (nitrate plus nitrite), "anoxic" to the absence of free oxygen but the presence of oxidized nitrogen, and "aerobic" to the presence of free oxygen (with or without oxidized nitrogen). A / O A T P Bardenpho bio-P B O D C O D 2,4-DNP D . O . G C G C / M S H B H R T M L S S M L V S S M U C T 1 1. INTRODUCTION 1.1. Eutrophication and Phosphorus Removal The eutrophication of lakes and rivers presents a major surface water quality problem. Its cause has been attributed to an excessive supply of nutrients which support the excessive growth of aquatic plants and algae. This growth has detrimental effects to aquatic life and to human use such as municipal (drinking water), recreational (aesthetics), industrial (cooling or process water) and agricultural (irrigation, drinking water for cattle). Carbon, nitrogen and phosphorus are the elements responsible for eutrophication. It was identified that in many cases, nitrogen and phosphorus were the limiting nutrients. When nitrogen limits microbial growth in a water body, blue-green algae that can fix nitrogen from the air, can introduce more nitrogen into the system. Algal blooms have been attributed to the excessive proliferation of floating mats of blue-green algae. Phosphorus, however, was identified as a much better element to remove for eutrophication control because of its only source being from the earth crust and of its limited solubility which make it the limiting nutrient in many natural aquatic systems. Control of phosphorus enrichment in a water body requires a nutrient budget in order to determine important sources and determine the best strategy for nutrient removal. In some instances, the nutrient contribution from non-point sources, such as urban runoff or agricultural land drainage may be so great that the benefits from point sources nutrient removal becomes insignificant. In numerous cases, however, nutrient removal from point sources such as industrial or municipal wastewater has proven beneficial (Vallentyne, 1974; W P C F , 1983). Phosphorus removal from wastewater is referred to as tertiary or advanced treatment, in contrast with primary treatment by sedimentation of coarse solids, and secondary treatment for carbonaceous (BOD) removal. Primary treatment achieves about 5 to 10% phosphorus removal mainly by enmeshment with particulate materials while secondary treatment achieves about 10 to 20% phosphorus removal by enmeshment and biomass growth (WPCF, 1983). The most commonly used methods for phosphorus removal from wastewater rely on chemical precipitation with iron, aluminum or calcium salts (for reviews see U S E P A , 1974; Bowker and Stensel, 1987; W P C F , 1983; Wiechers, 1987). Biological phosphorus removal (bio-P), however, relies on phosphate accumulation in excess of metabolic requirements for growth and is achieved in an activated sludge process modified by the addition of one or more unaerated zones. Operational and cost advantages of bio-P removal over chemical 2 precipitation are attributable to lower (if any) chemical requirements and lower sludge production. A general comparison for Canada was made between chemical and bio-P removal by Canviro (1986) and a specific study of eight plants by Morrison (1988). Morrison concluded that bio-P removal was more cost effective than chemical precipitation especially for new plants and for retrofitting medium to large plants where the cost of chemicals or of sludge management were high. 1.2. Bio-P Removal Bio-P removal can be achieved by modification of the conventional activated sludge process. Essentially, a continuous flow bio-P removal process uses a bioreactor in which the aerobic zone is preceded by an anaerobic zone. With the addition of wastewater to the anaerobic zone, the soluble Pi concentration increases as a result of Pi release by the biomass. In the subsequent aerobic zone, the soluble Pi concentration decreases as a result of Pi uptake by the biomass in excess of metabolic requirements. The "excess" Pi is accumulated intracellular^ as polyphosphates (polyP). The %P (based on dry solids) of non bio-P sludges is normally in the range of 1.5 to 2.0% whereas that of bio-P sludge can range from 3% to 10% in full-scale plants (Pitman et al, 1983; Oldham and Stevens, 1984; Arvin, 1985; Randall et al, 1988). Bio-P removal was reported as early as 1959 (Srinath et al.) and in the late 60's, Barnard was largely responsible for popularizing the Bardenpho process to remove nitrogen and phosphorus (Barnard, 1974). Scarce water supplies in South Africa were also responsible for stimulating much research in that country. After some controversy over the actual mechanism for excess phosphorus removal, it became clear that the removal mechanism was mainly biological with the possibility of some chemical co-precipitation with hard waters (see reviews by Arvin, 1983 and Marais et al, 1983). In the biological mechanism proposed by Comeau et al. (1986), polyP-accumulating microorganisms derive an advantage of storing polyP under aerobic conditions by using that polyP for substrate storage as poly-/3-hydroxybutyrate (PHB) under anaerobic conditions. This stored carbon reserve then provides exclusive access of this PHB to polyP-accumulating microorganisms under aerobic conditions where carbon availability limits microbial growth. From this explanation was derived the beneficial effect of the addition of simple carbon substrates to the anaerobic zone and of removing nitrate and oxygen from any stream feeding into the anaerobic zone in order to favor maximum anaerobic PHB accumulation that, in turn, favors maximum aerobic polyP accumulation. 3 2. L I T E R A T U R E R E V I E W 2.1. Historical Developments and Bio-P Processes 2.1.1 Historical developments For a more complete historical review of bio-P research, the reader is referred to Marais et al. (1983) and Ekama et al. (1984). In the following section, it will be shown that contributions to the understanding of the mechanisms for bio-P removal evolved from a) the recognition of the requirement of an anaerobic zone upstream of the aerobic zone, to b) the exclusion of electron acceptors from the anaerobic zone, and finally to c) the need for simple substrates addition into the anaerobic zone. a) Importance of an anaerobic zone upstream of the aerobic zone Srinath et al. (1959) and Alarcon (1961) were the first researchers to report the occurrence of bio-P removal. Both observed that rapid Pi uptake took place when.sludge samples taken from some types of plants were combined with raw wastewater and aerated. However, they offered no explanation for this phenomenon. Later, Levin and Shapiro.(1965) observed that Pi release took place when sludge from a full-scale plant was kept under anaerobic conditions or aerated for a prolonged period of time. Pi uptake at a faster rate than required for biomass growth was also observed when glucose or succinate was added to the sludge. They also showed that the rate of aeration could limit the rate of Pi uptake. No explanation was offered to explain anaerobic Pi release but the first bio-P process was developed from this research, namely the PhoStrip process. . Until the early 1980's, a controversy existed over the biological versus chemical nature of excess phosphorus removal. With a biological mechanism, Pi would be accumulated as polyP whereas with a chemical mechanism, Pi would be precipitated with calcium and magnesium. The biological nature of excess Pi removal found its first support in the observations of Shapiro et al. (1967) and of Wells (1969) that Pi released anaerobically could be taken up aerobically. The inhibition of aerobic Pi uptake by 2,4-DNP (Levin and Shapiro, 1965; Yall et ah, 1970; Fuhs and Chen, 1975; Rensink et al, 1981) also provided a strong argument for the proponents of a biological mechanism for the phenomenon of enhanced phosphorus removal. Reviews of these opposing concepts were presented by Arvin and Kristensen (1983) and Marais et al. (1983). 4 Empirical design guidelines were proposed by Vacker et al. (1967), Scalf et al. (1969) and Milbury et al. (1971) that provided an important basis for future investigations. It was recognized that the following measures favored efficient bio-P removal: a plug flow regime with the wastewater added at the head end of the process, reversed tapered aeration with sufficient residual D.O. at the downstream end of the process, and avoidance of nitrification. These measures pointed to the importance of cycling the sludge under alternating anaerobic/aerobic conditions, and of the presence of substrates in the anaerobic zone. Fuhs and Chen (1975) were the first to recognize the importance of the anaerobic/aerobic sequence. They observed that when aerobic sludge taken from a full-scale plant was aerated after an anaerobic period that lasted overnight, it contained more polyP than a sludge that had not been aerated. They were also the first to establish the presence of polyP-accumulating Acinetobacter in their sludge. They proposed that Acinetobacter could flourish in such a process because they could grow aerobically on simple carbon compounds produced by fermentative bacteria under anaerobic conditions. They were also.first to report PHB accumulation in a bio-P sludge. Although they could not attribute any specific role to this reserve material, they proposed that stored PHB could have been used for aerobic polyP accumulation. b) Electron acceptors Barnard (1974), in developing the Bardenpho process, proposed that to obtain efficient phosphorus removal, the sludge had to be subjected to an anaerobic state of such intensity that Pi release would occur. Barnard identified that the presence of nitrates in the anaerobic zone had an adverse effect on the efficiency of phosphorus removal. From this recognition, he proposed a modification of the Bardenpho process into the Phoredox process (see section 2.1.2; Barnard, 1976). Experimentation at lab-, pilot- and full-scale later confirmed the. detrimental effect of nitrate addition to the anaerobic zone (Simpkins and McLaren, 1978; Nicholls and Osborn, 1979; Hascoet and Florentz, 1985). The UCT and M U C T process configuration (see section 2.1.2) were also proposed to prevent any nitrate recirculation to the anaerobic zone (Rabinowitz and Marais, 1980; Siebritz et al., 1983). Furthermore, the entrainment of oxygen from air into the anaerobic zone was identified as detrimental, by a rational extension of the effects of nitrates (Paepcke, 1983; Pitman, 1984). To minimize air entrainment into the anaerobic zone, it was proposed to prevent excessive mbdng in the anaerobic zone itself and to reduce turbulence in the incoming streams such as from aerated grit channels, cascades, screw pumps, etc. 5 c) Simple substrates Nicholls and Osborn (1979) proposed a biochemical model involving carbon reserves and polyP. They proposed that under anaerobic "stressing" conditions, simple substrates would be stored as PHB and that, somehow, this mechanism would be linked to Pi release. Under aerobic conditions, PHB degradation would produce energy available for polyP storage.. Following the observation of Fuhs and Chen (1975) that Acinetobacter could metabolize simple substrates but not sugars, they recommended primary sludge digestion as a means of increasing VFA addition to the anaerobic zone. The importance of the availability of simple substrates in the anaerobic zone was recognized by researchers at the University of Cape Town (S.A.) in their mathematical modelling of the activated sludge process. To predict anaerobic Pi release they proposed the use of the "anaerobic capacity", defined as the mass of nitrate entering the unaerated reactor minus the reactor's denitrification potential (Ekama et al, 1979; Siebritz et al, 1983). They, also recognized the importance of maximizing the availability of simple carbon compounds (readily biodegradable COD) in the anaerobic zone to increase the phosphorus removal capacity of the process (Siebritz et al, 1983; Ekama et al, 1983). From this work, Marais et al. (1983) proposed a biochemical model for bio^P removal in which polyP provided energy for PHB storage under anaerobic conditions. This storage of carbon would then favor the selective proliferation of bio-P bacteria capable of sequestering simple carbon substrates as PHB with the assistance of polyP. Thus, the role of the anaerobic zone shifted from one to create "stress" conditions to one in which simple substrate storage would take place. Comeau et al. (1986, 1987b) proposed biochemical mechanisms to explain how anaerobic polyP degradation could be used for the transport and energization of simple substrates such as acetate for PHB storage. A simplified representation of the model is shown in Figures 2.1-A and 2.1-B. Batch experiments also indicated that acetate and propionate were: the "preferred" substrates to stimulate anaerobic Pi release (Potgieter and Evans, 1983; Siebritz al, 1983; Arvin and Kristensen, 1985; Comeau et al, 1987a). To maximize VFA addition to the anaerobic zone, longer anaerobic retention time (to favor fermentation), primary sludge fermentation, and the addition of waste material from certain food and chemical industries were proposed (Pitman, 1984). 6 acetate and available carbon substrates Pi Figure 2.1 Simplified model for anaerobic conditions and B. aerobic or anoxic conditions. 7 2.1.2 Bio-P processes A number of processes that are referred to in the literature on bio-P removal are shown in Figure 2.2. The first process developed for aerobic polyP accumulation was the PhoStrip process (Levin and Shapiro, 1965; Fig. 2.2-A-(i)). In this process, the anaerobic zone, called the "stripper", receives only a fraction of the return sludge (the stripper tank processes 15 to 35% of the total influent flowrate; Levin and Sala, 1987). A fairly long anaerobic retention time of 8 to 12 hours is generally required to obtain sufficient Pi release (WPCF, 1983). A modified PhoStrip process for phosphorus and nitrogen removal was proposed by Matsch and Drevnich (1978); Fig. 2.2-A-(ii)). Ludzack and Ettinger (1962; Fig. 2.2-A-(iii)) in studying nitrogen removal, proposed a process where biodegradable matter from the influent would be used for denitrification. A partial connection between the anoxic and aerobic reactors, however, resulted in variable denitrification performance. The separation of the anoxic from the aerobic zone was termed the Modified Ludzack and Ettinger process by van Haandel et al. (1981; Fig. 2.2-A-(iv)). An essentially similar process is marketed under the name A / O (Anoxic/Oxic) by Air Products and Chemicals Inc. Wuhrmann (1964; Fig. 2.2-A-(v)) proposed the use of endogenous biodegradable material in a post-denitrification reactor. Barnard (1974) proposed a complete separation of the anoxic pre-denitrification reactor and the use of an internal recycle from the aerobic to the anoxic zone to increase the denitrification capacity. A second anoxic zone provided essentially complete denitrification. The final "flash" aeration reactor was used to strip any nitrogen gas bubbles and nitrify any ammonia that could have been released in the anoxic zone. This configuration was called the Bardenpho process (BARnard DENitrification PHOsphorus removal process; Fig. 2.2-A-(vi)) for nitrogen and phosphorus removal. He later found that the exclusion of nitrate from the anaerobic zone improved phosphorus removal efficiency. This development resulted in the Modified Bardenpho or Phoredox process because of the lower redox potential that could be achieved in the anaerobic zone (Barnard, 1976; Fig. 2.2-A-(vii)). On the basis of slow denitrification efficiency, the exclusion of the two downstream reactors from the process configuration resulted in the Modified Phoredox process (Barnard, 1976; Fig. 2.2-B-(i)). An essentially similar process is marketed under the name A 2 / 0 by Air Products and Chemicals Inc. To counteract the detrimental effect of nitrate recirculation to the anaerobic zone, researchers at the University of Cape Town developed the U C T process (Siebritz er al, 1983; Fig. 2.2-B-(ii)). In this process, positive exclusion of nitrate from the anaerobic zone 8 (ii)- PhoStrip for P and N removal (1978) (Mi)- Ludzaek and Ettinger (1962) (v)- Modified Ludzack and Ettinger (A/0) (1975) (vii)- Modified Bardenpho (Phoredox) (1976) Figure 2.2-A Configuration of processes for biological nitrogen and phosphorus removal. 9 (0- Modified Phoredox ( A 9 / 0 ) ( 1 9 7 6 ) (ill)- Modified UCT (1983) (iv)- Biodenipho (1983) T bio-P process (v)- Primary sludge fermentation (19 84) Figure 2.2-B Configuration of processes for biological nitrogen and phosphorus removal. 10 is provided by denitrifying the return sludge prior to recirculation into the anaerobic zone. A further separation of the return sludge from the aerobic sludge denitrification zones resulted in the Modified UCT process (Siebritz et al, 1983 Fig. 2.2.B-(iii)), A comparison of the operational characteristics of the above processes was presented by Ekama et al. (1983, 1984). Concepts presented above have been applied in a variety of ways to achieve bio-P removal. The Biodenipho process, for example, (Fig. 2.2-B-(iv)) makes use of one anaerobic reactor in which the wastewater and the return sludge are combined, and of two similar, intermittently operated reactors. These reactors operate alternatively in an anoxic and aerobic mode for denitrification/nitrification (Bundgaard et al, 1983; Bundgaard, 1988). Oxidation ditches are typically used as aerobic/anoxic reactors. Sequencing batch reactors have also been used successfully for bio-P removal (Irvine, 1985; Manning and Irvine, 1985; Raper et al, 1985; Ketchum et al, 1987; Vlekke et al., 1988). In this case, the sequence of anaerobic/anoxic/aerobic conditions is achieved over time in a given reactor rather than by flowing from one reactor to the next. Fixed film systems for bio-P removal have also been tested with a rotating biological contactor (RBC) (Simm, 1988) that was alternatively submerged and exposed to air, and with a trickling filter serving as the first aeration zone in a Bardenpho type of process (Kelly, 1987). Realizing that simple substrate addition to the anaerobic reactor could improve the efficiency of bio-P removal, primary sludge fermentation to produce volatile fatty acids (VFA) was used successfully (Fig. 2.2-B-(v)). Types of fermenter used include primary clarifiers (Barnard, 1984; Schonberger and Hegemann, 1987; Rabinowitz et al, 1988), unmixed sludge thickeners (Oldham, 1985), and completely mixed fermenters (Rabinowitz and Oldham, 1986). The stream that contains VFA can either be returned to the influent stream (especially if it contains solids from a completely mixed fermenter), to a separate clarifier, or directly to the anaerobic reactor to prevent any reduction of the VFA produced. To achieve VFA production, the use of extended retention time in the anaerobic zone of a bio-P process has also been suggested (Gerber and Winter, 1985). . Design guidelines for a number of bio-P removal processes were presented by Simpkins and Gerber (1981), Barnard (1983), Ekama and Marais (1984), Ekama et al. (1984), Keay (1984), Barnard et al (1985), Krichten et al. (1987) and Bowker and Stensel (1987). ( 11 2.2. Microbial Aspects of Bio-P Removal 2.2.1. Microbial Metabolism a) PolyP metabolism The metabolism of polyP in microorganisms has been reviewed by Harold (1966), Dawes and Senior (1973) and Kulaev and Vagabov (1983). Harold (1966) indicated that two fundamental mechanisms of polyP accumulation were recognized: "luxury uptake" and "overplus accumulation". In the luxury uptake mechanism,'polyP accumulation takes place during the deprivation of another nutrient such as nitrogen or sulfur. Upon addition of the nutrient, growth resumes and polyP reserves are depleted to be used- in the synthesis of nucleic acids. In the overplus accumulation the deprivation of Pi is followed by a sudden exposure of the microorganisms to Pi which causes the microorganisms to rapidly accumulate polyP. PolyP reserves are then slowly degraded. The occurrence of polyP in microorganisms is not uncommon. Dawes and Senior (1973) and Osborn and Nicholls (1978) list more than 20 genera of microorganisms in which the presence of polyP has been reported. The biosynthesis of polyP occurs mainly by the transfer of a Pi from ATP to a growing chain of polyP via the enzyme polyP kinase (see Figure 2.3). The transfer of one Pi group from 1,3-diphosphoglycerate (an intermediate of the glycolysis pathway) to polyP has also been reported (Kulaev, 1975). PolyP degradation usually occurs by simple hydrolysis with the enzyme polyphosphatase and results in the loss of the energy contained in the Pi bond. At low ATP/ADP ratios, however, polyP has been shown to transfer its energy back to ATP in a reversal action of the enzyme polyP kinase (Kornberg, 1957). A number of other pathways have been shown in which the high energy Pi is transferred from AMP to ADP, from N A D + to NADP, from glucose to glucose 6-phosphate, and from 3-phosphoglycerate to 1,3-diphosphoglycerate (Kulaev and Vagabov, 1983). PolyP is found in various locations in the cell, in fractions of different molecular weights, and responds differently to different physiological states of the cell (Kulaev and Vagabov, 1983). PolyP forms a useful reserve of activated Pi that is used, in particular, in the metabolism of nucleic acids and carbohydrates. Harold (1966) suggested that polyP may have been the ancestral form of ATP as an energy carrier for earlier living organisms in the evolution of life. The presence of polyP in bio-P processes'has long been established by staining (with methylene blue or toluidine blue) (Levin and Shapiro, 1965; Fuhs and Chen, 1975; Lotter et al, 1986), electron dispersive X-ray analysis (EDAX) (Buchan, 1983), by 31P-nuclear magnetic resonance (NMR) (Florentz and Granger, 1983; Florentz et al, 1984; Suresh et al, 1985; Halvorson et al, 1986), and by chemical fractionation (Mino et al, 1984). The 12 _ t alkaline _ _ . P esters • Pi phosphatase POLYPHOSPHATES V polypho-sphatase polyP kinase • P i — A T P • nucleic acids membrane Figure 2.3 Polyphosphate metabolism (adapted from Harold, 1966). CH 3-C ~ SCoA ocetyl CoA CH, O C - C H 3 - C ^ S C o A ocetoocetyl CoA CH, .HSCoA NADH synthesis HO-CH-CH 2-C~SCoA P-hydroxybutyryl CoA HSCoA CH, 9 ? H 3 ? H 3 / p 0=C-CH 2-C-0H ocetoocetate NAD + CH, o HO-CH-CH rC-OH fi-hydroxybutyrote (vH 2 0 0 C H 3 degradation HO-CH-CH 2-C-0-CH-CH 2-C-0-CH-CH 2-C / /-0. poly- /I-hydroxybutyrote Figure 2.4 Poly-£-hydroxybutyrate metabolic pathways (adapted from Starrier et al, 1976). 13 high percentage of phosphorus content of the sludge, 3 to 12% compared to a normal 1.5 to 2.0% dry weight content also gives an indirect indication of the amount of polyP in the sludge. Enzymatic studies on an Acinetobacter strain isolated from a bio-P plant have shown that polyP could transfer its energy to AMP for ADP formation via the polyP:AMP phosphotransferase enzyme. Two ADP would then produce ATP plus AMP via the adenylate kinase enzyme. The activity of these enzymes in samples from bio-P plants was later found to be directly correlated to the bio-P removal efficiency of these plants (Groenestijn et al. 1987a; Groenestijn, 1988). b) PHB and PHV A review of PHB metabolism was presented by Dawes and Senior (1973). Poly-y9-hydroxybutyrate (PHB) is a polymer of D(-)-/3-hydroxybutyrate. Its role is more of an energy source than of a carbon source for carbon skeletons, when starvation conditions occur (Dawes and Senior, 1973). Deposits of PHB are readily visible with the light microscope, occurring as dark granules of variable size scattered through the cell. Sudan black is a selective stain for these granules. The major pathways of synthesis and degradation are shown in Figure 2.4. Other pathways of secondary importance are reviewed by Dawes and Senior (1973). The synthesis of PHB involves the condensation of two acetyl CoA and a reduction with N A D H . This synthesis is unique among energy storage compounds in not requiring the direct participation of ATP. Reducing power in the form of N A D H is essential, however, and PHB formation may be regarded as a quasi-fermentation process permitting the reoxidation of N A D H into NAD + . Such a process is particularly useful under conditions of oxygen limitation, which prevent the re-oxidation of N A D H by the electron transport chain, or under conditions of nitrogen starvation which result in intracellular accumulation of NADH, since ATP is not utilized for protein synthesis. Therefore, PHB reserves will accumulate when cells are limited in oxygen or in nitrogen but still have a carbon source available (Dawes and Senior, 1973). PHB can be seen as the procaryotic reserve material equivalent to fat in higher organisms (Stanier et al, 1970). The assimilated carbon stored in cells may accumulate until it represents as much as 50% of the cellular dry weight (Dawes and Senior, 1973). Essentially, PHB synthesis represents a device for carbon accumulation in a form that is "osmotically inert". Indeed, the free carboxyl group of the acid precursors is eliminated through the formation of an ester bond between the subunits of the polymer. The degradation of PHB results in the production of two acetyl CoA and one N A D H . This degradation occurs when the internal concentration of N A D + and free CoA 14 increases while the concentration of acetyl CoA is low. For example, PHB is degraded in the presence of oxygen when the external carbon sources are limited. However, if both oxygen and an external carbon source are present, PHB should not be degraded (Dawes and Senior, 1973). The presence of poly-/?-hydroxyalkanoates (PHA) other than PHB by an activated sludge biomass was first documented by Wallen and Davis (1972), and Wallen and Rohwedder (1974). They found that what was referred to as PHB, was in fact a microbial copolymer consisting of a mixture of C4, C5, C6 and C7 components with the C5 (poly-/3-hydroxyvalerate, PHV) and C4 (PHB) being the predominant forms in a 5:1 ratio (83% of PHA in the form of PHV). Crystallographic analyses of PHA obtained from their activated sludge confirmed the heteropolymer nature of PHA (Marchessault et al, 1984). Wallen and Rohwedder (1974) also mentioned that they could not produce PHA in vitro from species isolated from activated sludge and speculated that precursors formed in a complex biomass might be essential to PHA accumulation. Findlay and White (1983) reported the accumulation of a complex mixture of 11 types of PHA in marine sediments. PHB (30% of PHA), PHV (30%), the C7 (10%) and C8 (14%) were the most abundant PHA fractions. In a pure culture of Bacillus megaterium, they found a mixture of 6 polymers with 95% of the PHA consisting of PHB and PHV. A pathway for the bacterial synthesis of PHV was suggested to be via a condensation of acetyl CoA and propionyl CoA (Holmes, 1981). The presence of PHB in bio-P sludges has been confirmed by staining (Fuhs and Chen, 1975; Lawson and Tonhazy, 1980; Buchan, 1981; Lotter et al, 1986). PHB quantification was also done by a spectrophotometric technique (Somiya et al, 1988; Fukase et al, 1982, 1984) or by gas chromatography (GC) (Deinema et al, 1980; Potgieter and Evans, 1983; Lotter, 1987; Comeau et al, 1986, 1987a, 1987b). Comeau et al (1987a) found that PHB and PHV could be stored as PHA in a bio-P sludge (see Results section). The chemical structure of PHB and PHV is shown in Figure 2.5. C H 3 C H 2 C H 3 _(CH-CH 2 -C-0 -)n _ _(CH-CH 2 -C-0-) n poly-/3-hydroxybutyrate (PHB) poly-0-hydroxyvalerate (PHV) Figure 2.5 Chemical structure of PHB and PHV. 15 c) Glycogen Glycogen is the other major form of carbon reserve for microorganisms (see Dawes and Senior, 1973 for a review, of metabolism). This storage product is made of glucose subunits. Brock et al. (1984) reported that glycogen granules are usually smaller than PHB granules and can only be. seen by an electron microscope. The presence of glycogen, however, can be detected by light microscopy when cells are treated with dilute iodine because of the glycogen-iodine reaction that turns the cells red-brown. The accumulation of glycogen normally occurs when growth is limited by the supply of utilizable nitrogen and that there is an abundant supply of carbon. In general, glycogen synthesis is stimulated by a high adenylate energy charge (similar to a high ATP/ADP ratio) whereas a low adenylate energy charge promotes degradation and inhibits synthesis (Dawes and Senior, 1973). In bio-P processes, the presence of glycogen has been reported when a significant proportion of the added carbon was in the form of glucose (Fukase et al, 1982; Mino et al, 1987; Tsuno et al, 1987; Somiya et al, 1988; Arun et al, 1988; Manoharan; 1988). Nicholls and Osbbrn (1979) reported that no glycogen accumulation could be detected in a fullrscale bio-P treatment plant treating municipal wastewater. d) Bacterial bioenergetics A major aspect Of bacterial bioenergetics is concerned with the maintenance by bacteria of a proton motive force (pmf). The pmf is a chemiosmotic gradient across the bacterial cytoplasmic membrane that can be considered to comprise two distinct components. One component is an electrical potential (expressed as interior negative) arising from a net negative charge on the cytoplasmic side of the cell membrane as compared to the outside. The other component is a pH gradient caused by the higher alkalinity of the bacterial cytoplasm. Thus, the proton concentration outside the cytoplasmic membrane would be greater than the proton concentration in the cytoplasm, creating a driving for protons to come back into the cell. Translocation of protons outside the cell membrane thus increases both components of the pmf (Harold,. 1977; Brock et al, 1984). ; , : - Major roles of the pmf. are in the production of ATP by the membrane-bound ATP-ase enzyme complex,; and for the transport of substrates, as discussed in the next section. Three major mechanisms are used by. most bacteria to translocate protons and maintain a pmf (see Figure 2.6). The first one is of major importance and makes use of the cytoplasmic membrane-bound electron transport chain to expel H + from the cell when carbon substrates and an electron acceptor, mainly oxygen or oxidized nitrogen, is present. This process is part of aerobic or anaerobic respiration; (Note that anaerobic respiration with nitrate occurs under "anoxic" conditions, a term used in engineering to describe the Bacteria E.T.C. NADH- ' tronshydrogenase -ATP-ase (reversible) Figure 2.6 Overview of bacterial bioenergetics (from Comeau etal, 1986). pcptfdogtycM layar (25 nm) pcriplaamtc spac* plasma msmprana paptidoglycaai laysr (3 nm) ouMf sugar-Pi \ g l u U m i n * A T p phospha-ADP pyruvate ' • p y r u v a i glutamliM sugars (glueoaa, IruetoM, mannoaa, laetoaa) (negatively chargatf amine acids) Figure 2.7 Overview of bacterial membrane transport (from Comeau, 1984). 17 presence of oxidized nitrogen but absence of free oxygen. The term "anaerobic" conditions is used in engineering to describe the absence of both oxidized nitrogen and free oxygen.) Substrates can be processed via glycolysis and/or the T C A cycle to produce N A D H which then acts as a donor, for the electron transport chain to result in proton expulsion. In the absence of electron acceptors, this mode of proton expulsion will be inoperative and the accumulation of NADH will inhibit further N A D H production from metabolic pathways. Under such conditions, a second mechanism which consists of ATP breakdown at the ATP-ase site can be used to translocate protons. This process is essentially a reversal of the production of ATP from the pmf. A third mechanism makes use of the enzyme NADH-transhydrogenase to break down N A D H into NAD + in order to translocate H + (Harold, 1977). Bacteria will tend to maintain a fairly constant pmf. If the external pH is decreased, for example, the high H + concentration outside the cell will cause the pH gradient to increase.- To maintain a constant pmf, the charge gradient could be reduced by cation expulsion. Potassium can be used for that purpose since its concentration in cells is relatively high (200 mM in Escherichia coli). Conversely, at a high external pH, cation accumulation by the cell takes place (Bakker and Mangerich, 1981). The various components of the pmf can be neutralized individually or simultaneously by the addition of a toxicant. To neutralize only the pH gradient, acetate or other weak acids can be used (Kaback, 1976). Indeed, such acids form a neutral molecule before diffusing through the membrane. Once in the cell, the acids dissociate because of the relatively high pH in the cytoplasm of the cell (e.g. pH 7.6; Schuldiner and Padan, 1982) and remain in their ionic form, thus, trapped inside. For each molecule of acetate taken up, one H + is transported into the cell, effectively decreasing the pH gradient. To affect both the charge and the pH gradient, 2,4-dinitrophendl (DNP) can be used. .This inhibitor shuttles H + across the membrane such that the gradient of H + is dissipated. H + movement influences both the pH gradient (since pH represents the free proton concentration) and the charge gradient (since protons are charged). Inhibitors like 2,4-DNP are called "uncouplers" since they uncouple ATP formation at the ATP-ase site from respiration (which uses oxygen or oxidized nitrogen at the electron transport chain) by dissipating the pmf. \ e) Bacterial membrane transport The following concepts of membrane transport are summarized in Figure 2.7. Bacteria have a cytoplasmic membrane that acts as a permeability barrier for hydrophilic and charged, molecules. A peptidoglycan layer that surrounds the cytoplasmic membrane confers rigidity and shape to bacteria: In gram-negative bacteria, an additional 18 outer membrane serves as a barrier to large hydrophilic and to hydrophobic molecules (Hancock, 1984). There are three kinds of bacterial membrane transport: passive diffusion, facilitated diffusion and active (energized) transport (Harold, 1977; Brock et al, 1984). Passive diffusion is a transport mechanism by which neutral molecules tend to equilibrate across the membrane. Water, oxygen and carbon dioxide are transported by passive diffusion across the cytoplasmic membrane. In the case of facilitated diffusion, the permeating molecule combines with a membrane carrier and is transported inside the cell along its concentration gradient. Non-specific bacterial porins, which are channels of the outer membrane, are an example of facilitated diffusion. There are three well-recognized categories of active transport: ATP-dependent, group translocation and transport coupled to the pmf. For active transport, a specific carrier is generally required for each solute. The solute can be accumulated such that its internal concentration exceeds its external concentration. In ATP-dependent transport, the hydrolysis of ATP drives the internal accumulation of solutes such as negatively charged amino acids. In group translocation, the solute is modified during its transport (e.g. sugars phosphorylated by phosphoenolpyruvate). In transport coupled to the pmf, cations, anions or neutral molecules can be co-transported with protons or other cations such that the molecule is neutral or carries a net positive charge when it crosses the membrane. For neutral molecules, such as sugars or amino acids, the carrier proteins effectively transfer a positively charged molecule where protons are bound to the carrier for its activation. 2.2.2. Microbiology A summary of bacterial species isolated from bio-P sludges is given in Table 2.1. Predominant genera found in characterized bio-P sludge were Acinetobacter, Pseudomonas and Aeromonas. A number of researchers have proposed that species from the genera Acinetobacter (most importantly) and also Pseudomonas were largely responsible for polyP accumulation while Aeromonas species could be responsible for V F A production by fermentation. In this thesis, the non-restrictive term bio-P bacteria will be used to describe bacteria responsible for polyP accumulation in a bio-P process. Essential and minimum characteristics of bio-P bacteria are that they are aerobes (probably strict aerobes) and capable of both polyP and PHA accumulation. Nitrate was shown to induce Pi uptake from bio-P sludges (Florentz, 1982; Menoret, 1984; Iwema and Meunier, 1985; Comeau et al, 1985 Vlekke et al, 1988), and from Acinetobacter pure cultures (Groenestijn and Deinema, 1985; Lotter , 1985). Thus, at least 19 Table 2.1 Genera of bacteria isolated from bio-P sludges. Genus References Acinetobacter 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 22, 24, 25, 26, 27, 28, 29; Aeromonas 2, 3, 4, 5, 6, 18, 20,21,22, 23, 28; Alcaligenes 4,22; Bacillus 10, 27; Chromobacterium 2; Citrobacter 2, 3, 6, 10, 18, 20; Enterobacter 2, 6, 18; Erwinia 2; Escherichia 2, 3, 4, 6,18; Flavobacterium 2, 4, 6, 18, 20, 23; Klebsiella 2, 3, 6, 18; Micro thrix 1,2,16; Moraxella-Mima 2, 22; Nocardia 2, 16; Neisseria 22; Pasteurella 2, 3, 4, 6, 20 Proteus 2; Pseudomonas 2, 4, 5, 6, 10, 22, 27, 28, 29; Salmonella 2; • Serratia 2; Shigella 6,20 Yersinia 2, 18, 20; Xanthomonas 22; References: (1) Best et al, 1985; (2) Brodisch and Joyner, 1983; (3) Brodisch, 1985; (4) Buchan, 1983; (5) Chow, 1988; (6) Cloete et al, 1985; (7) Deinema et al, 1980; (8) Deinema et al, 1983; (9) Deinema et al, 1985; (10) Florentz and Hartemann, 1984; (11) Fuhs and Chen, 1975; (12) Groenestijn, 1988; (13) Groenestijn and Deinema, 1985; (14) Groenestijn and Deinema, 1987; (15) Hao and Chang, 1987; (16) Hart, 1985; (17) Lawson and Tonhazy, 1980; (18) Le Flohic, 1985; (19) Lotter, 1985; (20) Lotter and Murphy, 1985; (21) Lotter et al, 1986; (22) Meganck, 1987; (23) Meganck et al, 1985; (24) Murphy and Lotter, 1986; (25) Osborn and Nicholls, 1978; (26) Rensink et al, 1981; (27) Suresh et al, 1985; (28) T'Seyen et al, 1985; (29) Wentzell et al, 1988. 