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Preliminary exploration of estuarine ecosystem structure at low trophic levels with a controlled microcosm Wu, Yong 1985

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PRELIMINARY EXPLORATION OF ESTUARINE ECOSYSTEM STRUCTURE AT LOW TROPHIC LEVELS WITH A CONTROLLED MICROCOSM by Yong Wu B . S c , Xiamen U n i v e r s i t y , Xiamen, China, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Oceanography) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1985 (c) Yong Wu, 1985 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of Oceanography The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date 3 March, 1986 A b s t r a c t I n a l a b o r a t o r y m i c r o c o s m , two k i n d s o f s a l i n i t y g r a d i e n t s w e r e c r e a t e d t o s i m u l a t e t h e p r o c e s s o c c u r r i n g i n e s t u a r i n e c i r c u l a t i o n w i t h m i x i n g s e a w a t e r and f r e s h w a t e r . The b e h a v i o u r o f t h e f r e s h w a t e r a n d s e a w a t e r e c o s y s t e m c o m p o n e n t s i n r e l a t i o n t o t h e s a l i n i t y g r a d i e n t s w e r e i n v e s t i g a t e d . S e v e n p a r a m e t e r s w e r e c h o s e n a s i n d i c a t o r s o f t h e d i f f e r e n t t r o p h i c l e v e l s i n t h e e c o s y s t e m o r o f t h e e n v i r o n m e n t a l c o n d i t i o n s . The i n t e r a c t i o n among t h e d i f f e r e n t t r o p h i c l e v e l s was r e f l e c t e d i n t h e d e v e l o p m e n t p a t t e r n o f p h y t o p l a n k t o n , n u t r i e n t s , b a c t e r i a , a n d n a n o z o o f l a g e l l a t e s . The i n t e r a c t i o n among t h e e c o s y s t e m a nd e n v i r o n m e n t a l c o n d i t i o n s w e r e r e f l e c t e d i n t h e d i f f e r e n c e b e t w e e n t h e d i f f e r e n t e x p e r i m e n t s w i t h d i f f e r e n t s a l i n i t y g r a d i e n t s a n d d i f f e r e n t e c o s y s t e m o r i g i n . I n s t a g e I f r e s h w a t e r p h y t o p l a n k t o n w e r e t e s t e d on two d i f f e r e n t s a l i n i t y g r a d i e n t s . The d a t a showed t h a t t h e f r e s h w a t e r b i o t a c o u l d n o t p a s s t h r o u g h t h e s a l i n i t y g r a d i e n t . M o s t o f them d i e d o r w e r e i n h i b i t e d d u r i n g t h e m i x i n g p r o c e s s . The a u t o t r o p h i c c o m p o nent i n f r e s h w a t e r c o u l d no l o n g e r f u n c t i o n a s a a u t o t r o p h i c c omponent b u t s e r v e d a s an o r g a n i c s u b s t r a t e c o n t r i b u t o r . The i n h i b i t i o n o f p h y t o p l a n k t o n g r o w t h by t h e s a l i n i t y g r a d i e n t p r o v i d e d a c o n d i t i o n i n w h i c h t h e b a c t e r i a c o u l d c o n c u r r e n t l y d e v e l o p i n t h e e c o s y s t e m . I n a homogenous c o n d i t i o n w i t h c o n t r o l l e d f l a s k s , t h e d e v e l o p m e n t o f t h e a u t o t r o p h i c c o m p o nent was s e p a r a t e d f r o m h e t e r o t r o p h i c i i i b a c t e r i a o v e r t i m e . I n s t a g e I I , a s e a w a t e r e c o s y s t e m h a d a d i f f e r e n t r e s p o n c e f r o m t h a t o f f r e s h w a t e r . W i t h an i n c r e a s i n g s a l i n i t y , t h e g r o w t h o f p h y t o p l a n k t o n c o u l d be l i m i t e d a t l o w s a l i n i t i e s . T h i s r e s u l t e d i n a d e l a y o f t h e maximum p h y t o p l a n k t o n b i o m a s s a n d p r o v i d e d t h e f i r s t p e r i o d o f t i m e f o r b a c t e r i a l d e v e l o p m e n t b e f o r e t h e b l o o m o f a u t o t r o p h i c c o m p o n e n t s . Thus b a c t e r i a f o r m e d a p e a k b e f o r e p h y t o p l a n k t o n d e v e l o p e d . S e a w a t e r p h y t o p l a n k t o n c o u l d , on t h e o t h e r h a n d , a c t i v e l y a n d q u i c k l y r e s p o n d i n t h e i r g r o w t h , on t h e s a l i n i t y g r a d i e n t . Thus t h e s e a w a t e r a u t o t r o p h i c c o mponent may p l a y t h e m a j o r r o l e i n p r i m a r y p r o d u c t i o n i n a p h y t o p l a n k t o n b a s e d e s t u a r i n e e c o s y s t e m . The b e h a v i o r o f a u t o t r o p h i c c o m p o n e n t s i n b o t h s y s t e m s c a n h a v e a s t r o n g e f f e c t on t h e r e s t o f t h e c o m p o n e n t s i n t h e s y s t e m ; t h e i r c h a n g e s c o u l d c a u s e a g r e a t c h a n g e i n t h e w h o l e s y s t e m s t r u c t u r e . D i f f e r e n t d e v e l o p m e n t a l p a t t e r n s o f p h y t o p l a n k t o n a n d b a c t e r i a i n d i f f e r e n t e x p e r i m e n t s w e r e e x p l a i n e d w i t h a c o n c e p t u a l d i a g r a m w h i c h s u m m a r i z e s t h e i d e a o f e n e r g y s t a t e s o f an e c o s y s t e m a n d t h e f u n c t i o n o f p h y t o p l a n k t o n a n d b a c t e r i a i n e c o s y s t e m d y n a m i c s . T e m p o r a l d e v e l o p m e n t p a t t e r n s o f t h e e c o s y s t e m c o m p o n e n t s i n o u r e x p e r i m e n t s may be e x t r a p o l a t e d i n t o s p a t i a l d i s t r i b u t i o n s i f a b o d y o f w a t e r i s m o v i n g s e a w a r d i n an e s t u a r y . Thus a c o n c e p t u a l m o d e l i s p r e s e n t e d t o e x p l a i n t h e s p a t i a l d i s t r i b u t i o n o f many b i o l o g i c a l l y i m p o r t a n t c o m p o n e n t s w h i c h h a v e o f t e n b e e n r e p o r t e d i n many f i e l d i n v e s t i g a t i o n s o f e s t u a r i e s . i v TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES V LIST OF FIGURES v i ACKNOWLEDGEMENTS i x 1. INTRODUCTION 1 2. EXPERIMENTAL DESIGN 10 2.1 PRECONSIDERATION 10 2.2 DESIGN OF THE EXPERIMENT 14 2.3 CONDITION OF EXPERIMENT 18 3. METHODS OF ANALYSIS OF SAMPLES 22 3.1 NUTRIENTS 22 3.2 TOTAL BACTERIA NUMBERS, BIOVOLUMES AND BIOMASS 23 3.3 CALCULATION OF SALINITY GRADIENT 25 3.4 MICROFLAGELLATES AND PHYTOPLANKTON 25 3.5 STANDING STOCK OF PHYTOPLANKTON 26 4. RESULTS 28 4.1 EXPERIMENT STAGE I 28 4.11 INCREASING SALINITY GRADIENT 28 4.12 DECREASING SALINITY GRADIENT 48 4.2 EXPERIMENT STAGE II 60 4.21 INCREASING SALINTIY GRADIENT 60 4.22 DECREASING SALINITY GRADIENT 69 5. DISCUSSION 84 6. LITERATURE CITED 109 V L i s t of Tables Table page 1. The procedures of the experiment -in the two sta g e s . 17 2. The s a l i n i t y of i n i t i a l waters c o l l e c t e d a t d i f f e r e n t times f o r the l a b o r a t o r y experiments 22 3. C o r r e l a t i o n matrix o b t a i n e d from the 7 v a r i a b l e s X 20 ob s e r v a t i o n s i n experiment stage I, p a r t 1 d u r i n g J u l y , 1985, between time (day), F l u o r e s c e n c e ( f l u o ) , N0~3, NH+4, F l a g e l l a t e s ( f i g ) , B a c t e r i a (bac), and S a l i n i t y . P r o b a b i l i t y : * p<0.1, ** p < 0.05, *** p < 0.02, **** p <0.01 41 4. C o r r e l a t i o n matrix o b t a i n e d from the 7 v a r i a b l e s X 20 ob s e r v a t i o n s i n experiment stage I, p a r t 2 d u r i n g J u l y , 1985, between time (day), F l u o r e s c e n c e ( f l u o ) , NO~3, NH +4, F l a g e l l a t e s ( f i g ) , B a c t e r i a (bac), and S a l i n i t y . P r o b a b i l i t y : * p<0.1, ** p < 0.05, *** p < 0.02, **** p <0.01 41 5. C o r r e l a t i o n matrix o b t a i n e d from the 6 v a r i a b l e s X 12 o b s e r v a t i o n s i n experiment stage I I , p a r t 1 d u r i n g Aug., 1985, between time (day), F l u o r e s c e n c e ( f l u o ) , N0~3, F l a g e l l a t e s ( f i g ) , B a c t e r i a (bac), and s a l i n i t y . P r o b a b i l i t y : * p<0.1, ** p < 0.05, *** p < 0.02, **** p <0.01 68 6. C o r r e l a t i o n matrix o b t a i n e d from the 6 v a r i a b l e s X 12 o b s e r v a t i o n s i n experiment stage I I , p a r t 2 du r i n g Aug., 1985. between time (day), F l u o r e s c e n c e ( f l u o ) , NO~3, F l a g e l l a t e s ( f i g ) , B a c t e r i a (bac), and S a l i n i t y , p r o b a b i l i t y : * p<0.1, ** p < 0.05, *** p < 0.02, **** p <0.01 68 v i L I S T OF FIGURES 1. F l o w d i a g r a m f o r t h e u p p e r l a y e r box o f w a t e r i n a two d i m e n s i o n a l m o d e l o f e s t u a r i n e c i r c u l a t i o n 11 2. S a m p l i n g s i t e s f o r l a b o r a t o r y e x p e r i m e n t s a n d s u r f a c e s a l i n i t y d i s t r i b u t i o n i n F r a s e r e s t u a r y a n d pl u m e 17 3. D e s i g n o f t h e e x p e r i m e n t s w i t h t h r e e u n i t s 20 4. The t e m p o r a l s a l i n i t y p a t t e r n i n t h e e x p e r i m e n t s t a g e I , I I p a r t 1 w i t h an i n c r e a s i n g s a l i n i t y g r a d i e n t 29 5 a . The d e v e l o p m e n t p a t t e r n o f a u t o t r o p h i c c o m p o nent a nd n u t r i e n t i n t h e e x p e r i m e n t s t a g e I , p a r t 1 d u r i n g J u l y , 1985 30 5b. The d e v e l o p m e n t p a t t e r n o f NH +4 i n t h e e x p e r i m e n t s t a g e I , p a r t 1 d u r i n g J u l y , 1985 31 6 a . The d e v e l o p m e n t p a t t e r n o f a u t o t r o p h i c c o m p o nent a nd n u t r i e n t i n t h e e x p e r i m e n t s t a g e I , p a r t 1 d u r i n g F e b r u a r y , 1985 32 6b. The d e v e l o p m e n t p a t t e r n o f NH +4 i n t h e e x p e r i m e n t s t a g e I , p a r t 1 d u r i n g F e b r u a r y , 1985 33 7. The d e v e l o p m e n t p a t t e r n f o r b o t h h e t e r o t r o p h i c b a c t e r i a and n a n o z o o f l a g e l l a t e s i n e x p e r i m e n t s t a g e I p a r t 1 d u r i n g J u l y , 1985 36 8. G r o w t h o f f r e s h w a t e r b a c t e r i a on p l a t e m e d i a a t d i f f e r e n t s a l i n i t y c o n c e n t r a t i o n s .36 9. The t e m p o r a l s a l i n i t y p a t t e r n i n e x p e r i m e n t s t a g e I & I I , p a r t 2 . w i t h a d e c r e a s i n g s a l i n i t y g r a d i e n t 43 1 0 a . The d e v e l o p m e n t p a t t e r n o f a u t o t r o p h i c c omponent a n d n u t r i e n t s i n S t a g e I , p a r t 2. w i t h a d e c r e a s i n g s a l i n i t y g r a d i e n t i n J u l y , 1985 44 10b. The d e v e l o p m e n t p a t t e r n o f NH +4 c o n c e n t r a t i o n i n S t a g e I , p a r t 2 w i t h a d e c r e a s i n g s a l i n i t y g r a d i e n t i n J u l y 1985 45 1 1 a . The d e v e l o p m e n t p a t t e r n o f a u t o t r o p h i c c o m p o nent a nd n u t r i e n t s i n S t a g e I , p a r t 2 w i t h a d e c r e a s i n g s a l i n i t y g r a d i e n t i n F e b r u a r y , 1985 46 V l l l i b . The development p a t t e r n of NH +4 c o n c e n t r a t i o n i n Stage I, P a r t 2 with a d e c r e a s i n g s a l i n i t y g r a d i e n t i n February, 1985 47 12. The development p a t t e r n of b a c t e r i a and n a n o z o o f l a g e l l a t e s i n Stage I, p a r t 2 with a de c r e a s i n g s a l i n i t y g r a d i e n t 49 13. The development p a t t e r n of a u t o t r o p h i c component, n u t r i e n t and b a c t e r i a i n a c o n t r o l l e d system with o r i g i n a l seawater d u r i n g J u l y , 1985 53 14. The development p a t t e r n of a u t o t r o p h i c component n u t r i e n t and b a c t e r i a i n a c o n t r o l l e d system wi t h o r i g i n a l freshwater d u r i n g J u l y , 1985 54 15. The development p a t t e r n of phytoplankton and c o n c e n t r a t i o n of n u t r i e n t s i n stage I I p a r t 1 on i n c r e a s i n g s a l i n i t y g r a d i e n t d u r i n g Aug. 1985 61 16a. The development p a t t e r n of a u t o t r o p h i c component and c o n c e n t r a t i o n of n u t r i e n t s i n stage I I p a r t 1 on an i n c r e a s i n g s a l i n i t y g r a d i e n t d u r i n g A p r i l , 1985 62 16b. The development p a t t e r n of NH +4 i n stage I I , p a r t 1 on an i n c r e a s i n g s a l i n i t y g r a d i e n t d u r i n g A p r i l , 1985 63 17. The development p a t t e r n of b a c t e r i a and n a n o z o o f l a g e l l a t e s i n stage I I , p a r t 1 with i n c r e a s i n g s a l i n i t y g r a d i e n t d u r i n g Aug., 1985 65 18. Development of a u t o t r o p h i c component i n subsample c u l t u r e s e r i e s at temp o r a l l y constant s a l i n i t y i n stage I I , p a r t 1 d u r i n g Aug., 1985 66 19. The development p a t t e r n of a u t o t r o p h i c component and c o n c e n t r a t i o n of n u t r i e n t s i n stage I I , p a r t 2 on a de c r e a s i n g s a l i n i t y g r a d i e n t d u r i n g Aug. 1985 70 20a. The development p a t t e r n of a u t o t r o p h i c component and c o n c e n t r a t i o n of n u t r i e n t s i n stage I I , p a r t 2 on a de c r e a s i n g s a l i n i t y g r a d i e n t d u r i n g A p r i l , 1985 71 20b. The development p a t t e r n of NH +4 i n stage I I , p a r t 2 on a d e c r e a s i n g s a l i n i t y g r a d i e n t d u r i n g A p r i l , 1985 72 21. The development p a t t e r n of b a c t e r i a and n a n o z o o f l a g e l l a t e s i n stage I I , p a r t 2 on a d e c r e a s i n g s a l i n i t y g r a d i e n t d u r i n g Aug., 1985 75 v i i i 22. Development of a u t o t r o p h i c component i n a subsample c u l t u r e s e r i e s a t te m p o r a l l y constant s a l i n i t y i n stage I I , p a r t 1 d u r i n g Aug., 1985 76 2 3 a . The d e v e l o p m e n t p a t t e r n o f a u t o t r o p h i c c omponent i n a c o n t r o l l e d s y s t e m w i t h o r i g i n a l s e a w a t e r d u r i n g Aug. 1985 82 23b. The d e v e l o p m e n t p a t t e r n o f a u t o t r o p h i c c omponent i n a c o n t r o l l e d s y s t e m w i t h o r i g i n a l f r e s h w a t e r d u r i n g Aug. 1985 83 24. The d y n a m i c s o f e c o s y s t e m e n e r g e t i c s t a t e i n t h e e n e r g y f i e l d 88 25. The a r r a n g e m e n t o f e c o s y s t e m c o m p o n e n t s i n an e s t u a r i n e p h y s i c a l f o r c e f i e l d . 105 i x A c k n o w l e d g e m e n t T h i s t h e s i s w o u l d n o t h a v e b e e n p o s s i b l e w i t h o u t t h e h e l p o f m a n y p e o p l e . I am v e r y g r a t e f u l t o my t h e s i s s u p e r v i s o r , D r . T . R. P a r s o n s , f o r h i s e n c o u r a g e m e n t , i n s p i r a t i o n i n t h e i n i t i a t i o n a n d t h e f r e e d o m o f e x p l o r a t i o n t h r o u g h o u t t h e s t u d y . I t i s h e w h o d i r e c t e d me i n t h i s f i e l d a n d I t h i n k h i s i n f l u e n c e i n t h i n k i n g o f e c o s y s t e m s w i l l b e w i t h me f o r m a n y y e a r s t o c o m e . I am g r a t e f u l t o D r . P . J . H a r r i s o n f o r h i s c o n s t r u c t i v e s u g g e s t i o n s , h e l p f u l d i s c u s s i o n a s w e l l a s t h e k i n d n e s s h e s h o w e d t o me d u r i n g t h e p a s t t w o y e a r s , a n d a l s o f o r p r o v i d i n g t h e u s e o f h i s l a b o r a t o r y . S p e c i a l t h a n k s a r e d u e t o D r . A . G . L e w i s f o r h i s v e r y h e l p f u l d i s c u s s i o n a n d c o m m e n t s a t t h e b e g i n n i n g o f my l a b o r a t o r y w o r k . T h a n k s a r e a l s o d u e t o D r . F . J . R . T a y l o r , f o r h i s c o m m e n t s a n d a l l o w i n g me t o u s e h i s l a b o r a t o r y . H e r e , t h a n k s a r e e x t e n d e d t o M i s s J u d y A c r e m a n f o r h e r h e l p i n my i d e n t i f i c a t i o n o f s p e c i e s . M u c h a p p r e c i a t i o n i s e x t e n d e d t o D r . C a r o l L a l l i f o r h e r h a r d w o r k i n i m p r o v i n g my m a n u s c r i p t . I am g r a t e f u l t o D r . R . J . A n d e r s e n , w h o s a t o n my c o m m i t t e e a n d f o r a p p r o v a l o f my p r o p o s a l a n d r e a d i n g my t h e s i s . I l i k e t o t h a n k D r . P . H . L e B l o n d a n d D r . R . W . B u r l i n g f o r t h e i r h e l p f u l d i s c u s s i o n o n t h e e s t u a r i n e c i r c u l a t i o n . M y a p p r e c i a t i o n i s a l s o g i v e n t o m a n y f e l l o w s , f a c u l t y a n d s e c r e t a r i e s a t t h e D e p a r t m e n t o f O c e a n o g r a p h y f o r t h e i r k i n d n e s s a n d t h e h e l p t h e y h a v e g i v e n me w h e n e v e r I n e e d e d i t . S p e c i a l t h a n k s t o M i s s H e a t h e r D o v e y f o r h e r f r i e n d s h i p a n d m u c h a s s i s t a n c e t h r o u g h o u t t h e s t u d y a n d h e r a s s i s t a n c e i n t y p i n g t h i s m a n u s c r i p a n d i n d r a w i n g t h e f i g u r e s . S p e c i a l t h a n k s t o P e t e r T h o m p s o n f o r h i s k i n d a s s i s t a n c e d u r i n g my u s e o f D r . H a r r i s o n ' s l a b o r a t o r y e q u i p m e n t a n d t h e h e l p f u l d i s c u s s i o n d u r i n g my e x p e r i m e n t . T h a n k s a r e d u e t o M . P . S t o r m f o r h i s a s s i s t a n c e i n m e a s u r i n g s a l i n i t i e s f o r my e x p e r i m e n t s . M a n y t h a n k s t o my f e l l o w g r a d u a t e s t u d e n t s , f o r t h e i r f r i e n d s h i p a n d g e n e r a l h e l p w h i c h g a v e me a g o o d e n v i r o n m e n t i n t h e p a s t t w o y e a r s , a n d m a n y t h a n k s t o s t u d e n t s i n D r . P . J . H a r r i s o n ' s g r o u p f o r t h e i r g e n e r a l h e l p . T o d e t a i l a l l o f t h e s u p p o r t I h a v e r e c e i v e d i s i m p o s s i b l e . I w o u l d l i k e t o t h a n k a l l my f r i e n d s i n C a n a d a a n d i n C h i n a f o r t h e i r c o n t i n u o u s e n c o u r a g e m e n t , c a r e a n d h e l p w h i c h d i r e c t l y a n d i n d i r e c t l y a i d e d me i n my t w o y e a r s o f s t u d y . I w i l l a l w a y s b e g r a t e f u l t o my p a r e n t s a n d my w i f e f o r t h e i r c o n s t a n t m o r a l s u p p o r t a n d t h e c o n f i d e n c e t h e y p l a c e d i n m e ; t o t h e m I d e d i c a t e t h i s t h e s i s . F i n a l l y , I a c k n o w l e d g e t h a t I w a s s u p p o r t e d b y t h e N a t i o n a l S c h o l a r s h i p o f T h e P e o p l e s ' R e p u b l i c o f C h i n a a n d a n a w a r d f r o m t h e I n t e r n a t i o n a l D e v e l o p m e n t R e s e a r c h C e n t r e o f C a n a d a . 1. I n t r o d u c t i o n 1 E s t u a r i n e e c o s y s t e m s a r e a t r a n s i t i o n b e t w e e n f r e s h w a t e r a n d m a r i n e s y s t e m s a n d , a s s u c h , a r e h i g h l y d y n a m i c a n d r e l a t i v e l y v e r y c o m p l e x . W i t h i n a n e s t u a r y f r e s h w a t e r a n d s e a w a t e r m i x t o g e t h e r t o p r o d u c e a d e n s i t y d i f f e r e n c e o v e r s p a c e w h i c h d r i v e s t h e v a r i o u s e s t u a r i n e c i r c u l a t i o n p a t t e r n s . P r i t c h a r d ( 1 9 5 5 ) c l a s s i f i e d f o u r p a t t e r n s , w i t h t h e mos t t y p i c a l b e i n g a p a r t i a l m i x i n g e s t u a r y w h i c h h a s t h e most t y p i c a l c i r c u l a t i o n p a t t e r n a s s o c i a t e d w i t h a d i s t i n c t i v e s p a t i a l a n d t e m p o r a l s a l i n i t y g r a d i e n t . M o r e o v e r , many b i o l o g i c a l l y a n d e c o l o g i c a l l y i m p o r t a n t p a r a m e t e r s w h i c h a r e composed o f e c o l o g i c a l c o n d i t i o n s a r e r e l a t e d t o t h i s g r a d i e n t . T h e r e f o r e , t h e r e must be a c o n d i t i o n a l g r a d i e n t a l o n g t h i s t r a n s i t i o n f o r b o t h s o u r c e s y s t e m s . T h i s a s s o c i a t i o n p r o v i d e s a c o n v e n i e n t way o f u s i n g t h e c o n s e r v a t i v e n a t u r e o f s a l i n i t y f o r i d e n t i f y i n g t h e d y n a m i c s p a t i a l a n d t e m p o r a l b e h a v i o u r o f t h e e c o s y s t e m s a l o n g t h i s t r a n s i t i o n z o n e f r o m b o t h e x t r e m e s o f s a l i n i t y ( 0°/oo t o 29°/oo). T h i s d y n a m i c g r a d i e n t f u n c t i o n s a s a b i o l o g i c a l f i l t e r ( K e n n e d y , 1984 ) m o s t o f t h e b i o l o g i c a l p a r a m e t e r s r e a c t on t h i s f i l t e r . O b v i o u s l y , when f r e s h w a t e r f l o w s i n t o t h e e s t u a r y on t h e s u r f a c e l a y e r a n d c a u s e s a l a n d w a r d f l o w o f s e a w a t e r u n d e r n e a t h , t h e e c o s y s t e m c o m p o n e n t s w h i c h a r e s u s p e n d e d i n b o t h w a t e r b o d i e s w i l l be s u b j e c t t o t h e m i x i n g p r o c e s s on t h e c o n d i t i o n a l g r a d i e n t a l o n g t h e t r a n s i t i o n . T h e r e f o r e , one may a d d r e s s a v e r y b r o a d 2 q u e s t i o n o f w h a t i s t h e e f f e c t o f t h e s a l i n i t y g r a d i e n t , o r i n w h a t ways d o e s t h i s c o n d i t i o n a l g r a d i e n t a c t a s a d y n a m i c f u n c t i o n a l f i l t e r on e i t h e r e c o s y s t e m by p e r m i t t i n g o r l i m i t i n g t h e p a s s a g e o f e c o l o g i c a l c o m p o n e n t s f r o m one t y p e o f e n v i r o n m e n t t o a n o t h e r ? How do m i x i n g p r o c e s s e s a n d c i r c u l a t i o n p a t t e r n s c o u p l e two v e r y d i f f e r e n t e c o s y s t e m s t o f o r m a h y b r i d e c o s y s t e m ? T h i s g r a d i e n t f i l t e r may f u n c t i o n a s a c o n v e r t e r , s o one s y s t e m ' s c o m p o n e n t may be c h a n g e d i n t o a n o t h e r k i n d o f c o m p o nent ( s u c h a s POC t o DOC ) when t h e y p a s s t h r o u g h . T h i s w o u l d r e s u l t i n a d i f f e r e n t d i s t r i b u t i o n o f e n e r g y a n d s t r u c t u r e c h a n g e i n t h e e c o s y s t e m . The q u a l i t y a n d q u a n t i t i e s o f t h e e c o l o g i c a l c o m p o n e n t s e x p o r t e d f r o m t h e e s t u a r y may be d i f f e r e n t f r o m t h o s e t h e e s t u a r y r e c e i v e s . I t i s i n t e r e s t i n g t h a t w h i l e b o t h m a r i n e a n d f r e s h w a t e r s y s t e m s u n d e r g o e x t r e m e e n v i r o n m e n t a l c h a n g e s t h e r e s u l t i n g h y b r i d e s t u a r i n e s y s t e m s a r e more p r o d u c t i v e t h a n e i t h e r o f t h e o r i g i n a l w a t e r b o d i e s ( L a u f f , 1 9 6 7 ) . T h i s i s t r u e e v e n w i t h o u t man made e n r i c h m e n t f r o m sewage o r o t h e r a d d e d p o l l u t a n t s . E s t u a r i e s h a v e been u s e d a s i m p o r t a n t n u r s e r y a r e a s f o r many e c o n o m i c a l l y i m p o r t a n t f i s h s p e c i e s a n d t h i s i s a r e f l e c t i o n o f t h e i r h i g h p r i m a r y p r o d u c t i v i t y . An i n c r e a s i n g demand f o r a c o m p r e h e n s i v e u n d e r s t a n d i n g o f t h e s t r u c t u r e , f u n c t i o n , a nd d y n a m i c s o f e s t u a r i e s h a s s t i m u l a t e d a number o f c o n f e r e n c e s and s y m p o s i a s u m m a r i z i n g k n o w l e d g e o f t h e n a t u r a l c h a r a c t e r i s t i c s o f e s t u a r i e s ( L a u f f , 1 9 6 7 ) , t h e i n t e r a c t i o n s o f t h e s e s y s t e m s ( W i l e y , 1 9 7 6 ) , t h e c o m p a r i s o n o f t h e d i f f e r e n t p r o c e s s e s i n t h e s e e s t u a r i n e 3 s y s t e m s ( K e n n e d y , 1 9 8 2 ) , t h e f u n c t i o n s o f e s t u a r i e s ( K e n n e d y , 1984 ) , t h e d y n a m i c s o f c h e m i c a l n u t r i e n t s ( N e i l s o n & C r o n i n , 1 9 7 9 ) . M i c r o b i a l e c o l o g y a n d p h y s i o l o g y a s a s p e c t s o f b a s i c e s t u a r i n e e c o l o g y a l s o h a v e r e c e i v e d p r e l i m i n a r y a t t e n t i o n f r o m S t e v e n s o n a n d C o l w e l l ( 1 9 7 3 ) a n d w e r e i n c l u d e d i n a c o m p r e h e n s i v e d i s c u s s i o n by R h e i n h e i m e r ( 1 9 7 7 ) . R e c e n t l y , numerous p a p e r s a b o u t e s t u a r i e s h a v e a p p e a r e d w h i c h i n c r e a s e o u r u n d e r s t a n d i n g a n d k n o w l e d g e a b o u t t h i s u n i q u e e c o s y s t e m . However, e s t u a r i e s a r e v e r y d y n a m i c s y s t e m s and i t i s p r o v i n g d i f f i c u l t t o c o n s t r u c t an o v e r a l l u n d e r s t a n d i n g a b o u t t h e s y s t e m f r o m s t u d i e s d e a l i n g w i t h s p e c i f i c a s p e c t s . S t u d i e s w h i c h t r e a t e s t u a r i e s a s w h o l e e c o s y s t e m s o r w h i c h l o o k a t w h o l e p r o c e s s e s may be more h e l p f u l i n o b t a i n i n g a b e t t e r p i c t u r e o f t h e d y n a m i c s t r u c t u r e i n v o l v e d . To f u l l y u n d e r s t a n d t h e e c o l o g i c a l s t r u c t u r e o f e s t u a r i e s we h a v e t o f o c u s o u r p e r s p e c t i v e a t t h e s c a l e w i t h i n an e s t u a r y . S e v e r a l s t u d i e s h a v e f o u n d a s p a t i a l p a t t e r n o f most e c o l o g i c a l p a r a m e t e r s a l o n g a n e s t u a r i n e t r a n s i t i o n z o n e o r s a l i n i t y g r a d i e n t ( F o e s t e r , 1 9 7 6 ; H e l d e r , 1 9 8 3 ; P a l u m b o a n d F e r g u s o n , 1 9 7 8 ; S e l i g e r e t a l . , 1 9 8 1 ; W r i g h t , 1 9 7 8 ) . M o r r i s ( 1 9 7 8 ) f o u n d s t r o n g h e t e r o t r o p h i c a c t i v i t y a t t h e t u r b i d i t y maximum z o n e ( o r l o w s a l i n i t y r e g i o n s ) ( A l l b r i g h t , 1 9 7 7 , 1 9 8 3 a , b , ; B e n t a n d G o u l d e r 1 9 8 1 ; J o i n t e t a l . 