UBC Theses and Dissertations

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UBC Theses and Dissertations

The trophic relationships between suspended marine bacteria and the suspension-feeders Mytilus edulis… McKeag, Maura Anne 1983

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THE TROPHIC RELATIONSHIPS BETWEEN SUSPENDED MARINE BACTERIA AND THE SUSPENSION-FEEDERS MYTILUS EPULIS AND ARTEMIA SALINA by MAURA ANNE MCKEAG B . S c , Queens U n i v e r s i t y , K i n g s t o n , 1980 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 a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA May 1983 (c) Maura Anne McKeag, 1983 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) i i ABSTRACT The t r o p h i c r e l a t i o n s h i p s , i n terms of growth e f f i c i e n c i e s , between suspended marine b a c t e r i a and the s u s p e n s i o n f e e d e r s , the b l u e m u s s e l , M y t i l u s e d u l i s and the b r i n e s hrimp, A r t e m i a  s a l i n a , were e s t a b l i s h e d . M y t i l u s e d u l i s c o u l d not be s u p p o r t e d on suspended b a c t e r i a . The growth e f f i c i e n c y of A . s a l i n a was dependent upon both the c o n c e n t r a t i o n of b a c t e r i a i n the c u l t u r e p r o v i d e d as a food source and on the s i z e of the b r i n e shrimp. At c o n c e n t r a t i o n s l e s s than 1.5 X 10 s c e l l s / m l , young shrimp, l e s s than 1 mm i n l e n g t h , d i e d w i t h i n a few days ( z e r o growth e f f i c i e n c y ) . C o r r e s p o n d i n g l y , low f i l t e r i n g r a t e s ( l e s s than 1 ml/hour/organism) and low consumption r a t e s ( l e s s than 0.1 Mg/hour/organism) were o b s e r v e d f o r t h e s e organisms a t such low c o n c e n t r a t i o n s . For b r i n e shrimp g r e a t e r i n l e n g t h than 1.0 mm, a b a c t e r i a l c o n c e n t r a t i o n of 2.5 X 10 6 c e l l s / m l was r e q u i r e d b e f o r e p o s i t i v e growth e f f i c i e n c i e s were o b t a i n e d . As the food c o n c e n t r a t i o n i n c r e a s e d beyond t h i s c o n c e n t r a t i o n , growth e f f i c i e n c i e s s t e a d i l y i n c r e a s e d . An upper l i m i t f o r the c o n c e n t r a t i o n of b a c t e r i a l c e l l s t h a t c o u l d be c o n v e r t e d i n t o the biomass of A . s a l i n a was not d e t e c t e d ; the growth e f f i c i e n c i e s c o n t i n u e d t o i n c r e a s e t o a maximum of 60% as the b a c t e r i a l c o n c e n t r a t i o n s s u p p l i e d i n c r e a s e d t o 10 7 c e l l s / m l . The growth e f f i c i e n c i e s were maximal when A r t e m i a s a l i n a o b t a i n e d a l e n g t h of 2.5 mm, a t which time the h i g h e s t consumption r a t e s and growth r a t e s were a l s o o b s e r v e d . Growth e f f i c i e n c i e s f o r organisms l a r g e r or s m a l l e r than 2.5 mm were l o w e r , B a c t e r i a l d e n s i t i e s , e x p r e s s e d i n terms of both c e l l s per ml and the amount of ATP per ml, s u p p o r t e d from v a r i o u s t y p e s of o r g a n i c s u b s t r a t e s , were d e t e r m i n e d under v a r y i n g i n o r g a n i c n u t r i e n t and oxygen regimes. The s u b s t r a t e s s t u d i e d i n c l u d e d the seaweeds, U l v a l a c t u c a and Fucus v e s i c u l o s u s , and the v a s c u l a r p l a n t s , Z o s t e r a marina and wood c h i p s . Under n u t r i e n t - r i c h c o n d i t i o n s (30 MM NO 3",6.0 MM POi," 3 ), b a c t e r i a l d e n s i t i e s s u p p o r t e d from 1 g ( d r y weight) of U l v a l a c t u c a reached a maximum of 2 X 10 7 c e l l s / m l or 12 X 10" 3 nq ATP/ml. Based on the e s t a b l i s h e d t r o p h i c r e l a t i o n s h i p s , i t was c a l c u l a t e d t h a t the amount of suspended b a c t e r i a per gram d r y weight of s u b s t r a t e grown under t h e s e c o n d i t i o n s can s u s t a i n a maximum weight of 46 jug of A r t e m i a s a l i n a or a d u l t b r i n e shrimp. i v TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS x INTRODUCTION 1 Overview 1 The Study A n i m a l s 2 T r o p h i c R e l a t i o n s h i p s 3 I n d i c a t o r s of B a c t e r i a l D e n s i t i e s 6 ATP A n a l y s i s 6 E p i f l u o r e s c e n c e A n a l y s i s 10 B a c t e r i a l D e n s i t i e s ' 11 Purpose of Study 16 METHODS and MATERIALS 17 B a c t e r i a l Growth 17 C u l t u r e Techniques 17 E p i f l u o r e s c e n c e A n a l y s i s 19 ATP A n a l y s i s 21 U n i t s f o r B a c t e r i a l D e n s i t y 23 S t a t i s t i c a l A n a l y s i s 23 De c o m p o s i t i o n Rates 24 Growth and F e e d i n g of Sus p e n s i o n - F e e d e r s 25 Growth E x p e r i m e n t s 25 ( i ) The Source of Food 25 V ( i i ) M y t i l u s e d u l i s 25 ( i i i ) A r t e m i a s a l i n a 26 Fe e d i n g Experiments 27 ( i ) C a l c u l a t i o n of F i l t e r i n g Rates 27 ( i i ) M y t i l u s e d u l i s 27 ( i i i ) A r t e m i a s a l i n a 28 Growth E f f i c i e n c i e s 29 RESULTS 30 B a c t e r i a l Growth and D e n s i t i e s 30 The Growth Curve 30 The N u t r i e n t Content of the S u b s t r a t e s 30 The E f f e c t of D i f f e r e n t O r g a n i c S u b s t r a t e s on B a c t e r i a l D e n s i t i e s 31 ( i ) S i m i l a r S i z e P a r t i c l e s , Same S'ubtrate P r e p a r a t i o n 31 ( i i ) S i m i l a r S i z e P a r t i c l e s , D i f f e r e n t S u b s t r a t e P r e p a r a t i o n s 31 ( i i i ) D i f f e r e n t S i z e P a r t i c l e s , Same S u b s t r a t e P r e p a r a t i o n 32 The E f f e c t of S p e c i f i c E x p e r i m e n t a l C o n d i t i o n s 32 Dec o m p o s i t i o n Rates 33 Growth of B a c t e r i a l G r a z e r s 34 M y t i l u s e d u l i s 34 A r t e m i a s a l i n a 34 DISCUSSION 37 B a c t e r i a l Growth and D e n s i t i e s 37 E p i f l u o r e s c e n c e v e r s u s ATP 37 v i The Growth P a t t e r n 38 The E f f e c t of D i f f e r e n t O r g a n i c S u b s t r a t e s on B a c t e r i a l Growth and D e n s i t i e s 39 ( i ) S i m i l a r S i z e P a r t i c l e s , Same S u b s t r a t e P r e p a r a t i o n 39 ( i i ) D i f f e r e n t S i z e P a r t i c l e s , Same S u b s t r a t e P r e p a r a t i o n 41 ( i i i ) S i m i l a r S i z e P a r t i c l e s , D i f f e r e n t S u b s t r a t e P r e p a r a t i o n 42 The E f f e c t of S p e c i f i c E x p e r i m e n t a l C o n d i t i o n s 43 ( i ) I n o r g a n i c N u t r i e n t s 43 ( i i ) Oxygen 44 Deco m p o s i t i o n Rates 45 The Growth of Su s p e n s i o n - F e e d e r s 46 M y t i l u s e d u l i s 46 A r t e m i a s a l i n a 47 Summary 51 CONCLUSIONS 52 REFERENCES 55 TABLES 70 FIGURES 77 LIST OF TABLES T a b l e 1. The s u r f a c e a r e a of the s u b s t r a t e p a r t i c l e s '71 Tab l e 2. A summary of the c o n d i t i o n s of the b a c t e r i a l growth e x p e r i m e n t s 72 T a b l e 3. The e x p e r i m e n t a l c o n d i t i o n s used t o det e r m i n e the growth r a t e s of M y t i l u s e d u l i s 73 T a b l e 4. The r e l a t i v e p e r c e n t s of t o t a l c a r b o n , hydrogen and n i t r o g e n w i t h i n the s u b s t r a t e s . 74 Tab l e 5. D e c o m p o s i t i o n r a t e s 75 v i i i LIST OF FIGURES F i g u r e 1. A t y p i c a l b a c t e r i a l growth c u r v e 78 F i g u r e 2. The suspended b a c t e r i a l biomass i n uqC over time 80 F i g u r e 3. B a c t e r i a l d e n s i t i e s per gram of s u b s t r a t e t h a t can be s u p p o r t e d w i t h d i f f e r e n t amounts of Z o s t e r a marina 82 F i g u r e 4. The e f f e c t of a u t o c l a v e d s u b s t r a t e s on the number of b a c t e r i a 84 F i g u r e 5. The e f f e c t of a u t o c l a v e d s u b s t r a t e s on b a c t e r i a l ATP 86 F i g u r e 6. The e f f e c t of d r i e d s u b s t r a t e s on b a c t e r i a l d e n s i t i e s 88 F i g u r e 7. The e f f e c t of a u t o c l a v e d and d r i e d , l a r g e s u b s t r a t e p a r t i c l e s on the number of b a c t e r i a 90 F i g u r e 8. The e f f e c t of a u t o c l a v e d and d r i e d , l a r g e s u b s t r a t e p a r t i c l e s on b a c t e r i a l ATP 92 F i g u r e 9. The e f f e c t of a u t o c l a v e d and d r i e d , s m a l l s u b s t r a t e p a r t i c l e s on b a c t e r i a l d e n s i t i e s 94 F i g u r e 10. The e f f e c t of s m a l l and l a r g e , a u t o c l a v e d s u b s t r a t e p a r t i c l e s on b a c t e r i a l d e n s i t i e s 96 F i g u r e 11. The e f f e c t of s m a l l and l a r g e , d r i e d s u b s t r a t e p a r t i c l e s on b a c t e r i a l d e n s i t i e s 98 F i g u r e 12. The e f f e c t of n u t r r e n t c o n d i t i o n s w i t h a u t o c l a v e d s u s t r a t e s on b a c t e r i a l d e n s i t i e s 101 F i g u r e 13. The e f f e c t of n u t r i e n t - d e p l e t e d c o n d i t i o n s on i x the number of b a c t e r i a s u p p o r t e d from v a r i o u s s u b s t r a t e s 1 04 F i g u r e 14. The e f f e c t of a e r o b i c and a n a e r o b i c c o n d i t i o n s on t h e number of b a c t e r i a s u p p o r t e d from v a r i o u s s u b s t r a t e s 106 F i g u r e 15. F i l t e r i n g Rates of M y t i l u s e d u l i s 108 F i g u r e 16. Growth of A r t e m i a s a l i n a when s u p p l i e d w i t h b a c t e r i a l c u l t u r e s of c e l l c o n c e n t r a t i o n s g r e a t e r than l o g 6.2 ..110 F i g u r e 17. Growth of A r t e m i a s a l i n a when s u p p l i e d w i t h b a c t e r i a l c u l t u r e s of c e l l c o n c e n t r a t i o n s l e s s than l o g .6.2 1 1 2 F i g u r e 18. Growth Rates of A r t e m i a s a l i n a 114 F i g u r e 19. F i l t e r i n g Rates (FR) of A r t e m i a s a l i n a 116 F i g u r e 20. Growth E f f i c i e n c i e s of A r t e m i a s a l i n a 118 F i g u r e 21. Consumption Rates of A r t e m i a s a l i n a .....120 F i g u r e 22. The l e n g t h of A r t e m i a s a l i n a t h a t can be o b t a i n e d when s u p p l i e d w i t h d i f f e r e n t c o n c e n t r a t i o n s of suspended b a c t e r i a 122 F i g u r e 23. The r e l a t i o n s h i p between t o t a l n i t r o g e n c o n t e n t of the s u b s t r a t e and the number of b a c t e r i a s u p p o r t e d ..124 F i g u r e 24. The weight of A r t e m i a s a l i n a t h a t can be o b t a i n e d from b a c t e r i a l c u l t u r e s grown under v a r i o u s e x p e r i m e n t a l c o n d i t i o n s 126 X ACKNOWLEDGEMENTS I would l i k e t o extend my s i n c e r e thanks t o my s u p e r v i s o r , Dr. T.R. . Parsons,- f o r h i s guidance and a d v i c e throughout the s t u d y . My thanks a r e a l s o extended t o Dr. John P a r s l o w f o r h i s e x p l a n a t i o n s on the s t a t i s t i c a l a n a l y s i s . I w i s h t o thank the Department of Zoology f o r p r o v i d i n g me w i t h T e a c h i n g A s s i s t a n t s h i p s and my s u p e r v i s o r f o r h i s f i n a n c i a l a s s i s t a n c e . F i n a l l y I owe a s p e c i a l thank you t o Andy Thomas f o r h i s c o n t i n u o u s encouragement. H i s h e l p f u l computer t i p s and p a t i e n t e d i t i n g work were g r e a t l y a p p r e c i a t e d . 1 INTRODUCTION Overview The b a c t e r i a l component of the d e t r i t a l food c h a i n i s r e c o g n i z e d as an i m p o r t a n t f a c t o r c o n t r o l l i n g the o v e r a l l p r o d u c t i v i t y of an e s t u a r i n e ecosystem ( S i b e r t and Naiman,1980). By a s s i m i l a t i n g o r g a n i c matter and c o n v e r t i n g i t i n t o biomass, marine b a c t e r i a c o n s t i t u t e an i m p o r t a n t r o u t e f o r the f l o w of matter and energy through marine food webs (Azam and Hodson,l977) and by g r a z i n g on t h i s b a c t e r i a l biomass, many marine organisms meet t h e i r n u t r i t i o n a l r e q u i r e m e n t s (Jorgensen,1966; Boucherand and Chamroux,1976). B a c t e r i a can e x i s t as c e l l s a t t a c h e d t o p a r t i c l e s , as a g g r e g a t e s , or as suspended c e l l s (Jones and Jannasch,1956; R i l e y , 1 9 6 3 ) . The importance of the a t t a c h e d b a c t e r i a as a food source f o r many g r a z e r s has been w e l l e s t a b l i s h e d . By c o l o n i z i n g d e t r i t a l p a r t i c l e s , b a c t e r i a i n c r e a s e the n u t r i t i o n a l v a l u e of the p a r t i c l e s f o r the consumer (Newell,1965; F e n c h e l , 1 970; Odum et e/1.,1973). D e t r i t u s has minor food v a l u e , and the a t t a c h e d b a c t e r i a form the major source of h i g h p r o t e i n food (Frankenberg and Smith,1967; Fenchel,1972; M e y e r - R e i l and Faubel,1980; F i n d l a y and Tenore,1982). T h i s has been shown t o be the case f o r many d e t r i t i v o r e s such as o l i g o c h a e t e s ( G i e r e , l 9 7 5 ; C O U 1 1 , 1 9 7 3 ) and p o l y c h a e t e s (Tenore e t a l . , 1 9 7 9 ; Tenore and Hanson,1980). B a c t e r i a l a g g r e g a t e s have a l s o been shown t o be an i m p o r t a n t food s o u r c e f o r many p r i m a r y consumers 2 such as z o o p l a n k t o n ( P r o v a s o l i et a l . , 1 9 5 9 ; S e k i , 1 9 6 6 a ; S e k i and Kennedy,1969; S o r o k i n e t a l . , 1 9 7 0 ; R i e p e r , 1 9 7 8 ) , sponges ( R e i s w i g , 1 9 7 5 ) , g a s t r o p o d s ( S o r o k i n , 1 9 6 8 ) , mussels ( S c h l e y e r , 1 9 8 1 ) , and o y s t e r s ( S o r o k i n , 1 9 6 8 ) . The importance of suspended b a c t e r i a as a food source f o r many organisms has not been i n v e s t i g a t e d as t h o r o u g h l y as t h a t of a t t a c h e d and aggr e g a t e d b a c t e r i a . In t h i s s tudy the term f r e e - l i v i n g b a c t e r i a r e f e r s t o suspended b a c t e r i a and s h o u l d not be c o n f u s e d w i t h n o n - s y m b i o t i c b a c t e r i a . R e s e a r c h e r s r e c o g n i z e t h a t i n t e r a c t i o n s between suspended b a c t e r i a and b a c t e r i a l g r a z e r s e x i s t , but emphasis i s p l a c e d on s u c c e s s i o n a l changes i n the number of b a c t e r i a l c e l l s and b a c t e r i a l g r a z e r s over t i m e , not on the s p e c i f i c t r o p h i c r e l a t i o n s h i p s (Hamilton,1973; Fenchel,1982a,1982b). The Study A n i m a l s Both M y t i l u s e d u l i s ( L ) and A r t e m i a s a l i n a ( L ) a r e s u s p e n s i o n - f e e d e r s and o b t a i n t h e i r food by f i l t e r i n g water and r e t a i n i n g the suspended p a r t i c l e s ( J o r gensen,1966). A r t e m i a  s a l i n a i s c l a s s i f i e d as a n o n - f i l t e r i n g s u s p e n s i o n - f e e d e r s i n c e the p r o d u c t i o n of water c u r r e n t s and food uptake a re performed by the same org a n s . M y t i l u s e d u l i s i s a f i l t e r i n g s u s p e n s i o n -f e e d e r as the s e a c t i v i t i e s a r e performed by d i f f e r e n t appendages ( S e k i , 1 9 8 2 ) , A c o n t r o v e r s y e x i s t s i n the l i t e r a t u r e r e g a r d i n g the a b i l i t y of M . e d u l i s t o su p p o r t i t s e l f w i t h suspended marine b a c t e r i a as i t s o n l y source of f o o d . H o l l i b a u g h e t a l . ( l 9 8 0 ) 3 showed t h a t the b l u e mussel was c a p a b l e of f e e d i n g on f r e e -l i v i n g b a c t e r i a as some b a c t e r i a l r e t e n t i o n was observed over t i m e . However, Wright e t a l . ( l 9 8 2 ) demonstrated t h a t t h i s o r ganism c o u l d not e x i s t beyond a few days when s u p p l i e d w i t h f r e e - l i v i n g b a c t e r i a as i t s o n l y food s o u r c e . S e k i (1966b) d e v e l o p e d a chemostat c o n s i s t i n g of d i s s o l v e d o r g a n i c m a t t e r (DOM), b a c t e r i a and p r o t o z o a which were s u p p l i e d t o A . s a l i n a . In t h i s p a r t i c u l a r system, the b r i n e shrimp reached s e x u a l m a t u r i t y i n 20 days. S e k i (1966b) suggested t h a t the b r i n e shrimp were n o u r i s h e d c h i e f l y by g r a z i n g the p r o t o z o a . However, the b a c t e r i a may have a l s o s u s t a i n e d the growth of s u s p e n s i o n - f e e d e r s as L i n l e y and N e w e l l (1981) sug g e s t e d . S e k i e t a l . (1968) d i d , however, demonstrate t h a t suspended b a c t e r i a , grown on ba c t o p e p t o n e , e n a b l e d b r i n e shrimp t o grow, but the e x t e n t of the growth dependency was not e s t a b l i s h e d . S i n c e t r o p h i c r e l a t i o n s h i p s between f r e e - l i v i n g b a