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UBC Theses and Dissertations

Potential of spent sulfite liquor as raw material for production of vitamin B12 by certain species of… Truong, Thieu Long 1973

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POTENTIAL AS  RAW  OF  MATERIAL BY  LACTOBACILLUS,  SPENT  FOR  SULFITE  PRODUCTION  CERTAIN  LIQUOR  CF  SPECIES  VITAKIN  E12  OF  PROPIONIBACTERIA,  AND  STREFTCWYCES  by jTRUONG B . S C A .  et  Diplome  THIEU  d * I n g e n i e u r ,  LONG  E c c l e  P c l y t e c h n i q u e ,  1968 H . S c ,  A  U n i v e r s i t y  THESIS THE  of  SUBMITTED  B r i t i s h  IN  PARTIAL  REQUIREMENTS MASTER  i n  OF  C o l u m b i a ,  FCR  THE  APPLIED  the  FULFILMENT DEGREE  1972  OF  CF  SCIENCE  Department of  CHEMICAL  We  a c c e p t  -this  ENGINEERING  t h e e i s  r ^ j a l r e d  THE  UNIVfRSITY  OF  August  as  conforming  to  standard  BRITISH 1973  COLUMBIA  the  M o n t r e a l ,  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y  a v a i l a b l e f o r r e f e r e n c e and  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e  c o p y i n g of t h i s  study. thesis  f o r s c h o l a r l y purposes may be g r a n t e d by the Head of my Department o r by h i s r e p r e s e n t a t i v e s .  I t i s understood t h a t c o p y i n g or p u b l i c a t i o n  of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my written permission.  Department of  Chemical Engineering  The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada  Date  December 1 s t , 1973  i  ABSTRACT  Spent  s u l f i t e  P r o p i o n i b a c t e r i a L a c t o b a c i l l u s Streptomyces L.  i n  i n  of  SSL  as  two  the  i n  s t a g e s ,  the  f i r s t  source  and  optimum  y i e l d  i n  i s  of  and  s u i t a b l e The  B12  one  g o a l  of  are  of  s h e r m a n i i ,  Streptomyces  s t a g e of  and  g r i s e u s  and  t o g e t h e r , w i t h  mixed  producing  of  the  i n  c u l t u r e ,  v i t a m i n  these  of  v i t a m i n  to  f o r  be  of  b r o t h . the  of  i s  They  B12  B12  from  a  net  as  1.5mg/l,  a c i d s .  t o  F i s h  be  as  p l a n t  The  p r e c u r s o r .  a c i d s .  i n  e s s e n t i a l  accompanied  the  done  a e r o b i c .  e x t r a c t  i o n s  v o l a t i l e  show  should  second  yeast  b e n e f i c i a l  y e a s t  the  f r e u d e n r e i c h i i  and  B12  v o l a t i l e  of  producer  c o b a l t  moles/1  found  a  P.  5 g / l  v i t a m i n  r e d u c t i o n by  good  a n a e r o b i c  replacement  a c c o m p l i s h e d  of  w i t h  1ppm  0.14  b e n z i m i d a z o l e v i t a m i n  of  alone.  value  n i t r o g e n  of  or  as  SSL  market  r e q u i r e m e n t s  p r o d u c t i o n  f o r  f r e u d e n r e i c h i i  u l t i m a t e  not  of  optimum  a  s t a g e s  i s  Fermentations  two  not  P.  f r e u d e n r e i c h i i  i n c r e a s e  growth  q u a n t i t y .  s h e r m a n i i P.  f o r  P r o p i o n i b a c t e r i a  but  and  s e r v i n g  s u b s t a n t i a l P.  s u i t a b l e  o l i v a c e u s .  p l a n t a r i u m  poor  i s  f r e u d e n r e i c h i i ,  p l a n t a r i u m  f e r m e n t a t i o n s are  l i q u o r  The  by  the  5 , 6 - d i m e t h y l  y i e l d s  of  e f f l u e n t  c e l l s , i s  not  e x t r a c t .  p o l l u t i n g  f e r m e n t a t i o n s , A b s t r a c t  p o t e n t i a l i s  o n l y  about  of 145?  SSL, of  the  i i  t o t a l removal c e l l s  c h e m i c a l of and  p o l l u t i n g  about  oxygen  demand.  70*  the  v o l a t i l e p o t e n t i a l  of  a c i d s . of  20*  T h i s  r e d u c i n g These  of  the  sugars t o t a l  A b s t r a c t  r e d u c t i o n s u g a r s ,  i s  to  the  transformed  t c  o r i g i n a l l y COD  to  SSL.  due  c c n t r i t u t e  a  i i i  TABLE  OF  CONTENTS  Page ABSTRACT TABLE  i  OF CONTENTS  i i i  LIST  OF TABLES  v i  LIST  OF  i x  FIGOBES  ABBREVIATIONS  x i v  ACKNOWLEDGMENTS  XV  Chapter 1  INTRODUCTION 1.1  T  Spent  S u l f i t e  1.1.1  C o m p o s i t i o n  1.1.2  P o l l u t i o n  1.1.3  Treatments  1.2  V i t a m i n  1.3  Microorganisms  1.4  L i q u o r  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  1 1  Caused  by  Spent  S u l f i t e  L i q u o r  . . . . . .  2  and Uses  8  B12  10 Used  17  1.3.1  L a c t o b a c i l l u s  P l a n t a r i u m  1.3.2  P r o p i o n i b a c t e r i a  1.3.3  Streptomyces  . . . . . . . . . . . . . . . . . . . . . .  F r e u d e n r e i c h i i  G r i s e u s  and Shermanii  and O l i v a c e u s  O b j e c t i v e  . . . . . . . . . . . .  17 .  17 20 21  Table  o f  Contents  i v  Chapter 2  EXPERIMENTAL  TECHNIQUES  2.1  P r e p a r a t i o n  2.2  Treatments  2.3  2.4  3  Page  o f  Inoculum  P r i o r  t o  2.2.1  P r e p a r a t i o n  2 . 2 . 2  N u t r i e n t  F e r m e n t a t i o n  23 23  Fermentation  o f  Spent  24  S u l f i t e  L i g u c r  24  A d d i t i o n  31  and Apparatus  . . . . . . . . . . . . . . . . . . . . . . . . . .  32  2.3.1  Apparatus  32  2 . 3 . 2  P r e f e r m e n t a t i o n  33  2 . 3 . 3  F e r m e n t a t i o n  37  A n a l y s i s  Methods  38  2.4.1  Measurement  o f  B a c t e r i a l  2.4.2  Measurement  o f  Sugar  2 . 4 . 3  Measurement  o f  V i t a m i n  2.4.4  Measurement  o f  A c i d s  2.4.5  D e t e r m i n a t i o n  EXPERIMENTS  of  Growth  39  C o n c e n t r a t i o n  45  B12 C o n c e n t r a t i o n  C o n c e n t r a t i o n  C h e m i c a l  Oxygen  45  . . . . . . . . . . . .  Demand  . . . . . . .  AND RESULTS  3.1  F e r m e n t a t i o n  3.2  Growth  3.3  P r e l i m i n a r y  3.4  Yeast  3.5  F i s h  3.6  P r e l i m i n a r y  3.7  F e r m e n t a t i o n s  3.8  Fermentation  o f  with  L a c t o b a c i l l u s  E x t r a c t P l a n t  from  E f f l u e n t  on  i n  D i f f e r e n t  o f  The 71 S o l u t i o n  Table  o f  . . . . . . . . . . . . . . . .  Sources  Erlenmeyer  69  79 F l a s k s  . . . . . . . .  80  Fermentors  . . . . . . . .  89  . . . . . . . . . . . . . . . . .  157  Glucose  Contents  69  77  Source  Microferm o f  . . . . . . . . . . . . . . . . . . .  E x t r a c t  a s N i t r o g e n i n  63  X y l o s e  Yeast  Experiments i n  56 63  Streptomyces  S t u d i e s  48  V  Chapter 4  5  Page  DISCUSSIONS  159  4.1  Fermentation  with  Streptomyces  4.2  Fermentation  with  I.  4.3  Fermentation  with  Mixed  4.4  Fermentation  with  P.  Shermanii  4.5  Fermentation  with  P .  F r e u d e n r e i c h i i  4.5.1  Ose o f  Sugars  164  4.5.2  E f f e c t  o f  pfl  165  4.5.3  E f f e c t  o f  A i r  4.5.4  E f f e c t  of  A g i t a t i o n  4 . 5 . 5  E f f e c t  o f  l e a s t  4.5.6  E f f e c t  o f  C o b a l t  4 . 5 . ?  E f f e c t  o f  5 , 6 - d i m e t h y l  P l a n t a r i u m  159  . . . . . . . . . . . . . . . . . . . . .  160  . . . . . . . . . . . . . . . . . . . . .  162 164 •  . . . . . . . . . . . . . . . . .  P r o d u c t i o n  of  A c i d s  4.7  P r o d u c t i o n  o f  V i t a m i n  4.8  N u t r i e n t  4.9  Fermentation  by  Requirements  and Volume  E x t r a c t I o n  P .  Broth  . . . . . . .  167  . . . . . . . . .  169  . . . . . . . . . . . .  170  C o n c e n t r a t i o n  C o n c e n t r a t i o n  B e n z i m i d a z c l e  170  F r e u d e n r e i c h i i  B12 by  P.  and C o s t s  S o l u t i o n  of  o f  F r e u d e n r e i c h i i  171 . . . . . .  173  . . . . . . . . . . . . . . . . . . . . .  175  Glucose  177  CONCLUSIONS  178  BIBLIOGRAPHY LIST  164  . . . . . 1 6 6  4.6  of  C u l t u r e  . . . . . . . . . . . . . . . . . . . . . .  180  OF OCCURENCES  OF REFERENCES  APPENDIX  I  :  Tabulated  APPENDIX  I I  :  A n a l y s i s  APPENDIX  I I I  :  S o l u t i o n s  R e s u l t s  190  . . . . . . . . . . . . . . . . . . . . . . . . . . .  Procedures  . . . . . . . . . . . . . . . . . . . . . . . . .  and C h e m i c a l s  Table  of  Contents  193 213  . . . . . 2 2 6  v i  LIST  OF  TABLES  Table  Page  1.1  The Percentage  1.2  Y i e l d  o f  Sugars 1.3  1.4 1.5  Composition  P o t e n t i a l  o f  Reducing  . . . . . . . . .  2  Fermentable 3  4  Composition  o f  5  A  Accepted  G e n e r a l l y  S u l f i t e  T y p i c a l  1.7  Treatments  1.8  P r o c e s s e s  1.9  A n t i b i o t i c - p r o d u c i n g V i t a m i n  o f  f o r  V i t a m i n  1.11  V i t a m i n  11.12  R e i n f o r c e d  11.13  Waksman's  . . . . . . . .  The Composition  Spent  t o  S u l f i t e  S y n t h e s i s  i n  L i q u o r  o f  6 7 9  B12 . . . .  13  . . . . . . . . . . . . . . . . . . . . . . . . . . .  16  Commercial  Vitamin  with  P r o d u c t i o n 16  i n  C l o s t r i d i a l  The United  s t a t e s  . . . . . . . . . .  Medium  16 23  N o . 3 Medium o f  1  o f  SSL . . . . . . . . . . . . . . . .  F e r m e n t a t i o n s  B12 P r o d u c e r s  24  V a r i o u s  Methods  of  Removal  o f  D i o x i d e  C o n c e n t r a t i o n f o r  Used  c f  L i q u o r s  B12  E f f e c t i v e n e s s S u l f u r  o f  By-product  Microorganisms of  Exposed  M i c r o b i a l  B12 a s  S u l f i t e  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  Salmon  and Uses  Spent  p i c t u r e  L i q u o r  M o r t a l i t i e s  11.15  and  Hoods  Hoods  1.6  11.14  C e r t a i n  The Percentage Composition o f Sugars of T y p i c a l Spent S u l f i t e L i q u o r s a n d Heed H y d r o l y z a t e s . . . . . . . . .  Spent  1.10  of  27 o f  N u t r i e n t s  P r o p i o n i b a c t a r i a  L i s t  i n SSL  and L a c t o b a c i l l u s  o f  Tables  . . . . . . . . . . . . . .  31  v i i  Table 11.16  11.17  Page E f f e c t o f Submitted Manual A c i d s  V a r i o u s P r e t r e a t m e n t s on C e l l s t o V i t a m i n B 1 2Assay . . . . . . . . . . . . . . . . . . . . . .  Ether from  E x t r a c t i o n  a n Aqueous  R e s u l t s  o f  11.19  R e s u l t s  o f C o r r e c t i n g  11.20  R e l i a b i l i t y  111.21  Fermentations  o f  111.22  Fermentations  o f D i l u t e d  111.23  Summary  111.24  111.25  Measures  D i l u t e d  Summary  c n COD T e s t  . . . . . . . . . . ,.  S S Lby S . g r i s e u s  . . . . . . . . . .  S S Lb y S . o l i v a c e u s  o n f e r m e n t a t i o n s  C o b a l t  Cost  R e s u l t s  P r o p i o n i b a c t e r i a  59 64 65  o fS S L  . . . . . . . . . . . . . . .  S o l u t i o n s  c f Fermentations F r e u d e n r e i c h i i  Fermentation o f S S Lw i t h i n Erlenmeyer F l a s k s  124  -  I o n C o n c e n t r a t i o n  o f Aqueous  a n dP r o f i t  58  90  o f O v e r a l l o f  51 57  Summary o f O v e r a l l R e s u l t s E f f e c t o f Yeast E x t r a c t C o n c e n t r a t i o n  IV.27  AI.31  o n 3 Samples  o f i n f o r m a t i o n  Fermentation  AI.30  . . . . . . . . . . . . . . . . . . . . . .  o f T h e CODT e s t  111.26  AI.29  a n d l a c t i c  7 1 Fermentors  E f f e c t  AI.28  V o l a t i l e  S o l u t i o n  11.18  i n  CODT e s t s  o f  48  L .  125 c f Glucose  . . . . . . . .  158  . . . . . . . . . . . . . . . . . . . . .  175  with  plantarium 196  Fermentation o f S S Lwith P . f r e u d e n r e i c h i i i n Erlenmeyer F l a s k s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  196  Fermentation o f S S Lwith i n Erlenmeyer F l a s k s  197  P .  shermanii  Fermentation o f S S Lwith A Mixed C u l t u r e of L . p l a n t a r i u m a n d P . f r e u d e n r e i c h i i i n Erlenmeyer F l a s k s  197  AI.32  Run F P 1 . 1 .  Fermentation  with  L .  plantarium  . . . . . . . . . .  198  AI.33  R u n FP1.2.  Fermentation  with  L .  plantarium  . . . . . . . . . .  199  L i s t  o f  Tables  viii  Table AI.34 AI*35 AI.36 AI.37 AI.38  Page Run FP2.1. F e r m e n t a t i o n w i t h P. f r e u d e n r e i c h i i on L a c t i c A c i d a s Main S u b s t r a t e  200  Run FP2.2. F e r m e n t a t i o n w i t h P. f r e u d e n r e i c h i i on L a c t i c A c i d a s Main S u b s t r a t e  201  Run FP3. F e r m e n t a t i o n w i t h Mixed C u l t u r e o f L. p l a n t a r i u m and P. f r e u d e n r e i c h i i  202  Run F15.22a. F e r m e n t a t i o n E f f e c t o f pH  with  203  Run F15.22b. F e r m e n t a t i o n i n One A n a e r o b i c S t a g e  with  P. P.  freudenreichii. freudenreichii 204  AI.39  Run F05.22. F e r m e n t a t i o n w i t h P. f r e u d e n r e i c h i i . Y e a s t E x t r a c t C o n c e n t r a t i o n a s V a r i a b l e . . . . . . . . . . . . . 205  AI.40  Run F10.22. F e r m e n t a t i o n w i t h P. f r e u d e n r e i c h i i . Y e a s t E x t r a c t C o n c e n t r a t i o n as V a r i a b l e  206  Run F20.22. F e r m e n t a t i o n w i t h P. f r e u d e n r e i c h i i . Y e a s t E x t r a c t C o n c e n t r a t i o n as V a r i a b l e  207  Run F30.22. F e r m e n t a t i o n w i t h P. f r e u d e n r e i c h i i . Yeast E x t r a c t C o n c e n t r a t i o n as V a r i a b l e .............  208  AI.41 AI.42 AI.43  Run F20.00. F e r m e n t a t i o n w i t h P. f r e u d e n r e i c h i i . C o b a l t Ion C o n c e n t r a t i o n as V a r i a b l e  ,. 209  AI.44  Run F20.09,. F e r m e n t a t i o n w i t h P. f r e u d e n r e i c h i i . C o b a l t I o n C o n c e n t r a t i o n a s V a r i a b l e . . . . . . . . . . . . . . . . 210  AI.45  Run F20.22.126. F e r m e n t a t i o n DMBZ w i t h P. f r e u d e n r e i c h i i  AI.46  RunS20.22. Fermentation  List  with  o f SSL  Containing 211  P. s h e r m a n i i  of Tables  ..........  212  ix  LIST  OF  FIGURES  F i g u r e  Page  1.1  S t r u c t u r e  1.2  L.  p l a n t a r i u m  i n  R e i n f o r c e d  1.3  L.  p l a n t a r i u m  i n  Spent  1.4  P.  f r e u d e n r e i c h i i  i n  R e i n f o r c e d  1.5  P.  f r e u d e n r e i c h i i  i n  Spent  1.6  P.  s h e r m a n i i  i n  R e i n f o r c e d  1.7  P«  s h e r m a n i i  i n  ,Spent  1.8  S.  g r i s e u s  2.9  S t r i p p i n g  2.10  of  i n of  I n c u b a t i o n , D i l u t e d  V i t a m i n  of  H a l f  2.11  Right  2.12  Side  2.13  Top  View  2.14  The  pfl  2.15  S t e r i l i z a t i o n  2.16  Foaming  2.17  R e l a t i o n  View  of of  of  S.  i n  The The  NBS  . . . . . .  C l o s t r i d i a l  Medium  ..  18  Medium  19  L i q u o r  19  Medium by  and  19  B o i l i n g S.  M i c r o f e r m  Fermentor  and  o l i v a c e u s  A e r a t i o n  .  Twin  i n  The  71  between  29  i n  unit  34 ,.  Head  34 34  Head  35  C o n t r o l l e r of  18  L i q u o r  I n c u b a t o r - s h a k e r  Fermentor  18 18  C l o s t r i d i a l  Mo.3  Medium  L i q u o r  S u l f i t e  D i o x i d e  BBS  C l o s t r i d i a l  S u l f i t e  g r i s e u s  The  The  11  S u l f i t e  Waksman's S u l f u r  SSL,  B12  36 The  71  Fermentor  36  Fermentor M o d i f i e d  L i s t  of  and  36 Standard  F i g u r e s  T u r b i d i t y  ....  41  X  F i g u r e 2.18  2.19  2.20  Page R e l a t i o n between M o d i f i e d T u r b i d i t y and C r y C e l l Height o f 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 . . . . . . . . . . .  12  R e l a t i o n between M o d i f i e d T u r b i d i t y and Dry Weight of P r o p i o n i b a c t e r i a Shermanii  C e l l 43  R e l a t i o n  C e l l  Weight  between  of  L a c t o b a c i l l u s  2.21  L a c t o b a c i l l u s  2.22  P a r t i t i o n  2.23  S p e c i a l with  M o d i f i e d  of  Glassware  C o n c e n t r a t e d  The Ether  2.25  Recovery  2.26  Ether E x t r a c t i o n Recovery o f L a c t i c Ether  E x t r a c t o r A c i d s  Cry  Bacto  44 B12 C u l t u r e  between  A c i d i f i c a t i o n  S u l f u r i c  2.24  o f  i n  A c i d s  f o r  and  P l a n t a r i u m  L e i c h m a n i i V o l a t i l e  T u r b i d i t y  A c i d  Water of  Agar  ..  46  .  49  .  46  and Ether  Samples  A  . . . . . . . . . . . . . . . . .  ,  . . . . . . . . . . . . .  from  S S L by  A c i d  from  52  Continuous SSL by  54  Continuous  E x t r a c t i o n  55  3.27  Growth  o f  Streptomyces  G r i s e u s  3.28  Growth  of  Streptomyces  O l i v a c e u s  3.29  Growth  o f  L.  3.30  and G l u c o s e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E f f e c t of Yeast E x t r a c t C o n c e n t r a t i o n on Growth o f L. p l a n t a r i u m i n SSL i n T e s t Tubes  3.31  E f f e c t P.  3.32  o f  p l a n t a r i u m  Yeast  E x t r a c t  f r e u d e n r e i c h i i  E f f e c t of Yeast P. s h e r m a n i i i n f o r  i n  i n  i n  D i l u t e d i n  D i l u t e d  S o l u t i o n s  T e s t  Numbered  Growth  o n Growth .,  3.34  Growth o f L. p l a n t a r i u m a s Measurement of E f f e c t i v e n e s s o f Yeast E x t r a c t from V a r i o u s o f  SSL with  L .  3.36  F e r m e n t a t i o n s i n F l a s k s  o f  SSL with  P.  67  68 71  o f 73  Legend  F e r m e n t a t i o n s  S S L . . . . .  X y l o s e  3.33  3.35  from  on  67  Tubes  E x t r a c t C o n c e n t r a t i o n SSL i n Test Tubes  F i g u r e s  o f  C o n c e n t r a t i o n  SSL i n  S S L . . . . . . .  3 . 3 0 t o  p l a n t a r i u m  o f 75  3 . 3 2 . . . . . . .  i n  Sources F l a s k s  76  .  . . .  78 82  f r e u d e n r e i c h i i 84  L i s t  o f  F i g u r e s  x i  Figure  Page  3.37  Fermentations  o f  SSL with  P.  3.38  Fermentations  o f  SSL with  A  L.  and P .  p l a n t a r i u m  s h e r m a n i i M i x t u r e  o f  f r e u d e n r e i c h i i  i n  i n  F l a s k s  F l a s k s  . . . .  86  . . . . . . .  88  3.39  Run F P 1 . 1 .  Fermentation  with  L .  p l a n t a r i u m  . . . . . . . . . .  93  3.40  Run F P 1 . 2 .  Fermentation  w i t h  L .  p l a n t a r i u m  . . . . . . . . . .  95  3.41  Run F P 2 . 1 .  Fermentation  w i t h  P.  3.42  on L a c t i c A c i d a s Main S u b s t r a t e Run F P 2 . 2 . Fermentation w i t h P. on L a c t i c Acid a s Main S u b s t r a t e  3.43  3.44  3.45  3.47  f r e u d e n r e i c h i i  97 99  Run F P 3 . Fermentation with Mixed C u l t u r e of L . p l a n t a r i u m and P. f r e u d e n r e i c h i i  101  Run F15.22a. Fermentation with P. f r e u d e n r e i c h i i . E f f e c t o f pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  103  Run F15.22b. i n  3.46  f r e u d e n r e i c h i i  Fermentation  One Anaerobic  with  P.  f r e u d e n r e i c h i i  Stage  105  Run F 0 5 . 2 2 . . Fermentation with Y e a s t E x t r a c t C o n c e n t r a t i o n as  P. f r e u d e n r e i c h i i . V a r i a b l e . . . . . . . . . . . . .  Run F 1 0 . 2 2 . . Fermentation with P. f r e u d e n r e i c h i i . Yeast E x t r a c t C o n c e n t r a t i o n a s V a r i a b l e  107  109  3.48  Run F 2 0 . 2 2 . . Fermentation with Yeast E x t r a c t C o n c e n t r a t i o n a s  3.49  Run F30.22. Fermentation with P. f r e u d e n r e i c h i i . Yeast E x t r a c t C o n c e n t r a t i o n a s V a r i a b l e . . . . . . . . . . . . . 1 1 3  3.50  Run F20.00. F e r m e n t a t i o n C o b a l t I o n C o n c e n t r a t i o n  with P. f r e u d e n r e i c h i i . a s V a r i a b l e . . . . . . . . . . . . . . . .  115  Run F20.09. Fermentation C o b a l t I o n C o n c e n t r a t i o n  with P. f r e u d e n r e i c h i i . a s V a r i a b l e . . . . . . . . . . . . . . . .  117  3.51  3.52  3.53  Run F20^22. 126. Fermentation DMBZ w i t h P . f r e u d e n r e i c h i i Run S20.22.  Fermentation  L i s t  o f  w i t h  P. f r e u d e n r e i c h i i . V a r i a b l e . . . . . 1 1 1  o f  SSL  C o n t a i n i n g 119  P.  F i g u r e s  s h e r m a n i i  . . . . . . . . . .  121  x i i  F i g u r e  Page  3.54  Legend f o r A l l F i g u r e s , L i s t e d i n Table I I I . 2 3 , from 3 . 3 9 t o 3 . 5 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 2  3.55  P r o d u c t i o n E f f e c t  3.56  3.57  3.58  o f  of  V i t a m i n  Yeast  B12 by  P .  f r e u d e n r e i c h i i . . . . . . . . . . . . . . . .  127  P r o d u c t i o n of V i t a m i n B12 by P. f r e u d e n r e i c h i i . E f f e c t o f C o b a l t I o n C o n c e n t r a t i o n . . . . . . . . . . . . . . . . . .  129  P r o d u c t i o n o f V o l a t i l e A c i d s by P. f r e u d e n r e i c h i i . E f f e c t of Yeast E x t r a c t C o n c e n t r a t i o n . . . . . . . . . . . . . . .  131  P r o d u c t i o n o f V o l a t i l e A c i d s by P. E f f e c t o f C o b a l t I o n C o n c e n t r a t i o n  133  Legend  3.60  Hun F P 1 . 1 .  Fermentation  Rate  of  L .  3.61  Run F P 2 . 2 .  Fermentation  Rate  of  P.  3.62  3.63  3.64  3.65  L a c t i c  3.67  3.68  a s  Main  from  3 . 5 5 t o  3 . 5 8 . . .  134  . . . . . . .  136  p l a n t a r i u m  f r e u d e n r e i c h i i  S u b s t r a t e  . . . . . . . . . . . . . . . . . .  138  140  Run F10.22. Fermentation Rate of P. f r e u d e n r e i c h i i . Yeast E x t r a c t C o n c e n t r a t i o n a s V a r i a b l e . . . . . . . . . . . . .  142  Run F20.22. Fermentation Rate of P. f r e u d e n r e i c h i i . Yeast E x t r a c t C o n c e n t r a t i o n a s V a r i a b l e . . . . . . . . . . . . .  144  Run F30.22.  Fermentation  E x t r a c t  Rate  C o n c e n t r a t i o n  o f  a s  P.  f r e u d e n r e i c h i i .  V a r i a b l e  146  Run F20.00. Fermentation C o b a l t I o n C o n c e n t r a t i o n  Rate of P. a s V a r i a b l e  f r e u d e n r e i c h i i .  Run F20.09. Fermentation C o b a l t Ion C o n c e n t r a t i o n  Rate o f P. a s V a r i a b l e  f r e u d e n r e i c h i i . . . . . . . . . . . . . . . . .  Run F20.22.126. w i t h  A d d i t i o n  3.69  Run S20.22.  3.70  Legend  3.71  A c i d  Numbered  f r e u d e n r e i c h i i . . . . . . . . . . . . . . . . . . .  Run F05.22. Fermentation Rate of P. f r e u d e n r e i c h i i . Yeast E x t r a c t C o n c e n t r a t i o n a s V a r i a b l e  Yeast 3.66  A l l F i g u r e s  C o n c e n t r a t i o n  3.59  with  f o r  E x t r a c t  Fermentation o f  o f  PF  152  Fermentation  R e l a t i v e  C a p a c i t y  SSL  L a c t i c  o f  A c i d L i s t  150  i n SSL  DMBZ  f o r A l l F i g u r e s  w i t h  Rate  148  Rate  of  Numbered  PS a n d PF a s o f  Main  P.  s h e r m a n i i  from  3 . 6 0 t o  i n  154  3 . 6 9 . . .  155  . . . . . . . . . . . . . .  156  Fermenting  S u b s t r a t e  F i g u r e s  . . . . . . .  x i i i  F i g u r e  Page  4.72  L .  p l a n t a r i u m  A2.73  D r y i n g  A2.74  T y p i c a l  Standard  Curve  f o r The Reducing  A2.75  T y p i c a l  Standard  Curve  f o r The V i t a m i n  o f  a n d P .  Aluminum  f r e u d e n r e i c h i i  Dishes  L i s t  o f  i n  i n  S S I (Bun FP3)  The Isotemp  F i g u r e s  Oven Sugar  .  163  . . . . . . .  215  Test  ..  216  . . . .  220  E12 Assay  x i v  ABBREVIATIONS  BOD  -  B i o c h e m i c a l  GOD  -  C h e m i c a l  DHBZ  -  5,6-dimethyl  L.  -  L a c t o b a c i l l u s .  LP  -  L a c t o b a c i l l u s  P.  -  PF  -  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 .  PS  -  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 .  S.  -  SSL  -  -  Oxygen  Demand.  Demand.  B e n z i m i d a z o l e .  P l a n t a r i u m .  P r o p i o n i b a c t e r i a .  Streptomyces. Spent (also  00D  Oxygen  S u l f i t e c a l l e d  U l t i m a t e  L i q u o r  Waste  Oxygen  o r  S u l f i t e  S u l f i t e  Spent  L i q u o r ) .  Demand.  A b b r e v i a t i o n s  L i q u o r .  XV  ACKNOWLEDGMENTS  Sheer ae  through  within  this  f o r m u l a t e my t h a n k s  project.  all,  this  work was u n d e r t a k e n ,  f o rtheir  who  helped  p a r t o f , my i n d e b t e d n e s s :  D r . R i c h a r d B r a n i o n and D r .  direction  to those  N e v e r t h e l e s s , words a r e t h e o n l y means  my r e a c h t o e x p r e s s , a t l e a s t  -To  of  words c a n n o t  Ken  Pinder,  f o rtheir  patience throughout  under  guidance,  whose  and most  t h e l e n g t h o f time  of t h i s  work. -To Canada  D r . George S t r a s d i n e o f t h e F i s h e r i e s for technical  cultures -To  of bacteria  advice  and f i s h  as  well  plant  the various i n d i v i d u a l s  Research  as f o r p r o v i d i n g  Syeklocha  in  of the f a c u l t y  microbiological  f o r her i n s t r u c t i o n  assay, t o Br. B i l l  in  stock  effluent. and s t a f f  Department o f M i c r o b i o l o g y , namely, t o Mrs. 0. V o l k o f f instruction  Board o f  techniques, performing  to  the  c f the  f o r her Dr.  Delfa  vitamin  B12  Page f o r h i s m a i n t e n a n c e o f s t o c k c u l t u r e s o f  Streptcmyces. -To  the  ITT  Rayonier  I n c . f o r the spent s u l f i t e  liquor  supplied. -To  t h e Department o f Energy,  Resources University -And  Division, of B r i t i s h finally,  the  M i n e s and R e s o u r c e s  National  Columbia  Research  f o rtheir  t o a l l my f r i e n d s  who  Acknowledgments  Water  C o u n c i l and t h e  financial have  -  assistance.  come,  and  most  xvi probably  gone,  leaving  behind  u n f o r g e t t a b l e souvenirs of a l l those those  "physical  individual the  education  them, social  in  my  memory,  the  get-togethers  and  l e s s o n s " where t h e p r e s e n c e o f e v e r y  had made a p r e c i o u s c o n t r i b u t i o n  joy o f the events.  Acknowledgments  t o the  success  and  r ....luan.an.chi.co.mot.ma.mucn.tang.bao.nhieu.nguoi..... . ...tang.cho.tat.ca.nhung.nguoi.da.true.tiep.hay ....gian.tiep.khuyen.khich.viec.hoan.thanh.them.mot..,. ....chang.duong.hoc.van.  . ... k i n h . t a n g . p h u . m a u . . . . . . . . m e n . t a n g . c h a u . . . . . . . . . . . . . ....tang.be.diem........tang.be.mat.hat.de............. ....tang.chi.kirn..........tang.be.van.thanh............  •  Potential as  Raw  o f Spent S u l f i t e  Material f o r Production by C e r t a i n  Lactobacillus,  liguor  I  o f V i t a m i n B12  Species of  Propionibacteria,  |  and S t r e p t o m y c e s  | | j  . ... t a n g , v a n . h o a . t i e n ....tang.nguoi.ban.than......tang.van.khanh . ....tang a.francine...................  P o t e n t i a l  of  Spent  S u l f i t e  Chapter  Liquor  1  INTRODUCTION  1.1  SPENT_SULFITE_II2U0R  1.1.1  Composition In  t h e  pressure a  s u l f i t e  and  a t  s o l u t i o n  ammonium, a c i d ,  with i s  v a r i e s  c o m p o s i t i o n  of  and,  i n  c a s e s  where  h y d r o l y z a t e s d i f f e r e n t s u l f i t e from  ITT  s p e c i e s  of  data  woods. from  used  t h e  not  r e p r e s e n t a t i o n  the  a v a i l a b l e  Table  1.4  d i f f e r e n t t h i s of  from  of  cooking  t h e  s o u r c e s , r e s e a r c h .  (SSL)  used)  s u l f i t e  the  sugars S S L ,  the  pulping i n  of  from  composition  of  t h e  Nevertheless s u l f i t e  SSL wood  sugars)  i n c l u d i n g  t o  t h e  A l s o ,  t h e  of  .  spent  on  of  of  or  s p e c i e s  used.  c o m p o s i t i o n shows  This  one  f o r  with  sodium;  depending  t h e c o m p o s i t i o n  I n t r o d u c t i o n  160<>c)  weed  of  wood  under  l i g u o r  of  c o n d i t i o n s  s i m i l a r  i n  50??  another  t h e c o m p o s i t i o n  i s  have  s u l f i t e  d i f f e r e n t  c f  heated  t o  d i o x i d e .  (about  t o  source  shows  140°c  t h e c o m p o s i t i o n  m i l l  on  are  magnesium,  s u l f u r spent  i s  1.2),  and  l i q u o r s  Rayonier,  approximate  wood  one  (which  spent  of  and  1.3  of  as  c h i p s  (about  components  SSL depends  Table  wood  c a l c i u m ,  excess  wood  from  s p e c i e s  of  discharged  1.1  p a r t i c u l a r  p r o c e s s .  an  c o m p o s i t i o n  (Tables  p r o c e s s ,  temperature  b i s u l f i t e  d i s s o l v e d  t h e  another l i q u o r  o f  used,  c o n t a i n i n g As  e l e v a t e d  u s u a l l y  once  p u l p i n g  one an  spent  P o t e n t i a l  of  Spent  S u l f i t e  Table THE • I |Wood  PERCENTAGE  L i q u o r  2  1.1  COMPOSITION  OF  CERTAIN  WOODS  :  [99]  :  s p e c i e s  HC  L  AC  Heme  P  T  U  H  A  | i  i i  i  1 |White s p r u c e |Red spruce j E a s t e r n hemlock (Balsam f i r |Jack pine (Aspen (Willow (Maple (White  26. 6 26. 31. 30. 27. 17. 22. 23. 24.  oak  73. 72. 68. 69. 72. 82. 78. 76. 75.  6 5 1 2 3 0 5 1  3 9 5 9 5 5 3 3 4  49.5  23.8  48 48 44 49 50  24 20 25 23 31  . . . . .  3 2 0 5 7  50.0 49.5  . . . . .  10. 9 11. 6 10. 0 10. 3 12. 8 23. 5  6 3 9 0 8  2. 3. 3. 3.  26.3 25.9  —  68 20 40 08  2. 35 2 . 50 1. 87 2 . 24  2 . 92 4 . 28  1. 92 4. 65  --  --  I— L BC  : :  from  (Table  t h i s  u s e f u l i n  i s  of  more  waste,  i s i s  g e n e r a l l y  away  as  i n d u s t r y  s e r i o u s .  In  d i s c h a r g e  t h e i r  high  p a r t i c u l a r waste  accepted  d i s c h a r g e d  Caused_by  with  75 | 93 |  —  I 1  A l p h a c e l l u l o s e H e m i c e l l u l o s e Pentosans Dronic a c i d anhydride A c e t y l Methoxyl i n carbohydrate  s u g a r s ,  thrown  P o l l u t i o n The  i t s  p i c t u r e  amount  SSL,  1.1.2  1.5)  70| 92| 84| 41(  L i g n i n H o l o c e l l u l o s e  AC : HemC: P : U : A : M :  l i q u o r  0., 0, 0,, 0. 0.. 0.  about  without 2 0 *  of  [ 6 4 , 1 1 0 ] . r e c o v e r y ,  the  t o t a l  If an  the  SSL  a p p r e c i a b l e  d i s s o l v e d  s o l i d s  waste.  Spent grows,  S u l f i t e ^ I i g u g r the  more  the  s t r e a m - p o l l u t i n g pulp  l i q u o r  m i l l s ,  with  a  I n t r o d u c t i o n  using 5-day  d i s p o s a l  problem  p o t e n t i a l , the  s u l f i t e  b i o c h e m i c a l  of  becomes p r o c e s s , oxygen  P o t e n t i a l  of  Spent  Table YIELD  OF  POTENTIAL  Wood  SEDUCING  AND  S u l f i t e  L i q u o r  3  1.2  FERMENTABLE  s p e c i e s  SUGARS  PRS  OF  WOODS  [99]  PFS  F  Hardwoods American Beech Aspen B i r c h Maple Red Oak Sweetgum Yellow P o p l a r  70. 1 75.1 69.9 68.2  75. 76. 67. 71. 63. 73. 76.  63.6 66. 4 70.9  52. 57. 47. 48.  1 3 8 0 0  6 3 4 4  40. 2 49. 0 54. 0  8 1  Softwoods Douglas E a s t e r n Hemlock  F i r White  Ponderosa Pine Redwood S i t k a Spruce Southern Y e l l o w Sugar P i n e  demand two  (BOD)  s u l f i t e a l l  SSL  being  of  a q u a t i c owners  Pine  P o t e n t i a l r e d u c i n g F e r m e n t a b i l i t y {%)  PFS  :  P o t e n t i a l  of  at i n  l e a s t B r i t i s h t h e i r  p o i s o n o u s ,  f a u n a ' s that  l i f e the  l b s  waste  [ 1 0 9 ] . of  at  i n t o  1 3 0 4  {%)  sugars  ton  of  s e v e r a l  the  s e a .  the  other  a  few  f i s h  hand, i s  i n  p u l p .  The  Washington  E c o l o g i s t s  extremely  On  {%)  s u l f i t e  and  l e a s t  I n t r o d u c t i o n  2  sugars  per  Columbia  or  l o s s  82. 77. 85. 82. 82.  fermentable  400  57. 4 57. 4 58. 3 55. 9 40. 4 59. 8 53. 2 53. 0  86.2 86.3 88. 2  2.4 0.1 4.8 4.3  : :  d i s c h a r g e  argue  5 7 6 6  6 5 1 0  PRS F  m i l l s  s t a t e  66. 66. 66. 68.  Pine  accuse  harmful, some  pulp  to m i l l  i n c o n s e q u e n t i a l  P o t e n t i a l  of  Spent  S u l f i t e  Table THE  P E R C E N T A G E C O M P O S I T I O N OF L I Q U O R S AND HOOD  i""' ™"  I  " ••  L i q u o r  "•"  1  L i q u o r  4  1.3  SUGARS OF T Y P I C A L HYDRGLYZATES [ 9 9 ]  SPENT  SULFITE  *•"  1  —  Type  SSL  T o t a l H y d r o l y z a t e s  SSL  i  P a r t i a l H y d r o l y z a t e s — i  |  Wcod  Type  S  HF  wS  SP  bP  j  B j i  |  Sugars  j | | | |  Hexose  | | |  Pentose X y l o s e Arabinose  1 |  Glucose Mannose G a l a c t o s e F r u c t o s e  G a l a c t u r o n i c U n i d e n t i f i e d  15 48 10 2  28.9 42.7 4.2 4.0 17.0  15 6  A c i d  29.3  53.7 23.8 2.9  37.7 6.4  67.6 14. 1 6.2  61.9 24.7 4.0 1.4  13.3 5.4  — —  8.9 3.2  12.3 7.7  8.0  --  3.2  —  4  67. 7 1. 8 0. 0 —  30. 1 0. 4  7.9  «  . i  compared hundreds t h a t the  the  HF S wS  : : :  8595 w e s t e r n h e m l o c k Spruce White s p r u c e  SP jP  : :  Spruce and Jack pine  bP B  : :  Bordeaux B i r c h  to of  the  e x t r a  d i s s o l v e d  from  A p o l o g i s t s  may  white  f i r  pine  carbohydrate  oxygen  151  pine  b e n e f i t s  workers.  plus  have  go  a  l a r g e  even  source  I n t r o d u c t i o n  f u r t h e r  and  b e n e f i c i a l  development  the  to  s l i g h t  e f f e c t s  on  employing  c l a i m  [110]  l o w e r i n g the  of  growth  P o t e n t i a l  of  Spent  S u l f i t e  Table COMPOSITION  OF  TYPICAL  L i q u o r  5  1.4 SPENT  SULFITE  IIQUCBS  . —  1—  I  T h i s  Source  Work  [49]  [70]  [100]  [25]  |  ...  |  Wood  S p e c i e s  j | j  S u l f u r T o t a l Free  D i o x i d e  |  S o l i d s  (g/1)  t j | | | | j | | | |  S o l i d Components {% o f t o t a l s o l i d s ) . . . L i g n o s u l f o n i c A c i d s T o t a l Reducing Sugars 32 Fermentable Sugars 22 Non-fermentable Sugars 10 Calcium (as CaO) Organic A c i d s Sugar A c i d s Ash Other .  . . .  95 : 5 Hemlock & White F i r  . . .  Spruce  Spruce  1  8 2 126  128  100  62 29 18 11  65 20 15 5 7  9  52 23  55 20 14 •6  8  microorganisms, Arguments out  to  hence,  cannot  experiment,  than  few  day.  Some  o y s t e r s prove  the  compound  of  [ 1 7 , 3 7 , 5 0 , 1 1 3 ] .  as  yet  by  harm been  p r o d u c t i o n sheer  these on  on  s i t e ,  the  Table caused  of  words,  p o i n t s .  l a b o r a t o r y  c o n c e n t r a t e d  p o s s i b l e  the  conducted  p r o j e c t s  have  has  won  i n v e s t i g a t e  thorough a  be  on  have  e f f e c t s  of  1.6  s e r v e s  to  f i s h  by  i s o l a t e d  from  SSL.  I n t r o d u c t i o n  j  w e l l .  as  have  r e p o r t e d ,  SSL  The  as  _  a p p a r e n t l y  seen  SSL.  i  I I I  r e s e a r c h e r s  been  s c a l e  |  4  Though  has  I I  9  f i s h  so  | |  5  7 10 10 3  -  1 |  120-160  100-150  ,  s e t  |  (g/1)  i.  of  J  i  the on  no more  l i g h t f i s h  and  i l l u s t r a t i o n  However,  no  danger  t h a t  of  to  t o x i c t h i s  P o t e n t i a l  of  Spent  S u l f i t e  T a b l e A  GENERALLY  ACCEPTED  PICTURE  OF  L i q u o r  6  1.5 THE  COMPOSITION  CF  SSL  £64,114]  1  C o n c e n t r a t i o n  Components  (%  M i s c e l l a n e o u s  Organic  Compounds  ...  of  t o t a l  s o l i d s )  5-8  L  l i q u o r of  imposes  at  l e a s t  r e c e i v i n g  water,  p r o s p e c t s r a t e  these  of  e x t r a  t h e i r  i n  s o u r c e  happy  i s the  to  a l s o g i l l  r e c e i v i n g problem, s e t t l i n g ,  stream, however, or  as  w e l l  of  s i n c e  f i s h  or  c r e a t i n g can  e a s i l y  i t .  