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Optimization of cultural factors influencing the production of extracellular vesicles and proteinase… Myhara, Robert Michael 1989

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O p t i m i z a t i o n o f C u l t u r a l F a c t o r s I n f l u e n c i n g t h e P r o d u c t i o n of. E x t r a c e l l u l a r V e s i c l e s and P r o t e i n a s e by Pseudomonas f r a g l ATCC 49 73. by R o b e r t M i c h a e l Myhara B . S c , The U n i v e r s i t y o f M a n i t o b a , 1974 M . S c , The U n i v e r s i t y o f M a n i t o b a , 1983 A t h e s i s p r e s e n t e d l n p a r t i a l f u l f i l l m e n t o f t h e r e q u i r e m e n t s f o r t h e d e g r e e o f D o c t o r o f P h i l o s o p h y in THE FACULTY OF GRADUATE STUDIES The Department o f Food S c i e n c e We a c c e p t t h i s t h e s i s as c o n f o r m i n g o t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 6, 1989 © R o b e r t M i c h a e l Myhara, 1989. In presenting t h i s thesis in p a r t i a l f u l f i l l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Food Science The University of B r i t i s h Columbia Vancouver, Canada Date A p r i l 28, 1989 A B S T R A C T Myhara, R o b e r t M. The U n i v e r s i t y o f B r i t i s h C o l u m b i a . A p r i l 6, 1989. O p t i m i z a t i o n o f C u l t u r a l F a c t o r s I n f l u e n c i n g t h e P r o d u c t i o n o f E x t r a c e l l u l a r V e s i c l e s and P r o t e i n a s e by Pseudomonas f r a g i ATCC 4973. Pseudomonas f r a g i ATCC 4973 was grown i n t r y p t i c a s e s o y b r o t h ( T S B ) , on a t r y p t i c a s e s o y b r o t h + 1.5% a g a r (TSA) s u r f a c e , and i n a d e f i n e d c i t r a t e b r o t h . The c i t r a t e b r o t h c o n t a i n e d g l u t a m i n e as t h e s o l e n i t r o g e n s o u r c e , Pseudomonas f r a g i grown i n TSB s t a r t e d p r o t e i n a s e p r o d u c t i o n a t 24 h, d u r i n g t h e l a t e l o g a r i t h m i c e a r l y s t a t i o n a r y g r o w t h p h a s e . Pseudomonas f r a g i grown on TSA s u r f a c e s i n i t i a t e d p r o t e i n a s e p r o d u c t i o n a t 4 h, 20 h o u r s e a r l i e r t h a n i n l i q u i d medium. E l e c t r o n m i c r o g r a p h s o f P. f r a g i grown on TSA r e v e a l e d e x t r a c e l l u l a r v e s i c l e s c a . 20 nm i n d i a m e t e r " b l e b b i n g " o f f t h e s u r f a c e o f t h e c e l l s . These v e s i c l e s were a b s e n t f r o m t h e s u r f a c e o f P. f r a g i c e l l s grown i n TSB, a l t h o u g h v e s i c l e s c o u l d be i s o l a t e d f r o m t h e c u l t u r e s u p e r n a t a n t . I s o l a t e d e x t r a c e l l u l a r v e s i c l e s were c a . 20 nm i n d i a m e t e r and c o n t a i n e d a p r o t e i n a s e s i m i l a r t o t h a t f o u n d i n t h e s u p e r n a t a n t . E l e c t r o p h o r e t i c a n a l y s i s showed t h e v e s i c l e s and o u t e r c e l l membrane o f P. f r a g i t o s h a r e s i m i l a r i t i e s i n t h e i r c o m p o s i t i o n . Use o f t h e c e n t r o i d s e a r c h t e c h n i q u e o f A i s h i m a and N a k a i , showed t h e optimum i i c u l t u r a l conditions for proteinase production by P. f r a g i , in defined c i t r a t e broth to be: incubation temperature, 12.5 C; incubation time, 38 h; i n i t i a l pH, 6.8; organic nitrogen concentration, 314 mmole nitrogen/L (glutamine); a gas mixture containing 16.4% oxygen flowing over the medium (7.42 ppm dissolved oxygen). Oxygen was the major factor influencing proteinase production by P. f r a g i . A comparison of optimization techniques suitable for microbiological experiments showed that the centroid search technique of Aishima and Nakai, the modified super simplex of Nakai and Kaneko and the simplex technique of Morgan and Deming a l l required similar time and experiment numbers to obtain the optimum point. i i i CONTENTS ABSTRACT II CONTENTS IV LIST OF TABLES VIII LIST OF FIGURES X ACKNOWLEDGEMENT XIII INTRODUCTION 1 LITERATURE REVIEW 3 SPOILAGE OF MEAT SURFACES 3 DEFINITION 3 MICROBIAL FLORA 3 Temperature 4 Oxygen 4 Nutrient a v a i l a b i l i t y 5 SPOILAGE OF DAIRY PRODUCTS 6 PROCESSING 6 SPOILAGE MICROORGANISMS 6 EXTRACELLULAR PROTEINASES 7 PSEUDOMONADS 8 EXTRACELLULAR PROTEINASE 9 EXTRACELLULAR VESICLES 12 INTRODUCTION 12 BACTERIOCINS 12 H i s t o r i c a l perspectives 14 Detection of bacteriocins ..15 Unrelated antagonists 16 Factors a f f e c t i n g production of bacteriocins 17 Bacteriocin induction 18 Physical properties of bacteriocins 19 Chemical composition 20 Mode of action of bacteriocins 21 Bacteriocin induced c e l l damage 23 OPTIMIZATION 23 INTRODUCTION 23 LINEAR PROGRAMMING 2 5 DIRECT SEARCH 25 i v EVOLUTIONARY OPERATION TECHNIQUES.. 26 Response surface methodology 26 Simplex methods 27 Morgan-Deming simplex 27 Modified super simplex 28 Centroid search 29 MATERIALS & METHODS 30 BACTERIOCIN 30 ORGANISMS 30 The host 30 The indicators 30 Storage 30 GROWTH OF ORGANISM 31 The media 31 Washed c e l l suspension 31 Detection plates 31 DETECTION OF BACTERIOCINOGENICITY 31 Solid culture 31 INDUCTION. 32 Radiation induction 32 Mitomycin C 33 Spi r a l plate detection 34 GROWTH OF P. f r a g i ON SOLID AND LIQUID MEDIUM 34 INOCULUM GROWTH 3 4 INCUBATION ON SOLID MEDIUM 35 INCUBATION IN LIQUID MEDIUM 35 ENUMERATION OF CELLS 3 5 PROTEINASE ACTIVITY 36 DATA PRESENTATION 37 ELECTRON MICROSCOPY 37 Sample f i x a t i o n 37 Sample preparation 37 Microscopy 38 OPTIMIZATION 39 THE MEDIA 39 CELL NUMBER ENUMERATION 39 CELL SUSPENSION 39 I n i t i a l broth culture 39 Working inoculum 40 EXPERIMENTAL CONDITIONS 40 Gas mixtures 40 PROTEINASE ACTIVITY 4 4 ELECTRON MICROSCOPY 4 4 PROTEIN ASSAY 4 4 ISOELECTRIC FOCUSING (IEF) 45 Proteinase l o c a l i z a t i o n 45 OPTIMIZATION PROCEDURES 45 OPTIMIZATION MODELS 47 CENTROID OPTIMIZATION 55 Step 1 - I n i t i a l simplex ...55 Step 2 - Evaluate the centroids 57 v Step 3 - Calculate new vertices and ranges 61 Step 4 - Evaluate new simplex 62 Step 5 - Evaluate new centroids 62 Step 6 - Mapping 6 3 NEW SIMPLEX 66 CELL AND GROWTH MEDIUM FRACTIONATION 72 BROTH CULTURE 7 2 SUPERNATANT PREPARATION 7 2 EXTRACELLULAR VESICLE PREPARATION 72 Electron microscopy 73 Buoyant density 73 Carbohydrate assay 74 OUTER CELL MEMBRANE PREPARATION 74 SOLUBLE CELL MATERIAL PREPARATION 75 PROTEINASE ACTIVITY 75 PROTEIN ASSAY 75 ELECTROPHORETIC TECHNIQUES 76 Electrophoresis 7 6 I s o e l e c t r i c focusing (IEF) 77 Proteinase l o c a l i z a t i o n 77 RESULTS 79 GROWTH OF P. fra g i ON SOLID AND LIQUID MEDIUM 79 PRODUCTION OF PROTEINASE BY P. fr a g i 79 ELECTRON MICROSCOPY/EXTRACELLULAR VESICLE PRODUCTION BY P. fra g i 8 4 Scanning electron microscopy 84 Thin section transmission electron microscopy 89 VESICLE SHEDDING VERSUS PROTEINASE PRODUCTION BY P. fragi 107 CELL AND GROWTH MEDIUM FRACTIONATION 107 ISOLATION OF EXTRACELLULAR VESICLES 10 7 Electron microscopy 108 OUTER-CELL MEMBRANE PREPARATION 10 8 BUOYANT DENSITY I l l ISOELECTRIC FOCUSING (IEF) I l l Ext r a c e l l u l a r v e s i c l e s and supernatant I l l OUTER-CELL MEMBRANE/EXTRACELLULAR VESICLE COMPONENT PROFILES I l l Ext r a c e l l u l a r vesicle/outer c e l l membrane enzyme l o c a l i z a t i o n 118 COMPOSITION OF THE COMPONENTS 119 BACTERIOCINS 121 EXTRACELLULAR VESICLES 121 BACTERIOCIN INDUCTION 121 OPTIMIZATION 122 OPTIMIZATION OF PROTEINASE PRODUCTION 122 Fractional f a c t o r i a l 122 Centroid search optimization 126 Mapping 129 v i Thin section transmission electron microscopy 138 Is o e l e c t r i c focusing (IEF) 144 MODELING 144 Description of centroid search 144 COMPARISON OF OPTIMIZATION TECHNIQUES 149 DISCUSSION 160 GROWTH OF P. fra g i ON SOLID AND LIQUID MEDIUM 160 CULTURE GROWTH 160 PROTEINASE PRODUCTION 160 ELECTRON MICROSCOPY 161 Scanning electron microscopy... 161 Thin section transmission electron microscopy 162 CELL AND GROWTH MEDIUM FRACTIONATION 162 BACTERIOCINS 169 OPTIMIZATION 171 DISCUSSION OF FACTORS STUDIED 172 Glutamine 172 pH 176 Time 177 Temperature 177 Oxygen 179 THIN SECTION ELECTRON MICROSCOPY 180 OXYGEN LEVEL AND THE DAIRY INDUSTRY 181 OPTIMIZATION TECHNIQUE 182 Centroid search 182 Mapping 185 OPTIMIZATION COMPARISONS 185 Morgan Deming simplex 186 Modified super simplex 187 Centroid search 187 Response surface methodology 188 Comparison of simplex methods 188 Response surface methodology 189 OPTIMIZATION IN MICROBIOLOGICAL SYSTEMS 190 CONCLUSIONS 192 LITERATURE CITED 194 v i i LIST OF TABLES Table I MATHEMATICAL MODELS USED TO EVALUATE OPTIMIZATION TECHNIQUES 54 II EXPERIMENTS GENERATED BY CENTROID SEARCH AND RESPONSE VALUES RETURNED BY MODEL #1 60 III EXPERIMENTS GENERATED BY CENTROID SEARCH AND EVALUATED BY MODEL #1 - NEW SIMPLEX 6 7 IV COMPOSITION OF VESICLES, SOLUBLE CELL MATERIAL AND SUPERNATANT 12 0 V FRACTIONAL FACTORIAL EXPERIMENTS 123 Via ANALYSIS OF FRACTIONAL FACTORIAL GROWTH DATA VARIANCE (FACTORS) 124 VIb ANALYSIS OF FRACTIONAL FACTORIAL GROWTH DATA VARIANCE (REPLICATES) 124 V i l a ANALYSIS OF FRACTIONAL FACTORIAL PROTEINASE DATA VARIANCE (FACTORS) 125 VI lb ANALYSIS OF FRACTIONAL FACTORIAL PROTEINASE DATA VARIANCE (REPLICATES) ...125 V i l l a INITIAL SPENDLEY MATRIX EXPERIMENTS AND RESPONSE VALUES 127 V I l i b ANALYSIS OF SPENDLEY PROTEINASE DATA VARIANCE (REPLICATES) 127 IXa CENTROID EXPERIMENTS AND THEIR RESPONSE VALUES 128 v i i i IXb ANALYSIS OF CENTROID PROTEINASE DATA VARIANCE (REPLICATES) 128 X SUGGESTED OPTIMUM VALUES FOR PROTEINASE PRODUCTION BY P. FRAGI 130 XI SIMULTANEOUS SHIFT EXPERIMENTS AND THEIR RESPONSE VALUES 131 XII OPTIMIZATION TRIALS SELECTED FOR ELECTRON MICROSCOPY 139 XIII COMPARISON OF OPTIMIZATION TECHNIQUES USING MATHEMATICAL MODELS 157 XIVa COMPARISON OF OPTIMIZATION TECHNIQUES WITH RESPECT TO TIME REQUIRED 158 XIVb COMPARISON OF OPTIMIZATION TECHNIQUES WITH RESPECT TO EXPERIMENT NUMBER REQUIRED 159 i x L I S T . OF JFIGURES F i g u r e 1 DIAGRAM OF GAS MIXTURE CONTROL AND GYRATORY WATER BATH SETUP . 4 2 2 THREE DIMENSIONAL PLOT OF MODEL #1 52 3 TOPOGRAPHICAL PLOT OF MODEL #1 WITH CENTROID GENERATED EXPERIMENTS... 59 4 MAP OF FACTORS FROM MODEL #1 65 5 TOPOGRAPHICAL MAP OF MODEL #1 AND EXPERIMENTS GENERATED BY CENTROID SEARCH - NEW SIMPLEX 6 9 6 MAP OF FACTORS FROM MODEL #1 - NEW SIMPLEX 71 7 P. f r a g i GROWTH AND PROTEINASE PRODUCTION IN L I Q U I D MEDIUM 81 8 P. f r a g i GROWTH AND PROTEINASE PRODUCTION ON SOLID MEDIUM 83 9 SCANNING ELECTRON MICROGRAPH OF P. f r a g i GROWN IN L I Q U I D MEDIUM FOR 4 HOURS 86 10 SCANNING ELECTRON MICROGRAPH OF P. f r a g i GROWN IN L I Q U I D MEDIUM FOR 27 HOURS 86 11 SCANNING ELECTRON MICROGRAPH OF P. f r a g i GROWN IN L I Q U I D MEDIUM FOR 76 HOURS 88 12 SCANNING ELECTRON MICROGRAPH OF P. fragi GROWN ON SOLID MEDIUM FOR 4 HOURS 88 13 SCANNING ELECTRON MICROGRAPH OF P. f r a g i GROWN ON SOLID MEDIUM FOR 24 HOURS 91 x 14 TRANSMISSION ELECTRON MICROGRAPH OF P. fragi GROWN IN LIQUID MEDIUM FOR 4 HOURS 91 15 TRANSMISSION ELECTRON MICROGRAPH OF P. fra g i GROWN IN LIQUID MEDIUM FOR 16 HOURS 9 3 16 TRANSMISSION ELECTRON MICROGRAPH OF P. fra g i GROWN IN LIQUID MEDIUM FOR 16 HOURS 95 17 TRANSMISSION ELECTRON MICROGRAPH OF P. fr a g i GROWN IN LIQUID MEDIUM FOR 44 HOURS : 95 18 TRANSMISSION ELECTRON MICROGRAPH OF P. fra g i GROWN IN LIQUID MEDIUM FOR 92 HOURS .98 19 TRANSMISSION ELECTRON MICROGRAPH OF P. fr a g i GROWN ON SOLID MEDIUM FOR 4 HOURS 100 20 TRANSMISSION ELECTRON MICROGRAPH OF P. fr a g i GROWN ON SOLID MEDIUM FOR 20 HOURS SHOWING PRESENCE OF VESICLES 102 21 TRANSMISSION ELECTRON MICROGRAPH OF P. fra g i GROWN ON SOLID MEDIUM FOR 32 HOURS 104 22 TRANSMISSION ELECTRON MICROGRAPH OF P. fra g i GROWN ON SOLID MEDIUM FOR 56 HOURS 10 6 23 TRANSMISSION ELECTRON MICROGRAPH OF NEGATIVELY STAINED ISOLATED EXTRACELLULAR VESICLES 110 2 4 IEF SEPARATION OF CULTURE SUPERNATANT AND EXTRACELLULAR VESICLE PROTEINS 113 2 5 ZYMOGRAM OF IEF SEPARATED CULTURE SUPERNATANT AND EXTRACELLULAR VESICLES 115 2 6 SDS-PAGE SEPARATION OF EXTRACELLULAR VESICLE AND OUTER CELL MEMBRANE PROTEINS 117 x i 27 OPTIMIZATION MAP OF FACTORS TIME AND TEMPERATURE 133 28 OPTIMIZATION MAP OF FACTORS GLUTAMINE AND PH 13 5 29 OPTIMIZATION MAP OF FACTOR OXYGEN 137 30 TRANSMISSION ELECTRON MICROGRAPHS OF P. fr a g i USED IN THE OPTIMIZATION EXPERIMENTS 141 31 TRANSMISSION ELECTRON MICROGRAPHS OF P. fr a g i USED IN THE OPTIMIZATION EXPERIMENTS 143 32 SUPERNATANT FROM OPTIMIZATION EXPERIMENTS T3 AND T4 SEPARATED ON IEF GELS 146 33 TOPOGRAPHICAL PLOT OF MODEL #1 WITH CENTROID GENERATED EXPERIMENTS 148 34 TOPOGRAPHICAL MAP OF MODEL #1 AND EXPERIMENTS GENERATED BY CENTROID SEARCH - NEW SIMPLEX 151 35 TOPOGRAPHICAL PLOT OF MODEL #2 CENTROID GENERATED EXPERIMENTS 153 36 TOPOGRAPHICAL PLOT OF MODEL #2 CENTROID GENERATED EXPERIMENTS-NEW SIMPLEX 155 X i i ACKNOWLEDGEMENT I would l i k e t o t a k e t h i s o p p o r t u n i t y t o e x p r e s s my a p p r e c i a t i o n and g r a t i t u d e t o my a d v i s o r Dr. B . J . S k u r a , f o r h i s g u i d a n c e and s u p e r v i s i o n d u r i n g t h e c o u r s e o f t h i s s t u d y . I would a l s o l i k e t o t h a n k Dr. T. P a t e l o f M e m o r i a l U n i v e r s i t y o f New f o u n d l a n d f o r t h e use o f h i s f a c i l i t i e s . I would l i k e t o thank t h e members o f t h e r e v i e w i n g c o m m i t t e e : Dr. B. M c B r i d e and Dr. C. B e l l D e partment o f M i c r o b i o l o g y ; Dr. S. N a k a i , Dr. W.D. P o w r i e , and Dr. P.M. T o w n s l e y , Department o f Food S c i e n c e . The a u t h o r would l i k e t o g r a t e f u l l y t h a nk M i c h a e l W e i s s , Department o f B o t a n y who h e l p e d w i t h t h e e l e c t r o n m i c r o s c o p e . To o t h e r s t a f f members and f e l l o w s t u d e n t s , I thank them f o r t h e i r h e l p and g u i d a n c e d u r i n g my s t a y a t The U n i v e r s i t y o f B r i t i s h C o l u m b i a . T h i s t h e s i s i s d e d i c a t e d t o my w i f e B r e n d a . x i i i INTRODUCTION P s y c h r o t r o p h i c m i c r o o r g a n i s m s a r e p r e s e n t i n n e a r l y a l l raw f o o d p r o d u c t s . They a r e common c o n t a m i n a n t s o f p r o c e s s e d f o o d i t e m s s u c h as p a s t e u r i z e d m i l k o r p o s t -s l a u g h t e r meat s u r f a c e s . They a r e a major f a c t o r i n d e t e r m i n i n g t h e k e e p i n g q u a l i t y o f t h e s e f o o d s . The g r o w t h and m e t a b o l i s m o f t h e s e m i c r o o r g a n i s m s has been s e l e c t e d by t h e d e v e l o p m e n t o f s y s t e m s l e a d i n g t o e x t e n d e d r e f r i g e r a t e d s t o r a g e o f f o o d s . As a p a r t o f t h e i r m e t a b o l i s m , p s y c h r o t r o p h i c b a c t e r i a p r o d u c e e x t r a c e l l u l a r enzymes, p r i m a r i l y l i p a s e s and p r o t e i n a s e s , w h i c h a r e major c o n t r i b u t o r s t o a d v a n c e d s t a g e s o f m i c r o b i a l s p o i l a g e o f r e f r i g e r a t e d f o o d s o f a n i m a l o r i g i n . Many o f t h e p r o t e i n a s e s , e s p e c i a l l y t h o s e o f t h e p s y c h r o t r o p h i c pseudomonads, a r e e x t r e m e l y h e a t s t a b l e . The h e a t s t a b l e p r o t e i n a s e s a r e a b l e t o w i t h s t a n d p a s t e u r i z a t i o n and o t h e r h e a t t r e a t m e n t s , and r e m a i n a c t i v e i n t h e r m a l l y p r o c e s s e d f o o d s . C o n s e q u e n t l y , t h e y a r e o f c o n s i d e r a b l e c o m m e r c i a l i m p o r t a n c e , s i n c e t h e p r o d u c t i o n o f s h e l f - s t a b l e f o o d i t e m s d e p e n d s , i n p a r t , on t h e d e s t r u c t i o n o f d e l e t e r i o u s enzymes. The o b j e c t i v e o f t h i s s t u d y was t o d e t e r m i n e how e n v i r o n m e n t a l and n u t r i t i o n a l f a c t o r s a f f e c t e d p r o t e i n a s e p r o d u c t i o n by one o f t h e s e p s y c h r o t r o p h i c o r g a n i s m s , P. f r a g i ATCC 4 9 7 3 . I n p a r t , t h e s t u d y compared v a r i o u s t e c h n i q u e s t h a t c o u l d be us e d t o examine t h e r e l a t i o n s h i p s 1 between the factors and proteinase production. F i n a l l y , t h i s study attempted to understand and explain the function of e x t r a c e l l u l a r v e s i c l e s produced by P. f r a g i . S p e c i f i c a l l y t h i s study focussed on the re l a t i o n s h i p between e x t r a c e l l u l a r v e s i c l e s , with bacteriocin or proteinase production. 2 SPOILAGE OF MEAT SURFACES Meat, s i m p l y s t a t e d , i s t h e f l e s h o f a s l a u g h t e r e d a n i m a l w h i c h may be us e d as f o o d . I m m e d i a t e l y a f t e r s l a u g h t e r , t h e meat u n d e r g o e s d e g r a d a t i o n w h i c h may u l t i m a t e l y s p o i l t h e meat. M i c r o b i a l s p o i l a g e i s a p r o c e s s i n w h i c h m i c r o o r g a n i s m s p r o d u c e p r o t e i n a s e s c a p a b l e o f d e g r a d i n g f u n c t i o n a l and s t r u c t u r a l p r o t e i n s . Some p r o t e i n s may be d e g r a d e d by c a t h e p s i n s d u r i n g t h e p r o c e s s o f a g i n g . I t i s i m p o r t a n t t o be a b l e t o d i s t i n g u i s h m i c r o b i a l e n z y m i c breakdown o f p r o t e i n s f r o m t h e p r o t e i n d e c o m p o s i t i o n by endogenous enzymes. To d e l a y s p o i l a g e , a d r e s s e d a n i m a l c a r c a s s i s c h i l l e d b e l o w 25 C and s t o r e d a t t e m p e r a t u r e s between -1 and +5 C. M j c y o p j a l f l p r a , The e s t a b l i s h m e n t o f a m i c r o b i a l f l o r a on a meat c a r c a s s depends upon t h e e n v i r o n m e n t i n w h i c h t h e o r g a n i s m s a r e f o r c e d t o grow. As s u c h , t h e o r g a n i s m s i n v o l v e d a r e s u b j e c t t o c h a n g e s i n oxygen t e n s i o n , w a t e r a c t i v i t y and pH. The s e f a c t o r s a r e a c o n s e q u e n c e o f t h e p o s t m o r t e m b i o c h e m i c a l c h a n g e s w h i c h t a k e p l a c e i n t h e m u s c l e t i s s u e s . B e s i d e s t h o s e f a c t o r s i n f l u e n c e d by b i o c h e m i c a l c h a n g e s i n t h e meat i t s e l f , t h e m i c r o b i a l f l o r a may a l s o be i n f l u e n c e d by n u t r i e n t a v a i l a b i l i t y and m i c r o b i a l a n t a g o n i s m . 3 Temperature At c h i l l temperatures (temperatures between -1 and +5 C) the growing b a c t e r i a l f l o r a consists of cold tolerant organisms only. G i l l and Newton (1980) found strains of Pseudomonas, Mozaxella, Acinetobacter, Lactobacillus, Brochothzix thermosphacta and some psychrotrophic Enterobacteriaceae to be the most common b a c t e r i a l types found on s p o i l i n g meat, oxygen Under normal meat storage conditions, where animal carcasses are hung in a cooler, spoilage i s a surface phenomenon. If the humidity of the cooler a i r is high, a s u p e r f i c i a l layer of slime w i l l develop. This slime layer i s primarily produced by bacteria of the Pseudomonas genus (Ingram and Dainty, 1971). G i l l and Newton (1980) found that Pseudomonas species grew faster than the other bacteria present on meat surfaces from 2 to 15 C, and that the difference in growth rates increased with a decrease in temperature. These investigators found that Enterobacter sp. and B. thermosphacta grew more slowly when competing with Pseudomonas sp. which had attained maximum c e l l d ensities on a meat surface. They suggested that the maximum c e l l density attainable by Pseudomonas sp* was determined by the rate at which oxygen became available to the c e l l s . The s t r i c t l y aerobic pseudomonads would have a greater requirement for oxygen than either Enterobacter sp. or M. thermosphactum. The l a t t e r two organisms must obtain 4 energy from the less e f f i c i e n t g l y c o l y t i c pathway. Available nutrient u t i l i z a t i o n between the competing organisms was not as great a factor as oxygen a v a i l a b i l i t y . Nutrient a v a i l a b i l i t y Advanced microbial spoilage of meat involves the production of proteinases by the s p o i l i n g microorganisms. Meat spoiled at warm temperatures by C l o s t r i d i a c l e a r l y involved p r o t e o l y t i c breakdown of the tissues. It appeared, however, that i n i t i a l spoilage at intermediate and c h i l l temperatures involved the breakdown of low molecular weight water-soluble peptides and not larger s t r u c t u r a l or functional proteins. Borton et al. (1970a) inoculated minced beef muscle with P. f r a g i and incubated the mixture at 2 C. These researchers found an i n i t i a l decrease in soluble proteinaceous material. Sodium dodecyl sulfate polyacrylamide electrophoresis studies (Borton et a l . , 1970b) showed that, after eight days of incubation, no major destruction of s a l t soluble m y o f i b r i l l a r proteins could be detected. Those changes which did appear at t h i s time were caused by endogenous enzymic a c t i v i t y . After 20 days incubation, however, considerable amounts of large molecular weight proteins disappeared. They concluded that the organism p r e f e r e n t i a l l y u t i l i z e d those compounds most e a s i l y assimilated, and that proteolysis occurred only after these compounds were destroyed. 5 SPOILAGE OF DAIRY PRODUCTS P r o c e s s i n g F l u i d m i l k i n Canada i s a l m o s t e x c l u s i v e l y s t o r e d a t 4 t o 6 C. T y p i c a l l y , m i l k p r o d u c e r s s t o r e m i l k f o r p i c k - u p e v e r y s e c o n d d a y . The r e f r i g e r a t e d b u l k t a n k t r u c k w i l l c o l l e c t and mix m i l k f r o m s e v e r a l d a i r y f a r m s . M i l k i s t h e n u s u a l l y d e l i v e r e d t o t h e p r o c e s s i n g p l a n t t h e same day. The d e l i v e r e d m i l k w i l l be pumped t o l a r g e b u l k t a n k s , f o r r e f r i g e r a t e d s t o r a g e , p r i o r t o p a s t e u r i z a t i o n . The t i m e p e r i o d between p r o d u c t i o n and p a s t e u r i z a t i o n may v a r y up t o 1 2 0 h ( 6 . 5 d a y s ) d e p e n d i n g upon s t o r a g e c a p a c i t y and f l u i d m i l k demand ( C o u s i n , 1 9 8 1 ) . C o m m e r c i a l raw m i l k i n g e n e r a l i s composed o f a b o u t 9 0 % m o i s t u r e , 3 % f a t and 3 . 2 % p r o t e i n . The p r o t e i n component i s p r i m a r i l y c a s e i n . S p o i l a g e m i c r o o r g a n i s m s C o n s t a n t a g i t a t i o n d u r i n g s t o r a g e i s n e c e s s a r y t o p r e v e n t m i l k and c r e a m s e p a r a t i o n , and t o m a i n t a i n u n i f o r m t e m p e r a t u r e . C o n s e q u e n t l y , m i l k i s w e l l a e r a t e d d u r i n g s t o r a g e . As a r e s u l t o f t h i s a e r a t i o n , m i c r o o r g a n i s m s r e s p o n s i b l e f o r m i l k s p o i l a g e t e n d t o be a e r o b i c or f a c u l t a t i v e l y a n a e r o b i c . These m i c r o o r g a n i s m s g e n e r a l l y p r o d u c e e x t r a c e l l u l a r enzymes, w h i c h a r e c a p a b l e o f h y d r o l y z l n g t h e l i p i d and t h e p r o t e i n a c e o u s components o f t h e m i l k . When raw m i l k i s h e l d a t a c o n s t a n t r e f r i g e r a t i o n t e m p e r a t u r e , m e s o p h i l i c a e r o b i c b a c t e r i a l s p o i l a g e c a n be 6 reduced considerably. These conditions, however, s e l e c t i v e l y favor the growth of aerobic psychrotrophic bacteria (Cousin and Marth, 1977). The most frequently found psychrotrophic bacteria in raw milk are gram negative rods, e s p e c i a l l y those of the Pseudomonas species (Kwan and Skura, 1985; J u f f s , 1973). Pseudomonas fluorescens has been shown to be the species most often associated with milk spoilage (McKellar and Cholette, 1985; McKellar and Cholette, 1987; Patel et a l . , 1983a; Jackman et a l . , 1983; F a i r b a i r n and Law, 1987; McKellar, 1982; Rowe and Gilmour, 1982). Kwan and Skura (1985) studying psychrotrophic spoilage pseudomonads from raw milk, found only half of the organisms belonged to the fluorescens species with P. f r a g i making up the bulk of the remaining species. This study found P. f r a g i and P. f r a g i - l i k e organisms to reside in a c l o s e l y related cluster when subjected to numerical taxonomy. Isolation of P. f r a g i from commercial raw milk samples has also been reported by Law et a l . (1976), Samagh and Cunningham (1972), and Overcast (1968). E x t r a c e l l u l a r p r o t e i n a s e s Growth of these pseudomonads in raw refrigerated milk is accompanied by the production of e x t r a c e l l u l a r proteinases. These enzymes have been shown to produce b i t t e r off flavors in milk due to peptide formation, and poor f u n c t i o n a l i t y of milk protein in cheese making, due to p a r t i a l protein degradation (Speck and Adams, 1976; Law et a l . , 1976; Law, 1979; Cousin, 1982; Hicks et a l . , 1986). 7 These proteinases tend to be heat stable ( G r i f f i t h s et a l . , 1981), able to withstand commercial high temperature short time (HTST), and u l t r a high temperature (UHT) treatments. Subsequent to heat treatment, these enzymes have been responsible for breakdown of protein components in milk subject to long term storage. Typical defects have included b i t t e r off flavors and milk gelation. Since heat treatment results in milk unsuitable for manufacturing into products requiring formation of a curd, milk must be stored u n t i l needed in an unpasteurized form. Elimination of the microorganisms through other means would be d i f f i c u l t . Control of environmental factors, influencing the production of the proteinase by the microorganisms, may help to eliminate the problems associated with long term refrigerated raw milk storage. PSEUPQMQNADS The term pseudomonad can be used to describe a group of bacteria belonging to the family Pseudomonadaceae (Bergey's Manual, 1984). This family i s composed of straight or curved gram negative rods, motile by polar f l a g e l l a , capable of growing at temperatures between 4 and 43 C. The organisms are s t r i c t aerobes ( i . e . possess a respiratory metabolism) and are never fermentative. Organisms belonging to the genus Pseudomonas predominate ln the family with over 265 species described. The Pseudomonas genus i s ubiquitous. It has been associated with the decomposition of a wide 8 v a r i e t y of substrates, r e f l e c t i n g the v e r s a t i l i t y of the genus. Pseudomonas fluorescens belongs to a group of organisms including P. aeruginosa, P. putida, P. chlororaphis, and P. aureofaciens which produce a fluorescent pigment (Bergey's Manual, 1984). Pseudomonas fr a g i on the other hand i s a non-fluorescent Pseudomonas species described as a species in Bergey's Manual (1984). Using competitive DNA-rRNA hybridization studies P a l l e r o n i , et al. (1973), and De Vos and De Ley (1983) divided the genus Pseudomonas into fi v e d i s t i n c t RNA homology groups. Members of the f i r s t RNA group included P. aeruginosa, P. fluorescens, P. putida, P. c i c h o r i i , P. syringae, P. savastanoi, P. phaseolicola, P. mori, P. glycinea, P. tomato, P. viridiflava, P. stutzerl, P. stanier i, P. mendoclna and P. pseudoalcaligenes. Recent DNA-DNA hybridization experiments conducted by Ursing (1986), Kiprianova, et al. (1987) and Kiprianova, et a l . (1988) revealed that P. f r a g i (ATCC 4973) belonged to this f i r s t rRNA group which Included P. fluorescens. EXTRACELLULAR PROTEINASE Ex t r a c e l l u l a r proteinases produced by the genus Pseudomonas a l l appear to be c l o s e l y related. The enzymes generally f a l l into the class of proteinases referred to as neutral metalloproteinases. As such, they are most active at neutral pH values and are inactivated by metal chelating 9 agents, since metalloproteinases contain a divalent metal such as Zn at their active s i t e (Mihalyi, 1978). The enzymes are s p e c i f i c for peptide bonds whose imino group contains a hydrophobic or bulky residue. Moreover, the proteinases are active against large peptides only (>4 residues) and thus do not display esterase a c t i v i t y (Morihara, 1968). The proteinases have molecular weights between 35,000-36,000 daltons. They are most active at temperatures between 30 and 45 C, although many show extensive a c t i v i t y at 4 C. They display considerable thermal s t a b i l i t y . Jackman et a l . (1983) examined proteinases produced by several P. fluorescens s t r a i n s . A l l st r a i n s produced only one proteinase. These proteinases a l l had a molecular weight of 42,000 ± 1,500. The proteinases were inactivated by chelating agents. Rabbit antiserum was prepared from each of the proteinases isolated from the d i f f e r e n t s t r a i n s . I n h i b i t i o n of proteinases by the antisera, and gel p r e c i p i t i n bands of antisera and proteinases, showed that a l l the proteinases shared antigenic determinants. This suggested that the proteinases were similar in structure and amino acid content. Patel et a l . (1983a; 1983b) p u r i f i e d e x t r a c e l l u l a r proteinases from Pseudomonas species isolated from raw milk. A l l i s o l a t e s produced only one major proteinase. The proteinases ranged in molecular weight from 39,000 to 44,000. They were neutral metalloproteinases, being most 10 active at pH 7.2 to 7.4, and inactivated by EDTA. The enzymes were heat r e s i s t a n t , being able to retain p a r t i a l a c t i v i t y after heat treatment of 120 C for 10 min. Malik and Mathur (1984) p u r i f i e d a proteinase from Pseudomonas sp. B-25, an organism Isolated from refrigerated butter samples. The enzyme, with a molecular weight of 41,200, was a neutral metalloproteinase since i t was most active at pH 6-10, and was inactivated by EDTA. It was most active at a temperature of 65 C. The enzyme retained 26% of i t s a c t i v i t y after 10 minutes at 70 C. Stepaniak and Fox (1985) and Yan et a l . (1985) both p u r i f i e d proteinases from Pseudomonas species isolated from raw milk. The enzymes ranged in molecular weight from 44,000 to 46,000, were active at pH 7.0 to 7.5 and were inactivated by EDTA. The proteinase from Pseudomonas AFT 21 (Stepaniak and Fox, 1985) was most active at temperatures between 45 and 47 C, but retained 20% of i t s a c t i v i t y at 4 C. The proteinase examined by Yan et al. (1985) was heat r e s i s t a n t , retaining 50% of i t s a c t i v i t y after 30 min at 63 C. Porzio and Pearson (1975) p u r i f i e d and characterized a proteinase produced by P. f r a g i ATCC 4973. The proteinase had a molecular weight of 40,000 to 50,000 and was most active at pH leve l s of 7.0 to 7.5. The enzyme was a metalloproteinase, since i t was inactivated by EDTA. The enzyme had an i s o e l e c t r i c point of pH 5.2. Thompson et a l . (1985a) also examined the proteinase produced by P. f r a g i . 