@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Science, Faculty of"@en, "Earth, Ocean and Atmospheric Sciences, Department of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "McKeag, Maura Anne"@en ; dcterms:issued "2010-04-21T19:16:30Z"@en, "1983"@en ; vivo:relatedDegree "Master of Science - MSc"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description "The trophic relationships, in terms of growth efficiencies, between suspended marine bacteria and the suspension feeders, the blue mussel, Mytilus edulis and the brine shrimp, Artemia salina, were established. Mytilus edulis could not be supported on suspended bacteria. The growth efficiency of A.salina was dependent upon both the concentration of bacteria in the culture provided as a food source and on the size of the brine shrimp. At concentrations less than 1.5 X 10⁶ cells/ml, young shrimp, less than 1 mm in length, died within a few days (zero growth efficiency). Correspondingly, low filtering rates (less than 1 ml/hour/organism) and low consumption rates (less than 0.1 μg/hour/organism) were observed for these organisms at such low concentrations. For brine shrimp greater in length than 1.0 mm, a bacterial concentration of 2.5 X 10⁶ cells/ml was required before positive growth efficiencies were obtained. As the food concentration increased beyond this concentration, growth efficiencies steadily increased. An upper limit for the concentration of bacterial cells that could be converted into the biomass of A.salina was not detected; the growth efficiencies continued to increase to a maximum of 60% as the bacterial concentrations supplied increased to 10⁷ cells/ml. The growth efficiencies were maximal when Artemia salina obtained a length of 2.5 mm, at which time the highest consumption rates and growth rates were also observed. Growth efficiencies for organisms larger or smaller than 2.5 mm were lower, Bacterial densities, expressed in terms of both cells per ml and the amount of ATP per ml, supported from various types of organic substrates, were determined under varying inorganic nutrient and oxygen regimes. The substrates studied included the seaweeds, Ulva lactuca and Fucus vesiculosus, and the vascular plants, Zostera marina and wood chips. Under nutrient-rich conditions (30 μM NO₃⁻,6.0 μM PO₄⁻³), bacterial densities supported from 1 g (dry weight) of Ulva lactuca reached a maximum of 2 X 10⁷ cells/ml or 12 X 10⁻³ μg ATP/ml. Based on the established trophic relationships, it was calculated that the amount of suspended bacteria per gram dry weight of substrate grown under these conditions can sustain a maximum weight of 46 μg of Artemia salina or adult brine shrimp."@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/23974?expand=metadata"@en ; skos:note "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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Some e c o l o g i c a l a s p e c t s of marine b a c t e r i a i n the K u r o s h i o c u r r e n t . B u l l . M i s a k i Mar. B i o l . I n s t i t . Kyoto U n i v . 12:65-76. Tenore, K.R. 1977a. U t i l i z a t i o n of aged d e t r i t u s d e r i v e d from d i f f e r e n t s o u r c e s by the p o l y c h a e t e C a p t i e l l a c a p i t a t a . Mar. B i o l . 44:51-55. 68 Tenore, K.R. 1 9 7 7 b . Growth of the p o l y c h a e t e C a p i t e l l a c a p i t a t a c u l t u r e d under d i f f e r e n t l e v e l s of d e t r i t u s d e r i v e d from v a r i o u s s o u r c e s . L i m n o l . Oceanogr. 2 2 : 9 3 6 - 9 4 1 . Tenore, K.R. 1983. What c o n t r o l s the a v a i l a b i l i t y t o a n i m a l s of d e t r i t u s d e r i v e d v a s c u l a r p l a n t s : o r g a n i c n i t r o g e n enrichment of c a l o r i c a v a i l a b i l i t y ? Mar. E c o l . P r o g . S e r . 10:307-309. Tenore, K.R. and R.B. Hanson. 1980. 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 "@en ; edm:hasType "Thesis/Dissertation"@en ; edm:isShownAt "10.14288/1.0053213"@en ; dcterms:language "eng"@en ; ns0:degreeDiscipline "Oceanography"@en ; edm:provider "Vancouver : University of British Columbia Library"@en ; dcterms:publisher "University of British Columbia"@en ; dcterms:rights "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en ; ns0:scholarLevel "Graduate"@en ; dcterms:title "The trophic relationships between suspended marine bacteria and the suspension-feeders Mytilus edulis and Artemia salina"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/23974"@en .