a l s o  c l i m b s , from  as  f i s h e s ,  s u f f e r  adequately  may  turn  out  f i b r e may  or  b l a n k e t  a n a e r o b i c s o l v e d  f l o t a t i o n . I n t r o d u c t i o n  may the  i n  t h e i r  thus  of growth  T h e r e f o r e  from  a  l a c k  p r o v i d e d , to  be,  of t h i s  i n d e e d ,  matter  cause  i n  i r r i t a t i o n  bottom  s c r e e n i n g ,  the  e x c e e d i n g  a i r .  c o n d i t i o n s . by  BOD  supply  suspended  bark  high  present  Hence,  oxygen  i s  i t s  i n c r e a s e d  Furthermore,  be  to  of  oxygen  l i f e ) .  problem,  t i s s u e  oxygen  c a r b o h y d r a t e  a q u a t i c a  i n  due  a l r e a d y  the  consume  d i s s o l u t i o n  i f  probably  with  and  demand  f a c t , of  i s  M i c r o o r g a n i s m s ,  SSL,  n a t u r a l  (but  b e n e f i c i a l  of  are  from  and  l i f e  ppm.  m i c r o o r g a n i s m s ,  oxygen  SSL  a q u a t i c  20,000  c a r b o h y d r a t e s  the  on  of  T h i s  the l a s t  f i l t r a t i o n ,  P o t e n t i a l  of  Spent  Table MORTALITIES r  •  :  | I  |  OF  S u l f i t e  L i q u o r  7  1.6  SALMON  EXPOSED  TO  SSL  —  "•i  M o r t a l i t y (estimate of 1  Salmon  j  Chinook  |  P i n k  |  S i l v e r  T h r e s h o l d I n d i c e s approxima t e l y a  S p e c i e s  Age (days)  [ 113 ] K i l l )  5%  T h r e s h o l d I n d i c e s (ppm o f SSL)  38 280  1175 600  305 102 198 351  560 1550 530 1230 1015  412  j i ( M o r t a l i t i e s |  of  2 0 - d a y -o l d C h u m S a l m o n E x p o s e d Spent S u l f i t e L i q u o r [37]  to  Calcium  Ease  i 1 |  Average  f | | | | j | | | |  of SSL (ppm) Standard D e v i a t i o n (ppm) Average T o t a l S u l f u r D i o x i d e (ppm) Average D i s s o l v e d Oxygen (ppm) Average pH D u r a t i o n of Experiments (days)  C o n c e n t r a t i o n  |  P e r c e n t  j  M o r t a l i t y *  0  558  1071  1754  2498  —  95.6  128. 1  381. 1  115.5  • —  2.6  5.3  9. 1  13.2  8. 3 7. 3  8.6 7.0  8. 1 6.8  9. 1 7. ?  8.7 7.6  30  30  30  30  10  3  1  27  91  100  of  •  t  • M o r t a l i t y due to l e t h a l q u a l i t i e s d i s s o l v e d oxygen c o n c e n t r a t i o n was minimum a l l o w a b l e l e v e l  I n t r o d u c t i o n  cf SSL a l o n e , maintained above  as the  the 5ppm  P o t e n t i a l  Some  a u t h o r s  components  found  a c e t a l d e h y d e , s u l f u r j u s t  not  doubts  of  d i o x i d e ,  high  i n  [ 1 0 7 ] .  SSL  enough  the  [34]  But  than  wood  200ppm)  to  c e r t a i n  [ 2 9 ] ,  a r e ,  or  more  and  d i l u t e d  be  [ 8 1 , 1 1 0 ] ,  t u r p e n t i n e  e f f e c t s ,  when  c f  t h i o s u l f a t e s  c o n c l u s i o n s  SSL  8  p o t e n t i a l  as  of  i n h i b i t i n g in  l i q u o r  t o x i c such  t h e i r  content  (less  S u l f i t e  d e r i v a t i v e s  p o s s i b l e i t s  Spent  s t u d i e d  f u r f u r a l ,  d i o x i d e  s u l f u r  have  of  i n  l e s s ,  the  i n t o  and  case  streams  of i s  harmful.  I 1.1.3  Treatments If  pulp  the  m i l l s  have  o u t c r i e s  to  p o l l u t i o n  c l o s e  made  s u b s t a n c e s  to  with  any  p o s s i b l e  i s  p r a c t i c a l  s c a l e  i s  at  e i t h e r  monetary  the  p r a c t i c e .  p r o c e s s e s  which  the  produced f u l l y  provide  recovery  new  of  the  oxygen  p o t e n t i a l  at  high  has  cause  s u l f i t e  s u b f i e l d  or  from  the  reached  the  that  to  remove  SSL.  f a r  exceeding  b y - p r o d u c t s ,  and  i n  attempts  to  c o s t ,  marketing  s c a l e ,  t h a t  a  most  adequate  by  not  open  l i s t s  n u t r i e n t s  one  p r o c e s s e s ,  commercial  t o  1.7  commercial  only  d i d  did  so  t r i c k l i n g  none  f i l t e r  s e m i - c o m m e r c i a l  a l l .  Some  demand,  they  oxygen-demanding  r e q u i r e  on  e n v i r o n m e n t a l i s t s  T a b l e  high  Treatments  of  down,  s t u d i e s .  been  p r o c e s s  and_Uses  hence,  problem i n  a  f u r n i s h e d .  p r o c e s s e s by  l a r g e  as  the  l i g n i n  caused  r a t h e r  SSL  However,  use  these  using  as  raw  m a t e r i a l ,  products a  d e r i v e d  component  cannot  m i l l i o n s q u a n t i t y ,  S u g a r - f e r m e n t i n g I n t r o d u c t i o n  be of but  have  ajre  tons that  of  from  i n  regarded  as  SSL.  f a c e  i n t o  v a r i o u s  very  market  p r o c e s s e s  gone  a  l i m i t e d s o l u t i o n  V a n i l l i n i s  i s  a l r e a d y an  e x t r a  P o t e n t i a l  o f  Spent  Table  «—-—: |  AND USES  :  l i q u o r  9  1 . 7  OF SPENT  SULFITE  LIQUOR  TREATMENTS  \ I I |  TREATMENTS  S u l f i t e  \  A c t i v a t e d sludge C o n t a c t a e r a t i o n T r i c k l i n g f i l t e r  [ 5 1 , 8 2 , 8 3 ] (Hays p r o c e s s ) [ 3 5 , 8 0 , 9 8 ]  [ 3 6 ]  USES Heat  | I | I | | I |  Uses  | | j I | I | I | | | |  P r o d u c t s from f e r m e n t a t i o n o f sugars - E t h a n o l * £99,106] - A c e t o n e - B u t a n o l £112] - P r o p i o n i c - A c e t i c a c i d s £2,23,59,70] - L a c t i c a c i d £46,93] , - B u t y r i c a c i d £18] - G l y c e r o l £6 5 ] - 2 , 3 - B u t a n e d i o l £66] - A n t i b i o t i c s £20] -Methane £8,9] - L i q u i d carbon d i o x i d e £64] - F a t yeast £6 4 ]  I |  *0n  I j | |  j  i  :1  r e c o v e r y  1  [ 6 4 ]  o f l i g n o s u l f o n a t e * £64] -Cement o r c e r a m i c a d d i t i v e - L i g n o l e u m p a s t e , i n d u s t r i a l a d h e s i v e , t e m p o r a r y road pavement,road spray t o prevent dust - B i n d e r f o r foundry c o r e s -Tanning agent - S o i l f e r t i l i z e r o r s t a b i l i z e r £1,76,88,89] - V a n i l l i n  - V i t a m i n B12 £2,23,59] -Food y e a s t * £99,111]  \ I | I I I I | | | j | | j | j | | I | | | 1  ,  commercial  |  s c a l e  I n t r o d u c t i o n  P o t e n t i a l  problem,  the  e f f l u e n t . reached from  complete  A l c o h o l  l i m i t e d  yeast  w i t h  p l a n t  SSL  i n  Dufrenoy r e l a t i o n s h i p s t r u c t u r e l i g n i n  1.2  but  raw  S u l f i t e  unused yeast the  m a t e r i a l ,  and  P r a t t  [20] the  SSL,  a l c o h o l  e x i s t e n c e i n  North  presented p e n i c i l l i n  and  are  and of  the  SSI  have  from  with  c o m p e t i t i o n  the  l a t t e r  only  America  a  3  has  a l c o h o l  and  only  p o s s i b l e  and  one  of  molecular  cannct  use  the  promising  with  c a r b o h y d r a t e s  and  e c o n o m i c a l l y  s o u r c e s  g e n e r i c  l i g n i n  f e r m e n t a t i o n s  not  c o n c e n t r a t e d  n i t r o g e n o u s  meets  i n t o  S t a t e s .  a n t i b i o t i c - p r o d u c i n g of  10  f e r m e n t a t i o n s  former  the  L i g u o r  l i g n o s u l f o n a t e  s y n t h e t i c  e x p l a i n s  U n i t e d  more  n u t r i e n t .  VITAMIN_BJ.2 Vitamin  B 1 2 , f i r s t  c o m p l i c a t e d  v i t a m i n  c o n t a i n i n g  a  r e q u i r e d  a l l  i s  of  between  to  o r g a n i c  as  of  But  the  p o r t i o n  r e s p e c t  s c a l e .  That  Spent  fcod  p r o d u c t i o n  markets.  p l a n t s ,  l o s s  and  commercial  cheaper  of  by  found  pork,  i n  v e a l ,  s y n t h e s i z e d  by  during  metabolism. the  The  known  animals  i n  T h i s  I t  i s  gut  i s  not  the  the  such  r a t h e r  then  l a r g e s t  only 1.1).  found  m i l k .  e x c l u s i v e  i s  (Figure  foods  and  i t  the  but i n  cheese,  a n i m a l s ,  f e r m e n t a t i o n  1948,  c o b a l t  q u a n t i t i e s  lamb,  i n  d i s c o v e r e d .  i o n ,  higher  f u n g a l  among  yet  metal  s m a l l  i s o l a t e d  i n as  beef  by  B12,  p l a n t s ,  l i v e r , i t  most  v i t a m i n  higher  produced  m i c r o b i a l  known  Vitamin  However,  absorbed  and  beef,  i s  not  b a c t e r i a l  and  or  c o n c e n t r a t e d  s y n t h e s i s  i s  unique  v i t a m i n s .  b i o c h e m i c a l  r o l e  of  v i t a m i n  I n t r o d u c t i o n  B12  has  not  been  c o m p l e t e l y  Potential  FIGURE  1.1  of  Spent  Sulfite  : Structure  C H  2  of  O H  Introduction  Liquor  Vitamin  11  B12  P o t e n t i a l  e l u c i d a t e d ,  i t  has  d e f i c i e n c y  - A hence  but  t h i s  of  v i t a m i n  of  Spent  been  shown  v i t a m i n  i s  the  S u l f i t e  12  t h a t :  B12  o n l y  L i q u o r  produces  c u r a t i v e  p e r n i c i o u s  anemia,  f a c t o r  f o r  t h a t  d i s e a s e  c o n s t i t u e n t  of  the  animal  [ 1 1 5 ] . - V i t a m i n p r o t e i n  B12  f a c t o r  i s  (APF)  - V i t a m i n  an  e s s e n t i a l  f o r  a n i m a l  B12  i s  such  as  u s e f u l  d i s o r d e r s  [60]  endocrine  d i s t u r b a n c e s ,  l i v e r  s k i n  and  V i t a m i n  was  B12 i n  of i n  SSL  been  has  [ 2 3 , 7 0 ] .  The  completed [ 1 1 5 ] . s i n c e during  i t a  treatment  anemia, growth,  c f  a  number  m u l t i p l e  r e t a r d e d  the  t h i s  r e q u i r e d p e r i o d  of  Table  of  s c l e r o s i s ,  pregnancy,  i n  a r t i f i c i a l (in  the 11  e f f o r t s  At  and  at  p r e s e n t ,  f o r  l i s t s  some  of  s y n t h e s i s  vitamin  i s of  s t i l l 99  years.  I n t r o d u c t i o n  of  from  workers  v i t a m i n B12  F e r g u s o n ' s  Woodward  f a r  s p e c i e s  commercial  v i t a m i n  and  by  V i t a m i n  these  used  P r o d u c t i o n  1973)  of  a n t i b i o t i c - p r o d u c i n g  are 1.11  s p e c i e s  f i r s t  N i s h i k a w a ' s  January  s y n t h e s i s  d i f f e r e n t  in  I.10  Table  S t a t e s .  r e p o r t e d  f i r s t  i n  by  1.8).  A p p a r e n t l y ,  B12.  United  (Table  b y - p r o d u c t  1.9).  v i t a m i n  s y n t h e s i z e d  media a  l i s t e d  r e c e n t l y  But  been  as  (Table  producers  retarded  v a r i o u s  microorganisms  p r o d u c t i o n  the  m a c r o c y t i c  has  produced  f e r m e n t a t i o n s of  i n  d i s o r d e r s .  microorganisms B12  growth.  B12  has  B12 from  theses been  and  Eschenmoser  being  c o m p e t i t i v e  from  19  c o u n t r i e s  P o t e n t i a l  of  Spent  Table PROCESSES  FOR  MICROBIAL  S u l f i t e  L i q u o r  13  1.8  SYNTHESIS  OF  VITAMIN  E12  [ 2 3 , 7 0 , 7 3 , 7 5 ] 1—  J  ._  -—  Microorganisms  \  Media  Oxygen  j j |  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  Temp. <°C)  Y i e l d (mg/l) II  |  1  !  2  Anaerobic  I  2  M i c r o A e r o b i c  30  |  3  I I I  3  Anaerobic A e r o b i c  4 5  30 30 30 30  I I I  6 7 8  | |  |  Time (hrs)  |  Anaerobic M i c r o A e r o b i c  -  30 30  96 100 Cent. 100 Cont. 89 2ft 132 100  P r o p i o n i b a c t e r i a Shermanii  |10  | |  12.2  |  0.875 1.3 25.0 3.0  ! j  Streptomyces  |  G r i s e u s  | |  -  2.4  |  168 280  1.5 2.3  | |  Anaerobic  30  160  1.5  |  Anaerobic then A e r o b i c  |  | |  30 30  -  I 12 113  1  Anaerobic A n a e r o b i c then A e r o b i c  Anaerobic Anaerobic  |11  | | ,i  4.5 0.6  —  .  ,,  -  | |  1  30 30 —  30  80 288  -  200  _  ,  i  1  1  23.0 17.6 3.0 0.8  mm  —  — —  | | | |  — ,mm mm — —  0.3  |  I  | | j  •  Streptomyces o l i v a c e u s  ^  j  115  —  —  -  3.3  |  • - JL .  ..  ,.,..  Continued  I n t r o d u c t i o n  J  P o t e n t i a l  of  Table  i  1  |  Spent  I.8  S u l f i t e  14  l i q u o r  (continued)  .  Microorganisms  i |  Oxygen  Media  Temp. (o ) C  Time (hrs)  Y i e l d (mg/1) j  |  Streptomyces  |  S p e c i e s  |16 25  I  A e r o b i c  28-34  96  2.0 5.7 j  I  -  0.7  | 1  Streptomyces F r a d i a e  J |  Pseudomonas D e n i t r i f i c a n s  118  Aerobic  31.5  70  5.8  |  B a c i l l u s  | 19  A e r o b i c  55  18  6.0  |  Coagulans  1 j  B a c i l l u s Megaterium  17  —  '• _  i i  l  -  20  -H —  _  0.45 j i  J j  Agrobacterium  |  S p e c i e s  | j  | j  121  A e r o b i c  F l a v o b a c t e r i u m S o l a r e  |22  -  F l a v o b a c t e r i u m Devorans  |23  -  t  Pseudomonas S p e c i e s and P r o t e u s S p e c i e s  4.3  —  _  0.6  —  0.6  0.4  |24  . .i  96  1  ! | j I  28  ,„,  i Continued  I n t r o d u c t i o n  P o t e n t i a l  of  Table Media 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15  Spent  1.8  S u l f i t e  L i q u o r  15  (continued)  Components 12% b e e t molasses, 10% y e a s t e x t r a c t , i n v e r t a s e 10% g l u c o s e , a u t o l y s e d p e n i c i l l i n mycelium(0.43% S), 2.75 ppm c o b a l t 4% g l u c o s e , 2% y e a s t e x t r a c t , 5 ppm c o b a l t , 2% c a l c i u m carbonate 10% g l u c o s e , 6.6% cornsteep l i q u o r , 20ppm sodium c o b a l t i c ethylene diamine t e t r a a c e t a t e , DMBZ g l u c o s e , yeast e x t r a c t , c c b a l t s a l t , c a s e i n h y d r o l y s a t e , l a c t i c a c i d g l u c o s e , y e a s t , peptone, c o b a l t s a l t , d i p c t a s s i u m hydrogen phosphate S S L ( 1 0 g / l of reducing s u g a r s ) , 1% y e a s t e x t r a c t , 5ppm c o b a l t c h l o r i d e , potassium dihydrogen phosphate S S L ( 3 0 g / l of red 6.3ppm c o b a l t i o SSL ( 4 0 g / l of red 2ppm c o b a l t i o n , 10% g l u c o s e , cor  ucing s u g a r s ) , 6% y e a s t e x t r a c t , n , phosphate, carbonate ucing s u g a r s ) , 0.5% yeast e x t r a c t , phosphate nsteep l i q u o r (0.32% N), 5ppm c o b a l t  1% g l u c o s e ( c o n s t a n t c o n c e n t r a t i o n ) , c a s e i n h y d r o l y s a t e ( 0 . 1 % N), c o b a l t s a l t , 0.02% DMBZ g l u c o s e , yeast e x t r a c t , c o b a l t s a l t S S L ( 4 0 g / l of reducing s u g a r s ) , 2% y e a s t e x t r a c t , 2ppm c o b a l t i o n , phosphate g l u c o s e , soybean meal, c o b a l t s a l t g l u c o s e , soybean meal, d i s t i l l e r ' s s o l u b l e s , c o b a l t s a l t  16 17  g l u c o s e , animal s t i c k l i q u o r , c o b a l t s a l t g l u c o s e , brewer's y e a s t , soybean meal, c o b a l t i n o r g a n i c s a l t s  18  8% 2%  19  5% c o r n s t e e p l i q u o r , 0.002% c o b a l t ( a s c o b a l t s u l p h a t e ) , 0.3% t r i e t h a n c l a m i n e  20  beet molasses, ammonium i n o r g a n i c s a l t s  21  1% 3%  22  beet molasses, s a l t s i n c l u d i n g b e t a i n e , 0.0005% DMBZ  g l u c o s e , s u c r o s e ,  1% 3%  meat malt  phosphate,  0.5% betaine h y d r o c h l o r i d e g l u c o s e , yeast e x t r a c t , malt r e s i d u e , i n o r g a n i c s a l t s g l u c o s e , soybean meal, s a l t , buffer s a l t s  24 25  soybean meal 2.2 g l u c o s e ,  or 3%  other p r o t e i n soybean meal,  c o b a l t  s a l t ,  1% c o r n s t e e p s o l i d s , 15ppm c o b a l t , e x t r a c t ,  cornsteep  I n t r o d u c t i o n  (40ppm),  sodium c y a n i d e , 0.004% 0.1% c i t r i c a c i d ,  e x t r a c t , s p r o u t s ,  23  c o b a l t  s a l t ,  p e n i c i l l i n  l i q u o r ,  m a t e r i a l s 5ppm c o b a l t ,  c o b a l t  s a l t s  mash  P o t e n t i a l  of  Spent  S u l f i t e  Table ANTIBIOTIC-PRODUCING  L i q u o r  16  1.9  FERMENTSTICKS  HITH  VITAMIN  E12  AS  BY-PRODUCT  -  S t r e p t o m y c i n Neomycin Aureomycin ( C h l o r t e t r a c y c l i n e ) T y r o t h r i c i n T r i c h o m y c i n 0  Table MICROORGANISMS  USED  FOR  1.10  COMMERCIAL  -  B a c i l l u s megaterium Streptomyces o l i v a c e u s Streptomyces s p e c i e s  -  A mixed Proteus  PRODUCTION  VITAMIN  c u l t u r e of Pseudomonas s p e c i e s , S t r e p t c c c c u s v u l g a r i s , and C l o s t r i d i u m p u t r i f i c u m  Table VITAMIN  B12  PRODUCERS  1.11 IN  THE  Commercial S o l v e n t s C o r p o r a t i o n Dawe's L o b o r a t o r i e s , Inc. L e d e r l e L a b o r a t o r i e s , D i v i s i o n of MerckSCo, Inc. Pubst Chas. U. S.  OF  L a b o r a t o r i e s , P a c i f i c Yeast P f i z e r S C o , Inc. I n d u s t r i a l Chemicals Co.  D i v i s i o n of N a t i o n a l D i s t i l l e r ' s Grain P r o c e s s i n g Corp.  UNITED  American P r o d u c t s ,  Corp.  I n t r o d u c t i o n  STATES  Cyanamid Inc.  Co.'  B12  b c v i s ,  P o t e n t i a l  1«3  Spent  S u l f i t e  17  L i g u c r  MIGBOgBGAHISHS_DSED  1.3.1  L a c t o b a c i l l u s , P l a n t a r i u m L a c t o b a c i l l u s  a b l e to  t c ferment i n a c t i v e  i n a c t i v e  HEXOSE  1  PENTOSE  t o  =—=>  2  pentoses a c i d  ( x y l o s e ,  i n a  molar  ACID  ACETIC  ACID  •  1 LACTIC  p y r u v i c  a c i d ,  g a l a c t o s e )  arabinose) r a t i o  o f  t o 1:1  t h e n ,  being  ACID  (1.2)  pathway  a n a e r o b i c ,  t o  c o n v e r t  reduce  p y r u v i c  a c i d .  found  o f  S S L  u s i n g  i n s a u e r k r a u t , 1 . 2  p l a n t a r i u m  t o be  (1.1)  t h e Embden-Heyerhof-Parnas  F i g u r e s  [ 1 0 , 2 6 , 7 4 ]  mannose,  f o l l o w  F e r m e n t a t i o n s  1.3.2  shown  r e a c t i o n s :  LACTIC 1  been  f r u c t o s e ,  a n d  a n d a c e t i c  ==*=>  t o a c e t i c  organism  a c i d  a c i d  h a s  (glucose,  t h e f o l l o w i n g  1  hexoses a c i d  l a c t i c  t o  They  p l a n t a r i u m  hexoses  l a c t i c  a c c o r d i n g  L.  o f  i n  a n d  have  1.3  c l o s t r i d i a l  a r e  Embden-Heyerhof-Parnas  thought  pathway  t o  M  a  s p e c i e s  o f  reported [ 4 6 ] ,  enlarged  F r e u d e n r e i c h i i  P r o p i o n i b a c t e r i a  p l a n t a r i u m ,  been  show  r e i n f o r c e d  P r o p i o n i b a c t e r i a  L .  photographs  medium  a n d  M  and i n  o f  S S L .  S h e r m  [ 2 3 , 8 1 ]  ferment  t o  hexoses  f o l l o w t o  t h e  p y r u v i c  a c i d : 6[0]  •  Then, 2  3  HEXOSE  they  PYBUVIC  ====>  o x i d i z e ACID  •  6 PYRUVIC  t h i s  p y r u v i c  2 [ 0 ] ====>  2 +  In  order  t o  s u p p l y  oxygen  ACID  +  BATES  (1.3)  a c i d  t o a c e t i c  a c i d :  ACETIC 2  CABBON  6  ACID DIOXIDE  t o r e a c t i o n s  I n t r o d u c t i o n  (1.4)  (1.3) a n d  (1.4),  Potential of Spent S u l f i t e FIGUBE 1.2 : L . plantarium in Synthetic Medium  FIGURE 1.3 in SSL  j  Scale : |  Liquor : L . plantarium  i  ( • 12 microns  Introduction  18  Potential  o f Spent  FIGURE 1.6 : P. s h e r m a n i i i n S y n t h e t i c Medium  Sulfite FIGURE i n SSL  Liquor  1.7  : P.  L  19  shermanii  j  Scale  : }•  4 = 12  Introduction  microns  P o t e n t i a l  they  reduce 4  p y r u v i c  PYRUVIC  And 3  a c i d  ACID  +  t h e o v e r a l l  HEXOSE  ====>  4  i s  mechanism  n o t w e l l  v i t a m i n Rhem  known.  B12  [ 7 8 ] .. I n  n u c l e o t i d e produce need  ====>  r e a c t i o n  would  2  t h i s  been  which o f  1.1)  v i t a m i n  PROPIONIC •  2  WATER  s t e p s  oxygen,  ACID (1.6)  produce  o f  t h e  v i t a m i n  B 1 2  s y n t h e s i s  o f  [ 3 0 ] , M i l l e t  P r o p i o n i b a c t e r i a  5 , 6 - d i m e t h y l  [ 6 2 ] ,a n d need  b e n z i m i d a z o l e  s l i g h t  a e r a t i o n ,  t a s k ,  however  (DMBZ)  they  high  t h e t o  do  n o t  l e v e l s  o f  growth.  which  may b e f o u n d a n d Ferguson  1.4 t o media  1 . 7 show a n d i n  P .  i n Emmenthal [ 2 3 , 7 0 ]  cheese  t o ferment  have  SSL  a n d  These  s o i l  a c t i n o m y c e t e s  Streptomyces  microorganism  used  f r e u d e n r e i c h i i  have  [ 3 1 , 7 3 ] .  v i t a m i n  B12  C e r t a i n [ 2 1 , 7 3 ] ,  been i s  commercial  1 . 1 0 ) [ 7 5 ] , a n d i s o n e o f  s p e c i e s  P.  s h e r m a n i i  . O l i y a c e u s  o l i v a c e u s i n  a n d  S S L .  Stre£tomyces,Griseus.and  (Table  4  DIOXIDE  t h a t  +8[0] (1,5)  b e :  +  B 1 2 . With  ACID  v i t a m i n B 1 2 .  s y n t h e t i c  B12.,  PROPIONIC  by Greenberg  o f  20  a c i d :  4  t h e known  f o r  t h e i r  L i q u o r  P r o p i o n i b a c t e r i a  o u t l i n e d  by N i s h i k a w a  F i g u r e s  1.3.3  ACID  CARBON  Some  b a c t e r i a  used  produce  2  n u c l e o t i d e  i n h i b i t  These  ACETIC  by  (Figure  S u l f i t e  t c p r o p i o n i c  t h e absence  complete  a e r a t i o n  i n  a r e  Spent  HATER  + The  o f  s t r a i n s b u t  known one  o f  p r o d u c t i o n t h e best  o f  a l s o  I n t r o d u c t i o n  S .  t h e of  v i t a m i n  g r i s e u s  a r e  t o produce  s p e c i e s v i t a m i n  o f B 1 2  B12-producing  n o t only  c a p a b l e  v i t a m i n  o f  produce producing  P o t e n t i a l of Spent S u l f i t e L i q u o r Streptomycin  [103,104].  F i g u r e 1.8 synthetic  21  p r e s e n t s a m i c r o s c o p i c p i c t u r e of S. g r i s e u s , i n  media,  with  the  typical  hair-like  feature  of  actinomycetes.  1.4  OBJECTIVE OF THIS WORK The o v e r a l l o b j e c t i v e of t h i s p r o j e c t i s to i n v e s t i g a t e the  p o s s i b i l i t y of d e v e l o p i n g a method f o r the economical of  vitamin  B12  as  a  means  of  reducing  p o l l u t i o n problem caused t c streams This  goal  is  approached  with  by  the  production  or e l i m i n a t i n g the  spent  sulfite  liquor.  project  limited  to  the  following sub-objectives: 1. D e t e r m i n a t i o n component  for  a  of the s u i t a b i l i t y of  medium  capable  of  SSL  as  the  s u p p o r t i n g the growth of  S t r e p t o m y c e s . T h i s d e t e r m i n a t i o n i s l i m i t e d to c n l y two S. o l i v a c e u s , one of the b e s t v i t a m i n B12  producer  species:  species,  S. g r i s e u s , f o r the p o s s i b l e p r o d u c t i o n of S t r e p t o m y c i n , q u a n t i t y , which may  major  h e l p the r e c o v e r y c f v i t a m i n B12  and  in tiny  i n very  low  concentration. 2. O b s e r v a t i o n p o t e n t i a l of l a c t i c carbohydrate  source  of  the  acid, for  SSL acid  consists  to  vitamin  B 12-producing  hexose,  as  the  main  with P r o p i o n i b a c t e r i a .  v i a two s e r i e s of f e r m e n t a t i o n s .  The  of two s t a g e s , L. p l a n t a r i u m c o n v e r t s sugars i n  to l a c t i c a c i d , then thus  compared  fermentations  T h i s p o t e n t i a l i s observed first  relative  produced  Propionibacteria  t o v o l a t i l e a c i d s and Introduction  ferment  the  v i t a m i n B12  lactic (Leviton  I  P o t e n t i a l  and  Hargrove  r e p o r t e d  f e r m e n t a t i o n s L.  p l a n t a r i u m 3.  4. P.  to  P.  Spent  i n c r e a s e s  [ 3 2 , 4 7 , 6 7 ] ) . and  P.  Determination  compared  of  for  f r e u d e n r e i c h i i  i n  The  y i e l d  l i g u e r  of  second  22  v i t a m i n  uses  a  E12  mixed  in  such  c u l t u r e  of  f r e u d e n r e i c h i i . of  the  r e l a t i v e  f r e u d e n r e i c h i i ,  Search  S u l f i t e  cheap  and  f o r  I n v e s t i g a t i o n  of  as  a  a b i l i t y  v i t a m i n  n u t r i e n t the  as  optimum  a  of  812  P.  producer.  n i t r o g e n  r e g u i r e d  s h e r m a n i i ,  source  amount  of  f o r t h i s  n u t r i e n t . 5. and  the 6.  p r o d u c t i v i t y I n v e s t i g a t i o n  p r e c u r s o r s e f f i c i e n c y  on of  the P.  of  the  P.  of r a t e  e f f e c t  of  a e r a t i o n  cn  the  growth  f r e u d e n r e i c h i i .  the of  e f f e c t s growth  f r e u d e n r e i c h i i .  I n t r o d u c t i o n  of  c o b a l t  and  i o n s  and  p r o d u c t i o n ,  DMEZ  and  cn  as the  P o t e n t i a l  of  Spent  S u l f i t e  Chapter EXPERIMENTAL  2.1  Type  c u l t u r e s  were  C u l t u r e  (ATCC-8262),  obtained  2 TECHNIQUES  o r i g i n a l l y  C o l l e c t i o n  f r e u d e n r e i c h i i  (ATCC)  (ATCC-6207),  and  OXOID  L a c t o b a c i l l u s  CM149  serve  as  the  t r a n s f e r  5 5 3 3 1 0.5 0.5  C h l o r i d e e E x t r a c t A c e t a t e e S t a r c h n e H y d r o c h l o r i d e  I n o c u l a  e x p o n e n t i a l an  (ATCC-8014)  were  c l o s t r i d i a l of  growth  a l i q u o t  of  grown  MEDIUM  these phase f r e s h  E x p e r i m e n t a l  Source Lab-Lemco  Sodium Glucos Yeast Sodium S o l u b l C y s t e i Agar  t o  s h e r m a n i i  C o n c e n t r a t i o n s ( g / 1 )  r e i n f o r c e d  American  P r c p i c n i b a c t e r i a  CLOSTRIDIA!  10 10  i n o c u l u m .  the  11.12  Beef E x t r a c t Peptone  s e m i - s o l i d  [ 3 ] .  p l a n t a r i u m  REINFORCED  Components  from  P r o p i o n i b a c t e r i a  Table  i n  23  PREPARATION_OF_INOCUL A l l  i n  L i q u o r  medium  c u l t u r e s (36  to  medium)  Techniques  (Table  were 96  OXOID  L37  OXOID  L20  II.12)  u s u a l l y hours  when  taken  a f t e r  the  to  the  growth  (measured 1 . 5 g / l P.  a s  o f  d r y  c e l l  weight)  f o r  L.  p l a n t a r i u m ,  of  inoculum  r e s p e c t i v e l y  s h e r m a n i i .  f e r m e n t a t i o n s Erlenmeyer Fresh  The volume i n  o f  o l i v a c e u s was  prepared  every  inoculum  2.2 2.2.1  o f  one lump  t h i s  about  t o  2  and  100ml  drops  f o r  i n  250ml  (ATCC-10137)  and  tubes.  g r i s e u s were  maintained The  on  s l a n t e d  medium  used  was  o f  was  added  1 1 . 1 3  No.  3  MEDIUM  (Table  11.13)  medium  t o  7 - l i t e r  i n  f r e u d e n r e i c h i i ,  1 t e s t  0 . 5 , 2 . 0 ,  10 5 5 5  ( i n , e x p o n e n t i a l i n  P.  o f  C o n c e n t r a t i o n s (g/1)  N o . 3 medium l i t e r  24  t h e v i c i n i t y  b r o t h .  lucose eptone eef E x t r a c t odium C h l o r i d e  f e r m e n t a t i o n s l e a s t  i n  i n  L i q u o r  was u s u a l l y  (ATCC-3335)  Components  to  i n  Streptomyces  WAKSMAN»S  Haksman's  i s  f e r m e n t o r s ,  Table  G P B S  S u l f i t e  and one l o o p f u l  c u l t u r e s  Inoculum  Spent  7 - l i t e r  f l a s k s ,  Streptomyces agar.  P o t e n t i a l  250ml  make  growth)  j a r s  2g  s l a n t s .  was u s u a l l y  and i n  Erlenmeyer  [ 1 0 3 ] ,  B e l l c o  f l a s k s  o r  agar The  volume  o f  100ml  f o r  about  f e r m e n t o r s , i n  t e s t  and  a t  tubes.  TREATMENT_OF_SPENT_SU ££g£aration SSL  h a s been  o f  S p e n t _ S u l f i t e _ I i g u g r  used  i n  v a r i o u s  E x p e r i m e n t a l  f e r m e n t a t i o n s Techniques  (Table  1.7)  and  P o t e n t i a l  was  not  known  d i o x i d e ,  to  which  microorganisms used  (about  200ppm however s u l f u r  T h i s  Ferguson  treatment  on  that V a r i o u s  S u l f u r  d i o x i d e  powdered and  w i t h  s t r i p p i n g a d d i t i o n a  has 0.2%  c h l o r i n e  In a  by  w i t h  s l i g h t *  s u l f u r i c  a c i d  [ 4 3 ] .  as  w e l l of  removal  f o r  then  a d d i t i o n lime  reagent)  n e u t r a l i z a t i o n  and  steam  i n i t i a l l y  has  even  untreated  only  ammonium commercial  [ 4 4 ] ,  been  S u l f u r  SSI,  Steam  or  with  done  with  s t r i p p i n g ,  been  as  [ 5 5 , 5 6 ] ,  [ 5 2 , 5 7 , 9 9 ] .  a l s o  of  s u l f u r  [ 1 , 2 0 ] .  m o d i f i c a t i o n  of  purpose.  of  (in  has  It  of  s a l t s  It  [95],  and  t h i s  barium and  e f f e c t  p r e c i p i t a t e d  s t r i p p i n g  used  both  the  made  SSL  l i m i t  i n h i b i t i n g  with  steam  the  of  l i t e r a t u r e ,  i s  e c o n o m i c a l  removed  of  f e r m e n t a t i o n , a e r a t i o n  be been  c o m b i n a t i o n  with  most  the  f i r s t  a e r a t i o n or  i n  r e c o g n i z e d i n  s u l f u r  s p e c i e s  the  [ 7 1 ] ,  [ 3 9 ] ,  v a r i o u s  So,  been  carbonate  than  d i o x i d e  the  n e u t r a l i z e d ,  subsequent  the  a l s o  of  be  the  vague  r e c e i v e have  25  other  usual  s t u d i e d  should  may  of  the  f r e u d e n r e i c h i i .  s u l f a t e  being  can  has  L i q u o r  s u l f u r  somewhat  approaches  magnesium  d i o x i d e  [23]  with  of  exceeds  i s  P.  growth  c o n t e n t  f a r  S u l f i t e  s u b s t a n c e ,  the  l i m i t  sodium  c h a l k  p r a c t i c e ,  The  SSL  d i o x i d e .  s u l p h i t e s  any  i n h i b i t s used.  d i o x i d e  Spent  c o n t a i n  7000ppm)  [ 1 0 7 ] .  of  done  with  and  during  c o n t r o l l e d  [54]. s e a r c h  comparison  n e u t r a l i z a t i o n  of  a  s u i t a b l e  has and  been  method  made  of  p r e c i p i t a t i o n ,  for 3  s u l f u r  d i o x i d e  d i f f e r e n t  pseudo  steam  removal,  methods,  namely  s t r i p p i n g ,  and  a e r a t i o n . The  f i r s t  one  has  been  E x p e r i m e n t a l  adopted  by  Techniques  Nishikawa  [ 7 0 ] ,  and  P o t e n t i a l  c o n s i s t s 1.  o f  o f  and  T h i s  t h e r e s u l t i n g 2.  s u l f u r i c  I t  i s  p r o c e s s  water  i s  e q u i v a l e n t  done  evaporated help  of  i n  r e p l a c e d l e v e l .  i n v e s t i g a t i o n .  best  f o r  a  71  and  Although 1  t h i s  t r e a t e d t o  i s  Microferm  w i t h  t h e a i r  F i g u r e  2 . 9  purpose.  Nishikawa»s T h e  of  o f  blown  with  p r e c i p i t a t i o n  removal  b o i l e d  and t h e  s u l f u r  d i o x i d e  water  through  f e r m e n t o r ,  makes  t h i s  S S L .  T h i s  s o  t h e l o s s  minimized  of  with  t h e  o f  t h i s  l i n e .  show  t h e  alone  complete  f i n a l  6 - 7  p r o c e s s .  was  n e u t r a l i z a t i o n  removed  second  SSL i s  t h e a i r o u t l e t  removal,  t h e  t h e content  s i m p l y  as  c a l c i u m  S S L .  c a l l  s t r i p p i n g  removed  adding  t o  The r e p l e n i s h m e n t  NBS  on  by  r e f e r r e d  by  S S L .  t o  be  5 . 5 )  n e u t r a l i z a t i o n ,  h a s i t s pH r e a d j u s t e d  u n t i l  a i r  as  then  n o n - t r e a t e d  s t r i p p i n g .  steam  i s  t o  i s  w i l l  12.  1.5  The p r e c i p i t a t e  steam  c a r r i e d  1 1 . 1 4 .  d i o x i d e  t o  a p p r o p r i a t e  process  t h e  water  s u l f u r  N i s h i k a w a ' s  t o  l i g n o s u l f o n a t e  stage  t o  t h i r d  r e f e r r e d  a s N i s h i k a w a ' s  t h e condenser  Table  a s  t h e pH  than  b o i l i n g  was  w i l l  o f  more  t h e d e s i r e d  t h e  26  (pH f r o m  be  l i q u i d  r a t h e r  d i o x i d e  stage  T h i s  reaches  In  L i q u o r  The p r e c i p i t a t e  f i l t r a t e  b o i l i n g  evaporated  s u l f u r  and t h e f i l t r a t e  a c i d .  t h e f i n a l  S u l f i t e  carbonate.  and b r i n g i n g  f i l t r a t i o n  and  o f  P r e c i p i t a t i o n  hydroxide by  c a l c i u m  f i l t r a t i o n .  Spent  s t e p s :  N e u t r a l i z a t i o n  a d d i t i o n by  two  o f  r e s u l t s  i s  n o t  adequate  treatment  t r e a t e d  SSL  i s  i s  f o r t h e  c l e a r .  0 n l y free s u l f u r d i o x i d e has d i r e c t i n h i b i t i n g e f f e c t s on t h e growth o f b a c t e r i a , s o u n l e s s o t h e r w i s e s p e c i f i e d , f r e e s u l f u r d i o x i d e would be r e f e r r e d t o a s s u l f u r d i o x i d e . 1  E x p e r i m e n t a l  Techniques  P o t e n t i a l  of  Spent  Table EFFECTIVENESS  OF  VARIOUS  S u l f i t e  L i g u o r  27  11.14  METHODS  OF  REMOVAL  OF  SULFUR  DIOXIDE  t  j  , Type  of  SSL  S u l f u r Dioxidg T o t a l ppm  Sugar Cone.  Free ppm  Reduction i n Sugar  Reduction i n Volume  Cone.  %  g / i  T o t a l | % l o s s ) of Sugar  %  | | j 1  | |  As Received N o n - t r e a t e d  5477 6534  | | j | j | |  Treated Aerated (40hrs) Aerated <pH 7 , 48hrs) B o i l e d (8hrs) B o i l e d (pH 8 . 3 , 18hrs)  545 4228 3588  1890  39.6  897 64  36.6 14.0  7.5 65.0  None 63  7.5 87.0  96 288  —•-  |  36.3  7.5  None  7.5  }  96 224  36.9 32. 1  7.0 12.5  None  7.0  |  None  12.5  j  1185 2978  —  —  ,  j  assumingly a l s o  f r e e  i n d u c e s  presumably with l a r g e  t h i s  a  of  l i g n o s u l f o n a t e .  t o t a l  i n  t r e a t m e n t .  of  f i l t e r i n g  a  of  a  not  87%  cf  the  p r e c i p i t a t e  method  q u a n t i t y  U n f o r t u n a t e l y ,  as  of w e l l  of  s l i m y  l i q u i d .  The  necessary  f o r  t h i s  sugar,  or  which  removal  a l s c  as  imposing  waste  and  e x t r a  the  treatment which  p r e c i p i t a t e s r e q u i r e s  a  problem  of  the  the  i s  problem  of  p r e c i p i t a t i o n  cf  f e r m e n t a t i o n s  s t u d i e d  as  b e l i e v e d .  A e r a t i o n  r e q u i r e s  answer,  convenient  the  T h i s  foamy i s  of  c h e m i c a l s  l a r g e  l i g n o s u l f o n a t e Nishikawa  l o s s  r e t a i n e d  amount  d i s p o s a l  best  | |  than  though  too  much  on  l a b o r a t o r y  a e r a t i o n  as  i t  t i m e .  B o i l i n g s c a l e  r e q u i r e s  E x p e r i m e n t a l  seems  b o i l i n g  c o n s t a n t  Techniques  to i s  be  the  much  l e s s  s u r v e i l l a n c e  to  28  Potential  of Spent  Sulfite  Liquor  LEGEND  TREATMENT  ALKALI  ADDED  +  AERATION  YES  •  AERATION  NO  X  BOILING  YES  A  BOILING  NO  Ei A D D I T I O N O F ALKALI AFTER TREATMENT  TOTAL FREE  SULFUR SULFUR  Experimental  DIOXIDE DIOXIDE  Techniques  Potential  of Spent S u l f i t e Liguor  29  FIGURE 2*9 ^ S t r i p p i n g of Sulfur Dioxide by B o i l i n g and Aeration  0.0  10-0  20.0  30.0  40.0  DURATION OF TREATMENT M R S ) Experimental Techniques  50.0  P o t e n t i a l  of  from  b o i l i n g  over  s t a i n l e s s  s t e e l ,  prevent even over  i t  7)  was  s u l f u r  t e s t e d  d i o x i d e  e f f e c t i v e ,  as  by  the  200ppm,  nor  content  of  s u l f u r  not  have  does  with  was  f i n a l  the  damage as  to  an  n o n - r e a d j u s t e d a d j u s t e d of  p r i o r  r e l e a s i n g  d i o x i d e  s t u d i e s d i o x i d e  f r e e  of  T h i s  of  before  steam  the  of  the  pH  w i l l  of  by  be  SSL  does i t  no  not  (to  have the  NaOH  treatment  to  remove  s t a r t  such of  of  and as  E x p e r i m e n t a l  second  a e r a t i o n  c a p i t a l  energy  should  two-stage the  be  pH  i t s  pH  to  The  the  of  s u l f u r  the  a  has  c o s t  of  s o u r c e .  accounted  Techniques  a l l o w  a e r a t i o n s u l f u r  b o i l i n g s t a g e .  to  be  i t s  with  However,  made  eguipment, The  to  used  p o t e n t i a l  i n  be  avoid  been  shown  would  on  undertaken  jars)  with  l a t e r  e f f e c t  l o o s e l y - b o u n d  a d j u s t e d  high  shown  i s  best  under  the  has  has  means  not  SSL  g l a s s  from  i s  removal  was  fermentors)  removal,  d i o x i d e  (to  experiments  (in If  pH  Howver,  pH-readjusted  d i o x i d e  s t r i p p i n g  s t r i p p i n g  d i o x i d e  as  m e t a l ,  p o t e n t i a l  a v a i l a b i l i t y  steam  i s  i n h i b i t i n g  i n  p r o j e c t .  