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Proteolytic enzymes from fermentation of fish plant wastes Wah-On, Howard Christopher 1974

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PROTEOLYTIC ENZYMES FROM FERMENTATION OF FISH PLANT WASTES by HOWARD CHRISTOPHER WAH-ON B. Eng., M c G i l l U n i v e r s i t y , 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE i n the Department of CHEMICAL ENGINEERING We accept this, t h e s i s as conforming to, -the r e q u i t e d standard. THE UNIVERSITY OF BRITISH COLUMBIA August, 1974 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e H e a d o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t 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 V a n c o u v e r 8 , C a n a d a i i ABSTRACT A c l a r i f i e d , nonheat coagulable f i s h medium de r i v e d from condensed f i s h s o l u b l e s (B.C. Packers, Richmond, B.C.) was explored f o r i t s p o t e n t i a l use as the major component of a fermentation medium f o r the p r o d u c t i o n of p r o t e o l y t i c enzymes. The organism used was a pure s t r a i n of Sorangium. F a c t o r s a f f e c t i n g the b a c t e r i a l metabolic a c t i v i t y and consequently the r a t e and extent of protease formation were s t u d i e d . S t a t i s t i c a l l y designed experiments showed that both calcium and glucose had a s i g n i f i c a n t e f f e c t on growth and protease formation, the l a t t e r supplementary n u t r i e n t b e i n g the more i n f l u e n t i a l . Of the s i x carbohydrate sources (glucose, mannose, g a l a c t o s e , x y l o s e , arabinose, and s o l u b l e s t a r c h ) t e s t e d , only glucose and mannose were u t i l i z e d f o r f u r t h e r growth. N e i t h e r i n i t i a l pH ( i n the range 4 to 8) nor inoculum age (16 to 24 hrs) s i g n i f i c a n t l y a f f e c t the maximum y i e l d of protease. K i n e t i c s t u d i e s w i t h the optimum medium i n 250 ml Erlenneyer f l a s k s and i n a 7 - l i t r e fermentor revealed that growth was b i p h a s i c w i t h an i n i t i a l f a s t growth f o l l o w e d by a slower secondary growth. The staged growth was a t t r i b u t e d to the i n i t i a l , f a s t u t i l i z a t i o n of e a s i l y a v a i l a b l e n u t r i e n t s (such as amino acids) o r i g i n a l l y present i n the medium, followed by a second, slower r e a c t i o n i n which the l a r g e r p o l y p e p t i d e u n i t s i n the medium were d i g e s t e d . Since the i n f l e c t i o n i n the growth curve was e n t i r e l y e l i m i n a t e d by i n c r e a s i n g the a g i t a t i o n and/or a e r a t i o n r a t e ( s ) , i t was thought that d i s s o l v e d oxygen c o n c e n t r a t i o n and r a t e of oxygen i i i t r a n s f e r might be the r a t e l i m i t i n g f a c t o r s . K i n e t i c a n a l y s i s of the data showed that product formation was not a s s o c i a t e d by any d i r e c t mechanism w i t h carbohydrate u t i l i z a t i o n . Protease formation was hi g h e s t w i t h an a g i t a t i o n r a t e of 500 rpm and an a e r a t i o n r a t e of 2 l i t r e of a i r per minute, which corresponded to an oxygen t r a n s f e r c o e f f i c i e n t of 0.805 m i l l i m o l e s O^/atm. min. i. Recommendations f o r f u r t h e r work are suggested. i v ACKNOWLEDGEMENTS I wish to express s i n c e r e a p p r e c i a t i o n to Dr. Richard Branion, under whose d i r e c t i o n t h i s work was undertaken, f o r h i s support, kindness, and encouragement i n a l l phases of t h i s work. Thanks are a l s o due to Dr. George St r a s d i n e of the F i s h e r i e s Research Board of Canada f o r suggesting t h i s t o p i c , f o r h i s t e c h n i c a l advice, and f o r p r o v i d i n g s t a r t i n g r e f e r e n c e m a t e r i a l s , stock c u l t u r e s of Sorangium and condensed f i s h s o l u b l e s . A p p r e c i a t i o n i s a l s o extended to Mr. T.L. Truong and Mr. D. Ferguson f o r t h e i r i n v a l u a b l e i n s t r u c t i o n s i n the use of some of the equipment . Furthermore I wish to thank Mrs. Y.S. Choo f o r t y p i n g t h i s t h e s i s . L a s t , but not l e a s t , I wish to thank Environment Canada, F i s h e r i e s and Marine S e r v i c e , and the N a t i o n a l Research C o u n c i l of Canada, f o r p r o v i d i n g f i n a n c i a l support. V TABLE OF CONTENTS Page LIST OF TABLES v i i i LIST OF FIGURES x NOMENCLATURE x i i Chapter 1 INTRODUCTION 1 1.1 Nature of the Problem 1 1.2 F i s h S t i c k w a t e r and F i s h Solubles 3 1.3 Fermentation C u l t u r e Medium 4 1.3.1 Salmon-Canning Wastewater as a M i c r o b i a l Growth Medium 7 1.3.2 F i s h S t i c k w a t e r as a M i c r o b i a l Medium 11 1.3.3 F i s h S o lubles as a M i c r o b i a l Medium 12 1.4 M i c r o b i a l Proteases 16 1.5 The Micro-organism Used - Sorangium 495 17 1.6 N u t r i t i o n of Myxobacteria 20 1.7 O b j e c t i v e of t h i s Work 22 Chapter 2 EXPERIMENTAL TECHNIQUES 24 2.1 General 24 2.2 P r e p a r a t i o n of C u l t u r e Medium 24 2.3 Inoculum P r e p a r a t i o n 28 2.4 Apparatus 29 2.5 Sampling Technique 31 v i TABLE OF CONTENTS (Contd) Page 2.6 Measurement of B a c t e r i a l Growth 32 2.7 Measurement of Sugar Concentrat ion 34 I . Phenol - S u l f u r i c A c i d Reagent Method 34 I I . D i n i t r o s a l i c y l i c (DNS) Method 35 2.8 Measurement of P r o t e o l y t i c A c t i v i t y 38 2.9 Measurement of P r o t e i n Content 41 1. UV Absorpt ion 43 2. C o l o r i m e t r i c Methods 43 Chapter 3 RESULTS AND DISCUSSION 47 3.1 E f f e c t of Medium Const i tuents 47 3.2 E f f e c t of Glucose Concentrat ion 52 3.3 E f f e c t of I n i t i a l pH 57 3.4 E f f e c t of Condensed F i s h Solubles Concentrat ion 57 3.5 E f f e c t of Carbohydrate Source 63 3.6 E f f e c t of Inoculum Age 67 3.7 7 - l i t r e Fermentation Studies 71 3 .7 .1 E f f e c t of A g i t a t i o n and Aerat ion on the Course of Fermentation 72 3.7.2 E f f e c t of A g i t a t i o n and A e r a t i o n on Ult imate Y i e l d s 84 3 .7 .3 E f f e c t of A g i t a t i o n and Aerat ion on Rate of Oxygen Transfer 87 v i i TABLE OF CONTENTS (Contd) Page Chapter 4 FERMENTATION KINETICS 91 Chapter 5 CONCLUSIONS 107 Chapter 6 RECOMMENDATIONS 110 BIBLIOGRAPHY 112 Appendix I EXPERIMENTAL DATA 119 Appendix I I ANALYSIS PROCEDURES 139 11.1 Measurement of Glucose Concentration 140 11.2 Measurement of P r o t e o l y t i c A c t i v i t y 144 11.3 P r o t e i n E s t i m a t i o n w i t h the B i u r e t Reagent 148 Appendix I I I COMPUTATION OF SUM OF SQUARES 151 v i i i LIST OF TABLES Table Page 1.1 PARAMETERS OF VARIOUS FISH PROCESSING PLANT EFFLUENTS 2 1.2 TYPICAL ANALYSIS OF FISH STICKWATER 6 1.3 TYPICAL ANALYSIS OF CONDENSED FISH SOLUBLES 6 1.4 CELL YIELD AS A FUNCTION OF NITROGEN SOURCE FOR VARIOUS SPECIES OF BACTERIA 9 1.5 CELL YIELD OF SALMON-CANNING WASTE WATER AS A FUNCTION OF CELL NUMBER PER MILLILITER 10 1.6 AMINO ACID ANALYSIS OF CONDENSED FISH SOLUBLES 13 1.7 TYPICAL VITAMIN CONTENT OF WEST COAST FISH SOLUBLES 14 1.8 TYPICAL ANALYSIS OF FISH SOLUBLES ASH CONSTITUENTS 14 1.9 ALTERNATE PROTEIN SUPPLY SOURCES 15 2.10 TIME VARIATION IN pH AND DRY WEIGHT OF REFRIGERATED CFS 25 2.11 ANALYSIS OF FILTERED CFS AT VARIOUS DILUTIONS 27 3.12 RESULTS OF FACTORIAL EXPERIMENTS 49 3.13 ANALYSIS OF VARIANCE 51 3.14 EFFECT OF INITIAL pH 58 3.15 EFFECT OF AGITATION AND AERATION ON ULTIMATE YIELDS 86 3.16 K VALUES ATTAINED IN 7-L FERMENTATION RUNS 89 v 4.17 7-LITRE FERMENTATION - CFS RUN NO. 3 VOLUMETRIC RATES OF 92 4.18 7-LITRE FERMENTATION - CFS RUN NO. 3 SPECIFIC RATES OF 93 4.19 7-LITRE FERMENTATION - CFS RUN NO. 5 VOLUMETRIC RATES OF 94 i x LIST OF TABLES (Contd.) Table Page 4.20 7-LITRE FERMENTATION - CFS RUN NO. 5 SPECIFIC RATES OF 95 4.21 7-LITRE FERMENTATION - CFS RUN NO. 7 VOLUMETRIC RATES OF 96 4.22 7-LITRE FERMENTATION - CFS RUN NO. 7 SPECIFIC RATES OF 97 A I . l EFFECT OF GLUCOSE CONCENTRATION (FILTERED CFS) 120 AI .2 EFFECT OF GLUCOSE CONCENTRATION (UNFILTERED CFS) 121 AI . 3 EFFECT OF CFS CONCENTRATION 122 AI .4 EFFECT OF HEXOSE SUGARS 125 AI .5 EFFECT OF PENTOSES AND STARCH 127 AI .6 EFFECT OF INOCULUM AGE 128 AI .7 7-LITRE FERMENTATION - NB - GLUCOSE RUN 130 AI .8 7-LITRE FERMENTATION - CFS RUN NO. 1 131 AI .9 7-LITRE FERMENTATION - CFS RUN NO. 2 132 AI.10 7-LITRE FERMENTATION - CFS RUN NO. 3 133 A I . l l 7-LITRE FERMENTATION - CFS RUN NO. 4 134 AI.12 7-LITRE FERMENTATION - CFS RUN NO. 5 135 AI.13 7-LITRE FERMENTATION - CFS RUN NO. 6 136 AI.14 7-LITRE FERMENTATION - CFS RUN NO. 7 137 AI.15 SHAKE FLASK FERMENTATION - GLUCOSE DEFICIENT MEDIUM 138 X LIST OF FIGURES Figure Page 1.1 FISH RENDERING PROCESS 5 2.2 SCHEMATIC DRAWING OF 7-LITRE FERMENTOR 30 2.3 TURBIDITY VS DRY CELL WEIGHT AT VARIOUS WAVELENGTHS 33 2.4 COMPARISON OF DNS AND PHENOL-H„SO. ASSAYS 37 2 4 2.5 PROTEOLYTIC ACTIVITY VS ENZYME CONCENTRATION 42 3.6 EFFECT OF GLUCOSE CONCENTRATION ON ACTIVITY -UNFILTERED CFS 54 3.7 EFFECT OF GLUCOSE CONCENTRATION ON GROWTH -FILTERED CFS 55 3.8 EFFECT OF GLUCOSE CONCENTRATION ON ACTIVITY -FILTERED CFS 56 3.9 EFFECT OF CONDENSED FISH SOLUBLES CONCENTRATION ON GROWTH 60 3.10 EFFECT OF CONDENSED FISH SOLUBLES CONCENTRATION ON PROTEASE FORMATION 61 3.11 EFFECT OF PROTEIN CONCENTRATION ON ULTIMATE PROTEASE YIELD AND ON MAXIMUM RATE OF PROTEASE FORMATION 62 3.12 EFFECT OF HEXOSES ON PROTEASE FORMATION 64 3.13 EFFECT OF SOLUBLE STARCH AND PENTOSES ON PROTEASE FORMATION 65 3.14 EFFECT OF CARBOHYDRATE SOURCE ON GROWTH 66 3.15 EFFECT OF INOCULUM AGE ON GROWTH 68 3.16 EFFECT OF INOCULUM AGE ON GLUCOSE UTILIZATION 69 3.17 EFFECT OF INOCULUM AGE ON PROTEASE FORMATION 70 3.18 7-LITRE FERMENTOR NB - GLUCOSE RUN 73 3.19 7-LITRE FERMENTOR CFS RUN NO. 1 74 3.20 7-LITRE FERMENTOR CFS RUN NO. 2 75 x i LIST OF FIGURES (Contd) Figure Page 3.21 7-LITRE FERMENTOR CFS RUN NO. 3 76 3.22 7-LITRE FERMENTOR CFS RUN NO. 4 77 3.23 7-LITRE FERMENTOR CFS RUN NO. 5 78 3.24 7-LITRE FERMENTOR CFS RUN NO. 6 79 3.25 7-LITRE FERMENTOR CFS RUN NO. 7 80 3.26 SHAKE FLASK FERMENTATION - GLUCOSE DEFICIENT MEDIUM 85 3.27 ACTIVITY AND CELL YIELD VS SUGAR USED FOR VARIOUS 7-LITRE FERMENTOR RUNS 88 3.28 MAXIMUM YIELD AND MAXIMUM RATE OF ENZYME PRODUCTION AS A FUNCTION OF K v 4.29 VOLUMETRIC RATES VS TIME FOR FERMENTOR RUN NO. 3 98 4.30 SPECIFIC RATES VS TIME FOR FERMENTOR RUN NO. 3 99 4.31 VOLUMETRIC RATES VS TIME FOR FERMENTOR RUN NO. 5 100 4.32 SPECIFIC RATES VS TIME FOR FERMENTOR RUN NO. 5 101 4.33 VOLUMETRIC RATES VS TIME FOR FERMENTOR RUN NO. 7 102 4.34 SPECIFIC RATES VS TIME FOR FERMENTOR RUN NO. 7 103 4.35 SPECIFIC RATE OF GLUCOSE UTILIZATION VS SPECIFIC RATE OF GROWTH FOR VARIOUS FERMENTOR RUNS 105 All.36 GLUCOSE TESTS STANDARD CURVES 142 All.37 TYROSINE TEST STANDARD CURVE 147 All.38 PROTEIN TEST STANDARD CURVE 150 x i i NOMENCLATURE Abbreviat ions BSA Bovine Serum Albumin CFS Condensed F i s h Solubles . DNS D i n i t r o s a l i c y l i c A c i d FPC F i s h P r o t e i n Concentrate NB Nutr i en t Broth NBS New Brunswick S c i e n t i f i c OD O p t i c a l Density SCE Salmon-canning E f f l u e n t SCP S i n g l e - c e l l P r o t e i n Symbols df S t a t i s t i c a l number of degrees of freedom E Enzyme e Enzyme concentrat ion ES Enzyme - Substrate complex F F - t e s t s t a t i s t i c k^ Forward r e a c t i o n rate constant Reverse r e a c t i o n rate constant k^ Reaction rate constant k Michael i s -Menten constant m Kv Volumetric t r a n s f e r c o e f f i c i e n t x i i i Symbols (contd) M M o l a r i t y MS S t a t i s t i c a l mean square N Normali ty P Product S Substrate s Concentrat ion of t o t a l substrate SS S t a t i s t i c a l sum of squares SSE Sum of squares of the r e s i d u a l e r r o r a f t e r some mathematical model has been app l i ed to the experimental data t Time, u s u a l l y i n hours v Rate of product formation x C e l l mass 1 Chapter 1 INTRODUCTION 1.1 Nature of the problem The salmon-canning and the h e r r i n g process ing operat ions (e i ther for reduct ion to meal and o i l , or for food purposes) on the West Coast cause major problems of l i q u i d waste d i s p o s a l . Average e f f luent flow from these process ing operations are of the order of 200 to 1000 U . S . G . P . M . for most p lants [1] . These large wastewater flows r e s u l t from the b u t c h e r i n g , washing and process ing of the product . The major p o l l u t a n t s i n these e f f luents are f l e s h p a r t i c l e s , s c a l e s , b l o o d , s l ime and s o l u b l e p r o t e i n s . When o i l y f i s h , such as menhaden, s a r d i n e s , mackerel , and h e r r i n g are processed, free o i l i n the form of an emulsion i s a lso present as a contami-nant i n these wastewaters. Table 1.1, which i s taken from Claggett [1] shows t y p i c a l parameters of these wastewaters. P r e v i o u s l y , these e f f luents were discharged to waterways, or to sewage treatment p l a n t s ; the proteinaceous matter decomposed r a p i d l y , evo lv ing CC^, and other gases, thus g i v i n g r i s e to ser ious p o l l u t i o n problems. Owing to i n c r e a s i n g p o l l u t i o n c o n t r o l l e g i s l a t i o n , other methods of d i spos ing of these wastes have been s t u d i e d . The more u s e f u l methods descr ibed so far have concentrated on recover ing as much of the proteinaceous and o i l y m a t e r i a l as p o s s i b l e . Claggett [1, 2, 3, 4, 5] apparently the most v e r s a t i l e i n v e s t i g a t o r i n th i s area , r ecent ly descr ibed a method i n which Table 1.1 PARAMETERS OF VARIOUS FISH PROCESSING PLANT EFFLUENTS SPECIES PROCESSED BOD SUSPENDED SOLIDS TOTAL SOLIDS mg/ l #/1000# f i s h mg/ l #/1000# f i s h mg/ l ///1000// f i s h Ha l ibut 200 4 350 7.2 500 10.3 Grey Cod 435 2.2 300 1.5 600 3.0 Lingcod 460 4.1 235 2.2 550 5.2 Sole 200 1.4 125 0.8 300 1.9 Ocean Perch 75 0.7 50 1.3 85 2.2 Salmon Canning 3500 28 1500 12 3000 24 Food H e r r i n g 3850 22 3000 21 6000 42 ho 3 the BOD load of salmon canning wastewaters could be reduced by over 90%. This method invo lves chemical treatment and a i r f l o t a t i o n . The sludge from th i s process i s used for the product ion of a meal which could rep lace the convent ional f i sh-meal i n animal feed. One of the by-products from the f i s h meal product ion process i s f i s h s t i c k w a t e r . At present t h i s s t ickwater i s used to make f i s h so lub les which are used as v i tamin supplements i n animal feed. In view of the r i c h p r o t e i n content of the f i s h waste, another poss ib l e o u t l e t i s to use i t as an inducer for the product ion of m i c r o b i a l proteases . Such a process would be qu i te contemporary and i t has the p o t e n t i a l of k i l l i n g two b i r d s wi th one stone; i t can serve as a p o l l u t i o n abatement system as w e l l as an enzyme product ion process . The s c a r c i t y of m i c r o b i a l enzymes together with t h e i r r i s i n g importance i n indus try provide the source of optimism for the development of an economical ly f e a s i b l e process . 1.2 F i s h St ickwater and F i s h Solubles An exce l l en t account of the o r i g i n and proper t i e s of f i s h so lubles i s given by Soderquist [6] . A summary of the s a l i e n t po ints i s presented here . F i s h so lub les are made from f i s h s t ickwater which i s a by-product from f i s h - r e n d e r i n g processes . The f i s h and/or f i s h waste are steam-cooked to denature the f i s h p r o t e i n . This m a t e r i a l i s then separated under pressure 4 i n t o an aqueous e x t r a c t and f i s h pulp. The f i s h pulp i s dehydrated to a meal, c o n t a i n i n g about 8% moisture, which can be s o l d as a f e r t i l i z e r or as an animal feed. The press water i s screened to remove any s o l i d s and then passed through a g r a v i t y separator or a c e n t r i f u g e . The sludge from t h i s process i s returned to the press cake and the c l a r i f i e d press l i q u o r i s passed to an o i l c e n t r i f u g e . The o i l c e n t r i f u g e y i e l d s two products, namely, the f i s h o i l and the s t i c k w a t e r . The s t i c k w a t e r i s t r e a t e d w i t h a c i d i n h o l d i n g tanks and then t r a n s f e r to an evaporator. The evaporator reduces the water content from 95% to 50%. " S o l u b l e s " are produced i n the form of a brown, somewhat viscous l i q u i d w i t h a m i l d , f i s h y odour. Figure 1.1 (taken from Soderquist) shows the process as d e s c r i b e d . The approximate composition of s t i c k w a t e r and condensed f i s h s o l u b l e s are given i n Tables 1.2 and 1.3 r e s p e c t i v e l y [ 7 ] . 1.3 Fermentation C u l t u r e Medium The n u t r i t i o n a l requirements of micro-organisms are d i v e r s e because they d i f f e r i n h e r e n t l y i n a b i l i t y to s y n t h e s i z e e s s e n t i a l growth f a c t o r s from simple n u t r i e n t s . However, a l l micro-organisms demand water, carbon, n i t r o g e n and m i n e r a l elements, as w e l l as access to hydrogen and oxygen [ 8 ] . The development and f o r m u l a t i o n of c u l t u r e medium i s one of the most d i v e r s e steps of a fermentation programme; i t r e q u i r e s a l o t of f r i g g i n g around. The type of medium used depends upon s e v e r a l i n t e r r e l a t e d 5 Figure 1.1 FISH RENDERING PROCESS PROCESS WASTES DISPOSAL Table 1.2 TYPICAL ANALYSIS OF FISH STICKWATER PARAMETER VALUE T o t a l s o l i d s . 5.6% Ash 0.95% Fat ty substances 0.60% Crude p r o t e i n (N x 6.25) 3.5% Table 1.3 TYPICAL ANALYSIS OF CONDENSED FISH SOLUBLES PARAMETER VALUE T o t a l s o l i d s 50.43% Ash 8.86% Fat 4.8% Crude p r o t e i n (N x 6.25) 33.85% Sp. g r . at 20°C 1.20 pH 4.5 7 v a r i a b l e s . Cost i s almost always a f a c t o r i n determining the s u i t a b i l i t y of any i n d u s t r i a l fermentation medium. When large q u a n t i t i e s of c u l t u r e medium are r e q u i r e d , the exce l l en t and covenient types of s tandardized laboratory media as supp l i ed by D i f c o , Oxoid , BBL, e t c . are very uneconomical, i t i s therefore necessary to f ind a l t e r n a t i v e sources of n u t r i e n t s . Obviously such m a t e r i a l s , because they must be cheaper, cannot be as pure or as s tandardized as t h e i r l aboratory counterparts . I n d u s t r i a l fermentation media are therefore u s u a l l y complex [9] . T h e i r compositions are u s u a l l y formulated to promote the des ired metabol ic patterns of the micro-organism under examination; however, s ince knowledge of the l a t t e r i s u s u a l l y l i m i t e d , development i s done on an adhoc b a s i s , although under ly ing p r i n c i p l e s can sometimes be i n c o r p o r a t e d . A protease i s an enzyme that hydrolyses prote ins and therefore the fermentation medium must supply the n u t r i t i o n a l s t imulus to cause i t to do so. The fo l lowing sect ions discuss the s u i t a b i l i t y of salmon-canning wastewaters and f i s h e r i e s by-products as the major component of a fermenta-t i o n medium for protease p r o d u c t i o n . 1.3.1 Salmon-Canning Wastewater as a M i c r o b i a l Growth Medium Strasdine and M e l v i l l e [10] showed that salmon-canning e f f l u e n t (SCE) can support the growth of s i x species of b a c t e r i a when used both as a component of a complex medium and as the so le source of a v a i l a b l e n i t r o g e n . Tables 1.4 and 1.5, are adapted from Strasd ine and M e l v i l l e [10]. Table 1.4 ind ica te s the t o t a l c e l l y i e l d for each of the s i x species i n media 8 containing the var ious n i t rogen sources together wi th K^HPO^ (0.2%), MgS0 4 .7H 2 0 (0.02%), F e S 0 4 . 