11 They found the organism to produce a single proteinase with a molecular weight of 48,000 + 1,200. Electron micrographs of P. fragi stained to l o c a l i z e the proteinase revealed high concentrations of the enzyme near the c e l l wall. No enzyme could be detected in the cytoplasm. They hypothesized that the enzyme would also appear in e x t r a c e l l u l a r v e s i c l e s , which the organism may produce on occasion. EXTRACELLULAR VESICLES Introduction Pseudomonads are the predominant psychrotrophic microflora of refrigerated raw milk (Stradhouder, 1975) and meat (Shaw and Latty, 1982). Pseudomonas f r a g i has been isolated from raw milk (Kwan and Skura, 1985) and spoiled meat (Shaw and Latty, 1982). Pseudomonas f r a g i i s commonly involved in the psychrotrophic spoilage of meat and dairy products. During the growth of P. fr a g i ATCC 4973, electron micrographs ( Lee Wing et a l . , 1983; Lee Wing 1984; Thompson et a l . , 1985a) revealed the presence of e x t r a c e l l u l a r v e s i c l e - l i k e structures. These vesic l e s appeared to emanate from the surface of the bacterium by a process of exocytosis and accumulated in the supernatant. Bacteriocins Some pseudomonads, spontaneously or under the influence of inducing agents, produce antimicrobial factors known as bacteriocins (Bradley, 1967). These bacte r i o c i d a l agents, which may emanate from the c e l l surface, are primarily 12 active against bacteria of the same genus and species, although antagonism towards d i f f e r e n t genera can occur. Austin-Prather and Booth (1984) reported an active bacteriocin found in association with e x t r a c e l l u l a r membrane vesi c l e s released from whole Bacteroides uniformis c e l l s . These membrane ve s i c l e s were similar in size and shape to those produced by P. f r a g i ATCC 4973. Greer (1982) studied bacteriophage s e n s i t i v i t y of several meatborne pseudomonads and found that phage extended the lag phase of some bacteria under psychrotrophic conditions, r e s u l t i n g in the selection of phage resis t a n t mutants. Patel and Jackman (1986) showed that pseudomonads Isolated from milk were susceptible to bacteriophages active against meat derived pseudomonads. It is possible that P. fr a g i may produce membrane ve s i c l e bound bacteriocins which could have ef f e c t s s i m i l a r to that of bacteriophage in l i m i t i n g competition between bacteria of d i f f e r i n g s t r a i n s , species, or genera. Bacteriocins are a group of e n t i t i e s produced by some "host" bacteria, under some conditions, which may be l e t h a l to other "susceptible" bacteria of a d i f f e r e n t s t r a i n , species, genus or order. They are not c l i n i c a l l y important in destroying pathogenic bacteria. Bacteriocins, however, may a f f e c t the pathogenicity of some bacteria, and have been studied In t h i s context. Of p a r t i c u l a r interest to t h i s study, are the ecological ramifications of bacteriocins as they a f f e c t 13 competition between d i f f e r i n g s t r a i n s , species, or genera of bacteria contained in a dynamic mixed culture such as in s p o i l i n g foods, p a r t i c u l a r l y f l u i d milk and muscle tissues. Both commodities, when stored under r e f r i g e r a t i o n conditions, soon develop a microflora composed of a small number of related organisms, mainly of the pseudomonad group. These organisms est a b l i s h themselves at the expense of other organisms. The mechanisms of t h i s competition is poorly understood and may involve antagonistic chemical compounds such as bacteriocins. H i s t o r i c a l perspectives The study of bacteriocin has been for the most part centred upon c o l i c i n , the a n t i b i o t i c agent produced by Escherichia c o l i . Several reasons for t h i s r e s t r i c t i o n applY/ such as the concomitant detailed study of th i s organism; but the main reason stems from the i n i t i a l work of Gratia (1925). A s t r a i n of E. coli V (named V for vir u l e n t ) produced a substance bacteriocidal towards E. coli <t>. A f i l t e r s t e r i l i z e d culture supernatant of the v i r u l e n t E. coli s t r a i n proved only l e t h a l towards susceptible strains of E. c o l i . It d i f f e r e d from bacteriophage in that the p r i n c i p a l agent did not reproduce i t s e l f in a fashion si m i l a r to bacteriophage. The antagonistic agent was stable af t e r 1 h at 100 C and % h at 120 C and was active after exposure to chloroform vapors. Jacob et a l . (1953), in an attempt to define the term bacteriocin, suggested that these substances should be 14 protein in nature, produced by l e t h a l biosynthesis, adsorbed onto s p e c i f i c receptor s i t e s and active against organisms within the same species. Considerable complications have since confused t h i s d e f i n i t i o n since many such substances; " c l a s s i c a l " a n t i b i o t i c s , metabolic substances, enzymes and defective bacteriophage, have at one point or another been ca l l e d bacteriocins. Hamon and Peron (1963) found many bacteriocins of gram positive bacteria did not f i t the c l a s s i c a l d e f i n i t i o n . In p a r t i c u l a r , they found a much wider spectrum of a c t i v i t y , not r e s t r i c t e d to one species. The precise meaning of the term bacteriocin then, seems elusive. In general terms, the presence of a b i o l o g i c a l l y active p r o t e i n - l i k e substance possessing a bacte r i o c i d a l property can be considered b a c t e r i o c i n - l i k e . Detection of bacteriocins For screening,>the general test for antagonism involves growth on s o l i d medium. Antagonism i s displayed by a reduction or elimination in the growth of an indicator (passive) s t r a i n by the test (active) host s t r a i n . There are two general methods used to detect antagonism: the d i r e c t and the deferred procedure. The host and indicator are placed on the agar plate together. For example, the host may be stabbed into an agar plate seeded with the indicator organism. Both grow at the same time, and the i n h i b i t o r diffuses through the agar, i n h i b i t i n g the indicator organism (Gratia, 1946). 15 In the deferred procedure, developed by Fredericq (1948), the host organism is grown on an agar plate for some period of time, following which the c e l l s are k i l l e d with chloroform vapors. A second layer of sloppy agar medium (growth medium containing ca. 1.0% agar) seeded with the indicator organism i s poured over the f i r s t agar layer. Zones of i n h i b i t i o n , observed around the dead host colonies, indicate bacteriocin a c t i v i t y . The deferred procedure has been shown to be more sensit i v e at detecting antagonistic behavior than the d i r e c t method (Tagg et a l . , 1976). With the deferred procedure, incubation time and conditions of the host or indicator organism can be independently varied, thus accommodating differences in growth rate. Unrelated antagonists When investigating the occurrence of a bacteriocin, i t is important to eliminate other substances unrelated to but similar in action to bacteriocin. In many cases, the discovery of bacteriocins from s p e c i f i c species was the by-product of investigations into the lysogeny of the organism due to bacteriophage. Bacteriophage are v i r a l p a r t i c l e s which may infect s p e c i f i c b a c t e r i a l c e l l s , causing l y s i s . Unlike bacteriophage, bacteriocins cannot be propagated from one culture to another, due to the lack of necessary genetic material. Since bacteriocins cannot reproduce through b a c t e r i a l l y s i s , bacteriocins may be di l u t e d to the point where no zone of clearing occurs. 16 Bacteria can produce a variety of low-molecular weight i n h i b i t o r s and b a c t e r i o l y t i c enzymes which may possess b a c t e r i o c i n - l i k e properties. An example of the former i s the c l a s s i c a l a n t i b i o t i c n i s i n produced e x t r a c e l l u l a r l y by stra i n s of Streptococcus l a c t i s (Tagg et a l . , 1976). Lysostaphin from Staphylococcus (Schendler and Schuhardt, 1965), possessing a n t i b i o t i c - l i k e a c t i v i t y , i s an example of the l a t t e r . C l a s s i c a l methods of bacteriocin detection, whether d i r e c t or i n d i r e c t , can only detect antagonistic substances produced by bacteria. Other methods must be used to determine the class of substances produced. Factors a f f e c t i n g production of bacteriocins in general terms, i f one looks long enough at any given species, one may find a b a c t e r i o c i n - l i k e substance produced by the host organism antagonistic towards some indicator organism (Tagg et a l . , 1976). The d i f f i c u l t y l i e s in finding the indicator. No special growth media or c u l t u r a l conditions may be needed to produce bacteriocins, other than optimal growth conditions for the host (Bradley, 1967). Maximum bacteriocin production, however, does depend upon some important factors. Kelstrup and Gibbons (1969) cu l t i v a t e d Streptococcus sp. of human and rodent oral cavity o r i g i n in trypticase medium (2%) plus 0.2% glucose. Supplementation of th i s growth medium with either agar (0.005% to 0.1%) or starch (0.05% to 1.0%) increased bacteriocin production. S i m i l a r l y , Jetten et a l . (1972) found that S. epidermidis produced ca. 20 times more 17 b a c t e r i o c i n a c t i v i t y on a s e m i s o l i d medium, t h a n i n l i q u i d c u l t u r e o f t h e same volum e . V i d a v e r e t al. (1972) f o u n d t h a t P. phaseolicola and P. s y r i n g a e p r o d u c e d b a c t e r i o c i n o n l y on s o l i d medium. Growth phase d o e s seem t o a f f e c t b a c t e r i o c i n p r o d u c t i o n . S t r e p t o c o c c u s s p . p r o d u c e d maximum s t r e p t o c o c c i n d u r i n g t h e e x p o n e n t i a l p h a s e , t h e n d e c l i n e d s h a r p l y as t h e c u l t u r e e n t e r e d s t a t i o n a r y p h a s e . In c o n t r a s t , S t r e p t o c o c c u s A - F F 2 2 s t a r t e d b a c t e r i o c i n p r o d u c t i o n i n t h e l a t e l o g a r i t h m i c p hase and d e c r e a s e d s l o w l y as t h e c u l t u r e a g ed (Tagg e t a l . 1 9 7 6 ) . V i d a v e r e t al. (1972) f o u n d t h a t i n c r e a s i n g i n c u b a t i o n t i m e w e l l p a s t t h e e x p o n e n t i a l phase (up t o 72 h) r e s u l t e d i n an i n c r e a s e i n b a c t e r i o c i n p r o d u c t i o n f r o m Pseudomonas s p . B a c t e r i o c i n i n d u c t i o n B a c t e r i a may or may n o t p r o d u c e b a c t e r i o c i n s p o n t a n e o u s l y , d e p e n d i n g upon t h e s p e c i e s . I f t h e b a c t e r i o c i n must be i n d u c e d , i t s p r o d u c t i o n u s u a l l y i s a s s o c i a t e d w i t h c e l l l y s i s ( B r a d l e y , 1 9 6 7 ) . B o t h p h y s i c a l i n d u c i n g a g e n t s s u c h as u l t r a v i o l e t (UV) r a d i a t i o n and c h e m i c a l i n d u c i n g a g e n t s s u c h as m i t o m y c i n C t e n d t o be m e t a b o l i c d i s r u p t e r s . Hamon e t a l . (1961) I n d u c e d s e v e r a l b a c t e r i o c i n s f r o m Pseudomonas s p . u s i n g u l t r a v i o l e t r a d i a t i o n a t 254 nm. S i m i l a r l y , C r o w l e y and DeBoer (1980) i n d u c e d b a c t e r i o c i n i n Erwinia s p . w i t h r a d i a t i o n o f t h e same w a v e l e n g t h . 18 M i t o m y c i n C was u t i l i z e d as a c h e m i c a l i n d u c i n g a g e n t by T a k e y a e t a l . (1969) and Haag and V i d a v e r (1974) t o p r o d u c e b a c t e r i o c i n f r o m Pseudomonas s p . The m i t o m y c i n C c o n c e n t r a t i o n v a r i e d f r o m 0.2 t o 2.0 Hg/mL i n t h e s e s t u d i e s . P h y s i c a l p r o p e r t i e s o f b a c t e r i o c i n s B a c t e r i o c i n - 1 i k e s u b s t a n c e s p r o d u c e d by b a c t e r i a have b e e n c l a s s i f i e d b y B r a d l e y (1967) i n t o two g r o u p s , d e s i g n a t e d as low ( c a . 10,000 t o 100,000 d) or h i g h (1.0 x 10 6 t o >1.0 x 10 7 d) m o l e c u l a r w e i g h t . The h i g h m o l e c u l a r w e i g h t s u b s t a n c e s i n c l u d e p a r t i c l e s r e s e m b l i n g b a c t e r i o p h a g e t a i l s , or o t h e r w i s e d e f e c t i v e b a c t e r i o p h a g e . These p a r t i c l e s f r e q u e n t l y a r e i n d u c i b l e upon e x p o s u r e t o U.V. r a d i a t i o n or m i t o m y c i n C, and a r e o f t e n a s s o c i a t e d w i t h c e l l l y s i s . K e l s t r u p and G i b b o n s (1969) d e s c r i b e d a low m o l e c u l a r w e i g h t b a c t e r i o c i n p r o d u c e d by S t r e p t o c o c c u s s p . I t was d l a l i z a b l e , s u g g e s t i n g a m o l e c u l a r w e i g h t o f c a . 12,000. J e t t e n e t a l . (1972) d e s c r i b e d t h e p r o d u c t i o n o f a b a c t e r i o c i n p r o d u c e d by Staphylococcus e p i d e r m i d i s w i t h a m o l e c u l a r w e i g h t o f 150,000 t o 400,000, composed o f 20,000 d s u b u n i t s . I n b o t h o f t h e s e c a s e s , n e i t h e r U.V. r a d i a t i o n n o r m i t o m y c i n C i n c r e a s e d b a c t e r i o c i n p r o d u c t i o n . The l i t e r a t u r e c o n t a i n s many examples o f b a c t e r i o c i n -l i k e p a r t i c l e s p r o d u c e d by b a c t e r i a . T h e s e p a r t i c l e s may or may n o t be c l o s e l y a s s o c i a t e d w i t h t h e c e l l o u t e r l a y e r s . A u s t i n - P r a t h e r and B o o t h (1984) f o u n d a b a c t e r i o c i n o f B. u n i f o r m i s t o be a s s o c i a t e d w i t h membrane v e s i c l e s a b o u t 100 19 nm in diameter, which were released by bleb formation from the outer surface of the c e l l s . Hamon et a l . (1961) induced b a c t e r i o c i n - l i k e p a r t i c l e s with U.V. radiation from several Pseudomonas sp. including " f l u o c i n " from a P. fluorescens s t r a i n . Takeya et a l . (1969) described a rod shaped b a c t e r i o c i n - l i k e p a r t i c l e induced with mitomycin C from P. aeruginosa P28. This bacteriocin, l a b e l l e d "pyocln" 28, varied in size from 9.0 to ca 400 nm in size when viewed under the electron microscope. Similar studies of such p a r t i c l e s (Crowley and DeBoer, 1980; Haag and Vidaver, 1974) also indicated the need for either U.V. or mitomycin C induction. Chemical composition Various bacteriocins d i f f e r s i g n i f i c a n t l y in their chemical compositions, r e f l e c t i n g t h e i r diverse physical properties. Generally speaking, the low molecular weight bacteriocins are composed primarily of protein, consequently they are susceptible to attack by trypsin or other similar proteinases. The high molecular weight bacteriocins, r e f l e c t i n g a more diverse composition, are r e l a t i v e l y immune to trypsin attack but are e a s i l y inactivated by high temperatures. Both bacteriocin types may or may not contain carbohydrate or l i p i d , but a l l must contain protein. Kelstrup and Gibbons (1969) described an example of a low molecular weight bacteriocin produced from Streptococcus sp. with a molecular weight less than 12,000. Four proteinases, including trypsin, t o t a l l y inactivated the 20 bacteriocin. The substance was, however, stable after heating at 80 C for 45 min. Gagliano and H i n s d i l l (1970) and Dandeu (1971) found staphylococcin and c o l i c i n respectively to be chemically similar to the c e l l wall of the originating bacterium. In p a r t i c u l a r , the l a t t e r study found the active protein component to be integral with the lipopolysaccharide on the surface of the producer c e l l . In contrast to low molecular weight bacteriocin, Takeya et a l . (1969) found high molecular weight rod shaped pyocin from P. aeruginosa to be resist a n t to treatment by trypsin and nagarase (a b a c t e r i a l proteinase). It was, however, inactivated by exposure for 10 minutes at 60 C. Haag and Vidaver (1974) found similar results from a study of syringacln from P. syringae. Mode of action of bacteriocins Most of the o r i g i n a l information regarding the mode of action of bacteriocins upon bacteria i s based on studies of c o l i c i n s . Research has tended to focus on two aspects of the interaction between bacteriocin and the susceptible bacteria. These are the kinetics of the physical interaction between the two, and the development of some f a t a l disruption in the biochemical process in an affected c e l l . The widely accepted hypothesis regarding the mode of action of bacteriocins i s that i t is a two step process. In the f i r s t step adsorption of the bacteriocin to the surface of the c e l l i s f a c i l i t a t e d by receptors on the surface of the c e l l . This f i r s t stage i s thought to be a reversible 21 step. No damage i s done to the c e l l since removal or destruction of the bacteriocin at t h i s stage, with trypsin digestion, saved the c e l l from destruction and l e f t no permanent damage. At a d e f i n i t e time period, following the f i r s t step, permanent damage begins to become evident through i r r e v e r s i b l e biochemical changes. The adsorption of bacteriocin can be demonstrated by a drop in bacteriocin t i t r e in a suspension with excess susceptible bacteria. Elution of bacteriocin from treated c e l l s after elimination of free bacteriocin proved that the bacteriocin was, in fact, removed from solution or suspension and not enzymatically inactivated by the b a c t e r i a l c e l l s (Tagg et a l . , 1976). It i s thought that the receptor s i t e s on the b a c t e r i a l c e l l surface s p e c i f i c for bacteriocins are the same s i t e s to which bacteriophage also attach. This r e l a t i o n s h i p between the two binding s i t e s was strengthened when i t was found that r e s i s t a n t mutants (to c o l i c i n ) were resist a n t to both bacteriocin and bacteriophage attack. Sabet and Schnaltman (1973) isolated c o l i c i n E3-CA38 receptor from the c e l l surface of E. coll by treating the c e l l wall with Triton X-100 in the presence of ethylenediaminetetracetic acid (EDTA). It was found to be a glycoprotein with a molecular weight of 60,000. It is thought to be situated in the outer membrane. The p u r i f i e d receptor inactivated c o l i c i n . S i g n i f i c a n t binding of c o l i c i n to the isolated receptors was shown to prevent bacteriophage binding. The binding of 22 c o l i c i n and b a c t e r i o p h a g e s t o common r e c e p t o r s s u g g e s t s t h a t b o t h have s i m i l a r a n c e s t r y . B a c t e r i o c i n induced c e l l damage The p h y s i o l o g i c a l s t a t e of t h e i n d i c a t o r c e l l seems t o be important i n b a c t e r i o c i n a c t i v i t y . A c t i v e l y growing c e l l s a r e most s u s c e p t i b l e t o c e l l damage. K i n e t i c s t u d i e s have shown t h a t t h e l e t h a l a c t i o n i s of a s i n g l e h i t . That i s , one molecule w i l l , with a c e r t a i n p r o b a b i l i t y , k i l l a b a c t e r i a l c e l l . However, due t o t h e p r e s e n c e of more than one a c t i v e binding s i t e , more t h a n one b a c t e r i o c i n molecule i s l i k e l y t o bind and k i l l one b a c t e r i a l c e l l . S p e c i f i c t a r g e t s of bioc h e m i c a l a t t a c k c e n t r e around e n e r g y p r o d u c t i o n , macromolecule p r o d u c t i o n and membrane t r a n s p o r t and p e r m e a b i l i t y . C o l i c i n s A, B, E l , D and S a r e s i m i l a r i n t h e i r a c t i o n s i n c e e n e r g y dependent s y n t h e s e s and t r a n s p o r t p r o c e s s e s a r e i n h i b i t e d . S e v e r a l e n e r g y dependent t r a n s p o r t p r o c e s s e s , s u c h as t h e t r a n s p o r t of amino a c i d s , a r e i n h i b i t e d , l e a d i n g t o l o s s of p r o t e i n s y n t h e s i s . The membranes a r e a l t e r e d allowing r e l e a s e of K + and t h e f r e e r movement of Mg 2 + and C a 2 + t h r o u g h t h e membrane. Since t h e c e l l a t t e m p t s t o maintain a p r o p e r K + c o n c e n t r a t i o n a c r o s s t h e membrane t h r o u g h a c t i v e t r a n s p o r t , ATP l e v e l s drop. OPTIMIZATION I n t r o d u c t i o n O p t i m i z a t i o n t e c h n i q u e s c a n be u s e d t o improve t h e e f f i c i e n c y o f most p r o c e s s e s when more t h a n one f a c t o r i s i n v o l v e d . I n t h e c a s e o f c h e m i c a l p r o c e s s e s , o p t i m i z a t i o n 23 may be required to improve the e f f i c i e n c y of an i n d u s t r i a l or a n a l y t i c a l method. Major improvements in the productivity of microbiological fermentation may also be achieved by c o n t r o l l i n g incubation conditions or culture medium compositions. The objectives of any optimization strategy are two-f o l d : (1) to improve the o v e r a l l e f f i c i e n c y of the process under study (frequently the process under study is referred to as the objective function); (2) to a t t a i n t h i s gain in e f f i c i e n c y in as few experiments (or in the least time) as possible. Few "wasted" experiments (that i s experiments which do not contribute to improvement of the objective function) should have to be evaluated (Hendrix, 1980). The measure of the objective function can vary widely, depending upon the nature of the function. It may be a measure of some chemical, the result of a chemical process. In microbiological systems it may be a measure of enzymatic activity, or of some chemical metabolite. The factors involved in influencing the objective function can also vary greatly. In microbiological systems, the factors tend to be cultural conditions such as pH, incubation temperature, or oxygen level or culture medium composition differences (Greasham and Inamine, 1986). As such, certain constraints may have to be placed upon the factors. For example, pH values have to be constrained for microorganisms growing in a narrow pH range. As a result, the optimization technique used must be able to work satisfactorily within the boundaries set up by these constraints. Several 24 optimization techniques have been used which can, to varying extents, satisfy the above criterion. Two such techniques include linear programming and direct search methods. Linear programming Linear programming techniques can and have been used to optimize process control or product formulation (Harper and Wanninger, 1970). This technique e n t a i l s solving a series of simultaneous equations, usually through zeroing of row functions. Linear programming techniques can handle boundaries well, since the technique depends upon constrained factors. Unfortunately, linear programming requires that each factor be represented by a linear equation. This s i t u a t i o n is ra r e l y , i f ever, encountered in a chemical or microbiological system. Linear programming may therefore be of limited use. Direct search Linear programming techniques used to optimize processes r e l y upon solving an ov e r a l l v a r i a t i o n in a defined series of linear equations. When the behavior of each factor as applied to the objective function Is unknown ( i . e . when the linear functions are undefined), then a more d i r e c t approach must be taken. Direct search techniques (Saguy, 1983) r e l y upon the evaluation of the objective function at a sequence of t r i a l points, points made up by a v a r i a t i o n of factor levels within the constraint boundaries. Progress towards the optimum point i s assured i f the search i s forced to move to the region of optimum response. Simply 25 s t a t e d , i f t h e s e t o f f a c t o r v a l u e s r e s u l t s l n an improvement o f t h e o b j e c t i v e f u n c t i o n , t h e n t h e t e c h n i q u e w i l l move i n t h a t " f a v o r e d " d i r e c t i o n . The t e r m f a v o r e d d i r e c t i o n , a s u s e d h e r e , r e f e r s t o an improvement i n t h e d e s i r a b i l i t y o f t h e o b j e c t i v e . As s u c h , o p t i m i z a t i o n t e c h n i q u e s may be us e d t o maximize or m i n i m i z e t h e o b j e c t i v e f u n c t i o n . T h i s t e c h n i q u e o f d i r e c t s e a r c h and e v o l u t i o n a r y o p e r a t i o n s (EVOP) was f i r s t f o r m u l a t e d by Box (1957). C e n t r a l t o t h e c o n c e p t was e v a l u a t i o n o f t r i a l s , w i t h an improvement o f t h e o b j e c t i v e f u n c t i o n i n mind. E v o l u t i o n a r y o p e r a t i o n t e c h n i q u e s Response s u r f a c e m e t h o d o l o g y The i n i t i a l d e s i g n o f Box (1957) was c e n t r e d a r o u n d an e s t a b l i s h e d f u l l - s c a l e p r o c e s s . T h a t i s , t h e e x p e r i m e n t a l l e v e l s o f e a c h f a c t o r were e q u a l l y s p a c e d and t h e e x p e r i m e n t s e v a l u a t e d . When c o m p l e t e , t h e d a t a were u s e d t o c o n s t r u c t , by m u l t i l i n e a r r e g r e s s i o n a n a l y s i s , a r e s p o n s e s u r f a c e . F u l l s c a l e f