1 9 8 2 ; V a l d e s e t a l . 1 9 8 0 , 1 9 8 1 ; W r i g h t , 1 9 8 3 ) a n d m a x i m i z a t i o n o f t h e p h y t o p l a n k t o n s t a n d i n g s t o c k o f t e n a p p e a r s a t t h e mouth o f an e s t u a r y ( o r t h e s e a end o f t h e g r a d i e n t ) ( P a r s o n s , 1 9 6 9 ; Cadee 19 7 8 , 1 9 8 4 ; S t o c k n e r e t 4 a l . , 1979; H a r r i s o n , 1983; Peterson, 1984). Almost a l l other e c o l o g i c a l parameters a l s o show t h i s type of s p a t i a l dynamic d i s t r i b u t i o n (Ducklow, 1982; Kemp et a l . , 1982; N e i l s o n et a l . , 1979; Peterson, 1975; Sharp et a l . , 1982; Cadee, 1984; Carpenter, 1985). So f a r , some attempts have been made to understand the mechanisms behind these phenomena e i t h e r through mathematical modeling (Loern and Cheng, 1981; Parsonsand k e s s l e r , 1985; Peterson a t a l . , 1984) or s i m u l a t i o n i n the l a b o r a t o r y (Margalef, 1967; Cooper et a l . , 1973; Spies & Parsons, 1984), but a c l e a r p i c t u r e i s s t i l l l a c k i n g . Ecosystem s t r u c t u r e i s the r e s u l t of r e a c t i o n s w i t h i n and between the p h y s i c a l system and ecosystem. F i r s t , the i n h e r e n t ( i n t r i n s i c ) i n t e r r e l a t i o n s h i p s among those ecosystem components i n terms of m a t e r i a l c y c l i n g and energy flow determine the c o v a r i a n c e of those components among t r o p h i c l e v e l s . Second, the p h y s i c a l f o r c i n g f i e l d may arrange and modify the s p a t i a l and temporal p o s i t i o n o f e c o l o g i c a l components w i t h i n t h i s f i e l d . A good understanding of the s t r u c t u r e and dynamics of e s t u a r i n e ecosystem f u n c t i o n s r e q u i r e s knowledge of both p h y s i c a l mechanisms and e c o l o g i c a l mechanisms. In an e s t u a r i n e flow, t h e r e are two major l e v e l s of e c o l o g i c a l compartments c o n t a i n e d i n the water masses: 1 ). substances; i n c l u d i n g i o n s , i n o r g a n i c n u t r i e n t s , b i o l o g i c a l t r a c e metals, d i s s o l v e d o r g a n i c m a t e r i a l and p a r t i c u l a t e ( d e t r i t u s ) m a t e r i a l s , a n d 2 ) . l i v i n g organisms which mainly i n c l u d e d phytoplankton, b a c t e r i a , f l a g e l l a t e s , c i l i a t e s and 5 macrozooplankton (the l a t t e r are not i n c l u d e d i n our experiments ). The concept about the s t r u c t u r e and i n t e r a c t i o n s of these e c o l o g i c a l compartments has changed d r a m a t i c a l l y as a r e s u l t of new evidence which comfirms the important r o l e of b a c t e r i a and f l a g e l l a t e s i n ecosystems(Sorokin, 1971,1975). As e a r l y as 1963, Parsons p o i n t e d out t h a t p a r t i c u l a t e o r g a n i c carbon(POC) i n the ocean amounts t o about 2 X lO^-^ g which i s 5 times more than t h a t of the phytoplankton biomass and 10 times l e s s than t h a t of d i s s o l v e d o r g a n i c carbon. So t h e r e i s a very l a r g e bioenergy p o o l i n the a q u a t i c ecosystem. In e s t u a r i e s , the (DOC+POC) i s much higher than t h a t i n the ocean ( N e i l s o n et a l . , 1979; B i l l e n e t a l . , 1980; Wright, 1984; W i l l i a m , 1975; Turner,1978). B a c t e r i a have been known as users of DOC and POC f o r a long time. B a c t e r i a are e f f i c i e n t scavengers of i n o r g a n i c n u t r i e n t s (Rhee, 1972; Parker, 1975), they can compete s u c c e s s f u l l y with phytoplankton f o r i n o r g a n i c phosphorus and othe r n u t r i e n t s (Brown, 1975,1981; Fredrickson,1977; Saks, 1979; Vaccaro e t a l . , 1969). Both b a c t e r i a l growth r a t e s and biomass can reach a s i g n i f i c a n t l y high q u a n t i t y (Biddanda, 1985; Williams,1981).But the p o s i t i o n of b a c t e r i a i n e s t u a r i n e ecosystems o n l y r e c e n t l y has been f i r m l y e s t a b l i s h e d (Wright, 1985). They are a t l e a s t as important as net plan k t o n and l a r g e organisms. B a c t e r i a p l a y an equal r o l e with phytoplankton i n terms of energy flow and n u t r i e n t r e c y c l i n g ( S o r o k i n , 1977; S i e b u r t h e t a l . , 1978; King e t a l . , 1980). The biomass of the b a c t e r i a i n the t o t a l system may approach t h a t of the e n t i r e fauna ( Bratbak, 1984, 1985; Fuhrman & Azam,1982 Newell,1981; 6 Watson, 1979). Since the turnover time of b a c t e r i a i s much f a s t e r than t h a t of macroorganism biomass, i t f o l l o w s t h a t f l u x of energy and m a t e r i a l i s l i k e l y t o be g r e a t ( M a r i t a , 1978). The very high c o n v e r s i o n e f f i c i e n c i e s i n b a c t e r i a (50%-70%) (Calow, 1977) l e a v e s a gap i n r e m i n e r a l i z a t i o n of n u t r i e n t s . Because d i r e c t e x c r e t i o n of i n o r g a n i c n u t r i e n t s a p p a r e n t l y are low , t h i s i s not a major mode f o r r e m i n e r a l i z a t i o n ( W i l l i a m s , 1981). However many s t u d i e s have demonstrated t h a t probably 80 t o 90% of n u t r i e n t s i n the e u p h o t i c zone of p e l a g i c water are s u p p l i e d by r e g e n e r a t i o n (Eppely and Peterson, 1979). T h i s d i f f e r e n c e can be e x p l a i n e d by an abundance of evidence t h a t phagotrophic m i c r o f l a g e l l a t e s are the main g r a z e r s of b a c t e r i a i n the sea (Azam e t a l . , 1983; Andersen et a l . 1985; Caron and Goldman, 1985; Davis & S i e b u r t h , 1984; Fenchel, 1982a, b, c,; Goldman & Caron, 1985; Haas & Weebs, 1979). The g r a z i n g process opens a pathway f o r o r g a n i c substances to be r e c y c l e d back to i n o r g a n i c form. A new and very important component i n the ecosystem, "the m i c r o b i a l l o o p " , i s thus e s t a b l i s h e d . F o l l o w i n g the d i s c o v e r y of t h i s energy pathway, the d i s t r i b u t i o n and s t a t e of the energy flow s t a r t i n g from p h o t o s y n t h e s i s i n the ecosystem became a very i n t e r e s t i n g s u b j e c t f o r f u r t h e r study and r e c o n s t r u c t i o n . I t has been estimated t h a t 50% of carbon f i x e d by phytoplankton i s immediately r e l e a s e d as DOM (Thomas, 1971; Larsson & Hagstrom, 1982,-Smith and Wiebe 1976). B a c t e r i a use t h i s energy source both from d i s s o l v e d o r g a n i c substances and d e t r i t u s t o c r e a t e a l a r g e biomass f l u x and energy flow (Cobeney, 1982; C o l e , e t a l . 1982; L a n c e l o r , 1979). 7 T h r o u g h t h i s " m i c r o b i a l l o o p " , p a r t o f t h e b a c t e r i a l b i o m a s s s e r v e s t o r e p a c k a g e e n e r g y i n t o c e l l s a c c e s s i b l e t o f i l t e r f e e d i n g z o o p l a n k t o n (Haas a n d Webb, 1979) a n d p a r t o f i t i s r e c y c l e d b a c k t o i n o r g a n i c s u b s t a n c e s ( i . e . m i n e r i a l i z e d ) ( C a r o n a n d G o l d m a n , 1 9 8 5 ) . Many i n v e s t i g a t i o n s ( L i n l e y e t a l . , 1 9 8 3 ; W r i g h t , 1 9 8 4 ) h a v e shown t h a t e s t u a r i e s s u p p o r t h i g h p o p u l a t i o n s o f b a c t e r i a a n d h i g h r a t e s o f b a c t e r i a l a c t i v i t y h a v e been f o u n d i n t h i s s y s t e m f o r b o t h i n o r g a n i c n u t r i e n t s a n d o r g a n i c compounds ( F a u s t , 1976; F a u s t & C o n r e l l , 1 9 7 7 ) . So t h e m i c r o b i a l l o o p may p l a y a more i m p o r t a n t r o l e i n t h i s e c o s y s t e m . B e c a u s e t h e s e two p a t h w a y s o f e n e r g y f l o w e x i s t , t h e y must h a v e d i f f e r e n t f u n c t i o n s f o r e n e r g y f l o w . The d i s t r i b u t i o n s o f t h e e n e r g y i n d i f f e r e n t p a t h w a y s i s a r e s p o n s e o f t h e e c o s y s t e m t o t h e e n v i r o n m e n t a l c o n d i t i o n s . The d y n a m i c b e h a v i o u r o f t h e s e two c o m p o n e n t s a n d t h e d i f f e r e n t r e s p o n s e t o t h e e n v i r o n m e n t a l c o n d i t i o n s may r e f l e c t t h e i r f u n c t i o n a l d i f f e r e n c e s a n d t h e s i g n i f i c a n c e o f t h e i r c o e x i s t e n c e . The t i m e e v o l u t i o n d i f f e r e n c e o f t h e s e two c o m p o n e n t s i n t e r m s o f t h e i r g r o w t h p a t t e r n s w h i c h r e s u l t f r o m d i f f e r e n c e s i n a d a p t a t i o n c a p a b i l i t y , i n t e r a c t i n g , c o m m e n s a l i s m , c o m p e t i t i o n a n d p r e d a t i o n w i l l r e v e a l t h e i n t e r r e l a t i o n s h i p o f t h e two p a t h w a y s a n d t h e i r f u n c t i o n a l r e s p o n s e t o t h e e n v i r o n m e n t . I t i s p o s s i b l e t o make some r e a l i s t i c e s t i m a t e s o f c a r b o n f l o w t h r o u g h t h e two d i f f e r e n t p a t h w a y s , p r o v i d e d t h a t s i m u l t a n e o u s m e a s u r e m e n t s a r e made o f e a c h o f t h e c o m p o n e n t s o f t h e s t a n d i n g s t o c k i n t h e same w a t e r c o l u m n . T h i s p r o v i d e s t h e b a s i c c o n c e p t i n o u r e x p e r i m e n t . 8 The q u e s t i o n s addressed here are about some b a s i c and e s s e n t i a l p rocesses i n e s t u a r i n e ecosystems: 1) What i s the behaviour and response of some freshwater and seawater ecosystem components e.g., phytoplankton, f l a g e l l a t e s , b a c t e r i a and n u t r i e n t s t o the s a l i n i t y g r a d i e n t and to the dynamic s t a t e of the e s t u a r i n e environment i n terms of the d i l u t i o n process? 2) What are the dynamic i n t e r a c t i o n s among these major compartments when they go through the s a l i n i t y g r a d i e n t at a constant d i l u t i o n r a t e ? 3) Do a l l the components behave i n the same manner on the temporal s a l i n i t y g r a d i e n t as they d i d i n the o r i g i n a l environment? 4) What i s the s a l i n i t y e f f e c t on the growth of phytoplankton assemblages i n both f r e s h water and sea water? 5) How can the d i f f e r e n c e i n growth p a t t e r n s of d i f f e r e n t components over time be e x t r a p o l a t e d f o r s p a t i a l s i g n i f i c a n c e ( i n c o n s i d e r i n g an e s t u a r i n e c i r c u l a t i o n p a t t e r n ) ? To attempt t o answer these q u e s t i o n s , I have t o e l i m i n a t e a l l o ther a s s o c i a t e d f a c t o r s i n nature such as: the h o r i z o n t a l l i g h t g r a d i e n t ; temperature d i f f e r e n c e ; sediment i n f l u e n c e s , e t c . These a r t i f i c i a l c o n d i t i o n s can be achieved i n l a b o r a t o r y microcosm experiments and an apparatus was c o n s t r u c t e d t o achieve the o b j e c t i v e . The purpose of t h i s work was to make a p r e l i m i n a r y i n v e s t i g a t i o n of the time e v o l u t i o n of growth p a t t e r n s of two primary t r o p h i c l e v e l s along a temporal s a l i n i t y g r a d i e n t . 9 E s t u a r i n e ecosystems are such complicated systems encompassing so many f e a t u r e s of other a q u a t i c systems t h a t l a b o r a t o r y experiments on a microcosm s c a l e can not be a d u p l i c a t i o n of t h i s system. The purpose here i s not to i m i t a t e nature but, on the c o n t r a r y , t o s i m p l i f y the s i t u a t i o n i n order t o i d e n t i f y o p e r a t i o n a l mechanisms and some major processes found i n a wide v a r i e t y of e s t u a r i e s . For example, p r e v i o u s work has shown t h a t low phytoplankton p r o d u c t i o n a t the low s a l i n i t y segment of an e s t u a r y i s due to the high t u r b i d i t y and high l i g h t e x t i n c t i o n r a t e . So we may ask the q u e s t i o n : i f the t u r b i d i t y e f f e c t i s e l i m i n a t e d , does t h a t area become one of high p r o d u c t i o n ? I f not, then the t u r b i d i t y e f f e c t i s o n l y a s u b e f f e c t which masks the r e a l mechanism. In order to observe p h y s i o l o g i c a l e f f e c t s (due t o s a l i n i t y or to other unknown f a c t o r s ) and p h y s i c a l e f f e c t s (due to d i l u t i o n r a t e and to time s c a l e ) , two d i f f e r e n t temporal s a l i n i t y g r a d i e n t s are s i m u l a t e d by c o n s t a n t entrainment of s a l i n e n u t r i e n t - r i c h deep water or d i l u t i o n by f r e s h water over time w i t h i n one microcosm; the experimental development and d e s i g n are d e s c r i b e d i n Chapter I I . The e v o l u t i o n p a t t e r n and the s t r u c t u r e produced by growth d i f f e r e n c e s of the b i o t a are d e s c r i b e d i n Chapter I I I . The immediate purpose of these experiments was to understand some of the i n t e r a c t i n g f e a t u r e s of the b i o t a , growth p a t t e r n arrangement and c e r t a i n c h a r a c t e r i s t i c s p e r t a i n i n g t o the s t r u c t u r e and f u n c t i o n s of e s t u a r i n e ecosystems which happened d u r i n g the mixing of two very d i f f e r e n t water masses. 10 2. Experimental design 2.1. P r e c o n s i d e r a t i o n The experimental model s i m u l a t e s the process i n a two l a y e r flow e s t u a r y with entrainment from the bottom (average t i d a l e f f e c t ) . I t i s assumed t h a t the exchange of water between the e s t u a r y and sea takes p l a c e e n t i r e l y by a d v e c t i o n . H o r i z o n t a l d i f f u s i o n being n e g l i g i b l e , the r a t e of flow can be c a l c u l a t e d from a knowledge of the mean s a l i n i t y of the i n f l o w and outflow l a y e r s and freshwater i n f l u x ( P r i t c h a r d , 1969). The s a l i n i t y of the deep l a y e r remains almost unchanged, so t h a t o n l y the upper l a y e r i s c o n s i d e r e d . I t i s assumed t h a t the depth of the upper l a y e r i s constant at the steady s t a t e of the e s t u a r i n e c i r c u l a t i o n . I f the body of water a t the head of the e s t u a r y i s c o n s i d e r e d as one element which flows to the mouth of the e s t u a r y a t average speed (see F i g . 1), the entrainment of high s a l i n i t y seawater i n c r e a s e s the s a l i n i t y of the water w i t h i n t h a t element. As the requirement of c o n t i n u i t y , mixed water flows out from t h a t element. Thus t h e r e are two major processes happening i n t h a t body of water; s a l i n i t y i n c r e a s e s over time and the o r i g i n a l water becomes d i l u t e d . Since o n l y a d v e c t i o n i s c o n s i d e r e d and the whole c i r c u l a t i o n i s i n dynamic e q u i l i b r i u m , these two processes a l s o happen over space. The temporal e v o l u t i o n of a l l the c o n s t i t u e n t s c o n t a i n e d i n the water mass w i l l correspond to the one s p a t i a l dimension. T h i s means t h a t i n a body of water with entrainment of sea water a t a c e r t a i n d i l u t i o n r a t e , the p a t t e r n of temporal e v o l u t i o n of the processes can r e f l e c t the s p a t i a l p a t t e r n of those processes i n an e s t u a r i n e system • because a l l the parameters c a r r i e d by S e a R i v e r • i i F i g u r e 1. F l o w d i a g r a m f o r t h e u p p e r l a y e r b o x o f n u m b e r n i n a t w o d i m e n s i o n a l m o d e l o f e s t u a r i n e c i r c u l a t i o n w i t h v e r t i c a l m i x i n g ( a d v e c t i o n o n l y ) . S , s a l i n i t y ; Q , v o l u m e o f f l u x r a t e ; S v s a l i n i t y a t l o w e r l a y e r , Q v , v o l u m e o f f l u x f r o m t h e b o t t o m l a y e r , b e l o w : t y p i c a l s a l i n i t y p r o f i l e s ; 0, l o w s a l i n i t y e n d a n d S , h i g h s a l i n i t y e n d . . 12 t h i s m o v i n g b o d y o f w a t e r a c t u a l l y s t a y i n a s t a t e o f r e s t r e l a t i v e t o t h e i r c a r r i e r . T h i s p r o v i d e s u s w i t h t h e p o s s i b i l i t y o f u s i n g a s e t t l e d b o d y o f w a t e r i n t h e l a b o r a t o r y t o i n v e s t i g a t e t h e t e m p o r a l e v o l u t i o n o f d e s i r e d b i o l o g i c a l f e a t u r e s t h r o u g h c o n s t a n t e n t r a i n m e n t o f s e a w a t e r t o r e p r o d u c e a s a l i n i t y g r a d i e n t a n d t h e d i l u t i o n p r o c e s s . T h e t e m p o r a l e v o l u t i o n p a t t e r n o f s o m e e c o l o g i c a l c o m p o n e n t s h o u l d r e f l e c t t h e s p a t i a l d i s t r i b u t i o n o f t h e s e c o m p o n e n t s i n t h e e s t u a r i e s . O n e p o i n t w h i c h i s e s s e n t i a l i n t h e l a b o r a t o r y e x p e r i m e n t s i s t h a t t h e r e m u s t b e a n e q u a l s t a r t i n g p o i n t f o r b o t h e c o s y s t e m s i n t h e e s t u a r y a n d i n t h e l a b o r a t o r y . I n l a b o r a t o r y c o n d i t i o n s , t h e s t a r t i n g p o i n t o f t h e w h o l e p r i m a r y p r o d u c t i o n s y s t e m i s a r t i f i c i a l l y c o n t r o l l e d b y o p e r a t i o n o f t h e l i g h t s o u r c e . L i g h t e n e r g y a c t i v a t e s t h e p h o t o s y n t h e t i c p r o c e s s a n d t h e t e m p o r a l e v o l u t i o n o f t h e b i o l o g i c a l f e a t u r e s b e g i n s . B u t i n a r e a l e s t u a r y , w e a r e n o t s u r e t h a t t h e h e a d o f t h e e s t u a r y ( w h e r e w e c o n s i d e r t h e p r o c e s s e s t o s t a r t ) i s t h e b e g i n n i n g o f m a j o r p h y t o p l a n k t o n d e v e l o p m e n t w h i c h d r i v e s t h e e c o s y s t e m . T h e a n a l y s i s o f a n e s t u a r i n e c i r c u l a t i o n p a t t e r n t e l l s u s t h a t t h e s e a w a t e r s p e c i e s o f p h y t o p l a n k t o n a r e c a r r i e d i n t o t h e e s t u a r y f r o m t h e d e e p e r w a t e r l a y e r . T h i s s u g g e s t s t h a t b e c a u s e o f l i g h t l i m i t a t i o n u n d e r n e a t h t h e l a y e r , t h e s e m a r i n e p h y t o p l a n k t o n c a n n o t s t a r t p h o t o s y n t h e t i c g r o w t h u n t i l t h e y r e a c h t h e e u p h o t i c z o n e t h r o u g h e n t r a i n m e n t i f t h e e s t u a r y e c o s y s t e m i s a s e a w a t e r p h y t o p l a n k t o n d o m i n a t e d s y s t e m . T h e e v o l u t i o n o f t h e c o m p o n e n t s u n d e r o u r l a b o r a t o r y c o n d i t i o n s w i l l n o t e n t i r e l y c o i n c i d e w i t h t h i s e s t u a r y s y s t e m . 13 On the other hand, what i s the s t a t e of the freshwater ecosystem components upon r e a c h i n g the e s t u a r y ? What happens to them a t the s a l i n i t y g r a d i e n t ? Does t h i s s a l i n i t y g r a d i e n t f i l t e r out phytoplankton components or other l i v i n g components? The e s t u a r y may be a c t u a l l y a grave f o r some of freshwater components. the f i r s t experiment i s designed t o look at the response of the freshwater ecosystem when i t encounters the s a l i n i t y g r a d i e n t . A n a t u r a l water column captured i n a l a r g e c o n t a i n e r has been used t o analyse some of e c o l o g i c a l components; t h i s i s known as a "mesocosm" and they have been d e s c r i b e d by others ( G r i c e & Reeve, 1982). T h i s method has been suggested as an important t o o l i n f u r t h e r i n g our understanding of food c h a i n t r a n s f o r m a t i o n mechanisms (Parsons e t a l . , 1978). For the purpose of microphytoplankton s t u d i e s , the volume of water i n an "Mesocosm" e n c l o s u r e i s u n n e c e s s a r i l y l a r g e f o r these organisms, and too expensive and d i f f i c u l t t o be manipulated. So, f o r t h i s l e v e l of study, the s i z e of the c o n t a i n e r i s reduced t o t h a t of a "microcosm" which can be more e a s i l y manipulated and the c o n d i t i o n s can more a c c u r a t e l y be c o n t r o l l e d at w i l l . However, "batch c u l t u r e " type microcosms are i s o l a t e d water bodies which can not s i m u l a t e the e s t u a r i n e p h y s i c a l c o n d i t i o n s and the e c o l o g i c a l p r o c e s s e s . On the other hand, chemostats have long been used as powerful t o o l s i n m i c r o b i a l ecology (Jannasch e t a l . 