4 H 2 0 (0.002%), MnS0 4 .4H 2 0 (0.002%) and dextrose (0.5%). Table 1.5 i n d i c a t e s the a b i l i t y of SCE to serve i n the capaci ty of a complete medium i n which no other addi t ions were necessary. Based on these r e s u l t s , the authors suggest two p o s s i b l e uses for salmon-canning wastewaters, namely : (1) as an inexpensive source of a v a i l a b l e n i t rogen for the m i c r o b i a l degradation and/or u t i l i z a t i o n of n i t r o g e n - d e f i c i e n t wastes, and (2) as a m i c r o b i o l o g i c a l medium per se for the product ion of crude enzyme prepara t ions , and of s i n g l e - c e l l p r o t e i n s . The f i r s t suggest ion has been worked on to a c e r t a i n extent by Truong [11], whose main i n t e r e s t was i n the product ion of Vi tamin B ^ 2 and v o l a t i l e acids (Propionic and a c e t i c i n h i s case) v i a fermentation of s u l f i t e spent l i q u o r . He was unsuccessful i n growing L a c t o b a c i l l u s plantarum, P r o p i o n i -b a c t e r i a F r e u d e n r e i c h i i and P r o p i o n i b a c t e r i a Shermanii i n a medium i n which yeast extract was replaced by f i s h s t ickwater as the so le source of n i t r o g e n . No work has been reported yet on the crude enzyme product ion p o t e n t i a l of salmon-canning wastewaters. Why not? The major d i f f i c u l t i e s which w i l l obvious ly a r i s e from the use of f i s h process ing wastewaters as a fermentation substrate are (1) large volume of e f f luent to be processed, (2) r a p i d b i o d e g r a d a b i l i t y of the wastes, (3) l arge f l u c t u a t i o n s i n composit ion, and (4) seasonal a v a i l a b i l i t y . The f i r s t two c h a r a c t e r i s t i c s give r i s e to p r a c t i c a l d i f f i -c u l t i e s of storage whi le the t h i r d give r i s e to d i f f i c u l t i e s i n media r e p r o d u c i b i l i t y . These problems are l e ss apparent when the more concentrated Table 1.4 CELL YIELD AS A FUNCTION OF NITROGEN SOURCE FOR VARIOUS SPECIES OF BACTERIA N-SOURCE SPECIE SCE TRYPTICASE CELLS/ML OF MEDIA (loj ACIDICASE POLYPEPTONE '10 ) PEPTONE PHYTONE MEAT EXTRACT Sorangium sp . 10.08* 6.99 9.51 9.37 9.23 8.29 8.68 P. putre fac iens 9.32 6.38 7.00 8.15 5.30 8.36 8.28 L . plantarum 8.18 7.88 5.30 8.11 9.18 7.26 6.46 A. aerogenes 8.67 7.96 7.79 9.63 9.00 9.04 9.57 B a c i l l u s sp . 7.00 8.65 8.15 8.08 7.63 7.83 7.18 S t . f a e c a l i s 7.79 7.04 7.30 8.69 7.72 8.23 7.63 Under l ined values i n d i c a t e maximum c e l l y i e l d . Table 1.5 CELL YIELD OF SALMON-CANNING WASTE WATER AS A FUNCTION OF CELL NUMBER PER MILLILITER CELLS/ML d o g 1 0 ) % OF MAXIMUM FROM TABLE 1.4 MEDIA GIVING MAXIMUM GROWTH Sorangium sp. 9.34 19.1 SCE - S A. aerogenes 8.60 9.3 polypeptone - S Bacillus sp. 7.20 3.6 trypticase - S P. putrefaciens 6.83 0.3 SCE - S St. faecalis 6.76 1.2 polypeptone - S L. plantarum 6.74 0.3 peptone - S S : salts 11 f i s h s t ickwater and so lubles are used. 1.3.2 F i s h St ickwater as a M i c r o b i a l Medium F i s h s t i ckwater , as s tated e a r l i e r , i s an aqueous ex trac t r e s u l t i n g from the press ing of cooked f i s h dur ing f i s h meal p r o d u c t i o n . St ickwater or presswater, as i t sometimes c a l l e d , i s a l so the raw m a t e r i a l for the manufacture of condensed f i s h s o l u b l e s . St ickwater e x h i b i t s p r o p e r t i e s which are c h a r a c t e r i s t i c of both f i s h muscle ex trac t s and the g e l a t i n and g lue -conta in ing extracts of bones, c a r t i l a g e , s k i n , and other connective t i s sues [12]. A t y p i c a l ana lys i s of some of the main const i tuents of s t i c k -water i s shown i n Table 1.2. The n i t r o g e n - c o n t a i n i n g substances i n s t i ckwater c o n s i s t l a r g e l y of noncoagulable, water - so lub le proteoses , peptones, h i g h l y d ispersed p r o t e i n p a r t i c l e s , and nitrogenous e x t r a c t i v e s [6, 7 ] . These e x t r a c t i v e s inc lude such compounds as ammonia, mono-, d i , and tr imethylamine, urea , amino a c i d s , guanidine d e r i v a t i v e s (creat ine and arg in ine) and imidozole d e r i v a t i v e s ( h i s t i d i n e and c a r n o s i n e ) . I f the propor t ion of these e x t r a c t i v e s , which are not considered to be food p r o t e i n sources , i s unduly h i g h , the conver-s ion fac tor of 6.25, which assumes that the average p r o t e i n contains 16.0% of n i t r o g e n , gives a f a l s e l y h igh value for the p r o t e i n content . The f a t t y substances are h i g h l y d i spersed and are i n t i m a t e l y t i e d to the p r o t e i n p a r t i c l e s to give the s t ickwater an opaque or milky appearance. These suspended p a r t i c l e s can be p r e c i p i t a t e d by adding a f l o c c u l a n t l i k e a lumi -nium s u l f a t e or by a change i n the pH of the s t i ckwater . 12 Although the s t i c k w a t e r contains many d e s i r a b l e b i o l o g i c a l growth f a c t o r s , i t s h i g h l y p e r i s h a b l e nature, even at very low temperatures, makes i t very unpleasant to work w i t h , and very d i f f i c u l t to s t o r e . I t i s a w e l l recognised, but rarely-mentioned, f a c t that f o r a s u b s t r a t e to be indus-t r i a l l y acceptable, the s u b s t r a t e must not only be appealing to the microbes but i t must a l s o not create an adverse employee r e a c t i o n . 1.3.3 F i s h Solubles as a M i c r o b i a l Medium F i s h s o l u b l e s , as mentioned e a r l i e r , i s a c i d i f i e d and concentrated s t i c k w a t e r . D i g e s t i o n w i t h a c i d degrades the p r o t e i n s present i n s t i c k w a t e r to s m a l l e r molecules, thus making i t more d e s i r a b l e as a b a c t e r i o l o g i c a l c u l t u r e media. A c i d i f i c a t i o n a l s o reduces the problem of s p o i l a g e ; concen-t r a t i o n reduces storage space and enables a wider range of c o n c e n t r a t i o n to be examined. Tables 1.6, 1.7, 1.8, adapted from Lassen [ 7 ] , r e f l e c t the average content of some of the more important amino a c i d s , vitamins and ash c o n s t i -tuents r e s p e c t i v e l y . These, together w i t h some other u n i d e n t i f i e d growth f a c t o r s [13] r e v e a l the reason why condensed f i s h s o l u b l e s have a t t a i n e d such prominence i n the f i e l d of animal n u t r i t i o n . However not much revenue i s obtained f r o n t h i s use. Table 1.9 compares the p r i c e s of commercially a v a i l a b l e p r o t e i n sources which can be used as the major component of a fermentation medium f o r protease production. Most obvious i s the r e l a t i v e l y low cost of Table 1.6 AMINO ACID ANALYSIS OF CONDENSED FISH SOLUBLES % CRUDE PROTEIN ASSAY METHOD AMINO ACID (N x 6.25) A r g i n i n e 4. 84 Mb. a H i s t i d i n e 5. 79 6 Chem. Lysi n e 4. 87 Mb. Leucine 4. 67 Mb. I s o l e u c i n e 2. 73 Mb. Phenylalanine 2. 33 Chem. Tryptophan 0. 35 Mb. Methionine 1. 51 Mb. Threonine 2. 55 Chem. Cys t i n e 0. 58 Chem. Glutamic a c i d 8. 44 Chem. P r o l i n e 6. 70 Chem. Mb. = m i c r o b i o l o g i c a l . Chem. = chemical. Table 1.7 TYPICAL VITAMIN CONTENT OF WEST COAST FISH SOLUBLES VITAMIN Mg/g VITAMIN ug/g R i b o f l a v i n 22 P y r i d o x i n 12.5 Pantothenic A c i d 84 Choline 1100 Thiamine 7.5. F o l i c A c i d 0.23 N i a c i n 390 Vitamin B ^ 0.47 . Table 1.8 TYPICAL ANALYSIS OF FISH SOLUBLES ASH CONSTITUENTS CONSTITUENT % CONSTITUENT % Potassium (K) 1.93 Ir o n (Fe) 0 .0249 Sodium (Na) 1.87 Magnesium (Mg) 0 .016 Phosphorous (P) 0.85 Copper (Cu) 0 .007 Calcium (Ca) 0.0869 Iodine (I) 0 .007 Manganese (Mn) 0.0869 Aluminum ( Al) 0 .005 TOTAL ASH 8.86% 15 Table 1.9 ALTERNATE PROTEIN SUPPLY SOURCES [14, 15] MATERIAL PRICE/POUND* PRICE/POUND PROTEIN (cents) (cents) Soy Meal and Flour 3 .5-6 .5 8-14.8 Soy Protein Cone. 21.5 26.5-35 F i s h Solubles 2.50 7.8 F i s h Meal (feed grade) 6 .3-8 .5 10.5-14.2 F i s h Protein Cone. 10-16 13-20 Cottonseed Flour 11 20 Wheat F lour 6.6 60 Dry Skim Milk 14.4-21.0 40-60 Yeast - Torula ( s u l f i t e waste) 15-16 27-29 * Based on 1969 market. 16 condensed f i s h s o l u b l e s . For the reasons c i t e d above, t h i s work, which i s to i n v e s t i g a t e the protease production p o t e n t i a l of condensed f i s h s o l u b l e s , emerges. 1.4 M i c r o b i a l Proteases A l l p r o t e o l y t i c enzymes, or proteases, whether they are of animal, p l a n t , or m i c r o b i a l o r i g i n are c h a r a c t e r i z e d by t h e i r a b i l i t y to c a t a l y z e h y d r o l y t i c cleavage of peptide l i n k a g e s between amino acid s [16, 17, 18, 19]. These enzymes are g e n e r a l l y c l a s s i f i e d i n t o two d i s t i n c t groups [20, 21], namely, the proteinases (or endopeptidases) and the peptidases (or exopeptidases). The proteinases are enzymes which a t t a c k i n t e r n a l peptide bonds i n the high molecular weight pol y p e p t i d e p r o t e i n molecules; peptides such as proteoses and peptones are u s u a l l y formed from t h i s r e a c t i o n . The peptidases are enzymes which a t t a c k t e r m i n a l peptide bonds adjacent to f r e e p o l a r groups thus r e s u l t i n g i n the l i b e r a t i o n of f r e e amino a c i d s [17]. A l l micro-organisms which u t i l i z e p r o t e i n s as n u t r i e n t m a t e r i a l s produce p r o t e o l y t i c enzymes f o r the h y d r o l y s i s of the p r o t e i n s to the amino acids necessary i n t h e i r metabolism f o r the production of t h e i r own c e l l u l a r and enzyme p r o t e i n s . The m a j o r i t y of m i c r o b i a l enzymes s t u d i e d to date have been e x t r a c e l l u l a r , i s o l a t e d i n a c t i v e form from the c u l t u r e f i l t r a t e s of the appropriate organism. These e x t r a c e l l u l a r enzymes are predominantly endopeptidases and can be d i v i d e d i n t o three main groups, namely, a c i d , 17 n e u t r a l , or a l k a l i n e , depending upon the pH range i n which t h e i r a c t i v i t y i s greatest [22, 23, 24] . The few m i c r o b i a l proteases present ly a v a i l a b l e commercially are from b a c t e r i a l and fungal sources; they are u s u a l l y s o l d i n the crude from, which may conta in a v a r i e t y of other enzymes i n vary ing amounts. Hence t h e i r enzymatic a c t i o n i s extremely compl icated. Notwithstanding the i m p u r i t i e s present , these m i c r o b i a l enzymes are enjoying wide a p p l i c a t i o n s i n i n d u s t r y . Some of these a p p l i c a t i o n s [19, 20, 23] , most of which were developed from e m p i r i c a l , t r i a l and e r r o r methods, are i n bread making, meat t e n d e r i z i n g , spot removal i n c l ean ing , deha ir ing of h i d e s , c h i l l proof ing of beer , plaque removal from teeth , and p r o t e i n hydro lysa te manufacturing. In 1969, over 4 m i l l i o n d o l l a r s ' worth of p r o t e o l y t i c enzymes were marketed [25]. 1.5 The Micro-organism Used - Sorangium 495 The organism chosen for th i s study was a s o i l b a c t e r i a be longing to the genus Sorangium. This organism, designated i s o l a t e No. 495, was i s o l a t e d by G i l l e s p i e and Cook [26] i n 1964. I t was obtained for t h i s study from G . A . S t r a s d i n e , Vancouver l a b o r a t o r y , F i s h e r i e s Research Board of Canada, who, as mentioned e a r l i e r , showed that th i s specie gave the h ighest c e l l y i e l d i n media conta in ing SCE as the so le source of a v a i l a b l e n i t r o g e n . 18 A perusa l of "Sergey's Manual of Determinative Bac ter io logy" [27] shows that the genus Sorangium i s a member of the family Sorangiaceae which belongs to the order Myxobacterales . Members of th i s Order can be d i s t ingu i shed from a true bacterium by two p r i n c i p a l features : (1) i t s c h a r a c t e r i s t i c g l i d i n g , n o n - f l a g e l l a r movement, which occurs only i n contact with s o l i d sur faces , and (2) i t s f l e x i b i l i t y , due to the d e l i c a t e nature of the c e l l w a l l . The vegetat ive c e l l s of the Myxobacter ia are gram-negative rods which dur ing vegetat ive growth remain p e r f e c t l y independent of one another, although they tend to be he ld i n a s s o c i a t i o n by a t h i n layer of s l ime which they synthes ize dur ing movement and which keeps them together i n a loose colony. Such a myxobacter ia l colony can give r i s e to s tructures known as f r u i t i n g bodies , which are formed by the co -operat ive ac t i on of many thousands of vegetat ive c e l l s [28, 29] . D i r e c t photometric methods of measuring growth are therefore imposs ib le . The vegetat ive c e l l s of the family Sorangiaceae are s h o r t , r i g i d c e l l s with b lunt ends (1.2 x 2.5) , and thus d i f f e r from the members of the other fami l i e s (Myxococcaceae, Archangiaceae, and Polyangiaceae) which possess vegetat ive c e l l s of the long, f lexuous, tapered type (0.5 x 5 - 10) [30]. The Sorangium species are predominantly s o i l forms. Many i s o l a t e s are s t rong ly c e l l u l o l y t i c , although some do not possess th i s proper ty . The work of G i l l e s p i e and Cook [26] and l a t e r , Whitaker, Cook and G i l l e s p i e [31] showed that t h e i r i s o l a t e , Sorangium 495, was able to to produce at l e a s t two e x t r a c e l l u l a r enzymes which are separable by 19 chromatography on hydroxyapatite: (1) a l y t i c enzyme which e f f e c t s complete l y s i s of v a r i o u s species of Staphylococcus, B a c i l l u s , S a r c i n a , and A r t h r o b a c t e r , and p a r t i a l l y s i s of b a c t e r i a from s e v e r a l other genera; (2) a protease w i t h h y d r o l y t i c a c t i v i t y towards c a s e i n and denatured hemoglobin but without l y t i c a c t i v i t y towards the above organisms. G i l l e s p i e and Cook [26] s t u d i e d the change i n p r o t e o l y t i c a c t i v i t y w i t h changing pH f o r both c a s e i n and haemoglobin. T h e i r r e s u l t s showed that the pH optimum f o r both s u b s t r a t e s was at pH 8.5. Katznelson et a l [32] have a l s o observed that c e r t a i n s o i l nematodes were l y s e d by t h i s s t r a i n of Sorangium. Subsequent work by Whitaker [33], Jurasek and Whitaker [34], Whitaker et a l [35], T s a i e t a l [36], showed that there were two enzymes which were r e s p o n s i b l e f o r the high l y t i c a c t i v i t y . They c a l l e d these two enzymes a- and 3 - l y t i c proteases. Recently, owing to p o p u l a t i o n i n c r e a s e and land shortage, much concern has been expressed about a world p r o t e i n shortage i n the f u t u r e . M i c r o b i o l o g i c a l l y - s y n t h e s i z e d p r o t e i n , u s u a l l y termed s i n g l e c e l l p r o t e i n (SCP) due to the u n i c e l l u l a r nature of i t s parent micro-organism, i s one way of supplementing the conventional p r o t e i n s from a g r i c u l t u r e and the f i s h i n g i n d u s t r y [37, 38]. Micro-organisms have many a t t r a c t i o n s as p r o t e i n supplements: they m u l t i p l y r a p i d l y , are e a s i l y handled, and can be grown e c o n i m i c a l l y on inexpensive organic m a t e r i a l s such as s u l f i t e l i q u o r s , f i s h wastes, hydrocarbons, molasses, whey and other a g r i c u l t u r a l wastes [38]. However, before these SCP can be used as food f o r human consumption, the c e l l w a l l , which i s r e s i s t a n t to d i g e s t i o n , must be 20 d i s r u p t e d . L y t i c enzymes from s e l e c t e d micro-organisms such as Sorangium 495 may prove v a l u a b l e f o r t h i s purpose. Another p o s s i b l e use f o r l y t i c enzymes i s i n the study of c e l l w a l l s t r u c t u r e [39]. The enzyme used by G i l l e s p i e and Cook [26] i n t h e i r study was obtained from shake c u l t u r e s i n f l a s k s c o n t a i n i n g 50 - 500 ml. of medium, w h i l e that used by Whitaker and co-workers was produced i n a fermentor c o n t a i n i n g 100 - 130 l i t r e s of medium. The composition of the c u l t u r e medium ( i n gms/1) i n most cases was as f o l l o w s : Difco.Casamino A c i d s , 10; Glucose, 1; K 2HP0 4, 1; KN0 3, 0.5; MgS0 4 > 7H 20, 0.2; NaCl, 0.1; F e C l 3 . 6 H 2 0 , 0.01; and NaOH to adjust the pH to 7.0 - 7.1 i f necessary. The glucose was autoclaved s e p a r a t e l y , and the temperature of c u l t i v a t i o n was 25°C. No attempt was made by these authors to examine c o n d i t i o n s f o r maximal production of enzymes. The p r o t e i n source, Casamino A c i d s , was r a t h e r expensive. 1.6 N u t r i t i o n of Myxobacteria The myxobacteria are not very f a s t i d i o u s microorganisms [30, 40, 41]. They are ubiquitous i n h a b i t a n t s of normal s o i l , bark, and decaying p l a n t m a t e r i a l s . They are cosmopolitan, having been reported i n the s o i l s of Poland, Great B r i t a i n , A u s t r i a , Sweden, I n d i a , R u s s i a and Japan [42]. The myxobacteria are capable of h y d r o l y z i n g p a r t i c u l a t e i n s o l u b l e s u b s t r a t e s , such as s t a r c h , glycogen, p r o t e i n , c e l l u l o s e , and b a c t e r i a l c e l l w a l l s . Recently, M a r t i n and So [43] have shown that c e r t a i n s t r a i n s of myxobacter have the a b i l i t y to u t i l i z e autoclaved keratinaceous s u b s t r a t e s , i n the 21 form of feathers and wool , as so le sources of carbon and n i t r o g e n . I t has been suggested, i n the review by Dworkin [42], that the g l i d i n g m o t i l i t y and the n u t r i t i o n a l dependence of the organism on i t s a b i l i t y to s o l u b i l i z e i n s o l u b l e macromolecules are r e l a t e d to each o ther . He sa id : An organism whose e c o l o g i c a l niche i s an aquat ic one may depend on i t s f l a g e l l a m o t i l i t y and random c o l l i s i o n s to b r i n g i t i n t o contact wi th s o l u b l e substrate molecules . A t e r r e s t r i a l organism dependent on i n s o l u b l e macromolecules for i t s sustenance i s ob l iged to creep or crawl to i t s food. For b u i l d i n g p r o t e i n s , the f r u i t i n g myxobacteria r e q u i r e complex nitrogenous mater ia l s [41] or mixtures of amino acids [40] . Baur [44] reported that these organisms could be c u l t i v a t e d on media conta in ing peptone. A l so Loebeck [45], and Dworkin [40] have shown that although the e s s e n t i a l n u t r i t i o n a l requirements could be suppl i ed by free amino a c i d s , peptides and hydrolyzed prote ins were b e t t e r substrates for growth. According to Dworkin [40], a smal l number of sugars i n c l u d i n g f ruc tose , glucose and arabinose have n e g l i g i b l e e f f e c t on the growth ra te of some s t r a i n s of myxobacteria; however, McDonald and Peterson [41] reported that glucose was d i s t i n c t l y the carbon source of choice for the two species they examined, and that the d e l e t i o n of i t from the medium i n favor of any other carbon source r e s u l t e d i n marked i n h i b i t i o n of growth. Noren [46] has shown that h i s species could at tack a large number of carbohydrates and that growth was s t imulated by the presence of maltose and arabinose . Chase [47], on the other hand, was unable to demonstrate any e f f e c t of carbohydrates on the growth of h i s species i n a def ined 22 medium. I t appears therefore that there are s i g n i f i c a n t d i f f e r e n c e s i n responses to carbohydrate sources among s t r a i n s of myxobacteria. While such may be the case for carbohydrates , there seems to be unanimous agreement [40, 41, 47] that , i n genera l , the myxobacteria do not possess requirements for a p a r t i c u l a r v i t a m i n or amino a c i d , nor do they appear to have any p a r t i c u l a r l y exot ic requirements for growth. 