20 some of the bio-P bacteria should be capable of nitrate reduction. It should be pointed out that the classification proposed by Bergey's manual (1974;) and Juni (1978), states that. Acinetobacter is not capable of nitrate reduction. Considering that the name Acinetobacter covers a broad range of bacteria, it is conceivable that some Acinetobacter could be capable of nitrate reduction while some others could not. Yeoman et al. (1988) attempted a sludge "bio-enhancement" experiment by the addition of two types oi Acinetobacter species (A. Iwoffi, A. calcoaceticus) and Aeromonas species to lab-scale bio-P process. No increased efficiency in bio-P removal could be observed, however. An interesting insight into the selective proliferation of Acinetobacter in bio-P processes was presented by Wentzell et al. (1988). They developed an "enhanced" bio-P biomass by gradually increasing the proportion of acetate into the feed until it contributed 100% of the carbon added. As much as 500 mg COD/1 of acetate was added at sludge ages of 10 to 20 days, resulting in phosphorus removal capacities of 50 to 60 mg P/l. At one point of their study, however, they observed a gradual loss of phosphorus removal efficiency down to 10 mg P/l. Population studies with the Analytical Profile Index (API) method (Ayerst, 1977) indicated that the proportion oi Acinetobacter had decreased from 90% to 25% (these numbers may not correctly reflect true values because ofthe difficulty in separating individual bacteria from activated sludge floes) and that the proportion of Pseudomonas increased inversely. The commencement of the loss in phosphorus removal efficiency was associated with a process malfunction that could have resulted in the "leakage" of acetate (the sole carbon source used) into the downstream anoxic zone. It was suggested that although Pseudomonas could accumulate polyP, they did not achieve P removal with the same efficiency as efficiently as Acinetobacter. Thus, to favor Acinetobacter proliferation, the leakage of simple organic substrates should be prevented by providing a relatively long anaerobic retention time and that a cascade of two anaerobic reactors would be desirable. A 30% anaerobic mass fraction was recommended for a system fed with a high concentration of acetate as sole carbon source in order to avoid such "leakage". 21 3. RESEARCH OBJECTrVES AND APPROACH The objective of this research was to define the importance and the role of carbon storage in bio-P removal. For that purpose, a sensitive GC technique was developed for PHB quantification. This technique also allowed the quantification of poly-/3-hydroxyvalerate (PHV). Both PHB and PHV will be collectively referred to as poly-/3-hydroxyalkanoates (PHA). Since glycogen is another common carbon storage compound that has been detected in some bio-P plants, some glycogen determinations.were also made on the sludge of the UBC pilot plant. The importance of carbon storage was done by characterizing bio-P: sludge obtained from the UBC pilot plant in a number of batch experiments conducted under a sequence of anaerobic and aerobic conditions. The role of substrates such as acetate, propionate, and of other short chain fatty acids (SCFA) were investigated. One experiment was conducted with radioactive acetate to establish in which fraction the isotope would be metabolized. The "detrimental" effect of nitrate and of nitrite was also characterized by their anaerobic addition with or without other substrates. At lab-scale, four sequencing batch reactors (SBR) were operated in parallel to develop bio-P sludges acclimated to different levels of acetate addition (0, 15, 30 and 45 mg COD/l). These sludges were also characterized in a batch experiment. At pilot and full-scale, PHA monitoring was done on bio-P sludges taken from the UBC pilot plant over a five months period, and from the Kelowna full-scale treatment plant in two consecutive days. At these two plants, primary sludge fermentation was done to enhance the efficiency of bio-P removal resulting in the acclimation of these sludges to V F A addition, mainly in the form of acetate and propionate. Preliminary experiments (Comeau, 1984) had indicated that the metallic cations potassium, magnesium and calcium appeared to be co-transported along with phosphate in and out of bacteria under anaerobic and aerobic conditions. These observations were repeated and the effect of cations addition was also, tested. The response of the pilot plant sludge to toxicants was tested to help postulate biochemical mechanisms for bio-P removal. A table, in the Results section (Table 5.1), gives an overview of all the conditions tested and of the results presented for each batch experiment. From the results obtained, a biochemical model was proposed to rationalize the activity of bio-P bacteria under anaerobic, anoxic and aerobic conditions. Molar ratios were also given to verify possible pathways proposed. Finally, a summary of microbial 22 activity of bio-P bacteria and of other microorganisms present in a bio-P biomass was presented. Carbon storage as PHA was proposed to play a central role in explaining observations made in bio-P plants. 23 4. M A T E R I A L S A N D M E T H O D S 4.1. Experimental Setup 4.1.1. Batch experiments Batch experiments were performed in 2.8 1 Erlenmeyer flasks stirred with magnetic bars (Figure 4.1). At the start of an experiment, each Erlenmeyer flask was filled with mixed liquor by complete immersion in the aerobic reactor of the UBC pilot plant. This was defined as time zero hours. Within half an hour of sampling, each sealed flask was installed on a magnetic stirrer in the laboratory. For most experiments, an initial unaerated period of 3 to 6 hours was provided to ensure complete denitrification of the sludge before the addition of any carbon substrate. This prevented substrate consumption for denitrification. A rubber stopper seal was fitted with a septum through which a syringe needle, connected to a nitrogen or helium filled balloon, was inserted to replace the volume of sampled liquid and to maintain an inert atmosphere above the liquid. This septum was also used for the injection of chemicals. A clamped plastic sampling tube, and, for some reactors, an aluminum tube to which was attached an ORP probe, were also inserted in the stopper. Mixed liquor samples were withdrawn as required, filtered and preserved for the analysis of chemical components. For aeration, the stopper was removed and air was bubbled into solution through a 1.5 mm ID plastic tube. All experiments were conducted at room temperature (20#C % 1#C). Detailed influent, process and effluent characteristics at the time of each batch experiment are given in Appendix A-3. For most experiments, the sludge was completely denitrified prior to the addition of chemicals such as substrates or toxicants. Such complete denitrification was performed to ensure that nitrate (or nitrite) could not influence the effect of the added chemical. A minimum non-aerated denitrification time of 3 hours under completely mixed conditions was used for that purpose. In many batch experiments and SBR runs, an ORP probe was used to confirm the complete disappearance of nitrate (Koch and Oldham, 1985; see section 4.1.2 for a typical plot). ' For batch experiments with pH adjustment, pH values of 5 or 9 were maintained with the frequent addition of 0.5 N solutions of either HC1 or NaOH, after an initial pH adjustment done with 6 N solutions. 24 nitrogen balloon on syringe needle Figure 4.1 Batch experiment apparatus. 25 4.1.2. SBR Sequencing batch reactors (SBR) were chosen for lab-scale continuous operation because of their flexibility of operation and of their reproduction of perfect plug flow conditions which are favorable to bio-P removal and allow a direct comparison with results from batch experiments. An SBR schematic is shown in Figure 4.2. The tall shape of the reactors was chosen to minimize exposure to air during the unaerated periods. Four SBR were operated in parallel to compare the effect of various levels of acetate addition on steady-state operation. The operation of an SBR consisted of a sequence of six basic phases: fill, react I (unaerated), react II (aerated), settle, draw and idle (see Figure 4.3). The total cycle period was 8 hours. During a short unaerated fill phase of 5 minutes, wastewater was pumped from an influent container to the reactor. The react I phase lasted 3 hours and was unaerated. Denitrification was completed during the first 1.5 hour at which time an acetate solution was pumped in for a period of 5 minutes to add 0, 15, 30 and 35 mg of acetate per liter of influent to SBR no. 1, no. 2, no. 3, and no. 4, respectively. The react II aerated phase lasted 4 hours. Mixing and aeration were then stopped for a 50 minute SETTLE phase. Flushing of the air line prevented bubbles from rising through the settling sludge blanket. The last 10 minutes of the cycle were used for DRAWING off the supernatant and for any IDLE phase before the start of the next cycle. Once a day, just before the end of the react II phase, some mixed liquor was wasted to control the sludge retention time of the biomass, taking into account the amount of suspended solids lost by the effluent. Multiple head pumps were used for wastewater, acetate solution and solids wastage pumping. Two four-channel microprocessor timers (Chrontrol Timers from Lindburg Entreprises, San Diego, CA) were used to operate each controllable element. The duration of each phase, as given above, was eventually selected so that complete nitrification would be achieved in the aerated R E A C T II phase, complete denitrification during the first 1.5 hour of the REACT I phase, and complete uptake of acetate during the last 1.5 hour of the REACT I phase. Settling was always very good and a SETTLE phase period of 50 minutes was found sufficient. An oxidation reduction potential (ORP) probe connected to a strip chart recorder was used to determine the actual time of complete denitrification (Koch and Oldham, 1985; Comeau et al., 1987b). A typical ORP plot is shown in Figure 4.4. Domestic wastewater from a UBC family housing complex was used as feed. To ensure consistent characteristics of the wastewater over time, enough wastewater was collected in 24 liter carboys and stored at 4°C, to last for about three weeks (25 carboys were required). The strength of the wastewater, which was 265 mg COD/1 on average, was adjusted to 200 mg COD/1 by dilution with tap water when filling the SBR vessel. To 26 level switch M 5.4 I 12 cm dia Jr plexiglas — 4.8 I J max level 2.4 I min le sampling port pressure regulator B slow-speed mixer to ORP meter C O C O d o f 5.0 I overflow level overflow container solenoid ^ ^ - s valve S ^ effluent to sink pump AIR [>'<] r > r < ] Mdiffuser (!) M Compressed * ^ i AIR supply . . . . . _J for flushing o b daily sludge wastage acetate feed influent wastewater controlled by microprocessor-based timer Figure 4.2 Lab-scale SBR apparatus. Abbreviations: LS, level switch (that can stop the influent pump); M, mixer; P, pump; S, solenoid valve. 27 -REACT I Controllable element FILL Microprocessor circuit 0 (non-aerated) -REACT II • SETTLE & (aerated) DRAW/ IDLE Cycle Time (h) 3 4 5 wastewater pump A1 -(5 min) sludge wastage pump A2 acetate pump A3 act. sludge mixer A4 wastewater mixer B1 air solenoid B2 air flushing solenoid B3 effluent solenoid B4 (5 min) ~ (75 min) (4 min) •(6:55 h) "(4 hours) -(10 sec) _ ( 1 0 min) Figure 4.3 Schematic description of the automated operation of the SBRs by two four-channels microprocessor controllers. SBR Operation (SBR 30 acetate; week 9.5, May 1, 1986) 200 > E OL cc O 160 -120 ~ 80 40 -0 --40 ~ -80 -no Aeration • * anaerobic anoxic T-2 Aeration-aerobic 4 —r 6 S.D.I Cycle time (h) Figure 4.4 Use of ORP to define the time required for complete denitrification in an SBR. 28 ensure that Pi would not limit the extent of polyP accumulation by the biomass, 7 mg P/l (of a neutralized NaHyPO^ and Na2HP04 solution) was added to the influent wastewater. Mixed liquor sampling was done through a port located at about a third of the height of the reactor. Effluent sampling was done just before or during the draw period by the same sampling port (always above the level of the sludge blanket). 4.2. Characteristics of Treatment Plants Studied . Sludge samples for PHA analysis were taken from each cell of the bioreactor at the UBC pilot plant for a period of five months, and at the full-scale Kelowna plant for two consecutive days. Both plant configurations and average operating characteristics are described below. 4.2.1. UBC Pilot plant The process configuration for each parallel module of the pilot plant is shown in Figure 4:5. The influent to the pilot plant consisted of domestic wastewater from a two thousand person residential UBC complex. Every morning, two stirred storage tanks were filled with wastewater. Two parallel streams were studied to characterize the effect of primary sludge fermentation on the efficiency of phosphorus removal. The primary clarifier (500 1) was followed by a bioreactor (2500 1) divided into an anaerobic (l/7th), anoxic (2/7th) and aerobic zone (4/7th of the volume), followed by a secondary clarifier (500 1). The sludge from the primary clarifier was pumped into a 400 1 completely mixed fermenter from which it was returned to the primary clarifier. The actual retention times were 8.9 hours for the primary sludge fermenter, and 1.2, 1.4 and 4.0 hours for the anaerobic, anoxic arid aerobic zones, respectively. The pilot plant operating characteristics are given in Table 4.1. 4.2.2. Kelowna full-scale plant A detailed schematic of the Kelowna full-scale wastewater treatment plant is shown, in Figure 4.6. The configuration of one module of the bioreactor is shown in Figure 4.7-A and an "exploded" view in Figure 4.7-B. The bioreactor configuration is essentially that of a Modified Bardenpho (see Chapter 3) but differs from it by the introduction of a small anoxic zone in the secondary aerobic zone. This was done to minimize aerobic Pi release in the last aerobic section of the plant because of low carbon loading conditions at the time of sampling. The total volume of each bioreactor module was 9450 m 3, giving a total nominal hydraulic retention time of 21.3 hours. 29 UBC Pilot Plant Process Configuration Side "A" Side "B" • 1 mm mm,  * LEGEND ^ anaerobic 22 anoxic robic primary sludge, fermenter anoxic recycle 8 , u d S * wastage Figure 4.5 U B C pilot plant process configuration. *S"»ARMINUTOR MPLjpW MEASUREMENT PRIMARY LIFT PUMPS t4"*ARMINUTOR. [ QUIT  CHAMBERS QUIT TO DISPOSAL PRIMARY FLOW EQUALIZATION ' OA SIN PRMARY CLARIFERS North GRAVITY SLUDGE THICKENER m l ^ J W i ORIT TO > DISPOSAL JDWHTEORATOR DtSSOLVEO AIR FLOTATION THICKENINO UNITS •ARDCMPHO REACTOR South MODULE .MIXED LIOUOR / RECYCLE PUMPS STATIONARY SCREEN-STORAGE VAULT L E O E N D : 0 U l Q M I I C Flow M . t . l SawAQ* p i o » — • — S l w t f f l * p l p « OUTFLOW MEASUREMENT IDUAL MEDIA FILTERS BACKWASH TO ORAVITY SLUOOE THICKENER If SLUDGE RETURN PUMPS rIRRIGATION PUMP STATION .. TO WRKJATKJN *FORCE MAIN SCREW LIFT PUMP LAKE - OUTFALL PIPE "CHLORINE CONTACT •ASIN Figure 4.6 Schematic of the Kelowna bio-P wastewater treatment plant (adapted from Oldham and Stevens, 1984). 30 Table 4.1 Average U B C pilot plant process and sludge characteristics for the period studied (February 9 to July 6). Parameter Side "A" Side "B" Influent (primary effluent) (raw influent) COD (mg/l) 162 205 TP(mgP/l) 3.5 3.5 T K N (mg N/1) 20.5 20.5 Effluent COD (mg/l) 29 32 sol. TP (mg P/l) 0.5 1.9 sol. TKN (mg N/1) 0.9 1.1 N 0 3 " (mgN/1) 5.9 6.5 Process Sludge age (d) 26.3 26.3 MLSS (aerobic; mg/l) 2490 2320 %P (aerobic; %P/SS) 4.0 2.1 max Pi anaerobic (mg P/l) 11.3 4.9 Table 4.2 Average Kelowna full-scale plant process and sludge characteristics for July 15 and 16,1987. Parameter North module South module Influent COD (mg/l) 203 203 TP (mg P/l) 3.85 3.85 T K N (mgN/1) 18.5 18.5 Effluent (of secondary clarifier) BOD (mg/l) < 5 < 5 sol. Pi (mg P/l) 0.9 0.1 sol. N H 4 + (mg N/1) 0.6 1.0 . N 0 3 " (mgN/1) 0.35 0.35 Process Sludge age (d) 15 15 MLSS (aerobic; mg/l) 1230 1390 %P (aerobic; %P/SS) 4.62 4.3 max Pi anaerobic (mg P/l) 10.5 10.3 31 Kelowna full-scale treatment plant - July 15 & 16, 1987 B to -clarifier primary •ffluent North (South) row 1 (6) row 2 (5) row 3 (4) thickener supernatant 0.08 Q 1.0 Q primary •ffluent 5.0 Q [60% (40% aerobic recycle LEGEND anaerobic anoxic aerobic secondary clarifier return sludge Figure 4.7 Process configuration of the Kelowna full-scale bio-P treatment plant: A. modules configuration and IL linear configuration of a module. 32 A difference in the operation of the two sides at the time of the experiment was that some alum (100 kg/day) was added to the effluent from the last cell of side A (cell Al) to reduce the effluent Pi concentration. This chemical addition considerably reduced foaming in the A side. Operating characteristics at the time of sampling are summarized in Table 4.2. 4.3. Analytical Methods 4.3.1. Standard analytical techniques The analysis of soluble components was performed on samples filtered through 0.45 nm membrane filters or Whatman 4 filters. Sample preservation, when needed, and the analyses listed in Table 4.3 were conducted according to "Standard Methods" (A.P.H.A. et al., 1985). Total Kjeldahl nitrogen (TKN) and total phosphorus (TP) analyses were done on samples digested by heating in the presence of sulfuric acid, K 2 S 0 4 and HgS0 4 for two and a half hours on a block digester (Technicon BD40). The residue was then cooled and •analyzed by automated methods for ammonia (Technicon Method No. 325-74W), and for Pi (Technicon Method No. 327-74W). Oxidation-reduction potential (ORP) was monitored with a combined platinum (or gold) and reference (Ag/AgCl) electrode obtained from Broadley-James. Instruments used were: for metals analyses, a Jarrell Ash Model No. 810 or a Thermo Jarrell Ash Model Video 22 aa/ae atomic absorption spectrophotometer; for phosphorus and nitrogen automated analyses, a Technicon AutoAnalyzer II; for D.O., a YSI Model 54A Oxygen Meter; and for pH, a Beckman 44pH, or a Fisher Accumet model 210 or 320. 4.3.2. Radioactivity One experiment was conducted with uniformly labelled 14C-acetate (added with 50 mg COD/1 of unlabelled acetate; see section 5.2.1.e.i). Sub-samples taken from a reactor were filtered on a 0.45 membrane filter (Sartorius) to separate the soluble and particulate fractions. The C 0 2 fraction was trapped by acidifying (0.2 ml of 5 N H 2 S0 4 ) a 10 ml sub-sample placed in a 25 ml Erlenmeyer flask fitted with a small cup holding an absorbent paper wetted with a 0.2 ml phenethylamine solution. All fractions were dissolved in a PCS scintillation solution (Amersham-Searle) and counted on a Nuclear Chicago Isocap scintillation counter using an external standard for quench correction (Hall et al, 1972, for method; A.P.H.A. et al, 1985, for standard addition technique). Reported 33 Table 4.3 Standard analytical techniques. Parameter Analysis Reference No. MLSS total'filtrable residue at 104°C 209 C MLVSS total volatile and fixed residue at 550 C 209 E sludge volume index (SVI) SVI 213 C magnesium, potassium, sodium1 L atomic absorption in 303 A air-acetylene flame calcium1 A A . in nitrous oxide-acetylene flame 303 C ammonia automated phenate method 417 F nitrate automated cadmium reduction method 418.F nitrite automated method (no reduction) 418 F dissolved oxygen membrane electrode method 421 F pH value PH 423-soluble phosphorus (Pi) automated ascorbic acid 424 G reduction method chemical oxygen demand (COD) dichromate reflux 508 A *a lanthanum solution was added such that 0.1% of Lsr+ served as a radiation buffer for all metal solutions. 34 soluble fraction results were corrected by subtracting the radioactive counts of C 0 2 from the counts of the soluble fraction. 4.3.3. VFA Acetate, propionate, butyrate and valerate were analyzed on aqueous acidified (with phosphoric acid) samples by gas chromatography on a Hewlett Packard Model 5880A, using a Hewlett Packard 7672A programmable auto-sampler. The glass column was 0.91 m long, 4 mm internal diameter, packed with 60/80 Carbopack C coated with 0.3% Carbowax/0.1% H 3 P 0 4 (Supelco, 1982). Lactate was analyzed from 1 ml filtered samples to which 250 pi of 50% H 2 S 0 4 and 2.0 ml of methanol had been added. After 24 hours of incubation at 25 °C, 1 ml of chloroform was added for extraction of the methylated lactate (Grenier, 1986). A 10 (A aliquot of the chloroform phase was injected into the G C (HP 5880A) under the same conditions as used for P H A analysis (see section 4.3.5). Formate was assayed by a colorimetric method in which 50 (A of the enzyme formate dehydrogenase (8 units /ml) was added to 100 /A of sample in the presence of 100 (A of 0.02 •M nicotinamide adenine dinucleotide ( N A D ) and 250 pi of 0.5 M Pi buffer at a p H of .7.0. After six hours of incubation at 37°C, absorbance was measured at 340 nm on a Spectronics 88 photometer (Grenier, 1986). Technical problems encountered with the formate assay could have been due to partially inactivated enzymes. 4.3.4. Total carbohydrates and glycogen The procedure for the analysis of total carbohydrates consisted in the preparation of diluted glucose standards and samples such that they contained 10 to 100 ng glucose/ml, the addition of 5.0 ml of cold anthrone reagent (prepared daily with 200 mg of anthrone to 5 ml of absolute ethanol, made up to 100 ml with 75%, vol/vol, H 2 S 0 4 ) , followed by heating of the samples in a boiling water bath for precisely 10 min, and the cooling of the samples in an ice-water bath. Absorbance, resulting from the reaction of anthrone with dehydrated monosaccharides, was measured at 625 nm (Gerhardt, 1981). Glycogen, like other polysaccharides, is resistant to hydrolysis by alkali, but is readily soluble in water and insoluble in ethanol. These properties are used for the isolation of glycogen from most other carbohydrates. Sub-samples (100 mg) of centrifuged, o washed and lyophilized sludge pellets were incubated at 100 C with 1 ml pf 30% K O H for 3 hours. When cool, 3 ml of distilled water and 8 ml of ethanol were added to precipitate the glycogen. After centrifugation, the precipitate was washed with 8 ml of 60% ice-cold ethanol, dried in a vacuum dessicator, and analyzed for glucose by the anthrone method (Gerhardt, 1981). . 35 4.3.5. P H A An improved method for the extraction and quantification of PHA was devised as an adaptation of the method of Braunegg et al. (1973) and reported by Comeau et al. (1988). a) PHA extraction and quantification The principle of the method for PHA extraction consists in the depolymerization of PHA by sulfuric acid, conversion into volatile methyl esters with methanol, and recovery in chloroform for injection into a GC. For PHA analysis, activated sludge samples (10 to 15 ml) were centrifuged, and the sludge pellet frozen and lyophilized (lyophilizers used were a Virtis 10-234, or a Multi-Dry by FTS Systems Inc.). A weighed amount (about 20 mg) of lyophilized sludge was combined with 2 ml of acidified methanol (3% H 2 S0 4 ) , containing benzoic acid as the internal standard, and 2 ml of chloroform. D-L, 3-hydroxybutyric (HB) acid sodium salt dissolved in acidified methanol, was used as a standard. Samples and standards were heated for 3.5 hours at 100°C in Pyrex test tubes (volume of 15 ml) with Teflon-lined cap. Further purification was achieved by transferring 1.9 ml ofthe denser chloroform phase to another Pyrex tube (volume of 10 ml) containing 0.5 ml of distilled water. This re-extraction step greatly improved the reliability of the method by removing acids and particulate debris which caused inaccuracies and premature degradation of the G C column. After 5 min of vigorous shaking and centrifugation (1,500 x g for 3 min), the chloroform phase containing the PHA methyl esters was transferred to a G C vial for split injection of a 1-^ 1 sample into a Hewlett-Packard 5880A GC. The G C was equipped with a programmable autosampler (Hewlett-Packard 7672A) and a megabore J&W Scientific Co. capillary column (length, 15 m; inside diameter, 0.52 mm; coated with 1.0 /mi of DB-Wax). At 200°C the linear velocity was 20 to 30 cm/s with helium as the carrier gas. The temperature of the injection port was set at 210°C and of the flame ionization detector port at 220°C. The following temperature profile was used: 1 minute at 50°C, followed by a temperature increase rate of 8°C/min, of 5 minutes at 160°C, a post-run of 4 minutes at 200°C, and an equilibrium time of 3 minutes at 50°C. This temperature program provided the essential separation of /3-hydroxyvalerate from its close neighbor 4-oxo-valerate (see section 5.1, and Comeau et al, 1988). No standards were available to directly calibrate the H V response from the GC. Thus, it was assumed that the relative response factors for the methyl esters of hydroxybutyric and hydroxyvaleric acid would be similar to the relative response factors for the methyl esters of butyric and valeric acid (Willard et al, 191 A). Thus, valerate and butyrate standards were extracted and injected on the GC. The chromatograph, flame ionization detector, and operating conditions were the same as for PHA, but a lower 36 polarity J&W Scientific Co. capillary column (30 m long, 0.32 mm ID, coated with 1 pm DB-5) was used. The observed ratio of response factors for valeric acid to butyric acid was calculated as 1.211 ± 0.029 (n = 14). Thus, the relative amount of PHV was estimated from a calibration curve of HB standards and divided by .1.21 to give the corresponding amount of PHV. In addition, correction factors were applied to PHB and PHV amounts calculated as a function of the weight of lyophilized sludge extracted: PHB correction factor = 1.000 + 0.00361 x lyophilized weight; PHV correction factor = 1.000 + 0.00780 x lyophilized weight (see section 5.1). The analysis of a batch of 80 samples, excluding lyophilization time, required approximately 5.5 h of direct attention and 9 hours to complete. b) GC/MS Gas chromatography/mass spectrometry (GC/MS) was used to identify the components that were separated by gas liquid chromatography of the PHA extract. The same column and similar operating conditions as for PHA separation were used. The instrument, a Hewlett Packard Model 5985B, was autotuned with perfluorotributylamine (PFTBA) and operated at a 70 MeV electron impact ionization energy. Compound match was established by probability base peak search with the U.S. EPA/NIH library file (U.S. EPA/NIH, 1978). 4.4 Statistical Techniques Average, standard deviation, coefficient of variation, and linear regression were calculated by the software program Symphony (release 1.2) of Lotus Development Corporation (Cambridge, MA). Analysis of variance and curvilinear regression was performed with the software program Statpak (release 4.1) of Northwest Analytical, Inc., Portland, OR. These methods are explained in Walpole (1982). 37 .5. RESULTS In this section, data obtained to validate the P H A method is first presented. Then, results from a number of batch experiments done under anaerobic/aerobic conditions with acetate, other substrates and toxicants are given. Finally, P H A storage results obtained from continuous bio-P lab-, pilot-and full-scale plants are presented. 5.1. PHA Determination in Activated Sludge 5.1.1. PHA quantification A typical G C chromatogram obtained from a sludge sample is shown in Figure 5.1 The identity of the compounds was confirmed by G C / M S . No standards were available to directly calibrate the H V response from the G C . Thus, it was assumed that the relative response factors for the methyl esters of hydroxybutyric and hydroxyvaleric acid would be similar to the relative response factors for the methyl esters of butyric and valeric acid (Willard et al, 1974). Since valerate contains more carbon atoms per mass of compound than butyrate, the response factor on the G C flame ionization detector was expected to be higher for the same mass (Willard et al, 1974). Valerate and butyrate standards were extracted such that triplicate samples of 46, 92, 185, 369 and 738 ng of valeric acid, and 26, 52, 104, 209 and 418 ng of butyric acid were injected in the G C . The observed ratio of response factors for valeric acid to butyric acid was calculated as 1.211 ± 0.029 (n = 14). 5.1.2. Reproducibility, recovery and sensitivity Reproducibility with standards was estimated with six replicates each of 50, 100 and 200 ng H B / m l . Reproducibility with sludge samples was tested by extracting triplicates of 5, 15, 25, 35, 45 and 55 mg of one large lyophilized sludge sample. The coefficients of variation were calculated as 2.0% and 3.0%, respectively. When samples were spiked with H B standards and compared to unspiked samples, the recovery gave 99.4 ± 2.4% for lyophilized samples weighing 5, 25 and 45 mg (n = 3 for each). To test the effect of the weight of lyophilized sludge on the extraction efficiency, replicate sub-samples ranging from 5 to 55 mg (n = 3 for 15, 35, 55 mg, and n = 6 for 5, 25, 45 mg) were used. In this instance, however, it was found that the recovery decreased as larger amounts of sludge were extracted. Based on these results, correction factors were calculated for P H B and P H V such that extrapolation to a weight of 0.00 mg gave a factor of 1.000 (Figure 5.2). The following equations were obtained: P H B correction factor = 1.000 + 0.00361 x lyophilized weight, and P H V correction factor = 1.000 + 0.00780 x lyophilized 38 1 234 CD CO c o Q. CO © IX CD > CD cn 0 5 10 15 Retention Time (min) Figure 5.1 Chromatogram of methyl esters derivatives extracted from a bio-P activated sludge sample. Components identified by GC/mass spectrometry were methyl esters of /9-hydroxybutyric acid (peak 1), 0-hydroxyvaleric acid (peak 2), 4-oxo-valeric acid (peak 3), and benzoic acid (peak 4) which was used as an internal standard. Peaks a and b were not identified. 39 Figure 5.2 Correction factors for PHB and IL PHV as a function of the weight of lyophilized sludge extracted. Average and error bars for the standard deviation are shown. 40 weight. The correlation coefficients (/-) obtained were 0.95 and 0.98 for PHB and PHV, respectively. For example, the mass of PHA measured in a 10 mg sample of lyophilized sludge should be increased by 3.6% for PHB and 7.8% for PHV. The fact that the extraction efficiency decreased as the weight of sludge extracted increased might have been due to competing interactions with extraneous chemicals present in the samples. The difference in slopes between the two compounds, however, suggested a lower extraction efficiency of the solvent system for PHV than for PHB, as if PHV had a higher affinity for particulate materials found in activated sludge. With HB standards, the detection limit was estimated at IO"5 g of HB per liter. This limit is comparable to that of the method of Braunegg et al. (1973). However, we observed that when activated sludge was extracted without lyophilization, the sensitivity of the test was found to be reduced by one order of magnitude. Water has been reported to reduce the extraction efficiency of PHA from aqueous samples (Odham et al, 1986) explaining why sample lyophilization allowed an improved standards recovery. The weight of lyophilized sludge correlated directly to the weight of suspended solids in a ratio of 1.000 ± 0.019 mg lyophilized weight per mg of suspended solids (n = 9). .5.2. Laboratory Batch Experiments Batch experiments were used to characterize the sludge at the moment it was taken from a given treatment plant. In' most cases, sludge was taken from side "A" (with the primary sludge fermenter) of the UBC pilot plant but, in other cases, from side "B" (without a primary sludge fermenter), a continuous lab-scale unit or the SBR. Process configuration and sludge characterization data are given for each batch experiment in Appendix 3. Experiments reported in this section are grouped by type of chemical added and chronologically for a type of chemical. Table 5.1 presents a summary of the conditions tested and data reported for each batch experiment presented below. Unless specified otherwise, the sludge was denitrified for 3 to 6 hours of non-aerated conditions, prior to chemical addition under anaerobic conditions. This was done to avoid a reduction in the amount of substrate added as a result of consumption for denitrification. In a number of experiments, ORP monitoring was used to establish the time of denitrification. A minimum period of 3 hours of anaerobic conditions was then used to give sufficient time for the the added chemical to react. In many experiments, a subsequent aerobic period of at least 3 hours was also used. Molar ratios and rates of reaction obtained from the experiments presented in this section will be compared in the Discussion. Table 5.1-A Overview of batch experiments (B.E.). Section (and title) Conditions tested Data reported 5.2.1 ANAEROBIC addition of acetate a) PHB and PHV storage i. B.E. of October 23,1985 ii. B.E. of August 19, 1986 b) Reproducibility i. B.E. of February 24, 1987 c) Excess acetate i. B.E. of April 24, 1984 ii. B.E. of May 25, 1986 25, 50 or 75 mg COD/1 to sludge and wastewater (75/25 v/v) 31 mg COD/1 (in duplicates) 30 mg COD/1 (four replicates) 25, 50, 75 or 100 mg COD/1 normal 15, 30 or 45 mg COD/1 to SBR sludges excess 200 mg COD/1 to SBR sludges d) Metallic cations (added with 10 mg P/l) i. B.E. of June 4, 1987 30 mg COD/1 plus Na, K, Mg or Ca e) Radioactive acetate i. B.E. of November 4, 1985 50 mg COD/1 with 14C-acetate to sludge and wastewater (75/25 v/v) - Pi, Ac, PHB & PHV cone, vs time - Summary Table1 - Pi, nitrate, PHB & PHV cone, vs time - Pi, acetate & pH vs time; - Net Pi rel. vs Ac cons. - Aeration time for minimum Pi cone vs Ac cons. - Pi, PHB & PHV cone, vs time - Pi, acetate, PHB & PHV cone, vs time - Pi rel./PHA stor. vs SBR number - Pi, PHB & PHV cone, vs time - Pi, PHB & PHV cone, vs time - % of label detected vs time Abbreviations: acet.: acetate; B.E.: batch experiment; cone: concentration; cons.: consumed; prop.: propionate; rel.: released; SCFAs: short chain fatty acids; Nummary Table includes Anaerobic Pi release, PHA storage and %(PHA stored as PHV), and Aerobic Pi uptake and PHA consumed; Table 5.1-B Overview of batch experiments (B.E.). Section (and title) Conditions tested Data reported 5.2.2 AEROBIC addition of acetate and/or propionate i. B.E.of July 8, 1987 20, 40, 60 or 100 mg COD/1 (acet) ii. B.E. of August 6, 1987 20/20 acet/prop, 40 acet or 40 prop mg COD/1 5.2.3 Propionate with or without acetate (anaerobic addition) i. B.E. of August 19, 1986 0.55 mM of acet,.prop or acet/prop ii. B.E. of March 3, 1987 30 acet, 20/10,10/20 acet/prop or 30 prop mg COD/1 Pi, acetate, PHB & PHV cone, vs time Pi vs time ( 10 days) Pi, PHB & PHV cone, vs time iii. B.E. of July 8, 1987 20/20 acet/prop, 20 or 40 prop mg COD/1 Summary Table Pi, PHB & PHV cone, vs time Net PHA stor vs Substrate uptake Pi,acet/prop,PHB & PHV cone vs time 5.2.4 Other substrates (anaerobic addition) a) VFA and fermented primary sludge i. B.E. of August 19, 1986 0.55 mM of formate, acet, prop, butyrate, iso-butyrate, valerate, iso-valerate or hexanoate ii. B.E. of March 3, 1987 30 mg COD/1 of valerate, formate, fermented primary sludge, lactate or butyrate b) Other substrates i. B.E. of August 19, 1986 0.55 mM of acetate, lactate, glycine, citrate, ethanol or glucose ii. B.E. of October 28, 1986 0.5 mM of acet, B-hydroxybutyrate or succinate - Summary Table - Summary Table - Pi, SCFAs, PHB & PHV cone, vs time - Summary Table 5.2.5 Wastewater characterization by its ability to induce PHA storage: Penticton i. B.E. of June 25, 1987 anaerobic conditions for 4 wastewaters 5.2.6 Metallic cations i.B.E.of April 24, 1984 25, 50, 75 or 100 mg COD/1 of acetate Pi, PHB & PHV cone, vs time - K + , M g 2 + , C a 2 + & N a + cone, vs time - K + , M g 2 + , C a 2 + cone, vs Pi cone. Table 5.1-C Overview of batch experiments (B.E.). Section (and title) Conditions tested Data reported 5.2.7 Nitrate and nitrite i. B.E. of November 13, 1984 3, 6, 9,12 mg/1 (plus 50 mg COD/1 of acetate) ii. B.E. of March 11, 1987 0 2 (with air) or 10 mg N/1 of N 0 3 or N 0 2 5.2.8 Toxicants (all added anaerobically) a)2,4-DNP 0.5 mM 2,4-DNP 1.0 mM 2,4-DNP (& 31 mg COD/I of acet) 0.5 or 5.0 mM 2,4-DNP (with or without acet) 0.1, 1.0 or 10 mM 2,4-DNP (& 30 mg COD/1 of acet) (at neutral pH) i. B.E. of December 14, 1984 ii. B.E. of August 19, 1986 iii. B.E. of November 11, 1986 iv. B.E. of March 11, 1987 b) pH i. B.E. of June 26, 1984 ii. B.E. of December 14, 1984 iii. B.E. of March 11, 1987 c) Cyanide, fluoride i. B.E. of August 19, 1986 d) C0 2 , H 2 S i. B.E. of August 22, 1985 pH 8.6 with 25, 50, 75, 100 mg COD/1 of acet pH4or9 pH 5 or 9 (& also 30 mg COD/1 of acet) 0.55 mM of cyanide or fluoride C 0 2 or H 2S gas bubbling Pi, nitrate, acetate cone, vs time Total Pi release vs Nitrate added Pi,N0 3 /N0 2 ,PHB &PHV cone vs time - Pi cone, vs time - Summary Table - Summary Table - Pi, PHB, PHV pH vs time - K + , M g 2 + , C a 2 + & Na+conc. vs time - K + , M g 2 + , C a 2 + & Na + conc. vs Pi cone - Pi & acet cone, vs time - Pi cone, vs time - Pi, PHB, PHV and pH vs time - K + , M g 2 + , C a 2 + & Na+conc. vs time - K + , M g 2 + , C a 2 + & Na+conc. vs Pi cone - Summary Table - Pi cone, vs time 44 5.2.1. ANAEROBIC addition of acetate A series of experiments was conducted to characterize the effects of anaerobic acetate addition on PHB and PHV storage, on the reproducibility of the results, on the effect of excess acetate addition, on the effect of metallic cations, and on the possible pathways of acetate utilization using radioactive acetate. a) PHB and PHV storage i. Batch experiment of October 23, 1985 The objective of this experiment was to quantify the storage of PHB and PHV by bio-P sludge as a result pf acetate addition. Four reactors were used to which pilot plant aerobic mixed liquor (75% by volume) was combined with wastewater (25% by volume). At time six hours 0, 25, 50, or 75 mg COD/1 of acetate was added. Three hours later, air was turned on. The concentration over time of soluble phosphate (Pi), acetate, PHB and PHV are shown in Figure 5.3 The anaerobic addition of increasing amounts of acetate resulted in correspondingly larger amounts of Pi release and of PHB/PHV storage. Under subsequent aerobic conditions, the sludge that contained more PHA took up more Pi. These basic observations, that were repeatedly made subsequently, supported the concept of polyP serving as an energy source for bio-P bacteria under anaerobic conditions for acetate storage as PHA. Under aerobic conditions, PHA consumption would serve for Pi uptake and, presumably, polyP storage (as documented by others; see Literature Review). In the Control reactor (zero acetate addition), Pi release and PHA storage took place mostly in the form of PHV (76% versus 20% for the reactors with added acetate). If we suppose that PHV formation requires the combination of acetate and propionate molecules (see Literature Review), the composition of the added wastewater (25% v/v) and the rate of fermentation must have been such that it favored PHV over PHB formation. The molar ratio of Pi release/PHA stored was 7.0 moles P/mole PHA for the Control but 2.5 moles P/mole PHA for all reactors to which acetate was added. A more complete discussion and comparison of molar ratios obtained from different experiments, will be made in the next chapter. ii. Batch experiment of August 19, 1986 The objective of this experiment was to determine the effect of the addition of 0.55 mmole/1 of various chemicals on anaerobic Pi release and PHA storage, and on aerobic Pi uptake and PHA consumption. The following chemicals were added: formate, acetate &/or propionate, butyrate, iso-butyrate, valerate, iso-valerate, hexanoate, glycine, ethanol; 45 Batch •xpariment of October 23, 1985 0 -I 1 . 