1974) and l a t e l y have been used i n many s t u d i e s of phytoplankton ecology. A s e r i e s of chemostats connected i n sequence has been used by Cooper and 14 C o p e l a n d ( 1 9 7 3 ) f o r s t u d y i n g t h e g r o w t h o f p h y t o p l a n k t o n i n a s p a t i a l s a l i n i t y g r a d i e n t c r e a t e d i n t h e s e r i e s o f c h e m o s t a t f l a s k s . I n an e s t u a r y , t h e p h y s i c a l g r a d i e n t ( e . g . g r a v i t a t i o n a l g r a d i e n t , e t c . ) i s i n e q u i l i b r i u m . H o wever, f o r e c o s y s t e m s , t h e e s t u a r y i s a p r o c e s s t e n d i n g t o w a r d s e q u i l i b r i u m , l i k e a c o n t i n u o u s c u l t u r e b e f o r e r e a c h i n g s t e a d y s t a t e . I n t e r m s o f s t a b i l i t y o f an e c o s y s t e m , i t may be b r i e f l y i n a s t a t e o f dampened h a r m o n i c o s c i l l a t i o n . The s a l i n i t y g r a d i e n t i s a s s o c i a t e d w i t h a c o n s t a n t d i l u t i o n r a t e , s o t h e n a t u r e o f t h e s a l i n i t y g r a d i e n t i s common f o r t h e m i c r o b i a l e c o s y s t e m i n b o t h t h e e s t u a r i n e f i e l d a n d t h e l a b o r a t o r y e x p e r i m e n t a l c o n d i t i o n s . Our r e s u l t s a n d i n t e r p o l a t i o n s a r e o n l y v a l i d i n r e s p e c t t o t h e p r o c e s s e s o f a d m i x t u r e o f s e a and f r e s h w a t e r w h i c h i s common i n b o t h s y s t e m s ( L e f f l e r , 1 9 80) and n o t t o o t h e r e s t u a r i n e p r o p e r t i e s ( e . g . s e d i m e n t a t i o n , e t c . ) . 2.2 . D e s i g n o f t h e E x p e r i m e n t S i n c e t h e e x p e r i m e n t a l a p p a r a t u s was d e s i g n e d t o s i m u l a t e i m p o r t a n d e x p o r t e x c h a n g e c h a r a c t e r i s t i c s a n d a s a l i n i t y g r a d i e n t , t h e " o p e n n e s s " o f t h e s y s t e m f i r s t h a d t o be i n c o r p o r a t e d i n t o t h e " b a t c h c u l t u r e " . An e s t u a r i n e e c o s y s t e m c a n be c o n s i d e r e d t o be an "o p e n " s y s t e m f o r t h e u p p e r w a t e r l a y e r i n w h i c h i m p o r t o f w a t e r o f a v e r y d i f f e r e n t c h a r a c t e r a nd e x p o r t o f m i x e d w a t e r a r e o f p a r a m o u n t i m p o r t a n c e . An open c o n t i n u o u s s y s t e m w o u l d a p p e a r t o r e s e m b l e e s t u a r i n e d y n a m i c s more c l o s e l y ( C o o p e r e t a l . , 1 9 7 3 ; M a r g a l e f , 1 9 6 7 ) . B a s e d on t h i s c o n s i d e r a t i o n , I a d o p t e d an a p p r o a c h b e t w e e n b a t c h c u l t u r 15 t i m e a r o u n d 6 d a y s ) was a c h i e v e d i n f o u r , 6 l f l a s k s b y a c o n s t a n t i n p u t o f s e a w a t e r o r f r e s h w a t e r a nd a c o n s t a n t o u t p u t o f m i x e d w a t e r o v e r t i m e . T h i s e s t a b l i s h e d an i n c r e a s i n g o r d e c r e a s i n g s a l i n i t y g r a d i e n t o v e r t i m e i n t h e f l a s k s . The s i z e o f c o n t a i n e r s u s e d f o r o u r e x p e r i m e n t s r e p r e s e n t e d a s u i t a b l e v o l u m e o f w a t e r f o r s t u d y i n g b a c t e r i a , m i c r o f l a g e l l a t e s a n d p h y t o p l a n k t o n a n d t h e a n a l y s i s c o u l d be managed by one p e r s o n . C o n s t a n t t e m p e r a t u r e a n d c o n s t a n t l i g h t i n t e n s i t y e l i m i n a t e d t h e e f f e c t s o f n a t u r a l f l u c t u a t i o n . A 20 l i t r e b o t t l e was u s e d a s a r e s e r v o i r t o h o l d o r i g i n a l u n f i l t e r e d w a t e r o r f i l t e r e d w a t e r . T h e s e w e r e c o v e r e d w i t h b l a c k p l a s t i c b a g s t o s i m u l a t e d a r k l o w e r w a t e r l a y e r i n e s t u a r i e s . E v e r y s e t o f e x p e r i m e n t s was p e r f o r m e d i n two s t a g e s . W i t h e a c h s t a g e h a v i n g t h r e e u n i t s o f two f l a s k s e a c h . I n t h e f i r s t s t a g e , I t e s t e d t h e f r e s h w a t e r e c o s y s t e m c o m p o n e n t s . I n u n i t o n e , t h e s y s t e m was i n i t i a t e d w i t h u n f i l t e r e d f r e s h w a t e r a nd f i l t e r e d s e a w a t e r was a l l o w e d t o r u n i n t o t h e f l a s k s o t h a t t h e s a l i n i t y i n t h e f l a s k i n c r e a s e d o v e r t i m e . I n u n i t t w o , u n f i l t e r e d f r e s h w a t e r was r u n i n t o f i l t e r e d s e a w a t e r w h i c h was t h e i n i t i a l w a t e r s o t h a t t h e s a l i n i t y d e c r e a s e d o v e r t i m e . U n i t t h r e e f o r b o t h s t a g e I a n d I I a r e t h e same. E a c h o f t h e f l a s k s h e l d o r i g i n a l s e a w a t e r a n d f r e s h w a t e r u n d e r c o n s t a n t e n v i r o n m e n t a l c o n d i t i o n s t o p r o d u c e a " c o n t r o l c u l t u r e " . I n s t a g e t w o , u s i n g s e a w a t e r a s t h e o r i g i n a l t e s t w a t e r , t h e same p r o c e d u r e a s t h a t i n s t a g e one was f o l l o w e d t o o b s e r v e t h e i s e a w a t e r e c o s y s t e m b e h a v i o r on t h e s a l i n i t y g r a d i e n t ( s e e T a b l e 1 ) . New medium was pumped i n t h r o u g h Tygon t u b i n g o f 8 mm 16 diameter at a r a t e of 1 1/day ( d i l u t i o n r a t e of 0.I6 d a y - 1 , r e s i d e n c e time 6 d a y s ) . One l i t r e of mixed c u l t u r e medium was pumped out b e f o r e the new medium was pumped i n . The outflow water was c o l l e c t e d f o r a n a l y s i s and s u b c u l t u r e d at 24 h i n t e r v a l s . ( F i g . 2) Because the experiments were n e c e s s a r i l y l i m i t e d i n s i z e and scope , many parameters were not monitored. Only the most important and e s s e n t i a l v a r i a b l e s f o r each ecosystem component were measured as i n d i c a t o r s of the dynamics of the growth p a t t e r n . For h e t e r o t r o p h i c b a c t e r i a , o n l y the number and the average s i z e of the b a c t e r i a were measured f o r biovolume c o n c e n t r a t i o n and biocarbon c o n v e r s i o n . At the phagotrophic l e v e l , the number and the average s i z e f o r non-pigmented m i c r o f l a g e l l a t e s were determined to o b t a i n the r e l a t i v e number and biomass. The biomass of the a u t o t r o p h i c l e v e l components was measured with the i n v i v o a u t o f l u o r e s c e n c e method ( K i e f e r , 1973). The reason f o r choosing t h i s method i s t h a t a l l the subsamples needed to be c u l t u r e d f u r t h e r under "batch c u l t u r e " c o n d i t i o n s . Even though t h e r e are s u b s t a n t i a l v a r i a t i o n s per c e l l i n t h i s method f o r n a t u r a l assemblages of phytoplankton with environmental c o n d i t i o n s , c o n t r o l experiments have shown t h a t the e x p o n e n t i a l i n c r e a s e of _in v i v o f l u o r e s c e n c e a c c u r a t e l y r e f l e c t s the e x p o n e n t i a l i n c r e a s e i n c e l l abundance a f t e r the c u l t u r e s have a c c l i m a t e d i n a c o n s t a n t environment (Brand e t al.,1981). Since i n my experiments the g e n e r a l c o n d i t i o n s were always the same, the community composition d i d not show much d i f f e r e n c e between experiments and the c o r r e l a t i o n between in v i v o f l u o r e s c e n c e control f l a s k part 1 experimental flasks col t action f l a s k part 2 experimental flasks control f l a s k raservlors c o l l e c t i o n f l a s k F i g u r e 2. Design of the experiments with t h r e e u n i t s , EXPERIMENT Stage I storage supply experimental Part I f i l t e r e d „ sea water (29 °/00) fresh water (0 °/oo) Part II fresh water (0 °/oo) f i l t e r e d , sea water C29 °/oo) Control o r i g i n a l water control vessels storage supply Stage II •xperlaental (29 °/oo) f i l t e r e d fresh water (0 °/oo) f i l t e r e d fresh water (0 /oo) sea water (29 /oo) o r i g i n a l water control vessels Table 1. The procedures of the experiment i n the two 6tages. 18 and e x t r a c t e d c h l o r o p h y l l a was l i n e a r ( r 2 = 0.98). For i n o r g a n i c components, two kinds of nitrogenous n u t r i e n t s were measured: the dynamics of NC>3~ c o n c e n t r a t i o n i s a process of d i l u t i o n and a u t o t r o p h i c u t i l i z a t i o n and the c o n c e n t r a t i o n of NH4 + i s a r e s u l t of u t i l i z a t i o n by phytoplankton and m i n e r a l i z a t i o n by b a c t e r i a ; both are a l s o a f f e c t e d by d i l u t i o n . 2.3 C o n d i t i o n of Experiment N a t u r a l sea water or f r e s h water were f i l t e r e d with a 0.8 urn M i l l i p o r e f i l t e r t o remove the phytoplankton seed p o p u l a t i o n s and m i c r o f l a g e l l a t e s but to l e t some f i n e p a r t i c l e s and d i s s o l v e d substances (<0.45 urn) pass through. T h i s r e t a i n e d the much of the o r i g i n a l chemical p r o p e r t i e s of the water i n order t o i n v e s t i g a t e i t s p r o p e r t i e s i n the s a l i n i t y g r a d i e n t . The f r e s h water (0°/oo s a l i n i t y ) was c o l l e c t e d from the bank of the F r a s e r R i v e r at New Westminster, approximately 32 km upstream from Steveston Harbour, a t low t i d e . A f t e r c o l l e c t i o n , the water was s t o r e d f o r two days i n a temperature c o n t r o l l e d room where the experiment was performed f o r a d a p t a t i o n . The water was d i v i d e d i n t o two p a r t s . One p a r t was s t o r e d u n f i l t e r e d and one p a r t was f i l t e r e d through 0.8 um AA M i l l i p o r e f i l t e r . High s a l i n i t y water was pumped from a depth of about 25 m and 200 m away from a dock a t the West Vancouver l a b o r a t o r y of the Department of F i s h e r i e s and Oceans ( F i g . 3). T h i s water was covered w i t h b l a c k p l a s t i c and immediately t r a n s p o r t e d to a temperature c o n t r o l l e d room (15°C) . Some of the sea water a l s o was f i l t e r e d through 0.8 um M i l l i p o r e f i l t e r s . These two kinds of water were s t o r e d i n a p r e c l e a n e d 20 1 Pyrex f l a s k as r e s e r v o i r or i n 6 1 f l a s k s as i n i t i a l e xperimental water 19 under l i g h t u n t i l the experiment began. I n t a c t sea water was a l s o s t o r e d i n dark r e s e r v o i r s covered with black p l a s t i c . A l l experiments were performed a t 15°Cin a temperature c o n t r o l l e d room. The experimental (6 l i t e r ) f l a s k s were p o s i t i o n e d i n f r o n t of banks of f o u r , c o o l white, f l u o r e s c e n t tubes of i r r a d i a n c e of 200-250 uE*m~2s _ 1 measured i n the middle of the f l a s k p o s i t i o n . T h i s i s a s a t u r a t i n g i r r a d i a n c e f o r diatom s p e c i e s and s m a l l e r d i n o f l a g e l l a t e s (Chan, 1978). The f l a s k s were c l o s e d by caps p e r m i t t i n g a i r exchange but p r e v e n t i n g contamination. A magnetic s t i r r e r was used to e l i m i n a t e h e t e r o g e n e i t y over space and s i n k i n g . A l l experiments were performed on a 12:12 LD c y c l e . N i t r a t e - N (20 ug-at I " 1 ) , phosphate-P (2 ug*at 1~1) and s i l i c a t e - S i (50 ug-at l - 1 ) were added to the i n i t i a l water. I n i t i a l n u t r i e n t enrichment was thought to be necessary f o r four reasons. F i r s t , t o decrease the seasonal v a r i a b i l i t y of both n u t r i e n t c o n c e n t r a t i o n s and major n u t r i e n t r a t i o s i n the source water. Second, to c r e a t e c o n d i t i o n s f a v o u r a b l e f o r the growth of diatoms which have requirement f o r s i l i c a t e . T h i r d , t o c o n t r o l the i n c u b a t i o n time t o approximately the same time f o r each experiment. Fourth , a c c o r d i n g t o the R e d f i e l d r a t i o (e.g. 106 C : 16 N : 1 P by atoms), t o make sure t h a t n i t r o g e n would be the l i m i t i n g n u t r i e n t f o r a u t o t r o p h i c growth as i s the case of n a t u r a l marine environments ( A n t i a et a l . , 1963; Ryther & Dunstan, 1971). Every p a r t of the experiment was run as one u n i t of two d u p l i c a t e s under e x a c t l y the same c o n d i t i o n and the experiment F i g u r e 3 . Sampling s i t e s f o r l a b o r a t o r y experiments and m s u r f a c e s a l i n i t y d i s t r i b u t i o n i n F r a s e r e s t u a r y and ° plum 21 was repeated a t d i f f e r e n t seasons of the year. The purpose was not t o study the v a r i a b i l i t y between d u p l i c a t e f l a s k s and d i f f e r e n t seasons of the year, but t r y to ensure t h a t r e l a t i v e l y the same p a t t e r n of events o c c u r r e d i n the same experimental c o n d i t i o n s and t r y t o q u a l i t a t i v e l y compare the s i m i l a r i t y of the e v o l u t i o n p a t t e r n between the two d u p l i c a t e f l a s k s . Thus w h i l e they may show some d i f f e r e n c e i n q u a n t i t a t i v e data, the q u a l i t a t i v e r e a c t i o n should be the same. Haque e t a l . (1980) p o i n t e d out t h a t some parameters i n two i d e n t i c a l microcosms may e x h i b i t s i m i l a r form, but be s h i f t e d i n phase. When data are compared at any one i n s t a n c e , the a n a l y s i s of the v a r i a n c e would l i k e l y i n f e r t h a t the two microcosms d i d not r e p l i c a t e . C a l c u l a t i o n of a u t o c o r r e l a t i o n f u n c t i o n s or power spectrum f u n c t i o n s , however, may e l i m i n a t e the e f f e c t s of phasing. In summary, the major i n t e r e s t s here were not how much q u a n t i t a t i v e v a r i a n c e e x i s t e d between the two same v a r i a b l e s i n d u p l i c a t e f l a s k s a t any one i n s t a n c e , or i n two experiments a t d i f f e r e n t times of the year, but the q u a l i t a t i v e f e a t u r e of how the growth p a t t e r n dynamics and how much the d i f f e r e n t parameters may vary q u a n t i t a t i v e l y at one i n s t a n c e due to i n t e r a c t i o n among them w i t h i n one system . I d e n t i c a l experiments was c a r r i e d out at d i f f e r e n t times d u r i n g of 1984-1985. Because of the complexity of e s t u a r i n e c o n d i t i o n s , any change i n b i o l o g i c a l l y important c o n s t i t u e n t s may dominate the dynamic s t a t e of e s t u a r i n e ecosystems. Seasonal v a r i a t i o n s i n an e s t u a r y r e s u l t s i n dramatic d i f f e r e n c e s i n s p e c i e s composition as w e l l as q u a l i t y and 22 q u a n t i t y of biomass and t r o p h i c l e v e l i n t e r a c t i o n . Does the dynamic s a l i n i t y g r a d i e n t have the same e f f e c t on the ecosystem w i t h i n the seasonal v a r i a t i o n ? Does water c o l l e c t e d at d i f f e r e n t seasons which c o n t a i n d i f f e r e n t s e asonal s p e c i e s assemblages show more or l e s s the same growth p a t t e r n being s u b j e c t e d t o the same c o n d i t i o n ( c o n d i t i o n a l c o n t r o l l e d chamber) which may or may not be q u i t e d i f f e r e n t from t h e i r o r i g i n a l c o n d i t i o n s ? The s a l i n i t i e s of the o r i g i n a l water c o l l e c t e d at d i f f e r e n t times of the year are almost the same. Table 2 shows the s a l i n i t y of the water used f o r the experiments at d i f f e r e n t times of the year. Table 2. S a l i n i t y of i n i t i a l waters c o l l e c t e d at d i f f e r e n t times f o r the l a b o r a t o r y experiments. S a l i n i t y o/oo New Westminster West Vancouver Feb. 5, 1985 0.000 29.488 Feb. 29, 1985 0.000 28.614 A p r i l 26, 1985 0.000 29.958 J u l y 3, 1985 0.000 27.490 J u l y 23, 1985 0.000 26.630 3. Methods of A n a l y s i s of samples 3.1 N u t r i e n t s As major l i m i t i n g n u t r i e n t s , both n i t r a t e and ammonium were measured. A l l samples c o l l e c t e d i n l a b o r a t o r y experiments were immediately f i l t e r e d through precombusted (at 450 ° C f o r 24 h) g l a s s f i b e r f i l t e r s (Whatman 934-AH, 2.4 cm d i a m e t e r ) , 23 s t o r e d i n Nalgene b o t t l e s and f r o z e n t o -20°C. Stored samples were q u i c k l y thawed before a n a l y s i s . Automated d e t e r m i n a t i o n f o r n i t r a t e p l u s n i t r i t e was done as d e s c r i b e d by Armstrong et a l . (1967) with Technicon Autoanalyzer I I . The range of n i t r a t e c o n c e n t r a t i o n s was 0-50 ug-at-N 1~1. Ammonium (0-4.5 ug*at-N 1~1) were measured c o n c u r r e n t l y (Parsons et a l . , 1984). 3.2 T o t a l b a c t e r i a l numbers, biovolumes and biomass A l i q u o t s of 10 ml were taken from each experimental f l a s k f o r enumeration of t o t a l b a c t e r i a l numbers. Subsamples were p l a c e d i n t o g l a s s v i a l s and immediately f i x e d with 2.5 ml of f i l t e r e d (0.22 um) formaldehyde (37%) . Samples were s t o r e d i n the dark a t -20°C and q u i c k l y thawed be f o r e f u r t h e r treatment. Because of the attachment of b a c t e r i a t o d e t r i t u s and phytoplankton f r u s t u l e s and the formation of b a c t e r i a l aggregates, the water samples of the b a c t e r i a must be p r e t r e a t e d i n order t o have a random d i s t r i b u t i o n f o r cou n t i n g purposes. The method f o r d i s a g g r e g a t i o n of b a c t e r i a was taken from V e l j e (1984). Samples were incubated i n 0.001 M te t r a s o d i u m pyrophosphate suspended i n 0.44 M sodium c h l o r i d e s o l u t i o n f o r 15 min and then t r e a t e d with s o n i f i c a t i o n at 100'W f o r 1 min ( i n my experiment). Then a one ml a l i q u o t was taken from the t r e a t e d sample water with an automatic p i p e t t e and put i n t o 9 ml of f i l t e r e d d i s t i l l e d water t o get a f i n a l d i l u t i o n of 10 times. F i n a l l y , 4 ml of d i l u t e d sample were f i l t e r e d through Nuclepore p o l y c a r b o n a t e membrane f i l t e r s ( pore s i z e of 0.2 um and diameter 25 mm which had been p r e s t a i n e d 24 h i n 0.2% s o l u t i o n of I r g a l a n b l a c k BGL i n 2% a c e t i c a c i d ) ; afterwards 0.4 24 ml a c r i d i n e orange was added t o s t a i n the b a c t e r i a f o r 3-5 min and then the sample was f i l t e r e d . S l i d e s were made and counting was c a r r i e d out with a Z e i s s compound microscope equipped with an e p i f l u o r e s c e n t i l l u m i n a t i o n system (Hobbie e t a l . , 1977). Depending on the d i s p e r s i v e s t a t e of b a c t e r i a on the f i l t e r , 9-14 f i e l d s and a t o t a l of a t l e a s t 200-400 b a c t e r i a were counted ( V e n r i c k , 1978). T o t a l numbers were c a l c u l a t e d a c c o r d i n g t o the e q u a t i o n : , . s t a i n e d area of f i l t e r x X x 10 x 1.37 C e l l s / m l • area of counting g r i d x 4 X = mean number of c e l l s of 9 or more f i e l d s , 4 = s t a i n e d water on the f i l t e r membrane, 10 = d i l u t i o n index, 1.37 = c o r r e c t i o n f o r added formaldehyde and t e t r a s o d i u m pyrophosphate s o l u t i o n , s t a i n e d area of f i l t e r = 2.0 x 10& um2, area of c o u n t i n g g r i d = 10,000 um2, For the biovolume of the b a c t e r i a , the l e n g t h (L) and width (W) of the c e l l was taken as the average of 50 c e l l s i n each sample and the volume of c e l l s was c a l c u l a t e d as : Biovolume = (pi/4)W 2(L-W/3) T h i s formula a p p l i e s t o both rods (L>W) and c o c c i (L=W) (Bratbak, 1985). B a c t e r i a carbon content was taken from the l i t e r a t u r e . 2.0 x 1 0 " 1 3 g c c e l l - 1 f o r b a c t e r i a was r e p o r t e d by Robinson e t a l . (1982) and average biovolume to s p e c i f i c carbon content of the c e l l i s 2.4 x l O " 1 ^ g C um~3 (Bowden, 1977; Watson, 1979; Hagstrom, 1979); 5 . 6 x l 0 " 1 3 g C um~3 was suggested by Bratbak (1985). 25 Biovolume/ ml = b a c t e r i a number/ml x average volume B a c t e r i a carbon/ml = biovolume/ml x carbon c o n v e r s i o n 3.3 Measurement of s a l i n i t y and c a l c u l a t i o n of s a l i n i t y  g r a d i e n t and n u t r i e n t d i l u t i o n change The i n i t i a l s a l i n i t i e s were measured i n the p h y s i c a l oceanography l a b o r a t o r y i n our department immediately a f t e r the c o l l e c t i o n of water. During the experiment, i n t e r m e d i a t e s a l i n i t i e s f o r each day were c a l c u l a t e d a c c o r d i n g to c o n s e r v a t i o n and c o n t i n u i t y of the s a l t u sing the f o l l o w i n g equation d e a l i n g with a two dimensional box model ( P r i t c h a r d , 1969): V l ' S i - ! 4 V 2 • S 0 S i « • V l • v 2 V i = volume of experimental water, V 2 = volume of water i n f l o w from r e s e r v o i r (1 l i t r e ) , S i = s a l i n i t y of t h a t day i n experimental water, S Q = s a l i n i t y of r e s e r v o i r , S i _ i = s a l i n i t y of p r e v i o u s day, The c a l c u l a t i o n was done with a s m a l l computer program. The dynamic changes i n n u t r i e n t c o n c e n t r a t i o n r e s u l t from u t i l i z a t i o n by phytoplankton b i o t a and b a c t e r i a and d i l u t i o n e f f e c t by mixing with two d i f f e r e n t waters of d i f f e r e n t n u t r i e n t c o n c e n t r a t i o n s . For the pure d i l u t i o n e f f e c t i t was assumed t h a t n u t r i e n t c o n c e n t r a t i o n i s a c o n s e r v a t i v e p r o p e r t y and thus change was c a l c u l a t e d as f o r s a l i n i t y . 3.4 M i c r o f l a g e l l a t e s and phytoplankton Many methods have been r e p o r t e d f o r enumeration and i d e n t i f i c a t i o n of m i c r o f l a g e l l a t e s ( F e n c h e l , 1975; S o rokin, 26 1979; Sherr e t a l . , 1983 and Hewes, 1983). The method used here was d i r e c t c o u n t i n g using r o u t i n e l i g h t microscopy a t 4 0 x m a g n i f i c a t i o n . 150 ml water samples were p r e s e r v e d with 5 drops of Lugol's s o l u t i o n (200 g KI + 100 g I 2 i n 2000 ml H 2 O + 190 ml g l a c i a l C H 3 C O O H ) . Counting and i d e n t i f i c a t i o n were done i n a i n v e r t e d microscope base p l a t e chamber (volume = 2 m l ) . I d e n t i f i c a t i o n of h e t e r o t r o p h i c andautotrophic m i c r o f l a g e l l a t e s was a c c o r d i n g t o the pink and brown c o l o u r . A l l the non-pigmented f l a g e l l a t e s were not i n c l u d e d i n the co u n t i n g , o n l y the m i c r o z o o f l a g e l l a t e s i z e between 2-10 um were counted because those t h a t are l a r g e r than t h i s may have a higher t r o p h i c f u n c t i o n (Fenchel, 1982b). C i l i a t e s were found i n the samples but the low number made i t very d i f f i c u l t t o o b t a i n meaningful counts of t h i s group. C i l i a t e s were not counted, but t h e i r presence suggests they must p l a y a important r o l e i n c o n t r o l l i n g f l a g e l l a t e numbers. For a n a l y s i s of phytoplankton o n l y major dominant s p e c i e s were i d e n t i f i e d t o genus or s p e c i e s , a l l other minor s p e c i e s were counted as one group. Because I c o n s i d e r e d the microcosm system based on t r o p h i c l e v e l , the s p e c i e s composition was not regarded as being of major importance. 