1.7 Object ives of th i s work The objec t ives of the present work are : 1. To determine the s u i t a b i l i t y of condensed f i s h so lub les as the major component of a fermentation medium for the growth of a pure s t r a i n of Sorangium 495. 2. To determine the pre-treatment requ ired before the Sorangium specie w i l l grow w e l l on the f i s h waste medium. 3. To determine the amounts of supplementary n u t r i e n t s (g lucose , ca lc ium, phosphate) required for optimum growth •and protease product ion . 4. To determine which of the carbohydrate sources (hexoses, pentoses, and starch) can be u t i l i z e d by the micro-organism for growth and protease p r o d u c t i o n . 5. To determine the e f f ec t s of a g i t a t i o n and a e r a t i o n on the rates of growth and of product ion . 23 6. To determine from an ana lys i s of the batch k i n e t i c data the r e l a t i o n s h i p s between rates of growth, substrate u t i l i z a t i o n and protease product ion of the Sorangium spec i e . 24 Chapter 2 EXPERIMENTAL TECHNIQUES 2.1 General B i o l o g i c a l experiments are known for t h e i r large number of v a r i a b l e s . Due to the tedious and time consuming nature of these e x p e r i -ments, i t i s i m p r a c t i c a l , although p o s s i b l e , to examine the e f f ec t s of a l l the v a r i a b l e s one can conceive , even wi th the help of f a c t o r i a l experimen-t a t i o n . The procedure adopted here was as fol lows : a f t e r us ing f a c t o r i a l design [48, 49, 50] to determine the most i n f l u e n t i a l supplementary n u t r i e n t among those known to a f f ec t protease produc t ion , th i s important n u t r i e n t was then f u r t h e r s tudied at a d d i t i o n a l l e v e l s . In subsequent experiments, other process v a r i a b l e s were s tudied one at a t ime. Such experiments assume that there i s no i n t e r a c t i o n between the v a r i a b l e s [49]. 2.2 P r e p a r a t i o n of C u l t u r e Medium A l l work has been c a r r i e d out wi th a s i n g l e batch of condensed f i s h s o l u b l e s . This m a t e r i a l was supp l i ed by B . C . Packers , Richmond, B . C . When not i n use , the so lubles were r e f r i g e r a t e d at 6 ° C . Under these condi t ions spo i lage was l a r g e l y prevented. Table 2.10 shows the dry weight and pH of the so lub les measured over a per iod of seven months. The n e g l i g i b l e change i n pH value supported the view that there had been n e g l i g i b l e decomposition of the s o l u b l e s . 25 Table 2.10 TIME VARIATION IN pH AND DRY WEIGHT OF REFRIGERATED CFS DATE pH wt/vol ,% 23/9/73 3.65 52.76 12/10/73 3.66 52.21 28/10/73 3.78 52.63 28/10/73 3.78 51.61 8/11/73 3.80 52.47 21/11/73 3.75 52.10 4/12/73 3.92 52.45 7/1/74 3.75 51.74 31/1/74 3.65 51.80 12/2/74 3.75 51.87 26/2/74 3.80 52.81 Average 3.75 52.20 a ± 0.0836 ± 0.43 26 The condensed f i s h so lubles used i n the fermentations were prepared by d i l u t i o n wi th tap water. Almost a l l the runs were made wi th d i l u t e d f i s h so lubles that had been f i l t e r e d through Reeve-Angel glass f i b r e f i l t e r papers to give a c l ear media conta in ing l i t t l e or no coagulable proteinaceous m a t e r i a l s . The average so lub le p r o t e i n content (measured as Bovine Serum Albumin equivalent) at var ious d i l u t i o n s i s shown i n Table 2.11. The table a l so shows the average glucose content at these d i l u t i o n s . I t was determined e a r l y i n th i s study that the d i l u t e d so lub les ( f i l t e r e d or u n f i l t e r e d ) could be used as a complete medium without any other a d d i t i o n s . However, the a d d i t i o n of glucose as a carbon source increases the protease y i e l d (Sect ion 3 . 2 ) . The pH of the medium was adjusted to 7.0 wi th 2.5N or 6N Sodium Hydroxide before s t e r i l i z a t i o n . I n i t i a l l y , s t e r i l i z a t i o n was accomplished by autoc lav ing the fermentat ion mixture i n d i r e c t contact wi th steam at 15psig . f or 15 minutes when smal l volumes (lOOmls or l e s s ) were used, and for 30 minutes when large volumes (4 l i t r e s ) were used. Later on, when the "Energy Conservat ion Act" came i n t o e f f e c t (around December 1973), the maximum steam pressure a t t a i n a b l e i n the s t e r i l i z e r was 10 p s i g ; under such a c r i s i s the s t e r i l i z a t i o n time was increased by 15 minutes i n both cases. There was a s l i g h t drop i n the pH of the medium during s t e r i l i z a t i o n . This i s probably due to the loss of v o l a t i l e bases . No attempt was made to re -adjus t the pH before i n o c u l a t i o n . T a b l e 2.11 A N A L Y S I S OF F I L T E R E D C F S A T V A R I O U S D I L U T I O N S D I L U T I O N ( v / v ) P R O T E I N G L U C O S E DRY WEIGHT mg B S A / m l m g / m l m g / m l 10/1000 1.87 20/1000 3.85 0.062 7.9 30/1000 5.22 - 10.8 40/1000 6.75 0.087 14.0 50/1000 8.66 - 18.06 60/1000 10.20 0.105 21.44 28 2.3 Inoculum Preparat ion Cul tures of the Sorangium species were maintained on a B r a i n Heart Infus ion agar s l a n t prepared by d i s s o l v i n g 37 gm of B r a i n Heart In fus ion agar (Difco) and 15 gm Agar i n 1,000 ml d i s t i l l e d water. A f t e r 2 - 3 days growth at 3 0 ° C , the cu l tures were s tored at r e f r i g e r a t i o n temperature ( 6 ° C ) . The organism was grown at 30°C i n two stages i n 250 ml Erlenmeyer f l a s k s , conta in ing 100 ml of a Nutr i en t Broth (Peptone, 0.5%; Beef E x t r a c t , 0.3%) medium, to provide the inoculum for p r e l i m i n a r y s tudies i n shake f lasks and for k i n e t i c s tudies i n 7 - l i t r e fermentors. The inoculum for the f i r s t stage was prepared by a t r a n s f e r from the r e f r i g e r a t e d c u l t u r e . In th i s s tage , the organism was induced to grow i n the Nutr i en t Broth medium. When heavy growth was obtained (a f ter about 1 - 2 days) , 1 ml o f t h i s a c t i v e l y growing c u l t u r e was used as the inoculum for the second stage which employed the same medium as the f i r s t s tage . The inoculum for the f i s h medium was taken from the c u l t u r e r e s u l t i n g from the second t r a n s f e r . The t r a n s f e r was made a f t e r 15 - 24 hrs growth, when the organism was i n the exponent ia l growth phase. The amount of inoculum used was, i n r a t i o of inoculum to fresh medium, 1 : 100 for Erlenmeyer f l a sks and 1 : 40 for 7 - l i t r e fermentors. Inoculum prepared i n th i s way reduced the chance of contaminat ion. This i s e s p e c i a l l y important for myxobacteria because, even though s e v e r a l of the myxobacteria ( i n c l u d i n g the specie used i n th i s work) produce 29 a n t i b a c t e r i a l and ant i funga l substances [42], they are poor competitors and can therefore be overgrown by the more r a p i d l y growing eubacter ia when both are p laced on a r i c h medium [30]. 2.4 Apparatus Most of the p r e l i m i n a r y experiments to tes t the in f luence of medium composition on y i e l d s of c e l l s and enzymes were c a r r i e d out i n standard 250 ml Erlenmeyer f l a sks stoppered wi th non-absorbent co t ton . The micro-organisms were c u l t i v a t e d on a New Brunswick S c i e n t i f i c (NBS) incubator-shaker working at 140 rpm wi th a gyrotory rad ius of 1/2 i n c h , at a constant temperature of 3 0 ° C . For the study of a g i t a t i o n and aera t ion e f f e c t s , a NBS model MF-207 bench top fermentor was used. This model cons i s t s of two 7 - l i t r e , c y l i n d r i c a l glass growth v e s s e l s , each 15 cm i n diameter and 45 cm h i g h . Four s t a i n l e s s - s t e e l b a f f l e - p l a t e s , each 42 x 2 cm, were f ixed to the head p l a t e . They were jo ined together by s t a i n l e s s - s t e e l tubing at the bottom. The b a f f l e p la tes were p laced at r i g h t angles with t h e i r surfaces perpendicu lar to the fermentor wal l s and t h e i r lower ends 3 cm above the bottom of the v e s s e l . The b a f f l e assembly was u t i l i z e d for the i n t r o d u c t i o n of a i r . An a g i t a t o r shaft with two adjustable impe l l er s was c e n t r a l l y mounted through the head p l a t e . The impe l l ers were made of s i x 3/4 x 3/4 cm s t a i n l e s s - s t e e l b lades , set 6 0 ° apart wi th t h e i r f l a t surfaces perpendicu lar to the bottom of the v e s s e l . F igure 2.2, adapted from Ferguson [51], i s a schematic drawing of the fermentor 30 F i g u r e 2 . 2 S C H E M A T I C DRAWING OF 7 - L I T R E FERMENTOR 15 cm. I D fermentor jar pH electrodes 31 descr ibed above. With th i s u n i t , i t was p o s s i b l e to ferment up to 4 l i t r e s of b r o t h ; c o n t r o l the temperature to w i t h i n 1°C of a set po in t w i t h i n a range of 10°C to 5 0 ° C ; c o n t r o l the a g i t a t i o n rate from 10 to 900 rpm; and to c o n t r o l the a i r flow rate from 1 to 16 l i t r e / m i n . [51, 52] . In a d d i t i o n , the fermentor could be s t e r i l i z e d wi th the medium i n i t ; samples coulb be removed and addi t ions could be made, under asept i c c o n d i t i o n s , at any time during the fermentation [51]. 2.5 Sampling Technique 1. Shake F l a s k s Each treatment i n an experiment was c a r r i e d out i n d u p l i c a t e , samples of the two r e p l i c a t e s were mixed at harvest t ime, the mixture was centr i fuged to remove the organisms. The f i l t r a t e was saved for l a t e r assays. 2. 7-1 Fermentor A NBS model S20 sampl ing - inocu la tor was used to withdraw samples at various time i n t e r v a l s . This device cons i s t s of a removable glass c o l l e c t i n g tube and a b u i l t - i n a i r f i l t e r chamber. I t can be s t e r i -l i z e d with the Fermentor or s epara te ly . 32 2.6 Measurement of B a c t e r i a l Growth Severa l methods for the q u a n t i t a t i v e es t imat ion of b a c t e r i a l growth are a v a i l a b l e . Among them are : t u r b i d i t y [53], dry c e l l weight [54], d i l u t i o n or p l a t e count [30], and ATP content . V i a b l e counts are measures of c e l l concentrat ions (that i s , number of i n d i v i d u a l c e l l s per u n i t v o l . of c u l t u r e ) , and are e s s e n t i a l only i n problems where c e l l d i v i s i o n i s of i n t e r e s t . T u r b i d i t y and dry c e l l weight are measures of b a c t e r i a l dens i ty ( that i s , dry weight of c e l l s per u n i t volume of c u l t u r e ) , and, according to Monod [55] are not equiva lent to the c e l l concentrat ion; t h i s i s because the average s i zes of the c e l l s vary cons iderably from one phase to another of a growth c y c l e . Since the f i s h medium was c l a r i f i e d p r i o r to c u l t u r e , b a c t e r i a l dens i ty i s s u f f i c i e n t as a measurement of growth for the purposes of th i s s tudy. Due to the formation of s l imy matter, and a l so s ince there was cons iderable c o l o r v a r i a t i o n i n the medium during growth, d i r e c t t u r b i d i m e t r i c methods could not be used to fo l low growth. Although a l i n e a r r e l a t i o n s h i p was shown to e x i s t between the t u r b i d i t y of a washed c e l l suspension and the dry c e l l weight at var ious wavelengths (Figure 2 .3 ) , a l l the measurements reported here were made by d i r e c t weighing of the dry c e l l s . The dry b a c t e r i a c e l l s were obtained as fo l lows . A sample of fermentation bro th was centr i fuged i n an IEC i n t e r -n a t i o n a l Centr i fuge - U n i v e r s a l Model UV, f i t t e d with an I n t e r n a t i o n a l Mult i speed Attachment, for 10 minutes at 22,000 x G . The supernatant from th i s c e n t r i f u g a t i o n was decanted and saved for l a t e r sugar ana lys i s 33 Figure 2.3 TURBIDITY VS DRY CELL WEIGHT AT VARIOUS WAVELENGTHS — — — : v Note :- No attempt was made to t e s t the r e l i a b i l i t y of t h i s r e l a t i o n s h i p . The samples used f o r these measurements were taken at one p a r t i c u l a r stage of growth, and the experiment was not repeated. 34 and p r o t e o l y t i c a c t i v i t y assay. The c e l l s were re-suspended i n d i s t i l l e d water us ing a vortex mixer and recentr i fuged for 10 minutes at 22,000 x G to remove the wash water . This washing and c e n t r i f u g i n g procedure was done twice. A f t e r the f i n a l washing, the c e l l s were d i spersed i n d i s t i l l e d water and were then t rans ferred to pre-weighted alumimum d i s h e s . These were then d r i e d at 9 5 ° C i n a F i s h e r Isotemp oven for at l e a s t 12 hours . 2.7 Measurement of Sugar Concentrat ion Many c o l o r i m e t r i c tests for reducing sugars and po lysacchar ides are a v a i l a b l e [56]. The two methods tested and used i n th i s work were : I . Phenol - S u l f u r i c A c i d Reagent Method This method, developed by Dubois et a l [56], i s based on the fac t that simple sugars and t h e i r d e r i v a t i v e s give an orange-yel low c o l o r when treated with phenol and concentrated s u l f u r i c a c i d . The authors claimed that : The method i s s imple , r a p i d , and s e n s i t i v e , and gives reproduc ib le r e s u l t s . The reagent i s inexpensive and s t a b l e , and a given s o l u t i o n requires only one standard curve for each sugar. The c o l o r produced i s permanent and i t i s unnecessary to pay s p e c i a l a t t e n t i o n to the c o n t r o l of the c o n d i t i o n s . The above claims have been confirmed dur ing the course of th i s work. However, i n order to get reporduc ib le r e s u l t s , i t was necessary to 35 make the fo l lowing caut ionary notes : (1) Owing to the h igh v i s c o s i t y of the concentrated s u l f u r i c a c i d , fas t d e l i v e r y of the ac id r e s u l t e d i n a s i g n i f i c a n t amount of the a c i d being l e f t behind on the wa l l s of the p ipe t (or b u r e t t e ) . Care must be taken to ensure that the exact amount of a c i d i s in t roduced . (2) In as much as the r e a c t i o n i s brought about by the heat developed when the concentrated a c i d and water so lu t ions are mixed, the shape and s i z e of the r e a c t i o n tube are important . Tubes wi th an i n t e r n a l diameter of 16 mm. were used. This diameter w i l l al low good mixing without d i s s i p a t i n g the heat too r a p i d l y [55]. Good mixing i s obtained by d i r e c t i n g the stream of a c i d against the l i q u i d surface ra ther than against the s ide of the tes t tube. (3) The s o l u t i o n must be poured s lowly i n t o the Spectronic 20 tubes j u s t before the readings are made. Rapid t r a n s f e r r e s u l t e d i n entrained gas bubbles wi th low r i s e v e l o c i t i e s , these bubbles , i f not removed, w i l l i n t e r f e r e with the spectrophotometer readings . I I . D i n i t r o s a l i c y l i c (DNS) Method This method was developed by Sumner [57] i n 1925 to detect reducing sugars i n d i a b e t i c u r i n e . In th i s method, the sugar s o l u t i o n was allowed to react with d i n i t r o s a l i c y l i c ac id reagent (Appendix I I . 1 ) . This reagent contains sodium hydroxide , d i n i t r o s a l i c y l i c a c i d , Rochel le s a l t , phenol and sodium b i s u l f i t e . According to Sumner [57], the Rochel le 36 s a l t prevented the d i s s o l u t i o n of oxygen; t h i s was d e s i r a b l e because the d i s s o l v e d can destroy p a r t of the sugar. Excess phenol was added so as to enhance the c o l o r produced by the sugar. A d d i t i o n of sodium b i s u l f i t e helped to s t a b i l i z e the c o l o r produced when the DNS - phenol s o l u t i o n was heated w i t h glucose. F i g u r e 2.4 i s a comparison of the phenol - s u l f u r i c a c i d and the DNS t e s t s f o r sugars. Although the agreement between the two methods was q u i t e good, most of the sugar t e s t r e s u l t s reported i n t h i s work were done by the DNS method f o r the f o l l o w i n g reasons : (1) The phenol - s u l f u r i c a c i d method r e q u i r e s the h a n d l i n g of a h i g h l y c o r r o s i v e reagent. This n e c e s s i t a t e s the o b s e r v a t i o n of c e r t a i n obvious precautions which tends to slow down the process. (2) The phenol - s u l f u r i c a c i d method i s four times more s e n s i -t i v e than the DNS method. Since the fermentation c o n c e n t r a t i o n s of sugar were u s u a l l y i n the m i l l i g r a m / m l range, more d i l u t i o n s were r e q u i r e d w i t h the phenol - s u l f u r i c a c i d method (range i n ug/ml), r e s u l t i n g i n a greater r i s k of e r r o r . (3) When many samples were simultaneously measured, i t was found that the DNS method was f a s t e r and e a s i e r to handle. The d e t a i l s of the two t e s t methods and the standard curves are given i n Appendix I I . 1 . 37 F i g u r e 2.4 COMPARISON OF DNS AND PHENOL-H-SO. ASSAYS 2 4 2 4 6 8 10 S U G A R by PHENOL-H 2 S0 4 (mg/ml ) 38 2.8 Measurement of P r o t e o l y t i c A c t i v i t y An absolute q u a n t i t a t i v e determinat ion of crude enzyme preparat ions by chemical or p h y s i c a l a n s l y s i s i s not f e a s i b l e because : (1) the s t r u c t u r e of enzymes i s genera l ly not w e l l known, (2) most enzymes occur only i n trace amounts, (3) most enzymes simply give tests for p r o t e i n s . Such tests do not d i s t i n g u i s h them from other p r o t e i n s . As a r e s u l t enzyme quant i ty cannot be expressed i n mi l l i grams or moles of the substance as i s v a l i d for other d i f f e r e n t p r o t e i n s . The amount of enzyme produced i s i n v a r i a b l y measured by the r e s u l t s i t produces, that i s , by i t s a c t i v i t y . Enzyme a c t i v i t y i s expressed i n a r b i t r a r y un i t s where the u n i t has been determined under s p e c i f i e d condi t ions of substrate concentra t ion , temperature, and pH, that i s , under condi t ions where a l l f a c t o r s which are known to in f luence enzyme a c t i v i t y are maintained constant i f p o s s i b l e [58]. There often e x i s t s e v e r a l kinds of un i t s for one and the same enzyme depending upon whose method i s employed; consequently, i n many cases , i t i s not pos s ib l e to compare measurements of one and the same enzyme because the d i f f e r e n t condi t ions of assay do not al low the use of equivalence c a l c u l a t i o n f a c t o r s . With a view to s t a n d a r d i z i n g the enzyme u n i t , the Enzyme Commission of the I n t e r n a t i o n a l Union of Biochemistry [59] proposed the fo l lowing d e f i n i t i o n of an enzyme u n i t : 39 One u n i t of any enzyme i s def ined as that amount which cata lyzes the transformation of one micromole of substrate per minute, or where more than one bond of a more complex substrate (eg. p r o t e i n , p o l y - s a c c h a r i d e , e t c . ) i s attacked one micro-equiva lent of the group concerned per minute under w e l l def ined c o n d i t i o n s . The temperature should be s ta t ed , and i t i s suggested that where p r a c t i c a b l e i t should be 2 5 ° C . The other c o n d i t i o n s , i n c l u d i n g pH and substrate concentra t ion , shou ld , where p r a c t i c a b l e , be o p t i m a l . I t was a lso recommended that enzyme assays be based, where p r a c t i c a b l e , upon measurements of i n i t i a l ra te of r e a c t i o n , i n order to avoid compl i ca -t ions due, for i n s t a n c e , to r e v e r s i b i l i t y of reac t ions or to formation of i n h i b i t o r y products . The substrate concentrat ion should be, whenever p o s s i b l e , s u f f i c i e n t for s a t u r a t i o n of the enzyme, so that the k i n e t i c s i n the assay approach zero order . Concentrat ion of an enzyme i n a s o l u t i o n should be expressed as uni ts per m i l l i l i t r e . Most of the methods which are used to determine the a c t i v i t y of p r o t e o l y t i c enzymes, and are s u i t a b l e for rout ine ana lys i s are based on a q u a n t i t a t i v e determinat ion of the low - molecular weight products (amino acids and peptides) formed by the enzymatic h y d r o l y s i s of the substrate p r o t e i n . Various modi f i ca t ions of Anson's c o l o r i m e t r i c method [60] are c u r r e n t l y enjoying wide a p p l i c a t i o n i n research . In th i s method, the enzyme a c t i v i t y i s assessed from the amount of tyros ine contained i n the p r o t e o l y s i s products which are not p r e c i p i t a t e d by t r i c h l o r o a c e t i c a c i d . Tysosine i s determined from the depth of c o l o r which i t produces with AO F o l i n ' s phenol reagent [61]. In view of the large number of samples to be analyzed, a simple and r a p i d method i s d e s i r a b l e . A f t e r cons ider ing var ious a l t e r n a t i v e s , the m o d i f i c a t i o n of the Anson method used by Petrova and V i n t s y u n a i t e [62] was decided upon. The d e t a i l s of th i s method are given i n Appendix I I . 