c t e r i a and the s u s p e n s i o n - f e e d e r s , A r t e m i a s a l i n a (L) and M y t i l u s  e d u l i s ( L ) , have not been w e l l e s t a b l i s h e d t h e s e organisms were chosen f o r t h i s s t u d y . T r o p h i c R e l a t i o n s h i p s The term t r o p h i c r e l a t i o n s h i p used i n t h i s s tudy r e f e r s t o the t r a n s f e r of energy between organisms w i t h i n s u c c e s s i v e t r o p h i c l e v e l s of the food web. A v a l u a b l e t o o l i n s t u d i e s of t r o p h i c r e l a t i o n s h i p s i s 'Growth E f f i c i e n c y ' (GE). GE measures the e f f i c i e n c y w i t h which an organism c o n v e r t s the food a v a i l a b l e i n t o i t s own body t i s s u e (Reeve,1963a). The energy o b t a i n e d 4 from the a v a i l a b l e food i s a s s i m i l a t e d i n t o the organism's biomass ( P a r s o n s et a l . , 1 9 7 7 ) . The a s s i m i l a t e d energy i s d i r e c t e d i n t o pathways f o r growth, m e t a b o l i c r e q u i r e m e n t s , and e x c r e t i o n . Growth i s d e f i n e d i n t h i s s tudy as the i n c r e a s e i n body weight over time and t r o p h i c r e l a t i o n s h i p s w i l l be de t e r m i n e d o n l y i n terms of g r o s s GEs which o n l y account f o r the energy used f o r growth. I t has been w e l l e s t a b l i s h e d t h a t GE depends on the c o n c e n t r a t i o n of the suspended p a r t i c l e s s u p p l i e d t o the organism. The e f f i c i e n c y of the c o n v e r s i o n of the food i n t o the biomass of the s u s p e n s i o n - f e e d e r i s a f u n c t i o n of the food i n t a k e , s i n c e t h i s d e t e r m i n e s the amount of food energy a v a i l a b l e f o r growth. Food i n t a k e w i l l v a r y w i t h the f i l t e r i n g r a t e of the s u s p e n s i o n - f e e d e r . F i l t e r i n g r a t e s measure the volume of medium t h a t passes the f o o d - c a t c h i n g a p p a r a t u s w i t h i n a c e r t a i n p e r i o d of time and, t h e r e f o r e , w i l l a f f e c t the number of p a r t i c l e s t h a t p o t e n t i a l l y can be r e t a i n e d (Gauld,1951). A minimum number of p a r t i c l e s may be r e q u i r e d b e f o r e f i l t e r i n g commences ( F r o s t , 1 9 7 5 ) and the e f f i c i e n c y of p a r t i c l e r e t e n t i o n a t low food c o n c e n t r a t i o n s may be poor, as observed f o r Daphnia  magna ( R i g l e r , 1 9 6 1 ) and f o r A r t e m i a s a l i n a (Reeve,1963b,c). T h e o r e t i c a l l y , as the food c o n c e n t r a t i o n i n c r e a s e s , f i l t e r i n g r a t e s d e c l i n e . T h i s was shown t o be the case f o r copepods f e e d i n g on p h y t o p l a n k t o n c e l l s ( M a r s h a l l and O r r , l 9 5 5 ; Conover,1966; M u l l i n , l 9 6 3 ; F r o s t , 1 9 7 2 ) . As the f i l t e r i n g r a t e d e c l i n e s , the r a t e of food i n t a k e or consumption r a t e may e i t h e r remain c o n s t a n t or d e c l i n e ( P a rsons e t a l . , 1 9 7 7 ) . However, as 5 Gaudy (1974) c l e a r l y shows, a s s i m i l a t i o n r a t e may i n c r e a s e w i t h d e c r e a s i n g food i n t a k e , p r o b a b l y because a t the low food c o n c e n t r a t i o n s , the suspended c e l l s remain i n the gut l o n g e r due t o the lower f l u s h i n g r a t e of the i n t e s t i n e (Reeve,1963d). The l o n g e r the food s t a y s i n the g u t , the g r e a t e r the d i g e s t i v e e f f i c i e n c y , and p o s s i b l y growth a s s i m i l a t i o n . T h e r e f o r e , a t lower food c o n c e n t r a t i o n s peak GEs a r e ob s e r v e d . T h i s r e l a t i o n s h i p between food c o n c e n t r a t i o n s and GEs i s found f o r bot h A r t e m i a s a l i n a ( G i l b o r , 1 9 5 7 ; Reeve,1963a) and Myt i l u s  e d u l i s (Jorgensen,1952; S c h u t t l e , 1 9 7 5 ) as w e l l as o t h e r s u s p e n s i o n - f e e d e r s , such as e u p h a u s i i d s (Lasker,1960) and copepods ( M a r s h a l l and O r r , l 9 5 5 ; Conover,1961) which were f e d p h y t o p l a n k t o n . GEs a l s o v a r y , w i t h .. t h e s i z e of the organism, g e n e r a l l y d e c r e a s i n g w i t h age (Brody,1945; Richman,1958). The amount of energy used f o r growth i n young and t h e r e f o r e s m a l l e r organisms i s g r e a t e r than the amount used i n l a r g e r and o l d e r organisms (P a r s o n s et a l . , 1 9 7 7 ) . A p e r i o d of maximum GEs d u r i n g the a n i m a l ' s l i f e , may be observed (Makarova and Ye Z a i k a , l 9 7 1 ) . At t h i s p o i n t a minimum amount of energy i s r e q u i r e d f o r m e t a b o l i c f u n c t i o n s and maximum growth r a t e s can be o b t a i n e d . As the organis m approaches s e x u a l m a t u r i t y , the energy used f o r growth approaches z e r o , hence GE d e c r e a s e s and approaches z e r o . The p e r i o d of i n c r e a s i n g GE t o a maximum may a l s o be due t o the m o r p h o l o g i c a l changes t a k i n g p l a c e i n the organism over t i m e . As the s u s p e n s i o n - f e e d e r i n c r e a s e s i n s i z e , the f i l t e r i n g appendages become more complex and more e f f i c i e n t i n r e t a i n i n g 6 p a r t i c l e s (Gauld,1959). However, once the energy r e q u i r e d f o r body maintenance of the organism exceeds t h a t f o r growth, GEs w i l l d e c r e a s e d e s p i t e the e f f i c i e n c y of the f i l t e r i n g a p p a r a t u s . P u b l i s h e d GEs f o r b r i n e shrimp and b l u e mussels have been p r i m a r i l y d e t e r m i n e d u s i n g a p h y t o p l a n k t o n food s o u r c e ( G i l b o r , 1 9 5 7 ; Reeve,1963a; H o l l i b a u g h et a l . , 1 9 8 0 ; Wright et a l . , 1 9 8 2 ) . L i t t l e emphasis has been p l a c e d on the importance of suspended b a c t e r i a i n c o n t r o l l i n g growth. I n d i c a t o r s of B a c t e r i a l D e n s i t i e s S e v e r a l methods a r e a v a i l a b l e t o d e t e r m i n e b a c t e r i a l d e n s i t i e s ; however, the suspended b a c t e r i a l d e n s i t i e s i n t h i s s t u d y were d e t e r m i n e d from the amount of c e l l u l a r adenosine t r i p h o s p h a t e (ATP) and by d i r e c t c o u n t i n g under a m i c r o s c o p e . ATP A n a l y s i s ATP has been used e x t e n s i v e l y as an i n d i c a t o r of m i c r o b i a l d e n s i t y s i n c e i t s development by Holm-Hansen and Booth i n 1966. The a n a l y s i s i s based on a c h e m i c a l r e a c t i o n which i n v o l v e s the e m i s s i o n of l i g h t . I n the presence of l u c i f e r i n and the enzyme l u c i f e r a s e , ATP i s h y d r o l y z e d . For every m o l e c u l e of ATP p r e s e n t , one photon of l i g h t i s e m i t t e d . The r e a c t i o n proceeds as i n d i c a t e d i n the f o l l o w i n g e q u a t i o n : 7 l u c i f e r a s e LUCIFERIN + ATP + 0 2 -> LUCIFERIN + AMP + P-P + hv (reduced) M g 2 + ( o x i d i z e d ) When a l l r e a g e n t s a re i n e x c e s s , the l i g h t i n t e n s i t y , hv, i s d i r e c t l y p r o p o r t i o n a l t o the c o n c e n t r a t i o n of ATP. Magnesium i s added t o extend the time of peak l i g h t i n t e n s i t y which d e c l i n e s e x p o n e n t i a l l y (McElroy and S t r e h l e r , 1 9 4 9 ) . In o r d e r t o o b t a i n r e p r o d u c e a b l e r e s u l t s , the i n t e n s i t y of l i g h t produced from the samples s h o u l d be measured a f t e r a c o n s t a n t time (Holm-Hansen,1973). The amount of ATP i n the sample i s de t e r m i n e d by comparison w i t h a s e t a s t a n d a r d s . When u s i n g the crude enzyme e x t r a c t of the f i r e f l y , the l i m i t of d e t e c t i o n i s 10"" uq ATP/ml (Holm-Hansen and Booth,1966). F i r e f l y l u c i f e r a s e was o r i g i n a l l y thought t o be s p e c i f i c f o r ATP (McEl r o y and S t r e h l e r , 1 9 4 9 ) y e t i t i s l i k e l y t h a t o t h e r m o l e c u l e s c o n t a i n i n g h i g h - e n e r g y phosphate bonds a l s o cause some l i g h t e m i s s i o n . Adenosine d i p h o s p h a t e (ADP), g u a n o s i n e t r i p h o s p h a t e (GTP), coenzyme A, p l u s a v a r i e t y of o t h e r n u c l e o s i d e t r i p h o s p h a t e s emit some l i g h t i n the presence of the f i r e f l y e x t r a c t (Holm-Hansen and Booth,1966); however, the a u t h o r s c o n c l u d e t h a t t h e s e compounds a r e not of g r e a t importance when e s t i m a t i n g b a c t e r i a l d e n s i t i e s by t h i s t e c h n i q u e s i n c e they a r e i n low c o n c e n t r a t i o n s . The r e l i a b i l i t y of the method i s based on s e v e r a l assumpt i o n s . 1. ATP i s a c o n s t i t u e n t of a l l l i v i n g c e l l s . 8 ATP i s one of the most im p o r t a n t low m o l e c u l a r weight compounds and i s u b i q u i t o u s i n a l l l i v i n g organisms. The m o l e c u l e undergoes d e p h o s p h o r y l a t i o n t o r e l e a s e energy f o r such m e t a b o l i c p r o c e s s e s as g l y c o l y s i s , f a t t y a c i d s y n t h e s i s and o x i d a t i o n , p r o t e i n and n u c l e i c a c i d s y n t h e s i s ( B u l l e i d , 1 9 7 7 ) and i s a l s o r e s p o n s i b l e f o r the e n e r g y - t r a n s f e r p r o c e s s a s s o c i a t e d w i t h l i g h t p r o d u c t i o n i n b i o l u m i n e s c e n t o r g anisms. 2. ATP i s not a s s o c i a t e d w i t h dead c e l l s . Holm-Hansen (1973) found t h a t when ATP was added t o a c u l t u r e of dead c e l l s and the s o l u t i o n was f i l t e r e d , ATP was not d e t e c t e d on the f i l t e r . He, t h e r e f o r e , c o n c l u d e d not o n l y t h a t ATP was not p r e s e n t i n dead c e l l s , but t h a t i t d i d . not adsorb onto d e t r i t a l p a r t i c l e s . 3. The c e l l u l a r c o n c e n t r a t i o n of ATP i s c o n s t a n t d u r i n g the a n a l y t i c a l p r o c e d u r e . Changes i n the ATP p o o l of an organism can occur when c e l l s a r e s t r e s s e d . Hodson and Azam (1977) suggest t h a t one form of s t r e s s c o u l d be f i l t r a t i o n and t h a t t o m i n i m i z e t h i s , the f i l t r a t i o n p e r i o d s h o u l d be q u i t e s h o r t . T h i s i s a c h i e v e d by f i l t e r i n g s m a l l volumes of medium ( l e s s than a l i t r e ) ( S u t c l i f f e and O r r , 1 9 7 6 ) . L y s i n g , a n o t h e r form of s t r e s s , i s m i n i m i z e d by e n s u r i n g t h a t the f i l t e r i n g vacuum p r e s s u r e does not exceed 8 atm (Holm-Hansen,1973). The e x t r a c t i o n p r o c e d u r e i s d e s i g n e d t o ensure t h a t no changes i n the l e v e l s of ATP o c c u r . TRIS ( t r i s hydroxymethyl-aminomethane) i s t h e most e f f e c t i v e 9 e x t r a c t i n g reagent f o r seawater samples (Holm-Hansen and Booth,1966; Hamilton,1973; S u t c l i f f e and O r r , l 9 7 6 ; Hodson et a l . , 1 9 7 6 ) . TRIS b u f f e r e f f e c t i v e l y p r e v e n t s the breakdown of ATP by d e n a t u r i n g any p h o s p h o r y l a t i n g enzymes t h a t a r e p r e s e n t ( H a m i l t o n and Holm-Hansen,1967). I t i s e s s e n t i a l t h a t the l i v i n g c e l l s a re k i l l e d i n s t a n t l y . In o r d e r t o do t h i s the b u f f e r must be c l o s e t o 100°C i n o r d e r t o r u p t u r e the c e l l w a l l s and r e l e a s e i n t r a c e l l u l a r ATP ( B u l l e i d , 1 9 7 7 ; Jones and Simon,1977). Holm-Hansen e t a l . ( l 9 6 8 ) suggest t h a t the pH of the b u f f e r must be m i l d l y a l k a l i n e (pH 7.7) t o p r e v e n t the p r e c i p i t a t i o n of the TRIS b u f f e r which l o w e r s the luminescence ( B u l l e i d , 1 9 7 8 ; P e r r y e t a l . , 1 9 7 9 ) . 4. The carbon t o ATP r a t i o i s c o n s t a n t . T h i s i s the most c r i t i c i z e d a s s u m p t i o n . The c e l l u l a r weight r a t i o of carbon t o ATP f o r b a c t e r i a has been shown t o v a r y c o n s i d e r a b l y between t a x a , c u l t u r e c o n d i t i o n s and s t a g e s of growth. The r a t i o can v a r y as much as 50% between d i f f e r e n t s p e c i e s of b a c t e r i a ( K a r l , 1 9 8 0 ) but i n marine b a c t e r i a t h e r a t i o i s around 250±25 (Banse,1980). N u t r i e n t - d e f i c i e n t c u l t u r e s t e n d t o have lower amounts of c e l l u l a r ATP, hence the r a t i o i s h i g h e r (Holm-Hansen,1969). ATP c o n c e n t r a t i o n s a l s o v a r y depending on the s t a g e of growth. F o r r e s t (1965) found t h a t the amount of ATP i n S t r e p t o c o c c u s f a e c a l i s reached a maximum d u r i n g e x p o n e n t i a l growth and the carbon t o ATP r a t i o per c e l l reached a minimum. However, when ATP c o n c e n t r a t i o n i s e x p r e s s e d i n terms of c e l l mass, no change i n the r a t i o i s observed 10 throughout the v a r i o u s phases of growth (Franzen and B i n k l e y , 1 9 6 1 ; A t k i n s o n and Walton,1967; Chapman and A t k i n s o n , 1 977 ) . E p i f l u o r e s c e n c e A n a l y s i s D i r e c t c o u n t i n g i s s t i l l b e l i e v e d by many r e s e a r c h e r s t o be the most r e l i a b l e method f o r e s t i m a t i n g b a c t e r i a d e n s i t y . The b e s t r e s o l u t i o n i s o b t a i n e d when a f l u o r e s c e n t s t a i n i s used and the c e l l s a r e viewed w i t h 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 (Daley and Hobbie,1975). In 1979, Daley m o d i f i e d and r e f i n e d the A c r i d i n e Orange D i r e c t C o u n t i n g (AODC) t e c h n i q u e and v e r i f i e d t h a t t h i s method i s the b e s t a v a i l a b l e procedure f o r a c c u r a t e l y c o u n t i n g n a t i v e a q u a t i c b a c t e r i a . To ensure r e p r o d u c e a b l e r e s u l t s the f i l t e r must remove a l l the c e l l s (Bowden,1977). Hobbie et a l . (1972) recommend u s i n g p o l y c a r b o n a t e N u c l e p o r e f i l t e r s (0.2 urn) because they have a u n i f o r m pore s i z e and a f l a t s u r f a c e t h a t r e t a i n s a l l the b a c t e r i a on top of the f i l t e r . A l l the b a c t e r i a l c e l l s must be v i s i b l e . A c r i d i n e Orange r e a c t s w i t h n u c l e i c a c i d s p r o d u c i n g a red-orange glow when i t b i n d s w i t h RNA, a compound more abundant i n a c t i v e l y growing c e l l s , and a green glow when i t b i n d s t o DNA, found i n i n a c t i v e c e l l s (Hobbie et a l . , 1 9 7 2 ) . The s t a i n e d c e l l s become c l e a r l y v i s i b l e when p l a c e d a g a i n s t a dark background and f o r t h i s reason I r a g a l a n B l a c k i s used t o s t a i n the N u c l e p o r e f i l t e r s ( D a l e y , 1 9 7 9 ) . The c e l l s must be non-aggregated p r i o r t o c o u n t i n g as a c c u r a t e c o u n t s can o n l y be o b t a i n e d i f the b a c t e r i a are e v e n l y d i s t r i b u t e d on the f i l t e r . Bowden .