3.5 Standing stock of phytoplankton In v i v o f l u o r e s c e n c e was measured to g i v e an i n d i c a t i o n of biomass of the phytoplankton assemblages i n the c u l t u r e s . Samples were h e l d i n 25 x 150 mm c u l t u r e tube which were i n s e r t e d i n t o a Turner 10-000 fluorometer every day at a given time. The subsamples were then c u l t u r e d i n sequence at con s t a n t s a l i n i t y and at the same c o n d i t i o n and time p e r i o d as i n 27 experimental f l a s k s . For the f i r s t experiment, c h l o r o p h y l l a and _in v i v o f l u o r e s c e n c e were measured simultaneouslyand then the c o r r e l a t i o n and r e g r e s s i o n between them c a l c u l a t e d . The r e g r e s s i o n equation f o r in v i v o f l u o r e s c e n c e and the f l u o r e s c e n c e with e x t r a c t e d c h l o r o p h y l l a i n 90 °/o acetone was Y = 0.70X + 1.73, ( r 2 = 0.98, N = 6) The r e g r e s s i o n equation f o r c h l o r o p h y l l a (ug/1) and f l u o r e s c e n c e (of e x t r a c t e d value) was : Y = 0.167X + 1.74, ( r 2 = 1.00, N = 10) ac c o r d i n g t o these two equations c h l o r o p h y l l a and biomass of phytoplankton can be c a l c u l a t e d i n the experiment. The r a t i o f o r B a c i l l a r i o p h y c e a e are not very d i f f e r e n t . ( K i e f e r , 1973; Parsons, 1969; Parsons et a l . , 1984). 28 4. R e s u l t s 4.1 Experiment stage I T h i s stage of the experiment was designed to answer the q u e s t i o n of what i s the behavior of f r e s h water e c o l o g i c a l components on the s a l i n i t y g r a d i e n t . Because the e x t r a p o l a t i o n of temporal e v o l u t i o n of an e s t u a r i n e ecosystem to e s t u a r y s p a t i a l d i s t r i b u t i o n i s based on the p r e c o n d i t i o n of the f r e s h water a u t o t r o p h i c components which stop f u n c t i o n i n g a t the head of the e s t u a r y , the e v o l u t i o n curve i s a c t u a l l y t h a t of a sea water p o p u l a t i o n of phytoplankton. So i n t h i s experiment sea water was f i l t e r e d by a 0.8 um f i l t e r t o remove the sea water phytoplankton p o p u l a t i o n and m i c r o f l a g e l l a t e s and then I used i t e i t h e r as d i l u t i o n water or i n i t i a l experimental water t o c r e a t e e i t h e r an i n c r e a s i n g s a l i n i t y i n p a r t 1 or a d e c r e a s i n g s a l i n i t y g r a d i e n t i n p a r t 2. The response of f r e s h water components on these s a l i n i t y g r a d i e n t s were i n v e s t i g a t e d . 4.11 I n c r e a s i n g s a l i n i t y g r a d i e n t In experiment p a r t one, an i n c r e a s i n g s a l i n i t y g r a d i e n t was maintained by adding f i l t e r e d seawater (29°/oo) to f r e s h water. The s a l i n i t y change d u r i n g the experimental p e r i o d i s shown i n F i g . 4. An i n c r e a s i n g temporal s a l i n i t y g r a d i e n t was c l e a r l y c o n s t r u c t e d i n the Stage I, p a r t 1. The r e s u l t a n t curve of phytoplankton and n u t r i e n t s (N0~3, NH +4> ( F i g . 5a) shows the i n t e r a c t i o n between these two components on the s a l i n i t y g r a d i e n t as the combined e f f e c t s of the s a l i n i t y and the d i l u t i o n . A s m a l l i n c r e a s e on day 1 and 2 was due to f r e s h water a l g a l growth; the major s p e c i e s was T h a l a s s i o s i r a spp.. On F i g u r e 4. The temporal s a l i n i t y p a t t e r n i n a l l of the experiment stage I or I I , p a r t 1 with an i n c r e a s i n g s a l i n i t y g r a d i e n t . F i g u r e 5a. The development p a t t e r n of a u t o t r o p h i c component and n u t r i e n t s i n the experiment stage I, p a r t 1 during J u l y , 1985. O 2.5 *-o 1 2.0 1.5 E 1.0 3 C w E 0.5 E < 0.0 .-A I 0 T " 5 T 10 D a y s 15 20 F i g u r e 5b. The development p a t t e r n of NH+4 i n the experiment stage I, p a r t 1 d u r i n g J u l y , 1985. 0.30 4 0 Z 30-1 - 20 ? 104 10 Days 15 1-0.25 g 1-0.20 J hO.I5 | u . I-0.10 f-0.05 «5 0.00 K 20 Figure 6a. The development p a t t e r n of a u t o t r o p h i c component and n u t r i e n t s i n the experiment stage I, p a r t 1 during February, 1985. o 1 2.0 3 . 1.5 E 1.0 O E 0.5 E < 0.0 % 4 T 5 10 D a y s T " 15 —i 20 F i g u r e 6b. The development p a t t e r n of NH +4 i n the experiment stage 1, p a r t 1 d u r i n g February, 1985. 34 day 3, when the s a l i n i t y i n c r e a s e d t o about 15°/oo, the growth of algae were depressed u n t i l day 6. Most of the f r e s h water diatoms d i e d or were depressed when s a l i n i t y reached 10°/oo (Blanc e t al.,1969). Some of the diatoms showed very d i s t i n c t i v e m o r p h o l o g i c a l changes and some other s t r u c t u r a l changes (such as a decrease i n c h l o r o p l a s t s ) and auxospores were formed w i t h i n 48 h ( F o e s t e r , 1971). The p h y s i o l o g i c a l s t a t e of these diatoms decreased with i n c r e a s i n g s a l i n i t y (Qasim, 1972; Paasche,1975; W e t h e r e l l , 1961). An i n c r e a s e i n i n v i v o f l u o r e s c e n c e a f t e r day 6 was caused by a mixed p o p u l a t i o n . At the beginning of growth, T h a l a s s i o s i r a spp. o c c u r r e d and were then r e p l a c e d by a s m a l l round diatom ( u n i d e n t i f i e d ) . The d e c l i n e of the l a t t e r p o p u l a t i o n at day 16 may have been due to even higher s a l i n i t y (>25°/oo). The s a l i n i t y range i n which the second group of diatoms grew was 15 to 25 °/oo and i t appears t h e r e f o r e t h a t the o r i g i n a l freshwater was i n f l u e n c e d by the s a l t w a t e r . I t may have been contaminated by spores of e s t u a r i n e s p e c i e s which have an o p t i m a l growth s a l i n i t y around 10-20°/oo (Blanc et a l . , 1969; Mahoney, 1979; Qasim, 1972; Paasche, 1975). The experiment c a r r i e d out i n February showed no i n c r e a s e of phytoplankton d u r i n g the whole experimental time and a l s o no decrease of n u t r i e n t s ( F i g . 6a). Ammonium which i s f r e q u e n t l y more r a p i d l y taken up than n i t r a t e decreased before the decrease of N0~3 ( F i g . 5b, 6b). Even though i n the experiment c a r r i e d out i n Feburary, 1985 t h e r e was no growth of phytoplankton, the c o n c e n t r a t i o n of NH +4 s t i l l showed a d e c l i n e d u r i n g the f i r s t p e r i o d of the experiment ( F i g . 6b). 35 T h i s i n d i c a t e d a consumption which was not due to the a u t o t r o p h i c component and was most l i k e l y due t o f r e e l i v i n g b a c t e r i a . In t h i s work, I found t h a t T h a l a s s i o s i r a spp. appeared at the two ends of the s a l i n i t y range. These two groups may belong to e n t i r e l y two d i f f e r e n t s p e c i e s . U n f o r t u n a t e l y they were not i d e n t i f i e d t o the s p e c i e s l e v e l , but there was no c o n n e c t i o n between the two groups i n themiddle range of s a l i n i t y . T h i s dominant s p e c i e s i s the major s p e c i e s i n the annual s p r i n g phytoplankton bloom i n the S t r a i t of G e o r g i a . In the F r a s e r R i v e r e s t u a r y n i t r o g e n i n the s u r f a c e l a y e r i s s u p p l i e d from seawater entrainment ( T u l l y and Dodimead, 1957; Parsons e t a l . , 1980). R i v e r water i s r i c h i n B 1 2 ( C a t t e l l , 1973) and m i c r o n u t r i e n t s (Cross and Sunda, 1977). The i n f l o w of n u t r i e n t r i c h water should s t i m u l a t e the growth of phytoplankton. However, comparing the f r e s h water c o n t r o l ( F i g . 14) with the experimental f l a s k and c o n s i d e r i n g the n u t r i e n t dynamics, the phytoplankton growth was depressed and delayed i n the experimental f l a s k by osmotic s t r e s s . T h e r e f o r e , the d i l u t i o n ( i . e . the s a l i n i t y g r a d i e n t ) f u n c t i o n s as a f i l t e r e s s e n t i a l l y f i l t e r i n g out a l l the freshwater s p e c i e s . M o r r i s (1978) suggested t h a t one mechanism of oxygen d e p l e t i o n i n an e s t u a r y i s due to a mass m o r t a l i t y of f r e s h water halophobic phytoplankton which r e s u l t s from the osmotic changes o c c u r r i n g i n the low s a l i n i t y r e g i o n ; the concommitant r e l e a s e of o r g a n i c m a t e r i a l and i t s a s s i m i l a t i o n by the h e t e r o t r o p h i c microbes would cause decreased O 2 . The e f f e c t of t h i s p rocess on the h e t e r o t r o p h i c component w i l l be d i s c u s s e d i n the next s e c t i o n . 36 9 20 M 8 15 to I '° i o 10 Days 15 K aof 1.0 ao o 20 Figure 7. The development pattern of heterotrophic bacteria and nanozooflagellates in experiment stage I part 1 during July, 1985. 300 250 © 200 1 u 150 e 0 100 500 0.00 OO 10 20 30 Salinity <%•) To F i g u r e 8. Growth of freshwater b a c t e r i a on p l a t e media at d i f f e r e n t s a l i n i t i e s . 37 H e t e r o t r o p h i c and phagotrophic component B a c t e r i a , as the lowest h e t e r o t r o p h i c component i n an ecosystem, u t i l i z e o r g a n i c compounds and p a r t i c u l a t e o r g a n i c m a t e r i a l as energy sources. They e x i s t i n n a t u r a l sea water i n two forms; a c t i v e or dormant b a c t e r i a l c e l l s . A c t i v e b a c t e r i a has a high V m f o r uptake of o r g a n i c s u b s t r a t e (Parsons et a l . 1977) and a very high growth r a t e (Ammerman, 1984; Hagstrom e t al.,1984; Van-Wambeke, 1985; Landry, 1984; H o l l i b a u g h , 1980). T h e s e p r o p e r t i e s combined with others p r o v i d e the b a c t e r i a with the p h y s i o l o g i c a l c a p a b i l i t y t o complement t h e i r e c o l o g i c a l r o l e . The b a c t e r i a l numbers i n our experiment are i n the range 4 x 1 0 5 t o 4 x l O ^ c e l l s / m l , which i s c o n s i s t e n t with Watson's work (1.5 x 1 0 4 to 6.29 x 10 6; Watson,1979). Most of the c e l l s are s m a l l - s i z e d and c o c c i shaped a t the beginni n g . Z o o f l a g e l l a t e s are the major consumers of b a c t e r i a i n p e l a g i c systems ( L i g h t h a r t , 1969; Haas and Webb, 1979; Fe n c h e l , 1982b). They appeared o v a l shaped and b i f l a g e l l a t e d . Most of them f a l l i n the s i z e range 2-10 um. The i d e n t i f i c a t i o n of s p e c i e s was not thoroughly c a r r i e d out but a c c o r d i n g t o Kudo (1966), Fenchel (1982b) and Haas and Webb (1979), they are mostly Monadidae, Bodonidae and Amphimonadidae (Sherr and Sherr, 1982). They are very abundant i n the a q u a t i c environment; u s u a l l y from 0.4 to 13 x 10^ c e l l s / m l i n v a r i o u s p a r t s of the world oceans (S o r o k i n , 1981). T h e i r d e n s i t y i s c l o s e t o one. The s i z e d i s t r i b u t i o n i s toward the sm a l l s i z e f r a c t i o n (2-4 um) and the biomass d i s t r i b u t i o n was more even (101.3-198.3 ug wet weight/ml) (S o r o k i n , 1981). These f l a g e l l a t e s may grow a t 1.5 x 10 2 -38 2.2 x 1 0 2 c e l l s / m l / h w i t h d o u b l i n g times of 9.7-18.2 h (Sherr & Sherr, 1982). These d o u b l i n g times are q u i t e high f o r n a t u r a l waters, i n d i c a t i n g m i c r o f l a g e l l a t e s are capable of responding t o the dynamics of b a c t e r i a l p o p u l a t i o n s under n a t u r a l c o n d i t i o n s a t c e r t a i n r a t e . The q u a l i t a t i v e i n t e r a c t i o n between b a c t e r i a and n a n o z o o f l a g e l l a t e s i n t r o p h i c r e l a t i o n s have been c l e a r l y r e f l e c t e d i n these data ( F i g . 7). The low counts (4 x 10 5 c e l l s / m l ) and s m a l l s i z e of b a c t e r i a on day 1 are due to the h i g h zoof l a g e l l a t e g r a z i n g (13.8 x 10-* c e l l s / m l ) which i s s e l e c t i v e towards b i g b a c t e r i a a t high c o n c e n t r a t i o n s (Fenchel, 1980a,b). T h i s p o i n t may r e p r e s e n t the lowest t h r e s h o l d of f l a g e l l a t e g r a z i n g on a s u b s t r a t e l i m i t e d b a c t e r i a l p o p u l a t i o n . The s l i g h t i n c r e a s e i n b a c t e r i a l numbers on day 2 and day 6 may be due to enrichment of the b a c t e r i a from the i n f l o w of s a l i n e water and the r e l e a s e of o r g a n i c s u b s t r a t e by osmotic impact on freshwater organisms. In a d d i t i o n i t r e s u l t e d i n the optimum s a l i n i t y f o r b a c t e r i a as i n d i c a t e d i n my l a b o r a t o r y work ( F i g . 8). During the i n i t i a l p e r i o d , m i c r o z o o f l a g e l l a t e s d e c l i n e d s h a r p l y . The reason i s not c l e a r ; one p o s s i b l e e x p l a n a t i o n i s the i n c r e a s e i n s a l i n i t y may s t r o n g l y a f f e c t the z o o f l a g e l l a t e p o p u l a t i o n or i n a l i m i t e d s i t u a t i o n , a z o o f l a g e l l a t e p o p u l a t i o n can be s t r o n g l y a f f e c t e d by d i l u t i o n . U n f o r t u n a t e l y , t h e r e i s no work on the t o l e r a n c e of z o o f l a g e l l a t e s to s a l i n i t y . The second i n c r e a s e i n b a c t e r i a p o p u l a t i o n seems to be due to the r e l e a s e of e x t r a c e l l u l a r o r g a n i c products or some s t i m u l a t i n g products by phytoplankton growth ( B e l l and Sakshaug, 39 1980). Because the s t a n d i n g stock of c h l o r o p h y l l a was low compared with t h a t of b a c t e r i a , i t seems t h a t the b a c t e r i a consumed a carbon source which may not have come e n t i r e l y from phytoplankton. Even though we assume t h a t growth of the a u t o t r o p h i c component under osmotic s t r e s s may have a high p r o p o r t i o n of e x c r e t e d o r g a n i c p r o d u c t s , t h e r e appears to be a gap i n terms of the carbon budget. The d i v i s i o n of o r g a n i c carbon among the t h r e e t r o p h i c l e v e l s i n d i c a t e s t h a t under t h i s c o n d i t i o n , the h e t e r o t r o p h i c component makes a very l a r g e c o n t r i b u t i o n to the energy flow. The h i g h e s t v a l u e of b a c t e r i a l s t a n d i n g carbon was 314 ug C / l . The average s i z e of the z o o f l a g e l l a t e s was ca. 4.1 um. I f I assume t h a t a l l c e l l s are s p h e r i c a l then V = l / 6 p i D 3 = l / 6 p i ( 4 . 1 ) 3 =36.1 u m 3 / c e l l the carbon c o n v e r s i o n c o e f f i c i e n t = 0.75 x 1 0 - 1 1 g C / c e l l f o r the 4.5 um diameter c e l l s ( Fenchel, 1982b). So the h i g h e s t v a l u e f o r the z o o f l a g e l l a t e carbon standing stock was 106 ugC/1 (14.1 x 10 3 c e l l s / m l ) . The c h l o r o p h y l l a maximum was 4.32 ugChla/1; i f 30 i s used as a C / c h l c o n v e r s i o n f a c t o r (Parsons, et a l . , 1969), the phytoplankton s t a n d i n g stock o n l y accounts f o r 129.6 ugC/1; however the r a t e of a l l the t h r e e components' a c t i v i t i e s has not been measured. I have no i d e a of the energy f l u x i n the system which would be a more meaningful index than j u s t carbon s t a n d i n g s t o c k . N e v e r t h e l e s s , the h i g h growth r a t e of b a c t e r i a (Ammerman, 1984) and z o o f l a g e l l a t e s under c e r t a i n c o n d i t i o n s (Fenchel, 1982a) and the high g r a z i n g r a t e (20% of the b a c t e r i a p o p u l a t i o n per day F e n c h e l , 1982b; and a 40 b a c t e r i a l p o p u l a t i o n s of 2 x 10^ c e l l s / m l / d a y to 7.5 x 10 6 c e l l s / d a y i n a 7 x 10 6 c e l l s / m l p o p u l a t i o n i n an estuary; Wright, 1983), both suggest t h a t these two components may have g r e a t e r p o t e n t i a l c a p a b i l i t y to c o n t r i b u t e to the carbon energy flow i n the whole ecosystem. C i l i a t e s as a t h i r d higher l e v e l of t r o p h i c i n t e r a c t i o n (Banse, 1982) e x i s t e d i n the experimental medium, re a c h i n g a r e l a t i v e l y high abundance (main s p e c i e s was T i n t i n n i d a e spp.). T h i s i m p l i e s a high l e v e l of t r a n s f o r m a t i o n and i n s h a r i n g the s t a n d i n g stock of carbon (Heinbokel, 1978). Thus the dynamic r a t e and p h y s i o l o g i c a l s t a t e probably c o n t a i n more s i g n i f i c a n t i n f o r m a t i o n f o r the r e v e l a t i o n of the f u n c t i o n a l s t r u c t u r e and energy pathways i n the ecosystem. These r e s u l t s suggest t h a t f r e s h water phytoplankton can not p l a y the same r o l e i n an e s t u a r i n e ecosystem as they d i d i n the o r i g i n a l water. They cannot pass through the s a l i n i t y g r a d i e n t without changing t h e i r r o l e . T h e i r e c o l o g i c a l r o l e i s not as a primary producer, but an o r g a n i c c o n t r i b u t o r . Data a n a l y s i s was f a c i l i t a t e d by the use of a s t a t i s t i c a l software package, "SYSTAT", and an IBM p e r s o n a l computer. C o r r e l a t i o n matrixes were c a l c u l a t e d f o r each p a r t of the experiment from 7 v a r i a b l e s and 20 o b s e r v a t i o n s . The i n t e r r e l a t i o n s h i p s between the d i f f e r e n t v a r i a b l e s were assessed by comparing p a i r s of v a r i a b l e s . The c o r r e l a t i o n c o e f f i c i e n t s between p a i r s of v a r i a b l e s are shown i n Table 3. The high c o r r e l a t i o n of some v a r i a b l e s such as s t a n d i n g s t o c k s of phytoplankton, NO~3, NH +4 and b a c t e r i a with time can be 4\ Table 3. C o r r e l a t i o n matrix o b t a i n e d from the 7 v a r i a b l e s X 20 o b s e r v a t i o n s i n experiment stage I, p a r t 1 duri n g J u l y , 1985, between time (day), F l u o r e s c e n c e ( f l u o ) , N0~3, NH +4, F l a g e l l a t e s ( f i g ) , B a c t e r i a (bac), and s a l i n i t y . P r o b a b i l i t y : * p<0.1, ** p < 0.05, *** p < 0.02, **** p <0.01. | DAY 1 FLUO | N03 I NH4 I FLG | BACT DAY | 1.000 | FLUO | 0.903**** 1.000 | 1 N03 1-0.726 -0.835****1 1. 000 ! NH4 j-0.688**** -0.608****| 0. 626**** 1 .000 1 I FLG 1-0.244 0.040 | 0. 027 0 .285 1.000 | BACT | 0.857**** 0.723****1 -0. 760**** -0 .757**** -0.397 |1 .000 SAL | 0.952**** 0.740****| -0. 580*** -0 .658**** -0.475*10 .845**** Table 4. C o r r e l a t i o n matrix o b t a i n e d from the 7 v a r i a b l e s X 20 o b s e r v a t i o n s i n experiment stage I, p a r t 2 duri n g J u l y , 1985, between time (day), F l u o r e s c e n c e ( f l u o ) , N0~3, NH+4, F l a g e l l a t e s ( f i g ) , B a c t e r i a (bac), and s a l i n i t y . P r o b a b i l i t y : * p<0.1, ** p < 0.05, *** p < 0.02, **** p <0.01. 1 I DAY FLUO N03 NH4 FLG 1 BACT DAY I 1.000 | FLUO I 0.917**** 1. 000 | N03 1-0.967**** -0. 855**** 1.000 | NH4 1-0.859**** -0. 909**** 0.785**** 1.000 J FLG I 0.036 0. 052 -0.051 0.219 1.000 | BACT | 0.868**** 0. 930**** -0.835**** -0.837**** -0.026 I 1.000 SAL 1-0.948**** -0. 782**** 0.967**** 0.774**** 0.078 1-0.765**** 42 e a s i l y e x p l a i n e d by the time e v o l u t i o n of these components. However, i f we c o n s i d e r a body of water moving seaward at the s u r f a c e l a y e r i n an e s t u a r y , these c o r r e l a t i o n s can be i n t e r p r e t e d as o c c u r r i n g on a s p a t i a l s c a l e s i n c e time i s p r o p o r t i o n a l to d i s t a n c e d u r i n g the movement of the water. T h i s assumes t h a t : (1) the f r e s h water organisms stop f u n c t i o n i n g as a u t o t r o p h i c components at the head of the e s t u a r y , and (2) marine phytoplankton are the major a u t o t r o p h i c component i n the t r a n s i t i o n zone, but o n l y begin to f u n c t i o n a f t e r r e a c h i n g the e u p h o t i c zone. N i t r a t e , n i t r i t e and ammonium were n e g a t i v e l y c o r r e l a t e d with c h l o r o p h y l l a because the phytoplankton remove nitrogenous n u t r i e n t s to s y n t h e s i z e new biomass with n i t r o g e n compounds being mainly i n c o r p o r a t e d i n t o p r o t e i n . B a c t e r i a a l s o take up n i t r o g e n as a n u t r i e n t source, but such uptake may be l i m i t e d t o o r g a n i c s u b s t r a t e s which serve as the b a c t e r i a l energy source. H e t e r o t r o p h i c b a c t e r i a were p o s i t i v e l y c o r r e l a t e d with phytoplankton s t a n d i n g stock because a t the beginning of the experiment, b a c t e r i a l p o p u l a t i o n s were l i m i t e d by o r g a n i c s u b s t r a t e . The o r g a n i c products r e l e a s e d d u r i n g growth of phytoplankton are u t i l i z e d by b a c t e r i a as the o n l y source (Rheinheimer, 1977; Hoppe, 1978; Larsson and Hagstrom, 1979; Wolter, 1982). M i c r o f l a g e l l a t e s and b a c t e r i a were not s i g n i f i c a n t l y c o r r e l a t e d , even though t h e i r t r o p h i c i n t e r a c t i o n s are v e r y c l e a r on the c o v a r i a t i o n p r o c e s s . The c y c l e s between these two components correspond very c l o s e l y and o f t e n , having completed s e v e r a l i n t e r a c t i o n c y c l e s d u r i n g the experimental Pigure 9. The temporal s a l i n i t y pattern in experiment stage I t> II,art 2.with a decreasing s a l i n i t y gradient ua c n n> Q) 3 a 3 C rr o Oi 03 •"I 01 •-3 3" a> a CD < m 3 rr f0 3 rr 0 Qi CO TJ 3 3 3 rr CO h- rr 3 0) TJ Q Q rr C rr t—' t—l ft> •< •» >1 - 3 TJ h-> fl) o *fl T fti oo rr ui rr • fo zr • r o C 0) H- C rr rr 3" O rr O O,TJ (D 3" O >-» 0) a> o o 3 3 TJ 03 O 3 fO 3 rr o R e l a t i v e F l u o r e s c e n c e o P o o J . g s fi - J - o J. P to o a . —-I 1 1 1 1 1— .cj — — in> ro oi o o 0 1 © m o N i t r a t e ( p g - a t / l ) ° D a y s F i g u r e 10b. The development p a t t e r n of NH +4 c o n c e n t r a t i o n i n Stage I, p a r t 2 with a d e c r e a s i n g s a l i n i t y g r a d i e n t i n J u l y 1985. o D a y s F i g u r e 1 1 a . The d e v e l o p m e n t p a t t e r n o f t h e a u t o t r o p h i c c o m p o n e n t and n u t r i e n t s i n S t a g e I , p a r t 2 w i t h a d e c r e a s i n g s a l i n i t y g r a d i e n t i n F e b r u a r y , 1 9 8 5 . 1.2 ^ 1.0 1 I 0.8 ~ 0.6 H £ | 0.4 o | 0.2 < 0.0 * 0 * * *4 T -5 10 Days 15 20 Figure l i b . The development p a t t e r n of NH +4 c o n c e n t r a t i o n i n Stage I, Part 2 with a d e c r e a s i n g s a l i n i t y g r a d i e n t i n February, 1985. —i 48 time, the c o r r e l a t i o n s o f f s e t each o t h e r . 4.12 Decreasing s a l i n i t y g r a d i e n t In the second p a r t of the experiment, stage I, 5 1 of f i l t e r e d (0.8 um) seawater was used as the experimental water and f r e s h water was pumped i n t o i t . The f r e s h water e c o l o g i c a l component c o n t a i n e d i n the i n f l o w as a inoculum encountered a g r a d u a l l y d e c r e a s i n g s a l i n i t y g r a d i e n t ( F i g . 9). The h e t e r o t r o p h i c b a c t e r i a i n the sea water a l s o encountered a d e c r e a s i n g s a l i n i t y g r a d i e n t . Freshwater phytoplankton i n c u l t u r e d i d not s t a r t growing u n t i l day 6 ( F i g . 10a, 11a). The i n c r e a s e i n c h l o r o p h y l l a s t a r t e d a f t e r day 6 (10°/oo on t h a t day). The growth r a t e of phytoplankton i n c r e a s e d as the s a l i n i t y decreased. C a l c u l a t e d biomass ( c h l o r o p h y l l a) i n c r e a s e d q u i c k l y r e a c h i n g about 1.88 ug Chla/1 a t day 13-15 u n t i l the experiment was terminated. Phytoplankton carbon s t a n d i n g stock reached a maximum of 56.40 ug C / l (c o n v e r s i o n f a c t o r 30 which i s f o r h e a l t h y c e l l s under r i c h n i t r o g e n c o n d i t i o n s ( A n t i a e t a l . 