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UBC Theses and Dissertations

Urea and selenium nutrition of marine phytoplanton : a physiological and biochemical study Price, Neil Martin 1987

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U R E A A N D S E L E N I U M N U T R I T I O N O F M A R I N E P H Y T O P L A N K T O N : A P H Y S I O L O G I C A L A N D B I O C H E M I C A L S T U D Y by NEIL MARTIN PRICE B . S c , The Univers i ty of New Brunswick, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR PHILOSOPHY in The Faculty of Graduate Studies (Department of Botany) We accept th i s thes is as conforming to the required standard. The Un ivers i ty of B r i t i s h Columbia March 1987 © N e i l Martin P r i c e , 1987 71 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of B o t a n y  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) i i ABSTRACT Laboratory and f i e l d experiments measured urea uptake and 14 15 a s s i m i l a t i o n with C- and N-urea and by disappearance of d i s s o l v e d urea. A m o d i f i e d d i a c e t y l monoxime method was developed, which a c c u r a t e l y and p r e c i s e l y determined d i s s o l v e d urea c o n c e n t r a t i o n s i n seawater. In the S t r a i t of Georgia, c h l o r o p h y l l a ( c h i a) s p e c i f i c uptake r a t e s of ammonium (NH^ +) and urea were g r e a t e s t i n s t r a t i f i e d water; whereas, c h i a s p e c i f i c uptake r a t e s of n i t r a t e (NO^ ) were g r e a t e s t i n f r o n t a l water. Ammonium and urea r e g e n e r a t i o n r a t e s were c a l c u l a t e d by a mass balance method and the r a t e s were s i m i l a r . D i f f e r e n c e s between measurements of p a r t i c u l a t e + 15 n i t r o g e n , d i s s o l v e d NH^ , NO^ and urea, and N uptake were used to e x p l a i n the dominant N t r a n s f o r m a t i o n s i n f r o n t a l and 1 4 s t r a t i f i e d seawater. Uptake r a t e s measured by C-urea were 1 5 ca. 1.4 times f a s t e r than those determined by N-urea i n the Sargasso Sea. Turnover times of urea i n the surface-mixed l a y e r were ca. 12 h. Within some seawater samples, phytoplankton u t i l i z e d urea at r a t e s which approximated the maximum r a t e s of u t i l i z a t i o n . In a n i t r a t e - s u f f i c i e n t c u l t u r e of Thai assi osi ra pseudonana (clone 3H) (Hustedt) Hasle and Heimdal, urea uptake r a t e s measured by t h r e e methods dis a g r e e d ; whereas, no d i s c r e p a n c i e s o c c u r r e d i n a n i t r a t e -s t a r v e d c u l t u r e . NH^ + was r e l e a s e d from c e l l s a f t e r urea was taken up and was l a t e r reabsorbed. A model of urea uptake and a s s i m i l a t i o n by T. pseudonana i s proposed. An o b l i g a t e selenium (Se) requirement f o r growth of T. ps eudonana was demonstrated i n axenic c u l t u r e i n a r t i f i c i a l -9 2-seawater. The a d d i t i o n of 10 M SeO^ to c u l t u r e medium was 2-s u f f i c i e n t f o r good growth of t h i s a l g a ; SeO^ was only - 7 e f f e c t i v e a t c o n c e n t r a t i o n s greater than 10 M. To e l u c i d a t e the b i o c h e m i c a l r o l e of Se i n T. pseudonana, c e l l s were . . -9 75 c u l t u r e d i n medium c o n t a i n i n g 10 M Na2 SeO^. Two s o l u b l e 7 5 p o l y p e p t i d e s of 21 and 29 kD contained Se. G l u t a t h i o n e p e r o x i d a s e was d e t e c t e d on non-denaturing p o l y a c r y l a m i d e g e l s 75 . and Se co-migrated with the enzyme. I t was concluded that Se i s an e s s e n t i a l element f o r growth of T. ps eudonana due, i n p a r t , t o the presence of the selenoenzyme g l u t a t h i o n e p e r o x i d a s e . i v TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES x i LIST OF FIGURES xv ACKNOWLEDGEMENTS xxv i i i PART I : UREA 1 INTRODUCTION 1 Overview and o b j e c t i v e s 1 Nitrogen l i m i t a t i o n of phytoplankton p r o d u c t i v i t y 2 Sources and s i n k s of urea i n seawater 4 Phytoplankton growth on urea 6 Laboratory measurements of urea uptake 7 Urea metabolism 8 Test organism 10 CHAPTER 1. A COMPARISON OF METHODS FOR THE MEASUREMENT OF DISSOLVED UREA CONCENTRATIONS IN SEAWATER 12 Background 12 M a t e r i a l s and Methods 13 Urease method 13 D i a c e t y l monoxime method 15 Experimental methods 18 R e s u l t s 19 Standard curves 19 pH e f f e c t s 23 Heating time 23 Enzyme c o n c e n t r a t i o n 23 V S p e c i f i c i t y of the d i a c e t y l monoxime method .. 29 N a t u r a l seawater samples 33 D i s c u s s i o n 38 pH e f f e c t s 38 Urea h y d r o l y s i s 44 S p e c i f i c i t y of methods 47 A n a l y s i s of n a t u r a l seawater 48 Summary 51 CHAPTER 2. TIME COURSE OF UPTAKE OF ORGANIC AND INORGANIC NITROGEN BY PHYTOPLANKTON IN THE STRAIT OF GEORGIA: COMPARISON OF FRONTAL AND STRATIFIED COMMUNITIES 53 Background 53 M a t e r i a l s and Methods 56 Sample c o l l e c t i o n 56 Chemical, p h y s i c a l and b i o l o g i c a l a n a l y s es ... 56 Ni t r o g e n uptake 60 C a l c u l a t i o n s 61 R e s u l t s 62 S t a t i o n p r o f i l e s 62 Plankton community s t r u c t u r e 63 1 5 N uptake and n i t r o g e n disappearance 68 Uptake p e r i o d i c i t y 73 Chi a s p e c i f i c uptake r a t e s 74 Comparison of r a t e s 74 Dark/Light uptake 80 Regeneration r a t e s 82 Mass balance 82 v i D i s c u s s i o n 85 Experimental c o n s i d e r a t i o n s - 85 Simultaneous uptake of n i t r o g e n compounds .... 86 V a r i a t i o n s i n n i t r o g e n uptake r a t e 88 E f f e c t s of l i g h t / d a r k regime on n i t r o g e n uptake 89 NH 4 + and urea r e g e n e r a t i o n 90 P a r t i c u l a t e n i t r o g e n balance 93 Summary 99 CHAPTER 3. UREA UPTAKE BY SARGASSO SEA PHYTOLANKTON: SATURATED AND IN SITU UPTAKE RATES 101 Background 101 M a t e r i a l s and Methods 102 Sample c o l l e c t i o n 102 P h y s i c a l , chemical and b i o l o g i c a l analyses ... 102 Sat u r a t e d uptake experiments 105 1 5 N - I a b e l l e d s u b s t r a t e s 106 1 4 C - l a b e l l e d s u b s t r a t e s 107 In situ urea uptake r a t e s 108 R e s u l t s 109 Environmental parameters 109 1 5N uptake 113 1 4 C - u r e a uptake 118 In situ urea uptake r a t e s 121 D i s c u s s i o n 125 Environmental parameters 125 1 5 Sat u r a t e d N uptake r a t e s 127 Urea uptake r a t e s : 1 4 C and 1 5 N 130 v i i Urea turnover times 133 Urea uptake: s a t u r a t i n g / t r a c e r a t e s 137 Summary 139 CHAPTER 4. FATE OF UREA-C AND N DURING UREA UPTAKE BY THE COASTAL MARINE DIATOM THALASSIOSIRA PSEUDONANA 141 Background 141 M a t e r i a l s and Methods 143 C u l t u r i n g procedure 143 Urea p u r i t y 144 Urea uptake 145 Rate measurements 145 Change i n d i s s o l v e d urea c o n c e n t r a t i o n .. 145 1 5N-urea uptake 146 1 4 C - u r e a uptake 147 R e s u l t s 148 C u l t u r e c o n d i t i o n s 148 N i t r a t e - s u f f i c i e n t c u l t u r e s 148 N i t r a t e - s t a r v e d c u l t u r e 159 D i s c u s s i o n 166 I n t e r p r e t a t i o n of r e s u l t s 166 Metho d o l o g i c a l c o n s i d e r a t i o n s 170 N i t r a t e - s u f f i c i e n t c u l t u r e s 171 Release of DON 172 Pa t t e r n of urea uptake 173 Urea pools and e f f l u x 174 N i t r a t e - s t a r v e d c u l t u r e 175 Ammonium e f f l u x 177 v i i i A model f o r urea uptake by Thai as si osi r a pseudonana 179 I m p l i c a t i o n s f o r urea uptake measurements i n nature 182 Summary 184 PART I I : SELENIUM . 186 INTRODUCTION 186 Overview and o b j e c t i v e s 186 H i s t o r i c a l p e r s p e c t i v e 187 Selenium requirements i n p l a n t s 188 Chemical i n t e r a c t i o n s of selenium 191 S p e c i f i c s e l e n i u m - c o n t a i n i n g macromolecules .. 192 Se l e n o m e t a b o l i t e s i n p l a n t s 193 G l u t a t h i o n e p e r o x i d a s e 194 CHAPTER 5. SELENIUM: AN ESSENTIAL ELEMENT FOR GROWTH OF THE COASTAL MARINE DIATOM THALASSIOSIRA PSEUDONANA 196 Background 196 M a t e r i a l s and Methods 198 A l g a l c u l t u r e 198 C u l t u r e medium and f l a s k s 198 C u l t u r e c o n d i t i o n s and growth measurements ... 200 R e s u l t s 201 Selenium requirement and recovery 201 M o r p h o l o g i c a l changes 206 Growth on s e l e n i t e and s e l e n a t e 211 D i s c u s s i o n 220 S e l e n i u m - l i m i t a t i o n and recovery 220 ix Growth on s e l e n i t e compared with s e l e n a t e .... 222 Mo r p h o l o g i c a l changes 226 E c o l o g i c a l c o n s i d e r a t i o n s 227 The r o l e of selenium 228 Summary 230 CHAPTER 6. SPECIFIC SELENIUM-CONTAINING POLYPEPTIDES IN THE MARINE DIATOM THALASSIOSIRA PSEUDONANA 232 Background 232 M a t e r i a l s and Methods 234 C u l t u r e c o n d i t i o n s 234 75 Measurement of Se 234 C o l l e c t i o n of c e l l s 242 Biochemical a n a l y s i s 242 Gel f i l t r a t i o n 244 E l e c t r o p h o r e s i s 245 G l u t a t h i o n e peroxidase a c t i v i t y 246 R e s u l t s 2 47 Uptake of 7 5 S e 247 75 Biochemical d i s t r i b u t i o n of Se 247 Gel f i l t r a t i o n and e l e c t r o p h o r e s i s 251 G l u t a t h i o n e peroxidase a c t i v i t y 255 D i s c u s s i o n 261 Selenium uptake 261 75 I n t r a c e l l u l a r d i s t r i b u t i o n of Se 267 S p e c i f i c s e l e n i u m - c o n t a i n i n g molecules 271 G l u t a t h i o n e peroxidase 274 G l u t a t h i o n e peroxidase a c t i v i t y on g e l s 276 Selenium n u t r i t i o n 276 X Summary 277 GENERAL CONCLUSIONS 279 REFERENCES 283 x i L I S T OF TABLES Table I. Measured urease a c t i v i t y of enzyme stock s o l u t i o n s compared to t h e o r e t i c a l a c t i v i t y determined by manufacturer 28 Table I I . Organic and i n o r g a n i c compounds t e s t e d f o r i n t e r f e r e n c e with the d i a c e t y l monoxime method. S o l u t i o n s were prepared i n d e i o n i z e d d i s t i l l e d water 32 Table I I I . An Ocean S t a t i o n P seawater sample was spik e d with 2.00 uM ammonium and d i v i d e d i n t o two p o r t i o n s . To one p o r t i o n , urease was added to giv e a f i n a l c o n c e n t r a t i o n of 2.3 U-ml 1; the other p o r t i o n served as a c o n t r o l . The samples were heated at 50°C f o r 20 min and the c o n c e n t r a t i o n of ammonium was measured immediately (0 min) and a f t e r the samples had cooled (55 min). Incubations were conducted i n 125 ml Erlenmeyer f l a s k s covered with aluminum f o i l 34 Table IV. C o n c e n t r a t i o n (uM) of d i s s o l v e d urea i n seawater samples c o l l e c t e d from the S t r a i t of Georg i a , as determined by the d i a c e t y l monoxime and urease methods. Each value represents a s i n g l e measurement 37 Table V. L o c a t i o n of s t a t i o n and time of sampling 59 Table VI. I n i t i a l e nvironmental c o n d i t i o n s of seawater c o l l e c t e d f o r time course experiments 66 x i i Table V I I . Phytoplankton community composition i n f r o n t a l and s t r a t i f i e d water 67 Table V I I I . Zooplankton community s t r u c t u r e i n f r o n t a l and s t r a t i f i e d water 69 Table IX. C h l o r o p h y l l a s p e c i f i c uptake r a t e s of NH 4 +, NC>2 and urea i n f r o n t a l (A5) and s t r a t i f i e d (T4) water. The dark p e r i o d occurs d u r i n g the 12 to 18 h time i n t e r v a l 77 Table X. R a t i o of dark to l i g h t uptake r a t e s ( V D : V L ^ of NH 4 +, NC>3 and urea f o r f r o n t a l and s t r a t i f i e d water 81 Table XI. Regeneration r a t e s of NH 4 + and urea i n f r o n t a l and s t r a t i f i e d water 83 Table X I I . Changes over time i n measured DIN and urea c o n c e n t r a t i o n ( A P ^ ) , p a r t i c u l a t e n i t r o g e n 1 5 (APON), and amount of N-nitrogen accumulated i n the p a r t i c u l a t e matter ( $ V T ) i n f r o n t a l and s t r a t i f i e d water 84 Table X I I I . Biomass data from water samples c o l l e c t e d f o r n i t r o g e n uptake experiments 110 15 14 Table XIV. Comparison of N-urea and C-urea uptake r a t e s determined d u r i n g 24 h i n c u b a t i o n s . Rates of 1 4 C-urea uptake were converted to e q u i v a l e n t n i t r o g e n uptake r a t e s as d e s c r i b e d in the t e x t . 1 5 Values i n bracke t s are d a i l y N-urea uptake r a t e s (ug at N - l ~ 1 - d" 1 ) 119 Table XV. R a t i o of 1 4 C - u r e a uptake r a t e s (ug at urea-C ' l _ 1 * h ~ 1 ) determined d u r i n g 6 and 24 h x i i i i n c u b a t i o n s at three s t a t i o n s (S1, S2, and S3) i n water samples c o l l e c t e d from 6 depths. Values i n 1 4 br a c k e t s are the C-urea uptake r a t e s d u r i n g 6 h i n c u b a t i o n s (ug at urea-C-1 1- h 1) 120 Table XVI. Turnover times of d i s s o l v e d urea c o n c e n t r a t i o n i n surface-mixed and deep water at S t a t i o n s 1, 2 and 3. The c o r r e l a t i o n c o e f f i c i e n t f o r the e x p o n e n t i a l equations i s gi v e n . D i s s o l v e d urea c o n c e n t r a t i o n s are the mean of d u p l i c a t e s (± 1 SD) 124 1 4 Table XVII. Uptake r a t e s of C-urea determined d u r i n g 6 h i n c u b a t i o n s i n seawater samples c o l l e c t e d from the 50 and 3% s u r f a c e l i g h t depths at three s t a t i o n s (S1, S2, and S3), and spi k e d with t r a c e and s a t u r a t i n g c o n c e n t r a t i o n s . Rates are expressed i n terms of urea-N uptake 126 Table XVIII. Urea turnover times i n o l i g o t r o p h i c ocean waters 135 Table XIX. Summary of c u l t u r e c o n d i t i o n s at the beginning of each experiment 151 Table XX. Summary of urea uptakes r a t e s measured by C- and N - l a b e l l e d urea and by disappearance i n n i t r a t e - s u f f i c i e n t and n i t r a t e - s t a r v e d c u l t u r e s . N i t r a t e uptake r a t e measured i n the n i t r a t e -s u f f i c i e n t c u l t u r e i s a l s o given, as are the measured r a t e s of change of the p a r t i c u l a t e n i t r o g e n over the i n c u b a t i o n 169 Table XXI. Trace elements t e s t e d f o r t h e i r a b i l i t y to x i v support growth of T. ps eudonana i n Se-deplete a r t i f i c i a l seawater (ESAW). The form of the -9 element added i s gi v e n , and 10 M was added i n each case. Each treatment was performed i n d u p l i c a t e . No growth (-), good growth ( + ) 212 7 5 Table XXII. P a r t i t i o n i n g of Se between p r o t e i n , p o l y s a c c h a r i d e s and n u c l e i c a c i d s , l i p i d s and low molecular weight compounds i n e x p o n e n t i a l l y growing T. pseudonana, as determined by s o l v e n t e x t r a c t i o n t e chniques. Values are r e p o r t e d as dpm: 25 ml of c u l t u r e was e x t r a c t e d . Each measurement i s the mean of q u a d r u p l i c a t e 3 d e t e r m i n a t i o n s + 1 SD, and u n i t s are dpm•10 250 75 Table XXIII. D i s t r i b u t i o n of Se between s o l u b l e and membrane p r o t e i n s of T. pseudonana. Average c o n c e n t r a t i o n s ( c a l c u l a t e d from q u a d r u p l i c a t e measurements) of p r o t e i n are r e p o r t e d ± 1 SD 252 Table XXIV. G l u t a t h i o n e peroxidase a c t i v i t y measured in c e l l - f r e e e x t r a c t s of T. ps eudonana. Enzyme a c t i v i t y i s expressed as the r a t e of NADPH2 o x i d i z e d and i t was normalized to p r o t e i n . The r e a c t i o n temperature was 25°C. Bracketed v a l u e s are the number of d i f f e r e n t p r o t e i n p r e p a r a t i o n s assayed, and GSH-Px a c t i v i t y i n each p r e p a r a t i o n was measured i n d u p l i c a t e ; average v a l u e s are reported ± 1 SD 258 X V L I S T OF FIGURES (8) F i g u r e 1. Schematic diagram of Autoanalyzer 4* system fo r urea a n a l y s i s by the d i a c e t y l monoxime method. Composition of reagents i s given i n the " M a t e r i a l s and Methods - D i a c e t y l monoxime method". The debubbler l i n e p u l l e d 0.60 ml-min 1 to prevent any l a r g e a i r bubbles from e n t e r i n g the system. A b o i l i n g water bath gave a more constant i n c u b a t i o n temperature and helped e l i m i n a t e the o s c i l l a t i n g b a s e l i n e that was seen when l e s s constant and lower temperatures were used 16 F i g u r e 2. Standard curves f o r urea determined by (A) automated d i a c e t y l monoxime method and (B) urease method, with urea standards in d e i o n i z e d d i s t i l l e d water or 3% NaCl (A), i n a r t i f i c i a l seawater (•) and i n c u l t u r e f i l t r a t e (O) 21 F i g u r e 3. H y d r o l y s i s of urea i n a r t i f i c i a l seawater (dashed l i n e ) and c u l t u r e f i l t r a t e (continuous l i n e ) by urease, as a f u n c t i o n of seawater pH. Urease c o n c e n t r a t i o n was twice that recommended by McCarthy (1970). T o t a l amount of urea was determined by the d i a c e t y l monoxime method, and c o n c e n t r a t i o n s determined by the urease method are expressed as a percentage of t h i s v a l u e . E r r o r bars i n d i c a t e the range of d u p l i c a t e s 24 F i g u r e 4. Urea h y d r o l y z e d as a f u n c t i o n of h e a t i n g time of the urease-seawater samples at 50°C. A x v i 1.00 uM urea standard was prepared i n 3% NaCl (•) and i n seawater samples c o l l e c t e d from the S t r a i t of Georgia (O). Samples had c o o l e d to room temperature before the NH^ + measurements were made. C o n c e n t r a t i o n of urea i n each water sample was determined by the d i a c e t y l monoxime method, and v a l u e s are r e p o r t e d as a percentage of t h i s v a lue 26 F i g u r e 5. Urea h y d r o l y z e d in u r e a s e - t r e a t e d seawater samples, as a f u n c t i o n of f i n a l enzyme a c t i v i t y in the urease-seawater samples. Samples were c o l l e c t e d from Ocean S t a t i o n P and spiked with 1.00 uM urea. Incubations were conducted at 50°C f o r 20 min, and samples were c o o l e d f o r 0 (•), 32 (O), and 76 min (•). Amount of urea h y d r o l y z e d was expressed as a percentage of the c o n c e n t r a t i o n determined by the d i a c e t y l monoxime method 30 F i g u r e 6. H y d r o l y s i s of 0.50 uM 1 4 C - u r e a i n 3% NaCl (•) and Ocean S t a t i o n P seawater (O), as a f u n c t i o n of enzyme c o n c e n t r a t i o n . Samples were 50 ml and were heated f o r 20 min at 50°C. The r e a c t i o n was terminated once the samples had c o o l e d to room temperature. The amount of urea h y d r o l y z e d i s expressed as a percentage of the 1 4 C-urea added, which was determined by l i q u i d s c i n t i l l a t i o n c o unting 35 F i g u r e 7. Time course of h y d r o l y s i s of a 1.00 uM urea standard prepared i n n a t u r a l seawater c o l l e c t e d from E n g l i s h Bay, Vancouver, B r i t i s h Columbia (A), and S t r a i t of Georgia ( S t a t i o n T3; 49°50.4'N; 125°00.5'W) at 15:47 (O) and 22:30 hrs (•) and from Ocean S t a t i o n P (•). Urease-seawater samples were heated f o r 20 min at 50°C, and time on the a b s c i s s a r e f e r s to time f o l l o w i n g h e a t i n g , when samples were c o o l i n g at room temperature. C o n c e n t r a t i o n of urea i n each water sample was determined using the d i a c e t y l monoxime method, and values measured by the urease method are r e p o r t e d as a percentage of t h i s v a l u e . E r r o r bars i n d i c a t e the range d u p l i c a t e s F i g u r e 8. Comparison between the d i a c e t y l monoxime and urease methods for measuring urea i n a n a t u r a l seawater sample (Ocean S t a t i o n P) spiked with known c o n c e n t r a t i o n s of urea. Urea c o n c e n t r a t i o n s were measured using the d i a c e t y l monoxime method (•) and the urease method (•rO) a f t e r heated urease-seawater samples were allowed to c o o l at room temperature f o r 37 (O) and 104 (•) min F i g u r e 9. S t a t i o n l o c a t i o n s f o r Time Course 1 (T3, f r o n t a l s t a t i o n ) , Time Course 2 (A5, f r o n t a l s t a t i o n ) and Time Course 3 (T4, s t r a t i f i e d s t a t ion ) F i g u r e 10. Depth p r o f i l e s of temperature, in vivo f l u o r e s c e n c e , and NO^ c o n c e n t r a t i o n . (A) F r o n t a l s t a t i o n A5, Time Course 2. (B) S t r a t i f i e d s t a t i o n T4, Time Course 3 F i g u r e 11. Time course measurements at f r o n t a l s t a t i o n (A5), Time Course 2. (A) D a i l y i n c i d e n t i r r a d i a n c e d u r i n g experiment. (B, D, F) N atom % excess i n p a r t i c u l a t e matter f o r l i g h t and dark b o t t l e i n c u b a t i o n s f o l l o w i n g a d d i t i o n of 6 ug at N * l 1 of (B) NH 4 +, (D) NC>3~ and (F) urea ( e r r o r bars represent the range of d u p l i c a t e s ) . (C, E, G) Corresponding measurements of d i s s o l v e d NH^ + (•) , N0 3~ (O) and urea (A) i n (C) NH 4 +, (E) NC>3~, and (G) urea-s p i k e d samples. Dashed l i n e i n d i c a t e s no measurements of d i s s o l v e d urea at 3 and 6 h Fi g u r e 12. As F i g . 11 except at s t r a t i f i e d s t a t i o n (T4), Time Course 3 Fi g u r e 13. N i t r o g e n - s p e c i f i c uptake r a t e s of NH 4 + (•), NC>3 (O) and urea (A) i n (A) f r o n t a l and (B) s t r a t i f i e d water. Rates determined f o r 3 or 6 h i n t e r v a l s ; each p o i n t i n d i c a t e s a ra t e c a l c u l a t e d over the time i n t e r v a l between i t and the next p o i n t on the curve. Shaded area on the a b s c i s s a d e l i m i t s the dark p e r i o d 1 5 Fi g u r e 14. Nitrogen uptake r a t e s determined by N atom % excess accumulation i n the p a r t i c u l a t e s (•) , change i n d i s s o l v e d n i t r o g e n c o n c e n t r a t i o n (o) and by change i n the p a r t i c u l a t e n i t r o g e n c o n c e n t r a t i o n (A) over 6, 12, 18 and 24 h time i n t e r v a l s . (A) NH 4 +, (B) NC>3~, and (C) urea-spi k e d samples in f r o n t a l water and (D) NH 4 +, (E) NO., and (F) urea-sp i k e d samples i n s t r a t i f i e d x i x water . 78 F i g u r e 15. Schematic diagram of n i t r o g e n c y c l i n g i n the euphotic zone of f r o n t a l and s t r a t i f i e d water. Arrows i n d i c a t e major pathways of n i t r o g e n t r a n s f o r m a t i o n between the v a r i o u s p o o l s ; other pathways are excluded s i n c e they were not found to be dominant in these experiments. The d i s s o l v e d o r g a n i c n i t r o g e n pool i n c l u d e s amino a c i d s , p r o t e i n s and other n i t r o g e n c o n t a i n i n g macromolecules. E x c r e t i o n may i n v o l v e a c t i v e and p a s s i v e processes 97 F i g u r e 16. L o c a t i o n of s t a t i o n s i n the northwest A t l a n t i c Ocean. SI i s i n c o n t i n e n t a l slope water, S2 and S3 are i n the Sargasso Sea 103 F i g u r e 17. D i s s o l v e d NH 4 +, N0 3 + N0 2 and urea c o n c e n t r a t i o n (ug at N-1 1 ) measured i n f i l t e r e d f r o z e n samples c o l l e c t e d from (A) S t a t i o n 1 ( c o n t i n e n t a l slope water), (B) S t a t i o n 2 (Sargasso Sea), and (C) S t a t i o n 3 (Sargasso Sea) 111 1 5 F i g u r e 18. Time course of i n c o r p o r a t i o n of N-urea i n t o p a r t i c u l a t e matter in water samples c o l l e c t e d from 6.6 m at S t a t i o n 1 (•), and 15 m at S t a t i o n s 2 (•) and 3 (O). Incubation times were measured from the s t a r t i n g time of the i n c u b a t i o n . The dark p e r i o d d u r i n g the time course at S t a t i o n s 1 and 3 occurred between 8.5 and 18 h and between 10.5 and 20 h at S t a t i o n 2 114 XX F i g u r e 19. D a i l y n i t r o g e n uptake r a t e s determined with 15 + -s a t u r a t i n g a d d i t i o n s of N - l a b e l l e d NH 4 , N0 3 and urea by plankton i n seawater samples c o l l e c t e d at (A) S t a t i o n 1, (B) S t a t i o n 2 and (C) S t a t i o n 3. E r r o r bars are the range of d u p l i c a t e samples 116 14 . . . F i g u r e 20. The amount of C-urea remaining i n seawater d u r i n g 6 h time course experiments. Water was c o l l e c t e d from the 55 (O) and 3% (•) sea-surface l i g h t depths at (A) S t a t i o n 1 ( c o n t i n e n t a l slope water), (B) S t a t i o n 2 (Sargasso Sea), and (C) S t a t i o n 3 (Sargasso Sea). P a r t i c u l a t e matter was removed from samples by f i l t r a t i o n , and the f i l t r a t e was a c i d i f i e d to 14 . . • • remove C0 2- The remaining a c t i v i t y was measured. E r r o r bars are the range of d u p l i c a t e subsamples from a s i n g l e sample b o t t l e 122 F i g u r e 21. Growth of T. pseudonana measured by in vivo c h l o r o p h y l l a f l u o r e s c e n c e (O) , and the c o n c e n t r a t i o n of d i s s o l v e d NO^ i n the c u l t u r e medium (•) measured over time. The arrows l a b e l l e d 1 and 2 i n d i c a t e the times at which urea uptake experiments were i n i t i a t e d f o r the n i t r a t e -s u f f i c i e n t and n i t r a t e - s t a r v e d c u l t u r e , r e s p e c t i v e l y 149 F i g u r e 22. D i s s o l v e d urea (•) and n i t r a t e (O) c o n c e n t r a t i o n i n d u p l i c a t e samples ( s o l i d and dashed l i n e s ) of a n i t r a t e - s u f f i c i e n t c u l t u r e of T. pseudonana s p i k e d with 10 ug at urea~N*l 1 . x x i Urea disappearance was constant over the i n c u b a t i o n ; the r e g r e s s i o n c o e f f i c i e n t s f o r the 2 2 two l i n e s were: r = 0.996 (dashed l i n e ) and r 0.998 ( s o l i d l i n e ) 153 F i g u r e 23. (A) 1 4 C - u r e a uptake rate measured i n d u p l i c a t e samples ( • f O ) of a n i t r a t e - s u f f i c i e n t c u l t u r e of T. pseudonana. (B) 1 4 C - u r e a uptake by n i t r a t e - s u f f i c i e n t T. pseudonana determined i n a separate experiment. A s i n g l e uptake de t e r m i n a t i o n was made on two r e p l i c a t e c u l t u r e s ( • , O ) , and sampling times were of s h o r t e r d u r a t i o n d u r i n g the f i r s t 5 min of the experiment. (C) 1 4C accumulation i n T. pseudonana. (D) 1 4 C 0 2 r e l e a s e d i n t o the medium. A l l r a t e s were c a l c u l a t e d between s u c c e s s i v e sampling p o i n t s and are p l o t t e d a g a i n s t the average i n c u b a t i o n time ... 155 1 5 Fi g u r e 24. N-urea uptake r a t e measured i n d u p l i c a t e samples (• r O ) of a n i t r a t e - s u f f i c i e n t c u l t u r e of 1 5 T. pseudonana. (A) N accumulation expressed as 1 5 N atom percent excess i n the c e l l s determined 1 5 over time. (B) N-urea uptake r a t e d u r i n g each i n c u b a t i o n p e r i o d p l o t t e d a g a i n s t the average i n c u b a t i o n time 157 F i g u r e 25. D i s s o l v e d urea c o n c e n t r a t i o n measured i n d u p l i c a t e ( • , O ) samples of a n i t r a t e - s t a r v e d c u l t u r e of T. pseudonana spiked with 10 ug at u r e a - N - l ~ 1 160 1 4 F i g u r e 26. C-urea uptake r a t e measured i n d u p l i c a t e xx i i samples ( • , O ) of a n i t r a t e - s t a r v e d c u l t u r e of T. F i g u r e 27. pseudonana N-urea uptake r a t e measured i n d u p l i c a t e 1 62 samples ( • , O ) of a n i t r a t e - s t a r v e d c u l t u r e of T. 1 5 ps eudonana. (A) N accumulation i n the c e l l s 1 5 over time. (B) N-urea uptake r a t e d u r i n g each i n c u b a t i o n p e r i o d p l o t t e d a g a i n s t the average i n c u b a t i o n time 1 64 Fi g u r e 28. D i s s o l v e d ammonium c o n c e n t r a t i o n in (A) n i t r a t e - s u f f i c i e n t , and (B) n i t r a t e - s t a r v e d c u l t u r e s of 7". ps eudonana, f o l l o w i n g the a d d i t i o n of 10 ug at urea-N-1 1 . The ammonium c o n c e n t r a t i o n i n the c u l t u r e s p r i o r to the a d d i t i o n i s i n d i c a t e d by the arrows 167 Fi g u r e 29. Diagrammatic r e p r e s e n t a t i o n of urea uptake, and a s s i m i l a t i o n of urea-C and urea-N by Thai as s i os i r a ps eudonana. S t i p p l e d region r e p r e s e n t s c e l l membrane. The arrows p a s s i n g through open c i r c l e s i n the membrane represent t r a n s p o r t by membrane p o r t e r s ; and arrows p a s s i n g d i r e c t l y through the membrane represent d i f f u s i o n . GS/GOGAT i s Glutamine synthetase/glutamate o x o k e t o g l u t a r a t e amino t r a n s f e r a s e . Losses of C r^O and R-NH2 are shown to occur by d i f f u s i o n and a c t i v e or mediated t r a n s p o r t processes; the a c t u a l mechanism i s not known. Consult the t e x t f o r c l a r i f i c a t i o n of r a t e s and processes 180 F i g u r e 30. Growth of T. pseudonana over three-s u c c e s s i v e t r a n s f e r s in a r t i f i c i a l seawater (ESAW) in the absence of Se (O) and i n ESAW supplemented - 9 with 10 M Na 2Se0 3 (•). The growth curves f o r the f i r s t , second and t h i r d t r a n s f e r s begin at 0, 114 and 212 h, r e s p e c t i v e l y . When the c e l l s were in l a t e e x p o n e n t i a l growth a p o r t i o n of the c u l t u r e was t r a n s f e r r e d i n t o f r e s h medium. Growth of the c u l t u r e was then f o l l o w e d by monitoring in vivo c h l o r o p h y l l a f l u o r e s c e n c e ( s o l i d l i n e s ) . The arrows i n d i c a t e when the c u l t u r e s were t r a n s f e r r e d . During the t h i r d t r a n s f e r , c e l l numbers (dashed l i n e ) were a l s o determined i n the S e - r e p l e t e c u l t u r e F i g u r e 31. E a r l y s t a t i o n a r y phase T. pseudonana was t r a n s f e r r e d from Se-deplete ESAW i n t o 2 c u l t u r e tubes c o n t a i n i n g the same medium. At 120 h ( i n d i c a t e d by the arrow) one tube was spiked with 10 1 0 M Na2SeC>3 (•) and the other tube served as the c o n t r o l (O) Fi g u r e 32. Average c e l l volume of T. pseudonana f o l l o w i n g t r a n s f e r of c e l l s from an ammonium-l i m i t e d chemostat c u l t u r e growing on Se-deplete ESAW i n t o Se-deplete (O) or S e - r e p l e t e (•) ESAW. The i n i t i a l c e l l volume was determined in d u p l i c a t e , and the range of the value s was l e s s than the width of the symbol. The experiment was done i n t r i p l i c a t e but only the f i n a l c e l l volumes x x i v of the other two c u l t u r e s are given 207 Fi g u r e 33. Photomicrographs of v e g e t a t i v e c e l l s of T. - 9 pseudonana grown i n ESAW e n r i c h e d with 10 M Na2Se0 3; (a) g i r d l e view and (b) valve view and i n Se-deplete ESAW; (c) g i r d l e view. Sc a l e bar = 5 um 209 Fi g u r e 34. Growth of T. pseudonana as a f u n c t i o n of Na2SeC>3 c o n c e n t r a t i o n . An a l i q u o t of c e l l s from an e a r l y s t a t i o n a r y phase c u l t u r e growing i n Se-de p l e t e ESAW was added to ESAW e n r i c h e d with; (A) 10~ 7, 10" 8, 10~ 9 (O), 1 0 ~ 1 0 (•), B. 10~ 1 1 (•) or - 1 5 10 M (O) Na 2Se0 3. E r r o r bars i n d i c a t e the range of d u p l i c a t e s and where absent the range was l e s s than the width of the symbol 213 Fi g u r e 35. D u p l i c a t e c u l t u r e tubes e n r i c h e d with - 1 2 10 M Na 2Se0 3 were i n o c u l a t e d with Se-depleted 7. pseudonana. Growth was monitored to examine long term e f f e c t s of exposure to l w c o n c e n t r a t i o n s ofSe 216 Fi g u r e 36. Maximum e x p o n e n t i a l growth rate as a f u n c t i o n of Na 2Se0 3 (•) and Na2SeC>4 (O) c o n c e n t r a t i o n . Growth r a t e s are the mean of four r e p l i c a t e s and e r r o r bars i n d i c a t e + 1 SD 218 7 5 F i g u r e 37. Energy spectrum of Se as measured by l i q u i d s c i n t i l l a t i o n counting with Aquasol II as f l u o r 236 75 F i g u r e 38. R e l a t i o n s h i p between the amount of Se measured by l i q u i d s c i n t i l l a t i o n counting (DPM) X X V 75 and the amount of Se added to s c i n t i l l a t i o n v i a l s c o n t a i n i n g Aquasol I I 238 75 F i g u r e 39. Quench curve f o r Se determined by using the H-Number method of quench m o n i t o r i n g . Acetone was used as the quenching agent. The equation of the l i n e i s y = -3.253x + 1.104; r 2 = 0.997 240 F i g u r e 40. Growth of 7". pseudonana as measured by i n v i v o c h l o r o p h y l l a f l u o r e s c e n c e (•) over the d u r a t i o n of the experiment. The accumulation of 75 Se by c e l l s (O) was measured i n 5 ml p o r t i o n s of c u l t u r e c o l l e c t e d by f i l t r a t i o n and r i n s e d with 5 ml of p a r t i c l e f r e e seawater. The arrow i n d i c a t e s when the c u l t u r e was harvested 248 7 5 F i g u r e 41. (A) E l u t i o n p a t t e r n of Se from Sephadex G-150 column. Arrows i n d i c a t e where the molecular weight standards e l u t e d from the column: AD, a l c o h o l dehydrogenase; BSA, bovine serum albumin; CA, c a r b o n i c anhydrase; CC, cytochrome c. I n s e r t i s a p l o t of the l o g molecular weight of standard p r o t e i n s versus the volume of b u f f e r r e q u i r e d to e l u t e the p r o t e i n from the column expressed r e l a t i v e to the v o i d volume measured using Dextran Blue 2000. Peak height was used to determine e l u t e d volume (Ve) and v o i d volume (Vo). The 2 equation of the l i n e i s y = - 1 . 2 5 1 X + 7.269; r = 0.996. (B) E l u t i o n p a t t e r n of p r o t e i n from Sephadex G-150 as d e t e c t e d s p e c t r o p h o t o m e t r i c a l l y F i g u r e 42. (A) Coomassie blue s t a i n e d SDS-PAGE of s o l u b l e and membrane p r o t e i n s from T. pseudonana. Standard p r o t e i n s ( f o r d e s c r i p t i o n see " M a t e r i a l s and Methods") were run i n lane S; the number beside each standard i s the mol e c u l a r weight i n kD. V a r y i n g amounts of s o l u b l e and membrane p r o t e i n e x t r a c t were a p p l i e d to the g e l ; lane 1 r e c e i v e d 60 or 50 mg of s o l u b l e or membrane p r o t e i n , r e s p e c t i v e l y . Lanes 2 and 3 were loaded with twice the amount of p r o t e i n i n lane 1, and lane 4 c o n t a i n e d four times the p r o t e i n of lane 1. (B) Autoradiograms of g e l exposed to X-ray f i l m f o r 100 h. Two s o l u b l e p r o t e i n s of 29 and 21 kD 75 were l a b e l l e d with Se F i g u r e 43. G l u t a t h i o n e peroxidase a c t i v i t y d e t e c t e d on a p o l y a c r y l a m i d e g e l f o l l o w i n g 6 h of e l e c t r o -p h o r e s i s . Two bands showing enzyme a c t i v i t y (NADPH 2 o x i d a t i o n r e s u l t i n g i n d e f l u o r e s c e n c e ) were ev i d e n t on the g e l . However, due to the i n t e n s i t y of the r e a c t i o n i n both bands, the s e p a r a t i o n between them i s not obvio u s . Sample b u f f e r , g l y c e r o l and bromphenol blue (bpb) were run as a c o n t r o l i n lane 1, and c e l l - f r e e e x t r a c t i n b u f f e r with g l y c e r o l and bpb were run i n lane 2 F i g u r e 44. G l u t a t h i o n e peroxidase a c t i v i t y d e t e c t e d on p o l y a c r y l a m i d e g e l s u s i n g (lane 1) tBOOH and (lane xxvi i 2) H2C>2 as a s u b s t r a t e a f t e r e l e c t r o p h o r e s i s f o r 9 h. The upper band (a) was not d e t e c t e d on the g e l t r e a t e d with assay s o l u t i o n c o n t a i n i n g H 2 0 2 . Band (b) r e a c t e d with both p e r o x i d e s 262 Fig u r e 45. (A) G l u t a t h i o n e peroxidase a c t i v i t y d e t e c t e d on a p o l y a c r y l a m i d e g e l u s i n g H 2 0 2 as 75 s u b s t r a t e . (B) Amount of Se i n the g e l (d p m - s l i c e " 1 ) 264 xxvi i i ACKNOWLEDGEMENTS I g r a t e f u l l y acknowledge the support of my academic s u p e r v i s o r and b e n e f a c t o r , Dr. P.J. H a r r i s o n , who provided a s t i m u l a t i n g atmosphere that was conducive to re s e a r c h . Thanks i s a l s o extended to members of my s u p e r v i s o r y committee: Drs. N.J. A n t i a , R.E. DeWreede, A.D.M. G l a s s and L. O l i v e i r a . The rese a r c h presented i n t h i s t h e s i s b e n e f i t t e d from d i s c u s s i o n s with my c o l l e a g u e s : Drs. J.A. Parslow and C A . S u t t l e , W.P. Cochlan, G.J. Doucette and P.A. Thompson. I am very g r a t e f u l to W.P. Cochlan who d r a f t e d the f i g u r e s i n t h i s t h e s i s , and for h i s c o n t r i b u t i o n s to the experiments presented i n Chapter 2. P.A. Thompson and P. Yu a s s i s t e d w i t h aspects of the selenium r e s e a r c h i n Chapter 5 and P.J. C l i f f o r d and G.J. Doucette helped with photography. I a l s o thank the f a c u l t y of the Departments of Botany and Oceanography, many of whom I i n t e r a c t e d with d u r i n g my tenure, as a graduate student a t U.B.C.; n o t a b l y , Drs. E. Camm and E.V. G r i l l . Dr. R. R i v k i n generously p r o v i d e d the opp o r t u n i t y for me to p a r t i c i p a t e i n a r e s e a r c h c r u i s e to the Sargasso Sea. I e s p e c i a l l y thank N.J. Smith f o r her support, encouragement and p a t i e n c e d u r i n g every p a r t of t h i s work. F i n a n c i a l support was p r o v i d e d by s c h o l a r s h i p s from NSERC and the K i l l a m Foundation, and t e a c h i n g a s s i s t a n t s h i p s . The rese a r c h was supported by funds from a NSERC o p e r a t i n g grant (58-0128) to Dr. P.J. H a r r i s o n . 1 PART I: UREA  Introduction Overview and objectives The f i r s t p art of t h i s t h e s i s examines urea uptake and a s s i m i l a t i o n by phytoplankton communities i n c o a s t a l and open ocean environments and by phytoplankton grown i n l a b o r a t o r y c u l t u r e . Two methods f o r measuring d i s s o l v e d urea c o n c e n t r a t i o n s i n seawater were r e f i n e d , and are compared i n the f i r s t chapter. A mo d i f i e d d i a c e t y l monoxime method gave the best r e s u l t s f o r urea a n a l y s i s ; t h i s technique was used i n subsequent chapters to determine urea uptake r a t e s by phytoplankton, and to measure urea c o n c e n t r a t i o n s i n seawater samples d u r i n g f i e l d experiments. In Chapter 2 , urea uptake r a t e s by phytoplankton i n the S t r a i t of Georgia, i n n i t r a t e -r e p l e t e and n i t r a t e - d e p l e t e seawater, were measured and compared to r a t e s of n i t r a t e and ammonium uptake. S p e c i f i c q u e s t i o n s of t h i s study addressed phytoplankton n i t r o g e n p r e f e r e n c e s , the d i e l p a t t e r n of n i t r o g e n uptake, and ammonium and urea r e g e n e r a t i o n . These r e s u l t s p r o v i d e d unique i n f o r m a t i o n regarding n i t r o g e n uptake a n d . i t s t r a n s f o r m a t i o n i n these two c o n t r a s t i n g c o a s t a l environments. Experiments conducted i n the Sargasso Sea determined in situ and maximum 14 15 urea uptake r a t e s , and eva l u a t e d C- and N - l a b e l l e d urea uptake. These r e s u l t s are presented i n Chapter 3 . D i s c r e p a n c i e s between the urea uptake r a t e s measured by the two i s o t o p e s were e v i d e n t . I t i s argued that phytoplankton i n 2 these r e g i o n s are adapted to the low ambient urea c o n c e n t r a t i o n s and, w i t h i n some communities, u t i l i z e urea at ra t e s which approximate the maximum r a t e s of u t i l i z a t i o n . L a boratory experiments examined urea uptake and a s s i m i l a t i o n i n an attempt to e x p l a i n r e s u l t s o b t a i n e d from experiments conducted i n the Sargasso Sea. Chapter 4 presents the r e s u l t s of urea uptake by the marine diatom Thai assiosi ra pseudonana measured by three d i f f e r e n t methods d u r i n g n i t r a t e - s u f f i c i e n t growth and n i t r a t e - s t a r v a t i o n . On the b a s i s of these r e s u l t s , a model f o r urea uptake and a s s i m i l a t i o n by T. ps eudonana i s proposed. Support f o r t h i s model i s found i n the l i t e r a t u r e , and the model i s c o n s i s t e n t with other r e s u l t s r e p o r t e d i n t h i s t h e s i s . Nitrogen limitation of phytoplankton productivity S u b s t a n t i a l evidence has accumulated to suggest that n i t r o g e n i s f r e q u e n t l y a g r o w t h - l i m i t i n g f a c t o r f o r phytoplankton i n many nearshore waters (Ryther and Dunstan 1971, Goldman et al . 1973, Thomas ,et al . 1974, Goldman 1976). The r a t e of supply of n i t r o g e n to phytoplankton i n the upper mixed l a y e r of the ocean, by r e g e n e r a t i v e processes and by all o c h t h o n o u s i n p u t s , r e g u l a t e s phytoplankton p r o d u c t i v i t y . N e v e r t h e l e s s , numerous arguments have been advanced to suggest that phytoplankton i n the open ocean are not n i t r o g e n d e f i c i e n t (Thomas 1970, M o r r i s et al. 1971, Goldman et al . 1979, D i T u l l i o and Laws 1983, Laws et al. 1984, Kanda et al . 1985); by no means i s t h i s q u e s t i o n r e s o l v e d . In the mixed l a y e r of the ocean, i n l a t e s p r i n g and 3 summer, n i t r o g e n c o n c e n t r a t i o n s are o f t e n near or below our l i m i t s of d e t e c t i o n ( l i m i t s of d e t e c t i o n of n i t r a t e [NO^ ], 50 ng at N-1 1 , ammonium [ N H 4 + ] , 30 ng at N-1 1 ; urea, 50 ng at N - l ~ 1 ) (McCarthy 1980, Raymont 1980, Garside 1985). During these p e r i o d s , phytoplankton p r i m a r i l y u t i l i z e r egenerated forms of n i t r o g e n , such as ammonium and urea, f o r growth (Dugdale and Goering 1967, McCarthy et al. 1977, G l i b e r t et al . 1982a, Cochlan 1986), and n i t r a t e and n i t r i t e are of l e s s importance. Nitrogen uptake and r e g e n e r a t i o n are envisaged to be t i g h t l y coupled i n these r e g i o n s (Goldman 1984). C l o s e c o u p l i n g between supply and u t i l i z a t i o n of ammonium i s e v i d e n t i n the r e s u l t s of a number of s t u d i e s conducted i n c o a s t a l and oceanic r e g i o n s . These s t u d i e s r e p o r t that ammonium re g e n e r a t i o n by zooplankton, and i n p a r t i c u l a r those organisms l e s s than 10 um i n diameter, can supply most, i f not a l l , of the phytoplankton ammonium requirements f o r growth ( H a r r i s o n 1978, Caperon et al. 1979, G l i b e r t 1982, H a r r i s o n et al. 1983, Cochlan 1986). Much l e s s i s known r e g a r d i n g urea r e g e n e r a t i o n and u t i l i z a t i o n by plankton communities. Recent r e p o r t s by Laws et al. (1985) and Wheeler and Kirchman (1986) q u e s t i o n our c u r r e n t concepts of n i t r o g e n c y c l i n g i n marine systems. These i n v e s t i g a t o r s p r e s e n t e d r e s u l t s that suggested that i n the o l i g o t r o p h i c ocean, and i n c o a s t a l waters, most of the ammonium i s taken up by h e t e r o t r o p h i c b a c t e r i a and not by phytoplankton. S i n c e much s i g n i f i c a n c e i s given to ammonium as a n i t r o g e n source f o r phytoplankton growth, these r e s u l t s are profound. I f 4 u t i l i z a t i o n by h e t e r o t r o p h i c b a c t e r i a i s the primary f a t e of ammonium, then other n i t r o g e n compounds, p r e v i o u s l y overlooked or given l e s s importance, must supply the phytoplankton n i t r o g e n requirements f o r growth: In some marine environments, urea i s a major form of n i t r o g e n taken up by phytoplankton (Kaufman et al. 1983, H a r r i s o n et al. 1985). Sources and sinks of urea in seawater A s i g n i f i c a n t source of urea i n seawater i s from e x c r e t i o n by i n v e r t e b r a t e s and f i s h (e.g. Corner and Newell 1967, McCarthy and Whitledge 1972, Whitledge 1981). As much as 50% of the t o t a l ammonium and urea excreted by a mixed macrozooplankton assemblage was urea (Eppley et al . 1973). McCarthy (1971) found that urea e x c r e t i o n r a t e s of Cal anus hel gol andi cus were roughly 30% of the NH^ + e x c r e t i o n r a t e s . A s i m i l a r p r o p o r t i o n of the e x c r e t a from f i s h i s made up of urea. B a c t e r i a l p r o d u c t i o n of urea from d i s s o l v e d organic matter i n seawater has r e c e i v e d l e s s a t t e n t i o n . Maita et al . (1973) argued t h a t b a c t e r i a l decomposition of a r g i n i n e was the major source of urea i n seawater. They f a i l e d to address the s i g n i f i c a n c e of t h i s pathway in situ, and i n s t e a d they only demonstrated the p o t e n t i a l f o r urea production from a r g i n i n e . In freshwater l a k e s , Satoh (1980) and Satoh et al . (1980) r e p o r t e d that decomposition of phytoplankton by b a c t e r i a was a major source of ur e a . The extent and importance of t h i s process i n the ocean i s unknown, but decomposition of zooplankton by marine b a c t e r i a does produce urea (Mitamura and S a i j o 1980b). Nearshore waters may r e c e i v e urea v i a 5 t e r r e s t r i a l drainage (Remsen et a l . 1974), and atmospheric p r e c i p i t a t i o n has a l s o been i d e n t i f i e d as a urea source (Timperley et al . 1985), which presumably c o n t r i b u t e s some urea to seawater. S i z e f r a c t i o n a t i o n s t u d i e s have been conducted to q u a n t i f y the r e l a t i v e importance of d i f f e r e n t types of phytoplankton and the r o l e of b a c t e r i a i n urea u t i l i z a t i o n , i n seawater. The i n i t i a l r e s u l t s of Remsen et al . (1972) i m p l i c a t e d phytoplankton as the most important component of the plankton r e s p o n s i b l e f o r urea decomposition i n c o a s t a l waters and a s s o c i a t e d e s t u a r i e s of Geo r g i a , U.S.A. A d d i t i o n a l i n v e s t i g a t i o n s by Mitamura and S a i j o (1975) and Webb and Haas (1976) confirmed these r e s u l t s i n d i f f e r e n t ocean environments, and B i l l e n (1984) a l s o concluded that phytoplankton were p r i m a r i l y r e s p o n s i b l e f o r u t i l i z i n g urea i n seawater. These s t u d i e s were based, f o r the most p a r t , on urea u t i l i z a t i o n by c o a s t a l p l a n k t o n . Remsen et a l . (1974) found that i n c o a s t a l waters o f f Georgia, 86% of the urea uptake c o u l d be a t t r i b u t e d to organisms g r e a t e r than 10 um i n diameter; whereas, only 49% of the urea uptake was by plankton of t h i s s i z e i n the North A t l a n t i c . These r e s u l t s cannot be used as evidence to support a g r e a t e r r o l e of h e t e r o t r o p h i c b a c t e r i a i n urea uptake i n the open ocean because phytoplankton would c e r t a i n l y be a l a r g e component of the less-than-10 um s i z e f r a c t i o n . R e c e n t l y , Wheeler and Kirchman (1986) found n e g l i g i b l e uptake of urea by Gulf Stream and c o a s t a l organisms l e s s than 1 um i n diameter. I t i s 6 concluded, from the r e s u l t s of these s t u d i e s , that phytoplankton are r e s p o n s i b l e f o r most, i f not a l l , of the urea uptake i n the ocean. U t i l i z a t i o n of urea as a n i t r o g e n s u b s t r a t e f o r growth of phytoplankton i n the ocean has r e c e i v e d renewed i n t e r e s t s i n c e the i n i t i a l s t u d i e s of McCarthy, Remsen and coworkers (McCarthy, 1970, 1972a, Remsen 1971, Carpenter et al . 1972a, McCarthy and Eppley 1972, McCarthy and Kamykowski 1972, Remsen et al . 1972, 1974). T h i s i s a r e s u l t , at l e a s t i n p a r t , of our growing r e a l i z a t i o n of the importance of d i s s o l v e d o r g a n i c n i t r o g e n i n phytoplankton n u t r i t i o n (e.g. F i s h e r and Cowdell 1982). Current e s t i m a t e s i n d i c a t e that urea c o n s t i t u t e s a s i g n i f i c a n t f r a c t i o n (20 to 50%) of the t o t a l n i t r o g e n u t i l i z e d by phytoplankton i n c o a s t a l (McCarthy 1972a, Harvey and Caperon 1976, McCarthy et al. 1977, Kaufman et al . 1983, K r i s t i a n s e n 1983, H a r r i s o n et al . 1985) and oceanic (Eppley et al . 1973, 1977) environments. Furthermore, r a p i d turnover times of urea (Herbland 1976, Savidge and Hutley 1977) i n seawater emphasizes the s t r a t e g i c r o l e of urea i n the marine n i t r o g e n c y c l e . Phytoplankton growth on urea Numerous r e p o r t s confirmed that urea i s an e x c e l l e n t source of n i t r o g e n f o r growth of many phytoplankton ( G u i l l a r d 1963, McCarthy 1971, A n t i a et al. 1975, 1977, Turner 1979, N e i l s e n and La r s s o n 1980, F i s h e r and Cowdell 1982). However, in these s t u d i e s , there were some s p e c i e s that were unable to grow or that showed poor growth with urea. A n t i a et al. 7 (1977) were able to c u l t u r e some of these s p e c i e s by i n c r e a s i n g the urea c o n c e n t r a t i o n i n the medium. Most r e c e n t l y , O l i v e i r a and A n t i a (1984, 1986b) demonstrated that 2 + by supplementing the c u l t u r e medium with Ni , some alg a e , p r e v i o u s l y unable to u t i l i z e urea, grew w e l l with urea as the so l e n i t r o g e n source. These r e s u l t s extend the l i s t of phytoplankton known to be able to use urea as a n i t r o g e n source for growth. The e a r l y r e s e a r c h of Harvey (1940) documented p r e f e r e n t i a l u t i l i z a t i o n of urea by p a r t i c u l a r phytoplankton s p e c i e s . He showed t h a t the phytoplankton community that developed i n seawater samples e n r i c h e d with urea d i f f e r e d from the community i n ammonium-enriched samples. The i m p l i c a t i o n of h i s r e s u l t s was th a t some s p e c i e s are be t t e r able to sequester and u t i l i z e urea than o t h e r s . Laboratory studies of urea uptake Uptake of urea by marine and freshwater phytoplankton has been the su b j e c t of a number of i n v e s t i g a t i o n s . These s t u d i e s examined the k i n e t i c s of urea uptake by phytoplankton grown i n l a b o r a t o r y batch c u l t u r e u sing a v a r i e t y of i s o t o p i c t r a c e r s 1 5 i n c l u d i n g N-urea (McCarthy 1972b, C o l l o s and Slawyk 1979, Horrigan and McCarthy 1981, 1982), 1 4 C - u r e a (Carpenter et al. 1972a, b, W i l l i a m s and Hodson 1977, K i r k and K i r k 1978a, b, Bekheet and S y r e t t 1979, Rees and S y r e t t 1979a, H o r r i g a n and McCarthy 1981, 1982, S y r e t t et al. 1986), 3 5 S - t h i o u r e a ( S y r e t t and Bekheet 1977, Rees and S y r e t t 1979b), and by chemical measurements of d i s s o l v e d urea c o n c e n t r a t i o n (Healey 1977). 