(1977) s u g g e s t s t h a t 11 a g g r e g a t i o n can be reduced by p l a c i n g a M i l l i p o r e f i l t e r , type GS, under the N u c l e p o r e f i l t e r . B a c t e r i a l D e n s i t i e s I t has been w e l l e s t a b l i s h e d t h a t suspended b a c t e r i a a r e n u t r i t i o n a l l y dependent upon d i s s o l v e d o r g a n i c matter (DOM) ( K h a i l o v and B u r l a k o v a , 1 9 6 9 ; S c h l e y e r , 1 9 8 1 ) and t h a t the amount and type of DOM c o n t r o l s b a c t e r i a l d e n s i t y (Jannasch,1967; Taga,l968; S i e b u r t h , 1 9 7 1 ) . By h a r v e s t i n g c o n s i d e r a b l e v. q u a n t i t i e s of DOM, b a c t e r i a may c o n v e r t i t i n t o t h e i r own biomass ( P a e r l , 1 9 7 4 , 1 9 7 8 ) . DOM c o n s i s t s of a p o o l of p r e d o m i n a n t l y macromolecular and c o l l o i d a l m a t e r i a l which i s v e r y r e s i s t a n t t o m i c r o b i a l d e g r a d a t i o n as w e l l as a much s m a l l e r p o o l of lower m o l e c u l a r weight compounds which can be used d i r e c t l y by b a c t e r i a ( F e n c h e l and B l a c k b u r n , 1 9 7 9 ) . T h i r t y p e r c e n t of the DOM p r e s e n t i n an e s t u a r i n e environment c o n s i s t s of c a r b o h y d r a t e s , 70% of which i s r e s i s t a n t t o m i c r o b i a l d e g a d a t i o n ( V e r l i m r o v e t a l . , 1 9 8 1 ) . G r e a t e r than 50% of the DOM i s r e p r e s e n t e d by compounds such as amino a c i d s and n o n - s t r u c t u r a l p r o t e i n s which are r e a d i l y u t i l i z e d by b a c t e r i a ( R i c e and Tenore,1981). The two major s o u r c e s of DOM f o r b a c t e r i a l uptake a r e p a r t i c u l a t e o r g a n i c matter (POM) and z o o p l a n k t o n . The POM r e l e a s e s DOM p r i m a r i l y t h r o u g h l e a c h i n g and b a c t e r i a l d e c o m p o s i t i o n and the z o o p l a n k t o n e x p e l DOM by e x c r e t i o n . L e a c h i n g i s the major p r o c e s s f o r DOM r e l e a s e ( H a r r i s o n and Mann,1975a,b) and t a k e s p l a c e i n the f i r s t few weeks a f t e r the 12 s u b s t r a t e i s p l a c e d i n seawater ( R i c e and Tenore,1981). B a c t e r i a l d e c o m p o s i t i o n becomes the most i m p o r t a n t p r o c e s s f o r DOM r e l e a s e when l e a c h i n g has s u b s i d e d . The amount of DOM r e l e a s e d by l e a c h i n g depends on the t y p e , age, s t a t e , and s i z e of the POM. A l g a l d e t r i t u s i s more s u s c e p t i b l e t o l e a c h i n g than v a s c u l a r p l a n t d e t r i t u s ( 0 1 h a , l 9 7 2 ; Tenore,1977b; Ric e , 1 9 7 9 ; Tenore and Hanson,1980). M a r i n e macroalgae l o s e about 60% of t h e i r g r o s s p r o d u c t i o n as DOM by l e a c h i n g ( K h a i l o v and Burlakova,1969) compared t o o n l y 40% f o r v a s c u l a r p l a n t s ( O t s u k i and Wetzel,1974) and 30% f o r deciduous l e a f l i t t e r (Cummins,1974). The age, s t a t e , and s i z e of the d e t r i t u s a l s o d e t e r m i n e s s u b s t r a t e - s u s c e p t i b i l i t y t o l e a c h i n g . H a r r i s o n and Mann (1975a) show t h a t young Z o s t e r a marina l o s e s more o r g a n i c m atter per day, e x p r e s s e d as a p e r c e n t a g e of t o t a l o r g a n i c m atter (TOM) than o l d Z.marina and o l d e e l g r a s s l o s e s more than dead p a r t i c l e s . K h a i l o v and B u r l a k o v a (1979) r e p o r t s i m i l a r f i n d i n g s f o r macroalgae. D r i e d Z o s t e r a marina r e l e a s e s more DOM than f r e s h Z o s t e r a mar i n a ( H a r r i s o n and Mann,1975b) and the amount of DOM e x p e l l e d from s m a l l p a r t i c l e s i s much g r e a t e r than t h a t r e l e a s e d from l a r g e r p a r t i c l e s ( G o s s e l i n k and K i r b y , l 9 7 4 ; H a r r i s o n and Mann,1975b). Once most of the l e a c h i n g has c e a sed, a l g a l d e t r i t u s i s decomposed more e a s i l y by m i c r o b e s than i s v a s c u l a r d e t r i t u s (Cummins et a l . , 1 9 7 3 ; G o s s e l i n k and K i r b y , l 9 7 4 ; Tenore,1977a; Tenore and Hanson,1980). The r e d seaweed G r a c i l a r i a f o l i i f e r a 13 i s r e a d i l y decomposed compared t o the d e c a y - r e s i s t a n t v a s c u l a r p l a n t , S p a r t i n a a l t e r n i f l o r a . (Tenore,1977b). R i c e and Tenore ( 1 9 8 1 ) r e p o r t e d the t o t a l o r g a n i c carbon (TOC) l o s t a f t e r 150 days when v a r i o u s types of s u b s t r a t e s were p l a c e d i n s t e r i l e s eawater. Red a l g a e l o s t 65%, the marshgrass, S p a r t i n a  a l t e r n i f l o r a , l o s t 20% and brown a l g a e l o s t an i n t e r m e d i a t e amount of around 35%. These d i f f e r e n c e s may be a t t r i b u t e d t o the amounts of l i g n i n , t o t a l n i t r o g e n , and p h e n o l i c r e s i d u e s ( N e w e l l and L U C U S , 1 9 8 1 ) . The c e l l w a l l of the o r g a n i c s u b s t r a t e i s the major d e t e r m i n a n t of the r e s i s t a n c e or s u s c e p t i b i l i t y t o m i c r o b i a l d e c o m p o s i t i o n (Gunnison and A l e x a n d e r , 1 9 7 5 a ) . R e s i s t a n c e i n c r e a s e s w i t h i n c r e a s i n g amounts of l i g n i n p r e s e n t i n the c e l l w a l l (Gunnison and A l e x a n d e r , 1 9 7 5 b ) . T y p i c a l l y , v a s c u l a r p l a n t s c o n t a i n l e s s t o t a l n i t r o g e n than seaweeds ( T e n o r e , 1 9 8 3 ) . S u b s t r a t e s which are h i g h i n t o t a l n i t r o g e n e n a b l e a g r e a t e r i n i t i a l r a t e of b a c t e r i a l d e c o m p o s i t i o n (R i c e , 1 9 7 9 ; Tenore et a l . , 1 9 7 9 ) . The r e m i n e r a l i z a t i o n of o r g a n i c n i t r o g e n i s c o n s i d e r a b l y more r a p i d than t h a t of o r g a n i c phosphorus; b o t h , however, are r e g e n e r a t e d more r a p i d l y than o r g a n i c carbon ( S e k i e t a l . , 1 9 6 8 ) . T h e r e f o r e , s u b s t r a t e s h i g h i n o r g a n i c n i t r o g e n e n a b l e the m i c r o b e s t o decompose the s u b s t r a t e a t a g r e a t e r i n i t i a l r a t e than s u b s t r a t e s low i n o r g a n i c n i t r o g e n ; hence t o t a l b a c t e r i a l d e n s i t i e s a s s o c i a t e d w i t h n i t r o g e n - r i c h s u b s t r a t e s w i l l be g r e a t e r (Tenore,1983). K i n g and Heath (1967), Suberkropp et a l . d 9 7 6 ) and K aushik 14 and Hynes (1971) r e p o r t t h a t p h e n o l i c r e s i d u e s a l s o a f f e c t m i c r o b i a l a t t a c k . P o l y p h e n o l s a r e a l k a l i - or a l c o h o l -e x t r a c t a b l e polyhydroxybenzene d e r i v a t i v e s formed by the o x i d a t i o n of l i g n i n s (Hedges and Mann,1979). These compounds are e x t r e m e l y decay r e s i s t a n t ( F e n c h e l and Blackburn,1979) and are p r e s e n t i n l a r g e q u a n t i t i e s i n woody p l a n t m a t e r i a l s . S m a l l e r q u a n t i t i e s are found i n non-woody v a s c u l a r p l a n t s and the l e a s t amount i s found among n o n - v a s c u l a r s u b s t r a t e s (Hedges and Mann,1979). W a t e r - s o l u b l e e x t r a c t s from d e t r i t u s c o n t a i n i n g p h e n o l i c compounds have a l s o been shown t o i n h i b i t the growth of many marine b a c t e r i a ( H a r r i s o n , 1 9 8 2 ) . S m a l l p a r t i c l e s have a g r e a t e r s u r f a c e a r e a which enhances m i c r o b i a l a c t i v i t y ( F enchel,1970; Hargrave,1972; G o s s e l i n k and K i r b y , l 9 7 4 ) . P a r t i c l e s i z e t h u s p l a y s an i m p o r t a n t r o l e i n d e t e r m i n i n g the r e l e a s e of of DOM by m i c r o b i a l d e c o m p o s i t i o n . As b a c t e r i a l a c t i v i t y i n c r e a s e s , more DOM i s r e l e a s e d (Odum and de l a C r u z , 1 9 6 7 ) . D r y i n g of the s u b s t r a t e s causes s t r u c t u r a l and c h e m i c a l changes which a f f e c t m i c r o b i a l a t t a c k . H a r r i s o n and Mann (1975b) f i n d t h a t the e e l g r a s s , Z o s t e r a m a r i n a , decomposed l e s s r a p i d l y when d r i e d but Zieman (1968) f i n d s t h a t d r y i n g of a s i m i l a r l y s t r u c t u r e d v a s c u l a r p l a n t , T h a l a s s i a t e s t u d i n u m , p e r m i t s e a s i e r e n t r a n c e by m i c r o b e s , and hence, f a s t e r d e c o m p o s i t i o n . In systems which i n c l u d e p r i m a r y consumers such as z o o p l a n k t o n , i t has been shown t h a t the a s s o c i a t e d b a c t e r i a l p o p u l a t i o n remains i n an a c t i v e m e t a b o l i c s t a t e ( F e n c h e l and 1 5 H a r r i s o n , 1 9 7 6 ; Fenchel,1977; L e v i n g t o n , 1 9 8 0 ) . One reason t h i s may r e s u l t i s t h a t the g r a z e r s a r e e x c r e t i n g DOM which becomes a v a i l a b l e t o the microbes ( Z o b e l and Feltham,1938; Johannes,1968; Newell,1965; Hargrave,1970; Fenchel,1970,1975). H e t e r o t r o p h i c b a c t e r i a r e q u i r e a r a t i o of carbon t o n i t r o g e n t o phosphorus by weight of 200 t o 10 t o 1 f o r t h e i r c o n v e r s i o n t o m i c r o b i a l p r o t o p l a s m (Thayer,1976). When the mi c r o o r g a n i s m s are s u p p o r t e d on n u t r i e n t - p o o r m a t t e r , t h e i r demands f o r n i t r o g e n and phosphorus are met by a s s i m i l a t i n g d i s s o l v e d i n o r g a n i c n i t r o g e n and phosphorus ( F e n c h e l and Jorgensen,1977). Thus the c o n c e n t r a t i o n of d i s s o l v e d i n o r g a n i c n u t r i e n t s can i n f l u e n c e b a c t e r i a l d e n s i t i e s . The a d d i t i o n of n i t r a t e and phosphate s t i m u l a t e s m i c r o b i a l growth thus i n c r e a s i n g t o t a l biomass ( C a r l u c c i , 1 9 7 1 ; F e n c h e l and H a r r i s o n , 1 9 7 6 ; Fenchel,1977; F e n c h e l and B l a c k b u r n , 1 9 7 9 ) . As the b a c t e r i a decompose and a s s i m i l a t e the s t r u c t u r a l compounds of the d e t r i t u s , they u t i l i z e the d i s s o l v e d i n o r g a n i c n u t r i e n t s p r e s e n t (Tenore,1977b). The amount of d i s s o l v e d i n o r g a n i c m a t t e r i n i t i a l l y d e c r e a s e s as the m i c r o b i a l p o p u l a t i o n grows, but once the s u b s t r a t e i s a t t a c k e d by the m i c r o b e s , the c o n c e n t r a t i o n of the d i s s o l v e d i n o r g a n i c n u t r i e n t s w i l l i n c r e a s e as they a r e r e l e a s e d t h r o u g h b a c t e r i a l m i n e r a l i z a t i o n ( F e n c h e l and H a r r i s o n , 1 9 7 6 ) . These f a c t o r s , which a f f e c t b a c t e r i a l d e n s i t i e s , a r e those which o p e r a t e i n an a e r o b i c system. In an a n a e r o b i c environment, the p r o c e s s of b a c t e r i a l m i n e r a l i z a t i o n can be d i v i d e d i n t o a s e r i e s of m e t a b o l i c s t e p s w i t h each s t e p 16 r e q u i r i n g a p h y s i o l o g i c a l l y d i f f e r e n t type of organism ( F e n c h e l and Jorgensen,1977). The i n i t i a l p r o c e s s ( h y d r o l y s i s of p a r t i c u l a t e matter i n t o amino a c i d s , d i s a c c h a r i d e s and l o n g c h a i n f a t t y a c i d s , by e x t r a c e l l u l a r enzymes) i s s i m i l a r t o a e r o b i c d e g r a d a t i o n . However, i n the absence of oxygen, t h e s e m o l e c u l e s a r e then c o n v e r t e d t o o t h e r compounds by f e r m e n t a t i o n . These compounds, which i n c l u d e l a c t i c , f o r m i c , a c e t i c , p r o p i o n i c and b u t y r i c a c i d s , then s e r v e as s u b s t r a t e s f o r f u r t h e r b a c t e r i a l m i n e r a l i z a t i o n ( F e n c h e l and B l a c k b u r n , 1 9 7 9 ) . A l t h o u g h the mechanisms of a n a e r o b i c and a e r o b i c d e g r a d a t i o n d i f f e r , the b a c t e r i a l d e n s i t i e s s u p p o r t e d on the o r g a n i c matter and the r a t e of decay are not s i g n i f i c a n t l y d i f f e r e n t ( S e k i and Yokohama,1978). Purpose of Study "This study examines the the t r o p h i c r e l a t i o n s h i p s , i n terms of GEs, between suspended marine b a c t e r i a and the s u s p e n s i o n -f e e d e r s , the b l u e m u s s e l , M y t i l u s e d u l i s ( L ) , and the b r i n e s h rimp, A r t e m i a s a l i n a ( L ) . The e x p e r i m e n t a l c o n d i t i o n s n e c e s s a r y t o support b a c t e r i a l d e n s i t i e s which enable t h e s e s u s p e n s i o n - f e e d e r s , of v a r i o u s s i z e s , t o o b t a i n the h i g h e s t o b s e r v e d GEs w i l l then be d e t e r m i n e d . The h y p o t h e s i s t o be t e s t e d i s t h a t suspended b a c t e r i a can be c o n v e r t e d i n t o the biomass of M . e d u l i s and A . s a l i n a a t some measureable, optimum growth e f f i c i e n c y . 1 7 METHODS AND MATERIALS B a c t e r i a l Growth C u l t u r e Techniques Four t y p e s of o r g a n i c s u b s t r a t e were c o l l e c t e d on e s t u a r i n e beaches around Vancouver, B r i t i s h Columbia. Fucus  v e s i c u l o s u s L. and wood c h i p s were o b t a i n e d from P o i n t Grey and Z o s t e r a mar i n a L. and U l v a l a c t u c a L. were c o l l e c t e d from the f e r r y j e t t y a t Tsawwassen. For each e x p e r i m e n t , the s u b s t r a t e was washed w i t h f r e s h water t o remove a t t a c h e d sediment p a r t i c l e s and e p i p h y t e s and then was d i v i d e d i n t o two p o r t i o n s of e q u a l wet w e i g h t . One p o r t i o n was d r i e d i n an oven a t 50°C f o r 12 h t o determine i t s d r y weight and the o t h e r underwent v a r i o u s p r e p a r a t i o n s depending on the e x p e r i m e n t a l d e s i g n . The e x p e r i m e n t a l sample e i t h e r remained wet or was d r i e d . I t was then e i t h e r l e f t i n t a c t or ground i n t o p a r t i c l e s i n a b l e n d e r . Wet e x p e r i m e n t a l s u b s t r a t e s were a u t o c l a v e d . The s i z e s of the s u b s t r a t e p a r t i c l e s which were determined under a microscope a r e r e p o r t e d i n T a b l e 1. Table 2 shows the v a r i o u s t r e a t m e n t s t h a t were conducted. A sample of each s u b s t r a t e was sent t o 'The Canadian M i c r o a n a l y t i c a l S e r v i c e ' , i n Vancouver, where the r e l a t i v e amounts of t o t a l c a r b o n , hydrogen, and n i t r o g e n were d e t e r m i n e d . The p r e p a r e d p a r t i c u l a t e matter was added t o a 6 1 f l a s k c o n t a i n i n g 5 1 of a u t o c l a v e d s y n t h e t i c seawater (50 g MgSOfl 7H 20, 0.25 g NaHC0 3 H 20 and 155 g NaCl i n 10 1 of 18 d i s t i l l e d w a t e r ) . The media had a pH of a p p r o x i m a t e l y 7.5 (S.D.= 0.5) measured by a pH meter (model#320,Fisher Co.) and a s a l i n i t y of 28 p a r t s per thousand (ppt) measured by a YSI s a l i n o m e t e r (model#33). Two hundred m i l l i l i t e r s of a n a t u r a l seawater sample, c o l l e c t e d from the s h o r e l i n e of P o i n t Grey, was f i l t e r e d t h r o u g h a 0.45 um M i l l i p o r e f i l t e r ( t y p e HA, d i a m e t e r of 47 mm) and added t o the f l a s k . Three l e v e l s of i n o r g a n i c n u t r i e n t c o n c e n t r a t i o n s used i n the e x p e r i m e n t s were c r e a t e d from the a d d i t i o n of n i t r a t e -n i t r o g e n and phosphate-phosphorous. N u t r i e n t - r i c h media c o n t a i n e d 6.0 yM phosphate and 30 iM n i t r a t e , n u t r i e n t - p o o r media were a t c o n c e n t r a t i o n s of 1.2 IJM phosphate and 10 pM n i t r a t e and n u t r i e n t - d e p l e t e d media had no i n o r g a n i c n u t r i e n t s added. The f l a s k s were then p l a c e d i n a c o l d room at 12°C and kept i n d a r k n e s s . A i r was i n j e c t e d i n t o the f l a s k s f o r the a e r o b i c e x p e r i m e n t s by i n s e r t i n g a i r s t o n e s which were c o n n e c t e d t o a l a b o r a t o r y a i r s u p p l y . For the a n a e r o b i c e x p e r i m e n t s , the f l a s k s c o n t a i n i n g the a u t o c l a v e d s y n t h e t i c seawater were bubbled w i t h n i t r o g e n gas from a tank t o remove a l l oxygen. The redox p o t e n t i a l was m o n i t o r e d d a i l y d u r i n g t h i s p e r i o d . When the e l e c t r i c a l p o t e n t i a l (Eh) reached -200 mv (measured by a pH meter,model#150, F i s h e r C o . ) , which took a p p r o x i m a t e l y 7 days, the s u b s t r a t e s were added t o the f l a s k s . The medium was purged w i t h n i t r o g e n gas f o r an a d d i t i o n a l two days. The redox p o t e n t i a l was checked p e r i o d i c a l l y throughout the e x p e r i m e n t a l p e r i o d t o ensure t h a t the media were s t i l l a n a e r o b i c (Eh was 19 l e s s than -200 mv, W e t z e l , 1 9 7 5 ) . B a c t e r i a l growth over time was de t e r m i n e d from a water sample t h a t was taken e i t h e r d a i l y or every second day f o r a p e r i o d of 1 8 days. The f l a s k s under a n a e r o b i c c o n d i t i o n s were t i g h t l y s e a l e d w i t h a rubber p l u g w i t h t h r e e p r o j e c t i n g t u b e s . One tube s e r v e d as an e n t r a n c e f o r the n i t r o g e n gas. The second tube e n a b l e d e x c e s s n i t r o g e n gas t o escape ( t h i s tube was c l o s e d once n i t r o g e n p u r g i n g c e a s e d ) . The t h i r d tube was d e s i g n e d t o enable c o l l e c t i o n of water samples w i t h o u t a l l o w i n g a i r t o e n t e r . . T h i s was a c h i e v e d by h a v i n g a c o n t i n u a l vacuum i n the tube. The sample was then e x t r a c t e d from the f l a s k by opening a s t o p cock i n the vacuum t u b i n g and b l e e d i n g o f f the sample. Water samples from the oxygenated f l a s k s were o b t a i n e d by p o u r i n g the d e s i r e d q u a n t i t y of the medium i n t o a beaker. One p o r t i o n of