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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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P O T E N T I A L O F S P E N T S U L F I T E L I Q U O R A S R A W M A T E R I A L F O R P R O D U C T I O N C F V I T A K I N E 1 2 B Y C E R T A I N S P E C I E S O F L A C T O B A C I L L U S , P R O P I O N I B A C T E R I A , A N D S T R E F T C W Y C E S by j T R U O N G T H I E U L O N G B . S C A . e t D i p l o m e d * I n g e n i e u r , E c c l e P c l y t e c h n i q u e , M o n t r e a l , 1 9 6 8 H . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1 9 7 2 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F C R T H E D E G R E E C F M A S T E R O F A P P L I E D S C I E N C E i n t h e D e p a r t m e n t o f C H E M I C A L E N G I N E E R I N G We a c c e p t - t h i s t h e e i s a s c o n f o r m i n g t o t h e r ^ j a l r e d s t a n d a r d T H E U N I V f R S I T Y O F B R I T I S H C O L U M B I A A u g u s t 1 9 7 3 In presenting 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 of the requirements 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 that 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 reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying 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 gain s h a l l not be allowed without my w r i t t e n 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 A B S T R A C T S p e n t s u l f i t e l i q u o r i s s u i t a b l e f o r g r o w t h 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 , 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 , a n d L a c t o b a c i l l u s p l a n t a r i u m b u t n o t f o r S t r e p t o m y c e s g r i s e u s a n d S t r e p t o m y c e s o l i v a c e u s . 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 t o g e t h e r , i n f e r m e n t a t i o n s o f t w o s t a g e s o r o f o n e s t a g e w i t h m i x e d c u l t u r e , a r e p o o r i n s e r v i n g t h e u l t i m a t e g o a l o f p r o d u c i n g v i t a m i n B 1 2 i n s u b s t a n t i a l q u a n t i t y . P . s h e r m a n i i i s n o t a s g o o d a p r o d u c e r o f v i t a m i n B 1 2 f r o m S S L a s P . f r e u d e n r e i c h i i a l o n e . F e r m e n t a t i o n s o f S S L w i t h P . f r e u d e n r e i c h i i s h o w a n e t i n c r e a s e i n m a r k e t v a l u e o f t h e b r o t h . T h e y s h o u l d b e d o n e i n t w o s t a g e s , t h e f i r s t a n a e r o b i c a n d t h e s e c o n d a e r o b i c . T h e o p t i m u m r e q u i r e m e n t s a r e 5 g / l o f y e a s t e x t r a c t a s e s s e n t i a l n i t r o g e n s o u r c e a n d 1 p p m o f c o b a l t i o n s a s p r e c u r s o r . T h e o p t i m u m y i e l d i n v i t a m i n B 1 2 i s 1 . 5 m g / l , a c c o m p a n i e d b y t h e p r o d u c t i o n o f 0 . 1 4 m o l e s / 1 o f v o l a t i l e a c i d s . 5 , 6 - d i m e t h y l b e n z i m i d a z o l e i s f o u n d t o b e b e n e f i c i a l t o t h e y i e l d s o f c e l l s , o f v i t a m i n B 1 2 a n d o f v o l a t i l e a c i d s . F i s h p l a n t e f f l u e n t i s n o t a s u i t a b l e r e p l a c e m e n t f o r y e a s t e x t r a c t . T h e r e d u c t i o n o f t h e p o l l u t i n g p o t e n t i a l o f S S L , a c c o m p l i s h e d b y t h e s e f e r m e n t a t i o n s , i s o n l y a b o u t 145? o f t h e A b s t r a c t i i t o t a l c h e m i c a l o x y g e n d e m a n d . T h i s r e d u c t i o n i s d u e t o t h e r e m o v a l o f a b o u t 7 0 * o f t h e r e d u c i n g s u g a r s , t r a n s f o r m e d t c c e l l s a n d v o l a t i l e a c i d s . T h e s e s u g a r s o r i g i n a l l y c c n t r i t u t e a p o l l u t i n g p o t e n t i a l o f 2 0 * o f t h e t o t a l C O D t o S S L . A b s t r a c t i i i T A B L E O F C O N T E N T S P a g e A B S T R A C T i T A B L E O F C O N T E N T S i i i L I S T O F T A B L E S v i L I S T O F F I G O B E S i x A B B R E V I A T I O N S x i v A C K N O W L E D G M E N T S XV C h a p t e r 1 I N T R O D U C T I O N T 1 . 1 S p e n t S u l f i t e L i q u o r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 . 1 . 1 C o m p o s i t i o n 1 1 . 1 . 2 P o l l u t i o n C a u s e d b y S p e n t S u l f i t e L i q u o r . . . . . . 2 1 . 1 . 3 T r e a t m e n t s a n d U s e s 8 1 . 2 V i t a m i n B 1 2 1 0 1 . 3 M i c r o o r g a n i s m s U s e d 1 7 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 7 1 . 3 . 2 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 a n d S h e r m a n i i . 1 7 1 . 3 . 3 S t r e p t o m y c e s G r i s e u s a n d O l i v a c e u s . . . . . . . . . . . . 2 0 1 . 4 O b j e c t i v e 2 1 T a b l e o f C o n t e n t s i v C h a p t e r P a g e 2 E X P E R I M E N T A L T E C H N I Q U E S 2 3 2 . 1 P r e p a r a t i o n o f I n o c u l u m 2 3 2 . 2 T r e a t m e n t s P r i o r t o F e r m e n t a t i o n 2 4 2 . 2 . 1 P r e p a r a t i o n o f S p e n t S u l f i t e L i g u c r 2 4 2 . 2 . 2 N u t r i e n t A d d i t i o n 3 1 2 . 3 F e r m e n t a t i o n a n d A p p a r a t u s . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2 2 . 3 . 1 A p p a r a t u s 3 2 2 . 3 . 2 P r e f e r m e n t a t i o n 3 3 2 . 3 . 3 F e r m e n t a t i o n 3 7 2 . 4 A n a l y s i s M e t h o d s 3 8 2 . 4 . 1 M e a s u r e m e n t o f B a c t e r i a l G r o w t h 3 9 2 . 4 . 2 M e a s u r e m e n t o f S u g a r C o n c e n t r a t i o n 4 5 2 . 4 . 3 M e a s u r e m e n t o f V i t a m i n B 1 2 C o n c e n t r a t i o n 4 5 2 . 4 . 4 M e a s u r e m e n t o f A c i d s C o n c e n t r a t i o n . . . . . . . . . . . . 4 8 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 l O x y g e n D e m a n d . . . . . . . 5 6 3 E X P E R I M E N T S A N D R E S U L T S 6 3 3 . 1 F e r m e n t a t i o n w i t h S t r e p t o m y c e s 6 3 3 . 2 G r o w t h o f L a c t o b a c i l l u s i n X y l o s e . . . . . . . . . . . . . . . . . . . 6 9 3 . 3 P r e l i m i n a r y S t u d i e s o n Y e a s t E x t r a c t . . . . . . . . . . . . . . . . 6 9 3 . 4 Y e a s t E x t r a c t f r o m D i f f e r e n t S o u r c e s 7 7 3 . 5 F i s h P l a n t E f f l u e n t a s N i t r o g e n S o u r c e 7 9 3 . 6 P r e l i m i n a r y E x p e r i m e n t s i n E r l e n m e y e r F l a s k s . . . . . . . . 8 0 3 . 7 F e r m e n t a t i o n s i n T h e 7 1 M i c r o f e r m F e r m e n t o r s . . . . . . . . 8 9 3 . 8 F e r m e n t a t i o n o f S o l u t i o n o f G l u c o s e . . . . . . . . . . . . . . . . . 1 5 7 T a b l e o f C o n t e n t s V C h a p t e r P a g e 4 D I S C U S S I O N S 1 5 9 4 . 1 F e r m e n t a t i o n w i t h S t r e p t o m y c e s . . . . . . . . . . . . . . . . . . . . . . 1 5 9 4 . 2 F e r m e n t a t i o n w i t h I . P l a n t a r i u m . . . . . . . . . . . . . . . . . . . . . 1 6 0 4 . 3 F e r m e n t a t i o n w i t h M i x e d C u l t u r e . . . . . . . . . . . . . . . . . . . . . 1 6 2 4 . 4 F e r m e n t a t i o n w i t h P . S h e r m a n i i 1 6 4 • 4 . 5 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 . . . . . . . . . . . . . . . . . 1 6 4 4 . 5 . 1 O s e o f S u g a r s 1 6 4 4 . 5 . 2 E f f e c t o f p f l 1 6 5 4 . 5 . 3 E f f e c t o f A i r . . . . . 1 6 6 4 . 5 . 4 E f f e c t o f A g i t a t i o n a n d V o l u m e o f B r o t h . . . . . . . 1 6 7 4 . 5 . 5 E f f e c t o f l e a s t E x t r a c t C o n c e n t r a t i o n . . . . . . . . . 1 6 9 4 . 5 . 6 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 . . . . . . . . . . . . 1 7 0 4 . 5 . ? E f f e c t o f 5 , 6 - d i m e t h y l B e n z i m i d a z c l e 1 7 0 4 . 6 P r o d u c t i o n o f A c i d s b y P . F r e u d e n r e i c h i i 1 7 1 4 . 7 P r o d u c t i o n o f V i t a m i n B 1 2 b y P . F r e u d e n r e i c h i i . . . . . . 1 7 3 4 . 8 N u t r i e n t R e q u i r e m e n t s a n d C o s t s . . . . . . . . . . . . . . . . . . . . . 1 7 5 4 . 9 F e r m e n t a t i o n o f S o l u t i o n o f G l u c o s e 1 7 7 5 C O N C L U S I O N S 1 7 8 B I B L I O G R A P H Y 1 8 0 L I S T O F O C C U R E N C E S O F R E F E R E N C E S 1 9 0 A P P E N D I X I : T a b u l a t e d R e s u l t s . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 9 3 A P P E N D I X I I : A n a l y s i s P r o c e d u r e s . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 3 A P P E N D I X I I I : S o l u t i o n s a n d C h e m i c a l s . . . . . 2 2 6 T a b l e o f C o n t e n t s v i L I S T O F T A B L E S T a b l e P a g e 1 . 1 T h e P e r c e n t a g e C o m p o s i t i o n o f C e r t a i n H o o d s . . . . . . . . . 2 1 . 2 Y i e l d o f P o t e n t i a l R e d u c i n g a n d F e r m e n t a b l e S u g a r s o f H o o d s 3 1 . 3 T h e P e r c e n t a g e C o m p o s i t i o n o f S u g a r s o f T y p i c a l 1 S p e n t S u l f i t e L i q u o r s a n d H e e d H y d r o l y z a t e s . . . . . . . . . 4 1 . 4 C o m p o s i t i o n o f T y p i c a l S p e n t S u l f i t e L i q u o r s . . . . . . . . 5 1 . 5 A G e n e r a l l y A c c e p t e d p i c t u r e c f T h e C o m p o s i t i o n o f S p e n t S u l f i t e L i q u o r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1 . 6 M o r t a l i t i e s o f S a l m o n E x p o s e d t o S S L . . . . . . . . . . . . . . . . 7 1.7 T r e a t m e n t s a n d U s e s o f S p e n t S u l f i t e L i q u o r 9 1 . 8 P r o c e s s e s f o r M i c r o b i a l S y n t h e s i s o f V i t a m i n B 1 2 . . . . 1 3 1 . 9 A n t i b i o t i c - p r o d u c i n g F e r m e n t a t i o n s w i t h V i t a m i n B 1 2 a s B y - p r o d u c t . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6 1 . 1 0 M i c r o o r g a n i s m s U s e d i n C o m m e r c i a l P r o d u c t i o n o f V i t a m i n B 1 2 1 6 1 . 1 1 V i t a m i n B 1 2 P r o d u c e r s i n T h e U n i t e d s t a t e s . . . . . . . . . . 1 6 1 1 . 1 2 R e i n f o r c e d C l o s t r i d i a l M e d i u m 2 3 1 1 . 1 3 W a k s m a n ' s N o . 3 M e d i u m 2 4 1 1 . 1 4 E f f e c t i v e n e s s o f V a r i o u s M e t h o d s o f R e m o v a l o f S u l f u r D i o x i d e 2 7 1 1 . 1 5 C o n c e n t r a t i o n o f N u t r i e n t s i n S S L f o r P r o p i o n i b a c t a r i a a n d L a c t o b a c i l l u s . . . . . . . . . . . . . . 3 1 L i s t o f T a b l e s v i i T a b l e P a g e 1 1 . 1 6 E f f e c t o f V a r i o u s P r e t r e a t m e n t s o n C e l l s S u b m i t t e d t o V i t a m i n B 1 2 A s s a y . . . . . . . . . . . . . . . . . . . . . . 4 8 1 1 . 1 7 M a n u a l E t h e r E x t r a c t i o n o f V o l a t i l e a n d l a c t i c A c i d s f r o m a n A q u e o u s S o l u t i o n . . . . . . . . . . . . . . . . . . . . . . 5 1 1 1 . 1 8 R e s u l t s o f C O D T e s t s o n 3 S a m p l e s 5 7 1 1 . 1 9 R e s u l t s o f C o r r e c t i n g M e a s u r e s c n C O D T e s t . . . . . . . . . . 5 8 1 1 . 2 0 R e l i a b i l i t y o f T h e C O D T e s t , . 5 9 1 1 1 . 2 1 F e r m e n t a t i o n s o f D i l u t e d S S L b y S . g r i s e u s . . . . . . . . . . 6 4 1 1 1 . 2 2 F e r m e n t a t i o n s o f D i l u t e d S S L b y S . o l i v a c e u s 6 5 1 1 1 . 2 3 S u m m a r y o f i n f o r m a t i o n o n f e r m e n t a t i o n s o f S S L i n 7 1 F e r m e n t o r s 9 0 1 1 1 . 2 4 S u m m a r y o f O v e r a l l R e s u l t s -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 . . . . . . . . . . . . . . . 1 2 4 1 1 1 . 2 5 S u m m a r y o f O v e r a l l R e s u l t s -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 1 2 5 1 1 1 . 2 6 F e r m e n t a t i o n o f A q u e o u s S o l u t i o n s c f G l u c o s e . . . . . . . . 1 5 8 I V . 2 7 C o s t a n d P r o f i t c f F e r m e n t a t i o n s w i t h 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 7 5 A I . 2 8 F e r m e n t a t i o n o f S S L w i t h L . p l a n t a r i u m i n E r l e n m e y e r F l a s k s 1 9 6 A I . 2 9 F e r m e n t a t i o n 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 E r l e n m e y e r F l a s k s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 9 6 A I . 3 0 F e r m e n t a t i o n o f S S L w i t h P . s h e r m a n i i i n E r l e n m e y e r F l a s k s 1 9 7 A I . 3 1 F e r m e n t a t i o n o f S S L w i t h A M i x e d C u l t u r e o f 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 E r l e n m e y e r F l a s k s 1 9 7 A I . 3 2 R u n F P 1 . 1 . F e r m e n t a t i o n w i t h L . p l a n t a r i u m . . . . . . . . . . 1 9 8 A I . 3 3 R u n F P 1 . 2 . F e r m e n t a t i o n w i t h L . p l a n t a r i u m . . . . . . . . . . 1 9 9 L i s t o f T a b l e s v i i i T able Page AI.34 Run FP2.1. Fermentation with 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 as Main Substrate 200 AI*35 Run FP2.2. Fermentation with 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 as Main S u b s t r a t e 201 AI.36 Run FP3. Fermentation with Mixed C u l t u r e of L. plantarium and P. f r e u d e n r e i c h i i 202 AI.37 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 of pH 203 AI.38 Run F15.22b. Fermentation with P. f r e u d e n r e i c h i i i n One Anaerobic Stage 204 AI.39 Run F05.22. 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 as V a r i a b l e ............. 205 AI . 4 0 Run F10.22. 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 as V a r i a b l e 206 AI . 4 1 Run F20.22. 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 as V a r i a b l e 207 AI.42 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 as V a r i a b l e ............. 208 AI.43 Run F20.00. Fermentation with P. f r e u d e n r e i c h i i . Cobalt 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,. Fermentation with 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 ................ 210 AI.45 Run F20.22.126. Fermentation of SSL C o n t a i n i n g DMBZ with P. f r e u d e n r e i c h i i 211 AI.46 RunS20.22. Fermentation with P. shermanii .......... 212 L i s t of Tables ix L I S T O F F I G U R E S F i g u r e P a g e 1 . 1 S t r u c t u r e o f V i t a m i n B 1 2 11 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 C l o s t r i d i a l M e d i u m . . . . . . 1 8 1 . 3 L . p l a n t a r i u m i n S p e n t S u l f i t e L i q u o r 1 8 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 C l o s t r i d i a l M e d i u m . . 1 8 1 . 5 P . f r e u d e n r e i c h i i i n S p e n t S u l f i t e L i q u o r 1 8 1 . 6 P . s h e r m a n i i i n R e i n f o r c e d C l o s t r i d i a l M e d i u m 1 9 1 . 7 P« s h e r m a n i i i n , S p e n t S u l f i t e L i q u o r 1 9 1 . 8 S . g r i s e u s i n W a k s m a n ' s M o . 3 M e d i u m 1 9 2 . 9 S t r i p p i n g o f S u l f u r D i o x i d e b y B o i l i n g a n d A e r a t i o n . 2 9 2 . 1 0 I n c u b a t i o n , o f S . g r i s e u s a n d S . 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 N B S I n c u b a t o r - s h a k e r 3 4 2 . 1 1 R i g h t H a l f o f T h e B B S M i c r o f e r m T w i n u n i t , . 3 4 2 . 1 2 S i d e V i e w o f T h e F e r m e n t o r H e a d 3 4 2 . 1 3 T o p V i e w o f T h e F e r m e n t o r H e a d 3 5 2 . 1 4 T h e p f l C o n t r o l l e r 3 6 2 . 1 5 S t e r i l i z a t i o n o f T h e 7 1 F e r m e n t o r 3 6 2 . 1 6 F o a m i n g i n T h e 7 1 F e r m e n t o r 3 6 2 . 1 7 R e l a t i o n b e t w e e n M o d i f i e d a n d S t a n d a r d T u r b i d i t y . . . . 4 1 L i s t o f F i g u r e s X F i g u r e P a g e 2 . 1 8 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 C r y C e l l H e i g h t 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 . . . . . . . . . . . 1 2 2 . 1 9 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 C e l l W e i g h t o f 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 4 3 2 . 2 0 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 C r y C e l l W e i g h t 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 4 4 2 . 2 1 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 B 1 2 C u l t u r e A g a r . . 4 6 2 . 2 2 P a r t i t i o n o f V o l a t i l e A c i d s b e t w e e n W a t e r a n d E t h e r . 4 9 2 . 2 3 S p e c i a l G l a s s w a r e f o r A c i d i f i c a t i o n o f S a m p l e s w i t h C o n c e n t r a t e d S u l f u r i c A c i d . . . . . . . . . . . . . . . . . A , . 4 6 2 . 2 4 T h e E t h e r E x t r a c t o r . . . . . . . . . . . . . 5 2 2 . 2 5 R e c o v e r y o f A c i d s f r o m S S L b y C o n t i n u o u s E t h e r E x t r a c t i o n 5 4 2 . 2 6 R e c o v e r y o f L a c t i c A c i d f r o m S S L b y C o n t i n u o u s E t h e r E x t r a c t i o n 5 5 3 . 2 7 G r o w t h o f S t r e p t o m y c e s G r i s e u s i n D i l u t e d S S L . . . . . . . 6 7 3 . 2 8 G r o w t h o f S t r e p t o m y c e s O l i v a c e u s i n D i l u t e d S S L . . . . . 6 7 3 . 2 9 G r o w t h o f L . p l a n t a r i u m i n S o l u t i o n s o f X y l o s e a n d G l u c o s e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 8 3 . 3 0 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 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 7 1 3 . 3 1 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 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 7 3 3 . 3 2 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 P . s h e r m a n i i i n S S L i n T e s t T u b e s . , 7 5 3 . 3 3 L e g e n d f o r F i g u r e s N u m b e r e d f r o m 3 . 3 0 t o 3 . 3 2 . . . . . . . 7 6 3 . 3 4 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 Y e a s t E x t r a c t f r o m V a r i o u s S o u r c e s . 7 8 3 . 3 5 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 i n F l a s k s . . . 8 2 3 . 3 6 F e r m e n t a t i o n s 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 F l a s k s 8 4 L i s t o f F i g u r e s x i F i g u r e P a g e 3 . 3 7 F e r m e n t a t i o n s o f S S L w i t h P . s h e r m a n i i i n F l a s k s . . . . 8 6 3 . 3 8 F e r m e n t a t i o n s o f S S L w i t h A M i x t u r e o f 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 F l a s k s . . . . . . . 8 8 3 . 3 9 R u n F P 1 . 1 . F e r m e n t a t i o n w i t h L . p l a n t a r i u m . . . . . . . . . . 9 3 3 . 4 0 R u n F P 1 . 2 . F e r m e n t a t i o n w i t h L . p l a n t a r i u m . . . . . . . . . . 9 5 3 . 4 1 R u n F P 2 . 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 o n L a c t i c A c i d a s M a i n S u b s t r a t e 9 7 3 . 4 2 R u n F P 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 o n L a c t i c A c i d a s M a i n S u b s t r a t e 9 9 3 . 4 3 R u n F P 3 . F e r m e n t a t i o n w i t h M i x e d C u l t u r e o f 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 1 0 1 3 . 4 4 R u n F 1 5 . 2 2 a . 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 . E f f e c t o f p H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 3 3 . 4 5 R u n F 1 5 . 2 2 b . 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 i n O n e A n a e r o b i c S t a g e 1 0 5 3 . 4 6 R u n F 0 5 . 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 . 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 . . . . . . . . . . . . . 1 0 7 3 . 4 7 R u n F 1 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 . 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 1 0 9 3 . 4 8 R u n 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 . 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 . . . . . 1 1 1 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 . 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 . . . . . . . . . . . . . 1 1 3 3 . 5 0 R u n 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 . 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 . . . . . . . . . . . . . . . . 1 1 5 3 . 5 1 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 . 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 . . . . . . . . . . . . . . . . 1 1 7 3 . 5 2 R u n F 2 0 ^ 2 2 . 1 2 6 . F e r m e n t a t i o n o f S S L C o n t a i n i n g D M B Z w i t h P . f r e u d e n r e i c h i i 1 1 9 3 . 5 3 R u n S 2 0 . 2 2 . F e r m e n t a t i o n w i t h P . s h e r m a n i i . . . . . . . . . . 1 2 1 L i s t o f F i g u r e s x i i F i g u r e P a g e 3 . 5 4 L e g e n d f o r A l l F i g u r e s , L i s t e d i n T a b l e I I I . 2 3 , f r o m 3 . 3 9 t o 3 . 5 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 2 3 . 5 5 P r o d u c t i o n o f V i t a m i n B 1 2 b y P . f r e u d e n r e i c h i i . 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 . . . . . . . . . . . . . . . 1 2 7 3 . 5 6 P r o d u c t i o n o f V i t a m i n B 1 2 b y 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 . . . . . . . . . . . . . . . . . . 1 2 9 3 . 5 7 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 . 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 . . . . . . . . . . . . . . . 1 3 1 3 . 5 8 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 . 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 . . . . . . . . . . . . . . . . . . 1 3 3 3 . 5 9 L e g e n d f o r A l l F i g u r e s N u m b e r e d f r o m 3 . 5 5 t o 3 . 5 8 . . . 1 3 4 3 . 6 0 H u n F P 1 . 1 . F e r m e n t a t i o n R a t e o f L . p l a n t a r i u m . . . . . . . 1 3 6 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 w i t h L a c t i c A c i d a s M a i n S u b s t r a t e . . . . . . . . . . . . . . . . . . 1 3 8 3 . 6 2 R u n F 0 5 . 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 . 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 1 4 0 3 . 6 3 R u n F 1 0 . 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 . 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 . . . . . . . . . . . . . 1 4 2 3 . 6 4 R u n F 2 0 . 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 . 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 . . . . . . . . . . . . . 1 4 4 3 . 6 5 R u n F 3 0 . 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 . 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 1 4 6 3 . 6 6 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 1 4 8 3 . 6 7 R u n F 2 0 . 0 9 . 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 . . . . . . . . . . . . . . . . 1 5 0 3 . 6 8 R u n F 2 0 . 2 2 . 1 2 6 . F e r m e n t a t i o n R a t e o f P F i n S S L w i t h A d d i t i o n o f D M B Z 1 5 2 3 . 6 9 R u n S 2 0 . 2 2 . F e r m e n t a t i o n R a t e o f P . s h e r m a n i i . . . . . . . 1 5 4 3 . 7 0 L e g e n d f o r A l l F i g u r e s N u m b e r e d f r o m 3 . 6 0 t o 3 . 6 9 . . . 1 5 5 3 . 7 1 R e l a t i v e C a p a c i t y o f P S a n d P F i n F e r m e n t i n g S S L w i t h L a c t i c A c i d a s M a i n S u b s t r a t e . . . . . . . . . . . . . . 1 5 6 L i s t o f F i g u r e s x i i i F i g u r e P a g e 4 . 7 2 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 S S I ( B u n F P 3 ) . 1 6 3 A 2 . 7 3 D r y i n g o f A l u m i n u m D i s h e s i n T h e I s o t e m p O v e n . . . . . . . 2 1 5 A 2 . 7 4 T y p i c a l S t a n d a r d C u r v e f o r T h e R e d u c i n g S u g a r T e s t . . 2 1 6 A 2 . 7 5 T y p i c a l S t a n d a r d C u r v e f o r T h e V i t a m i n E 1 2 A s s a y . . . . 2 2 0 L i s t o f F i g u r e s x i v A B B R E V I A T I O N S B O D - B i o c h e m i c a l O x y g e n D e m a n d . G O D - C h e m i c a l O x y g e n D e m a n d . D H B Z - 5 , 6 - d i m e t h y l B e n z i m i d a z o l e . L . - L a c t o b a c i l l u s . L P - L a c t o b a c i l l u s P l a n t a r i u m . P . - P r o p i o n i b a c t e r i a . P 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 . P S - 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 . - S t r e p t o m y c e s . S S L - S p e n t S u l f i t e L i q u o r o r S u l f i t e S p e n t L i q u o r . ( a l s o c a l l e d W a s t e S u l f i t e L i q u o r ) . 0 0 D - U l t i m a t e O x y g e n D e m a n d . A b b r e v i a t i o n s ACKNOWLEDGMENTS XV Sheer words cannot formulate my thanks to those who helped ae through t h i s p r o j e c t . N e v e r t h e l e s s , words are the only means wi t h i n my reach to express, at l e a s t part o f, my indebtedness : -To Dr. Ric h a r d Branion and Dr. Ken Pind e r , under whose d i r e c t i o n t h i s work was undertaken, f o r t h e i r guidance, and most of a l l , f o r t h e i r p a t i e n c e throughout the length of time of t h i s work. -To Dr. George S t r a s d i n e of the F i s h e r i e s Research Board of Canada f o r t e c h n i c a l advice as well as f o r p r o v i d i n g stock c u l t u r e s of b a c t e r i a and f i s h p l a n t e f f l u e n t . -To the v a r i o u s i n d i v i d u a l s of the f a c u l t y and s t a f f c f the Department of M i c r o b i o l o g y , namely, to Mrs. 0. V o l k o f f f o r her i n s t r u c t i o n i n m i c r o b i o l o g i c a l t e c h n i q u e s , to Dr. D e l f a Syeklocha f o r her i n s t r u c t i o n i n performing the vitamin B12 assay, t o Br. B i l l Page f o r h i s maintenance of stock c u l t u r e s of Streptcmyces. -To the ITT Rayonier Inc. f o r the spent s u l f i t e l i q u o r s u p p l i e d . -To the Department of Energy, Mines and Resources - Water Resources D i v i s i o n , the N a t i o n a l Research C o u n c i l and the U n i v e r s i t y of B r i t i s h Columbia f o r t h e i r f i n a n c i a l a s s i s t a n c e . -And f i n a l l y , t o a l l my f r i e n d s who have come, and most Acknowledgments x v i probably gone, l e a v i n g behind them, i n my memory, the u n f o r g e t t a b l e s o u v e n i r s of a l l those s o c i a l g e t - t o g e t h e r s and those " p h y s i c a l education l e s s o n s " where the presence of every i n d i v i d u a l had made a prec i o u s c o n t r i b u t i o n to the success and the joy of the events. Acknowledgments 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. . ... kinh.tang.phu.mau........men.tang.chau............. ....tang.be.diem........tang.be.mat.hat.de............. ....tang.chi.kirn..........tang.be.van.thanh............ • P o t e n t i a l of Spent S u l f i t e l i g u o r | as Raw M a t e r i a l f o r Pro d u c t i o n of Vitamin B12 I by C e r t a i n Species of | L a c t o b a c i l l u s , P r o p i o n i b a c t e r i a , and Streptomyces | j . ... tang, van.hoa. t i e n ....tang.nguoi.ban.than......tang.van.khanh . ....tang a . f r a n c i n e . . . . . . . . . . . . . . . . . . . P o t e n t i a l o f S p e n t S u l f i t e L i q u o r C h a p t e r 1 I N T R O D U C T I O N 1 . 1 S P E N T _ S U L F I T E _ I I 2 U 0 R 1 . 1 . 1 C o m p o s i t i o n I n t h e s u l f i t e p u l p i n g p r o c e s s , w o o d c h i p s a r e h e a t e d u n d e r p r e s s u r e a n d a t e l e v a t e d t e m p e r a t u r e ( a b o u t 1 4 0 ° c t o 160<>c) w i t h a s o l u t i o n o f b i s u l f i t e o f c a l c i u m , m a g n e s i u m , s o d i u m ; o r a m m o n i u m , u s u a l l y w i t h a n e x c e s s o f s u l f u r d i o x i d e . T h i s c o o k i n g a c i d , o n c e u s e d , i s d i s c h a r g e d a s s p e n t s u l f i t e l i g u o r ( S S L ) c o n t a i n i n g d i s s o l v e d w o o d c o m p o n e n t s ( a b o u t 50?? o f w e e d u s e d ) . A s t h e c o m p o s i t i o n o f w o o d i s d i f f e r e n t f r o m o n e s p e c i e s t o a n o t h e r ( T a b l e s 1 . 1 a n d 1 . 2 ) , t h e c o m p o s i t i o n o f s u l f i t e s p e n t l i q u o r v a r i e s f r o m o n e m i l l t o a n o t h e r d e p e n d i n g o n t h e p a r t i c u l a r s p e c i e s a n d s o u r c e c f t h e w o o d u s e d . A l s o , t h e c o m p o s i t i o n o f S S L d e p e n d s o n t h e c o n d i t i o n s o f t h e p u l p i n g p r o c e s s . T a b l e 1 .3 s h o w s t h e c o m p o s i t i o n o f t h e s u g a r s i n S S L a n d , i n c a s e s w h e r e d a t a i s n o t a v a i l a b l e f o r S S L , o f w o o d h y d r o l y z a t e s ( w h i c h h a v e s i m i l a r c o m p o s i t i o n o f s u g a r s ) f r o m d i f f e r e n t s p e c i e s o f w o o d s . T a b l e 1 . 4 s h o w s t h e c o m p o s i t i o n o f s u l f i t e s p e n t l i q u o r s f r o m d i f f e r e n t s o u r c e s , i n c l u d i n g t h e o n e f r o m I T T R a y o n i e r , u s e d i n t h i s r e s e a r c h . N e v e r t h e l e s s a n a p p r o x i m a t e r e p r e s e n t a t i o n o f t h e c o m p o s i t i o n o f s u l f i t e s p e n t I n t r o d u c t i o n P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 2 T a b l e 1 . 1 T H E P E R C E N T A G E C O M P O S I T I O N O F C E R T A I N W O O D S [ 9 9 ] • : : T I | W o o d s p e c i e s i L H C A C H e m e P U A H | i i 1 | W h i t e s p r u c e 2 6 . 6 7 3 . 3 4 9 . 5 2 3 . 8 1 0 . 9 2 . 6 8 2 . 3 5 0 . i , 7 0 | | R e d s p r u c e 2 6 . 6 7 2 . 9 4 8 . 3 2 4 . 6 1 1 . 6 3 . 2 0 2 . 5 0 0 , 9 2 | j E a s t e r n h e m l o c k 3 1 . 5 6 8 . 5 4 8 . 2 2 0 . 3 1 0 . 0 3 . 4 0 1 . 8 7 0 , , 8 4 | ( B a l s a m f i r 3 0 . 1 6 9 . 9 4 4 . 0 2 5 . 9 1 0 . 3 3 . 0 8 2 . 2 4 0 . 4 1 ( | J a c k p i n e 2 7 . 2 7 2 . 5 4 9 . 5 2 3 . 0 1 2 . 8 2 . 9 2 1 . 9 2 0 . . 7 5 | ( A s p e n 1 7 . 3 8 2 . 5 5 0 . 7 3 1 . 8 2 3 . 5 4 . 2 8 4 . 6 5 0 . 9 3 | ( W i l l o w 2 2 . 0 7 8 . 3 ( M a p l e 2 3 . 5 7 6 . 3 5 0 . 0 2 6 . 3 ( W h i t e o a k 2 4 . 1 7 5 . 4 4 9 . 5 2 5 . 9 — -- -- — I I— 1 L : L i g n i n B C : H o l o c e l l u l o s e A C : A l p h a c e l l u l o s e H e m C : H e m i c e l l u l o s e P : P e n t o s a n s U : D r o n i c a c i d a n h y d r i d e A : A c e t y l M : M e t h o x y l i n c a r b o h y d r a t e l i q u o r ( T a b l e 1 . 5 ) i s g e n e r a l l y a c c e p t e d [ 6 4 , 1 1 0 ] . I f t h e S S L f r o m t h i s p i c t u r e i s d i s c h a r g e d w i t h o u t r e c o v e r y , a n a p p r e c i a b l e u s e f u l a m o u n t o f s u g a r s , a b o u t 2 0 * o f t h e t o t a l d i s s o l v e d s o l i d s i n S S L , i s t h r o w n a w a y a s w a s t e . 1 . 1 . 2 P o l l u t i o n C a u s e d _ b y S p e n t S u l f i t e ^ I i g u g r T h e m o r e i n d u s t r y g r o w s , t h e m o r e t h e d i s p o s a l p r o b l e m o f i t s w a s t e , w i t h h i g h s t r e a m - p o l l u t i n g p o t e n t i a l , b e c o m e s s e r i o u s . I n p a r t i c u l a r p u l p m i l l s , u s i n g t h e s u l f i t e p r o c e s s , d i s c h a r g e t h e i r w a s t e l i q u o r w i t h a 5 - d a y b i o c h e m i c a l o x y g e n I n t r o d u c t i o n P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 3 T a b l e 1 . 2 Y I E L D O F P O T E N T I A L S E D U C I N G A N D F E R M E N T A B L E S U G A R S O F WOODS [ 9 9 ] W o o d s p e c i e s P R S F P F S H a r d w o o d s A m e r i c a n B e e c h 7 0 . 1 7 5 . 1 5 2 . 6 A s p e n 7 5 . 1 7 6 . 3 5 7 . 3 B i r c h 6 9 . 9 6 7 . 8 4 7 . 4 M a p l e 6 8 . 2 7 1 . 0 4 8 . 4 R e d O a k 6 3 . 6 6 3 . 0 4 0 . 2 S w e e t g u m 6 6 . 4 7 3 . 8 4 9 . 0 Y e l l o w P o p l a r 7 0 . 9 7 6 . 1 5 4 . 0 S o f t w o o d s D o u g l a s F i r 6 6 . 6 8 6 . 2 5 7 . 4 E a s t e r n W h i t e P i n e 6 6 . 5 8 6 . 3 5 7 . 4 H e m l o c k 6 6 . 1 8 8 . 2 5 8 . 3 P o n d e r o s a P i n e 6 8 . 0 8 2 . 2 5 5 . 9 R e d w o o d 5 2 . 4 7 7 . 1 4 0 . 4 S i t k a S p r u c e 7 0 . 1 8 5 . 3 5 9 . 8 S o u t h e r n Y e l l o w P i n e 6 4 . 8 8 2 . 0 5 3 . 2 S u g a r P i n e 6 4 . 3 8 2 . 4 5 3 . 0 P R S : P o t e n t i a l r e d u c i n g s u g a r s {%) F : F e r m e n t a b i l i t y {%) P F S : P o t e n t i a l f e r m e n t a b l e s u g a r s {%) d e m a n d ( B O D ) o f a t l e a s t 4 0 0 l b s p e r t o n o f s u l f i t e p u l p . T h e t w o s u l f i t e m i l l s i n B r i t i s h C o l u m b i a a n d s e v e r a l i n W a s h i n g t o n s t a t e a l l d i s c h a r g e t h e i r w a s t e i n t o t h e s e a . E c o l o g i s t s a c c u s e S S L o f b e i n g p o i s o n o u s , o r a t l e a s t e x t r e m e l y h a r m f u l , t o a q u a t i c f a u n a ' s l i f e [ 1 0 9 ] . O n t h e o t h e r h a n d , s o m e p u l p m i l l o w n e r s a r g u e t h a t t h e l o s s o f a f e w f i s h i s i n c o n s e q u e n t i a l I n t r o d u c t i o n P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 4 T a b l e 1 . 3 T H E P E R C E N T A G E C O M P O S I T I O N O F S U G A R S O F T Y P I C A L S P E N T S U L F I T E L I Q U O R S A N D H O O D H Y D R G L Y Z A T E S [ 9 9 ] i""' ™" " ••  1 "•"1 *•" I L i q u o r T y p e S S L S S L T o t a l P a r t i a l — i H y d r o l y z a t e s H y d r o l y z a t e s j | W c o d T y p e H F S w S b P S P B — i j | S u g a r s i j H e x o s e | G l u c o s e 1 5 2 8 . 9 2 9 . 3 5 3 . 7 6 7 . 6 6 1 . 9 6 7 . 7 | M a n n o s e 4 8 4 2 . 7 3 7 . 7 2 3 . 8 1 4 . 1 2 4 . 7 1 . 8 | G a l a c t o s e 1 0 4 . 2 6 . 4 2 . 9 6 . 2 4 . 0 0 . 0 | F r u c t o s e 2 4 . 0 1 . 4 — | P e n t o s e 1 7 . 0 | X y l o s e 1 5 — 1 3 . 3 1 2 . 3 8 . 9 8 . 0 3 0 . 1 | A r a b i n o s e 6 — 5 . 4 7 . 7 3 . 2 -- 0 . 4 1 G a l a c t u r o n i c A c i d — 3 . 2 | U n i d e n t i f i e d « 4 7 . 9 . i H F : 8595 w e s t e r n h e m l o c k p l u s 1 5 1 w h i t e f i r S : S p r u c e w S : W h i t e s p r u c e S P : S p r u c e a n d p i n e j P : J a c k p i n e b P : B o r d e a u x p i n e B : B i r c h c o m p a r e d t o t h e b e n e f i t s f r o m a l a r g e d e v e l o p m e n t e m p l o y i n g h u n d r e d s o f w o r k e r s . A p o l o g i s t s g o e v e n f u r t h e r t o c l a i m [ 1 1 0 ] t h a t t h e e x t r a c a r b o h y d r a t e s o u r c e a n d t h e s l i g h t l o w e r i n g o f t h e d i s s o l v e d o x y g e n m a y h a v e b e n e f i c i a l e f f e c t s o n t h e g r o w t h I n t r o d u c t i o n P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 5 T a b l e 1 . 4 C O M P O S I T I O N O F T Y P I C A L S P E N T S U L F I T E I I Q U C B S 1— . — I S o u r c e T h i s W o r k [ 7 0 ] [ 4 9 ] [ 1 0 0 ] [ 2 5 ] | . . . J | W o o d S p e c i e s 9 5 : 5 . . . . . . S p r u c e i S p r u c e | H e m l o c k & W h i t e F i r j S u l f u r D i o x i d e ( g / 1 ) | T o t a l 8 1 j F r e e 2 1 | S o l i d s ( g / 1 ) 1 2 6 1 2 8 1 0 0 1 0 0 - 1 5 0 1 2 0 - 1 6 0 | t S o l i d C o m p o n e n t s j {% 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 . . . 6 2 6 5 5 5 5 2 | | T o t a l R e d u c i n g S u g a r s 3 2 2 9 2 0 2 0 2 3 | | F e r m e n t a b l e S u g a r s 2 2 1 8 1 5 1 4 I | N o n - f e r m e n t a b l e S u g a r s 1 0 1 1 5 •6 I j C a l c i u m ( a s C a O ) 7 5 | | O r g a n i c A c i d s 7 9 i | S u g a r A c i d s - 1 0 I | A s h 1 0 I | O t h e r . 9 8 3 4 I i. , _ j o f m i c r o o r g a n i s m s , h e n c e , o n t h e p r o d u c t i o n o f f i s h a s w e l l . A r g u m e n t s c a n n o t b e w o n b y s h e e r w o r d s , s o r e s e a r c h e r s h a v e s e t o u t t o i n v e s t i g a t e t h e s e p o i n t s . T h o u g h a p p a r e n t l y n o t h o r o u g h e x p e r i m e n t , c o n d u c t e d o n s i t e , h a s b e e n r e p o r t e d , m o r e t h a n a f e w p r o j e c t s o f l a b o r a t o r y s c a l e h a v e s e e n t h e l i g h t o f d a y . S o m e h a v e c o n c e n t r a t e d o n t h e e f f e c t s o f S S L o n f i s h a n d o y s t e r s [ 1 7 , 3 7 , 5 0 , 1 1 3 ] . T a b l e 1 . 6 s e r v e s a s i l l u s t r a t i o n t o p r o v e t h e p o s s i b l e h a r m c a u s e d t o f i s h b y S S L . H o w e v e r , n o t o x i c c o m p o u n d h a s a s y e t b e e n i s o l a t e d f r o m S S L . T h e d a n g e r t h a t t h i s I n t r o d u c t i o n P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 6 T a b l e 1 . 5 A G E N E R A L L Y A C C E P T E D P I C T U R E O F T H E C O M P O S I T I O N C F S S L £ 6 4 , 1 1 4 ] 1 C o m p o n e n t s C o n c e n t r a t i o n (% o f t o t a l s o l i d s ) L M i s c e l l a n e o u s O r g a n i c C o m p o u n d s . . . 5 - 8 l i q u o r i m p o s e s o n a q u a t i c l i f e i s p r o b a b l y d u e t o i t s h i g h B O D o f a t l e a s t 2 0 , 0 0 0 p p m . M i c r o o r g a n i s m s , a l r e a d y p r e s e n t i n t h e r e c e i v i n g w a t e r , a r e h a p p y w i t h t h e i n c r e a s e d s u p p l y o f c a r b o h y d r a t e s f r o m S S L , a n d c o n s u m e i t . H e n c e , t h e i r g r o w t h p r o s p e c t s a n d t h e i r d e m a n d i n o x y g e n a l s o c l i m b s , t h u s e x c e e d i n g t h e r a t e o f n a t u r a l d i s s o l u t i o n o f o x y g e n f r o m a i r . T h e r e f o r e t h e s e m i c r o o r g a n i s m s , a s w e l l a s f i s h e s , s u f f e r f r o m a l a c k o f o x y g e n ( b u t i n f a c t , i f o x y g e n i s a d e q u a t e l y p r o v i d e d , t h i s e x t r a s o u r c e o f c a r b o h y d r a t e m a y t u r n o u t t o b e , i n d e e d , b e n e f i c i a l t o a q u a t i c l i f e ) . F u r t h e r m o r e , s u s p e n d e d m a t t e r i n S S L i s a l s o a p r o b l e m , s i n c e f i b r e o r b a r k m a y c a u s e i r r i t a t i o n o f t h e g i l l t i s s u e o f f i s h o r m a y b l a n k e t t h e b o t t o m o f t h e r e c e i v i n g s t r e a m , c r e a t i n g a n a e r o b i c c o n d i t i o n s . T h i s l a s t p r o b l e m , h o w e v e r , c a n e a s i l y b e s o l v e d b y s c r e e n i n g , f i l t r a t i o n , s e t t l i n g , o r f l o t a t i o n . I n t r o d u c t i o n P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 7 T a b l e 1 . 6 M O R T A L I T I E S O F S A L M O N E X P O S E D T O S S L r • : — " • i | M o r t a l i t y 1 T h r e s h o l d I n d i c e s [ 1 1 3 ] I ( e s t i m a t e o f a p p r o x i m a t e l y a 5% K i l l ) | S a l m o n S p e c i e s A g e T h r e s h o l d I n d i c e s ( d a y s ) ( p p m o f S S L ) j C h i n o o k 3 8 1 1 7 5 2 8 0 6 0 0 3 0 5 5 6 0 | P i n k 1 0 2 1 5 5 0 1 9 8 5 3 0 | S i l v e r 3 5 1 1 2 3 0 4 1 2 1 0 1 5 j ( M o r t a l i t i e s o f 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 t o C a l c i u m E a s e i | S p e n t S u l f i t e L i q u o r [ 3 7 ] i | A v e r a g e C o n c e n t r a t i o n 1 f o f S S L ( p p m ) 0 5 5 8 1 0 7 1 1 7 5 4 2 4 9 8 | S t a n d a r d | D e v i a t i o n ( p p m ) — 9 5 . 6 1 2 8 . 1 3 8 1 . 1 1 1 5 . 5 | A v e r a g e T o t a l | S u l f u r D i o x i d e ( p p m ) • — 2 . 6 5 . 3 9 . 1 1 3 . 2 j A v e r a g e D i s s o l v e d | O x y g e n ( p p m ) 9 . 1 8 . 7 8 . 3 8 . 6 8 . 