1 > , , , —I 4 6 8 10 12 Tim* (h) Figure 5.3 Effects of anaerobic acetate addition to denitrified bio-P sludge and of aeration (Ai Pi, IL acetate, £ . PHB and D. PHV concentrations). Various amounts of acetate were added at time 6 h to a 75/25 (vol/vol) mixture of aerobic sludge and wastewater. 46 lactate, citrate and glucose. Both the acid and sodium salt form of acetate, propionate and butyrate were added to different reactors. The results of this batch experiment will be presented in subsequent appropriate sections. At time 0 hour, 6 liters of aerobic mixed liquor were taken from the pilot plant. This volume was subdivided into 23 small Erlenmeyer flasks (125 ml). At time 6 hours, 22 chemical solutions (neutralized to pH 7.0 with NaOH or HQ) were added to different flasks, one of them serving as Control. Three hours later, air was bubbled into each of the flasks except for two that received either a nitrate or nitrite solution. The aeration period lasted 3 hours. Samples were taken in duplicates from each reactor at the end of the anaerobic period (time 7 h) and at the end of the aerobic period (time 10 h). The Control reactor was sampled just before chemical addition. The pH values measured were very similar for all reactors and started at an average pH value of 7.8 just before chemical addition, decreased to an average value of 7.4 at the end of the anaerobic period, and increased to an average value of 8.4 at the end ofthe aerobic period. Some exceptions will be pointed out where appropriate. Aerobic C 0 2 stripping can probably explain the aerobic pH increase. The effects of the addition of 0.55 mmole/1 of acetate (35 mg COD/l) on Pi and PHA are summarized in Table 5.2. No difference was obtained between acetate prepared from the sodium salt or the acid neutralized with NaOH. Other observations regarding the magnitude of Pi release or uptake and PHA storage or consumption are very similar to those reported in the previous experiment. A more detailed comparison of all the results from this chapter will be presented in the Discussion. Table 5.2 Effects of 0.55 mM (31 mg COD/1) acetate addition on anaerobic Pi release and PHA storage, and aerobic Pi uptake and PHA consumption. Batch experiment of August 19, 1986. Substrate Pi rel . Pi upt PHA sto %PHVsto/ PHA cons PHA sto (mg/0 . (mg/1) (mgHB/1) (%) (mgHB/1) Control 3.8 2.1 6.5 76 5.4 NaAc 27.6 15.0 52.5 35 36.1 HAc 24.8 16.5 51.3 33 37.4 Abbreviations (common to all subsequent Tables of this Chapter; see also List of Abbreviations): NaAc: sodium acetate; cons: consumed; rel: released; sto: stored; upt: taken up. 47 b) Reproducibility i. Batch experiment of February 24, 1987 Reproducibility of results was assessed with eight batch reactors. Four were used as Control and four received 30 mg COD/1 of acetate during the anaerobic period. The concentration profiles versus time of Pi, N0 3", PHB and PHV are shown in Figure 5.4. Average and standard deviation are shown for each point. In the Control reactor, as was reported in the previous experiment, Pi release was associated only with PHB and not with PHV storage. Subsequent aerobic Pi release and PHV consumption were observed. c) Excess acetate i. Batch experiment of April 24, 1984 The objective of this experiment was to correlate anaerobic Pi release and acetate uptake. For that purpose, various amounts of acetate were added. To ensure that aerobic Pi accumulation would not be limited by soluble Pi availability, 50 mg P/l of neutralized sodium Pi solution was added just before aeration. The pH of the Pi solution was adjusted to 6.8. Aeration was started 6.7 hours after the start of the experiment. The following parameters were monitored: Pi, acetate, pH (Figure 5.5), potassium, magnesium, calcium and sodium (see Figure 5.23 further). PHB and PHV were not monitored in this experiment. As observed before, the addition of larger amounts of acetate resulted in greater Pi release from the biomass during the anaerobic period. From the reactors to which 75 or 100 mg COD/1 of acetate were added, however, a maximum level of Pi release was reached and only 66 mg HAc/1 (71 mg COD/1) of acetate was accumulated by the biomass, and the excess acetate remained in solution. These observations suggested that a depletion of the intracellular polyP reserves (or that fraction of polyP available as energy for acetate uptake and storage) had limited further acetate uptake. A plot of net Pi release (subtracting the corresponding Control Pi concentration) versus acetate taken up gave a linear relationship with a slope of 0.77 mg P/mg HAc which corresponds to a molar ratio of 1.49 mole P/mole HAc (Figure 5.6). The significance of these molar ratios will be elaborated in the Discussion. As soon as air was turned on, residual acetate consumption and Pi uptake were observed. A slow Pi release resumed in the Control reactor about 1 hour after the start of aeration. This time was 2.1 hours for the reactor with 25 mg COD/1 of acetate, 3.1 hours for the one with 50 mg COD/1, and 4.4 hours for the 75 and 100 mg COD/1 reactors 48 Batch • xperimant of February 24, 19B7 o. E Z o> E a x o> E 10 X 0. a x Ol E > x 30 20 10 2 -0 40 30 20 10 0 15 10 Logand • control A 30 mg C O D / I of acatati l r-5 7 Tim» (h) 11 Figure 5.4 Assessment of experimental reproducibility for anaerobic acetate addition (A. Pi, B. nitrate, £. PHB and B^  PHV concentrations). At time 4.65 h, 30 mg COD/l of acetate was added to 4 reactors; 4 others served as Control. Average and bars for standard deviation are shown. 49 Batch »xp»rlm»nt o» April 24, 1*64 a. a E a o u 100 -100 ao eo 40 20 Logond • Control • 25 mg COD/I * 50 mg COD/I * 78 mg COO/I « 100 mg COD/I 7.6 7.0 6.6 H 1 1 1 1 1 1 1 1 I 3 6 7 8 11 Tbno(h) Figure 5.5 Limitation of anaerobic acetate uptake by internal polyP reserves availability, and prolonged aerobic Pi release with increasing amounts of acetate added (A. Pi and g. acetate concentrations, and £ pH). After complete denitrification, at time 3.5 hours, various levels of acetate were added to each reactors. At time 6.3 hours, 50 mg P/l of a sodium Pi solution was added to all reactors. 50 Batch experiment of April 24, 19 84 a E A z 60 50 40 30 20 -10 -Legend • Control + 25mgCOD/l ° 50mgCOD/l A 75mgC0D/l x 100 mg COD/I Net Pi rei • -0.8 • 0.770 x Acet cons (r - 0.995) 20 40 6 0 Acetate consumed (mg HAc/l) Figure 5.6 Relationship between net P i release and acetate consumed. Batch experiment of April 24, 19 84 o c o u a E 3 E o a E c o « 2 -20 40 Acetate consumed (mg HAc/l) Figure 5.7 Aeration time required to reach a minimum P i concentration as a function of the amount of acetate consumed. 51 (Figure 5.7). Thus, for each 20 mg C O D / 1 of acetate added, Pi uptake was prolonged by 1 hour. Although no P H A were monitored for this batch experiment, it was hypothesized that larger reserves of P H A allowed bio-P bacteria to accumulate Pi over a longer aeration period. The p H of each reactor showed a step increase of about 1.0 p H unit (0.75 for the Control reactor) upon aeration. This p H rise may be attributed to C 0 2 air stripping. i i . Batch experiment of May 25, 1986 The sludge for this batch experiment was taken from each one of the four sequencing batch reactors (SBR) on the last day of their operation (see section 5.3.1). Each reactor was acclimated to a different level of acetate (0, 15, 30 and 45 mg acetate added expressed as COD/1 of influent to SBR 1, 2, 3 and 4, respectively). On that last day, the SBR operation cycle was started as usual by pumping the wastewater into the reactors. However, just after pumping, enough sludge was taken from each of the four SBR to fill up completely four one liter Erlenmeyer flasks. The normal SBR cycle was then allowed to continue with acetate addition at time 1.5 hour, but at time 3 hours, no aeration was allowed and the SBR were mixed under unaerated conditions for a period of 25 hours. In the four corresponding one-liter Erlenmeyer flasks, an excess of acetate (171 mg of acetate as C O D per liter of reactor) was added at time 1.5 hours and the sludge was also kept unaerated for 25 hours. Detailed sludge characteristics can be found in section 5.3.1. Results of the concentration over time of soluble Pi , acetate, P H B and P H V are given in Figure 5.8 for SBR 1 to 4, and in Figure 5.9 for the corresponding flasks to which an excess of acetate was added (reactors l A c , 2Ac, 3Ac and 4Ac). A comparison of the two Pi plots showed that after 25 hours of anaerobic conditions, the maximum concentration reached was virtually the same for reactors with identical sludge (SBR 0 acetate with 0 or 200 mg COD/1 acetate addition; SBR 15 with 15 or 200; SBR 30 with 30 or 200; and SBR 45 with 45 or 200 mg COD/1 of acetate). Thus, with or without an excess of acetate added, a maximum of Pi was released after an unaerated period of 25 hours. The major difference was that with acetate, the maximum Pi concentration was reached more rapidly. The maximum amount of Pi released represented 17% of the total phosphorus sludge content for the Control SBR, and 35% for the other three SBR (nos. 2, 3 and 4) to which acetate was added. This proportion provides an indirect estimation of the amount of polyP degraded by bio-P bacteria. Since only a fraction of the sludge population is made of bio-P bacteria, the actual percentage of phosphorus in the form of polyP in these bacteria should be even greater. A n excess of acetate always remained in solution in each flask l A c , 2Ac, 3Ac and 4Ac (Figure 5.9-B). The amount of P H V and especially of P H B accumulated by the sludge was much greater in the reactors to which an excess of acetate was added. The total 52 Figure 5.8 Normal acetate addition to bio-P sludge obtained from four SBRs acclimated to 0,15, 30 or 45 mg COD/1 acetate addition (A. Pi, B. PHB and C, PHV concentrations). Data plotted at time 10 hours was obtained at time 25 hours. 53 Batch • xparimont of March 26, 1986 0 -I 1 1 1 1 1 1 1 1 1—'y— 0 2 4 6 B (26) Tim* (h) Figure 5.9 Excess acetate addition (200 mg COD/1) to bio-P sludge obtained from four SBR acclimated to 0, 15, 30 or 45 mg COD/1 acetate addition (A. Pi, B. acetate, C. PHB and D. PHV concentrations). amount of PHB stored was as much as 5 times greater for SBR 30 with 200'than with 30 mg COD/1 of acetate, and for SBR 45 with 200 than with 45 mg COD/1 of acetate. These two SBR were acclimated to the highest levels of acetate addition studied. The corresponding amount of PHV stored was about 2 to 3 times larger. These extra amounts of carbon stored in the reactors with an excess of acetate compared to those that received the normal level of acetate addition, must have originated from acetate. However, since PHV formation requires propionate, it would appear that acetate addition somehow stimulated the propionate production (by fermentation) by the sludge. The ratio of PHV to PHA stored was 50% in reactors 1 and lAc, and about 20% for all others. The molarratios of Pi released to PHA stored for each flask with excess acetate was only 30 to 60% of the value of the corresponding reactors (Figure 5.10). In other words, for the same amount of polyP degraded, more PHA storage took place when an excess of acetate remained in solution. d) Metallic cations i. Batch experiment of June 4, 1987 The objective of this experiment was to test the effect of metallic cations (sodium, potassium, magnesium and calcium) on Pi release and uptake. Cations addition was done as salts of phosphate such that 10 mg P/l was added to ensure that Pi availability would not limit aerobic Pi uptake. The molar ratio of metal to Pi added was 1.5:1.0. Sodium dihydrogeh Pi was used to provide phosphorus. Metallic cations were obtained from sodium chloride (for Na + ), magnesium chloride (for Mg 2 + ) , and calcium chloride (for Ca 2 + ) . For potassium, the salts potassium dihydrogen Pi and potassium chloride were used. Neutralization was done with sodium hydroxide to obtain a pH of 7.0. After 5 hours.of unaerated conditions, 10 mg P/l and 30 mg HAc/1 (acetate added as sodium salt) were added. Three hours later aeration was started for a period of 7 hours. Figure 5.11 shows the concentration profiles of Pi, PHB and PHV. No difference between the Pi, PHB or PHV concentration profiles were seen with the metallic salt additions. Therefore, it was concluded that the natural availability of sodium, potassium, magnesium or calcium in solution did not limit Pi uptake. 55 Batch experiment of March 26, 1986 2.0 -i SBR number Figure 5.10 Molar ratio of anaerobic Pi release over PHA storage for each SBR with normal and excess acetate addition. Numbers 1 to 4 represent the SBR number and "Ac" represents that these reactors received an excess of acetate (200 mg COD/1). 56 Figure 5.11 Effect of metallic cations (potassium, magnesium, calcium or sodium) on Pi release/uptake, and PHA storage/consumption (A. Pi, B_. PHB and C. PHV concentrations). 57 e) Radioactive acetate i. Batch experiment of November 4, 1985 The objective of this experiment was to trace the fate of radioactive acetate under a sequence of anaerobic/aerobic conditions. For that purpose, three 2.8 1 reactors were used to which a mixture of 75% aerobic mixed liquor and 25% wastewater was added. The first reactor received no acetate and served as Control. The second "dead" reactor received a formaldehyde solution (100 mg/l) 20 min prior to acetate addition at time 5.5 hours. To both the second and third ("active") reactors were added solutions of 50 mg COD/1 of acetate and 34 pCi of a uniformly labelled 14C-acetate solution having a specific activity of 57.6 mCi/mmol resulting in the addition of 35.8 pg acetate per reactor. Aeration of all three reactors was initiated at time 8.5 hours. The concentration profiles of Pi, PHB and PHV are shown in Figure 5.12. The percent of * 4 C label detected in the various fractions of the "active" reactor samples versus time is shown in Figure 5.13 (average of duplicate samples). The Control reactor showed a relatively rapid rate of Pi release and PHV storage as can be explained from the fermentation of simple compounds present in the added wastewater. The PHV profile under anaerobic conditions reached a plateau, however, which did not correspond to a plateau in the Pi profile. Such an observation is peculiar in that in most other experiments, anaerobic Pi release was always accompanied by PHV (and some PHB) storage. Lysis of bio-P bacteria at a rate fast enough to result in such Pi accumulation seems improbable. The leakage of air into the reactor could have explained this plateau. However, the ORP profile did not indicate any such entrainment. This irregularity remained unexplained. Microbial activity was effectively stopped in the "dead" reactor to which formaldehyde was added, as shown by the absence of Pi release or uptake, the absence of PHB/PHV storage or consumption (see Fig. 5.12), and by the absence of uptake of radioactive acetate (all of the labelled acetate was found to remain in the soluble fraction). In the "active" reactor to which radioactive and non-radioactive acetate were added, rapid Pi release, PHB and PHV storage were observed as expected. An average of 60% of the radioactivity added to the reactor was accounted for in the three components measured. Quenching by inert material may explain this incomplete recovery. Rapid soluble acetate uptake corresponded very closely to particulate accumulation by the biomass. A close correspondence with the PHB/PHV profiles suggested that the added acetate was stored as PHB and PHV. A more sophisticated separation technique would be required to quantify the proportion of the added acetate that ended up as PHB and as PHV. Anaerobic C 0 2 production corresponded to acetate uptake and storage and accounted for about 7% of the added radioactivity. Considering that the pathway of 58 Batch oxporlmont of Novombor 4, 198S 40 H - aoratlon-0) X a E X Cv acotat* Tim* (h) o o > 10 H 10 H Figure 5.12 Radioactive 14C-acetate addition (A. Pi, B. PHB and C. PHV concentrations). 59 Batch experiment of November 4, 1 9 8 5 Time (h) Figure 5.13 Fate of radioactive 14C-acetate into dissolved, particulates and C 0 2 fractions. Average of duplicate samples are shown. 60 PHB/PHV storage requires N A D H formation (see Literature Review), the T C A cycle may have resulted in the consumption of some acetate for N A D H formation at a rate just sufficient to satisfy the demand for PHA storage (since the T C A cycle is normally inoperative for aerobic microorganisms under anaerobic conditions because of N A D H repression - see Literature Review). • Under aerobic conditions in the "active reactor", PHB and PHV are practically all consumed but the relatively high radioactive particulate fraction suggested that a significant fraction of the PHA was used for cellular material and not just for energy production. The low percentage of label accounted for as C 0 2 may simply reflect the loss of 1 4 C - C 0 2 to the atmosphere by air stripping (the pH went up from 6.8 to 7.2 upon aeration). The apparent accumulation of label in the soluble fraction may also be an indirect result of the loss of C 0 2 by air stripping since the actual number of counts did not increase (an apparent increase was obtained only when the results were expressed as percentage of the total amount of radioisotope detected). 5.2.2. AEROBIC presence or addition of acetate and/or propionate i. Batch experiment of July 8, 1987 The objective of this experiment was to determine the sources of energy used by bio-P bacteria under aerobic conditions. For that purpose, various levels of acetate were added (0, 20, 40, 60 and 100 mg COD/I) under anaerobic conditions and aeration was started three hours later that would last for an extended period of 10 days. A 10 mg P/l solution of Pi was added at the same time as acetate to try to ensure that aerobic Pi uptake would not be limited by the availability of soluble Pi. This Pi addition explains the sudden increase in Pi concentration in the Control reactor at the time of acetate addition. Figure 5.14 shows the concentration profiles of Pi, acetate, PHB and PHV for the period between 8 and 20 hours and Figure 5.15 shows the Pi concentration profile for the period between 0 and 250 hours. During anaerobic conditions, Pi was released to a maximum level of about 53 mg P/l in the reactors to which 60 and 100 mg COD/1 of acetate had been added. The acetate plot shows that only 45 to 50 mg HAc/1 was taken up by the biomass in these reactors, presumably because of a limitation in polyP reserves. Thus, at the beginning of the aeration period, 11 and 42 mg HAc/1 of acetate was remaining in the reactors to which 60 and 100 mg COD/1 of acetate had been added, respectively. As air was turned on, PHB and PHV accumulation was observed for a short time until the remaining acetate disappeared from solution, after which PHB/PHV consumption started. During that short period when acetate was still in solution, Pi uptake took place, instead of Pi release (see next experiment). Since the Pi concentration profiles before aeration were similar for the reactors to which 40, 60 or 100 mg COD/1 were added and 61 Batch oxporimont of July B, 1887 a o> E u < X o> E a E c o V ffi X a. m X o> E o u > X a. Figure 5.14 Aerobic PHA storage in the 60 and 100 mg COD/1 reactors to which an excess of acetate was added anaerobically. PolyP reserves were depleted in these reactors (A Pi, B. acetate, £ PHB and D PHV concentrations). 62 Batch experiment of July 8, 1987 a E o c o o 60 50 -i — acetate 40 -30 -20 - -!h 10 -aeration-Legend • Control + 20 Acetate o 40 Acetate A 60 Acetate x 100 Acetate Time (h) Figure 5.15 Slow aerobic polyP degradation over a 10-day period. Soluble Pi degradation gives an indirect estimation of polyP reserves degradation. 63 that all three reached a plateau, and that no more acetate was taken up, it can be assumed that polyP reserves were completely depleted in the 100 mg COD/1 reactor. Even though 10 mg P/l of Pi had been added to all reactors, the Pi concentration in the reactors with 60 and. 100 mg COD/1 of acetate reached a very low concentration (0.2 to 0.5 mg P/l). Thus, the Pi uptake capacity exceeded the soluble Pi concentration, probably because ofthe presence of additional acetate under aerobic conditions. Prolonged aeration resulted in a slow Pi release over a 10-day period. Even after that extended aeration period, the maximum Pi concentration did not reach the 53 mg P/l level reached in 3 hours after a high dosage of acetate. Furthermore, the final Pi concentration was inversely proportional to the amount of acetate added, suggesting that polyP degradation had been retarded by the availability of stored PHA. ii. Batch experiment of August 6, 1987 The objective of this experiment, as for the previous experiment, was to determine what sources of energy bio-P bacteria use under aerobic conditions. The procedure followed was very similar to the one used for the previous experiment with the difference that, first, 20 mg P/l of phosphate was added along with 20 mg COD/1 of acetate under anaerobic conditions. Second, just prior to aeration, acetate (40 mg COD/1), propionate (40 mg COD/1) and combinations of acetate and propionate (20 mg COD/1 each) were added to the sludge. The level of 40 mg COD/1 was chosen because it corresponded closely to the acetate concentration remaining just before aeration in the reactor to which 100 mg COD/1 had been added in the previous experiment. Pi, PHB and PHV concentration profiles are shown for the period close to the start of aeration (10 to 16 hours) in Figure 5.16. Even though the first sample was taken only 0.5 hour after substrate addition, Pi release and PHB/PHV storage were observed upon aerobic acetate and/or propionate addition. More frequent sampling would have been desirable to establish the maximum Pi and PHA concentrations reached. A dashed line was shown to represent expected aerobic Pi release upon acetate or propionate addition (as documented by Gerber et al, 1986, 1987a, 1987b). Acetate addition resulted mainly in PHB formation whereas propionate or acetate plus propionate resulted mainly in PHV formation. Therefore, with the availability of polyP reserves in bio-P bacteria (the initial addition of 20 mg COD/1 of acetate apparently did not deplete these reserves), some of the energy required for PHA storage upon acetate and/or propionate addition originated from polyP degradation. Some energy required for PHA storage must have also originated from acetate degradation coupled to oxidative phosphorylation. Glucose and glycine (an acetate molecule with an - N H 2 group on the alpha carbon) were also added just prior to aeration in other reactors during the same experiment (results 64 Batch experiment of August 6, 1987 a. 01 X a E x a. •a x > x 50 - aeration-acetate &/or 1 propionate addition! PI release (extrapolated) Tim* (h) Figure 5.16 Aerobic Pi release and PHA storage with aerobic acetate and/or propionate addition (A. Pi, B. PHB and £ , PHV concentrations). 65 not shown). The Pi and PHA concentration profiles of these reactors followed the Control reactor very closely and thus, it appeared that glucose or glycine could not induce aerobic Pi release and PHA storage in the time period of these experiments. This observation suggested that, for the sludge used, anaerobic glucose addition will result in Pi release and PHA storage only because of glucose fermentation into acetate and propionate (see section 5.2.4). 5.2.3. Propionate with or without acetate i. Batch experiment of August 19, 1986. The general procedure followed for this batch experiment was described in section 5.2.1.a.ii. The objective of this experiment was to compare the effect of the addition of 0.55 mM of acetate (35 mg COD/1), propionate (57.5 mg COD/1) and of an equimolar combination of acetate and propionate (totalling 0.55 mM) on Pi release/uptake and PHA storage/consumption. Propionate was added both as a sodium salt and an acid neutralized to pH 7 by NaOH. A summary of results is presented in Table 5.3. Table 5.3 Effects of 0.55 m M acetate and/or propionate addition on Pi and PHA. Batch experiment of August 19,1986. Substrate Pi rel Pi upt PHA sto %PHVsto/ PHA cons PHA sto (mg/1) (mg/1) (mg HB/1) (%) (mgHB/1) Control 3.8 2.1 . 6.5 76 5.4 NaAc 27.6 15.0 52.5 35 36.1 NaProp 27.3 14.1 26.9 83 19.4 HProp 26.8 11.0 27.4 88 . 20.9 NaAc+NaProp 37.3 1.4 35.8 17 -3.4 The magnitude of anaerobic Pi release was very similar for each reactor to which either acetate or propionate was added but higher for the reactor that received both acetate and propionate. The Pi uptake in that reactor, however, was unexpectedly low (1.4 mg P/l). Acetate addition resulted in approximately twice the level of PHA storage as did propionate. The proportion of PHA stored as PHV was greatest when propionate alone (88%) was added. With acetate, 65% of the PHA was as PHB. The addition of acetate or propionate as the sodium salt or as an acid neutralized to pH with NaOH resulted in no significant difference as could be expected. 66 ii. Batch experiment of March 3, 1987 The objective of this experiment was to verify the preliminary results of the previous experiment on the effects of acetate and propionate addition on Pi release/uptake and PHA storage/consumption. Five 2.8 1 Erlenmeyer flasks were filled with aerobic mixed liquor and kept unaerated for 8 hours. At time 4.5 hours, about 30 mg COD/1 of substrate was added to each reactor: acetate (28.8 mg COD/1), acetate + propionate (19.2 + 9.6 mg COD/1), acetate + propionate (9.6 + 19.2 mg COD/1) and propionate (28.7 mg COD/1). Aeration was started at time 8 hours. Pi, PHB and PHV concentration profiles are shown in Figure 5.17. The pH decreased from 7.4 at the start of the unaerated period to 7.2 at the end, and rose to 7.9 upon aeration for all flasks. From V F A analyses it was estimated that acetate and propionate, when added separately, were taken up in 33 minutes. When added jointly, these substrates were removed in only 22 minutes. The maximum amount of PHV was obtained when both acetate and propionate were added together whereas maximum PHB formation resulted from the addition acetate alone. The addition of propionate alone resulted in the < formation of only a small amount of PHV (see Figure 5.18). With the addition of propionate alone, PHA storage took place only in the form of PHV. Since acetate is also required for PHV formation and that none was added, the required acetate must have originated from either (or both) the degradation of some propionate into acetate or from the fermentation of other substrates present in solution. It was estimated that the number of carbon molecules stored as PHA corresponded to 51% of the carbon added as propionate (see summary Table in the Discussion). Thus, with propionate addition, the rate-limiting factor for PHA storage could presumably be that of the formation of acetate. It can be noted that with propionate, the rate of Pi release and PHA storage was slightly slower than with acetate or combinations of acetate and propionate. Even though a small amount of PHA was accumulated when propionate alone was added, practically the same amount of Pi release and uptake took place in all reactors to which either acetate and/or propionate had been added. Assuming that the only energy available under aerobic conditions was in the form of PHA, it could be estimated that 55% of the energy available as PHA was used for polyP accumulation in the propionate reactor as opposed to only 20% in the acetate reactor (refer to the Discussion for energy balance estimations). It is not clear why propionate would have the same effect as acetate on Pi release (and Pi uptake) but result in only a third of PHA storage. 67 Figure 5.17 PHA storage with anaerobic acetate and/or propionate addition (A. Pi, B. PHB and C. PHV concentrations). A total of about 30 mg COD/1 was added. 68 Batch experiment of March 3, 1987 30 30 acet 20ac+10prop 10ac*20prop 30 prop Substrate taken up (30 mg COD/I) Figure 5.18 Maximum amounts of net PHB and PHV storage in the anaerobic zone for a total of 30 mg COD/1 of acetate and/or propionate addition. 69 iii. Batch experiment of July 8, 1987 The objective of this experiment was to confirm the results of the previous experiment that propionate did not result in PHB (but only PHV) storage and that acetate and propionate resulted in both PHB and PHV storage. For that purpose, solutions of acetate plus propionate (20 mg COD/1 of each in the same reactor), and propionate (20 or 40 mg COD/1 in different reactors) were added under anaerobic conditions to denitrified sludge in four 2.8 1 Erlenmeyer flasks (one serving as Control). A 10 mg P/l solution was added at the same time as the substrates to ensure that aerobic Pi uptake would not be limited by the availability of soluble Pi. Aeration was started 3 hours after the addition of substrate and Pi. Figure 5.19 shows the concentration profiles of Pi, acetate or propionate, PHB and PHV. Patterns of Pi release/uptake and PHA storage/consumption were very similar to what had been observed in the previous experiment. Again, no PHB was accumulated anaerobically with the addition of only propionate whereas PHB and PHV accumulation were highest in the reactor to which both acetate and propionate had been added. Using the data obtained 20 minutes after substrate addition, an estimation of the proportion of carbon added as substrate to that stored as PHA can be made. The amount of PHA stored in the Control reactor was subtracted from that value to obtain a "net" amount of PHA storage due to simple substrate addition alone. The above ratio was estimated at 112% for the reactor with acetate plus propionate (20 + 20 mg COD/1), 57% and 32% for the reactors with propionate alone (20 and 40 mg COD/1, respectively). Thus, the storage of a combination of acetate and propionate appeared to be much more efficient than that of propionate alone. It is unclear why there was such a difference in the fraction of carbon stored between acetate + propionate and propionate while the amount of Pi release is similar between the reactors to which 40 mg COD/1 were added. 5.2.4. Other substrates In this section the results of experiments that were conducted with the following carbon substrates will be described: formate, acetate, propionate (data repeated for acetate and propionate), butyrate, valerate, hexanoate, fermented primary sludge, lactate, /9-hydroxybutyrate, succinate, iso-butyrate, iso-valerate, citrate, ethanol, glycine, and glucose. Refer to Appendix A - l for the chemical structure of these compounds. i. Batch experiment of August 19, 1986 In this batch experiment the effect of various chemicals addition (0.55 mmole/1 for each, and neutralized to pH 7.0 before addition) on Pi release/uptake and PHA storage/consumption was tested. Details on how the experiment was conducted can be found in section 5.2.l.a.ii. Table 5.4 summarizes the results obtained for various V F A of 70 Figure 5.19 PHA storage with anaerobic acetate and/or propionate addition (A. Pi, B. acetate or propionate, & PHB and D .PHV concentrations). 71 chain length varying between 1 and 6 carbon atoms. Substrates are listed in the Table by decreasing order of Pi release (except for the Control which is given first). Table 5.4 Effects of the addition of 0.55 mM of various substrates. Substrate Pi rel Pi upt PHA sto %PHVsto/ PHA sto PHA cons (mg/0 . (mg/1) (mgHB/1) (%) (mg HB/1) Control 3.8 2.1 ' 6.5 77 5.4 HButyric 29.4 19.4 35.3 55 0.6 NaButyric 28.3 16.0 28.6 46 3.5 NaAc 27.6 15.0 52.5 35 36.1 NaProp 27.3 14.1 26.9 83 19.4 lactate 21.1 12.1 57.8 39 47.5 HIso-Butyric 16.3 6.1 28.6 31 2.4 glycine 13.3 3.2 16.1 82 10.8 citrate 10.6 4.4 14.0 71 8.3 ethanol 9.6 4.8 17.0 65 -15.8 HHexanoic 8.3 6.9 28.4 40 27.0 HValeric 7.9 4.4 38.5 92 18.6 glucose 6.2 5.0 21.2 67 16.1 HFormic 3.6 1.4 7.2 71 5.8 HIso-Valeric 1.8 0.7 10.0 50 -9.9 Maximum Pi release was obtained with the addition of acetate, propionate, butyrate and lactate. The first three of these substrates are those normally detected in the primary sludge fermentation stream added to the bioreactor at the UBC pilot plant from where the sludge was taken. Acetate and propionate alone represented 95% of the V F A produced by primary sludge fermentation. Thus, the sludge could have responded to these substrates because of its previous acclimation. The high response to lactate addition could suggest that this substrate was also present in the fermented primary sludge stream although its quantification was never attempted. For all reactors, only the equivalent of 35 to 70% of the released Pi was taken up under aerobic conditions. The aeration period may have been too short to allow complete Pi uptake. Maximum PHA storage was observed with the addition of acetate and lactate. Minimum PHA storage resulted from the addition of formate and iso-valerate. Again, acclimation of the sludge to the substrate may have been an important factor. 