t h i s water sample was used f o r e p i f l u o r e s c e n c e a n a l y s i s and a second f o r ATP d e t e r m i n a t i o n . E p i f l u o r e s c e n c e A n a l y s i s The e p i f l u o r e s c e n c e or A c r i d i n e Orange D i r e c t C o u n t i n g (AODC) method was f o l l o w e d a c c o r d i n g t o the proc e d u r e o u t l i n e d by Hobbie et a l . (1977). F i v e m i l l i l i t e r s of the sample was p r e s e r v e d w i t h 0.25 ml of f o r m a l i n (37% f o r m a l d e h y d e ) . The sample was c o v e r e d and p l a c e d i n a r e f r i d g e r a t o r a t 10°C u n t i l the a n a l y s i s was t o be c o n d u c t e d . The a n a l y s i s i n v o l v e d t h r e e s t e p s : s t a i n i n g of the c e l l s f o l l o w e d by t h e i r f i l t r a t i o n and d i r e c t c o u n t i n g under a m i c r o s c o p e . The f l u o r e s c e n t dye used was 1 mg a c r i d i n e orange 20 (AO) ( F i s h e r S c i e n t i f i c Company) per ml of d i s t i l l e d w a t e r . Once p r e p a r e d , t h i s s o l u t i o n was f i l t e r e d t h r o u g h a 0.22 um M i l l i p o r e f i l t e r , type GS. A volume of 0.1 ml or 0.2 ml of t h i s a c r i d i n e orange s o l u t i o n was added t o 1 ml or 2 m l , r e s p e c t i v e l y , of the water sample. A f t e r two m i n u t e s , the sample was f i l t e r e d under a vacuum p r e s s u r e of a p p r o x i m a t e l y 0.8 atm. The f i l t r a t i o n a p p a r a t u s c o n s i s t e d of a 0.45 um M i l l i p o r e f i l t e r ( t y p e HA, 25 mm i n d i a m e t e r ) which was p l a c e d underneath a N u c l e p o r e f i l t e r (0.2 um pore s i z e , 25 mm i n d i a m e t e r ) . The N u c l e p o r e f i l t e r was p r e v i o u s l y dyed i n I r a g a l a n B l a c k (2 g I r a g a l a n i n 1 1 of 2% a c e t i c a c i d ) . When the f i l t r a t i o n of the sample was c o m p l e t e , the N u c l e p o r e f i l t e r was p l a c e d on a c l e a n g l a s s m i c r o s c o p e s l i d e . The blank had o n l y the f i l t e r e d AO s t a i n . One drop of immersion o i l was added and a c o v e r s l i p was then p l a c e d on t o p . S l i d e s were kept i n the dark u n t i l ready f o r c o u n t i n g (always w i t h i n 24 h o u r s ) . B a c t e r i a w i t h i n 10 f i e l d s of view were counted u s i n g a Z e i s s m i c r o s c o p e w i t h e p i f l u o r e s c e n c e a t t a c h m e n t s . The s i z e of the f i e l d v a r i e d so t h a t a t l e a s t 20 c e l l s per f i e l d were p r e s e n t . The f i l t e r c o m b i n a t i o n s of t h i s m icroscope were i d e n t i c a l t o those used by Hobbie e t a l . (1977). The number of b a c t e r i a per ml was d e t e r m i n e d u s i n g the f o l l o w i n g f o r m u l a : 21 c e l l s / m l = (the mean b a c t e r i a l count of 10 f i e l d s ) (the a r e a c o v e r e d by f i l t e r e d c e l l s , 2.011 X 10 8 Mm2) (the a r e a of the f i e l d , i n M ^ 2 ) " 1 ( t h e volume of sample f i l t e r e d i n m l ) " 1 . ATP A n a l y s i s The ATP c o n t e n t was d e t e r m i n e d i n a manner s i m i l a r t o the method d e s c r i b e d by Holm-Hansen (1966). The t e c h n i q u e i n v o l v e d two s t e p s , the e x t r a c t i o n of ATP and the q u a n t i t a t i v e a n a l y s i s . E i t h e r d a i l y or every second day, a sample f o r a n a l y s i s was e x t r a c t e d from the b a c t e r i a l c u l t u r e s and f r o z e n u n t i l analyzed.. Ten m i l l i l i t e r s of a u t o c l a v e d TRIS b u f f e r (0.02 M t r i s h y d r o x y m e t h y l aminomethane (THAM), a d j u s t e d t o pH 7.7 w i t h 1 N HCL) was heated t o b o i l i n g (98°C) i n a t e s t tube by means of a b l o c k h e a t e r . A volume of the sample, which v a r i e d between 10 ml and 100 ml, depending on the d e n s i t y of the c u l t u r e , was f i l t e r e d onto a 0.22 um M i l l i p o r e f i l t e r ( type GS, diameter of 47 mm). The f i l t e r was then r o l l e d and p l a c e d i n the b o i l i n g t r i s b u f f e r f o r 7 minutes. A f t e r t h i s p e r i o d , the s o l v e n t was decanted i n t o a c l e a n t e s t tube and c o l d t r i s b u f f e r was added t o b r i n g the t o t a l volume t o 10 ml. The t e s t tube was then p l a c e d on i c e u n t i l the s o l u t i o n reached room temperature (18°C). The samples were c o v e r e d and f r o z e n u n t i l the time of the a n a l y s i s . Two r e p l i c a t e s were p r o c e s s e d from each e x t r a c t . Blank or c o n t r o l samples c o n s i s t e d of the e x t r a c t o b t a i n e d from h e a t i n g a M i l l i p o r e f i l t e r i n the TRIS b u f f e r . The pe r c e n t a g e of ATP r e c o v e r e d from t h i s e x t r a c t i o n 22 p r o c e d u r e averaged between 85-95% f o r a l l the e x p e r i m e n t s . T h i s p e r c e n t a g e was d e t e r m i n e d by comparing the ATP c o n t e n t of a sample s p i k e d w i t h a known amount of ATP w i t h t h a t of a non-s p i k e d sample u s i n g the f o l l o w i n g r e l a t i o n s h i p : % Recovery = 100 [ ( s p i k e + sample) - s a m p l e ] ( s p i k e ) " 1 (Geesey and C o s t e r t o n , 1 9 7 9 ) . Q u a n t i t a t i v e a n a l y s i s of ATP was d e t e r m i n e d u s i n g a Chem-Glow Photometer (model# J4-7441 ) and an I n t e g r a t o r Timer (model# J4-74622), both of which were purchased from the American Instrument Company.- The f i r e f l y e x t r a c t (Sigma Chemical Company) was r e c o n s t i t u t e d i n 5 ml of d i s t i l l e d water t o o b t a i n a s o l u t i o n a t pH 7.4 c o n t a i n i n g 0.05 M p o t a s s i u m a r s e n a t e and 0.02 M magnesium s u l p h a t e . T h i s s o l u t i o n was then p l a c e d i n the dark a t 12°C f o r 2 t o 3 hours t o o b t a i n s t a b i l i z e d a c t i v i t y . F o l l o w i n g t h i s p e r i o d the f i r e f l y e x t r a c t was d i l u t e d w i t h ~ 5 " n r l ~ of 0.1 M sodium a r s e n a t e and 5 ml of 0.04 M magnesium c h l o r i d e , b o th of which had a pH of 7.4. Immediately a f t e r the a d d i t i o n of t h e s e d i l u t a n t s , t h i s enzyme s o l u t i o n was mixed and p l a c e d on i c e . When the sample e x t r a c t s thawed, 0.2 ml of t h i s e x t r a c t and 0.2 ml of the enzyme were i n j e c t e d i n t o a c u v e t t e , mixed and p l a c e d i n the photometer c e l l h o l d e r . T h i s p r o c e d u r e took p l a c e i n e x a c t l y 15 s e c . The number of l i g h t I n t e n s i t y U n i t s (IU) which accumulated i n a p e r i o d of 10 sec. was r e c o r d e d . In o r d e r t o c o n v e r t IU i n t o nq of ATP, a s e t of ATP s t a n d a r d s was p r e p a r e d r a n g i n g i n IU s i m i l a r t o those o b s e r v e d 23 from the e x t r a c t s . The amount of ATP per e x t r a c t was then c o n v e r t e d t o nq of ATP per ml of the e x p e r i m e n t a l sample u s i n g c o n v e r s i o n s based on d i r e c t p r o p o r t i o n s . U n i t s f o r B a c t e r i a l D e n s i t y For most ex p e r i m e n t s b a c t e r i a l d e n s i t y was d e t e r m i n e d i n c e l l s / m l and nq ATP/ml, and then e x p r e s s e d per gram dry weight of the s u b s t r a t e . These v a l u e s were c o n v e r t e d t o nqC f o r o n l y one experiment by assuming the mass of one b a c t e r i a l c e l l was 2.2 X 10- 7 MgC ( D a l e , 1 9 7 4 ) . and t h a t the carbon t o ATP r a t i o was 250 (Holm-Hanson,1960). S t a t i s t i c a l A n a l y s i s For each s u b s t r a t e under n u t r i e n t - p o o r c o n d i t i o n s , b a c t e r i a l d e n s i t i e s were m o n i t o r e d i n d u p l i c a t e e x p e r i m e n t a l f l a s k s . A one-way a n a l y s i s of v a r i a n c e was performed t o d e t e r m i n e the v a r i a n c e a s s o c i a t e d w i t h e x p e r i m e n t a l e r r o r . T h i s was e s s e n t i a l i n o r d e r t o determine s i g n i f i c a n t d i f f e r e n c e s a t the 95% c o n f i d e n c e l e v e l f o r the b a c t e r i a l d e n s i t i e s o b t a i n e d under the d i f f e r e n t e x p e r i m e n t a l c o n d i t i o n s . T h i s v a r i a n c e a s s o c i a t e d w i t h e x p e r i m e n t a l e r r o r was then assumed t o be c o n s t a n t f o r a l l e x p e r i m e n t s undertaken i n t h i s s t u d y . To determine s i g n i f i c a n t d i f f e r e n c e s between v a l u e s of b a c t e r i a l d e n s i t y , o b t a i n e d from the v a r i o u s t r e a t m e n t s , an F-t e s t was performed u s i n g the v a r i a n c e c a l c u l a t e d from the above e x p e r i m e n t a l e r r o r ; The n u l l h y p o t h e s i s s t a t e d t h a t the 24 v a r i a n c e between ex p e r i m e n t s was e q u a l t o the v a r i a n c e a s s o c i a t e d w i t h e r r o r or the v a r i a n c e w i t h i n the e x p e r i m e n t s . I f F . 0 5 [ x , y ] , d e f i n e d as the mean square among e x p e r i m e n t s d i v i d e d by the mean square w i t h i n e x p e r i m e n t s (x and y r e f e r t o the degrees of freedom a s s o c i a t e d w i t h the tr e a t m e n t and w i t h the e x p e r i m e n t a l e r r o r , r e s p e c t i v e l y ) , was g r e a t e r than or e q u a l t o t he r e p o r t e d F . 0 5 [ x , y ] then the n u l l h y p o t h e s i s was r e j e c t e d ( S o k a l and R o h l f , l 9 6 9 ) . The c o n c l u s i o n i n t h i s case would then have been t h a t the d i f f e r e n c e among e x p e r i m e n t a l r e p l i c a t e s was due t o the f i x e d t r e atment e f f e c t . D e c o m p o s i t i o n Rates The p e r c e n t a g e weight l o s t by the s u b s t r a t e s d u r i n g each experiment was used as an i n d i c a t i o n . o f d e c o m p o s i t i o n r a t e . T h i s l o s s was computed by d i v i d i n g the i n i t i a l d r y weight of the s u b s t r a t e i n t o the d i f f e r e n c e of the i n i t i a l and f i n a l d r y w e i g h t s and m u l t i p l y i n g by 100. 25 Growth and F e e d i n g of S u s p e n s i o n - F e e d e r s  Growth Experiments (_i) The Source of Food The food s u p p l i e d f o r the growth e x p e r i m e n t s on A r t e m i a  s a l i n a and M y t i l u s e d u l i s c o n s i s t e d of the suspended b a c t e r i a l c u l t u r e s t h a t were h a r v e s t e d from the b a c t e r i a l growth e x p e r i m e n t s . These s u s p e n s i o n s were f i l t e r e d t h r o u g h a g l a s s f i b r e f i l t e r t o attempt t o remove the b a c t e r i a a t t a c h e d t o the s u b s t r a t e p a r t i c l e s . The c o n c e n t r a t i o n of the b a c t e r i a l c u l t u r e s used f o r the growth e x p e r i m e n t s ranged from 10 5 c e l l s / m l t o 10 7 c e l l s / m l . D u n a l i e l l a t e r t i o l e c t a B u t c h e r was a l s o p r o v i d e d as a food s o u r c e f o r the growth e x p e r i m e n t s w i t h M y t i l u s e d u l i s . T h i s c u l t u r e was o b t a i n e d from the N o r t h e a s t P a c i f i c C u l t u r e C o l l e c t i o n (#1) a t the U n i v e r s i t y of B r i t i s h C olumbia. ( i i ) M y t i l u s e d u l i s The b l u e m u s s e l , M y t i l u s e d u l i s , was c o l l e c t e d on r o c k s i n the i n t e r t i d a l a r e a on the n o r t h shore of P o i n t Grey, B r i t i s h C o lumbia. A s e r i e s of growth e x p e r i m e n t s were conducted i n which t h r e e food s o u r c e s were p r o v i d e d , b a c t e r i a s u p p o r t e d from U l v a l a c t u c a , Fucus v e s i c u l o s u s and D u n a l i e l l a t e r t i o l e c t a . T w e n t y - f i v e mussels were p l a c e d i n 1400 ml of the b a c t e r i a l or p h y t o p l a n k t o n c u l t u r e s . The b a c t e r i a l c u l t u r e s were of two 26 d i f f e r e n t s a l i n i t i e s , 14 ppt and 28 p p t . C o n t i n u o u s a i r was s u p p l i e d by i n s e r t i n g a i r s t o n e s , which were c o n n e c t e d t o an a i r s u p p l y , and the growth e x p e r i m e n t s were conducted a t 12°C. The mean l e n g t h and w i d t h of th e s e mussels were d e t e r m i n e d on the i n i t i a l and f i n a l days of the e x p e r i m e n t s . E v e r y t h i r d day, 100 ml of the food source was added t o the f l a s k s . The t o t a l amount of food added (the sum of a l l food p r o v i d e d throughout the e x p e r i m e n t ) was d e t e r m i n e d by e p i f l u o r e s c e n c e f o r the b a c t e r i a l c u l t u r e s , and by a C o u l t e r - C o u n t e r (model ZB1) f o r the p h y t o p l a n k t o n c u l t u r e s . The d e t a i l s of the e x p e r i m e n t a l c o n d i t i o n s are- shown i n T a b l e 2. Mean growth r a t e s of the m u s s e l s , i n u n i t s of mm/day, were a l s o d e t e r m i n e d . ( i i i ) A r t e m i a s a l i n a Dry eggs of A . s a l i n a ( L ) , p urchased from the C a r o l i n a B i o l o g i c a l Supply Company, were added t o an aquarium c o n t a i n i n g 2 t o 3 1 of s y n t h e t i c seawater (28 p p t ) . T h i s tank was w e l l a e r a t e d and kept a t room temperature (18°C). A desk lamp was p l a c e d a t one end of the t a n k . The p o s i t i v e p h o t o t a c t i c response of the hatched b r i n e shrimp (Sogeloos,1973) a l l o w e d t h e i r easy removal w i t h a p i p e t t e . Between 100 and 150 shrimp ( a l l were i n the f i r s t or second i n s t a r of growth) were added t o a s e r i e s of a e r a t e d f l a s k s c o n t a i n i n g 50 ml of the b a c t e r i a l c u l t u r e s . B a c t e r i a l d e n s i t i e s were m o n i t o r e d over time and ranged from 6.8 X 10 s c e l l s / m l (S.D.=1.5 X 10 5) t o 9.33 X 10 6 c e l l s / m l (S.D.=0.80 X 1 0 6 ) . F i v e b r i n e shrimp were removed d a i l y , as of day 0, from 27 each f l a s k and the mean l e n g t h and the 95% c o n f i d e n c e l i m i t s were d e t e r m i n e d i n mm under a d i s s e c t i n g m i c r o s c o p e . The ex p e r i m e n t s l a s t e d between 4 and 16 days. F e e d i n g E x p e r i m e n t s (_i ) C a l c u l a t i o n of F i l t e r i n g Rates F i l t e r i n g r a t e s were c a l c u l a t e d as f o l l o w s : ml/hr = 2 . 3 0 3 V ( l o g C o - l o g C t ) ( t ) " 1 , where C 0 and C« e q u a l the number of c e l l s / m l p r e s e n t b e f o r e and a f t e r the s p e c i f i e d t i m e , t ( i n hour s ) and V ( i n ml) i s e q u a l t o the volume of medium per organism ( H e n t i g , 1 9 7 1 ) . ( i i ) M y t i l u s e d u l i s F i l t e r i n g r a t e s were d e t e r m i n e d o n l y f o r the mussels f e e d i n g on p h y t o p l a n k t o n . The reason f o r t h i s w i l l become e v i d e n t i n the d i s c u s s i o n . One or two m u s s e l s , of a s i m i l a r s i z e , were p l a c e d i n a s e r i e s of beakers c o n t a i n i n g 100 ml of the c u l t u r e of D u n a l i e l l a t e r t i o l e c t a . The c u l t u r e s were d i l u t e d w i t h f i l t e r e d s y n t h e t i c seawater (28 p p t ) t o g i v e f i n a l c o n c e n t r a t i o n s r a n g i n g from 30,000 t o 40,000 c e l l s / m l . The mussels used t o determine f i l t e r i n g r a t e s were th o s e p r e v i o u s l y f e d w i t h D u n a l i e l l a t e r t i o l e c t a i n the growth e x p e r i m e n t s . F i l t e r i n g r a t e e x p e r i m e n t s were conducted under the same c o n d i t i o n s as the growth e x p e r i m e n t s . C o n t r o l f l a s k s , w i t h o u t 28 mu s s e l s , were m o n i t o r e d t o determine n a t u r a l i n c r e a s e s i n c e l l number d u r i n g the e x p e r i m e n t a l p e r i o d . The number of c e l l s , b e f o r e and a f t e r the f i l t r a t i o n p e r i o d of 3 hours was det e r m i n e d u s i n g the C o u l t e r - C o u n t e r . ( i i i ) A r t e m i a s a l i n a One t o s i x b r i n e shrimp v a r y i n g i n s i z e from 0.5 mm t o 3.0 mm were p l a c e d i n a s e r i e s of f l a s k s c o n t a i n i n g 50 ml of a b a c t e r i a l c u l t u r e r a n g i n g i n c o n c e n t r a t i o n from 10 5 c e l l s / m l t o 10 7 c e l l s / m l . In a l l but one of the e x p e r i m e n t s , the c u l t u r e s were i n o c u l a t e d w i t h a b a c t e r i a l - a n t i b i o t i c 24 hours p r e v i o u s to. the b e g i n n i n g of the e x p e r i m e n t . The 2 ml inoculum used was tak e n from a s o l u t i o n c o n t a i n i n g 100 mg of p e n i c i l l i n and 50 mg of s t r e p t o m y c i n d i s s o l v e d i n 10 ml of d i s t i l l e d water (Judy Acreman, p e r s o n a l c o m m u n i c a t i o n ) . T h i s inoculum was f i r s t f i l t e r e d t h r o u g h a 0.22 ym M i l l i p o r e f i l t e r b e f o r e b e i n g added t o the b a c t e r i a l c u l t u r e . The number of b a c t e r i a l c e l l s b e f o r e and a f t e r the e x p e r i m e n t a l time was det e r m i n e d u s i n g the A c r i d i n e Orange D i r e c t C o u n t i n g Technique. The f i l t r a t i o n e x p e r i m e n t s v a r i e d between 3 and 4 ho u r s . Changes i n b a c t e r i a l numbers w i t h i n the c o n t r o l f l a s k s , c o n t a i n i n g no organisms, were taken i n t o a c c o u n t . 