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Urea metabolism by the marine cryptomonad, Rhodomonas Lens Johnson, Sherran Ruth 1975

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UREA METABOLISM BY THE MARINE CRYPTOMONAD, RHODOMONAS LENS by SHERRAN RUTH JOHNSON B . S c , U n i v e r s i t y o f O t t a w a , 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e D e p a r t m e n t o f B i o l o g y We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA O c t o b e r , 1975 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced deg ree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r ee t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y pu rpo se s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depa rtment The U n i v e r s i t y o f B r i t i s h Co l umb i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 ABSTRACT i The e f f e c t o f u r e a on a m a r i n e c r y p t o m o n a d , Rhodomonas  l e n s , was I n v e s t i g a t e d . C e l l s f r o m a n i t r a t e e n r i c h e d , n a t u r a l s e a w a t e r medium were a c c l i m a t e d t o s y n t h e t i c s e a w a t e r w i t h e i t h e r n i t r a t e ( c o n t r o l medium) o r u r e a ( e x p e r i m e n t a l medium) as t h e s o l e n i t r o g e n s o u r c e . C e l l s a c c l i m a t e d r e a d i l y t o t h e c o n t r o l medium; a c c l i m a t i o n t o t h e e x p e r i m e n t a l medium was e r r a t i c . C o n t r o l a n d e x p e r i m e n t a l c e l l s were t a k e n f r o m m e d i a on w h i c h good g r o w t h h a b i t u a l l y o c c u r r e d . T e s t e d o p t i m a l g r o w t h c o n d i t i o n s o c c u r r e d a t 22°C and a 16:8 h o u r , l i g h t : d a r k c y c l e w i t h f l u o r e s c e n t l i g h t a t a p p r o x i m a t e l y 150 f o o t - c a n d l e s . G r owth was f o l l o w e d t u r b i d i m e t r i c a l l y a n d by d i r e c t c e l l c o u n t . C o n t r o l c u l t u r e s e x h i b i t e d t h e t y p i c a l l a g , l o g a r i t h m i c and s t a t i o n a r y p h a s e s . The s t a t i o n a r y p h a s e merged i n t o a g r a d u a l d e c l i n e p h a s e . C u l t u r e c o l o u r p r o c e e d e d f r o m a r e d - p i n k d u r i n g e a r l y p h a s e s t o a r e d - o r a n g e , t h e n g r e e n d u r i n g s t a t i o n a r y - d e c l i n e p h a s e . I n i t i a l l y , u l t r a s t r u c t u r e was t y p i c a l o f c r y p t o m o n a d s : c e l l s e x h i b i t e d a b i - l o b e d c h l o r o p l a s t h a v i n g t h y l a k o i d s , o f t e n a r r a n g e d i n bands o f two, w i t h a l o c u l u s s i z e o f 110-260 A. O c c a s i o n a l l i p i d d r o p l e t s were p r e s e n t w i t h i n t h e c y t o p l a s m . S t a t i o n a r y - d e c l i n e p h a s e showed an a c c u m u l a t i o n o f a l i p i d - l i k e m a t e r i a l . T h y l a k o i d s were a r r a n g e d s i n g l y ; l o c u l u s s i z e b e i n g d e c r e a s e d t o 70-150 A. E x p e r i m e n t a l c u l t u r e s a t c o n c e n t r a t i o n s l e s s t h a n 1.25 mM u r e a e x h i b i t e d s i m i l a r g r o w t h t o c o n t r o l c u l t u r e s . At u r e a c o n c e n t r a t i o n s e q u a l t o o r g r e a t e r t h a n 1.25 mM u r e a , c e l l s d u r i n g l a g and e a r l y l o g a r i t h m i c p h a s e s showed a l i p i d - l i k e a c c u m u l a t i o n ; d u r i n g l a t e r l o g a r i t h m i c p h a s e , l i p i d d r o p l e t s were o b s e r v e d . S t a t i o n a r y p h a s e was s h o r t and c e l l s p r o c e e d e d d i r e c t l y i n t o a r a p i d d e c l i n e p h a s e . C o n c o m i t a n t l y , an i n c r e a s e i n pH o c c u r r e d and c u l t u r e s became a creamy w h i t e i n c o l o u r . ' To t e s t e l e c t r o l y t i c and pH e f f e c t s , NaOH a n d NH^OH were a d d e d t o a h e a l t h y c o n t r o l c u l t u r e . I n c r e a s e d pH w i t h NaOH had no g r o s s e f f e c t on t h e c u l t u r e s : i n c r e a s e d pH w i t h NH^OH c a u s e d a l y t i c e f f e c t s i m i l a r t o l y s i s o b s e r v e d d u r i n g d e c l i n e p h a s e o f e x p e r i m e n t a l c u l t u r e s . O b s e r v a t i o n s on c o n t r o l c e l l s d u r i n g s t a t i o n a r y - d e c l i n e p h a s e s s u g g e s t a l a c k o f a v a i l a b l e n i t r o g e n , p r o b a b l y due t o d e p l e t i o n : o b s e r v a t i o n s on e x p e r i m e n t a l c e l l s d u r i n g l a g and e a r l y l o g a r i t h m i c p h a s e s a r e s i m i l a r , t h u s s u g g e s t i n g a l a c k o f n i t r o g e n . As s u f f i c i e n t u r e a i s p r e s e n t , i t i s s u g g e s t e d t h a t t h e u p t a k e a n d / o r m e t a b o l i s m o f u r e a i n e x p e r i m e n t a l c e l l s i s i n e f f i c i e n t , c a u s i n g n i t r o g e n s t r e s s . I n c r e a s e i n pH p r i o r t o d e c l i n e p h a s e , and t h e r e s u l t s o f e l e c t r o l y t i c • e x p e r i m e n t s s u g g e s t t h a t u r e a i s c o n v e r t e d i n t o ammonia and e x c e s s ammonia i s r e l e a s e d i n t o t h e medium. The i n c r e a s i n g ammonia c o n c e n t r a t i o n becomes t o x i c a n d a c c e l e r a t i n g c e l l l y s i s t h u s r e s u l t s i n a d e c l i n e p h a s e . i i i TABLE OP CONTENTS Page ABSTRACT i TABLE OP CONTENTS i i i L I S T OF TABLES v L I S T OF PLATES AND FIGURES v i ACKNOWLEDGEMENTS v i i i INTRODUCTION 1 METHODS AND MATERIALS 13 I . C u l t u r e s 13 I I . G e n e r a l Methods 13 1. Growth measurement's 13 2. U r e a a n a l y s i s 17 3. M i c r o s c o p y 17 I I I . E x p e r i m e n t a l Methods 19 1. A c c l i m a t i o n o f c u l t u r e s t o m e d i a 19 2. L i g h t and t e m p e r a t u r e 22 3. U r e a e f f e c t s 22 4. pH e f f e c t s 23 RESULTS 25 1. A c c l i m a t i o n o f c u l t u r e s t o m e d i a 25 2. L i g h t and t e m p e r a t u r e 27 3. Growth o f c o n t r o l 27 ( i ) Measurement 27 ( i i ) U l t r a s t r u c t u r e 27 l j . U r e a e f f e c t s 32 i v ( i ) Growth measurements 32 ( i i ) U l t r a s t r u c t u r e 39 ( i i i ) E l e c t r o l y t i c e f f e c t s " 39 DISCUSSION 46 CONCLUSION 59 PLATES AND EXPLANATION 60 FOOTNOTES 7^ REFERENCES ' 77 ADDENDUM 86 LIST OP TABLES v EXPLANATION OF TABLES Table Page 1. Enriched s y n t h e t i c sea water media. 14 2 . S t e r i l i t y media 15 3 . The determination of urea i n sea water. 18 4. F i x a t i o n and dehydration schedule I . 20 5 . F i x a t i o n and dehydration schedule I I . 21 6 . A c c l i m a t i o n of the c o n t r o l c u l t u r e to enriched s y n t h e t i c sea water plus 1 .25 mM urea. 26' 7 . A comparison of growth i n c o n t r o l media at fo u r temperatures and two photoperiods. 28 8 . ' A comparison of c u l t u r e colour and M-co n c e n t r a t i o n during growth of experimental c u l t u r e s at s i x concentrations of urea. 35 9. A comparison of growth, by d i v i s i o n , of c u l t u r e s at i n c r e a s i n g concentrations of urea. - 42 1 0 . V a r i a t i o n i n pH and urea catabolism among i n d i v i d u a l c u l t u r e s . 43 11. The e f f e c t s of pH on healthy c o n t r o l c u l t u r e s . 45 v i L I S T OF PLATES AND FIGURES E x p l a n a t i o n o f F i g u r e s Page F i g u r e 1. Growth p a t t e r n o f c u l t u r e i n c u b a t e d i n c o n t r o l medium. 29 PLATE 1. 61 F i g u r e 2. C o n t r o l c e l l . F i g u r e 3. C o n t r o l c e l l , a r r a n g e m e n t o f o r g a n e l l e s PLATE 2. 63 F i g u r e 4. F l a g e l l u m , f i b r o u s m a s t i g o n e m e s . F i g u r e 5. F l a g e l l a , t u b u l a r m a s t i g o n e m e s PLATE 3. 65 F i g u r e 6. A r r a n g e m e n t o f c h l o r o p l a s t . F i g u r e 7. A r r a n g e m e n t . o f t h y l a k o i d s . PLATE 4. 67 F i g u r e 8. C o n t r o l c e l l s , l a t e s t a t i o n a r y p h a s e . F i g u r e 9. C o n t r o l c e l l s , l a t e s t a t i o n a r y p h a s e . F i g u r e 10. C o n t r o l c e l l , l a t e s t a t i o n a r y p h a s e . F i g u r e 11. Growth p a t t e r n o f c u l t u r e s a t s e v e r a l c o n c e n t r a t i o n s o f u r e a b a s e d on t u r b i d i m e t r i c d e t e r m i n a t i o n s . 33 F i g u r e 12. Growth p a t t e r n o f e x p e r i m e n t a l c e l l s a t s e v e r a l c o n c e n t r a t i o n s o f u r e a . 36 F i g u r e 13. The r e l a t i o n s h i p b e t w e e n t h e g e n e r a t i o n t i m e o f a c u l t u r e and t h e c o n c e n t r a t i o n o f u r e a i n w h i c h i t has been i n c u b a t e d . 40 PLATE 5 69 F i g u r e 14. E x p e r i m e n t a l c e l l , l o g a r i t h m i c p h a s e . F i g u r e 15. E x p e r i m e n t a l c e l l , l o g a r i t h m i c p h a s e . PLATE 6. F i g u r e 16. E x p e r i m e n t a l c e l l s , e a r l y l o g a r i t h m i c p h a s e . F i g u r e 17. E x p e r i m e n t a l c e l l s , e a r l y l o g a r i t h m i c p h a s e . PLATE 7. F i g u r e s 18 t o 25. C o n t r o l c e l l s u n d e r t o x i c ammonia c o n d i t i o n s a t i n c r e a s e d pH. F i g u r e 26. M e t a b o l i c i n t e r r e l a t i o n s h i p s . v \ i i ACKNOWLEDGEMENTS I w o u l d l i k e t o t h a n k Dr. T. B i s a l p u t r a f o r h i s g u i d a n c e d u r i n g t h e s e s t u d i e s , and t h e members o f my co m m i t t e e : D r . N. A n t i a , Dr. P . J . H a r r i s o n , Dr. F.J.R. T a y l o r a n d Dr. R. S c a g e l , f o r t h e comments and s u g g e s t i o n s t h r o u g h o u t t h e p r o j e c t . The u n t i m e l y d e a t h o f Dr. B. T r e g u n n a when t h i s t h e s i s was n e a r l y c o m p l e t e v/as a deep p e r s o n a l l o s s . I a p p r e c i a t e d h i s i n v o l v e m e n t i n t h e d i f f i c u l t i e s r e l a t e d t o t h i s p r o j e c t and h i s r e a d y g e n e r o s i t y w i t h b o t h h e l p and encou r a g e m e n t . I w o u l d e s p e c i a l l y l i k e t o t h a n k t h o s e f r i e n d s who h e l p e d i n t h e p r e p a r a t i o n o f t h i s t h e s i s : B e r l O a k l e y i n d i s c u s s i n g f i x a t i o n and c u l t u r e methods, S u s a n S h i n n who e d i t e d and t y p e d t h e m a n u s c r i p t , C h r i s t o p h e r K r e i s i n d i s c u s s i n g b i o c h e m i c a l p r o b l e m s r e l a t e d t o t h e p r o j e c t and who h e l p e d e d i t t h e m a n u s c r i p t , and Thomas Mumford who h e l p e d p r e p a r e t h e p l a t e s and s l i d e s and p r o o f r e a d t h e m a n u s c r i p t . I w o u l d a l s o l i k e t o t h a n k Dr. D o n a l d 0. A n d e r s o n and J o h n and Wendy Hardy f o r t h e i r p r a y e r , s u p p o r t and encouragement t h r o u g h o u t t h i s p r o j e c t . INTRODUCTION N i t r o g e n i s an e s s e n t i a l e l e m e n t f o r p l a n t g r o w t h . I t i s o f t e n t h e c o n c e n t r a t i o n o f t h i s n u t r i e n t w h i c h l i m i t s p h y t o p l a n k t o n g r o w t h ( S p o e h r a n d M i l n e r , 19^9; Holmes, W i l l i a m s and E p p l e y , 1967; D u g d a l e and G o e r i n g , 1970; Thomas and Owen, 1971; R y t h e r a n d D u n s t a n , 1971). N i t r o g e n o c c u r s i n a v a r i e t y o f combined f o r m s i n s e a w a t e r . Of t h e s e ammonium and n i t r a t e a r e t h e most a b u n d a n t a n d a r e t h e commonest s o u r c e s o f n i t r o g e n f o r p l a n t n u t r i t i o n — i n many s i t u a t i o n s ammonium i s known t o be u t i l i z e d b e f o r e n i t r a t e ( S y r e t t , 1962; G r a n t , Madgwick and D a l P o n t , 1967; S t r i c k l a n d , Holm-Hansen, E p p l e y and L i n n , 1969; G o e r i n g , W a l l e n , a n d Nauman, 1970; N a y l o r , 1970). Of t h e o r g a n i c n i t r o g e n compounds, amino a c i d s and u r e a a r e f o u n d a t c o n c e n t r a t i o n s i n s e a w a t e r w h i c h s u g g e s t t h a t t h e y may c o n s t i t u t e a b i o l o g i c a l l y s i g n i f i c a n t s o u r c e o f n i t r o g e n . E x t e n s i v e a n a l y s i s o f m a r i n e s u r f a c e w a t e r s by H o b b i e , C r a w f o r d , a n d Webb (1964), Degens ( 1 9 6 8 ) , and L i t c h f i e l d a n d P r e s c o t t (1970) show c o n c e n t r a t i o n s o f f r e e amino a c i d s r a n g i n g f r o m 38 t o 70 jag l i t e r \ Of t h e s e , a s p a r t i c a c i d i s f o u n d i n v a r y i n g c o n c e n t r a t i o n s i n a l l w a t e r s s a m p l e d ; g l y c i n e , s e r i n e and o r n i t h i n e are- f o u n d i n t h e g r e a t e s t c o n c e n t r a t i o n and a r e p r e s e n t f r e q u e n t l y . The p r e s e n c e o f t h e s e amino a c i d s may be s i g n i f i c a n t , n o t o n l y as n u t r i e n t s f o r c e r t a i n p h y t o p l a n k t e r s ( L e w i n , 1962; G u i l l a r d , 1963; S h i h i r a and K r a u s s , 1965; C a i n , 1965; H e l l e b u s t a n d G u i l l a r d , 1967; 2 N o r t h a n d S t e p h e n s ; 1 9 6 9 , 1971) b u t a l s o as compounds d i r e c t l y o r i n d i r e c t l y c o n n e c t e d v i a t h e " u r e a c y c l e . " The d i s t r i b u t i o n a n d c o n c e n t r a t i o n o f u r e a f o u n d i n s u r f a c e w a t e r s i s v a r i a b l e , and p r o b a b l y d e p e n d e n t on t h e n a t u r e o f t h e b i o l o g i c a l c o n d i t i o n s i n t h e i m m e d i a t e e n v i r o n m e n t . O b s e r v e d c o n c e n t r a t i o n s r a n g e b e t w e e n 0 . 5 4 t o _1 1 .00 ^ g - a t o m u r e a - N l i t e r o f f L a J o l l a , C a l i f o r n i a ( M c C a r t h y , 1970) and o f f t h e c o n t i n e n t a l s h e l f b e t w e e n Panama and C a l l a o (Remsen, 1 9 7 1 ) , 0 . 