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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 t h e Department of CHEMICAL ENGINEERING  We a c c e p t t h i s , t h e s i s as c o n f o r m i n g to, -the r e q u i t e d standard.  THE UNIVERSITY OF BRITISH COLUMBIA August, 1974  In  presenting  an  advanced  the I  Library  further  for  degree shall  agree  scholarly  by  his  of  this  written  this  thesis  in  at  University  the  make  that  it  purposes  for  freely  permission may  representatives. thesis  partial  be  It  financial  for  of  Columbia,  British  gain  Depa r t m e n t Columbia  for  extensive by  the  understood  permission.  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  of  available  granted  is  fulfilment  shall  Head  be  requirements  reference copying  that  not  the  of  agree  and  of my  I  this  or  allowed  without  that  study. thesis  Department  copying  for  or  publication my  ii  ABSTRACT  A c l a r i f i e d , nonheat c o a g u l a b l e f i s h medium d e r i v e d from condensed f i s h s o l u b l e s  (B.C. P a c k e r s , Richmond, B.C.)  was  explored for  i t s p o t e n t i a l use as the major component of a f e r m e n t a t i o n medium f o r the p r o d u c t i o n o f p r o t e o l y t i c enzymes. Sorangium.  The organism used was  a p u r e s t r a i n of  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 m e t a b o l i c a c t i v i t y  consequently  and  the r a t e and e x t e n t o f p r o t e a s e f o r m a t i o n were s t u d i e d .  S t a t i s t i c a l l y designed experiments  showed t h a t b o t h c a l c i u m and  g l u c o s e had a s i g n i f i c a n t e f f e c t on growth and p r o t e a s e f o r m a t i o n , 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  ( g l u c o s e , mannose, g a l a c t o s e , x y l o s e , a r a b i n o s e , and  s o l u b l e s t a r c h ) t e s t e d , o n l y g l u c o s e and mannose were u t i l i z e d f o r f u r t h e r growth.  Neither i n i t i a l  (16  24  to  hrs)  pH  ( i n the range  4  to  8) n o r i n o c u l u m  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 o f p r o t e a s e .  K i n e t i c s t u d i e s w i t h the optimum medium i n f l a s k s and i n a  250 ml  7 - l i t r e f e r m e n t o r r e v e a l e d t h a t growth was  an i n i t i a l f a s t growth f o l l o w e d by a s l o w e r secondary growth was  age  Erlenneyer biphasic with  growth.  The  a t t r i b u t e d t o the i n i t i a l , f a s t u t i l i z a t i o n o f e a s i l y  staged  available  n u t r i e n t s (such as amino a c i d s ) o r i g i n a l l y p r e s e n t i n the medium, f o l l o w e d by a second, s l o w e r r e a c t i o n i n w h i c h medium were d i g e s t e d .  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  S i n c e the i n f l e c t i o n i n the growth c u r v e  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 t h a t 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  iii  t r a n s f e r might be t h e r a t e l i m i t i n g  factors.  K i n e t i c a n a l y s i s of t h e d a t a showed t h a t p r o d u c t f o r m a t i o n not a s s o c i a t e d by any d i r e c t mechanism w i t h c a r b o h y d r a t e  utilization.  P r o t e a s e f o r m a t i o n was h i g h e s t w i t h an a g i t a t i o n r a t e o f  500 rpm  aeration rate of  2  was  and an  l i t r e o f a i r p e r m i n u t e , w h i c h c o r r e s p o n d e d t o an  oxygen t r a n s f e r c o e f f i c i e n t o f  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 a r e s u g g e s t e d .  iv  ACKNOWLEDGEMENTS  I w i s h t o e x p r e s s s i n c e r e a p p r e c i a t i o n to D r . R i c h a r d under whose d i r e c t i o n t h i s work was u n d e r t a k e n , f o r h i s s u p p o r t ,  Branion, kindness,  and encouragement i n a l l phases o f t h i s work.  Thanks a r e a l s o due t o Dr. George S t r a s d i n e o f t h e F i s h e r i e s Research Board o f Canada f o r s u g g e s t i n g  this topic , for his technical  a d v i c e , 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 , s t o c k c u l t u r e s o f Sorangium and condensed f i s h  solubles.  A p p r e c i a t i o n i s a l s o extended t o 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 t h e use o f some o f t h e equipment . Furthermore I w i s h t o 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 , b u t n o t l e a s t , I w i s h t o thank Environment Canada, F i s h e r i e s and M a r i n e S e r v i c e , and t h e N a t i o n a l Research C o u n c i l o f Canada, f o r providing financial  support.  V TABLE OF CONTENTS Page LIST OF TABLES  viii  LIST OF FIGURES  x  NOMENCLATURE Chapter  1  x i i INTRODUCTION  1  1.1  Nature o f the Problem  1  1.2  F i s h S t i c k w a t e r and F i s h S o l u b l e s  3  1.3  F e r m e n t a t i o n 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 l u b l e s 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  1.5  The M i c r o - o r g a n i s m Used - Sorangium  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 o f t h i s Work  22  Chapter  2  16 495  EXPERIMENTAL TECHNIQUES  17  24  2.1  General  24  2.2  P r e p a r a t i o n o f 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  vi  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 C o n c e n t r a t i o n  34  I.  34  P h e n o l - S u l f u r i c A c i d Reagent Method  II.  Dinitrosalicylic  (DNS) Method  35  2.8  Measurement o f P r o t e o l y t i c  2.9  Measurement o f P r o t e i n Content  41  1.  UV A b s o r p t i o n  43  2.  C o l o r i m e t r i c Methods  43  3  47  Chapter  Activity  38  RESULTS AND DISCUSSION  3.1  E f f e c t o f Medium C o n s t i t u e n t s  47  3.2  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  52  3.3  E f f e c t of I n i t i a l  57  3.4  Effect  o f 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  57  3.5  Effect  of C a r b o h y d r a t e 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  3.7.2  3.7.3  pH  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 Course of F e r m e n t a t i o n  the 72  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 Ultimate Yields  84  E f f e c t o f A g i t a t i o n and A e r a t i o n on Rate o f Oxygen T r a n s f e r  87  vii  TABLE OF CONTENTS  (Contd) Page  Chapter  4  FERMENTATION KINETICS  Chapter  5  CONCLUSIONS  107  Chapter  6  RECOMMENDATIONS  110  BIBLIOGRAPHY  91  112  Appendix  I  EXPERIMENTAL DATA  119  Appendix  II  ANALYSIS PROCEDURES  139  11.1  Measurement o f G l u c o s e C o n c e n t r a t i o n  140  11.2  Measurement o f 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 t h e B i u r e t Reagent  148  Appendix I I I  COMPUTATION OF SUM OF SQUARES  151  viii  LIST OF TABLES Table 1.1  Page 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  1.5  VARIOUS SPECIES OF BACTERIA CELL YIELD OF SALMON-CANNING WASTE WATER AS A  9  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  25  CFS  2.11  ANALYSIS OF FILTERED  3.12  RESULTS OF FACTORIAL EXPERIMENTS  49  3.13  ANALYSIS OF VARIANCE  51  3.14  EFFECT OF I N I T I A L  58  3.15  EFFECT OF AGITATION AND AERATION ON ULTIMATE YIELDS  3.16  K  CFS  AT VARIOUS DILUTIONS  pH  VALUES ATTAINED IN 7-L FERMENTATION RUNS  27  86 89  v  4.17 4.18 4.19  7-LITRE FERMENTATION - CFS RUN NO. 3 VOLUMETRIC RATES OF 7-LITRE FERMENTATION - CFS RUN NO. 3 SPECIFIC RATES OF 7-LITRE FERMENTATION - CFS RUN NO. 5 VOLUMETRIC RATES OF  92 93 94  ix  LIST OF TABLES (Contd.) Table 4.20 4.21  4.22  Page 7-LITRE FERMENTATION - CFS RUN NO. 5 SPECIFIC RATES OF  95  7-LITRE FERMENTATION - CFS RUN NO. 7 VOLUMETRIC RATES OF  96  7-LITRE FERMENTATION - CFS RUN NO. 7 SPECIFIC RATES OF  97  AI.l  EFFECT OF GLUCOSE CONCENTRATION (FILTERED CFS)  120  AI.2  EFFECT OF GLUCOSE CONCENTRATION (UNFILTERED CFS)  121  AI.3  EFFECT OF  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  AI.ll  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  CFS  CONCENTRATION  SHAKE FLASK FERMENTATION - GLUCOSE DEFICIENT MEDIUM  138  X LIST OF FIGURES Figure  Page  1.1  FISH RENDERING PROCESS  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 2 4 PROTEOLYTIC ACTIVITY VS ENZYME CONCENTRATION  37  2.5 3.6 3.7  3.8  5  42  EFFECT OF GLUCOSE CONCENTRATION ON ACTIVITY UNFILTERED CFS  54  EFFECT OF GLUCOSE CONCENTRATION ON GROWTH FILTERED CFS EFFECT OF GLUCOSE CONCENTRATION ON ACTIVITY  55 -  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  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  3.11  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  3.19 3.20  FERMENTOR  NB -  GLUCOSE RUN  73  7-LITRE FERMENTOR  CFS  RUN NO.  1  74  7-LITRE  CFS  RUN NO.  2  75  FERMENTOR  xi  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  3.27  ACTIVITY AND CELL YIELD VS SUGAR USED FOR VARIOUS 7-LITRE FERMENTOR RUNS  85  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  xii  NOMENCLATURE  Abbreviations  BSA  B o v i n e Serum Albumin  CFS  Condensed F i s h  . DNS  Solubles  D i n i t r o s a l i c y l i c Acid  FPC  F i s h P r o t e i n Concentrate  NB  Nutrient Broth  NBS  New Brunswick  OD  Optical  SCE  Salmon-canning E f f l u e n t  SCP  Single-cell  Scientific  Density  Protein  Symbols  df  Statistical  number of degrees o f  E  Enzyme  e  Enzyme c o n c e n t r a t i o n  ES  Enzyme -  F  F-test  k^  Forward r e a c t i o n r a t e  constant  Reverse r e a c t i o n r a t e  constant  Substrate  statistic  k^  Reaction rate  k  Michaelis-Menten  m  K v  complex  constant constant  Volumetric transfer  coefficient  freedom  xiii  Symbols  (contd)  M  Molarity  MS  S t a t i s t i c a l mean square  N  Normality  P  Product  S  Substrate  s  C o n c e n t r a t i o n of  SS  Statistical  SSE  Sum of squares o f the r e s i d u a l e r r o r a f t e r some m a t h e m a t i c a l model has been a p p l i e d to the experimental data  t  Time, u s u a l l y i n hours  v  Rate of p r o d u c t f o r m a t i o n  x  C e l l mass  total  sum of  substrate  squares  1  Chapter  1  INTRODUCTION  1.1  Nature of  the problem  The s a l m o n - c a n n i n g and the h e r r i n g p r o c e s s i n g o p e r a t i o n s for  r e d u c t i o n to meal and o i l , o r f o r food purposes)  cause major problems o f l i q u i d waste d i s p o s a l . these p r o c e s s i n g o p e r a t i o n s for  most p l a n t s  [1].  are o f  These l a r g e wastewater  washing and p r o c e s s i n g o f the p r o d u c t . effluents  are f l e s h p a r t i c l e s , s c a l e s ,  When o i l y f i s h , processed,  Previously,  Table 1.1,  CC^,  to  result  1000  The major p o l l u t a n t s i n these b l o o d , s l i m e and s o l u b l e  which i s  and o t h e r g a s e s ,  were d i s c h a r g e d to waterways,  thus g i v i n g r i s e  these wastes have been s t u d i e d .  investigator  i n this  are  taken from C l a g g e t t  the p r o t e i n a c e o u s m a t t e r decomposed  o i l y m a t e r i a l as p o s s i b l e .  versatile  proteins.  a l s o p r e s e n t as a c o n t a m i [1]  Claggett  [1,  2,  5]  to  pollution  o t h e r methods  The more u s e f u l  3, 4,  or  rapidly,  to s e r i o u s  d e s c r i b e d so f a r have c o n c e n t r a t e d on r e c o v e r i n g as much of and  U.S.G.P.M.  from the b u t c h e r i n g ,  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 ,  d i s p o s i n g of  from  wastewaters.  these e f f l u e n t s  sewage treatment p l a n t s ;  of  flows  f r e e o i l i n the form of an e m u l s i o n i s  shows t y p i c a l parameters o f these  problems.  200  flow  such as menhaden, s a r d i n e s , m a c k e r e l , and h e r r i n g  nant i n these wastewaters.  evolving  on the West C o a s t  Average e f f l u e n t  the o r d e r of  (either  the  methods proteinaceous  a p p a r e n t l y the most  a r e a , r e c e n t l y d e s c r i b e d a method i n which  Table  1.1  PARAMETERS OF VARIOUS FISH PROCESSING PLANT EFFLUENTS  SPECIES PROCESSED  BOD mg/l  SUSPENDED SOLIDS #/1000#  fish  mg/l  #/1000#  fish  TOTAL SOLIDS mg/l  ///1000//  Halibut  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  75  0.7  50  1.3  85  2.2  Ocean Salmon  Perch Canning  Food H e r r i n g  3500  28  1500  12  3000  24  3850  22  3000  21  6000  42  fish  ho  3  the  BOD  l o a d of salmon canning wastewaters c o u l d be reduced by over  T h i s method i n v o l v e s  c h e m i c a l treatment  from t h i s p r o c e s s  used f o r the p r o d u c t i o n of a meal which c o u l d  the c o n v e n t i o n a l  is  f i s h - m e a l i n animal feed.  f i s h meal p r o d u c t i o n p r o c e s s is  is  used to make f i s h s o l u b l e s  animal  and a i r f l o t a t i o n .  One of  fish stickwater.  90%.  The s l u d g e replace  the b y - p r o d u c t s from the At present  this  stickwater  which a r e used as v i t a m i n supplements  in  feed.  In view of possible  outlet  proteases.  is  the r i c h p r o t e i n content  to use i t  of  the f i s h w a s t e ,  another  as an i n d u c e r f o r the p r o d u c t i o n of m i c r o b i a l  Such a p r o c e s s would be q u i t e  contemporary and i t has  potential  of k i l l i n g  two b i r d s w i t h one s t o n e ; i t  abatement  system as w e l l as an enzyme p r o d u c t i o n p r o c e s s .  the  can s e r v e as a p o l l u t i o n The s c a r c i t y  of  m i c r o b i a l enzymes t o g e t h e r w i t h t h e i r r i s i n g importance i n i n d u s t r y p r o v i d e the s o u r c e o f optimism f o r the development  o f an e c o n o m i c a l l y  feasible  process.  1.2  F i s h S t i c k w a t e r and F i s h  Solubles  An e x c e l l e n t account of is  g i v e n by S o d e r q u i s t  [6].  the o r i g i n and p r o p e r t i e s  A summary of  the s a l i e n t  points  of is  fish  solubles  presented  here.  Fish solubles  a r e made from f i s h s t i c k w a t e r which i s  from f i s h - r e n d e r i n g p r o c e s s e s . to denature  the  fish protein.  The f i s h a n d / o r f i s h waste a r e This m a t e r i a l i s  a by-product steam-cooked  then s e p a r a t e d under  pressure  4  i n t o an aqueous e x t r a c t and f i s h p u l p . meal, c o n t a i n i n g about as an a n i m a l f e e d .  8%  The f i s h p u l p i s dehydrated  to a  m o i s t u r e , w h i c h can be s o l d as a f e r t i l i z e r o r  The p r e s s w a t e r i s s c r e e n e d t o remove any s o l i d s and  then passed through a g r a v i t y s e p a r a t o r o r a c e n t r i f u g e .  The s l u d g e from  t h i s p r o c e s s i s r e t u r n e d t o t h e p r e s s cake and t h e c l a r i f i e d p r e s s 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 p r o d u c t s ,  namely, the f i s h o i l and t h e 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 t o an e v a p o r a t o r . reduces t h e w a t e r c o n t e n t from  95%  to  50%.  The e v a p o r a t o r  " S o l u b l e s " a r e produced i n  the form o f a brown, somewhat v i s c o u s l i q u i d w i t h a m i l d , f i s h y  odour.  F i g u r e 1.1 ( t a k e n from S o d e r q u i s t ) shows t h e p r o c e s s as d e s c r i b e d .  The approximate c o m p o s i t i o n o f s t i c k w a t e r and condensed  fish  s o l u b l e s a r e g i v e n i n T a b l e s 1.2 and 1.3 r e s p e c t i v e l y [ 7 ] .  1.3  F e r m e n t a t i o n C u l t u r e Medium  The n u t r i t i o n a l r e q u i r e m e n t s o f m i c r o - o r g a n i s m s  are diverse  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 t o s y n t h e s i z e e s s e n t i a l f a c t o r s from s i m p l e n u t r i e n t s .  However, a l l m i c r o - o r g a n i s m s  growth  demand w a t e r ,  c a r b o n , n i t r o g e n and m i n e r a l elements, as w e l l as a c c e s s t o hydrogen and oxygen [ 8 ] .  The development and f o r m u l a t i o n o f c u l t u r e medium i s one o f t h e most d i v e r s e s t e p s o f a f e r m e n t a t i o n programme; i t r e q u i r e s a l o t o f f r i g g i n g around.  The type o f medium used depends  upon s e v e r a l  interrelated  5  Figure  1.1  FISH RENDERING PROCESS  PROCESS  WASTES  DISPOSAL  Table  1.2  TYPICAL ANALYSIS OF FISH STICKWATER  PARAMETER  VALUE  Total solids  . 5.6%  Ash  0.95%  F a t t y substances  0.60%  Crude p r o t e i n  3.5%  (N x 6.25)  Table  1.3  TYPICAL ANALYSIS OF CONDENSED FISH SOLUBLES  PARAMETER  VALUE  Total solids  50.43%  Ash  8.86%  Fat  4.8%  Crude p r o t e i n Sp. pH  (N x 6.25)  g r . at 2 0 ° C  33.85% 1.20 4.5  7  variables.  Cost i s  almost always  a f a c t o r i n d e t e r m i n i n g the  o f any i n d u s t r i a l f e r m e n t a t i o n medium.  When l a r g e q u a n t i t i e s  medium are r e q u i r e d , the e x c e l l e n t and c o v e n i e n t  types of  l a b o r a t o r y media as s u p p l i e d by D i f c o , O x o i d , B B L , e t c . it  is  therefore  necessary  to f i n d  alternative  sources  their laboratory counterparts.  media a r e t h e r e f o r e u s u a l l y complex  [9].  under e x a m i n a t i o n ; however, development  is  nutrients. cannot be as p u r e  Industrial  of  fermentation  are usually  the m i c r o - o r g a n i s m  s i n c e knowledge o f the l a t t e r  done on an adhoc b a s i s ,  culture  standardized  Their compositions  formulated to promote the d e s i r e d m e t a b o l i c p a t t e r n s  of  are very uneconomical,  of  O b v i o u s l y such m a t e r i a l s , because they must be c h e a p e r , or as s t a n d a r d i z e d as  suitability  is  usually  limited,  although underlying 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 the  is  an enzyme t h a t h y d r o l y s e s  and  f e r m e n t a t i o n medium must s u p p l y the n u t r i t i o n a l s t i m u l u s  do s o .  The f o l l o w i n g s e c t i o n s d i s c u s s  wastewaters and f i s h e r i e s t i o n medium f o r p r o t e a s e  1.3.1  proteins  the s u i t a b i l i t y o f  therefore  to cause i t  to  salmon-canning  b y - p r o d u c t s as the major component of  a fermenta-  production.  Salmon-Canning Wastewater as a M i c r o b i a l Growth Medium  S t r a s d i n e and M e l v i l l e [10]  showed  that salmon-canning  effluent  (SCE) can s u p p o r t the growth of s i x s p e c i e s of b a c t e r i a when used b o t h a component of a complex medium and as T a b l e s 1.4 indicates  and 1.5, the  total  the s o l e source of a v a i l a b l e  a r e adapted from S t r a s d i n e and M e l v i l l e [ 1 0 ] . c e l l y i e l d f o r each o f  the s i x  s p e c i e s i n media  as  nitrogen. Table  1.4  8  containing  the v a r i o u s n i t r o g e n s o u r c e s t o g e t h e r w i t h  M g S 0 . 