a c t o r i a l d e s i g n s a r e i m p r a c t i c a l f o r l a r g e numbers o f f a c t o r s (Greasham and Inamine, 1986). As a r e s u l t , Box and Behnken (1960) f o r m u l a t e d a s y s t e m o f t h r e e l e v e l d e s i g n s f o r r e d u c i n g t h e t o t a l numbers o f t r i a l s r e q u i r e d . T h i s c o m b i n a t i o n o f f r a c t i o n a l f a c t o r i a l w i t h m u l t i l i n e a r r e g r e s s i o n a n a l y s i s i s commonly r e f e r r e d t o as r e s p o n s e s u r f a c e m e t h o d o l o g y ( M c D a n i e l e t a l . , 1976). N a k a l (1982) f o u n d , however, t h a t r e s p o n s e s u r f a c e m e t h o d o l o g y became i n a c c u r a t e when c o n s t r a i n t b o u n d a r i e s d i d n o t c o v e r 26 the factor levels of the true function, or i f i n i t i a l boundaries were set too narrow. Simplex methods Shortly after Box (1957) formulated the concept of EVOP, Spendley et a l . (1962) suggested a system of EVOP which was t r u l y evolutionary. Rather than formulate a set number of t r i a l s , these researchers proposed an i t e r a t i v e procedure. The t r i a l s were formulated from a simplex, a geometric figure containing one more than the number of factors involved in the optimization. The simplex was derived from a Spendley matrix. By forcing the search to move to the region of optimum response, the simplex figure would work towards the optimum objective function value. This was achieved by a series of r e f l e c t i o n s , where the worst function point was abandoned and that point r e f l e c t e d towards a favored d i r e c t i o n . MoKgan-Pewting s i m p l e ^ F l e x i b l e as the simplex technique appears, several problems remain. The simplex figure moved in a favorable d i r e c t i o n , at a constant rate, regardless of the steepness of the ascent. Morgan and Deming (1974) proposed that expansion and contraction factors be used to accelerate movement in a more favorable d i r e c t i o n , while moving away from a less desirable d i r e c t i o n . This procedure tended to speed up the optimization rate. To further improve the e f f i c i e n c y of the simplex technique, Routh et a l . (1977) introduced a quadratic curve f i t t i n g routine into the 27 / s i m p l e x d e s i g n . The q u a d r a t i c c u r v e f i t t e d p o i n t r e p l a c e d t h e w o r s t p o i n t o f t h e p r e v i o u s t r i a l . T h i s p r o c e d u r e t e n d e d t o f u r t h e r improve t h e s i m p l e x t e c h n i q u e . However, b o t h t h e m o d i f i e d s i m p l e x o f Morgan and Deming (1974) and t h e s u p e r m o d i f i e d s i m p l e x t e c h n i q u e o f R o u t h e t a l . (1977) o c c a s i o n a l l y s t a l l e d n e a r t o or a t t h e c o n s t r a i n t b o u n d a r y . M o d i f i e d s u p e r s i m p l e x In an a t t e m p t t o improve t h e b e h a v i o r o f t h e s i m p l e x t e c h n i q u e a r o u n d c o n s t r a i n t b o u n d a r i e s , N a k a i (1982) p r o p o s e d t h e m o d i f i e d s u p e r s i m p l e x . T h i s t e c h n i q u e was a b a s i c s u p e r m o d i f i e d s i m p l e x ( R o u t h e t a l . , 1977) w i t h t h e a d d i t i o n o f a " q u a d r a t i c f a c t o r i a l r e g r e s s i o n a n a l y s i s " s t e p . The new p r o c e d u r e was shown t o be more e f f i c i e n t , t h a n t h e two above m e n t i o n e d t e c h n i q u e s , when t h e q u a d r a t i c f a c t o r i a l r e g r e s s i o n e q u a t i o n f i t t e d t h e e x p e r i m e n t a l r e s p o n s e s u r f a c e . When t h e e q u a t i o n d i d n o t f i t t h e r e s p o n s e s u r f a c e , however, no i n c r e a s e d e f f i c i e n c y o c c u r r e d . To o b t a i n a f u r t h e r improvement i n e f f i c i e n c y , u nder a l l s i t u a t i o n s , N a k a i e t a l . (1984) i n t r o d u c e d a mapping p r o c e d u r e t o t h e s u p e r m o d i f i e d s i m p l e x ( R o u t h e t a l . , 1977). T h i s t e c h n i q u e shows, i n a two d i m e n s i o n a l p l o t , t h e r e l a t i o n s h i p s one s e p a r a t e f a c t o r has w i t h a l l t h e o t h e r f a c t o r s i n v o l v e d i n t h e o b j e c t i v e f u n c t i o n . T hese r e s e a r c h e r s f o u n d a s i g n i f i c a n t improvement i n e f f i c i e n c y o v e r t h e m o d i f i e d s u p e r s i m p l e x . I n a l l o p t i m i z a t i o n t e c h n i q u e s , a d a n g e r e x i s t s t h a t t h e i d e n t i f i e d optimum p o i n t i s i n f a c t a l o c a l optimum and 28 that the global maximum has been missed. A solution to t h i s problem, developed by Nakai et a l . (1984), was to s h i f t simultaneously a l l the factor l e v e l s away from the i d e n t i f i e d optimum point, towards new target values. S h i f t i n g i s continued u n t i l a worse response value i s obtained. Centroid search Simplex techniques are e f f i c i e n t at finding optimum points because they are evolutionary operations. As such, new t r i a l s are suggested based upon the results of the previous t r i a l . By a succession of such t r i a l s , at smaller and smaller ranges, the optimum point i s eventually found. This technique i s e f f i c i e n t when individual experiments can be conducted quickly. In the case of lengthy experiments, such as in microbial fermentation where incubation times can take several days, the i t e r a t i v e nature of the simplex technique can be very time consuming. In an attempt to speed up the optimization, Aishima and Nakai (1986) proposed centroid search, an innovation of the c l a s s i c simplex procedure. With the centroid search technique, a series of t r i a l s can be evaluated simultaneously. In t h i s way, the time required for optimization may be s u b s t a n t i a l l y reduced. The centroid search technique includes evaluation of Spendley matrix, centroid and simultaneous search experiments. 29 M A T E R I A L S & M E T H O D S BACTERIOCIN Organisms The host Pseudomonas fragi ATCC 4973 was purchased from the American Type Culture C o l l e c t i o n (Rockville, M D ) . A l l subsequent studies used th i s bacterium. The indicators Streptococcus f a e c a l l s ATCC 27286 and 12984; P. fluorescens ATCC 948; P. aureofaciens ATCC 13985; P. fluorescens biotype "A" A T C C 17397; P. acidovorans ATCC 15668; P. lemmonieri ATCC 12983; P. pseudoalcaligenes ATCC 17440; P. putida ATCC 12633; P. s t u t z e r i ATCC 17588; P. mendocina ATCC 25411; and E. c o l i ATCC 25922 were purchased from the American Type Culture C o l l e c t i o n . The following organisms, isolated by Kwan and Skura (1985) from raw milk and c l a s s i f i e d by numerical taxonomy, were also used as indicators: s i x d i f f e r e n t strains of P. f r a g i 4-1, 6-3, 8-4, 10-0, 10-7, A14; and six d i f f e r e n t strains of P. fluorescens 0-0, 6-0, 10-0, 10-6, B16, C10. Storage A l l organisms were suspended In trypticase soy broth (TSB) (BBL, Baltimore, M D ) and stored frozen in l i q u i d N 2. Cultures were thawed and incubated in TSB at 21 C for 18 h, and streaked onto trypticase soy agar (TSA, BBL) slants. 30 These stock cultures were stored at 4 C and replaced at 4 week Intervals. Growth of organism  The media A l l cultures were grown in TSB. Bacteriocin a c t i v i t y was determined on TSA plates. Washed c e l l suspension A 24 h broth culture (TSB, 28 C) was centrifuged (10,400 x g, 10 min, 4 C) and the supernatant discarded. An equal volume of 0.1% peptone water was used to resuspend the p e l l e t and the procedure repeated. The f i n a l suspension in 0.1% peptone contained 1.0 x 10 8 cfu/mL. Detection plates Plates for stab inoculation were prepared from 15 mL molten and cooled (45 C) TSA poured into a p e t r i plate on a l e v e l surface and allowed to s o l i d i f y (ca. 10 min), with the tops o f f , in a laminar flow hood. Lawns of P. f r a g i were also prepared by mixing 0.2 mL washed c e l l suspension with 15 mL of TSA (45 C) prior to pouring. Detection of bacterlocinogenlcity  Solid culture The procedure of Crowley and DeBoer (1980) was used to detect antagonistic behavior. The host, P. f r a g i ATCC 4973, was stab inoculated onto TSA plates and incubated (28 C, 48 h). The incubated plates were inverted over chloroform soaked f i l t e r paper for 20 min, the colony material scraped o f f , and the plates treated an additional 20 min. Residual 31 chloroform vapors were allowed to dissipate by exposure to s t e r i l e a i r in a laminar flow hood (Model BM6-2A, Canadian Cabinets Inc., Nepean, ON) for 15 min. Indicator organisms were prepared by mixing 0.1 mL aliquots of washed c e l l suspension in 5.0 mL of 0.6% agar containing 1% peptone at 45 C. The resultant suspension was mixed and poured over the previously prepared host test plates and incubated (28 C, 24 h). Induction Radiation induction Shake broth cultures of P. f r a g i , grown to the mid-logarithmic growth phase (28 C, 18 h), were poured into s t e r i l e glass pans (13 x 21 cm) to a depth of 2.0 mm. The pan was placed 15 cm below an u l t r a v i o l e t l i g h t source (254 nm) with a dose rate, calculated by potassium f e r r i o x a l a t e actinometry (Jagger, 1967), of 35.57 u.J/cm2/s. Total dose was 2000 ergs/mm2. Alternately, a mid-logarithmic broth culture was irr a d i a t e d with 30, 90 and 160 Gy of gamma radiation in a Gammacell 220 i r r a d i a t o r (Atomic Energy of Canada, Kanata, ON). Both' ionizing and non-ionizing dose levels r e f l e c t e d those used by Lwoff et a l . (1950) to induce bacteriophage in Bacillus megaterium. Subsequent to i r r a d i a t i o n , the cultures were incubated a further 4 h at 28 C. After incubation, the b a c t e r i a l c e l l s were removed by centrifugation (10,400 x g, 10 min, 4 C) and the supernatant f i l t e r - s t e r i l i z e d by passage through a 0.45um ce l l u l o s e 32 acetate membrane f i l t e r ( Millipore Corp., Malton, ON). Supernatant was l e f t "as i s " , or was concentrated (x 20) in an u l t r a f i l t r a t i o n c e l l with a 10,000 MW cut-off membrane (Amicon, Danvers, MA). The test for antagonistic a c t i v i t y involved spotting 0.02 mL drops of "as i s " or concentrated supernatant onto the surface of TSA plates containing indicator organisms. A positive test would be revealed by a zone of clearing in the ba c t e r i a l lawn where the supernatants were applied. Alternately, indicator organisms were grown in 10.0 mL TSB for 18 h followed by the addition of 2.0 mL "as i s " supernatant. The absorbance, at 600 nm, of the cultures was followed over a further 24 h period using a spectrophotometer (Shimadzu S c i e n t i f i c Instr. Inc., Columbia, MD, model UV-160). Mitomycin C A mid-logarithmic broth culture of P. fr a g i (450 mL) was centrifuged (10,400 x g, 10 min, 4 C) and the p e l l e t resuspended in TSB. After incubation (21 C, 4 h), the chemical inducer mitomycin C (Sigma Chemical Co., St. Louis, MO) was added at a concentration of 1.0 to 20.0 )ig/mL and the c e l l suspension incubated a further 6 h at 21 C. Subsequent to the second incubation period, the culture was again centrifuged, the supernatant co l l e c t e d and f i l t e r -s t e r i l i z e d . Some supernatant was used "as i s " and some was dialyzed against d i s t i l l e d water. Twenty mL of retentate was c o l l e c t e d . The retentate and "as i s " supernatant were 33 s p o t t e d on i n d i c a t o r p l a t e s t o t e s t f o r a n t a g o n i s t i c a c t i v i t y . The a b s o r b a n c e o f i n d i c a t o r b r o t h c u l t u r e s t r e a t e d w i t h b o t h s u p e r n a t a n t s , as d e s c r i b e d e a r l i e r , was d e t e r m i n e d . S p i r a l p l a t e d e t e c t i o n In a d d i t i o n t o s p o t p l a t i n g , t h e s p i r a l p l a t i n g t e c h n i q u e was us e d t o d e t e c t p o s s i b l e a n t a g o n i s m f r o m P. fragi i n d u c e d by i o n i z i n g r a d i a t i o n and m i t o m y c i n C. The t e c h n i q u e i n v o l v e d t h e c o n c o m i t a n t use o f t h e u n i f o r m and v a r i a b l e d e p o s i t i o n modes o f a model DU s p i r a l p l a t e r ( S p i r a l Systems I n c . , C i n c i n n a t i , OH). I n i t i a l l y , a d i l u t e d washed c e l l s u s p e n s i o n o f i n d i c a t o r o r g a n i s m s (1.0 x 10 5 cfu/mL) was d e p o s i t e d , u s i n g t h e u n i f o r m cam, o n t o a TSA p l a t e ( i n o c u l u m d e n s i t y 1.3 cfu/mm 2 o f t r a c k ) and t h e l i q u i d a l l o w e d t o d i f f u s e i n t o t h e a g a r . R a d i a t i o n or m i t o m y c i n C i n d u c e d P. f r a g i c u l t u r e s u p e r n a t a n t s , i n b o t h r e g u l a r or c o n c e n t r a t e d f o r m s , were t h e n d e p o s i t e d , u s i n g t h e v a r i a b l e cam. The s u p e r n a t a n t s were d e p o s i t e d d i r e c t l y on t o p o f t h e b a c t e r i a , f o l l o w i n g t h e same t r a c k . A t o t a l o f 0.05 mL o f s u p e r n a t a n t was d e p o s i t e d p e r p l a t e . R e g u l a r or c o n c e n t r a t e d TSB was d e p o s i t e d on s e p a r a t e i n d i c a t o r p l a t e s w h i c h were u s e d as c o n t r o l s . GROWTH OF P. f r a g i ON SOLID AND LIQUID MEDIUM i n o c u l u m g r o w t h Pseudomonas f r a g i was i n c u b a t e d , w i t h a g i t a t i o n , a t 21 C f o r 24 h i n TSB. 34 Incubation on s o l i d medium A 0.01 mL aliquot of inoculum containing ca. 4.8 x 10^ cfu was mixed with 100 mL 0.1% peptone water and the suspension drawn through a 47 mm polycarbonate, membrane f i l t e r (0.45u.m, Nucleopore Corp., Pleasanton, CA). The membrane, together with the coll e c t e d c e l l s , was placed onto the surface of 15 mL TSB + agar, contained in a 50 mm p e t r i plate, and incubated at 21 C at 4 h time intervals up to 60 h. These experiments were repeated three times. Incubation in l i q u i d medium Separate 250 mL flasks containing 150 mL TSB were inoculated with ca. 1.5 x 10 6 cfu washed c e l l s and incubated, with agita t i o n (150 RPM), at 21 C up to 95 h. These experiments were also repeated three times. Enumeration of c e l l s After incubation, the membrane f i l t e r was removed from the agar surface and the P. frag i c e l l s on the membrane f i l t e r were dispersed into 100 mL of 0.1% peptone water with a Stomacher 400 (Cooke Laboratory Products, Alexander, VA). Mixing time was 2 min. Surface pla t i n g on TSA using the s p i r a l plating technique (Spiral Systems Inc) was used for enumeration of P. fr a g i grown on s o l i d and in l i q u i d medium. Duplicates of a l l counts were conducted. Plates were incubated at 21 C for 24-48 h. 35 Proteinase a c t i v i t y The substrate was prepared by adding 10.0 g Hammersten casein (Chemical Dynamics Corp., New Jersey, NJ) to 187.5 mL 0.4 M Na 2HP0 4 followed by b o i l i n g for 30 min to s o l u b i l i z e the casein. After cooling, 312.5 mL of 0.4 M NaH 2P0 4 was added and the mixture diluted with d i s t i l l e d water to 1 L. For determination of proteinase a c t i v i t y in l i q u i d medium, 0.2 mL of culture supernatant, c l a r i f i e d by centrifugation (10,400 x g, 10 min, 4 C) and f i l t e r s t e r i l i z e d , was added to 2.0 mL of substrate. The supernatant was added to the substrate within 30 min of cell/supernatant separation. Proteinase a c t i v i t y of whole c e l l s grown in l i q u i d medium was determined by adding 0.2 mL u n c l a r i f i e d culture medium to 2.0 mL substrate. Proteinase a c t i v i t y in s o l i d medium was determined by adding the agar medium contained within the 50 mm p e t r i plate (15.0 mL agar), without the membrane f i l t e r , to 150.0 mL substrate, followed by blending for 2 min with a Stomacher 400. The agar was added to the substrate within 30 min. In a l l cases, 2.2 mL test mixture aliquots were incubated at 40 C for time periods ranging up to 2 h. For incubation times in excess of 1 h, 0.02% NaNg (Sigma) was added to the substrate. After Incubation, the reaction was stopped with 2.0 mL 24% t r i c h l o r o a c e t i c acid (TCA), and the mixture centrifuged (2,000 x g, 10 min). Absorbance, at 280 nm, of the supernatants was determined using a spectrophotometer (Shimadzu). One enzyme unit of proteinase was defined as 36 t h e q u a n t i t y o£ p r o t e i n a s e t h a t l i b e r a t e d TCA s o l u b l e amino a c i d s and p e p t i d e s e q u i v a l e n t t o 0.001 )*Mole o f t y r o s i n e p e r min. The a b s o r b a n c e o f u n i n c u b a t e d c o n t r o l s was d e d u c t e d f r o m i n c u b a t e d s a m p l e s t o compensate f o r n o n p r o t e i n a c e o u s U.V. a b s o r b i n g m a t e r i a l p r e s e n t i n t h e s u b s t r a t e . I n a l l c a s e s d u p l i c a t e p r o t e i n a s e d e t e r m i n a t i o n s were p e r f o r m e d . D a t a p r e s e n t a t i o n At e a c h t i m e i n t e r v a l t h e mean o f t h e s i x p r o t e i n a s e d e t e r m i n a t i o n s , f r o m t h e t h r e e r e p e a t e d P. f r a g i c e l l g r o w t h e x p e r i m e n t s ( b o t h l i q u i d and s o l i d ) , was c a l c u l a t e d . The means and t h e i r r e s u l t a n t s t a n d a r d d e v i a t i o n s were p l o t t e d . E l e c t r o n m i c r o s c o p y  Sample f i x a t i o n C e l l s f r o m l i q u i d medium were c e n t r i f u g e d (2,000 x g, 10 m i n ) , washed i n 0.05 M p h o s p h a t e b u f f e r (PB) pH 7.0, r e c e n t r i f u g e d , and f i x e d w i t h 2.5% g l u t a r a l d e h y d e ( J . B . EM S e r v i c e s , D o r v a l , PQ) i n PB a t room t e m p e r a t u r e f o r 1 h. F i x e d c e l l s were washed i n PB and p o s t - f i x e d w i t h 1% w/v osmium t e t r o x i d e ( J . B . EM S e r v i c e s ) i n PB a t room t e m p e r a t u r e f o r 1 h. F i x a t i o n o f P. f r a g i c e l l s grown on s o l i d medium was a c h i e v e d by p l a c i n g t h e e n t i r e p o l y c a r b o n a t e membrane f i l t e r ( N u c l e o p o r e ) i n t o t h e f i x a t i v e s . S a m p l e p r e p a r a t i o n S c a n n i n g e l e c t r o n m i c r o s c o p e s a m p l e s were d e h y d r a t e d on t h e s u r f a c e o f p o l y c a r b o n a t e membrane f i l t e r s ( N u c l e o p o r e ) t h r o u g h a g r a d e d s e r i e s o f aqueous e t h a n o l s o l u t i o n s . The 37 f i l t e r s were immersed i n 30, 50, 70 and 80% e t h a n o l , f o r 5 min f o r e a c h c o n c e n t r a t i o n , f o l l o w e d by two c h a n g es o f 90% e t h a n o l f o r 10 min e a c h , and e n d i n g w i t h two c h a n g e s o f 100% e t h a n o l f o r 20 min e a c h . Samples were c r i t i c a l p o i n t d r i e d i n a Parr-bomb ( P a r r I n s t r u m e n t s Co., M o l i n e , I L ) u s i n g C0 2 a s t h e t r a n s i t i o n f l u i d . D r i e d membrane f i l t e r s were mounted on aluminum s t u b s w i t h s i l v e r p a s t e ( J . B . EM S e r v i c e s I n c . ) and s p u t t e r c o a t e d w i t h g o l d (SEMPREP I I S p u t t e r C o a t e r , N a n o t e c h L t d . , P r e s w i c h , E n g l a n d ) . F i x e d c e l l s , t o be examined by t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y , were s u s p e n d e d l n a m i x t u r e o f 1.5% a g a r i n PB. A f t e r s o l i d i f i c a t i o n , 1 mm** b l o c k s were c u t and immersed i n a g r a d e d s e r i e s o f aqueous e t h a n o l s o l u t i o n s as d e s c r i b e d f o r s c a n n i n g e l e c t r o n m i c r o s c o p y . Samples were i n f i l t r a t e d w i t h EPON 812 ( J . B . EM S e r v i c e s I n c . ) , u s i n g p r o p y l e n e o x i d e as a t r a n s i t i o n s o l v e n t , and p o l y m e r i z e d a t 60 C f o r 36 h. Embedded s a m p l e s were s e c t i o n e d w i t h a P o r t e r - B l u m u l t r a m i c r o t o m e ( I v a n S o r v a l l I n c . , N o r w a l k , CN). S e c t i o n s were mounted on 3 mm c o p p e r g r i d s and s t a i n e d w i t h 2% w/v u r a n y l a c e t a t e and R e y n o l d ' s l e a d c i t r a t e ( R e y n o l d , 1963). M i c r o s c o p y S c a n n i n g e l e c t r o n m i c r o s c o p i c e x a m i n a t i o n o f c e l l s was done w i t h a Cambridge S t e r e o s c a n 250 o p e r a t e d a t 40 Kv. T r a n s m i s s i o n e l e c t r o n m i c r o s c o p y was done w i t h a Z e i s s EM-10 a t an a c c e l e r a t i n g v o l t a g e o f 80 Kv. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h s o f P. f r a g i c e l l s grown i n TSB + a g a r f o r 20, 32, 40 and 56 h were e x a m i n e d . 38 The t o t a l of the c e l l perimeters for each electron micrograph was calculated, in Um. In addition, the t o t a l number of e x t r a c e l l u l a r v e s i c l e s appearing on these perimeters were counted. With t h i s information the number of v e s i c l e s per um of c e l l perimeter was calculated. At each time i n t e r v a l four electron micrographs were examined. Mean values and standard deviations were determined. The media Trypticase soy broth (TSB), trypticase soy agar (TSA) and Kosers c i t r a t e medium were from BBL. Nitrogen free c i t r a t e medium was composed of Nal^PO^ (1.0 g/L), Na 2HP0 4 (1.0 g/L), MgS04-7H20 (0.2 g/L) and sodium c i t r a t e (2.94 g/L). A l l were obtained from Sigma. Nitrogen free c i t r a t e medium was supplemented with 1-glutamine (Sigma), added on a mmole nitrogen/L basis. C e l l number enumeration Pseudomonas fr a g i suspensions were enumerated on TSA plates using the S p i r a l plating technique. Plates were incubated at 21 C for 46 h. Colony forming units were counted with a Model 500A bacteria colony counter (Spiral Systems Inc.) interfaced with a S p i r a l Systems Computer Assisted S p i r a l Bioassay data system. C e l l suspension I n i t i a l broth culture An i n i t i a l broth culture of P. f r a g i was prepared in 150 mL of TSB, incubated at 21 C for 18 h. After incubation, the c e l l s were harvested by centrifugation 39 (10,400 x g, 10 min, 4 C) and r e s u s p e n d e d i n 150 mL n i t r o g e n - f r e e c i t r a t e medium. W o r k i n g i n o c u l u m K o s e r s c i t r a t e medium (150 mL) was i n o c u l a t e d w i t h 7.5 mL washed i n i t i a l b r o t h c u l t u r e . The c u l t u r e s were i n c u b a t e d a t 21 C f o r 18 h i n a g y r a t o r y s h a k i n g i n c u b a t o r (120 RPM). A f t e r i n c u b a t i o n , t h e c e l l s were h a r v e s t e d by c e n t r i f u g a t i o n (10,400 x g, 10 min, 4 C) and r e s u s p e n d e d i n 150 mL n i t r o g e n - f r e e c i t r a t e medium. The r e s u l t a n t w o r k i n g i n o c u l u m c o n t a i n e d 5.7 x 10 8 cfu/mL. E x p e r i m e n t a l c o n d i t i o n s Growth o f P. f r a g i d u r i n g t h e e x p e r i m e n t s was c a r r i e d o u t i n 300 mL s q u a r e - p a k f l a s k s ( A m e r i c a n S t e r i l i z e r Co., E r i e , PN) ( F i g u r e 1). E a c h f l a s k c o n t a i n e d r u b b e r septum t y p e c l o s u r e s t h r o u g h w h i c h h y p o d e r m i c n e e d l e s c a r r i e d gas m i x t u r e s and b a c t e r i a l i n o c u l u m . E a c h e x p e r i m e n t was r e p e a t e d t w i c e . Gas m i x t u r e s Gas m i x t u r e s c o n s i s t e d o f U.S.P g r a d e N 2 and 0 2 ( U n i o n C a r b i d e C o r p . , T o r o n t o , ON). Gas p r e s s u r e s were c o n t r o l l e d w i t h oxygen and n i t r o g e n two s t a g e gas r e g u l a t o r s ( L i n d e , U n i o n C a r b i d e C o r p . ) . Gas m i x t u r e f l o w was c o n t r o l l e d by f l o w m e t e r s ( C o l e - P a r m e r I n s t r u m e n t Co., C h i c a g o , I L ) . P a s s a g e o f t h e gas m i x t u r e t h r o u g h p r e s t e r i l i z e d 37 mm b a c t e r i a l a i r v e n t s (0.45 Hm; Gelman S c i e n c e I n c . , Ann A r b o r , MI) a s s u r e d gas m i x t u r e s t e r i l i t y . Gas m i x t u r e f l o w 40 FIGURE 1. Diagram of gas mixture control and gyratory water bath setup. Each Square Pak flask contained 50.0 mL nitrogen free c i t r a t e medium plus varying amounts of glutamine. The flasks were closed with rubber septums. Gases were supplied by two stage regulator valves, and the mixtures controlled by flow meters. The gas mixtures were s t e r i l i z e d by passage through b a c t e r i a l f i l t e r s (0.45 Um). 41 0 , N . i n Lfl. BOTTLED GASES FLOW METERS BACTERIAL FILTER WATER BATH A - A FLASK NO. 1 FLASK NO. 2 rate was controlled at 400 mL/min. Dissolved oxygen in the experimental c i t r a t e medium was measured with a dissolved oxygen meter (Cole-Parmer). Experiments showed that t h i s flow rate maintained a constant dissolved oxygen l e v e l during growth of P. f r a g i . Oxygen content of the gas mixture was expressed as percent 0 2 present in the mixture. Dissolved oxygen present in the culture medium was expressed as ppm 0 2 . For experimental design purposes, percent oxygen was used a s t h e factor l e v e l , although dissolved oxygen was dependant upon incubation temperature. Each experimental unit consisted of two square-pak fl a s k s , each containing 50 mL of experimental c i t r a t e medium (Figure 1). The gas mixture entered flask #1 which acted as a humidifier and experimental control. Humidified gas mixture then was passed by f l e x i b l e tubing to the second fl a s k . The second flask acted as the active experimental chamber. Gas mixture exited through a b a c t e r i a l a i r vent. The experimental unit was contained in a gyratory shaking water bath incubator (Lab-Line Instruments, Melrose Park, IL). Water bath temperature was monitored and recorded with a Microtemp model 811 temperature monitor/recorder (Intelicom Systems Inc., North Vancouver, BC). The experimental units were equilibrated at the indicated Incubation temperature and gas mixture for 24 h pr i o r to inoculation. 43 S u b s e q u e n t t o e q u i l i b r i a t i o n , 2.5 mL o f w o r k i n g i n o c u l u m was i n t r o d u c e d i n t o f l a s k #2 o f t h e e x p e r i m e n t a l u n i t t h r o u g h t h e r u b b e r septum ( p r e v i o u s l y s t e r i l i z e d w i t h a 70% e t h a n o l swab) by h y p o d e r m i c i n j e c t i o n . I n i t i a l i n o c u l u m l e v e l , was 2.8 x 1 0 7 cfu/mL. I n o c u l a t e d f l a s k s were s h a k e n a t 120 RPM d u r i n g i n c u b a t i o n . A f t e r i n c u b a t i o n , P. f r a g i c e l l numbers were e n u m e r a t e d , i n d u p l i c a t e , by s p i r a l p l a t i n g . The b a c t e r i a l c e l l s were t h e n h a r v e s t e d by c e n t r i f u g a t i o n (10,400 x g, 10 min, 4 c) and t h e s u p e r n a t a n t f i l t e r s t e r i l i z e d by p a s s a g e t h r o u g h a 0.45 Vim membrane f i l t e r ( M i l l i p o r e ) . P r o t e i n a s e a c t i v i t y P r o t e i n a s e a c t i v i t y o f t h e c u l t u r e s u p e r n a t a n t s was d e t e r m i n e d , as p r e v i o u s l y d e s c r i b e d . E l e c t r o n m i c r o s c o p y The h a r v e s t e d c e l l s were examined by e l e c t r o n m i c r o s c o p y , as p r e v i o u s l y d e s c r i b e d . P r o t e i n a s s a y P r o t e i n a s s a y was p e r f o r m e d u s i n g t h e b i c i n c h o n i n l c a c i d (BCA) p r o t e i n a s s a y r e a g e n t ( P i e r c e C h e m i c a l Co., R o c k f o r d , I L ) . B r i e f l y , 0.1 mL o f sample was mixed w i t h 2.0 mL o f w o r k i n g BCA r e a g e n t and i n c u b a t e d a t 37 C f o r 30 min. A f t e r c o o l i n g t h e a b s o r b a n c e was measured w i t h a s p e c t r o p h o t o m e t e r (Shimadzu) a t 562 nm. The a b s o r b a n c e o f a b l a n k c o n t a i n i n g BCA w o r k i n g r e a g e n t was s u b t r a c t e d f r o m t h e sample a b s o r b a n c e . B o v i n e serum a l b u m i n (BSA, Sigma) was u s e d as a s t a n d a r d . 