2 5 t o 1 1 .2 ^ g - a t o m u r e a - N l i t e r ^ b e t w e e n Cape Cod a n d Cape May ( i n c l u s i v e o f New Y o r k H a r b o u r w a t e r s ) (Remsen, 1 9 7 1 ) , 0 . 0 0 t o 0 . 6 7 _1 /ag-atom u r e a - N l i t e r a l o n g t h e P e r u v i a n c o a s t ( M c C a r t h y , 1 9 7 0 ) , and 3 . 0 7 ;ag-atom u r e a - N l i t e r 1 i n t h e E n g l i s h C h a n n e l ( N e w e l l , 1 9 6 7 ) . The h i g h f i g u r e f o r t h e a r e a i n c l u s i v e o f New Y o r k H a r b o u r s u g g e s t s t h a t much o f t h i s u r e a i s t e r r i g e n o u s . The s o u r c e becomes o b v i o u s f r o m e c o l o g i c a l s t u d i e s . C r o n i n (1967) o b s e r v e d a s h a r p i n c r e a s e o f sewage o u t f a l l s i n t o c o a s t a l w a t e r s i n 'the p a s t f i f t e e n y e a r s ' . I n t h e Potomac R i v e r e f f l u e n t l o a d s v a r y s e a s o n a l l y f r o m 5 t o 40% o f r i v e r volume ( S h a p i r o a n d R i b e i r o , 1 9 6 5 ) ; 16$ o f t h e summer f l o w o f t h e Hudson R i v e r a t Y o n k e r s , b e f o r e e f f l u e n t s f r o m New Y o r k C i t y e n t e r , i s m u n i c i p a l sewage e f f l u e n t ( H o w e l l s , K n e i p e and E i s e n b u d , 1 9 7 0 ) . Man i s a g e n e r o u s c o n t r i b u t o r . t o c o a s t a l o r g a n i c n i t r o g e n . C o a s t a l b i r d s may a l s o c o n t r i b u t e i n a l e s s e r q u a n t i t y . U r i c a c i d c o m p r i s e s a p p r o x i m a t e l y 70 t o 80% o f t h e n i t r o g e n e x c r e t e d by b i r d s (Needham, 1 9 3 1 ) ; t h i s i s r a p i d l y b r o k e n down t o u r e a i n s e a w a t e r ( M c C a r t h y , 1971). I n t h e open o c e a n , m a r i n e mammals i n h i g h d e n s i t y w o u l d be c o n t r i b u t o r s i n i s o l a t e d i n s t a n c e s . D u r i n g t h i s p e r i o d t h e y may e x c r e t e s i g n i f i c a n t q u a n t i t i e s o f u r e a ( M c C a r t h y , 1971)• F i s h a n d p l a n k t o n i c i n v e r t e b r a t e s a r e t h e l a r g e s t o f f - s h o r e c o n t r i b u t o r s t o u r e a r e g e n e r a t i o n ( M c C a r t h y , 1970), a r e g e n e r a t i o n f u r t h e r e d by m i c r o b i a l a c t i o n (Remsen, 1971)• T h u s , u r e a i s p r e s e n t i n m a r i n e s u r f a c e w a t e r s — i t s s o u r c e s i n open w a t e r s b e i n g r e l a t i v e l y s t a b l e and i n c o a s t a l w a t e r s b e i n g c o n t i n u a l l y augmented. Can m a r i n e p h y t o p l a n k t o n u t i l i z e u r e a a t t h e c o n c e n t r a t i o n s n a t u r a l l y a v a i l a b l e ? H a r v e y (19^0) s u g g e s t e d t h a t m a r i n e p h y t o p l a n k t o n p r o b a b l y were c a p a b l e o f u t i l i z i n g u r e a as a n i t r o g e n s o u r c e . A l t h o u g h a l l p h y t o p l a n k t o n do n o t u t i l i z e u r e a as a s o l e n i t r o g e n s o u r c e , many have b e e n o b s e r v e d t o do so i n c u l t u r e ( B i r d s e y and L y n c h , 1962; E p p l e y e t a l . , 1971; M c C a r t h y , 1971; C a r p e n t e r e t a l . , 1972), sometimes i n p r e f e r e n c e t o n i t r a t e ( G r a n t e t a l . , 1967) o r e v e n w i t h c o m p l e t e r e j e c t i o n o f n i t r a t e ( A n t i a and C h o r n e y , 1968). H a r v e y (19^0), u s i n g a p h y t o p l a n k t o n i c community hea v y i n d i a t o m s , o b s e r v e d good g r o w t h on u r e a and on u r i c a c i d . The r a t e o f g r o w t h was s l o w e r compared t o t h a t on ammonium. E p p l e y e t a l . (1971), w i t h s h i p b o a r d c u l t u r e s , and C a r p e n t e r e t a l . (1972) , w i t h C h a e t o c e r o s s p . a n d S k e l e t o n e m a s p . , o b s e r v e d no d r a m a t i c d i f f e r e n c e s i n g r o w t h r a t e o r q u a l i t y o f g r o w t h between c e l l s grown on n i t r a t e , ammonium, o r u r e a . I n a l l p u b l i s h e d work w i t h c u l t u r e s , t h e 4 n i t r o g e n c o n c e n t r a t i o n g r e a t l y e x c e e d s t h a t m e a s u r e d f o r n a t u r a l s e a w a t e r . A l t h o u g h o c e a n i c p h y t o p l a n k t e r s p o s s e s s mechanisms f o r m a x i m i z i n g t h e u t i l i z a t i o n o f v e r y low l e v e l s o f n i t r o g e n o u s n u t r i e n t s ( E p p l e y , R o g e r s and M c C a r t h y , 1 9 6 9 ) , i n c u l t u r e t h e y a p p e a r t o n e e d much h i g h e r l e v e l s . ' ' " I n u n i c e l l u l a r a l g a e t h e c a t a b o l i s m o f u r e a may p r o c e e d v i a one o f two enzyme s y s t e m s . U r e a s e ( u r e a amido h y d r o l a s e , EC 3 . 5 . 1 . 5 ) c a t a l y z e s t h e h y d r o l y t i c c l e a v a g e o f u r e a ; C0(NH ) + H O >C0 + 2NH . 2 2 2 2 3 T h o u g h t t o . b e n e a r l y u b i q u i t o u s , u r e a s e has b e e n f o u n d i n r e p r e s e n t a t i v e s o f t h e B a c i l l a r i o p h y c e a e , C h r y s o p h y c e a e , C y a n o p h y c e a e , E u g l e n o p h y c e a e , P r a s i n o p h y c e a e , a n d X a n t h o p h y c e a e ( A l l i s o n e t a l . , 1954; B e r n s , H o l o h a n a n d S c o t t , 1 9 6 6 ; L u i and R o e l s , 1 9 7 0 ; Roon and L e v e n b e r g , 1 9 7 0 , 1972; P e c o r a , 1 9 7 2 ; L e f t l e y and S y r e t t , 1 9 7 3 ) . R e p r e s e n t a t i v e s o f t h e C h l o r o p h y c e a e a n d c e r t a i n y e a s t s , a l t h o u g h a b l e t o m e t a b o l i z e u r e a , c o n t a i n no d e t e c t a b l e u r e a s e a c t i v i t y ( W a l k e r , 1952; H a t t o r i , 1957; B a k e r a n d Thompson, 1 9 6 2 ; Domnas, 1962; K a t i n g , 1 9 6 2 ; Cook and B o u l t e r , 1 9 6 4 ) . T h e s e o r g a n i s m s p o s s e s s an e n e r g y - r e q u i r i n g s y s t e m — A T P : u r e a a m i d o l y a s e ( U A L a s e ) . O r i g i n a l l y r e p o r t e d t o be one enzyme (Roon and L e v e n b e r g , 1968) , UALase i s now c o n s i d e r e d t o be e i t h e r two d i s t i n c t enzymes (Thompson and M u e n s t e r , 1971) o r a n enzyme complex ( W h i t n e y and C o o p e r , 1 9 7 2 , a, b ) . The two p o s s i b l e enzymes a r e u r e a c a r b o x y l a s e ( u r e a : CO l i g a s e (ADP), EC 6 . 3 - 4 . 6 ) and a l l o p h a n a t e amido h y d r o l a s e (EC 3.5.1.13). They c a t a l y z e t h e r e a c t i o n s : The f i n a l p r o d u c t o f b o t h enzyme s y s t e m s i s ammonia. No t r a c e o f t h i s p r o d u c t was o b s e r v e d i n C h l o r e l l a c e l l s c u l t u r e d on u r e a by H a t t o r i (1958) o r S y r e t t (1953). More r e c e n t l y , however, Hodson an d Thompson (1969) o b s e r v e d an a c c u m u l a t i o n o f ammonia when C h l o r e l l a v u l g a r i s was c u l t u r e d on u r e a i n t h e p r e s e n c e o f c y a n i d e . C y a n i d e i n h i b i t s t h e m e t a b o l i s m o f ammonia t o a much g r e a t e r e x t e n t t h a n i t i n h i b i t s u r e a m e t a b o l i s m ( H a t t o r i , 1957). T h e i r r e s u l t s s u g g e s t t h a t b o t h n i t r o g e n atoms f r o m u r e a f o r m ammonia. The ammonia i s p r o b a b l y v e r y r a p i d l y m e t a b o l i z e d , a c c o u n t i n g f o r i t s l a c k o f d e t e c t i o n i n p r e v i o u s work. A f u r t h e r i n d i c a t i o n t h a t ammonia i s a p r o d u c t o f u r e a m e t a b o l i s m i s t h e e x c r e t i o n o f ammonia by o r g a n i s m s grown on u r e a as t h e s o l e n i t r o g e n s o u r c e . L i t t l e and Mah (1970) f o u n d t h a t when C h l o r e l l a e l l i p s o i d e a was c u l t u r e d on u r e a p l u s l i m i t i n g amounts o f g l u c o s e , ammonia was e x c r e t e d : r e c e n t l y U c h i d a (1975) o b s e r v e d t h e d i n o f l a g e l l a t e P r o r o c e n t r u m m i c a n s e x c r e t e d ammonia when c u l t u r e d i n m edia w i t h u r e a as t h e s o l e n i t r o g e n s o u r c e . A l t h o u g h no u n i q u e pathway f o r u r e a n i t r o g e n has b e e n d e s c r i b e d (Thompson e t a l . , 1966), t h e c o n c e n t r a t i o n o f s e v e r a l amino a c i d s i s o b s e r v e d t o i n c r e a s e c o n c o m i t a n t w i t h t h e a s s i m i l a t i o n o f u r e a i n n i t r o g e n - p o o r c e l l s . u r e a + ATP + HCO 3 •> a l l o p h a n a t e + ADP + P i a l l o p h a n a t e A f t e r up t o 30 m i n u t e s e x p o s u r e t o u r e a , t h e r e i s a s u b s t a n t i a l i n c r e a s e i n g l u t a m i c a c i d and a s i g n i f i c a n t i n c r e a s e i n g l u t a m i n e . An i n i t i a l d e c r e a s e i s f o l l o w e d by a s l i g h t i n c r e a s e i n a l a n i n e ( B a k e r and Thompson, 1 9 6 2 ) . The c o n t r o l f o r t h i s work was e x p o s e d t o NH C l . T h e s e c e l l s e x h i b i t e d a s u b s t a n t i a l i n c r e a s e i n a l a n i n e a n d a s i g n i f i c a n t i n c r e a s e i n g l u t a m i n e . H a t t o r i ( 1 9 5 7 3 1958) w i t h C h l o r e l l a e l l i p s o i d e a o b s e r v e d an i n c r e a s e i n a r g i n i n e c o r r e s p o n d i n g t o t h e r a t e o f u r e a c a t a b o l i s m . Thomas an d K r a u s s ( 1955) w i t h Scenedesmus o b l i q u u s and W a l k e r (1952) w i t h C h l o r e l l a p y r e n o i d o s a a l s o o b s e r v e d an i n c r e a s e i n a r g i n i n e p r o d u c t i o n when t h e s e o r g a n i s m s were c u l t u r e d on u r e a . Thus g l u t a m a t e i s p r o b a b l y an e a r l y p r o d u c t o f u r e a m e t a b o l i s m , f o l l o w e d a t a l o n g e r t i m e p e r i o d , by a r g i n i n e . The f a t e o f t h e u r e a c a r b o n i s a m b i g u o u s . I t was n o t f o u n d i n c o r p o r a t e d i n t o a r g i n i n e by A l l i s o n e t a l . (1954) o r H a t t o r i (I960), b u t B a k e r and Thompson (1962) o b s e r v e d some i n c o r p o r a t i o n o f t h e u r e a c a r b o n i n t o most amino a c i d s , w i t h t h e g r e a t e s t c o n c e n t r a t i o n s i n g l u t a m a t e , a s p a r t a t e and s e r i n e ( i n d e c r e a s i n g o r d e r ) . T h e r e i s e v i d e n c e t h a t a b o u t k0% o f t h e u r e a c a r b o n e n t e r s t h e CO : c a r b o n a t e c y c l e 2 J d u r i n g m e t a b o l i s m ( H a t t o r i , I960) and B a k e r and Thompson (1962) c o n c l u d e d t h a t t h e d i r e c t p r o d u c t o f u r e a c a t a b o l i s m i s C 0 2 . A s u g g e s t e d mechanism i s : 7 n i t r o g e n 2NH 3 * g l u t a m a t e - a r g i n i n e u r e a c e l l s u b s t a n c e c a r b o n *C0 I t h a s b e e n shown: t h a t u r e a i s a n d w i l l c o n t i n u e t o be a s i g n i f i c a n t component o f m a r i n e s u r f a c e w a t e r s ; t h a t t h e p r e s e n t c o n c e n t r a t i o n i s s u f f i c i e n t f o r t h e n i t r o g e n o u s n u t r i t i o n o f p h y t o p l a n k t o n ; a n d t h a t a v a r i e t y o f p h y t o p l a n k t e r s c o n t a i n s y s t e m s w h i c h m e t a b o l i z e u r e a . E v i d e n c e h a s b e e n p r e s e n t e d on two modes o f u r e a c a t a b o l i s m a n d t h e p r o b a b l e p r o d u c t s o f a s s i m i l a t i o n . Work on t h e r e l a t i o n s h i p o f u r e a a n d m a r i n e p h y t o p l a n k t o n h a s p r o v i d e d a q u a n t i t y o f d a t a . U n f o r t u n a t e l y , t h e r e s u l t s o f v a r i o u s w o r k e r s h a v e n o t b e e n i n t e g r a t e d a n d o f t e n t h e e x p e r i m e n t a l o r g a n i s m s a r e d i s s i m i l a r . ( M e t a b o l i c d a t a i s l a r g e l y b a s e d on t h e C h l o r o p h y t a , enzyme d a t a on C h l o r o p h y t a a n d y e a s t , a n d g r o w t h d a t a f r o m a v a r i e t y o f p h y t o p l a n k t e r s b u t p r i m a r i l y i n t h e B a c i l l a r i o p h y t a . ) To augment p r e s e n t k n o w l e d g e a n d t o h e l p i n t e g r a t e t h e d i v e r s i t y o f e x i s t i n g d a t a , i t was p r o p o s e d t o s t u d y t h e c y t o l o g i c a l a n d p h y s i o l o g i c a l e f f e c t s o f u r e a on a member o f a n e g l e c t e d g r o u p o f m a r i n e p h y t o p l a n k t o n . 