29 Growth E f f i c i e n c i e s Growth e f f i c i e n c i e s of A r t e m i a s a l i n a when f e d suspended b a c t e r i a were de t e r m i n e d as f o l l o w s : Growth E f f i c i e n c y ( % ) = I 0 0 ( t h e change i n d r y weight of A . s a l i n a per hour) (the d r y weight of the b a c t e r i a l c e l l s consumed per h o u r ) " 1 Reeve, 1963a The d r y weight of A . s a l i n a was d e t e r m i n e d u s i n g the e x p o n e n t i a l r e l a t i o n s h i p between b r i n e shrimp l e n g t h , i n mm, and dry w e i g h t , i n yg, r e p o r t e d by Reeve(1963a). The changes i n dry weight of b r i n e shrimp per hour a t v a r i o u s b a c t e r i a l c o n c e n t r a t i o n s were de t e r m i n e d from the growth r a t e s t h a t were ob s e r v e d a t those c o n c e n t r a t i o n s . C o r r e s p o n d i n g l y , the c e l l s consumed by organisms of t h i s s i z e were c a l c u l a t e d from the f i l t e r i n g r a t e — e x p e r i m e n t s . One b a c t e r i a l c e l l was assumed t o be 2.2 X 10~ 7 ygC ( D a l e , 1974) and the carbon t o d r y weight r a t i o was e q u a l t o 0.344 (Ferguson and Rublee,1975). 30 RESULTS B a c t e r i a l Growth and D e n s i t i e s The Growth Curve A f t e r the b a c t e r i a l inoculum was added t o each f l a s k the b a c t e r i a l c o n c e n t r a t i o n on day 0 v a r i e d i n c e l l s / m l from 10 2 t o 10 3 , and i n ng ATP/ml from 10" 7 t o 10~ 6. A p a t t e r n was g e n e r a l l y observed i n a l l e x p e r i m e n t s . The i n i t i a l growth phase was r e p r e s e n t e d by a r a p i d 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 t o a peak which was f o l l o w e d by a p l a t e a u i n d e n s i t y which was e i t h e r a t the same l e v e l as the peak or lower. F i g u r e 1 i s an example of t h i s g e n e r a l p a t t e r n . When b a c t e r i a l biomass was e x p r e s s e d i n terms of ixq of carbon per ml, the c o r r e l a t i o n between the e p i f l u o r e s c e n c e and ATP da t a was poor ( F i g u r e 2 ) . B a c t e r i a l d e n s i t i e s were t h e r e f o r e d e t e r m i n e d i n terms of 10 6 c e l l s per ml and ng ATP per ml which were both e x p r e s s e d per gram dry weight of the s u b s t r a t e added t o each e x p e r i m e n t a l f l a s k . The s u b s t r a t e amount had no e f f e c t on b a c t e r i a l d e n s i t i e s when e x p r e s s e d i n thes e l a t t e r u n i t s ( F i g u r e 3 ) . The N u t r i e n t Content of the S u b s t r a t e s T a b l e 4 i n d i c a t e s the r e l a t i v e p e r c e n t of t o t a l c a r b o n , hydrogen and n i t r o g e n found w i t h i n the v a r i o u s s u b s t r a t e s . Fucus v e s i c u l o s u s and Z o s t e r a marina had s i m i l a r amounts of the 31 el e m e n t s . U l v a l a c t u c a p o s s e s s e d s i g n i f i c a n t l y more n i t r o g e n and l e s s carbon than the o t h e r t h r e e s u b s t r a t e s and wood c h i p s c o n t a i n e d the l e a s t n i t r o g e n but the most of c a r b o n . The E f f e c t of D i f f e r e n t O r g a n i c S u b s t r a t e s on B a c t e r i a l  D e n s i t i e s (_i) S i m i l a r S i z e P a r t i c l e s , Same S u b t r a t e P r e p a r a t i o n When a u t o c l a v e d s u b s t r a t e s were added t o media under n u t r i e n t - r i c h c o n d i t i o n s , b a c t e r i a l . d e n s i t i e s were s i g n i f i c a n t l y h i g h e r d u r i n g the growth p e r i o d when the s u b s t r a t e was U . l a c t u c a than when i t was F . v e s i c u l o s u s or Z.marina. A l l of these s u b s t r a t e s s u p p o r t e d g r e a t e r b a c t e r i a l d e n s i t i e s than wood c h i p s ( F i g u r e 4 a ) . T h i s was the g e n e r a l p a t t e r n o b s e r v e d under n u t r i e n t - p o o r c o n d i t i o n s ( F i g u r e 4b). The ATP d a t a s u p p o r t e d t h i s r e s u l t ( F i g u r e 5 ) . T h i s same g e n e r a l t r e n d was a l s o found when the s u b s t r a t e s were d r i e d ( F i g u r e 6 ) . ( i i ) S i m i l a r S i z e P a r t i c l e s , P i f f e r e n t S u b s t r a t e P r e p a r a t i o n s The e p i f l u o r e s c e n c e d a t a i n d i c a t e d t h a t i n f l a s k s w i t h e i t h e r a u t o c l a v e d or d r i e d , l a r g e s u b s t r a t e p a r t i c l e s under n u t r i e n t - p o o r c o n d i t i o n s , b a c t e r i a l numbers were not s i g n i f i c a n t l y d i f f e r e n t t h roughout most of the growth c u r v e ( F i g u r e 7 ) . However, f o r the same e x p e r i m e n t s , the amount of ATP was g r e a t e r i n f l a s k s w i t h the d r i e d s u b s t r a t e s ( F i g u r e 8 ) . When the s u b s t r a t e p a r t i c l e s were s m a l l , s i g n i f i c a n t 32 d i f f e r e n c e s o c c u r r e d a t the peak phase of growth; a u t o c l a v e d p a r t i c l e s s u p p o r t e d g r e a t e r d e n s i t i e s than d r i e d p a r t i c l e s ( F i g u r e 9 ) . ( i i i ) P i f f e r e n t S i z e P a r t i c l e s , Same S u b s t r a t e P r e p a r a t i o n When the s u b s t r a t e s were a u t o c l a v e d , the s i z e of the p a r t i c l e s had no e f f e c t on b a c t e r i a l d e n s i t i e s nor the shape of the growth c u r v e . Both the e p i f l u o r e s c e n c e and ATP data s u p p o r t e d t h i s r e s u l t ( F i g u r e 10). When the s u b s t r a t e s were d r i e d , o n l y b a c t e r i a l d e n s i t i e s a s s o c i a t e d w i t h l a r g e p a r t i c l e s of U l v a l a c t u c a were g r e a t e r than those a s s o c i a t e d w i t h s m a l l p a r t i c l e s d u r i n g the peak phase of growth. No s i g n i f i c a n t d i f f e r e n c e s were o b s e r v e d , however, once the p o p u l a t i o n reached the p l a t e a u s t a g e of growth ( F i g u r e 11).. The Ef f e c t of Spec i f i c E x p e r i m e n t a l C o n d i t i o n s I n o r g a n i c n u t r i e n t s ^ a p p e a r e d t o have a s i g n i f i c a n t e f f e c t on the number of b a c t e r i a l c e l l s s u p p o r t e d by U l v a l a c t u c a and Fucus v e s i c u l o s u s o n l y ( F i g u r e 1 2 ) ) . The a d d i t i o n of n u t r i e n t s a l s o p r o l o n g e d the p e r i o d of maximal b a c t e r i a l d e n s i t i e s o b t a i n e d w i t h U . l a c t u c a and F . v e s i c u l o s u s . M e d i a w i t h v a r y i n g c o n c e n t r a t i o n s of i n o r g a n i c n u t r i e n t s had no s i g n i f i c a n t e f f e c t on the q u a n t i t y of b a c t e r i a s u p p o r t e d by e i t h e r Z.marina or wood c h i p s throughout the e n t i r e growth p e r i o d ( F i g u r e s 12). Without the a d d i t i o n of i n o r g a n i c n u t r i e n t s , b a c t e r i a l 33 p o p u l a t i o n s s u p p o r t e d by U . l a c t u c a and F . v e s i c u l o s u s s t e a d i l y d e c l i n e d f o l l o w i n g the peak phase t o the l o w e s t v a l u e s o b t a i n e d i n any e x p e r i m e n t . I n i t i a l l y , more b a c t e r i a were s u p p o r t e d by U . l a c t u c a , but by the time the p o p u l a t i o n l e v e l l e d i n d e n s i t y , t h e r e was no s i g n i f i c a n t d i f f e r e n c e i n the q u a n t i t y of b a c t e r i a o b s e r v e d ( F i g u r e 13). S i g n i f i c a n t d i f f e r e n c e s i n d e n s i t i e s were not observed f o r Z.marina and wood c h i p s throughout the e n t i r e growth p e r i o d . D u r i n g the f i n a l s t age of growth, b a c t e r i a l numbers s u p p o r t e d from the v a r i o u s s u b s t r a t e s under a n a e r o b i c c o n d i t i o n s were not s i g n i f i c a n t l y d i f f e r e n t than those o b t a i n e d from the a e r o b i c e x p e r i m e n t s ( F i g u r e 14). D e c o m p o s i t i o n Rates D e c o m p o s i t i o n r a t e s , e x p r e s s e d as p e r c e n t of dry weight l o s t d u r i n g the e x p e r i m e n t s , were a f u n c t i o n of the s u b s t r a t e type and s i z e , and the c o n c e n t r a t i o n of i n o r g a n i c n u t r i e n t s w i t h i n the medium. U l v a l a c t u c a decomposed f a s t e r than Fucus  v e s i c u l o s u s and Z o s t e r a m a r i n a , which i n t u r n had a h i g h e r d e c o m p o s i t i o n r a t e than wood c h i p s ( T a b l e 5 ) . The presence of i n o r g a n i c n u t r i e n t s enhanced d e c o m p o s i t i o n . S m a l l p a r t i c l e s decomposed f a s t e r than l a r g e p a r t i c l e s . These o b s e r v a t i o n s were found f o r a l l s u b s t r a t e s except wood c h i p s which demonstrated s i m i l a r d e c o m p o s i t i o n r a t e s r e g a r d l e s s of p a r t i c l e s i z e or n u t r i e n t a d d i t i o n . 34 Growth of B a c t e r i a l G r a z e r s M y t i l u s e d u l i s M y t i l u s e d u l i s was unable t o s u r v i v e when s u p p l i e d w i t h suspended b a c t e r i a as t h e i r s ource of f o o d . Both s m a l l and l a r g e mussels p r o v i d e d w i t h b a c t e r i a c u l t u r e d w i t h Fucus  v e s i c u l o s u s and U l v a l a c t u c a d i e d a f t e r 3 and 4 days, r e s p e c t i v e l y , r e g a r d l e s s of the s a l i n i t y or b a c t e r i a l c o n c e n t r a t i o n of the medium (Table 3 ) . M u s s e l s of s i m i l a r s i z e s s u p p l i e d w i t h D u n a l i e l l a t e r t i o l e c t a f i l t e r e d the c e l l s ( F i g u r e 15). T h e i r growth r a t e s and t o t a l number of c e l l s consumed w i t h i n the growth p e r i o d a r e i n d i c a t e d i n T a b l e 3. A r t e m i a s a l i n a The growth of A r t e m i a s a l i n a was a f u n c t i o n of the b a c t e r i a l c o n c e n t r a t i o n s u p p l i e d . A . s a l i n a grown i n a medium c o n t a i n i n g h i g h e r c o n c e n t r a t i o n s of b a c t e r i a a c h i e v e d a l o n g e r l e n g t h d u r i n g the e x p e r i m e n t a l p e r i o d than those grown i n a medium c o n t a i n i n g lower b a c t e r i a l c o n c e n t r a t i o n s . However s i g n i f i c a n t d i f f e r e n c e s i n l e n g t h o c c u r r e d o n l y a f t e r a week of growth ( F i g u r e 16). When the c o n c e n t r a t i o n of b a c t e r i a was l e s s than 1.5 X 10 s c e l l s / m l , l o g 6.2, A r t e m i a s a l i n a was not a b l e t o s u r v i v e beyond a c o u p l e of days ( F i g u r e 17). Growth r a t e s were dependent upon the c o n c e n t r a t i o n of b a c t e r i a s u p p l i e d ; as the i n i t i a l b a c t e r i a l c o n c e n t r a t i o n i n c r e a s e d , growth r a t e s i n c r e a s e d ( F i g u r e 18). Growth r a t e s 35 a l s o v a r i e d w i t h the s i z e of the shrimp; as the organism o b t a i n e d a l o n g e r l e n g t h , the growth r a t e i n c r e a s e d t o a maximum and then d e c r e a s e d ( F i g u r e 18). The type of o r g a n i c s u b s t r a t e used t o support the b a c t e r i a l c u l t u r e s had no e f f e c t on these p a t t e r n s . A r t e m i a s a l i n a demonstrated f i l t e r i n g r a t e s t h a t were a f u n c t i o n of the i n i t i a l b a c t e r i a l c o n c e n t r a t i o n as w e l l as the age of the b r i n e shrimp. F i l t e r i n g r a t e s i n c r e a s e d t o a maximum as the c o n c e n t r a t i o n of the b a c t e r i a l c u l t u r e s p r o v i d e d i n c r e a s e d , but then s t e a d i l y d e c r e a s e d as the b a c t e r i a l c o n c e n t r a t i o n f u r t h e r i n c r e a s e d . These f i l t e r i n g r a t e s were g r e a t e r w i t h l a r g e r b r i n e shrimp ( F i g u r e 19). The growth e f f i c i e n c i e s of A r t e m i a s a l i n a , when s u p p l i e d w i t h suspended b a c t e r i a , i n c r e a s e d t o a maximum as the c o n c e n t r a t i o n of the b a c t e r i a i n c r e a s e d beyond 2.5 X 10 6 c e l l s / m l , l o g 6.4 ( F i g u r e 2 0 ) . As the body l e n g t h of A . s a l i n a i n c r e a s e d , the shrimp was a b l e t o c o n v e r t b a c t e r i a l c e l l s i n t o i t s own biomass w i t h a g r e a t e r e f f i c i e n c y . T h i s growth e f f i c i e n c y reached a peak a t a l e n g t h of 2.5 mm and a f t e r t h i s , s t e a d i l y d e c l i n e d ( F i g u r e 2 0 ) . T h i s peak c o r r e l a t e d w i t h the number of c e l l s consumed. B r i n e shrimp of 2.5 mm consumed more c e l l s than organisms e i t h e r s m a l l e r or g r e a t e r i n s i z e ( F i g u r e 2 1 ) . When b r i n e shrimp were s u p p l i e d w i t h b a c t e r i a a t c o n c e n t r a t i o n s of l e s s than 2.5 X 10 6 c e l l s / m l , l o g 6.4, growth e f f i c i e n c i e s dropped t o z e r o ( F i g u r e 2 0 ) . The maximum s i z e of b r i n e shrimp t h a t c o u l d be s u p p o r t e d by the b a c t e r i a l c u l t u r e s i n c r e a s e d i n d i r e c t p r o p o r t i o n t o the 36 c o n c e n t r a t i o n of the b a c t e r i a ( F i g u r e 22) and t h e b a c t e r i a l c e l l c o n c e n t r a t i o n was d i r e c t l y p r o p o r t i o n a l to the t o t a l n i t r o g e n c o n t e n t of the s u b s t r a t e ( F i g u r e 23). The w e i g h t s of A r t e m i a  s a l i n a t h a t c o u l d be o b t a i n e d i f s u p p l i e d w i t h the c o n c e n t r a t i o n s of b a c t e r i a s u p p o r t e d from 1 g of s u b s t r a t e d u r i n g the p l a t e a u phase of the b a c t e r i a l growth e x p e r i m e n t s a r e r e p o r t e d i n F i g u r e 24. 