1 | A v e r a g e p H 7 . ? 7 . 6 7 . 3 7 . 0 6 . 8 | D u r a t i o n o f | E x p e r i m e n t s ( d a y s ) 3 0 3 0 3 0 3 0 1 0 | P e r c e n t o f j M o r t a l i t y * • 3 1 2 7 9 1 1 0 0 t • M o r t a l i t y d u e t o l e t h a l q u a l i t i e s c f S S L a l o n e , a s t h e d i s s o l v e d o x y g e n c o n c e n t r a t i o n w a s m a i n t a i n e d a b o v e t h e 5 p p m m i n i m u m 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 P o t e n t i a l o f S p e n t S u l f i t e l i q u o r 8 S o m e a u t h o r s h a v e s t u d i e d t h e t o x i c p o t e n t i a l c f c e r t a i n c o m p o n e n t s f o u n d i n S S L [ 3 4 ] s u c h a s t h i o s u l f a t e s [ 8 1 , 1 1 0 ] , a c e t a l d e h y d e , f u r f u r a l , d e r i v a t i v e s o f w o o d t u r p e n t i n e [ 2 9 ] , a n d s u l f u r d i o x i d e [ 1 0 7 ] . B u t t h e i r c o n c l u s i o n s a r e , m o r e o r l e s s , j u s t d o u b t s o f p o s s i b l e i n h i b i t i n g e f f e c t s , a n d i n t h e c a s e o f s u l f u r d i o x i d e , i t s c o n t e n t i n S S L w h e n d i l u t e d i n t o s t r e a m s i s n o t h i g h e n o u g h ( l e s s t h a n 2 0 0 p p m ) t o b e h a r m f u l . I 1 . 1 . 3 T r e a t m e n t s a n d _ U s e s I f t h e o u t c r i e s o f e n v i r o n m e n t a l i s t s d i d n o t c a u s e s u l f i t e p u l p m i l l s t o c l o s e d o w n , t h e y d i d o p e n a n e w s u b f i e l d i n p o l l u t i o n s t u d i e s . T a b l e 1 . 7 l i s t s m o s t o f t h e a t t e m p t s t h a t h a v e b e e n m a d e t o e i t h e r p r o v i d e a d e q u a t e o x y g e n t o o r t o r e m o v e s u b s t a n c e s w i t h h i g h o x y g e n - d e m a n d i n g p o t e n t i a l f r o m S S L . T r e a t m e n t s r e q u i r e n u t r i e n t s a t h i g h c o s t , f a r e x c e e d i n g a n y p o s s i b l e m o n e t a r y r e c o v e r y b y m a r k e t i n g b y - p r o d u c t s , s o n o n e i s p r a c t i c a l o n c o m m e r c i a l s c a l e , a n d t h e t r i c k l i n g f i l t e r p r o c e s s i s t h e o n l y o n e t h a t h a s r e a c h e d t h e s e m i - c o m m e r c i a l s c a l e a t a l l . S o m e p r o c e s s e s , u s i n g S S L a s r a w m a t e r i a l , h a v e g o n e i n t o c o m m e r c i a l p r a c t i c e . H o w e v e r , t h e p r o d u c t s d e r i v e d f r o m v a r i o u s p r o c e s s e s w h i c h u s e l i g n i n a s a c o m p o n e n t a j r e i n v e r y l i m i t e d d e m a n d , h e n c e , t h e s e p r o c e s s e s c a n n o t b e r e g a r d e d a s a s o l u t i o n t o t h e p r o b l e m c a u s e d b y m i l l i o n s o f t o n s o f S S L . V a n i l l i n i s p r o d u c e d i n a r a t h e r l a r g e q u a n t i t y , b u t t h a t m a r k e t i s a l r e a d y f u l l y f u r n i s h e d . S u g a r - f e r m e n t i n g p r o c e s s e s f a c e a n e x t r a I n t r o d u c t i o n P o t e n t i a l o f S p e n t S u l f i t e l i q u o r 9 T a b l e 1 . 7 T R E A T M E N T S A N D U S E S O F S P E N T S U L F I T E L I Q U O R «—-—: : : 1 | T R E A T M E N T S \ \ A c t i v a t e d s l u d g e [ 5 1 , 8 2 , 8 3 ] I I C o n t a c t a e r a t i o n ( H a y s p r o c e s s ) [ 3 6 ] j I T r i c k l i n g f i l t e r [ 3 5 , 8 0 , 9 8 ] | | U S E S | j H e a t r e c o v e r y 1 [ 6 4 ] | | U s e s o f l i g n o s u l f o n a t e * £ 6 4 ] \ I - C e m e n t o r c e r a m i c a d d i t i v e I | - 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 | I r o a d p a v e m e n t , r o a d s p r a y t o p r e v e n t d u s t I | - B i n d e r f o r f o u n d r y c o r e s I | - T a n n i n g a g e n t I I - 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 , 7 6 , 8 8 , 8 9 ] I | - V a n i l l i n | | P r o d u c t s f r o m f e r m e n t a t i o n o f s u g a r s | | - E t h a n o l * £ 9 9 , 1 0 6 ] | j - A c e t o n e - B u t a n o l £ 1 1 2 ] j I - P r o p i o n i c - A c e t i c a c i d s £ 2 , 2 3 , 5 9 , 7 0 ] | | - L a c t i c a c i d £ 4 6 , 9 3 ] , | I - B u t y r i c a c i d £ 1 8 ] j | - G l y c e r o l £ 6 5 ] | I - 2 , 3 - B u t a n e d i o l £ 6 6 ] j | - A n t i b i o t i c s £ 2 0 ] | | - M e t h a n e £ 8 , 9 ] | | - L i q u i d c a r b o n d i o x i d e £ 6 4 ] I | - F a t y e a s t £ 6 4 ] | I - V i t a m i n B 1 2 £ 2 , 2 3 , 5 9 ] | | - F o o d y e a s t * £ 9 9 , 1 1 1 ] | i , 1 * 0 n c o m m e r c i a l 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 o f S p e n t S u l f i t e L i g u o r 1 0 p r o b l e m , t h e c o m p l e t e l o s s o f u n u s e d l i g n o s u l f o n a t e i n t o t h e e f f l u e n t . A l c o h o l a n d f c o d y e a s t f e r m e n t a t i o n s f r o m S S I h a v e r e a c h e d c o m m e r c i a l s c a l e . B u t t h e f o r m e r m e e t s w i t h c o m p e t i t i o n f r o m c h e a p e r p r o d u c t i o n o f s y n t h e t i c a l c o h o l a n d t h e l a t t e r h a s l i m i t e d m a r k e t s . T h a t e x p l a i n s t h e e x i s t e n c e o f o n l y 3 a l c o h o l p l a n t s , w i t h S S L a s r a w m a t e r i a l , i n N o r t h A m e r i c a a n d o n l y o n e y e a s t p l a n t i n t h e U n i t e d S t a t e s . D u f r e n o y a n d P r a t t [ 2 0 ] p r e s e n t e d a p o s s i b l e g e n e r i c r e l a t i o n s h i p b e t w e e n t h e p e n i c i l l i n a n d l i g n i n m o l e c u l a r s t r u c t u r e b u t a n t i b i o t i c - p r o d u c i n g f e r m e n t a t i o n s c a n n c t u s e t h e l i g n i n p o r t i o n o f S S L , a n d a r e n o t e c o n o m i c a l l y p r o m i s i n g w i t h r e s p e c t t o m o r e c o n c e n t r a t e d s o u r c e s o f c a r b o h y d r a t e s a n d o r g a n i c n i t r o g e n o u s n u t r i e n t . 1 . 2 V I T A M I N _ B J . 2 V i t a m i n B 1 2 , f i r s t i s o l a t e d i n 1 9 4 8 , i s t h e l a r g e s t a n d m o s t c o m p l i c a t e d v i t a m i n y e t d i s c o v e r e d . I t i s t h e o n l y k n o w n v i t a m i n c o n t a i n i n g a m e t a l i o n , c o b a l t ( F i g u r e 1 . 1 ) . V i t a m i n B 1 2 , r e q u i r e d b y a l l h i g h e r a n i m a l s b u t n o t f o u n d i n h i g h e r p l a n t s , i s f o u n d i n s m a l l q u a n t i t i e s i n f o o d s s u c h a s b e e f l i v e r , b e e f , p o r k , v e a l , l a m b , c h e e s e , a n d m i l k . H o w e v e r , i t i s n o t s y n t h e s i z e d b y a n i m a l s , i t i s r a t h e r p r o d u c e d b y b a c t e r i a l o r f u n g a l f e r m e n t a t i o n i n t h e g u t t h e n a b s o r b e d a n d c o n c e n t r a t e d d u r i n g m e t a b o l i s m . T h i s e x c l u s i v e m i c r o b i a l s y n t h e s i s i s u n i q u e a m o n g t h e k n o w n v i t a m i n s . T h e b i o c h e m i c a l r o l e o f v i t a m i n B 1 2 h a s n o t b e e n c o m p l e t e l y I n t r o d u c t i o n P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 11 FIGURE 1.1 : S t r u c t u r e o f V i t a m i n B12 C H 2 O H I n t r o d u c t i o n P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 1 2 e l u c i d a t e d , b u t i t h a s b e e n s h o w n t h a t : - A d e f i c i e n c y o f v i t a m i n B 1 2 p r o d u c e s p e r n i c i o u s a n e m i a , h e n c e t h i s v i t a m i n i s t h e o n l y c u r a t i v e f a c t o r f o r t h a t d i s e a s e [ 1 1 5 ] . - V i t a m i n B 1 2 i s a n e s s e n t i a l c o n s t i t u e n t o f t h e a n i m a l p r o t e i n f a c t o r ( A P F ) f o r a n i m a l g r o w t h . - V i t a m i n B 1 2 i s u s e f u l i n t h e t r e a t m e n t c f a n u m b e r o f d i s o r d e r s [ 6 0 ] s u c h a s m a c r o c y t i c a n e m i a , m u l t i p l e s c l e r o s i s , e n d o c r i n e d i s t u r b a n c e s , r e t a r d e d g r o w t h , r e t a r d e d p r e g n a n c y , a n d l i v e r a n d s k i n d i s o r d e r s . V i t a m i n B 1 2 h a s b e e n s y n t h e s i z e d b y d i f f e r e n t s p e c i e s o f m i c r o o r g a n i s m s i n v a r i o u s m e d i a ( T a b l e 1 . 8 ) . A t f i r s t V i t a m i n B 1 2 w a s p r o d u c e d a s a b y - p r o d u c t i n a n t i b i o t i c - p r o d u c i n g f e r m e n t a t i o n s ( T a b l e 1 . 9 ) . A p p a r e n t l y , a t p r e s e n t , t h e s e s p e c i e s o f m i c r o o r g a n i s m s l i s t e d i n T a b l e I . 1 0 a r e u s e d f o r c o m m e r c i a l p r o d u c t i o n o f v i t a m i n B 1 2 . T a b l e 1 . 1 1 l i s t s s o m e v i t a m i n B 1 2 p r o d u c e r s i n t h e U n i t e d S t a t e s . P r o d u c t i o n o f v i t a m i n B 1 2 f r o m S S L h a s b e e n r e p o r t e d i n N i s h i k a w a ' s a n d F e r g u s o n ' s t h e s e s [ 2 3 , 7 0 ] . T h e f i r s t a r t i f i c i a l s y n t h e s i s o f v i t a m i n B 1 2 h a s b e e n c o m p l e t e d r e c e n t l y ( i n J a n u a r y 1 9 7 3 ) b y W o o d w a r d a n d E s c h e n m o s e r [ 1 1 5 ] . B u t t h i s s y n t h e s i s i s s t i l l f a r f r o m b e i n g c o m p e t i t i v e s i n c e i t r e q u i r e d t h e e f f o r t s o f 9 9 w o r k e r s f r o m 1 9 c o u n t r i e s d u r i n g a p e r i o d o f 1 1 y e a r s . I n t r o d u c t i o n P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 1 3 T a b l e 1 . 8 P R O C E S S E S F O R M I C R O B I A L S Y N T H E S I S O F V I T A M I N E 1 2 [ 2 3 , 7 0 , 7 3 , 7 5 ] 1— - — . _ 1 J M i c r o o r g a n i s m s \ M e d i a O x y g e n T e m p . T i m e Y i e l d | j <°C) ( h r s ) ( m g / l ) | II ,i j P r o p i o n i b a c t e r i a | 1 3 0 9 6 1 4 . 5 | | F r e u d e n r e i c h i i ! 2 A n a e r o b i c 3 0 1 0 0 0 . 6 | | C e n t . I 2 M i c r o - 3 0 1 0 0 1 2 . 2 | | A e r o b i c C o n t . | 3 A n a e r o b i c 3 0 8 9 0 . 8 7 5 | I 3 A e r o b i c 3 0 2ft 1 . 3 | I 4 A n a e r o b i c 3 0 1 3 2 2 5 . 0 | I 5 M i c r o - 3 0 1 0 0 3 . 0 | | A e r o b i c I 6 - — . - 2 . 4 | I 7 A n a e r o b i c 3 0 1 6 8 1 . 5 | I 8 A n a e r o b i c 3 0 2 8 0 2 . 3 | | t h e n A e r o b i c A n a e r o b i c 3 0 1 6 0 1 . 5 | ,, , i | P r o p i o n i b a c t e r i a | 1 0 A n a e r o b i c 3 0 8 0 - 1 1 2 3 . 0 | | S h e r m a n i i | 1 1 A n a e r o b i c 3 0 2 8 8 1 7 . 6 | I 1 2 - — - 3 . 0 | 1 1 3 A n a e r o b i c 3 0 2 0 0 0 . 8 | | t h e n ! A e r o b i c j S t r e p t o m y c e s _ mm — — — — ,mm mm — — ^ 0 . 3 | | G r i s e u s I | j | S t r e p t o m y c e s 1 1 5 — — - 3 . 3 | j o l i v a c e u s • • - JL . .. , . , . . J C o n t i n u e d I n t r o d u c t i o n P o t e n t i a l o f S p e n t S u l f i t e l i q u o r 1 4 T a b l e I . 8 ( c o n t i n u e d ) i 1 . | M i c r o o r g a n i s m s | M e d i a O x y g e n T e m p . T i m e ( o C ) ( h r s ) Y i e l d ( m g / 1 ) i j | S t r e p t o m y c e s | S p e c i e s | 1 6 I 2 5 A e r o b i c 2 8 - 3 4 9 6 2 . 0 5 . 7 j | S t r e p t o m y c e s 1 F r a d i a e I 1 7 i - — '• _ 0 . 7 i J P s e u d o m o n a s | D e n i t r i f i c a n s 1 1 8 A e r o b i c 3 1 . 5 7 0 5 . 8 | B a c i l l u s | C o a g u l a n s | 1 9 A e r o b i c 5 5 1 8 6 . 0 -H j 1 B a c i l l u s j M e g a t e r i u m l 2 0 - — _ 0 . 4 5 J i j A g r o b a c t e r i u m | S p e c i e s 1 2 1 A e r o b i c 2 8 9 6 4 . 3 | F l a v o b a c t e r i u m j S o l a r e | 2 2 - — _ 0 . 6 | F l a v o b a c t e r i u m j D e v o r a n s | 2 3 - — 0 . 6 1 ! | P s e u d o m o n a s j S p e c i e s a n d I P r o t e u s S p e c i e s t | 2 4 . . i ,„, 0 . 4 i C o n t i n u e d I n t r o d u c t i o n P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 1 5 T a b l e 1 . 8 ( c o n t i n u e d ) M e d i a C o m p o n e n t s 1 1 2 % b e e t m o l a s s e s , 1 0 % y e a s t e x t r a c t , i n v e r t a s e 2 1 0 % 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 m y c e l i u m ( 0 . 4 3 % S ) , 2 . 7 5 p p m c o b a l t 3 4% g l u c o s e , 2 % y e a s t e x t r a c t , 5 p p m c o b a l t , 2 % c a l c i u m c a r b o n a t e 4 1 0 % g l u c o s e , 6 . 6 % c o r n s t e e p l i q u o r , 2 0 p p m s o d i u m c o b a l t i c e t h y l e n e d i a m i n e t e t r a a c e t a t e , D M B Z 5 g l u c o s e , y e a s t 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 6 g l u c o s e , y e a s t , p e p t o n e , c o b a l t s a l t , d i p c t a s s i u m h y d r o g e n p h o s p h a t e 7 S S L ( 1 0 g / l o f r e d u c i n g s u g a r s ) , 1% y e a s t e x t r a c t , 5 p p m c o b a l t c h l o r i d e , 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 8 S S L ( 3 0 g / l o f r e d u c i n g s u g a r s ) , 6% y e a s t e x t r a c t , 6 . 3 p p m c o b a l t i o n , p h o s p h a t e , c a r b o n a t e 9 S S L ( 4 0 g / l o f r e d u c i n g s u g a r s ) , 0 . 5 % y e a s t e x t r a c t , 2 p p m c o b a l t i o n , p h o s p h a t e 1 0 1 0 % g l u c o s e , c o r n s t e e p l i q u o r ( 0 . 3 2 % N ) , 5 p p m c o b a l t 1 1 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 . 0 2 % D M B Z 1 2 g l u c o s e , y e a s t e x t r a c t , c o b a l t s a l t 1 3 S S L ( 4 0 g / l o f r e d u c i n g s u g a r s ) , 2 % y e a s t e x t r a c t , 2 p p m c o b a l t i o n , p h o s p h a t e 1 4 g l u c o s e , s o y b e a n m e a l , c o b a l t s a l t 1 5 g l u c o s e , s o y b e a n m e a l , 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 1 6 g l u c o s e , a n i m a l s t i c k l i q u o r , c o b a l t s a l t 1 7 g l u c o s e , b r e w e r ' s y e a s t , s o y b e a n m e a l , c o b a l t s a l t , i n o r g a n i c s a l t s 1 8 8% b e e t m o l a s s e s , s a l t s i n c l u d i n g c o b a l t ( 4 0 p p m ) , 2 % b e t a i n e , 0 . 0 0 0 5 % D M B Z 1 9 5 % c o r n s t e e p l i q u o r , 0 . 0 0 2 % s o d i u m c y a n i d e , 0 . 0 0 4 % c o b a l t ( a s c o b a l t s u l p h a t e ) , 0 . 1 % c i t r i c a c i d , 0 . 3 % t r i e t h a n c l a m i n e 2 0 b e e t m o l a s s e s , a m m o n i u m p h o s p h a t e , c o b a l t s a l t , i n o r g a n i c s a l t s 2 1 1% g l u c o s e , 1% m e a t e x t r a c t , 1% c o r n s t e e p s o l i d s , 3 % s u c r o s e , 3 % m a l t s p r o u t s , 1 5 p p m c o b a l t , 0 . 5 % b e t a i n e h y d r o c h l o r i d e 2 2 g l u c o s e , y e a s t e x t r a c t , m a l t e x t r a c t , p e n i c i l l i n m a s h r e s i d u e , i n o r g a n i c s a l t s 2 3 g l u c o s e , s o y b e a n m e a l , c o r n s t e e p l i q u o r , c o b a l t s a l t , b u f f e r s a l t s 2 4 s o y b e a n m e a l o r o t h e r p r o t e i n m a t e r i a l s 2 5 2 . 2 g l u c o s e , 3 % s o y b e a n m e a l , 5 p p m c o b a l t , s a l t s I n t r o d u c t i o n P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 1 6 T a b l e 1 . 9 A N T I B I O T I C - P R O D U C I N G F E R M E N T S T I C K S H I T H V I T A M I N E 1 2 A S B Y - P R O D U C T - S t r e p t o m y c i n 0 - N e o m y c i n - A u r e o m y c i n ( 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 T a b l e 1 . 1 0 M I C R O O R G A N I S M S U S E D F O R C O M M E R C I A L P R O D U C T I O N O F V I T A M I N B 1 2 - B a c i l l u s m e g a t e r i u m - S t r e p t o m y c e s o l i v a c e u s - S t r e p t o m y c e s s p e c i e s - A m i x e d c u l t u r e o f P s e u d o m o n a s s p e c i e s , S t r e p t c c c c u s b c v i s , P r o t e u s v u l g a r i s , a n d C l o s t r i d i u m p u t r i f i c u m T a b l e 1 . 1 1 V I T A M I N B 1 2 P R O D U C E R S I N T H E U N I T E D S T A T E S C o m m e r c i a l S o l v e n t s C o r p o r a t i o n D a w e ' s L o b o r a t o r i e s , I n c . 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 o f A m e r i c a n C y a n a m i d C o . ' M e r c k S C o , I n c . P u b s t L a b o r a t o r i e s , P a c i f i c Y e a s t P r o d u c t s , I n c . C h a s . P f i z e r S C o , I n c . U . S . I n d u s t r i a l C h e m i c a l s C o . D i v i s i o n o f N a t i o n a l D i s t i l l e r ' s C o r p . G r a i n P r o c e s s i n g C o r p . I n t r o d u c t i o n P o t e n t i a l o f S p e n t S u l f i t e L i g u c r 1 7 1«3 M I G B O g B G A H I S H S _ D S E D 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 p l a n t a r i u m h a s b e e n s h o w n [ 1 0 , 2 6 , 7 4 ] t o b e a b l e t c f e r m e n t h e x o s e s ( g l u c o s e , f r u c t o s e , m a n n o s e , g a l a c t o s e ) t o i n a c t i v e l a c t i c a c i d a n d p e n t o s e s ( x y l o s e , a r a b i n o s e ) t o i n a c t i v e l a c t i c a c i d a n d a c e t i c a c i d i n a m o l a r r a t i o o f 1 : 1 a c c o r d i n g t o t h e f o l l o w i n g r e a c t i o n s : 1 H E X O S E = — = > 2 L A C T I C A C I D ( 1 . 1 ) 1 P E N T O S E = = * = > 1 A C E T I C A C I D • 1 L A C T I C A C I D ( 1 . 2 ) T h e y f o l l o w t h e E m b d e n - H e y e r h o f - P a r n a s p a t h w a y t o c o n v e r t h e x o s e s t o p y r u v i c a c i d , t h e n , b e i n g a n a e r o b i c , r e d u c e p y r u v i c a c i d t o a c e t i c a c i d . F e r m e n t a t i o n s o f S S L u s i n g L . p l a n t a r i u m , a s p e c i e s o f o r g a n i s m f o u n d i n s a u e r k r a u t , h a v e b e e n r e p o r t e d [ 4 6 ] , F i g u r e s 1 . 2 a n d 1 . 3 s h o w e n l a r g e d p h o t o g r a p h s o f L . p l a n t a r i u m i n r e i n f o r c e d c l o s t r i d i a l m e d i u m a n d i n S S L . 1 . 3 . 2 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 M a n d M S h e r m P r o p i o n i b a c t e r i a a r e t h o u g h t [ 2 3 , 8 1 ] t o f o l l o w t h e E m b d e n - H e y e r h o f - P a r n a s p a t h w a y t o f e r m e n t h e x o s e s t o p y r u v i c a c i d : 6 [ 0 ] • 3 H E X O S E ====> 6 P Y R U V I C A C I D + 6 B A T E S ( 1 . 3 ) T h e n , t h e y o x i d i z e t h i s p y r u v i c a c i d t o a c e t i c a c i d : 2 P Y B U V I C A C I D • 2 [ 0 ] = = = = > 2 A C E T I C A C I D + 2 C A B B O N D I O X I D E ( 1 . 4 ) I n o r d e r t o s u p p l y o x y g e n t o r e a c t i o n s ( 1 . 3 ) a n d ( 1 . 4 ) , I n t r o d u c t i o n Potential of Spent Sulfite Liquor 18 FIGUBE 1.2 : L . plantarium FIGURE 1.3 : L . plantarium in Synthetic Medium in SSL j i Scale : | ( • 12 microns Introduction P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 19 FIGURE 1.6 : P. s h e r m a n i i FIGURE 1.7 : P. s h e r m a n i i i n S y n t h e t i c Medium i n SSL L j S c a l e : }• 4 = 12 m i c r o n s I n t r o d u c t i o n P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 2 0 t h e y r e d u c e p y r u v i c a c i d t c p r o p i o n i c a c i d : 4 P Y R U V I C A C I D + 4 H A T E R ====> 4 P R O P I O N I C A C I D + 8 [ 0 ] ( 1 , 5 ) A n d t h e o v e r a l l r e a c t i o n w o u l d b e : 3 H E X O S E ====> 2 A C E T I C A C I D + 4 P R O P I O N I C A C I D + 2 C A R B O N D I O X I D E • 2 W A T E R ( 1 . 6 ) T h e m e c h a n i s m b y w h i c h P r o p i o n i b a c t e r i a p r o d u c e v i t a m i n B 1 2 i s n o t w e l l k n o w n . S o m e o f t h e k n o w n s t e p s o f t h e s y n t h e s i s o f v i t a m i n B 1 2 a r e o u t l i n e d b y G r e e n b e r g [ 3 0 ] , M i l l e t [ 6 2 ] , a n d R h e m [ 7 8 ] . . I n t h e a b s e n c e o f o x y g e n , P r o p i o n i b a c t e r i a n e e d t h e n u c l e o t i d e ( F i g u r e 1 . 1 ) 5 , 6 - d i m e t h y l b e n z i m i d a z o l e ( D M B Z ) t o p r o d u c e c o m p l e t e v i t a m i n B 1 2 . W i t h s l i g h t a e r a t i o n , t h e y d o n o t n e e d t h i s n u c l e o t i d e f o r t h a t t a s k , h o w e v e r h i g h l e v e l s o f a e r a t i o n i n h i b i t t h e i r g r o w t h . T h e s e b a c t e r i a w h i c h m a y b e f o u n d i n E m m e n t h a l c h e e s e h a v e b e e n u s e d b y N i s h i k a w a a n d F e r g u s o n [ 2 3 , 7 0 ] t o f e r m e n t S S L a n d p r o d u c e v i t a m i n B 1 2 . F i g u r e s 1 . 4 t o 1 . 7 s h o w P . f r e u d e n r e i c h i i a n d P . s h e r m a n i i i n s y n t h e t i c m e d i a a n d i n S S L . 1 . 3 . 3 S t r e £ t o m y c e s , G r i s e u s . a n d . O l i y a c e u s T h e s e s o i l a c t i n o m y c e t e s h a v e b e e n k n o w n t o p r o d u c e v i t a m i n B 1 2 . , S t r e p t o m y c e s o l i v a c e u s i s o n e o f t h e s p e c i e s o f m i c r o o r g a n i s m u s e d i n c o m m e r c i a l p r o d u c t i o n o f v i t a m i n B 1 2 ( T a b l e 1 . 1 0 ) [ 7 5 ] , a n d i s o n e o f t h e b e s t v i t a m i n B 1 2 - p r o d u c i n g s p e c i e s [ 3 1 , 7 3 ] . C e r t a i n s t r a i n s o f S . g r i s e u s n o t o n l y p r o d u c e v i t a m i n B 1 2 [ 2 1 , 7 3 ] , b u t a l s o a r e c a p a b l e o f p r o d u c i n g I n t r o d u c t i o n P o t e n t i a l of Spent S u l f i t e Liquor 21 Streptomycin [103,104]. Figure 1.8 presents a microscopic p i c t u r e of S. g r i s e u s , i n s y n t h e t i c media, with the t y p i c a l h a i r - l i k e 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 developing a method f o r the economical production of vitamin B12 as a means of reducing or e l i m i n a t i n g the p o l l u t i o n problem caused t c streams by spent s u l f i t e l i q u o r . This goal i s approached with the p r o j e c t l i m i t e d to the f o l l o w i n g s u b - o b j e c t i v e s : 1. Determination of the s u i t a b i l i t y of SSL as the major component f o r a medium capable of supporting the growth of Streptomyces. This determination i s l i m i t e d to c n l y two s p e c i e s : S. o l i v a c e u s , one of the best vitamin B12 producer s p e c i e s , and S. g r i s e u s , f o r the p o s s i b l e production of Streptomycin, i n t i n y q u a n t i t y , which may help the recovery cf vitamin B12 i n very low c o n c e n t r a t i o n . 2. Observation of the r e l a t i v e vitamin B 12-producing p o t e n t i a l of l a c t i c a c i d , compared to hexose, as the main carbohydrate source f o r fermentations with P r o p i o n i b a c t e r i a . This p o t e n t i a l i s observed v i a two s e r i e s of fermentations. The f i r s t c o n s i s t s of two stages, L. plantarium converts sugars i n SSL to l a c t i c a c i d , then P r o p i o n i b a c t e r i a ferment the l a c t i c a c i d thus produced to v o l a t i l e acids and vitamin B12 (Leviton I n t r o d u c t i o n I P o t e n t i a l o f S p e n t S u l f i t e l i g u e r 2 2 a n d H a r g r o v e r e p o r t e d i n c r e a s e s i n y i e l d o f v i t a m i n E 1 2 i n s u c h f e r m e n t a t i o n s [ 3 2 , 4 7 , 6 7 ] ) . T h e s e c o n d u s e s a m i x e d c u l t u r e o f 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 . 3 . D e t e r m i n a t i o n o f t h e r e l a t i v e a b i l i t y o f P . s h e r m a n i i , c o m p a r e d t o P . f r e u d e n r e i c h i i , a s a v i t a m i n 8 1 2 p r o d u c e r . 4 . S e a r c h f o r c h e a p n u t r i e n t a s a n i t r o g e n s o u r c e f o r P . f r e u d e n r e i c h i i a n d f o r t h e o p t i m u m r e g u i r e d a m o u n t o f t h i s n u t r i e n t . 5 . I n v e s t i g a t i o n o f t h e e f f e c t o f a e r a t i o n c n t h e g r o w t h a n d t h e p r o d u c t i v i t y o f P . f r e u d e n r e i c h i i . 6 . I n v e s t i g a t i o n o f t h e e f f e c t s o f c o b a l t i o n s a n d D M E Z a s p r e c u r s o r s o n t h e r a t e o f g r o w t h a n d p r o d u c t i o n , a n d c n t h e e f f i c i e n c y o f P . 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 P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 2 3 C h a p t e r 2 E X P E R I M E N T A L T E C H N I Q U E S 2 .1 P R E P A R A T I O N _ O F _ I N O C U L A l l c u l t u r e s w e r e o b t a i n e d o r i g i n a l l y f r o m t h e A m e r i c a n T y p e C u l t u r e C o l l e c t i o n ( A T C C ) [ 3 ] . P r c p i c n i b a c t e r i a f r e u d e n r e i c h i i ( A T C C - 6 2 0 7 ) , 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 ( A T C C - 8 2 6 2 ) , a n d L a c t o b a c i l l u s p l a n t a r i u m ( A T C C - 8 0 1 4 ) w e r e g r o w n T a b l e 1 1 . 1 2 O X O I D C M 1 4 9 R E I N F O R C E D C L O S T R I D I A ! M E D I U M C o m p o n e n t s C o n c e n t r a t i o n s ( g / 1 ) B e e f E x t r a c t 1 0 P e p t o n e 1 0 S o d i u m C h l o r i d e 5 G l u c o s e 5 Y e a s t E x t r a c t 3 S o d i u m A c e t a t e 3 S o l u b l e S t a r c h 1 C y s t e i n e H y d r o c h l o r i d e 0 . 5 A g a r 0 . 5 S o u r c e L a b - L e m c o O X O I D L 3 7 O X O I D L 2 0 i n s e m i - s o l i d r e i n f o r c e d c l o s t r i d i a l m e d i u m ( T a b l e I I . 1 2 ) t o s e r v e a s i n o c u l u m . I n o c u l a o f t h e s e c u l t u r e s w e r e u s u a l l y t a k e n i n t h e e x p o n e n t i a l g r o w t h p h a s e ( 3 6 t o 9 6 h o u r s a f t e r t h e t r a n s f e r t o a n a l i q u o t o f f r e s h m e d i u m ) w h e n t h e g r o w t h E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 2 4 ( m e a s u r e d a s d r y c e l l w e i g h t ) i s i n t h e v i c i n i t y o f 0 . 5 , 2 . 0 , 1 . 5 g / l r e s p e c t i v e l y f o r L . p l a n t a r i u m , P . f r e u d e n r e i c h i i , a n d P . s h e r m a n i i . T h e v o l u m e o f i n o c u l u m w a s u s u a l l y a b o u t 1 0 0 m l f o r f e r m e n t a t i o n s i n 7 - l i t e r f e r m e n t o r s , 1 t o 2 d r o p s i n 2 5 0 m l E r l e n m e y e r f l a s k s , a n d o n e l o o p f u l i n t e s t t u b e s . F r e s h c u l t u r e s o f S t r e p t o m y c e s g r i s e u s ( A T C C - 1 0 1 3 7 ) a n d S t r e p t o m y c e s o l i v a c e u s ( A T C C - 3 3 3 5 ) w e r e m a i n t a i n e d o n s l a n t e d a g a r . I n o c u l u m w a s p r e p a r e d i n b r o t h . T h e m e d i u m u s e d w a s T a b l e 1 1 . 1 3 WAKSMAN»S N o . 3 M E D I U M C o m p o n e n t s C o n c e n t r a t i o n s ( g / 1 ) G l u c o s e 1 0 P e p t o n e 5 B e e f E x t r a c t 5 S o d i u m C h l o r i d e 5 H a k s m a n ' s N o . 3 m e d i u m ( T a b l e 1 1 . 1 3 ) [ 1 0 3 ] , 2 g o f a g a r w a s a d d e d t o e v e r y l i t e r o f t h i s m e d i u m t o m a k e s l a n t s . T h e v o l u m e o f i n o c u l u m ( i n , e x p o n e n t i a l g r o w t h ) w a s u s u a l l y a b o u t 1 0 0 m l f o r f e r m e n t a t i o n s i n 7 - l i t e r j a r s a n d i n B e l l c o f e r m e n t o r s , a n d a t l e a s t o n e l u m p i n 2 5 0 m l E r l e n m e y e r f l a s k s o r i n t e s t t u b e s . 2 . 2 T R E A T M E N T _ O F _ S P E N T _ S U 2 . 2 . 1 £ £ g £ a r a t i o n o f S p e n t _ S u l f i t e _ I i g u g r S S L h a s b e e n u s e d i n v a r i o u s f e r m e n t a t i o n s ( T a b l e 1 . 7 ) a n d E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 2 5 w a s n o t k n o w n t o c o n t a i n a n y s u b s t a n c e , o t h e r t h a n s u l f u r d i o x i d e , w h i c h i n h i b i t s t h e g r o w t h o f t h e v a r i o u s s p e c i e s o f m i c r o o r g a n i s m s u s e d . T h e c o n t e n t o f s u l f u r d i o x i d e i n t h e S S L u s e d ( a b o u t 7 0 0 0 p p m ) f a r e x c e e d s t h e u s u a l r e c o g n i z e d l i m i t o f 2 0 0 p p m [ 1 0 7 ] . T h i s l i m i t i s s o m e w h a t v a g u e i n t h e l i t e r a t u r e , h o w e v e r F e r g u s o n [ 2 3 ] h a s s t u d i e d t h e i n h i b i t i n g e f f e c t o f s u l f u r d i o x i d e o n P . f r e u d e n r e i c h i i . S o , t h e f i r s t a n d o n l y t r e a t m e n t t h a t S S L s h o u l d r e c e i v e i s t h e r e m o v a l o f s u l f u r d i o x i d e . V a r i o u s a p p r o a c h e s h a v e b e e n m a d e f o r t h i s p u r p o s e . S u l f u r d i o x i d e m a y b e n e u t r a l i z e d , t h e n p r e c i p i t a t e d a s s u l p h i t e s w i t h s o d i u m c a r b o n a t e [ 7 1 ] , b a r i u m s a l t s [ 5 5 , 5 6 ] , p o w d e r e d c h a l k w i t h s u b s e q u e n t a e r a t i o n a n d a d d i t i o n o f a m m o n i u m a n d m a g n e s i u m s u l f a t e [ 3 9 ] , o r w i t h l i m e ( i n c o m m e r c i a l p r a c t i c e , b e i n g t h e m o s t e c o n o m i c a l r e a g e n t ) [ 1 , 2 0 ] . S u l f u r d i o x i d e c a n a l s o b e r e m o v e d b y s t e a m s t r i p p i n g [ 5 2 , 5 7 , 9 9 ] . S t e a m s t r i p p i n g h a s b e e n u s e d w i t h s l i g h t * m o d i f i c a t i o n [ 4 4 ] , o r w i t h a d d i t i o n o f 0 . 2 % s u l f u r i c a c i d [ 4 3 ] . I t h a s a l s o b e e n d o n e w i t h a c o m b i n a t i o n o f b o t h n e u t r a l i z a t i o n a n d s t e a m s t r i p p i n g , a n d w i t h c h l o r i n e a s w e l l [ 9 5 ] , I t h a s e v e n b e e n d o n e d u r i n g f e r m e n t a t i o n , o f i n i t i a l l y u n t r e a t e d S S I , w i t h c o n t r o l l e d a e r a t i o n [ 5 4 ] . I n s e a r c h o f a s u i t a b l e m e t h o d f o r s u l f u r d i o x i d e r e m o v a l , a c o m p a r i s o n h a s b e e n m a d e o f 3 d i f f e r e n t m e t h o d s , n a m e l y n e u t r a l i z a t i o n a n d p r e c i p i t a t i o n , p s e u d o s t e a m s t r i p p i n g , a n d a e r a t i o n . T h e f i r s t o n e h a s b e e n a d o p t e d b y N i s h i k a w a [ 7 0 ] , a n d E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 2 6 c o n s i s t s o f t w o s t e p s : 1 . N e u t r a l i z a t i o n o f s u l f u r d i o x i d e ( p H f r o m 1 . 5 t o 5 . 5 ) b y a d d i t i o n o f c a l c i u m c a r b o n a t e . T h e p r e c i p i t a t e i s t h e n r e m o v e d b y f i l t r a t i o n . T h i s s t a g e w i l l b e r e f e r r e d t o a s n e u t r a l i z a t i o n , a n d t h e r e s u l t i n g f i l t r a t e a s N i s h i k a w a ' s n o n - t r e a t e d S S L . 2 . P r e c i p i t a t i o n o f l i g n o s u l f o n a t e b y a d d i n g c a l c i u m h y d r o x i d e a n d b r i n g i n g t h e p H t o 1 2 . T h e p r e c i p i t a t e i s r e m o v e d b y f i l t r a t i o n a n d t h e f i l t r a t e h a s i t s p H r e a d j u s t e d t o 6 - 7 w i t h s u l f u r i c a c i d . T h i s s t a g e w i l l b e r e f e r r e d t o a s p r e c i p i t a t i o n a n d t h e f i n a l l i q u i d a s N i s h i k a w a ' s t r e a t e d S S L . I t i s r a t h e r m o r e a p p r o p r i a t e t o c a l l t h e s e c o n d r e m o v a l p r o c e s s b o i l i n g t h a n s t e a m s t r i p p i n g . S S L i s b o i l e d a n d t h e e v a p o r a t e d w a t e r i s r e p l a c e d u n t i l t h e c o n t e n t o f s u l f u r d i o x i d e r e a c h e s t h e d e s i r e d l e v e l . T h e r e p l e n i s h m e n t o f w a t e r m a k e s t h i s b o i l i n g e q u i v a l e n t t o a s t e a m s t r i p p i n g p r o c e s s . I n t h e t h i r d p r o c e s s a i r i s s i m p l y b l o w n t h r o u g h S S L . T h i s w a s d o n e i n t h e 7 1 N B S M i c r o f e r m f e r m e n t o r , s o t h e l o s s o f e v a p o r a t e d w a t e r c a r r i e d w i t h t h e a i r w a s m i n i m i z e d w i t h t h e h e l p o f t h e c o n d e n s e r o n t h e a i r o u t l e t l i n e . T a b l e 1 1 . 1 4 . a n d F i g u r e 2 . 9 s h o w t h e r e s u l t s o f t h i s i n v e s t i g a t i o n . A l t h o u g h n e u t r a l i z a t i o n a l o n e i s n o t a d e q u a t e f o r s u l f u r d i o x i d e 1 r e m o v a l , N i s h i k a w a » s c o m p l e t e t r e a t m e n t i s t h e b e s t f o r t h i s p u r p o s e . T h e f i n a l t r e a t e d S S L i s c l e a r . 1 0 n l y f r e e s u l f u r d i o x i d e h a s d i r e c t i n h i b i t i n g e f f e c t s o n t h e g r o w t h 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 w o u l d b e 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 . E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e L i g u o r 2 7 T a b l e 1 1 . 1 4 E F F E C T I V E N E S S O F V A R I O U S M E T H O D S O F R E M O V A L O F S U L F U R D I O X I D E t j T y p e o f S S L S u l f u r S u g a r R e d u c t i o n R e d u c t i o n , T o t a l | D i o x i d g C o n e . i n S u g a r i n V o l u m e % l o s s ) T o t a l F r e e C o n e . o f | p p m p p m g / i % % S u g a r | j | A s R e c e i v e d 5 4 7 7 1 8 9 0 3 9 . 6 1 | N o n - t r e a t e d 6 5 3 4 8 9 7 3 6 . 6 7 . 5 N o n e 7 . 5 | | T r e a t e d 5 4 5 6 4 1 4 . 0 6 5 . 0 6 3 8 7 . 0 | | A e r a t e d ( 4 0 h r s ) 4 2 2 8 9 6 — — —•- | j A e r a t e d 3 5 8 8 2 8 8 3 6 . 3 7 . 5 N o n e 7 . 5 } | <pH 7 , 4 8 h r s ) j B o i l e d ( 8 h r s ) 1 1 8 5 9 6 3 6 . 9 7 . 0 N o n e 7 . 0 | | B o i l e d 2 9 7 8 2 2 4 3 2 . 1 1 2 . 5 N o n e 1 2 . 5 j | ( p H 8 . 3 , 1 8 h r s ) , j a s s u m i n g l y f r e e o f l i g n o s u l f o n a t e . U n f o r t u n a t e l y , t h i s t r e a t m e n t a l s o i n d u c e s a t o t a l l o s s o f 8 7 % c f t h e s u g a r , w h i c h i s p r e s u m a b l y r e t a i n e d i n t h e p r e c i p i t a t e o r w h i c h p r e c i p i t a t e s w i t h t h i s t r e a t m e n t . T h i s m e t h o d o f r e m o v a l a l s c r e q u i r e s a l a r g e a m o u n t o f c h e m i c a l s a s w e l l a s i m p o s i n g t h e p r o b l e m o f d i s p o s a l o f a l a r g e q u a n t i t y o f s l i m y w a s t e a n d t h e p r o b l e m o f f i l t e r i n g a f o a m y l i q u i d . T h e e x t r a p r e c i p i t a t i o n c f l i g n o s u l f o n a t e i s n o t n e c e s s a r y f o r t h e f e r m e n t a t i o n s s t u d i e d a s N i s h i k a w a b e l i e v e d . A e r a t i o n r e q u i r e s t o o m u c h t i m e . B o i l i n g s e e m s t o b e t h e b e s t a n s w e r , t h o u g h o n l a b o r a t o r y s c a l e b o i l i n g i s m u c h l e s s c o n v e n i e n t t h a n a e r a t i o n a s i t r e q u i r e s c o n s t a n t s u r v e i l l a n c e t o E x p e r i m e n t a l T e c h n i q u e s 28 P o t e n t i a l of Spent S u l f i t e L i q u o r L E G E N D T R E A T M E N T A L K A L I A D D E D + A E R A T I O N Y E S • A E R A T I O N N O X B O I L I N G Y E S A B O I L I N G N O Ei A D D I T I O N O F A L K A L I A F T E R T R E A T M E N T T O T A L S U L F U R D I O X I D E F R E E S U L F U R D I O X I D E E x p e r i m e n t a l T e c h n i q u e s Potential of Spent Sulf i te Liguor 29 FIGURE 2*9 ^Str ipping of Sulfur Dioxide by Boiling and Aeration 0.0 10-0 2 0 . 0 30.0 4 0 . 0 D U R A T I O N O F T R E A T M E N T M R S ) E x p e r i m e n t a l T e c h n i q u e s 5 0 . 0 P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 30 p r e v e n t i t f r o m b o i l i n g o v e r o r b o i l i n g d r y . S S L a t t a c k s m e t a l , e v e n s t a i n l e s s s t e e l , a t l o w p H , s o S S L w i t h r e a d j u s t e d p H ( t o o v e r 7 ) w a s t e s t e d w i t h a e r a t i o n a n d b o i l i n g . T h e r e m o v a l o f s u l f u r d i o x i d e b y t h e s e a l t e r n a t i v e m e t h o d s i s b y n o m e a n s e f f e c t i v e , a s t h e f i n a l c o n t e n t o f s u l f u r d i o x i d e w a s n o t u n d e r 2 0 0 p p m , n o r w a s t h e t i m e r e q u i r e d s a t i s f a c t o r y . H o w v e r , t h e h i g h c o n t e n t o f s u l f u r d i o x i d e o f a b o u t 2 8 8 p p m a s w i l l b e s h o w n l a t e r d o e s n o t h a v e a n y p e r c e p t i b l e i n h i b i t i n g e f f e c t o n P r o p i o n i b a c t e r i a a n d L a c t o b a c i l l u s i n t h e e x p e r i m e n t s u n d e r t a k e n i n t h i s p r o j e c t . S o a e r a t i o n o f p H - r e a d j u s t e d S S L ( t o a v o i d d a m a g e t o s t a i n l e s s s t e e l p a r t s o f t h e f e r m e n t o r s ) h a s b e e n u s e d a s a n a l t e r n a t i v e t o b o i l i n g ( i n g l a s s j a r s ) o f p H n o n - r e a d j u s t e d S S L i n t h i s p r o j e c t . I f S S L d o e s n o t h a v e i t s p H a d j u s t e d p r i o r t o s u l f u r d i o x i d e r e m o v a l , i t h a s t h e p o t e n t i a l o f r e l e a s i n g f r e e s u l f u r d i o x i d e f r o m l o o s e l y - b o u n d s u l f u r d i o x i d e c o m p o u n d s w h e n i t s p H i s a d j u s t e d w i t h N a O H t o a l l o w b a c t e r i a l g r o w t h . T h i s p o t e n t i a l i s s h o w n i n t h e a e r a t i o n s t u d i e s ( F i g u r e 2 . 9 ) . T h e b e s t t r e a t m e n t t o r e m o v e s u l f u r d i o x i d e e f f e c t i v e l y t h e n w o u l d b e a t w o - s t a g e b o i l i n g w i t h a d j u s t m e n t o f p H b e f o r e t h e s t a r t o f t h e s e c o n d s t a g e . H o w e v e r , c h o i c e b e t w e e n s t e a m s t r i p p i n g a n d a e r a t i o n h a s t o b e m a d e w i t h n u m e r o u s c o n s i d e r a t i o n s s u c h a s c a p i t a l c o s t o f e g u i p m e n t , c o s t o f o p e r a t i o n , a v a i l a b i l i t y o f e n e r g y s o u r c e . T h e s t e r i l i z i n g e f f e c t o f s t e a m s t r i p p i n g s h o u l d b e a c c o u n t e d t o i t s c r e d i t . E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 3 1 2 . 2 . 2 N u t r i e n t _ A d d i t i o n 2 . 2 . 2 . 1 F e r m e n t a t i o n w i t h P r o p i o n i b a c t e r i a a n d L a c t o b a c i l l u s T a b l e 1 1 . 1 5 C O N C E N T R A T I O N O F N U T R I E N T S I N S S L F O R P R O P I O N I B A C T E R I A A N D L A C T O B A C I L L U S 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 4 g / 1 C o b a l t I o n 0 - 2 . 2 p p m y e a s t E x t r a c t 0 - 1 0 0 g / 1 5 , 6 - D i m e t h y l B e n z i m i d a z o l e 0 - 0 . 1 2 6 g / 1 F i s h P l a n t E f f l u e n t 0 - 0 . 5 1/1 T h e r a n g e s o f t h e a m o u n t s o f n u t r i e n t s a d d e d t o S S L a r e l i s t e d i n T a b l e 1 1 . 1 5 . Y e a s t e x t r a c t s u p p l i e s a m i n e a c i d s a s a n i t r o g e n s o u r c e t o t h e s e m i c r o o r g a n i s m s . A t t e m p t s t o r e p l a c e y e a s t e x t r a c t b y f i s h p l a n t e f f l u e n t , a m u c h c h e a p e r ( a t a l m o s t n o c o s t ) s o u r c e o f n i t r o g e n , w e r e a l s o m a d e . C o b a l t c h l o r i d e i n s o l u t i o n w a s a d d e d t o s u p p l y t h e c o b a l t i o n n e e d e d ( a t l e a s t 4 . 3 5 p a r t s f o r e v e r y 1 0 0 p a r t s o f v i t a m i n B 1 2 p r o d u c e d ) . A n a m o u n t o f 0 . 1 2 6 g o f D H B Z p e r l i t e r c f b r o t h w a s a d d e d t o o n e b a t c h . 2 . 