44 I s o e l e c t r i c focusing (IEF) F i l t e r s t e r i l i z e d supernatants were drop dialyzed on a 25 mm, 50 nm membrane f i l t e r (Millipore) as described by Marusyk and Sergeant (1980) and separated on IEF polyacrylamide gels pH 3.7 to 6.2 as described by Righetti (1987). Samples were applied onto the gel using f i l t e r paper squares. Duplicate gels were made and either overlayed with a casein-agar mixture or stained with s i l v e r s t a i n . Proteinase l o c a l i z a t i o n Active proteinase was l o c a l i z e d within IEF gels by overlaying a 1.5% agar suspension, containing (1%) casein substrate, over the gel. The casein substrate was the same preparation that was used for the proteinase assay. B r i e f l y , the s o l u b i l i z e d casein-agar mixture was heated to b o i l i n g , cooled to ca 45 C and poured over the IEF g e l , s t i l l on i t s glass backing plate. The IEF gel and glass backing plate had previously been placed within an aluminum template to maintain a 2 mm thick casein-agar gel layer. The IEF gel-casein gel composite was incubated for periods up to 24 h at room temperature. Hydrolyzed areas in the casein gels were v i s u a l i z e d by immersion, for 10 min, in a 4% s u l f o s a l i c y l i c acid, 12.5% t r i c h l o r o a c e t i c acid, 30% methanol solution. Optimization procedures The centroid search technique was used to optimize the production of proteinase from P. fr a g i under given c u l t u r a l 45 conditions. The conditions and the i r ranges were: incubation temperature 1 to 40 C; incubation time 4 to 72 h; i n i t i a l pH 5 to 10; glutamine concentration 200 to 650 mmole nitrogen/L; oxygen concentration 0 to 50%. The response value was enzyme units/mL. Prior to optimization, a f r a c t i o n a l f a c t o r i a l design experiment was conducted in order to evaluate the importance of each factor. The f r a c t i o n a l f a c t o r i a l design used in th i s study was an Lg (2 7) orthogonal array, as described by Taguchi (1957). It consisted of eight experiments at two factor l e v e l s . The fi v e factors and the i r two levels were as follows: temperature 20 and 30 C; time 32 and 72 h; pH 7 and 9; glutamine concentration 7 and 125 mmole nitrogen/L; oxygen 10 and 30%. Each experiment was run twice. A nested or h i e r a r c h i a l design (Snedecor and Cochran, 1971) analysis of variance of the data obtained from the Taguchi (1957) f r a c t i o n a l f a c t o r i a l was performed on the individual experiments versus experiment r e p l i c a t e s for both proteinase determinations and c e l l number enumerations ( t o t a l of two analyses of variance). A nested design analysis of variance was also performed on the individual experiments versus experiment r e p l i c a t e s for proteinase determinations from both the Spendley matrix and centroid experiments. Each experimental unit consisted of two re p l i c a t e s , each done in duplicate. These analyses were performed in order to determine whether re p l i c a t e s contributed s i g n i f i c a n t l y to the variance. Analysis of 46 v a r i a n c e was a l s o p e r f o r m e d on i n d i v i d u a l f a c t o r s f r o m t h e f r a c t i o n a l f a c t o r i a l e x p e r i m e n t . One a n a l y s i s o f v a r i a n c e was p e r f o r m e d u s i n g enzyme u n i t s / m L and one u s e d c o l o n y f o r m i n g u n i t s / m L . The mean o f t h e p r o t e i n a s e d e t e r m i n a t i o n f o r e a c h e x p e r i m e n t a l u n i t o f t h e f r a c t i o n a l f a c t o r i a l and c e n t r o i d s e a r c h ( S p e n d l e y m a t r i x , c e n t r o i d and t h e s i m u l t a n e o u s s h i f t e x p e r i m e n t s ) was d e t e r m i n e d . The s t a n d a r d d e v i a t i o n o f e a c h o f t h e f r a c t i o n a l f a c t o r i a l e x p e r i m e n t a l u n i t means was c a l c u l a t e d . The s t a n d a r d e r r o r o f e a c h o f t h e S p e n d l e y m a t r i x and c e n t r o i d e x p e r i m e n t s e x p e r i m e n t a l u n i t means was c a l c u l a t e d . The mean o f t h e means, and i t s a s s o c i a t e d s t a n d a r d e r r o r , o f t h e i n d i v i d u a l e x p e r i m e n t a l u n i t s f o r e a c h o f t h e f r a c t i o n a l f a c t o r i a l , S p e n d l e y m a t r i x and c e n t r o i d e x p e r i m e n t s were d e t e r m i n e d . O p t i m i z a t i o n models The p u r p o s e o f t h e f o l l o w i n g s e c t i o n i s t o p r o v i d e t h e d e r i v a t i o n o f m a t h e m a t i c a l models u s e d , i n t h i s s t u d y , t o e v a l u a t e t h e e f f i c i e n c y o f s e v e r a l p o p u l a r o p t i m i z a t i o n t e c h n i q u e s . I t c a n g e n e r a l l y be assumed t h a t an o r g a n i s m w i l l p r o d u c e a s p e c i f i c enzyme, or g r o u p o f s i m i l a r enzymes, o v e r a wide r a n g e o f c u l t u r a l c o n d i t i o n s . I f a s p e c i f i c enzyme c a n be i d e n t i f i e d , t h e n t h e r e i s most l i k e l y some u n i q u e s e t o f c u l t u r a l c o n d i t i o n s where t h e maximum y i e l d o f enzyme would o c c u r . 47 The r a t e a t w h i c h t h e enzyme i s p r o d u c e d a t s u b o p t i m a l c u l t u r e c o n d i t i o n s may change s m o o t h l y , r i s i n g a t a c o n s t a n t r a t e o f change f a r f r o m t h e optimum p o i n t , and e n d i n g a t t h e optimum p o i n t . T h i s i d e a l s i t u a t i o n i n a l l p r o b a b i l i t y d o e s n o t o c c u r . I t i s more l i k e l y t h a t t h e r e i s a v a r i a b l e r a t e o f change i n enzyme p r o d u c t i o n . I f one c o u l d i m a g i n e a r e s p o n s e s u r f a c e on w h i c h m i c r o b i a l enzyme p r o d u c t i o n v a r i e s o v e r c h a n g i n g c u l t u r e c o n d i t i o n s , one would e x p e c t h i l l s , v a l l e y s and p l a t e a u s t o o c c u r as t h e enzyme y i e l d r e s p o n d s t o c h a n g e s i n c u l t u r e c o n d i t i o n s . T h i s p i c t u r e would become i n c r e a s i n g l y c o m p l i c a t e d i f a g r o u p o f s i m i l a r enzymes were b e i n g m o n i t o r e d , r a t h e r t h a n one s p e c i f i c enzyme. Such s i t u a t i o n s may o c c u r when one or more i s o e n z y m e s a r e p r o d u c e d . Under t h e s e c i r c u m s t a n c e s , l o c a l maxima may p o p u l a t e t h e t e r r a i n . A m a t h e m a t i c a l model d e s i g n e d t o e v a l u a t e o p t i m i z a t i o n t e c h n i q u e s as t h e y a r e a p p l i e d t o p r o t e i n a s e p r o d u c t i o n by an o r g a n i s m , s u c h as P. f r a g i , p r e s e n t s more of a c h a l l e n g e t h a n s i m p l e c o n t i n u o u s f u n c t i o n s c a n p r o v i d e . S e v e r a l t r i g o n o m e t r i c f u n c t i o n s e x i s t w h i c h c a n p r o d u c e d i s c o n t i n u o u s r e s p o n s e s u r f a c e s . T hese f u n c t i o n s a r e u s e f u l , b u t t h e r e s p o n s e e x t r e m e s a r e h a r d t o p r e d i c t and t h e c o m p u t a t i o n s c a n be c o m p l i c a t e d , e s p e c i a l l y w i t h f u n c t i o n s c o n t a i n i n g s e v e r a l f a c t o r s . S i m p l e c o n t i n u o u s f u n c t i o n s c a n , however, be e a s i l y m a n i p u l a t e d s u c h t h a t t h e p o i n t o f maximum r e s p o n s e c a n be 4 8 c o n t r o l l e d b o t h i n p o s i t i o n and v a l u e . I n t h e f o l l o w i n g f u n c t i o n : f ( X 1 / X 2 ) = { D - ( X 1 ± A ) 2 * C 1 - ( X 2 ± B ) 2 * C 2 } ( I ) The maximum ( o r minimum) p o i n t w i l l o c c u r a t t h e p o i n t X^ = A, X 2 = B. The s l o p e o f t h e r e s p o n s e s u r f a c e c a n be c o n t r o l l e d by C± and C 2 . As an example: Y 1 = { 2 5 0 - ( X 1 - 3 ) 2 * 3 0 - ( X 2 - 7 ) 2 * 5 } / 2 . 5 ( I I ) The maximum r e s p o n s e (100) w i l l o c c u r at = 3 , X 2 = 7 . I f a n o t h e r c o n t i n u o u s f u n c t i o n i s s u p e r i m p o s e d on t h e o r i g i n a l f u n c t i o n by s i m p l e a d d i t i o n , t h e n t h e r e s u l t i n g r e s p o n s e s u r f a c e w i l l r e f l e c t t h i s . I f t h e maximum ( o r minimum) p o i n t s o f t h e two f u n c t i o n s d i f f e r , t h e n t h e r e s u l t a n t r e s p o n s e s u r f a c e becomes d i s c o n t i n u o u s . F o r example, i f e q u a t i o n I I I w i t h a maximum r e s p o n s e v a l u e o f 60 a t X1 = 8, X 2 = 2 : Y 2 = ( 3 2 5 - ( X 1 - 8 ) 2 * 3 0 - ( X 2 - 2 ) 2 * 5 } / 5 . 4 ( I I I ) were added t o I I s u c h t h a t : Z=Y 1+Y 2 ( I V ) ( v a l u e s o f Y± o r Y 2 i f <0 a r e changed t o 0) 49 the response surface would have two maxima, one at = 3, X 2 = 7 with a value of 100, and one at X^ = 8, X 2 = 2 with a value of 60. A three dimensional plot of factors X-^ , X 2, and Z i s shown in Figure 2. This same technique may be used to construct discontinuous response surfaces in dimensions greater than two. For example, two continuous three factor functions (V) and (VI) both designed to y i e l d only one maximum response value are shown below: Yj^ ={ 200 + 4 5X1-50X2 + 30X3 + 140X 1X 2-10X 1X 3 + 61X2X3 (V) +45(X 1) 2-245(X 2) 2-11(X 3) 2}/10 Y2={20 0+4 5X 1-50X 2+30X 3+140X 1X 2-10X 1X 3+61X 2X 3 (VI) +45(X 1) 2-123(X 2) 2-43(X 3) 2}/10 Functions (V) and (VI) can be combined in (VII). Z = Y 1 + Y 2 (VII) (same rules as IV) Function (VII) would form a discontinuous four dimensional response surface. Equations V and VI were formulated according to the method of Bowman and Gerard (1967) and Saguy (1983) such that: (1) for a minimum response value Dj>0 for a l l i=l,2,3, ,n (VIII) (2) for a maximum response value Di<0 for a l l odd i (1=1,3,5....) Di>0 for a l l even 1 (1=2,4,6...) where: 50 FIGURE 2. Three dimensional plot of model #1. A three dimensional plot of the following equations: Y 1={250-(X 1-3) 2*30-(X 2-7) 2*5}/2.5 and Y 2 = {325-(X 1-8) 2*30-(X 2-2) 2*5}/5 .4 Z=Y 1 +Y 2 (values of Y^ or Y 2 i f <0 are changed to 0) Factor Xj^ is plotted on the X-axis and factor X 2 is plotted on the Y-axis. Factor Z i s plotted on the Z-axis. Two maxima, one at X^ = 3, X 2 = 7, and one at X^ = 8, X 2 = 2 can be seen. 51 52 £ 1 1 £ 12 f 1 3 f 2 1 £ 22 f 2 3 £ 3 1 £ 32 £ 33 (IX) £11 £12 £13 £ l n £11 £12 £13 £ l n f l l £12 £13 £ l n n l rn2 rn3 rnn A l l the models constructed were used to evaluate the centroid search optimization technique (models are shown in Table I ) . The modified simplex technique (MDS) of Morgan and Denting (1974), the modified super simplex technique (MSS) with mapping (Nakai and Kaneko, 1985) and the response surface methodology (RS) derived from a Box and Behnken (1960) f a c t o r i a l design were compared against the centroid search technique (CS), using a l l models. Multiple regression analysis of the Box and Behnken (1960) f a c t o r i a l was performed on an IBM-XT compatible computer using the multiple regression procedure provided by the s t a t i s t i c a l graphing system "Statgraphics" ( S t a t i s t i c a l Graphics Corp., Baltimore, MD). A l l models were created with a range of 10. A l l four optimization techniques were then applied to each model. Further runs of a l l the techniques were conducted using ranges of less than 10. These ranges were selected using a random number generator. A t o t a l of four runs were conducted. The stopping c r i t e r i o n was two successive 53 TABLE I. Mathematical models used to evaluate optimization techniques. MODEL #1 Y 1=(250-(X 1-3) 2*30-(X 2-7) 2*5)/2.5 Y 2=(325-(X 1-8) 2*30-(X 2-2) 2*5)/5.4 MODEL #2 Y 1=(250-(X 1-3) 2*12-(X 2-7) 2*5)/2.5 Y 2=(325-(X 1-8) 2*12-(X 2-7) 2*5)/3.25 MODEL #3 Yx=(200+45*X1-50*X2+30*X3+140*X1*X2-10*X1*X3+61*X2*X3+4 5X 1 2-245*X 2 2-11*X 3 2)/10 Y2=(200+45*X1-50*X2+30*X3+140*X1*X2-10*X1*X3+61*X2*X3+45X12-123*X 2 2-43*X 3 2)/10 54 r e s p o n s e v a l u e s >98% o f t h e t r u e r e s p o n s e v a l u e s . M e a s u r i n g t h e amount o f t i m e r e q u i r e d t o c o m p l e t e t h e o p t i m i z a t i o n was i m p o r t a n t i n t h i s s t u d y . C o n s e q u e n t l y , one i n d e p e n d e n t f a c t o r was c h o s e n t o r e p r e s e n t t i m e ( i n h o u r s ) . An a n a l y s i s o f v a r i a n c e o f t h e d a t a o b t a i n e d f r o m t h e c o m p a r i s o n s o f t h r e e o p t i m i z a t i o n t e c h n i q u e s (Morgan Deming s u p e r s i m p l e x , m o d i f i e d s u p e r s i m p l e x and c e n t r o i d s e a r c h ) v e r s u s t h e t h r e e m o d els (model #1, model #2 and model #3) was p e r f o r m e d ( S n e d e c o r and C o c h r a n , 1 9 7 1 ) . The d a t a c o n s i s t e d o f means o f e x p e r i m e n t number and means o f e x p e r i m e n t t i m e . A m u l t i p l e r a n g e t e s t ( l e a s t s i g n i f i c a n t d i f f e r e n c e ) was u s e d t o compare d i f f e r e n c e s between means. C e n t r o i d o p t i m i s a t i o n The o b j e c t o f t h e f o l l o w i n g s e c t i o n i s t o d e m o n s t r a t e t h e c e n t r o i d s e a r c h and mapping p r o c e d u r e s u s i n g f u n c t i o n ( I V ) , h e r e a f t e r r e f e r r e d t o as model #1. S t e p 1 - I n i t i a l s i m p l e x An i n i t i a l s i m p l e x , a g e o m e t r i c f i g u r e d e f i n e d by e x p e r i m e n t a l p o i n t s e q u a l t o one more t h a n t h e number o f i n d e p e n d e n t v a r i a b l e s ( t h e number o f i n d e p e n d e n t v a r i a b l e s b e i n g n ) , was c r e a t e d . The v e r t i c e s o f t h e s i m p l e x were o b t a i n e d f r o m a S p e n d l e y m a t r i x once an o r i g i n and s c a l e o f d i m e n s i o n had been c h o s e n . The o r i g i n i s t h e l o w e r l i m i t ( L L ) and t h e o r i g i n + t h e s c a l e i s t h e u p p e r l i m i t ( U L ) . I n model #1 t h e o r i g i n i s z e r o and t h e s c a l e (S) i s 10. The S p e n d l e y m a t r i x s u g g e s t s v e r t i c e s ( D x ) w h i c h have r e g u l a r i t y 55 of spacing such that the values of the independent variables are of equal in t e r e s t . The derivation i s as follows: D1 = (X 1,X 2,X 3 / ,Xn) D2 = <Pn + xl'<In + x2'<in + x3' '% + xn> D 3 = (q n+X 1 / P n+X 2,q n+X 3, , .<3n+xn> <x> * • • * D n 4 l = < q n + x l ^ n + x 2 ^ n + x 3 ' 'Pn+Xn> where: p = — ; [Cn - 1 ) + Yn + 1 1 * S CXI> nV 2 ' 1 J ' and: q - — ; C Y n + l - l 1 * S CXH) nV 2 ' S = sca le fac tor D •= vertex X n = number of Independent v a r i a b l e s For n=2, and S=10, p=9.659 and q=2.588. The resultant v e r t i c e s of the simplex were: D l = X1' X2 = 0,0 D2 = Pn + Xl'<ln + X2 56 = 9.659,2.588 D3 = % X 1 / P n + X 2 = 2.588,9.659 The S p e n d l e y m a t r i x g e n e r a t e d v e r t i c e s , w h i c h a r e p l o t t e d i n F i g u r e 3, show t h e X^ f a c t o r r a n g i n g f r o m 0 t o 10 on t h e X a x i s , w i t h t h e X 2 f a c t o r r a n g i n g f r o m 0 t o 10 on t h e Y a x i s . The r e s p o n s e v a l u e s a r e shown as l i n e s o f c o n t o u r , much l i k e e l e v a t i o n s on a t o p o g r a p h i c a l map. V e r t i c e s a r e d e s c r i b e d a s x , y p a i r s w h i c h have c o r r e s p o n d i n g r e s p o n s e v a l u e s . The e x p e r i m e n t s and t h e i r r e s p o n s e s a r e shown i n T a b l e I I . T h e s e r e s u l t s show t h a t v e r t e x 3 p r o d u c e d t h e b e s t r e s u l t , w i t h v e r t e x 2 p r o d u c i n g t h e n e x t b e s t r e s u l t . V e r t e x 1 p r o d u c e d t h e w o r s t r e s p o n s e . S t e p 2 - E v a l u a t e t h e c e n t r o l d s The c e n t r o i d s between t h e b e s t and t h e n e x t b e s t v e r t i c e s were s e a r c h e d . The c e n t r o i d i s t h e b i s e c t i o n p o i n t between t h e b e s t ( X D ) and t h e n e x t b e s t ( X n ) v e r t e x ( p o i n t 4 i n F i g u r e 3). The optimum r e s p o n s e i s a c h i e v e d by moving i n t h e d i r e c t i o n o f i m p r o v e d r e s p o n s e . I f t h e c e n t r o i d has a h i g h e r r e s p o n s e t h a n e i t h e r t h e b e s t or t h e n e x t b e s t v e r t e x t h e n t h e s e a r c h i s i n f a c t moving i n t h e d i r e c t i o n o f i m p r o v e d r e s p o n s e . I f t h i s c o n d i t i o n i s met, t h e n t h i s p o i n t ( p o i n t 4) becomes t h e n e x t b e s t v e r t e x . I f on t h e o t h e r hand, t h i s c o n d i t i o n I s n o t met ( i . e . i f p o i n t 4 has a l o w e r r e s p o n s e v a l u e t h a n e i t h e r p o i n t 2 o r p o i n t 3), t h e n t h e s e a r c h i s 57 FIGURE 3. Topographical plot of model #1 with centroid generated experiments. A topographical map of model 1. Factor i s plotted on the X-axis and factor X? i s plotted on the Y-axis. Lines shown correspond to equal values for the response value (Z). Vertices 1, 2 and 3 are points generated by the i n i t i a l Spendley matrix. Vertex 3 produced the best r e s u l t , with vertex 2 producing the next best r e s u l t . Vertex 1 produced the worst r e s u l t . The centroid (point 4) between the best and the next best r e s u l t s was evaluated and found to have a lower r e s u l t than vertex 3. A new simplex with vertices 5, 6 and 7 was calculated. Centroids (points 8, 9 and 10) were evaluated. Point 10 came to within 99% of the true optimum value. 58 TABLE II. Experiments generated by centroid search and response values returned by model #1. EXPERIMENTS FACTOR RESPONSE X l x2 VALUE i n i t i a l VERTEX 1 0.000 0.000 0.000 VERTEX 2 9.659 2.588 44.570 VERTEX 2.588 9.659 83.820 CENTRD 4(1) 6.123 6.123 24.870 new VERTEX 5 2.588 9 .659 83.820 VERTEX 6 9 .418 7.829 17.550 VERTEX 7 4.418 2.829 41.070 CENTRD 8 3.503 6.244 95.820 CENTRD 9 3.045 7.951 98.160 CENTRD 10 3.274 7 .097 99 .080 ( 1 ) Centroid 60 not moving in the d i r e c t i o n of improved response. This search l i n e must be abandoned. Figure 3 and Table II show that t h i s response was worse than the best from the i n i t i a l simplex. This search l i n e was abandoned and a new simplex with new ranges calculated. Step 3 - Calculate new vertices and ranges New vertices and ranges were calculated according to the rules set out by Aishima and Nakai (1986). (1) When the responses of X D and X n ( i . e . Y D and Y n) are close to one another: ! Y b ~ Y n l < l Y b " Y e l Where: Y D = best response value Y n = next best response value Y e = estimated best response value then X n i s replaced by Xn+1 the t h i r d best vertex. (2) When the difference between the best (X D) and the next best (X n) vertex i s large: |Xb - X n| > JUL - LL| / 2 X b and X n are used as the new lower and upper l i m i t s . (3) When the difference between the best (X D) and the next best (X n) vertex i s small: l xb " xn! < l U L " L L I / 2 0 the new lower l i m i t = X D -the new upper l i m i t = X D + UL - LL UL - LL / 20 / 20 (4) When the difference between the best (X D) and the next best (X n) vertex i s small but X D Is near the boundary (UL or LL) then: the new lower l i m i t = X D - q * jX 5 - X n| 61 new upper l i m i t = xb + lxb -Xn{ / 1 - q A new S p e n d l e y m a t r i x was p r o d u c e d a t t h i s p o i n t . The f a c t o r l e v e l s a r e shown i n T a b l e I I ( v e r t i c e s 5,6,7). S t e p 4 - E v a l u a t e new s i m p l e x R esponse v a l u e s a r e shown i n T a b l e I I . V e r t e x 5 ( F i g u r e 3) was t h e b e s t v e r t e x , w h i l e v e r t e x 7 was t h e n e x t b e s t . S t e p 5 - E v a l u a t e new c e n t r o l d s The new c e n t r o i d s a r e t h o s e p o i n t s 8, 9 and 10 between t h e b e s t and t h e n e x t b e s t v e r t i c e s . The r e s p o n s e v a l u e s a r e shown i n T a b l e I I . The v a l u e o f t h e f i r s t c e n t r o i d ( p o i n t 8) was h i g h e r t h a n t h e b e s t f r o m t h e new s i m p l e x ( p o i n t 5), t h u s s e a r c h i n g was c o n t i n u e d . A new c e n t r o i d ( p o i n t 9), h a l f way between t h e b e s t and t h e new n e x t b e s t p o i n t , i s e v a l u a t e d . C e n t r o i d s 9 and 10 y i e l d e d y e t h i g h e r r e s p o n s e v a l u e s s u g g e s t i n g t h a t t h i s s e a r c h l i n e may be c l o s e t o t h e optimum r e s p o n s e v a l u e . A t t h i s p o i n t i t i s w i s e t o s t o p f u r t h e r s e a r c h e s . C e n t r o i d s e a r c h i n g i n v o l v e s a r e l a t i v e l y s m a l l s e a r c h a r e a . As a r e s u l t , l o c a l t o p o g r a p h y i s e v a l u a t e d , w h i l e g l o b a l t r e n d s t e n d t o be i g n o r e d . To remedy t h i s s i t u a t i o n a method must be u s e d t o map t r e n d s i n t h e d a t a and v i e w t h i s o b j e c t i v e l y . I n t h i s way i t i s p o s s i b l e t o move t o o t h e r a r e a s where t h e t r u e maximum may r e s i d e , and n o t g e t "bogged down" a t a l o c a l maximum, t h e r e s u l t o f c e n t r o i d s e a r c h ' s " m yopic" n a t u r e . 62 Step y - Mapping Mapping ( N a k a i e t al., 1984) i s an a p p r o x i m a t e v i s u a l i z a t i o n o f the e x p e r i m e n t a l r e s p o n s e s u r f a c e . The p u r p o s e o f mapping i s t o d e m o n s t r a t e t r e n d s t o w a r d t h e t r u e optimum. E a c h f a c t o r was p l o t t e d w i t h t h e f a c t o r r a n g e on t h e X a x i s and t h e r e s p o n s e v a l u e s on t h e Y a x i s . F o r e a c h f a c t o r n o t b e i n g compared, t h e r a n g e o f v a l u e s , as d e t e r m i n e d by t h e r a n g e o f t h e r e s p o n s e s u r f a c e , was d i v i d e d i n t o f o u r e q u a l s e c t i o n s known as t h e l a r g e l i m i t . S e a r c h e s were made f o r two or more i d e n t i c a l d a t a p o i n t s a p p e a r i n g i n a t l e a s t one s e c t i o n o f e a c h f a c t o r . Those d a t a p o i n t s f o u n d were t h e n e n t i t l e d t o be j o i n e d i n a map o f t h e c o m p a r i s o n f a c t o r . A f t e r a l l t h e f a c t o r s were i n t u r n compared u s i n g t h e l a r g e l i m i t , a s m a l l l i m i t was t h e n s e l e c t e d and t h e p r o c e s s r e p e a t e d . The s m a l l l i m i t was d e t e r m i n e d f r o m t h e p l o t o f t h e c o m p a r i s o n f a c t o r , i n c l u d i n g o n l y t h o s e p o i n t s s u g g e s t e d by t h e u s e r as b e i n g o f major i m p o r t a n c e . A medium l i m i t was t h e n u s e d t o e v a l u a t e a l l t h e c o m p a r i s o n f a c t o r s . The medium l i m i t was c a l c u l a t e d as t h e a v e r a g e o f t h e v a l u e s o f t h e l a r g e and s m a l l l i m i t s . A f t e r a l l t h e matched d a t a p o i n t s f r o m a l l t h r e e l i m i t s were p l o t t e d and l i n k e d , q u a d r a t i c c u r v e f i t t i n g was p e r f o r m e d , i f p o s s i b l e . F i g u r e 4a shows t h e mapping o f f a c t o r X^, w h i l e F i g u r e 4b shows t h e mapping o f f a c t o r X 2 . The X v a l u e s a r e shown on t h e a b s c i s s a w h i l e t h e r e s p o n s e v a l u e s a r e shown on t h e o r d i n a t e . D a t a p o i n t s c o r r e s p o n d i n g t o f a c t o r v a l u e s and 63 FIGURE 4. Map of factors from model #1. The range for each factor i s plotted against response value. The data points represent the v e r t i c e s and centroids generated by centroid search. Data points are grouped into four groups. Those points e n t i t l e d to be joined belong to the same group of a l l the other factors. The matched points are linked and quadratic curve f i t t i n g performed, i f possible. Short l i n e segments represent quadratic curves that have been f i t t e d , but cannot be joined within the bounds of the map. (a) Map of factor Optimum response i s between 2.5 and 3.5. (b) Map of factor X 2 Optimum response i s between 6.5 and 9.0. 64 t h e i r respective response values are plotted. Figure 4a c l e a r l y shows an optimum response at X l 7 values between 2.5 and 3.5, while Figure 4b shows an optimum response at X 2, values between 6.5 and 9.0. New simplex The centroid search procedure was repeated with either the new ranges suggested by the previous optimization procedure or by the mapping technique. The new vertices and th e i r response values are shown in Table III and Figure 5. For brevity the verti c e s and the i r responses are given without comment. UPPER AND LOWER LIMITS X l LL 2.5 UL 3.5 x2 LL 6.5 UL 9.0 Mapping of the vertices for factor X-^  i s shown in Figure 6a, while mapping of factor X 2 i s shown in Figure 6b. Axes have been contracted to "zoom i n " on the optimum points. Optimum points are close to vertex 4 (Figure 5), i. e . X x = 2.982, X 2 = 6.823. 66 TABLE III. Experiments generated by centroid search and evaluated by model #1 - new simplex. EXPERIMENTS FACTOR RESPONSE XJL X 2 VALUE VERTEX 1 2.500 6.500 96.500 VERTEX 2 3.465 7.147 97.360 VERTEX 3 2.758 8.914 91.970 CENTRD 4 2.982 6.823 99.930 CENTRD 5 3.224 6.985 99.390 CENTRD 6 3.103 6.904 99.850 67 FIGURE 5. Topographical map of Model #1 and experiments generated by centroid search - new simplex. Factor X^ i s plotted on the X-axis and factor X 2 i s plotted on the Y-axis. Lines shown correspond to equal values for the response value (Z). Using ranges suggested from the i n i t i a l mapping of model #1, a second centroid search optimization was performed. Points 1, 2 and 3 are verti c e s generated by the Spendley matrix. Point 2 i s the best response and point 1 the next best. Centroid 4 came to within 99.93% of the true optimum. 68 FIGURE 6. Map o£ factors from model #1 - new simplex. The range for each factor i s plotted against response value. The data points represent the v e r t i c e s and centroids generated by centroid search. The range for each factor has narrowed, converging towards the optimum point. (a) Map of factor Xi Optimum response i s close to vertex 4 (2.982) (b) Map of factor X? Optimum response i s close to vertex 4 (6.823). 70 CELL AND GROWTH MEDIUM FRACTIONATION B r o t h c u l t u r e S e p a r a t e 250 mL f l a s k s c o n t a i n i n g 150 mL TSB were i n o c u l a t e d w i t h c a . 