2 Rhodomonas l e n s , a r e p r e s e n t a t i v e o f t h e C r y p t o p h y c e a e , was c h o s e n as t h e e x p e r i m e n t a l o r g a n i s m . The C r y p t o p h y c e a e a r e "a s m a l l b u t w e l l d e f i n e d g r o u p o f g e n e r a l l y o v o i d . . . o r g a n i s m s c h a r a c t e r i z e d by t h e two e q u a l o r s u b e q u a l 8 homodynamic f l a g e l l a and a f u r r o w , g r o o v e o r t u b u l a r g u l l e t on one s u r f a c e . " ( B u t c h e r , 1967) A number o f u n i q u e c h a r a c t e r i s t i c s c a n be s e e n w i t h t h e l i g h t m i c r o s c o p e . P e n a r d (1921) s t u d i e d t h e n a t u r e o f t r i c h o c y s t s i n Cryptomonas o v a t a . T h e s e t r i c h o c y s t s , o r e j e c t o s o m e s , ( d i f f e r i n g f r o m t h e t r i c h o c y s t s o f o t h e r f l a g e l l a t e s ) a r e - f o u n d l i n i n g t h e s i n g l e a n t e r i o r g u l l e t . The p e r i p l a s t c o n t a i n s t h e c e l l and i s o c c a s i o n a l l y s t r i a t e ( B u t c h e r , 1967) . E a c h c e l l c o n t a i n s one o r two c h l o r o p l a s t s and g e n e r a l l y one p y r e n o i d . A l a r g e v a r i a t i o n i n c o l o u r o c c u r s , d e p e n d e n t upon c u l t u r e c o n d i t i o n s and t h e age o f t h e c u l t u r e ( B u t c h e r , 1967). S t a r c h and l i p i d a r e p r o d u c e d as s t o r a g e p r o d u c t s . E l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s have augmented t h e knowledge o b t a i n e d f r o m u s e o f t h e l i g h t m i c r o s c o p e . D e t a i l e d f i n e s t r u c t u r a l d e s c r i p t i o n s a r e g i v e n by Dodge ( 1 9 6 9 ) , L u c a s ( 1 9 7 0 ) , H i b b a r d e t a l . ( 1 9 7 D , T a y l o r and Lee ( 1 9 7 D , and A n t i a e t a l . ( 1 9 7 3 ) . The c e l l s u r f a c e o f Chroomonas s p . i s c o v e r e d w i t h a f i b r o u s ' f u z z ' ( G a n t t , 1 9 7 1 ) ; u n d e r t h i s l a y e r t h e c e l l i s c o m p l e t e l y e n c l o s e d by a p e r i p l a s t . R e c e n t l y , s e v e r a l w o r k e r s have e x a m i n e d t h e C r y p t o m o n a d p e r i p l a s t i n d e t a i l . I t i s composed o f e i t h e r two o r t h r e e d i s t i n c t l a y e r s , one o f w h i c h i s a l w a y s t h e p l a s m a membrane (Dodge, 1 9 6 9 ; G a n t t ; 1971 , F a u s t , 1 9 7 4 ) . Chroomonas m e s o s t i g m a t i c a has a t r i p l e l a y e r e d p e r i p l a s t w i t h a b o u t 7 o b l i q u e g r o o v e s a d j a c e n t t o w h i c h t h e s m a l l e j e c t o s o m e s a r e l o c a t e d (Dodge, 1 9 6 9 ) ; Chroomonas s p . has a 9 t r i p a r t i t e p e r i p l a s t w i t h a t h i c k n e s s o f a b o u t 250 A i n c r o s s s e c t i o n ( G a n t t , 1 9 7 1 ) ; C r y p t o m o n a s o v a t a v a r . p a l u s t r i s has a b i p a r t i t e p e r i p l a s t composed o f p o l y g o n a l p l a t e s w h i c h a r e d e l i n e a t e d by s h a l l o w r i d g e s ( F a u s t , 1 9 7 4 ) . The p e r i p l a s t , t h e n , c o n s i s t s o f t h e p l a s m a membrane and f u r t h e r l a y e r s w h i c h a r e a p p r e s s e d t o i t s o u t e r and i n n e r s u r f a c e s , e x c e p t o v e r t h e f l a g e l l a r a nd g u l l e t r e g i o n where t h e p l a s m a membrane i s n o t c o v e r e d ( G a n t t , 1971; F a u s t , 1 9 7 4 ) . The i n n e r l a y e r i s composed o f p l a t e s and i t i s t h e a r r a n g e m e n t and p o s s i b l y t h e shape o f t h e s e p l a t e s a n d t h e a s s o c i a t e d e j e c t o s o m e s w h i c h g i v e t h e c e l l i t s d i s t i n c t s u r f a c e s t r u c t u r e ( B u t c h e r , 1967; Dodge, 1 9 6 9 ; L u c a s , 1970; G a n t t , 1971 ; F a u s t , 1 9 7 4 ) . The e j e c t o s o m e s have b e e n d e s c r i b e d by D r a g e s c o ( 1 9 5 1 ) , A n d e r s o n ( 1 9 6 2 ) , J o y o n ( 1 9 6 3 ) , H o v a s s e , M i g n o n and J o y o n ( 1 9 6 7 ) , and S c h u s t e r ( 1 9 7 0 ) . They a r e b o d i e s o f two d i s t i n c t s i z e s h a v i n g a r e g u l a r g e o m e t r i c shape and a complex i n t e r n a l s t r u c t u r e ( A n d e r s o n , 1 9 6 2 ; H o v a s s e e t a l . , 1 9 6 7 ) . H o v a s s e e t a l . (1967) s u g g e s t e d t h a t t h e y c o n s i s t o f two u n e q u a l c o mponents, e a c h p o s s i b l y c u r l e d i n a t i g h t s p i r a l and e n c l o s e d by a t h i n membrane. The l a r g e e j e c t o s o m e s a r e a b o u t 500 nm i n d i a m e t e r a n d 400 nm deep and a r e f o u n d w i t h i n t h e a n t e r i o r f u r r o w ( B u t c h e r , 1 9 6 7 ; H o v a s s e e t a l . , 1967; Dodge, 19.69; L u c a s , 1 9 7 0 ) . The s m a l l e r e j e c t o s o m e s , a b o u t 200 nm i n d i a m e t e r , a r e p a r t o f th e p e r i p l a s t a nd a p p e a r t o be on t h e e x t e r i o r o f t h e c e l l , s e p a r a t e d f r o m t h e c y t o p l a s m by t h e p l a s m a membrane ( L u c a s , 1 9 7 0 ; G a n t t , 1 9 7 D . The s i n g l e c h l o r o p l a s t i s b i l o b e d , t h e 10 two l o b e s b e i n g c o n n e c t e d by a g r a n u l a r p y r e n o i d ( G i b b s , 1962, a, b; Dodge, 1969; L u c a s , 1970; G a n t t , Edwards and P r o v a s o l i , 1971). The t h y l a k o i d s a r e g e n e r a l l y a s s o c i a t e d i n bands o f two o r more ( C r y p t o m o n a s c r y o p h i l a has an a t y p i c a l c h l o r o p l a s t i n w h i c h t h e t h y l a k o i d s a r e a r r a n g e d i n l a y e r s o f v a r i a b l e numbers, T a y l o r a n d L e e , 1971.) s e p a r a t e d by a s p a c e o f 3-8 nm ( G i b b s , 1962, a, b; Wehrmeyer, 1970; G a n t t , Edwards and P r o v a s o l i , 1971; B i s a l p u t r a , 1974) a n d a r e o f t e n f o u n d p e n e t r a t i n g o r t r a v e r s i n g t h e p y r e n o i d ( G i b b s , 1962, a, b; Wehrmeyer, 1970; G a n t t e t a l . , 1971). The t h y l a k o i d l o c u l u s r a n g e s f r o m 10-50 nm, d e p e n d e n t on t h e genus a n d c o n t a i n s - - a f i n e g r a n u l a r , e l e c t r o n opaque m a t r i x (Wehrmeyer, 1970; G a n t t e t a l . , 1971). G a n t t e t a l . (197D and F a u s t and G a n t t (1973) s u g g e s t t h a t t h i s m a t r i x c o n t a i n s t h e p h y c o b i l i n p r o t e i n s . They f o u n d no e v i d e n c e o f p h y c o b i l i s o m e -l i k e a g g r e g a t e s . The c h l o r o p l a s t a n d p y r e n o i d a r e e n c l o s e d by a double-membrane e n v e l o p e a n d , o u t s i d e o f i t , by a s a c o f e n d o p l a s m i c r e t i c u l u m w h i c h i s c o n t i n u o u s w i t h t h e n u c l e a r e n v e l o p e ( G i b b s , 1962 a; Wehrmeyer, 1970; G a n t t e t a l . , 1971)• S t a r c h g r a i n s a r e f o r m e d a r o u n d t h e p y r e n o i d and w i t h i n t h e p e r i c h l o r o p l a s t i c m a t r i x ( G i b b s , 1962 a, b; Wehrmeyer, 1970; G a n t t e t a l . , 1971; B i s a l p u t r a , 1974). The i n t e r p h a s e n u c l e u s i s l o c a t e d p o s t e r i o r l y a n d c o n t a i n s a p r o m i n e n t n u c l e o l u s and many c h r o m a t i n a r e a s (Dodge, 1969; L u c a s , 1970; T a y l o r and L e e , 1971; O a k l e y , 1974). I t i s f r e q u e n t l y t r a n s e c t e d by a n a r r o w t o n g u e o f c y t o p l a s m (Dodge, 1969; T a y l o r a n d L e e , 1971). P r i o r t o 11 c e l l d i v i s i o n t h e n u c l e u s moves t o a more c e n t r a l p o s i t i o n ( O a k l e y , 1 9 7 4 ) . The m i t o c h o n d r i a a r e l a r g e and o f v a r i a b l e shape w i t h u n i f o r m f l a t t e n e d c r i s t a e (Dodge, 1969; L u c a s , 1970; T a y l o r and L e e , 1971 ; T a y l o r , 1971)• The C o r p s de Maupas, d e s c r i b e d by L u c a s (1970) as a r o u g h l y s p h e r i c a l body c o n t a i n i n g s h o r t s t a c k s o f f i b r i l s , numerous s m a l l v e s i c l e s and s h o r t l e n g t h s o f membrane e n c l o s e d i n a s i n g l e smooth membrane, i s u s u a l l y s i t u a t e d n e a r t h e g o l g i a p p a r a t u s (Dodge, 1 9 6 9 ; L u c a s , 1 9 7 0 ; T a y l o r and Lee,"" 1 9 7 1 ) . The two f l a g e l l a a r e a l i k e b u t may be e q u a l o r u n e q u a l i n l e n g t h ( B u t c h e r , 1967; Dodge, 1 9 6 9 ; L u c a s , 1970; T a y l o r a n d L e e , 1 9 7 1 ) . M a s t i g o n e m e s a r e f o u n d o v e r t h e e n t i r e f l a g e l l a r l e n g t h ( L u c a s , 1 9 7 0 ; T a y l o r and L e e , 1 9 7 1 ) . The u s u a l 9 + 2 axoneme a r r a n g e m e n t i s p r e s e n t : a t a p o i n t j u s t p r i o r t o f l a g e l l a r i n s e r t i o n i n t h e mouth o f t h e g u l l e t t h e s h e a t h i s c o n s t r i c t e d so t h a t i t t o u c h e s t h e 9 o u t e r d o u b l e t s w h i l e a t t h e same p o i n t t h e c e n t r a l 2 t u b u l e s p a s s t h r o u g h a d i s c o r t r a n s v e r s e septum (Dodge, 1969; L u c a s , 1 9 7 0 ) . About 100 nm n e a r e r t o t h e c e l l t h e c e n t r a l t u b u l e s t e r m i n a t e a t a s e c o n d d i s c (Dodge, 1 9 6 9 ; L u c a s , 1 9 7 0 ) . W i t h i n t h e c e l l t h e 9 d o u b l e t s become t r i p l e t s . The two b a s a l b o d i e s a r e c o n n e c t e d by a ' b l o c k o f d e n s e l y s t a i n i n g m a t e r i a l ' (Dodge, 1969) and l i e c l o s e t o g e t h e r and a l m o s t p a r a l l e l (Dodge, 1969; L u c a s , 1 9 7 0 ) . S m a l l g r o u p s o f m i c r o t u b u l e s r u n f r o m t h e f l a g e l l a r b a s e s p o s t e r i o r l y t o w a r d t h e n u c l e u s (Dodge, 1969; O a k l e y , 1 9 7 4 ) . 12 The c y t o p l a s m a l s o c o n t a i n s many r i b o s o m e s , r o u g h e n d o p l a s m i c r e t i c u l u m , s m a l l v e s i c l e s , a n d o c c a s i o n a l l i p i d g l o b u l e s (Dodge, 1969; L u c a s , 1970; T a y l o r and L e e , 1971; A n t i a e t a l . , 1973; O a k l e y , 197*0. METHODS AND MATERIALS I . C u l t u r e s The c u l t u r e o f Rhodomonas l e n s , P a s c h e r and R u t t n e r , 3 was o b t a i n e d f r o m Dr. N. A n t l a o f t h e F i s h e r i e s R e s e a r c h B o a r d o f Canada, V a n c o u v e r , B.C. One ml o f t h i s c u l t u r e was a s e p t i c a l l y t r a n s f e r r e d t o e x p e r i m e n t a l f l a s k s c o n t a i n i n g e n r i c h e d s y n t h e t i c s e a w a t e r . The c o n t e n t s p e r l i t e r o f t h i s medium a r e g i v e n i n T a b l e 1. The medium i s w i t h o u t combined n i t r o g e n . N i t r o g e n was a d d e d as e i t h e r p o t a s s i u m n i t r a t e o r u r e a . M e d i a were s t e r i l i z e d by f i l t r a t i o n . S t o c k c u l t u r e s were m a i n t a i n e d on two t y p e s o f m e d i a , one w i t h 0.5 mM KNO^ and one w i t h 1.25 mM u r e a . To t e s t f o r b a c t e r i a l c o n t a m i n a n t s , p e r i o d i c a l l y two d r o p s o f t h e s t o c k c u l t u r e s were ad d e d a s e p t i c a l l y t o e i t h e r s t e r i l i t y medium STP o r ST^ ( s e e T a b l e 2). T h e s e were k e p t i n t h e d a r k a t room t e m p e r a t u r e (22°C) and were o b s e r v e d a f t e r one week f o r t u r b i d i t y i n t h e l i q u i d m e d i a as e v i d e n c e o f b a c t e r i a l g r o w t h . C u l t u r e s were a l s o e x a m i n e d m i c r o s c o p i c a l l y a t e a c h t r a n s f e r f o r c o n t a m i n a n t s . I I . G e n e r a l Methods 1. Growth Measurements Growth o f t h e c u l t u r e s was f o l l o w e d by t u r b i d i m e t r i c d e t e r m i n a t i o n a t 600 nm on a S a r g e n t - W e l c h SM s p e c t r o p h o t o m e t e r i n 13 mm p r e c a l i b r a t e d t u b e s a n d / o r by d i r e c t c e l l c o u n t u s i n g a S p e n c e r B r i g h t L i n e i m p r o v e d 14 Table 1. Enriched synthetic sea water medium. NaH 2P0 4.H 20 Na 2Si0 3.9H 20 Vitamins: Thiamin.HCI B i o t i n B 1 2 Buffer: TRIS.HCI 6.9 mg 84.0 mg 1.25 mg 2.50 mg 5.00 mg 200 mis Trace metals (chelated) 16.2 mg Na EDTA.2H 0 2 2 FeCl .6H 0 3 2 MnSO,.4H 0 4 2 ZnSO..7H 0 4 2 NaJMoO., .2H 0 2 4 2 CuS0^.5H20 CoSO,.5H 0 4 2 (41.3 mM, pH 6.8) Synthetic sea water (to one l i t e r ) 'Synthetic sea water ( S a l i n i t y = 28 parts ^oo.) 5.4 mg 2.25 mg 1.15 mg 0.586 mg 0.050 mg 0.028 mg NaCl MgCl .6H 0 a 2 2 Na 2S0 l j CaCl 2.6H 20 KCI NaHC0_ 23.48 gms 10.13 gms 3.92 gms 2.17 gms 0.66 gms 0.19 gms .KBr., • .. 96 mg .' 26 mg 40 mg NaF 3 mg d i s t i l l e d water 950 mis H 3B0 3 SrCl .6H 0 2 2. 15 T a b l e 2. S t e r i l i t y m e d i a . ( T a t e w a k i and P r o v a s o l i , 1964) STP ST, s e a w a t e r 80 ml J 70 ml H 2 0 15 ml 25 ml s o i l e x t r a c t 5 ml 5 ml NaNO 3 20 mg 5 mg K 2 H P 0 i | 1 mg N a ^ g l y c e r o p h o s p h a t e 1 mg Hy-Case ( S h e f f i e l d C h e m i c a l ) 2 mg Y e a s t a u t o l y s a t e (N.B.C.) 20 mg Y e a s t e x t r a c t ( D i f c o ) 1 mg L i v e r o x o i d L-25 (Oxo, L t d . ) 2 mg B 1 2 0.01 ug V i t a m i n mix 8A 0. 1 ml 0.10 ml S u c r o s e 100 mg 2 C - s o u r c e Mix I I 2 ml N a H - g l u t a m a t e 50 mg D L - a l a n i n e 10 mg T r y p t i c a s e (BBL) 20 mg G l y c i n e 10 mg G l y c y l g l y c i n e 40 mg PH 7-5- 7.6 7.9 One ml o f V i t a m i n mix 8A c o n t a i n s : t h i a m i n e . H C 1 0.2 mg; n i c o t i n i c a c i d 0.1 mg; p u t r e s c i n e . 2 H C 1 0.04 mg; c o b a l t p a n t o t h e n a t e 0.1 mg; r i b o f l a v i n 5 ug; p y r i d o x i n e . 2 H C 1 0.04 mg; 16 T a b l e 2. ( c o n t i n u e d ) pyridoxamine. 2 H C 1 0.02 mg; p - a m i n o b e n z o i c a c i d 0.01 mg; b i o t i n 0.05 ug; c h o l i n e h y d r o g e n c i t r a t e 0.5 mg; i n o s i t o l 1.0 mg; t h y m i n e 0 .8 mg; o r o t i c a c i d 0.26 mg; B 0.05 P-g', f o l i c a c i d 2.5 Jig; f o l i n i c a c i d 0.2 jag. 2 C - s o u r c e s Mix I I c o n t a i n s : g l y c i n e 100 mg; D L - a l a n i n e 100 mg; L - a s p a r a g i n e 100 mg; s o d i u m a c e t a t e . 3H 0 200 mg; g l u c o s e 200 mg; L - g l u t a m i c a c i d 200 mg; H^O 100 m l . 1 7 Neubauer h a e m o c y t o m e t e r . The c e l l s were i m m o b i l i z e d f o r d i r e c t c e l l c o u n t s by a d d i n g one d r o p o f 0.5% g l u t a r a l d e h y d e t o a one ml a l i q u o t o f c u l t u r e . The g e n e r a t i o n t i m e was c a l c u l a t e d f r o m t h e e q u a t i o n s : n = l o g i o y ~ l o g i o x 301 and g = t , n where n i s t h e number o f g e n e r a t i o n s , y i s t h e l a r g e r ' number o f o r g a n i s m s a t t h e end o f a t i m e p e r i o d t , x i s t h e s m a l l e r number o f o r g a n i s m s a t t h e b e g i n n i n g o f a t i m e p e r i o d t , and g i s t h e g e n e r a t i o n t i m e . 2. U r e a A n a l y s i s The amount o f u r e a i n t h e m e d i a was d e t e r m i n e d by t h e method o f N e w e l l e t a l . ( s e e T a b l e 3 ) . A f i v e ml a l i q u o t o f t h e c u l t u r e t o be a n a l y s e d was c e n t r i f u g e d a t 6500 x g i n 7 a n I n t e r n a t i o n a l HN c e n t r i f u g e f o r f i v e m i n u t e s . ' The s u p e r n a t a n t was r e s e r v e d and f r o z e n . When s e v e r a l s a m p l e s had a c c u m u l a t e d t h e s a m p l e s were thawed and t h e a n a l y s i s i m m e d i a t e l y c o n d u c t e d . 