7 H 0 (0.02%), 4  2  dextrose  (0.5%).  c a p a c i t y of  F e S 0 . 4 H 0 (0.002%), 4  2  T a b l e 1.5  indicates  K^HPO^ (0.2%),  M n S 0 . 4 H 0 (0.002%) 4  and  2  the a b i l i t y of  SCE  to s e r v e i n  a complete medium i n which no o t h e r a d d i t i o n s were n e c e s s a r y .  Based on these r e s u l t s ,  the authors  f o r salmon-canning w a s t e w a t e r s , namely  :  (1)  s u g g e s t two p o s s i b l e as an i n e x p e n s i v e  uses  source  available nitrogen  f o r the m i c r o b i a l d e g r a d a t i o n a n d / o r u t i l i z a t i o n  nitrogen-deficient  wastes,  and  (2)  and o f s i n g l e - c e l l  acids  ( P r o p i o n i c and a c e t i c i n h i s  liquor.  He was u n s u c c e s s f u l  and  2  case) v i a f e r m e n t a t i o n  for  proteins.  s u g g e s t i o n has been worked on to a c e r t a i n e x t e n t by Truong  whose main i n t e r e s t was i n the p r o d u c t i o n of V i t a m i n B ^  of  of  as a m i c r o b i o l o g i c a l medium per se  the p r o d u c t i o n of crude enzyme p r e p a r a t i o n s , The f i r s t  the  [11],  volatile  of s u l f i t e  spent  i n growing L a c t o b a c i l l u s p l a n t a r u m , 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 S h e r m a n i i i n a medium i n which y e a s t e x t r a c t was r e p l a c e d by f i s h s t i c k w a t e r No work has been r e p o r t e d y e t  as  the s o l e source  of  nitrogen.  on the crude enzyme p r o d u c t i o n 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 o b v i o u s l y  arise  from  the use of f i s h p r o c e s s i n g wastewaters as a f e r m e n t a t i o n  substrate  (1)  biodegradability  of  l a r g e volume of e f f l u e n t the wastes,  availability.  (3)  large  The f i r s t  c u l t i e s of s t o r a g e w h i l e reproducibility.  to be p r o c e s s e d ,  fluctuations  (2)  rapid  i n composition,  and  two c h a r a c t e r i s t i c s  give r i s e  the  to d i f f i c u l t i e s  t h i r d give r i s e  (4)  are  seasonal  to p r a c t i c a l  diffi-  i n media  These problems are l e s s apparent when the more  concentrated  Table  1.4  CELL YIELD AS A FUNCTION OF NITROGEN SOURCE FOR VARIOUS SPECIES OF BACTERIA  CELLS/ML OF MEDIA N-SOURCE  (loj '10  )  PHYTONE  ACIDICASE  POLYPEPTONE  PEPTONE  10.08*  6.99  9.51  9.37  9.23  8.29  8.68  9.32  6.38  7.00  8.15  5.30  8.36  8.28  SPECIE  Sorangium P.  sp.  putrefaciens  MEAT EXTRACT  TRYPTICASE  SCE  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  sp.  7.00  8.65  8.15  8.08  7.63  7.83  7.18  faecalis  7.79  7.04  7.30  8.69  7.72  8.23  7.63  Bacillus St.  Underlined values indicate  maximum c e l l  yield.  Table 1.5  CELL YIELD OF SALMON-CANNING WASTE WATER AS A FUNCTION OF CELL NUMBER PER MILLILITER  CELLS/ML  % OF MAXIMUM  dog  FROM TABLE  1 0  )  1.4  MEDIA GIVING MAXIMUM GROWTH  Sorangium sp.  9.34  19.1  A. aerogenes  8.60  9.3  polypeptone - S  B a c i l l u s sp.  7.20  3.6  trypticase - S  P. putrefaciens  6.83  0.3  SCE - S  St. f a e c a l i s  6.76  1.2  polypeptone - S  L. plantarum  6.74  0.3  peptone - S  S : salts  SCE - S  11  fish stickwater  1.3.2  and s o l u b l e s  are  used.  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  Fish stickwater,  as s t a t e d e a r l i e r ,  is  an aqueous  extract  from the p r e s s i n g of cooked f i s h d u r i n g f i s h meal p r o d u c t i o n . or p r e s s w a t e r ,  as i t  sometimes c a l l e d ,  manufacture o f  condensed  is  fish solubles.  tissues water i s  [12].  extracts  of bones,  A t y p i c a l analysis  shown i n T a b l e  Stickwater exhibits  cartilage, of some o f  skin,  particles,  and o t h e r  and n i t r o g e n o u s  proteoses,  extractives  (creatine  and c a r n o s i n e ) .  are not c o n s i d e r e d  [6,  peptones, 7].  of n i t r o g e n ,  If  and a r g i n i n e )  which assumes t h a t  gives a f a l s e l y  high value  to g i v e the s t i c k w a t e r  suspended p a r t i c l e s nium s u l f a t e  stick-  unduly h i g h ,  the  which conver-  the average p r o t e i n c o n t a i n s f o r the p r o t e i n c o n t e n t . t i e d to the  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 the  stickwater.  acids,  derivatives  these e x t r a c t i v e s ,  is  protein  include  u r e a , amino  an opaque o r m i l k y appearance.  o r by a change i n the pH o f  largely  highly dispersed  and i m i d o z o l e  substances a r e h i g h l y d i s p e r s e d and are i n t i m a t e l y particles  consist  These e x t r a c t i v e s  the p r o p o r t i o n of  to be food p r o t e i n s o u r c e s ,  s i o n f a c t o r of 6.25,  and  connective  the main c o n s t i t u e n t s of  such compounds as ammonia, mono-, d i , and t r i m e t h y l a m i n e ,  (histidine  properties  1.2.  water-soluble  guanidine d e r i v a t i v e s  the  and the g e l a t i n  The n i t r o g e n - c o n t a i n i n g s u b s t a n c e s i n s t i c k w a t e r of n o n c o a g u l a b l e ,  Stickwater  a l s o the raw m a t e r i a l f o r  which a r e c h a r a c t e r i s t i c o f b o t h f i s h muscle e x t r a c t s glue-containing  resulting  The  16.0% fatty  protein These like  alumi-  12  Although  the s t i c k w a t e r c o n t a i n s 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 n a t u r e , even a t v e r y low t e m p e r a t u r e s , i t very unpleasant  to work w i t h , and v e r y d i f f i c u l t  to s t o r e .  makes  It i s a well  r e c o g n i s e d , b u t r a r e l y - m e n t i o n e d , f a c t t h a t f o r a s u b s t r a t e t o be  indus-  t r i a l l y a c c e p t a b l e , the s u b s t r a t e must not o n l y be a p p e a l i n g to the m i c r o b e s b u t i t must a l s o not c r e a t e an adverse  1.3.3  employee r e a c t i o n .  F i s h S o l u b l e s 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 stickwater.  concentrated  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 p r e s e n t i n s t i c k w a t e r  to s m a l l e r m o l e c u l e s ,  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 p r o b l e m o f s p o i l a g e ; concen-  t r a t i o n reduces  s t o r a g e space and e n a b l e s a w i d e r range o f c o n c e n t r a t i o n  to be examined.  T a b l e s 1.6,  1.7,  1.8,  adapted from L a s s e n  [ 7 ] , r e f l e c t the average  c o n t e n t of some o f the more i m p o r t a n t amino a c i d s , v i t a m i n s and ash tuents r e s p e c t i v e l y .  These, t o g e t h e r w i t h some o t h e r 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 r e a s o n 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 o f a n i m a l n u t r i t i o n . i s obtained f r o n this  T a b l e 1.9  consti-  However n o t much revenue  use.  compares the p r i c e s o f c o m m e r c i a l l y  available protein  s o u r c e s w h i c h can be used as the major component o f a f e r m e n t a t i o n medium for  protease production.  Most obvious i s the r e l a t i v e l y low c o s t o f  Table  1.6  AMINO ACID ANALYSIS OF CONDENSED FISH SOLUBLES  % CRUDE PROTEIN AMINO ACID  (N x  ASSAY METHOD  6.25)  Arginine  4. 84  Histidine  5. 79  Lysine  4. 87  Mb.  Leucine  4. 67  Mb.  Isoleucine  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 acid  8. 44  Chem.  Proline  6. 70  Chem.  Mb. = m i c r o b i o l o g i c a l . Chem. = c h e m i c a l .  Mb.  a  6 Chem.  T a b l e 1.7  TYPICAL VITAMIN CONTENT OF WEST COAST FISH SOLUBLES  VITAMIN  VITAMIN  Mg/g  Riboflavin  22  Pyridoxin  Pantothenic Acid  84  Choline  Thiamine  7.5.  Niacin  390  Folic  12.5 1100  Acid  Vitamin  .  ug/g  0.23  B^  0.47  T a b l e 1.8  TYPICAL ANALYSIS OF FISH SOLUBLES ASH CONSTITUENTS  CONSTITUENT  %  P o t a s s i u m (K)  1.93  I r o n (Fe)  0 .0249  Sodium (Na)  1.87  Magnesium (Mg)  0 .016  Phosphorous (P)  0.85  Copper (Cu)  0 .007  C a l c i u m (Ca)  0.0869  Iodine ( I )  0 .007  Manganese (Mn)  0.0869  Aluminum ( A l )  0 .005  TOTAL ASH  CONSTITUENT  8.86%  %  15  Table  1.9  ALTERNATE PROTEIN SUPPLY SOURCES [14,  MATERIAL  PRICE/POUND* (cents)  Soy Meal and F l o u r  3.5-6.5  Soy P r o t e i n Cone.  21.5  F i s h Solubles  2.50  F i s h Meal (feed grade) F i s h P r o t e i n Cone. Cottonseed Flour  Dry Skim  Y e a s t - Torula ( s u l f i t e waste)  *  Based on  1969  market.  (cents)  8-14.8 26.5-35 7.8 10.5-14.2  10-16  13-20  6.6  Milk  PRICE/POUND PROTEIN  6.3-8.5  11  Wheat F l o u r  15]  20 60  14.4-21.0  40-60  15-16  27-29  16  condensed f i s h  For  solubles.  the reasons c i t e d above, t h i s work, w h i c h i s t o i n v e s t i g a t e  the p r o t e a s e p r o d u c t i o n  1.4  p o t e n t i a l of condensed f i s h s o l u b l e s , emerges.  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, o r p r o t e a s e s , whether they a r e a n i m a l , p l a n t , o r m i c r o b i a l o r i g i n a r e c h a r a c t e r i z e d by  of  their ability  to  c a t a l y z e h y d r o l y t i c c l e a v a g e o f p e p t i d e l i n k a g e s between amino a c i d s [16, 17, 18, 1 9 ] .  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  groups [20, 2 1 ] , namely, the p r o t e i n a s e s peptidases (or exopeptidases).  The  ( o r e n d o p e p t i d a s e s ) and  proteinases  distinct the  a r e enzymes w h i c h  i n t e r n a l p e p t i d e bonds i n the h i g h m o l e c u l a r w e i g h t p o l y p e p t i d e  attack  protein  m o l e c u l e s ; p e p t i d e s such as p r o t e o s e s and peptones are u s u a l l y formed from this reaction.  The  p e p t i d a s e s are enzymes w h i c h a t t a c k t e r m i n a l  peptide  bonds a d j a c e n t 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 m i c r o - o r g a n i s m s w h i c h u t i l i z e p r o t e i n s as n u t r i e n t 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 a c i d s n e c e s s a r y i n t h e i r m e t a b o l i s m f o r the p r o d u c t i o n and  enzyme p r o t e i n s .  The  majority  materials  to the amino  of t h e i r own  of m i c r o b i a l enzymes s t u d i e d  cellular  t o 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 the a p p r o p r i a t e  organism.  e n d o p e p t i d a s e s and  These e x t r a c e l l u l a r enzymes are  of  predominantly  can be d i v i d e d i n t o t h r e e main groups, namely, a c i d ,  17  neutral, is  or a l k a l i n e ,  greatest  [22,  23,  depending upon the pH range i n which t h e i r  activity  24].  The few m i c r o b i a l p r o t e a s e s p r e s e n t l y from b a c t e r i a l and f u n g a l s o u r c e s ;  available  c o m m e r c i a l l y are  they a r e u s u a l l y s o l d i n the crude  from,  which may c o n t a i n a v a r i e t y of o t h e r enzymes i n v a r y i n g amounts.  Hence  t h e i r enzymatic a c t i o n i s  the  impurities present, in industry. developed  extremely  complicated.  these m i c r o b i a l enzymes are e n j o y i n g wide  Some o f  these a p p l i c a t i o n s  from e m p i r i c a l , t r i a l  [19,  s p o t removal i n c l e a n i n g ,  proofing of beer,  plaque removal from t e e t h ,  In 1969,  enzymes were marketed  1.5  23],  applications  most of w h i c h were  a r e i n b r e a d making,  dehairing of h i d e s ,  chill  and p r o t e i n h y d r o l y s a t e  over 4 m i l l i o n d o l l a r s ' worth of  proteolytic  [25].  The M i c r o - o r g a n i s m Used - Sorangium 495  The organism chosen f o r t h i s to  20,  and e r r o r methods,  meat t e n d e r i z i n g ,  manufacturing.  Notwithstanding  the genus Sorangium.  This organism, designated  i s o l a t e d by G i l l e s p i e and Cook [26] study  study was a s o i l b a c t e r i a b e l o n g i n g  i n 1964.  i s o l a t e No. 495,  I t was o b t a i n e d f o r  was this  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 R e s e a r c h Board  o f Canada, who,  as mentioned e a r l i e r ,  h i g h e s t c e l l y i e l d i n media c o n t a i n i n g nitrogen.  showed t h a t SCE  as  this  s p e c i e gave  the s o l e s o u r c e of  the available  18  A p e r u s a l of shows that  "Sergey's Manual of D e t e r m i n a t i v e B a c t e r i o l o g y "  the genus Sorangium i s  a member of  which b e l o n g s to the o r d e r M y x o b a c t e r a l e s . distinguished  gliding, non-flagellar  with s o l i d surfaces,  n a t u r e of the c e l l w a l l . gram-negative  family  Members of  from a t r u e b a c t e r i u m by two p r i n c i p a l  characteristic contact  the  and  movement,  (2)  its  although  :  (1)  due to  known as  in  the  methods o f measuring growth a r e t h e r e f o r e  The v e g e t a t i v e c e l l s o f  [28,  by a t h i n them  29].  co-operative  Direct  the f a m i l y S o r a n g i a c e a e  Archangiaceae,  the l o n g ,  to  photometric  impossible.  x 2.5) , and thus d i f f e r  (Myxococcaceae,  possess v e g e t a t i v e c e l l s o f  independent  and w h i c h keeps  w h i c h a r e formed by the  of v e g e t a t i v e c e l l s  c e l l s w i t h b l u n t ends (1.2  delicate  Such a m y x o b a c t e r i a l c o l o n y can g i v e r i s e  f r u i t i n g bodies,  a c t i o n of many thousands  its  the M y x o b a c t e r i a a r e  they tend to be h e l d i n a s s o c i a t i o n  together i n a loose colony.  families  features  flexibility,  l a y e r of s l i m e which they s y n t h e s i z e d u r i n g movement  other  t h i s Order can be  rods which d u r i n g v e g e t a t i v e growth remain p e r f e c t l y  o f one a n o t h e r ,  structures  Sorangiaceae  which o c c u r s o n l y  The v e g e t a t i v e c e l l s o f  [27]  are short,  from the members of  and P o l y a n g i a c e a e )  flexuous,  rigid  tapered  type  (0.5  the  which x 5 -  [30].  The Sorangium s p e c i e s are p r e d o m i n a n t l y s o i l i s o l a t e s are s t r o n g l y property.  cellulolytic,  [31]  to produce at l e a s t  showed that  Many  a l t h o u g h some do not possess  The work of G i l l e s p i e and Cook [26]  and G i l l e s p i e  forms.  their isolate,  two e x t r a c e l l u l a r  and l a t e r ,  this  W h i t a k e r , Cook  Sorangium 495,  was a b l e  enzymes which are s e p a r a b l e  by  to  10)  19  chromatography  on h y d r o x y a p a t i t e :  (1)  complete l y s i s o f v a r i o u s s p e c i e s o f  a l y t i c enzyme w h i c h  effects  Staphylococcus, B a c i l l u s ,  Sarcina,  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 o f b a c t e r i a from s e v e r a l o t h e r g e n e r a ; (2)  a p r o t e a s e 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 d e n a t u r e d  hemoglobin b u t w i t h o u t l y t i c a c t i v i t y towards t h e above o r g a n i s m s . G i l l e s p i e and Cook [26] s t u d i e d t h e 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 b o t h c a s e i n and haemoglobin.  T h e i r r e s u l t s showed t h a t the  pH optimum f o r b o t h s u b s t r a t e s was a t pH 8.5.  K a t z n e l s o n e t a l [32] have a l s o observed t h a t c e r t a i n nematodes were l y s e d by t h i s s t r a i n o f Sorangium.  soil  Subsequent work by  W h i t a k e r [ 3 3 ] , J u r a s e k and W h i t a k e r [ 3 4 ] , W h i t a k e r e t a l [ 3 5 ] , T s a i e t a l [36],  showed t h a t t h e r e were two enzymes w h i c h were r e s p o n s i b l e f o r t h e  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 p r o t e a s e s .  R e c e n t l y , owing t o p o p u l a t i o n i n c r e a s e and l a n d s h o r t a g e , much c o n c e r n has been e x p r e s s e d about a w o r l d p r o t e i n s h o r t a g e i n t h e 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 (SCP)  protein  due t o t h e u n i c e l l u l a r n a t u r e o f i t s p a r e n t m i c r o - o r g a n i s m , i s one  way o f s u p p l e m e n t i n g t h e c o n v e n t i o n a l p r o t e i n s from a g r i c u l t u r e and t h e fishing industry  [37, 3 8 ] . M i c r o - o r g a n i s m s 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 , a r e e a s i l y h a n d l e d , and can  be grown e c o n i m i c a l l y on i n e x p e n s i v e o r g a n i c m a t e r i a l s s u c h as s u l f i t e l i q u o r s , f i s h w a s t e s , h y d r o c a r b o n s , m o l a s s e s , whey and o t h e r a g r i c u l t u r a l wastes  [ 3 8 ] . However, b e f o r e these  SCP  can be used as food f o r human  consumption, the c e l l w a l l , w h i c h i s r e s i s t a n t t o d i g e s t i o n , must be  20  disrupted. 495 may  L y t i c enzymes from s e l e c t e d m i c r o - o r g a n i s m s  prove v a l u a b l e f o r t h i s purpose.  such as Sorangium  Another p o s s i b l e use f o r l y t i c  enzymes i s i n the s t u d y of c e l l w a l l s t r u c t u r e [ 3 9 ] .  The enzyme used by G i l l e s p i e and Cook [26] i n t h e i r s t u d y  was  o b t a i n e d 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 m l . of medium, w h i l e t h a t used by W h i t a k e r and co-workers was produced c o n t a i n i n g 100 - 130 l i t r e s  of medium.  medium ( i n gms/1) i n most cases was 10;  2  F e C l . 6 H 0 , 0.01; 3  was  25°C.  3  and NaOH  2  The g l u c o s e was  4  The c o m p o s i t i o n o f the c u l t u r e  as f o l l o w s :  Glucose, 1; K H P 0 , 1; K N 0 , 0.5;  i n a fermentor  MgS0  4>  Difco.Casamino  7H 0, 0.2;  t o a d j u s t t h e pH to  2  7.0  NaCl,  - 7.1  Acids, 0.1;  i f necessary.  a u t o c l a v e d s e p a r a t e l y , and the temperature o f c u l t i v a t i o n  No attempt was  made by t h e s e a u t h o r s to examine c o n d i t i o n s f o r  maximal p r o d u c t i o n of enzymes.  The p r o t e i n s o u r c e , Casamino A c i d s , was  rather expensive.  1.6  N u t r i t i o n of M y x o b a c t e r i a  The m y x o b a c t e r i a a r e n o t v e r y f a s t i d i o u s m i c r o o r g a n i s m s 41].  They a r e u b i q u i t o u s i n h a b i t a n t s o f normal s o i l , b a r k , and  plant materials. of  [30, 40, decaying  They are c o s m o p o l i t a n , h a v i n g been r e p o r t e d i n the s o i l s  P o l a n d , 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 [ 4 2 ] .  The m y x o b a c t e r i a a r e c a p a b l e o f 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 , g l y c o g e n , 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 R e c e n t l y , M a r t i n and So have the a b i l i t y  walls.  [43] have shown t h a t c e r t a i n s t r a i n s o f myxobacter  to u t i l i z e  a u t o c l a v e d k e r a t i n a c e o u s s u b s t r a t e s , i n the  21  form of f e a t h e r s been s u g g e s t e d ,  and w o o l ,  as s o l e sources  i n the review by Dworkin [ 4 2 ] ,  and the n u t r i t i o n a l dependence i n s o l u b l e macromolecules  of  depend on i t s bring i t  are r e l a t e d  niche i s  sustenance i s  For b u i l d i n g p r o t e i n s , materials  reported that  [41]  the  essential  n u t r i t i o n a l requirements  an a q u a t i c one may  substrate  macromolecules food.  f r u i t i n g m y x o b a c t e r i a r e q u i r e complex  and Dworkin [40]  [40] .  have shown t h a t  c o u l d be s u p p l i e d by f r e e  A c c o r d i n g to Dworkin [ 4 0 ] ,  substrates  Baur  of m y x o b a c t e r i a ; however,  r e p o r t e d t h a t g l u c o s e was d i s t i n c t l y and t h a t  effect  although  including  on the growth  McDonald and P e t e r s o n  of i t  has shown t h a t h i s  rate  [41]  the c a r b o n s o u r c e o f c h o i c e the d e l e t i o n  the  amino a c i d s ,  for  the  from the medium i n  f a v o r o f any o t h e r carbon s o u r c e r e s u l t e d i n marked i n h i b i t i o n o f Noren [46]  [44]  f o r growth.  a s m a l l number of sugars  g l u c o s e and a r a b i n o s e have n e g l i g i b l e  two s p e c i e s they examined,  to  molecules.  on i n s o l u b l e  and h y d r o l y z e d p r o t e i n s were b e t t e r  of some s t r a i n s  :  c o u l d be c u l t i v a t e d on media c o n t a i n i n g  A l s o Loebeck [45],  fructose,  He s a i d  or m i x t u r e s o f amino a c i d s  these organisms  has  solubilize  o b l i g e d to creep or c r a w l to i t s  peptone.  peptides  a b i l i t y to  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  i n t o contact with soluble  It  the g l i d i n g m o t i l i t y  to each o t h e r .  A t e r r e s t r i a l organism dependent for i t s  that  the organism on i t s  An organism whose e c o l o g i c a l  nitrogenous  of c a r b o n and n i t r o g e n .  growth.  s p e c i e s c o u l d a t t a c k a l a r g e number o f  c a r b o h y d r a t e s and t h a t growth was s t i m u l a t e d by the p r e s e n c e  of  and a r a b i n o s e .  demonstrate  any e f f e c t  Chase [ 4 7 ] ,  on the o t h e r hand, was unable to  of c a r b o h y d r a t e s on the growth of h i s  maltose  species i n a defined  22  medium.  It  appears  therefore  that  responses to c a r b o h y d r a t e sources  t h e r e are s i g n i f i c a n t among s t r a i n s  such may be the case f o r c a r b o h y d r a t e s , agreement  [40,  requirements  41,  47]  that,  While  i n general,  the m y x o b a c t e r i a do not  possess  f o r a p a r t i c u l a r v i t a m i n or amino a c i d , n o r do they appear  Objectives  of  to  f o r growth.  t h i s work  The o b j e c t i v e s  1.  of m y x o b a c t e r i a .  in  t h e r e seems to be unanimous  have any p a r t i c u l a r l y e x o t i c requirements  1.7  differences  of  To determine  the p r e s e n t work a r e :  the s u i t a b i l i t y o f condensed  fish solubles  as  the major component of a f e r m e n t a t i o n medium f o r the growth o f a pure s t r a i n of Sorangium 495.  2.  To determine  Sorangium s p e c i e  3. calcium,  pentoses, protease  required before  the  w i l l grow w e l l on the f i s h waste medium.  To determine  phosphate)  4.  the p r e - t r e a t m e n t  the amounts o f supplementary n u t r i e n t s  r e q u i r e d f o r optimum growth •and p r o t e a s e  To determine which of  and s t a r c h )  the c a r b o h y d r a t e sources  (glucose,  production.  (hexoses,  can be u t i l i z e d by the m i c r o - o r g a n i s m f o r growth and  production.  5.  To determine  the e f f e c t s  r a t e s of growth and of p r o d u c t i o n .  of a g i t a t i o n  and a e r a t i o n on  the  23  6.  To determine  the r e l a t i o n s h i p s  from an a n a l y s i s  between r a t e s o f growth,  p r o t e a s e p r o d u c t i o n of  of  the b a t c h k i n e t i c  substrate  the Sorangium s p e c i e .  data  u t i l i z a t i o n and  24  Chapter  2  EXPERIMENTAL TECHNIQUES  2.1  General  B i o l o g i c a l experiments variables. ments,  it  Due to the is  tedious  and time consuming n a t u r e o f these e x p e r i -  i m p r a c t i c a l , although p o s s i b l e ,  the v a r i a b l e s one can c o n c e i v e , tation. design  a r e known f o r t h e i r l a r g e number o f  to examine the e f f e c t s  49,  50]  to determine  among those known to a f f e c t  :  after using f a c t o r i a l  the most i n f l u e n t i a l supplementary n u t r i e n t  protease  production, this  was then f u r t h e r s t u d i e d at a d d i t i o n a l l e v e l s .  important n u t r i e n t  I n subsequent  other process  v a r i a b l e s were s t u d i e d one at a t i m e .  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  2.2  P r e p a r a t i o n o f C u l t u r e Medium  When not i n u s e ,  assume  [49].  condensed  T h i s m a t e r i a l was s u p p l i e d by B . C . P a c k e r s , Richmond, B . C . the s o l u b l e s  were r e f r i g e r a t e d at 6 ° C .  c o n d i t i o n s s p o i l a g e was l a r g e l y p r e v e n t e d . and  experiments,  Such experiments  A l l work has been c a r r i e d out w i t h a s i n g l e b a t c h o f fish solubles.  all  even w i t h the h e l p o f f a c t o r i a l e x p e r i m e n -  The p r o c e d u r e adopted h e r e was as f o l l o w s [48,  of  pH o f the s o l u b l e s  T a b l e 2.10  Under these  shows the dry weight  measured o v e r a p e r i o d o f seven months.  negligible  change i n pH v a l u e s u p p o r t e d the view t h a t  negligible  decomposition of  the  solubles.  The  t h e r e had been  25  Table  2.10  TIME VARIATION IN pH AND DRY WEIGHT OF REFRIGERATED CFS  wt/vol,%  DATE  pH  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 s o l u b l e s used i n the f e r m e n t a t i o n s  p r e p a r e d by d i l u t i o n w i t h tap w a t e r . diluted fish solubles fibre  f i l t e r papers  proteinaceous  Almost a l l the runs were made w i t h  t h a t had been f i l t e r e d  through Reeve-Angel g l a s s  to g i v e a c l e a r media c o n t a i n i n g l i t t l e  materials.  The average s o l u b l e  Bovine Serum Albumin e q u i v a l e n t )  at various d i l u t i o n s at  other a d d i t i o n s . increases  The  pH  of  (measured  shown i n T a b l e  yield  (Section  the d i l u t e d  were u s e d ,  and f o r  30  to  7.0  Initially,  with  2.5N  sterilization  was 10 p s i g ; 15  minutes  15  minutes when s m a l l volumes  minutes when l a r g e volumes  the maximum steam p r e s s u r e  under such a c r i s i s i n both cases.  medium d u r i n g s t e r i l i z a t i o n . bases.  any  the s t e r i l i z a t i o n  There was a s l i g h t This i s  was  were u s e d .  i n the  (around sterilizer  time was i n c r e a s e d by  drop i n the  pH  of  p r o b a b l y due to the l o s s o f  No attempt was made to r e - a d j u s t  the  pH  6N  (lOOmls o r l e s s )  (4 l i t r e s )  attainable  or  contact  L a t e r o n , when the "Energy C o n s e r v a t i o n A c t " came i n t o e f f e c t December 1973),  solubles  3.2).  the medium was a d j u s t e d  for  2.11.  dilutions.  accomplished by a u t o c l a v i n g the f e r m e n t a t i o n m i x t u r e i n d i r e c t 15psig.  as  the a d d i t i o n o f g l u c o s e as a carbon s o u r c e  Sodium H y d r o x i d e b e f o r e s t e r i l i z a t i o n .  w i t h steam a t  coagulable  c o u l d be used as a complete medium w i t h o u t  However,  the p r o t e a s e  is  these  I t was determined e a r l y i n t h i s study t h a t or u n f i l t e r e d )  or no  p r o t e i n content  The t a b l e a l s o shows the average g l u c o s e content  (filtered  were  before  the volatile  inoculation.  Table  ANALYSIS  DILUTION  (v/v)  OF FILTERED  PROTEIN mg B S A / m l  2.11  C F S A TVARIOUS  GLUCOSE  DILUTIONS  DRY  WEIGHT  mg/ml  mg/ml  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 P r e p a r a t i o n  C u l t u r e s of  the Sorangium s p e c i e s were m a i n t a i n e d on a B r a i n  H e a r t I n f u s i o n agar s l a n t p r e p a r e d by d i s s o l v i n g I n f u s i o n agar ( D i f c o ) After 2 - 3  Beef E x t r a c t , 0.3%)  In  flasks  the f i r s t this  c o n t a i n i n g 100 ml  and f o r k i n e t i c  stage,  i n two s t a g e s i n  (after  the  15 -  of a N u t r i e n t B r o t h (Peptone,  fermentors.  about 1 - 2  days),  The i n o c u l u m  1 ml  of  the inoculum f o r the second f i r s t stage.  growth p h a s e .  culture.  this stage  The inoculum f o r  The amount o f i n o c u l u m used was, f o r Erlenmeyer f l a s k s  and  the  transfer. in  in ratio 1 : 40  fermentors.  Inoculum p r e p a r e d i n t h i s way reduced the  of  0.5%;  24 h r s growth, when the organism was  inoculum to f r e s h medium, 1 : 100  This i s  250 ml  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 was made a f t e r  7-litre  refrigeration  the organism was induced to grow i n the N u t r i e n t B r o t h medium.  growing c u l t u r e was used as  the e x p o n e n t i a l  for  water.  s t a g e 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  f i s h medium was  of  30°C  studies i n 7 - l i t r e  which employed the same medium as  The  ml d i s t i l l e d  medium, to p r o v i d e the inoculum f o r p r e l i m i n a r y s t u d i e s  When heavy growth was o b t a i n e d actively  1,000  the c u l t u r e s were s t o r e d a t  organism was grown at  Erlenmeyer f l a s k s ,  for  30°C,  Agar i n  (6°C).  The  shake  15 gm  days growth at  temperature  in  and  37 gm o f B r a i n H e a r t  especially  the m y x o b a c t e r i a  chance of  important for myxobacteria because,  contamination.  even though  ( i n c l u d i n g the s p e c i e used i n t h i s work)  several  produce  29  a n t i b a c t e r i a l and a n t i f u n g a l substances  [42],  they are poor  and can t h e r e f o r e be overgrown by the more r a p i d l y growing when b o t h a r e p l a c e d on a r i c h medium  2.4  competitors eubacteria  [30].  Apparatus  Most of  the p r e l i m i n a r y experiments  medium c o m p o s i t i o n on y i e l d s standard  250 ml  to t e s t the i n f l u e n c e  o f c e l l s and enzymes were c a r r i e d out  Erlenmeyer f l a s k s  stoppered with non-absorbent  at a c o n s t a n t  140 rpm  temperature o f  with a gyrotory radius of  and a e r a t i o n e f f e c t s ,  MF-207 bench top fermentor was u s e d . c y l i n d r i c a l g l a s s growth v e s s e l s ,  at  the bottom.  each  surfaces  a  NBS  15 cm  i n d i a m e t e r and  each  42 x 2 cm,  fermentor w a l l s  the v e s s e l .  i n t r o d u c t i o n of a i r .  7-litre, 45 cm fixed tubing  p l a t e s were p l a c e d a t r i g h t a n g l e s w i t h  p e r p e n d i c u l a r to the  above the bottom of  inch,  model  were  They were j o i n e d t o g e t h e r by s t a i n l e s s - s t e e l  The b a f f l e  1/2  T h i s model c o n s i s t s of two  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 ,  to the head p l a t e .  (NBS)  30°C.  F o r the study of a g i t a t i o n  high.  in  cotton.  The m i c r o - o r g a n i s m s 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 i n c u b a t o r - s h a k e r w o r k i n g at  of  The b a f f l e  and t h e i r lower ends  their 3 cm  assembly was u t i l i z e d f o r  An a g i t a t o r s h a f t w i t h two a d j u s t a b l e  impellers  c e n t r a l l y mounted through the head p l a t e .  The i m p e l l e r s were made of  six  3/4  set  flat  surfaces  x 3/4  cm  s t a i n l e s s - s t e e l blades,  p e r p e n d i c u l a r to  adapted from Ferguson [51],  is  the bottom o f a schematic  60°  apart with  the v e s s e l .  drawing of  the  Figure  their 2.2,  fermentor  the was  30 Figure  2.2  15 cm. I D fermentor jar  pH  electrodes  SCHEMATIC  D R A W I N G OF 7 - L I T R E  FERMENTOR  31  d e s c r i b e d above. of b r o t h ; range o f  With t h i s  unit,  c o n t r o l the temperature to w i t h i n 10°C  to  50°C;  the  of a s e t  rate  from  1 to 16 l i t r e / m i n .  any time d u r i n g the  litres  10 to 900 rpm; [51,  c o u l d be made, under a s e p t i c  fermentation  4  point within a  52].  fermentor c o u l d be s t e r i l i z e d w i t h the medium i n i t ;  coulb be removed and a d d i t i o n s  2.5  from  to ferment up to  1°C  c o n t r o l the a g i t a t i o n  and to c o n t r o l the a i r flow r a t e addition,  i t was p o s s i b l e  In samples  conditions,  at  [51].  Sampling Technique  1.  Shake F l a s k s  Each treatment samples  of  the  centrifuged  i n an experiment was c a r r i e d out i n d u p l i c a t e ,  two r e p l i c a t e s  were mixed at h a r v e s t  to remove the o r g a n i s m s .  time,  the m i x t u r e was  The f i l t r a t e was saved f o r  later  assays.  2.  A samples  7-1 Fermentor  NBS model  at v a r i o u s  glass c o l l e c t i n g  S20  s a m p l i n g - i n o c u l a t o r was used to withdraw  time i n t e r v a l s .  This device  tube and a b u i l t - i n a i r f i l t e r  l i z e d w i t h the Fermentor or  separately.  c o n s i s t s of a removable chamber.  It  can be  steri-  32  2.6  Measurement of B a c t e r i a l Growth  S e v e r a l methods growth are a v a i l a b l e . [54],  Among them are  d i l u t i o n or p l a t e  measures  of  cell  count  [30],  concentrations  u n i t v o l . of c u l t u r e ) , division is  f o r the q u a n t i t a t i v e  and  (that i s ,  a c c o r d i n g to Monod [55]  this  is  to c u l t u r e , b a c t e r i a l d e n s i t y  and a l s o s i n c e  only i n problems where  of  this  Since is  the  per  cell of  culture),  concentration;  f i s h medium was  sufficient  study.  t h e r e was  to the c e l l  are  the c e l l s v a r y c o n s i d e r a b l y from one  phase to another o f a growth c y c l e .  matter,  V i a b l e counts  number of i n d i v i d u a l c e l l s  are not e q u i v a l e n t  average s i z e s of  growth f o r the purposes  content.  d r y c e l l weight  dry w e i g h t of c e l l s p e r u n i t volume of  and,  prior  ATP  of b a c t e r i a l  T u r b i d i t y and dry c e l l weight are measures  (that i s ,  because the  t u r b i d i t y [53],  and are e s s e n t i a l  of i n t e r e s t .  b a c t e r i a l density  :  estimation  clarified  as a measurement  Due to the f o r m a t i o n o f  considerable  d u r i n g growth, d i r e c t t u r b i d i m e t r i c methods  of  slimy  c o l o r v a r i a t i o n i n the medium  c o u l d not be used to  follow  growth.  A l t h o u g h 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 t u r b i d i t y o f a washed c e l l s u s p e n s i o n wavelengths  (Figure 2.3),  d i r e c t w e i g h i n g of follows.  a l l the measurements  the dry c e l l s .  A sample of  and the dry c e l l weight at  the  various  r e p o r t e d h e r e were made by  The dry b a c t e r i a c e l l s were o b t a i n e d  f e r m e n t a t i o n b r o t h was c e n t r i f u g e d i n an  IEC  inter-  n a t i o n a l C e n t r i f u g e - U n i v e r s a l Model UV, f i t t e d w i t h an I n t e r n a t i o n a l M u l t i s p e e d Attachment, from t h i s  for  10  minutes  c e n t r i f u g a t i o n was decanted  at  22,000 x G .  The s u p e r n a t a n t  and saved f o r l a t e r sugar  as  analysis  33 F i g u r e 2.3 TURBIDITY VS DRY CELL WEIGHT AT VARIOUS WAVELENGTHS —  Note :-  —  —  No attempt was made to t e s t t h e r e l i a b i l i t y  :  of t h i s  v  relationship.  The samples used f o r these measurements were taken a t one p a r t i c u l a r s t a g e of growth, and the experiment was n o t r e p e a t e d .  34  and p r o t e o l y t i c a c t i v i t y a s s a y .  The c e l l s were r e - s u s p e n d e d i n d i s t i l l e d  water u s i n g a v o r t e x mixer and r e c e n t r i f u g e d f o r to remove the wash w a t e r . twice.  After  10 minutes a t 22,000 x G  T h i s washing and c e n t r i f u g i n g p r o c e d u r e was done  the f i n a l w a s h i n g ,  the c e l l s were d i s p e r s e d i n d i s t i l l e d  water and were then t r a n s f e r r e d to p r e - w e i g h t e d alumimum d i s h e s . were then d r i e d at  2.7  95°C  i n a F i s h e r Isotemp oven f o r at l e a s t  These 12 h o u r s .  Measurement of Sugar C o n c e n t r a t i o n  Many c o l o r i m e t r i c t e s t s f o r r e d u c i n g sugars and p o l y s a c c h a r i d e s are a v a i l a b l e  [56].  I.  The two methods  t e s t e d and used i n t h i s work were  P h e n o l - S u l f u r i c A c i d Reagent Method  T h i s method, developed by Dubois e t fact  t h a t s i m p l e sugars and t h e i r d e r i v a t i v e s  al  [56],  i s b a s e d on the  g i v e an o r a n g e - y e l l o w  when t r e a t e d w i t h p h e n o l and c o n c e n t r a t e d s u l f u r i c a c i d . claimed that  color  The a u t h o r s  :  The method i s  simple,  reproducible r e s u l t s . stable,  and i t  rapid,  and s e n s i t i v e ,  The reagent i s  and g i v e s  i n e x p e n s i v e and  and a g i v e n s o l u t i o n r e q u i r e s o n l y one s t a n d a r d  curve f o r each s u g a r . is  c o n t r o l of  unnecessary the  The c o l o r produced i s  permanent  to pay s p e c i a l a t t e n t i o n  to  the  conditions.  The above c l a i m s have been confirmed d u r i n g the course o f work.  :  However, i n o r d e r to get  reporducible results,  this  i t was n e c e s s a r y  to  35  make the  f o l l o w i n g c a u t i o n a r y notes :  (1) acid,  fast  Owing t o the h i g h v i s c o s i t y  d e l i v e r y of  acid being l e f t  (2)  that  concentrated s u l f u r i c  the a c i d r e s u l t e d i n a s i g n i f i c a n t  b e h i n d on the w a l l s  be taken to ensure  o f the  o f the p i p e t  (or b u r e t t e ) .  the e x a c t amount o f a c i d i s  In as much as  diameter o f  16 mm. were u s e d .  w i t h o u t d i s s i p a t i n g the heat  This diameter w i l l  too r a p i d l y  by d i r e c t i n g the stream o f a c i d a g a i n s t against  the s i d e o f  (3)  the t e s t  [55].  