8 The g e n e r a l r e s u l t s from t h i s work can be summarized as f o l l o w s : Urea uptake i s a c o n s t i t u t i v e p r o p e r t y of phytoplankton growing on n i t r a t e as the s o l e n i t r o g e n source, but an i n d u c t i o n p e r i o d i s r e q u i r e d before ammonium-grown c e l l s take up urea. Phytoplankton possess a urea uptake system t h a t f o l l o w s s a t u r a t i o n k i n e t i c s . Phytoplankton are a b l e to accumulate urea i n t r a c e l l u l a r l y at a r a t e , and to a c o n c e n t r a t i o n , t h a t suggests that uptake i s mediated by an a c t i v e t r a n s p o r t p r o c e s s . Although the energy requirements f o r urea t r a n s p o r t are not w e l l documented, uptake i s s t i m u l a t e d by l i g h t and i n h i b i t e d by the presence of proton ionophores. In t o t a l , these and other experiments have e s t a b l i s h e d t h a t ATP i s necessary f o r urea t r a n s p o r t by m i c r o a l g a e . F o l l o w i n g a p e r i o d of n i t r o g e n d e p r i v a t i o n , phytoplankton take up urea at r a t e s which are i n excess of those r e q u i r e d to meet t h e i r n i t r o g e n demands d u r i n g balanced growth. Urea metabolism The d e g r a d a t i o n of urea occurs by one of two enzymes, urease (EC 3.5.1.5) or ATP:urea amidolyase. The ATP:urea amidolyase enzyme complex c o n s i s t s of two d i s t i n c t enzyme a c t i v i t i e s , urea c a r b o x y l a s e (EC 6.3.4.6) and an a l l o p h a n a t e h y d r o l a s e (EC 3.5.1.13); together they c a t a l y z e the d e g r a d a t i o n of urea to C0 2 and NH^ (Whitney and Cooper 1972). Urease and ATP:urea amidolyase are mutually e x c l u s i v e i n a l g a e , and ATP:urea amidolyase appears c o n f i n e d to a small group of c h l o r o p h y l l b c o n t a i n i n g microalgae ( S y r e t t and A l 9 Houty 1984). Urease of some higher p l a n t s i s a metalloenzyme c o n t a i n i n g n i c k e l as an i n t e g r a l component (Dixon et al . 1975a). Evidence p r o v i d e d by S y r e t t (1981), Rees and Bekheet (1982) and O l i v e i r a and A n t i a (1984, 1986a) i n d i c a t e s that t h i s i s probably t r u e f o r microalgae as w e l l . Depending on the a l g a l s p e c i e s , and the d u r a t i o n of the uptake experiment, once urea i s t r a n s p o r t e d through the c e l l membrane i t may remain i n t a c t or i t may be degraded to C0 2 and 2NH^. Phaeodact yl um tricornutum c o n c e n t r a t e s urea i n t r a c e l l u l a r l y , and du r i n g 10 min uptake experiments 80% of 1 4 the C r e t a i n e d by the c e l l s remains as urea (Rees and S y r e t t , 1979a). In other a l g a e , such as Chl or el I a fusca var. vacuolata, Skeletonema costatum and Thai assiosira pseudonana, 50-98% of the C from urea i s r e l e a s e d from the c e l l s d u r i n g 5 min i n c u b a t i o n s (Bekheet and S y r e t t 1979, H o r r i g a n and McCarthy 1981 ). E l l n e r and S t e e r s (1955) showed that urea carbon was i n c o r p o r a t e d i n t o c e l l u l a r c o n s t i t u e n t s i n Chl or el I a pyrenoidosa and Scenedesmus basilensis, and much of the 1 4 a c t i v i t y of C was present i n guanine. Webster et al. (1955) confirmed that urea carbon can be u t i l i z e d as a carbon source. T h e i r r e s u l t s showed, as d i d those of A l l i s o n et al. (1954), that a s i m i l a r p a t t e r n of l a b e l l i n g of amino a c i d s occurred i n p l a n t s s u p p l i e d with b i c a r b o n a t e ( H 1 4 C 0 3 ) and NH 4 + as with 1 4 C-urea. These r e s u l t s suggested that urea carbon i s eq u i v a l e n t to HCO^ . N e v e r t h e l e s s , d i f f e r e n c e s i n the types 1 4 of amino a c i d s s y n t h e s i z e d by bean l e a v e s exposed to C-urea 10 1 4 -compared wit h H CO^ and ammonium, as w e l l as g r e a t e r l e v e l s 14 • • of C a c t i v i t y i n c e r t a i n amino a c i d s i n the u r e a - s p i k e d p l a n t s , p r o v i d e d evidence that urea carbon i s not n e c e s s a r i l y a s s i m i l a t e d by p h o t o s y n t h e t i c pathways. Since the s p e c i f i c 1 4 a c t i v i t y of C from urea was l e s s than i n the p l a n t s s u p p l i e d 1 4 -w i t h H CO-j , a much grea t e r p r o p o r t i o n of C from urea was 1 4 f i x e d i n t o amino a c i d s than was C from H CO^ . Webb and Haas (1976) and Mitamura and S a i j o (1975) concluded that l i g h t -d r i v e n p h o t o s y n t h e t i c c a r b o x y l a t i n g r e a c t i o n s were not the 1 4 s o l e mechanism f o r C-urea i n c o r p o r a t i o n by n a t u r a l communities of phytoplankton. In white spruce s e e d l i n g s , Durzan (1973) r e p o r t e d that carbamyl compounds (carbamyl 1 4 a s p a r t a t e and c i t r u l l m e ) were formed more r e a d i l y from C-1 4 urea than from H CO^ . In h i s proposed scheme, urea was p r e f e r e n t i a l l y i n c o r p o r a t e d i n t o carbamyl phosphate, the p r e c u r s o r of other carbamyl compounds. Some of the urea-N was a l s o i n c o r p o r a t e d i n t o organic n i t r o g e n without f i r s t being degraded to NH^; independent evidence e x i s t s which i n d i c a t e s t h a t urea-N may be a s s i m i l a t e d i n t o amino a c i d s p r i o r to i t s c o n v e r s i o n to NH^ ( K i t o h and Hori 1977). Metabolism of NH^, produced by urea degradation, i n t o amino a c i d s i s by n i t r o g e n a s s i m i l a t o r y pathways which operate when NH^ + i s s u p p l i e d to the c e l l s (see T u r p i n and H a r r i s o n 1978, S y r e t t 1981). Tes t or gani sm In three chapters of t h i s t h e s i s the u n i c e l l u l a r c e n t r i c marine diatom Thai assi osi ra pseudonana (clone 3H) (Hustedt) Hasle and Heimdal was used to i n v e s t i g a t e p h y s i o l o g i c a l and 11 b i o c h e m i c a l a s p e c t s of phytoplankton n u t r i t i o n . T h i s clone was i s o l a t e d from Moriches Bay, Long I s l a n d i n 1958 by R.R.L. G u i l l a r d . In 1970, i t was o b t a i n e d from N.J. A n t i a and maintained i n the Northeast P a c i f i c C u l t u r e C o l l e c t i o n at the U n i v e r s i t y of B r i t i s h Columbia. A complete morphological d e s c r i p t i o n of t h i s a l g a was p u b l i s h e d by Hasle and Heimdal (1970) and Hasle (1976). Thai assi osi ra pseudonana has been the ch o i c e of many i n v e s t i g a t o r s as an experimental organism f o r s t u d i e s examining n u t r i e n t uptake (Goldman and McCarthy 1978, Dortch et al . 1982, Parslow et al . 1984a, b, 1985) t r a c e metal requirements and i n t e r a c t i o n s (Murphy et al. 1981, Sunda and Huntsman 1983), and temperature and s a l i n i t y e f f e c t s ( G u i l l a r d and Ryther 1962, Brand et al. 1981). Thai assiosira pseudonana grows r a p i d l y , has been e x t e n s i v e l y s t u d i e d , and i s e a s i l y maintained i n axenic c u l t u r e . These c r i t e r i a make i t an i d e a l organism i n which to examine p h y s i o l o g i c a l and biochemical p r o c e s s e s . 1 2 C H A P T E R 1. A C O M P A R I S O N O F M E T H O D S F O R T H E M E A S U R E M E N T O F  D I S S O L V E D U R E A C O N C E N T R A T I O N S I N S E A W A T E R Background The c o n c e n t r a t i o n of d i s s o l v e d urea i n oceanic environments i s g e n e r a l l y l e s s than 0.50 uM (Eppley et al. 1977, Mitamura and S a i j o 1980a, McCarthy 1980); whereas, i n c o a s t a l and e s t u a r i n e environments, c o n c e n t r a t i o n s tend to be hig h e r and more v a r i a b l e (Remsen et al. 1974, McCarthy 1980a). V a r i a b i l i t y i n urea c o n c e n t r a t i o n i s a l s o observed i n v e r t i c a l p r o f i l e s of marine waters (Remsen 1971, McCarthy 1972) and can be a t t r i b u t e d to patchy d i s t r i b u t i o n of u r e a - r e g e n e r a t i n g organisms. In f a c t , McCarthy and Kamykowski (1972) r e p o r t e d temporal v a r i a t i o n i n urea c o n c e n t r a t i o n s which was a s s o c i a t e d with a shark i n f e s t a t i o n . Measurement of d i s s o l v e d urea c o n c e n t r a t i o n s r e v e a l s l i t t l e r e g a r d i n g the r o l e of urea i n n i t r o g e n c y c l i n g or phytoplankton n u t r i t i o n . However, a c c u r a t e and p r e c i s e measurements are r e q u i r e d to determine uptake and e x c r e t i o n r a t e s of urea and to provide an i n d i c a t i o n of the n i t r o g e n s t a t u s of phytoplankton communities. Throughout t h i s t h e s i s , the u n i t s uM (umol-1 1) and ug at N-1 1 are used i n t e r c h a n g e a b l y . The reader i s reminded that 1 uM urea i s e q u i v a l e n t to 2 ug at urea-N-1 1 because urea c o n t a i n s two n i t r o g e n atoms per molecule. For compounds c o n t a i n i n g a s i n g l e n i t r o g e n atom, such as ammonium (NH^ +), n i t r a t e and n i t r i t e , v a l u e s expressed as uM or ug at N-1 1 are i d e n t i c a l . C u r r e n t l y , two techniques f o r measuring the c o n c e n t r a t i o n 1 3 of d i s s o l v e d urea i n seawater are i n use. The urease method (McCarthy 1970) i n v o l v e s enzymatic h y d r o l y s i s of urea, by -urease, to carbon d i o x i d e and ammonia. Subsequently, the l i b e r a t e d ammonia i s assayed by the procedure of Solorzano (1969), as o u t l i n e d i n McCarthy (1970), and the c o n c e n t r a t i o n of urea n i t r o g e n i s determined. The d i a c e t y l monoxime method i s a c o l o u r i m e t r i c a n a l y s i s which d i r e c t l y determines the c o n c e n t r a t i o n of d i s s o l v e d urea. The chemistry of t h i s r e a c t i o n has been examined ( B u t l e r et al . 1981). The d i a c e t y l monoxime method was f i r s t adapted f o r use i n seawater by Newell et al . (1967) and has s i n c e been m o d i f i e d by DeManche et al . (1973), Whitledge et al . (1981), Aminot and Kerouel (1982) and K o r o l e f f (1983). T h i s chapter e v a l u a t e s and compares the d i a c e t y l monoxime and urease methods f o r measuring the c o n c e n t r a t i o n of d i s s o l v e d urea i n a r t i f i c i a l seawater, phytoplankton c u l t u r e f i l t r a t e and both n a t u r a l and u r e a - s p i k e d seawater samples from c o a s t a l and oceanic environments. Mater ia l s and Methods Urease met hod Urea c o n c e n t r a t i o n was determined by the urease method of McCarthy (1970). Worthington URC and Sigma Type III urease were used as enzyme sources as suggested by McCarthy (1970) and McCarthy and Kamykowski (1972), r e s p e c t i v e l y . The a c t i v i t y of Worthington URC urease was 72 u n i t s (U) mg-solid 1 and f o r Sigma Type II I urease i t was 40 U mg-solid 1 . I 1 4 determined urease a c t i v i t i e s independently of the manufacturers' s p e c i f i c a t i o n s , by measuring r a t e s of 1 4 C 0 2 . 1 4 o pr o d u c t i o n from C-urea i n phosphate b u f f e r , pH 7.6, at 25 C. Incubations were conducted i n acid-washed 25 ml Erlenmeyer f l a s k s s e a l e d with rubber s e p t a . Small g l a s s v i a l s , c o n t a i n i n g f i l t e r paper impregnated with 50 u l phenethylamine 1 4 to t r a p e n z y m a t i c a l l y r e l e a s e d C 0 2 ' w e r e suspended i n s i d e the f l a s k s . The r e a c t i o n was t e r m i n a t e d a f t e r 5 min by the a d d i t i o n of 0.5 ml 6N HC1, and recovery was 100%. S i m i l a r procedures were employed to examine the e f f e c t of urease 1 4 enzyme c o n c e n t r a t i o n on C-urea h y d r o l y s i s i n seawater. With the e x c e p t i o n of experiments designed to examine the e f f e c t s of temperature and i n c u b a t i o n d u r a t i o n on urea h y d r o l y s i s , urease-seawater samples were heated i n a temperature-regulated water bath at 50°C f o r 20 min. Ammonium c o n c e n t r a t i o n was measured manually as d e s c r i b e d by McCarthy (1970) and by automated a n a l y s i s on a Technicon (R) A u t o a n a l y z e r ^ f o l l o w i n g the procedure of Slawyk and Maclsaac (1972). These two methods gave i d e n t i c a l r e s u l t s f o r NH 4 + d e t e r m i n a t i o n s i n s o l u t i o n s of d e i o n i z e d d i s t i l l e d water (DDW), DDW c o n t a i n i n g 3% NaCl (w/v) ( r e f e r r e d to h e r e a f t e r as 3% NaCl), a r t i f i c i a l seawater (ESAW; H a r r i s o n et al . 1980) and n a t u r a l seawater. Both NH^ + t e c h n i q u e s were employed f o r urea a n a l y s i s u s i n g the urease method. They gave i d e n t i c a l r e s u l t s , and consequently both N H 4 + a n a l y s e s have been used i n t h i s study. Standard s o l u t i o n s of urea spanned the range of values encountered i n the a n a l y s e s . U n l e s s s t a t e d otherwise, standards were prepared i n 3% NaCl. For comparison I have c a l c u l a t e d a f a c t o r , F, d e f i n e d by McCarthy (1970) as: F = c o n c e n t r a t i o n of urea standard (absorbance of standard-absorbance of blank) f o r d i f f e r e n t c o n c e n t r a t i o n s of urea. The background NH 4 + c o n c e n t r a t i o n s i n the Worthington URC and Sigma Type I I I urease p r e p a r a t i o n s , when added to seawater in c o n c e n t r a t i o n s suggested by McCarthy (1970) and McCarthy and Kamykowski (1972), were 0.17 and 0.25 uM, r e s p e c t i v e l y . The p r e c i s i o n (+ 1 SD) of r e p l i c a t e samples c o n t a i n i n g 0.50 uM urea, prepared i n DDW, was 0.01 uM urea (n=5). Diacetyl monoxime method Urea c o n c e n t r a t i o n was measured using the d i a c e t y l monoxime method of Rahmatullah and Boyde (1980). T h i s method (R) was m o d i f i e d and automated f o r a Technicon A u t o a n a l y z e r w by Whitledge et al . (1981). F u r t h e r m o d i f i c a t i o n s to t h i s procedure were made, and f o r c l a r i f i c a t i o n I have i n c l u d e d these d e t a i l s ( F i g . 1). The compositions of the reagents are as f o l l o w s - Reagent 1: 5 ml B r i j - 3 5 (Technicon) i n 500 ml DDW; Reagent 2: 300 ml H 2 S 0 4 and 100 ml H 3P0 4 i n 600 ml DDW, add 50.0 mg FeCl^-6H 20 when the s o l u t i o n has c o o l e d ; Reagent 3: 5.0 g d i a c e t y l monoxime and 50.0 mg t h i o s e m i c a r b i z i d e i n 250 ml DDW. To determine the i d e n t i t y of p o t e n t i a l l y i n t e r f e r i n g substances, 29 o r g a n i c and in o r g a n i c n i t r o g e n compounds were t e s t e d . Concentrated s o l u t i o n s of each 16 F i g . 1. Schematic diagram of A u t o a n a l y z e r ^ system f o r urea a n a l y s i s by the d i a c e t y l monoxime method. Composition of reagents i s given i n the " M a t e r i a l s and Methods -D i a c e t y l monoxime method". The debubbler l i n e p u l l e d 0.60 ml-min 1 to prevent any l a r g e a i r bubbles from e n t e r i n g the system. A b o i l i n g water bath gave a more constant i n c u b a t i o n temperature and helped e l i m i n a t e the o s c i l l a t i n g b a s e l i n e that was seen when l e s s c o n s t a n t and lower temperatures were used. DEBUBBLER SAMPLE AIR REAGENT I REAGENT 2 REAGENT 3 WASTE ml m i n 0.60 0.23 0.32 0.10 0.60 0.16 0.42 PUMP KEY ///// 10-TURN COIL 0 ONE-WAY VALVE WASTE -HtiihH///hH//n WASTE RECORDER WATER BATH IOO°C length = 6m i.d. -2 mm COLORIMETER 50 mm F/C 520 nm OVERFLOW 18 compound were prepared i n u r e a - f r e e DDW, and samples were analyzed f o r urea. In s o l u t i o n s where urea was found to be present, subsamples were t r e a t e d with urease and then reanalyzed by the d i a c e t y l monoxime method. T h i s was done to determine whether the compound mimicked urea and r e a c t e d with the c o l o u r reagents, or i f the s o l u t i o n c o n t a i n e d contaminating urea which was i n t r o d u c e d with the compound being t e s t e d . S o l u t i o n s used to set the b a s e l i n e on the a u t o a n a l y z e r had the i d e n t i c a l composition (minus urea) to the samples being analyzed. However, f o r n a t u r a l seawater samples urea-f r e e NaCl or ESAW s o l u t i o n s were used f o r the b a s e l i n e , and urea standards were prepared i n these same s o l u t i o n s . As suggested by Aminot and Kerouel (1982), a Chelex 100 (Bio-Rad L a b o r a t o r i e s , Richmond, C a l i f o r n i a , USA) column was p o s i t i o n e d i n the sample l i n e to remove p o t e n t i a l l y i n t e r f e r i n g metals; however, i n the n a t u r a l seawater samples I examined, there were no b e n e f i c i a l e f f e c t s of the column and i t was not used f o r subsequent a n a l y s i s . In a separate experiment, d i s s o l v e d amino a c i d s were removed from seawater samples by 1igand-exchange chromatography ( S i e g a l and Degens 1966), and the seawater samples were r e a n a l y z e d f o r urea. The p r e c i s i o n (± 1 SD) of r e p l i c a t e samples of 0.50 uM urea prepared i n DDW, was 0.01 uM urea (n=5) and the lower l i m i t of d e t e c t i o n was 0.025 uM urea. Experimental methods Urea c o n c e n t r a t i o n s i n s o l u t i o n s of DDW, 3% NaCl, ESAW 1 9 and phytoplankton c u l t u r e f i l t r a t e were measured s i m u l t a n e o u s l y by the d i a c e t y l monoxime and urease methods. Phytoplankton c u l t u r e f i l t r a t e ( r e f e r r e d to h e r e a f t e r as " c u l t u r e f i l t r a t e " ) was obtained by growing Thai assi osi ra pseudonana ( c l o n e 3H) i n ESAW. The c e l l s were h a r v e s t e d i n e a r l y s t a t i o n a r y phase by f i l t r a t i o n through combusted Whatman GF/F f i l t e r s (460°C f o r 4 h ) , and the c u l t u r e f i l t r a t e was c o l l e c t e d i n an a c i d - r i n s e d s t e r i l e side-arm f l a s k . N a t u r a l seawater samples were c o l l e c t e d from the S t r a i t of G e o r g i a , B r i t i s h Columbia and E n g l i s h Bay, B r i t i s h Columbia (49°18'N; 123°12'W) i n August, 1984. Samples were f i l t e r e d through combusted Whatman GF/F f i l t e r s and s t o r e d f r o z e n at -20°C i n p o l y p r o p y l e n e b o t t l e s . There was no e f f e c t of f r e e z i n g samples of f i l t e r e d seawater or samples s p i k e d with known c o n c e n t r a t i o n s of urea for p e r i o d s up to one year ( P r i c e , u n p u b l i s h e d ) . Seawater c o l l e c t e d at 10 m from Ocean S t a t i o n P, c e n t r a l North P a c i f i c (49°55'N; 144°40'W) was . (R) screened through 5 um N i t e x w n e t t i n g and s t o r e d i n 2 0 - l i t r e p o l y e t h y l e n e carboys i n a dark, cold-room. The seawater was l a t e r r e f i l t e r e d through combusted Whatman GF/F f i l t e r s i n the l a b o r a t o r y . To determine the accuracy with which urea was measured, i n t e r n a l standards were run i n the n a t u r a l seawater samples. R e s u l t s St andar d cur v es Urea s o l u t i o n s prepared i n DDW, 3% NaCl, ESAW and c u l t u r e f i l t r a t e were analyzed by the d i a c e t y l monoxime ( F i g . 2A) and urease ( F i g . 2B) methods. Using the d i a c e t y l monoxime method, c a l i b r a t i o n curves were i n d i s t i n g u i s h a b l e and l i n e a r (r = 1.00) over a range of urea c o n c e n t r a t i o n s prepared i n DDW, 3% NaCl, ESAW and c u l t u r e f i l t r a t e . By c o n t r a s t , the slopes of the r e g r e s s i o n l i n e s u s ing the urease method were s i g n i f i c a n t l y d i f f e r e n t (p < 0.001). These urea standard curves were a l s o l i n e a r (r = 1.00) f o r urea s o l u t i o n s prepared i n DDW, 3% NaCl, ESAW and c u l t u r e f i l t r a t e . An average f a c t o r , F, c a l c u l a t e d f o r c o n c e n t r a t i o n s of d i s s o l v e d urea prepared i n DDW or 3% NaCl, was 6.23. In ESAW and c u l t u r e f i l t r a t e , the f a c t o r F was 12.4 and 24.6, r e s p e c t i v e l y . These d i s c r e p a n c i e s were not the r e s u l t of e r r o r s i n the NH 4 + a n a l y s i s . I t was shown i n subsequent experiments that ammonium standard curves prepared i n DDW, ESAW and c u l t u r e f i l t r a t e were i d e n t i c a l with urea standard curves prepared i n DDW or 3% NaCl. T h e r e f o r e , the urease method a c c u r a t e l y measured d i s s o l v e d urea c o n c e n t r a t i o n s only in DDW or 3% NaCl. A 2.50 uM urea standard prepared i n c u l t u r e f i l t r a t e was analyzed by the d i a c e t y l monoxime method, and a c o n c e n t r a t i o n of 2.52 ± 0.01 uM (h=3) urea was measured. The same standard was a l s o a n a l y z e d by the urease method; the urea c o n c e n t r a t i o n was determined to be 1.07 ± 0.02 uM (n=3). Small a l i q u o t s of the u r e a s e - t r e a t e d standard were reanalyzed by the d i a c e t y l monoxime method. I t was found t h a t urea was present i n these samples (1.55 + 0.05 uM; n=3), i n d i c a t i n g that the urease had not completely h y d r o l y z e d the urea to 21 F i g . 2 . Standard curves f o r urea determined by (A) automated d i a c e t y l monoxime method and (B) urease method, with urea standards i n d e i o n i z e d d i s t i l l e d water or 3% NaCl (A), i n a r t i f i c i a l seawater (•) and i n c u l t u r e f i l t r a t e (O). ammonium. pH effects The high pH of the c u l t u r e f i l t r a t e (pH 9.2) c o u l d e x p l a i n the incomplete h y d r o l y s i s of urea i n the samples a n a l y z e d by the urease method. The e f f e c t of pH on h y d r o l y s i s of urea i n ESAW and c u l t u r e f i l t r a t e , u s i ng the urease method, i s shown i n F i g u r e 3. The c o n c e n t r a t i o n of urease added to these samples was twice the c o n c e n t r a t i o n recommended by McCarthy (1970). H y d r o l y s i s of urea proceeded most completely at pH 7.2 i n both s o l u t i o n s ; however, there was l e s s urea h y d r o l y s i s i n c u l t u r e f i l t r a t e compared with ESAW. There was no e f f e c t of sample pH on the d i a c e t y l monoxime method. Heating time Time-course measurements of urea c o n c e n t r a t i o n a n a l y z e d by the urease method, as a f u n c t i o n of h e a t i n g time, are shown in F i g u r e 4. Complete h y d r o l y s i s of a 1.00 uM urea standard prepared i n 3% NaCl occ u r r e d a f t e r 5 min of h e a t i n g . Only 85% of the urea added to the n a t u r a l seawater samples was hy d r o l y z e d a f t e r 45 min h e a t i n g . Enzyme concentration To determine the e f f e c t of enzyme s t r e n g t h on urea h y d r o l y s i s , the a c t i v i t y of the enzyme s o l u t i o n was i n i t i a l l y determined (Table I ) . R e s u l t s demonstrated that the a c t i v i t i e s of the Worthington URC enzyme p r e p a r a t i o n s were l e s s than the manufacturer's s p e c i f i e d a c t i v i t i e s and a l l 2 4 F i g . 3. H y d r o l y s i s of urea i n a r t i f i c i a l seawater (dashed l i n e ) and c u l t u r e f i l t r a t e (continuous l i n e ) by urease, as a f u n c t i o n of seawater pH. Urease c o n c e n t r a t i o n was twice that recommended by McCarthy (1970). T o t a l amount of urea was determined by the d i a c e t y l monoxime method, and c o n c e n t r a t i o n s determined by the urease method are expressed as a percentage of t h i s v a l u e . E r r o r bars i n d i c a t e the range of d u p l i c a t e s . 2 5 26 F i g . 4. Urea hydrolyzed as a f u n c t i o n of h e a t i n g time of the urease-seawater samples at 50°C. A 1.00 uM urea standard was prepared i n 3% NaCl (•) and i n seawater samples c o l l e c t e d from the S t r a i t of Georgia (O). Samples had coo l e d to room temperature before the NH^ + measurements were made. C o n c e n t r a t i o n of urea i n each water sample was determined by the d i a c e t y l monoxime method, and values are repo r t e d as a percentage of t h i s v a l u e . 2 7 Table I Measured urease a c t i v i t y of enzyme stock s o l u t i o n s compared to t h e o r e t i c a l a c t i v i t y determined by manufacturer. Enzyme Type Date Date Measured T h e o r e t i c a l prepared measured a c t i v i t y a c t i v i t y (U-ml~ 1) (U-ml - 1) Worthington URC 15 .11 . 1 984 21 .XI . 1 984 500 720 Worthington URC 8 .VII.1984 21 .XI . 1 984 480 720 Worthington URC 1 6 .X. 1 984 21 .XI . 1 984 470 720 Sigma Type III 20 .1 . 1 983 21 .XI .1984 840 950 Sigma Type III 26 .XI . 1 984 27 .XI . 1 984 1860 2 One u n i t (U) of urease r e l e a s e s 1 umol NH^-min ; see " M a t e r i a l s and Methods" and " D i s c u s s i o n " f o r assay c o n d i t i o n s . Not a v a i l a b l e . 29 three batches were s i m i l a r . The enzyme p r e p a r a t i o n s were s t a b l e for p e r i o d s up to 9 months. In the case of the Sigma product, urease a c t i v i t y was s i m i l a r to the t h e o r e t i c a l a c t i v i t y f o r up to 22 mo. Using a range of urease c o n c e n t r a t i o n s (Sigma Type I I I ) , h y d r o l y s i s of a s i n g l e urea standard s o l u t i o n was measured ( F i g . 5). Incomplete h y d r o l y s i s of the urea standard was e v i d e n t at a l l enzyme a c t i v i t i e s . Upon c o o l i n g the samples, i t was e v i d e n t from the NH 4 + measurements that urea was s t i l l being h y d r o l y z e d ; i n no case was a l l of the urea-NH 4 + measured. At h i g h e r urease c o n c e n t r a t i o n s (1.9 to 2.3 U-ml 1 seawater), i t appeared as i f l i t t l e urea was being h y d r o l y z e d when the samples c o o l e d . In seawater samples c o n t a i n i n g lower enzyme c o n c e n t r a t i o n s ( l e s s than 1.4 U-ml 1 seawater), urea c o n t i n u e d to be h y d r o l y z e d du r i n g c o o l i n g ( F i g . 5). Specificity of diacetyl monoxime method To examine the s p e c i f i c i t y of the d i a c e t y l monoxime method, 29 organic and i n o r g a n i c n i t r o g e n compounds of v a r y i n g c o n c e n t r a t i o n were analyzed (Table I I ) . C i t r u l l i n e was the only compound which gave s i g n i f i c a n t i n t e r f e r e n c e . Three amino a c i d s gave p o s i t i v e r e a c t i o n s with the d i a c e t y l monoxime reagents; however, a f t e r t r e a t i n g these s o l u t i o n s with urease, urea c o u l d no longer be d e t e c t e d . A p p a r e n t l y , these samples had been contaminated with urea, and there was no evidence that the amino a c i d s were i n t e r f e r i n g with the t e c h n i q u e . 30 F i g . 5. Urea h y d r o l y z e d i n u r e a s e - t r e a t e d seawater samples, as a f u n c t i o n of f i n a l enzyme a c t i v i t y i n the urease seawater samples. Samples were c o l l e c t e d from Ocean S t a t i o n P and s p i k e d with 1.00 uM urea. I n c u b a t i o n s were conducted at 50°C f o r 20 min, and samples were c o o l e d for 0 (•) , 32 (O), and 76 min (•). Amount of urea h y d r o l y z e d was expressed as a percentage of the c o n c e n t r a t i o n determined by the d i a c e t y l monoxime method. 3 2 Table II Organic and i n o r g a n i c compounds t e s t e d f o r i n t e r f e r e n c e with the d i a c e t y l monoxime method. S o l u t i o n s were prepared i n d e i o n i z e d d i s t i l l e d water. Compound t e s t e d C o n c e n t r a t i o n Measured Urea t e s t e d (uM) cone. (uM) A l a n i n e 10 .0 < 0 . 0 2 5 Ammonium 5 0 0 . 0 < 0 . 0 2 5 A r g i n i n e 10 .0 < 0 . 0 2 5 Asparagine 10 .0 < 0 . 0 2 5 A s p a r t a t e 10 .0 < 0 . 0 2 5 C i t r u l l i n e 10 .0 11 . 7 ( 1 0 . 2 / Creat ine 1 0 0 0 . 0 0.64 C y s t e i n e 10 .0 < 0 . 0 2 5 Glutamine 10 .0 0 • 18 ( < 0 . 0 2 5 ) 1 Glutamate 10 .0 < 0 . 0 2 5 G l u t a t h i o n e 10 .0 < 0 . 0 2 5 Glyc ine 10 .0 < 0 . 0 2 5 H i s t i d i n e 10 .0 < 0 . 0 2 5 H y d r o x y p r o l i n e 10 .0 < 0 . 0 2 5 I s o l e u c ine 10 .0 0 . 1 5 ( < 0 . 0 2 5 ) L y s i n e 10 .0 < 0 . 0 2 5 Methionine 10 .0 < 0 . 0 2 5 N i t r a t e 5 5 0 0 . 0 < 0 . 0 2 5 N i t r i t e 10 .0 < 0 . 0 2 5 O r n i t h i n e 1 0 0 0 . 0 < 0 . 0 2 5 ( < 0 . 0 2 5 V P h e n y l a l a n i n e 10 .0 0 . 0 7 Potassium cyanate 1 0 0 0 . 0 0 . 6 8 S e r i n e 10 .0 < 0 . 0 2 5 Tr is(hydroxymethy1)amino- 8 0 0 0 . 0 < 0 . 0 2 5 methane Threonine 10 .0 < 0 . 0 2 5 Tryptophan 10 .0 < 0 . 0 2 5 T y r o s i n e 10 .0 < 0 . 0 2 5 Urac i 1 1 0 0 . 0 0.47 U r i c a c i d 1 0 0 . 0 0 . 5 0 C o n c e n t r a t i o n of urea measured a f t e r t r e a t i n g sample with urease. 33 Natural seawater samples Ammonium a n a l y s i s of samples of Ocean S t a t i o n P seawater (pH 8.32), spiked with v a r i o u s urease c o n c e n t r a t i o n s , i n d i c a t e d that some NH 4 + was l o s t from the samples dur i n g h e a t i n g . There was a l s o evidence t h a t the higher urease c o n c e n t r a t i o n s i n h i b i t e d the NH 4 + measurement (Table I I I ) . A urease c o n c e n t r a t i o n of 3 U-ml 1 seawater d i d not a l t e r the seawater pH and only s l i g h t l y a f f e c t e d the pH of the r e a c t i o n mixture i n the manual NH 4 + a n a l y s i s . There was no e f f e c t on the pH of the r e a c t i o n mixture i n the automated procedure. To a v o i d any confounding e f f e c t s from the N H 4 + determination step 1 4 of the urease assay, C-urea h y d r o l y s i s by urease was measured. 3% NaCl and Ocean S t a t i o n P seawater were spiked 14 14 with 0.5 uM C-urea, and the amount of CO2 r e l e a s e d by urease h y d r o l y s i s was determined. The r e s u l t s ( F i g . 6) i n d i c a t e d that i n 3% NaCl urea h y d r o l y s i s by urease was complete at enzyme c o n c e n t r a t i o n s g r e a t e r than 1.0 U-ml 1 ; c o n s i s t e n t with the p r e v i o u s r e s u l t . In the Ocean S t a t i o n P sample, urea h y d r o l y s i s i n c r e a s e d with i n c r e a s i n g urease c o n c e n t r a t i o n and was almost complete at the h i g h e s t enzyme c o n c e n t r a t i o n (3.0 U-ml 1 seawater). Seawater samples c o l l e c t e d from the S t r a i t of Georgia were analyzed by the d i a c e t y l monoxime and urease methods (Table I V ) . Standards were prepared i n 3% NaCl. The n u l l h y p o t h e s i s , i . e . , the c o n c e n t r a t i o n of urea determined by the urease method equals the c o n c e n t r a t i o n of urea determined by the d i a c e t y l monoxime method, was r e j e c t e d (p < 0.05, 34 Table III An Ocean S t a t i o n P seawater sample was spi k e d with 2.00 uM ammonium and d i v i d e d i n t o two p o r t i o n s . To one p o r t i o n , urease was added to give a f i n a l c o n c e n t r a t i o n of 2.3 U-ml 1 ; the other p o r t i o n served as a c o n t r o l . The samples were heated at 50°C f o r 20 min and the c o n c e n t r a t i o n of ammonium was measured immediately (0 min) and a f t e r the samples had c o o l e d (55 min). Incubations were conducted i n 125 ml Erlenmeyer f l a s k s covered with aluminum f o i l . Urease cone. Ammonium cone. (uM) (U-ml 1) 0 min 55 min 0.0 2.3 1 .91 1 .58 1 .84 1 .55 3 5 F i g . 6. H y d r o l y s i s of 0.50 uM C-urea i n 3% NaCl(») and Ocean S t a t i o n P seawater (O), as a f u n c t i o n of enzyme c o n c e n t r a t i o n . Samples were 50 ml and were heated f o r 20 min at 50°C. The r e a c t i o n was terminated once the samples had cooled to room temperature. The amount of urea h y d r o l y z e d i s expressed as a percentage of the 1 4 C -urea added, which was determined by l i q u i d s c i n t i l l a t i o n count i n g . T a b l e IV C o n c e n t r a t i o n (uM) of d i s s o l v e d urea i n seawater samples c o l l e c t e d from the S t r a i t of G e o r g i a , as determined by the d i a c e t y l monoxime and urease methods. Each value r e p r e s e n t s a s i n g l e measurement. S t a t i o n L o c a t i o n Depth Cone, as determined by (m) D i a c e t y l Urease monoxime method method T8 49°48.6'N; 0 0.28 0.25 0 2 1.01 0.82 124U50.7'W 3.5 0.46 0.26 7.5 0.57 0.56 18.5 0.52 0.33 T10 49°55.6'N; 0 0.33 0.12 0 1.5 0.16 0.15 125°02.3'W 5 0.55 0.29 A530b 49°53.0'N; 0 - 0.42 0.31 0 2 0.43 0.28 125U05.9'W 7.5 0.30 0.21 15 0.34 0.10 20 0.21 0.43 T14 49°53.4'N; 0 0.19 0.09 n 1.5 0.40 0.09 125 05.7'W 7 0.43 0.29 12 0.17 0.12 Tl83b 49°46.7'N; 2.5 0.25 0.21 _ 4.5 0.19 0.14 124U53.5'W 9 0.26 0.32 12 0.30 0.16 T8lb 49°48.6'N; 0 0.37 0.25 _ 2 0.37 0.26 124°50.7'W 4 0.15 0.13 13 0.08 0.25 Wilcoxon-matched-pair-signed-rank t e s t ) . The d i a c e t y l monoxime method measured s i g n i f i c a n t l y h i g h e r c o n c e n t r a t i o n s of urea i n these n a t u r a l seawater samples. Measurement of urea i n n a t u r a l seawater samples, s p i k e d with a known c o n c e n t r a t i o n of urea, demonstrated t h a t sampling l o c a t i o n and time of c o l l e c t i o n were important v a r i a b l e s a f f e c t i n g urea d e t e r m i n a t i o n by the urease method ( F i g . 7). With the exception of one seawater sample from E n g l i s h Bay, urea h y d r o l y s i s was incomplete i n the samples; moreover, i t was evident that as the samples c o o l e d urea was s t i l l b eing h y d r o l y z e d by the urease. A n a l y s i s of the same n a t u r a l seawater samples by the d i a c e t y l monoxime method demonstrated that urea was a c c u r a t e l y measured i n these samples r e g a r d l e s s of seawater type. Ocean S t a t i o n P seawater was spiked with v a r y i n g c o n c e n t r a t i o n s of urea, and the c o n c e n t r a t i o n s were determined by the d i a c e t y l monoxime and the urease methods ( F i g . 8 ) . S i n g l e d eterminations by the d i a c e t y l monoxime method were i n e x c e l l e n t agreement with the amount of urea added; whereas, the urease method underestimated the urea c o n c e n t r a t i o n s . Discussion pH effects The pH dependence of enzymes i s w e l l known, and f o r urease the pH optimum appears to be a f u n c t i o n of the b u f f e r system (Lynn 1967). F i s h b e i n (1969a) r e p o r t e d the pH optimum of jack bean urease to be 7.0; however, broad maxima have been 3 9 F i g . 7. Time course of h y d r o l y s i s of a 1.00 uM urea standard prepared i n n a t u r a l seawater c o l l e c t e d from E n g l i s h Bay, Vancouver, B r i t i s h Columbia ( A ) , and S t r a i t of Georgia ( S t a t i o n T3; 49°50.4'N; 125°00.5'W) at 15:47 (O ) and 22:30 hrs (•) and from Ocean S t a t i o n P (•). Urease-seawater samples were heated f o r 20 min at 50°C, and time on the a b s c i s s a r e f e r s to time f o l l o w i n g h e a t i n g , when samples were c o o l i n g at room temperature. C o n c e n t r a t i o n of urea i n each water sample was determined u s i n g the d i a c e t y l monoxime method, and values measured by the urease method are r e p o r t e d as a percentage of t h i s v a l u e . E r r o r bars i n d i c a t e the range of d u p l i c a t e s . 40 41 F i g . 8 . C o m p a r i s o n b e t w e e n t h e d i a c e t y l m o n o x i m e a n d u r e a s e m e t h o d s f o r m e a s u r i n g u r e a i n a n a t u r a l s e a w a t e r s a m p l e ( O c e a n S t a t i o n P ) s p i k e d w i t h k n o w n c o n c e n t r a t i o n s o f u r e a . U r e a c o n c e n t r a t i o n s w e r e m e a s u r e d u s i n g t h e d i a c e t y l m o n o x i m e m e t h o d (•) a n d t h e u r e a s e m e t h o d (*,o). T h e h e a t e d u r e a s e - s e a w a t e r s a m p l e s w e r e a l l o w e d t o c o o l a t r o o m t e m p e r a t u r e f o r 37 (o) a n d 104 (•) m i n . Urea Concentration Measured (pM) 43 r e p o r t e d ( F i s h b e i n et al. 1965), and the e x i s t e n c e of urease isozymes may be one e x p l a n a t i o n f o r these r e s u l t s . In ESAW, maximum urease a c t i v i t y occurred at pH 7.2 and, although t h i s was the lowest pH t e s t e d , an examination of lower pH v a l u e s was not warranted s i n c e they would be much l e s s than the normal pH of seawater. The pH of seawater i s l a r g e l y determined by the b i c a r b o n a t e / b o r a t e c o n c e n t r a t i o n . In the open ocean, the pH of s u r f a c e water i s between 8.1 and 8.3 (Sverdrup et al . 1942). While i n c o a s t a l r e g i o n s , pH values are more v a r i a b l e as a r e s u l t of freshwater i n f l u e n c e s and g r e a t e r primary p r o d u c t i v i t y ; they are t y p i c a l l y between 7.5 and 8.5. Recent measurements by Z i r i n o et al . (1983) r e v e a l e d pH v a l u e s as h i g h as 8.91 i n the Peruvian upwelling zone, and T u l l y and Dodimead (1957) recorded pH values of 8.8 i n s u r f a c e water of the S t r a i t of Georgia. T u l l y and Dodimead (1957) a l s o noted s p a t i a l v a r i a t i o n i n pH, and they a t t r i b u t e d these o b s e r v a t i o n s to patchy b i o l o g i c a l a c t i v i t y and complex mixing p a t t e r n s i n the s t r a i t . The r e s u l t s of t h i s study f i r m l y e s t a b l i s h the importance of pH i n the h y d r o l y s i s of urea i n a r t i f i c i a l seawater s o l u t i o n s . The o b s e r v a t i o n s imply that pH may be an important parameter i n f l u e n c i n g urea h y d r o l y s i s i n n a t u r a l seawater samples. S t r i c k l a n d and Parsons (1972, p. 91-95) p o i n t e d out the p o t e n t i a l problem of high pH i n freshwater, and suggested that the pH should be a d j u s t e d i f necessary. I concur with t h i s s u g g e s t i o n ; however, in order to a v o i d i n h i b i t i o n of the indophenol blue r e a c t i o n used to determine NH. +, s p e c i a l c a r e 44 should be taken i n a d j u s t i n g the pH s i n c e t h i s r e a c t i o n i s h i g h l y pH-dependent (Harwood and Huyser 1970). Tris® b u f f e r i n t e r f e r e s with the p h e n o l - h y p o c h l o r i t e method and i s not s u i t a b l e f o r b u f f e r i n g (McCarthy 1970). To my knowledge, there are no p u b l i s h e d r e p o r t s of the e f f e c t s of Good's b u f f e r s (Good et al . 1966) on the p h e n o l - h y p o c h l o r i t e method, but MOPS [3-(N-morpholino) p r o p a n e s u l f o n i c a c i d ] causes no i n t e r f e r e n c e at the 5 mM l e v e l ( C A . S u t t l e , p e r s o n a l communication). Urea hydrolys i s Urea was inc o m p l e t e l y h y d r o l y z e d i n ESAW, and the amount of urea h y d r o l y z e d was not c o n s i s t e n t between samples (see F i g s . 2 and 3). The c o n c e n t r a t i o n of urease added to the samples was d i f f e r e n t between these experiments; b e t t e r r e s u l t s were o b t a i n e d with a higher enzyme c o n c e n t r a t i o n . T h i s d i s c r e p a n c y can a l s o be a t t r i b u t e d to d i f f e r e n c e s i n sample pH and r a t e s of c o o l i n g between the samples, important f a c t o r s I was unaware of i n i t i a l l y . I t has a l s o been reported that the s p e c i f i c a c t i v i t y of a c o n c e n t r a t e d urease s o l u t i o n , d i l u t e d with the same s o l v e n t , i n c r e a s e s with time a f t e r d i l u t i o n (Peterson et al. 1948). T h i s was not c o n t r o l l e d d u r i n g the experiments. T h i s o b s e r v a t i o n suggests that the amount of urea h y d r o l y z e d may depend upon the time the working urease p r e p a r a t i o n {sensu McCarthy 1970) has been prepared, and when i t i s added to the seawater sample. Urease i s s e n s i t i v e to heavy metals (Hughes et al. 1969, Olson and C h r i s t e n s e n 1982) and i s a l s o i n h i b i t e d by Na + and K + (Fasman and Niemann 1951) at c o n c e n t r a t i o n s s i m i l a r to those found i n seawater. The l a t t e r o b s e r v a t i o n c o u l d e x p l a i n the incomplete h y d r o l y s i s of urea i n ESAW, except that urea h y d r o l y s i s proceeds q u i c k l y i n 3% NaCl s o l u t i o n s . An examination of the i n d i v i d u a l reagent-grade s a l t s of a r t i f i c i a l seawater demonstrated that urea h y d r o l y s i s proceeds most sl o w l y and i n c o m p l e t e l y i n s o l u t i o n s of MgCl 2 and C a C l 2 ( P r i c e , 2 + u n p u b l i s h e d r e s u l t s ) . Urease i n h i b i t i o n by Ca has been p r e v i o u s l y r e p o r t e d (Olson and C h r i s t e n s e n 1982). U s i n g the urease method, h y d r o l y s i s of a 1.00 uM urea standard, prepared i n n a t u r a l seawater, was incomplete f o l l o w i n g 20 min at 50°C. These data are i n disagreement with McCarthy (1970) who reported complete h y d r o l y s i s of 7.5 uM urea under the same assay c o n d i t i o n s ; the composition of h i s sample s o l u t i o n was not r e p o r t e d . The r e s u l t s from t h i s study i n d i c a t e t h a t only urea standards prepared i n DDW or 3% NaCl are completely hydrolyzed under these c o n d i t i o n s . The e f f i c i e n c y of recovery of i n t e r n a l standards of urea from n a t u r a l seawater was dependent upon seawater type. T h i s o b s e r v a t i o n i n v a l i d a t e s the p r e v i o u s assumption that there i s a c o n s t a n t recovery of urea from seawater samples. To c o r r e c t f o r t h i s , i t i s advised that i n t e r n a l standards be run with each sample. If i n t e r n a l standards are p r o p e r l y used, i t may be p o s s i b l e to determine a c c u r a t e l y d i s s o l v e d urea c o n c e n t r a t i o n s i n seawater with the urease method. The r e s u l t s from t h i s study have a l s o shown that there i s a time-dependence of the urease method, which i s a r e s u l t of. the 46 continued h y d r o l y s i s of urea i n u r e a s e - t r e a t e d samples d u r i n g c o o l i n g . In my experimental p r o t o c o l , samples were allowed to c o o l as suggested by McCarthy (1970). Depending on a i r c u r r e n t s and other f a c t o r s , samples that c o o l e d p a s s i v e l y reached room temperature ca. 30 min a f t e r t h e i r removal from the h e a t i n g bath. The independent e s t i m a t e s of urease a c t i v i t y are i n c l o s e agreement with the manufacturers' s p e c i f i c a t i o n s and are evidence of an a c t i v e enzyme p r e p a r a t i o n . The d i s c r e p a n c i e s , p a r t i c u l a r l y with the Worthington URC product, are most l i k e l y a r e s u l t of d i f f e r e n t enzyme assay c o n d i t i o n s . Worthington determines urease a c t i v i t y i n a glutamate dehydrogenase-coupled system i n phosphate b u f f e r , pH 7.6, at 25°C; whereas, 14 14 the enzyme assay I used measured C 0 2 r e l e a s e from C-urea. McCarthy and Kamykowski (1972) have a l s o r e p o r t e d r e c e i v i n g two batches of the Worthington product which y i e l d e d l e s s a c t i v i t y and a higher blank than i n the i n i t i a l p u b l i c a t i o n of McCarthy (1970). F o l l o w i n g t h i s , they suggested using Sigma Type II I urease as the enzyme source f o r the urease method. Both types of urease enzyme p r e p a r a t i o n s were u n s a t i s f a c t o r y f o r o b t a i n i n g complete h y d r o l y s i s of urea i n seawater. However, complete r e c o v e r y of urea i n one seawater sample c o l l e c t e d from E n g l i s h Bay was demonstrated, and i d e n t i c a l r e s u l t s were o b t a i n e d by the d i a c e t y l monoxime technique. In four seawater samples from the S t r a i t of Georgia, urea c o n c e n t r a t i o n s were s i m i l a r when determined by both, techniques (Table IV: S t a t i o n T8, 0 and 7.5 m; S t a t i o n T10, 1.5 m; S t a t i o n T 8 l b , 4 m), and i n three other samples the urease method y i e l d e d higher c o n c e n t r a t i o n s of urea than the d i a c e t y l monoxime method (Table IV: S t a t i o n A530b, 20 m; S t a t i o n Tl83b, 9 m; S t a t i o n T 8 l b , 13 m). Reasons f o r t h i s l a t t e r o b s e r v a t i o n are not c l e a r , but h y d r o l y s i s of d i s s o l v e d o r g a n i c n i t r o g e n compounds in the seawater by urease may be one e x p l a n a t i o n (see below " S p e c i f i c i t y of methods"). Sped fi ci t y of methods Although the d i a c e t y l monoxime method i s not s p e c i f i c f o r urea, there was no evidence of i n t e r f e r e n c e from c i t r u l l i n e i n seven c o a s t a l seawater samples ( S t a t i o n T8, 3.5, 7.5, and 18 m; S t a t i o n A530b, 0 and 2 m; S t a t i o n T14, 1.5 and 7 m). These data support a s i m i l a r c o n c l u s i o n made by Aminot and Keroeul (1982) who analyzed 20 seawater samples from the E n g l i s h Channel. Urease, long c o n s i d e r e d to be an a b s o l u t e l y s p e c i f i c enzyme f o r urea, i s now known to c a t a l y z e h y d r o l y s i s of other compounds producing NH 4 + as an end product ( F i s h b e i n et al . 1965, F i s h b e i n 1969b, Sundaram and L a i d l e r 1970). Of the many urease i n h i b i t o r s (see f o r example Olson and C h r i s t e n s e n 1982), hydroxamic a c i d s are extremely potent (Kobashi et al . 1962, Gale and A t k i n s 1969, Dixon et al . 1975b). A number of organisms are known to produce hydroxamic a c i d s , i n c l u d i n g c y a n o b a c t e r i a (Estep et a l . 1975, Armstrong and Van Baalen 1979) and e u k a r y o t i c phytoplankton ( T r i c k et a l . 1983). However, the presence or absence of these compounds as d i s s o l v e d c o n s t i t u e n t s i n seawater i s u n c l e a r . In phytoplankton c u l t u r e f i l t r a t e , measured urea 4 8 c o n c e n t r a t i o n s were l e s s than i n ESAW even a f t e r c o r r e c t i o n s were made f o r pH ( F i g . 3). A p o s s i b l e e x p l a n a t i o n i s that e x t r a c e l l u l a r m e t a b o l i t e s produced by the phytoplankton (or b a c t e r i o p l a n k t o n ) may have i n h i b i t e d the urease. A number of a d d i t i v e f a c t o r s are most l i k e l y r e s p o n s i b l e f o r the low a c t i v i t y - o f urease i n seawater, u l t i m a t e l y r e s u l t i n g i n the incomplete h y d r o l y s i s of urea d u r i n g the urease assay. Analysis of natural seawater I n c r e a s i n g the urease c o n c e n t r a t i o n ' d i d not r e s u l t i n complete r e c o v e r y of urea added to n a t u r a l seawater samples. 14 14 A n a l y s i s of CC>2 e v o l v e d from C-urea demonstrated that urea h y d r o l y s i s proceeds more co m p l e t e l y at the. higher urease c o n c e n t r a t i o n s than i s i n d i c a t e d by the NH^ + measurement. Nonetheless, by u s i n g g r e a t e r enzyme c o n c e n t r a t i o n s I f a i l e d to measure a l l of the urea which had been added to the sample. These data suggest t h a t the h i g h urease enzyme c o n c e n t r a t i o n s are r e s p o n s i b l e f o r a f f e c t i n g the NH^ + determination step i n the urease assay. D i r e c t measurement of NH^+ i n the presence of high l e v e l s of urease confirmed t h i s , although I found the r e s u l t s were v a r i a b l e . Loss of NH 4 + d u r i n g heating was a minor f a c t o r c o n t r i b u t i n g to the lower urea measurements by the urease assay. Using an e m p i r i c a l l y d e r i v e d s t a b i l i t y constant of the e q u i l i b r i u m , NH^ + H + = NH 4 + (Johansson and Wedborg 1980), i t was determined that ca. 40% of t o t a l NH^ and NH 4 + i n a 50°C-heated seawater sample, pH 8.3, w i l l be i n the NH_ form. T h i s NH-. gas, d i s s o l v e d i n the seawater, r e p r e s e n t s a s i g n i f i c a n t p o r t i o n of the t o t a l NH 3 and NH 4 +. The NH 3 has the p o t e n t i a l to d i f f u s e out of s o l u t i o n , thereby d e c r e a s i n g the NH 3 and NH 4 + c o n c e n t r a t i o n i n the water. The use of screw-capped tubes with l i t t l e a i r space above the seawater would help d i m i n i s h l o s s of NH 3. F i n a l l y , i t has been noted by s e v e r a l i n v e s t i g a t o r s that urease a c t i v i t y i s dependent upon enzyme c o n c e n t r a t i o n . In d i l u t e s o l u t i o n s , the s p e c i f i c a c t i v i t y per u n i t weight of enzyme i s g r e a t e r than i n more concentrated s o l u t i o n s (Hofstee 1948, Peterson et al . 