2 . Condit ions i n the r e a c t i n g mixture were maintained as constant as p o s s i b l e during the assays. The r e a c t i o n was c a r r i e d out i n a constant temperature water bath capable of automat ica l ly c o n t r o l l i n g temperatures w i t h i n very c lose to lerances ; a l l reagents were brought to the des ired temperature before the r e a c t i o n was s t a r t e d by mixing them. Adequate b u f f e r i n g was provided by a T r i s - HC1 b u f f e r prepared as o u t l i n e d i n Appendix I I . 2 . The r e a c t i o n temperature was 3 0 ° C . The pH was buf fered at 8.5 - the optimum found by G i l l e s p i e and Cook [26] for the p r o t e o l y t i c enzymes produced by the Sorangium specie used i n th i s work. For a f ixed i n i t i a l substrate concentrat ion and vary ing i n i t i a l enzyme concentrat ions , the amount of substrate transformed i n a given per iod of time may not be p r o p o r t i o n a l to the amount of enzyme. This i s a consequence of the elementary Michael i s -Menten scheme which, i n genera l , may be expressed as [8] : k k E + S —> ES —> E + P The rate of product formation, v , i n the enzyme r e a c t i o n depicted above i s given by : 41 k^es k^e v = k + s = 1 + k /s ' m m When condi t ions are such that s » k , then v = k „ e and the r e a c t i o n m 3 rate i s d i r e c t l y p r o p o r t i o n a l to the enzyme concentra t ion . In such cases the r e a c t i o n ra te i s of the f i r s t order as r e l a t e d to the enzyme concentra-t i o n and of zero order as to the substrate concentrat ion . I t i s therefore e s s e n t i a l to e s t a b l i s h the l i m i t of enzyme concentrat ion beyond which, maximum rate of breakdown of the enzyme-substrate complex i s u n a t t a i n a b l e . F igure 2.5 i s a p l o t of amount of tyros ine formed i n 10 minutes versus crude enzyme concentra t ion . I t i s apparent from th i s f igure that under the condi t ions of th i s assay, beyond an enzyme concentrat ion equiva lent to 120 yg t y r o s i n e , the substrate concentrat ion used was not s u f f i c i e n t to maintain a constant maximum r e a c t i o n rate during the whole incubat ion time. For tunate ly , an absorbance reading of 0 .9 , the upper l i m i t on the r e l i a b l e p o r t i o n of the spectrophotometer, corresponds to a tyros ine value of 70 yg; thus by d i l u t i n g h igher enzyme concentrat ions to give c o l o r values w i t h i n the r e l i a b l e p o r t i o n of the Spectronic 20 w i l l a lso ensure s a t u r a t i o n of the enzyme. 2.9 Measurement of P r o t e i n Content Many methods for the q u a n t i t a t i v e es t imat ion of p r o t e i n i n s o l u -t ion are a v a i l a b l e . Among them are : 42 F igure 2.5 PROTEOLYTIC ACTIVITY VS ENZYME CONCENTRATION Enzyme s o l u t i o n : 10 days growth i n shake f l a s k - O - 2% glucose - A - 1% glucose E N Z Y M E CONC.Cml of sample) 43 1. UV Absorpt ion Most prote ins e n h i b i t a d i s t i n c t u l t r a v i o l e t l i g h t absorpt ion maximum at 280 mu, due p r i m a r i l y to the presence of tyros ine and tryptophan res idues [16, 63 ] . S ince the abundance of t y r o s i n e and tryptophan v a r i e s from p r o t e i n to p r o t e i n , th i s method i s i n general not r e l i a b l e . However reasonably r e l i a b l e r e s u l t s can be obtained wi th very heterogeneous mixtures of p r o t e i n s , because, i n t h i s case, i t i s u n l i k e l y that the mixture would be s t r i k i n g l y r i c h or poor i n any p a r t i c u l a r amino a c i d s . Nevertheless , d i r e c t photometry i s not as s p e c i f i c as c o l o r i m e t r i c methods, and i s more l i a b l e to inaccuracy due to t u r b i d i t y . 2. C o l o r i m e t r i c Methods There are b a s i c a l l y two c o l o r i m e t r i c methods which are widely used for p r o t e i n e s t imat ion . The chemical bases of these assays are : A . F o l i n Phenols and phenol ic amino acids such as tyros ine react with F o l i n ' s phenol reagent [61] i n a l k a l i n e s o l u t i o n to form a reduced phosphomolybdic - phosphotungstic a c i d complex which i s blue i n c o l o r . In the presence of copper i o n s , a d d i t i o n a l co lor i s produced through a b i u r e t complex formation and through other mechanisms not p r e s e n t l y understood [34]. This method s u f f e r s form the same disadvantages as the UV absorpt ion method i n that any v a r i a t i o n i n tyros ine content of the 44 samples w i l l i n v a l i d a t e the method. B . B i u r e t When b i u r e t , whose s t r u c t u r a l formula i s ^NCONHCONH^, i s t r e a -ted wi th an a l k a l i n e potassium copper t a r t r a t e s o l u t i o n , two b i u r e t molecules are jo ined to form the fo l lowing complex v i o l e t co lored compound [63] : OH OH I I CONH. Cu NH„CO I 2 ' I NH NH I I C0NH o -K K-NH„CO I 2 I 2OH OH Any compound that has i n i t s molecular s t r u c t u r e p a i r s of carbamyl groups (-CONH^) l i n k e d through n i t rogen or carbon (or pept ide l inkages) w i l l show the above r e a c t i o n [65]. Since p r o t e i n conta in these l i n k a g e s , they react to give the c h a r a c t e r i s t i c b i u r e t c o l o r . I t i s b e l i e v e d [16] that the chromophore, or l i g h t absorbing centre , i s a complex between the pept ide backbone and c u p r i c ions : 45 0=C NH HN NH HN I I I I Since peptide bonds occur with approximately the same frequency per gram of m a t e r i a l for most p r o t e i n s , dev ia t ions are encountered l ess frequent ly than with the F o l i n - phenol reagent. However, s ince the F o l i n reagent i s about 100 times more s e n s i t i v e than the b i u r e t r e a c t i o n [66], more m a t e r i a l i s r equ ired for assay by the l a t t e r method. C. Chemical Method One of the most widely used methods of p r o t e i n determinat ion i s the K j e l d a h l method, which i s based on the q u a n t i t a t i v e e s t imat ion of t o t a l n i t r o g e n . The method, as descr ibed i n the O f f i c i a l Methods of A n a l y s i s [67], involves d i g e s t i o n of the p r o t e i n with concentrated s u l f u r i c ac id i n the presence of copper s u l f a t e , mercuric s u l f a t e , or some other s u i t a b l e c a t a l y s t . The ammonia formed may be q u a n t i t a t i v e l y determined 46 by removal from the d i g e s t i o n mixture through steam d i s t i l l a t i o n fol lowed by t i t r a t i o n or a d d i t i o n of N e s s l e r ' s reagent (mercuric potassium i o d i d e i n aqueous sodium hydrox ide ) . The l a t t e r treatment permits ammonia to be determined c o l o r i m e t r i c a l l y . For the photometric and c o l o r i m e t r i c methods, the p r o t e i n content i s obtained by comparison against a standard s o l u t i o n of serum p r o t e i n , u s u a l l y c r y s t a l l i n e Bovine Serum Albumin; i n the K j e l d a h l method, the average p r o t e i n i s c a l c u l a t e d by assuming a n i t rogen content of 16%. The d e t a i l s of the B i u r e t method, which was used throughout t h i s work, are given i n Appendix II .3 . 47 Chapter 3 RESULTS AND DISCUSSION 3.1 E f f e c t of Medium Const i tuents P r e l i m i n a r y s tudies revealed that a medium c o n s i s t i n g of d i l u t e d , condensed f i s h so lubles alone could s u f f i c e as a substrate for good growth and protease p r o d u c t i o n . However, i t i s a w e l l known fact that the i n c l u s i o n of c e r t a i n ingred ient s i n a fermentation medium may have an enhancing e f f ec t on p r o t e o l y t i c enzyme p r o d u c t i o n . M e r r i l l and Clark [68] and Haines [69] found that ca lc ium was necessary for good protease y i e l d s , whi le Fukumoto and Negoro [70] showed that phosphate(s) could s t imulate protease product ion a l s o . Fukumoto et a l [71] have shown that the e f f e c t of phosphates on enzyme product ion was very pronounced when carbohydrates were used as carbon sources . In the present i n v e s t i g a t i o n , the f a c t o r i a l approach to e x p e r i -mentation was used to determine which of the above three ingred ien t s were s i g n i f i c a n t for fur ther t e s t i n g . Such an approach enables (1) v a r i a t i o n of severa l fac tors at once i n accordance wi th a pre-arranged p a t t e r n , thereby obta in ing more informat ion with p o s s i b l y fewer runs , and (2) eva luat ion of i n t e r a c t i o n s between v a r i a b l e s , i f any e x i s t . In the present i n v e s t i g a t i o n , the fo l lowing l e v e l s of the three factors were used : 48 Factor L e v e l 1 L e v e l 2 L e v e l 3 A. Glucose concentrat ion 0 5 10 (gm/lOOml) B . Phosphate concentrat ion 0 0.3 0.7 (ml 1M s o l n . K^HPC^) C. Calcium concentrat ion 0 0.5 1.0 (ml 1M s o l n . C a C l 2 added) One run was made at each combination of f a c t o r s , thus g i v i n g r i s e to 27 experimental c o n d i t i o n s , each of which cons is ted of 100 mis of a f i l t e r e d , 1 : 50 v / v d i l u t e d , condensed f i s h so lubles medium made up according to s p e c i f i c a t i o n s . A white p r e c i p i t a t e was formed when calc ium c h l o r i d e and potassium hydrogen phospate at the h igher l e v e l s were added to the medium. No experiments were designed to examine what e f f e c t t h i s might have on enzyme y i e l d ; i n other words, the d i f f e r e n c e i n enzyme product ion were a t t r i b u t e d s o l e l y to the a d d i t i o n or d e l e t i o n of the p a r t i c u l a r i n g r e d i e n t . The order for running the 27 experiments was completely rando-mized by withdrawing numbers from a beaker. A f t e r 3 days growth at 3 0 ° C and 140 rpm i n the incubator - shaker , the p r o t e o l y t i c a c t i v i t y of the c u l t u r e f i l t r a t e s was measured. The r e s u l t s are given i n Table 3.12. The model assumed for the ana lys i s of th i s experiment i s given by [72, 73] : ^IjlcA = y + ° i + B j + Y k + ^hj + < « Y ) l k + <PY) j k + ( a B Y > i j k Table 3.12 RESULTS OF FACTORIAL EXPERIMENTS SUGAR LEVEL (A) 1 2 3 PHOSPHATE CALCIUM LEVEL (C) LEVEL (B) 1 2 3 1 2 3 1 2 3 1 3.66* 1.39 1.41 2.04 0.95 0.57 0.0 0.13 0.0 2 2.92 1.47 1.79 1.64 1.06 0.73 0.0 0.0 0.0 3 2.19 1.06 0.89 2.38 0.98 0.63 0.0 0.08 0.0 Values are i n enzyme un i t s / 10 cc . 50 where y . ., represents the observat ion taken at the i ^ l e v e l of fac tor A , j 1 " l e v e l of f a c t o r B, and the k**1 l e v e l of f a c t o r C; u t i l i s the o v e r a l l mean, a. the e f f e c t of the i l e v e l of f a c t o r A, g . i 3 th. the e f f e c t of the j l e v e l of fac tor B, and the e f f e c t of the ttl k l e v e l of f a c t o r C . e . . . measures the d e r i v a t i o n s of the observed y. values i n the ( i j k ) c n c e l l from the popula t ion mean u . The other terms stand for i n t e r a c t i o n s between the main fac tors a , (3, and y> I t i s u s u a l l y assumed that the sums of the main e f f ec t s as w e l l as the sums of the two - way i n t e r a c t i o n e f fec t s summed on e i t h e r s u b s c r i p t 3 equal zero for any value of the other s u b s c r i p t ( i . e . £ (cB) . . = 0, e t c . ) i = l 1 J and that the sum of the three - way i n t e r a c t i o n e f f ec t s summed on any one of the subscr ip t s i s zero for any values of the other two subscr ip t s ( i . e . 3 £ (aBy) . = 0, e t c . ) . These r e s t r i c t i o n s w i l l assure unique estimates i = l 1 J R for the parameters of the model. In order that v a l i d s i g n i f i c a n c e tests can be made, i t i s a l so assumed that the errors are values of independent and normally d i s t r i b u t e d 2 random v a r i a b l e s , each wi th zero mean and common var iance a . The complete ana lys i s of var iance for th i s t h r e e - f a c t o r experiment i s given i n Table 3.13. The computational procedures for obta in ing the sums of squares are s p e l l e d out i n Appendix I I I . Each treatment combination defines a c e l l . I f there are n r e p l i c a t i o n s , each c e l l contains n observat ions . Table 3.13 ANALYSIS OF VARIANCE SOURCES OF VARIATION SS df MS=SS/df COMPUTED F Main E f f e c t s A 15.72 2 7.86 131.0* B 0.23 2 0.12 2.0 C 5.13 2 2.57 42.83* Two Factor I n t e r a c t i o n s AB 0.90 4 0.23 3.83 AC 2.99 4 0.75 12.50* BC 0.25 4 0.06 1.00 Three Factor I n t e r a c t i o n s ABC 0.51 8 0.06 Note SS(ABC) was taken as SSE F2,8,0.05 = 4 ' 4 6 F2,8,0.01 = 8 ' 6 5 F4,8,0.05 " 3 , 8 4 F4,8,0.01 " 7 ' 0 1 * These factors are s i g n i f i c a n t at the 0.01 l e v e l . 52 Note that s ince only one observat ion was made at each set of cond i t ions , a w i t h i n - c e l l mean square cannot be used as an estimate of the true var iance of the system. Instead, the ABC i n t e r a c t i o n was assumed to be n e g l i g i b l e and that SS(ABC) represents v a r i a t i o n due s o l e l y to experimental e r r o r and thereby provides an est imate of the e r r o r v a r i a n c e . The F s t a t i s t i c i n the A n a l y s i s of Variance tab le was c a l c u l a t e d us ing M S ^ . The fo l lowing conclusions can be made from the F r a t i o i n the A n a l y s i s of Variance tab le : (1) Sugar and calc ium e f fec t s are s i g n i f i c a n t at the 0.01 l e v e l . (2) I n t e r a c t i o n ex i s t s between the d i f f e r e n t sugar concentrat ions and d i f f e r e n t calc ium concentrat ions . (3) The F - r a t i o for glucose - phosphate i n t e r a c t i o n i s b a r e l y below the 0.05 c r i t i c a l va lue; there i s not enough evidence to conclude that i n t e r a c t i o n between these two fac tors does not e x i s t . (4) None of the other F - r a t i o s are s i g n i f i c a n t at e i t h e r the 0.05 or 0.01 l e v e l . 3 .2 . E f f e c t of Glucose Concentrat ion The ana lys i s of var iance table i n d i c a t e d that the amount of glucose present i n the medium i s the most important fac tor a f f e c t i n g enzyme 53 y i e l d . The e f f ec t of th i s ingred ient was fur ther studied i n an attempt to develop a medium y i e l d i n g maximal prote inase p r o d u c t i o n . The media used for th is study were both f i l t e r e d and u n f i l t e r e d , 1 : 50 v / v d i l u t e d , condensed f i s h so lubles without added calc ium s a l t s or phosphates. Figures 3 .6 , 3.7 and 3.8 show the changes occur ing dur ing the course of fermentation when the glucose concentrat ion was v a r i e d between 0 and 40 gm/1 i n both f i l t e r e d and u n f i l t e r e d media. Since there was no easy method to separate the i n s o l u b l e prote ins from the bugs, the growth i n the u n f i l t e r e d media was not measured. The data for these f igures are tabulated i n Tables A I . l , AI .2 of Appendix I . Each reported value i s the average of d u p l i c a t e runs (Sect ion 2-5) . The r e s u l t s i n d i c a t e tha t , f o r both f i l t e r e d and u n f i l t e r e d media, the y i e l d of protease was maximum i n medium conta in ing 1% glucose , whi le bes t growth was obtained with medium conta in ing 2% glucose . I t i s c l e a r l y demonstrated that the e f f e c t of the added carbohydrate was one of increased growth. I t i s a l so apparent that protease product ion lags growth. The changes i n the pH of the medium during incubat ion suggest that protease product ion i s h igher i n media developing an a l k a l i n e r e a c t i o n , whi le growth i s genera l ly h igher i n media developing an ac id r e a c t i o n . The d r a s t i c drop i n enzyme y i e l d on the e ight day with media conta in ing 3% or 4% glucose may be due to d e s t r u c t i o n of the enxyme when exposed to such low pH values (3.4 to 4 .1 ) . Comparison of F igure 3.6 and 3.8 i n d i c a t e s that the f i l t e r e d f i s h so lubles i s a b e t t e r medium for protease product ion . 54 F i g u r e 3 .6 E F F E C T OF G L U C O S E C O N C E N T R A T I O N ON A C T I V I T Y - U N F I L T E R E D CFS TIME ( d a y s ) Figure 3.7 EFFECT OF GLUCOSE CONCENTRATION ON GROWTH - FILTERED CFS Figure 3.8 EFFECT OF GLUCOSE CONCENTRATION ON ACTIVITY - FILTERED CFS 57 I t should be pointed out here that autoclaved glucose can i n h i b i t the growth of c e r t a i n species of b a c t e r i a [74]. A l l the experiments i n t h i s work were c a r r i e d out w i t h autoclaved glucose. The p o s s i b l e i n h i b i t o r y e f f e c t s of autoclaved sugars on the growth of Sorangium 495 were not taken i n t o c o n s i d e r a t i o n . 3.3 E f f e c t of I n i t i a l pH The e f f e c t s of i n i t i a l pH ( v a r y i n g from 5 to 8) on growth and protease p r o d u c t i o n were s t u d i e d on f i l t e r e d 1 : 50. v/v d i l u t e d , condensed f i s h s o l u b l e s and c o n t a i n i n g 1% glucose. The i n i t i a l pH of the medium was adjusted to v a r i o u s values by the a d d i t i o n of 2N sodium hydroxide. During the course of i n c u b a t i o n , the pH was not adjusted but l e f t to seek i t s own l e v e l , the f i n a l pH value (at the end of 8 days) r e p r e s e n t i n g chemical s t a b i l i z a t i o n of the system. The dry c e l l weight and the p r o t e o l y t i c a c t i v i t y of the b r o t h were measured at the f o u r t h and eighth day of growth. The r e s u l t s are shown i n Table 3.14. The r e s u l t s i n d i c a t e that the i n i t i a l l y e s t a b l i s h e d pH tends to r i s e (probably due to the formation of ammonia from the decomposition of p r o t e i n ) during the course of growth; the f i n a l pH being s l i g h t l y above 8.0. The data show that an i n i t i a l pH of 7.0 i s best f o r enzyme pr o d u c t i o n , w h i l e an i n i t i a l pH of 8.0 i s best f o r growth. 3.4 E f f e c t of Condensed F i s h Solubles Concentration I t has been reported [75, 76, 77] that many organisms produce 58 Table 3.14 EFFECT OF INITIAL pH INITIAL pH AFTER 4 DAYS AFTER 8 DAYS pH DRY CELL WEIGHT gm/£ PROTEOLYTIC ACTIVITY uni t s /10cc pH DRY CELL WEIGHT gm/£ PROTEOLYTIC ACTIVITY u n i t s / l O c c 5 7.5 1.59 3.40 8.1 1.50 7.51 6 7.58 1.46 3.76 8.2 1.51 7.96 7 7.62 1.43 4.13 8.35 1.29 8.84 8 7.50 1.83 3.98 8.30 1.58 7.12 59 enzymes i n response to the presance of a p a r t i c u l a r substrate i n the c u l t u r e medium - "adaptive product ion of enzymes." This evidence l ed to the b e l i e f that a medium r i c h i n p r o t e i n i s requ ired for the product ion of h igh y i e l d s of p r o t e o l y t i c enzymes. F igures 3.9 and 3.10, which show the e f f ec t of i n c r e a s i n g the amount of condensed f i s h so lub les on the time course of growth and protease product ion r e s p e c t i v e l y , demonstrate that concentrat ions of f i l t e r e d so lubles having an average so lub le p r o t e i n content higher than 3.85 mg BSA/ml were markedly i n h i b i t o r y for enzyme product ion . The data for these f igures are tabulated i n Table A I . 