1963). In my experiment the growth of phytoplankton c e l l s was under the impact of s a l i n i t y change. The c o n d i t i o n was not a normal c o n d i t i o n so t h i s c o n v e r s i o n v a l u e may c o n t a i n a l a r g e d e v i a t i o n . The experiment was c a r r i e d out d u r i n g February, 1985. I t showed the same p a t t e r n except there was an jln v i v o f l u o r s c e n c e peak by day 2-3, which then decreased as the s a l i n i t y decreased. T h i s may have been caused by the growth of some seawater autotrophs which were l e s s than 0.8 um and c o u l d be i n h i b i t e d by a d e c r e a s i n g s a l i n i t y . Pigure 12. The development p a t t e r n of b a c t e r i a and n a n o z o o c l a g e l l a t e s i n Stage I, p a r t 2 with d e c r e a s i n g s a l i n i t y g r a d i e n t i n J u l y 1985. a 50 Table 4 shows the c o r r e l a t i o n c o e f f i c i e n t between 8 v a r i a b l e s i n the experiment p a r t two. The c o r r e l a t i o n between most of the parameters can be e x p l a i n e d by the development of phytoplankton, except z o o f l a g e l l a t e s . A l l the v a r i a b l e s which are d i r e c t l y r e l a t e d t o each other show a c l o s e c o r r e l a t i o n ; f o r example, n u t r i e n t s and phytoplankton, and, b a c t e r i a and phytoplankton are c l o s e l y c o r r e l a t e d , time r e l a t e s t o the e c o l o g i c a l components because of the development course of the phytoplankton. Time and s a l i n i t y c o r r e l a t e d with each other because i t i s the requirement of the experiment. The n a n o z o o f l a g e l l a t e s were not c o r r e l a t e d with other v a r i a b l e s d u r i n g g i v e n time course and t h i s suggests t h a t the t r o p h i c r e l a t i o n of n a n o z o o f l a g e l l a t e s i s not c l o s e l y and d i r e c t l y r e l a t e d t o primary p r o d u c t i o n i n terms of t r o p h i c r e l a t i o n s h i p s . Hobbie and Rublee (1977) demonstrated a c l o s e r e l a t i o n s h i p between phytoplankton primary p r o d u c t i o n and the p o t e n t i a l b a c t e r i a l uptake of o r g a n i c substances. Recent r e s e a r c h has confirmed t h i s r e l a t i o n s h i p . The development of phytoplankton i n t h i s microecosystem r e s u l t s i n the p r o d u c t i o n of phytoplankton biomass and p r o v i d e s d i s s o l v e d o r g a n i c substances which s t i m u l a t e b a c t e r i a l p o p u l a t i o n s . The d i s t r i b u t i o n s of carbon and energy flow between these two pathways depends upon many f a c t o r s . These i n c l u d e the s p e c i e s composition of the a l g a l f l o r a (Fogg e t a l . , 1965; Wolter,1982) and the p h y s i o l o g i c a l s t a t e of the p o p u l a t i o n (Sharp,1977); m i c r o f l a g e l l a t e s a l s o may have an e f f e c t (Wolter, 1982; Goldman et a l . , 1985). In our experiment, p o s s i b l y a l l of these f a c t o r s 51 a f f e c t e d the p r o d u c t i o n of e x t r a c e l l u l a r o r g a n i c carbon (EOC). The p r o d u c t i o n of EOC and i t s u t i l i z a t i o n as a b a c t e r i a l food source i s determined both by the s t a n d i n g stock of biomass and by the r e l a t i v e r a t e of r e l e a s e of d i f f e r e n t p h o t o s y n t h e t i c p r o d u c t s . However, i n t h i s experiment the phytoplankton can not pass through the f u l l spectrum of the s a l i n i t y g r a d i e n t , so the primary p r o d u c t i o n c o n t r i b u t i o n to s t a n d i n g carbon i s very s m a l l (45.40 ug C / l ) . I t i s not l i k e l y t h a t phytoplankton r e l e a s e d EOC can s u s t a i n the b a c t e r i a l requirement. So some other -source of o r g a n i c s u b s t r a t e s must have been u t i l i z e d . The i n t e r e s t i n g t h i n g i s t h a t a c l o s e c o v a r i a n c e was maintained between phytoplankton and b a c t e r i a . H e t e r o t r o p h i c b a c t e r i a and m i c r o f l a g e l l a t e s F i g u r e 12 shows the development of b a c t e r i a and m i c r o f l a g e l l a t e s . B a c t e r i a l d e n s i t y a t day 1 (8.3 x 10^ c e l l s / m l ) was higher comparing t h a t i n the stage I, p a r t one. T h i s l e v e l may r e p r e s e n t an upper boundary of the o r g a n i c s u b s t r a t e l i m i t a t i o n because the i n i t i a l water was f i l t e r e d w ith a 0.8 um f i l t e r . Most of the b a c t e r i v o r o u s m i c r o f l a g e l l a t e s would have been e l i m i n a t e d from the medium. T h i s would have r e l e a s e d the g r a z i n g e x p l o i t a t i o n of b a c t e r i a by n a n o f l a g e l l a t e s (Gak e t a l . , 1972; Fuhrman and Azam, 1980; Wright, 1984). With the i n f l o w of f r e s h water inoculum, the z o o f l a g e l l a t e s would have o c c u r r e d i n a r e l a t i v e l y higher b a c t e r i a medium. Thus the new m i c r o z o o f l a g e l l a t e p o p u l a t i o n may have grown very q u i c k l y . As the m i c r o f l a g e l l a t e s i n c r e a s e d , b a c t e r i a showed a s l i g h t decrease and then an i n c r e a s e to a s m a l l peak. During t h i s p e r i o d , the b a c t e r i a may have grown a c t i v e l y due to the enrichment by o r g a n i c substances r e s u l t i n g from mass m o r t a l i t y of f r e s h water organisms when they flow i n t o the high s a l i n i t y water (29°/oo). Some of the b a c t e r i a l biomass may be transformed i n t o z o o f l a g e l l a t e biomass by g r a z i n g d u r i n g one day. I f we assume t h a t b a c t e r i a l biomass doubled d u r i n g one day (Ammerman et a l . , 1984) from day 1 t o day 2 under a c e r t a i n o r g a n i c s u b s t r a t e supply r a t e , then the b a c t e r i a p o p u l a t i o n c o u l d have i n c r e a s e d to 16 x 10^ c e l l s / m l . However o n l y 6 x 1 0 5 c e l l s / m l were pr e s e n t a t day 2, suggesting t h a t 10 x 10^ c e l l s / m l were removed from the p o p u l a t i o n . I f the b a c t e r i a / z o o f l a g e l l a t e y i e l d f a c t o r i s taken as 100 B/F.(Laak et al.,1984), then 10 x 10 5 b a c t e r i a l c e l l s / m l may produce 10 x 10^ z o o f l a g e l l a t e s / m l . T h i s value i s i n agreement with my experimental data. The number of f l a g e l l a t e s i n c r e a s e d from 8.4 x 10^ c e l l s / m l on day 1 to 18.5 x 10^ c e l l s / m l on day 2. Thus the sudden i n c r e a s e of n a n o z o o f l a g e l l a t e s can be e x p l a i n e d by the t r a n s f o r m a t i o n of b a c t e r i a l biomass i n t o m i c r o z o o f l a g e l l a t e s . I f t h i s c a l c u l a t i o n i s j u s t i f i a b l e , i t s magnitude i s s i m i l a r t o t h a t observed by o t h e r s . Kopylov and Moiseyev (1980) observed f l a g e l l a t e d o u b l i n g times of 0.67 t o 1.44 days with a consumption of 50% of the d a i l y p r o d u c t i o n of b a c t e r i a . Wright (1983) observed 7.5 x 10^ c e l l s / m l were removed per day by z o o f l a g e l l a t e s from a p o p u l a t i o n of 7 x 10^ c e l l s / m l . T h i s evidence i n d i c a t e s t h a t the carbon f l u x through t h i s pathway can be very f a s t and s i g n i f i c a n t i n p r o p o r t i o n to other p r o c e s s e s . But i n the ecosystem because of the f a s t N i t r a t e ( ^ g - a t / l ) • _ - . N N 0 o» O Ul O Oi 1 I I 1 I I B a c t e r i a N o . ( x l O * ) * ^ oi j i R e l a t i v e F l u o r e s c e n c e o B a c t e r i a (xlO6) C 0 3 ^ C >-3 ill T (C 3 a) a> 01 3 CD t— rr < Ml D) H "1 3 O 0> CLTJ 0) 3 3" O" fD € 01 3 0) O rr rr rr fD (D TJ 1 1 D) m- r* a a» c h- 3 3 iQ 0* rr 0> 3 t4 o con \-> 3 3" "< n- a> - *1 O 01 M M C vO rr ao it o Ui Qi rr • n CO O >< TJ W 3" rr h-(D O 3 o < o h. 3 rrTj 3- O 3 a> 3 rr o — ro I 1 L . Oi L> 45-— I Relative Fluorescence o o o — — ro o ro o (x> o Nitrate (/jg-at/l) • 7S 55 uptake r a t e and g r e a t growth r a t e of b a c t e r i a , the b a c t e r i a can exhaust t h e i r s u b s t r a t e over long t i m e p e r i o d s and i n g e n e r a l , maximum growth r a t e s w i l l o n l y occur over s h o r t p e r i o d s . These two components may l i m i t e d t o e x p l o r e t h e i r p o t e n t i a l c a p a b i l i t y over long time p e r i o d . The r e s u l t s i n the experiments i s a combination of the e f f e c t of both f a c t o r s ( growth l i m i t a t i o n and g r a z i n g ) . From day 6, the number of b a c t e r i a i n c r e a s e d c o n s t a n t l y and, c o i n c i d e n t a l l y , the i n c r e a s e of b a c t e r i a was simultaneous with the i n c r e a s e i n c h l o r o p h y l l a (see F i g . 1 0 a ) . However, c a l c u l a t i n g the phytoplankton carbon biomass, i t i s a l s o not l i k e l y t h a t the carbon i n c o r p o r a t e d i n t o b a c t e r i a l biomass i s t o t a l l y from the r e l e a s e of p h o t o s y n t h e t i c p r o d u c t s . The maximum b a c t e r i a l biomass i s 314 ug C / l and the maximum of phytoplankton biomass o n l y reaches 46.40 ug C / l . There are two p o s s i b l e e x p l a n a t i o n s : (1) t h a t under s t r e s s e d c o n d i t i o n s , most of the p h o t o s y n t h e t i c products were r e l e a s e d as o r g a n i c substances and the i n f l o w of f r e s h water a l s o brought i n some o r g a n i c m a t e r i a l , o r , (2) phytoplankton may produce some other m a t e r i a l s which can a c t u a l l y s t i m u l a t e b a c t e r i a l use of another source of DOC which may be u n a v a i l a b l e i n the absence of the s t i m u l a n t r e l e a s e d by phytoplankton. T h i s may suggest t h a t b a c t e r i a may be l i m i t e d by some other r e q u i r e d n u t r i e n t . M i c r o f l a g e l l a t e s on day 2 reached 18.5 x 10 3 c e l l s / m l and then d e c l i n e d to 9.4 x 10 3 c e l l s / m l . T h i s d e c r e a s i n g p o p u l a t i o n shows a response to the i n c r e a s e of b a c t e r i a a t day 4-5. T h i s i n d i c a t e s t h a t t h i s f l a g e l l a t e p o p u l a t i o n was food source l i m i t e d and then was a l s o removed by other causes. There 56 are two obvious causes f o r the d e c l i n e : ( D a non-growing p o p u l a t i o n may be s t r o n g l y a f f e c t e d by d i l u t i o n and (2) some l a r g e c i l i a t e s such as T i n t i n n i d a e spp. were common i n the experiment. They may have been be r e s p o n s i b l e f o r the d e c l i n e i n the f l a g e l l a t e s ( S p i t t l e r 1973; Stoecker, 1981). on day 15, as t h e n a n o z o o f l a g e l l a t e s i n c r e a s e d , the b a c t e r i a decreased. Summary of experiment Stage I Both f r e s h water and sea water phytoplankton grew very w e l l i n the c o n t r o l l e d f l a s k s and e x p o n e n t i a l growth appeared a t day 2. There was almost no l a g phase; N0~3 decreased s h a r p l y to zero. The d e c l i n e of biomass was caused by n u t r i e n t l i m i t a t i o n ( F i g . 13, 14). The s p e c i e s composition was i n agreement with o t h e r r e s e a r c h ( S p i e s , 1984; Stockner, 1977). Dominant s p e c i e s i n the f r e s h water and sea water are t o t a l l y d i f f e r e n t . In the sea water c o n t r o l , almost 90% of the biomass i s a t t r i b u t e d t o Skeletonema costatum. In f r e s h water, the major s p e c i e s was T h a l a s s i o s i r a spp. The maximum c h l o r o p h y l l a i n sea water and f r e s h water were q u i t e d i f f e r e n t ; t h i s was mainly due to the i n i t i a l n u t r i e n t c o n c e n t r a t i o n s being d i f f e r e n t ( f r e s h water, 8.83 ug NO"3/l; sea water, 24.19 ugNO~3/l). However, both systems developed phytoplankton blooms and the development p a t t e r n s were very s i m i l a r . But the de v e l o p i n g p a t t e r n of b a c t e r i a i n the freshwater system showed an i n t e r e s t i n g d i f f e r e n c e d u r i n g the f i r s t p a r t of the experiment (e.g. d u r i n g e x p o n e n t i a l growth phase of the phytoplankton) compared with t h a t of the seawater system. The b a c t e r i a l p o p u l a t i o n showed a sma l l i n c r e a s e , not completely i n h i b i t e d by the growth of 57 autotrophs as they d i d i n the seawater system, even though both of them were maximal a f t e r the d e g r a d a t i o n of phytoplankton. T h i s may have been due to s p e c i e s composition being d i f f e r e n t i n the two systems. Comparing experimental v e s s e l s with the c o n t r o l , i t becomes obvious t h a t the growth p a t t e r n s were d i f f e r e n t . Not o n l y was the biomass very low d u r i n g the same time p e r i o d s i n p a r t 1 and 2, but a v e r y l o n g l a g p e r i o d a l s o appeared i n both due to the s a l i n i t y impact and to d i l u t i o n p r o c e s s . The r e s u l t s i n both p a r t s 1 and 2 i n d i c a t e t h a t freshwater s p e c i e s can not continue growing or s t a r t growing at s a l i n i t i e s higher than 10°/oo. I t i s s a f e to conclude t h a t f r e s h water phytoplankton can not a c t i v e l y f u n c t i o n on the s a l i n i t y g r a d i e n t as the primary a u t o t r o p h i c component; i n s t e a d they may undergo l y s i s and r e l e a s e of c e l l u l a r c o n t e n t s , or they may form r e s t i n g spores or be predated. Fresh water s p e c i e s may s t i l l be c u l t u r e d a f t e r some ad a p t a t i o n s as, f o r example, i n W e t h e l l ' s work (1961). However, most p a r t i a l l y mixed e s t u a r i e s such as the F r a s e r R i v e r e s t u a r y have r e s i d e n c e times of l e s s than 10 days. Fresh water phytoplankton may q u i c k l y go through a l l the s a l i n i t y c o n c e n t r a t i o n s and t h i s sudden impact may i n h i b i t s p e c i e s a d a p t a t i o n and germination and r e s u l t i n high m o r t a l i t y . T h i s suggests t h a t e s t u a r i e s may f u n c t i o n to c o n v e r t most f r e s h water p l a n k t o n i c organisms (except h e t e r o t r o p h i c b a c t e r i a ) to h e t e r o t r o p h i c u t i l i z a b l e components which then go through the b a c t e r i a l pathway. Data f o r h e t e r o t r o p h i c components f o r two d i f f e r e n t 58 s a l i n i t y g r a d i e n t s seems t o suggest t h a t b a c t e r i a p o p u l a t i o n s are mainly c o n t r o l l e d by two parameters: 1) by u t i l i z a b l e o r g a n i c s u b s t r a t e s (both c o n c e n t r a t i o n s and r a t e s of pr o d u c t i o n ) and 2) by h e t e r o t r o p h i c m i c r o f l a g e l l a t e s . The a c t i v i t y of the h e t e r o t r o p h i c component may not be as i n f l u e n c e d by s a l i n i t y as t h a t of f r e s h water phytoplankton. T h i s may be due t o a c l o s e s u c c e s s i o n i n growth between f r e s h water and sea water b a c t e r i a or because f r e s h water b a c t e r i a u s u a l l y growing b e t t e r i n i n t e r m e d i a t e s a l i n i t i e s ( S p i e s , 1984). The development and arrangement of the e c o l o g i c a l components are very d i f f e r e n t i n experimental systems ( i . e . changing s a l i n i t y ) compared with the c o n t r o l l e d systems ( i . e . s t a b l e s a l i n i t y ) . C o n t r o l l e d systems are a homogenous e c o l o g i c a l system, i n which the c o n d i t i o n i s a prime s t a t e c o n t a i n i n g the lowest system energy. I t i s a s t a b l e s t a t e f o r a system. T h i s c o n d i t i o n favours phytoplankton growth with a d e c l i n e of b a c t e r i a d u r i n g a phytoplankton bloom sugge s t i n g a slow down of energy and m a t e r i a l s f l o w i n g through the h e t e r o t r o p h i c pathway. In the c o n t r o l systems, b a c t e r i a d i d not show a c o - i n c r e a s e with phytoplankton e x p o n e n t i a l growth as they d i d i n the experimental v e s s e l s , but the maximum b a c t e r i a l biomass s h i f t e d t o the degr a d a t i o n phase of the phytoplankton ( F i g . 13,14). T h i s s e p a r a t i o n of two components over time p r o v i d e s the phytoplankton with the l a r g e s t space and m a t e r i a l s supply ( i . e . econiche) i n the system. T h i s c r e a t e s the l a r g e s t i n p u t of l i g h t energy ( e x t e r n a l energy) i n t o ecosystem (system energy). However, i n experimental v e s s e l s , the s a l i n i t y 59 g r a d i e n t c r e a t e s a heterogeneous c o n d i t i o n over time. Phytoplankton growth was i n h i b i t e d a t some p a r t of the g r a d i e n t . T h i s l e f t some ecosystem space which was used by h e t e r o t r o p h i c b a c t e r i a . As a r e s u l t , the b a c t e r i a showed a c o e v o l u t i o n with phytoplankton. These two d i f f e r e n t s t r u c t u r e s under d i f f e r e n t p h y s i c a l environmental c o n d i t i o n s may r e f l e c t the f u n c t i o n a l response of an ecosystem to achieve the l a r g e s t ecosystem e f f i c i e n c y . The e c o l o g i c a l s i g n i f i c a n c e of the d i f f e r e n t behaviour of phytoplankton and h e t e r o t r o p h i c components on the s a l i n i t y g r a d i e n t (or a heterogeneous c o n d i t i o n a l g r a d i e n t ) suggests an e c o l o g i c a l e v o l u t i o n of e s t u a r i n e ecosystems. The phytoplankton can not occupy a c e r t a i n space i n the g r a d i e n t . However, i f t h i s space c o n t a i n s a high system energy (e.g. o r g a n i c substances) the h e t e r o t r o p h i c components may s u c c e s s f u l l y u t i l i z e i t . T h i s i s what happens at the head of many e s t u a r i e s i n which t h e r e i s more o r g a n i c input than i n any other a q u a t i c environment. The ecosystem should have a s t r a t e g y to e x p l o i t t h i s energy source. So, the h e t e r o t r o p h i c component must p l a y a more important r o l e than i n any other k i n d of a q u a t i c environment. 60 4.2 Experiment Stage I I As was shown i n experimental stage I , the f r e s h water a u t o t r o p h i c components are not the major f u n c t i o n a l a u t o t r o p h i c group on the s a l i n i t y g r a d i e n t i n the e s t u a r i n e ecosystem. Thus i f e s t u a r i n e systems remain a phytoplankton energy input pathway (e.g. phytoplankton based food c h a i n ) , the sea water phytoplankton should p l a y a dominant r o l e i n e s t u a r i e s p rovided t h a t the l i g h t i n t e n s i t y g r a d i e n t (determined by sedimental l o a d i n g and r e s i d e n c e time coupled with e s t u a r i n e c i r c u l a t i o n ) permits the growth of the phytoplankton. The experiment stage I I was designed to observe the behaviour of marine e c o l o g i c a l components on a temporal s a l i n i t y g r a d i e n t . The o r i g i n a l marine e c o l o g i c a l components go along the g r a d i e n t a t a constant d i l u t i o n r a t e s i m u l a t i n g the process which happens t o the sea water e c o l o g i c a l components on the s u r f a c e l a y e r d u r i n g e s t u a r i n e mixing. In the p r o c e s s , marine ecosystem p l a n k t o n i c components are brought up to the e u p h o t i c l a y e r by entrainment from below where i t i s dark. Simultaneously, a s a l i n i t y g r a d i e n t e x i s t s i n the s u r f a c e water over time and over d i s t a n c e . The experiments were a l s o designed i n t h r e e p a r t s . 4.21 I n c r e a s i n g s a l i n i t y g r a d i e n t In p a r t 1, which was designed with an i n c r e a s i n g s a l i n i t y g r a d i e n t , o r i g i n a l sea water was pumped i n t o l i g h t e d e xperimental v e s s e l s (volume, 61) from a covered r e s e r v o i r to s i m u l a t e the i n c r e a s i n g s a l i n i t y g r a d i e n t over time which occurs i n a p a r t i a l l y mixed e s t u a r y . A sea water seed p o p u l a t i o n was allowed t o flow i n t o a body of f i l t e r e d f r e s h « 0.20 o c U 0.15-1 1 0.10-1 u . J 0.05] k. OjOO o * * a C / \ ^ m 6 Days 8 10 2 0 ^ H 5 ° 10 I Oi 12 •50 J z 0.0 F i g u r e 15. The development p a t t e r n of phytoplankton and co n c e n t r a t i o n of n u t r i e n t s i n stage II p a r t 1 on an i n c r e a s i n g s a l i n i t y g r a d i e n t d u r i n g Aug. 1985. F i g u r e 16a. The development p a t t e r n of the a u t o t r o p h i c component and c o n c e n t r a t i o n of n u t r i e n t s i n stage II o> pa r t 1 i n an i n c r e a s i n g s a l i n i t y g r a d i e n t d u r i n g A p r i l 1985. 10 —T" 15 — i 20 5 D a y s F i g u r e 16b. The development p a t t e r n of NH +4 i n stage II pa r t 1 i n an i n c r e a s i n g s a l i n i t y g r a d i e n t d u r i n g A p r i l , 1985. 64 water i n an i n c r e a s i n g s a l i n i t y g r a d i e n t a t a c e r t a i n d i l u t i o n r a t e ( D = 0.16 / day ) ( F i g . 4) The e v o l u t i o n p a t t e r n of the a u t o t r o p h i c and n u t r i e n t (NO~3 + NO~2> components are i n d i c a t e d i n F i g . 15. During the f i r s t two days, the high c h l o r o p h y l l a v a l u e was a r e s u l t of some smal l f r e s h water a u t o t r o p h i c organisms (<0.8 um) which grew because of the e l i m i n a t i o n of g r a z i n g by z o o f l a g e l l a t e s d u r i n g f i l t r a t i o n . T h i s s m a l l c h l o r o p h y l l a peak decreased to zero with the i n f l o w of sea water. S e v e r a l mechanisms may work i n t h i s p r o c e s s : osmotic s t r e s s , sedimentation due t o a change i n s u r f a c e charge, and d i l u t i o n by sea water c o n t a i n i n g low b a c t e r i a and high n a n o f l a g e l l a t e c o n c e n t r a t i o n s . C h l o r o p h y l l a d i d not i n c r e a s e u n t i l day 5 ( s a l i n i t y 15°/oo). T h i s prolonged l a g phase compared wi t h " c o n t r o l v e s s e l s " can be a t t r i b u t e d t o the i n h i b i t i o n e f f e c t of low s a l i n i t y on the germination of seed p o p u l a t i o n s and on the growth of the growing p o p u l a t i o n as w e l l as to the d i l u t i o n r a t e . F i g u r e 18 shows the l o g transformed data of c h l o r o p h y l l a i n c r e a s i n g at d i f f e r e n t constant s a l i n i t i e s . The growth of marine phytoplankton was a p p a r e n t l y i n h i b i t e d a t low s a l i n i t y . As the s a l i n i t y i n c r e a s e d , toward an o p t i m a l c o n d i t i o n , the growth r a t e became f a s t e r by day 5. The c h l o r o p h y l l a s t a n d i n g stock i n c r e a s e d e x p o n e n t i a l l y t h e r e a f t e r . By t h i s time, the s a l i n i t y g r a d i e n t spectrum was w i t h i n the o p t i m a l range f o r marine phytoplankton growth, the g r a d i e n t no longer s u s t a i n e d a heterogeneous c o n d i t i o n so the whole system s h i f t e d t o a phytoplankton p r i o r i t y system. At the beginning, ( F i g . 