37 DISCUSSION B a c t e r i a l Growth and D e n s i t i e s E p i f l u o r e s c e n c e v e r s u s ATP The poor c o r r e l a t i o n between the e p i f l u o r e s c e n c e and ATP d a t a when c o n v e r t e d t o u n i t s of jug carbon was p o s s i b l y due t o the assumptions made i n making t h e s e c o n v e r s i o n s . In o r d e r t o c o n v e r t c e l l s / m l t o jugC/ml, a c o n s t a n t weight f o r the b a c t e r i a l c e l l s was assumed. However, n a t u r a l v a r i a t i o n i n both b a c t e r i a l c e l l s i z e s and w e i g h t s occur i n time and space (Watson et a l . , 1 9 7 7 ) . S i n c e the e p i f l u o r e s c e n c e d a t a were lower than the ATP d a t a i n terms of jugC/ml, one might s p e c u l a t e t h a t the average weight of one b a c t e r i a l c e l l was g r e a t e r than 2.2 X 10" 7 MgC, the weight assumed f o r t h i s s t u d y , i f the ATP d a t a were c o r r e c t . For the c o n v e r s i o n of nq ATP t o nq C, i t was assumed t h a t the carbon t o ATP r a t i o was c o n s t a n t . As mentioned p r e v i o u s l y , t h i s i s the most c r i t i c i z e d assumption of the a n a l y s i s ( K a r l , 1 9 8 0 ) . In o r d e r f o r t h i s r a t i o t o be c o n s t a n t d u r i n g growth, c e l l u l a r ATP must i n c r e a s e p r o p o r t i o n a l l y w i t h the i n c r e a s i n g c e l l u l a r c a r b o n , and the p r o d u c t i o n r a t e of ATP must e q u a l the r a t e of u t i l i z a t i o n ( F o r r e s t , 1 9 6 5 ) . S i n c e the b a c t e r i a l jug C c a l c u l a t e d from ATP d a t a exceeded the jugC c a l c u l a t e d from e p i f l u o r e s c e n c e d a t a , one might s p e c u l a t e t h a t the carbon t o ATP r a t i o of 250 was too h i g h or t h a t the r a t e of p r o d u c t i o n of ATP d u r i n g the time of measurement exceeded the 38 r a t e of u t i l i z a t i o n , thus p r o d u c i n g more ATP per c e l l . C a u t i o n s h o u l d t h e r e f o r e be used when d e t e r m i n i n g b a c t e r i a l biomass i n carbon u n i t s from ATP a n a l y s i s . However, the use of ATP as a r e l a t i v e i n d i c a t i o n of b a c t e r i a growth i s a c c e p t a b l e s i n c e t h i s s t udy demonstrated t h a t the same b a c t e r i a l growth t r e n d s were ob s e r v e d w i t h both e p i f l u o r e s c e n c e and ATP t e c h n i q u e s . The Growth P a t t e r n The s t a n d a r d p a t t e r n of b a c t e r i a l growth over time o b t a i n e d i n t h i s study was a l s o found by Hargrave (1970), who used the oxygen consumption t e c h n i q u e , by F e n c h e l (1970,1972,1977) . and S e k i and Yokohama (1978), who used the e p i f l u o r e s c e n c e t e c h n i q u e , and by H a m i l t o n and Holm-Hansen (1967) who used the ATP t e c h n i q u e . In a l l of t h e s e s t u d i e s the b a c t e r i a l p o p u l a t i o n rose t o a peak w i t h i n a p e r i o d of 1 or 2 days, and then e i t h e r m a i n t a i n e d h i g h d e n s i t i e s or d e c r e a s e d t o a p l a t e a u by 6 t o 8 days. B a c t e r i a l d e n s i t i e s may have i n c r e a s e d t o a maximum w i t h i n a few days because the b a c t e r i a were u t i l i z i n g the DOM t h a t was i n i t i a l l y l e a c h e d from the s u b s t r a t e and a s s i m i l a t i n g t h e s e r e a d i l y a v a i l a b l e compounds i n t o t h e i r own biomass ( H a r r i s o n and Mann,1975a). I f the amount of t h e s e r e a d i l y a v a i l a b l e n u t r i e n t s was r e d u c e d , the p o p u l a t i o n would m a i n t a i n s t e a d y s t a t e . At t h i s p o i n t , most of the DOM u t i l i z e d was p r o b a b l y produced by b a c t e r i a l d e c o m p o s i t i o n of b o t h dead b a c t e r i a l c e l l s and s u b s t r a t e (Robinson e t a l . , 1 9 8 2 ) . T h i s p l a t e a u or f i n a l phase was the s t a g e a t which the b a c t e r i a l c u l t u r e s were s u p p l i e d t o 39 the s u s p e n s i o n f e e d e r s as the food s o u r c e . The E f f e c t of D i f f e r e n t O r g a n i c S u b s t r a t e s on B a c t e r i a l Growth and D e n s i t i e s (_i) S i m i l a r S i z e P a r t i c l e s , Same S u b s t r a t e P r e p a r a t i o n The observed d i f f e r e n c e s i n b a c t e r i a l d e n s i t i e s s u p p o r t e d from the v a r i o u s s u b s t r a t e s c o u l d be a t t r i b u t e d t o d i f f e r e n c e s i n the q u a l i t y and q u a n t i t y of the DOM t h a t was i n i t i a l l y l e a c h e d from the o r g a n i c m a t t e r . A l g a l d e t r i t u s has been shown t o be more s u s c e p t a b l e t o l e a c h i n g than v a s c u l a r p l a n t d e t r i t u s (Tenore,1977a; R i c e , 1 9 7 9 ; Tenore and Hanson,1980) and t h e r e f o r e r e l e a s e s _ more DOM f o r subsequent b a c t e r i a l uptake (Tenore and R i c e , 1 9 8 1 ) . T h i s would account f o r the g r e a t e r d e n s i t i e s w i t h U l v a l a c t u c a compared t o those w i t h Z o s t e r a marina and wood c h i p s (Tenore and R i c e , 1 9 8 1 ) . D i s s o l v e d o r g a n i c compounds may have been r e a d i l y taken up by the b a c t e r i a e n a b l i n g the p o p u l a t i o n t o r e a c h g r e a t e r d e n s i t i e s when grown on U . l a c t u c a . P h e n o l i c r e s i d u e s may a l s o have a f f e c t e d the b a c t e r i a numbers and ATP c o n c e n t r a t i o n s as t h e s e compounds a r e a n t i b i o t i c (Conover and S i e b u r t h , 1 9 6 3 ; P r a k a s h et a l . , 1 9 7 2 ) . Both Z o s t e r a  marina and wood c h i p s r e l e a s e t h e s e compounds (Hedges and Mann,1979). The lower b a c t e r i a l d e n s i t i e s s u p p o r t e d by t h e s e s u b s t r a t e s c o u l d be a r e s u l t of the presence of these compounds. One might s u s p e c t Fucus v e s i c u l o s u s , a l s o a seaweed, t o have s u p p o r t e d m o r e — b a c t e r i a than Z o s t e r a mar i n a , but as t h i s s tudy demonstrated d e n s i t i e s were not s i g n i f i c a n t l y d i f f e r e n t 40 throughout most of the growth p e r i o d . A p o s s i b l e e x p l a n a t i o n f o r t h i s i s t h a t F . v e s i c u l o s u s c o n t a i n s and r e l e a s e s s i m i l a r amounts of. p o l y p h e n o l i c compounds as Z.marina (Hedges and Mann,1979). The c o n c e n t r a t i o n of t o t a l n i t r o g e n may a l s o have d i c t a t e d the amount of b a c t e r i a t h a t c o u l d be s u p p o r t e d by the d e t r i t u s . D e t r i t u s h i g h i n t o t a l n i t r o g e n may r e l e a s e more d i s s o l v e d t o t a l n i t r o g e n i n t o the medium which would become a v a i l a b l e f o r m i c r o b i a l uptake and subsequent m i c r o b i a l growth (Tenore et a l . , 1 9 7 9 ) . U l v a l a c t u c a not o n l y was the most r e a d i l y l e a c h e d s u b s t r a t e , but a l s o c o n t a i n e d the most t o t a l n i t r o g e n . B a c t e r i a l p o p u l a t i o n s s u p p o r t e d by U . l a c t u c a t h e r e f o r e were c a p a b l e of o b t a i n i n g maximal l e v e l s . Fucus v e s i c u l o s u s and Z o s t e r a marina had s i m i l a r , but l o w e r , amounts of t o t a l n i t r o g e n and t h e r e f o r e s u p p o r t e d lower b a c t e r i a l d e n s i t i e s . Wood c h i p s c o n t a i n e d t h e l e a s t amount of n i t r o g e n and s u p p o r t e d the l o w e s t amount of b a c t e r i a . The amount of o r g a n i c n i t r o g e n w i t h i n the s u b s t r a t e appeared t o be an i m p o r t a n t f a c t o r c o n t r o l l i n g the q u a n t i t y of b a c t e r i a . The b a c t e r i a l d e n s i t i e s i n f l a s k s w i t h s u b s t r a t e s t h a t were e i t h e r a u t o c l a v e d or d r i e d were i n d i r e c t p r o p o r t i o n t o the amount of s u b s t r a t e n i t r o g e n . U l v a l a c t u c a s u p p o r t e d g r e a t e r m i c r o b i a l d e n s i t i e s than Fucus v e s i c u l o s u s or Z o s t e r a  m a r i n a , both of which s u p p o r t e d g r e a t e r d e n s i t i e s than wood c h i p s . 41 ( i i ) D i f f e r e n t S i z e P a r t i c l e s , Same S u b s t r a t e P r e p a r a t i o n Based on the l i t e r a t u r e , one would expect t o f i n d more b a c t e r i a per u n i t s u r f a c e a r e a a s s o c i a t e d w i t h s m a l l p a r t i c l e s than l a r g e p a r t i c l e s . The s m a l l e r the p a r t i c l e s , the l a r g e r the t o t a l s u r f a c e a r e a a v a i l a b l e f o r m i c r o b i a l a c t i v i t y and attac h m e n t , and hence g r e a t e r p o t e n t i a l DOM r e l e a s e (Odum and de l a Cruz,1967; Fenchel,1970; H a r g r a v e , 1 9 7 2 ) . However, when the s u b s t r a t e s were d r i e d , the suspended b a c t e r i a l d e n s i t i e s were g r e a t e r f o r a l o n g e r p e r i o d of time a t the peak growth phase i n f l a s k s c o n t a i n i n g l a r g e p a r t i c l e s of U . l a c t u c a • The reason f o r t h i s c o u l d be t h a t more b a c t e r i a l c e l l s were a t t a c h e d t o the s m a l l e r p a r t i c l e s than t o the l a r g e r p a r t i c l e s meaning t h a t a l t h o u g h t o t a l b a c t e r i a l d e n s i t i e s were p r o b a b l y g r e a t e r i n the f l a s k s w i t h the s m a l l e r p a r t i c l e s , as the r e s e a r c h e r s s u g g e s t , the d e n s i t i e s of f r e e - l i v i n g b a c t e r i a which were measured i n t h i s study were a c t u a l l y l e s s . T h i s i d e a i s s u p p o r t e d by the f a c t t h a t d u r i n g the i n i t i a l and p l a t e a u phases of growth, the amount of b a c t e r i a s u p p o r t e d from l a r g e and s m a l l p a r t i c l e s was not s i g n i f i c a n t l y d i f f e r e n t . I n i t i a l l y , the amount of DOM r e l e a s e d from l a r g e and s m a l l p a r t i c l e s was the same, thus g i v i n g r i s e t o a s i m i l a r b a c t e r i a l d e n s i t i e s . However, as time p r o g r e s s e d , more c e l l s c o l o n i z e d the s m a l l e r p a r t i c l e s and the number of suspended c e l l s d e c l i n e d t o a c o n c e n t r a t i o n l e s s than t h a t a s s o c i a t e d w i t h the l a r g e r p a r t i c l e s . Once the s m a l l p a r t i c l e s were c o v e r e d by the a t t a c h e d b a c t e r i a , more DOM was r e l e a s e d , which e n a b l e d the number of f r e e - l i v i n g b a c t e r i a t o i n c r e a s e t o v a l u e s t h a t were no l o n g e r s i g n i f i c a n t l y d i f f e r e n t 42 than those s u p p o r t e d from the l a r g e r p a r t i c l e s . The s i z e of p a r t i c l e s of U l v a l a c t u c a had no e f f e c t on the b a c t e r i a l numbers when a u t o c l a v e d . I t would appear t h a t a u t o c l a v i n g e n a b l e d more DOM t o be r e l e a s e d from the s m a l l p a r t i c l e s m a i n t a i n i n g the f r e e - l i v i n g b a c t e r i a l p o p u l a t i o n a t h i g h c o n c e n t r a t i o n s r e g a r d l e s s of m i c r o b i a l p a r t i c l e a t tachment. No d i f f e r e n c e s i n b a c t e r i a l d e n s i t i e s were observed f o r Fucus v e s i c u l o s u s , wood c h i p s , and Z o s t e r a marina of d i f f e r e n t p a r t i c l e s i z e s . T h i s was p r o b a b l y because b a c t e r i a tend not t o adhere t o v a s c u l a r p l a n t s or woody t i s s u e s as r e a d i l y as t o a l g a l d e t r i t u s (Tenore and Hanson,1980) and the p o s s i b i l t y t h a t t h e s e s u b s t r a t e s r e l e a s e p h e n o l i c compounds r e g a r d l e s s of t h e i r s t a t e . ( i i i ) S i m i l a r S i z e P a r t i c l e s , D i f f e r e n t S u b s t r a t e P r e p a r a t i o n For s m a l l p a r t i c l e s g r e a t e r b a c t e r i a l d e n s i t i e s were o b t a i n e d when Fucus v e s i c u l o s u s and Z o s t e r a marina were a u t o c l a v e d as opposed t o d r i e d . T h i s s u p p o r t s the p r e v i o u s l y s t a t e d t h e o r y t h a t a u t o c l a v i n g r e l e a s e s more DOM i n t o the medium e n a b l i n g the p o p u l a t i o n t o peak a t g r e a t e r c o n c e n t r a t i o n s . However, a u t o c l a v i n g may not have been an e f f e c t i v e d e v i c e t o r e l e a s e DOM from the l a r g e p a r t i c l e s as no s i g n i f i c a n t d i f f e r e n c e s were obse r v e d i n b a c t e r i a l d e n s i t i e s a s s o c i a t e d w i t h l a r g e p a r t i c l e s of a u t o c l a v e d and d r i e d F . v e s i c u l o s u s and Z.mar i n a . 43 The E f f e c t of S p e c i f i c E x p e r i m e n t a l C o n d i t i o n s (_i) I n o r g a n i c N u t r i e n t s The change of n u t r i e n t c o n d i t i o n s from poor t o r i c h e n a b l e d b a c t e r i a l p o p u l a t i o n s , s u p p o r t e d from the seaweeds F . v e s i c u l o s u s and U . l a c t u c a , t o i n c r e a s e i n c o n c e n t r a t i o n by one o r d e r of magnitude. T h i s r e s u l t s u g g e s t s t h a t suspended b a c t e r i a are c a p a b l e of r a p i d uptake of i n o r g a n i c n u t r i e n t s , as F e n c h e l (1977) found, as w e l l as e f f i c i e n t c o n v e r s i o n of i n o r g a n i c n u t r i e n t s i n t o t h e i r own biomass. These d i f f e r e n c e s were s t i l l o b served a t the f i n a l growth phase even though the n u t r i e n t s were p r o b a b l y d e p l e t e d by t h i s t i m e , as shown by F e n c h e l (1977) and F e n c h e l and B l a c k b u r n (1979). T h i s i m p l i e s t h a t the b a c t e r i a l p o p u l a t i o n s s u s t a i n e d t h e i r d i f f e r e n c e s i n d e n s i t i e s by u t i l i z i n g the DOM r e l e a s e d from b a c t e r i a l d e c o m p o s i t i o n . Under n u t r i e n t - d e p l e t e d c o n d i t i o n s , the p o p u l a t i o n s s u p p o r t e d on U l v a l a c t u c a and Fucus v e s i c u l o s u s d e c l i n e d t o the l o w e s t v a l u e s o b s e r v e d i n a l l the e x p e r i m e n t s . T h i s d emonstrates the importance of d i s s o l v e d i n o r g a n i c n u t r i e n t s i n c o n t r o l l i n g b a c t e r i a l d e n s i t i e s s i n c e the DOM component a l o n e , under n u t r i e n t - d e p l e t e d c o n d i t i o n s , was not c a p a b l e of m a i n t a i n i n g c o n c e n t r a t i o n s g r e a t e r than 10 6 c e l l s / m l . The c o n c e n t r a t i o n of i n o r g a n i c n u t r i e n t s had no e f f e c t on b a c t e r i a l d e n s i t i e s o b t a i n e d from Z o s t e r a marina and wood c h i p s . G e n e r a l l y , i n o r g a n i c n u t r i e n t s i n the medium are i m p o r t a n t t o b a c t e r i a s u p p o r t e d on n u t r i e n t - p o o r d e t r i t u s ( F e n c h e l and J o r g e n s e n , 1 9 7 7 ) . B a c t e r i a then r e q u i r e an exogenous s u p p l y of 44 n u t r i e n t s . S i n c e both Z o s t e r a marina and wood c h i p s c o n t a i n e d low amounts of t o t a l n i t r o g e n , i t may be suggested t h a t t h e s e s u b s t r a t e s l i m i t e d the number of b a c t e r i a from the low amount of DOM they r e l e a s e d . The c o n c e n t r a t i o n of i n o r g a n i c n u t r i e n t s added may not have been enough t o o b t a i n d e t e c t a b l e d i f f e r e n c e s . ( i i ) Oxygen D i s s o l v e d o r g a n i c compounds r e l e a s e d under a e r o b i c c o n d i t i o n s may be d i f f e r e n t from those r e l e a s e d under a n a e r o b i c c o n d i t i o n s . T h i s i s because a l g a l components m i n e r a l i z e c o m p l e t e l y i n an a e r o b i c environment and not i n an a n a e r o b i c environment and l i g n i n s , waxes, p h e n o l s , and a r o m a t i c compounds demand the presence of oxygen f o r b i o l o g i c a l l y c a t a l i z e d c l e a v a g e ( F e n c h e l and B l a c k b u r n , 1 9 7 9 ) . D e s p i t e t h e s e d i f f e r e n c e s i n the type of DOM r e l e a s e d from the s u b s t r a t e , i t appeared t h a t a e r o b i c and a n a e r o b i c c o n d i t i o n s had no e f f e c t on the q u a n t i t y of b a c t e r i a s u p p o r t e d . The r e s u l t s of S e k i and Yokohama (1968) s u p p o r t t h e s e d a t a . T h e r e f o r e , one might expect t h a t DOM was e q u a l l y a v a i l a b l e t o the b a c t e r i a i n the oxygenated and deoxygenated f l a s k s (as Knauer and A y e r s , l 9 7 7 ) suggest) and t h e r e f o r e s i m i l a r c o n c e n t r a t i o n s of suspended b a c t e r i a may be s u p p o r t e d . 