72 In general, the proportion of PHA stored as PHV was. larger with substrates containing an odd number of carbon atoms per molecule: valerate (92%).and propionate (83%). The proportion of PHA stored as PHB was generally larger with substrates containing an even number of carbon atoms per molecule (lowest %PHV/PHA): iso-butyrate (31%), acetate (35%), hexanoate (40%) and butyrate (46%). The anaerobic degradation of these substrates into acetate and propionate was probably required before storage as PHB/PHV explaining why substrates containing an odd number of carbons could favor propionate formation. However, since lactate favored PHB formation (39% PHV/PHA) while being a 3-carbon compound could indicate that this substrate was preferentially broken down into acetate before storage as PHA. The high proportion of PHV storage with glycine (81%) could suggest a different degradation pathway for this chemical. The amount of aerobic PHA consumption was generally related to PHA storage. Notable exceptions were observed for butyrate, iso-butyrate and iso-valerate addition where the PHA consumption was much lower than the corresponding amount of PHA stored. With iso-valerate and ethanol, even aerobic PHA storage was observed. This last unusual observation should be interpreted with care as it was not reproduced. ii. Batch experiment of October 28, 1986 This preliminary experiment was performed to test the individual effects of the addition of £-hydroxybutyrate and of succinate (0.50 mM) on Pi release/uptake and PHA storage/consumption. The experiment was conducted in the same way as on August 19, 1986 for which experimental details can be found in section 5.2.1.a.ii. Table 5.5 summarizes results for Pi and PHA data. A low percentage of PHA storage as PHV (23%) was observed for the Control reactor. For most other experiments, this value was in the range 65% to 85% (see Discussion). It is suspected that, at least for this reactor, some air might have been entrained inside, possibly because of an imperfect seal between the rubber stopper and its attachments. Therefore, fermentation activity, which is responsible for acetate and propionate production required for PHV formation, could have been inhibited by the presence of air, or fermentation products could have been consumed instead of being stored as PHV. Comparing the effects of /3-hydroxybutyrate addition with that of acetate, indicated a similar Pi release/uptake but a much greater PHA storage/consumption. In both cases most of the PHA were stored as PHB (99%). Succinate resulted in a high degree of Pi release/uptake but in almost negligible "net" PHA storage/consumption-(subtracting the Control values). The possibility of air entrainment into the reactor (see paragraph above) suggested caution in the interpretation 73 of these results. Nevertheless, it was unusual that so much Pi release would be associated with so little PHA storage while resulting in good subsequent Pi uptake. Table 5.5 Pi release/uptake and P H A storage/consumption for batch experiment of October 28,1986. Substrate Pi rel Pi upt PHA sto %PHVsto/ PHA cons PHA sto (mg/1) (mg/1) (mg HB/1) (%) (mgHB/1) Control 9 11 4.4 23 5.7 acetate 25 23 26.8 1 23.5 : /9-hydroxybutyrate 34 30 58.4 . 1 38.3 succinate . 32 25. 6.4 80 6.0 Note: "total" values are reported in this table (as opposed to "net" values that would be corrected for the Control, as for other experiments) iii. Batch experiment of March 3, 1987 The objective of this experiment was to determine the effect of the addition of various fatty acids (valerate, formate, lactate, butyrate, and fermented primary sludge) on Pi release/uptake and PHA storage/consumption. As opposed to the previous preliminary experiment, more intensive sampling was performed during the course of the test to determine rates of reaction. Analysis of individual fatty acids was also done. Six 2.8 1 Erlenmeyer flasks were filled with aerobic mixed liquor and kept unaerated for 8 hours. At time 4.5 hours, about 30 mg COD/1 of substrate was added to the denitrified sludge in five ofthe reactors in the following forms: valerate, formate, lactate, butyrate, and fermented primary sludge. VFA accounted for 56% of the soluble COD in fermented primary sludge of which 40% was as acetate and 57% as propionate. Aeration was started at time 8 hours. Pi, fatty acid, PHB and PHV concentration profiles are shown in Figure 5.20. Despite unreliable results for. the quantification of formate, it was estimated that approximately 25% of the formate added originally, was left in solution at the end of the anaerobic period. With fermented primary sludge addition, the rapid rate of Pi release and PHB/PHV storage can presumably be attributed to its acetate and propionate content. The concentration of these two substrates decreased rapidly in this reactor (see Figure 5.20-B). With all the other substrates, the rate of Pi release, fatty acid consumption and PHB/PHV storage was slower than with acetate and/or propionate addition (see section 5.2.3.ii). For example, the rate of substrate uptake ranged between 40 to 80 mg COD/l /h 74 Batch experiment of March 3, 1987 0 H 1 1 1 1 1 1 1 1 1 r-1 3 5 7 9 11 Tlmo (h) Figure 5.20 PHA storage with anaerobic short chain fatty acids (SCFA ) addition (A. Pi, . B. SCFA , £ PHB and D PHV concentrations). 75 for fermented primary sludge, acetate and/or propionate whereas it was only 3.5 to 7 mg C O D / l / h for all other fatty acids. This large difference suggests that acetate and propionate were stored directly as PHA whereas the other fatty acids had to be metabolized first, probably by non bio-P fermentative bacteria. With the addition of various types of fatty acids it was hypothesized that some other types of PHA compounds could be found that would be of different composition than PHB or PHV. However, a careful analysis of all the peaks from the chromatograms did not indicate any significant quantitative change in any peaks other than PHB and PHV. Figure 5.21 presents a visual comparison of the amounts and proportions of PHB/PHV storage with the addition of various substrates tested in this experiment (data from this section and section 5.2.3.ii). Amounts of PHB/PHV storage were corrected for the Control values and corrected for a 30 mg COD/1 substrate addition. This bar graph shows that acetate, 20/10 acetate/propionate and valerate resulted in the greatest amounts of PHA storage. Also the high proportion of PHB storage with acetate addition and of PHV storage with propionate, formate and valerate are readily seen. 5.2.5. Wastewater characterization by its ability to induce PHA storage: Penticton wastewater i. Batch experiment of June 25, 1987 The objective of this experiment was to assess the detrimental effect of air entrainment (see Literature Review) in the headworks of an existing full-scale wastewater treatment plant. The plant of Penticton, B.C., was studied in view of its pending upgrading to a bio-P plant. Four samples taken from different locations in the headworks were compared: upstream of the screw pumps in the influent sump well (wastewater-1), downstream of the screw pumps (wastewater-2), downstream of the rotor strainers (wastewater-3), and downstream of the primary clarifier (wastewater-4). It was recognized that soluble BOD, soluble COD or soluble VFA analyses would not be sensitive enough to determine the effect of air entrainment between these points of sampling. In the proposed upgrading of the plant to a bio-P removal process, the installation of primary sludge fermenters was included. Therefore, a biological test that could assess a reduction of the "fermentability" of the wastewater, because of air entrainment, was explored. For that purpose, it was decided to combined a given volume of wastewater sample with a given volume of activated sludge taken from the UBC pilot plant. It was expected that as V F A would be produced from the wastewater by the fermentative microorganisms present in the UBC pilot plant sludge, Pi release and PHA accumulation would be observed. The effect of air entrainment would be to: reduce the total amount of Pi release 76 Figure 5.21 Net PHB/PHV anaerobic storage corresponding to the addition of 30 mg COD/1 of various substrates. "Net" refers to values corrected for the Control values. 77 and of PHA storage. A comparison between the various wastewater samples would be possible by monitoring Pi, PHB and PHV over a given period of time. The collection of the wastewater samples was done to obtain a quasi-composite sample by combining five 200 ml aliquots taken at each hour between 8:00 h and 12:00 h. Primary effluent samples were collected between 9:00 and 13:00 h to account for a hydraulic lag due to the hydraulic retention time in the primary clarifier. Samples were sent in an ice-chest by airplane and wastewater samples were mixed with the pilot plant sludge at 20:30 h the same day. Activated sludge samples were taken from the UBC pilot plant. After 7 hours under non-aerated conditions, some denitrified sludge was withdrawn (845 ml) to allow the later addition of 885 ml of wastewater at time 8.5 hours. No sludge wastage was done from the Control reactor. The anaerobic transfer of such large volumes required special care. For sludge wastage, positive pressure from a helium gas cylinder was used to force the sludge out of the reactor into a graduated cylinder. Similarly, wastewater addition was achieved by exerting positive pressure with helium into a sealed Erlenmeyer flask to expel its wastewater content into the sampling tube of the 2.8 liter reactor. Syringe needles inserted through the septum of the 2.8 liter flask allowed the gas to escape as the reactor filled up. The flask contents were kept unaerated for a total of 24 hours. Table 5.6 summarizes CODs and VFA concentration for each wastewater sample. These samples did not contain any propionate or butyrate. However, wastewater samples 1, 2 and 3 appeared to contain a significant amount of ethanol (from a winery), as indicated by an early peak on the G C trace used for VFA analyses. The total amount of unfiltered COD added per liter of reactor was 128, 137, 132 and 80 mg COD/1 for reactors with wastewater-1, -2, -3 and -4 respectively. Figure 5.22 presents the Pi, PHB and PHV concentration profiles over time. Table 5.6 C O D and acetate concentration of the four Penticton wastewater samples. Sample COD COD Acetate total filtered filtered (mg/l) (mg/l) (mg HAc/l) wastewater-1 396 219 3.5 wastewater-2 . 427 228 3.3 wastewater-3 405 214 2.1 wastewater-4 . 2 4 9 184 3.7 78 Bitch *xp*rlm*ht el Jun* 25, 1987 a. E X n E x B E > x 1« H L*g*nd Control + wait*wat*r- 1 0 wi i t iw i t t r - 2 o waat*wat*r- 3 X wait*w*t*r- 4 Tim* (h) Figure 5.22 Characterization of a wastewater fermentability by its ability to induce anaerobic Pi release and PHA storage by a bio-P sludge (A. Pi, PHB and C PHV concentrations). 79 The rapid increase in PHB concentration just after wastewater addition may have reflected the availability of acetate and possibly of ethanol from the wastewater. The similarity between the Pi, PHB and PHV profiles for the four wastewater samples suggested that the "fermentability" of these samples was not significantly different. Thus, it was concluded that, for practical purposes, the effect of air entrainment in the headworks could be efficiently counteracted by primary sludge fermentation. In light of the results from this experiment and of other factors it was eventually decided that it was not justified to change the newly installed and reliable screw pumps (in 1982 at the cost of about $500,000) for non air-entraining pumps. Modifications that were expected to achieve about 50% reduction in air entrainment in the headworks, included covering the screw pumps, bypassing the maintenance-intensive and air-entraining rotor strainers with medium size bar screens and submerging the primary clarifier weirs. 5.2.6. Metallic cations In a number of batch experiments, the concentration of filtrable metallic cations was monitored. A definite relationship was repeatedly established between Pi and cations concentrations. The results from only one "typical" experiment will be reported here and a summary Table of molar ratios obtained in other experiments will be given in the Discussion. i. Batch experiment of April 24, 1984 The Pi, acetate and pH concentration profiles were shown previously for this experiment on the effect of various levels of acetate addition (see Figure 5.5). Potassium, magnesium, calcium and sodium were also monitored during this experiment (Figure 5.23). Cadmium, manganese, iron and aluminum were also measured on the filtered samples. The concentration of these cations did not change for any one of the samples analyzed suggesting that they did not play an important role in Pi release/uptake. . Molar ratios for the anaerobic and the aerobic periods were derived from plots of metal versus Pi concentration (Figure 5.24; see also the summary in the Discussion). Two distinct and almost parallel lines were obtained because of the addition of 50 mg P/l just before aeration. From the slope of these lines, the total molar ratios of positive charges to Pi was 0.94 for anaerobic conditions and 1.02 for aerobic conditions. Thus, on average, one negative charge of a Pi molecule was neutralized by a positive charge originating from one of potassium, magnesium or calcium for either uptake or release. At a pH of 6.35 (the average anaerobic pH value), a Pi molecule should have a negative charge of 1.12, and at a pH of 7.2 (the average aerobic pH value), a negative Pi charge of 1.50 (pl<2 for phosphate of 7.2). Thus, even though the difference in pH could result in a net negative charge of 0.38 (1.50 - 1.12) per Pi molecule, the actual difference in the number of positive charges co-transported with Pi was only 0.08 (1.02 - 0.94). Metallic 80 Batch experiment of April 24, 1884 i i 1 1 i 1 1 r \ 1 • ^ . — — r ' • • -: i - T ~ 3 6 7 8 11 Time (h) Figure 5.23 Metallic cations concentration profiles in an experiment with various levels of anaerobic acetate addition (A. potassium, B. magnesium, Q. calcium and D. sodium concentrations). 81 Bitch experiment of April 24, 1984 o 0. a E w O c o o E a • c a • S a E 28 24 20 18 12 -•lope • 0.280 mg/mg (0.222 mole/mole) (for PI relenee) •lop* • 0.30S mg/mg (0.242 mole/mole) (for Pi uptake) —i 1 r-20 40 80 80 PI cone, (mg P/l) 100 120 12 -10 -8 -8 -4 -2 0 IB 10 8 -y / •lop* » 0.181 mg/mg « / * / (0.244 mole/mole) * * / (for PI release) / e y x / / + o / m + X ' /* / X u/% alop* • 0.176 mg/mg *fc (0.224 mole/mole) (for Pi uptake) / e*6*/ 20 40 60 80 PI eonc. (mg P/l) 100 120 •lop* • 0.11B mg/mg (0.091 mole/mole) (for Pi r*l*as*) g (for PI uptake) (0.119 mole/mole) 0 alop* • 0.164 mg/mg Legend e Control * 25 acet • 60 acet » 50 acet » 100 acet 20 40 60 80 PI cone, (mg P/l) 100 120 Figure 5.24 Molar ratios of metallic cations and Pi transport for anaerobic and aerobic conditions for acetate addition (concentration of At potassium B. magnesium and C. calcium versus Pi concentration). 82 cations released anaerobically could have remained bound to Pi in solution for subsequent aerobic uptake by bio-P bacteria. 5.2.7. Nitrate and nitrite i. Batch experiment of November 13, 1984 Acetate consumption for denitrification is documented to compete with acetate uptake for storage as PHA and thus, to reduce bio-P removal efficiency (see Literature Review). Therefore, the objective of this experiment was to quantify the "detrimental" effect of nitrate on Pi release. For that purpose, 50 mg COD/1 of acetate and various amounts of nitrate (0, 3, 6, 9 and 12 mg N/1) were added simultaneously to denitrified sludge at time 3.5 hours. Aeration was initiated at time 6.7 hours. Figure 5.25 shows the concentration profiles of Pi, nitrate and of acetate. In all reactors the rate of denitrification in the presence of acetate was quite rapid, about 15 mg N , r L h" 1 . The rate of acetate uptake was 66 mg HAc ,l" 1 ,h" 1 for the Control reactor and was 110 mg H A c i " 1 ^ * 1 for all the other reactors to which nitrate had been added. Since the rate of acetate uptake in the Control reactor should only reflect acetate storage by bio-P bacteria, the rate of acetate consumption for denitrification could be estimated, by subtraction, to be 44 mg HAc*]"1^" 1. Thus, for the sludge used, the rate of acetate uptake for storage by bio-P bacteria was 50% greater than that for denitrification by all denitrifiers (66/44). The extent of Pi release was reduced in direct proportion to the amount of nitrate added. However, Pi release did not occur only after the complete disappearance of nitrate from solution but as soon as acetate was added. Thus, the two reactions of acetate uptake by bio-P bacteria and of uptake by denitrifiers occurred simultaneously. Thus, the effect of nitrate on acetate uptake by bio-P bacteria is probably one of competition rather than of inhibition. To measure the effect of nitrate addition on Pi release, a plot was prepared that shows total Pi release versus nitrate added (Figure 5.26). The slope of the straight portion of the curve indicates that for each mg of nitrate (as N) added, Pi release was reduced by 3 mg P/l. In the reactors to which 9 or 12 mg N/1 of nitrate was added, Pi uptake under unaerated conditions was observed after acetate disappearance while some nitrate still remained in solution. After nitrate disappearance, a slow rate of Pi release was observed as for all other reactors. Therefore, it appeared that nitrate could be used as an electron 83 Batch •xp*rim*nt ol Nov*mb*r 13, 1884 a. z 0) E 6 c < x B E 60 60 40 H 30 H 20 i o H nltraU * 60 mg COD/I of aotat* atrition-10 11 L*g*nd • Control * 3mgN03-N/l - » 6 mg N03-N/I t 8 mg N03-N/I x 12mgN03-N/l -Figure 5.25 Reduction in the magnitude of anaerobic Pi release with acetate addition, due to increasing amounts of nitrate addition (A. Pi, B. nitrate and Q. acetate concentrations). 84 Figure 5.26 Quantification of the effect of nitrate addition on anaerobic Pi release. 85 acceptor by bio-P bacteria in place of oxygen. Future experiments also confirmed this observation. , ii. Batch experiment of March 11, 1987 The objective of this experiment was to confirm the observation of a previous (non reported) preliminary experiment that the anaerobic addition of 1, 3 or 10 mg N/1 of nitrite did not result in any Pi release, PHB or PHV storage. A total of 10 mg N/1 of nitrite or of nitrate was added to denitrified sludge after 9 hours. Aeration was started in the Control reactor at that time. The results for Pi, PHB and PHV are shown in Figure 5.27. Nitrite disappeared from solution in 26 hours after the start of the experiment. The pH dropped from 7.3 to 7.15 during the first nine hours. With nitrite or nitrate addition, the pH rose to 7.2-7.3 whereas aeration of the Control reactor resulted in a pH of 7.8 (probably due to C 0 2 stripping). The graphs show that Pi uptake and PHB/PHV consumption took place in the presence of nitrate or of oxygen, but not of nitrite. Therefore, at least for the bio-P activated sludge studied, bio-P bacteria were capable of using oxygen or nitrate as electron acceptors but not nitrite. PHV accumulation stopped after the addition of nitrite while PHB accumulation continued. This observation suggested that only propionate and not acetate production by fermentative microorganisms was inhibited by nitrite addition. Considering that nitrite was gradually removed from solution (by non bio-P bacteria) it is possible that propionate-producing fermentative microorganisms switched their metabolism from fermentation to nitrite reduction. 5.2.8. Toxicants a) 2,4-DNP i. Batch experiment of December 14, 1984 The objective of this experiment was to verify the effect of 2,4-dinitrophenol (2,4-DNP) addition on Pi release. 2,4-DNP affects the energized state of cellular membranes by acting as a proton "shuttle" and thus, cancelling the proton gradient (see Literature Review). At time 6.25 h, 1.0 mM 2,4-DNP was added to denitrified sludge. Aeration of the reactors was started at time 10.5 hours (see Figure 5.28; results on the effect of pH is also shown - refer to the subsequent section 5.2.8.b.ii). 86 Batch •xp*rim«nt of March 11, 1987 2 • | i • • T t • t I I I T r 14 -12 -0 -I 1 1 i 1 1 1 1 1 r — 3 6 7 8 11 13 Time (h) Figure 5.27 Comparison of oxygen, nitrate and nitrite as electron acceptors for Pi uptake and P H A consumption (A^ Pi, & nitrate or nitrite, C* PHB and D PHV concentrations). 87 Batch experiment of December 14, 1984 Figure 5.28 Effect of 2,4-DNP, high pH (9.0) and low pH (4.0) on anaerobic Pi release. The 2,4-DNP solution was not neutralized before addition. 88 Anaerobic 2,4-DNP addition resulted in Pi release (it even continued under subsequent aerobic conditions). This observation suggested that polyP could play a role in the regulation of the pH gradient (see Discussion). ii. Batch experiment of August 19, 1986 This experiment was a preliminary one in which the effect of a number of chemicals on Pi release/uptake and PHA storage/consumption was tested. Details on the execution of the experiment can be found in section 5.2.1.a.ii. Table 5.7 summarizes the results obtained for the addition of 1.0 mM 2,4-DNP, and of 0.55 mM of acetate (31 mg GOD/1, given for comparison). The pH of the reactor decreased from 7.8 to 7.1 following 2,4-DNP addition (the concentrated 2,4-DNP solution had a pH of 3.6) and increased to 8.3 after aeration. Table 5.7 Effects of 2,4-DNP addition. Batch experiment of August 19,1986. Substrate Pi rei Pi upt PHA sto %PHVsto/ PHA cons PHA sto (mg/l) (mg/l) (mg HB/1) (%) (mgHB/1) Control 3.8 2.1 6.5 76 5.4 NaAc 27.6 15.0 52.5 35 36.1 2,4-DNP 13.1 1.6 38.4 22 33.7 For the addition of 2,4-DNP, anaerobic Pi release was observed as,in the previous experiment, but this time some small amount of aerobic Pi uptake took place. PHA were both stored anaerobically and consumed aerobically. This observation is interesting since no substrates were added to this reactor. Thus, 2,4-DNP addition may have stimulated V F A production by fermentative bacteria. Such stimulation could occur by the negative effect of 2,4-DNP on the pH gradient of fermentative bacteria that would require the fermentation of more substrates to obtain the energy used to re-establish the pH gradient (much like polyP would be degraded by bio-P bacteria). Thus, this experiment indicated that 2,4-DNP could have stimulated Pi release by an increase in V F A production by fermentative bacteria (as evidenced by PHA storage at a less-toxic 1.0 mM level of 2,4-DNP added), and/or by the direct effect of 2,4-DNP on the pH gradient of bio-P bacteria. 89 iii. Batch experiment of November 11, 1986 This experiment was similar to the previous ones with 2,4-DNP except that the levels of 2,4-DNP added were 0.5 mM and 5.0 mM. The effects of the addition of 2,4-DNP combined with 28 mg COD/1 of acetate were also tested. Duplicate reactors were used for a total of 10 reactors. Chemicals were added simultaneously to denitrified sludge at time 4.5 hours, and aeration was started at time 7 hours. A summary of Pi and PHA data is given in Table 5.8. Table 5.8 Effects of 0.5 and 5.0 m M 2,4-DNP addition. Batch experiment of November 11, 1986. Substrate Pi rel Pi upt PHA sto %PHVsto/ PHA cons PHA sto (mg/0 (mg/1) (mgHB/1) (%) (mgHB/1) Control 5.7 10.0 5.5 64 12.9 0.5 mM DNP 12.7 0.6 6.2 68 9.1 5.0 mM DNP 22,0 (-3.2) 0.3 0 0.6 0.5 DNP+Acet 18.9 17.9 . 27.4 39 18.9 5.0 DNP + Acet 13.2 (-4.0) 2.5 68 1.3 Note: P H V / P H A percentages based on total amounts of PHB and PHV stored. As in the previous experiments, Pi release was stimulated by 2,4-DNP addition, and PHA storage/consumption was observed but only in the reactors to which 0.5 mM 2,4-DNP was added. With 5.0 mM addition, no PHA storage or consumption took place with or without acetate addition. Therefore, with sufficient amounts of 2,4-DNP, acetate storage as PHA was effectively inhibited and Pi release indicated that polyP degradation could produce energy to re-establish the pH gradient (see Discussion). The pH of the sludge initially collected was 6.6. In the Control reactor, the pH increased to 6.8 by the end of the anaerobic period, and to 7.2 after aeration. In the reactors in which a low level of 2,4-DNP (0.5 mM) was added, the pH decreased to 6.2 by the end of the anaerobic period and increased to 6.9 at the end of the aerobic period. A much sharper drop was observed in the reactors with a high level of 2,4-DNP (5.0 mM) in which the pH decreased to 4.7 at the end of the anaerobic and remained at this level until the end of the aerobic period. 90 Nitrite accumulated in the reactor with 0.5 mM 2,4-DNP but not in the reactor to which acetate was also added. Nitrification was completely inhibited in the reactor that received 5.0 mM 2,4-DNP. iv. Batch experiment of March 11, 1987 In this last experiment with 2,4-DNP, the effect of neutralized (pH 7.0) solutions of 0.1, 1.0 and 10.0 mM 2,4-DNP on Pi release/uptake.and PHA storage/consumption were determined. Chemicals were added to denitrified sludge at time 5.5 hours and aeration was started at time 9 hours. Figure 5.29 presents the Pi, PHB, PHV and pH results. Curves corresponding to the addition of 30 mg COD/1 of acetate are also shown for comparison purposes. With all three levels of 2,4-DNP addition, Pi release under anaerobic conditions was somewhat stimulated but not to the extent observed in other experiments (e.g. see Figure 5.28). The effect of a neutralized 2,4-DNP solution, in which less of the toxic unionized species would be found, may explain this difference. Aerobic Pi uptake, however, was inhibited by the addition of either 1.0 or 10.0 mM 2,4-DNP. PHB storage was negligible compared to that resulting from the addition of 30 mg COD/1 of acetate. PHV storage, however, was even stimulated for the reactor with 0.1 mM 2,4-DNP as was Pi release in this reactor. At this low concentration, this toxicant may have played a stimulatory effect on VFA production by fermentative bacteria by depleting their pH gradient which would have favored an increased rate of fermentation. With 1.0 mM and 10.0 mM 2,4-DNP, PHV storage and consumption was lower. The effects of 2,4-DNP on metallic cations movement (K + , Mg 2 t and C a 2 + ) is shown in Figure 5.30. Using the Control and the reactor with acetate for visual comparison, it can be seen that a significant amount of potassium, less of magnesium and a small amount of calcium were released into solution following 2,4-DNP addition. Plots of metal concentration against Pi concentration permitted a determination of whether 2,4-DNP addition stimulated an extra release of metals (Figure 5.31). Using the reactor with acetate as a reference to compare the number of moles of each metal released per Pi, it was estimated that the addition of 10.0 mM 2,4-DNP resulted in an extra (but transient) release of 20 mg/1 of potassium over a period of about 1.0 hour. Similarly, with 1.0 mM 2,4-DNP, an extra 14 mg/1 of potassium was released in less than 0.7 hour. Some extra magnesium and calcium may also have been released with the addition of 10 mM 2,4-DNP but no definite relationship could be established. Therefore, potassium release from cells (not necessarily only by bio-P bacteria) could have assisted in re-establishing the charge gradient component of the proton motive force that was affected by the presence of 2,4-DNP. 91 Batch experiment ol March 11, 1987 40 -, 6.5 -i 1 1 1 1 1 1 1 i 1 3 6 7 8 11 ' 13 Time (h) Figure 5.29 Effect of the addition of a neutralized 2,4-DNP solution on anaerobic Pi. release and P H A storage. The effect of 30 mg COD/1 of acetate addition is shown for comparison (A» Pi, & PHB, C± PHV concentrations and D pH). 92 Figure 5.30 Effect of 2,4-DNP addition on metallic cations. The effect of 30 mg COD/1 of acetate addition is shown for comparison (A. potassium B. magnesium and calcium concentrations). 93 Batch •xparimant ot March 11, 1987 30 Figure 5.31 Molar ratios of metallic cations and Pi transport for anaerobic and aerobic conditions for 2,4-DNP addition (concentration of A. potassium B. magnesium and C. calcium versus Pi concentration). 94 b ) p H i . Batch experiment of June 26, 1984 The objective of this experiment was to test the effect of a high p H (9.0) on Pi release in the presence and absence of acetate. For all five reactors used, the p H was maintained at a targeted value of 9.0. At time 3 hours and 40 minutes, the p H of all reactors was adjusted to 8.5 (observed value) with a 1 N N a O H solution. At time 4 hours, the following levels of acetate were added to different reactors: 0, 25, 50, 75 and 100 mg COD/1 . Aeration was started at time 7 hours. Figure 5.32 shows the concentration profiles of Pi and acetate. Actual p H values were maintained at close to 8.3, 8.4, 9.0, 8.4 and 8.8 for the 0, 25, 50, 75 and 100 mg COD/1 reactors, respectively. The rate of Pi release in the Control reactor averaged about 10 mg P T ^ h " 1 , ' a value higher than an usual 1.5 mg PT 1 *! !" 1 in the absence of substrate. For all reactors to which acetate was added, the final Pi concentration was in the range of 50 to 55 mg P / l . Normally, different levels of acetate addition would result in different levels of Pi release (ranging between 25 and 75 mg P/ l ) . Additionally, the molar ratios of Pi released to acetate taken up averaged 3.8 mole P/mole acetate. Without p H adjustment this value is normally around 1.4 mole P/mole acetate (see Discussion). Thus, the magnitude and rate of acetate uptake was probably limited by availability of polyP reserves. Larger amounts of acetate resulted in only marginally higher P i release. Even though no P H A measurements were taken, it was thought that P H A storage and consumption were low, as indicated by low amounts of acetate uptake during the anaerobic period, and that a significant fraction of the Pi release was caused by a high p H . A high p H could have induced polyP degradation by requiring energy from polyP for proton expulsion, much like the ATP-ase enzyme can do by reversing its normal mode of operation (see Literature Review). Aerobic P i uptake was only marginal in all reactors even though a significant amount of anaerobic Pi release had occurred previously. This minimal Pi uptake was in agreement with the minimal amount of acetate taken up (and supposed minimal P H A storage). A portion of the energy produced aerobically from external and internal P H A reserves was probably used to counteract the p H gradient resulting in less energy being available for aerobic Pi uptake. It should be noted that in the reactors with 50, 75 and 100 mg COD/1 of acetate, the availability of acetate at the beginning of the aerobic period did not result in Pi release as was seen in section 5.2.2.ii. PolyP reserves depletion may explain this observation as had been the case in the batch experiment reported in section 5.2.2.L 95 Batch experiment of June 26, 1984 pH adjustment 1» Time (h) Figure 5.32 Anaerobic Pi release and limitation of acetate uptake at high pH (8.3 to 9.0) (A. Pi and B. acetate concentrations). 96 ii. Batch experiment of December 14, 1984 The objective of this experiment was to test the effect of not only high but also.low pH on Pi release. From time 6.25 hours, the pH was continuously adjusted by the addition of 0.5 N solutions of either HC1 or NaOH to pH 4 or pH 9. A pH probe was inserted through the rubber bung and was in contact with the sludge. Aeration of the reactors was started at time 10.5 hours (see Figure 5.28; shown before for a simultaneous 2,4-DNP experiment). • Significant Pi release was observed at pH 9 under anaerobic conditions. Only a small amount of Pi was transiently released in the pH 4 reactor. This observation may-be explained by the solubilization of some loosely bound Pi molecules from the surface of activated sludge floes. Under aerobic conditions, only the Control reactor showed some Pi uptake, with the normal metabolic activity of the biomass in the other reactors being presumably inhibited. iii. Batch experiment of March 11, 1987 The objective of this experiment was to test the effect of high and low pH adjustment, as in the previous experiment, but this time with more detailed information on Pi, PHA and metals concentration. At time 5.5 hours pH adjustment was started for reactors with pH 5 or 9, or 30 mg COD/1 of acetate was added (results given for comparison). Aeration started at time 9 hours. Figure 5.33 shows the concentration profiles of Pi, PHB, PHV and pH, and Figure 5.34 those of potassium, magnesium and calcium. Figure 5.35 gives the concentration of these three metals against Pi concentration. As in the previous experiment, Pi release occurred at a higher rate at pH 9 and only a transient increase occurred at pH 5. Some Pi uptake was later observed more in the pH 9 than the pH 4 reactor. Compared to the addition of acetate, PHB storage or consumption at either pH 5 or 9 was negligible. PHV storage and consumption, however, took place to a very similar extent to what had occurred in the Control reactor. As proposed before, a high pH may have affected the pH gradient of bio-P bacteria that responded by polyP degradation, possibly to maintain a constant pH gradient. The concentration profiles of metal versus time and the plots of metal concentration versus Pi concentration allowed a comparison of the amount of metal released or taken up relatively to what normally takes place in the presence of acetate. At pH 5, for each Pi molecule released or taken up, 2.7 times as much potassium and 1.6 times as much magnesium were transported than in the Control reactor. A rapid but only transient Pi and calcium release was observed, presumably from the resolubilization of loosely-bound molecules. Thus, the bio-P sludge responded to low pH adjustment by a transient release of Pi and calcium and by an increased rate of potassium 97 Batch axperiment of March 11, 1987 40 T : : — ; 5 " j T T T T ' | | 1 1 3 6 7 . 9 11 13 Tima(h) Figure 5.33 Effect of high pH (9.0) and low pH (5.0) on anaerobic Pi release. The effect of 30 mg COD/1 of acetate addition is shown for comparison (A. Pi, B. PHB, C. PHV concentrations and D pH). 98 Batch *xp*rlm*nt of March 11, 1987 Tim* (h) T 1 1 1 1 1 1 1 r 3 5 7 9 11 13 Tim* (h) Figure 5.34 Effect of high and low pH on metallic cations. The effect of 30 mg COD/l of acetate addition is shown for comparison (A. Pi, B. PHB, d PHV concentrations and D PH). 99 Figure 5.35 Molar ratios of metallic cations and Pi transport at high and low pH (concentration of A. potassium JL magnesium and Cj calcium versus Pi concentration). 100 and magnesium release. It was unclear why potassium and magnesium were released at low pH. At pH 9, however, even though more Pi was released and taken up, the ratios of potassium and calcium co-transported did not change much compared to the reactor that .received acetate. Some extra potassium was released just after the initial pH adjustment. The ratio of magnesium to Pi, however, was reduced. Thus, the bio-P sludge responded to high pH adjustment mainly by a transient potassium release, an increased rate of Pi release and a slower rate of magnesium release. Considering that a high pH affects .the energized state of bacterial membranes by reducing their pH gradient, it is suggested that Pi release reflects the degradation of polyP to re-establish the pH gradient. At a high pH it would seem that less magnesium was used for Pi transport. c) Cyanide, Fluoride i. Batch experiment of August 19, 1986 The effect of cyanide and fluoride (added as neutral solutions) on Pi release/uptake and PHA storage/consumption was tested in a preliminary experiment. Details on how the experiment was conducted can be found in section 5.2.1.a.ii. Table 5.9 summarizes the results obtained for acetate (given for comparison), cyanide (10.0 mM) and fluoride (10.0 mM). Table 5.9 Effects of cyanide (5.0 mM) and fluoride (10 mM). Batch experiment of August 19,1986. Substrate Pi rel Pi upt PHA sto %PHVsto/ PHA cons PHA sto (mg/1) (mg/1) (mg HB/1) (%) (mgHB/1) Control 3.8 2.1 6.5 76 5.4 NaAc 27.6 15.0 52.5 35 36.1 NaCyanide 29.1 -9.8 13.8 53 1.4 NaFluoride 10.0 6.6 13.9 72 8.0 Cyanide addition stimulated anaerobic Pi release as much as the addition of 31 mg COD/1 of acetate. Some PHA storage also took place. Under aerobic conditions, no Pi uptake and even Pi release was observed, with minimal PHA consumption taking place. The effects of fluoride addition were not as marked as with cyanide. Some extra Pi release and PHA storage took place, but no significant inhibition of Pi uptake and PHA consumption was observed for aerobic conditions. 101 d) C 0 2 , H 2 S i. Batch experiment of August 22, 1985 The objective of this experiment was to test the effect on anaerobic Pi release of H 2S and C 0 2 addition. The biomass was obtained by combining aerobic sludge from a laboratory-scale plant with wastewater in a 75:25 proportion (see plant characterization data in Appendix 3). Starting at time 5.0 hours, H 2S or C 0 2 gas was bubbled into the reactor contents. The concentration profile of Pi is shown in Figure 5.36. The initial pH of the solution was 6.5 for all reactors before gas addition. The pH of the reactors that received H 2 S or C 0 2 decreased and rapidly stabilized at 4.5. Pi release was rapid with H 2S but only marginally greater with C 0 2 than in the Control reactor. Possible explanations are proposed in the Discussion. 5.3. Studies on Continuous bio-P Wastewater Treatment Plants 5.3.1. Sequencing Batch Reactors (SBR) The objective of this experiment was to characterize the effect of the addition of various levels of acetate on PHA storage by a biomass in a continuously operating process. Four laboratory scale SBR were used to which 0, 15, 30 or 45 mg/l of acetate (as COD) were added. The SBR were operated for a period of 13 weeks at an average reactor o temperature of 19 C. To quantify the effect of acetate on PHA accumulation but avoid nitrate interference, complete denitrification was achieved by keeping the sludge unaerated for a period of 1.5 hours before acetate addition (see Materials and Methods). Complete denitrification was indicated by a drop (a "knee") in the ORP profile (see Figure 4.3) (Koch and Oldham, 1985; Comeau et ai, 1987b). In the case shown in Figure 4.3, the time required for complete denitrification was only 0.65 hour, because of incomplete nitrification (effluent filtered ammonia of 4.9 mg N/1 instead of 0.5 mg/l with complete nitrification). With complete nitrification, total denitrification normally required between 1.2 and 1.5 hours. Average influent, reactor and effluent characteristics are summarized for a relatively stable five week operation period (week 9 to 13) in Table 5.10. Profiles over an eight-hour SBR cycle for Pi, nitrate, PHB and PHV (Figure 5.37) are shown for data obtained on week 11. Under anaerobic conditions, Pi release and PHA storage were stimulated by acetate addition. Under aerobic conditions, Pi uptake, and PHA consumption were observed. From the nitrate graph, it appeared that denitrification was slower in the Control reactor, and that nitrification was incomplete in SBR 4 (45 mg 102 Batch experiment of August 22, 1985 Time (h) Figure 5.36 Anaerobic Pi release with H 2 S and C 0 2 addition. 