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Nitrogen uptake by marine phytoplankton : the effects of irradiance, nitrogen supply and diel periodicity Cochlan, William Patrick 1989

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NITROGEN UPTAKE BY MARINE PHYTOPLANKTON: THE EFFECTS OF IRRADIANCE, NITROGEN SUPPLY AND DIEL PERIODICITY by WILLIAM PATRICK COCHLAN B.Sc. (Hons.)/ University of B r i t i s h Columbia, 1978 M.Sc, Dalhousie University, 1982 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Oceanography) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December 198 9 © William Patrick Cochlan, 1989 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 Oc<&<aoo<qraph' The University of British Columbia Vancouver, Canada Date DE-6 (2/88) ABSTRACT D i e l p a t t e r n s o f n i t r o g e n ( N O 3 - , NH^"1", u r e a ) u p t a k e w e r e i n v e s t i g a t e d i n n a t u r a l a s s e m b l a g e s o f p h y t o p l a n k t o n f r o m n e r i t i c a n d o c e a n i c e n v i r o n m e n t s o f f t h e c o a s t o f B r i t i s h C o l u m b i a . T h i s i s t h e f i r s t s t u d y t o r e p o r t n i t r o g e n u p t a k e r a t e s a n d e x t e n s i v e m e a s u r e m e n t s o f a m b i e n t N H ^ + a n d u r e a c o n c e n t r a t i o n s i n t h e s e w a t e r s . C a l c u l a t e d r a t e s o f N u p t a k e , 15 b a s e d on N i n c o r p o r a t i o n i n t o p a r t i c u l a t e m a t t e r d u r i n g t i m e c o u r s e e x p e r i m e n t s , w e r e m a x i m a l d u r i n g t h e d a y a n d m i n i m a l a t n i g h t . B e s i d e s t h e o b v i o u s e f f e c t s o f i r r a d i a n c e , t h e a m p l i t u d e o f t h e p e r i o d i c i t y i n u p t a k e r a t e was i n f l u e n c e d by p h y t o p l a n k t o n c o m m u n i t y c o m p o s i t i o n , a m b i e n t n i t r o g e n c o n c e n t r a t i o n , f o r m s o f n i t r o g e n a v a i l a b l e , a n d d e p t h o f s a m p l i n g . U p t a k e o f n i t r o g e n d u r i n g t h e n i g h t a n d i n a r t i f i c i a l d a r k n e s s w e r e m e a s u r a b l e p r o p o r t i o n s o f d a y t i m e a n d l i g h t u p t a k e r a t e s , w i t h t h e i m p o r t a n c e o f d a r k u p t a k e g e n e r a l l y i n c r e a s i n g w i t h i n c r e a s i n g N l i m i t a t i o n . T h i s i s t h e f i r s t s t u d y o f d i e l u r e a u p t a k e b y m a r i n e p h y t o p l a n k t o n i n t h e f i e l d . The r a t i o s o f d a r k t o l i g h t u r e a u p t a k e o v e r a d i e l c y c l e w e r e more s i m i l a r t o t h o s e o f N O 3 - t h a n t h o s e o f t h e o t h e r r e d u c e d N f o r m , NH^ +. R a t e s o f N O 3 - a n d u r e a u p t a k e b y p h y t o p l a n k t o n i n t h e s h a l l o w a n d d e e p c h l o r o p h y l l l a y e r s o f t h e S t r a i t o f G e o r g i a w e r e m e a s u r e d o v e r a g r a d i e n t o f i r r a d i a n c e s a n d r e s u l t s o f t h e s e e x p e r i m e n t s c o u l d be f i t t e d w i t h a h y p e r b o l i c f u n c t i o n s i m i l a r t o t h e M i c h a e l i s - M e n t e n e q u a t i o n . H a l f - s a t u r a t i o n c o n s t a n t s ( K L T ) f o r l i g h t - d e p e n d e n t u p t a k e o f u r e a a n d N O 3 -r a n g e d f r o m 0 t o 14% o f t h e s u r f a c e i r r a d i a n c e a n d d a r k u p t a k e was a v a r i a b l e , b u t o f t e n s u b s t a n t i a l (> 50%) p o r t i o n o f t h e t o t a l ( l i g h t + d a r k ) u p t a k e . The u p t a k e r e s p o n s e o f n i t r a t e - r e p l e t e a n d - s t a r v e d p o p u l a t i o n s o f t h e p i c o f l a g e l l a t e , Micromonas p u s i l l a ( B u t c h . ) M a n t o n e t P a r k e , t o u r e a , NH^"1" a n d N O 3 - p e r t u r b a t i o n s was d e t e r m i n e d b y b o t h a c c u m u l a t i o n a n d n u t r i e n t d i s a p p e a r a n c e f r o m t h e c u l t u r e medium. Maximum s p e c i f i c u p t a k e r a t e s ( V m a x ) o f NH^"1" w e r e 0.13 h ~ ^ , more t h a n 2 t i m e s t h e V m a x o f N O 3 - o r u r e a ( c a . 0.05 h - ^ ) . The h a l f - s a t u r a t i o n c o n s t a n t s ( K s ) f o r u r e a , NH^ + a n d N O 3 - w e r e w i t h i n ± 0.1 uq-at N-L--'- o f e a c h o t h e r ; t h e a v e r a g e v a l u e o f 0.41 uq-at. N-L-"*" i s w i t h i n t h e r a n g e r e p o r t e d f o r s m a l l , o c e a n i c d i a t o m s . N O 3 - u p t a k e was c o m p l e t e l y i n h i b i t e d f o l l o w i n g NH^ "*" a d d i t i o n (1-10 uq-at. N-L -"'"), w h e r e a s u r e a a d d i t i o n r e s u l t e d i n o n l y a 2 8 % r e d u c t i o n i n N O 3 - u p t a k e . S t a r v e d c u l t u r e s o f M. p u s i l l a e x h i b i t e d v a r i a b l e u p t a k e o f NH^ + a n d u r e a a s a f u n c t i o n o f t i m e , w i t h a n i n i t i a l " s u r g e " u p t a k e r e s p o n s e . T h i s i s t h e f i r s t l a b o r a t o r y s t u d y o f N u p t a k e by a n e u c a r o y o t i c p i c o p l a n k t e r a n d d e m o n s t r a t e s t h a t many o f t h e t r a n s i e n t u p t a k e r e s p o n s e s r e p o r t e d f o r d i a t o m s , w i t h w h i c h i t c o m p e t e s i n t h e f i e l d , a r e common t o t h i s p i c o p l a n k t e r . D i e l p e r i o d i c i t y o f n i t r o g e n u p t a k e a n d a s s i m i l a t i o n w e r e m e a s u r e d i n N - r e p l e t e b a t c h c u l t u r e s o f M. p u s i l l a a n d a l s o i n N - l i m i t e d c y c l o s t a t c u l t u r e s (14L:10D) a t t h r e e g r o w t h r a t e s c o r r e s p o n d i n g t o c a . 7 5 , 50 a n d 2 5 % o f i t ' s m a x i m a l g r o w t h r a t e . N i t r a t e u p t a k e was c o n t i n u o u s a n d i n d e p e n d e n t o f t h e i v L:D c y c l e i n the c y c l o s t a t c u l t u r e s at the lowest d i l u t i o n r a t e , but N O 3 - uptake r a t e s e x h i b i t e d pronounced p e r i o d i c i t y i n the batch and high e r d i l u t i o n r a t e c u l t u r e s , a response s i m i l a r t o t h a t seen i n p r e v i o u s s t u d i e s of c y c l o s t a t c u l t u r e s of some diatoms. D i e l p a t t e r n s i n c e l l d i v i s i o n , mean c e l l volume, p o t e n t i a l uptake r a t e s and i n t e r n a l p o o l s of NC>3~ were a l s o observed and are d i s c u s s e d w i t h r e s p e c t t o the n u t r i t i o n a l s t a t u s of the c e l l s . The e f f e c t of i r r a d i a n c e on the uptake of NH^ "1" and N O 3 - by M. p u s i l l a was a l s o d e s c r i b e d by Michaelis-Menten k i n e t i c s ; with i n c r e a s i n g N l i m i t a t i o n the importance of l i g h t f o r n i t r o g e n uptake decreased and dark uptake i n c r e a s e d from 5-20% t o 21-39% of N O 3 - and N H 4 4 " uptake r a t e s , r e s p e c t i v e l y , at s a t u r a t i n g i r r a d i a n c e . V TABLE OF CONTENTS ABSTRACT i i L I S T OF TABLES i x L I S T OF FIGURES x i i ACKNOWLEDGEMENTS x i x INTRODUCTION O v e r v i e w 1 T h e s i s o b j e