i t s  then  c o n s i d e r a t i o n s  o p e r a t i o n ,  e f f e c t  when  2 . 9 ) .  pH  methods  288ppm  b o i l i n g  s u l f u r  growth.  between  numerous of  s u l f u r  e f f e c t i v e l y  adjustment  t h i s  The  s a t i s f a c t o r y .  of  a t t a c k s  r e a d j u s t e d  b o i l i n g .  p e r c e p t i b l e  parts  SSL  with  s u l f u r  about  to  i n  to  (Figure  of  s t e e l  compounds  b a c t e r i a l  c h o i c e  SSL  of  L a c t o b a c i l l u s  a l t e r n a t i v e  SSL  and  30  Liquor  dry.  a l t e r n a t i v e  a e r a t i o n  s t a i n l e s s  so  r e q u i r e d  any  So  p r o j e c t .  pH,  content  d i o x i d e  i n  S u l f i t e  b o i l i n g  a e r a t i o n  time  and  or  low  these  P r o p i o n i b a c t e r i a t h i s  at  Spent  with c o s t  s t e r i l i z i n g c r e d i t .  P o t e n t i a l  2 . 2 . 2  of  Spent  S u l f i t e  L i q u o r  31  N u t r i e n t _ A d d i t i o n  2.2.2.1  Fermentation  w i t h  P r o p i o n i b a c t e r i a Table  CONCENTRATION  OF  NUTRIENTS  and  L a c t o b a c i l l u s  11.15  IN  SSL  FOR  PROPIONIBACTERIA  AND  LACTOBACILLUS  Potassium Dihydrogen Phosphate Cobalt Ion yeast E x t r a c t 5 , 6 - D i m e t h y l B e n z i m i d a z o l e F i s h P l a n t E f f l u e n t  The l i s t e d  ranges  i n  n i t r o g e n yeast no  Table  cost)  s o l u t i o n  p a r t s  amount  of  by  s o u r c e was  the  11.15.  source  e x t r a c t  4.35  of  0.126g  Y e a s t t h e s e  f i s h  plant  added f o r  amounts  to  of  of  DHBZ  n u t r i e n t s s u p p l i e s  microorganisms. e f f l u e n t ,  supply  every  of  e x t r a c t  n i t r o g e n , to  4 0 0 0 0  were the  100  a l s o  much  l i t e r  of c f  added  amine  Attempts cheaper Cobalt  ion  needed  v i t a m i n broth  to  B12  was  SSL  a c i d s  made.  c o b a l t  p a r t s  per  a  g/1 -2.2 ppm -100 g/1 -0.126 g/1 -0.5 1/1  as  are a  to  r e p l a c e  (at  almost  c h l o r i d e (at  l e a s t  produced). added  i n  to  An one  b a t c h .  2 . 2 . 2 . 2  Fermentation  A l l except  components  g l u c o s e  a c c l i m a t i z a t i o n medium  i n  were the  d i f f e r e n t  with of  Streptomyces Waksman's  added SSL  to so  SSL  t r e a t e d  No.3 i n  the  was  mixed  p r o p o r t i o n s . E x p e r i m e n t a l  medium  Techniques  same with  (Table amounts. the  11.13) For  s y n t h e t i c  P o t e n t i a l  2.3  FERMENTATION  2.3.1  Spent  S u l f i t e  Liquor  32  APPARATUS  A£_par a t u s Most  250ml  o f  t h e  experiments  Erlenmeyer  S c i e n t i f i c i n  AND  of  t h e  (NBS)  NBS  f l a s k s  u s i n g  Streptomyces  shaken  a t  i n c u b a t o r - s h a k e r  fermentor  200rpm  (Figure  d e s c r i b e d  were  i n  2 . 1 0 ) .  below,  and  done  i n  a  New  Brunswick  A  few  were  i n  done  B e l l c o  g l a s s  fermentors. For  t h e  L a c t o b a c i l l u s , were  c a r r i e d  f l a s k s . w h i l e  most  o u t  Some  o t h e r s  f e r m e n t a t i o n s of  i n  o f  f l a s k s  l e f t  t e s t  submitted  tubes, t o  a c t i v i t i e s Hicroferm shows  o f  to  w i t h i n  to  900rpm,  The  a i r  of  s t e r i l i z e d  l i n e  The w i t h  pH  f l o w  r a t e s  be  has  a d j u s t e d ,  u n i t 10<>C  up  heated a  t o and  condenser  fermentor t h e  of  i s  the  o r t e s t  shaken  NBS  used.  a  2 . 1 3  a g i t a t i o n  through  minimize  i n  added,  Technigues  l o s s  s e a l e d , i t .  twin 2.11  show  temperature  f i l t e r e d  which  Figure  2 . 1 2 ,  50<>C,  medium  whenever  with  time,  a  tubes.  i n  1 6 , 0 0 0 c c / m i n u t e .  t o  i n  experiments,  a l l o w s  inoculum  E x p e r i m e n t a l  i n c u b a t o r - s h a k e r  with  F i g u r e s  t o  Erlenmeyer  i n c u b a t o r  was  a s e p t i c a l l y  f e r m e n t a t i o n and  same  and  experiments  250ml  the  f o l l o w e d  and  range  i n  main  u n i t ,  a i r  o u t l e t  t h e  were  T h i s  and  v i g o r o u s l y  the  and  c a n  F o r  s c r e e n i n g  along  [ 7 2 ] ,  i n  a i r  the  MF-07  1°C  t h e  i n  model  fermentors.  e v a p o r a t i o n .  removed,  t e s t s .  t h e  o u t l e t  shaken  were  f e r m e n t a t i o n s  s i d e  were  however,  fermentor,  one  d e t a i l s  and  of  and  tubes  i n c u b a t o r ,  t u r b i d i t y  P r o p i o n i b a c t e r i a  t e s t  unshaken  c o n v e r t e d - r e f r i g e r a t o r These  p r e l i m i n a r y  13x100mm  t h e  were  t h e  with  the  c o n t r o l speeds  up  The  i n l e t  g l a s s  wool.  c f  water  by  c a n  be  and Samples  under  c a n  be  a s e p t i c  P o t e n t i a l  c o n d i t i o n s In NBS  at  the  any  l a t e  a u t o m a t i c  e l e c t r o d e s F i g u r e s  2 . 3 . 2  time  are  of  t h i s  t o  S u l f i t e  l i g u o r  33  f e r m e n t a t i o n s . p r o j e c t ,  pH  (Figure  2.'1'4).  c o n t r o l l e r  a t t a c h e d  2.12,  Spent  during  s t a g e s pH  of  the  was  fermentor  c o n t r o l l e d I t s  head  by  an  s t e r i l i z a b l e as  shown  i n  2.13.  P r e f e r m e n t a t i o n A f t e r  medium  the  was  together  a d d i t i o n  a d j u s t e d  w i t h  i t s  s t e r i l i z a t i o n broth  i n  (15  t e s t  t u b e s ) .  the  broth  medium through with  was  the  a  i n o c u l a t i o n  even  w i t h  a s e p t i c  p i p e t t e s  or  i n  and  F i n a l l y , open  the  t r a n s f e r , loop  15 in  When  the  2 . 1 5 ) .  the  so  syringe-method  i t  was  f l a s k s  and  the  The  pH  of  of  the  e i t h e r port  volume  awkward  d i s c o v e r e d  and  t h a t  For  l e s s  f l a s k s  glassware  of and  g u i c k  P r o p i o n i b a c t e r i a  s a f e  Technigues  20  inoculum  needle.  adopted.  f c r  i n t r o d u c e d  the  was  This  f e r m e n t a t i o n  (121°C)  the  with  medium,  temperature  was  through  n e c k - f l a m e d  E x p e r i m e n t a l  (Figure  the  Erlenmeyer  inoculum  pert  f e r m e n t a t i o n  Then,  p s i g  i n o c u l a t o r .  was  the  s t e r i l i z a t i o n ,  growth  w i t h ,  at  or  inoculum  sampling  7.  hypodermic  rendered  of  a u t o c l a v i n g  7.  l e v e l ,  e x p o n e n t i a l  platinum  to  i n o c u l a t o r ,  100ml)  through  by  during  s y r i n g e  pH  above  volumes,  a d j u s t e d  d e s i r e d  r i s k y .  L a c t o b a c i l l u s  normal  the  the  s t e r i l i z e d  steam  s m a l l  dropped  again  (about  was  with  f c r  sampling  a s e p t i c a l l y  than  pH  s t e r i l i z e d  inoculum  s l i g h t l y  accomplished  minutes  reached  to  c o n t a c t  The  n u t r i e n t s ,  c o n t a i n e r ,  was  d i r e c t  minutes  of  or  cumbersome and  and  tubes, P a s t e u r  Potential  of Spent  Sulfite  Liquor  F I G U R E 2.10 : I n c u b a t i o n , o f S. c j r i s e u s a n d f>. o l i v a c e u s i n D i l u t e d S S L , i n T h e NBS Incubator-Shaker (scale : y -j = m . 6 cm)  L  F I G U R E 2.12 (see F i g u r e  : S i d e V i e w o f T h e F e r m e n t o r Head 2.13 f o r l e g e n d , s c a l e : \—7cm—-j )  Experimental  Techniques  P o t e n t i a l o f Spent S u l f i t e L i q u o r  35  FIGURE 2.13 : Top View o f The F e r m e n t o r Head (scale : —4cm—A\) r  —i  LEGEND i  Exhaust A i r Condenser Inoculation Port Thermometer W e l l  .... ....  Sampling I n o c u l a t o r .  |  I I I I I  1 1 1 1  1  1 2 3 4 5 6 7 3 9 10  Experimental  11 12 13 14 15 16 17 18 19 20  Inlet Lines Hater O u t l e t Thermistor Well Foam P r o b e Air Outlet A g i t a t o r S h a f t ....... A i r Nozzle D i s t r i b u t o r Heat Exchanger Baffle pH A d d i t i o n s Agitator  Techniques  Potential  of Spent  Sulfite  T  Liquor  F I G U R E 2.14 : ( l e f t ) T h e pH C o n t r o l l e r (scale : | 4 = 18.6 cm)  F I G U R E 2.15 : ( b e l o w ) S t e r i l i z a t i o n o f The 71 F e r m e n t o r (scale : 1 -J = 1 6 . 2 cm)  F I G U R E 2.16 : ( a b o v e ) F o a m i n g i n The 71 Fermentor (scale : | 4 = 8.6 cm)  Experimental  j  Techniques  P o t e n t i a l  2 . 3 . 3  With  f e r m e n t a t i o n case  t h e  through  condensed  by  a  fermentor  f i l t e r e d water  NBS  c o l d  head.  through  a  l i n e ,  s p e c i a l  magnet  magnetic however,  i m p l i c i t l y The  these  water  was  done  provided  a n d 25°C  temperatures  by  then  w i t h  was  t h e  fermentor, with  bulb  a i r  a i r  g l a s s  a x l e  Erlenmeyer  from  was  only  a s a  l e s s  o f  t r a p  on  a s e p t i c a l l y  t h e B e l l c o  g l a s s  l i n e  w c c l ,  condenser  r e p l e n i s h e d  i n  a g i t a t o r .  was preheated,  t h e f e r m e n t a t i o n s  S.  was a t  o l i v a c e u s ,  a s optima.  growth.  be  an  t h e  fermentor, a c t i v a t e d  with  f l a s k s ,  a t by  by  a a  S t r e p t o m y c e s , and  a i r  was  37°C  f o r  s h a k i n g .  f o r  Streptomyces  A l l  o f  a  temperature  a n d 30°C  2 . 3 . 3 . 2  t o  250ml  temperatures,  t h i s  p r o v i d e d ,  Host i n  g l a s s  by  through  t h e a i r c u t l e t  f i l l e d  had t o  b a r a t t a c h e d  on  B e l l c c  50ml  a i r  e n t r a i n e d  condenser  passage a  and mixed t h i s  Water  t h e  by  f e r m e n t a t i o n  g r i s e u s  wool.  s h o r t  s t i r r e r . were  fermentor,  F o r  but  A g i t a t i o n  at  37  L i q u o r  s c a i r was bubbled  t h e bottom,  water  was minimized  o u t l e t  a e r o b i c ,  from  g l a s s  times.  S.  a r e  medium  o f  f i l t e r e d  the  S u l f i t e  Streptomyces  Streptomyces  the  Spent  f e r m e n t a t i o n  2 . 3 . 3 . 1  In  o f  proved  a s  However, t o  be  A l l subsequent  f i r s t  f i x e d  Bergey growth  much  at  [ 1 0 ]  recommended  was peer  more  experiments  a t  these  f a v o r a b l e were  t o  c o n t r o l l e d  temperature.  With  P r o p i o n i b a c t e r i a  f e r m e n t a t i o n s  microorganisms  were  done  i n  and L a c t o b a c i l l u s t h e  w i t h  SBS  f e t m e n t o r s  pH c o n t r o l l e d  E x p e r i m e n t a l  Techniques  a t  with  these  a p p r o x i m a t e l y  7.  P o t e n t i a l  Whenever and  the  pH  c o n t r o l l e r  c o n s t a n t l y  c o n d i t i o n s . i n t o  the  The  from  F e r m e n t a t i o n s  with So  of  was  P r o p i o n i b a c t e r i a  beginning the  of  s t a t i o n a r y A l l  under were  other  continuous The  w i t h i n  these  2.4  A8ALYSIS A l l  l a t e r  i n  flow  necessary remained  c o n s t a n t .  under  where  f o r  a n a e r o b i c  they  are  w e l l  f e r m e n t a t i o n s  of  a i r  i n t e r v a l s  f e r m e n t a t i o n s  growth  (in  c o n d i t i o n s  to  f e r m e n t a t i o n s  c o n d i t i o n s . flow  r a t e s  with  r a t e are  was  with  To  a v o i d  l i m i t e d  net  to  b e n e f i c i a l  varying phase  from to  6  hours  about  48  at  hours  the in  a  second  f l a s k s  f o r  stage  the w i t h  and  f i r s t a i r  tubes) s t a g e .  were A  provided  few  by  of  done them  o c c a s i o n a l  s h a k i n g .  T h i s  f o r  at  e x p o n e n t i a l  temperature  1°C.  a i r  to  manually  phase.  a n a e r o b i c s u b j e c t e d  stage  the  pH  cf  a e r o b i c  2.16)  was  done  (up  a u t o m a t i c a l l y  [ 2 3 ] ,  taken  the  were  phase)  under  High  was  not  the  stage  38  i t  was  s i n c e  second  (Figure  were  a c i d  f i r s t  done  pH  otherwise  growth  2 0 0 0 c c / m i n .  Samples  or  the  L i q u o r  a v a i l a b l e ,  c o n t r o l  The  problems  S u l f i t e  L a c t o b a c i l l u s  e x p o n e n t i a l  maximum  to  pH  was  P r o p i o n i b a c t e r i a  a  was  l a c t i c  P r o p i o n i b a c t e r i a .  foaming  Spent  c o n t r o l l e d ,  s p o t - c o n t r o l l e d . f e r m e n t a t i o n  of  used  f o r  temperature  i n c u b a t i o n was  was  recommended  30°C, as  c o n t r o l l e d  optimum  to  [ 1 0 , 7 7 ]  m i c r o o r g a n i s m s .  HETHOES  d e t a i l e d Appendix  procedures II  and  and  Appendix  E x p e r i m e n t a l  III.  c h e m i c a l s Only  Technigues  used  the  are  o u t l i n e d  p r i n c i p l e s  of  P o t e n t i a l  the  methods,  s p e c i a l  t h e  a p p a r a t u s  Dry  c e l l  measurement  used  more  e l a b o r a t e  method  p i e c e  Moreover,  the i n  v  p r o d u c t i o n  of  L i q u o r  e n c o u n t e r e d ,  presented  was  of  of  39  t h e i r  s o l u t i o n s ,  and  here.  s u f f i c i e n t  growth, v i a b l e  so (or  eguipment  amount  t h i s  S u l f i t e  B a c t e r i a l ^ G r o w t h '  b a c t e r i a l  e x p e n s i v e  i n t e r e s t  of  are  weight  of  Spent  d i f f i c u l t i e s  Measurement  2.4.1  of  of  p r o j e c t c e l l s  f o r  no  r e c o u r s e  plate)  known  dry  c e l l  s i n c e  i t  count  as  matter s e r v e s  c o n t a i n i n g  t h i s  a  work  was  made  [92]  or  C o u l t e r  produced as  v i t a m i n  a  a  to  the  to  that  c o u n t e r . of  d i r e c t f o r  B12  i s  as  primary  index  use  as  of  a n i m a l s  f e e d . D i r e c t p r o j e c t  weighing  w i t h o u t  method.  T h i s  t u r b i d i t y sample,  t e s t  p r e c i s e  c e l l s  was  s u b s t i t u t i o n was  based  somewhat  same  the  amount  sample  r e l a t i v e  on  by the  used  the  throughout  standard  t h i s  t u r b i d i m e t r y  f a c t  t h a t ,  f a s t e r  and  needs  a  of  and  time  i n  although  the  s m a l l e r  s i z e  manual  m a n i p u l a t i o n s  l e s s  dry  weight  g r e a t e r  r i s k  r e l i a b l e  (the  an  a n a l y t i c a l  A  modified  being  s m a l l e r ,  the  f i r s t  i n t r o d u c e s  more  r e q u i r e s  more  e r r o r .  than  a  work  s i z e ,  f a s t e r  hence  - I s than  the  s t e p ;  - I s  water)  i s  dry  t e s t :  - B e g u i r e s  s i g n i f i c a n t  any  d e c i s i o n  t h i s  washing  of  i n  time,  but  ( r e s u s p e c s i c n  of  i t in  a  known  i s  l e s s  volume  of  e r r o r .  spectrophotometer  r e l i a b l e  b a l a n c e ) .  t u r b i d i m e t r y  method  E x p e r i m e n t a l  was  t e s t e d  Techniques  f o r  r e l i a b i l i t y .  In  t h i s  water. were  P o t e n t i a l  of  method  the  were  T h e i r  t u r b i d i t y  s t i l l  suspended  brown  c o l o r ,  same  medium, as  i t  used  be  c o n t r i b u t e d  measured  c e l l s  P.  In  0.05  a  i n  0.15  more  cannot  be  becomes  P.  of  of  to  the  P.  as  t h i s  a  dark  so  the  r e f r i g e r a t e d ,  c e l l s ,  weighing. w i t h  not  to  by  the  2.18,  of  2.19,  and the  f o r  L.  s e n s i t i v i t y  t h i s the  growth  or  d i r e c t  E x p e r i m e n t a l  1.2,  i n  (up  dry  t u r b i d i t y  0.4,  c e l l s  of  1 0 g / l  a of  c e l l P.  f o r  on  the  d e n s i t y s h e r m a n i i  spectrophotometer  s t a n d a r d i z a t i o n medium t e s t  was  used  tubes  where  Techniques  most  to  f a l l s  Hence  the  t e s t  of  r e s p e c t i v e l y , to  prepared  t e s t  the  0.01  of  0 . 7 g / l  of  s p e c i a l l y d i r e c t  t u r b i d i t y  p l a n t a r i u m  c o m p l i c a t e d  growth  to  2.20)  s c a l e .  t u r b i d i t y  spectrophotometer  L a c t o b a c i l l u s )  t u r b i d i t y  A  0.5  The  However,  the  of  t u r b i d i m e t r i c  the  2 . 1 7 ) .  on  growth  with  T h e r e f o r e , of  c e l l  (Figure  a  l e v e l .  f o r  standard  f r e u d e n r e i c h i i  the  r e q u i r e d  s t u d i e s  dry  s c a l e  2 g / l  then  '  has  c o n j e c t u r e d  the  r e l a t i o n s h i p  r e s t  s t e p  p r e l i m i n a r y  than  s h e r m a n i i ,  d i l u t i o n  c o n c e n t r a t i o n s .  was of  they  SSL  and  i n  w h i l e  blank,  u n i n o c u l a t e d  than  (Figures  d i f f i c u l t be  as  f a s t e r  water  and  3 g / l  at  kept  d i r e c t l y  water  d e n s i t y  the  The  measured n u l l  use  resuspended  S i n c e  the  i n  p o r t i o n  than  b r o t h .  by  f a s t e r  of  P r o p i o n i b a c t e r i a  of  o r i g i n a l  t u r b i d i t y  only  g/1  SSL.  u n r e l i a b l e  measured  to  40  nor  the  l i n e a r  f r e u d e n r e i c h i i ,  grown  washed  way  corresponds to  L i q u o r  s i m p l y  been  t h i s  much  p o r t i o n  (0.05-0.7) and  had  resuspended  r e l i a b l e  the  not  i m p o s s i b l e  much  has  was  S u l f i t e  medium.  i s  hence  i n  to  the  t e s t  method, so  blank.  of  T h i s  was  which  was  v a r i a t i o n  c e l l s  Spent  at only the  and  a  would lower f o r sample  Potential of Spent S u l f i t e FIGURE 2.17  Liquor  U1  : Relation between Modified and Standard Turbidity  not drawn  Regression l i n e at 0.95 confidence x ( ± 0 . 0 2 5 ) = 0.9 ( ± 0 . 0 2 ) y - 0.0 12 ( ± 0 . 0 1 5 )  2.0  •  1.6 cc LL!  CD CD  CD  m  s— CC  i—t  •  _j _J  •  LU  C_>  CD  (ZD  •  &  O.B  U_ ° >I— »—«  •  Q  i—< OD  •  0.4  • 0.0  0.4 TURBIDITY  ±  0.8 OF CELLS  1.2  1.6  IN SYNTHETIC  Experimental  o.  ±  Techniques  MEDIA  2 .0  ZD  I—  Potential  of Spent S u l f i t e  Liquor  FIGURE 2.18 : R e l a t i o n b e t w e e n M o d i f i e d T u r b i d i t y a n d D r y Weight of 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 — —  1 — —  —  Regression y(±0.137) = y(±0. 214) = y (±0. 124) =  DRY  lines 1. 100 0.430 0.092  a t 0.95 c o n f i d e n c e (±0. 130) x + 0 . 0 9 4 (±0.094) (±0.074)x - 0. 110 (±0.098) (±0.037) x + 0. 9 2 0 (±0. 2 6 8 )  CELL  WEIGHT  (G/L)  Experimental Techniques  42 Cell  Potential FIGURE 2.19  — — _ _ _ _ _  o f Spent S u l f i t e  Liquor  : R e l a t i o n between M o d i f i e d T u r b i d i t y and Dry Weight o f P r o p i o n i b a c t e r i a Shermanii Regression lines y ( ± 0 . 144) = 1.230 y(±1.040) = 0.578 y ( ± 0 . 1 1 7 ) •= 0. 103  DRY  CELL  a t 0.95 c o n f i d e n c e (±0. 157) x + 0. 11 3 (±0. 1 00) (±0. 6 9 4 ) x - 0. 159 (±0. 9 4 9 ) (±0.033) x + 1. 0 0 0 (±0. 2 3 0 )  WEIGHT  (G/L)  Experimental Techniques  43 Cell  Potential FIGURE 2.20  _  _  of Spent S u l f i t e  44  Liquor  : R e l a t i o n between M o d i f i e d T u r b i d i t y Weight o f L a c t o b a c i l l u s P l a n t a r i u m  and Dry  R e g r e s s i o n l i n e s a t 0.95 c o n f i d e n c e y ( ± 0 . 179) = 2 . 0 5 0 (±0.669) x - 0 . 0 0 5 (±0. 1 7 9 ) y ( ± 0 . 0 6 4 ) = 0 . 6 6 7 (±0.057) x - 0. 19 3 (±0. 0 6 8 )  DRY  CELL  WEIGHT  (G/L)  Experimental Techniques  Cell  P o t e n t i a l  s i z e  r e q u i r e d  a l s o  used  between  2.4.2  f o r  f o r  c u p r i c  of  r e d u c i n g  copper,  when  T h i s of  S u l f i t e  net to  L i q u e t  be  45  accommodated.  determine  It  adequate  was  i n t e r v a l s  an  the  i n t e r p o l a t i o n r e d u c i n g  i s  sugar the  f o r  of  The  of  i s  be  molybdenum  known  the  sugar  used  i n  i n  be  the  amount standard  measured  along  c o n c e n t r a t i o n .  d e t e r m i n a t i o n  sugar  t h i s  should  samples,  blue  added.  Therefore  unknown  reduced  determined  s t o i c h i o m e t r i c .  those  of  the  terms  the  Then  c o n c e n t r a t i o n  of  the  e q u i v a l e n t  standard  s o l u t i o n s ,  work.  V i t a m i n B 1 2 _ C o n c e n t r a t i c n -  v i t a m i n growth  compared  can  of  cepper,  and  method  l e i c h m a n i i  f o r  i s  reduced  p r e s e n t ,  amount  i s  copper  c o m p l e t e ,  m i c r o b i o l o g i c a l  L a c t o b a c i l l u s  the  s c l u t i c n  not  a l l o w i n  a l k a l i n e  The  reagent  r e a c t i o n  p a r t i c u l a r  Measurement  i s  of  of  2.4.3  sample  o x i d e .  samples w i l l  an  t a r t r a t e s ,  c o n c e n t r a t i o n s  g l u c o s e  B12.  i n  d i f f e r e n t  was  v i t a m i n  by  amount  not  sugars  the  requirement  heated  arsenomonclybdate  which  The  are  c o l o r i m e t r i c a l l y  of  of  C o n c e n t r a t i o n  cuprous  p r e c i p i t a t i o n  s o l u t i o n s  weight  as the  p r e c i p i t a t e  with  Sugar  s t a b i l i z e d  to  measuring  formed  c o u l d  c h e c k s  sugars  p r e c i p i t a t e d  p r o p o r t i o n a l  of  quick  Spent  s a m p l i n g s .  When  by  weighing  Measurement  and  of  to  B12 of i t s  was  (Figure and  growth  E x p e r i m e n t a l  It  2 . 2 1 ) ,  has  t h i s  used.  been  on  c o n s i s t s  which a  microorganism i n  s y n t h e t i c  Techniques  has  of an  a b s o l u t e  r e s t r i c t e d cn  the  medium  growing  d i e t  of  unknown c o n t a i n i n g  P o t e n t i a l of Spent S u l f i t e L i q u o r FIGURE  2.21  FIGURE  2.23  : L a c t o b a c i l l u s L e i c h m a n i i i n B a c t o B12 ( s c a l e : f4 = 12 microns)  : S p e c i a l Glasswares f o r A c i d i f i c a t i o n with Concentrated S u l f u r i c Acid ( s c a l e : f4 = 6.6 cm)  Experimental  Techniques  46 C u l t u r e Agar  of Samples  P o t e n t i a l  known the  q u a n t i t i e s  sample  can  of  be  as  true  an by  determine t h i s had L.  c o u l d  S u l f i t e  and  from  were  the  a  b e l i e v e d  product.  T h i s  t h i s  However,  no  assay.  whether  L.  used  or  l e i c h m a n i i product.  without  any  c o u l d  whether  break  the  t e s t  c e l l  b e l i e f  B12  medium  produce  was  proved  had  a b i l i t y  to  been  to  to  be  made  to  consume  as  get  the  of  v i t a m i n  m i c r o b i o l o g i c a l  w a l l  and  content  c u r v e .  whatsoever,  c e l l  of  47  to  attempt  standard  the  w a l l  v i t a m i n  [ 2 3 , 7 0 ]  the  q u e s t i o n ,  a t t a c k  the  had  The  l i q u o r  c a l i b r a t i o n  i n t r a c e l l u l a r  l e i c h m a n i i  i n s i d e ,  B12  determined  i n t r a c e l l u l a r been  Spent  v i t a m i n  P r o p i o n i b a c t e r i a B12  of  at  method  to  whether  the  v i t a m i n  treatment  P r o p i o n i b a c t e r i a  to  a l l  thereof  r e l e a s e  the  v i t a m i n . Answers  to  e x p e r i m e n t s . to  break.  these  q u e s t i o n s  However,  U l t r a s o n i c  the  c e l l  h o l e s ,  treatment  i n  months,  were  cause  f o r  breakage  a u t o c l a v i n g g l a s s  beads  degree  of  but  was  very  d i d  not  w a l l  of  to  was  French  with  no  much.  only  p r e s s ,  of  e s t i m a t e d  s u s p e n s i o n  with  50%  from  with  a c i d ,  through  about  i s  s u c t i o n  Treatment  S u c t i o n  c e l l - b r e a k i n g  P r o p i o n i b a c t e r i a  Treatment  w a l l s  with  a c e t o n e ,  a v a i l .  e f f e c t i v e .  break  breakage  sought  v i b r a t i o n s ,  m i c r o s c o p i c water  were  t i n y of  the  crude  hard  through of  c e l l s  a c i d f o l l o w e d holes c e l l s .  did by with The  m i c r o s c o p i c  o b s e r v a t i o n s . The the so  standard  adjustment the  c e l l s  t r e a t m e n t .  of  are A  method pH  of  the  expected t e s t  on  f o r  v i t a m i n  samples to  be  a l i q u o t s  E x p e r i m e n t a l  to  B12  d e t e r m i n a t i o n  4.5  broken of  an  Techniques  before by  r e g u i r e s  s t e r i l i z a t i o n ,  t h i s  unknown  l i g h t sample  a c i d was  P o t e n t i a l  o f  Spent  Table EFFECT  OF  VARIOUS  S u l f i t e  1 1 . 1 6  PRETREATMENTS ON C E L L S B12 ASSAY  Treatments  SUBMITTED  (equivalence  i n  TO  VITAMIN  A c t i v i t y V i t a m i n B12)  N o n - t r e a t e d Sample Sample Sucked with G l a s s Beads Sample w i t h pH A d j u s t e d t o 1.0  0 . 6 5 1.00  mg/1 mg/1  before S t e r i l i z a t i o n Sample Tested w i t h D e s c r i b e d Standard Method (pH A d j u s t e d t o 4 . 5 before S t e r i l i z a t i o n )  2 . 9 0 3 . 9 0  mg/1 mg/1  performed.  Each  c e l l - b r e a k i n g t e s t .  a l i q u o t methods.  The s t a n d a r d  was  a f t e r  g r e a t e s t  a l l  have  been  e a r l i e r  method,  t h e  B12  t h e  sample  due t o  phase  r e s u l t s  from  was o f  t h e were  4 . 5  e x t r a  from pH  1  v a r i o u s  t h e r e s u l t s  of  c e l l - b r e a k i n g L .  t h e  l e i c h m a n i i  c e l l s .  was not  o f  with  e f f o r t s , with  F u r t h e r  b e n e f i c i a l ;  v i t a m i n  t h i s  B12  t h e a c i d  t h i s  may  when  pH  i s  37°C.  In  t h e  [38,101 ].  recommended  t h e  p r o j e c t ,  i n c u b a t i o n  with due  i n c u b a t i o n t o  was done  t h e a t  30°C  a t  u n a v a i l a b i l i t y f o r  t h e t e s t ,  o f and  poor.  Measurement,o| V o l a t i l e  t o  shows  provided  t h e i n s t a b i l i t y  f a r  t e s t  without t h a t  of  prepared  I I . 16  one  v i t a m i n  i n c u b a t o r s ,  2.4.4  Table  o f  s i g n i f i c a n t l y The  was d i f f e r e n t l y  amount  treatment  the  L i q u o r  a c i d s ,  A c i d s ^ C o n c e n t r a t i o n produced  by  E x p e r i m e n t a l  P r o p i o n i b a c t e r i a , Techniques  assumed  t o  be  Potential FIGURE 2.22  of Spent S u l f i t e Liguor  49  : P a r t i t i o n of V o l a t i t e Acids between Water and Ether Regression l i n e at 0.95 confidence y ( ± 0 . 4 7 8 ) = 0.427 ( ± 0 . 0 3 9 ) x + 0. 94 5 (±0. 628)  0.0  20.0  VOLATILE  ACIDS  40.0 IN  60.0  AQUEOUS  Experimental  PHASE  80.0  300.0  (MM0LES/L)  Techniques  P o t e n t i a l  a  mixture  e a s i l y  of  2:1  steam  p r o j e c t .  However,  [ 7 , 5 8 , 6 7 ]  between were  in  the  from  products  of  c o n s e c u t i v e f o r  the  manual the  a l r e a d y  steam  These  v o l a t i l e more  the  by  a c i d s ,  r e l i a b l e the  where  of the  than  the  of  graph  v o l a t i l e  were  tc  the  end  the of  with  with  be  l a c t i c  to  a c i d s  t h i s  e x t r a c t i o n  3  adequate But  t h i s  a c i d  from  s u p p o r t i n g l a c t i c  the  a c i d  replacement  c o n t i n u o u s  manual  batch  e x t r a c t i o n  d i s t i l l a t i o n ,  carbon  method  fermented  proved  p r i o r  d i o x i d e  i n  s o l u t i o n s  of  d i s t i l l a t i o n  i s  d i l u t e d  of  f o r  a c i d s  r e s u l t s  steam  of  very end  Data  i l l u s t r a t e s  however,  a c i d  v o l a t i l e  time-consuming  presence  t i t r a t i o n s and  more  the  e x t r a c t i o n .  s e p a r a t i n g  f e r m e n t a t i o n s ,  l a c t i c  c o n c e n t r a t i o n .  e r r a t i c  are  c f  c o l o r i m e t r i c  ether  a c i d s  11.17  cf  a l r e a d y  volumes  f o r  s t a g e s  e x t r a c t i o n s  SSL  i n  the  [ 1 1 , 2 3 , 7 0 ] ,  e a r l y  v o l a t i l e  of  50  of  phase.  the  by  a  e t h e r  Manual  v o l a t i l e  e a r l y  by  e t h e r  equal  Table  to  e x p l a i n s  For  c o n s i d e r a b l e , to  i s  phase.  much  broth  d i s t i l l a t i o n  at  i n  p a r t i t i o n  f r e u d e n r e i c h i i .  i n t r o d u c e d  during  s o l u t i o n s .  e x t r a c t i o n  e x t r a c t i o n .  the  manual  inadequate  manual  done  aguecus  e x t r a c t i o n  T h i s  a c i d s  made  of  c o n c e n t r a t i o n  e r r o r  s i n g l e  e x t r a c t i o n s  aqueous  a c e t i c  the  the  L i q u o r  d e t e r m i n a t i o n  d e t e r m i n a t i o n  r e s u l t s .  of  and  aqueous  P r o p i o n i b a c t e r i a  was  p r e s e n t s  from  S u l f i t e  f e r m e n t a t i o n  ether  the  was  d i r e c t  r e c o u r s e  2.22  c o l l e c t e d  a c i d s  the  and  T h i s  when  f a i l e d ,  F i g u r e  Spent  p r o p i o n i c  d i s t i l l e d .  c o n c e n t r a t i o n  of  i s  where  the  the  a i r  a c i d s ,  i s  d i f f i c u l t  judge. The  low  compared  to  p a r t i t i o n t h a t  i n  c o e f f i c i e n t the  aqueous  E x p e r i m e n t a l  of  phase  l a c t i c f o r c e d  Technigues  a c i d the  i n  ether  d e t e r m i n a t i o n  1  P o t e n t i a l  of  Spent  Table MANUAL  i |  ETHER  S u l f i t e  l i g u o r  1 1 . 1 7  EXTRACTION* OF V O L A T I L E AND L A C T I C AQUEOUS SOLUTION  :  51  ACIDS  FROM  AN  —  1  E x t r a c t i o n s  F i r s t  Second  T h i r d  T o t a l  — |  V o l a t i l e  | j  I n i t i a l C o n c e n t r a t i o n  | | ]  E x t r a c t e d (moles/1) Residue % E x t r a c t e d  |  %  •  i  A c i d s  (moles/1)  0. 601 0 . 196 0. 405 32 .5  Non-recovered  0 .405 0 . 130 0 .275 32. 1  0.275 0.079 0.196 28. 6  0. 601 0. 405 0 . 172 67 . 4 4 .0  0 .579 0 .028 0 .551 4.8  0.551 0.026 0.525 4.7  0 . 591 0. 066 0. 493 11 . 2 5 .4  i r j  L a c t i c  A c i d  | j  I n i t i a l C o n c e n t r a t i o n  j |  E x t r a c t e d Residue  1 |  95 %  (moles/1)  0. 0. 0. 2  (moles/1)  E x t r a c t e d Non-recovered  591 012 579 .0  j  «  *20ml o f t h e o r i g i n a l c o n s e c u t i v e e x t r a c t i o n s , u with a p o r t i o n of 20ml s e p a r a t e d from t h e agueous volume of water, and t t i t r a t e d w i t h NaOH a t t h e  of  a c i d  c o n c e n t r a t i o n  F i r s t , s p e c i a l a c i d s  c o n c e n t r a t e d  produced a c i d .  done  by  s u l f u r i c  (Figure by  u b j e c t e d t o 3 a t o r y f u n n e l , ether phase i s i n a l a r g e e s i d u e i s a l s o  N  be  c o n c e n t r a t e d  glassware  vapor  t o  aqueous s o l u t i o n i s s s i n g a pear shaped s e p a r of e t h e r each time. The phase, then d i s p e r s e d i t r a t e d w i t h NaOH. The r e n d .  t h e  Then,  2.23) heat t h e  E x p e r i m e n t a l  continuous a c i d  t o  i s  prevent  batch  e x t r a c t i o n .  added t h e  t o  l o s s  c f  r e l e a s e d  by  t h e  mixture  i s  c o o l e d  Techniques  SSL  i n  v o l a t i l e  a d d i t i o n down  o f and  Potential  of  Spent  Sulfite  F I G U R E 2.24 : T h e E t h e r  Experimental  Liquor  Extractor  Techniques  P o t e n t i a l  f i l t e r e d .  The  E t h e r (1),  w i t h  f i l t r a t e  i s a  b o i l e d  ground  temperature. with  two  ether  f u n n e l of  in  i n  which  the i s  top  tube  i s  suspended  i t  almost  above.  The  ether  f u l l  i n  f i l t r a t e ,  the  gather  to  e v e n t u a l l y and  the  back  a c i d s  remain  i s  r e c o v e r y  a c i d s  T h i s  of at  c o n t i n u o u s  a c c u r a c y . (2  p r o p i o n i c  were  added  Known : to  1  and  l i g u i d  f i l t r a t e  ether the to  i s  top  a c e t i c ) a l i g u o t s  w a l l  ether i n  e x c e s s  cf  of  E x p e r i m e n t a l  a c i d  Then  i n  t h i s  Techniques  and  d e s c r i b e d tube  and  through  the  a c i d s  and  up  ether  i s  almost  of  of  in  with  e x t r a c t i o n  the  NaOH.  t e s t e d  v o l a t i l e  v a r i o u s  the  complete)  c o l l e c t e d water,  tube  r e c y c l e d  d u r a t i o n (or  ether  r e s i d u e  t h r o r o u g h l y  mixtures  l a c t i c SSL.  was  the  The  phase  r e s i d u e  supernatant  The  complete  end,  j o i n t ,  as  journey  of  end  c l o s e d  lower  p i c k  t i n y  other  r e s i d u e  The  c o n t a i n e r .  e x t r a c t i o n  and  i t .  c o n t a i n e r .  The  amounts  the ether  of  outer  t i t r a t e d ,  batch  of  the  a  The  prepared  i n  along  ensure  SSL.  SSL  t h i s  the vapor  cnto  the  (2)  to  The  (3). the  tube  connected  (4), i n  c o n s t a n t  vapor  drops  tube  c o n t a i n e r  at  (6).  e t h e r  above  of  drops  on  the  tube  suspended  t h e r e i n ,  (5)  one  p i e r c e d  tube,  i s  bath  condenser  h o l e s ,  the  from end  a  the  a d j u s t e d  the  to  along  i n  t u b e - l i k e  ends,  vapor  l a y e r  t e s t  through  two  53  e x t r a c t i o n .  water  r i s e s at  d i s p e r s e d  t h i n  a  a  r e s i d u e  tube  l i q u i d  i n  the  the  L i q u o r  to  d i s p e r s i o n  t i n y  the  i n t o  e x t r a c t i o n  c o n t a i n e r  i n  o v e r f l o w s  drops  and  a  the  through  f i n e l y  form  of  with  the  i n  vapor  Four  d i s p e r s i o n  submerged  neck,  condenser,  c l o s e d .  the  2.24)  other  part  S u l f i t e  s u b j e c t e d  openings  the  d i s p e r s e  i s  (Figure  j o i n t  the  Spent  then  ether  c o n t a i n e r ,  condenses  i s  g l a s s  The  ground  of  f o r  a c i d s  p r o p o r t i o n s technique  Potential FIGURE 2.25  of Spent S u l f i t e Liquor  : Recovery of Acids from SSL by Continuous Extraction  Ether  Regression l i n e at 0.95 confidence y ( ± 0 . 0 1 ) = 0.879 ( ± 0 . 0 3 5 ) x + 0. 09 6 (±0. 009)  0.5  0.0  0.3  0.2 RCIDS  0.3  fiDDED  Experimental  (MDLES/U  Techniques  0.4  0.5  Potential of Spent S u l f i t e Liquor FIGURE 2.26  55  : Recovery of L a c t i c Acid from SSL Continuous Ether Extraction Regression l i n e at 0.95 confidence y ( ± 0 . 0 1 4 ) = 1.000 ( ± 0 . 0 9 3 ) x + 0. 029 (±0. 0 11)  0.5  LACTIC  ACID  ADDED  (M0LES/L)  Experimental Techniques  P o t e n t i a l  was  used  to  r e c o v e r  d u p l i c a t e . t i t r a t i o n  L a c t i c of  F i g u r e s with  the  the  the a c i d  and  of  check  c o r r e c t n e s s  c o n c e n t r a t i o n s c o n t e n t other  of  t h i s  thus  SSL  method).  a t t r i b u t i o n d i f f e r e n c e s a t i s f i e s a c i d s  by  l i n e  i s  show  of  not  f e r m e n t a t i o n . above  and  t h e r e  were  d e t e r m i n a t i o n ,  95*  the by  l e a s t  v o l a t i l e  i n  a c i d s  The  t e s t  No  was  attempt value  by  of  of  made  to  the  determined  a c c u r a t e l y  the SSL  determining  e x p e r i m e n t a l  a c i d  t e s t  the  r e c o v e r y r e c o v e r y  no  i n  the  SSL  case  e x p e r i m e n t a l  t h i s  l i n e ,  produced  by  not  data  (in  the  c o m p l e t e l y d e v i a t e  f e r m e n t a t i o n s  the f a r  as T h i s of  s t r a i g h t  t h e o r e t i c a l  i s  any  p r o d u c t i o n  the  l i n e  by  c o n c e r n s .  to  the  a c i d s  as  p a r a l l e l a c i d s  a c i d s  c o n c l u s i v e l y  to i n  along  proofs  (absolute  a l l o w  i n  t h i s as  a c c u r a c y  i n t e r e s t  of  method  r e s u l t s  c o n c e n t r a t i o n s , a c i d s  at  c o n f i d e n c e  the  nor  high  but  from  a b s o l u t e  c o n c e n t r a t i o n s  primary  samples  r e s u l t s  method.  