103 Table 5.10 SBR average influent, reactor and effluent characteristics for weeks 9 to 13. Parameter Concentration (mg/1) I N F L U E N T Unfiltered C O D 202 T P 1 10.0 T K N 16.9 Filtered C O D 96 P i 1 8.2 N H 4 + 10.3 SBR Number 1 2 3 4 R E A C T O R acetate added (as C O D ) 0.0 15.9 29.4 43.8 M L S S 1650 1695 1930 2148 SVI (ml/g) 111 90 89 94 aerobic % P (%P/SS) 3.1 3.8 4.3 4.7 anaerobic Pi (as P ) . 9.2 13.3 18.2 23.3 aerobic Pi (as P) 6.8 6.2 5.6 5.5 P H A storage (as H B ) 2.0 7.8 17.2 23.0 E F F L U E N T Unfiltered * SS 16 6 7 8 Filtered T K N 2 1.5 1.8 1.8 1.2 N 0 3 " 2 5.6 5.8 5.0 4.9 Notes: 1the equivalent of 7.0 mg P / l of a concentrated neutral sodium salt solution was added to the influent container; 2average effluent T K N and nitrate values exclude results from periods of incomplete nitrification. - for all SBR, the average %(VSS/SS) was 72%, average SRT was 20.4 days, and average effluent C O D was 31 mg/1. 104 SBRs - cycle from week 1 1 (May 12, 1986) o. co E z E c o o Z m x O) E c o o a X a. a> x Ol E u c o u > X a C y c l e time (h) Figure 5.37 Typical concentration profiles of Pi, B. nitrate, C. PHB and D PHV over an 8-hour cycle in the four SBR . Data from week 11 was used. 105 COD/1 acetate), as shown by an effluent ammonia concentration of 4.8 mg N/1 instead of a value of 0.5 mg N/1 with full nitrification. The percentage of PHA stored as PHV averaged 25% for the SBR with acetate added and 32% for the Control SBR. True steady-state conditions were not reached with this SBR experimentation. Aeration control proved to be a major difficulty, because of the eight hours required to see the effect of an air flow adjustment. Thus, aeration was sometimes insufficient to allow complete Pi uptake and full nitrification. Good correlations with the amount of acetate added were obtained for parameters characterizing the sludge, such as anaerobic Pi release (Figure 5.38-A), phosphorus content of aerobic sludge (Figure 5.38-B), and anaerobic PHA storage (Figure 5.38-C) for weeks 9 to 13. A good correlation was also obtained between the amount of Pi release and the amount of PHA stored (Figure 5.39). Molar ratios of the slope of the best-fit line for these plots indicated that for one acetate molecule added, 1.28 molecule of Pi was released, that 0.44 molecule of PHB was stored, and 0.19 molecule of PHV was stored. The sum of these PHA values indicate that for 100 carbon atoms added in the form of acetate, 108 carbon atoms were stored as PHA. Anaerobic fermentation probably provided the extra carbon stored. A molar ratio of 2.3 molecules of Pi released for each molecule of PHA stored was obtained. For the design of bio-P plants it could be useful to know the amount of acetate required to increase the sludge phosphorus content by one percent. Figure 5.38-B relates the difference between the aerobic sludge phosphorus content of a reactor minus that of the Control to the amount of acetate added. The slope of the best-fit line gave a one percent increase in sludge phosphorus content for the addition of 32 mg of acetate as COD per liter of influent, or of 25 mg/1 of acetate if the best-fit line is forced through the origin (as should be expected since the Control reactor did not receive any acetate). 5.3.2. PHA at the UBC Pilot Plant To quantify the extent of PHA accumulation in a continuous process, activated sludge samples were taken for PHA analysis from the UBC pilot plant. Both sides of the pilot plant were monitored such that the effect of primary sludge fermentation on PHA accumulation could be characterized. It was also desired to see if a preliminary comparison between PHA and other parameters that were continuously monitored, could suggest some correlations that could be used for future modelling purposes. PHA monitoring was performed between February 9 and July 6, 1987. A table can be found in Appendix A-4 for detailed PHA data obtained from the pilot plant. During that period, studies were conducted to assess the effect of primary sludge fermentation on 106 Figure 5.38 Correlations between the amount of acetate added and A» Pi release, IL %P content and £» PHA concentration. Data obtained from the four SBR between week 9 and 13. On graph £ * "plus" signs refer to PHV, "rectangles" to PHB, and "losanges" to PHA data. 107 SBRs for weeks 9 to 13 0 4 8 12 16 20 24 28 PHA stored (mg HB/I) Figure 5.39 Correlation between the amounts of Pi release and PHA stored. Data obtained from the four SBR between week 9 and 13. UBC Pilot Plant - January to August 1987 M cn E o c o o o 5000 4000 3000 -2000 1000 -period of PHAs data collection period of data used for side B Legend • Side A + Side B — 3-day moving avg — i 1 1 1 1 1 1 1 1 1 1 1 Jan 1 Feb 9 Mar 20 Apr 29 Jun 8 Jul 18 Aug 27 Date Figure 5.40 MLSS concentration at the UBC pilot plant in sides "A" and "B" between January and September 1987. 108 the efficiency of bio-P removal. The mode of operation of the treatment plant remained the same throughout this period except that on May 1, 1987 the fermenter volume was reduced from 400 1 to 250 1. The influent temperature (measured in the primary clarifier) o o o increased steadily 1 C/month during the period studied from 14.5 C to 19.5 C. Both of these changes did not appear to have a significant effect on any of the parameters analyzed. An operational perturbation on the B side occurred during the sampling period. On May 14, the effluent outlet of the B side was rebuilt without installing a vent on the effluent line. As a result, solids loss occurred to the point where the MLSS concentration decreased from 2700 to 500 mg/l (see Figure 5.40). Therefore, to allow a meaningful comparison between both sides, only the data obtained prior to May 11 was used for side B. After repairing the effluent line defect, and reducing solids wastage, the B side process resumed normal operation in early September. Operating characteristics presented in Table 4.1 (see Materials and Methods) showed that primary sludge fermentation (in A side) improved the phosphorus removal efficiency, as indicated by a lower soluble effluent phosphorus concentration. Also, the phosphorus content of the sludge from the A side was significantly higher than that of in the B side (4.0 vs 2.1%). The anaerobic soluble Pi concentration was also much higher on the A side than on the B side. These results are in agreement with literature information which states that greater anaerobic Pi release is associated with greater phosphorus removal in a bio-P plant. The difference in the magnitude of phosphorus release and uptake between the two sides can be seen in a Pi mass balance bar chart (Figure 5.41 -A; average for the period studied). The extent of Pi release and uptake was 3.5 to 4.5 times higher on side A than on side B. This chart also shows that, for both sides, 25% of the Pi uptake occurred in the anoxic zone. The PHA content of the sludge, which varied inversely to its Pi content, increased markedly in the anaerobic zone where simple "storable" substrates were added (Table 5.11). In the aerobic zone, where PHA were consumed, the PHA content reached a low value of 2.0 to 2.5 mg PHA as HB/g SS. Fermenter sludge samples showed a PHA content of 2.4 mg PHA as HB/g SS. Assuming that no PHA storage took place in the fermenter sludge, this level could be considered "background" level. The percent of the sludge PHA that consisted of PHV was higher on the A side, where fermented primary sludge was added (38 to 50%), than on the B side (19 to 23%). With the fermenter, an average amount of 8.3 mg V F A (as HAc) per liter of influent was added to the A side. Of this amount 63% consisted of acetate, 36% of propionate and about 1% of butyrate, iso-butyrate and valerate. Since PHV requires both acetate and propionate for its synthesis, the larger proportion of PHV in the A side sludge 109 Figure 5.41 Average, over the monitoring period, of A. Pi release and uptake and B. PHA storage and consumption in each reactor of the UBC pilot plant. 110 (with the fermenter) could be explained by a greater availability of propionate per acetate on the A side than on the B side. A mass balance on PHA concentration allowed determination of how much PHA storage or consumption took place in each zone (see Figure 5.41-B). PHA storage in the anaerobic zone and PHA consumption in the aerobic zone was 2.5 times larger in the A than in the B side. PHA consumption took place in both the aerobic and anoxic zones. As much as 20 to 30% (B and A side, respectively) of the PHA consumption took place in the anoxic zone. Table 5.11 UBC pilot plant average PHA content and %(PHV/PHA). February 9 to July 6 for A side, and February 9 to May 11,1987 for B side. Zone Anaerobic Anoxic Aerobic Fermenter avg (s) avg (s) avg (s) avg (s) PHA Content (mg HB/g SS) A side (n = 35) 10.8 (2.2) B side (n = 20) 5.0 (0.8) 4.6(1.1) 2.9 (0.5) 2.5 (0.5) 2.0 (0.3) 2.4 (0.9) % (PHV/PHA) (%) A side 50 (5) B side 23 (4) 43 (5). 22 (5) 38 (5) 19 (6) 41(6) A mass balance on Pi concentration indicated that 25% of the Pi uptake took place in the anoxic zone. The role of PHA storage at the pilot plant could only be investigated with "passively" obtained monitoring data. As a result, the effect of various factors on PHA storage and consumption could only be qualitatively estimated. Nevertheless, some significant correlations could be derived by pooling the results obtained on both sides. First, a very significant relationship (r = 0.98) could be drawn between Pi uptake and 'total Pi release (see Figure 5.42-A). Mass balance data were used for this graph with Pi uptake calculated as the sum of anoxic and aerobic uptake. The plot shows a direct association between anaerobic Pi release and total Pi uptake. Second, direct relationships were observed between anaerobic Pi release and PHA storage (r = 0.86) (Figure 5.42-B), and between total Pi uptake and PHA consumption (r = 0.84) (Figure 5.42-C). I l l UBC Pilot Plant - February to July 1887 • — I — i — i — i — i — i — I — i — i — i — i — i — i — i — i — r — i — i — i — 0 4 8 12 16 20 PI rolsas* ((mg P/l) / Oln) 30 o 20 Logond • Sid* A SldoB 10 slop* • 2.3 molss/molo i i 30 10 20 PHAs storsg* ((mg P/t) / Oln) 30 JE O a 20 -10 -slop* • 2.S mol*s/mol* i 1 1 1 i — 10 20 30 PHAs consumed ((mg HB/I) / Oln) Figure 5.42 Relationship and molar ratios for A* Pi uptake versus Pi release, B. Pi release versus PHA storage, and Q. Pi uptake versus PHA consumed for data obtained at the U B C pilot plant. 112 From the monitoring data available, however, only a poor correlation could be established between PHA storage and parameters estimating the supply of carbon in the influent: COD (r = 0.24), TOC (r = 0.10) and VFA (r = 0.57). An experiment designed where various amounts of simple substrates would be added could probably give a stronger correlation. In summary, results presented above indicated that PHB and PHV accumulation did take place in a continuous flow process treating domestic wastewater. The side where primary sludge fermentation was used to increase the supply of simple carbon substrates (A side), showed better phosphorus removal and more PHA storage than the side without primary sludge fermentation (B side). It was also shown, from the limited data available, that PHA storage could be predicted reasonably well from Pi release, and PHA consumption from Pi uptake. 5.3.3. Glycogen at the UBC Pilot Plant The basic requirement for bacteria to perform bio-P removal is that they should be able to accumulate both polyP and carbon reserves. These carbon reserves have been reported to be as PHA and sometimes in the form of glycogen. For glycogen to accumulate, however, it is expected that simple sugars such as soluble glucose, which can be readily stored as glycogen, be present in the feed (see Literature Review) and that acclimation of the biomass to the feed had occurred. No glycogen accumulation was expected at the UBC pilot plant because of the absence of sugar addition to the wastewater. Nevertheless, it was decided to verify this assumption by analyzing the glycogen and total carbohydrates content of the UBC pilot plant sludge. Samples (n = 3) were collected from each zone and stream of the pilot plant and analyzed for total carbohydrates, soluble carbohydrates and glycogen (see Table 5.12). The concentration of total carbohydrates in the influent indicated that primary sludge fermentation reduced the content of particulate carbohydrates by as much as 85 percent. This reduction could be correlated to the production of VFA in the fermenter. The amount of glycogen accounted for only 2 percent of the total carbohydrate content of the sludge, suggesting that total carbohydrates quantification may not always be sensitive enough to estimate indirectly the amount of glycogen (as is done by some researchers). No significant difference was found between the two sides of the pilot plant for the glycogen or soluble carbohydrates data. A significant difference was estimated between the two sides for total carbohydrates, however, even though the trends between reactors were not the same on each side and were also inconsistent with aerobic glycogen storage reported in the literature. Diurnal fluctuations in the wastewater particulate carbohydrates 113 concentration coupled with a lag due to the hydraulic retention time in each zone may explain this difference. Table 5.12 UBC bio-P pilot plant carbohydrates and glycogen content (December 7,1987). Content (mg glucose/g SS) Stream/ Zone total CHO sol. CHO glycogen Influent (concentration in mg glucose/1) fermented influent 7.2 9.2 raw influent 42.2 6.7 -Side -A- (with fermented influent) anaerobic 239 4.7 3.6 anoxic 236 4.3 3.7 aerobic 199 3.2 4.0 return Sludge 267 3.5 4.1 Side -B- (with raw influent) anaerobic 244 2.6 3.5 anoxic 290 3.0 3.3 aerobic 258 4.3 3.5 return Sludge 285 4.1 3.5 Note: sol. CHO stands for soluble carbohydrates. Therefore, the amounts of glycogen and of total carbohydrates did not appear to change as the sludge was exposed to anaerobic, anoxic and aerobic conditions through the UBC pilot plant. 5.3.4. PHA at the Kelowna, B.C. Full-scale Plant Characterization of PHA storage at the Kelowna plant was done by sampling each cell on two consecutive days (July 15 and 16, 1987). Soluble Pi sub-samples were collected from the same samples as those for PHA on July 15 (no Pi data on July 16). Data for nitrate concentration was that obtained on July 13 by plant staff. Similarly, MLSS values were interpolated at 1230 mg/l for the North side (cell IA), and at 1390 mg/l for the South side (cell 6A), from values obtained on July 14 and 16. The concentration profiles of soluble Pi, nitrate, and of the PHA content of the sludge are shown in Figures 5.43-A and 5.43-B for the North and South side, respectively. The range of percent PHV to PHA was between 34 and 46%. 114 Kelowna full-seal* treatment plant - July 15 & 16, 1987 10 -.. e i * o w o a i < 5 ¥ 1 . c C o X o A. a < 1 5 6 -4 -North Side A A A A LEGEND • PI * NO3 • PHA , July 15 A PHA , July 16 © o A A A -1—1—r ~i 1 1 1 1 1 1 1 1 1 1 1—1—T—1—r A3A2B2C2D2E2F2G2G3F3E3D3C3B3 G1F1E1D1C1B1A1 AN AX AX 0 2 AX 0 2 Reactors cell ID (see Fig. 4.7) and conditions (AN: anaerobic; AX: anoxic; 0 2 : aerobic) 2 » o w 0 a « ^ v Z " ? E 1 • . c C o v o 0. D> < £ 2 10 8 -8 -4 -South Side LEGEND • PI + NO 3 • PHA .July 15 * PHA , July 16 * * 8 * o « . 8 8 ' A A O ©• A A © © A A 1—1—1—1—r-i—1—1—1—1—f—T—•—1—T—1—1—1—1—r A4A5B5C5D5E5F5G5G4F4E4D4C4B4 G6F6E6D6C6B6A6 -T-f-AN AX AX 0 2 AX 0 2 Reactors cell ID (see Fig. 4.7) and conditions (AN: anaerobic; AX: anoxic; 0^. aerobic) Figure 5.43 Pi or nitrate concentration, PHA content for the North side and Eu the South side of the Kelowna full-scale bio-P treatment plant. 115 By looking at concentration data, it is difficult to establish whether Pi release or uptake, and whether PHA storage or consumption took place in a given cell. Mass balance calculations circumvent this difficulty by considering all inputs into and outputs from one cell on a mass basis, thus also accounting for different flow rates. To express the results relative to the influent flow rate, results were divided by the influent flow rate value of 127 1/s. Figure 5.44-A presents the Pi mass balance results. As expected, the largest amount of Pi release took place in the anaerobic zone. In the first anoxic cells (cells A2 to F2), net Pi uptake was observed, although some Pi release appeared to have occurred on the North side in cells E2 and F2. In the aerobic cells Pi uptake took place until no more Pi could be accumulated from solution. PHA mass balance results are shown in Figure 5.44-B and 5.44-C for the North and South side, respectively. Some data for the PHA mass balance plots were obtained by interpolation from Figure 5.43. Maximum PHA storage was observed in the anaerobic cell, for both days and in both sides of the bioreactor. In the anoxic cells, mixed reproducibility of the results suggested that either net PHA storage or consumption could take place. V F A production in these anoxic cells may explain why PHA storage could have taken place. In the aerobic zone, however, PHA consumption dominated. These general observations made on a full-scale bio-P treatment plant concur with more detailed batch experiments results. A plot of the mass balance for Pi versus that for PHA is presented in Figure 5.45. Pi release was generally correlated with PHA storage, and Pi uptake with PHA consumption. The slope of Pi over PHA gave 0.66 mg Pi/mg PHA which translates into a molar ratio of 2.2 moles Pi/mole PHA. This result was in close agreement with ratios found in batch experiments (see Discussion). 116 PHA* from Kilowm (July 18 and 16, 1*87) X o B X E < x a. a E 4 --4 -12 Legend • July 18 + July 16 estimated for day* 1 and 2 -16 -I—i—i—i—i—i—i—i—i—i—i—i i i—i—i—i—i—i—r-A 3 A 2 B 2 C 2 D 2 E 2 F 2 G . 2 a 3 F 3 E 3 D 3 C 3 B 3 Q 1 F 1 E 1 D 1 C 1 B l A 1 Reactor cell ID •16 H—i—i—i—i—i—i—r—i—i—i—i—i—i—p—i—i i i i A4A6B6C6D6E6F6G6G4F4E4D4C4B4G6F6EeD6C6B6A6 Reactor cell ID Figure 5.44 Mass balance for A. Pi, B. PHA (North module) and C. PHA (South module) data collected at the Kelowna full-scale bio-P treatment plant. Pi data was collected only on July 15, 1987. 117 Figure 5.45 General relationship between Pi and P H A for data obtained at the Kelowna full-scale bio-P treatment plant. 118 6. DISCUSSION Experimental results will first be integrated into a biochemical model summarizing anaerobic, aerobic and anoxic activity of bio-P bacteria. Then, results from a number of experiments will be compared, analyzed and their significance discussed. Molar ratios and rates of reaction will be used for that purpose. Difficulties of the model to explain some observations, a summary of microbial activity in a bio-P biomass, and finally, the implication of the concepts for full-scale design will be discussed. 6.1. Biochemical Model The biochemical model presented here for anaerobic and aerobic activity of bio-P bacteria is based on that presented by Comeau et al. (1986). Extensions include the proposition that propionate as well as acetate is a "storable" substrate, and that PHV as well as PHB is a stored carbon reserve. For bio-P biomasses acclimated to an influent wastewater containing other types of substrates, such as butyrate or glucose, Manoharan (1988) showed that these substrates could also become readily storable. There is always a difficulty in proposing biochemical pathways based on macroscopic observations made on a mixed culture activated sludge biomass. Nevertheless, the purpose of presenting this biochemical model is to integrate the information available from this thesis and the literature into a framework to allow better engineering applications and to provide hypotheses for future investigations. Difficulties of the model to explain some observations will be discussed further. 6.1.1. Anaerobic model a. Postulated model Figure 6.1-A summarizes the concepts presented below for a biochemical model of bio-P bacteria subjected to anaerobic conditions. An observation that is consistently made is that the addition of simple carbon substrates to a sample of bio-P sludge results in Pi release. From the results presented in this thesis, the two substrates that played the most central role were acetate and propionate. Their addition always resulted in the fastest rate of substrate uptake and of Pi release. Other simple substrates are proposed to be first "fermented" to acetate and propionate which bio-P bacteria can then use. The activated sludge used to draw these conclusions was taken from the UBC pilot plant that treated domestic wastewater (and not synthetic sewage largely composed of glucose, for example) and, thus, should be reasonably representative of the activity of bio-P bacteria in municipal full-scale plants. 119 acatat* and PfM* carbon Figure 6.1 Biochemical model of bio-P bacteria for A. anaerobic and B. aerobic (and anoxic) conditions. Abbreviations: Ac", acetate; E.T.C., electron transport chain; M + or Met + , metallic cation; PHAs, poly-/3-hydroxyalkanoates; Pi", Pi; pmf, proton motive force; Prop", propionate; T C A cycle, tricarboxylic acid cycle. 120 To be stored as PHA, substrates must first be transported into the cell. Several monocarboxylic acids are transported neutrally across the membrane if an appropriate pH gradient exists (Kaback, 1976). With acetate transport into the cell followed by intracellular acetate dissociation, a decrease in the pH gradient of about one H + for each acetate transported is expected. This decrease in the pH gradient will reduce the pmf which cells tend to maintain at a fairly constant level (Bakker and Mangerich, 1981; Schuldiner and Padan, 1982). Unless bacteria regenerate the pH gradient, acetate uptake by the pH gradient-dependent mechanism, and consequently the ability to increase carbon storage as PHA, will quickly cease. Three mechanisms presented in the bioenergetics section of the Literature Review are available to bacteria to re-establish the pH gradient. Under,aerobic conditions, H + can be expelled by the electron transport chain, a mechanism that cannot function under anaerobic conditions. Another way to eject H + involves breaking down ATP by the membrane-bound ATP-ase enzyme (Harold, 1977). With acetate as substrate, however, ATP could not be regenerated by fermentation and any cellular ATP would be rapidly depleted. A third mechanism involves the utilization of N A D H at the membrane-bound transhydrogenase enzyme to expel H + (Harold, 1977). N A D H could be provided by feeding acetyl GoA into the TCA cycle as can be done by anaerobic bacteria. Acetyl CoA, in turn, could be produced from the energization of acetate by polyP as will be discussed later. This mechanism may be available to bio-P bacteria and should be kept in mind in future investigations. However, the utilization of acetyl CoA to produce N A D H for the regeneration of the pH gradient does not give a direct role to polyP reserves and cannot explain why so much Pi is released upon substrate addition. Finally, as a fourth mechanism, it is proposed that polyP is used to expel protons across the cytoplasmic membrane by a translocating enzyme. Evidence for this mechanism arose from the observations that polyP was degraded in the presence of 2,4-DNP (section 5.2.8.a), at a high pH (section 5.2.8.b), and in the presence of high levels of acetate (or propionate) under any of anaerobic, anoxic or aerobic conditions (section 5.2.1, 5.2.2, Gerber et al, 1986, 1987a, 1987b). As mentioned in the section on bioenergetics, ATP breakdown can be used to expel protons by reversing the normal function of the ATP-ase enzyme (Harold, 1977). ATP could be synthesized from polyP under anaerobic conditions as a result of the low intracellular ATP/ADP ratio (Kornberg, 1957; Kulaev, 1975). Alternatively, an enzyme similar to the ATP-ase could use polyP directly for proton translocation. It is possible that such an enzyme would be induced under "stringent" conditions of energy limitation when the synthesis of other enzymes is repressed, as is the case for enzymes produced when bacteria are starved for amino acids (Cozzone, 1981; Nierlich, 1978). In fact, Barsky et al (1975) and Moyle et al. (1972) reported that 121 pyrophosphate could be used by some bacteria for proton translocation. Thus, it is conceivable that longer chains of polyP could also play a similar role to pyrophosphate although such enzymes have yet to be detected. Mino et al. (1985) reported that both low and high molecular weight polyP occurred in bio-P bacteria and they suggested that the low molecular weight polyP functioned as an energy pool under anaerobic conditions while high molecular weight polyP functioned as reserves mainly for nucleic acid synthesis. PolyP degradation for energy results in Pi accumulation in the cell. Although bacteria have inorganic Pi pools (Rae and Strickland, 1975), like any other unused metabolite, Pi would build up to a certain intracellular level above which it would be released in solution along its concentration gradient. To avoid wastage, inorganic Pi could be "sensed" by the cell to be non-essential under anaerobic conditions by having a Pi carrier protein that would be pH gradient-sensitive. With a reduced pH gradient, as is the case for bio-P bacteria under conditions of energy limitation, inorganic Pi could not be used for synthetic processes and would be released from the cell if the intracellular concentration exceeded a certain level. Conversely, under conditions of favorable pH gradient (such as under aerobic conditions), Pi release by this carrier would not occur. For the carrier enzyme to be pH gradient-sensitive would not be a unique phenomenon. For example, transport proteins have been reported to respond to changes of the pH gradient for inward transport of potassium (Bakker and Mangerich, 1981), to changes of the charge gradient for outward movement of sodium (Sorensen and Rosen, 1982), and to changes to the proton motive force for sugar uptake (Peterkofsky and Gazdar, 1979; Reider et al, 1979). Therefore, Pi release in itself is suggested to play a passive role in bio-P removal and the rate of Pi release would simply reflect the rate of polyP utilization by bio-P bacteria. It is also expected that the maximum extent of acetate accumulation by bio-P bacteria would be limited by the availability of polyP that could be used to re-establish the pH gradient and allow more acetate uptake. Indeed, it was shown that when an excess of acetate was added to a batch of sludge, Pi release took place up to a maximum level beyond which no further Pi release was observed, while any excess unstored acetate would remain in solution. Potassium, magnesium and calcium are proposed to be co-transported with Pi. Experiments showed that at neutral pH, about one cationic charge was provided per Pi molecule by these cations. Little information is available from the literature on Pi expulsion from bacteria as this is a relatively unusual phenomenon. Pi uptake information, however, indicates that a Pi-specific carrier would transport Pi as a neutral molecule with protons used to neutralize the negative charges of Pi (Harold, 1977; Kaback, 1979). Cations that accumulate intracellular^ because of polyP breakdown (cations stabilizing the polyP chains by binding to the negative charges of Pi) could then be expelled from cells by 122 cation/proton antiport systems (Sorensen and Rosen, 1982). The net effect would be the apparent co-transport of Pi and metallic cations. Alternatively, potassium, magnesium and calcium used to neutralize Pi during its transport across the cell membrane, could remain bound together while in solution, a hypothesis supported by the fact that the metals to Pi ratios for both uptake and release are very similar. A second use for the energy produced by polyP would be for the storage of acetate and propionate as PHA (see section on PHB metabolism in Literature Review). Acetyl CoA (and propionyl CoA) can be produced either directly from acetate (or propionate) or via the formation of acetyl Pi (or propionyl Pi). In both cases, energy in the form of ATP is required. The enzyme that catalyses the reaction from acetyl Pi to acetyl CoA is called phosphotransacetylase (Thauer et al, 1977). Lotter (1985) reported a significant activity of this enzyme in full-scale bio-P treatment plants. ATP could be generated from polyP under conditions of low energy (low ATP/ADP ratio). It is also conceivable that polyP could be involved directly in the energization of acetate. Finally, a source of NADH is required for the condensation of two acetyl CoA to form acetoacetyl CoA, and propionyl CoA and acetyl CoA to form propionoacetyl CoA. Feeding some acetyl CoA into the TCA cycle could provide N A D H at a rate just sufficient to resupply the amount utilized. Some C 0 2 production would then be expected from the added acetate. Such C 0 2 production was reported in a test conducted with radioactive 14C-acetate (see section 5.2.1.e; and Bordacs and Chiesa, 1987) that provides support for this possible mechanism. These precursors would then be attached to a growing chain of PHA (PHB and PHV) co-polymers (Holmes, 1981). PHB and PHV were the only two PHA compounds found. In summary, it is postulated that under anaerobic conditions, acetate and propionate will be transported as neutral molecules, decreasing the pH gradient of bacteria. Bio-P bacteria will be able to utilize their polyP reserves to re-establish the pH gradient possibly directly or via the production of ATP. PolyP can also provide energy for the formation of acetyl CoA and propionyl CoA. Storage as PHB and PHV requires NADH, which the T C A cycle can produce anaerobically. Pi expulsion takes place because of the excess of Pi molecules accumulating in the cell. A pH gradient-sensitive carrier could "sense" that Pi cannot be used for synthesis. Metallic cations would be co-transported with Pi. b. Anaerobic energy balance for bio-P bacteria Based on the above biochemical model, and principles of bacterial transport and bioenergetics (see Literature Review), a table of energy balance for anaerobic PHB storage of acetate by bio-P bacteria was prepared (Table 6.1). Numbers derived from this table will be used in the following sections to predict molar ratios under conditions of 123 anaerobic acetate addition. It is expected that similar numbers would apply for acetate and propionate storage as PHV. Table 6.1 Proposed energy balance for anaerobic P H B storage with acetate as substrate (Comeau et al., 1987a). Reaction Energy consumed N A D H produced Storage of 4 PHB monomers: A) HB storage (4 times): 8 Ac"--> 8 HAc (transport) 8HAc--> 8 AcCoA 8 AcCoA -> 4HBCoA 8 x 0.5 ATP (assuming 2H + /ATP) 8 ATP 3 NADH + 1FADH„ B) N A D H (and FADH 2 ) production by the TCA cycle 1 Ac" -- > 1 HAc (transport) 0.5 ATP 1 HAc--> 1 AcCoA 1ATP 1 AcCoA--> TCA cycle 3 N A D H +1 F A D H n NET BALANCE: Ac" transport: 13.5 ATP (that is, 3.4 ATP/HB) Abbreviations: Ac": acetate ion; AcCoA: acetyl CoA; ATP: adenosine triphosphate; F A D H 2 : reduced flavin adenine dinucleotide; HAc: acetic acid; H + : proton (hydrogen ion); HB: /3-hydroxybutyrate; HBCoA: /3-hydroxybutyryl CoA; N A D H : reduced nicotinamide adenine dinucleotide; TCA: tricarboxylic acid (cycle). 6.1.2. Aerobic model a. Postulated model Figure 6.1-B summarizes the concepts presented below for a biochemical model of bio-P bacteria subjected to aerobic conditions. This aerobic model was simpler to propose than the anaerobic model for bio-P bacteria because no new metabolic pathways had to be proposed to explain some observations. Essentially, upon entering the aerobic zone, bio-P bacteria will have accumulated PHA but have limited polyP reserves. With oxygen available they can now generate energy by the consumption of available external carbon substrates and internal PHA reserves. A major pathway of energy production will be via the TCA cycle for N A D H production. This 124 N A D H can be used at the electron transport chain, in the presence of an electron acceptor, to create a proton motive force. The proton motive force, in turn, can be used for Pi transport and ATP production. ATP can then be used for synthesis and growth, as well as for polyP accumulation. Metallic cations would be co-transported with Pi to stabilize growing polyP chains. In section 6.3, it will be discussed that a high concentration of substrate could result in concurrent PHA storage and Pi release in the presence of oxygen (or nitrate) when the soluble substrate concentration is high enough. b. Aerobic energy balance for bio-P bacteria Based on the biochemical model presented above, an energy balance can be proposed for the aerobic consumption of PHB by bio-P bacteria (Table 6.2). It was assumed, in constructing this table, that each NADH would yield 3 ATP molecules. Thus, each acetyl CoA could produce 12 ATP. It is known, however, that some bacteria can only produce 2 ATP per molecule of NADH. Thus, a total of 9 ATP would then be generated for each acetyl CoA. The aerobic consumption of PHV should give an energy balance similar to that of PHB. Numbers derived from this Table will be used in the following section to predict molar ratios. It is assumed that similar numbers would apply for aerobic PHV degradation. Table 6.2 Proposed energy balance for aerobic PHB consumption (Comeau et al., 1987a) Reaction Energy consumed Energy produced PHB degradation: PHB--> HB HB --> 2 AcCoA 2 AcCoA -> T C A cycle 0 2 ATP 0 l N A D H - > 3 ATP 2 x 12 ATP NET BALANCE: 25 ATP PolyP accumulation: Pi transport Pi --> ATP--> polyP 0.5 ATP (assuming 1 H + per Pi) 1.0 ATP NET BALANCE: 1.5 ATP Abbreviations: see Table 6.1. 125 6.1.3. Anoxic model The anoxic model for bio-P bacteria is quite similar to the aerobic model and differs only in that nitrate instead of oxygen could be used by those capable of nitrate reduction. This difference is shown in Figure 6.1-B. Under anoxic conditions, the activity of bio-P bacteria unable to use nitrate would be similar to that of bio-P bacteria subjected to anaerobic conditions. Thus, the net effect on Pi accumulation or release into solution would depend on the relative mass and activity of these two groups of bio-P bacteria as a function of substrate and nitrate availability. It was shown that nitrite could not induce Pi uptake and thus, that it could not serve as an electron acceptor. Nevertheless, nitrite reducing bio-P bacteria could possibly be found in bio-P treatment plants where significant amounts of nitrite are present in the influent or produced by the sludge. 6.2. Phosphate and PHA In this section, a summary of results will first be presented. Molar ratios and rates of reaction obtained for a number of experiments will then be compared, and their significance discussed. 6.2.1 Synthesis of results a) Methods i) PHA quantification A G C method was developed by adapting that of Braunegg et al. (1978). This method allowed the quantification of PHB and of PHV in bio-P sludges. An indirect technique was developed to quantify PHV (see Materials and Methods). ii) PolyP estimation It is proposed that the anaerobic addition of an excess of acetate can give an estimation of the amount of polyP available for substrate uptake and storage as PHA. Samples can be taken at various times until no more increase is observed in the soluble Pi concentration (after 4 hours for example). Some residual acetate (e.g. minimum of 20 mg COD/1) should always remain in solution to ensure that acetate is non-limiting. The total amount of Pi released should give an estimate of the amount of polyP available for substrate transport and storage. For example, in the case of a reactor that released a maximum of 62 mg P/l, it was estimated that the polyP used for substrate uptake made up 1.8% of the sludge mass and 126 that this polyP fraction represented 53% of the total aerobic P content of the sludge which was 3.4% P/SS. It was shown that complete aerobic Pi release without any substrate addition could take more than 10 days (Figure 5.15). With acetate addition, however, this time can be reduced to just a few hours. With sludge containing as much as 12% phosphorus (Randall, 1988), the amount of acetate required for complete polyP degradation could be as high as a few hundreds mg COD/1. In wastewaters containing cations that could precipitate some of the released Pi, the polyP content estimated by this procedure could possibly underestimate the actual polyP content, depending on the pH. If the sludge was completely denitrified prior to acetate addition, the maximum rate of Pi release for acetate uptake could be calculated and compared to results obtained here (see further, Figure 6.5-B). For this calculation to be meaningful, it would be important to use a reactor system that ensures positive air exclusion with a properly sealed vessel, and to use a sampling technique that precludes any air entrainment. Finally, this technique would only provide an estimate of the amount of polyP available for acetate (and other substrates) accumulation. Other polyP reserves could be used strictly as Pi reserves for cellular material (Groenestijn 1988; Mino et al, 1984, 1985). iii) Wastewater characterization by its fermentability It is difficult to measure the "fermentability" of some wastewaters (or primary sludge) because a portion of the produced VFA are further metabolized and, thus, cannot be detected. An indirect method to characterize wastewater fermentability was used in which the wastewater to be characterized was combined with some bio-P sludge containing fermentative microorganisms (see section 5.2.5). The presence of significant numbers of fermentative microorganisms can be ensured by the presence of a primary sludge fermenter in the process that is used as a source of bio-P sludge. In the experiment reported, no VFA were detected in solution, but significant Pi release over 18 hours was observed which was attributed to the production of VFA and their rapid uptake by bio-P bacteria. This technique could be used to characterize the fermentability of various municipal or industrial wastewaters. b) Anaerobic substrate addition The anaerobic addition of a variety of substrates resulted in Pi release, as reported by a large number of authors, and also in the accumulation of PHB and PHV. No other PHA could be found from the GC chromatograms or by GC/MS. Experiments in which an excess of acetate was added indicated that polyP reserves were finite and that they limited the amount of acetate or other substrate that could be taken up (see section 5.2.l.c). This characteristic was proposed as a tentative method to assess the amount of polyP available for substrate accumulation (see section 6.2.1.a.ii). 