3. M i c r o s c o p y M a t e r i a l f o r l i g h t m i c r o s c o p y was p r e p a r e d by a d d i n g one d r o p o f 0.5% g l u t a r a l d e h y d e t o one ml o f c u l t u r e . M i c r o g r a p h s o f t h e f r e s h l y f i x e d c u l t u r e were o b t a i n e d u s i n g a Z e i s s P h o t o m i c r o s c o p e w i t h phase c o n t r a s t o r N o m a r s k i 18 T a b l e 3 . The d e t e r m i n a t i o n o f u r e a i n s e a w a t e r ( N e w e l l , Morgan,' and Cundy, 1967) R e a g e n t s : A) D i s s o l v e 85 gm Nal^PO^.2 H 0 i n 1 l i t e r o f c o n c e n t r a t e d H SO,. B) D i s s o l v e 5 gm d i - a c e t y l monoxime 2 4 i n 100 ml w a t e r . Warm t o a s s i s t s o l u t i o n . C o o l , and add 0 . 0 6 gm s e m i - c a r b a z i d e HC1. C) D i s s o l v e 200 gm MnCl .4H 0 a n d 4 gm KNO i n 500 ml w a t e r . D) Mix e q u a l q u a n t i t i e s o f B) and C ) . c u l t u r e 1 c e n t r i f u g e s u p e r n a t a n t p e l l e t d i l u t e ( 5 - 1 0 ugN/1) 1 add 1 ml t o 29 ml d i s t i l l e d w a t e r i n t e s t t u b e , i add 5 gm NaCI, 4 ml r e a g e n t A, 1 ml r e a g e n t D \ mix w e l l 1 o immerse t e s t t u b e i n w a t e r b a t h a t ' 70 C f o r 90 m i n . 1 c o o l r a p i d l y 1 measure a b s o r b a n c e i n a 13 mm t u b e a t 5200 A A c a l i b r a t i o n f a c t o r i s d e t e r m i n e d i n 20$ NaCI s o l u t i o n f r o m a s t o c k u r e a s o l u t i o n c o n t a i n i n g 500 ug N p e r ml a n d d i l u t e d 100 t i m e s j u s t b e f o r e u s e . Reagent b l a n k s a r e a l s o d e t e r m i n e d i n 20% NaCI s o l u t i o n . i n t e r f e r e n c e i l l u m i n a t i o n s y s t e m . M a t e r i a l f o r t h i n s e c t i o n i n g was c o n c e n t r a t e d by c e n t r i f u g a t i o n a t 3 6 0 0 x g f o r f i v e m i n u t e s i n an I n t e r n a t i o n a l HN c e n t r i f u g e . The f i x a t i o n and d e h y d r a t i o n s c h e d u l e s a r e shown i n T a b l e s 4 and 5 . A f t e r d e h y d r a t i o n t h e c e l l s were i n f i l t r a t e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f S p u r r ' s embedding medium ( S p u r r , 1 9 6 9 ) i n e t h a n o l and c u r e d i n 100% r e s i n . S e c t i o n s were c u t on e i t h e r a R e i c h e r t OMU 3 u l t r a m i c r o t o m e o r a P o r t e r B l u m MT 3 u l t r a m i c r o t o m e u s i n g a duPont diamond k n i f e a n d were p o s t - s t a i n e d f o r 20 m i n u t e s w i t h 5% u r a n y l a c e t a t e i n 50% e t h a n o l and t h e n f o r 10 m i n u t e s w i t h R e y n o l d ' s l e a d c i t r a t e ( R e y n o l d s , 1 9 6 3 ) . S e c t i o n s were v i e w e d w i t h e i t h e r a Z e i s s EM 9S e l e c t r o n m i c r o s c o p e o r an AEI-6B e l e c t r o n m i c r o s c o p e . I I I . E x p e r i m e n t a l Methods 1. A c c l i m a t i o n o f C u l t u r e s t o M e d i a The o r g a n i s m s were a d a p t e d f r o m a n i t r a t e e n r i c h e d , n a t u r a l s e a w a t e r medium t o e n r i c h e d s y n t h e t i c s e a w a t e r p l u s 0 . 5 mM KNO^ a n d t h e n t o 1 . 2 5 mM u r e a i n e n r i c h e d s y n t h e t i c s e a w a t e r . T h i s was a c h i e v e d by a s e p t i c a l l y t r a n s f e r r i n g a one ml a l i q u o t o f t h e o r i g i n a l c u l t u r e t o d u p l i c a t e 50 ml E r l e n m e y e r f l a s k s c o n t a i n i n g 20 mis o f e n r i c h e d s y n t h e t i c s e a 1 w a t e r p l u s 0 . 5 mM KNO^. When good g r o w t h was o b s e r v e d , a one ml a l i q u o t f r o m t h e s e f l a s k s was t r a n s f e r r e d t o a d u p l i c a t e s e t o f f l a s k s a n d s i m i l a r l y a t h i r d t r a n s f e r was made. When t h r e e t r a n s f e r s r e s u l t i n g 20 T a b l e 4. F i x a t i o n ( G a n t t , Edwards, and P r o v a s o l i , 1971) and d e h y d r a t i o n s c h e d u l e I Medium Time 2% g l u t a r a l d e h y d e added d i r e c t l y t o l i q u i d c u l t u r e i n 1 : 1 p r o p o r t i o n 1 h o u r 0.1M s o d i u m c a c o d y l a t e b u f f e r w i t h 2.5% s u c r o s e (pH 7.4) 15 m i n u t e s 0.1M s o d i u m c a c o d y l a t e b u f f e r w i t h 1.0% s u c r o s e 15 m i n u t e s 0.1M s o d i u m c a c o d y l a t e b u f f e r 15 m i n u t e s 1.0% OsO^, b u f f e r 1 h o u r b u f f e r 10 m i n u t e s b u f f e r 10 m i n u t e s 20$ m e t h a n o l 15 m i n u t e s 50% m e t h a n o l 15 m i n u t e s 70$ m e t h a n o l 15 m i n u t e s 90% m e t h a n o l 15 m i n u t e s 100$ m e t h a n o l 30 m i n u t e s 100$ m e t h a n o l 30 m i n u t e s 21 T a b l e 5- F i x a t i o n and d e h y d r a t i o n s c h e d u l e I I ( O a k l e y , p e r s o n a l c o m m u n i c a t i o n ) Medium Time 1% g l u t a r a l d e h y d e , b u f f e r , 0.3 M 0.125 M s u c r o s e , p h o s p h a t e pH 7.4 1 h o u r b u f f e r j 0.15 M s u c r o s e 15 m i n u t e s b u f f e r 10 m i n u t e s 1% OsO^, b u f f e r 1 h o u r b u f f e r 10 m i n u t e s d i s t i l l e d w a t e r 10 m i n u t e s 25% e t h a n o l 30 m i n u t e s 50% e t h a n o l 30 m i n u t e s 75$ e t h a n o l 30 m i n u t e s a b s o l u t e e t h a n o l 30 m i n u t e s a b s o l u t e e t h a n o l 30 m i n u t e s a b s o l u t e e t h a n o l 30 m i n u t e s 22 i n good g r o w t h were c o m p l e t e d , t h e c u l t u r e was c o n s i d e r e d t o be a c c l i m a t e d t o KNO i n e n r i c h e d s y n t h e t i c s e a w a t e r . T h e s e a r e t e r m e d t h e " c o n t r o l " c u l t u r e s and m e d i a . T h i s c u l t u r e was t h e n a c c l i m a t e d t o 1.25 a n d 12.5 mM u r e a i n e n r i c h e d s y n t h e t i c s e a w a t e r u s i n g a s i m i l a r p r o c e d u r e . D u r i n g t h e a c c l i m a t i o n t o t h e use o f u r e a , t r a n s f e r s were made a t one week i n t e r v a l s e v e n i f good g r o w t h was n o t v i s i b l e . When good g r o w t h was o b s e r v e d f o l l o w i n g t h r e e o r more t r a n s f e r s , t h e c u l t u r e s were c o n s i d e r e d t o be a c c l i m a t e d t o t h e u r e a m e d i a . T h e s e a r e t e r m e d t h e " e x p e r i m e n t a l " c u l t u r e s and m e d i a . 2. L i g h t a n d . T e m p e r a t u r e To d e t e r m i n e t h e o p t i m a l t e m p e r a t u r e and l i g h t c o n d i t i o n s f o r good g r o w t h , a one ml i n n o c u l u m f r o m c o n t r o l c u l t u r e s was a s e p t i c a l l y t r a n s f e r r e d t o 50 ml E r l e n m e y e r f l a s k s c o n t a i n i n g 20 mis o f c o n t r o l medium. D u p l i c a t e f l a s k s were i n c u b a t e d a t 7°, 10°, 15°, a n d 22°C a n d a 16:8 h o u r , l i g h t : d a r k c y c l e , and a t 22°C w i t h c o n t i n u o u s l i g h t . Q I l l u m i n a t i o n was w i t h f l u o r e s c e n t l i g h t a t a p p r o x i m a t e l y 150 f o o t - c a n d l e s . S u b j e c t i v e c o m p a r i s o n s were made b e t w e e n t h e g r o w t h and p i g m e n t a t i o n o f t h e c e l l s i n e a c h p a i r o f f l a s k s a f t e r one and two weeks o f i n c u b a t i o n . 3. U r e a E f f e c t s U r e a was added t o s y n t h e t i c s e a w a t e r t o g i v e f i n a l c o n c e n t r a t i o n s o f 0.10, 0.25, 0.50, 1.25, 6.25, and 12.50 23 mM u r e a . A one ml i n n o c u l u m f r o m t h e e x p e r i m e n t a l s t o c k c u l t u r e was a s e p t i c a l l y t r a n s f e r r e d t o 125 ml E r l e n m e y e r f l a s k s c o n t a i n i n g 50 mis o f t h e e x p e r i m e n t a l medium. D u p l i c a t e f l a s k s were p r e p a r e d f o r e a c h c o n c e n t r a t i o n : o t h e s e were i n c u b a t e d a t 22 C and a 16:8 h o u r , l i g h t : d a r k c y c l e . T u r b i d i m e t r i c r e a d i n g s and d i r e c t c e l l c o u n t s were made a t a p p r o x i m a t e l y 24 h o u r i n t e r v a l s . The pH change d u r i n g g r o w t h was f o l l o w e d u s i n g a R a d i o m e t e r PHM 63 pH m e t e r a n d t h e amount o f u r e a p r e s e n t i n t h e c u l t u r e s a t 1.25 and 12.50 mM u r e a was d e t e r m i n e d a t e a c h t i m e i n t e r v a l ( s e e T a b l e 3, p . l 8 ) . C u l t u r e s a t 1.25 and 12.50 mM u r e a were s a m p l e d a t 48 h o u r i n t e r v a l s and t h e m a t e r i a l p r e p a r e d f o r e l e c t r o n m i c r o s c o p y ( s e e T a b l e s 4 and 5 S pp.20, 21. ). S i m i l a r d e t e r m i n a t i o n s were made on c o n t r o l c u l t u r e s a t 0.50 and 2.50 mM KNO^ i n e n r i c h e d s y n t h e t i c s e a w a t e r . 4. pH E f f e c t s The e n z y m a t i c breakdown o f one mole o f u r e a y i e l d s one mole o f CO and two moles o f NH . E x c e s s i v e c a t a b o l i s m o f 2 3 u r e a c o u l d l e a d t o ammonia p o i s o n i n g a n d / o r a l e t h a l change o f pH. T h e r e f o r e , t h e s e e f f e c t s were s i m u l a t e d i n a h e a l t h y c o n t r o l c u l t u r e . The e f f e c t s o f a h i g h pH were o b s e r v e d by a r t i f i c i a l l y a d j u s t i n g t h e pH between 8.2 and 9-2 a t i n t e r v a l s o f 0.2 pH u n i t s w i t h NaOH. The e f f e c t s o f an i n c r e a s e d pH a n d an i n c r e a s e d c o n c e n t r a t i o n o f ammonia were o b s e r v e d by a d j u s t i n g t h e pH t o t h e same pH i n t e r v a l s w i t h NH^OH. P e r i o d i c a l l y an a l i q u o t f r o m e a c h was removed and g r o s s s t r u c t u r a l c h a n g e s were o b s e r v e d on t h e Z e i s s P h o t o m i c r o s c o p e . The t i m e r e q u i r e d a t e a c h pH i n t e r v a l f o r a change i n p i g m e n t a t i o n and f o r c u l t u r e l y s i s t o o c c u r was m e a s u r e d . RESULTS 1. A c c l i m a t i o n o f C u l t u r e s t o M e d i a The r e s u l t s f o r t h e a c c l i m a t i o n o f t h e o r i g i n a l c u l t u r e t o t h e e x p e r i m e n t a l m e d i a a r e summarized i n T a b l e 6. The c a t e g o r i e s u s e d a r e b r o a d as t h e r e was v a r i a t i o n i n t h e M c o n c e n t r a t i o n ( t h e t o t a l p o p u l a t i o n o f c e l l s p e r ml a t t h e end o f s t a t i o n a r y p h a s e ) and i n t h e c u l t u r e p i g m e n t a t i o n among i n d i v i d u a l c u l t u r e s w h i c h were u n d e r a p p a r e n t l y i d e n t i c a l c u l t u r e c o n d i t i o n s . T h e s e c a t e g o r i e s a r e as f o l l o w s : ( i ) +++, t h e c u l t u r e was t u r b i d when s h a k e n a n d a p p e a r e d r e d d i s h - p i n k ; ( i i ) ++, t h e c u l t u r e was t u r b i d when s h a k e n and a p p e a r e d y e l l o w - o r a n g e ; ( i i i ) + , t h e c u l t u r e was v i s i b l e b e f o r e . s h a k i n g a s t i n y p i n - p r i c k s p o t s on t h e b o t t o m o f t h e f l a s k and a f t e r s h a k i n g p r o d u c e d v e r y l i t t l e t u r b i d i t y and a p p e a r e d c o l o u r l e s s ; ( i v ) MV, v i a b l e ( M o t i l e c e l l s o n l y were a r b i t r a r i l y t a k e n as a v i s i b l e s i g n o f v i a b i l i t y d u r i n g m i c r o s c o p i c e x a m i n a t i o n . ) c e l l s were v i s i b l e o n l y w i t h m i c r o s c o p i c e x a m i n a t i o n a n d t h e c u l t u r e a p p e a r e d c l e a r a n d c o l o u r l e s s ; ( v) -, no v i a b l e c e l l s were o b s e r v e d . W h i l e a c c l i m a t i o n o f t h e o r i g i n a l c u l t u r e t o c o n t r o l m e d i a p r e s e n t e d no d i f f i c u l t i e s and was e a s i l y d u p l i c a t e d (Good g r o w t h was o b s e r v e d i n e a c h o f t h r e e d i f f e r e n t e x p e r i m e n t a l r u n s . ) , t h e a c c l i m a t i o n o f t h e c o n t r o l c u l t u r e t o e x p e r i m e n t a l m e d i a was d i f f i c u l t a nd t h e d u p l i c a t i o n o f t h e r e s u l t s was e r r a t i c ( s e e T a b l e 6). T a b l e 6 . A c c l i m a t i o n o f t h e c o n t r o l c u l t u r e t o e n r i c h e d s y n t h e t i c s e a w a t e r p l u s 1.25 mM u r e a . The r e s u l t s o f s e v e r a l d i f f e r e n t e x p e r i m e n t a l r u n s a r e c o m p i l e d u n d e r e a c h e x p e r i m e n t . ( E x p e r i m e n t 3 i s a s y n t h e s i s o f 50 d i f f e r e n t t e s t t u b e s , t h e o t h e r two e x p e r i m e n t s were r u n w i t h d u p l i c a t e t e s t t u b e s . ) A f t e r an i n c u b a t i o n p e r i o d o f one week t h e t y p e o f g r o w t h was s u b j e c t i v e l y d e t e r m i n e d and an i n n o c u l u m t r a n s f e r r e d t o f r e s h m e d i a . The c u l t u r e was c o n s i d e r e d a c c l i m a t e d when good g r o w t h (+++) r e s u l t e d a f t e r t h r e e t r a n s f e r s . E x p e r i m e n t Growth i n c o n t r o l m e d i a Growth i n e x p e r i m e n t a l m e d i a T r a n s f e r 1 T r a n s f e r 2 T r a n s f e r 3 1 +++ ++ M V M V 2 +++ ++ ** — 3 +++ ++ +++ L e g e n d : +++, t h e c u l t u r e i s t u r b i d when s h a k e n a n d a p p e a r s r e d d i s h - p i n k ; ++, t h e c u l t u r e i s t u r b i d when s h a k e n and a p p e a r s y e l l o w - o r a n g e ; + , t h e c u l t u r e i s v i s i b l e b e f o r e s h a k i n g as t i n y p i n - p r i c k s p o t s on t h e b o t t o m o f t h e f l a s k and a f t e r s h a k i n g p r o d u c e s v e r y l i t t l e t u r b i d i t y and a p p e a r s c o l o u r l e s s ; M V , v i a b l e c e l l s a r e v i s i b l e o n l y w i t h l i g h t m i c r o s c o p i c e x a m i n a t i o n and t h e c u l t u r e a p p e a r s c o l o u r l e s s ; -, no v i a b l e c e l l s a r e o b s e r v e d . C u l t u r e i n t e r m e d i a t e between + and ++ i n t h a t y e l l o w -o r a n g e p i n - p r i c k c o l o n i e s a r e o b s e r v e d . 