1 948, Ambrose et al . 1950). M o d i f i c a t i o n s to the urease method of McCarthy (1970) have been p u b l i s h e d (McCarthy and Kamykowski 1972, S t r i c k l a n d and Parsons 1972, Parsons et al. 1984b). These procedures have with other changes, i n c r e a s e d the amount of enzyme added to the sample. U n f o r t u n a t e l y , there has been no emphasis on using a s p e c i f i e d a c t i v i t y of urease f o r the h y d r o l y s i s of urea. Enzymes purchased from Worthington and Sigma may vary in a c t i v i t y by as much as t w o - f o l d between batches. For t h i s reason, the a c t i v i t y of d i a l y z e d urease may vary s i m i l a r l y . I n a t t e n t i o n to t h i s f a c t may r e s u l t i n enzyme s o l u t i o n s of extremely low a c t i v i t y , and consequently, urea h y d r o l y s i s i n the seawater samples w i l l be very much reduced. When urease was added at c o n c e n t r a t i o n s suggested by McCarthy (1970) {ca. 0.7 U-ml~ 1) i t c o n t r i b u t e d 0.17 to 0.25 uM NH 4 + to the seawater samples. These c o n c e n t r a t i o n s f o r the NH^ + blank are s l i g h t l y g r e a t e r than those o b t a i n e d by McCarthy (1970). I f higher c o n c e n t r a t i o n s of urease are added to the samples, f o r example 2.5 U-ml 1 , the amount of 5 0 background NH 4 + w i l l i n c r e a s e p r o p o r t i o n a t e l y to 0.6 to 0 . 9 uM NH 4 +. U n c e r t a i n t y i n the urea measurement w i l l a l s o i n c r e a s e , because t h i s NH^ + c o n c e n t r a t i o n , which may be many times g r e a t e r than the a c t u a l urea c o n c e n t r a t i o n , must be s u b t r a c t e d to c a l c u l a t e the urea c o n c e n t r a t i o n . A number of r e s e a r c h e r s have supported the use of e i t h e r the d i a c e t y l monoxime (Carpenter et al. 1972a, Nakas and L i t c h f i e l d 1977) or urease (McCarthy 1970, S t r i c k l a n d and Parsons 1972, Parsons et al . 1984) methods f o r measuring urea c o n c e n t r a t i o n s ; however, t h e i r c o n c l u s i o n s were not based on p u b l i s h e d d a t a . K r i s t i a n s e n (1983) found no s i g n i f i c a n t d i f f e r e n c e between the d i a c e t y l monoxime method of Newell et al. (1967) and the urease method of McCarthy (1970), but he only t e s t e d seawater from two s t a t i o n s w i t h i n O s l o f j o r d , and he d i d not run i n t e r n a l standards. These data demonstrate t h a t , i n g e n e r a l , the urease method measures lower c o n c e n t r a t i o n s of d i s s o l v e d urea, i n a v a r i e t y of seawater samples, than the d i a c e t y l monoxime method. In c o n c l u s i o n , the de t e r m i n a t i o n of urea c o n c e n t r a t i o n s i n seawater by the d i a c e t y l monoxime procedure i s acc u r a t e and r e p r o d u c i b l e . T h i s method i s recommended i n pr e f e r e n c e to the urease method. The d i a c e t y l monoxime method i s s u i t a b l e f o r urea measurements i n a r t i f i c i a l and n a t u r a l seawater samples. I t i s only s u b j e c t to i n t e r f e r e n c e by c i t r u l l i n e , an organic n i t r o g e n compound t h a t appears to be absent from the c o a s t a l seawater samples I examined. The method d e s c r i b e d i s an automated procedure, a l t h o u g h a manual a p p l i c a t i o n of t h i s 51 method i s p u b l i s h e d (Rahmatullah and Boyde 1980). The urease method r e q u i r e s a more complex b l a n k i n g c o r r e c t i o n , and the r e s u l t s from t h i s i n v e s t i g a t i o n suggest that i n t e r n a l standards must be run with each sample. Other f a c t o r s are a l s o important f o r measuring urea by the urease technique and they must be r i g o r o u s l y c o n t r o l l e d . I n c r e a s i n g the h e a t i n g time or the c o n c e n t r a t i o n of urease improves the r e s u l t s . These data demonstrate that a constant r e c o v e r y of urea cannot be assumed with the urease technique, and that seawater type and pH are important f a c t o r s a f f e c t i n g the a c t i v i t y of urease i n t h i s assay. S u m m a r y A comparison between the d i a c e t y l monoxime and urease methods f o r measuring d i s s o l v e d c o n c e n t r a t i o n s of urea i n seawater was conducted i n a r t i f i c i a l seawater, phytoplankton c u l t u r e f i l t r a t e and both n a t u r a l and u r e a - s p i k e d f i e l d samples from c o a s t a l and oceanic environments d u r i n g 1984. The urease technique underestimated urea c o n c e n t r a t i o n s i n unbuffered phytoplankton c u l t u r e f i l t r a t e . Incomplete h y d r o l y s i s of urea i n these samples was a r e s u l t of the i n h i b i t i o n of the urease enzyme. F a c t o r s r e s p o n s i b l e f o r i n h i b i t i n g urease i n c l u d e d pH, seawater i o n s , and p o s s i b l y e x t r a c e l l u l a r m e t a b o l i t e s produced i n u n i a l g a l c u l t u r e s . Seawater type and time of sample c o l l e c t i o n were important v a r i a b l e s a f f e c t i n g urea measurement by the urease method, and recovery of i n t e r n a l standards ranged from 40 to 100%. In c r e a s i n g the h e a t i n g time of the urease assay, or the c o n c e n t r a t i o n of urease added to the seawater samples, i n c r e a s e d the amount of urea determined by the urease method. However, measured v a l u e s were s t i l l l e s s than the c o n c e n t r a t i o n of the urea i n t e r n a l standards. The d i a c e t y l monoxime method was s u i t a b l e f o r urea d e t e r m i n a t i o n s i n a l l the seawater samples examined; i t was e a s i l y automated, and the r e s u l t s were a c c u r a t e and r e p r o d u c i b l e . T h i s m o d i f i e d technique i s recommended f o r measuring d i s s o l v e d c o n c e n t r a t i o n s of urea i n seawater. 53 C H A P T E R 2. T I M E C O U R S E O F U P T A K E O F I N O R G A N I C A N D O R G A N I C  N I T R O G E N BY P H Y T O P L A N K T O N I N T H E S T R A I T O F G E O R G I A :  C O M P A R I S O N O F F R O N T A L A N D S T R A T I F I E D C O M M U N I T I E S B a c k g r o u n d Shallow sea f r o n t s are areas of hig h primary p r o d u c t i v i t y (Pingree et al . 1975, Parsons et a l . 1981, 1983, H o l l i g a n et al. 1984) l o c a t e d at the boundary between mixed and s t r a t i f i e d water. These r e g i o n s are c h a r a c t e r i z e d by having h i g h phytoplankton biomass i n s u r f a c e water, with measurable c o n c e n t r a t i o n s of d i s s o l v e d n i t r a t e , and a shallow p y c n o c l i n e which extends to the s u r f a c e at the f r o n t a l boundary (e.g. Simpson and Pingree 1978). In the S t r a i t of Geor g i a , a c o a s t a l b a s i n o f f the west co a s t of Canada, s e v e r a l t i d a l l y -induced f r o n t a l r e g i o n s have been d e s c r i b e d (Parsons et al. 1981). The n i t r o g e n dynamics of f r o n t a l r e g i o n s has r e c e i v e d l i t t l e a t t e n t i o n . Floodgate et al. (1981) measured urea decomposition r a t e s i n f r o n t a l waters of the I r i s h Sea. These r a t e s were g r e a t e r than r a t e s i n mixed and s t r a t i f i e d water, and were concomitant with higher d i s s o l v e d urea c o n c e n t r a t i o n s . High r a t e s of carbon and n i t r a t e uptake have been observed i n the p r o x i m i t y of a f r o n t by Parsons et al. (1984a). H o l l i g a n et al. (1984) c a l c u l a t e d t hat ammonium e x c r e t i o n by zooplankton c o u l d account f o r g r e a t e r than 50% of the p o t e n t i a l phytoplankton requirements on the s t r a t i f i e d s i d e of a f r o n t , but n e i t h e r uptake nor r e g e n e r a t i o n were measured. I t i s apparent that a d d i t i o n a l i n f o r m a t i o n i s r e q u i r e d i n order to d e s c r i b e and understand the n i t r o g e n 54 c y c l i n g between d i s s o l v e d and p a r t i c u l a t e components i n these a r e a s . A s u r f a c e t r a n s e c t normal to a f r o n t a l boundary p r o g r e s s e s from a r e g i o n of high n i t r a t e c o n c e n t r a t i o n on the mixed s i d e to n i t r o g e n - d e p l e t e d water on the s t r a t i f i e d s i d e . T h i s t r a n s e c t r e p r e s e n t s a g r a d i e n t of n i t r o g e n a v a i l a b i l i t y and hence phytoplankton n u t r i t i o n a l s t a t e s . Most of the n i t r o g e n demands of phytoplankton i n n i t r o g e n - i m p o v e r i s h e d water are s u p p l i e d by ammonium and urea from r e g e n e r a t i v e p r o c e s s e s ; whereas, i n n i t r o g e n - r i c h areas n i t r o g e n compounds appear to be u t i l i z e d at r a t e s p r o p o r t i o n a l to t h e i r a v a i l a b i l i t y (e.g. Dugdale and Goering 1967, McCarthy et al . 1977). In the n i t r o g e n - r i c h r e g i o n s , n i t r a t e s u p p l i e s most of the phytoplankton n i t r o g e n requirements by v i r t u e of it's abundance. Experiments using l a b o r a t o r y c u l t u r e s of phytoplankton have demonstrated that the p r e c o n d i t i o n i n g n i t r o g e n s u b s t r a t e a f f e c t s the response of phytoplankton to the a d d i t i o n s of d i f f e r e n t forms of n i t r o g e n (Horrigan and McCarthy 1981,1982, Dortch and Conway 1984). In n i t r o g e n - s t a r v e d phytoplankton, the a b i l i t y to take up n i t r a t e may be l o s t and must be induced (Dortch et al . 1982, review by C o l l o s 1983, Parslow et a l . 1984b). These o b s e r v a t i o n s suggest that phytoplankton communities i n f r o n t a l and s t r a t i f i e d water may d i f f e r i n t h e i r response to p e r t u r b a t i o n s of n i t r o g e n by t h e i r p r e f e r e n c e f o r , and uptake r a t e s of, d i f f e r e n t n i t r o g e n s u b s t r a t e s . 55 Previous n i t r o g e n uptake experiments have i n v o l v e d s i n g l e 1 5 end-point measurements of accumulated N - l a b e l l e d s u b s t r a t e s in p a r t i c u l a t e matter over long time i n t e r v a l s (Goldman et al . 1981, review by H a r r i s o n 1983a). In theory, these experiments p r o v i d e important i n f o r m a t i o n c o n c e r n i n g d a i l y r a t e s of n i t r o g e n u t i l i z a t i o n , as they i n v a r i a b l y take i n t o account d i e l p a t t e r n s of uptake ( p r o v i d i n g they are of 24 h d u r a t i o n ) . The e x i s t e n c e of uptake p e r i o d i c i t y has been rep o r t e d f o r c y c l o s t a t c u l t u r e s of Skeletonema cost at um (Eppley et al . 1971b) and Chaet oceros sp. (Malone et al. 1975), and n a t u r a l phytoplankton communities (e.g. Goering et al . 1964, Eppley et al . 1970, 1971a, McCarthy and Eppley 1972, Maclsaac 1978, F i s h e r et al. 1982). The experiments p r e s e n t e d i n t h i s chapter were designed to examine the time course of n i t r o g e n uptake by phytoplankton from n i t r a t e - d e p l e t e s t r a t i f i e d water and n i t r a t e - r e p l e t e f r o n t a l water, over a 24 h c y c l e . The response of the phytoplankton to a d d i t i o n s of ammonium, n i t r a t e , and urea was measured i n these two r e g i o n s . From measurements of nit r o g e n 1 5 . uptake r a t e u s i n g N i s o t o p e i n c o r p o r a t i o n , and changes i n the c o n c e n t r a t i o n of d i s s o l v e d n i t r o g e n over time, r e g e n e r a t i o n r a t e s of ammonium and urea by i n t a c t plankton communities were c a l c u l a t e d . Measured p a r t i c u l a t e n i t r o g e n c o n c e n t r a t i o n s were compared with t h e o r e t i c a l values c a l c u l a t e d from the changes i n the c o n c e n t r a t i o n s of 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 urea. These r e s u l t s are d i s c u s s e d w i t h i n the c u r r e n t concepts of n i t r o g e n c y c l i n g i n marine p l a n k t o n i c ecosystems. 56 M a t e r i a l s and Methods Sampl e c o l l e c t i o n Three 24 h time course experiments were conducted i n the S t r a i t of Georgia, B.C., Canada aboard the C.S.S. 'Vector' ( J u l y 1984); s t a t i o n l o c a t i o n s f o r Time Courses 1, 2 and 3 are shown i n F i g u r e 9, and a d e s c r i p t i o n of the sample s t a t i o n s i s give n i n Table V. At approximately 0900 h, water samples were c o l l e c t e d from a depth c o r r e s p o n d i n g to 50% of the s e a - s u r f a c e i r r a d i a n c e u s ing 5 - l i t r e PVC N i s k i n b o t t l e s and t r a n s f e r r e d i n t o a 2 0 - l i t r e Nalgene carboy. Chemical, physical and b i o l o g i c a l analyses Subsamples fo r n u t r i e n t a n a l y s e s were f i l t e r e d through combusted (460°C fo r 4 h) Whatman GF/F f i l t e r s using an a c i d -washed s y r i n g e and a 25 mm M i l l i p o r e Swinex w f i l t e r h o l d e r . Samples were g e n t l y f i l t e r e d i n t o acid-washed p o l y e t h y l e n e b o t t l e s and were e i t h e r analyzed immediately f o r 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 (DIN) as ammonium (NH 4 +) and n i t r a t e (NO^ + NC>2 ), and urea c o n c e n t r a t i o n s or f i l t e r e d samples were s t o r e d f r o z e n (-20°C), and analyzed w i t h i n 24 h. NH^ + and N0 3 — R + N0 2 were measured with a Technicon Autoanalyzer II f o l l o w i n g the procedures o u t l i n e d i n Slawyk and Maclsaac (1972) and Wood et al . (1967), r e s p e c t i v e l y . Urea was determined by the d i a c e t y l monoxime t h i o s e m i c a r b i z i d e technique d e s c r i b e d i n Chapter 1. D u p l i c a t e samples f o r c h l o r o p h y l l a ( c h i a) ( c o e f f i c i e n t of v a r i a t i o n , CV, = 4.4 ± 9. S t a t i o n l o c a t i o n s f o r Time Course 1 (T3, f r o n t a l s t a t i o n ) , Time Course 2 (A5, f r o n t a l s t a t i o n ) and Time Course 3 (T4, s t r a t i f i e d s t a t i o n ) . 58 59 Tabl e V L o c a t i o n of s t a t i o n s and time of sampling, Sta t ion and Locat ion D e s c r i p t i o n Date Sample Sample time depth (m) T3 49 50.42'N; F r o n t a l 24.VII.1984 0730 125^00.54'W A5 49 53.02'N; F r o n t a l 28.VII.1984 1000 125^05.48'W T4 49 U55.30'N; S t r a t i f i e d 29.VII.1984' 0800 124^55.30'W 4.1%; 5 data p a i r s ) were c o l l e c t e d on Whatman GF/F f i l t e r s and st o r e d frozen i n a d e s i c c a t o r . Chi a was e x t r a c t e d i n 90% acetone and analyzed by in vitro fluorometry ( S t r i c k l a n d and Parsons 1972) using a Turner Designs model 10 f l u o r o m e t e r . P a r t i c u l a t e organic carbon and n i t r o g e n (POC and PON) (CV = 5.2 + 4.8% and 3.8 + 4.1%; 7 d a t a . p a i r s ) , c o l l e c t e d on combusted Whatman GF/F f i l t e r s , were s t o r e d s i m i l a r l y and analyzed l a t e r with a P e r k i n Elmer model 240 elemental a n a l y z e r . V e r t i c a l p r o f i l e s of temperature and s a l i n i t y were obtained from continuous p r o f i l e s , run p r i o r to b o t t l e sampling, using an Interocean CTD system. In vi vo c h i a f l u o r e s c e n c e was measured simultaneously with a Turner model 111 fluorometer. I n c i d e n t s o l a r i r r a d i a n c e (P.A.R.) was monitored c o n t i n u o u s l y with a Lambda Instruments Ll-185 l i g h t meter equipped with a LI-190S Surface Quantum Sensor. Phytoplankton s p e c i e s samples (250 ml) were p r e s e r v e d i n Lugol's s o l u t i o n and 10 ml subsamples were s e t t l e d and counted on an i n v e r t e d microscope; 100 ml subsamples were examined fo r microzooplankton. Nitr ogen uptake Within 1 h of c o l l e c t i o n , water was t r a n s f e r r e d i n t o 500 ml Wheaton g l a s s b o t t l e s ( c l e a r : l i g h t b o t t l e s , or darkened with black tape: dark b o t t l e s ) with t e f l o n - l i n e d caps and s a t u r a t i n g a d d i t i o n s of e i t h e r 1 5NH 4C1, N a 1 5 N 0 3 or C O ( 1 5 N H 2 ) 2 ( a l l 99 at % 1 5N) were added. In Time course 1, 2 ug at N - l ~ 1 1 5 - 15 of N0 3 or C0( N H 2^2 w a s a d d e d a n d i n T i m e Courses 2 and 3, 6 ug at N - l ~ 1 of 1 5 N H 4 + , 1 5 N 0 3 ~ or C O ( 1 5 N H 2 ) 2 was added. The 61 p r e c i s i o n (+ 1 SD) of the n u t r i e n t d e terminations f o r the time-zero samples was ± 0.09 ug-at N-1 1 (n=5) f o r NH 4 +, ± 0.07 ug-at N - 1 - 1 (n=5) f o r N0 3~ and + 0.02 ug at N - l ~ 1 (n=4) for urea. L i g h t and dark b o t t l e uptake r a t e s of each n i t r o g e n s u b s t r a t e were measured over the time course, and a l l sample b o t t l e s were mixed h o u r l y . Time-zero samples f o r d i s s o l v e d n i t r o g e n were withdrawn immediately and analyzed f o r NH 4 +, NC>2 and urea c o n c e n t r a t i o n s i n a l l b o t t l e s . Incubations were conducted under n a t u r a l l i g h t i n c l e a r Plexiglas® deck i n c u b a t o r s , c o o l e d with s u r f a c e seawater and covered with n e u t r a l d e n s i t y s c r e e n i n g to simulate the i r r a d i a n c e at the 50% l i g h t depth. At 3 h i n t e r v a l s , p a r t i c u l a t e matter from d u p l i c a t e samples was c o l l e c t e d by f i l t r a t i o n (vacuum l e s s than 125 mm Hg) onto combusted Whatman GF/F f i l t e r s and s t o r e d f r o z e n i n a d e s i c c a t o r . Samples f o r d i s s o l v e d n i t r o g e n c o n c e n t r a t i o n s were taken c o n c u r r e n t l y , and samples f o r c h l a, and POC and PON were taken every 6 h. I t i s important to note that samples were taken from randomly s e l e c t e d i n c u b a t i o n b o t t l e s and c h l a and POC and PON samples were taken from 1 5 b o t t l e s d i s t i n c t from those analyzed f o r N atom % excess i n p a r t i c u l a t e matter and d i s s o l v e d n i t r o g e n c o n c e n t r a t i o n s . Calculations N i t r o g e n i n the p a r t i c u l a t e samples was converted to N 2 (g) by the micro-Dumas dry combustion technique as d e s c r i b e d 1 5 by LaRoche (1983). Samples were analyzed f o r N enrichment with a JASCO model N-150 emission spectrometer ( F i e d l e r and Proksch 1975). N i t r o g e n uptake r a t e s were c a l c u l a t e d a c c o r d i n g to the equations of Dugdale and Goering (1967) and are presented as n i t r o g e n - s p e c i f i c (h 1) and a b s o l u t e (ug at N-1 1-h 1) r a t e s . The r a t i o of n i t r o g e n uptake i n the dark b o t t l e (continuous darkness) to that i n the l i g h t b o t t l e (exposed to the n a t u r a l l i g h t c y c l e ) (V D:V" L) i s a l s o r e p o r t e d . Ammonium and urea r e g e n e r a t i o n r a t e s (d) have been determined using the approach of F i s h e r et al . (1981). These r a t e s were c a l c u l a t e d u sing the Blackburn-Caperon equation (Blackburn 1979, Caperon et al . 1979) d = P/t + i where P = change i n c o n c e n t r a t i o n of NH^* or urea (ug at N-1 1) over time i n t e r v a l t (h), and i = n i t r o g e n uptake rate — 1 — 1 1 c (ug at N-1 -h ) c a l c u l a t e d from N accumulation i n the p a r t i c u l a t e matter (Dugdale and Goering 1967). Disappearance uptake r a t e s (V ) were c a l c u l a t e d from the change i n co n c e n t r a t i o n of d i s s o l v e d n i t r o g e n per u n i t time (AP/t) and, 15 i l i k e the n i t r o g e n - s p e c i f i c and a b s o l u t e N uptake r a t e s (V ), are reported f o r the time i n t e r v a l s over which they have been c a l c u l a t e d . Results St ati on p r o f i l e s The v e r t i c a l p r o f i l e s of temperature, r e l a t i v e in vivo c h i a f l u o r e s c e n c e , and NO^ c o n c e n t r a t i o n f o r the f r o n t a l water s t a t i o n s (Time Course 1 [T3]; and Time Course 2 [A5]) 63 showed almost i d e n t i c a l trends with depth; thus only the sy n o p t i c p r o f i l e of Time Course 2 i s presented ( F i g . 10A). -Throughout the R e s u l t s and D i s c u s s i o n , r e f e r e n c e to f r o n t a l water p e r t a i n s t o Time Course 2 unless s p e c i f i e d otherwise. The r e s u l t s of Time Course 1 are d i s c u s s e d only b r i e f l y because of the p a u c i t y of data. The d i a g n o s t i c f e a t u r e s of the f r o n t a l water were the shallow thermocline and hig h f l u o r e s c e n c e at the depth of the n i t r a c l i n e (3 to 7 m). Time Course 3 was conducted i n s t r a t i f i e d water at S t a t i o n T4, and the depth p r o f i l e ( F i g . 10B) demonstrated a subsurface f l u o r e s c e n c e maximum (ca. 10 m) which was o v e r l a i n by n i t r a t e -d e p l e t e d mixed water. A summary of the i n i t i a l biomass data and environmental c o n d i t i o n s f o r each s t a t i o n i s given i n Table VI. PIankt on community composition The s p e c i e s composition of the phytoplankton community i n the f r o n t a l and s t r a t i f i e d water was very d i f f e r e n t (Table V I I ) . In the f r o n t a l water, chain-forming diatoms of the genus Chaet oceros formed aggregates, l e s s than or equal to 1 mm, which c o n t a i n e d some pennate diatoms belonging to Navicula spp. and Nitzschia spp. Because of the s i z e of the diatom f l o e s , the water samples were not screened through Nitex® n e t t i n g i n order to minimize macrozooplankton p r e d a t i o n d u r i n g i n c u b a t i o n s . In s p i t e of the absence of these aggregates i n s t r a t i f i e d water, to remain c o n s i s t e n t , these water samples were not passed through Nitex® n e t t i n g . Small f l a g e l l a t e s l e s s than 5 um were the most common phytoplankton i n the 10. Depth p r o f i l e s of temperature, in vivo f l u o r e s c e n c e , and N0 3 c o n c e n t r a t i o n . (A) F r o n t a l s t a t i o n A5, Time Course 2. (B) S t r a t i f i e d s t a t i o n T4, Time Course 3. 65 6 6 Table VI I n i t i a l environmental c o n d i t i o n s of seawater c o l l e c t e d f o r time course experiments. S t a t i o n D i s s o l v e d n u t r i e n t C h i a PON POC c o n c e n t r a t i o n (ug-1 ' M u g a t N-1 M u g at C - l ) NH 4 + N 0 3 ~ urea (ug at N - 1 - 1 ) 1 -- 2 . 9 9 0 . 1 8 6 . 5 5 1 4 . 8 1 0 6 2 0 . 2 7 4 . 5 5 0 . 6 0 2 . 1 2 7 . 2 8 4 7 . 3 3 0 . 1 9 < . 0 5 0 . 3 3 0 . 3 9 3 . 5 7 3 1 . 4 67 Table VII Phytoplankton community composition i n f r o n t a l and s t r a t i f i e d water. S t a t i o n Phytoplankton (10 c e l l s - 1 ) Diatoms D i n o f l a g e l l a t e s F l a g e l l a t e s F r o n t a l A5 S t r a t i f i e d T4 2.3 0.43 0.023 0.049 1 .6 1 .6 s t r a t i f i e d water. Chaet oceros spp., C. social is and Skel et onema cost at um were the most abundant of the diatoms,-whereas d i n o f l a g e l l a t e s were almost e x c l u s i v e l y Gymnodinium spp. Water samples were not o r i g i n a l l y taken f o r zooplankton s p e c i e s enumeration. The abundance of these organisms, as seen i n the phytoplankton samples, suggested that they c o u l d have been important g r a z e r s and n i t r o g e n r e m i n e r a l i z e r s . As f i r s t approximation, the numbers and types of these organisms i n samples were determined (Table V I I I ) . ^ N upt ake and nitrogen di s appear a nee During the time course experiments, changes i n the c o n c e n t r a t i o n of d i s s o l v e d NH^ +, N0 3 and urea, and the 15 i n c o r p o r a t i o n of N - l a b e l l e d n i t r o g e n i n t o the p a r t i c u l a t e matter were measured. Both approaches y i e l d d i f f e r e n t i n f o r m a t i o n c oncerning n i t r o g e n u t i l i z a t i o n by the phytoplankton. Changes i n d i s s o l v e d n i t r o g e n c o n c e n t r a t i o n represent net community f l u x of th a t n u t r i e n t and encompass 1 5 r e g e n e r a t i v e and uptake p r o c e s s e s . By c o n t r a s t , N-isotope accumulation g i v e s a measure of the gros s uptake by the 15 15 phytoplankton p r o v i d i n g there i s no r e c y c l i n g of N, and N enrichment remains c o n s t a n t . R e s u l t s from Time Course 2 ( f r o n t a l water) and Time Course 3 ( s t r a t i f i e d water) experiments are shown i n F i g u r e s 11 and 12, r e s p e c t i v e l y . Data from Time Course 2 demonstrate m u l t i p l e n i t r o g e n s u b s t r a t e u t i l i z a t i o n by phytoplankton; s p e c i f i c a l l y f o r NH^ + N0 3 and urea ( F i g . 11C, E) and N0 3 and urea ( F i g . 11G). By v i r t u e of the high ambient NO., c o n c e n t r a t i o n i n the f r o n t a l Table VIII Zooplankton community s t r u c t u r e i n f r o n t a l and s t r a t i f i e d water. Zooplankton (1 ) S t a t i o n T i n t i n n i d s C a l a n o i d C i l i a t e s e x c l . Others copepods t i n t i n n i d s F r o n t a l A5 470 50 730 280 S t r a t i f i e d T4 180 60 140 300 50 to 60% were Noctiluca sp., the remainder were Oikopleura sp. and u n i d e n t i f i e d zooplankton. 70 F i g . 11. Time course measurements at f r o n t a l s t a t i o n (A5), Time Course 2. (A) D a i l y i n c i d e n t i r r a d i a n c e d u r i n g 1 5 experiment. (B, D, F) N atom % excess in p a r t i c u l a t e matter f o r l i g h t and dark b o t t l e i n c u b a t i o n s f o l l o w i n g a d d i t i o n of 6 ug at N - l ~ 1 of (B) NH 4 +, (D) N0 3~ and (F) urea ( e r r o r bars r e p r e s e n t the range of d u p l i c a t e s ) . (C, E, G) Corresponding measurements of d i s s o l v e d NH 4 + (•), N0 3" (O) and urea (A) i n (C) NH 4 +, (E) NC>3~, and (G) ur e a - s p i k e d samples. Dashed l i n e i n d i c a t e s no measurements of d i s s o l v e d urea at 3 and 6 h. F i g . 12. As F i g . 12 except at s t r a t i f i e d s t a t i o n (T4), Time Course 3. 71 waters, disappearance uptake r a t e s of NC>3 i n the NH^ and urea - s p i k e d samples were determined. Disappearance uptake r a t e s f o r n i t r a t e were s i m i l a r i n the presence (V n_,, = 0.521 0-6h ug at N - l ~ 1 - h ~ 1 ) and absence ( V Q _ g h = 0.567 ug at N - l _ 1 - h - 1 ) of urea but were reduced i n the N H 4 + - s p i k e d samples (Vg.g^ = -1 -1 15 0.267 ug at N-1 -h ). The N-urea atom % accumulation r a t e was constant over the f i r s t 15 h, but p r i o r to the end of the dark p e r i o d i t i n c r e a s e d and remained constant u n t i l the end of the i n c u b a t i o n ( F i g . 11F). The increase i n urea uptake r a t e c o i n c i d e d with the d e p l e t i o n of e x t e r n a l NC>3 ; moreover, the change i n urea c o n c e n t r a t i o n was minimal over the f i r s t 6 h, when NC>3 c o n c e n t r a t i o n s were high (4.55 to 1.4 ug at N - l ~ 1 ) and N0 3~ was being taken up ( F i g . 1 1G) . 15N-NC>3~ and 1 5 + . N-NH^ i n c o r p o r a t i o n was not constant d u r i n g the i n c u b a t i o n , but s u b s t r a t e e x h a u s t i o n d i d not occur u n t i l the 21 to 24 h time i n t e r v a l . Time Course 1 was conducted i n p h y t o p l a n k t o n - r i c h water and NC>3 d e p l e t i o n o c c u r r e d i n l e s s than 3 h. N i t r o g e n -s p e c i f i c uptake r a t e of NC>3 ^ v o-3h = 0.070 ^ ^ w a s t n e highest of any n i t r o g e n s u b s t r a t e measured i n a l l time course experiments. The disappearance uptake rate over the same time i n t e r v a l ( vg-3h^ w a s 1 • ^ u 9 a t N ' l 1 ' h 1 , and using a time averaged p a r t i c u l a t e n i t r o g e n , c a l c u l a t e d from the amount of NC>3 taken up and the i n i t i a l measured p a r t i c u l a t e n i t r o g e n , the n i t r o g e n - s p e c i f i c uptake r a t e ( vn-3h = 0*081 h 1) was i n 1 5 f a i r agreement with the r a t e determined by N uptake. As a consequence of s u b s t r a t e exhaustion, both techniques y i e l d e d r a t e s which were underestimates. Simultaneous uptake of NC>3 73 and urea was evident i n the ur e a - s p i k e d samples. The maximum disappearance r a t e of urea ( V3_gj 1 55 0 . 3 1 4 ug at N-1 1- h 1 ) - was l e s s than the NC>3~ r a t e ( V Q _ 6 h = 0.441 ug at N - l ~ 1 - h ~ 1 ) . 1 5 The p a t t e r n of N uptake by the phytoplankton i n the s t r a t i f i e d water was s i m i l a r i n the NH 4 +, N0 3 and ur e a - s p i k e d samples ( F i g . 12B, D, F ) . Uptake was l i n e a r over the f i r s t 9 to 12 h and was subsequently reduced d u r i n g the dark p e r i o d and i n c r e a s e d again i n the e a r l y morning. S u b s t r a t e d e p l e t i o n d i d not occur i n these experiments, and u t i l i z a t i o n of ni t r o g e n was minimal i n the NH 4 +, N0 3 and u r e a - s p i k e d samples (23, 18 and 10%, r e s p e c t i v e l y ) . Upt ake periodicity C l e a r i n d i c a t i o n s of urea r e g e n e r a t i o n , and to a l e s s e r extent NH 4 + r e g e n e r a t i o n , were ev i d e n t from i n c r e a s e s i n t h e i r c o n c e n t r a t i o n s over the time course i n a l l 3 experiments ( F i g . 12C, E, G). Furthermore, the p a t t e r n of NH 4 + and urea production i n the samples suggested that there was a p e r i o d i c i t y i n uptake and/or r e g e n e r a t i o n p r o c e s s e s . S i m i l a r r e s u l t s were seen i n Time Course 2 ( F i g . 11C, E, G) p a r t i c u l a r l y f o r urea p r o d u c t i o n over the 15 to 21 h time i n t e r v a l . R e s u l t s from both f r o n t a l and s t r a t i f i e d time course experiments demonstrate that simultaneous u t i l i z a t i o n of two or more n i t r o g e n s u b s t r a t e s occurs even when the c o n c e n t r a t i o n of one of the n i t r o g e n s u b s t r a t e s ( N H 4 + , N0 3 , or urea) i s i n excess of the o t h e r ( s ) . T h i s suggests t h a t such a phenomenon may n a t u r a l l y occur i n these communities. 15 + -The p a t t e r n of N - l a b e l l e d NH. , NO., and urea uptake r a t e s suggests the e x i s t e n c e of a d i e l p e r i o d i c i t y i n n i t r o g e n u p t a k e , . i n both f r o n t a l and s t r a t i f i e d water ( F i g . 1 3 ) . The decrease i n uptake of NH^ + and NO^ from 21 to 24 h i n Time Course 2 was due to s u b s t r a t e exhaustion (see F i g . 11C, E ) . In the f r o n t a l community, uptake r a t e s of NO^ were g r e a t e s t throughout the time course, and there was s i g n i f i c a n t dark uptake of NO^ . In comparison, NH^ + uptake r a t e s were h i g h e s t i n the s t r a t i f i e d community, and NO^ and urea uptake r a t e s were s i m i l a r but of a l e s s e r magnitude. In both time courses there was a tendency f o r n i t r o g e n uptake r a t e s to i n c r e a s e p r i o r to the onset of the l i g h t p e r i o d ; t h i s was most evident i n the u r e a - s p i k e d samples. Chl a specific uptake rates Uptake r a t e s n o r m a l i z e d per u n i t c h l a showed that NH^ + and urea uptake were on average 2 and 2.4 times higher i n the s t r a t i f i e d water; whereas, NO^ uptake r a t e s were on average 1.6 times h i g h e r i n f r o n t a l water (Table I X ) . C hl a s p e c i f i c uptake r a t e s f o r each n u t r i e n t , when compared between s t a t i o n s , were most s i m i l a r over the dark p e r i o d (12 to 18 h), while the g r e a t e s t d i s p a r i t y was found over the f i r s t 6 h. Comparison of rates 15 The N uptake r a t e , disappearance uptake r a t e and the ra t e of change of the PON, c a l c u l a t e d from the d i f f e r e n c e between measured v a l u e s , are presented i n F i g u r e 14. A l l r a t e s were c a l c u l a t e d over 6, 12, 18, and 24 h time i n t e r v a l s , 7 5 F i g . 13. N i t r o g e n - s p e c i f i c uptake r a t e s of NH^ + (•), N0 3 (o) and urea (A) i n (A) f r o n t a l and (B) s t r a t i f i e d water. Rates determined f o r 3 or 6 h i n t e r v a l s ; each p o i n t i n d i c a t e s a r a t e c a l c u l a t e d over the time i n t e r v a l between i t and the next p o i n t on the c u r v e . Shaded area on the a b s c i s s a d e l i m i t s the dark p e r i o d . 76 77 Table IX C h l o r o p h y l l a s p e c i f i c uptake r a t e s of NH 4 +, NO^ and urea i n f r o n t a l (A5) and s t r a t i f i e d (T4) water. The dark p e r i o d occurs d u r i n g the 12 to 18 h time i n t e r v a l . N i trogen Time i n t e r v a l C h i a s p e c i f i c N-uptake r a t e s u b s t r a t e (h) [ug a t N (ug c h l a ) • h )] F r o n t a l s t n S t r a t i f i e d stn NH 4 + N0 3 Urea 0 -- 6 0.091 0.261 6 -- 12 0.060 0. 1 33 12 -- 18 0.025 0.030 18 -- 24 0.028 0.047 0 -- 6 0. 1 62 0.098 6 -- 12 0.075 0.082 12 -- 18 0.042 0.019 18 -- 24 0.068 0.039 0 -- 6 0.040 0. 127 6 -- 12 0.028 0. 1 25 12 -- 18 0.026 0.019 18 -- 24 0.050 0.053 78 F i g . 14. Nitrogen uptake r a t e s determined by N atom % excess accumulation in the p a r t i c u l a t e s (•), change i n d i s s o l v e d n i t r o g e n c o n c e n t r a t i o n (o) and by change i n the p a r t i c u l a t e n i t r o g e n c o n c e n t r a t i o n (A) over 6, 12, 18 and 24 h time i n t e r v a l s . (A) NH 4 +, (B) N0 3 , and (C) urea-s p i k e d samples i n f r o n t a l water and (D) NH 4 +, (E) NC>3 and (F) urea-spiked samples i n s t r a t i f i e d water. SL 8 0 and t h i s approach was taken, r a t h e r than c a l c u l a t i n g the r a t e s over s u c c e s s i v e 6 h i n t e r v a l s , to minimize f l u c t u a t i o n s due to sample v a r i a b i l i t y . Comparison of data from the f r o n t a l s t a t i o n i n d i c a t e s that i n the NH 4 + and urea-sp i k e d samples, the r a t e of change of the p a r t i c u l a t e n i t r o g e n i s g r e a t e r than 1 5 the accumulation of N or the disappearance of e i t h e r n u t r i e n t ( F i g . 14A, C). In the n i t r a t e - s p i k e d samples ( F i g . 14B), the ra t e of n i t r a t e uptake as determined by the 1 5 -disappearance of N0 3 , the i n c o r p o r a t i o n of N-NC>3 and the change i n PON are s i m i l a r . A general f e a t u r e of the data from the s t r a t i f i e d s t a t i o n i s the more r a p i d change i n PON than 1 5 the N uptake or disappearance uptake r a t e s ( F i g . 14D, E, F ) . Furthermore, the disappearance r a t e s of NH^ + and urea are 1 5 c o n s i s t e n t l y l e s s than the N uptake r a t e s . Dark/Light uptake 1 5 The r a t i o of dark to l i g h t N uptake r a t e (VT.:VT ) f o r NH^ +, N0 3 and urea i s given i n Table X. At both s t a t i o n , N H 4 + dark uptake r a t e s were a s i g n i f i c a n t f r a c t i o n of the l i g h t uptake r a t e s throughout the e n t i r e time c o u r s e . The VD: V L f o r NH 4 + i n f r o n t a l water was constant (38%) and was l e s s than the r a t i o i n s t r a t i f i e d water (52 to 102%). I n i t i a l dark uptake r a t e s of urea were 60 to 81% of the l i g h t r a t e s i n a l l t h ree time course experiments. Dark urea uptake i n the s t r a t i f i e d water was always a measurable f r a c t i o n of the l i g h t r a t e and appeared l e s s l i g h t dependent than at the f r o n t a l s t a t i o n s . The l i g h t dependency of N0 3 uptake was more s i m i l a r to that of urea than ammonium i n both s t r a t i f i e d and 81 T a b l e X R a t i o of dark to l i g h t uptake r a t e s (V^:VT ) of NH. + , NO " JJ Jj 4 o and urea f o r f r o n t a l and s t r a t i f i e d water. S t a t i o n Time i n t e r v a l (h) NH, NO. Urea F r o n t a l T3 0 6 1 1 18 6 1 1 18 24 0.81 0. 37 0 F r o n t a l A5 0 6 1 2 18 6 1 2 18 24 0.37 0.39 0.37 0.39 0 . 0 8 0 0 0 0.60 0 0 0 S t r a t i f i e d T4 0 9 18 9 18 24 0.58 1 .02 0.52 0.18 0.60 0 0.66 0.24 0.06 82 f r o n t a l water. Regeneration r a t e s The r e g e n e r a t i o n r a t e s of NH 4 + and urea i n the f r o n t a l water were s i m i l a r (Table X I ) . Note that the change i n 1 5 s u b s t r a t e c o n c e n t r a t i o n was much g r e a t e r than the N uptake r a t e over the 18 to 24 and 6 to 12 h time p e r i o d s f o r NH 4 + and urea, r e s p e c t i v e l y . As a r e s u l t , negative r a t e s of rege n e r a t i o n have been c a l c u l a t e d . A c o n s i s t e n t p a t t e r n was seen for the c a l c u l a t e d values of NH 4 + and urea r e g e n e r a t i o n r a t e s i n s t r a t i f i e d water. The disappearance r a t e s of both 1 5 n u t r i e n t s surpassed the N uptake r a t e s i n the 12 to 18 and 18 and 24 h time i n t e r v a l s . Urea r e g e n e r a t i o n r a t e s were approximately 5 and 2 times g r e a t e r than the c o r r e s p o n d i n g NH 4 + r e g e n e r a t i o n r a t e s f o r the f i r s t two 6 h i n t e r v a l s . Mas s balance Changes i n the PON over 6, 12, 18 and 24 h time i n t e r v a l s f o r each set of n i t r o g e n - s p i k e d samples were c a l c u l a t e d (Table X I I ) . By way of comparison, the change i n the t o t a l DIN and urea ( AP^,) over the same time i n t e r v a l and the amount of ni t r o g e n accumulated in p a r t i c u l a t e matter (^V^,), as 1 5 determined by N atom percent excess data, are r e p o r t e d . The r e s u l t s i n d i c a t e that the f r o n t a l and s t r a t i f i e d communities were very d i f f e r e n t . The change i n P^ , i n the f r o n t a l water samples c o n s i s t e n t l y overestimated the change in the PON f o r a l l 3 n u t r i e n t s . However, the op p o s i t e was true i n the s t r a t i f i e d water samples where the change i n PON was always 8 3 Table XI Regeneration r a t e s of NH^ + and urea i n f r o n t a l and s t r a t i f i e d water. S t a t i o n N i t r o g e n Time a d d i t i o n (h) Change i n cone, of added N 1 5 N-uptake Regeneration rate r a t e (ug at N-1 1 •h" 1) F r o n t a l NH + 0 -- 6 . 1 77 .224 .047 A5 ft 6 -- 12 .206 .232 .026 12 -- 18 .086 . 141 .055* 18 -• 24 .420 .174 -.246 Urea 0 -- 6 .040 .094 •054* 6 -- 12 .398 .094 -.305 12 -- 18 .116 . 1 32 .016 18 -- 24 .287 .308 .021 S t r a t i f i e d NH + 0 -- 6 .085 .103 .018 T4 6 -- 12 .052 .073 .021* 12 -- 18 .039 .025 -.014* 18 -- 24 .074 .053 -.021 Urea 0 -- 6 -.044 .050 .094 6 -- 12 .023 .066 .043 1 2 -- 18 .026 .014 -.012 18 -- 24 .088 .050 -.033 1 E q u i v a l e n t : to V. ; n e g a t i v e value i n d i c a t e s an i n c r e a s e i n s u b s t r a t e c o n c e n t r a t i o n over the i n c u b a t i o n p e r i o d . Regenerative f l u x e s were c a l c u l a t e d using a mass balance; see " M a t e r i a l s and Methods". I n d i c a t e s t h a t disappearance of d i s s o l v e d n u t r i e n t was 1 5 g r e a t e r than uptake r a t e s c a l c u l a t e d from N. 8 4 Table XII Changes over time i n measured DIN and urea c o n c e n t r a t i o n ( A P T ) , p a r t i c u l a t e n i t r o g e n ( A PON), and amount of 15 N-n i t r o g e n accumulated i n the part i c u l a t e matter ( ^V^, ) in f r o n t a l and s t r a t i f i e d water • S t a t i o n N i t r o g e n Time A Pm A PON addi t ion (h) 1 T (ug at N• r 1 ) F r o n t a l NH. + 0 - 6 3.51 1 .94 1 . 38 A5 4 0 - 1 2 5.79 4.86 2.76 0 - 18 5.59 7.24 3.62 0 - 24 10.10 8. 19 4.67 NO ~ 0 - 6 3.41 2.66 2. 58 0 - 1 2 5.36 4.99 4.27 0 - 18 7.74 5.58 5.46 0 - 24 1 0.58 9. 18 7.82 Urea 0 - 6 3.21 2.97 0. 57 0 - 12 6.56 3.65 1.13 0 - 18 7.70 5.13 1 . 93 0 - 24 9.62 7.60 3.79 S t r a t i f i e d NH + 0 - 6 -0.01 0.98 0.62 T4 4 0 - 1 2 0.58 1 .01 1 . 06 0 - 18 0.66 1 .42 1.21 0 - 24 1.51 1 . 47 1 .53 NO," 0 - 6 -1 .08 0.21 0.23 0 - 12 0.0 1 1 .28 0.51 0 - 18 0.16 1 .55 0.61 0 - 24 0.65 1 . 66 0 .85 Urea 0 - 6 -0.24 0.57 0.29 0 - 1 2 -0.08 1.15 0.69 0 - 18 0.08 1.19 0. 78 0 - 24 0.59 1 .60 1 .08 8 5 1 c g r e a t e r than AP T« Summation of the N accumulation i n the p a r t i c u l a t e matter over time i n d i c a t e s that t h i s n i t r o g e n c o n t r i b u t i o n cannot account f o r the change i n the p a r t i c u l a t e n i t r o g e n except i n the n i t r a t e - s p i k e d time course i n f r o n t a l water and the N H 4 + - s p i k e d time course i n s t r a t i f i e d water. D i s c u s s i o n Experimental considerations In these experiments, s a t u r a t i n g a d d i t i o n s of each n i t r o g e n compound (NH 4 +, NO^ and urea) were r e q u i r e d to ensure that s u b s t r a t e exhaustion d i d not occur during, the time course. T h i s approach was chosen, rather than c o l l e c t i n g water samples at v a r i o u s times and determining in situ r a t e s of n i t r o g e n uptake, i n order to e l i m i n a t e p o t e n t i a l c o m p l i c a t i n g f a c t o r s such as d i e l m i g r a t i o n of phytoplankton (Blasco 1978), s u r f a c e water advection and problems a s s o c i a t e d 1 5 with adding t r a c e r amounts of N-substrate (Goldman el al. 1981, G l i b e r t et al. 1982b). Consequently, these r a t e s of n i t r o g e n uptake are p o t e n t i a l r a t e s (with the exce p t i o n of NO^ uptake in f r o n t a l s t a t i o n s ) , as they w i l l only be r e a l i z e d under c o n d i t i o n s where the ni t r o g e n s u b s t r a t e c o n c e n t r a t i o n i s e l e v a t e d to a l e v e l s u f f i c i e n t to s a t u r a t e the uptake system. E m p i r i c a l o b s e r v a t i o n s , such as deep water i n j e c t i o n (Walsh et al . 1977), s o l i t o n enrichment ( H o l l i g a n etl. 1985), d i e l m i g r a t o r y behavior ( C u l l e n and Horrigan 1981), phytoplankton s i n k i n g (Bienfang et al . 1982) and patchy e x c r e t i o n (Lehman and Sca v i a 1982) plus t h e o r e t i c a l 86 c o n s i d e r a t i o n s (McCarthy and Goldman 1979, Parslow et al . 1985) l e n d credence to t h i s approach. More i m p o r t a n t l y , t h i s approach has enabled me to d e r i v e a d d i t i o n a l i n f o r m a t i o n concerning the p h y s i o l o g i c a l s t a t e of, and the n i t r o g e n c y c l i n g w i t h i n , the plankton community of these two types of c o a s t a l ecosystems. Simultaneous uptake of nitrogen compounds Simultaneous u t i l i z a t i o n of NH 4 + and NO^ i s w e l l documented ( C o l l o s and Lewin 1974, Eppley and Renger 1974, Bienfang 1975, Conover 1975, Caperon and Ziemann 1976, Conway 1977, M a e s t r i n i et al . 1982), and the r e s u l t s of t h i s study not only demonstrate dual n i t r o g e n s u b s t r a t e u t i l i z a t i o n but that NH 4 +, NO^ and urea may be taken up c o n c u r r e n t l y . As poi n t e d out by C o l l o s (unpubl.), m u l t i p l e n i t r o g e n s u b s t r a t e r u t i l i z a t i o n w i l l r e s u l t i n a r e d u c t i o n of the n i t r o g e n -1 5 s p e c i f i c uptake r a t e of the N - l a b e l l e d compound compared to the n i t r o g e n - s p e c i f i c uptake r a t e determined when only the 1 5 N - l a b e l l e d compound i s being taken up. I c a l c u l a t e d the absolute uptake r a t e s u s i n g the f i n a l PON, determined at the end of an i n c u b a t i o n , which g i v e s an a c c u r a t e measure of the 15 uptake r a t e of the N - l a b e l l e d n u t r i e n t i n t o the phytoplankton and avoids p o t e n t i a l a r t i f a c t s caused by the 1 5 i n c o r p o r a t i o n of non- N - l a b e l l e d n i t r o g e n . R e c e n t l y , M a e s t r i n i et al . (1982) demonstrated t h a t m i c r o a l g a e of oyster ponds took up NH 4 + and NO^ at the same r a t e once the NH 4 + c o n c e n t r a t i o n had decreased to ca. 7 ug at N-1 1 . The r e s u l t s from the f r o n t a l community demonstrated the s i m i l a r i t y of NH. + 87 and NC>2 disappearance uptake r a t e s , i n the N H 4 + - s p i k e d samples. However, the NO^ disappearance uptake r a t e was reduced by 50% i n the N H 4 + - s p i k e d samples by comparison to the n i t r a t e - s p i k e d samples. S i m i l a r NH^ + suppression of NO^ uptake has been r e p o r t e d f o r both l a b o r a t o r y (e.g. Conway 1977, C r e s s w e l l and S y r e t t 1979) and n a t u r a l phytoplankton assemblages (e.g. B l a s c o and Conway 1982). NO^ and urea uptake i n t e r a c t i o n s from two time course experiments i n f r o n t a l water are c o n t r a d i c t o r y . In Time Course 1, there was a 70% r e d u c t i o n i n the NO^ disappearance uptake r a t e i n the presence of urea, but the NO^ disappearance uptake r a t e was u n a f f e c t e d or s l i g h t l y enhanced i n the presence of urea i n Time Course 2. The reasons f o r t h i s d i s c r e p a n c y are not apparent, n o n e t h e l e s s , v a r i a t i o n i n phytoplankton community s t r u c t u r e , r e l a t i v e growth r a t e s and i n t e r n a l n i t r o g e n s t a t u s may be important d i f f e r e n c e s between the two s t a t i o n s . These v a r i a b l e s have been i d e n t i f i e d as a f f e c t i n g uptake i n t e r a c t i o n s among n i t r o g e n compounds (Dortch and Conway 1984). In Time Course 2, the apparent slow disappearance uptake r a t e of urea, over the f i r s t 6 h, may be e x p l a i n e d by r e g e n e r a t i o n of urea over t h i s p e r i o d . A l t e r n a t i v e l y , McCarthy and Eppley (1972) have r e p o r t e d NO^ i n h i b i t i o n of urea uptake i n n a t u r a l seawater samples. I r r e s p e c t i v e of the c o n c e n t r a t i o n of NH^ +, NO^ or urea ( l e s s than equal to.6 ug at N-1 1 ) , phytoplankton i n both the f r o n t a l and s t r a t i f i e d water are capable of u t i l i z i n g low c o n c e n t r a t i o n s of regenerated n i t r o g e n (NH. + and u r e a ) . 88 Variations in nitrogen uptake rate In these experiments, sampling i n t e r v a l s were long r e l a t i v e to phytoplankton r a p i d uptake responses seen i n the l a b o r a t o r y (Conway et a l . 1976, Parslow et al . 1984a, b) and the f i e l d (e.g. G l i b e r t and Goldman 1981); t h e r e f o r e , short term v a r i a t i o n s i n uptake r a t e were not e v i d e n t . Enhanced uptake of NH 4 + and urea by N0 3 s u f f i c i e n t phytoplankton has been r e p o r t e d (Horrigan and McCarthy 1981, 1982, Parslow et al . 1984b). In the l i g h t of the slower long-term r a t e s of NH 4 + and urea uptake i n the f r o n t a l s t a t i o n , r e l a t i v e to NO^ , i t appears u n l i k e l y that such a p r o c e s s o c c u r r e d on time s c a l e s s h o r t e r than these measurements. On long time s c a l e s , changes i n uptake r a t e due to d i e l p e r i o d i c i t y were evident i n the r e s u l t s . B o t t l e confinement e f f e c t s have been shown to le a d to s e r i o u s underestimates of r a t e p r o c e s s e s (Venrick et al . 1977), but the constant r a t e s of c h l a and POC and PON s y n t h e s i s i n d i c a t e no such a r t i f a c t s i n these experiments. Olson and Chisholm (1983) have shown t h a t c e l l d i v i s i o n p a t t e r n s of n i t r o g e n - l i m i t e d phytoplankton c u l t u r e s may be e n t r a i n e d by NH^ + p u l s e s . Although samples were spiked with s a t u r a t i n g a d d i t i o n s of each n i t r o g e n compound, there i s evidence from an e a r l i e r c r u i s e i n the S t r a i t of Georgia (August 1983) of uptake p e r i o d i c i t y at ambient c o n c e n t r a t i o n s of d i s s o l v e d n i t r o g e n (Parslow et al. u n p u b l . ) . Uptake p e r i o d i c i t y was a l s o e v i d e n t i n n i t r o g e n - s u f f i c i e n t f r o n t a l water. 89 Effects of light/dark regime on nitrogen uptake The constancy of V D:V L f o r NH 4 + i n f r o n t a l water, when NH 4 + uptake r a t e s of phytoplankton exposed to the n a t u r a l l i g h t / d a r k c y c l e were p e r i o d i c , suggests that NH^ + uptake i s c i r c a d i a n ; i n absence of the l i g h t / d a r k c y c l e the rhythm i s f r e e running (see Chisholm 1981). T h i s c o n c l u s i o n i s supported by Goering et al. (1964) who found rhythmic v a r i a t i o n i n both NH 4 + and NO^ uptake by n a t u r a l communities under continuous l i g h t . The r e s u l t s f o r NO^ and urea demonstrate the dependency of uptake on l i g h t , and i n t h i s r e s p e c t both n u t r i e n t s are comparable. The l i g h t dependence of uptake of both n u t r i e n t s i s w e l l e s t a b l i s h e d (Maclsaac and Dugdale 1972, Mitamura and S a i j o 1975, 1980a, Harvey and Caperon 1976, Webb and Haas 1976, Nelson and Conway 1979). Other r e p o r t s have shown that n i t r o g e n - d e p r i v e d phytoplankton have hi g h e r dark uptake r a t e s of n i t r o g e n than n i t r o g e n -r e p l e t e phytoplankton (e.g. S y r e t t 1962, Eppley and Coatsworth 1968, Malone et al. 1975, Rees and S y r e t t 1979b). In t h i s study, dark n i t r o g e n uptake r a t e s normalized to c h l a were h i g h e s t i n the n i t r o g e n - d e p l e t e d s t r a t i f i e d water, i n agreement with these o b s e r v a t i o n s . Dark uptake r a t e s were a l s o a g r e a t e r p r o p o r t i o n of the l i g h t r a t e s f o r NH 4 +, N0 3 and urea i n s t r a t i f i e d water. The higher c h l a s p e c i f i c uptake r a t e s of N H 4 + and urea i n s t r a t i f i e d water and of NO^ i n f r o n t a l water are c o n s i s t e n t with the way we envisage n i t r o g e n supply to these areas. Regenerated n i t r o g e n ( N H 4 + and urea) has been shown to supply most of the phytoplankton n i t r o g e n 90 demand i n n i t r o g e n - d e p l e t e d waters and as the c o n c e n t r a t i o n of ambient NO^ i n c r e a s e s so does the r e l a t i v e importance of NO^ for the phytoplankton n i t r o g e n r a t i o n (e.g. McCarthy et al . 