3 of Appendix I . The maximum protease product ion rates (slope of exponent ia l phase of the product ion curves i n F igure 3.10) i n un i t s /10 c c . h r . are p l o t t e d against p r o t e i n concentrat ion i n F igure 3.11. A l s o p l o t t e d i n the same f i gure i s the maximum a c t i v i t y a t ta ined i n each medium. I t i s apparent from th i s f i gure that at low p r o t e i n l e v e l s , protease product ion i s l i m i t e d by a v a i l a b i l i t y of p r o t e i n n i t rogen (the f i n a l pH value of 5.9 a l so support th is view); at h igh p r o t e i n l e v e l s , ra te of product ion and y i e l d of enzyme are l i m i t e d by substrate i n h i b i t i o n . That the de tr imenta l e f f e c t of the higher so lubles concentrat ions on protease formation could be a t t r i b u t e d to i n h i b i t o r y concentrat ions of p r o t e i n or perhaps, of the organic n i t rogen compounds, i s i n d i c a t e d i n Figure 3 .9 . This f i gure shows that growth at the h igher p r o t e i n concentrat ions was as good as, or b e t t e r than, that obtained at the optimum concentrat ion for protease format ion. Figure 3.9 EFFECT OF CONDENSED FISH SOLUBLES CONCENTRATION ON GROWTH 40 80 120 T IME (hrs.) 160 61 F i g u r e 3.10 E F F E C T OF CONDENSED F I S H S O L U B L E S C O N C E N T R A T I O N ON P R O T E A S E F O R M A T I O N 12 10 o 2 8 N CO -4—• c o < V O L . C F S i n I O O O cc H^O 20CC 30 CC 60C C 40 80 120 T I M E (hrs.) 160 62 F i g u r e 3.11 E F F E C T OF P R O T E I N C O N C E N T R A T I O N ON U L T I M A T E P R O T E A S E Y I E L D AND ON MAXIMUM R A T E OF P R O T E A S E FORMATION 2 4 6 8 10 P R O T E I N C O N C . ( mg. BSA/ml.) 63 The r e s u l t s form these s tudies i n d i c a t e that enzyme y i e l d i s a f fec ted by the r a t i o of carbon to n i t rogen i n the medium. Best r e s u l t s with Sorangium 495 were obtained i n a f i s h so lubles medium conta in ing n i t rogen equivalent to 3.85 mg BSA/ml , and 10 gm/1 of g lucose . 3.5 E f f e c t of Carbohydrate Source Glucose i s commonly used as the energy and carbon source i n m i c r o b i o l o g i c a l media for laboratory growth experiments. However, i n d u s t r i a l wastes h igh i n carbohydrates are more s u i t a b l e for commercial purposes. These wastes usua l ly conta in a mixture of monosaccharides; for example, i n spent s u l f i t e l i q u o r , there are the hexoses - g lucose , mannose and galactose , and the pentoses - xylose and arabinose . I t was therefore thought to be u s e f u l to i n v e s t i g a t e the performance of cu l tures i n c o r p o r a t i n g these d i f f e r e n t sugars as the carbon and energy sources . Since the McCurdy medium [78], a medium used for c u l t i v a t i n g a wide spectrum of myxobacteria spec ies , contains a h igh amount of so lub le s t a r c h , the s u i t a b i l i t y of th i s po lysacchar ide as an energy supplement for the f i s h media was a l so eva luated . The medium used i n th i s experiment contained f i l t e r e d , 2% v / v condensed f i s h s o l u b l e s , and 1 gm/1 of carbohydrate . The r e s u l t s are graphed i n Figures 3.12, 3.13 and 3.14, and tabulated i n Tables A I . 4 and AI .5 of Appendix I . These r e s u l t s i n d i c a t e that the organism grew more r e a d i l y on mannose and glucose than on ga lac tose , s t a r c h or pentoses. F igure 3.14 i n d i c a t e s that b a c t e r i o l y s i s i s more pronounced i n media conta in ing s tarch or pentoses. The data demonstrate qui te c l e a r l y that Figure 3.12 EFFECT OF HEXOSES ON PROTEASE FORMATION TI ME (hrs.) 65 Figure 3.13 EFFECT OF SOLUBLE STARCH AND PENTOSES ON PROTEASE FORMATION T I M E (hrs. ) 6 6 Figure 3.14 EFFECT OF CARBOHYDRATE SOURCE ON GROWTH T I M E (hrs.) 67 good growth i s necessary for h igh y i e l d s of enzyme. I t i s a l so evident that glucose i s b e t t e r for protease format ion, and mannose i s b e t t e r for growth. The b i p h a s i c nature of the protease product ion curve i n medium conta in ing mannose was not f u r t h e r i n v e s t i g a t e d . The b i p h a s i c growth i n glucose w i l l be discussed i n l a t e r s e c t i o n s . Aga in , i t should be noted that the e f f e c t of s t e r i l i z a t i o n upon the d i f f e r e n t sugars was not taken i n t o account i n the above d i s c u s s i o n . According to Davis and Rogers [79], autoc lav ing sugar s o l u t i o n s can cause changes i n (1) o p t i c a l a c t i v i t y , (2) pH, (3) caramel i za t ion and (4) reducing power. The degree of change d i f f e r s from one sugar to another, the most s e n s i t i v e b e i n g , amongst o thers , dextrose and arabinose . 3.6 E f f e c t of Inoculum Age Inoculum age between 16 and 24 h r s . , which inc luded ages from ear ly l og phase to d e c e l e r a t i o n phase of growth, was tested for e f f e c t on y i e l d and rate of product ion . The changes occur ing dur ing the course of incubat ion i n "optimal" medium ( f i l t e r e d , 2% v / v d i l u t e d condensed f i s h so lubles with 1% glucose) are shown i n F igures 3.15, 3.16 and 3.17 and are tabulated i n Table AI .6 of Appendix I . These r e s u l t s i n d i c a t e no s i g n i f i c a n t e f f e c t on growth, protease product ion , and glucose u t i l i z a t i o n . Figure 3.15 EFFECT OF INOCULUM AGE ON GROWTH Figure 3.16 EFFECT OF INOCULUM AGE ON GLUCOSE UTILIZATION A G E 0 4 0 80 120 160 T I M E ( h o u r s ) 70 F i g u r e 3.17 EFFECT OF INOCULUM AGE ON PROTEASE FORMATION 71 3.7 7 - l i t r e Fermentation Studies A l l the above experiments were c a r r i e d out i n 250 ml Erlenmeyer f lasks conta in ing 100 ml of f i s h medium. On the bas i s of these r e s u l t s , a set of "optimal" c u l t u r a l condi t ions was chosen for maximum protease product ion . These "optima" are , at bes t , approximations, s ince i t was not p o s s i b l e to study f u l l y the i n t e r a c t i o n s of the various fac tors that a f f e c t protease y i e l d s ; a l so i t was not p o s s i b l e to study the e f f ec t s of a g i t a t i o n and a e r a t i o n . In th i s s e c t i o n , experiments to i n v e s t i g a t e the in f luence of these two process v a r i a b l e s on enzyme y i e l d s are d e s c r i b e d . The 7 - l i t r e fermentor descr ibed i n Sect ion 2.4 was used. A medium c o n s i s t i n g of 4 l i t r e s of f i l t e r e d , 2% v / v d i l u t e d condensed f i s h so lubles wi th 1% glucose , and i n i t i a l pH adjusted to 7.0, was used. E ight 7 - l i t r e fermentations were r u n . One of these used a Nutr i en t Broth - glucose medium, the composition of which was, i n g m / £ , peptone, 5; Di fco Beef E x t r a c t , 3; and glucose, 10. The r e s u l t s of th i s run are p r e -sented i n F igure 3.18 and Table A I . 7 of Appendix I . The remainder of the runs were made with the f i s h so lubles medium; a g i t a t i o n ra te was v a r i e d from 300 rpm to 750 rpm. F ive of these runs were made at an aera t ion rate of 1 &/min whi le the other two were performed at an aera t ion ra te of 2 £ / m i n . In no case was an e n t i r e run repeated. Although an NBS automatic pH c o n t r o l l e r was a v a i l a b l e at the time these experiments were performed, no attempt was made to regulate the pll by the automatic a d d i t i o n of ac id or base during the course of the 72 fermentation. I t was thought that the high p r o t e i n content of the medium provided the necessary b u f f e r ; the r a t i o of carbohydrate to p r o t e i n was such as to keep the pH of the medium w i t h i n ± 1 of n e u t r a l i t y . Due to the high content of surface a c t i v e agents i n the medium, there was consi d e r a b l e foaming. Since an automatic antifoam a d d i t i o n system was not a v a i l a b l e at the time, s m a l l amounts of defoaming agent i n the form of a 2% s o l u t i o n of A n t i f o r m B was added, whenever necessary, through the inoculum port of the fermentor head w i t h a s t e r i l i z e d s y r i n g e and hypodermic needle. 3.7.1 E f f e c t of A g i t a t i o n and A e r a t i o n on the Course of Fermentation The time v a r i a t i o n s of pH, sugar u t i l i z a t i o n , p r o t e o l y t i c a c t i v i t y and dry c e l l weight f o r the 7 - l i t r e fermentations are presented i n Figures 3.19 to 3.25 and are tabulated i n Tables AI.8 to AI.14 of Appendix I . These r e s u l t s i n d i c a t e that the r e l a t i v e amounts and times to maximum as w e l l as the shape of the growth curve are a f f e c t e d by a g i t a t o r speed and v o l u m e t r i c a i r flow r a t e . Sugar disappeareance, pH, and protease formation appear to be i n t e r r e l a t e d . An i n d u c t i o n p e r i o d was noted during which sugar u t i l i z a t i o n i s s m a l l , pH r i s e s , and protease formation has not yet begun. A f t e r t h i s i n i t i a l p e r i o d , sugar c o n c e n t r a t i o n * decreases at a nonuniform r a t e toward a zero value. In the same i n t e r v a l , the pH i s The f i g u r e s are a c t u a l l y p l o t t e d i n terms of sugar used r a t h e r than as sugar c o n c e n t r a t i o n . 73 F i g u r e 3.18 7-LITRE FERMENTOR NB - GLUCOSE RUN ( A g i t a t o r speed : 400 rpm; A i r flow r a t e : 1 £/min) TI ME (hrs.) 74 Table 3.19 7-LITRE FERMENTOR CFS RUN NO. 1 ( A g i t a t o r speed : 300 rpm; A i r flow r a t e : 1 £/min) 9 o o o £ 1 2 1 "E D >• > § 8 Q UJ C/) Z ) L U C/) O O D _ j O X Q . • © -• O -• A -• • -pH CELLS ACTIVITY GLUCOSE ® 80 TI M E (hrs.) 120 .8 C O .4 0 U J O 75 Figure 3.20 7-LITRE FERMENTOR CFS RUN NO. 2 (Agi ta tor speed : 400 rpm; A i r flow rate : 1 £ / m i n ) O O o \ CO © © e £ 12 c 10 o < Q L U C O D L U CO O O _ l O o -A — • — PH CELLS ACTIVITY GLUCOSE o-o H 9 X a 1.2 8 u> GO •4 ^ 4 a o 40 80 T I M E (hrs.) 120 m CO G L U C O S E USED (g/1 ); ACTIVITY(units/iocc.) O - N CO to o CO o 00 o C E L L S ( g / l ) \ i 9 I ® J / \ e MO pH > H-rt P> rr O H cn T) ro ro a. U l o o H 3 > i-i l-tl t—' § 3 I tr1 H O n C/2 2! O U> CK) e i-! ON G L U C O S E USED(g/ l ) ; ACT IV ITY (units/iocc.) 78 Figure 3.23 7-LITRE FERMENTOR CFS RUN NO. 5 ( A g i t a t o r speed : 750 rpm; A i r flow r a t e : 1 2,/min) O o • / L / o • / o / • p / ' I L e O A - 7 T A - O -- A — - • — PH C E L L S ACTIVITY GLUCOSE) 8 X Q _ 1.6 Cf) UJ .8 O 0 0 20 40 TIME (hrs.) 60 79 Figure 3.24 7-LITRE FERMENTOR CFS RUN NO. 6 ( A g i t a t o r speed : 400 rpm; A i r flow r a t e : 2 Jl/min) 0 20 40 6 0 T I M E (hrs.) 80 Figure 3.25 7-LITRE FERMENTOR CFS RUN NO. 7 (Agitator speed : 500 rpm; Air flow rate : 2 fc/min) TIM E ( hrs) 81 noted to reach a maximum and then decrease to a minimum. The concentrat ion of enzyme r i s e s i n a s igmoidal fashion u n t i l a peak i s reached. In a l l cases , the protease product ion curve lags behind the growth i n a fashion s i m i l a r to shake f l a s k c u l t u r a t i o n . The growth curve of F igure 3.19 can be d i v i d e d up i n t o 4 phases, namely: (1) phase of i n i t i a l growth, (2) f i r s t s t a t i o n a r y or death phase, (3) a phase of secondary growth and (4) a f i n a l d e c e l e r a t i o n and s ta t ionary phase. The durat ion of the second phase was great ly decreased, from 40 hours to 20 hours , as the a g i t a t o r speed increased from 300 rpm to 400 rpm (Figures 3.19 and 3 .20) . At s t i l l h igher a g i t a t i o n r a t e s , 500 rpm and 600 rpm, the death phase of the second p o r t i o n was completely e l iminated; at the h ighest a g i t a t i o n r a t e , 750 rpm, the f i r s t and t h i r d phase meshed to give a s igmoidal growth curve . Increas ing the a e r a t i o n ra te from 1 £ / m i n to 2 &/min a l so completely e l iminated the second phase (Figures 3.24, 3 .25) . The e f f e c t was therefore thought to be r e l a t e d to mass t rans fer l i m i t a t i o n . However F igure 3.18 (Table AI .7 of Appendix I) ind ica ted that the nature of the n i t rogen source may have contr ibuted to the o v e r a l l e f f e c t . As mentioned e a r l i e r , F igure 3.18 represents the r e s u l t s of a run made with peptone and meat e x t r a c t as the n i t rogen source; the a g i t a t i o n rate was 400 rpm and the aera t ion rate was 1 £ / m i n . This f i gure i n d i c a t e s that m u l t i p l i c a t i o n proceeded up to the 15 hour, a f t e r which extensive l y s i s occurred , and very l i t t l e glucose was u t i l i z e d . The r i s e i n the pH of the cu l ture broth was obviously due to the products of n i trogen 82 metabolism. The l a r g e amount of unused glucose may have c o n t r i b u t e d to the extensive l y s i s observed a f t e r growth ceased [80, 81]. N i t r o g e n appears to be the l i m i t i n g f a c t o r f o r growth and protease p r o d u c t i o n i n t h i s case. The f i r s t and second p o r t i o n s of the growth curve of F i g u r e s 3.19 and 3.20 correspond approximately, to the growth i n the N u t r i e n t Broth -glucose medium. These two p o r t i o n s of the growth curve may be i n t e r p r e t e d as r e p r e s e n t i n g the d e p l e t i o n of f r e e amino acid s and nitrogenous bases i n i t i a l l y present i n the condensed f i s h s o l u b l e s medium. A f t e r the deple-t i o n of the i n i t i a l simple nitrogenous m a t e r i a l s , b a c t e r i a l metabolism appears to be l i m i t e d by the r a t e at which the p r o t e o l y t i c enzyme can convert the l a r g e r polypeptides to s i m p l e r forms. Apparently, the r a t e of p r o t e i n h y d r o l y s i s depends on the r a t e of enzyme p r o d u c t i o n , which i s i n turn determined by the l e v e l of a g i t a t i o n . Although these r e a c t i o n s occur at d i f f e r e n t r a t e s , they a l l proceed simultaneously. The d i f f e r e n c e i n r a t e s coupled w i t h the i n i t i a l presence of an e a s i l y a v a i l a b l e n i t r o g e n source caused the staged nature of the growth k i n e t i c s . These r e s u l t s are c o n s i s t e n t w i t h Sperry and Rettger's [82] c o n c l u s i o n s , which are quoted below : The i n a b i l i t y of b a c t e r i a to decompose n a t i v e p r o t e i n s i s not l i m i t e d to aerobes and f a c u l t a t i v e anareobes, but even well-known and extremely a c t i v e p u t r e f a c t i v e anaerobes are devoid of t h i s property. S o l u t i o n s of n a t i v e p r o t e i n s may undergo complete h y d r o l y s i s , however, i f they cont a i n peptone or some other nitrogenous food m a t e r i a l which 83 r e a d i l y f u r n i s h e s the necessary n i t r o g e n f o r b a c t e r i a l development. In such instances the p r o t e o l y s i s of the n a t i v e p r o t e i n i s the immediate r e s u l t of the a c t i o n of an enzyme which has been elaborated by the b a c t e r i a during the process of r a p i d m u l t i p l i c a t i o n . This m u l t i p l i c a t i o n i s made p o s s i b l e by the n i t r o g e n - c o n t a i n i n g m a t e r i a l which i s present along w i t h the n a t i v e p r o t e i n . The r e s i s t a n c e of n a t i v e p r o t e i n s to d i r e c t decomposi-t i o n by b a c t e r i a i s not due to any a n t i s e p t i c p r o p e r t i e s of the p r o t e i n s , but to a c o n s t r u c t i o n of the molecule which renders i t r e l a t i v e l y s t a b l e , the component p a r t s being so f i r m l y bound together that strong cleavage - producing agents, such as extreme heat, strong acids and/or a l k a l i e s , and enzymes, are r e q u i r e d to change them so that b a c t e r i a may u t i l i z e t h e i r products f o r c e l l n u t r i t i o n . R e t t g e r , Berman and Sturges [83] c a r r i e d the matter a stage f u r t h e r when they showed that heat - coagulated albumin, and sometimes peptones are not a v a i l a b l e f o r d i r e c t u t i l i z a t i o n u n t i l f u r t h e r decomposed by enzyme a c t i o n . Of i n t e r e s t a l s o are the f i n d i n g s of Archer et a l [84]. While studying the s o l u b i l i z a t i o n of f i s h p r o t e i n concentrate (FPC) by B a c i l l u s S u b t i l i s protease (Monzyme), they found that the o v e r a l l k i n e t i c s could be described by a sequence of two f i r s t - o r d e r processes - an i n i t i a l , f a s t r e a c t i o n i n which l o o s e l y bound polypeptide chains were cleaved, and a second, slower r e a c t i o n i n which more compacted p r o t e i n was d i g e s t e d . S i m i l a r b i p h a s i c k i n e t i c s were obtained by M i h a l y i and H a r r i n g t o n [85]. 84 Figure 3.26 shows that when the Sorangium specie was grown i n a f i s h so lub les medium without added glucose , staged k i n e t i c s were not detected. A comparison of th i s f i gure with F igure 3.19 i n d i c a t e s that the maximum enzyme concentrat ion a t ta ined i n the glucose - d e f i c i e n t media corresponds to the value at the end of the second phase of growth shown i n F igure 3.19. The r e s u l t s therefore suggest that the organism was unable , i n the absence of a source of carbohydrate, to b r i n g about breakdown of the l a r g e r polypept ide uni t s present i n the medium. The data points of th i s f i gure are tabulated i n Table A I . 1 5 . Shake f l a s k c u l t i v a t i o n was used to obta in these r e s u l t s . 3 .7.2 E f f e c t of A g i t a t i o n and A e r a t i o n on Ult imate Y i e l d s The e f fec t s of a g i t a t i o n and aera t ion on u l t imate y i e l d s are summarized i n Table 3.15. The h ighest protease product ion occurred at 500 rpm with 1 l i t r e of a i r introduced per min. However, more s i g n i f i c a n t l y at the same a g i t a t o r speed and 2 l i t r e s of a i r per minute, almost the same u l t imate protease y i e l d was obtained but at a much f a s t e r r a t e . Other a g i t a t i o n rates r e s u l t e d i n lower p r o d u c t i v i t y . The percentage of i n i t i a l sugar u t i l i z e d at the end of fermentation was noted to be approximately the same at a l l l e v e l s of a g i t a t i o n and aera t ion tested; however the h ighest sugar consumption rate occurred at 750 rpm with 1 l i t r e of a i r per minute. Aera t ion rates and a g i t a t i o n rates are interdependent i n t h e i r e f f ec t on c e l l metabolism - for example, at 600 rpm and an a i r flow of 1 5,/min, t o t a l sugar consumption, protease product ion , and growth are almost Figure 3.26 SHAKE FLASK FERMENTATION - GLUCOSE DEFICIENT MEDIUM T IME (hrs.) Table 3.15 EFFECT OF AGITATION AND AERATION ON ULTIMATE YIELDS MEDIA AIR FLOW RATE ( £ / m i n ) AGITATOR SPEED rpm DRY MAX (g/*) WEIGHT TIME TO MAX (hrs) PROTEOLYTIC ACTIVITY MAX TIME TO MAX (un i t s / lOcc ) (hrs) SUGAR MAX (%init ia l ) UTILISATION TIME TO MAX (hrs) 300 1.00* 3 113f 8.84 134 96.3 134 FILTERED 2% v / v CONDENSED 1 400 500 0.85* 1.29 94 9.95 10.52 1 1 6 | 95.0 95.4 1 1 6 | 77 FISH 600 1.62 35 7.82 4 94.4 4 SOLUBLES + 750 2.05 4 6.94 45 92.4 45 1% GLUCOSE 2 400 1.55 4 7.74 47 94.3 56 i 500 1.55 16 9.86 45 94.1 4 ABOVE MEDIUM minus 1% SHAKER INCUBATOR 1.00 32 2.85 45 - -GLUCOSE NB plus 1% 1 400 0.83 15 2.28 45 16.0 34 GLUCOSE G l o b a l maximum 87 i d e n t i c a l to those at an a i r flow of 2£/min and 400 rpm. F igure 3.27 i s a p l o t of c e l l s produced, and p r o t e o l y t i c a c t i v i t y against glucose used. No r e l a t i o n s h i p between the three v a r i a b l e s i s apparent. 