27 2. L i g h t and T e m p e r a t u r e The s h o r t e s t l a g p e r i o d and t h e b e s t c u l t u r e c o l o u r were o b s e r v e d a t 22°C w i t h a 16:8 h o u r , l i g h t : d a r k c y c l e ( s e e T a b l e 7). As s e e n f r o m T a b l e 7, an i n c r e a s e d l a g p e r i o d o c c u r r e d w i t h i n c u b a t i o n a t t h e l o w e r t e m p e r a t u r e s and p o o r c u l t u r e p i g m e n t a t i o n was o b s e r v e d w i t h c o n t i n u o u s i l l u m i n a t i o n . 3 . Growth ( i ) M e a s urements A g r o w t h p a t t e r n t y p i c a l o f t h e c o n t r o l c u l t u r e s i n c u b a t e d a t 22°C w i t h a 16:8 h o u r , l i g h t : d a r k c y c l e i s shown i n F i g u r e 1. The s h o r t l a g p h a s e was f o l l o w e d by an e x t e n d e d p e r i o d o f l o g a r i t h m i c g r o w t h , t h e n by a s t a t i o n a r y p h a s e . D u r i n g t h e s t a t i o n a r y p h a s e v e r y l i t t l e c e l l d i v i s i o n o c c u r r e d and t h e c u l t u r e c o l o u r c h a n g e d f r o m a c l e a r r e d d i s h - p i n k t o a d u l l e r r e d d i s h - o r a n g e . Many c e l l s a p p e a r e d t o be n o n - m o t i l e b u t no o t h e r g r o s s s t r u c t u r a l a b n o r m a l i t i e s were o b s e r v e d . G r a d u a l l y t h e d e a t h o r d e c l i n e 9 p h a s e b e g a n d u r i n g w h i c h t h e c u l t u r e p i g m e n t a t i o n o c c a s i o n a l l y t u r n e d g r e e n and e v i d e n c e o f c e l l l y s i s s u c h as s t a r c h g r a i n s and membrane f r a g m e n t s c o u l d be s e e n w i t h t h e l i g h t m i c r o s c o p e . ( i i ) U l t r a s t r u c t u r e The u l t r a s t r u c t u r e o f c o n t r o l c e l l s d u r i n g t h e l a g and l o g a r i t h m i c p h a s e s was s i m i l a r t o t h a t d e s c r i b e d f o r t h e 28 T a b l e 7- A c o m p a r i s o n o f g r o w t h i n c o n t r o l m e d i a a t v a r i o u s t e m p e r a t u r e s a n d l i g h t p e r i o d s . P a r a m e t e r s u s e d a r e l a g p e r i o d a n d p i g m e n t a t i o n o f t h e c u l t u r e , d u r i n g l o g a r i t h m i c p h a s e . A d a s h i n d i c a t e s no g r o w t h . Growth < T e m p . ' ° C c o n d i t i o n s I l l u m i n a t i o n L a g p e r i o d P i g m e n t a t i o n d u r i n g l o g p h a s e 7 16:8, l i g h t : d a r k 10 — --15 ti 3 weeks c l e a r r e d - p i n k 22 II 3 days c l e a r r e d - p i n k 22 c o n t i n u o u s 2 d ays y e l l o w - o r a n g e g u r e 1. Growth p a t t e r n o f c u l t u r e i n c u b a t e d i n c o n t r o l m e d i a . C i r c l e s i n d i c a t e 0.5 mM KNO^; t r i a n g l e s , i n d i c a t e 1.0 mM KNO^. S o l i d s ymbols a r e t u r b i d i m e t r i c d e t e r m i n a t i o n s , open s y m b o l s i n d i c a t e c e l l c o u n t . 30 o O 0 • N o «n «r " c< .-0 o o o o o o o 1 i i i i i J L o o o o o o o o <0 N « 10 « «*> c« "" 1 W / I N O O D 113D C r y p t o p h y t a ( s e e page7 )• The c e l l s o f Rhodomonas l e n s a r e s m a l l e r t h a n t h o s e d e s c r i b e d by B u t c h e r (1967) and a v e r a g e 8-10 p. d u r i n g i n t e r p h a s e . P l a t e s 1-3 i l l u s t r a t e t h e a s y m m e t r i c c e l l s h a p e , t h e C o r p s de Maupas (CM), t h e t r i c h o c y s t s ( T ) , a n d t h e two u n e q u a l f l a g e l l a (F) i n s e r t i n g n e a r t h e a n t e r i o r g u l l e t (G) w h i c h a r e t y p i c a l o f t h i s o r g a n i s m ( B u t c h e r , 1967). The f l a g e l l a a r e c o v e r e d w i t h r e g u l a r l y a r r a n g e d m a s t i g o n e m e s (M, P l a t e 2). A b i l o b e d c h l o r o p l a s t (CHL, P l a t e 3) f o l l o w s t h e c e l l shape and i s c l o s e t o b u t n o t a p p r e s s e d t o t h e p e r i p l a s t ( P ) . The t h y l a k o i d s , u s u a l l y a s s o c i a t e d i n bands o f two ( a r r o w s , P l a t e s 1 and 3), a r e o f t e n a r r a n g e d i n p a r a l l e l s t a c k s . They do n o t t r a v e r s e t h e p y r e n o i d ( P y , P l a t e 1). S t a r c h g r a i n s ( S , P l a t e 1) a r e f o u n d a r o u n d t h e p y r e n o i d a n d o c c a s i o n a l l y w i t h i n t h e p e r i c h l o r o p l a s t i c m a t r i x . When p r e s e n t t h e s m a l l l i p i d b o d i e s a r e l o c a t e d p o s t e r i o r l y . N e a r t h e c h l o r o p l a s t and o f t e n b e t w e e n t h e c h l o r o p l a s t and t h e p e r i p l a s t a r e numerous m i t o c h o n d r i a (M, P l a t e s 1 and 3). The p e r i p l a s t a p p e a r s b i p a r t i t e ( P l a t e s 1 and 3, w h i t e a r r o w s ) , c o n s i s t i n g o f t h e p l a s m a membrane (PM) and a n o t h e r i n n e r l a y e r ( I L ) . The p l a s m a membrane i s c o n t i n u o u s o v e r t h e e n t i r e c e l l : t h e i n n e r l a y e r i s a b s e n t o v e r t h e f l a g e l l a and t h e g u l l e t r e g i o n . D u r i n g s t a t i o n a r y and d e c l i n e p h a s e s i n t h e c o n t r o l c u l t u r e s some c e l l d i v i s i o n o c c u r s , and c e l l s w h i c h a r e u s e d as i n n o c u l a f r o m t h e s e p h a s e s r e a d i l y p r o d u c e good g r o w t h i n f r e s h m e d i a . The u l t r a s t r u c t u r e o f s u c h c e l l s shows d i s t i n c t d i f f e r e n c e s f r o m t h a t o f e a r l i e r p h a s e s . P l a t e 4 i l l u s t r a t e s t h e s e d i f f e r e n c e s . The c h l o r o p l a s t o c c u p i e s a l e s s e r a r e a o f t h e c y t o p l a s m w i t h t h e t h y l a k o i d s o c c u r r i n g s i n g l y ( a r r o w s ) r a t h e r t h a n i n a s s o c i a t e d p a i r s . The s t r o m a l a r e a (SA) has i n c r e a s e d s i g n i f i c a n t l y a n d l a r g e s t a r c h g r a i n s a r e f o u n d w i t h i n t h e p e r i c h l o r o p l a s t i c m a t r i x M i t o c h o n d r i a a p p e a r o n l y n e a r t h e b a s e o f t h e f l a g e l l a . L a r g e b o d i e s o f l i p i d - l i k e ( L L ) m a t e r i a l o c c u p y much o f t h e c y t o p l a s m ; i t i s t h i s m a t e r i a l w h i c h a p p e a r s t o be e x t r u d e d f i r s t when l y s i s o c c u r s . 4.. U r e a E f f e c t s ( i ) Growth Measurements P r e l i m i n a r y e x p e r i m e n t s , i n w h i c h t u r b i d i m e t r i c r e a d i n g s were u s e d as a measurement o f c u l t u r e g r o w t h , i n d i c a t e d l i t t l e v a r i a n c e i n g r o w t h among t h e c u l t u r e s a t t h e s e v e r a l c o n c e n t r a t i o n s o f u r e a ( F i g u r e 11). A l o n g s t a t i o n a r y p h a s e and a v e r y g r a d u a l d e c l i n e p h a s e was o b s e r v e d i n c u l t u r e s grown i n m e d i a c o n t a i n i n g 0.25, 0.50, 1.25 and 2.50 mM u r e a . T a b l e 8 shows t h e c o m p a r a t i v e c h a n g e s i n p i g m e n t a t i o n and M - c o n c e n t r a t i o n a t v a r y i n g l e v e l s o f u r e a . The r e s u l t s f r o m c u l t u r e s grown i n m e d i a c o n t a i n i n g 6.25, a n d 12.50 mM u r e a show a v e r y s h o r t s t a t i o n a r y p h a s e f o l l o w e d by a s h a r p d e c l i n e p h a s e . I n l a t e r e x p e r i m e n t s b o t h t u r b i d i m e t r i c r e a d i n g s a n d d i r e c t c e l l c o u n t s were u s e d as measurements o f g r o w t h , t h e two methods a c t i n g a s a c h e c k , one f o r t h e o t h e r . F i g u r e 12 g u r e 11. Growth p a t t e r n o f c u l t u r e s a t s e v e r a l c o n c e n t r a t i o n s o f u r e a b a s e d on t u r b i d i m e t r i c d e t e r m i n a t i o n s . C l o s e d s q u a r e s = 0.25 mM u r e a ; open c i r c l e s = 0.50 mM u r e a ; c l o s e d t r i a n g l e s = 1.25 mM u r e a ; o p e n t r i a n g l e s = 2.50 mM u r e a ; c l o s e d c i r c l e s = 6.25 mM u r e a ; open s q u a r e s = 12.50 mM u r e a . 34 2. DAYS 35 T a b l e 8. C o m p a r i s o n o f c u l t u r e c o l o u r and M - c o n c e n t r a t i o n d u r i n g g r o w t h o f e x p e r i m e n t a l c u l t u r e s a t v a r y i n g c o n c e n t r a t i o n s o f u r e a . C o n c e n t r a t i o n P i g m e n t a t i o n P i g m e n t a t i o n M-o f u r e a d u r i n g d u r i n g C o n c e n t r a t i o n (mM) l o g a r i t h m i c p h a s e s t a t i o n a r y p h a s e n X 10 .25 y e l l o w - o r a n g e y e l l o w - g r e e n 135 .50 y e l l o w - o r a n g e y e l l o w - g r e e n — 1. 25 c l e a r r e d - p i n k o r a n g e - r e d 150 2. 50 c l e a r r e d - p i n k o r a n g e - r e d 115 6.25 c l e a r r e d - p i n k 58 12. 50 c l e a r r e d - p i n k ** 52 c o n t r o l c l e a r r e d - p i n k o r a n g e - r e d 80 (.5 mM NO ) 3 M - c o n c e n t r a t i o n i s t h e t o t a l p o p u l a t i o n o f c e l l s p e r ml a t t h e end o f s t a t i o n a r y p h a s e . No s t a t i o n a r y p h a s e o b s e r v e d . g u r e 1 2 . Growth p a t t e r n o f e x p e r i m e n t a l c e l l s a t s e v e r a l c o n c e n t r a t i o n s o f u r e a . C l o s e d c i r c l e s show g r o w t h as d e t e r m i n e d by d i r e c t c e l l c o u n t ; open c i r c l e s , by t u r b i d i m e t r i c d e t e r m i n a t i o n s . D A Y S 0 0 9 ao C E L L C O U N T / M L X 1 0 4 i l l u s t r a t e s g r o w t h m e a s u r e d i n t h i s way. D u r i n g t h e l a g and l o g a r i t h m i c p h a s e s o f c u l t u r e s grown a t c o n c e n t r a t i o n s e q u a l t o o r g r e a t e r t h a n 1.25 mM u r e a t h e g r o w t h as m e a s u r e d by e i t h e r method was r o u g h l y e q u i v a l e n t . However, once s t a t i o n a r y p h a s e b e g a n , t h e t u r b i d i m e t r i c r e a d i n g s i n d i c a t e d a s u b s t a n t i a l l y g r e a t e r d e g r e e o f g r o w t h t h a n was o b s e r v e d by d i r e c t c e l l c o u n t s . D a t a f r o m t h e s e two t y p e s o f measurement were n o t s i m i l a r f o r c u l t u r e s grown i n 0.25 mM u r e a . The c a l c u l a t e d g e n e r a t i o n t i m e s f o r c u l t u r e s a t e a c h c o n c e n t r a t i o n o f u r e a a r e g i v e n i n T a b l e 9. F i g u r e 13 shows t h e l o g a r i t h m i c d e c r e a s e i n g e n e r a t i o n t i m e w h i c h o c c u r s w i t h i n c r e a s i n g u r e a c o n c e n t r a t i o n . ( i i ) U l t r a s t r u c t u r e The u l t r a s t r u c t u r e o f e x p e r i m e n t a l c e l l s a t a l l l e v e l s o f u r e a d u r i n g l a g and l o g a r i t h m i c p h a s e s i s s i m i l a r t o t h a t o f t h e c o n t r o l c e l l s d u r i n g t h e s e p h a s e s ( P l a t e s 1-3). I n c r e a s e d l i p i d a c c u m u l a t i o n (L) was o b s e r v e d i n a l l e x p e r i m e n t a l c e l l s d u r i n g l a g a n d e a r l y l o g a r i t h m i c p h a s e s ( P l a t e 5) b u t most o f t h i s d i s a p p e a r e d a t t h e o n s e t o f r a p i d d i v i s i o n . Some l i p i d was u s u a l l y o b s e r v e d i n d i s c r e t e d r o p s n e a r t h e c e l l p e r i p h e r y ( P l a t e 6). ( i i i ) E l e c t r o l y t i c E f f e c t s The pH r e c o r d e d d u r i n g g r o w t h v a r i e d c o n s i d e r a b l y between i n d i v i d u a l c u l t u r e s ( s e e T a b l e 10). I n Run 1 t h e l y s i s o f c e l l s s h o r t l y a f t e r t h e o n s e t o f l o g a r i t h m i c 40 F i g u r e 13. The r e l a t i o n s h i p b etween t h e g e n e r a t i o n t i m e o f a c u l t u r e and t h e c o n c e n t r a t i o n o f u r e a i n w h i c h i t has b e e n i n c u b a t e d . m M UREA Table 9. A comparison of growth by d i v i s i o n of cultures at increasing concentrations of urea. Concentration No. of generations. Time Generation time of urea (mM) a n (hrs) b g c o n t r o l 0 (0.5 mM NO ) 1. 74 24 13.8 0.25 1.42 24 17.0 1.25 1. 51 24 15.9 2 .50 0.79 24 30.4 6.25 1.92 24 12.5 12.50 2.50 24 9.6 This figure was calculated from the equation n = ^ o g l Q y ~ l o S l 0 x , where n i s the number of generations, y .301 i s the larger number of organisms at the end of a time period t, and x i s the smaller number of organisms at the beginning of the time period t . This figure i s calculated from the equation g = t_, n where g i s the generation time, t i s the time period, and n i s the number of generations, c The control was grown i n media without urea, and with 0.5 mM KN0o added. 43 T a b l e 10. V a r i a t i o n i n pH a n d u r e a c a t a b o l i s m among i n d i v i d u a l c u l t u r e s . C u l t u r e Time C e l l no. p e r pH ji g - a t o m u r e a - N ( d a y s ) m l , x 10 4 p e r ml m e d i a C o n t r o l (0.5 mM NO ) 6 54 7.6 — 3 11 70 7-6 — 1.2 5 mM u r e a a ~ b Run 1 0 e s t i m a t e d 7.4 80 2 3.5 8.0 — 6 14.8 8.5 83 11 l y s e d 8.7 17 1.25 mM u r e a Run 2 0 e s t i m a t e d 7.4 80 2 1.6 7.4 72 6 29.6 7.4 79 78.0 7.6 82 12.5 mM u r e a Run 1 0 • e s t i m a t e d 7.4 750 2 10. 