1.5 x 10*> c f u P. f r a g i and i n c u b a t e d w i t h a g i t a t i o n (150 RPM) a t 21 C f o r 82 h. A f t e r i n c u b a t i o n , f l a s k c o n t e n t s were p o o l e d and c e n t r i f u g e d (10,400 x g, 10 min, 4 C ) . B o t h c e l l p e l l e t and s u p e r n a t a n t were c o l l e c t e d . S e p a r a t e 150 mL c u l t u r e u n i t s were us e d t o m a i n t a i n a e r o b i c c o n t i n u i t y . S u p e r n a t a n t p r e p a r a t i o n P. f r a g i was grown i n 1 L TSB a t 21 C f o r 82 h. C e l l s were removed by c e n t r i f u g a t i o n (10,400 x g, 10 min, 4 C) and t h e s u p e r n a t a n t f i l t e r s t e r i l i z e d . The s u p e r n a t a n t was c o n c e n t r a t e d i n u l t r a f i l t r a t i o n c e l l s w i t h 10,000 MW c u t - o f f membranes ( A m i c o n ) . The c o n c e n t r a t e d s u p e r n a t a n t was t h e n d i a l y z e d a g a i n s t 4 L o f wa t e r a t 4 C and f u r t h e r c o n c e n t r a t e d by u l t r a f i l t r a t i o n . T h i s p r o c e d u r e would e l i m i n a t e s a l t s w h i c h would i n t e r f e r e w i t h e l e c t r o p h o r e t i c s e p a r a t i o n o f p r o t e i n a c e o u s m a t e r i a l p r e s e n t I n t h e s u p e r n a t a n t . E x t r a c e l l u l a r v e s i c l e p r e p a r a t i o n E x t r a c e l l u l a r v e s i c l e s were p r e c i p i t a t e d f r o m 1 L c u l t u r e s u p e r n a t a n t b y s l o w a d d i t i o n ( o v e r a 1 h p e r i o d ) o f 240 g [NH4] 2S0 4 (40% s a t u r a t i o n ) a t 4 C, w i t h a g i t a t i o n . The r e s u l t a n t s u s p e n s i o n was c e n t r i f u g e d (20,000 x g, 40 min, 4 C) and t h e p e l l e t r e s u s p e n d e d i n 150 mL o f 50 mM 72 T r i s buffer pH 6.8. The suspension was dialyzed for 24 h in 4 L of the same buffer. After d i a l y s i s , the retentate was collected and centrifuged (27,000 x g, 40 min, 4 C) . The p e l l e t was suspended in 30 mL water and again centrifuged. The p e l l e t was resuspended in 3.0 mL of water and the resultant preparation stored at -20 C for subsequent electron microscopy, or freeze-dried for chemical analysis. Electron microscopy A sample of e x t r a c e l l u l a r v e s i c l e suspension was deposited onto Formvar coated 3 mm copper electron microscopy grids and negative stained with 2% aqueous uranyl acetate. Negatively stained grids were examined by TEM, as previously described. Buoyant density The buoyant density of the e x t r a c e l l u l a r v e s i c l e s was determined by high speed centrifugation in P e r c o l l (Pharmacia) plus 0.25 M sucrose. The P e r c o l l working solution consisted of 15.0 mL 2.5 M sucrose, 127.5 mL P e r c o l l (from the bottle) and water to make up to a t o t a l of 150 mL. The density of the resultant so l u t i o n was 1.14 g/mL. Twenty mL of the P e r c o l l working so l u t i o n was placed in 30 mL high speed polycarbonate centrifuge tubes (Sorvall) and a 0.5 mL sample (suspended in 0.25 M sucrose) layered on top of the P e r c o l l . The centrifuge tubes were spun at 60,000 x g in a T865 fix e d -angle rotor head (Sorvall) for 45 min. Density marker beads 73 (Pharmacia), as well as refractometry, were used to measure the generation of the linear density gradient. The density ranged from 1.097 to 1.165 g/mL. Carbohydrate assay Total carbohydrate of the e x t r a c e l l u l a r vesicles was determined by the Phenol-sulfuric acid reaction as described by Dubois, et al. (1956). Dextrose was used as a standard. Outer c e l l membrane preparation Outer c e l l membrane preparations were obtained by adaptations of the methods described by F l l i p et a l . (1973) , Hancock and Carey, (1979), and Anwar, et al. (1985). The c e l l p e l l e t , c o l l e c t e d by centrifugation (10,400 x g, 10 min, 4 C), was suspended in 20 mL 10 mM T r i s buffer pH 8.0 containing 20% sucrose, and frozen. After thawing, 1000 u.g DNase I (Sigma) was added to the mixture and Incubated at room temperature for 15 min. After Incubation, the c e l l s were u l t r a s o n i c a l l y disrupted (Tekmar sonic disruptor Model TM500, Tekmar Inc., New York, NY) by 4 x 30 s pulses at 280 W. The mixture was maintained in an ice bath during treatment. Subsequent to disruption, the mixture was centrifuged (3,000 x g, 10 min, 4 C) and the p e l l e t discarded. To the supernatant 0.4 g sodium l a u r y l sarcosinate (Sigma) was added and the mixture incubated 30 min at room temperature. After incubation, the mixture was ultracentrifuged (100,000 x g, 1 h, 4 C) (Sorvall model OTD-50 u t i l i z i n g a T865 fixed-angle rotor head, (Ingram and B e l l S c i e n t i f i c , Weston, ON) and the resultant p e l l e t washed 74 twice with d i s t i l l e d water. The outer membrane was suspended in 3.0 mL water and stored at -20 C. Soluble c e l l material preparation Soluble c e l l material was prepared in a manner similar to that of outer c e l l membrane material. In the preparation of soluble c e l l material no sodium l a u r y l sarcosinate was used to s o l u b i l i z e membrane structures. Subsequent to the i n i t i a l high speed centrifugation, the supernatant and the salmon colored p e l l e t were reserved. The supernatant was the soluble c e l l material. Proteinase a c t i v i t y The proteinase a c t i v i t y of the supernatant, e x t r a c e l l u l a r v e s i c l e s , outer c e l l membrane and soluble c e l l material was determined using casein substrate, as previously described. An aliquot of the e x t r a c e l l u l a r v e s i c l e suspension, maintained in an ice bath, was disrupted (s o l u b i l i z e d ) by a sonic disruptor (Tekmar) for 2 min at 280 W. The resultant disrupted mixture was centrifuged at 10,400 x g for 10 min at 4 C and the proteinase a c t i v i t y of the supernatant determined. Protein assay Protein assays were performed on the supernatant, e x t r a c e l l u l a r v e s i c l e s , outer c e l l membrane and soluble c e l l material using the BCA protein assay reagent (Pierce), as previously described. E x t r a c e l l u l a r v e s i c l e s and outer c e l l membrane material were s o l u b i l i z e d with 1.0% Triton X-100 (Pierce) prior to protein determination. 75 Electrophoretic techniques  Electrophoresis The proteinaceous components of the e x t r a c e l l u l a r v e s i c l e s and the outer c e l l membrane preparation were separated on horizontal slab sodium dodecyl sulfate containing polyacrylamide gels (SDS-PAG) using the buffer system of Laemmli (1970). The acrylamide concentration of the separating gel was 7.5% and that of the stacking gel was 4.0%. Samples were s o l u b i l i z e d by heating at 100 C for 2.0 min in a sample buffer containing 2.0% SDS and 5.0% 2-mercaptoethanol (Pharmacia). T h i r t y - s i x u.L of sample were added to the sample wells. Gels were run at 50 mA constant current u n t i l the bromophenol blue tracking dye reached the end of the gel. Gels were fixed in 11.4% t r i c h l o r o a c e t i c acid, 3.4% s u l p h o s a l i c y l i c acid, 30.0% methanol, and stained with 0.25% Coomassie Blue R250 in 50.0% methanol. The proteinase p r o f i l i n g technique described by Kelleher and Juliano (1984) was used in an attempt to p r o f i l e the proteinases present in the v e s i c l e s . In t h i s technique, casein i s covalently bound to glutaraldehyde-activated linear polyacrylamide. The conjugated casein, subsequent to incorporation ln SDS-Polyacrylamide gels, does not migrate during electrophoresis and remains susceptible to enzymlc attack following regeneration of enzyme a c t i v i t y . Enzyme regeneration requires complete removal of SDS. The gel i s i n i t i a l l y washed with water, followed by 60 min incubation in 10 mM T r i s buffer pH 6.6 containing 1.0% 76 T r i t o n X-100 (Sigma). The washed gels are transferred to development buffer (50 mM T r i s buffer pH 6.6) containing 10 mM C a + + and incubated for 24 h. After incubation, gels are stained with Coomassie Blue. I s o e l e c t r i c focusing (IEF) The supernatant and three preparations of the e x t r a c e l l u l a r v e s i c l e s were separated using 2.0 mm IEF polyacrylamide gels pH 4.0 to 6.0. One v e s i c l e preparation was unaltered, one was sonicated, as previously described and one was s o l u b i l i z e d with 2.0% Triton X-100 (Pierce). I s o e l e c t r i c focusing was performed on a LKB 2117 Multiphor II Electrophoresis system (Pharmacia Inc., Dorval, PQ). Samples were applied onto the gel using f i l t e r paper squares. Runs were conducted at 10 C for 2.5 h at 25 w. Power was supplied by a LKB MacroDrive 5 power supply (Pharmacia). Duplicate gels were made and either overlayed with previously poured 0.5 mm casein-agar gels, or stained with Coomassie Blue as described by Righetti (1987). Proteinase l o c a l i z a t i o n Active proteinase was l o c a l i z e d within IEF gels by overlaying the gel with a 0.5 mm 1.5% agar (Noble, BBL) gel containing 1.0% casein substrate. The casein substrate was the same preparation that was used for the proteinase assay. B r i e f l y , the s o l u b i l i z e d casein-agar mixture was heated to b o i l i n g , cooled to ca. 70 C and poured into a preheated (70 C) gel mould and cooled. The cooled casein-agar gel supported by a LKB GelBond (Pharmacia) f i l m was placed onto 77 the IEF gel and a weight applied in order to ensure contact between the two gels. The casein-agar gel was incubated for 24 h at 25 C. After incubation, hydrolyzed areas in the casein-agar gel were vi s u a l i z e d by immersion, for 10 min, in a 3.5% s u l f o s a l i c y l i c acid, 11.5% t r i c h l o r o a c e t i c acid s o l u t i o n . 78 RESULTS GROWTH OF P. f r a o i ON SOLID AND LIQUID MEDIUM In l i q u i d medium (Figure 7a), the P. fr a g i population increased from the i n i t i a l 2.0 x 10 5 cfu/mL to 2.0 x 10 9 cfu/mL after 90 h. The bacteria entered an exponential growth rate at 8 h that continued up to 20 h, followed by a stationary growth phase. The population began to decline somewhat after 56 h, but began to increase again after 76 h. On s o l i d medium (Figure 8a), the P. f r a g i population increased from the i n i t i a l 2.8 x 10 5 cfu/cm 2 to 8.2 x 10 9 cfu/cm 2 after 60 h. The growth rate became exponential at 4 h. From 16 to 32 h a much reduced rate of growth occurred. After 32 h, the culture entered a stationary growth phase. C e l l colonies on the membrane surface became v i s i b l e after 8 h. By 24 h a thick mat of c e l l s could be seen growing on the membrane surface. At 60 h the mat was several millimeters thick. Production of proteinase by P. fraqri Proteinase production, by P. f r a g i c e l l s grown in l i q u i d medium (Figure 7b), was f i r s t detected in the culture supernatant at 26 h and increased s t e a d i l y to a maximum at ca. 82 h. At t h i s point proteinase production began to decline. 79 FIGURE 7. P. f r a g i growth and proteinase production in l i q u i d medium. P. f r a g i were grown in TSB at 21 C for time periods up to 95 h. (a) Log P. f r a g i colony forming units/mL. The data points represent the geometric mean of six data points. Bars represent ranges. (b) Proteinase enzyme units/mL. The data points represent the arithmetic mean of six data points. Bars represent standard deviation. 80 TIME (hours) •J 50.00 cr s f ITl 40.00 r r . 81 FIGURE 8. P. fr a g i growth and proteinase production on s o l i d medium. P. fr a g i were grown on TSB + agar surfaces at 21 C for time periods up to 60 h. (a) Log P. f r a g i colony forming units/cm 2. The data points represent the geometric mean of s i x data points. Error bars represent ranges. (b) Proteinase enzyme units/mL. The data points represent the arithmetic mean of six data points. Error bars represent standard deviation. (c) Vesicles per unit perimeter. Transmission electron micrographs of P.fragi c e l l s at 20, 32, 40 and 56 h were examined. C e l l perimeters were calculated, and vesicles appearing on the perimeters were counted. Mean values of ve s i c l e s per um were plotted versus time. Error bars represent sample mean standard deviation. 82 3vESICLES/um PERIMETER | ^ ENZYME UNITS/mL o P fragi ( l o g c f u / c m 2 ) P r o t e i n a s e p r o d u c t i o n o f whole P. f r a g i c e l l s grown i n l i q u i d medium c o u l d n o t be d e m o n s t r a t e d a t t i m e p e r i o d s b e f o r e 26 h. P r o t e i n a s e p r o d u c t i o n , by P. f r a g i c e l l s grown on a s o l i d s u r f a c e o f a g a r g r o w t h medium, f i r s t a p p e a r e d a t a b o u t 4 h ( f i g u r e 8b), a p p r o x i m a t e l y 22 h s o o n e r t h a n i n l i q u i d c u l t u r e . The p r o d u c t i o n d e c l i n e d somewhat a t 8 h b u t began t o i n c r e a s e q u i c k l y a f t e r 16 h. A t 20 h t h e i n c r e a s e i n p r o d u c t i o n s l o w e d somewhat, but d i d i n c r e a s e u n t i l 32 h. A t t h i s p o i n t , p r o d u c t i o n d e c l i n e d s h a r p l y and d i d n o t r i s e s u b s t a n t i a l l y f o r 8 h. A t 40 h p r o t e i n a s e began t o i n c r e a s e s t e a d i l y f o r 16 h t o a maximum a t t h e 56 h mark. A t 56 h p r o d u c t i o n a g a i n d e c l i n e d t o t h e end o f t h e e x p e r i m e n t . E l e c t r o n m i c r o s c o p y / e x t r a c e l l u l a r v e s i c l e p r o d u c t i o n by P. f r a g i S c a n n i n g e l e c t r o n m i c r o s c o p y At 4 and 27 h t h e s u r f a c e o f P. f r a g i c e l l s grown i n l i q u i d medium a p p e a r e d w i t h o u t g l o b u l e s ( F i g u r e s 9 and 10). The c e l l s u r f a c e s d i d n o t change a p p r e c i a b l y u n t i l t h e end o f t h e l o g a r i t h m i c p h a s e . A t 76 h, d u r i n g t h e s t a t i o n a r y g r o w t h p h a s e , g l o b u l e s , c a . 100-200 nm In d i a m e t e r , were o b s e r v e d on t h e c e l l s u r f a c e s ( F i g u r e 11). T h i s p e r i o d c o i n c i d e d w i t h t h e c u l t u r e ' s maximum p r o t e i n a s e p r o d u c t i o n . On s o l i d medium, a t 4 h, g l o b u l e s were n o t o b s e r v e d on t h e P. f r a g i c e l l s u r f a c e s ( F i g u r e 12). A t 24 h, d u r i n g t h e l a t e l o g a r i t h m i c e a r l y s t a t i o n a r y p h a s e , t h e c e l l s u r f a c e s 84 FIGURE 9. Scanning electron micrograph of P. f r a g i grown in l i q u i d medium for 4 hours. No globules appear on the c e l l surface. FIGURE 10. Scanning electron micrograph of P. f r a g i grown in l i q u i d medium for 27 hours. No globules appear on the c e l l surface. 85 FIGURE 11. Scanning electron micrograph of P. f r a g i grown in l i q u i d medium for 76 hours. Globules approximately 100-200 nm in diameter were present on the c e l l surfaces. FIGURE 12. Scanning electron micrograph of P. f r a g i grown on s o l i d medium for 4 hours. Globules were not observed on the c e l l surfaces. 87 88 had a r a g g e d l o o k w i t h an a c c u m u l a t i o n o£ g l o b u l e s c a . 40-190 nm i n d i a m e t e r ( F i g u r e 13). T h i n s e c t i o n t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h s o f P. f r a g i grown i n l i q u i d medium f o r 4 h showed t h e o u t e r c e l l m a r g i n as a c omplex s t r u c t u r e composed o f s e v e r a l l a y e r s ( F i g u r e 14), t h e o u t e r most p o r t i o n b e i n g h i g h l y c o n v o l u t e d . The c y t o p l a s m i c p o r t i o n (CP) o f a P. f r a g i c e l l grown f o r 16 h ( F i g u r e 15) was s u r r o u n d e d by an i n n e r c y t o p l a s m i c membrane (CM). T h i s membrane was i n t u r n c o n t a i n e d by a c e l l w a l l (CW) s t r u c t u r e . T o g e t h e r t h e c y t o p l a s m i c membrane and t h e c e l l w a l l c o n t a i n t h e c y t o p l a s m and d e f i n e t h e o v e r a l l s hape o f t h e c e l l . Beyond t h e s e s t r u c t u r e s t h e p e r i p l a s m i c gap (PG) s e p a r a t e s t h e c e l l w a l l f r o m t h e o u t e r c e l l membrane (OM). I t was t h i s o u t e r membrane, c o m p l e t e l y s u r r o u n d i n g t h e c e l l , w h i c h gave t h e c o n v o l u t e d a p p e a r a n c e t o t h e c e l l o u t e r m a r g i n . The o u t e r c e l l membrane a t t i m e s a p p e a r e d t o b r e a c h t h e gap between t h e o u t e r c e l l membrane and t h e c e l l w a l l ( F i g u r e 15) . Young c e l l s , s u c h as t h o s e d e p i c t e d i n F i g u r e 16 (16 h ) , c o n t a i n e d a more or l e s s homogeneous c y t o p l a s m w i t h no o b v i o u s i n c l u s i o n s o r v o i d s . The components w h i c h make up t h e c e l l m a r g i n s were d i s t i n c t and w e l l d e f i n e d . As t h e c e l l s a g ed, s u c h as t h a t d e p i c t e d i n F i g u r e 17 (44 h ) , t h e p e r i p l a s m i c gap w i d e n e d and t h e o u t e r c e l l membrane 89 FIGURE 13. Scanning electron micrograph of P. fr a g i grown on s o l i d medium for 24 hours. An accumulation of globules, approximately 40-190 nm in diameter, were observed on the c e l l surfaces. FIGURE 14. Transmission electron micrograph of P. f r a g i grown in l i q u i d medium for 4 hours. C e l l s were fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide and suspended in agar. Agar blocks were dehydrated in ethanol and i n f i l t r a t e d with EPON 812. Embedded c e l l s were sectioned, and stained with uranyl acetate and lead c i t r a t e . 90 91 FIGURE 15. Transmission electron micrograph of P. f r a g i grown in l i q u i d medium for 16 hours. (OM) outer membrane; (PG) periplasmic gap; (CP) cytoplasm; (CM) cytoplasmic membrane; (CW) c e l l wall. 92 FIGURE 16. Transmission electron micrograph of P. f r a g i grown in l i q u i d medium for 16 hours. FIGURE 17. Transmission electron micrograph of P. f r a g i grown in l i q u i d medium for 44 hours. Note thickening of the outer membrane and increase in periplasmic gap. 94 thickened. This gap widening and membrane thickening became extreme at 92 h (Figure 18). Although the outer c e l l membrane was highly convoluted on c e l l s grown in l i q u i d medium, no evidence of ex t r a c e l l u l a r v e s i c l e s or globule formation could be seen. Transmission electron micrographs of P. fr a g i grown on a s o l i d surface for 4 h (Figure 19), again showed the periplasmic gap separating the outer c e l l membrane from the cytoplasmic membrane-cell wall structures. At 20 h (Figure 20) most c e l l s were surrounded by many round v e s i c l e s more or less bound to the c e l l surface. A l l the v e s i c l e s , between 30 and 40 nm ln diameter, appeared to originate from the outer c e l l membrane. In some instances the ve s i c l e s appeared to be attached to the c e l l by a stalk. The individual v e s i c l e s , when examined in section, were surrounded with a membrane similar in appearance to that of the outer c e l l membrane surrounding in t a c t c e l l s . Those c e l l s a c t i v e l y shedding p a r t i c l e s appeared to have intact c e l l wall-cytoplasmic membrane structures. The c e l l wall did not appear to contribute to v e s i c l e formation. The ve s i c l e s began appearing between 4 and 20 h. The vesicl e s continued to be present on the c e l l surfaces at 32 h (Figure 21) and at 56 h (Figure 22). In a sim i l a r fashion to P. f r a g i grown in l i q u i d medium, the outer c e l l membrane of c e l l s grown on a s o l i d surface thickened as the culture aged. 96 FIGURE 18. Transmission electron micrograph of P. fr a g i grown in l i q u i d medium for 92 hours. Note continued thickening of the outer membrane and increased periplasmic gap widening. The cytoplasm seems to contain voids or inclusions not seen in e a r l i e r c e l l s . 97 FIGURE 19. Transmission electron micrograph of P. f r a g i grown on s o l i d medium for 4 hours. Note the absence of e x t r a c e l l u l a r v e s i c l e s . 99 100 FIGURE 20. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h o f P. f r a g i grown on s o l i d medium f o r 20 h o u r s s h o w i n g p r e s e n c e o f v e s i c l e s . F i r s t o b s e r v e d o c c u r r e n c e o f e x t r a c e l l u l a r v e s i c l e s on c e l l s u r f a c e s . 101 102 FIGURE 21. Transmission electron micrograph of P. f r a g i grown on s o l i d medium for 32 hours. Vesicles continued to be present on the c e l l surfaces. 103 104 FIGURE 22. Transmission electron micrograph of P. f r a g i grown on s o l i d medium for 56 hours. Maximum numbers of ve s i c l e s per unit perimeter of c e l l s . 105 106 V e s i c l e s h e d d i n g v e r s u s p r o t e i n a s e p r o d u c t i o n by P.  f r a g i SEM o f P. f r a g i c e l l s grown i n l i q u i d medium r e v e a l e d g l o b u l e s ( F i g u r e 11) 100 t o 200 nm i n d i a m e t e r . These g l o b u l e s were v i s i b l e o n l y a f t e r 76 h, t h e a p p r o x i m a t e t i m e p e r i o d when p r o t e i n a s e p r o d u c t i o n was a t a maximum ( F i g u r e 7b). T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h s o f c e l l s grown i n l i q u i d medium f a i l e d t o d i s p l a y e v i d e n c e o f v e s i c l e s h e d d i n g a t a ny o f t h e t i m e p e r i o d s e x amined. SEM r e v e a l e d g l o b u l e s , a p p r o x i m a t e l y 40 t o 190 nm i n d i a m e t e r , on P. f r a g i c e l l s grown f o r 24 h on s o l i d medium ( F i g u r e 13). T h i s t i m e p e r i o d c o i n c i d e d w i t h i n c r e a s e d p r o t e i n a s e p r o d u c t i o n by c e l l s grown on s o l i d medium ( F i g u r e 8b). TEM r e v e a l e d t h a t e x t r a c e l l u l a r v e s i c l e s a p p e a r e d a t 20 h ( F i g u r e 20). T h i s p o i n t i n t i m e c o r r e s p o n d e d w i t h a d r a m a t i c i n c r e a s e i n t o t a l p r o t e i n a s e ( F i g u r e 8 b ) . A c o u n t o f t h e e x t r a c e l l u l a r v e s i c l e s p e r u n i t p e r i m e t e r o f c e l l s grown on s o l i d medium f u r t h e r s u p p o r t e d t h e a s s o c i a t i o n o f v e s i c l e a p p e a r a n c e and p r o t e i n a s e p r o d u c t i o n ( F i g u r e 8c). CELL AND GROWTH MEDIUM FRACTIONATION I s o l a t i o n o f e x t r a c e l l u l a r v e s i c l e s Pseudomonas f r a g i was grown t o t h e s t a t i o n a r y g r o w t h phase i n l i q u i d c u l t u r e (82 h a t 21 C) and t h e c u l t u r e s u p e r n a t a n t c o l l e c t e d . Ammonium s u l f a t e p r e c i p i t a t i o n and 107 subsequent d i a l y s i s <of duplicates isolates yielded a mean of 11.55 + 1.60 mg (mean of duplicate i s o l a t i o n s + one half the range) of p a r t i c l e s per l i t r e of culture supernatant. The p a r t i c l e s were not a r t i f a c t s , since uninoculated culture medium did not y i e l d such p a r t i c l e s . After ammonium sulfate p r e c i p i t a t i o n and subsequent d i a l y s i s , the p a r t i c l e s r e a d i l y associated together producing a loosely packed, black coloured precipitate which formed after less than one minute. This tendency to flocc u l a t e made p a r t i c l e separation by low speed centrlfugation from the suspending menstrum quite easy. The black pr e c i p i t a t e was e a s i l y resuspended by shaking. Electron microscopy Examination of the vesi c l e preparation mounted and stained on Formvar coated grids (Figure 23) revealed the presence of a myriad of spherical v e s i c l e s ca. 20 nm in diameter. These ve s i c l e s were the same size as those p a r t i c l e s observed on the surface of P. f r a g i , grown on s o l i d medium, when the sectioned c e l l s were examined by TEM (Figure 21). A l l of the vesicl e s appeared to be consistent in s i z e . No evidence of p i l i could be seen. Outer-cell membrane preparation Outer-cell membrane material was isolated by selective s o l u b i l i z a t i o n of DNA and cytoplasmic membrane. High speed centrlfugation yielded a j e l l y - l i k e p e l l e t . The outer c e l l membrane material contained approximately 5.91 ± 0.83 mg protein/mL. 108 FIGURE 23. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h o f n e g a t i v e l y s t a i n e d i s o l a t e d e x t r a c e l l u l a r v e s i c l e s . E x t r a c e l l u l a r v e s i c l e s were i s o l a t e d by ammonium s u l f a t e p r e c i p i t a t i o n o f c u l t u r e s u p e r n a t a n t . P. f r a g i was grown a t 21 C . Washed v e s i c l e s were p l a c e d on t h e s u r f a c e o f Formvar c o a t e d E.M. g r i d s and s t a i n e d w i t h u r a n y l a c e t a t e . V e s i c l e s were a p p r o x i m a t e l y 20 nm i n d i a m e t e r . 109 100 n m 110 B u o y a n t d e n s i t y The b u o y a n t d e n s i t y o f t h e e x t r a c e l l u l a r v e s i c l e s was 1.153 g/mL. I s o e l e c t r i c f o c u s i n g ( I E F ) E x t r a c e l l u l a r v e s i c l e s and s u p e r n a t a n t A p p r o x i m a t e l y 30 U L o f s u p e r n a t a n t , and 50 U L o f u n t r e a t e d e x t r a c e l l u l a r v e s i c l e s , e x t r a c e l l u l a r v e s i c l e s s o l u b i l i z e d by T r i t o n X-100 and e x t r a c e l l u l a r v e s i c l e s s o l u b i l i z e d by s o n i c a t i o n were s e p a r a t e d by I E F . S e v e r a l components w i t h i s o e l e c t r i c p o i n t s between pH 4.0 and 6.0 were r e s o l v e d w i t h C o o m a s s i e B l u e s t a i n ( F i g u r e 24). The s u p e r n a t a n t p r o d u c e d t h e most bands i n t h e g e l , f o l l o w e d by t h e s o n i c a t e d v e s i c l e s , t h e T r i t o n X-100 t r e a t e d v e s i c l e s and t h e u n t r e a t e d v e s i c l e s . A c a s e i n - a g a r g e l was o v e r l a i d on a d u p l i c a t e I E F g e l . A p p r o x i m a t e l y 60 ]XL o f s u p e r n a t a n t and 50 UL o f e a c h o f t h e t h r e e e x t r a c e l l u l a r v e s i c l e p r e p a r a t i o n s were a p p l i e d . V i s u a l i z a t i o n w i t h TCA, s u l p h o s a l i c y l i c a c i d f i x a t i v e , r e v e a l e d a r e g i o n o f h y d r o l y s i s a t a p o i n t e q u i v a l e n t t o pH 5.2 on t h e I E F g e l , w h i c h c o r r e s p o n d e d t o a major band on a l l f o u r s a m p l e s ( F i g u r e 25). O u t e r - c e l l m e m b r a n e / e x t r a c e l l u l a r v e s i c l e component p r o f i l e s E l e c t r o p h o r e s i s , on SDS-PAG, o f t h e e x t r a c e l l u l a r v e s i c l e s and t h e i s o l a t e d P. f r a g i o u t e r membrane ( F i g u r e 26) r e v e a l e d s e v e r a l p r o t e i n bands w i t h m o l e c u l a r w e i g h t s 111 FIGURE 24. IEF separation of culture supernatant and ex t r a c e l l u l a r v e s i c l e proteins. Proteins were separated on 5.0% polyacrylamide gel containing pH 4-6 ampholyte. Bands were vi s u a l i z e d with Coomassie Blue protein s t a i n . (A) Culture supernatant. Approximately 448 u.g of protein was applied to the gel; (B) Untreated e x t r a c e l l u l a r v e s i c l e s . Approximately 225 ug of protein was applied to the gel; (C) E x t r a c e l l u l a r v e s i c l e s s o l u b i l i z e d in 2.0% Triton X-100. Approximately 225 Jig of protein was applied to the gel; (D) E x t r a c e l l u l a r v e s i c l e s s o l u b i l i z e d by sonication. Approximately 225 }ig of protein was applied to the gel; (E) Stained protein bands with the same pi and possessing proteinase a c t i v i t y . 112 113 FIGURE 25. Zymogram of IEF separated culture supernatant and ex t r a c e l l u l a r v e s i c l e s . Proteins were separated on 5.0% polyacrylamide gel containing pH 4-6 ampholyte. Areas containing active proteinase were v i s u a l i z e d by overlaying the IEF gel with a casein-agar gel and incubating for 24 h at 25 C. Gel was developed with a TCA/sulphosalicylic acid f i x a t i v e . The zymogram was b a c k l i t and photographed against a black background. Due to the f i x a t i v e , the casein was opaque and appeared grey, while the translucent hydrolyzed areas around pH 5.2 (indicated by an E) revealed the black background. (A) Untreated e x t r a c e l l u l a r v e s i c l e s . Approximately 225 ug of protein was applied to the gel; (B) E x t r a c e l l u l a r v e s i c l e s s o l u b i l i z e d in 2.0% Triton X-100. Approximately 225 ug of protein was applied to the gel; (C) E x t r a c e l l u l a r v e s i c l e s s o l u b i l i z e d by sonication. Approximately 225 ug of protein was applied to the gel; (D) Culture supernatant. Approximately 900 Ug of protein was applied to the gel. 114 115 FIGURE 26. SDS-PAGE separation of e x t r a c e l l u l a r v e s i c l e and outer c e l l membrane proteins. Proteins were separated on a 7.5% acrylamide gel. Stacking gel consisted of 4.0% acrylamide. Samples were s o l u b i l i z e d in a sample buffer containing 2.0% SDS and 5.0% 2-mercaptoethanol. (EM) e x t r a c e l l u l a r v e s i c l e material. Approximately 225 Hg of protein was applied to the gel; (OM) P. f r a g i outer c e l l membrane, approximately 216 u.g of protein was applied to the g e l . 