known  the  a c i d s  the  at  s a t i s f a c t o r y  of the  supposing a c i d s  of  these  56  e x t r a c t s .  determined  But  L i g u o r  d i s t i n g u i s h e d  e x t r a c t i n g  was  S u l f i t e  from  l i n e s  r e l i a b i l i t y the  was  2.26  r e g r e s s i o n  Spent  a c i d s  b o i l e d  2.25  of  c u r v e ,  of  t o t a l  p a r a l l e l ) .  c o n s i d e r a b l y never  reach  At from  these  c o n c e n t r a t i o n s .  2 . 4 . 5  D e t e r m i n a t i o n _ o f _ C h e m i c a 1 _ 0 x e n _ D e m a n d The  the  c h e m i c a l  b i o c h e m i c a l  measure The  of  oxygen oxygen  demand  (COD)  demand  d e t e r m i n a t i o n ,  (BOD)  t e s t ,  was  i n s t e a d t e s t e d  of as  a  p o l l u t i o n . p r i n c i p l e  i n v o l v e d  i n  E x p e r i m e n t a l  t h i s  t e s t  Technigues  c o n s i s t s  of  the  P o t e n t i a l  of  Spent  Table BESOLTS  r  OF  COD  S u l f i t e  L i q u o r  57  11.18  TESTS  ON  3  SAMPLES  .  —i COD  (ppm)  — i |  Without C o r r e c t i o n  Sample  With C o r r e c t i o n by D i f f e r e n c e  i  With C o r r e c t i o n by B a t i c  i I  186560 157699 203280 186960 285360  1  224500 182500 187000  249967 189239 183335 203511 207143  201250 228700  .  —t I  195888 .205216 184800 206640 305040  2  183338 200000 225000  187300 196200 238900  221429  248400  j  j  —1  I  158576 149248 221760  3  187000 233150 212000  275520  199975 240000 195349  i-,.  i  o x i d i z a t i o n  of  dichromate  and  f e r r o u s  o r g a n i c the  ammonium  The  COD  m a t e r i a l  u s i n g  was  f e r m e n t a t i o n s .  However,  s c a t t e r e d  c o l l e c t e d ^  data  a  of  the  t i t r a t i o n  s u l f a t e  t e s t  with  f e r r c i n  performed i t  was  amount  cf  potassium  r e s i d u a l . d i c h r o m a t e  with  i n d i c a t o r . a l l  samples  i m p o s s i b l e  B e s u l t s  E x p e r i m e n t a l  on  known  c c u l d  Techniques  not  to  of  e a r l y  c o r r e l a t e be  the  r e p r o d u c e d ; v  P o t e n t i a l  of  Spent  S u l f i t e  Table RESULTS  r  OF  CORRECTING  -—  —  —  l i g u o r  58  11.19 MEASURES  ON  COD  TEST  — Without C o r r e c t i o n  with by  C o r r e c t i o n  D i f f e r e n c e  by  R a t i o  j j  Sample  1  j | | |  Mean (ppm) Standard D e v i a t i o n (ppm) E s t i m a t e d D e v i a t i o n * A b s o l u t e (ppm)  I  R e l a t i v e  |  Sample  203972 48369  204790 21131  206639 26140  60047 29.4  26233 12. 8  32452 15.6  219517 48601  207442 19496  217700 30432  60336 27.5  19496 14.6  30432 22.7  {%)  2  |  Mean  | j  Standard D e v i a t i o n (ppm) E s t i m a t e d D e v i a t i o n *  (ppm)  I |  A b s o l u t e R e l a t i v e  (ppm) {%)  -1 |  Sample  3  | |  Mean Standard  I | |  E s t i m a t e d A b s o l u t e R e l a t i v e  D e v i a t i o n  (ppm)  201276 59054  210717  211775  23102  24553  93953 47. 1  57392 26. 3  60441  D e v i a t i o n * (ppm) (%)  28.5  i(  »At  a  degree  d e v i a t i o n s much  of  from  c a u t i o n ,  r e l i a b i l i t y  c o n f i d e n c e  of  one  run  to  e f f o r t  and  c a r e  of  the  t e s t  0 . 9 5 ,  another were was  E x p e r i m e n t a l  by  were paid  then  t - t e s t  enormous, to  matter  m a n i p u l a t i o n s .  immediately  Techniques  no  how The  q u e s t i o n e d .  P o t e n t i a l  of  Spent  Table RELIABILITY  | |  Sugar C o n c e n t r a t i o n The Aqueous S o l u t i o n  S u l f i t e  l i q u o r  59  11.20  OF  THE  COD  TEST  c f (ppm)  30  100  50  300 ,  | j |  Sumber of T e s t s Measured COD M e a n Standard D e v i a t i o n  | | | | j  E s t i m (9595 A b s o l R e l a t D e v i a  | |  from True Value (95) Extreme D e v i a t i o n (ppm)  l  a t e d D e v i a t i o n Confidence) u t e (ppm) i v e (95) t i o n of Mean  r e s u l t s  s o l u t i o n s  of  Table  11.20  the  i s s u e .  whole  t e s t s  s e r i e s  were  done  s i m u l t a n e o u s l y 300ppm  i n  Table  so  had  i n  of  COD  checks  mislead A  run  1 94.5  —  2. 5 9. 8  —  —  —  —  —  14. 1 7. 6  39 287.3 19.1  2.0  18.0 6.3  5.5  4.2 132.0 J  agueous  COD  1 49  :  P r e l i m i n a r y  d i d  6 25. 8 2. 4  (ppm) (ppm)  i  l  of  agueous 11.18  the  same  c o r r e c t i n g  sample measured  was and  and  made  sugar, f o r  of  on  on  but  COD  t e s t s on  3  of  these  s o l u t i o n  of  sugar.  sugar  c o r r e c t e d  analyzed  s o l u t i o n were with  t h e o r e t i c a l  the  t e s t  point  50  100ppm  and  designed  three The i n  of  w i d e l y  measured  COD  of  Techniques  the  sugar)  out.  Tests  were  one  are  from  or  of  t e s t CCD  sugar  sample  t a b u l a t e d  The  the  (see  c a r r i e d  and  11.19.  with  checks  SSI.  r e s u l t s  d i f f e r e n c e  (true)  and  samples  Table  The  e a r l i e r  s i n g l e  samples  v a r i e s  adopted.  E x p e r i m e n t a l  of  was  two  f u r t h e r  the  good  s o l u t i o n s  r e p e a t e d l y  measures  the  been  measured to  t e s t ,  of  r a t i o  every of  s o l u t i o n  the of  N  P o t e n t i a l  known  c o n c e n t r a t i o n .  b e t t e r .  These  (Table  11.19),  d e c r e a s i n g the  was  c o n c e n t r a t i o n t e s t  i s  t i m e s  ( i n  extreme  h a s been  1ml  (to  i n  each  case  i n t r o d u c e d  by  standard  such  reach  mere  than  20%  s o l u t i o n s ,  e r r o r  caused  than  a  heating  a s d i l u t i o n a r e  f e w  reasons  o f  which  o f  t o o f  i f  would  i s  known  t h i s  w i t h i n o f  a s  30% o r  t o  t h e w i t h i n  any  drop  e x p l a i n s  sources  i t  10%  c o n s i d e r a b l e  T h i s  and  COD many  t e s t ,  condensing  made  l a r g e  by t h e excess  account.  t h e t e s t ,  A  repeated  l i m i t e d  other  r a t e ,  i n t o  c f  more  10%.  h a l f with  o f  t h i s  be  samples  taken  t h e abandonment  In  be  t r i p l i c a t e ,  c o n c e n t r a t i o n  i n t r o d u c e d  c a n  o f  i s e r r o r  95% c o n f i d e n c e .  alone  v a r i a t i o n s  manipulations  t h e exact  e r r o r  o f  i n  s o l u t i o n s  t h e t e s t  e x p e r i m e n t a l  t c  s h a r p l y  done  estimated  t h e e r r o r  down  The r e l i a b i l i t y  i s r e q u i r e d  t o  95% c o n f i d e n c e .  sugar  I f  t h e  from  a t  T h e r e f o r e ,  e r r o r  11.20.  t e s t s  t i t r a t i o n s  t i t r a t i o n  p o s s i b l e  with  300ppm).  39)  d e v i a t i o n  2  a v o i d  magnitude).  30% a t  about  by Table  seems  i n c r e a s e d  h i g h ,  o f  60  c u t t h e v a r i a t i o n  v a r i a t i o n  done  L i q u o r  d i f f e r e n c e  With  observed)  o f  d i d  by  r e p e t i t i o n s .  (mostly  t h i s  d i f f e r e n c e  t h e  were  presented  (though more  o f  t e s t s  S u l f i t e  The c o r r e c t i o n  b u t  number  o f  Spent  c o r r e c t i o n s  d e v i a t i o n  number  o f  o f  rate  t h e e r r o r ,  and i n  p r e p a r a t i o n T h i s  o f  i n e v i t a b l e  unveiled r e s u l t  more  c o l l e c t e d  i n s i g n i f i c a n t : 1. then the much  I t  n o t same  takes  more  p r a c t i c a l  sample.  a s 30% ( o r  than t o  Hence more)  3  have  hours more  t h e r e s u l t s from  t o  t h e i r  E x p e r i m e n t a l  complete  than  one  r u n ,  i t  2 COD d e t e r m i n a t i o n s  a r e expected mean. Techniques  t o  d e v i a t e  i s f o r a s  P o t e n t i a l  2. (when and  On  a l l  the U0g/1  c e l l s  are  the  That  means  that  even  a l l o w  the  a c i d  t o t a l  The  r e q u i r e s  the  used)  COD  p o t e n t i a l s o u r c e s  of  4.  be  i n  SSL  b a c t e r i a SSL,  r e a d i l y  so  the  i s  sugar  c o n c e n t r a t i o n  of  COD same  with  a  to  t h i s  a  products 40,000ppm  the  to  t e s t  not  measured. f e r m e n t a t i o n  a  broth  f i l t r a t i o n ,  o r i g i n a l then  Hence  any  r e d u c t i o n  those  extraneous  s u l f u r i c  p r e c i p i t a t e .  the  SSL.  20*).  do  (concentrated  of  i n  the  amounts  of  i n t r o d u c e s  volume  r e d u c t i o n  about  (that  the  a c i d  COD  a l l  only  be  from  d i s s o l v e d  a l l  SSL  and  r e l i a b i l i t y  strong  the  i s  61  Steam sample.  i n t r o d u c e s  i t  of  i s  not  the  an  l i k e l y  p o l l u t i n g  c h e m i c a l s  and  e x t r a  are  b e l i e v e d  r e d u c t i o n from  of  the  d i r e c t l y SSL  can  to  consume  u l t i m a t e sugar  oxygen  demand  consumption  p r o p o r t i o n a l  e a s i l y  only  serve  to  as  an  (UOD)  d a t a .  the  UOD,  index  the  so of  The the i t s  p o t e n t i a l . the  l a s t  the'  BOD  t r o u b l e  measurements accuracy  cf  a c i d s  c a l c u l a t e d  consumption  For  consumed  of  used  sugar  p o l l u t i n g  and  r e v e a l  with  i s  r e d u c t i o n  hence  can  p o s s i b l e  COD  of  e t h e r  maximum  200,000ppm  r e q u i r e s  of  L i g u o r  e r r o r .  The  sugars  SSL  S u l f i t e  f e r m e n t a t i o n s  changes  t e s t  of  the  a c c u r a c y  and  a l s o  amount  the  of  maximum  e x t r a c t i o n  unknown  can  COD  i n  by  a d d i t i o n  d i s t i l l a t i o n  t h a t  sugar  removal  the  was  Ether  of  Spent  hand,  removed)  from  3.  other  of  of  than  two  reasons  t e s t . that  attempt  Furthermore COD  d i s s o l v e d the  no  d i d , oxygen  measurements E x p e r i m e n t a l  the  as  was  BOD  made  t e s t  d i l u t i o n do  not  r e q u i r e d Techniques  t c  may of  r e p l a c e run  i n t o  samples,  guarantee  any  by  t e s t .  the  CCD  the the and more EOE  P o t e n t i a l  c o n t r i b u t e d about  by  30%-UO*  of  fermentable of  the  t o t a l  Spent  S u l f i t e  sugars  i n  BOD  SSL  of  E x p e r i m e n t a l  SSL  l i q u o r  has  been  [ 1 0 0 , 1 1 0 ] .  Techniques  62  reported  to  be  P o t e n t i a l  o f  Spent  S u l f i t e  Chapter EXPERIMENTS  3.1  63  3  AND RESULTS  FERMENTATION.«ITH_STREPT Repeated  o l i v a c e u s ) the  attempts  i n  experiments  condenser  d i d  e v a p o r a t i o n .  t o  ended  w i t h  c o n t a m i n a t i o n  j o b  i n  a  up  poor  The numerous through  5  During  t h a t  the  a c t i n o m y c e t e s  f l a s k s with  and i n  t h e 71  a s  long  a s  Fermentation g r i s e u s  [ 1 0 ] .  and  B u t then  Streptomyces medium, s p e c i e s . s t i l l ,  no  T h i s  b u t  h a l f  very  was a  S.  much  whatsoever c o u l d  fermentors g l a s s  l o s s  o f  c o u l d  n o t  i n  be  period  be  by kept  (more  than  growth  250ml  u s u a l l y  bulb  water  n o t i c e a b l e  c o u l d  were  c o n t r o l l e d  o l i v a c e u s ,  p o o r l y ,  subsequent  f a i l u r e  was no  t h e  and  o f  Erlenmeyer  d i d n o t end up observed  a f t e r  month.  was d i s c o v e r e d  grew  growth  f o r  water  fermentors  g l a s s  as  fermenting  growth  (griseus  B e l l c o  Experiments  no  a  o f  t h e r e  temperatures  i t  Hence,  p e r i o d  In  p r e v e n t i n g  t h e long  Microferm  25°C  a n d 30°C  a v a i l .  a d d i t i o n s  i n o c u l a t e d .  c o n t a m i n a t i o n ,  p e r i o d s  no  Streptomyces  t o  a s e p t i c  d a y s ) .  grow  S S L were  a b s o l u t e l y  S.  L i g u o r  t h a t , even  more  i n  c o u l d  Experiments  a t  be  were  and  these  done  R e s u l t s  t o  by  f o r  Bergey  temperatures, N o . 3  temperature  d e t e c t e d  a t t r i b u t e d  37°C  recommended  Waksman*s  f a v o r a b l e  experiments  be  a s  a t  a t  s y n t h e t i c f o r  t h e s e  30°C.  B u t  i n S S L . e i t h e r  t h e  l a c k  o f  P o t e n t i a l  o f  Spent  Table FERMENTATIONS r j  OF  S u l f i t e  L i q u o r  64  I I I . 2 1  DILUTED  S S L BY S .  GRISEUS  — Sample  C o n c e n t r a t i o n (ml o f S S L / 1 of S o l u t i o n )  Dry C e l l Height  F e r m e n t a t i o n Time (hours)  (g/i)  Inoculum from  | j  j  i 1 G0.1 | GO.2 | GO. 3 | G 3 . 1  I  G 3 . 2 | G 2 5 . 1 | G25.2 | G25.3* | GA25.4* | G 5 0 . 1 | G50.2 | GA50.3* | GA50.4* | G100.1  0000 0000 0000 0003 0003 0025 0025 0025 0025 0050 0050 0050 0050 0100  | |  G100.2 G100.3  0100 0100  I |  G100.4 G100.5  0100 0100  | | | |  G1 GA GA GA  0.6 0Q.7* 00.1* 00.2*  0100 0100  | |  GA300.2* G500.1  0300 0500  | | |  GA500.2* GA500.3* G1000.1  0500 0500 1000  |  G1000.2  1000 1000  0 1 2 2  } G10Q0.32  1.661 1.341 1.457 1.783 1.554 1.945 1.512 2.085 2.130 0.590 1.382 2.850 2.880 0.035 0.034  0200 0200  110 110 126 110 110 110 110 126 126 110 110 117 117 110  GO.3 GO. 3  | |  GA25.4 GA25.4  | |  110  0.003  126  0.000 0.000 0.000  126 204 204  2. 3. 3. 0. 0.  216 253 253 204  GA50.4  |  GA100.7 GA100.7 GA200.1  | | |  0.000 0.000  126 204 204  GA200.1 GA200.1  | |  0.000 0.000 0.000  126 110  455 388 165 000 000  110  L .._  J  C h a r a c t e r i s t i c s  o f  r e s p e c t i v e l y  of  f r e e  ^ R e s u l t s f o r SSL t r e a t e d  f e r m e n t a t i o n s by a c c l i m a t i z a t i o n w i t h Hishikawa»s n e u t r a l i z a t i o n a n d  Z  SSL used  :  3 7 g / l  and t o t a l  Experiments  o f  s u l f u r  and  s u g a r ,  96ppm  and  1200ppm  d i o x i d e  R e s u l t s  p r e c i p i t a t i o n  Potential  o f Spent S u l f i t e Table  65  Liquor  III.22  FEBMENTATIONS OF DILUTED SSL BY S. OLIVACEUS r |  _— — Concentration (ml o f S S L / 1 of S o l u t i o n )  Sample  I 00.1  J | | | I | | | | | | | | \ | | | | | f | | | | | | | |  00.2 00.3 03.1 025.1 025.2 OA25.3* 0A25.4* 050.1 050.2 OA50.3* OA50.4* O100.1 0100.2 O100.3 0100.4 0100.5 0100.6 OA100.7* OA100.8 OA200.1* OA300.1* OA300.2* 0500.1 O500.2 OA500.3* OA500.4* O1000.1 01000.2  | 01000.32 t  ;  0000  oooo oooo  0003 0025 0025 0025 0025 0050 0050 0050 0050 0100 0100 0100 0100 0100 0100 0100 0100 0200 0300 0300 0500  0500  0500 0500 1000 1000  1000  Dry C e l l Height (g/i)  Fermentation Time (hours)  0.924 1.054 2.205 0.829 1.634 1.035 2.085 2. 130 0.892 0.648 2.490 2.570 0.164 0.590 0.078 0.000 0.338 0.000 2.213 2. 135 2.678 0.000 0.000 0. 152 0.000 0.000 0.000 0.000 0.000 0.000  110 110 126 110 110 110 126 126 110 110 117 117 110 110 126 126 204 204 216 216 253 204 204 126 204 204 204 110 126 110  Inoculum from  00.3  O0.3 0A25.4 OA25.4  OA50.4 OA50.4 OA100.8 OA200.1 GA20,0. 1 -  OA200.1 CA200.1  . . .  C h a r a c t e r i s t i c s o f SSL used : 3 7 g / l o f s u g a r , 96ppm and 1200ppra r e s p e c t i v e l y o f f r e e and t o t a l s u l f u r d i o x i d e 1 2  B e s u l t s f o r f e r m e n t a t i o n s by a c c l i m a t i z a t i o n S S L t r e a t e d w i t h N i s h i k a w a » s n e u t r a l i z a t i o n and p r e c i p i t a t i o n E x p e r i m e n t s and R e s u l t s  P o t e n t i a l  necessary t o x i c  to  n u t r i e n t s  Streptomyces  So,  o b v i o u s l y  n u t r i e n t s , of  fill  Waksman's  n u t r i e n t s  did  next  p o s s i b l e  ^but  added  to  the  step*  as  b a c t e r i a  them  s l o w l y  Growth  was  help  of was  time  was  to  However,  growth  cf  d i l u t e d  to  i n  The  SSL.  was  the  these lower  before  the  was  medium.  d i l u t i o n s .  show  then  a c c l i m a t i z a t i o n ,  through i n  of  more  to  and  the  more  Waksman's i n  new  of  same  of  these  medium  10,  Hence,  20,  of with  N u t r i e n t s  were  However,  when  per  100ml  of  improvement.  that by  s y n t h e t i c 5,  the  dene  any  c o n c e n t r a t e d  No.3  m i x t u r e s  SSL  not  of SSL,  c o n c e n t r a t i o n  to  used  i n  i n  Streptomyces.  ceased  get  a d d i t i o n  a d d i t i o n  d i l u t i o n No.3  the  SSL,  Streptomyces to  components  abundant  of  SSL/100ml  i s ,  g i v i n g  t r a n s f e r r i n g  (in  SSL)  media.  medium, 30,  then  50,100ml  of  m i x t u r e .  R e s u l t s  of  a c c l i m a t i z a t i o n  3.27  which  be  10ml  s u c c e s s i v e l y  F i g u r e s  would  added  11.13.  made  66  c e r t a i n  over  continued  of  were  reached  of  step  sugar,  above,  L i q u o r  both.  Waksman»s  SSL  to  s t a r t e d  R e s u l t s  but  the  w i t h  of  or  subsequent  Table  SSL  S u l f i t e  presence  SSL,  d e s c r i b e d  growth  Recourse the  the  s u b s t a n c e s  c o n c e n t r a t i o n  m i x t u r e ,  i n  r a t h e r  SSL  Spent  medium  not  t o x i c  water  i n  the  No.3  l i s t e d  the  to  components,  q u a n t i t i e s  i n  or  of  these  are  l i s t e d  experiments and  experiments  with  i n  Table  d i l u t i o n  with I I I . 2 1 can  be  3.28.  Experiments  and  R e s u l t s  d i l u t i o n and  and  Table  v i s u a l l y  with I I I . 2 2 .  observed  i n  Potential  FIGURE  3.27  : Growth  of Spent S u l f i t e  Liquor  of Streptomyces Griseus  i n Diluted  67 SSL  T o p r o w : G 0 . 1 , G 3 . 1 , 6 2 5 . 1, G 1 0 0 0 . 3 B o t t o m r o w : GO.2, G 3 . 2 , G 2 5 . 2 , G 5 0 . 2 (see T a b l e I I I . 2 1 f o r d e t a i l s )  F I G U R E 3.28  : Growth  of Streptomyces Olivaceus  Top row : 0 0 . 1 , 0 2 5 . 1 , 0 5 0 . 1 , 01000.3 Bottom row : O0.2, 0 2 5 . 2 , 0 5 0 . 2 , 0 1 0 0 . 1 (see T a b l e I I I . 2 2 f o r d e t a i l s )  Scale  : y  -J = 6.4  E x p e r i m e n t s and  cm  Results  i n Diluted  SSL  Potential of Spent S u l f i t e  Liquor  68  FIGDRE 3.29 : Growth of L . plantarium i n Solutions of Xylose and Glucose •—--—=—--  Regression l i n e s at 0.95 confidence y ( ± 0 . 1 4 7 ) = 0.0550 ( ± 0 . 0 1 1 ) x + 0. 108 (±0. 1 14) y ( ± 0 . 0 8 2 ) = 0.0015 ( ± 0 . 0 0 6 ) x + 0. 185 (±0. 064)  YEfiST  EXTRACT  CONCENTRATION  Experiments and Results  (G/LJ  P o t e n t i a l  3.2  o f  Spent  p l a n t a r i u m  Experiments tubes 50ml  i n  were  h a s  125ml  been  known  t o  Erlenmeyer  f l a s k s  and  conducted  under  and 15ml r e s p e c t i v e l y  medium  c o n s i s t s  potassium  a t  t h i s  s e r i e s  o f  Ferguson  o f  s y n t h e t i c of  phosphate,  69  and  t o  t o  7  these  r e p e a t i n g  t e s t s  [ 1 0 , 7 7 ]  a l s o  backs  PRELIMINARY T h i s  The  o f  was  measurement  o f  of  c o b a l t  added  phosphate, from  0  These s h a k i n g  t o  1 0 0 g / l ,  f o l l o w e d ,  were  were  (needed  hours.  F e r g u s o n ' s  s a l t .  f o r  Hence  was done  Only  added  t o  u  i n  o f  o f  yeast  was  p l a n t a r i u m  pentoses p o i n t  30°C  CONCENTRATION  13x100mm  t e s t  e n t i r e l y  tubes. by  medium  o c c a s i o n a l  t u r b i d i t y )  a r e summarized  i n  F i g u r e s  and  R e s u l t s  f r e e  dihydrogen  p a s t e ,  c f  t h e  was  potassium  with  i n  s u b j e c t .  ranging  t h e s u l f u r - d i o x i d e - s t r i p p e d a t  by  l i t e r a t u r e  t h i s  e x t r a c t  o f  3 . 2 9 .  The  measurement  Experiments  t h e medium  EXTRACT  time,  g / l  o r  was no  on  The SSL f e r m e n t a t i o n  i n c u b a t e d  The r e s u l t s  OF,YEAST  glucose  of  there  4 g / l o f  The r e s u l t s  F i g u r e  c o n c l u s i o n s  a g a i n s t  amounts  every  on  with  s y n t h e t i c  L .  tubes.  P r o p i o n i b a c t e r i a .  experiments  and v a r i o u s  tubes  on  t u r b i d i t y .  pH o f  and t e s t  STUDIES,ON,EFFECT  s e r i e s  growth  up  The  t e s t  30°C  powder,  e i t h e r  t h e n o n f e r m e n t a b i l i t y  f r e u d e n r e i c h i i .  a t  T h i s  e x t r a c t  s u g a r ,  pentose.  16x150mm  medium.  a r e presented  P r o p i o n i b a c t e r i a these  i n  s t e r i l i z a t i o n .  f l a s k s  e x p e r i m e n t s [ 2 3 ] showed  a f t e r  ferment  c o n d i t i o n s  yeast  c o n c e n t r a t i o n s .  r e a d j u s t e d  i n o c u l a t e d  a n a e r o b i c  1 0 0 g / l  v a r i o u s  a s e p t i c a l l y were  o f  dihydrogen  x y l o s e ,  350  L i q u o r  GROHTH_OF_LACTQBACILLDS_IN_XYLOSE L.  3.3  S u l f i t e  f o r  S S L .  thorough up  t o  3 . 3 0 , 3 . 3 1 , and  70  Potential  of Spent S u l f i t e  Liguor  LEGEND  YEfiST EXTRACT CONCENTRATION (PASTE. G/L)  •  0 5  +  10  X  20 40  +  60  K  70  Y  80  H  100  Experiments  and  Results  Potential FIGURE 3 , 3 0 1.60  o f Spent  Sulfite  71  Liguor  E f f e c t of Yeast E x t r a c t Concentration L. p l a n t a r i u m i n S S L i n T e s t T u b e s  on Growth o f  X  1.40  X  1.20 X  1.00  X  Y  Y  X  JR  0Q  •  X  X  X  Y  *  X  Y  X  Y  X  X  Jft  Y X  X  X  X  X  •  *  *  *  * •  •  •  X  Y  o  •  • •  •  X X Y X X  o.ao  >-  X  •  X  OQ  o a: CD  X  •  0.60 X  X X  X X  0.40  X  X  X  X  Y  X  +  4-  +  +  +  X  0.20  <t> + ,+ + v .m  T  •  m  AA  0.0 0.0  70.0  140.0  210.0  FERMENTATION TIME(HRS) E x p e r i m e n t s and R e s u l t s  1  A  280.0  CP 350.0  72  P o t e n t i a l of Spent S u l f i t e Liquor  LEGEND  YERST EXTRACT CONCENTRATION (PASTE. G/L)  •  0 5  +  10  X  20 40 60  K  70  Y  80  W  Experiments  100  and R e s u l t s  Potential  o f Spent S u l f i t e  73  Liquor  F I G U R E 3.31 : E f f e c t o f Y e a s t E x t r a c t C o n c e n t r a t i o n o n G r o w t h o f j gg P. f r e u d e n r e i c h i i i n S S L i n T e s t T u b e s  1.40  •  x  x  £  • •  X  X  YX  ¥ V.Y  »  X  X  1.20  1.00  x  CQ  Cd <P X  OQ  o  X +  m+  A A  0.80  X  +  ++  55 X Y  a  A  A  0.60  (-Q  *X  0.40  A  0 0.20  0.0  L  • + A  ll I m  8  .0.0  X  •  70.0  +  •  CD  •  •  •  •  a 140.0  210.0  280.0  FERMENTATION TIME(HRS) E x p e r i m e n t s and R e s u l t s  350.0  Potential of Spent S u l f i t e  Liquor  LEGEND  YEAST EXTRACT CONCENTRATION (PASTE. G/L)  •  0 5  +  10  X  20 40  +  60  K  70  Y  80  W  100  Experiments and Results  Potential of Spent S u l f i t e FIGURE 3.32 0 80  Liquor  75  : Effect of Yeast Extract Concentration on Growth of * i i i SSL i Test Tubes P  s n e r r n a n  n  n  0.70  SC  0.60  Y  0.50  0  Y Y  4  X  X  Q 1—4  CD  •  Y Y  0.40 CD  o cr: CD  Y  W  H  *  H  X  M  0.30  Y  <!>•  X XX  X X X  X  x  if X X +  0.20  *X  +  ++  Y  • * + 2 X  0.10  I  .  x 8+ J Y  A A A  SJJA D 6  * A  a  CD CD A  A  A CD  A A  £ C D ° CD A  •  CD  CD  A •  CD  0.0  JL  0.0  CD  CD  70.0  340.0  210.0  280.0  FERMENTATION TIME(MRS) Experiments and Results  350.0  Potential of Spent S u l f i t e FIGURE 3.33  Liquor  : Legend for Figures Numbered from 3.30 to  LEGEND YERST EXTRACT CONCENTRATION (PASTE. G/L) 0  •  5  +  10  X  20 40 60  K  70  Y  80  «  Experiments  100  and  Results  76 3.32  P o t e n t i a l  3.32  for  L.  of  p l a n t a r i u m ,  P.  r e s p e c t i v e l y .  The  r e s u l t s  P.  the  growth  s h e r m a n i i ,  y e a s t not  e x t r a c t  0 P.  n o t i c e a b l y  s h a r p l y  to  with  5 g / l ,  f r e u d e n r e i c h i i  that  up  to  of  yeast  s l a c k e n s  r e g u i r e  l e s s  1.  g r a d u a l l y  For  then  P.  yeast  P.  s h e r m a n i i  p l a n t a r i u m  and  with  i n c r e a s e s  the  growth  growth  c o n c e n t r a t i o n  pace.  e x t r a c t  from  O b v i o u s l y  than  the  of does  f r e u d e n r e i c h i i ,  e x t r a c t i t s  77  and  f o r  6 0 g / l ,  anymore.  i t  L i g u o r  f r e u d e n r e i c h i i ,  i n c r e a s e s  i n c r e a s e  then  S u l f i t e  shew  c o n c e n t r a t i o n  i n c r e a s e  r i s e s  Spent  then,  other  two  s p e c i e s .  3.4  EFFECTIVENESS Though  main  D i f c o  e x p e r i m e n t s ,  d i f f e r e n t e x t r a c t i n  OF  d i f f e r e n t  y e a s t then  due  stages  checked  experiments used  potassium  with  was  an  aqueous  vigorous  shaking  f o r  /Growth  of  130  e q u i v a l e n c e  L.  p r o j e c t .  Yeast  i n  s o l u t i o n  were  hours. of  e x t r a c t  p l a n t a r i u m  with  The  the  used  Experiments  on  125ml  a  most  L.  of  varying  incubated  were  used  of  the  at  g l u c o s e ,  30<>C i n  compared  yeast  e x t r a c t  These  of  on  a  paste  any 3.34,  the  dry  of  yeast  without  of  The  4 g / l  F i g u r e  value  was  f l a s k s .  amount  r e s u l t s ,  R e s u l t s  yeast  p l a n t a r i u m .  of  from  b a c t e r i a  erlenmeyer  4 0 g / l  the  and  paste)  growth  of  e x t r a c t s  powder  was  and  f o r  e f f e c t i v e n e s s  using  of  SOURCES  yeast  t o t a l  n u t r i e n t  paste  with  of  The  s o u r c e s  on  f l a s k s  was  e x t r a c t  s o l i d s / g  phosphate  These  used.  yeast  of  c a r r i e d  added.  e x t r a c t s  the  DIFFERENT  powder  f e r m e n t a t i o n s  dihydrogen  the  NB  FROM  a v a i l a b i l i t y ,  d i f f e r e n t  e x t r a c t  u n v e i l  t h e i r  0.77g of  from  were  to  EXTRACT  e x t r a c t  namely  (with  e x t r a c t  medium  yeast  s o u r c e s , paste  YEAST  yeast b a s i s .  d e v i a t e d  P o t e n t i a l o f Spent  Sulfite  78  Liquor  FIGURE 3.34 : G r o w t h o f L. p l a n t a r i u m a s M e a s u r e m e n t o f E f f e c t i v e n e s s o f Yeast E x t r a c t from V a r i o u s Sources R e g r e s s i o n l i n e a t 0.95 c o n f i d e n c e y(±0.066) = 0.013 (±0.002)x + 0.037 (±0.066)  YEAST 0 0  EXTRACT  PASTE  10.0 ,  CQNC.  (G/L.DRY  20.0  30.0  40.0  1  1  1  BASIS) 50.CJ  I  YEAST  0 0 YEAST  10.0  20.0  EXTRACT  POWDER  30.0  40.0  CONCENTRATION  E x p e r i m e n t s and R e s u l t s  50.0 CG/L)  Q  P o t e n t i a l  somewhat p a s t e  from  was  y e a s t  on  e f f o r t paste  was was  common  used  growth  expressed  3.5  the  only and  made i t  on  wet  REPLACEMENT SSL,  i n  e f f l u e n t  at  the  f i s h  w i t h  any  supernatant the  s o l u b l e  s l i m y of  and  s o l i d s  weight. but  no  growth was  growth  was  hard d i d  to  not  or  made  of  was  yeast  s t u d i e s  on  e x t r a c t  e f f e c t s  so  Whenever  the  was  no  of  f u r t h e r  yeast  c o n c e n t r a t i o n  was  e x t r a c t w i l l  be  4 g / l  of  and  f o r  steam  d u r i n g  c o u l d  be  namely  gave  a c i d s .  and  but  s i g n s  p r o h i b i t e d as  w e l l  by  R e s u l t s  as  c e n t r i f u g e d .  The  P a r t  They  reduced by  c o n t e n t c e l l growth,  measurements cf  of were  dry  p o s s i b l e  I d e n t i f i c a t i o n with  a l l  f i s h  growth of  high  T h i s  The  of  and  the  e x p e r i m e n t s .  o f f .  a f f i r m e d  f e r m e n t a t i o n s Experiments  and  then  O r i g i n a l l y ,  s t e r i l i z a t i o n .  measurement  p l a n t  5:5)  o b s e r v a t i o n s .  subsequent  o b s e r v a t i o n s  300C.  t u r b i d i t y ,  c e n t r i f u g e  the  at  soluble)  m i c r o s c o p i c  f i s h  6:4,  r e c e i v e d ,  or  with  with  f r e u d e n r e i c h i i  f l a s k s ,  2 0 g / l  with  8 : 2 ,  P.  t o t a l , weight  EFFLUENT  mixed  (9:1,  as  c r  PLANT  d i o x i d e ,  added  used  prevent  growth  FISH  added,  cocked  f i l t e r  f o r  BY  s u l f u r  p r e c i p i t a t e d  p r o d u c t s ,  only  and  Erlenmeyer  by  then  M i c r o s c o p i c p o s i t i v e  p o i n t .  p r o p o r t i o n s  p o s s i b l e  l i g u i d s o l i d s  of  ( 7 2 g / l  easy,  e f f l u e n t  s t a t e d ,  EXTRACT  250ml  e f f l u e n t  of  However,  f e r m e n t a t i o n s ,  p l a n t a r i u m  s o l i d s  measurements  p l a n t  be  phosphate  L.  i n  of  b a r r i n g  t h i s  d i f f e r e n t  p l a n t  c o n t e n t  c l e a r  79  L i g u o r  p r e l i m i n a r y  e a r l y  s t r i p p e d  dihydrogen  s h e r m a n i i  l i n e .  few  YEAST  potassium  P.  the  S u l f i t e  b a s i s .  OF  steam  fermented  a  w i l l  Spent  growth i n  t o  used a  of  of  p r o d u c t s  P r o p i o n i b a c t e r i a ,  as  P o t e n t i a l  v o l a t i l e a c i d s  a c i d s  may  c o n t e n t  e x t r a c t i o n amino  i n  the  n i t r o g e n  s o u r c e  p r o t e i n  f i s h of  to  S u l f i t e  l i q u o r  d i s t i n g u i s h e d  e a r l i e r  the  hope  Spent  e a s i l y  the  d e s c r i b e d  a c i d s  d e s t r o y  of  be  of  i n  the  cannot  p l a n t  u s i n g r e p l a c e  from  s e p a r a t e  p l a n t  the  the  e f f l u e n t .  e f f l u e n t .  f i s h  80  amino  The  e t h e r  l a c t i c  These  more  a c i d  from  experiments  e f f l u e n t  much  high  as  a  cheap  expensive  yeast  e x t r a c t .  3.6  PRELIMINARY,EXPERIMENTS Only  two  a v a i l a b l e c o u l d  t o  be  f o l l o w e d o t h e r  done  w i l l  be  c h o i c e  these  experiments  the  range i n  R e i n f o r c e d c o n c e n t r a t i o n s 100g/l)  were  o n l y f o r  the  of  i n  v a r i o u s  the  which  were  f e r m e n t a t i o n  needed  to  l i v e  reasons  f o r  t h i s i n  regarded  as  then  t c  used  v a r i a b l e s  t o  to  Most  Erlenmeyer  found  were  experiments  f a i l e d  d i s c u s s i o n s j u s t  u n i t ,  f e r m e n t c r s .  250ml  experiments  i n  t w i n  f e r m e n t a t i o n s these  done  then  be of  f l a s k s . up  to  f a i l u r e  the  next  p r e l i m i n a r y help  be  i n  used  i n  the the  fermentors.  of  yeast  used  f o r  c o n c e n t r a t i o n  s i m u l t a n e o u s  The  c l o s t r i d i a l  f r e u d e n r e i c h i i ,  two  Thus  l a t t e r  71  o n l y  then  r e s u l t s  the  M i c r o f e r m  were  were  of  the  saved  s h o r t l y  These  of  were  purpose.  presented  of  so  t h e s e .  time  intended  e x p e r i m e n t s ,  The  i n  a g a i n s t  c h a p t e r .  P.  p r o j e c t ,  f e r m e n t a t i o n s  t h e i r  ERLENMEYER,FLASKS  7 1 - f e r m e n t o r s ,  t h i s  U n f o r t u n a t e l y ,  IN  P. of  medium  e x t r a c t  and  SSL  powder  (0,  f e r m e n t a t i o n s  s h e r m a n i i ,  and  potassium  Experiments  and  a  medium 10,  with  mixture  dihydrogen R e s u l t s  with 20, L.  c f  the  30,  v a r i o u s 40,  50,  p l a n t a r i u m , f i r s t  phosphate  was  two. kept  Potential of Spent S u l f i t e Liquor  LEGEND + GROWTH. DRY C E L L WEIGHT (G/L) x  Y  A  L A C T I C ACID PRODUCTION  (MOLES/L)  SUGAR CONSUMPTION ( G / L ) V O L A T I L E ACIDS PRODUCTION  Experiments a n d R e s u l t s  (M0LE5/L)  Potential  o f Spent  F I G U R E 3.35 : F e r m e n t a t i o n s  Sulfite  o f SSL with  Liquor  L. p l a n t a r i u m  82 i n Flasks  30.0  2.4  0.0  20.0 YEAST  40.0 EXTRACT  60.0  CONCENTRATION  Experiments and Results  80.0 (G/L)  100.0  P o t e n t i a l of  S p e n t S u l f i t e Liquor  LEGEND + GROWTH. LACTIC  x  DRY ACID  Y  SUGAR  A  VOLATILE  CELL  WEIGHT  PRODUCTION  CONSUMPTION ACIDS  Experiments  (G/L) (MOLES/L)  (G/L)  PRODUCTION  and  Results  (MOLES/L)  Potential  o f Spent  FIGURE 3.36 : F e r m e n t a t i o n s  Sulfite  Liquor  84  o f S S L w i t h P. f r e u d e n r e i c h i i i n Flasks  30.0  Y  27.5  Y  + 2.0 j -  25.0  Y  + 1.8 j  22.5  I  +  + 2.2! -  -  -  Y  + 1.8 j  20.0  Y  I.4!  17.5  Y  + 1.2!  15.0  Y  12.5  Y  10.0  Y  +  x o ° x  0-  8  !  0 6  i  x 0-04 i X0.02J  1  +  _ r/A  •  M—  vo.ooij 0.0  X  0.0G . A  +  0.04 A 0.02 1  20.0 YEAST  40.0  EXTRACT  60.0  CONCENTRATION  Experiments and Results  1  80.0 (G/L)  0.00 100.0  1 1 1  |A I 3 1  1 1 !  A A  Potential of Spent S u l f i t e Liquor  85  LEGEND  + x  _ „  Y  A  GROWTH. LACTIC SUGAR  DRY ACID  CELL  PRODUCTION  CONSUMPTION  VOLATILE  WEIGHT  ACIDS  (G/L) (MOLES/L)  IG/L)  PRODUCTION  Experiments  and R e s u l t s  (MOLES/L)  Potential FIGURE  of Spent  3.37 : F e r m e n t a t i o n s  Sulfite  o f SSL  with  Liguor  P.  shermanii  86  i n Flasks  30.0 ,  0.0  20.0  40.0  60.0  YEflST EXTRACT CONCENTRATION  Experiments  and  Results  80.0  (G/L)  JOO.O  Y  Potential of Spent S u l f i t e Liquor  87  LEGEND + x  Y A  GROWTH. LACTIC SUGAR  DRY ACID  CELL  PRODUCTION  CONSUMPTION  VOLATILE  WEIGHT  ACIDS  (G/L) (MOLES/L)  (G/L)  PRODUCTION  Experiments  and R e s u l t s  (MOLES/L)  Potential  o f Spent  Sulfite  Liquor  F I G U R E 3.38 : F e r m e n t a t i o n s o f S S L w i t h L. p l a n t a r i u m a n d P. f r e u d e n r e i c h i i  88  A Mixture of i n Flasks  30.0  2.4 2.2 + 2-0 1.8  IL  + 1-6 1.4 1.2 X 0.10  X  o.oa  X 0.06  L  X 0.04  X  x  0.02|L 0 . 0 0 # 0.0  20.0  40.0  60.0  YERST EXTRRCT C0NCENTRRTI0N Experiments  and  Results  80.0  (G/L)  o.oo  100.0  A  Potential c o n s t a n t i n  at  SSL.  ftg/1,  Every  250ml  and  100ml  a s e p t i c a l l y Only  the  the  of  Erlenmeyer  necessary  products during  s u f f i c i e n t  for  Spent  Sulfite  c o n c e n t r a t i o n  the  d e s i r e d  f l a s k  before  r e a d j u s t e d  end  of  a f t e r were  medium  c o b a l t was  These  f l a s k s  pH  except  i n t e r e s t ,  so  and  ions  i n  no  100ml  were  at  2.2ppm  s t e r i l i z e d  i n c c u l a t i o n .  f e r m e n t a t i o n ,  a n a l y s i s .  of  s t e r i l i z a t i o n of  89  Liquor  i n  was  always  cne  s e r i e s .  sampling was  then  a  was  more  than  incubated  at  i 30°C  f o r  100  i n o c u l a t e d  w i t h  a f t e r  the  second  s t a g e .  The  3.36,  A I . 3 0 ,  AI.31  80-100  these  3 . 3 7 ,  3.38  i n  Appendix  IN  runs  are  f e r m e n t a t i o n s ,  SSL  or  phosphate  lower.  was  the  and  a d d i t i o n  and  was  were  of  f l a s k s  which  c o n t i n u o u s l y  i n c u b a t i o n ,  are  t a b u l a t e d  MICROFERM i n  steam  N u t r i e n t s  of  these  during  t a b l e s  been  shaken  presented i n  had  only  the  a e r o b i c  i n  F i g u r e s  A I . 2 8 ,  A I . 2 9 ,  I.  l i s t e d  yeast  of  experiments  THE.7L  c o n c e n t r a t i o n  c o n c e n t r a t i o n  Most  hours  of  FERMENTATIONS  200ppm  hours.  P r o p i o n i b a c t e r i a  r e s u l t s  The  150  f i r s t  3.35,  3.7  to  then  kept  e x t r a c t  DMBZ.  Table  I I I . 2 3 .  s t r i p p e d  were  was  FERMENTORS  of  added;  c o n s t a n t  s u l f u r  potassium  to  Ug/1,  c o n c e n t r a t i o n  These  runs  can  be  In  were  these  d i o x i d e  dihydrogen c o b a l t  v a r i a b l e s ,  d i v i d e d  to  i n t o  U  ion as main  c a t e g o r i e s : 1. two  Fermentation  with  L.  p l a n t a r i u m  and  P.  f r e u d e n r e i c h i i  i n  s t a g e s . 2.  