1977, H a r r i s o n 1 980, G l i b e r t et al . 1982a, Cochlan 1986). NH * and urea regeneration These estimates of NH^ + r e g e n e r a t i o n r a t e s are i n agreement with p r e v i o u s l y p u b l i s h e d r a t e s f o r c o a s t a l waters (0.01 to 0.31 ug at N - l ~ 1 - h _ 1 ) (Caperon et al . 1979, Cochlan 1982, G l i b e r t 1982, G l i b e r t et al . 1982b, Paasche and K r i s t i a n s e n 1982, LaRoche 1983). The method used to c a l c u l a t e r e g e n e r a t i o n r a t e s i s i n f e r i o r by comparison to the isotope d i l u t i o n method (Blackburn 1979, Caperon et al. 1979). U n l i k e 1 5 experiments employing t r a c e a d d i t i o n s of N, the c o n c e n t r a t i o n of regenerated n i t r o g e n was small r e l a t i v e to 15 15 the added N s u b s t r a t e s , and thus the N enrichment f a c t o r remained constant over the i n c u b a t i o n . Consequently, there i s l i t t l e e r r o r a s s o c i a t e d with the uptake measurement. The r a t e s of urea r e g e n e r a t i o n are of s i m i l a r magnitude to the NH^ r e g e n e r a t i o n r a t e s , and the r a t e s are comparable i n both communities. These experiments have enabled me to q u a n t i f y urea r e g e n e r a t i o n by an i n t a c t p l a n k t o n community. These r e s u l t s are an improvement over p r e v i o u s attempts which have q u a n t i f i e d urea p r o d u c t i o n by s p e c i e s , or s i z e f r a c t i o n a t e d assemblages, of zooplankton. The p a t t e r n s of NH^ + and urea p r o d u c t i o n over the time course experiments i n d i c a t e a p e r i o d i c i t y which i s not a r e s u l t of reduced uptake r a t e s . T h i s c o r r o b o r a t e s data of Caperon et al. (1979) and G l i b e r t 91 (1982) who reported higher rates of NH^ + regeneration at night and ear ly morning. A d d i t i o n a l l y , Co l los and Lewin (1974) and H a t t o r i (1982) have shown d i e l v a r i a t i o n s in d i s so lved NH^ + concentration in coastal waters. Unlike the d i s so lved ni trogen concentration measurements, the ca l cu la ted regeneration rates do not show the same p e r i o d i c i t y , s ince the time scales over which they were ca lcu la ted are much greater than these phys io log ica l processes. Regeneration of nitrogen has long been recognized as a 1 5 poss ib le a r t i f a c t in determining N uptake rates (Dugdale and Goering 1967), and recently i t has been shown that these rates 1 5 may be underestimated by a factor of ca. 2 when a constant N atom % enrichment i s assumed (Gl iber t et al. 1982b). I have not corrected these uptake rates for isotope d i l u t i o n , but as I w i l l now show, regeneration of NH^ + and urea in these experiments, has l i t t l e ef fect on uptake rates c a l c u l a t e d with the i n i t i a l enrichment; however., such a process may dramat ica l ly af fect the disappearance uptake rates . I n i t i a l addit ions of nitrogen were 6.0 ug at N-1 1 , c o r r e c t i n g for background and p u r i t y of the substrate th i s 15 -1 represents 5.92 ug at N 1 . When no regeneration occurs 1 5 . and N is conserved; P t - P Q - v 1 (t) and P - P. = V 1 o t t where PQ and Pfc = i n i t i a l and f i n a l c o n c e n t r a t i o n s of i 15 d i s s o l v e d n i t r o g e n ; V = N uptake r a t e ; t = time; and 1 5 disappearance uptake r a t e equals N uptake r a t e . It i s 1 5 obvious that l a r g e a d d i t i o n s of N to samples cause the i s o t o p e enrichment f a c t o r (R) to be r e l a t i v e l y i n s e n s i t i v e to a d d i t i o n s of regenerated n i t r o g e n . For example, i f R changes from 0.9394 to 0.8500 and I assume that the pulse of regenerated 1 4 N (0.66 ug at N-1 1) i s added immediately a f t e r time zero, disappearance uptake r a t e s w i l l be underestimated 1 5 by 62%, while N uptake r a t e s w i l l decrease by only 10%. T h e r e f o r e , r e g e n e r a t i v e p r o c e s s e s are of l e s s e r consequence to 15 • 15 N-uptake r a t e c a l c u l a t i o n s when the c o n c e n t r a t i o n of N i s l a r g e ; i f the t o t a l c o n c e n t r a t i o n of d i s s o l v e d n i t r o g e n becomes low, f o r example toward the end of a time-course 1 4 experiment, r e g e n e r a t i o n of N w i l l have a g r e a t e r e f f e c t on 15 N uptake r a t e c a l c u l a t i o n s . With t h i s l i n e of r e a s o n i n g and as d i s c u s s e d e a r l i e r , I have i n t e r p r e t e d d i s c r e p a n c i e s between the disappearance 15 uptake r a t e and N uptake r a t e as i n d i c a t i v e of r e g e n e r a t i o n . In the NH^ + and u r e a - s p i k e d samples from s t r a t i f i e d water, r e g e n e r a t i o n i s e v i d e n t ; however, the disappearance uptake 1 5 r a t e s are i n c l o s e r agreement with the N uptake r a t e s at the end of the time course compared with the beginning ( F i g . 14). In f r o n t a l water, the d i s c r e p a n c i e s between disappearance + 15 + uptake r a t e s of NH^ and NH 4 uptake r a t e s may be adequately e x p l a i n e d by r e g e n e r a t i o n . The e x c e p t i o n i s the f i n a l 6 h 1 5 p e r i o d where changes i n d i s s o l v e d c o n c e n t r a t i o n s exceed N i n c o r p o r a t i o n r a t e s . The urea disappearance r a t e s are g r e a t e r 1 5 t h a n N i n c o r p o r a t i o n r a t e s a f t e r t h e f i r s t 6 h , a n d t h e v e r y f a s t d i s a p p e a r a n c e r a t e s f r o m 6 t o 12 h m a k e t h e 0 t o 18 a n d 0 t o 2 4 r a t e s h i g h a s w e l l . Particulate nitrogen balance I n a t w o - c o m p a r t m e n t s y s t e m c o n s i s t i n g o f D I N + u r e a a n d P O N , r e g a r d l e s s o f t h e f l u x r a t e s b e t w e e n t h e t w o p o o l s , c h a n g e s i n t h e c o n c e n t r a t i o n o f o n e c o m p o n e n t s h o u l d b e r e f l e c t e d b y c o r r e s p o n d i n g c h a n g e s i n t h e o t h e r . U s i n g t h i s a p p r o a c h , n i t r o g e n w i l l b e c o n s e r v e d p r o v i d i n g t h e s y s t e m i s c l o s e d . A d d i t i o n a l l y , b y i n c l u d i n g N H 4 + , NO^ a n d u r e a a s p a r t o f t h e d i s s o l v e d n i t r o g e n p o o l I am a b l e t o a c c o u n t f o r c i r c u m s t a n c e s w h e n r e g e n e r a t e d n i t r o g e n d i f f e r s f r o m t h e a s s i m i l a t e d f o r m . T h e c o r o l l a r y o f t h i s i s t h a t t h e r e g e n e r a t e d n i t r o g e n i s i n t h e f o r m o f N H 4 + a n d / o r u r e a . I n s u m m a r y , t h i s r e l a t i o n m a y b e e x p r e s s e d a s : P O N Q + P T Q = P O N t + P T T a n d A P O N = A P T w h e r e P O N Q a n d P O N f c = i n i t i a l a n d f i n a l p a r t i c u l a t e n i t r o g e n c o n c e n t r a t i o n s ; P T q a n d P T T = i n i t i a l a n d f i n a l D I N + u r e a c o n c e n t r a t i o n s ; A P O N = P O N f c - P O N Q ; A P T = P T Q - P T F C . D e v i a t i o n s f r o m t h i s m o d e l a r e i n s t r u c t i v e s i n c e t h e y p r o v i d e i n f o r m a t i o n c o n c e r n i n g n i t r o g e n c y c l i n g a n d i t s t r a n s f o r m a t i o n i n a q u a t i c s y s t e m s . F r o m t h e r e s u l t s o f t h i s s t u d y , i t i s a p p a r e n t t h a t a d d i t i o n s a n d l o s s e s o f n i t r o g e n a r e o c c u r r i n g 94 and that t h i s t r e n d i s c o n s i s t e n t w i t h i n the f r o n t a l and s t r a t i f i e d communities. The d i s c r e p a n c i e s between A P O N and A P M (Table XII) i n f r o n t a l water i n d i c a t e that n i t r o g e n i s being l o s t from the system. Given the p r e c i s i o n and accuracy for determining the c o n c e n t r a t i o n of the d i f f e r e n t d i s s o l v e d n i t r o g e n f r a c t i o n s (see " M a t e r i a l s and Methods"), I argue that n i t r o g e n l o s s e s are o c c u r r i n g from the P O N compartment. Changes i n P O N f o r 1 5 the N - n i t r a t e - s p i k e d samples from f r o n t a l water, as 15 i p r e d i c t e d by N uptake {^.Vj) are not s i g n i f i c a n t l y d i f f e r e n t form the measured v a l u e s ( A P O N ) ( p a i r e d t - t e s t , p > 0.05). 1 5 T h e r e f o r e , the i n c o r p o r a t i o n of N i n t o p a r t i c u l a t e matter accounts f o r the i n c r e a s e i n P O N . The d i s c r e p a n c i e s between A P O N and 5: Vy i n the N H 4 + and urea-spiked samples are, i n p a r t , a consequence of the simultaneous uptake of u n l a b e l l e d N O ^ and i t s c o n t r i b u t i o n to P O N . Further s t a t i s t i c a l 1 5 a n a l y s i s of the data from the N - n i t r a t e - s p i k e d samples showed that the hypotheses A P M = and A P T = A P O N must be r e j e c t e d (p < 0.05 and p < 0.05). Therefore, because A P T > A P O N a n d ^ V j , I conclude that the l o s t P O N i s l a b e l l e d with 1 5 N and that i t has the same i s o t o p i c composition as the d i s s o l v e d N O ^ . Furthermore, these r e s u l t s suggest that n i t r o g e n l o s s e s from the P O N pool can be most e a s i l y e x p l a i n e d by e x c r e t i o n or g r a z i n g l o s s e s to a d i s s o l v e d organic pool ( D O N ) . The a l t e r n a t i v e e x p l a n a t i o n s , that P O N or D O N i s l o s t d i r e c t l y v i a m e t h o d o l o g i c a l a r t i f a c t s , are untenable f o r the f o l l o w i n g reasons. The e f f e c t i v e r e t e n t i o n s i z e of a Whatman GF/F f i l t e r (0.7 um) i s s u f f i c i e n t to have caught a l l of the 95 l a r g e chain-forming diatoms which dominated the f r o n t a l water. Phytoplankton samples c o l l e c t e d on 0.2 um Nuclepore f i l t e r s and examined with a Z e i s s e p i f l u o r e s c e n c e microscope showed that there were no c h l o r o p h y l l - c o n t a i n i n g organisms l e s s than 2 um and that b a c t e r i o p l a n k t o n were ca. 1 um. Secondly, the low f i l t r a t i o n p r e ssure d i f f e r e n t i a l ( l e s s than 125 mm Hg) would have minimized c e l l l y s i s on the f i l t e r , and i n the s t r a t i f i e d water, dominated by s o f t - b o d i e d f l a g e l l a t e s , l o s s of n i t r o g e n was not seen. Feeding zooplankton have been shown to c o n t r i b u t e to the d i s s o l v e d organic carbon pool by l o s s of phytoplankton c e l l c ontents d u r i n g h a n d l i n g and f e e d i n g (Lampert 1978). The high zooplankton biomass i n the f r o n t a l and s t r a t i f i e d s t a t i o n s suggests that Such p r o c e s s e s may have c o n t r i b u t e d to the l o s s of PON, as DON, d u r i n g the i n c u b a t i o n s . I t i s not c l e a r why s i m i l a r l o s s e s were not seen in the s t r a t i f i e d s t a t i o n . E x c r e t i o n of DON by phytoplankton has been rep o r t e d (Newell et al . 1972, Mague et al . 1980). The i n t e r p r e t a t i o n of these r e s u l t s i s c o n s i s t e n t with the 1 5 a n a l y s i s by Laws (1984) that l o s s e s of N seen i n these data and from p r e v i o u s l y p u b l i s h e d r e s u l t s c o u l d be a t t r i b u t e d to 1 5 l o s s e s of DO N, at l e a s t f o r experiments l a s t i n g 6 h. In s t r a t i f i e d water, the d i f f e r e n c e s between APON and A P T are e x a c t l y o p p o s i t e to the r e s u l t s from the f r o n t a l s t a t i o n . Anomalously high PON values i n d i c a t e the phytoplankton must be u t i l i z i n g a d d i t i o n a l n i t r o g e n sources other than those accounted f o r in the i n i t i a l mass balance. These compounds are most probably DON as the n i t r o g e n f i x i n g 1 5 + microorganisms were absent from the samples. In the NH 4 -s p i k e d samples, ^ V j i s i n good agreement with APON sug g e s t i n g that no a d d i t i o n a l n i t r o g e n was r e q u i r e d to account f o r the i n c r e a s e i n PON; a l s o , at the end of the experiment, A P T i s the same as APON. T h i s i s c e r t a i n l y not true f o r the 15 - 15 NO^ and N-urea-spiked samples but reason(s) f o r t h i s d i f f e r e n c e are not apparent. Since NH 4 + i n h i b i t s uptake of NO^ and urea, i t may a l s o i n h i b i t DON u t i l i z a t i o n by phytoplankton. C h a r a c t e r i z a t i o n of seawater DON remains an enigma and c u r r e n t e s t i m a t e s suggest that f r e e and combined amino a c i d s and humic a c i d s can account fo r only 50% of the DON pool (Sharp 1983). Wheeler et al. (1974) and Geesey and M o r i t a (1979) have shown that these types of compounds can be u t i l i z e d by marine phytoplankton and b a c t e r i a , r e s p e c t i v e l y . S i m i l a r l y , H o l l i b a u g h (1978) has reported degradation of s e v e r a l amino a c i d s i n n a t u r a l seawater samples incubated i n the dark. Support f o r in s i t u DON u t i l i z a t i o n i s s c a r c e , but i n d i r e c t evidence from depth p r o f i l e s i n the Indian Ocean (Fraga 1966) and work by Armstrong et al . (1966) showed sea-s u r f a c e d e p l e t i o n of DON r e l a t i v e to deep samples. More s i g n i f i c a n t l y , F i s h e r and Cowdell (1982) repo r t e d that e i g h t diatom c l o n e s were ab l e to u t i l i z e at l e a s t some n a t u r a l DON. A schematic r e p r e s e n t a t i o n of s i m p l i f i e d n i t r o g e n c y c l e s in f r o n t a l and s t r a t i f i e d water i s presented i n F i g u r e 15. It i s i m p o s s i b l e to d i s t i n g u i s h between d i r e c t u t i l i z a t i o n of DON or i n d i r e c t u t i l i z a t i o n v i a regenerated NH 4 + and/or urea in s t r a t i f i e d water. 9 7 F i g . 15. Schematic diagram of n i t r o g e n c y c l i n g i n the euphotic zone of f r o n t a l and s t r a t i f i e d water. Arrows i n d i c a t e major pathways of n i t r o g e n t r a n s f o r m a t i o n between the v a r i o u s p o o l s ; other pathways are excluded s i n c e they were not found to be dominant i n these experiments. The d i s s o l v e d o r g a n i c n i t r o g e n p o o l i n c l u d e s amino a c i d s , p r o t e i n s and other n i t r o g e n c o n t a i n i n g macromolecules. E x c r e t i o n may i n v o l v e a c t i v e and p a s s i v e p r o c e s s e s . U P T A K E Frontal Water Stratified Water 00 9 9 Summary In both f r o n t a l and s t r a t i f i e d water of the S t r a i t of Ge o r g i a , changes i n d i s s o l v e d n i t r o g e n c o n c e n t r a t i o n s p r o v i d e d evidence f o r the simultaneous uptake of ammonium, n i t r a t e and urea by a summer phytoplankton community. C h l o r o p h y l l a s p e c i f i c uptake r a t e s of ammonium and urea were ca. 2 and 2.4 times g r e a t e r i n s t r a t i f i e d water than i n f r o n t a l water; whereas, c h l o r o p h y l l a s p e c i f i c n i t r a t e uptake r a t e s were ca. 1.6 times g r e a t e r i n f r o n t a l water. Ammonium and urea r e g e n e r a t i o n r a t e s , c a l c u l a t e d using a mass balance approach, were s i m i l a r i n f r o n t a l water, but urea r e g e n e r a t i o n r a t e s were 2 to 5 times g r e a t e r i n the s t r a t i f i e d water d u r i n g the f i r s t 12 h of the experiment. Increases i n p a r t i c u l a t e n i t r o g e n c o u l d not be accounted for by corresp o n d i n g decreases in t o t a l 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 i t r o g e n and 15 urea, or by N accumulation i n the p a r t i c u l a t e s . In f r o n t a l water, the change i n t o t a l 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 urea c o n s i s t e n t l y overestimated the change i n p a r t i c u l a t e n i t r o g e n , while i n s t r a t i f i e d water, the change i n t o t a l 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 urea c o n s i s t e n t l y underestimated the change i n p a r t i c u l a t e n i t r o g e n . These data suggest that the plankton community i n f r o n t a l water was l o s i n g n i t r o g e n i n the form of d i s s o l v e d o r g a n i c n i t r o g e n . By c o n t r a s t , the plankton community i n s t r a t i f i e d water took up n i t r o g e n compounds which were not measured as pa r t of the t o t a l d i s s o l v e d i n o r g a n i c and urea n i t r o g e n , but were most l i k e l y d i s s o l v e d o r g a n i c n i t r o g e n compounds. These r e s u l t s 1 00 s t r e s s the importance of de t e r m i n i n g uptake r a t e s of a l l three n i t r o g e n s u b s t r a t e s (NH^ +, NO^ and urea) u s i n g 1 5 N isotopes and by s i m u l t a n e o u s l y measuring the change i n c o n c e n t r a t i o n of these compounds throughout the i n c u b a t i o n p e r i o d . CHAPTER 3. UREA UPTAKE BY SARGASSO SEA PHYTOPLANKTON: SATURATED AND IN SITU UPTAKE RATES Background R e s u l t s of recent s t u d i e s have c o n t r a d i c t e d our c o n v e n t i o n a l view of phytoplankton n i t r o g e n n u t r i t i o n , i n which the m a j o r i t y of the phytoplankton n i t r o g e n requirements are met by ammonium ( N H 4 + ) . In f a c t , these s t u d i e s showed th a t urea i s q u a n t i t a t i v e l y as important as, i f not more important than, NH 4 + f o r phytoplankton n u t r i t i o n i n c e r t a i n ocean environments (Kaufman et al . 1983, H a r r i s o n et a l . 1985) . Urea uptake by phytoplankton i n the o l i g o t r o p h i c ocean has been p o o r l y s t u d i e d . Rapid urea turnover times of l e s s than one day were measured by Herbland (1976) i n the t r o p i c a l South A t l a n t i c Ocean. N e v e r t h e l e s s , s i n c e 1 4 C - u r e a was used i n these experiments, the c o n t r i b u t i o n of urea-N to phytoplankton growth was not addressed. Without e x c e p t i o n , i n other o l i g o t r o p h i c environments, turnover times of urea are on the order of s e v e r a l days to months (Remsen et a l . 1974, Eppley et al. 1973, 1977, Mitamura and S a i j o 1980a, Kanda et al . 1986). These r e s u l t s imply that urea i s not an important n i t r o g e n compound in these regions or that the organisms which u t i l i z e urea are growing s l o w l y . T h i s study examines urea uptake by phytoplankton communities i n the western North A t l a n t i c Ocean. S a t u r a t i n g 14 15 c o n c e n t r a t i o n s of C- and N-urea were added to seawater samples to determine maximum urea uptake r a t e s and to compare 1 02 the uptake of both i s o t o p e s . Rates of n i t r a t e and ammonium uptake were measured c o n c u r r e n t l y . The r a t i o of maximum urea uptake rate// n situ urea uptake r a t e was used to evaluate p h y s i o l o g i c a l d i f f e r e n c e s between phytoplankton communities i n these waters. Mater ia l s and Methods Sample collection Experiments were performed on board the R/V Cape Hatt e r a s d u r i n g August 1985 ( C r u i s e : C o r s a i r 10) at one s t a t i o n i n c o n t i n e n t a l slope water, SI (36°30'N; 74°35*W) and two s t a t i o n s i n the Sargasso Sea, S2 (33°12'N; 66°28'W) and S3 (33°54'N; 70°17'W) ( F i g . 16). Water samples were c o l l e c t e d w i t h 5 - l i t r e G l o - F l o b o t t l e s from depths c o r r e s p o n d i n g to 100, 55, 30, 10, 3 and 1% of the sea- s u r f a c e i r r a d i a n c e . A l i g h t e x t i n c t i o n c o e f f i c i e n t o f 0.09 m 1 and 0.04 m 1 , measured on a p r e v i o u s c r u i s e (R. R i v k i n , p e r s . comm.), was used to determine these depths i n c o n t i n e n t a l slope water and the Sargasso Sea, r e s p e c t i v e l y . Samples were c o l l e c t e d from S1 at 11:30, S2 at 09:30 and S3 at 11:20 h. Water from each depth was di s p e n s e d i n t o 2 0 - l i t r e (R) Nalgene^ carboys covered with black p l a s t i c to shade the samples c o l l e c t e d from low l i g h t . Physical, chemical and biological analyses V e r t i c a l p r o f i l e s of temperature and s a l i n i t y were obt a i n e d c o i n c i d e n t l y with the b o t t l e c a s t s with a N e i l Brown 16. L o c a t i o n of s t a t i o n s i n the western North A t l a n t i c Ocean. S1 i s i n c o n t i n e n t a l slope water, S2 and S3 are i n the Sargasso Sea. 1 04 105 CTD attached to the r o s e t t e sampler. Samples f o r n u t r i e n t a n a l y s i s were f i l t e r e d through combusted (460°C f o r 4h) Whatman GF/F f i l t e r s u s i n g an acid-washed s y r i n g e and a 25 mm (R) M i l l i p o r e Swinex 4^ f i l t e r h o lder i n t o acid-washed p o l y p r o p y l e n e sample b o t t l e s and s t o r e d f r o z e n (-20°C). Ammonium (NH^ +) c o n c e n t r a t i o n was determined i n f r e s h samples taken from a separate b o t t l e c a s t at the same s t a t i o n u s ing the manual method of Solorzano (1969). The f r o z e n samples were a n a l y z e d f o r NH 4 + and n i t r a t e p l u s n i t r i t e (N0 3 + N0 2 ) with a Technicon Autoanalyzer-^ II f o l l o w i n g the procedure of Slawyk and Maclsaac (1972) and Wood et al . (1967), r e s p e c t i v e l y . Urea was determined by the d i a c e t y l monoxime t h i o s e m i c a r b i z i d e technique d e s c r i b e d i n Chapter 1. C h l o r o p h y l l a ( c h i a) samples (1.0 l i t r e ) were f i l t e r e d onto Whatman GF/F f i l t e r s and s t o r e d frozen i n a d e s i c c a t o r . Samples were e x t r a c t e d i n 90% acetone and analyzed by in vitro fluorometry ( S t r i c k l a n d and Parsons 1972) using a Turner Designs model 111 fluorometer. P a r t i c u l a t e o rganic carbon and n i t r o g e n (POC and PON) samples (1.0 l i t r e ) , c o l l e c t e d on combusted Whatman GF/F f i l t e r s , were s t o r e d s i m i l a r l y and an a l y z e d with a C a r l o Erba Elemental Analyzer model 1106. Saturated uptake experiments 1 5 + Experiments were conducted u s i n g N - l a b e l l e d NH^ and 15 14 NO^ and both N- and C - l a b e l l e d urea. A l l n i t r o g e n uptake experiments were i n i t i a t e d w i t h i n 1 h of sampling. (R) Incubations were conducted on deck i n c l e a r P l e x i g l a s 4 ^ i n c u b a t o r s , c o o l e d with s u r f a c e seawater. Where necessary, 1 06 i n c u b a t i o n b o t t l e s were covered with n e u t r a l d e n s i t y s c r e e n i n g to s i m u late the in situ l i g h t regime at each sample depth. ^N-labelled substrates 15 + S a t u r a t i n g a d d i t i o n s of N - l a b e l l e d NH^ , NO^ or urea 1 5 ( a l l 99 at % N) were added to separate 1200 ml Wheaton g l a s s b o t t l e s , and measurements were d u p l i c a t e d f o r a l l N s u b s t r a t e s at each depth. N H 4 + and urea uptake r a t e s were determined at s i x depths, but NO^ uptake was only measured at the 55 and 3% se a - s u r f a c e l i g h t depths. Water was not prescreened through N i t e x ^ n e t t i n g because of the abundance of Tri chodesmi um c o l o n i e s at 2 of the 3 s t a t i o n s . In water c o l l e c t e d from the 1 5 55% s e a - s u r f a c e l i g h t depth, i n c o r p o r a t i o n of N-urea was measured at 6 h i n t e r v a l s over the 24 h in c u b a t i o n to t e s t f o r constant uptake at each s t a t i o n . Experiments were terminated by f i l t r a t i o n (vacuum l e s s than 100 mm Hg) onto combusted Whatman GF/F f i l t e r s , and the samples were s t o r e d f r o z e n i n a d e s i c c a t o r . N i t r o g e n i n the p a r t i c u l a t e matter was converted to N 2 (g) by the micro-Dumas dry combustion technique as 1 5 d e s c r i b e d by LaRoche (1983), and then analyzed f o r N en-richment with a JASCO model N-150 emission spectrometer 1 5 ( F i e d l e r and Proksch 1975). N uptake r a t e s were c a l c u l a t e d a c c o r d i n g to the equ a t i o n s of Dugdale and Goering (1967) except that i n i t i a l PON measurements were used i n p l a c e of f i n a l PON. C o r r e c t i o n f o r changes i n the i n i t i a l PON due to uptake of added N d u r i n g the i n c u b a t i o n were made as d e s c r i b e d by C o l l o s (1984). 107 14 C-l abelI ed substrates 1 5 Concurrent with the s a t u r a t i n g N uptake experiments, 1 4 C - u r e a (Amersham) was added at s a t u r a t i n g (10 ug at N-1 1 ; 0.072 GBq-mmol~ 1) and t r a c e (5 ng at N - l ~ 1 ; 2.04 GBq-mmol~1) c o n c e n t r a t i o n s . A subsample (100 ml) from one of the d u p l i c a t e 1200 ml Wheaton g l a s s i n c u b a t i o n b o t t l e s was taken a f t e r 6 h of i n c u b a t i o n , and samples were taken again from 1 4 both b o t t l e s a f t e r 24 h. Uptake of C-urea was determined by 1 4 measuring CC>2 r e l e a s e d i n t o the seawater, as a r e s u l t of 1 4 C - u r e a h y d r o l y s i s , and by measuring the q u a n t i t y of 1 4 C i n the p a r t i c u l a t e matter r e t a i n e d by a Whatman GF/F f i l t e r . The p a r t i c u l a t e matter was c o l l e c t e d by gen t l e f i l t r a t i o n , and the f i l t e r s were r i n s e d w i t h f i l t e r e d (0.45 um M i l l i p o r e f i l t e r ) seawater. To c o r r e c t f o r a b i o t i c degradation of urea, f o r m a l i n - k i l l e d (1 ml, 20% forma l i n ) c o n t r o l s were run with each set of samples. Immediately f o l l o w i n g the a d d i t i o n of 1 4 C-urea, samples were withdrawn and f i l t e r e d to determine the amount of f i l t e r a d s o r p t i o n . To determine the amount of 1 4 C 0 2 i n the f i l t r a t e , s m a l l g l a s s v i a l s c o n t a i n i n g f i l t e r paper impregnated with 50 u l phenethylamine were suspended i n s i d e 250 ml Erlenmeyer f l a s k s c o n t a i n i n g 100 ml of the sample. The f l a s k s were s e a l e d w i t h rubber septa, and 0.5 ml 6N HCl was added to the f i l t r a t e . A f t e r 24 h, the f i l t e r s were removed from the f l a s k s and p l a c e d i n s c i n t i l l a t i o n v i a l s c o n t a i n i n g 1 4 10 ml Aquasol I I . Recovery of C0 2 was determined to be 62.5%. Uptake of urea was c a l c u l a t e d by adding the amount of 1 4 CC>2, which was produced d u r i n g the in c u b a t i o n and the amount 108 1 4 of C r e t a i n e d by the f i l t e r minus the c o n t r o l s . These uptake r a t e s are a c t u a l l y urea h y d r o l y s i s r a t e s and represent 1 4 a measurement based on C and not n i t r o g e n . To convert these 1 4 r a t e s to n i t r o g e n uptake r a t e s , I assumed that f o r each C appearing i n C0 2, or i n the p a r t i c u l a t e f r a c t i o n , 2 n i t r o g e n atoms were taken up (see D i s c u s s i o n ) . In si t u urea upt ake rat es 1 4 Trace a d d i t i o n s of C-urea were added to 1200 ml water samples c o l l e c t e d from the 55 and 3% sea-surface l i g h t depths. D u p l i c a t e 5 ml samples taken from each b o t t l e at the s t a r t of the experiment were added to s c i n t i l l a t i o n v i a l s c o n t a i n i n g 10 ml of f l u o r . At 2, 4, and 6 h, d u p l i c a t e 100 ml samples were f i l t e r e d through Whatman GF/F f i l t e r s , and the f i l t e r s were r i n s e d with 15 ml f i l t e r e d seawater. P r e c a u t i o n was taken to ensure t h a t the f i l t e r s d i d not become dry p r i o r to r i n s i n g . The f i l t r a t e (25 ml) was dispensed i n t o 50 ml Erlenmeyer 14 14 f l a s k s . To remove CC>2 produced from C-urea h y d r o l y s i s , the f i l t r a t e was a c i d i f i e d by adding 0.5 ml 6H HC1 and allowed to degas i n a vacuum d e s i c c a t o r under reduced pressure f o r 12 1 4 h. T h i s procedure removed a l l C0 2 from the samples. The data were f i t t e d to an e x p o n e n t i a l equation to determine the r a t e c o e f f i c i e n t . The r e c i p r o c a l of t h i s c o e f f i c i e n t i s the turnover time of d i s s o l v e d urea measured i n 14 • hours. Trace C-urea uptake r a t e s were determined from the product of the turnover time and the ambient d i s s o l v e d urea 1 4 c o n c e n t r a t i o n . A l l C samples were counted by Packard T r i -Carb 460c l i q u i d s c i n t i l l a t i o n c o u nter, and the standard d e v i a t i o n of each count was l e s s than 2%. Re s u l t s Environmental parameters I n i t i a l biomass data f o r the three s t a t i o n s are presented in Table X I I I . POC:PON was s i m i l a r i n c o n t i n e n t a l slope water and the Sargasso Sea; however, PON/chl a measurements i n d i c a t e d that there was c o n s i d e r a b l y more d e t r i t a l N i n the Sargasso Sea samples. T h i s was p a r t i c u l a r l y e v i d e n t i n the deep samples, where the PON/chl a was 8 times g r e a t e r than p a r t i c u l a t e matter i n slope water c o l l e c t e d from a s i m i l a r l i g h t regime. S i z e f r a c t i o n a t e d measurements i n d i c a t e d that ca. 90% of the c h l a was i n the l e s s than 3 um s i z e f r a c t i o n (R. R i v k i n , unpublished r e s u l t s ) . D i s s o l v e d NO^ + N0 2 were d e t e c t a b l e throughout the surface-mixed l a y e r at S t a t i o n s 1 and 2, but were undetectable at S t a t i o n 3 except at 115 m ( F i g . 17). These a n a l y s e s d i d not d i f f e r e n t i a t e between NO^ and N0 2 ; s i n c e N0 2 i s g e n e r a l l y i n lower abundance, I have r e f e r r e d to these measurements as NO^ c o n c e n t r a t i o n s . NH^ + c o n c e n t r a t i o n was determined i n f r e s h l y c o l l e c t e d samples from an e a r l i e r c a s t at the same s t a t i o n . These val u e s were o f t e n d e t e c t a b l e ; with the e x c e p t i o n of S t a t i o n 3, they were lower than the NH 4 + c o n c e n t r a t i o n s measured i n the f r o z e n samples. Data are reported f o r the f r o z e n samples. Urea c o n c e n t r a t i o n was undetectable i n the s u r f a c e and at 15 m at S t a t i o n s 2 and 3. Table XIII Biomass data from water samples c o l l e c t e d f o r n i t r o g e n uptake experiments. Mixed S t a t i o n Date l a y e r Sample Chl a PON POC:PON depth depth (m) (m) ( u g - 1 H u g at N-1 ) (atoms) 1 4/VIII/85 18 2 8/VIII/85 25 3 13/VIII/85 28 0 0.12 0.75 11.7 6.6 0.13 0.75 11.3 13 0.19 0.82 9.6 26 1 .65 1 .93 7.1 38 0.33 0.64 8.1 51 0.13 0.42 9.9 0 0.028 0.33 11.7 1 5 0.030 0.44 10.5 30 0.044 0.48 10.2 58 0.15 0.48 8.1 88 0.14 0.35 11.8 1 1 5 0.067 0.32 11.5 0 0.037 0.29 11.4 1 5 0.034 0.42 9.9 30 0.042 0.44 9.2 58 0.071 0.47 9.6 88 0.13 0.36 9.4 1 1 5 0.047 0.31 13.0 111 F i g . 17. D i s s o l v e d NH^ +, NO^ + N0 2 and urea c o n c e n t r a t i o n (ug at N-1 1) measured i n f i l t e r e d f r o z e n samples c o l l e c t e d from (A) S t a t i o n 1 ( c o n t i n e n t a l slope water), (B) S t a t i o n 2 (Sargasso Sea), and (C) S t a t i o n 3 (Sargasso Sea). 1 13 upt ake 1 5 Time course measurements of N-urea uptake by plankton c o l l e c t e d from the 50% s e a - s u r f a c e l i g h t depth demonstrated 1 5 that i n c o r p o r a t i o n of N was cons t a n t over the 24 h i n c u b a t i o n ( F i g . 18). S i m i l a r time course experiments were not run f o r NH 4 + or NC>3 . 1 5 Sat u r a t e d N uptake r a t e s were hi g h e r i n slope water than i n the Sargasso Sea, because phytoplankton biomass (measured as c h i a) was an order of magnitude gre a t e r ( F i g . 19A). At a l l depths i n slope water, NH 4 + uptake r a t e s were 2-3 times g r e a t e r than urea uptake r a t e s and 3-3.5 times g r e a t e r than NO^ uptake r a t e s . N i t r o g e n - s p e c i f i c uptake r a t e s of NH 4 + ranged from 0.15 to 0.70 d 1 . N0 3 uptake r a t e (0.031 + 0.012 ug at N - l ~ 1 - d ~ 1 ) i n the 38 m water sample was r e p r e s e n t a t i v e of the in situ uptake r a t e , s i n c e ambient N0 3 c o n c e n t r a t i o n s were high,(5.3 ug at N-1 1 ) . At S t a t i o n 2, urea uptake r a t e s were l e s s than N H 4 + uptake r a t e s ( F i g . 19B). Urea uptake r a t e s were the same at the three depths w i t h i n the surface-mixed l a y e r ; however, i n the upper p o r t i o n of the c h l o r o p h y l l maximum, the urea uptake rate decreased. In the s u r f a c e waters of S t a t i o n 3, l i t t l e uptake of NH 4 + was evi d e n t , but NH 4 + uptake i n c r e a s e d with depth ( F i g . 19C). The s a t u r a t e d urea uptake r a t e s were 5 times g r e a t e r than the NH 4 + uptake r a t e s , and N0 3 uptake r a t e s were a l s o h i g h e r . Below the p y c n o c l i n e , NH 4 + uptake r a t e s i n c r e a s e d and were g r e a t e r than both NO- and urea uptake r a t e s . 18. Time course of i n c o r p o r a t i o n of N-urea i n t o p a r t i c u l a t e matter i n water samples c o l l e c t e d from 6.6 m at S t a t i o n 1 (•), and 15 m at S t a t i o n s 2 (•) and 3 (O). Incubation times were measured from the s t a r t i n g time of the i n c u b a t i o n . The dark p e r i o d d u r i n g the time course at S t a t i o n s 1 and 3 o c c u r r e d between 8.5 and 18 h and between 10.5 and 20 h at S t a t i o n 2. 1 5 N - UPTAKE (atom % excess) 91 L 1 1 6 F i g . 19. D a i l y n i t r o g e n uptake r a t e s determined with 15 + -s a t u r a t i n g a d d i t i o n s of N - l a b e l l e d NH^ , NO^ and urea by plankton i n seawater samples c o l l e c t e d at (A) S t a t i o n 1, (B) S t a t i o n 2 and (C) S t a t i o n 3. E r r o r bars are the range of d u p l i c a t e samples. NITROGEN UPTAKE RATE (yg otN- l"'d"') 0.3 0 777777* H 7777H 10 -I t-D-20 30 40 60 ////////////////A—I. 0.6 0.9 -I 1 b 1.2 0 0.02 0.04 0.06 0.08 0.02 0.04 0.06 0.08 £3 U R E A | | AMMONIUM • N I T R A T E 20 40 60 80 100 ////////////////77\—H 7////7/~J//////A—\ "i r 77>A 6 3F 77777/77///77/7/ 20 40 60 80 100 I Z Z Z Z p ^ Z Z Z Z Z Z Z Z Z Z Z Z Z H ZZZZ2& "i r ///;/////////////7\—i ////////s////////\ 7//////Jt-{ 1 18 ^^C-urea uptake 14 14 I n c o r p o r a t i o n of. C, from C-urea, i n t o the p a r t i c u l a t e matter was n e g l i g i b l e i n a l l water samples except i n the c h l o r o p h y l l maximum (ca. 10% s u r f a c e l i g h t d e p t h ) . At S t a t i o n 1 4 . 1, C r e t a i n e d by the f i l t e r accounted f o r 63% of the t o t a l urea taken up a f t e r 6 h. There was no f u r t h e r i n c o r p o r a t i o n 14 of C durin g the l a s t 18 h, but some urea c o n t i n u e d to be 1 4 taken up. C i n c o r p o r a t i o n i n t o the p a r t i c u l a t e matter over the 24 h i n c u b a t i o n amounted to 3 and 4% at S t a t i o n s 2 and 3, 1 4 r e s p e c t i v e l y ; no measurable r e t e n t i o n of C o c c u r r e d d u r i n g 14 14 the f i r s t 6 h. A l l of the C i n C-urea was r e l e a s e d i n t o 1 4 the seawater as c®2' 14 15 The C-urea uptake r a t e s were g r e a t e r than N-urea uptake r a t e s , determined over the same i n c u b a t i o n p e r i o d , at the three s t a t i o n s (Table XIV). There were no apparent t r e n d s 1 4 with depth or between s t a t i o n s . C-urea uptake r a t e s were on 1 5 average 1.4 times g r e a t e r than N-urea uptake r a t e s , but at 15 14 two depths N-urea uptake r a t e s exceeded C-urea uptake r a t e s . 15 14 By c o n t r a s t to N-urea uptake, C-urea uptake was not constant over the 24 h i n c u b a t i o n p e r i o d . On an h o u r l y b a s i s , 14 the C-urea uptake r a t e d u r i n g the f i r s t 6 h g r e a t l y exceeded the rat e measured over the 24 h i n c u b a t i o n , i n extreme cases by as much as 25 times (Table XV). In seven of the twelve 1 4 samples from the Sargasso Sea, l e s s C-urea uptake was measured d u r i n g the 24 h i n c u b a t i o n than over the f i r s t 6 h. 1 4 . 1 4 . Since only the C0 2 i n the seawater and the C r e t a i n e d by 1 19 Table XIV 15 14 Comparison of N-urea and C-urea uptake r a t e s determined over 24 h i n c u b a t i o n s . Rates of 1 4 C - u r e a uptake were c o n v e r t e d to e q u i v a l e n t n i t r o g e n uptake r a t e s as 1 5 d e s c r i b e d i n the t e x t . Values i n brackets are d a i l y N-urea uptake r a t e s (ug at N-1 1 - d 1 ) . N Uptake Rate/ C Uptake Rate L i g h t Depth SI S2 S3 (% of s u r f a c e i r r a d i a n c e ) 100 0.54 (0.22) 0.62 (0.038) 0.25 (0.023) 55 0.68 (0.15) 0.78 (0.041) 0.63 (0.043) 30 1.30 (0.24) 0.32 (0.046) 0.88 (0.052) 10 0.76 (0.62) 0.06 (0.006) 0.67 (0.048) 3 0.86 (0.041) 0.84 (0.036) 1.63 (0.043) 1 0.48 (0.022) 0.60 (0.024) 0.62 (0.018) 1 20 Table XV R a t i o of 1 4 C - u r e a uptake r a t e s (ug at urea-C-1 1- h 1 ) determined d u r i n g 6 and 24 h i n c u b a t i o n s at t h r e e s t a t i o n s (S1, S2, and S3) i n water samples c o l l e c t e d from 6 depths. 1 4 Values i n b r a c k e t s are the C-urea uptake r a t e s d u r i n g 6 h i n c u b a t i o n s (ug at urea-C-1 1-h 1 ) . 6 h Incubation/24 h I n c u b a t i o n L i g h t Depth S1 S2 S3 (% of s u r f a c e i r r a d i a n c e ) 100 0.32 (0.0055) 25.0 (0.065) 4.68 (0.018) 55 1.39 (0.013) 2.49 (0.0054) 4.54 (0.013) 30 1.64 (0.013) 16.3 (0.090) 4.78 (0.012) 10 1.06 (0.036) 1.76 (0.0075) 3.90 (0.012) 3 1.69 (0.0034) 13.0 (0.023) 2.81 (0.0031) 1 1.28 (0.0024) 11.9 (0.020) 2.92 (0.0035) the p a r t i c u l a t e matter was accounted f o r d u r i n g these uptake 1 4 experiments, i t i s p o s s i b l e that some C was l o s t to a compartment which was not measured by these methods. At S t a t i o n 1, the r a t e s c a l c u l a t e d from the 6 and 24 h i n c u b a t i o n s were i n f a i r agreement, and the 6 h i n c u b a t i o n r a t e was on average 1.2 times f a s t e r . In the s u r f a c e water sample, there was an i n i t i a l l a g i n urea uptake. At S t a t i o n 2, the h o u r l y uptake r a t e s d u r i n g the 6 and 24 h i n c u b a t i o n s were most d i s s i m i l a r . In n e i t h e r of these s t a t i o n s were any depth r e l a t e d t r e n d s apparent. T h i s c o n t r a s t s the r e s u l t s from S t a t i o n 3 where the g r e a t e s t d i s p a r i t y between the r a t e s measured d u r i n g the 6 and 24 h i n c u b a t i o n s o c c u r r e d i n the 1 4 surface-mixed l a y e r . Below 28 m, the 6 and 24 h C-urea uptake r a t e s were i n b e t t e r agreement. In situ urea uptake rates 1 4 The disappearance of C-urea from seawater samples 1 4 sp i k e d with 5 nM C-urea i s shown i n F i g u r e 20. The slopes of these c u r v e s , c a l c u l a t e d from an e x p o n e n t i a l r e g r e s s i o n , equal the turnover r a t e s of urea. The average turnover time of urea i n the surface-mixed l a y e r of the Sargasso Sea was 13 h; t h i s was s l i g h t l y f a s t e r than the tur n o v e r time i n slope water (Table XVI). At the 3% su r f a c e l i g h t depth, the turnover times of urea were slower than i n s u r f a c e samples. In situ urea uptake r a t e s , r e f e r r e d to here as urea t r a c e uptakes or V t r a c e , c o u l d not be a c c u r a t e l y computed i n instances where the ambient urea c o n c e n t r a t i o n was below d e t e c t i o n . In these cases, I assumed 1 22 F i g . 20. The amount of C-urea remaining i n seawater dur i n g 6 h time course experiments. Water was c o l l e c t e d from the 55 (O) and 3% (•) s e a - s u r f a c e l i g h t depths at (A) S t a t i o n 1 ( c o n t i n e n t a l slope water), (B) S t a t i o n 2 (Sargasso Sea), and (C) S t a t i o n 3 (Sargasso Sea). P a r t i c u l a t e matter was removed from samples by f i l t r a t i o n , and the f i l t r a t e was a c i d i f i e d to remove 14 . . CC>2 • The remaining a c t i v i t y was measured. E r r o r bars are the range of d u p l i c a t e subsamples from a s i n g l e sample b o t t l e . 1 24 Table XVI Turnover times of d i s s o l v e d urea c o n c e n t r a t i o n i n surface-mixed and deep water at S t a t i o n s 1, 2 and 3. The c o r r e l a t i o n c o e f f i c i e n t f o r each e x p o n e n t i a l equation i s gi v e n . D i s s o l v e d urea c o n c e n t r a t i o n s are the mean of d u p l i c a t e s (± 1 SD). 2 S t a t i o n Depth H y d r o l y s i s Turnover r Urea (m) r a t e time (h) c o n c e n t r a t i o n ( d ~ 1 ) (ug at N - l " 1 ) 6.6 38 1 5 88 15 88 1 .67 0.66 1 .86 0.61 1.81 0.70 21.1 0.953 0.18 + 0.02 58.5 0.834 0.12 + 0.02 12.6 0.923 <0.05 43.6 0.992 0.14+ 0.03 13.3 0.957 <0.05 34.3 0.924 0.14+ 0.07 t h a t the urea c o n c e n t r a t i o n was equal to the l i m i t of d e t e c t i o n : 50 ng at N-1 1 . As a r e s u l t , these estimates of V t r a c e are an upper l i m i t . A comparison of urea uptake r a t e s determined using s a t u r a t i n g and t r a c e a d d i t i o n s of s u b s t r a t e i s g i v e n i n Table XVII. S a t u r a t i n g uptake r a t e s of urea were i n excess of in situ uptake r a t e s at a l l samples with the ex c e p t i o n of the 88 m sample from S t a t i o n 3. In t h i s sample, one of the d i s s o l v e d urea samples was twice as high as the ot h e r , consequently the mean value may be too h i g h . T h i s r e s u l t s i n an overestimate of the a c t u a l uptake r a t e . Discussion Environmental parameters In o l i g o t r o p h i c waters, c o n c e n t r a t i o n s of d i s s o l v e d NO^ + NC>2 measured by c o l o u r i m e t r i c techniques are u s u a l l y below our l i m i t s of d e t e c t i o n (Garside 1985). T h i s was onl y t r u e i n t h i s study at S t a t i o n 3. Although the samples were f i l t e r e d and s t o r e d frozen before a n a l y s i s , MacDonald and McLaughlin (1982) found t h i s procedure had no e f f e c t on the storage of NC>2 samples. Cochlan (1982) measured an average NO^ + N0 2 c o n c e n t r a t i o n of 0.15 ug at N-1 1 i n the mixed l a y e r at a s i n g l e s t a t i o n i n the northern Sargasso Sea. On a separate c r u i s e d u r i n g J u l y 1986, a n a l y s i s of f r e s h l y c o l l e c t e d water samples from the Sargasso Sea i n d i c a t e d the presence of low, but d e t e c t a b l e c o n c e n t r a t i o n s of NO^ + N0 2 ( P r i c e , u n p u b l i s h e d r e s u l t s ) . The estimates of the c o n t r i b u t i o n of d e t r i t u s to POC and PON measurements i n t h i s study are' o n l y Table XVII 1 4 Uptake r a t e s of C-urea determined dur i n g 6 h i n c u b a t i o n s i n seawater samples c o l l e c t e d from the 50 and 3% s u r f a c e l i g h t depths at three s t a t i o n s (SI, S2, and S3), and sp i k e d with t r a c e and s a t u r a t i n g c o n c e n t r a t i o n s . Rates are expressed i n terms of urea-N uptake. S t a t i o n Depth V t r a c e Vsat V s a t / V t r a c e (m) (ug at N - l " 1 - h" 1 ) 1 6.6 0.0085 0.0127 1.5 38 0.0021 0.00335 1.6 2 15 0.0039 0.00545 1.4 88 0.0032 0.0233 7.3 3 15 0.0038 0.0130 3.4 88 0.0041* 0.00312 0.8* * One of the d u p l i c a t e measurements of ambient d i s s o l v e d urea c o n c e n t r a t i o n was 2 times the c o n c e n t r a t i o n of the other; consequently, the mean val u e that was used i n the c a l c u l a t i o n may be too h i g h . T h i s would r e s u l t i n an overestimate of the a c t u a l uptake r a t e , and an underestimate of the V s a t / V t r a c e r a t i o . r e l a t i v e . The PON/chl a values r e p o r t e d here are higher than those r e p o r t e d by McCarthy and Nevins (1986) i n a warm-core r i n g and G l i b e r t and McCarthy (1984) i n the Caribbean and the Sargasso Sea. T h i s i n d i c a t e s t hat there was a g r e a t e r d e t r i t a l contamination of the samples c o l l e c t e d i n t h i s study. There was no r e l a t i o n s h i p between PON or POC and c h i a at e i t h e r of the Sargasso Sea s t a t i o n s . Other i n v e s t i g a t o r s have found good r e l a t i o n s h i p s between POC and c h i a and are a b l e to estimate the p r o p o r t i o n of d e t r i t u s i n t h e i r samples from a l i n e a r r e g r e s s i o n of these v a r i a b l e s . In the o l i g o t r o p h i c North P a c i f i c , the p r o p o r t i o n of l i v i n g POC, as estimated from c e l l volumes and numbers and from ATP measurements (Sharp et al. 1980, Winn and K a r l 1984), was ca. 30% of the t o t a l POC. Laws et al. (1985) determined from ATP measurements that 42% of the t o t a l p a r t i c u l a t e C was l i v i n g i n o l i g o t r o p h i c Hawaiian waters. In these s t u d i e s , the PON/chl a i n the s u r f a c e waters ( l e s s than 30 m) were l e s s than the samples I c o l l e c t e d w i t h i n the mixed l a y e r . From these data, I c o n s e r v a t i v e l y estimate d e t r i t a l contamination of POC and PON to be g r e a t e r than 50% of the t o t a l POC and PON present i n the Sargasso Sea s t a t i o n s . Saturated uptake rates 15 L i n e a r i n c o r p o r a t i o n of N over long i n c u b a t i o n times (24 h) has been r e p o r t e d f o r : NO^ and NH 4 +, i n c o a s t a l waters of the S c o t i a n Shelf (Cochlan 1986) and i n a warm-core r i n g (McCarthy and Nevins 1986); NH 4 +, i n Vi n e y a r d Sound (Goldman et al . 1981), and NO^ , NH 4 + and urea i n the e a s t e r n Canadian A r c t i c ( H a r r i s o n 1983b). However, there are g e n e r a l l y 1 28 pronounced d i e l p a t t e r n s i n n i t r o g e n uptake, with r a t e s being g r e a t e s t d u r i n g the d a y l i g h t hours ( C o l l o s and Slawyk 1976, Maclsaac 1978, Olson 1980). Although the 1 5N-urea uptake r a t e s were l i n e a r over the time course, i t i s not known whether NH^ + and NO^ uptake r a t e s were a l s o l i n e a r because time course experiments were not run. Since uptake of NO^ i s known to be more l i g h t dependent than the uptake of e i t h e r + . . 15 NH 4 or urea, i t seems u n l i k e l y that NO^ i n c o r p o r a t i o n was constant over the time c o u r s e . The measured r a t e s of N0 3 uptake at the 3% se a - s u r f a c e l i g h t depth at S t a t i o n s 1 and 2 are in situ uptake r a t e s because ambient NO^ c o n c e n t r a t i o n s were alr e a d y s a t u r a t i n g . N H 4 + and urea uptake r a t e s were g r e a t e r than NO^ uptake r a t e s . I t i s p o s s i b l e t h a t t h i s i s a r e s u l t of reduced NO^ uptake i n the dark, a l t h o u g h Horrigan and McCarthy (1981) found that n i t r a t e - s u f f i c i e n t c u l t u r e s of phytoplankton had enhanced uptake r a t e s f o r N H 4 + and urea. S a t u r a t e d uptake r a t e s of NH 4 +, NO^ and urea demonstrate the p o t e n t i a l c a p a c i t y f o r n i t r o g e n uptake by the plankton communities. These r e s u l t s i n d i c a t e the s p a t i a l v a r i a b i l i t y i n n i t r o g e n uptake by phytoplankton assemblages at d i f f e r e n t depths and between s t a t i o n s . G e n e r a l l y , NH 4 + uptake r a t e s exceeded urea uptake r a t e s ; however, the d i f f e r e n c e s depended upon the depth from which the samples were c o l l e c t e d . The v e r t i c a l d i s t r i b u t i o n of phytoplankton i n ocean water p r o v i d e s evidence f o r v a r i a t i o n i n the community composition with depth. In the Sargasso Sea, maximum c e l l d e n s i t i e s of c h r o o c o c c o i d c y a n o b a c t e r i a occur i n s u r f a c e waters ( l e s s than 40 m) , and the e u k a r y o t i c phytoplankton are found i n g r e a t e s t numbers below t h i s depth (Murphy and Haugen 1985). Most of these p i c o p l a n k t o n have not been t e s t e d f o r t h e i r a b i l i t y to use urea under l a b o r a t o r y c o n d i t i o n s . The u t i l i z a t i o n of urea by the plankton community r e p r e s e n t s an i n t e g r a t e d response of the i n d i v i d u a l s p e c i e s of phytoplankton and b a c t e r i o p l a n k t o n i n the sample. S e l e c t i v e u t i l i z a t i o n of c e r t a i n types of ni t r o g e n compounds by d i f f e r e n t s i z e s of plankton was shown i n s i z e - f r a c t i o n a t e d n i t r o g e n uptake experiments i n the Benguela upwelling and the A n t a r c t i c Ocean (Probyn 1985, Probyn and P a i n t i n g 1985). Although the r e s u l t s p r e s e n t e d here do not allow the same r e s o l u t i o n as obtained by Probyn, they demonstrate that plankton assemblages i n the d i f f e r e n t water samples possess the c a p a b i l i t y to u t i l i z e d i f f e r e n t n i t r o g e n sources to v a r y i n g degrees. Plankton c o l l e c t e d at S t a t i o n 2 from the c h l o r o p h y l l 1 5 maximum had the lowest urea uptake r a t e s measured by N compared with plankton c o l l e c t e d from other depths. NH 4 + uptake r a t e s were maximum at t h i s depth. R e s u l t s from the sur f a c e waters of S t a t i o n 3 are unique: urea uptake r a t e s were much g r e a t e r than NH 4 + uptake r a t e s . T h i s appears r e l a t e d to a low c a p a c i t y f o r NH 4 + uptake, s i n c e urea uptake r a t e s were s i m i l a r to r a t e s seen at S t a t i o n 2; NO^ uptake r a t e s were a l s o g r e a t e r than NH 4 + uptake r a t e s . T h i s response by the phytoplankton has not been p r e v i o u s l y r e p o r t e d , and i s s u r p r i s i n g i n l i g h t of the almost u n i v e r s a l p r e f e r e n c e of phytoplankton f o r NH. + . Not only does the p r e c o n d i t i o n i n g 1 30 n i t r o g e n source e f f e c t the a b i l i t y of phytoplankton to u t i l i z e other n i t r o g e n sources (Horrigan and McCarthy 1981, 1982), but growth r a t e (Dortch and Conway,1984) and l i g h t regime (Bates 1976) may a l s o be important f a c t o r s . Urea uptake: and tracers 1 4 The d i s c r e p a n c y between C-urea uptake r a t e s d u r i n g 6 1 5 and 24 h i n c u b a t i o n s c o n t r a s t s the l i n e a r uptake of N-urea at the three s t a t i o n s . These r e s u l t s provide new i n f o r m a t i o n r e g a r d i n g urea c y c l i n g i n seawater and i t s uptake and a s s i m i l a t i o n by p l a n k t o n i c organisms. Measured over short time i n t e r v a l s (on the order of minutes), the i n i t i a l uptake 14 15 r a t e s of C- and N-urea represent i n f l u x of urea. As i n c u b a t i o n times i n c r e a s e the t r a n s p o r t e d s u b s t r a t e i s metabolized, and the r a t e of metabolism may r e g u l a t e the 14 15 uptake r a t e . A comparison of the u t i l i z a t i o n of C- and N-l a b e l l e d urea i s confounded by the f a c t that urea i s degraded r a p i d l y by the p l a n k t o n and the r a d i o a c t i v e ( 1 4 C ) and s t a b l e 1 5 ( N) i s o t o p e l a b e l s are metabolized by d i f f e r e n t pathways. 