127 The addition of radioactive acetate showed that acetate was removed from solution and found as particulate matter (presumably PHA; it would have been interesting to quantify the proportion of isotope channelled into PHB and PHV). Some radioactive C 0 2 was also found which supported the concept of the TCA cycle being used anaerobically for N A D H production (see biochemical model, section 6.1.1.a). The combined addition of acetate and propionate resulted in the highest rates of substrate uptake and PHA storage (see also section 6.2.3). These observations provided support to the proposition of Holmes (1981) that PHV, a 5-carbon compound, required the condensation of derivatives of acetate and propionate (acetyl CoA and propionyl CoA) (see Figure 6.2). The energy for the transport and energization of acetate and propionate was proposed to originate from polyP degradation. The addition of propionate alone resulted in PHV formation. Since the storage of a PHV monomer required the condensation of one acetate with one propionate molecule, some of the added propionate was probably degraded to acetate. The slower rate of PHV storage with propionate addition when compared to acetate + propionate addition could indicate that PHV storage was rate-limited by propionate degradation to acetate. The magnitude of PHA storage with propionate addition was only about one third the amount of PHA stored with acetate addition (see Figure 5.18). For both substrates, however, the magnitude of subsequent aerobic uptake was the same, although at a slower rate with propionate. It is unclear why so little PHA storage occurred with propionate, but, as observed by Manoharan (1988), it is expected that acclimation to propionate could have resulted in a much greater magnitude of PHA storage. The addition of acetate or propionate under aerobic conditions resulted in Pi release unless polyP reserves had been depleted (see section 5.2.2). This result was also observed in pure cultures Acinetobacter to which acetate had been added (Wentzellef al, 1986). Gerber et al (1986, 1987a), studying the addition of various substrates to mixed cultures, showed that only acetate or propionate could induce aerobic or anoxic Pi release. Graphical results presented by Gerber et al (1986) indicated that formate could also result in some anoxic Pi release. Other compounds such as ethanol, glucose, lactate, butyrate, succinate, 2,3-butanediol, methanol and citrate could only induce anaerobic and not aerobic or anoxic Pi release. c) Sequencing batch reactors The operation of four SBR at continous levels of acetate addition (0, 15, 30 and .45 mg COD/1) confirmed the correlation between the storage/consumption of PHB/PHV with the degradation/accumulation of Pi. A one percent increase in the phosphorus content of the sludge required the extra addition of 25 to 30 mg acetate as COD per litre of influent. 128 A c A c A c ^ -hydroxybutyra te (PHB) Prop b io -P bacter ia j j - h y d r o x y v a l e r a t e (PHV) : V p o l y - |j -hydroxyalkanoates (PHA ) ( c o - p o l y m e r ) ^ •energy f rom polyP Figure 6.2 Proposed simplified pathway of carbon storage as PHB and PHV. 129 The same apparatus operated by Vlekke (Vlekke et al, 1988) showed that nitrate could be used as the sole electron acceptor for bio-P removal. He also confirmed that PHA storage/consumption and Pi release/uptake took place with nitrate as with oxygen. d) UBC pilot plant and Kelowna full-scale plant PHA monitoring at the UBC pilot plant and at Kelowna confirmed the anaerobic storage/consumption of PHB and PHV under anaerobic/aerobic conditions (sections 5.3.2 and 5.3.4). At the pilot plant, mass balance data indicated that about 25% of the Pi uptake and of the PHA consumption took place in the anoxic zone. For the five months period studied, the presence of a fermenter (A side) compared to the Control side, resulted in the addition of 8.3 mg VFA/1 of influent (expressed as acetate and composed of 63% acetate, 36% propionate), an increased total phosphorus removal of 1.3 mg P/l (2.7 - 1.4), an extra 2.0 %P aerobic sludge content (4.0 - 2.0), and in an extra 12.9 mg HB/1 of PHA storage or consumption (18.1 - 5.2). Thus, a 1.0 %P increase required an extra 8.7 mg COD/1 of V F A for the pilot plant, 15 mg COD/1 of acetate in a continuous lab-scale bio-P plant (Manoharan, 1988), and 27 mg COD/1 of acetate for the SBR. Considering that 1.5% phosphorus represents the "normal" background phosphorus content of a biomass, the 4.5% phosphorus measured at the Kelowna bio-P plant (see Table 4.2) represented a 3.0% excess P storage. Predictions of the amounts of COD added as VFA would then be 26 mg COD/1 from the pilot plant's data, 45 mg COD/1 from Manoharan's, and 81 mg COD/1 from the SBR's data. Two factors could contribute to explain such differences. First, the amount of COD required, as predicted from the pilot plant data, could be too low because the amount of storable compounds actually added at the pilot plant could exceed that actually measured as VFA. Dead zones where fermentation would take place within the anaerobic reactor could also support this concept. Alternatively, lab-scale data could overestimate the amount of VFA required because of experimental difficulties due to air entrainment, which would result in the consumption of some of the added VFA. More investigation on this aspect would certainly be desirable considering its importance to process design. No glycogen or total carbohydrates storage/consumption could be seen at the UBC pilot plant which treated a wastewater that was strictly residential (section 5.3.3). e) Metallic cations The metallic cations potassium, magnesium and calcium appeared to be co-transported with Pi both for import and export from the cells. Molar ratios obtained from a number of experiments (see next section) indicated that, at a near neutral pH, an average of 0.91 charges were contributed by these cations. Potassium accounted for about 25 to 130 30%, magnesium for about 50% and calcium for about 15% of the neutralizing charges (see section 6.2.2.e). With 10 mM 2,4-DNP addition (added as a neutral solution) an initial but transient release of potassium of 20 mg/1 was observed. In this case, the release of potassium could be attributed to the tendency of bacteria to counteract the decrease in the proton gradient by increasing the charge gradient with the expulsion of potassium. In the pH 5 reactor an initial and transient release of 2.5 mg/1 of calcium was observed. Since the bacterial cell wall contains stabilizing divalent cations (Hancock, 1984), this transient calcium release could be attributed to the solubilization of some of these stabilizing calcium ions. Besides metallic cation co-transport, Gerber et al. (1987a) recently pointed out that sulfate was also co-transported along with Pi. He did not propose any explanation for this observation, however. Unfortunately, sulfate was not monitored in this research. f) Oxidized nitrogen It was shown that nitrate but not nitrite could be used for Pi uptake and PHA consumption. A number of researchers have also reported that nitrate could be used as an electron acceptor to induce Pi uptake (Florentz et al, 1984; Hascoet and Florentz, 1985, Iwema and Meunier, 1985, with mixed cultures; Groenestijn and Deinema, 1985, with an Acinetobacter culture). Vlekke et al. (1988) also showed that a bio-P process could operate with nitrate as sole electron acceptor. Thus, at least some of the bio-P bacteria are facultative in that they are capable of nitrate reduction. The relative desirability of Pi uptake in the presence of nitrate in an anoxic zone, or with oxygen in an aerobic zone, can be considered from a theoretical standpoint for stored carbon consumption. With N A D H as electron donor, only 2 ATP are expected to be produced with nitrate as an electron acceptor instead of 3 ATP with oxygen (Brock et al, 1984). Painter (1970) and Payne (1981) also reviewed that the growth yield (wt. of cells/wt. of substrate) was lower with nitrate than with oxygen, the extra energy being wasted as heat. Thus, from the consumption of a given amount of stored carbon, less energy will be obtained from nitrate and it could be expected that less Pi could be taken up. For optimum bio-P removal, it would appear that denitrification could best be limited to the prevention of nitrate recirculation into the anaerobic zone (Comeau et al, 1987b). g) Toxicants In comparison to corresponding Control reactors, an increased rate of anaerobic Pi release was observed with 2,4-DNP, cyanide, or H 2S addition, and by the maintenance of a high pH. Pi release under anaerobic conditions indicated that polyP degradation could play a role in the regulation of the pH gradient. However, a low pH (4.0 or 5.0), 2,4-DNP at a neutral pH, C 0 2 , or fluoride addition did not result in an increased rate of Pi release. 131 With 2,4-DNP addition, protons are channelled across the membrane into bio-P bacteria via this toxicant, and polyP degradation could be used to re-establish the proton gradient by providing energy to the ATP-ase enzyme (see Figure 6.3). This mechanism of polyP degradation to re-establish the pH gradient could occur via ATP or maybe via the direct energization by polyP of the ATP-ase (or a related) enzyme. Bio-P sludge responded to high pH adjustment (pH 9) mainly by a transient potassium release, an increased rate of Pi release and a slower rate of magnesium release. Considering that a high pH affects the energized state of bacterial membranes by reducing their pH gradient, it was suggested that Pi release reflected the degradation of polyP to re-establish the pH gradient (see Figure 6.4). At a high pH, less magnesium appeared to be used for Pi transport. Rapid Pi release with H 2S could not be explained by the resulting low pH because of the observed negligible effect of low pH on Pi release. However, it is expected that H 2 S diffusion inside cells could reduce the internal pH by ionic equilibration. Thus, proton expulsion by the ATP-ase enzyme with polyP providing energy could explain the observed Pi release as well as the rapid and transient initial Pi release following H 2 S addition. This mechanism of energy consumption for proton expulsion to re-establish the pH gradient would be similar in the case of a high pH. Since sulfide has the chemical tendency to react with divalent cations to form stable precipitates, it is possible that other mechanisms could also be involved to explain the observed Pi release. Pi release with C 0 2 was only marginally greater than in the Control reactor. Fuhs and Chen (1975), however, reported a sudden Pi release with C 0 2 addition. They proposed that the low pH resulting from these conditions would resolubilize precipitated Pi molecules. In our experiments, we observed that only a limited release of Pi occurred with C 0 2 addition, and that transient Pi and calcium release took place at a low pH. At a low pH (with HC1 addition), only an increased rate of potassium and magnesium export was reported, however. Thus, it can be proposed that the solubilization of externally bound Pi and, calcium could explain the transient Pi and calcium release that we observed, and that the maintenance of a constant proton motive force by cation expulsion could explain potassium and magnesium expulsion. Assuming that C 0 2 diffused inside bacteria and affected the pH gradient, a similar mechanism of proton expulsion to re-establish the pH gradient could explain the extra Pi release. The slower rate could be attributed to the relatively slow reaction of C 0 2 hydration. Based on the above observations on the effects of 2,4-DNP, high pH and H 2S addition on anaerobic Pi release, it was proposed that a pH gradient-sensitive carrier could have "sensed" a decrease in pH gradient and resulted in the coupling of polyP degradation with proton expulsion (either directly or via ATP and the ATP-ase enzyme). 132 Figure 6.3 Proposed model of the effect of 2,4-DNP on bio-P bacteria. H2° Pi Figure 6.4 Proposed model of the effect of a high pH on bio-P bacteria. 133 Anaerobic Pi release by cyanide addition was also observed in one batch experiment but could not be explained by the above mechanism. The physiological effect of cyanide on bacteria is to inhibit electron transport by interfering with the action of electron carriers (see Literature Review). Cyanide binds tightly to the iron of the porphyrin ring of the cytochromes and prevents its oxidation and reduction (Brock et al, 1984). On this basis, aerobic energy production should be severely limited and it can be understood why aerobic Pi uptake and PHA consumption was inhibited. However, under anaerobic conditions, in the absence of electron acceptors, no electron carrier activity is expected and it is unclear why so much Pi release and PHA storage took place. The increased PHA storage also suggests that cyanide stimulated acetate and propionate production by fermentative bacteria. Some increased Pi release and PHA storage was observed with 10.0 mM fluoride addition. The physiological effect of fluoride is to inhibit glycolysis by preventing the production of phosphoenolpyruvate from 2-phosphoglycerate. The enzyme catalyzing this reaction requires magnesium which is tightly bound by the fluorophosphate ion, the true inhibitory agent that is made from fluoride plus Pi (Lehninger, 1982). Based on the observed increased PHA storage, it is possible that fluoride could have stimulated acetate and propionate production by fermentative bacteria which could also explain the extra Pi release. It is unclear, however, why fluoride would have stimulated V F A production. Considering that Pi uptake and PHA consumption were not inhibited by fluoride, it can be suggested that aerobic energy production from PHA by bio-P bacteria did not depend on the glycolysis pathway, as expected from the pathway of PHB degradation into acetyl CoA. Formaldehyde (at 100 mg/1) was found to be a very effective and complete inhibitor of microbial activity. Its addition under anaerobic conditions prevented any Pi release. These characteristics were useful in an experiment with radioactive 14C-acetate to assess the degree of adsorption of the added isotope by inert biomass (see Section 5.2.1.e.i). The toxicity of various metallic cations was studied in batch experiments by Hascoet et al (1985). Various levels of metal were added at the beginning of a 3 hour anaerobic period along with 300 mg COD/1 of beef extract. Metal toxicity was evaluated by the inhibition of subsequent aerobic Pi uptake. They reported maximum admissible doses of 1 mg/1 for copper, 10 mg/1 for cadmium, 5 mg/1 for nickel and 5 mg/1 for zinc. No effect was found with 100 mg/1 of lead. 6.2.2. M o l a r ratios a) Summary of values Molar ratios were calculated for a number of experiments and are summarized in Table 6.3-A, -B and -C. 134 Table 6.3-A Summary of molar ratios for batch experiments. ANAEROBIC AEROBIC Section/ P i ^ P H V S T 0 / P U E L / P w / P H A c S T 0 / P i p p T / P i u p T / Date/ P H A S T 0 P H A S T 0 Subsu p T SubsCW P i R Er PHA C 0 N S COD added of (net) (net) (net) (net) (net) (total) (total) substrate __, _ _ _ (mgP/1) (%) (mol/mol) (mol/mol) (%) (%) (mol/mol) theoretical values for - 0 3.4 1.69 89 700 16 acetate (see sections 6.1.l.b, 6.1.2.b) (max) (max) (max) (max) 5.2;l.a.i; October 23,1985 (75% aerobic sludge + 25% wastewater) Control (10) (76) (7.0) n/a n/a 173 5.2 25 acetate 11 14 2.3 0.9 81 125 3.1 50 acetate 22 20 1.8 2.1 250 108 2.1 75 acetate 29 16 1.7 1.4 177 102 1.9 5.2.1.b.i; February 23, 1987 Control (5) (85) (5.0) n/a n/a 100 3.4 30 acetate 25 15 1.8 1.2 138 101 3.1 5.2.1.c.i; April 24,1984 Control (12) n/a n/a n/a n/a 110 n/a 25 acetate 21 n/a n/a 1.2 n/a 110 n/a 50 acetate 35 n/a n/a 2.0 n/a 125 n/a 75 acetate 48 n/a n/a 0.5 n/a 116 n/a 100 acetate 48 n/a n/a 0.5 n/a 87 n/a 5.2.1.C.H; May 26,1986 (SBR) Control (7) (57). (2.5) n/a n/a n/a n/a 15 acetate 15 7 2.0 0.9 92 n/a n/a 30 acetate 24 44 3.6 2.8 175 n/a n/a 45 acetate 30 30 3.3 2.8 180 n/a n/a 5.2.1.c:ii; May 26,1986 (SBR with acetate added in excess) exc. ac to Contr. 5 18 0.5 0.1 36 n/a n/a exc. ac to 15 Ac 13 8 2.4 0.4 36 n/a n/a exc. ac to 30 Ac 25 18 1.1 1.1 199 n/a n/a exc. ac to 45 Ac 29 18 0.8 0.7 181 n/a n/a (excess acetate added to sludge from each SBR) 5.2.1.e.i; November 4,1985 (75% aerobic sludge + 25% wastewater) Control (14) (91) (15.7) n/a n/a 151 6.3 50 acetate 28 15 1.6 1.1 141 114 2.0 135 Table 6.3-B Summary of molar ratios for batch experiments. Section/ Date/ COD added of substrate Pi R E L (net) A N A E R O B I C / P I R E L Subs (net)"\ (neif~ (net) P H V S T 0 / P i R E L PHAgrpQ PHAgrpQ / A E R O B I C P H A C S T 0 / P i y p T / Pi (net) U P T SubsCyP T P i R E T PHA" UPT' (total) CONS (total) (nig P/l) (%) (mol/mo!) (mol/mol) (%) (%) (mol/mol) 5.2.2.i; July 8, 1987 Control (10) (57) (10.4) n/a n/a 92 4.4 20 acetate 22 15 2.6 1.4 112 111 4.1 60 acetate 41 12 3.9 2.3 122 122 3.3 100 acetate 42 11 3.2 3.9 251 115 3.4 5.2.3.i (and 5.2.1.a.ii); August 19, 1986 (0.55 mM of pH 7.0 solutions added) Control (4) (77) (2.0) n/a n/a 55 1.3 35 sodium acet 24 29 1.7 1.4* 172* 54 1.4 35 acetic acid 21 27 1.6 1.2* 167* 67 1.5 62 Na propion 24 84 3.9 1.4* 58* 52 2.4 62 prop acid 23 91 3.7 1.3* 60; 41 1.8 18 acet+ 31 prop 34 4 3.8 2.0* 83* 4 assuming complete substrate uptake in 3.5 hours of anaerob ic conditions 5.2.3.H; March 3, 1987 Control (4) (68) (7.2) n/a n/a 63 2.1 30 acetate 26 16 2.4 1.6 142 102 3.2 20 acet+lOprop 23 59 2.0 2.0 193 104 3.3 10 acet+ 20 prop 22 87 2.1 1.3 117 105 3.6 30 propionate 21 97 5.0 1.4 45 94 6.7 5.2.3.iii; July 8, 1987 Control (10) - (57) (10.4) n/a n/a 92 4.4 20 acet+ 20 prop 33 58 2.0 ' 1.2 107 113 4.8 20 propionate 18 98 6.1 1.7 47 100 7.8 40 propionate 30 115 13.6 1.8 22 112 8.0 5.2.3 .iv; March 3,1987 Control (4) (68) (7.2) n/a n/a 63 2.1 30 valerate 8 99 1.9 3.5 178 108 2.9 29 formate 10 91 3.8 0.31 321 53 2.3 22 ferm.prim.sl. 6 70 1.3 0.52 702 115 3.2 34 lactate 20 84 3.6 2.13 923 88 3.3 28 butyrate 20 47 6.0 3.8 71 93 5.9 hht determination of formate could be inaccurate; Supposing that only acetate and propionate made up the 22 mg COD/1 (9 and 13 mg COD/1 of acetate and propionate, respectively); s^upposing that lactate cone, at time 8 h was 4.5 mg HLact/1 136 Table 6.3-C Summary of molar ratios for batch experiments and continuous operation. ANAEROBIC AEROBIC Section/ Piwa. P H V S T 0 / P i R E L / P i R E L / P H A c S T 0 / P i U P T / P i U P T / Date/ P H A S T 0 P H A S T 0 Subsu p T SubsC;™ P i R E r PHA COD added of (net) (net) (net) (net) (net) (total) (total) substrate (mgP/1) (%) (mol/mol) (mol/mol) (%) (%) (mol/mol) 5.2.4.b.i; August 19, 1986 Control (4) (77) (2.0) 9 Hformic 0 13 -1.0 88 butyrate 25 37 3.7 88 Hbutyric 26 50 3.0 114 Hvaleric 4 96 0.4 141 Hhexanoic 5 29 0.7 88 Hiso-butyr 13 18 1.9 114 Hiso-valer -2 0 -1.9-45 Hlactic 17 35 1.1 79 citrate 7 67 3.0 53 ethanol 6 58 1.8 35 glycine 10 86 3.3 106 glucose . 2 63 0.5 Notes: - 0.55 mM of pH 7.0 solutions added; 5.2.4.b.ii; October 28, 1986 Control (9) (23) (6.9) 72 0-OH butyrate 30 0 1.6 56 succinate 28 205 39. 5.3.1.a; SBR weeks 9-13,1986 for all SBR — 17 2.4 5.3.2; UBC pilot plant; February to June 1987 side "A" -- 43 2.3 side "B" — 23 2.35 0.0, 1.4 1.5* 0.21 0-3, 0-7 -0.1 1.0 0.4, 0-3, 0.6 0.f n/a 1.5 1.33 n/a n/a n/a, «: 57. 4s°: 130. 10, 42. 27. 20 n/a, 103. 6 n/a n/a 55 39 57 66 56 83 37 39 57 42 50 24 81 122 88 78 100 110 110 1.3 0.8 9.0 8.5 0.8 0.9 8.3 1.8 0.9 1.8 1.9 1.0 1.1 6.5 2.6 14.0 0.56 2.5 2.5 5.3.4; Kelowna full-scale plant, July 13 and 14, 1987 North & South -- 40 2.2 .n/a n/a n / a 2.2 'assuming complete substrate uptake during the anaerobic period (3.0 to 3.5 h). 137 Ratios for the anaerobic period were calculated with "net" values corrected for the Control. For example, if the Pi release was 30 mg P/l in the reactor to which acetate was added and was 4 mg P/l in the Control reactor for the corresponding period, the "net" value used in calculations for the acetate reactor was 26 mg P/l. The same concept was applied to PHB and PHV storage. Ratios given for a Control reactor were based on "total" values and are reported between parentheses. All ratios for the aerobic period, however, were based on "total" values and thus, were not corrected for the corresponding Control value. It was reasoned that once aeration started, bio-P bacteria will not make use of PHA reserves accumulated from substrate addition any differently than PHA from fermentation. Thus, reported ratios were based on "total" values to indicate the efficiency of Pi uptake and PHA consumption. Amounts of substrate added were expressed as mg COD per liter of reactor. A Table of the relationship between mg acid/1 and mg COD/1 can be found in Appendix A-2 for all.substrates used. For the anaerobic period, the net amount of Pi released from the time of chemical addition to the end of the anaerobic period is given to indicate the magnitude of Pi release. Molar ratios are given for the percentage of PHA stored as PHV ( P H V ^ Q / P H A ^ Q ) , for the number of moles of Pi released per mole of PHA stored ( P I ^ L / P H A ^ Q ) , for the number of moles of Pi released per mole of substrate taken up (Pij^/SubSypp), and for the number of moles of carbon stored as PHA per mole of carbon taken up from the added substrate (PHAC S T O/SubsCU P T). When a measured substrate concentration was not available for this calculation, it was assumed that all of the added substrate was consumed by the end of the anaerobic period. It is in fact possible that all of the substrate had not been taken up, and by that time, some Pi release and PHA storage could have taken place that was not due to the added chemical. To indicate these possible inaccuracies, an asterisk (*) is shown beside such ratios. For the aerobic period, ratios are given for the percentage of Pi taken up per Pi released (Pijjpp/Pij^) and for the number of moles of Pi taken up per mole of PHA consumed ( P i U F r / P H A C O N S ) . A theoretical estimation of each ratio was made with acetate on the basis of the proposed energy balance for anaerobic and aerobic activity (see previous sections). Predicted values are reported in the first line of the Table. b) Acetate Average values from many experiments in which acetate was added, were calculated by using only the values obtained with the sludge from the pilot plant (not using the data from the SBR sludge). For each experiment with acetate, only one value was calculated and used in the text below (for average and standard deviation calculations) and in Figure 6.5. 138 A1 + Net Pi release (mg P/l) Figure 6.5 Molar ratio of PHA storage over Pi release calculated from many experiments with acetate addition. In A. the data used was that obtained shortly (usually 0.5 hour) after acetate addition, whereas in B. the data used was that obtained at the end of the anaerobic period, after usually 3 hours. The point identified as A l was one for which data was only obtained at the end of the anaerobic period. Points identified as W l to W4 corresponded to the reactor in which wastewater composed 25% of the reactor volume at the beginning of the experiment. Only one data point was obtained from a given reactor in an experiment. 139 The percentage of PHA stored as PHV is expected to be 0% since acetate should only be stored as PHB. However, the ratio averaged 17% (s = 6%). Considering that ratios were calculated as "net" values (correcting for PHA storage in the Control reactor), the addition of acetate appeared to have stimulated increased propionate production by fermentative microorganisms. The predicted molar ratio of Pi released per PHA stored was estimated at 3.4, assuming that polyP degradation yielded one ATP per Pi hydrolyzed. Figure 6.5-A gives a visual summary for the data obtained to verify this ratio (its inverse in fact). Only one data point was obtained per reactor for many experiments in which acetate was added. The top two data points were excluded from the regression because of their significant (and unexplainable) deviation from the rest of the points. The best-fit line was forced through the origin because any amount of PHA storage was always associated with a corresponding amount of Pi release. The slope of the regression line was 1.39 mg PHA/mg P which corresponds to 2.4 moles P/mole of PHA, a value lower than the predicted 3.4 moles P/mole PHA (or 1.00 mg PHA as HB/mg P). Thus, energy use for PHA storage appeared to have been more efficient than predicted by the model. There was no obvious difference between the experimental conditions that existed in the reactors from which the three excluded points were obtained compared to conditions in the other reactors; that is denitrification was complete, pH was in the same range, etc. In two experiments, however, the combination of wastewater and sludge resulted in an increase of efficiency of PHA storage. The plot of Figure 6.5-B was prepared with data from the same experiments as for Figure 6.5-A with the difference that the data used were collected at the end of the anaerobic period (later than 3 hours after substrate addition) instead of shortly after substrate addition, when some substrate was still left in solution (usually at 0.5 hours after substrate addition). In Figure 6.5-A, the points identified Wl, W2, W3 and W4 were closer to the regression line than in Figure 6.5-B, where these points departed significantly from the best-fit line. Thus, the presence of wastewater in the sludge appeared to favor a more efficient PHA storage possibly because of the greater availability of fermentable products. The slope of the best-fit line was lower in Figure 6.5-B (1.09) than in Figure 6.5-A (1.39). As will be discussed in the next section, PHA storage was less efficient in the Control reactor than in the reactors that received acetate (0.47 mg PHA as HB/mg P which corresponds to an energy expense of 7.1 moles P/mole PHA). Thus, keeping the sludge under prolonged anaerobic conditions beyond that required for complete acetate uptake appeared to be detrimental to the overall efficiency of PHA storage. As seen earlier, however, prolonged anaerobic conditions in which fermentation of wastewater took place resulted in an improved efficiency of PHA storage. Therefore, it can be suggested that 140 PHA storage under prolonged anaerobic conditions can be favorable in the presence of fermentable substrates but unfavorable in their absence. In an experiment where an excess of acetate was added to sludge taken from four different SBR, the molar ratios of Pi released to PHA stored for each flask with an excess of acetate was only 30 to 60% of the value of the corresponding SBR to which the standard amount of acetate had been added. In fact, the extent of Pi release was similar between corresponding reactors, but the major difference was in the total amount of PHA stored. Thus, an excess of acetate in solution appeared to favor PHA storage, possibly because of lower energy requirements for acetate transport (one of the two proposed functions of s polyP in PHA storage). The predicted molar ratio of Pi released per acetate added was estimated at 1.69 (13.5/8). The average value obtained was 1.09 (s = 0.42) suggesting again a more efficient acetate storage than predicted. Data reported by a number of researchers were also used to estimate this ratio. Values ranged between 0.24 to 0.52 (Jones et al, 1985; Wentzell et al., 1985), 0.9 to 1.1 (Fukase et al., 1982; Potgieter and Evans, 1983; Menoret, 1984; Jones et al, 1985; Lotter, 1985; de Vries and Rensink, 1985; Meganck, 1987), and 1.2 to 1.5 (Arvin and Kristensen, 1985 - estimated from COD and corrected for denitrification; Iwema and Meunier, 1985). The lower values (0.24 to 0.52) could reflect the presence of air or nitrate left at the beginning of the test which would result in acetate consumption not related to Pi release. The other values are in closer agreement with the result obtained in this work and with the predicted value. The predicted value for the molar ratio of the number of carbons added as acetate to the number of carbons stored as PHA was 89% because of 11% of the acetate being degraded to C 0 2 by the T C A cycle for NADH production. The average value obtained experimentally was 111% (s = 39%). As suggested before, the addition of acetate may have stimulated the formation of VFA, resulting in more carbons stored than were added. An experiment conducted with 14C-acetate indicated that 7% of the carbon added anaerobically was found as 1 4 C - C 0 2 (section 5.2.1.e.i). In a similar type of experiment, Bordacs and Chiesa (1987) found a similar value of 8%. The reason for a difference between the observed (7%) and the theoretical value (11%) is not known but could be explained by factors such as other intracellular sources of NADH, experimental error, or incorrect assumptions in estimating a theoretical value. The percentage of Pi taken up aerobically to Pi released anaerobically could theoretically be as high as 700% if all of the energy stored as PHA was used strictly for Pi uptake (100% x (25 - 1.5) x 4 / 13.5 = 700%). The average value observed was 104% (s = 19%). Of course, some of the energy available must have been used for cell growth. Pi availability must certainly have limited Pi uptake. 141 Finally, the maximum amount of Pi that could be taken up if all of the energy from PHA were used strictly for Pi uptake would be 16 moles P/mole PHA. The average value obtained was 2.6 (s = 0.8) which means that, as expected, bio-P bacteria used some of the energy from PHA for synthesis and cell growth. Ratios calculated with the data from the SBR, the UBC pilot plant and the Kelowna full-scale plant, were in close agreement with the values reported for the batch experiments (see Table 6.3). At pilot- and full-scale, however, the proportion of PHA stored as PHV was greater, probably because the presence of propionate added by the fermented primary sludge. In summary, anaerobic acetate addition appeared to stimulate V F A production by the biomass which resulted in more carbon stored as PHA than added as acetate. PHA storage was predominantly in the form of PHB and aerobic PHA consumption indicated that about 16% (2.6/16) of the total energy available as PHA was used for Pi uptake (assuming no other source of energy than PHA). c) Other substrates Calculations and comparisons with theoretical values could be made for other substrates as were made for acetate, but since actual pathways and resultant energy balances i n mixed cultures are not known with enough certainty, the discussion will be limited to a comparison of the results with acetate and of the results among themselves. All values reported are for "net" values which were corrected for the Control, except for Control values and for molar ratios calculated for the aerobic period ( P i U P T / P i R E L a n c * ^>1urr/^>^i^coNs)* In the Control reactors, to which no substrates were added, PHA storage took place preferentially in the form of PHV (72%, s = 12). Assuming that acetate and propionate were the only V F A produced, this ratio means that propionate constituted 57% of the COD produced, a proportion close to that observed in the primary sludge fermenter at the pilot plant. Thus, it can be suggested that fermentation activity took place in the Control reactors (as well as all others to which substrates were added) because of the seeding of the sludge, with fermentative microorganisms from the primary sludge fermenter. The efficiency of PHA storage i n terms of Pi .release per PHA stored was much less i n the Control reactors than i n the reactors with acetate: 7.1 (s = 4.0) instead of 2.4 moles P released per mole PHA stored. Thus, more energy from polyP appeared to be spent for PHA storage i n the absence of acetate than in its presence. Similarly, under aerobic conditions, a greater proportion of the energy available from PHA was spent for polyP storage i n the Control reactors than in the reactors that had received acetate (4.1, ^ = 1.7, instead of 2.6 moles P taken up per mole of PHA consumed). 142 The percentage of PHA stored as PHV gives an indication of the metabolic activity resulting from the addition of substrate in solution. With concurrent acetate and propionate addition, PHV was favored over PHB formation (see Figure 5.18). The largest amount of PHV storage took place when propionate constituted two thirds of the COD. With 100% COD from propionate, only PHV was stored but the total amount of storage was only one third of that obtained with acetate addition alone. For other substrates, a general tendency was that substrates composed of an odd number of carbon atoms tended to favor PHV over PHB storage. For example, the highest proportion of PHV storage were obtained with valerate (96%, 99%), propionate (91%, 97%, 98%, 115% [artificially above 100% because less PHB was stored than in the Control]), glycine (86%) and lactate (84%; although only 35% in another experiment). These observations can probably be explained by the pathways of degradation of these substrates by fermentative microorganisms that resulted in propionate formation, which would favor PHV over PHB storage. Formate also resulted mainly in PHV formation (91%) but in no PHA accumulation in another batch experiment. The direct synthesis of acetate or propionate from formate in the case where PHA storage took place, is expected to be quite unfavorable energetically. However, formate has been reported to serve as an electron donor (Thauer et al, 1977) and may, in fact, have stimulated the production of acetate and propionate by fermentative microorganisms. Such a case was specifically documented for bacteria found in the mouth of people affected by periodontal diseases (Tanner et al, 1981). In contrast, the addition of substrates composed of an even number of carbons generally favored PHB storage over PHV. For example, the highest proportions of PHB storage were obtained with ^-hydroxybutyrate (0% as PHV), acetate (23%), iso-butyrate (18%), hexanoate (29%) and butyrate (37%, 50%, 50%). With such substrates, acetate production by fermentative microorganisms were probably favored over propionate formation. Exceptions to this tendency of storage of substrates composed of odd or even number of carbons were observed for iso-valerate (0% as PHV), ethanol (58%), glucose (63%), glycine (86%) and succinate (205%; see discussion below). The actual pathways by which acetate and propionate were synthesized from these substrates might explain these differences. Succinate was a notable exception in that the net percentage of PHV stored was calculated as 205% (greater than 100% because more PHB was stored in the Control than in the reactor with succinate). Succinate also triggered an unusual response of the sludge in that very little PHA was stored for a high degree of Pi release (the molar ratio of Pi release to PHA stored was the highest obtained at 39 moles per mole). Obviously, bio-P bacteria "spent" significant amounts of energy from polyP following the addition of 143 succinate but apparently not much of this energy supported PHA storage. More research on the effects of succinate on bio-P bacteria could certainly provide interesting information on their metabolic activities and on PHA storage in the presence of this substrate which is also a T C A and glyoxylate cycle intermediate. The effect of malate, another TCA/glyoxylate cycle intermediate, could also be tested. The ratio of Pi released to PHA stored provides evidence of the amount of energy "spent" from polyP degradation for PHA storage with a number of substrates. Figure 6.6-A gives a visual summary of "net" PHA storage against "net" Pi release for many experiments. For each experiment, only one value was used from a given reactor and reported in Figure 6.6. The regression line obtained with acetate is shown for reference. With acetate, the degree of Pi release could be reasonably well predicted from the amount of Pi released by using the regression line. The slope of a best-fit line (not shown) for propionate, butyrate and combinations of acetate and propionate (referring to all data points without numbers) would give a lower value than for acetate, suggesting a lower efficiency of PHA storage per Pi molecule. For the other types of substrates, no clear relationship could be drawn. In terms of "energy from polyP spent", some substrates were less efficient for PHA storage than acetate (less PHA storage/Pi released), notably succinate. Other substrates, to the contrary, were more efficient than acetate; e.g. valerate, 0-hydroxybutyrate, lactate and hexanoate. Also, acetate addition to an activated sludge pre-mixed with wastewater resulted in more efficient PHA storage. The efficiency of the sludge to store some substrates as PHA is probably dependent on the activity of fermentative microorganisms and of bio-P bacteria. This activity would probably be affected by the environmental conditions of the microorganisms, such as characteristics of the influent and plant operation. The ratio of Pi released to substrate taken up, like the previous ratio of Pi released to PHA storage, gives an idea of the amount of energy spent for energy storage but in an indirect way. The advantage of this ratio over the previous one is that it only requires measurement of the concentration of substrate left in solution instead of the more time consuming PHA determination. In some experiments, the substrate concentration was not measured and it was assumed that all of the added substrate was taken up during the anaerobic period, an assumption that was probably not always correct and would have resulted in a lower ratio. In these cases, as mentioned earlier, an asterisk (*) was added beside the reported ratio. These data were not used in the following discussion. With the addition of propionate or combinations of acetate and propionate (including primary fermented sludge) the ratio was in the range 1.3 to 1.8 with values averaging 1.6. Similar values were obtained by others researchers (see Table 6.4). These results were close to the predicted value of 1.69 for acetate. 