17) b a c t e r i a had a high d e n s i t y F i g u r e 17. The development p a t t e r n of b a c t e r i a and n a n o z o o f l a g e l l a t e s i n stage I I , p a r t 1 with i n c r e a s i n g s a l i n i t y g r a d i e n t d u r i n g Aug., 1985. 66 F i g u r e 18. Developement of the a u t o t r o p h i c component i n a subsample c u l t u r e s e r i e s at t e m p o r a l l y constant s a l i n i t y i n stage I I , p a r t 1 d u r i n g Aug. 1985. which may be composed of high c y a n o b a c t e r i a as i n d i c a t e d by high c h l o r o p h y l l a valu e s . A f t e r day 2 i t d e c l i n e d . B a c t e r i a l d e n s i t y reached i t s peak d u r i n g the f i r s t p e r i o d before phytoplankton s t a r t e d growing (by day 6 ) . The maximum d e n s i t y appeared a t day 4, then s l o w l y decreased u n t i l the end of the experiment. B a c t e r i a d i d not show a c l o s e c o r r e l a t i o n with the growth of phytoplankton a f t e r day 6 (see Table 3) as i n a l l other experiments. But the p a t t e r n may be e x p l a i n a b l e by the c o v a r i a t i o n of the m i c r o f l a g e l l a t e e v o l u t i o n p a t t e r n ( F i g . 17) and the i n h i b i t i o n e f f e c t before a f u l l phytoplankton bloom as i n the c o n t r o l l e d experiment ( F i g . 13). The lower d e n s i t y of n a n o z o o f l a g e l l a t e s i s expected a t the beginning because of the e f f e c t of f i l t r a t i o n . F l a g e l l a t e s s t a r t e d growing from day 2 u n t i l day 6, when the f i r s t peak was reached. The e x p o n e n t i a l i n c r e a s e i n the m i c r o f l a g e l l a t e p o p u l a t i o n i n d i c a t e d a gre a t b a c t e r i a l biomass demand. To meet t h i s demand, b a c t e r i a should a l s o have undergone a g r e a t i n c r e a s e i n biomass, i n d i c a t i n g a high b a c t e r i a l a c t i v i t y i n the f i r s t p e r i o d . The second i n c r e a s e of m i c r o f l a g e l l a t e s from day 7 to the end of the experiment may be r e s p o n s i b l e f o r the l a c k of c o r r e l a t i o n of b a c t e r i a with phytoplankton growth. The i n c r e a s e of b a c t e r i a by u t i l i z a t i o n of the p h o t o s y n t h e t i c products r e l e a s e d by phytoplankton may be o f f s e t by the n a n o z o o f l a g e l l a t e g r a z i n g . The i n c r e a s e i n b a c t e r i a l d e n s i t y i s i n d i r e c t l y r e f l e c t e d i n the i n c r e a s e i n n a n o f l a g e l l a t e abundance. The d i e l and p e r i o d i c r e l e a s e of o r g a n i c products by phytoplankton (Burney e t a l . , 1981, 1982; E b e r l e i n , 1983; Hammer, 1983; Kaplan, 1982; Sournia, 68 Table 5. C o r r e l a t i o n matrix o b t a i n e d from the 6 v a r i a b l e s X 12 o b s e r v a t i o n s i n experiment stage I I , p a r t 1 d u r i n g Aug., 1985, between time (day), F l u o r e s c e n c e ( f l u o ) , N0~3, F l a g e l l a t e s ( f i g ) , B a c t e r i a (bac), and s a l i n i t y . P r o b a b i l i t y : * p<0.1, ** p < 0.05, *** p < 0.02, **** p <0.01. I DAY 1 FLUO J N03 I FLG 1 1 BACT DAY | 1.000 i j j j FLUO 0.539* 1.000 N03 -0.938**** -0.553* 1.000 FLG 0.500* -0.230 -0.417 1.000 BACT -0.381 -0.409 0.411 0.249 1 .000 SAL 0.983**** 0.380 -0.900**** 0.610** -0 .319 Table 6. C o r r e l a t i o n matrix o b t a i n e d from the 6 v a r i a b l e s X 12 o b s e r v a t i o n s i n experiment stage I I , p a r t 2 d u r i n g Aug., 1985, between time (day), F l u o r e s c e n c e ( f l u o ) , N0~3, F l a g e l l a t e s ( f i g ) , B a c t e r i a (bac), and s a l i n i t y . P r o b a b i l i t y : * p<0.1, ** p < 0.05, *** p < 0.02, **** p <0.01. DAY FLUO N03 FLG BACT DAY 1.000 FLUO 0.857**** 1.000 NO 3 -0.934**** -0.816**** 1.000 FLG -0.495* -0.264 0.654*** 1 .000 BACT -0.177 0.136 0.018 0 .067 1.000 SAL -0.983**** -0.821**** 0.971**** 0 .630** 0.166 69 1974) may c r e a t e s i t u a t i o n s i n which b a c t e r i a are t e m p o r a r i l y l i m i t e d by s u b s t r a t e , so the e x p l o i t a t i o n by n a n o z o o f l a g e l l a t e s may show a net r e d u c t i o n e f f e c t . T able 5 shows the c o r r e l a t i o n c o e f f i c i e n t between the v a r i a b l e s ; b a c t e r i a d i d not show any s i g n i f i c a n t c o r r e l a t i o n c o e f f i c i e n t with f l u o r e s c e n c e as i n stage I experiment. 4.22 Decreasing s a l i n i t y g r a d i e n t In p a r t 2, the o r i g i n a l sea water was h e l d i n experimental v e s s e l s and d i l u t e d with f i l t e r e d f r e s h water. T h i s r e s u l t e d i n a d e c r e a s i n g s a l i n i t y g r a d i e n t over time f o r the marine e c o l o g i c a l m i c r o b i a l component and f r e s h water b a c t e r i a . The development of marine phytoplankton ( F i g . 19) shows a two day l a g phase, compared wi t h the " c o n t r o l l e d v e s s e l s " . The e x t r a one day l a g may have been due to d i l u t i o n (D = 0.16 d - 1 ) , and the p e r t u r b a t i o n of d e c r e a s i n g s a l i n i t y may a l s o a f f e c t the germination of seed p o p u l a t i o n s . The op t i m a l s a l i n i t y f o r many seawater s p e c i e s , i n c l u d i n g Skeletonema was r e p o r t e d a t a lower s a l i n i t y range so the growth at the beginning may be slower r e l a t i v e t o the d i l u t i o n r a t e . The e x p o n e n t i a l growth phase s t a r t e d a t day 3. Biomass i n c r e a s e d very q u i c k l y and reached i t s peak by day 6. The d e p l e t i o n of n i t r o g e n by day 6 stopped the phytoplankton biomass i n c r e a s e d ( F i g . 19) and the osmotic s t r e s s • and d i l u t i o n may have caused the d e c l i n e of c h l a sta n d i n g stock through the death and l y s i s of the c e l l s . T h i s i n t u r n may have speeded up the b a c t e r i a l a c t i v i t y . The i n f l o w of f r e s h water i n t r o d u c e d some n u t r i e n t s (17.5 ug-at N t o 6 1 of water), which may have caused the prolonged p l a t e a u phase Figure 19. The development p a t t e r n of the a u t o t r o p h i c component and c o n c e n t r a t i o n of n u t r i e n t s i n stage I I , p a r t 2 ona de c r e a s i n g s a l i n i t y g r a d i e n t d u r i n g Aug. 1985. o D a y s Pigure 20b. The development p a t t e r n of NH+4 i n stage I I , pa r t 2 on a d e c r e a s i n g s a l i n i t y g r a d i e n t d u r i n g A p r i l , 1985. 73 observed d u r i n g the Aug. 1985 experiment ( F i g . 19), but the slow d e c l i n e r a t e compared with the " c o n t r o l v e s s e l s " i n d i c a t e s t h a t at l e a s t p a r t of the biomass was i n a chemostat s t a t e , i f the n u t r i e n t f l u x was l a r g e enough as i n the experiment c a r r i e d out i n A p r i l , i n which f r e s h water c o n t a i n e d much higher n u t r i e n t s ( F i g . 20a). The l a r g e biomass s t a n d i n g stock may be s u s t a i n e d i n a t e m p o r a l l y balanced s t a t e . T h i s i m p l i e s t h a t , i n the mouth of an e s t u a r y , c e r t a i n c o n d i t i o n s may e x i s t b r i e f l y as i n a n a t u r a l chemostat environment which p r o v i d e s longer p e r i o d s of high s t a n d i n g stock biomass; t h i s may b e n e f i t the zooplankton l a r v a l f e e d i n g and may promote the carbon t r a n s p o r t e f f i c i e n c y r e s u l t i n g i n a high secondary p r o d u c t i o n (Parsons e t a l . , 1984). H e t e r o t r o p h i c b a c t e r i a have a very s t r o n g a b i l i t y t o a s s i m i l a t e i n o r g a n i c n u t r i e n t s ( N i c h o l a s , 1963; P a i n t e r , 1970). With a s u f f i c i e n t source of o r g a n i c carbon b a c t e r i a can s u c c e s s f u l l y compete with the autotrophs f o r i n o r g a n i c n u t r i e n t s . Most of the o r g a n i c products r e l e a s e d d u r i n g p h o t o s y n t h e s i s are carbohydrates so b a c t e r i a need nitrogenous n u t r i e n t s f o r the s y n t h e s i s of amino a c i d s . The g r e a t amount of o r g a n i c carbon r e l e a s e d a t the p l a t e a u phase may q u i c k l y be used by h e t e r o t r o p h i c b a c t e r i a (Larson and Hagstrom, 1979; M a r t i n , 1980; Wolter, 1982). T h i s i n t u r n speeds up the c o m p e t i t i o n f o r i n o r g a n i c n u t r i e n t s and the death of phytoplankton. T h i s c h a i n r e a c t i o n a f t e r e x p o n e n t i a l growth favours b a c t e r i a l a c t i v i t y and i n c r e a s e s the r a t e of m a t e r i a l s r e c y c l i n g . The e v o l u t i o n of n i t r o g e n (NO~3 + NO~2) was not at f i r s t v e ry t i g h t l y coupled with the growth of phytoplankton and 74 with the d i l u t i o n . The d e c l i n e of (N0~3 + N0~2> was l a r g e r than the d i l u t i o n e f f e c t . The b a c t e r i a l growth d u r i n g t h i s p e r i o d may have caused the d e c l i n e of n i t r o g e n n u t r i e n t s but t h i s uptake by b a c t e r i a seemed t o be stopped by the d e p l e t i o n o f a v a i l a b l e o r g a n i c carbon s o u r c e s . The continued d e c l i n e i n n i t r o g e n c o n c e n t r a t i o n was o b v i o u s l y due to phytoplankton growth from day 3 to day 6. By day 6, nitrogenous n u t r i e n t s (N0~3 + N0 _2> were d e p l e t e d . Some phytoplankton s p e c i e s were prese n t i n the experimental water, such as N i t z s c h i a spp., T h a l a s s i o s i r a spp., Ditylum spp., Chaetoceros spp. and A s t e r i o n e l l a spp., but a l l were r a r e . The major s p e c i e s was Skeletonema costatum, which became e s p e c i a l l y dominant at the end of the experiment. T h i s may have been due to c o m p e t i t i o n and d i l u t i o n r a t e s e l e c t i o n e f f e c t s as shown by H a r r i s o n and Davis (1978). Subsamples were taken every day and c u l t u r e d i n 25 x 150 mm b o r o s i l i c a t e c u l t u r e tubes i n s e r i e s i n order t o d e t e c t the growth or germination of these phytoplankton under a constant s a l i n i t y i n a batch c u l t u r e s t a t e . F i g u r e 22 i n d i c a t e s t h a t the n a t u r a l assemblage of phytoplankton had an op t i m a l s a l i n i t y of about 15°/oo i n our experiments. T h i s i s c o n s i s t e n t with the r e s u l t s of ot h e r s which i n d i c a t e t h a t most c o a s t a l s p e c i e s of phytoplankton from d i f f e r e n t areas of the world show maximum growth a t low s a l i n i t i e s , (15 - 25°/oo) i . e . lower than normal sea water. (Brand, 1984, 1982; Qasim e t a l . , 1972). Skeletonema i s a common e u r y h a l i n e p l a n k t o n i c diatom, o p t i m a l growth seems t o occur a t s a l i n i t i e s of (15-25%) (Braarud, 1951; o o o CO F i g u r e 21. The development p a t t e r n of b a c t e r i a and n a n o z o o f l a g e l l a t e s i n stage I I , p a r t 2 on a de c r e a s i n g s a l i n i t y g r a d i e n t d u r i n g Aug., 1985. 77 Paasche, 1975; Brand, 1984), which i s c o n s i s t e n t with my data. F i g u r e 21 shows the c o e v o l u t i o n curve of h e t e r o t r o p h i c b a c t e r i a and n a n o z o o f l a g e l l a t e s . The higher n a n o f l a g e l l a t e and lower b a c t e r i a l biomass at the beginning was q u i t e o b v i o u s l y c o n s i s t e n t with t h a t of other experiments. T h i s may i n d i c a t e the lowest t h r e s h o l d of g r a z i n g on b a c t e r i a by n a n o z o o f l a g e l l a t e s . B a c t e r i a l biomass reached i t s f i r s t peak before phytoplankton s t a r t e d growing but decreased s h a r p l y when the phytoplankton began e x p o n e n t i a l growth. T h i s sharp decrease of b a c t e r i a was a l s o shown i n Spies* work (1984) and the data i n the c o n t r o l v e s s e l ( F i g . 13, 14). These s i m i l a r r e s u l t s i n d i f f e r e n t s t u d i e s a t d i f f e r e n t times suggest t h a t the r e s u l t s i n e v i t a b l y occur when fa v o u r a b l e c o n d i t i o n s l e a d t o phytoplankton blooms. Low n a n o z o o f l a g e l l a t e d e n s i t i e s which occur d u r i n g t h i s p e r i o d do not seem to be r e s p o n s i b l e the f o r r e d u c t i o n i n b a c t e r i a . The mutual c o n t r o l of b i o l o g i c a l a c t i v i t y by e x t e r n a l m e t a b o l i t e s i s an important r e l a t i o n s h i p between phytoplankton and b a c t e r i a . The s p e c i f i c i n t e r a c t i o n may be mediated through p r o d u c t i o n of s t i m u l a t i v e substances, the e x c r e t i o n of i n h i b i t o r y substances, or through c o m p e t i t i o n ( S i e b u r t h , 1959, 1968). B e l l e t a l . (1974) r e p o r t e d a s e l e c t i v e s t i m u l a t i o n and i n h i b i t i o n of b a c t e r i a by Skeletonema costatum. Kogure (1979) confirmed t h i s r e s u l t with d i r e c t evidence t h a t S. costatum has an i n h i b i t o r y e f f e c t on Pseudomonas and V i b r i o spp. and a s t i m u l a t o r y e f f e c t on F l a v o b a c t e r i u m spp. d u r i n g e x p o n e n t i a l phase. T h i s a n t i b a c t e r i a l a c t i o n b a s i c a l l y depends upon the a l g a l growth c o n d i t i o n s . The s t r o n g e s t a n t i b a c t e r i a l a c t i v i t y was observed d u r i n g the e x p o n e n t i a l 78 phase of a l g a l growth. M a r t i n (1980) showed t h a t the b a c t e r i a p o p u l a t i o n d u r i n g the e v o l u t i o n may s h i f t i t s composition with a s u c c e s s i o n of d i f f e r e n t g e n e r i c groups t o adapt t o the d i f f e r e n t p h y s i o l o g i c a l s t a t e s of phytoplankton (Skeletonema  costatum). The dominant b a c t e r i a l s p e c i e s d u r i n g the e x p o n e n t i a l growth phase of phytoplankton and the beginning of the c h l a p l a t e a u was d i f f e r e n t from t h a t found d u r i n g the phytoplankton death p e r i o d . M a r t i n found t h a t V i b r i o - l i k e organisms are more important l a t e r , p a r t i c u l a r l y when plankton m o r t a l i t y o c c u r s . T h i s i s i n agreement with work done by S i e b u r t h (1968) and Simidu et a l . (1971). C o n s i d e r i n g the n u t r i t i o n a l mode, the e x t r a c e l l u l a r products of phytoplankton d u r i n g e x p o n e n t i a l growth are mostly carbohydrates ( E b e r l e i n , e t a l . , 1984). B a c t e r i a which use these as t h e i r o n l y carbon source must take up n i t r o g e n . During t h i s p e r i o d , the n i t r o g e n sources were not d e p l e t e d . However, the b a c t e r i a which grow a f t e r the p l a t e a u phase have no ambient m i n e r a l n i t r o g e n source and most l i k e l y u t i l i z e amino a c i d s (Amano e t a l . , 1982, B r i g h t , 1983). T h i s a l s o suggests t h a t a d i f f e r e n t n u t r i t i o n a l mode may d i c t a t e d i f f e r e n t s p e c i e s groups. In my experiment, b a c t e r i a growing d u r i n g the f i r s t p e r i o d were l a r g e l y f r e e - l i v i n g w h i l e a t t a c h e d b a c t e r i a dominated a f t e r phytoplankton e x p o n e n t i a l growth. The l a t t e r were mostly a t t a c h e d t o diatom f r u s t l e s , and were b i g and rod shaped. In n a t u r a l waters, f r e e - l i v i n g b a c t e r i a and a t t a c h e d b a c t e r i a may have d i f f e r e n t m o r p h o l o g i c a l p r o p e r t i e s and a l s o may r e q u i r e d i f f e r e n t n u t r i t i o n a l modes of d i s s o l v e d o r g a n i c s u b s t r a t e s (Fukami e t a l . , 1985, 1983,1981). The two peaks which developed i n t h i s p a r t of the experiment may r e f l e c t t h i s s h i f t i n c o mposition. Phytoplankton may e x c r e t e i n h i b i t o r y substances to decrease f r e e - l i v i n g b a c t e r i a which may be p o t e n t i a l n u t r i e n t c ompetitors and to s t i m u l a t e another s t r a i n of b a c t e r i a l growth. The f a c t t h a t b a c t e r i a l d e n s i t y decreased before n u t r i e n t d e p l e t i o n i n d i c a t e s t h a t n u t r i e n t c o m p e t i t i o n was not the d i r e c t mechanism f o r the b a c t e r i a l d e c l i n e before the phytoplankton bloom. Kogure (1979) showed t h a t the a d d i t i o n of N and P d i d not i n f l u e n c e the i n h i b i t o r y e f f e c t and concluded t h a t s u p p r e s s i o n of b a c t e r i a l growth was not a r e s u l t of c o m p e t i t i o n . He a l s o found t h a t higher and lower l i g h t i n t e n s i t i e s decreased the a n t i b a c t e r i a l a c t i v i t y . T h i s may be due to the l i g h t i n h i b i t i o n and l i g h t l i m i t a t i o n which reduce the growth of p hytoplankton. T h i s may e x p l a i n why the i n h i b i t o r y e f f e c t d i d not occur i n the stage I experiment i n which the growth of phytoplankton was i n h i b i t e d by the s a l i n i t y g r a d i e n t . Thus th e r e must e x i s t a more complex dynamic s t a t e of i n t e r a c t i o n s between b a c t e r i a and phytoplankton. T h i s phenomena i n d i c a t e s t h a t under the c o n d i t i o n s of an a u t o t r o p h i c bloom, h e t e r o t r o p h i c pathways are shut down to g i v e way to the u t i l i z a t i o n of m utually r e q u i r e d n u t r i e n t s f o r a u t o t r o p h i c p r o d u c t i o n . Then the whole system becomes an a u t o t r o p h i c p r i o r i t y system. What i s the e f f e c t of t h i s i n t e r a c t i o n on m i c r o f l a g e l l a t e s ? 80 There i s no immediate answer t o t h i s q u e s t i o n , s i n c e they are a t a higher t r o p h i c l e v e l and depend on b a c t e r i a , they must be a f f e c t e d . The constant low l e v e l of n a n o z o o f l a g e l l a t e s u n t i l day 6 i s q u i t e d i f f e r e n t from other experiments. The d e c l i n e of the b a c t e r i a l p o p u l a t i o n ( F i g . 21) d u r i n g f i r s t p e r i o d may prevent the r e s t o r a t i o n of the n a n o z o o f l a g e l l a t e s . The r e s u l t s i n t h i s experiment show the q u i t e d i f f e r e n t responses of seawater phytoplankton p o p u l a t i o n s t o a s a l i n i t y g r a d i e n t . Seawater p o p u l a t i o n s may have a wide range of ad a p t a t i o n s t o s a l i n i t y but they can be i n h i b i t e d on the low s a l i n i t y range (<10°/oo). Subsample c u l t u r e s show t h a t below 10 o/oo of s a l i n i t y , the seed p o p u l a t i o n d i d not grow durin g the same p e r i o d of time as i n the experimental f l a s k s . They can not a c t i v e l y f u n c t i o n or germinate i n the low s a l i n i t y p o r t i o n of the g r a d i e n t . T h i s suggests t h a t seawater p o p u l a t i o n s can f l o u r i s h o n l y a t the higher end of s a l i n i t y spectrum. The r e s u l t s i n these two p a r t s of the experiments suggest t h a t b a c t e r i a occupy the f i r s t p e r i o d of the experiment and phytoplankton occupy the l a s t p a r t . In other words, b a c t e r i a adapted t o the low p a r t of the s a l i n i t y g r a d i e n t , w h i l e phytoplankton adapted t o the high p a r t of s a l i n i t y g r a d i e n t . In e x t r a p o l a t i n g t h i s r e s u l t t o an e s t u a r y , i t means t h a t the h e t e r o t r o p h i c b a c t e r i a occupy the low s a l i n i t y water at the head of the e s t u a r y , and phytoplankton s t a n d i n g stock appears g r e a t e r at the mouth of the e s t u a r y . T h i s i s i n agreement with many f i e l d o b s e r v a t i o n s ( A l b r i g h t , 1983; B e l l e t a l . , 1981; Wright, 81 1984, 1985). C o r r e l a t i o n c a l c u l a t i o n (Table 6) shows a higher s i g n i f i c a n t c o e f f i c i e n t between time, f l u o , N0~3, and s a l i n i t y . T h i s i s expected because of the development of the phytoplankton. However, the c o r r e l a t i o n between b a c t e r i a and phytoplankton d i d not appear i n t h i s p a r t of the experiment. The c o n t r o l s had two c u l t u r e v e s s e l s c o n t a i n i n g o r i g i n a l f r e s h water and sea water r e s p e c t i v e l y . The e v o l u t i o n p a t t e r n of phytoplankton assemblages were s i m i l a r i n these two f l a s k s ( F i g . 23a,b). The major s p e c i e s of the c h l o r o p h y l l maximum were Skeletonema costatum and T h a l a s s i o s i r a spp. i n sea water and f r e s h water r e s p e c t i v e l y . The maxima of biomass i n these two f l a s k s are q u i t e d i f f e r e n t ; t h i s may r e s u l t from the i n i t i a l n i t r a t e c o n c e n t r a t i o n d i f f e r e n c e . Sea water c o n t a i n s 35 ug-at N/1, while f r e s h water c o n t a i n s 17.5 ug-at N/1. The d e c l i n e of standing stock a f t e r n u t r i e n t d e p l e t i o n i s due t o s i n k i n g t o the bottom (even though I used a s t r o n g magnetic s t i r r e r ) and a l s o due to deg r a d a t i o n by h e t e r o t r o p h i c b a c t e r i a ( F i g . 13,14). o Q£ 0»0 i i i i i i I i I i i » i I i ' 0 5 10 |5 20 D a y s Figure 23a. The development p a t t e r n of the a u t o t r o p h i c component i n a c o n t r o l l e d system with o r i g i n a l seawater dur i n g Aug. 1985. 0 3.0i ;ence 25 20 u. \& > 1.0 05' rr 00 10 D a y s 15 20 Figure 23b. The development p a t t e r n of the a u t o t r o p h i c component i n a c o n t r o l l e d system with o r i g i n a l freshwater d u r i n g Aug. 1985. 00 84 D i s s c u s s i o n There i s s u r p r i s i n g l y l i t t l e i n f o r m a t i o n on the ecosystem s t r u c t u r e of f r e s h water e n t e r i n g the sea or of the e c o l o g i c a l r o l e of the f r e s h water phytoplankton component i n an e s t u a r i n e ecosystem. Some work has been r e p o r t e d on f r e s h water phytoplankton i n e s t u a r i e s and s a l i n e water environments (Blanc et a l . , 1968; W e t h e r e l l , 1961; F o e s t e r , 1973), but they focus more on the p h y s i o l o g i c a l s t a t e of these phytoplankton groups than on the e c o l o g i c a l r o l e i n the e s t u a r i n e ecosystem. Blanc et a l . (1969) concluded t h a t f r e s h water phytoplankton i n d i l u t e d sea water (8°/oo - 26 °/oo) were mostly dead or almost dead. F o e s t e r (1973) i n a more d e t a i l e d study, concluded t h a t freshwater phytoplankton can s u r v i v e i n d i l u t e d s a l i n e waters. I t i s an important matter whether freshwater phytoplankton can s t i l l p l a y an a u t o t r o p h i c r o l e i n e s t u a r i n e ecosystems. Can they pass through the s a l i n i t y g r a d i e n t with some normal f u n c t i o n s ? I f not , what i s the e f f e c t of t h i s d i s f u n c t i o n of phytoplankton on the whole ecosystem s t r u c t u r e . The r e s u l t s i n my experiment (stage I ) c l e a r l y demonstrated t h a t freshwater phytoplankton can not a c t i v e l y f u n c t i o n as an a u t o t r o p h i c component i n an e s t u a r i n e ecosystem. The l i v i n g biomass c o n t r i b u t i o n i n experimental systems by freshwater phytoplankton was very s m a l l ( F i g . 