the r e a d i n g s a r e made.  the S p e c t r o n i c 20  Rapid t r a n s f e r r e s u l t e d these b u b b l e s ,  if  in not  readings.  D i n i t r o s a l i c y l i c (DNS) Method  r e d u c i n g sugars  in diabetic urine.  I n t h i s method,  i n 1925  contains  to  the sugar  was allowed to r e a c t w i t h d i n i t r o s a l i c y l i c a c i d reagent  salt,  obtained  tube.  T h i s method was developed by Sumner [57]  T h i s reagent  the  a l l o w good m i x i n g  Good m i x i n g i s  i n t e r f e r e w i t h the s p e c t r o p h o t o m e t e r  II.  are mixed,  the l i q u i d s u r f a c e r a t h e r than  e n t r a i n e d gas b u b b l e s w i t h low r i s e v e l o c i t i e s , removed, w i l l  heat  Tubes w i t h an i n t e r n a l  The s o l u t i o n must be poured s l o w l y i n t o  tubes j u s t b e f o r e  Care must  the r e a c t i o n i s b r o u g h t about by the  the r e a c t i o n tube are i m p o r t a n t .  the  introduced.  developed when the c o n c e n t r a t e d a c i d and water s o l u t i o n s shape and s i z e o f  amount o f  solution  (Appendix I I . 1 ) .  sodium h y d r o x i d e , d i n i t r o s a l i c y l i c a c i d ,  phenol and sodium b i s u l f i t e .  detect  A c c o r d i n g to Sumner [57],  Rochelle the  Rochelle  36  s a l t p r e v e n t e d the d i s s o l u t i o n o f oxygen; dissolved  t h i s was d e s i r a b l e because t h e  can d e s t r o y p a r t o f t h e s u g a r .  Excess p h e n o l was added so  as t o enhance the c o l o r produced by the s u g a r . h e l p e d t o s t a b i l i z e the c o l o r produced when t h e  A d d i t i o n o f sodium  bisulfite  DNS - p h e n o l 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 o f t h e p h e n o l - s u l f u r i c a c i d and t h e DNS  tests f o r sugars.  A l t h o u g h t h e agreement between the two methods was  q u i t e good, most o f the s u g a r t e s t r e s u l t s r e p o r t e d i n t h i s work were done by t h e  DNS  method f o r the f o l l o w i n g reasons :  (1)  The p h e n o l - 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 o f  a h i g h l y corrosive reagent.  T h i s n e c e s s i t a t e s the o b s e r v a t i o n o f c e r t a i n  o b v i o u s p r e c a u t i o n s w h i c h tends t o s l o w down the p r o c e s s .  (2) t i v e t h a n the  The p h e n o l - s u l f u r i c a c i d method i s f o u r times more s e n s i 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 p h e n o l - s u l f u r i c a c i d method (range i n  ug/ml),  resulting i n a  greater r i s k of e r r o r .  t h a t the  (3)  When many samples were s i m u l t a n e o u s l y measured, i t was  DNS  method was f a s t e r and e a s i e r t o h a n d l e .  found  The d e t a i l s o f the two t e s t methods and the s t a n d a r d c u r v e s a r e g i v e n i n Appendix I I . 1 .  37  F i g u r e 2.4  COMPARISON OF  DNS  AND PHENOL-H-SO. 2  2  4  ASSAYS  4  6  8  10  S U G A R by P H E N O L - H S 0 ( m g / m l ) 2  4  38  2.8  Measurement o f P r o t e o l y t i c A c t i v i t y  An a b s o l u t e q u a n t i t a t i v e d e t e r m i n a t i o n of c r u d e enzyme p r e p a r a t i o n s by c h e m i c a l o r p h y s i c a l a n s l y s i s (1)  the s t r u c t u r e o f enzymes  enzymes  i s g e n e r a l l y not w e l l known,  o c c u r o n l y i n t r a c e amounts,  for p r o t e i n s .  i s n o t f e a s i b l e because  (3)  most enzymes  of  the substance  as i s v a l i d f o r o t h e r d i f f e r e n t  has been determined under s p e c i f i e d t e m p e r a t u r e , and  proteins. it  conditions  of substrate  concentration,  enzyme a c t i v i t y are m a i n t a i n e d c o n s t a n t  i t i s not p o s s i b l e  to compare measurements  the d i f f e r e n t  calculation  conditions  if  possible  f o r one and the same  enzyme depending upon whose method i s employed; c o n s e q u e n t l y ,  equivalence  produces,  i n a r b i t r a r y u n i t s where the u n i t  There o f t e n e x i s t s e v e r a l k i n d s o f u n i t s  enzyme because  The amount  p H , t h a t i s , under c o n d i t i o n s where a l l f a c t o r s w h i c h  are known to i n f l u e n c e  cases,  proteins.  activity.  Enzyme a c t i v i t y i s expressed  [58].  tests  i n m i l l i g r a m s o r moles  o f enzyme produced i s i n v a r i a b l y measured by the r e s u l t s t h a t i s , by i t s  most  simply give  Such t e s t s do n o t d i s t i n g u i s h them from o t h e r  As a r e s u l t enzyme q u a n t i t y cannot be e x p r e s s e d  (2)  :  i n many  o f one and the same  o f assay do not a l l o w the use o f  factors.  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 o f the I n t e r n a t i o n a l Union of B i o c h e m i s t r y [59] proposed the f o l l o w i n g d e f i n i t i o n o f an enzyme u n i t :  39  One u n i t of any enzyme i s catalyzes  the  substrate  per m i n u t e ,  is  t h a t amount which  t r a n s f o r m a t i o n o f one micromole of or where more than one bond of  a more complex s u b s t r a t e etc.)  d e f i n e d as  attacked  (eg.  protein,  one m i c r o - e q u i v a l e n t of  concerned p e r minute under w e l l d e f i n e d The temperature s h o u l d be s t a t e d , t h a t where p r a c t i c a b l e i t conditions,  poly-saccharide,  including  and i t  s h o u l d be  pH  the group  conditions. is  25°C.  and s u b s t r a t e  suggested The o t h e r  concentration,  s h o u l d , where p r a c t i c a b l e , be o p t i m a l .  I t was a l s o recommended t h a t enzyme assays be b a s e d , where p r a c t i c a b l e , upon measurements tions  due,  of i n i t i a l  for instance,  inhibitory products. possible,  sufficient  r a t e of r e a c t i o n ,  to r e v e r s i b i l i t y o f r e a c t i o n s  The s u b s t r a t e  Most of  as u n i t s  acids  protein.  for routine analysis  the a c t i v i t y  in  formed by the enzymatic h y d r o l y s i s of  the  of Anson's c o l o r i m e t r i c method  a s s e s s e d from the amount of  (amino  substrate [60]  In t h i s method,  tyrosine  of  a r e b a s e d on a  the low - m o l e c u l a r weight p r o d u c t s  c u r r e n t l y e n j o y i n g wide a p p l i c a t i o n i n r e s e a r c h . is  the k i n e t i c s  per m i l l i l i t r e .  Various modifications  enzyme a c t i v i t y  so t h a t  whenever  C o n c e n t r a t i o n o f an enzyme i n a s o l u t i o n  and a r e s u i t a b l e  d e t e r m i n a t i o n of  and p e p t i d e s )  the enzyme,  the methods which are used to determine  p r o t e o l y t i c enzymes, quantitative  o r to f o r m a t i o n o f  concentration should be,  for s a t u r a t i o n of  the assay approach zero o r d e r . should be expressed  i n o r d e r to a v o i d c o m p l i c a -  are the  contained i n  proteolysis  p r o d u c t s 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  Tysosine i s  determined from the depth of c o l o r which i t  produces  the  acid.  with  AO  F o l i n ' s p h e n o l reagent  In view of and r a p i d method i s  [61].  the l a r g e number of samples desirable.  After  to be a n a l y z e d ,  a simple  considering various a l t e r n a t i v e s ,  m o d i f i c a t i o n o f the Anson method used by P e t r o v a and V i n t s y u n a i t e d e c i d e d upon.  The d e t a i l s  of this  method are g i v e n i n Appendix  C o n d i t i o n s i n the r e a c t i n g m i x t u r e were m a i n t a i n e d as d u r i n g the a s s a y s .  The r e a c t i o n was c a r r i e d out i n a c o n s t a n t  water b a t h c a p a b l e of a u t o m a t i c a l l y close tolerances; before  constant  [62]  was  II.2.  as  possible  temperature  c o n t r o l l i n g temperatures w i t h i n v e r y  a l l reagents were b r o u g h t to the d e s i r e d  the r e a c t i o n was s t a r t e d by m i x i n g them.  temperature  Adequate b u f f e r i n g was  p r o v i d e d by a T r i s - HC1 b u f f e r p r e p a r e d as o u t l i n e d i n Appendix  II.2.  The r e a c t i o n temperature was  -  30°C.  the  The  optimum found by G i l l e s p i e and Cook [26]  pH  was b u f f e r e d at  f o r the p r o t e o l y t i c  8.5  the  enzymes produced  by the Sorangium s p e c i e used i n t h i s work.  For a fixed i n i t i a l substrate enzyme c o n c e n t r a t i o n s , p e r i o d of  the amount of s u b s t r a t e  time may not be p r o p o r t i o n a l to  consequence  of  This i s  the elementary M i c h a e l i s - M e n t e n scheme w h i c h , i n  general,  as  [8]  :  k  k E + S  The r a t e of p r o d u c t f o r m a t i o n , g i v e n by :  transformed i n a given  the amount o f enzyme.  may be e x p r e s s e d  is  c o n c e n t r a t i o n and v a r y i n g i n i t i a l  —>  v,  ES  —> E + P  i n the enzyme r e a c t i o n d e p i c t e d  above  a  41  k^es v  When c o n d i t i o n s rate is  k  =  are such t h a t  t i o n and o f essential  of  the  zero o r d e r as  1 + k /s m  s »  k , m  F i g u r e 2.5  to e s t a b l i s h  '  v = k„e 3  and the  is  conditions  of  this  to  It  tyrosine  is  the enzyme  concentration.  the s u b s t r a t e  r e l i a b l e p o r t i o n of  formed i n  concentra-  is  therefore  unattainable.  10 minutes figure  versus  t h a t under  maximum r e a c t i o n r a t e d u r i n g 0.9,  the s p e c t r o p h o t o m e t e r ,  the whole  v a l u e s w i t h i n the r e l i a b l e p o r t i o n of  incubation  the upper l i m i t on corresponds  the S p e c t r o n i c  to g i v e will  to time.  the  to a t y r o s i n e  20  the  to  c o n c e n t r a t i o n used was not s u f f i c i e n t  thus by d i l u t i n g h i g h e r enzyme c o n c e n t r a t i o n s  the  It  complex i s  apparent from t h i s  an absorbance r e a d i n g of  s a t u r a t i o n of  I n such cases  a s s a y , beyond an enzyme c o n c e n t r a t i o n e q u i v a l e n t  maintain a constant  70 yg;  o r d e r as r e l a t e d  the enzyme-substrate  a p l o t of amount of  tyrosine,  Fortunately,  first  reaction  the l i m i t of enzyme c o n c e n t r a t i o n beyond w h i c h ,  crude enzyme c o n c e n t r a t i o n .  2.9  then  to the s u b s t r a t e  maximum r a t e of breakdown of  of  + s  d i r e c t l y p r o p o r t i o n a l to the enzyme c o n c e n t r a t i o n .  the r e a c t i o n r a t e i s  120 yg  m  k^e =  value  color  also  ensure  enzyme.  Measurement of P r o t e i n Content  Many methods t i o n are a v a i l a b l e .  f o r the q u a n t i t a t i v e  Among them are :  estimation  of p r o t e i n i n s o l u -  42  Figure  2.5  PROTEOLYTIC ACTIVITY VS ENZYME CONCENTRATION  Enzyme s o l u t i o n : 10 days growth i n shake - O-  2% g l u c o s e  -A-  1% g l u c o s e  flask  E N Z Y M E C O N C . C m l of sample)  43  1.  UV A b s o r p t i o n  Most p r o t e i n s maximum a t residues  enhibit a distinct  280 mu, due p r i m a r i l y  [16,  63].  ultraviolet light  to the presence  absorption  o f t y r o s i n e and t r y p t o p h a n  S i n c e the abundance o f t y r o s i n e and t r y p t o p h a n v a r i e s  from p r o t e i n to p r o t e i n , t h i s method i s reasonably r e l i a b l e r e s u l t s mixtures of p r o t e i n s ,  i n g e n e r a l not r e l i a b l e .  However  can be o b t a i n e d w i t h v e r y heterogeneous  because,  i n this  case,  it  is  u n l i k e l y that  the  m i x t u r e would be s t r i k i n g l y r i c h o r poor i n any p a r t i c u l a r amino a c i d s . Nevertheless, and i s  d i r e c t photometry i s  more l i a b l e  2.  not as s p e c i f i c  as c o l o r i m e t r i c methods,  to i n a c c u r a c y due t o t u r b i d i t y .  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 a r e w i d e l y used f o r p r o t e i n e s t i m a t i o n .  A.  The c h e m i c a l bases o f  Folin  Phenols and p h e n o l i c amino a c i d s such as F o l i n ' s phenol reagent  [61]  in alkaline solution  phosphomolybdic - p h o s p h o t u n g s t i c In the presence biuret  of  copper i o n s ,  tyrosine react with  to form a reduced  a c i d complex which i s b l u e i n c o l o r .  additional color is  produced through a  complex f o r m a t i o n and through o t h e r mechanisms not  understood UV  these assays a r e :  [34].  T h i s method s u f f e r s  presently  form the same d i s a d v a n t a g e s  a b s o r p t i o n method i n t h a t any v a r i a t i o n i n t y r o s i n e c o n t e n t  of  as  the  the  44  samples w i l l i n v a l i d a t e  B.  the method.  Biuret  When b i u r e t , whose s t r u c t u r a l formula i s ted w i t h an a l k a l i n e potassium are j o i n e d  to form the  copper t a r t r a t e s o l u t i o n ,  following  complex v i o l e t  OH  OH  I  I  CONH.  I  Cu  NH  I  c o l o r e d compound [63]  NH  I  2  2  OH  OH  Any compound t h a t has i n i t s  molecular structure p a i r s  (-CONH^) l i n k e d through n i t r o g e n o r carbon (or p e p t i d e reaction  [65].  absorbing centre,  backbone and c u p r i c i o n s  :  of carbamyl  linkages)  S i n c e p r o t e i n c o n t a i n these l i n k a g e s ,  to g i v e 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 .  chromophore, o r l i g h t  :  I  I  react  two b i u r e t m o l e c u l e s  K-NH„CO  o  show the above  trea-  I  C0NH -K  groups  is  NH„CO  '  2  ^NCONHCONH^,  is  It  is believed  [16]  a complex between the  will  they that  peptide  the  45  0=C  NH  HN  NH  HN  I  I  I  I  S i n c e p e p t i d e bonds occur w i t h a p p r o x i m a t e l y p e r gram of m a t e r i a l f o r most p r o t e i n s , frequently  than w i t h  reagent i s  about  more m a t e r i a l i s  C.  the F o l i n - p h e n o l r e a g e n t .  100  times more s e n s i t i v e  total nitrogen.  less Folin  than the b i u r e t r e a c t i o n  [66],  method.  the most w i d e l y used methods o f p r o t e i n d e t e r m i n a t i o n  The method, involves  a c i d i n the presence o f suitable  are encountered  However, s i n c e the  r e q u i r e d f o r assay by the l a t t e r  the K j e l d a h l method, which i s  [67],  frequency  C h e m i c a l Method  One o f  Analysis  deviations  the same  catalyst.  based on the q u a n t i t a t i v e  estimation  as d e s c r i b e d i n the O f f i c i a l  digestion  of  mercuric s u l f a t e ,  of  Methods o f  the p r o t e i n w i t h c o n c e n t r a t e d  copper s u l f a t e ,  is  sulfuric  o r some o t h e r  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  m i x t u r e through steam d i s t i l l a t i o n  followed  by t i t r a t i o n o r a d d i t i o n of N e s s l e r ' s r e a g e n t ( m e r c u r i c potassium i n aqueous sodium h y d r o x i d e ) . determined  treatment  permits  ammonia to be  colorimetrically.  F o r the p h o t o m e t r i c is  The l a t t e r  iodide  and c o l o r i m e t r i c methods,  o b t a i n e d by comparison a g a i n s t  a standard s o l u t i o n  usually  c r y s t a l l i n e B o v i n e Serum A l b u m i n ;  average  protein is  The d e t a i l s o f  the p r o t e i n  of serum p r o t e i n ,  i n the K j e l d a h l method,  c a l c u l a t e d by assuming a n i t r o g e n c o n t e n t of  the B i u r e t method, which was used  are g i v e n i n Appendix  II.3.  content  throughout  the  16%.  t h i s work,  47  Chapter  3  RESULTS AND DISCUSSION  3.1  E f f e c t o f Medium C o n s t i t u e n t s  Preliminary studies revealed condensed  fish solubles  and p r o t e a s e of  production.  certain ingredients  effect [69]  alone  t h a t a medium c o n s i s t i n g  could s u f f i c e  However, i t  is  found t h a t c a l c i u m was n e c e s s a r y  production a l s o . phosphates  a w e l l known f a c t  diluted,  f o r good growth that  the  inclusion  i n a f e r m e n t a t i o n medium may have an enhancing  on p r o t e o l y t i c enzyme p r o d u c t i o n .  Fukumoto and Negoro  as a s u b s t r a t e  of  [70]  Merrill  and C l a r k  f o r good p r o t e a s e  showed t h a t p h o s p h a t e ( s )  Fukumoto et  al  [71]  [68]  yields,  and H a i n e s while  could stimulate  have shown t h a t  the e f f e c t  protease of  on enzyme p r o d u c t i o n was v e r y pronounced when c a r b o h y d r a t e s  were used as carbon s o u r c e s .  In  the p r e s e n t  investigation,  the f a c t o r i a l approach to  mentation was used to determine which of the above significant of  several  for further testing. factors  three i n g r e d i e n t s  Such an approach e n a b l e s  fewer  evaluation of i n t e r a c t i o n s  any  the p r e s e n t  f a c t o r s were used :  (1)  were  variation  at once i n accordance w i t h a p r e - a r r a n g e d p a t t e r n ,  thereby o b t a i n i n g more i n f o r m a t i o n w i t h p o s s i b l y  In  experi-  between v a r i a b l e s ,  investigation,  the  if  following  r u n s , and  (2)  exist.  l e v e l s of  the  three  48  Factor  Level 1  Level 2  Level 3  A.  Glucose concentration (gm/lOOml)  0  5  10  B.  Phosphate c o n c e n t r a t i o n (ml 1M s o l n . K^HPC^)  0  0.3  0.7  C.  Calcium concentration (ml 1M s o l n . C a C l added)  0  0.5  1.0  2  One r u n was made a t each c o m b i n a t i o n of experimental c o n d i t i o n s , 1 : 50 v / v  diluted,  specifications.  condensed  fish solubles  at  the d i f f e r e n c e  to the a d d i t i o n or d e l e t i o n  i n the i n c u b a t o r - s h a k e r ,  culture f i l t r a t e s  was measured.  The model assumed 73]  filtered, to  i n enzyme p r o d u c t i o n were of  the p a r t i c u l a r i n g r e d i e n t .  After  3  days  the p r o t e o l y t i c  The r e s u l t s  f o r the a n a l y s i s  completely  growth a t  a c t i v i t y of  are g i v e n i n T a b l e  of  this  rando-  experiment  =  y  +  °i  +  B  j  +  Y  k  +  ^hj  +  <«Y)  + l k  <PY)  j k  +  30°C  the  3.12.  is  given  :  ^IjlcA  27  t h i s might have on  27 experiments was  mized by w i t h d r a w i n g numbers from a b e a k e r .  [72,  100 mis o f a  medium made up a c c o r d i n g  t o examine what e f f e c t  The o r d e r f o r r u n n i n g the  by  to  the h i g h e r l e v e l s were added to the medium.  