Fermentation  with  a  Experiments  mixture and  of  R e s u l t s  L.  plantarium  and  P o t e n t i a l  of  Spent  Table SUMMARY  OF  r I S e r i e s  INFORMATION  Run  B a c t e r i a S p e c i e s  ON  Yeast E x t r a c t Cone.  1.1  I  1.2  L i g u o r  90  I I I . 2 3  FERMENTATIONS  (g/i) |  S u l f i t e  C o b a l t i o n Cone. (ppm)  OF  SSL  pH C o n t r o l  IN  7L  FERMENTORS  Figures  Table:  FP1.1 FP1.2  LP  20 20  2.2  LP  2.2  Automatic Spot  3 . 39 3.40  AI.32 A I . 3 3  FP2.1  (LPthen)  20  2.2  No  3.41  AI.34  FP2.2  PF (LPthen)  20  2.2  No  3.42  AI.35  20  2.2  Automatic  3.43  A I . 3 6  PF  1 I  I  1  1 1  2 3.1  3.2  3.3  3.4 4  FP3  LP  6  F15.22a  PF  20 (paste)  2.2  No  3.44  A I . 3 7  F15.22b  PF  20 (paste)  2. 2  No  3.45  A I . 3 8  F05.22 F10.22 F20.22  PF  05 10 20  2.2 2.2 2.2  Automatic  PF PF  Automatic Automatic  3.46 3.47 3.48  AI.39 A I . 4 0 AI.41  F30.22  PF  30  2.2  Automatic  3.49  A I . 4 2  F20.00 F20.09  PF PF  20 20  0.0 0.9  Automatic Automatic  3.50 3.51  AI.4 3 AI.44  F20.22.126*  PF  20  2.2  Automatic  3.52  AI.45  S20.22  PS  20  2.2  Automatic  3.53  AI.46  S e r i e s  Type  1 1.1  Fermentation Fermentation  1.2 2 3 3.1 3.2 3.3 3.4 4  F F F E F F A F  *126ppm  PF  e e e a e e d e  of  w i t h L. p l a n t a r i u m of Sugar to L a c t i c  and P. A c i d by  f r e u d e n r e i c h i i L. p l a n t a r i u m  rmentation of L a c t i c A c i d to V o l a t i l e A c i d s rmentation with a M i x t u r e of LP and PF rmentation with P. f r e u d e n r e i c h i i r l y F e r m e n t a t i o n s rmentation w i t h Yeast E x t r a c t as V a r i a b l e r m e n t a t i o n with C o b a l t Ion C o n c e n t r a t i o n as d i t i o n of 5 6 - D i m e t h y l B e n z i m i d a z o l e rmentation w i t h P. s h e r m a n i i f  DMBZ  was  added  to  SSL  Experiments  p r i o r and  to  i n o c u l a t i o n  R e s u l t s  by  PF  V a r i a b l e  P o t e n t i a l  P.  L i q u o r  Fermentation  w i t h  P.  f r e u d e n r e i c h i i .  4.  F e r m e n t a t i o n  with  P.  s h e r m a n i i .  the  f i r s t  to  l a c t i c  c o n t r o l l e d  a t  c o b a l t  7.  stage  In  ion  reached  e x t r a c t  added  was  were  s t e r i l i z e d  The  second  run  FP1.2  t h i s  stage  the  f i n a l  amount  of  the  s e r i e s  of  experiments  which  are  amounts  of  yeast  fermented  SSL  a s e p t i c a l l y E i t h e r  P.  In c o b a l t  1,  s e r i e s  i n  91  s e r i e s  were  t h i s  f l a s k  from  was 2,  end  blown SSL, was  a i r 3,  of  the  with  at  with  tun  one  i n  f e r m e n t a t i o n s Experiments  the  of  t h i s  stage  of  were and  yeast  T h i s  broth  for  the  another c f  v a r i o u s  100ml  was  then  from  r e s u l t s  with  of  t r a n s f e r r e d s t e r i l i z e d .  i n o c u l a t e d  to  f e r m e n t a t i o n s  of  e x t r a c t  with  dene  of  2000cc/min.*  yeast  r a t e  the  s u b s t a n t i a l  were  of  When  s e r i e s ,  broth  were  2.2ppm  broth  A  f l a s k s  L.  Near a  f i n a l  f l a s k s , In  was  f r e u d e n r e i c h i i .  a s i d e  f e r m e n t o r ) ,  r a t e  at  P.  set  (the  second  2 0 g / l  b r o t h .  F P 1 . 1 .  was  to  i n o c u l a t e d  blown  f i n a l  used  3.71.  s i m u l t a n e o u s l y . was  the  s h e r m a n i i  a  and  more  added  P.  e x t r a c t  20g  SSL  a u t o m a t i c a l l y  phase,  FP1.1  the  pH  the  b r o t h .  Erlenmeyer  or  i n  with  in  f e r m e n t a t i o n  from  but  sugar  yeast  FP2.2  F i g u r e  A l l  the  of  of  run  each.  i o n ,  f e r m e n t a t i o n ,  one  i n  Near  a i r  Run  of  the  i n o c u l a t e d  250ml  e x t r a c t  f r e u d e n r e i c h i i  In  i n  f r e u d e n r e i c h i i  f l a s k s .  s e r i e s  to  the  broth  presented  the  l i t e r  began.  FP2.1  2 0 g / l  to  then  1,  p l a n t a r i u m  s t a t i o n a r y  every  then  run  L.  added  to  and  s e r i e s  by  a g a i n ,  s t a g e  of  a c i d  were  f e r m e n t a t i o n  P.  S u l f i t e  3.  fermented  the  Spent  f r e u d e n r e i c h i i .  In  of  of  end  of  of and the  2 0 0 0 c c / m i n .  with  R e s u l t s  2.2ppm  p l a n t a r i u m  the of  and  P.  f r e u d e n r e i c h i i  92  Potential  of Spent  Sulfite  Liquor  Run F P 1 . 1 2 0 q / l o f y e a s t e x t r a c t powder 2.2ppm o f c o b a l t i o n Uq/1 o f p o t a s s i u m d i h y d r o g e n p h o s p h a t e pH a u t o m a t i c a l l y c o n t r o l l e d  LEGEND  * +  .__  y A  CONCENTRATION  GROWTH. LACTIC  x  ^  flCJDS  SUGAR  DRY ACID  CELL  WEIGHT  (G/L)  CONCENTRATION  CONCENTRATION  VOLATILE  (MOLES/L)  ACIDS  (MOLES/L)  (G/L)  CONCENTRATION  Experiments  and  Results  (MOLES/L)  P o t e n t i a l o f Spent S u l f i t e L i g u o r FIGURE 3.39 : Run F P 1 . 1 . F e r m e n t a t i o n  0.0  25.0  50.0  FERMENTATION  w i t h L. p l a n t a r i u m  75.0 TIME  93  100.0 (HOURS)  E x p e r i m e n t s and R e s u l t s  125.0  Potential  94  o f Spent  Sulfite  Liquor  r Run FP1.2 2 0 g / l o f y e a s t e x t r a c t powder 2.2ppm o f c o b a l t i o n 4 g / l of potassium dihydrogen phosphate pH m a n u a l l y s p o t c o n t r o l l e d  LEGEND . . . M  .  flCJDS  x  CONCENTRATION  + GROWTH, x  Y  A  LACTIC SUGAR  DRY ACID  CELL  WEIGHT  (G/L)  CONCENTRATION  CONCENTRATION  VOLATILE  (MOLES/L)  ACIDS  (M0LE5/L)  (G/L)  CONCENTRATION  Experiments  and  Results  (MOLES/L)  Potential  FIGURE 3.40  o f Spent  Sulfite  : Run FP1.2. F e r m e n t a t i o n  with  L. p l a n t a r i u m  42.0  0.36  35.0  Y  28.0 L  + 0.9  0.G  + 0.3  95  Liquor  L  0.0 0.0  25.0  50.0  FERMENTATION  Experiments  75.0 TIME  100.0  (HOURS)  and R e s u l t s  I Potential  o f Spent  •j  Sulfite  Liquor  1  Run  I  FP2.1  20g/l o f y e a s t e x t r a c t powder 2.2ppm o f c o b a l t i o n Ug/1 o f p o t a s s i u m d i h y d r o g e n p h o s p h a t e pH n o t c o n t r o l l e d  LEGEND  m  + x  Y  n  A  ACIDS  CONCENTRATION  GROWTH. LACTIC SUGRR  DRY ACID  CELL  (MOLES/L)  WEIGHT  CONCENTRATION  CONCENTRATION  VITAMIN VOLATILE  B12  (MOLES/L)  (G/L)  CONCENTRATION  ACIDS  (G/L)  (MG/L)  CONCENTRATION  E x p e r i m e n t s and R e s u l t s (  (MOLES/L)  I I | I  Potential  o f Spent  Sulfite  F I G U R E 3.41 : R u n F P 2 . 1 . F e r m e n t a t i o n L a c t i c A c i d as Main  97  Liquor  w i t h P. f r e u d e n r e i c h i i o n Substrate  ANAEROBIC  AEROBIC  0.30  J  A  X  X  • A  n  o.o  40.0  80.0  FERMENTATION Experiments  1Z0.0 TIME  160.0 (HOURS)  and R e s u l t s  £  0.2B 0.26  $  0.24  ^  0.22  a  0.20  A  0.18  A  0.16  A  0.14  A.  0.12  x  0.30  x  0.08  x  0.06 200 .0  x  98  Potential  o f Spent  Sulfite  Liquor  Run F P 2 . 2 2 0 g / l o f y e a s t e x t r a c t powder 2.2ppm o f c o b a l t i o n 4g/l of potassium dihydrogen phosphate pH n o t c o n t r o l l e d  LEGEND  * +  _.._„..  CONCENTRATION  GROWTH. LACTIC  x  Y  SUGAR  DRY ACID  A  CELL  VOLATILE  BI2  (MOLES/L)  WEIGHT  (G/L)  CONCENTRATION  CONCENTRATION  VITAMIN  n  -  ACIDS  (G/L)  CONCENTRATION  RCIDS  (MOLES/L)  (MG/L)  CONCENTRATION  Experiments  and  Results  (MOLES/L)  Potential  o f Spent  Sulfite  F I G U R E 3.42 : R u n F P 2 . 2 . F e r m e n t a t i o n L a c t i c Acid a s Main  99  Liquor  w i t h P. f r e u d e n r e i c h i i o n Substrate  REROBIC  ANAEROBIC  0.0G  0.0  40.0  80.0  FERMENTATION  Experiments  120.0  TIME  160.0  (HOURS)  and R e s u l t s  x  Potential  Run  of Spent  Sulfite  Liguor  FP3  2 0 g / l o f y e a s t e x t r a c t powder 2.2ppm o f c o b a l t i o n Ug/1 o f p o t a s s i u m d i h y d r o g e n p h o s p h a t e pH a u t o m a t i c a l l y c o n t r o l l e d  LEGEND .  +  x  Y  m  A  GROWTH. LRCTIC SUGAR  DRY ACID  CELL  WEIGHT  CONCENTRATION  CONCENTRATION  VITAMIN VOLATILE  B12  (G/L)  (G/L)  CONCENTRATION  ACIDS  (M0LE5/L) (MG/L)  CONCENTRATION  Experiments  and  Results  (MOLES/L)  Potential  101  of Spent S u l f i t e L i q u o r  FIGURE 3 . 4 3 : Run F P 3 . F e r m e n t a t i o n w i t h Mixed C u l t u r e o f L. p l a n t a r i u m and P. f r e u d e n r e i c h i i  ANAEROBIC  Y  36.0  Y  28.0  L  20.0  L  Y  •  0.30  •  0.20  •  0.10  •  0.00  L  0.04 0.0  40.0  80.0  FERMENTATION  120.0 TIME  160.0  (HOURS)  E x p e r i m e n t s and R e s u l t s  x  102  P o t e n t i a l  Run  Spent  S u l f i t e  L i q u o r  F15.22a  2 0 g / l  o f  2.2ppm Ug/1 pH  o f  o f  yeast  o f  e x t r a c t  c o b a l t  i o n  potassium  r e a d j u s t e d  a t  paste  dihydrogen t i m e s ,  n o t  phosphate c o n t r o l l e d  I  ;  LEGEND  x  +  ACIDS  CONCENTRATION  GROWTH.  DRY  CELL  (MOLES/L)  WEIGHT  • PH  Experiments  a n d R e s u l t s  (G/L)  I  Potential  o f Spent  Sulfite  F I G U R E 3.44 : R u n F l 5 . 2 2 a . F e r m e n t a t i o n E f f e c t o f pH  Liguor  with  P.  FERMENTATION TIME (HOURS)  Experiments  and R e s u l t s  103  freudenreichii.  104  P o t e n t i a l  o f  Spent  S u l f i t e  L i q u o r  r Bun  F15.22b  2 0 g / l  o f  2.2ppm 4 g / l pH  o f  n o t  yeast  o f  e x t r a c t  c o b a l t  paste  i o n  potassium  dihydrogen  phosphate  c o n t r o l l e d  LEGEND  -  -  +  GROWTH.  *  pH  Y  • — „  A  SUGAR  DRY  CELL  WEIGHT  CONCENTRATION  VITAMIN VOLATILE  B12  (G/L)  (G/L)  CONCENTRATION  ACIDS  (MG/L)  CONCENTRATION  Experiments  a n d  R e s u l t s  (MOLES/L)  Potential  o f Spent  Sulfite  Liquor  FIGURE 3.45 : Run F 1 5 . 2 2 b . F e r m e n t a t i o n w i t h i n One A n a e r o b i c S t a g e  P.  105  freudenreichii  0.36  0.30  J0.24  JO.IB  J0.4  J0.2  0.0  100.0  200.0  FERMENTATION  Experiments  300.0 TIME  400.0 (HOURS)  and R e s u l t s  0.0 500.0  A  A  A  A  n  a  a  Potential of Spent S u l f i t e  106  Liquor  r  Run F05.22 05g/l of yeast extract powder 2.2ppm of cobalt ion 4 g / l of potassium dihydrogen phosphate pH automatically controlled  LEGEND  + ._ _  x  Y  D  A  GROWTH. LACTIC SUGAR  DRY ACID  CELL  WEIGHT  CONCENTRATION  CONCENTRATION  VITAMIN VOLATILE  B12  (MOLES/L)  (G/L)  CONCENTRATION  ACIDS  (G/L)  (MG/L)  CONCENTRATION  Experiments  and R e s u l t s  (MOLES/L)  Potential of Spent S u l f i t e FIGURE 3.46  9.0  Liquor  107  : Run F05.22. Fermentation with P. f r e u d e n r e i c h i i . Yeast Extract Concentration as Variable  AEROBIC  ANAEROBIC  7.5  A  6.0  •  •  •  0.9  0.6  0.3  T*  40.0  s J  80.0  120.0  160.0  FERMENTATION TIME (HOURS) Experiments  and R e s u l t s  44.  Y  41.  Y  38.  Y  35.  Y  -A 32'.  Y  0.14  A  0.12  A  0.10  A  0.08  A  0.06  X  0.04  X  0.02  X  0.00  X  .0  108  P o t e n t i a l  Run  Spent  S u l f i t e  L i g u o r  F10.22  1 0 g / l  o f  2.2ppai 4 g / l pH  o f  y e a s t  o f  o f  e x t r a c t  c o b a l t  potassium  a u t o m a t i c a l l y  powder  i o n d i h y d r o g e n  phosphate  c o n t r o l l e d  LEGEND  + x  Y  —  n  A  GROWTH. LACTIC SUGAR  DRY ACID  CELL  WEIGHT  CONCENTRATION  CONCENTRATION  VITAMIN VOLATILE  B12  (G/L)  (G/L)  CONCENTRATION  ACIDS  (MOLES/L)  (MG/L)  CONCENTRATION  Experiments  and  R e s u l t s  (MOLES/L)  Potential of Spent S u l f i t e FIGURE 3.17  +  +  Liquor  : Run F10.22. Fermentation with P. f r e u d e n r e i c h i i . Yeast Extract Concentration as Variable  ANAEROBIC  9.0  109  AEROBIC  7.5  44.  Y  41.  Y  38.  Y  35.  Y  32.  Y  0.14  A  0.12  A  0.10  A  0.08  A  0.06  x  0.04  x  0.02  x  0.00  x  . +.-+  6.0  • 0.9  0.6  •  0.3  • 0.0  -X-X-,  o-  o.o  :/ •  x  x  \  • 40.0  I  80.0  FERMENTATION  Experiments  \ 120.0  TIME  160.0 (HOURS)  and R e s u l t s  200.0  110  P o t e n t i a l of Spent S u l f i t e  Liquor  Run F20.22 20g/l of yeast extract powder 2.2ppm of cobalt ion 4 g / l of potassium dihydrogen phosphate pH automatically controlled  LEGEND + x  _  Y  • „  A  GROWTH. LACTIC SUGAR  DRY ACID  CELL  WEIGHT  CONCENTRATION  CONCENTRATION  VITAMIN VOLATILE  B12  (MOLES/L)  (G/L)  CONCENTRATION  ACIDS  (G/L)  (MG/L)  CONCENTRATION  Experiments  and R e s u l t s  (MOLES/L)  Potential  o f Spent  Sulfite  111  Liquor  FIGURE 3.48 : Run F 2 0 . 2 2 . F e r m e n t a t i o n w i t h P. f r e u d e n r e i c h i i . Yeast Extract Concentration as Variable  AEROBIC  ANAEROBIC  9.0  7.5  I  — . - A 6.0  +5 /  \  V Y  •  0.9  l  li  A  A A  •  V  •  •  i  A  -~  0.6  /  m  V  //  //V  0.3  X  r  ^ Y _ Y  44.  Y  41.  Y  38.  Y  35.  Y  32.  Y  -|0.14  A  0.12  A  0.10  A  0.08  A  0.06  x  0.04  x  0.02  X  ~ - Y « —  _L  fTjo.o[Si_-in 0.0  40.0  80.0  FERMENTATION  Experiments  120.0 TIME  160.0 (HOURS)  and R e s u l t s  0.00 200.0  x  Potential of Spent S u l f i t e  112  Liquor  Run F30.22 30g/l of yeast extract powder 2.2ppm of cobalt ion 4 g / l of potassium dihydrogen phosphate pH automatically controlled  LEGEND  + x  .  _  Y  n  A  GROWTH. LACTIC SUGAR  DRY ACID  CELL  WEIGHT  CONCENTRATION  CONCENTRATION  VITAMIN VOLATILE  B12  (MOLES/L)  (G/L)  CONCENTRATION  ACIDS  (G/L)  (MG/L)  CONCENTRATION  Experiments  and R e s u l t s  (MOLES/L)  Potential  FIGURE  o f Spent  Sulfite  Liquor  113  3 . 4 9 : R u n F 3 0 . 2 2 . F e r m e n t a t i o n w i t h P. f r e u d e n r e i c h i i . Yeast E x t r a c t Concentration as V a r i a b l e  + 9.0  ANAEROBIC  AEROBIC  44.  Y  43.  + 7.5  + 6.0  \/ Y /  G)0.6  —x--Jc'-\f/"x **  m  G3 3 . 3  x  \ Y  40.0  38.  Y  35.  Y  32.  Y  0.14  A  0.12  A  0.10  A  0.08  A  0.0G  x  0.04  x  0.02  x  •>  \ • 0.0$ 0.0  Y  Y -  Y  .  80.0  FERMENTATION  Experiments  _L  120.0 TIME  160.0  (HOURS)  and R e s u l t s  0.00 200 .0  x  Potential of Spent S u l f i t e  114  Run  Liquor  F20.00  20g/l of yeast extract powder O.Oppm of cobalt ion 4 g / l of potassium dihydrogen phosphate pH automatically controlled  LEGEND + x  _  Y  n  A  GROWTH, LACTIC SUGAR  DRY ACID  CELL  WEIGHT  CONCENTRATION  CONCENTRATION  VITAMIN VOLATILE  B12  (MOLES/L)  (G/L)  CONCENTRATION  ACIDS  (G/L)  (MG/L)  CONCENTRATION  Experiments and Results  (MOLES/L)  Potential  o f Spent S u l f i t e  115  Liquor  F I G U R E 3.50 : Run F 2 0 . 0 0 . F e r m e n t a t i o n w i t h P. f r e u d e n r e i c h i i . Cobalt I o n Concentration as V a r i a b l e  RER08IC  A"  44.  Y  43.  Y  38.  Y  35.  Y  32.  Y  0.14  A  0.12  A  0.10  A  0.08  A  0.06  X  0.04  X  0.02  X  0.00 200.0  X  Y —L  40.0  80.0  FERMENTATION  160.0  120.0 TIME  (HOURS)  Experiments and Results  116  Potential  of Spent  Sulfite  Liquor  Run F 2 0 . 0 9 2 0 g / l o f y e a s t e x t r a c t powder 0.9ppm o f c o b a l t i c n Ug/1 o f p o t a s s i u m d i h y d r o g e n p h o s p h a t e pH a u t o m a t i c a l l y c o n t r o l l e d  LEGEND  + x  Y  • „  A  GROWTH. LACTIC SUGAR  DRY ACID  CELL  WEIGHT  CONCENTRATION  CONCENTRATION  VITAMIN VOLATILE  B12  (MOLES/L)  (G/L)  CONCENTRATION  ACIDS  (G/L)  (MG/L)  CONCENTRATION  Experiments  and  Results  (MOLES/L)  Potential  of Spent  Sulfite  117  Liquor  F I G U R E 3.51 : R u n F 2 0 . 0 9 . F e r m e n t a t i o n w i t h P. f r e u d e n r e i c h i i . Cobalt I o n Concentration as V a r i a b l e  ANAEROBIC  9.0  AEROBIC  + 7.5  / 6.0  •  0.9  CD 0.6  Xf—'— Q  0.3  CD 0. 40.0  80.0  FERMENTATION  Experiments  120.0 TIME  160.0 (HOURS)  and R e s u l t s  44.  Y  41.  Y  38.  Y  35.  Y  32.  Y  0.14  A  0.12  A  0.10  A  0.08  A  0.06  X  0.04  X  0.02  X  0.00 200.0  X  118  Potential  o f Spent  Sulfite  Liquor  r Run F 2 0 . 2 2 . 1 2 6 2 0 g / l o f y e a s t e x t r a c t powder 2.2ppm o f c o b a l t i o n Ug/1 o f p o t a s s i u m d i h y d r o g e n phosphate pH a u t o m a t i c a l l y c o n t r o l l e d 0 . 1 2 6 g / l o f DMBZ  LEGEND + „  x  y n  „  A  GROWTH. LACTIC SUGAR  DRY ACID  CELL  WEIGHT  CONCENTRATION  CONCENTRATION  VITAMIN VOLATILE  812  (G/L)  (G/L)  CONCENTRATION  ACIDS  (M0LE5/L)  (MG/L)  CONCENTRATION  Experiments  and  Results  (MOLES/L)  Potential  o f Spent S u l f i t e  Liquor  FIGURE 3.52 : Run F 2 0 . 2 2 . 1 2 6 . F e r m e n t a t i o n o f SSL C o n t a i n i n g DMBZ w i t h P. f r e u d e n r e i c h i i  0.0  40.0  80.0  FERMENTATION  Experiments  120.0 TIME  160.0 (HOURS)  and Results  119  Potential of Spent S u l f i t e  Liquor  Run S20.22 20g/l of yeast extract powder 2.2ppm of cobalt ion 4g/l of potassium dihydrogen phosphate pH automatically controlled  LEGEND  + x  -  GROWTH. LACTIC  ACID  y  SUGAR  •  VITAMIN  A  DRY  CELL  WEIGHT  CONCENTRATION  CONCENTRATION  VOLATILE  B12  (G/L)  (G/L)  CONCENTRATION  ACIDS  (MOLES/L)  (MG/L)  CONCENTRATION  Experiments and  Results  (MOLES/L)  Potential  o f Spent  FIGURE 3.53 : Run S 2 0 . 2 2 .  Sulfite  Fermentation  Liquor  with  P.  121  shermanii  AEROBIC  44.  Y  41.  Y  38.  Y  35.  Y  32.  Y  0.14  A  0.12  A  0.10  A  0.08  A  0.06 X 0.04 X 0.02 X 40.0  80.0  FERMENTATION  Experiments  120.0 TIME  160.0  (HOURS)  and Results  0.00 X 200.0  Potential  F I G U R E 3.54 : L e g e n d  o f Spent  Sulfite  Liquor  f o rA l l Figures, Listed f r o m 3.39 t o 3.53  122  i n Table  III.23,  LEGEND  RCIDS —  CONCENTRATION  GROWTH.  +  X  LACTIC  DRY ACID  CELL  (MOLES/L)  WEIGHT  (G/L)  CONCENTRATION  (MOLES/L)  ( L A C T I C A C I D P R O D U C T I O N FOR F E R M E N T A T I O N S IN FLASKS. F I G U R E S 3 . 3 5 TO 3 . 3 8 )  +  pH  Y  SUGAR  CONCENTRATION  (G/L)  (SUGAR CONSUMPTION FOR F E R M E N T A T I O N S IN FLASKS. F I G U R E S 3 . 3 5 TO 3 . 3 8 ) —  Q  A  VITAMIN VOLATILE  B12  CONCENTRATION  ACIDS  (MG/L)  CONCENTRATION  ( V O L A T I L E ACIDS PRODUCTION F E R M E N T A T I O N S IN FLASKS. F I G U R E S 3 . 3 5 TO 3 . 3 8 )  Experiments  and  Results  FOR  (MOLES/L)  P o t e n t i a l  a l o n e .  For  v a r i e d kept  the  (5,  10,  20,  c o n s t a n t  at  and  c o n c e n t r a t i o n  was  2 0 g / l , only  y e a s t  and  were  yeast  One c o b a l t  i o n ,  P.  r e s u l t s  numbered  from  i n  Oppm  to  of  s u b s e r i e s was  kept  DHBZ  i s  f o r  was  phosphate ion  then  r a i s e d  c o n s t a n t  remained  3 . 1 .  was  c c b a l t  0.9ppm  was  f e r m e n t a t i o n s ,  paste  dihydrogen 3 . 3 ,  c o n c e n t r a t i o n  at  4 g / l .  l i s t e d  which  In  The under  the  the  at  a n a l y s e s  l a s t  c i t e d  used.  SSL,  with  2 0 g / l  u s u a l ,  with  4 g / l  e f f i c i e n c y  of  3.39  Appendix  consumption,  to  from  c o n c e n t r a t i o n c o n c e n t r a t i o n  s u b s e r i e s  vary  123  of of  yeast  e x t r a c t ,  phosphate,  was  2.2ppm  of  i n o c u l a t e d  s h e r m a n i i .  The  i n  as  c o b a l t  potassium  a d d i t i o n  appear  l i q u o r  e x t r a c t  c o n c e n t r a t i o n  e a r l y  of  and  and  to  with  e x t r a c t  batch  w h i l e  phosphate  The  i n c o m p l e t e ,  s u b s e r i e s  with  made  S u l f i t e  yeast  For  e x t r a c t  again  3.4.  3 0 g / l )  U g / 1 .  f e r m e n t a t i o n  s u b s e r i e s  3 . 2 ,  2.2ppm,  at  2.2ppm,  Spent  s u b s e r i e s  c o n c e n t r a t i o n  to  of  I  to  of  these  3.54  (see  and  are  a l l  f o u r  and  Table  i n  the  I I I . 2 3 ) .  p r o d u c t i o n s  p l o t t e d  s e r i e s  a g a i n s t  of  appear  Tables  ftl.32  Rates  v i t a m i n  of  the  time  F'igures  through  growth,  B12  f e r m e n t a t i o n  i n  A I . 4 6  of  sugar  and  a c i d s ,  and  in  Figures  3.60  3.70. A  I I I . 2 4  summary and  producing F i g u r e s  of  I I I . 2 5 . v o l a t i l e  3.55,  3.56,  the The a c i d s 3.57,  o v e r a l l  r e s u l t s  performance and and  Experiments  v i t a m i n  i s  of  i n  Tables  P r o p i o n i b a c t e r i a B12  3.58.  and  presented  R e s u l t s  are  summarized  i n i n  P o t e n t i a l  of  Spent  Table  S u l f i t e  Yeast  |  C o n c e n t r a t i o n  124  I I I . 2 4  SUMMARY OF OVERALL EFFECT OF YEAST EXTRACT  I  L i q u o r  RESULTS CONCENTRATION  E x t r a c t 5  (g/1)  10  20  30 _  | |  Sugar % of  1 -  Consumption I n i t i a l Sugar  9/1  |  _  _  i  67.6 25.0  72.4 21.0  72.6 27.0  71.4 21.0  | |  0.13 8.0  | |  |  V o l a t i l e  1 | |  Produced (moles/1) Dry C e l l Weight (g/1) Growth Bate ( g / l / h r )  0.14 7.0 0.10  0.13 6.5 0.10  0 . 14 8. 5 0.21  1 |  P r o d u c t i o n of v i t a m i n B12 (mg/1)  0.15  I  1.50  1.40  0.95  1.00  |  j j j |  Y i e l d o f Dry C e l l s -based on Sugar Used - b a s e d on Yeast E x t r a c t Added (g/g)  0.28  0.31  0.32  0.38  |  1.40  0.65  0.43  0.27  |  I |  Y i e l d of V i t a m i n - b a s e d on Sugar  0.060 0.214  0.067 0.209  0.035 0. 112  0.048 0.125  | |  0.0202 0.512  0.0192 0.536  0.0165 0.467  0.0163 C.558  | |  —  j  1 Used | - b a s e d  A c i d s  (mg/g) on Growth  j  Y i e l d  | j  Based on Growth E f f i c i e n c y  of  V o l a t i l e  Potassium  B12  (g/g)  1  (mg/g) A c i d s (moles/g)  Cobalt Ion C o n c e n t r a t i o n : 2 . 2 ppm Dihydrogen Phosphate C o n c e n t r a t i o n  Experiments  and  R e s u l t s  :  4  g/1  P o t e n t i a l  o f Spent  Table  S u l f i t e  L i q u o r  125  I I I . 2 5  SUMMARY OF OVERALL RESULTS E F F E C T OF COBALT ION CONCENTRATION -  1—  | |  .  I n i t i a l Cobalt I o n C o n c e n t r a t i o n (ppm)  1  0.00  0.90  2.20  2.20*  f , 1  |  Sugar  1 -  %  1 -  9 / 1  1  Consumption  o f I n i t i a l  Sugar  j | \ | | |  V o l a t i l e A c i d s Produced (moles/1) D r y C e l l Weight (g/1) Growth Rate ( g / l / h r ) P r o d u c t i o n o f Vitamin B 1 2 (mg/l)  | j  Y i e l d Based  | | j |  Y i e l d o f V i t a m i n B12 -based on Sugar Used (mg/g) - b a s e d on Growth (mg/g)  j | |  Y i e l d o f V o l a t i l e A c i d s Based on Growth (moles/g) E f f i c i e n c y  o f Dry C e l l s on Sugar Used  ( g / g )  73.6 27.6  66.4 23. 1  72.6 27. 1  64.5 18.0  | |  0.16 7.8 0. 156  0.15 8.5 0.133  0 . 14 8.5 0.053  0 . 13 6.9 0.087  | | |  0.40  1.30  0.95  1.00  |  0.283  0.368  0.315  0.383  |  0.014 0.051  0.056 0 . 152  0.035 0 . 112  0.056 0.145  | |  0.0200 0.509  0.0180 0.597  0.0165 0.467  0.0188 0.650  | |  Yeast E x t r a c t C o n c e n t r a t i c n : 2 0 g/1 Potassium Dihydrogen Phosphate C o n c e n t r a t i c n : *With  i  0 . 1 2 6 g / l  o f  DMBZ  added  Experiments  a n d R e s u l t s  4 g/1  126  Potential of Spent S u l f i t e  Liquor  LEGEND  CONCENTRATIONS OF : YEAST COBALT EXTRACT ION (G/L) (PPM) 5  n  05  2.2  10  A  10  2.2  20  •  20  2.2  30  +  30  2.2  ANAEROBIC —  AEROBIC  Experiments  STAGE  STAGE  and R e s u l t s  P o t e n t i a l o f Spent S u l f i t e FIGURE  3.55 : P r o d u c t i o n o f V i t a m i n E f f e c t o f Yeast E x t r a c t  1.60  1.40  L  1.20  L  Liguor  127  B12 by P. f r e u d e n r e i c h i i . Concentration  CD CM  1.00  CD  0.80  o  L  0.60  CJ Q O  CC Q_  0.40 L  0.20  0.0  L  0.  40.  80.  120.  360.  FERMENTATION TIME(HOURS) Experiments and R e s u l t s  200.  Potential of Spent S u l f i t e Liguor  128  LEGEND CONCENTRATIONS OF : COBALT YEAST ION EXTRACT (PPMJ (G/L) 0 X 0.9 K 2.2  •  DMBZ X PS Y  0.0  20  0.9  20  2.2  20  2.2  20  DMBZ ADDED  2.2  20  WITH  ANAEROBIC STAGE AEROBIC STAGE  Experiments  and R e s u l t s  PS  Potential  FIGURE 3.56 1.60  of Spent S u l f i t e  Liquor  129  P r o d u c t i o n o f V i t a m i n B12 by P. f r e u d e n r e i c h i i . E f f e c t of C o b a l t Ion C o n c e n t r a t i o n  130  Potential  of Spent S u l f i t e  Liquor  LEGEND  C O N C E N T R A T I O N S OF : YEAST COBALT EXTRACT ION (G/L) (PPM) 5 10  •  05  2.2  A  10  2.2  20  2.2  30  2.2  20 30  +  ANAEROBIC AEROBIC  Experiments  STAGE  STAGE  and  Results  Potential FIGURE  0.16  3.57  of  Spent  Sulfite  Liguor  131  P r o d u c t i o n o f V o l a t i l e A c i d s by P. freudenreichii. E f f e c t of Yeast E x t r a c t C o n c e n t r a t i o n  132  Potential of Spent S u l f i t e Liquor  LEGEND  0 X 0.9 K 2.2 •  D (vl B Z PS Y  CONCENTRATIONS OF : COBALT YEAST ION EXTRACT (PPM) (G/L) 20 0.0 20 0.9 20 2.2 20 DMBZ ADDED 2.2 20 WITH PS 2.2 ANAEROBIC STAGE AEROBIC STAGE  Experiments  and  Results  Potential FIGURE 3 . 5 8  Sulfite  133  Liquor  P r o d u c t i o n o f V o l a t i l e A c i d s b y P. f r e u d e n r e i c h i i . Effect of Cobalt Ion Concentration  0.16  -J  o f Spent  0.14  L  0.12  U  tn  LU  g  o.io  cr  ec _J o > Lu O  0.08  L  0.06  L  0.04  L  0.02  L  O  ZD  a o Q: a.  0.0 40.  80.  120.  160.  FERMENTflTION TIME(HOURS) Experiments and R e s u l t s  200.  Potential of Spent S u l f i t e Liquor FIGURE 3.59  : Legend for A l l Figures Numbered from 3.55 to  LEGEND C O N C E N T R A T I O N S OF : YEAST COBALT EXTRACT ION (G/L) (PPM)  •  05  2.2  10  10  2.2  20  20  2.2  30  2.2  5  30  +  C O N C E N T R A T I O N S OF : COBALT YEAST ION EXTRACT (PPM) (G/L) 0  X  0.0  20  0.9  W  0.9  20  •  2.2  20  X Y  2.2  20  DMBZ A D D E D  2.2  20  WITH  2.2 DMBZ PS  ANAEROBIC AEROBIC  STAGE  STAGE  Experiments  and R e s u l t s  PS  13U 3.58  135  Potential  of Spent  Sulfite  Run F P 1 . 1 20g/l of yeast extract 2.2ppm o f c o b a l t i o n 4g/l of potassium dihydrogen pH a u t o m a t i c a l l y c o n t r o l l e d  Liquor  phosphate  LEGEND  o +  EFFICIENCY GROWTH  RATE  (MG O F DRV C E L L 5 / L / H R J x  LACTIC RATE  ACID PRODUCTION (MMOLES/L/HR)  v  SUGAR CONSUMPTION (G/L/HR)  Experiments  and  RATE  Results  Potential of Spent S u l f i t e Liquor FIGURE 3.60  : Run FP1.1. Fermentation Rate of L . plantarium  Experiments  and R e s u l t s  136  37  P o t e n t i a l o f Spent  Sulfite  Liquor  Run F P 2 . 2 2 0 g / l o f y e a s t e x t r a c t powder 2.2ppm o f c o b a l t i o n 4g/l of potassium dihydrogen phosphate pH n o t c o n t r o l l e d  LEGEND  <»  EFFICIENCY  +  GROWTH (MG  OF  RATE  DRY  CELLS/L/HR)  X VARIATION  •  RATE CONCENTRATION  OF L A C T I C A C I D (MM0LE5/L/HR)  VITAMIN 812 PRODUCTION RATE (MICROGRAMS/L/HR) V O L A T I L E ACIDS PRODUCTION RATE (MMOLES/L/HR)  Experiments  and R e s u l t s  Potential  o f Spent  Sulfite  Liguor  138  F I G U R E 3 . 6 1 : R u n F P 2 . 2 . F e r m e n t a t i o n R a t e o f P. f r e u d e n r e i c h i i with L a c t i c Acid a s Main S u b s t r a t e  REROBIC  RNREROBIC  5.  4.  A  3.  A  2.  A  1. X  A  0.  X  X  X XI  • 3.0  - i .  X  -2.  X  2.0  30.0  +  • l.o  20.0  +  10.0  +  n  /  <> j l.o  /  <$> 4> <*> 0 <s>  ^  <»<!>«> A  <J>  .ft.  /  +  *  0.0  <3> 0.0 0.0  40.0  80.0  FERMENTRTI0N  120.0  TIME  160.0  (HOURS)  Experiments a n d R e s u l t s  200.0  +  Potential of Spent S u l f i t e Liquor  139  r  Run F05.22 05g/l of yeast extract powder 2.2ppm of cobalt ion Ug/1 of potassium dihydrogen phosphate pH automatically controlled  LEGEND <$• E F F I C I E N C Y  —  +  GROWTH (MG  y  • """""  RATE  O F DRY  SUGAR CONSUMPTION (G/L/HRJ  RATE  VITAMIN B12 PRODUCTION RATE (MICROGRAMS/L/HR) VOLATILE  A  CELLS/L/HR)  RATE  ACIDS  PRODUCTION  (MMOLES/L/HR)  Experiments  and Results  Potential  P.  o f Spent  Sulfite  mo  Liquor  F I G U R E 3.62 : Run F 0 5 . 2 2 . F e r m e n t a t i o n Rate o f f r e u d e n r e i c h i i . Yeast Extract Concentration as Variable  AEROBIC  ANAEROBIC  0.75  Y  0.50  Y  0.25  Y  0.00  Y  120.0  +  80.0  40.0  0.0  • o.o  20.0  60.0  100.0  FERMENTATION  Experiments  140.0 TIME  180.0  (HOURS)  and R e s u l t s  220.0  +  141  Potential of Spent S u l f i t e Liquor  r Run F10.22 10g/l of yeast extract powder 2.2ppm of cobalt ion 4 g / l of potassium dihydrogen phosphate pH automatically controlled  LEGEND  <>  EFFICIENCY  +  GROWTH  y  SUGAR CONSUMPTION (G/L/HRJ  • A  (MG  RATE  O F DRY  CELLS/L/HRJ RATE  VITAMIN B12 PRODUCTION RATE (MICROGRAMS/L/HRJ VOLATILE RATE  ACIDS  PRODUCTION  (MMOLES/L/HR)  Experiments  and R e s u l t s  Potential  P.  o f Spent  Sulfite  Liquor  142  F I G U R E 3.63 : R u n F 1 0 . 2 2 . F e r m e n t a t i o n Rate o f f r e u d e n r e i c h i i . Yeast Extract Concentration as Variable  AEROBIC  J  0.0  40.0  80.0  FERMENTATION  Experiments  120.0 TIME  150.0  (HOURS)  and R e s u l t s  200.0  40.0  +  0.0  +  143  Potential  o f Spent  Sulfite  Liguor  Run F20.22 20g/l of yeast extract powder 2.2ppm of cobalt ion 4 g / l of potassium dihydrogen phosphate pH automatically controlled  LEGEND <$> E F F I C I E N C Y — . — -  +  GROWTH (MG  -  RRTE  O F DRY  CELLS/L/HR)  Y SUGAR CONSUMPTION R A T E  •  (G/L/HR)  VITAMIN B l 2 PRODUCTION RATE (MICR0GRAM5/L/HR) VOLATILE RATE  ACIDS  PRODUCTION  (MMOLES/L/HR)  E x p e r i m e n t s and R e s u l t s  Potential  P.  o f Spent  Sulfite  144  Liquor  F I G U R E 3.64 : Run F 2 0 . 2 2 . F e r m e n t a t i o n Rate o f f r e u d e n r e i c h i i . Yeast Extract Concentration as Variable  RNRERGBIC >/  It  AEROBIC 1,  .+  0.75  8  J0.50  Y Y  0.25 0.00  J  160.0  +  J  120.0  +  80.0  +  40.0  40.0  80.0  FERMENTATION  Experiments  120.0 TIME  160.0 (HOURS)  and R e s u l t s  200.0  Potential of Spent S u l f i t e Liquor  145  Run F30.22 30g/l of yeast extract powder 2.2ppin of cobalt ion 4 g / l of potassium dihydrogen phosphate pH automatically controlled  LEGEND  EFFICIENCY — -  —  -  +  GROWTH (MG  y  • —  A  RRTE  O F DRY  CELLS/L/HRJ  SUGAR CONSUMPTION (G/L/HRJ  RATE  V I T A M I N B12 PRODUCTION RATE (MICROGRAMS/L/HR) VOLATILE RATE  ACIDS  PRODUCTION  (MMOLES/L/HR)  Experiments  and R e s u l t s  Potential  P.  o f Spent  Sulfite  Liquor  FIGURE 3.65 : Run F 3 0 . 2 2 . F e r m e n t a t i o n Rate o f f r e u d e n r e i c h i i . Yeast Extract Concentration as Variable  ANAEROBIC/ A -° 6  146  AEROBIC  A-  •  0.75 -J0.50 0.25 0.00 120.0  Y  Y  Y  +  80.0  40.0  0.0  +  o.o  0.0  40.0  80.0  FERMENTATION Experiments  120.0 TIME  160.0 (HOURS)  and R e s u l t s  200.0  +  Potential of Spent S u l f i t e Liquor  147 t  Run F20.00 20g/l of yeast extract powder O.Oppm of cobalt ion 4 g / l of potassium dihydrogen phosphate pH automatically controlled  LEGEND  <j> E F F I C I E N C Y  —  +  GROWTH  Y  SUGAR  •  (MG  RATE  O F DRY  CELLS/L/HRJ  CONSUMPTION  RATE  (6/L/HRJ VITAMIN 012 PRODUCTION RATE (MICR0GRAM5/L/HRJ VOLATILE RATE  ACIDS  PRODUCTION  (MMOLES/L/HR)  Experiments  and  Results  Potential  o f Spent  Sulfite  148  Liquor  F I G U R E 3.66 : R u n F 2 0 . 0 0 . F e r m e n t a t i o n R a t e o f P. f r e u d e n r e i c h i i . C o b a l t I o n C o n c e n t r a t i o n a s V a r i a b l e  REROBIC  «> o.o  o.o  40.0  80.0  120.0  FERMENTATION  Experiments  TIME  and  160.0  (HOURS)  Results  200.0  149  Potential  of  Spent S u l f i t e  Liguor  Run F 2 0 . 0 9 2 0 g / l o f y e a s t e x t r a c t powder 0.9ppm o f c o b a l t i o n 4g/l of potassium dihydrogen phosphate pH a u t o m a t i c a l l y c o n t r o l l e d  LEGEND o EFFICIENCY GROWTH RATE (MG OF DRY CELLS/L/HR) „ SUGAR CONSUMPTION RATE (G/L/HR) m VITAMIN 812 PRODUCTION RATE (MICROGRAMS/L/HR) . „ A VOLATILE ACIDS PRODUCTION RATE (MMOLES/L/HR) +  Y  Experiments  and  Results  Potential  P.  OO.O  o f Spent  Sulfite  150  Liquor  FIGURE 3.67 : Run F 2 0 . 0 9 . F e r m e n t a t i o n R a t e o f freudenreichii. Cobalt Ion Concentration as Variable  I  0 0  L_J  40.0  1  80.0  FERMENTATION  Experiments  120.0 TIME  160.0  (HOURS)  and R e s u l t s  1  1  200.0  151  Potential  o f Spent  Sulfite  Liguor  i  Run F 2 0 . 2 2 . 1 2 6 2 0 g / l o f y e a s t e x t r a c t powder 2.2ppm o f c o b a l t i o n Ug/1 o f p o t a s s i u m d i h y d r o g e n phosphate pH a u t o m a t i c a l l y c o n t r o l l e d 0 . 1 2 6 g / l o f DMBZ  LEGEND  <> j EFFICIENCY —  +  GROWTH (MG O F  RATE  DRY  CELLS/L/HRJ  —  Y SUGAR CONSUMPTION R A T E  — —  a VITAMIN B12 PRODUCTION RATE (MICROGRAMS/L/HRJ  (G/L/HRJ  A  VOLATILE RATE  ACIDS PRODUCTION (MMOLES/L/HRJ  Experiments  and R e s u l t s  Potential  o f Spent  Sulfite  Liguor  FIGURE 3.68 : Run F 2 0 . 2 2 . 1 2 6 . F e r m e n t a t i o n w i t h A d d i t i o n o f DMBZ  Rate  152 o f PF i n SSL  AEROBIC  40.0  80.0  FERMENTATION  Experiments  120.0 TIME  1 60.0 (HOURS)  and R e s u l t s  0.75  200.0  Y  Potential of Spent S u l f i t e  Liguor  1  Run S20.22 20g/l of yeast extract powder 2.2ppm of cobalt ion 4 g / l of potassium dihydrogen phosphate pH automatically controlled  LEGEND  o .  . +  Y  ,  •  EFFICIENCY GROWTH (MG  RATE  O F DRY  SUGAR  CELL5/L/HRJ  CONSUMPTION  (G/L/HRJ  RATE  V I T A M I N B12 P R O D U C T I O N RATE (MICROGRAMS/L/HRJ VOLATILE  RATE  ACIDS  PRODUCTION  (MMOLES/L/HR)  /  Experiments  and Results  1 I I | J  Potential  FIGURE  o f Spent  3 . 6 9 : Run S 2 0 . 2 2 .  Sulfite  Fermentation  Liguor  Rate  o f P. s h e r m a n i i  ANAEROBIC  A 5.0  154  AEROBIC  0.75  Y  A 4.0  0.50  Y  A 3-0  -| 0.25  Y  0.00  Y  320.0  +  80.0  +  40.0  +  ~Y_  A 2-0  .•A  A LO •  25.0  a  20.0  •  15.0  n  10.0  •  5.0  A  ......  O l.o  0.0  0 0.5 <j> 0 . 0 23.0  X  63.0  X  103.0  FERMENTATION  Experiments  X  143.0 TIME  183.0  (HOURS)  and R e s u l t s  223.0  +  Potential  GURE 3 . 7 0 : L e g e n d  o f Spent  Sulfite  f o r A l l Figures  Liguor  Numbered  from  3.60 t o 3.  LEGEND  <j> E F F I C I E N C Y  —  +  GROWTH  x  VARIATION RATE CONCENTRATION  y  SUGAR C O N S U M P T I O N (G/L/HR)  (MG  RATE  OF  DRY  CELL5/L/HRJ OF L A C T I C A C I D (MMQLES/L/HR) RATE  CD V I T A M I N  B12 PRODUCTION (MICROGRAMS/L/HRJ  A  VARIATION  ACIDS  RATE  OF  VOLATILE  CONCENTRATION  Experiments  and  RATE  (MMOLES/L/HR)  Results  Potential  o f Spent  Sulfite  Liquor  156  F I G U R E 3.71 : R e l a t i v e C a p a c i t y o f PS a n d P F i n F e r m e n t i n g SSL w i t h L a c t i c A c i d a s M a i n Substrate F S N  For Propionibacteria Freudenreichii For P r o p i o n i b a c t e r i a Shernianii F o r p. f r e u d e n r e i c h i i w i t h o u t S t e r i l i z a t i o n  J  I  0.0  I  10.0  YERST  Experiments  ADDED  and  Stages  !  20.0  EXTRACT  between  (G/L)  Results  0.02  P o t e n t i a l  3.8  Spent  S u l f i t e  L i q u o r  157  IERMENTATION_OF_AQUEOUS Table  done  i n  were  250ml  of  D i f c o was  I I I . 2 6  e x t r a c t  powder  n e a r l y  to  was  y e a s t  show  out/  potassium  added  the  the  the  the  was  and  In no  v a r i e d  and  the  t h i r d  0  value  ion to  s e r i e s  P.  i n  In one,  5 0 g / l .  o f  the  and  U0g/1  2  of  2.2ppm NB  c f  Here  again  R e s u l t s  These g l u c o s e , s e r i e s  c o b a l t  yeast  The  yeast  cf these  added.  same.  Experiments  f e r m e n t a t i o n s  of  s e r i e s , was  cf  f r e u d e n r e i c h i i .  s o l u t i o n s  used,  from  3  phosphate.  c o b a l t  n u t r i t i v e  c f  with  aqueous  dihydrogen  b r o t h .  used  r e s u l t s  f l a s k s  w i t h  powder  e x t r a c t  t h a t  l i s t s  Erlenmeyer  c a r r i e d  U g / l  of  of  ion  e x t r a c t  c o n c e n t r a t i o n  e x t r a c t  the  r e s u l t s  powders  was  P o t e n t i a l  of  Spent  Table FERMENTATION  OF  S u l f i t e  L i q u o r  158  I I I . 