116 8 6 K 117 between 20,000 and 86,000 d a l t o n s . The o u t e r membrane c o n t a i n e d about 9 major bands and some minor bands. The major bands o c c u r r e d between 20,000 and 47,000 D. The e x t r a c e l l u l a r v e s i c l e s showed f o u r major bands and s e v e r a l minor bands. A comparison of the e l e c t r o p h o r e t i c bands showed the s m a l l bands of the e x t r a c e l l u l a r v e s i c l e s c o r r e s p o n d e d t o major bands i n the I s o l a t e d o u t e r c e l l membrane m a t e r i a l . The e x t r a c e l l u l a r v e s i c l e s may, a t l e a s t i n p a r t , be made up of o u t e r c e l l membrane m a t e r i a l . E x t r a c e l l u l a r v e s i c l e / o u t e r c e l l membrane enzyme  l o c a l i z a t i o n . L o c a l i z a t i o n of the a c t i v e p r o t e i n a s e was und e r t a k e n i n normal S D S - p o l y a c r y l a m i d e g e l or S D S - p o l y a c r y l a m i d e g e l bound c o v a l e n t l y by g l u t a r a l d e h y d e t o c a s e i n . Subsequent t o e l e c t r o p h o r e s i s , r e g e n e r a t i o n of enzyme a c t i v i t y was attempted i n the presence of Ca + + i o n a f t e r removal of SDS w i t h a n o n - i o n i c d e t e r g e n t wash. The r e g e n e r a t e d enzyme was the n f r e e t o h y d r o l y z e the c a s e i n : i n the case of normal S D S - p o l y a c r y l a m i d e g e l , p r e s e n t i n a c a s e i n - a g a r g e l o v e r l a y ; i n the case of the c o v a l e n t l y bound g e l , found i n the c a s e i n - p o l y a c r y l a m i d e g e l i t s e l f . For e l e c t r o p h o r e s i s , samples were p r e p a r e d w i t h o u t m e r c a p t o e t h a n o l or he a t . In both i n s t a n c e s , enzyme c o u l d not be l o c a l i z e d d e s p i t e s e v e r a l a t t e m p t s t o r e j u v e n a t e the enzyme, i n c l u d i n g e x t e n s i v e washings t o e l i m i n a t e SDS. 118 Composition of the components The culture supernatant contained 34.71 enzyme units/mL (Table IV). The protein content was 0.31 mg/mL. The s p e c i f i c a c t i v i t y of the supernatant was 111.97 enzyme units/mg protein. The t o t a l amount of proteinase in one L was 34,710.00 enzyme units. The e x t r a c e l l u l a r v e s i c l e s contained 51.92 enzyme units/mL. The protein content was 0.45 mg/mL. The s p e c i f i c a c t i v i t y of the vesicl e s was 115.38 enzyme units/mg protein. The t o t a l amount of proteinase present in the ves i c l e s associated with one L of P. fr a g i culture was 103.80 enzyme units. The soluble c e l l material contained 16.32 enzyme units/mL. The protein content of the material was 11.71 mg/mL. The s p e c i f i c a c t i v i t y of the soluble material was 1.39 enzyme units/mg protein. The t o t a l amount of proteinase present in c e l l s from one L was 244.80 enzyme units. A freeze-dried e x t r a c e l l u l a r v e s i c l e preparation contained 11.7% (dry weight) protein. After sonic disruption of the o r i g i n a l p a r t i c l e suspension, the soluble portion of the resultant mixture contained 1.7% (dry weight) prote i n . The proteinase content of the sonicated v e s i c l e s was 58.52 enzyme units/mL. The protein content of the soluble material l e f t after sonication was 0.09 mg/mL. 119 Table IV. Composition of v e s i c l e s , soluble c e l l material and supernatant. ANALYSIS VESICLES VESICLES SOLUBLE SUPERNATANT SONICATED CELL MAT-ERIAL PROTEINASE^ 51 .92 + 2 .40 58.52+1 .12 16.32±1.80 34.71±2. 82 PROTEIN8 0 .45±0 .05 0.09+0 .00 11.71+0.46 0.31+0. 03 SPECIFIC ACTIVITY 0 115 .38 680.50 1.39 111.97 TOTAL PROTEINASE 103 .80 117.00 244.80 34710.00 DRY WEIGHT0 8 .56±1 .15 CARBOHYDRATE0 1 .04 + 0 .04 DRY WEIGHTE 11 .55±1 .60 Determinations are the res u l t of duplicate experiments, grown in one L quantities for 82h at 21 C. Results are means of duplicate experiments + one half the range. A Enzyme units/mL. B Milligrams/mL. c Enzyme units/mg protein. 0 Milligrams/milligram protein. E Milligrams. 120 The s p e c i f i c a c t i v i t y of the crude v e s i c l e preparation was 115.38 enzyme units/mg protein while that of the sonicated preparation was 680.50 enzyme units/mg protein. E x t r a c e l l u l a r v e s i c l e s contained 1.04 mg carbohydrate/mg protein (Table IV). BACTERIOCINS Ext r a c e l l u l a r v e s i c l e s , The electron micrographs of P. f r a g i grown to the stationary growth phase (48 h) on TSB + agar revealed spherical membrane ve s i c l e s ca. 20 nm in diameter. These v e s i c l e s , bound to the c e l l surface, or free in the culture medium, bore some resemblance to bacteriocin p a r t i c l e s produced by B. uniformis and described by Austin-Prather and Booth (1984). Supernatant, containing p a r t i c l e s produced by P. fr a g i and described in t h i s study, f a i l e d to e l i c i t zones of i n h i b i t i o n when applied to TSA plates containing indicator organisms. In a l i k e fashion, i t did not a l t e r the growth rate of indicator organisms as measured by absorbance over a 24 h period. Bacteriocin induction. Pseudomonas f r a g i ATCC 4973 also f a i l e d to show bac t e r i o c i d a l a c t i v i t y towards the indicator organisms despite attempts to force bacteriocin production with U.V. or gamma ionizing radiation, or mitomycin C inducing agents. Culture supernatant from P. f r a g i ATCC 4973 grown in TSB for 48 h, when treated with 20.0 u.g/mL mitomycin C 121 d i s p l a y e d c l e a r z o n e s on TSA p l a t e s c o n t a i n i n g i n d i c a t o r o r g a n i s m s and a l t e r e d t h e growth r a t e o f i n d i c a t o r o r g a n i s m s , as measured b y a b s o r b a n c e o v e r t i m e . T h i s i n h i b i t o r y e f f e c t was due e n t i r e l y t o m i t o m y c i n C, and n o t b a c t e r i o c i n , s i n c e c o n t r o l s c o n s i s t i n g o f u n i n o c u l a t e d c u l t u r e medium c o n t a i n i n g 20 u.g/mL m i t o m y c i n C d i s p l a y e d i d e n t i c a l i n h i b i t o r y p r o p e r t i e s . When P. f r a g i c u l t u r e s u p e r n a t a n t s were d i a l y z e d t o remove m i t o m y c i n C t h e y l o s t t h e i r a b i l i t y t o a l t e r g r o w t h r a t e s o f t h e i n d i c a t o r o r g a n i s m s . OPTIMIZATION O p t i m i z a t i o n o f p r o t e i n a s e p r o d u c t i o n F r a c t i o n a l f a c t o r i a l P r i o r t o o p t i m i z a t i o n , a T a g u c h i (1957) f r a c t i o n a l f a c t o r i a l d e s i g n e x p e r i m e n t was c o n d u c t e d t o d e t e r m i n e t h e i m p o r t a n c e o f e a c h f a c t o r . The e x p e r i m e n t s shown i n T a b l e V were e v a l u a t e d . A n a l y s i s o f t h e g r o w t h d a t a i n T a b l e V i a r e v e a l e d t h a t a l l t h e f a c t o r s s i g n i f i c a n t l y a f f e c t e d g r o w t h o f P. f r a g i (P<.01). Time was t h e most s i g n i f i c a n t f a c t o r , f o l l o w e d by oxygen, g l u t a m i n e , t e m p e r a t u r e and pH. R e p l i c a t e s e f f e c t s were n o t s i g n i f i c a n t (P>.05) ( T a b l e V l b ) . A n a l y s i s o f p r o t e i n a s e r e s p o n s e d a t a ( T a b l e V i l a ) r e v e a l e d t h a t o xygen l e v e l , g l u t a m i n e c o n c e n t r a t i o n , i n c u b a t i o n t e m p e r a t u r e and t i m e were a l l s i g n i f i c a n t f a c t o r s (P<.01). I n i t i a l pH was s i g n i f i c a n t a t t h e 5% c o n f i d e n c e l e v e l . Oxygen was t h e most s i g n i f i c a n t f a c t o r . I n g e n e r a l h i g h e r o xygen l e v e l s (>10%, or 4.2ppm) f a v o r e d g r e a t e r 122 r TABLE V. F r a c t i o n a l f a c t o r i a l experiments EXP TEMP TIME pH GLUT < 3 * OXYGEN PROTEINASE P . f r a g i # (C) (h) (mmo1e) (%) (ppm) (units/mL) (cfu/mL) T l ( 2 ) 2 0 32 7 7 10 4.2<1) 5.2 (0.0)*6.9X10 8 T2 20 32 9 125 30 12.9 6.2 (1.0) 3.8X10 9 T3 20 72 7 7 30 12.9 6.8 (0.8) 1.1X10J T4 20 72 9 125 10 4.2 3.0 (0.0) 7.7X10 7 T5 30 32 7 125 30 10.7 5.1 (1.9) 3.2X10 9 T6 30 32 9 7 10 3.6 2.6 (0.5) 1.4X10 9 T7 30 72 7 125 10 3.6 1.9 (0.3) 2.7X10 7 T8 30 72 9 7 30 10.7 6.9 (0.3) 4.1X10 5 (1) P a r t s per m i l l i o n of d i s s o l v e d oxygen contained l n the growth medium. (2) "T" experiments r e f e r to Taguchi experiments and are synonymous with f r a c t i o n a l f a c t o r i a l experiments. (3) GLUT = glutamine (mmole n i t r o g e n / L ) . * Standard d e v i a t i o n . F i g u r e s are the r e s u l t s of 4 experiments. 123 TABLE Via. Analysis of f r a c t i o n a l f a c t o r i a l growth data variance (Factors). Source of va r i a t i o n ss df ms s i g n i f temp C. time PH glutamine oxygen % error 1.26xl0 1 8 2.80xl0 1 9 2.82xl0 1 7 1.97xl0 1 9 1.98xl0 1 9 2.00xl0 1 7 1 1 1 1 1 26 1.26xl0 1 8 163.85 2.80xl0j- 9 3641.09 2.82xl0 1 7 36.67 1.97xl0 1 9 2561.77 1.98xl0 1 9 2574.77 7.69xl0 1 5 ** ** ** t o t a l (**) P<.01 6.92x10 19 31 TABLE VIb. Analysis of f r a c t i o n a l f a c t o r i a l growth data variance (Replicates), Source of va r i a t i o n ss df ms F s i g n i f . Experiments Replicates error 6.84xl0 1 9 4.97xl0 1 6 6.86xl0 1 7 7 8 16 9.77xl0 1 8 6.22xl0 1 5 4.29xl0 1 6 1570. 0. 74 15 ** ns t o t a l 6.91xl0 1 9 31 (**) P<.01 (ns) not s i g n i f i c a n t P>.05 F s t a t i s t i c c a l c u l a t i o n for experiments = m s ( e x p e r i m e n t s ) / m s ( r e p l i c a t e s ) 124 TABLE V i l a . A n a l y s i s o f f r a c t i o n a l f a c t o r i a l p r o t e i n a s e d a t a v a r i a n c e ( F a c t o r s ) . Source of va r i a t i o n ss df ms F s i g n i f . temp C. 15. ,68 1 15, ,68 50. , 58 ** 11 me h 3, ,25 1 3, .25 10. ,48 ** pH 2. .18 1 2, .18 7. ,03 * glutamine 17. .19 1 17, .19 55. ,45 ** oxygen % 77, ,30 1 77, .30 249. ,35 ** error 7. .94 26 0, .31 t o t a l 123, .54 31 (**) P<.01 (*) P<.05 TABLE V l l b . A n a l y s i s o f f r a c t i o n a l f a c t o r i a l p r o t e i n a s e d a t a v a r i a n c e ( R e p l i c a t e s ) . Source of var i a t i o n ss df ms F s i g n i f . Experiments 107. 34 7 15.33 109 . 50 ** Replicates 1. 15 8 0.14 0. 15 ns error 15. 05 16 0.94 t o t a l 123.54 31 (experiments) ^ (**) P<.01 (ns) not s i g n i f i c a n t P>.05 F s t a t i s t i c c a l c u l a t i o n for experiments = m s (replicates) 125 p r o t e i n a s e p r o d u c t i o n . R e p l i c a t e e f f e c t s were n o t s i g n i f i c a n t (P>.05) ( T a b l e s V l l b ) . C e n t r o i d s e a r c h o p t i m i z a t i o n The same f a c t o r s u s e d i n t h e f r a c t i o n a l f a c t o r i a l e x p e r i m e n t s were u s e d i n t h e c e n t r o i d s e a r c h p r o c e d u r e . The r e s p o n s e f a c t o r was a g a i n enzyme u n i t s / m L . The e x p e r i m e n t s ( T a b l e V i l l a ) were e v a l u a t e d f o r p r o t e i n a s e p r o d u c t i o n and c e l l numbers. The b e s t r e s p o n s e (7.7 enzyme u n i t s / m L ) o c c u r r e d w i t h e x p e r i m e n t number S3. T h i s e x p e r i m e n t a l s o s u p p o r t e d t h e b e s t c e l l g r o w t h . The n e x t b e s t r e s p o n s e was e x p e r i m e n t S4 (5.4 enzyme u n i t s / m L ) , w h i l e e x p e r i m e n t S I was t h e w o r s t (0.0 enzyme u n i t s / m L ) . From t h e r e s u l t s o f t h e s i x S p e n d l e y e x p e r i m e n t s ( T a b l e V i l l a ) , s i x c e n t r o i d s were s u g g e s t e d . The e x p e r i m e n t a l c o n d i t i o n s f o r f o u r c e n t r o i d s were e v a l u a t e d and t h e r e s p o n s e v a l u e s a r e shown i n T a b l e I X a . A n a l y s i s o f v a r i a n c e o f t h e S p e n d l e y m a t r i x e x p e r i m e n t s and c e n t r o i d e x p e r i m e n t s ( T a b l e V H I b and T a b l e I X b ) , showed t h a t b o t h e x p e r i m e n t a l u n i t s and r e p l i c a t e s c o n t r i b u t e d s i g n i f i c a n t l y t o t h e o v e r a l l v a r i a n c e (P<.01). S i n c e t h e i n i t i a l c e n t r o i d r e s p o n s e v a l u e ( c e n t r o i d C7) was g r e a t e r t h a n o r e q u a l t o t h e b e s t r e s p o n s e v a l u e o b t a i n e d f r o m t h e s t a r t i n g S p e n d l e y m a t r i x , e v a l u a t i o n o f a m a j o r i t y o f t h e r e m a i n i n g c e n t r o i d s was c a r r i e d o u t . C e n t r o i d 11 w i t h a r e s p o n s e v a l u e o f 8.4 enzyme u n i t s / m L was t h e h i g h e s t r e s u l t o b t a i n e d f r o m a l l e x p e r i m e n t s , i n c l u d i n g 126 TABLE Villa. I n i t i a l Spendley matrix experiments and response values. EXP TEMP TIME pH GLUT B OXYGEN PROTEINASE P.fragi (C) (h) (mmole) (%) (ppm) (units/mL) (cfu/mL) SI 1.0 4 5.0 200 00.0 0.0 0.0 ( 0 . 0 ) * 5.4X10? S2 36.6 18 6.0 292 10.3 3.1 4 .7 (1.1) 2.0x10; 2.1X10 9 S3 9.0 66 6.0 292 10.3 5.4 7.7 (0.6) S4 9.0 18 9.6 292 10.3 5.4 5.4 (0.0) 3 .6X10 7 2.1X10 8 S5 9.0 18 6.0 610 10.3 5.4 4.0 (0.7) S6 9.0 18 6.0 292 45.6 25.2 4.8 (0 .4) 3 .5X10 8 S 7 A 9.0 18 6.0 292 10.3 5.4 6.2 (0.4) 3 . 4 X 1 0 8 Mean value for the seven experiments 4.7. Standard error of £he mean 2.2. Standard error of the mean. Figures are the re s u l t of 4 experiments. A This experiment was an additional experiment not meant to be included in the optimization. It was included in the ANOVA only. B GLUT = glutamine (mmole nitrogen / L ) . TABLE VHIb. Analysis of Spendley proteinase data variance (Replicates). Source of va r i a t i o n ss df ms F s i g n i f . Experiments 138.00 6 23.00 16.79 ** Replicates 9 .58 7 1. 37 1957.14 ** error 0.01 14 7.00xl0" 4 t o t a l 147.59 27 (**) P<.01 F s t a t i s t i c c a l c u l a t i o n for experiments = ms (experiments) ^ m s (replicates) 127 TABLE IXa. Centroid experiments and their response values EXP TEMP TIME PH GLUT A OXYGEN PROTEINASE P.fragi # (C) (h) (mmole) (%) (ppm) (units/mL) (cfu/mL) C7 14.5 27.6 6.7 356 17.3 8.1 7.7 (0.0) * 4.4X109 C8 15.6 29.5 6.9 305 18.7 8.8 5.0 (0.0) 4.4X109 CIO 11.9 34.6 7.3 311 15.4 7.6 5.9 (0.5) 3.5X109 C l l 12.5 37.9 6.8 314 16.4 7.4 8.4 (0.0) 3.2X109 Mean value for the four experiments 6.8. Standard error of £he mean 1.4. Standard error of the mean. Figures are the resu l t of 4 experiments. A GLUT = glutamine (mmole nitrogen / L ) . TABLE IXb. Analysis of centroid proteinase data variance (Replicates). Source of va r i a t i o n ss df ms F s i g n i f . Experiments 30.39 3 10.13 42.21 ** Replicates 0.95 4 0.24 80.00 ** error 0.02 8 3.00x10-3 t o t a l 31.36 15 (**) P<.01 F s t a t i s t i c c a l c u l a t i o n for experiments = m s(experiments) ' m s ( r e p l i c a t e s ) 128 those of the f r a c t i o n a l f a c t o r i a l . The factor levels for centroid 11 are shown in Table X. The f r a c t i o n a l f a c t o r i a l experiments showed that oxygen concentration was the most s i g n i f i c a n t factor. Response values at 30% (10.7-13.0 ppm) oxygen were higher than at 10% (3.1-5.4 ppm) (Table V). The Spendley experiments showed that at 45.6% (25.2 ppm) oxygen, response values were lower than those at 30% oxygen (Table V i l l a ) . The centroid experiments showed that 16.4% (7.4 ppm) oxygen had the highest response value overal l (Table iXa). To test whether or not oxygen at t h i s concentration was close to the optimum response value, oxygen levels were shi f t e d to bracket t h i s projected optimum point. These experimental conditions and the responses obtained are shown in Table XI. In general, r a i s i n g or lowering the oxygen l e v e l away from 16.4% (7.4 ppm) oxygen resulted in lower proteinase production. Mapping Data points from the above experiments, mapped in Figures 27, 28 and 29, show the approximate values of the factors where the optimum response may e x i s t . Broken lines represent curves manually added to estimate proposed directions determined by quadratic curve f i t t i n g . These curves could not be plotted because of limited map space. These maps indicate that the above values ( i . e . centroid 11) are close to the maximum projected response in each factor. 129 TABLE X. Suggested optimum values for proteinase production by P. fr a g i FACTORS LEVELS Temperature 12.5 C Time 37.9 h pH 6.8 Glutamine 314.0 mmole Nitrogen/L Oxygen 16.4% (7.4 ppm) 130 TABLE XI. Simultaneous s h i f t experiments and the i r response values. EXP TEMP TIME PH GLUTA OXYGEN PROTEINASE P .fragi # (C) (h) (mmole) (%) (ppm) (units/mL) (cfu/mL) SSI 10. 4 62 7 468 19.2 10.1 7.3 (0.5) *1.9X109 SS2 8. 0 68 7 468 10.2 5.5 7.9 (0.1) 3.1X109 A GLUT = glutamine (mmole nitrogen / L). Standard deviation. Figures are the resu l t of 4 experiments. 131 FIGURE 27. Optimization map o£ factors time and temperature. (a) Optimum time appears ca. 45 h. (b) Optimum temperature appears ca. 13 C. 132 FIGURE 28. Optimization map of factors glutamine and pH. (a) Optimum glutamine l e v e l appears ca. 300 mmole nltrogen/L. (b) Optimum pH appears ca. pH 7. 134 FIGURE 29. Optimization map of factor oxygen. Optimum oxygen l e v e l appears ca. 16.4% oxygen (7.4 ppm). 136 As optimization continues, new factor ranges, as suggested by centroid search and mapping, w i l l continue to decrease. The maps shown in Figures 27, 28 and 29 show data points c l u s t e r i n g about a narrow range corresponding to the optimum point. Continuation of optimization would resu l t in a further narrowing of the factor ranges. In p a r t i c u l a r , continuation of optimization would require a temperature factor range of less than one degree Celsius. This fine temperature control was not possible with the equipment ava i l a b l e . As a r e s u l t , a p r a c t i c a l l i m i t had been reached, and thus further optimization t r i a l s were not carried out. Thin section transmission electron microscopy A representative sample of P. f r a g i c e l l s from the optimization t r i a l s , shown in Table XII, were selected for microscopic examination. Transmission electron micrographs (Figure 30a;b;c and Figure 31a;b) revealed the presence of e x t r a c e l l u l a r v e s i c l e s on the surface of some c e l l s grown in l i q u i d culture. At high temperatures c e l l s were found both with (experiment T6, Figure 31a) and without (experiment T8, Figure 31b) e x t r a c e l l u l a r v e s i c l e s on the c e l l surface. The same i s true for P. f r a g i grown at low temperatures. Experiment S6 (Figure 30c) shows no v e s i c l e s attached to the c e l l s , while experiments S5 and S3 (Figure 30b;a) show attached v e s i c l e s . 138 TABLE XII. Optimization t r i a l s selected for electron microscopy. EXP TEMP TIME PH GLUTA OXYGEN 1 (C) (h) (mmole) (%) (ppm) T8 30 72 9 7 30.0 10.7 T6 30 32 9 7 10.0 3.6 S5 9 18 6 610 10.3 5.4 S6 9 18 6 292 45.6 25.2 S3 9 66 6 292 10.3 5.4 GLUT = glutamine (mmole nitrogen / L ) . 139 FIGURE 30. Transmission electron micrographs of P. fr a g i used in the optimization experiments. (a) S3-Cells grown at 9 C; 66 h; pH 6; glutamine concentration 292 mmole nitrogen/L; 10.3% oxygen (5.4 ppm). Vesicles were present. (b) S5-Cells grown at 9 C; 18 h; pH 6; glutamine concentration 610 mmole nitrogen/L; 10.3% oxygen levels (5.4 ppm). Vesicles were present. (c) S6-Cells grown at 9 C; 18 h; pH 6; glutamine concentration 292 mmole nitrogen/L; 45.6% oxygen levels (25.2 ppm). Vesicles were not present. 140 141 FIGURE 31. Transmission electron micrographs of P. fragi used in the optimization experiments. (a) T6-Cells grown at 30 C; 32 h; pH 9; glutamine concentration 7 mmole nitrogen/L; 10.0% oxygen levels (3.6 ppm). Vesicles were present. (b) T8-Cells grown at 30 C; 72 h; pH 9; glutamine concentration 7 mmole nitrogen/L; 30.0% oxygen levels (10.7 ppm). Vesicles were not present. 1 4 2 143 Those experiments with long incubation times were found both with (experiment S3) and without (experiment T8) v e s i c l e s attached to P. f r a g i c e l l surfaces. Experiments with a shorter incubation time also revealed c e l l s with (experiment S5) and without (experiment S6) attached v e s i c l e s . These trends continued with both high and low pH levels and glutamine concentrations (Table XII). The oxygen l e v e l did a f f e c t the presence of vesi c l e s on the c e l l surfaces. At high oxygen levels (experiments T8, S6) no e x t r a c e l l u l a r v e s i c l e s were present on the c e l l surfaces. Those c e l l s grown at lower oxygen levels c l e a r l y showed attached e x t r a c e l l u l a r v e s i c l e s . This trend was evident regardless of the levels of the other growth factors studied. i s o e l e c t r i c f o c u s i n g ( I E F ) I s o e l e c t r i c focusing of culture supernatants c o l l e c t e d from optimization t r i a l s T4 and T3 and stained with s i l v e r s t a i n (Figure 32) showed the presence of several protein bands. One of these bands, as revealed on the duplicate gel overlaid with casein-agar, possessed proteinase a c t i v i t y . The pi of t h i s proteinase was pH 5.2. Modeling Description of centroid search In model #1 (Figure 33), the f i r s t centroid (point 4) had a worse response than any of the ori g i n a t i n g v e r t i c e s . 144 FIGURE 32. Supernatant from optimization experiments T3 and T4 separated on IEF gels. (a) Zymogram of IEF separated culture supernatant. Proteins were separated on 5.0% polyacrylamide gel containing pH 3.7-6.4 ampholyte. Areas containing active proteinase were vi s u a l i z e d by overlaying the IEF gel with a casein-agar gel and incubating for 24 h at 20 C. Gel was developed with a TCA/sulphosalicylic acid f i x a t i v e . The zymogram was b a c k l i t and photographed against a white background. Due to the f i x a t i v e , the casein was opaque and appeared dark, while the translucent hydrolyzed areas around pH 5.2 (arrow) revealed the white background. (b) Culture supernatant from optimization experiments T3 and T4 separated on IEF gel pH 3.7-6.4. Arrow indicates protein bands around pH 5.2. The gel was stained with s i l v e r s t a i n . 145 3.9-4.0 -4.3-4.6-4.9-4.1 4.2 4.5 4.8 5.1 5.1 5.3 5.2 - 5.4 5.5 - 5.7 5.8- 6.1 T3 T4 T3 T4 1 4 6 FIGURE 33. Topographical plot of model #1 with centroid generated experiments. A topographical map of model 1. Factor i s plotted on the X-axis and factor Xo i s plotted on the Y-axis. Lines shown correspond to equal values for the response value (Z). Vertices 1, 2 and 3 are points generated by the i n i t i a l Spendley matrix. Vertex 3 produced the best r e s u l t , with vertex 2 producing the next best r e s u l t . Vertex 1 produced the worst r e s u l t . The centroid (point 4) between the best and the next best r e s u l t was evaluated and found to have a lower r e s u l t than vertex 3. A new simplex with vertices 5, 6 and 7 was calculated. Centroids (points 8, 9 and 10) were evaluated. Point 10 came to within 99% of the true optimum value. 147 As a r e s u l t , continuing searches along l i n e 2,3 were abandoned and a new simplex was generated. The response values of a l l the r e s u l t i n g centroids of t h i s simplex continued to improve. Mapping was carried out and new ranges derived. The f i r s t centroid of a new set of simplices (Figure 34) came within 99.93% of the th e o r e t i c a l optimum. Subsequent centroids had lower response values, and as such further searches were abandoned. Centroid search was able to Ignore the l o c a l maximum at X2=8, X2=2, and found the true maximum, which was centred on a simple ascending peak. In model #2 (Figure 35), the response value of 112.7, for the f i r s t centroid (point 4), was better than a l l three of the originating simplices. Centroid two (point 5) had a lower response value (91.7). As a r e s u l t , the search of li n e 2,3 was abandoned. By combining the results of the optimization with mapping of the data points, new ranges were obtained, r e s u l t i n g in the new simplex shown in Figure 36. The f i r s t centroid (point 4) came within 99.64% of the the o r e t i c a l optimum point which was 126.13. Subsequent centroids (points 5 and 6) had lower response values and further searches were abandoned. Comparison of optimization techniques Three d i f f e r e n t response models were used to compare the optimization techniques. The three models were evaluated with an i n i t i a l range of 10. Three rep e t i t i o n s , 149 FIGURE 34. Topographical map of Model #1 and experiments generated by centroid search - new simplex. Factor i s plotted on the X-axis and factor Xo i s plotted on the Y-axis. Lines shown correspond to equal values for the response value (Z). Using ranges suggested from the i n i t i a l mapping of model #1, a second centroid search optimization was performed. Points 1, 2 and 3 are vertices generated by the Spendley matrix. Point 2 i s the best response and point 1 the next best. Centroid 4 came to within 99.93% of the true optimum. 150 FACTOR X2 c n cn> -<z o o FIGURE 35. Topographical plot of model #2 centroid generated experiments. A topographical map of model #2. Factor i s plotted on the X-axis and factor X? i s plotted on the Y-axis. Lines shown correspond to equal values for the response value (Z). The response value of the f i r s t centroid (point 4) was 112.7. The response value of the second centroid (point 5), at 91.7, was lower than point 4 . As a re s u l t further searches were abandoned. 152 FIGURE 36. T o p o g r a p h i c a l p l o t o f model #2 c e n t r o i d g e n e r a t e d e x p e r i m e n t s - n e w s i m p l e x . A t o p o g r a p h i c a l map o f model #2. F a c t o r i s p l o t t e d on t h e X - a x i s and f a c t o r X? i s p l o t t e d on t h e Y - a x i s . L i n e s shown c o r r e s p o n d t o e q u a l v a l u e s f o r t h e r e s p o n s e v a l u e (Z). The f i r s t c e n t r o i d ( p o i n t 4), a t 125.7, was w i t h i n 99.64% o f t h e t h e o r e t i c a l optimum p o i n t . 154 u s i n g s m a l l e r r a n g e s , were a l s o e v a l u a t e d ( T a b l e X I I I ) . A n a l y s i s o f t h e c o m p a r i s o n d a t a showed t h a t , f o r e x p e r i m e n t t i m e ( T a b l e X l V a ) and e x p e r i m e n t number ( T a b l e X l V b ) r e q u i r e d t o r e a c h t h e optimum, t h e r e was no s i g n i f i c a n t d i f f e r e n c e between t h e t h r e e o p t i m i z a t i o n t e c h n i q u e s . Response s u r f a c e m e t h o d o l o g y was u n a b l e t o p r e d i c t t h e optimum r e s p o n s e v a l u e f o r model #1 and model #3. O n l y model #2 was c o r r e c t l y e v a l u a t e d by RS, where t h e p r e d i c t e d r e s p o n s e v a l u e came w i t h i n 4.3% o f t h e t r u e optimum v a l u e . 156 TABLE XIII. Comparison o£ optimization techniques using mathematical models. MODEL #1 Y 1=(250-(X 1-3) 2*30-(X 2-7) 2*5)/2.5 Y 2=(325-(X 1-8) 2*30-(X 2-2) 2*5)/5.4 MDS ( 1 ) MSS ( 2 ) C S ( 3 ) 23 exp 143.6 h 13 exp 57.3 h 10 exp 33.4 h 17 exp 105.7 h 23 exp 62.5 h 15 exp 64.1 h 8 exp 52.8 h 13 exp 87.1 h 18 exp 90.5 h 18 exp 133.9 h 18 exp 137.7 h 15 exp 92.0 h MODEL #2 Y 1=(250-(X 1-3) 2*12-(X 2-7) 2*5)/2.5 Y 2=(325-(X 1-8) 2*12-(X 2-7) 2*5)/3.25 MDS MSS CS 21 exp 108.4 h 25 exp 88.0 h 11 exp 26.9 h 31 exp 114.1 h 28 exp 111.0 h 19 exp 73.2 h 10 exp 62.4 h 29 exp 162.9 h 13 exp 70.2 h 16 exp 84.6 h 14 exp 93.6 h 12 exp 73.0 h MODEL #3 Yx=(200+45*X1-50*X2+30*X3+140*X1*X2-10*X1*X3+61*X2*X3+4 5X 1 2-245*X 2 2-11*X 3 2)/10 Y2=(200+45*X1-50*X2+30*X3+140*X1*X2-10*X1*X3+61*X2*X3+4 5X 1 2-123*X 2 2-43*X 3 2)/10 MDS MSS CS 23 exp 191.6 h 23 exp 91.5 h 34 exp 118.4 h 16 exp 76.9 h 14 exp 52.6 h 25 exp 100.2 h 8 exp 33.7 h 14 exp 54.3 h 27 exp 90.0 h 23 exp 112.4 h 15 exp 73.5 h 17 exp 34.3 h (1) MDS Morgan-Demlng simplex (2) MSS Modified super simplex (3) CS Centroid search 157 TABLE XlVa. Comparison of optimization techniques with respect to time required. Analysis of variance (time required) Source of v a r i a t i o n ss df ms F s i g n i f . technique 5266. .44 2 2633. ,22 1. ,81 ns model 70. .51 2 35. ,26 0. .02 ns r e p l i c a t e 1679. ,90 3 559. ,97 0. ,39 ns error 40767. .18 28 1455, .97 t o t a l 47784.03 35 (ns) not signigicant (P>.05) Table of means (time required) * Techniques Mean (time) L S D > 0 5 = 31.94** MDS 101.67 a MSS 89.33 a CS 72.18 a * MDS, Morgan and Deming simplex; MSS, modified super simplex; CS, centroid search. ** Means followed by the same l e t t e r do not d i f f e r s i g n i f i c a n t l y from one another. 158 TABLE XlVb. Comparison of optimization techniques with respect to experiment number required. Analysis of variance (experiment number) Source of v a r i a t i o n ss df ms F s i g n i f . technique 11.06 model 106.89 re p l i c a t e 196.31 error 1215.39 2 5.53 0.13 2 53.44 1.23 3 65.44 1.51 28 43.41 ns ns ns t o t a l 1529.64 35 (ns) not signigicant (P>.05) Table of means (experiment number) Techniques* Mean L S D 0 5 = 5. 51** MSS CS MDS 19.08 a 18.00 a 17.83 a * MDS, Morgan and Deming simplex; MSS, modified super simplex; CS, centroid search. ** Means followed by the same l e t t e r do not d i f f e r s i g n i f i c a n t l y from one another. 