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A microbiological study of the enhanced biological phosphate removal process Chu, Angus 1990

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A MICROBIOLOGICAL STUDY OF THE ENHANCED BIOLOGICAL PHOSPHATE REMOVAL PROCESS By ANGUS CHU B.Sc, The Un i v e r s i t y of B r i t i s h Columbia, 1988 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Microbiology) We accept t h i s t hesis as conformed to the required standards THE UNIVERSITY OF BRITISH COLUMBIA September, 1990 ©Angus Chu, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT The research objective i n t h i s thesis i s to define and c l a r i f y s p e c i f i c f u n c t i o n a l properties of sewage treatment process biomass. The s p e c i f i c o b j e c t i v e i s to determine whether the b a c t e r i a recovered from sludge correspond to the organisms with the r e a l f unctional c a p a b i l i t y to sequester phosphate w i t h i n the sewage ecosystem. This objective has been broken down into three categories. . The f i r s t category deals with these organisms i n terms of t h e i r recovery and v i a b i l i t y . The second deals with t h e i r i d e n t i f i c a t i o n . The t h i r d deals with the phosphate accumulating a b i l i t y of the i s o l a t e d organisms. To address the question of recovery, the microscopic numbers of b a c t e r i a i s o l a t e d from sludge were compared to the organisms recovered from p l a t i n g . The r e s u l t s from t h i s comparison was s i m i l a r to previously published data (1-10% recovery). The 8 um f r a c t i o n a t i o n assay allowed the f r a c t i o n a t i o n of sewage into two separate components ( f i l t r a t e and f i l t e r ) and the a b i l i t y to look at the f r a c t i o n s i n terms of recovery and phosphate accumulating capacity. From t h i s assay i t was shown that the f l o e s were responsible f o r the majority of the phosphate accumulation. Microscopic autoradiography also confirmed this observation. The cross p l a t i n g technique was an attempt to improve recovery from s o l i d media and to look at whether d i f f e r e n t organisms were selected under d i f f e r e n t n u t r i e n t conditions. I d e n t i f i c a t i o n of two of the populations i s o l a t e d from d i f f e r e n t media suggested that the type of media played a c e n t r a l r o l e i n recovery. Microplating was attempted i n order to look at colony development at the microscopic l e v e l to answer some fundamental questions regarding recovery from f l o e s and free b a c t e r i a . To answer the question of which b a c t e r i a are responsible f o r the phenomenon of phosphate accumulation, a pure culture autoradiographic assay was developed to screen organisms i s o l a t e d from sludge for t h e i r p o t e n t i a l to. accumulate excessive amounts of phosphates. Autoradiography of pure' cultures a f t e r growth i n plate conditions mimicking the n u t r i t i o n a l parameters of the Bio-P process allowed the c o r r e l a t i o n of the p o t e n t i a l to accumulate phosphate to i n d i v i d u a l i s o l a t e d b a c t e r i a . From these r e s u l t s i t was shown.that, contrary to what many investigators believe, Acinetobacter may not be the only_;genus i n t e r t i a r y sewage....treatment that i s responsible for phosphate accumulation. The r e s u l t s i n d i c a t e that there are s p e c i f i c groups within the genus Acinetobacter and other genera that have the p o t e n t i a l to accumulate phosphate under these conditions. TABLE OF CONTENT ABSTRACT . i TABLE OF CONTENT . . . . i i LIST OF TABLES i v LIST OF FIGURES v i ACKNOWLEDGEMENT v i i i INTRODUCTION 1 A. Bacter i a and the Bio-P process 1 B. Bio-P removal 4 C. Thesis objective 7 MATERIALS AND METHODS 8 A. Recovery and v i a b i l i t y 8 1. Crossplating......... 8 2. F r a c t i o n a t i o n of microscopic vs. pl a t e counts 9 3. Microplating 9 4. I s o l a t i o n of s t r i c t anaerobes 10 B. B a c t e r i a l i d e n t i f i c a t i o n 10 C. Plants included i n the study 13 D. 3 2P accumulation 13 1. In s i t u incorporation 13 a. Uptake of l a b e l 13 b. Microscopic autoradiography 14 2. Pure culture autoradiography 15 RESULTS 17 A. Recovery and v i a b i l i t y 17 1. Cross p l a t i n g . . . . 17 2. 8 um f r a c t i o n a t i o n of microscopic vs. plate counts 20 3. Microplating and microscopic autoradiography 21 B. .Bacterial i d e n t i f i c a t i o n 26 1. Biolog and API/NFT i d e n t i f i c a t i o n systems 26 2. Population p r o f i l e s 29 3. Acinetobacter genospecies c l a s s i f i c a t i o n 33 4. Anaerobic growth 35 C. 3 2P accumulation 37 1. In s i t u incorporation ....37 2. Pure cu l t u r e autoradiography ...37 3. Optimized autoradiography for screening Bio-P 52 DISCUSSION .64 A. Recovery and v i a b i l i t y . 64 1. Enumeration 64 2. Cross p l a t i n g 65 3. Microscopic vs. plate counts 66 4. Microplating and microscopic autoradiography ...68 5. I s o l a t i o n of s t r i c t anaerobes 69 B. B a c t e r i a l i d e n t i f i c a t i o n 70 i i i 1. API/NFT and Biolog i d e n t i f i c a t i o n 70 2. Population p r o f i l e s of a l l plants 71 C. Pure culture autoradiography 73 1. Optimization of autoradiographic assay 73 2. Pure culture autoradiographic screening 74 D. Biochemical model for phosphate accumulation 78 E. Comparison between Biolog's and Bouvet's system on Acinetobacter genospecies c l a s s i f i c a t i o n 80 CONCLUSION 83 REFERENCES . 84 LIST OF TABLES Table I: Genera of b a c t e r i a i s o l a t e d from enhanced phosphorus removal plants. Table I I : Plants sampled i n the study. Table I I I : Genus l e v e l comparison between PYE and CGY media fo r population s e l e c t i o n from the UBC p i l o t plant. Table IV: Microscopic versus plate counts for the 8 urn f r a c t i o n a t i o n assay. Table V: Comparison between API/NFT and Biolog systems of b a c t e r i a l i d e n t i f i c a t i o n . Table VI: P i l o t plant sample comparison between API/NFT and Biolog systems of b a c t e r i a l i d e n t i f i c a t i o n . Table VII: Samples used i n the study and t h e i r health i n terms of phosphate accumulating capacity. Table VIII: Genus l e v e l comparison between Kelowna, p i l o t plant and Squamish samples. Table IX: Genus l e v e l comparison between p i l o t plant samples. Table X: Evaluation of Bouvet's method for Acinetobacter genospecies c l a s s i f i c a t i o n f o r autoradiographic method 1. Table XI: Evaluation of Bouvet's method of Acinetobacter genospecies c l a s s i f i c a t i o n f o r autoradiographic method 2. Table XII: Anaerobic growth a f t e r 15 days. Table XIII: Summary of the optimum conditions for the pure culture autoradiographic assay. Table XIV: Accumulators from Kelowna and p i l o t plant samples screened with autoradiographic method 1. Table XV: Accumulators from Kelowna, p i l o t plant and Squamish samples for autoradiographic method 2. Table XVI: Autoradiographic method 1 for Kelowna and p i l o t plant (P,R) populations. Table XVII: Autoradiographic method 2 for Kelowna and p i l o t plant (P,R) populations. Table XVIII: Autoradiographic method 2 genus level comparison of a l l genera Table XIX: Accumulators within i n d i v i d u a l genera screened with autoradiographic method 1. Table XX: Accumulators within i n d i v i d u a l genera screened with autoradiographic method 2. Table XXI: Species l e v e l comparison between accumulators and nonaccumulators screened with autoradiographic method 1. Table XXII: Species l e v e l comparison between accumulators and nonaccumulators screened with autoradiographic method 2. LIST OF FIGURES Figure 1: Biochemical model of phosphate accumulation (Comeau, 1988). (A)anaerobic zone, (B)aerobic zone. Figure 2: Plant process configuration for the Kelowna (A) and fo r the UBC p i l o t plant (B). Figure 3: Plant process configuration f o r the plants at Squamish and Mamqualm (A) and Salmon Arm (B) sewage treatment plant. Figure 4: Cross p l a t i n g f o r the various media. Figure 5: 3 2P uptake of f i l t e r f r a c t i o n versus f i l t r a t e f r a c t i o n . Figure 6: Microplating of samples of sewage. Microcolony development can be followed f o r up to 12 hours. (A)Time 0, (B)6 hours, (C)12 hours at room temperature. Figure 7: (A)Microscopic autoradiography on samples of sludge. The dark areas are l o c a l i z e d areas of radioactive phosphate. (B)A combination of microplating and microscopic autoradiography. Figure 8: 3 2P uptake p r o f i l e s of a l l the treatment plants sampled. Figure 9: Percent accumulators from the Kelowna population versus the v a r i a b l e s tested. (A)anaerobic incubation time, (B) change i n pH, (C)pre-assay incubation time. Figure 10: Percent accumulators from Kelowna versus the v a r i a b l e tested. (A)radiation exposure l e v e l s , ( B )radiation exposure times, (C)temperature. Figure 11: Average accumulation r a t i o f o r the accumulator group from Kelowna versus v a r i a b l e s tested. (A)anaerobic incubation time, (B)changing the pH, (C)pre-assay incubation time. Figure 12: Average accumulation r a t i o for the accumulator group from Kelowna versus v a r i a b l e s tested. (A)radiation exposure l e v e l s , (B)radiation exposure time, (C)temperature. Figure 13: Average normalized counts (CPM/A562) of the accumulator population from Kelowna versus the v a r i a b l e s tested. (A)anaerobic incubation time, (B)changing the pH, (C) pre-assay incubation time. Figure 14: Average normalized counts (CPM/A562) of the accumulator population from Kelowna versus the v a r i a b l e s tested. (A) r a d i a t i o n exposure l e v e l s , (B)radiation exposure times, (C)temperature. Figure 15: Percent accumulators from Kelowna versus the va r i a b l e s tested at the genus l e v e l . (A)anaerobic incubation time, (B)changing the pH, (C)pre-assay incubation time. Figure 16: Percent accumulators from Kelowna versus the va r i a b l e s tested at the genus l e v e l . (A)radiation exposure l e v e l , (B)radiation exposure time, (C)temperature. Figure 17: Average accumulation r a t i o for accumulators from Kelowna versus the v a r i a b l e s tested at the genus l e v e l . (A)anaerobic incubation times, (B)changing the pH, (C)pre-ass incubation times. Figure 18: Average accumulation r a t i o for accumulators from Kelowna versus the variables tested at the genus l e v e l . ( A ) r a diation exposure l e v e l s , (B)radiation exposure times, (C)temperature. Figure 19: A photograph of an autoradiograph done on a n i t r o c e l l u l o s e d i s c . v i i i ACKNOWLEDGEMENT I g r a t e f u l l y acknowledge the following persons f o r t h e i r guidance and support during my studies and research at The Un i v e r s i t y of B r i t i s h Columbia: Dr. William Ramey f o r supervising my research and encouraging me throughout my studies, Dr. William Oldham, Professor and Head of the C i v i l Engineering Department and coordinator and founder of the Bio-P research group for h i s v i s i o n , support and f i n a n c i a l assistance, Dr. Barry McBride, Professor and Dean of Science and Dr. Barbara D i l l for serving on my committee and providing valuable recommendations. Frederick Koch, research associate with the Bio-P research group for enlightening discussions and f o r operating the UBC p i l o t plant from which samples were supplied for my work, Terrance Mah, fellow graduate student f o r h i s help and contribution i n performing some of the experiments, and James Low, Andrew Wong, Homa Ahmad and Shelley Pearlman, summer students working i n the lab. 1 INTRODUCTION A. Bact e r i a and the Bio-P process Eutrophication i s a water p o l l u t i o n problem which r e s u l t s i n the overabundant growth of algae and aquatic plants. Carbon, nitrogen and phosphorus have been recognized as the elements responsible f o r eutophication. In almost a l l cases nitrogen and phosphorus have been i d e n t i f i e d as the l i m i t i n g nutrients. Phosphorus has been recognized as a be t t e r element to remove than nitrogen for eutrophication control because atmospheric nitrogen can be f i x e d by blue-green algae. Activated sludge treatment plants modified for enhanced phosphate removal e x i s t now i n at l e a s t 8 countries around the world. These countries include A u s t r a l i a , B r a z i l , Canada, France, New Zealand, South A f r i c a , the United States and Zimbabwe (Toerien et al., 1990). Conventional activated sludge processes remove only 20 to 30% of the phosphorus i n sewage (Nesbitt, 1969; Schaak et al. , 1985). However removal of 90% or greater i s necccessary to control eutrophication problems ( i e . 0.5-1.0 mg/L phosphorus i n e f f l u e n t ) . This e f f i c i e n c y can be achieved by ei t h e r chemical p r e c i p i t a t i o n , enhanced b i o l o g i c a l accumulation or both. Chemical removal involves ad d i t i o n of m e t a l l i c s a l t s such as calcium, i r o n or aluminum (Wiechers,1987). However t h i s technique suffers from high costs of chemicals and the accumulation of large quantities of chemical waste sludge. A comparison has been made between chemical versus b i o l o g i c a l processes f o r the removal of phosphates from wastewater (Canviro, 1986; Morrison, 1988). I t was concluded that Bio-P removal was more cost e f f e c t i v e than chemical p r e c i p i t a t i o n . Under many conditions t h i s conclusion l e d to an i n t e n s i f i c a t i o n of research on b i o l o g i c a l methods of phosphorus removal ( Y a l l et al., 1970; 2 Levin, 1975; Fukase et al. , 1985). Enhanced phosphate removal from these systems seems to be governed by a b i o l o g i c a l mechanism and i n order to optimize this process a thorough understanding of the physiology and ecology of the system i s required. Table I i s a l i s t of the genera that have been i s o l a t e d from various phosphorus removal plants. To date the techniques used to investigate the f u n c t i o n a l properties of the i s o l a t e d organisms from sewage i n terms of t h e i r phosphate accumulating a b i l i t y are inadequate. In most studies population i d e n t i f i c a t i o n was the extent of the analysis. When they were tested i t was by e i t h e r screening the b a c t e r i a f or phosphate uptake as a response to growth on a r i c h medium or to induce phosphate accumulation by s t a r v a t i o n (Suresh et al., 1985; Fuhs and Chen, 1975; Strechan et al., 1990). Both of these methods were d i f f i c u l t to r e l a t e to plant operational parameters. The development of a technique f o r screening Bio-P p o t e n t i a l at the l e v e l of the i n d i v i d u a l bacterium might resolve or avoid some of the contradictions and assumptions made i n previous studies. Fuhs and Chen (1975) believed that b a c t e r i a from the genus AcLnetobacter were the p r i n c i p l e agents i n the Bio-P process. Since then many researchers have implicated t h i s genus along with other genera as being important i n the phenomenon of phosphate accumulation (Brodish, 1985; Cloete, 1985; Buchan, 1983). On the other hand, the o v e r a l l contribution of the genus AcLnetobacter has been questioned (Cloete and Steyn, 1988; Stephenson, 1987). Some plants with a high percentage of AcLnetobacter work poorly while other plants operate well without any detectable AcLnetobacter population. In many of these reports on the microbiology of phosphorus removal plants, the authors can be c r i t i c i z e d over the methodology used for sampling, i s o l a t i n g and i d e n t i f y i n g t h e i r populations as well as a l i m i t e d c o r r e l a t i o n to the Bio-P process. Most of the studies i n v o l v i n g AcLnetobacter have r e l i e d on c h a r a c t e r i s t i c s which f a i l to recognize 3 Table I: Genera of b a c t e r i a i s o l a t e d from enhanced phosphorous removal plants. Genus References AcLnetobacter 1,5,6,8 ,9 ,10,11,12 ,13 ,14,16 ,17 ,18 ,19 , 21,22,24,27,29,30,31,32,33,34,35,36,37 