4 8.3 — 6 23.0 9.1 338 11 l y s e d 8.7 14 12.5 mM u r e a Run 2 0 e s t i m a t e d 7.4 750 2 1.3 7.4 — 6 45.9 7.5 749 8 c 79.0 7.6 756 A one ml i n n o c u l u m c o n t a i n i n g a p p r o x i m a t e l y 15.4 x 10 c e l l s ml was a d d e d t o e a c h 20 mis o f m e d i a . T h i s f i g u r e i s c a l c u l a t e d f r o m t h e known amount o f u r e a a d d e d t o t h e m e d i a , c B a c t e r i a l c o n t a m i n a t i o n was o b s e r v e d a t 10 d a y s , t h e r e f o r e t h e r u n was t e r m i n a t e d . p h a s e c o i n c i d e d w i t h an i n c r e a s e d pH and a d i s a p p e a r a n c e o f u r e a i n t h e m e d i a . I n t h e s e c o n d ( a n d i d e n t i c a l ) r u n a n o r m a l i n c r e a s e ( as p e r F i g u r e 12) i n c e l l number p e r ml was o b s e r v e d and no s i g n i f i c a n t i n c r e a s e i n pH o r d e c r e a s e i n u r e a o c c u r r e d . I n o r d e r t o c l a r i f y t h i s d i s c r e p a n c y i n r e s u l t s t h e pH e x p e r i m e n t s were i n i t i a t e d . T a b l e 11 summarizes t h e s e e x p e r i m e n t s . The a r t i f i c i a l i n c r e a s e i n pH w i t h NaOH d i d n o t i n d u c e any g r o s s s t r u c t u r a l c h a n g e s i n t h e c e l l s a nd c u l t u r e g r o w t h c o n t i n u e d . When t h e pH o f t h e c u l t u r e was a d j u s t e d by t h e a d d i t i o n o f NH^OH, c u l t u r e d e a t h was o b s e r v e d w i t h pH l e v e l s e q u a l t o o r g r e a t e r t h a n 8.4 ( T a b l e 1 1 ) . P l a t e 7 i l l u s t r a t e s t h e s e q u e n t i a l c h a n g e s f r o m n o r m a l c e l l s t r u c t u r e a t t h e p o i n t o f pH change t o c u l t u r e d e a t h . At pH l e v e l s b e t w e e n 8.4 a n d 8.65, c e l l s i n i t i a l l y l o s e t h e i r c h a r a c t e r i s t i c shape and become r o t u n d ( F i g u r e 18, 1 9 ) ; t h e c u p - s h a p e d . c h l o r o p l a s t becomes f l a t t e n e d t o w a r d s t h e p o s t e r i o r o f t h e c e l l ( F i g u r e s 20, 2 1 ) ; t h e n p r o t r u s i o n o f t h e c e l l c o n t e n t s and c y t o p l a s m i c v a c u o l a t i o n o c c u r s ( F i g u r e s 22 t o 2 4 ) . F i n a l l y , one o r more o f t h e p r o t r u s i o n s b u r s t s , a n d t h e c e l l c o n t e n t s a r e r e l e a s e d ( F i g u r e 2 5 ) . A t pH l e v e l s g r e a t e r t h a n 8.65 c u l t u r e d e a t h o c c u r s v e r y r a p i d l y . . The c e l l c o n t e n t s p r o t r u d e , e j e c t e d t r i c h o c y s t - l i k e t h r e a d s a r e r e l e a s e d e x t e r n a l l y , and one o r more o f t h e p r o t r u s i o n s b u r s t s . T a b l e 1 1 . The e f f e c t s o f pH on a h e a l t h y c o n t r o l c u l t u r e . (The pH was a d j u s t e d w i t h e i t h e r NaOH o r NH OH.) PH NH cone 3(mM) Time r e q u i r e d f o r c u l t u r e l y s i s w i t h : NH, OH 4 NaOH 7.4 8.0 8.2 8.4 8.65 8.8 9.0 9.2 0.8 2.4 4.0 5.6 8.0 10.30 13-50 no l y s i s no l y s i s no l y s i s 24 h o u r s 8 h o u r s 4-6 h o u r s 1 h o u r 1 h o u r no l y s i s DISCUSSION 46 Rhodomonas l e n s i s a p h o t o a u t o t r o p h . S uch o r g a n i s m s u s u a l l y p o s s e s s a l a t e n t c a p a c i t y t o use v a r i o u s n u t r i e n t s o u r c e s v i a t h e i n d u c t i o n o f t h e enz y m e ( s ) a p p r o p r i a t e t o m e t a b o l i z e t h e compound. The u p t a k e o f n i t r a t e i n many s p e c i e s o f m a r i n e p h y t o p l a n k t o n i s m e d i a t e d by a membrane-bound (NO^ , C l ) - a c t ' i v a t e d a d e n o s i n e t r i p h o s p h a t a s e ( F a l k o w s k i , 1975 a, b) and t h e m e t a b o l i s m o f n i t r a t e r e q u i r e s t h e enzyme n i t r a t e r e d u c t a s e w h i c h c a t a l y z e s t h e r e d u c t i o n o f n i t r a t e - t o n i t r i t e a n d t h e enzyme n i t r i t e r e d u c t a s e , w h i c h c a t a l y z e s t h e r e d u c t i o n o f n i t r i t e t o ammonia. Ammonia i s u t i l i z e d v i a t r a n s a m i n a t i o n r e a c t i o n s . N i t r a t e r e d u c t a s e has b e e n shown t o be i n d u c i b l e i n t h e p r e s e n c e o f n i t r a t e a n d molybdenum ( S c h r a d e r e t a l . , 1967) • N i t r i t e r e d u c t a s e i s a l s o known t o be i n d u c i b l e ( S c h r a d e r e t a l . , 1 9 6 7 ) . The m e t a b o l i s m o f u r e a i s c a t a l y z e d by e i t h e r u r e a s e o r UALase. B o t h o f t h e s e enzymes have been shown t o be i n d u c e d i n t h e p r e s e n c e o f u r e a ( J e f f r i e s , 1964; B e r n s , H o l o h a n and S c o t t , 1 9 6 6 ; Roon, a n d L e v e n b e r g , 1 9 7 0 ) . T h e s e enzymes s e r v e t o c o n v e r t a l t e r n a t i v e s o u r c e s o f n i t r o g e n t o ammonia, w h i c h c a n t h e n be m e t a b o l i z e d i n t h e c e n t r a l m e t a b o l i c p athway. The r e s u l t s i n d i c a t e t h a t Rhodomonas l e n s r e a d i l y a c c l i m a t e d t o t h e n i t r a t e medium. However, t h e a c c l i m a t i o n o f Rhodomonas l e n s t o u r e a medium was v e r y e r r a t i c a l t h o u g h e x p e r i m e n t a l c o n d i t i o n s were a p p a r e n t l y i d e n t i c a l . I t was hi assumed t h a t when g r o w t h o c c u r r e d t h e enzyme"^ ha d b e e n i n d u c e d , s i n c e e x p e r i m e n t a l r e s u l t s i n d i c a t e d t h e i m p r o b a b i l i t y o f a c c l i m a t i o n r e s u l t i n g f r o m change o r m u t a t i o n : ( T a b l e 6). The c a u s e o f t h e e r r a t i c b e h a v i o u r o f t h i s o r g a n i s m i n u r e a i s unknown. A n t i a ( p e r s o n a l ^ c o m m u n i c a t i o n ) o b s e r v e d some e r r a t i c g r o w t h w i t h Rhodomonas  l e n s i n a u r e a e n r i c h e d , n a t u r a l medium. As t h e c u l t u r e was o r i g i n a l l y i s o l a t e d f r o m t h e G u l f S t r e a m , i t i s p o s s i b l e t h a t t h i s o r g a n i s m r e q u i r e s a h i g h e r s a l i n i t y - ' - 1 t h a n t h a t p r o v i d e d by t h e c u l t u r e medium: i f t h e s a l i n i t y o f t h e medium was a t t h e edge o f i t s minimum r e q u i r e m e n t , e r r a t i c g r o w t h w o u l d be e x p e c t e d . T h i s c o u l d be t e s t e d by i n o c u l a t i n g c o n t r o l c e l l s i n t o e x p e r i m e n t a l m e d i a a d j u s t e d t o s e v e r a l l e v e l s o f s a l i n i t y . ( T e m p e r a t u r e and l i g h t o p t i m a were c h e c k e d . ) A n o t h e r p o s s i b l e v a r i a b l e I s t h e u r e a m e d i a . The m e d i a was n o t a l w a y s p r e p a r e d d i r e c t l y b e f o r e : use a n d e s p e c i a l l y I n c a s e s where i t was a l l o w e d t o s i t f o r s e v e r a l d a y s a t room t e m p e r a t u r e , t h e p r e s e n c e o f breakdown p r o d u c t s i n h i b i t o r y t o c e l l g r o w t h c a n n o t / b e d i s c o u n t e d . T h i s w i l l be d i s c u s s e d i n d e t a i l l a t e r . The r o l e o f n i t r o g e n i n a b a l a n c e d m e t a b o l i s m I s i l l u s t r a t e d i n F i g u r e 26 ( l e f t s i d e ) . The r a t e o f s y n t h e s i s o f e a c h t y p e o f m a c r o m o l e c u l e i s g e a r e d so t h a t one does n o t o v e r g r o w a n o t h e r . G e n e r a l l y , i f an i m b a l a n c e l e a d i n g t o u n d e r p r o d u c t i o n o f a m e t a b o l i t e o c c u r s , e i t h e r l y s i s o r c u l t u r e s t a s i s w o u l d be e x p e c t e d . However, d e p e n d e n t on F i g u r e 2 6 . 1 9 . ) M e t a b o l i c i n t e r r e l a t i o n s h i p s . ( L e h n i n g e r , 48 The C e l l N u c l e u s M i t o c h o n d r i a C h l o r o p l a s t s Enzyme c o m p l e x e s Ribosomes C o n t r a c t i l e s y s t e m s t  N u c l e i c a c i d s Mono-n u c l e o t i d e s P r o t e i n s Amino a c i d s P o l y -s a c c h a r i d e s S i m p l e s u g a r s L i p i d s F a t t y a c i d s , g l y c e r o l R i b o s e , c a r b a m y l p h o s p h a t e t - k e t o a c i d s CO. P h o s p h o -p y r u v a t e , m a l a t e A c e t a t e , m a l o n a t e N ' 49 t h e n u t r i e n t , t h i s w o u l d o c c u r i n v a r y i n g d e g r e e s . I f n i t r o g e n was d e p l e t e d f r o m t h e medium, t h e s y n t h e s i s o f amino a c i d s , n u c l e o t i d e s and h e x o s a m i n e s w o u l d s t o p as w o u l d a l l p o l y m e r s d e r i v e d f r o m them. R e f e r r i n g t o F i g u r e 26, i t w i l l be n o t e d t h a t o n l y t h e p o l y s a c c h a r i d e s a n d n o n - p r o t e i n l i p i d s w o u l d c o n t i n u e t o be s y n t h e s i z e d . I f n i t r o g e n was n o t d e p l e t e d b u t t h e c u l t u r e was s u b j e c t e d t o 12 some d e g r e e o f n i t r o g e n s t r e s s , a g a i n l i p i d s and p o l y s a c c h a r i d e s w o u l d c o n t i n u e t o be s y n t h e s i z e d n o r m a l l y b u t t h e s y n t h e s i s o f a l l m o l e c u l e s c o n t a i n i n g n i t r o g e n w o u l d p r o c e e d a t t h e r a t e a t w h i c h n i t r o g e n was made a v a i l a b l e . T h i s w o u l d r e s u l t i n an u n b a l a n c e d m e t a b o l i s m , t h e most o b v i o u s symptoms b e i n g an a c c u m u l a t i o n o f s t o r a g e p r o d u c t s , a l a c k o f any n o n - v i t a l m o l e c u l e s c o n t a i n i n g n i t r o g e n , a n d a r a t e o f ^ d i v i s i o n d i r e c t l y r e l a t e d t o t h e r a t e a t w h i c h n i t r o g e n was made a v a i l a b l e . Rhodomonas l e n s p r o d u c e s d i s p e n s i b l e , n i t r o g e n -c o n t a i n i n g p r o d u c t s , t h e p h y c o b i l i n p i g m e n t s , w h i c h can be u s e d a s a s u b j e c t i v e i n d i c a t i o n o f t h e n u t r i t i o n a l w e l l - b e i n g o f t h e c e l l s . T h r e e c l a s s e s o f p i g m e n t s a r e f o u n d in. t h e C r y p t o p h y t a : t h e c h l o r o p h y l l s , t h e c a r o t e n o i d s , a n d t h e p h y c o b i l i n s . C h l o r o p h y l l i s t h e p r i m a r y l i g h t - t r a p p i n g m o l e c u l e ; t h e c a r o t e n o i d s and t h e p h y c o b i l i n s p r o b a b l y s e r v e as s u p p l e m e n t a r y l i g h t r e c e p t o r s f o r t h a t p o r t i o n o f t h e v i s i b l e s p e c t r u m n o t c o m p l e t e l y c o v e r e d by c h l o r o p h y l l . The c a r o t e n o i d s p r o b a b l y a l s o f u n c t i o n t o p r o t e c t c h l o r o p h y l l f r o m d e g r a d a t i v e a t t a c k by m o l e c u l a r 50 oxygen. The p h y c o b i l i n s a r e o p e n - c h a i n t e t r a p y r r o l e s w h i c h a r e c o n j u g a t e d t o s p e c i f i c p r o t e i n s : COOH COOH H H H H H H 2 P h y c o e r y t h r o b i l i n The p h y c o b i l i n s a r e d e g r a d e d d u r i n g c o n d i t i o n s o f • n u t r i t i o n a l d e f i c i e n c y w i t h t h e i r components, s p e c i f i c a l l y n i t r o g e n , b e i n g u t i l i z e d f o r more v i t a l c e l l p r o c e s s e s ( A n t i a e t a l . , 1973). S i n c e e a c h c l a s s o f p i g m e n t g i v e s 13 t h e c u l t u r e a s p e c i f i c c o l o u r a t i o n , when one c l a s s i s d e g r a d e d t h e c u l t u r e changes c o l o u r . The c o l o u r change has a l w a y s been o b s e r v e d i n t h e f o l l o w i n g o r d e r : c l e a r r e d - p i n k t o r e d d i s h - o r a n g e t o g r e e n t o cream. As t h e o n l y v a r i a b l e b etween t h e c o n t r o l a nd e x p e r i m e n t a l media was t h e n i t r o g e n s o u r c e , t h e d i f f e r e n c e s w h i c h o c c u r r e d a r e a t t r i b u t a b l e t o t h e s o u r c e o f t h i s 51 n u t r i e n t . The c o n t r o l c u l t u r e s d u r i n g l a g and l o g a r i t h m i c p h a s e s e x h i b i t e d t h e c l e a r r e d - p i n k c o l o u r c h a r a c t e r i s t i c o f t h e p h y c o b i l i n p i g m e n t ( s ) and d e d u c t i v e l y , i n d i c a t i v e o f s u f f i c i e n t n i t r o g e n . M i c r o s c o p y a l s o p o i n t e d t o h e a l t h y , b a l a n c e d m e t a b o l i s m : l 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 showed a n o r m a l d e g r e e o f m o t i l i t y , t h e t y p i c a l a s y m m e t r i c c e l l s h a p e , and some e v i d e n c e o f v a r i o u s s t a g e s o f c e l l d i v i s i o n . The amount o f r o u g h e n d o p l a s m i c r e t i c u l u m , a n d numerous r i b o s o m e s and g o l g i v e s i c l e s s u g g e s t a c t i v e p r o t e i n s y n t h e s i s , w h i l e a b a l a n c e d m e t a b o l i s m was i n d i c a t e d by t h e l a c k o f l i p i d o r o t h e r s t o r a g e p r o d u c t s . (The c h l o r o p l a s t was a c t i v e l y p r o d u c i n g s t a r c h , b u t t h i s p r o d u c t was f o u n d o n l y w i t h i n t h e p e r i c h l o r o p l a s t i c m a t r i x a n d n o t a c c u m u l a t e d i n t h e c y t o p l a s m . ) S h o r t l y a f t e r t h e o n s e t o f s t a t i o n a r y p h a s e t h e c u l t u r e c o l o u r c h a n g e d t o a n o r a n g e - r e d . T h i s a p p a r e n t d e g r a d a t i o n o f t h e p h y c o b i l i n s s u g g e s t s t h a t n i t r o g e n had become l i m i t i n g ; t h e c e l l s ' r e s p o n s e t o a l a c k o f n i t r o g e n i n t h e medium b e i n g t h e m o b i l i z a t i o n o f t h e n i t r o g e n components f r o m t h e p h y c o b i l i n s 1 ^ . C y t o l o g i c a l e v i d e n c e c o r r o b o r a t e s t h i s . The l o c u l u s c o n t a i n s l i p i d s , p r o t e i n s a n d s o l u b l e p i g m e n t s . A d e p l e t i o n o f any o f t h e s e c o u l d c o n c e i v a b l y c a u s e a change i n l o c u l u s s i z e . I n t h e c o n t r o l c e l l s d u r i n g l o g a r i t h m i c p h a s e t h e l o c u l u s m e a s u r e s a b o u t 110-260 A i n d i a m e t e r ; d u r i n g s t a t i o n a r y p h a s e , when t h e t h y l a k o i d s a r e a r r a n g e d s i n g l y , t h e l o c u l u s r a n g e s between 70 a n d 150 A. I t c a n be a r g u e d t h a t one o r