1 5 The l e n g t h of the i n c u b a t i o n time r e q u i r e d to measure N i n c o r p o r a t i o n i n the o l i g o t r o p h i c ocean i s of s u f f i c i e n t d u r a t i o n that the t r a n s p o r t e d s u b s t r a t e i s metabolized. Wheeler et al. (1982) demonstrated that g r e a t e r than 80% of 15 + the NH 4 taken up by Chesapeake phytoplankton was i n c o r p o r a t e d i n t o p r o t e i n w i t h i n 15 minutes. Even i f r a t e processes are slower i n open ocean water compared to c o a s t a l 15 r e g i o n s , N uptake may a l s o be sub j e c t to r e g u l a t i o n by 131 feedback mechanisms a s s o c i a t e d with enzymatic n i t r o g e n 1 4 a s s i m i l a t i o n . C-urea uptake r a t e s are determined from ra t e s of urea h y d r o l y s i s , which c e r t a i n l y cannot be confused with a transmembrane f l u x . The o b s e r v a t i o n of n e g l i g i b l e r e t e n t i o n of 1 4 C by the p a r t i c u l a t e matter i s i n agreement with Herbland (1976), but c o n t r a s t s with the r e s u l t s of Mitamura and S a i j o (1975). T h i s may be e x p l a i n e d by the much g r e a t e r phytoplankton biomass in the c o a s t a l water samples of Mitamura and S a i j o (1975). The 1 4 only i n s t a n c e where i n c o r p o r a t i o n of C i n t o the p a r t i c u l a t e f r a c t i o n was s i g n i f i c a n t was i n a water sample which contained one to two o r d e r s of magnitude more c h l a. The f a t e of urea-N i s u n c l e a r from the 1 4 C - u r e a t r a c e r uptake s t u d i e s ; however, from these r e s u l t s and those of H a r r i s o n et al. 1985, i t appears that o n l y 50-80% of the n i t r o g e n from urea i s i n c o r p o r a t e d i n t o p a r t i c u l a t e matter r e t a i n e d by GF/F f i l t e r s . R e s u l t s from t h i s study a l s o demonstrate that there are temporal d i f f e r e n c e s i n the f a t e of 14 15 14 the C and N t r a c e r s . I n i t i a l r a t e s of C-urea h y d r o l y s i s i n d i c a t e that urea i s r a p i d l y h y d r o l y z e d to CC>2 and NH 3. The o b s e r v a t i o n that i n i t i a l r a t e s of urea h y d r o l y s i s were g r e a t e s t at S t a t i o n 2 compared with the other 2 s t a t i o n s may be a r e s u l t of the e a r l i e r s t a r t i n g time of these i n c u b a t i o n s . 1 4 The only p l a u s i b l e e x p l a n a t i o n to account f o r the l o s s of C during the 24 h i n c u b a t i o n s i s i t s l o s s to a f r a c t i o n not measured by these methods, such as DOC. The i n i t i a l r a p i d h y d r o l y s i s of urea, i n d i c a t e d by the 1 4 l i b e r a t i o n of CO- and the slower r a t e of urea-N uptake, 132 might be a n t i c i p a t e d i f urease were an e x t r a c e l l u l a r enzyme. However, our c u r r e n t knowledge suggests that urease i s an i n t r a c e l l u l a r enzyme i n e u k a r y o t i c microalgae ( L e f t l e y and S y r e t t 1973), c y a n o b a c t e r i a (Berns et al. 1966), and h e t e r o t r o p h i c b a c t e r i a (McLean et al . 1985). Consequently, h y d r o l y s i s r a t e s of urea, when measured over the time i n t e r v a l s such as used i n these experiments, occur on time s c a l e s s i m i l a r to those f o r urea uptake, s i n c e urea must be taken i n t o the c e l l s before i t i s degraded. These r a t e s may be c o n s i d e r e d gross urea uptake r a t e s . To account f o r the lower urea-N uptake r a t e s , I p o s t u l a t e that e i t h e r the N from urea i s l o s t as DON, i n c o r p o r a t e d i n t o p a r t i c u l a t e matter not r e t a i n e d by GF/F f i l t e r s , or that i t d i f f u s e s out of the c e l l s as NH-j p r i o r to being a s s i m i l a t e d i n t o amino a c i d s and 1 5 p r o t e i n s . During n i t r o g e n uptake experiments, l o s s of N has 1 5 been a t t r i b u t e d to DO N e x c r e t i o n or l o s s from the p a r t i c u l a t e matter (Laws 1984, P r i c e et al. 1985), although d i r e c t evidence i s l a c k i n g . Both L i et al . (1983) and Cuhel et al. (1983) demonstrated that GF/F f i l t e r s r e t a i n the small p h o t o s y n t h e t i c p i c o p l a n k t o n . Laboratory evidence f o r NH 4 + r e l e a s e was r e p o r t e d by Uchida (1976). He observed that Pr or oce nt r um minimum excreted NH 4 + when grown in u r e a - e n r i c h e d c u l t u r e medium. Rees (1979) a l s o r e p o r t e d NH^ + r e l e a s e by urea-grown Phaeodact yl um tricornutum. These r e s u l t s c o n t r a s t the f i n d i n g s of H o r r i g a n and McCarthy (1981). They observed 14 15 . t h a t uptake r a t e s of C- and N-urea were i d e n t i c a l i n two diatom s p e c i e s . S i n c e t h e i r measurements were made over much 133 s h o r t e r time i n t e r v a l s than the s t u d i e s of Uchida and Rees, i t i s p o s s i b l e that NH 4 + l o s s from the c e l l s may not have been evident i n t h e i r r e s u l t s . I propose t h a t , when phytoplankton are exposed to e l e v a t e d urea c o n c e n t r a t i o n s , urea i s r a p i d l y h y d r o l y z e d by urea degrading enzymes. The C 0 2 from urea d i f f u s e s out of the c e l l s , and although some NH 4 + i s i n c o r p o r a t e d i n t o macromolecules, some NH 4 + d i f f u s e s out of the c e l l s as NH^. Urea turnover times The turnover time of urea i n seawater depends upon the ambient d i s s o l v e d urea c o n c e n t r a t i o n , phytoplankton biomass and n u t r i t i o n a l s t a t e , and water temperature. Previous i n v e s t i g a t o r s found that urea turnover times i n o l i g o t r o p h i c ocean environments were g e n e r a l l y slow (Table X V I I I ) ; however, the r e s u l t s of Herbland (1976) are an e x c e p t i o n . He reported r a p i d turnover times ( c a . 1.2 d) i n t r o p i c a l South A t l a n t i c waters. In i n s t a n c e s where turnover times were not d i r e c t l y r e p o r t e d by the i n v e s t i g a t o r , I c a l c u l a t e d r a t e s from t h e i r data. The r e s u l t s presented i n t h i s study are the most r a p i d turnover times of urea measured i n an o l i g o t r o p h i c oceanic gyre; these r a t e s are as f a s t or f a s t e r than turnover times of urea i n c o a s t a l waters. The turnover time of urea, determined by a v a r i e t y of techniques i n s e v e r a l c o a s t a l waters, v a r i e s between 5-10 d (Carpenter et al . 1972, Remsen et al . 1974, Mitamura and S a i j o 1975, 1 980a, Savidge and Hutley 1977, H a r r i s o n et al . 1985). In two e x c e p t i o n a l s t u d i e s , K r i s t i a n s e n (1983) and T u r l e y (1985) measured urea 134 turnover times on the order of a few hours i n O s l o f j o r d and at a f r o n t i n the western I r i s h Sea, r e s p e c t i v e l y . Some of the d i f f e r e n c e between the Sargasso Sea data and data c o l l e c t e d by other i n v e s t i g a t o r s a r i s e s because of the method used f o r measuring urea turnover and uptake. The f o l l o w i n g example serves to demonstrate t h i s p o i n t . Urea turnover times, c a l c u l a t e d from data given i n Eppley et al . (1977), averaged 52 d. Turnover times were c a l c u l a t e d from the ambient d i s s o l v e d urea c o n c e n t r a t i o n s and in situ urea uptake r a t e s 1 5 measured with N-urea. Using data c o l l e c t e d on the same c r u i s e s of Eppley et al . , Sharp et al. (1980) r e p o r t e d an average urea turnover time of 22 d. They used urea uptake measurements determined over a range of s u b s t r a t e c o n c e n t r a t i o n s , and c a l c u l a t e d turnover times from l i n e a r i z e d p l o t s of the Michaelis-Menten equation. T h i s method, f i r s t used by Parsons and S t r i c k l a n d (1962) f o r measuring glucose turnover i n seawater, r e q u i r e s no knowledge of the ambient d i s s o l v e d urea c o n c e n t r a t i o n . Eppley et al . (1977) reco g n i z e d that the c o n c e n t r a t i o n s of urea they r e p o r t e d were i n e r r o r ; t h i s c o u l d be r e s p o n s i b l e f o r the d i f f e r e n c e s between the two methods f o r c a l c u l a t i n g turnover times. Many of the e a r l y s t u d i e s measuring urea uptake by phyto-p l a n k t o n , which u l t i m a t e l y l e d to our r e c o g n i t i o n of the importance of urea f o r phytoplankton n u t r i t i o n i n the ocean, were d e r i v e d from long i n c u b a t i o n s with s u b t r a t e a d d i t i o n s s i m i l a r to ambient l e v e l s . These uptake r a t e s may be b i a s e d . 1 5 The l i m i t a t i o n s of the N method are now widely known, and Table XVIII Urea turnover times i n o l i g o t r o p h i c ocean waters. Region Sample T(°C) Chi a [Urea] Turnover Ref depth time (m) (ug-1 ) (ug at N-1 1) (d) N A t l a n t i c - - - 0.62 98 1 0.28 59.2 1 T r o p i c a l <40 28.4 0.4 - 1.44 2 S A t l a n t i c <15 27 0.1 - 0.71 2 <10 25 0.1 - 1.91 2 C e n t r a l <85 21 0.07 0.28 52 a N P a c i f i c N P a c i f i c <30 - - 0.37 140 S u b a r c t i c <30 - - 0.23 90 Pac i f i c Sargasso 15 25.5 0.32 0.09 0.54 Sea N A t l a n t i c 6.6 26 0.13 0.15 0.88 5 Slope a Sharp et al . 1980 c a l c u l a t e d an average turnover time of 22 d u s i n g data c o l l e c t e d by Eppley, but not reported i n Eppley et al . 1977. 1 Remsen et al. 1974 2 Herbland 1976 3 Eppley et al . 1977 4 Mitamura and S a i j o 1980a 5 T h i s study are r e c e n t l y reviewed by Dugdale and Wilkerson (1986). The 1 4 use of C-urea i n these experiments allowed a c c u r a t e measurements of in situ urea turnover r a t e s . These r a t e s were measured d u r i n g short i n c u b a t i o n s and without s i g n i f i c a n t l y p e r t u r b i n g the plankton community by the a d d i t i o n of the t r a c e r . Herbland (1976) used a s i m i l a r methodology to that r e p o r t e d here. Current methodology i s i n s u f f i c i e n t to determine urea r e g e n e r a t i o n r a t e s i n a manner s i m i l a r to that used f o r NH 4 +. As was d i s c u s s e d e a r l i e r , there are many sources of urea i n seawater; there i s a s i g n i f i c a n t p o t e n t i a l f o r urea r e g e n e r a t i o n i n seawater samples. In Chapter 2, a mass balance method was used to q u a n t i f y urea r e g e n e r a t i o n by pla n k t o n i n a c o a s t a l environment. The r e s u l t s showed that urea r e g e n e r a t i o n r a t e s were s i m i l a r i n magnitude to NH 4 + r e g e n e r a t i o n r a t e s . By comparison to other n i t r o g e n compounds, the r a p i d urea turnover times i n the Sargasso Sea are s i m i l a r . G l i b e r t and McCarthy (1984) measured NH 4 + turnover times i n the Sargasso Sea of ca. 11 h. These r a t e s were c a l c u l a t e d from in situ uptake r a t e data and d i s s o l v e d ammonium c o n c e n t r a t i o n s . When c o n c e n t r a t i o n s were u n d e t e c t a b l e , they were set equal to 30 nM: the l i m i t of d e t e c t i o n . There was no s i g n i f i c a n t d i f f e r e n c e between the turnover times of ammonium i n the surface-mixed l a y e r , or at the 5% se a - s u r f a c e l i g h t depth, but the median value f o r the NH 4 + turnover time i n s u r f a c e water was l e s s . Although most of the phytoplankton biomass was below the th e r m o c l i n e , I found f a s t e r turnover times of urea i n s u r f a c e waters, i n agreement with r e s u l t s of Herbland (1976). These r a p i d turnover times of urea i n d i c a t e t h a t urea reg e n e r a t i o n r a t e s are of s i m i l a r magnitude to NH^ "*" r e g e n e r a t i o n r a t e s . T h e r e f o r e , c a l c u l a t i o n of in situ urea uptake r a t e s , using 14 15 C- or N-urea, must take t h i s i n t o c o n s i d e r a t i o n . F a i l u r e to do t h i s w i l l r e s u l t i n an underestimate of the in situ urea uptake r a t e . Urea uptake: saturating/trace rates Harvey and Caperon (1976) compared r a t e s of urea uptake by phytoplankton p o p u l a t i o n s i n Kaneohe Bay with s a t u r a t i n g (8 ug at N-1 1) and t r a c e (0.8 ug at N-1 1) a d d i t i o n s . They found that s a t u r a t e d uptake r a t e s were on average 1.25 times g r e a t e r than in situ urea uptake r a t e s , but s t a t i s t i c a l a n a l y s i s of t h e i r data i n d i c a t e d t h a t the uptake r a t 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 . T h i s r e s u l t implied that urea uptake r a t e s were s a t u r a t e d at ambient in situ c o n c e n t r a t i o n s . K r i s t i a n s e n (1983) measured urea uptake r a t e s i n O s l o f j o r d f o l l o w i n g 1 and 10 ug at N-1 1 a d d i t i o n s , although he d i d not compare the two r a t e s . G l i b e r t and McCarthy (1984) suggested comparing these measurements, u s i n g NH^ + as the ni t r o g e n source, as a means of q u a n t i f y i n g the n i t r o g e n s t a t u s of phytoplankton communities. My r e s u l t s are too few to show any apparent p a t t e r n s . Trace and s a t u r a t i n g urea uptake r a t e s were i n f a i r l y good agreement i n c o n t i n e n t a l slope water, with s a t u r a t i n g r a t e s o n l y 1.5 times the in situ r a t e s . The d i s s o l v e d urea c o n c e n t r a t i o n s were 0.12 and 0.18 ug at N-1 1 i n s u r f a c e and deep water, r e s p e c t i v e l y . Laboratory s t u d i e s 1 38 on a l i m i t e d number of p h y t o p l a n k t o n . s p e c i e s show that these c o n c e n t r a t i o n s are near the Ks f o r uptake of urea, so the -sma l l d i s c r e p a n c y seen between the two r a t e s i s expected. In the Sargasso Sea V s a t / V t r a c e i s more v a r i a b l e , the g r e a t e s t d i s p a r i t y between the r a t e s o c c u r r e d i n water c o l l e c t e d from 88 m at S t a t i o n 2. McCarthy and Nevins (1986) found a s i m i l a r v a r i a b i l i t y i n V s a t / V t r a c e f o r NH 4 + uptake i n t h e i r warm-core r i n g s study. In the deep water sample from S t a t i o n 3, V t r a c e was s i m i l a r to Vsat, however, V t r a c e may be o v e r e s t i m a t e d because one of the d u p l i c a t e urea samples was two times the c o n c e n t r a t i o n of the o t h e r . In i n s t a n c e s where urea c o n c e n t r a t i o n s were below the l i m i t of d e t e c t i o n , I a r b i t r a r i l y chose the l i m i t of d e t e c t i o n as the ambient c o n c e n t r a t i o n . In c o n c l u s i o n , the s i m i l a r i t y between Vsat and V t r a c e demonstrates that i n the Sargasso Sea there i s both h o r i z o n t a l and v e r t i c a l v a r i a b i l i t y i n the urea-N s t a t u s of the p l a n k t o n communities. In slope water, in situ urea uptake r a t e s were in c l o s e agreement with s a t u r a t e d uptake r a t e s . The r a p i d turnover times of urea i n the Sargasso Sea and the low ambient c o n c e n t r a t i o n s suggests that urea i s s u p p l i e d by r e g e n e r a t i v e processes at r a t e s which are c l o s e to the r a t e s of u t i l i z a t i o n . In some phytoplankton communities i n the Sargasso and i n c o n t i n e n t a l s lope water, urea i s u t i l i z e d a t r a t e s which approximate the maximum r a t e s of u t i l i z a t i o n , i mplying that the phytoplankton are a b l e to e f f e c t i v e l y sequester nanomolar c o n c e n t r a t i o n s of urea from seawater. 1 39 Summary Uptake r a t e s , determined with s a t u r a t i n g a d d i t i o n s (10 ug at -1 1 5 N-1 ) of N - l a b e l l e d ammonium, n i t r a t e and urea, were measured at two s t a t i o n s i n the western Sargasso Sea and at one s t a t i o n over the c o n t i n e n t a l slope o f f Cape Hatteras i n August 1985. D a i l y r a t e s of n i t r o g e n uptake were determined d u r i n g 24 h in c u b a t i o n s i n water samples c o l l e c t e d w i t h i n the euphotic zone. 1 5 Urea uptake, as measured by N i s o t o p e s , was constant over the i n c u b a t i o n at a l l s t a t i o n s . Throughout the euphotic zone i n slope water, NH^ + uptake r a t e s were 2-3 times gr e a t e r than urea uptake r a t e s and 3-3.5 times g r e a t e r than NO^ uptake r a t e s . In the s u r f a c e waters of one Sargasso Sea s t a t i o n , urea uptake r a t e s were 5 times g r e a t e r than NH^ + uptake r a t e s , but below the 30 m, uptake r a t e s of NH 4 + were g r e a t e r than urea uptake r a t e s . At the other Sargasso s t a t i o n , N H 4 + uptake r a t e s were g e n e r a l l y g r e a t e r than urea uptake r a t e s . 14 15 Urea uptake r a t e s measured by C- and N-urea were not 1 4 e q u i v a l e n t : r a t e s measured by C-urea were on average 1.4 f a s t e r 1 5 than those determined with N-urea. In situ urea turnover times were determined by t r a c e 1 4 a d d i t i o n s of C-urea. In the surface-mixed l a y e r of the Sargasso Sea, urea turnover times were ca. 12 h, and they were ca. 2 d i n water samples c o l l e c t e d from the base of the euphotic zone. The r a t i o of s a t u r a t e d uptake r a t e / / n situ uptake r a t e was near u n i t y i n 4 of the 6 samples. These r e s u l t s provide evidence that urea uptake r a t e s in situ are near the maximum p o t e n t i a l uptake r a t e s , s uggesting that the phytoplankton i n these regions have a hig h a f f i n i t y f o r d i s s o l v e d urea. 141 CHAPTER 4. FATE OF UREA-C AND N DURING UREA UPTAKE BY THE  COASTAL MARINE DIATOM THALASSIPS IRA PSEUDONANA Background Measurements of urea uptake r a t e s by phytoplankton i n nature r e l y on the use of r a d i o a c t i v e and s t a b l e i s o t o p e 14 15 t r a c e r s of urea: C-urea and N-urea. Since the r o l e of urea-N i n s u p p o r t i n g phytoplankton growth and i t s c o n t r i b u t i o n to phytoplankton n i t r o g e n requirements i s of g r e a t e s t 1 5 i n t e r e s t , N-urea has been f r e q u e n t l y used to measure urea uptake r a t e s (Eppley et al. 1971a, McCarthy 1972a, Eppley et al . 1973, 1977, McCarthy et al. 1977, Kaufman et al . 1983, K r i s t i a n s e n 1983, H a r r i s o n et al. 1985, Probyn 1985, Probyn 1 4 and P a i n t i n g 1985). Many s t u d i e s have a l s o u t i l i z e d C-urea to determine urea uptake r a t e s (Carpenter et al . 1972a, Remsen et al. 1974, Mitamura and S a i j o 1975, 1980a, Harvey and Caperon 1976, Webb and Haas 1976, Herbland 1976, Savidge and Hutley 1977, Floodgate et al . 1981, Ignatiades 1986, T u r l e y 1985, 1986). D i s c r e p a n c i e s between these two methods were f i r s t r e p o r t e d by H a r r i s o n et al. (1985). They found that 1 4 uptake r a t e s of C-urea were gre a t e r than i n c o r p o r a t i o n r a t e s 1 5 of N-urea by pl a n k t o n i n the eastern Canadian A r c t i c . In the r e s u l t s of the l a s t chapter, there were d i s c r e p a n c i e s between uptake r a t e s of urea c a l c u l a t e d with 14 15 . . . C-urea and N-urea and d i f f e r e n c e s i n the time course of 1 5 uptake of both l a b e l l e d s u b s t r a t e s . N-urea i n c o r p o r a t i o n by phytoplankton was c o n s t a n t over the 24 h i n c u b a t i o n p e r i o d , 14 • while C-urea uptake was most r a p i d over the f i r s t 6 h of the i n c u b a t i o n . During 24 h i n c u b a t i o n s , some C was l o s t to a compartment not measured i n the a n a l y s e s . In c o n t r a s t to these r e s u l t s , Mitamura and S a i j o (1986) found no d i f f e r e n c e 14 15 between C- and N-urea uptake r a t e s i n Lake Biwa. 1 4 D i s c r e p a n c i e s between C-urea uptake and disappearance of d i s s o l v e d urea from seawater were e v i d e n t i n the data of Harvey and Caperon (1976). T h i s might be expected i f urea r e g e n e r a t i o n i s an important p r o c e s s , and the d i f f e r e n c e between the two measurements should favour h i g h e r uptake r a t e s 1 4 determined with C-urea. T h i s was not the case i n Kaneohe Bay, as Harvey and Caperon (1976) r e p o r t e d t h a t r a t e s of urea disappearance were g e n e r a l l y g r e a t e r than 1 4 C - u r e a uptake r a t e s . In l a b o r a t o r y experiments with u n i a l g a l c u l t u r e s of Thai assi osi ra pseudonana and Skeletonema costatum, Horrigan and McCarthy (1981) found no d i f f e r e n c e i n urea uptake r a t e s 14 15 determined by C- and N-urea. However, t h e r e was evidence i n t h e i r r e s u l t s to suggest that the two i s o t o p e s were not measuring the same pr o c e s s . Ammonium completely i n h i b i t e d 15 N-urea i n c o r p o r a t i o n by T. ps eudonana; whereas, i n a 1 4 separate experiment C-urea uptake r a t e was o n l y reduced by 60% i n the presence of 10 ug at NH 4 +-N-1~ 1. In t h i s study, uptake of urea by axenic c u l t u r e s of 14 15 Thal assi osi ra pseudonana was measured by C-urea, N-urea and by chemical a n a l y s i s of d i s s o l v e d urea. Urea uptake r a t e s were determined i n n i t r a t e - s u f f i c i e n t and n i t r a t e - s t a r v e d batch c u l t u r e s , f o l l o w i n g the a d d i t i o n of 10 ug at urea-N-1 1 . 1 43 Mater ia ls and Methods Culturing procedure An axenic c u l t u r e of Thai as si osira pseudonana c l o n e 3H, obta i n e d from the Northeast P a c i f i c C u l t u r e C o l l e c t i o n , Dept. Oceanography, U.B.C., was e s t a b l i s h e d with a n t i b i o t i c treatment using a procedure s i m i l a r t o t h a t o u t l i n e d by Droop (1967). The absence of b a c t e r i a was v e r i f i e d by e p i f l u o r e s c e n c e microscopy (Hobbie et a l . 1977), and by the use of s t e r i l i t y t e s t medium ( P r o v a s o l i et a l . 1957). Batch c u l t u r e s of T. pseudonana were grown i n m o d i f i e d a r t i f i c i a l seawater medium (ESAW) of H a r r i s o n et al. (1980), d e s c r i b e d i n d e t a i l i n Chapter 5, with N0 3~ (50 ug at N-1 - 1) as the ni t r o g e n source. C u l t u r e s were grown i n a 2.5 l i t r e p olycarbonate Fernbach f l a s k or 250 ml Erlenmeyer f l a s k s under - 2 -1 continuous i l l u m i n a t i o n at an i r r a d i a n c e of 120 uE-m -s and 18°C. C u l t u r e s were c o n t i n u o u s l y s t i r r e d by t e f l o n - c o a t e d magnetic s t i r bars. C e l l growth was monitored by in vivo c h l o r o p h y l l a f l u o r e s c e n c e measured on a Turner Designs model 10 (R) fluorometer, and by c e l l counts u s i n g a C o u l t e r Counter 4^ model TA I I . D i s s o l v e d NO^ + N0 2 and NH 4 + were measured with a (R) Technicon A u t o a n a l y z e r ^ II u s i n g the methods of Wood et al. (1967) and Slawyk and Mad saac (1972), r e s p e c t i v e l y . D i s s o l v e d urea c o n c e n t r a t i o n was an a l y z e d by the mo d i f i e d d i a c e t y l monoxime method d e s c r i b e d i n Chapter 1. P a r t i c u l a t e n i t r o g e n (PON) samples were c o l l e c t e d on combusted Whatman GF/F f i l t e r s , oven d r i e d at 60°C and measured on a C a r l o Erba Elemental A n a l y z e r . Urea purity The p u r i t y of the urea stock s o l u t i o n s used d u r i n g uptake s t u d i e s was v e r i f i e d by a n a l y z i n g them f o r ammonium and other 1 4 p o t e n t i a l contaminants. C r y s t a l l i n e C-urea purchased from Amersham ( s p e c i f i c a c t i v i t y 55 uCi-umol ^ ; 2.04 GBq-mmol 1) was r e p o r t e d by the manufacturer to be 99% pure. A stock s o l u t i o n was prepared i n s t e r i l e d e i o n i z e d d i s t i l l e d water, and s t o r e d f r o z e n at -30°C. T h i s s o l u t i o n was ana l y z e d f o r NH^ + to determine i f any urea had decomposed d u r i n g storage. -1 i 4 R e s u l t s showed t h a t i n a 10 ug at N-1 s o l u t i o n of C-urea, NH^ + was u n d e t e c t a b l e . Urease (1860 u n i t s - m l 1) was added to -1 1 4 10 ml samples of 10 ug at N-1 C-urea stock i n a f i n a l c o n c e n t r a t i o n of 9.3 u n i t s - m l 1 . A f t e r i n c u b a t i o n at room temperature f o r 1 h, the samples were a c i d i f i e d and allowed to 1 4 degas o v e r n i g h t . A l l but 0.16% of the i n i t i a l C a c t i v i t y was l o s t from s o l u t i o n , i n d i c a t i n g t h e r e was n e g l i g i b l e 1 4 contamination by C - c o n t a i n i n g compounds other than urea. 15 15 The N-urea purchased from Kor Isotopes was 99% N. A stock s o l u t i o n was prepared i n a manner s i m i l a r to that 14 + d e s c r i b e d f o r C-urea; no NH 4 was d e t e c t e d i n a 10 ug at N-1 1 s o l u t i o n . A n a l y t i c a l reagent grade urea (ACS approved), purchased from F i s h e r Chemical company, was used i n the p r e p a r a t i o n of the " c o l d " urea stock s o l u t i o n . I t s p u r i t y was confirmed by chemical a n a l y s i s , and no contaminating NH. + was de t e c t e d i n a 1 45 10 ug a t urea-N-1 1 s o l u t i o n . Urea uptake Urea uptake experiments were performed with T. ps eudonana d u r i n g n i t r a t e - s u f f i c i e n t growth and a f t e r 24 h of n i t r a t e -s t a r v a t i o n . A l i q u o t s of phytoplankton c u l t u r e (100 ml) were a s e p t i c a l l y added to s t e r i l e 250 ml polycarbonate Erlenmeyer 1 5 f l a s k s . Uptake r a t e s were determined i n d u p l i c a t e with N-1 4 urea, C-urea, and by measuring the change i n urea c o n c e n t r a t i o n i n the medium by chemical a n a l y s i s . A l l uptake experiments were i n i t i a t e d w i t h i n 45 min of each o t h e r , f o l l o w i n g the a d d i t i o n of a s a t u r a t i n g c o n c e n t r a t i o n (10 ug at N-1 1) of u r e a . Urea uptake r a t e s were measured d u r i n g 2 h i n c u b a t i o n s f o r the n i t r a t e - s u f f i c i e n t c u l t u r e s and 1 h f o r the n i t r a t e - s t a r v e d c u l t u r e s . Average uptake r a t e s are r e p o r t e d ± 1 SD. Rate measurements Change in dissolved urea concentration At d e s i g n a t e d time i n t e r v a l s (1, 5, 15, 30, 60 and 120 (R) min), samples were withdrawn and f i l t e r e d through Swinex*^ f i l t e r h o l d e r s c o n t a i n i n g combusted (4 h at 460°C) Whatman GF/F f i l t e r s . P r e v i o u s l y acid-washed sample cups were r i n s e d once with sample. The f i l t e r e d samples were immediately a n a l y z e d f o r NH 4 +, NO^ + N0 2 and urea. The r a t e of decrease of d i s s o l v e d urea c o n c e n t r a t i o n was c a l c u l a t e d from the slope of a l i n e a r r e g r e s s i o n through the data p o i n t s . T h i s r a t e i s the disappearance uptake rate or net urea uptake r a t e expressed as ug at N-1 1- h 1 . ^N- ur ea upt ake 1 5 Samples f o r N-urea uptake (10 ml) were c o l l e c t e d by f i l t r a t i o n onto combusted Whatman GF/F f i l t e r s and r i n s e d with 10 ml f i l t e r e d ESAW (0.2 um Nuclepore f i l t e r ) . F i l t r a t i o n p r e s s u r e s were always l e s s than 100 mm Hg. Nitrogen i n the p a r t i c u l a t e samples was c o n v e r t e d to N 2 (g) by the micro-Dumas dry combustion technique, as d e s c r i b e d by LaRoche (1983), and 1 5 then analyzed f o r N enrichment with a JASCO model N-150 emission spectrometer ( F i e d l e r and Proksch 1975). Uptake r a t e s were c a l c u l a t e d a c c o r d i n g to the equations of Dugdale and Goering (1967) and are p r e s e n t e d as absolute uptake r a t e s (ug at N-1 1-h 1) determined u s i n g the f i n a l PON. 1 5 The d i f f e r e n c e i n the i n i t i a l and f i n a l N atom percent excess (ape) i n s u c c e s s i v e samples was d i v i d e d by the length of the time i n t e r v a l , and uptake r a t e s (V) were c a l c u l a t e d as: V = a p e f - ape i (PON f) ( t f - t t ) R where PON i s the p a r t i c u l a t e n i t r o g e n , t i s the time, s u b s c r i p t s i and f d e s i g n a t e i n i t i a l and f i n a l measurements, r e s p e c t i v e l y , and R i s the enrichment f a c t o r . These r a t e s are p l o t t e d a g a i n s t the average i n c u b a t i o n time. The PON at the end of each time i n t e r v a l was c a l c u l a t e d from an e x p o n e n t i a l r e g r e s s i o n equation through PON v a l u e s measured at the s t a r t , middle and end of the experiment. A l i n e a r r e g r e s s i o n was 1 47 used f o r the n i t r a t e - s t a r v e d c u l t u r e s . C-urea uptake Subsamples of c u l t u r e (10 ml) were f i l t e r e d through combusted Whatman GF/F f i l t e r s , and the f i l t e r s r i n s e d before running dry with 10 ml f i l t e r e d ESAW. F i l t r a t i o n p r e s s u r e s were always l e s s than 100 mm Hg. The f i l t e r s were added to s c i n t i l l a t i o n v i a l s c o n t a i n i n g 10 ml of Aquasol I I . The 1 4 f i l t r a t e was r e t a i n e d , and the C0 2 recovered as d e s c r i b e d i n 1 4 Chapter 1. The amount of C i n the phytoplankton samples, 1 4 and the amount r e l e a s e d as C0 2 was determined by l i q u i d s c i n t i l l a t i o n c o unting on an Isocap/300 ( S e a r l e A n a l y t i c a l I n c . ) . Samples were counted u n t i l the standard d e v i a t i o n of each count was 2%. 1 4 C-urea uptake r a t e s were c a l c u l a t e d over s u c c e s s i v e 1 5 time i n t e r v a l s i n a manner s i m i l a r to the N-urea uptake c a l c u l a t i o n s . The t o t a l amount of urea taken up was 1 4 c a l c u l a t e d by summing the amount of C r e t a i n e d on the 1 4 f i l t e r s and the amount of C0 2 r e l e a s e d by the c e l l s . 1 4 Recovery of known q u a n t i t i e s of C-HCO^ from seawater samples was 91.5% (n=4), and uptake r a t e s were c o r r e c t e d a c c o r d i n g l y . Rates-are expressed as n m o l - c e l l 1- min 1 , but they are a l s o given i n a b s o l u t e r a t e s f o r comparison with other d a t a . R e s u l t s Culture conditions Growth of Thai as s i os i r a pseudonana, and c o n c e n t r a t i o n of d i s s o l v e d NO^ i n the medium are given i n Figu r e 21. The ex p o n e n t i a l growth r a t e was 1.63 d 1, and the i n i t i a l biomass parameters f o r each experiment are summarized i n Table XIX. Ni t rat e-suffi ci ent culture Urea disappearance r a t e from the c u l t u r e medium averaged 1.88 + 0.08 ug at N-1 - 1- h ~ 1 , and was constant. N i t r a t e c o n c e n t r a t i o n measured i n the same c u l t u r e s i n d i c a t e d that NC>2 uptake was not constant over the 2 h in c u b a t i o n ( F i g . 22). During the f i r s t 15 min, no NO^ uptake was d e t e c t e d . Between the 15-60 min i n t e r v a l , NC>3 uptake rate averaged 1.14 + 0.08 ug at N-1 1 - h 1 , but over the f i n a l hour of the in c u b a t i o n t h i s r a t e decreased to 0.29 ± 0.14 ug at N-1 1- h 1 . From these chemical measurements, an average t o t a l n i t r o g e n uptake r a t e ( i . e . NO^ and urea) was 2.78 ug at N-1 1-h 1 . Th i s n i t r o g e n uptake r a t e exceeded the growth requirements of T. pseudonana d u r i n g t h i s stage of the growth c y c l e . Nitrogen demand, c a l c u l a t e d from the growth r a t e (1.63 d 1) and an ex p o n e n t i a l average of the p a r t i c u l a t e n i t r o g e n over the in c u b a t i o n (28.7 ug at N - l ~ 1 ) , e q u a l l e d 1.95 ug at N - l ~ 1 - h ~ 1 . 1 4 Uptake of C-urea v a r i e d over the in c u b a t i o n p e r i o d ( F i g . 23A). The apparent decrease i n c e l l u l a r uptake r a t e of urea, which was ev i d e n t d u r i n g the 5-15 min time i n t e r v a l , was confirmed i n a separate repeat experiment using s h o r t e r 1 49 F i g . 21. Growth of T. pseudonana measured by in vivo c h l o r o p h y l l a f l u o r e s c e n c e (O), and the c o n c e n t r a t i o n of d i s s o l v e d NO^ i n the c u l t u r e medium (•) measured over time. The arrows l a b e l l e d 1 and 2 i n d i c a t e the times at which urea uptake experiments were i n i t i a t e d f o r the n i t r a t e - s u f f i c i e n t and n i t r a t e - s t a r v e d c u l t u r e , respect i v e l y . 091 Table XIX Summary of c u l t u r e c o n d i t i o n s at the beg i n n i n g of each experiment. i C e l l -C u l t u r e [N0 3 ] PON Numbers Q D e s c r i p t i o n _ _ „ _ (ug at N-1 ')(ug at N-1 1)(10°-1 ')(fmol N - c e l l ) N i t r a t e -s u f f i c i e n t 22.0 26.8 3.14 85.4 N i t r a t e -s t a r v e d < 0.05 42.6 8.3 51.3 P a r t i c u l a t e n i t r o g e n C e l l n i t r o g e n quota sampling times ( F i g . 23B). A high i n i t i a l uptake r a t e o c c u r r e d d u r i n g the 0.5-2 min time i n t e r v a l and was p r i m a r i l y 1 4 the r e s u l t of i n c o r p o r a t i o n of the C l a b e l i n t o the c e l l s . . . 14 ( F i g . 23C). The amount of C0 2 r e l e a s e d by the c e l l s over t h i s time was only 37% of the t o t a l urea taken up ( F i g . 23D). H y d r o l y s i s of urea, and subsequent r e l e a s e of 1 4CC> 2 became a g r e a t e r f r a c t i o n of the t o t a l urea taken up as the i n c u b a t i o n p r o g r e s s e d . For comparison, c e l l u l a r uptake r a t e s were converted to r a t e s of urea disappearance from the c u l t u r e medium. In t h i s 1 4 c a l c u l a t i o n , I assumed that f o r each C t r a n s p o r t e d a c r o s s the c e l l membrane and r e t a i n e d by the c e l l or r e l e a s e d as 1 4 . . C0 2 two n i t r o g e n atoms are t r a n s p o r t e d i n t o the c e l l . The 14 -1 -1 average C-urea uptake r a t e was 1.03 ug at N-1 • h 1 5 The i n c o r p o r a t i o n of N-urea i n t o the phytoplankton was not constant over the i n c u b a t i o n ( F i g . 24A). The r a t e of 1 5 i n c r e a s e of c e l l u l a r N decreased markedly a f t e r 30 min, and 1 5 the i n i t i a l r a t e of N-urea i n c o r p o r a t i o n was l e s s than the 1 5 maximum N uptake r a t e . During the 30-60 and 60-120 min i n t e r v a l s , the r a t e s of urea-N uptake were l e s s than 30% of 1 5 the maximum N-urea uptake rate ( F i g . 24B). The t o t a l amount of urea-N r e t a i n e d by the phytoplankton d u r i n g the i n c u b a t i o n -1 . . 15 was 0.59 ± 0.05 ug at N-1 . T h i s y i e l d s an average N-urea uptake r a t e of 0.29 ± 0.03 ug at N - l ~ 1 - h ~ 1 . Over the i n c u b a t i o n p e r i o d , c e l l u l a r n i t r o g e n , as measured by elem e n t a l a n a l y s i s , i n c r e a s e d at an average r a t e of 1.5 + 0.1 ug at N-1 1- h 1 . T h i s i n c r e a s e was i d e n t i c a l w ith the decrease i n NO- c o n c e n t r a t i o n measured i n the 1 5 3 F i g . 22. Dissolved urea (•) and n i t r a t e (O) concentrat ion in dupl icate samples ( s o l i d and dashed l ines ) of a n i t r a t e -s u f f i c i e n t cu l ture of T. ps eudonana spiked with 10 ug at urea-N-1 1 . Urea disappearance was constant over the incubation; the regression c o e f f i c i e n t s for the two l ines were: r 2 = 0.996 (dashed l ine ) and r 2 = 0.998 ( s o l i d l i n e ) . 1 55 F i g . 23. (A) C-urea uptake r a t e measured i n d u p l i c a t e samples (• ,0 ) of a n i t r a t e - s u f f i c i e n t c u l t u r e of T. pseudonana. (B) 1 4 C - u r e a uptake by n i t r a t e - s u f f i c i e n t T. pseudonana determined i n a separate experiment. A s i n g l e uptake d e t e r m i n a t i o n was made on two r e p l i c a t e c u l t u r e s ( • , O ), and sampling times were of s h o r t e r d u r a t i o n d u r i n g the f i r s t 5 min of the experiment. (C) 1 4 C accumulation i n T. pseudonana. (D) 1 4 C 0 2 r e l e a s e d i n t o the medium. A l l r a t e s were c a l c u l a t e d between s u c c e s s i v e sampling p o i n t s and are p l o t t e d a g a i n s t the average i n c u b a t i o n time. , 4 C - U R E A UPTAKE RATE (nmol-cell"-min") o r o A c n c o o o — ro oi A 9S I 1 57 F i g . 24. N-urea uptake r a t e measured i n d u p l i c a t e samples ( • , O ) of a n i t r a t e - s u f f i c i e n t c u l t u r e of 7". pseudonana. 15 15 (A) N accumulation expressed as N atom percent excess 1 5 i n the c e l l s determined over time. (B) N-urea uptake r a t e d u r i n g each i n c u b a t i o n p e r i o d p l o t t e d against the average i n c u b a t i o n time. 1 58 0 20 40 60 80 I00 120 TIME (min) unperturbed c u l t u r e d u r i n g the same time p e r i o d (1.5 ug at N ' l " 1 - h ~ 1 ) . Ni t r at e-s t ar v ed culture An uptake r a t e of 5.5 ± 0.1 ug at N-1 1-h 1 was determined by chemical a n a l y s i s of d i s s o l v e d urea, and t h i s r a t e was constant over the i n c u b a t i o n ( F i g . 25). Many of the samples were l o s t due to an instrument m a l f u n c t i o n , n e v e r t h e l e s s , uptake r a t e s of the r e p l i c a t e c u l t u r e s were comparable. 1 4 Uptake of C-urea was c o n s t a n t over the i n c u b a t i o n , although the s h o r t e s t time i n t e r v a l was only 1-5 min ( F i g . 26). These c e l l u l a r r a t e s were s i m i l a r to those found f o r n i t r a t e - s u f f i c i e n t c e l l s . The urea uptake rate was determined to be 5.3 ± 0.04 ug at N-1 1- h 1 , and was i n e x c e l l e n t agreement with the r a t e c a l c u l a t e d by chemical a n a l y s i s of urea disappearance from the medium. 1 5 . I n c o r p o r a t i o n of N-urea i n t o T. ps eudonana i s shown i n 1 5 F i g u r e 27A. E l e v a t e d N-urea uptake r a t e s occurred d u r i n g 1 5 the 5-15 min time i n t e r v a l . F o l l o w i n g t h i s , the N-urea uptake r a t e decreased to the i n i t i a l r a t e , although i t appeared that the uptake r a t e i n c r e a s e d d u r i n g the f i n a l 1 5 i n c u b a t i o n p e r i o d s ( F i g . 27B). An average N-urea uptake rate was 5.18 ± 0.04 ug a t N-1 1 - h 1 and was i n good agreement with the r a t e s measured by the other methods. From the d i r e c t measurement of the c e l l u l a r PON, at the beginning and end of the experiment, a n i t r o g e n uptake r a t e of 5.3 + 0.05 ug at N-1 1 - h 1 was c a l c u l a t e d . 1 6 0 F i g . 25. D i s s o l v e d urea c o n c e n t r a t i o n measured i n d u p l i c a t e (• , O) samples of a n i t r a t e - s t a r v e d c u l t u r e of T. pseudonana spiked with 10 ug at urea-N-1 \ 1 62 1 4 F i g . 26. C-urea uptake r a t e measured i n d u p l i c a t e samples (•, O) of a n i t r a t e - s t a r v e d c u l t u r e of T. ps eudonana. o 27. N-urea uptake rate measured i n d u p l i c a t e samples ( • ,0 ) of a n i t r a t e - s t a r v e d c u l t u r e of T. ps eudonana. 15 15 (A) N accumulation i n the c e l l s over time. (B) l 3N-urea uptake r a t e d u r i n g each i n c u b a t i o n p e r i o d p l o t t e d a g a i n s t the average i n c u b a t i o n time. 166 C o n c e n t r a t i o n of d i s s o l v e d N H 4 + i n c r e a s e d i n the c u l t u r e medium a f t e r the a d d i t i o n of urea to the n i t r a t e - s u f f i c i e n t and n i t r a t e - s t a r v e d c u l t u r e s ( F i g . 28). Then the NH 4 + c o n c e n t r a t i o n decreased d u r i n g the remainder of the experiment. A l l r e s u l t s f o r the n i t r a t e - s u f f i c i e n t and n i t r a t e - s t a r v e d c u l t u r e s are summarized i n Table XX. D i s c u s s i o n Int er pr et at i on of measurements 15 14 Urea uptake r a t e s measured by N-urea, C-urea and by chemical a n a l y s i s of d i s s o l v e d urea d i f f e r depending on the ni t r o g e n s t a t u s of the c u l t u r e , and the times over which they were determined. Throughout t h i s d i s c u s s i o n , I w i l l r e f e r to urea uptake r a t e measured by urea disappearance from the c u l t u r e medium as the net urea uptake r a t e . The change i n d i s s o l v e d urea c o n c e n t r a t i o n over time p r o v i d e s an unambiguous measure of net urea uptake by phytoplankton, s i n c e i t i s the 1 4 d i f f e r e n c e between the i n f l u x and e f f l u x r a t e s . With C- and 1 5 N - l a b e l l e d urea, assumptions must be made r e g a r d i n g the f a t e of the l a b e l once i t i s i n s i d e the c e l l . F a i l u r e to account fo r a p o r t i o n of the l a b e l w i l l r e s u l t i n an underestimate of the uptake r a t e and may obscure the t r u e . p h y s i o l o g i c a l p rocess. T h i s very problem was p o i n t e d out by Stephens and North (1971) i n t h e i r study of 1 4C-amino a c i d uptake by marine phytoplankton. They found t h a t not onl y are amino a c i d s metabolized a f t e r they are taken up by phytoplankton, but a p o r t i o n of the amino a c i d carbon s k e l e t o n i s r e l e a s e d to the 28. D i s s o l v e d ammonium c o n c e n t r a t i o n i n (A) n i t r a t e -s u f f i c i e n t , and (B) n i t r a t e - s t a r v e d c u l t u r e s of T. pseudonana, f o l l o w i n g the a d d i t i o n of 10 ug at urea-N-1 1 . The ammonium c o n c e n t r a t i o n i n the c u l t u r e s p r i o r to the a d d i t i o n of urea i s i n d i c a t e d by the arrows. 1 2 0 TIME (min) Table XX 169 Summary of urea uptakes r a t e s measured by C- and N-l a b e l l e d urea and by disappearance i n n i t r a t e - s u f f i c i e n t and n i t r a t e - s t a r v e d c u l t u r e s . N i t r a t e uptake r a t e measured i n the n i t r a t e - s u f f i c i e n t c u l t u r e i s a l s o g i v e n , as are the measured r a t e s of change of the p a r t i c u l a t e n i t r o g e n over the i n c u b a t i o n . C u l t u r e Parameter Measured Rate Measured (ug at N-1~ 1•h~ 1) N i t r a t e - Urea Disappearance 1.88 ± 0.08 s u f f i c i e n t N i t r a t e Disappearance 0.90 ± 0.10 1 4 C - u r e a Uptake 1.03 ± 0.05 l 5N-urea Uptake 0.29 ± 0.10 P a r t i c u l a t e N i t r o g e n 1.50 ± 0.10 N i t r a t e - Urea Disappearance 5.5 ± 0.1 s t a r v e d C-urea Uptake 5.3 ± 0.04 l 5N-urea Uptake 5.2 ± 0.04 P a r t i c u l a t e N i t r o g e n 5.3 ± 0.03 A l l r a t e s are averaged over the i n c u b a t i o n p e r i o d and are r e p o r t e d on an ho u r l y b a s i s ± 1 SD. medium as C0 2 and a c i d - s t a b l e p r o d u c t s . Methodological considerations The d i s c r e p a n c y between methods for measuring urea uptake r a t e s are only e v i d e n t i n the n i t r a t e - s u f f i c i e n t c u l t u r e . These r e s u l t s w i l l be e x p l a i n e d l a t e r by d i s s o l v e d o r g a n i c carbon (DOC) and d i s s o l v e d o r g a n i c n i t r o g e n (DON) e x c r e t i o n . 1 4 Other i n v e s t i g a t o r s have r e p o r t e d l o s s of C - l a b e l l e d DOC from phytoplankton, i n f i l t e r e d samples which were r i n s e d with seawater a f t e r the f i l t e r s have d r i e d (Goldman and Dennett 1985). But i n g e n e r a l , l o s s e s are not seen when the r i n s e s o l u t i o n i s added before the f i l t e r s go dry, and l i t t l e or no l o s s i s seen when us i n g g l a s s f i b r e f i l t e r s . I am c o n f i d e n t that the f i l t r a t i o n technique used i n t h i s study prevented 14 15 l o s s of e i t h e r C- or N - l a b e l l e d m e t a b o l i t e s from T. pseudonana. At a l l times d u r i n g these experiments, f i l t r a t i o n s were done u s i n g vacuum p r e s s u r e s l e s s than 100 mm of Hg, and the r i n s e s o l u t i o n was added p r i o r to the f i l t e r d r y i n g . A d d i t i o n a l evidence r e f u t i n g the l o s s of l a b e l l e d m e t a b o l i t e s d u r i n g r i n s i n g i s p r o v i d e d i n the data from the n i t r a t e - s t a r v e d c u l t u r e s p u l s e d with urea. Goldman and Dennett (1985) showed that N - l i m i t e d phytoplankton pulsed with + 14 NH^ at the s t a r t of a C uptake experiment l o s t 1 4 s i g n i f i c a n t l y g r e a t e r DO C than phytoplankton which d i d not r e c e i v e NH^ +. I f any l o s s e s were t o occur as an a r t i f a c t of f i l t r a t i o n d u r i n g experiments i n t h i s study, I expect they would have been most obvious i n the n i t r a t e - s t a r v e d c u l t u r e s . But i n the r e s u l t s from the n i t r a t e - s t a r v e d c u l t u r e s a l l 171 methods for measuring urea uptake were i n e x c e l l e n t agreement, and the increase i n PON was e x a c t l y balanced by the uptake of u r e a - n i t r o g e n . Nitrate-sufficient cultures In the n i t r a t e - s u f f i c i e n t c u l t u r e s , the disappearance r a t e of urea, and the simultaneous uptake of NO^ , r e s u l t e d i n a t o t a l n i t r o g e n f l u x i n t o the c e l l s of 2.78 ug at N-1 1-h 1 . However, by c o n t r a s t to the urea disappearance r a t e , i t was 1 5 . e v i d e n t that N-urea i n c o r p o r a t i o n was not constant and o n l y represented 15% of the net urea uptake r a t e . Since the change i n PON over the i n c u b a t i o n p e r i o d c o u l d be accounted f o r by - 15 NOg uptake and N-urea i n c o r p o r a t i o n (Table 2), these data i n d i c a t e that most of the urea n i t r o g e n was l o s t from the c e l l s to a n i t r o g e n pool not measured by the methods used i n t h i s study. 1 5 The reduced r a t e s of N-urea uptake are not due to 15 . . 1 4 -d i l u t i o n of N-urea by c o i n c i d e n t NO^ uptake. As i t was p o i n t e d out i n Chapter 2, and by Dugdale and Wilkerson (1986), 1 4 simultaneous u t i l i z a t i o n of N - l a b e l l e d n i t r o g e n s u b s t r a t e s 1 5 and a N - l a b e l l e d compound w i l l r e s u l t i n a decrease i n the 1 5 N - s p e c i f i c uptake r a t e of the N - l a b e l l e d compound. In t h i s 1 5 study, the N-urea uptake r a t e s were c a l c u l a t e d u s i n g the PON a t the end of the i n c u b a t i o n thereby a v o i d i n g t h i s problem. S i n c e the r e l e a s e d NH^/NH^* was l a r g e l y r e a s s i m i l a t e d by the c e l l s , I conclude that most of the urea-N i s l o s t from the 15 . . . n i t r a t e - s u f f l c i e n t c e l l s as DO N. There i s ample evidence i n 172 the l i t e r a t u r e to support t h i s theory. Release of DON The r e l e a s e of d i s s o l v e d f r e e amino a c i d s (DFAA) by phytoplankton d u r i n g n i t r a t e - s u f f i c i e n t growth has been u n e q u i v o c a l l y demonstrated i n axenic c u l t u r e by HPLC a n a l y s i s (Admiraal et al. 1986). Although DFAA e x c r e t i o n i s s p e c i e s s p e c i f i c , and r e l a t e d to the stage of the growth c y c l e , i n the extreme case of CoscI nodi scus grant i as much as 3% of a s s i m i l a t e d NO^ i s e x c r e t e d as amino a c i d s . F a i l u r e to account f o r 85% of the u r e a - n i t r o g e n taken up by the T. ps eudonana suggests that t h i s n i t r o g e n must be l o s t from the 1 5 c e l l s as DO N. O b s e r v a t i o n s by other i n v e s t i g a t o r s support t h i s i n t e r p r e t a t i o n of the r e s u l t s . Furthermore, estimates of PON, c a l c u l a t e d from the amount of N0 3 taken up by T. ps eudonana d u r i n g e x p o n e n t i a l growth, overestimated the a c t u a l PON determined by elemental a n a l y s i s . T h i s i s c o n s i s t e n t with Admiraal's o b s e r v a t i o n s that phytoplankton r e l e a s e DON which i s not r e u t i l i z e d . I t i s w e l l known, that i n axenic c u l t u r e , phytoplankton are capable of u t i l i z i n g many forms of DON; but some amino a c i d s may not be u t i l i z e d ( G u i l l a r d 1963, Wheeler et al . 1974, A n t i a et al . 