3.7.3 E f f e c t of A g i t a t i o n and A e r a t i o n on Rate of Oxygen Transfer The funct ions of a g i t a t i o n and aera t ion i n aerobic fermentors are mainly to create a large a i r - l i q u i d i n t e r f a c i a l area and to reduce the res i s tance to oxygen d i f f u s i o n by reducing the thickness of the l i q u i d f i l m surrounding each bubble [86]. The c o r r e l a t i o n of Yamada et a l [87] was used to c a l c u l a t e the values of K ^ , the vo lumetr ic oxygen t r a n s f e r c o e f f i c i e n t , shown i n Table 3.16. An attempt to c o r r e l a t e the values wi th the maximum y i e l d and the maximum rate of protease formation i s shown i n F i g u r e 3.28. This method of c o r r e l a t i o n was unsuccessful i n b r i n g i n g together data for the various systems s t u d i e d . I t appears therefore that mass t r a n s f e r i s not the only c o n t r o l l i n g f a c t o r i n the product ion of p r o t e o l y t i c enzyme or that Yamada's c o r r e l a t i o n for K i s not a p p l i c a b l e to our fermentation v system, and that a fur ther i n v e s t i g a t i o n of th i s subject i s necessary. 88 F i g u r e 3 . 2 7 A C T I V I T Y AND C E L L Y I E L D VS SUGAR U S E D FOR V A R I O U S 7 - L I T R E FERMENTOR RUNS 89 T a b l e 3.16 K VALUES ATTAINED IN 7-L FERMENTATION RUNS v AIR FLOW RATE £/min AGITATOR SPEED rpm K V m i l l i m o l e s / a t m . min. l i t r e (dp/dt)max u n i t s / l O O c c . h r 1 300 0.177 1.20 400 0.369 1.40 500 0.654 3.50 600 1.043 3.70 750 1.847 5.65 2 400 0.452 3.60 500 0.805 6.90 C o r r e l a t i o n u s e d was : K = 8.06 x I O - 8 x v°- 3 x N 2 ' 5 6 v T h i s e m p i r i c a l r e l a t i o n s h i p was o b t a i n e d by Yamada e t a l (87) u s i n g a s t a i n l e s s s t e e l , 8 - 1 c a p a c i t y f e r m e n t o r f i t t e d w i t h a s i n g l e h o l e s p a r g e r and a d i s k t y p e a g i t a t o r . 90 F i g u r e 3.28 MAXIMUM YIELD AND MAXIMUM RATE OF ENZYME PRODUCTION AS A FUNCTION OF K v K v> mmoles/atm. min. litre 91 Chapter 4 FERMENTATION KINETICS Batch k i n e t i c data are needed not only to develop b a s i c under-standing of fermentation processes but a l so to permit r a t i o n a l des ign of continuous fermentation systems. The fermentation of f i s h so lubles to produce enzymes has so f a r not rece ived any q u a n t i t a t i v e treatment; consequently no k i n e t i c ana lys i s of t h i s fermentation can be found i n the l i t e r a t u r e . This chapter attempts to analyse fur ther some of the r e s u l t s presented i n the previous s e c t i o n s . In p a r t i c u l a r , i t i s the hope of t h i s chapter to determine the r e l a t i o n s h i p s , i f any, between the d i f f e r e n t rates and to see how these rates vary with respect to a g i t a t i o n and a e r a t i o n . Instantaneous (or volumetric) rates of growth, sugar u t i l i s a t i o n , and enzyme product ion were obtained by the g r a p h i c a l d i f f e r e n t i a t i o n [88] of curves of f igures showing the time changes i n these v a r i a b l e s . The s p e c i f i c rates were determined by d i v i d i n g the vo lumetr ic rates by the c e l l mass at that t ime. The s p e c i f i c rates are more u s e f u l for k i n e t i c ana lys i s s ince they put everything on a comparable b a s i s , i e . per u n i t mass of c e l l s . Complete ra te pa t t erns , on both vo lumetr ic and s p e c i f i c rate bases, for fermentations at 500 rpm at 1 Jl /min , 750 rpm at 1 £ / m i n and 500 rpm at 2 Jl /min , are presented i n Tables 4.17 to 4.22 and graphed i n Figures 4.29 to 4.34. They were c a l c u l a t e d from the data presented i n Figures 3.21, 3.23 and 3.25. 92 T a b l e 4.17 7-LITRE FERMENTATION - CFS RUN NO. 3* VOLUMETRIC RATES OF GROWTH SUGAR UTILIZATION PROTEASE FORMATION TIME ( h r s ) d x / d t (g/«,.hrxlO) TIME ( h r s ) d s / d t ( g M . h r x l O ) TIME ( h r s ) dp/dt ( u n i t s / c c . h r x l O O ) 2.5 0.44 4.0 0.70 7.0 0.0 5.0 0.84 10.5 1.10 12.0 0.6 7.0 1.24 20.0 1.55 16.0 0.8 8.5 1.60 30.5 1.90 24.0 1.6 9.5 0.32 40.0 2.10 29.5 2.0 10.5 0.18 48.5 1.50 34.0 2.4 14.0 0.0 61.5 0.50 40.5 2.85 18.0 0.10 80.0 0.10 47.0 3.35 23.0 0.44 92.0 0.00 51.0 3.80 26.5 0.29 53.5 2.10 29 .5 0.20 62.5 0.40 35.0 0.08 75.0 0.00 44.0 0.02 55.0 0.00 A g i t a t o r speed : 500 rpm A i r f l o w r a t e : 1 &/min. 93 Table 4.18 7-LITRE FERMENTATION - CFS RUN NO. 3* SPECIFIC RATES OF GROWTH SUGAR UTILIZATION PROTEASE FORMATION 1 dx 1 ds 1 dp TIME x dt x dt x dt (hrs) (g/g.hrxlO) (g/g.hrxlO) (units/mg.hrxlOO) 2.5 4.89 5.56 0.78 5.0 3.11 2.78 0.74 7.0 2.53 1.83 -8.5 2.22 1.35 -10.0 0.33 1.28 0.55 14.0 0.0 1.49 0.83 18.0 0.12 1.69 1.16 20.0 0.22 1.74 1.31 23.0 0.46 - 1.46 27.5 0.27 1.62 1.67 30.0 0.19 1.65 1.78 32.5 - - 1.91 35.0 0.07 1.65 2.07 40.0 0.03 1.65 2.38 45.0 - 1.46 2.64 50.0 - 1.11 2.72 55.0 - 0.79 1.61 60.0 - 0.56 0.75 65.0 - 0.36 0.32 75.0 0.12 0.0 A g i t a t o r speed : 500 rpm A i r flow rate : 1 £ / m i n . 94 Table 4.19 7-LITRE FERMENTATION - CFS RUN NO. 5* VOLUMETRIC RATES OF GROWTH SUGAR UTILIZATION PROTEASE FORMATION TIME dx/dt TIME ds/dt TIME dp/dt (hrs) (g/JUhrxlO) (hrs) (g/£.hrxlO) (hrs) (units/cc.hrxlOO) 2.3 0.24 7.4 1.0 12.8 0.6 6.5 0.81 12.0 2.2 21.3 2.05 9.0 1.80 18.5 3.05 25.3 5.65 10.7 2.34 24.4 3.70 28.0 4.80 13.5 1.12 30.0 3.00 30.5 2.80 16.7 0.64 36.5 0.95 36.0 0.80 20.7 0.39 45.0 0.25 . 42.5 0.35 26.8 0.10 55.0 0.0 47.5 0.20 32.5 0.0 52.5 0.0 * Agitator speed : 750 rpm Air flow rate : 1 A/min. 95 Table A.20 7-LITRE FERMENTATION - CFS RUN NO. 5* SPECIFIC RATES OF GROWTH SUGAR UTILIZATION PROTEASE FORMATION 1 dx 1 ds_ _1 dp_ TIME x dt x dt x dt (hrs) (g/g.hrxlO) (g/g.hrxlO) (units/mg.hrxlOO) 2.5 5.75 5.75 0.0 5.0 A.A3 A.A3 0.0 7.5 3.57 3.1A 0.0 10.0 2.59 2.13 0.19 12.5 1.14 1.70 0.3A 15.0 0.57 1.69 0.A7 17.5 0.3A 1.75 0.68 20.0 0.21 1.78 0.96 22.5 0.13 1.80 1.37 25.0 0.07 1.79 2.A6 25.5 - - 2.77 27.5 O.OA 1.65 2.A2 30.0 0.02 1.A5 1.7A 32.5 0.0 0.89 0.89 35.0 0.0 0.61 0.61 AO.O 0.0 0.28 0.28 A5.0 0.0 0.10 • 0.10 A g i t a t o r speed : 750 rpm A i r flow r a t e : 1 £./min. 96 Table 4.21 7-LITRE FERMENTATION - CFS RUN NO. 7* VOLUMETRIC RATES OF GROWTH SUGAR UTILIZATION PROTEASE FORMATION TIME dx/dt TIME ds/dt TIME dp/dt (hrs) (g/£.hrxlO) (hrs) ( g / £ . h r x l 0 ) (hrs) (units/cc.hrxlOO) 2.7 6.8 9.5 11.5 12.5 12.8 15.0 18.0 0.26 0.86 1.71 3.10 1.38 0.97 0.25 0.0 2.5 6.5 11.0 17.6 26.0 35.0 42.0 47.5 52.2 55.0 0.85 1.55 2.10 2.50 2.30 2.10 1.65 0.80 0.20 0.0 12.6 21.5 29.0 34.5 38.8 42.0 43.7 44.7 48.5 52.5 0.15 1.10 2.57 3.85 5.10 6.90 3.80 2.30 0.60 0.0 A g i t a t o r speed : 500 rpm A i r flow r a t e : 2 £/min. 97 Table 4.22 7-LITRE FERMENTATION - CFS RUN NO. 7* SPECIFIC RATES OF TIME (hrs) GROWTH I dx x dt (g /g .hrx lO) SUGAR UTILIZATION 1 ds x dt (g /g .hrx lO) PROTEASE FORMATION x dt (units /mg.hrxlOO) 1.5 2.5 5.0 7.5 10.0 11.5 12.5 15.0 17.5 20.0 25.0 30.0 35.0 40.0 45.0 50.0 7.0 6.0 4.46 3.05 2.41 2.87 1.00 0.19 0.0 20.0 16.8 9.6 5.0 2.88 2.08 1.70 1.61 1.62 1.62 1.56 1.49 1.39 1.20 0.84 0.31 0.07 0.16 0.32 0.52 1.10 1.82 2.69 3.90 1.75 0.31 A g i t a t o r speed : 500 rpm A i r flow rate : 2 Jl/min. 98 F i g u r e 4 . 2 9 VOLUMETRIC RATES VS TIME FOR FERMENTOR RUN NO. 3 A 2.01 1.6 ,8 •o-• • -A -A GROWTH, g/(l.io.hr; B ENZYME SYNTHESIS.units/ioocc.hr C SUGAR UTILIZATION, g/|johr. 0 40 Tl ME (hrs) ^4 B,C 6 0 99 F igure 4.30 SPECIFIC RATES VS TIME FOR FERMENTOR RUN NO. 3 - o - A GROWTH , g./g.io hr. - • - B ENZYME SYNTHESIS, units/mg.ioohr. A - A - C SUGAR UTILIZATION, g./g.io hr. o TIMEChr.) F i g u r e 4.31 VOLUMETRIC RATES VS TIME FOR FERMENTOR RUN NO. 5 - o - A G R O W T H , g/(1.10 hr) I -•- B E N Z Y M E S Y N T H E S I S . u n i t s / i o o c c h r . ] - A - C S U G A R U T I L I Z A T I O N , g / | - 1 0 h r . • 0 20 40 60 T IME (hrs.) F i g u r e 4.32 SPECIFIC RATES VS TIME FOR FERMENTOR RUN NO. 5 -o- A GROWTH , g./g.iohr. - • - B E N Z Y M E S Y N T H E S I S , u n i t s / m g . i o o h i : - A - C S U G A R U T I L I Z A T I O N , g . / g . i o h r . T I M E (hrs . ) Figure 4.33 VOLUMETRIC RATES VS TIME FOR FERMENTOR RUN NO. 7 - o - A G R O W T H , Q / ( l . i o . h r J T I M E (hrs . ) 103 Figure 4.34 SPECIFIC RATES VS TIME FOR FERMENTOR RUN NO. 7 o - A GROWTH , g . / g . i o h r . - • - B E N Z Y M E , S Y N T H E S I S , u n i t s / m g . i o o l i r . - A - C S U G A R U T I L I Z A T I O N , g . / g . i o hr. A,B| 12 r-A 8 ho oi , nWr* 0 2 0 40 6 0 T IME ( h r s . ) 104 The rate curves of these f igures show two d i s t i n c t phases i n the fermentation system under c o n s i d e r a t i o n . In the i n i t i a l phase, sugar i s used p r i m a r i l y for c e l l m u l t i p l i c a t i o n wi th p r a c t i c a l l y no product ion of enzyme; i n the second phase, when the growth rate has decreased to a zero va lue , enzyme accumulation i s maximum. This i s most evident i n the s p e c i f i c rate p l o t s . Another p o i n t , a lso demonstrated qui te c l e a r l y i n the s p e c i f i c rate pa t t erns , i s the c lose coincidence of growth and sugar u t i l i z a t i o n rates i n the i n i t i a l stages (10-15 h o u r s ) . During the p e r i o d of enzyme produc t ion , both the dry c e l l weight and the s p e c i f i c ra te of carbohydrate u t i l i z a t i o n remained constant . The r e l a t i o n s h i p between the s p e c i f i c ra te 1 ds 1 dx of glucose u t i l i z a t i o n ( — — ) and the s p e c i f i c rate of growth (— — ) x dt x dt i s furthered demonstrated i n Figure 4.35. This p l o t suggests that dur ing the e a r l y stages of the fermentation (up to the 10 ^  hour) the rate of carbohydrate consumption per c e l l i s d i r e c t l y p r o p o r t i o n a l to the s p e c i f i c rate of growth. The constant of p r o p o r t i o n a l i t y depends more on the a i r flow rate than on the a g i t a t o r speed. From the 10 ^  hour to the e a r l y stages of the f i n a l s t a t i o n a r y growth phase, the ra te of glucose consumption i s constant at 1.682 g /g . hr x 10. This va lue , obtained by averaging a l l 1 ds values of — — between 1.6 and 1.8, was the same for a l l three cases x dt ' examined. During the per iod of constant s p e c i f i c glucose u t i l i z a t i o n , glucose consumption and protease product ion had j u s t entered t h e i r l o g a r i -thmic phases (Figures 3.21, 3.23 and 3 .25) . No d i r e c t r e l a t i o n s h i p between carbohydrate o x i d a t i o n rate and protease product ion rate i s apparent. One can therefore speculate that one of the purposes served by the glucose Figure 4.35 SPECIFIC RATE OF GLUCOSE UTILIZATION VS SPECIFIC RATE OF GROWTH FOR VARIOUS FERMENTOR RUNS Specific Rate of Growth (g./g.iohr) 106 was to supply the energy required for the endergonic r e a c t i o n of syn thes i s . A comparison of the s p e c i f i c rates of enzyme product ion for the three cases examined i n d i c a t e s that (1) a g i t a t o r speed has a greater in f luence on th i s rate than does a i r flow r a t e . For example, at a s t i r r i n g ra te of 500 rpm and 1 Jl /min , the rate of enzyme product ion per gm dry c e l l weight increased s lowly and s t e a d i l y to i t s maximum value a f t e r 50 hours , and at 750 rpm and the same aera t ion r a t e , the maximum was a t ta ined a f t e r 25 hours , whi le at 500 rpm and 2 £ / m i n , time to maximum was 40 hours; and (2) The metabol ic a c t i v i t y of the organism during the e a r l y part of the fermentations i s h ighes t at an aera t ion ra te of 2 l i t r e a i r per minute. The k i n e t i c patterns obtained i n th i s study are qu i te complex. A d d i t i o n a l s tudies on the e f f ec t s of d i s s o l v e d oxygen concentrat ion and rate of oxygen t r a n s f e r to the medium are needed to e l u c i d a t e the var ious l i m i t i n g s teps . 107 Chapter 5 CONCLUSIONS This study has shown that the batch p r o d u c t i o n of e x t r a c e l l u l a r protease by Soranguim 495 can be achieved i n a medium c o n t a i n i n g condensed f i s h s o l u b l e s as i t s major component. The condensed f i s h s o l u b l e s , obtained from B.C. Packers, Richmond, B.C., was d i l u t e d and any s o l i d m a t e r i a l f i l t e r e d o f f . The f i l t e r e d media was found to give b e t t e r protease y i e l d s than the u n f i l t e r e d media. A d i l u t i o n r a t i o of 20 cc condensed f i s h s o l u b l e s to 1000 cc of water, c o n t a i n i n g s o l u b l e p r o t e i n e q u i v a l e n t to 3.85 mg BSA/ml , was found to be the optimum f o r enzyme y i e l d . P r o l i f i c growth of the organism was obtained i n both f i l t e r e d and u n f i l t e r e d d i l u t e d condensed f i s h s o l u b l e s . S t a t i s t i c a l l y designed e x p e r i -ments showed th a t w h i l e a d d i t i o n a l phosphate has no s i g n i f i c a n t e f f e c t on protease p r o d u c t i o n , t h i s was not so f o r glucose and calcium. The amount of supplementary glucose was found to have a l a r g e i n f l u e n c e on both growth and protease pr o d u c t i o n . The one t a i l e d F - t e s t a l s o i n d i c a t e d that there was s i g n i f i c a n t i n t e r a c t i o n between the d i f f e r e n t sugar concentrations and the d i f f e r e n t calcium c o n c e n t r a t i o n s . The complete a n a l y s i s of v a r i a n c e i s given i n Table 3.13. F u r t h e r experimental runs w i t h medium c o n t a i n i n g s e v e r a l l e v e l s of glucose (0% to 4%) i n d i c a t e d that a two f o l d i n c r e a s e i n c o s e i n o l y t i c a c t i v i t y was obtained by i n c r e a s i n g the i n i t i a l sugar content from 0.062 108 mg per ml, o r i g i n a l l y present i n the medium, to 10 mg per ml. That the a d d i t i o n a l glucose exerted i t s i n f l u e n c e by i n c r e a s i n g the growth of the organism was r e a d i l y apparent from v i s u a l observation and from the two phase nature of the growth curve. The data a l s o i l l u s t r a t e d that a d d i t i o n of glucose retarded protease formation. L a t e r r e s u l t s d i s c l o s e d that adequate a g i t a t i o n and/or a e r a t i o n not only e l i m i n a t e d the staged growth but a l s o i n c r e a s e d both the y i e l d and r a t e of protease formation. Glucose and mannose supported the growth of the organism. Best growth was achieved w i t h mannose. Best protease y i e l d occurred w i t h glucose. The medium c o n t a i n i n g mannose gave a b i p h a s i c enzyme pro d u c t i o n curve w h i l e the medium c o n t a i n i n g glucose gave a b i p h a s i c growth curve. S o l u b l e s t a r c h , g a l a c t o s e , x y l o s e and arabinose were not u t i l i z e d . I n i t i a l pH ( i n the range 4 to 8 ) d i d not s i g n i f i c a n t l y a f f e c t the maximum y i e l d of proteases nor d i d inoculum age (16 to 24 hou r s ) . Reproducible r e s u l t s have been obtained w i t h shake f l a s k c u l t i v a t i o n . 7 - l i t r e fermentation s t u d i e s i n d i c a t e d that the i n f l e c t i o n i n the growth curve observed at the lower a g i t a t i o n and a e r a t i o n r a t e s and i n shake f l a s k c u l t i v a t i o n may be a complex r e s u l t of i n i t i a l amino a c i d (and p o s s i b l y s m a l l polypeptides) u t i l i z a t i o n on one hand and enzymatic d i g e s t i o n of l a r g e r p o l y p e p t i d e u n i t s plus enzyme s e c r e t i o n and oxygen l i m i t a t i o n on the other. Highest p r o d u c t i v i t y was obtained w i t h an a g i t a t i o n r a t e of 500 rpm and an a e r a t i o n r a t e of 2 l i t r e of a i r per minute; t h i s c o r r e s -ponded to an oxygen absorption c o e f f i c i e n t of 0.805 m i l l i m o l e s O^/atm.min.I. 109 This value was obtained using the c o r r e l a t i o n of Yamada et a l [ 8 7 ] . When the k i n e t i c data were presented on a per u n i t of c e l l mass bas is (that i s s p e c i f i c ra te b a s i s ) , i t t r a n s p i r e d that the two processes , growth and protease synthes i s , were independent. During the phase of enzyme product ion , the s p e c i f i c rate of glucose u t i l i z a t i o n remained at a constant value of 1.682 g /g . hr x 10. I t was therefore apparent that product formation was not assoc iated by any d i r e c t mechanism with carbohy-drate u t i l i z a t i o n . Enzyme product ion was a f fec ted more by ra te of a g i t a t i o n than a e r a t i o n ; the reverse holds for sugar consumption. The k i n e t i c patterns are qui te complex, and fur ther work i s necessary to e l u c i d a t e the var ious mechanisms i n v o l v e d . 110 Chapter 6 RECOMMENDATIONS (1) A g r i c u l t u r a l wastes h igh i n carbohydrate, such as f r u i t wastes, should be i n v e s t i g a t e d wi th the view to r e p l a c i n g glucose as a supplementary n u t r i e n t . The ac id hydrolyzed condensed f i s h so lub les contains a large amount of p r o t e i n hydrolysates and other e a s i l y a v a i l a b l e n i t rogen compounds which are not present i n the s t ickwater that Truong [11] used, unsuccess fu l ly , as a replacement for yeast e x t r a c t . I t i s therefore recommended that condensed f i s h so lubles and not s t i ckwater should be used as a replacement for yeast e x t r a c t . (2) A d d i t i o n a l s tudies on the e f fec t s of d i s s o l v e d oxygen concentrat ion and rate of oxygen t r a n s f e r to the medium should be c a r r i e d out . Hopeful ly data from these r e s u l t s w i l l c l a r i f y the major features of the mechanism of the system so that a working t h e o r e t i c a l model can be formulated. I t i s recommended that these s tudies be c a r r i e d out with , b e t t e r pH and foam c o n t r o l . (3) The e f f e c t of f i l t e r - s t e r i l i z e d and autoclaved sugars on growth and enzyme producing a b i l i t y of the Sorangium specie should be i n v e s t i g a t e d . (4) The e f f e c t of calc ium ions on protease y i e l d s should be further i n v e s t i g a t e d . I l l (5) In order to develop u s e f u l a p p l i c a t i o n s of the Sorangium protease, i t s s p e c i f i c i t y and optimum c o n d i t i o n s of a c t i o n should be s t u d i e d . In t h i s connection, i t i s suggested that the enzyme be t e s t e d f o r i t s a b i l i t y to hydrolyze f i s h to produce FPC f o r human consumption and to produce f i s h h y d r o l y s a t e s f o r use i n b a c t e r i o l o g i c a l c u l t u r e media. A more meaningful cost estimate f o r an enzyme production process can be made when such i n f o r m a t i o n i s obtained. (6) Experimentation w i t h other microorganisms, such as those a s s o c i a t e d w i t h i n d u s t r i a l fermentation processes ( f o r example, B a c i l l u s s u b t i l i s , and A s p e r g i l l u s oryzae ), i s recommended. 112 BIBLIOGRAPHY [I] C lagge t t , F . G . , 1972. " C l a r i f i c a t i o n of F i sh Process ing P lant e f f luent s by Chemical Treatment and A i r F l o t a t i o n , " F i s h e r i e s Research Board of Canada, T e c h n i c a l Report No. 343. [2] Clagget t , F . G . , 1971. "Demonstration Wastewater Treatment U n i t . Interium Report 19 71 Salmon Season," F i s h e r i e s Research Board of Canada, Vancouver Laboratory , T e c h n i c a l Report No. 286. [3] C lagge t t , F . G . , 1970. "A Proposed Demonstration P lant for T r e a t i n g F i s h Process ing P lant Wastewater," F i s h e r i e s Research Board of Canada, Vanocuver Laboratory , T e c h n i c a l Report No. 197. [4] C lagge t t , F . G . and J . Wong, 1969. "Salmon Canning Wastewater C l a r i f i c a t i o n . Par t I I . A Comparison of Various Arrangements for F l o t a t i o n and some observat ions concerning Sedimentation and H e r r i n g Pump Water C l a r i f i c a t i o n , " F i s h e r i e s Research Board of Canada, Vancouver Laboratory , C i r c u l a r No. 42. [5] C lagge t t , F . G . and J . Wong, 1968. "Salmon-Canning C l a r i f i c a t i o n . Part I . F l o t a t i o n by T o t a l Flow P r e s s u r i z a t i o n , " F i s h e r i e s Research Board of Canada, Vancouver Laboratory , C i r c u l a r No. 38. [6] Soderquis t , M . R . , 1970. "Current P r a c t i c e i n Seafoods Process ing Waste Treatment," Department^ of Food Sc ience , Oregon State U n i v e r s i t y , C o r v a l i s , Oregon. Report prepared for EPA Water Q u a l i t y O f f i c e , Washington, D . C . [7] Lassen, S . , 1965. "Fish S o l u b l e s , " i n F i s h As Food, V o l . I l l , G . Borgstrom ( e d . ) , Academic Press , N . Y . , pp. 281-299. [8] A i b a , S . , A . E . Humphrey, and N . F . M i l l i s , 1956. Biochemical  Eng ineer ing , Academic Press , N . Y . [9] Solomons, G . C . , 1969. M a t e r i a l s and Methods i n Fermentat ion, Academic Press , London and N . Y . pp. 115-131. [10] S t r a s d i n e , G . A . and J . M . M e l v i l l e , 1972. "Salmon-Canning Waste Water as a M i c r o b i a l Growth Medium," Journa l F i s h e r i e s Research Board of Canada, V o l . 