2 6  AQUEOUS  SOLUTIONS  OF  GLUCOSE  Glucose C o n c e n t r a t i o n : 4 0 g / l Potassium Dihydrogen Phosphate C o n c e n t r a t i o n : C o b a l t Ion C o n c e n t r a t i o n : Oppm i n S e r i e s 1 and 3,  4 g / l 2.2ppm  S e r i e s 2 D i f c o Y e a s t E x t r a c t Powder Used i n S e r i e s 1 and NB Y e a s t E x t r a c t P o w d e r ^ U s e d i n S e r i e s 3  j Dry  j  I i I | 1  1  | |  I  I C e l l  weight}  (9/1) „  1 ,. _  t  Sugar Residue C o n c e n t r a t i o n  | j  ,«  (g/i)  1  1  V i t a m i n B12 C o n c e n t r a t i o n  (mg/D  J j  s e r i e s  1 2 3 1 2 3  Y e a s t  E x t r a c t  C o n c e n t r a t i o n  0  20  30  40  0 0 0  5.62 5.62  7.17 6.60 7.42  9.39  44. 4 44. 4 44. 4  5.42 18.5 21.6 24.9  13.5 17.55 17.4 0.208  _  -  -  1  —  —  1  0 0 0  0. 0. 0.  •  V o l a t i l e A c i d s C o n c e n t r a t i o n (moles/1)  J | |  2 3  | |  L a c t i c A c i d C o n c e n t r a t i o n  | |  1 2  0 0  0.082 0.074  |  3  0  0.064  0. 620 0.090 0 . 107  (moles/1)  8.3 7.5 11.1  0. 176 0.228  | | |  |  9.28  0 0 0  139 102 101  0.202  0.  0.188  0. 195  0.  0 . 142 0. 093 0. 136  168  -  114 -  0.  116  j j |  1 2 3  0 0 0  0.221 0 . 176 0 . 165  0.309 0.248 0.275  317  j j  F i n a l  | j  1 2 3  6. 3 6. 3 6. 3  3.8 4.1  3.8 4.0  3.9  3.9  3.8  3.9  |  Experiments  and  6 .6 6.4 9 .15  0.149 0. 158  T o t a l A c i d s C o n c e n t r a t i o n (moles/1)  I  10 . 4 1 10 . 4 3 9 .97  0. 420 0. 370 0. 480  —  | | |  pH  (g/i) 50  -  1 2 3  2  R e s u l t s  -  0.304  -  0. 213 0. 203  0. 355 0. 296 0. 331 4. 0 4. 2 4. 0  i n  P o t e n t i a l  of  Spent  S u l f i t e  Chapter  L i g u o r  159  4  DISCUSSICHS  4.1  FERMENTATION S.  g r i s e u s  I I I . 2 1 of  and  seemed  i n  enjoy  Waksman's  No.3  over  T h i s  5 0 m l / l  s u l f u r and  nor  d i o x i d e  120ppm  Nishikawa almost  f r e e  capable  improve exceed  SSL  of  of  on  but  t o t a l  i s  of  On  i n  mixed  growth t h a t  l i m i t  medium  over  s i g n i f i c a n t l y of  due  a  when  2095. D i s c u s s i o n s  mixed  with  c o n c e n t r a t i o n growing. by  s u l f u r  and  s o l u t i o n  were  about  10  t r e a t e d  by  when  hand, and  SSL  supposed  t o t a l  to  growth  be  e i t h e r .  to  develop  by  a c c l i m a t i z a t i o n .  the  c o n c e n t r a t i o n  volume,  SSL  some  f r e e  t e s t e d  i n  by  Streptomyces  caused  Streptomyces  2095  lack  the  other  f a i l e d  the  stopped  not  SSL  (Tables  to  when  reached  Streptomyces  a l s o  was  109?  was  SSL  caused  SSL  d i l u t i o n ,  favor  SSL  a c c l i m a t i z a t i o n  i n  SSL  the  not  not  r a t h e r  p r e c i p i t a t i o n ,  these  r e p r o d u c t i o n  the  of  i n  Streptomyces  With  c o n c e n t r a t i o n s  did  i s  d e f i n i t e l y  l i g n o s u l f o n a t e .  and  grow  microorganisms.  the  s o l u t i o n ,  e f f e c t  l i g n i n ,  i s  sugar  when  r e s p e c t i v e l y .  of  It  of  not  problem  these  abundance  medium,  d i d  the  medium.  of  the  a b i l i t y  However, i n  However,  n e u t r a l i z a t i o n  The  SSL  by  o l i v a c e u s  the  i n h i b i t i n g  d i o x i d e  STREPTOMYCES  S.  e f f e c t  to  of  and  I I I . 2 2 ) .  n u t r i e n t s  i n h i b i t i n g  WITH  though  c o n c e n t r a t i o n  a  s t r a i n  of  i t  d i d  did  net  P o t e n t i a l  I t have  i s b e l i e v e d  been  much  more  a  d i f f e r e n c e s a l l o w e d  that  success  s l o w l y . i n  o f  these i f  That  i s  develop with  L i q u o r  a c c l i m a t i z i n g  i f  mixtures  160  experiments s t e p s  had been  and these  more  more  S u l f i t e  t h e i n t e r m e d i a t e  c o n c e n t r a t i o n  t o  c o n c e n t r a t i o n  Spent  h a d been  made  with  a c t i n o m y c e t e s  g e n e r a t i o n s  i n  would taken s m a l l e r  had  each  been  l e v e l  o f  t r a n s f e r s .  4.2 Z^iMllhlI^-RIl^-lis.^RLMMiM^ The r e v e a l  t h e c u r i o u s  (Figure based  f e r m e n t a t i o n s  3 . 2 9 ) ,  L.  pentosus,  o r i g i n a l l y  I,  o f  between  be used  a l  that a n d I.  McCoy's  Probably  [ 2 6 , 7 4 ] ,  a n d L.  a r a b i n o s u s ,  component, i n  stage  t h e i r x y l o s e ,  o f  t h e f e r m e n t a t i o n s a s  Bergey,  was  ferment  a n d  x y l o s e  was  under  with  group  a n dh i s  t h e name  i n  t h i s  c a l l e d  d i f f e r e n c e  x y l o s e .  pentose  was  C ,  Peterson  grouped t c ferment  n o t  ATCC-8014,  major  S S L  x y l o s e  deduction  s t r a i n  The  t h e 3091 o f o f  (by  was t h e s t r a i n  a r a b i n o s u s .  a b i l i t y  t o  1 7 - 5 ,  i t  p l a n t a r i u m  .  plantarium  that  pentosus  i n  known  L .  n o t ferment  t h i s  s t r a i n  L .  l i e s  does  fermentations)  used,  with  arabinose  c a l l e d  p l a n t a r i u m ,  f i r s t  ferment  ordered,  s t r a i n  x y l o s e  s t r a i n  d i s c o v e r e d  e t  F e r m e n t a t i o n s , the  t h i s  o f  have  p l a n t a r i u m ,  not  other  by h e r [ 3 , 5 3 ] ,  a l s o  major  o f  does  E l i z a b e t h  P e t e r s o n  the  L.  The from  L .  i t  was  s o l u t i o n s  that  t h e s t r a i n  a r a b i n o s u s  coworkers  L.  though  i t  [ 1 0 , 2 6 , 7 4 ] ,  L.  f a c t  on t h e r e s u l t s Then  o f  So  o f  then,  S S L could s t r a i n  o f  i n t e n d e d . done  t o convert  i n  two s t a g e s ,  sugar  ( i t would  D i s c u s s i o n s  with  L.  plantarium  have  been  b e t t e r  i n with  P o t e n t i a l  L-  pentosus,  l a c t i c to  as  a c i d ,  i t  can  then  convert  of  c o n v e r t  with  l a c t i c  Spent  P.  a c i d  S u l f i t e  x y l o s e  L i g u o r  as  w e l l  p r o p i o n i b a c t e r i a to  v o l a t i l e  i n  161  as  hexoses)  the  second  a c i d s ,  o f f e r  to stage  c e r t a i n  conveniences: 1.  L.  plantarium  P r o p i o n i b a c t e r i a . manner, s t a g e ,  or pH  remained pH  perhaps  should  might  be 3.  being  With  the  L a c t o b a c i l l u s  as  the  a c i d s  and  p o t e n t i a l  of  by  much  ( 1 . 6 g / l  ( e f f i c i e n c y P r o b a b l y , carbohydrate  compared  of  1  b u i l d  l a c t i c c e l l s  compared  source  a c i d i n  to  l i v e to  needed,  v i t a m i n  l a c t i c  a c i d  stage  would  a l l o w  r e d u c t i o n  more  a c i d ,  the  second t h a t  one  pH  s i n g l e  s u f f i c i e n t . l a c t i c  a c i d  v o l a t i l e  a c i d s  a  maximum cannot  c e l l s , on  so  fermented b e t t e r the  do  when  of  they  as  the  reproduce  as  do  l a c t i c there  e x t r a c t ,  and  a c i d s .  However,  not  D i s c u s s i o n s  s u g a r s ,  not  yeast  i n t r a c e l l u l a r  i n  p o l l u t i n g  e f f i c i e n c y use  y i e l d  by  s u g a r s .  i n s t e a d  though  a  of  r e d u c i n g  8 g / l ) ,  v o l a t i l e the  to  pentoses  P r o p i o n i b a c t e r i a  s o l e l y  B12,  e x t r a  to  be  than  c o a r s e r  show  and  probably  pH a  In  did  the  l a c t i c  b u i l d  abundance,  not  i n  s t a g e .  r e s u l t s  would  from  of  to  then  to  product.  P r o p i o n i b a c t e r i a  P r o p i o n i b a c t e r i a the  s o u r c e ,  the  then  g r e a t e r  w i t h  done  f i r s t  and  s t a g e ,  use  s e n s i t i v e  be  the  s t a g e s ,  f i r s t  the  U n f o r t u n a t e l y , carbohydrate  i s  e x t r a  a  may  and  a c i d s  an  second  v o l a t i l e  main  two  l e s s  i n  much,  c o n v e r s i o n  i n  SSL  a l l  c o n t r o l  v o l a t i l e  the  i n  at  vary  pH  recovered  1:1  not  between  B e s i d e s  much  adjustments  not  c o n s t a n t .  adjustment, 2.  pH  i s  harder  of  0.65).  a c i d  are  product  work  no  as  s u g a r s ,  convert being then  a  a l l  able  to  cannot  P o t e n t i a l  be  made  very  i n  low  q u a n t i t y .  p r o d u c t i o n  v i t a m i n  B12  (0. 135mg/g)  i t  S u l f i t e  i s  v i t a m i n  (based  compared  Spent  Hence,  of  y i e l d  f e r m e n t a t i o n s  of  to  with  on  dry  the  not  s u r p r i s i n g  to  d i s c o v e r  B12  (0.22mg/l  maximum).  c e l l  weight)  not  y i e l d  sugars  162  l i q u o r  as  c f  i s  the  low  same  n u t r i e n t  source  h o p e f u l l y ,  improve  a  The at  a l l  product  i n  (0.112  to  0.214mg/g). I. of  p l a n t a r i u m  v i t a m i n  B12  f e r m e n t a t i o n s e x t r a c t ,  an  e x t r a c t  F i g u r e  3.30).  4.3  gave  was  not  of  and  with  e x t r a  an  which  y e a s t  second  pursued  two  v i t a m i n  f o r  more  the  L.  with i s  s p e c i e s  observed  mixed  c u l t u r e  So,  But  these yeast  (the  be  had  y i e l d  more  s t a g e  would  s t a g e . f u r t h e r .  s t a g e s .  r e g u i r e d  e x t r a c t  any  very  t h e i r  main  p l a n t a r i u m .  Once  t h e i r  c a r b o h y d r a t e  (known  s t a r t  the  i n t e r e s t i n g to  p l a n t a r i u m  from  resume  a  appears  through  f r e u d e n r e i c h i i  (deduced L.  y i e l d  n u t r i e n t ,  to  f r e u d e n r e i c h i i  when P.  low  i n  the  optimum  about t c  4 5 g / l ,  be  t h i s  added  s e r i e s  of  FB^MENTATION_WITH_HIXE  i n d i v i d u a l be  f e r m e n t a t i o n s  Furthermore,  F e r m e n t a t i o n P.  t o ,  c o n c e n t r a t i o n  3.71)  experiments  chosen  with  e x p e n s i v e  y e a s t  (Figure  was  growth, s o u r c e .  to  p l a n t a r i u m but F i g u r e  very  they curve)  time  4.72  shows  l e a v i n g  using the  i s  P.  and  of  each  growth  3 . 4 3 ) .  s t o p  through,  t h i s  D i s c u s s i o n s  (Figure  growth  T h e i r  slower)  then  are  p l a n t a r i u m  the  d i s t i n c t .  grow  a c i d s  L.  because  p r o d u c t s  growing,  v o l a t i l e L.  be  of  At  can  f i r s t ,  not  ready,  t h e i r  growth  the  broth  to  f r e u d e n r e i c h i i  l a c t i c  c o e x i s t e n c e  a c i d of  as  these  Potential  FIGURE 4.72  two  that P.  Liquor  163  p l a n t a r i u m and P. f r e u d e n r e i c h i i (from Run FP3) ( s c a l e : \. \ * 12 m i c r o n s )  direct  (0.5mg/l) i s s t i l l  SSL  lower  than  SSL  by  p r o d u c t i o n , however, a l c n g  with  fermentations  freudenreichii  alone.  This  g r o w t h , i s much i m p r o v e d  successive stages  in  FP3.  p r o d u c t i o n o f v i t a m i n B12  of  total  Sulfite  : L.  s p e c i e s i n run The  of Spent  with  these  from  two  of  sugars  in  t h a t of f e r m e n t a t i o n s  species  (Figures  cf  3.43,  two 3.55,  3.56). Fermentations  of  f l a s k s gave r e s u l t s L. p l a n t a r i u m P.  medium,  where  turn a c i d i c , component,  similar  alone.  freudenreichii  the and  SSL  That  t o low  the  pH.  nature  final  with to  mixed those  fact  culture for  affirms  in  fermentations the  product  t h e growth was  had  medium volatile  has  less  acids  of g r e a t e r m a g n i t u d e  Discussions  with  sensitivity  However, i n r e i n f o r c e d o f the  Erlenmeyer  of  clostridial potential as than  a  to  major that in  P o t e n t i a l  f e r m e n t a t i o n s that  a  not  reproduce  4.4  too  and  to  growth  L.  of  P.  f r e u d e n r e i c h i i .  s l i g h t l y 3 . 5 3 ,  l o w e r ,  Table  (Figure  i n  3.7.1).,On  the  e x t r a c t  f o r  f r e u d e n r e i c h i i  e x p e r i m e n t s  4.5  Use, of No P.  (mean  matter  The  hexoses  the  7095  (Tables  s t r a i n  of  w e l l  i n  SSL  as  then  P.  be  deduced  f r e u d e n r e i c h i i  i s  to  out  of  sugars  c f  (Figure  f e r m e n t a t i o n s  c f  of  optimum  l i k e l y  P.  of  t h a t E12  3 . 3 1 ,  with  t c  v i t a m i n  the  i s  s i m i l a r  p r o d u c t i o n  i n  hand  to  l a c t i c  a c i d  reguirement  be  3 . 3 2 ) .  SSL  higher So  i s  i n  than  that  of  the  most  f r e u d e n r e i c h i i .  FREUDENREICHII  i t  of  d i d  the  L.  i n i t i a l  always  make  with  30%  a p p a r e n t l y  a l l o w s  f e r m e n t a t i o n s  (Figures  s t a n d a r d  p e n t o s e s ,  r e m a i n i n g  than  what  f r e u d e n r e i c h i i  that  can  p l a n t a r i u m .  t h e i r  s h e r m a n i i  f._  It  161  SHERMANII  other  c a r r i e d  pH  L.  s h e r m a n i i  as  a l o n e .  L i q u o r  ..-.Sugars  69.78,  deduce and  P.  FERMENTATION_ WITH  1-5*1  i s ,  f o r  were  P.  d i r e c t  A I . 4 6 ) ,  S u l f i t e  i n  with  However,  yeast P.  change  c o - e x i s t  WITH  Spent  p l a n t a r i u m  abrupt  FERMENTATION The  P.  with  of  up  70%  r e d u c i n g  of  as  o t h e r s u g a r s .  about  the  of  7091  3 . 1 8 ) .  If,  t o t a l  major  I I I . 2 4 ,  p l a n t a r i u m  ferment  up  d e v i a t i o n  x y l o s e 1.4,  use  c o n c e n t r a t i o n  r e d u c i n g of  i s  t h i s *  f a i r l y  sugar s a f e  r e d u c i n g  component,  sugar  to  s u g a r s ,  make  up  the  x y l o s e  but  I I I . 2 5 ) .  used  did  pentoses  not as  Presumably  a r a b i n o s e . D i s c u s s i o n s  i t  ferment can  t h i s  ferment  s t r a i n  more  ferments  P o t e n t i a l  4.5.2  Spent  s i g n i f i c a n t mass  f o r they  and  c o - a u t h o r s  v a r i a t i o n  i n  the  P r o p i o n i b a c t e r i a give  v i t a m i n  L i q u o r  B12  a  narrower  pH  of  P r o p i o n i b a c t e r i a  or  lower.  In  t h i s  The  e f f e c t  of  f o l l o w e d . t h i s  r e s u l t s low When  point  repeated.  pH  t h e  The  run  (Figure  pH.  In  165  pH  t h i s  [ 2 3 ]  show  to  7.5)  showed  that  the pH  growth  and  was  to  pH  5.5  to  not  7.  curves  a  7 . 5 ,  optimum  a t  t e s t  no c e l l  i n h i b i t i o n  o c c u r s  to  i s  and  f c r  pH  t h e  o b v i o u s l y  came  r e a d j u s t e d  there  b a c t e r i a l  between  was done  r u n ,  5,  product  i s  (6.25  that  of  f r e u d e n r e i c h i i  3.44)  was  growth,  c f  F15.22a  approached pH  the  Ferguson  work,  found  when  range  p r o d u c t i o n .  have  y i e l d  growth  pH.  [ 6 9 ]  u l t i m a t e  the  of  S u l f i t e  Ef|ect_Of_£H Neronova  but  of  of 4.9  e f f e c t  i n h i b i t i n g  c o n t r o l l e d ,  but  s t a n d s t i l l .  A t  T h i s  procedure  a r e  of  the  was same  rhythm. A  d e d u c t i o n ,  r e s u l t s  then  should  of  a l l  f e r m e n t a t i o n s  tubes  a r e  not  v a l i d  e f f e c t  of  almost  c e r t a i n  low  pH  l i m i t i n g as  a  be  (with  kept  as  a c i d s  besides  s o l u t i o n c o n s t a n t  a c i d - n e u t r a l i z i n g  I t  pH  i n  be  with  a  an  substance  f o r  D i s c u s s i o n s  t c  t h a t ,  and  t e s t  study as  i t  a s e p t i c a l l y ,  by  e f f e c t s  t h e  yeast  lack  The  of  e x t r a c t  the i s such of the a c t s  v a l u e .  these  as  a r e  that  a p p r o p r i a t e such  done  i n h i b i t i n g  they  noting  f l a s k s  means  f l a s k s  n u t r i t i v e  found  were  This  the  as  worth  c o n c l u s i o n :  Erlenmeyer  such  by  produced) i s  t h i s  experiments  a f f e c t e d  having  may  i n  s u b t r a t e s .  a d j u s t  much  s u b s t r a t e .  b u f f e r A  a r e  t o  done  these  l i m i t i n g  i m p o s s i b l e  experiments  i f  f o l l o w  f e r m e n t a t i o n s b u f f e r ,  l i m e s t o n e .  or  i f  pH  c a n  with  an  P o t e n t i a l  4 . 5 . 3  E f f e c t A i r ,  i s  of  d e t r i m e n t a l  a  has  the  e x p o n e n t i a l c l e a r l y  on  o t h e r  i n  t h e i r  s t a t i o n a r y  growth  as  (Figure  of  a i r  The i s  For very  a i r  to  of  to of  that  of  become a i r on  and  on  d r a s t i c a l l y .  B12 an i n  of  B12.  run  e x c e p t i o n the  h i g h .  T h i s  F15.22b to  t h i s  s t a t i o n a r y  T h i s  was  f e r m e n t a t i o n  of  the  sugar  i s  more  has  the  no  3.50, and  But,  e f f e c t  i n on  e f f e c t  l a t e  r e v e r s e  seen  are  i n  b e n e f i c i a l  marked  (Figures  3 . 5 2 ) .  taken  added  consumption  v i t a m i n  u s u a l l y  i s  It  a l s o  due  which  by  the  the e f f e c t  e f f e c t  of  3 . 5 1 ) .  a c i d s  p r o d u c t i o n  the  a i r  but to was  low  Table was  the  e f f e c t  of  a i r  i s  Sparging  a i r  i n t o  the  p r o d u c t i o n  of  a£  r a t e  r a i s e s  3.45,  as  phase,  the  b e n e f i c i a l .  shown  r u l e ,  B12,  r a i s e s  (Figure  probably b r o t h ,  a i r  has  i n t o  be  b a c t e r i a  n u t r i e n t s  b a c t e r i a ,  remarkably  v i t a m i n  v i t a m i n  i f  However,  can  3.49,  a i r  yet  w e l l  e f f e c t  the  r a t e s ,  not  phase. are  3.47,  then  l i m i t i n g  of  medium  of  growth  low  have  growth  when  at  growth.  f e r m e n t a t i o n B12  (Figures  added  by  they  b e n e f i c i a l  c e r t a i n  growth  p r o d u c t i o n  marked  T h i s  cf,  even  P r o p i o n i b a c t e r i a  runs i s  masked  about  the  i f  i f  Furthermore,  be  e f f e c t  s i m i l a r  c e r t a i n  i f  e x p o n e n t i a l  phase.  phase  phase  may  s u b s t r a t e s  e f f e c t  d e f i c i e n c y  3 . 4 8 ) .  e x p o n e n t i a l  166  l i g u o r  b r o t h ,  f r e u d e n r e i c h i i ,  growth  hand,  the  S u l f i t e  f e r m e n t a t i o n  developed  b e n e f i c i a l  q u i t e  the  P.  w e l l  a i r  the  i n t o to  a  Spent  A i r  mixed  e s t a b l i s h e d  of  the  u l t i m a t e  p r o d u c t i o n A I . 3 8 ) .  mixed  i n t o  p r o d u c t i o n  the never  D i s c u s s i o n s  d i s s o l v e d measured,  p r o d u c t i o n of  B a t c h the  of  v i t a m i n F20.22  batch  v i t a m i n oxygen  and  the  i s only  B12 i n  i s the  a g i t a t i o n  P o t e n t i a l  r a t e  might  oxygen  have  than  to  Foaming 2000cc/min. a i r  any  No  Again, on  and  o t h e r  e f f e c t  the  the  A g i t a t i o n any  fermentor. and  no  high  t h a t  as f o r a i r of  But  a l l  the  runs.  at  d i s t r i b u t i o n  flow cn  run  with  more  r a t e  the  t c  a  maximum  p o s s i b i l i t y  of  of  any  r a t e .  a i r  of  c o u l d  the  of  made  on  v i t a m i n  B12  p r o d u c t i o n  out  in  Erlenmeyer  l i t t l e  value  as  be  much  c a r r i e d  too  f e r m e n t a t i o n  a i r  but  they  are  present  i n  perhaps  not  were speed and  was  hand, of  to  mass  the  was  would  j u s t  cn  the  very  d i s p e r s e d of  a d j u s t e d  bottom  c o n s t a n t  e f f e c t  the  r a t e  i n t e r f a c e .  The  almost  e x p e c t e d , of  the  e f f e c t a n d / o r  of  of  h o p e f u l l y ,  d i s s o l u t i o n of  of  a i r  i n  m i c r o o r g a n i s m s ,  the  as i f  the  t h i s  only  at  was  kept  same  value  d i s s o l u t i o n c o n s t a n t  to  a g i t a t i o n  D i s c u s s i o n s  at  a g i t a t o r  almost  of  to  a g i t a t i o n ,  c o m p l e t e l y  the to  s u f f i c i e n t  v i g o r o u s  a g i t a t i o n  r o t a t i o n  r a t e  foods  l e v e l  r e q u i r e d  keep  of  i t  a  c e l l  then  l i q u i d - a i r  same  VolujSg..gf_,Fermentation_Broth  t h i s  t o  A g i t a t i o n  the  and  t i m e s ,  The  a g i t a t i o n  o t h e r  of  a d j u s t e d  C e l l s  a p p r o x i m a t e l y  there  A g i t a t i o n  p o s s i b l e ,  through  made  probably  beginning  was  times.  as  are  d e p o s i t i o n  l e v e l .  low  was  a i r  f e r m e n t a t i o n s  Thus  was  attempt  sampling  t h i s  end.  Ef f e e t_gf_  prevent  the  of  at  4 . 5 . 4  l i m i t e d  e f f e c t  s o l u t i o n  167  b a t c h .  marked  tubes  L i g u o r  supply  f l o w  means  t e s t  to  higher  t h r o u g h o u t .  a t  enough  S u l f i t e  at  a e r o b i c  enough  Spent  i n v e s t i g a t i o n  the  growth  f l a s k s  high  problems  i n h i b i t i n g  and  teen  of  generate  of  r a t e the  a l l  runs.  On  an  agent  f o r  was  not  any,  P o t e n t i a l  at  a l l  v a r i e d  i n i t i a l r a t h e r  o b s e r v a t i o n  f l a s k s  and  was  i n  Without v a r i a b l e  of t e s t  by  volume  the  of  The  was  a  run  to  than  above  a  are  Erlenmeyer e f f e c t s  i n t o  growth  run,  and  d i s c u s s e d  s t u d i e d .  no  It  s y s t e m a t i c  here  the  was  not  uniform,  p i c t u r e ,  but  with  r a t i o  to  i n  r e a f f i r m Erlenmeyer  high  of  hence  shaking  Furthermore,  exposed  the  more  a i r  so  growth  haphazard,  s u r f a c e  one  which  to  a i r  i n  may  be  over  the  b r o t h . of on in  the  f e r m e n t a t i o n  the  r e s u l t s .  Erlenmeyer reasons  to  then one  f e r m e n t a t i o n s  a l l  {Table these  nature p o i n t  n e u t r a l  l o w e r i n g  e f f e c t  and  e x p e c t i n g  e f f e c t s  the  broth  of  should  of  as  i s  of  the  a  be  much a c i d s  of  D i s c u s s i o n s  of  are  to  than  s o l u t i o n s  SSL  i n  combined  t o g e t h e r  t c  be  r e s u l t s  glucose  g l u c o s e  b u f f e r ,  c i t e d  to  complex,  compared  produced,  cause  growth  c o n s i d e r e d  b e t t e r  a  with  there  seems  good  the  n o t e ,  have  r e s u l t s .  s o l u t i o n s  medium  worthy  pH  tubes,  i n d i v i d u a l  However,  c a u s e s  a l s o  t e s t  the  not  f e r m e n t a t i o n s  erroneous  from  aqueous  SSL  i n  does  c o n c l u s i o n  drawn  I I I . 2 6 ) .  f o r  each  the  of  i t s e l f  However,  f l a s k s  f o r  s e p a r a t e  Another to  v a r i a b l e  f e r m e n t a t i o n s  the  u n f a v o r a b l e  tendency  from  e x t r e m e l y  f l a s k s  of  g l u c o s e .  the  c o n s i d e r e d ,  f o r  not  though,  was  c o n t r a d i c t o r y o b t a i n e d  i s ,  f l a s k s  few  the  It  growth.  volume  volumes  pH  broth  the  e f f e c t  If  the  of  168  tubes.  i n h i b i t e d  e x p l a i n e d  the  L i q u o r  r e s u l t s  s h a k i n g ,  Erlenmeyer  more  S u l f i t e  from  made.  e n t e r e d  d i s s o l v e d  d i r e c t  volume  widely  i n v a l i d i t y  s m a l l  Spent  i n v e s t i g a t e d .  The  the  of  with  with of i t s  c o u n t e r a c t with  i t s  P o t e n t i a l  a c i d i c  4.5.5  f f f ec t_of  yeast  i n i t i a l and  P.  i s  These  in  e x t r a c t  s h e r m a n i i  l i m i t i n g  of  i s  a  the  up  u l t i m a t e  c e l l s  mass.  any,  the  e x t r a c t the  as  other  e x t r a c t  e x t r a c t  the  s e r i e s  p i c t u r e : r a t e  of  i n t e r f a c e ,  and  a e r o b i c  be  second  of the  these  been  l e a s t  a  those an  very  P.  f r e u d e n r e i c h i i  i t  s t i l l  from  c l e a r  masked  with  the the  a  8 0 g / l .  i n  yeast  i n c r e a s e  e x t r a c t  by  be  f e r m e n t a t i o n s  r e s u l t s  yeast  and  an  the  can  of  i n c r e a s e  these of  to  c o n c e n t r a t i o n s , but  that  a c i d s  at  c o n c e n t r a t i c n  with  c l e a r l y  i n  the  cannct  be  as  l i m i t i n g  pH  l o w e r i n g  s i d e  e f f e c t  of  b u f f e r .  as  r e s u l t s to  a was  from  deduce  l i m i t i n g not  i n i t i a l  time An  when  the any  At  c o n t e n t  c o n c l u s i o n  cut  as  l e a s t of  oxygen  a i r  was cf  D i s c u s s i o n s  3  the  the  e x t r a c t  to  yeast  v a r i a b l e  oxygen  a i r  71  e f f e c t  v a r i a b l e s  i n t r o d u c e d yeast  i n  the  s o l e  other  from  on  Indeed,  d i s s o l v e d  of  i n c r e a s e  f e r m e n t a t i o n s  s u b s t r a t e .  s i n g l e d  experiments.  stage..  demonstrate  f o r  nature,  e f f e c t  of  d i s s o l u t i o n the  Above  b r i n g  have  used  c o n c e n t r a t i o n  those the  pH hand,  of  15g/l  c o n f i r m  The  to  and  U n f o r t u n a t e l y ,  would  a  cannot  yeast  to  p r o d u c t i o n  fermentors  i n  169  t i o n  seem  i n i t i a l  would  c o n c l u s i v e .  i f  of  On  i n  L i q u o r  s u b s t r a t e  together  seem  c o n c e n t r a t i o n  s u b s t r a t e ,  an  r e s u l t s  f l a s k s  as  4 g / l  d r a s t i c  to  e x t r a c t  regarded  tubes  l i m i t i n g  of  l e s s  tube  Erlenmeyer  e f f e c t  t e s t  r e s p e c t i v e l y .  s u b s t r a t e  t e s t  yeast  S u l f i t e  Yeasty Ex t r a c t _ C o n c e n t r a  c o n c e n t r a t i o n  e f f e c t  i n  Spent  c h a r a c t e r .  Fermentations that  of  i n  were SSL,  through  the  s t a r t  the  c o n c e n t r a t i o n  P o t e n t i a l  d i d  b r i n g  However,  about  t h i s  A l l  on  p r a c t i c a l more  T h i s the  e f f e c t  t h e  i s  l e s s  c o n c l u s i o n i n c r e a s e  j u s t i f y  o f  t h e  expensive  paragraphs  4 . 6  o f  u l t i m a t e c o s t  drawn  o f  an  mass  o f  o f  t h e  be  a r e o f  e x t r a c t  t h e i s  o f  only 5 g / l  s t u d i e d .  sense  that  n o t enough  amount  e x t r a c t  w i l l  r e s u l t s  c o n d i t i o n s i n  though.  I I I . 2 4 ) .  y e a s t  produced  yeast  B12 and a c i d s  mass  from  a d d i t i o n a l  o f  c e l l  c o n c e n t r a t i o n  t h e growth,  c e l l  170  (Table  t h e range  on  The e f f e c t  v i t a m i n  be  c o n c e n t r a t i o n  only  based  e i t h e r  yeast  i s  L i g u o r  t h e u l t i m a t e  c a n  w i t h i n  n u t r i e n t . o f  i n  optimum,  high  p r o d u c t i o n  which  i s , a  t h e  S u l f i t e  n o t c l e a r  e f f e c t  u s e . That  o r  Spent  i n c r e a s e  c o n c l u s i o n s  c o l l e c t e d  i s  an  o f  such  an  c o n c e n t r a t i o n  on  d i s c u s s e d  o f  t o  s h o r t l y  i n  and 4 . 7 .  i 4.5.6  E f f e c t _ o f _ C o b a l t Cobalt  i o n s  d i d have  P r o p i o n i b a c t e r i a . r a t e ,  i t  4 . 5 . 7  and  t o  i n h i b i t i n g t h i s  a f f e c t  e f f e c t  e f f e c t  on  was only  t h e u l t i m a t e  c e l l  t h e growth on  t h e  mass.  of  growth  See  a l s o  4 . 6 , 4 . 7 .  x  T h i s  p r e c u r s o r  d i d  improve  d i d improve t h e y i e l d  U n f o r t u n a t e l y  l i m i t e d  c e l l  an  Effect_of_5 6-dij§ethj[l_Benzimida  used.  of  C o n c e n t r a t i o n  However  d i d n o t seem  s e c t i o n s  Ion  r e s u l t s  t h e mass  broth t o  o f  these a  was s o  back  up  s i n g l e  o f  2ole  t h e growth c e l l s  p e r  c o n c l u s i o n s  r a t e unit were  r u n . The i n i t i a l  l o w that t h e b e l i e f  there o f  c o u l d  (Table o f  n o t  sugar  from  t h e  c o n c e n t r a t i o n  be an  t h e b e n e f i c i a l  D i s c u s s i o n s  r e d u c i n g  drawn  sugar  I I I . 2 5 )  i n c r e a s e  e f f e c t  o f  i n t h i s  P o t e n t i a l  p r e c u r s o r  4.6  on  the  PBODUCTION The  mixture works  by  u l t i m a t e  OF  ACIDS  v o l a t i l e  a c i d s  of  1 1 , 2 3 , 7 0 ] ) .  The  u l t i m a t e  of  a  e f f e c t  those  i n  e f f e c t e d  by  the  the  b r o t h , which  of  and may  v a r i a t i o n s F i g u r e  the  the  the  p r o d u c t i o n  upward). of  of  same  curve  inoculum.  c o i n c i d e n c e  may  5  have  However, t h a t  the  of  the  f a c t  An  i n c r e a s e  been  caused would  r e s u l t s  by have  were  D i s c u s s i o n s  net  more  of  l a g  seem  or  the  t h i s  c o b a l t  ion  the  the  because  i n  the  a l l  of own  c u r v e s  p o i n t s  yeast  phase  i n f a l l of  e x t r a c t  ( c o n s i d e r a b l y  v a r i a t i o n  i n  the  independence  i n  been  a l i g n e d  be  t h e i r  moderately the  to  b r c t h ,  of  over  s t a g e ,  with  a l l  a c i d s .  and  the  nature  slope  t h a t  the  cf  d i s s o l v e d  to  a f f i r m  1 0 g / l ,  i t  a i r  be  second  do  reduce  nor  v a r i a b l e s  3.58  d i d  a f f e c t e d  c e r t a i n d i d  2:1  p r e v i o u s  not  e x t r a c t  the  by  to  i n f l u e n c e  But  a  appears  o r i g i n a l l y  own  the  was  c o n c l u s i v e  of  be  c o n d i t i o n  a e r o b i c  oxygen  n o t ) .  to  in  yeast  of  t h e i r  v o l a t i l e  from  i n be  Figure  though,  i n c r e a s e T h i s  in  a c i d s  T h i s  to  checked  p r o d u c t i o n  not  furthermore,  of  added.  d i s s o l u t i o n  or  (as  v o l a t i l e  a c i d  of  c o n t r i b u t e d  c o n c e n t r a t i o n , an  amount  r a t e  and  on  with  s t a r t  a c i d s  v a r i a t i o n s  of  c o u l d  (measured  3.57,  the  r a t e  T h i s  the  have  by  assumed  f e r m e n t a t i o n  v a r i a t i o n s  i m p r e c i s e  u n c e r t a i n t y  of  any  171  >  were  a c e t i c  n u t r i e n t  The  c o n c e n t r a t i o n s . of  any  o t h e r s .  and  L i g u o r  FBEUDENBIICBII  produced  of  c o n c e n t r a t i o n  S u l f i t e  mass.  P.  p r o d u c t i o n  v a r i a t i o n  c a n c e l l i n g  Spent  c e l l  BY  p r o p i o n i c  £  the  of  from i n  1 0 g / l the  too  much  of  t h i s  manner.  age a The  P o t e n t i a l  inoculum  was  always  f e r m e n t a t i o n p o s s i b l e .  of  The u n i t  when  the  t a b l e s  of  r e l a t i v e  them  c o l l e c t e d  v a r i a t i o n d i d  show,  not  show  n e i t h e r  a c i d s of  per  the  amount glucose)  the  u n i t  sugar of  e x t r a c t i o n  l i m i t e d ,  the  does  u n c e r t a i n  a c i d s  o b t a i n e d  t c  more  t r e n d , of  over  the  the t r y i n g  was  high.  t o x i c  i o n s  data  thus  the  did  c o l l e c t i v e  a l l  w i t h  as  y i e l d  over  a They  improve  c a l c u l a t e d  produced  These  c o b a l t  the  i n  a  lower  c o n c e n t r a t i o n s .  DMBZ  measured  when  was  on  was  d e t e r i o r a t i o n ,  ion  of  It  as  that  f r e u d e n r e i c h i i  c o b a l t  not  the  age  I I I . 2 5 ) ,  However,  nor  consumed.  a c i d s  c o n v e r t e d  during  was  I I I . 2 U ,  cf  c l e a r  a d d i t i o n  v o l a t i l e  f r e u d e n r e i c h i i a c i d  the  e f f i c i e n c y of  P.  presence  and  r e l a t i v e  t h i n k  n u t r i e n t s  improvement  e x t r a c t  that  v o l a t i l e are  P. l a c t i c  The  amount of  of  yeast  though,  of  any  to  (Tables w i t h  same  to  f a s t e r .  p r o d u c t i v i t y .  d i d  e f f i c i e n c y .  a c i d s  172  i n t r o d u c e d  b a c t e r i a  medium,  the  lower  be  the  the  t h a t  have  to  to  gets  new  l i g u o r  l o g i c a l  a v a i l a b i l i t y  show  i n  i s  b a s i s ,  to  e f f i c i e n c y ,  i t  the  mass  prepared c l o s e  v o l a t i l e  c e l l  a l s o  causes  of  to  S u l f i t e  as  n u t r i e n t s  a d a p t i n g y i e l d  per  at  Furthermore, of  Spent  c a r e f u l l y  broth  a v a i l a b i l i t y period  of  cf as  the  r e d u c i n g  t h i s  v o l a t i l e the  r a t i o  t h e o r e t i c a l sugars  (as  a c i d s .  seems a e r o b i c not  i n c r e a s e  to  produce  s t a g e .  However,  a l l o w  a  i n  l a c t i c  the  D i s c u s s i o n s  a  f i r m  l i m i t e d the  c l a i m a c i d  amount  a c c u r a c y f o r  t h i s  c o n c e n t r a t i o n .  of of very  P o t e n t i a l  4.7  PRODUCTION F i g u r e s  r e f e r e n c e s The  OF  f o r  a i r  the  and  complete  f o r  The i n c r e a s e s  in  c o b a l t  up  a d d i t i o n a l any  yeast that  of  to  0.9ppm.  and  from  of  other  However,  An by  a  of  of  v i t a m i n  serve  as  v i t a m i n  E12  the  B12  n e c e s s i t y  to  the  true  not  i n c r e a s e  decrease  i n  r e v e r s e  c o b a l t i n  t h i s  d i f f e r e n c e  then to  ion the  to  more at  caused  the  seem  t c  and  i n  However,  or  t h i s  2 0 g / l  b e l i e v e d the  d i f f e r e d and  of  of then,  r a t e  of  s i g n i f i c a n t l y  the  l a t e  t h i s  s t a r t  batch  to  of have  the  f e r m e n t a t i o n  production  than  0.9ppm  of  of  broth  v i t a m i n  c o b a l t  i c n s  i s E12. to  a l l .  e x t r a c t  u l t i m a t e  i s  and  o t h e r s .  u l t i m a t e  adding  yeast  have  the  r a t e s ,  batch  T h i s  not  (with  It  with  upwards,  e f f e c t .  i o n ) .  1 0 g / l  did  F20.22  runs.  may  to  p r o d u c t i o n  run  oxygen  up  p r e c u r s o r  somehow,  of  the  the  of  n o t i c e a b l e i n  and  a  i n c r e a s e d  c o n c e n t r a t i o n s  b r o t h ,  o p t i m i z e  was  these  B12  e x t r a c t  the  c o b a l t  e f f e c t  v i t a m i n  with  compared  of  to the  broth  [ 2 3 , 7 0 , 7 9 ]  y e a s t  had  o n l y  stage  behaviour,  A d d i t i o n  the  to  second  of  even  d i s s o l v e d  a i r  of  on  2.2ppm  of  e s s e n t i a l  I I I . 2 5  p r o d u c t i o n  i n c o m p l e t e  n u t r i e n t  appeared  i n i t i a l  abnormal  of  e f f e c t  they  e x t r a c t  a e r o b i c  the  r e p o r t  From  of  d i s s o l u t i o n  the  the  production  amounts  those  I I I . 2 4 ,  d i s c u s s i o n .  c o n c e n t r a t i o n  e f f e c t  the  173  FRBUDBN^BICHII  Tables  a f f e c t e d  c o n v e r s i o n  c a s e s ,  r e v e r s e  a i r  c o n f i r m s  b e n e f i c i a l  c e r t a i n  and  P.  L i g u o r  one.  r a t e  i o n  of  S u l f i t e  BY  f o l l o w i n g  presence T h i s  have  3.56,  the  enormously.  Spent  VITAHIN,B12  3.55,  f o r  of  c o n c e n t r a t i o n  p r o d u c t i o n  D i s c u s s i o n s  of  was  v i t a m i n  accompanied B12.  It  was  P o t e n t i a l  a l s o  accompanied  based  both  t h e n ,  of  c e l l s  per  acids)  r e d u c i n g  the u n i t  w e l l  an  the  consumption of  of  used)  but  as  (in  i n  y i e l d i n g  t h e i r  l i f e  causes v i t a m i n  c o l l e c t i v e  174  c f and  n u t r i e n t s  t h e i r  sugar  L i g u o r  y i e l d  enjoy  B12. f o r  s p e c i e s ,  as  y i e l d  the  i n c r e a s e s ,  along  B12,  an  w i t h  b e n e f i c i a l  p r o d u c t i o n of  c e l l s )  the  with  of  P.  sugar)  and  i n  c e l l s  the  of  v i t a m i n on  (in  terms  of  (increased a  T h i s yeast  amounts  decrease B12  B12,  growth.  