Aeromonas 3,4,5 ,6 ,7 ,23,25,26,27,28,36 Alcaligenes 5,27 Bacillus 13,35 Chromobacterium 3 Citrobacter 3,4,7,13,23,25 Enterobacter 3,7,23 Erwinia 3 Escherichia 3,4,5,7,23 Flavobacterium 3,5,7,23,25,28 Klebsiella 3,4,7,15,23 Microthrix 2,3,20 Moraxella 3,27,28 Nocardia 3,20 Neisseria 27 Pasteurella 3,4,5,7,25 Proteus 3 Pseudomonas 3,5,6,7,13,27,28,35,36,37 Salmonella 3 Serratia 3 Shigella 7,25 Yersinia 3,23,25 Xanthomonas 27 References: (l)Beacham et al.,1990; (2)Best et al. , 1985; (3)Brodisch and Joyner 1983; (4)Brodisch, 1985; (5)Buchan,1983; (6)Chow, 1988; (7)Cloete et a l . , 1985 (8)Cloete'and Steyn, 1988; (9)Deinema et al., 1980; (lO)Deinema et al., 1985 (ll)Deinema et al.,1985; (12)Duncan et al.,1988; (13)Floretz. and Hartman, 1984 (14)Fuhs and Chen, 1975; (15)Gersberg and Allen,1985; (16)Groenestijn, 1988 (17)Groenestijn and Deinema, 1985; (18) Groenestijn and Deinema, 1987; (19)Hao and Chang, 1987; (20)Hart, 1985; (21)Hiraishi et al., 1989; (22)Lawson and Tonhazy, 1980; (23)Le F l o h i c , 1985; (24)Lotter, 1985; (25)Lotter and Murphy, 1985; (26)Lotter et al. , 1986; (27)Meganck, 1987; (28)Meganck et al., 1985; (29)Murphy and Lotter, 1986; (30)0sborn and N i c h o l l s , 1978; (31)0sborn et al., 1986; (32)Rensink et al. , 1981; (33)Stephenson, 1987; (34)Streichan et al. 1990; (35)Suresh et al. 1985; (36)T'Seyen et al., 1985; (37)Wentzell et al., 1988. h the taxonomic problems associated with t h i s genus. Some of the discrepancies concerning the p o t e n t i a l of t h i s genus i n the systems might be due to d i f f e r e n t unrecognized species of Acinetobacter i n the d i f f e r e n t systems. U n t i l recently, the genus Acinetobacter consisted of a si n g l e species calcoaceticus divided into 2 morphologically and biochemically d i s s i m i l a r biotypes, anitratus and lwoffii. Organisms of t h i s genus are widespread i n the environment and have been i s o l a t e d from s o i l , water, sewage, and mucosal and outer surfaces of humans. Some are opportunistic pathogens which are considered to be a problem i n nosocomial infe c t i o n s (Bouvet and Grimont, 1987). These 2 species of Acinetobacter were simply c l a s s i f i e d as being non-motile oxidase p o s i t i v e rods. More recent studies have shown that t h i s genus contains at l e a s t 12 d i s t i n c t genetic groups (Bouvet and Grimont, 1986). The paradox concerning the r o l e of Acinetobacter i s due to a l i m i t e d understanding of the c a p a b i l i t y of the d i f f e r e n t species i n the Bio-F process. Consequently, our understanding of the extent of the d i v e r s i t y which exi s t s within t h i s genus i n the context of phosphate removal from wastewater and the consequences of plant performance i s poor. B. Bio-P removal The process of b i o l o g i c a l phosphorous removal i s a modification of a conventional acti v a t e d sludge process. A continuous flow Bio-P process uses an aerobic reactor preceded by an anaerobic reactor. In the anaerobic zone the soluble phosphorus concentration increases as a r e s u l t of release by the biomass. In the subsequent aerobic zone, the soluble phosphorus concentration decreases as a r e s u l t of phosphorus accumulation by the biomass i n excess of metabolic requirements. There have been many models developed i n an attempt to explain these observations (Toerien et al. , 1990). Despite the many successful a p p l i c a t i o n s of enhanced phosphorus removal plants, the r o l e of the organisms responsible f o r phosphate removal and the mechanisms involved are s t i l l unclear (Gerber et al., 1986). N i c h o l l s and Osborn (1979) postulated that the anaerobic zone caused stress i n the microbial population, which res u l t e d i n the eventual production of poly-phosphate. Fuhs and Chen (1975) believed that the anaerobic zone functioned as a fermentation zone converting organic matter to fermentation end products which could i n turn be converted to storage products f o r subsequent use i n the aerobic zone to drive the accumulation of phosphate. In the model proposed by Comeau et al. (1986) and Wentzel et al. (1986), the b a c t e r i a responsible f o r phosphate accumulation are believed to derive an advantage over other b a c t e r i a by exchanging poly-P for the accumulation and storage of polyhydroxyalkanoate (PHA), e s p e c i a l l y poly-B-hydroxybutyrate (PHB), i n the anaerobic zone. This stored carbon reserve i n turn provides energy to drive the accumulation of phosphate under aerobic conditions where carbon a v a i l a b i l i t y l i m i t s m i c r o b i a l growth. This model explains the b e n e f i c i a l e f f e c t s of simple acetate and propionate additions to the anaerobic zone since these short chain f a t t y acids favor the anaerobic accumulation of PHB's which would i n turn favor the aerobic accumulation of phosphates (Potgieter and Evans,1983; S i e b r i t z et al., 1983; A r v i n and Kristensen, 1985). Thus with t h i s model the r o l e of the anaerobic zone s h i f t e d from one to create a stress condition to one i n which anaerobic carbon storage could occur for subsequent u t i l i z a t i o n i n the carbon poor aerobic stage. A s i m p l i f i e d representation of the model i s shown i n Figure 6 acatat* and A. available carbon substrates Pi Figure 1: Biochemical model of phosphate accumulation (Comeau, 1988). (A)anaerobic zone, (B)aerobic zone. 7 C. Thesis objective The general goal of the research as a whole was to gain a better understanding of the process of b i o l o g i c a l phosphate removal from wastewater. The s p e c i f i c objective was to determine whether the ba c t e r i a i s o l a t e d from mixed l i q u o r correspond to the organisms with the r e a l functional c a p a b i l i t i e s to sequester phosphate within the sewage ecosystem. This objective has been broken down into three s p e c i f i c areas of i n v e s t i g a t i o n . One deals with the i s o l a t i o n of s p e c i f i c organisms and t h e i r subsequent recovery and v i a b i l i t y , another deals with organism i d e n t i f i c a t i o n , and the l a s t deals with the phosphate accumulating a b i l i t y of the i s o l a t e d organisms. I have investigated the factors influencing the recovery and v i a b i l i t y of b a c t e r i a from sewage, t h e i r i d e n t i f i c a t i o n and the phosphate accumulating a b i l i t y of selected organisms. To answer the fundamental question of which b a c t e r i a are responsible f o r the phenomenon of phosphate accumulation, a pure culture autoradiographic assay was developed to screen organisms i s o l a t e d from sludge f o r t h e i r p o t e n t i a l to accumulate phosphate. MATERIALS AND METHODS A. Recovery and v i a b i l i t y 1. Cross p l a t i n g In an attempt to f i n d a better medium f o r the growth of sewage organisms and determine whether the populations from several media could be combined to improve the t o t a l v i a b l e recovery of microorganisms from the sewage, b a c t e r i a were i s o l a t e d from the 10"5 d i l u t i o n of samples plated on 9 d i f f e r e n t growth media. The growth media were: 1. Lu r i a : 10 g tryptone, 5 g yeast extract, 5 g sodium c h l o r i d e , and 2 g glucose i n 1 L of water (pH 7). 2. Nutrient: 5 g pepticase, 3 g beef extract, 5 g sodium c h l o r i d e , and 2 g glucose i n 1 L of water (pH 7.3). 3. CGY: 5 g casitone, 1 g yeast extract and 10 ml g l y c e r o l i n 1 L of water (pH 7). 4. PYE: 2 g peptone, 1 g yeast extract, 0.2 g MgSo<*7H20, and 0.1 g CaCl 2*2H 20 i n 1 L of water (Poindexter, 1964). 5. Sabouraud: 10 g neopeoptone and 20 g dextrose (or maltose) i n 1 L of water ( PH5.6). 6. M9 Medium: 0.5 g NaCl, 7.0 g Na2HP0A, 3.0 g KH2P0<,, 1.0 g NHAC1 and 0.2 g MgS0«*7H 20 and 0.2% (W/V) glucose i n 1 L of water with the following combinations of a d d i t i v e s , (a) 40 ug/ml u r a c i l , 10 ug/ml cysteine (M9S), (b) 40 ug/ml u r a c i l , lOug/ml cysteine, 20 ug/ml tryptophan (M9T), (c) 20ug/ml tryptophan, 40 ug/ml u r a c i l (M9U), (d) 20ug/ml tryptophan, 10 ug/ml cysteine (M9R) and (e) no 9 additives (M9Q). These organisms were streaked i n a g r i d pattern and r e p l i c a p lated to each of the other media. The growth of the tra n s f e r r e d organisms were followed under various conditions (10 and 25° C as well as anaerobiosis) and various incubation times (2, 5, 9 and 12 days) to determine whether the selected populations were s i g n i f i c a n t l y d i f f e r e n t . 2. Fra c t i o n a t i o n of microscopic versus plate counts Some samples of sewage were counted microscopically i n a Petroff-Hausser and Helber b a c t e r i a l counting chamber, while comparable samples were plated onto CGY medium for v i a b l e colony counts and incubated at room temperature for up to 5 days. Other samples were f r a c t i o n a t e d into two separate components. To accomplish t h i s the sample was passed though a f i l t e r with a s i z e c u t o f f of 8 um (either M i l l i p o r e HAUP 800 n i t r o c e l l u l o s e or Nucleopore SN: 110614 polycarbonate) . Microscopic counts, p l a t e counts onto CGY medium at room temperature f or up to 5 days and 3 2P uptake assays were then performed on both the f i l t e r retentate (floes) and the f i l t r a t e (free p a r t i c l e s ) f r a c t i o n s . 3. Microplating To assess the v i a b i l i t y of microscopically v i s i b l e organisms i n the samples, a microplating procedure was used. Microscope s l i d e s and cover s l i p s were s t e r i l i z e d i n 75% ethanol. 0.5 ml of molten CGY agar (1.2%) medium was pipeted onto one end of the s t e r i l e glass s l i d e . The s l i d e was placed on s t e r i l e Whatman Number 1 f i l t e r paper moistened with 1.0 ml of s t e r i l e water i n a s t e r i l e P e t r i 10 plate to s o l i d i f y . Samples of sewage were spread onto the agar and covered with the glass cover s l i p and incubated at room temperature. Microcolony development can then be observed f o r up to 12 hours (Torello and Monta, 1981). 4. I s o l a t i o n of s t r i c t anaerobes To assess the possible s i g n i f i c a n c e of the obligate anaerobic population wi t h i n the t o t a l b a c t e r i a l population, anaerobic cultures were c o l l e c t e d by immersing an Erlenmyer f l a s k gassed with anaerobic mixture (5% hydrogen, 5% carbon dioxide, and 90% nitrogen) into the anaerobic zone of the UBC p i l o t plant. The sample was placed i n a glove bag gassed with the anaerobic mixture and plated on CGY agar with resazurin and incubated at room temperture for 15 days. B. B a c t e r i a l i d e n t i f i c a t i o n A t o t a l of 6 b a c t e r i a l populations have been i d e n t i f i e d , four populations from the UBC p i l o t plant, one population from Kelowna and one population from Squamish. 2 have been i d e n t i f i e d with the API/NFT i d e n t i f i c a t i o n system Analylab products (1977) and a l l 6 have been i d e n t i f i e d with the Biolog i d e n t i f i c a t i o n system (Bockner, 1989). The i s o l a t e s i d e n t i f i e d as Acinetobacter were further tested by the transformation assay (Juni, 1972) and the a b i l i t y to u t i l i z e 23 d i f f e r e n t carbon substrates which allow p r a c t i c a l delineation of t h i s genus into 12 separate genospecies (Bouvet and Grimont [1986]). 11 thickener supernatant primary effluent aerobic recycle trough return sludge LEGEND S3 anaerobic anoxic aerobic secondary clarlfier Figure 2: Plant process configurations f or the Kelowna (A) and the UBC p i l o t plants (B). 12 LEGEND anaerobic anoxic aerobic sludge wastage B. Figure 3: Plant process configurations for the plants at Squamish and Mamqualm (A) and Salmon Arm(B) sewage treatment plant. 13 Table I I : Plants sampled i n the study. Plants Type Configuration Kelowna t e r t i a r y Squamish secondary Modified UCT Conventional activ a t e d sludge Modified UCT t r i c k l i n g f i l t e r conventional acti v a t e d sludge UBC p i l o t plant Salmon Arm Mamqualm t e r t i a r y t e r t i a r y secondary Note: ' ; UCT stands f o r The Un i v e r s i t y of Cape Town, South A f r i c a . C. Plants included i n the study The municipal sewage treatment plants that were sampled are l i s t e d i n Table I I . The process configuration of each of these plants i s shown i n Figures 2 and 3. 3 2P uptake assays were done on each of the plants l i s t e d . Population i d e n t i f i c a t i o n s and pure culture autoradiography were done on samples from Kelowna, Squamish and the UBC p i l o t plant. D. 3 2P accumulation 1. In s i t u incorporation a. Uptake of l a b e l The effectiveness of the samples to sequester soluble phosphate was measured by monitoring the uptake of radioactive phosphate. The assay involves taking a sample of anaerobic sewage and allowing i t to s i t undisturbed for 2 hours. A 3 ml sample i n a 16x150 mm acid washed t e s t tube was then spiked with radioactive ortho-phosphate (approximately 0.04 uCi) and 10 mg/1 of cold phosphate c a r r i e r . The spiked samples were then aerated on a shaking platform at room temperature. 14 At times 0, 15, 30, 45, 60, 75, and 90 minutes, 150 u l aliquots were withdrawn from the incubating tubes and centrifuged i n a microcentrifuge tube for 3 minutes. 100 u l of supernatant was then immediately removed and absorbed onto a f i l t e r disk. These disks were then d r i e d and counted i n a s c i n t i l l a t i o n counter f o r t o t a l counts to measure the t o t a l soluble 3 2P remaining i n the supernatant. b. Microscopic autoradiography Microscopic autoradiography was used to te s t the l o c a l i z a t i o n of the incorporated phosphate wi t h i n the sewage f l o e s . A sample of labeled sewage from the standard 3 2P uptake assay was e i t h e r spread onto a glass s l i d e , a i r dried and heat f i x e d , or plated onto microplates and grown for 6 hours at room temperature and then d r i e d at 80° C e l s i u s . These s l i d e s were then dipped into a mixture of I l f o r d nuclear research emulsion (K5) d i l u t e d by an equal volume of d i s t i l l e d deionized water heated to 50° Celsius. The coated s l i d e s were drained i n the v e r t i c a l plane and d r i e d at room temperature for 30 minutes. The d r i e d s l i d e s were then exposed i n the dark f o r up to two weeks and developed at regular i n t e r v a l s . To develop the emulsion, the s l i d e s were immersed i n X-dol developer for 10 minutes, t r a n s f e r r e d to a 1% aqueous acetic a c i d stop s o l u t i o n for 1 minute, then f i x e d i n a s o l u t i o n of 30% sodium t h i o s u l f a t e for twice the time i t takes the emulsion to c l e a r ( B e r l i n and Rylander, 1963). 35 mm photographic s l i d e s were taken of the developed autoradiographic s l i d e s with the Olympus Vanox-S microscope system. 