c t i v e s 7 T h e s i s o u t l i n e 8 E x p e r i m e n t a l o r g a n i s m 9 CHAPTER 1. EFFECTS OF D I E L P E R I O D I C I T Y ON NITROGEN UPTAKE BY NATURAL ASSEMBLAGES OF PHYTOPLANKTON I n t r o d u c t i o n 12 M a t e r i a l s a n d M e t h o d s * G e n e r a l 15 S a m p l e c o l l e c t i o n 15 A n a l y t i c a l m e t h o d s 18 T r a c e r e x p e r i m e n t s 2 0 E x p e r i m e n t a l p r o c e d u r e s 21 R e s u l t s P h y s i c a l o b s e r v a t i o n s 24 B i o l o g i c a l o b s e r v a t i o n s 31 N i t r o g e n u p t a k e r a t e s 36 S u b a r c t i c P a c i f i c O cean 36 S t r a i t o f G e o r g i a 36 O f f s h o r e w a t e r s 43 v i D i s c u s s i o n E x p e r i m e n t a l c o n s i d e r a t i o n s 55 S i m u l t a n e o u s u p t a k e o f n i t r o g e n compounds 58 E f f e c t s o f l i g h t / d a r k r e g i m e on n i t r o g e n u p t a k e ...60 CHAPTER 2. EFFECTS OF IRRADIANCE ON NITROGEN UPTAKE BY PHYTOPLANKTON: COMPARISON OF FRONTAL AND S T R A T I F I E D COMMUNITIES I n t r o d u c t i o n 67 M a t e r i a l s a n d M e t h o d s G e n e r a l 71 E x p e r i m e n t a l 73 K i n e t i c p a r a m e t e r s o f n i t r o g e n u p t a k e 76 R e s u l t s a n d D i s c u s s i o n G e n e r a l d e s c r i p t i o n o f s t a t i o n s 7 7 E f f e c t o f l i g h t on n i t r o g e n u p t a k e r a t e s 81 K i n e t i c p a r a m e t e r s o f n i t r o g e n u p t a k e 85 D a r k n i t r o g e n u p t a k e 92 Summary 100 CHAPTER 3. NITROGEN UPTAKE BY THE EUCARYOTIC PICOPLANKTER, MICROMONAS PUSILLA AND THE EFFECTS OF N DEPRIVATION ON UPTAKE RESPONSE I n t r o d u c t i o n 102 M a t e r i a l s a n d M e t h o d s C u l t u r i n g 106 A n a l y t i c a l m e t h o d s 107 E x p e r i m e n t a l p r o c e d u r e s K i n e t i c p a r a m e t e r s f o r N u p t a k e 108 v i i S u b s t r a t e i n t e r a c t i o n 110 E f f e c t o f NH^ + c o n c e n t r a t i o n on N O 3 - u p t a k e r a t e I l l U p t a k e o f n i t r o g e n b y N 0 3 ~ - s t a r v e d c e l l s ....112 E s t i m a t i o n o f k i n e t i c p a r a m e t e r s 113 R e s u l t s U p t a k e k i n e t i c s 115 S u b s t r a t e i n t e r a c t i o n 118 N i t r o g e n - s t a r v e d c e l l s 121 D i s c u s s i o n U p t a k e k i n e t i c s 131 C e l l u l a r p h y s i o l o g i c a l s t a t e 133 S u b s t r a t e i n t e r a c t i o n 138 E c o l o g i c a l s i g n i f i c a n c e 141 CHAPTER 4. EFFECTS OF IRRADIANCE AND D I E L P E R I O D I C I T Y ON NITROGEN U T I L I Z A T I O N I N MICROMONAS PUSILLA I n t r o d u c t i o n 143 M a t e r i a l s a n d M e t h o d s C u l t u r i n g 146 A n a l y t i c a l p r o c e d u r e s 148 E x p e r i m e n t a l p r o c e d u r e s D i e l c y c l e s o f u p t a k e a n d g r o w t h 14 9 D i e l v a r i a t i o n i n maximum N u p t a k e r a t e s ....151 E f f e c t o f PPFD on N 0 3 ~ a n d N H 4 + u p t a k e 152 R e s u l t s N i t r a t e - r e p l e t e c u l t u r e s . 155 N i t r a t e - l i m i t e d c u l t u r e s 157 v i i i P o t e n t i a l N u p t a k e r a t e s 170 I n f l u e n c e o f l i g h t on N u p t a k e r a t e s 175 D i s c u s s i o n 180 GENERAL CONCLUSIONS 190 REFERENCES 193 APPENDIX 1. E q u a t i o n s u s e d t o c a l c u l a t e -^N u p t a k e r a t e s ..222 APPENDIX 2. G r o w t h - i r r a d i a n c e c u r v e o f Micromonas pusilla .226 APPENDIX 3. C o m p a r i s o n o f t h e i n c r e a s e s i n in vivo f l u o r e s c e n c e a n d c e l l c o n c e n t r a t i o n d u r i n g e x p o n e n t i a l g r o w t h o f Micromonas pusilla 22 9 APPENDIX 4. C o m p a r i s o n s o f t h e r a t e s o f p a r t i c u l a t e n i t r o g e n p r o d u c t i o n a n d i n o r g a n i c n i t r o g e n d i s a p p e a r a n c e 232 APPENDIX 5. 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 d i s a p p e a r a n c e c u r v e d u r i n g g r o w t h o f M.pusilla 235 APPENDIX 6. P r e c i s i o n o f a n a l y t i c a l t e c h n i q u e s 238 i x L I S T OF TABLES T a b l e 1.1 I n i t i a l e n v i r o n m e n t a l c o n d i t i o n s o f s e a w a t e r c o l l e c t e d f o r t i m e c o u r s e e x p e r i m e n t s o f n i t r o g e n u p t a k e b y n a t u r a l p h y t o p l a n k t o n a s s e m b l a g e s . S t a t i o n s a r e F: N o r t h e a s t P a c i f i c O c e a n ; A 5 : S t r a i t o f G e o r g i a - f r o n t a l ; T4: S t r a i t o f G e o r g i a -s t r a t i f i e d ; 24: u p w e l l i n g p l u m e o f f s o u t h w e s t c o a s t o f V a n c o u v e r I s l a n d ; o f f s h o r e o f w e s t e r n C a n a d i a n c o n t i n e n t a l s h e l f 17 T a b l e 1.2 P l a n k t o n c o m m u n i t y c o m p o s i t i o n i n f r o n t a l a n d s t r a t i f i e d w a t e r o f S t r a i t o f G e o r g i a , B.C., ( s e e F i g . 1.1 B) 33 T a b l e 1.3 R a t i o o f d a r k t o l i g h t u p t a k e r a t e s ( V D : V ) o f NH 4 +, NO " a n d u r e a f o r f r o n t a l a n d s t r a t i f i e d w a t e r o f t h e S t r a i t o f G e o r g i a , B.C., ( s e e F i g , 1.1 B) 44 T a b l e 1.4 C h l o r o p h y l l a s p e c i f i c u p t a k e r a t e s o f NH 4 +, N0 3" an d u r e a i n f r o n t a l (A5) a n d s t r a t i f i e d (T4) w a t e r o f t h e S t r a i t o f G e o r g i a . , ( s e e F i g . 1.1 B ) . The d a r k p e r i o d o c c u r s d u r i n g t h e 12 t o 18 h t i m e i n t e r v a l 45 T a b l e 1.5 I n i t i a l e n v i r o n m e n t a l c o n d i t i o n s o f s e a w a t e r c o l l e c t e d f o r n i t r o g e n u p t a k e e x p e r i m e n t s d u r i n g t i m e c o u r s e 4 47 T a b l e 1.6 I n i t i a l e n v i r o n m e n t a l c o n d i t i o n s d u r i n g t i m e c o u r s e 5 c o n d u c t e d o f f t h e w e s t c o a s t o f V a n c o u v e r I s l a n d on A u g u s t 2 5 - 2 6 , 1986 52 T a b l e 2.1 I n i t i a l e n v i r o n m e n t a l c o n d i t i o n s o f s e a w a t e r c o l l e c t e d i n t h e S t r a i t o f G e o r g i a f o r N u p t a k e v e r s u s i r r a d i a n c e e x p e r i m e n t s 79 T a b l e 2.2 P h y t o p l a n k t o n c o m m u n i t y c o m p o s i t i o n i n f r o n t a l a n d s t r a t i f i e d w a t e r o f S t r a i t o f G e o r g i a , B.C.. 80 T a b l e 2.3 P a r a m e t e r s d e s c r i b i n g t h e c h a r a c t e r i s t i c s o f n i t r o g e n u p t a k e , a s a f u n c t i o n o f PPFD, f o r p h y t o p l a n k t o n a s s e m b l a g e s i n t h e S t r a i t o f G e o r g i a , B.C. D e f i n i t i o n s a r e g i v e n i n t h e t e x t , s t a n d a r d e r r o r s o f p a r a m e t e r s i n p a r e n t h e s e s . . . . . 