159 DISCUSSION GROWTH OF P. f r a g i ON SOLID AND LIQUID MEDIUM Culture growth Pseudomonas f r a g i , in general, grew equally well in l i q u i d and on s o l i d culture medium. The form of the culture medium had l i t t l e e f f e c t on i t s rate of growth (Figure 7a & 8a). Following a short (4 h) lag phase, both cultures in l i q u i d and on s o l i d media grew exponentially u n t i l the beginning of the stationary growth phase (ca. 24 h). The cultures did continue to increase in c e l l numbers, however, to the end of the experiments. Proteinase production In l i q u i d medium, proteinase production was f i r s t detected at 26 h during the late logarithmic, early stationary growth phase (Figure 7b). The c e l l s were not producing measurable amounts of proteinase, since p r o t e o l y t i c a c t i v i t y could not be demonstrated in whole c e l l s grown in l i q u i d medium at 8 and 12 h. These observations were in agreement with previous research (McKellar, 1982; Thompson et a l . , 1985a). Several explanations regarding t h i s behavior have been postulated Including depletion of soluble nitrogenous nutrients (Lee Wing, 1984), or depletion of dissolved oxygen (Rowe and Gilmour, 1982). P r i e s t (1983), however, suggested that nitrogen metabolism i s related to e x t r a c e l l u l a r proteinase 1 6 0 production. B a s i c a l l y , the enzymes are constitutive in nature and are produced in greater amounts only in the presence of some inducer. In the context of t h i s study, i t is possible that the proteinase was present,,albeit in small quantities, but the enzyme assay lacked s u f f i c i e n t s e n s i t i v i t y to detect i t s presence. On s o l i d medium, proteinase f i r s t appeared at 4 h, approximately 22 h sooner than that of l i q u i d culture (Figure 8b). Obviously the type of culture ( s o l i d versus l i q u i d ) influenced proteinase production. As with those c e l l s grown in l i q u i d culture, some sort of growth factor depletion may have been involved in stimulating proteinase production. E l e c t r o n m i c r o s c o p y Scanning electron microscopy Scanning electron micrographs of P. fr a g i c e l l s in l i q u i d medium, grown to the lag and beginning stationary growth phases, showed c e l l surfaces apparently devoid of globules (Figures 9 and 10). At 76 h, c e l l s grown In l i q u i d medium showed the presence of globules approximately 100-200 nm in diameter (Figure 11). Globules also appeared on c e l l s grown on s o l i d medium (Figure 13), but at 24 h rather than at 76 h. Lee Wing (1984) also described c e l l surface globule formation by P. f r a g i c e l l s grown on s o l i d culture medium. The globules observed In t h i s study and those described by Lee Wing (1984), were much larger than the e x t r a c e l l u l a r v e s i c l e s v i s i b l e on the surface of P. f r a g i 161 c e l l s and described as "blebs" by Lee Wing (1984) and Thompson, et a l . (1985a). It i s possible that globules found here and also reported by Lee Wing et a l . (1983) were composed of coalesced e x t r a c e l l u l a r v e s i c l e s . Further research i s needed to determine the nature of these globules. Thin section transmission electron microscopy Transmission electron micrographs of P. f r a g i grown in liquid medium for 16 h (Figure 15), revealed structures t y p i c a l of gram negative bacteria as described by Costerton et a l . (1974) and Michaelis and Beckwith (1982). Pseudomonas f r a g i grown for 20 h on s o l i d growth medium (Figure 20) displayed many e x t r a c e l l u l a r p a r t i c l e s attached to the c e l l surface. These p a r t i c l e s , i d e n t i c a l in size to those described by Lee Wing (1984 ) and Thompson et al. (1985a), were present while the c e l l s were producing increased quantities of proteinase (Figures 8b and 8c). Larger numbers of v e s i c l e s appeared on the c e l l surfaces at 32 and 56h. This coincided with the time periods at which large amounts of proteinase production occurred. This would be expected i f ve s i c l e s were a means of enzyme transport through the outer c e l l membrane. CELL AND GROWTH MEDIUM FRACTIONATION Isolation of e x t r a c e l l u l a r v e s i c l e s , produced by P. f r a g i in l i q u i d culture by [NH^^SO^ p r e c i p i t a t i o n , revealed p a r t i c l e s (Figure 23) similar in size and shape to those revealed by TEM on the surface of P. f r a g i c e l l s (Figure 162 21). The p a r t i c l e s bore a resemblance to p a r t i c l e s produced by other gram negative bacteria, such as B. g i n g i v a l i s (Grenier and Mayrand, 1987), E. coli (Hoekstra et a l . , 1976) and Aezomonas hydzophila (Maclntyre et a l . , 1980). Sodium dodecyl sulfate polyacrylamide electrophoretic separation (Figure 26) showed the vesi c l e s to contain some small protein bands with m o b i l i t i e s similar to protein bands of the outer c e l l membrane of P. f r a g i . This indicated that the vesi c l e s are associated with, and may have the i r o r i g i n in the outer c e l l membrane. Hoekstra et a l . (1976) found membranes surrounding e x t r a c e l l u l a r v e s i c l e s produced by E. coli to c l o s e l y resemble the outer membrane of that organism. Markers i d e n t i f y i n g the cytoplasmic membrane or the accompanying c e l l wall were absent. Only one proteinase with an i s o e l e c t r i c point of pH 5.2 could be detected i n the supernatant from P. f r a g i c e l l s grown in l i q u i d culture (Figure 24 and 25). I s o e l e c t r i c focusing showed that vesi c l e s contained only one major proteinase. This proteinase, l i k e that found in culture supernatant of P. f r a g i c e l l s grown in l i q u i d culture, had an i s o e l e c t r i c point of pH 5.2. The evidence strongly supports the theory that the same proteinase was present in both the supernatant and the v e s i c l e s . Analysis of the l i q u i d culture supernatant, e x t r a c e l l u l a r v e s i c l e s and the soluble c e l l material, showed that the majority of the proteinase could be found ln the supernatant (34,710.00 enzyme u n i t s ) . Smaller amounts were 163 associated with the e x t r a c e l l u l a r v e s i c l e s , s o l u b i l i z e d by sonication (117.00 enzyme u n i t s ) , and with the soluble c e l l material (244.80 enzyme units) (Table IV). Analysis of the s p e c i f i c a c t i v i t y of these components, however, showed that the proteinase was concentrated in the vesic l e s (680.50 enzyme units/mg protein) (Table IV). Jensen et a l . (1980) found that the majority of the proteinase in a late logarithmic culture of P. aeruginosa was in the culture supernatant. The t o t a l quantity of proteinase found in the supernatant (34,710.0 enzyme units) was the resu l t of growth for 82 h (Table IV). The t o t a l enzyme associated with the ex t r a c e l l u l a r v e s i c l e s ( s o l u b i l i z e d by sonication) was 117.0 enzyme units. If e x t r a c e l l u l a r v e s i c l e s were the only means by which P. f r a g i could export proteinase outside the c e l l , i t would have to produce 117.0 enzyme units once every 16.6 min., and produce (assuming 11.55 mg of vesic l e s in the supernatant at any one time) ca. 41.8 mg of vesi c l e s / h . Enzymes destined for the c e l l exterior may be synthesized in the c e l l cytoplasm by ribosomes attached to or free of the cytoplasmic membrane (Randall et a l . , 1987; Pr i e s t , 1983). Transport through the cytoplasmic membrane may involve interaction between hydrophobic portions of the enzyme and hydrophobic portions of the cytoplasmic membrane (Wlckner, 1979). The enzyme could appear in the cytoplasm as an active enzyme or as an inactive enzyme precursor (Pr i e s t , 1983). It could also appear in the periplasm as an 164 active or inactive enzyme. An inactive form of enzyme, associated with cytoplasmic membrane material was found In the periplasm of P. aeruginosa (Fecycz and Campbell,.1985). Active proteinase has been shown to be present in the i n t e r i o r of e x t r a c e l l u l a r vesicles of P. f r a g i (Thompson et a l . , 1985a). Whatever the state, proteinase residing in the periplasm must transverse the outer c e l l membrane. Fecycz and Campbell (1985) determined that transport through the outer membrane was not the same as that through the cytoplasmic membrane. Indeed, the small channels of pseudomonad porin molecules (Fairbairn and Law, 1986) would not allow a f u l l y formed active enzyme with a molecular weight of ca. 45,000 through the outer c e l l membrane. It seems l i k e l y that some other form of transport would be required. Winkler and Stuckmann (1979) hypothesized a mechanism for the sel e c t i v e stimulation of exolipase formation from Serratia marcescens by a v a r i e t y of exogenous polysaccharides. In that study, exolipase y i e l d increased with an increase in either exogenous Serratia polysaccharide or glycogen concentration. The theory suggests hypothetical " s i t e s " , on or near the c e l l surface, where newly formed exolipase molecules accumulate. Exolipase enhancing polysaccharide detaches the exolipase from the c e l l surface by competing for these s i t e s , or by changing the conformation of the enzyme. The glycogen probably mimics the exolipase enhancing properties of the b a c t e r i a l 165 exopolysaccharide. An alternate hypothesis proposed by these authors suggested that the exogenous polysaccharides protect newly-formed enzymes from proteolysis during secretion. In either theory, the authors proposed that exoenzymes which are glycoproteins would p r e f e r e n t i a l l y bind to the c e l l surface and respond to enzyme-enhancing polysaccharides. In the case of P. f r a g i , proteinase is associated with outer c e l l membrane material. This material may complex with the proteinase, forming a v e s i c l e . The v e s i c l e then "blebs" off the c e l l surface. The resultant v e s i c l e may contain polysaccharide material of outer c e l l membrane o r i g i n loosely bound with proteinase. The P. f r a g i v e s i c l e s did contain carbohydrate (1.04 mg carbohydrate/mg protein). Maclntyre et a l . (1980) found that v e s i c l e s produced by Aeromonas salmonicida also contained carbohydrate (4.1 mg carbohydrate/mg protein). When the e x t r a c e l l u l a r v e s i c l e s were disrupted by ultrasonic disruption, a 338% increase in s p e c i f i c proteinase a c t i v i t y occurred. If the proteinase had merely adsorbed to the v e s i c l e surface, no such increase in s p e c i f i c proteinase a c t i v i t y would have been observed. The l i t e r a t u r e (Porzio and Pearson, 1975); (Noreau and Drapeau, 1979) does not Indicate whether the proteinase produced by P. f r a g i i s a glycoprotein. F a i r b a i r n and Law (1986) described the proteinases produced by Pseudomonas 145-2 and P. aeruginosa s t r a i n 34362 to be glycoproteins. If the proteinase produced by P. f r a g i i s a glycoprotein, then the theory of 166 Winkler and Stuckmann (1979) may apply. Further experimentation w i l l be necessary to support these hypotheses. The possible appearance of proteinase with outer c e l l membrane material may be related to transport of the enzyme through the membrane. The continued association of membrane material along with the proteinase in the form of a v e s i c l e i s , however, not r e a d i l y apparent. Why would the c e l l allow a substantial portion of i t s e l f to be released into the supernatant along with the proteinase, when reclamation of the membrane material would appear to be more conservative? Winkler and Stuckmann (1979) proposed that the b a c t e r i a l exopolysaccharide associated with the exoenzyme, i s recycled back to the c e l l surface, l i b e r a t i n g more exoenzyme. Encapsulation of the proteinase may afford some advantage which the enzyme and ultimately the c e l l may benefit from. Lee Wing et a l . (1983) postulated that P. f r a g i v e s i c l e s may serve to channel the proteinase produced by the c e l l , towards the nutrient source for which they were produced. Grenier and Mayrand (1987) echoed t h i s hypothesis, suggesting that e x t r a c e l l u l a r v e s i c l e s expelled by B. g i n g i v a l i s may serve as a transport medium, conducting packages of highly concentrated enzymes, from the c e l l to the nutrient source. Indeed, since P. f r a g i i s known to colonize meat surfaces, the vesi c l e s may serve to transport these concentrated enzyme packages, between and around i n t e r c e l l u l a r ( i . e . meat) spaces just as the vesicles of B. 1 6 7 g i n g i v a l i s may carry i t s proteinases to areas of tissue not normally accessible to them. This process which acts as a factor in B. g i n g i v a l i s ' s attack on the gingival cavity t i s s u e s , may be similar to the attack of P. fra g i on a s p o i l i n g meat surface. The two situations are s i m i l a r . This discussion suggests that e x t r a c e l l u l a r v e s i c l e s may be vehicles for the transport of proteinase only. A survey of the l i t e r a t u r e has revealed e x t r a c e l l u l a r v e s i c l e s of other organisms to be complexed with many e x t r a c e l l u l a r bound molecules Including Aeromonas hydrophila acyltransferase (Maclntyre and Buckley, 1978; Maclntyre et a l . , 1980), and B. uniformis bacteriocin (Austin-Prather and Booth, 1984). Apparently, e x t r a c e l l u l a r v e s i c l e formation takes place in several gram negative bacteria, acting to transport a wide var i e t y of molecules through the outer c e l l membrane. The association of enzymes with outer c e l l membrane material may have a s t a b i l i z i n g e f f e c t on the enzyme molecule. While the enzyme resides in the periplasm, i t is in e f f e c t shielded from denaturing physical or chemical agents. While encapsulated by outer c e l l membrane material the enzyme may be afforded s t r u c t u r a l s t a b i l i t y , perhaps through actual complex formation, or by concentration of s t a b i l i z i n g ions. The proteinase of P. f r a g i i s a metalloproteinase and does require Z n + + for a c t i v i t y and C a + + for s t a b i l i t y (Porzio and Pearson, 1975). 168 This encapsulation creates a microenvironment which would d i f f e r from the surrounding menstrum. It would screen the enzyme and protect i t from damage by physical and chemical means. The thermostability of proteinases produced by psychrotrophic pseudomonads has been well documented ( G r i f f i t h s et a l . , 1981; Malik and Mathur, 1984; Stepaniak and Fox, 1985). The mechanism of t h i s s t a b i l i t y i s poorly understood. It i s possible that at least part of t h i s s t a b i l i t y may be due to exopolysaccharide-proteinase complex formation. Porzio and Pearson (1975) found that P. f r a g i proteinase retained approximately 30% of i t s a c t i v i t y after 10 min at 60 C, in the presence of C a + + . Pseudomonad proteinases tend to be further s t a b i l i z e d in the presence of milk and milk products (Patel et a l . , 1983b). BACTERIOCINS Several researchers have found bacteriocinogenic pseudomonads, mainly of phytopathogenic (Cuppels et a l . , 1978; Vidaver et a l . , 1972) or phytopathogenic and c l i n i c a l o r i g i n (Gonzalez and Vidaver, 1979). Few studies have detected bacteriocinogenicity in pseudomonads responsible for the spoilage of food products in cold storage. Hamon et al. (1961) described a s t r a i n of P. fluorescens to be bacterIocinogenic, active mainly against other P. fluorescens s t r a i n s . Pseudomonas fluorescens has been implicated in spoilage of raw meat products (Greer, 1982), and raw and pasteurized milk (McKellar and Cholette, 1987). Smirnov et a l . (1984) described a bacteriocinogenic P. 169 f r a g i , of unknown o r i g i n , which inhibited the growth of P. lemonnieri, P. putida, P. s t u t z e r i , P. menclocinaf P. pseudoalcaligenes, and P. acidovorans, but not other f r a g i s t r a i n s . In t h i s study, P. f r a g i ATCC 4973 did not i n h i b i t the growth of the above species nor six d i f f e r e n t P. f r a g i s t r a i n s , nine d i f f e r e n t fluorescens st r a i n s (including P. aureofaciens ATCC 13985), E. coli, and two Streptococcus s t r a i n s . P. f r a g i did produce e x t r a c e l l u l a r membrane vesicl e s similar in size and shape to those bacteriocin containing p a r t i c l e s of 8. uniformis (Austin-Prather and Booth, 1984). The p a r t i c l e s present in P. f r a g i culture supernatant did not have bacteriocin a c t i v i t y . This lack of bacteriocinogenicity seen In P. f r a g i ATCC 4973 is in variance with the findings of Smirnov et a l . (1984) . Those researchers found that a P. f r a g i s t r a i n (not ATCC 4973) did produce bacteriocins i n h i b i t o r y towards d i f f e r e n t species of Pseudomonas. The species was, however, limited in i t s range of antagonism. In t h i s study, indicator organisms similar ( i . e . same species, d i f f e r e n t strains) to those used by Smirnov et a l . (1984) were not i n h i b i t e d by P. f r a g i ATCC 4973. Some of the indicator organisms employed in t h i s study, isolated by Kwan and Skura (1985) , were implicated in milk spoilage. It appears that P. f r a g i ATCC 4973 may not gain a competitive advantage over other milk or meat spoilage bacteria due to bacteriocin 170 production. Such a n t i b a c t e r i a l properties could not be demonstrated under the conditions used in t h i s study. OPTIMIZATION There have been several attempts by previous researchers (Fairbairn and Law, 1987; G r i f f i t h s and P h i l l i p s , 1984; Malik et al., 1985; McKellar, 1982) to determine optimal culture conditions for the production of proteinase from pseudomonads (primarily P. fluorescens). A l l these studies, however, examined only one factor at a time. McKellar (1982) and Fairbairn and Law (1987) looked at nitrogen and carbon source, and the i r e f f e c t upon proteinase production. G r i f f i t h s and P h i l l i p s (1984) and Malik et a l . (1985) looked at culture aeration, and oxygen's e f f e c t upon proteinase production. As well, Malik et a l . , (1985) studied pH, time and nutrient source. By using optimization techniques, several c u l t u r a l factors were examined simultaneously in t h i s study to determine their e f f e c t upon the production of proteinase by P. f r a g i . Since the optimization approach had not been previously used, decisions regarding appropriate factors, and their value ranges had to be made subjectively, using knowledge gained from previously published research. There are undoubtedly many c u l t u r a l factors involved in the regulation of proteinase production from pseudomonads. Some of these factors seem to be of minor influence while others have been found to be of fundamental Importance. The object of thi s study was, in part, to determine, from a selection of 171 c u l t u r a l factors, which were of major and which were of minor importance for the production of proteinase by P. f r a g i . The factors chosen were ones that could be regulated by process control in an i n d u s t r i a l food environment. In addition, i t was the object of th i s study to determine what the optimum factor values were. The factors evaluated in th i s study were organic nitrogen l e v e l , i n i t i a l culture pH, incubation time, incubation temperature, and culture oxygen l e v e l s . Discussion of factors studied Glutamine McKellar (1982) found P. fluorescens capable of producing proteinase with glutamine as the sole nitrogen source at 7.0 mmole nitrogen/L. In t h i s study, P. fr a g i was also found capable of producing proteinase when grown in the presence of glutamine as the sole nitrogen source. The glutamine levels used for optimization ranged from 7 to 610 mmole nitrogen/L. The upper glutamine l e v e l was close to the maximum s o l u b i l i t y of the compound in culture medium (an equivalent of 650 mmole nitrogen/L). Glutamine was also used because i t was shown to influence the system of nitrogen metabolism and enzyme regulation in pseudomonads (Fairbairn and Law, 1986). Both proteins or amino acids may contribute carbon and nitrogen to the production of proteinase. The amount of nitrogen present may, however, control the production of proteinase by a process of feedback i n h i b i t i o n . Nitrogen required for c e l l u l a r biosynthesis may be acquired by P. fr a g i through N metabolism and ammonia assimi l a t i o n . Two pathways of ammonia assimilation may be involved (Fairbairn and Law, 1986). In the f i r s t , 2-oxoglutarate is converted to glutamate, mediated by glutamate dehydrogenase (GDH). Ammonia contributes the nitrogen with NADPH as the terminal electron acceptor: GDH 2-oxoglutarate + NH3 + NADPH + H + ^glutamate + NADP+ + H 20 For the above reaction to occur, an association between GDH and NADPH must e x i s t . This association can occur only during periods of high NH3 l e v e l s . Alternately, glutamate may be converted to glutamine, mediated by glutamine synthetase (GS). Ammonia again functions as the nitrogen source: GS NH3 + glutamate + ATP >glutamine + ADP + Pj The GS system produces a pool of glutamine, allowing the o v e r a l l amination of 2-oxoglutarate to glutamate by the glutamine: 2-oxoglutarate aminotransferase (GOGAT) system: GOGAT glutamine + 2-oxoglutarate + NADPH + H + •—^glutamate + NADP+ + H 20 Comparing the two groups of reactions, the GS/GOGAT system has a much higher a f f i n i t y for ammonia than the GDH 173 s y s t e m . The GS/GOGAT doe s n o t r e q u i r e f o r m a t i o n o f a c omplex, s u c h as GDH-NADPH, and t h u s i s a l l o w e d t o o c c u r a t l o w NH3 l e v e l s . The GS/GOGAT s y s t e m i s i r r e v e r s i b l e and i s r e p r e s s e d d u r i n g g r o w t h on good n i t r o g e n s o u r c e s . Most o f t h e n i t r o g e n a s s i m i l a t e d by t h e GS/GOGAT s y s t e m i s s t o r e d as g l u t a m i n e . G l u t a m i n e n o t o n l y a c t s as an i n t e r m e d i a t e i n t h e a m i n a t i o n o f 2 - o x o g l u t a r a t e , b u t may c o n t r i b u t e n i t r o g e n t o o t h e r b i o s y n t h e t i c p r o c e s s e s . Thus g l u t a m i n e i s an i m p o r t a n t a s p e c t o f n i t r o g e n m e t a b o l i s m . The mechanism o f n i t r o g e n c a t a b o l i t e r e p r e s s i o n o f GS was d e s c r i b e d by S h a p i r o and s t a d t m a n ( 1 9 7 0 ) , and T y l e r ( 1 9 7 8 ) . GS c a n be m o d i f i e d by a d d i t i o n o f a d e n i n e t o t h e m o l e c u l e , s u c h t h a t t h e enzyme becomes l e s s a c t i v e . A d e n y l y l a t i o n o f GS i s m e d i a t e d by a d e n y l y l t r a n s f e r a s e ( A T ) . The a c t i v i t y o f AT i s i t s e l f c o n t r o l l e d by a p r o t e i n ( P J J ) w h i c h may e x i s t i n two f o r m s . The u n m o d i f i e d P T I c o m p l e x e s w i t h AT, c a t a l y z i n g t h e t r a n s f e r o f a d e n i n e t o GS. The p r o t e i n may be m o d i f i e d by a d d i t i o n o f u r i d i n e , m e d i a t e d by u r i d y l y l t r a n s f e r a s e ( U T ) . U r i d y l y l t r a n s f e r a s e i s s t i m u l a t e d by 2 - o x o g l u t a r a t e and ATP, w h i l e i n h i b i t e d by g l u t a m i n e , and i n o r g a n i c p h o s p h a t e : 174 AT [ P n ] -GS-AD GS ^ [ P I T U R ] UT [2-oxoglutarate / ATP] tglutamine / P^] The modified P J J U R stimulates the deadenylylation of GS to a more active form. In summary, high levels of glutamine with low levels of 2-oxoglutarate, the r e s u l t of a r i c h nitrogen substrate, renders the GS enzyme in a less active glutamine l e v e l s , with high 2-oxoglutarate l e v e l s , renders the GS enzyme more active, allowing more NH3 a s s i m i l a t i o n . Since proteins are sources of nitrogen, P. f r a g i produces e x t r a c e l l u l a r proteinases, allowing the c e l l access to t h i s nutrient. But since such proteinase production would contribute to the nitrogen pool, i n h i b i t i o n of the enzyme through nitrogen catabolic repression would seem l o g i c a l . It seems clear that glutamine would act as the perfect defined organic nitrogen source in order to study the e f f e c t of nutrient l e v e l on proteinase production. Being the only nitrogen source, glutamine must be deaminated, and the resultant NH3 assimilated. Glutamine must stimulate proteinase production, since Kosers c i t r a t e medium (containing inorganic nitrogen) does not support proteinase production from P. f r a g i (Thompson et a l . , 1985a). At some state. As a result' less NH3 w i l l be assimilated. Low 175 l e v e l , however, glutamine must also act to l i m i t proteinase production by N catabolic repression. At t h i s point, maximum proteinase production would occur. This study indicates that glutamine at a l e v e l equivalent to approximately 300 mmole nitrogen/L culture medium, does act in t h i s way. Previous researchers (Lee Wing et a l . , 1983, Borton et a l . , 1970a; b) postulated that proteinase had been stimulated when psychrotrophic bacteria were subjected to l i m i t a t i o n of nutrients. This e f f e c t would be more acute for c e l l s grown on a s o l i d medium, since the area immediately surrounding the c e l l s would be quickly depleted of nutrients. Proteinase production, in t h i s study, was c l e a r l y stimulated at lower nitrogen l e v e l s . This may explain the e a r l i e r occurrence of proteinase when P. f r a g i was grown on a TSB + agar surface (Figure 8b), as compared to those c e l l s grown in l i q u i d TSB medium (Figures 7b). A similar occurrence may have existed when Yada and Skura (1981) observed stimulation of proteinase from P. f r a g i , grown on sarcoplasmic depleted s t e r i l e beef tissue. As colony size on the beef surface increased, digested areas on the beef surface increased in si z e , p a r t i c u l a r l y when P. fragi entered the late logarithmic, early stationary phase of growth (Yada and Skura, 1982). pH Malik et al. (1985) found maximum proteinase production from P. fluorescens at pH 7.0 ± 0.5. Fai r b a i r n and Law 176 (1987) found l i t t l e proteinase production below pH 6.7, but maximum proteinase at pH 6.8. In t h i s study, pH values from 5.0 to 9.0 were used to determine the optimum proteinase production from P. f r a g i . F a i r b a i r n and Law (1987) postulated that an increase in pH would r e s u l t in a decrease in proteinase production. This study found pH 6.8 to be optimal for proteinase production. The e f f e c t of pH repression of proteinase production was weak, however, since large amounts of proteinase were produced by P. f r a g i at both pH 6.0 (experiment #S3) (Table V i l l a ) and at pH 9.0 (experiment #T8) (Table V). Time Previous research had found proteinase production by P. fluorescens (McKellar, 1982) and P. f r a g i (Yada and Skura, 1981; Thompson et al., 1985b; Stead, 1987) to commence at the late logarithmic, early stationary phase. For P. f r a g i in the present study, proteinase production in TSB (Figure 7b) commenced at approximately 30 h, which corresponded to the late logarithmic, early stationary phase. Production continued and extended to ca. 82 h at 21 C. The time range used for optimization was from 4 to 72 h. Extending the time range beyond 72 h made p r a c t i c a l optimization experiments impossible. For P. f r a g i , the optimum time for proteinase production was 37.9 h. Temperature Previous research had shown that 20 C (McKellar, 1982), 22 C (Malik et a l . , 1985), and 20 C (Fairbairn and Law, 177 1987) were t h e optimum t e m p e r a t u r e s f o r p r o t e i n a s e p r o d u c t i o n by P. f l u o r e s c e n s . The t e m p e r a t u r e r a n g e c h o s e n f o r o p t i m i z a t i o n o f p r o t e i n a s e p r o d u c t i o n by P. f r a g i i n t h i s s t u d y was 1 C t o 40 C. M c K e l l a r (1982) f o u n d maximum p r o t e i n a s e p r o d u c t i o n o c c u r r e d a t 20 C. F i f t y - f i v e p e r c e n t o f maximum p r o d u c t i o n was o b s e r v e d a t 5 C. A d d i t i o n a l l y , M c K e l l a r and C h o l e t t e (1987) f o u n d t h a t a s h i f t f r o m 20 t o 30 C h a l t e d p r o t e i n a s e p r o d u c t i o n by P. f l u o r e s c e n s . T h i s s