144 From many batch •xperiments 60 to X o> E O) CO < a z 50 -40 30 -20 10 Legend + acet • prop o propionate x butyrate A other substrates 12 6 15 acetate regression line.-5 11 6 3 1 A 2 A9 10 13 14 6 10 20 Net Pi release (mg P/l) — i — 30 40 co X o> E CB o tt < X CL 50 40 30 -20 -10 10 Legend o 2,4-DNP A others 1 mM, pH not ajusted acetate regression line A 10 mM F o 0.1 mM, pH 7 pH 9 10 mM CN p H 5 °1mM, P H 7 o 10 mM, pH 7 -i 1 1 r 8 12 16 20 Net Pi release (mg P/l) 24 28 gure 6.6 Absence of a general relationship between PHA storage and Pi release for a variety of A. substrates and B. toxicants. Numbers on A. plot identify substrates as follows: (1) iso-valerate, (2, 10) formate, (3) glucose, (4, 8) valerate, (5) hexanoate, (6) ethanol, (7) citrate, (8) valerate, (9) glycine, (10) formate, (11) iso-butyrate, (12, 13) lactate, (14) succinate, (15) £-hydroxybutyrate. 145 For lactate, valerate and butyrate, higher ratios were obtained (2.1, 3.5 and 3.8) which suggested that the storage of these substrates was less efficient than for acetate. With /3-hydroxybutyrate, the ratio of percentage of carbon stored to that added as substrate (next ratio to be discussed) indicated complete substrate uptake, although no actual measurements of /3-hydroxybutyrate confirmed this hypothesis. Since with /3-hydroxybutyrate no N A D H is required, as would be needed for the combination of two acetate molecules, a ratio of 1.5 was predicted (0.5 ATP for transport and 1.0 ATP for energization per molecule instead of 1.69 for acetate). A close value of 1.6 was obtained. A number of ratios were calculated from data presented in the literature (Table 6.4). In many cases the ratios obtained were quite low and even sometimes zero. Such low values could be explained by two factors. First, in most cases the substrate concentration was not actually measured but only assumed to be completely consumed. With high levels of substrate that would not be all taken up, the Pi/substrate ratio would then be underestimated. Also the presence of any air would result in aerobic substrate consumption and in a smaller release of Pi by bio-P bacteria. Thus, it is difficult to meaningfully compare ratios calculated from the literature with those obtained from our experiments. Table 6.4 M o l a r ratios of Pi released over substrate taken up. Substrate Pi rel/Substrate taken up (mole/mole) Reference f propionate 1.8 . 1 II 1.4-2.1 2 II 1.0-2.2 3 butyrate 0.38 1 it 2.6-3.8 2 succinate 0.57 4 /3-hydroxybutyrate 0.25 1 glucose 0.28 1 it 1.1-1.7 2 lactose 1.0-2.0 2 ribose 0.0 1 glycerol 0.0 1 ethanol 0.65-5.3 2 it 0.8 5 II 0.0 5 146 References: (1) Potgieter and Evans (1983); (2) Arvin and Kristensen (1983); (3) Meganck (1987); (4) Lotter (1985); (5) Vries and Rensink (1985). The percentage of carbon atoms added as substrate that were stored as PHA gives an indication of the suitability for PHA storage of various substrates. With combinations of acetate and propionate, the percentage calculated varied between 73% and 117% (with one value of 193%). For comparison, the calculated value for acetate averaged 119% whereas the predicted value was 89%. Thus, much like acetate, the combined addition of acetate and propionate appeared to stimulate VFA production by the sludge. Valerate had a similar effect (178%). Propionate alone, however, resulted in only 22% to 60% carbon storage. Since PHA storage requires the combined storage of acetate and propionate, acetate formation by fermentative microorganisms must have resulted in the consumption of some of the added propionate. It is peculiar, however, that Pi release and uptake was as high as when acetate alone was added. This point will be discussed in more details in section 6.3.4. The proportion calculated for /3-hydroxybutyrate was 104%. Even though the residual concentration of the substrate was not actually measured, /3-hydroxybutyrate appeared to be stored very efficiently as could be expected from its chemical structure which is that of a PHB monomer. This fact probably explains why ^ -hydroxybutyrate was only stored as PHB and not at all as PHV. The percentage of aerobic Pi uptake over anaerobic Pi release gives an indication of the efficiency of the consumption of stored PHA for Pi uptake. It is mainly useful to indicate if inhibition of Pi uptake by toxicants took place (see following section). In general, the ratio of Pi uptake to Pi release was greater for the reactors to which some substrate was added than for the Control which received no substrate. Care should be taken to compare this ratio only to values of the corresponding Control of the same experiment because some Pi was often released before the substrate addition by which time some PHA storage had already taken place which could subsequently be used aerobically for Pi uptake. In Control reactors, to which no substrate had been added, Pi release and PHV storage were repeatedly observed whereas only negligible PHB storage took place (see Figs. 5.3 and 5.4 for example). The observed Pi release could be explained by the degradation of polyP serving for PHV storage, or by the lysis of bio-P bacteria. Since a significant fraction of the anaerobically released Pi was subsequently taken up aerobically (see Table 6.3), it would seem more logical that bio-P bacteria had not lyzed but simply consumed some of their polyP reserves for substrate storage. When wastewater was combined with sludge at the beginning of an experiment, the rate of Pi release and PHV 147 storage was greater than when sludge alone was used (see Figs. 5.3 and 5.4). With wastewater, more fermentable organic matter would remain in solution than with sludge alone. Thus, the rate of Pi release and P H V storage could be directly related to the rate of substrate fermentation. Finally, the ratio of Pi uptake over PHA consumed gives an idea of the amount of energy from P H A that was used for Pi storage as polyP (neglecting that some of the Pi taken up was used for growth). The theoretical maximum was estimated at 16 moles Pi per mole of P H A consumed, a lower ratio indicating that the rest of the energy from P H A was used for other metabolic functions than polyP accumulation such as synthesis and growth. For acetate and the Control reactors, averages of 4.1 and 2.6 moles Pi taken up per mole of P H A consumed were obtained, respectively. For most substrates, the. values obtained were in a similar range, namely 2.0 to 5.0. Propionate and butyrate gave higher ratios mainly between 5.0 and 9.0 with a couple of lower values for propionate (1.8 and 2.4). Succinate had a ratio of 14.0. For these three substrates, the high ratios were obtained with high Pi uptake values while the P H A consumption was relatively small. If P H A were the sole source of energy, it would appear that a large fraction of the available energy was used for Pi uptake. It is possible that some other sources of energy were available to bio-P bacteria under aerobic conditions in the presence of these substrates. Careful investigations of the G C chromatograms from P H A analyses failed to reveal any fluctuations of peaks other than those corresponding to P H B and P H V . Maybe some other compounds can serve as energy reserves under aerobic conditions in the presence of these substrates but their identity could not be established by the isolation technique used in this work. d) Toxicants Toxicants added under anaerobic conditions were 2,4-DNP, cyanide, fluoride, HC1 to maintain p H 5 and N a O H to maintain pH.9. In some experiments with toxicants, P H A measurements were made from which a plot of net P H A stored against net P i release could be prepared (Figure 6.6-B). The maintenance of p H 5 by HC1 addition did not affect P H A storage and Pi release (in comparison to the Control; see Figures 5.28, 5.33 and 5.34). Fluoride resulted in some P H A storage and Pi release in a ratio close to that normally observed with acetate (see Figure 6.6-B). The addition of a neutralized 2,4-DNP solution (0.1, 1.0 or 10 mM) did not result in much Pi release or P H A storage (some P H A consumption was observed in fact). When 1.0 m M of non-neutralized (pH 3.8) 2,4-DNP was added, however, significant Pi release and extra P H A storage were observed (see also Tables 5.7 and 5.8, Figures 5.28 and 5.29). A 148 low p H by itself was shown not to affect Pi release and P H A storage. Thus, 2,4-DNP could have stimulated V F A production by fermentative microorganisms by affecting their p H gradient which would have required them to produce more energy (and V F A ) to re-establish their p H gradient. The maintenance of p H 9 by N a O H addition, resulted in extra Pi release with minimal corresponding P H A storage. It was proposed that an unfavorable p H gradient could have resulted in polyP degradation to assist the maintenance of a constant pmf (see also Section 5.2.8.b). Cyanide, like a high p H , resulted in a significant amount of Pi release with minimal P H A storage. The normal mode of action of cyanide is to inhibit electron transport by binding to the iron of porphyrin rings of cytochromes composing the electron transport chain (Brock et al, 1984). Under anaerobic conditions, however, this mechanism is not expected to be of much significance. It is unclear why cyanide resulted in so much Pi release. e) Metallic cations Potassium, magnesium and calcium were repeatedly found to be released and taken up from solution along with Pi . Table 6.5 summarizes the cation/P molar ratios from a number of experiments and from the literature. Sodium was found to increase not as a function of P i concentration but in steps, according to the amounts of chemicals added as sodium salts (acetate, N a O H , etc.). No concentration changes were observed for aluminum, iron, cadmium and manganese (see section 5.2.6). The similarity between the concentration profiles of potassium, magnesium, calcium and Pi (see Figures 5.5 and 5.23) indicated that these metallic cations could be co-transported with Pi for both its release and uptake by bio-P bacteria. The sudden but transient increase in calcium just before aeration may have resulted from the solubilization and re-binding of some calcium bound to the external cell wall (Hancock, 1984) upon the addition of sodium Pi . It was estimated that with the addition of acetate or of other substrates, the sum of charges of cations per molecule of Pi transported averaged about 0.91. Potassium, magnesium and calcium accounted for about 25 to 30%, 50% and 15% of the neutralizing charges, respectively (see also section 6.2.1.e). Literature data correspond reasonably well with these values (see Table 6.5). For each Pi group in a polyP chain, one negative charge has to be neutralized for charge stability. With electron dispersive X-ray analyses ( E D A X ) Buchan (1981) showed that calcium was an important polyP stabilizing cation in Acinetobacter isolated from bio-P sludge. With the same technique, Groenestijn (1988) found that both calcium and magnesium were major polyP counterions. Observed co-transport ratios, however, do not necessarily reflect the proportion in which a cation serves as a polyP counterion. Indeed, Table 6.5 M o l a r ratios of concentration changes of cations and P i 149 Description and K + /P M g 2 + / P C a 2 + / P sum of rel or reference charges/P upt (all units in moles cation/mole P) batch experiment with acetate (section 5.2.1 and ref "b") and other substrates (section 5.2.3, 5.2.4 and ref. T ) : Sect. 5.2.1.b.i 0.31 0.22 0.03 0.81 r&u Sect. 5.2.1.c.i, 5.2.6, (a) 0.34 0.24 0.06 0.94 r&u Sect. 5.2.1.d.ii 0.23 0.261 0.07 0.89 rel t i i t 0.23 0.262 0.11 0.97 upt (b) 0.20 0.28 0.09 0.94 rel I I I I 0.23 0.27 0.12 1.01 upt Sect. 5.2.3.ii, 5.2.4.iv3 0.34 0.20 0.03 0.80 r&u (c)4 0.23 0.32 0.06 0.99 r&u continuous operation with municipal wastewater: (d) n.d. 0.31 n.d. n.d. r&u (d) n.d. 0.33 n.d. n.d. r&u (e) 0.27 0.26 0.00 0.79 rel (0 0.25 0.25 0.00 0.75 r&u continuous operation with synthetic wastewater (supplemented with acetate): (g) 0.31 0.28 0.05 0.97 rel Acinetobacter culture: (n)5 0.32 0.26 .0.01 0.85 rel toxicants: Sect. 5.2.8.a.iv: 2,4-DNP6 0.29 0.35 0.08 1.15 anaero (only for 10 mM) 0.29 0.17 0.00 0.63 aero Sect. 5.2.8.b.iii: pH 57 0.84 0.44 0.05 1.82 anaero t i t i 0.84 0.00 0.00 0.84 aero Sect. 5.2.8.b.iii: pH 98 0.31 0.14 0.02 0.63 anaero M 11 0.31 0.07 0.00 0.45 aero References: (a) Comeau et al. (1985); (b) Comeau (1984); (c) Arvin and Kristensen (1985); (d) Olson and Connell (1971); (e) Miyamoto-Mills et al. (1983); (f) Gerber and Winter (1985); (g) Fukase et al. (1983); (h) Groenestijn (1988); 150 Abbreviations: aero, aerobic; anaerob, anaerobic; n.d.: no data available; rei, release; upt, uptake. 1 Mg/P ratio for Pi release was lower (0.11) just before acetate addition; 2 Mg/P ratio for Pi uptake was lower (0.20) after pH increase from 7.2 to 7.8 (pH increase probably due to C 0 2 stripping); 3results are averages for Pi release and uptake for the following substrates: acetate, propionate, acetate + propionate,.fermented primary sludge, butyrate, valerate, lactate and formate; 4results given by Arvin and Kristensen are averages of experiments conducted with sludge taken from three different bio-P plants. The following substrates were tested: acetate, propionate, butyrate, lactate, ethanol and glucose; 5pure cultures of Acinetobacter also gave an anaerobic release of 0.09 mole of ammonia per mole of Pi; 6results with 2,4-DNP are given only for the reactor to which 10 mM were added. Under anaerobic conditions, Pi release was associated with a transient (for 1 hour) potassium release of 20 mg/l. Under aerobic conditions, Pi release was associated with a transient (for 1 hour) magnesium and calcium uptake of 1.0 and 1.3 mg/l, respectively; 7at pH 5 under anaerobic conditions, an initial release of 2.5 mg/l of calcium was observed; 8at pH 9 under aerobic conditions, an initial uptake of magnesium and of calcium of 1.3 and 1.1 mg/l were observed, respectively. 151 intracellular potassium and magnesium concentrations are normally much greater than that of calcium. Thus, with polyP breakdown, calcium ions may remain "trapped" in the cell while potassium and magnesium are preferentially co-transported outside the cell. Thus, it is proposed that the three cations potassium, magnesium and calcium provided neutralizing charges during Pi import or export from bio-P bacteria. These cations would appear to be co-transported with Pi but the actual transport mechanisms could possibly involve a Pi molecule neutralized by protons coupled with proton/metallic cations antiport systems. 6.2.3. Rates of reaction a) Substrate uptake The rate of acetate uptake was maximum right after its addition to the sludge. For anaerobic sludge from the U B C pilot plant (MLSS of 2400 mg/l), the maximum acetate uptake rate was about 55 mg C O D l ' ^h" 1 . This net rate of acetate uptake could be attributed to P H A storage alone. With an excess of acetate addition to bio-P sludge, negligible rates of acetate uptake and Pi release were observed about 2 hours after anaerobic acetate addition. The depletion of polyP reserves could probably not allow any more substrate uptake and storage. When both acetate and propionate were added jointly (see Figure 5.17), the maximum rate of substrate uptake increased to 79 mg C O D T ^ - h ' * versus 52 mg 1 i C O D T 1 - h " A when added separately. Thus, P H A storage appeared to be more effective when both acetate and propionate were added together. This observation was made with sludge taken from the U B C pilot plant which was acclimated to fermented primary sludge. Similar experiments conducted with sludge acclimated to different substrates, such as glucose (Fukase et al., 1982; Manoharan, 1988) or butyrate (Manoharan, 1988), could give a different response. In that regard, the U B C pilot plant sludge did not assimilate other substrate as efficiently as acetate and propionate. It was estimated that butyrate, valerate, lactate and 1 1 formate were taken up at a rate of 5 to 10 mg C O D - l " 1 ^ " 1 which was 5 to 10 times lower than the rate of acetate and propionate uptake. Based on the much slower rate of Pi release and P H A formation, it was suggested that these substrates were first degraded into acetate and propionate by fermentative microorganisms before being stored as P H A by bio-P bacteria. Thus, for the U B C pilot plant studied, acetate and propionate were the two substrates that could best be stored as P H A to serve for bio-P removal. With sludge from South African full-scale plants, Gerber et al. (1986, 1987a, 1987b) also found that acetate and propionate caused the greatest anaerobic Pi release and 152 aerobic Pi uptake. Formate also resulted in a rapid anaerobic Pi release but only in poor aerobic Pi uptake. Butyrate and lactate also favored high Pi release (at a slower rate than acetate or propionate) and high Pi uptake. In the presence of oxygen or nitrate the rate of acetate uptake increased . For the batch experiment of section 5.2.7.i, in which nitrate and acetate addition were added simultaneously, maximum rates of acetate uptake were 62 and 41 mg COD-l" 1 ^" 1 for PHA storage and for denitrification, respectively (a total of 103 mg CODT^'h"* was observed with both acetate and nitrate). In the presence of both oxygen and acetate, the rate of acetate uptake was estimated to be greater than 98 mg CODT^'h"-* (Figure 5.16). In a previous experiment, the rate of acetate uptake in the presence of both oxygen and acetate had been only 55 mg C O D - r^ h " ! when all polyP reserves had been depleted by excess acetate addition under anaerobic conditions (Figure 5.14). Thus, the difference between these two acetate uptake rates, as well as the observed aerobic Pi release and PHA storage, clearly indicated that polyP reserves could assist in acetate accumulation under aerobic or anoxic as well as anaerobic conditions. The maximum rate of substrate uptake should not be used as the only criterion in sizing the anaerobic zone. As pointed out by Wentzell et al. (1988) from their work with acetate as the sole carbon substrate, the hydraulic retention time of the anaerobic zone should exceed the minimum required for complete substrate uptake. With an anaerobic mass fraction of only 10%, the overall phosphorus removal was 5 times less efficient than with a mass fraction of 50%. This reduced efficiency was attributed to the "leaching" of acetate from the anaerobic reactor to the downstream anoxic reactor which resulted in a shift in bacterial population from Acinetobacter to Pseudomonas that'did not accumulate polyP as efficiently. A recommendation stemming from this experience could be to use at least two completely mixed anaerobic reactors in series in preference to a single reactor to prevent substrate "leaching" to the downstream zone, especially when high concentrations of substrates would be added to the anaerobic zone (as was the case in WentzelPs work with as high as 500 mg COD/1 of acetate). b) Pi release and uptake i. Pi release For a given sludge, the rate of Pi release under anaerobic conditions was highest just after substrate addition. As the Pi concentration increased, the rate of Pi release gradually decreased. Data gathered from a number of experiments in which an excess of acetate was added, in order to ensure that some acetate always remained in solution, was used to prepare the plot shown in Figure 6.7-A. One reactor provided one "line" of data, and for From 3 batch experiments 153 E a> o 0 20 40 60 Pi cone (mg P/l) 70 0 20 40 60 estimated maximum polyP content (mg P/l) Figures 6.7 Rate ( A J and maximum rate (B.) of anaerobic Pi release. Note: P.P.: pilot plant. 154 each line, the maximum extent of Pi release was used to give an indication of the maximum amount of polyP that can be used as energy for acetate accumulation by bio-P bacteria. Extrapolating the curves to the origin allowed to estimate the maximum rate of Pi release for a given amount of polyP content (see Figure 6.7-B). Thus, the maximum amount of polyP available for energy increased as a function of the maximum rate of Pi release (Figure 6.7-B). Conversely, as more of the acetate added was taken up and Pi released, the residual amount of polyP left, decreased and the rate of Pi release also decreased. Eventually, the rate of Pi release became negligible, presumably when polyP reserves were depleted. It was estimated that the rate of Pi release declined at a rate of about 6.5 rng-l"1^"1 for each 10 mg P/l of Pi released. The observation that the rate of Pi release declines as the amount of Pi release increases can also be seen from the graphical data of a number of authors (e.g. Arvin and Kristensen, 1983; Meganck, 1987; Gerber et al, 1987b). Wentzell et al. (1987), however, suggested that, for modelling purposes, the rate of Pi release was zero order with respect to acetate concentration. It is believed that this observation will appear to be true only when a small amount of acetate will be taken up by the sludge compared to the maximum amount that polyP reserves could allow to accumulate. With the addition of an excess of acetate, the reaction should not be zero order but first order with respect to polyP reserves left for acetate uptake. ii. Pi uptake Figure 6.8 shows a graph of Pi uptake rate obtained from a number of experiments. Residual amounts of acetate and/or propionate greatly increased the rate of Pi uptake by the sludge; as high as 60°mg P-l" 1^" 1 compared to about 10 mg ,l"1,h"1. These high rates of Pi uptake were obtained from experiments in which an excess of substrate had been added under anaerobic conditions that resulted in a complete depletion of polyP reserves. When air was turned on, some substrate was still in solution. Thus, the highest rates of Pi uptake were observed when some substrate was left in solution and when polyP reserves were depleted (otherwise more Pi release would have occurred; see section 6.3.1). When wastewater was combined with sludge at the beginning of an experiment (see full boxes in Figure 6.8), the resulting rate of aerobic Pi uptake was also quite high: 20 to 30 mg P-r^h"1. Residual organic matter at the beginning of the aerobic period may explain this observation. A tentative correlation was drawn between the amount of PHA in the sludge and the rate of aerobic Pi uptake. The slope indicated that the rate of Pi uptake increased by 0.4 mg P-l" 1^" 1 for each mg HB/1 of PHA. The minimum amount of PHA below which no more Pi was taken up was about 5 mg HB/1, a possible "background" level. 155 Figure 6.8 Rate of aerobic Pi uptake versus PHA content of the sludge. Only one data point was obtained from a given reactor at the beginning of the aerobic period in an experiment. Points identified with the letters "Pr", "A&P" or "Ac" indicated that some propionate, acetate and propionate, or acetate was left at the beginning of the aerobic period and that polyP reserves were depleted, resulting in an increased rate of aerobic Pi uptake. Full square symbols indicated that wastewater composed 25% of the reactor volume at the beginning of the experiment. 156 It was shown that Pi uptake could be prolonged by 1 hour for each acetate increment of 20 mg COD/1 (see Figure 5.7). Assuming that 100% of the acetate was stored as PHA, this translated into a 1 hour prolongation for each 17 mg HB/1 of PHA (PHA were not measured in that particular experiment). 6.3. Difficulties of the proposed model to explain some observations 6.3.1. Aerobic or anoxic Pi release upon substrate addition a. Proposed explanation for aerobic Pi release;: polyP degradation regulated by the pH gradient The observation that Pi is released upon acetate or propionate addition under aerobic or anoxic conditions is in apparent contradiction with the postulated biochemical model presented above. Gerber et al. (1986, 1987a, 1987b;) also reported that aerobic, anoxic or anaerobic Pi release would occur if acetate, propionate or formate (but not methanol, ethanol, citrate, glucose or 2,3-butanediol) were added aerobically. Data reported in section 5.2.2 (Figures 5.14, 5.16) indicated that PHA storage would take place upon acetate or propionate addition under aerobic conditions. Aerobic Pi release, however, was observed only when polyP reserves had not been depleted by previous excess acetate addition. In a first experiment in which an excess of acetate added anaerobically (100 mg COD/1) resulted in an excess of about 40 to 45 mg COD/1 remaining in solution before the start of aeration, the observed maximum aerobic rate of acetate uptake was 55 mg COD l'^h"1 (see Figure 5.14). In this experiment, polyP reserves were depleted because of the excess of acetate added previously, explaining why no Pi release was observed since no polyP reserves were available for that purpose. To the contrary, very rapid Pi uptake was observed at a rate six times as high as when only PHA was used for polyP storage (60 mg P l " 1 ^ " 1 instead of 10 mg Pi"1-]!"1'; see section 6.2.3.b.ii). In another experiment in which only some acetate (20 mg COD/1) had been added under previous anaerobic conditions, the addition of acetate and/or propionate at the start of aeration resulted in Pi release (as also reported by Gerber 1986, 1987a, 1987b) and PHB/PHV storage (see Figure 5.16). In this case, the observed rate of acetate uptake was greater than 98 mg CODi" 1-]!" 1, nearly twice higher than in the previous experiment. In this case, however, polyP reserves had not been depleted by the addition of an. excess of acetate under aerobic conditions and, thus, could have contributed energy for acetate uptake. 157 The following discussion will concentrate on explaining possible mechanisms with acetate addition under aerobic conditions resulting in Pi release. Nevertheless, similar mechanisms are proposed to apply with acetate or propionate addition under either aerobic or anoxic conditions. With the addition of acetate in the presence of oxygen, the biochemical model predicts that available substrates (normally scarcely available under aerobic conditions) and PHA would be consumed for energy production and polyP accumulation. However, should the acetate concentration suddenly become high, PHA accumulation rather than its degradation can be expected because of the high intracellular levels of both acetyl CoA and N A D H . The degradation of polyP rather than its accumulation, however, appears to be more unusual since energy was available which could have resulted in polyP accumulation (Figure 5.16; Gerber 1986, 1987a, 1987b). This observation also suggested that ATP production from acetyl CoA consumption via the TCA cycle and electron transport phosphorylation, was not used alone to satisfy aerobic energy requirements for bio-P bacteria. However, the model proposed that energy from polyP was used not only for acetate energization into acetyl CoA (probably via acetyl Pi) but also to re-establish the proton gradient. Therefore, it is proposed that aerobic Pi release upon acetate addition is regulated by the pH gradient. Thus, with a high level of acetate, the pH gradient would be lowered enough for both polyP and electron transport to be used for protons expulsion. PolyP could expel protons via a pump similar to the membrane-bound ATP-ase enzyme, either via ATP production or possibly directly, as has been reported for pyrophosphate (Moyle et al, 1972; Barsky et al, 1975). This second role of polyP was initially derived from the observation that Pi release could be stimulated from the maintenance of a high pH or by 2,4-DNP addition, two conditions that would reduce the pH gradient (see section 5.2.8). The fact that acetate and propionate can also trigger this response simply suggests a similar active transport mechanism for acetate and propionate (see Literature Review) that affects the pH gradient of bio-P bacteria. When no more acetate would remain in solution, the pH gradient would not be "depleted" by the substrate any more, and polyP would cease to be degraded and would resume a "normal" Pi accumulation as a result of Pi and ATP availability. Thus, the regulation of the storage and degradation of polyP should involve a pH gradient-sensitive enzyme. The above observations and discussion, highlighted again the importance and central role of carbon in relation to polyP metabolism in bio-P removal. On the basis of the observations reported above, it can be proposed that under aerobic conditions, bio-P bacteria would obtain energy from the following sources in the preferred sequence: 1) 158 soluble substrate (acetate, propionate and oth ers that can be consumed aerobically by bio-P bacteria), 2) stored P H A , 3) polyP, then 4) cellular material (e.g. proteins and R N A consumed by "endogenous" respiration). Experiments conducted here showed that the time required to completely consume these sources of energy would be in the order of 0.5 hour for acetate soluble substrate (see Figure 5.14), of 3 hours for P H A (see Figure 5.5), of more than 10 days for polyP (see Figure 5.15), and longer than 10 days for endogenous decay which could ultimately results in cell lysis. Depending on the availability and need of bacteria for energy, more than one source could probably be used at a given time. b. Anoxic Pi release The effects of nitrate added jointly with acetate on Pi, nitrate and acetate was shown in Figure 5.25. Initially, when both nitrate and acetate were still in solution, three types of bacteria would have been active: bio-P non-denitrifiers (degrading polyP for acetate storage as P H A ) , bio-P denitrifiers (using both nitrate and polyP to obtain energy for acetate storage as P H A and acetate consumption into CO2), and non-bio-P denitrifiers (using nitrate and oxidizing acetate into CO9). Thus, anoxic acetate addition should result in an increase in Pi concentration and in a decrease in nitrate and acetate concentration. When some acetate but no more nitrate (anaerobic conditions) would remain in solution (reactors with 0, 3 or 6 mg NO3-N/I), polyP degradation alone would provide energy for acetate storage as P H A by bio-P bacteria. When some nitrate but no acetate would remain in solution (reactors with 9 or 12 mg NO3-N/I), polyP degradation for P H A storage would stop and the reverse, polyP storage, would take place as stored P H A would be consumed by bio-P bacteria. A n indirect evidence for P H A storage even in the presence of nitrate ( P H A storage was not actually measured in that experiment) is that the concentration of Pi reached and remained at a very low level in all reactors at time 11 hours, even in the reactors with high level of nitrate, whereas aerobic Pi release would normally have occurred earlier (see Figure 5.5). c. Implications of aerobic substrate addition for full-scale treatment It was shown that P H A storage could take place under aerobic conditions in the presence of a high concentration of acetate or propionate and that, once these substrates were consumed, polyP accumulation started. Thus, it could be tempting to save money by removing,the anaerobic basin from the process train of a bio-P plant since its most important role of P H A accumulation appears to be achievable in an aerobic (or anoxic) zone. However, it is believed that such a measure would not promote bio-P removal and may in fact be quite detrimental for a number of reasons. First, it should be recognized that aerobic Pi release occurred in batch experiments when high concentrations of acetate or propionate (e.g. 20 mg C O D / 1 ) were artificially 159 added in solution, an unlikely situation in most full-scale plants. Thus, these observations are more of interest to characterize bio-P bacteria from a physiological point of view than from a plant operation point of view. In the same way, creating pH 4 conditions or adding 10 mM of 2,4-DNP can provide physiological information for bio-P bacteria without providing a meaningful representation of full-scale operating conditions. The presence of substrate under anoxic or aerobic conditions could occur in full-scale plants from the "leakage" of substrates from the anaerobic zone to the downstream zone. Such cases would be possible when there would be a high load of storable substrates available in the anaerobic zone but only a short actual anaerobic HRT that would not allow all of the storable substrates to be taken up by bio-P bacteria. The actual effect of acetate "leakage" on a continuous basis has been documented by Wentzell et al. (1988). They operated a lab-scale bio-P process in which they fed acetate as sole carbon source (plus some yeast extract and micro-nutrients for essential metabolites) in order to develop an enhanced culture of bio-P microorganisms in a U C T and a modified Bardenpho process. Two operational conditions led to the "leakage" of acetate into the downstream anoxic zone; first, when acetate addition was performed in "slugs" instead of in the usual continuous manner, and second, when the hydraulic retention time in the anaerobic zone was kept too short because of a small anaerobic reactor. Shifting from a favorable continuous to an unfavorable "slug" mode of acetate addition led to a decrease in the phosphorus removal capacity of the process from 50 mg P/l (0.1 mg P/mg COD) down to 10 mg P/l. Sludge population analyses indicated a marked shift from Acinetobacter to Pseudomonas predominance. Thus, if Pseudomonas could accumulate polyP, they did not appear to be able to promote the same level of efficiency as Acinetobacter species. Experimentation with various anaerobic sludge mass fractions led Wentzell et al. (1988) to recommend that 30% of the sludge be kept under anaerobic conditions when acetate is the sole source of carbon added. This proportion was greatly in excess of what was theoretically essential to store all of the added acetate. Operation at 10% anaerobic sludge mass fraction, however, resulted in unfavorable acetate "leakage". Second, the factor that limits biomass growth in activated sludge plants that operate at long SRT, is carbon availability. It is also for that reason that an effluent of good quality that exerts a low BOD can be obtained. Thus, if the influent wastewater was added to an anoxic or aerobic zone, bio-P bacteria would not have the competitive advantage of first access to the "readily storable" substrate which could then be consumed by any heterotrophs. Faster metabolizing non bio-P heterotrophs could eventually predominate in the biomass, and the process would not remove Pi in excess of metabolic requirements, an observation repeatedly documented in plants where only aerobic or anoxic zones are present. 