86 X Table 2.4 Indices of N uptake dependency on PPFD for phytoplankton i n the S t r a i t of Georgia: the r a t i o of dark to light-saturated uptake rate (V *V ) , the PPFD at which half of t o t a l N uptake occurs (K L T', K LT " ) * , r a t i o of uptake under 1% I q to 55% I Q (V :V 5 5 %). The K L T values are expressed as PPFD values and as a percentage of surface PPFD ( I Q ) which i s shown i n parentheses 88 Table 2.5 Table 2.6 Table 3.1 Table 3.2 Comparison of half-saturation constants (K L T) for inorganic n i t r a t e transport i n various aquatic ecosystems 89 Summary of l i t e r a t u r e values of dark:light nitrogen s p e c i f i c ( V D/V L) or absolute(P D/p L) uptake rates, determined during daytime, i n natural phytoplankton communities 94 Kinetic parameters for n i t r a t e , urea and ammonium uptake of N-replete Micromonas p u s i l l a . Michaelis-Menten parameters, Kg (half-saturation constant) and V m a x (maximum uptake ve l o c i t y ) were estimated from a d i r e c t nonlinear curve f i t t i n g model 1 and Hanes-Woolf l i n e a r transformation 2 of the data obtained from r e p l i c a t e cultures (1 or 2) and the cultures treated together (1 + 2) 117 Average n i t r a t e uptake rates (h - 1) for N03~-starved Micromonas p u s i l l a . Rates determined from least-squares l i n e a r regression of p a r t i c u l a t e 15N enrichment or the decrease i n the external concentration of N03~ + NO " versus time and reported as ± 1 standard deviation (in parentheses) of the mean of duplicate cultures. 123 Table 3.3 Average N uptake rates V (h - 1) for NO "-starved Micromonas p u s i l l a . Rates determined from l e a s t -squares l i n e a r regression of p a r t i c u l a t e 15 N enrichment or the decrease i n the external concentration of dissolved nitrogen versus time and reported as ± 1 standard deviation (in parentheses) of the mean of duplicate cultures. 123 Table 3.4 Summary of culture conditions at the beginning of each experiment 130 x i T a b l e 4.1 T a b l e 4.2 T a b l e 4.3 Mean l i g h t a n d d a r k s p e c i f i c n i t r a t e u p t a k e r a t e s ( h _ 1 ) a n d t h e i r r a t i o s ( d a r k : l i g h t ) f o r Micromonas pusilla grown on a 14:10 l i g h t - d a r k c y c l e i n b a t c h (*) a n d c y c l o s t a t c u l t u r e s . The s t a n d a r d d e v i a t i o n s o f s e p a r a t e ( 5 - 7 ) r a t e m e a s u r e m e n t s d u r i n g t h e l i g h t o r d a r k p e r i o d a r e g i v e n i n p a r e n t h e s e s 168 Summary o f c y c l o s t a t c u l t u r e c o n d i t i o n s a t t h e b e g i n n i n g o f e a c h e x p e r i m e n t 171 N i t r o g e n s p e c i f i c u p t a k e r a t e s ( h _ 1 ) , d e t e r m i n e d o v e r 2 h i n l i g h t a n d d a r k n e s s , a n d t h e i r r a t i o s (D/L) f o r Micromonas pusilla p r e v i o u s l y g r o w n a t 0.24, 0.49, 0.74 d" 1 i n NO " - l i m i t e d c y c l o s t a t c u l t u r e s on a 14 h l i g h t : 1 0 h d a r k i l l u m i n a t i o n c y c l e ( l i g h t s o n : 0800 h, l i g h t s o f f : 2200 h ) . 174 T a b l e 4.4 P a r a m e t e r s d e s c r i b i n g t h e c h a r a c t e r i s t i c s o f N s p e c i f i c u p t a k e ( h " 1 ) , a s a f u n c t i o n o f PPFD f o r c y c l o s t a t c u l t u r e s o f Micromonas pusilla ( F i g . 4 . 1 1 ) . D a r k u p t a k e (V ) , maximum s p e c i f i c l i g h t u p t a k e ( V ) , t h e h a l f - s a t u r a t i o n c o n s t a n t ( K T m ) i _ max . _ v L T ' a n d t h e s l o p e o f i n i t i a l p o r t i o n o f N u p t a k e v s PPFD c u r v e ( a = V /K T m) . E s t i m a t e d s t a n d a r d max L T ' e r r o r s o f p a r a m e t e r s a r e g i v e n i n p a r e n t h e s e s 177 T a b l e 4.5 I n d i c e s o f N u p t a k e d e p e n d e n c y on PPFD f o r c y c l o s t a t c u l t u r e s o f Micromonas pusilla: t h e r a t i o , o f d a r k t o l i g h t - s a t u r a t e d u p t a k e r a t e ( V D : V L ) , t h e PPFD a t w h i c h h a l f t h e t o t a l N u p t a k e o c c u r s ( K L T ' , K " ) * a n d t h e r a t i o o f N u p t a k e a t 1% I t o N u p t a k e a t 100% I ( V 1 % : V ). S a t u r a t e d PPFD a n d I a r e t h e g r o w t h PPFD (120 uE m ^ s - 1 ) . . . 178 x i i LIST OF FIGURES F i g u r e 1.1 F i g u r e 1.2 F i g u r e 1.3, F i g u r e 1.4 F i g u r e 1.5, F i g u r e 1.6 F i g u r e 1.7 F i g u r e 1.8. S t a t i o n l o c a t i o n s f o r time c o u r s e experiments of n i t r o g e n uptake. (A) T C . l a t s t n F; TC.4 at s t n 24; TC.5 a t s t n 85. (B) TC.2 a t s t n A5; TC.3 a t s t n T4. Panel B i s an enlargement of the area d e l i m i t e d by dashed l i n e s i n pane l A. 16 Depth p r o f i l e s of temperature ( T ) , s a l i n i t y ( S ) , i n v i v o f l u o r e s c e n c e ( F ) , and n i t r a t e p l u s n i t r i t e c o n c e n t r a t i o n (N) f o r t h r e e s t a t i o n s sampled f o r containment time c o u r s e experiments. (A) Oceanic s t a t i o n F, T C . l . (B) F r o n t a l s t a t i o n A5, TC.2. (C) S t r a t i f i e d s t a t i o n T4, TC.3. The shallow t h e r m o c l i n e step i s i n d i c a t e d by the arrow l a b e l l e d 'sT' i n pan e l A 25 Depth p r o f i l e s of temperature (T) and s a l i n i t y (S) f o r the two s t a t i o n s r e p e a t e d l y sampled d u r i n g drogue-type time course experiments. A: s t n 24 (beginning of TC.4). B: s t n 49 (end of TC.4). C: s t n 84 (beginning of TC.5). D: s t n 98 (end of TC.5) 27 Depth p r o f i l e s of N03~ and NH 4 + a t 6 h i n t e r v a l s d u r i n g time course 4 28 Depth p r o f i l e s of S i 0 4 - 4 and P0 4" 3 a t 6 h i n t e r v a l s d u r i n g time course 4 29 A: Depth p r o f i l e s of N0 3 _ (•) and NH 4 + (C) a t 2 h i n t e r v a l s d u r i n g time c o u r s e 5. B: Depth p r o f i l e s of S i 0 4 " 4 (•) and P0 4" 3 (•) a t 2 h i n t e r v a l s d u r i n g time course 5. 30 Composition of the phyto p l a n k t o n community, A: a t the b e g i n n i n g ( s t n 24) and end ( s t n 49) of time course 4 B: b e g i n n i n g ( s t n 84) and end ( s t n 98) of time course 5 35 Time course measurements a t o c e a n i c s t a t i o n F., Time Course 1. (A) D a i l y i n c i d e n t s u r f a c e i r r a d i a n c e d u r i n g experiment. (B) 1 5N atom % excess i n p a r t i c u l a t e matter f o r l i g h t b o t t l e i n c u b a t i o n s ( e r r o r bars r e p r e s e n t ± 1 S.D. of t r i p l i c a t e s ) p l o t t e d a g a i n s t e l a p s e d time measured a f t e r the a d d i t i o n of 1.0 pg-at N-N0 3*L - 1. (C) N i t r o g e n s p e c i f i c uptake r a t e s of 1 5N0 3" c a l c u l a t e d f o r 3 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 x i i i F i g u r e 1.9. F i g u r e 1.10 F i g u r e 1.11 i n t e r v a l b e t w e e n i t a nd t h e p r e v i o u s p o i n t on t h e c u r v e a n d p l o t t e d a g a i n s t a v e r a g e i n c u b a t i o n t i m e b e t w e e n s a m p l i n g 37 T i m e c o u r s e m e a s u r e m e n t s a t f r o n t a l s t a t i o n ( A 5 ) , T i m e C o u r s e 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 e x p e r i m e n t ( B , D, F) 1 5N at o m % e x c e s s i n p a r t i c u l a t e m a t t e r f o r l i g h t a n d d a r k 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 o f 6 / j g - a t N - L - 1 o f (B) NH 4 +, (D) N 0 3 _ a n d ( F ) u r e a ( e r r o r b a r s r e p r e s e n t t h e r a n g e o f d u p l i c a t e s ) . ( C , E, G) C o r r e s p o n d i n g m e a s u r e m e n t s o f d i s s o l v e d NH 4 + ( • ) , N0 3" ( o ) a n d u r e a ( A ) i n (C) NH 4 +, (E) N0 3~, a n d (G) u r e a - s p i k e d s a m p l e s . D a s h e d l i n e i n d i c a t e s no m e a s u r e m e n t s o f d i s s o l v e d u r e a a t 3 a n d 6 h ; . ( l e f t s i d e o f p a g e ) 40 A s F i g u r e 1.9 e x c e p t a t s t r a t i f i e d s t a t i o n ( T 4 ) , T ime C o u r s e 3; ( r i g h t s i d e o f p a g e ) . 