t u d y f o u n d 12.5 C t o be t h e optimum t e m p e r a t u r e f o r p r o t e i n a s e p r o d u c t i o n by P. f r a g i . Pseudomonas f r a g i d i d p r o d u c e p r o t e i n a s e a t h i g h t e m p e r a t u r e s ( i n c u b a t i n g a t 36.6 C p r o d u c e d 4.70 enzyme u n i t s / m L ) . I n c u b a t i o n a t t e m p e r a t u r e s l o w e r t h a n t h e optimum, i n d u c e d p r o d u c t i o n o f l a r g e amounts o f p r o t e i n a s e , b u t o n l y a t l o n g e r i n c u b a t i o n t i m e s . P r o t e i n a s e p r o d u c t i o n by P. f r a g i was f a v o r e d a t l o w e r t e m p e r a t u r e s . As t e m p e r a t u r e s d e c l i n e , however, m e t a b o l i c a c t i v i t y i n t h e c e l l a l s o d e c l i n e s , t o t h e p o i n t where t h e c e l l i s u n a b l e t o p r o d u c e p r o t e i n a s e . As was t h e c a s e w i t h P. f l u o r e s c e n s ( M c K e l l a r and C h o l e t t e , 1987), t e m p e r a t u r e s h i g h e r t h a n 30 C d i s c o u r a g e d p r o t e i n a s e p r o d u c t i o n by P. f r a g i . A t t h e s e t e m p e r a t u r e s c e l l g r o w t h a p p e a r e d t o be g r e a t l y a c c e l e r a t e d , and a t r u e d e a t h phase had e v i d e n t l y o c c u r r e d . S i n c e p r o t e i n a s e p r o d u c t i o n o c c u r s p r i m a r i l y between t h e end o f t h e l o g a r i t h m i c phase and t h e b e g i n n i n g o f t h e d e a t h p h a s e , a n y f a c t o r w h i c h t e n d s t o p r o l o n g t h i s s t a t i o n a r y p h a s e , would promote i n c r e a s e d t o t a l p r o t e i n a s e p r o d u c t i o n . D e c r e a s i n g 178 temperatures do favor a longer stationary growth phase, allowing more proteinase to accumulate. Oxygen L i t t l e information, regarding the optimum oxygen levels required for proteinase production from P. f r a g i , exists in the l i t e r a t u r e . Ambient oxygen levels centre around 18% (8.4 ppm maximum dissolved oxygen in water). The upper l i m i t (50%) and the lower l i m i t (0%) used for optimization of proteinase production, contain t h i s ambient value. Results of the f i r s t preliminary experiments showed oxygen levels to be the most important factor in determining proteinase production. Results of the f r a c t i o n a l f a c t o r i a l experiment showed that oxygen levels above 10% oxygen (>3.6 ppm) strongly favored proteinase production. Malik et a l . (1985) found l i q u i d culture aeration stimulated the production of proteinase by Pseudomonas sp. B-25. Continuing on t h i s theme, Murray et a l . (1983) and Skura et a l . (1986) observed that psychrotrophic microorganisms in raw milk did not produce proteinase when the milk was purged of oxygen by flushing with N 2. Rowe and Gilmour (1982), however, found proteinase production by P. fluorescens could be induced by f o r c i b l y decreasing the oxygen tension of the growth medium. In contrast, G r i f f i t h s and P h i l l i p s (1984) showed that accumulation of proteinases and lipases of psychrotrophic bacteria in raw milk could be suppressed by maintaining the milk in an aerated state. The results of t h i s study indicated that optimum proteinase production 179 occurred when the gas mixture flowing over the growth medium contained 16.4% oxygen (7.4 ppm oxygen dissolved in the medium). This dissolved oxygen l e v e l was somewhat lower than that found when a i r was used as the gas (ca. 8.4 ppm). When Rowe and Gilmour (1982) reduced t h e i r oxygen l e v e l , the action i n i t i a l l y may have resulted in a stimulation of proteinase production. Thin section electron microscopy Transmission electron micrographs of P. fr a g i c e l l s , grown under various oxygen le v e l s , revealed e x t r a c e l l u l a r v e s i c l e s on the surfaces of c e l l s grown under reduced oxygen level s only (Figure 30a, b; 31a). C e l l s grown under richer oxygen levels were devoid of vesic l e s (Figure 30c; 31b). Vesicles were found associated with c e l l s grown close to the oxygen le v e l optimal for proteinase production. These observations tend to support the hypothesis that vesi c l e s are a means of transport for proteinase through the outer membrane. Pseudomonas f r a g i c e l l s grown in TSB l i q u i d cultures (Figures 14,15,16,17,18) were devoid of accumulated e x t r a c e l l u l a r v e s i c l e s , while the surfaces of c e l l s grown on TSB + agar medium for more than 20 h (Figures 20,21,22) were studded with these p a r t i c l e s . The p a r t i c l e s were produced by c e l l s grown in TSB l i q u i d culture, since v e s i c l e recovery from culture supernatant was possible. It appears l i k e l y that, of the factors examined in t h i s study, dissolved oxygen concentrations mediate ves i c l e detachment from the 180 c e l l surface. Dissolved oxygen concentrations in l i q u i d agitated cultures would be higher than those in the immediate v i c i n i t y of c e l l s grown on s o l i d medium. Pseudomonas fragi c e l l s grown on s o l i d medium attained a considerable mat thickness quickly, which probably cut off oxygen supplies to those c e l l s nearest the nutrient source. Oxygen l e v e l and the dairy industry Presently, l i t t l e attention i s paid to the oxygen levels of raw milk, stored on the dairy farm, in the bulk tank truck, or in the processing plant s i l o . Optimum proteinase production occurs when P. f r a g i i s grown at 0 2 levels s l i g h t l y below that of the atmosphere. It would seem advantageous to store milk containing P. fr a g i at oxygen levels either well below, or well above atmospheric l e v e l s . Dramatic flavor changes due to l i p i d oxidation would rule out storage of milk at very high oxygen l e v e l s . On the other hand, considerable reduction in proteinase production could be achieved by eliminating, possibly by N 2 flushing, oxygen from raw milk (Murray et a l . , 1983; Skura et a l . , 1986; Rowe and Gilmour, 1982). Rowe and Gilmour (1986) suggested that oxygen tension measurements may be a means of detecting proteinase and lipase mediated spoilage of raw milk by psychrotrophic organisms. Presently, raw milk in s i l o s i s often agitated by gentle release of a i r from the tank bottom. This practice may tend to stimulate proteinase production by continually supplying a small amount of oxygen to the psychrotrophic bacteria in the raw milk. 181 O p t i m i z a t i o n t e c h n i q u e S i n c e o p t i m i z a t i o n t e c h n i q u e s had n o t p r e v i o u s l y been u s e d t o o p t i m i z e p r o t e i n a s e p r o d u c t i o n f r o m P. f r a g i , t h e i m p o r t a n c e o f e a c h c u l t u r e f a c t o r , and t h e i r i n f l u e n c e on t h e o p t i m i z a t i o n p r o c e s s were unknown. C o n s e q u e n t l y , an e i g h t e x p e r i m e n t f r a c t i o n a l f a c t o r i a l d e s i g n e x p e r i m e n t u s i n g f i v e f a c t o r s was c o n d u c t e d t o g i v e some i n d i c a t i o n o f t h e i r i m p o r t a n c e . A l l f a c t o r s were e v a l u a t e d a t two s e t f a c t o r l e v e l s ( T a b l e V ) . A f r a c t i o n a l f a c t o r i a l e x p e r i m e n t e m p l o y i n g 3 f a c t o r l e v e l s (27 e x p e r i m e n t s ) would have p r o v i d e d more i n f o r m a t i o n , b u t would have d r a m a t i c a l l y i n c r e a s e d t h e number o f e x p e r i m e n t s r e q u i r e d . A n a l y s i s o f t h e v a r i a n c e o f t h e d a t a c o l l e c t e d f r o m t h e f r a c t i o n f a c t o r i a l e x p e r i m e n t ( T a b l e V i l a ) , showed t h a t o x y g e n l e v e l was t h e most s i g n i f i c a n t f a c t o r , o f t h e f a c t o r s s t u d i e d , i n r e g u l a t i n g p r o t e i n a s e p r o d u c t i o n f r o m P. f r a g i . T h i s f a c t may be o f i m p o r t a n c e t o t h e d a i r y i n d u s t r y i n t h e i r e f f o r t s t o c o n t r o l p s y c h r o t r o p h i c s p o i l a g e o f raw m i l k . C e n t r o i d s e a r c h E v a l u a t i o n o f t h e p r o g r e s s o f t h e o p t i m i z a t i o n p r o c e s s c o u l d be c h e c k e d by c o m p a r i n g t h e s t a n d a r d e r r o r o f t h e mean f o r t h e S p e n d l e y m a t r i x s e t o f e x p e r i m e n t s w i t h t h a t o f t h e c e n t r o i d s e a r c h e x p e r i m e n t s . I t was assumed t h a t as o p t i m i z a t i o n c o n t i n u e d , t h e v a r i a n c e between t h e e x p e r i m e n t a l u n i t s would d e c r e a s e . T h e o r e t i c a l l y , when t h e 182 true optimum value was reached the variance between experimental units would be zero. P r a c t i c a l l y , however, t h i s point could not be reached because of random error. In t h i s study, the standard error of the mean for enzyme units/mL declined from 2.2 for the Spendley matrix set of experiments to 1.4 for the centroid experiments mean. If optimization could have been continued, i t would be expected that the standard error of the mean would continue to decline. Subsequent to the i n i t i a l Spendley matrix evaluation, s i x centroids were generated (Table IXa). Four of these, including centroid #1 (experiment C7) were evaluated. The strength of the centroid search technique l i e s , in part, in i t s a b i l i t y to evaluate a l l centroids simultaneously, rather than one at a time as other i t e r a t i v e procedures do. This can res u l t in a considerable saving of time, i f the f i r s t centroid response value i s better than that of the best response of the i n i t i a l Spendley matrix. If on the other hand, the f i r s t centroid response value i s less than the best response of the Spendley matrix, then the continued search of the remaining f i v e experiments would be a waste of time. The optimum factor response would not l i e on that search l i n e . It was therefore decided that only a portion of the centroids should i n i t i a l l y be searched. If thi s i n i t i a l search showed continued improvement of response value, then the remaining centroids would be evaluated, otherwise the search l i n e would be abandoned. It was hoped 183 that a search of s i x t y percent of the centroids would s t r i k e a balance between too few and too many searches. In the present s i t u a t i o n four of six centroids were searched. Three centroids were selected randomly (only three were selected since the f i r s t centroid must always be evaluated). The selection proved fortuitous since the i n i t i a l centroid response value (experiment C7, Table IXa) was not lower than the best response from the Spendley matrix (a t i e was deemed an improvement). The second centroid (experiment C8) response value was lower than that of the f i r s t centroid, indicating that further searches along that l i n e may not re s u l t in the optimum. At that point, a new Spendley matrix with a new (and narrower) range of factor levels was generated. Continued search of a new set of vertices with a narrower range of factor levels was not possible since the equipment used to maintain incubation temperature was not s u f f i c i e n t l y precise to maintain the lower temperature differences suggested by the new Spendley matrix. As a re s u l t , further searching was stopped. According to the centroid search procedure of Aishima and Nakai (1986) once the suspected area of optimal response value has been found, simultaneous factor s h i f t could be used to move the factors quickly towards other areas on the response surface. The danger here is that centroid search has "homed i n " on a l o c a l maximum and has missed the true maximum area. Subsequent to centroid search evaluation, factor levels were shi f t e d to suspected areas of increased 184 response v a l u e s . Continued s h i f t i n g of f a c t o r s were continued u n t i l the r e s u l t a n t response value became worse than the preceding one. In t h i s study, simultaneous s h i f t p r o v i d e d the s e t of f a c t o r l e v e l s given i n Table XI. These experiments r e s u l t e d i n lower response v a l u e s . F u r t h e r simultaneous s h i f t s were t h e r e f o r e not performed. M a p p i n g Data from the Spendley matrix, c e n t r o i d searches and simultaneous s h i f t searches were used to c o n s t r u c t i n i t i a l data maps. The maps (Fig u r e s 27, 28, 29 ), of the f a c t o r s time, temperature, glutamine c o n c e n t r a t i o n , pH and oxygen l e v e l , show a c l u s t e r i n g of p o i n t s , i n d i c a t i n g a c l e a r optimum response v a l u e . Mapping searches f o r data p o i n t s present i n groups, over a l l f a c t o r s . The o b j e c t i v e of the o p t i m i z a t i o n technique used, be i t c e n t r o i d search, or some other technique, i s to organize f a c t o r values together i n such groups. O p t i m i z a t i o n comparisons The o p t i m i z a t i o n technique i n g e n e r a l can be used to a l t e r two or more independent v a r i a b l e s ( f a c t o r s ) , i n an organized f a s h i o n , such t h a t an improvement i n the dependent v a r i a b l e (response value) can be achieved. Without such o r g a n i z a t i o n , improvement of the response value can o n l y be gained by l u c k y or chance f a c t o r value changes. Of the many o p t i m i z a t i o n techniques a v a i l a b l e , o n l y the simplex o p t i m i z a t i o n techniques and the response s u r f a c e methodology technique w i l l be d i s c u s s e d . With the simplex 185 d e s i g n , e v o l u t i o n a r y o p e r a t i o n s (EVOP) a r e f o l l o w e d , c o n s t r u c t i n g t h e e x p e r i m e n t s as i t p r o c e e d s . R e s p o n s e s u r f a c e m e t h o d o l o g y r e l i e s on a f o r m a l s e t f a c t o r i a l e x p e r i m e n t d e s i g n e d b e f o r e h a n d . B o t h p r o c e s s e s a t t e m p t t o d e s c r i b e , i n as few e x p e r i m e n t s as p o s s i b l e , enough o f a r e s p o n s e s u r f a c e , t o p r e d i c t where t h e t r u e optimum p o i n t may l i e . Morgan and Deminq s i m p l e x The Morgan and Deming (1974) s i m p l e x p r o c e d u r e i s b a s e d , f o r t h e most p a r t , upon t h e EVOP a p p r o a c h d e s c r i b e d by S p e n d l e y e t a l . (1962). The p r o c e d u r e i s begun by d e f i n i n g r a n g e s f o r t h e f a c t o r s . A f t e r s u c h s c a l i n g , an i n i t i a l s i m p l e x c o n t a i n i n g n+1 v e r t i c e s i s g e n e r a t e d . , S u b s e q u e n t t o e v a l u a t i o n a t e a c h v e r t e x , t h e v e r t e x w i t h t h e w o r s t r e s p o n s e v a l u e ( l o w e s t v a l u e f o r m a x i m i z a t i o n , h i g h e s t f o r m i n i m i z a t i o n ) i s r e p l a c e d by i t s r e f l e c t i o n t h r o u g h t h e c e n t r o i d r e s i d i n g on t h e l i n e c o n n e c t i n g t h e r e m a i n i n g n v e r t i c e s , c r e a t i n g a new s i m p l e x . The new v e r t e x i s e v a l u a t e d and t h e p r o c e s s c o n t i n u e d . I f t h e new v e r t e x c o n t a i n s t h e w o r s t r e s p o n s e v a l u e , r e f l e c t i o n o f t h e n e x t b e s t v e r t e x i s done t o a v o i d o s c i l l a t i o n s . T h i s i s t h e b a s i c s i m p l e x d e s i g n . M o d i f i c a t i o n o f t h e above p r o c e d u r e , c o n t a i n e d w i t h i n t h e MDS (Morgan and Deming (1974) a l l o w s a more r a p i d c o n v e r g e n c e t o w a r d s t h e optimum p o i n t . I f t h e r e f l e c t e d v e r t e x r e s p o n s e v a l u e i s b e t t e r t h a n t h a t o f t h e p r e v i o u s b e s t v e r t e x , movement i n t h i s d i r e c t i o n i s f a v o r a b l e . As a 186 r e s u l t , an e x p a n s i o n f a c t o r i s us e d t o e x t e n d t h i s new b e s t p o i n t f u r t h e r i n t h e i n d i c a t e d d i r e c t i o n c r e a t i n g a new v e r t e x f o r e v a l u a t i o n . I f , on t h e o t h e r hand, t h e r e f l e c t e d v e r t e x r e s p o n s e v a l u e i s worse t h a n t h e p r e v i o u s b e s t v e r t e x , b u t b e t t e r t h a n t h e p r e v i o u s w o r s t v e r t e x , t h e n t h i s d i r e c t i o n i s n o t as d e s i r a b l e . A c o n t r a c t i o n f a c t o r i s us e d t o c o n s t r u c t a new v e r t e x away f r o m t h e i n d i c a t e d d i r e c t i o n . R e f l e c t i o n , e x p a n s i o n and c o n t r a c t i o n p r o c e d u r e s a r e c o n t i n u e d u n t i l some r e s p o n s e v a l u e c r i t e r i o n i s met. Such a s t o p p i n g c r i t e r i o n may be t h e r e s p o n s e v a l u e s t a n d a r d d e v i a t i o n , or a p r e s e t r e s p o n s e v a l u e o b j e c t i v e . M o d i f i e d s u p e r s i m p l e x The m o d i f i e d s u p e r s i m p l e x p r o c e d u r e o f N a k a i and Kaneko (1985) r e l i e s upon t h e same g e n e r a l p r o c e d u r e as o u t l i n e d a b o v e . I n a d d i t i o n , a q u a d r a t i c f a c t o r i a l r e g r e s s i o n a n a l y s i s p r o c e d u r e was i n c l u d e d i n t h e p r o c e s s . The i n c l u s i o n o f t h i s p r o c e d u r e r e s u l t s i n t h e p r o c e s s g e n e r a t i n g a new r e f l e c t e d v e r t e x , a c e n t r o i d , and a c u r v e f i t t e d p o i n t g e n e r a t e d by q u a d r a t i c r e g r e s s i o n a n a l y s i s . The m o d i f i e d s i m p l e x p r o c e d u r e , t o g e t h e r w i t h mapping ( p r e v i o u s l y d e s c r i b e d i n t h e M a t e r i a l s and M e t h o d s ) , c o n s t i t u t e s t h e MSS. C e n t r o i d s e a r c h C e n t r o i d s e a r c h a l s o r e l i e s upon an i n i t i a l s i m p l e x g e n e r a t i o n . S u b s e q u e n t c e n t r o i d s e a r c h e s a r e u n d e r t a k e n as d e s c r i b e d i n t h e M a t e r i a l and Methods s e c t i o n . 187 Response surface methodology Response surface methodology as used in t h i s study i s not an EVOP, but rather a "batch type" operation. The RS technique begins with scaling of the independent factors. Subsequently, a symmetrical 3-level f a c t o r i a l design experiment is constructed as described by Box and Behnken (1960). Each experimental point i s then evaluated, and the resultant data analyzed by multiple regression analysis. From such an analysis, the regression c o e f f i c i e n t value for each factor i s u t i l i z e d to construct a response surface using the following response function: Y=b 0+b 1X 1+b 2X2+b 3X 1X2+b 4(X 1) 2+b 5(X 2) 2 where: bn = constant D1' D2 = linear c o e f f i c i e n t s b 3 = cross product c o e f f i c i e n t b 4,b5 = quadratic c o e f f i c i e n t s x l ' x 2 = levels of the two variables Response surfaces can be generated with a suitable graphing procedure. Comparison of simplex methods Both MDS and MSS are i t e r a t i v e procedures, with each new experimental point being generated one at a time. As a r e s u l t , generation of the next experimental point must wait u n t i l the previous experiment i s evaluated. Centroid search, however, generates n+1 experiments (centroids) simultaneously. A l l the centroids may be evaluated in one experimental run reducing the t o t a l time required to obtain the optimum point. This may be of considerable advantage to microbiological systems where incubation times may be long. 188 Analysis of variance of the comparison data showed, however, that none of the optimization techniques d i f f e r e d s i g n i f i c a n t l y in their a b i l i t y to at t a i n the optimum response value (Tables XlVa and b). This was true for both experiment time as well as for experiment number. The CS technique did require su b s t a n t i a l l y , but not s i g n i f i c a n t l y less time to a t t a i n the optimum response value than the MDS technique (Table XlVa). Theoretically, centroid search should have required less time to a t t a i n the optimum response value. Centroid search i s , however, confined to searching centroids only. The search area tends to be much smaller than the search area available to MDS or MSS. This smaller search area tends to reduce the e f f i c i e n c y of the technique. The advantage of simultaneous centroid searching, then, i s pa r t l y negated by a loss of e f f i c i e n c y due to a reduced search area. Response surface methodology Response surface methodology, in most cases, did not accurately predict the optimum response value. Since RS i s a batch type process, experiments cannot be altered "mid stream" to take advantage of previously evaluated experimental points. Response surface methodology can, however, predict over a l l response surface shapes, thus allowing subsequent EVOP techniques to pinpoint the true optimum point. This philosophy was previously adopted (Greasham and Inamine, 1986) to optimize cephamycin C 189 f e r m e n t a t i o n by s t r e p t o m y c e t e s . M o d i f i e d s u p e r s i m p l e x o r CS t e c h n i q u e s i n c o m b i n a t i o n w i t h t h e mapping p r o c e d u r e may be a b l e t o e l i m i n a t e t h e RS s t e p , however, a l l o w i n g f o r a s u b s t a n t i a l i n c r e a s e i n o p t i m i z a t i o n e f f i c i e n c y . O p t i m i z a t i o n i n m i c r o b i o l o g i c a l s y s t e m s In a n y s y s t e m an i n c r e a s e i n a r e s p o n s e v a l u e , may be g a i n e d by a l t e r i n g one or more f a c t o r v a l u e s . An i n c r e a s e i n a r e s p o n s e v a l u e i s d e f i n e d as an i n c r e a s e i n t h e d e s i r e d d i r e c t i o n , e i t h e r p o s i t i v e ( m a x i m i z a t i o n ) , or n e g a t i v e ( m i n i m i z a t i o n ) . The i n t u i t i v e a p p r o a c h w i l l most l i k e l y a l t e r one f a c t o r , w h i l e k e e p i n g a l l t h e o t h e r f a c t o r s c o n s t a n t . Not o n l y i s t h i s t e c h n i q u e i n e f f i c i e n t , b u t i t i s u n l i k e l y t o r e s u l t i n a t t a i n m e n t o f t h e optimum s e t o f f a c t o r l e v e l s . A b e t t e r a p p r o a c h would be t o a l t e r a l l t h e f a c t o r s s i m u l t a n e o u s l y , i n a random f a s h i o n . I t i s p o s s i b l e u s i n g t h i s t e c h n i q u e t o a p p r o a c h t h e t r u e optimum. A random s e a r c h i s i n e f f i c i e n t , however, b e c a u s e a l l p o s s i b l e f a c t o r l e v e l c o m b i n a t i o n s would have t o be e v a l u a t e d , i n o r d e r t o e n s u r e a t t a i n m e n t o f t h e optimum r e s p o n s e v a l u e . A more e f f i c i e n t t e c h n i q u e would be t o e v a l u a t e f a c t o r l e v e l c o m b i n a t i o n s , u s i n g a p r e s c r i b e d s e a r c h s t r a t e g y . One s u c h s e a r c h t e c h n i q u e i s s i m p l e x o p t i m i z a t i o n . T h i s t e c h n i q u e f o r c e s t h e s e a r c h t o move t o t h e r e g i o n o f optimum r e s p o n s e , and i s i t e r a t i v e i n n a t u r e . A p p l y i n g t h i s t o a m i c r o b i a l s y s t e m c o u l d r e s u l t i n a l e n g t h y s e a r c h due t o l o n g i n c u b a t i o n t i m e s . The c e n t r o i d s e a r c h s y s t e m may be more e f f e c t i v e under t h e s e c o n d i t i o n s , s i n c e t h e v e r t i c e s c r e a t e d 190 by t h e t e c h n i q u e c a n be e v a l u a t e d s i m u l t a n e o u s l y . Whatever t h e o p t i m i z a t i o n t e c h n i q u e u s e d ( i . e . s i m p l e x o r c e n t r o i d ) i t w i l l be more e f f i c a c i o u s t h a n e i t h e r i n t u i t i o n , or random s e a r c h e s . O p t i m i z a t i o n t e c h n i q u e s s u c h as s i m p l e x , or c e n t r o i d s e a r c h c a n i n i t i a l l y a p p e a r t o be c o m p l i c a t e d , and c o n f u s i n g . A l t h o u g h t h e m a t h e m a t i c s i n v o l v e d w i t h t h e g e n e r a t i o n o f new e x p e r i m e n t s i s n o t complex, i t c a n be t e d i o u s . U t i l i z a t i o n o f c o m p u t e r s c a n e l i m i n a t e most o f t h i s t e d i u m . The p e r f o r m a n c e o f t h e s e a r c h t e c h n i q u e may s u f f e r , i f t h e c h o i c e o f t h e s t a r t i n g f a c t o r l e v e l s a r e c l o s e t o t h e a c t u a l optimum. In r e l a t i v e t e r m s , t h e i n c r e a s e i n t h e r e s p o n s e v a l u e s w i l l be low. T h i s i s n o t a f a i l i n g o f t h e s e a r c h t e c h n i q u e , however, s i n c e a t t a i n m e n t o f t h e optimum r e s p o n s e v a l u e i s t h e p u r p o s e f o r e m p l o y i n g t h e o p t i m i z a t i o n method and what t h e s y s t e m has a c c o m p l i s h e d i s a q u i c k and v a l i d means o f v e r i f y i n g t h i s optimum. I n a m i c r o b i a l s y s t e m t h e a b s o l u t e r e s p o n s e v a l u e s o f t h e i n d i v i d u a l e x p e r i m e n t s must be l a r g e enough f o r t h e t e c h n i q u e t o o p e r a t e p r o p e r l y . In t h i s s t u d y g l u t a m i n e i n d u c e d r e l a t i v e l y s m a l l amounts o f p r o t e i n a s e , when compared t o o t h e r s u b s t r a t e s s u c h as TSB. A t t h e s e low l e v e l s , o p t i m i z a t i o n i s d i f f i c u l t . 191 CONCLUSIONS (1) When grown on s o l i d culture medium P. f r a g i started proteinase production ca. 20 h e a r l i e r than when grown in l i q u i d medium. (2) E x t r a c e l l u l a r v e s i c l e s present on the surface of P. f r a g i c e l l s grown on s o l i d surfaces, were absent from those c e l l s grown in l i q u i d culture. (3) Isolated e x t r a c e l l u l a r v e s i c l e s were ca. 20 nm in diameter. The evidence is persuasive but not conclusive that the ve s i c l e s contained the same proteinase as that found in the culture supernatant. The vesic l e s may be similar in composition to the outer c e l l membrane of P. f r a g i , suggesting they are associated with the outer c e l l membrane. (4) Counts done of the e x t r a c e l l u l a r v e s i c l e s on the surface of P. f r a g i c e l l s grown on s o l i d medium revealed an association between numbers of ve s i c l e s and proteinase production. (5) Optimization of c u l t u r a l conditions promoting proteinase production by P. f r a g i showed the optimum conditions to be: (i) temperature, 13 C; ( i i ) time, 38h; ( i i i ) pH, 6.8; (iv) organic nitrogen concentration, 314 mmole nitrogen/L; (v) oxygen, 16.4% oxygen In the gas mixture (7.4 ppm dissolved oxygen in the culture medium). 192 (6) Of the factors examined, oxygen was the largest contributing factor to proteinase production by P. f r a g i grown in a chemically defined medium. (7) The centroid search optimization technique was successfully applied in optimizing proteinase production by P. f r a g i in a defined c i t r a t e medium. (8) The MSS of Nakai and Kaneko (1985), the CS of Aishima and Nakai (1986) and the MDS of Morgan and Deming (1974) had similar e f f i c i e n c y in both experiment numbers and experimental time. The CS of Aishima and Nakai (1986) required s u b s t a n t i a l l y , but not s i g n i f i c a n t l y less time to a t t a i n the optimum response value than the MDS of Morgan and Deming (1974). Response surface methodology was unable, in most instances, to obtain the optimum point. (9) Pseudomonas f r a g i , under the conditions tested, did not produce bacteriocins active against the indicator organisms used in t h i s study. The e x t r a c e l l u l a r v e s i c l e s did not appear to possess bacteriocin a c t i v i t y . 193 LITERATURE CITED Aishima, T., and Nakai, S. 1986. Centroid mapping optimization: a new e f f i c i e n t optimization for food research and processing. J. Food S c i . 51:1297-1300 Anwar, H., Shand, G.H., Ward, K.H., Brown, M.R.W., Alpar, K.E., and Gowar, J. 1985. Antibody response to acute Pseudomonas aeruginosa i n f e c t i o n in a burn wound. FEMS Microbiol. Lett. 29:225-230. Austin-Prather, S.L., and Booth S.J. 1984. 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