29, No. 12. [II] Truong L . T . , 1973. " P o t e n t i a l of Spent S u l f i t e L i q u o r as Raw M a t e r i a l for Product ion of Vi tamin B^„ by C e r t a i n Species of L a c t o b a c i l l u s , P r o p i o n i b a c t e r i a , and Streptomyces," M . A . S c . Thes i s , U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B . C . 113 [12] Burgess, G.H.O., C.L. C u t t i n g , J.A. Lovern, and J . J . Waterman (Eds.), 1965. F i s h Handling and Pr o c e s s i n g , Her Majesty's S t a t i o n e r y O f f i c e , Edinburgh. [13] Camp, A.A., H.T. C a r t r i t e , J.H. Quisenberry, and J.R. Couch, 1955. "Further Information Concerning U n i d e n t i f i e d Chick Growth F a c t o r s , " P o u l t r y Science, V o l . 34, pp. 559-566. [14] M e l l e r , F.H. 1969. "Conversion of Organic S o l i d Wastes i n t o Yeast; an Economic E v a l u a t i o n , " U.S. Dept. of H e a l t h , Education and Welfare. Bureau of S o l i d Waste Management. [15] Keay, L. 1972. "Proteases of the Genus B a c i l l u s , " Proc. IV IFS: Ferment. Technol. Today, pp. 289-298. [16] Cantarow, A. and B. Schepartz, 1954. Biochemistry. W.B. Saunders, P h i l a d e l p h i a , pp. 234-241. [17] Neurath, H., 1964. " P r o t e i n - D i g e s t i n g Enzymes," S c i e n t i f i c American, V o l . 211.6, pp. 68-79. [18] Arima, K., 1964. " M i c r o b i a l Enzyme P r o d u c t i o n , " i n G l o b a l Impacts  of A p p l i e d M i c r o b i o l o g y , M.P. S t a r r , Ed., John W i l l e y & Sons, N.Y. pg. 277. [19] U n d e r k o f l e r , L.A. and R.L. Charles, 1960. " P r o t e o l y s i s by M i c r o b i a l P r o t e i n a s e s , " i n Developments i n I n d u s t r i a l M i c r o b i o l o g y , V o l . 1, Proc. 16th Meeting, S o c i e t y Ind. M i c r o b i o l g y , Plenum P r e s s , N.Y. pp. 125. [20] Stephenson, M., 1939. B a c t e r i a l Metabolism. Longmans, Green & Co., London pp. 122. [21] Matsubara, H. and J . Feder, 1971. "Other B a c t e r i a l , Mold and Yeast Proteases," i n The Enzymes, 3rd ed., V o l . 3, P.D. Boyer, Ed., Academic P r e s s , N.Y. pp. 721. [22] Wang, D.I.C., 1969. "Biochemical Engineering," Chemical Engineering, December 15, Mc Graw H i l l , pp. 108-120. [23] Keay, L., 1971. " M i c r o b i a l Proteases," Process Biochem., V o l . 6, No. 8, pp. 17. [24] Keay, L., M.H. Moselsy, R.S. Anderson, R.J. O'Connor and B.S. W i l d i , 1972. "Production and I s o l a t i o n of M i c r o b i a l Proteases," B i o t e c h n o l & Bioeng. Symp. No. 3, pp. 63-92. [25] De Ley, J . , 1969. " M i c r o b i a l Enzymes and t h e i r I n d u s t r i a l A p p l i c a t i o n s , " i n Symp. on Pharmaceutical Enzymes and Their Assay, U n i v e r s i t y of Ghent, May 24, 1968, R. Ruyssen, Ed., U n i v e r s i t a i r e Pers, Belgium, pp. 95. 114 [26] G i l l e s p i e , D.C. and F.D. Cook, 1965. " E x t r a c e l l u l a r Enzymes from S t r a i n s of Sorangium," Can. J. M i c r o b i o l . , V o l . I I , pp 109-118. [27] Breed, R.S., E.G.O. Murray and N.R. Smith, 1957. Bergey's Manual  of Determinative B a c t e r i o l o g y , '7th ed., The W i l l i a m s and W i l k i n s Co., Ba l t i m o r e , Md. [28] S t a n i e r , R.Y., M. Doudoroff, and E.A. Adelberg, 1963. The M i c r o b i a l World, 2nd ed., P r e n t i c e H a l l , N.J. [29] Carpenter, P.L., 1972. M i c r o b i o l o g y , 3rd ed., W.B. Saunders Co., Toronto. [30] H e n r i c i , A.T. and E.J. Ordal, 1948. The B i o l o g y of B a c t e r i a , 3rd ed., D.C. Heath and Co., Boston. [31] Whitaker, D.R., F.D. Cook, and D.C. G i l l e s p i e , 1965. " L y t i c Enzymes of Sorangium Sp. Some Aspects of Enzyme P r o d u c t i o n i n Submerged C u l t u r e , " Can. J . Biochem., V o l . 43, pp 1927-1933. [32] Katznelson, H., D.C. G i l l e s p i e , and F.D. Cook, 1964. "Studies on the R e l a t i o n s h i p s between Nematodes and Other S o i l Microorganisms. I I I . L y t i c A c t i o n of S o i l Myxobacters on C e r t a i n Species of Nematodes," Can. J . M i c r o b i o l . , V o l . 10, pp 699-704. [33] Whitaker, D.R., 1965. " L y t i c Enzymes of Sorangium Sp. I s o l a t i o n and Enzymatic P r o p e r t i e s of the a-and 8 - L y t i c Proteases," Can. J . Biochem., V o l . 43, pp. 1935-1954. [34] Jurasek, L. and D.R. Whitaker, 1965. " L y t i c Enzymes of Sorangium Sp. A Comparison of Some P h y s i c a l P r o p e r t i e s of the a- and 8 - L y t i c Proteases," Can. J . Biochem., V o l . 43, 1055-1960. [35] Whitaker, D.R., C. Roy, C.S. T s a i , and L. Jurasek, 1965. Can. J . Biochem., V o l . 43, pg 1961-1970. [36] T s a i , C.S., D.R. Whitaker, L. Jurasek, and D.C. G i l l e s p i e , 1965. Can. J . Biochem., V o l . 43, pp. 1971. [37] Whittenbury, R., 1969. " M i c r o b i a l U t i l i z a t i o n of Methane," Process Biochem., V o l . 4, No. 1, pp. 51-56. [38] V i l e n c h i c h , R. and W. Akhtar, 1971. " M i c r o b i o l o g i c a l Synthesis of P r o t e i n s , " Process Biochem., V o l . 6, No. 2 pp. 41-44. [39] S t o l p , H. and M.P. S t a r r , 1965. " B a c t e r i o l y s i s , " Ann. Rev. M i c r o b i o l . , V o l . 19, pp. 79-104. 115 ] Dworkin, M., 1962. " N u t r i t i o n a l Requirements f o r V e g e t a t i v e Growth of Myxococcus Xanthus," J . B a c t e r i d . , V o l . 84, pp. 250-257. ] McDonald, J.C. and J.E. Peterson, 1962. " L i q u i d C u l t u r e of Two Members of the Higher F r u i t i n g Myxobacteria," Mycologia, V o l . 54, pp. 368-373. ] Dworkin, M., 1966. "Biology of the Myxobacteria," Ann. Rev. M i c r o b i o l . , V o l . 20, pp. 75-106. ] M a r t i n , S.M. and V. So, 1969. " S o l u b i l i z a t i o n of Autoclaved Feathers and Wool by Myxobacteria," Can. J . M i c r o b i o l . , V o l . 15, pp. 1393-97. ] Baur, E., 1905. "Myxobakterien - Studien," Arch. P r o t i s t e n k , V o l . 5, pp. 92-121. ] Loebeck, M.E., 1954. "Studies on the T e r r e s t r i a l F r u i t i n g Myxobacteria, Ph.D Thesis U n i v e r s i t y of Washington, S e a t t l e , Washington. ] Noren, B., 1955. "Studies on Myxobacteria. I I I . Organic Factors i n N u t r i t i o n , " B o taniska N o t i s e r , V o l . 108, pp. 81-134. ] Chase, J.M., 1965. " N u t r i t i o n of Some Aquatic Myxobacteria," Master's Thesis, U n i v e r s i t y of Wash., S e a t t l e , Wash. ] Andersen, L.B., 1963. "How to Apply S t a t i s t i c s I n Design of Experiments," CHEMICAL ENGINEERING, McGraw H i l l , N.Y., August 5, pp. 113-116. ] Andersen, L.B., 1963. " F a c t o r i a l Design of Experiments," CHEMICAL ENGINEERING, McGraw H i l l , N.Y., Sept. 2, pp. 99-105. ] S c h u l t z , J.S., Reihard, D., and E. L i n d , 1960. " S t a t i s t i c a l Methods i n Fermentation Development," Ind. Eng. Chem., V o l . 52, No. 10, pp. 827-830. ] Ferguson, D.K., 1972. "Manufacture of Vitamin B- 2^ from S u l f i t e Spent L i q u o r , " M.A.Sc Th e s i s , U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B.C. ] New Brunswick S c i e n t i f i c Co., Inc., 1969. "Operating Manual -Modular Microferm, Bench Top Fermentor, S e r i e s MF-100 and MF-200," Dec. 17, New Brunswick, N.J. ] Gavin, J . J . , 1957. " M i c r o b i o l o g i c a l Process Report. A n a l y t i c a l M i c r o b i o l o g y . I I I . T u r b i d i m e t r i c Methods," Appl. M i c r o b i o l . , V o l . 5, pp. 235-242. ] S o c i e t y of American B a c t e r i o l o g i s t s , 1957. Manual of M i c r o b i o l o g i c a l  Methods, McGraw H i l l , N.Y. 116 [55] Monod, J . 1949. "The Growth of B a c t e r i a l C u l t u r e s , " Ann. Rev. of M i c r o b i o l o g y , V o l . 3, pp. 371. [56] Dubois, M., K.A. G i l l e s , J.K. Hamilton, P.A. Rebers, and F. Smith, 1956. " C o l o r i m e t r i c Method f o r Determination of Sugars and Related Substances," A n a l . Chemistry, V o l . 28, pp. 350. [57] Sumner, J.B., 1925. " D i n i t r o s a l i c y l i c Method f o r Glucose," J . B i o l . Chem., V o l . 65, pp. 393. [58] Ruyssen, R. 1969. "Assay Methods of the F.I.P. Commission on Enzymes," i n Symp. on Pharm. Enzymes and Their Assay, R. Ruyssen, Ed., U n i v e r s i t a i r e Pers, Belgium, pp. 95. [59] Report of the Commission on Enzymes of the U.I.B., 1961. Oxford, Pergamon Press . [60] Anson, M.L. 1938. "The E s t i m a t i o n of Pepsi n , T r y p s i n , Papain, and Cathepsin w i t h Hemoglobin," J . Gen. P h y s i o l . , V o l . 22, pp. 79. [61] F o l i n , 0. and V. C i o c a l t e u , 1927. "On Tyrosine and Tryptophane Determinations i n P r o t e i n s , " J . B i o l . Chem., V o l . 73, pp. 627. [62] Petrova, I.S. and M.M. V i n t s y u n a i t e , 1966. "Determination of P r o t e o l y t i c A c t i v i t y of Enzyme P r e p a r a t i o n s of M i c r o b i a l O r i g i n , " P r i k l . Biokhim. i M i k r o b i o l . ( A p p l i e d Biochemistry and M i c r o b i o l o g y ) , V o l . 2, No. 3, pp. 322-327. [63] Mahler, H.R. and E.H. Cordes, 1971. B i o l o g i c a l Chemistry, 2nd ed., N.Y. Harper & Row. [64] Lowry, D.H., N.J. Rosebrough, A.L. F a r r , and R.J. R a n d a l l , 1951. J . B i o l . Chem., V o l . 193, pp. 265. [65] Laboratory Manual, 1974. Dept. of Biochem., F a c u l t y of Medicine, U n i v e r s i t y of B r i t i s h Columbia. [66] Layne, E., 1957. "Spectrophotometric and T u r b i d i m e t r i c Methods f o r Measuring P r o t e i n s , " i n Methods i n Enzymology, V o l . I l l S.P. Colowick and N.O. Kaplan, Eds., Academic P r e s s , N.Y. pp. 447. [67] A s s o c i a t i o n of O f f i c i a l A g r i c u l t u r a l Chemists, 1965. O f f i c i a l Methods  of A n a l y s i s of the A s s o c i a t i o n of O f f i c i a l A g r i c u l t u r a l Chemists. 10th ed., A s s o c i a t i o n of O f f i c i a l A g r i c u l t u r a l Chemists, Washington, D.C. pp. 744-745. [68] M e r r i l l , A.T. and W. M a n s f i e l d C l a r k , 1928. "Some Conditions A f f e c t i n g the Production of G e l a t i n a s e by Proteus B a c t e r i a , " J . Bact., V o l . 15, pp. 267. 117 ] Haines , R . B . 1931. "The Formation of B a c t e r i a l Proteases , E s p e c i a l l y i n Synthet ic Media ," Biochem. J . , V o l . 25, pp. 1851. See Also : Biochem. J . , V o l . 27, pp. 466. ] Fukumoto, J . and H. Negoro. 1952. Symposia on Enzyme Chem. (Japan), V o l . 7. pp. 8. ] Fukumoto, J . , Yamamoto, and D. Tsuru , 1957. J . A g r . Chem. Soc. Japan, V o l . 31, pp. 425. ] H i c k s , C R . 1964. Fundamental Concepts i n the Design of Experiments , H o l t , Rinehart and Winston, I n c . , N . Y . ] M i l l e r , I . and J . E . Freund, 1965. P r o b a b i l i t y and S t a t i s t i s t i c s for  Engineers , P r e n t i c e - H a l l , N . J . ] S t a n i e r , R . Y . 1942. "The Cytophaga Group : A C o n t r i b u t i o n to the Bio logy of Myxobacter ia ," B a c t e r i o l o g i c a l Reviews, V o l . 6, pp. 143-196. ] Dubos, R . J . 1940. "The Adaptive Product ion of Enzymes by B a c t e r i a , " B a c t e r i o l o g i c a l Reviews, V o l . 4, pp. 1-16. ] Ga le , E . F . 1943. "Factors In f luenc ing the Enzymic A c t i v i t i e s of B a c t e r i a , " B a c t e r i o l o g i c a l Reviews, V o l . 7, pp. 139-173. ] Mandelstam, J . and K . M c Q u i l l e n , E d s . , 1968. Biochemistry of B a c t e r i a l Growth. B l a c k w e l l S c i e n t i f i c P u b l i c a t i o n s , Oxford and Edinburgh. ] McCurdy, H . D . , J r . , 1963. Can. J . M i c r o b i o l . , V o l . 9, pp. 282-85. ] Davis , J . G . and H . J . Rogers, 1939. "The E f f e c t of S t e r i l i z a t i o n Upon Sugars ," Zentr . B a k t e r i o l . , Abt I I , V o l . 101, pp. 102-110. ] Holme, T . and R. Brookes, 1969. "The Inf luence of D i f f e r e n t Energy Sources on B a c t e r i a l Growth and Synthes i s ," i n Fermentation  Advances, D. Perlman, E d . , Academic Press , N . Y . , pp. 145. ] Holme, T . , S. A r v i d s o n , B . Lindholm, and B. Pav lu , 1970. "Enzymes -Laboratory - Scale P r o d u c t i o n , " Process Biochem., V o l . 5, No. 9, pp. 62. ] Sperry , J . A . and L . F . Ret tger , 1915. "The Behaviour of B a c t e r i a towards P u r i f i e d Animal and Vegetable P r o t e i n s , " J . B i o l . Chem., V o l . 20, pp. 445. 118 [83] Rettger, L.F., N. Berman, and W.S. Sturges, 1916. "Further Studies on B a c t e r i a N u t r i t i o n : The U t i l i z a t i o n of P r o t e i n & Non-P r o t e i n N i t r o g e n , " J . Bact., V o l . 1, pp. 15. See Also : J . Bact., V o l . 3, pp. 389. [84] Archer, M.C., J.O. Ragnarsson, S.R. Tannenbaum, and D.I.C. Wang, 1973. "Enzymatic S o l u b i l i z a t i o n of an I n s o l u b l e S u b s t r a t e , F i s h P r o t e i n Concentrate : Process and K i n e t i c C o n s i d e r a t i o n s , " B i o t e c h . & Bioeng., V o l . XV, pp. 181-196. [85] M i h a l y i , E. and W.F. H a r r i n g t o n , 1959. Biochim. Biophys. A c t a , V o l . 36, pp. 449. [86] F i n n , R..K. 1967. " A g i t a t i o n and A e r a t i o n , " i n Biochemical and  B i o l o g i c a l Engineering Science, V o l . 1, N. Blakebrough, Ed., Academic Press, N.Y. [87] Yamada, K., J . Takahasi and H. Okada, 1952. "Fundamental Studies on the Aerobic Fermentation. P a r t 2. Determination of an E m p i r i c a l Formula on the E f f i c i e n c y of Oxygen Supply of Fermentor," J . A g r i c u l t u r a l Chemical Soc. of Japan, V o l . 27, pp. 704-708. [88] Levens, A.S. 1962. Graphics - With an I n t r o d u c t i o n to Conceptual  Design, John Wiley & Sons, N.Y. pp. 316-320. Appendix I EXPERIMENTAL DATA 120 Table AI.l EFFECT OF GLUCOSE CONCENTRATION (FILTERED CFS) GLUCOSE CONCEN- TIME (days) PH DRY CELL WEIGHT TYROSINE PROTEOLYTIC ACTIVITY TRATION (gm/&) (ug/ml) (units/10cc) 2 7.9 0.70 96.0 4 .24 0% 4 8.9 0.30 90.5 4.00 6 8.9 0.32 74.0 3.27 8 8.9 0.38 60.3 2.67 2 7.5 1.38 60.2 2.66 0.5% 4 8.3 1.16 131.0 5.79 6 8.4 0.59 135.0 5.97 8 8.9 0.53 119.5 5.28 2 7.42 1.20 46.8 2.07 4 7.66 1.52 135.0 5.97 1.0% 6 7.80 2 . 44 202.5 8.96 8 8.25 1.28 204.5 9.04 10 8.20 0.80 164.5 7.18 2 7.32 1.18 42.1 1.86 4 7.31 1.76 100.0 4.42 2% 6 6.70 2.60 183.0 7.98 8 5.80 3.20 180.0 7.85 10 7.25 3.17 118.5 5.08 12 7.60 - 128.0 5.66 2 7.25 1.10 40.0 1.77 3% 4 6.71 1.60 92.5 4.09 6 5.95 2.28 127.0 5.60 8 4.10 2.30 20.3 0.90 2 7.20 1.06 35.8 1.58 4% 4 6.21 1.60 84.0 3.71 6 5.25 2.11 92.0 4.07 8 3 .40 1.87 6.0 0.26 Inoculum age : 24 hours -Dry c e l l weight at t = 0 : 0.0085 gm/l 121 Table AI.2 EFFECT OF GLUCOSE CONCENTRATION (UNFILTERED CFS) GLUCOSE TIME pH TYROSINE PROTEOLYTIC ACTIVITY CONCENTRATION (days) (yg/ml) ( u n i t s / l O c c ) 0% 0.5% 1.0% 2.0% 3.0% 4.0% 3 5 8 3 5 8 10 12 3 5 8 10 12 3 5 8 10 12 3 5 8 10 12 3 5 8 10 12 8.0 8.75 8.90 7.55 8.20 9.10 8.90 8.75 7.45 7.40 8.12 8.50 8.61 7.25 7.0 6.2 6.2 7.61 7.15 6.60 6.10 6.00 5.10 7.09 6.40 6.15 5.95 33.0 37.8 31.5 33.0 59.2 80.5 92.5 77.0 26.2 63.5 107.5 129.5 128.0 26.2 74.2 100.0 102.5 103.0 21.5 54.0 66.9 73.0 42.8 20 48.1 59.6 44.0 1.46 1.67 1.39 1.46 2.62 3.56 4.09 3.40 1.158 2.807 4.752 5.724 5.658 1.158 3.280 4.420 4.531 4.56 0.950 2.387 2.957 3.227 1.892 0.884 2.126 2.634 1.945 Table AI .3 EFFECT OF CFS CONCENTRATION I n i t i a l pH F i n a l pH 7.0 5.9 DILUTION : 1:100 v / v DILUTION : 2:100 v / v DRY PROTEOLYTIC DRY PROTEOLYTIC TIME WEIGHT TYROSINE ACTIVITY WEIGHT TYROSINE ACTIVITY (hrs) (gmA) (yg/ml) ( u n i t s / l O c c ) (gm/*) (ug/ml) (uni ts / lOOcc) 7 0.12 1.2 0.053 0.16 1.2 0.053 19 0.86 18.9 0.835 0.92 9.4 0.415 30 1.06 39.0 1.724 1.00 24.8 1.096 43 1.20 73.3 3.240 1.22 45.0 1.989 55 1.68 100.0 4.420 1.36 91.7 4.053 67 1.96 122.5 5.414 1.58 94.0 4.155 2.02 110.5 4.884 1.78 146.0 6.453 «* 2.18 96.0 4.243 1.96 162.5 7.183 102^ 4 2.02 91.3 4.035 2.18 209.5 9.260 i 4 2.26 73.0 3.227 2.46 252.5 11.161 1 2 5 | 2.08 58.6 2.590 2.52 271.5 12.000 138 2.12 60.0 2.652 2.44 265.5 11.735 150 1.74 56.8 2.511 2.18 249.0 11.006 I n i t i a l pH F i n a l pH 7.0 8.0 h- 1 CO N3 Table AI.3 (Contd) EFFECT OF CFS CONCENTRATION DILUTION : 3:100 v / v DILUTION : 4:100 v / v TIME (hrs) DRY WEIGHT (gm/Jl) TYROSINE (ug/ml) PROTEOLYTIC ACTIVITY ( u n i t s / l O c c ) DRY WEIGHT (gm/Jl) TYROSINE (ug/ml) PROTEOLYTIC ACTIVITY (un i t s / lOcc ) 7 0.16 0.5 0.022 0.20 1.6 0.071 19 0.84 7.1 0.314 0.90 4.5 0.199 30 0.98 15.6 0.690 0.98 12.0 0.530 43 1.30 32.0 1.414 1.34 21.8 0.964 55 1.64 56.0 2.475 1.52 31.8 1.406 67 1.84 72.5 3.205 1.80 63.3 2.798 1.86 86.5 3.823 1.90 66.7 2.948 90^  4 2.00 107.0 4.729 2.08 78.3 3.461 102^  4 2.16 127.0 5.613 2.16 94.0 4.155 2.44 131.5 5.812 2.28 99.7 4.407 125y 2.50 150.0 6.630 2.38 112.3 4.964 138 2.44 172.5 7.625 2.34 138.7 6.130 150 2.12 172.5 7.625 2.34 127.0 5.613 I n i t i a l pH : 7.0 F i n a l pH : 8.4 I n i t i a l pH : 7. F i n a l pH : 8. 