40 N i t r o g e n - s p e c i f i c u p t a k e r a t e s c f NH + ( • ) , N0 3" ( o ) a n d u r e a ( A ) i n (A) f r o n t a l a n d (B) s t r a t i f i e d water.. R a t e s d e t e r m i n e d f o r 3 o r 6 h i n t e r v a l s ; e a c h 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 o v e r t h e t i m e i n t e r v a l b e t w e e n i t a n d t h e p r e v i o u s p o i n t on t h e c u r v e . S h a d e d a r e a o n t h e a b s c i s s a d e l i m i t s t h e d a r k p e r i o d . 42 F i g u r e 1.12 F i g u r e 1.13 F i g u r e 1.14 Time course measurements a t upw e l l e d plume s t a t i o n s 24-49, time course 4. (A) D a i l y i n c i d e n t s u r f a c e i r r a d i a n c e d u r i n g experiment. (B) N i t r a t e and (C) ammonium s p e c i f i c uptake r a t e s a t 100% I O ( O ) , 30% l " o (•) and 1% I O (A) c a l c u l a t e d over 4 h i n c u b a t i o n p e r i o d s and p l o t t e d a g a i n s t average i n c u b a t i o n p e r i o d . . . 49 Time course measurements a t upw e l l e d plume s t a t i o n s 24-49, time course 4. (A) D a i l y i n c i d e n t s u r f a c e i r r a d i a n c e d u r i n g experiment. (B) N i t r a t e and (C) ammonium a b s o l u t e uptake r a t e s a t 100% I Q ( O ) , 30% I q ( • ) and 1% I q (A) c a l c u l a t e d over 4 h i n c u b a t i o n p e r i o d s and p l o t t e d a g a i n s t average i n c u b a t i o n p e r i o d . . . 50 Time course measurements a t s t a t i o n s 85-98, time course 5. (A) D a i l y i n c i d e n t s u r f a c e i r r a d i a n c e d u r i n g experiment. (B) N i t r o g e n s p e c i f i c uptake r a t e s of n i t r a t e a t 100% I Q ( O ) and 1% ( • ) c a l c u l a t e d over 4 h i n c u b a t i o n p e r i o d s and p l o t t e d a g a i n s t average i n c u b a t i o n p e r i o d 54 x i v F i g u r e 2.1. S t a t i o n l o c a t i o n s f o r n i t r o g e n uptake experiments. F r o n t a l (T14), s h a l l o w s t r a t i f i e d (A5) and deeply s t r a t i f i e d (T8) s t a t i o n s i n the S t r a i t of Georgia, B.C 72 F i g u r e 2.2. Depth p r o f i l e s of temperature ( T ) , s a l i n i t y ( S ) , i n v i v o f l u o r e s c e n c e (F) and n i t r a t e p l u s n i t r i t e c o n c e n t r a t i o n (N) f o r the t h r e e s t a t i o n s sampled (T14: f r o n t a l ; A5: s h a l l o w s t r a t i f i e d ; and T8: d e e p l y s t r a t i f i e d ) 78 F i g u r e 2.3. N i t r a t e uptake of the s u r f a c e ( o ) and DCM ( • ) phytoplankton communities of the S t r a i t of G e o r g i a . The curved p l o t s are f i t t e d d i r e c t l y t o the Michaelis-Menten e q u a t i o n ; the l i n e a r - (dashed l i n e ) P P F D - i n h i b i t e d p o r t i o n s were not i n c l u d e d i n the c a l c u l a t i o n s . S t a t i o n s are T14 ( f r o n t a l ) , A5 ( s h a l l o w s t r a t i f i e d ) and T8 (deeply s t r a t i f i e d ) 83 F i g u r e 2.4. Urea uptake of the s u r f a c e ( O) and DCM ( • ) phytoplankton communities of the S t r a i t of G e o r g i a . The curved p l o t s are f i t t e d d i r e c t l y t o the Michaelis-Menten e q u a t i o n ; the l i n e a r (dashed l i n e ) P P F D - i n h i b i t e d p o r t i o n s were not i n c l u d e d i n the c a l c u l a t i o n s . S t a t i o n s are A5 ( s h a l l o w s t r a t i f i e d ) and T8 (deeply s t r a t i f i e d ) 84 F i g u r e 3.1. N i t r o g e n s p e c i f i c uptake r a t e s (V) determined over 10 min a f t e r the a d d i t i o n of 0.2, 0.4, 0.8, 1.6, 2.4, 4.2 and 10 uq-at N-L"1 of N0 3 _ (A), NH 4 + (B) or urea (C) t o d u p l i c a t e n i t r a t e -r e p l e t e c u l t u r e s (0,«) of Micromonas pusilla. Rates (h _ 1) are p l o t t e d v e r s u s the average s u b s t r a t e c o n c e n t r a t i o n d u r i n g the 10 min i n t e r v a l . Curve c a l c u l a t e d by computer programme (see t e x t f o r d e t a i l s ) 116 F i g u r e 3.2. Comparison of n i t r o g e n s p e c i f i c uptake r a t e s f o r n i t r a t e - r e p l e t e c u l t u r e s of Micromonas pusilla determined over 10 and 60 min i n c u b a t i o n p e r i o d s . C u l t u r e s are numbered and v a l u e s are the mean (n = 2) of d u p l i c a t e i n c u b a t i o n s , * d e s i g n a t e s no r e p l i c a t e . Bar r e p r e s e n t s ± 1 S.D 119 F i g u r e 3.3. N i t r o g e n uptake by r e p l i c a t e c u l t u r e s of n i t r a t e - r e p l e t e Micromonas pusilla over a 4 h i n c u b a t i o n p e r i o d . A. D i s s o l v e d N03" + N02" c o n c e n t r a t i o n (•) and 1 5N-atom % excess (O) a f t e r 10 uq-at N-urea-L" 1 a d d i t i o n . B. D i s s o l v e d N03~ + N02" c o n c e n t r a t i o n (0,A) a f t e r no and 10 uq-at N- N0 3"«L _ 1 a d d i t i o n , X V F i g u r e 3.4 r e s p e c t i v e l y . D i s s o l v e d NO ~ + N0 2" c o n c e n t r a t i o n (•,•) a f t e r 10 L/g-at N*L" a d d i t i o n o f NH 4 + a n d u r e a , r e s p e c t i v e l y . D i s s o l v e d N H + c o n c e n t r a t i o n (•) a f t e r a d d i t i o n o f 10 / j g - a t N-NH + • L" 1. 120 D i s s o l v e d NO " + N0 2" c o n c e n t r a t i o n w i t h o u t ( O ) , a n d w i t h (•) , 5 ( A ) , 2 ( B ) , a n d 1 ( C ) L-g-at N - L - 1 [ 1 5N] - NH 4 + e n r i c h m e n t ; 1 5NH 4 + a t o m % e x c e s s i n p a r t i c u l a t e s (•) p l o t t e d v e r s u s t i m e ( m i n ) . A r r o w s d e s i g n a t e t i m e o f NH 4 + a d d i t i o n 122 F i g u r e 3.5 N i t r a t e u p t a k e b y n i t r a t e - ^ s t a r v e d Micromonas pusilla a f t e r t h e a d d i t i o n o f 15 j j g - a t N-NOq NO -l t o d u p l i c a t e c u l t u r e s . A. D i s s o l v e d + NO " (•,•) i n t h e c u l t u r e medium; 15 NO, 3 2 v ~ , - , — ' " - 3 a t o m % e x c e s s i n p a r t i c u l a t e m a t t e r ( 0 , 9 ) . B. N i t r a t e u p t a k e r a t e d e t e r m i n e d f r o m N0 3" + N0 2" d i s a p p e a r a n c e t e c h n i q u e . C. [ 1 5N] n i t r a t e u p t a k e r a t e . V a l u e s i n A a r e p l o t t e d a g a i n s t e l a p s e d t i m e m e a s u r e d a f t e r e n r i c h m e n t a n d u p t a k e r a t e s (B,C) a r e p l o t t e d a g a i n s t a v e r a g e i n c u b a t i o n t i m e 125 F i g u r e 3.6 F i g u r e 3.7 F i g u r e 4.1 U r e a u p t a k e b y n i t r a t e - s t a r v e d Micromonas pusilla a f t e r t h e a d d i t i o n o f 10 / j g - a t N - u r e a - L " 1 t o d u p l i c a t e c u l t u r e s ( O , * ) . A. 1 5 N - u r e a a t o m % e x c e s s i n p a r t i c u l a t e m a t t e r i s p l o t t e d a g a i n s t e l a p s e d t i m e m e a s u r e d a f t e r a d d i t i o n o f u r e a . B. [ 1 5N] u r e a u p t a k e r a t e p l o t t e d a g a i n s t a v e r a g e i n c u b a t i o n t i m e . ... 