5a, 6a, 10a, 11a ) compared with the p r o d u c t i o n i n the c o n t r o l l e d systems ( F i g . 13, 14). The a c t i v i t y of the freshwater phytoplankton was r e s t r i c t e d i n a narrow range of s a l i n i t y . Along the whole s a l i n i t y g r a d i e n t , 85 most of the freshwater phytoplankton biomass was converted i n t o the o r g a n i c d e t r i t u s p o o l . T h i s change r e s u l t e d i n a s t r u c t u r a l change f o r the whole system which s h i f t e d the energy flow towards a h e t e r o t r o p h i c pathway. Th e r e f o r e the h e t e r o t r o p h i c component became the major c o n t r i b u t i o n i n the f r e s h water component of the e s t u a r i n e ecosystem. Experiment stage I I showed t h a t the sea water phytoplankton can q u i c k l y f u n c t i o n i n a s a l i n i t y g r a d i e n t . T h i s i s probably because the e v o l u t i o n of phytoplankton has been i n the same d i r e c t i o n as the e v o l u t i o n of the s a l i n i t y g r a d i e n t from low to high over time and space. So the ecosystem i s i n a c o n d i t i o n of always being c l o s e t o a sea water s t a t e . The time s e r i e s of b i o l o g i c a l change i n the p o p u l a t i o n of freshwater organisms i n sea water (Stage I, P a r t 1) and the seawater organisms i n the same g r a d i e n t (Stage I I , P a r t 1) together g i v e a p i c t u r e of what may occur i n a r e a l e s t u a r y ( F i g . 5a, 6a; F i g . 15,16a). At the phytoplankton l e v e l , the sea water p o p u l a t i o n i s dominant i n an e s t u a r i n e system. T h i s dominance g r a d u a l l y reaches i t s maximum at the high s a l i n i t y end of the g r a d i e n t . T h i s s a l i n i t y g r a d i e n t c r e a t e s a heterogeneous c o n d i t i o n which c l e a r l y separates the phytoplankton niches over time. Marine s p e c i e s were i n h i b i t e d at the low end of the s a l i n i t y g r a d i e n t ( F i g . 15), as the g r a d i e n t moves c l o s e t o a s p e c i e s s u i t a b l e range, phytoplankton grow very q u i c k l y and the biomass i n c r e a s e s e x p o n e n t i a l l y . The maximum development of biomass i s almost the same as i n the c o n t r o l l e d v e s s e l s , but the delayed growth phase r e s u l t s from the s a l i n i t y g r a d i e n t . I f we c o n s i d e r the same process happening i n an element of water moving seaward on the s u r f a c e l a y e r 86 of an e s t u a r y the temporal e v o l u t i o n p a t t e r n of the e c o l o g i c a l components i n t h a t element w i l l become a s p a t i a l d i s t r i b u t i o n p a t t e r n . T h i s model may be a reasonable e x p l a n a t i o n of why e s t u a r i n e ecosystems show t h i s s p a t i a l d i s t r i b u t i o n p a t t e r n . The f a c t t h a t the i n c r e a s e of c h l a along a s a l i n i t y g r a d i e n t reaches i t s maximum o f t e n a t some d i s t a n c e from the mouth of the e s t u a r y i s an e v o l v i n g p a t t e r n which c o i n c i d e s with the s p a t i a l s c a l e because of the movement of the water mass. The dynamic p a t t e r n of nitrogenous n u t r i e n t s i n a l l our experimental systems was a f u n c t i o n of e v o l u t i o n of phytoplankton and b a c t e r i a l dynamics. T h i s i s to be expected because the n u t r i t i o n a l r e l a t i o n s h i p decided the c o v a r i a n c e between l i v i n g components and n u t r i e n t s . That i s , the growth process i s a process of i n c o r p o r a t i n g m a t e r i a l i n t o the l i v i n g biomass of the components (phytoplankton and b a c t e r i a ) . S t a t i s t i c a l c a l c u l a t i o n s show a s t r o n g n e g a t i v e c o r r e l a t i o n between C h l a and N0~3, and a n e g a t i v e c o r r e l a t i o n between N0~3 and time which can be e a s i l y e x p l a i n e d as a r e s u l t of phytoplankton development over time. T h i s k i n d of d i s t r i b u t i o n of necessary n u t r i e n t s along an e s t u a r i n e s a l i n i t y g r a d i e n t i s a l s o shown i n a l l works i n which an i n c r e a s i n g biomass of phytoplankton i s found along an e s t u a r i n e t r a n s i t i o n zone. In e s t u a r i n e ecosystems, seawater phytoplankton and some e s t u a r i n e p o p u l a t i o n s are the major a u t o t r o p h i c components. Because the temporal e v o l u t i o n of the component from the head of the e s t u a r y c o i n c i d e d with s p a t i a l s c a l e by continuous outflow, the i n c r e a s e of biomass along an e s t u a r y should be a standard phenomena as 87 the phytoplankton d i s t r i b u t i o n s p a t i a l p a t t e r n and the primary c o n s e r v a t i v e n u t r i e n t s a l s o have a corresponding s p a t i a l p a t t e r n . However, the dynamics of ammonimum i n the system has a q u i t e d i f f e r e n t behaviour p a t t e r n . The dynamics of ammonimum c o n c e n t r a t i o n i s a combination of many processes which happen i n the ecosystem. Phytoplankton and b a c t e r i a can u t i l i z e NH +4 as a n i t r o g e n source. The a s s i m i l a t i o n of n i t r a t e and ammonium i s the major b i o l o g i c a l process by which these two components c o v e r t i n o r g a n i c n i t r o g e n to o r g a n i c n i t r o g e n , although t h e i r r e l a t i v e c o n t r i b u t i o n t o t o t a l n i t r o g e n a s s i m i l a t i o n i s extremely v a r i a b l e i n time and space (Dugdale, 1967 McCarthy, 1972; Walsh e t a l . , 1980). I t i s w e l l known t h a t i n the presence of NH +4, u n i c e l l u l a r algae p r e f e r e n t i a l l y take up t h i s n u t r i e n t (Blasco and Conway, 1982; McCarthy & Eppley, 1972). The gen e r a t i o n time of NH +4 and N0~3 i s very d i f f e r e n t . T h i s may r e s u l t i n q u i t e d i f f e r e n t dynamic p a t t e r n s . B a c t e r i a can u t i l i z e amino a c i d as energy source and then be grazed by m i c r o f l a g e l l a t e s which may d i r e c t l y r e l e a s e NH +4 (Adernson, 1985; Goldman and Caron 1985). I t i s i n t e r e s t i n g t h a t i n my experiments the dynamics of NH +4 showed d i f f e r e n t p a t t e r n s with d i f f e r e n t system components but not with d i f f e r e n t s a l i n i t y g r a d i e n t s ( F i g . 5b,6b, 16b and 20b). In the freshwater components t e s t i n g experiment, the c o n c e n t r a t i o n of NH +4 shows a decrease from i t s i n i t i a l v a l u e t o a value c l o s e t o zero i n the f i r s t p e r i o d o f the experiment, before the i n c r e a s e of C h i a. There was no i n d i c a t i o n t h a t t h i s decrease was due to uptake by phytoplankton ( t h i s i s most c l e a r i n the X « c 6 thermodynamlcally unstable organic based system (high I n t e r n a l energy) I A i < J n u t r i e n t s I I < I h e t e r o t r o p h i c p r i o r i t y systems s t a t e e x t e r n a l ( l i g h t ) energy x i n u t r i e n t s shortwave r a d i a t i o n •photosynthetl process. A ±= i (Concurrent' ! s t a t e 1 ' ) , , T - , n u t r i e n t s | e t e r o t r d p phlc process e x t e r n a l (heat) energy r e s p i r a t i o n longwave r a d i a t i o n a u t o t r o p h i c p r i o r i t y systems s t a t e , , { n u t r i e n t s J « ( • Cheriaodynamlcally s t a b l e Inorganic based system ! (low I n t e r n a l energy) I t L energy flow ground s t a t e oo 00 F i g u r e 2 4 . The e n e r g e t i c dynamic s t a t e of an ecosystem i n the energy l e v e l f i e l d . 8 9 experiment d u r i n g A p r i l ( F i g . 5b,6b)). There was no decrease of N0~3 over the whole experimental p e r i o d but the NH +4 s t i l l showed a d e c l i n e over time. The h e t e r o t r o p h i c b a c t e r i a p r o d u c t i o n showed an i n c r e a s e of biomass which may p o s s i b l y u t i l i z e NH +4 ( F i g . 7 ) . T h i s may suggest t h a t the b a c t e r i a which grow i n the f i r s t p e r i o d of the experiment were most l i k e l y a t the expense a t carbohydrate, and thus they need an e x t r a nitrogenous n u t r i e n t to s y n t h e s i z e amino a c i d s . In the experiments i n v o l v i n g the seawater b i o t a , the dynamics of the c o n c e n t r a t i o n of NH +4 are q u i t e low but had i n c r e a s i n g peaks a f t e r phytoplankton was degraded, r e g a r d l e s s of the d i f f e r e n t s a l i n i t y g r a d i e n t s . The NH +4 peaks may i n d i c a t e the m i n e r a l i z a t i o n of phytoplankton d e t r i t a l o r g a n i c m a t e r i a l s by the h e t e r o t r o p h i c components. These two d i f f e r e n t p a t t e r n s of NH +4 uptake may r e s u l t from d i f f e r e n c e s i n the r a t i o of C/N i n sea water and f r e s h water or a d i f f e r e n c e i n the nature of the b a c t e r i a l p o p u l a t i o n s i n e i t h e r water ( i f we assume t h a t the b a c t e r i a i n both the i n i t i a l waters had a l r e a d y reached maximum biomass as determined by some other f a c t o r s ) . In experiment stage I the i n f l o w of f r e s h water and sea water r e s u l t i n an osmotic s t r e s s which may cause the r e l e a s e of or g a n i c s u b s t r a t e from f r e s h water organisms. T h i s a d d i t i o n of or g a n i c n u t r i e n t s may s t i m u l a t e the growth of b a c t e r i a . However, i n the seawater system, the water may c o n t a i n l i t t l e carbohydrate because of the long h i s t o r y of u t i l i z a t i o n by b a c t e r i a i n the a p h o t i c zone. The sea water p o p u l a t i o n had a higher i n i t i a l c e l l c o n c e n t r a t i o n which may have r e s u l t e d i n 9 0 r a p i d u p t a k e o f N H + 4 b e f o r e t h e s t a r t o f e x p o n e n t i a l g r o w t h . T h e h i g h m e t a b o l i c r a t e a n d f a s t m i n e r a l i z a t i o n o f h e t e r o t r o p h i c c o m p o n e n t s s u g g e s t t h a t t h e s y s t e m h a s a t e n d a n c y t o g o b a c k t o t h e a u t o t r o p h i c p r i o r i t y s y s t e m , t h a t i s w h e n t h e i n t e r n a l e n e r g y i s a t i t s l o w e s t l e v e l , a n d t h e s y s t e m i s d o m i n a t e d b y i n o r g a n i c s u b s t a n c e s a n d h a v i n g t h e l a r g e s t p o t e n t i a l b i o e n e r g y c a r r y i n g c a p a c i t y . T h e r e f o r e t h e w h o l e s y s t e m a d j u s t s s o t h a t i t i s b a s e d o n a l o w e n e r g y c o n t a i n i n g c o m p o n e n t w h i c h m a y b e m o r e s t a b l e . F r o m my e x p e r i m e n t , t h e r e s u l t s c l e a r l y s h o w t h a t t h e b e h a v i o u r o f f r e s h w a t e r a u t o t r o p h i c c o m p o n e n t a n d s e a w a t e r a u t o t r o p h i c c o m p o n e n t i s t o t a l l y d i f f e r e n t i n t h e s a l i n i t y g r a d i e n t . I n t h e r e a l e s t u a r y , w i t h o n e d i r e c t i o n c i r c u l a t i o n ( t i d e a v e r a g e ) t h e r e i s o n l y a n i n c r e a s i n g s a l i n i t y g r a d i e n t . T o f r e s h w a t e r c o m p o n e n t s , t h i s g r a d i e n t w o r k s a s a f i l t e r ; i t f i l t e r s o u t m o s t o f t h e f r e s h w a t e r p h y t o p l a n k t o n a s t h e y p a s s t o h i g h s a l i n i t y a n d a r e c o n v e r t e d i n t o o r g a n i c m a t e r i a l . S o m e e u r y h a l i n e p h y t o p l a n k t o n m a y k e e p t h e i r f u n c t i o n a t i n t e r m e d i a t e r a n g e s o f t h e s a l i n i t y g r a d i e n t , b u t t h e i r c o n t r i b u t i o n c o m p a r e d t o t h e s e a w a t e r p o p u l a t i o n i s v e r y l o w . T h e s e a w a t e r p h y t o p l a n k t o n h a v e a d i f f e r e n t b e h a v i o u r . T h e y c a n b e i n h i b i t e d a t l o w s a l i n i t i e s . H o w e v e r , t h e c o n t i n u a l m o v e m e n t a l o n g t h e g r a d i e n t m o v e s t h e m c l o s e t o t h e i r o p t i m a l e n v i r o n m e n t . S o t h e y f u n c t i o n m o r e a c t i v e l y a s t h e o u t f l o w g r a d u a l l y p r o v i d e s m o r e f a v o u r a b l e c o n d i t i o n s . T h e m o s t i m p o r t a n t f e a t u r e o f a n e c o s y s t e m i s t h e d y n a m i c s 91 of the phytoplankton p r o d u c t i o n which must have a r a d i c a l e f f e c t on the whole s t r u c t u r e of the ecosystem. The most c l o s e l y r e l a t e d e c o l o g i c a l components to phytoplankton are the i n o r g a n i c n u t r i e n t p o o l , the o r g a n i c substance p o o l and the h e t e r o t r o p h i c components. Because h e t e r o t r o p h i c b a c t e r i a are a second l e v e l energy t r a n s f o r m e r , so t h e i r a c t i v i t y and p r o d u c t i o n i s p r i m a r i l y dependent on the a v a i l a b i l i t y of o r g a n i c substance i n the water. There are two d i f f e r e n t foundations based on these two components which d e c i d e d i f f e r e n t energy routes i n the ecosystem. In a l l our experimental systems i t i s shown t h a t b a c t e r i a l p r o d u c t i v i t y i s not a f f e c t e d by the s a l i n i t y g r a d i e n t as much as t h a t of phytoplankton. For two d i f f e r e n t s a l i n i t y g r a d i e n t s having two d i f f e r e n t h e t e r o t r o p h i c components, biomass and growth of b a c t e r i a d i d not show a c r i t i c a l range of s a l i n i t y as d i d the phytoplankton. T h i s may be due t o the presense of b a c t e r i a i n both seawater and freshwater. And a l s o e i t h e r seawater or freshwater b a c t e r i a show a t o l e r a n c e t o a range of s a l i n i t i e s . T h e i r dynamics are much more t i g h t l y coupled with the amount and q u a l i t y of o r g a n i c s u b s t r a t e s and the s t a t e of the a u t o t r o p h i c component. I f one c o n s i d e r s the energy s t a t e of a ecosystem, the system can have two energy s t a t e s , one i s a high i n t e r n a l energy s t a t e which i s thermodynamically u n s t a b l e . There i s a g r e a t amount of o r g a n i c substances i n the system. T h i s system tends to r e l e a s e i n t e r n a l energy out of the system which goes back to an i n o r g a n i c substance based system; t h i s i s a low i n t e r n a l energy s t a t e and i s more thermodynamically s t a b l e . B a c t e r i a p l a y a major r o l e i n t h i s energy r e l e a s e p r o c e s s . B a c t e r i a have a very high uptake r a t e (V m) f o r both o r g a n i c substances and i n o r g a n i c m i n e r a l n u t r i e n t s as w e l l as a high growth r a t e (u) which can s u c c e s s f u l l y compete over the a u t o t r o p h i c component when the energy s t a t e i s high i n the system. A l s o when the system energy s t a t e decreases t o a c e r t a i n l e v e l the b a c t e r i a become l i m i t e d by the energy source. The p h y s i o l o g i c a l p r o p e r t i e s of the b a c t e r i a a c t u a l l y are c o n s i s t e n t with t h e i r n a t u r a l e c o l o g i c a l r o l e . The b a c t e r i a h e t e r o t r o p h i c process i s l i m i t e d by the system's i n t e r n a l energy. The oth e r s t a t e i s a low i n t e r n a l energy s t a t e which i s thermodynamically s t a b l e . T h i s s t a t e can be e x c i t e d by e x t e r n a l energy through the p h o t o s y n t h e t i c p r o c e s s . As the l i g h t energy flows i n t o the ecosystem, the energy s t a t e of the system i n c r e a s e s to a c e r t a i n l e v e l which i s determined by the energy c a r r y i n g c a p a c i t y of the system; t h i s c a p a c i t y i s mainly c o n t r o l l e d by the l i m i t i n g n u t r i e n t s . T h i s means t h a t a high energy s t a t e system ( l i v i n g system) always needs energy i n p u t from out s i d e of the ecosystem otherwise i t w i l l go back t o ground s t a t e through a h e t e r o t r o p h i c process (mainly b a c t e r i a ) . Between these two p r o c e s s e s , h e t e r o t r o p h i c p rocesses are d o w n h i l l p r o c e s s e s ; t h e i r p o t e n t i a l may be g r e a t e r than a u t o t r o p h i c p rocesses which are u p h i l l p r o c e s s e s , because they needs e x t e r n a l energy support. T h i s i d e a i s summarized i n a diagram ( F i g . 24). The p h y s i o l o g i c a l and e n e r g e t i c p r o p e r t i e s of b a c t e r i a p r o v i d e b a c t e r i a the p r i v i l e g e of u t i l i z i n g i t s energy source as q u i c k l y and as completely as p o s s i b l e to accomplish i t s e c o l o g i c a l r o l e i n an ecosystem, a l l o w i n g the system t o go back q u i c k l y to a s t a b l e s t a t e . The f a s t response mechanism i s a l s o r e f l e c t e d i n the dormant nature of the b a c t e r i a l p o p u l a t i o n . When o r g a n i c n u t r i e n t s become l i m i t i n g , b a c t e r i a do not immediatly go through d e p l e t i o n as phytoplankton d i d , but go i n t o a dormant stage which suggests t h a t t h i s storage nature p r o v i d e s a high background c o n c e n t r a t i o n of b a c t e r i a . T h i s means th a t b a c t e r i a keep a c e r t a i n storage biomass so they can q u i c k l y respond to the i n c r e a s e i n energy s t a t e of the system. T h i s process has the a b i l i t y t o l e t an ecosystem go back to a s t a b l e s t a t e as soon as p o s s i b l e . The whole system would tend to s t a y a t a low i n t e r n a l energy s t a t e (phytoplankton p r i o r i t y systems) i f t h e r e i s no f u r t h e r energy i n p u t . B a c t e r i a i n t h i s system s t a t e should be l i m i t e d by o r g a n i c substances. F u r t h e r , t h e r e i s no e q u i l i b r i u m between b a c t e r i a and m i c r o f l a g e l l a t e s . T h i s means t h a t n e i t h e r the amount of o r g a n i c s u b s t r a t e s can ever be e f f e c t i v e l y u t i l i z e d , (so the system c o u l d not go back to s t a b l e s t a t e ) , nor can the m i c r o z o o f l a g e l l a t e s ever become f u l l y developed due t o t h e i r own f e e d i n g l i m i t a t i o n on the b a c t e r i a l food source. I m p l i c i t i n t h i s theory i s t h a t b a c t e r i a may have a low K m f o r o r g a n i c m a t e r i a l a t low n u t r i e n t c o n c e n t r a t i o n ( s i n c e the DOM c o n c e n t r a t i o n i n s eawateris low). Morever, t h i s K m can change with the changing of c o n d i t i o n s (Azam e t a l . , 1981; Vaccaro e t a l . , 1967,1969). In a number of experiments inwhich s u b s t r a t e has been added t o seawater (Parsons e t a l . , 1977,1981) the b a c t e r i a showed a very high K m. T h i s suggests t h a t b a c t e r i a may s h i f t the K m w i t h i n the p o p u l a t i o n from low to very high depending the s u b s t r a t e c o n c e n t r a t i o n so i t s growth r a t e can always c a t c h up with the a d d i t i o n of o r g a n i c substances. T h i s means t h a t the growth of b a c t e r i a can l a r g e l y exceed the g r a z i n g of z o o f l a g e l l a t e s under high c o n c e n t r i t i o n of o r g a n i c s u b s t r a t e s . In my experiment, b a c t e r i a l biomass always i n c r e a s e d with the i n p u t of o r g a n i c substances by phytoplankton growth. The m i c r o f l a g e l l a t e s c o u l d not balance the growth of b a c t e r i a when the o r g a n i c substance was not l i m i t e d . The m i c r o f l a g e l l a t e s may o n l y o f f s e t the b a c t e r i a at a c e r t a i n r a t e of comsumption where b a c t e r i a l growth i s l i m i t e d by o r g a n i c n u t r i e n t s . Some work i n d i c a t e s t h a t the abundance of m i c r o f l a g e l l a t e s i s a f u n c t i o n of b a c t e r i a l c e l l numbers r e g a r d l e s s of i t s p h y s i o l o g i c a l s t a t e (e.g. l i v i n g or dormant). T h e r e f o r e b a c t i v o r o u s f l a g e l l a t e s can not l i m i t the u t i l i z a t i o n of o r g a n i c substances by b a c t e r i a and can not c o n t r o l the upper l i m i t of the b a c t e r i a l d e n s i t y . B a c t e r i a l c o n c e n t r a t i o n upper l i m i t i s d e c i d e d o n l y by a v a i l a b i l i t y of n u t r i e n t s and a space d e n s i t y f a c t o r . A f t e r b a c t e r i a go i n t o a l i m i t a t i o n s t a t e , g r a z i n g d e c i d e s the lower d e n s i t y l i m i t , which i s the g r a z i n g t h r e s h o l d of the m i c r o f l a g e l l a t e p o p u l a t i o n s . M i c r o f l a g e l l a t e s are more an e x p l o i t e r of b a c t e r i a than a l i m i t e r . The e q u i l i b r i u m between o r g a n i c substances, growth of b a c t e r i a and g r a z i n g of m i c r o f l a g e l l a t e s doesn't e x i s t as suggested by some authors (Wright, 1978, 1982, 1984). This e q u i l i b r i u m being a mechanism which i s a g a i n s t the a c t u a l f u n c t i o n of b a c t e r i a i n the ecosystem. 95 The p r o p e r t i e s of b a c t e r i a as an e c o l o g i c a l f u n c t i o n a l requirement p r o v i d e b a c t e r i a the a b i l i t y o f q u i c k l y responding to the a d d i t i o n of o r g a n i c n u t r i e n t s and i t can o f t e n i n c r e a s e s e v e r a l f o l d w i t h i n a 24 hour p e r i o d (Wright, 1978, 1983). The dynamics of the b a c t e r i a i s a c t u a l l y a r e f l e c t i o n of the dynamics of the o r g a n i c p o o l i n the experiments. In the absence of phytoplankton d u r i n g the f i r s t p e r i o d of experiment stage I, I I , p a r t 1, the i n c r e a s e of b a c t e r i a should i n d i c a t e the o r g a n i c substance a d d i t i o n or r e s u l t i n g from the mixing of two kinds of water which p r o v i d e a mutual b e n e f i t f o r the b a c t e r i a l growth. T h i s c o u l d be some m i c r o n u t r i e n t s which may be s h o r t i n one type of water but r i c h i n the o t h e r . I f we assume t h a t before the experiment s t a r t e d the i n i t i a l waters were both i n an o r g a n i c l i m i t e d s t a t e f o r b a c t e r i a , the mixing process must then cause some t r a n s f o r m a t i o n of o r g a n i c substance t o a l a b i l e source, o r, i f the r e i s some l a b i l e o r g a n i c source, i t s u t i l i z a t i o n must be l i m i t e d by some other f a c t o r s which c o u l d be r e l e a s e d by mixing the two kinds of water. In an i s o l a t e d system, the o r g a n i c source must be from the a u t o t r o p h i c component. T h e r e f o r e b a c t e r i a depend on the a u t o t r o p h i c component which can e n t i r e l y c o n t r o l the h e t e r o t r o p h i c a c t i v i t y through the supply of s u b s t r a t e . However, i n an e s t u a r i n e ecosystem, the c o n t r o l of b a c t e r i a by phytoplankton i s pe r t u r b e d by a l l o c h t h o n o u s o r g a n i c substance from the t e r r e s t r i a l environment. T h e r e f o r e the b a c t e r i a may i n c r e a s e the importance of t h e i r r o l e i n the ecosystem due to 96 the independent course of s u b s t r a t e supply. They w i l l p l a y a much bigger r o l e than i n a autochthonous system i n terms of energy flow and matter r e c y c l i n g . T h e i r q u a n t i t i v e r o l e depends on the r a t i o of a v a i l a b l e o r g a n i c substances and the m i n e r a l n u t r i e n t . The ecosystem s t r u c t u r e may show three forms: 1. Phytoplankton p r i o r i t y systems. In these systems, the c o n d i t i o n s favour phytoplankton growth. B a c t e r i a may be more or l e s s l i m i t e d by o r g a n i c substances. The f o u n d a t i o n of the system i s t h a t i n o r g a n i c substances dominate. The q u i c k growth of phytoplankton may i n h i b i t any p r e v i o u s e x i s t i n g b a c t e r i a l p o p u l a t i o n s so t h a t m a t e r i a l and energy flow through the b a c t e r i a l pathway i s shut down. A l l the p o s s i b l e m i n e r a l n u t r i e n t p o o l goes to p r o v i d e a u t o t r o p h i c p r o d u c t i o n s as i s shown i n our experiment, stage I I p a r t 2 and a l l " c o n t r o l v e s s e l s " ( F i g . 