more ' f i l l e r ' s u b s t a n c e ( s ) w h i c h h e l p e d t o m a i n t a i n t h e o r i g i n a l l o c u l u s s i z e has b e en d e p l e t e d somewhat: t h e o b v i o u s s u b s t a n c e w o u l d be t h e p h y c o b i l i n p i g m e n t s . Thus t h e change i n l o c u l u s s i z e may be an i n d i c a t i o n t h a t t h e p h y c o b i l i n s have b e e n d e p l e t e d . E l e c t r o n m i c r o s c o p y a l s o shows c o p i o u s amounts o f a l i p i d - l i k e m a t e r i a l a n d numerous s t a r c h g r a n u l e s . W h i l e A n t i a e t a l . (1973), s u g g e s t t h a t t h e s e may be symptoms o f s e n e s c e n c e , b o t h B e i j e r i n c k (1904) i n d i a t o m s an d S p o e h r and M i l n e r (1949) i n C h l o r e l l a n o t e d an i n c r e a s e i n l i p i d c o n t e n t w i t h an e x i g u o u s s u p p l y o f n i t r o g e n . I t i s p r o b a b l e t h a t t h e ' a g i n g ' i s d i r e c t l y a t t r i b u t a b l e t o t h e d e p l e t i o n o f n i t r o g e n f r o m t h e m e d i a . E v e n t u a l l y , d u r i n g a p r o l o n g e d s t a t i o n a r y - d e c l i n e p h a s e , t h e c u l t u r e may t u r n g r e e n a n d g e n e r a l l y s h o r t l y t h e r e a f t e r , a c r e a m y - w h i t e . The b reakdown o f much o f t h e c a r o t e n o i d s i n d i c a t e s t h a t t h e c u l t u r e i s i n s e r i o u s t r o u b l e n u t r i t i o n a l l y a n d t h i s i s c o r r o b o r a t e d by t h e s u b s e q u e n t c o l o u r - c h a n g e o b s e r v e d as t h e c e l l s l y s e . The f i n e s t r u c t u r e o f e x p e r i m e n t a l c e l l s d u r i n g l a t e l a g a n d e a r l y l o g a r i t h m i c p h a s e s a p p r o x i m a t e s t h a t o f t h e c o n t r o l c e l l s d u r i n g s t a t i o n a r y p h a s e when t h e y e x h i b i t s i g n s o f n i t r o g e n s t r e s s . A s l i g h t l y l e s s e r a c c u m u l a t i o n o f l i p i d i s f o u n d i n t h e e x p e r i m e n t a l c e l l s d u r i n g t h e s e p h a s e s . The o t h e r two p a r a m e t e r s , p i g m e n t a t i o n and r a t e o f d i v i s i o n , a r e d i f f i c u l t t o a p p l y a t t h i s s t a g e . A t r e n d t o w a r d n i t r o g e n s t r e s s i s n o t e d d u r i n g l o g a r i t h m i c p h a s e : s m a l l l i p i d d r o p l e t s a r e o f t e n s e e n w i t h i n t h e c y t o p l a s m a n d a d i v i s i o n r a t e somewhat l o w e r 1 ^ t h a n t h a t o f t h e c o n t r o l i s o b s e r v e d . The d i r e c t i n c r e a s e o f d i v i s i o n r a t e w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f u r e a s u g g e s t s t h a t i t i s t h e u p t a k e a n d / o r m e t a b o l i s m o f t h i s compound w h i c h d i r e c t l y a f f e c t s t h e d i v i s i o n r a t e . T h e r e f o r e , a l t h o u g h i t i s v e r y u n l i k e l y t h a t t h e c u l t u r e i s a c t u a l l y m e t a b o l i z i n g a l l t h e u r e a i n t h e m e d i a , t h e e x p e r i m e n t a l c u l t u r e s a p p e a r t o be u n d e r g o i n g n i t r o g e n s t r e s s d u r i n g l a g a n d e a r l y l o g a r i t h m i c p h a s e s a n d t o a l e s s e r d e g r e e d u r i n g m i d - a n d l a t e l o g a r i t h m i c p h a s e . As p r e v i o u s l y d i s c u s s e d , t h i s w o u l d a c c o u n t f o r an a c c u m u l a t i o n o f s t o r a g e p r o d u c t s , e s p e c i a l l y d u r i n g t h e e x t e n d e d p e r i o d o f t i m e when t h e c e l l s a r e r e p l e n i s h i n g t h e i r i n t e r n a l s u p p l y o f i n t e r m e d i a t e s and m a n u f a c t u r i n g e s s e n t i a l m e t a b o l i t e s and enzymes p r e p a r a t o r y t o m u l t i p l i c a t i o n . To a l e s s e r e x t e n t t h i s a c c u m u l a t i o n w o u l d o c c u r d u r i n g l o g a r i t h m i c g r o w t h , n i t r o g e n s t r e s s b e i n g r e f l e c t e d more i n d i r e c t l y i n t h e d i v i s i o n r a t e . D e c l i n e p h a s e i s marked by a c o m p l e t e l o s s o f p i g m e n t a t i o n and a d e f i n i t e s e r i e s o f m o r p h o l o g i c a l c h a n g e s w h i c h t e r m i n a t e i n c u l t u r e a u t o l y s i s . T h e s e c h a n g e s a r e c h a r a c t e r i s t i c o f c e l l s s u f f e r i n g f r o m o s m o t i c i m b a l a n c e . The i n c r e a s e i n pH l e v e l s p r i o r t o t h e o n s e t o f d e c l i n e p h a s e s u g g e s t s t h a t t h e change i s c a u s e d by o r a s s o c i a t e d w i t h a b a s i c i o n . The c h a n g e s a r e n o t p r e c i p i t a t e d by a s i m p l e pH e f f e c t ; r a t h e r t h e c e l l s a r e t o l e r a n t o f h i g h pH l e v e l s . When t h e pH o f t h e c u l t u r e medium was r a i s e d a r t i f i c i a l l y w i t h NaOH, t h e c e l l s e x h i b i t e d no s t r u c t u r a l a b n o r m a l i t i e s a n d c o n t i n u e d t o d i v i d e , e v e n a t a pH o f 9.2. However, when pH l e v e l s were a d j u s t e d w i t h NH^OH, m o r p h o l o g i c a l c h a n g es s i m i l a r t o t h o s e o b s e r v e d d u r i n g t h e d e c l i n e p h a s e o f t h e e x p e r i m e n t a l c u l t u r e s o c c u r r e d . S h i l o a n d S h i l o (1962) r e p o r t t h e i n d u c t i o n o f s i m i l a r m o r p h o l o g i c a l c h a n g e s i n Prymnesium u s i n g ammonia and a c e t i c a c i d as weak e l e c t r o l y t e s a s s o c i a t e d w i t h pH l e v e l s b e t w e e n 8.0 a n d 9.0.. A c o n s i d e r a t i o n o f t h e e x p e r i m e n t a l c u l t u r e m e d i a s u g g e s t s t h a t t h e most l i k e l y s o u r c e o f b a s i c i o n s i s u r e a . U r e a i s e n z y m a t i c a l l y c a t a b o l i z e d g i v i n g 2 m o l e s o f ammonia and one mole o f c a r b o n d i o x i d e . The d e c o m p o s i t i o n o f u r e a i n m e d i a i s unknown b u t i n d i s t i l l e d w a t e r 8.0 M u r e a decomposes s l i g h t l y , g i v i n g 20 mM ammonium c y a n a t e . T h a t t h e d e c l i n e p h a s e o c c u r s o n l y above a c e r t a i n c o n c e n t r a t i o n o f u r e a (1.25 mM) a n d t h a t t h e o n s e t o f t h e d e c l i n e p h a s e i s more r a p i d a t i n c r e a s i n g c o n c e n t r a t i o n s o f u r e a , i s a f u r t h e r i n d i c a t i o n t h a t t h e f a c t o r w h i c h i n d u c e s t h i s p h a s e i s a p r o d u c t o f u r e a d e c o m p o s i t i o n , p r o b a b l y ammonia. Two q u e s t i o n s a r i s e f r o m t h e s e o b s e r v a t i o n s : 1. Why do c e l l s c a p a b l e o f m e t a b o l i z i n g u r e a as a n i t r o g e n s o u r c e show s i g n s o f n i t r o g e n s t r e s s i n m e d i a c o n t a i n i n g e x c e s s u r e a ? 2. Why do t h e same c e l l s d u r i n g l o g a r i t h m i c p h a s e d e v e l o p s i g n s o f ammonia p o i s o n i n g and l y s e ? The ^ig-atom N l i t e r - c o n c e n t r a t i o n i s 2 t o 50 t i m e s g r e a t e r i n t h e u r e a m e d i a t h a n i n t h e n i t r a t e m e d i a . N i t r o g e n s t r e s s must, t h e r e f o r e , e i t h e r be t h e r e s u l t o f an i n e f f i c i e n t a s s i m i l a t i o n o f u r e a o r an i n e f f i c i e n t m e t a b o l i s m o f u r e a by t h e c e l l . As t h e r e s u l t s i n d i c a t e a l a t e r a c c u m u l a t i o n o f a b a s i c i o n , p r o b a b l y ammonia, i t i s s u g g e s t e d t h a t t h e u r e a - N pathway i s i n h i b i t e d s u c h t h a t t h e ammonia p r o d u c e d i s n o t e f f i c i e n t l y a s s i m i l a t e d b u t r e l e a s e d i n t o t h e m e d i a . U c h i d a (1975) f o u n d t h a t t h e d i n o f l a g e l l a t e P r o r o c e n t r u m  m i c a n s t o o k up u r e a when grown i n a medium w i t h u r e a as t h e s o l e n i t r o g e n s o u r c e , and t h e n r e l e a s e d ammonia i n t o t h e medium. T h i s o c c u r r e d d u r i n g a p r o l o n g e d l a g p h a s e . When a ' c r i t i c a l ' l e v e l o f ammonia i n t h e medium was a t t a i n e d , t h e o r g a n i s m e n t e r e d l o g a r i t h m i c p h a s e and a p p a r e n t l y m a i n t a i n e d t h i s p h a s e u n t i l much o f t h e ammonia was a s s i m i l a t e d . The o r g a n i s m t h e n r e p e a t e d t h e p r o c e s s , t h e s e c o n d ' c r i t i c a l ' l e v e l o f ammonia b e i n g l o w e r t h a n t h e f i r s t . The g r o w t h c u r v e t h u s a p p e a r s t y p i c a l o f d i a u x i c g r o w t h . I n two e x p e r i m e n t a l r u n s w i t h Rhodomonas l e n s , u r e a was d e p l e t e d r a p i d l y w i t h a c o n c o m i t a n t i n c r e a s e i n pH ( i n d i c a t i n g a n i n c r e a s e i n b a s i c i o n s — p r o b a b l y NH^ +) ( T a b l e 10): t h u s F i g u r e 12, a t c o n c e n t r a t i o n s o f 0.25-2.50 mM, c o u l d be drawn t o i n c l u d e a l l p o i n t s ( r a t h e r t h a n d r a w i n g t h e b e s t l i n e among a l l p o i n t s ) . T h i s c u r v e w o u l d t h e n be i n d i c a t i v e o f d i a u x i c g r o w t h . T h e r e f o r e , i t i s 56 p o s s i b l e t h a t Rhodomonas l e n s i s e x h i b i t i n g g r o w t h on u r e a s i m i l a r t o t h a t shown w i t h P r o r o c e n t r u m m i c a n s . S p e c u l a t i v e l y , i f Rhodomonas l e n s i s b e h a v i n g i n t h i s manner and i f t h e amount o f ammonia e x c r e t e d 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 c o n c e n t r a t i o n o f u r e a i n t h e medium i t i s p r o b a b l e t h a t a t h i g h e r l e v e l s o f u r e a (6.25 and 12.50 mM) t h e amount o f ammonia e x c r e t e d w o u l d r e a c h t o x i c l e v e l s w h i c h , w i t h t h e i n c r e a s e d pH, w o u l d c a u s e l y s i s o f t h e c u l t u r e . A p o s s i b l e a l t e r n a t i v e answer t o t h e q u e s t i o n s p o s e d i s t h a t b r e a kdown p r o d u c t s o f u r e a i n s o l u t i o n a r e i n h i b i t i v e t o some p a r t o f t h e n i t r o g e n m e t a b o l i c p a t h w a y . H a t t o r i (1957), w i t h C h l o r e l l a , f o u n d t h a t c y a n i d e i n a c o n c e n t r a t i o n o f 4.0 mM a l m o s t c o m p l e t e l y h a l t e d ammonia a s s i m i l a t i o n , w h i l e i t was a l m o s t c o m p l e t e l y w i t h o u t e f f e c t on u r e a a s s i m i l a t i o n . C y a n i d e a t a c o n c e n t r a t i o n o f 0.4 mM i n h i b i t e d 78$ o f ammonia a s s i m i l a t i o n ( H a t t o r i , 1957). Hodson and Thompson (1969) a l s o u s e d c y a n i d e a t a c o n c e n t r a t i o n (10 mM) w h i c h i n h i b i t e d ammonia u p t a k e c o m p l e t e l y b u t u r e a u p t a k e o n l y s l i g h t l y i n C h l o r e l l a ; u n d e r t h e s e c o n d i t i o n s t h e y f o u n d an a c c u m u l a t i o n o f ammonia i n t h e medium. A n o t h e r s u b s t a n c e c a u s i n g s i m i l a r r e s u l t s i s a r s e n i t e ( H a t t o r i , 1957; Hodson and Thompson, 1969). A s u r v e y o f t h e l i t e r a t u r e r e v e a l e d t h a t u r e a decomposes i n aqueous s o l u t i o n . F a w s i t t (1902) a n d W a l k e r and Hambly (1895) b o t h d e m o n s t r a t e d t h a t an i n t e r m e d i a t e 57 c y a n a t e s t a g e f o r m e d when aqueous s o l u t i o n s o f u r e a a r e h e a t e d above 70°C. Werner (1918) showed t h a t t h e ' h y d r o l y s i s ' o f u r e a u n d e r a l l c o n d i t i o n s i n v o l v e d t h e s i m p l e d i s s o c i a t i o n o f t h e u r e a m o l e c u l e i n t o ammonia a n d f r e e c y a n i c a c i d : CON 2H l j >.NH3 + HNCO. P e a r o n (1923) d e t e c t e d t h e p r e s e n c e o f c y a n i c a c i d i n h i s u r e a / u r e a s e s y s t e m a l t h o u g h he w r o n g l y a t t r i b u t e d i t t o an e n z y m a t i c b r e akdown p r o d u c t o f u r e a . More r e c e n t l y , A n t i a and Landymore (197*0 f o u n d t h a t u r i c a c i d i s c h e m i c a l l y u n s t a b l e i n s e a w a t e r p h y t o p l a n k t o n c u l t u r e medium. U r i c a c i d showed a s l o w d e g r a d a t i o n i n d a r k n e s s : t h i s was i n c r e a s e d s e v e r a l - f o l d by i l l u m i n a t i o n . A m a j o r f a c t o r i n t h e a c c e l e r a t i o n o f u r i c a c i d b r e a kdown was f o u n d t o be f r e e t r a c e - m e t a l i o n s , whose i m p o r t a n c e has b e e n a t t e s t e d t o by many o r g a n i c c h e m i s t s . U n f o r t u n a t e l y , t h e breakdown p r o d u c t s were n o t i d e n t i f i e d . I f u r e a decomposes t o c y a n a t e a n d ammonium i n aqueous s o l u t i o n , i t i s p r o b a b l e t h a t i t a l s o does so t o a g r e a t e r e x t e n t u n d e r i l l u m i n a t i o n a n d i n t h e p r e s e n c e o f f r e e t r a c e m e t a l i o n s . The e f f e c t o f c y a n a t e on n i t r o g e n m e t a b o l i s m i s unknown. The amount o f ammonia p r o d u c e d by s u c h a d e c o m p o s i t i o n i s u n l i k e l y t o p r o d u c e t h e t o x i c e f f e c t s n o t e d i n t h e e x p e r i m e n t a l c u l t u r e s . I f , however, c y a n a t e r e a c t e d s i m i l a r l y t o c y a n i d e i n i n h i b i t i n g ammonia a s s i m i l a t i o n , a 58 v e r y l o w l e v e l o f c y a n a t e i n t h e m e d i a c o u l d c a u s e a d e g r e e o f n i t r o g e n s t r e s s c o n c o m i t a n t t o a n i n c r e a s i n g a c c u m u l a t i o n o f ammonia. A t a c e r t a i n p o i n t , d e p e n d e n t upon t h e c o n c e n t r a t i o n o f u r e a , t h e l e v e l o f ammonia w o u l d become t o x i c and t h e e l e c t r o l y t i c e f f e c t s n o t e d w o u l d o c c u r . The p r e s e n c e o f c y a n a t e i n u r e a c u l t u r e m e d i a c o u l d be e v a l u a t e d by r e m o v i n g a l i q u o t s o f a s t e r i l e sample o f s u c h m e d i a o v e r a p e r i o d o f t i m e and t r e a t i n g t h e s e a l i q u o t s w i t h u r e a s e . T h i s w o u l d g i v e a q u a n t i t a t i v e i n d i c a t i o n o f t h e amount o f u r e a n o t decomposed. The u r e a s e c o u l d , t h e n be r e m o v e d by means o f a l u m i n u m h y d r o x i d e a n d a s i l v e r n i t r a t e t e s t f o r c y a n a t e employed••; ( F e a r o n , 1923) The e f f e c t o f c y a n a t e on n i t r o g e n m e t a b o l i s m s h o u l d a l s o be e v a l u a t e d . T h i s c o u l d be d e t e r m i n e d by t h e a d d i t i o n o f v a r y i n g c o n c e n t r a t i o n s o f c y a n a t e t o c u l t u r e s grown i n u r e a , n i t r a t e and ammonia m e d i a . The r a t e o f u p t a k e o f t h e n i t r o g e n compound and t h e amount o f ammonia i n t h e m e d i a o v e r a p e r i o d o f t i m e s h o u l d be a s c e r t a i n e d . The ammonia m e d i a w o u l d g i v e a d i r e c t i n d i c a t i o n , o f t h e e f f e c t o f c y a n a t e on ammonia a s s i m i l a t i o n . The u r e a m e d i a w o u l d g i v e a d i r e c t i n d i c a t i o n o f t h e e f f e c t o f c y a n a t e on u r e a a s s i m i l a t i o n a n d i n h i b i t i o n o f t h e s u b s e q u e n t m e t a b o l i c s t e p . The n i t r a t e m e dia w o u l d a c t as a c h e c k , a s n o r m a l l y no ammonia.would be o b s e r v e d i n t h e m e d i a u n l e s s t h e m e t a b o l i c pathway were i n h i b i t e d . 