i n o t h e r words,  attributed solely  140 rpm  of  giving r i s e  A w h i t e p r e c i p i t a t e was formed when c a l c i u m c h l o r i d e and  No experiments were d e s i g n e d  and  thus  each of which c o n s i s t e d  potassium hydrogen phospate  enzyme y i e l d ;  factors,  (aBY>  i j k  Table  3.12  RESULTS OF FACTORIAL EXPERIMENTS  SUGAR LEVEL (A) 2  1  PHOSPHATE  3  CALCIUM LEVEL (C) 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  LEVEL (B)  V a l u e s a r e i n enzyme u n i t s / 10 c c .  50  where  y . .,  represents  of  factor A, j "  is  the o v e r a l l mean,  1  level  the  o b s e r v a t i o n taken a t the  of f a c t o r B , and the  a.  the e f f e c t  of  the  k** til  level  l e v e l of f a c t o r C;  1  i  i ^  l e v e l of  u  factor A,  g. 3  i the e f f e c t  of  the  j  th.  level  of factor  B , and  the e f f e c t  of  the  ttl  k  l e v e l of factor C.  y.  values  i n the  e...  (ijk)  measures cell  c n  the d e r i v a t i o n s o f  the  from the p o p u l a t i o n mean  o t h e r terms s t a n d f o r i n t e r a c t i o n s between the main f a c t o r s It  is  u s u a l l y assumed t h a t  the sums o f the main e f f e c t s  observed u.  a,  The  (3, and  as w e l l as  y>  the  sums of the two - way i n t e r a c t i o n e f f e c t s  summed on e i t h e r s u b s c r i p t 3 e q u a l zero f o r any v a l u e o f the o t h e r s u b s c r i p t ( i . e . £ (cB) . . = 0, e t c . ) i=l and t h a t the sum o f the t h r e e - way i n t e r a c t i o n e f f e c t s summed on any one 1  of  the s u b s c r i p t s  is  zero f o r any v a l u e s  J  o f the o t h e r two s u b s c r i p t s  (i.e.  3 £ i  =  l  (aBy) . J 1  R  = 0,  etc.).  f o r the parameters o f  These r e s t r i c t i o n s w i l l  the m o d e l .  In o r d e r t h a t v a l i d s i g n i f i c a n c e assumed t h a t  the e r r o r s are v a l u e s  random v a r i a b l e s ,  t e s t s can be made, i t  analysis  of variance f o r t h i s  given i n Table 3.13.  o b t a i n i n g the sums of squares  is  also  o f independent and n o r m a l l y d i s t r i b u t e d  each w i t h zero mean and common v a r i a n c e  The complete experiment i s  a s s u r e unique e s t i m a t e s  2  a .  three-factor  The c o m p u t a t i o n a l p r o c e d u r e s  for  are s p e l l e d out i n Appendix I I I .  Each treatment c o m b i n a t i o n d e f i n e s each c e l l c o n t a i n s n observations.  a cell.  If  there are  n  replications,  T a b l e 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*  AB  0.90  4  0.23  3.83  AC  2.99  4  0.75  12.50*  BC  0.25  4  0.06  1.00  0.51  8  0.06  Two F a c t o r I n t e r a c t i o n s  Three Factor I n t e r a c t i o n s ABC  Note  *  SS(ABC)  was t a k e n as  F  2,8,0.05 = '  F  4,8,0.05 "  SSE  4 6  F  2,8,0.01 =  8  '  6 5  3 , 8 4  F  4,8,0.01 "  7  '  0 1  4  These f a c t o r s a r e s i g n i f i c a n t a t the  0.01  level.  52  Note t h a t s i n c e conditions,  o n l y one o b s e r v a t i o n was made at each s e t  a w i t h i n - c e l l mean square cannot be used as an e s t i m a t e o f  the t r u e v a r i a n c e of  the system.  assumed to be n e g l i g i b l e  Instead,  and t h a t  SS(ABC)  to e x p e r i m e n t a l e r r o r and thereby p r o v i d e s variance. using  of  The  F  the  ABC  i n t e r a c t i o n was  represents  v a r i a t i o n due  an e s t i m a t e of  the  solely  error  s t a t i s t i c i n the A n a l y s i s of V a r i a n c e t a b l e was  calculated  M S ^ .  The f o l l o w i n g  conclusions  A n a l y s i s of V a r i a n c e t a b l e  can be made from the  F  ratio in  the  :  (1)  Sugar and c a l c i u m e f f e c t s  are s i g n i f i c a n t  (2)  I n t e r a c t i o n e x i s t s between the d i f f e r e n t  at  the  0.01  level.  and d i f f e r e n t  calcium  (3) below the  0.05  The F - r a t i o f o r g l u c o s e - phosphate c r i t i c a l value;  0.01  None of  there i s  interaction is  not enough e v i d e n c e does not  or  3.2.  E f f e c t of Glucose C o n c e n t r a t i o n  to  barely conclude  exist.  the o t h e r F - r a t i o s are s i g n i f i c a n t  0.05  at e i t h e r  the  level.  The a n a l y s i s glucose present  concentrations  concentrations.  t h a t i n t e r a c t i o n between these two f a c t o r s  (4)  sugar  of v a r i a n c e t a b l e  i n the medium i s  indicated  that  the amount of  the most important f a c t o r a f f e c t i n g  enzyme  53  yield.  The e f f e c t  to develop  of  t h i s i n g r e d i e n t was f u r t h e r s t u d i e d  a medium y i e l d i n g maximal p r o t e i n a s e  i n an  production.  The media used f o r t h i s study were b o t h f i l t e r e d 1  : 50 v / v d i l u t e d ,  or phosphates. the course of between  0  condensed  Figures 3.6,  f i s h s o l u b l e s without 3.7  and 3.8  40 gm/1  added c a l c i u m s a l t s  show the changes o c c u r i n g d u r i n g  i n both f i l t e r e d  there was no easy method to s e p a r a t e  the i n s o l u b l e  proteins  the average  of d u p l i c a t e  The r e s u l t s i n d i c a t e media,  runs ( S e c t i o n  that,  c l e a r l y demonstrated  of  increased  growth.  growth.  It  that  for both f i l t e r e d  is  The changes i n the  that p r o t e a s e p r o d u c t i o n i s growth i s  the e f f e c t  of  or  4%  to such low  and u n f i l t e r e d  2%  indicates  that  production.  the  (3.4  filtered  glucose,  glucose.  It one  a l s o apparent t h a t p r o t e a s e p r o d u c t i o n l a g s pH  of  the medium d u r i n g i n c u b a t i o n suggest  h i g h e r i n media d e v e l o p i n g  g l u c o s e may be due to d e s t r u c t i o n values  1%  the added c a r b o h y d r a t e was  g e n e r a l l y h i g h e r i n media d e v e l o p i n g  pH  these  Each r e p o r t e d  an a l k a l i n e  an a c i d  The d r a s t i c drop i n enzyme y i e l d on the e i g h t day w i t h media 3%  bugs,  2-5).  w h i l e b e s t growth was o b t a i n e d w i t h medium c o n t a i n i n g  while  from the  the y i e l d o f p r o t e a s e was maximum i n medium c o n t a i n i n g  is  Since  The d a t a f o r  are t a b u l a t e d i n T a b l e s A I . l , A I . 2 o f Appendix I .  value i s  varied  and u n f i l t e r e d m e d i a .  the growth i n the u n f i l t e r e d media was not measured. figures  and u n f i l t e r e d ,  f e r m e n t a t i o n when the g l u c o s e c o n c e n t r a t i o n was  and  attempt  to 4 . 1 ) .  of  reaction. containing  the enxyme when exposed  Comparison of F i g u r e 3.6  fish solubles is  reaction,  a b e t t e r medium f o r  and  3.8  protease  54  Figure  EFFECT  OF  GLUCOSE CONCENTRATION  3.6  ON A C T I V I T Y  TIME  -  (days)  UNFILTERED  CFS  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 s h o u l d be p o i n t e d out h e r e t h a t a u t o c l a v e d the growth o f c e r t a i n s p e c i e s o f b a c t e r i a [ 7 4 ] . t h i s work were c a r r i e d out w i t h a u t o c l a v e d  g l u c o s e can  inhibit  A l l the e x p e r i m e n t s i n  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 o f a u t o c l a v e d s u g a r s on the growth o f Sorangium 495 were not  taken  into consideration.  3.3  E f f e c t of I n i t i a l The  pH  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  p r o t e a s e 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 f i s h s o l u b l e s and was  1%  glucose.  The  initial  a d j u s t e d to v a r i o u s v a l u e s by the a d d i t i o n of  During its  containing  1 : 50. v/v  own  the c o u r s e  o f i n c u b a t i o n , the  l e v e l , the f i n a l  chemical s t a b i l i z a t i o n  pH  pH  was  2N  pH  o f the system.  tends t o r i s e  from the d e c o m p o s i t i o n pH  pH  o f the medium  sodium  hydroxide.  v a l u e ( a t the end o f 8 days) r e p r e s e n t i n g  r e s u l t s a r e shown i n T a b l e 3.14.  established  d i l u t e d , condensed  n o t a d j u s t e d b u t l e f t to seek  The d r y c e l l w e i g h t and  a c t i v i t y o f the b r o t h were measured a t the f o u r t h and The  The  the p r o t e o l y t i c  e i g h t h day o f growth.  r e s u l t s i n d i c a t e t h a t the  ( p r o b a b l y due  8.0.  The  initially  t o the f o r m a t i o n o f ammonia  of p r o t e i n ) d u r i n g the c o u r s e o f growth;  b e i n g s l i g h t l y above  and  d a t a show t h a t an i n i t i a l  i s b e s t f o r enzyme p r 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  the pH  final  of  i s best f o r  growth.  3.4  E f f e c t o f Condensed F i s h S o l u b l e s  I t has been r e p o r t e d  Concentration  [75, 76, 77]  7.0  t h a t many organisms produce  58  Table  3.14  EFFECT OF INITIAL  AFTER INITIAL  pH  pH  4  pH  DAYS  AFTER  DRY CELL WEIGHT  PROTEOLYTIC ACTIVITY  gm/£  units/10cc  pH  8  DAYS  DRY CELL WEIGHT  PROTEOLYTIC ACTIVITY  gm/£  units/lOcc  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 c u l t u r e medium the b e l i e f  of a p a r t i c u l a r s u b s t r a t e i n  " a d a p t i v e p r o d u c t i o n of enzymes."  t h a t a medium r i c h i n p r o t e i n i s  high yields  of p r o t e o l y t i c  enzymes.  the amount o f  This evidence led  F i g u r e s 3.9  course  of growth and p r o t e a s e p r o d u c t i o n r e s p e c t i v e l y ,  content higher  than 3.85  production. Appendix  filtered  condensed  and 3 . 1 0 ,  of i n c r e a s i n g  of  fish  BSA/ml  The d a t a f o r these f i g u r e s  i n F i g u r e 3.10)  against protein concentration is  the maximum a c t i v i t y  from t h i s  figure  by a v a i l a b i l i t y  are tabulated  i n Figure 3.11.  that  protein  i n T a b l e A I . 3 of  attained  of p r o t e i n n i t r o g e n  (the  i n each medium.  of  the h i g h e r s o l u b l e s  to i n h i b i t o r y c o n c e n t r a t i o n s  compounds,  is  indicated  at  plotted  i n the same is  apparent  v a l u e o f 5.9  limited  also  That the d e t r i m e n t a l  effect  on p r o t e a s e f o r m a t i o n c o u l d be of p r o t e i n or p e r h a p s ,  i n Figure 3.9.  the h i g h e r p r o t e i n c o n c e n t r a t i o n s  that obtained  pH  phase  r a t e o f p r o d u c t i o n and y i e l d  enzyme are l i m i t e d by s u b s t r a t e i n h i b i t i o n .  attributed  It  are  protease production is  final  at high p r o t e i n l e v e l s ,  concentrations  (slope of exponential  Also plotted  of  growth at  demonstrate  i n units/10 c c . h r .  t h a t at low p r o t e i n l e v e l s ,  support t h i s view);  nitrogen  time  I.  the p r o d u c t i o n curves  figure  s o l u b l e s on the  the  were markedly i n h i b i t o r y f o r enzyme  The maximum p r o t e a s e p r o d u c t i o n r a t e s of  which show  s o l u b l e s h a v i n g an average s o l u b l e  mg  to  r e q u i r e d f o r the p r o d u c t i o n of  effect  concentrations  the  the optimum c o n c e n t r a t i o n  This f i g u r e  was as  good a s ,  for protease  of  the  organic  shows t h a t or b e t t e r  formation.  than,  Figure  3.9  EFFECT OF CONDENSED FISH SOLUBLES CONCENTRATION ON GROWTH  40  80 120 T I M E (hrs.)  160  61  EFFECT  Figure  3.10  OF CONDENSED  FISH  ON P R O T E A S E  SOLUBLES  CONCENTRATION  FORMATION  12  VOL.  CFS  I O O O cc  10  in H^O  20CC  o  2  8  N  30 CC  CO  -4—•  c  o <  60C C  40  80 120 T I M E (hrs.)  160  62  Figure EFFECT  OF P R O T E I N  CONCENTRATION  AND ON M A X I M U M R A T E  2 4 PROTEIN  3.11  OF P R O T E A S E  6 CONC.  ON U L T I M A T E  PROTEASE  YIELD  FORMATION  8 10 ( mg. BSA/ml.)  63  The r e s u l t s affected  form these s t u d i e s i n d i c a t e  t h a t enzyme y i e l d  by the r a t i o of carbon to n i t r o g e n i n the medium.  w i t h Sorangium 495 were o b t a i n e d i n a f i s h s o l u b l e s nitrogen equivalent  3.5  to 3.85  mg B S A / m l ,  Best  results  medium c o n t a i n i n g  and 10 gm/1 of  glucose.  E f f e c t of C a r b o h y d r a t e Source  Glucose i s  commonly used as  the energy and c a r b o n s o u r c e  m i c r o b i o l o g i c a l media f o r l a b o r a t o r y growth e x p e r i m e n t s . wastes h i g h i n c a r b o h y d r a t e s are more s u i t a b l e  i n spent s u l f i t e galactose, thought  liquor,  f o r commercial p u r p o s e s .  these d i f f e r e n t medium [78], species,  t h e r e a r e the hexoses - g l u c o s e ,  and the pentoses - x y l o s e and a r a b i n o s e .  to be u s e f u l  to i n v e s t i g a t e  sugars  mannose and  I t was  as the carbon and energy s o u r c e s .  example,  therefore incorporating  S i n c e the McCurdy  a medium used f o r c u l t i v a t i n g a wide spectrum of m y x o b a c t e r i a  contains  a h i g h amount o f s o l u b l e  The medium used i n t h i s fish solubles,  graphed i n F i g u r e s 3 . 1 2 , o f Appendix I .  f o r the  experiment  and  1 gm/1  3.13  and 3.14,  These r e s u l t s  starch,  indicates  contained f i l t e r e d ,  of carbohydrate.  this  evaluated.  2% v / v  The r e s u l t s  are  and t a b u l a t e d i n T a b l e s A I . 4 and  indicate  that b a c t e r i o l y s i s  c o n t a i n i n g s t a r c h or p e n t o s e s .  the s u i t a b i l i t y o f  f i s h media was a l s o  that  r e a d i l y on mannose and g l u c o s e than on g a l a c t o s e , F i g u r e 3.14  for  the performance o f c u l t u r e s  p o l y s a c c h a r i d e as an energy supplement  condensed  in  However, i n d u s t r i a l  These wastes u s u a l l y c o n t a i n a m i x t u r e o f monosaccharides;  AI.5  is  the organism grew more s t a r c h or pentoses.  i s more pronounced i n media  The d a t a demonstrate  quite  clearly  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.)  66  Figure 3.14  EFFECT OF CARBOHYDRATE SOURCE ON GROWTH  T I M E (hrs.)  67  good growth i s  necessary  that glucose i s growth.  better  for high y i e l d s  for protease  The b i p h a s i c n a t u r e of  of enzyme.  the p r o t e a s e  Again, the d i f f e r e n t  it  in later  [79],  optical activity,  The degree of  sensitive being,  for  The b i p h a s i c growth i n  the e f f e c t  of s t e r i l i z a t i o n upon discussion.  a u t o c l a v i n g sugar s o l u t i o n s  (2)  change d i f f e r s  amongst o t h e r s ,  evident  p r o d u c t i o n curve i n medium  sugars was not taken i n t o account i n the above  changes i n (1)  3.6  also  sections.  s h o u l d be noted t h a t  A c c o r d i n g to D a v i s and Rogers  power.  is  f o r m a t i o n , and mannose i s b e t t e r  c o n t a i n i n g mannose was not f u r t h e r i n v e s t i g a t e d . g l u c o s e w i l l be d i s c u s s e d  It  p H , (3)  c a r a m e l i z a t i o n and (4)  from one s u g a r to a n o t h e r ,  dextrose  can cause reducing  the most  and a r a b i n o s e .  E f f e c t of Inoculum Age  Inoculum age between 16 and 24 h r s . , which i n c l u d e d ages from e a r l y l o g phase  to d e c e l e r a t i o n  on y i e l d and r a t e of p r o d u c t i o n .  phase of g r o w t h , was  w i t h 1%  glucose)  2% v / v  effect  diluted  are shown i n F i g u r e s 3 . 1 5 ,  and are t a b u l a t e d i n T a b l e A I . 6 of Appendix I . significant  effect  The changes o c c u r i n g d u r i n g the  o f i n c u b a t i o n i n " o p t i m a l " medium ( f i l t e r e d , fish solubles  tested for  on growth, p r o t e a s e  condensed  3.16  These r e s u l t s  course  and  3.17  indicate  no  p r o d u c t i o n , and g l u c o s e 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  AGE  0  40 80 TIME(hours)  120  160  70  Figure  3.17  E F F E C T OF INOCULUM AGE ON PROTEASE FORMATION  71  3.7  7-litre  Fermentation Studies  All flasks a set  the above experiments were c a r r i e d out i n  containing  100 ml  of  f i s h medium.  On the b a s i s  of  Erlenmeyer  these  results,  o f " o p t i m a l " c u l t u r a l c o n d i t i o n s was chosen f o r maximum p r o t e a s e  production. possible  These "optima" a r e , at b e s t ,  to study f u l l y the i n t e r a c t i o n s  protease y i e l d s ; and a e r a t i o n .  approximations, since of the v a r i o u s f a c t o r s  a l s o i t was not p o s s i b l e  In t h i s  section,  to study the e f f e c t s  experiments  to i n v e s t i g a t e  these two p r o c e s s v a r i a b l e s on enzyme y i e l d s  litres  of f i l t e r e d ,  glucose,  2%  and i n i t i a l  pH  Eight 7 - l i t r e Broth -  d i l u t e d condensed f i s h s o l u b l e s  a d j u s t e d to  7.0,  3;  was  f e r m e n t a t i o n s were r u n .  i n F i g u r e 3.18  and g l u c o s e , and T a b l e A I . 7  runs were made w i t h the f i s h s o l u b l e s 300 rpm  rate of 2 £/min.  to  1 &/min  750 rpm.  F i v e of  10.  that of  affect  agitation  with  4 1%  used.  One o f these used a N u t r i e n t  The r e s u l t s  of Appendix I . medium;  of  The 7 - l i t r e  A medium c o n s i s t i n g o f  g l u c o s e medium, the c o m p o s i t i o n o f which was,  D i f c o Beef E x t r a c t , sented  v/v  i t was not  the i n f l u e n c e  are d e s c r i b e d .  fermentor d e s c r i b e d i n S e c t i o n 2.4 was u s e d .  from  250 ml  in of  gm/£, this  peptone,  r u n are  The remainder o f  prethe  a g i t a t i o n r a t e was v a r i e d  these runs were made at an a e r a t i o n  w h i l e the o t h e r two were performed at an a e r a t i o n r a t e o f  In no case was an e n t i r e run r e p e a t e d .  A l t h o u g h an  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 p e r f o r m e d , no attempt was made to r e g u l a t e  the  pll  5;  NBS  automatic  pH  by the automatic a d d i t i o n of a c i d or base d u r i n g the course o f  the  72  fermentation.  I t was thought t h a t t h e h i g h p r o t e i n c o n t e n t o f t h e medium  provided the necessary b u f f e r ; such as t o keep t h e  pH  the r a t i o o f c a r b o h y d r a t e t o p r o t e i n was  o f the medium w i t h i n  ±1  of n e u t r a l i t y .  Due t o  the h i g h c o n t e n t o f s u r f a c e a c t i v e agents i n the medium, t h e r e was c o n s i d e r a b l e foaming.  