15 2. Pure culture autoradiography Two methods were developed to screen the i s o l a t e d organisms f o r the p o t e n t i a l to sequester phosphate i n an a l t e r n a t i n g anaerobic/aerobic condition. In method 1 the i s o l a t e s were grown up overnight i n g r i d patterns on s o l i d CGY medium at room temperature before the assay i s started. In method 2 the pure cultures of b a c t e r i a were i s o l a t e d from a sample of sewage onto CGY medium and grown up i n CGY l i q u i d media overnight at room temperature. Two u l of each i s o l a t e was pipetted onto 2 plates of CGY media i n a g r i d pattern and grown up overnight. In e i t h e r method a p a i r of colony l i f t s were then done by pressing a sheet of Schleicher and Schuell BA88 n i t r o c e l l u l o s e against the colonies i n the g r i d pattern on separate p l a t e s . Both f i l t e r s were then l i f t e d o f f and placed, colony side up, onto fresh CGY for 2 hours. The f i l t e r s were then washed by 4 consecutive transfers i n 1% agar and placed onto 2 plates of minimal media containing acetate as the sole carbon source (1.0 g/1 NHAC1, 0.7 mg/1 Na2HP0A, 0.2 g/1 KH2P04, 0.2 g/1 MgS04 and 2 mg/1 acetate pH 6.2). One f i l t e r acted as cont r o l and remained aerobic, the other was placed i n an anaerobic j a r gassed with anaerobic mix (5% hydrogen, 5% carbon dioxide and 90% nitrogen) f or 4 hours. Both f i l t e r s were then washed 4 successive times by leaving them on 1% agar for 1 minute, then placed onto minimal media plates containing r a d i o a c t i v e phosphate and no carbon source f o r 30 minutes (1 g/1 NHAC1, 0.03 mg/1 Na2HP04, 8.8 mg/1 KH2P0/, and 10 uCi radioactive ortho-phosphate) . A f t e r 30 minutes the f i l t e r s were removed and washed 4 times by leaving them on 1% agar to d i l u t e out the unincorporated background radiation,, then dri e d at 90° Ce l s i u s . A f t e r autoradiography, colonies were punched out of the f i l t e r s with a 6 mm hole punch and counted i n a s c i n t i l l a t i o n counter for t o t a l counts. Bicinchoninic protein 16 assays (BCA) were then done on a l l f i l t e r samples and used to normalize the counts. Each experiment was done i n t r i p l i c a t e . The assay was optimized by t e s t i n g several v a r iables which included: 1. The exposure time of the f i l t e r s to radioactive plates (10, 30, 60, 120 minutes). 2. The phosphate concentrations i n radioactive plates (5, 10, 50 and 100 uCi same r a t i o of r a d i o a c t i v i t y to col d phosphate). 3. The number of anaerobic and aerobic recycles (2 hours anaerobic to 1 hour aerobic f o r 2 c y c l e s ) . 4. The extent of anaerobic exposure (1, 2, 4 and 8 hours). 5. The preassay incubation times before the anaerobic exposure.(0, 2, 4 and 8 hours) 6. The a c i d i t y of the medium during anaerobic exposure (pH 6, 7 and 8). 7. The temperature during the assay (12, 25 and 37° C) . In each t e s t , the autoradiographic assay was repeated by changing one variable at a time and holding a l l other v a r i a b l e s constant. In order to assess the data some fu n c t i o n a l parameters must be defined. The anaerobic counts/protein i s defined as the amount of counts incorporated by a single colony i n the anaerobic s h i f t condition normalized to the amount protein i n that colony (CPM/A562) . The aerobic counts/protein i s defined as the amount of counts incorporated by a s i n g l e colony i n the aerobic control condition normalized to the amount of p r o t e i n i n that colony. The accumulation r a t i o i s defined as the anaerobic counts/protein divided into the aerobic counts/protein. An accumulator i s defined as an i s o l a t e that has an accumulation r a t i o of 1.2 or greater. A nonaccumulator i s defined as an i s o l a t e that has an accumulation r a t i o of less than 1.2. c 17 RESULTS A. Recovery and v i a b i l i t y 1. Cross p l a t i n g I t i s a w e l l established f a c t that p l a t e counts of b a c t e r i a i n enhanced phosphorus removal plants give values that are only 1-10% of those obtained by microscopic counts (Cloete and Steyn, 1987) . This e f f e c t was not due to a s i g n i f i c a n t proportion of obligate anaerobic organisms. The anaerobic plate counts were approximately 100 f o l d l e s s than the aerobic plate counts. When these primary anaerobic colonies were subsequently streaked onto the same medium and grown a e r o b i c a l l y the majority of these b a c t e r i a turned out to be f a c u l t a t i v e organisms. To check whether a broader range of media would s e l e c t f o r a greater v a r i e t y of b a c t e r i a (Sorheim et al., 1989), sewage microbes were i s o l a t e d from 10 d i f f e r e n t media and tested f o r growth on a l l of the other media. The growth of these i s o l a t e s was further tested at 10° C, 25° C and under anaerobic conditions to determine whether a s i g n i f i c a n t number of unique i s o l a t e s were capable of growth on p a r t i c u l a r media. Figure 4 depicts the a b i l i t y of the t o t a l organisms i s o l a t e d o f f of a l l primary media to grow on each i n d i v i d u a l medium. For example the population of organisms i s o l a t e d from CGY was tested for i t s a b i l i t y to grow on each of the other media i n c l u d i n g CGY. The same procedure was applied to the other media and the t o t a l growth on each medium graphed. Approximately 95% of the organisms i s o l a t e d from the various culture 18 Figure 4: Cross p l a t i n g for the various media. The bars represent the t o t a l percentage of i s o l a t e s from a l l the media tested that grew on each p a r t i c u l a r medium. Primary (Prm.), nutr i e n t (Nut.), PYE (Cau.), Sabouraud (Yst.). A. 25° C incubation temperature, B. 12° C incubation temperature. Table I I I : Genus l e v e l comparison between PYE and CGY media f o r population s e l e c t i o n from the UBC p i l o t plant. Genus PYE(%) CGY(%) Acinetobacter 54. 2 a 16.7 Klebsiella 8. 3 5.6 Aeromonas 4. 2 16.7 Vibrio 12 .5 22.2. Enterobacter 4. 2 0.0 Pseudomonas 8. 3 27.8 CDC groups 0. 0 5.6 Pasteurella-Actinobacillus 4. 2 5.6 Note: aPercent of that genus recovered from the sample Table IV: Microscopic versus plate counts f or the 8 urn f r a c t i o n a t i o n assay. microscopy concentration (particles/ml) (xl(T 8) Fractions Salmon arm p i l o t plant Kelowna Squamish free 1.75 0.40 0.15 0.45 p a r t i c l e s t o t a l f l o e s 6.93 1.17 4.06 7.80 t o t a l 8.68 1.57 4.21 8.25 p l a t i n g concentration (cfu/ml) (xl(T 6) f i l t r a t e 0.23 2.88 3.40 4.20 f i l t e r 2.63 2.80 2.40 78.0 t o t a l 9.60 12.20 6.30 104.0 20 conditions could grow on either CGY or PYE media, implying that the populations i s o l a t e d from the various media were not appreciably d i f f e r e n t . The r e s u l t s suggested that there was no appreciable s e l e c t i v e difference between the media (under these l i m i t e d parameters), although subsequent i d e n t i f i c a t i o n of two of the populations i s o l a t e d from CGY and PYE.media using both API/NFT and Biolog i d e n t i f i c a t i o n systems showed an apparent difference i n terms of species composition. A genus l e v e l comparison between the populations (Table III) showed that PYE medium appears to s e l e c t f o r a higher percentage of Acinetobacter and Enterobacter than does CGY medium (54 and 4% compared to 17 and 0%) and a lower percentage of Aeromonas, CDC groups and Pseudomonas (4, 0 and 12% compared to 17, 6 and 28%). Since these populations came from the same sample, t h i s difference must be due to a s e l e c t i v i t y of these two types of media which was not picked up by the cross p l a t i n g . This apparent difference also appears at the species l e v e l . Out of the genus Acinetobacter, 79% of t h i s genus i s o l a t e d from PYE medium were from genospecies 7 while the number f o r CGY medium was only 50%. This d i f f e r e n c e i n selected populations might be explained as differences i n competitive growth rates between b a c t e r i a within a f l o e or the s u r v i v a l rates on the medium. In e i t h e r case, the choice of media seemed to bias the i s o l a t i o n but not increase the t o t a l i s o l a t a b l e population. For t h i s reason most of the subsequent studies used CGY medium i n order to f a c i l i t a t e comparison to r e s u l t s generated i n other studies. 2. 8 urn f r a c t i o n a t i o n of microscopic versus plate counts To further t e s t whether the problem of recovery was due to c e l l clumping within f l o e s rather than l i m i t e d c a p a b i l i t y of organisms to grow on plates, samples of sewage were counted microscopically and plated f o r v i a b l e colony counts (Table IV). These r e s u l t s showed that approximately 1-10% of the microscopic count gave r i s e to colony forming u n i t s ( c f u ) . The 8 um f i l t e r assay was developed i n an attempt to fract i o n a t e the sewage and look at i t s components i n terms of recovery and phosphate accumulating capacity. In t h i s assay the samples were separated into a f r a c t i o n which contained i n d i v i d u a l c e l l s and a f r a c t i o n which contained f l o e s . Each sample was counted microscopically, plated fo r v i a b l e counts and tested for 3 2 P uptake. The r e s u l t s showed that approximately 0.3-10% of the free p a r t i c l e s gave r i s e to c f u and 100% of the f l o e s gave r i s e to c f u (table V). Figure 5 represents the r e s u l t s of a 3 ZP uptake assay performed on the 8 um f r a c t i o n s . From t h i s figure i t i s c l e a r that the f i l t e r f r a c t i o n which constitutes the f l o e f r a c t i o n of the sewage i s responsible f o r the majority of the phosphate accumulation. I f we normalize f o r the amount of p r o t e i n i n each f r a c t i o n and express phosphate accumulation per mg of p r o t e i n , the r e s u l t i s the opposite of what was observed e a r l i e r (data not shown). The f i l t r a t e f r a c t i o n i s now better than the f i l t e r f r a c t i o n i n 3 ZP uptake per amount of protein. This implies that the phosphate sequestering organisms are not l i m i t e d to f l o e s , even though the majority of the organisms seem to be associated with the f l o e s . 3. Mi c r o p l a t i n g and microscopic autoradiography To further t e s t the v i a b i l i t y and function of i n d i v i d u a l organisms i n the samples, aliquots of sewage were spread onto agar set on a microscope s l i d e and covered with a glass cover s l i p . Microcolony development can then be observed fo r up to 12 hours, and co r r e l a t e d to the recovered v i a b l e counts of the 8um f i l t e r assay. Figure 6 depicts a time course experiment at 0, 6, and 12 hours. A l l the f l o e s observed gave r i s e to microcolonies which should i n turn give r i s e to colonies on agar plates while only approximately 10% of the free p a r t i c l e s gave r i s e to microcolonies. I t i s i n t e r e s t i n g that more than one microcolony can a r i s e from a sing l e f l o e but we do not see any mixed colonies when primary colonies from plates are streaked f o r p u r i t y on a second nut r i e n t plate. This observation implies that microcolonies either stop growing or are simply overwhelmed by f a s t e r growing more p r o l i f i c neighbouring microcolonies. To look at the fun c t i o n a l properties of phosphate accumulation capacity, microscopic autoradiography of d i r e c t l y l a b e l l e d sewage samples was attempted. Figure 7 contains photograph of l a b e l l e d sewage samples a f t e r microscopic autoradiography. As one can p l a i n l y see the floes of b a c t e r i a contain the majority of l a b e l when compared to the free p a r t i c l e s . Panel B shows microscopic autoradiography performed on l a b e l l e d samples plated onto CGY agar set onto microscope s l i d e s incubated f o r 12 hours at room temperature. From these photographs i t i s evident that the majority of the 3 2P w i t h i n the fl o e s i s not d i s t r i b u t e d evenly throughout the microcolony. The organisms d i r e c t l y responsible f o r phosphate accumulation i n the sewage eit h e r do not grow under our nu t r i e n t conditions or microcolonies a r i s e from a few b a c t e r i a within each flo e such that r a d i o a c t i v i t y associated with the o r i g i n a l c e l l s are d i l u t e d to an extent which renders i t undetectable. c. F i g u r e 6: M i c r o p l a t i n g o f samples o f sewage. M i c r o c o l o n y development can be f o l l o w e d f o r up t o 12 h o u r s . (A)Time 0, (B)6 h o u r s , (C)12 h o u r s a t room t e m p e r a t u r e . Figure 7: (A)Microscopic autoradiography on 2 samples of sludge. The dark areas are l o c a l i z e d areas of radioactive phosphate. (B)A combination of microplating and microscopic autoradiography (under br i g h t f i e l d microscopy on l e f t side and phase contrast microscopy on the r i g h t s i d e ) . 26 B. B a c t e r i a l i d e n t i f i c a t i o n 1. Biolog and API/NFT I d e n t i f i c a t i o n Systems A t o t a l of 6 populations have been i d e n t i f i e d using these i d e n t i f i c a t i o n systems, 4 from the UBC p i l o t plant, one from Kelowna and one from Squamish. Two have been i d e n t i f i e d with the t r a d i t i o n a l API/NFT i d e n t i f i c a t i o n system and a l l s i x have been i d e n t i f i e d with the recently developed Biolog i d e n t i f i c a t i o n system. This l a t t e r assay i s based on d i f f e r e n t p r i n c i p l e s than ei t h e r the t r a d i t i o n a l biochemical responses of API or the growth responses of NFT. I t r e l i e s on the reduction of a single redox dye, tetrazolium c h l o r i d e , to detect the increased r e s p i r a t i o n that occurs i n a c e l l o x i d i z i n g a given carbon source. The dye reduction patterns are then r e l a t e d to expected patterns for a v a r i e t y of species stored i n an extensive computer database. The Biolog system has some d i s t i n c t advantages over both the API and NFT systems. I t has more environmental species i n i t s database. I t can d i f f e r e n t i a t e more species of AcLnetobacter and the i d e n t i f i c a t i o n r e l i e s on many more nutri e n t t e s t s . Because t h i s new method i s d i f f e r e n t from standard biochemical methods, r e l a t i n g the responses generated by t h i s technique to biochemical responses i n t r a d i t i o n a l taxonomic assays i s d i f f i c u l t . Therefore the two populations that were i d e n t i f i e d with both systems were compared for discrepancies (Tables V and VI) . Out of the 12 AcLnetobacters i n Table V that had been i d e n t i f i e d with the API/NFT systems, 11 could be i d e n t i f i e d by the Biolog system while out of the 18 AcLnetobacters i d e n t i f i e d with the Biolog system only 11 could be i d e n t i f i e d with API/NFT. This trend i s s i m i l a r f o r Aeromonas, Comamonas, KlebsLeila, and Pseudomonas. Within the AcLnetobacter, Biolog delineated 4 of the possible 13 genospecies while API/NFT 27 Table V: Comparison between API/NFT and Biolog systems of b a c t e r i a l i d e n t i f i c a t i o n . Genera Biolog on API/NFT API/NFT on Biolog Acinetobacter l l / 1 2 a 11/18 Aeromonas 3/4 3/11 Bordetella 0/1 Buttiaxella 0/1 CDC group II-H 0/1 0/1 Comamonas 0/3 Enterobacter 0/1 Erwinia 0/1 Klebsiella 3/3 3/5 Horaxella 0/1 Pasteurella 0/1 Pseudomonas 7/7 7/9 Sphingobacterium 0/1 Vibrio 1/5 1/4 Note: ' ' \ aThe number of i s o l a t e s i d e n t i f i e d to that genus l e v e l by the f i r s t i d e n t i f i c a t i o n system out of the number of i s o l a t e s i d e n t i f i e d by the second. Table VII: Samples used i n the accumulating capacity. study and t h e i r health i n terms of phosphate Samples Code Functional/nonfunctional UBC p i l o t plant Ra it P + CI II C2 + Kelowna K + Squamish S + Note: - are nonfunctional samples and + are functional samples i n terms of phosphate uptake. aThe code r e f e r s to the l e t t e r given to each sample. 