45 D e c o m p o s i t i o n Rates D e c o m p o s i t i o n r a t e s v a r i e d w i t h s u b s t r a t e type p o s s i b l y due t o the d i f f e r e n c e s i n the amount of o r g a n i c n i t r o g e n ( S e k i et a l . , 1 9 6 8 ) , l i g n i n (Gunnison and Alexander,1975a) and p o l y p h e n o l s ( K i n g and Heath,1967) c o n t a i n e d w i t h i n them. U l v a l a c t u c a was more r e a d i l y decomposed compared t o Z o s t e r a mar i n a s i n c e m i c r o b e s p r o c e s s a l g a l d e t r i t u s more e a s i l y than v a s c u l a r d e t r i t u s (Tenore,1975a; Ri c e , 1 9 7 9 ; Tenore and Hanson,1980). Fucus v e s i c u l o s u s and Z o s t e r a mar i n a decomposed at s i m i l a r r a t e s p o s s i b l y because of the s i m i l a r amounts of o r g a n i c n i t r o g e n and p o l y p h e n o l i c compounds t h a t were r e l e a s e d from t h e s e s u b s t r a t e s ; both may have been decomposed more s l o w l y than U l v a l a c t u c a because of the g r e a t e r amount of o r g a n i c n i t r o g e n and no p h e n o l i c r e s i d u e s r e l e a s e d from t h i s s u b s t r a t e . S i m i l a r d e c o m p o s i t i o n r a t e s were obse r v e d f o r wood c h i p s f o r a l l t r e a t m e n t s . T h i s was p r o b a b l y due t o the h i g h amount of l i g n i n t h a t was p r e s e n t i n the c e l l w a l l s (Gunnison and Alexander,1975a) which p r e v e n t e d m i c r o b i a l a t t a c k . The d e c o m p o s i t i o n r a t e s were a r e l a t i v e i n d i c a t i o n of the amount of suspended b a c t e r i a t h a t was s u p p o r t e d by the v a r i o u s s u b s t r a t e s under d i f f e r e n t e x p e r i m e n t a l c o n d i t i o n s . E x c e p t i o n s t o t h i s o b s e r v a t i o n were s m a l l p a r t i c l e s of U l v a l a c t u c a which had a 2 - f o l d i n c r e a s e i n d e c o m p o s i t i o n r a t e over l a r g e p a r t i c l e s , y e t the suspended b a c t e r i a l d e n s i t i e s s u p p o r t e d by the l a r g e and s m a l l p a r t i c l e s were s i m i l a r . H i g her d e c o m p o s i t i o n r a t e s a s s o c i a t e d w i t h s m a l l p a r t i c l e s have a l s o o b s e r v e d by G o s s e l i n k and K i r b y (1974), Hargrave (1972) and 46 H a r r i s o n and Mann (1975a). F e n c h e l (1977) a t t r i b u t e s the enhanced d e c o m p o s i t i o n of s m a l l p a r t i c l e s t o g r e a t e r m i c r o b i a l a t t a c h m e n t . As suggested p r e v i o u s l y , the t o t a l m i c r o b i a l biomass ( a t t a c h e d and suspended b a c t e r i a ) a s s o c i a t e d w i t h the s m a l l p a r t i c l e s may have been g r e a t e r than t h a t w i t h l a r g e p a r t i c l e s , and t h e r e f o r e c o u l d have g i v e n r i s e t o t h i s g r e a t e r d e c o m p o s i t i o n r a t e . The Growth of S u s p e n s i o n - F e e d e r s M y t i l u s e d u l i s T o x i c i t y , l a b o r a t o r y c o n d i t i o n s , or s t a r v a t i o n a r e p o s s i b l e reasons f o r the obser v e d death of M y t i l u s e d u l i s when s u p p l i e d w i t h the suspended b a c t e r i a l c u l t u r e s as t h e i r o n l y source of fo o d . P o l y p h e n o l s a r e t o x i c m e t a b o l i t e s r e l e a s e d by Fucus  v e s i c u l o s u s (Tenore and R i c e , 1 9 8 0 ) . The presence of the s e compounds i n the b a c t e r i a l c u l t u r e medium s u p p o r t e d by F . v e s i c u l o s u s c o u l d have caused the mussels t o d i e . However, i t was u n l i k e l y t h a t a t o x i c response was the o n l y cause of de a t h s i n c e M . e d u l i s a l s o d i e d when s u p p l i e d w i t h a c u l t u r e of suspended b a c t e r i a grown from U l v a l a c t u c a , a s u b s t r a t e which does not produce t h e s e t o x i c m e t a b o l i t e s . The l a b o r a t o r y c o n d i t i o n s t o which the mussels were adapted were not r e l a t e d t o t h e i r d e a t h . S i m i l a r l y c o n d i t i o n e d mussels were c a p a b l e of growing and f e e d i n g on the p h y t o p l a n k t o n c u l t u r e , D u n a l i e l l a  t e r t i o l e c t a . W right e t a l . ( 1982) d e s c r i b e the b l u e m u s s e l , M y t i l u s e d u l i s , as h a v i n g a "course f i l t e r i n g a p p a r a t u s " as t h i s 47 organism has g r e a t e r spaces between the c i r r i a l o n g i t s g i l l f i l a m e n t s than o t h e r m o l l u s c s . Geukensia demissa and M y t i l i s  c a l i f o r n i a n u s a r e both c a p a b l e of f i l t e r i n g f r e e - l i v i n g b a c t e r i a ( V a h l , l 9 7 2 ; J o r gensen,1975). Because of t h i s m o r p h o l o g i c a l f e a t u r e , M . e d u l i s may not have been a b l e t o remove b a c t e r i a from the s u s p e n s i o n w i t h any measureable e f f i c i e n c y . T h e i r d e a t h was p r o b a b l y a r e s u l t of s t a r v a t i o n . I t cannot be i m p l i e d t h a t no b a c t e r i a l c e l l s were f i l t e r e d , s i n c e some b a c t e r i a l r e t e n t i o n may have taken p l a c e as found by H o l l i b a u g h e t a l . ( l 9 8 0 ) . However, even i f t h e r e was some r e t e n t i o n , the energy a s s i m i l a t e d from t h i s s ource of food was s u f f i c i e n t t o s u p p o r t the mussels f o r o n l y a few days. The c o n c e n t r a t i o n of f r e e -l i v i n g b a c t e r i a l c e l l s s u p p l i e d as a source of food was not a c o n t r i b u t i n g f a c t o r i n the mussels' death as media of s i m i l a r b a c t e r i a l c o n c e n t r a t i o n s have been shown t o s u p p o r t M y t i l i s  c a l i f o r n i a n u s f o r s e v e r a l months ( Z o b e l and Feltham,1938). A r t e m i a s a l i n a The GEs of A r t e m i a s a l i n a were a f u n c t i o n of the c o n c e n t r a t i o n of suspended b a c t e r i a s u p p l i e d as f o o d . A minimum c o n c e n t r a t i o n of 2.5 X 10 s c e l l s / m l ( l o g 6.4) was r e q u i r e d t o o b t a i n a d e t e c t a b l e GE. I t c o u l d be t h a t c o n c e n t r a t i o n s l e s s than t h i s v a l u e d i d not e n a b l e the p a r a p o d i a of the b r i n e shrimp t o e f f i c i e n t l y f i l t e r the medium c l e a r of c e l l s , as P r o v a s o l i and D'Agostino (1969) s u g g e s t e d , s i n c e low f i l t e r i n g r a t e s were ob s e r v e d . The number of p a r t i c l e s c o l l e c t e d and then consumed was not g r e a t enough t o enable the organism t o grow, and hence 48 GEs were z e r o . Once b a c t e r i a l c o n c e n t r a t i o n s exceeded 2.5 X 10 6 c e l l s / m l , the shrimp e f f i c i e n t l y f i l t e r e d the media, r e t a i n e d and d i g e s t e d the suspended b a c t e r i a and were then a b l e t o c o n v e r t the food i n t o t h e i r own biomass. Observed GEs then i n c r e a s e d above z e r o because of the enhanced consumption r a t e s . As the food c o n c e n t r a t i o n i n c r e a s e d , f i l t e r i n g r a t e s d e c r e a s e d , which e n a b l e d e f f i c i e n t r e t e n t i o n of b a c t e r i a . S i n c e growth r a t e s i n c r e a s e d i t seems r e a s o n a b l e t o assume t h a t most of the i n g e s t e d food was a s s i m i l a t e d f o r growth. Belkemishev (1954) and Raymont and G r o s s ( l 9 5 4 ) o b s e r v e d an upper l i m i t t o the number of p a r t i c l e s t h a t c o u l d be c o n v e r t e d i n t o the biomass of an organism. At h i g h food c o n c e n t r a t i o n s , the d i g e s t i v e e f f i c i e n c y of b r i n e shrimp has been obse r v e d ' t o de c r e a s e (Reeve,1963b) due t o the g r e a t e r p r e s s u r e on the p a r t i c l e s as they pass t h r o u g h the gut. C e l l s a r e t h e r e f o r e e x p e l l e d b e f o r e they c o u l d be d i g e s t e d (Reeve,1963d). T h i s upper l i m i t was not obser v e d d u r i n g the study and may t h e r e f o r e o c c u r a t c e l l c o n c e n t r a t i o n s above 10 7 c e l l s / m l , the h i g h e s t c o n c e n t r a t i o n used i n the growth e x p e r i m e n t s . D e s p i t e the f a c t t h a t t h i s upper l i m i t f o r GEs o c c u r r e d a t c e l l c o n c e n t r a t i o n s g r e a t e r than 10 7 c e l l s / m l , maximum consumption o c c u r r e d a t c o n c e n t r a t i o n s l e s s than t h i s v a l u e . Other r e s e a r c h e r s have a l s o found a maximum t h r e s h o l d f o r prey consumption (Parsons and Le B r a s s e u r , 1 9 7 0 ; F r o s t , 1 9 7 5 ) . In a d d i t i o n , however, t h i s study found t h a t t h i s peak i n consumption was f o l l o w e d by a d e c l i n e as pr e y c o n c e n t r a t i o n was f u r t h e r i n c r e a s e d ; a t r e n d a l s o observed 49 by M u l l i n (1963) and Nassogne (1970). T h i s d e c l i n e may be e x p l a i n e d by examining the f e e d i n g p r o c e s s e s of b r i n e shrimp t h a t ' w e r e o b s e r v e d by Reeve (1963b). As b r i n e shrimp f i l t e r the medium, a clump of food p a r t i c l e s accumulate b e h i n d the labrum b e f o r e b e i n g passed t o the m a x i l l a e . At h i g h food c o n c e n t r a t i o n s , the b a l l of c e l l s i n c r e a s e s i n s i z e too q u i c k l y t o be brought t o the mouth f o r consumption and i t i s d i s p o s e d of by the f i r s t t h o r a c i c l i m b s . T h i s p r o c e s s of d i s c a r d i n g the e x c e s s c o l l e c t e d c e l l s may account f o r the lower consumption r a t e s observed a t h i g h b a c t e r i a l c o n c e n t r a t i o n s . However, c e l l s t h a t were consumed must have been e f f i c i e n t l y d i g e s t e d as the shrimp c o n t i n u e d t o grow. T h i s gave r i s e t o the i n c r e a s e d GEs obser v e d a t h i g h food c o n c e n t r a t i o n s . GEs a l s o i n c r e a s e d as the s i z e of A r t e m i a s a l i n a i n c r e a s e d . However, s i g n i f i c a n t d i f f e r e n c e s o c c u r r e d o n l y a f t e r the organism o b t a i n e d a l e n g t h of 1 mm. Once the b r i n e shrimp were l a r g e r than 1 mm, h i g h e r f i l t e r i n g r a t e s were o b s e r v e d . T h i s p r o b a b l y o c c u r r e d because t h e i r f i l t e r i n g appendages were more complex and t h e r e f o r e more e f f i c i e n t i n r e t a i n i n g b a c t e r i a c e l l s . G a u l d (1959) noted t h a t the number of t h o r a c i c l i m b s of A r t e m i a s a l i n a i n c r e a s e d and the set a e became more f i n e l y d e v e l o p e d i n the 4 t h and 6th i n s t a r s , the growth s t a g e s which c o r r e s p o n d t o b r i n e shrimp of s i z e s 1 mm and 1.5 mm, r e s p e c t i v e l y . The marked i n c r e a s e i n GEs c o n t i n u e d as the organism i n c r e a s e d t o a l e n g t h of 2.5 mm but then d e c r e a s e d . T h i s c o u l d be due t o the organism's energy r e q u i r e m e n t s f o r growth and body maintenance. Young A . s a l i n a have a lower energy 50 c o s t f o r body maintenance and most of the i n g e s t e d f o o d i s c o n v e r t e d d i r e c t l y i n t o biomass. As they c o n t i n u e t o grow, the f e e d i n g appendages become more complex and t h e r e f o r e more e f f i c i e n t i n c o l l e c t i n g food p a r t i c l e s , thus i n c r e a s i n g GEs. However, once the organism reaches 2.5 mm, a b a l a n c e c o u l d be reached between the energy r e q u i r e m e n t s f o r growth, m e t a b o l i s m , and p h y s i c a l a c t i v i t i e s . T h e r e f o r e , l e s s energy would be c o n v e r t e d i n t o t h e i r own biomass, and GEs would d e c r e a s e . The lower GEs f o r l a r g e r shrimp may a l s o have been a r e s u l t of the lower consumption r a t e s o b s e r v e d . C o r r e s p o n d i n g f i l t e r i n g r a t e s , however, were h i g h e r , meaning t h a t the volume of medium passed by the f i l a m e n t s was g r e a t e r but c e l l r e t e n t i o n was l e s s . The d e c r e a s e d r a t i o n might be a r e s u l t of the f r e e - l i v i n g b a c t e r i a l c e l l s b e i n g too s m a l l f o r the l a r g e b r i n e shrimp t o r e t a i n . The d i s t a n c e between the f i l t e r i n g s e t u l e s of a d u l t b r i n e shrimp are 5.7 Mm ( U s s i n g , 1 9 3 8 ) , and the b a c t e r i a l c e l l s , were 2-4 Mm i n d i a m e t e r . The GEs c a l c u l a t e d f o r A r t e m i a s a l i n a s u p p o r t e d from suspended b a c t e r i a a r e comparable t o those r e p o r t e d by G i l b o r (1957) and Reeve (1963a) f o r A . s a l i n a f e d w i t h p h y t o p l a n k t o n c u l t u r e s . A maximum growth e f f i c i e n c y of 65% was found by Reeve (1963a) a t a c o n c e n t r a t i o n of 3X10' c e l l s / m l . Such an e f f i c i e n c y was o b t a i n e d i n t h i s s tudy a t a c o n c e n t r a t i o n of 10 7 b a c t e r i a l c e l l s / m l . F r e e - l i v i n g b a c t e r i a and p h y t o p l a n k t o n c e l l s a r e t h e r e f o r e comparable food s o u r c e s i n terms of p o t e n t i a l GEs f o r A . s a l i n a . 51 Summary GEs f o r the b l u e m u s s e l , M y t i l u s e d u l i s were not measureable; however, GEs were d e t e c t e d f o r A r t e m i a s a l i n a s u p p o r t e d on t h i s food source and d e f i n i t e r e l a t i o n s h i p s e x i s t e d between growth and r a t i o n . The l e n g t h of A . s a l i n a t h a t was s u p p o r t e d or m a i n t a i n e d on suspended b a c t e r i a l c u l t u r e s was l i n e a r l y p r o p o r t i o n a l t o b a c t e r i a l c o n c e n t r a t i o n as shown i n F i g u r e 22. S i n c e e x p e r i m e n t a l c o n d i t i o n s as w e l l as s u b s t r a t e type d i c t a t e d b a c t e r i a l d e n s i t i e s o b t a i n e d per gram (dry w e ight) of the s u b s t r a t e p r o v i d e d , the w e i g h t , i n Mg, of one b r i n e shrimp t h a t can be s u p p o r t e d from these b a c t e r i a l c u l t u r e s was d e t e r m i n e d . The r e s u l t s of t h i s c a l c u l a t i o n are r e p o r t e d i n F i g u r e 24. B a c t e r i a s u p p o r t e d by 1 g (dry w e i g h t ) U l v a l a c t u c a under n u t r i e n t - r i c h c o n d i t i o n s can s u p p o r t one a d u l t b r i n e shrimp which weighs 46 Mg. F r e e - l i v i n g b a c t e r i a l c u l t u r e s s u p p o r t e d by wood c h i p s can o n l y m a i n t a i n A . s a l i n a t h a t a r e i n the f i r s t i n s t a r growth s t a g e or weigh 0.5 Mg. S i m i l a r , y e t i n t e r m e d i a t e , s i z e s of b r i n e shrimp c o u l d be s u s t a i n e d from b a c t e r i a l c u l t u r e s grown w i t h Fucus v e s i c u l o s u s and Z o s t e r a  m a r i n a . Under n u t r i e n t - r i c h c o n d i t i o n s , b a c t e r i a a r e a t s u f f i c i e n t c o n c e n t r a t i o n s t o s u p p o r t b r i n e shrimp g r e a t e r i n weight than 2 Mg or 2.0 mm i n l e n g t h . B a c t e r i a l c u l t u r e s grown under n u t r i e n t - p o o r c o n d i t i o n s w i l l o n l y s u s t a i n A . s a l i n a t h a t are l e s s than 2Mg. 52 CONCLUSIONS Based on t h e f i n d i n g s of t h i s s t u d y , the f o l l o w i n g c o n c l u s i o n s can be made. 1 . E p i f l u o r e s c e n c e and ATP t e c h n i q u e s are comparable i n d i c a t o r s of b a c t e r i a l growth. 2. The growth of b a c t e r i a when s u p p l i e d w i t h a d e t r i t a l s u bstance f o l l o w s a d i s t i n c t p a t t e r n . I t i s suggested t h a t t h i s growth p a t t e r n i s a r e s u l t of the amount of DOM r e l e a s e d from the d e t r i t u s by l e a c h i n g and b a c t e r i a l d e c o m p o s i t i o n . 3. The type of the o r g a n i c s u b s t r a t e s u p p l i e d t o seawater i s the most i m p o r t a n t f a c t o r c o n t r o l l i n g suspended b a c t e r i a l d e n s i t i e s t h a t can be s u p p o r t e d ; U l v a l a c t u c a s u p p o r t s more than Fucus v e s i c u l o s u s and Z o s t e r a marina which support more than wood c h i p s . The amounts of o r g a n i c n i t r o g e n , p o l y p h e n o l i c compounds, and l i g n i n , a r e p r o b a b l y r e s p o n s i b l e f o r t h e s e o b s e r v e d d i f f e r e n c e s . 4. D e c o m p o s i t i o n r a t e s of o r g a n i c s u b s t r a t e s g e n e r a l l y r e f l e c t the suspended b a c t e r i a l biomass t h a t i s a s s o c i a t e d w i t h the s u b s t r a t e . 5. The a d d i t i o n of i n o r g a n i c n u t r i e n t s t o media w i t h the seaweeds U l v a l a c t u c a and Fucus v e s i c u l o s u s e n a b l e s g r e a t e r q u a n t i t i e s of suspended b a c t e r i a t o be o b t a i n e d . However, 53 i n o r g a n i c n u t r i e n t s have no e f f e c t on b a c t e r i a l biomass s u p p o r t e d from Z o s t e r a marina and wood c h i p s . 6. A e r o b i c and a n e r o b i c c o n d i t i o n s y i e l d b a c t e r i a l c u l t u r e s t h a t a r e not s i g n i f i c a n t l y d i f f e r e n t i n d e n s i t i e s and t h e r e f o r e are not c o n s i d e r e d i m p o r t a n t i n d e t e r m i n i n g suspended b a c t e r i a l d e n s i t i e s . 7. B a c t e r i a l d e n s i t i e s s u p p o r t e d by U l v a l a c t u c a under n u t r i e n t - r i c h a e r o b i c c o n d i t i o n s e n a b l e A . s a l i n a t o o b t a i n an o p t i m a l GE of 60%. Under th e s e c o n d i t i o n s , 1 g (dry weight) of U l v a l a c t u c a can support 10 7 c e l l s / m l , which i n t u r n can s u s t a i n the growth of a d u l t b r i n e shrimp which are 3 mm i n l e n g t h . B a c t e r i a grown on 1 g of l a r g e or s m a l l p a r t i c l e s of Fucus  v e s i c u l o s u s or Z o s t e r a m a r i n a , e i t h e r a u t o c l a v e d or d r i e d , a r e o n l y c a p a b l e of s u p p o r t i n g b r i n e shrimp l e s s than 2 mm i n l e n g t h . B a c t e r i a l d e n s i t i e s o b t a i n e d from b a c t e r i a l c u l t u r e s grown w i t h wood c h i p s can o n l y support b r i n e shrimp which a r e l e s s than 0.5 mm i n l e n g t h . 8. M y t i l u s e d u l i s cannot c o n v e r t suspended b a c t e r i a i n t o t h e i r own biomass w i t h any measureable e f f i c i e n c y . F u r t h e r m o r e , they are not c a p a b l e of e x i s t i n g w i t h suspended b a c t e r i a as t h e i r o n l y source of f o o d . I t i s suggested t h a t t h i s i s a r e s u l t of t h e i r i n a b i l i t y t o f i l t e r the s m a l l suspended b a c t e r i a l c e l l s w i t h t h e i r r e l a t i v e l y c o u r s e f i l t e r i n g a p p a r a t u s . 