126 Ammonium u p t a k e b y n i t r a t e - s t a r v e d Micromonas pusilla a f t e r t h e a d d i t i o n o f 15 uq-at N-NH 4 +*L _ 1 t o d u p l i c a t e c u l t u r e s . A. D i s s o l v e d N H 4 + c o n c e n t r a t i o n i n t h e c u l t u r e medium ( ( ) , • ) ; 1 5N-NH 4 atom % e x c e s s i n p a r t i c u l a t e m a t t e r (•,•) p l o t t e d a g a i n s t e l a p s e d t i m e a f t e r e n r i c h m e n t . B. Ammonium u p t a k e r a t e , d e t e r m i n e d b y NH 4 + d i s a p p e a r a n c e t e c h n i q u e . C. [ N] NH 4 u p t a k e r a t e . V a l u e s i n B a n d C p l o t t e d a g a i n s t a v e r a g e i n c u b a t i o n t i m e . 128 C e l l c o n c e n t r a t i o n a s a f u n c t i o n o f t i m e f o r n i t r a t e - l i m i t e d c y c l o s t a t c u l t u r e s o f Micromonas pusilla g r o w n i n a 14 h : 1 0 h L:D c y c l e a t (O) 0.74, (©) 0.49 a n d (A) 0.24 d _ 1 d i l u t i o n r a t e s . E x p e r i m e n t s w e r e c o n d u c t e d o n d a y s 2,7,8,11,13 a n d 16 154 F i g u r e 4.2 C e l l c o n c e n t r a t i o n ( A ) , g r o w t h r a t e (B) a n d mean c e l l v o l u m e (C) v e r s u s e l a p s e d t i m e s i n c e l i g h t s on i n d u p l i c a t e (<D,#) b a t c h c u l t u r e s o f x v i Micromonas pusilla grown on a 14h:10h L:D i l l u m i n a t i o n c y c l e . Dashed l i n e i n d i c a t e s onset of dark p e r i o d denoted by dark bar. Growth r a t e p l o t t e d a g a i n s t average time between sampling 156 F i g u r e 4.3. D i s s o l v e d n i t r a t e c o n c e n t r a t i o n i n c u l t u r e medium (A), s p e c i f i c n i t r a t e uptake r a t e (B), and i n t r a c e l l u l a r n i t r a t e c o n c e n t r a t i o n (C) of d u p l i c a t e , batch c u l t u r e s of Micromonas pusilla grown on a 14h:10h L:D i l l u m i n a t i o n c y c l e . Dashed l i n e i n d i c a t e s onset of dark p e r i o d denoted by dark bar. N i t r a t e c o n c e n t r a t i o n s p l o t t e d a g a i n s t e l a p s e d time s i n c e l i g h t s on; n i t r a t e uptake p l o t t e d a g a i n s t average time between sampling 158 F i g u r e 4.4. C e l l c o n c e n t r a t i o n s of d u p l i c a t e n i t r a t e -l i m i t e d c y c l o s t a t s of Micromonas pusilla grown i n a 14h:10h L:D c y c l e a t 0.74 d _ 1 (A), 0.48 d _ 1 (B),.and 0.24 d _ 1 (C) d i l u t i o n r a t e s . C o n c e n t r a t i o n p l o t t e d a g a i n s t e l a p s e d time s i n c e l i g h t s on. Dashed l i n e i n d i c a t e s onset of dark p e r i o d denoted by dark bar 159 F i g u r e 4.5. C e l l d i v i s i o n r a t e of d u p l i c a t e n i t r a t e - l i m i t e d c y c l o s t a t s of Micromonas pusilla grown i n a 14h:10h L:D c y c l e a t 0.74 d" 1 (A), 0.48 d _ 1 (B), and 0.24 d" 1 (C) d i l u t i o n r a t e s . C e l l d i v i s i o n p l o t t e d a g a i n s t average time between sampling, dashed h o r i z o n t a l l i n e i n d i c a t e s d i l u t i o n r a t e i n h" 1. Dashed v e r t i c a l l i n e i n d i c a t e s onset of dark p e r i o d denoted by dark bar 161 F i g u r e 4.6. Mean c e l l volume of d u p l i c a t e n i t r a t e - l i m i t e d c y c l o s t a t s of Micromonas pusilla grown i n a 14h:10h L:D c y c l e a t 0.74 d _ 1 (A), 0.48 d" 1 (B), and 0.24 d" 1 (C) d i l u t i o n r a t e s p l o t t e d a g a i n s t e l a p s e d time s i n c e l i g h t s on. Dashed l i n e i n d i c a t e s onset of dark p e r i o d denoted by dark bar 162 F i g u r e 4.7. D i s s o l v e d n i t r a t e ( O, • ) and n i t r i t e ( A , • ) c o n c e n t r a t i o n s i n the medium of d u p l i c a t e n i t r a t e - l i m i t e d c y c l o s t a t s of Micromonas pusilla grown i n a 14h:10h L:D c y c l e a t 0.74 d - 1 (A), 0.48 d" 1 (B), and 0.24 d _ 1 (C) d i l u t i o n r a t e s p l o t t e d a g a i n s t e l a p s e d time s i n c e l i g h t s on. Dashed l i n e i n d i c a t e s onset of dark p e r i o d denoted by dark bar 165 F i g u r e 4.8. S p e c i f i c n i t r a t e uptake r a t e s of d u p l i c a t e n i t r a t e - l i m i t e d c y c l o s t a t s of Micromonas pusilla grown i n a 14h:10h L:D c y c l e a t (A): x v i i 0.74 d" 1 ( 0 . 0 3 1 h " 1 ) ; B: 0.48 d" 1 ( 0 . 0 2 0 h _ 1 ) ; a n d C: 0.24 d" 1 ( O . O l O h - 1 ) d i l u t i o n r a t e s . R a t e s p l o t t e d a g a i n s t a v e r a g e t i m e b e t w e e n s a m p l i n g . D a s h e d l i n e i n d i c a t e s o n s e t o f d a r k p e r i o d d e n o t e d by d a r k b a r 166 F i g u r e 4.9. I n t r a c e l l u l a r n i t r a t e c o n c e n t r a t i o n s o f d u p l i c a t e n i t r a t e - l i m i t e d c y c l o s t a t s o f Micromonas pusilla g r o w n i n a 1 4 h : 1 0 h L:D c y c l e a t 0.74 d" 1 ( A ) , 0.48 d" 1 ( B ) , a n d 0.24 d _ 1 (C) d i l u t i o n r a t e s p l o t t e d a g a i n s t e l a p s e d t i m e s i n c e l i g h t s o n . D a s h e d l i n e i n d i c a t e s o n s e t o f d a r k p e r i o d d e n o t e d b y d a r k b a r 169 F i g u r e 4.10. Maximum s p e c i f i c u p t a k e r a t e s ( h - 1 ) o f n i t r a t e ( A ) , u r e a ( O )/ a n d ammonium ( • ) d e t e r m i n e d i n 2 h i n c u b a t i o n s o f s a m p l e s f r o m n i t r a t e -l i m i t e d c y c l o s t a t c u l t u r e s o f Micromonas pusilla ( 1 4 h : 1 0 h L:D c y c l e ) g r o w n a t 0.74 d _ 1 ( A ) , 0.49 d _ 1 ( B ) , a n d 0.24 d _ 1 (C) d i l u t i o n r a t e s . S p e c i f i c r a t e s a r e p l o t t e d a g a i n s t a v e r a g e t i m e o f i n c u b a t i o n p e r i o d . D a s h e d l i n e i n d i c a t e s o n s e t o f d a r k p e r i o d d e n o t e d by d a r k b a r 172 F i g u r e 4 . 1 1 . N i t r o g e n s p e c i f i c u p t a k e r a t e s , d e t e r m i n e d o v e r 2 h, a f t e r s a t u r a t i n g e n r i c h m e n t o f 1 5NH 4 + (•) o r 1 5N0 3" (o) t o n i t r a t e - l i m i t e d c y c l o s t a t c u l t u r e s o f Micromonas pusilla ( 1 4 h : 1 0 h L:D c y c l e ) p r e v i o u s l y g r o w n a t 0.77 d" 1 ( A ) , 0.52 d" 1 ( B ) , and 0.24 d" 1 (C) d i l u t i o n r a t e s . U p t a k e r a t e s ( h _ 1 ) a r e p l o t t e d a g a i n s t i n c i d e n t PPFD, c u r v e d p l o t s a r e f i t t e d d i r e c t l y t o t h e M i c h a e l i s - M e n t e n e q u a t i o n b y c o m p u t e r programme ( s e e t e x t f o r d e t a i l s ) 176 F i g u r e A . l . S p e c i f i c g r o w t h r a t e (p) i n d ~ ^ a s a f u n c t i o n o f PPFD f o r M. pusilla g r o w n on N O 3 - . B a r s r e p r e s e n t ± 1 S.D. (n = 2 - 1 3 ) . E r r o r b a r s a r e s m a l l e r t h a n s y m b o l s w h e r e n o t v i s i b l e 228 F i g u r e A . 