2 . 2 . 2 F e r m e n t a t i o n w i t h S t r e p t o m y c e s A l l c o m p o n e n t s o f W a k s m a n ' s N o . 3 m e d i u m ( T a b l e 1 1 . 1 3 ) e x c e p t g l u c o s e w e r e a d d e d t o S S L i n t h e s a m e a m o u n t s . F o r a c c l i m a t i z a t i o n t h e S S L s o t r e a t e d w a s m i x e d w i t h t h e s y n t h e t i c m e d i u m i n d i f f e r e n t p r o p o r t i o n s . E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 32 2 . 3 F E R M E N T A T I O N A N D A P P A R A T U S 2 . 3 . 1 A£_par a t u s M o s t o f t h e e x p e r i m e n t s u s i n g S t r e p t o m y c e s w e r e d o n e i n 2 5 0 m l E r l e n m e y e r f l a s k s s h a k e n a t 2 0 0 r p m i n a N e w B r u n s w i c k S c i e n t i f i c ( N B S ) i n c u b a t o r - s h a k e r ( F i g u r e 2 . 1 0 ) . A f e w w e r e d o n e i n t h e N B S f e r m e n t o r d e s c r i b e d b e l o w , a n d i n B e l l c o g l a s s f e r m e n t o r s . F o r t h e f e r m e n t a t i o n s w i t h P r o p i o n i b a c t e r i a a n d L a c t o b a c i l l u s , m o s t o f t h e p r e l i m i n a r y a n d s c r e e n i n g e x p e r i m e n t s w e r e c a r r i e d o u t i n 1 3 x 1 0 0 m m t e s t t u b e s a n d 2 5 0 m l E r l e n m e y e r f l a s k s . S o m e o f t h e f l a s k s w e r e s h a k e n i n t h e i n c u b a t o r - s h a k e r w h i l e o t h e r s w e r e l e f t u n s h a k e n i n t h e s a m e i n c u b a t o r o r i n a c o n v e r t e d - r e f r i g e r a t o r i n c u b a t o r , a l o n g w i t h t h e t e s t t u b e s . T h e s e t e s t t u b e s , h o w e v e r , w e r e v i g o r o u s l y s h a k e n w h e n e v e r s u b m i t t e d t o t u r b i d i t y t e s t s . F o r t h e m a i n e x p e r i m e n t s , i n w h i c h a c t i v i t i e s o f f e r m e n t a t i o n s w e r e f o l l o w e d w i t h t i m e , a N B S t w i n H i c r o f e r m f e r m e n t o r , m o d e l M F - 0 7 [ 7 2 ] , w a s u s e d . F i g u r e 2 . 1 1 s h o w s o n e s i d e o f t h e u n i t , a n d F i g u r e s 2 . 1 2 , 2 . 1 3 s h o w t h e d e t a i l s o f t h e f e r m e n t o r s . T h i s u n i t a l l o w s t e m p e r a t u r e c o n t r o l t o w i t h i n 1 °C i n t h e r a n g e o f 10<>C t o 50<>C, a g i t a t i o n s p e e d s u p t o 9 0 0 r p m , a n d a i r f l o w r a t e s u p t o 1 6 , 0 0 0 c c / m i n u t e . T h e i n l e t a n d o u t l e t a i r c a n b e h e a t e d a n d f i l t e r e d t h r o u g h g l a s s w o o l . T h e a i r o u t l e t l i n e h a s a c o n d e n s e r t o m i n i m i z e l o s s c f w a t e r b y e v a p o r a t i o n . T h e f e r m e n t o r i s a s e p t i c a l l y s e a l e d , a n d c a n b e s t e r i l i z e d w i t h t h e f e r m e n t a t i o n m e d i u m i n i t . S a m p l e s c a n b e r e m o v e d , p H a d j u s t e d , a n d i n o c u l u m a d d e d , u n d e r a s e p t i c E x p e r i m e n t a l T e c h n i g u e s P o t e n t i a l o f S p e n t S u l f i t e l i g u o r 3 3 c o n d i t i o n s a t a n y t i m e d u r i n g f e r m e n t a t i o n s . I n t h e l a t e s t a g e s o f t h i s p r o j e c t , p H w a s c o n t r o l l e d b y a n N B S a u t o m a t i c p H c o n t r o l l e r ( F i g u r e 2 . ' 1 ' 4 ) . I t s s t e r i l i z a b l e e l e c t r o d e s a r e a t t a c h e d t o t h e f e r m e n t o r h e a d a s s h o w n i n F i g u r e s 2 . 1 2 , 2 . 1 3 . 2 . 3 . 2 P r e f e r m e n t a t i o n A f t e r t h e a d d i t i o n o f n u t r i e n t s , t h e p H o f t h e f e r m e n t a t i o n m e d i u m w a s a d j u s t e d t o s l i g h t l y a b o v e 7 . T h e n , t h e m e d i u m , t o g e t h e r w i t h i t s c o n t a i n e r , w a s s t e r i l i z e d ( F i g u r e 2 . 1 5 ) . T h i s s t e r i l i z a t i o n w a s a c c o m p l i s h e d b y a u t o c l a v i n g t h e f e r m e n t a t i o n b r o t h i n d i r e c t c o n t a c t w i t h s t e a m a t 1 5 p s i g ( 1 2 1 ° C ) f c r 2 0 m i n u t e s ( 1 5 m i n u t e s f c r s m a l l v o l u m e s , i n E r l e n m e y e r f l a s k s a n d t e s t t u b e s ) . T h e p H d r o p p e d d u r i n g s t e r i l i z a t i o n , s o t h e p H o f t h e b r o t h w a s a g a i n a d j u s t e d t o 7 . W h e n t h e t e m p e r a t u r e o f t h e m e d i u m r e a c h e d t h e d e s i r e d l e v e l , i n o c u l u m w a s i n t r o d u c e d e i t h e r t h r o u g h t h e s a m p l i n g i n o c u l a t o r , o r t h r o u g h t h e i n o c u l u m p o r t w i t h a s t e r i l i z e d s y r i n g e a n d h y p o d e r m i c n e e d l e . T h e v o l u m e o f i n o c u l u m ( a b o u t 1 0 0 m l ) r e n d e r e d t h e s y r i n g e - m e t h o d a w k w a r d a n d a s e p t i c a l l y r i s k y . F i n a l l y , i t w a s d i s c o v e r e d t h a t g u i c k i n o c u l a t i o n t h r o u g h o p e n i n o c u l u m p e r t w i t h P r o p i o n i b a c t e r i a o r L a c t o b a c i l l u s i n e x p o n e n t i a l g r o w t h w a s s a f e a n d l e s s c u m b e r s o m e t h a n e v e n w i t h t h e s a m p l i n g i n o c u l a t o r . F o r f l a s k s a n d t u b e s , n o r m a l a s e p t i c t r a n s f e r , w i t h n e c k - f l a m e d g l a s s w a r e a n d P a s t e u r p i p e t t e s o r p l a t i n u m l o o p , w a s a d o p t e d . E x p e r i m e n t a l T e c h n i g u e s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r FIGURE 2.10 : I n c u b a t i o n , o f S. c j r i s e u s and f>. o l i v a c e u s i n D i l u t e d SSL, i n The NBS I n c u b a t o r - S h a k e r ( s c a l e : y -j = m.6 cm) L FIGURE 2.12 : S i d e View o f The F e r m e n t o r Head (see F i g u r e 2.13 f o r l e g e n d , s c a l e : \—7cm—-j ) E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l of Spent S u l f i t e L i q u o r 35 FIGURE 2.13 : Top View o f The Fermentor Head ( s c a l e : r — 4 c m — A \ ) — i I n l e t L i n e s Hater O u t l e t T h e r m i s t o r W e l l Foam Probe A i r O u t l e t A g i t a t o r S h a f t ....... A i r N o z z l e D i s t r i b u t o r Heat Exchanger B a f f l e pH A d d i t i o n s A g i t a t o r LEGEND i 1 Exhaust A i r Condenser | 1 11 I 2 12 I 3 13 I n o c u l a t i o n P o r t .... I 4 14 Thermometer W e l l .... I 5 15 I 6 16 1 7 17 1 3 18 Sampling I n o c u l a t o r . 1 9 19 1 10 20 E x p e r i m e n t a l Techniques P o t e n t i a l o f S p e n t S u l f i t e L i q u o r T FIGURE 2.14 : ( l e f t ) The pH C o n t r o l l e r ( s c a l e : | 4 = 18.6 cm) FIGURE 2.15 : (below) 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 ( s c a l e : 1 -J = 16.2 cm) FIGURE 2.16 : (above) F o a m i n g i n The 71 F e r m e n t o r ( s c a l e : | 4 = 8.6 cm) j E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 37 2 . 3 . 3 f e r m e n t a t i o n 2 . 3 . 3 . 1 W i t h S t r e p t o m y c e s S t r e p t o m y c e s a r e a e r o b i c , s c a i r w a s b u b b l e d t h r o u g h t h e f e r m e n t a t i o n m e d i u m f r o m t h e b o t t o m , a n d m i x e d b y a n a g i t a t o r . I n c a s e o f t h e N B S f e r m e n t o r , t h i s a i r w a s p r e h e a t e d , t h e n f i l t e r e d t h r o u g h g l a s s w o o l . W a t e r e n t r a i n e d w i t h t h e a i r w a s c o n d e n s e d b y a c o l d w a t e r c o n d e n s e r o n t h e a i r c u t l e t l i n e f r o m t h e f e r m e n t o r h e a d . F o r t h e B e l l c c f e r m e n t o r , a i r w a s o n l y f i l t e r e d t h r o u g h a s h o r t p a s s a g e f i l l e d w i t h g l a s s w c c l , l e s s o f w a t e r w a s m i n i m i z e d b y a 5 0 m l g l a s s b u l b c o n d e n s e r a s a t r a p o n t h e o u t l e t l i n e , b u t w a t e r h a d t o b e r e p l e n i s h e d a s e p t i c a l l y a t t i m e s . A g i t a t i o n w a s p r o v i d e d , i n t h e B e l l c o f e r m e n t o r , b y a s p e c i a l m a g n e t b a r a t t a c h e d t o a g l a s s a x l e a c t i v a t e d b y a m a g n e t i c s t i r r e r . H o s t o f t h e f e r m e n t a t i o n s w i t h S t r e p t o m y c e s , h o w e v e r , w e r e d o n e i n 2 5 0 m l E r l e n m e y e r f l a s k s , a n d a i r w a s i m p l i c i t l y p r o v i d e d b y s h a k i n g . T h e f e r m e n t a t i o n t e m p e r a t u r e w a s a t f i r s t f i x e d a t 3 7 ° C f o r S . g r i s e u s a n d 2 5 ° C f o r S . o l i v a c e u s , a s B e r g e y [ 1 0 ] r e c o m m e n d e d t h e s e t e m p e r a t u r e s a s o p t i m a . H o w e v e r , g r o w t h w a s p e e r a t t h e s e t e m p e r a t u r e s , a n d 3 0 ° C p r o v e d t o b e m u c h m o r e f a v o r a b l e t o S t r e p t o m y c e s g r o w t h . A l l s u b s e q u e n t e x p e r i m e n t s w e r e c o n t r o l l e d a t t h i s t e m p e r a t u r e . 2 . 3 . 3 . 2 W i t h P r o p i o n i b a c t e r i a a n d L a c t o b a c i l l u s A l l f e r m e n t a t i o n s i n t h e S B S f e t m e n t o r s w i t h t h e s e m i c r o o r g a n i s m s w e r e d o n e w i t h p H c o n t r o l l e d a t a p p r o x i m a t e l y 7 . E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 3 8 W h e n e v e r t h e p H c o n t r o l l e r w a s a v a i l a b l e , p H w a s a u t o m a t i c a l l y a n d c o n s t a n t l y c o n t r o l l e d , o t h e r w i s e i t w a s m a n u a l l y s p o t - c o n t r o l l e d . T h e p H c o n t r o l w a s n o t n e c e s s a r y f o r f e r m e n t a t i o n f r o m l a c t i c a c i d s i n c e t h e p H r e m a i n e d c o n s t a n t . F e r m e n t a t i o n s w i t h L a c t o b a c i l l u s w e r e d o n e u n d e r a n a e r o b i c c o n d i t i o n s . S o w a s t h e f i r s t s t a g e ( u p t o w h e r e t h e y a r e w e l l i n t o t h e e x p o n e n t i a l g r o w t h p h a s e ) c f f e r m e n t a t i o n s w i t h P r o p i o n i b a c t e r i a . T h e s e c o n d s t a g e o f f e r m e n t a t i o n s w i t h P r o p i o n i b a c t e r i a w a s d o n e u n d e r a e r o b i c c o n d i t i o n s . T o a v o i d f o a m i n g p r o b l e m s ( F i g u r e 2 . 1 6 ) t h e a i r f l o w r a t e w a s l i m i t e d t o a m a x i m u m o f 2 0 0 0 c c / m i n . H i g h a i r f l o w r a t e s a r e n e t b e n e f i c i a l t o P r o p i o n i b a c t e r i a [ 2 3 ] , S a m p l e s w e r e t a k e n a t i n t e r v a l s v a r y i n g f r o m 6 h o u r s a t t h e b e g i n n i n g o f t h e e x p o n e n t i a l g r o w t h p h a s e t o a b o u t 4 8 h o u r s i n t h e s t a t i o n a r y p h a s e . A l l o t h e r f e r m e n t a t i o n s ( i n f l a s k s a n d t u b e s ) w e r e d o n e u n d e r a n a e r o b i c c o n d i t i o n s f o r t h e f i r s t s t a g e . A f e w o f t h e m w e r e s u b j e c t e d t o a s e c o n d s t a g e w i t h a i r p r o v i d e d b y o c c a s i o n a l o r c o n t i n u o u s s h a k i n g . T h e t e m p e r a t u r e u s e d f o r i n c u b a t i o n w a s 3 0 ° C , c o n t r o l l e d t o w i t h i n 1 ° C . T h i s t e m p e r a t u r e w a s r e c o m m e n d e d a s o p t i m u m [ 1 0 , 7 7 ] f o r t h e s e m i c r o o r g a n i s m s . 2 . 4 A 8 A L Y S I S H E T H O E S A l l d e t a i l e d p r o c e d u r e s a n d c h e m i c a l s u s e d a r e o u t l i n e d l a t e r i n A p p e n d i x I I a n d A p p e n d i x I I I . O n l y t h e p r i n c i p l e s o f E x p e r i m e n t a l T e c h n i g u e s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 39 t h e m e t h o d s , t h e d i f f i c u l t i e s e n c o u n t e r e d , t h e i r s o l u t i o n s , a n d s p e c i a l a p p a r a t u s u s e d a r e p r e s e n t e d h e r e . 2.4.1 M e a s u r e m e n t o f B a c t e r i a l ^ G r o w t h ' D r y c e l l w e i g h t w a s s u f f i c i e n t f o r t h i s w o r k a s a m e a s u r e m e n t o f b a c t e r i a l g r o w t h , s o n o r e c o u r s e w a s m a d e t o t h e m o r e e l a b o r a t e m e t h o d o f v i a b l e ( o r p l a t e ) c o u n t [ 9 2 ] o r t o t h a t e x p e n s i v e p i e c e o f e g u i p m e n t k n o w n a s a C o u l t e r c o u n t e r . M o r e o v e r , t h e a m o u n t o f d r y c e l l m a t t e r p r o d u c e d i s o f p r i m a r y i n t e r e s t i n v t h i s p r o j e c t s i n c e i t s e r v e s a s a d i r e c t i n d e x o f p r o d u c t i o n o f c e l l s c o n t a i n i n g v i t a m i n B12 f o r u s e a s a n i m a l s f e e d . D i r e c t w e i g h i n g o f d r y c e l l s w a s u s e d t h r o u g h o u t t h i s p r o j e c t w i t h o u t a n y s u b s t i t u t i o n b y t h e s t a n d a r d t u r b i d i m e t r y m e t h o d . T h i s d e c i s i o n w a s b a s e d o n t h e f a c t t h a t , a l t h o u g h t h e t u r b i d i t y t e s t i s s o m e w h a t f a s t e r a n d n e e d s a s m a l l e r s i z e s a m p l e , t h i s t e s t : - B e g u i r e s t h e s a m e a m o u n t o f w o r k a n d t i m e i n t h e f i r s t w a s h i n g s t e p ; t h e s a m p l e s i z e , b e i n g s m a l l e r , i n t r o d u c e s m o r e s i g n i f i c a n t r e l a t i v e e r r o r . - I s f a s t e r t h a n d r y w e i g h t i n t i m e , b u t i t r e q u i r e s m o r e p r e c i s e m a n u a l m a n i p u l a t i o n s ( r e s u s p e c s i c n i n a k n o w n v o l u m e o f w a t e r ) h e n c e a g r e a t e r r i s k o f e r r o r . - I s l e s s r e l i a b l e ( t h e s p e c t r o p h o t o m e t e r i s l e s s r e l i a b l e t h a n a n a n a l y t i c a l b a l a n c e ) . A m o d i f i e d t u r b i d i m e t r y m e t h o d w a s t e s t e d f o r r e l i a b i l i t y . E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 4 0 I n t h i s m e t h o d t h e c e l l s w e r e n o t w a s h e d n o r r e s u s p e n d e d i n w a t e r . T h e i r t u r b i d i t y w a s s i m p l y m e a s u r e d d i r e c t l y w h i l e t h e y w e r e s t i l l s u s p e n d e d i n t h e o r i g i n a l b r o t h . S i n c e S S L h a s a d a r k b r o w n c o l o r , i t w a s i m p o s s i b l e t o u s e w a t e r a s b l a n k , s o t h e s a m e m e d i u m , w h i c h h a d b e e n k e p t u n i n o c u l a t e d a n d r e f r i g e r a t e d , w a s u s e d a s b l a n k . I n t h i s w a y t h e t u r b i d i t y w a s c o n j e c t u r e d t o b e c o n t r i b u t e d t o o n l y b y t h e d e n s i t y o f c e l l s , n o t b y t h e v a r i a t i o n o f t h e m e d i u m . T h i s t e s t i s m u c h f a s t e r t h a n t h e s t a n d a r d t u r b i d i m e t r i c m e t h o d , h e n c e m u c h f a s t e r t h a n d r y c e l l w e i g h i n g . T h e t u r b i d i t y s o m e a s u r e d h a s a l i n e a r r e l a t i o n s h i p w i t h t h e t u r b i d i t y o f c e l l s r e s u s p e n d e d i n w a t e r ( F i g u r e 2 . 1 7 ) . H o w e v e r , t h e m o s t r e l i a b l e p o r t i o n o f t h e s c a l e o n t h e s p e c t r o p h o t o m e t e r ( 0 . 0 5 - 0 . 7 ) c o r r e s p o n d s t o a g r o w t h o f 0 . 5 t o 1 . 2 , 0 . 0 1 t o 0 . 4 , a n d 0 . 0 5 t o 0 . 1 5 g / 1 ( F i g u r e s 2 . 1 8 , 2 . 1 9 , 2 . 2 0 ) o f d r y c e l l s o f P . f r e u d e n r e i c h i i , P . s h e r m a n i i , a n d L . p l a n t a r i u m r e s p e c t i v e l y , g r o w n i n S S L . T h e r e s t o f t h e g r o w t h ( u p t o 1 0 g / l f o r P r o p i o n i b a c t e r i a a n d 2 g / l f o r L a c t o b a c i l l u s ) f a l l s o n t h e u n r e l i a b l e p o r t i o n o f t h e t u r b i d i t y s c a l e . H e n c e a c e l l d e n s i t y o f m o r e t h a n 3 g / l o f P . f r e u d e n r e i c h i i o r 0 . 7 g / l o f P. s h e r m a n i i c a n n o t b e m e a s u r e d a s t h e s e n s i t i v i t y o f t h e s p e c t r o p h o t o m e t e r b e c o m e s n u l l a t t h i s l e v e l . A c o m p l i c a t e d s t a n d a r d i z a t i o n a n d a d i f f i c u l t d i l u t i o n s t e p w i t h s p e c i a l l y p r e p a r e d m e d i u m w o u l d t h e n b e r e q u i r e d f o r t h i s d i r e c t t u r b i d i t y t e s t a t l o w e r c o n c e n t r a t i o n s . T h e r e f o r e , t h e d i r e c t t e s t w a s u s e d o n l y f o r p r e l i m i n a r y s t u d i e s o f g r o w t h i n t e s t t u b e s w h e r e t h e s a m p l e ' E x p e r i m e n t a l T e c h n i q u e s Potential of Spent Sulfite Liquor U1 FIGURE 2.17 : Relation between Modified and Standard Turbidity Regression l ine at 0.95 confidence not drawn x ( ± 0 . 0 2 5 ) = 0.9 ( ± 0 . 0 2 ) y - 0.0 12 (±0.015) 2.0 • & • CD CD 1.6 cc L L ! s— C C i—t • CD m • CD _j _ J L U C_> (ZD • U_ O.B ° • >-I — »—« Q i—< OD 0.4 Z D I — • ± ± 0.0 0.4 0.8 1.2 1.6 2 T U R B I D I T Y O F C E L L S I N S Y N T H E T I C M E D I A o. .0 E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 42 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 C e l l W e i g h t 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 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 — 1 — y(±0.137) = 1. 100 (±0. 130) x + 0.094 (±0.094) — — — y(±0. 214) = 0.430 (±0.074)x - 0. 110 (±0.098) y (±0. 124) = 0.092 (±0.037) x + 0. 920 (±0. 268) D R Y C E L L W E I G H T ( G / L ) E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 43 FIGURE 2.19 : 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 C e l l W e i g h t o f 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 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. 144) = 1.230 (±0. 157) x + 0. 11 3 (±0. 1 00) _ _ _ _ _ y(±1.040) = 0.578 (±0. 694) x - 0. 159 (±0. 949) y(±0.117) •= 0. 103 (±0.033) x + 1. 000 (±0. 230) D R Y C E L L W E I G H T ( G / L ) E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 44 FIGURE 2.20 : 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 and D r y C e l l W e i g h t 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 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) = 2.050 (±0.669) x - 0.005 (±0. 179) _ _ y(±0.064) = 0.667 (±0.057) x - 0. 19 3 (±0. 068) D R Y C E L L W E I G H T ( G / L ) E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e L i q u e t 4 5 s i z e r e q u i r e d f o r w e i g h i n g c o u l d n e t b e a c c o m m o d a t e d . I t w a s a l s o u s e d f o r q u i c k c h e c k s t o d e t e r m i n e a d e q u a t e i n t e r v a l s b e t w e e n s a m p l i n g s . 2 . 4 . 2 M e a s u r e m e n t o f S u g a r C o n c e n t r a t i o n W h e n r e d u c i n g s u g a r s a r e h e a t e d i n a n a l k a l i n e s c l u t i c n o f c u p r i c c o p p e r , s t a b i l i z e d b y t a r t r a t e s , t h e c o p p e r i s r e d u c e d a n d p r e c i p i t a t e d a s c u p r o u s o x i d e . T h e r e d u c e d c e p p e r , p r o p o r t i o n a l t o t h e a m o u n t o f s u g a r p r e s e n t , c a n b e d e t e r m i n e d b y m e a s u r i n g c o l o r i m e t r i c a l l y t h e a m o u n t o f m o l y b d e n u m b l u e f o r m e d w h e n a n a r s e n o m o n c l y b d a t e r e a g e n t i s a d d e d . T h i s p r e c i p i t a t i o n r e a c t i o n i s n o t c o m p l e t e , a n d t h e a m o u n t o f p r e c i p i t a t e i s n o t s t o i c h i o m e t r i c . T h e r e f o r e k n o w n s t a n d a r d s o l u t i o n s o f d i f f e r e n t c o n c e n t r a t i o n s s h o u l d b e m e a s u r e d a l o n g w i t h t h e s a m p l e s o f u n k n o w n s u g a r c o n c e n t r a t i o n . T h e n i n t e r p o l a t i o n w i l l a l l o w t h e d e t e r m i n a t i o n o f t h e c o n c e n t r a t i o n o f r e d u c i n g s u g a r s i n t h o s e s a m p l e s , i n t e r m s o f t h e e q u i v a l e n t w e i g h t o f t h e p a r t i c u l a r s u g a r u s e d i n t h e s t a n d a r d s o l u t i o n s , w h i c h w a s g l u c o s e f o r t h i s w o r k . 2 . 4 . 3 M e a s u r e m e n t o f V i t a m i n - B 1 2 _ C o n c e n t r a t i c n T h e m i c r o b i o l o g i c a l m e t h o d w a s u s e d . I t c o n s i s t s o f g r o w i n g L a c t o b a c i l l u s l e i c h m a n i i ( F i g u r e 2 . 2 1 ) , w h i c h h a s a n a b s o l u t e r e q u i r e m e n t f o r v i t a m i n B 1 2 a n d h a s b e e n o n a r e s t r i c t e d d i e t o f v i t a m i n B 1 2 . T h e g r o w t h o f t h i s m i c r o o r g a n i s m c n t h e u n k n o w n s a m p l e i s c o m p a r e d t o i t s g r o w t h i n s y n t h e t i c m e d i u m c o n t a i n i n g E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l of Spent S u l f i t e L i q u o r 46 F I G U R E 2.21 : 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 Bacto B12 C u l t u r e Agar ( s c a l e : f- 4 = 12 microns) F I G U R E 2.23 : S p e c i a l G l a s s w a r e s f o r A c i d i f i c a t i o n of Samples w i t h C o n c e n t r a t e d S u l f u r i c A c i d ( s c a l e : f- 4 = 6.6 cm) E x p e r i m e n t a l Techniques P o t e n t i a l o f S p e n t S u l f i t e l i q u o r 4 7 k n o w n q u a n t i t i e s o f v i t a m i n B 1 2 a n d t h e v i t a m i n B 1 2 c o n t e n t o f t h e s a m p l e c a n b e d e t e r m i n e d f r o m a c a l i b r a t i o n c u r v e . P r o p i o n i b a c t e r i a w e r e b e l i e v e d [ 2 3 , 7 0 ] t o p r o d u c e v i t a m i n B 1 2 a s a n i n t r a c e l l u l a r p r o d u c t . T h i s b e l i e f w a s p r o v e d t o b e t r u e b y t h i s a s s a y . H o w e v e r , n o a t t e m p t h a d b e e n m a d e t o d e t e r m i n e w h e t h e r L . l e i c h m a n i i h a d t h e a b i l i t y t o c o n s u m e a l l t h i s i n t r a c e l l u l a r p r o d u c t . T h e s t a n d a r d m i c r o b i o l o g i c a l m e t h o d h a d b e e n u s e d w i t h o u t a n y q u e s t i o n , w h a t s o e v e r , a s t o w h e t h e r L . l e i c h m a n i i c o u l d a t t a c k t h e c e l l w a l l t o g e t a t t h e v i t a m i n i n s i d e , o r w h e t h e r t h e t e s t m e d i u m a n d t h e t r e a t m e n t t h e r e o f c o u l d b r e a k t h e c e l l w a l l o f P r o p i o n i b a c t e r i a t o r e l e a s e t h e v i t a m i n . A n s w e r s t o t h e s e q u e s t i o n s w e r e s o u g h t w i t h c e l l - b r e a k i n g e x p e r i m e n t s . H o w e v e r , t h e c e l l w a l l o f P r o p i o n i b a c t e r i a i s h a r d t o b r e a k . U l t r a s o n i c v i b r a t i o n s , F r e n c h p r e s s , s u c t i o n t h r o u g h m i c r o s c o p i c h o l e s , t r e a t m e n t w i t h a c e t o n e , s u s p e n s i o n o f c e l l s i n w a t e r f o r m o n t h s , w e r e t o n o a v a i l . T r e a t m e n t w i t h a c i d d i d c a u s e b r e a k a g e b u t n o t m u c h . T r e a t m e n t w i t h a c i d , f o l l o w e d b y a u t o c l a v i n g w a s v e r y e f f e c t i v e . S u c t i o n t h r o u g h t i n y h o l e s w i t h g l a s s b e a d s d i d b r e a k w a l l s o f a b o u t 5 0 % o f t h e c e l l s . T h e d e g r e e o f b r e a k a g e w a s o n l y e s t i m a t e d f r o m c r u d e m i c r o s c o p i c o b s e r v a t i o n s . T h e s t a n d a r d m e t h o d f o r v i t a m i n B 1 2 d e t e r m i n a t i o n r e g u i r e s t h e a d j u s t m e n t o f p H o f t h e s a m p l e s t o 4 . 5 b e f o r e s t e r i l i z a t i o n , s o t h e c e l l s a r e e x p e c t e d t o b e b r o k e n b y t h i s l i g h t a c i d t r e a t m e n t . A t e s t o n a l i q u o t s o f a n u n k n o w n s a m p l e w a s E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r T a b l e 1 1 . 1 6 E F F E C T O F V A R I O U S P R E T R E A T M E N T S O N C E L L S S U B M I T T E D T O V I T A M I N B 1 2 A S S A Y T r e a t m e n t s A c t i v i t y ( e q u i v a l e n c e i n V i t a m i n B 1 2 ) N o n - t r e a t e d S a m p l e 0 . 6 5 m g / 1 S a m p l e S u c k e d w i t h G l a s s B e a d s 1 . 0 0 m g / 1 S a m p l e w i t h p H A d j u s t e d t o 1 . 0 b e f o r e S t e r i l i z a t i o n 2 . 9 0 m g / 1 S a m p l e T e s t e d w i t h D e s c r i b e d S t a n d a r d M e t h o d 3 . 9 0 m g / 1 ( p H A d j u s t e d t o 4 . 5 b e f o r e S t e r i l i z a t i o n ) p e r f o r m e d . E a c h a l i q u o t w a s d i f f e r e n t l y p r e p a r e d w i t h v a r i o u s c e l l - b r e a k i n g m e t h o d s . T a b l e I I . 1 6 s h o w s t h e r e s u l t s o f t h i s t e s t . T h e s t a n d a r d m e t h o d , w i t h o u t e x t r a c e l l - b r e a k i n g e f f o r t s , w a s a f t e r a l l t h e o n e t h a t p r o v i d e d L . l e i c h m a n i i w i t h t h e g r e a t e s t a m o u n t o f v i t a m i n B 1 2 f r o m t h e c e l l s . F u r t h e r a c i d t r e a t m e n t o f t h e s a m p l e t o p H 1 w a s n o t b e n e f i c i a l ; t h i s m a y h a v e b e e n d u e t o t h e i n s t a b i l i t y o f v i t a m i n B 1 2 w h e n p H i s s i g n i f i c a n t l y f a r f r o m 4 . 5 [ 3 8 , 1 0 1 ] . T h e t e s t w a s r e c o m m e n d e d w i t h i n c u b a t i o n a t 3 7 ° C . I n t h e e a r l i e r p h a s e o f t h e p r o j e c t , d u e t o t h e u n a v a i l a b i l i t y o f i n c u b a t o r s , t h e i n c u b a t i o n w a s d o n e a t 3 0 ° C f o r t h e t e s t , a n d t h e r e s u l t s w e r e p o o r . 2 . 4 . 4 M e a s u r e m e n t , o | A c i d s ^ C o n c e n t r a t i o n V o l a t i l e a c i d s , p r o d u c e d b y P r o p i o n i b a c t e r i a , a s s u m e d t o b e E x p e r i m e n t a l T e c h n i q u e s Potential of Spent Sulf i te Liguor 49 FIGURE 2.22 : Partit ion of Volatite Acids between Water and Ether Regression line at 0.95 confidence y ( ± 0 . 4 7 8 ) = 0.427 (±0.039) x + 0. 94 5 (±0. 628) 0.0 20.0 40.0 60.0 80.0 300.0 V O L A T I L E A C I D S IN AQUEOUS P H A S E ( M M 0 L E S / L ) Experimental Techniques P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 5 0 a m i x t u r e o f 2 : 1 p r o p i o n i c a n d a c e t i c a c i d s [ 1 1 , 2 3 , 7 0 ] , a r e e a s i l y s t e a m d i s t i l l e d . T h i s w a s d o n e i n t h e e a r l y s t a g e s c f t h e p r o j e c t . H o w e v e r , w h e n d i r e c t d e t e r m i n a t i o n c f l a c t i c a c i d c o n c e n t r a t i o n i n t h e f e r m e n t a t i o n b r o t h b y a c o l o r i m e t r i c m e t h o d [ 7 , 5 8 , 6 7 ] f a i l e d , r e c o u r s e w a s m a d e t o e t h e r e x t r a c t i o n . F i g u r e 2 . 2 2 p r e s e n t s t h e p a r t i t i o n o f v o l a t i l e a c i d s b e t w e e n t h e e t h e r a n d t h e a g u e c u s p h a s e . D a t a f o r t h i s g r a p h w e r e c o l l e c t e d f r o m s i n g l e m a n u a l e t h e r e x t r a c t i o n s o f v o l a t i l e a c i d s f r o m a q u e o u s s o l u t i o n s . T h e s e v o l a t i l e a c i d s w e r e t h e e n d p r o d u c t s o f t h e s t e a m d i s t i l l a t i o n o f S S L a l r e a d y f e r m e n t e d w i t h 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 . M a n u a l e t h e r e x t r a c t i o n w i t h 3 c o n s e c u t i v e e x t r a c t i o n s a t e q u a l v o l u m e s p r o v e d t c b e a d e q u a t e f o r t h e d e t e r m i n a t i o n o f 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 . B u t t h i s m a n u a l e x t r a c t i o n i s i n a d e q u a t e f o r s e p a r a t i n g l a c t i c a c i d f r o m t h e a q u e o u s p h a s e . T a b l e 1 1 . 1 7 i l l u s t r a t e s t h e s u p p o r t i n g r e s u l t s . T h i s e x p l a i n s t h e e r r a t i c r e s u l t s o f l a c t i c a c i d c o n c e n t r a t i o n i n e a r l y f e r m e n t a t i o n s , p r i o r t o t h e r e p l a c e m e n t o f m a n u a l e x t r a c t i o n b y t h e m o r e t i m e - c o n s u m i n g c o n t i n u o u s b a t c h e x t r a c t i o n . F o r v o l a t i l e a c i d s , h o w e v e r , m a n u a l e x t r a c t i o n i s a l r e a d y m u c h m o r e r e l i a b l e t h a n s t e a m d i s t i l l a t i o n , w h e r e t h e e r r o r i n t r o d u c e d b y t h e p r e s e n c e o f c a r b o n d i o x i d e i n t h e a i r d u r i n g t h e t i t r a t i o n s o f v e r y d i l u t e d s o l u t i o n s o f a c i d s , i s c o n s i d e r a b l e , a n d w h e r e t h e e n d o f t h e d i s t i l l a t i o n i s d i f f i c u l t t o j u d g e . T h e l o w p a r t i t i o n c o e f f i c i e n t o f l a c t i c a c i d i n e t h e r c o m p a r e d t o t h a t i n t h e a q u e o u s p h a s e f o r c e d t h e d e t e r m i n a t i o n E x p e r i m e n t a l T e c h n i g u e s 1 P o t e n t i a l o f S p e n t S u l f i t e l i g u o r 5 1 T a b l e 1 1 . 1 7 M A N U A L E T H E R E X T R A C T I O N * O F V O L A T I L E A N D L A C T I C A C I D S F R O M A N A Q U E O U S S O L U T I O N i : — | E x t r a c t i o n s F i r s t S e c o n d T h i r d T o t a l 1 • | V o l a t i l e A c i d s — i | I n i t i a l j C o n c e n t r a t i o n ( m o l e s / 1 ) 0 . 6 0 1 0 . 4 0 5 0 . 2 7 5 0 . 6 0 1 | E x t r a c t e d ( m o l e s / 1 ) 0 . 1 9 6 0 . 1 3 0 0 . 0 7 9 0 . 4 0 5 | R e s i d u e 0 . 4 0 5 0 . 2 7 5 0 . 1 9 6 0 . 1 7 2 ] % E x t r a c t e d 3 2 . 5 3 2 . 1 2 8 . 6 6 7 . 4 | % N o n - r e c o v e r e d i 4 . 0 r j L a c t i c A c i d | I n i t i a l j C o n c e n t r a t i o n ( m o l e s / 1 ) 0 . 5 9 1 0 . 5 7 9 0 . 5 5 1 0 . 5 9 1 j E x t r a c t e d ( m o l e s / 1 ) 0 . 0 1 2 0 . 0 2 8 0 . 0 2 6 0 . 0 6 6 | R e s i d u e 0 . 5 7 9 0 . 5 5 1 0 . 5 2 5 0 . 4 9 3 1 95 E x t r a c t e d 2 . 0 4 . 8 4 . 7 1 1 . 2 | % N o n - r e c o v e r e d 5 . 4 « j * 2 0 m l o f t h e o r i g i n a l a q u e o u s s o l u t i o n i s s u b j e c t e d t o 3 c o n s e c u t i v e e x t r a c t i o n s , u s i n g a p e a r s h a p e d s e p a r a t o r y f u n n e l , w i t h a p o r t i o n o f 2 0 m l o f e t h e r e a c h t i m e . T h e e t h e r p h a s e i s s e p a r a t e d f r o m t h e a g u e o u s p h a s e , t h e n d i s p e r s e d i n a l a r g e v o l u m e o f w a t e r , a n d t i t r a t e d w i t h N a O H . T h e r e s i d u e i s a l s o t i t r a t e d w i t h N a O H a t t h e e n d . N o f a c i d c o n c e n t r a t i o n t o b e d o n e b y c o n t i n u o u s b a t c h e x t r a c t i o n . F i r s t , c o n c e n t r a t e d s u l f u r i c a c i d i s a d d e d t o S S L i n s p e c i a l g l a s s w a r e ( F i g u r e 2 . 2 3 ) t o p r e v e n t t h e l o s s c f v o l a t i l e a c i d s v a p o r p r o d u c e d b y t h e h e a t r e l e a s e d b y t h e a d d i t i o n o f c o n c e n t r a t e d a c i d . T h e n , t h e m i x t u r e i s c o o l e d d o w n a n d E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r FIGURE 2.24 : The E t h e r E x t r a c t o r E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 5 3 f i l t e r e d . T h e f i l t r a t e i s t h e n s u b j e c t e d t o e x t r a c t i o n . E t h e r i s b o i l e d ( F i g u r e 2 . 2 4 ) i n a t e s t t u b e - l i k e c o n t a i n e r ( 1 ) , w i t h a g r o u n d g l a s s n e c k , i n a w a t e r b a t h ( 5 ) a t c o n s t a n t t e m p e r a t u r e . T h e e t h e r v a p o r r i s e s t h r o u g h t h e v a p o r t u b e ( 2 ) w i t h t w o g r o u n d j o i n t o p e n i n g s a t t w o e n d s , o n e c o n n e c t e d t o t h e e t h e r c o n t a i n e r , t h e o t h e r t o a c o n d e n s e r ( 6 ) . T h e v a p o r c o n d e n s e s i n t h e c o n d e n s e r , a n d l i g u i d e t h e r d r o p s c n t o a t i n y f u n n e l i n t h e t o p p a r t o f t h e d i s p e r s i o n t u b e ( 4 ) , t h e o t h e r e n d o f w h i c h i s c l o s e d . F o u r t i n y h o l e s , p i e r c e d i n t h i s c l o s e d e n d , d i s p e r s e t h e e t h e r t h r o u g h t h e r e s i d u e t u b e ( 3 ) . T h e r e s i d u e t u b e i s s u s p e n d e d i n t h e v a p o r t u b e , a b o v e t h e l o w e r j o i n t , a n d i t i s a l m o s t f u l l w i t h t h e f i l t r a t e o f S S L p r e p a r e d a s d e s c r i b e d a b o v e . T h e d i s p e r s i o n t u b e i s s u s p e n d e d i n t h e r e s i d u e t u b e a n d s u b m e r g e d i n t h e l i q u i d t h e r e i n , a l o n g t h e j o u r n e y t h r o u g h t h e f i l t r a t e , t h e f i n e l y d i s p e r s e d d r o p s o f e t h e r p i c k u p a c i d s a n d g a t h e r t o f o r m a t h i n l a y e r o n t o p o f i t . T h e s u p e r n a t a n t e t h e r e v e n t u a l l y o v e r f l o w s a l o n g t h e o u t e r w a l l o f t h e r e s i d u e t u b e a n d d r o p s b a c k i n t o t h e e t h e r c o n t a i n e r . T h e e t h e r i s r e c y c l e d a n d t h e a c i d s r e m a i n i n t h e c o n t a i n e r . T h e d u r a t i o n o f t h e e x t r a c t i o n i s a d j u s t e d t o e n s u r e c o m p l e t e ( o r a l m o s t c o m p l e t e ) r e c o v e r y o f a c i d s f r o m S S L . T h e e t h e r p h a s e c o l l e c t e d i n t h e c o n t a i n e r a t t h e e n d i s t i t r a t e d , i n e x c e s s w a t e r , w i t h N a O H . T h i s c o n t i n u o u s b a t c h e x t r a c t i o n w a s t h r o r o u g h l y t e s t e d f o r a c c u r a c y . K n o w n a m o u n t s o f m i x t u r e s o f v o l a t i l e a c i d s ( 2 p r o p i o n i c : 1 a c e t i c ) a n d l a c t i c a c i d i n v a r i o u s p r o p o r t i o n s w e r e a d d e d t o a l i g u o t s c f S S L . T h e n t h i s e x t r a c t i o n t e c h n i q u e E x p e r i m e n t a l T e c h n i q u e s Potential of Spent Sulfite Liquor FIGURE 2.25 : Recovery of Acids from SSL by Continuous Ether Extraction Regression l ine at 0.95 confidence y ( ± 0 . 0 1 ) = 0.879 (±0.035) x + 0. 09 6 (±0. 009) 0.5 0 . 0 0 .3 0 . 2 0 . 3 0 . 4 0 . 5 R C I D S fiDDED ( M D L E S / U Experimental Techniques Potential of Spent Sulfite Liquor 55 FIGURE 2.26 : Recovery of Lactic Acid from SSL Continuous Ether Extraction Regression l ine at 0.95 confidence y ( ± 0 . 0 1 4 ) = 1.000 ( ± 0 . 0 9 3 ) x + 0. 029 (±0. 0 11) 0.5 L A C T I C A C I D ADDED ( M 0 L E S / L ) Experimental Techniques P o t e n t i a l o f S p e n t S u l f i t e L i g u o r 5 6 w a s u s e d t o r e c o v e r t h e a c i d s f r o m t h e s e s a m p l e s a t l e a s t i n d u p l i c a t e . L a c t i c a c i d w a s d i s t i n g u i s h e d f r o m v o l a t i l e a c i d s b y t i t r a t i o n o f t h e b o i l e d e x t r a c t s . F i g u r e s 2 . 2 5 a n d 2 . 2 6 s h o w t h e r e s u l t s o f t h i s t e s t a l o n g w i t h t h e r e g r e s s i o n l i n e s a t 9 5 * c o n f i d e n c e a s p r o o f s o f r e l i a b i l i t y o f t h i s e x t r a c t i n g m e t h o d . N o a t t e m p t w a s m a d e t o c h e c k t h e c o r r e c t n e s s o f t h e a b s o l u t e v a l u e o f t h e a c i d s c o n c e n t r a t i o n s t h u s d e t e r m i n e d b y t h e m e t h o d ( a b s o l u t e a c i d s c o n t e n t o f S S L w a s n o t k n o w n n o r d e t e r m i n e d a c c u r a t e l y b y a n y o t h e r m e t h o d ) . B u t t h e r e s u l t s a l l o w c o n c l u s i v e l y t h e a t t r i b u t i o n o f s a t i s f a c t o r y a c c u r a c y t o t h e t e s t a s f a r a s d i f f e r e n c e o f a c i d s c o n c e n t r a t i o n s i n S S L c o n c e r n s . T h i s s a t i s f i e s t h e p r i m a r y i n t e r e s t i n d e t e r m i n i n g t h e p r o d u c t i o n o f a c i d s b y f e r m e n t a t i o n . T h e e x p e r i m e n t a l a c i d r e c o v e r y s t r a i g h t l i n e i s a b o v e a n d p a r a l l e l t o t h e t h e o r e t i c a l r e c o v e r y c u r v e , s u p p o s i n g t h e r e w e r e n o a c i d s i n t h e S S L ( i n t h e c a s e o f t o t a l a c i d s d e t e r m i n a t i o n , t h e l i n e i s n o t c o m p l e t e l y p a r a l l e l ) . A t h i g h c o n c e n t r a t i o n s , e x p e r i m e n t a l d a t a d e v i a t e c o n s i d e r a b l y f r o m t h i s l i n e , b u t a c i d s p r o d u c e d b y f e r m e n t a t i o n s n e v e r r e a c h t h e s e 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 T h e c h e m i c a l o x y g e n d e m a n d ( C O D ) d e t e r m i n a t i o n , i n s t e a d o f t h e b i o c h e m i c a l o x y g e n d e m a n d ( B O D ) t e s t , w a s t e s t e d a s a m e a s u r e o f p o l l u t i o n . T h e p r i n c i p l e i n v o l v e d i n t h i s t e s t c o n s i s t s o f t h e E x p e r i m e n t a l T e c h n i g u e s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 5 7 T a b l e 1 1 . 1 8 B E S O L T S O F C O D T E S T S O N 3 S A M P L E S r . — i C O D ( p p m ) i | S a m p l e W i t h o u t W i t h C o r r e c t i o n W i t h C o r r e c t i o n — i C o r r e c t i o n b y D i f f e r e n c e b y B a t i c i I 1 1 8 6 5 6 0 2 2 4 5 0 0 2 4 9 9 6 7 1 5 7 6 9 9 1 8 2 5 0 0 1 8 9 2 3 9 2 0 3 2 8 0 1 8 7 0 0 0 1 8 3 3 3 5 1 8 6 9 6 0 2 0 1 2 5 0 2 0 3 5 1 1 2 8 5 3 6 0 2 2 8 7 0 0 2 0 7 1 4 3 . j I 2 1 9 5 8 8 8 — t . 2 0 5 2 1 6 1 8 3 3 3 8 1 8 7 3 0 0 1 8 4 8 0 0 2 0 0 0 0 0 1 9 6 2 0 0 2 0 6 6 4 0 2 2 5 0 0 0 2 3 8 9 0 0 3 0 5 0 4 0 2 2 1 4 2 9 2 4 8 4 0 0 j — 1 I 3 1 5 8 5 7 6 1 4 9 2 4 8 1 8 7 0 0 0 1 9 9 9 7 5 2 2 1 7 6 0 2 3 3 1 5 0 2 4 0 0 0 0 2 7 5 5 2 0 2 1 2 0 0 0 1 9 5 3 4 9 i - , . i o x i d i z a t i o n o f o r g a n i c m a t e r i a l w i t h a k n o w n a m o u n t c f p o t a s s i u m d i c h r o m a t e a n d t h e t i t r a t i o n o f t h e r e s i d u a l . d i c h r o m a t e w i t h f e r r o u s a m m o n i u m s u l f a t e u s i n g f e r r c i n i n d i c a t o r . T h e C O D t e s t w a s p e r f o r m e d o n a l l s a m p l e s o f e a r l y f e r m e n t a t i o n s . H o w e v e r , i t w a s i m p o s s i b l e t o c o r r e l a t e t h e s c a t t e r e d d a t a c o l l e c t e d ^ B e s u l t s c c u l d n o t b e r e p r o d u c e d ; E x p e r i m e n t a l T e c h n i q u e s v P o t e n t i a l o f S p e n t S u l f i t e l i g u o r 5 8 T a b l e 11.19 R E S U L T S O F C O R R E C T I N G M E A S U R E S ON C O D T E S T r — -— — — W i t h o u t w i t h C o r r e c t i o n C o r r e c t i o n b y D i f f e r e n c e b y R a t i o j j S a m p l e 1 j M e a n ( p p m ) 2 0 3 9 7 2 2 0 4 7 9 0 2 0 6 6 3 9 | S t a n d a r d D e v i a t i o n ( p p m ) 4 8 3 6 9 2 1 1 3 1 2 6 1 4 0 | 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 ( p p m ) 6 0 0 4 7 2 6 2 3 3 3 2 4 5 2 I R e l a t i v e {%) 2 9 . 4 1 2 . 8 1 5 . 