28 Table VI: P i l o t plant sample comparison between API/NFT and Biolog i d e n t i f i c a t i o n systems. Species from Biolog AcLnetobacter genospecLes 12 AcLnetobacter JohnsonLL/genospecLes 7 AcLnetobacter lwoffLL/genospecLes 8 Aeromonas hydrophLla Aeromonas hydrophLla DNA group 1 Aeromonas hydrophLla DNA group 2 Aeromonas media Bordetella-like specLes ButtLauxella agrestLs CDC group II-H Comamonas terrLgena Comamonas testosteronL ErwLnLa amylovora KlebsLeila oxytoca KlebsLella pneumonLae KlebsLella pneumonLae sub.C KlebsLella terrLgena KlebsLella trevLsaLL Moraxella osloensLs Pasteurella anatlpestLfer Pseudomonas corrugata Pseudomonas delafLeldLL Pseudomonas fluorescens Pseudomonas fragL Pseudomonas margLnalLs Pseudomonas putLda sub.B Pseudomonas-unLdentLfLed fluorescent SphLngobacterLum MultLvorum-ILke VLbrLo anguLllarum VLbrLo cholera VLbrLo fluvLalLs VLbrLo vulnLfLcus sub.A Species from API/NFT AcLnetobacter calcoacetLcus/lwoffLL Aaeromonas hydrophLla CDC groupll F Enterobacter agglomerans KlebsLella oxytoca , KlebsLella pneumonLae Pseudomonas aerugLnosa Pseudomonas fluorescens Pseudomonas putLda VLbrLo fluvLalLs 29 could only i d e n t i f y 1 out of the 2 possible biotypes (Table V) . This holds true f o r many of the other genera. The r e s u l t s c l e a r l y show that the B i o l o g system has much more r e s o l v i n g p o t e n t i a l than the previously used systems. Biolog can d i f f e r e n t i a t e 18 species for Aeromonas, 71 for Pseudomonas and 12 f o r Moraxella while API/NFT can only d i f f e r e n t i a t e 5, 17, and 4 r e s p e c t i v e l y . As shown i n Table VI B i o l o g d i f f e r e n t i a t e s a t o t a l of 32 species compared to the 10 species i d e n t i f i e d by API/NFT. 2. Population P r o f i l e s B a c t e r i a l populations from 6 samples have been i d e n t i f i e d with the BIOLOG i d e n t i f i c a t i o n system. Tables VIII and IX are genus l e v e l comparisons between populations from the Kelowna, UBC p i l o t and Squamish treatment plants. Kelowna and the UBC p i l o t plant have s i m i l a r process configurations and are both t e r t i a r y treatment plants while the Squamish plant i s a secondary treatment plant. Figure 8 shows that Kelowna, Squamish (although only secondary treatment) and p i l o t plant samples (C1,C2,P) possess biomass with phosphate accumulating capacity (although CI and C2 are only semi-functional). I t also shows that the p i l o t plant sample (R) i s a collapsed nonfunctional system (Table VII). With t h i s pattern of functions i n mind i t i s c l e a r from Tables VIII and IX that the genus Acinetobacter accounts f o r a major percentage of the population i n a l l of the f u n c t i o n a l Bio-P plants (24-41%) except for Squamish and i s low i n the collapsed plant (R). However, i n the case of Squamish, even though i t only constitutes 4% of the population, the t o t a l number of AcLnetobacter i s much higher than any of the other plants. In the case of Squamish the CDC group made up an abnormally high 82% of the t o t a l population. I f t h i s abnormal group was deleted from the 0 15 30 45 60 75 90 Time ( m i n . ) A S q u a m i s n ( S ) x P i l o t p l a n t ( C l , C 2 ) v UBC p i l o t p l a n t ( P ) • K e l o v n a ( K ) i- N e g a t i v e c o n t r o l o UBC p i l o t p l a n t ( R ) Time (min.) • S a l m o n Arm + Mamqualm o N e g a t i v e c o n t r o l Figure 8: 3 2P uptake p r o f i l e s of a l l the treatment plants sampled. 31 Table V I I I : Genus l e v e l comparisons between Kelowna, p i l o t plant and Squamish samples. Genera p i l o t plant Kelowna Squamish R,collapsed K,functional S,functional cells/mL xicr* % t o t . cells/mL xlO" 4 % tot. cells/mL xlO" 6 % Tot. Unknown 195 9 77 5 18 1 AcLnetobacter 90b 4a 373 24 68 4 ActLnobacLllus 40 2 0 0 0 0 Aeromonas 202 9 306 20 65 4 Agrobacterium 0 0 20 1 0 0 Alcaligenes 60 3 0 0 o o Buttiuaxella 0 0 10 1 0 0 Campnocytophaga 10 0 0 0 11 1 CDC groups 100 5 0 0 1255 82 CLtrobacter 10 0 0 0 0 0 ClavLbacter 0 0 o 0 11 1 Comamonas 0 0 0 0 6 0 Enterobacter 10 0 91 6 0 0 EscherLcLa 30 1 20 1 1 0 FlavobacterLum 1 0 0 0 0 0 GLlardLa 520 24 5 0 0 0 Haemophilus 117 5 40 3 2 0 Kingella 1 0 1 0 10 1 Klebsiella 10 0 42 3 12 1 Kluyvera 0 0 10 1 0 0 Listonella 0 0 0 0 1 0 Methlobacterium 10 0 0 Q 0 0 Moraxella 570 27 201 13 14 1 Neisseria 10 0 10 1 0 0 Oligella . 0 0 0 0 1 0 Pasteurella 0 0 0 0 55 4 Pseudomonas 120 6 225 15 3 0 Psychrobacter 0 0 28 2 1 0 Seratia 10 0 0 0 0 0 Shewanella 0 0 1 0 1 0 Vibrio 211 10 140 9 13 1 Yersinia 0 0 1 0 1 0 Note: ; 1 ; aPercent of the t o t a l population as based on cfu. bConcentration of that genus within the t o t a l population. 32 Table IX: Genus l e v e l comparisons between p i l o t plant samples. Genera p i l o t plant p i l o t plant p i l o t plant P,functional CI,functional C2,functional cells/mL xlO"* % t o t . cells/mL xlO-* % t o t . cells/mL xl0" A % tot. Unknown 35 10 30 9 10 4 Acinetobacter 90 b 35 a 140 41 60 26 Actinobacillus 0 0 0 0 0 0 Aeromonas 60 23 60 18 70 30 Agrobacterium 10 4 0 0 0 0 Alcaligenes 0 0 0 0 0 0 Buttiuaxella 0 0 0 0 0 0 Campnocytdphaga 0 0 0 0 0 0 CDC 0 0 10 3 0 0 Citrobacter 0 0 0 0 0 0 Clavibacter 0 0 0 0 0 0 Comamonas 0 0 20 6 10 4 Enterobacter 0 0 0 0 0 0 Eschericia 0 0 0 0 0 0 Flavobacterium 10 4 0 0 0 0 Gilardia 0 0 0 0 0 0 Haemophilus 0 0 0 0 0 0 Kingella 0 0 0 0 0 0 Klebsiella 40 15 40 12 10 4 Kluyvera 0 0 0 0 0 0 Listonella 0 0 0 0 0 0 Methylobacterium 0 0 0 0 0 0 Moraxella 10 4 20 6 0 0 Neisseria 0 0 0 0 0 0 Oligella 0 0 0 0 0 0 Pasteurella 0 0 0 0 0 0 Pseudomonas 40 15 30 9 60 26 Psychrobacter 0 0 0 0 0 0 Seratia 0 0 0 0 0 0 Shewanella o 0 0 0 0 0 Vibrio 0 0 20 6 20 9 Yersinia 0 0 0 0 0 0 Note: ; ; " aPercent of the t o t a l population as based on cfu. C o n c e n t r a t i o n of that genus within the t o t a l population. 33 c a l c u l a t i o n s the percentage of AcLnetobacter would increase to approximately 20% of the remaining population and would resemble the other working plants. The t o t a l number of AcLnetobacter i n the collapsed system, R i s also higher than C2 although i t accounts f o r only 4% of the population. Many of these AcLnetobacter might not be f u n c t i o n a l Bio-P organisms. This evidence provides no good c o r r e l a t i o n between the numbers of t o t a l AcLnetobacter and phosphate accumulating a b i l i t y . There i s however a c o r r e l a t i o n between the percent of that genus out of the t o t a l population (% t o t a l ) and the accumulation that occurs under plant operating conditions except i n the case of Squamish. 3. AcLnetobacter genospecies c l a s s i f i c a t i o n A l l i s o l a t e s i d e n t i f i e d as AcLnetobacter with either API/NFT or Biolog methods of i d e n t i f i c a t i o n were grouped and were tested f or t h e i r a b i l i t y to u t i l i z e 19 d i f f e r e n t carbon sources which allow p r a c t i c a l d e l i n e a t i o n of this genus into 12 separate genospecies (Bouvet and Grimont 1986) . Out of the 19 carbon substrates 3 showed some i n t e r e s t i n g r e s u l t s (Tables X,XI). When compared to the i s o l a t e s a b i l i t y to accumulate phosphate, the proportion of s t r a i n s which used trans-aconitate, L-aspartate, and malonate showed a diffe r e n c e between accumulators and nonaccumulators f o r both autoradiographic methods. From both methods 1 and 2 i t i s c l e a r that i s o l a t e s that were c l a s s i f i e d as accumulators had a c o n s i s t e n t l y lower proportion of p o s i t i v e substrate u t i l i z a t i o n tests than those c l a s s i f i e d as nonaccumulators. For example from Table XI out of the t o t a l AcLnetobacter population 20% of the accumulators were p o s i t i v e f o r trans-aconitate u t i l i z a t i o n compared to 75% of the nonaccumulators. The basis for this d i f f e r e n c e i s not c l e a r . I t appears that e i t h e r the accumulator s t r a i n s came Table X: Evaluation of Bouvet's method f o r Acinetobacter genospecies c l a s s i f i c a t i o n f o r autoradiographic method 1. s t r a i n aut. (n) aco. asp. mal. TOTAL + 17 0 00 a 0 .00 0 .00 1 ~ 11 0 .18 0 .18 0 .18 Acinetobacter + 9 0. 00 0. 00 0. 00 johnsonii/genosp. 7 7 0. 14 0. 14 0. 14 Acinetobacter + 6 0. 00 0. 00 0. 00 lwoffii/genosp. 8 3 0. 33 0. 33 0. 33 "Proportion of the population that i s p o s i t i v e f o r that t e s t . aut.- Autoradiographic r e s u l t s , + i s accumulators, =,- are nonaccumulators aco.-aconitic acid, asp.L-aspartic acid, mal.-malonic acid. Table XI: Evaluation of Bouvet's method of Acinetobacter genospecies c l a s s i f i c a t i o n f o r autoradiographic method 2. st r a i n s aut. (n) aco. asp. mal. TOTAL + 10 0. 20 a 0 .00 0 .20 = 12 0 .36 0 .07 0 .36 - 12 0 .75 0 .25 0 .75 Acinetobacter + 8 0. 25 0. 00 0. 25 johnsonii/genosp.7 = 4 0. 25 0. 00 0. 25 - 8 0. 88 0. 13 0. 88 Acinetobacter + 1 0. 00 0. 00 0. 00 lwoffii/genosp.8 = 7 0. 29 0. 00 0. 29-- 3 0. 67 0. 33 0. 67 P r o p o r t i o n of the population that i s p o s i t i v e f o r that t e s t . aut.- autoradiographic r e s u l t s , + i s accumulators, = i s ne u t r a l accumulator i s nonaccumulators. aco.-aconitic acid, asp.L-aspartate, mal.-malonic a c i 35 from a l i m i t e d group within each species or that there i s a s e l e c t i v e pressure i n the system to l i m i t the u t i l i z a t i o n of these nutrients i n each accumulator s t r a i n . Another possible explanation l i e s i n the f a c t that these carbon u t i l i z a t i o n tests were chosen for t h e i r a b i l i t y to d i f f e r e n t i a t e genetic groups wit h i n the genus Acinetobacter. I f the phenomenon of phosphate accumulation i s associated with a p a r t i c u l a r genospecies, i t could p o s s i b l y mean that these tests could d i f f e r e n t i a t e t h i s functional property. 4. Anaerobic growth The model of phosphate accumulation proposed by Comeau et a l (1986,1987) assumes that a l l accumulators are s t r i c t aerobes. The organisms that were tested with the autoradiographic assay were also tested for t h e i r a b i l i t y to grow anaerobically. This was done by r e p l i c a p l a t i n g the b a c t e r i a onto a g r i d pattern and looking f o r growth a f t e r a 15 day incubation period i n an anaerobic j a r gassed with anaerobic mixture at room temperature. Table XII consists of a l l the major genera broken down into accumulators and nonaccumulators. A l l the accumulators of the major genera with the exception of K l e b s i e l l a and CDC group II-H were obligate aerobes. The minor genera were grouped together but the percent of f a c u l t a t i v e organisms between accumulators and nonaccumulators were not s i g n i f i c a n t l y d i f f e r e n t . The CDC group II.-H organisms were i s o l a t e d from sample from Squamish which i s a secondary treatment plant. In t h i s case, since the biochemical model does not apply, the predictions of that model may not be appl i c a b l e . For the minor genera and the l i m i t e d s t r a i n s of functional Klebsiella there could possibly e x i s t a d i f f e r e n t b i o l o g i c a l mechanism governing t h e i r phosphate accumulating capacity. 36 Table XII: Anaerobic growth a f t e r 15 days Genus accumulation T o t a l # % growth AcLnetobacter + 15 0 a - 42 17 Aeromonas + 7 0 - 34 35 CDC group II-H + 15 33 -• 13 46 KlebsLella + 1 100 - 14 93 ' Moraxella + 5 0 - 12 17 Pseudomonas + 14 0 - 15 33 VLbrLo + 4 0 - 10 20 Other genera + 22 27 - 35 23 T o t a l + 84 14 - 18.5 32 Note: apercent growth of the population. + are accumulators according to pure culture autoradiography - are nonaccumulators. Table XIII: Summary of the optimum conditions for the pure culture autoradiographic assay. 1 Variables Optimum conditions % accumulators Average Average anaerobic accumulation r a t i o 3 counts/protein b Anaerobic time 4 4 4 (h) pH s h i f t 8 to 7 C 8 to 7 8 to 7 Pre- incubation 2 8 • 4 time (h) Isotope added 10 10 50 (uCi) Radioactive 30 90 90 exposure time (min.) Temperature (°C) 25 37 37 "Ratio of the anaerobic counts/protein divided by the aerobic counts/protein. bAnaerobic counts/protein measured as CPM/A562. cRefers to. a pH s h i f t from an anaerobic environment to an aerobic environment. 37 C. 3 2P accumulation 1. In s i t u incorporation A sample of anaerobic sewage was spiked with radioactive ortho-phosphate. The incorporation of l a b e l was followed over a 90 minute period. This allows us to look at the he a l t h of the sewage i n terms of i t s phosphate accumulating capacity. Samples from the UBC p i l o t plant and treatment plants at Salmon Arm, Kelowna, Squamish, and Mamqualm have been analyzed. Figure 8 shows the uptake p r o f i l e s of a l l the plants that were sampled. The samples from. Salmon Arm, Kelowna, Squamish, Mamqualm and the UBC p i l o t plant (P) are c l a s s i f i e d as f u n c t i o n a l Bio-P samples. P i l o t plant samples CI and C2 are categorized as semi-f u n c t i o n a l samples. P i l o t plant sample R was c l a s s i f i e d as collapsed or non-fu n c t i o n a l (Table V I I ) . A note should be made concerning 3 of the samples (Squamish, Mamqualm and p i l o t p l a n t [ C ] ) . Although the municipal plants at Squamish and Mamqualm only have a secondary treatment process configuration they both had extremely good uptake p r o f i l e s i n terms of amount of phosphate accumulated and thus were c l a s s i f i e d as functional Bio-P plants. Even though the p i l o t plant (CI) population was i s o l a t e d from a functional sample, the s e l e c t i v e conditions of the i s o l a t i o n procedure were d i f f e r e n t ( i e . PYE medium was used as the i s o l a t i o n medium rather than CGY medium). 2. Pure culture autoradiography To answer the question of phosphate accumulating a b i l i t y , an autoradiographic assay to screen sewage samples for organisms with the p o t e n t i a l 38 to accumulate excessive amounts of phosphates was developed. A l l of the i s o l a t e s were screened using t h i s autoradiographic assay. The parameters evaluated i n order to optimize t h i s assay included: 1. Exposure times to radioactive plates (10, 30, 60, 90 minutes). 2. Phosphate l e v e l s i n radioactive plates (5, 10, and 50 Uci with the same r a t i o of r a d i o a c t i v i t y to c o l d phosphate). \ 3. Anaerobic times (1, 2, 4 and 7 hours). 4. Preassay incubation times before the anaerobic exposure(0, 2, 4 and 8 hours). 5. pH e f f e c t s (pH s h i f t 6-7,constant pH 7 and pH s h i f t 8-7). 6. Temperature e f f e c t s (12, 25 and 37 degrees). In each t e s t , the autoradiographic assay was repeated by changing one v a r i a b l e at a time and holding a l l other v a r i a b l e s constant. One hundred i s o l a t e s from the Kelowna municipal sewage plant were used i n t h i s study. For example, i f the temperature e f f e c t s were being tested, a l l the i s o l a t e s were subjected to control conditions except f o r the temperature, which was var i e d from 12 to 37° C. The experiment consisted of an aerobic c o n t r o l f i l t e r and an anaerobic s h i f t f i l t e r . Both counts and BCA p r o t e i n assays were done on each i s o l a t e from each f i l t e r . Ratios were then c a l c u l a t e d from counts per amount of p r o t e i n of the aerobic con t r o l d i v i d e d by counts per amount of pr o t e i n of the anaerobic t e s t ( i e . accumulation r a t i o ) . The population was then divided into accumulators and non-accumulators . An accumulator was defined as an i s o l a t e that had an accumulation r a t i o of 1.2 or greater, i . e . i t took up at le a s t 20% more phosphate when subjected to anaerobic/aerobic conditions. For a summary of Figures 9-18 refer to Table XIII . Figure 9 and 10 are graphs showing percentages of accumulators i n the population versus the s p e c i f i c v a r i a b l e tested. Figures 11 and 12 show the e f f e c t the v a r i a b l e tested had on the average accumulation r a t i o for 39 accumulators. Figures 13 and 14 show the e f f e c t of the v a r i a b l e tested on the average anaerobic counts/protein r a t i o . Figure 9A appears to have a peak at 4 hours anaerobic time f o r the greatest percentage of accumulators but there i s no strong trend. However, Figures 11A and 13A support the assumption that 4 hours anaerobic seems to be the optimum condition. The average anaerobic counts/protein f o r the population i s greatest at 4 hours. Figure 9B shows that pH s h i f t i n g i s better than constant pH when looking at percentage of accumulators. Although i t i s not c l e a r which i s the bet t e r s h i f t c ondition from Figure 9B, Figure 11B shows a better r e s u l t f o r pH s h i f t of 8 to 7, i n producing a higher average accumulation r a t i o for accumulators. Figure 13B c l e a r l y demonstrates that a pH s h i f t of 8-7 i s the most e f f e c t i v e i n increasing the actual average anaerobic counts/protein f o r the population. From t h i s evidence a pH s h i f t from 8 to 7 seems to be best f o r the optimization of phosphate uptake. Pre-incubation time was one parameter that gave v a r i a b l e r e s u l t s . Figure 9C shows that the highest percentage of accumulators occurs with a pre-incubation time of 2 hours. However, Figure 11C shows that the average accumulation r a t i o f o r accumulators i s best at 8 hours pre-incubation time. Figure 13C shows that the absolute amount represented by the average anaerobic counts/protein i s best f o r accumulators at 4 hours pre-incubation time. Optimization f o r t h i s v a r i a b l e then becomes very d i f f i c u l t because i t must then be decided which i s the most important aspect of the three; whether % accumulators, average accumulation r a t i o or actual amount of phosphate accumulated i s the most important c r i t e r i a to base the decision. Figures 10A, 12A and 14A agree i n presenting evidence f o r 10 uCi being the optimum r a d i a t i o n l e v e l f o r the assay. Beyond 10 uCi, there appears to be no ex t r a b e n e f i t to adding more radioactive phosphate to the plat e s i n increasing the percentage of accumulators or the accumulation r a t i o of both the 40 \ a c c u m u l a t o r s v s . a n a e r o b i c t i m e 2 4 6 a n a e r o b i c t i m e ( b ) t a c c u m u l a t o r s v s . pH s h i f t % a c c u m u l a t o r s v s . p i e • i n c u b a t i o n t i m e Figure 9: Percent accumulators from the Kelowna population versus the variables tested. (A)anaerobic incubation time, (B)change i n pH, (C)pre-assay incubation time. 41 c u m u l a t o i s v s . i n c r e a s i n g i a d i a t i o n 0 20 40 60 80 100 r a d i o a c t i v e e x p o s u r e t i m e | i i n . ) c c u m u l a t o r s v s . t e m p e i a t u r e 2 0 4 0 t e m p e r a t u r e (C) Figure 10: Percent accumulators from Kelowna versus the v a r i a b l e tested. ( A ) r a d i a t i o n exposure l e v e l s , (B)radiation exposure times, (C)temperature. 