1975). Admiraal et al . (1986) showed that some phytoplankton r e l e a s e DFAA which they are unable to r e a s s i m i l a t e , at l e a s t while NO^ i s prese n t . They d i d not examine n i t r o g e n - s t a r v e d c u l t u r e s , but e x t r a c e l l u l a r o r g a n i c phosphates, which are produced during e x p o n e n t i a l growth of some c o a s t a l diatoms, are incompletely reabsorbed d u r i n g phosphorus s t a r v a t i o n (Admiraal and Werner 1983). 173 Pattern of urea uptake 1 4 The p a t t e r n of C-urea uptake observed i n the f i r s t experiment was confirmed and c l a r i f i e d i n a separate experiment using s h o r t e r sampling times. The time course of urea uptake i n v o l v e s an i n i t i a l r a p i d uptake p e r i o d f o l l o w e d by a shutdown and a r e t u r n to an int e r m e d i a t e r a t e , which i s maintained throughout the remainder of the experiment. The . . . 14 i n i t i a l r a t e of C-urea uptake, determined d u r i n g the 0.5-2 min time i n t e r v a l from the data presented i n F i g u r e 23B, — 8 — 1 -1 averages 7.9 + 1.9-10 n m o l - c e l l -min . Although the average 1 4 C - u r e a uptake r a t e (over the 2 h i n c u b a t i o n ) was only 55% of the net urea uptake r a t e (5.0 + 0.3-10 nmol-c e l l 1-min 1 ) , the i n i t i a l 1 4 C urea uptake r a t e was a c t u a l l y 1 4 g r e a t e r . The i n i t i a l C-urea uptake r a t e i s the most accurate measurement of the urea i n f l u x r a t e . As uptake proceeds, e f f l u x of urea becomes a more s i g n i f i c a n t f r a c t i o n 1 4 of the i n f l u x , and e v e n t u a l l y C-urea uptake r a t e should equal the net urea uptake r a t e . The d i s c r e p a n c y between net 1 4 urea uptake r a t e and the C-urea uptake r a t e can o n l y be 1 4 r e c o n c i l e d by proposing that some of the C i s l o s t to a compartment not accounted f o r i n these a n a l y s e s . T h i s compartment may be DOC. Since urea d e g r a d a t i o n o c c u r s very r a p i d l y i n T. pseudonana, as evidenced by the r a p i d appearance 1 4 -of C0 2, i n i t i a l uptake measurements w i l l be l e a s t a f f e c t e d by metabolic p r o c e s s e s . In f a c t , i t was observed t h a t the 1 4 i n i t i a l C-urea uptake r a t e was predominantly a r e s u l t of the 174 1 4 accumulation of C-urea by T. ps eudonana. T h i s probably r e p r e s e n t s the f i l l i n g of an i n t r a c e l l u l a r urea p o o l . The 1 4 l a c k of the r a p i d i n i t i a l C-urea uptake i n F i g u r e 23A and i n the n i t r a t e - s t a r v e d c u l t u r e s i s s o l e l y a consequence of the long sampling times r e l a t i v e to the time over which t h i s p r ocess i s o c c u r r i n g . Long times between sample p o i n t s w i l l tend to smooth out t h i s t r a n s i e n t , p o o l - f i l l i n g phase, and only the constant long term uptake rate w i l l be e v i d e n t . Urea pools and efflux The c o n c e n t r a t i o n of urea that accumulated i n t r a c e l l u l a r l y was est i m a t e d d u r i n g the 0.5-2 min time 14 -8 i n t e r v a l as f o l l o w s : Using the C-urea data, 2.4-10 nmol 1 4 - 1 C - c e l l was r e t a i n e d by the phytoplankton d u r i n g t h i s p e r i o d , and the average c e l l volume was 43 f e m t o l i t r e s - c e l l 1 . 1 4 If 80% of the l a b e l i s present as C-urea, s i m i l a r to that which i s found i n Phaeodact yl um tricornutum (Rees and S y r e t t 1979a), the average i n t e r n a l d i s s o l v e d urea c o n c e n t r a t i o n i s 0.45 mM. I suggest t h a t the decrease i n urea uptake r a t e and 14 i n t r a c e l l u l a r C observed d u r i n g the 2-5 min i n t e r v a l i s due, in p a r t , to urea e f f l u x . Using a urea p e r m e a b i l i t y — 6 — 1 c o e f f i c i e n t of 5-10 cm-s (Raven 1980), the urea e f f l u x _ D _1 _ 1 r a t e i s 8.5-10 n m o l - c e l l -min . P r o v i d i n g the i n i t i a l — 8 -1 -1 i n f l u x r a t e (ca. 8-10 n m o l - c e l l -min ) remains constant throughout the experiment, i t i s obvious from a comparison of the i n f l u x and e f f l u x r a t e s that net urea uptake should equal z e r o . Since net uptake i s measurable, urea e f f l u x must decrease subsequently. T h i s proposed r e d u c t i o n i n urea e f f l u x 175 can be e x p l a i n e d as a r e s u l t of a r e d u c t i o n of the i n t r a c e l l u l a r urea c o n c e n t r a t i o n by urea d e g r a d a t i o n and a s s i m i l a t i o n . Evidence f o r t h i s i s p r o v i d e d by the observed 1 4 incr e a s e i n C0 2 e v o l u t i o n from the c e l l s ( F i g . 23D) . F o l l o w i n g the i n i t i a l t r a n s i e n t i n urea uptake, net uptake i s reduced to 60% of urea i n f l u x r a t e (0.5-2 min). T h i s r e s u l t s i n a continuous e f f l u x r a t e of 3 - 10 nmol-c e l l 1- min 1 . The i n t r a c e l l u l a r c o n c e n t r a t i o n r e q u i r e d to support t h i s r a t e , when the e x t e r n a l urea c o n c e n t r a t i o n equals 10 ug at N-1 \ i s 0.16 mM. A comparison of t h i s average c e l l c o n c e n t r a t i o n with the Km f o r urease i n Phaeodactyl um tricor nutum (0.46 mM) ( S y r e t t and L e f t l e y 1976) suggests t h a t , a l l t h i n g s being equal, to account f o r the r a p i d metabolism of urea, urea must be compartmentalized i n T. pseudonana. Dagestad et al . (1981) p o s t u l a t e d that urea i s s t o r e d i n the c h l o r o p l a s t of Chi amydomonas , but not me t a b o l i z e d t h e r e . Ni t r at e-s t ar v ed culture 14 15 Urea uptake r a t e s measured by C- and N - l a b e l l e d urea and by chemical a n a l y s i s of d i s s o l v e d urea c o n c e n t r a t i o n were in e x c e l l e n t agreement over the d u r a t i o n of the experiment i n the n i t r a t e - s t a r v e d c u l t u r e . However, th e r e were t r a n s i e n t p a t t e r n s i n the uptake data which were c o n s i s t e n t with the r e s u l t s from the n i t r a t e - s u f f i c i e n t c u l t u r e . 1 5 During the 5-15 min time i n t e r v a l , the r a t e of N-urea uptake (7.7 ± 0.7 ug at N-1 1 - h 1) was ca. 2 times g r e a t e r than the i n i t i a l and subsequent r a t e . T h i s r a t e was a l s o i n 176 excess of the net uptake rate measured by disappearance (5.5 t 0.1 ug at N - l ~ 1 - h ~ 1 ) . The i n i t i a l r a t e of 1 5 N - u r e a uptake, c a l c u l a t e d over the f i r s t 5 min was 3.7 ug at N-1 ^ h 1 , and was s i g n i f i c a n t l y l e s s than the net uptake r a t e . T h i s 15 15 + d i f f e r e n c e can be accounted f o r by the r e l e a s e of N H 3 / N H 4 1 5 i n t o the medium by the phytoplankton (see F i g . 28). N i n c o r p o r a t i o n r a t e over the 5-15 min time i n t e r v a l i s g r e a t e r 1 4 than net urea uptake, and the C-urea uptake r a t e . 1 5 T h e r e f o r e , r e l e a s e d N must be taken back up over t h i s time i n t e r v a l . During t h i s time i n t e r v a l approximately 0.7 ug at 1^N-1 1 must be taken up at the same time as 1^N-urea to 1 5 account f o r the i n c r e a s e d N i n c o r p o r a t i o n r a t e . R e s u l t s from McCarthy (1972b) are e n t i r e l y c o n s i s t e n t with these o b s e r v a t i o n s and the i n t e r p r e t a t i o n of the r e s u l t s i n t h i s study. He r e p o r t e d that the long term (3.5-7 h) 1 5 uptake r a t e of N-urea by Cyclotella nana (3H) ( s i c ) (renamed Thai assi osira pseudonana) was 1.8 times g r e a t e r than the r a t e measured over 10 min. Although the p r e c i s e p h y s i o l o g i c a l s t a t e of the c u l t u r e s i n McCarthy's experiments i s unknown, urea uptake experiments were s t a r t e d as soon as n i t r o g e n (NC>2 ) c o u l d no longer be dete c t e d i n the medium. T h i s would suggest t h a t the c e l l s were i n e a r l y stages of n i t r o g e n -s t a r v a t i o n (see a l s o Parslow et al. 1984b). The t o t a l amount of NH^ r e l e a s e d i n t o the medium i s too great to be accounted f o r s o l e l y on the b a s i s of u r e a - n i t r o g e n e x c r e t i o n . Although p r e c a u t i o n s were taken to ensure that the sample c o n t a i n e r s were NH^ +-free, contamination may have o c c u r r e d . T h i s may e x p l a i n why such high c o n c e n t r a t i o n s of NH^ + were i n i t i a l l y measured. An a l t e r n a t i v e e x p l a n a t i o n , which has not r e c e i v e d c o n s i d e r a t i o n , i s th a t d u r i n g uptake of urea i n t r a c e l l u l a r 1 4 N H 3 / 1 4 N H 4 + i s r e l e a s e d as w e l l as 15 15 + NH.j/ NH^ from urea. If urea uptake i s co u p l e d to c o t r a n s p o r t of H +, as shown i n Chi or el I a fusca (Rees and S y r e t t 1984), or Na + as suggested f o r Phaeodact yl um tricornutum (Rees et al . 1980), then perhaps NH 4 + may serve as a counter ion to maintain change balance d u r i n g urea uptake i n T. pseudonana. In higher p l a n t s , i t i s observed that e f f l u x 2+ . . . of Ca i s a n o n - s p e c i f i c response of low s a l t p l a n t s to monovalent c a t i o n uptake ( S i d d i q i and G l a s s 1984). I t i s argued that these f l u x e s are d i r e c t e d towards m a i n t a i n i n g the charge balance w i t h i n the c e l l s . In n i t r a t e - s u f f i c i e n t T. pseudonana, there i s a l a r g e i n t r a c e l l u l a r NH^ + p o o l . Dortch et al . (1984) found that the i n t r a c e l l u l a r c o n c e n t r a t i o n of NH 4 + i n n i t r a t e - s u f f i c i e n t T. pseudonana was 0.3% of the t o t a l PON. For the c u l t u r e s i n t h i s study, t h i s amounts to 0.80 ug at NH 4 +- 1 1 or 6 mM on a c e l l volume b a s i s . Although Dortch et al . (1984) r e p o r t e d that f o l l o w i n g 4 d of N0 3 s t a r v a t i o n NH 4 + p o o l s were below d e t e c t i o n , no data were giv e n f o r sho r t e r p e r i o d s of NO^ s t a r v a t i o n . Ammoni a efflux D i r e c t measurement of NH^ e f f l u x from a l g a l c e l l s i s l a c k i n g , although on t h e o r e t i c a l grounds, a f i n i t e leakage i s unavoidable (Raven 1980). C i r c u m s t a n t i a l evidence i n d i c a t e s that NH-. l o s s from c e l l s i s common. Castorph and K l e i n e r (1984) found that the ammonium t r a n s p o r t mutant of Klebsiella pneumoniae l a c k s the a b i l i t y to accumulate NH^ + i n t r a c e l l u l a r l y , but c o n s t a n t l y l o s e s NH^ by d i f f u s i o n . They propose t h a t e f f l u x i s the norm, and that a c t i v e t r a n s p o r t a c t s to circumvent t h i s l o s s . Pulse chase experiments by Wheeler (1980), Wheeler and H e l l e b u s t (1981) and Balch (1986) showed t h a t methylamine e f f l u x ,and by analogy NH^ e f f l u x , o c curs i n marine phytoplankton. The appearance of NH 4 + i n the medium f o l l o w i n g the a d d i t i o n of urea, and i t s r e a s s i m i l a t i o n , i s e n t i r e l y c o n s i s t e n t with the r a p i d e f f l u x of NH^ produced d u r i n g urea metabolism i n T. pseudonana. T h i s response i s seen i n n i t r a t e - s u f f i c i e n t and n i t r a t e - s t a r v e d phytoplankton. C o n f i r m a t i o n of these o b s e r v a t i o n s and the pro p o s a l of NH^ e f f l u x d u r i n g urea uptake by T. ps eudonana i s found i n exaggerated form i n the r e s u l t s of Uchida (1976). He observed that Prorocentrum mi cans , p r e v i o u s l y d e p r i v e d of n i t r o g e n , e x c r e t e d NH^ i n t o the medium a f t e r the a d d i t i o n of 210 ug at urea-N-1 1 . As much as 40 ug at NH 4 +-N-1 1 accumulated i n the medium; i t was then r e a s s i m i l a t e d by the c e l l s : L i t t l e urea was taken up d u r i n g t h i s time. Once the e x t e r n a l NH^ + c o n c e n t r a t i o n decreased below 7 ug at N * l 1 , urea uptake i n c r e a s e d , and NH^ + was excreted back i n t o the medium and then reabsorbed. 1 5 The t r a n s i e n t i n c r e a s e i n N urea uptake by T. ps eudonana can onl y be e x p l a i n e d on the b a s i s of simultaneous 15 uptake of a d d i t i o n a l N compounds with urea. I suggest that 15 + the c e l l s reabsorb excreted NH. produced from urea h y d r o l y s i s . A model for urea uptake by ThalassI osI ra pseudonana On the b a s i s of these r e s u l t s , I propose a model of urea uptake i n the diatom Thai assi osi ra pseudonana. The major events a s s o c i a t e d with urea t r a n s p o r t , and the a s s i m i l a t i o n of urea-C and urea-N are d e p i c t e d i n F i g u r e 29. Regardless of the n i t r o g e n s t a t u s of the c e l l s , the i n i t i a l stages of urea uptake i n v o l v e a r a p i d i n f l u x and the accumulation of an i n t r a c e l l u l a r urea p o o l . Urea uptake i s reduced a f t e r t h i s i n i t i a l surge, and t h i s apparent shutdown can be e x p l a i n e d as a r e s u l t of e f f l u x of urea. A decrease i n the i n t r a c e l l u l a r urea c o n c e n t r a t i o n , as a r e s u l t of me t a b o l i c degradation, reduces the urea pool s i z e , and consequently the e f f l u x r a t e . Resumption of urea uptake, a l b e i t at a lower r a t e than seen i n i t i a l l y , i s evidence f o r t h i s . T h i s i n t r a c e l l u l a r urea pool i s r a p i d l y m e t a b o l i z e d by a urea-degrading enzyme to C0 2 and 2NH^. I t seems l i k e l y t h a t t h i s enzyme i s urease, s i n c e i t i s report e d i n other diatoms ( L e f t l e y and S y r e t t 1973, O l i v e i r a and Ant i a 1986a). During n i t r o g e n - s u f f i c i e n c y , when c e l l s are growing at 1 4 rat e s which are not l i m i t e d by a b i o t i c f a c t o r s , C - l a b e l l e d organic products d e r i v e d from urea-C are e x c r e t e d i n t o the medium (see e a r l i e r D i s c u s s i o n ) . The net l o s s of D0 1 4C i s approximately 45% of the t o t a l urea-C t r a n s p o r t e d i n t o the c e l l s . Under n i t r a t e - s u f f i c i e n c y and n i t r a t e - s t a r v a t i o n , there i s an e f f l u x of NH-j/NH* d e r i v e d from urea, and the 29. Diagrammatic r e p r e s e n t a t i o n of urea uptake, and a s s i m i l a t i o n of urea-C and urea-N by Thai assiosi ra pseudonana. S t i p p l e d region r e p r e s e n t s c e l l membrane. The arrows passi n g through open c i r c l e s i n the membrane repre s e n t t r a n s p o r t by membrane p o r t e r s ; and arrows p a s s i n g d i r e c t l y through the membrane represent d i f f u s i o n . GS/GOGAT i s Glutamine synthetase/glutamate o x o k e t o g l u t a r a t e amino t r a n s f e r a s e . Losses of (C^O) and R-NH2 are shown to occur by d i f f u s i o n and a c t i v e or mediated t r a n s p o r t processes; the a c t u a l mechanism i s not known. Consult the text f o r c l a r i f i c a t i o n of r a t e s and pr o c e s s e s . CO(NH2)2 CO, ; = = ^ CO (NH2)2 '•* • *•* '• .•-*•.»•••'• •'•'.*••>*•;• ?. H,0 CARBOHYDRATE NH,+ H%r=* NH; CARBOXYLASE (CH20)n V" •T't',! ; -y • • j :"t'-i /•'••V.'- {••'•'• r-'; V- iV *7-G % O G AT R-NH2 PROTEIN (CH20)n R-NH 2 CD 182 1 5 i n i t i a l r a t e of N-urea i n c o r p o r a t i o n i s low. Within 5-15 min, t h i s e f f l u x e d NH^ + i s c o i n c i d e n t l y taken back up by the phytoplankton, with urea. The c e l l membrane becomes l e s s permeable to NH 4 + and i n f l u x and e f f l u x of NH^ + are balanced. A f u r t h e r c o m p l i c a t i o n a r i s e s i n N - s u f f i c i e n t c u l t u r e s where a l a r g e f r a c t i o n of the urea-N i s r e l e a s e d from the c e l l s i n the form of organic n i t r o g e n . T h i s DON i s not r e a s s i m i l a t e d d u r i n g the short term. There was no evidence of DON r e l e a s e by n i t r a t e - s t a r v e d phytoplankton. N i t r a t e - r e p l e t e T. ps eudonana d i d not have enhanced uptake r a t e s f o r urea, c o n t r a r y to the r e s u l t s of H o r r i g a n and McCarthy (1981, 1982). N i t r o g e n - s p e c i f i c uptake r a t e was a v a r i a b l e and averaged 0.26 d 1 under n i t r o g e n - s u f f i c i e n c y , but i n c r e a s e d to 3.2 d 1 i n the n i t r a t e - s t a r v e d c u l t u r e s . T h i s was a consequence of a r e d u c t i o n i n the n i t r o g e n c e l l quota and the r e t e n t i o n of a l l the u r e a - n i t r o g e n by the n i t r a t e - s t a r v e d c e l l s . From a comparison of net urea uptake r a t e s , determined from chemical measurements, i t i s apparent t h a t i n T. ps eudonana there i s no i n c r e a s e i n urea uptake r a t e upon n i t r a t e s t a r v a t i o n . S i m i l a r r e s u l t s have been found i n Chl or el I a pyrenoi dosa (Bekheet and S y r e t t 1979); however, S y r e t t et al. (1986) r e p o r t e d i n c r e a s e d urea uptake r a t e s upon i n c r e a s i n g n i t r o g e n s t a r v a t i o n i n Phaeodactyl um tricornutum. Implications for urea uptake measurements in nature 14 15 There are advantages i n usi n g of C- and N - l a b e l l e d urea f o r measuring urea uptake by phytoplankton. The most obvious advantage i s the gr e a t e r s e n s i t i v i t y of both 183 techniques compared to chemical a n a l y s i s of d i s s o l v e d urea c o n c e n t r a t i o n s . T h i s i s p a r t i c u l a r l y true in nature because c e l l d e n s i t i e s are u s u a l l y too low to allow accurate measurements of urea disappearance to be made; s i n c e urea c o n c e n t r a t i o n s are low i n the ocean, isotopes are the only a l t e r n a t i v e . As s t r e s s e d e a r l i e r , urea disappearance from the c u l t u r e medium r e p r e s e n t s the net uptake of urea, s i n c e e f f l u x of urea a l s o o c c u r s . These r e s u l t s demonstrate that 1 4 C - and 1^N-urea do not n e c e s s a r i l y measure the same process, and 1 4 C -urea uptake cannot be equated with simultaneous i n c o r p o r a t i o n 1 5 of urea-N. There i s l i t t l e doubt that N-urea i n c o r p o r a t i o n by phytoplankton r e p r e s e n t s the o v e r a l l c o n t r i b u t i o n of urea-N to phytoplankton n i t r o g e n requirements. What these r e s u l t s have shown, though, i s th a t under n i t r a t e - s u f f i c i e n c y t h i s c o n t r i b u t i o n can be very much l e s s than the t o t a l urea-n i t r o g e n taken up by the c e l l s . I f a s i m i l a r mechanism of urea uptake i s present i n other phytoplankton, the outcome of the r e s u l t s of urea uptake measurements conducted i n r e g i o n s of d i f f e r i n g n i t r o g e n s t a t u s may d i f f e r . Under c o n d i t i o n s of n i t r a t e - s u f f i c i e n c y , urea 14 15 uptake r a t e s measured by C~ and N-urea should be most d i v e r g e n t , while the converse w i l l be true i n n i t r a t e - s t a r v e d phytoplankton. A p o r t i o n of the urea-N i s r e l e a s e d by T. ps eudonana as NH^/NH^ and l a t e r reabsorbed. The 14 15 d i s c r e p a n c i e s between measurements of C- and N-urea uptake i n f i e l d experiments, r e p o r t e d i n Chapter 3, provide evidence that some urea-N i s l o s t from phytoplankton and i s not reabsorbed. A p o r t i o n of t h i s urea-N may be NH 4 +. The a b i l i t y of algae to sequester t h i s NH 4 + w i l l depend upon the ambient c o n c e n t r a t i o n of NH 4 +, and i t s r a t e of r e c y c l i n g . When the e x t e r n a l N H 4 + c o n c e n t r a t i o n i s g r e a t , the amount of 15 + ure a - d e r i v e d NH 4 that i s r e a s s i m i l a t e d w i l l be smal l because of i t s d i l u t i o n by the ambient NH 4 + p o o l . Experiments designed to determine the e f f e c t s of NH 4 + on urea uptake must a l s o take t h i s i n t o c o n s i d e r a t i o n . The true e f f e c t of ammonium on urea uptake can be determined by urea disappearance measurements. 1 5 These r e s u l t s p r o v i d e c i r c u m s t a n t i a l evidence f o r DO N 1 5 l o s s from n i t r a t e - s u f f i c l e n t phytoplankton s p i k e d with N-urea. D i r e c t measurement of DON p r o d u c t i o n i s now r e q u i r e d to s u b s t a n t i a t e t h i s c l a i m . I t i s p o s s i b l e that the f a i l u r e to 1 5 account f o r a l l of the N d u r i n g f i e l d experiments (see f o r 1 c example Laws 1984, P r i c e et al . 1985) may be due to DO N l o s s . Other processes may a l s o c o n t r i b u t e to t h i s l o s s , such as n i t r i f i c a t i o n and d e n i t r i f i c a t i o n , but t h e i r s i g n i f i c a n c e i s undetermined. Summary Urea uptake r a t e s were measured i n n i t r a t e - s u f f i c i e n t and n i t r a t e - s t a r v e d batch c u l t u r e s of Thai assi osira pseudonana (clone 3H) by 1^N-urea, 1 4 C - u r e a , and by measuring the disappearance of d i s s o l v e d urea from the medium. In n i t r a t e - s u f f i c i e n t c u l t u r e s , urea uptake was determined by adding 10 ug at urea-N-1 1 , and uptake was measured d u r i n g a 2 h i n c u b a t i o n . I n i t i a l urea i n f l u x r a t e measured by C-urea was g r e a t e r than the net urea uptake r a t e measured by disappearance of urea from the medium du r i n g the 1 4 f i r s t 2 min. But the long term average C-urea uptake r a t e was only 60% of the net uptake r a t e . N i t r a t e uptake c o n t i n u e d in the presence of 10 ug at urea-N-1 1 at a reduced r a t e . Only 15% of the urea-N was r e t a i n e d by the phytoplankton, and the i n c r e a s e i n the p a r t i c u l a t e n i t r o g e n was accounted f o r by the t o t a l NO^ and urea-N uptake. In 24 h n i t r a t e - s t a r v e d c u l t u r e s , the average urea uptake r a t e s measured by a l l methods were i n e x c e l l e n t agreement with the i n c r e a s e i n the p a r t i c u l a t e n i t r o g e n d u r i n g the 15 i n c u b a t i o n . N-urea uptake r a t e s were not consta n t , and maximum r a t e s were measured 5-15 min a f t e r the a d d i t i o n of 1 4 urea. Uptake of C-urea and the net uptake r a t e were constant d u r i n g the i n c u b a t i o n . A model of urea uptake and a s s i m i l a t i o n by Thai assi osi ra ps eudonana t h a t i n v o l v e s urea-N e f f l u x as NH^ and i t s r a p i d r e a b s o r p t i o n i s proposed. These r e s u l t s e x p l a i n e a r l i e r o b s e r v a t i o n s i n the l i t e r a t u r e and have i m p l i c a t i o n s f o r urea-N u t i l i z a t i o n and c y c l i n g by phytoplankton in nature. PART I I : SELENIUM INTRODUCTION Overview and objectives In the second p a r t of t h i s t h e s i s , the selenium (Se) n u t r i t i o n of the c o a s t a l marine diatom, Thai assi osira pseudonana, was i n v e s t i g a t e d . T h i s a l g a was used d u r i n g the i n i t i a l stages of t h i s t h e s i s f o r urea uptake experiments. Thai as si osi ra pseudonana grew w e l l i n the a r t i f i c i a l seawater medium r o u t i n e l y used i n our l a b o r a t o r y to c u l t u r e s u c c e s s f u l l y other s p e c i e s . Suddenly, f o r no apparent reason, i t became i n c r e a s i n g l y d i f f i c u l t to c u l t u r e T. ps eudo nana i n t h i s medium; f i n a l l y i t stopped growing. The r e s e a r c h d e s c r i b e d i n the forthcoming c h a p t e r s was i n i t i a t e d to e l u c i d a t e the m i s s i n g f a c t o r ( s ) , which prevented growth of t h i s a l g a . As the re s e a r c h progressed, i t became apparent that Se was an e s s e n t i a l element f o r growth of T. ps eudonana, and that i t was absent from the c u l t u r e medium. R e s u l t s presented i n Chapter 5 document the e s s e n t i a l growth requirement of T. ps eudo nana f o r selenium. The growth promoting p r o p e r t i e s of two forms of i n o r g a n i c Se present i n seawater, s e l e n a t e and s e l e n i t e , were examined. Ob s e r v a t i o n s of morphological f e a t u r e s d i a g n o s t i c of s e l e n i u m - d e f i c i e n t c e l l s were r e p o r t e d . The s p e c i f i c n u t r i t i o n a l r o l e of selenium i n a l g a e , and i n p l a n t s i n g e n e r a l , i s not understood. C o l l a b o r a t i v e research with G.J. Doucette (Doucette et al. 1987), which examined u l t r a s t r u c t u r a l a l t e r a t i o n s i n S e - d e f i c i e n t T. pseudonana, p r o v i d e d a working hypothesis to e x p l a i n the selenium requirement i n T. ps eudonana. By t e s t i n g t h i s h y p o t h e s i s , I sought to e x p l a i n the biochem i c a l b a s i s f o r the o b l i g a t e selenium requirement i n t h i s a l g a . These r e s u l t s are pre s e n t e d i n Chapter 6. An axenic c u l t u r e of T. ps eudonana 75 was grown i n the presence of Na 2 SeO^; the d i s t r i b u t i o n of selenium among the bi o c h e m i c a l c o n s t i t u e n t s , measured by s o l v e n t e x t r a c t i o n procedures, was determined. Gel e l e c t r o p h o r e s i s was used to i d e n t i f y s p e c i f i c s e l e n o p r o t e i n s , and c e l l e x t r a c t s were assayed f o r the selenoenzyme, g l u t a t h i o n e p e r o x i d a s e . Since the p l a n t b i o l o g y of selenium i s so p o o r l y understood and comparatively l i t t l e work has been done i n t h i s f i e l d , a d e t a i l e d account of our c u r r e n t knowledge of Se b i o l o g y i s pr o v i d e d . Historical perspective B i o l o g i c a l selenium (Se) re s e a r c h began i n the 1930's when p o i s o n i n g among l i v e s t o c k was connected to high Se content of f o o d s t u f f s (Robinson 1933, Byers 1935). Franke (1934) demonstrated e x p e r i m e n t a l l y that g r a i n grown i n c e r t a i n types of s o i l was t o x i c to animals, and these r e s u l t s l e d to the d i s c o v e r y t h a t Se d e r i v e d from p l a n t s was the c a u s a t i v e agent of a l k a l i d i s e a s e i n l i v e s t o c k . In t h e i r surveys of p l a n t s and s o i l s of Wyoming, Beath and co-workers (Beath et al . 1 934, 1935) observed that c e r t a i n genera were r e s t r i c t e d to s o i l s c o n t a i n i n g h i g h l e v e l s of Se. Some of these so-c a l l e d s e l e n i u m - i n d i c a t o r , or accumulator p l a n t s , s p e c i e s of 188 Astragalus, accumulated up to 10,000 ppm Se from s o i l c o n t a i n i n g l e s s than 10 ppm Se. Selenium t o x i c i t y was of overwhelming concern at t h i s time because of i t s impact on a g r i c u l t u r e , and i t s p o t e n t i a l as a p u b l i c h e a l t h hazard. Perhaps i t was f o r these reasons, t h a t the r e p o r t s by T r e l e a s e and T r e l e a s e (1938a, b ) , which s t a t e d that some i n d i c a t o r p l a n t s r e q u i r e d Se f o r growth, went l a r g e l y unnoticed. Even now the Se requirements of p l a n t s remain a p o o r l y s t u d i e d aspect of Se n u t r i t i o n . The b e n e f i c i a l e f f e c t s of Se i n animals were r e a l i z e d i n the l a t e 1950's. Schwarz and F o l t z (1957) reported that Se was an e s s e n t i a l t r a c e element, which was completely e f f e c t i v e in p r e v e n t i n g l i v e r n e c r o s i s i n r a t s grown on S e - d e f i c i e n t d i e t . S i m u l taneously, P a t t e r s o n et al. (1957) and Schwarz et al . (1957) p u b l i s h e d r e s u l t s a t t r i b u t i n g exudative d i a t h e s i s i n v i t a m i n E d e f i c i e n t c h i c k s to Se d e f i c i e n c y . T h e r e a f t e r , a number of r e s e a r c h e r s d i s c o v e r e d t h a t some p r e v i o u s l y unexplained i l l n e s s i n l i v e s t o c k were a r e s u l t of the lack of Se i n the d i e t (Muth et al . 1958, P r o c t o r et al. 1958): Suddenly Se had gained a r e p u t a b l e s t a t u s . Selenium requirements in plants U n l i k e the une q u i v o c a l r e s u l t s demonstrating Se e s s e n t i a l i t y i n animals, much c o n t r o v e r s y , a l b e i t based on l i t t l e study, surrounds the r o l e of Se i n p l a n t s . A c o n s i d e r a b l e p e r i o d of time e l a p s e d between the p u b l i c a t i o n s of T r e l e a s e and T r e l e a s e (1938a, b) and other r e s e a r c h 189 i n v e s t i g a t i n g the growth promoting p r o p e r t i e s of Se i n p l a n t s . R o s e n f e l d and Beath (1964) found the dry weight of Astragalus racemosus grown i n the presence of s e l e n i t e or s e l e n a t e was twice that of the c o n t r o l p l a n t s which were not s u p p l i e d with Se. But Broyer et al. (1966) f a i l e d to observe any s t i m u l a t o r y e f f e c t s of Se added to the non-accumulators, Medicago sativa and Trifolium subt er aneum. Even though c o n t r o l p l a n t s i n the study of Broyer et al. (1966) were grown i n S e - f r e e c u l t u r e ( a c t u a l l y 50 ng Se was present i n the n u t r i e n t s o l u t i o n s of each p l a n t ) they each c o n t a i n e d 1.5 ug Se when the experiments were terminated. Measurements of Se accumulated i n the carbon f i l t e r used to p u r i f y the green house a i r confirmed the presence of atmospheric Se; t h i s l e d Broyer et al. (1966) to p o s t u l a t e that p l a n t s were ab l e to d e r i v e Se from the a i r . The la c k of methodology a v a i l a b l e to grow p l a n t s i n a Se-free environment appears to be one of the major shortcomings of t h i s and s i m i l a r r e s e a r c h . In a l l s t u d i e s conducted to date, the f a c t that the c o n t r o l p l a n t s were able to grow i n the absence of Se enrichments confounds the i n t e r p r e t a t i o n of the r e s u l t s and q u e s t i o n s the v a l i d i t y of c o n c l u s i o n s that Se i s an e s s e n t i a l element f o r p l a n t s . True e s s e n t i a l i t y can only be demonstrated i f i n the absence of an element, p l a n t growth i s abnormal, the p l a n t s l i f e c y c l e can not be completed, or i f an e s s e n t i a l m e t a b o l i t e c o n t a i n s the element as an i n t e g r a l component ( S a l i s b u r y and Ross 1978). C l e a r l y , the r e s u l t s f o r higher p l a n t s f a l l s h o r t of s a t i s f y i n g these c r i t e r i a . To make matters worse, Broyer et al . (1972a, b) re p o r t e d that the r e s u l t s of T r e l e a s e and 190 T r e l e a s e (1938a, b) c o u l d be e x p l a i n e d as an a r t i f a c t caused by phosphate t o x i c i t y i n the c o n t r o l p l a n t s . Broyer et a l . (1972a) argued that the a d d i t i o n s of Se as SeC>3 or SeC>4 a m e l i o r a t e d the t o x i c e f f e c t s of phosphate and thereby c r e a t e d the impression that Se s t i m u l a t e d growth. Ne v e r t h e l e s s , the a d d i t i o n of 5 ug at Se-1 1 , two o r d e r s of magnitude l e s s than the phosphate c o n c e n t r a t i o n , i n c r e a s e d the dry weight of the p l a n t s by t w o - f o l d and reduced the t o t a l p l a n t phosphorus c o n c e n t r a t i o n by o n e - t h i r d . T h i s i s a pronounced e f f e c t f o r such a r e l a t i v e l y s m a l l a d d i t i o n of Se. Ziebus and S h r i f t (1971) found no evidence f o r Se e s s e n t i a l i t y i n c a l l u s t i s s u e of Astragalus s p e c i e s . F i n a l l y , Anderson and Scarf (1983) documented a s t i m u l a t o r y e f f e c t of Na 2Se0 3 on the growth of Trifolium repens and Neptunia amphexi caulis , but i n l i g h t of the evidence put f o r t h by Broyer et al. (1972b) reg a r d i n g . . . 2-l o n i c i n t e r a c t i o n s of S e0 3 , one cannot r u l e out t h i s c r i t i c i s m of t h e i r work. Although r e s e a r c h with h i g h e r p l a n t s has l e f t many unanswered q u e s t i o n s , s t u d i e s on algae have given more c o n c l u s i v e r e s u l t s . In two phytoplankton s p e c i e s , the o b l i g a t e growth requirements f o r Se are w e l l e s t a b l i s h e d (Lindstrom and Rodhe, 1978, Wehr and Brown 1985), and in. other s p e c i e s s t i m u l a t o r y e f f e c t s of Se on growth are r e p o r t e d . T h i s t h e s i s examines the selenium n u t r i t i o n of a marine a l g a , but I w i l l f o rgo f u r t h e r d i s c u s s i o n of t h i s aspect of Se b i o l o g y u n t i l Chapter 5. Chemical interactions of selenium Chemical i n t e r a c t i o n s of Se with other ions i n the growth medium, and at the membrane l e v e l , may a c t to a l t e r the a v a i l a b i l i t y of e i t h e r ion to the p l a n t . Through t h i s mechanism the b e n e f i c i a l e f f e c t s of Se may not be m a n i f e s t e d i n the u t i l i z a t i o n of selenium per se, but r a t h e r selenium may a l t e r the a v a i l a b i l i t y of the other i o n ( s ) to the p l a n t . The data of Broyer et al. (1972b) pr o v i d e one example. They found that the amount of Mn i n p l a n t t i s s u e v a r i e s a c c o r d i n g to the 2-amount of Se s u p p l i e d in the c u l t u r e medium. Since SeO^ i s - known to adsorb to Fe oxyhydroxides ( H a r r i s o n and B e r k h e i s e r 1982), i t s a d s o r p t i o n to Mn oxides may reduce the a v a i l a b i l i t y of Mn to the p l a n t and r e s u l t i n low Mn l e v e l s i n p l a n t t i s s u e . S i m i l a r l y , i f chemical i n t e r a c t i o n s o f Se with a t o x i c element reduces the t o x i c i t y of t h i s element to the organism in q u e s t i o n , then the b e n e f i c i a l e f f e c t s of Se may i n c o r r e c t l y be viewed as those a t t r i b u t e d to e s s e n t i a l elements. The r e s u l t s of G o t s i s (1982) demonstrate t h a t Se/Hg and Se/Cu antagonisms r e s u l t i n an e l i m i n a t i o n of the i n h i b i t o r y e f f e c t s seen when these elements are s u p p l i e d i n d i v i d u a l l y . Whether t h i s i n t e r a c t i o n o ccurs i n s o l u t i o n or i n t r a c e l l u l a r l y i s not apparent,, although h i g h Se l e v e l s i n the t i s s u e s of animals p r o t e c t s a g a i n s t the t o x i c e f f e c t s of heavy metals (Dip l o c k 1976). By c o n t r a s t to these i n d i r e c t e f f e c t s of Se, a number of s p e c i f i c S e - c o n t a i n i n g macromolecules are known i n a d i v e r s e group of organisms. Sped fi c s eI e ni um-cont ai ni ng macr omol e cul es P r i o r to the d i s c o v e r y that Se was an e s s e n t i a l element f o r animals, P i n s e n t (1954) p r o v i d e d the f i r s t example of a s p e c i f i c c a t a l y t i c r o l e f o r Se. She demonstrated that t r a c e amounts of Se, i n a d d i t i o n to Mo and Fe, were necessary f o r e x p r e s s i o n of a c t i v e formate dehydrogenase a c t i v i t y i n Escherichia c o l i . But i t wasn't u n t i l 23 years l a t e r t h a t Se was shown to be an i n t e g r a l component of t h i s enzyme i n E. coli (Enoch and L e s t e r 1975). Since then, Se-dependent formate dehydrogenases have been d e s c r i b e d i n s e v e r a l anaerobic b a c t e r i a (Jones et al. 1979). Other s e l e n o p r o t e i n s i n b a c t e r i a are w e l l c h a r a c t e r i z e d . The best known i s g l y c i n e reductase of Clostridium stricklandii (Turner and Stadtman 1973). N i c o t i n i c a c i d hydroxylase of Clostridium barkeri (Imhoff and Andreesen 1979) and xanthine dehydrogenases from Clostridium acidiurici and C. c yl i ndr os por um show enhanced a c t i v i t y when s e l e n i t e i s added to the medium (Wagner and Andreesen 1979). In C. barkeri, D i l w o r t h (1980) found that 75 Se and n i c o t i n i c a c i d h ydroxylase a c t i v i t y co-migrate d u r i n g e l e c t r o p h o r e s i s . F i n a l l y , t h i o l a s e i s o l a t e d from Clostridium kluyveri a l s o appears to be a s e l e n o p r o t e i n (Hartmanis 1980). Not on l y are s e l e n o p r o t e i n s common i n b a c t e r i a , s p e c i f i c s e l e n o n u c l e o s i d e s of tRNA's from Clostridium stricklandii (Chen and Stadtman 1980), Met hanococcus vannielii (Ching et al. 1984), Escherichia coli (Wittwer 1983) and c u l t u r e d mouse leukemia c e l l s (Ching 1984) have been i d e n t i f i e d . In Clostridium stricklandii, one Se - c o n t a i n i n g tRNA i s the major glutamate-accepting s p e c i e s among a l l the tRNA's, and i t s aminoacylation a c t i v i t y i s dependent upon the presence of Se (Ching and Stadtman 1982). The number of s e l e n o p r o t e i n s i d e n t i f i e d i n mammals has inc r e a s e d d r a m a t i c a l l y s i n c e the demonstration t h a t g l u t a t h i o n e peroxidase i s a selenoenzyme (Rotruck et al. 1972, 1973, Flohe et al. 1973). C u r r e n t l y , as many as 11 unique s e l e n o p r o t e i n s have been d e t e c t e d i n mice mammary e p i t h e l i a l c e l l s (Danielson and Medina 1986), and i n c l u d i n g d i f f e r e n t i s o e l e c t r i c forms d e t e c t e d by 2-D SDS-PAGE, a t o t a l of 25 pol y p e p t i d e s c o n t a i n Se s t a b l y a s s o c i a t e d with the p r o t e i n i n the form of s e l e n o c y s t e i n e . The s p e c i f i c enzymatic or biochemical f u n c t i o n s of these p r o t e i n s i s unknown. Sel e nomet abol i t e s in plants No s p e c i f i c S e - c o n t a i n i n g compounds have been i s o l a t e d from higher p l a n t s , but the accumulation of s e l e n o m e t a b o l i t e s has been w e l l examined i n the S e - i n d i c a t o r s p e c i e s . N e a r l y 80% of Se i n Astragalus i s hot-water e x t r a c t a b l e and i s present i n low-molecular weight compounds, which are f o r the most part Se analogs of the S - c o n t a i n i n g amino a c i d s (Horn and Jones 1940, T r e l e a s e et al. 1960) In n o n - S e - i n d i c a t o r p l a n t s , such as Atriplex sp. and Machaerant hera ramosa, Se i s accumulated i n the form of SeO^ (Ro s e n f e l d and Beath 1964); whereas, Peterson and B u t l e r (1962) found i n Lolium perenne, Tr i t i cum vulgare, Trif ol i um r e pens and T. pratense t h a t greater than 60% of c e l l u l a r Se was i n c o r p o r a t e d i n t o p r o t e i n . Recent evidence of the enzyme g l u t a t h i o n e p e r o x i d a s e i n c u l t u r e d p l a n t c e l l s (Drotar et al . 1985) and e a r l i e r r e p o r t s .of t h i s enzyme i n s p i n a c h c h l o r o p l a s t s (Flohe and Menzel 1971) and i t s p o s s i b l e e x i s t e n c e i n p l a n t t i s s u e (Neubert et al. 1962) have p r o v i d e d a new l i n e of evidence to address the q u e s t i o n of Se e s s e n t i a l i t y i n p l a n t s . Although these r e s u l t s c o n f l i c t with o b s e r v a t i o n s of Smith and S h r i f t (1979), i t i s apparent t h a t a g l u t a t h i o n e peroxidase does e x i s t i n some p l a n t s ; whether i t i s a Se-dependent enzyme remains to be r e s o l v e d . Glutathione peroxidase Oxygen r a d i c a l s and r e l a t e d products are d e t o x i f i e d i n t r a c e l l u l a r l y by a number of enzymes i n c l u d i n g superoxide dismutase, c a t a l a s e , and g l u t a t h i o n e p e r o x i d a s e . These r a d i c a l s are a b l e to i n i t i a t e f r e e r a d i c a l c h a i n r e a c t i o n s l e a d i n g to o r g a n i c and l i p i d hydroperoxide formation ( H a l l i w e l l 1974, Tappel 1977, Burton and Ingold 1986). P r o d u c t i o n of r a d i c a l s i n c e l l s may occur by a number of pathways: Superoxide, f o r example, i s formed d u r i n g o x i d a t i o n of s e v e r a l reduced compounds, such as f e r r e d o x i n and hemoproteins; by o x i d a t i v e enzymes, i n c l u d i n g xanthine oxidase; and by c h l o r o p l a s t s and mitochondria ( F r i d o v i c h , 1984). P o l y u n s a t u r a t e d f a t t y a c i d s , components of c e l l membranes, are p a r t i c u l a r l y s u s c e p t i b l e to o x i d a t i o n by p e r o x y r a d i c a l s . S i n c e c e l l membranes are e s s e n t i a l f o r c ompartmentalizing b i o c h e m i c a l processes and r e g u l a t i n g the flow of m a t e r i a l s i n t o and out of the c e l l , d e s t r u c t i o n of 195 membrane i n t e g r i t y u l t i m a t e l y l e a d s to c e l l death. G l u t a t h i o n e peroxidase (GSH-Px) i s a enzyme which c a t a l y z e s the r e d u c t i o n of a v a r i e t y of hydroperoxides (Flohe et al . 1979), but reduced g l u t a t h i o n e (GSH) i s the only reductant with which i t i s a c t i v e (Flohe 1976). T h i s enzyme p l a y s an important p r o t e c t i v e r o l e i n the c e l l removing i n j u r i o u s hydroperoxides. Two major types of GSH-Px have been found. In mammalian and a v i a n t i s s u e s , GSH-Px (EC 1.11.1.9) i s a selenoenzyme which i s a c t i v e with hydrogen peroxide and a v a r i e t y of or g a n i c p e r o x i d e s . A selenium-independent enzyme showing GSH-Px a c t i v i t y was i s o l a t e d from r a t t i s s u e (Lawrence and Burk 1976), but s i m i l a r i t i e s between t h i s enzyme and g l u t a t h i o n e - S - t r a n s f e r a s e s (EC 2.5.1.18) i n d i c a t e that they are probably the same enzyme (Prohaska 1980). The g l u t a t h i o n e - S - t r a n s f e r a s e s can be d i f f e r e n t i a t e d from true GSH-Px's by t h e i r i n a b i l i t y to reduce H 202 and t h e i r lack of Se. The molecular weight of g l u t a t h i o n e p e r o x i d a s e (EC 1.11.1.9) from a v a r i e y of sources ranges from 76,000 to 96,000 d a l t o n s (Stadtman 1980a). The enzyme i s a tetramer composed of i d e n t i c a l s ubunits (19,000 to 23,000 d a l t o n s ) each c o n t a i n i n g one s e l e n o c y s t e i n y l r e s i d u e . In the l i t e r a t u r e , enzymes which produce o x i d i z e d g l u t a t h i o n e i n the presence of organic hydroperoxides are o f t e n e r r o n e o u s l y r e f e r r e d to as GSH-Px, when they may be GSH-S-transferases. In t h i s t h e s i s , GSH-Px r e f e r s s p e c i f i c a l l y to the selenoenzyme GSH-Px (EC 1.11.1.9), but enzymes showing "GSH-Px a c t i v i t y " may or may not be true GSH-Px; they may a l s o be GSH-S-transferases. CHAPTER 5. SELENIUM: AN ESSENTIAL ELEMENT FOR GROWTH OF THE COASTAL MARINE DIATOM THALASSI OSIRA PSEUDONANA Background T h e n u t r i t i o n a l i m p o r t a n c e o f s e l e n i u m ( S e ) f o r a l g a l g r o w t h i s b e c o m i n g i n c r e a s i n g l y a p p a r e n t . P i o n e e r i n g w o r k b y P i n t n e r a n d P r o v a s o l i ( 1 9 6 8 ) d o c u m e n t e d t h e s t i m u l a t o r y e f f e c t s o f S e o n t h e g r o w t h o f t h r e e m a r i n e Chrysochromul i na s p p . S i n c e t h e n , t w o o t h e r s t u d i e s h a v e s h o w n , u s i n g a x e n i c c u l t u r e s , t h a t Peridinium cinctum f a . west i I ( L i n d s t r o m a n d R o d h e 1 9 7 8 ) a n d Chrysochromuli na b r e v i t u r r i t a ( W e h r a n d B r o w n 1 9 8 5 ) h a v e a n a b s o l u t e g r o w t h r e q u i r e m e n t f o r S e . O t h e r i n v e s t i g a t o r s h a v e d e m o n s t r a t e d t h a t S e a d d i t i o n s t o n a t u r a l a n d a r t i f i c i a l s e a w a t e r m e d i a s t i m u l a t e s t h e g r o w t h o f a x e n i c p h y t o p l a n k t o n ( W h e e l e r et al. 1 9 8 2 ) , a x e n i c m a c r o a l g a e ( F r i e s 1 9 8 2 ) , a n d x e n i c p h y t o p l a n k t o n ( v . S t o c h 1 9 8 0 , L i n d s t r o m 1 9 8 3 , 1 9 8 5 , K e l l e r et al . 1 9 8 4 ) c u l t u r e s . I n t h e s e r e p o r t s , t h e a l g a e w e r e a b l e t o g r o w w i t h o u t S e e n r i c h m e n t s , a l t h o u g h t h e b a c k g r o u n d c o n c e n t r a t i o n s o f S e i n t h e g r o w t h m e d i a w e r e n o t k n o w n . T o d a t e , r e p r e s e n t a t i v e s , f r o m s i x c l a s s e s o f a l g a e h a v e b e e n s h o w n t o h a v e a r e q u i r e m e n t f o r S e w h i c h i s e s s e n t i a l f o r , o r w h i c h m a r k e d l y s t i m u l a t e s , g r o w t h . T h e c o n c e n t r a t i o n o f S e r e q u i r e d t o s u p p o r t g r o w t h o f a l g a e d e p e n d s u p o n t h e c h e m i c a l f o r m o f t h e e l e m e n t , a n d t h e r e q u i r e m e n t i s s p e c i e s s p e c i f i c . I n s e a w a t e r , d i s s o l v e d S e i s 2 - 2 -p r e s e n t a s s e l e n i t e ( S e 0 3 , S e I V ) , s e l e n a t e ( S e 0 4 , S e V I ) a n d o r g a n i c S e . I n g e n e r a l , i t a p p e a r s t h a t s e l e n i t e i s t h e m o r e b i o l o g i c a l l y a c t i v e f o r m o f i n o r g a n i c S e ; h o w e v e r , t h e growth promoting p r o p e r t i e s of o r g a n i c Se compounds have r e c e i v e d much l e s s study. The importance of s e l e n i t e i n phytoplankton n u t r i t i o n i s f u r t h e r supported by the f i n d i n g of Wrench and Measures (1982). They observed that decreases i n Se IV , but not Se VI, c o n c e n t r a t i o n were i n v e r s e l y c o r r e l a t e d with i n c r e a s e s i n phytoplankton biomass and p a r t i c u l a t e Se i n a c o a s t a l seawater environment. Selenomethionine and s e l e n o c y s t i n e may be u t i l i z e d by some phytoplankton, and at l e a s t three s p e c i e s grow as w e l l on these o r g a n i c forms of Se as on Na 2Se0 3 (Lindstrom 1983, Wehr and Brown 1985). Chrysochromul ina brevi turrit a has a l s o been shown to u t i l i z e d imethyl s e l e n i d e (DMSe) as a Se source (Wehr and Brown 1985). T h i s a b i l i t y may be r e l e v a n t to Se c y c l i n g i n a q u a t i c systems, as DMSe and other v o l a t i l e Se compounds can be produced by m i c r o b i a l assemblages i n lake sediments (Chau et al., 1976). Current a t t e n t i o n to Se t o x i c i t y i n organisms from marine and freshwater environments i s warranted. Increases i n anthropogenic i n p u t s of Se, from a c i d r a i n and a g r i c u l t u r a l r u n - o f f , to a q u a t i c systems i s w e l l documented ( G i s s e l - N i e l s e n and G i s s e l - N i e l s e n 1973, Andren et al . 1975, Fur r et al . 1977, Parekh and Husain 1981) and p u b l i c i z e d ( M a r s h a l l 1985, 1986). However, l i t t l e i n f o r m a t i o n i s a v a i l a b l e on the requirements of Se by marine phytoplankton or on the e f f e c t s of e l e v a t e d c o n c e n t r a t i o n s of t h i s element on a l g a l growth. The present study documents the e s s e n t i a l requirement of Se f o r growth of the c o a s t a l marine diatom Thai as si osi ra pseudonana (clone 3H). T. pseudonana c o u l d not be maintained i n a r t i f i c i a l seawater medium without the a d d i t i o n of 1 98 nanomolar q u a n t i t i e s of Se. S e - l i m i t a t i o n and S e - s t a r v a t i o n i n T. ps eudonana r e s u l t e d i n a r e d u c t i o n i n c e l l growth r a t e and subsequent c e s s a t i o n of c e l l d i v i s i o n and a pronounced i n c r e a s e i n c e l l s i z e . M a t e r i a l s and Methods Al gal cul t ur e Thai assi osI ra pseudonana (clone 3H) was obtained from the Northeast P a c i f i c C u l t u r e C o l l e c t i o n (N.E.P.C.C. #58), Department of Oceanography, U n i v e r s i t y of B r i t i s h Columbia, where i t was maintained on n u t r i e n t e n r i c h e d n a t u r a l seawater. Axenic c u l t u r e s of T. pseudonana were e s t a b l i s h e d by repeated t r a n s f e r s on agar medium (1% agar, w/v), and the absence of b a c t e r i a was v e r i f i e d by a c r i d i n e orange e p i f l u o r e s c e n t microscopy (Hobbie et al. 1977). S t a i n e d samples were viewed under a Z e i s s model D-7802 e p i f l u o r e s c e n t microscope. Axenic c u l t u r e s were used i n i n i t i a l experiments ( F i g s . 