6 | S a m p l e 2 | M e a n ( p p m ) 2 1 9 5 1 7 2 0 7 4 4 2 2 1 7 7 0 0 | S t a n d a r d D e v i a t i o n ( p p m ) 4 8 6 0 1 1 9 4 9 6 3 0 4 3 2 j E s t i m a t e d D e v i a t i o n * I A b s o l u t e ( p p m ) 6 0 3 3 6 1 9 4 9 6 3 0 4 3 2 | R e l a t i v e {%) 2 7 . 5 1 4 . 6 2 2 . 7 | S a m p l e 3 | M e a n 2 0 1 2 7 6 2 1 0 7 1 7 2 1 1 7 7 5 | S t a n d a r d D e v i a t i o n ( p p m ) 5 9 0 5 4 2 3 1 0 2 2 4 5 5 3 I 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 ( p p m ) 9 3 9 5 3 5 7 3 9 2 6 0 4 4 1 | R e l a t i v e (%) i -4 7 . 1 2 6 . 3 2 8 . 5 -1 ( » A t a d e g r e e o f c o n f i d e n c e o f 0 . 9 5 , b y t - t e s t d e v i a t i o n s f r o m o n e r u n t o a n o t h e r w e r e e n o r m o u s , n o m a t t e r h o w m u c h c a u t i o n , e f f o r t a n d c a r e w e r e p a i d t o m a n i p u l a t i o n s . T h e r e l i a b i l i t y o f t h e t e s t w a s t h e n i m m e d i a t e l y q u e s t i o n e d . E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e l i q u o r 5 9 T a b l e 1 1 . 2 0 R E L I A B I L I T Y O F T H E C O D T E S T | S u g a r C o n c e n t r a t i o n c f | T h e A q u e o u s S o l u t i o n ( p p m ) 3 0 5 0 1 0 0 3 0 0 , i | S u m b e r o f T e s t s 6 1 1 3 9 l j M e a s u r e d C O D M e a n ( p p m ) 2 5 . 8 4 9 9 4 . 5 2 8 7 . 3 | S t a n d a r d D e v i a t i o n ( p p m ) 2 . 4 — — 1 9 . 1 | E s t i m a t e d D e v i a t i o n | (9595 C o n f i d e n c e ) | A b s o l u t e ( p p m ) 2 . 5 — — 1 8 . 0 | R e l a t i v e (95) 9 . 8 — — 6 . 3 j D e v i a t i o n o f M e a n | f r o m T r u e V a l u e (95) 1 4 . 1 2 . 0 5 . 5 4 . 2 | E x t r e m e D e v i a t i o n ( p p m ) 7 . 6 1 3 2 . 0 l : J P r e l i m i n a r y c h e c k s h a d b e e n m a d e o n t h e t e s t e a r l i e r w i t h a g u e o u s s o l u t i o n s o f s u g a r , b u t g o o d s i n g l e p o i n t c h e c k s ( s e e N r e s u l t s i n T a b l e 1 1 . 2 0 f o r s o l u t i o n s o f 5 0 a n d 1 0 0 p p m o f s u g a r ) d i d m i s l e a d t h e i s s u e . A w h o l e s e r i e s o f C O D t e s t s w a s d e s i g n e d a n d c a r r i e d o u t . C O D t e s t s w e r e d o n e r e p e a t e d l y o n 3 s a m p l e s o f S S I . T e s t s w e r e r u n s i m u l t a n e o u s l y o n t w o o f t h e s e t h r e e s a m p l e s a n d o n e s a m p l e o f 3 0 0 p p m a g u e o u s s o l u t i o n o f s u g a r . T h e r e s u l t s a r e t a b u l a t e d i n T a b l e 1 1 . 1 8 a n d f u r t h e r a n a l y z e d i n T a b l e 1 1 . 1 9 . T h e m e a s u r e d C O D o f t h e s a m e s u g a r s o l u t i o n v a r i e s w i d e l y f r o m t e s t t o t e s t , s o c o r r e c t i n g m e a s u r e s w e r e a d o p t e d . T h e m e a s u r e d C C D o f e v e r y s a m p l e w a s c o r r e c t e d w i t h t h e d i f f e r e n c e o r t h e r a t i o o f t h e m e a s u r e d a n d t h e t h e o r e t i c a l ( t r u e ) C O D o f t h e s u g a r s o l u t i o n o f E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 6 0 k n o w n c o n c e n t r a t i o n . T h e c o r r e c t i o n b y d i f f e r e n c e s e e m s t o b e b e t t e r . T h e s e c o r r e c t i o n s d i d c u t t h e v a r i a t i o n d o w n t c h a l f ( T a b l e 1 1 . 1 9 ) , b u t t h e v a r i a t i o n i n c r e a s e d s h a r p l y w i t h d e c r e a s i n g n u m b e r o f r e p e t i t i o n s . W i t h t e s t s d o n e i n t r i p l i c a t e , t h e d e v i a t i o n w a s h i g h , a b o u t 3 0 % a t 9 5 % c o n f i d e n c e . A l a r g e n u m b e r o f t e s t s w e r e d o n e w i t h s u g a r s o l u t i o n s o f k n o w n c o n c e n t r a t i o n ( m o s t l y o f 3 0 0 p p m ) . T h e r e l i a b i l i t y c f t h i s C O D t e s t i s p r e s e n t e d b y T a b l e 1 1 . 2 0 . I f t h e t e s t i s r e p e a t e d m a n y t i m e s ( i n t h i s c a s e 3 9 ) t h e e s t i m a t e d e r r o r i s w i t h i n 1 0 % ( t h o u g h e x t r e m e d e v i a t i o n f r o m t h e e x a c t c o n c e n t r a t i o n o f 3 0 % o r m o r e h a s b e e n o b s e r v e d ) a t 9 5 % c o n f i d e n c e . I n t h i s t e s t , t h e d i f f e r e n c e o f 2 t i t r a t i o n s i s r e q u i r e d t o b e l i m i t e d t o w i t h i n 1 m l ( t o a v o i d e x p e r i m e n t a l e r r o r o f m o r e c o n s i d e r a b l e m a g n i t u d e ) . T h e r e f o r e , t h e e r r o r i n t r o d u c e d b y t h e e x c e s s d r o p i n e a c h t i t r a t i o n a l o n e c a n r e a c h 1 0 % . T h i s e x p l a i n s t h e p o s s i b l e e r r o r o f m e r e t h a n 2 0 % i f o t h e r s o u r c e s o f e r r o r , i n t r o d u c e d b y v a r i a t i o n s o f h e a t i n g r a t e , c o n d e n s i n g r a t e a n d i n m a n i p u l a t i o n s s u c h a s d i l u t i o n o f s a m p l e s a n d p r e p a r a t i o n o f s t a n d a r d s o l u t i o n s , a r e t a k e n i n t o a c c o u n t . T h i s i n e v i t a b l e e r r o r c a u s e d t h e a b a n d o n m e n t o f t h e t e s t , a s i t u n v e i l e d m o r e t h a n a f e w r e a s o n s w h i c h w o u l d m a d e a n y r e s u l t c o l l e c t e d i n s i g n i f i c a n t : 1 . I t t a k e s m o r e t h a n 3 h o u r s t o c o m p l e t e o n e r u n , i t i s t h e n n o t p r a c t i c a l t o h a v e m o r e t h a n 2 C O D d e t e r m i n a t i o n s f o r t h e s a m e s a m p l e . H e n c e t h e r e s u l t s a r e e x p e c t e d t o d e v i a t e a s m u c h a s 3 0 % ( o r m o r e ) f r o m t h e i r m e a n . E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e L i g u o r 6 1 2 . O n t h e o t h e r h a n d , t h e m a x i m u m p o s s i b l e C O D r e d u c t i o n ( w h e n a l l U 0 g / 1 o f s u g a r i n S S L i s c o n s u m e d a n d a l l t h e p r o d u c t s a n d c e l l s a r e r e m o v e d ) b y f e r m e n t a t i o n s i s o n l y a b o u t 4 0 , 0 0 0 p p m f r o m t h e t o t a l C O D o f 2 0 0 , 0 0 0 p p m c f S S L ( t h a t a m o u n t s t o 2 0 * ) . T h a t m e a n s t h a t t h e a c c u r a c y a n d r e l i a b i l i t y o f t h e t e s t d o n o t e v e n a l l o w t h e m a x i m u m C O D r e d u c t i o n t o b e m e a s u r e d . 3 . T h e r e m o v a l o f a c i d s f r o m t h e f e r m e n t a t i o n b r o t h r e q u i r e s t h e a d d i t i o n o f a s t r o n g a c i d ( c o n c e n t r a t e d s u l f u r i c a c i d w a s u s e d ) a n d h e n c e t h i s i n t r o d u c e s a p r e c i p i t a t e . S t e a m d i s t i l l a t i o n a l s o c h a n g e s t h e v o l u m e o f t h e o r i g i n a l s a m p l e . E t h e r e x t r a c t i o n r e q u i r e s a f i l t r a t i o n , t h e n i n t r o d u c e s a n u n k n o w n a m o u n t o f e t h e r d i s s o l v e d i n S S L . H e n c e i t i s n o t l i k e l y t h a t t h e C O D t e s t c a n r e v e a l a n y r e d u c t i o n o f t h e p o l l u t i n g p o t e n t i a l o f S S L w i t h a l l t h o s e e x t r a n e o u s c h e m i c a l s a n d e x t r a s o u r c e s o f e r r o r . 4 . T h e b a c t e r i a u s e d a r e b e l i e v e d t o c o n s u m e o n l y t h e s u g a r s i n S S L , s o t h e r e d u c t i o n o f u l t i m a t e o x y g e n d e m a n d ( U O D ) c a n b e r e a d i l y c a l c u l a t e d f r o m t h e s u g a r c o n s u m p t i o n d a t a . T h e s u g a r c o n s u m p t i o n i s d i r e c t l y p r o p o r t i o n a l t o t h e U O D , s o t h e s u g a r c o n c e n t r a t i o n o f S S L c a n e a s i l y s e r v e a s a n i n d e x o f i t s p o l l u t i n g p o t e n t i a l . F o r t h e l a s t t w o r e a s o n s n o a t t e m p t w a s m a d e t c r e p l a c e t h e C O D w i t h t h e ' B O D t e s t . F u r t h e r m o r e t h e B O D t e s t m a y r u n i n t o t h e s a m e t r o u b l e t h a t C O D d i d , a s d i l u t i o n o f s a m p l e s , a n d m e a s u r e m e n t s o f d i s s o l v e d o x y g e n d o n o t g u a r a n t e e a n y m o r e a c c u r a c y t h a n t h e m e a s u r e m e n t s r e q u i r e d b y t h e C C D t e s t . E O E E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e l i q u o r 6 2 c o n t r i b u t e d b y f e r m e n t a b l e s u g a r s i n S S L h a s b e e n r e p o r t e d t o b e a b o u t 3 0 % - U O * o f t h e t o t a l B O D o f S S L [ 1 0 0 , 1 1 0 ] . E x p e r i m e n t a l T e c h n i q u e s P o t e n t i a l o f S p e n t S u l f i t e L i g u o r 6 3 C h a p t e r 3 E X P E R I M E N T S A N D R E S U L T S 3 . 1 F E R M E N T A T I O N . « I T H _ S T R E P T R e p e a t e d a t t e m p t s t o g r o w S t r e p t o m y c e s ( g r i s e u s a n d o l i v a c e u s ) i n S S L w e r e t o n o a v a i l . I n B e l l c o g l a s s f e r m e n t o r s t h e e x p e r i m e n t s e n d e d u p w i t h c o n t a m i n a t i o n a s t h e g l a s s b u l b c o n d e n s e r d i d a p o o r j o b i n p r e v e n t i n g l o s s o f w a t e r b y e v a p o r a t i o n . T h e n u m e r o u s a d d i t i o n s o f w a t e r c o u l d n o t b e k e p t a b s o l u t e l y a s e p t i c t h r o u g h t h e l o n g f e r m e n t i n g p e r i o d ( m o r e t h a n 5 d a y s ) . D u r i n g t h a t p e r i o d t h e r e w a s n o n o t i c e a b l e g r o w t h o f t h e a c t i n o m y c e t e s i n o c u l a t e d . E x p e r i m e n t s i n 2 5 0 m l E r l e n m e y e r f l a s k s a n d i n t h e 71 M i c r o f e r m f e r m e n t o r s u s u a l l y d i d n o t e n d u p w i t h c o n t a m i n a t i o n , b u t n o g r o w t h c o u l d b e o b s e r v e d a f t e r p e r i o d s a s l o n g a s h a l f a m o n t h . F e r m e n t a t i o n t e m p e r a t u r e s w e r e c o n t r o l l e d a t 3 7 ° C f o r S . g r i s e u s a n d 2 5 ° C f o r S . o l i v a c e u s , a s r e c o m m e n d e d b y B e r g e y [ 1 0 ] . B u t t h e n i t w a s d i s c o v e r e d t h a t , a t t h e s e t e m p e r a t u r e s , S t r e p t o m y c e s g r e w v e r y p o o r l y , e v e n i n W a k s m a n * s N o . 3 s y n t h e t i c m e d i u m , a n d 3 0 ° C w a s a m u c h m o r e f a v o r a b l e t e m p e r a t u r e f o r t h e s e s p e c i e s . H e n c e , s u b s e q u e n t e x p e r i m e n t s w e r e d o n e a t 3 0 ° C . B u t s t i l l , n o g r o w t h w h a t s o e v e r c o u l d b e d e t e c t e d i n S S L . T h i s f a i l u r e c o u l d b e a t t r i b u t e d t o e i t h e r t h e l a c k o f E x p e r i m e n t s a n d R e s u l t s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 6 4 T a b l e I I I . 2 1 F E R M E N T A T I O N S O F D I L U T E D S S L B Y S . G R I S E U S r — j S a m p l e C o n c e n t r a t i o n D r y C e l l F e r m e n t a t i o n I n o c u l u m | ( m l o f S S L / 1 H e i g h t T i m e f r o m j o f S o l u t i o n ) ( g / i ) ( h o u r s ) j 1 G 0 . 1 0 0 0 0 1 . 6 6 1 1 1 0 i | G O . 2 0 0 0 0 1 . 3 4 1 1 1 0 | G O . 3 0 0 0 0 1 . 4 5 7 1 2 6 | G 3 . 1 0 0 0 3 1 . 7 8 3 1 1 0 I G 3 . 2 0 0 0 3 1 . 5 5 4 1 1 0 | G 2 5 . 1 0 0 2 5 1 . 9 4 5 1 1 0 | G 2 5 . 2 0 0 2 5 1 . 5 1 2 1 1 0 | G 2 5 . 3 * 0 0 2 5 2 . 0 8 5 1 2 6 G O . 3 | | G A 2 5 . 4 * 0 0 2 5 2 . 1 3 0 1 2 6 G O . 3 | | G 5 0 . 1 0 0 5 0 0 . 5 9 0 1 1 0 | G 5 0 . 2 0 0 5 0 1 . 3 8 2 1 1 0 | G A 5 0 . 3 * 0 0 5 0 2 . 8 5 0 1 1 7 G A 2 5 . 4 | | G A 5 0 . 4 * 0 0 5 0 2 . 8 8 0 1 1 7 G A 2 5 . 4 | | G 1 0 0 . 1 0 1 0 0 0 . 0 3 5 1 1 0 | G 1 0 0 . 2 0 1 0 0 0 . 0 3 4 1 1 0 | G 1 0 0 . 3 0 1 0 0 0 . 0 0 3 1 2 6 I G 1 0 0 . 4 0 1 0 0 0 . 0 0 0 1 2 6 | G 1 0 0 . 5 0 1 0 0 0 . 0 0 0 2 0 4 | G 1 0 0 . 6 0 1 0 0 0 . 0 0 0 2 0 4 | G A 1 0 Q . 7 * 0 1 0 0 2 . 4 5 5 2 1 6 G A 5 0 . 4 | | G A 2 0 0 . 1 * 0 2 0 0 3 . 3 8 8 2 5 3 G A 1 0 0 . 7 | | G A 2 0 0 . 2 * 0 2 0 0 3 . 1 6 5 2 5 3 G A 1 0 0 . 7 | | G A 3 0 0 . 2 * 0 3 0 0 0 . 0 0 0 2 0 4 G A 2 0 0 . 1 | | G 5 0 0 . 1 0 5 0 0 0 . 0 0 0 1 2 6 | G A 5 0 0 . 2 * 0 5 0 0 0 . 0 0 0 2 0 4 G A 2 0 0 . 1 | | G A 5 0 0 . 3 * 0 5 0 0 0 . 0 0 0 2 0 4 G A 2 0 0 . 1 | | G 1 0 0 0 . 1 1 0 0 0 0 . 0 0 0 1 1 0 | G 1 0 0 0 . 2 1 0 0 0 0 . 0 0 0 1 2 6 } G 1 0 Q 0 . 3 2 1 0 0 0 0 . 0 0 0 1 1 0 L .._ J C h a r a c t e r i s t i c s o f S S L u s e d : 3 7 g / l o f s u g a r , 9 6 p p m a n d 1 2 0 0 p p m r e s p e c t i v e l y o f f r e e a n d t o t a l s u l f u r d i o x i d e ^ R e s u l t s f o r f e r m e n t a t i o n s b y a c c l i m a t i z a t i o n Z S S L t r e a t e d w i t h H i s h i k a w a » s n e u t r a l i z a t i o n a n d p r e c i p i t a t i o n E x p e r i m e n t s a n d R e s u l t s P o t e n t i a l of Spent S u l f i t e L i q u o r 65 Table III.22 FEBMENTATIONS OF DILUTED SSL BY S. OLIVACEUS r _ — — | Sample C o n c e n t r a t i o n (ml of SSL/1 of S o l u t i o n ) Dry C e l l Height (g/i) Fermentation Time (hours) Inoculum from I 00.1 0000 0.924 110 J 00.2 oooo 1.054 110 | 00.3 oooo 2.205 126 | 03.1 0003 0.829 110 | 025.1 0025 1.634 110 I 025.2 0025 1.035 110 | OA25.3* 0025 2.085 126 00.3 | 0A25.4* 0025 2. 130 126 O0.3 | 050.1 0050 0.892 110 | 050.2 0050 0.648 110 | OA50.3* 0050 2.490 117 0A25.4 | OA50.4* 0050 2.570 117 OA25.4 | O100.1 0100 0.164 110 | 0100.2 0100 0.590 110 \ O100.3 0100 0.078 126 | 0100.4 0100 0.000 126 | 0100.5 0100 0.338 204 | 0100.6 0100 0.000 204 | OA100.7* 0100 2.213 216 OA50.4 | OA100.8 0100 2. 135 216 OA50.4 f OA200.1* 0200 2.678 253 OA100.8 | OA300.1* 0300 0.000 204 OA200.1 | OA300.2* 0300 0.000 204 GA20,0. 1 | 0500.1 0500 0. 152 126 | O500.2 0500 0.000 204 -| OA500.3* 0500 0.000 204 OA200.1 | OA500.4* 0500 0.000 204 CA200.1 | O1000.1 1000 0.000 110 | 01000.2 1000 0.000 126 | 01000.32 t ; .. 1000 0.000 110 C h a r a c t e r i s t i c s of SSL used : 37g/l of sugar, 96ppm and 1200ppra r e s p e c t i v e l y of fr e e and t o t a l s u l f u r d i o x i d e 1 B e s u l t s f o r fermentations by a c c l i m a t i z a t i o n 2SSL t r e a t e d with Nishikawa»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 Experiments and R e s u l t s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 6 6 n e c e s s a r y n u t r i e n t s o r t o t h e p r e s e n c e o f c e r t a i n c o m p o n e n t s t o x i c t o S t r e p t o m y c e s i n S S L , o r b o t h . S o , o b v i o u s l y t h e s u b s e q u e n t s t e p w o u l d b e t h e a d d i t i o n o f n u t r i e n t s , fill c o m p o n e n t s , b u t s u g a r , w h i c h w a s a b u n d a n t i n S S L , o f W a k s m a n ' s N o . 3 m e d i u m w e r e a d d e d t o S S L , i n t h e s a m e q u a n t i t i e s l i s t e d i n T a b l e 1 1 . 1 3 . H o w e v e r , t h e a d d i t i o n o f t h e s e n u t r i e n t s d i d n o t h e l p t h e g r o w t h c f t h e s e S t r e p t o m y c e s . H e n c e , i n t h e n e x t s t e p * S S L w a s d i l u t e d t o l o w e r t h e c o n c e n t r a t i o n o f p o s s i b l e t o x i c s u b s t a n c e s i n S S L . T h e d i l u t i o n w a s n o t d e n e w i t h w a t e r ^ b u t r a t h e r w i t h Waksman»s N o . 3 m e d i u m . N u t r i e n t s w e r e a d d e d t o S S L a s d e s c r i b e d a b o v e , b e f o r e d i l u t i o n s . H o w e v e r , w h e n t h e c o n c e n t r a t i o n o f S S L r e a c h e d o v e r 1 0 m l o f S S L p e r 1 0 0 m l o f m i x t u r e , g r o w t h o f S t r e p t o m y c e s c e a s e d t o s h o w a n y i m p r o v e m e n t . R e c o u r s e w a s t h e n m a d e t o a c c l i m a t i z a t i o n , t h a t i s , g i v i n g t h e b a c t e r i a t i m e t o g e t u s e d t o t h e n e w m e d i u m b y t r a n s f e r r i n g t h e m s l o w l y t h r o u g h m o r e a n d m o r e c o n c e n t r a t e d ( i n S S L ) m e d i a . G r o w t h w a s s t a r t e d i n W a k s m a n ' s N o . 3 s y n t h e t i c m e d i u m , t h e n c o n t i n u e d s u c c e s s i v e l y i n m i x t u r e s o f 5 , 1 0 , 2 0 , 3 0 , 5 0 , 1 0 0 m l o f S S L / 1 0 0 m l o f m i x t u r e . R e s u l t s o f t h e s e e x p e r i m e n t s w i t h d i l u t i o n a n d w i t h a c c l i m a t i z a t i o n a r e l i s t e d i n T a b l e I I I . 2 1 a n d T a b l e I I I . 2 2 . R e s u l t s o f e x p e r i m e n t s w i t h d i l u t i o n c a n b e v i s u a l l y o b s e r v e d i n F i g u r e s 3 . 2 7 a n d 3 . 2 8 . E x p e r i m e n t s a n d R e s u l t s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 67 FIGURE 3.27 : G r o w t h o f S t r e p t o m y c e s G r i s e u s i n D i l u t e d SSL Top row : G0.1, G3.1, 6 2 5 . 1, G1000.3 B o t t o m row : GO.2, G3.2, G25.2, G50.2 (see T a b l e I I I . 2 1 f o r d e t a i l s ) FIGURE 3.28 : G r o w t h o f S t r e p t o m y c e s O l i v a c e u s i n D i l u t e d SSL Top row : 0 0 . 1 , 0 2 5 . 1 , 0 5 0 . 1 , 01000.3 B o t t o m 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 ) S c a l e : y -J = 6.4 cm E x p e r i m e n t s and R e s u l t s Potential of Spent Sulf i te Liquor 68 FIGDRE 3.29 : Growth of L . plantarium in Solutions of Xylose and Glucose Regression lines at 0.95 confidence •—--—=—-- y ( ± 0 . 1 4 7 ) = 0.0550 (±0.011) x + 0. 108 (±0. 1 14) y (±0.082) = 0.0015 (±0 .006) x + 0. 185 (±0. 064) YEf i ST E X T R A C T C O N C E N T R A T I O N (G/L J Experiments and Results P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 6 9 3 . 2 G R O H T H _ O F _ L A C T Q B A C I L L D S _ I N _ X Y L O S E L . p l a n t a r i u m h a s b e e n k n o w n t o f e r m e n t p e n t o s e . E x p e r i m e n t s i n 1 2 5 m l E r l e n m e y e r f l a s k s a n d i n 1 6 x 1 5 0 m m t e s t t u b e s w e r e c o n d u c t e d u n d e r a n a e r o b i c c o n d i t i o n s a t 3 0 ° C w i t h 5 0 m l a n d 1 5 m l r e s p e c t i v e l y o f s y n t h e t i c m e d i u m . T h i s s y n t h e t i c m e d i u m c o n s i s t s o f 1 0 0 g / l o f y e a s t e x t r a c t p o w d e r , 4 g / l 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 , a n d s u g a r , e i t h e r g l u c o s e o r x y l o s e , a t v a r i o u s c o n c e n t r a t i o n s . T h e p H o f t h e m e d i u m w a s a s e p t i c a l l y r e a d j u s t e d t o 7 a f t e r s t e r i l i z a t i o n . L . p l a n t a r i u m w e r e i n o c u l a t e d t o t h e s e f l a s k s a n d t e s t t u b e s . T h e r e s u l t s o f t h i s s e r i e s o f e x p e r i m e n t s a r e p r e s e n t e d o n F i g u r e 3 . 2 9 . F e r g u s o n [ 2 3 ] s h o w e d t h e n o n f e r m e n t a b i l i t y o f p e n t o s e s b y 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 . H e n c e t h e r e w a s n o p o i n t i n r e p e a t i n g t h e s e t e s t s o n P r o p i o n i b a c t e r i a . T h e l i t e r a t u r e [ 1 0 , 7 7 ] a l s o b a c k s u p F e r g u s o n ' s c o n c l u s i o n s o n t h i s s u b j e c t . 3 . 3 P R E L I M I N A R Y S T U D I E S , O N , 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 T h i s s e r i e s o f e x p e r i m e n t s w a s d o n e i n 1 3 x 1 0 0 m m t e s t t u b e s . T h e g r o w t h w a s f o l l o w e d , a g a i n s t t i m e , e n t i r e l y b y t h e m e a s u r e m e n t o f t u r b i d i t y . T h e S S L f e r m e n t a t i o n m e d i u m w a s f r e e o f a d d e d c o b a l t s a l t . O n l y u g / l 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 , a n d v a r i o u s a m o u n t s o f y e a s t e x t r a c t p a s t e , r a n g i n g f r o m 0 t o 1 0 0 g / l , w e r e a d d e d t o t h e s u l f u r - d i o x i d e - s t r i p p e d S S L . T h e s e t u b e s w e r e i n c u b a t e d a t 3 0 ° C w i t h o c c a s i o n a l t h o r o u g h s h a k i n g ( n e e d e d f o r e v e r y m e a s u r e m e n t c f t u r b i d i t y ) f o r u p t o 3 5 0 h o u r s . T h e r e s u l t s a r e s u m m a r i z e d i n F i g u r e s 3 . 3 0 , 3 . 3 1 , a n d E x p e r i m e n t s a n d R e s u l t s 70 P o t e n t i a l of Spent S u l f i t e L i g u o r L E G E N D YE f i ST E X T R A C T C O N C E N T R A T I O N ( P A S T E . G / L ) • 0 5 + 10 X 2 0 4 0 + 6 0 K 70 Y 8 0 H 100 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 o f S p e n t S u l f i t e L i g u o r 71 FIGURE 3 , 3 0 1.60 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 on G r o w t h o f L. p l a n t a r i u m i n SSL i n T e s t T u b e s  1.40 1.20 1.00 0Q >-OQ o a : CD o.ao 0.60 0.40 0.20 X X X Y X X X X Y X X • Y X Y * X * X X X Jft * * Y * X • • X Y Y X JR • • X • • X X Y X X • X X X • • o • X X X X X X X X X Y X X + 4- + + + <t> + ,+ + v . m T m • 0.0 A A 1 A 0.0 70.0 140.0 210.0 2 8 0 . 0 F E R M E N T A T I O N T I M E ( H R S ) Experiments and R e s u l t s CP 350.0 7 2 Potential of Spent Sulfite Liquor L E G E N D Y E R S T E X T R A C T C O N C E N T R A T I O N ( P A S T E . G / L ) • 0 5 + 10 X 2 0 4 0 6 0 K 70 Y 8 0 W 100 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 o f S p e n t S u l f i t e L i q u o r 73 FIGURE 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 on 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 SSL i n T e s t T u b e s 1.40 1.20 1.00 CQ Cd OQ o (-Q 0.80 0.60 0.40 0.20 L 0.0 * X A 0 X + • + A • • x x £ a • • X X Y X ¥ V.Y » X X + 55 X Y X + x m + + + A A A <P X A X CD l l I8 • • • • a m .0.0 • 70.0 140.0 210.0 280.0 F E R M E N T A T I O N T I M E ( H R S ) E x p e r i m e n t s and R e s u l t s 350.0 Potential of Spent Sulf i te Liquor L E G E N D 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 ( P A S T E . G / L ) • 0 5 + 10 X 2 0 4 0 + 6 0 K 7 0 Y 8 0 W 100 E x p e r i m e n t s and R e s u l t s Potential of Spent Sulf i te Liquor 75 FIGURE 3.32 : Effect of Yeast Extract Concentration on Growth of 0 80 P * s n e r r n a n i i i n SSL i n Test Tubes 0 .70 0 . 6 0 0 .50 Q 1—4 CD CD o cr: CD 0 .40 0.30 0.20 0.10 0 .0 <!>• *X Y • * + 2 Y if X X X . I x 8 + * 6 J Y A A A S J J A D A a • Y Y Y W H H * M X X X x + + A A CD JL CD C D A C D • CD CD CD SC Y 0 X Y Y X X X X X X + + A CD A £ A A C D ° • CD 0 . 0 7 0 . 0 340.0 210.0 280 .0 F E R M E N T A T I O N T I M E ( M R S ) E x p e r i m e n t s and R e s u l t s 4 350.0 Potential of Spent Sulfite Liquor 76 FIGURE 3.33 : Legend for Figures Numbered from 3.30 to 3.32 L E G E N D Y E R S T E X T R A C T C O N C E N T R A T I O N ( P A S T E . G / L ) • 0 5 + 10 X 2 0 4 0 6 0 K 70 Y 8 0 « 100 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 o f S p e n t S u l f i t e L i g u o r 7 7 3 . 3 2 f o r L . p l a n t a r i u m , P . f r e u d e n r e i c h i i , a n d P . s h e r m a n i i r e s p e c t i v e l y . T h e r e s u l t s s h e w t h a t f o r 1 . p l a n t a r i u m a n d P . s h e r m a n i i , t h e g r o w t h i n c r e a s e s g r a d u a l l y w i t h i n c r e a s e s 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 u p t o 6 0 g / l , t h e n t h e g r o w t h d o e s n o t i n c r e a s e n o t i c e a b l y a n y m o r e . F o r P . f r e u d e n r e i c h i i , g r o w t h r i s e s s h a r p l y w i t h i n c r e a s e 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 f r o m 0 t o 5 g / l , t h e n i t s l a c k e n s i t s p a c e . O b v i o u s l y t h e n , P . f r e u d e n r e i c h i i r e g u i r e l e s s y e a s t e x t r a c t t h a n t h e o t h e r t w o s p e c i e s . 3 . 4 E F F E C T I V E N E S S O F Y E A S T E X T R A C T F R O M D I F F E R E N T S O U R C E S T h o u g h D i f c o y e a s t e x t r a c t p o w d e r w a s u s e d f o r m o s t o f t h e m a i n e x p e r i m e n t s , d u e t o t h e i r a v a i l a b i l i t y , y e a s t e x t r a c t s f r o m d i f f e r e n t s o u r c e s , n a m e l y N B y e a s t e x t r a c t p o w d e r a n d y e a s t e x t r a c t p a s t e ( w i t h 0 . 7 7 g o f s o l i d s / g o f t o t a l p a s t e ) w e r e u s e d i n d i f f e r e n t s t a g e s o f t h e p r o j e c t . T h e e f f e c t i v e n e s s o f t h e y e a s t e x t r a c t f r o m d i f f e r e n t s o u r c e s o n g r o w t h o f b a c t e r i a w a s t h e n c h e c k e d w i t h f e r m e n t a t i o n s u s i n g L . p l a n t a r i u m . T h e s e e x p e r i m e n t s w e r e c a r r i e d o n i n 1 2 5 m l e r l e n m e y e r f l a s k s . T h e m e d i u m u s e d w a s a n a q u e o u s s o l u t i o n o f 4 0 g / l o f g l u c o s e , 4 g / l 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 w i t h a v a r y i n g a m o u n t o f y e a s t e x t r a c t a d d e d . T h e s e f l a s k s w e r e i n c u b a t e d a t 30<>C w i t h o u t a n y v i g o r o u s s h a k i n g f o r 1 3 0 h o u r s . T h e r e s u l t s , i n F i g u r e 3 . 3 4 , u n v e i l t h e e q u i v a l e n c e o f t h e n u t r i e n t v a l u e o f t h e y e a s t e x t r a c t s u s e d . Y e a s t e x t r a c t p a s t e w a s c o m p a r e d o n a d r y b a s i s . / G r o w t h o f L . p l a n t a r i u m w i t h y e a s t e x t r a c t p a s t e d e v i a t e d E x p e r i m e n t s a n d R e s u l t s P o t e n t i a l of Spent S u l f i t e L i q u o r 78 FIGURE 3.34 : Growth o f L. p l a n t a r i u m as Measurement o f E f f e c t i v e n e s s of Yeast E x t r a c t from V a r i o u s S o u r c e s 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) Y E A S T E X T R A C T P A S T E C Q N C . ( G / L . D R Y B A S I S ) 0 0 10.0 20.0 30.0 40.0 50.CJ Q , 1 1 1 I Y E A S T 0 0 10.0 2 0 . 0 30 .0 4 0 . 0 5 0 . 0 Y E A S T E X T R A C T POWDER C O N C E N T R A T I O N CG/L ) 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 o f S p e n t S u l f i t e L i g u o r 79 s o m e w h a t f r o m t h e c o m m o n g r o w t h l i n e . H o w e v e r , y e a s t e x t r a c t p a s t e w a s u s e d o n l y i n t h e p r e l i m i n a r y s t u d i e s o n e f f e c t s o f y e a s t o n g r o w t h a n d i n a f e w e a r l y f e r m e n t a t i o n s , s o n o f u r t h e r e f f o r t w a s m a d e t o c l e a r t h i s p o i n t . W h e n e v e r y e a s t e x t r a c t p a s t e w a s u s e d i t w i l l b e s t a t e d , a n d t h e c o n c e n t r a t i o n w i l l b e e x p r e s s e d o n a w e t b a s i s . 3 . 5 R E P L A C E M E N T O F Y E A S T E X T R A C T B Y F I S H P L A N T E F F L U E N T S S L , s t e a m s t r i p p e d o f s u l f u r d i o x i d e , w i t h 4 g / l 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 a d d e d , w a s m i x e d w i t h f i s h p l a n t e f f l u e n t a t d i f f e r e n t p r o p o r t i o n s ( 9 : 1 , 8 : 2 , 6 : 4 , 5 : 5 ) t h e n f e r m e n t e d 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 a n d P . s h e r m a n i i i n 2 5 0 m l E r l e n m e y e r f l a s k s , a t 3 0 0 C . O r i g i n a l l y , t h e f i s h p l a n t e f f l u e n t w a s a d d e d a s r e c e i v e d , b u t t h e h i g h c o n t e n t o f s o l i d s ( 7 2 g / l t o t a l , 2 0 g / l s o l u b l e ) p r o h i b i t e d a l l m e a s u r e m e n t s o f g r o w t h b y w e i g h t o r t u r b i d i t y , a s w e l l a s b a r r i n g a n y e a s y , p o s s i b l e m i c r o s c o p i c o b s e r v a t i o n s . T h i s f i s h p l a n t e f f l u e n t w a s t h e n c o c k e d w i t h s t e a m a n d c e n t r i f u g e d . T h e s u p e r n a t a n t l i g u i d w a s u s e d f o r s u b s e q u e n t e x p e r i m e n t s . P a r t o f t h e s o l u b l e s o l i d s p r e c i p i t a t e d d u r i n g s t e r i l i z a t i o n . T h e y w e r e s l i m y a n d h a r d t o f i l t e r c r c e n t r i f u g e o f f . T h e r e d u c e d c o n t e n t o f s o l i d s d i d n o t p r e v e n t t h e m e a s u r e m e n t o f g r o w t h b y d r y c e l l w e i g h t . M i c r o s c o p i c o b s e r v a t i o n s g a v e s i g n s o f p o s s i b l e g r o w t h , b u t n o p o s i t i v e g r o w t h c o u l d b e a f f i r m e d b y m e a s u r e m e n t s o f g r o w t h o r o f p r o d u c t s , n a m e l y a c i d s . I d e n t i f i c a t i o n c f p r o d u c t s w a s m a d e o n l y f o r f e r m e n t a t i o n s w i t h P r o p i o n i b a c t e r i a , a s E x p e r i m e n t s a n d R e s u l t s P o t e n t i a l o f S p e n t S u l f i t e l i q u o r 8 0 v o l a t i l e a c i d s m a y b e e a s i l y d i s t i n g u i s h e d f r o m t h e h i g h a m i n o a c i d s c o n t e n t o f t h e p r o t e i n i n t h e e f f l u e n t . T h e e t h e r e x t r a c t i o n d e s c r i b e d e a r l i e r c a n n o t s e p a r a t e l a c t i c a c i d f r o m a m i n o a c i d s i n t h e f i s h p l a n t e f f l u e n t . T h e s e e x p e r i m e n t s d e s t r o y t h e h o p e o f u s i n g f i s h p l a n t e f f l u e n t a s a c h e a p n i t r o g e n s o u r c e t o r e p l a c e t h e m u c h m o r e e x p e n s i v e y e a s t e x t r a c t . 3 . 6 P R E L I M I N A R Y , E X P E R I M E N T S I N E R L E N M E Y E R , F L A S K S O n l y t w o 7 1 - f e r m e n t o r s , o f t h e M i c r o f e r m t w i n u n i t , w e r e a v a i l a b l e t o t h i s p r o j e c t , s o o n l y t w o s i m u l t a n e o u s e x p e r i m e n t s c o u l d b e d o n e i n t h e s e . T h u s o n l y f e r m e n t a t i o n s n e e d e d t o b e f o l l o w e d a g a i n s t t i m e w e r e s a v e d f o r t h e s e f e r m e n t c r s . M o s t o f o t h e r f e r m e n t a t i o n s w e r e t h e n d o n e i n 2 5 0 m l E r l e n m e y e r f l a s k s . U n f o r t u n a t e l y , t h e s e l a t t e r e x p e r i m e n t s f a i l e d t o l i v e u p t o t h e i r i n t e n d e d p u r p o s e . T h e v a r i o u s r e a s o n s f o r t h i s f a i l u r e w i l l b e p r e s e n t e d s h o r t l y i n t h e d i s c u s s i o n s f o u n d i n t h e n e x t c h a p t e r . T h e s e w e r e t h e n j u s t r e g a r d e d a s p r e l i m i n a r y e x p e r i m e n t s , t h e r e s u l t s o f w h i c h w e r e t h e n u s e d t c h e l p i n t h e c h o i c e o f r a n g e o f t h e f e r m e n t a t i o n v a r i a b l e s t o b e u s e d i n t h e e x p e r i m e n t s i n t h e 7 1 f e r m e n t o r s . R e i n f o r c e d c l o s t r i d i a l m e d i u m a n d S S L m e d i u m w i t h v a r i o u s c o n c e n t r a t i o n s o f y e a s t e x t r a c t p o w d e r ( 0 , 1 0 , 2 0 , 3 0 , 4 0 , 5 0 , 1 0 0 g / l ) w e r e u s e d f o r f e r m e n t a t i o n s w i t h L . p l a n t a r i u m , P . f r e u d e n r e i c h i i , P . s h e r m a n i i , a n d a m i x t u r e c f t h e f i r s t t w o . T h e c o n c e n t r a t i o n 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 w a s k e p t E x p e r i m e n t s a n d R e s u l t s Potential of Spent Sulf i te Liquor LEGEND + GROWTH. DRY CELL WEIGHT (G/L) x LACTIC ACID PRODUCTION (MOLES/L) Y SUGAR CONSUMPTION (G/L) A VOLATILE ACIDS PRODUCTION (M0LE5/L) Experiments and R e s u l t s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 82 FIGURE 3.35 : F e r m e n t a t i o n s o f SSL w i t h L. p l a n t a r i u m i n F l a s k s 2.4 30.0 0.0 20.0 40.0 60.0 80.0 100.0 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 (G/L ) Experiments and Results P o t e n t i a l of Spent S u l f i t e Liquor L E G E N D + GROWTH. DRY C E L L WEIGHT (G/L ) x L A C T I C A C I D PRODUCT ION ( M O L E S / L ) Y SUGAR CONSUMPT ION ( G / L ) A V O L A T I L E A C I D S PRODUCT ION ( M O L E S / L ) 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 o f S p e n t S u l f i t e L i q u o r 84 FIGURE 3.36 : F e r m e n t a t i o n s o f SSL w i t h P. f r e u d e n r e i c h i i i n F l a s k s + I 30 .0 Y + 2.2! - - 2 7 . 5 Y + 2.0 j - 25 .0 Y + 1.8 j - 2 2 . 5 Y + 1.8 j + I .4 ! + 1 . 2 ! 20 .0 17.5 15 .0 Y Y Y x o ° 8 ! x 0 - 0 6 i x 0 -04 i X 0 . 0 2 J v o . o o i j - 1 _ r/A • M— + X + A 1 1 12 .5 10.0 0.0G 0 . 0 4 0 .02 0 .00 Y Y . A 1 1 1 | A I 3 1 1 A 1 ! A 0 .0 20 .0 40.0 60 .0 80.0 100.0 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 (G/L ) Experiments and Results 85 Potential of Spent Sulfite Liquor L E G E N D + GROWTH. DRY C E L L WEIGHT (G/L ) x L A C T I C A C I D PRODUCT ION ( M O L E S / L ) _ Y SUGAR CONSUMPT ION IG/L ) „ A V O L A T I L E A C I D S PRODUCT ION ( M O L E S / L ) 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 o f S p e n t S u l f i t e L i g u o r 8 6 F I G U R E 3.37 : F e r m e n t a t i o n s o f SSL w i t h P. s h e r m a n i i i n F l a s k s 30.0 , Y 0.0 20.0 40.0 60.0 80.0 JOO.O YEflST EXTRACT CONCENTRATION (G/L) E x p e r i m e n t s and R e s u l t s 87 Potential of Spent Sulf i te Liquor L E G E N D + GROWTH. DRY C E L L WEIGHT (G/L ) x L A C T I C A C I D PRODUCT ION ( M O L E S / L ) Y SUGAR CONSUMPT ION (G/L ) A V O L A T I L E A C I D S PRODUCT ION ( M O L E S / L ) 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 o f S p e n t S u l f i t e L i q u o r 88 FIGURE 3.38 : F e r m e n t a t i o n s o f SSL w i t h A M i x t u r e o f 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 F l a s k s 2 . 4 2 .2 + 2 -0 3 0 . 0 1.8 I L + 1-6 1.4 1.2 X X X X 0.10 o.oa 0.06 L 0 .04 X 0 . 0 2 | L x 0 . 0 0 # 0 .0 20.0 40 .0 60 .0 80.0 YERST EXTRRCT C0NCENTRRTI0N (G/L) o.oo A 100.0 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 o f S p e n t S u l f i t e L i q u o r 8 9 c o n s t a n t a t ftg/1, a n d t h e c o n c e n t r a t i o n o f c o b a l t i o n s a t 2 . 2 p p m i n S S L . E v e r y 1 0 0 m l o f t h e d e s i r e d m e d i u m w a s s t e r i l i z e d i n a 2 5 0 m l E r l e n m e y e r f l a s k b e f o r e i n c c u l a t i o n . p H w a s a l w a y s a s e p t i c a l l y r e a d j u s t e d a f t e r s t e r i l i z a t i o n e x c e p t i n c n e s e r i e s . O n l y t h e e n d p r o d u c t s w e r e o f i n t e r e s t , s o n o s a m p l i n g w a s n e c e s s a r y d u r i n g f e r m e n t a t i o n , a n d 1 0 0 m l w a s m o r e t h a n s u f f i c i e n t f o r a n a l y s i s . T h e s e f l a s k s w e r e t h e n i n c u b a t e d a t i 3 0 ° C f o r 1 0 0 t o 1 5 0 h o u r s . M o s t o f t h e s e f l a s k s w h i c h h a d b e e n i n o c u l a t e d w i t h P r o p i o n i b a c t e r i a w e r e c o n t i n u o u s l y s h a k e n o n l y a f t e r t h e f i r s t 8 0 - 1 0 0 h o u r s o f i n c u b a t i o n , d u r i n g t h e a e r o b i c s e c o n d s t a g e . T h e r e s u l t s o f t h e s e e x p e r i m e n t s a r e p r e s e n t e d i n F i g u r e s 3 . 3 5 , 3 . 3 6 , 3 . 3 7 , 3 . 3 8 a n d t a b u l a t e d i n t a b l e s A I . 2 8 , A I . 2 9 , A I . 3 0 , A I . 3 1 i n A p p e n d i x I . 3.7 F E R M E N T A T I O N S I N T H E . 7 L M I C R O F E R M F E R M E N T O R S T h e r u n s a r e l i s t e d i n T a b l e I I I . 2 3 . I n t h e s e f e r m e n t a t i o n s , S S L w a s s t e a m s t r i p p e d o f s u l f u r d i o x i d e t o 2 0 0 p p m o r l o w e r . N u t r i e n t s w e r e t h e n a d d e d ; 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 c o n c e n t r a t i o n w a s k e p t c o n s t a n t t o U g / 1 , c o b a l t i o n c o n c e n t r a t i o n a n d 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 w e r e v a r i a b l e s , a s w a s t h e a d d i t i o n o f D M B Z . T h e s e r u n s c a n b e d i v i d e d i n t o U m a i n c a t e g o r i e s : 1 . F e r m e n t a t i o n 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 i n t w o s t a g e s . 2 . F e r m e n t a t i o n w i t h a m i x t u r e o f L . p l a n t a r i u m a n d E x p e r i m e n t s a n d R e s u l t s P o t e n t i a l o f S p e n t S u l f i t e L i g u o r 90 T a b l e I I I . 2 3 S U M M A R Y O F I N F O R M A T I O N O N F E R M E N T A T I O N S O F S S L I N 7 L F E R M E N T O R S r I S e r i e s R u n B a c t e r i a Y e a s t C o b a l t p H F i g u r e s T a b l e : S p e c i e s E x t r a c t i o n C o n t r o l C o n e . C o n e . ( g / i ) ( p p m ) | 1 . 1 F P 1 . 1 L P 2 0 2 . 2 A u t o m a t i c 3 . 3 9 A I . 3 2 F P 1 . 2 L P 2 0 2 . 2 S p o t 3 . 4 0 A I . 3 3 I 1 . 2 F P 2 . 1 ( L P t h e n ) 2 0 2 . 2 N o 3 . 4 1 A I . 3 4 P F F P 2 . 2 ( L P t h e n ) 2 0 2 . 2 N o 3 . 4 2 A I . 3 5 P F 1 2 F P 3 L P 6 P F 2 0 2 . 2 A u t o m a t i c 3 . 4 3 A I . 3 6 I 3 . 1 F 1 5 . 2 2 a P F 2 0 2 . 2 N o 3 . 4 4 A I . 3 7 ( p a s t e ) F 1 5 . 2 2 b P F 2 0 2 . 2 N o 3 . 4 5 A I . 3 8 ( p a s t e ) I 3 . 2 F 0 5 . 2 2 P F 0 5 2 . 2 A u t o m a t i c 3 . 4 6 A I . 3 9 F 1 0 . 2 2 P F 1 0 2 . 2 A u t o m a t i c 3 . 4 7 A I . 4 0 F 2 0 . 2 2 P F 2 0 2 . 2 A u t o m a t i c 3 . 4 8 A I . 4 1 F 3 0 . 2 2 P F 3 0 2 . 2 A u t o m a t i c 3 . 4 9 A I . 4 2 1 3 . 3 F 2 0 . 0 0 P F 2 0 0 . 0 A u t o m a t i c 3 . 5 0 A I . 4 3 F 2 0 . 0 9 P F 2 0 0 . 9 A u t o m a t i c 3 . 5 1 A I . 4 4 1 3 . 4 F 2 0 . 2 2 . 1 2 6 * P F 2 0 2 . 2 A u t o m a t i c 3 . 5 2 A I . 4 5 1 4 S 2 0 . 2 2 P S 2 0 2 . 2 A u t o m a t i c 3 . 5 3 A I . 4 6 S e r i e s T y p e 1 F e r m e n t a t i o n 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 1 . 1 F e r m e n t a t i o n o f S u g a r t o L a c t i c A c i d b y L . p l a n t a r i u m 1 . 2 F e r m e n t a t i o n o f L a c t i c A c i d t o V o l a t i l e A c i d s b y P F 2 F e r m e n t a t i o n w i t h a M i x t u r e o f L P a n d P F 3 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 3 . 1 E a r l y F e r m e n t a t i o n s 3 . 2 F e r m e n t a t i o n w i t h Y e a s t E x t r a c t a s V a r i a b l e 3 . 3 F e r m e n t a t i o n w i t h 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 3 . 4 A d d i t i o n o f 5 f 6 - D i m e t h y l B e n z i m i d a z o l e 4 F e r m e n t a t i o n w i t h P . s h e r m a n i i * 1 2 6 p p m o f D M B Z w a s a d d e d t o S S L p r i o r t o i n o c u l a t i o n E x p e r i m e n t s a n d R e s u l t s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 9 1 P . f r e u d e n r e i c h i i . 3 . 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 . 4 . F e r m e n t a t i o n w i t h P . s h e r m a n i i . I n t h e f i r s t s t a g e o f s e r i e s 1 , t h e s u g a r i n t h e S S L w a s f e r m e n t e d t o l a c t i c a c i d b y L . p l a n t a r i u m w i t h p H a u t o m a t i c a l l y c o n t r o l l e d a t 7 . I n t h i s s t a g e 2 0 g / l o f y e a s t e x t r a c t a n d 2 . 2 p p m o f c o b a l t i o n w e r e a d d e d t o t h e f e r m e n t a t i o n b r o t h . W h e n t h e f e r m e n t a t i o n r e a c h e d t h e s t a t i o n a r y p h a s e , 2 0 g m o r e o f y e a s t e x t r a c t w e r e a d d e d t o e v e r y l i t e r o f t h e f i n a l b r o t h . T h i s b r o t h w a s s t e r i l i z e d a g a i n , t h e n i n o c u l a t e d w i t h P . f r e u d e n r e i c h i i . T h e s e c o n d s t a g e t h e n b e g a n . R u n F P 2 . 