42 a n a e i o b i c t i n e 2 4 6 a n a e i o b i c i n c u b a t i o n t i m e ( h ) Figure 11: Average accumulation r a t i o for the accumulator group from Kelowna versus v a r i a b l e s tested. (A)anaerobic incubation time, (B)changing the pH, (C)pre-assay incubation time. 43 a vg . a c c u n . r a t i o v e. r a d i a t i o n l e v e l s 0 20 4 0 6 0 r a d i a t i o n l e v e l e x p o s u r e { u C i ) a c c u n . r a t i o v s t a d . e x p o s u r e t i n e 0 20 40 6 0 SO 100 r a d i o a c t i v e e x p o s u r e t i n e ( n i a . ) Figure 12: Average accumulation r a t i o f o r the accumulator group from Kelowna versus v a r i a b l e s tested. (A)radiation exposure l e v e l , (B)radiation exposure time, (C)temperature. . 44 g. a n a e r o b i c c/p v s . a n a e r o b i c t i m e A. a v g . a n a e r o b i c c/p v s . pH s h i f t o » o.s -a 0 . 4 -0 . 2 -S . l 6.2 6.6 7 7.4 7.1 8.2 pB a a y g . a n a e r o b i c c/p v s . p i e - l n c . t l a e • 1 . 4 -C . o «, U 1 , ! 1 , , 1 1 I 1 0 2 4 6 e ' p r e - i n c u b a t i o n t i n e {ht Figure 13: Average normalized counts (CPM/A562) of the accumulator population from Kelowna versus the v a r i a b l e s tested. (A)anaerobic incubation time, (B)changing the pH, (C)pre-assay incubation time. 45 0 r—1 1 ••• i r - i 1 1 0 20 4 0 6 0 , r a d i a t i o n l e v e l ( u C i ) a v g . a n a e r o b i c c/p v s r a d . e x p o s u r e t i m e B. 0 20 40 CO 10 100 r a d i o a c t i v e e x p o a u i e t i a e ( t i n . ) . i m t l o b l c c / p v . t e a p g i a t m c o . < -0 . 4 -0.2 -0 -1 1 1 1 1 1 1 1 1 1 1 1 1 1 n X 0 II I t 22 It i t }< 31 t i i p t n t u i e (C) Figure 14: Average normalized counts (CPM/A562) of the accumulator population from Kelowna versus the v a r i a b l e s tested. ( A ) r a d i a t i o n exposure l e v e l s , (B)radiation exposure times, (C)temperature. 46 t a c c u m u l a t o r s v s . . a n a e r o b i c t i n e 2 4 6 A n a e r o b i c t i n e (h) X a c c u m u l a t o r s v s . pH s h i f t \ 10 0 accumulators vs. pre•incubjtion time Figure 15: Percent accumulators from Kelowna versus the va r i a b l e s tested at the genus l e v e l . (A)anaerobic incubation times, (B)changing the pH, (C)pre-assay incubation times. 47 1 0 0 t a c c u m u l a t o r s v s . r a d i a t i o n l e v e l 0 2 0 4 0 R a d i a t i o n l e v e l e x p o s u r e ( u C i ) % a c c u m u l a t o r s v s . r a d . e x p o s u r e t i m e 10 0 0 2 0 4 0 6 0 r a d i o a c t i v e e x p o s u r e t i m e 8 0 10 0 ( m i n . ) % a c c u m u l a t o r s v s . t e m p e r a t u r e 14 18 2 2 2 6 30 34 T e m p e r a t u r e ( C ) Figure 16: Percent accumulators from Kelowna versus the v a r i a b l e s tested at the genus l e v e l . ( A ) radiation exposure l e v e l , (B)radiation exposure time, (C)temperature. 48 anaerobic tine 2 4 « anaerobic tine (h) pH s h i f t 0 -I 1 1 1 1 1 1 1 r 0 2 4 « 8 p i e - i i c u b a t i o n t i n e ( h ) Figure 17: Average accumulation r a t i o f o r accumulators from Kelowna versus the v a r i a b l e s tested at the genus l e v e l . (A)anaerobic incubation times, (B)changing the pH, (C)pre-assay incubation times. 49 Figure 18: Average accumulation r a t i o f o r accumulators from Kelowna versus the variables tested at the genus l e v e l . (A)radiation exposure l e v e l s , (B)radiation exposure times, (C)temperature. 50 Figure 19: A photograph of an autoradiograph done on n i t r o c e l l u l o s e d i s c s . The Dark spots represent the amount of isotope accumulated by a p a r t i c u l a r colony. (A) Control f i l t e r which has remained aerobic for the length of the assay. (B) S h i f t condition which has undergone an anaerobic-aerobic s h i f t . The arrow corresponds to a p o t e n t i a l phosphate accumulator. 51 accumulators and non-accumulators. Figure 14A shows what was expected, i . e . i f more phosphate i s added, more phosphate i s incorporated. The p l a t shown i n Figure 10B i s not conclusive, but i t appears that 30 minutes exposure time i s the optimal condition f o r the autoradiographic assay. However, a s i m i l a r problem e x i s t s to the one encountered with pre-incubation time. Figure 12B shows that the accumulators accumulate the best at 90 minutes ra d i o a c t i v e exposure time. Figures 14B shows that as the length of radioactive exposure time increases, the actual amount of radioactive phosphate accumulated increases. Similar to the var i a b l e s of pre-incubation time and radioactive exposure time, Figure 10C shows the highest percent of accumulators at 25° C but Figure 12C shows that the accumulators accumulate the best at 37° C and Figure 14C shows that the amount accumulated i s highest also at 37° C. The data was then sorted by genus and the s i x genera with the la r g e s t number of representatives i n the population were graphed. Figures 15 and 16 show the percentage of accumulators versus the s p e c i f i c v a r i a b l e tested. Figures 17 and 18 show the e f f e c t of the v a r i a b l e tested on the average accumulation r a t i o of both accumulators and non-accumulators. Klebsiella tended to behave very d i f f e r e n t l y from the other 5 genera and often d i d not follow the trends of the others. Figure 15A shows very l i t t l e evidence conclusively. Figure 15B once again shows that any pH s h i f t i s more favourable than not having one at a l l . Whether pH 8 to pH 7 or pH 6 to pH 7 was better depended on the genus, so a genus l e v e l e f f e c t may e x i s t . Figure 15C follows Figure 9C very c l o s e l y , with most of the genera ihaving the highest percentage of accumulators at 2 hours pre-incubation time except for Klebsiella. Figure 16A shows that even on a genus l e v e l , there i s r e a l l y no b e n e f i t to increasing r a d i a t i o n l e v e l s beyond 10 uCi to optimize the assay. Figure 16B shows that the optimal radioactive exposure time i s 30 minutes. Figure 16C shows 52 that, s i m i l a r to the en t i r e population, the greatest percentage of accumulators i n each genus occurs at 25° C. In terms of average accumulation r a t i o when looking at the anaerobic incubation time i n d i v i d u a l genera exhibited d i f f e r e n t behavior (fi g u r e 17A). Klebsiella had a maximum r a t i o at 2 hours, Acinetobacter at 4 hours, Pseudomonas at 7 hours and Moraxella and Aeromonas remained unchanged. The pH s h i f t v a r i a b l e ( f i g u r e 17B) shows that every genus except f o r Moraxella and Pseudomonas gave better r a t i o s when subjected to pH s h i f t s . For pre-incubation time Pseudomonas and Aeromonas had a maximum r a t i o at 8 hours while the others remained unchanged (fig u r e 17C) . The optimal l e v e l of r a d i a t i o n f o r a l l genera except Pseudomonas and Klebsiella seems to be at 10 uCi ( f i g u r e 18A). For r a d i a t i o n exposure time and temperature e f f e c t s (figures 18B,C) a l l genera behaved co n s i s t e n t l y and had a maximum r a t i o at 90 minutes and 37° C r e s p e c t i v e l y with the exception of Moraxella. Though the percent accumulators was picked as the most important parameter i n the determination of optimal conditions, i t i s not c l e a r that i t i s a c t u a l l y the most important. Since i t i s not c l e a r which parameters are more important i t becomes very d i f f i c u l t to chose the optimum conditions f or t h i s assay. In other words, how important are the accumulation r a t i o s and anaerobic count/protein when deciding optimum conditions. 3. Optimized autoradiography f o r screening Bio-P p o t e n t i a l To date we have i d e n t i f i c a t i o n and autoradiographic data on 4 populations from the p i l o t plant, one from the Kelowna treatment plant and one from the Squamish treatment plant. Autoradiographic method 1 has been used to screen populations' from Kelowna, P i l o t plant (R) and P i l o t plant (P) samples. A l l 6 populations were screened with method 2. A photograph of an autoradiograph i s 53 Table XIV: Accumulators from Kelowna and p i l o t plant samples screened with autoradiographic method 1. Kelowna(k) P i l o t ( R ) s 2.78 66 10547 2.31 0.57 45 5454 2.40 P i l o t ( P ) (cells/rnDxlO - 8 % prevalence 1 5 normalized counts (CPM/A 5 6 2) C Average accumulation r a t i o Note: aThe values represent the accumulator f r a c t i o n only. bPrevalance i s percent of t o t a l c e l l s / m l . cNormalized counts i s expressed as counts per minute divided by amount of p r o t e i n expressed as Absorbance units (562 nm). Nonaccumulators not shown. 1.69 65; 10199 3.03 Table XV: Accumulators from Kelowna, p i l o t plant and Squamish samples f o r autoradiographic method 2. Kelowna (K) P i l o t (R) a P i l o t (P) P i l o t (CI) P i l o t (C2) Squamish (S) (cells/mDxlO - 8 1.89 0.51 0.59 0.46 0.77 6.60 % prevalence b 45 40 . 23 18 30 80 normalized counts (CPM/A 5 6 2) e 772 1439 1356 1236 1746 1255 Average 2.13 1.90 2.24 1.76 1.88 2.06 accumulation r a t i o Note: aThe values represent the accumulator f r a c t i o n only. bPrevalance i s percent of t o t a l c e l l s / m l . cNormalized counts i s expressed as counts per minute divided by amount of p r o t e i n expressed as Absorbance units (562 nm). Nonaccumulators not shown. 54 included i n Figure 19. The dark spots represent the amount of isotope accumulated by a p a r t i c u l a r colony. The Kelowna, Squamish, and p i l o t plant samples (P, CI, and C2) are from functional Bio-P treatment plants. The only collapsed system i s the p i l o t plant (R) sample. Although Squamish i s only c l a s s i f i e d as a secondary treatment plant i t does e x h i b i t good phosphate accumulation p o t e n t i a l according to our 3 2P uptake assay. This f a c t should be considered when making cor r e l a t i o n s to other samples. Table XIV consists of accumulators and nonaccumulators for the three plants screened with autoradiographic method 1. I t i s c l e a r from t h i s table that the percent of accumulators to nonaccumulators i n a f u n c t i o n a l system i s higher than i n a collapsed system (66 and 65% vs. 45%). When the percent i s converted into concentration, the concentration of accumulators i n a fu n c t i o n a l plant i s on the order of 5 f o l d higher than the concentration of accumulators i n a collapsed plant (2.78xl0 8 c e l l s / m l and 1.69xl0 8 c e l l s / m l compared to 0.57xl0 8 c e l l s / m l ) . In addition, the normalized count, of the accumulators, which i s the average counts per absorbance u n i t (A 5 6 2) of protein, i s 2 f o l d higher i n functional versus nonfunctional plant samples. Table XV shows r e s u l t s of autoradiographic method 2 f o r a l l 6 populations. In t h i s Table the percent of accumulators to nonaccumulators within a population does not c o r r e l a t e well to 3 2P uptake. Some plants with a low percentage of accumulators work well and other plants with a high percentage of accumulators work poorly i n terms of phosphate accumulation capacity. This paradox becomes cl e a r e r when att e n t i o n i s drawn to the concentration values. The actual concentration of accumulators i n the population from fu n c t i o n a l treatment plants i s higher than the concentration of accumulators from the collapsed plant with the exception of p i l o t plant (CI) . Since the CI population was o r i g i n a l l y i s o l a t e d from PYE medium whereas the others were Table XVI: Autoradiographic method 1 f o r Kelowna and P i l o t plant (P,R) populations. Genus %accum. t o t a l # avg. accum. avg. nonaccum. Unknown 54a 46 2.3b 0.8C Acinetobacter 82 28 3.2 1.0 Aeromonas 75 36 2.1 1.0 Agrobacterium 100 2 2.6 ' N/A Alcaligenes 50 4 3.2 0.7 Bordetella 100 1 1.5 N/A Brucella 100 1 2.3 N/A Capnocytophaga 0 2 N/A 0.7 CDC group II-H 33 3 1.3 0.8 Citrobacter 29 7 1.3 0.9 Clavibacter 80 5 2.0 0.7 Comamonas 50 2 2.9 0.6 Enterobacter 40 5 1.7 0.9 Erwinia 0 1 N/A 0.6 Escherichia 0 2 N/A 0.8 Flavimonas N/A N/A N/A N/A Flavobacterium 50 2 2.5 0.5 Haemophilus 80 5 2.0 0.8 Hydrogenophaga 0 2 N/A 1.0 Kingella 100 1 1.3 N/A K l e b s i e l l a 53 15 2.3 0.9 Kluyvera 0 1 N/A 1.2 Methylobacterium 0 2 N/A 0.7 Moraxella 43 21 2.8 0.8 Neisseria 50 2 5.7 0.8 Pseudomonas 43 21 2.8 0.9 Psychrobacter 100 1 1.5 N/A Shewanella 100 1 1.2 N/A Sphingobacterium 100 3 2.9 N/A V i b r i o 36 11 3.8 0.9 Yersinia 33 3 1.9 0.7 N o t e : ' ' aPercent of the genus that are accumulators bAverage accumulation ra t i o , f o r accumulators. °Average accumulation r a t i o f o r nonaccumulators. Table XVII: Autoradiographic method 2 for Kelowna and P i l o t plant (P,R) populations. Genus %accum. Tota l # avg. accum. avg. nonaccum. Unknown 47 a 34 2.6b 0.8C AcLnetobacter 43 30 1.8 0.9 Aeromonas 29 31 2.0 0.8 Agrobacterium 50 2 2.2 1.1 Alcaligenes 50 4 2.0 0.7 Bordetella 100 1 1.4 N/A Brucella 0 1 N/A 1.0 Capnocytophaga 50 2 1.7 0.8 CDC group II-H 100 3 1.7 N/A Citrobacter 50 2 1.3 0.6 Clavibacter 40 5 2.3 0.8 Comamonas 50 2 1.4 1.0 Enterobacter 75 4 2.4 1.1 Erwinia N/A N/A N/A N/A EschericLa 0 2 N/A 0.5 Flavimonas 0 1 N/A 0.6 FlavobacterLum 0 1 N/A 1.2 Haemophilus 67 3 2.5 1.0 Hydrogenophaga 0 1 N/A 0.8 Kingella N/A N/A N/A N/A K l e b s i e l l a 11 9 1.7 0.7 Kluyvera 0 1 N/A 1.0 Methylobacterium 50 2 2.1 1.1 Moraxella 44 18 1.8 0.8 Neisseria 0 1 N/A 0.9 Pseudomonas 44 18 2.3 0.8 Psychrobacter 100 1 4.0 N/A Shewanella N/A N/A N/A N/A Sphingobacterium 67 3 1.9 0.7 V i b r i o 57 7 2.0 0.9 Yersinia 67 3 2.4 0.8 Note: aPercent of the genus that are accumulators bAverage accumulation r a t i o f o r accumulators. cAverage accumulation r a t i o for nonaccumulators. 