0 32 Table AI .3 (Contd) EFFECT OF CFS CONCENTRATION DILUTION : 5:100 v / v DILUTION : 6:100 v / v DRY PROTEOLYTIC DRY PROTEOLYTIC TIME WEIGHT TYROSINE ACTIVITY WEIGHT TYROSINE ACTIVITY (hrs) (gm/Jc.) (yg/ml) ( u n i t s / l O c c ) ( g m / £ ) (yg/ml) (un i t s / lOcc ) 7 0.14 0.07 0.003 0.06 - -19 0.98 3.5 0.155 0.94 3.3 0.146 30 1.10 4.2 0.186 1.06 4.5 0.199 43 1.38 10.3 0.455 1.34 7.8 0.345 55 1.64 13.6 0.601 1.64 7.8 0.345 67 1.92 21.4 0.946 1.80 15.0 0.663 2.04 26.9 1.189 1.88 19.8 0.875 9 0 | 2.10 29.5 1.304 2.18 19.5 0.862 1 0 2 ± 2.06 - - 2.28 20.9 0.924 2.30 41.5 1.834 2.52 11.8 -1 2 5 | -2.22 40.5 1.790 2.54 29.1 1.286 138 2.20 53.4 2.360 2.54 28.4 1.255 150 2.24 51.2 2.263 2.42 29.3 1.295 I n i t i a l pH : 7.0 F i n a l pH : 8.40 I n i t i a l pH : 7.0 F i n a l pH : 8.40 Table AI.4 EFFECT OF HEXOSE SUGARS (GLUCOSE) TIME (hrs) DRY WEIGHT (gm/Jl) GLUCOSE CONCENTRATION (gm/A) TYROSINE (yg/ml) PROTEOLYTIC ACTIVITY ( u n i t s / l O c c ) 0 0.0085 10.4 — — 8 0.24 10.4 - -4 0.76 - 3.5 0.16 30 1.02 9.2 13.1 0.58 42 1.36 8.45 23.8 1.05 4 - 7.50 37.2 1.64 70 1.38 6.55 59.8 2.64 80 1.50 5.40 76.8 3.40 ioo | 1.74 3.10 124.5 5.50 115 2.03 1.40 175.0 7.74 125 2.10 0.60 202.5 8.95 139 2.06 0.50 202.5 8.95 149 - 0.50 209.0 9.24 I n i t i a l pH : 7.0 F i n a l pH : 8.12 Inoculum age = 24 hours Dry c e l l weight at t = 0 : 0.0085 gm/£ H-1 ro Table AI .4 (Contd) EFFECT OF HEXOSE SUGARS GALACTOSE MANNOSE TIME (hrs) DRY WEIGHT ( g m / £ ) TYROSINE (yg/ml) PROTEOLYTIC ACTIVITY ( u n i t s / l O c c ) DRY WEIGHT ( g m / £ ) TYROSINE (yg/ml) PROTEOLYTIC ACTIVITY ( u n i t s / l O c c ) 8 0.30 - - 0.18 - -4 0.74 3.6 0.159 0.70 2.1 0.093 30 1.09 20.8 0.919 1.00 16.3 0.720 42 1.25 36.8 1.626 1.54 34.6 1.529 4 1.22 41.6 1.839 1.67 42.8 1.892 70 1.52 48.2 2.130 2.04 50.8 2.245 80 - 47.8 2.113 - 49.6 2.192 ioo| 1.48 56.5 2.497 2.38 71.3 3.151 115 1.46 58.8 2.599 2.54 115.5 5.105 125 1.46 63.8 2.820 2.48 135.7 5.998 139 1.50 69.0 3.00 2.55 111.5 -149 1.40 63.8 2.820 2.48 137.5 6.078 I n i t i a l pH : 7.00 F i n a l pH : 7.30 I n i t i a l pH : 7.00 F i n a l pH : 8.12 127 Table AI.5 EFFECT OF PENTOSES AND STARCH XYLOSE ARABINOSE TIME (hrs) DRY WEIGHT (gm/ic.) TYROSINE (yg/ml) PROTEOLYTIC ACTIVITY (units/lOcc) DRY WEIGHT (gm/£) TYROSINE (yg/ml) PROTEOLYTIC ACTIVITY (units/lOcc) 8 0 - - 0.02 - -4 0.32 1.3 0.057 0.50 1.9 0.084 30 0.85 10.0 0.442 0.82 22.4 0.990 42 0.94 38.3 1.693 0.92 39.4 1.741 4 0.70 47.0 2.077 0.60 44.0 1.945 70 0.54 50.8 2.245 0.70 43.8 1.936 80 0.40 48.0 2.122 0.50 41.6 1.839 ioo | - 47.0 2.077 - 40.0 1.768 I n i t i a l pH : 7.00 Final pH : 8.90 I n i t i a l pH : 7.00 Final pH : 8.90 STARCH 8 0.42 - -4 0.68 7.3 0.323 30 1.25 34.6 1.529 42 1.31 58.1 2.568 4 0.95 68.0 3.006 70 0.85 68.0 3.006 80 0.57 70.6 3.121 ioo | - 68.8 3.041 I n i t i a l pH : 7.00 Final pH : 9.10 128 Table AI .6 EFFECT OF INOCULUM AGE (1) Inoculum Age : 16 hours TIME DRY GLUCOSE PROTEOLYTIC (hrs) WEIGHT CONCENTRATION l i K U S l I N f c i ACTIVITY (gm/A) (grn/i) (yg/ml) ( u n i t s / l O c c ) 0 0.007 10.3 - -8 0.30 10.3 - -20 0.76 10.3 1.7 0.075 32 1.04 9.4 13.3 0.588 45 1.32 8.45 22.6 0.999 56 1.06 7.70 35.2 1.556 68 1.04 6.65 48.8 2.157 92 1.88 3.97 106.7 4.716 10 ly 2.21 2.40 123.5 5.459 115j 2.44 0.78 165.5 7.315 126 2.32 0.50 180.0 7.956 140 2.25 0.45 190.0 8.398 150 2.00 0.42 192.5 8.508 I n i t i a l pH : 7.0 F i n a l pH : 8.1 Table AI.6 (Contd) EFFECT OF INOCULUM AGE (2) Inoculum age : 20 hours 129 TIME DRY (hrs) WEIGHT (gm/A) GLUCOSE CONCENTRATION (gm/4) TYROSINE PROTEOLYTIC ACTIVITY (yg/ml) ( u n i t s / l O c c ) 0 0.0085 10.3 - -8 0.18 10.3 - 0 20 0.86 10.0 3.2 0.141 32 1.14 9.2 12.6 0.557 45 1.40 7.8 22.2 0.981 56 1.20 7.45 29.4 1.300 68 1.10 6.00 47.0 2.077 92 1.82 3.63 104.3 4.610 10l| 2.06 2.46 124.5 5.503 n s f 2.26 0.70 170.0 7.514 126 2.28 0.52 180.5 7.978 140 2.03 0.49 190.0 8.398 150 1.80 0.44 187.0 8.265 I n i t i a l pH F i n a l pH 7.0 8.1 130 Table AI .7 7-LITRE FERMENTATION - NB - GLUCOSE RUN (A g i t a t o r speed : 400 rpm; A i r flow r a t e : 1 &/min) TIME pH DRY PROTEOLYTIC GLUCOSE (hrs) WEIGHT rYKOSlNi. ACTIVITY CONCENTRATION (gm/£) (yg/ml) ( u n i t s / l O c c ) (gm/£) 0 8 12 15 18 21 34 41 45 7.00 7.10 7.40 7.50 7.62 7.66 7.87 8.35 8.22 8.45 0.023 0.28 0.34 0.83 0.71 0.50 0.37 0.12 0.22 0.07 4.0 8.2 14.3 25.5 43.6 50.2 51.5 0.177 0.362 0.632 1.127 1.927 2.219 2.276 8.1 8.1 7.8 8.1 7.6 7.0 6.8 6.8 7.4 131 Table AI.8 7-LITRE FERMENTATION - CFS RUN NO. 1 (Ag i ta tor speed : 300 rpm-; A i r flow rate : 1 Jl/min) TIME DRY TYRn« JTOT PROTEOLYTIC GLUCOSE (hrs) P WEIGHT U K U & i n r , ACTIVITY CONCENTRATION (gm/Jl) (yg/ml) ( u n i t s / l O c c ) (gm/Jl) 0 6.82 0.023 - - 10.00 3 6.92 0.090 - - -4 7.38 0.40 - - -14 7.53 0.57 1.5 0.066 9.40 16 7.62 0.66 - - -18 7.70 0.58 5.4 0.239 9.60 20j 7.75 0.65 - - -24 7.80 0.65 7.6 0.336 9.20 27 7.83 0.65 - - -35 7.85 0.51 21.0 0.928 8.70 40 7.80 0.52 - - -7.85 0.48 39.0 1.724 7.90 49 7.92 0.48 54.5 2.409 7.40 4 7.78 0.46 75.0 3.315 5.80 4 7.70 0.72 114.0 5.039 4.10 91 7.70 0.74 126.0 5.569 3.50 97 7.62 0.77 137.5 6.078 2.70 109y 7.52 0.94 167.5 7.400 1.70 3 113y-4 7.48 1.00 184.2 8.132 0.99 7.82 0.95 199.5 8.012 0.44 13 l y -4 8.10 0.88 197.0 8.702 0.39 134 8.10 0.80 200.0 8.819 0.37 132 Table AI .9 7-LITRE FERMENTATION - CFS RUN NO. 2 (Ag i ta tor speed : 400 rpm; A i r flow rate L 1 Jl/min) TIME DRY PROTEOLYTIC GLUCOSE (hrs) P WEIGHT iiRUblMt, ACTIVITY CONCENTRATION (gm/Jl) (yg/ml) ( u n i t s / l O c c ) (gm/Jl) 0 7.0 0.023 - — 9.00 6 7.1 0.15 - - 9.00 15 7.85 0.59 4.8 0.212 8.95 4 . 7 . 8 5 0.66 9.5 0.420 8.55 4 7.95 0.66 21.5 0.950 7.85 23 7.92 0.75 30.2 1.335 7.85 29 8.20 0.46 37.0 1.635 6.90 41 8.22 0.49 54.0 2.387 6.25 54 8.25 0.61 93.5 4.133 5.00 68 8.12 0.71 126.5 5.591 4.00 8.00 0.80 151.0 6.674 3.27 84 7.75 0.81 190.0 8.398 2.20 94 7.61 0.85 211.5 9.348 1.64 102 7.36 0.78 207.5 9.172 0.77 I l l y 7.83 0.82 217.0 9.591 0.50 1 1 6 | 7.98 0.83 225.0 9.945 0.45 133 Table A L I O 7-LITRE FERMENTATION - CFS RUN NO. 3 ( A g i t a t o r speed : 500 rpm; A i r flow r a t e : 1 £/min) TIME DRY PROTEOLYTIC GLUCOSE (hrs) P WEIGHT IxRUbllNE ACTIVITY CONCENTRATION (gm/£) (ug/ml) ( u n i t s / l O c c ) (gm/£) 0 6.88 0.023 - - 9.9 5 7.20 0.270 - - 9.2 7 7.42 0.49 - - 9.9 9 7.65 0.86 1.0 0.044 8.9 4 8.15 0.81 15.5 0.685 7.95 22 8.15 0.91 26.2 1.158 7.50 25 8.15 1.08 39.0 1.724 6.90 32 8.03 1.25 60.5 2.674 5.30 42 7.70 1.20 127.0 5.613 3.53 49± 7.40 1.18 183.5 8.111 2.20 4 7.08 1.29 211.5 9.348 1.48 4 6.80 1.10 231.0 10.210 1.07 6&| 6.92 1.25 252.5 11.14 0.54 77 7.20 1.20 238.0 10.52 0.46 81 7.28 1.20 235.5 10.41 0.42 4 7.33 1.20 235.5 10.41 0.37 94 7.35 1.21 231.0 10.21 0.37 134 Table A I . l l 7-LITRE FERMENTATION -• CFS RUN NO. 4 (Ag i ta tor speed : 600 rpm; A i r flow rate : 1 Jl/min) TIME (hrs) PH DRY WEIGHT (gm/A) TYROSINE (yg/ml) PROTEOLYTIC ACTIVITY ( u n i t s / l O c c ) GLUCOSE CONCENTRATION (gm/A) 0 6.9 0.023 - - 8.7 4 7.0 0.18 - - 8.7 8 7.25 0.45 - - 8.7 10 7.45 0.96 0.7 0.031 8.5 20 7.70 1.20 20.7 0.915 6.2 23 7.65 1.44 30.3 1.340 5.6 26 7.60 1.55 45.6 2.016 5.2 28 7.50 1.56 59.2 2.617 4.4 35 7.20 1.62 122.5 5.415 3.2 44 6.15 1.50 172.5 7.624 1.5 48 5.72 1.56 171.0 7.558 0.8 «! 5.85 1.65 177.0 7.823 0.55 "! 6.05 1.59 178.5 7.890 0.49 68| 6.40~ 1.55 184.0 8.133 0.47 76 6.70 1.58 177.5 7.846 0.45 83± 6.85 1.54 178.5 7.890 0.43 135 Table AI.12 7-LITRE FERMENTATION - CFS RUN NO. 5 ( A g i t a t o r speed : 750 rpm; A i r flow r a t e : 1 X/min) TIME (hrs) pH DRY WEIGHT (gm/Jl) TYROSINE (yg/ml) PROTEOLYTIC ACTIVITY ( u n i t s / l O c c ) GLUCOSE CONCENTRATION (gm/Jl) 0 6.80 0.023 - - 9.6 5 6.98 0.120 - - 9.4 7.30 0.57 0.7 0.031 9.2 10 7.40 1.06 1.0 0.044 9.0 12 7.44 1.40 8.0 0.354 8.65 14 7.65 1.51 8.2 0.362 7.65 16 7.70 1.50 14.8 0.654 7.05 18 7.62 1.72 15.8 0.698 6.55 20 7.50 1.93 23.0 1.017 5.80 24 7.40 1.97 46.2 2.042 4.70 26 7.22 2.23 68.0 3.006 4.00 29— y2 7.00 2.05 118.6 5.242 2.87 32— J 2 6.60 2.14 135.5 5.989 1.84 41 6.08 1.93 152.0 6.718 0.82 45 6.25 2.01 157.0 6.939 0.73 50 6.75 1.99 157.0 6.939 0.72 54| 6.82 1.83 152.5 6.741 0.67 57 6.90 1.86 157.0 6.939 0.65 136 T a b l e AI.13 7-LITRE FERMENTATION - CFS RUN NO. 6 ( A g i t a t o r speed : 400 rpm; A i r f l o w r a t e : 2 l/mln) ME DRY PROTEOLYTIC GLUCOSE r s ) P WEIGHT ACTIVITY CONCENTRATION (gm/2.) (yg/ml) ( u n i t s / l O c c ) (gm/il) 0 7.20 0.023 0 0 10.55 8y - 0.55 0.7 0.031 9.90 18 8.02 1.12 15.0 0.663 8.20 22 8.02 1.40 29.5 1.304 6.80 3 l | 7.70 1.55 89.0 3.934 4.27 42 7.22 1.44 146.5 6.475 2.12 47 6.75 1.16 175.0 7.735 1.05 56 6.80 1.10 175.0 7.735. 0.60 68 7.30 1.27 172.5 7.624 0.51 137 Table AI.14 7-LITRE FERMENTATION - CFS RUN NO. 7 (Agitator speed : 500 rpm; Air flow rate : 2 2,/min) TIME DRY PROTEOLYTIC GLUCOSE (hrs) P WEIGHT l ^ K U b l N H ACTIVITY CONCENTRATION (gm/Jl) (yg/ml) (units/lOcc) (gm/Jl) 0 6.70 0.023 - - 9.75 8 7.30 0.35 - - 8.15 10 7.50 0.73 - - 7.80 12 7.70 1.34 5.2 0.230 8.00 14 7.70 1.51 11.8 0.522 7.50 16 7.80 1.55 18.5 0.818 6.70 19 7.90 - 22.2 0.981 6.35 22 7.88 1.53 28.5 1.260 6.00 25 7.90 1.50 32.0 1.414 4.63 33 7.70 1.52 74.8 3.300 3.10 37 7.50 - 110.0 4.862 2.50 40 7.38 - 152 6.718 1.70 45 6.90 1.42 223 9.857 0.88 4 6.90 1.33 205 9.061 0.58 58 7.10 - 229 10.122 0.52 62 7.20 - 203 8.973 0.56 66 7.30 — 220 9.724 0.49 138 Table AI.15 SHAKE FLASK FERMENTATION - GLUCOSE DEFICIENT MEDIUM TIME DRY CELL WEIGHT PROTEOLYTIC ru \ t /ON ACTIVITY (hrs) (gm/£) units/lOcc 0 0.008 0 8 0.41 .009 20 0.88 .559 32 1.00 2.20 45 0.83 2.85 56 0.40 2.78 139 Appendix I I Analysis Procedures : 11.1 Measurement of Glucose Concentration 11.2 Measurement of Proteolytic Activity 11.3 Protein Estimation with the Biuret Reagent 140 Appendix II I I . 1 Measurement of Glucose Concentrat ion I I . 1 . 1 Phenol - S u l f u r i c Ac id Reagent Method [56] Reagents : (1) S u l f u r i c A c i d - Reagent grade 95.5%, conforming to ACS s p e c i f i c a t i o n s , s p e c i f i c g r a v i t y 1.84. (2) Phenol - 5% s o l u t i o n i n water. Apparatus : (1) Fast d e l i v e r y 5-ml p i p e t , to d e l i v e r 5 ml of concentrated s u l f u r i c a c i d i n 10 to 20 seconds. Can be prepared by c u t t i n g a p o r t i o n o f f the t i p of a standard 5 ml p i p e t . (2) Set of tubes with i n t e r n a l diameter between 16 and 20 mm. (3) Spec tron ic 20 tubes or a set of matched co lor imeter tubes. Procedure : Standard Curve (1) Prepare 1 ml glucose s o l u t i o n s conta in ing between 10 and 70 ug of g lucose . (2) Add 1 ml of 5% phenol s o l u t i o n to each. (3) Add 5 ml of concentrated t^SO^ r a p i d l y ; the stream of ac id i s d i r e c t e d against the l i q u i d surface rather than the s ide of the 1 4 1 t e s t t u b e i n o r d e r t o o b t a i n g o o d m i x i n g . ( 4 ) A l l o w t o s t a n d f o r 10 m i n u t e s a t r o o m t e m p e r a t u r e . ( 5 ) S h a k e a n d p l a c e i n w a t e r b a t h s e t a t 30°C f o r 10 t o 20 m i n u t e s . ( 6 ) T r a n s f e r s l o w l y t o c o l o r i m e t e r t u b e s a n d m e a s u r e t h e a b s o r b a n c e a t 4 9 0 my i n s p e c t r o n i c 2 0 . t o t h e d a t a , a s s h o w n i n F i g u r e A l l . 3 6 . U n k n o w n R e p e a t s t e p s 2 t o 6 w i t h u n k n o w n s a m p l e , a n d o b t a i n t h e s u g a r c o n c e n t r a t i o n f r o m F i g u r e A l l . 3 6 . I I . 1 . 2 D i n i t r o s a l i c y l i c M e t h o d [ 5 7 ] R e a g e n t s : D i n i t r o s a l i c y l i c a c i d r e a g e n t p r e p a r e d a s f o l l o w s : T o 3 0 0 m l o f 4 . 5 p e r c e n t s o d i u m h y d r o x i d e , a n d 8 8 0 m l o f 1 p e r c e n t d i n i t r o s a -l i c y l i c a c i d a d d 2 5 5 g o f R o c h e l l e s a l t . T o 10 g o f c r y s t a l l i n e p h e n o l , a d d 2 2 m l o f 10 p e r c e n t s o d i u m h y d r o x i d e . A d d w a t e r t o d i s s o l v e . D i l u t e t o 1 0 0 m l a n d m i x . To 69 m l o f t h i s s o l u t i o n a d d 6 . 9 g o f s o d i u m b i s u l f i t e a n d a d d t o t h e d i n i t r o s a l i c y l i c a c i d s o l u t i o n . M i x w e l l u n t i l a l l o f t h e R o c h e l l e s a l t h a s d i s s o l v e d . K e e p t i g h t l y s t o p p e r e d i n w e l l f i l l e d b o t t l e s . T h e r e a g e n t w i l l l a s t f o r a t l e a s t o n e y e a r . T h r e e m l o f t h e r e a g e n t s h o u l d c o n t a i n b y t i t r a t i o n , w i t h ( 7 ) P l o t A '490 v e r s u s y g g l u c o s e a n d m a k e a l e a s t s q u a r e f i t 142 Figure A l l . 3 6 GLUCOSE TESTS STANDARD CURVES G L U C O S E , 143 phenolphthale in as i n d i c a t o r , the equivalent of 5 to 6 ml of tenth normal sodium hydrox ide . Apparatus : (1) Set of test tubes with i n t e r n a l diameter between 16 and (2) B o i l i n g water ba th . (3) Spectronic 20 tubes or a set of matched co lor imeter tubes. Procedure : Standard Curve (1) Prepare 1 ml glucose so lu t ions conta in ing between 0.04 (2) Add 3 ml of the d i n i t r o s a l i c y l i c a c i d reagent to each, mix (3) Capped and heat for 5 minutes i n the b o i l i n g water . (4) Cool immediately i n running water for 3 minutes. (5) Transfer to co lor imeter tubes and measure absorbance at 20 mm. and 0.3 mg of g lucose . w e l l . 520 my. (6) P l o t A 520 versus mg of glucose and make a l e a s t square f i t to the data , as shown i n Figure A l l . 3 6 . 144 Unknown Repeat steps 2 to 5 with the unknown sample, and obta in the sugar concentrat ion from F igure A l l . 3 6 . I I . 2 . Measurement of P r o t e o l y t i c A c t i v i t y [62] Reagents : (1) 5% s o l u t i o n of t r i c h o l o a c e t i c a c i d (CC13C00H) (2) 6% s o l u t i o n of sodium carbonate, ^200^ (3) F o l i n ' s reagent prepared as fol lows : 100 gm of sodium tungstate and 25 gm of sodium molybdenate are put i n t o a 2 - l i t r e round -bottomed f l a s k f i t t e d with a r e f l u x condenser wi th a ground - g lass j o i n t . 700 ml of water, 50 ml of 85% orthophosphoric a c i d (sp. g r . 1.689), and 100 ml of concentrated h y d r o c h l o r i c a c i d are added. The s o l u t i o n i s b o i l e d for 10 h r . A f t e r c o o l i n g , 150 gm of l i t h i u m s u l f a t e , 50 ml of water, and s e v e r a l (5-6) drops of bromine water are added. The open f l a s k i s heated u n t i l the excess bromine evaporates . The s o l u t i o n should then be ye l low. I f the s o l u t i o n i s green, the treatment with bromine water i s repeated. The cooled s o l u t i o n i s brought to 1 l i t r e with water. The reagent should be 2N i n a c i d . The a c i d concentrat ion i s checked by t i t r a t i o n of t e n f o l d - d i l u t e d F o l i n ' s reagent with 0 .IN NaOH s o l u t i o n using phenolptha le in as an i n d i c a t o r . The prepared reagent i s s tored i n a dark b o t t l e . The working s o l u t i o n i s prepared before the a c t i v i t y determinations by adding four volumes of water to one volume of F o l i n ' s 145 reagent. Note that the concentrated F o l i n ' s reagent may be obtained commercially from F i s h e r S c i e n t i f i c Co. (4) 0.025M t r i s - H C l B u f f e r , pH 8.5 prepared as f o l l o w s : P i p e t t e 25 ml of 0.2M stock t r i s (24.24 gm of t r i s (hydroxymethyl) aminomethane i n 1000 ml of H^O) i n t o 200 ml v o l u m e t r i c f l a s k . Add 0.1N HC1 to i t u n t i l the pH f a l l s to 8.5. Make up to 200 mis w i t h water. (5) 2% Casein s o l u t i o n prepared as f o l l o w s : Suspend 2 gm of "Hammarsten" case i n i n about 45 mis of the 0.025M t r i s - H C l b u f f e r . S t i r v i g o r o u s l y w i t h a magnetic s t i r r e r . The pH of the suspension drops to about 7; add a s m a l l amount of 6% sodium carbonate s o l u t i o n to e l e v a t e the pH to about 8 so as to f a c i l i t a t e the d i s s o l u t i o n of the c a s e i n . Heat the suspension i n a water bath at 65°-70°C t i l l the s o l i d p a r t i c l e s d i s s o l v e . A f t e r c o o l i n g , b r i n g the pH of the s o l u t i o n to 8.5 and make up to 100 mis w i t h the t r i s — H C 1 b u f f e r . (6) Tyrosine s o l u t i o n : 10 mg of t y r o s i n e i s d i s s o l v e d i n 5 ml of 0.1N a l k a l i , t r a n s f e r r e d to a 25-ml measuring f l a s k , and brought to the mark w i t h water. This s o l u t i o n , c o n t a i n i n g 400 yg of t y r o s i n e per ml, i s used to prepare s o l u t i o n s of lower c o n c e n t r a t i o n c o n t a i n i n g 5 to 100 yg of t y r o s i n e per ml. Procedure Co n s t r u c t i o n of C a l i b r a t i o n Curve 146 One ml batches of tyros ine so lu t ions of d i f f e r e n t concentrat ion are taken and to these are added 4 ml of 6% sodium carbonate s o l u t i o n and 1 ml of f i v e - f o l d - d i l u t e d F o l i n ' s reagent. These so lu t ions are immediately mixed and l e f t for the c o l o r to develop - for 30 min at room temperature (20 to 2 5 ° C ) . A c o n t r o l i s s imultaneously set up for the reagents; i n the c o n t r o l the tyros ine s o l u t i o n i s replaced by water . The depth of c o l o r i s determined on a photocolorimeter at wavelength 660 mu i n co lor imeter tubes. P l o t the data and draw a l e a s t square s t r a i g h t l i n e through the p o i n t s , as shown i n F i g u r e A l l . 3 7 . Determination of A c t i v i t y (1) B r i n g 2 mis of 2% case in s o l u t i o n and 1 ml of enzyme s o l u t i o n ( cu l ture f i l t r a t e ) , i n separate tes t tubes, to 3 0 ° C . (2) Add the case in s o l u t i o n to the enzyme s o l u t i o n , mixed, and immediately s t a r t stop watch. Incubate for 10 mins at 3 0 ° C . (3) Stop the r e a c t i o n by adding 5 ml of 5% t r i c h l o r o a c e t i c ac id s o l u t i o n as r a p i d l y as p o s s i b l e ; mixed and placed i n 3 0 ° C bath for about 2 to 5 minutes. (4) F i l t e r the mixture through Whatman #3 f i l t e r paper, to remove the white p r e c i p i t a t e . (5) Determine the amount of tyros ine i n the f i l t r a t e us ing the method o u t l i n e d i n "Construct ion of C a l i b r a t i o n Curve" - 1 ml of f i l t r a t e i s used ins tead of 1 ml of tyros ine s o l u t i o n . 147 F i g u r e A l l . 3 7 T Y R O S I N E T E S T S T A N D A R D C U R V E 148 (6) A c o n t r o l i s s imultaneously set up by adding f i r s t 5 ml of t r i c h l o r o a c e t i c a c i d to the enzyme s o l u t i o n and then 2 ml of case in s o l u t i o n . The s o l u t i o n i s mixed, f i l t e r e d , and 1 ml of the f i l t r a t e ins tead of 1 ml of water i s used as the blank i n the tyros ine assay. C a l c u l a t i o n of a c t i v i t y of enzvme preparat ions In th is method the enzyme u n i t i s def ined ( fo l lowing the recommendations of the Enzyme Commission of the Inter - Union of Biochemistry) as the amount which i n one minute forms p r o t e o l y s i s products which are not p r e c i p i t a t e d wi th t r i c h l o r o a c e t i c a c i d and contain one microequalent of t y r o s i n e . Therefore the number of enzyme un i t s per ml of the i n v e s t i g a t e d s o l u t i o n w i l l be : E = (a x 8)/(181 x 10) = a x 0.00442 . Where E i s the enzyme u n i t (yeq. t y r o s i n e / m l . min); a i s the number of ug of tyros ine found from the c a l i b r a t i o n curve; 8 i s the d i l u t i o n fac tor a f t er p r e c i p i t a t i o n with t r i c h l o r o a c e t i c a c i d ; 181 i s the microgram molecular weight of tyros ine and 10 i s the p r o t e o l y s i s per iod i n minutes. I I . 3 P r o t e i n Es t imat ion with the B i u r e t Reagent [66] Reagents (1) Standard s o l u t i o n of Bovine Serum Albumin (BSA). D i s so lve 0.25 gm of c r y s t a l l i n e BSA i n 25 mis of H^O. This s o l u t i o n , conta in ing 10 mg of BSA per ml , i s used to prepare so lu t ions of lower concentrat ion , 149 c o n t a i n i n g 1 to 10 mg/ml. (2) B i u r e t Reagent (may be obtained commercially from BBL) : D i s s o l v e 1.50 g of c u p r i c s u l f a t e (CuSO^.SI^O) and 6.0 g of sodium potassium t a r t r a t e (NaKC.H 0 .4H 0) i n 500 ml of water. Add, w i t h 4 4 6 2 constant s w i r l i n g , 300 ml of 10% sodium hydroxide. D i l u t e to 1 l i t r e w i t h water, and s t o r e i n a p a r a f f i n - l i n e d b o t t l e . This reagent should keep i n d e f i n i t e l y but must be discarded i f , as a r e s u l t of contamination or of f a u l t y p r e p a r a t i o n , i t shows signs of d e p o s i t i n g any b l a c k or reddish p r e c i p i t a t e . Procedure To 1.0 ml of a s o l u t i o n c o n t a i n i n g 1 to 10 mg of p r o t e i n per ml add 4.0 ml of b i u r e t reagent, mix by s w i r l i n g , and al l o w to stand f o r 30 minutes at room temperature (20 to 25°C). Measure the per cent transmission at 540 my, w i t h the Spect r o n i c 20, against a blank c o n s i s t i n g of 4.0 ml of b i u r e t reagent plus 1.0 ml of water. The c o n c e n t r a t i o n of p r o t e i n i n the sample i s obtained by reference to a c a l i b r a t i o n curve p r e v i o u s l y e s t a b l i s h e d w i t h a c l e a r s o l u t i o n of serum p r o t e i n (Figure A l l . 3 8 . ) 151 Appendix I I I COMPUTATION OF SUM OF SQUARES The treatment sum of squares* i s subdivided i n t o the three main e f f e c t sums of squares SSA, SSB, SSC, the three two-way i n t e r a c t i o n  sums of squares SS(AB), SS(AC), and SS(BC), and the three-way i n t e r -ac t ion sum of squares SS(ABC). To f a c i l i t a t e the c a l c u l a t i o n of these sums of square the fo l lowing two-way tables of t o t a l s and subtota l s were constructed : B A 1 2 3 T o t a l 1 6.46 6.18 4.14 16.78 2 3.56 3.43 3.99 10.98 3 0.13 0.0 0.08 0.21 T o t a l 10.15 9.61 8.21 27.97 C A 1 2 3 T o t a l 1 8.77 3.92 4.09 16.78 2 6.06 2.99 1.93 10.98 3 0.0 0.21 0.0 0.21 T o t a l 14.83 7.12 6.02 27.97 When there is no replication, the total sum of squares is used. C B 1 2 3 T o t a l 1 5.70 2.47 1.98 10.15 2 4.56 2.53 2.52 9.61 3 4.57 2.12 1.52 8.21 T o t a l 14.83 7.12 6.02 27.97 C o r r e c t i o n term, CT (27.97)' 27 28.97 SST = 3 .66 2 + 1 .39 2 + . . . + 0 . 0 8 2 - CT = 25.73 SSA = | - (16 .78 2 + 10 .98 2 + 0 .21 2 ) - CT = 15.72 SSB = | - (10 .15 2 + 9 . 6 1 2 + 8 .21 2 ) - CT = 0.23 SSC = ^(14 .83 2 + 7 .12 2 + 6 .02 2 ) - CT = 5.13 SS(AB) = j ( 6 . 4 6 2 + 3 .56 2 + . . . + 0 .08 2 ) - CT - SSA - SSB 0.90 SS(AC) = 3 ( 8 - 7 7 2 + 6 - 0 6 2 + • • • + O.O 2 ) - CT - SSA - SSC = 2.99 - j (5 .70 2 + 4 . 5 6 2 + . . . + 1.52 2 ) - CT - SSB - SSC = 0.25 SS(BC) SS(ABC) = SST - SSA - SSB - SSC - SS(AB) - SS(AC) - SS(BC) = 0.510 

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