13,14, 23a,b). 2. Phytoplankton and h e t e r o t r o p h i c c o e v o l u t i o n systems. Provi d e d b a c t e r i a are l i m i t e d by t h e i r energy source, most of the o r g a n i c n u t r i e n t must come from r e l e a s e of phytoplankton exudate d u r i n g p h o t o s y n t h e t i c p r o d u c t i o n . But i f the growth of the a u t o t r o p h i c component i s r e s t r i c t e d f o r some other reason, the a u t o t r o p h i c component can not make f u l l use of the system. T h i s system has e x t r a s u b s t r a t e f o r b a c t e r i a and so the b a c t e r i a can show a c o e v o l u t i o n p a t t e r n . The i n h i b i t i n g e f f e c t of phytoplankton on b a c t e r i a i s not obvious. However, the use of r e l e a s e d m a t e r i a l from phytoplankton by b a c t e r i a i s i n d i c a t e d i n c o r r e l a t i o n c a l c u l a t i o n s . These c l e a r l y show i n our experiment stage I and I I ( F i g . 7, 12) t h a t the s a l i n i t y g r a d i e n t c o n d i t i o n depressed the phytoplankton growth. So i n t h i s case the carbon budget showed two c o n c u r r e n t pathways. 3. H e t e r o t r o p h i c p r i o r i t y system. The system a t the beginning may have a high a l l o c h t h o n o u s o r g a n i c substance a d d i t i o n , such as o f t e n happens i n an e s t u a r y and some p o l l u t e d water. Then the c o n t r o l of b a c t e r i a by the phytoplankton a u t o t r o p h i c component through energy supply i s o v e r r u l e d by the a d d i t i o n of an a l l o c h t h o n o u s energy source. In . t h i s case, very s t r o n g m e t a b o l i c growth of the b a c t e r i a under c o n d i t i o n s of enough energy p r o v i d e b a c t e r i a with the a b i l i t y t o dominate i n the system. Most of the m a t e r i a l goes i n t o the h e t e r o t r o p h i c pathway. The a u t o t r o p h i c p r o d u c t i o n i s then depressed, as shown by Parsons (1977, 1981) and Spies (1984). T h i s i s a homeostatic mechanism which occurs r a p i d l y and e l i m i n a t e s the d i s s o l v e d o r g a n i c matter through b a c t e r i a l biomass and m i n e r a l i z a t i o n . Then the system r e t u r n s from a high energy s t a t e t o a low energy s t a t e and p r o v i d e a v a i l a b l e substance f o r e x t e r n a l energy i n p u t t o the system which i s a phytoplankton p r i o r i t y s t a t e and more s t a b l e i n terms of ecosystem s t r u c t u r e . The above d i s c u s s i o n attempts to look at the i n t e r a c t i o n s w i t h i n an ecosystem. I f we c o n s i d e r t h i s c y c l e of energy i n a time s e r i e s of the same p h y s i c a l space, we have a s u c c e s s i o n of b a c t e r i a — phytoplankton — b a c t e r i a . However, at the p o p u l a t i o n l e v e l , the two b a c t e r i a l maxmia may belong to d i f f e r e n t groups. In my experiments most of a u t o t r o p h i c 98 c o m p o n e n t d e v e l o p m e n t s h o w e d t w o d i s t i n c t i v e p e r i o d s . T h a t i s , t h e b a c t e r i a g r o w i n g d u r i n g t h e f i r s t p e r i o d a r e q u i t e d i f f e r e n t f r o m t h a t i n t h e s e c o n d p e r i o d . A s d i s c u s s e d i n t h e p r e v i o u s s e c t i o n s , t h e f i r s t g r o u p o f b a c t e r i a a r e m o s t l i k e l y t o u t i l i z e c a r b o h y d r a t e a s a n e n e r g y s o u r c e a n d t a k e u p m i n e r a l n u t r i e n t s d u r i n g b i o s y n t h e s i s . T h i s g r o u p o f b a c t e r i a p o t e n t i a l l y c o m p e t e f o r t h e i n o r g a n i c n u t r i e n t p o o l w i t h t h e p h y t o p l a n k t o n , s o t h e y h a v e a s t r o n g i n t e r a c t i o n w i t h t h e a u t o t r o p h i c c o m p o n e n t . T h e d e c l i n e o f N H + 4 i n t h e e x p e r i m e n t b e f o r e p h y t o p l a n k t o n g r o w t h m a y b e d u e t o t h i s u p t a k e . T h e y m a y b e c o m e a c o m p e t i t o r w i t h p h y t o p l a n k t o n d u r i n g p h o t o s y n t h e s i s b e c a u s e t h o s e b a c t e r i a a r e n o r m a l l y l i m i t e d b y a n e n e r g y s o u r c e i n a n a t u r a l s y s t e m . T h e r e l e a s e o f o r g a n i c s u b s t a n c e m a y a l l o w t h e m t o g r o w a n d c o m p e t e f o r t h e i n o r g a n i c n u t r i e n t . P h y t o p l a n k t o n m a y i n h i b i t t h i s g r o u p o f h e t e r o t r o p h i c b a c t e r i a b e f o r e o r d u r i n g e x p o n e n t i a l g r o w t h . T h e s e c o n d g r o u p o f b a c t e r i a a p p e a r d u r i n g t h e d e g r a d a t i o n p e r i o d . A t t h i s t i m e a l l t h e a v a i l a b l e n u t r i e n t s h a v e b e e n i n c o r p o r a t e d i n t o o r g a n i c s u b s t a n c e s . T h e d e p l e t i o n o f n u t r i e n t s c a u s e s t h e g r o w t h o f a u t o t r o p h i c c o m p o n e n t s t o e n d . T h e s y s t e m e x h a u s t s i t s a u t o t r o p h i c e n e r g y c a r r y i n g c a p a b i l i t y . T h e b a c t e r i a l i v i n g a t t h e t i m e a r e d i f f e r e n t f r o m t h o s e i n t h e f i r s t p e r i o d . T h e r e a r e n o t e n o u g h n u t r i e n t s l e f t f o r t h e m t o t a k e u p . T h e y a r e n o t c o m p e t i t o r s w i t h p h y t o p l a n k t o n . T h e y m o s t l i k e l y u t i l i z e d e g r a d e d p h y t o p l a n k t o n m a t e r i a l w h i c h c o n t a i n s a l l t h e n e c e s s a r y b i o l o g i c a l e l e m e n t s , s u c h a s a m i n o a c i d s c o n t a i n i n g n i t r o g e n o r o r g a n i c p h o s p h o r u s c o m p o u n d s . As the experiment shows, i f the a u t o t r o p h i c component i s r e s t r i c t e d by some f a c t o r s , the system can have two pathways f o r energy flow s i m u l t i n e o u s l y . Because of the high growth r a t e and high a d a p t i v e a b i l i t y , the b a c t e r i a may occupy many d i f f e r e n t environments which are not a v a i l a b l e to autotrophs, as long as t h e r e i s enough i n t e r n a l system energy. In my r e s u l t s , b a c t e r i a can s u c c e s s f u l l y occupy the s a l i n i t y g r a d i e n t range i n which autotrophs are i n h i b i t e d . Combining the experiment I, p a r t 1 and stage I I , p a r t 1 ( F i g . 5a & F i g . 15a), which i s an i n c r e a s i n g s a l i n i t y g r a d i e n t f o r f r e s h and seawater e c o l o g i c a l components, i t i s apparent t h a t the freshwater a u t o t r o p h i c component d i d not s u r v i v e the s a l i n i t y g r a d i e n t . The seawater phytoplankton i n c r e a s e d from the middle of t h i s g r a d i e n t but mainly occupy the high s a l i n i t y end. However, the h e t e r o t r o p h i c b a c t e r i a showed a p o t e n t i a l to be a c t i v e d u r i n g f i r s t p e r i o d of experiment be f o r e phytoplankton grow. T h i s i n d i c a t e s t h a t the s a l i n i t y g r a d i e n t c r e a t e s a heterogenous c o n d i t i o n t o t e m p o r a l l y separate these two e c o l o g i c a l components along the s a l i n i t y g r a d i e n t . I f t h i s temporal d i s t r i b u t i o n of two d i f f e r e n t components happens i n a body of water moving i n the s u r f a c e l a y e r of the e s t u a r y i t can be expected to show a s p a t i a l d i s t r i b u t i o n . Numerous s t u d i e s have i n d i c a t e d that h e t e r o t r o p h i c a c t i v i t y and b a c t e r i a l numbers are g r e a t i n the low s a l i n i t y range of an e s t u a r y (Stevenson and Erkenbrecher, 1976; Wright, 1978; Wright e t a l . , 1983). Wright (1983) concludes t h a t there i s a c l e a r p a t t e r n of s p a t i a l d i s t r i b u t i o n of b a c t e r i a i n an e s t u a r y . E s t u a r i e s develop a b a c t e r i a l 100 f l o r a s e v e r a l f o l d higher i n c o n c e n t r a t i o n than e i t h e r the f r e s h water or sea water at e i t h e r end of the e s t u a r y (Palumbo and Ferguson, 1978). The b a c t e r i a are o n l y b i o l o g i c a l l y a c t i v e i n the upper e s t u a r y . Wright (1978) i n d i c a t e d the s p e c i f i c a c t i v i t y of b a c t e r i a d e c l i n e d s i g n i f i c a n t l y i n the t r a n s i t i o n t o s a l t water. T h i s i s s i m i l i a r t o the ecosystem s t r u c t u r e over time shown i n our experiments. T h e r e f o r e the two phenomena, high phytoplankton biomass at the mouth and high b a c t e r i a p o p u l a t i o n a t the head, a c t u a l l y are r e l a t e d to each other and are a r e s u l t of the dynamics of the whole system i n respones to the environmental c o n d i t i o n s . The p a r a l l e l i n c r e a s e of b i o l o g i c a l energy flow through these two pathways seems obvious. However, under which c o n d i t i o n s the s t a n d i n g stock of b a c t e r i a , m i c r o f l a g e l l a t e s and phytoplankton can show a c o r r e l a t i o n i s a combination of the i n t e r a c t i o n s and i n t e r r e l a t i o n s among a l l ecosystem compartments. T h e r e f o r e o n l y by s i m u l t a n e o u s l y measuring both r e l a t i o n s between s t a n d i n g s t o c k and f l u x r a t e can we estimate the f u n c t i o n a l r o l e they p l a y i n the energy flow and carbon c y c l i n g i n an ecosystem. That i s , under what c o n d i t i o n s does the a u t o t r o p h i c pathway slow down to g i v e p r i o r i t y t o h e t e r o t r o p h i c growth as many workers have i n d i c a t e d i n the f i e l d (Parker, 1975), i n b i g c o n t a i n e r s of n a t u r a l water (Parsons, 1982) and i n l a b o r a t o r y c u l t u r e s ( S p i e s , 1984)? A l s o , under what c o n d i t i o n s does h e t e r o t r o p h i c p r o d u c t i o n slow down? The i n o r g a n i c environment g e n e r a l l y f a v o u r s an a u t o t r o p h i c pathway through the i n h i b i t o r y e f f e c t s of phytoplankton on b a c t e r i a (Lucas, 101 1955; S i e b u r t h , 1959; B e l l e t a l . , 1974; Kogure e t a l . , 1979; S p i e s , 1984). C o n s i d e r i n g the l i m i t a t i o n of the r e s i d e n c e time f o r b i o l o g i c a l p r o d u c t i o n i n an e s t u a r y , t h e r e should be a low phytoplankton biomass at low s a l i n i t y and i n c r e a s i n g amounts along the t r a n s i t i o n r e a c h i n g a maximum at high s a l i n i t y a t the mouth of the e s t u a r y . I f the ecosystem i s a phytoplankton based system, at the head of the e s t u a r y , they may be not e x p l o r e d by phytoplankton. In a h i g h l y p r o d u c t i v e area t h i s seems unreasonable. However, c o n s i d e r i n g the behaviour of h e t e r o t r o p h i c component, they show a c t i v e behaviour a l l along the s a l i n i t y g r a d i e n t at l e a s t on the p o p u l a t i o n l e v e l . Many c o n d i t i o n s a t the head of e s t u a r y i n d i c a t e b a c t e r i a may f u l l y u t i l i z e t h i s space. The c o n d i t i o n s which are f a v o u r a b l e to h e t e r o t r o p h i c b a c t e r i a are high o r g a n i c n u t r i e n t supply by r i v e r d i s c h a r g e ( i n c l u d e s a l l p o s s i b l e sources, e.g. r e l e a s e from marsh base, s o i l e t c . ) higher t u r b i d i t y r e s u l t i n g i n low l i g h t i n t e n s i t y , slow growth of phytoplankton i . e . low p r o d u c t i o n of b a c t e r i a l i n h i b i t o r s and higher n u t r i e n t c o n c e n t r a t i o n as w e l l as the p h y s i o l o g i c a l nature of b a c t e r i a such as high uptake r a t e of n u t r i e n t s , high response speed to i n p u t of substances, high s a t u r a t i o n c o e f f i c i e n t and high growth r a t e p r o v i d e b a c t e r i a with an advantage to overcome the time l i m i t a t i o n by c i r c u l a t i o n . In a r e a l e s t u a r y , a temporal e v o l u t i o n of e c o l o g i c a l components c a r r i e d seaward i n a moving water mass of the s u r f a c e l a y e r i s always accompanied by an i n c r e a s i n g s a l i n i t y 102 g r a d i e n t . For t h i s p rocess and on the m i c r o b i a l ecosystem l e v e l , the system i n our experiment and i n r e a l e s t u a r y are i n common, However, t h i s common nature may be o v e r r i d d e n i n a r e a l e s t u a r y by many f a c t o r s (e.g. d i f f e r e n t c i r c u l a t i o n p a t t e r n s , t i d a l e f f e c t s , e t c . ) . In an e s t u a r y more all o c h t h o n o u s o r g a n i c substance may c o n t r i b u t e than t h a t i n our experiment. For example, sewage outflow, landward counter c u r r e n t flow which may b r i n g the s i n k i n g phytoplankton p a r t i c l e s a t the e s t u a r y mouth back t o the head of the es t u a r y to e n r i c h maximum t u r b i d i t y , and marsh based c o a s t a l r e l e a s e of o r g a n i c as w e l l as sedimentation and bottom e f f e c t s a l l c o n t r i b u t e t o the e s t u a r i n e trophodynamics. Most of these processes happen at the head of an e s t u a r y so the h e t e r o t r o p h i c p o t e n t i a l a c t i v i t y should be higher than i n our sim u l a t e d system at low s a l i n i t y range. Another d i s c r e p a n c y between our experiment and f i e l d s t u d i e s i s t h a t i n our experiment a t the high s a l i n i t y end, a f t e r phytoplankton d e g r a d a t i o n , the b a c t e r i a numbers i n c r e a s e g r e a t l y and most of them are b i g rods and are atta c h e d b a c t e r i a . T h i s i s w e l l known and i t i s reasonable t h a t a l a r g e amount of dead phytoplankton should p r o v i d e more o r g a n i c substance a f t e r the bloom. We should expect v i g o r o u s a c t i v i t y of at t a c h e d b a c t e r i a i n an e s t u a r y as I found i n the l a s t p e r i o d of our experiment. In c o n t r a s t , p r e v i o u s r e s e a r c h e r s have not found t h i s zone. B e l l and A l b r i g h t (1981) found i n the F r a s e r R i v e r e s t u a r y t h a t the at t a c h e d b a c t e r i a decreased along the s a l i n i t y g r a d i e n t . T h i s may be a reasonable phenomenon i n a r e a l e s t u a r y . In the 103 e s t u a r y , when the water body reaches the f r o n t a l zone at the mouth, the flow speed slows down. The dead phytoplankton d e t r i t u s w i l l s i n k out of the upper l a y e r as the flow speed decreases i n the plume area (Lorenzen e t a l . , 1983; Smetacek, 1985;). The b a c t e r i a a t t a c h e d on the p a r t i c u l a t e m a t e r i a l w i l l a l s o f o l l o w the host s i n k i n g out of the s u r f a c e l a y e r , but the f r e e l i v i n g b a c t e r i a would not s i n k . Thus the attached b a c t e r i a s i n k out of the s u r f a c e l a y e r . T h i s s i n k i n g component can be c a r r i e d landward to the head of the e s t u a r y (LeBlond, p e r s . comm.). T h i s a c t u a l l y i n c r e a s e s the number of attached b a c t e r i a i n the low s a l i n i t y range i f t h i s t r a n s p o r t a t i o n process i s s h o r t e r than the time f o r b a c t e r i a to compelete ths d e g r a d a t i o n or i n c r e a s e the necessary n u t r i e n t s i f the d e g r a d a t i o n has been completed d u r i n g t h i s t r a n s p o r t i n g p r o c e s s . In our experiments we d i d not have t h i s p r o c e s s . The a t t a c h e d b a c t e r i a l p o p u l a t i o n can not s i n k out of the experimental f l a s k s . However we d i d f i n d t h a t a f t e r the growth p l a t e a u phase, the phytoplankton showed a g r e a t tendency to sediment. Even though we used a magnetic s t i r r e r , s t i l l i t was impossible to stop the s i n k i n g and a g r e a t amount of dead phytoplankton sedimented on the bottom. T h i s a l s o happened i n Spies' work (1984). T h e r e f o r e combining my experiment with the e s t u a r i n e c i r c u l a t i o n , the s p a t i a l s e p a r a t i o n between h e t e r o t r o p h i c p r o d u c t i o n and phytoplankotn p r o d u c t i o n along the s a l i n i t y g r a d i e n t i s a r e s u l t of i n t e r a c t i o n i n both e c o s y s t e m s t r u c t u r e and n a t u r a l p h y s i c a l s t r u c t u r e . In e s t u a r i e s , a sea water phytoplankton based ecosystem 104 mostly occuppies the high range of s a l i n i t y g r a d i e n t w h i l e the low range of s a l i n i t y i s occuppied by h e t e r o t r o p h i c b a c t e r i a . The h e t e r t r o p h i c p o p u l a t i o n ( b a c t e r i a ) may be made up of both sea water and f r e s h water. These mixed p o p u l a t i o n s have an a d a p t a t i o n f o r the whole s a l i n i t y spectrum. T h e i r e v o l u t i o n a r y p a t t e r n s are mainly d e c i d e d by the s t a t e of o r g a n i c substance p o o l . At l i m i t e d c o n c e n t r a t i o n s of o r g a n i c substance, b a c t e r i a are s t r o n g l y a f f e c t e d by the p h y s i o l o g i c a l s t a t e of phytoplankton i n terms of the amount of o r g a n i c s u b s t r a t e r e l e a s e d as w e l l as the phytoplankton bloom i n terms of i t s i n h i b i t i o n e f f e c t . C o n s i d e r i n g the p h y s i c a l c i r c u l a t i o n and other f a c t o r s i n the p h y s i c a l f i e l d and the e v o l u t i o n of ecosystem components i n a e c o l o g i c a l space, and combining these two systems t o g e t h e r , I propose a c o n c e p t u a l model i n summary to demonstrate the s t r u c t u r e of e s t u a r i n e ecosystem and i t s arrangement i n the p h y s i c a l space by the c i r c u l a t i o n process and the c o n d i t i o n a l g r a d i e n t ( F i g . 25). F i g u r e 25. The arrangement of ecosystem components in an e s t u a r i n e c i r c u l a t i o n f i e l d . ^ o Summary 106 1. In an e s t u a r y , the temporal development of an ecosystem i n the upper l a y e r of water moving seaward c o i n c i d e s with the s p a t i a l d i s t r i b u t i o n due t o the movement of the water mass. T h e r e f o r e , c e r t a i n s p a t i a l d i s t r i b u t i o n s may occur i n an e s t u a r y as an r e s u l t of t h i s p r o c e s s . 2. Freshwater phytoplankton can not pass through a s a l i n i t y g r a d i e n t . Most of them d i e as the s a l i n i t y i n c r e a s e s , and consequently mass m o r t a l i t y may occur i n an e s t u a r y . Freshwater phytoplankton can not f u n c t i o n as a u t o t r o p h i c components i n an e s t u a r i n e ecosystem but they c o n t r i b u t e to o r g a n i c substances. 3. Seawater phytoplankton can q u i c k l y respond and grow i n a s a l i n i t y g r a d i e n t and a maximum i n biomass appears a t the high end of the s a l i n i t y g r a d i e n t . As a r e s u l t , a u t o t r o p h i c p r o d u c t i o n i n an e s t u a r y i s p r i m a r i l y the r e s u l t of marine s p e c i e s and maximum p r o d u c t i o n occurs at some d i s t a n c e from the mouth of an e s t u a r y . 4. The energy flow through the m i c r o b i a l pathway becomes r e l a t i v e l y important i f the ecosystem has a high l e v e l of i n t e r n a l energy which can be c r e a t e d by a l l o c h t h o n o u s o r g a n i c substances. The m i c r o b i a l pathway which i s made up of b a c t e r i a and f l a g e l l a t e s may have a g r e a t p o t e n t i a l a b i l i t y t o c o n t r i b u t e to the carbon energy flow and a l l o w the whole ecosystem to r e t u r n t o a low energy s t a t e as soon as p o s s i b l e . 107 5. A S a l i n i t y g r a d i e n t does not seem t o have a str o n g e f f e c t on b a c t e r i a l growth. M i c r o f l a g e l l a t e s can a f f e c t the b a c t e r i a l p o p u l a t i o n dynamics. B a c t e r i a may s h i f t t h e i r s p e c i e s composition w i t h i n a p o p u l a t i o n t o o b t a i n a c l o s e s u c c e s s i o n and to adapt to d i f f e r e n t c o n d i t i o n s . 6. The b a c t e r i a l component i s mainly d e c i d e d by org a n i c substances which a l l o w growth t o an upper l i m i t , and they are then e x p l o i t e d by m i c r o z o o f l a g e l l a t e s and a lower l i m i t i s set by a f e e d i n g t h r e s h o l d . There i s no dynamic e q u i l i b r i u m between these two process when o r g a n i c energy sources are not l i m i t e d . 7. A h y p o t h e t i c a l model of energy l e v e l f i e l d theory i s proposed t o e x p l a i n the dynamic s t r u c t u r e of the ecosystem and the f u n c t i o n of a u t o t r o p h i c and h e t e r o t r o p h i c components i n an ecosystem. 8. H e t e r o t r o p h i c p r o d u c t i o n occurs maximally near the mouth of the e s t u a r y and i s the r e s u l t of al l o c h t h o n o u s substances as w e l l as the decay of freshwater phytoplankton i n the sea. 9. H e t e r o t r o p h i c p r o d u c t i o n i s more u b i q u i t o u s i n the system, being made up of both freshwater and marine s p e c i e s . A b a c t e r i a l maximum may be expected at the area of the phytoplankton bloom but a l s o a t c e r t a i n depths as a r e s u l t of s i n k i n g of o r g a n i c m a t e r i a l s . 10. The e s t u a r i n e c i r c u l a t i o n t r a n s p o r t s m a t e r i a l s along a s a l i n i t y g r a d i e n t t o connect the ecosystem components of d i f f e r e n t t r o p h i c f u n c t i o n s which are separated by the s a l i n i t y g r a d i e n t over space. T h i s r e s u l t s i n a complete c y c l i n g p r o c e s s . 108 11. In the mouth of an e s t u a r y , c e r t a i n c o n d i t i o n s may e x i s t b r i e f l y as i n a n a t u r a l chemostat environment which p r o v i d e s longer p e r i o d s of high s t a n d i n g stock biomass. 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