57 i s o l a t e d o f f of CGY medium, the difference may p o s s i b l y r e f l e c t the difference i n species s e l e c t i v i t y by PYE medium rather than a true difference i n the plant. I t i s i n t e r e s t i n g that i n both autoradiographic methods the c u t o f f of the concentration of accumulators from a fu n c t i o n a l compared to a collapsed plant i s approximately 5.5xl0 7 c e l l s / m l . Tables XVI and XVII are genus l e v e l comparisons between autoradiographic methods 1 and 2 f o r the Kelowna and p i l o t plant (R and P) samples divided into accumulators and nonaccumulators. In general, the percentage of accumulators for the major genera decreased i n a comparison between methods 1 and 2. The genus Acinetobacter went from 82% to 43% accumulators, Aeromonas went from 75% to 29% accumulators and K l e b s i e l l a went from 53% to 11%. The genera Pseudomonas, Moraxella, and Vibrio remained f a i r l y constant. In addition, the average accumulation r a t i o f o r accumulators f o r many of the genera had decreased between methods 1 and 2. When Squamish and P i l o t plant (CI and C2) samples are included f or autoradiographic method 2 (Table XVIII), there i s a s i g n i f i c a n t decrease i n the percent accumulators f o r the genera Acinetobacter, Moraxella, and Vibrio (43-29%, 44-33%, and 57-41%). To answer the question of how important each genus i s within each i n d i v i d u a l sample the major genera were separated according to the o r i g i n of each sample. Table XIX i s comprised of accumulators versus nonaccumulators within i n d i v i d u a l genera for Kelowna and p i l o t plant (P and R) screened with autoradiographic method 1. The data suggests that between 89 and 100% of the Acinetobacter are accumulators according to t h i s method. The percent of accumulators for the genus Aeromonas i s also quite high f o r a l l three samples (72-89%). This method however does not d i f f e r e n t i a t e c l e a r l y between fun c t i o n a l and collapsed Bio-P plants [Kelowna and p i l o t plant (P) are f u n c t i o n a l ] . Table XX Is s i m i l a r to Table XIX except these i s o l a t e s were screened with autoradiographic method 2 and i t contains a l l 6 samples. In t h i s Table XVIII: Autoradiographic method 2 genus l e v e l comparison of a l l genera. Genera %accum. Total # Avg. accum. Avg. nonaccum. Unknown 40 a 35 2.4b 0.8C AcLnetobacter 29 62 1.7 0.8 Aeromonas 24 54 1.9 0.7 AgrobacterLum 33 3 2.2 0.8 Alcaligenes 50 4 2.0 0.6 Bordetella 50 2 1.4 0.8 Brucella 0 1 N/A 1.0 Capnocytophaga 0 1 N/A 0.7 CDC group II-H 25 4 1.7 0.8 CLtrobacter 53 28 1.9 0.8 ClavLbacter 33 3 1.3 0.8 Comamonas 42 7 2.2 0.8 Enterobacter 42 7 1.6 0.8 ErvrLnia 50 6 2.3 0.8 Escherichia, 0 3 N/A 0.5 FlavLmonas 0 1 N/A 0.6 FlavobacterLum 0 1 N/A 1.1 Haemophilus 50 4 2.5 0.8 Hydrogenophaga 0 1 N/A 0.8 Kingella 0 1 N/A 0.4 Klebsiella 12 16 2.9 0.7 Kluyvera 0 1 N/A 1.0 Listonella 0 1 N/A 1.1 Methylobacteri um 50 2 2.1 1.1 Moraxella 33 24 1.8 0.7 Oligella 0 1 N/A 0.5 Pasteurella 33 6 1.8 0.7 Pseudomonas 46 32 2.5 0.9 Psychrobacter 100 2 3.0 N/A Shewanella 0 1 N/A 0.6 Sphingobacterium 50 4 1.8 0.6 Vibrio 41 17 1.8 0.8 Yersinia 50 4 2.4 0.8 Note: aPercent of the genus that are accumulators. bAverage accumulation r a t i o f o r accumulators. cAverage accumulation r a t i o f o r nonaccumulators. 59 Table XIX: Accumulators within with autoradiographic method 1. i n d i v i d u a l genera screened Kelowna P i l o t P i l o t (K)(%) (R)(%) (P)(%) Acinetobacter 89 a 89 100 Aeromonas 72 85 89 CDC groups 0 50 0 Escherichia 0 0 0 Klebsiella 100 0 33 Pseudomonas 42 33 100 Psychrobacter 100 0 0 Vibrio 36 28 100 Note: "Percent accumulators out of the genus s p e c i f i e d . Nonaccumulators not included. Table XX: Accumulators within i n d i v i d u a l genera screened with autoradiographic method 2. Kelowna (K)(%) P i l o t (R)(%) P i l o t (P)(%) P i l o t ( C l ) ( % ) P i l o t (C2)(%) SqjanLsh (S)(%) Acinetobacter 18 a 2 29 7 50 30 Aeromonas 66 5 0 17 14 0 CDC group II N/A 100 N/A N/A N/A 96 Escherichia 0 0 N/A N/A N/A 0 Klebsiella 2 0 0 0 0 0 Moraxella 59 84 N/A N/A N/A 0 Pseudomonas 63 43 50 100 33 0 Psychrobacter 100 N/A N/A N/A N/A 100 Vibrio 9 33 N/A 0 0 8 Note: aPercent accumulators out of the genus s p e c i f i e d . Nonaccumulators not included. 60 Table there i s a c l e a r e r d i f f e r e n t i a t i o n between fun c t i o n a l and collapsed plants. This dif f e r e n c e i s most evident i n the cases of Acinetobacter and Aeromonas. In the collapsed p i l o t plant (R) sample, 2% of the genus Acinetobacter were accumulators compared to a range of 18 to 50% accumulators f o r Kelowna, P i l o t plant (P,C2), and Squamish samples. The P i l o t plant (C2) sample had 7% accumulators but as was mentioned e a r l i e r t h i s population was i s o l a t e d from a d i f f e r e n t medium which might be b i a s i n g the r e s u l t s . I t i s i n t e r e s t i n g to point out that i n both Tables XIX and XX, a l l Psychrobacters were considered accumulators according to ei t h e r autoradiographic method. The members of the genus Psychrobacter were formerly considered a subset of the genus Moraxella. I t comprises many of the psychrophilic Moraxella (Juni and Heym, 1986) and may be s i g n i f i c a n t i n plants operating i n colder climates. Tables XXI and XXII are species l e v e l comparisons between accumulators and nonaccumulators screened with e i t h e r autoradiographic methods 1 or 2. Method 1 does not d i f f e r e n t i a t e between accumulators and nonaccumulators at the species l e v e l with the exception of perhaps Pseudomonas. A l l Pseudomonas putida were accumulators while the majority of the other Pseudomonas spp. were nonaccumulators. Since method 1 can d i f f e r e n t i a t e between functional and collapsed plants at the t o t a l population l e v e l why then can t h i s method not d i f f e r e n t i a t e between the species within the major genera? A possible explanation for t h i s anomaly i s that the phenomenon of phosphate accumulation with t h i s method i s a population dependant response. Using the genus Acinetobacter as an example the numbers of t h i s genus from a f u n c t i o n a l plant i s much higher than from a collapsed one and t h i s i s the reason that we see the d i f f e r e n t i a t i o n between f u n c t i o n a l and nonfunctional plants at the t o t a l population l e v e l but not at the genus l e v e l or species l e v e l . In other words t h i s method cannot resolve the differences between fun c t i o n a l and 61 Table XXI: Species l e v e l comparison between accumulators and nonaccumulators screened with autoradiographic method 1. Species #accum. #nonaccu. avg. avg. accum nonaccum AcLnetobacter 3 1 2.23a l . l l b calco./genosp.1 AcLnetobacter genosp. 12 0 1 N/A 0.82 AcLnetobacter 10 2 3.46 0.97 johnsonnLL/genosp.7 AcLnetobacter 8 1 3.33 1.14 IwoffLL/genosp.8 Aeromonas hydrophLla 14 6 1.54 1.08 Aer. hydrophLla DNA 5A 1 1 1.40 1.00 Aer. hydrophLla DNA 1 3 0 2.40 N/A Aeromonas cavaie 3 0 2.85 N/A Aer. hydrophLla DNA 12 2 2 1.75 0.81 Aeromonas medLa 3 0 3.60 N/A CDC group II-H 2 5 2.47 1.10 Moraxella atlantae 1 1 1.60 0.34 Moraxella bovLs 5 5 2.83 0.91 Moraxella osloensLs 2 3 3.20 0.84 Pseudomonas putLda 4 0 3.46 N/A Pseudomonas vesLcularLs 1 3 1.70 0.83 Psuedomonas delafLeldLL 1 2 1.90 0.72 Pseudomonas facilLs 1 2 2.50 0.76 Pseudomonas dLmLnuta 0 0 N/A N/A Psychrobacter LmmobLlLs 1 0 1.50 N/A VLbrLo anguLllarum 2 3 3.37 1.00 VLbrLo cholera 0 2 N/A 0.80 YersLnLa enterocolLtLca 0 1 N/A 0.60 YersLnLa LntermedLa 1 1 1.90 0.80 Note: aAverage accumulation r a t i o f o r accumulators. bAverage accumulation r a t i o f o r nonaccumulators. Table XXII: Species l e v e l comparison between accumulators and nonaccumulators screened with autoradiographic method 2. Species #accum. #nonacc. avg. accum. avg. nonaccu Acinetobacter calco./genosp. 1 Acinetobacter genosp. 12 Acinetobacter johnsonnii/genosp. 7 Acinetobacter lwoffii/genosp. 8 Aeromonas hydrophila Aeromonas cavaie Aer. hydrophila DNA 5A Aer. hydrophila DNA 1 Aer. hydrophila DNA 12 Aeromonas media CDC group II-H Moraxella atlantae Moraxella bovis Moraxella qsloensis Psuedomonas delafieldii Pseudomonas facilis Pseudomonas diminuta . Pseudomonas vesicularis Pseudomonas putida sub.B Psychrobacter immobilis Vibrio anguillarum Vibrio cholera Yersinia enterocolitica Yersinia intermedia 1 10 4 1 0 1 3 4 13 2 4 2 2 2 2 1 5 2 4 1 2 0 1 21 18 26 2 2 3 4 6 11 1 5 8 2 2 1 4 2 0 4 3 0 2 2.53a 1.30 1.70 1.39 1.57 1.35 N/A 1.40 2.14 2.22 1.92 2.22 1.68 1.46 1.34 1.92 4.70 2.70 2.40 3.00 1.73 1.50 2.42 N/A 0.70b 1.00 0.72 0.90 0.71 0.70 0.65 0.69 0.92 0.91 0.76 0.76 0.68 0.88 0.73 0.67 0.80 1.00 0.76 N/A 0.79 0.91 N/A 0.84 Note: aAverage accumulation r a t i o f o r accumulators. bAvera'ge accumulation r a t i o f o r nonaccumulators. 63 nonfunctional AcLnetobacter. Method 2 (Table XXII) showed more encouraging r e s u l t s . This method was able to d i f f e r e n t i a t e accumulators and nonaccumulators within species of AcLnetobacter, Aeromonas, Moraxella, and Pseudomonas. AcLnetobacter johnsonii/genospecLes 7 had a higher f r a c t i o n of accumulators than AcLnetobacter lwoffii/genospecLes 8 (32% compared to 14%). Aeromonas medLa and Aeromonas hydrophLla DNA group 12 had a higher percentage of accumulators than Aeromonas hydrophLla (41% vs. 13%). Moraxella bovis and Pseudomonas putLda subgroup B have higher percentages of accumulators than Moraxella osloensLs and Pseudomonas vesLcularLs (44 and 71% compared to 20 and 20%) . Is the stringency of d i f f e r e n t i a t i o n f o r autoradiographic method 1 s t r i c t enough? Is i t more a t t r a c t i v e to have an autoradiographic assay that can d i f f e r e n t i a t e between accumulators and nonaccumulators within a single genus? The v a l i d i t y of each method w i l l be evaluated i n the following discussion. 64 DISCUSSION A.Recovery and v i a b i l i t y 1. Enumeration There are generally two approaches to assessing the importance of b a c t e r i a i n t h e i r environments. The f i r s t approach i s p r i m a r i l y one of d i r e c t microscopy whereby the b a c t e r i a are stained and observed without a r t i f i c i a l enrichment. The second i s the i n d i r e c t approach which i s o l a t e s p a r t i c u l a r b a c t e r i a from t h e i r h a b i t a t using a prescribed set of culture conditions, then tests these b a c t e r i a f o r s p e c i f i c properties or features. Activated sludge with a l t e r n a t i n g anaerobic-aerobic treatment systems are characterized by t h e i r capacity for e f f i c i e n t phosphate removal from wastewater (Barnard, 1976). This phosphate removal from wastewater i s a b i o l o g i c a l process and i s thought to be due to the presence of c e r t a i n b a c t e r i a capable of phosphate accumulation. The study of the b a c t e r i a l populations from these systems i s a subject of great concern. Over the past 20 years, studies on the population structure of modified a c t i v a t e d sludge systems have received considerable attention but as yet are f a r from being completely understood. Most of these studies have attempted to use p l a t i n g methodology in' which populations of sludge organisms are recovered from a s o l i d medium (Lotter and Murphy, 1985; Brodisch and Joyner, 1983; Cloete et al. , 1985; Buchan, 1983). Prakasam and Dondero (1967) were the f i r s t to report a higher number of c e l l s under microscopic enumeration as compared to several d i f f e r e n t kinds of p l a t i n g techniques. Cloete and Stern (1987) reported that l e s s than 10% of the organisms enumerated by microscopic counts could be accounted for by plate 65 enumeration. The r e s u l t s presented i n t h i s t h e s i s are consistent with the body of knowledge concerning recoverable b a c t e r i a . The v i a b l e c f u from p l a t i n g onto CGY medium ranged anywhere from 1 to 10% of the microscopic counts depending on the sample used. This lower recovery i s probably due to b a c t e r i a l f l o c c u l a t i o n problems. One area of recovery that has not received much a t t e n t i o n i s the area concerning f l o e dynamics. The t r a d i t i o n a l methodology with respect to f l o e s i s the attempt on the part of researchers to break up the f l o e s into i n d i v i d u a l organisms. The two methods most often used to achieve t h i s goal are d i f f e r e n t types and degrees of sonication and the use of chemical dispersing agents such as detergents (Banks and Walker, 1977; Y i n and Moyer, 1968). ; Both methods have s i g n i f i c a n t drawbacks associated with them i n terms of c e l l v i a b i l i t y but at present there are no better techniques when dealing with t h i s problem. 2. Cross p l a t i n g Eight d i f f e r e n t media were tested f o r t h e i r a b i l i t y to recover d i f f e r e n t populations of b a c t e r i a from sewage. Most of the organisms that were i s o l a t e d from CGY medium could grow on PYE medium and v i c e versa. Although the r e s u l t s suggested that there was no appreciable s e l e c t i v e d i f f e r e n c e between the media (under these l i m i t e d parameters), i d e n t i f i c a t i o n of two of the populations i s o l a t e d from CGY medium and PYE medium y i e l d e d an apparent d i f f e r e n c e i n terms of species composition (Table I I I ) . This puzzling anomaly was answered by looking at the microplate assay developed by Postgate et a l (1961) and extended by Fry and Z i a (1982). The r e s u l t s . i n d i c a t e d that the same f l o e can give r i s e to d i f f e r e n t colonies depending on the type of medium employed. Consequently, the same f l o e , i f plated onto 2 d i f f e r e n t media, could give r i s e to 2 d i f f e r e n t 66 colonies, since the clone that arises from a f l o e has to out compete a l l other microcolonies that are t r y i n g to e s t a b l i s h themselves. This competition i s a contest for many fac t o r one of which i s nutrients. The nutrient used w i l l impart a s e l e c t i v e pressure on the floes f or the bacterium that can most r e a d i l y u t i l i z e the components of the medium. Figure 6 shows that i n the early stages of colony development, many microcolonies are competing f o r domination of the f l o e . I t i s conceivable that depending on the medium used, d i f f e r e n t microcolonies w i l l have the advantage when i t comes to competition and therefore w i l l give d i f f e r e n t colonies. 3. Microscopic versus plate counts The 8 um f r a c t i o n a t i o n assay was developed i n an attempt to answer some fundamental questions concerning recovery of organisms. The novel approach consists of an assay i n which i t i s possible to f r a c t i o n a t e the sewage into components by passing samples through a c u t o f f f i l t e r (8um) and observe each i n d i v i d u a l f r a c t i o n with respect to d i f f e r e n t parameters. The two that were investigated were recovery on agar plates and phosphate accumulating a b i l i t y . From Table IV less than 10% of the free p a r t i c l e s enumerated by microscopic counts were recovered onto CGY medium. This observation i s consistent with many other reported cases (Cloete and Steyn, 1987; Brodish, 1985). In the f i l t e r f r a c t i o n 100% of the f l o e s gave r i s e to cfu. Each f l o e contains a substantial number of b a c t e r i a (anywhere from 100-1000 b a c t e r i a / f l o c ) and the colony that a r i s e s from each f l o e on agar plates i s pure when streaked onto a second plate. These r e s u l t s meant that at l e a s t one species of bacterium from each f l o e was v i a b l e . Therefore, the microplating technique was performed to look at 67 development i n the very e a r l y stages of colony growth (Fry and Zia, 1982; Postgate et al, 1961) and assess the v i a b l e number of c e l l s within the f l o e s . As was expected from the photographs i n Figure 7B, many microcolonies could be observed growing out of f l o e s . Since these f l o e s developed into pure colonies, i t may be assumed that i n the early stages of colony development many v i a b l e b a c t e r i a w i t h i n f l o e s were out competed f o r growth by a f a s t e r growing, more p r o l i f i c organisms and thus were not recoverable, although they were v i a b l e and could have grown on the medium i n the absence of competition from other b a c t e r i a . This e f f e c t could be a problem, since i t means that the proportions of any one i s o l a t a b l e species i n a population i s dependent on the t o t a l composition of the other organisms w i t h i n that f l o e . In terms of phosphate accumulation p o t e n t i a l , the f i l t e r f r a c t i o n was responsible for the majority of the uptake (Figure 5), except when the accumulation i s normalized and expressed per amount of protein. In t h i s case the opposite trend i s seen. The f i l t r a t e f r a c t i o n now i s better than the f i l t e r f r a c t i o n i n i t s a b i l i t y to take up phosphate (ranges from 4-7x b e t t e r ) . This e f f e c t probably means that f l o e s contain a large proportion of i n e r t material and i n a c t i v e b a c t e r i a that may contribute to the o v e r a l l protein measurement but make no co n t r i b u t i o n to phosphate accumulation. Another i n t e r e s t i n g observation i s that samples from Kelowna and the UBC p i l o t plant which have almost i d e n t i c a l process configurations (Figure 2) have an equal proportion of recoverable b a c t e r i a from f i l t r a t e to f i l t e r f r a c t i o n s while Squamish, which i s a secondary treatment plant and Salmon Arm which has a d i f f e r e n t process configuration than e i t h e r Kelowna or the p i l o t plant, have a f i l t r a t e f r a c t i o n one tenth of the f i l t e r f r a c t i o n . I t appears from t h i s difference that d i f f e r e n t processes seem to have an e f f e c t on the f r a c t i o n of f i l t r a t e to f i l t e r organisms. 