2 u s e d t h e f i n a l b r o t h f r o m r u n F P 1 . 2 a n d r u n F P 2 . 1 t h e o n e f r o m t u n F P 1 . 1 . A s u b s t a n t i a l a m o u n t o f t h e f i n a l b r o t h o f r u n F P 1 . 1 w a s s e t a s i d e f o r a n o t h e r s e r i e s o f e x p e r i m e n t s i n 2 5 0 m l E r l e n m e y e r f l a s k s , t h e r e s u l t s c f w h i c h a r e p r e s e n t e d i n F i g u r e 3 . 7 1 . I n t h i s s e r i e s , v a r i o u s a m o u n t s o f y e a s t e x t r a c t w e r e a d d e d t o f l a s k s w i t h 1 0 0 m l o f f e r m e n t e d S S L i n e a c h . A l l b u t o n e ( t h e b r o t h w a s t r a n s f e r r e d a s e p t i c a l l y t o t h i s f l a s k f r o m t h e f e r m e n t o r ) , w e r e s t e r i l i z e d . E i t h e r P . f r e u d e n r e i c h i i o r P . s h e r m a n i i w e r e t h e n i n o c u l a t e d t o t h e f l a s k s . N e a r t h e e n d o f t h e s e c o n d s t a g e o f f e r m e n t a t i o n s o f s e r i e s 1 , a i r w a s b l o w n i n a t a r a t e o f 2 0 0 0 c c / m i n . * I n s e r i e s 2 , S S L , w i t h 2 0 g / l o f y e a s t e x t r a c t a n d 2 . 2 p p m o f c o b a l t i o n , w a s i n o c u l a t e d 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 s i m u l t a n e o u s l y . N e a r t h e e n d o f t h e f e r m e n t a t i o n , a i r w a s b l o w n i n a t a r a t e o f 2 0 0 0 c c / m i n . I n s e r i e s 3 , f e r m e n t a t i o n s w e r e d e n e w i t h P . 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 a n d R e s u l t s 92 P o t e n t i a l o f S p e n t S u l f i t e L i q u o r Run FP1.1 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 L E G E N D * flCJDS C O N C E N T R A T I O N ( M O L E S / L ) + GROWTH. DRY C E L L WEIGHT (G/L ) .__ x L A C T I C A C I D C O N C E N T R A T I O N ( M O L E S / L ) ^ y SUGAR C O N C E N T R A T I O N (G/L ) A 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 ( M O L E S / L ) 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 o f Spent S u l f i t e L i g u o r 93 FIGURE 3.39 : Run FP1.1. F e r m e n t a t i o n w i t h L. p l a n t a r i u m 0.0 25.0 50.0 75.0 100.0 125.0 F E R M E N T A T I O N T I M E (HOURS) E x p e r i m e n t s and R e s u l t s 94 r P o t e n t i a l of Spent S u l f i t e L i q u o r Run FP1.2 20g/l of yeast e x t r a c t powder 2.2ppm of c o b a l t i o n 4 g / l of potassium dihydrogen phosphate pH manually spot c o n t r o l l e d L E G E N D . x flCJDS C O N C E N T R A T I O N ( M O L E S / L ) . + GROWTH, DRY C E L L WEIGHT (G/L ) . x L A C T I C A C I D C O N C E N T R A T I O N ( M 0 L E 5 / L ) M Y SUGAR C O N C E N T R A T I O N (G/L ) . A 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 ( M O L E S / L ) 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 of Spent S u l f i t e L i q u o r 9 5 FIGURE 3.40 : Run FP1.2. Fermentation with L. plantarium 42.0 0.36 35.0 Y 28.0 L + 0.9 0.G + 0.3 L 0.0 0.0 25.0 50.0 75.0 100.0 F E R M E N T A T I O N T I M E (HOURS) Experiments and R e s u l t s I •j P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 1 Run FP2.1 I 2 0 g / l o f y e a s t e x t r a c t powder I 2.2ppm o f c o b a l t i o n I 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 I L E G E N D m A C I D S C O N C E N T R A T I O N ( M O L E S / L ) + GROWTH. DRY C E L L WEIGHT (G/L ) x L A C T I C A C I D C O N C E N T R A T I O N ( M O L E S / L ) Y SUGRR C O N C E N T R A T I O N (G/L ) n V I T A M I N B 1 2 C O N C E N T R A T I O N (MG/L ) A 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 ( M O L E S / L ) 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 o f S p e n t S u l f i t e L i q u o r 97 FIGURE 3.41 : Run F P 2.1. 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 M a i n S u b s t r a t e A N A E R O B I C o.o A E R O B I C A X X • n A 0.30 £ J 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 40.0 80.0 1Z0.0 160.0 F E R M E N T A T I O N T I M E (HOURS) 200 0.06 x .0 E x p e r i m e n t s and R e s u l t s 98 P o t e n t i a l o f S p e n t S u l f i t e L i q u o r Run FP2.2 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 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 L E G E N D * A C I D S C O N C E N T R A T I O N ( M O L E S / L ) + GROWTH. DRY C E L L WEIGHT (G/L ) _ . . _ „ . . x L A C T I C A C I D C O N C E N T R A T I O N ( M O L E S / L ) Y SUGAR C O N C E N T R A T I O N (G/L ) n V I T A M I N B I 2 C O N C E N T R A T I O N (MG/L ) - A V O L A T I L E R C I D S C O N C E N T R A T I O N ( M O L E S / L ) 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 o f S p e n t S u l f i t e L i q u o r 99 FIGURE 3.42 : Run F P 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 on L a c t i c A c i d a s M a i n S u b s t r a t e A N A E R O B I C R E R O B I C 0 . 0 G x 0 . 0 4 0 . 0 8 0 . 0 1 2 0 . 0 1 6 0 . 0 F E R M E N T A T I O N T I M E (HOURS) 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 of Spent S u l f i t e L i g u o r Run FP3 20g/l of yeast e x t r a c t powder 2.2ppm of c o b a l t ion Ug/1 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 L E G E N D . + GROWTH. DRY C E L L WEIGHT (G/L ) x L R C T I C A C I D C O N C E N T R A T I O N ( M 0 L E 5 / L ) Y SUGAR C O N C E N T R A T I O N ( G / L ) m V I T A M I N B12 C O N C E N T R A T I O N (MG/L) A 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 ( M O L E S / L ) Experiments and R e s u l t s P o t e n t i a l of Spent S u l f i t e L i q u o r 101 FIGURE 3.43 : 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 Y 36.0 A N A E R O B I C Y 28.0 L Y 2 0 . 0 L • 0.30 • 0.20 L • 0.10 • 0.00 0.04 x 0 .0 40.0 80 .0 120.0 160.0 F E R M E N T A T I O N T IME (HOURS) Experiments and R e s u l t s 102 P o t e n t i a l o f S p e n t S u l f i t e L i q u o r R u n F 1 5 . 2 2 a 2 0 g / l o f y e a s t e x t r a c t p a s t e 2 . 2 p p m o f c o b a l t i o n U g / 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 p H r e a d j u s t e d a t t i m e s , n o t c o n t r o l l e d I ; I L E G E N D x A C I D S C O N C E N T R A T I O N ( M O L E S / L ) + GROWTH. DRY C E L L WEIGHT (G/L ) • PH E x p e r i m e n t s a n d R e s u l t s P o t e n t i a l o f S p e n t S u l f i t e L i g u o r 103 FIGURE 3.44 : Run F l 5 . 2 2 a . 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 . E f f e c t o f pH FERMENTATION TIME (HOURS) E x p e r i m e n t s and R e s u l t s 1 0 4 r P o t e n t i a l o f S p e n t S u l f i t e L i q u o r B u n F 1 5 . 2 2 b 2 0 g / l o f y e a s t e x t r a c t p a s t e 2 . 2 p p m o f c o b a l t i o n 4 g / l 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 p H n o t c o n t r o l l e d L E G E N D + GROWTH. DRY C E L L WEIGHT (G/L ) * p H - - Y SUGAR C O N C E N T R A T I O N (G/L ) • V I T A M I N B 1 2 C O N C E N T R A T I O N (MG/L ) — „ A 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 ( M O L E S / L ) E x p e r i m e n t s a n d R e s u l t s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 105 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 P. f r e u d e n r e i c h i i i n One A n a e r o b i c S t a g e 0.36 A 0.0 100.0 200.0 300.0 400.0 F E R M E N T A T I O N T I M E (HOURS) 0.30 A J 0 . 2 4 A J O . I B A J 0 . 4 n J 0 . 2 a 0.0 a 500.0 E x p e r i m e n t s and R e s u l t s 106 r Potential of Spent Sulf i te Liquor Run F05.22 05g/l of yeast extract powder 2.2ppm of cobalt ion 4g/ l of potassium dihydrogen phosphate pH automatically controlled L E G E N D + GROWTH. DRY C E L L WEIGHT (G/L ) ._ x L A C T I C A C I D C O N C E N T R A T I O N ( M O L E S / L ) _ Y SUGAR C O N C E N T R A T I O N (G/L ) D V I T A M I N B 1 2 C O N C E N T R A T I O N (MG/L ) A 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 ( M O L E S / L ) E x p e r i m e n t s and R e s u l t s Potential of Spent Sulfite Liquor 107 FIGURE 3.46 : Run F05.22. Fermentation with P. freudenreichii . Yeast Extract Concentration as Variable 9.0 ANAEROBIC 7 .5 6 .0 • 0.9 • 0 .6 • 0 .3 AEROBIC T* s J A -A 40.0 80.0 120.0 160.0 FERMENTATION TIME (HOURS) 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 .0 X E x p e r i m e n t s and R e s u l t s 108 P o t e n t i a l o f S p e n t S u l f i t e L i g u o r R u n F 1 0 . 2 2 1 0 g / l o f y e a s t e x t r a c t p o w d e r 2 . 2 p p a i o f c o b a l t i o n 4 g / l 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 p H a u t o m a t i c a l l y c o n t r o l l e d L E G E N D + GROWTH. DRY C E L L WEIGHT (G/L ) x L A C T I C A C I D C O N C E N T R A T I O N ( M O L E S / L ) Y SUGAR C O N C E N T R A T I O N (G/L ) — n V I T A M I N B 1 2 C O N C E N T R A T I O N (MG/L) A 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 ( M O L E S / L ) E x p e r i m e n t s a n d R e s u l t s Potential of Spent Sulfite Liquor 109 FIGURE 3.17 : Run F10.22. Fermentation with P. freudenreichii . Yeast Extract Concentration as Variable + 9 .0 A N A E R O B I C + 7 .5 6 .0 • 0.9 . +.-+ 0 .6 • 0 .3 • 0.0 o --X-X-, : / • x x • \ I \ A E R O B I C o.o 40.0 80.0 120.0 160.0 F E R M E N T A T I O N T I M E (HOURS) 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 200.0 E x p e r i m e n t s and R e s u l t s 110 Potential of Spent Sulfite Liquor Run F20.22 20g/l of yeast extract powder 2.2ppm of cobalt ion 4g/ l of potassium dihydrogen phosphate pH automatically controlled L E G E N D + GROWTH. DRY C E L L WEIGHT (G/L ) x L A C T I C A C I D C O N C E N T R A T I O N ( M O L E S / L ) _ Y SUGAR C O N C E N T R A T I O N (G/L ) • V I T A M I N B 1 2 C O N C E N T R A T I O N (MG/L) „ A 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 ( M O L E S / L ) 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 o f S p e n t S u l f i t e L i q u o r 111 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 . 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 9.0 A N A E R O B I C 7.5 6.0 • 0.9 A E R O B I C I A — . - A - ~ i \ li +5 / A • 0.6 0.3 fTjo.o[Si_-in Y l A A V V • / • m / / V / / V r ^ Y _ Y X ~ - 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 _L 0.00 x 0.0 40.0 80.0 120.0 160.0 F E R M E N T A T I O N T I M E (HOURS) 200.0 E x p e r i m e n t s and R e s u l t s 1 1 2 Potential of Spent Sulfite Liquor Run F30.22 30g/l of yeast extract powder 2.2ppm of cobalt ion 4g/ l of potassium dihydrogen phosphate pH automatically controlled L E G E N D + GROWTH. DRY C E L L WEIGHT (G/L ) x L A C T I C A C I D C O N C E N T R A T I O N ( M O L E S / L ) . _ Y SUGAR C O N C E N T R A T I O N (G/L ) n V I T A M I N B12 C O N C E N T R A T I O N (MG/L) A 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 ( M O L E S / L ) 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 o f S p e n t S u l f i t e L i q u o r 113 FIGURE 3.49 : Run 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 . 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 + 9.0 A N A E R O B I C A E R O B I C + 7.5 + 6.0 G)0.6 G3 3 .3 • 0.0$ \/ Y / — x - - J c ' - \ f / " x x ** m •> \ \ Y Y - Y . _L 0.0 40.0 80.0 120.0 160.0 F E R M E N T A T I O N T I M E (HOURS) 200 44. Y 43. Y 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 0.00 x .0 E x p e r i m e n t s and R e s u l t s 114 Potential of Spent Sulf i te Liquor Run F20.00 20g/l of yeast extract powder O.Oppm of cobalt ion 4g/ l of potassium dihydrogen phosphate pH automatically controlled L E G E N D + GROWTH, DRY C E L L WEIGHT (G/L ) x L A C T I C A C I D C O N C E N T R A T I O N ( M O L E S / L ) _ Y SUGAR C O N C E N T R A T I O N (G/L ) n V I T A M I N B 1 2 C O N C E N T R A T I O N (MG/L ) A 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 ( M O L E S / L ) Experiments and Results P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 115 FIGURE 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 . 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 R E R 0 8 I C A " Y — L 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 X 40.0 80.0 120.0 160.0 F E R M E N T A T I O N T I M E (HOURS) 200.0 Experiments and R e s u l t s 116 P o t e n t i a l o f S p e n t S u l f i t e L i q u o r Run F20.09 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 L E G E N D + GROWTH. DRY C E L L WEIGHT (G/L ) x L A C T I C A C I D C O N C E N T R A T I O N ( M O L E S / L ) Y SUGAR C O N C E N T R A T I O N (G/L ) • V I T A M I N B 1 2 C O N C E N T R A T I O N (MG/L) „ A 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 ( M O L E S / L ) 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 o f S p e n t S u l f i t e L i q u o r 117 FIGURE 3.51 : Run 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 . C o b a l t I o n C o n c e n t r a t i o n as V a r i a b l e 9.0 A N A E R O B I C + 7.5 / 6.0 A E R O B I C • 0.9 CD 0.6 Xf—'— Q 0 . 3 CD 0. 40.0 80.0 120.0 160.0 F E R M E N T A T I O N T I M E (HOURS) 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 200.0 E x p e r i m e n t s and R e s u l t s 118 r P o t e n t i a l o f S p e n t S u l f i t e L i q u o 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 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 0 . 1 2 6 g / l o f DMBZ L E G E N D + GROWTH. DRY C E L L WEIGHT (G/L ) „ x L A C T I C A C I D C O N C E N T R A T I O N ( M 0 L E 5 / L ) y SUGAR C O N C E N T R A T I O N (G/L ) n V I T A M I N 8 1 2 C O N C E N T R A T I O N (MG/L ) „ A 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 ( M O L E S / L ) 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 o f S p e n t S u l f i t e L i q u o r 119 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 80.0 120.0 160.0 F E R M E N T A T I O N T I M E (HOURS) E x p e r i m e n t s a n d R e s u l t s Potential of Spent Sulfite Liquor Run S20.22 20g/l of yeast extract powder 2.2ppm of cobalt ion 4g/l of potassium dihydrogen phosphate pH automatically controlled L E G E N D + GROWTH. DRY C E L L WEIGHT (G/L ) x L A C T I C A C I D C O N C E N T R A T I O N ( M O L E S / L ) - y SUGAR C O N C E N T R A T I O N (G/L ) • V I T A M I N B12 C O N C E N T R A T I O N (MG/L) A 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 ( M O L E S / L ) E x p e r i m e n t s a n d R e s u l t s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 121 FIGURE 3.53 : Run S 2 0 . 2 2 . F e r m e n t a t i o n w i t h P. s h e r m a n i i A E R O B I C 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 40.0 80.0 120.0 160.0 F E R M E N T A T I O N T I M E (HOURS) 200.0 E x p e r i m e n t s a n d R e s u l t s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 122 FIGURE 3.54 : L e g e n d f o r A l l F i g u r e s , L i s t e d i n T a b l e I I I . 2 3 , f r o m 3.39 t o 3.53 L E G E N D R C I D S C O N C E N T R A T I O N ( M O L E S / L ) — + GROWTH. DRY C E L L WEIGHT (G/L ) X L A C T I C A C I D C O N C E N T R A T I O N ( M O L E S / 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 F L A S K S . F I G U R E S 3 . 3 5 TO 3 . 3 8 ) + Y pH SUGAR C O N C E N T R A T I O N (G/L ) (SUGAR CONSUMPT ION FOR F E R M E N T A T I O N S IN F L A S K S . F I G U R E S 3 . 3 5 TO 3 . 3 8 ) — Q V I T A M I N B 1 2 C O N C E N T R A T I O N (MG/L ) A 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 ( M O L E S / L ) ( V O L A T I L E A C I D S PRODUCT ION FOR F E R M E N T A T I O N S IN F L A S K S . F I G U R E S 3 . 3 5 TO 3 . 3 8 ) 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 o f S p e n t S u l f i t e l i q u o r 1 2 3 a l o n e . F o r t h e s u b s e r i e s 3 . 2 , 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 w a s v a r i e d ( 5 , 1 0 , 2 0 , a n d 3 0 g / l ) w h i l e c o b a l t c o n c e n t r a t i o n w a s k e p t c o n s t a n t a t 2 . 2 p p m , a n d 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 c o n c e n t r a t i o n a t U g / 1 . F o r s u b s e r i e s 3 . 3 , c c b a l t i o n c o n c e n t r a t i o n w a s m a d e t o v a r y f r o m O p p m t o 0 . 9 p p m t h e n r a i s e d t o 2 . 2 p p m , 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 w a s k e p t c o n s t a n t a t 2 0 g / l , a n d a g a i n p h o s p h a t e c o n c e n t r a t i o n r e m a i n e d a t 4 g / l . T h e o n l y 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 D H B Z i s l i s t e d u n d e r s u b s e r i e s 3 . 4 . T h e e a r l y f e r m e n t a t i o n s , f o r w h i c h t h e a n a l y s e s w e r e i n c o m p l e t e , a p p e a r i n s u b s e r i e s 3 . 1 . I n t h e l a s t c i t e d s u b s e r i e s y e a s t e x t r a c t p a s t e w a s u s e d . O n e b a t c h o f S S L , w i t h 2 0 g / l o f y e a s t e x t r a c t , 2 . 2 p p m o f c o b a l t i o n , a n d a s u s u a l , w i t h 4 g / l o f p h o s p h a t e , w a s i n o c u l a t e d w i t h P . s h e r m a n i i . T h e r e s u l t s o f a l l t h e s e f o u r s e r i e s a p p e a r i n t h e F ' i g u r e s n u m b e r e d f r o m 3 . 3 9 t o 3 . 5 4 a n d i n t h e T a b l e s ftl.32 t h r o u g h A I . 4 6 i n A p p e n d i x I ( s e e T a b l e I I I . 2 3 ) . R a t e s o f g r o w t h , o f s u g a r c o n s u m p t i o n , a n d o f p r o d u c t i o n s o f v i t a m i n B 1 2 a n d a c i d s , a n d e f f i c i e n c y a r e p l o t t e d a g a i n s t f e r m e n t a t i o n t i m e i n F i g u r e s 3 . 6 0 t o 3 . 7 0 . A s u m m a r y o f t h e o v e r a l l r e s u l t s i s p r e s e n t e d i n T a b l e s I I I . 2 4 a n d I I I . 2 5 . T h e p e r f o r m a n c e o f P r o p i o n i b a c t e r i a i n p r o d u c i n g v o l a t i l e a c i d s a n d v i t a m i n B 1 2 a r e s u m m a r i z e d i n F i g u r e s 3 . 5 5 , 3 . 5 6 , 3 . 5 7 , a n d 3 . 5 8 . E x p e r i m e n t s a n d R e s u l t s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 1 2 4 T a b l e I I I . 2 4 S U M M A R Y O F O V E R A L L R E S U L T S 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 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 ( g / 1 ) 5 1 0 2 0 3 0 | _ _ _ i | S u g a r C o n s u m p t i o n | - % o f I n i t i a l S u g a r 6 7 . 6 7 2 . 4 7 2 . 6 7 1 . 4 | 1 - 9 / 1 2 5 . 0 2 1 . 0 2 7 . 0 2 1 . 0 | | V o l a t i l e A c i d s 1 P r o d u c e d ( m o l e s / 1 ) 0 . 1 4 0 . 1 3 0 . 1 4 0 . 1 3 | | D r y C e l l W e i g h t ( g / 1 ) 7 . 0 6 . 5 8 . 5 8 . 0 | | G r o w t h B a t e ( g / l / h r ) 0 . 1 0 0 . 1 0 0 . 2 1 0 . 1 5 I 1 P r o d u c t i o n o f | v i t a m i n B 1 2 ( m g / 1 ) 1 . 5 0 1 . 4 0 0 . 9 5 1 . 0 0 | j Y i e l d o f D r y C e l l s j - b a s e d o n S u g a r U s e d ( g / g ) 0 . 2 8 0 . 3 1 0 . 3 2 0 . 3 8 | j - b a s e d o n Y e a s t | E x t r a c t A d d e d ( g / g ) 1 . 4 0 0 . 6 5 0 . 4 3 0 . 2 7 | I Y i e l d o f V i t a m i n B 1 2 1 | - b a s e d o n S u g a r 1 U s e d ( m g / g ) 0 . 0 6 0 0 . 0 6 7 0 . 0 3 5 0 . 0 4 8 | | - b a s e d o n G r o w t h ( m g / g ) 0 . 2 1 4 0 . 2 0 9 0 . 1 1 2 0 . 1 2 5 | j Y i e l d o f V o l a t i l e A c i d s | B a s e d o n G r o w t h ( m o l e s / g ) 0 . 0 2 0 2 0 . 0 1 9 2 0 . 0 1 6 5 0 . 0 1 6 3 | j E f f i c i e n c y 0 . 5 1 2 0 . 5 3 6 0 . 4 6 7 C . 5 5 8 | — j C o b a l t I o n C o n c e n t r a t i o n : 2 . 2 p p m 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 C o n c e n t r a t i o n : 4 g / 1 E x p e r i m e n t s a n d R e s u l t s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 1 2 5 T a b l e I I I . 2 5 SUMMARY OF OVERALL RESULTS EFFECT OF COBALT ION CONCENTRATION 1— - . 1 | I n i t i a l C o b a l t I o n | C o n c e n t r a t i o n ( p p m ) 0 . 0 0 0 . 9 0 2 . 2 0 2 . 2 0 * f , i | S u g a r C o n s u m p t i o n 1 - % o f I n i t i a l S u g a r 1 - 9 / 1 7 3 . 6 2 7 . 6 6 6 . 4 2 3 . 1 7 2 . 6 2 7 . 1 1 1 6 4 . 5 | 1 8 . 0 | j V o l a t i l e A c i d s | P r o d u c e d ( m o l e s / 1 ) \ D r y C e l l W e i g h t ( g / 1 ) | G r o w t h R a t e ( g / l / h r ) | P r o d u c t i o n o f | V i t a m i n B 1 2 ( m g / l ) 0 . 1 6 7 . 8 0 . 1 5 6 0 . 4 0 0 . 1 5 8 . 5 0 . 1 3 3 1 . 3 0 0 . 1 4 8 . 5 0 . 0 5 3 0 . 9 5 0 . 1 3 | 6 . 9 | 0 . 0 8 7 | 1 . 0 0 | | Y i e l d o f D r y C e l l s j B a s e d o n S u g a r U s e d ( g / g ) 0 . 2 8 3 0 . 3 6 8 0 . 3 1 5 0 . 3 8 3 | | Y i e l d o f V i t a m i n B 1 2 | - b a s e d o n S u g a r j U s e d ( m g / g ) | - b a s e d o n G r o w t h ( m g / g ) 0 . 0 1 4 0 . 0 5 1 0 . 0 5 6 0 . 1 5 2 0 . 0 3 5 0 . 1 1 2 0 . 0 5 6 | 0 . 1 4 5 | j Y i e l d o f V o l a t i l e A c i d s | B a s e d o n G r o w t h ( m o l e s / g ) | E f f i c i e n c y 0 . 0 2 0 0 0 . 5 0 9 0 . 0 1 8 0 0 . 5 9 7 0 . 0 1 6 5 0 . 4 6 7 0 . 0 1 8 8 | 0 . 6 5 0 | Y e a s t E x t r a c t C o n c e n t r a t i c n : 2 0 g / 1 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 C o n c e n t r a t i c n : 4 g / 1 * W i t h 0 . 1 2 6 g / l o f D M B Z a d d e d E x p e r i m e n t s a n d R e s u l t s 126 Potential of Spent Sulfite Liquor L E G E N D C O N C E N T R A T I O N S OF : Y E A S T C O B A L T E X T R A C T ION (G/L ) (PPM) 5 n 0 5 2 . 2 10 A 10 2 . 2 20 • 20 2 . 2 30 + 3 0 2 . 2 A N A E R O B I C S T A G E — A E R O B I C S T A G E 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 of Spent S u l f i t e L i g u o r 127 F I G U R E 3.55 : 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 . 1.60 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 1.40 L 1.20 L CD CM 1.00 CD 0.80 L o 0.60 C J Q O CC Q _ 0.40 L 0.20 L 0.0 0. 40. 80. 120. 360. F E R M E N T A T I O N T I M E ( H O U R S ) E x p e r i m e n t s and R e s u l t s 200. 128 Potential of Spent Sulfite Liguor LEGEND CONCENTRATIONS OF : COBALT ION (PPMJ YEAST EXTRACT (G/L) 0 X 0 . 0 20 0.9 K 0 . 9 20 2.2 • 2 . 2 20 DMBZ X 2 .2 20 DMBZ ADDED PS Y 2 . 2 20 WITH PS ANAEROBIC STAGE AEROBIC STAGE 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 of Spent S u l f i t e L i q u o r 129 FIGURE 3.56 1.60 P r o d u c t i o n of Vitamin 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 P o t e n t i a l of Spent S u l f i t e L i q u o r L E G E N D C O N C E N T R A T I O N S OF : Y E A S T E X T R A C T (G/L) C O B A L T ION (PPM) 5 • 0 5 2 . 2 10 A 10 2 . 2 20 20 2 . 2 30 + 3 0 2 . 2 A N A E R O B I C S T A G E A E R O B I C S T A G E 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 o f S p e n t S u l f i t e L i g u o r 131 FIGURE 3.57 0.16 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 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 132 Potential of Spent Sulf i te Liquor LEGEND CONCENTRATIONS OF : COBALT YEAST ION EXTRACT (PPM) (G/L) 0 X 0.0 20 0.9 K 0.9 20 2.2 • 2.2 20 D (vl B Z 2.2 20 DMBZ ADDED PS Y 2.2 20 WITH PS ANAEROBIC STAGE AEROBIC STAGE 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 o f S p e n t S u l f i t e L i q u o r 1 3 3 F I G U R E 3 . 5 8 0.16 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 o f C o b a l t I o n C o n c e n t r a t i o n 0.14 L - J 0.12 U tn L U g o.io cr 0.08 L ec _ J o > L u O O ZD a o Q: a. 0.06 L 0.04 L 0.02 L 0.0 4 0 . 8 0 . 120. 160. F E R M E N T f l T I O N T I M E ( H O U R S ) E x p e r i m e n t s and R e s u l t s 200. Potential of Spent Sulfite Liquor 13U FIGURE 3.59 : Legend for A l l Figures Numbered from 3.55 to 3.58 L E G E N D C O N C E N T R A T I O N S OF : Y E A S T C O B A L T E X T R A C T ION ( G / L ) (PPM) 5 • 0 5 2 . 2 10 10 2 . 2 20 2 0 2 . 2 30 + 3 0 2 . 2 C O N C E N T R A T I O N S OF : C O B A L T Y E A S T ION E X T R A C T (PPM) ( G / L ) 0 X 0 . 0 2 0 0.9 W 0 . 9 2 0 2.2 • 2 . 2 2 0 DMBZ X 2 . 2 2 0 DMBZ ADDED PS Y 2 . 2 2 0 WITH P S A N A E R O B I C S T A G E A E R O B I C S T A G E E x p e r i m e n t s and R e s u l t s 135 P o t e n t i a l o f S p e n t S u l f i t e L i q u o r Run FP1.1 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 i o n 4 g / l 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 L E G E N D o E F F I C I E N C Y + G R O W T H R A T E ( M G O F D R V C E L L 5 / L / H R J x L A C T I C A C I D P R O D U C T I O N R A T E ( M M O L E S / L / H R ) v S U G A R C O N S U M P T I O N R A T E ( G / L / H R ) E x p e r i m e n t s and R e s u l t s Potential of Spent Sulf i te Liquor 136 FIGURE 3.60 : Run FP1.1. Fermentation Rate of L. plantarium E x p e r i m e n t s and R e s u l t s 37 P o t e n t i a l o f S p e n t S u l f i t e L i q u o r Run FP2.2 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 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 L E G E N D <» E F F I C I E N C Y + GROWTH R A T E (MG OF DRY C E L L S / L / H R ) X V A R I A T I O N R A T E OF L A C T I C A C I D C O N C E N T R A T I O N ( M M 0 L E 5 / L / H R ) • V I T A M I N 8 1 2 PRODUCT ION R A T E (M ICROGRAMS/L/HR ) V O L A T I L E A C I D S PRODUCT ION R A T E (MMOLES/L/HR ) 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 o f S p e n t S u l f i t e L i g u o r 138 FIGURE 3.61 : Run 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 w i t h L a c t i c A c i d a s M a i n S u b s t r a t e RNREROB IC • 3.0 n2.0 • l.o <j> l.o <3> 0.0 / / ^ <»<!>«> A <J> <$> 4> <*> 0 <s> / * R E R O B I C X . f t . X A XI + 5. 4. 3. 2. 1. 0. A A A X X - i . X -2. X 30.0 + 20.0 + 10.0 + 0.0 + 0.0 40.0 80.0 120.0 160.0 F E R M E N T R T I 0 N T I M E (HOURS) 200.0 Experiments and Results 139 r Potential of Spent Sulfite Liquor Run F05.22 05g/l of yeast extract powder 2.2ppm of cobalt ion Ug/1 of potassium dihydrogen phosphate pH automatically controlled L E G E N D <$• E F F I C I E N C Y — + GROWTH R A T E (MG OF DRY C E L L S / L / H R ) y SUGAR CONSUMPT ION R A T E (G/L/HRJ • V I T A M I N B12 PRODUCT ION R A T E (M ICROGRAMS/L/HR ) " " " " " A V O L A T I L E A C I D S PRODUCT ION R A T E (MMOLES/L/HR ) 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 o f S p e n t S u l f i t e L i q u o r mo FIGURE 3.62 : Run F 0 5 . 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 . 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 A N A E R O B I C A E R O B I C • o.o 0.75 Y 0.50 Y 0.25 Y 0.00 Y 120.0 + 80.0 40.0 + 0.0 20.0 60.0 100.0 140.0 180.0 F E R M E N T A T I O N T I M E (HOURS) 220.0 E x p e r i m e n t s and R e s u l t s 141 r Potential of Spent Sulf i te Liquor Run F10.22 10g/l of yeast extract powder 2.2ppm of cobalt ion 4g/l of potassium dihydrogen phosphate pH automatically controlled L E G E N D <> E F F I C I E N C Y + GROWTH R A T E (MG OF DRY C E L L S / L / H R J y SUGAR CONSUMPT ION R A T E (G/L/HRJ • V I T A M I N B12 PRODUCT ION R A T E (M ICROGRAMS/L/HR J A V O L A T I L E A C I D S PRODUCT ION R A T E (MMOLES/L/HR ) 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 o f S p e n t S u l f i t e L i q u o r 142 FIGURE 3.63 : Run F 1 0 . 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 . 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 A E R O B I C J 40.0 + 0.0 + 0.0 40.0 80.0 120.0 150.0 F E R M E N T A T I O N T I M E (HOURS) 200.0 E x p e r i m e n t s and R e s u l t s 143 P o t e n t i a l o f S p e n t S u l f i t e L i g u o r Run F20.22 20g/l of yeast extract powder 2.2ppm of cobalt ion 4g/l of potassium dihydrogen phosphate pH automatically controlled L E G E N D <$> E F F I C I E N C Y — . — - + GROWTH R R T E (MG OF DRY C E L L S / L / H R ) - Y SUGAR CONSUMPT ION R A T E (G/L/HR ) • V I T A M I N B l 2 PRODUCT ION R A T E ( M I C R 0 G R A M 5 / L / H R ) V O L A T I L E A C I D S PRODUCT ION R A T E (MMOLES/L/HR ) Experiments and Re s u l t s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 144 FIGURE 3.64 : Run F 2 0 . 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 . 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 RNRERGB IC > / 1, 8 It .+ A E R O B I C 0.75 Y J 0 . 5 0 Y 0.25 0.00 J 160.0 + J 120.0 + 80.0 + 40.0 40.0 80.0 120.0 160.0 F E R M E N T A T I O N T I M E (HOURS) 200.0 E x p e r i m e n t s and R e s u l t s 145 Potential of Spent Sulf i te Liquor Run F30.22 30g/l of yeast extract powder 2.2ppin of cobalt ion 4g/l of potassium dihydrogen phosphate pH automatically controlled L E G E N D E F F I C I E N C Y — - — - + GROWTH R R T E (MG OF DRY C E L L S / L / H R J y SUGAR CONSUMPT ION R A T E (G/L/HRJ • V I T A M I N B12 PRODUCT ION R A T E (M ICROGRAMS/L/HR ) — A V O L A T I L E A C I D S PRODUCT ION R A T E (MMOLES/L/HR ) 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 o f S p e n t S u l f i t e L i q u o r 146 FIGURE 3.65 : Run F 3 0 . 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 . 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 A N A E R O B I C / A 6-° • A-A E R O B I C o.o + 0.0 40.0 80.0 120.0 160.0 F E R M E N T A T I O N T I M E (HOURS) 0.75 -J0.50 Y 0.25 Y 0.00 Y 120.0 + 80.0 40.0 + 0.0 200.0 E x p e r i m e n t s and R e s u l t s 147 t Potential of Spent Sulfite Liquor Run F20.00 20g/l of yeast extract powder O.Oppm of cobalt ion 4g/l of potassium dihydrogen phosphate pH automatically controlled L E G E N D <j> E F F I C I E N C Y + GROWTH R A T E (MG OF DRY C E L L S / L / H R J Y SUGAR CONSUMPT ION R A T E (6/L/HR J — • V I T A M I N 0 1 2 PRODUCT ION R A T E (M ICR0GRAM5/L/HR J V O L A T I L E A C I D S PRODUCT ION R A T E (MMOLES/L/HR ) 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 o f S p e n t S u l f i t e L i q u o r 148 FIGURE 3.66 : Run 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 R E R O B I C «> o.o o.o 40.0 80.0 120.0 160.0 F E R M E N T A T I O N T I M E (HOURS) 200.0 E x p e r i m e n t s and R e s u l t s 149 P o t e n t i a l o f S p e n t S u l f i t e L i g u o r Run F20.09 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 4 g / l 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 o EFFICIENCY + GROWTH RATE (MG OF DRY CELLS/L/HR) „ Y SUGAR CONSUMPTION RATE (G/L/HR) m VITAMIN 812 PRODUCTION RATE (MICROGRAMS/L/HR) . „ A VOLATILE ACIDS PRODUCTION RATE (MMOLES/L/HR) 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 o f S p e n t S u l f i t e L i q u o r 150 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 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 OO.O I L_J 1 1 1 0 0 40.0 80.0 120.0 160.0 200.0 F E R M E N T A T I O N T I M E (HOURS) E x p e r i m e n t s and R e s u l t s 151 i P o t e n t i a l o f S p e n t S u l f i t e L i g u o 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 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 0 . 1 2 6 g / l o f DMBZ L E G E N D <j> E F F I C I E N C Y — + GROWTH R A T E (MG OF DRY C E L L S / L / H R J — Y SUGAR CONSUMPT ION R A T E (G/L/HRJ — — a V I T A M I N B12 PRODUCT ION R A T E (M ICROGRAMS/L/HR J A V O L A T I L E A C I D S PRODUCT ION R A T E (MMOLES/L/HRJ 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 o f S p e n t S u l f i t e L i g u o r 152 FIGURE 3.68 : Run F 2 0 . 2 2 . 1 2 6 . F e r m e n t a t i o n R a t e o f PF i n SSL w i t h A d d i t i o n o f DMBZ A E R O B I C 0.75 Y 40.0 80.0 120.0 1 60.0 F E R M E N T A T I O N T I M E (HOURS) 200.0 E x p e r i m e n t s and R e s u l t s Potential of Spent Sulf i te Liguor 1 Run S20.22 1 20g/l of yeast extract powder I 2.2ppm of cobalt ion I 4g/l of potassium dihydrogen phosphate | pH automatically controlled J L E G E N D o E F F I C I E N C Y . . + GROWTH R A T E (MG OF DRY C E L L 5 / L / H R J Y SUGAR CONSUMPT ION R A T E (G/L/HRJ , • V I T A M I N B12 PRODUCT ION R A T E (M ICROGRAMS/L/HRJ V O L A T I L E A C I D S PRODUCT ION R A T E (MMOLES/L/HR ) / 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 o f S p e n t S u l f i t e L i g u o r 154 FIGURE 3.69 : Run S 2 0 . 2 2 . F e r m e n t a t i o n R a t e o f P. s h e r m a n i i A 5 . 0 A 4 . 0 A 3 - 0 A 2 - 0 A L O • 25 .0 a 20 .0 • 15.0 n 10.0 • 5 . 0 O l . o 0 0 .5 <j> 0 .0 A N A E R O B I C A E R O B I C ~Y_ .•A 0 .50 Y - | 0 .25 Y 0 .00 Y 320.0 + A . . . . . . X X X 0 .75 Y 80 .0 + 40 .0 + 0 .0 + 23.0 63.0 103.0 143.0 183.0 223 .0 F E R M E N T A T I O N T I M E (HOURS) 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 o f S p e n t S u l f i t e L i g u o r GURE 3.70 : L e g e n d f o r A l l F i g u r e s Numbered f r o m 3.60 t o 3. L E G E N D <j> E F F I C I E N C Y + GROWTH R A T E (MG OF DRY C E L L 5 / L / H R J x V A R I A T I O N R A T E OF L A C T I C A C I D C O N C E N T R A T I O N (MMQLES/L/HR ) y SUGAR CONSUMPT ION R A T E (G/L/HR ) — CD V I T A M I N B12 PRODUCT ION R A T E (M ICROGRAMS/L/HRJ A V A R I A T I O N R A T E OF 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 (MMOLES/L/HR ) 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 o f S p e n t S u l f i t e L i q u o r 156 FIGURE 3.71 : R e l a t i v e C a p a c i t y o f PS and PF 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 Main S u b s t r a t e F F o r 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 S F o r P r o p i o n i b a c t e r i a S h e r n i a n i i N 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 b e t w e e n S t a g e s J I I ! 0.02 0.0 10.0 20.0 Y E R S T E X T R A C T ADDED ( G / L ) 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 o f S p e n t S u l f i t e L i q u o r 1 5 7 3 . 8 I E R M E N T A T I O N _ O F _ A Q U E O U S T a b l e I I I . 2 6 l i s t s t h e r e s u l t s c f 3 s e r i e s c f f e r m e n t a t i o n s d o n e i n 2 5 0 m l E r l e n m e y e r f l a s k s w i t h P . f r e u d e n r e i c h i i . T h e s e w e r e c a r r i e d o u t / w i t h a q u e o u s s o l u t i o n s o f U 0 g / 1 c f g l u c o s e , U g / l 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 . I n 2 o f t h e s e s e r i e s D i f c o e x t r a c t p o w d e r w a s u s e d , a n d i n o n e , 2 . 2 p p m c f c o b a l t i o n w a s a d d e d t o t h e b r o t h . I n t h e t h i r d s e r i e s , N B y e a s t e x t r a c t p o w d e r w a s u s e d a n d n o c o b a l t i o n w a s a d d e d . T h e c o n c e n t r a t i o n o f y e a s t e x t r a c t v a r i e d f r o m 0 t o 5 0 g / l . H e r e a g a i n t h e r e s u l t s s h o w t h a t t h e n u t r i t i v e v a l u e o f t h e y e a s t e x t r a c t p o w d e r s w a s n e a r l y t h e s a m e . E x p e r i m e n t s a n d R e s u l t s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 1 5 8 T a b l e I I I . 2 6 F E R M E N T A T I O N O F A Q U E O U S S O L U T I O N S O F G L U C O S E G l u c o s e C o n c e n t r a t i o n : 4 0 g / l 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 C o n c e n t r a t i o n : 4 g / l C o b a l t I o n C o n c e n t r a t i o n : O p p m i n S e r i e s 1 a n d 3 , 2 . 2 p p m i n S e r i e s 2 D i f c o 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 1 a n d 2 N B 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 I s e r i e s 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 ( g / i ) 0 2 0 3 0 4 0 5 0 j D r y C e l l w e i g h t } 1 0 5 . 6 2 7 . 1 7 9 . 3 9 1 0 . 4 1 I ( 9 / 1 ) 1 2 0 5 . 6 2 6 . 6 0 - 1 0 . 4 3 i „ ,« ,. _ t 3 0 5 . 4 2 7 . 4 2 9 . 2 8 9 . 9 7 I 1 | S u g a r R e s i d u e | 1 4 4 . 4 1 8 . 5 1 3 . 5 8 . 3 6 . 6 1 C o n c e n t r a t i o n j 2 4 4 . 4 2 1 . 6 1 7 . 5 5 7 . 5 6 . 4 1 ( g / i ) 1 3 4 4 . 4 2 4 . 9 1 7 . 4 1 1 . 1 9 . 1 5 | V i t a m i n B 1 2 J 1 0 0 . 1 7 6 0 . 2 0 8 _ 0 . 4 2 0 | C o n c e n t r a t i o n j 2 0 0 . 2 2 8 - - 0 . 3 7 0 I ( m g / D 1 3 0 — — • — 0 . 4 8 0 | V o l a t i l e A c i d s J 1 0 0 . 1 3 9 0 . 1 4 9 0 . 2 0 2 0 . 2 1 3 | C o n c e n t r a t i o n | 2 0 0 . 1 0 2 0 . 1 5 8 - 0 . 2 0 3 | ( m o l e s / 1 ) | 3 0 0 . 1 0 1 0 . 1 6 8 0 . 1 8 8 0 . 1 9 5 | L a c t i c A c i d | 1 0 0 . 0 8 2 0 . 6 2 0 0 . 1 1 4 0 . 1 4 2 | C o n c e n t r a t i o n | 2 0 0 . 0 7 4 0 . 0 9 0 - 0 . 0 9 3 | ( m o l e s / 1 ) | 3 0 0 . 0 6 4 0 . 1 0 7 0 . 1 1 6 0 . 1 3 6 | T o t a l A c i d s j 1 0 0 . 2 2 1 0 . 3 0 9 3 1 7 0 . 3 5 5 | C o n c e n t r a t i o n j 2 0 0 . 1 7 6 0 . 2 4 8 - 0 . 2 9 6 | ( m o l e s / 1 ) | 3 0 0 . 1 6 5 0 . 2 7 5 0 . 3 0 4 0 . 3 3 1 j F i n a l p H | 1 6 . 3 3 . 8 3 . 8 3 . 9 4 . 0 j j 2 6 . 3 4 . 1 4 . 0 - 4 . 2 I | 3 6 . 3 3 . 9 3 . 8 3 . 9 4 . 0 E x p e r i m e n t s a n d R e s u l t s P o t e n t i a l o f S p e n t S u l f i t e L i g u o r 1 5 9 C h a p t e r 4 D I S C U S S I C H S 4 . 1 F E R M E N T A T I O N W I T H S T R E P T O M Y C E S S . g r i s e u s a n d S . o l i v a c e u s d i d n o t g r o w i n S S L ( T a b l e s I I I . 2 1 a n d I I I . 2 2 ) . H o w e v e r , t h e p r o b l e m i s n o t d u e t o t h e l a c k o f n u t r i e n t s i n t h e m e d i u m . I t i s r a t h e r c a u s e d b y s o m e i n h i b i t i n g e f f e c t o f S S L o n t h e s e m i c r o o r g a n i s m s . S t r e p t o m y c e s s e e m e d t o e n j o y t h e a b u n d a n c e o f s u g a r i n S S L w h e n m i x e d w i t h W a k s m a n ' s N o . 3 m e d i u m , b u t w h e n t h e S S L r e a c h e d a c o n c e n t r a t i o n o f o v e r 5 0 m l / l o f t o t a l s o l u t i o n , S t r e p t o m y c e s s t o p p e d g r o w i n g . T h i s i n h i b i t i n g e f f e c t i s d e f i n i t e l y n o t c a u s e d b y s u l f u r d i o x i d e n o r b y l i g n o s u l f o n a t e . W i t h d i l u t i o n , t h e f r e e a n d t o t a l s u l f u r d i o x i d e c o n c e n t r a t i o n s o f 109? S S L s o l u t i o n w e r e a b o u t 1 0 a n d 1 2 0 p p m r e s p e c t i v e l y . O n t h e o t h e r h a n d , S S L t r e a t e d b y N i s h i k a w a n e u t r a l i z a t i o n a n d p r e c i p i t a t i o n , a n d s u p p o s e d t o b e a l m o s t f r e e o f l i g n i n , d i d n o t f a v o r S t r e p t o m y c e s g r o w t h e i t h e r . T h e a b i l i t y o f t h e s e S t r e p t o m y c e s t o d e v e l o p a s t r a i n c a p a b l e o f r e p r o d u c t i o n i n S S L w a s t e s t e d b y a c c l i m a t i z a t i o n . H o w e v e r , a c c l i m a t i z a t i o n a l s o f a i l e d w h e n t h e c o n c e n t r a t i o n o f S S L i n t h e m i x e d m e d i u m w a s o v e r 2095 i n v o l u m e , t h o u g h i t d i d i m p r o v e g r o w t h s i g n i f i c a n t l y w h e n S S L c o n c e n t r a t i o n d i d n e t e x c e e d t h a t l i m i t o f 2 0 9 5 . D i s c u s s i o n s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 1 6 0 I t i s b e l i e v e d t h a t t h e s e a c c l i m a t i z i n g e x p e r i m e n t s w o u l d h a v e b e e n a s u c c e s s i f t h e i n t e r m e d i a t e s t e p s h a d b e e n t a k e n m u c h m o r e s l o w l y . T h a t i s i f m i x t u r e s h a d b e e n m a d e w i t h s m a l l e r d i f f e r e n c e s i n c o n c e n t r a t i o n a n d t h e s e a c t i n o m y c e t e s h a d b e e n a l l o w e d t o d e v e l o p m o r e g e n e r a t i o n s i n e a c h l e v e l o f c o n c e n t r a t i o n w i t h m o r e t r a n s f e r s . 