31 and 32) to co n f i r m t h a t the s t i m u l a t o r y e f f e c t s of Se oc c u r r e d i n the absence of b a c t e r i a . T h e r e a f t e r , u n i a l g a l c u l t u r e s were used, but p r e c a u t i o n s were taken to minimize b a c t e r i a l c o n t a m i n a t i o n . Selenium was an e s s e n t i a l element f o r growth of Thal assi osi ra pseudonana i n xenic and axenic c u l t u r e . Culture medium and flasks C u l t u r e s of T. ps eudonana were grown i n f i l t e r - s t e r i l i z e d (0.22 um M i l l i p o r e f i l t e r s ) n u t r i e n t e n r i c h e d a r t i f i c i a l seawater based on ESAW (Harr i s o n et al. 1980). Axenic 199 c u l t u r e s were grown i n a u t o c l a v e d ESAW with f i l t e r - s t e r i l i z e d .vitamins and NaHCO^ added a f t e r the medium had co o l e d . Reagent grade chemicals were used throughout, and n u t r i e n t enrichment s o l u t i o n s were prepared i n d e i o n i z e d d i s t i l l e d water (DDW). In some experiments, the seawater s a l t s of ESAW were p u r i f i e d of c a t i o n i c t r a n s i t i o n metals by the use of Chelex 100, f o l l o w i n g the procedure of Morel et al . (1979). M o d i f i c a t i o n s to ESAW i n c l u d e d r e p l a c i n g F e N H ^ S G ^ ^ - 6 H 2 ° *>y an equimolar c o n c e n t r a t i o n of F e C l 3 - 6 H 2 0 , and by adding a l l the Fe to a Na 2EDTA s o l u t i o n to g i v e an EDTA:Fe molar r a t i o of 1.6. The remaining Na 2EDTA added to ESAW was in c l u d e d with the t r a c e metal stock s o l u t i o n , and 0.0126 g-1 1 Na 2Mo0 4 and 0.0059 g - l ~ 1 N i C l 2 - 6 H 2 0 were a l s o added. N a 2 g l y c e r o P 0 4 was re p l a c e d with an equimolar c o n c e n t r a t i o n of Na 2HP0 4. N a 2 S i 0 3 - 9 H 2 0 was prepared and added as d e s c r i b e d by S u t t l e et al. (1986). Se was added to ESAW, when r e q u i r e d , as aqueous s o l u t i o n s of Na2SeC>3 or Na 2Se0 4, and these s o l u t i o n s were f r e s h l y prepared f o r each experiment. Glassware and pol y c a r b o n a t e f l a s k s were used f o r c u l t u r i n g algae and s t o r i n g ESAW. They were soaked overnight i n 10% (v/v) HC1 and were a u t o c l a v e d b e f o r e use. A l t e r n a t i v e l y , glassware were soaked i n 1M HNC>3 and r i n s e d (R) thoroughly i n DDW, f o l l o w i n g 24 h of soaking i n DDW. P y r e x ^ (R) and Kimax^ 50-ml screw-capped tubes with t e f l o n l i n e r s were used f o r c u l t u r i n g T. pseudonana, and they were t r e a t e d s i m i l a r l y except they were f i l l e d w ith f r e s h DDW and aut o c l a v e d twice p r i o r to. use. The tubes were then 200 r e a u t o c l a v e d without DDW and used immediately. Culture conditions and growth measurements C u l t u r e s were c o n t i n u o u s l y i l l u m i n a t e d from two s i d e s by Vita-Lite® UHO and Sylvania®VHO d a y l i g h t f l u o r e s c e n t tubes. The l i g h t was f i l t e r e d through a 3 mm t h i c k sheet of blue CR) P l e x i g l a s w (No. 2069, Rohm and Hass), and the i r r a d i a n c e , measured at the sur f a c e of the c u l t u r e v e s s e l s , was 120 uE-m ^-s 1 . Growth temperature was maintained at 18°C i n a temperature r e g u l a t e d water bath. A l l experiments were conducted i n batch c u l t u r e s . In one experiment, a subsample from a continuous c u l t u r e was used as an inoculum source tor the batch c u l t u r e s . C e l l growth was monitored by in vivo c h l o r o p h y l l a f l u o r e s c e n c e measured by a Turner Designs model 10 fluorometer CR) and by c e l l counts on a C o u l t e r C o u n t e r ^ model TA I I . Average c e l l volumes were computed from the c e l l d i s t r i b u t i o n i n the (R) v a r i o u s channels of the C o u l t e r Counter , u s i n g a 70 um (R) a p e r t u r e sample tube. The C o u l t e r C o u n t e r 7 was c a l i b r a t e d with microspheres of 5.07 um i n diameter. In experiments -designed to examine the e f f e c t s of Na^eO^ and N a 2 S e 0 4 c o n c e n t r a t i o n on the growth r a t e of T. pseudonana, c e l l s were p r e c o n d i t i o n e d i n the medium f o r at l e a s t 10 c e l l d o u b l i n g s . Water mounts of T. pseudonana were prepared f o r l i g h t microscopy and were examined with a Z e i s s Photomicroscope II l i g h t microscope using Nomarski i n t e r f e r e n c e o p t i c s and b r i g h t f i e l d i l l u m i n a t i o n . 201 R e s u l t s Selenium requirement and recovery Thai assi osi ra pseudonana was i n o c u l a t e d from n u t r i e n t e n r i c h e d n a t u r a l seawater (ESNW) i n t o ESAW c o n t a i n i n g no added Se (-Se) or ESAW e n r i c h e d with 10~ 9 M Na 2Se0 3 (+Se) ( F i g . 30). The c u l t u r e s were s e r i a l l y d i l u t e d to maintain the c e l l s i n ex p o n e n t i a l growth. The inoculum was d i l u t e d 1000-fold with f r e s h medium to minimize any c a r r y over of d i s s o l v e d Se from the ESNW. The i n i t i a l c e l l c o n c e n t r a t i o n i n each c u l t u r e was ca. 3400 c e l l s - m l 1 . E x p o n e n t i a l growth r a t e s of T. ps eudonana were i d e n t i c a l f o r the -Se and +Se c u l t u r e s d u r i n g the f i r s t t r a n s f e r . However, the c e l l s i n the -Se ESAW had a reduced growth r a t e and f l u o r e s c e n c e y i e l d by comparison to the +Se c u l t u r e f o r the second t r a n s f e r . When T. ps eudonana was t r a n s f e r r e d f o r the t h i r d time i n t o -Se ESAW the c e l l s f a i l e d to grow. The +Se c u l t u r e maintained the same growth ra t e over the three t r a n s f e r s . C e l l counts of T. pseudonana v e r i f i e d t h a t f l u o r e s c e n c e was a c c u r a t e l y r e p r e s e n t i n g growth of t h i s a l g a and these have been i n c l u d e d , f o r comparison, with the f l u o r e s c e n c e data ( F i g . 30). Fur t h e r evidence f o r a Se requirement by T. ps eudonana i s presented i n F i g u r e 31. The a d d i t i o n of 10 1 ^ M Na2SeC>3 to a Se-deplete s t a t i o n a r y phase c u l t u r e caused a resumption i n growth of the c e l l s . C e l l counts and microscopic o b s e r v a t i o n confirmed t h a t the i n c r e a s e i n in vivo f l u o r e s c e n c e was a t t r i b u t e d to an i n c r e a s e i n c e l l numbers and was not j u s t an in c r e a s e i n c h l o r o p h y l l per c e l l or a change i n the 202 F i g . 30. Growth of T. ps eudonana over three s u c c e s s i v e t r a n s f e r s in a r t i f i c i a l seawater (ESAW) i n the absence of - 9 Se (O) and in ESAW supplemented with 10 M Na2SeC>3 (•). The growth curves f o r the f i r s t , second and t h i r d t r a n s f e r s begin at 0, 114 and 212 h, r e s p e c t i v e l y . When the c e l l s were in l a t e e x p o n e n t i a l growth, a p o r t i o n of the c u l t u r e was t r a n s f e r r e d i n t o f r e s h medium. Growth of the c u l t u r e was then f o l l o w e d by mo n i t o r i n g in vivo c h l o r o p h y l l a f l u o r e s c e n c e ( s o l i d l i n e s ) . The arrows i n d i c a t e when the c u l t u r e s were t r a n s f e r r e d . During the t h i r d t r a n s f e r , c e l l numbers (dashed l i n e ) were a l s o determined in the S e - r e p l e t e c u l t u r e . 2 0 3 2 0 4 F i g . 31. E a r l y s t a t i o n a r y phase T. pseudonana was t r a n s f e r r e d from Se-deplete ESAW i n t o two c u l t u r e tubes c o n t a i n i n g the same medium. At 120 h ( i n d i c a t e d by the arrow) one tube was spiked with 10 1 ^  M Na2SeC>3 (•), and the other tube served as the c o n t r o l (O) . p h o t o s y n t h e t i c e f f i c i e n c y . Morphological changes An ammonium ( N H 4 + ) - l i m i t e d chemostat c u l t u r e of T. pseudonana was grown at a d i l u t i o n r a t e of 1.0 d 1 . The medium r e s e r v o i r c o n t a i n e d ESAW without added Se, and the c u l t u r e s t a r t e d to wash out. At t h i s time, a l i q u o t s of the chemostat c u l t u r e were t r a n s f e r r e d to tubes e n r i c h e d with 25 + . -9 uM NH 4 , with and without 10 M Na 2SeC> 3. S i g n i f i c a n t l y g r e a t e r growth r a t e s were seen i n the +Se c u l t u r e s (mean value ± 1 SD) (1.49 + 0.08 d ~ 1 , n=4) compared to the -Se c u l t u r e s (1.03 ± 0.07 d ~ 1 , n=4). The C o u l t e r Counter® c e l l s i z e d i s t r i b u t i o n i n d i c a t e d that the average c e l l volume of the -Se c u l t u r e s i n c r e a s e d t w o - f o l d over the d u r a t i o n of the experiment; the c e l l s growing i n ESAW +Se decreased i n volume ( F i g . 32). M i c r o s c o p i c examination v e r i f i e d t h a t changes i n c e l l volume were not a t t r i b u t e d to c e l l clumping or to the formation of c e l l c h a i n s (see D i s c u s s i o n ) . C e l l s of J . ps e udo nana s t a r v e d f o r Se were g r e a t l y elongated by comparison to c e l l s growing i n S e - r e p l e t e ESAW ( F i g . 33a, c ) . C e l l l e n g t h along the p e r v a l v a r a x i s was as great as 60 um i n the -Se grown c u l t u r e s ; e x p e r i m e n t a l - g c u l t u r e s grown i n the presence of 10 M Na 2 S e 0 3 c o n t a i n e d c e l l s ca. 4-5 um i n l e n g t h ( F i g . 33a). There was no d i f f e r e n c e i n the v a l v e diameter between the -Se and +Se grown c e l l s ( F i g . 33b, c ) . Other m o r p h o l o g i c a l changes seen i n some Se-starved c e l l s i n c l u d e d bent and t w i s t e d c e l l s and c e l l s with cytoplasmic p r o t r u s i o n s from the g i r d l e r e g i o n . To 207 F i g . 32. Average c e l l volume of T. pseudonana, f o l l o w i n g t r a n s f e r of c e l l s from an ammonium-limited chemostat c u l t u r e growing on Se-deplete ESAW i n t o Se-deplete (O) or S e - r e p l e t e (•) ESAW. The i n i t i a l c e l l volume was determined i n d u p l i c a t e , and the range of the value s was l e s s than the width of the symbol. The experiment was done i n t r i p l i c a t e , but only the f i n a l c e l l volumes of the other two c u l t u r e s are gi v e n . 2 0 8 3 3 . Photomicrographs of v e g e t a t i v e c e l l s of T. - 9 ps eudonana grown i n ESAW e n r i c h e d with 10 M Na 2SeO (a) g i r d l e view and (b) va l v e view and i n Se-deplete ESAW; (c) g i r d l e view. S c a l e bar = 5 um. 210 e l i m i n a t e the p o s s i b i l i t y of t r a c e metal t o x i c i t y i n the c u l t u r e s , excess c h e l a t o r was added to the medium. The a d d i t i o n of 100 uM Na 2EDTA to ESAW -Se f a i l e d to a l l e v i a t e the growth i n h i b i t i o n seen i n the -Se c u l t u r e s of T. pseudonana. Moreover, these c e l l s underwent the same m o r p h o l o g i c a l changes seen i n the Se-deplete c u l t u r e s c o n t a i n i n g normal ESAW c o n c e n t r a t i o n s of EDTA (20 uM). S i m i l a r r e s u l t s were o b t a i n e d with T. ps eudonana grown i n Se-deplete chelexed ESAW. A l s o , i n c r e a s i n g the c o n c e n t r a t i o n of t r a c e metals added to ESAW by 5 times y i e l d e d r e s u l t s which were i d e n t i c a l to those o b t a i n e d by growing c e l l s i n ESAW -Se with normal t r a c e metal c o n c e n t r a t i o n s . Eleven t r a c e elements were t e s t e d f o r t h e i r a b i l i t y to support growth of T. pseudonana i n the absence of Se (Table XXI). The r e s u l t s demonstrated that Se was the only element which s t i m u l a t e d growth and that Na 2Se0 3, but not - 9 Na 2Se0 4, was e f f e c t i v e at a c o n c e n t r a t i o n of 10 M. Growth on selenite and selenate V a r i o u s c o n c e n t r a t i o n s of Na 2Se0 3 were t e s t e d to determine the lower l i m i t of requirement and the upper l i m i t of t o l e r a n c e f o r t h i s compound ( F i g . 34). E x c e l l e n t growth - 9 - 6 was o b t a i n e d with Na2SeC>3 c o n c e n t r a t i o n s of 10 to 10 M. An i n c r e a s e d l a g p e r i o d was seen with 10 1 ^ M Na 2Se0 3; however, the ex p o n e n t i a l growth r a t e was the same as that - 9 - 6 o b t a i n e d with 10 to 10 M. Thai assi osi ra pseudonana grew at a much reduced growth r a t e i n ESAW e n r i c h e d with 10 1 1 M N a 2 S e 0 3 and the l a g p e r i o d was ca. 175 h. There was c o n s i d e r a b l y more v a r i a t i o n between the d u p l i c a t e c u l t u r e s 212 Table XXI Trace elements tested for the i r a b i l i t y to support growth of T. ps eudonana in Se-deplete a r t i f i c i a l seawater (ESAW). - g The form of the element added i s given, and 10 M was added in each case. Each treatment was performed in dup l i ca te . No growth (-) , good growth (+). Element Growth Response A l - A 1 2 ( S 0 4 ) 3 -Ba- BaCl2> 2H 20 -C r - K 2 C r 0 4 -Cu- CuS0 4 -5H 2 0 -L i - L i C l -N i - N i C l 2 - 6 H 2 0 -Rb- RbCl -s - N a 2 S 0 3 -Se- Na 2 Se0 3 + Se- Na 2 Se0 4 -Te- K 2 T e 0 3 -V- NH 4 V0 3 -213 F i g . 34. Growth of T. ps eudonana as a f u n c t i o n of Na^eO^ c o n c e n t r a t i o n . An a l i q u o t of c e l l s from an e a r l y s t a t i o n a r y phase c u l t u r e growing in Se-deplete ESAW was added to ESAW e n r i c h e d with; (A) 10~ 7, 10~ 8, 10~ 9 (O) , 10 ~ 1 0 (•), (B) 10" 1 1 (•) or 1 0 " 1 5 M (O) Na 2Se0 3. E r r o r bars i n d i c a t e the range of d u p l i c a t e s and where absent the range was l e s s than the width of the symbol. 214 215 growing on 10 1 1 M Na 2Se0 3 than there was at the hi g h e r c o n c e n t r a t i o n s of Na 2SeC> 3. Thai assi osi ra pseudonana f a i l e d to -1 5 grow when the Na 2Se0 3 c o n c e n t r a t i o n was 10 M. C e l l growth resumed f o l l o w i n g a l a g time of 400 h, i n ESAW c o n t a i n i n g 1 0 ~ 1 2 M Na 2Se0 3 ( F i g . 35). Selenium was s u p p l i e d as s e l e n a t e i n the form of N a 2 S e 0 4 and the growth r a t e of T. ps eudonana was measured as a fu n c t i o n of Na 2Se0 4 c o n c e n t r a t ion ( F i g . 36). Thalassiosira _ q ps eudonana f a i l e d to grow i n ESAW supplemented with 10 and 10 M N a 2 S e 0 4 > The maximum growth r a t e of 1.45 d , observed -4 in c u l t u r e s growing with 10 M Na 2Se0 4, was l e s s than the maximum growth r a t e seen i n c u l t u r e s growing on Na 2SeC> 3. -3 -2 Concentr a t i o n s of 10 arid 10 M Na 2 S e 0 4 were i n h i b i t o r y to growth. As a comparison, growth r a t e of T. ps eudonana over a range of Na2SeC>3 c o n c e n t r a t i o n s i s a l s o i n c l u d e d ( F i g . 36). Thalassiosira pseudonana grew w e l l at 10 and 10 M Na2SeC>3, and the growth r a t e s were only s l i g h t l y l e s s f o r c e l l s exposed -3 • -1 to 10 M compared with the maximum growth r a t e of 1.7 d . A c r y s t a l l i n e p r e c i p i t a t e formed on the bottom of the c u l t u r e . . . . -2 tubes c o n t a i n i n g ESAW e n r i c h e d with 10 Na 2Se0 3. While the composition of t h i s m a t e r i a l i s unknown, i t may have been a 2+ +2 2-Ca and/or Mg s a l t of Se0 3 . I f t h i s i s true then the 2-c o n c e n t r a t i o n of d i s s o l v e d Se0 3 - i n these tubes was l e s s than -2 10 M. To t e s t f o r p o s s i b l e Se-phosphate i n t e r a c t i o n s , the growth rate of T. ps eudonana was measured i n the Se-deplete and S e - r e p l e t e (10 M Na 2Se0 3) ESAW c o n t a i n i n g three d i f f e r e n t d i s s o l v e d phosphorus (Na 2HP0 4) c o n c e n t r a t i o n s . 3-Thalassiosira pseudonana f a i l e d to grow at any of he P 0 4 35. D u p l i c a t e c u l t u r e s tubes e n r i c h e d with 10 M Na2SeC>2 were i n o c u l a t e d with Se-depleted T. ps eudonana. Growth was monitored to examine long term e f f e c t s of exposure to low c o n c e n t r a t i o n s of Se. 217 TIME (h) 218 F i g . 36. Maximum e x p o n e n t i a l growth rate as a f u n c t i o n of Na 2 S e 0 3 (•) and Na2SeC>4 (O) c o n c e n t r a t i o n . Growth r a t e s are the mean of four r e p l i c a t e s and e r r o r bars i n d i c a t e i 1 SD. SELENIUM CONC. (M) 220 c o n c e n t r a t i o n s (100, 21 and 0.5 uM) i n Se-deplete ESAW. In -9 ESAW e n r i c h e d with 10 M Na^eO^, T. pseudonana grew at the -1 3 -maximum growth r a t e of 1.7 d at a l l PC>4 c o n c e n t r a t i o n s . In ESAW, phosphate i s added at 20 uM and t h i s was used i n a l l other experiments. D i s c u s s i o n SeIenium-Iimitati on and recovery In ESAW unenriched with Se, T. ps eudonana was a b l e to undergo ca. 9 c e l l d i v i s i o n s p r i o r to Se s t a r v a t i o n and the c e s s a t i o n of growth ( F i g . 30). T h i s growth should r e p r e s e n t a r e d u c t i o n of i n t r a c e l l u l a r Se c o n c e n t r a t i o n to the minimum c e l l quota and the time r e q u i r e d f o r c e l l s to express Se l i m i t a t i o n i s a f u n c t i o n of t h e i r Se p r e c o n d i t i o n i n g . The i n i t i a l inoculum f o r these experiments had been maintained on n a t u r a l seawater, c o l l e c t e d from E n g l i s h Bay, Vancouver, which was e n r i c h e d with ES n u t r i e n t s o l u t i o n s ( H a r r i s o n et al . 1980) or the i n o c u l a were from c u l t u r e s growing on ESAW supplemented -9 with 10 M Na^eO^. The background c o n c e n t r a t i o n of Se i n ESAW i s unknown, however, the growth data ( F i g . 33) i n d i c a t e t h a t i t must be l e s s than 10 1 1 M. At times d u r i n g the experiments, I encountered problems with Se contamination of the Se-deplete ESAW. T h i s appeared to r e s u l t , at l e a s t i n p a r t , from an i n a b i l i t y to remove Se from glassware used f o r c u l t u r i n g the a l g a e . A more r i g o r o u s procedure was adopted where glassware were soaked i n 1 N HCl fo r 24 h and were then autoclaved twice c o n t a i n i n g f r e s h DDW. 221 T h i s was a s u c c e s s f u l means of c l e a n i n g glassware. I a l s o t e s t e d HNO^, a st r o n g o x i d i z i n g a c i d , i n an e f f o r t to convert the Se present i n the glassware to the more s o l u b l e -2 s p e c i e s of Se0 4 , but t h i s proved no more e f f e c t i v e than HC1. Autoclaved Se-deplete ESAW gave e r r a t i c r e s u l t s , and I n o t i c e d that T. ps eudonana was o f t e n a b l e to grow i n t h i s medium without Se enrichments. I suspect t h a t the autoclave was contaminating the Se-deplete ESAW w i t h s u f f i c i e n t Se to allow the a l g a to grow. In f i l t e r - s t e r i l i z e d ESAW, T. ps eudonana was never a b l e to grow without Se enrichments. Se a d d i t i o n s , i n the form of Na^eO^, have been observed to s t i m u l a t e the growth of Peri di ni um cinctum f a . westii at -1 3 c o n c e n t r a t i o n s as low as 2.5-10 M (Lindstrom and Rodhe 1978). Other a l g a l s p e c i e s r e q u i r e c o n s i d e r a b l y higher c o n c e n t r a t i o n s of Na 2Se0 3, such as Platymonas spp. which grew b e t t e r than c o n t r o l c u l t u r e s o n l y when c o n c e n t r a t i o n s g r e a t e r than 1.3*10 ^ M were added to the medium (Wheeler et al . 1982). Thai as si osi ra pseudonana grew at i t s maximum growth ra t e (1.7 d~ 1) i n ESAW c o n t a i n i n g 1 0 ~ 1 0 M Na 2Se0 3, although there was a s l i g h t l y i n c r e a s e d l a g p e r i o d r e l a t i v e to those c e l l s exposed to higher c o n c e n t r a t i o n s of N a 2 S e 0 3 < When 10 1 ^ M Na 2Se0 3 was added to a Se s t a r v e d c u l t u r e ( F i g . 32) there was a g r e a t e r l a g p e r i o d than t h a t observed with the p r e c o n d i t i o n e d c e l l s , and the growth r a t e was reduced to 0.4 d 1 . T h i s f u r t h e r emphasizes the importance of the c e l l s ' p r e c o n d i t i o n i n g to Se. I t a l s o demonstrates that c e l l growth can resume once the l i m i t i n g n u t r i e n t (Se) i s r e s u p p l i e d to the c e l l s . The use of f l u o r e s c e n c e i s too i n s e n s i t i v e to give 222 an a c c u r a t e measure of the recovery time of the c e l l s from Se-s t a r v a t i o n . Roughly 100 h elapsed before there was any evidence of c e l l growth. T h i s recovery time i s an upper 1 4 -l i m i t , and i n the f u t u r e the use of H CO^ may be one method to i n c r e a s e r e s o l u t i o n . The morphological changes seen i n the S e - l i m i t e d and s t a r v e d T. ps eudonana were not u n l i k e those seen i n phytoplankton s u b j e c t e d to high c o n c e n t r a t i o n s of t o x i c metals or s i l i c a d e f i c i e n c y (Thomas et al . 1980, Jahnke and Baumann 1983). However, the same growth i n h i b i t i o n and abnormal c e l l s of T. ps eudonana were observed i n Se-deplete chelexed ESAW and Se-deplete ESAW supplemented with 100 uM Na 2EDTA. Only i n ESAW c o n t a i n i n g Se c o u l d c e l l s grow. I t was demonstrated that other t r a c e elements, perhaps present as contaminants i n the Se stock s o l u t i o n s or made more a v a i l a b l e by the a d d i t i o n of Se, c o u l d not a l l e v i a t e the growth requirement f o r Se. In 3 -these experiments, there was no evidence f o r P 0 4 t o x i c i t y i n T. pseudonana grown i n S e - r e p l e t e ESAW e n r i c h e d with 20 and 3 -0.5 uM PO^ . In the absence of Se, phosphate has been observed to be t o x i c to some higher p l a n t s (Broyer et a l . 1972b). The f a c t that T. pseudonana was unable to grow i n . 3-medium l a c k i n g Se, r e g a r d l e s s of the P0 4 c o n c e n t r a t i o n , f u r t h e r i n d i c a t e s that growth i n h i b i t i o n was a r e s u l t of Se . . 3-d e f i c i e n c y and not P0 4 t o x i c i t y . Growth on selenite compared with selenate O v e r a l l , Na~SeO~ supported b e t t e r growth of T. pseudonana 223 than N a 2Se0 4. The minimum c o n c e n t r a t i o n of Na 2Se0 4 r e q u i r e d f o r growth of T. pseudonana was 10,000 times g r e a t e r than N a 2Se0 3. The s m a l l e s t a d d i t i o n of Na2SeC>3 which supported good growth of T. ps eudonana was 10 1 ^ M. T h i s c o n c e n t r a t i o n may be an underestimate because the background c o n c e n t r a t i o n of Se 2 -i n ESAW i s unknown. Moreover, i t has been shown that SeO^ 2-and Se0 4 can adsorb to m i n e r a l s u r f a c e s such as FeOOH (Hingston et al . 1968, H a r r i s o n and Berkheiser 1982) which are present i n seawater. The o b s e r v a t i o n s of Peterson and B u t l e r (1962) c o n f i r m that t h i s can occur; t h i s may have the e f f e c t of reducing the b i o a v a i l a b i l i t y of Se. U n c e r t a i n t y i n the amount of b i o l o g i c a l l y a v a i l a b l e Se suggests that f u t u r e work n e c e s s i t a t e s the development of a c h e m i c a l l y - d e f i n e d c u l t u r e medium where the a c t i v i t i e s of the v a r i o u s Se forms may be re g u l a t e d and determined. Such an approach has al r e a d y been taken by Morel et a l . (1979) f o r c a t i o n i c t r a c e metal s p e c i a t i o n i n a r t i f i c i a l seawater. In some a l g a e , N a 2 S e 0 4 i s as e f f e c t i v e l y u t i l i z e d as Na 2Se0 3 ( F r i e s 1982, Lindstrom 1985, Wehr and Brown 1985) and when these Se compounds are s u p p l i e d at the same c o n c e n t r a t i o n , a l g a l growth r a t e and f i n a l biomass y i e l d are i d e n t i c a l . T h i s was not observed f o r T. pseudonana, although - 5 10 M Na 2 S e 0 4 was not t e s t e d f o r growth (Na2SeC>3 data not shown). Higher c o n c e n t r a t i o n s of Na 2Se0 4 were r e q u i r e d to support growth of T. ps eudonana. T h i s i s c o n s i s t e n t with the r e s u l t s of Lindstrom (1983). He found that long a d a p t a t i o n times were r e q u i r e d f o r a l g a l growth on Na 2Se0 4, r e l a t i v e to Na 9SeO~, but I saw no evidence f o r t h i s . Experiments were not 224 designed to determine long term a d a p t a t i o n times of T. 2-ps eudonana exposed to low c o n c e n t r a t i o n s of SeO^ . However, i t was n o t i c e d f o r one experiment that growth resumed a f t e r -1 2 400 h of exposure to 10 M Na 2SeC> 3. At t h i s c o n c e n t r a t i o n , I d i d not t e s t whether repeated s u b c u l t u r i n g would reduce the long l a g p e r i o d f o r growth, but when c e l l s which had been adapted to 10~ 1 1 and 1 0 ~ 1 0 M Na2SeC>3 f o r at l e a s t 10 c e l l d i v i s i o n s were t r a n s f e r r e d to new medium, the l a g p e r i o d s were s t i l l observed. T h i s does not p r e c l u d e the p o s s i b i l i t y of c e l l u l a r a d a p t a t i o n over s t i l l longer p e r i o d s of time, and that t h i s can occur i n c e l l s grown i n i n c r e a s i n g concen-t r a t i o n s of Na2SeC>4 (Kumar 1964) i n d i c a t e s that a d a p t a t i o n to low c o n c e n t r a t i o n s may a l s o occur. S e l e n i t e has been shown to be more i n h i b i t o r y to growth of c y a n o b a c t e r i a than e q u i v a l e n t c o n c e n t r a t i o n s of N a 2 S e 0 4 and organic Se (Kumar and Prakash 1971). S i e l i c k i and Burnham — 5 (1973) have a l s o found t h a t c o n c e n t r a t i o n s of Na 2Se0 3 of 10 M and greater were t o x i c to Phormidium luridum v a r . olivacea. T h i s a l g a reduced Na 2Se0 3 to elemental Se at hig h N a 2 S e 0 3 c o n c e n t r a t i o n s . Thalassiosira ps eudonana i s t o l e r a n t of -3 Na2SeC>3 c o n c e n t r a t i o n s g r e a t e r than 10 M and there i s on l y a s l i g h t but s i g n i f i c a n t (p < 0.05) r e d u c t i o n i n c e l l growth r a t e . Wehr and Brown (1985) showed a r e d u c t i o n i n growth and y i e l d of Chrysochromul i na brevi turrit a when Na2SeC>3 -4 c o n c e n t r a t i o n s were g r e a t e r than 10 M. In other organisms, Na2SeC>4 i s the more t o x i c form of i n o r g a n i c Se (Brown and Smith 1979). T h i s appears to be the case i n T. pseudonana, 225 and Na2SeC>4 completely i n h i b i t e d c e l l growth when s u p p l i e d at - 3 - 2 10. and 10 M. Even when c e l l s which had been maintained f o r g r e a t e r than 20 g e n e r a t i o n s i n ESAW supplemented with 10~ 4 M Na2SeC>4 were used as an inoculum f o r the higher Se c o n c e n t r a t i o n s , growth was i n h i b i t e d . The t o x i c e f f e c t s of Se are b e l i e v e d to a r i s e as the r e s u l t of the i n d i s c r i m i n a n t i n c o r p o r a t i o n of Se i n t o s u l f u r (S) c o n t a i n i n g macromolecules through the S a s s i m i l a t o r y pathways (Stadtman 1979). I t i s . . 2-not s u r p r i s i n g , t h e r e f o r e , t h a t Se0 4 , which i s c h e m i c a l l y 2- 2-more s i m i l a r to SC>4 than i s SeO^ , i s the more potent of 2 -these two Se forms. S h r i f t (1954) p o i n t e d out that SC>4 and 2-Se0 4 compete f o r a common p o r t e r i n Chi or el I a, and Brown and S h r i f t (1980a) have a l s o demonstrated t h i s to be true i n a 2 -bacterium. A d d i t i o n a l l y , i t appears that uptake of SeC>3 i s 2- 2-d i s t i n c t from S e0 4 and S 0 4 uptake i n b a c t e r i a (Brown and S h r i f t 1980a, Hudman and Glenn, 1984, 1985). In T. pseudonana, the growth r a t e data, i n response to d i f f e r e n t c o n c e n t r a t i o n s of Na 2SeC> 4 and Na 2Se0 3, demonstrate that the uptake and/or a s s i m i l a t i o n of these Se compounds i s d i s s i m i l a r . N e v e r t h e l e s s , the f a c t that both Se forms can support the growth of T. ps eudonana i n d i c a t e s that there are some s i m i l a r i t i e s . The l a c k of Na 2Se0 3 t o x i c i t y suggests that T. ps eudonana i s capable of r e g u l a t i n g the i n f l u x or e f f l u x of t h i s ion or i s ab l e to d e t o x i f y i t i n t r a c e l l u l a r l y . F u r t h e r work i s necessary to r e s o l v e the d i f f e r e n c e s between the c e l l u l a r metabolism of these Se compounds i n alg a e . 226 Morphological changes C e l l volumes of T. ps eudonana based on C o u l t e r Counter® data are underestimates, s i n c e the o r i e n t a t i o n of the c e l l s p a s s i n g through the ape r t u r e w i l l e f f e c t the measurement. M i c r o s c o p i c o b s e r v a t i o n s demonstrated t h a t S e-depleted c e l l s were up to 10 times the l e n g t h of the S e - r e p l e t e c e l l s , and a s i m i l a r i n c r e a s e would a l s o be expected i n c e l l volume. Roughly 90% of the c e l l s i n the Se-deplete ESAW became elongated, but t h i s depended on the stage of the growth c y c l e . E a r l y evidence of Se l i m i t a t i o n was observed by an i n c r e a s e i n the number of c e l l s which f a i l e d to separate f o l l o w i n g c y t o k i n e s i s . Thalassiosira pseudonana i s a u n i c e l l u l a r diatom when grown under n u t r i e n t s u f f i c i e n c y . As the growth r a t e of T. ps eudo nana became reduced i n the ESAW -Se, c e l l s of 2-4 times the s i z e of the +Se c e l l s were seen i n c h a i n s or as s o l i t a r y c e l l s . F i n a l l y , the c e l l s became more e l o n g a t e d and some of the c e l l s became t w i s t e d and bent. M i c r o s c o p i c o b s e r v a t i o n of c e l l s s t a i n e d with a c r i d i n e orange showed that the elongate c e l l s from the -Se c u l t u r e were, f o r the most p a r t , u n i n u c l e a t e and no c e l l s c o n t a i n e d m u l t i p l e n u c l e i . Although b i n u c l e a t e c e l l s were observed they were no more frequent than i n the +Se c u l t u r e . Thus, Se s t a r v a t i o n prevents n u c l e a r d i v i s i o n and c y t o k i n e s i s ; however, c e l l s are s t i l l a b le to i n c r e a s e i n s i z e i n s p i t e of the blockage of these events. Doucette et al . (1987) have d e s c r i b e d the m o r p h o l o g i c a l changes a s s o c i a t e d with Se d e f i c i e n c y i n more d e t a i l . I t i s 227 i n t e r e s t i n g t h a t p r e v i o u s r e s e a r c h examining Se n u t r i t i o n has r e p o r t e d i n c r e a s e s i n a l g a l c e l l s i z e f o l l o w i n g exposure of-c e l l s to h i g h c o n c e n t r a t i o n s of Se ( S h r i f t 1954), but no such changes were e v i d e n t i n T. pseudonana. By c o n t r a s t , Lindstrom (1983) has r e p o r t e d an i n c r e a s e i n the c e l l diameter of Peridinium cinctum s t a r v e d f o r Se. Although h i s r e s u l t s were l e s s dramatic than the r e s u l t s with T. pseudonana, i t i s tempting to s p e c u l a t e that Se may pl a y a s i m i l a r r o l e i n these d i n o f lage H a t e s . Ecological considerations By comparison with n a t u r a l seawater c o n c e n t r a t i o n s of Se, the minimum amount of Na 2Se0 3 r e q u i r e d to support growth of T. pseudonana i s very s i m i l a r to the c o n c e n t r a t i o n measured i n n a t u r a l seawater. In s u r f a c e seawater of the P a c i f i c and Indian Oceans, the c o n c e n t r a t i o n of Na 2Se0 3 i s ca. 5-10 1 1 M and c o n c e n t r a t i o n s i n the A t l a n t i c are 30-40% lower (Measures et a l . 1983). Comparable r e s u l t s have been obtained by C u t t e r and Bruland (1984), and many of t h e i r measurements were at or below the l i m i t of d e t e c t i o n (10 1 1 mol-Kg 1 ) . I t i s p o s s i b l e that i n nature, growth of T. ps eudonana c o u l d be l i m i t e d by t h i s c o n c e n t r a t i o n of Na 2Se0 3. There i s some independent c o n f i r m a t i o n of t h i s p r o p o s a l . Wehr and Brown (1985) demonstrated t h a t Se can l i m i t growth of Chrysochromul i na breviturrit a in s i t u , and K e l l e r et al . (1984) have found that Se enrichments to Sargasso seawater are necessary f o r the maintenance of marine u l t r a p l a n k t o n clones i s o l a t e d from o l i g o t r o p h i c oceanic environments. Although the c o n c e n t r a t i o n 228 of Na2Se0 4 i n seawater i s g r e a t e r than Na2Se0 3 (Measures et al . 1980, 1983, C u t t e r and Bruland 1984), i t i s presen t i n too low a c o n c e n t r a t i o n to be u t i l i z e d by T. pseudonana. In c o a s t a l seawater, the t o t a l Se c o n c e n t r a t i o n i s g r e a t e r than in oceanic water, but va l u e s are s t i l l very low and they are more v a r i a b l e (0.38-1.8 • 10~ 9 M) ( S c h u l t z and T u r e k i a n 1965, Sugimura et al . 1976, Measures and Burton 1980, C u t t e r 1982, Takayanagi and Wong 1984). The c o n c e n t r a t i o n of t o t a l Se i n o l i g o t r o p h i c oceanic s u r f a c e seawater averages 5-10 ^ mol-Kg 1 , and 80% of t h i s i s d i s s o l v e d o r g a n i c s e l e n i d e (Cutter and Bruland 1984); at l e a s t a p o r t i o n of the s e l e n i d e i s b e l i e v e d to be i n the form of amino a c i d s and p o l y p e p t i d e s . Phytoplankton are capable of u t i l i z i n g some forms of organic Se and t h i s l a r g e pool of org a n i c Se i n seawater may be d i r e c t l y important i n phytoplankton n u t r i t i o n . However, as a note of c a u t i o n , i t was r e p o r t e d that a v a r i a b l e p o r t i o n of the t o t a l d i s s o l v e d Se i n lake water i s u n a v a i l a b l e f o r phytoplankton growth (Lindstrom 1980). The b i o a v a i l a b i l i t y of d i s s o l v e d forms of Se i n seawater has not been examined. The r ol e of s el eni um The b i o c h e m i c a l b a s i s f o r the requirement of Se i n algae remains unknown. N a t u r a l l y o c c u r r i n g s e l e n o p r o t e i n s have been i d e n t i f i e d i n b a c t e r i a (reviewed by Stadtman 1980a, b) and the e s s e n t i a l requirement of Se i n mammals and b i r d s i s a t t r i b u t e d to the S e - c o n t a i n i n g enzyme g l u t a t h i o n e p e r o x i d a s e . In higher p l a n t s , demonstration of an o b l i g a t e Se requirement remains e q u i v o c a l . In some higher p l a n t s , i t i s apparent that Se i s an important c e l l u l a r c o n s t i t u e n t . F u r t h e r experimentation i s r e q u i r e d to e s t a b l i s h whether Se i s e s s e n t i a l f o r growth of a l l h igher p l a n t s . Enhanced g l u t a t h i o n e p e r o x i d a t i o n by c e l l e x t r a c t s of Dunaliella I er t i ol ect a and Porphyri di um cruentum was evident when these two alga e were c u l t u r e d i n medium c o n t a i n i n g Na2SeC>2 (Gennity et al . 1985). However, i t was concluded that the selenoenzyme g l u t a t h i o n e peroxidase was absent and that hydroperoxide dependent o x i d a t i o n of g l u t a t h i o n e was non-enzymatic i n na t u r e . In Euglena gracilis, i t has been shown that there e x i s t s two types of g l u t a t h i o n e p e r o x i d a s e s , one of which i s a Se-independent form of the enzyme (Overbaugh and F a l l 1982, 1985). Thus f a r I am unable to a s c r i b e a s p e c i f i c metabolic r o l e f o r Se i n T. pseudonana. I t i s evident from these data, and i t i s f u r t h e r c o r r o b o r a t e d by r e s u l t s of Doucette et al . (1987), t h a t Se i s an i n d i s p e n s a b l e t r a c e element f o r the growth of T. pseudonana. The development of u l t r a - c l e a n methods f o r the c u l t u r e of al g a e , through the use of c h e m i c a l l y d e f i n e d media prepared with u l t r a p u r e water and chemicals, has i n c r e a s e d our knowledge of the n u t r i t i o n a l requirements of these important primary p r o d u c e r s . I t has a l s o made c u l t u r i n g of some phytoplankton more d i f f i c u l t . P r e v i o u s l y , i n some i n s t a n c e s we have r e l i e d on contaminating sources of known and unknown e s s e n t i a l elements i n a r t i f i c i a l seawater to f u l f i l l the growth requirements of many phytoplankton (e.g. O l i v e i r a and A n t i a 1984). However, with the use of u l t r a - c l e a n a r t i f i c i a l seawater i t may now become i n c r e a s i n g l y d i f f i c u l t to c u l t u r e s u c c e s s f u l l y some of the more d i f f i c u l t - t o - g r o w phytoplankton u n t i l we have e l u c i d a t e d other e s s e n t i a l growth requirements of these organisms. S u m m a r y An o b l i g a t e requirement f o r selenium i s demonstrated in axenic c u l t u r e of the c o a s t a l marine diatom Thalassiosira ps eudonana (clone 3H) (Hustedt) Hasle and Heimdal grown.in a r t i f i c i a l seawater medium. Selenium d e f i c i e n c y was c h a r a c t e r i z e d by a r e d u c t i o n i n growth r a t e and e v e n t u a l l y by a c e s s a t i o n i n c e l l d i v i s i o n . The a d d i t i o n of 10 1 ^ M Na 2Se0 3 to n u t r i e n t e n r i c h e d a r t i f i c i a l seawater r e s u l t e d i n e x c e l l e n t growth of T. pseudonana. Only a s l i g h t i n h i b i t i o n of growth -3 -2 occur r e d with Na 2Se0 3 c o n c e n t r a t i o n s of 10 and 10 M. By c o n t r a s t , N a 2 S e 0 4 f a i l e d to support growth of T. ps eudonana -7 when s u p p l i e d at c o n c e n t r a t i o n s l e s s than 10 M, and the growth r a t e at t h i s c o n c e n t r a t i o n was onl y one q u a r t e r of the -3 -2 maximum growth r a t e . The a d d i t i o n of 10 and 10 M Na 2Se0 4 to the c u l t u r e medium was t o x i c and c e l l growth was completely i n h i b i t e d . Eleven t r a c e elements were t e s t e d f o r t h e i r a b i l i t y to r e p l a c e the selenium requirement by t h i s a l g a and a l l were without e f f e c t . In s e l e n i u m - d e f i c i e n t and s e l e n i u m - s t a r v e d c u l t u r e s of T. ps eudonana, c e l l volume i n c r e a s e d as much as 1 0 - f o l d as a r e s u l t of an i n c r e a s e i n c e l l l e n g t h (along the p e r v a l v a r a x i s ) , but c e l l width was constant. I t i s concluded that selenium i s an i n d i s p e n s a b l e element f o r the growth of T. ps eudonana; i t should be i n c l u d e d as a n u t r i e n t enrichment a r t i f i c i a l seawater medium when c u l t u r i n g t h i s a l g a . 232 CHAPTER 6. SPECIFIC SELENIUM-CONTAINING POLYPEPTIDES IN THE  MARINE DIATOM THALASSIOSIRA PSEUDONANA Background The b i o c h e m i c a l b a s i s f o r the o b l i g a t e growth requirement f o r selenium (Se) i n some marine and f r e s h water phytoplankton i s unknown. Although a number of s e l e n o p r o t e i n s are c h a r a c t e r i z e d i n animals and p r o k a r y o t i c h e t e r o t r o p h s , no s p e c i f i c S e - c o n t a i n i n g macromolecules have been i s o l a t e d from p h o t o s y n t h e t i c organisms. In the prec e e d i n g chapter, i t was demonstrated that growth of the marine diatom Thai as si osira pseudonana was dependent upon Se. T h i s requirement was s p e c i f i c f o r Se; no other elements were a b l e to s u b s t i t u t e f o r t h i s growth requirement. To date, the onl y i n v e s t i g a t i o n s of S e - c o n t a i n i n g macromolecules i n algae have examined s p e c i e s which have not been shown to have an o b l i g a t e Se requirement f o r growth. In two phytoplankton s p e c i e s , Tetraselmis tetrethele and 7 5 Dunaliella mi nut a, Wrench (1978) observed that ca. 55% of Se a s s o c i a t e d with the c e l l s was p r o t e i n bound. A f r a c t i o n of t h i s was present as hydrogen s e l e n i d e , and the remaining Se was i n the form of selenoamino a c i d s . Gennity et al. (1984) found that Se was a s s o c i a t e d with l i p i d i n Dunaliella primolecta and Porphyridium cruentum, when these algae were c u l t u r e d i n the presence of hig h c o n c e n t r a t i o n s of s e l e n i t e (0.13 mM). However, they found no evidence that Se was i n c o r p o r a t e d i n t o the c o v a l e n t s t r u c t u r e of any l i p i d s . U l t r a s t r u c t u r a l a l t e r a t i o n s to the membranes of Se-d e f i c i e n t T. ps eudonana were s i m i l a r to changes seen i n Se-d e f i c i e n t mammalian t i s s u e (Doucette et al. 1987). In mammals, Se i s known to be r e q u i r e d f o r the selenoenzyme g l u t a t h i o n e peroxidase (Rotruck et al . 1972, 1973, Flohe et al . 1973), and S e - d e f i c i e n c y r e s u l t s i n decreased l e v e l s of t h i s enzyme (Chow and Tappel 1974). In the absence of Se, c h l o r o p l a s t t h y l a k o i d s of T. pseudonana were d i s r u p t e d and i n some i n s t a n c e s formed b a l l o o n - l i k e s t r u c t u r e s . S i m i l a r o b s e r v a t i o n s were made i n spinach c h l o r o p l a s t s , which were induced to s w e l l i n high l i g h t (Murakami and Nobel 1967). The r e s u l t s of Murakami and Nobel (1967) were a t t r i b u t e d to l i p i d p e r o x i d a t i o n and the accumulation of l i p i d p e r oxides i n the membranes. The l i p i d peroxides caused changes i n the i n t e g r i t y of the membranes and thereby a l t e r e d t h e i r p h y s i o c h e m i c a l p r o p e r t i e s (Heath and Packer 1965, Murakami 1968). G l u t a t h i o n e induces s w e l l i n g of mi t o c h o n d r i a , and l i p i d p eroxides accumulate c o n c u r r e n t l y (Hunter et al. 1959, Lehninger and Schneider 1959). Neubert et al . ( 1 962) showed t h a t g l u t a t h i o n e peroxidase reversed t h i s s w e l l i n g by p r e v e n t i n g the accumulation of l i p i d p e r o x i d e s . In both m i t o c h o n d r i a and c h l o r o p l a s t s , i t appears that g l u t a t h i o n e p e r o x i d a s e a c t s to d e t o x i f y i n j u r i o u s l i p i d p e r o x i d e s , or t h e i r p r e c u r s o r s , and maintains membrane i n t e g r i t y (Flohe and Zimmerman 1970). On the b a s i s of these o b s e r v a t i o n s , I hyp o t h e s i z e d that the selenoenzyme g l u t a t h i o n e peroxidase o c c u r s i n T. ps eudonana. Although some enzymes showing g l u t a t h i o n e peroxidase a c t i v i t y have been d e t e c t e d i n a l g a e , t h e r e i s no evidence to i n d i c a t e whether or not they are Se-234 dependent enzymes. The purpose of t h i s chapter i s two- f o l d : 1) to d e s c r i b e Se uptake and the d i s t r i b u t i o n of Se w i t h i n the biochemical c o n s t i t u e n t s of T. pseudonana, and 2) to assay c e l l - f r e e e x t r a c t s f o r g l u t a t h i o n e p e r o x i d a s e a c t i v i t y . Mater ia ls and Methods Culture conditions Thalassiosira pseudonana was grown i n modified a r t i f i c i a l seawater medium (ESAW) of H a r r i s o n et al. (1980) as d e s c r i b e d i n Chapter 5. An axenic inoculum of T. pseudonana was t r a n s f e r r e d i n t o 8 l i t r e s of a u t o c l a v e d ESAW supplemented with 10 M Na 2 Se0 3 (Amersham; 5.72 mCi-mg ; 0.21 GBq-mg ). C e l l s were grown at 17°C under a continuous i r r a d i a n c e of 145 - 2 - 1 (R) uE-m • s s u p p l i e d by V i t a - L i t e ^ f l u o r e s c e n t bulbs, and growth was monitored by measuring in vivo c h l o r o p h y l l a (R) f l u o r e s c e n c e . A Spencer B r i g h t - L i n e ^ hemacytometer was used to count c e l l s . The c u l t u r e was c o n t i n u o u s l y s t i r r e d by a 75 t e f l o n - c o a t e d magnetic s t i r bar. I n c o r p o r a t i o n of Se i n t o the phytoplankton was determined by f i l t e r i n g 5 ml subsamples of c u l t u r e through Whatman GF/C f i l t e r s . The f i l t e r s were r i n s e d with 5 ml of f i l t e r - s t e r i l i z e d seawater (0.2 um M i l l i p o r e f i l t e r ) , and the amount of r a d i o a c t i v i t y r e t a i n e d by the f i l t e r s was measured. 7 5 Measurement of Se Selenium-75, a r a d i o a c t i v e i s o t o p e of selenium, undergoes e l e c t r o n capture decay with a h a l f - l i f e of 119.8 d. During 75 r a d i o a c t i v e decay, energy i s r e l e a s e d from Se atoms i n the form of ele c t r o m a g n e t i c (gamma rays and X-rays) and c o r p u s c u l a r ( i n t e r n a l - c o n v e r s i o n - e l e c t r o n t r a n s i t i o n s and Auger e l e c t r o n s ) r a d i a t i o n (NCRP Report No. 58 1985),. Gamma 75 c o u n t i n g i s the c o n v e n t i o n a l method f o r measuring Se, however, the isotope may a l s o be d e t e c t e d u s i n g l i q u i d s c i n t i l l a t i o n t echniques. L i k e beta p a r t i c l e s , Auger e l e c t r o n s cause l i q u i d f l u o r e s c e n t m a t e r i a l s to emit l i g h t which can be dete c t e d by s e n s i t i v e p h o t o - m u l t i p l i e r tubes present i n l i q u i d s c i n t i l l a t i o n c o u n t e r s . Since the use of 75 t h i s procedure to measure Se i s not r e p o r t e d i n the l i t e r a t u r e , the p r e l i m i n a r y experiments used to v a l i d a t e t h i s 125 methodology are re p o r t e d here. I, which a l s o decays by e l e c t r o n capture, can be counted e q u a l l y w e l l u s i n g gamma or 51 l i q u i d s c i n t i l l a t i o n c o unting (Helman and T i n g 1973); Cr can be counted more e f f i c i e n t l y by l i q u i d s c i n t i l l a t i o n c o u n t i n g than by gamma counting (Sheppard and Marlow 1971, Helman and Tin g 1973). The energy d i s t r i b u t i o n of the Auger e l e c t r o n s e m i t t e d by 75 . Se i s shown i n F i g u r e 37. The t o t a l number of cpm measured by l i q u i d s c i n t i l l a t i o n c o u n t i n g , with Aquasol,II as s c i n t i l l a t i o n f l u o r , was l i n e a r l y r e l a t e d to the amount of 7 5 Se added to each sample ( F i g . 38). A quench curve was c o n s t r u c t e d u s i n g acetone as the quenching agent ( F i g . 39). In a l l cases, quenching by phytoplankton samples was n e g l i g i b l e , and coun t i n g e f f i c i e n c y was approximately 93%. 37. Energy spectrum of Se as measured by l i q u i d s c i n t i l l a t i o n c o u n t i n g with Aquasol II as f l u o r . 38. R e l a t i o n s h i p between the amount of Se measured by l i q u i d s c i n t i l l a t i o n counting (cpm) and the amount of 7 5 . Se added to s c i n t i l l a t i o n v i a l s c o n t a i n i n g Aquasol I I . 240 75 F i g . 39. Quench curve f o r Se determined by u s i n g the H-Number method of quench monitoring. Acetone was used as the quenching agent. The equation of the l i n e i s y = -3.253x + 1.104; r 2 = 0.997. 241 AON3IOIJd3 39VlN30H3d Collection of cells When the c u l t u r e was i n l a t e e x p o n e n t i a l growth phase, the c e l l s were harvested by f i l t r a t i o n onto Whatman GF/C f i l t e r s . C e l l s were removed from the f i l t e r s with a s p a t u l a , (R> p l a c e d i n C r y o v i a l s * ^ (Simport P l a s t i c s L t d . , Quebec), and immediately f r o z e n i n l i q u i d n i t r o g e n . Phytoplankton c e l l s were l y o p h i l i z e d i n the c e n t r e - w e l l of a f r e e z e - d r i e r ( V i r t i s Company Inc., N.Y.). The l y o p h i l i z e d c e l l s were s t o r e d under vacuum i n a d e s i c c a t o r at -30°C. A s i m i l a r procedure i s d e s c r i b e d by A n t i a and Kripps (1978) f o r the p r e p a r a t i o n and sto r a g e of a l g a l powders. They found that when a l g a l samples were t r e a t e d i s t h i s manner, the enzymes remained a c t i v e with no l o s s i n a c t i v i t y f o r p e r i o d s up to one year. During e x p o n e n t i a l growth, d u p l i c a t e 25 ml subsamples of 75 c u l t u r e were c o l l e c t e d f o r a n a l y s i s of Se i n v a r i o u s b i o c h e m i c a l c o n s t i t u e n t s . Biochemical analysis B i o c h e m i c a l c o n s t i t u e n t s , determined d u r i n g e x p o n e n t i a l growth, were separated i n t o l i p i d s (CHCl-j f r a c t i o n ) , p r o t e i n s ( T C A - i n s o l u b l e f r a c t i o n ) , p o l y s a c c h a r i d e s and n u c l e i c a c i d s (TCA-soluble f r a c t i o n ) and low molecular weight compounds (KCl-MeOH-I^O f r a c t i o n ) a c c o r d i n g to the procedure of T e r r y et al . (1983). S i n c e t h i s procedure i s d i s c u s s e d l a t e r , the exp e r i m e n t a l p r o t o c o l i s o u t l i n e d here. D u p l i c a t e subsamples of c u l t u r e were c o l l e c t e d by f i l t r a t i o n and r i n s e d with 5 ml S e - f r e e f i l t e r - s t e r i l i z e d ESAW. The f i l t e r s were immediately p l a c e d i n t e s t tubes c o n t a i n i n g 3 ml c o l d CHCl 3"MeOH (2:1; v/v) and mixed on a v o r t e x generator u n t i l the f i l t e r was d i s r u p t e d . The co n t e n t s of the t e s t tube were r e f i l t e r e d , and the t e s t tube and m a t e r i a l on the f i l t e r were r i n s e d three times with 3 ml of CHCl 3-MeOH. The CHCl 3"MeOH f r a c t i o n s were combined and e x t r a c t e d with 3 ml 0.88% KC1 s o l u t i o n and then r e - e x t r a c t e d with 3 ml MeOH-H 20-(1:1; v / v ) . The KC1 and MeOH-H 20 f r a c t i o n s were combined i n a l i q u i d s c i n t i l l a t i o n v i a l and evaporated to dryness at room temperature i n a fume hood. The CHC1 3 f r a c t i o n was added to a s c i n t i l l a t i o n v i a l and was t r e a t e d s i m i l a r l y . The f i l t e r s c o n t a i n i n g the CHCl 3~MeOH r e s i d u a l matter were added to a t e s t tube c o n t a i n i n g 3 ml 5% TCA and p l a c e d i n a b o i l i n g water bath f o r 30 min. The contents were f i l t e r e d , and the t e s t tube and f i l t e r were r i n s e d twice with 2 ml of c o l d 5% TCA. The TCA s o l u b l e f r a c t i o n s were combined i n a s c i n t i l l a t i o n v i a l , and the remaining f i l t e r was p l a c e d i n a separate s c i n t i l l a t i o n v i a l . Samples were counted by l i q u i d s c i n t i l l a t i o n techniques, and the counts were c o r r e c t e d f o r quenching. Samples were counted u n t i l the standard d e v i a t i o n of each count was 2%. L y o p h i l i z e d c e l l s were suspended i n i c e c o l d b u f f e r c o n t a i n i n g 65 mM T r i s - H C l (pH 6.8) ( U l t r a Pure, Schwarz/Mann) and s o n i c a t e d on i c e f o r 1 min with a Branson S o n i f i e r w c e l l d i s r u p t e r (model 200) at 60% output and 47% duty c y c l e . I t was determined by m i c r o s c o p i c o b s e r v a t i o n that no c e l l s remained i n t a c t f o l l o w i n g t h i s treatment. The sample was c e n t r i f u g e d at 180,000 g (TI-50 r o t o r , 40,000 rpm) f o r 2 h at 4°C i n a Beckman® L8-M U l t r a c e n t r i f u g e to p e l l e t the membranes and c e l l w a l l s . The supernatant was decanted and kept on i c e . The membrane p e l l e t was resuspended i n an equal volume of T r i s - H C l b u f f e r . P r o t e i n was measured i n each f r a c t i o n by the Lowry method (Lowry et al . 1951) f o l l o w i n g the procedure of Markwell et al.(1978). P r o t e i n was c o - p r e c i p i t a t e d with 50 u l of 25 mg-ml 1 s o l u b l e yeast RNA (Polachek and Cabib 1981), and the p r e c i p i t a t e p e l l e t e d by c e n t r i f u g a t i o n at 25,000 rpm f o r . . 