68 4. Microplating and microscopic autoradiography E c o l o g i c a l studies of microorganisms i n t h e i r complex environmental systems are l i m i t e d by the number and v a l i d i t y of the methods a v a i l a b l e . For many years the v i a b i l i t y of aquatic b a c t e r i a has been assessed from the r a t i o of p l a t i n g and t o t a l counting procedures. T o r e l l a and Morita i n 1981 and Fry and Z i a i n 1982 developed a method f o r estimating v i a b i l i t y of aquatic b a c t e r i a by s l i d e c ulture. Their method was an adaptation of another method developed i n 1961 by Postgate et al. We have attempted to use t h i s assay i n conjunction with a microscopic autoradiographic assay (Meyer-Reil, 1978; Faust and C o r r e l l , 1977) to answer some fundamental questions concerning phosphate accumulation w i t h i n the sewage environment. Autoradiography has become a u s e f u l t o o l i n e c o l o g i c a l studies, since i t enables investigators to r e l a t e a c t i v i t y or function to i n d i v i d u a l c e l l s or groups of c e l l s within the system. Figure 7 i s a photograph of l a b e l l e d sewage treatment plant biomass a f t e r microscopic autoradiography. The sample was taken at the end of a 3 2P uptake assay. I t i s c l e a r that the 3 2P concentrations shown on the photographs are consistent with the uptake done on the f r a c t i o n s from the 8 urn f r a c t i o n a t i o n assay. Both point to the f l o e s as being responsible f o r the majority of the accumulation. Some i n t e r e s t i n g observations do a r i s e when autoradiography i s done on l a b e l l e d samples plated onto CGY agar set onto a microscope s l i d e incubated f o r 12 hours at room temperature. The r a d i a t i o n i s l o c a l i z e d only to the floes and not spread into the microcolonies that were growing out of them (Figure 7B). -There are 2 possible explanations for t h i s observation. The f i r s t and e a s i e s t explanation i s that the organisms responsible f o r the removal of phosphate from wastewater j u s t do not grow under our culture conditions. The second and most l i k e l y explanation i s much more complicated. Microcolonies grow from the margin of the f l o e s where the density of b a c t e r i a i s the lowest. They do t h i s because of the ph y s i c a l space that i s required for growth. Because of space constraints within the f l o e , v i a b l e b a c t e r i a cannot grow. I f the b a c t e r i a within the f l o e are metabolically active and are accumulating phosphate t h i s a c t i v i t y could be an explanation of why r a d i a t i o n i s l o c a l i z e d w i t h i n the f l o e s . Even i f the ba c t e r i a occupying the margin of a f l o e are accumulating phosphate and can grow to give microcolonies, presumably t h i s r a d i a t i o n may be too d i l u t e d to be detected a f t e r many rounds of r e p l i c a t i o n . 5. I s o l a t i o n of s t r i c t anaerobes Since many t e r t i a r y treatment plants designed f o r phosphate removal incorporate an anaerobic zone p r i o r to the aerobic zone (Ekama et al, 1984), the obligate anaerobic population was investigated. From our fi n d i n g s , the majority of the population i s o l a t e d by anaerobic technique from the anaerobic zone of the UBC p i l o t plant turned out to be f a c u l t a t i v e organisms. This observation i s not s u r p r i s i n g due to the f a c t that these b a c t e r i a were i n a continuous recycle that cycled them back and f o r t h between the aerobic and anaerobic zones. I n t u i t i v e l y t h i s process would tend to s e l e c t f o r f a c u l t a t i v e organisms. The argument for the presence of obligate anaerobes came from the assumption that the oxygen tension w i t h i n a f l o e may be extremely low, so low that i t can support the growth of s t r i c t anaerobic organisms. Recent evidence has been published that deals with the actual structure of activated sludge f l o e s ( L i and Ganczarczyk, 1990). The authors report that within activated sludge f l o e s there are large water channels and open r e s e r v o i r s . Although i t i s s t i l l conceivable that parts of a 70 f l o e may indeed be anaerobic t h i s research has succeeded i n dealing with some of the misconceptions i n f l o e structure. B. B a c t e r i a l i d e n t i f i c a t i o n 1. API/NFT and Biolog i d e n t i f i c a t i o n The API 20E i s a standardized, miniaturized v e r s i o n of conventional procedure f o r the i d e n t i f i c a t i o n of a host of gram negative b a c t e r i a . I t consists of a microtube system designed f o r the performance of 23 standard biochemical t e s t s . The Rapid NFT system consists of a standardized 20 biochemical and a s s i m i l a t i o n tests f o r the i d e n t i f i c a t i o n of gram negative non-fermentative b a c t e r i a (Analytlab Products, 1977). Biolog has developed a technology which can be employed to characterize and i d e n t i f y i s o l a t e s on the basis of t h e i r carbon source u t i l i z a t i o n p r o f i l e s . The p r o f i l e s t e s t the a b i l i t y of a b a c t e r i a to metabolize or oxidize a set of chemical substrates. An i d e n t i f i c a t i o n can thus be generated by a computerized matching of the metabolic properties of that i s o l a t e to a database of p r o f i l e s (BIOLOG Reference manual, 1989; Bockner, 1989). Biolog has many advantages as well as some disadvantages compared to API and NFT\ In the context of i d e n t i f y i n g sewage i s o l a t e s , the B i o l o g system has d i s t i n c t advantages. There are on the order of 5x the number of tests i n the Biolog regime as compared to e i t h e r API or NFT. As a r e s u l t B i o l o g i s able to d i f f e r e n t i a t e a larger number of species. Biolog i s able to d i f f e r e n t i a t e at present 310 species and s t r a i n s of b a c t e r i a while API/NFT can only d i f f e r e n t i a t e between 60 and 120. From the information on Table VI, within every genus the Biolog system can give a more diverse separation of sewage species than both API and NFT systems. Its one major disadvantage i s that i t s tests are based on d i f f e r e n t reactions than that for standardized biochemical t e s t s . This makes i t extremely d i f f i c u l t to r e l a t e the data generated from t h i s method to the current body of knowledge i n the context of taxonomic c l a s s i f i c a t i o n . The background of information a v a i l a b l e on b a c t e r i a and t h e i r a b i l i t y to oxidize these sets of chemical substrates i s l i m i t e d and scarce (Bergey's Manual of Determinative Bacteriology, 1989). Despite these drawbacks, i t i s c l e a r that Biolog i s able to i d e n t i f y more species of organisms from sewage than both the combination of API and NFT. This system also improves r e s o l u t i o n between d i f f e r e n t species within c e r t a i n genera. The c o n f l i c t i n previous studies could have possibly been explained by species v a r i a t i o n which could not be picked up by the more conventional methods. 2. Population p r o f i l e s of d i f f e r e n t sewage treatment plants I t i s widely accepted that enhanced phosphate accumulation i n a c t i v a t e d sludge i s u s u a l l y governed by a b i o l o g i c a l mechanism. However, the methods i n which phosphate i s accumulated and the populations of organisms involved i n t h i s process are s t i l l l a r g e l y unknown. From the work of B u t t e r f i e l d (1935) , Zooglea ramigera was thought to be the most predominant and active organism i n activated sludge because of i t s a b i l i t y to f l o c c u l a t e . The f i r s t reported presence of other b a c t e r i a l species was made by A l l e n (1944) followed by McKinney and Weichlein (1953) and then Van G i l l s (1964). The presence and importance of the genus AcLnetobacter was f i r s t reported by Fuhs and Chen (1975). Since then a controversy has existed concerning the functional r o l e of t h i s genus i n enhanced Bio-P removal. Many authors have supported the importance of AcLnetobacter as 72 being mainly responsible f o r enhanced phosphorus removal (Deinema et al, 1985; L o t t e r and Murphy, 1985; Osborn et al, 1986; Duncan et al. , 1988; Beacham et al. , 1990). On the other hand, many authors have questioned the actual r o l e of t h i s group of organisms i n the enhanced phosphorus removal process (Hascoet et al, 1985; Cloete et al, 1985; Meganck et al, 1985; Suresh et al, 1985; H i r a i s h i et J al., 1989). A summary of b a c t e r i a i s o l a t e d from t e r t i a r y phosphorus removal plants are l i s t e d i n table I. In most of the studies the predominant genera found to characterize the sludge were Acinetobacter, Aeromonas, Klebsiella, Moraxella and Pseudomonas. The comparison of genera found i n other studies to those found i n t h i s study i s s i m i l a r , but with a few exceptions. The predominant genera from t h i s study were Acinetobacter, Aeromonas, and Pseudomonas. One exception was the presence of an abnormally high proportion of CDC group II-H organisms i s o l a t e d from the Squamish municipal sewage treatment plant (82%). Comparison of the population p r o f i l e s between functional and nonfunctional Bio-P plants at the genus l e v e l gave some i n t e r e s t i n g r e s u l t s (Tables VIII and IX). In terms of the percentage of the t o t a l population, the genus Acinetobacter c o n s t i t u t e d between 24% and 41% of the t o t a l biomass i n f u n c t i o n a l plants (with the exception of Squamish) , compared to only 4% f o r a collapsed system. When the t o t a l number of Acinetobacter i s considered rather than the percentage, the number f o r the Squamish sample i s now f a r better than any of the other functional plants, although the number f o r the collapsed system now i s more than the p i l o t p l a n t (C2) fu n c t i o n a l system. This means that plants must be assessed by examining the t o t a l number of fu n c t i o n a l organisms rather than simply percent of the population or t o t a l numbers within that population. However, the basic methodology that i s employed by investigators to look at the microbiology of enhanced phosphorus removal plants stops when the s t r a i n s are i d e n t i f i e d . 73 Consequently, improper conclusions may be drawn concerning the roles of the organisms by v i r t u e of t h e i r presence i n the system. Populations of b a c t e r i a from Bio-P plants were i s o l a t e d and i d e n t i f i e d by a number of commercially a v a i l a b l e i d e n t i f i c a t i o n systems and the presence of c e r t a i n organisms i n s u f f i c i e n t q u a n t i t i e s was assumed to be proof enough to v a l i d a t e t h e i r importance i n the phosphate removal process. In part these types of u n j u s t i f i e d conclusions have contributed to the controversy that e x i s t s w i t h i n t h i s f i e l d . From our data there i s not a 100% c o r r e l a t i o n between groups of b a c t e r i a and the plants Bio-P function. Therefore the s p e c i f i c i s o l a t e s must be tested to assess t h e i r f u nctional c a p a b i l i t i e s . C. Pure culture Autoradiography 1. Optimization of autoradiographic assay The v a r i a b l e s examined to optimize the autoradiographic assay were outlined e a r l i e r ; the parameters that were examined f o r each v a r i a b l e were percent accumulators, average accumulation r a t i o and average counts per amount of protein. The d e f i n i t i o n s of each of these parameters were l i s t e d e a r l i e r . When a l l three parameters were considered, the v a r i a b l e s that gave a consistent response were anaerobic exposure time, changing pH during the assay and supplied phosphate l e v e l s . A l l three parameters showed the same optimal l i m i t s (4 hour anaerobic time, pH s h i f t from 8-7 and 10 uCi l e v e l of r a d i a t i o n ) . The other three v a r i a b l e s were not so consistent (temperature e f f e c t s , pre-incubation time and r a d i a t i o n exposure time). These three parameters gave v a r i a b l e r e s u l t s as to which values were optimal (Table XIII). Optimization of these three variables 74 becomes more d i f f i c u l t . I t must then be decided which one of the three parameters i s the most important, the percent of accumulators, the average accumulation r a t i o or the actual amount of phosphate accumulated. The percentage of accumulators was chosen to be the most important parameter. According to t h i s parameter a temperature of 25°C, a pre-incubation time of 2 hours and a r a d i a t i o n exposure time of 30 minutes were chosen. The behavior of i n d i v i d u a l genera over these v a r i a b l e s was also investigated. The parameters that were looked at were percent accumulators and average accumulation r a t i o . In general the genus Klebsiella tended to behave very d i f f e r e n t l y from the other genera and often d i d not follow the trends of the others. This i s not s u r p r i s i n g since almost a l l the i s o l a t e s that belonged to the genus Klebsiella were found to be f a c u l t a t i v e organisms (Table XII) . Klebsiella would therefore behave d i f f e r e n t l y i n an assay that was designed to s e l e c t accumulators out of an obligate aerobic population. 