in  t h e i r  and  v o l a t i l e  i n  producing  e f f i c i e n c y  t h e i r  of  a d d i t i o n  e f f i c i e n c y  of  c o n c e n t r a t i o n f o r  0.045mg of P.  i c n no  DMBZ  every of  g i v e s  t h e i r  B12  2.2ppm  1mg  of  D i s c u s s i o n s  ion  v i t a m i n  t r u e  v i t a m i n  ( y i e l d  from  per  of  dry  when  the  0.9ppra.  reached  E12  the  u l t i m a t e  u n i t  has  T h i s  when  The  a l s o  the  produced,  used.  boost i n  of  decrease  c o b a l t  f r e u d e n r e i c h i i  consumed)  per  i s  b a c t e r i a l  e f f i c i e n c y  (yield  i s  product,  c o n c e n t r a t i o n .  of  a  p r o d u c t i o n  p r o d u c t i o n  2.2ppm.  reaches  c o b a l t  sugar  l o n g e r  reaches  v i t a m i n i o n  the  p a r t i c u l a r  of  the  p r o d u c t i v i t y  c o b a l t  A c t u a l l y ,  t h i s  i n  i s  f o r  i n t r a c e l l u l a r  c o b a l t  ion  ion  u n i t  f r e u d e n r e i c h i i ,  t o x i c  maximum  (per  i n c r e a s e of  c o b a l t  the  The  of  however,  Presumably, l e v e l .  metabolism  f a b r i c a t i n g  c o n c e n t r a t i o n  c o b a l t an  i n c r e a s e  of  need  v i t a m i n ,  of  e f f e c t ,  c o n c e n t r a t i o n  does  T h i s  needed  the  sugars  abundance  f r e u d e n r e i c h i i  v i t a m i n  a  i n  b a c t e r i a  probably  only  S u l f i t e  B12.  P.  u n i t  Spent  decrease  p r o d u c t i v i t y  as  v i t a m i n  a  that  helps  i n d i v i d u a l  of  on  c o n f i r m s  e x t r a c t )  by  of  to  the  p r o d u c t i v i t y  s y n t h e s i z i n g  vitamin  and E12.  P o t e n t i a l  4.8  N ^ T R I E Q The  to  SSL  c o b a l t  REQUIREMENTS  optimum  are  then  i o n .  of  amounts 5 g / l  Further  of  Spent  AND of  COSTS  r j  AND  yeast  a d d i t i o n  PROFIT  • N u t r i e n t  OF  FOB  n u t r i e n t s e x t r a c t of  the  Table COST  S u l f i t e  l i q u o r  175  FERMENTATIONS  and  p r e c u r s o r s  powder  and  expensive  WITH to  about  yeast  PF  be  -  added  1ppm  of  e x t r a c t  does  IV.27  FERMENTATIONS WITH FREUDENREICHII  PROPIONIBACTERIA  — or  i  Product  C o n c e n t r a t i o n  Assumed P r i c e  Value Eroth  (*/Kg)  (g/i)  i n  <*/l)  i | | |  Yeast E x t r a c t Powder Potassium Dihydrogen Phosphate  5  I | | |  C o b a l t C h l o r i d e C e l l s A c e t i c A c i d P r o p i o n i c A c i d  0.045 7.5 3.2 7.9  |  V i t a m i n  0.0015  60  4  B12  - 0 . 3  —  —  840  - 0 . 0 3 8 ~  —  20  0.064  33 800,000  0.261 1.2  i |  Net  •  Improvement  1.  l  187 —j  not  c o n t r i b u t e  p r o d u c t i v i t y  any  and  the  b e n e f i t  e f f i c i e n c y  encourage  t h i s  sugar  e f f i c i e n t l y .  more  s u f f i c i e n t nor  of  the  s p e c i e s  f o r  the  [23]  of  has  the of  However,  of  any  P.  b a c t e r i a  d e d u c t i o n  a f f i r m a t i o n  Ferguson  to  of  p r o c e s s .  to  and  produce  data optimum  t h a t  D i s c u s s i o n s  lowers DMBZ  economical  concluded  even  f r e u d e n r e i c h i i .  the the  It  more  c o l l e c t e d amount  to  the does  to  are be  use not added  improvement. t h i s  process  was  not  P o t e n t i a l  e c o n o m i c a l l y  f e a s i b l e  of  SSL)  d i l u t e d  cheaper, the  and  amount  e x o r b i t a n t process e x t r a c t  The  f e r m e n t a t i o n . e c o n o m i c a l l y used.  f o r  by  f e r m e n t a t i o n t i n y cost l i s t ,  c o b a l t  i t  of  c o b a l t  i s  a l s o  not  n e c e s s a r i l y  need  i t  to  c h l o r i d e  used  i s  p r o f i t a b l e .  I t  i s  f e a s i b i l i t y  of  the  p r i c e s  net  a t  what  was  was  SSL.  t h a t  the  o n l y  a  i s or  taken  from  t h i s  p r o c e s s rough  i s  and  p r o c e s s .  D i s c u s s i o n s  y e a s t  which  he  proved  to  p r o c e s s  i s  not  accounted  c o s t  cf  potassium  The  reason  behind  e s s e n t i a l ;  other  c o s t of  j u s t  of  needs  hand,  a the  p r i c e  phosphate.  about  immediately  t h i s  r e t a i l  c o n c l u s i o n  ready  of  was  Canlab  improvement  the  Ferguson  the  the  The  i n d u s t r i a l  t h i s  probably On  a  by  not  i t  cover  But  amount  guoted  by  of  5 0 g / l  t h a t  The  used.  to  the  which  0/1  enormous.  n u t r i e n t ,  i n c l u d e d .  a l l ,  b r i n g s  cf  added,  i n c l u d e d .  enough  then  l i t e r  mean  high  t a b l e ,  i s  was  p r o f i t a b l e  shows  c f  However,  reguirement,  a  n u t r i e n t  compared  p r o c e s s every  t h i s  e x t r a c t  term  s u b s t i t u t e d  over  IV.27  i o n  though,  that  probably  T h i s value  may  amount, of  i s  t h i s  of  phosphate,  e x c l u s i o n  In  SSL  when  from  i n  n i t r o g e n .  t h i s  a l l o w s  Table  f e a s i b l e . c o s t  yeast  be  of  to  t e n - f o l d  c e r t a i n l y  Ferguson,  dihydrogen t h i s  optimum  c o u l d  to  5 g / l  176  improvement  added  s o l e l y  to  L i g u o r  source  he  than  reduced  which  net  adequate,  more  low  S u l f i t e  e x t r a c t  e x t r a c t  Indeed,  The  yeast  a t t r i b u t e d  i s  5 g / l ,  were  yeast  Spent  n e g a t i v e  as  reduced  added  about  l e a s t  c o s t ,  i s  (a  u n l e s s  of  suggested. be  at  of  1iz  to  does  the not  c o m m e r c i a l l y  e s t i m a t e  of  the  P o t e n t i a l  4.9  of  these  i n c r e a s e  of  y e a s t  on  of  P  growth  v o l a t i l e then  S u l f i t e  i t  i s  a  177  L i q u o r  yeast  a c t  which  as  which  T h i s  pH  shows  of  t h a t  pH  of  hence  growing  s o l u t i o n s The  g l u c o s e ,  a d d i t i o n  even  on  the  a i r  f o r c e d  p r o d u c t i o n to  s u f f i c i e n t  c o b a l t  ion  f a c t o r s  to  i s the  the  i t s  masked  i n  using  2.2ppm of  v i t a m i n the  to  an  a e r o b i c  complete  to  of  the  second  used  v i t a m i n  B12.  D i s c u s s i o n s  to  pH  that  true s a i d  c f  v a l u e .  to  ion  broths  c l o s e  i n c r e a s e  by  to  a b i l i t y ,  the  of  a c i d i c g l u c o s e .  e x p e r i e n c e d i t  i n  adjustment. no  e f f e c t ,  c o n f i r m s  c o n v e r t  i s  growing  has  s t a g e ,  these  T h i s  pfi  a l l  n u t r i e n t  d i f f i c u l t y  f e r m e n t a t i o n  of  i s  the  s o l u t i o n s  T h i s  e f f e c t  was  broths  of  without  c o b a l t  be  t h i s  what  of  compared  B12.  i n  l i k e l y  the  f l a s k s of  If  n e u t r a l i z e d  great  SSL,  d i s s o l v e a l l o w  in  the  e x p l a i n s  a b i l i t y  or  t h a t  p r o d u c t i o n  to  e f f e c t  n u t r i t i v e  r e l a t i v e l y  when  of  to  to  P . f r e u d e n r e i c h i i of  b e n e f i c i a l  d i s t i n c t l y  the  E12.  magnitude  f i n a l  due  and  e x p l a i n s  v i t a m i n  t h e i r  f e r m e n t a t i o n  l e s s e n e d  a l s o  on  show  b e n e f i c i a l  a l l  not  up  as  a  however,  and  shows  had  s e r i e s ,  b u f f e r  SSL,  I I I . 2 6 )  c o n t r a d i c t o r y  The  the  i s  w e l l and  r a t h e r  c o n c e n t r a t i o n  was  nature  The  as  a c i d  t h i s  (Table  c o n c e n t r a t i o n  c h a p t e r .  of  d i s c r e p a n c i e s .  i n  l a c t i c  t h i s  experiments  4 . 0 ,  e x t r a c t  c o n c l u s i o n  i n  to  f e r m e n t a t i o n s  f r e u d e n r e i c h i i  a c i d s ,  e a r l i e r  not  Spent  FERMENTATION_OF_SO R e s u l t s  i n  of  by in  that s h a k i n g which  vitamin  not the i s the  E 1 2 - l i k e  P o t e n t i a l  of  Spent  S u l f i t e  Chapter  L i q u o r  178  5  CONCLUSIONS  SSL with  i s  P.  but  t h e s e not  s a t i s f a c t o r y  S.  g r i s e u s  Streptomyces  p l a n t a r i u m  thus  v i t a m i n  B12.  of  v i t a m i n  B12,  p o s s i b i l i t y  by  slow  P.  i n  of  but  for  and  cannot  f e r m e n t a t i o n s L.  grow  , p l a n t a r i u m .  i n  SSL.  a c c l i m a t i z a t i o n  B12.  s p e c i e s  i s  Growth p o s s i b l e  needed  as i s  s p e c i e s with an  yeast  p r e c u r s o r about  accompanied  by  i s  w i t h  r e q u i r e  T h i s  of  a  sugars  f e r m e n t a t i o n s  P r o p i o n i b a c t e r i a mixture  i n t e r e s t i n g y i e l d  r e d u c i n g  and  s t i l l  of  too  d i r e c t  of  the  l a c t i c  y i e l d  too  l i t t l e  L.  give  a  low  than  plantarium  b e t t e r to  and  p r o d u c t i o n  guarantee  f e r m e n t a t i o n s  of  any  SSL  with  a l o n e .  r e q u i r e s 5 g / l  by with  c o m p e t i t i o n  Experiments  more  subsequent  SSL,  the  s h e r m a n i i  v i t a m i n  use  the  are  f r e u d e n r e i c h i i  which  medium  s h e r m a n i i ,  o l i v a c e u s  SSL  Fermentations  f r e u d e n r e i c h i i  as  S.  i n  but  formed  P.  low  P.  can  P r o p i o n i b a c t e r i a ,  P.  and  s u i t a b l e  p r o m i s i n g .  L.  a c i d ,  and  f r e u d e n r e i c h i i ,  However, of  a  P.  cannot  e x t r a c t . for  n u t r i e n t s compete  the  a d d i t i o n One  optimum  The  p r o d u c t i o n  ppm  then of cf  0 . 14  C o n c l u s i o n s  P.  produce  e s s e n t i a l c o b a l t  of  m c l e s / 1  that  ion of  t h i s  n u t r i e n t i s  v o l a t i l e  as a l s o  vitamin  v i t a m i n of  l e s s  f r e u d e n r e i c h i i .  prove  production  p r o d u c t i o n of  and  with  f r e u d e n r e i c h i i  optimum  1.5mg/l. the  more  E12,  B12  i s  a c i d s .  P o t e n t i a l  With  these  very  e c o n o m i c a l l y A i r , P.  f r e u d e n r e i c h i i  the  to  a e r o b i c  and  the  s p e c i e s .  C o b a l t  i t s  of  f o r  s t a g e .  ion  a t  f o r  SSL.  not  r e d u c t i o n  sugars of  are  the  COD  BOD)  the COD  14%  only  t e s t ,  the  and  p r o c e s s  i s  P.  In  t h i s  an  i n  The  of  B12  B12,  growth and  amount  of t h e i r  amount  of i n  of  0.068mg one  l i t e r  improves  the  p l a n t  of  SSL  SSL  e f f l u e n t  i s  t e s t ,  r e l a t i v e  be  does  only So,  the  r e d u c i n g r e d u c t i o n  f o l l o w e d  The not  r e d u c t i o n  a  expected  where  best  sugars.  i s  sugars.  can  b a c t e r i a ,  these  C o n c l u s i o n s  b a c t e r i a l  optimum  F i s h  5 g / l  v i t a m i n  the  r e d u c i n g  SSL  .  over  t h i s  f e r m e n t a t i o n s , by  of  e x t r a c t .  COD  BOD  s m a l l  B12  of  v i t a m i n  a l l  of of  v i t a m i n  of  p o t e n t i a l  used  consumption  i s  p r e c u r s o r  by  such  p o t e n t i a l  of  of  yeast  t o t a l  but  reduce  f r e u d e n r e i c h i i .  the  s t a g e ,  p r o d u c t i v i t y  as  f o r  to  e x t r a c t  c a l c u l a t e d  p o l l u t i n g  of  of  f o r m a t i o n  2.2ppm.  DMBZ  d e t r i m e n t a l  f i r s t  to  1.5mg  components  probably  measure  of  i s  e f f i c i e n c y  t h e i r  caused  of  r a t e ,  yeast  the  the  of  of  f e r m e n t a t i o n s .  of  as  over  with  of  the  the  the  f o r  replacement  p o l l u t i n g  s i g n i f i c a n t  w e l l  SSL  about  o b s e r v a t i o n s the  i s  of  s t a r t s  p r o d u c t i o n  of  of  and  w e l l  a d d i t i o n  of  these  179  f e r m e n t a t i o n  p r o d u c t i o n  A d d i t i o n  needed  as  r e d u c t i o n  70%  from  i s  the  c o n c e n t r a t i o n  s u i t a b l e  The about  a  the  The  a  L i g u o r  a n a e r o b i c  e f f e c t  i o n  f e r m e n t a t i o n i s  t h i s  n o t i c e a b l e  needed  t o x i c  c o b a l t  needed  any  p r o d u c t i v i t y  e f f i c i e n c y , of  at  the  f r e u d e n r e i c h i i ,  1mg  S u l f i t e  requirements  i n  second  reduces  but  Spent  p r o m i s i n g .  i n t r o d u c e d  b e n e f i c i a l  P.  few  of  a c c u r a c y a l l o w i n  COD  by of any (or  B i b l i o g r a p h y  180  BIBLIOGRAPHY  [1]  A i a e r f e r , R. 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ABIPC, V o l . 34, No. 6, #3958, pp 52-96. ]  1~90  B i b l i o g r a p h y  LIST  Reference  C i t e d  OF OCCURENCES  OF  REFERENCES  i n  3 ^ 2 • 21• I' • • • [ 2 : I • • • •. . . . 1 . 1 . 3 [3; | . . . . 2. 1 , 4 . 2  •• • • • 1 • 1 •  n:  £ * : I • • • •. . . . I I . 6  [5;  I ••• •  [6n: | [8- | [9: | 10 | 11 •1 12 i i 13 •I 14 I 15" I 16 ) 17 | 18 J 19 ] 20 1 21 | 22 j i 23 |  II.6  •• • 2.4.3 .. . . 2.4.4 . . . .. . . . 1 . 1 . 3 . •.• 1.1.3 • • • •• •••1•3•1f 2•3•3. . . . .. . . . 2 . 4 . 4 , 4 . 6 .... ....2.4.5 • • •. . . . 1 . 1 . 2 • • •. . . . 1 . 1 . 1 • • •. . . . 2 . 4 . 3 • • •. . . . 2 . 4 . 3 • • • •. . . . 1 . 1 . 2 •** • 1.1.3 • • •. . . . 3 • • • • • ••* 1 • 1*3y2«2« 1 •• • • 1.3.3 ....1.1.3 • • . »• • * * 1 • 1 • 3 • 1 1. 3 • «22ff 2 « 2 « 1f2»3*3«2f2«4*3f2«^l«^4 r 6 3 • 2 • 4 • 5 • 24. f ,4.8 3 1 • • •. . . . 2 . 4 . 3 J • a • •. . . . 1 . 1 . 1 J ••• • 1.3.1,4.2 ]1 3 It ^ 2.4.4 J • • • *....1.1.2 j * • • •. . . . 1 . 3 . 2 J ••• * 1.3.3 j • • • •. . . . 1 . 4 ]1 . , .... 1.2 J • • • *....1.1.2 J • • • •. . . . 1 . 1 . 3 j • • • •. . . . 1 . 1 . 3 J ». • • • 1.1.2 1  1 1  1  l  l  J  24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 j * • • • . . . . I I . 4 39 J • • • • . . . . 2 . 2 . 1 40 i i . . . . . . . 2 . 4 . 4 41 1* . . . . . . . 1.2 9  #  #  M  #  M  t  Bibliography 42]* 43] 44 ] 45]* 46 ] 47] 48]* 49 ] 50] 51] 52 ] 53] 54] 55] 56] 57] 58] 59] 60] 61 ] 62] 63]* 64] 65] 66] 67] 68] 69] 70] 71 ] 72] 73] 74] 75] 76] 77^ 78] 79]* 80] 81 ] 82] 83] 84]* 85]* 86]* 87 ] 88] 89] 90]* 91] 92] 93] 94]  191  2.4.3 2.2.1 2.2. 1 .......1.1.3 1.1.3 ........1.4 1.2 1.1.1 .1.1.2 1.1.3 , 2.2. 1 ........4.2 ........2.2.1 ........2.2.1 ........2.2.1 ........2.2.1 2.4.4 1.1.3 1.2 .. 1.4 ........1.3.2 ..2.4.3 .1.1.1,1.1.3 1.1.3 1.1.3 2.4.4,11.3,11.5.3 ..II.3 ........4.5.2 1. 1. 1, 1. 1.3, 1.2, 1.3. 2,2. 2. 1, 2. 4. 3, 2.4. 4,4.6,11.6 2.2. 1 2.3.1 1.3.3 .1.3.1,4.2 ..1.3.3 1.7 *««««*«*2«3«3«2f3«2 1.3.2 1.2 1.1.3 1.1.2,1.3.2 . . . . . . . . 1 . 1.3 1.1.3 1.1.1 1.2 2.4.4 II.3 .1.1.3 ........1.1.3 2.2.1 ...II. 6 2.4.1 ..1.1.3 II.7  B i b l i o g r a p h y  [95] [96]* [97]* [98] [99] 100] 101 ] 102]* 103] 104] 105]* 106] 107] 108]* 109] 110] 111 ] 112] 113] 114]* 115] 116]*  *tiot  192  2.2.1 1.2 1. 1.2 ....1.1.3 ...... . . . 1 . 1 . 1,1. 1.3,2.2. 1 .... . . . . 1 . 1.1,2.4.5 .... II.4 ... ....2.4.4 . . ... 1.3.3,2.1 1.3.3 1. 1.2 .... ....1.1.3 .... ....1.1.2,2.2.1 ... 1.2 .... .... 1. 1.2 .... .... 1.1.1,1. 1.2,2.4.5 .........1.1.3 ....1.1.3 .... ....1.1.2 ... ....2.4.3 .... ....1.2 ... ....1.1 ... ...  c i t e d  anywhere  i d e n t i f i c a t i o n  i n  numbers  t e x t o f  b u t  which  r e l a t e d a r e  to  l i s t e d .  the  paragraphs  t h e  Appendix  I  :  Tabulated  APPENDIX Tabulated  A b b r e v i a t i o n s A i r  used r a t e  R e s u l t s  t h i s  appendix:  -  B  - V i t a m i n  B12  c o n c e n t r a t i o n ,  BR  -  V i t a m i n  B12  p r o d u c t i o n  C  -  Fermentation  2  1  not  2  2  s t a g e s ,  3  pH  unshaken,  -  Dry  E  -  E f f i c i e n c y .  c e l l  product  a c i d  shaken  r a t e  f o r  same  during  s t a r t i n g  shaken  p r o d u c t i o n  no  pH  weight  runs  sugar  GR  -  Growth  r a t e  L  -  L a c t i c  a c i d  s t a r t i n g  L a c t i c  a c i d  pH  7.  7. l a s t ,  a f t e r  s t e r i l i z a t i o n . a f t e r  s t e r i l i z a t i o n .  (g/1).  FP1.1  v o l a t i l e and  f o r (g  of  produced  ( l a c t i c  FP1.2).  t h e o r e t i c a l l y ( l a c t i c  a c i d s  a c i d  others) dry  obtained for  i s  runs  the  F P 2 . 1 ,  s u b s t r a t e F P 2 . 2 ,  and  consumed.  c e l l / l / h r ) .  c o n c e n t r a t i o n  consumption  i f  e x h a u s t i v e l y  by  (moles/1). -  .  E=a/b.  product  i n t e r e s t  pH  readjustment  o b t a i n e d -  r e d u c i n g  LC  (mg/l).  (micrograms/l/hr)  l a s t ,  during  readjustment  DCW  of  or  c o n d i t i o n s .  shaken,  s t a g e s ,  no  b:  (cc/min)  C o n d i t i o n s  0  a:  193  I  APR  C  flow  i n  R e s u l t s  (moles/1)  ether  e x t r a c t i o n  appendix  LCR  -  L a c t i c  a c i d  v a r i a t i o n  I  :  Tabulated  consumption  r a t e  of  -  L a c t i c  a c i d  p r o d u c t i o n  LR  -  L a c t i c  a c i d  p r o d u c t i o n  pH  -  F i n a l  r  -  R e i n f o r c e d  f o r  r a t e ,  i n  c o n c e n t r a t i o n  LP  pH  R e s u l t s  191  term  of  (mmoles/l/hr)  (moles/1), r a t e  f e r m e n t a t i o n s c l o s t r i d i a l  ( m m o l e s / l / h r ) . i n  f l a s k s  medium  used  as  f e r m e n t a t i o n  b r o t h , .  S  -  SC  Sugar  c o n c e n t r a t i o n  Sugar  consumption c o n c e n t r a t i o n  SR  -  Sugar  T  -  Fermentation  Ta  -  T o t a l  (g/1).  time  a c i d  (g/1)  v a r i a t i o n  r a t e  ( g / l / h r ) .  (hrs).  c o n c e n t r a t i o n  by  ether  e x t r a c t i o n  (moles/1). TI  -  TURB  -  V  -  Time  i n t e r v a l s  (hrs).  T u r b i d i t y V o l a t i l e  a c i d s  c o n c e n t r a t i o n  by  ether  e x t r a c t i o n  (moles/1). VP  -  V o l a t i l e  a c i d s  p r o d u c t i o n  VR  -  V o l a t i l e  a c i d s  p r o d u c t i o n  Y  -  Yeast  e x t r a c t  Ya  -  Y i e l d  of  a c i d s  YH  -  Y i e l d  of  dry  For  f e r m e n t a t i o n s  i d e n t i f i c a t i o n i n f o r m a t i o n . e x t r a c t  i n  ion  ppm  i n  The g / 1 . w i t h  number f i r s t The the  (moles/1) r a t e  c o n c e n t r a t i o n (moles/g  c e l l s  (g/g  ( m m o l e s / l / h r ) .  (g/1).  of  dry  c e l l s ) .  c f  reducing  i n  the  Hicroferm  of  the  run  number  second  i s  number  d e c i m a l  p o i n t  i s  the i s  the  71 used  sugar  f e r m e n t o r s , to  convey  c o n c e n t r a t i o n c o n c e n t r a t i c n  o m i t t e d .  The  consumed).  t h i r d  the  r e l a t e d  of  yeast  of  c o b a l t  number,  i f  Appendix  any,  i s  the  c o n c e n t r a t i o n  i d e n t i f i e s  the  inadequate  in  i n f o r m a t i o n  i s  obtained  I  from  :  of  b a c t e r i a l  Table  added  the  I I I . 2 3 .  195  The  used.  task  D e t a i l e d  R e s u l t s  DHBZ.  s p e c i e s  f u l f i l l i n g p r e s e n t e d .  T a b u l a t e d  s t a r t i n g  When  j u s t  i n f o r m a t i o n  t h i s  number  d e s c r i b e d , of  any  l e t t e r  run  i s f u l l  can  be  Appendix  I : Tabulated  Table FERMENTATION f™ 1 x t1 . I1 o r | 10 I 10 | 20 | 20 I 25 | 25 | 40 I 50 | 50 I is I 75 | 100  DCW 0.49 0.0 0. 14 0.39 1.00 0.83 1.30 1. 18 1.77 1.83 1.67 2.00 1.65 1.81  LP  VP  pH  7. 1 0.0 4.2 4.8 15.6 8.7  0.063 0.0 0.028 0.054 0. 119 0.089  0.039 0.0 0.0 0.007 0.004 0.007  19.4 23*4 26.7 24.0 27.0 21.3 24.0  0. 140 0. 158 0.175 0. 167 0. 151 0. 160 0. 133  0.035 0.017 0.027 0.029 0.025 0.028 0.034  Table FERMENTATION , Y r r 0 10 20 ; 20 20 25 40 50 75  , DCW  AI.28  OF SSL WITH L. PLANTARIOM  SC  196  Results  3.4 5.0 6.2 5.5 3. 1 4.9 3.0 2.8 3.7 3.8 2.9 3.6 3.4 3.8  IN ERLENMEYER  T  C  109 109 144 144 144 144 109 109 144 144 109 109 109 109  3 3 1 1 1 1 3 3 1 1 3 3 3 3  E  FLASKS i  YA  YW  | .j || | | I | | | | | | | j  1.300  0.210  0.600 1.140 0.710 0.994  0.2C0 0. 156 0. 123 0. 116  ,, , . , , 0.069 — 0.033 0.081 0.064 0.095  0.815 0.674 0.682 0.736 0.587 0.795 0.627  0. 149 0.099 0. 110 0. 117 0.088 0. 114 0.092  0.061 0.076 0.069 0.070 0.074 0.077 0.075  —  '  —  AI.29  OF SSL WITH P. FREUDENREICHII IN ERLENMEYER .  FLASKS  , SC  LP  VP  B  pH  T  7. 1 -.026 0.055 0.045 5. 0 156 2.46 1.76 7. 1 -.035 0.053 0.020 4. 1 156 0.0 0.0 0.0 0.0 0.0 4. 8 156 — 8. 1 0.050 0.020 0.37 4. 5 168 — 0.52 13.2 0.066 0.015 4. 7 168 — 0.47 10.5 0.073 0.016 4. 6 168 — 0.48 11.8 0.071 0.029 4. 9 137 0.68 4. 9 156 — 0.47 18.3 0.080 0.026 4. 9 168 0.84 18.9 0.058 0.034 — 5. 3 168 0.58 156  -  C 2 3 3 0 1 0 0 3 0 0 3  E  ^ YA  YW  0. 383 0.012 0.349 0.243 0.011 0.250 —  0.778 0. 580 0.764 0. 763  —  0. 192 0. 163 0. 189 0. 210 — 0.080 0. 522 0.225 0.438 0. 109  —  0.045 0.039 0.045 0.040 —  0.026 0.044  Appendix  I  :  T a b u l a t e d  Table FERMENTATION 1 1  *  | 1 |  r r 10  1.93 1.30 0.41  | | |  10 20 20 30  0.32 0.21 0 . 15 0.68 0.77 0.24  I  | | |  ECW  40 50 100  0.62  OF  SC 7. 1 7. 1 ' 6. 3 7. 8 —  —•  6. 0 9. 6 10. 8 —  SSL  WITH  LP  . . . .  0 0 0 0  3 3 4 6  x  5 6 5 6  SHERMANII  0.025 0.022 0.032 0.014 0.019 0.024 0.034  pH  0.038 0.020  0.055 0.071 0.019  — — — — .  —  r—  Y  OF  — —  SC  LP  VP 0.030  r r 0  0 . 53 0. 80 0. 0  7. 1 7. 1 0.0  0.003 - . 0 0 1  10 10 20  0. 98 0 . 14 0 . 52 1. 47  9.6  0.091 0.009 0.046  0.058 0.0 0.027 0.017 0.016  16.5 18. 1  0.131 0.133  0.017 0.009  19.2  0. 163 0.046  0.023 0.017 0.047  20 25 25 30 40 50 50 75 75 100  1. 32 1. 44 0. 75 1. 89 1. 88 1. 74 1. 93 1 . 96 2. 05  — —  —  27.0 26.1 22.4 22.0  0.0  0 . 199 0.201  21.6  0. 142 0 . 161 0. 165  22.2  0.115  B  :  ——  — — — —  —• — — —  6 0 3 8 7 4  pH  E  FLASKS  YA  ~1  YW  156 156 120  2 3 1  0.421 0. 230 0.629  0. 017 0. 014 0 . 107  0.274 0. 184 0.065  120 120 120 120  0 1 0 0  0.  0. 168 0 . 162 0. 304 0. 073  0.040  120 120  0 0  0. 516 0.576  120  0  612  — —  0.736  —  0. 072 0. 294 0 . 161  H  I  —  —  0. 113 0.080 0.022 —.  •  L. PLANTARIUM FLASKS  2 3  3. 8 6. 5 5. 9  156 156 156 120 120 120  3. 7 3. 0  120 156  2. 8 6. 5 3. 3 3. 4  156 120  0 2 3  120 120  0 0  0. 821 0.859  0 . 130 0 . 133  0.070 0.072  156 156  0 0  0. 743 0. 823  0 . 106 0 . 104  0.078 0.088  156  0 0  0.  156  0.048  — —  0.030 0. 109  —•  3. 3 3. 8 3. 4  —  4. 1  2 1 0 1  0  YA  YW  C  3. 8 3. 9 4. 7  E  AND  T  0.040 0.040  —  C  C U L T U R E OF ERLENMEYER  0.020 —  . . . . . .  ERLENMEYER  AI.31  S S L WITH A MIXED FREUDENREICHII IN  :  DCW  4 4 4 4 6 6  IN  T  6.3 6.3 6.1 6.3  —  Table FEBMENTATION  197  AI.30  B  VP  - . 0 2 2 - . 0 5 3 0.025 0.028 0.012 0.013 0 0 0 0  P.  R e s u l t s  0. 0.  421 728  0. 063 0. 072  0.074 0. 113  1.  100  0 . 120 0 . 186 0 . 119  0.  — —  0. 808 0.707 0. 873 —  813  0.909  0 . 100 0 . 108 0 . 128 0. 084  P.  -»  102  — —  0.089 0.073 0.075 —  0 . 100  0.091  0 . 109  0.092 1  appendix  I  :  T a b u l a t e d  Table  1  T  1 1 I  1  I I ! |  I I t  1 2 3 4 5  6 7 8 | 9 | 10 111 |12 | 13 |14 |15  TURB  0.45 0.44  0.0 0.011 0.015 0.009 0.0 0.022 0.516 0.602 0.796 0.770 0. 839 0.839 0.824 0.839 0.839  24.0 23.1 19.8 14.4  ****  59 67 72 79 83 95 107 110  9.6 8.1 7.5 4.0 4.5 4.8 4.8  ****  T  |  4 5  1 1  6 7 8  t |  9 10  1 | J  11 12 13  I I  DCH  26 32 43 56  2 3  I  S 39.6 25.5  |  I  F P 1 . 1 .  0 8 19  t '• •  I  RUN  14 15  0.36 0.37 0.41 0.38 0.91 1.22 1.37 1.52 1.50 1.59 1.39 1.49 1.60  8  14. 1  19 26  **** **** ****  32 43 56 59  *****  - 0 . 0 8 3 - 0 . 1 5 0  19.8 25.2 30.0  - 0 . 3 0 0 - 0 . 4 1 5 - 1 . 6 0 0  67 72  31.5  79 83 95 107  35.6 35.1 34.8  - 0 . 1 8 8 - 0 . 1 2 0 - 0 . 5 0 0 0.125 0.025  110  ****  ****  34.8  0.0  *****  L .  PLANTARIUM  B  V  L  0.195 0.146 0. 151  0.099 0.046 0 . 101  0.095 0.100 0.050  0.161 0.233 0.230 0.325  0.107 0.091 0.066 0.099  0.054  0. 0. 0. 0.  0. 0. 0. 0.  ***** *****  ***** *****  *****  *****  00 16 53 41  0.346  SR - 1 . 7 6 2  WITH  TA  3 3 3 3  198  AI.32  FERMENTATION  SC  R e s u l t s  036 100 107 111  0.013  E 0.03  **** **** ****  ***** ***** 0. 142 0.164 0.226  *****  0.264 0.216  0.246 0.230 0.333  **** **** **** **** **** **** **** **** **** **** **** **** **** **** ****  GR  LR  - . 0 0 0 6  0.625 - 4 . 5 4 5  - . 0 0 7 3 0.0007 0.0067  - 0 . 1 9 0 . 17  - . 0 0 2 3 0.0404  0.21 0.37  0.1033  ***** ***** 0. 167 6.769 7.333  0.0188 0.0300  7.750  0.43 0.31 0.39  - . 0 0 2 9 0.0225 - . 0 1 6 7  3. 167  0.35  0.0088 0.0367  ****  ****  *****  ***** 2.500  - 1 . 3 3 3 34.333  2 0 g / l o f Yeast E x t r a c t 2.2ppm o f C o b a l t Ions 4 g / l  o f  Potassium  Dihydrogen  Phosphate  pH 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0  BR  ***** ***** ***** ***** ***** ***** ***** ***** ***** ***** ***** ***** ***** *****  AFR  •H  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  TI 8 11 7 6 11 13 3 8 5 7 4 12 12 3  Appendix  I  :  Tabulated.  Table RUN  S  T 1 2 3 4 5 6 7 8 9  r I ir •--  0 30 45  23. 1  **** **** **** **** **** **** ****  48 53 60 66 73 107  8.5  T 2 3 4 5 6 7 8  30 45 48 53 60 66 73  9  107  F P 1 . 2 .  TURB  0.17 0.07  0.0 0. 0 0.456  0.92 1.57 1.25 1.55 1.56 1.53 1.47  SC  AI.33  FERMENTATION  DCW  0.721 0.602 0.886 0.824 0.839 0.839  SR  199  R e s u l t s  WITH  ***** ***** ***** 0. 190 0.258 0.245  *****  PLANTARIUM  V  L  ***** ***** ***** *****  0.075  Tft 0.125  L .  B  ***** ***** *****  0.050  0. 140  0.075  0. 175  0.258  E  **** **** **** **** **** **** ****  ****** ****** ****** ****** ****** ****** ******  **** **** **** **** **** **** ****  14.5  - 0 . 1 3 6  0.61  ***** *****  0.084  **** **** **** **** **** **** **** **** ****  ***** *****  0. 174  GR - . 0 0 3 5 0.0569 0.2173 - . 0 6 5 0 0.0436 0.0007 - . 0 0 4 7 - . 0 0 1 7  2 0 g / l o f Y e a s t E x t r a c t 2.2ppm o f C o b a l t Ions 4 g / l c f P o t a s s i u m D i h j d r c g e n  LR  ***** ***** ***** 1.226  ***** 2.692  *****  - 0 . 0 2 4  Phosphate  pH  AFR  7. 1 7. 1 4. 6 7. 4 6. 1 4. 6 7. 4 7. 3 7. 4  BR  ***** ***** ***** ***** ***** ***** ***** *****  0 0 0 0 0 0 0 0 0  TI 30 15 3 5 7 6 7 34  I J  Appendix  I  :  T a b u l a t e d  Table RON  F P 2 . 1 .  I  T  1 r 1  I  |  I I I I I I  7 8  9 |10  S  0 16 33  1 2 3 4 5 6  FERMENTATION  3. 1 3. 1 3. 1 3. 4 3. 4  144  58 68 82 116 155 178  2. 3. 3. 3. 3.  7 4 4 4 4  TURB  1 . 19 1.41 1.56  0.0 0 . 114  1.72 2 . 10 2.38 2.58 2.41 2.26 2.79  AI.34  WITH P. FREUDENREICHII MAIN SUBSTRATE  DCW  0 . 161 0. 229 0.310 0. 347 1.398 0.357 0.347 0. 481  0. 192 0. 188 0.184 0.202 0.244 .256 .214 .287 .237 .279  ON  LACTIC  V  L  B  0. 037 0. 039 0. 056 0. 068 0. 125 0 . 132 0. 100  0. 155 0 . 149 0. 128 0 . 134 0 . 119 0 . 124 0.114  0.01  0 . 152 0 . 122 0 . 129  0. 135 0. 115 0. 150  TA  0 0 0 0 0  200  R e s u l t s  0.01 0.03 0.04 0 0 0 0 0 0  .05 .06 .06 .07 .06 .C7  ACID  AS  AFR  PH 7.0 7.0 7.0 7.0 7.0  0 0  7.0 7.0 7.0 7.0 7.0  0 0  0 0 0  0 2000 2000  i i——  T  1 rI ,-• -  2 3 4  16 33 44  5 6 7  58  8 9 10  LC  LCR - 0 . 3 7 5 - 1 . 2 3 5  0.33 0.70  .021 .036 .031  1.48 2.44  82 116 155  .040  178  .005  1.522  .041 .020  VR  GR  .006 .027  0.545 - 1 . 0 7 1 0.500 - 0 . 7 1 4 0.618 - 0 . 5 1 3  68  E 0 0 0 0  3.06 1.54  . . . .  0 0 0 0  1 0 1 2  41 88 41 71  0.028 0.013 - . 0 0 4 - . 0 0 4  5.75 2 . 12 18.40  5 9 9 0  0.0233  BR  0 . 125 1. 000 1. 091 4. 071 0. 700 - 2 . 1. - 0 . 0.  TI  0.250 0.882 1.273 1.071 0.200  286 529 769 304  0.143 0.265 -• 0 . 2 5 6 0.739  with  20g  16 17 11 14 10 14 34 39 23  i  Inoculum : P. f r e u d e n r e i c h i i F e r m e n t a t i o n medium : F i n a l b r o t h e x t r a c t added per l i t e r A c i d s  c o n c e n t r a t i o n s determined  by  of  run  manual  FP1.1  e t h e r  of  e x t r a c t i o n  y e a s t  Appendix  I  :  T a b u l a t e d  Table RUN  FP2.2.'  FERMENTATION  S  T 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15  0 9  5.5  *** *** *** *** ***  17 25 33 41 49 58  4.6  ***  *** ***,  65 73 82 97 137 174  *** *** 5.5 5.1 4.8  197  TURB  1.40 1.40  0.0 0.022 0.004 0.066 0.086  0. 161 1.90 2 . 17 0 . 2 0 8 2.37 0.260 2.51 0.276 0. 314 2.65 2.70 0.310 2.69 0. 310 2.53 0.310 2.82 0.310 2.98 0.310  201  AI.35  WITH P . FREUDENREICHII MAIN SUBSTRATE  DCW  1.41 1.48 1.67  R e s u l t s  TA  V  0.285  0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0.  0.284  *****  ***** 0.300  ***** ***** ***** ***** ***** ***** ***** 0.290  E  L  0. 062 0 . 100  ***** *****  ON L A C T I C  079 086 104 129 161 180 202 231 223 222 232 217 223  0. 0. 0. 0. 0.  223 185 206  0.0  **** ****  **** ****  198 180  0.03 0.05  0. 155 0. 139  ****  0 . 120 0. 098 0. 069 0. 077 0. 078 0. 068 0. 083 0. 067  0.08  **** **** **** 0.08  **** 0.22  ACID  pH  AS  AFR  6. 5 6. 4 6. 4  0 0  6. 4 6. 2 6. 2 6. 1 6. 1 6. 1 6. 1 6. 1 6. 1 6. 1 6. 1 6. 1  0 0  0  0 0 0 0 0 0 0 2000 2000 2000 I  T 2 3 4 5 6 7 8 9 10 11 12  LCR  LC  9 17 25 33 41 49 58 65 73 82 97  13 14  137 174  15  197  . . 0 3 8 .017 .025 .043 .068 .084  - 4 . 2 2 2 -  2.625 1 . 0 0 0 2 . 2 5 0 3 . 1 2 5 2 . 0 0 0  .103 . 125 . 154 .146 . 145  - 2 . 111 - 3 . 1 4 3  .155 . 140 . 156  GR  E 1.00 1.0 0.9 0.9 0.9  0.0 0.0010  0 6 8 9  0.0095 0.0230 0.0296 0.0331  1.18 15 12 10 10  0.0222 0.0200  - 0 . 2 5 0  1. 10 1.10  - . 0 0 1 1 - . 0 0 4 1  0.405 - 0 . 6 9 6  1. 11 1.03  0.0081 0.0067  - 3 . 6 2 5 0.889 0.067  1. 1. 1. 1.  Inoculum : P. f r e u d e n r e i c h i i F e r m e n t a t i o n medium : F i n a l b r o t h e x t r a c t added p e r l i t e r  0.0175 0.0060  c f  VR  '  4.222 - 2 . 6 2 5 0.875 2.250 3. 125 4.000 2.111 3 . 143 3.625 - 0 . 8 8 9 - 0 . 0 6 7 0.250 - 0 . 4 0 5 0.261  r u n F P 1 . 1 with  BR  ***** ***** ***** *****  TI  0.854  9 8 8 8 8  1.625  *****  8  2.187  9 7  ***** ***** *****  8 9  0.0  ***** 2.250  20g o f  15 40 37 23  yeast  appendix  202  I : Tabulated Results T a b l e ai.36  RUN F P 3 . FERMENTATION WITH MIXED CULTURE OF L. PLANTARIUM AND P. FREUDENREICHII 1  T  1 I• —  -  1 1 2 3  I I I I I  *  5 6 | 7 I 8 I 9 |10 |11 |12 113 | 14 I 15 116 117  S 33.6 33. 3 32.7 29.4 25.8 16.5 12.7 12.6 12.9 12.7 13.1 11.6 11.8 10.6 9.6 8.7 8.5  0 12 24 31 39 46 51 57 63 69 75 87 99 114 129 142 176  DCW  TURB  0. 13 0.22 0.34 0.65 1.11 2.44 2.81 2.99 3.27 3.43 3.57 4.23 4.31 4.55 4.73 4.47 5.36  0.0 0. 013 0.048 0. 149 0.569 1.097 1. 187 1.301 1. 398 1.398 1. 398 1.222 1.523 1.602 1.523 1.523 1.523  Ta 0.113 0.126 0. 126 0.157 0.204 0.258 0. 324 0.304 0.310 0.296 0. 310 0.313 0. 323 0.320 0. 308 0.285 0.263  V  L  B  pH  AFR  0.056 0.066 0.079 0.100 0. 134 0.124 0. 137 0. 165 0. 181 0.207 0.243 0.250 0.247 0.263 0.250 0.228 0.218  0.057 0.060 0.047 0.057 0.070 0.134 0. 187 0.139 0. 129 0.089 0.067 0.063 0.076 0.057 0.058 0.057 0.044  0.0  6.7  0 0 0 0 0 0 0 0 0 0 0 2000 2000 2000 2000 2000 2000  **** 6.7 **** 6.7 **** 6.7  0.02 6.7 0.04 6.7 0.08 6.7 **** 6.7 **** 6.7 **** 6.7 0.30 6.7 0.38 6.7 0.45 6.7 0.38 6.7 **** 6.7 0.50 6.7 0.45 6.7  I  2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17  T  SC  12 24 31 39 46 51 57 63 69 75 87 99 114 129 142 176  0.3 0.9 4.2 7.8 17. 1 20.9 21.0 20.7 20.9 20.5 22.0 21.7 23.0 24.0 24.9 25.0  SR -0.025 -0.050 -0.471 -0.450 -1.329 -0.760 -0.017 0.050 -0.033 0.072 -0.127 0.021 -0.085 -0.065 -0.069 -0.004  E 3.00 2.30 0.94 0.90 0.36 0.35 0.47 0.54 0.65 0.82 0.79 0.79 0.81 0.73 0.62 0.58  GR  VR  BR  TI  0.0069 0.0103 0.0450 0.0575 0. 1899 0.0734 0.0300 0.0458 0.0273 0.0232 0.0551 0.0071 0.0157 0.0118 -.0200 0.0263  0.833 1.083 3.000 4.250 -1.429 2.600 4.667 2.667 4. 333 6.000 0.583 -0.250 1.067 -0.867 -1.692 -0.294  ***** ***** ***** 0.590 2.429 8.000 ***** ***** ***** 9. 167 6.250 6.250 -5.000 ***** 4.464 -1.471  12 12 7 8 7 5 6 6 6 6 12 12 15 15 13 34  20g/l of Yeast E x t r a c t 2.2ppm o f C o b a l t I o n s 4 g / l of Potassium Dihydrogen  Phosphate  Appendix  I  :  Tabulated  Table RUM  F15.22a.  |  T  j__  0 17 33 44 58 68 84 93 107 109 123 133 148 155 168 181 197 221 241 251 268 298 386 505  FERMENTATION  DCW  WITH  TURB  .  _—  0.60 1.60 2.00 2.30 2.55 2.56 2.53 2.41  0.0 0.31 0.96 1.15 1.30 1.40 1.40 1.40 1.30  2.29 2.49 3.00 3.24  1.30 1.35 1.40 1.40  3.42 3.49  1.52 1.70  3.51 3.68 3.63  1.60 1.70  TAM  3.51 3.29 3 . 18 3.00 6.00 6.50  FREUDENREICHII.  VA  .  H pB  0 . 108 0 . 147 0.267 0 . 140 0 . 177 0 . 193 0.200  0.090 0.104 0. 112 0.116 0. 116 0.130  A c i d  5.2 5.0 4.9 4.8  VA : V o l a t i l e (moles/1)  A c i d s  COD : Chemical E x t r a c t i o n  0.210  — — —  172  —  —  —  --  0.180 0. 170 0i170  —  0. 0.  — —  —  —  186 190  -  192 189 179 183 191 183  5.6 5.5 5.3 5.2 5. 1 5.1 6.6 6.6 6.6 6.4  —  —  225 193 210  6.1 6.0 5.6  —  A i r  i s  blown  i n  at  4000  by  -  196 240 210 157 138  -  6.9  Manual  C o n c e n t r a t i o n  Oxygen  Demand  c c / m i n  ]  265 238  4.8 4.8 6.7  0. 173 0.240  0.140 0. 152 0.152 0. 160 0.168  C o n c e n t r a t i o n  pH  290 220 216 216  180  2 0 g / l (15g/l dry b a s i s ) of Y e a s t E x t r a c t 2.2ppm of C o b a l t Ions 4 g / l of Potassium Dihydrogen Phosphate TAM : T o t a l (moles/1)  OF  CCD  6.8 6.4 5.5  —  —  •  EFFECT  4  0.062 0.062 0.080  1.52 1.52 1.52 1.52 1.52 1.70 1.70  203  AI.37 P.  0.  R e s u l t s  a f t e r  by (in  the  Ether  Steam lOOOppm),  f i r s t  300  P a s t e  E x t r a c t i o n D i s t i l l a t i o n a f t e r  hours  Ether  Appendix  I  :  Tabulated  Table RON  T  F l 5 . 2 2 b .  AI.38  FERMENTATION WITH P. FREUDENREICHII ANAEROBIC STAGE  DCW  TURB  0 22  —  0.0  —  33 70  —  98 106  —  0.02 0.04 0.08 0 . 13 0.27 0.40  115 131 146 165 177 190 363 470 495  204  R e s u l t s  —  0.68 1.00 1.21 1.82 2.61 3.44 3.45 3 . 10 —•  S  1.54 1.10 1.70 1.70  of  pH  — — —  -  •  —  —  3 3 2 2  8 6 2 4  . . . .  0 0 0 8  —•  3 2 2 2  3 4 3 1  . . . .  0 8 2 0  7.0 0.024  0.089 0.088  0.  0.118 0 . 180 0.278  —  basis) Ions  of  140  0.750 0. 950  0. 251 0.242 0.303 0.333  —  Potassium  Yeast  Dihydrogen  COD : C h e m i c a l E x t r a c t i o n  1000ppm) ,  (in  208 202 198  -  199 194 163 171 152  P a s t e  Phosphate At  Demand  7.1 7.1 6.8 6.4 5.9 5.5 5.3 5. 1 5.0 5.0  E x t r a c t  B : C o n c e n t r a t i o n of V i t a m i n B12 by A s s a y VA : V o l a t i l e A c i d s C o n c e n t r a t i o n by (moles/1) Oxygen  COD|  —  2 0 g / l (15g/l dry 2.2ppm of C o b a l t 4 g / l  ONE  1  VA  33.0  0.57 0.85 1 . 19 1.40  IN  30°C Steam  *  D i s t i l l a t i o n a f t e r  Ether  Appendix I : T a b u l a t e d  Table RON  S  T  1  DCW  TURB  0.64 1. 10 2.31 4.90 6.73 7.88 7.60 7.42 7.48  0.0 0. 260 0.824 1.301 1.420 1.495 1. 523 1.523 1.456  205  AI.39  F05.22. FERMENTATION YEAST EXTRACT  Results  WITH P.  FREUDENREICHII.  CONCENTRATION as TA  VARIABLE  V  L  B  0. 016 0. 016 0. 033 0. 087 0. 126 0. 157 0. 149 0. 161 0. 158  0.048 0.050 0.042 0.047 0.066 0.052 0.081 0.062 0.052  0.0  pH  AFR  L  r | I | I I I I I I  1 2 3 4 5 6 7 8 9  0 35 62 88 115 136 163 180 202  37.2 34.2 32.4 27.6 18.6 14.4 12.7 12.0 12.3  0.064 0.066 0.075 0.134 0. 192 0.209 0.230 0.223 0.210  ****  0. 12 0.50 1.05 1.50 1.40  **** ****  7.9 6.8 6.8 6.6 6.7 6.7 6.8 6.7 6.6  _j 0 0 0 1000 1000 1000 1000 1000 1C00  •  | I I | | | | | | «  r  I 2 3 4 5 6 7 8 9  L.  |  T  SC  35 62 88 115 136 163 180 202  3.0 4.8 9.6 18.6 22.8 24.4 25.2 24.9  SR -0.086 -0.067 -0.185 -0.333 -0.200 -0.061 -0.044 0.014  E 0.0 0. 