59 CONCLUSION One eff e c t of urea on Rhodomonas lens under.the imposed experimental conditions appears to be the induction of nitrogen stress during lag and logarithmic phases. This stress could be caused by an i n e f f i c i e n t uptake mechanism regulated perhaps by the s a l i n i t y of the media or by an i n e f f i c i e n t metabolism of urea. A decline phase during -growth of the•experimental organism occurs when concentra-tions' of urea are equal to or greater t h a n 0.50 mM. The appearance of c e l l s during t h i s phase i s si m i l a r to that of c e l l s e x h i b i t i n g ammonia to x i c i t y . . . It i s suggested that the pathway of urea metabolism i s inadequate, such, that the ammonia produced by the breakdown of urea i s s . i n e f f i c i e n t l y assimilated, with the excess ammonia being released into the medium. The increasing accumulation of ammonia i n the medium from the higher u r e a concentration, accelerates c e l l l y s i s , r e s u l t i n g i n a decline phase due to ammonia t o x i c i t y . A possible i n h i b i t o r y factor i n t h e urea u t i l i z a t i o n pathway i s a breakdown product of u r e a : urea > NH J | + cyanate. PLATE 1 . F i g u r e 2 . An o p t i c a l m i c r o g r a p h o f c o n t r o l c e l l s i n c u l t u r e u s i n g N o m a r s k i i n t e r f e r e n c e i l l u m i n a t i o n . N ote t h e a s y m m e t r i c c e l l shape and t h e i n s e r t i o n o f t h e f l a g e l l a ( F ) n e a r t h e a n t e r i o r g u l l e t ( G ) . X 2 9 2 0 . F i g u r e 3. A s l i g h t l y o b l i q u e t r a n s v e r s e s e c t i o n f r o m a c o n t r o l c u l t u r e d u r i n g l o g a r i t h m i c p h a s e . O r g a n e l l e s w h i c h c a n be s e e n a r e t h e n u c l e u s ( N ) , m i t o c h o n d r i a (M) , g o l g i body ( G o ) , l a r g e t r i c h o c y s t s (T) w h i c h l i n e t h e g u l l e t ( G ) , s m a l l t r i c h o c y s t s ( t ) n e a r t h e p e r i p l a s t ( P ) , and t h e c h l o r o p l a s t ( C H L ) . W i t h i n t h e c h l o r o p l a s t p a r t o f t h e p y r e n o i d (Py) and a s s o c i a t e d s t a r c h g r a i n s a r e v i s i b l e . The t h y l a k o i d s a r e a s s o c i a t e d i n bands o f two ( a r r o w s ) . Note t h e b i p a r t i t e n a t u r e o f t h e p e r i p l a s t ( open a r r o w s ) . X 1 2 , 0 0 0 . PLATE 2. C o n t r o l c e l l s . F i g u r e 4. A l o n g i t u d i n a l s e c t i o n o f a f l a g e l l u m s h o w i n g t h e r e g u l a r a r r a n g e m e n t o f t h e f i b r o u s m a s t i g o n e m e s ( a r r o w s ) T h e s e a r e s i m i l a r t o t h o s e o b s e r v e d i n Ochromonas 0 d a n i c a (Bouck, 1971) and measure a b o u t 50-70 A i n d i a m e t e r . Note a l s o t h e l a t e r a l r o o t l e t o f t h e f l a g e l l u m ( R ) . X 59,000. F i g u r e 5. An o b l i q u e s e c t i o n o f t h e f l a g e l l a ( F ) s h o w i n g t h e t u b u l a r m a s t i g o n e m e s (M), a b o u t 200 A i n d i a m e t e r , i n l o n g i t u d i n a l s e c t i o n . X 47,000. 65 PLATE 3. C o n t r o l c e l l s . F i g u r e 6. An o b l i q u e s e c t i o n s h o w i n g t h e a r r a n g e m e n t o f t h e c h l o r o p l a s t w i t h i n t h e c e l l . O r g a n e l l e s v i s i b l e a r e t h e C o r p s de Maupas (CM), m i t o c h o n d r i a (M), r o u g h e n d o p l a s m i c r e t i c u l u m (RER), and t h e l a r g e and s m a l l t r i c h o c y s t s (T & t ) . Note t h e b i p a r t i t e p e r i p l a s t ( a r r o w s ) . X 22,000. F i g u r e 7. A s e c t i o n o f t h e p e r i p h e r a l r e g i o n o f t h e c e l l s h o w i n g t h e a r r a n g e m e n t o f t h e t h y l a k o i d s ( a r r o w s ) w i t h i n t h e c h l o r o p l a s t . N ote t h e g r e a t e r e l e c t r o n o p a c i t y o f t h e l o c u l u s compared t o t h a t o f t h e s t r o m a l a r e a ( S A ) . The p l a s m a membrane (PM) and t h e i n n e r l a y e r ( I L ) o f t h e p e r i p l a s t a r e v i s i b l e . X 27,000. 65 PLATE 4. F i g u r e 8. C o n t r o l c e l l s d u r i n g l a t e s t a t i o n a r y p h a s e . Note t h e r e l a t i v e p o s i t i o n s o f t h e c h l o r o p l a s t (CHL) and t h e l i p i d - l i k e m a t e r i a l ( L L ) . L a r g e s t a r c h g r a i n s (S) c a n be s e e n w i t h i n t h e p e r i c h l o r o p l a s t i c m a t r i x and n e a r t h e p y r e n o i d ( P y ) . X 7310. F i g u r e 9. A l o n g i t u d i n a l s e c t i o n , d u r i n g l a t e s t a t i o n a r y p h a s e s h o w i n g t h e amount o f l i p i d - l i k e m a t e r i a l w i t h i n t h e c e l l . X 10,200. F i g u r e 10. A s l i g h t l y o b l i q u e l o n g i t u d i n a l s e c t i o n n e a r t h e c e l l p e r i p h e r y s h o w i n g t h e t h y l a k o i d a l a r r a n g e m e n t ( a r r o w s ) . N ote t h e s t a r c h g r a i n s ( S ) , s m a l l t r i c h o c y s t s ( t ) , and t h e C o r p s de Maupas (CM). X 29,400. PLATE 5. F i g u r e 14. E x p e r i m e n t a l c e l l d u r i n g l o g a r i t h m i c p h a s e . L i p i d d r o p l e t s ( L ) a r e s e e n between t h e p e r i p l a s t a n d t h e c h l o r o p l a s t ( C H L ) . X 33,000. F i g u r e 15. An o b l i q u e s e c t i o n n e a r t h e p o s t e r i o r o f t h e c e l l s h o w i n g some l i p i d ( L ) a c c u m u l a t i o n a n d t h e c h l o r o p l a s t (CHL) w i t h p l a s t o g l o b u l i ( P l ) . X 19,200. « 9 PLATE 6. F i g u r e 16. A s e c t i o n s h o w i n g s e v e r a l e x p e r i m e n t a l c e l l s f i x e d d u r i n g e a r l y l o g a r i t h m i c p h a s e . Note t h e a c c u m u l a t i o n o f l i p i d ( L ) i n e a c h c e l l . X 6800. F i g u r e 17. A l o n g i t u d i n a l s e c t i o n o f an e x p e r i m e n t a l c e l l f i x e d i n e a r l y l o g a r i t h m i c p h a s e s h o w i n g t h e l a r g e l i p i d d r o p l e t ( L) and t h e a r r a n g e m e n t o f t h y l a k o i d s i n t h e c h l o r o p l a s t ( C H L ) . X 22,800. PLATE 7-F i g u r e s 18 t o 25. O p t i c a l m i c r o g r a p h s o f c o n t r o l c e l l s u n d e r t o x i c ammonia c o n d i t i o n s a t i n c r e a s e d pH u s i n g N o m a r s k i i n t e r f e r e n c e i l l u m i n a t i o n . X 2830. F i g u r e 18. A n o r m a l . c e l l a t pH 7-6 and z e r o t i m e . F i g u r e 19. pH 8.65, lh h o u r s . C e l l has begun t o r o u n d . F i g u r e 20. pH 8.65, 3 h o u r s . C e l l i s r o t u n d : c h l o r o p l a s t i s s l i g h t l y f l a t t e n e d p o s t e r i o r l y . F i g u r e 21. pH 8.65, h o u r s . C e l l i s r o t u n d : c h l o r o p l a s t i s much more f l a t t e n e d p o s t e r i o r l y . F i g u r e 22. pH 8.65, 5h h o u r s . C e l l c o n t e n t s b e g i n t o p r o t r u d e : f l a g e l l a a r e s t i l l p r e s e n t . F i g u r e 23. pH 8.4, 8 h o u r s . P r o t r u s i o n s v e r y p r o n o u n c e d F i g u r e 24. pH 8.5, 8 h o u r s . C e l l s w e l l i n g i s n o t i c e a b l e a c c o m p a n y i n g v a c u o l a t i o n a n d i n c r e a s i n g p r o t r u s i o n s . F i g u r e 25. pH 8.5, 8 h o u r s . The c e l l has l y s e d a n d t h e c o n t e n t s a r e e x t r u d e d i n t o t h e m e d i a . 74 FOOTNOTES ''"Oceanic phytoplankton probably compete f o r the use of urea: blue-green algae (Berns, Holohan and S c o t t , 1966) and many types of b a c t e r i a ( J e f f r i e s , 1964) are known to cont a i n urease and grow w e l l i n urea media. 2 Butcher (1967) does not recognize Rhodomonas as a genus, nor does h i s d e s c r i p t i o n of Rhodomonas lens a c c u r a t e l y describe the organism used i n t h i s work. However, publ i s h e d work (Gibbs, 1962 a, b; Cheng and A n t i a , 1970; Gantt, Edwards and P r o v a s o l i , 1971; Oakley, 1974) w i t h t h i s organism used t h i s c l a s s i f i c a t i o n . Therefore, pending f u r t h e r c l a r i f i c a t i o n , i n t h i s t h e s i s the organism w i l l be c a l l e d Rhodomonas l e n s . Rhodomonas lens was o r i g i n a l l y i s o l a t e d from the Gulf Stream by R. Lasker. Dr. N. A n t i a obtained i t from Haskins L a b o u r a t o r i e s , New York. 4 Two m i l l i p o r e s i z e s were used c o n s e c u t i v e l y : M i l l i p o r e Type HA f o l l o w e d by M i l l i p o r e GSWP 047 00. (Pores: .45u,.22^i). 5 Compounds f o r s y n t h e t i c sea water medium were e l u c i d a t e d by Lyman and Fleming: the medium was pu b l i s h e d by A n t i a and Cheng (1970). Enrichment media was pu b l i s h e d by A n t i a and Chorney (1968). Formulae are taken from Eagon's Advanced General M i c r o b i o l o g y , page 27. 7 Some c e l l l y s i s may occur with t h i s method. A small amount of c e l l l y s i s may have occurred during c e n t r i f u g a t i o n 75 w i t h an a c c o m p n a y i n g r e l e a s e o f i n t r a c e l l u l a r u r e a a n d / o r u r e a s e . o . . S y l v a n i a " c o o l w h i t e " f l u o r e s c e n t l i g h t s . (.#P48T12/CW). were u s e d . m > 0 • 035 t •z 1 (i .DUO 2 r-Ul t-O 0 u. .025 n r • 020 J M r <r •oi5 l- o 0 • oio bl 0. cn 3 • ooi5 250 35o -ISO 5.50 ^ T h i s was n o t a l w a y s o b s e r v e d . O f t e n no d e c l i n e p h a s e was n o t e d a n d t h e s t a t i o n a r y p h a s e was s t i l l p r e s e n t a f t e r s e v e r a l weeks. <;- "^The enzyme u t i l i z e d t o c a t a l y z e t h e breakdown o f u r e a i n Rhodomonas l e n s i s p r o b a b l y u r e a s e ( L e f t l e y , p e r s o n a l ' c o m m u n i c a t i o n ) . ~^The s a l i n i t y o f t h e m e d i a was 28/*oo. S a . l i n i . t y o f o t h e G u l f S t r e a m s u r f a c e w a t e r s i s a p p r o x i m a t e l y 36/00. 12 N i t r o g e n s t r e s s , as u s e d i n t h i s t h e s i s , i s n o t u s e d i n a s t r i c t p h y s i o l o g i c a l s e n s e , b u t i s b a s e d on one p a r a m e t e r , t h e c y t o l o g i c a l e v i d e n c e o f l i p i d a c c u m u l a t i o n ( S p o e r and M i l n e r , 1949; V a n B a a l e n , 1963). 13 C h l o r o p h y l l p l u s c a r o t e n o i d s g i v e s a g r e e n o r a r e d - o r a n g e c o l o u r a t i o n ; c h l o r o p h y l l , c a r o t e n o i d s a n d p h y c o b i l i n s , a c l e a r r e d - p i n k c o l o u r (Cheng, D o n - P a u l 76 and A n t i a , 1974). 1 4 C e r t a i n accessory pigments may be n o n - e s s e n t i a l ; t h e r e f o r e , during a n u t r i t i o n a l - c r i s i s t h e i r components can be u t i l i z e d i n the synthesis of compounds v i t a l to.the c e l l 15 Comparatively, a 1 0 - f o l d increase i n ug-atom N - l i t e r i n the experimental media i s r e q u i r e d to a t t a i n an equal d i v i s i o n r a t e to that of c o n t r o l c u l t u r e s . 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T h ey s u g g e s t , due t o t h e e f f i c i e n c y and f a c i l i t y w i t h " w h i c h 85$ o f t h e t e s t e d s p e c i e s u t i l i z e d u r e a , t h a t " u r e a may be t h e most i m p o r t a n t s o u r c e o f o r g a n i c n i t r o g e n a v a i l a b l e f o r g r o w t h by m a r i n e p h y t o p l a n k t o n i n t h e o c e a n s . " The medium u s e d was e n r i c h e d s e a w a t e r , t h e s e a w a t e r b e i n g p r o c u r e d f r o m a M e d i t e r r a n e a n c o a s t a l i n l e t , w i t h a o f i n a l s a l i n i t y o f 31/oo a n d a n i t r o g e n l e v e l o f 50Q: ug-atom .. -1 ., -1 l i t e r I I . Z g u r o v s k a y a and K u s t e n k o (1-968.) o b s e r v e d a s i m i l a r c o n c e n t r a t i o n o f ammonia n i t r o g e n t o x i c i t y f o r t h e t e s t e d o r g a n i s m s t o t h a t n o t e d i n t h i s t h e s i s . 109. Z g u r o v s k a y a , L.N. a n d N.G. K u s t e n k o . 1968. 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