S i n c e an a u t o m a t i c a n t i f o a m a d d i t i o n system was n o t  a v a i l a b l e a t the t i m e , s m a l l amounts o f defoaming 2%  agent i n t h e form o f a  s o l u t i o n o f A n t i f o r m B was added, whenever n e c e s s a r y , through t h e  i n o c u l u m p o r t o f t h e f e r m e n t o r 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 o f A g i t a t i o n and A e r a t i o n on t h e Course o f F e r m e n t a t i o n The time v a r i a t i o n s o f  pH,  sugar u t i l i z a t i o n ,  proteolytic  a c t i v i t y and d r y c e l l w e i g h t f o r t h e 7 - l i t r e f e r m e n t a t i o n s a r e p r e s e n t e d i n F i g u r e s 3.19 t o 3.25 Appendix  I.  and a r e t a b u l a t e d i n T a b l e s AI.8 t o AI.14 o f  These r e s u l t s i n d i c a t e t h a t the r e l a t i v e amounts and times t o  maximum as w e l l as t h e shape of the growth curve a r e 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 f l o w r a t e . f o r m a t i o n appear  t o be i n t e r r e l a t e d .  which sugar u t i l i z a t i o n i s s m a l l , y e t begun.  Sugar d i s a p p e a r e a n c e ,  pH  pH,  and p r o t e a s e  An i n d u c t i o n p e r i o d was n o t e d d u r i n g r i s e s , and p r o t e a s e f o r m a t i o n has n o t  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 * d e c r e a s e s a t a  n o n u n i f o r m r a t e toward a zero v a l u e .  I n t h e same i n t e r v a l , t h e pH i s  The f i g u r e s a r e a c t u a l l y p l o t t e d i n terms o f 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 f l o w r a t e : 1 £/min)  TI M E  (hrs.)  74  T a b l e 3.19  7-LITRE FERMENTOR  CFS  RUN NO. 1  ( A g i t a t o r speed : 300 rpm; A i r f l o w r a t e : 1 £/min)  9 X  o o  Q.  o  £121  "E  •©-  D  •O-  >•  • A-  >  §  •• -  8  ®  pH CELLS ACTIVITY GLUCOSE  Q UJ  .8  C/)  CO  Z )  UJ  L U  C/)  .4 O  O O D _j  O  80  TI M E (hrs.)  120  0  75  Figure  7-LITRE FERMENTOR ( A g i t a t o r speed  3.20  CFS  RUN NO. 2  : 400 rpm; A i r flow r a t e  : 1 £/min)  H 9 ©  O O  ©  e  X a  o  \ CO  £ 12 c 10  o <  PH o - CELLS A — ACTIVITY • — GLUCOSE  1.2 Q L U  o-o  CO  D  8 u> GO  L U  CO  O O  •4 ^  O  o  4  _l  40  80  T I M E (hrs.)  120  a  GLUCOSE O  U S E D (g/1 ); ACTIVITY(units/iocc.) -N  CO  to  \  >  i  H-  /  m  J  1  rt  9  o  I tr  P>  I  rr O H  ®  cn  T)  ro ro a.  H O  o o  n  Ul  H  CO  CK)  e i-!  C/2  3  \  CO  o  >  i-i l-tl t—'  2! O U>  § e  3  00  CELLS(g/l)  MO  o  pH  ON  G L U C O S E USED(g/l);  A C T I V I T Y (units/iocc.)  78  F i g u r e 3.23  7-LITRE FERMENTOR  CFS  RUN NO. 5  ( A g i t a t o r speed : 750 rpm; A i r f l o w r a t e : 1 2,/min)  8 X Q_  e  O  o  /  L  /  •  o •  0  /  '  I  20  1.6  • A  - 7 T  A  Cf)  /  /  UJ  .8 O  PH  o  p  O  -  O  -  -  A —  -  •  —  CELLS ACTIVITY  GLUCOSE)  L  40 T I M E (hrs.)  60  0  79 F i g u r e 3.24  7-LITRE FERMENTOR  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 Jl/min)  0  20  40  T I M E (hrs.)  60  80  Figure  7-LITRE FERMENTOR  CFS  3.25  RUN  (Agitator speed : 500 rpm;  TIM E ( hrs)  NO.  7  A i r flow rate : 2 fc/min)  81  noted to reach a maximum and then decrease of  enzyme r i s e s  cases,  to a minimum.  i n a sigmoidal fashion u n t i l  the p r o t e a s e  a peak i s  The c o n c e n t r a t i o n  reached.  In a l l  p r o d u c t i o n curve l a g s b e h i n d the growth i n a f a s h i o n  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 namely: (3)  (1)  growth curve o f F i g u r e 3.19  phase of i n i t i a l  a phase o f secondary growth and  s t a t i o n a r y phase. from to  growth,  40 hours 400 rpm  500 rpm  to  20 h o u r s ,  ( F i g u r e s 3.19  and  eliminated;  The d u r a t i o n o f  (Figures 3.24,  to  3.25).  2 &/min  The e f f e c t was t h e r e f o r e However F i g u r e 3.18  rates, completely and t h i r d  aeration  thought to be r e l a t e d  phase to  ( T a b l e A I . 7 o f Appendix I)  the n i t r o g e n s o u r c e may have c o n t r i b u t e d  to  effect.  400 rpm  as  and the a e r a t i o n r a t e was  o c c u r r e d , and v e r y l i t t l e  represents  the r e s u l t s  the n i t r o g e n s o u r c e ;  t h a t m u l t i p l i c a t i o n proceeded up to the  of  the  300 rpm  a l s o c o m p l e t e l y e l i m i n a t e d the second  made w i t h peptone and meat e x t r a c t  lysis  the f i r s t  Increasing  phase,  decreased,  the second p o r t i o n was 750 rpm,  phases,  and  At s t i l l higher a g i t a t i o n  As mentioned e a r l i e r , F i g u r e 3.18  r a t e was  deceleration  as the a g i t a t o r speed i n c r e a s e d from  agitation rate,  the n a t u r e o f  4  s t a t i o n a r y or death  a final  a s i g m o i d a l growth c u r v e .  mass t r a n s f e r l i m i t a t i o n . indicated that  first  the second phase was g r e a t l y  and 3 . 2 0 ) .  at the h i g h e s t  1 £/min  the o v e r a l l  (4)  600 rpm, the death phase o f  phase meshed to g i v e r a t e from  (2)  can be d i v i d e d up i n t o  15  1 £/min.  This  agitation  figure  indicates  h o u r , a f t e r which e x t e n s i v e  g l u c o s e was u t i l i z e d .  the c u l t u r e b r o t h was o b v i o u s l y due to  the  o f a run  The r i s e i n the  the p r o d u c t s o f  nitrogen  pH  82  metabolism.  The  l a r g e amount of unused g l u c o s e may  have c o n t r i b u t e d  the e x t e n s i v e  l y s i s observed a f t e r growth ceased [80, 8 1 ] .  appears to be  the l i m i t i n g f a c t o r f o r growth and  to  Nitrogen  protease production  in  t h i s case.  The and  3.20  f i r s t and second p o r t i o n s of the growth c u r v e of F i g u r e s  c o r r e s p o n d a p p r o x i m a t e l y , to the growth i n the N u t r i e n t B r o t h  g l u c o s e medium. as r e p r e s e n t i n g initially  These two p o r t i o n s of the growth c u r v e may  be  -  interpreted  the d e p l e t i o n of f r e e amino a c i d s and n i t r o g e n o u s  p r e s e n t i n the condensed f i s h s o l u b l e s medium.  3.19  bases  A f t e r the  deple-  t i o n of the i n i t i a l s i m p l e n i t r o g e n o u s m a t e r i a l s , b a c t e r i a l m e t a b o l i s m appears t o be l i m i t e d by the r a t e a t w h i c h the p r o t e o l y t i c enzyme convert  the l a r g e r p o l y p e p t i d e s  to s i m p l e r forms.  Apparently,  can  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 , w h i c h i s i n t u r n 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  a t d i f f e r e n t r a t e s , they a l l p r o c e e d s i m u l t a n e o u s l y . rates coupled w i t h  The  difference i n  the i n i t i a l p r e s e n c e 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  s o u r c e caused the s t a g e d n a t u r e of the growth k i n e t i c s . c o n s i s t e n t w i t h S p e r r y and R e t t g e r ' s  [82]  These r e s u l t s are  c o n c l u s i o n s , w h i c h are quoted  below :  The  i n a b i l i t y of b a c t e r i a t o decompose n a t i v e p r o t e i n s i s  n o t l i m i t e d t o aerobes and well-known and are d e v o i d may  occur  f a c u l t a t i v e anareobes, b u t  extremely a c t i v e p u t r e f a c t i v e  of t h i s p r o p e r t y .  even  anaerobes  S o l u t i o n s of n a t i v e  undergo complete h y d r o l y s i s , however, i f they  proteins contain  peptone or some o t h e r n i t r o g e n o u s food m a t e r i a l w h i c h  83  r e a d i l y f u r n i s h e s the n e c e s s a r y n i t r o g e n f o r b a c t e r i a l development.  I n such i n s t a n c e s  the p r o t e o l y s i s o f  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 w h i c h has been e l a b o r a t e d by  the b a c t e r i a  during  the p r o c e s s of r a p i d m u l t i p l i c a t i o n . T h i s 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 w h i c h  i s p r e s e n t a l o n g 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 t o d i r e c t decomposi-  t i o n by b a c t e r i a i s not due the p r o t e i n s , b u t  to any  antiseptic properties  of  t o a c o n s t r u c t i o n of the m o l e c u l e w h i c h  r e n d e r s 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 b e i n g f i r m l y bound t o g e t h e r  so  that strong cleavage - producing  a g e n t s , such as extreme h e a t , s t r o n g a c i d s 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 t h a t 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  R e t t g e r , Berman and  nutrition.  S t u r g e s [83] c a r r i e d the m a t t e r a s t a g e  f u r t h e r when they showed t h a t h e a t - c o a g u l a t e d  albumin,  and  sometimes  peptones are n o t 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 A r c h e r e t a l [ 8 4 ] . 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 c o n c e n t r a t e  B a c i l l u s S u b t i l i s p r o t e a s e (Monzyme), c o u l d be d e s c r i b e d by a sequence of two  (FPC)  by  they found t h a t the o v e r a l l k i n e t i c s f i r s t - o r d e r p r o c e s s e s - an  f a s t r e a c t i o n i n w h i c h l o o s e l y bound p o l y p e p t i d e  by  initial,  c h a i n s were c l e a v e d ,  a second, s l o w e r r e a c t i o n i n w h i c h more compacted p r o t e i n was S i m i l a r b i p h a s i c k i n e t i c s were o b t a i n e d  While  and  digested.  M i h a l y i and H a r r i n g t o n  [85].  84  F i g u r e 3.26 fish solubles detected.  shows that when the Sorangium s p e c i e was grown i n a  medium w i t h o u t  A comparison of  added g l u c o s e ,  this  staged k i n e t i c s were  f i g u r e w i t h F i g u r e 3.19  the maximum enzyme c o n c e n t r a t i o n a t t a i n e d i n the g l u c o s e media corresponds to the v a l u e a t the end o f  indicates -  the second phase o f  The r e s u l t s  u n a b l e , i n the absence  o f a s o u r c e o f c a r b o h y d r a t e , to b r i n g  points  the l a r g e r p o l y p e p t i d e u n i t s p r e s e n t  of this  about The d a t a  Shake f l a s k  cultivation  results.  of a g i t a t i o n and a e r a t i o n on u l t i m a t e y i e l d s  summarized i n T a b l e 3 . 1 5 . 500 rpm  with  The h i g h e s t  ultimate protease a g i t a t i o n rates  protease  2 litres  However, more  y i e l d was o b t a i n e d but at a much f a s t e r  the end o f  Aeration rates  and a g i t a t i o n  on c e l l metabolism -  rates  f o r example,  at  1 5,/min, t o t a l sugar consumption, p r o t e a s e  with  the  same  Other of  initial  to be a p p r o x i m a t e l y  same at a l l l e v e l s o f a g i t a t i o n and a e r a t i o n t e s t e d ; 750 rpm  rate.  The p e r c e n t a g e  f e r m e n t a t i o n was noted  sugar consumption r a t e o c c u r r e d at  significantly  o f a i r p e r m i n u t e , almost  r e s u l t e d i n lower p r o d u c t i v i t y .  sugar u t i l i z e d at  are  p r o d u c t i o n o c c u r r e d at  1 l i t r e of a i r introduced per min.  at the same a g i t a t o r speed and  effect  the organism was  E f f e c t o f A g i t a t i o n and A e r a t i o n on U l t i m a t e Y i e l d s  The e f f e c t s  minute.  growth  i n the medium.  f i g u r e are t a b u l a t e d i n T a b l e A I . 1 5 .  was used to o b t a i n these  3.7.2  t h e r e f o r e suggest t h a t  that  deficient  shown i n F i g u r e 3 . 1 9 .  breakdown o f  not  however  the  the  highest  1 l i t r e of a i r per  are interdependent 600 rpm  in  their  and an a i r flow o f  p r o d u c t i o n , and growth are  almost  Figure 3.26  SHAKE FLASK FERMENTATION - GLUCOSE DEFICIENT MEDIUM  T I M E (hrs.)  Table  3.15  EFFECT OF AGITATION AND AERATION ON ULTIMATE YIELDS  MEDIA  AIR FLOW RATE (£/min)  AGITATOR SPEED rpm  DRY WEIGHT MAX  (g/*)  TIME TO MAX (hrs)  CONDENSED  1  FISH  2  8.84  134  96.3  134  400  0.85*  94  9.95  116|  95.0  116|  500  1.29  95.4  77  600  1.62  35  7.82  4  94.4  4  750  2.05  4  6.94  45  92.4  45  400  1.55  4  7.74  47  94.3  56  1.55  16  9.86  45  94.1  4  1.00  32  2.85  45  -  -  0.83  15  2.28  45  500 ABOVE MEDIUM minus 1% GLUCOSE NB p l u s 1% GLUCOSE  SHAKER INCUBATOR  1  G l o b a l maximum  TIME TO MAX (hrs)  3 113f  1% GLUCOSE  MAX TIME TO MAX (%initial) (hrs)  1.00*  SOLUBLES +  MAX (units/lOcc)  SUGAR UTILISATION  300 FILTERED 2% v / v  PROTEOLYTIC ACTIVITY  400  10.52  16.0  i  34  87  identical  to those a t an a i r flow of  F i g u r e 3.27 against  glucose  is  used.  2£/min  and  400 rpm.  a p l o t of c e l l s p r o d u c e d , and p r o t e o l y t i c  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  activity is  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 T r a n s f e r  The f u n c t i o n s are mainly to c r e a t e the r e s i s t a n c e  of a g i t a t i o n and a e r a t i o n i n a e r o b i c  a l a r g e a i r - l i q u i d i n t e r f a c i a l a r e a and to  to oxygen d i f f u s i o n by r e d u c i n g the t h i c k n e s s  f i l m s u r r o u n d i n g each b u b b l e  of  Table 3.16.  K^,  al  [87]  to c o r r e l a t e the  y i e l d and the maximum r a t e o f p r o t e a s e  It  or that Yamada's c o r r e l a t i o n f o r  the  shown i n  shown i n F i g u r e  i n b r i n g i n g together  appears t h e r e f o r e  data  t h a t mass t r a n s f e r  not the o n l y c o n t r o l l i n g f a c t o r i n the p r o d u c t i o n of p r o t e o l y t i c  system,  liquid  v a l u e s w i t h the maximum  formation i s  This method o f c o r r e l a t i o n was u n s u c c e s s f u l the v a r i o u s systems s t u d i e d .  the  was used to c a l c u l a t e  the v o l u m e t r i c oxygen t r a n s f e r c o e f f i c i e n t ,  An attempt  of  reduce  [86].  The c o r r e l a t i o n of Yamada e t values  fermentors  3.28. for is  enzyme  K i s not a p p l i c a b l e to our f e r m e n t a t i o n v and t h a t a f u r t h e r i n v e s t i g a t i o n of t h i s s u b j e c t i s n e c e s s a r y .  88  Figure  ACTIVITY  AND  FOR VARIOUS  3.27  CELL YIELD  VS  SUGAR  7 - L I T R E FERMENTOR  USED  RUNS  89  T a b l e 3.16  K  AIR  FLOW RATE  v  VALUES ATTAINED I N 7-L FERMENTATION RUNS  AGITATOR SPEED  £/min  rpm  K  (dp/dt)max V  millimoles/atm.  min. l i t r e  units/lOOcc.hr  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  400  0.452  3.60  500  0.805  6.90  1  2  C o r r e l a t i o n u s e d was :  K  This  = 8.06  x IO  - 8  x v°-  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  stainless steel,8-1 and  v  a  capacity  d i s k type a g i t a t o r .  3  x  N ' 2  5  6  b y Yamada e t a l (87) u s i n g  fermentor f i t t e d w i t h  a single hole  a  sparger  90  F i g u r e 3.28  MAXIMUM Y I E L D AND MAXIMUM RATE OF ENZYME PRODUCTION AS A FUNCTION OF  K v  K > mmoles/atm. min. litre v  91  Chapter  4  FERMENTATION KINETICS  Batch k i n e t i c d a t a are needed n o t o n l y to develop b a s i c u n d e r s t a n d i n g of  fermentation processes  continuous f e r m e n t a t i o n systems.  but also  to p e r m i t r a t i o n a l d e s i g n of  The f e r m e n t a t i o n o f  fish solubles  produce enzymes has so f a r not r e c e i v e d any q u a n t i t a t i v e c o n s e q u e n t l y no k i n e t i c a n a l y s i s literature.  This  of t h i s  c h a p t e r attempts  and  and of  (or volumetric)  it  is  any, between  rates  the  curves o f  figures  r a t e s were determined by d i v i d i n g  mass at t h a t t i m e . since  showing the time changes  The s p e c i f i c  rates  the hope of  bases,  for fermentations  500 rpm  at  at  the d i f f e r e n t  of  i n these v a r i a b l e s .  2 Jl/min,  graphed i n F i g u r e s 4.29  presented i n Figures 3.21,  3.23  The  ie.  for k i n e t i c  1 Jl/min,  750 rpm  at  to 4.22  3.25.  cells.  rate 1 £/min and  They were c a l c u l a t e d from the d a t a and  cell  analysis  p e r u n i t mass o f  are p r e s e n t e d i n T a b l e s 4.17  to 4 . 3 4 .  [88]  the v o l u m e t r i c r a t e s by the  are more u s e f u l  at  rates  growth, sugar u t i l i s a t i o n ,  on b o t h v o l u m e t r i c and s p e c i f i c  500 rpm  this  to a g i t a t i o n and a e r a t i o n .  they p u t e v e r y t h i n g on a comparable b a s i s ,  Complete r a t e p a t t e r n s ,  the  results  enzyme p r o d u c t i o n were o b t a i n e d 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  specific  and  if  to see how these r a t e s v a r y w i t h r e s p e c t  Instantaneous  f u r t h e r some o f  In p a r t i c u l a r ,  c h a p t e r to determine the r e l a t i o n s h i p s ,  treatment;  f e r m e n t a t i o n can be found i n  to a n a l y s e  p r e s e n t e d i n the p r e v i o u s s e c t i o n s .  to  92  Table  4.17  7-LITRE FERMENTATION - CFS  RUN NO.  3*  VOLUMETRIC RATES OF  SUGAR U T I L I Z A T I O N  GROWTH TIME (hrs)  TIME (hrs)  dx/dt (g/«,.hrxlO)  ds/dt (gM.hrxlO)  PROTEASE TIME (hrs)  FORMATION  dp/dt (units/cc.hrxlOO)  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 Air  flow rate  :  500  rpm  1 &/min.  93  4.18  Table  7-LITRE FERMENTATION - CFS  RUN NO. 3*  SPECIFIC RATES OF  GROWTH  SUGAR UTILIZATION  1 dx x dt (g/g.hrxlO)  TIME (hrs)  1 ds x dt (g/g.hrxlO)  PROTEASE FORMATION  1 dp x dt (units/mg.hrxlOO)  2.5 5.0  4.89 3.11  5.56 2.78  0.78 0.74  7.0 8.5  2.53 2.22  1.83 1.35  -  10.0 14.0  0.33 0.0  1.28 1.49  0.55  18.0  0.12  20.0  0.22  1.69 1.74  23.0  0.46  -  1.31 1.46  27.5  0.27  1.62  1.67  30.0  0.19  1.65  1.78  32.5  -  -  1.91  35.0 40.0  0.07 0.03  1.65 1.65  2.07 2.38  45.0  -  1.46  2.64  50.0  -  1.11  2.72  55.0 60.0  -  0.79 0.56  1.61 0.75  65.0 75.0  -  0.36 0.12  0.32  -  Agitator Air  speed : 500  flow r a t e  :  1  rpm £/min.  -  0.83 1.16  0.0  94  Table  4.19  7-LITRE FERMENTATION - CFS  RUN NO. 5*  VOLUMETRIC RATES OF  SUGAR UTILIZATION  GROWTH TIME (hrs)  dx/dt (g/JUhrxlO)  TIME (hrs)  ds/dt (g/£.hrxlO)  PROTEASE FORMATION TIME (hrs)  dp/dt (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  A i r flow rate : 1 A/min.  95  T a b l e A.20  7-LITRE FERMENTATION -  CFS  RUN NO. 5*  SPECIFIC RATES OF  GROWTH TIME (hrs)  SUGAR UTILIZATION  1 dx x dt (g/g.hrxlO)  1 ds_ x dt (g/g.hrxlO)  PROTEASE FORMATION _1 dp_ x dt (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  -  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 : Air  flow rate  :  750 rpm 1 £./min.  2.77  96  4.21  Table  7-LITRE FERMENTATION - CFS  RUN NO.  7*  VOLUMETRIC RATES OF  GROWTH TIME (hrs)  SUGAR UTILIZATION  dx/dt (g/£.hrxlO)  TIME (hrs)  ds/dt (g/£.hrxl0)  PROTEASE FORMATION TIME (hrs)  dp/dt (units/cc.hrxlOO)  2.7  0.26  2.5  0.85  12.6  0.15  6.8  0.86  6.5  1.55  21.5  1.10  9.5  1.71  11.0  2.10  29.0  2.57  11.5  3.10  17.6  2.50  34.5  3.85  12.5  1.38  26.0  2.30  38.8  5.10  12.8  0.97  35.0  2.10  42.0  6.90  15.0  0.25  42.0  1.65  43.7  3.80  18.0  0.0  47.5  0.80  44.7  2.30  52.2  0.20  48.5  0.60  55.0  0.0  52.5  0.0  A g i t a t o r speed : A i r flow rate :  500 rpm 2 £/min.  97  Table  4.22  7-LITRE FERMENTATION - CFS  RUN NO. 7*  SPECIFIC RATES OF  SUGAR  GROWTH TIME (hrs)  (g/g.hrxlO)  flow r a t e  :  PROTEASE FORMATION  x dt (units/mg.hrxlOO)  20.0  7.0 6.0 4.46 3.05 2.41 2.87 1.00 0.19 0.0  :  ds  x dt  (g/g.hrxlO)  A g i t a t o r speed Air  1  I dx x dt  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  UTILIZATION  16.8  9.6 5.0 2.88 2.08  500  rpm  2 Jl/min.  1.70  0.07  1.61  0.16  1.62  0.32  1.62  0.52  1.56  1.10  1.49  1.82  1.39  2.69  1.20  3.90  0.84  1.75  0.31  0.31  98  Figure 4.29  VOLUMETRIC RATES VS TIME FOR FERMENTOR RUN NO.  