160 Finally, any fermentation that could have taken place in the anaerobic zone, and could have resulted in increased PHA storage, would not take place. Therefore, an anaerobic zone in which a maximum of "storable carbon" is added, and where a sufficient H R T is provided (e.g. 1 hour nominal HRT; Barnard, 1983), are proposed to be indispensable for the efficient operation of any bio-P process. d. Implications of aerobic substrate addition for pure culture work As was pointed out by Gerber et al. (1987b), an excess of acetate in culture media, such as is commonly used in the Fuhs and Chen (1975) medium, contains many grams per liter of acetate, and could lead to a depletion of polyP reserves under anaerobic and aerobic conditions. Therefore, it would be advisable in pure culture work to develop conditions that are truly characteristic of bio-P treatment plants. Thus, under aerobic conditions, the availability of simple carbon compounds should be kept low and under anaerobic conditions they should be maintained at a "reasonable" level so that little or no substrate remains at the beginning of the aerobic period. 6.3.2. Glycogen storage No glycogen was detected in the sludge of the UBC pilot plant (see Table 5.12). Some researchers, however, noted important glycogen accumulations in their bio-P sludges (Fukase et al, 1982; Tsuno et al, 1987; Mino et al 1987; Arun et al. 1988; and Manoharan, 1988). In all these cases, the wastewater fed to these lab-scale plants contained either only glucose (in the first four papers) or a significant proportion of glucose (25 to 45% of the COD in Manoharan's work). Despite some contradictory observations, glycogen appeared to be stored under aerobic conditions and consumed under anaerobic conditions. Mino et al (1987) and Arun et al. (1988) proposed that glycogen was stored and consumed by bio-P bacteria and that its anaerobic degradation served for ATP and N A D H production. Thus, according to these authors, bio-P bacteria would be capable of polyP, PHA and glycogen accumulation. Glycogen storage under aerobic conditions may appear surprising because glucose was added under anaerobic conditions. Thus, glucose must have been carried over, possibly in a soluble form, into the aerobic zone for storage as glycogen. The storage of glycogen in bio-P bacteria as proposed by Mino et al. (1987) and Arun et al. (1988) remains to be confirmed as it may not constitute an essential component of the bio-P removal mechanism in bio-P bacteria. Glucose addition in a batch experiment with non glucose-acclimated sludge did not result in as high Pi release and PHA storage as when acetate was added to the same sludge (see Section 5.2.4; Potgieter and Evans, 1983; Arvin and Kristensen, 1985). Thus, in these cases, glucose could have simply been fermented and the released fatty acids stored as PHA. With glucose-acclimated sludge, 161 glycogen storage and consumption could take place in other types of microorganisms and anaerobic glycogen degradation could provide fatty acids for PHA storage. Radioisotope experiments with acetate and glucose addition to glucose-acclimated sludge and to pure cultures could be used to clarify the actual mechanisms. Thus, it appears that glycogen could play a role in bio-P removal if the wastewater contained a significant fraction of sugars. This situation is not to be expected for most municipal wastewaters but may become significant with the contribution of a sugar-rich industrial wastewater. It would be interesting to compare the efficiency of a bio-P plant operating with the direct addition of the sugar-rich wastewater to the anaerobic zone of the process to that of a plant in which a pre-acidification step (in a fermenter) would transform these sugars into V F A prior to addition to the anaerobic zone. 6.3.3. Biochemical mechanisms for the PhoStrip process The PhoStrip process, (a proprietary process of Biospherics of Rockville, MA) uses an anaerobic zone, the "stripper" tank, to which about 50% of the secondary sludge is added and where Pi release takes place (see Literature Review). The Pi stripped sludge is then returned to the bioreactor and the supernatant sidestream is treated chemically with lime. The treated supernatant is returned to the influent wastewater and the lime-Pi sludge is disposed of. Because there are ho readily storable substrates left in the secondary sludge to induce Pi release it can be questioned whether or not the biochemical mechanisms proposed in the model above, especially the applicability of the concept of P H A storage, still holds. Although, to the author's knowledge, PHA data was not reported for PhoStrip plants, evidence will be reviewed which suggests that the postulated biochemical model presented above, also holds for the PhoStrip process as it does for other mainstream bio-P processes. Design data on the PhosTrip process indicated that to promote sufficient Pi release in the stripper tank, the sludge retention time (SRT) needs to be as long as 8 to 12 hours (but could range from 5 to 20 hours; Bowker and Stensel, 1987) as opposed to an anaerobic hydraulic detention time of 0.5 to 2 hours in mainstream bio-P processes (Arvin, 1985). If nitrification takes place during aerobic treatment, the stripper S R T will need to be increased (Weston Inc., 1985). A modified process termed the PhoStrip II promotes the addition of BOD-containing primary effluent to increase the rate of Pi release in the stripper tank. The S R T could then be reduced by as much as 50% (Levin and Sala, 1987). These observations correspond very well with those made in the Control reactor of batch experiments with the UBC bio-P pilot plant sludge (see the Control of Figures 5.3 and 5.4, respectively). Figure 5.4 illustrates that no Pi release took place as long as nitrate 162 remained in solution. When denitrification was completed, Pi release started and PHA storage took place. It was suggested that fermentation products became available for PHA storage when denitrification was completed. Complete denitrification with endogenous carbon took about 4 hours. In Figure 5.3, for which the experiment was conducted with aerobic sludge mixed with influent wastewater (75%/25%, vol/vol), denitrification was completed in 3 hours. The rate of Pi release and of PHA storage were increased by 80% and 20%, respectively in comparison to those of Figure 5.4. This increase in activity could be attributed to the availability of extra simple substrates from the wastewater for fermentation, denitrification and PHA storage. In the PhoStrip process, if bio-P bacteria can proliferate in the biomass as a result of Pi release under anaerobic conditions, they have to gain some competitive advantage from this Pi release. The advantage proposed is that polyP degradation allows these bio-P bacteria to accumulate VFA in the stripper tank where they would be generated by fermentative microorganisms. VFA storage as PHA would then allow subsequent PHA consumption in the aerobic reactor. Tetreault et al. (1986) confirmed the presence of polyP granules in a PhoStrip process. Therefore, the same biochemical mechanism of dual storage of polyP and PHA would appear to apply for both mainstream and sidestream (such as PhoStrip) processes. The lack of substrate in the sludge to be "stripped" would explain why the anaerobic SRT requires a longer period than in mainstream processes. It can also be deduced that the addition of fermented primary sludge supernatant to the stripper would improve the rate of Pi release just as it does for a mainstream process. A theoretical comparison of the biochemical efficiency of PHA storage in a sidestream process versus a mainstream process can be made by assuming that the source of carbon is only from endogenous degradation for a sidestream process versus external carbon substrates. With acetate, propionate or fermented primary sludge addition, the ratio of moles of Pi released per mole of PHA stored ranged between 2.0 and 2.5. With only endogenous carbon sources, the molar ratios was as high as 5.0 to 10.0 moles of Pi released per mole of PHA stored. Thus, for the same amount of PHA stored, 3 to 4 times more Pi was released (and polyP degraded) when only endogenous carbon was relied upon, suggesting a lower process efficiency. For bio-P bacteria, this greater energy "cost" for PHA storage would probably not allow them to grow as efficiently. Thus, it is suggested that a process operating strictly with an anaerobic tank on the return sludge line would probably not be capable of achieving excellent Pi removal without requiring the chemical precipitation of a portion of the released Pi, as is done in a.PhoStrip process. In other words, PHA storage should be achieved in a reactor to which a maximum amount of simple substrates is added, instead of relying on the slow fermentation of endogenous products. 163 6.3.4. Mi n i m a l P H A storage with propionate or succinate addition When propionate made up all of the 30 mg COD/1 added to a batch of sludge, the extent of Pi release was similar to that obtained with acetate or combinations of acetate and propionate (see Figure 5.17). The net amount of PHA stored, however, was only about a third of that obtained with acetate addition ("net" refers to values corrected for the Control). Subsequent aerobic Pi uptake was comparable in both cases although a little slower for propionate. The slower rate of reaction may be explained by the requirement for the formation of acetate from propionate to allow PHV formation but it is unclear why so little PHA storage took place when such a high degree of Pi release was observed. With propionate, the high extent of Pi release and uptake could suggest that some other storage products than PHB and PHV were used by bio-P bacteria. A careful analysis of all the G C peaks from the PHA analysis, however, failed to indicate any storage in the form of shorter or longer chain PHA. It was expected that a component might be found that would correspond to poly-£-hydroxyhexanoate, a polymer that could be synthesized by the condensation of two propionate derivatives. No such compound could be found. The high degree of Pi uptake would have required as much as 40% (6.7/16) of the maximum amount of energy available from PHA, a proportion much greater than the 15 to 20% found in the case of acetate. Anaerobic addition of succinate, like propionate, resulted in very little PHA storage but in a high degree of Pi release (the molar ratio of Pi release to PHA stored was the highest obtained at 39 moles per mole). It appeared that bio-P bacteria "spent" significant amounts of their polyP energy as a result of succinate addition, but apparently not for PHA storage. Good Pi uptake (78% total Pi uptake/Pi release) was subsequently observed as for propionate addition. More research on the effects of succinate on PHA storage by bio-P bacteria could provide useful information about their metabolic activities and PHA storage. v 6.4. Microbial Activity In this section, a classification of the types of microorganisms present in a bio-P biomass, proposed sources of energy for bio-P bacteria under various conditions, and implications of PHA storage on full-scale design will be presented. 164 6.4.1. Classification and activity of microorganisms To understand and visualize the microbial activity taking place in a bio-P biomass, a classification of the activity and abundance of different types of microorganisms has been proposed (Table 6.6). As can be seen from Table 6.6, under anaerobic conditions, bio-P bacteria will store available simple carbon substrates (preferably acetate and propionate) as PHA, and fermentative microorganisms will produce more VFA which will also be stored by bio-P bacteria. Pi release will result from the storage of substrates as PHA by bio-P bacteria. Under anoxic conditions, both bio-P and non bio-P nitrate reducers would utilize nitrate and COD for growth. Nitrite could not be utilized by bio-P bacteria (in the biomass studied, at least). Pi uptake and PHA consumption would be performed by bio-P nitrate reducers. However, Pi release and PHA storage would continue to be performed by bio-P bacteria that are obligate aerobes as if they were under anaerobic conditions since they cannot use nitrate as an electron acceptor. The soluble COD would now be needed for both PHA storage and consumption by heterotrophs instead of being available only for storage as under anaerobic conditions. The net result of anoxic Pi release or uptake would depend on the relative number and activity of obligate aerobic bio-P bacteria (releasing Pi) and nitrate reducing bio-P bacteria (taking up Pi). Under aerobic conditions, all aerobic heterotrophs would use oxygen and available soluble organic compounds for growth. Bio-P bacteria would also have access to internal PHA reserves. Nitrifiers would use C 0 2 as a carbon source. The abundance of the various types of microorganisms was suggested, in Table 6.6, for the UBC pilot plant biomass. Although the categories should basically remain the same for various types and locations of bio-P processes, the relative proportion of microorganisms in each category could change with the local environmental conditions which create a pressure for the selection of the best-adapted types of microorganisms. For example, fermentative bacteria should not be as abundant in a plant where there is no primary sludge fermenter. Similarly, if nitrite were recycled to the anoxic zone, it is expected that nitrite reducing bio-P bacteria could develop. Once bio-P bacteria are established in an, activated sludge biomass, they should not be easily washed out, even if conditions are unfavorable to their growth. Manning and Irvine (1985) showed that a shift from a bio-P to a non bio-P mode of operation resulted in a lag period of 4 days with good bio-P removal followed by a gradual loss of the phosphorus removal capacity over 13 days. Conversely, a shift from a non favorable to a favorable bio-P mode resulted in a rapid resumption of the bio-P removal capacity in only 4 days. Similarly, Oldham (1985) showed in a full-scale plant that shifting from favorable fermented sludge addition to unfavorable conditions (without addition) resulted in a gradual reduction in phosphorus removal efficiency in a period of about 5 days. However, Table 6.6 Classification of microbial activity in the U B C bio-P pilot plant biomass Type of microorganism no NO x , no D.O. (anaerobic) Activity in the presence of: NO x , no D.O. (anoxic) D.O. (aerobic) Suggested abundance in UBC P.P. biomass A- Heterotrophic microorganisms (organic carbon source) 1- Bio-P bacteria (can use D.O., store polyP and store PHA) a) N0 3" reducers b) N02" reducers c) obligate aerobes Pi rel., PHA stor. growth: Pi upt., PHA & COD cons. Pi rel., PHA stor. growth: Pi upt., PHA & COD cons.*2 abundant negligible abundant I- Non bio-P microorganisms a) NO3" reducers none b) NO2" reducers none c) obligate aerobes none d) fermentative bact. VFA prod. growth: cons. COD i t tt none VFA prod. growth: cons. COD abundant non negligible abundant abundant*3 B- Autotrophic bacteria (inorganic carbon source) nitrifiers none none growth: use C0 2 abundant Abbreviations: COD: chemical oxygen demand (refers to carbonaceous substrates); cons.: consumed; D.O.: dissolved oxygen; N O x : oxidized nitrogen (N02" ro N03"); PHA: poly-s-hydroxyalkanoates (PHB and PHV); Pi: phosphate; P.P.: pilot plant; prod.: production; rel.: release; stor. storage; upt.: uptake. titrate reducers can produce nitrite of nitrogen gas; Hinder aerobic conditions, nitrate or nitrite reducers would use oxygen; 3non bio-P fermentative heterotrophs would be abundant in the biomass studied because of the primary sludge fermenter supernatant that continuously seeds the bioreactor. 166 shifting back to favorable operating conditions allowed a more rapid resumption of the phosphorus removal efficiency in about 2 to 3 days. 6.4.2. Energy for bio-P bacteria To clarify the roles of polyP and PHA,. sources of energy for bio-P bacteria will be discussed for anaerobic, anoxic and aerobic conditions for a variety of situations. The following concepts are only intended to provide a rational basis for predominant energy metabolism. Actual energy uses would depend on more complex cellular regulation. Under anaerobic conditions, polyP can be degraded to serve as an energy reserve for bio-P bacteria and for PHA accumulation, resulting in Pi release. The fastest rates of Pi release were observed with acetate and/or propionate addition. Slower rates were observed for substrates that were first fermented, presumably into acetate and propionate. Slowest rates of Pi release were observed when acetate and propionate were produced by the fermentation of more complex organic matter and possibly when some polyP was degraded as maintenance energy. If a bio-P sludge sample was kept under anaerobic conditions long enough for most of its polyP to be degraded, it is expected that the endogenous decay of proteins and other metabolites would then be used to provide energy. The term "secondary Pi release" was coined by Barnard (1984) and used by Gerber et al. (1987a) to describe slow anaerobic Pi release that was not associated with efficient subsequent aerobic Pi uptake. By contrast, "primary Pi release" would be associated with an subsequent efficient Pi uptake. According to the description above, secondary Pi release would, in fact, describe the consumption of polyP for maintenance energy which would not be associated with PHA storage. Primary Pi release would be associated with PHA storage of VFA that would be added or slowly produced by fermentation. Since polyP degradation for PHA storage represents an "investment" in the form of stored carbon that will allow subsequent uptake of Pi by bio-P bacteria, as opposed to polyP degradation for maintenance energy where the energy is not "invested" but spent, "secondary Pi release" should be minimized and "primary Pi release" maximized for optimum bio-P removal. Under anoxic conditions, nitrate can be used by nitrate-reducing bio-P bacteria as an electron acceptor for energy production by the oxidation of external substrates as well as of PHA reserves. When carbon supplies would become insufficient, polyP reserves could be degraded to serve as energy. Eventually, endogenous degradation of proteins and RNA could also be used. Bio-P bacteria unable to reduce nitrate would "feel" as if they were under anaerobic conditions. Under aerobic conditions, the situation would be essentially the same as under anoxic conditions for nitrate-reducing bio-P bacteria with the difference that oxygen instead of nitrate would be used as an electron acceptor. When carbon supplies from 167 external sources or PHA reserves become too low, polyP reserves could be used, resulting in Pi release. Finally, with the depletion of polyP reserves (it can take more than 10 days; see Figure 5.15) endogenous degradation could be used to provide energy. PolyP degradation for maintenance energy would presumably be used by bio-P bacteria as soon as the energy level (ATP/ADP) of the cell reaches a given low level. Such degradation would not occur when enough carbon from external sources or internal reserves is oxidized under anoxic or aerobic conditions. Under either anaerobic, anoxic or aerobic conditions, acetate or propionate addition could result in polyP degradation, provided that some polyP is available, to assist in substrate uptake and PHA storage. As discussed earlier, polyP degradation could be regulated by the pH gradient. 6.4.3. General considerations for full-scale design The research conducted and the biochemical model presented indicated that carbon storage as PHA emerges as playing a central role in explaining mechanisms of bio-P removal from wastewater. It is proposed that the "key" factor to optimize the efficiency of phosphorus removal in a bio-P process, is to maximize anaerobic PHA storage. For that purpose, two aspects need to be considered. First, the addition of simple "PHA storable" substrates (such as acetate and propionate) should be maximized. The addition of septic wastewater, fermented primary sludge, favorable industrial wastes, or acetate salts can all be beneficial. It would appear that the creation of a large anaerobic zone to allow VFA production would not be as efficient as VFA production in a primary sludge fermenter because of the potential degradation of polyP for maintenance energy. Second, the addition of electron acceptors such as nitrate or oxygen should be minimized in the anaerobic zone. The presence of these compounds would result in a reduction of PHA storage because of the consumption of "PHA storable" substrates instead of their storage by bio-P bacteria. Minimizing nitrate addition could be achieved by the denitrification of the mixed liquor and of the return sludge prior to its recycling into the anaerobic zone. Minimizing oxygen entrainment could be favored by the avoidance of vortexing as created by too vigorous mixing, of pumping sewage or return sludge with screw or air lift pumps, or by the use of aerated grit chambers. 168 7. CONCLUS IONS AND R E C O M M E N D A T I O N S 7.1 Conclusions This research consisted of a number of batch experiments, continuous operation of four lab-scale SBR, five months of PHA monitoring at the UBC bio-P pilot plant, and two consecutive days of PHA monitoring at the full-scale Kelowna bio-P treatment plant. Conclusions are the following: 1) Bio-P bacteria were defined as those that have the ability to store both polyP and carbon reserves. Other researchers have shown that mainly Acinetobacter and possibly Pseudomonas, found predominantly in bio-P sludges, are genera of bio-P bacteria; 2) Carbon reserves were found in the form of PHB and PHV, generically termed PHA. No significant glycogen storage took place in the UBC pilot plant sludge. Other researchers have shown, however, that glycogen could be stored aerobically and consumed anaerobically (maybe not by bio-P bacteria) when sugars such as glucose composed a significant fraction of the influent organic load. PHA reserves were proposed to play a central role in bio-P removal as the explanation of many mechanisms could be explained by substrate storage and consumption; 3) For optimum bio-P removal, PHA storage should be maximized in the anaerobic zone. For this purpose, the addition of readily storable substrates (such as acetate and propionate) should be maximized, and the addition of oxygen and oxidized nitrogen minimized; 4) Acetate and propionate were the most efficient substrates for PHA storage under anaerobic conditions by bio-P bacteria. It should be noted that the bio-P sludges studied were acclimated to acetate and propionate either from direct addition or from the addition of fermented primary sludge containing significant amounts of these two substrates. Other simple substrates such as butyrate, valerate and glucose, were taken up more slowly. Substrates made up of an odd number of carbons generally favored PHV formation whereas substrates made up of an even number of carbons favored PHB formation. Thus, it was proposed that these substrates were probably first degraded into acetate and propionate before being stored as PHA. Fermented primary sludge, that contained significant amounts of acetate and propionate, could efficiently stimulate anaerobic Pi release and PHA storage. Evidence was presented to support the fact that PHB is synthesized by the condensation of two acetate, and that PHV is synthesized by the 1 6 9 condensation of one acetate plus one propionate molecule to make up storage compounds of 4-carbon and 5-carbon monomers, respectively; 5) With 14C-acetate addition, 7% of the carbon added ended up as C 0 2 , providing support to the concept that some acetyl CoA is consumed in the T C A cycle under anaerobic conditions for NADH production; 6) PolyP degradation was proposed to play two roles as energy reserves under anaerobic conditions: first, for the transport, and second, for the energization of acetate and propionate, thus, allowing PHA storage. Under aerobic conditions polyP would be accumulated provided that there would be a sufficient availability of carbon (from an external source or from internal PHA reserves), and it would be degraded to provide energy when too little energy is produced from carbon oxidation. Under aerobic conditions, polyP could also be used as reserves for growth (e.g. for RNA or DNA synthesis); 7) The maximum Pi release rate, with the anaerobic addition of acetate, took place when polyP reserves (as estimated by total Pi release) were maximum. Thus, for each 1% P as polyP, the maximum rate of anaerobic Pi release was shown to increase by 20 mg P-l^'h"1 for a sludge of 2000 mg SS/1. The magnitude of Pi release, substrate uptake and of PHA storage were limited by polyP availability; 8) Enhanced Pi uptake (beyond that required for growth) by bio-P bacteria is proposed to be limited by the availability of stored PHA. It was shown that the rate of Pi uptake was negligible when the PHA concentration in the sludge entering the aerobic zone was about 5 mg HB/1 or lower; 9) Nitrate but not nitrite could be used by the tested bio-P bacteria as an alternative electron acceptor to oxygen. Nitrate could result in Pi uptake and PHA consumption. For the conditions of operation at the UBC pilot plant, 25% of the total Pi uptake and of the PHA consumption took place in the anoxic reactor; 10) The addition of acetate or propionate under either anaerobic, anoxic or aerobic conditions, resulted in Pi release and PHA storage. This phenomenon was explained by the regulation of polyP degradation by the pH gradient (which is reduced by the transport of the above substrates into bio-P bacteria under any of the three conditions). The continuous operation of a process with anoxic or aerobic substrate addition is not expected to favor optimum bio-P removal; 11) Potassium, magnesium and calcium were co-transported with Pi for both its release and uptake. Their contribution, expressed on a molar ratio basis, were about 0.28, 0.50 and 0.15 moles cationic charges/mole P, respectively, making up a total charge of about 0.91 cations/P for a pH near neutrality; 170 12) Biochemical models describing the activity of bio-P bacteria under anaerobic, anoxic and aerobic conditions (see section 6.1), were given significant experimental support by the experiments reported in this research; 13) Carbon storage as PHA was used to characterize the "fermentability" of a wastewater in batch tests. Excess acetate addition was also used as an indirect method to estimate the amount of polyP in a bio-P sludge; 14) A classification of the microbial activity of different groups of microorganisms present in a bio-P biomass was proposed. 7.2 Recommendations for Future Research Based on the findings of this research, it is recommended that the following areas be investigated: 1) Fundamental mechanisms remain to be explored to explain the regulation of polyP, PHA and glycogen storage and degradation, both with mixed and pure cultures. These mechanisms could be used to help develop meaningful and more accurate mathematical models for bio-P removal. It would also be useful to further investigate the pathways used for anaerobic PHA storage of various substrates with attention paid to the role and pathways of polyP degradation, and to the various degrees of PHA storage for given amounts of polyP degradation. The presence of lactate (in addition to mainly acetate and propionate as VFA) in fermented primary sludge could be quantified. The regulation of polyP degradation by the pH gradient could be confirmed in the presence of an excess of acetate or propionate, at a high pH or in the presence of 2,4-DNP or of other toxicants. An enzyme that uses polyP directly for proton expulsion (like the ATP-ase or some pyrophosphatases) may be found. N A D H production by the T C A cycle or other means could be confirmed. The effect of succinate resulting in Pi release but minimal PHA storage could be further quantified. The importance of glycogen storage when the wastewater contains high concentrations of sugars, could be studied. The simultaneous transport of sulfate with Pi release or uptake would need to be investigated and explained. Radioactive isotopes could be used for some of these investigations; 2) The isolation and characterization of the types of bio-P bacteria, and their importance and activities in various types of processes with different types of substrates (e.g. municipal wastewater, acetate, glucose) or electron acceptors (oxygen, nitrate, nitrite) could be investigated. The magnitude of fermentation activity within the anaerobic zone of a bio-P process could be established and its desirability compared to that occurring in a primary sludge fermenter. For microbiological investigations, dialysis bags immersed in SBR-types of reactors (where the unaerated/aerated sequence takes place in the same vessel) could be useful; 171 3) It would also be useful to determine which factors limit aerobic Pi uptake (such as PHA availability). It could be verified that aerobic polyP content of the sludge is the major factor that quantifies the aerobic Pi uptake capacity of a sludge. 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Bacteriol. 153, 371-374. A P P E N D I C E S 186 APPENDIX 1 Table A - l Chemical structure of compounds. Reserve polymers: polyP ~ ( p 0 3 ) n -~ poly-j8-hydroxybutyrate ~ ( C H ( C H 3 ) C H 2 C O O ) n ~ poly-/3-hydroxyvalerate ~ ( C H ( C H 2 C H 3 ) C H 2 C O O ) n ~ (Note: the /3-hydroxy oxygen is transformed into the hydroxy oxygen of the carboxyl group) poly-/?-hydroxyalkanoates co-polymer of PHB and PHV glycogen a glucose polymer with a-( 1- > 4) glycosyl linkages and occasional Q-(1-> 6) branch linkages 4-oxo-valeric acid (a carbon reserve?) C H 3 C O C H 2 C H 2 C O O H Substrates: formic acid acetic acid ethanol propionic acid lactic acid glycine butyric acid iso-butyric acid /3-hydroxybutyric acid succinic acid valeric acid iso-valeric acid hexanoic acid citric acid glucose HCOOH C H 3 C O O H C H 3 C H 2 O H C H 3 C H 2 C O O H C H 3 C H O H C O O H N H 2 C H 2 C O O H CH 3 (CH 2 ) 2 COOH (CH 3 ) 2 CHCOOH C H 3 C H O H C H 2 C O O H COOH(CH 2 ) 2 COOH CH 3 (CH 2 ) 3 COOH ( C H 3 ) 2 C H C H 2 C O O H CH 3 (CH 2 ) 4 COOH (COOH)CH 2 C(OH)(COOH)CH 2 COOH CHO(CHOH) 4 CH 2 OH Toxicants: fluoride cyanide 2,4-dinitrophenol F" CN-( N 0 2 ) 2 C 6 H 3 O H 187 APPENDIX 2 Table A-2 Molecular weight and C O D equivalence for substrates. Substrate (as acid) M.W. COD equivalence (mg/mmole) (mg/100 mg COD) formate 46.03 287.7 acetate 60.05 93.8 propionate 74.05 66.1 butyrate 88.11 55.1 iso-butyrate 88.11 55.1 valerate 102.13 49.1 iso-valerate 102.13 49.1 . hexanoate 116.16 45.3 lactate 90.08 93.8 ^-hydroxybutyrate 104.10 72.3 succinate 118.09 105.4 citrate 192.12 133.3 ethanol 46.07 47.9 glycine 75.07 117.3 glucose 180.16 93.9 Example of calculation of the number of mg acid/100 mg COD for acetate: Acetate C H 3 C O O H : balance for complete oxidation: C 2 H 4 0 2 +. 2 0 2 ---> 2 C 0 2 + 2 H 2 0 number of O: 2 4 4 2 M.W.: 60.05 64 (1.00 mM acetate) for 100 COD: 93.83 100 (1.56 mM acetate) 188 APPENDIX 3 Batch experiments - Treatment plants characterization data. The data presented in the following three tables summarize influent, process and effluent data that characterize the activated sludge used for each batch experiment. A process schematic of the plant at the time the sludge was taken is given in Figure A - l . Activated sludge samples were always taken from the aerobic zone. Because of the importance of the influent feed concentration on the activated sludge response for bio-P removal, and of the daily variability of the influent COD value, a weighted moving average is reported for influent COD. The weight factors used were 0.5, 1, 2 and 4 for the days preceding the date by three, two, one and zero (date of experiment) days. The sludge retention time was always maintained at 20 days. Occasional determinations of the MLVSS to MLSS ratio gave an average value of 0.79. The effluent suspended solids average concentration was about 10 mg/1. A three week study conducted to establish a relationship between B O D u (ultimate BOD) and COD gave a range of values from 0.75 to 0.95 with large variations from day to day. An average value of about 0.85 might be a reasonable estimate of the B O D u to COD ratio (Manoharan, 1988 - personal communication). 189 Process type: B Qin acetate i Qw 3.0 l/min 1 390 I A 790 I 1570 I Process type: C 1 0 Q 2.0 Q Ow Qin • 0.5 l/h 1.5 I ^ 2.25 I 4. 0 I 2.0 Q 1.0 I Process type: D and E Qin 1.0 Q 2.5 l/min 360 I f 715 I 1430 I 400 I 500 I 1.0 Q(typeD) 1.4 Q (type E) Figure A - l Process configuration at the time of sludge sampling for the batch experiments. Sludge was taken from the UBC bio-P pilot plant (process types "A", "B", "D" and "E") and from a lab-scale bio-P plant (process type "C"). Table A-3.1 Batch experiments - Plant characterization data (concentrations in mg/l). Charac- Oct 23, Oct 29 & teristics Dates: Apr 25/84 Nov 13/84 Dec 14/84 Aug 22/85 Nov4/85A Influent COD 253 + 354 + 24 (HAc) 50 (HAc) TP (as P) 3.8 5.5 TKN (as N) 22.2 36.2 Process type A B MLSS (aerobic) 3550 3450 aerobic %P 3.3 3.0 (%P/SS) Pi anaerobic 14.6 10.3 (as P) Effluent COD 26 51 TP (as P) 0.2 1.2 TKN (as N) 1.3 1.8 N0 3 - (as N) 6.7 13.1 300 + 300 + 360 29 (HAc) 60 (HAc) 64 (HAc) 5.0 4.6 4.8 30.6 27.5 25.0 A C D 4420 2340 5200 3.3 5.8 3.3 15.9 18.2 20.0 29 32 28 0.1 0.1 0.2 1.3 1.8 3.3 7.9 10.3 4.4 Note: xdata shown for Oct 23/85, Oct 29/85 and Nov 4/85 is an average of data obtained between November 14 to 25, 1985 (no earlier data was available). Table A-3.2 Batch experiments - Plant characterization data (concentrations in mg/1). Charac- Dates: Aug 19/86 Oct 8/86 Oct28/86 Novll/86 Feb 23/87 Mar 3/87 Mar 11/87 teristics Influent COD 236 154 201 264 194 184 182 TP (as P) 4.4 3.4 4.4 3.8 4.8 3.8 3.5 TKN (as N) 25.0 21.2 26.6 23.8 29.1 23.6 22.5 Process type E E E E E E E MLSS (aerobic) 2100 2580 2440 2670 2880 2860 2655 aerobic %P 4.1 3.8 4.3 3.5 4.3 3.8 3.9 (%P/SS) Pi anaerobic 14.0 13.0 15.7 16.5 14.3 12.2 12.3 (as P) Effluent COD 30 10 23 14 24 18 34 TP(asP) 0.8 0.1 0.2 0.1 0.7 1.9 0.3 TKN (as N) 1.1 0.7 5.2 1.1 1.5 1.2 1.6 NQ 3-(asN) 7.7 5.2 5.1 6.5 7.8 8.5 6.8 Table A-3.3 Batch experiments - Plant characterization data (concentrations in mg/I). Charac- Dates: June 4/87 June 25/87 July 8/87 Aug 6/87 teristics Influent COD 158 TP(asP) 3.1 TKN (as N) 17.2 Process type E MLSS (aerobic) 2480 aerobic %P 3.3 (%P/SS) Pi anaerobic 12.3 (as P) Effluent COD 26 TP (as P) 0.6 T K N (as N) 0.7 N0 3 " (as N) 5.8 181 98 240 2.2 1.9 4.5 15.4 13.5 30.0 E E E 2300 2410 2580 3.7 3.5 4.4 8.9 8.7 16.0 25 25 29 0.7 0,7 0.1 1.2 1.4 1.3 5.4 5.0 8.9 193 APPENDIX 4 Table A-4.1 UBC pilot plant PHB/PHV data; A side. A side Date PHB PHV (mg HB/g MLSS) (mg H V / g MLSS) (1987) F AN AX O F A N A X O 12-Feb 4.7 2.6 1.6 7.2 3.1 1.4 16-Feb 7.5 3.3 2.0 10.0 3.7 1.7 20-Feb 5.1 • 2.8 1.8 8.7 3.5 1.6 23-Feb 6.5 3.3 2.1 9.0 3.5 1.8 26-Feb 4.7 2.4 1.6 3.6 1.4 0.7 05-Mar 4.7 2.5 1.7 5.0 2.2 1.2 16-Mar 4.5 2.7 1.7 5.1 2.3 1.0 19-Mar 4.2 2.1 1.4 4.0 1.5 0.7 26-Mar 1.4 4.5 2.4 1.6 0.9 5.3 2.0 1.0 30-Mar 1.3 5.7 2.9 1.9 0.8 6.0 2.1 0.9 02-Apr 1.3 5.7 2.9 1.9 0.8 5.7 2.2 0.9 06-Apr 0.5 6.5 2.9 1.8 0.5 6.1 1.8 0.8 10-Apr 1.4 5.1 2.6 1.7 0.8 6.2 2.3 1.0 13-Apr 1.7 5.0 2.5 1.6 1.1 6.2 2.1 1.0 16-Apr 1.3 3.3 2.0 0.9 2.6 1.1 23-Apr 1.3 5.3 3.2 2.0 0.9 6.5 2.8 1.2 27-Apr 1.6 5.6 1.5 1.3 6.5 1.1 05-May 1.4 4.6 1.9 1.4 1.3 5.6 1.8 1.0 07-May 1.0 5.7 4.1 1.4 0.9 5.8 4.1 0.9 11-May 1.0 3.8 2.1 1.3 0.9 4.2 1.6 0.8 14-May .1.5 3.7 2.2 1.2 4.8 2.1 19-May 1.0 4.6 2.1 1.2 0.8 6.4 2.1 0.9 21-May 1.7 5.8 2.6 1.6 1.3 6.2 2.4 1.1 25-May 1.6 4.6 2.2 1.1 1.3 ' 5.4 2.0 0.9 28-May 1.4 5.6 2.4 1.4 1.1 6.7 2.2 1.0 01-Jun 1.0 5.0 2.5 1.4 0.8 5.4 2.2 1.2 05-Jun 2.3 6.4 2.6 1.1 1.1 8.8 3.0 1.2 08-J.un 0.2 5.9 2.5 1.0 0.3 8.0 2.5 0.9 11-Jun 0.4 5.0 2.1 1.0 0.3 5.1 1.8 1.0 15-Jun 2.4 6.2 2.6 1.6 1.6 7.1 2.4 1.1 18-Jun 1.7 5.1 2.6 1.5 1.3 6.0 2.9 1.3 23-Jun 0.9 3.7 1.8 0.9 1.0 4.4 1.4 0.8 25-Jun 2.2 8.3 3.4 2.0 1.7 5.5 2.2 1.2 29-Jun 0.9 9.2 3.6 1.0 7.0 2.3 02-Jul 3.0 8.3 3.4 1.8 1.9 5.7 2.2 1.0 06-Jul 2.5 4.3 2.6 1.6 1.5 4.8 1.9 1.0 avg s 1.4 0.6 5.5 1.3 2.7 0.5 1.6 0.3 1.1 0.4 6.1 1.4 2.3 0.6 1.1 0.3 194 Table A-4.2 UBC pilot plant PHB/PHV data; B side. B side Date PHB (mg HB/g SS) PHV (mg H V / g SS) (1987) A N AX O AN A X O 12-Feb 3.2 1.9 1.4 3.0 1.6 1.1 16-Feb 6.4 2.2 1.6 4.6 1.5 0.9 20-Feb 3.0 1.3 1.4 1.9 0.9 0.8 23-Feb 3.9 2.5 1.7 2.9 1.4 0.8 26-Feb 3.6 2.2 1.6 1.9 0.9 0.5 05-Mar 2.7 1.8 1.4 1.7 1.1 0.9 16-Mar 3.9 1.8 1.3 1.6 0.7 0.5 19-Mar 2.8 1.8 1.2 1.0 0.6 0.4 26-Mar 3.1 2.2 1.7 1.8 1.1 0.8 30-Mar 4.5 2.5 1.9 1.9 0.9 0.6 02-Apr 3.8 2.3 1.5 1.9 1.0 0.6 06-Apr 4.1 2.7 1.9 2.0 1.0 0.6 . 10-Apr 4.0 2.6 2.0 1.8 1.0 0.7 13-Apr 4.1 2.3 1.8 1.6 0.8 0.5 16-Apr 4.9 3.1 2.1 2.3 1.2 0.7 23-Apr 5.1 3.1 2.3 2.4 1.2 0.8 27-Apr 4.3 2.5 1.7 2.4 2.3 0.8 05-May 3.8 2.0 1.5 2.0 0.9 0.5 07-May 4.2 2.6 1.7 2.3 1.3 0.7 11-May 4.9 1.7 1.4 2.5 0.9 0.7 avg s 4.0 0.9 2.2 0.4 1.7 0.3 2.2 0.7 1.1 0.4 0.7 0.2 14-May 2.9 2.2 1.4 1.6 1.1 0.8 19-May 3.2 1.8 •1.4 1.7 0.8 0.6 21-May 4.4 2.4 1.6 2.4 1.3 0.8 25-May 3.1 1.9 1.7 2.2 1.2 0.8 28-May 3.9 2.5 1.6 2.3 1.4 0.8 01-Jun 3.4 2.1 1.6 2.0 1.2 1.0 05-Jun 4.0 2.4 1.4 3.0 1.7 1.0 11-Jun 5.1 1.9 1.7 2.4 1.1 1.0 15-Jun 3.9 2.6 1.2 3.0 1.5 1.0 18-Jun 4.2 2.7 1.5 2.6 1.8 1.1 23-Jun •2.7 2.1 1.6 2.8 1.1 1.2 29-Jun 15.4 8.0 4.2 9.6 7.5 3.6 02-Jul 11.3 7.5 2.9 6.3 5.3 2.1 06-Jul 3.3 1.8 2.4 1.1 The data corresponding to the period between May 14 and July 6 was not used because of an operational upset at the pilot plant. 

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