4.2 Z^iMllhlI^-RIl^-lis.^RLMMiM^ T h e f e r m e n t a t i o n s o f s o l u t i o n s o f x y l o s e w i t h L . p l a n t a r i u m r e v e a l t h e c u r i o u s f a c t t h a t t h i s s t r a i n d o e s n o t f e r m e n t x y l o s e ( F i g u r e 3 . 2 9 ) , t h o u g h i t d o e s f e r m e n t a r a b i n o s e ( b y d e d u c t i o n b a s e d o n t h e r e s u l t s o f o t h e r f e r m e n t a t i o n s ) . T h e n i t w a s d i s c o v e r e d t h a t t h i s s t r a i n w a s n o t L . p e n t o s u s , t h e s t r a i n o r d e r e d , a n d k n o w n t o f e r m e n t x y l o s e [ 1 0 , 2 6 , 7 4 ] , T h e s t r a i n u s e d , I . p l a n t a r i u m A T C C - 8 0 1 4 , w a s o r i g i n a l l y f r o m E l i z a b e t h M c C o y ' s s t r a i n 1 7 - 5 , a n d w a s c a l l e d L . a r a b i n o s u s b y h e r [ 3 , 5 3 ] , P r o b a b l y i t w a s t h e s t r a i n C , g r o u p I , o f P e t e r s o n e t a l [ 2 6 , 7 4 ] , t h a t B e r g e y , P e t e r s o n a n d h i s c o w o r k e r s a l s o h a v e c a l l e d L . a r a b i n o s u s . T h e m a j o r d i f f e r e n c e b e t w e e n L . p e n t o s u s a n d L . a r a b i n o s u s , g r o u p e d u n d e r t h e n a m e o f L . p l a n t a r i u m , l i e s i n t h e i r a b i l i t y t c f e r m e n t x y l o s e . S o t h e n , t h e m a j o r c o m p o n e n t , x y l o s e , o f t h e 3091 o f p e n t o s e i n S S L c o u l d n o t b e u s e d i n t h e f e r m e n t a t i o n s o f S S L w i t h t h i s s t r a i n o f L . p l a n t a r i u m , a s i n t e n d e d . F e r m e n t a t i o n s , d o n e i n t w o s t a g e s , w i t h L . p l a n t a r i u m i n t h e f i r s t s t a g e t o c o n v e r t s u g a r ( i t w o u l d h a v e b e e n b e t t e r w i t h D i s c u s s i o n s P o t e n t i a l o f S p e n t S u l f i t e L i g u o r 1 6 1 L - p e n t o s u s , a s i t c a n c o n v e r t x y l o s e a s w e l l a s h e x o s e s ) t o l a c t i c a c i d , t h e n w i t h P . p r o p i o n i b a c t e r i a i n t h e s e c o n d s t a g e t o c o n v e r t l a c t i c a c i d t o v o l a t i l e a c i d s , o f f e r c e r t a i n c o n v e n i e n c e s : 1 . L . p l a n t a r i u m i s m u c h l e s s s e n s i t i v e t o p H t h a n P r o p i o n i b a c t e r i a . p H a d j u s t m e n t s m a y b e d o n e t h e n i n a c o a r s e r m a n n e r , o r p e r h a p s n o t a t a l l i n t h e f i r s t s t a g e . I n t h e s e c o n d s t a g e , p H s h o u l d n o t v a r y m u c h , a n d t h e r e s u l t s d i d s h o w t h a t p H r e m a i n e d c o n s t a n t . p H c o n t r o l i s t h e n n o t n e e d e d , a n d o n e s i n g l e p H a d j u s t m e n t , b e t w e e n t w o s t a g e s , w o u l d p r o b a b l y b e s u f f i c i e n t . 2 . B e s i d e s v o l a t i l e a c i d s a n d v i t a m i n B 1 2 , l a c t i c a c i d m i g h t b e r e c o v e r e d a s a n e x t r a p r o d u c t . 3 . W i t h t h e c o n v e r s i o n f r o m l a c t i c a c i d t o v o l a t i l e a c i d s b e i n g 1 : 1 i n t h e s e c o n d s t a g e , t h e e x t r a p e n t o s e s f e r m e n t e d b y L a c t o b a c i l l u s i n t h e f i r s t s t a g e w o u l d a l l o w a b e t t e r y i e l d i n v o l a t i l e a c i d s a n d a g r e a t e r r e d u c t i o n o f t h e p o l l u t i n g p o t e n t i a l o f S S L b y t h e u s e o f m o r e r e d u c i n g s u g a r s . U n f o r t u n a t e l y , w i t h l a c t i c a c i d , i n s t e a d o f s u g a r s , a s t h e m a i n c a r b o h y d r a t e s o u r c e , P r o p i o n i b a c t e r i a d o n o t r e p r o d u c e a s m u c h ( 1 . 6 g / l c o m p a r e d t o 8 g / l ) , t h o u g h t h e y d o w o r k h a r d e r ( e f f i c i e n c y o f 1 c o m p a r e d t o a m a x i m u m e f f i c i e n c y o f 0 . 6 5 ) . P r o b a b l y , P r o p i o n i b a c t e r i a c a n n o t u s e l a c t i c a c i d a s a c a r b o h y d r a t e s o u r c e t o b u i l d c e l l s , s o w h e n t h e r e a r e n o s u g a r s , P r o p i o n i b a c t e r i a l i v e s o l e l y o n y e a s t e x t r a c t , a n d c o n v e r t a l l t h e l a c t i c a c i d t o v o l a t i l e a c i d s . H o w e v e r , n o t b e i n g a b l e t o b u i l d c e l l s i n a b u n d a n c e , t h e i n t r a c e l l u l a r p r o d u c t t h e n c a n n o t D i s c u s s i o n s P o t e n t i a l o f S p e n t S u l f i t e l i q u o r 162 b e m a d e i n q u a n t i t y . H e n c e , i t i s n o t s u r p r i s i n g t o d i s c o v e r a v e r y l o w p r o d u c t i o n o f v i t a m i n B 1 2 ( 0 . 2 2 m g / l m a x i m u m ) . T h e v i t a m i n B 1 2 y i e l d ( b a s e d o n d r y c e l l w e i g h t ) i s n o t l o w a t a l l ( 0 . 1 3 5 m g / g ) c o m p a r e d t o t h e y i e l d c f t h e s a m e p r o d u c t i n f e r m e n t a t i o n s w i t h s u g a r s a s n u t r i e n t s o u r c e ( 0 . 1 1 2 t o 0 . 2 1 4 m g / g ) . I . p l a n t a r i u m w a s c h o s e n t o , h o p e f u l l y , i m p r o v e t h e y i e l d o f v i t a m i n B 1 2 w i t h f e r m e n t a t i o n s i n t w o s t a g e s . B u t t h e s e f e r m e n t a t i o n s g a v e l o w y i e l d o f v i t a m i n a n d r e g u i r e d m o r e y e a s t e x t r a c t , a n e x p e n s i v e n u t r i e n t , w i t h a n e x t r a s t a g e ( t h e o p t i m u m 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 f o r w h i c h w o u l d b e a b o u t 4 5 g / l , F i g u r e 3 . 3 0 ) . F u r t h e r m o r e , m o r e y e a s t e x t r a c t h a d t c b e a d d e d ( F i g u r e 3 . 7 1 ) t o t h e s e c o n d s t a g e . S o , t h i s s e r i e s o f e x p e r i m e n t s w a s n o t p u r s u e d a n y f u r t h e r . 4 . 3 F B ^ M E N T A T I O N _ W I T H _ H I X E F e r m e n t a t i o n w i t h a m i x e d c u l t u r e o f 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 s v e r y i n t e r e s t i n g b e c a u s e t h e g r o w t h o f e a c h i n d i v i d u a l s p e c i e s a p p e a r s t o b e v e r y d i s t i n c t . T h e i r g r o w t h c a n b e o b s e r v e d t h r o u g h t h e i r m a i n p r o d u c t s ( F i g u r e 3 . 4 3 ) . A t f i r s t , w h e n L . p l a n t a r i u m ( k n o w n t o g r o w s l o w e r ) i s n o t r e a d y , P . f r e u d e n r e i c h i i s t a r t g r o w i n g , t h e n t h e y s t o p t h e i r g r o w t h ( d e d u c e d f r o m t h e v o l a t i l e a c i d s c u r v e ) l e a v i n g t h e b r o t h t o L . p l a n t a r i u m . O n c e L . p l a n t a r i u m a r e t h r o u g h , P . f r e u d e n r e i c h i i r e s u m e t h e i r g r o w t h , b u t t h i s t i m e u s i n g l a c t i c a c i d a s c a r b o h y d r a t e s o u r c e . F i g u r e 4 . 7 2 s h o w s t h e c o e x i s t e n c e o f t h e s e D i s c u s s i o n s P o t e n t i a l of Spent S u l f i t e L i q u o r 163 FIGURE 4.72 : L. plantarium and P. f r e u d e n r e i c h i i i n SSL (from Run FP3) ( s c a l e : \. \ * 12 microns) two s p e c i e s i n run FP3. The p r o d u c t i o n of vitamin B12 (0.5mg/l) i s s t i l l lower than t h a t of d i r e c t fermentations of sugars i n SSL by P. f r e u d e n r e i c h i i alone. T h i s p r o d u c t i o n , however, al c n g with t o t a l growth, i s much improved from that of fermentations c f two s u c c e s s i v e stages with these two s p e c i e s (Figures 3.43, 3.55, 3.56). Fermentations of SSL with mixed c u l t u r e i n Erlenmeyer f l a s k s gave r e s u l t s s i m i l a r to those f o r f e r m e n t a t i o n s with L. plantarium alone. That f a c t a f f i r m s the s e n s i t i v i t y of P. f r e u d e n r e i c h i i to low pH. However, i n r e i n f o r c e d c l o s t r i d i a l medium, where the nature of the medium has l e s s p o t e n t i a l to turn a c i d i c , the f i n a l product had v o l a t i l e a c i d s as a major component, and the growth was of g r e a t e r magnitude than t h a t i n D i s c u s s i o n s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 1 6 1 f e r m e n t a t i o n s w i t h L . p l a n t a r i u m a l o n e . I t c a n t h e n b e d e d u c e d t h a t a n o t t o o a b r u p t c h a n g e i n p H a l l o w s P . f r e u d e n r e i c h i i t o r e p r o d u c e a n d t o c o - e x i s t w i t h L . p l a n t a r i u m . 4 . 4 F E R M E N T A T I O N W I T H P . S H E R M A N I I T h e g r o w t h o f P . s h e r m a n i i i n S S L i s s i m i l a r t c t h a t o f P . f r e u d e n r e i c h i i . H o w e v e r , t h e i r p r o d u c t i o n o f v i t a m i n E 1 2 i s s l i g h t l y l o w e r , i n d i r e c t f e r m e n t a t i o n s o f s u g a r s c f S S L ( F i g u r e 3 . 5 3 , T a b l e A I . 4 6 ) , a s w e l l a s i n f e r m e n t a t i o n s c f l a c t i c a c i d ( F i g u r e 3 . 7 . 1 ) . , O n t h e o t h e r h a n d t h e o p t i m u m r e g u i r e m e n t i n y e a s t e x t r a c t f o r P . s h e r m a n i i i s l i k e l y t o b e h i g h e r t h a n t h a t f o r P . f r e u d e n r e i c h i i ( F i g u r e s 3 . 3 1 , 3 . 3 2 ) . S o m o s t o f t h e e x p e r i m e n t s w e r e c a r r i e d o u t w i t h P . f r e u d e n r e i c h i i . 4 . 5 F E R M E N T A T I O N _ W I T H f . _ F R E U D E N R E I C H I I 1 - 5 * 1 U s e , o f . . - . S u g a r s N o m a t t e r w h a t t h e i n i t i a l c o n c e n t r a t i o n o f r e d u c i n g s u g a r i s , P . f r e u d e n r e i c h i i a l w a y s u s e u p a b o u t 7091 o f t h i s * s u g a r ( m e a n 6 9 . 7 8 , s t a n d a r d d e v i a t i o n 3 . 1 8 ) . I f , i s f a i r l y s a f e t o d e d u c e t h a t h e x o s e s m a k e u p 7 0 % o f t h e t o t a l r e d u c i n g s u g a r s , a n d p e n t o s e s , w i t h x y l o s e a s m a j o r c o m p o n e n t , m a k e u p t h e r e m a i n i n g 3 0 % ( T a b l e s 1 . 4 , I I I . 2 4 , I I I . 2 5 ) . T h e s t r a i n o f L . p l a n t a r i u m u s e d d i d n o t f e r m e n t x y l o s e b u t a p p a r e n t l y i t d i d f e r m e n t o t h e r p e n t o s e s a s i t c a n f e r m e n t m o r e t h a n 7095 o f t h e r e d u c i n g s u g a r s . P r e s u m a b l y t h i s s t r a i n f e r m e n t s a r a b i n o s e . D i s c u s s i o n s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 1 6 5 4 . 5 . 2 E f | e c t _ O f _ £ H N e r o n o v a a n d c o - a u t h o r s [ 6 9 ] h a v e f o u n d t h a t t h e r e i s n o s i g n i f i c a n t v a r i a t i o n i n t h e u l t i m a t e y i e l d o f b a c t e r i a l c e l l m a s s f o r P r o p i o n i b a c t e r i a w h e n t h e p H i s b e t w e e n 5 . 5 a n d 7 . 5 , b u t t h e y g i v e a n a r r o w e r r a n g e c f p H ( 6 . 2 5 t o 7 . 5 ) f c r o p t i m u m v i t a m i n B 1 2 p r o d u c t i o n . F e r g u s o n [ 2 3 ] s h o w e d t h a t i n h i b i t i o n o f t h e g r o w t h 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 o c c u r s a t p H 4 . 9 o r l o w e r . I n t h i s w o r k , r u n F 1 5 . 2 2 a w a s d o n e t o t e s t t h e e f f e c t o f p H . T h e r e s u l t s ( F i g u r e 3 . 4 4 ) s h o w t h e o b v i o u s l y i n h i b i t i n g e f f e c t o f l o w p H . I n t h i s r u n , p H w a s n o t c o n t r o l l e d , b u t f o l l o w e d . W h e n p H a p p r o a c h e d 5 , g r o w t h c a m e t o a s t a n d s t i l l . A t t h i s p o i n t t h e p H w a s r e a d j u s t e d t o 7 . T h i s p r o c e d u r e w a s r e p e a t e d . T h e g r o w t h , p r o d u c t a n d p H c u r v e s a r e o f t h e s a m e r h y t h m . A d e d u c t i o n , t h e n s h o u l d f o l l o w t h i s c o n c l u s i o n : T h e r e s u l t s o f a l l f e r m e n t a t i o n s d o n e i n E r l e n m e y e r f l a s k s a n d t e s t t u b e s a r e n o t v a l i d i f t h e s e e x p e r i m e n t s w e r e d o n e t c s t u d y t h e e f f e c t o f c e r t a i n l i m i t i n g s u b t r a t e s . T h i s m e a n s t h a t , a s i t i s a l m o s t i m p o s s i b l e t o a d j u s t p H i n s u c h f l a s k s a s e p t i c a l l y , s u c h e x p e r i m e n t s a r e a s m u c h a f f e c t e d b y t h e i n h i b i t i n g e f f e c t s o f l o w p H ( w i t h a c i d s p r o d u c e d ) a s t h e y a r e b y t h e l a c k o f t h e l i m i t i n g s u b s t r a t e . I t i s w o r t h n o t i n g t h a t y e a s t e x t r a c t a c t s a s a b u f f e r b e s i d e s h a v i n g a n u t r i t i v e v a l u e . A s o l u t i o n m a y b e f o u n d f o r t h e s e f e r m e n t a t i o n s i f p H c a n b e k e p t c o n s t a n t w i t h a n a p p r o p r i a t e b u f f e r , o r w i t h a n a c i d - n e u t r a l i z i n g s u b s t a n c e s u c h a s l i m e s t o n e . D i s c u s s i o n s P o t e n t i a l o f S p e n t S u l f i t e l i g u o r 166 4 . 5 . 3 E f f e c t o f A i r A i r , m i x e d i n t o t h e f e r m e n t a t i o n b r o t h , e v e n a t l o w r a t e s , i s d e t r i m e n t a l t o P . f r e u d e n r e i c h i i , i f t h e y h a v e n o t y e t e s t a b l i s h e d a w e l l d e v e l o p e d e x p o n e n t i a l g r o w t h p h a s e . H o w e v e r , a i r h a s a b e n e f i c i a l e f f e c t i f P r o p i o n i b a c t e r i a a r e w e l l i n t o t h e e x p o n e n t i a l g r o w t h p h a s e . T h i s b e n e f i c i a l e f f e c t c a n b e s e e n q u i t e c l e a r l y i n c e r t a i n r u n s ( F i g u r e s 3 . 4 7 , 3 . 4 9 , 3 . 5 2 ) . B u t , o n t h e o t h e r h a n d , i f a i r i s a d d e d w h e n t h e b a c t e r i a a r e i n t h e i r s t a t i o n a r y p h a s e c f , g r o w t h t h e n a i r h a s n o e f f e c t o n g r o w t h a s t h e d e f i c i e n c y o f c e r t a i n n u t r i e n t s h a s t a k e n e f f e c t ( F i g u r e 3 . 4 8 ) . F u r t h e r m o r e , i f a i r i s a d d e d l a t e i n t h e e x p o n e n t i a l p h a s e o f g r o w t h o f b a c t e r i a , t h e b e n e f i c i a l e f f e c t o f a i r m a y b e m a s k e d b y t h e m o r e m a r k e d r e v e r s e e f f e c t o f s u b s t r a t e s a b o u t t o b e c o m e l i m i t i n g ( F i g u r e s 3 . 5 0 , 3 . 5 1 ) . T h e e f f e c t o f a i r o n s u g a r c o n s u m p t i o n a n d a c i d s p r o d u c t i o n i s s i m i l a r t o t h a t o n g r o w t h . F o r t h e p r o d u c t i o n o f v i t a m i n B 1 2 , t h e e f f e c t o f a i r i s v e r y m a r k e d a n d r e m a r k a b l y b e n e f i c i a l . S p a r g i n g a i r i n t o t h e f e r m e n t a t i o n m e d i u m u s u a l l y r a i s e s t h e r a t e a£ p r o d u c t i o n o f v i t a m i n B 1 2 d r a s t i c a l l y . I t a l s o r a i s e s t h e u l t i m a t e p r o d u c t i o n o f v i t a m i n B 1 2 . T h i s i s s h o w n b y t h e l o w p r o d u c t i o n o f v i t a m i n B 1 2 o f r u n F 1 5 . 2 2 b ( F i g u r e 3 . 4 5 , T a b l e A I . 3 8 ) . B a t c h F 2 0 . 2 2 i s a n e x c e p t i o n t o t h i s r u l e , a s a i r w a s m i x e d i n t o t h e b a t c h o n l y i n t h e s t a t i o n a r y p h a s e , b u t t h e p r o d u c t i o n o f v i t a m i n B 1 2 i s h i g h . T h i s w a s p r o b a b l y d u e t o t h e d i s s o l v e d o x y g e n i n t h e f e r m e n t a t i o n b r o t h , w h i c h w a s n e v e r m e a s u r e d , a n d t h e a g i t a t i o n D i s c u s s i o n s P o t e n t i a l o f S p e n t S u l f i t e L i g u o r 1 6 7 r a t e m i g h t h a v e t e e n h i g h e n o u g h t o s u p p l y t h i s r u n w i t h m o r e o x y g e n t h a n t o a n y o t h e r b a t c h . F o a m i n g p r o b l e m s l i m i t e d t h e a i r f l o w r a t e t c a m a x i m u m o f 2 0 0 0 c c / m i n . N o i n v e s t i g a t i o n w a s m a d e c n t h e p o s s i b i l i t y o f a n y a i r i n h i b i t i n g e f f e c t a t h i g h e r f l o w r a t e . A g a i n , t h e m a r k e d e f f e c t o f a i r o n v i t a m i n B 1 2 p r o d u c t i o n a n d o n g r o w t h m e a n s t h a t f e r m e n t a t i o n s c a r r i e d o u t i n E r l e n m e y e r f l a s k s a n d t e s t t u b e s a r e p r o b a b l y o f l i t t l e v a l u e a s t h e y a r e a e r o b i c t h r o u g h o u t . T h u s t h e r e c o u l d b e t o o m u c h a i r p r e s e n t i n s o l u t i o n a t t h e b e g i n n i n g o f t h e f e r m e n t a t i o n b u t p e r h a p s n o t e n o u g h a t t h e e n d . 4 . 5 . 4 E f f e e t _ g f _ A g i t a t i o n a n d V o l u j S g . . g f _ , F e r m e n t a t i o n _ B r o t h A g i t a t i o n w a s a d j u s t e d t o a l e v e l j u s t s u f f i c i e n t t o p r e v e n t a n y d e p o s i t i o n o f c e l l m a s s c n t h e b o t t o m o f t h e f e r m e n t o r . B u t a t t i m e s , t h i s r e q u i r e d v e r y v i g o r o u s a g i t a t i o n , a n d n o a t t e m p t w a s m a d e t o k e e p t h e a g i t a t i o n c o n s t a n t a t t h i s h i g h l e v e l . C e l l s w e r e t h e n d i s p e r s e d c o m p l e t e l y o n l y a t s a m p l i n g t i m e s . T h e s p e e d o f r o t a t i o n o f t h e a g i t a t o r w a s k e p t a s l o w a s p o s s i b l e , a n d i t w a s a d j u s t e d t o a l m o s t t h e s a m e v a l u e f o r a l l r u n s . A g i t a t i o n w o u l d e f f e c t t h e r a t e o f d i s s o l u t i o n o f a i r t h r o u g h t h e l i q u i d - a i r i n t e r f a c e . T h e a l m o s t c o n s t a n t r a t e o f a g i t a t i o n w a s e x p e c t e d , h o p e f u l l y , t o g e n e r a t e t h e a p p r o x i m a t e l y s a m e r a t e o f d i s s o l u t i o n o f a i r i n a l l r u n s . O n t h e o t h e r h a n d , t h e e f f e c t o f a g i t a t i o n a s a n a g e n t f o r d i s t r i b u t i o n o f f o o d s a n d / o r o f m i c r o o r g a n i s m s , i f a n y , w a s n o t D i s c u s s i o n s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 1 6 8 a t a l l i n v e s t i g a t e d . T h e i n i t i a l v o l u m e o f b r o t h w a s n o t a v a r i a b l e s t u d i e d . I t v a r i e d r a t h e r w i d e l y f r o m r u n t o r u n , a n d n o s y s t e m a t i c o b s e r v a t i o n w a s m a d e . I t i s , t h o u g h , d i s c u s s e d h e r e t o r e a f f i r m t h e i n v a l i d i t y o f r e s u l t s f r o m f e r m e n t a t i o n s i n E r l e n m e y e r f l a s k s a n d i n t e s t t u b e s . W i t h o u t s h a k i n g , t h e g r o w t h w a s n o t u n i f o r m , h e n c e o n e m o r e v a r i a b l e e n t e r e d i n t o t h e p i c t u r e , b u t w i t h s h a k i n g t h e a i r s o d i s s o l v e d i n h i b i t e d t h e g r o w t h . F u r t h e r m o r e , g r o w t h i n E r l e n m e y e r f l a s k s w a s e x t r e m e l y h a p h a z a r d , w h i c h m a y b e e x p l a i n e d b y t h e h i g h r a t i o o f s u r f a c e e x p o s e d t o a i r o v e r t h e v o l u m e o f t h e b r o t h . T h e v o l u m e o f t h e f e r m e n t a t i o n b r o t h i t s e l f d o e s n o t h a v e a d i r e c t e f f e c t o n t h e r e s u l t s . H o w e v e r , f o r f e r m e n t a t i o n s w i t h s m a l l v o l u m e s i n E r l e n m e y e r f l a s k s a n d i n t e s t t u b e s , t h e r e a r e m o r e t h a n a f e w r e a s o n s f o r e x p e c t i n g e r r o n e o u s r e s u l t s . I f t h e s e p a r a t e e f f e c t s o f e a c h i n d i v i d u a l c a u s e c i t e d a b o v e a r e c o n s i d e r e d , t h e n t h e c o n c l u s i o n s e e m s t o b e c o n t r a d i c t o r y t o t h e o n e d r a w n f r o m t h e g o o d g r o w t h r e s u l t s o b t a i n e d f o r f e r m e n t a t i o n s o f a q u e o u s s o l u t i o n s o f g l u c o s e i n E r l e n m e y e r f l a s k s { T a b l e I I I . 2 6 ) . H o w e v e r , t h e c o m p l e x , c o m b i n e d e f f e c t s o f a l l t h e s e c a u s e s s h o u l d b e c o n s i d e r e d t o g e t h e r w i t h t h e u n f a v o r a b l e n a t u r e o f S S L a s m e d i u m c o m p a r e d t o s o l u t i o n s o f g l u c o s e . A n o t h e r p o i n t a l s o w o r t h y o f n o t e , g l u c o s e w i t h i t s t e n d e n c y t o n e u t r a l p H i s a m u c h b e t t e r b u f f e r , t c c o u n t e r a c t t h e p H l o w e r i n g e f f e c t o f t h e a c i d s p r o d u c e d , t h a n S S L w i t h i t s D i s c u s s i o n s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 1 6 9 a c i d i c c h a r a c t e r . 4 . 5 . 5 f f f e c t _ o f Y e a s t y E x t r a c t _ C o n c e n t r a t i o n F e r m e n t a t i o n s i n t e s t t u b e s s e e m t o d e m o n s t r a t e c l e a r l y t h a t y e a s t e x t r a c t i s t h e l i m i t i n g s u b s t r a t e a t l e a s t t o a n i n i t i a l c o n c e n t r a t i o n o f 4 g / l a n d 1 5 g / l f o r P . f r e u d e n r e i c h i i a n d P . s h e r m a n i i r e s p e c t i v e l y . A b o v e t h e s e c o n c e n t r a t i o n s , t h e e f f e c t i s o f a l e s s d r a s t i c n a t u r e , b u t i t s t i l l c a n b e a l i m i t i n g s u b s t r a t e u p t o a n i n i t i a l c o n c e n t r a t i c n o f 8 0 g / l . T h e s e t e s t t u b e r e s u l t s t o g e t h e r w i t h t h o s e f r o m f e r m e n t a t i o n s i n E r l e n m e y e r f l a s k s s e e m t o c o n f i r m t h a t a n i n c r e a s e i n 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 w o u l d b r i n g a v e r y c l e a r i n c r e a s e i n t h e u l t i m a t e c e l l s m a s s . U n f o r t u n a t e l y , t h e s e r e s u l t s c a n n c t b e r e g a r d e d a s c o n c l u s i v e . T h e e f f e c t o f y e a s t e x t r a c t a s l i m i t i n g s u b s t r a t e , i f a n y , w o u l d h a v e b e e n m a s k e d b y t h e p H l o w e r i n g e f f e c t o f t h e p r o d u c t i o n o f a c i d s a n d w i t h t h e s i d e e f f e c t o f y e a s t e x t r a c t a s a p H b u f f e r . O n t h e o t h e r h a n d , r e s u l t s f r o m t h e f e r m e n t a t i o n s i n 7 1 f e r m e n t o r s c a n n o t b e u s e d t o d e d u c e a n y c o n c l u s i o n o n t h e e f f e c t o f y e a s t e x t r a c t a s a l i m i t i n g s u b s t r a t e . I n d e e d , t h e 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 w a s n o t s i n g l e d c u t a s t h e s o l e v a r i a b l e i n t h o s e s e r i e s o f e x p e r i m e n t s . A t l e a s t 3 o t h e r v a r i a b l e s w e r e i n t h e p i c t u r e : t h e i n i t i a l c o n t e n t o f d i s s o l v e d o x y g e n i n S S L , t h e r a t e o f d i s s o l u t i o n o f o x y g e n f r o m a i r t h r o u g h t h e i n t e r f a c e , a n d t h e t i m e w h e n a i r w a s i n t r o d u c e d t o s t a r t t h e a e r o b i c s e c o n d s t a g e . . A n i n c r e a s e c 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 D i s c u s s i o n s P o t e n t i a l o f S p e n t S u l f i t e L i g u o r 1 7 0 d i d b r i n g a b o u t a n i n c r e a s e i n t h e u l t i m a t e c e l l m a s s t h o u g h . H o w e v e r , t h i s e f f e c t i s n o t c l e a r e i t h e r ( T a b l e I I I . 2 4 ) . A l l c o n c l u s i o n s w h i c h c a n b e d r a w n f r o m t h e r e s u l t s c o l l e c t e d o n t h e e f f e c t o f y e a s t c o n c e n t r a t i o n a r e o f o n l y p r a c t i c a l u s e . T h a t i s , a c o n c e n t r a t i o n o f y e a s t e x t r a c t o f 5 g / l i s m o r e o r l e s s o p t i m u m , w i t h i n t h e r a n g e o f c o n d i t i o n s s t u d i e d . T h i s c o n c l u s i o n i s b a s e d o n l y o n t h e g r o w t h , i n t h e s e n s e t h a t t h e i n c r e a s e o f t h e u l t i m a t e c e l l m a s s p r o d u c e d i s n o t e n o u g h t o j u s t i f y t h e h i g h c o s t o f a n a d d i t i o n a l a m o u n t o f s u c h a n e x p e n s i v e n u t r i e n t . T h e 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 p r o d u c t i o n o f v i t a m i n B 1 2 a n d a c i d s w i l l b e d i s c u s s e d s h o r t l y i n p a r a g r a p h s 4 . 6 a n d 4 . 7 . i 4 . 5 . 6 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 C o b a l t i o n s d i d h a v e a n i n h i b i t i n g e f f e c t o n t h e g r o w t h o f P r o p i o n i b a c t e r i a . H o w e v e r t h i s e f f e c t w a s o n l y o n t h e g r o w t h r a t e , i t d i d n o t s e e m t o a f f e c t t h e u l t i m a t e c e l l m a s s . S e e a l s o s e c t i o n s 4 . 6 , 4 . 7 . 4 . 5 . 7 E f f e c t _ o f _ 5 x 6 - d i j § e t h j [ l _ B e n z i m i d a 2 o l e T h i s p r e c u r s o r d i d i m p r o v e t h e g r o w t h r a t e ( T a b l e I I I . 2 5 ) a n d d i d i m p r o v e t h e y i e l d o f c e l l s p e r u n i t o f r e d u c i n g s u g a r u s e d . U n f o r t u n a t e l y t h e s e c o n c l u s i o n s w e r e d r a w n f r o m t h e l i m i t e d r e s u l t s o f a s i n g l e r u n . T h e i n i t i a l s u g a r c o n c e n t r a t i o n o f t h e b r o t h w a s s o l o w t h a t t h e r e c o u l d n o t b e a n i n c r e a s e i n c e l l m a s s t o b a c k u p t h e b e l i e f o f t h e b e n e f i c i a l e f f e c t o f t h i s D i s c u s s i o n s P o t e n t i a l o f S p e n t S u l f i t e L i g u o r 1 7 1 p r e c u r s o r o n t h e u l t i m a t e c e l l m a s s . > 4 . 6 P B O D U C T I O N O F A C I D S B Y P . F B E U D E N B I I C B I I T h e v o l a t i l e a c i d s p r o d u c e d w e r e a s s u m e d t o b e a 2 : 1 m i x t u r e o f p r o p i o n i c a n d a c e t i c a c i d s ( a s c h e c k e d b y p r e v i o u s w o r k s £ 1 1 , 2 3 , 7 0 ] ) . T h e u l t i m a t e 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 w a s n o t a f f e c t e d b y t h e v a r i a t i o n o f a n y f e r m e n t a t i o n c o n d i t i o n n o r c f t h e c o n c e n t r a t i o n o f a n y n u t r i e n t a d d e d . T h i s a p p e a r s t o b e m o r e o f a c a n c e l l i n g e f f e c t b y t h e v a r i a t i o n s i n c e r t a i n v a r i a b l e s o v e r t h o s e i n o t h e r s . T h e r a t e o f a c i d p r o d u c t i o n d i d n e t s e e m t o b e e f f e c t e d b y t h e v a r i a t i o n s i n y e a s t e x t r a c t o r c o b a l t i o n c o n c e n t r a t i o n s . T h i s c o u l d n o t b e o f c o n c l u s i v e n a t u r e b e c a u s e o f t h e i m p r e c i s e s t a r t o f t h e a e r o b i c s e c o n d s t a g e , t h e u n c e r t a i n t y o f t h e a m o u n t o f o x y g e n o r i g i n a l l y d i s s o l v e d i n t h e b r o t h , a n d t h e r a t e o f d i s s o l u t i o n o f a i r t o t h e b r c t h , a l l o f w h i c h m a y h a v e c o n t r i b u t e d t h e i r o w n i n f l u e n c e w i t h t h e i r o w n v a r i a t i o n s ( m e a s u r e d o r n o t ) . B u t t h e s l o p e o f t h e c u r v e s i n F i g u r e 3 . 5 7 , a n d f u r t h e r m o r e , t h e f a c t t h a t a l l t h e p o i n t s f a l l o n t h e s a m e c u r v e i n F i g u r e 3 . 5 8 d o a f f i r m t h i s i n d e p e n d e n c e o f t h e 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 . A n i n c r e a s e i n 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 , t h o u g h , d i d r e d u c e t h e l a g p h a s e ( c o n s i d e r a b l y w i t h a n i n c r e a s e f r o m 5 t o 1 0 g / l , a n d m o d e r a t e l y f r o m 1 0 g / l u p w a r d ) . T h i s m a y h a v e b e e n c a u s e d b y t h e v a r i a t i o n i n t h e a g e o f i n o c u l u m . H o w e v e r , i t w o u l d h a v e b e e n t o o m u c h o f a c o i n c i d e n c e t h a t t h e r e s u l t s w e r e a l i g n e d i n t h i s m a n n e r . T h e D i s c u s s i o n s P o t e n t i a l o f S p e n t S u l f i t e l i g u o r 1 7 2 i n o c u l u m w a s a l w a y s c a r e f u l l y p r e p a r e d t o b e i n t r o d u c e d t o t h e f e r m e n t a t i o n b r o t h a t a s c l o s e t o t h e s a m e r e l a t i v e a g e a s p o s s i b l e . F u r t h e r m o r e , i t i s l o g i c a l t o t h i n k t h a t t h e a v a i l a b i l i t y o f n u t r i e n t s g e t s t h e b a c t e r i a o v e r t h e t r y i n g p e r i o d o f a d a p t i n g t o t h e n e w m e d i u m , f a s t e r . T h e y i e l d o f v o l a t i l e a c i d s ( T a b l e s I I I . 2 U , I I I . 2 5 ) , o n a p e r u n i t o f c e l l m a s s b a s i s , w i t h P . f r e u d e n r e i c h i i w a s l o w e r w h e n t h e r e l a t i v e a v a i l a b i l i t y o f n u t r i e n t s w a s h i g h . T h e s e t a b l e s a l s o s h o w t h a t t h e p r e s e n c e c f m o r e t o x i c c o b a l t i o n s c a u s e s t h e m t o h a v e l o w e r p r o d u c t i v i t y . H o w e v e r , t h e d a t a t h u s c o l l e c t e d d i d n o t s h o w a n y c l e a r t r e n d , i n t h e c o l l e c t i v e e f f i c i e n c y , n e i t h e r o f i m p r o v e m e n t n o r o f d e t e r i o r a t i o n , w i t h a v a r i a t i o n i n y e a s t e x t r a c t a n d c o b a l t i o n c o n c e n t r a t i o n s . T h e y d i d s h o w , t h o u g h , t h a t t h e a d d i t i o n o f D M B Z d i d i m p r o v e t h i s e f f i c i e n c y . T h e e f f i c i e n c y w a s n o t m e a s u r e d a s y i e l d c f v o l a t i l e a c i d s p e r u n i t o f s u g a r c o n s u m e d . I t w a s c a l c u l a t e d a s t h e r a t i o o f t h e a m o u n t o f v o l a t i l e a c i d s p r o d u c e d o v e r t h e t h e o r e t i c a l a m o u n t o f v o l a t i l e a c i d s o b t a i n e d w h e n a l l r e d u c i n g s u g a r s ( a s g l u c o s e ) a r e c o n v e r t e d t c a c i d s . P . f r e u d e n r e i c h i i s e e m s t o p r o d u c e a l i m i t e d a m o u n t o f l a c t i c a c i d d u r i n g t h e a e r o b i c s t a g e . H o w e v e r , t h e a c c u r a c y o f t h e e x t r a c t i o n d o e s n o t a l l o w a f i r m c l a i m f o r t h i s v e r y l i m i t e d , u n c e r t a i n i n c r e a s e i n t h e l a c t i c a c i d c o n c e n t r a t i o n . D i s c u s s i o n s P o t e n t i a l o f S p e n t S u l f i t e L i g u o r 1 7 3 4 . 7 P R O D U C T I O N O F V I T A H I N , B 1 2 B Y P . F R B U D B N ^ B I C H I I F i g u r e s 3 . 5 5 , 3 . 5 6 , a n d T a b l e s I I I . 2 4 , I I I . 2 5 s e r v e a s r e f e r e n c e s f o r t h e f o l l o w i n g d i s c u s s i o n . T h e p r e s e n c e o f a i r a f f e c t e d t h e p r o d u c t i o n o f v i t a m i n E 1 2 e n o r m o u s l y . T h i s c o n f i r m s t h e r e p o r t [ 2 3 , 7 0 , 7 9 ] o f t h e n e c e s s i t y f o r a i r f o r t h e c o n v e r s i o n o f i n c o m p l e t e v i t a m i n B 1 2 t o t h e t r u e a n d c o m p l e t e o n e . T h e r a t e o f p r o d u c t i o n o f v i t a m i n B 1 2 i n c r e a s e d w i t h i n c r e a s e s i n c o n c e n t r a t i o n o f y e a s t e x t r a c t u p t o 1 0 g / l a n d o f c o b a l t i o n u p t o 0 . 9 p p m . F r o m t h e s e c o n c e n t r a t i o n s u p w a r d s , t h e a d d i t i o n a l a m o u n t s o f n u t r i e n t a n d p r e c u r s o r d i d n o t s e e m t c h a v e a n y b e n e f i c i a l e f f e c t o n t h e p r o d u c t i o n r a t e s , a n d i n c e r t a i n c a s e s , t h e y e v e n h a d a r e v e r s e e f f e c t . H o w e v e r , t h i s r e v e r s e e f f e c t a p p e a r e d o n l y w i t h r u n F 2 0 . 2 2 ( w i t h 2 0 g / l o f y e a s t e x t r a c t a n d 2 . 2 p p m o f c o b a l t i o n ) . I t i s b e l i e v e d t h e n , t h a t t h e i n i t i a l d i s s o l v e d o x y g e n i n t h i s b a t c h o r t h e r a t e o f d i s s o l u t i o n o f a i r t o t h e b r o t h , s o m e h o w , d i f f e r e d s i g n i f i c a n t l y f r o m t h o s e o f o t h e r r u n s . T h i s d i f f e r e n c e a n d t h e l a t e s t a r t o f t h e a e r o b i c s e c o n d s t a g e m a y t h e n h a v e c a u s e d t h i s b a t c h t o h a v e a b n o r m a l b e h a v i o u r , c o m p a r e d t o t h e o t h e r s . A d d i t i o n o f c o b a l t i o n t o t h e f e r m e n t a t i o n b r o t h i s e s s e n t i a l t o o p t i m i z e t h e u l t i m a t e p r o d u c t i o n o f v i t a m i n E 1 2 . H o w e v e r , t h e e f f e c t o f a d d i n g m o r e t h a n 0 . 9 p p m o f c o b a l t i c n s t o t h e b r o t h w a s n o t n o t i c e a b l e a t a l l . A n i n c r e a s e i n 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 w a s a c c o m p a n i e d b y a d e c r e a s e i n t h e u l t i m a t e p r o d u c t i o n o f v i t a m i n B 1 2 . I t w a s D i s c u s s i o n s P o t e n t i a l o f S p e n t S u l f i t e L i g u o r 1 7 4 a l s o a c c o m p a n i e d b y a d e c r e a s e i n t h e y i e l d c f v i t a m i n B 1 2 , b a s e d b o t h o n r e d u c i n g s u g a r s c o n s u m p t i o n a n d o n g r o w t h . T h i s t h e n , c o n f i r m s t h a t a n a b u n d a n c e o f n u t r i e n t s ( i n t e r m s o f y e a s t e x t r a c t ) h e l p s t h e b a c t e r i a e n j o y t h e i r l i f e ( i n c r e a s e d a m o u n t s o f c e l l s p e r u n i t o f s u g a r u s e d ) b u t c a u s e s a d e c r e a s e i n t h e i r i n d i v i d u a l p r o d u c t i v i t y ( i n y i e l d i n g v i t a m i n B 1 2 a n d v o l a t i l e a c i d s ) a s w e l l a s i n t h e i r c o l l e c t i v e e f f i c i e n c y i n p r o d u c i n g v i t a m i n B 1 2 . P . f r e u d e n r e i c h i i d o e s n e e d c o b a l t i o n f o r t h e p r o d u c t i o n o f v i t a m i n B 1 2 . T h i s v i t a m i n , a n i n t r a c e l l u l a r p r o d u c t , i s p r o b a b l y n e e d e d f o r t h e m e t a b o l i s m o f t h i s p a r t i c u l a r b a c t e r i a l s p e c i e s , a s t h e y i e l d o f c e l l s ( p e r u n i t o f s u g a r c o n s u m e d ) a l s o i n c r e a s e s , a l o n g w i t h t h e i n c r e a s e i n t h e p r o d u c t i o n o f v i t a m i n B 1 2 , w i t h a n i n c r e a s e o f c o b a l t i c n c o n c e n t r a t i o n . T h i s b e n e f i c i a l e f f e c t , h o w e v e r , i s n o l o n g e r t r u e w h e n t h e c o n c e n t r a t i o n o f c o b a l t i o n r e a c h e s 2 . 2 p p m . T h e u l t i m a t e p r o d u c t i o n o f P . f r e u d e n r e i c h i i , t h e i r e f f i c i e n c y ( y i e l d p e r u n i t o f s u g a r ) a n d t h e i r p r o d u c t i v i t y ( y i e l d p e r u n i t o f d r y c e l l s ) i n f a b r i c a t i n g v i t a m i n B 1 2 d e c r e a s e w h e n t h e c o n c e n t r a t i o n o f c o b a l t i o n r e a c h e s 2 . 2 p p m f r o m 0 . 9 p p r a . P r e s u m a b l y , t h e c o n c e n t r a t i o n o f c o b a l t i o n h a s r e a c h e d t h e t o x i c l e v e l . A c t u a l l y , f o r e v e r y 1 m g o f v i t a m i n E 1 2 p r o d u c e d , o n l y a m a x i m u m o f 0 . 0 4 5 m g o f c o b a l t i s u s e d . T h e a d d i t i o n o f D M B Z g i v e s a b o o s t t o t h e p r o d u c t i v i t y a n d t h e e f f i c i e n c y o f P . f r e u d e n r e i c h i i i n s y n t h e s i z i n g v i t a m i n E 1 2 . D i s c u s s i o n s P o t e n t i a l o f S p e n t S u l f i t e l i q u o r 1 7 5 4 . 8 N ^ T R I E Q R E Q U I R E M E N T S A N D C O S T S F O B F E R M E N T A T I O N S W I T H P F -T h e o p t i m u m a m o u n t s o f n u t r i e n t s a n d p r e c u r s o r s t o b e a d d e d t o S S L a r e t h e n 5 g / l o f y e a s t e x t r a c t p o w d e r a n d a b o u t 1 p p m o f c o b a l t i o n . F u r t h e r a d d i t i o n o f t h e e x p e n s i v e y e a s t e x t r a c t d o e s T a b l e I V . 2 7 C O S T A N D P R O F I T O F F E R M E N T A T I O N S W I T H 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 r • — i j N u t r i e n t o r P r o d u c t C o n c e n t r a t i o n A s s u m e d V a l u e i n P r i c e E r o t h ( g / i ) ( * / K g ) < * / l ) • | Y e a s t E x t r a c t P o w d e r 5 6 0 - 0 . 3 i | 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 4 — — I C o b a l t C h l o r i d e 0 . 0 4 5 8 4 0 - 0 . 0 3 8 | C e l l s 7 . 5 — ~ | A c e t i c A c i d 3 . 2 2 0 0 . 0 6 4 | P r o p i o n i c A c i d 7 . 9 3 3 0 . 2 6 1 | V i t a m i n B 1 2 0 . 0 0 1 5 8 0 0 , 0 0 0 1 . 2 l | N e t I m p r o v e m e n t 1 . 1 8 7 i — j n o t c o n t r i b u t e a n y b e n e f i t t o t h e p r o c e s s . I t e v e n l o w e r s t h e p r o d u c t i v i t y a n d t h e e f f i c i e n c y o f P . f r e u d e n r e i c h i i . D M B Z d o e s e n c o u r a g e t h i s s p e c i e s o f b a c t e r i a t o p r o d u c e m o r e a n d t o u s e s u g a r m o r e e f f i c i e n t l y . H o w e v e r , t h e d a t a c o l l e c t e d a r e n o t s u f f i c i e n t f o r t h e d e d u c t i o n o f t h e o p t i m u m a m o u n t t o b e a d d e d n o r o f t h e a f f i r m a t i o n o f a n y e c o n o m i c a l i m p r o v e m e n t . F e r g u s o n [ 2 3 ] h a s c o n c l u d e d t h a t t h i s p r o c e s s w a s n o t D i s c u s s i o n s P o t e n t i a l o f S p e n t S u l f i t e L i g u o r 1 7 6 e c o n o m i c a l l y f e a s i b l e ( a n e g a t i v e n e t i m p r o v e m e n t i n t e r m c f 0 / 1 o f d i l u t e d S S L ) u n l e s s y e a s t e x t r a c t c o u l d b e s u b s t i t u t e d b y a c h e a p e r , a n d a t l e a s t a s a d e q u a t e , s o u r c e o f n i t r o g e n . H o w e v e r , t h e a m o u n t o f y e a s t e x t r a c t h e a d d e d t o S S L w a s e n o r m o u s . T h e e x o r b i t a n t c o s t , a t t r i b u t e d s o l e l y t o t h i s n u t r i e n t , o f t h e p r o c e s s i s r e d u c e d m o r e t h a n t e n - f o l d w h e n t h e a m o u n t o f y e a s t e x t r a c t a d d e d i s r e d u c e d t o 5 g / l f r o m o v e r 5 0 g / l w h i c h h e s u g g e s t e d . T h e l o w o p t i m u m y e a s t e x t r a c t r e g u i r e m e n t , p r o v e d t o b e a b o u t 5 g / l , w h i c h c e r t a i n l y a l l o w s a p r o f i t a b l e i n d u s t r i a l f e r m e n t a t i o n . I n d e e d , T a b l e I V . 