2 . G r o w t h c u r v e s o f d u p l i c a t e b a t c h c u l t u r e s o f M. pusilla grown on N O 3 - u n d e r s a t u r a t i n g PPFD. S e m i - l o g p l o t s o f r e l a t i v e i n v i v o f l u o r e s c e n c e ( O, • ) a n d c e l l c o n c e n t r a t i o n ( • , • ) v e r s u s t i m e 231 F i g u r e A . 3 . N i t r a t e u p t a k e by d u p l i c a t e b a t c h c u l t u r e s o f Micromonas pusilla. A. D e c r e a s e i n d i s s o l v e d N 0 o ~ + N02~ c o n c e n t r a t i o n i n t h e c u l t u r e medium. B. A c c u m u l a t i o n o f p a r t i c u l a t e o r g a n i c n i t r o g e n . C. R a t i o o f N O 3 - u p t a k e r a t e c a l c u l a t e d f r o m PON a c c u m u l a t i o n t o r a t e c a l c u l a t e d f r o m N 0 3 ~ + N O 2 - d i s a p p e a r a n c e f r o m x v i i i t h e medium. N i t r o g e n c o n c e n t r a t i o n s a r e p l o t t e d a g a i n s t e l a p s e d t i m e m e a s u r e d a f t e r c u l t u r e i n i t i a t i o n ; u p t a k e r a t e r a t i o s a r e p l o t t e d a g a i n s t a v e r a g e e l a p s e d t i m e b e t w e e n s u c c e s s i v e s a m p l i n g p e r i o d s 2 34 F i g u r e A.4. D i s s o l v e d N O 3 - and NC^ - c o n c e n t r a t i o n i n t h e c u l t u r e medium, d u r i n g b a t c h g r o w t h o f M. pusilla, p l o t t e d a g a i n s t t i m e o f s a m p l i n g . . . 237 ACKNOWLEDGEMENTS I g r a t e f u l l y a c k n o w l e d g e t h e g u i d a n c e a n d s u p p o r t o f my r e s e a r c h s u p e r v i s o r , D r . P . J . H a r r i s o n . H i s e n c o u r a g e m e n t t h r o u g h o u t a l l p h a s e s o f my t e n u r e a t U.B.C. h a s p r o v i d e d a s t i m u l a t i n g a t m o s p h e r e t h a t was c o n d u c t i v e t o r e s e a r c h a n d p e r s o n a l l y r e w a r d i n g . T h a n k s a r e a l s o e x t e n d e d t o t h e o t h e r members o f my s u p e r v i s o r y c o m m i t t e e , D r s . K.L. Denman, T.R. P a r s o n s a n d F.J.R. T a y l o r , f o r t h e i r c o n t r i b u t i o n s t o t h i s w o r k . The r e s e a r c h p r e s e n t e d i n t h i s d i s s e r t a t i o n h a s b e n e f i t t e d f r o m d i s c u s s i o n s a n d t e c h n i c a l a d v i c e f r o m my c o l l e a g u e s : D r s . Q. D o r t c h , G . J . D o u c e t t e , J.A. P a r s l o w , N.M. P r i c e , a n d C A . S u t t l e , a nd a l s o P . J . C l i f f o r d , L . J . J a c k s o n , M.E. L e v a s s e u r a n d P.A. Thompson. I am v e r y g r a t e f u l t o N.M. P r i c e f o r h i s c o n t r i b u t i o n s t o t h e S t r a i t o f G e o r g i a e x p e r i m e n t s p r e s e n t e d i n C h a p t e r s 1 a n d 2 a n d J.R. F o r b e s ( Ocean E c o l o g y , I.O.S.) f o r h i s a s s i s t a n c e a t s e a a n d t h e a c q u i s i t i o n o f p h y s i c a l ( t e m p e r a t u r e , s a l i n i t y ) a n d b i o l o g i c a l ( c h l o r o p h y l l a , p h y t o p l a n k t o n s p e c i e s c o m p o s i t i o n ) d a t a f o r e x p e r i m e n t s d e s c r i b e d i n C h a p t e r 1. A n a l y s t s f o r p h y t o p l a n k t o n i d e n t i f i c a t i o n a nd e n u m e r a t i o n w e r e R. W a t e r s a n d G . J . D o u c e t t e . A t s e a I a p p r e c i a t e d t h e a s s i s t a n c e by t h e o f f i c e r s a n d c r e w o f t h e C.S.S. P a r i z e a u a n d C.S.S. V e c t o r , t h e r e s e a r c h s t a f f o f Ocean E c o l o g y ( I . O . S . ) a n d f e l l o w s t u d e n t s i n t h e P.J.H. l a b o r a t o r y . F i n a n c i a l s u p p o r t was p r o v i d e d by a G r a d u a t e R e s e a r c h , E n g i n e e r i n g a n d T e c h n o l o g y (GREAT) s c h o l a r s h i p f r o m t h e p r o v i n c e o f B r i t i s h C o l u m b i a , a U n i v e r s i t y G r a d u a t e F e l l o w s h i p f r o m U . B . C , a n d s c h o l a r s h i p s f r o m C h e v r o n C a n a d a L t d . a n d S h e l l C a n a d a L t d . The r e s e a r c h was s u p p o r t e d by g r a n t s a w a r d e d t o D r . P . J . H a r r i s o n f r o m t h e N a t u r a l S c i e n c e s a n d E n g i n e e r i n g R e s e a r c h C o u n c i l o f C a n a d a a n d t h e C a n a d i a n F i s h e r i e s a n d O c e a n s S u b v e n t i o n programme. 1 INTRODUCTION Overview The g r o w t h o f m a r i n e p h y t o p l a n k t o n i s d i r e c t l y d e p e n d e n t on t h e i r a b i l i t y t o a s s i m i l a t e n u t r i e n t s a n d p h o t o s y n t h e s i z e i n a n e n v i r o n m e n t i n w h i c h n u t r i e n t s a n d / o r l i g h t a r e n o t n e c e s s a r i l y o p t i m a l f o r g r o w t h . Of t h e t h r e e m a j o r i n o r g a n i c n u t r i e n t s u t i l i z e d b y m a r i n e p h y t o p l a n k t o n ( n i t r o g e n , p h o s p h o r u s , s i l i c o n ) n i t r o g e n i s t h e n u t r i e n t whose s u p p l y i s most l i k e l y t o l i m i t p h y t o p l a n k t o n g r o w t h i n b o t h c o a s t a l ( e . g . , R y t h e r a n d D u n s t a n , 1971) a n d o c e a n i c w a t e r s ( e . g . , Thomas, 1966, 1969; Goldman e t a l . , 1 9 7 9 ) . P h o s p h o r u s , a l t h o u g h o f t e n f o u n d i n l o w c o n c e n t r a t i o n s , i s r e c y c l e d r a p i d l y a n d t h e r e f o r e i t i s l e s s l i k e l y t o l i m i t m a r i n e p r o d u c t i v i t y ( P e r r y a n d E p p l e y , 1981; S m i t h , 1 9 8 4 ) . S i l i c o n i s g e n e r a l l y r e c y c l e d s l o w l y ( e . g . , N e l s o n a n d G o e r i n g , 1 9 7 7 ) , h o w e v e r , i t i s n o t r e q u i r e d by a l l m a r i n e p h y t o p l a n k t o n . When i t i s l i m i t i n g , t h e g r o w t h o f d i a t o m s i s r e d u c e d a n d t h e r e f o r e t h e m a j o r e f f e c t i s on s p e c i e s c o m p o s i t i o n ( W a l s h , 1 9 8 1 ) . Of t h e n i t r o g e n (N) s o u r c e s i n t h e s e a t h a t a r e w e l l -c h a r a c t e r i z e d ( i . e . n o t i n c l u d i n g 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), ammonium ( N H 4 + ) , n i t r a t e ( N 0 3 ~ ) , n i t r i t e (NO2"") a n d u r e a (CO(NH2)2) a r e t h e most a b u n d a n t a n d , c o n s e q u e n t l y , most r e l e v a n t r e s e a r c h h a s c o n c e n t r a t e d on t h e i r u t i l i z a t i o n b y p h y t o p l a n k t o n . I n 1967, D u g d a l e a nd G o e r i n g i n t r o d u c e d a c o n c e p t u a l m o d e l t h a t d i s t i n g u i s h e d t h e r e l a t i v e i m p o r t a n c e o f t h e v a r i o u s s o u r c e s o f 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 f o r p h y t o p l a n k t o n g r o w t h . P r i m a r y p r o d u c t i o n s u p p o r t e d by 2 a l l o c h t h o n o u s N s o u r c e s , p r i n c i p a l l y n i t r a t e m i x e d i n t o s u r f a c e w a t e r s f r o m d e e p o c e a n r e s e r v e s , a n d s e c o n d a r i l y , N 2 -f i x a t i o n , r i v e r i n e i n f l o w a nd p r e c i p i t a t i o n , was t e r m e d "new p r o d u c t i o n " ; p r o d u c t i