32 0.67 0.53 0.56 0.49 0.52 0.51  GR  VR  BR  TI  0.0130 0.0448 0.0996 0.0678 0.0548 -.0104 -.0106 0.0027  0.0 0.630 2.077 1.444 1.476 •0.296 0.706 -0.136  ***** 1.935 14.615 20.370 21.429 -3.704 ***** *****  35 27 26 27 21 27 17 22  |  J  Appendix I : Tabulated Table  Results  206  AI.40  RUN F 1 0 . 2 2 . FERMENTATION WITH P. FREUDENREICHII. YEAST EXTRACT CONCENTRATION AS VARIABLE T  S  1 0 2 14 3 23 4 37 5 47 6 59 7 71 8 83 9 95 10 108 11 120  2 3 4 5 6 7 8 9 10 11  25.5 29.4 28.8 26.7 24.3 21.6 17.7 14.4 12.3 10.2 8.2  DCW  TURB  0.54 0.74 0.94 1.81 2.38 3.22 4. 10 5.26 6.59 7.07 7. 12  0.0 0. 125 0. 319 0.824 1.097 1. 155 1.301 1.398 1.523 1.523 1.523  T  SC  14 23 37 47 59 71 83 95 108 120  -3.9 -3.3 -1.2 1.2 3.9 7.8 11.1 13.2 15.3 17.3  SR 0.279 -0.067 -0.150 -0.240 -0.225 -0.325 -0.275 -0.175 - 0 . 162 - 0 . 162  TA 0.045 0.051 0.060 0.070 0.082 0.098 0. 139 0.162 0. 181 0.203 0. 204  V  L  B  PH  AFR  0. 007 0. 012 0. 020 0. 034 0. 045 0. 060 0. 083 0. 114 0. 132 0. 147 0. 132  0.038 0.039 0.040 0.036 0.037 0.038 0.056 0.048 0.049 0.056 0.071  0.0  7.7 7.4 7.2 7.3 7.2 7.2 7.3 7.1 7.2 7.3 7.4  0 0 0 0 0 0 500 500 500 500 500  **** 0.03  ****  0. 12  ****  0.23 0,80 1.30  **** 1.35  E  GR  VR  - 0 . 12 -0.35 -2.03 2.85 1.22 0.88 0.87 0.85 0.82 0.65  0.0143 0.0228 0.0618 0.0570 0.0700 0.0733 0.0971 0. 1104 0.0373 0.0038  0.357 0.889 1.000 1. 100 1.250 1.917 2.583 1.500 1.154 -1.250  BR  ***** 1.304  ***** 3.750  *****  4.583 47.500 41.667  ***** 2.000  _J  TI | 14 9 14 10 12 12 12 12 13 12  | I  | | | | | | | |  Appendix I : T a b u l a t e d  207  Results  T a b l e AI.41 RUN  F20.22. FERMENTATION YEAST EXTRACT S  T 0 1 9 2 17 3 4 25 33 5 41 6 7 49 8 55 9 60 10 65 73 11 82 12 13 97 14 137 15 174 16 197  37.2 37.2 36.0 34.5 32.7 28.8 24.0 22.5 20. 1 13.2 12.0 11.6 11.5 10.6 10.3 10.2  DCW  TURB  0. 14 0.28 0.41 0.71 1. 14 2.71 4.52 5.93 6.33 7.25 7.52 7.57 7.60 7.75 8.41 8.67  0.0 0. 034 0. 146 0.284 0.495 1.071 1. 398 1.456 1.699 1.699 1.699 1.824 1. 824 1.824 1.824 1.824  SC 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16  9 17 25 33 41 49 55 60 65 73 82 97 137 174 197  27.6 1.2 2.7 4.5 8.4 13.2 14.7 17. 1 24.0 25.2 25.6 25.6 26.5 26.8 27.0  WITH P. FREUDENREICHII.  CONCENTRATION AS VARIABLE V  L  B  0.076  0. 047  0.029  0.0  0.075 0.083 0. 101 0.093 0. 140 0. 169 0. 175 0.189 0. 191 0.199 0.225 0.233 0. 234 0.228  0. 046 0. 054 0. 072 0. 064 0. 111 0. 135 0. 141 0. 155 0. 136 0. 149 0. 175 0. 183 0. 186 0. 180  ***** *****  TA  ***** ' *****  SR 0.0 -0.150 -0. 188 •0.225 -0.488 -0.600 -0.250 -0.480 -1.380 -0.150 •0.044 •0.003 -0.022 •0.008 -0.007  **** -0.08 0.23 0.50 0. 18 0.44 0.54 0.50 0.41 0.32 0.36 0.45 0.46 0.47 0.44  7.0  **** 7.0  0.034  0.055 0.050  ***** ***** 0.048  *****  0.0158 0.0159 0.0381 0.0534 0.1961 0.2268 0.2343 0.0808 0. 1832 0.0337 0.0056 0.0020 0.0038 0.0180 0.0111  AFR  0 0 0 0.02 7.0 0 7.0 **** 0 0. 10 7.0 0 0.21 7.0 0 **** 7.0 0 0.45 7.0 0 **** 7.0 0 0.55 7.0 0 **** 7.0 0 0.80 7.0 0 0. 90 7.0 0.80 7.0 2000 0. 97 7.0 2C00 0.95 7.0 2000  ***** ***** ***** ***** ***** *****  GR  pH  VR  BR  *****  *****  -0.059 1.000 2.250 -1.000 5.875 4.000 1.200 2.800 •2.375 1.444 1.733 0. 200 0.081 •0.261  5. 187 13.750 ***** 17.143 ***** 10.000 ***** 14.706 6.667 -2.500 4.730 -1.087  1.000  *****  TI 9 8 8 8 8 8 6 5 5 8 9 15 40 37 23  ]  Appendix  I : Tabulated  Table RUN  F30.22.  FERMENTATION  Results  AI.42 WITH P. FREUDENREICHII.  YEAST EXTRACT CONCENTRATION |~ | | f | | | | | |  T 1 0 2 21 3 33 4 48 5 63 6 75 7 88 8 99 9 128  r | | | | | | | | |  2 3 4 5 6 7 8 9  S 29.4 28.2 23.1 16.8 12.4 9.2 8.9 8.7 8.4  DCW  TURB  0.42 1.36 2.90 5.32 7.72 8.10 8.41 8.49 8.34  0.0 • 0.081 0.770 0.108 1.301 0.122 1.523 0.162 1.699 0.200 1.699 0.211 1.699 0.224 1.699 0.242 1.699 0.236  T  SC  21 33 48 63 75 88 99 128  1.2 6.3 12.6 17.0 20.2 20.5 20.7 21.0  SR -0.057 -0.425 -0.420 -0.293 -0.267 -0.023 -0.018 -0.010  208  TA  AS VARIABLE  V  L  B  0.026 0.044 0.058 0.087 0.136 0.149 0.153 0.144 0.158  0.055 0.064 0.064 0.075 0.064 0.062 0.071 0.098 0.078  pH 0.0 0.11 0.22 0.38 1.00 **** 1.00 1.00 ****  AFR  6.9 0 6.2 0 6.8 0 6.8 1000 7.0 1000 7.0 1000 7.2 1000 7.0 1000 7.1 1000  :—: E  GR  VR  BR  1.35 0.46 0.44 0.58 0.55 0.56 0.51 0.57  0.0449 0.1281 0.1613 0.1600 0.0317 0.0238 0.0073 -.0052  0.857 1.167 1.933 3.267 1.083 0.308 -0.818 0.483  5.333 9.333 10.067 41.667 ***** 0.0 0.0 *****  ] \ \ | j | \ | \ |  1  TI | 21 12 15 15 12 13 11 29  1 | | | | | | |  appendix I : T a b u l a t e d Table RUN  F20.00, FERMENTATION  WITH P. FREUDENREICHII.  | 1  0 21 46 66 | 5 82 I 6 110 I 7 157  i  | |  j  |  AS VARIABLE ™  S  T  37.5 34.8 27.6 17.7 12.9 11.4 9.9  I 2 I 3 t *  J  209  AI.43  COBaLT ION CONCENTRaTION r  Results  2 3 4 5 6 7  DCW 0.27 1. 12 4.21 7.33 7.69 8.21 8.05  T  SC  21 46 66 82 110 157  2.7 9.9 19.8 24.6 26. 1 27.6  TORB 0.0 0. 509 1.301 1.523 1.523 1.602 1.553  SR -0.129 -0.288 -0.495 -0.300 -0.054 -0.032  V  TA 0.059 0.096 0. 128 0.184 0.207 0.195 0.232  E 0.43 0.54 0.57 0.51 0.51 0.51  B  L  0. 012 0. 025 0. 071 0. 138 0. 152 0. 159 0. 168  pH  AFR  0. 047 0.0 0 6.8 0 0. 071 . 0.06 6.9 0 0. 057 0.27 5.5 0 0.046 0.30 6. 1 **•* 6.7 1000 0. 055 0. 036 0.40 6.9 1000 **** 6.7 1000 0. 064  GR 0.0406 0.1236 0.1560 0.0225 0.0186 -.0034  I  VR 0 .619 1.840 3 .350 0 .875 0 .250 0 . 191  BR 2.857 8.320 1.600  *****  2.273  *****  TI  I .j  | | | |  "t  1 ji 21 25 20 16 28 47  Appendix I : T a b u l a t e d  Results  210  T a b l e AI.44 RUN  F20.09.  FERMENTATION  WITH P. FREUDENREICHII.  COBALT ION CONCENTRATION T  1 1 1  I 2 I 3 t  I 5 I 6  | 7  I 8 I 9 |10  S  0 28 49 57 72 88 102 114 126 138  34.8 38.4 29. 1 30.0 20.7 19.8 12.3 12.3 11.7 11.7  DCW  0.43 1. 24 3.92 5.06 7. 14 8.61 9.28 8.80 8.90 8.63  TURB  0.0 0. 585 1. 301 1.398 1.523 1.699 1.699 1.699 1.699 1.699  TA  AS VARIABLE  V  0.050 0.089 0. 136 0.146 0. 196 0.216 0. 266 0.252 0.226 0.241  B  L  0. 012 0. 030 0. 065 0. 095 0. 129 0. 156 0. 171 0. 184 0. 163 0. 165  0.038 0.059 0.071 0.051 0.067 0.059 0.095 0.068 0.063 0.076  pH  AFR . . .  0.0 0.07 0.36 0.34 0.52 1.17  6.9 0 6.8 0 6.7 0 6.7 1000 6.8 1000 6.9 1000 **** 7.0 1000 1.30 6.9 1000 **** 7.0 1000 **** 6.9 1000 _,  1 2 3 4 5 6 7 8 9 10  T  SC  28 49 57 72 88 102 114 126 138  -3.6 5.7 4.8 14.1 15.0 22.5 22.5 23.1 23.1  SR  0.129 -0.443 0. 113 -0.620 -0.056 -0.536 0.0 -0.050 0.0  |  i  j  1| 1 | | | | | | | J  E  GR  VR  BR  TI  -0.45 0.84 1.56 0.75 0.86 0.64 0.69 0.59 0.60  0.0289 0.1276 0. 1425 0.1387 0.0919 0.0479 -.0400 0.0083 -.0225  0.643 1.667 3.750 2.267 1.688 1.071 1.083 -1.750 0.167  2.429 13.905 -1.875 12.000 40.625 ***** 4.808 ***** *****  28 21 8 15 16 14 12 12 12  ]  Appendix  I : Tabulated  Table RUN  211  Results  AI.I45  F20.22.126,FERMENTATION OF S S I CONTAINING FREUDENREICHII  T  S  0 1 2 24 3 48 4 67 5 80 6 96 7 117 8 142  2 3 4 5 6 7 8  27.9 26.7 21.9 19.5 15.3 11. 1 10.8 9.9  DCW  TURB  0.65 0.94 2.34 3.97 5.66 7.30 7.67 7.52  0.0 0. 284 0.959 1.301 1. 398 1.523 1.523 1.523  T  SC  24 48 67 80 96 117 142  1.2 6.0 8.4 12.6 16.8 17.1 18.0  SR -0.050 -0.200 -0.126 -0.323 -0.262 -0.014 -0.036  TA 0.083 0.075 0.097 0.150 0. 156 0. 199 0.226 0.223  E 1.28 0.51 0.76 0.69 0.72 0.72 0.66  DMEZ WITH P.  V  L  B  pH  0.014 0.031 0.048 0.085 0. 110 0.148 0. 150 0. 146  0.069 0.044 0.049 0.076 0.046 0.051 0.076 0.077  0.0  7. 1  0.29 0.38 0.72 0.88 0.80 1.00  6.8 6.9 6.9 6.8 7.0 6.8  **** 6.7  AFR 0 0 0 2000 2G00 2000 2G00 2000  GR  VR  BR  TI |  0.0119 0.0587 0.0854 0.1302 0.1025 0.0176 -.0060  0.708 0.708 1.947 1.923 2.375 0.095 -0.160  ***** 6.042 4.737 26.154 1C.000 -3.810 8.000  24~* 24 19 13 16 21 25  Appendix I : Tabulated Table  Results  212  AI.46  BUN S 2 0 . 2 2 . FEBRENTATION WITH P. SHEBEANII T 1 2 3 4 5 6 7 8 9 10  S  0 17 58 82 97 120 143 169 193 221  38.4 37.8 36.0 30.0 28.5 21.0 17.7 14.8 11.2 11.2  T 2 3 4 5 6 7 8 9 10  17 58 82 97 120 . 143 169 193 221  DCW  TUBB  0.38 0.53 2.28 3.66 4.51 6. 18 7.01 8.09 8.58 8.67  0.0 0.208 1. 155 1. 301 1.523 1.699 1.745 1.824 1.921 1.824  SC 0.6 2.4 8.4 9.9 17.4 20.7 23.5 27. 1 27. 1  SB -0.035 -0.044 -0.250 -0.100 -0.326 -0.143 -0.110 -0.150 0.0  TA 0.060 0.063 0.102 0. 116 0.131 0. 176 0. 186 0. 213 0.221 0.208  E 0.90 1.01 0.61 0.64 0.41 0.49 0.50 0.43 0.45  V  I  E  0. 013 0. 019 0. 040 0. 070 0. 083 0. 093 0. 125 0. 145 0. 143 0. 150  0.047 0.044 0.062 0.046 0.048 0.083 0.061 0.068 0.078 0.058  pH  0.0 **** 0. 15 0.25 0.42 **** 0.57 **** 0.82 ****  GB  VH  0.0092 0.0426 0.0575 0.0567 0.0728 0.0359 0.0415 0.0204 0.0032  0.353 0.512 1.250 0.867 0.435 1.391 0.769 -0.083 0.250  6.8 6.4 6.9 6.8 6.8 6.8 5.6 6.7 6.9 6.7  AFB 0 0 0 1000 1000 1000 1000 1000 1000 1000  BB  ***** 2.586 4.375 11.3 33  ***** 3.261  *****  5.000  *****  TI |  j  1 17 41 | | 24 | 15 | 23 | 23 | 26 | 24 | 28 |  Appendix  II : Analysis  Procedures  213  APPENDIX I I Analysis  II.1  Procedures  SAMPLING Care  was t a k e n t h a t  samples  were g r e a t e r  than  were t h e n s e p a r a t e d from t h e f e r m e n t a t i o n b r o t h with d i s t i l l e d  w a t e r . The s e p a r a t i o n o f c e l l s  f r o m t h e washing 3000rpm.  The  electric pipets,  water  washing  was  done  by  thorough  shaker mixer. Three a l i g u o t s o f e v e r y sample,  well  destined  f o r t h e measurement o f g r o w t h . measurement  cell-free  broth  reducing  sugar  Streptomyces as no  from  was  used  and  Streptomyces,  could  be  pipetted  glassware,  so c e l l s  cell  was b a s e d  on t h e f i n a l  for  vitamin  performed.  weight  vitamin  Lactobacillus, of  one.  mixing  two o f t h e s e t h r e e Those'from B12  For only  B12  with  to t h i s  cell  aliguots  were  the t h i r d  fermentations  two a l i g u o t s  were  concentration  was  easily  o f the t o t a l  taken  concentration.  on t h e o t h e r hand, grew nor  w i t h an  one The  t h e d e t e r m i n a t i o n o f a c i d s and  concentrations.  measurement  not  of  centrifugation at  were s u b j e c t e d  process.  twice  t h e b r o t h and  o f 20ml e a c h ,  removal  were f o r t h e  Cells  mixed,  Cells  and washed  from  was done by a 30 minute  60ml.  removed  broth  on t h e o r i g i n a l  with needed, to  i n lumps,  from  were weighed  volume o f b r c t h  walls  be and of  and t h e d r y instead of  appendix  II.2  MEASUREMENT OF  11.2.1  Standard  The volume  : A n a l y s i s Procedures  T u r b i d i t y Method  made  up  resuspended  to  the  blank,  a spectrophotometer  wavelength  11.2.2  kept  of  540  i s then  volume o f measured,  (BauschEIomb  water,  the  the  broth.  The  with  water  as  Spectronic  20)  at  m i l i m i c r o n s . T u r b i d i t y i s then c a l c u l a t e d .  freshly  the  fermentation  broth,  prior  T h i s sample  serves  as  refrigerated.  transmittancy  measurements.  taken  milimicrons.  The  samples i s then  measured  turbidity  i s then  Turbidity =  2 - log10  (percent  i s net  to  innoculation,  blank  transmittancy  The  Turbidimetry of  solution  ^distilled  Modified_Turbiditj_Method  A sample is  of  in  original  of  the  this  are  transmittancy with  214  BACTERIA! GROWTH  washed c e l l s is  II  at a  suitable  subseguent  cf  well  wavelength  c a l c u l a t e d with of  for  the  mixed  of  915  eguation:  transmittance)  f o r measuring  growth  in  lumps  Streptomyces.  11.2.3  Crj_Cell_Weight  Aluminum heated  dishes  f o r at least  then  cooled  cells  are  down  then  ( F i g u r e A2.73) a r e two  hours at  to  room  the  same  cell  weight  drying  and  o v e n . The i s then  They  these  are  aluminum  then  dishes  c a l c u l a t e d by  weighing  after  i n a F i s h e r Isotemp  temperature  t r a n s f e r r e d to  50ml c e n t r i f u g e t u b e s .  110°C  preweighed  dried  dishes  overnight  then  times  from at  1100C  weighed a g a i n .  difference. This  i s a l w a y s done two  oven,  i n a d e s s i c a t o r . Washed  aluminum  are  being  the in Dry  seguence  of  successively.  The  Appendix FIGURE  A2.73  II : Analysis  Procedures  215  : D r y i n g o f Aluminum D i s h e s i n The Isctemp (scale : f -J » 13.2 cm)  i  i  average  values are  balance  a c c u r a t e to the t e n t h of a m i l l i g r a m .  II.3  Oven  used.  Weighing  was  dene  cn  a  precision  MEASUREMENT_CF_SUGAR_C This  adopted 25x200ml  is with size,  the  method  slight 1ml  described  modifications. of  a  sample,  by N e i s h [ 6 7 , 6 8 , 8 7 ] , Tc  each  containing  test  It is  tube,  of  up t c 0 . 2 q / l of  Appendix  FIGURE A2.74  : Typical  I I : Analysis  Procedures  Standard Curve Test  f o r The R e d u c i n g  216 Sugar  R e g r e s s i o n l i n e a t 0.95 c o n f i d e n c e y ( ± 0 . 0 3 9 ) = 0 . 0 0 7 (±0.0003)x + 0 . 0 3 3 (±0.034)  — 20.0 L  1 60.0  GLUCOSE  1 100.0  1 140.0  CONCENTRATION  I  380.0  (G/L)  I 0.0  appendix reducing sugar of  the  (preferably  Somogyi  closed  with caps  water  for water  The  addition  of  reagent  mixed  10  b a t h . Two  p r e p a r a t i o n . The is  and  solutions  minutes.  20ml  reaction  of  tubes are  then  heated  in  boiling  r e a g e n t i s mixed  Therefore, a  o f 660  thus  milimicrons,  for  solutions  a r e f r e s h l y made  known  typical  one  interpretation  from  a  standard  up  of  and  the  obtained, with  water and  solutions  for  on  Figure  a2.74,  by i n t e r p o l a t i o n . T h e s e  tests  solution  is  a r e needed f o r t h e  presented data  4  0.05,  conversion  of  on 1g/l  each of  T e s t s were a l w a y s done i n d u p l i c a t e .  s u g a r c o n c e n t r a t i o n was  the  known c o n c e n t r a t i o n ( 0 . 2 , 0.1,  the  reguired  in.  water, then, completes  i s not complete  curve,  series glucose  The  of kept  reducing  measured a s g l u c o s e .  KIASUREHENT_OF_VITAMIN^ T h i s method i s a s t a n d a r d b i o l o g i c a l a s s a y  II.4.1  [38,101].  Inoculum  Stock  culture  of  Lactobacillus  leichmanii  m a i n t a i n e d by s t a b t r a n s f e r , on B a c t o - D i f c o This  2ml  These t u b e s a r e then c o o l e d i n a  of d i s t i l l e d  standard  II.4  then  and  t e s t i s done s i m u l t a n e o u s l y f o r 12 s a m p l e s  0 . 0 2 g / l ) . The  refrigerated.  a d d e d . The  t r a n s m i t t a n c y of the s o l u t i o n s ,  glucose  samples  217  i s tranferred  ml o f a r s e n o m o l y b d a t e  of  empirical.  is  thoroughly  then measured a t a w a v e l e n g t h  as b l a n k . The  Procedures  over 0.02g/l)  copper  about  cold  II : analysis  transfer  B12  (aTCC-7830) i s culture  agar.  i s done t h r e e t i m e s a week. I n c u b a t i o n i s c a r r i e d  out a t 370C f o r a t l e a s t  12 h o u r s . O n l y c u l t u r e s  of l e s s  than  4  appendix d a y s e l d a r e used Stock  inoculum. culture at  transferred  are t r a n s f e r r e d ,  at  centrifuged,  another  inoculum. solution  least  16  separated  10ml.of s t e r i l e  10ml  of  The  cells  such  are  t h e b r o t h and t h e n  suspension  stock  incubated  aseptically  last  medium , t o  medium i s a c t u a l l y  strength. I t i s  a  resuspended i n  medium. Mix 1ml o f t h i s  sterile  5  f c r t h e p r e p a r a t i o n o f an  b r o t h . T h i s b r o t h i s then  from  The s u s p e n s i o n  for at least  w i t h a s t a b , from  hours.  suspension  at half  218 '  twice d a i l y  b e f o r e i t i s used  t o 10ml o f i n o c u l u m  37°C f o r  to  i s  transfers Cells  Procedures  f o r p r e p a r a t i o n of inoculum.  culture  successive  II : analysis  medium make t h e  a b a s a l medium s t o c k  autoclaved  at  121°C  fcr  10  minutes.  II.4.2  Procedure  Washed in  cells  from  20ml o f t h e sample o f f e r m e n t a t i o n b r o t h  q u e s t i o n a r e resuspended  in  water  meta-bisulfite  buffer  of  suspension of c e l l s  this diluted  for  10  minutes,  cobalamin The  added  aseptically This  t o make up a t o t a l  along  with  a  t o 5ml o f t h e same  standard  cobalamin  diluted  resulting  and  to  solution  a  is  t n e  ^  5ml  of  sodium  volume o f 50ml. Ten ml [ 1  sterilized  mixture  of  1ml  at  121 °C  c f standard  buffer.  solution,  so  prepared,  c o n c e n t r a t i c n o f 0.02 is  the  called  is  then  micrograms/1.  cyanocobalamin  working  solution. 5ml  of  16x150mm t e s t minutes  basal  stock  tubes. These  a t 121°C.  solution tubes  i s added  are  then  t c each  of s e v e r a l  sterilized  for  5  Appendix 1,  2,  working  3,  and  solution  duplicate. then  4  in  added t o s t e r i l e tubes.  Each  different  control  is  water  to  6  tubes  in  as d e s c r i b e d above,  are  so  water  a g a i n under a s e p t i c  t o make t h e conditions.  which c o n t a i n no  sample  in  0.04 finally  in  test  volumes a t 3  the chance  of  under-  duplicate.  volume o f a l l t u b e s This  nor  are  already with  aseptic  to  prepared  unknown c o n c e n t r a t i o n i s added  under  0.001  tubes  to minimize  cyanocobalamin  these  (from  Samples,  added  l e v e l s - presumably  tubes  to  distilled  concentration  219  the  b a s a l medium s t o c k s o l u t i o n ,  sample  Sterile 10ml,  with s t e r i l e  of  added  prepared  v i t a m i n B12.  or o v e r e s t i m a t i n g the  to  aseptically  suitable  micrograms/1)  respectively  samples,  properly diluted to  5ml  are  Sterile  conditions  II : A n a l y s i s Procedures  includes  cyanocobalamin  the  up 3  working  solution. One except then 24  drop  one  inoculum  of t h e c o n t r o l  thoroughly  is  aseptically  t u b e s . The  mixed. The  tubes  added  t c each  contents of a l l  are incubated  tube  tubes  are  a t 37°C f o r 16  to  hours. The  growth  millimicrons The of  of  is  w i t h one  standard  curve  measured c f the  by  inoculated  i s drawn w i t h  known c o n c e n t r a t i o n . A t y p i c a l  A2.75.  The  concentraticn  the  turbidity control  method a t  tube  measurements c f t h e curve  i s presented  of  samples  i s d i s c a r d e d when the  turbidity  is  as  600  blank.  solutions in  Figure  determined  by  interpolation. A test control than  0.2.  tubes,  with  the  B e s u l t s of the  u n i n o c u l a t e d one  test  are a l s o  of  the  inoculated  as b l a n k , i s g r e a t e r  disregarded i f  the  tube  Appendix  FIGURE A2.75  : Typical  II : Analysis  Standard Curve  Procedures  f o r The V i t a m i n B12  220  Assay  Appendix I I : a n a l y s i s  with  the  solution) not  highest content has a t u r b i d i t y  have a s t r a i g h t  Procedures  o f v i t a m i n B12 g r e a t e r than  portion,  or  i f  221  (added  v i a the working  0.5, o r i f t h e c u r v e  the  concentration  known s a m p l e i s o u t o f t h e r a n g e o f t h a t s t r a i g h t the  slope of t h i s  II.5  straight  a l l  phenolphthalein  11.5.1  portion,  the  or i f  portion i s too shallow.,  with  titrations  with  i n 50% e t h y l  a l c o h o l i s used  Steam D i s t i l l a t i o n  Ten a  sulfuric  ml o f c e l l  free  flat  bottom,  acid  i s stirred  Steam, g e n e r a t e d  flask,  is  vapor  bubbled  i s then  collected, until  of V o l a t i l e  i s  i n with from  through  below,  as i n d i c a t o r .  acids  then  to a  round  flask  10ml o f c o n c e n t r a t e d  water i n  the diluted  titrated  to  p r e c a u t i o n n o t t o l e t any  boiling  with  two c o n s e c u t i v e a l i g u o t s  11.5.2  referred  broth i s transferred  c o n d e n s e d . When a b o u t  i t  concentration  NaOH,  water i s added,  escape.  a  vapor  nearby  and a c i d i f i e d  round  b r o t h . The  50ml o f c o n d e n s a t e h a s  NaOH. T h e p r o c e s s  of condensate  are  been  i s continued of  the  same  i n acids.  Manual_Ether_Extraction  Ten vigorous  heat  of  MgaSUREMENT OF a C I D CONCENTRATION In  The  does  ml  of  mixing,  gaseous  t o 20ml o f c e l l  volatile  generated  condensation  concentrated  by  sulfuric free  acic  broth  is  added,  i n a closed  a c i d s which a r e b o i l e d  o f f the broth  this  a r e taken  as t h e v e s s e l  addition i s then  of acid cooled  with i c e .  with  vessel. by t h e  c a r e o f by  Appendix The r e s u l t i n g paper.  The  extracting  II : Analysis  solution  filtrate  is  Procedures  i s vacuum extracted  f u n n e l with a p o r t i o n  filtered 3  t o 50ml o f d i s t i l l e d  well  the  suction. with  20ml o f t h i s  solution  titrated the  of  ether  is  added  and a n o t h e r  water.  to prevent  acids  i n the  10  minutes  neutralized  t o t h e amount  20ml  broth.  e x t r a c t e d by e t h e r . T h i s was n o t t h e c a s e line  acid  to the  could  curve or a c a l i b r a t i o n  egual  of  in  content  would  so an e g u i l i b r i u m  and  acids,  i s proportional  original  linear  in  of l a c t i c  proportionality be t o t a l l y  be  is  going dry. T h i s  acids  20ml o f b r o t h . The d i f f e r e n c e  of v o l a t i l e  time. A l l  solution  for  i t from  the 2 samples s u b j e c t e d t o t i t r a t i o n s  amount  shaped  20ml o f t h e a g u e o u s phase a r e t h e n  sample i s p r o p o r t i o n a l  the o r i g i n a l  i n a pear  o f f with a g e n t l e  boiled  w i t h 0.05N HaOH. The c o n t e n t c f  boiled  filter  The  evaporated  agueous phase i s  water o c c a s i o n a l l y  boiled  in  then  times  through  o f 10ml o f e t h e r each  t h e e t h e r i s added mixed  222  The  t o 1 i f the a c i d s here,  a r e needed.  II.5.3 C o n t i n u o u s _ f l a t c h E m  The a p p a r a t u s , a d o p t e d are  from  N e i s h [ 6 7 ] , and  its  principle  i s added  t o 5ml o f  d e s c r i b e d i n 2.4.4. Three  broth  ml  of c o n c e n t r a t e d s u l f u r i c  i n a closed  vapors,  i f  extremely  any,  hot with  vessel  (Figure  evaporated the r e a c t i o n  added  solution  solution  the  resulting  is filtered.  the  heat  Five  to  2.23).  from  the i c e - s u r r o u n d e d v e s s e l .  acid  The liguid  generated,  ml o f d i s t i l l e d  The f i l t r a t e  after  volatile which  acids becomes  a r e condensed water  cooling.  i s volumetrically  is The  in then  final  transferred  appendix to t h e residue the  filtrate.  suspension then is and  water from  i s suspended  the  cooled  25ml o f e t h e r  container from  beginning,  to extraction.  then before  during  the  titrated  for  portions  One  of  replenished. of  the  second  first  3  hours  resulting  and v i g o r o u s  one sample  were was  in  gives  other  drops  extract  extract  water  titrated  the t o t a l  executed  phase t o f i n e  the  evaporated  was  was  was b o i l e d  occasionally  with  NaOH.  Result  acid concentration, the  concentraticn. was a d o p t e d .  extraction  f c r each s a m p l e . The e x t r a c t o f  i s cooled  titrated  A  i s needed o f f then  known  addition  again.  by  titration  of  titrated  water.  boiled The  In  for  excess of s u l f u r i c  e x t r a c t . I t i s then  titrated the  ether  an a l t e r n a t i v e method  concentration.  occasional  with  extracts  swirling  the ether  was f i n a l l y  the n o n - v o l a t i l e a c i d  Later only  titration  ether  0.05N NaOH. An e x c e s s o f water was  with  This extract  with  o f t h e same sample  the  to disperse  minutes  Hater  temperature of 7 0 O C .  two  the t i t r a t i o n ,  15  i s  going d r y .  b o i l e d o f f i n t h e same water b a t h . T h i s about  container.  t o share the load o f a c i d s  with  titration  The e x t r a c t o r  i n the ether  l a r g e volume o f w a t e r . The e t h e r  then  the vessel r e c e i v i n g  of t h e d i s p e r s i o n tube i n t o i t s e l f . with  223  i n the vapor tube a f t e r t h e  i n a water bath a t a c o n s t a n t  subjected  added  rinsing  Procedures  e x t r a c t i o n i s then r u n f o r about  In  the  tube  to prevent ether  boiled  the  This  to t h i s  The  in  tube with  assembled  added  II : Analysis  final  the  acid  for  this  15  total  acid  1N i s added t o minutes  bciled  The d e f i c i e n c y o f added s u l f u r i c  method,  with  solution i s  acid  revealed  i s o f t h e same magnitude o f t h e v o l a t i l e  acid  Appendix  content  II.6  of the o r i g i n a l  Procedures  224  sample.  HEASUREMENT_OF_CHEH1^A^ This  differ  test  from  Place flask  i s widely  one s o u r c e  with  ground  sulphate  and  in  boiled  joint  solution  silver  the f l a s k .  continuous  [4,5,70,91].  sample neck.  i n a 500ml Add  details  A good  25ml  mixing  f o r two h o u r s  Erlenmeyer of standard  of  mercuric  g l a s s beads a r e i n t r o d u c e d  75ml o f c o n c e n t r a t e d  s m a l l a l i g u o t s and w i t h  and r e f l u x e d  minor  (0.25N). Add 1g each  s u l p h a t e . Then  Finally  agitation.  Only  to another.  glass  dichromate  added,  used  50ml o f p r o p e r l y d i l u t e d  potassium  into  II : Analysis  slow  sulfuric  pouring  acid  is  a s w e l l as with  i s a s c e r t a i n e d . The f l a s k i s  with  a  Friedrich's  condenser  on a h o t p l a t e . A sample as  blank  consisting  o f 50ml o f d i s t i l l e d  i s simultaneously refluxed  water  instead of a  under c o n d i t i o n s as  identical  possible. The  two  standardized  flasks  a r e then  cooled  0.25N f e r r o u s ammonium  and t i t r a t e d  with  sulphate. Ferroin  freshly  i s used  as  indicator. COD i s c a l c u l a t e d  as f o l l o w s :  ppm COD =  (A-B) x 40  A,B=  of  titrations  ml  ferrous  ammonium  o f t h e s a m p l e and t h e b l a n k  sulphate  used  respectively.  for  the  Appendix I I : A n a l y s i s II.7  Procedures  225  2JASUREMENT_0F_SULFUR  11.7.1  Free_SuIfur_Dioxide  An  aliquot  Erlenmeyer until  of  flask.  5ml  Cold  of  the  sample  i s placed  water, o c c a s i o n a l l y with  t h e brown c o l o r o f SSL s a m p l e i s l i g h t  i n d i c a t o r c o l o r change. A t i t r a t i o n using  starch  as  i n d i c a t o r then  with  end  point  color.  The  only  concentration  iodine  f e l l o w s . The b l u e  p o i n t i s masked by t h e brown c o l o r o f i s recognized  ice, is  added  enough t o show t h e  0.1N  end  i n a 250ml  SSL,  solution  color c f the  therefore,  the  by t h e sudden change i n tone o f t h e of  sulfur  dioxide  i s c a l c u l a t e d as  follows: Sulfure  dioxide  concentration  (ppm) = ml o f 0.1N I o d i n e  x 640.6  11.7.2 T p t a l _ s u I f u r ^ d i o x i d e Total  sulfur  dioxide  loosely  combined  sulfur  combined  with  with  organic  consists cf free  sulfur  d i o x i d e . The l a t t e r  dioxide  i s sulfur  m a t t e r , which c a n be s p l i t  and  dioxide,  o f f by t r e a m e n t  NaOH. The  sample  same p r o c e d u r e a s d e s c r i b e d i s  submitted  t o a pretreatment  t e m p e r a t u r e f o r more t h a n followed  by  sulfuric  acid.  the  above  lowering  one of  hour. pH  with This  of  the  i s  used.  But the  5ml c f NaOH a t room treatment sample  is  with  then  dilute  Appendix I I I : S o l u t i o n s  and C h e m i c a l s  226  APPENDIX I I I Solutions  III.1  1.  SOLUTIONS  Solutions A  of Sulfuric  volume  approximately 1N  of  concentrated  The  Standard  Solutions  approximate to the desired  against  potassium  is  then  o f Sodium  Hydroxide  i s  diluted  (30ml t o 1 l i t e r f o r  standardized  against  in  weight,  of  NaOH  volume. T h e s o l u t i o n i s t h e n  biphthalate  as  standard  pellets i s standardized  substance  (0.8-0.9g  0.1N s o l u t i o n s ) .  Standard About  precision  S o l u t i o n o f 0.1N 12.7g o f r e s u b l i m e d balance  Erlenmeyer f l a s k water a r e added is  acid  concentration  solution  amount,  diluted  3.  sulfuric  NaOH.  An  for  Acid  to the desired  solutions).  standard  2.  and C h e m i c a l s  to  with  the  Iodine iodine nearest  glass stopper.  to the flask.  i s accurately tenth  makes 1 l i t e r  o f a miligram  i»0g o f KI and a b o u t  I t i s then  i n s o l u t i o n . The volume i s made up t o  iodine  weighed  shaken u n t i l 1  liter.  on a i n an  25ml o f  a l l iodine 12.692g  o f 0.1N s o l u t i o n . The s o l u t i o n i s k e p t  of in a  Appendix  brown b o t t l e  4. S t a r c h Two to  The  5.  with g l a s s  stopper.  Indicator grams o f s o l u b l e  starch  make a p a s t e . I t i s t h e n  boiling  point.  indicator  i s mixed  diluted  with s u f f i c i e n t  t o 200ml and h e a t e d  0.2 t o 0.4ml o f t o l u e n e i s added i s stored  water  t c the  as p r e s e r v a t i v e .  i n a glass-stoppered bottle.  Cobalt Chloride Solution 9.088g  liter  of  cobalt  of d i s t i l l e d  6. Somogyi C o p p e r  chloride  of  potassium  water. cupric 180g  100ml o f n o r m a l  phosphate  NaOH and t h e n  pentahydrate  stored  The volume i s t h e n  heptahydrate  are dissolve  in 1  and  40g  i n 700ml o f d i s t i l l e d  80ml  a r e added  o f anhydrous sodium s u l f a t e  solution.  i s dissolved  Reagent  sodium t a r t r a t e  sulfate  hexahydrate  water.  52.9g o f d i s c d i u m h y d r o g e n  7.  227  I I I : S o l u t i o n s and C h e m i c a l s  of  10%  solution  to the s o l u t i o n .  i s dissolved  in  made up t c 1 l i t e r .  the  of  Finally  resulting  The s o l u t i o n i s  i n a glass-stoppered bottle.  Arsenomolybdate Fifty  grams o f ammonium  distilled  water.  stirred  in.  heptahydrate, solution.  Reagent  42ml o f Six  The  final  concentrated  grams  dissolved  molybdate i s d i s s o l v e d  of  sulfuric  disodium  i n 50ml o f water, mixture  i s  then  i n 900ml o f  acid  hydrogen  i s then  added  is  then  arsenate t c the  i n c u b a t e d a t 37°C f o r 2  Appendix  I I I : Solutions  days. I t i s then f i l t e r e d  and C h e m i c a l s  and s t o r e d  228  i n a brown b o t t l e .  8. Sodium m e t a - B i s u l p h i t e b u f f e r One  liter  o f phosphate  24.36g o f d i s o d i u m citric  acid  toluene.  i s  added 25%  protected  (with  light  sterile  1.5g sulfate diluted  syringe This  refrigerated i s added,  of  and h y p o d e r m i c  solution  and t h e n d i s c a r d e d  just  under prior  needle) to  i s refrigerated,  when i t i s 6 months e l d .  Alcohol 95% c o n c e n t r a t i c n  concentration measured  Ferroin  is  and 11.47g o f  Solution  alcohol.  alcohol  indirectly  buffer  meta-bisulphite  Stock  25% S o l u t i o n o f E t h y l  the  heptahydrate  with  o f a s o l u t i o n o f 1OOmicrograms/ml o f v i t a m i n  ethyl  from  Ethyl  11.  Cobalamin  milliliter  until  The  (pH 4.5) i s made  100ml o f b u f f e r .  One  99ml o f  buffer  phosphate  One gram o f sodium  9. S t a n d a r d  10.  hydrogen  monohydrate.  to use, t o every  B12  citrate  with a  reaches  25%.  i s d i l u t e d with The  concentraticn  water is  hydrometer.  Indicator  o f 1,10-phenanthroline heptahydrate  t o 100ml.  are  m e n c h y d r a t e and 0.7g o f f e r r o u s  dissolved  in  distilled  water  and  a p p e n d i x I I I : S o l u t i o n s and C h e m i c a l s III.2  CHEMICALS  1. A c e t i c 2.  Acid  Glacial  - Allied  C h e m i c a l s 3019.  Agar B a c t o Agar - D i f c o  3. Ammonium 4. B e e f  0140-01-0.  M o l y b d a t e - B a k e r 0716.  Extract  Bacto Beef E x t r a c t  - Difco  0126-01.  5. C a l c i u m C a r b o n a t e , Powder - a l l i e d 6. C a l c i u m H y d r o x i d e - a l l i e d 7.  Citric  acid  Monchydrate,  8. C o b a l t C h l o r i d e 9. C u p r i c  C h e m i c a l s 1506.  C h e m i c a l s 1522.  Powder - F i s h e r  Hexahydrate,  Sulfate Pentahydrate - a l l i e d  11. E t h e r - B a k e r  C h e m i c a l s 1651.  E t h y l A l c o h o l , 95%.  13.  F e r r o u s Ammonium  14.  Ferrous  J590.  9240.  12.  Mallinckrodt  A-105.  C r y s t a l - Baker 1670.  10. 5 , 6 - d i m e t h y l B e n z i m i d a z o l e (DMBZ) - B a k e r  -  229  Sulfate  Hexahydrate  (Mohr»s  Salt),  Crystal  5064. Sulfate  Heptahydrate,  Granular,  Beagent - Baker  2070. . 15. F i s h  Plant  Besearch  B o a r d o f Canada.  16.  :(•)  Effluent  Glucose  - Vancouver  (Dextrose),  L a b o r a t o r y o f The  Anhydrous  33519. 17.  Iodine.  18.  Lactic  19.  Mercuric Sulsulphate - Fisher  Acid  - B a k e r 0194. M-190.  - British  Fisheries  Drug  Houses  Appendix  20.  III : Solutions  and  Chemicals  Peptone Bacto Peptone  - Difco  0118-01.  21.  1,10-phenanthroline Honohydrate  22.  Phenolphthalein -  23.  Potassium  Biphthalate  — Allied  24.  Potassium  Dichromate,  Crystal - Allied  25.  Potassium  Iodide,  26.  Potassium  Dihydrogen  Potassium  Phosphate,  Potassium  Sodium T a r t r a t e  27.  Sodium  allied  - Baker  Chemicals  T170.  2052.  Chemicals  C r y s t a l - Baker  2083.  Chemicals  3164.  Monobasic  - Mallinckrodt  (Rochelle  Propionic  29.  Reinforced  30.  Silver  S u l p h a t e - Baker  31.  Sodium  Arsenate, Dibasic,  32.  Sodium M e t a - b i s u l f i t e - F i s h e r  33.  Sodium C h l o r i d e Sodium  - Fisher  Clostridial  S-387.  A-258. Medium - O x o i d  CM149.  3436.  - Fisher  Hydroxide,  7100.  Salt)  Potassium T a r t r a t e , C r y s t a l - F i s h e r Acid  2088.,  Phosphate  28.  34.  230  G r a n u l a r - Baker  3486.  S-244.  S-271.  Electrolytic  Pellets  -  Fisher  S-318  (78575). 35.  Sodium  Phosphate,  (Disodium  Dibasic,  Heptahydrate  Hydrogen P h o s p h a t e  Sodium S u l f a t e , A n h y d r o u s - F i s h e r  37.  Spent  Liquor  S-421.  (SSL) - ITT R a y o n i e r  Inc., Port  Division. 38.  Starch,  Soluble  39.  Sulfuric Acid,  40.  Vitamin  B12  Powder - A l l i e d Reagent  3824.  Heptahydrate).  36.  Sulfite  - Baker  - Fisher  Chemicals A-300.  2352.  Angeles  Appendix Rubramin 41.  B12 A s s a y  Vitamin Bacto  43.  100mcg/ml - S q u i b b  V i t a m i n B12 A s s a y Bacto  42.  I I I : S o l u t i o n s and C h e m i c a l s  Bacto  Medium  Medium  U.S.P. - D i f c o  B12 I n o c u l u m  B12 Inoculum  B r o t h U.S.P. - D i f c o  45.  least  Extract  Paste  46.  Yeast  Extract  Powder  Bacto  Yeast  Extract  Yeast 4159.  0541-15.  Broth  D(+) X y l o s e - B r i t i s h  Corporation  0457-15  B12 C u l t u r e Agar  44.  NB  72240.  B12 C u l t u r e Agar O.S.P. - D i f c o  Vitamin  231  Extract  0542-15.  Drug Houses 227198.  - Oxoid  L-20.  Powder - D i f c o Powder  -  0127-01 Nutritional  Biochemicals  

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