2 7 s h o w s t h a t t h i s p r o c e s s i s e c o n o m i c a l l y f e a s i b l e . I n t h i s t a b l e , p r i c e s g u o t e d b y F e r g u s o n w e r e u s e d . T h e c o s t o f c o b a l t i o n a d d e d , w h i c h w a s n o t a c c o u n t e d f o r b y F e r g u s o n , i s a l s o i n c l u d e d . T h e c o s t c 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 , t h o u g h , i s n e t i n c l u d e d . T h e r e a s o n b e h i n d t h i s e x c l u s i o n i s t h a t t h i s n u t r i e n t i s n o t e s s e n t i a l ; t h i s f e r m e n t a t i o n m a y n o t n e e d i t a t a l l , o r i t p r o b a b l y j u s t n e e d s a t i n y a m o u n t , c o m p a r e d t o w h a t w a s u s e d . O n t h e o t h e r h a n d , t h e c o s t o f c o b a l t c h l o r i d e u s e d w a s t a k e n f r o m C a n l a b r e t a i l p r i c e l i s t , i t p r o b a b l y i s h i g h e n o u g h t o c o v e r t h e c o s t o f p h o s p h a t e . T h i s p r o c e s s t h e n b r i n g s i m p r o v e m e n t o f a b o u t 1iz t o t h e v a l u e o f e v e r y l i t e r c f S S L . B u t t h i s c o n c l u s i o n d o e s n o t n e c e s s a r i l y m e a n t h a t t h e p r o c e s s i s i m m e d i a t e l y c o m m e r c i a l l y p r o f i t a b l e . I t i s o n l y a r o u g h a n d r e a d y e s t i m a t e o f t h e f e a s i b i l i t y o f t h e p r o c e s s . D i s c u s s i o n s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 1 7 7 4 . 9 F E R M E N T A T I O N _ O F _ S O R e s u l t s o f t h e s e f e r m e n t a t i o n s ( T a b l e I I I . 2 6 ) s h o w t h a t t h e i n c r e a s e 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 h a d a b e n e f i c i a l e f f e c t o n g r o w t h o f P f r e u d e n r e i c h i i a s w e l l a s o n t h e i r 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 , l a c t i c a c i d a n d v i t a m i n E 1 2 . I f t h i s i s t r u e t h e n i t i s a c o n c l u s i o n r a t h e r c o n t r a d i c t o r y t o w h a t w a s s a i d e a r l i e r i n t h i s c h a p t e r . T h e m a g n i t u d e o f t h e p H c f a l l e x p e r i m e n t s o f t h i s s e r i e s , h o w e v e r , e x p l a i n s t h e s e d i s c r e p a n c i e s . T h e p H o f a l l f i n a l f e r m e n t a t i o n b r o t h s i s c l o s e t o 4 . 0 , w h i c h s h o w s t h a t t h e b e n e f i c i a l e f f e c t o f t h e i n c r e a s e i n y e a s t c o n c e n t r a t i o n i s d u e t o t h e a b i l i t y o f t h a t n u t r i e n t t o a c t a s p H b u f f e r a n d n o t t o i t s n u t r i t i v e v a l u e . T h i s a b i l i t y , w h i c h w a s l e s s e n e d a n d h e n c e m a s k e d o r n e u t r a l i z e d b y t h e a c i d i c n a t u r e o f S S L , s h o w s u p d i s t i n c t l y i n t h e s o l u t i o n s o f g l u c o s e . T h i s a l s o e x p l a i n s t h e r e l a t i v e l y g r e a t d i f f i c u l t y e x p e r i e n c e d i n g r o w i n g P . f r e u d e n r e i c h i i i n S S L , c o m p a r e d t o g r o w i n g i t i n s o l u t i o n s o f g l u c o s e , w h e n u s i n g f l a s k s w i t h o u t p f i a d j u s t m e n t . T h e a d d i t i o n o f 2 . 2 p p m o f c o b a l t i o n h a s n o e f f e c t , n o t e v e n o n t h e p r o d u c t i o n o f v i t a m i n B 1 2 . T h i s c o n f i r m s t h a t t h e a i r f o r c e d t o d i s s o l v e i n t h e f e r m e n t a t i o n b r o t h s b y s h a k i n g i s n o t s u f f i c i e n t t o a l l o w a n a e r o b i c s e c o n d s t a g e , i n w h i c h t h e c o b a l t i o n i s l i k e l y t o b e u s e d t o c o n v e r t v i t a m i n E 1 2 - l i k e f a c t o r s t o t h e c o m p l e t e v i t a m i n B 1 2 . D i s c u s s i o n s P o t e n t i a l o f S p e n t S u l f i t e L i q u o r 1 7 8 C h a p t e r 5 C O N C L U S I O N S S S L i s a s a t i s f a c t o r y a n d s u i t a b l e m e d i u m f o r f e r m e n t a t i o n s w i t h P . f r e u d e n r e i c h i i , P . s h e r m a n i i , a n d L . , p l a n t a r i u m . H o w e v e r , S . g r i s e u s a n d S . o l i v a c e u s c a n n o t g r o w i n S S L . G r o w t h o f t h e s e S t r e p t o m y c e s i n S S L b y s l o w a c c l i m a t i z a t i o n i s p o s s i b l e b u t n o t p r o m i s i n g . L . p l a n t a r i u m c a n u s e m o r e r e d u c i n g s u g a r s t h a n P r o p i o n i b a c t e r i a , b u t t h e s u b s e q u e n t f e r m e n t a t i o n s o f t h e l a c t i c a c i d , t h u s f o r m e d i n S S L , b y P r o p i o n i b a c t e r i a y i e l d t o o l i t t l e v i t a m i n B 1 2 . F e r m e n t a t i o n s w i t h a m i x t u r e o f 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 a r e i n t e r e s t i n g a n d g i v e a b e t t e r p r o d u c t i o n o f v i t a m i n B 1 2 , b u t t h e y i e l d i s s t i l l t o o l o w t o g u a r a n t e e a n y p o s s i b i l i t y o f c o m p e t i t i o n w i t h d i r e c t f e r m e n t a t i o n s o f S S L w i t h P . f r e u d e n r e i c h i i a l o n e . P . s h e r m a n i i r e q u i r e m o r e n u t r i e n t s a n d p r o d u c e l e s s v i t a m i n B 1 2 . T h i s s p e c i e s c a n n o t c o m p e t e w i t h P . 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 w i t h P . f r e u d e n r e i c h i i t h e n p r o v e t h a t t h i s s p e c i e s r e q u i r e s a n o p t i m u m a d d i t i o n o f e s s e n t i a l n u t r i e n t a s l o w a s 5 g / l o f y e a s t e x t r a c t . O n e p p m c f c o b a l t i o n i s a l s o n e e d e d a s p r e c u r s o r f o r t h e o p t i m u m p r o d u c t i o n o f v i t a m i n E 1 2 , w h i c h i s a b o u t 1 . 5 m g / l . T h e p r o d u c t i o n o f v i t a m i n B 1 2 i s a c c o m p a n i e d b y t h e p r o d u c t i o n o f 0 . 14 m c l e s / 1 o f v o l a t i l e a c i d s . C o n c l u s i o n s P o t e n t i a l o f S p e n t S u l f i t e L i g u o r 1 7 9 W i t h t h e s e v e r y f e w r e q u i r e m e n t s t h i s f e r m e n t a t i o n p r o c e s s i s e c o n o m i c a l l y p r o m i s i n g . A i r , i n t r o d u c e d a t a n y n o t i c e a b l e r a t e , i s d e t r i m e n t a l t o P . f r e u d e n r e i c h i i i n t h e a n a e r o b i c f i r s t s t a g e , b u t i s b e n e f i c i a l t o a n d n e e d e d f o r t h e p r o d u c t i o n o f v i t a m i n B 1 2 i n t h e a e r o b i c s e c o n d s t a g e . A d d i t i o n o f y e a s t e x t r a c t o f o v e r 5 g / l r e d u c e s t h e p r o d u c t i v i t y a n d t h e e f f i c i e n c y o f t h i s b a c t e r i a l s p e c i e s . C o b a l t i o n i s n e e d e d f o r t h e f o r m a t i o n o f v i t a m i n B 1 2 , b u t i t s t o x i c e f f e c t s t a r t s t o r e d u c e t h e g r o w t h o f P . f r e u d e n r e i c h i i , a s w e l l a s t h e i r p r o d u c t i v i t y a n d t h e i r e f f i c i e n c y , a t a c o n c e n t r a t i o n o f 2 . 2 p p m . T h e o p t i m u m a m o u n t o f 1 m g o f c o b a l t i o n i s w e l l o v e r t h e c a l c u l a t e d a m o u n t o f 0 . 0 6 8 m g n e e d e d f o r t h e p r o d u c t i o n o f 1 . 5 m g o f v i t a m i n B 1 2 i n o n e l i t e r o f S S L . T h e a d d i t i o n o f D M B Z a s p r e c u r s o r i m p r o v e s t h e f e r m e n t a t i o n o f S S L w i t h P . f r e u d e n r e i c h i i . F i s h p l a n t e f f l u e n t i s n o t a s u i t a b l e r e p l a c e m e n t f o r y e a s t e x t r a c t . T h e r e d u c t i o n o f t h e p o l l u t i n g p o t e n t i a l o f S S L i s o n l y a b o u t 7 0 % o f t h e C O D c a u s e d b y a l l r e d u c i n g s u g a r s . S o , a r e d u c t i o n o f a b o u t 1 4 % o f t h e t o t a l C O D o f S S L c a n b e e x p e c t e d f r o m t h e s e f e r m e n t a t i o n s . I n s u c h f e r m e n t a t i o n s , w h e r e r e d u c i n g s u g a r s a r e t h e o n l y c o m p o n e n t s u s e d b y b a c t e r i a , t h e r e d u c t i o n o f t h e p o l l u t i n g p o t e n t i a l o f S S L i s b e s t f o l l o w e d b y o b s e r v a t i o n s o f t h e c o n s u m p t i o n o f t h e s e s u g a r s . T h e a c c u r a c y o f t h e C O D t e s t , a n d p r o b a b l y o f a n B O D t e s t , d o e s n o t a l l o w a n y s i g n i f i c a n t m e a s u r e o f t h i s s m a l l r e l a t i v e r e d u c t i o n i n C O D ( o r B O D ) . C o n c l u s i o n s B i b l i o g r a p h y 1 8 0 B I B L I O G R A P H Y [ 1 ] A i a e r f e r , R . B . , H. F . G r i b b i n s a n d D . E . H a l e y . 1 9 4 4 . E f f e c t o f W a s t e S u l f i t e L i g u o r o n A g g r e g a t i o n o f S o i l P a r t i c l e s . I n d . a n d C h e m . , V o l . 3 6 , K o . 3 , p p 2 7 2 - 2 7 4 . [ 2 ] A m b e r g , H . R . f T . R . A s p i t a r t e , a n d J . F . C o r m a c k . 1 9 6 9 . F e r m e n t a t i o n o f S p e n t S u l f i t e L i q u o r f o r T h e 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 . W a t e r P o l l u t i o n C o n t r o l F e d e r a t i o n , V o l . 4 1 , H o . 1 1 , p p R 4 1 9 - R 4 3 0 . [ 3 ] A m e r i c a n T y p e C u l t u r e C o l l e c t i o n . 1 9 6 8 . C a t a l o g u e o f S t r a i n s , 8 t h E d i t i o n , R o c k v i l l e , M d . [ 4 ] A P H A , A W W A , a n d W P C F . 1 9 6 5 . S t a n d a r d M e t h o d s f o r T h e E x a m i n a t i o n o f W a t e r a n d W a s t e W a t e r , 1 2 t h E d . , A m e r i c a n P u b l i c H e a l t h A s s o c i a t i o n , I n c . , N e w Y o r k . [ 5 ] A S T M S t a n d a r d M e t h o d D 1 2 5 2 - 6 0 . 1 9 6 0 . C h e m i c a l O x y g e n D e m a n d o f I n d u s t r i a l W a s t e W a t e r . A m e r i c a n S o c i e t y f o r T e s t i n g a n d M a t e r i a l s , P h i l a d e l p h i a , p p 4 5 3 - 4 5 6 . [ 6 ] B a c h e r , F . A . , A . E . B o l e y a n d C . E . S h o n k . 1 9 5 4 . R a d i o A c t i v e T r a c e r A s s a y f o r V i t a m i n B 1 2 a n d O t h e r C o b a l a m i n s i n C o m p l e x M i x t u r e s . A n a l y t i c a l C h e m i s t r y , V o l . 2 6 , N o . 7 , p p 1 1 4 6 - 1 1 4 9 . [ 7 ] B a r k e r , S . B . a n d W. H . S u m m e r s o n . 1 9 4 1 . T h e C c l o r i m e t r i c D e t e r m i n a t i o n o f L a c t i c A c i d i n B i c l c g i c a l M a t e r i a l . J . B i o l . C h e m . , V o l . 1 3 8 , ^ p p 5 3 5 - 5 5 4 . [ 8 ] B e n s o n , H . K . a n d A . M . P a r t a n s k y . 1 9 3 4 . T h e R a t e a n d E x t e n t o f A n a e r o b i c D e c o m p o s i t i o n o f S u l p h i t e W a s t e L i q u o r b y B a c t e r i a o f S e a W a t e r M u d . I . S t u d y o f A n a e r o b i c F e r m e n t a t i o n s . P r o c . N a t l . A c a d . S c i . , V o l . 2 0 , p p 5 4 2 - 5 5 1 . [ 9 ] B e n s o n , H . K . a n d A . M . P a r t a n s k y . 1 9 3 6 . S u l f i t e w a s t e L i q u o r : L a b o r a t o r y S t u d y o f I t s A n a e r o b i c D e c o m p o s i t i o n w h e n D i s c h a r g e d i n t o W a t e r B o d i e s . I n d u s t r i a l a n d E n g i n e e r i n g C h e m i s t r y , V o l . 2 8 , N o . 6 , p p 7 3 8 - 7 4 0 . B i b l i o g r a p h y 18 r [ 1 0 ] B e r g e y , W. 1 9 5 7 . M a n u a l o f D e t e r m i n a t i o n B a c t e r i o l o g y , W i l l i a m s a n d W i l l i a m s , B a l t i m o r e . [ 1 1 ] B e r n h a u e r , K . 1 9 5 8 . C o l u m b i a C e l l . S t a t u s R e p o r t , P R - L S R - 3 5 6 , p . 5 . [ 1 2 ] B i o c h e m i c a l O x y g e n D e m a n d T e s t . I n s t r u c t i o n N o t e s f o r M i c r o b i o l o g y 3 0 7 l a b o r a t o r y . T h e . U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r . [ 1 3 ] B i s w a s , B . a n d J . B . L a i . 1 9 6 2 . I f f l u e n t P r o b l e m s i n I n d i a n P a p e r M i l l s . I n d i a n P u l p a n d P a p e r , V o l . 1 7 , N o . 1 , p p 6 5 - 7 2 . [ 1 1 ] B r a u n s , F . E . 1 9 5 2 . T h e C h e m i s t r y o f L i g n i n , a c a d e m i c P r e s s , N e w Y o r k . 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I . B r o w n , a n d F . B . B e i d . 1 9 3 6 . T h e ftmmoniation o f H a s t e S u l f i t e L i g u o r , J . A g r . R e s e a r c h , V o l . 5 3 , N o . 3 , p p 2 0 9 - 2 2 4 . [ 7 7 ] P r e v o t , a . R . , , A . T u r p i n , a n d P . K a i s e r . 1 9 6 7 . L e s B a c t e r i e s A n a e r o b i e s , D u n o d , P a r i s . [ 7 8 ] R h e m , H . j . 1 9 6 7 . I n d u s t r i e l l e M i k r o b i o l o g i e , S p r i n g e r V e r l a g , B e r l i n . [ 7 9 ] R i l e y , P . B . , P . W. J a c k s o n , 0 . R o s s a n d P . a . S a v a g e . 1 9 6 1 . P r o d u c t i o n o f V i t a m i n B 1 2 a n d a n a n a l o g u e b y C o n t i n u o u s F e r m e n t a t i o n . M o n o g r a p h N o . 1 2 , p p 1 2 7 - 1 3 9 . [ 8 0 ] R u d o l f s , W. a n d V . G u e r c i o . 1 9 4 7 . S a n d F i l t r a t i o n o f S o m e O r g a n i c H a s t e s . H a t e r a n d s e w a g e W o r k s , V o l . 9 4 , p p 3 4 9 - 3 5 1 . [ 8 1 ] S a m u e l s o n , 0 . a n d a . 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S v e n s k P a p p e r s t i d n i n g a r g . , S c a n d i n a v i a n P u l p , P a p e r a n d B o a r d T e s t i n g C o m m i t t e e , V o l . 6 8 , p p 3 5 1 - 3 5 3 . [ 8 5 ] S m i t h , E . L . 1 9 5 5 . I s o l a t i o n a n d C h e m i s t r y o f V i t a m i n B 1 2 . B i o c h e m . S o c . S y m p o s i a , V o l . 1 3 , p p 3 - 1 6 . [ 8 6 ] S n e l l a n d S n e l l . 1 9 5 3 . C o l o r i m e t r i c M e t h o d s o f a n a l y s i s , 3 r d E d i t i o n , D. V a n N o s t r a n d C o m p . , I n c . [ 8 7 ] S o m o g y i , M . 1 9 4 5 . A N e w B e a g e n t f o r T h e D e t e r m i n a t i o n o f S u g a r s . J . B i o l . C h e m . , V o l . 1 6 0 , p . 6 1 . B i b l i o g r a p h y 1 8 7 [ 8 8 ] S o w d e n , F . J . a n d H . j . A t k i n s o n . 1 9 4 8 . W a s t e S u l f i t e L i g u o r S o l i d s a s a S o i l T r e a t m e n t . S c . A g r . , V o l . 2 8 , p p 1 7 5 - 1 8 2 . [ 8 9 ] S o w d e n , F . J . a n d H . J . A t k i n s o n . 1 9 5 1 . E f f e c t o f W a s t e S u l f i t e L i g u o r S o l i d s o n T h e C h e m i c a l a n d P h y s i c a l P r o p e r t i e s o f S o i l , S c i . A g r . , V o l . 3 1 , p p 9 3 - 9 8 . [ 9 0 ] S p e a k m a n , H . B . 1 9 2 6 . F e r m e n t a t i o n s P r o d u c t s f r o m C e l l u l o s e . P u l p a n d P a p e r M a g a z i n e o f C a n a d a , V o l . 2 4 , p p 7 3 1 - 7 3 5 . [ 9 1 ] S t a n d a r d H . 3 P 9 1 9 6 6 . C h e m i c a l O x y g e n D e m a n d . S t a n d a r d M e t h o d s , T e c h n i c a l S e c t i o n , C a n a d i a n P u l p S P a p e r A s s o c i a t i o n . [ 9 2 ] S t a n i e r , B . Y . , M . D o u d o r o f f , a n d E . A . A l d e l b e r g . 1 9 6 3 . T h e M i c r o b i a l W o r l d , S e c o n d E d i t i o n , P r e n t i c e - B a l l , E n g l e w o o d C l i f f s , N e w Y o r k , p p 3 2 2 - 3 2 4 . [ 9 3 ] T a k e t o m i , N . , K . S h i o z a w a , a n d T . D e d a . 1 9 5 2 . P r e p a r a t i o n o f L a c t i c A c i d f r o m S u l f i t e W a s t e L i g u o r , J . C h e m . S o c , J a p a n , I n d . C h e m . S e c t . , V o l . 5 5 , p p 1 9 - 2 2 . [ 9 4 ] T a p p i S t a n d a r d s T 6 2 9 o s - 5 3 . 1 9 5 3 . T e c h n i c a l A s s o c i a t i o n o f T h e P u l p a n d P a p e r I n d u s t r y , N e w Y o r k . T h e C c l o r i n [ 9 5 ] T h i e r f e l d e n , K . 1 9 5 1 . P r e l i m i n a r y T r e a t m e n t c f S u l f i t e L i g u o r , G e r m a n P a t e n t 8 0 5 , 3 4 7 . [ 9 6 ] T o o h e y , J . I . a n d H . A . B a r k e r . 1 9 6 1 . I s o l a t i o n o f C o e n z y m e B 1 2 f r o m L i v e r . T h e J . o f E i c l . a n d C h e m . , V o l . 2 3 6 , N o . 2 , p p 5 6 0 - 5 6 3 . [ 9 7 ] T u l l y , J . P . 1 9 4 9 . P r e d i c t i o n o f P u l p M i l l P o l l u t i o n . P a p e r T r a d e J . , V o l . 1 2 9 , N o . 4 , p p 3 5 - 4 0 . [ 9 8 ] T y l e r , R . , W. M a s k e , a n d R . B r e w e r . 1 9 4 6 . T r e a t m e n t o f S u l f i t e W a s t e L i g u o r o r H i g h B a t e T r i c k l i n g F i l t e r s . S e w a g e W o r k s J . , V o l . 1 8 , p p 1 1 5 5 - 1 1 6 8 . [ 9 9 ] U n d e r k o f l e r , L . A . a n d R . J . H i c k e y ( e d . ) . 1 9 5 4 . I n d u s t r i a l F e r m m e n t a t i o n s , F i r s t E d i t i o n , C h a p . 4 6 5 , V o l . I , C h e m i c a l P u b l i s h i n g C o . , I n c . , N e w Y o r k , p p 9 5 - 1 7 1 . [ 1 0 0 ] V a s s e u r , E r i k . 1 9 6 6 . P r o g r e s s i n S u l f i t e P u l p P o l l u t i o n A b a t e m e n t i n S w e d e n . W a t e r P o l l u t i o n C o n t r o l F e d e r a t i o n , C o l . 3 8 , N o . 1 , p p 2 7 - 3 7 . B i b l i o g r a p h y 1 8 8 [ 1 0 1 ] V i t a m i n B 1 2 A s s a y . I n s t r u c t i o n N o t e s f o r H i c r o b i o l o g y 3 2 1 L a b o r a t o r y , T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r . [ 1 0 2 ] V o g e l , A . I . 1 9 6 1 . 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S t u d i e s cn The U t i l i z a t i o n of Wood Sugars by Fermentation. ] Hokkaido F o r e s t Products Research I n s t . Report, C. F. ABIPC, V o l . 34, No. 6, #3958, pp 52-96. B i b l i o g r a p h y 1~90 L I S T O F O C C U R E N C E S O F R E F E R E N C E S R e f e r e n c e C i t e d i n n : I' • • • • • • • • 1 • 1 • 3 ^ 2 • 2 • 1[ 2 : I • • • • . . . . 1 . 1 . 3 [ 3 ; | . . . . 2. 1,4.2 £* : I • • • • . . . . I I . 6 [ 5 ; I • • • • I I . 6 [6- 1 • • • 2 .4 .3 n : | . . . . 2 .4 .4 [8- | . . . . . . . . 1 . 1 . 3 [ 9 : | . • . • 1.1.3 10 | • • • • • •••1•3•1f 2•3• 3. 11 • 1 . . . . . . . . 2 . 4 . 4 , 4 . 6 12 i i . . . . . . . . 2 . 4 . 5 13 • I1 • • • . . . . 1 . 1 . 2 14 I1 • • • . . . . 1 . 1 . 1 15" I1 • • • . . . . 2 . 4 . 3 16 )l • • • . . . . 2 . 4 . 3 17 | • • • • . . . . 1 . 1 . 2 18 J • * * • 1.1.3 19 ] l • • • . . . . 3 20 1 • • • • • ••* 1 • 1*3y2«2« 1 21 | • • • • 1.3.3 22 j i . . . . 1 . 1 . 3 23 | • • . » • • * * 1 • 1 • 3 • 1 • 2 f . 3 • 2 • 4 • 5 • 2 f 4. 24 31 • • • . . . . 2 . 4 . 3 25 J • a • • . . . . 1 . 1 . 1 26 J • • • • 1 .3 .1 ,4 .2 27 ] 1 9 # M 3 28 I t # ^ # 2.4 .4 29 J • • • * . . . . 1 . 1 . 2 30 j * • • • . . . . 1 . 3 . 2 31 J • • • * 1.3.3 32 j • • • • . . . . 1 . 4 33 ] 1 . M , . . . . 1.2 34 J • • • * . . . . 1 . 1 . 2 35 J • • • • . . . . 1 . 1 . 3 36 j • • • • . . . . 1 . 1 . 3 37 J ». • • • 1.1.2 38 j * • • • . . . . I I . 4 39 J • • • • . . . . 2 . 2 . 1 40 i i . . . . . . . 2 . 4 . 4 41 1* . . . . . . . 1.2 3«2f2«2« J1f2»3*3«2f2«4*3f2«^l«^4 t r 6 ,4 .8 B i b l i o g r a p h y 191 42]* 2.4.3 43] 2.2.1 44 ] 2.2. 1 45]* .......1.1.3 46 ] 1.1.3 47] ........1.4 48]* 1.2 49 ] 1.1.1 50] .1.1.2 51] 1.1.3 52 ] , 2.2. 1 53] ........4.2 54] ........2.2.1 55] ........2.2.1 56] ........2.2.1 57] ........2.2.1 58] 2.4.4 59] 1.1.3 60] 1.2 61 ] .. 1.4 62] ........1.3.2 63]* ..2.4.3 64] .1.1.1,1.1.3 65] 1.1.3 66] 1.1.3 67] 2.4.4,11.3,11.5.3 68] ..II.3 69] ........4.5.2 70] 1. 1. 1, 1. 1.3, 1.2, 1.3. 2,2. 2. 1, 2. 4. 3, 2.4. 4,4.6,11.6 71 ] 2.2. 1 72] 2.3.1 73] 1.3.3 74] .1.3.1,4.2 75] ..1.3.3 76] 1.7 7 7 ^  *««««*«*2«3«3«2f3«2 78] 1.3.2 79]* 1.2 80] 1.1.3 81 ] 1.1.2,1.3.2 82] ........1. 1.3 83] 1.1.3 84]* 1.1.1 85]* 1.2 86]* 2.4.4 87 ] II.3 88] .1.1.3 89] ........1.1.3 90]* 2.2.1 91] . . . I I . 6 92] 2.4.1 93] ..1.1.3 94] II.7 B i b l i o g r a p h y 192 [ 9 5 ] 2.2.1 [ 9 6 ] * ... 1.2 [ 9 7 ] * ... 1. 1.2 [ 9 8 ] ....1.1.3 [ 9 9 ] ..... ....1.1. 1,1. 1.3,2.2. 1 100] .... . . . . 1 . 1.1,2.4.5 101 ] .... I I . 4 102]* ... ....2.4.4 103] . . ... 1.3.3,2.1 104] 1.3.3 105]* 1. 1.2 106] .... ....1.1.3 107] .... ....1.1.2,2.2.1 108]* ... 1.2 109] .... .... 1. 1.2 110] .... .... 1.1.1,1. 1.2,2.4.5 111 ] ..... ....1.1.3 112] ....1.1.3 113] .... ....1.1.2 114]* ... ....2.4.3 115] .... ....1.2 116]* ... ....1.1 * t i o t c i t e d a n y w h e r e i n t e x t b u t r e l a t e d t o t h e p a r a g r a p h s t h e i d e n t i f i c a t i o n n u m b e r s o f w h i c h a r e l i s t e d . A p p e n d i x I : T a b u l a t e d R e s u l t s 1 9 3 A P P E N D I X I T a b u l a t e d R e s u l t s A b b r e v i a t i o n s u s e d i n t h i s a p p e n d i x : A P R - A i r f l o w r a t e ( c c / m i n ) B - V i t a m i n B 1 2 c o n c e n t r a t i o n , o r p r o d u c t i o n ( m g / l ) . B R - V i t a m i n B 1 2 p r o d u c t i o n r a t e ( m i c r o g r a m s / l / h r ) . C - F e r m e n t a t i o n c o n d i t i o n s . C C o n d i t i o n s 0 2 s t a g e s , s h a k e n d u r i n g l a s t , s t a r t i n g p H 7 . 1 n o t s h a k e n , s t a r t i n g p H 7 . 2 2 s t a g e s , s h a k e n d u r i n g l a s t , n o p H r e a d j u s t m e n t a f t e r s t e r i l i z a t i o n . 3 u n s h a k e n , n o p H r e a d j u s t m e n t a f t e r s t e r i l i z a t i o n . DCW - D r y c e l l w e i g h t ( g / 1 ) . E - E f f i c i e n c y . E = a / b . a : p r o d u c t o b t a i n e d - v o l a t i l e a c i d s p r o d u c e d ( l a c t i c a c i d f o r r u n s F P 1 . 1 a n d F P 1 . 2 ) . b : s a m e p r o d u c t t h e o r e t i c a l l y o b t a i n e d i f t h e s u b s t r a t e o f i n t e r e s t ( l a c t i c a c i d f o r r u n s F P 2 . 1 , F P 2 . 2 , a n d r e d u c i n g s u g a r f o r o t h e r s ) i s e x h a u s t i v e l y c o n s u m e d . GR - G r o w t h r a t e ( g o f d r y c e l l / l / h r ) . L - L a c t i c a c i d c o n c e n t r a t i o n b y e t h e r e x t r a c t i o n ( m o l e s / 1 ) . L C - L a c t i c a c i d c o n s u m p t i o n ( m o l e s / 1 ) a p p e n d i x I : T a b u l a t e d R e s u l t s 1 9 1 L C R - L a c t i c a c i d c o n s u m p t i o n r a t e , i n t e r m o f v a r i a t i o n r a t e o f c o n c e n t r a t i o n ( m m o l e s / l / h r ) L P - L a c t i c a c i d p r o d u c t i o n ( m o l e s / 1 ) , L R - L a c t i c a c i d p r o d u c t i o n r a t e ( m m o l e s / l / h r ) . p H - F i n a l p H f o r f e r m e n t a t i o n s i n f l a s k s r - R e i n f o r c e d c l o s t r i d i a l m e d i u m u s e d a s f e r m e n t a t i o n b r o t h , . S - S u g a r c o n c e n t r a t i o n ( g / 1 ) S C S u g a r c o n s u m p t i o n ( g / 1 ) . S R - S u g a r c o n c e n t r a t i o n v a r i a t i o n r a t e ( g / l / h r ) . T - F e r m e n t a t i o n t i m e ( h r s ) . T a - T o t a l a c i d c o n c e n t r a t i o n b y e t h e r e x t r a c t i o n ( m o l e s / 1 ) . T I - T i m e i n t e r v a l s ( h r s ) . T U R B - T u r b i d i t y V - 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 b y e t h e r e x t r a c t i o n ( m o l e s / 1 ) . V P - V o l a t i l e a c i d s p r o d u c t i o n ( m o l e s / 1 ) V R - V o l a t i l e a c i d s p r o d u c t i o n r a t e ( m m o l e s / l / h r ) . Y - 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 ( g / 1 ) . Ya - Y i e l d o f a c i d s ( m o l e s / g o f d r y c e l l s ) . Y H - Y i e l d o f d r y c e l l s ( g / g c f r e d u c i n g s u g a r c o n s u m e d ) . F o r f e r m e n t a t i o n s i n t h e H i c r o f e r m 7 1 f e r m e n t o r s , t h e i d e n t i f i c a t i o n n u m b e r o f t h e r u n i s u s e d t o c o n v e y r e l a t e d i n f o r m a t i o n . T h e f i r s t n u m b e r i s t h e c o n c e n t r a t i o n o f y e a s t e x t r a c t i n g / 1 . T h e s e c o n d n u m b e r i s t h e c o n c e n t r a t i c n o f c o b a l t i o n i n p p m w i t h t h e d e c i m a l p o i n t o m i t t e d . T h e t h i r d n u m b e r , i f A p p e n d i x I : T a b u l a t e d R e s u l t s 1 9 5 a n y , i s t h e c o n c e n t r a t i o n o f a d d e d D H B Z . T h e s t a r t i n g l e t t e r i d e n t i f i e s t h e b a c t e r i a l s p e c i e s u s e d . W h e n t h i s n u m b e r i s i n a d e q u a t e i n f u l f i l l i n g t h e t a s k j u s t d e s c r i b e d , f u l l i n f o r m a t i o n i s p r e s e n t e d . D e t a i l e d i n f o r m a t i o n o f a n y r u n c a n b e o b t a i n e d f r o m T a b l e I I I . 2 3 . Appendix I : Tabulated R e s u l t s 196 Table AI.28 FERMENTATION OF SSL WITH L. PLANTARIOM IN ERLENMEYER FLASKS f™ 1 x t . DCW SC LP VP pH T C E YA i YW | 1 I r 0.49 7. 1 0.063 0.039 3.4 109 3 1.300 0.210 ,, , . , , . j 0.069 | 1 o 0.0 0.0 0.0 0.0 5.0 109 3 — ' — — | | 10 0. 14 4.2 0.028 0.0 6.2 144 1 0.600 0.2C0 0.033 | I 10 0.39 4.8 0.054 0.007 5.5 144 1 1.140 0. 156 0.081 | | 20 1.00 15.6 0. 119 0.004 3. 1 144 1 0.710 0. 123 0.064 I | 20 0.83 8.7 0.089 0.007 4.9 144 1 0.994 0. 116 0.095 | I 25 1.30 3.0 109 3 | 25 1. 18 19.4 0. 140 0.035 2.8 109 3 0.815 0. 149 0.061 | | 40 1.77 23*4 0. 158 0.017 3.7 144 1 0.674 0.099 0.076 | I 50 1.83 26.7 0.175 0.027 3.8 144 1 0.682 0. 110 0.069 | | 50 1.67 24.0 0. 167 0.029 2.9 109 3 0.736 0. 117 0.070 | I is 2.00 27.0 0. 151 0.025 3.6 109 3 0.587 0.088 0.074 | I 75 1.65 21.3 0. 160 0.028 3.4 109 3 0.795 0. 114 0.077 | | 100 1.81 24.0 0. 133 0.034 3.8 109 3 0.627 0.092 0.075 j Table AI.29 FERMENTATION OF SSL WITH P. FREUDENREICHII IN ERLENMEYER FLASKS , , . , ^ Y DCW SC LP VP B pH T C E YA YW r 2.46 7. 1 -.026 0.055 0.045 5. 0 156 2 0. 383 0.012 0.349 r 1.76 7. 1 -.035 0.053 0.020 4. 1 156 3 0.243 0.011 0.250 0 0.0 0.0 0.0 0.0 0.0 4. 8 156 3 — — — 10 0.37 8. 1 0.050 0.020 — 4. 5 168 0 0.778 0. 192 0.045 20 0.52 13.2 0.066 0.015 — 4. 7 168 1 0. 580 0. 163 0.039 ; 20 0.47 10.5 0.073 0.016 — 4. 6 168 0 0.764 0. 189 0.045 20 0.48 11.8 0.071 0.029 — 4. 9 137 0 0. 763 0. 210 0.040 25 0.68 4. 9 156 3 — 0.080 — 40 0.47 18.3 0.080 0.026 — 4. 9 168 0 0. 522 0.225 0.026 50 0.84 18.9 0.058 0.034 — 5. 3 168 0 0.438 0. 109 0.044 75 0.58 - 156 3 A p p e n d i x I : T a b u l a t e d R e s u l t s 1 9 7 T a b l e A I . 3 0 F E R M E N T A T I O N O F S S L W I T H P . S H E R M A N I I I N E R L E N M E Y E R F L A S K S 1 1 * ECW S C L P V P B p H T C E Y A YW | r 1 . 9 3 7 . 1 - . 0 2 2 0 . 0 5 5 0 . 0 3 8 4 . 6 1 5 6 2 0 . 4 2 1 0 . 0 1 7 0 . 2 7 4 1 r 1 . 3 0 7 . 1 - . 0 5 3 0 . 0 7 1 0 . 0 2 0 4 . 0 1 5 6 3 0 . 2 3 0 0 . 0 1 4 0 . 1 8 4 | 1 0 0 . 4 1 ' 6 . 3 0 . 0 2 5 0 . 0 1 9 — 4 . 3 1 2 0 1 0 . 6 2 9 0 . 1 0 7 0 . 0 6 5 I 1 0 0 . 3 2 7 . 8 0 . 0 2 8 0 . 0 2 5 — 4 . 8 1 2 0 0 0 . 6 1 2 0 . 1 6 8 0 . 0 4 0 | 2 0 0 . 2 1 — 0 . 0 1 2 0 . 0 2 2 — 6 . 7 1 2 0 1 — 0 . 1 6 2 — | 2 0 0 . 1 5 — • 0 . 0 1 3 x 0 . 0 3 2 — 6 . 4 1 2 0 0 — 0 . 3 0 4 — | 3 0 0 . 6 8 6 . 0 0 . 0 3 5 0 . 0 1 4 . — 6 . 3 1 2 0 0 0 . 7 3 6 0 . 0 7 3 0 . 1 1 3 | 4 0 0 . 7 7 9 . 6 0 . 0 3 6 0 . 0 1 9 — 6 . 3 1 2 0 0 0 . 5 1 6 0 . 0 7 2 0 . 0 8 0 | 5 0 0 . 2 4 1 0 . 8 0 . 0 4 5 0 . 0 2 4 — 6 . 1 1 2 0 0 0 . 5 7 6 0 . 2 9 4 0 . 0 2 2 | 1 0 0 0 . 6 2 — 0 . 0 6 6 0 . 0 3 4 — 6 . 3 1 2 0 0 — 0 . 1 6 1 — . • ~1 I H T a b l e A I . 3 1 F E B M E N T A T I O N O F S S L W I T H A M I X E D C U L T U R E O F 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 E R L E N M E Y E R F L A S K S r — : : -» Y DCW S C L P V P B p H T C E Y A YW r 0 . 5 3 7 . 1 0 . 0 0 3 0 . 0 3 0 —— 3 . 8 1 5 6 2 0 . 4 2 1 0 . 0 6 3 0 . 0 7 4 r 0 . 8 0 7 . 1 - . 0 0 1 0 . 0 5 8 0 . 0 2 0 3 . 9 1 5 6 3 0 . 7 2 8 0 . 0 7 2 0 . 1 1 3 0 0 . 0 0 . 0 0 . 0 0 . 0 — 4 . 7 1 5 6 2 1 0 0 . 9 8 9 . 6 0 . 0 9 1 0 . 0 2 7 — 3 . 8 1 2 0 1 1 . 1 0 0 0 . 1 2 0 0 . 1 0 2 1 0 0 . 1 4 — 0 . 0 0 9 0 . 0 1 7 — 6 . 5 1 2 0 0 — 0 . 1 8 6 — 2 0 0 . 5 2 — 0 . 0 4 6 0 . 0 1 6 — 5 . 9 1 2 0 1 — 0 . 1 1 9 — 2 0 1 . 4 7 1 6 . 5 0 . 1 3 1 0 . 0 1 7 — 3 . 7 1 2 0 0 0 . 8 0 8 0 . 1 0 0 0 . 0 8 9 2 5 1 . 3 2 1 8 . 1 0 . 1 3 3 0 . 0 0 9 — • 3 . 0 1 5 6 2 0 . 7 0 7 0 . 1 0 8 0 . 0 7 3 2 5 1 . 4 4 1 9 . 2 0 . 1 6 3 0 . 0 2 3 — 2 . 8 1 5 6 3 0 . 8 7 3 0 . 1 2 8 0 . 0 7 5 3 0 0 . 7 5 — 0 . 0 4 6 0 . 0 1 7 — 6 . 5 1 2 0 0 — 0 . 0 8 4 — 4 0 1 . 8 9 2 7 . 0 0 . 1 9 9 0 . 0 4 7 — 3 . 3 1 2 0 0 0 . 8 2 1 0 . 1 3 0 0 . 0 7 0 5 0 1 . 8 8 2 6 . 1 0 . 2 0 1 0 . 0 4 8 — 3 . 4 1 2 0 0 0 . 8 5 9 0 . 1 3 3 0 . 0 7 2 5 0 1 . 7 4 2 2 . 4 0 . 1 4 2 0 . 0 4 0 — 3 . 3 1 5 6 0 0 . 7 4 3 0 . 1 0 6 0 . 0 7 8 7 5 1 . 9 3 2 2 . 0 0 . 1 6 1 0 . 0 4 0 — 3 . 8 1 5 6 0 0 . 8 2 3 0 . 1 0 4 0 . 0 8 8 7 5 1 . 9 6 2 1 . 6 0 . 1 6 5 0 . 0 3 0 — • 3 . 4 1 5 6 0 0 . 8 1 3 0 . 1 0 0 0 . 0 9 1 1 0 0 2 . 0 5 2 2 . 2 0 . 1 1 5 0 . 1 0 9 — 4 . 1 1 5 6 0 0 . 9 0 9 0 . 1 0 9 0 . 0 9 2 1 a p p e n d i x I : T a b u l a t e d R e s u l t s 1 9 8 T a b l e A I . 3 2 R U N F P 1 . 1 . F E R M E N T A T I O N W I T H L . P L A N T A R I U M 1 1 1 T S D C H T U R B T A V L B p H A F R I 1 1 0 3 9 . 6 0 . 4 5 0 . 0 0 . 1 9 5 0 . 0 9 9 0 . 0 9 5 **** 7 . 0 0 I 2 8 2 5 . 5 0 . 4 4 0 . 0 1 1 0 . 1 4 6 0 . 0 4 6 0 . 1 0 0 **** 7 . 0 0 I 3 1 9 **** 0 . 3 6 0 . 0 1 5 0 . 1 5 1 0 . 1 0 1 0 . 0 5 0 **** 7 . 0 0 ! 4 2 6 2 4 . 0 0 . 3 7 0 . 0 0 9 ***** ***** ***** **** 7 . 0 0 | 5 3 2 2 3 . 1 0 . 4 1 0 . 0 ***** ***** ***** **** 7 . 0 0 I 6 4 3 1 9 . 8 0 . 3 8 0 . 0 2 2 0 . 1 6 1 0 . 1 0 7 0 . 0 5 4 **** 7 . 0 0 I 7 5 6 1 4 . 4 0 . 9 1 0 . 5 1 6 0 . 2 3 3 0 . 0 9 1 0 . 1 4 2 **** 7 . 0 0 t 8 5 9 9 . 6 1 . 2 2 0 . 6 0 2 0 . 2 3 0 0 . 0 6 6 0 . 1 6 4 **** 7 . 0 0 | 9 6 7 8 . 1 1 . 3 7 0 . 7 9 6 0 . 3 2 5 0 . 0 9 9 0 . 2 2 6 **** 7 . 0 0 | 1 0 7 2 7 . 5 1 . 5 2 0 . 7 7 0 ***** ***** ***** **** 7 . 0 0 1 1 1 7 9 4 . 0 1 . 5 0 0 . 8 3 9 0 . 3 0 0 0 . 0 3 6 0 . 2 6 4 **** 7 . 0 0 | 1 2 8 3 4 . 5 1 . 5 9 0 . 8 3 9 0 . 3 1 6 0 . 1 0 0 0 . 2 1 6 **** 7 . 0 0 | 1 3 9 5 4 . 8 1 . 3 9 0 . 8 2 4 0 . 3 5 3 0 . 1 0 7 0 . 2 4 6 **** 7 . 0 0 | 1 4 1 0 7 4 . 8 1 . 4 9 0 . 8 3 9 0 . 3 4 1 0 . 1 1 1 0 . 2 3 0 **** 7 . 0 0 | 1 5 1 1 0 **** 1 . 6 0 0 . 8 3 9 0 . 3 4 6 0 . 0 1 3 0 . 3 3 3 **** 7 . 0 0 •H t '• • T S C S R E G R L R B R T I I 2 8 1 4 . 1 - 1 . 7 6 2 0 . 0 3 - . 0 0 0 6 0 . 6 2 5 ***** 8 | 3 1 9 **** ***** **** - . 0 0 7 3 - 4 . 5 4 5 ***** 11 I 4 2 6 **** - 0 . 0 8 3 **** 0 . 0 0 0 7 ***** ***** 7 | 5 3 2 **** - 0 . 1 5 0 **** 0 . 0 0 6 7 ***** ***** 6 I 6 4 3 1 9 . 8 - 0 . 3 0 0 - 0 . 1 9 - . 0 0 2 3 0 . 1 6 7 ***** 1 1 1 7 5 6 2 5 . 2 - 0 . 4 1 5 0 . 1 7 0 . 0 4 0 4 6 . 7 6 9 ***** 1 3 1 8 5 9 3 0 . 0 - 1 . 6 0 0 0 . 2 1 0 . 1 0 3 3 7 . 3 3 3 ***** 3 t 9 6 7 3 1 . 5 - 0 . 1 8 8 0 . 3 7 0 . 0 1 8 8 7 . 7 5 0 ***** 8 | 1 0 7 2 **** - 0 . 1 2 0 **** 0 . 0 3 0 0 ***** ***** 5 1 1 1 7 9 3 5 . 6 - 0 . 5 0 0 0 . 4 3 - . 0 0 2 9 3 . 1 6 7 ***** 7 | 1 2 8 3 3 5 . 1 0 . 1 2 5 0 . 3 1 0 . 0 2 2 5 ***** ***** 4 J 1 3 9 5 3 4 . 8 0 . 0 2 5 0 . 3 9 - . 0 1 6 7 2 . 5 0 0 ***** 1 2 I 1 4 1 0 7 3 4 . 8 0 . 0 0 . 3 5 0 . 0 0 8 8 - 1 . 3 3 3 ***** 1 2 I 1 5 1 1 0 **** ***** **** 0 . 0 3 6 7 3 4 . 3 3 3 ***** 3 2 0 g / l o f Y e a s t E x t r a c t 2 . 2 p p m o f C o b a l t I o n s 4 g / l 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 A p p e n d i x I : T a b u l a t e d . R e s u l t s T a b l e A I . 3 3 R U N F P 1 . 2 . F E R M E N T A T I O N W I T H L . P L A N T A R I U M 1 9 9 T S DCW T U R B T f t V L B p H A F R 1 0 2 3 . 1 0 . 1 7 0 . 0 0 . 1 2 5 ***** 0 . 0 7 5 **** 7 . 1 0 2 3 0 **** 0 . 0 7 0 . 0 ***** ***** ***** **** 7 . 1 0 3 4 5 **** 0 . 9 2 0 . 4 5 6 ***** ***** ***** **** 4 . 6 0 4 4 8 **** 1 . 5 7 0 . 7 2 1 ***** ***** ***** **** 7 . 4 0 5 5 3 **** 1 . 2 5 0 . 6 0 2 0 . 1 9 0 0 . 0 5 0 0 . 1 4 0 **** 6 . 1 0 6 6 0 **** 1 . 5 5 0 . 8 8 6 0 . 2 5 8 ***** ***** **** 4 . 6 0 7 6 6 **** 1 . 5 6 0 . 8 2 4 0 . 2 4 5 0 . 0 7 5 0 . 1 7 5 **** 7 . 4 0 8 7 3 **** 1 . 5 3 0 . 8 3 9 ***** ***** ***** **** 7 . 3 0 9 1 0 7 8 . 5 1 . 4 7 0 . 8 3 9 0 . 2 5 8 0 . 0 8 4 0 . 1 7 4 **** 7 . 4 0 r I i T S C S R E G R L R B R T I I J r •-- 2 3 0 **** ****** **** - . 0 0 3 5 ***** ***** 3 0 3 4 5 **** ****** **** 0 . 0 5 6 9 ***** ***** 1 5 4 4 8 **** ****** **** 0 . 2 1 7 3 ***** ***** 3 5 5 3 **** ****** **** - . 0 6 5 0 1 . 2 2 6 ***** 5 6 6 0 **** ****** **** 0 . 0 4 3 6 ***** ***** 7 7 6 6 **** ****** **** 0 . 0 0 0 7 2 . 6 9 2 ***** 6 8 7 3 **** ****** **** - . 0 0 4 7 ***** ***** 7 9 1 0 7 1 4 . 5 - 0 . 1 3 6 0 . 6 1 - . 0 0 1 7 - 0 . 0 2 4 ***** 3 4 2 0 g / l o f Y e a s t E x t r a c t 2 . 2 p p m o f C o b a l t I o n s 4 g / l c f P o t a s s i u m D i h j d r c g e n P h o s p h a t e A p p e n d i x I : T a b u l a t e d R e s u l t s 2 0 0 T a b l e A I . 3 4 R O N F P 2 . 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 O N L A C T I C A C I D A S M A I N S U B S T R A T E I 1 T S DCW T U R B T A V L B P H A F R r 1 1 0 3 . 1 1 . 1 9 0 . 0 0 . 1 9 2 0 . 0 3 7 0 . 1 5 5 0 . 0 1 7 . 0 0 I 2 1 6 3 . 1 1 . 4 1 0 . 1 1 4 0 . 1 8 8 0 . 0 3 9 0 . 1 4 9 0 . 0 1 7 . 0 0 | 3 3 3 3 . 1 1 . 5 6 0 . 1 6 1 0 . 1 8 4 0 . 0 5 6 0 . 1 2 8 0 . 0 3 7 . 0 0 I 4 144 3 . 4 1 . 7 2 0 . 2 2 9 0 . 2 0 2 0 . 0 6 8 0 . 1 3 4 0 . 0 4 7 . 0 0 I 5 5 8 3 . 4 2 . 1 0 0 . 3 1 0 0 . 2 4 4 0 . 1 2 5 0 . 1 1 9 0 . 0 5 7 . 0 0 I 6 6 8 2 . 7 2 . 3 8 0 . 3 4 7 0 . 2 5 6 0 . 1 3 2 0 . 1 2 4 0 . 0 6 7 . 0 0 I 7 8 2 3 . 4 2 . 5 8 1 . 3 9 8 0 . 2 1 4 0 . 1 0 0 0 . 1 1 4 0 . 0 6 7 . 0 0 I 8 1 1 6 3 . 4 2 . 4 1 0 . 3 5 7 0 . 2 8 7 0 . 1 5 2 0 . 1 3 5 0 . 0 7 7 . 0 0 I 9 1 5 5 3 . 4 2 . 2 6 0 . 3 4 7 0 . 2 3 7 0 . 1 2 2 0 . 1 1 5 0 . 0 6 7 . 0 2 0 0 0 | 1 0 i 1 7 8 3 . 4 2 . 7 9 0 . 4 8 1 0 . 2 7 9 0 . 1 2 9 0 . 1 5 0 0 . C 7 7 . 0 2 0 0 0 i — — 1 I ,-• -T L C L C R E GR V R B R T I r2 1 6 . 0 0 6 - 0 . 3 7 5 0 . 3 3 0 . 0 1 4 1 0 . 1 2 5 0 . 2 5 0 1 6 3 3 3 . 0 2 7 - 1 . 2 3 5 0 . 7 0 0 . 0 0 8 8 1 . 0 0 0 0 . 8 8 2 1 7 4 4 4 . 0 2 1 0 . 5 4 5 1 . 4 8 0 . 0 1 4 1 1 . 0 9 1 1 . 2 7 3 1 1 5 5 8 . 0 3 6 - 1 . 0 7 1 2 . 4 4 0 . 0 2 7 1 4 . 0 7 1 1 . 0 7 1 1 4 6 6 8 . 0 3 1 0 . 5 0 0 3 . 0 6 0 . 0 2 8 5 0 . 7 0 0 0 . 2 0 0 1 0 7 8 2 . 0 4 1 - 0 . 7 1 4 1 . 5 4 0 . 0 1 3 9 - 2 . 2 8 6 0 . 1 4 3 1 4 8 1 1 6 . 0 2 0 0 . 6 1 8 5 . 7 5 - . 0 0 4 9 1 . 5 2 9 0 . 2 6 5 3 4 9 1 5 5 . 0 4 0 - 0 . 5 1 3 2 . 1 2 - . 0 0 4 0 - 0 . 7 6 9 -• 0 . 2 5 6 3 9 1 0 1 7 8 . 0 0 5 1 . 5 2 2 1 8 . 4 0 0 . 0 2 3 3 0 . 3 0 4 0 . 7 3 9 2 3 i I n o c u l u m : 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 m e d i u m : F i n a l b r o t h o f r u n F P 1 . 1 w i t h 2 0 g o f y e a s t e x t r a c t a d d e d p e r l i t e r A c i d s c o n c e n t r a t i o n s d e t e r m i n e d b y m a n u a l e t h e r e x t r a c t i o n A p p e n d i x I : T a b u l a t e d R e s u l t s 2 0 1 T a b l e A I . 3 5 R U N F P 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 O N L A C T I C A C I D A S M A I N S U B S T R A T E T S DCW T U R B T A V L E p H A F R 1 0 5 . 5 1 . 4 0 0 . 0 0 . 2 8 5 0 . 0 6 2 0 . 2 2 3 0 . 0 6 . 5 0 2 9 *** 1 . 4 0 0 . 0 2 2 ***** 0 . 1 0 0 0 . 1 8 5 **** 6 . 4 0 3 1 7 *** 1 . 4 1 0 . 0 0 4 ***** 0 . 0 7 9 0 . 2 0 6 **** 6 . 4 0 4 2 5 *** 1 . 4 8 0 . 0 6 6 0 . 2 8 4 0 . 0 8 6 0 . 1 9 8 **** 6 . 4 0 5 3 3 *** 1 . 6 7 0 . 0 8 6 ***** 0 . 1 0 4 0 . 1 8 0 **** 6 . 2 0 6 4 1 *** 1 . 9 0 0 . 1 6 1 ***** 0 . 1 2 9 0 . 1 5 5 0 . 0 3 6 . 2 0 7 4 9 4 . 6 2 . 1 7 0 . 2 0 8 0 . 3 0 0 0 . 1 6 1 0 . 1 3 9 0 . 0 5 6 . 1 0 8 5 8 *** 2 . 3 7 0 . 2 6 0 ***** 0 . 1 8 0 0 . 1 2 0 **** 6 . 1 0 9 6 5 *** 2 . 5 1 0 . 2 7 6 ***** 0 . 2 0 2 0 . 0 9 8 0 . 0 8 6 . 1 0 1 0 7 3 ***, 2 . 6 5 0 . 3 1 4 ***** 0 . 2 3 1 0 . 0 6 9 **** 6 . 1 0 1 1 8 2 *** 2 . 7 0 0 . 3 1 0 ***** 0 . 2 2 3 0 . 0 7 7 **** 6 . 1 0 1 2 9 7 *** 2 . 6 9 0 . 3 1 0 ***** 0 . 2 2 2 0 . 0 7 8 **** 6 . 1 0 1 3 1 3 7 5 . 5 2 . 5 3 0 . 3 1 0 ***** 0 . 2 3 2 0 . 0 6 8 0 . 0 8 6 . 1 2 0 0 0 1 4 1 7 4 5 . 1 2 . 8 2 0 . 3 1 0 ***** 0 . 2 1 7 0 . 0 8 3 **** 6 . 1 2 0 0 0 1 5 1 9 7 4 . 8 2 . 9 8 0 . 3 1 0 0 . 2 9 0 0 . 2 2 3 0 . 0 6 7 0 . 2 2 6 . 1 2 0 0 0 I T L C L C R E GR V R ' B R T I 2 9 . . 0 3 8 - 4 . 2 2 2 1 . 0 0 0 . 0 4 . 2 2 2 ***** 9 3 1 7 . 0 1 7 2 . 6 2 5 1 . 0 0 0 . 0 0 1 0 - 2 . 6 2 5 ***** 8 4 2 5 . 0 2 5 - 1 . 0 0 0 0 . 9 6 0 . 0 0 9 5 0 . 8 7 5 ***** 8 5 3 3 . 0 4 3 - 2 . 2 5 0 0 . 9 8 0 . 0 2 3 0 2 . 2 5 0 ***** 8 6 4 1 . 0 6 8 - 3 . 1 2 5 0 . 9 9 0 . 0 2 9 6 3 . 1 2 5 0 . 8 5 4 8 7 4 9 . 0 8 4 - 2 . 0 0 0 1 . 1 8 0 . 0 3 3 1 4 . 0 0 0 1 . 6 2 5 8 8 5 8 . 1 0 3 - 2 . 1 1 1 1 . 1 5 0 . 0 2 2 2 2 . 1 1 1 ***** 9 9 6 5 . 1 2 5 - 3 . 1 4 3 1 . 1 2 0 . 0 2 0 0 3 . 1 4 3 2 . 1 8 7 7 1 0 7 3 . 1 5 4 - 3 . 6 2 5 1 . 1 0 0 . 0 1 7 5 3 . 6 2 5 ***** 8 1 1 8 2 . 1 4 6 0 . 8 8 9 1 . 1 0 0 . 0 0 6 0 - 0 . 8 8 9 ***** 9 1 2 9 7 . 1 4 5 0 . 0 6 7 1 . 1 0 - . 0 0 1 1 - 0 . 0 6 7 ***** 1 5 1 3 1 3 7 . 1 5 5 - 0 . 2 5 0 1 . 1 0 - . 0 0 4 1 0 . 2 5 0 0 . 0 4 0 1 4 1 7 4 . 1 4 0 0 . 4 0 5 1 . 1 1 0 . 0 0 8 1 - 0 . 4 0 5 ***** 3 7 1 5 1 9 7 . 1 5 6 - 0 . 6 9 6 1 . 0 3 0 . 0 0 6 7 0 . 2 6 1 2 . 2 5 0 2 3 I n o c u l u m : 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 m e d i u m : F i n a l b r o t h c f r u n F P 1 . 1 w i t h 2 0 g o f y e a s t e x t r a c t a d d e d p e r l i t e r appendix I : T a b u l a t e d R e s u l t s 202 T a b l e ai.36 RUN FP3. FERMENTATION WITH MIXED CULTURE OF L. PLANTARIUM AND P. FREUDENREICHII 1 1 • T S DCW TURB Ta V L B pH AFR I — -1 1 0 33.6 0. 13 0.0 0.113 0.056 0.057 0.0 6.7 0 I 2 12 33. 3 0.22 0. 013 0.126 0.066 0.060 **** 6.7 0 I 3 24 32.7 0.34 0.048 0. 126 0.079 0.047 **** 6.7 0 I * 31 29.4 0.65 0. 149 0.157 0.100 0.057 **** 6.7 0 I 5 39 25.8 1.11 0.569 0.204 0. 134 0.070 0.02 6.7 0 I 6 46 16.5 2.44 1.097 0.258 0.124 0.134 0.04 6.7 0 | 7 51 12.7 2.81 1. 187 0. 324 0. 137 0. 187 0.08 6.7 0 I 8 57 12.6 2.99 1.301 0.304 0. 165 0.139 **** 6.7 0 I 9 63 12.9 3.27 1. 398 0.310 0. 181 0. 129 **** 6.7 0 |10 69 12.7 3.43 1.398 0.296 0.207 0.089 **** 6.7 0 |11 75 13.1 3.57 1. 398 0. 310 0.243 0.067 0.30 6.7 0 |12 87 11.6 4.23 1.222 0.313 0.250 0.063 0.38 6.7 2000 113 99 11.8 4.31 1.523 0. 323 0.247 0.076 0.45 6.7 2000 | 14 114 10.6 4.55 1.602 0.320 0.263 0.057 0.38 6.7 2000 I 15 129 9.6 4.73 1.523 0. 308 0.250 0.058 **** 6.7 2000 116 142 8.7 4.47 1.523 0.285 0.228 0.057 0.50 6.7 2000 117 176 8.5 5.36 1.523 0.263 0.218 0.044 0.45 6.7 2000 I T SC SR E GR VR BR TI 2 12 0.3 -0.025 3.00 0.0069 0.833 ***** 12 3 24 0.9 -0.050 2.30 0.0103 1.083 ***** 12 4 31 4.2 -0.471 0.94 0.0450 3.000 ***** 7 5 39 7.8 -0.450 0.90 0.0575 4.250 0.590 8 6 46 17. 1 -1.329 0.36 0. 1899 -1.429 2.429 7 7 51 20.9 -0.760 0.35 0.0734 2.600 8.000 5 8 57 21.0 -0.017 0.47 0.0300 4.667 ***** 6 9 63 20.7 0.050 0.54 0.0458 2.667 ***** 6 10 69 20.9 -0.033 0.65 0.0273 4. 333 ***** 6 11 75 20.5 0.072 0.82 0.0232 6.000 9. 167 6 12 87 22.0 -0.127 0.79 0.0551 0.583 6.250 12 13 99 21.7 0.021 0.79 0.0071 -0.250 6.250 12 14 114 23.0 -0.085 0.81 0.0157 1.067 -5.000 15 15 129 24.0 -0.065 0.73 0.0118 -0.867 ***** 15 16 142 24.9 -0.069 0.62 -.0200 -1.692 4.464 13 17 176 25.0 -0.004 0.58 0.0263 -0.294 -1.471 34 2 0 g / l 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 Potas s i u m Dihydrogen Phosphate A p p e n d i x I : T a b u l a t e d R e s u l t s 2 0 3 T a b l e A I . 3 7 R U M F 1 5 . 2 2 a . 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 . E F F E C T O F p H | T DCW T U R B T A M V A p H C C D ] j _ _ . . 4 B0 _ — 0 . 