A 2.01  •o-  ••A -  3  A G R O W T H , g/(l.io.hr; B  ENZYME SYNTHESIS.units/ioocc.hr  B,C 6  C SUGAR UTILIZATION, g/|johr.  ^4  1.6  ,8  0  40 Tl M E (hrs)  0  99  Figure  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.)  Figure  VOLUMETRIC  4.31  RATES VS TIME FOR FERMENTOR RUN NO.  g/(1.10 hr)  5  -o-  A  GROWTH,  -•-  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 / |  I  -  1  0  h r .  •  0  20 40 T I M E (hrs.)  60  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  - 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  - A - C  A,B|  S U G A R U T I L I Z A T I O N , g . / g . i o hr.  12  r-A  8 ho  oi 0  , nWr*  20  40 TIME (hrs.)  60  104  The r a t e curves of these f i g u r e s f e r m e n t a t i o n system under c o n s i d e r a t i o n . used p r i m a r i l y enzyme; value,  In the i n i t i a l  Another p o i n t , is  also  This i s  stages  p r o d u c t i o n , b o t h the d r y c e l l weight  utilization  1 ds ( —— ) x dt  is  f u r t h e r e d demonstrated i n F i g u r e 4 . 3 5 . the f e r m e n t a t i o n  The c o n s t a n t  constant of  examined. glucose  at 1 ds —— x dt  g/g.  between  From the  carbohydrate  This value,  1.6  1.8,  and  speculate  during  the r a t e  3.23  10 ^  specific  hour to the  air  early consumption  o b t a i n e d by a v e r a g i n g a l l  specific  glucose  and 3 . 2 5 ) .  cases  utilization,  p r o d u c t i o n had j u s t e n t e r e d  t h a t one of  of  was the same f o r a l l t h r e e  '  o x i d a t i o n r a t e and p r o t e a s e  One can t h e r e f o r e  hour)  the r a t e o f g l u c o s e  h r x 10.  consumption and p r o t e a s e (Figures 3.21,  10 ^  rate  1 dx (— — ) x dt  r a t e o f growth  of p r o p o r t i o n a l i t y depends more on the  D u r i n g the p e r i o d o f c o n s t a n t  thmic phases  specific  r a t e of carbohydrate  d i r e c t l y p r o p o r t i o n a l to the  s t a t i o n a r y growth phase,  1.682  specific  enzyme  This p l o t suggests that  (up to the  flow r a t e than on the a g i t a t o r s p e e d . the f i n a l  D u r i n g the p e r i o d o f  and the s p e c i f i c  c a r b o h y d r a t e consumption per c e l l i s r a t e of growth.  i n the  The r e l a t i o n s h i p between the s p e c i f i c  glucose  the e a r l y s t a g e s of  to a zero  c l e a r l y i n the  and the s p e c i f i c  of  stages o f  is  o f growth and sugar u t i l i z a t i o n  (10-15 h o u r s ) .  u t i l i z a t i o n remained c o n s t a n t .  most e v i d e n t  demonstrated q u i t e  the c l o s e c o i n c i d e n c e  i n the i n i t i a l  values  sugar  the  f o r c e l l m u l t i p l i c a t i o n w i t h p r a c t i c a l l y no p r o d u c t i o n of  enzyme a c c u m u l a t i o n i s maximum.  rate patterns,  is  phase,  i n the second phase, when the growth r a t e has d e c r e a s e d  rate p l o t s .  rates  show two d i s t i n c t phases i n  their l o g a r i -  No d i r e c t r e l a t i o n s h i p between production rate i s  the purposes  apparent.  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 s u p p l y the energy r e q u i r e d f o r the e n d e r g o n i c r e a c t i o n of A comparison of  the s p e c i f i c  examined i n d i c a t e s this  (1)  o f enzyme p r o d u c t i o n f o r the t h r e e  weight  and  1 Jl/min,  F o r example,  750 rpm  25 h o u r s , (2)  500 rpm  and  2 £/min,  The m e t a b o l i c a c t i v i t y o f  the f e r m e n t a t i o n s  is highest  Additional studies  r a t e o f oxygen t r a n s f e r to steps.  gm  dry c e l l 50 h o u r s ,  the maximum was a t t a i n e d time to maximum was  of  2 l i t r e a i r per minute.  s t u d y are q u i t e  o f d i s s o l v e d oxygen  after  40 h o u r s ;  the organism d u r i n g the e a r l y p a r t  obtained i n this  on the e f f e c t s  rate of  maximum v a l u e a f t e r  at an a e r a t i o n r a t e of  The k i n e t i c p a t t e r n s  limiting  to i t s  and the same a e r a t i o n r a t e ,  w h i l e at  at a s t i r r i n g  the r a t e of enzyme p r o d u c t i o n p e r  i n c r e a s e d s l o w l y and s t e a d i l y  and at  cases  a g i t a t o r speed has a g r e a t e r i n f l u e n c e on  r a t e than does a i r flow r a t e .  500 rpm  and  that  rates  synthesis.  complex.  c o n c e n t r a t i o n and  the medium are needed to e l u c i d a t e  the v a r i o u s  107  Chapter  5  CONCLUSIONS  T h i s s t u d y has  shown t h a t the b a t c h p r o d u c t i o n  p r o t e a s e by Soranguim 495  can be a c h i e v e d i n a medium c o n t a i n i n g  f i s h s o l u b l e s as i t s major component. obtained  from  B.C.  The  to  3.85  mg  1000  cc  BSA/ml , was  was  d i l u t e d and  solid  condensed equivalent  found t o be the optimum f o r enzyme y i e l d .  obtained  t h i s was  of supplementary g l u c o s e was The  i n both f i l t e r e d  no s i g n i f i c a n t e f f e c t on  not so f o r g l u c o s e and  calcium.  The  amount  found t o have a l a r g e i n f l u e n c e on b o t h growth one  t a i l e d F-test also i n d i c a t e d that  there  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 c o n c e n t r a t i o n s  the d i f f e r e n t c a l c i u m c o n c e n t r a t i o n s . i s given i n Table  a c t i v i t y was  (0%  The  complete a n a l y s i s of  and  variance  3.13.  Further experimental of glucose  and  S t a t i s t i c a l l y designed e x p e r i -  ments showed t h a t w h i l e a d d i t i o n a l phosphate has  was  20 cc  of w a t e r , c o n t a i n i n g s o l u b l e p r o t e i n  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 .  and p r o t e a s e p r o d u c t i o n .  any  found t o g i v e b e t t e r p r o t e a s e  A d i l u t i o n r a t i o of  P r o l i f i c growth of the o r g a n i s m was  protease production,  condensed  condensed f i s h s o l u b l e s ,  f i l t e r e d media was  y i e l d s than the u n f i l t e r e d media. f i s h s o l u b l e s to  The  P a c k e r s , Richmond, B.C.,  material f i l t e r e d off.  of e x t r a c e l l u l a r  to  obtained  4%) by  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  i n d i c a t e d t h a t a two  fold increase i n c o s e i n o l y t i c  i n c r e a s i n g the i n i t i a l sugar c o n t e n t from  0.062  108  mg p e r m l , o r i g i n a l l y p r e s e n t i n t h e medium, t o  10 mg p e r m l .  That t h e  a d d i t i o n a l g l u c o s e e x e r t e d i t s i n f l u e n c e by i n c r e a s i n g t h e growth o f t h e o r g a n i s m was r e a d i l y a p p a r e n t from v i s u a l o b s e r v a t i o n and from t h e two phase n a t u r e o f t h e growth c u r v e . of g l u c o s e r e t a r d e d p r o t e a s e  The d a t a a l s o i l l u s t r a t e d t h a t a d d i t i o n  formation.  Later 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 n o t o n l y e l i m i n a t e d t h e s t a g e d but a l s o i n c r e a s e d both  Glucose  the y i e l d and r a t e o f p r o t e a s e  and mannose s u p p o r t e d  growth  formation.  t h e growth o f t h e o r g a n i s m .  growth was a c h i e v e d w i t h mannose. B e s t p r o t e a s e y i e l d o c c u r r e d w i t h  Best 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 p r o d u c t i o n c u r v e w h i l e the medium c o n t a i n i n g g l u c o s e gave a b i p h a s i c growth c u r v e . galactose, xylose range  4 to 8 )  nor d i d i n o c u l u m  and a r a b i n o s e  were n o t u t i l i z e d .  Initial  Soluble starch, pH ( i n t h e  d i d n o t s i g n i f i c a n t l y a f f e c t t h e maximum y i e l d o f p r o t e a s e s age  (16 t o 24  o b t a i n e d w i t h shake f l a s k  hours).  Reproducible  r e s u l t s have been  cultivation.  7 - l i t r e f e r m e n t a t i o n s t u d i e s i n d i c a t e d t h a t the i n f l e c t i o n i n t h e growth c u r v e observed  a t t h e 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 o f 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 p o l y p e p t i d e s ) u t i l i z a t i o n on one hand and e n z y m a t i c l a r g e r p o l y p e p t i d e u n i t s p l u s enzyme s e c r e t i o n the o t h e r . 500  rpm  digestion of  and oxygen l i m i t a t i o n on  H i g h e s t p r o d u c t i v i t y was o b t a i n e d w i t h an a g i t a t i o n r a t e o f  and an a e r a t i o n r a t e o f  2 litre  of a i r p e r minute;  ponded t o an oxygen a b s o r p t i o n c o e f f i c i e n t of  this corres-  0.805 m i l l i m o l e s O^/atm.min.I.  109  T h i s v a l u e was o b t a i n e d u s i n g  the c o r r e l a t i o n of Yamada et  When the k i n e t i c d a t a were p r e s e n t e d basis  (that i s  specific  rate basis),  it  on a p e r u n i t o f  t r a n s p i r e d that  growth and p r o t e a s e s y n t h e s i s , were i n d e p e n d e n t . enzyme p r o d u c t i o n ,  the s p e c i f i c  constant  1.682  value  of  g/g.  r a t e of  h r x 10.  al  [87].  c e l l mass  the two p r o c e s s e s ,  D u r i n g the phase of  g l u c o s e u t i l i z a t i o n remained at I t was  therefore  apparent  p r o d u c t f o r m a t i o n was n o t a s s o c i a t e d by any d i r e c t mechanism w i t h drate u t i l i z a t i o n . than a e r a t i o n ;  Enzyme p r o d u c t i o n was a f f e c t e d  the r e v e r s e  holds  The k i n e t i c p a t t e r n s n e c e s s a r y to e l u c i d a t e  f o r sugar  are q u i t e  more by r a t e of  that carbohyagitation  consumption.  complex,  the v a r i o u s mechanisms  and f u r t h e r work  involved.  a  is  110  Chapter  6  RECOMMENDATIONS  (1) wastes,  A g r i c u l t u r a l wastes h i g h i n c a r b o h y d r a t e , such as  s h o u l d be i n v e s t i g a t e d  supplementary n u t r i e n t . contains  w i t h the view to r e p l a c i n g g l u c o s e as a  The a c i d h y d r o l y z e d condensed  a l a r g e amount o f p r o t e i n h y d r o l y s a t e s  n i t r o g e n compounds which are not p r e s e n t used,  unsuccessfully,  as a replacement  condensed  fish solubles  as a replacement  for yeast  extract.  and not s t i c k w a t e r  A d d i t i o n a l s t u d i e s on the e f f e c t s  H o p e f u l l y d a t a from these r e s u l t s  the mechanism of formulated. better  pH  It  solubles  and o t h e r e a s i l y  i n the s t i c k w a t e r  c o n c e n t r a t i o n and r a t e of oxygen t r a n s f e r out.  fish  for yeast extract.  recommended t h a t  (2)  fruit  available  t h a t Truong It  is  therefore  s h o u l d be  of d i s s o l v e d  [11]  used  oxygen  to the medium s h o u l d be c a r r i e d  w i l l c l a r i f y the major f e a t u r e s  the system so t h a t a w o r k i n g t h e o r e t i c a l model can be is  recommended t h a t  these s t u d i e s be c a r r i e d out w i t h ,  and foam c o n t r o l .  (3)  The e f f e c t  of  filter - sterilized  growth and enzyme p r o d u c i n g a b i l i t y of  and a u t o c l a v e d  sugars  the Sorangium s p e c i e s h o u l d be  investigated.  (4) further  of  The e f f e c t  investigated.  of calcium  ions  on p r o t e a s e  yields  s h o u l d be  on  Ill  (5)  I n o r d e r to develop u s e f u l a p p l i c a t i o n s o f the Sorangium  p r o t e a s e , 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 o f a c t i o n s h o u l d be s t u d i e d . I n t h i s c o n n e c t i o n , i t i s suggested a b i l i t y t o h y d r o l y z e f i s h t o produce  t h a t t h e enzyme be t e s t e d f o r i t s FPC  f o r human consumption and t o  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. meaningful  A more  c o s t e s t i m a t e f o r an enzyme p r o d u c t i o n p r o c e s s can be made when  such i n f o r m a t i o n i s o b t a i n e d . (6)  Experimentation w i t h other microorganisms,  associated w i t h i n d u s t r i a l fermentation processes s u b t i l i s , and A s p e r g i l l u s o r y z a e ) ,  such as those  ( f o r example, B a c i l l u s  i s recommended.  112  BIBLIOGRAPHY  [I]  C l a g g e t t , F . G . , 1972. " C l a r i f i c a t i o n of F i s h P r o c e s s i n g P l a n t e f f l u e n t s by Chemical Treatment and A i r F l o t a t i o n , " Fisheries Research Board o f Canada, T e c h n i c a l Report No. 343.  [2]  C l a g g e t t , F . G . , 1971. "Demonstration Wastewater Treatment U n i t . I n t e r i u m Report 19 71 Salmon S e a s o n , " F i s h e r i e s Research Board o f Canada, Vancouver L a b o r a t o r y , T e c h n i c a l Report No. 286.  [3]  C l a g g e t t , F . G . , 1970. 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" F a c t o r s I n f l u e n c i n g the Enzymic A c t i v i t i e s Bacteria," B a c t e r i o l o g i c a l Reviews, V o l . 7, p p . 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 E d i n b u r g h .  ]  McCurdy, H . D . , J r . , 1963.  ]  D a v i s , J . G . and H . J . Rogers, 1939. "The E f f e c t o f S t e r i l i z a t i o n Upon S u g a r s , " Z e n t r . B a k t e r i o l . , Abt I I , V o l . 101, p p . 102-110.  ]  Holme, T . and R. B r o o k e s , 1969. "The I n f l u e n c e of D i f f e r e n t Energy Sources on B a c t e r i a l Growth and S y n t h e s i s , " i n Fermentation Advances, D. P e r l m a n , E d . , Academic P r e s s , N . Y . , p p . 145.  ]  Holme, T . , S. A r v i d s o n , B . L i n d h o l m , and B . P a v l u , 1970. 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"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 C o n c e n t r a t e : P r o c e s s 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 . & B i o e n g . , 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. 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 B i o c h e m i c a l and B i o l o g i c a l E n g i n e e r i n g S c i e n c e , V o l . 1, N. B l a k e b r o u g h , Ed., Academic P r e s s , N.Y.  [87]  Yamada, K., J . T a k a h a s i and H. Okada, 1952. "Fundamental S t u d i e s on the A e r o b i c F e r m e n t a t i o n . P a r t 2. D e t e r m i n a t i o n o f an E m p i r i c a l Formula on the E f f i c i e n c y o f Oxygen Supply o f Fermentor," J . A g r i c u l t u r a l C h e m i c a l Soc. o f Japan, V o l . 27, pp. 704-708.  [88]  Levens, A.S. 1962. G r a p h i c s - W i t h an I n t r o d u c t i o n t o C o n c e p t u a l D e s i g n , John W i l e y & Sons, N.Y. pp. 316-320.  Biochim. Biophys. Acta,  Appendix  I  EXPERIMENTAL DATA  120  Table A I . l EFFECT OF GLUCOSE CONCENTRATION (FILTERED CFS)  GLUCOSE CONCENTRATION  0%  0.5%  1.0%  2%  3%  4%  DRY CELL WEIGHT (gm/&)  PROTEOLYTIC ACTIVITY  TYROSINE  TIME (days)  PH  2  7.9  0.70  96.0  4.24  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  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  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  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  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  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 : 24hours Dry c e l l weight a t t = 0  (ug/ml)  (units/10cc)  -  :  0.0085 gm/l  121 Table  AI.2  EFFECT OF GLUCOSE CONCENTRATION (UNFILTERED CFS)  GLUCOSE CONCENTRATION  TIME (days)  pH  TYROSINE (yg/ml)  PROTEOLYTIC ACTIVITY (units/lOcc)  3  8.0  33.0  1.46  5  8.75  37.8  1.67  8  8.90  31.5  1.39  0.5%  3 5 8 10 12  7.55 8.20 9.10 8.90 8.75  33.0 59.2 80.5 92.5 77.0  1.46 2.62 3.56 4.09 3.40  1.0%  3 5 8 10 12  7.45 7.40 8.12 8.50 8.61  26.2 63.5 107.5 129.5 128.0  1.158 2.807 4.752 5.724 5.658  7.25  26.2  1.158  7.0  74.2  3.280  2.0%  3 5 8 10 12  6.2  100.0  4.420  6.2  102.5  4.531  7.61  103.0  4.56  3  7.15  21.5  0.950  5  6.60  54.0  2.387  8  6.10  66.9  2.957  10  6.00  73.0  3.227  12  5.10  42.8  1.892  3  7.09  20  0.884  5  6.40  48.1  2.126  8  6.15  59.6  2.634  5.95  44.0  1.945  0%  3.0%  4.0%  10 12  Table EFFECT OF CFS  CONCENTRATION  DRY WEIGHT  TYROSINE  (gmA)  (yg/ml)  2:100 v / v  DILUTION :  1:100 v / v  DILUTION : TIME (hrs)  AI.3  PROTEOLYTIC ACTIVITY (units/lOcc)  DRY WEIGHT (gm/*)  TYROSINE (ug/ml)  PROTEOLYTIC ACTIVITY (units/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  i4  2.26  73.0  3.227  2.46  252.5  11.161  125|  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 hCO N3 1  I n i t i a l pH F i n a l pH  7.0 5.9  I n i t i a l pH F i n a l pH  7.0 8.0  Table  AI.3 (Contd)  EFFECT OF CFS  DILUTION TIME (hrs)  DRY WEIGHT  (gm/Jl)  :  3:100  CONCENTRATION  DILUTION :  v/v PROTEOLYTIC ACTIVITY (units/lOcc)  TYROSINE (ug/ml)  DRY WEIGHT  (gm/Jl)  4:100 v / v  TYROSINE (ug/ml)  PROTEOLYTIC ACTIVITY (units/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 F i n a l pH  : :  7.0 8.4  I n i t i a l pH F i n a l pH  : :  7. 0 8. 32  Table  A I . 3 (Contd)  EFFECT OF CFS CONCENTRATION  DILUTION :  5:100 v / v  DILUTION :  TIME (hrs)  DRY WEIGHT (gm/Jc.)  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  90|  2.10  29.5  1.304  2.18  19.5  0.862  102±  2.06  -  2.28  20.9  0.924  2.30  41.5  1.834  2.52  11.8  125|  -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  TYROSINE (yg/ml)  I n i t i a l pH : F i n a l pH :  PROTEOLYTIC ACTIVITY (units/lOcc)  -  7.0 8.40  DRY WEIGHT (gm/£)  6:100 v / v  TYROSINE (yg/ml)  I n i t i a l pH : F i n a l pH :  PROTEOLYTIC ACTIVITY (units/lOcc)  -  -  7.0 8.40  Table  AI.4  EFFECT OF HEXOSE SUGARS (GLUCOSE)  TIME (hrs)  DRY WEIGHT (gm/Jl)  GLUCOSE CONCENTRATION  TYROSINE  (gm/A)  (yg/ml)  PROTEOLYTIC ACTIVITY (units/lOcc)  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  Inoculum age = 24 hours Dry c e l l weight at t = 0 : 0.0085 g m / £  Hro  1  I n i t i a l pH : F i n a l pH :  7.0 8.12  Table  A I . 4 (Contd)  EFFECT OF HEXOSE SUGARS  MANNOSE  GALACTOSE TIME (hrs)  DRY WEIGHT (gm/£)  8  0.30  -  0.74  3.6  30  1.09  42  TYROSINE (yg/ml)  PROTEOLYTIC ACTIVITY (units/lOcc)  DRY WEIGHT (gm/£)  TYROSINE (yg/ml)  PROTEOLYTIC ACTIVITY (units/lOcc)  -  0.18  -  0.159  0.70  2.1  0.093  20.8  0.919  1.00  16.3  0.720  1.25  36.8  1.626  1.54  34.6  1.529  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  4 4  I n i t i a l pH : F i n a l pH :  -  7.00 7.30  I n i t i a l pH : F i n a l pH :  6.078 7.00 8.12  127  Table AI.5 EFFECT OF PENTOSES AND STARCH  ARABINOSE  XYLOSE TIME (hrs)  8  4  DRY WEIGHT (gm/ic.)  