2. Pure culture autoradiographic screening To go beyond the current state of knowledge concerning the b a c t e r i a responsible f o r the accumulation of phosphate i n enhanced Bio-P a c t i v a t e d sludge plants, an autoradiographic assay was developed. I t was developed i n an attempt to elucidate the r o l e of b a c t e r i a i s o l a t e d from activated sludge showing enhanced phosphorus removal. This method assays for the a b i l i t y of i n d i v i d u a l b a c t e r i a l i s o l a t e s to accumulate phosphate under an anaerobic/aerobic s h i f t condition compared to an aerobic c o n t r o l v The populations from each of the plants tested were then divided into accumulators and nonaccumulators (accumulators had, by d e f i n i t i o n , an accumulation r a t i o of 1.2 or greater, nonaccumulators had an accumulation r a t i o of less than 1.2). Two autoradiographic methods were 75 compared. From t h i s data i t i s not c l e a r which method i s be t t e r f o r the i d e n t i f i c a t i o n of phosphate accumulators. However, i t i s c l e a r that the minimum number of accumulators needed for a plant to be functional i s approximately 5.7xl0 7 c e l l s / m l . Plants that have accumulators i n excess of t h i s number are functional plants while plants that have accumulators below t h i s number are nonfunctional or collapsed plants. One sample from the UBC p i l o t plant does not adhere to t h i s rule [ p i l o t (CI)]. Although t h i s sample came from a f u n c t i o n a l plant i t was i s o l a t e d from a d i f f e r e n t medium (PYE medium) and was shown to s e l e c t f o r a d i f f e r e n t population of b a c t e r i a . P i l o t (CI) i s an i n t e r e s t i n g population. Forty-one percent of the population according to the Biolog i d e n t i f i c a t i o n method are Acinetobacter but only 7% of those Acinetobacter are phosphate accumulators, even though 18% of the population were accumulators. This i s a good example of how uninformative and misleading population i d e n t i f i c a t i o n s can be when conclusions are drawn s o l e l y from t h i s type of information. When i s o l a t e s from a l l the plants are compiled into one group and t h i s group i s divided into d i f f e r e n t genera, i t i s c l e a r that the genus Acinetobacter i s not s o l e l y responsible f or the phenomenon of Bio-P. Out of the t o t a l Acinetobacter population only 29% are c l a s s i f i e d as being accumulators. There are other genera that have a better percentage of accumulators than Acinetobacter (Pseudomonas, Vibrio, CDC group II-H). Within 3 populations [Kelowna, P i l o t plant (P, R) ] i t i s possible to compare both methods i n order to assess which method i s better i n i d e n t i f y i n g true accumulators. I t i s c l e a r that the genera do not behave c o n s i s t e n t l y from one method to another. The genera Acinetobacter, Klebsiella and Aeromonas both decrease i n the proportion of accumulators while the proportions of accumulators from the genera Pseudomonas, Moraxella and Vibrio stayed the same. Klebsiella i s an unexpected phosphate sequestering genus i n that a l l the i s o l a t e s i d e n t i f i e d as being from t h i s genus were found to be f a c u l t a t i v e organisms (Table XII) . According to the biochemical model of phosphate accumulation proposed by Comeau et al (1986,1987), phosphate accumulating b a c t e r i a should be obligate aerobes. The model thus predicts that Klebsiella would not be considered as a p o t e n t i a l accumulator. F i f t y - t h r e e percent of the genus Klebsiella were c l a s s i f i e d as accumulators with method 1 while only 11% were c l a s s i f i e d as accumulators with method 2. To gain a better understanding of which autoradiographic method i s better for the i d e n t i f i c a t i o n of accumulators i t i s necessary to look at the d i v i s i o n between f u n c t i o n a l and collapsed plants within the context of a genus l e v e l comparison (Tables XIV, XV). Autoradiographic method 1 i s not able to d i f f e r e n t i a t e between fun c t i o n a l and nonfunctional plants at the genus l e v e l . For example the proportion of Acinetobacter that are accumulators from a fu n c t i o n a l p l a n t are 89 and 100% compared to 89% accumulators f o r a collapsed plant. The same l i m i t a t i o n s apply f o r Aeromonas and Vibrio. Method 2 however seems to be able to d i f f e r e n t i a t e between functional and nonfunctional plants, although i t has a much lower proportion of accumulators to nonaccumulators compared to method 1. The collapsed plant has only 2% of i t s Acinetobacter i n the accumulator category while the other functional plants have percentages ranging from 18-50% with the exception of the p i l o t (CI) sample. The numbers f o r Aeromonas are s i m i l a r . Functional plants have 14-66% accumulators compared to 5% accumulators f o r a collapsed system. Method 2 has d e f i n i t e c l e a r e r d i f f e r e n t i a t i o n between working and nonworking plants i n terms of the genera Acinetobacter and Aeromonas. Using Acinetobacter as an example many inves t i g a t o r s have implicated t h i s genus as being the major contributor to phosphate accumulation (Brodisch, 1985; Kerdachi and Roberts, 1985). On the 77 other hand many inves t i g a t o r s have questioned the importance of t h i s genus i n Bio-P accumulation. Hascoet et al. (1985) could not c o n s i s t e n t l y i s o l a t e Acinetobacter from a p i l o t plant that was e f f i c i e n t l y removing phosphorous. At one f u l l scale plant, 56% of the b a c t e r i a l colonies which grew were i d e n t i f i e d as Acinetobacter; yet phosphorus removal was only 19% of the t o t a l i n f l u e n t phosphate l e v e l s (Lotter, 1985). Both schools of thought are correct. I t i s conceivable that the Genus Acinetobacter i s not the only genus responsible f o r the accumulation of phosphate and that a l l Acinetobacter spp. are not the same i i n terms of phosphate accumulation capacity. The conclusion that t h i s genus i s not important since 56% of the colonies i s o l a t e d from a nonfunctional Bio-P plant were Acinetobacter would then not be correct. I t i s also i n t e r e s t i n g that whenever Psychrobacter appears i t turns out to be an accumulator. The genus Psychrobacter comprises a group of psychrophilic Moraxella. I t i s d i f f i c u l t to draw fir m conclusions from t h i s because Psychrobacter shows up so infrequently i n these populations. The next step i s to look at the d i f f e r e n t i a t i o n between accumulators and nonaccumulators at the species l e v e l . In order to achieve t h i s d i f f e r e n t i a t i o n a l l populations had to be compiled to get enough numbers for a v a l i d comparison (Tables XXI and XXII). Method 2 d i f f e r e n t i a t e s species w i t h i n the genera Acinetobacter, Aeromonas, Moraxella and Pseudomonas. Method 1 only d i f f e r e n t i a t e s species within the genus Pseudomonas. In terms of i t s s e n s i t i v i t y method 2 seems to d i f f e r e n t i a t e better between accumulators and nonaccumulators at both the genus and species l e v e l s . Table XXII predicts that Acinetobacter johnsonii/genospecies 7 tends to have more phosphate accumulators than Acinetobacter lwoffii/genospecies 8. Recently investigators from A u s t r a l i a have published a study dealing with the genospecies d i v e r s i t y of Acinetobacter 78 i s o l a t e s obtained from activated sludge showing enhanced removal of phosphate (Beacham et al, 1990; Duncan et al., 1988). The study used the transformation assay of Juni (1972) f o r the confirmation of Acinetobacter s t r a i n s , and the c l a s s i f i c a t i o n regime of Bouvet and Grimont (1986) to c l a s s i f y the i s o l a t e s into t h e i r respective genospecies. These studies are considered sounder taxonomic approaches than those using generic and species dependent descriptions based on i d e n t i f i c a t i o n systems inappropriate for environmental, gram negative, nonfermentative b a c t e r i a (Schutte et al. , 1987). They showed genospecies 5, 7, 8 and 9 as being the predominant Acinetobacter i n t h e i r system but when they looked at genospecies i s o l a t e d from c l u s t e r s ( f l o e s ) by micromanipulation they found that genospecies 8/9 had disappeared. I f the f l o e s are responsible for the majority of the phosphate accumulation and the genus Acinetobacter i s important i n t h i s function then presumably genospecies 5 and 7 are important. The r e s u l t s from the autoradiographic assay suggest that genospecies 7 i s more important than genospecies 8 i n terms of phosphate accumulation. The major differ e n c e between the r e s u l t s from the A u s t r a l i a n study and t h i s study i s the presence of Acinetobacter junii/genospecies 5. Genospecies 5 was the most common Acinetobacter i s o l a t e d from t h e i r modified UCT system. On the other hand, we could not f i n d genospecies 5 i n any of the plants sampled. D. Biochemical model f o r phosphate accumulation Marais et al. (1983) stated that s i g n i f i c a n t advances into the use of enhanced b i o l o g i c a l phosphate removal from wastewater w i l l be contingent on a greater understanding of the biochemical mechanisms c o n t r o l l i n g the phenomenon. Bio-P removal can be achieved by a modification of a conventional activ a t e d sludge process. The process consists of incorporating an anaerobic zone before the aerobic zone. With the addition of wastewater to the anaerobic zone, the soluble phosphate concentration increases as a r e s u l t of release of phosphate from organisms within the sewage. In the subsequent aerobic zone the pool of soluble phosphate decreases as a r e s u l t of phosphate uptake' by the organisms. The phosphate that was released i n the anaerobic zone i s accumulated by the biomass i n addition to whatever phosphate was o r i g i n a l l y there. The current biochemical model f o r enhanced phosphate removal was postulated by Comeau et al. (1985, 1986). The model predicts that phosphate accumulating microorganisms derive an advantage of s t o r i n g poly-phosphate under aerobic conditions by using the poly-P under anaerobic conditions as an energy source to drive the substrate storage of PHB's. This stored carbon reserve then provides energy to phosphate accumulating b a c t e r i a i n the aerobic zone where carbon a v a i l a b i l i t y l i m i t s m i c r o b i a l growth. The model also predicts that a l l phosphate accumulating organisms must be obligate aerobes. Facultative organisms would tend to p r e f e r e n t i a l l y grow rather than accumulate carbon reserves i n the anaerobic zone. I f they do not accumulate reserves i n the anaerobic zone they would not be able to exchange these reserves to drive phosphate accumulation i n the aerobic zone. In order to v a l i d a t e the autoradiographic assay within the context of biochemical modelling the s t r a i n s i s o l a t e d from the various treatment plants were tested f o r t h e i r a b i l i t y to grow anaerobically (Table XII). From the r e s u l t s , a l l the major genera {Acinetobacter, Aeromonas, Moraxella, Pseudomonas and Vibrio) that are accumulators according to the autoradiographic assay were obligate aerobes. The f a c u l t a t i v e organisms ranged from ,17-33% for the nonaccumulator c l a s s . This trend breaks down when the CDC groups from Squamish are included as well as the other smaller genera. The operational configuration of Squamish i s quite 80 d i f f e r e n t than the other plants surveyed ( i e . secondary vs. t e r t i a r y ) . One consideration that must be addressed i s that of s e l e c t i v e pressures within a c t i v a t e d sludge systems. Since the CDC groups account f o r approximately 82% of the t o t a l i s o l a t e d population of the sample from Squamish i t i s conceivable that the organisms from t h i s plant operate according to a d i f f e r e n t biochemical mechanism that does not necessitate o b l i g a t e l y aerobic organisms. I f the biochemical model i s indeed true then i t i s l o g i c a l to assume that there must be some s o r t of s e l e c t i v e pressure f o r these types of organisms. The organisms selected f o r would then presumably make up a s i g n i f i c a n t proportion of the sludge population. This i s the case that i s seen with organisms from the Kelowna and UBC p i l o t plants. The organisms that are not selected f o r would then make up an i n s i g n i f i c a n t proportion of the population. The smaller groups of b a c t e r i a occupy a d i f f e r e n t environmental and biochemical niche and operate according to some other mechanism of phosphate accumulation that does not necessitate a b l i g a t e l y aerobic organisms. In the case of Squamish i t may indeed be a d i f f e r e n t biochemical system i n keeping with the p h y s i c a l c o n f i g u r a t i o n a l differences between t h i s plant and the Kelowna and the UBC p i l o t plants. I t i s u n r e a l i s t i c to assume that there i s only one biochemical mechanism that governs a l l the organisms i n each system. E. Comparison between Biolog's and Bouvet's system on Acinetobacter genospecies c l a s s i f i c a t i o n The genus Acinetobacter have frequently been implicated as a major contributor i n the mi c r o b i a l community to phosphorus accumulation i n nutrient removal a c t i v a t e d sludge plants (Wiechers,1983; Ramadori,1987). However t h e i r 81 o v e r a l l c o n t r i b u t i o n to t h i s process has often been questioned (Brodisch and Joyner, 1983; Stephenson, 1987; Cloete and Steyn, 1988). Many reports on the microbiology of phosphorus removal from wastewater can be c r i t i c i z e d over the methodology used f o r sampling, i s o l a t i n g and i d e n t i f y i n g t h e i r populations. Once i s o l a t e d , many of the systems used to i d e n t i f y Acinetobacter have f a i l e d to recognize the taxonomic problems associated with t h i s genus (Juni, 1984). Consequently, our understanding of the d i v e r s i t y which e x i s t s within t h i s genus i s poor. Recent advances into the taxonomy of Acinetobacter (Bouvet and Grimont, 1986) have made av a i l a b l e methods for the d e l i n e a t i o n of t h i s genus into at l e a s t 12 separate genetic groups. As a r e s u l t , researchers have been able to look at the d i s t r i b u t i o n of t h i s genus at a much more d e t a i l e d l e v e l of analysis within the context of phosphate removal from wastewater (Beacham et al.,1990; Duncan et a l . , 1988). The method of Bouvet and Grimont (1986) was used to look at Acinetobacter i s o l a t e d from the various treatment plants surveyed i n t h i s study. The te s t s c o n s i s t s of a regime of 23 carbon u t i l i z a t i o n tests designed to d i f f e r e n t i a t e the 12 genetic groups that e x i s t w i t h i n the genus Acinetobacter. Out of the 23 carbon substrates 3 of them were able to d i f f e r e n t i a t e between accumulators and nonaccumulators according to the autoradiographic assay (Aco n i t i c , L -aspartic, and malonic a c i d s ) . In each of the 3 cases the proportion of p o s i t i v e carbon u t i l i z a t i o n tests were lower f o r accumulators as compared to nonaccumulators. In other words there exists a tendency f o r accumulators within species of Acinetobacter to not be able to u t i l i z e these three carbon substrates but i t i s not absolute. Since a l l the substrates from t h i s c l a s s i f i c a t i o n method were chosen because of t h e i r a b i l i t y to d i f f e r e n t i a t e between genetic groups within the genus Acinetobacter, i t may be hypothesized that the phenomenon of phosphate accumulation i s l i n k e d to d i s t i n c t group subsets within the c l a s s i f i e d 82 genospecies. 83 CONCLUSION Enhanced Bio-P removal i s now a widely accepted and proven a l t e r n a t i v e i n eutrophication c o n t r o l . I t i s perhaps i r o n i c that t h i s phenomenon was discovered by accident and hundreds of m i l l i o n s of d o l l a r s have been invested i n i t without a f u l l comprehensive understanding of the basic b i o l o g i c a l mechanisms. I t i s recognized that the optimization of t h i s process i s dependant on a better understanding of the ecology and physiology of the organisms involved. According to the r e s u l t s presented i n t h i s thesis a v a r i e t y of organisms are capable of Bio-P p o t e n t i a l . This research has answered some basic fundamental questions concerning the r o l e of microorganisms i n the Bio-P process. A technique has been developed i n which organisms can be screened for t h e i r phosphate accumulating p o t e n t i a l . A p p l i c a t i o n of t h i s technique shows that contrary to what many inv e s t i g a t o r s believe, Acinetobacter may not be the only genus i n t e r t i a r y sewage treatment that i s responsible f o r phosphate accumulation. I t has also been demonstrated that other genera and organisms w i t h i n a genus can d i f f e r i n t h e i r capacity to accumulate phosphate. 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