75 30 min. The a c t i v i t y of Se was measured i n the membrane and s o l u b l e p r o t e i n and i n the supernatant of each f r a c t i o n . Gel f i I t r at i on Supernatant from the crude c e l l e x t r a c t was passed through a Sephadex G-150 column (1.6 • 45 cm) e q u i l i b r a t e d with T r i s - K C l b u f f e r (65 mM T r i s - H C l , 0.1 M KC1 (Bio-Rad); pH 6.8) i n a c o l d room (4°C). V o i d volume was determined by Blue Dextran 2000 (Pharmacia), and standards purchased from Sigma were used to c a l i b r a t e the column. The standard p r o t e i n s and t h e i r molecular weights ( d a l t o n s ; D) were; cytochrome c, 12,400; c a r b o n i c anhydrase, 29,000; bovine serum albumin, 66,000; and a l c o h o l dehydrogenase, 150,000. F r a c t i o n s (1.5 ml) were c o l l e c t e d with a G i l s o n m i c r o - f r a c t i o n a t o r , and p r o t e i n was det e c t e d s p e c t r o p h o t o m e t r i c a l l y (280 nm) on a 75 Bausch and Lomb Sp e c t r o n i c 2000. A c t i v i t y of Se i n the crude c e l l e x t r a c t s was measured i n 50 u l samples of each f r a c t i o n . Molecular weight was estimated from c a l i b r a t i o n p l o t s of l o g molecular weight versus e l u t e d volume/void volume (Cooper, 1977). Electrophoresis Membrane and s o l u b l e p r o t e i n s were separated on SDS-po l y a c r y l a m i d e g e l s u s i n g the procedure of Laemmli (1970). A l l e l e c t r o p h o r e t i c s e p a r a t i o n s were performed i n a c o l d room (4°C) with p r e c h i l l e d b u f f e r and g e l s . The sample b u f f e r c o n s i s t e d of 65 mM T r i s - H C l (pH 6.9), 5 mM d i t h i o t h r e i t o l , 1% l a u r y l s u l f a t e (SDS) (w/v), and 10% g l y c e r o l . The running b u f f e r c o n s i s t e d of 0.19 M g l y c i n e , 0.025 M T r i s , and 0.1% SDS (w/v). P r o t e i n s were d i s s o c i a t e d by hea t i n g i n the presence of the b u f f e r at 70°C f o r 2-5 min. P r o t e i n s were run i n the s t a c k i n g g e l with a c u r r e n t of 15 mA and separated i n the r e s o l v i n g g e l at 25 mA ( S e a r l e DC) Power Supply). Gels were s t a i n e d with 0.2% Coomassie B r i l l a n t Blue R i n 30% (v/v) methanol and 7% (v/v) a c e t i c a c i d , and des t a i n e d overnight i n 20% methanol and 7% a c e t i c a c i d . Standard p r o t e i n s obtained from Sigma (SDS-7) were a c e t y l a t e d a c c o r d i n g to Lane (1978). The standard p r o t e i n s and t h e i r molecular weights (D) were; bovine serum albumin, 66,000; ovalalbumin, 45,000; glyceraldehyde-3-phosphate dehydrogenase, 36,000; carbonic anhydrase, 29,000; t r y p s i n o g e n , 24,000; t r y p s i n i n h i b i t o r , 20,100; and a - l a c t a l b u m i n , 14,200. The e l e c t r o p h o r e t i c f r o n t was determined by adding bromphenol blue to the samples and standards. Autoradiograms were run to detect the l o c a t i o n of 75 . ' S e - l a b e l l e d p o l y p e p t i d e s . M o l e c u l a r weights of the sample p o l y p e p t i d e s were e s t i m a t e d from a c a l i b r a t i o n p l o t of l o g p r o t e i n molecular weight versus Rf ( d i s t a n c e of p r o t e i n m i g r a t i o n / d i s t a n c e of e l e c t r o p h o r e t i c f r o n t ) as d e s c r i b e d by 246 Weber and Osborne (1969). Glutathione peroxidase activity The g l u t a t h i o n e reductase-coupled assay d e s c r i b e d by Drotar et al. (1985) was used to measure g l u t a t h i o n e peroxidase (GSH-Px) i n T. pseudonana. G l u t a t h i o n e p e r o x i d a s e a c t i v i t y was assayed in c e l l - f r e e e x t r a c t s i n T r i s - H C l b u f f e r . The r e a c t i o n was i n i t i a t e d by the a d d i t i o n of 0.09 mM H 2 0 2 or 2 mM t e r t - b u t y l oxyhydroxide (tBOOH), and o x i d a t i o n of NADPH 2 was measured s p e c t r o p h o t o m e t r i c a l l y (340 nm) over 5 min. The o x i d a t i o n r a t e s determined i n c o n t r o l s l a c k i n g reduced g l u t a t h i o n e (GSH), H 2 0 2 or tBOOH ( c o l l e c t i v e l y ROOH), g l u t a t h i o n e reductase (GSH-reductase) or c e l l e x t r a c t were s u b t r a c t e d from the t o t a l o x i d a t i o n r a t e to c a l c u l a t e GSH-Px a c t i v i t y (Smith and S h r i f t 1979). G l u t a t h i o n e peroxidase a c t i v i t y was assayed on non-denaturing p o l y a c r y l a m i d e g e l s ( l a c k i n g SDS) by a method s i m i l a r to that d e s c r i b e d by B e u t l e r and West (1974). S o l u b l e p r o t e i n s i n c e l l - f r e e e x t r a c t s were separated by e l e c t r o p h o r e s i s on a 10% p o l y a c r y l a m i d e r e s o l v i n g g e l f o r 8 h with a c u r r e n t of 20 mA. The g e l was s l i c e d l o n g i t u d i n a l l y , and f i l t e r paper s t r i p s soaked i n the enzyme assay s o l u t i o n were placed on top of the g e l s l i c e s . The GSH-Px assay s o l u t i o n c o n s i s t e d of 0.2 M KH 2P0 4/K 2HP0 4 b u f f e r , pH 7.0; 10 mM GSH; 10 u n i t s - m l " 1 GSH-reductase; 1 mM NADPH2 and 5 mM EDTA. H 2 ° 2 ( ° * 7 a n d t B 0 0 H < 2 mM) were used as s u b s t r a t e s . C o n t r o l s were run i n the absence of GSH and ROOH. A f t e r 30 min at 25°C, the f i l t e r paper s t r i p s were removed, and the g e l s l i c e s were examined on a s h o r t wavelength (250 nm) UV l i g h t t a b l e (Fotodyne) u s i n g t r a n s m i t t e d UV l i g h t . Enzyme a c t i v i t y was evident i n bands l a c k i n g NADPH2 f l u o r e s c e n c e . Photographs of the g e l s were taken w i t h P o l a r o i d type 57 f i l m u s i n g an orange f i l t e r . The g e l s t r i p s were s l i c e d i n t o 0.25 cm 75 s e c t i o n s , and each s e c t i o n was counted to determine Se a c t i v i t y . R e s u l t s Uptake of "''~*Se Growth of Thalassiosira pseudonana, and i n c o r p o r a t i o n of 75 S e - s e l e m t e i n t o the c e l l s i s repor t e d i n Fi g u r e 40. The 75 . . rat e of Se uptake was i n f a i r agreement with the growth r a t e — 1 75 of the a l g a (1.27 and 1.56 d , r e s p e c t i v e l y ) , and Se uptake ceased once the c e l l s stopped growing. Assuming that 7 5 background l e v e l s of Se were n e g l i g i b l e compared to the Se a d d i t i o n (see Chapter 5 " D i s c u s s i o n " ) , approximately 12 nmol _ 1 p Se was taken up by the c e l l s . T h i s corresponds to 1.6-10 -1 5 -1 mol S e - c e l l or 9.8-10 atoms S e - c e l l 75 Biochemical distribution of Se 75 The m a j o r i t y of Se was present i n the p r o t e i n and the p o l y s a c c h a r i d e and n u c l e i c a c i d f r a c t i o n s (Table XXII). There was n e g l i g i b l e i n c o r p o r a t i o n of Se i n t o l i p i d s (CHCl^ f r a c t i o n ) , and only 11% of the l a b e l was a s s o c i a t e d with the 7 5 low molecular weight compounds (KCl-MeOH-H 20 f r a c t i o n ) . Se 40. Growth of T. pseudonana as measured by in vivo c h l o r o p h y l l a f l u o r e s c e n c e (•) over the d u r a t i o n of the 75 experiment. The accumulation of Se by c e l l s (O) was measured i n 5 ml p o r t i o n s of c u l t u r e c o l l e c t e d by f i l t r a t i o n and r i n s e d with 5 ml of f i l t e r - s t e r i l i z e d ESAW. The arrow i n d i c a t e s when the c u l t u r e was harvested. FLUORESCENCE m 7 5Se ACCUMULATION (dpm) ° to 250 Table XXII P a r t i t i o n i n g of Se between p r o t e i n , p o l y s a c c h a r i d e s and n u c l e i c a c i d s , l i p i d s and low molecular weight compounds in e x p o n e n t i a l l y growing T. pseudonana, as determined by s o l v e n t e x t r a c t i o n t e chniques. Values are r e p o r t e d as dpm: 25 ml of c u l t u r e was e x t r a c t e d . Each measurement i s the mean of 3 q u a d r u p l i c a t e d e t e r m i n a t i o n s ± 1 SD, and u n i t s are dpm-10 . P r o t e i n Polysacchar ides & N u c l e i c A c i d s L i p i d s Low Molecular Wt Compounds (TCA-insoluble) (TCA-soluble) (KCl-MeOH-H 20) 16.3 + 0.5 11.5 ± 0.7 0.59 ± 0.07 3.6 ± 0.09 was e q u a l l y d i s t r i b u t e d between the s o l u b l e and membrane-bound p r o t e i n s (Table X X I I I ) . Over 90% of the t o t a l c e l l u l a r 7 5 S e was e x t r a c t e d from the c e l l s i n the TCA p r e c i p i t a t e . T h i s p r e c i p i t a t e d m a t e r i a l i n c l u d e s p r o t e i n and n u c l e i c a c i d s . C e l l u l a r p r o t e i n comprised only 17% of the dry weight of the phytoplankton. T h i s value i s low because diatoms c o n t a i n a s i l i c e o u s c e l l w a l l which accounts f o r up to 35% of the dry weight (Parsons et al . 1961). There may a l s o have been an undetermined c o n t r i b u t i o n to the phytoplankton dry weight by sea s a l t s , which were not removed d u r i n g r i n s i n g . Gel filtration and'electrophoresis Gel f i l t r a t i o n of the c e l l - f r e e e x t r a c t through Sephadex 75 G-150 r e s o l v e d three d i s t i n c t peaks of Se a c t i v i t y ( F i g . 41A). The h i g h e s t molecular weight peak e l u t e d i n the v o i d volume, and was a s s o c i a t e d with p r o t e i n ( F i g . 42B); the two 7 5 peaks were not e x a c t l y c o i n c i d e n t . A broad peak of Se a c t i v i t y e l u t e d i n f r a c t i o n s 28 to 40. The average molecular weight of t h i s m a t e r i a l was 87 kD. Although l i t t l e p r o t e i n was d e t e c t e d i n these f r a c t i o n s , i t was not s u r p r i s i n g s i n c e the s e n s i t i v i t y of the method used to de t e c t p r o t e i n i s low and very l i t t l e p r o t e i n was a p p l i e d to the column (1.4 mg). 7 5 The t h i r d peak of Se a c t i v i t y had an apparent molecular weight of 4.6 kD. Other c e l l - f r e e e x t r a c t s gave i d e n t i c a l chromatograms. I t was noted, however, that i f the c e l l e x t r a c t was f r o z e n at -30°C and rerun through the column, 75 there was l i t t l e Se a s s o c i a t e d with higher molecular weight m a t e r i a l e l u t i n g from the column; as many as three separate Table XXIII 75 D i s t r i b u t i o n of Se between s o l u b l e and membrane p r o t e i n s of T. pseudonana. Average c o n c e n t r a t i o n s ( c a l c u l a t e d from q u a d r u p l i c a t e measurements) of p r o t e i n are repor t e d + 1 SD. P r o t e i n Selenium 1 (ng-mg dry wt c e l l s ) (ng-mg p r o t e i n ) Membrane 7 7 + 2 . 8 13 P r o t e i n S o l u b l e 95 + 3.6 9.9 P r o t e i n c a l c u l a t e d by assuming that Se was the on l y source of Se. 7 5 F i g . 41. (A) E l u t i o n p a t t e r n of Se from Sephadex G-150 column. Arrows i n d i c a t e where the molecular weight standards e l u t e d from the column: AD, a l c o h o l dehydrogenase; BSA, bovine serum albumin; CA, c a r b o n i c anhydrase; CC, cytochrome c. I n s e r t i s a p l o t of the l o g m o lecular weight of standard p r o t e i n s versus the volume of b u f f e r r e q u i r e d to e l u t e the p r o t e i n from the column expressed r e l a t i v e to the v o i d volume measured using Dextran Blue 2000. Peak height was used to determine e l u t e d volume (Ve) and v o i d volume (Vo). The equation of the l i n e i s y = -1.251x + 7.269; r 2 = 0.996. (B) E l u t i o n p a t t e r n of p r o t e i n from Sephadex G-150, as d e t e c t e d s p e c t r o p h o t o m e t r i c a l l y at 280 nm. 0 20 28 36 44 52 60 68 74 FRACTION NUMBER peaks of Se a c t i v i t y with molecular weights l e s s than 5 kD, c o u l d be r e s o l v e d . The membrane and s o l u b l e p r o t e i n s were separated by SDS-PAGE ( F i g . 42A). A f t e r s t a i n i n g , the g e l was exposed to X-ray f i l m f o r 100 h ( F i g . 42B). No S e - c o n t a i n i n g p o l y p e p t i d e s were d e t e c t e d i n the membrane f r a c t i o n , but two s o l u b l e 75 p o l y p e p t i d e s of 29 and 21 kD were h e a v i l y l a b e l l e d with Se. 75 These S e - l a b e l l e d p o l y p e p t i d e s were not the most abundant of the s o l u b l e p o l y p e p t i d e s . Gl ut at hi one peroxidase activity G l u t a t h i o n e peroxidase a c t i v i t y was d e t e c t e d i n the c e l l -f r e e e x t r a c t using H2C>2 and tBOOH as s u b s t r a t e s (Table XXIV) . Heat-denatured samples had no GSH-Px a c t i v i t y . The r a t e s of NADPH 2 o x i d a t i o n were s i m i l a r f o r both s u b s t r a t e s . G l u t a t h i o n e - S - t r a n s f e r a s e a c t i v i t y was a l s o assayed u s i n g 1-c h l o r o - 2 , 4 - d i n i t r o b e n z e n e as s u b s t r a t e (Habig et al. 1974), but i t c o u l d not be d e t e c t e d . G l u t a t h i o n e peroxidase a c t i v i t y was assayed on non-d e n a t u r i n g polyacrylamide g e l s . Two bands of enzyme a c t i v i t y were present using tBOOH as s u b s t r a t e ( F i g . 43). C o n t r o l s l a c k i n g GSH and tBOOH or both were run c o n c u r r e n t l y . In the absence of tBOOH, some d e f l u o r e s c e n c e o c c u r r e d at both bands, but i t was of much l e s s magnitude than the experimental treatments. To f a c i l i t a t e b e t t e r s e p a r a t i o n between the two bands of enzyme a c t i v i t y , the c e l l e x t r a c t was e l e c t r o p h o r e s e d under i d e n t i c a l c o n d i t i o n s f o r 9 h. The higher molecular weight band showed l e s s a c t i v i t y compared to the f i r s t g e l and 256 F i g . 42. (A) Coomassie blue s t a i n e d SDS-PAGE of s o l u b l e and membrane p r o t e i n s from T. ps eudonana. Standard p r o t e i n s ( f o r d e s c r i p t i o n see " M a t e r i a l s and Methods") were run i n lane S; the number beside each standard i s the molecular weight i n kD. D i f f e r e n t amounts of s o l u b l e and membrane p r o t e i n e x t r a c t were a p p l i e d t o the g e l ; lane 1 r e c e i v e d 60 or 50 mg of s o l u b l e or membrane p r o t e i n , r e s p e c t i v e l y . Lanes 2 and 3 were loaded with twice the amount of p r o t e i n i n lane 1, and lane 4 c o n t a i n e d four times the p r o t e i n as lane 1. (B) Autoradiograms of g e l exposed to X-ray f i l m f o r 100 h. Two s o l u b l e p r o t e i n s of 29 and 21 75 kD were l a b e l l e d w i t h Se. 257 258 Table XXIV G l u t a t h i o n e peroxidase a c t i v i t y measured in c e l l - f r e e e x t r a c t s of T. ps eudonana. Enzyme a c t i v i t y i s expressed as the r a t e of NADPH2 o x i d i z e d and was normalized to p r o t e i n . The r e a c t i o n temperature was 25°C. Bracketed values are the number of d i f f e r e n t p r o t e i n p r e p a r a t i o n s assayed, and GSH-Px a c t i v i t y i n each p r e p a r a t i o n was measured i n d u p l i c a t e ; average v a l u e s are r e p o r t e d ± 1 SD. Sub s t r a t e GSH-Px A c t i v i t y (nmol-min 1•mg p r o t e i n 1) H 2 ° 2 tBOOH 29.0 ± 1.0 (2) 36.0 Ci) 259 F i g . 43. G l u t a t h i o n e peroxidase a c t i v i t y d e t e c t e d on a polyacrylamide g e l f o l l o w i n g 6 h of e l e c t r o p h o r e s i s . Two bands showing enzyme a c t i v i t y (NADPP^ o x i d a t i o n r e s u l t i n g in d e f l u o r e s c e n c e ) were e v i d e n t on the g e l . However, the extent of the r e a c t i o n i n both bands r e s u l t e d i n the separate bands becoming i n d i s t i n c t . Sample b u f f e r , g l y c e r o l and bromphenol blue (bpb) were run as a c o n t r o l in lane 1, and c e l l - f r e e e x t r a c t i n b u f f e r with g l y c e r o l and bpb were run i n lane 2. 2 6 0 r e a c t e d l e s s s t r o n g l y than the l i g h t e r band. Both H 2 0 2 and tBOOH were used as s u b s t r a t e s , and the a p p r o p r i a t e c o n t r o l s were run f o r each. The higher molecular weight band was a c t i v e with tBOOH, but no a c t i v i t y was d e t e c t e d w i t h H 2 0 2 . The lower molecular weight band was a c t i v e with both s u b s t r a t e s ( F i g . 44). A p o r t i o n of the same g e l was assayed f o r GSH-Px a c t i v i t y using H 2 0 2 as s u b s t r a t e , and the g e l was 75 s l i c e d i n to 25 mm s e c t i o n s and counted f o r Se a c t i v i t y . 75 G l u t a t h i o n e peroxidase a c t i v i t y and Se co-migrated on the 7 5 g e l ( F i g . 45). There were a l s o other peaks of Se a c t i v i t y of lower molecular weight, and one small peak with a g r e a t e r weight. Discussion Selenium upt ake T h a l a s s i o s i r a pseudonana accumulated s e l e n i t e d u r i n g e x p o n e n t i a l growth, and uptake stopped once c e l l d i v i s i o n ceased. Sandholm et a l . (1973) found that Scenedesmus dimorphus d i d not i n c o r p o r a t e i n o r g a n i c , s e l e n i t e to an a p p r e c i a b l e extent d u r i n g 1 h i n c u b a t i o n s . While t h i s should not seem s u r p r i s i n g , because of the short time i n t e r v a l over which the uptake r a t e s were measured, i t was i n s t a r k c o n t r a s t to the uptake of selenomethionine. T h e i r r e s u l t s showed that 75 . . 60% of the Se-selenomethionine added to the medium was taken up i n 1 h. However, c a u t i o n must be e x e r c i s e d i n the i n t e r p r e t a t i o n of t h e i r r e s u l t s , s i n c e the phytoplankton c u l t u r e s were not a x e n i c . I t i s l i k e l y t h a t the r a p i d uptake 44. G l u t a t h i o n e peroxidase a c t i v i t y d e t e c t e d on p o l y a c r y l a m i d e g e l s using tBOOH (lane 1) and H 2 0 2 (lane 2) as a s u b s t r a t e , a f t e r e l e c t r o p h o r e s i s f o r 9 h. The upper band (a) was not detected on the g e l t r e a t e d with assay s o l u t i o n c o n t a i n i n g H 20 2. Band (b) r e a c t e d with both p e r o x i d e s . 263 45. (A) G l u t a t h i o n e peroxidase a c t i v i t y d e t e c t e d on a p o l y a c r y l a m i d e g e l using H2C>2 as s u b s t r a t e . (B) Amount 75 -1 of Se i n the g e l (dpm-slice ). of t h i s o r g a n i c compound can a l s o be a t t r i b u t e d to b a c t e r i a l u t i l i z a t i o n . The c e l l quota f o r Se re p o r t e d here i n v a r i a b l y i n c l u d e s some e x t r a c e l l u l a r l y bound Se that was not removed from the c e l l s d u r i n g r i n s i n g . Refinement of methods used to determine a c c u r a t e l y Fe c e l l quotas have r e s u l t e d i n a r e d u c t i o n of r e c e n t l y measured Fe quotas by comparison to the h i s t o r i c a l v a l u e s ( H a r r i s o n and Morel 1986). T h i s i s a r e s u l t of the removal of adsorbed metal oxides on the c e l l s u r f a c e by the use of a stro n g reductant, such as ascorbate (Anderson and Morel 1982). Whether i n o r g a n i c Se complexes, such as FeOOH/SeO^ , adsorb to the phytoplankton c e l l s u r f a c e i s unknown. A l s o , the Se c e l l quota r e p o r t e d here may i n c l u d e a "l u x u r y " component (sensu Droop 1975). The minimum Se c e l l quota can be estimated from data pr e s e n t e d i n Chapter 5. When c e l l s were t r a n s f e r r e d i n t o Se-d e p l e t e ESAW they undergo ca. 9 c e l l d i v i s i o n s ; at t h i s time -21 -1 the minimum Se c e l l quota would be 3.5-10 mol S e - c e l l I t i s i n t e r e s t i n g to note that t h i s quota i s very s i m i l a r to the s u b s i s t e n c e c e l l quota f o r vit a m i n B 1 2 r e p o r t e d by Droop (1975) i n a s i m i l a r - s i z e d phytoplankton, Monochrysis lutherii 3 - 1 ( s i c ) (28-52 um - c e l l ) (Burmaster 1979). These c a l c u l a t i o n s f u r t h e r emphasize why only the s l i g h t e s t contamination of Se in the medium w i l l allow T. pseudonana to grow. C e l l quotas f o r other e s s e n t i a l t r a c e elements, such as Fe and Mn, have been measured i n diatoms. The Mn c e l l quota of T. pseudonana and T. weissfl ogi i , growing i n n u t r i e n t - 1 4 - 1 4 - 1 s u f f i c i e n t medium i s 1.4*10 and 1.1 *10 mol M n - c e l l , 267 r e s p e c t i v e l y (Sunda and Huntsman 1983, H a r r i s o n and Morel 1986). While the minimum Fe c e l l quota r e q u i r e d to s u s t a i n -1 3 -1 maximum c e l l growth of T. weissflogii i s 1-10 mol F e - c e l l ( H a r r i s o n and Morel 1986). The Se c e l l quota of T. pseudonana, determined d u r i n g e x p o n e n t i a l growth, i s 4-5 orders of magnitude l e s s than these t r a c e metals. 7 5 Intracellular di s t r i but i on of Se Solvent e x t r a c t i o n techniques have been used to q u a n t i f y the b i o c h e m i c a l c o n s t i t u e n t s of p l a n t s , animals and microorganisms, and t h e r e are as many v a r i a t i o n s of these methods as there are i n v e s t i g a t o r s ( f o r the phytoplankton l i t e r a t u r e see: M o r r i s et a l . 1974, Kochert 1978, Wrench 1978, L i et al . 1980, Smith and M o r r i s 1980, , Hitchcock 1983, T e r r y et al. 1983, B o t t i n o et al. 1984, Dortch et al. 1984). From the work conducted with p r o k a r y o t e s , i t i s apparent that the v a r i o u s s o l v e n t f r a c t i o n s c o n t a i n a wide range of compounds, and that a s i n g l e c l a s s of b i o c h e m i c a l s ( f o r example p o l y s a c c h a r i d e s ) may not be e n t i r e l y e x t r a c t e d by one procedure ( S u t h e r l a n d and W i l k e r s o n 1971). Only two s t u d i e s have examined the e f f i c a c y of s o l v e n t e x t r a c t i o n procedures fo r b i o c h e m i c a l s e p a r a t i o n i n marine phytoplankton (Morris et al . 1974, H i t c h c o c k 1983). In a l l of these methods, the i s o l a t i o n of l i p i d s from b i o l o g i c a l samples i n v o l v e s CHCl^-MeOH e x t r a c t i o n : t h i s method was f i r s t d e s c r i b e d by F o l c h et al. (1957). R e - e x t r a c t i o n of the CHCl^-MeOH f r a c t i o n w i t h KC1 and MeOH removes p o l a r compounds, which i n c l u d e the f r e e amino a c i d s and other low molecular weight compounds such as carbohydrates. L i p i d s are r e t a i n e d i n the CHCl^ f r a c t i o n . Although i t i s g e n e r a l l y excepted that CHCl^-MeOH e x t r a c t i o n s o l u b i l i z e s a l l f r e e amino a c i d s , Wrench (1978) found t h i s was not tr u e d u r i n g e x t r a c t i o n of two green algae. Since h i s i n i t i a l s e p a r a t i o n s t e p i s i d e n t i c a l to what was used i n t h i s study, i t i s p o s s i b l e t h a t not a l l of the f r e e amino a c i d s i n T. ps eudonana were e x t r a c t e d i n t o the KCl-MeOH-H 20 f r a c t i o n ; some may a l s o be co n t a i n e d i n the TCA s o l u b l e f r a c t i o n . Whereas l i p i d e x t r a c t s of Dunaliella pr imol ect a and Porphyridium cruentum c o n t a i n e d h i g h l e v e l s of Se (Gennity et al. 1984), and were c o n f i n e d p r i m a r i l y to the c a r o t e n o i d s and xa n t h o p h y l l s , the r e s u l t s of t h i s i n v e s t i g a t i o n found very 75 l i t t l e Se a s s o c i a t e d with l i p i d i n T. pseudonana. S i m i l a r o b s e r v a t i o n s were made by Wrench (1978). He found t h a t the l i p i d s of Tetraselmis tetrethele and Dunaliella mi nut a c o n t a i n e d n e g l i g i b l e q u a n t i t i e s of Se when these a l g a e were 75 grown i n the presence of nanomolar q u a n t i t i e s of Se. The r e s u l t s of Gennity et al . (1984) appear more of an a r t i f a c t of the h i g h Se c o n c e n t r a t i o n added to t h e i r c u l t u r e medium (10 ppm Se as Na^eO^). Since SeO^ was shown to b i n d to l i p i d s d u r i n g e x t r a c t i o n , t h e i r r e s u l t s p r o v i d e d no proof f o r the e x i s t e n c e of S e - l i p i d a s s o c i a t i o n s in vivo. In f a c t , a l l e x t r a c t e d l i p i d s c o n t a i n i n g Se were unsaturated, and g e n t l e c a t a l y t i c hydrogenation with PtC>2 decreased the Se content of the l i p i d s by 95%. T h i s r e s u l t suggested that Se was a s s o c i a t e d with carbon double bonds ( s a t u r a t e d l i p i d s d i d not c o n t a i n Se), and there was no evidence f o r metabolic i n c o r p o r a t i o n of Se. M o r r i s et al . (1974) v e r i f i e d that b o i l i n g TCA hy d r o l y z e d p o l y s a c c h a r i d e s i n the CHClg-MeOH r e s i d u a l m a t e r i a l , and t h i s procedure gave s i m i l a r r e s u l t s to the more c o n v e n t i o n a l method of b o i l i n g s u l f u r i c a c i d h y d r o l y s i s f o r 3 h (Ca s s e l t o n and S y r e t t 1962). M o r r i s et al . (1974) made no mention of the s t a b i l i t y of p r o t e i n under these c o n d i t i o n s . The treatment with hot TCA h y d r o l y z e s n u c l e i c a c i d s as w e l l as p o l y s a c c h a r i d e s . H i t c h c o c k (1983) c r i t i c i z e d t h i s method on the b a s i s of r e s u l t s he o b t a i n e d from experiments u s i n g bovine serum albumin (BSA) sta n d a r d s . He found that only 20% of the BSA was recovered i n the T C A - i n s o l u b l e f r a c t i o n , but two other methods y i e l d e d 100% recovery of the BSA standards. In s p i t e of t h i s r e s u l t , a l l t h r e e methods e x t r a c t e d the same amount of p r o t e i n from two d i n o f l a g e l l a t e s p e c i e s , implying that there may have been something unique about the BSA standard which was not r e f l e c t e d i n the b i o l o g i c a l samples. I t should be po i n t e d out that the o b j e c t i v e of the biochemical f r a c t i o n a t i o n i n t h i s study was to determine the amount of 75 Se a s s o c i a t e d with the major groups of biochemicals i n T. pseudonana. N e v e r t h e l e s s , although these methods may be s u i t a b l e f o r i s o l a t i n g the or g a n i c molecules, they may d i s r u p t any loose a s s o c i a t i o n s of these compounds with Se. Because of i t s r e a c t i v i t y , some e x t r a c t i o n procedures, notably the hot TCA e x t r a c t i o n , may e l i m i n a t e Se from molecules i n which i t i s normally a c o n s t i t u e n t . 270 B o t t i n o et al . (1984) found most of the Se a s s o c i a t e d with Dunaliella pr i molect a and Chlorella sp. (73 and 98%, r e s p e c t i v e l y ) was e x t r a c t e d i n the f r e e amino a c i d and s o l u b l e carbohydrate f r a c t i o n ; whereas, 80% of the c e l l u l a r Se was bound to l i p i d s i n Porphyridium cruentum. The major c r i t i c i s m of t h i s work was that the l e v e l s of s e l e n i t e added to the _ 2 - 1 medium were u n r e a l i s t i c a l l y high (10 g-1 ). I t i s not c l e a r whether the r e s u l t s obtained by B o t t i n o et al . (1984) are r e p r e s e n t a t i v e of the Se p a r t i t i o n i n g among the c e l l u l a r c o n s t i t u e n t s of these organisms under more n a t u r a l c o n d i t i o n s . Rather, these S e - c o n t a i n i n g compounds maybe the end prod u c t s of metabolic pathways designed to d e t o x i f y high l e v e l s of Se, or the r e s u l t of i n d i s c r i m i n a t e i n c o r p o r a t i o n of Se i n t o macromolecules, by processes which may or may not be en z y m a t i c a l l y mediated. Protein-bound Se r e p r e s e n t s 51% of the t o t a l p a r t i c u l a t e Se i n T. pseudonana, and 36% of the Se was a s s o c i a t e d with p o l y s a c c h a r i d e s and n u c l e i c a c i d s . During measurements of the so l u b l e and membrane p r o t e i n c o n c e n t r a t i o n s , soluble"RNA was added to the samples to c o - p r e c i p i t a t e p r o t e i n i n the presence of TCA. The p r e c i p i t a t e , c o n t a i n i n g p r o t e i n and n u c l e i c a c i d s , and the supernatant from both f r a c t i o n s were a n a l y z e d 75 . . 75 . for Se a c t i v i t y . Over 90% of the Se was present i n the . . . 75 . . p r e c i p i t a t e . S ince only 51% of the t o t a l Se a c t i v i t y was a s s o c i a t e d with p r o t e i n , as measured by s o l v e n t e x t r a c t i o n 7 5 methods, the a d d i t i o n a l Se i n the p r o t e i n / n u c l e i c a c i d p r e c i p i t a t e can only be due to Se bound to n u c l e i c a c i d s . 75 T h i s argues t h a t , i n the TCA s o l u b l e f r a c t i o n , a l l of the Se was a s s o c i a t e d with n u c l e i c acids.and none with p o l y s a c c h a r i d e . S e l e n o n u c l e o t i d e s have r e c e n t l y been i d e n t i f i e d i n b a c t e r i a (Stadtman 1980b). N e v e r t h e l e s s , i f 7 5 b o i l i n g TCA i n a d v e r t a n t l y removed some l o o s e l y bound Se from 75 the p r o t e i n , the Se found i n the p o l y s a c c h a r i d e and n u c l e i c a c i d f r a c t i o n s may be an a r t i f a c t . Specific s el e ni um- cont ai ni ng molecules S e p a r a t i o n of s o l u b l e macromolecules by g e l f i l t r a t i o n 75 showed that some Se was a s s o c i a t e d with high molecular weight m a t e r i a l which e l u t e d i n the v o i d volume. The e x c l u s i o n . l i m i t of Sephadex G-150 i s 300 kD, and t h i s weight i s approximately the lower l i m i t of m a t e r i a l e l u t e d i n the v o i d volume. The two s o l u b l e p o l y p e p t i d e s i d e n t i f i e d i n the autoradiogram of the SDS-polyacrylamide g e l had Se s t a b l y i n c o r p o r a t e d i n t o t h e i r molecular s t r u c t u r e . The molecular weight of these p r o t e i n s , 21 and 29 kD, when compared to the 75 weight of the S e - l a b e l l e d m a t e r i a l e l u t i n g from the column, suggested that they are p o l y p e p t i d e s u b u n i t s . I t was n o t i c e d ' 75 . . that the Se i n f r a c t i o n s 28-40 d i d not e l u t e from the column i n a symmetrical peak. There was some i n d i c a t i o n that two 75 macromolecules c o n t a i n i n g Se with s i m i l a r molecular weights were e l u t e d from the column. In h i n d s i g h t , the molecular weights of the p o l y p e p t i d e subunits provides evidence that t h i s i s t r u e . I n t e g r a l m u l t i p l e s of the subunit molecular weights equal the mo l e c u l a r weight of the m a t e r i a l e l u t e d from the column i n f r a c t i o n s 28-40. These r e s u l t s p r o v i d e c i r c u m s t a n t i a l evidence that T. ps eudonana c o n t a i n s two Se-c o n t a i n i n g p o l y p e p t i d e s which have a n a t i v e molecular weight of approximately 87 kD. The s i m p l e s t e x p l a n a t i o n i s that one p r o t e i n i s a tetramer with a subunit molecular weight of 21 kD and the other i s a t r i m e r with a subunit molecular weight of 29 kD. The nature of the chemical form of Se i n these p r o t e i n s 75 was not addressed i n t h i s study. The f a c t t h a t the Se remained bound to the p r o t e i n s d u r i n g e l e c t r o p h o r e s i s suggests i t i s l i n k e d by a c o v a l e n t bond. In s e l e n o p r o t e i n s of b a c t e r i a , and i n mammalian and a v i a n GSH-Px, the S e - c o n t a i n i n g moiety i s s e l e n o c y s t e i n e ; the Se analogue of the S - c o n t a i n i n g amino a c i d c y s t e i n e (Cone et a l . 1976, Forstrom et al . 1978, Jones et al. 1979). T h i o l a s e i s a unique s e l e n o p r o t e i n i n which selenomethionine i s the S e - c o n t a i n i n g moiety (Hartmanis and Stadtman 1982). Although s p e c i f i c S e - c o n t a i n i n g p r o t e i n s have not been i d e n t i f i e d i n higher p l a n t s , chemical and enzymatic h y d r o l y s i s of crude p r o t e i n e x t r a c t s and t h e i r subsequent a n a l y s i s have shown the presence of selenoamino a c i d s i n these organisms. S e l e n o c y s t e i n e and i t s o x i d i z e d form s e l e n o c y s t i n e were i d e n t i f i e d i n corn and i n wheat g r a i n (Smith 1949: as c i t e d i n Rosenfeld and Beath 1964) and s e l e n o c y s t e i n e , selenomethionine, and t h e i r o x i d a t i o n products were present i n p r o t e i n s of c l o v e r and rye grass (Peterson and B u t l e r 1962). Stadtman (1974) c r i t i c i z e d the r e s u l t s of selenoamino a c i d composition of p r o t e i n obtained from a c i d h y d r o l y s i s . She argued, on the b a s i s of work conducted by Huber and C r i d d l e (1967), t h a t s e l e n o c y s t e i n e i s a l t e r e d upon a c i d h y d r o l y s i s , and a f t e r 6 h a l l the amino a c i d i s destroyed. From t h i s 7 5 o b s e r v a t i o n , the r e s u l t s of the p a r t i t i o n i n g of Se i n p r o t e i n of T . pseudonana may be underestimated as a consequence of the b o i l i n g TCA e x t r a c t i o n . A d d i t i o n a l l y , s i n c e a c i d v o l a t i l e Se was reported i n p r o t e i n e x t r a c t s of Tetraselmis tetrethele (Wrench 1978), suggesting the presence of hydrogen s e l e n i d e , p r o t e i n bound Se i n T. ps eudonana may have been l o s t d u r i n g t h i s treatment. Recently, Ng and Anderson (1979) r e p o r t e d the in vitro s y n t h e s i s of s e l e n o c y s t e i n e by c y s t e i n e synthases from a v a r i e t y of p l a n t s . Brown and S h r i f t (1980b) used a l k y l a t i n g agents to s t a b i l i z e the s e l e n o c y s t e i n y l r e s i d u e s and were able to u n e q u i v o c a l l y demonstrate the presence of s e l e n o c y s t e i n e i n p r o t e i n s of pea and bean. The l a c k of s p e c i f i c S e - l a b e l l e d membrane p r o t e i n ( s ) , i n s p i t e of e q u i v a l e n t a c t i v i t y . o n a weight b a s i s f o r both s o l u b l e and membrane p r o t e i n s , i s p e r p l e x i n g . A p o s s i b l e e x p l a n a t i o n may be that there was a n o n - s p e c i f i c i n c o r p o r a t i o n 75 of Se i n t o a l l the membrane p r o t e i n s , and the g e l was not exposed to the x-ray f i l m f o r a s u f f i c i e n t l e n g t h of time to d e t e c t these p r o t e i n s . T h i s f i r s t s c e n a r i o seems u n l i k e l y . If a l l the p r o t e i n s were e q u a l l y l a b e l l e d i t might be a n t i c i p a t e d t h a t some of the more abundant p r o t e i n s would have been d e t e c t e d i n the autoradiogram. If the degree of i n c o r p o r a t i o n of Se i s a f u n c t i o n of the amount of S-c o n t a i n i n g amino a c i d s i n a p r o t e i n , perhaps t h i s i s too s i m p l i s t i c a p r o p o s a l . I t i s known that s e l e n o l groups are e a s i l y o x i d i z e d , and the o x i d i z e d products w i l l undergo spontaneous e l i m i n a t i o n r e a c t i o n s producing elemental Se (Stadtman 1980a). The nature of the Se f u n c t i o n a l i t y i n membrane p r o t e i n s may favour t h i s type of r e a c t i o n . A l t e r n a t i v e l y , Schwarz and Sweeney (1964) r e p o r t e d that 75 Na SeO-j added to a c e l l e x t r a c t w i l l r e a d i l y b i n d to p r o t e i n . I t i s p o s s i b l e that the Se a s s o c i a t e d with the membrane p r o t e i n s was l o o s e l y bound and was r e l e a s e d when the p r o t e i n s were s o l u b i l i z e d and denatured p r i o r to e l e c t r o p h o r e s i s . D a n i e l s o n and Medina (1986) o f f e r e d t h i s e x p l a n a t i o n to 75 account f o r the lack of S e - l a b e l l e d membrane p o l y p e p t i d e s i n 75 t h e i r autoradiograms i n s p i t e of the h i g h a c t i v i t y of Se i n the membrane p r o t e i n e x t r a c t . Glutathione peroxidase Recent attempts to demonstrate the presence of GSH-Px i n Dunaliella primolecta and Porphyridium cruentum have f a i l e d (Gennity et al . 1985). But t h i s enzyme has been d e t e c t e d in p u r i f i e d c e l l - f r e e e x t r a c t s of a number of microalgae, i n c l u d i n g Euglena gracilis Z(UTEX 753), E. gracilis var. bacillaris (UTEX 884) and (W3BUL)., and Astasia I onga (Overbaugh and F a l l 1982, 1985). Overbaugh and F a l l (1985) found that GSH-Px i n Euglena gracilis was a Se-independent enzyme which was a c t i v e with both ^2^2 a n < ^ or g a n i c h y d r o p e r o x i d e s . T h i s was the f i r s t r e p o r t of a Se-independent GSH-Px which reduces H 2 ° 2 * T ^ e m ° l e c u l a r weight of t h i s enzyme i s much grea t e r than p r e v i o u s l y d e s c r i b e d Se-dependent GSH-Px. 275 G l u t a t h i o n e peroxidase a c t i v i t y was evident i n T. ps eudonana, and a c t i v i t y was observed with H 2 0 2 and tBOOH. Enzymatic a c t i v i t y was completely e l i m i n a t e d a f t e r h e a t i n g the c e l l e x t r a c t at 100°C f o r 5 min. T h i s o b s e r v a t i o n has r e l e v a n c e , s i n c e Gennity et al . (1985) concluded that o x i d a t i o n of g l u t a t h i o n e was non-enzymatic. They found that b o i l i n g the a l g a l e x t r a c t f o r 30 min d i d not e l i m i n a t e GSH-Px a c t i v i t y . The s p e c i f i c a c t i v i t y of GSH-Px i n crude e x t r a c t s of four p l a n k t o n i c e u g l e n o i d s ranged from 16-100 nmol NADPH2 o x i d i z e d -min 1«mg p r o t e i n 1 , with H 2 0 2 as s u b s t r a t e (Overbaugh and F a l l , 1982). S i m i l a r l e v e l s of t h i s enzyme were measured i n t i s s u e - c u l t u r e d p l a n t c e l l s (Drotar et a l . 1985). In both r e p o r t s , the a c t i v i t y of GSH-Px was g e n e r a l l y l e s s when orga n i c hydroperoxides (cumene hydroperoxide and tBOOH) were used as s u b s t r a t e s , but the measured a c t i v i t y with organic hydroperoxides compared with H 2 0 2 was g r e a t e r i n corn and s i m i l a r i n Lemna and E. gracilis v a r . bacillaris (W3BUL). I t i s i m p o s s i b l e to a s c r i b e these r a t e measurements to a s i n g l e enzyme s i n c e g l u t a t h i o n e - S - t r a n s f e r a s e s show GSH-Px a c t i v i t y when o r g a n i c hydroperoxides are pr e s e n t . Rates determined with H 2 0 2 r e p r e s e n t the a c t i v i t y of the true GSH-Px enzyme. The a c t i v i t y of GSH-Px measured i n T. ps eudonana was s l i g h t l y g r e a t e r with tBOOH (36.0 nmol«min 1•mg p r o t e i n 1) than H 2 0 2 (29.0 nmol-min 1-mg p r o t e i n 1 . Since g l u t a t h i o n e - S - t r a n s f e r a s e c o u l d not be d e t e c t e d i n T. pseudonana, both r a t e s may r e f l e c t GSH-Px a c t i v i t y . The l e v e l s of GSH-Px i n T. ps eudonana are very s i m i l a r to p u b l i s h e d v a l u e s f o r other p l a n t s and p r o t i s t s . Glutathione peroxidase activity on polyacrylamide gels Two enzymes e x h i b i t i n g GSH-Px a c t i v i t y with tBOOH were separated and assayed on non-denaturing polyacrylamide g e l s . The higher molecular weight p r o t e i n d i d not use H 2 0 2 as a s u b s t r a t e and d i d not show t r u e c h a r a c t e r i s t i c s of GSH-Px (EC 1.11.1.9). T h i s p r o t e i n appeared more l a b i l e than the lower molecular weight GSH-Px, and i t s a c t i v i t y was reduced f o l l o w i n g e l e c t r o p h o r e s i s f o r 9 h. The lower molecular weight enzyme demonstrated c h a r a c t e r i s t i c s of a true GSH-Px (EC 1.11.1.9); i t was a c t i v e with H 2 0 2 and tBOOH and contained Se. Se I enium nutrition The chemical s i m i l a r i t i e s between Se and S are w e l l known, and many enzymes which normally c a t a l y z e r e a c t i o n s of s u l f u r compounds f u n c t i o n e q u a l l y w e l l with the corresponding Se analogs (Stadtman 1979). The t o x i c e f f e c t s of Se are b e l i e v e d to be manifested i n organisms as a consequence of the i n d i s c r i m i n a n t i n c o r p o r a t i o n of Se i n t o e s s e n t i a l p r o t e i n s and macromolecules i n p l a c e of s u l f u r ( S ) . F o r t u n a t e l y , t h i s only occurs when organism are exposed to very high c o n c e n t r a t i o n s of Se r e l a t i v e to S. On the other hand, very s p e c i f i c pathways f o r the s y n t h e s i s of selenoenzymes f u n c t i o n i n the presence of ord e r s of magnitude g r e a t e r S c o n c e n t r a t i o n s . In the ocean, p l a n k t o n i c organisms are bathed i n a medium c o n t a i n i n g mM c o n c e n t r a t i o n s of S and pM c o n c e n t r a t i o n s of Se. Not o n l y must h i g h l y s p e c i f i c processes i n c o r p o r a t e Se i n t o n e c e s s a r y macromolecules, e q u a l l y s p e c i f i c membrane p o r t e r s must f u n c t i o n to overcome p o t e n t i a l c o m p e t i t i v e i n t e r a c t i o n s 2 - 2 - -w i t h s i m i l a r anions such as SO^ , SO^ and NC^ . The i o n i c c omposition of the c u l t u r e medium used in t h i s study i s r e p r e s e n t a t i v e of n a t u r a l c o n c e n t r a t i o n s of Se and S. I argue that any s e l e n o m e t a b o l i t e s produced by T. pseudonana, under the growth c o n d i t i o n s of these experiments, are normal c e l l u l a r c o n s t i t u e n t s , and are not a consequence of non-s p e c i f i c Se i n c o r p o r a t i o n . Summary These r e s u l t s are the f i r s t to p r o v i d e evidence of s p e c i f i c s e l e n i u m - c o n t a i n i n g p o l y p e p t i d e s i n a p h o t o s y n t h e t i c organism. G l u t a t h i o n e peroxidase a c t i v i t y was measured i n c e l l - f r e e e x t r a c t s by a g l u t a t h i o n e - r e d u c t a s e coupled assay and on non-denaturing p o l y a c r y l a m i d e g e l s . Two enzymes showing GSH-Px a c t i v i t y were present on p o l y a c r y l a m i d e g e l s . One enzyme was a c t i v e with ^2^2 a n d'tBOOH, c o n s i s t e n t with known Se-dependent g l u t a t h i o n e p e r o x i d a s e s . The other enzyme was o n l y a c t i v e with tBOOH. Co-migration of the GSH-Px that 7 5 was a c t i v e with ^2^2 a n d ^ e s u P P o r t s the enzymatic evidence that Thai assi osi ra pseudonana c o n t a i n s a Se-dependent 7 5 g l u t a t h i o n e peroxidase. The subunit molecular weight of Se-l a b e l l e d p o l y p e p t i d e s agrees w e l l with the weight of p r e v i o u s l y c h a r a c t e r i z e d GSH-Px from other sources. In 7 5 . . a d d i t i o n , the weight of S e - c o n t a i n i n g macromolecules, as measured by g e l f i l t r a t i o n , i s c o n s i s t e n t with the pro p o s a l that t h i s g l u t a t h i o n e p e r o x i d a s e i s a tetramer. E a r l i e r o b s e r v a t i o n s of u l t r a s t r u c t u r a l and morphological changes a s s o c i a t e d with S e - d e p l e t e d c e l l s support the c o n c l u s i o n that the o b l i g a t e requirement f o r Se i n Thalassiosira ps eudonana i s due, i n p a r t , to the presence of the selenoenzyme g l u t a t h i o n e p e r o x i d a s e . GENERAL CONCLUSIONS This thesis examined two aspects of marine phytoplankton physiology and n u t r i t i o n : urea and selenium. The s p e c i f i c f indings of th i s research are summarized below. 1. A modified d i a c e t y l monoxime method for urea analys i s in seawater gave superior resu l t s by comparison to the urease method. The urease method was plagued by a number of problems, which were a resu l t of the i n h i b i t i o n of the urease enzyme used in the assay. The d i a c e t y l monoxime method for urea analys i s i s recommended for measuring urea concentrations in seawater. 2 . Results of experiments conducted in the S t r a i t of Georgia , in n i t r a t e - r i c h f ronta l water and ni trogen-depleted s t r a t i f i e d water, were used to examine nitrogen c y c l i n g between the d isso lved and p a r t i c u l a t e components in these two d i f ferent coasta l regions. This was the f i r s t study of nitrogen c y c l i n g in th i s area . I argued that in f ronta l water plankton were los ing nitrogen in the form of d isso lved organic n i trogen, and in s t r a t i f i e d water phytoplankton ass imi la ted DON concurrent ly with inorganic nitrogen and urea. 3 . In both coasta l and oceanic regions, maximum uptake rates of urea and ammonium by phytoplankton were s i m i l a r . Ni trate uptake rates by n i t r a t e - s u f f i c i e n t phytoplankton communities in the S t r a i t of Georgia were faster than maximum uptake rates of ammonium and urea. 4 . In situ urea regeneration rates were determined by in tac t 280 p l a n k t o n communities. In c o a s t a l seawater, i n both f r o n t a l and s t r a t i f i e d communities, urea r e g e n e r a t i o n r a t e s were comparable to ammonium rege n e r a t i o n r a t e s . 5. Uptake and r e g e n e r a t i o n r a t e s of regenerated forms of n i t r o g e n are b e l i e v e d to be t i g h t l y coupled, but data only e x i s t f o r ammonium. T h i s t h e s i s has provided s u p p o r t i n g evidence f o r c o u p l i n g between urea uptake and re g e n e r a t i o n based on the f o l l o w i n g o b s e r v a t i o n s : D i s s o l v e d urea c o n c e n t r a t i o n s were low i n the Sargasso Sea; the turnover time of the ambient pool of urea was 12 h, and phytoplankton were ab l e to u t i l i z e these low c o n c e n t r a t i o n s of urea at r a t e s , which were comparable to the maximum p o s s i b l e r a t e s of u t i l i z a t i o n . These data represent the f a s t e s t turnover times of urea i n any o l i g o t r o p h i c ocean r e g i o n h i t h e r t o examined. 14 15 6. D i s c r e p a n c i e s between C-urea and N-urea uptake were evident i n data c o l l e c t e d from the Sargasso Sea. Uptake r a t e s determined by both i s o t o p e s were not e q u i v a l e n t . 1 4 C-urea uptake r a t e s were 1.4 times g r e a t e r than r a t e s 15 measured by N-urea. I t was proposed that urea-N i s l o s t from phytoplankton as ammonia/ammonium, which i s not taken back up, at l e a s t on the short term. Support f o r ammonia/ammonium e x c r e t i o n d u r i n g urea uptake by phytoplankton was found i n an axenic c u l t u r e of a marine diatom. 7. A model of urea uptake and a s s i m i l a t i o n by the c o a s t a l marine diatom Thai assi osira pseudonana (clone 3H) was 281 proposed. One s a l i e n t f e a t u r e of t h i s model i n c l u d e s urea-N e x c r e t i o n as ammonia/ammonium and i t s r a p i d r e a b s o r p t i o n . During n i t r a t e - s u f f i c i e n t growth, T. ps eudonana r e t a i n e d only 15% of the t o t a l urea-N taken up by the c e l l s , s uggesting that n i t r o g e n i s e x c r e t e d as d i s s o l v e d organic n i t r o g e n . Losses of urea-C were a l s o observed, but they were l e s s than those of urea-N. N i t r a t e - s t a r v e d c e l l s took up urea and d u r i n g a s s i m i l a t i o n r e t a i n e d a l l of the urea-N; the three methods used to measure urea uptake, 1 4 C - u r e a , 1^N-urea and the disappearance of d i s s o l v e d urea were i n e x c e l l e n t - agreement d u r i n g a 1 h urea uptake experiment with n i t r a t e - s t a r v e d c e l l s . 8. Selenium i s an e s s e n t i a l element f o r growth of Thalassiosira ps eudonana (clone 3H). Of the two 2-m o r g a n i c forms of Se examined, SeO^ was the most 2~ r e a d i l y u t i l i z e d f o r growth; SeO^ was only e f f e c t i v e i n supporting growth when c o n c e n t r a t i o n s were g r e a t e r than -7 . 2-10 M, four o r d e r s of magnitude g r e a t e r than SeO^ . No other element was abl e to s u b s t i t u t e f o r the Se requirement. 9. 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Urea uptake by n i t r a t e - l i m i t e d chemostat c u l t u r e s of T h a l a s s i o s i r a pseudonana (clone 3H). ASLO/PSA conference, Rhode I s l a n d . P r i c e , N.M., P.A. Thompson, G.J. Doucette and P.J. H a r r i s o n . 1986. A selenium requirement f o r T h a l a s s i o s i r a  pseudonana (clone 3H). ASLO/PSA conference, Rhode I s l a n d . P r i c e , N.M. and P.J. H a r r i s o n . 1986. Uptake and i n c o r p o r a t i o n of selenium: F u r t h e r evidence of a s p e c i f i c requirement f o r selenium i n the marine diatom T h a l a s s i o s i r a  pseudonana (clone 3H). 2nd Northwest A l g a l Symposium. Doucette, G.J., P r i c e , N.M. and P.J. H a r r i s o n . 1986. E f f e c t s of selenium d e f i c i e n c y on the u l t r a s t r u c t u r e of the marine diatom T h a l a s s i o s i r a pseudonana (clone 3H). 2nd Northwest A l g a l Symposium. P r i c e , N.M. and P.J. H a r r i s o n . 1986. Saturated n i t r o g e n uptake r a t e s of Sargasso Sea phytoplankton. ASLO Winter Meeting, San F r a n c i s c o . Eos 67(44):979. Cochlan, W.P., N.M. P r i c e and P.J. H a r r i s o n . 1986. L i g h t dependence of n i t r a t e and urea uptake by phytoplankton i n f r o n t a l and s t r a t i f i e d waters of the S t r a i t of Ge o r g i a . ASLO Winter Meeting, San F r a n c i s c o . Eos 67(44):980. N e i l M. Pr i ce March, 1987. Awards: Ki l l am Postdoctoral Fel lowship 1987 NSERC Postdoctoral Fel lowship 1987 NSERC V i s i t i n g Fel lowship (declined) 1987 B . C . Government Scholarship for Academic Excel lence 1986 Byrne Scholarship 1985 -1986 Edi th Ashton Memorial Scholarship 1984 -1985 K i l l a m Predoctoral Fel lowship 1982 -1985 G . L . P i c k a r d Scholarship in Oceanography 1983 -1984 NSERC Postgraduate Scholarship 1980 -1984 Norman S. Fraser Pr ize in Science 1980 Univers i ty Spec ia l Undergraduate Scholarship 1977 -1980 

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