TYROSINE (yg/ml)  -  0  PROTEOLYTIC ACTIVITY (units/lOcc)  -  DRY WEIGHT (gm/£)  TYROSINE (yg/ml)  0.02  -  PROTEOLYTIC ACTIVITY (units/lOcc)  -  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  4  0.94  38.3  1.693  0.92  39.4  1.741  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 F i n a l pH : 8.90  I n i t i a l pH : 7.00 F i n a l pH : 8.90  STARCH  -  0.42  -  0.68  7.3  0.323  30  1.25  34.6  1.529  42  4  1.31  58.1  2.568  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  8  4  I n i t i a l pH : 7.00 F i n a l pH : 9.10  128 Table AI.6 EFFECT OF INOCULUM AGE  (1) TIME (hrs)  Inoculum Age :  DRY WEIGHT (gm/A)  16 h o u r s  GLUCOSE CONCENTRATION  li  KUSlINfci  (grn/i)  (yg/ml)  PROTEOLYTIC ACTIVITY (units/lOcc)  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 F i n a l pH  : :  7.0 8.1  129  Table  AI.6 (Contd)  EFFECT OF INOCULUM AGE (2)  TIME (hrs)  Inoculum age : 20 h o u r s  DRY WEIGHT (gm/A)  GLUCOSE CONCENTRATION  TYROSINE  (gm/4)  (yg/ml)  PROTEOLYTIC ACTIVITY (units/lOcc)  0  0.0085  10.3  -  8  0.18  10.3  -  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  nsf  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  8.1  7.0  0  130  Table AI.7 7-LITRE FERMENTATION - NB - GLUCOSE RUN ( A g i t a t o r speed : 400 rpm; A i r f l o w r a t e : 1 &/min)  TIME (hrs)  pH  DRY WEIGHT (gm/£)  0  7.00  0.023  8.1  8  7.10  0.28  8.1  12  7.40  0.34  15  7.50  0.83  4.0  0.177  7.8  18  7.62  0.71  8.2  0.362  8.1  21  7.66  0.50  14.3  0.632  7.6  7.87  0.37  25.5  1.127  7.0  34  8.35  0.12  43.6  1.927  6.8  41  8.22  0.22  50.2  2.219  6.8  45  8.45  0.07  51.5  2.276  7.4  rYKOSlNi. (yg/ml)  PROTEOLYTIC ACTIVITY (units/lOcc)  GLUCOSE CONCENTRATION (gm/£)  131  Table  AI.8  7-LITRE FERMENTATION - CFS RUN NO. 1 : 3 0 0 rpm-; A i r flow r a t e : 1 Jl/min)  ( A g i t a t o r speed  TIME (hrs)  P  DRY WEIGHT (gm/Jl)  TYRn«JTOT UKU&inr,  (yg/ml)  PROTEOLYTIC ACTIVITY (units/lOcc)  7.38  0.023 0.090 0.40  -  0.57 0.66  1.5  16  7.53 7.62  18  7.70  0.58  7.75  0.65  0.239 -  9.60  20j  5.4 -  24  7.80  0.65  0.336  9.20  27  7.83  0.65  7.6 -  35 40  7.85 7.80  0.51 0.52  21.0  0.928 -  8.70 -  7.85  0.48  39.0  1.724  7.90  7.92  0.48  54.5  2.409  7.40  7.78  0.46  75.0  3.315  5.80  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 4  7.48  1.00  184.2  8.132  0.99  7.82  0.95  199.5  8.012  0.44  13 l 4 y-  8.10  0.88  197.0  8.702  0.39  134  8.10  0.80  200.0  8.819  0.37  0 3  4  14  49  4 4  113y-  6.82 6.92  -  -  -  GLUCOSE CONCENTRATION (gm/Jl)  0.066  -  -  10.00 -  9.40 -  -  -  132  Table  AI.9  7-LITRE FERMENTATION - CFS RUN NO. 2 ( A g i t a t o r speed  TIME (hrs)  P  DRY WEIGHT (gm/Jl)  :  iiRUblMt, (yg/ml)  -  400 rpm; A i r flow r a t e L  PROTEOLYTIC ACTIVITY (units/lOcc)  1 Jl/min)  GLUCOSE CONCENTRATION (gm/Jl)  —  9.00  -  9.00  0  7.0  0.023  6  7.1  0.15  -  7.85  0.59  4.8  0.212  8.95  0.66  9.5  0.420  8.55  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  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  Illy  7.83  0.82  217.0  9.591  0.50  116|  7.98  0.83  225.0  9.945  0.45  15  4 4  8  4  .7.85  133 Table  ALIO  7-LITRE FERMENTATION - CFS  RUN NO. 3  ( A g i t a t o r speed : 500 rpm; A i r f l o w r a t e : 1 £/min)  TIME (hrs)  P  DRY WEIGHT (gm/£)  IxRUbllNE  (ug/ml)  PROTEOLYTIC ACTIVITY (units/lOcc)  GLUCOSE CONCENTRATION (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  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  7.08  1.29  211.5  9.348  1.48  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  4  1.20  235.5  10.41  0.42  7.33  1.20  235.5  10.41  0.37  7.35  1.21  231.0  10.21  0.37  4  4 4  94  134 Table  AI.ll  4  7-LITRE FERMENTATION -• CFS RUN NO. ( A g i t a t o r speed  TIME (hrs)  P  H  :  600 rpm;  DRY WEIGHT  TYROSINE  (gm/A)  (yg/ml)  A i r flow  r a t e : 1 Jl/min)  PROTEOLYTIC ACTIVITY (units/lOcc)  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 f l o w r a t e  TIME (hrs)  pH  DRY WEIGHT (gm/Jl)  TYROSINE (yg/ml)  PROTEOLYTIC ACTIVITY (units/lOcc)  : 1 X/min)  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— 2  7.00  2.05  118.6  5.242  2.87  J  32— 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  y  136  Table  AI.13  7-LITRE FERMENTATION - CFS ( A g i t a t o r speed  ME rs)  0 8y  DRY WEIGHT (gm/2.)  P  7.20 -  RUN NO. 6  : 400 r p m ; A i r f l o w  (yg/ml)  PROTEOLYTIC ACTIVITY (units/lOcc)  rate  : 2  l/mln)  GLUCOSE CONCENTRATION (gm/il)  0.023  0  0  10.55  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  3l|  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; A i r flow rate : 2 2,/min)  TIME (hrs)  P  DRY WEIGHT (gm/Jl)  l^KUblNH  (yg/ml)  PROTEOLYTIC ACTIVITY (units/lOcc)  GLUCOSE CONCENTRATION (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  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  4  138  Table AI.15  SHAKE FLASK FERMENTATION - GLUCOSE DEFICIENT MEDIUM  TIME ru \ (hrs)  DRY CELL WEIGHT t /ON (gm/£)  PROTEOLYTIC ACTIVITY 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 P r o t e o l y t i c A c t i v i t y  11.3  Protein Estimation with the Biuret Reagent  140  Appendix  II. 1  II  Measurement o f G l u c o s e C o n c e n t r a t i o n  II.1.1  P h e n o l - S u l f u r i c A c i d Reagent Method  Reagents (1)  [56]  :  S u l f u r i c A c i d - Reagent  grade 95.5%, conforming to ACS  specifications, (2)  Phenol  -  specific  gravity  1.84.  5% s o l u t i o n i n w a t e r .  Apparatus : (1)  F a s t d e l i v e r y 5-ml p i p e t ,  sulfuric acid i n portion off  the  10  to  20  seconds.  t i p of a s t a n d a r d  (2)  Set o f  (3)  Spectronic  to d e l i v e r  5 ml  concentrated  Can be p r e p a r e d by c u t t i n g a  5 ml  pipet.  tubes w i t h i n t e r n a l d i a m e t e r between 20  of  tubes o r a s e t  16  and  of matched c o l o r i m e t e r  20 mm. tubes.  Procedure : Standard Curve (1) and  70 ug  acid is  of  Prepare  1 ml  glucose s o l u t i o n s  c o n t a i n i n g between  10  glucose.  (2)  Add  1 ml  of  (3)  Add  5 ml  of c o n c e n t r a t e d  directed against  5%  phenol s o l u t i o n  the l i q u i d s u r f a c e  t^SO^  to  each.  rapidly;  the stream of  r a t h e r than the s i d e of  the  141  test  tube  in  order  to  obtain  (4)  Allow  to  (5)  Shake  and  (6)  Transfer  good  stand  mixing.  for  place  10  in  minutes  water  bath  at  room  set  at  temperature. 30°C  for  10  to  20  square  fit  minutes.  absorbance  at  490  to  the  my  Plot  (7) data,  as  slowly in  in  colorimeter  spectronic versus  A '490  shown  to  Figure  tubes  and  measure  the  20.  yg  glucose  and  make  a least  All.36.  Unknown Repeat concentration  II.1.2  steps  2  to  from Figure  4.5  per  cent  licylic  acid  add  phenol,  add  22  dissolve. g  of  Mix w e l l stoppered year.  255 ml  sodium until in  Three  well ml  and  obtain  the  sugar  [57]  of 10  100  reagent  the  per  and  cent and  Rochelle bottles.  of  reagent  prepared 880  salt. sodium  mix.  add  filled the  and  Rochelle  ml  bisulfite of  acid  hydroxide,  g  of to  a l l  sample,  :  sodium  Dilute  unknown  All.36.  Dinitrosalicylic  6.9  with  D i n i t r o s a l i c y l i c Method  Reagents  of  6  to  salt The  should  ml  of  To  10  follows 1 per g  of  hydroxide.  To the  as  69  ml  of  contain  w i l l by  ml  crystalline  this  last  300  dinitrosa-  Add w a t e r  dissolved.  reagent  To  cent  dinitrosalicylic  has  :  to  solution acid  Keep for  titration,  add  solution.  tightly at  least  with  one  142  Figure  All.36  GLUCOSE TESTS STANDARD CURVES  GLUCOSE,  143  p h e n o l p h t h a l e i n as i n d i c a t o r ,  the e q u i v a l e n t  of  5  to  6 ml  of  tenth  normal sodium h y d r o x i d e .  Apparatus  :  (1)  Set of  test  tubes w i t h i n t e r n a l diameter between  (2)  B o i l i n g water b a t h .  (3)  Spectronic  16  and  20 mm.  Procedure  20 tubes  or a s e t  o f matched c o l o r i m e t e r  tubes.  :  Standard Curve  (1) 0.3 mg  and  Prepare of  1 ml  glucose solutions  c o n t a i n i n g between  0.04  glucose.  (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  (3)  Capped and heat f o r  (4)  C o o l immediately i n r u n n i n g water f o r  (5)  T r a n s f e r to c o l o r i m e t e r  (6)  Plot  to e a c h , mix  well. 5 minutes  i n the b o i l i n g w a t e r . 3 minutes.  tubes and measure absorbance  at  520 my.  fit  to the d a t a ,  A  520  versus  mg  of  g l u c o s e and make a l e a s t  as shown i n F i g u r e A l l . 3 6 .  square  144  Unknown  Repeat s t e p s  2 to 5  w i t h the unknown sample,  and o b t a i n  the  sugar c o n c e n t r a t i o n from F i g u r e A l l . 3 6 .  II.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  (2)  6%  s o l u t i o n o f sodium c a r b o n a t e ,  (3)  F o l i n ' s reagent  t u n g s t a t e and 25 gm bottomed f l a s k 700 ml and  is  o f sodium molybdenate  10 h r .  and s e v e r a l  If  repeated. reagent  3  ^200^  follows  :  100 gm  are put i n t o  50 ml  of  85%  After cooling, (5-6)  150 gm  of  green,  the  2N  in acid.  tenfold-diluted  determinations  gr.  joint.  50 ml  The open  1 l i t r e with water.  0.IN  prepared before  of water  is of  flask  The s o l u t i o n s h o u l d then be  the  is The  checked by  NaOH  The p r e p a r e d reagent  The w o r k i n g s o l u t i o n i s  -  1.689),  lithium sulfate,  F o l i n ' s reagent w i t h  by adding f o u r volumes  glass  The s o l u t i o n  The a c i d c o n c e n t r a t i o n i s  u s i n g p h e n o l p t h a l e i n as an i n d i c a t o r . a dark b o t t l e .  round  treatment w i t h bromine w a t e r  The c o o l e d s o l u t i o n i s b r o u g h t to  s h o u l d be  sodium  a 2-litre  drops of bromine water a r e added.  the s o l u t i o n i s  t i t r a t i o n of  of  orthophosphoric a c i d (sp.  heated u n t i l the excess bromine e v a p o r a t e s .  yellow.  (CC1 C00H)  o f c o n c e n t r a t e d h y d r o c h l o r i c a c i d are added.  boiled for water,  p r e p a r e d as  acid  f i t t e d w i t h a r e f l u x condenser w i t h a ground -  of water,  100 ml  tricholoacetic  is  solution stored  in  activity  to one volume o f  Folin's  145  reagent.  Note t h a t the c o n c e n t r a t e d  c o m m e r c i a l l y from F i s h e r S c i e n t i f i c (4) Pipette  0.025M  25 ml  of  aminomethane i n 0.1N  HC1  tris-HCl  0.2M  1000  F o l i n ' s r e a g e n t may  of  Buffer,  to i t u n t i l the  H^O) pH  obtained  Co. pH  8.5  s t o c k t r i s (24.24 gm  ml  be  into  of t r i s  200 ml  f a l l s to  8.5.  p r e p a r e d as f o l l o w s : (hydroxymethyl)  volumetric Make up  to  flask.  Add  200 mis  with  water. (5)  2%  C a s e i n s o l u t i o n p r e p a r e d as f o l l o w s :  o f "Hammarsten" c a s e i n i n about  45 mis  of the  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 . t o about 7;  add  a s m a l l amount o f  e l e v a t e the  pH  to about  casein.  pH  and make up t o (6) of  0.1N  Tyrosine  w i t h the t r i s — H C 1 solution :  o f the s u s p e n s i o n drops  10 mg  65°-70°C pH  to  100  yg  Procedure  Construction  of C a l i b r a t i o n Curve  the  solid  of t y r o s i n e i s d i s s o l v e d i n measuring f l a s k ,  This s o l u t i o n , c o n t a i n i n g  of t y r o s i n e p e r  till  the  o f the s o l u t i o n t o  t y r o s i n e per ml, i s used to p r e p a r e s o l u t i o n s of lower 5  buffer.  buffer.  a l k a l i , t r a n s f e r r e d to a 25-ml  b r o u g h t t o the mark w i t h w a t e r .  containing  tris-HCl  gm  so as t o f a c i l i t a t e the d i s s o l u t i o n of  A f t e r c o o l i n g , b r i n g the  100 mis  2  sodium c a r b o n a t e s o l u t i o n to  Heat the s u s p e n s i o n i n a w a t e r b a t h a t  particles dissolve.  5 ml  8  6%  The  0.025M  Suspend  ml.  400  and yg  concentration  of  8.5  146  One  ml  b a t c h e s of  tyrosine solutions  are taken and to these are added and  1 ml  of f i v e - f o l d  reagents;  (20  6%  f o r the c o l o r to develop  to 2 5 ° C ) .  A control is  colorimeter  through the p o i n t s ,  Plot  solution  These s o l u t i o n s  are  -  at  for  30 min  simultaneously  determined on a p h o t o c o l o r i m e t e r  tubes.  concentration  sodium carbonate  i n the c o n t r o l the t y r o s i n e s o l u t i o n i s  depth of c o l o r i s in  of  - d i l u t e d F o l i n ' s reagent.  immediately mixed and l e f t room temperature  4 ml  of d i f f e r e n t  set  up f o r  r e p l a c e d by w a t e r . at wavelength  The  660 mu  the d a t a and draw a l e a s t square s t r a i g h t  as shown i n F i g u r e  the  line  All.37.  D e t e r m i n a t i o n of A c t i v i t y  (1) solution  Bring  (culture (2)  2 mis  filtrate),  Add the c a s e i n  immediately s t a r t (3)  stop w a t c h .  of  2%  i n separate solution  for  Stop the r e a c t i o n by adding as r a p i d l y as p o s s i b l e ;  about  5  to  t e s t tubes,  (4)  of  used i n s t e a d  m i x e d , and  30°C.  5%  mixed and p l a c e d i n  trichloroacetic 30°C  Filter  the m i x t u r e through Whatman #3  bath  f i l t e r paper,  for  to  precipitate.  Determine the amount of  tyrosine  i n the  filtrate  method o u t l i n e d i n " C o n s t r u c t i o n of C a l i b r a t i o n Curve" - 1 ml is  enzyme  30°C.  10 mins a t 5 ml  of  minutes.  remove the white (5)  to  1 ml  to the enzyme s o l u t i o n ,  Incubate  acid solution 2  c a s e i n s o l u t i o n and  of  1 ml  of  tyrosine  solution.  using of  the  filtrate  147  Figure  All.37  TYROSINE TEST STANDARD  CURVE  148  (6)  A c o n t r o l i s simultaneously  trichloroacetic solution. instead  a c i d to the enzyme s o l u t i o n  The s o l u t i o n  of  s e t up by adding f i r s t  1 ml  i s mixed,  o f water  filtered,  and then and  1 ml  o f enzvme  the amount which i n one minute forms p r o t e o l y s i s  tyrosine.  casein  of the  filtrate  (following  o f the Enzyme Commission o f the I n t e r -  precipitated with t r i c h l o r o a c e t i c  of  assay.  preparations  I n t h i s method the enzyme u n i t i s d e f i n e d  as  of  i s used as the b l a n k i n the t y r o s i n e  C a l c u l a t i o n of a c t i v i t y  recommendations  2 ml  5 ml  the  Union o f B i o c h e m i s t r y )  p r o d u c t s which a r e n o t  a c i d and c o n t a i n one m i c r o e q u a l e n t of  T h e r e f o r e the number of enzyme u n i t s  p e r ml o f the  investigated  s o l u t i o n w i l l be :  E = (a x 8 ) / ( 1 8 1 x 10) = a x 0.00442 .  Where of  E  ug  is  the enzyme u n i t  of tyrosine  factor after  found from the c a l i b r a t i o n c u r v e ;  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  m o l e c u l a r weight o f t y r o s i n e  II.3  (yeq. t y r o s i n e / m l . min);  P r o t e i n Estimation with  and  10  is  acid;  a 8  the B i u r e t Reagent  is  181  the p r o t e o l y s i s  is  the number the d i l u t i o n  is  the microgram  period i n minutes.  [66]  Reagents (1) 0.25 gm 10 mg  of  Standard s o l u t i o n  of c r y s t a l l i n e BSA per m l ,  BSA i n i s used  of B o v i n e Serum Albumin 25 mis  of  (BSA).  H^O. T h i s s o l u t i o n ,  to p r e p a r e s o l u t i o n s o f lower  Dissolve containing  concentration,  149  containing  1 (2)  Dissolve  to  10 mg/ml.  B i u r e t Reagent (may be o b t a i n e d  1.50 g  commercially  o f c u p r i c s u l f a t e (CuSO^.SI^O)  potassium t a r t r a t e  (NaKC.H 0 .4H 0) 4 4 6  constant s w i r l i n g ,  300 ml  of  in  and  500 ml  from  6.0 g  of water.  BBL) :  o f sodium Add, w i t h  2  10%  sodium h y d r o x i d e .  w i t h w a t e r , 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 . keep i n d e f i n i t e l y b u t must be d i s c a r d e d i f ,  D i l u t e to  This reagent  as a r e s u l t o f  1  litre  should  contamination  or o f f a u l t y p r e p a r a t i o n , i t shows s i g n s o f d e p o s i t i n g any b l a c k o r reddish p r e c i p i t a t e . Procedure To per ml for  30  add  1.0 ml 4.0 ml  1  4.0 ml  to  10 mg  of protein  o f b i u r e t r e a g e n t , mix by s w i r l i n g , and a l l o w t o s t a n d  m i n u t e s a t room temperature (20 t o 25°C).  transmission at of  of a s o l u t i o n containing  540 my,  Measure t h e p e r c e n t  w i t h the S p e c t r o n i c 20, a g a i n s t a b l a n k c o n s i s t i n g  of b i u r e t r e a g e n t p l u s  1.0 ml  of water.  The c o n c e n t r a t i o n o f p r o t e i n i n t h e sample i s o b t a i n e d by r e f e r e n c e 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 o f serum p r o t e i n ( F i g u r e A l l . 3 8 . )  151  Appendix  III  COMPUTATION OF SUM OF SQUARES  The treatment sum of s q u a r e s * i s main e f f e c t  sums o f squares  sums o f squares  SS(AB),  a c t i o n sum o f squares  SSA,  SS(AC),  SS(ABC).  SSB, and  s u b d i v i d e d i n t o the  SSC,  three  the t h r e e two-way i n t e r a c t i o n  SS(BC),  and the three-way i n t e r -  To f a c i l i t a t e the c a l c u l a t i o n o f  sums o f square the f o l l o w i n g two-way t a b l e s  of t o t a l s  and s u b t o t a l s were  constructed : B A  1  2  3  Total  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  9.61  8.21  27.97  Total  10.15  C A  1  2  3  Total  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  Total  14.83  7.12  6.02  27.97  When there i s no r e p l i c a t i o n ,  these  the t o t a l sum of squares i s used.  C B  1  2  3  Total  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  14.83  7.12  6.02  27.97  Total  C o r r e c t i o n term,  (27.97)' 27  CT  + 1.39  28.97  SST  =  3.66  SSA  =  |-(16.78  2  + 10.98  SSB  =  |-(10.15  2  + 9.61  2  + 8.21 )  - CT =  0.23  SSC  =  ^(14.83  2  + 7.12  2  + 6.02 )  - CT =  5.13  2  SS(AB)  =  j(6.46  SS(AC)  =  3( -  SS(BC)  8  2  7 7 2  -j(5.70  2  + . . . + 0.08  2  + 0.21 )  2  2  2  2  + 3.56  +  6  -  0  6  2  - CT =  - CT =  + . . . + 0.08 ) 2  25.73  15.72  - CT - SSA - SSB  0.90  + • • • + O . O ) - CT - SSA - SSC =  2  + 4.56  2  2.99  2  2  + . . . + 1.52 ) 2  SS(ABC) = SST - SSA - SSB - SSC - SS(AB)  - CT - SSB - SSC  - SS(AC) -  SS(BC)  =  0.25  =  0.510  

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