54 9. A r t e m i a s a l i n a are c a p a b l e of c o n v e r t i n g suspended b a c t e r i a i n t o t h e i r own biomass. However, a minimum number of a v a i l a b l e c e l l s i s r e q u i r e d b e f o r e measureable growth e f f i c i e n c i e s are o b s e r v e d . Beyond t h i s c o n c e n t r a t i o n growth e f f i c i e n c i e s of A . s a l i n a depend on the c o n c e n t r a t i o n of a v a i l a b l e c e l l s ; GEs i n c r e a s e as the food a v a i l a b l e i n c r e a s e s . The upper l i m i t of the b a c t e r i a l c o n c e n t r a t i o n t h a t can be c o n v e r t e d i n t o A . s a l i n a biomass exceeds 10 7 c e l l s per ml. GEs a l s o v a r y w i t h the s i z e of A r t e m i a s a l i n a . Maximum GEs a r e o b s e r v e d f o r b r i n e shrimp t h a t are 2.5 mm i n l e n g t h . B r i n e shrimp of t h i s l e n g t h demonstrate h i g h e r growth r a t e s and consumption r a t e s than s m a l l e r or l a r g e r organisms. I t i s suggested t h a t t h i s r e s u l t s because the f i l t e r i n g a p p a r a t u s i s c o m p l e t e l y d e v e l o p e d d u r i n g t h i s stage of growth. 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A v a i l a b i l i t y of d e t r i t u s of d i f f e r e n t t y p e s and ages t o a p o l y c h a e t e consumer, C a p i t e l l a c a p i t a t a . L i m n o l . Oceanogr. 25:553-558. Tenore, K.R. and D.L.Rice. 1980. A review of the t r o p h i c f a c t o r s a f f e c t i n g secondary p r o d u c t i o n of d e p o s i t f e e d e r s . I n : Marine B e n t h i c Dynamics. K.R. Tenore and B.C. C o u l l ( e d s . ) , U n i v e r s i t y of South C a r o l i n a P r e s s , Columbia, pp. 325-340. Tenore, K.R., R.B. Hanson, B.E. D o r n s e i f and C.N. Wiederhod. 1979. The e f f e c t of o r g a n i c n i t r o g e n supplement on the u t i l i z a t i o n of d i f f e r e n t s o u r c e s of d e t r i t u s . L i m n o l . Oceanogr. 24:350-355. Thayer, G.W. 1 9 7 6 . I d e n t i t y and r e g u l a t i o n of n u t r i e n t s l i m i t i n g p h y t o p l a n k t o n p r o d u c t i o n i n the s h a l l o w e s t u a r i e s near B e a u f o r t , N.C. O e c o l o g i a J _ 4 : 7 5 - 9 2 . —• U s s i n g , H.H. 1938. The b i o l o g y of some i m p o r t a n t p l a n k t o n a n i m a l s i n f j o r d s of E a s t G r e e n l a n d . Medd. G r o n l a n d 100:1-108. V a h l , 0. 1972. E f f i c i e n c y of p a r t i c l e r e t e n t i o n i n M y t i l u s e d u l i s . O p h e l i a 10:17-25. V e r l i m r o v , B., J.A. O t t and R. Novak. 1981. M i c r o o r g a n i s m s on macrophyte d e b r i s : B i o d e g r a d a t i o n and i t s i m p l i c a t i o n i n the food web. K i e l e r M e e r e s f o r s c h . , S o n d e r t h . 5 : 3 3 3 - 3 4 4 . W e t z e l , R.G. 1 9 7 5 . L i m n o l o g y . W.B. Saunders Co., P h i l a d e l p h i a , pp. 2 4 8 - 2 5 6 . 69 W r i g h t , R.T., R.B. C o f f i n , C P . E r s i n g and D. P e a r s o n . 1982. F i e l d and l a b o r a t o r y measurements of b i v a l v e f i l t r a t i o n of n a t u r a l marine b a c t e r i o p l a n k t o n . L i m n o l . Oceanogr. 27:99-111. Zieman, J.C. 1968. A study of the growth and decompostion of the sea g r a s s T h a l a s s i a t e s t u d i n u m . M.S. T h e s i s , U n i v . Miami. 50p. Z o b e l , C.E. and C.B. Feltham. 1938. B a c t e r i a as food f o r c e r t a i n marine i n v e r t e b r a t e s . J . Mar. Res. 1:312-327. 70 TABLES 71 T a b l e 1. The s u r f a c e a r e a of the s u b s t r a t e p a r t i c l e s . These d a t a show the s u r f a c e a r e a , i n cm 2, of the v a r i o u s s u b s t r a t e s used i n the b a c t e r i a l growth e x p e r i m e n t s . S u r f a c e Area (cm 2) S u b s t r a t e U l v a sp. Fucus sp. E e l g r a s s Wood c h i p s Whole p l a n t 25-100 30-150 15-50 0.5-1.0 (wet or d r y ) wet 0.25-0.5 0.25-1.0 0.25-1.0 Blended P l a n t dry 0.02-0.08 0.24- 1.12 0.24-0.72 0.48-1.20 X10-* X10-" X10" 4 X10-" 72 T a b l e 2. A summary of the c o n d i t i o n s of the b a c t e r i a l growth 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 i z e s and s t a t e s of s u b s t r a t e s under d i f f e r e n t n u t r i e n t and oxygen regimes. S u b s t r a t e s (a) I n o r g a n i c N u t r i e n t s (b) D e t r i t u s S t a t e S i z e (c) (d) A e r a t e d U,F,E,WC U,F,E,WC U,F,E,WC U,F,E U,F,E,WC U,F,E,WC U,F,E,WC N-Nt N t t Nt Nt Nt Nt wet wet wet wet dry dry wet l a r g e l a r g e l a r g e s m a l l s m a l l l a r g e l a r g e yes yes yes yes yes yes no (a) U=Ulva sp. (b) N-=no i n o r g a n i c n u t r i e n t s added F=Fucus sp. N t = n u t r i e n t - p o o r E=Eelgrass N t t = n u t r i e n t - r i c h WC=Wood c h i p s (c) wet=autoclaved dr ied=oven-dr i e d (d) see Table 1 f o r s i z e s 73 T a b l e 3. The e x p e r i m e n t a l c o n d i t i o n s used t o determine the growth r a t e s of M y t i l u s e d u l i s when s u p p l i e d w i t h suspended b a c t e r i a l c u l t u r e s grown w i t h Fucus v e s i c u l o s u s and U l v a l a c t u c a , and the p h y t o p l a n k t o n , D u n a l i e l l a t e r t i o l e c t a . Food Source Fucus sp. U l v a sp. D u n a l i e l l a sp. S a l i n i t y (ppt) 28 1 4 28 14 28 28 Temperature (°C) 12 1 2 1 2 1 2 12 12 Length of Experiment (days) 3* 3* 4* 4* 1 6 16 i n i t i a l w i d t h (mm) S.D. 4.7 0.4 11.6 0.8 4.4 0.5 11.0 0.9 17.0 1 .2 7.7 0.6 i n i t i a l l e n g t h (mm) S.D. 1 .7 0.3 6.6 0.5 1 .6 0.2 6.6 « 0.6 10.1 0.8 4.5 0.4 C e l l s s u p p l i e d 5.28 ( 1 0 6 c e l l s / m l ) 4.98 6. 13 5.56 0.06 0.07 Growth Rate (mm/day ) ( l e n g t h / w i d t h ) S.D. 0/0 0/0 0/0 0/0 .01/ .03 .008/ .01 05/ 003 009/ 001 * l e n g t h of experiment i n d i c a t e s the day the mussels d i e S.D. r e p r e s e n t s S t a n d a r d D e v i a t i o n s T a b l e 4. The r e l a t i v e p e r c e n t s of t o t a l c a r b o n , hydrogen and n i t r o g e n w i t h i n the s u b s t r a t e s used i n t h i s s t u d y . S u b s t r a t e Carbon Hydrogen N i t r o g e n ( % ) ( % ) ( % ) U l v a sp. 29. ,96 5. .38 3.98 Fucus sp. 36. .20 5. .28 1.62 E e l g r a s s 36.60 5.86 1.82 Wood C h i p s 46.62 6.29 0.20 75 T a b l e 5. De c o m p o s i t i o n r a t e s i n terms of p e r c e n t d r y weight l o s t of l a r g e and s m a l l s u b s t r a t e p a r t i c l e s which a r e a u t o c l a v e d or d r i e d under d i f f e r e n t n u t r i e n t and oxygen regimes ( S t a n d a r d D e v i a t i o n s : wood chips=0.3, E e l g r a s s = 1 . 1 , Fucus sp.=0.8 and U l v a s p . = 2 . l ) . S u b s t r a t e I n o r g a n i c D e t r i t u s A e r a t e d % Weight N u t r i e n t s S t a t e S i z e L o s t (a) (b) (c) Wood C h i p s N- wet N+ wet N+ + wet E e l g r a s s N- wet N+ wet N+ + wet Fucus sp. N- wet N+ wet N+ + wet U l v a sp. N- wet N+ wet N++ wet l a r g e yes 1 .4 l a r g e yes 1 .2 l a r g e yes 1 .7 l a r g e yes 20.0 l a r g e yes 23.3 l a r g e yes 37.8 l a r g e yes 10.6 l a r g e yes 24.3 l a r g e yes 36.9 l a r g e yes 20.0 l a r g e yes 25.5 l a r g e yes 40.0 Wood c h i p s N+ + wet l a r g e yes 1 .7 E e l g r a s s N+ + wet l a r g e yes 37.8 Fucus sp. N++ wet l a r g e yes 36.9 U l v a sp. N+ + wet l a r g e yes 40.0 Wood c h i p s N+ d r i e d l a r g e yes 1 .1 N+ d r i e d s m a l l yes 1 .2 E e l g r a s s N+ d r i e d l a r g e yes 35. 1 N+ d r i e d s m a l l yes 33.7 Fucus sp. N+ d r i e d l a r g e yes 38.3 N+ d r i e d s m a l l yes 40.0 U l v a sp. N+ d r i e d l a r g e yes 29.9 N+ d r i e d s m a l l yes 66.4 76 Wood c h i p s N+ wet l a r g e no 1 .1 E e l g r a s s N+ wet l a r g e no 22.2 Fucus sp. N+ wet l a r g e no 28.3 U l v a sp. N+ wet l a r g e no 36.9 (a) N - = n u t r i e n t - d e p l e t e d (b) wet=autoclaved N+=nutrient-poor dr ied=oven-dr i e d N + + = n u t r i e n t - r i c h (c) see T a b l e 1 f o r s i z e s 77 FIGURES 78 F i g u r e 1. A t y p i c a l b a c t e r i a l growth c u r v e . These d a t a show b a c t e r i a l d e n s i t i e s over time when s u p p l i e d w i t h a u t o c l a v e d Z o s t e r a marina under n u t r i e n t - p o o r c o n d i t i o n s . 79 G DAYS 80 F i g u r e 2. The suspended b a c t e r i a l biomass i n nqC over time t h a t i s s u p p o r t e d from (a) U l v a l a c t u c a (b) Fucus v e s i c u l o s u s (ATP dataO; E p i f l u o r e s c e n c e d a t a * ) . 81 82 F i g u r e 3. B a c t e r i a l d e n s i t i e s per gram of s u b s t r a t e t h a t can be s u p p o r t e d w i t h d i f f e r e n t amounts of Z o s t e r a marina (a) E p i f l u o r e s c e n c e d a t a (b) ATP d a t a (20go; 60g«). 0 0 C o 84 F i g u r e 4. The e f f e c t of a u t o c l a v e d s u b s t r a t e s on the number b a c t e r i a (a) n u t r i e n t - r i c h c o n d i t i o n s (b) n u t r i e n t - p o o r c o n d i t i o n s ( U l v a l a c t u c a o ; Fucus v e s i c u l o s u s A ? Z o s t e r a marina A; wood c h i p s * ) . 86 F i g u r e 5. The e f f e c t of a u t o c l a v e d s u b s t r a t e s on b a c t e r i a l ATP under n u t r i e n t - p o o r c o n d i t i o n s ( U l v a l a c t u c a O ; Fucus  v e s i c u l o s u s A; Z o s t e r a marina A; wood c h i p s * ) . 87 88 F i g u r e 6 . The e f f e c t of d r i e d s u b s t r a t e s on b a c t e r i a l d e n s i t i e s under n u t r i e n t - p o o r c o n d i t i o n s (a) E p i f l u o r e s c e n c e d a t a (b) ATP d a t a ( U l v a l a c t u c a o ; Fucus v e s i c u l o s u s A; Z o s t e r a mar i n a A ; wood c h i p s * ) . 89 90 F i g u r e 7. The e f f e c t of a u t o c l a v e d and d r i e d , l a r g e s u b s t r a t e p a r t i c l e s on the number of b a c t e r i a under n u t r i e n t - p o o r c o n d i t i o n s (a) Fucus v e s i c u l o s u s (b) Z o s t e r a marina (c) U l v a l a c t u c a ( a u t o c l a v e d * ; d r i e d O ) . 91 D A Y S 92 F i g u r e 8. The e f f e c t of a u t o c l a v e d and d r i e d , l a r g e s u b s t r a t e p a r t i c l e s on b a c t e r i a l ATP under n u t r i e n t - p o o r c o n d i t i o n s (a) Fucus v e s i c u l o s u s (b) Z o s t e r a mar i n a (c) U l v a l a c t u c a ( a u t o c l a v e d * ; . d r i e d o ) . 93 . o i IE IB D A Y S 94 F i g u r e 9. The e f f e c t of a u t o c l a v e d and d r i e d , s m a l l s u b s t r a t e p a r t i c l e s on b a c t e r i a l d e n s i t i e s s u p p o r t e d under n u t r i e n t - p o o r c o n d i t i o n s (a) E p i f l u o r e s c e n c e d a t a f o r Fucus v e s i c u l o s u s (b) ATP d a t a f o r Fucus v e s i c u l o s u s (c) E p i f l u o r e s c e n c e d a t a f o r Z o s t e r a marina (d) ATP d a t a f o r Z o s t e r a marina ( a u o t o c l v e d * ; d r i e d O ) . 95 9d 96 F i g u r e 10. The e f f e c t of s m a l l and l a r g e , a u t o c l a v e d s u b s t r a t e p a r t i c l e s on b a c t e r i a l d e n s i t i e s under n u t r i e n t - p o o r c o n d i t i o n s (a) E p i f l u o r e s c e n c e d a t a f o r Fucus v e s i c u l o s u s (b) ATP d a t a f o r Fucus v e s i c u l o s u s (c) E p i f l u o r e s c e n c e d a t a f o r Z o s t e r a marina (d) ATP d a t a f o r Z o s t e r a marina ( s m a l l p a r t i c l e s O ; l a r g e p a r t i c l e s * ) . 97 10c 10 d 98 F i g u r e 11. The e f f e c t of s m a l l and l a r g e , d r i e d s u b s t r a t e p a r t i c l e s on b a c t e r i a l d e n s i t i e s under n u t r i e n t - p o o r c o n d i t i o n s (a) E p i f l u o r e s c e n c e d a t a f o r U l v a l a c t u c a (b) ATP da t a f o r U l v a l a c t u c a (c) E p i f l u o r e s c e n c e d a t a f o r Fucus v e s i c u l o s u s (d) ATP da t a f o r Fucus v e s i c u l o s u s (e) E p i f l u o r e s c e n c e d a t a f o r Z o s t e r a marina ( f ) ATP d a t a f o r Z o s t e r a marina (g) E p i f l u o r e s c e n c e d a t a f o r wood c h i p s (h) ATP d a t a f o r wood c h i p s ( s m a l l p a r t i c l e s O ; l a r g e p a r t i c l e s * ) . 99 11c 11d 100 101 F i g u r e 12. The e f f e c t of n u t r i e n t c o n d i t i o n s w i t h a u t o c l a v e d s u s t r a t e s on b a c t e r i a l d e n s i t i e s (a) E p i f l u o r e s c e n c e d a t a f o r U l v a l a c t u c a (b) E p i f l u o r e s c e n c e d a t a f o r Fucus v e s i c u l o s u s (c) E p i f l u o r e s c e n c e data f o r Z o s t e r a marina (d) E p i f l u o r e s c e n c e d a t a f o r wood c h i p s (e) ATP d a t a f o r wood c h i p s ( n u t r i e n t - r i c h * ; n u t r i e n t - p o o r O ; n u t r i e n t - d e p l e t e d ^ ) . 102 D A Y S 12C 12d D A Y S 12e 1 04 F i g u r e 13. The e f f e c t of n u t r i e n t - d e p l e t e d c o n d i t i o n s on the number of b a c t e r i a s u p p o r t e d from v a r i o u s s u b s t r a t e s ( U l v a l a c t u c a o ; Fucus v e s i c u l o s u s a; Z o s t e r a marina A ; wood c h i p s * ) . 1 06 F i g u r e 14. The e f f e c t of a e r o b i c and a n a e r o b i c c o n d i t i o n s on the number of b a c t e r i a s u p p o r t e d from v a r i o u s s u b s t r a t e s (a) Fucus v e s i c u l o s u s w o o d c h i p s * (b) U l v a l a c t u c a * ; Z o s t e r a marina • ( a e r o b i c 0+; a n a e r o b i c 0 - ) . 108 F i g u r e 15. F i l t e r i n g Rates of M y t i l u s e d u l i s as a f u n c t i o n of the c o n c e n t r a t i o n of the p h y t o p l a n k t o n c u l t u r e s u p p l i e d ( l a r g e m u s s e l s ! ; s m a l l m u s s e l s r ) . 110 F i g u r e 16. Growth of A r t e m i a s a i i n a when s u p p l i e d w i t h b a c t e r i a l c u l t u r e s of c e l l c o n c e n t r a t i o n s g r e a t e r than l o g 6.2 (the l o g c o n c e n t r a t i o n of the b a c t e r i a i s i n d i c a t e d b e s i d e the c u r v e s ; 95% C o n f i d e n c e I n t e r v a l s ). I l l 4 1 \ Y-O 6 12 IS DAYS 1 12 F i g u r e 17. Growth of A r t e m i a s a l i n a when s u p p l i e d w i t h b a c t e r i a l c u l t u r e s of c e l l c o n c e n t r a t i o n s l e s s than l o g 6.2 (the l o g c o n c e n t r a t i o n of the b a c t e r i a i s i n d i c a t e d b e s i d e the c u r v e s ; 95% C o n f i d e n c e I n t e r v a l s ). Hi J 4 1 \ i -O 6 12 IB DAYS 1 14 F i g u r e 18. Growth Rates of A r t e m i a s a l i n a (the l o g c o n c e n t r a t i o n of the b a c t e r i a s u p p l i e d and the s u b s t r a t e source i s i n d i c a t e d i n the b r a c k e t s b e s i d e the c u r v e s ; (F) Fucus v e s i c u l o s u s ; ( U ) U l v a l a c t u c a ; ( E ) E e l g r a s s . 115 1 16 F i g u r e 19. F i l t e r i n g Rates (FR) of A r t e m i a s a l i n a as a f u n c t i o n of the l o g c o n c e n t r a t i o n of the b a c t e r i a l c u l t u r e s u p p l i e d , (the l e n g t h , i n mm, i s i n d i c a t e d b e s i d e the c u r v e s ) 117 1 1 8 F i g u r e 20. Growth E f f i c i e n c i e s of A r t e m i a s a l i n a as a f u n c t i o n of the s i z e of the organism (the l o g c o n c e n t r a t i o n of the b a c t e r i a l c u l t u r e p r o v i d e d i s i n d i c a t e d b e s i d e the c u r v e s ) . 119 1 20 F i g u r e 21. Consumption Rates of A r t e m i a s a l i n a as a f u n c t i o n of the s i z e of the organism and the c o n c e n t r a t i o n of b a c t e r i a s u p p l i e d (the l e n g t h of A r t e m i a s a l i n a , i n mm, i s i n d i c a t e d b e s i d e the c u r v e s ) . 1 22 F i g u r e 22. The l e n g t h of A r t e m i a s a l i n a t h a t can be o b t a i n e d when s u p p l i e d w i t h d i f f e r e n t c o n c e n t r a t i o n s of suspended b a c t e r i a (95% C o n f i d e n c e L i m i t s ) . 1.23 124 F i g u r e 23. The r e l a t i o n s h i p between t o t a l n i t r o g e n c o n t e n t of the s u b s t r a t e and the number of b a c t e r i a s u p p o r t e d when s u s t r a t e s a r e a u t o c l a v e d ( A ) and d r i e d ( A ) ; (U) U l v a l a c t u c a , ( F ) Fucus v e s i c u l o s u s , ( E ) Eelgrass,(WC) Wood Chi p s 125 TOTAL NITROGEN (>) 1 26 F i g u r e 24. The weight of A r t e m i a s a l i n a t h a t can be o b t a i n e d from b a c t e r i a l c u l t u r e s grown under v a r i o u s e x p e r i m e n t a l c o n d i t i o n s . N- = n u t r i e n t - d e p l e t e d Wood C h i p s = Q N+ = n u t r i e n t - p o o r E e l g r a s s = © N++ = n u t r i e n t - r i c h Fucus sp. = El U l v a sp. = • DW = d r i e d s u b s t r a t e s l e f t i n t a c t DP = d r i e d s u b s t r a t e s of s m a l l p a r t i c l e s s i z e s ro -*J<3 B R I N E S H R I M P £ 0) • i: llllllllllllllllllllllllllll 

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