0 0 . 0 6 2 0 . 1 0 8 6 . 8 2 9 0 1 7 0 . 6 0 0 . 3 1 0 . 0 6 2 0 . 1 4 7 6 . 4 2 2 0 3 3 1 . 6 0 0 . 9 6 0 . 0 8 0 0 . 2 6 7 5 . 5 2 1 6 4 4 2 . 0 0 1 . 1 5 0 . 0 9 0 0 . 1 4 0 5 . 2 2 1 6 5 8 2 . 3 0 1 . 3 0 0 . 1 0 4 0 . 1 7 7 5 . 0 2 6 5 6 8 2 . 5 5 1 . 4 0 0 . 1 1 2 0 . 1 9 3 4 . 9 2 3 8 8 4 2 . 5 6 1 . 4 0 0 . 1 1 6 0 . 2 0 0 4 . 8 2 2 5 9 3 2 . 5 3 1 . 4 0 0 . 1 1 6 — 4 . 8 1 9 3 1 0 7 2 . 4 1 1 . 3 0 0 . 1 3 0 0 . 2 1 0 4 . 8 2 1 0 1 0 9 2 . 2 9 1 . 3 0 — 0 . 1 7 3 6 . 7 -1 2 3 2 . 4 9 1 . 3 5 0 . 1 4 0 0 . 2 4 0 6 . 1 1 9 2 1 3 3 3 . 0 0 1 . 4 0 0 . 1 5 2 — 6 . 0 1 8 9 1 4 8 3 . 2 4 1 . 4 0 0 . 1 5 2 — 5 . 6 1 7 9 1 5 5 3 . 4 2 1 . 5 2 0 . 1 6 0 — 5 . 6 1 8 3 1 6 8 3 . 4 9 1 . 7 0 0 . 1 6 8 — 5 . 5 1 9 1 1 8 1 3 . 5 1 1 . 6 0 0 . 1 7 2 — 5 . 3 1 8 3 1 9 7 3 . 6 8 1 . 7 0 — — 5 . 2 -2 2 1 3 . 6 3 1 . 5 2 — — 5 . 1 1 9 6 2 4 1 3 . 5 1 1 . 5 2 0 . 1 8 0 -- 5 . 1 2 4 0 2 5 1 3 . 2 9 1 . 5 2 0 . 1 7 0 — 6 . 6 2 1 0 2 6 8 3 . 1 8 1 . 5 2 0 i 1 7 0 — 6 . 6 1 5 7 2 9 8 3 . 0 0 1 . 5 2 • — 6 . 6 1 3 8 3 8 6 6 . 0 0 1 . 7 0 0 . 1 8 6 — 6 . 4 -5 0 5 6 . 5 0 1 . 7 0 0 . 1 9 0 — 6 . 9 1 8 0 2 0 g / l ( 1 5 g / l d r y b a s i s ) o f Y e a s t E x t r a c t P a s t e 2 . 2 p p m o f C o b a l t I o n s 4 g / l 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 T A M : T o t a l A c i d C o n c e n t r a t i o n b y M a n u a l E t h e r E x t r a c t i o n ( m o l e s / 1 ) V A : 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 b y S t e a m D i s t i l l a t i o n ( m o l e s / 1 ) C O D : C h e m i c a l O x y g e n D e m a n d ( i n l O O O p p m ) , a f t e r E t h e r E x t r a c t i o n A i r i s b l o w n i n a t 4 0 0 0 c c / m i n a f t e r t h e f i r s t 3 0 0 h o u r s A p p e n d i x I : T a b u l a t e d R e s u l t s 2 0 4 T a b l e A I . 3 8 R O N F l 5 . 2 2 b . 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 I N O N E A N A E R O B I C S T A G E T DCW T U R B S V A 0 — 0 . 0 3 3 . 0 — 2 2 — 0 . 0 2 — 3 3 — 0 . 0 4 — 7 0 — 0 . 0 8 — • 9 8 — 0 . 1 3 — — 1 0 6 0 . 6 8 0 . 2 7 3 8 . 0 — • 1 1 5 1 . 0 0 0 . 4 0 3 6 . 0 0 . 0 8 9 1 3 1 1 . 2 1 0 . 5 7 2 2 . 0 0 . 0 8 8 1 4 6 1 . 8 2 0 . 8 5 2 4 . 8 0 . 1 1 8 1 6 5 2 . 6 1 1 . 1 9 3 3 . 0 0 . 1 8 0 1 7 7 3 . 4 4 1 . 4 0 2 4 . 8 0 . 2 7 8 1 9 0 3 . 4 5 1 . 5 4 2 3 . 2 0 . 2 5 1 3 6 3 3 . 1 0 1 . 1 0 2 1 . 0 0 . 2 4 2 4 7 0 1 . 7 0 — 0 . 3 0 3 4 9 5 — • 1 . 7 0 — 0 . 3 3 3 0 . 0 2 4 0 . 1 4 0 0 . 7 5 0 0 . 9 5 0 1 p H C O D | 7 . 0 -7 . 1 7 . 1 2 0 8 6 . 8 2 0 2 6 . 4 1 9 8 5 . 9 -5 . 5 1 9 9 5 . 3 1 9 4 5 . 1 1 6 3 5 . 0 1 7 1 5 . 0 1 5 2 2 0 g / l ( 1 5 g / l d r y b a s i s ) o f Y e a s t E x t r a c t P a s t e 2 . 2 p p m o f C o b a l t I o n s 4 g / l 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 B : C o n c e n t r a t i o n o f V i t a m i n B 1 2 b y A s s a y A t 3 0 ° C V A : 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 b y S t e a m D i s t i l l a t i o n ( m o l e s / 1 ) C O D : C h e m i c a l O x y g e n D e m a n d ( i n 1 0 0 0 p p m ) , a f t e r E t h e r E x t r a c t i o n * Appendix I : Tabulated R e s u l t s 205 Table AI.39 RON F05.22. FERMENTATION WITH P. FREUDENREICHII. YEAST EXTRACT CONCENTRATION as VARIABLE 1 L T S DCW TURB TA V L B pH AFR | r | 1 0 37.2 0.64 0.0 0.064 0. 016 0.048 0.0 7.9 _j 0 | I 2 35 34.2 1. 10 0. 260 0.066 0. 016 0.050 **** 6.8 0 I | 3 62 32.4 2.31 0.824 0.075 0. 033 0.042 0. 12 6.8 0 I I 4 88 27.6 4.90 1.301 0.134 0. 087 0.047 0.50 6.6 1000 | I 5 115 18.6 6.73 1.420 0. 192 0. 126 0.066 1.05 6.7 1000 | I 6 136 14.4 7.88 1.495 0.209 0. 157 0.052 1.50 6.7 1000 | I 7 163 12.7 7.60 1. 523 0.230 0. 149 0.081 1.40 6.8 1000 | I 8 180 12.0 7.42 1.523 0.223 0. 161 0.062 **** 6.7 1000 | I 9 • 202 12.3 7.48 1.456 0.210 0. 158 0.052 **** 6.6 1C00 | « r I T SC SR E GR VR BR TI | 2 3 4 5 6 7 8 9 35 62 88 115 136 163 180 202 3.0 4.8 9.6 18.6 22.8 24.4 25.2 24.9 -0.086 -0.067 -0.185 -0.333 -0.200 -0.061 -0.044 0.014 0.0 0. 32 0.67 0.53 0.56 0.49 0.52 0.51 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 L . J Appendix I : T a b u l a t e d R e s u l t s 2 0 6 T a b l e AI.40 RUN F10.22. FERMENTATION WITH P. FREUDENREICHII. YEAST EXTRACT CONCENTRATION AS VARIABLE T S DCW TURB TA V L B PH AFR 1 0 25.5 0.54 0.0 0.045 0. 007 0.038 0.0 7.7 0 2 14 29.4 0.74 0. 125 0.051 0. 012 0.039 **** 7.4 0 3 23 28.8 0.94 0. 319 0.060 0. 020 0.040 0.03 7.2 0 4 37 26.7 1.81 0.824 0.070 0. 034 0.036 **** 7.3 0 5 47 24.3 2.38 1.097 0.082 0. 045 0.037 0. 12 7.2 0 6 59 21.6 3.22 1. 155 0.098 0. 060 0.038 **** 7.2 0 7 71 17.7 4. 10 1.301 0. 139 0. 083 0.056 0.23 7.3 500 8 83 14.4 5.26 1.398 0.162 0. 114 0.048 0,80 7.1 500 9 95 12.3 6.59 1.523 0. 181 0. 132 0.049 1.30 7.2 500 10 108 10.2 7.07 1.523 0.203 0. 147 0.056 **** 7.3 500 11 120 8.2 7. 12 1.523 0. 204 0. 132 0.071 1.35 7.4 500 _J T SC SR E GR VR BR TI | 2 14 -3.9 0.279 -0. 12 0.0143 0.357 ***** 14 | 3 23 -3.3 -0.067 -0.35 0.0228 0.889 1.304 9 I 4 37 -1.2 -0.150 -2.03 0.0618 1.000 ***** 14 | 5 47 1.2 -0.240 2.85 0.0570 1. 100 3.750 10 | 6 59 3.9 -0.225 1.22 0.0700 1.250 ***** 12 | 7 71 7.8 -0.325 0.88 0.0733 1.917 4.583 12 | 8 83 11.1 -0.275 0.87 0.0971 2.583 47.500 12 | 9 95 13.2 -0.175 0.85 0. 1104 1.500 41.667 12 | 10 108 15.3 - 0 . 162 0.82 0.0373 1.154 ***** 13 | 11 120 17.3 -0. 162 0.65 0.0038 -1.250 2.000 12 | Appendix I : Tabulated R e s u l t s 207 Table AI.41 RUN F20.22. FERMENTATION WITH P. FREUDENREICHII. YEAST EXTRACT CONCENTRATION AS VARIABLE T S DCW TURB TA V L B pH AFR 1 0 37.2 0. 14 0.0 0.076 0. 047 0.029 0.0 7.0 0 2 9 37.2 0.28 0. 034 ***** ' ***** ***** **** 7.0 0 3 17 36.0 0.41 0. 146 0.075 0. 046 ***** 0.02 7.0 0 4 25 34.5 0.71 0.284 0.083 0. 054 ***** **** 7.0 0 5 33 32.7 1. 14 0.495 0. 101 0. 072 ***** 0. 10 7.0 0 6 41 28.8 2.71 1.071 0.093 0. 064 ***** 0.21 7.0 0 7 49 24.0 4.52 1. 398 0. 140 0. 111 ***** **** 7.0 0 8 55 22.5 5.93 1.456 0. 169 0. 135 0.034 0.45 7.0 0 9 60 20. 1 6.33 1.699 0. 175 0. 141 ***** **** 7.0 0 10 65 13.2 7.25 1.699 0.189 0. 155 ***** 0.55 7.0 0 11 73 12.0 7.52 1.699 0. 191 0. 136 0.055 **** 7.0 0 12 82 11.6 7.57 1.824 0.199 0. 149 0.050 0.80 7.0 0 13 97 11.5 7.60 1. 824 0.225 0. 175 ***** 0. 90 7.0 0 14 137 10.6 7.75 1.824 0.233 0. 183 ***** 0.80 7.0 2000 15 174 10.3 8.41 1.824 0. 234 0. 186 0.048 0. 97 7.0 2C00 16 197 10.2 8.67 1.824 0.228 0. 180 ***** 0.95 7.0 2000 TI ] SC SR GR VR BR 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 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 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 ***** -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 ***** 1.000 ***** 5. 187 13.750 ***** 17.143 ***** 10.000 ***** 14.706 6.667 -2.500 4.730 -1.087 9 8 8 8 8 8 6 5 5 8 9 15 40 37 23 Appendix I : Tabulated R e s u l t s 208 Table AI.42 RUN F30.22. FERMENTATION WITH P. FREUDENREICHII. YEAST EXTRACT CONCENTRATION AS VARIABLE |~ T S DCW TURB TA V L B pH AFR ] | 1 0 29.4 0.42 0.0 • 0.081 0.026 0.055 0.0 6.9 0 \ | 2 21 28.2 1.36 0.770 0.108 0.044 0.064 0.11 6.2 0 \ f 3 33 23.1 2.90 1.301 0.122 0.058 0.064 0.22 6.8 0 | | 4 48 16.8 5.32 1.523 0.162 0.087 0.075 0.38 6.8 1000 j | 5 63 12.4 7.72 1.699 0.200 0.136 0.064 1.00 7.0 1000 | | 6 75 9.2 8.10 1.699 0.211 0.149 0.062 **** 7.0 1000 \ | 7 88 8.9 8.41 1.699 0.224 0.153 0.071 1.00 7.2 1000 | | 8 99 8.7 8.49 1.699 0.242 0.144 0.098 1.00 7.0 1000 \ | 9 128 8.4 8.34 1.699 0.236 0.158 0.078 **** 7.1 1000 | r :—: 1 | T SC SR E GR VR BR TI | | 2 21 1.2 -0.057 1.35 0.0449 0.857 5.333 21 1 | 3 33 6.3 -0.425 0.46 0.1281 1.167 9.333 12 | | 4 48 12.6 -0.420 0.44 0.1613 1.933 10.067 15 | | 5 63 17.0 -0.293 0.58 0.1600 3.267 41.667 15 | | 6 75 20.2 -0.267 0.55 0.0317 1.083 ***** 12 | | 7 88 20.5 -0.023 0.56 0.0238 0.308 0.0 13 | | 8 99 20.7 -0.018 0.51 0.0073 -0.818 0.0 11 | | 9 128 21.0 -0.010 0.57 -.0052 0.483 ***** 29 | appendix I : Tabulated R e s u l t s 209 Table AI.43 RUN F20.00, FERMENTATION WITH P. FREUDENREICHII. COBaLT ION CONCENTRaTION AS VARIABLE r ™ T S DCW TORB TA V L B pH AFR I . j | 1 0 37.5 0.27 0.0 0.059 0. 012 0. 047 0.0 6.8 0 I 2 21 34.8 1. 12 0. 509 0.096 0. 025 0. 071 . 0.06 6.9 0 | I 3 46 27.6 4.21 1.301 0. 128 0. 071 0. 057 0.27 5.5 0 t * 66 17.7 7.33 1.523 0.184 0. 138 0.046 0.30 6. 1 0 | | 5 82 12.9 7.69 1.523 0.207 0. 152 0. 055 **•* 6.7 1000 | I 6 110 11.4 8.21 1.602 0.195 0. 159 0. 036 0.40 6.9 1000 | I 7 157 9.9 8.05 1.553 0.232 0. 168 0. 064 **** 6.7 1000 i T SC SR E GR I VR BR TI "t 1 j J 2 21 2.7 -0.129 0.43 0.0406 0 .619 2.857 21 i | 3 46 9.9 -0.288 0.54 0.1236 1 .840 8.320 25 | 4 66 19.8 -0.495 0.57 0.1560 3 .350 1.600 20 j 5 82 24.6 -0.300 0.51 0.0225 0 .875 ***** 16 | 6 110 26. 1 -0.054 0.51 0.0186 0 .250 2.273 28 7 157 27.6 -0.032 0.51 -.0034 0 . 191 ***** 47 Appendix I : Tabulated R e s u l t s 210 Table AI.44 RUN F20.09. FERMENTATION WITH P. FREUDENREICHII. COBALT ION CONCENTRATION AS VARIABLE 1 T S DCW T U R B T A V L B pH AFR | . . . j 1 1 0 34.8 0.43 0.0 0.050 0. 012 0.038 0.0 6.9 i 0 | I 2 28 38.4 1. 24 0. 585 0.089 0. 030 0.059 0.07 6.8 0 1 I 3 49 29. 1 3.92 1. 301 0. 136 0. 065 0.071 0.36 6.7 0 1 t 57 30.0 5.06 1.398 0.146 0. 095 0.051 0.34 6.7 1000 | I 5 72 20.7 7. 14 1.523 0. 196 0. 129 0.067 0.52 6.8 1000 | I 6 88 19.8 8.61 1.699 0.216 0. 156 0.059 1.17 6.9 1000 | | 7 102 12.3 9.28 1.699 0. 266 0. 171 0.095 **** 7.0 1000 | I 8 114 12.3 8.80 1.699 0.252 0. 184 0.068 1.30 6.9 1000 | I 9 126 11.7 8.90 1.699 0.226 0. 163 0.063 **** 7.0 1000 | |10 138 11.7 8.63 1.699 0.241 0. 165 0.076 **** 6.9 1000 | _ , J 1 T S C SR E GR VR BR T I ] 2 28 -3.6 0.129 -0.45 0.0289 0.643 2.429 28 3 49 5.7 -0.443 0.84 0.1276 1.667 13.905 21 4 57 4.8 0. 113 1.56 0. 1425 3.750 -1.875 8 5 72 14.1 -0.620 0.75 0.1387 2.267 12.000 15 6 88 15.0 -0.056 0.86 0.0919 1.688 40.625 16 7 102 22.5 -0.536 0.64 0.0479 1.071 ***** 14 8 114 22.5 0.0 0.69 -.0400 1.083 4.808 12 9 126 23.1 -0.050 0.59 0.0083 -1.750 ***** 12 10 138 23.1 0.0 0.60 -.0225 0.167 ***** 12 Appendix I : Tabulated R e s u l t s 211 Table AI.I45 RUN F20.22.126,FERMENTATION OF SSI CONTAINING DMEZ WITH P. FREUDENREICHII T S DCW TURB TA V L B pH AFR 1 0 27.9 0.65 0.0 0.083 0.014 0.069 0.0 7. 1 0 2 24 26.7 0.94 0. 284 0.075 0.031 0.044 **** 6.7 0 3 48 21.9 2.34 0.959 0.097 0.048 0.049 0.29 6.8 0 4 67 19.5 3.97 1.301 0.150 0.085 0.076 0.38 6.9 2000 5 80 15.3 5.66 1. 398 0. 156 0. 110 0.046 0.72 6.9 2G00 6 96 11. 1 7.30 1.523 0. 199 0.148 0.051 0.88 6.8 2000 7 117 10.8 7.67 1.523 0.226 0. 150 0.076 0.80 7.0 2G00 8 142 9.9 7.52 1.523 0.223 0. 146 0.077 1.00 6.8 2000 T SC SR E GR VR BR TI | 2 24 1.2 -0.050 1.28 0.0119 0.708 ***** 24~* 3 48 6.0 -0.200 0.51 0.0587 0.708 6.042 24 4 67 8.4 -0.126 0.76 0.0854 1.947 4.737 19 5 80 12.6 -0.323 0.69 0.1302 1.923 26.154 13 6 96 16.8 -0.262 0.72 0.1025 2.375 1C.000 16 7 117 17.1 -0.014 0.72 0.0176 0.095 -3.810 21 8 142 18.0 -0.036 0.66 -.0060 -0.160 8.000 25 Appendix I : T a b u l a t e d R e s u l t s 212 T a b l e AI.46 BUN S20.22. FEBRENTATION WITH P. SHEBEANII T S DCW TUBB TA V I E pH AFB 1 0 38.4 0.38 0.0 0.060 0. 013 0.047 0.0 6.8 0 2 17 37.8 0.53 0.208 0.063 0. 019 0.044 **** 6.4 0 3 58 36.0 2.28 1. 155 0.102 0. 040 0.062 0. 15 6.9 0 4 82 30.0 3.66 1. 301 0. 116 0. 070 0.046 0.25 6.8 1000 5 97 28.5 4.51 1.523 0.131 0. 083 0.048 0.42 6.8 1000 6 120 21.0 6. 18 1.699 0. 176 0. 093 0.083 **** 6.8 1000 7 143 17.7 7.01 1.745 0. 186 0. 125 0.061 0.57 5.6 1000 8 169 14.8 8.09 1.824 0. 213 0. 145 0.068 **** 6.7 1000 9 193 11.2 8.58 1.921 0.221 0. 143 0.078 0.82 6.9 1000 10 221 11.2 8.67 1.824 0.208 0. 150 0.058 **** 6.7 1000 T SC SB E GB V H BB TI | j 2 17 0.6 -0.035 0.90 0.0092 0.353 ***** 1 17 | 3 58 2.4 -0.044 1.01 0.0426 0.512 2.586 41 | 4 82 8.4 -0.250 0.61 0.0575 1.250 4.375 24 | 5 97 9.9 -0.100 0.64 0.0567 0.867 11.3 33 15 | 6 120 . 17.4 -0.326 0.41 0.0728 0.435 ***** 23 | 7 143 20.7 -0.143 0.49 0.0359 1.391 3.261 23 | 8 169 23.5 -0.110 0.50 0.0415 0.769 ***** 26 | 9 193 27. 1 -0.150 0.43 0.0204 -0.083 5.000 24 | 10 221 27. 1 0.0 0.45 0.0032 0.250 ***** 28 | Appendix I I : A n a l y s i s Procedures APPENDIX I I A n a l y s i s Procedures 213 II.1 SAMPLING Care was taken that samples were g r e a t e r than 60ml. C e l l s were then separated from the f e r m e n t a t i o n broth and washed twice with d i s t i l l e d water. The s e p a r a t i o n of c e l l s from the broth and from the washing water was done by a 30 minute c e n t r i f u g a t i o n at 3000rpm. The washing was done by thorough mixing with an e l e c t r i c shaker mixer. Three a l i g u o t s of 20ml each, taken with p i p e t s , of every sample, w e l l mixed, were s u b j e c t e d to t h i s c e l l removal p r o c e s s . C e l l s from two of these three a l i g u o t s were d e s t i n e d f o r the measurement of growth. Those'from the t h i r d one were f o r the measurement of v i t a m i n B12 c o n c e n t r a t i o n . The c e l l - f r e e broth was used f o r the determination of a c i d s and reducing sugar c o n c e n t r a t i o n s . For f e r m e n t a t i o n s with Streptomyces and L a c t o b a c i l l u s , only two a l i g u o t s were needed, as no measurement of v i t a m i n B12 c o n c e n t r a t i o n was to be performed. Streptomyces, on the other hand, grew i n lumps, and cou l d not be p i p e t t e d nor e a s i l y removed from w a l l s of glassware, so c e l l s of the t o t a l broth were weighed and the dry c e l l weight was based on the o r i g i n a l volume o f br c t h i n s t e a d of on the f i n a l one. appendix I I : A n a l y s i s Procedures 214 II.2 MEASUREMENT OF BACTERIA! GROWTH 11.2.1 Standard T u r b i d i t y Method The washed c e l l s are resuspended i n ^ d i s t i l l e d water, the volume i s made up to the o r i g i n a l volume of the b r o t h . The t r a n s m i t t a n c y of t h i s s o l u t i o n i s then measured, with water as blank, with a spectrophotometer (BauschEIomb S p e c t r o n i c 20) at the wavelength of 540 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 . 11.2.2 M o d i f i e d _ T u r b i d i t j _ M e t h o d A sample of the f e r m e n t a t i o n broth, p r i o r to i n n o c u l a t i o n , i s kept r e f r i g e r a t e d . T h i s sample serves as blank f o r subseguent t r a n s m i t t a n c y measurements. The t r a n s m i t t a n c y cf w e l l mixed f r e s h l y taken samples i s then measured at a wavelength of 915 m i l i m i c r o n s . The t u r b i d i t y i s then c a l c u l a t e d with the e g u a t i o n : T u r b i d i t y = 2 - log10 (percent of transmittance) T u r b i d i m e t r y i s net s u i t a b l e f o r measuring growth i n lumps of Streptomyces. 11.2.3 C r j _ C e l l _ W e i g h t Aluminum dishes (Figure A2.73) are preweighed a f t e r being heated f o r a t l e a s t two hours at 110°C i n a F i s h e r Isotemp oven, then c o o l e d down to room temperature i n a d e s s i c a t o r . Washed c e l l s are then t r a n s f e r r e d to these aluminum dishes from the 50ml c e n t r i f u g e tubes. They are then d r i e d o v e r n i g h t at 1100C i n the same oven. The aluminum d i s h e s are then weighed a g a i n . Dry c e l l weight i s then c a l c u l a t e d by d i f f e r e n c e . T h i s seguence of d r y i n g and weighing i s always done two times s u c c e s s i v e l y . The Appendix II : A n a l y s i s Procedures 215 FIGURE A2.73 : Drying of Aluminum Dishes i n The Isctemp Oven (scale : f -J » 13.2 cm) i i average values are used. Weighing was dene cn a p r e c i s i o n balance accurate to the tenth of a m i l l i g r a m . II.3 MEASUREMENT_CF_SUGAR_C This i s the method described by Neish [67,68,87], I t i s adopted with s l i g h t m o d i f i c a t i o n s . Tc each t e s t tube, of 25x200ml s i z e , 1ml of a sample, c o n t a i n i n g up tc 0.2q/l of A p p e n d i x I I : A n a l y s i s P r o c e d u r e s 216 FIGURE A2.74 : T y p i c a l S t a n d a r d C u r v e f o r The R e d u c i n g S u g a r T e s t 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(±0.039) = 0.007 (±0.0003)x + 0.033 (±0.034) — L 1 1 1 I I 0.0 20.0 60.0 100.0 140.0 380.0 GLUCOSE CONCENTRATION (G/L) appendix I I : a n a l y s i s P r o c e d u r e s 217 r e d u c i n g sugar ( p r e f e r a b l y o v er 0.02g/l) i s t r a n f e r r e d and 2ml of the Somogyi copper r e a g e n t i s added. The tubes are then c l o s e d w i t h caps and mixed t h o r o u g h l y then heated i n b o i l i n g water f o r about 10 m i n u t e s . These tubes are then c o o l e d i n a c o l d water b a t h . Two ml of arsenomolybdate r e a g e n t i s mixed i n . The a d d i t i o n o f 20ml of d i s t i l l e d water, t h e n , completes the p r e p a r a t i o n . The t r a n s m i t t a n c y o f t h e s o l u t i o n s , thus o b t a i n e d , i s then measured a t a wavelength of 660 m i l i m i c r o n s , w i t h water as b l a n k . The 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 samples and 4 s o l u t i o n s of g l u c o s e of known c o n c e n t r a t i o n (0.2, 0.1, 0.05, 0 . 0 2 g / l ) . The r e a c t i o n i s not complete and the c o n v e r s i o n i s e m p i r i c a l . T h e r e f o r e , the known s o l u t i o n s are needed f o r the s t a n d a r d c u r v e , a t y p i c a l one p r e s e n t e d on F i g u r e a2.74, r e g u i r e d f o r i n t e r p r e t a t i o n o f data by i n t e r p o l a t i o n . These s o l u t i o n s a r e f r e s h l y made up f o r t e s t s on each s e r i e s o f samples from a s t a n d a r d s o l u t i o n of 1 g / l of g l u c o s e kept r e f r i g e r a t e d . T e s t s were always done i n d u p l i c a t e . The r e d u c i n g s u g a r c o n c e n t r a t i o n was measured as g l u c o s e . I I . 4 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 assay [ 3 8 , 1 0 1 ] . I I . 4 . 1 Inoculum Stock c u l t u r e o f L a c t o b a c i l l u s l e i c h m a n i i (aTCC-7830) i s 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 B12 c u l t u r e a g a r . T h i s t r a n s f e r 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 at 370C f o r a t l e a s t 12 hours. Only c u l t u r e s of l e s s than 4 appendix I I : a n a l y s i s Procedures 218 ' days e l d are used f o r p r e p a r a t i o n of inoculum. Stock c u l t u r e i s t r a n s f e r r e d twice d a i l y f o r a t l e a s t 5 s u c c e s s i v e t r a n s f e r s before i t i s used f c r the p r e p a r a t i o n of an inoculum. C e l l s are t r a n s f e r r e d , with a st a b , from such a stock c u l t u r e to 10ml of inoculum b r o t h . T h i s broth i s then incubated at 37°C f o r a t l e a s t 16 hours. The c e l l s are a s e p t i c a l l y c e n t r i f u g e d , separated from the broth and then resuspended i n 10ml.of s t e r i l e suspension medium. Mix 1ml of t h i s l a s t medium to another 10ml of s t e r i l e suspension medium , to make the inoculum. The suspension medium i s a c t u a l l y a b a s a l medium stock s o l u t i o n a t h a l f s t r e n g t h . I t i s autoclaved at 121°C f c r 10 minutes. II.4.2 Procedure Washed c e l l s from 20ml of the sample of fermentation broth i n q u e s t i o n are resuspended i n water and 5ml of the sodium m e t a - b i s u l f i t e b u f f e r to make up a t o t a l volume of 50ml. Ten ml of t h i s d i l u t e d suspension of c e l l s i s t n e^ [ 1 s t e r i l i z e d a t 121 °C f o r 10 minutes, along with a mixture of 1ml c f standard cobalamin added to 5ml of the same b u f f e r . The standard cobalamin s o l u t i o n , so prepared, i s then a s e p t i c a l l y d i l u t e d to a c o n c e n t r a t i c n of 0.02 micrograms/1. T h i s r e s u l t i n g s o l u t i o n i s c a l l e d cyanocobalamin working s o l u t i o n . 5ml of b a s a l stock s o l u t i o n i s added t c each of s e v e r a l 16x150mm t e s t tubes. These tubes are then s t e r i l i z e d f o r 5 minutes at 121°C. Appendix I I : A n a l y s i s Procedures 219 1, 2, 3, 4 and 5ml r e s p e c t i v e l y of the cyanocobalamin working s o l u t i o n are a s e p t i c a l l y added to these tubes i n d u p l i c a t e . S t e r i l e samples, prepared as d e s c r i b e d above, are then p r o p e r l y d i l u t e d with s t e r i l e d i s t i l l e d water under a s e p t i c c o n d i t i o n s to s u i t a b l e c o n c e n t r a t i o n (from 0.001 to 0.04 micrograms/1) i n vitamin B12. Samples, so prepared are f i n a l l y added to s t e r i l e b a s a l medium stock s o l u t i o n , a l r e a d y i n t e s t tubes. Each sample i s added to 6 tubes with volumes at 3 d i f f e r e n t l e v e l s - presumably to minimize the chance of under-or o v e r e s t i m a t i n g the unknown c o n c e n t r a t i o n - i n d u p l i c a t e . S t e r i l e water i s added to make the volume of a l l tubes up to 10ml, again under a s e p t i c c o n d i t i o n s . T h i s i n c l u d e s the 3 c o n t r o l tubes which c o n t a i n no sample nor cyanocobalamin working s o l u t i o n . One drop of inoculum i s a s e p t i c a l l y added t c each tube except one of the c o n t r o l tubes. The contents of a l l tubes are then thoroughly mixed. The tubes are incubated at 37°C f o r 16 to 24 hours. The growth i s measured by the t u r b i d i t y method at 600 m i l l i m i c r o n s with one c f the i n o c u l a t e d c o n t r o l tube as blank. The standard curve i s drawn with measurements c f the s o l u t i o n s of known c o n c e n t r a t i o n . A t y p i c a l curve i s presented i n F i g u r e A2.75. The c o n c e n t r a t i c n of samples i s determined by i n t e r p o l a t i o n . A t e s t i s d i s c a r d e d when the t u r b i d i t y of the i n o c u l a t e d c o n t r o l tubes, with the u n i n o c u l a t e d one as blank, i s g r e a t e r than 0.2. B e s u l t s of the t e s t are a l s o d i s r e g a r d e d i f the tube Appendix I I : A n a l y s i s Procedures 220 FIGURE A2.75 : T y p i c a l Standard Curve f o r The Vitamin B12 Assay Appendix I I : a n a l y s i s Procedures 221 with the hi g h e s t content of v i t a m i n B12 (added v i a the working s o l u t i o n ) has a t u r b i d i t y g r e a t e r than 0.5, or i f the curve does not have a s t r a i g h t p o r t i o n , or i f the c o n c e n t r a t i o n of the known sample i s out of the range of t h a t s t r a i g h t p o r t i o n , or i f the slope of t h i s s t r a i g h t p o r t i o n i s too shallow., I I . 5 MgaSUREMENT OF aCID CONCENTRATION In a l l t i t r a t i o n s with NaOH, r e f e r r e d to below, ph e n o l p h t h a l e i n i n 50% e t h y l a l c o h o l i s used as i n d i c a t o r . 11.5.1 Steam D i s t i l l a t i o n of V o l a t i l e a c i d s Ten ml of c e l l f r e e b roth i s t r a n s f e r r e d to a round f l a s k with a f l a t bottom, water i s added, then 10ml of concentrated s u l f u r i c a c i d i s s t i r r e d i n with p r e c a u t i o n not to l e t any vapor escape. Steam, generated from b o i l i n g water i n a nearby round f l a s k , i s bubbled through the d i l u t e d and a c i d i f i e d b r o t h . The vapor i s then condensed. When about 50ml of condensate has been c o l l e c t e d , i t i s t i t r a t e d with NaOH. The process i s continued u n t i l two c o n s e c u t i v e a l i g u o t s of condensate are of the same c o n c e n t r a t i o n i n a c i d s . 11.5.2 Manual_Ether_Extraction Ten ml of concentrated s u l f u r i c a c i c i s added, with vigorous mixing, to 20ml of c e l l f r e e broth i n a c l o s e d v e s s e l . The gaseous v o l a t i l e a c i d s which are b o i l e d o f f the broth by the heat generated by t h i s a d d i t i o n of a c i d are taken care of by condensation as the v e s s e l i s then cooled with i c e . Appendix I I : A n a l y s i s Procedures 222 The r e s u l t i n g s o l u t i o n i s vacuum f i l t e r e d through f i l t e r paper. The f i l t r a t e i s e x t r a c t e d 3 times i n a pear shaped e x t r a c t i n g f u n n e l with a p o r t i o n of 10ml of ether each time. A l l the ether i s added to 50ml of d i s t i l l e d water. The s o l u t i o n i s we l l mixed then the ether i s evaporated o f f with a g e n t l e s u c t i o n . 20ml of t h i s agueous phase i s b o i l e d f o r 10 minutes with water o c c a s i o n a l l y added to prevent i t from going dry. T h i s b o i l e d s o l u t i o n and another 20ml of the agueous phase are then t i t r a t e d with 0.05N HaOH. The content c f a c i d s n e u t r a l i z e d i n the b o i l e d sample i s p r o p o r t i o n a l to the amount of l a c t i c a c i d i n the o r i g i n a l 20ml of broth. The d i f f e r e n c e i n acids, content of the 2 samples s u b j e c t e d to t i t r a t i o n s i s p r o p o r t i o n a l to the amount of v o l a t i l e a c i d s i n the o r i g i n a l 20ml of bro t h . The p r o p o r t i o n a l i t y would be l i n e a r and egual to 1 i f the a c i d s could be t o t a l l y e x t r a c t e d by e t h e r . T h i s was not the case here, so an e g u i l i b r i u m curve or a c a l i b r a t i o n l i n e are needed. II.5.3 Continuous_flatch mE The apparatus, adopted from Neish [ 6 7 ] , and i t s p r i n c i p l e are d e s c r i b e d i n 2.4.4. Three ml of con c e n t r a t e d s u l f u r i c a c i d i s added to 5ml of broth i n a c l o s e d v e s s e l (Figure 2.23). The v o l a t i l e a c i d s vapors, i f any, evaporated from the l i g u i d which becomes extremely hot with the r e a c t i o n heat generated, are condensed i n the ice-surrounded v e s s e l . F i v e ml of d i s t i l l e d water i s then added to the r e s u l t i n g s o l u t i o n a f t e r c o o l i n g . The f i n a l s o l u t i o n i s f i l t e r e d . The f i l t r a t e i s v o l u m e t r i c a l l y t r a n s f e r r e d appendix I I : A n a l y s i s Procedures 223 to the r e s i d u e tube with r i n s i n g water from the v e s s e l r e c e i v i n g the f i l t r a t e . T h i s tube i s suspended i n the vapor tube a f t e r the suspension of the d i s p e r s i o n tube i n t o i t s e l f . The e x t r a c t o r i s then assembled with 25ml of eth e r i n the ether c o n t a i n e r . Hater i s added to t h i s c o n t a i n e r t o share the load of a c i d s with e t h e r and to prevent ether from going d r y . The e x t r a c t i o n i s then run f o r about 3 hours with ether b o i l e d i n a water bath at a constant temperature of 7 0 O C . In the beginning, two p o r t i o n s of the same sample were s u b j e c t e d to e x t r a c t i o n . One of the r e s u l t i n g e x t r a c t s was cooled then t i t r a t e d with 0.05N NaOH. An excess of water was added before the t i t r a t i o n , and vigorous s w i r l i n g was executed duri n g the t i t r a t i o n to d i s p e r s e the ether phase to f i n e drops i n l a r g e volume of water. The ether i n the other e x t r a c t was then b o i l e d o f f i n the same water bath. T h i s e x t r a c t was b o i l e d f o r about 15 minutes with evaporated water o c c a s i o n a l l y r e p l e n i s h e d . T h i s e x t r a c t was f i n a l l y t i t r a t e d with NaOH. Re s u l t of the f i r s t t i t r a t i o n g i v e s the t o t a l a c i d c o n c e n t r a t i o n , the second the n o n - v o l a t i l e a c i d c o n c e n t r a t i c n . L a t e r an a l t e r n a t i v e method was adopted. In t h i s method, only one e x t r a c t i o n i s needed f c r each sample. The e x t r a c t of the sample i s cooled o f f then t i t r a t e d f o r the t o t a l a c i d c o n c e n t r a t i o n . A known excess of s u l f u r i c a c i d 1N i s added to the t i t r a t e d e x t r a c t . I t i s then b o i l e d f o r 15 minutes with o c c a s i o n a l a d d i t i o n of water. The f i n a l b c i l e d s o l u t i o n i s t i t r a t e d again. The d e f i c i e n c y of added s u l f u r i c a c i d r e v e a l e d by the t i t r a t i o n i s of the same magnitude of the v o l a t i l e a c i d Appendix I I : A n a l y s i s Procedures 224 content of the o r i g i n a l sample. I I . 6 HEASUREMENT_OF_CHEH1^A^ T h i s t e s t i s widely used [4,5,70,91]. Only minor d e t a i l s d i f f e r from one source to another. P l a c e 50ml of p r o p e r l y d i l u t e d sample i n a 500ml Erlenmeyer f l a s k with ground g l a s s j o i n t neck. Add 25ml of standard potassium dichromate s o l u t i o n (0.25N). Add 1g each of mercuric s u l p h a t e and s i l v e r s u l p h a t e . Then g l a s s beads are i n t r o d u c e d i n t o the f l a s k . F i n a l l y 75ml of concentrated s u l f u r i c a c i d i s added, i n s m a l l a l i g u o t s and with slow pouring as well as with continuous a g i t a t i o n . A good mixing i s a s c e r t a i n e d . The f l a s k i s b o i l e d and r e f l u x e d f o r two hours with a F r i e d r i c h ' s condenser on a hot p l a t e . A blank c o n s i s t i n g of 50ml of d i s t i l l e d water i n s t e a d of a sample i s s i m u l t a n e o u s l y r e f l u x e d under c o n d i t i o n s as i d e n t i c a l as p o s s i b l e . The two f l a s k s are then cooled and t i t r a t e d with f r e s h l y s t a n d a r d i z e d 0.25N f e r r o u s ammonium s u l p h a t e . F e r r o i n i s used as i n d i c a t o r . 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= ml of f e r r o u s ammonium sulphate used f o r the t i t r a t i o n s of the sample and the blank r e s p e c t i v e l y . Appendix I I : A n a l y s i s Procedures 225 II.7 2JASUREMENT_0F_SULFUR 11.7.1 F r e e _ S u I f u r _ D i o x i d e An a l i q u o t of 5ml of the sample i s placed i n a 250ml Erlenmeyer f l a s k . Cold water, o c c a s i o n a l l y with i c e , i s added u n t i l the brown c o l o r of SSL sample i s l i g h t enough to show the 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 with 0.1N i o d i n e s o l u t i o n u s i n g s t a r c h as i n d i c a t o r then f e l l o w s . The blue c o l o r c f the end point i s masked by the brown c o l o r of SSL, t h e r e f o r e , the end p o i n t i s recog n i z e d only by the sudden change i n tone of the c o l o r . The c o n c e n t r a t i o n of s u l f u r d i o x i d e i s c a l c u l a t e d as f o l l o w s : S u l f u r e d i o x i d e c o n c e n t r a t i o n (ppm) = ml of 0.1N Iodine x 640.6 11.7.2 T p t a l _ s u I f u r ^ d i o x i d e T o t a l s u l f u r d i o x i d e c o n s i s t s c f f r e e s u l f u r d i o x i d e and l o o s e l y combined s u l f u r d i o x i d e . The l a t t e r i s s u l f u r d i o x i d e , combined with o r g a n i c matter, which can be s p l i t o f f by treament with NaOH. The same procedure as d e s c r i b e d above i s used. But the sample i s submitted to a pretreatment with 5ml c f NaOH at room temperature f o r more than one hour. T h i s treatment i s then f o l l o w e d by the lowering of pH of the sample with d i l u t e s u l f u r i c a c i d . Appendix I I I : S o l u t i o n s and Chemicals 226 APPENDIX I I I S o l u t i o n s and Chemicals I I I . 1 SOLUTIONS 1. S o l u t i o n s of S u l f u r i c Acid A volume of concentrated s u l f u r i c a c i d i s d i l u t e d approximately to the d e s i r e d c o n c e n t r a t i o n (30ml to 1 l i t e r f o r 1N s o l u t i o n s ) . The s o l u t i o n i s then s t a n d a r d i z e d a g a i n s t standard NaOH. 2. Standard S o l u t i o n s of Sodium Hydroxide An approximate amount, i n weight, of NaOH p e l l e t s i s d i l u t e d to the d e s i r e d volume. The s o l u t i o n i s then s t a n d a r d i z e d a g a i n s t potassium b i p h t h a l a t e as standard substance (0.8-0.9g f o r 0.1N s o l u t i o n s ) . 3. Standard S o l u t i o n of 0.1N Iodine About 12.7g of resublimed i o d i n e i s a c c u r a t e l y weighed on a p r e c i s i o n balance t o the nearest tenth of a miligram i n an Erlenmeyer f l a s k with g l a s s s t o p p e r . i»0g of KI and about 25ml of water are added to the f l a s k . I t i s then shaken u n t i l a l l i o d i n e i s i n s o l u t i o n . The volume i s made up to 1 l i t e r . 12.692g of i o d i n e makes 1 l i t e r of 0.1N s o l u t i o n . The s o l u t i o n i s kept i n a Appendix I I I : S o l u t i o n s and Chemicals 227 brown b o t t l e with g l a s s stopper. 4. Starc h I n d i c a t o r Two grams of s o l u b l e s t a r c h i s mixed with s u f f i c i e n t water to make a paste. I t i s then d i l u t e d to 200ml and heated tc the b o i l i n g p o i n t . 0.2 to 0.4ml of toluene i s added as p r e s e r v a t i v e . The i n d i c a t o r i s s t o r e d i n a g l a s s - s t o p p e r e d b o t t l e . 5. Cobalt C h l o r i d e S o l u t i o n 9.088g of c o b a l t c h l o r i d e hexahydrate i s d i s s o l v e d i n 1 l i t e r of d i s t i l l e d water. 6. Somogyi Copper Reagent 52.9g of discdium hydrogen phosphate heptahydrate and 40g of potassium sodium t a r t r a t e are d i s s o l v e i n 700ml of d i s t i l l e d water. 100ml of normal NaOH and then 80ml of 10% s o l u t i o n of c u p r i c s u l f a t e pentahydrate are added to the s o l u t i o n . F i n a l l y 180g o f anhydrous sodium s u l f a t e i s d i s s o l v e d i n the r e s u l t i n g s o l u t i o n . The volume i s then made up t c 1 l i t e r . The s o l u t i o n i s st o r e d i n a g l a s s - s t o p p e r e d b o t t l e . 7. Arsenomolybdate Reagent F i f t y grams of ammonium molybdate i s d i s s o l v e d i n 900ml of d i s t i l l e d water. 42ml of conc e n t r a t e d s u l f u r i c a c i d i s then s t i r r e d i n . S i x grams of disodium hydrogen arsenate heptahydrate, d i s s o l v e d i n 50ml of water, i s then added tc the s o l u t i o n . The f i n a l mixture i s then incubated at 37°C f o r 2 Appendix I I I : S o l u t i o n s and Chemicals 228 days. I t i s then f i l t e r e d and s t o r e d 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 l i t e r of phosphate c i t r a t e b u f f e r (pH 4.5) i s made with 24.36g of disodium hydrogen phosphate heptahydrate and 11.47g of c i t r i c a c i d monohydrate. The b u f f e r i s r e f r i g e r a t e d under toluene. One gram of sodium m e t a - b i s u l p h i t e i s added, j u s t p r i o r to use, to every 100ml of b u f f e r . 9. Standard Cobalamin Stock S o l u t i o n One m i l l i l i t e r of a s o l u t i o n of 1OOmicrograms/ml of vit a m i n B12 i s added (with s t e r i l e s y r i n g e and hypodermic needle) to 99ml of 25% e t h y l a l c o h o l . T h i s s o l u t i o n i s r e f r i g e r a t e d , p r o t e c t e d from l i g h t and then d i s c a r d e d when i t i s 6 months e l d . 10. 25% S o l u t i o n of E t h y l A l c o h o l E t h y l a l c o h o l of 95% c o n c e n t r a t i c n i s d i l u t e d with water u n t i l the c o n c e n t r a t i o n reaches 25%. The c o n c e n t r a t i c n i s i n d i r e c t l y measured with a hydrometer. 11. F e r r o i n I n d i c a t o r 1.5g of 1,10-phenanthroline menchydrate and 0.7g of f e r r o u s s u l f a t e heptahydrate are d i s s o l v e d i n d i s t i l l e d water and d i l u t e d to 100ml. appendix I I I : S o l u t i o n s and Chemicals 229 I I I . 2 CHEMICALS 1. A c e t i c Acid G l a c i a l - A l l i e d Chemicals 3019. 2. Agar Bacto Agar - D i f c o 0140-01-0. 3. Ammonium Molybdate - Baker 0716. 4. Beef E x t r a c t Bacto Beef E x t r a c t - D i f c o 0126-01. 5. Calcium Carbonate, Powder - a l l i e d Chemicals 1506. 6. Calcium Hydroxide - a l l i e d Chemicals 1522. 7. C i t r i c a c i d Monchydrate, Powder - F i s h e r A-105. 8. Co b a l t C h l o r i d e Hexahydrate, C r y s t a l - Baker 1670. 9. Cupr i c S u l f a t e Pentahydrate - a l l i e d Chemicals 1651. 10. 5,6-dimethyl Benzimidazole (DMBZ) - Baker J590. 11. E t h e r - Baker 9240. 12. E t h y l A l c o h o l , 95%. 13. Ferrous Ammonium S u l f a t e Hexahydrate (Mohr»s S a l t ) , C r y s t a l - M a l l i n c k r o d t 5064. 14. Ferrous S u l f a t e Heptahydrate, Granular, Beagent - Baker 2070. . 15. F i s h P l a n t E f f l u e n t - Vancouver Laboratory of The F i s h e r i e s Besearch Board of Canada. 16. :(•) Glucose (Dextrose), Anhydrous - B r i t i s h Drug Houses 33519. 17. Iodine. 18. L a c t i c Acid - Baker 0194. 19. Mercuric S u l s u l p h a t e - F i s h e r M-190. Appendix I I I : S o l u t i o n s and Chemicals 230 20. Peptone Bacto Peptone - D i f c o 0118-01. 21. 1,10-phenanthroline Honohydrate - Baker T170. 22. P h e n o l p h t h a l e i n - a l l i e d Chemicals 2052. 23. Potassium B i p h t h a l a t e — A l l i e d Chemicals 2083. 24. Potassium Dichromate, C r y s t a l - A l l i e d Chemicals 2088., 25. Potassium I o d i d e , C r y s t a l - Baker 3164. 26. Potassium Dihydrogen Phosphate Potassium Phosphate, Monobasic - M a l l i n c k r o d t 7100. 27. Potassium Sodium T a r t r a t e (Rochelle S a l t ) Sodium Potassium T a r t r a t e , C r y s t a l - F i s h e r S-387. 28. P r o p i o n i c A c i d - F i s h e r A-258. 29. R e i n f o r c e d C l o s t r i d i a l Medium - Oxoid CM149. 30. S i l v e r Sulphate - Baker 3436. 31. Sodium Arsenate, D i b a s i c , Granular - Baker 3486. 32. Sodium M e t a - b i s u l f i t e - F i s h e r S-244. 33. Sodium C h l o r i d e - F i s h e r S-271. 34. Sodium Hydroxide, E l e c t r o l y t i c P e l l e t s - F i s h e r S-318 (78575). 35. Sodium Phosphate, D i b a s i c , Heptahydrate - Baker 3824. (Disodium Hydrogen Phosphate Heptahydrate). 36. Sodium S u l f a t e , Anhydrous - F i s h e r S-421. 37. Spent S u l f i t e L i q u o r (SSL) - ITT Rayonier Inc., P o r t Angeles D i v i s i o n . 38. S t a r c h , S o l u b l e Powder - A l l i e d Chemicals 2352. 39. S u l f u r i c A c i d , Reagent - F i s h e r A-300. 40. Vitamin B12 Appendix I I I : S o l u t i o n s and Chemicals 231 Rubramin 100mcg/ml - Squibb 72240. 41. Vitamin B12 Assay Medium Bacto B12 Assay Medium U.S.P. - D i f c o 0457-15 42. Vitamin B12 C u l t u r e Agar Bacto B12 C u l t u r e Agar O.S.P. - D i f c o 0541-15. 43. Vitamin B12 Inoculum Broth Bacto B12 Inoculum Broth U.S.P. - D i f c o 0542-15. 44. D(+) Xylose - B r i t i s h Drug Houses 227198. 45. l e a s t E x t r a c t Paste - Oxoid L-20. 46. Yeast E x t r a c t Powder Bacto Yeast E x t r a c t Powder - D i f c o 0127-01 NB Yeast E x t r a c t Powder - N u t r i t i o n a l B i o c h e m i c a l s C o r p o r a t i o n 4159. 

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