o n r e s u l t i n g f r o m a u t o c h t h o n o u s N s o u r c e s , s u c h a s u r e a a n d ammonium f r o m a n i m a l e x c r e t i o n a n d m i c r o b i a l r e m i n e r a l i z a t i o n , was t e r m e d " r e g e n e r a t e d " p r o d u c t i o n . Numerous f i e l d a n d l a b o r a t o r y s t u d i e s h a v e d o c u m e n t e d t h e p r e f e r e n c e o f p h y t o p l a n k t o n f o r t h e more r e d u c e d f o r m s o f N (ammonium and u r e a ) r e l a t i v e t o t h e more o x i d i z e d f o r m s ( n i t r a t e a n d n i t r i t e ) ( e . g . , D u g d a l e a n d G o e r i n g , 1967; E p p l e y e t a l . , 1973; M c C a r t h y e t a l . , 1977, 1982) p r e s u m a b l y b e c a u s e r e d u c e d f o r m s r e q u i r e l e s s e n e r g y t o a s s i m i l a t e . N e v e r t h e l e s s t h e o x i d i z e d f o r m s , p a r t i c u l a r l y N O 3 - , may be q u a n t i t a t i v e l y i m p o r t a n t N s o u r c e s f o r p h y t o p l a n k t o n , ( e . g . , C a r p e n t e r and Dunham, 1985; P r o b y n , 1985; C o c h l a n , 1986; P i a t t e t a l . , 1 9 8 9 ) . I n t h e s u r f a c e w a t e r s o f c e n t r a l o c e a n i c g y r e s , n i t r o g e n c o n c e n t r a t i o n s a r e c o n s i s t e n t l y n e a r o r b e l o w c u r r e n t l i m i t s o f d e t e c t i o n ( g e n e r a l l y N O 3 " : 50 n g - a t N - L - 1 ; N H 4 + : 30 n g - a t N - L - 1 ; u r e a : 50 n g - a t N - L - 1 ) ( M c C a r t h y , 1980; Raymont, 1980; G a r s i d e , 1985; P r i c e a nd H a r r i s o n , 1987) a n d show no a p p a r e n t s e a s o n a l p a t t e r n ( M c C a r t h y , 1980; S h a r p , 1 9 8 3 ) . C o n c e n t r a t i o n s a r e i n v a r i a b l y h i g h e r a t d e p t h , p a r t i c u l a r l y f o r N O 3 - . N i t r a t e , t h e most a b u n d a n t f o r m o f n i t r o g e n ( e x c e p t f o r N 2 ) i n c o a s t a l r e g i o n s ( S h a r p , 1 9 8 3 ) , e x h i b i t s d i s t i n c t i v e s e a s o n a l t r e n d s a n d i s t h e most - i m p o r t a n t N s o u r c e i n h i g h l y p r o d u c t i v e a r e a s o f t h e w o r l d ' s o c e a n ( e . g . , E p p l e y a n d 3 P e t e r s o n , 1979; E p p l e y , 1 9 8 1; H a r r i s o n e t a l . , 1 9 8 7 ) . S u r f a c e n i t r a t e c o n c e n t r a t i o n s o f c o a s t a l w a t e r s a r e u s u a l l y e l e v a t e d d u r i n g t h e w i n t e r (> 20 uq-at N ' L - ^ ) a n d , f o l l o w i n g s t r a t i f i c a t i o n d u r i n g t h e s p r i n g a n d s u b s e q u e n t u t i l i z a t i o n by p h y t o p l a n k t o n , a r e g r a d u a l l y d e c r e a s e d t o d e t e c t i o n l i m i t s . A t t h i s p o i n t p h y t o p l a n k t o n g r o w t h i s s u p p o r t e d p r i m a r i l y b y r e g e n e r a t e d N, a s i n o l i g o t r o p h i c , o c e a n i c g y r e s , ( e . g . , M c C a r t h y e t a l . , 1977; G l i b e r t e t a l . , 1982b; C o c h l a n , 1 9 8 6 ) . N i t r o g e n u p t a k e a n d r e g e n e r a t i o n a r e e n v i s a g e d t o be t i g h t l y c o u p l e d ( G o l d m a n , 1984) and t h e o x i d i z e d N f o r m s a r e o f l i t t l e o r no i m p o r t a n c e . Forms, o f r e g e n e r a t e d N i n c o a s t a l r e g i o n s v a r y i n c o n c e n t r a t i o n b u t g e n e r a l l y do n o t e x c e e d 5 / j g - a t N * L - ^ a n d a r e f r e q u e n t l y b e l o w 0.5 uq-at N-L-"*" ( M c C a r t h y , 1980; S h a r p , 1983; A n t i a e t a l . , i n p r e s s ) . The u t i l i z a t i o n o f n i t r o g e n by p h y t o p l a n k t o n c a n be d i v i d e d i n t o two s t e p s ; t h e f i r s t s t e p , t e r m e d " u p t a k e " , d e s c r i b e s t h e a c t u a l t r a n s p o r t o f t h e p a r t i c u l a r f o r m o f N a c r o s s t h e c e l l ' s p l a s m a l e m m a , a n d t h e s e c o n d s t e p , t e r m e d " a s s i m i l a t i o n " , r e f e r s t o t h e s e q u e n c e o f m e t a b o l i c e v e n t s w i t h i n t h e c e l l i n w h i c h t h e i n o r g a n i c N i o n s a r e r e d u c e d t o N H 4 + ( i n t h e c a s e o f N O 3 - and NC^ -) and i n c o r p o r a t e d p r i m a r i l y i n t o a m i n o a c i d s a n d p r o t e i n s ( W h e e l e r , 1983; L o b b a n e t a l . , 1 9 8 5 ) . The u t i l i z a t i o n o f n i t r o g e n o u s n u t r i e n t s b y m a r i n e p h y t o p l a n k t o n i s i n f l u e n c e d by a number o f f a c t o r s , i n c l u d i n g t h e a m b i e n t N c o n c e n t r a t i o n , t h e r e l a t i v e a b u n d a n c e o f d i f f e r e n t N f o r m s , t h e p h y s i o l o g i c a l s t a t u s o f t h e p h y t o p l a n k t o n , t h e a v a i l a b i l i t y o f l i g h t a n d t e m p e r a t u r e . 4 U p t a k e i n t e r a c t i o n s b e t w e e n i n o r g a n i c N f o r m s h a v e b e e n t h e s u b j e c t o f many c u l t u r e a nd f i e l d s t u d i e s ( r e v i e w s by M c C a r t h y , 1981; S y r e t t , 1 9 81; U l l r i c h , 1987) w h i c h r e v e a l a r a n g e o f r e s p o n s e s w h i c h v a r y w i t h t h e p h y t o p l a n k t o n s p e c i e s a n d i t s n u t r i t i o n a l s t a t e . N i t r a t e u p t a k e h a s b e e n r e p o r t e d t o be i n h i b i t e d t o d i f f e r e n t d e g r e e s by NH 4 + r a n g i n g f r o m t o t a l s u p p r e s s i o n ( e . g . , S y r e t t a n d M o r r i s , 1963; M c C a r t h y a n d E p p l e y , 1972) t o s i m u l t a n e o u s a n d c o m p a r a b l e r a t e s o f N O 3 - a n d N H 4 + u p t a k e ( e . g . , Conway, 1977; M a e s t r i n i e t a l . , 1982, 1 9 8 6 ) . I n t e r a c t i o n s b e t w e e n u r e a and o t h e r N f o r m s h a v e a t t r a c t e d l e s s a t t e n t i o n p a r t l y b e c a u s e o n l y r e c e n t l y h a s t h e s i g n i f i c a n c e o f u r e a a s a s o u r c e o f N f o r t h e g r o w t h o f n a t u r a l p h y t o p l a n k t o n a s s e m b l a g e s b e e n g e n e r a l l y a c k n o w l e d g e d ( e . g . , M c C a r t h y , 1972,; Kaufman e t a l . , 1 983; K r i s t i a n s e n , 1983; H a r r i s o n e t a l . , 1985; T u r l e y , 1985, 1 9 8 6 ) . G e n e r a l l y , u r e a s u p p r e s s e s t h e u p t a k e o f N O 3 - , b u t a t a l o w e r l e v e l t h a n N H 4 + ( e . g . , G r a n t e t a l . , 1967; M o l l o y a n d S y r e t t , 1 9 8 8 b ) . The e f f e c t o f c e l l u l a r p h y s i o l o g i c a l s t a t e on N u p t a k e by p h y t o p l a n k t o n was f i r s t d e m o n s t r a t e d by S y r e t t ( 1 9 5 3) a n d H a r v e y (1953) who showed t h a t N H 4 + a n d N O 3 - u p t a k e by b a t c h c u l t u r e s o f " N - s t a r v e d " c e l l s was much more r a p i d t h a n by " n o r m a l " c e l l s t h a t w e r e N - r e p l e t e . More r e c e n t s t u d i e s h a v e a l s o shown t h a t N - s t a r v e d o r N - d e f i c i e n t p h y t o p l a n k t o n h a v e t h e a b i l i t y t o r a p i d l y t a k e up N H 4 + upon e x p o s u r e t o an e l e v a t e d c o n c e n t r a t i o n ( e . g . , Conway e t a l . , 197 6; Conway a n d H a r r i s o n , 1977; G l i b e r t a nd Goldman, 1 9 8 2 ) . I n c o n t r a s t , a f t e r a N O 3 - o r u r e a a d d i t i o n t o N - d e p l e t e c e l l s e n h a n c e d 5 u p t a k e may o r may n o t o c c u r ( e . g . , r e v i e w by C o l l o s , 1983; P r i c e a n d H a r r i s o n , 1 9 8 8 b ) . The i d e a t h a t t h e r a t e o f s t e a d y - s t a t e N u p t a k e by m a r i n e p h y t o p l a n k t o n c a n be d e s c r i b e d a s a h y p e r b o l i c f u n c t i o n o f t h e c o n c e n t r a t i o n o f t h e l i m i t i n g n u t r i e n t , s i m i l a r i n f o r m t o t h e M i c h a e l i s - M e n t e n e q u a t i o n f o r enzyme k i n e t i c s ( D u g d a l e 1967, E p p l e y a n d C o a t s w o r t h , 1968) h a s l e d t o numerous s t u d i e s w h i c h h a v e d e t e r m i n e d k i n e t i c p a r a m e t e r s f o r b o t h l a b o r a t o r y a n d n a t u r a l a s s e m b l a g e s o f p h y t o p l a n k t o n ( e . g . , r e v i e w s by M c C a r t h y , 1981; Goldman and G l i b e r t , 1 9 8 3 ) . I t a p p e a r s t h a t t h e h a l f - s a t u r a t i o n c o n c e n t r a t i o n s f o r u p t a k e a r e i n t h e r a n g e fi 7 o f 1 0 ~ D t o 1 0 - / M f o r m a r i n e p h y t o p l a n k t o n a n d t h a t s p e c i e s ( E p p l e y e t a l . , 1969; M a c l s a a c a n d D u g d a l e , 1969) a n d c l o n e s ( C a r p e n t e r a n d G u i l l a r d , 1971) w h i c h a r e commonly f o u n d i n e u t r o p h i c r e g i o n s show c o n s i s t e n t l y h i g h e r h a l f - s a t u r a t i o n c o n s t a n t s t h a n s p e c i e s o r c l o n e s i s o l a t e d f r o m o l i g o t r o p h i c w a t e r s . U n d e r N - l i m i t i n g c o n d i t i o n s , s p e c i e s w i t h l o w u p t a k e ( h a l f - s a t u r a t i o n ) c o n s t a n t s h o l d a c o m p e t i t i v e a d v a n t a g e o v e r s p e c i e s h a v i n g h i g h e r c o n s t a n t s . T h i s h a s b e e n u s e d t o e x p l a i n s p e c i e s d i s t r i b u t i o n i n r e l a t i o n t o N a v a i l a b i l i t y ( E p p l e y e t a l . , 1969; M a c l s a a c a n d D u g d a l e , 1 9 6 9 ) . L i g h t i n t e n s i t y a n d q u a l i t y e x h i b i t a w i d e r a n g e o f t e m p o r a l a n d s p a t i a l v a r i a t i o n . S e a s o n a l , l a t i t u d i n a l a n d p a r t i c u l a r l y d i e l ( d a y - n i g h t ) v a r i a t i o n s i n o v e r a l l i n t e n s i t y a r e v e r y p r o n o u n c e d . L i g h t a t t e n u a t e s e x p o n e n t i a l l y w i t h d e p t h , l i m i t i n g p h y t o p l a n k t o n g r o w t h t o t h e s u r f a c e w a t e r s ( e u p h o t i c z o n e ) o f t h e o c e a n . Many s t u d i e s h a v e shown t h a t N 6 u p t a k e i s r e l a t e d t o l i g h t i n t e n s i t y i n a h y p e r b o l i c f a s h i o n , s a t u r a t i o n o c c u r r i n g a t h i g h l i g h t i n t e n s i t i e s ( e . g . , H a t t o r i , 1962; G r a n t , 1967; E p p l e y a nd R o g e r s , 1 9 7 0 ) . I n n a t u r a l p h y t o p l a n k t o n c o m m u n i t i e s t h e d e p e n d e n c e o f N u p t a k e u p o n l i g h t i n t e n s i t y h a s b e e n d e s c r i b e d b y a r e c t a n g u l a r h y p e r b o l a o f t h e M i c h a e l i s - M e n t e n f o r m u l a t i o n ( e . g . , M a c l s a a c a n d D u g d a l e , 1972; F i s h e r e t a l . , 1 9 8 2 ) . S i n c e n e i t h e r membrane t r a n s p o r t n o r r e d u c t i o n o f o x i d i z e d N f o r m s r e q u i r e s l i g h t p e r s e , t h e d e p e n d e n c e o f N u p t a k e on l i g h t i s l i k e l y a n i n d i r e c t one ( S y r e t t , 1 9 8 1 ) . P r o d u c t i o n o f c o f a c t o r s f o r N O 3 - a n d NO2"" r e d u c t i o n a n d ATP f o r membrane t r a n s p o r t ( F a l k o w s k i , 1975a) a n d t h e a s s i m i l a t i o n o f N H 4 + and u r e a d e p e n d on p h o t o s y n t h e s i s ( S y r e t t , 1981) w h i c h may a c c o u n t f o r t h e o b s e r v a t i o n s o f l i g h t d e p e n d e n c e o f N u p t a k e . I n m ost e n v i r o n m e n t s , l i g h t i n t e n s i t y e x h i b i t s e x t r e m e v a r i a t i o n , i n a p e r i o d i c f a s h i o n e v e r y d a y . D i e l p h y s i o l o g i c a l r h y t h m s , c o u p l e d t o f l u c t u a t i o n s i n l i g h t i n t e n s i t y , h a v e b e e n d e t e c t e d i n c u l t u r e s a n d n a t u r a l p h y t o p l a n k t o n a s s e m b l a g e s a nd i n c l u d e numerous p r o c e s s e s ( r e v i e w by S o u r n i a , 1 9 7 4 ) , most n o t a b l y p h o t o s y n t h e s i s ( e . g . , D o t y a n d O g u r i , 1957; M a c C a u l l a nd P i a t t , 1 9 7 7 ) . The t e r m " d i e l " , u s e d a l m o s t e x c l u s i v e l y by o c e a n o g r a p h e r s , w i l l be u s e d t h r o u g h o u t t h i s d i s s e r t a t i o n t o d e s c r i b e any r h y t h m , whose p e r i o d i s a b o u t 24 h, o b s e r v e d i n n a t u r a l c o n d i t i o n s . C i r c a d i a n h a s t h e same m e a n i n g a s d i e l , b u t i s u s e d t o d e s c r i b e s u c h r h y t h m s w h i c h p e r s i s t u n d e r c o n s t a n t e n v i r o n m e n t a l c o n d i t i o n s ( i . e . e n d o g e n o u s c o n t r o l ) . The t e r m 7 " d i u r n a l " , b e i n g t h e o p p o s i t e o f " n o c t u r n a l " c a n n o t a p p l y t o a 24 h c y c l e , b u t o n l y t o an e v e n t w h i c h o c c u r s b e t w e e n s u n r i s e a n d s u n s e t ( S o u r n i a , 1974) a n d w i l l be u s e d t o d e s c r i b e r h y t h m s d u r i n g t h e d a y l i g h t h o u r s . E v i d e n c e o f t h e p e r i o d i c i t y o f N u p t a k e h a s b e e n r e p o r t e d i n c y c l o s t a t c u l t u r e s ( e . g . , E p p l e y e t a l . , 1971b; M a l o n e e t a l . , 1975) a n d n a t u r a l p h y t o p l a n k t o n c o m m u n i t i e s ( e . g . , E p p l e y e t a l . , 1970 , 19 7 1 a , M a c l s a a c , 1978; F i s h e r e t a l . , 1 9 8 2 ) . Maximum u p t a k e o c c u r s d u r i n g t h e d a y a n d minimum u p t a k e a t n i g h t . I n a n e a r l y s t u d y , G o e r i n g e t a l . , ( 1964) o b s e r v e d u p t a k e p e r i o d i c i t y o f NC^ - and N H 4 + by S a r g a s s o S e a p h y t o p l a n k t o n i n c u b a t e d u n d e r c o n s t a n t i l l u m i n a t i o n w h i c h s u g g e s t s t h a t t h e r h y t h m was c i r c a d i a n . A dampened a m p l i t u d e i n t h e d i e l p e r i o d i c i t y o f n i t r a t e u p t a k e h a s b e e n o b s e r v e d i n c u l t u r e a n d f i e l d s t u d i e s w i t h i n c r e a s e d N l i m i t a t i o n ( e . g . , M a l o n e e t a l . , 1975) o r N s t a r v a t i o n ( e . g . , H a r r i s o n , 1976; D o r t c h a n d M a s k e , 1 9 8 2 ) . Objectives The m a i n o b j e c t i v e s o f t h i s t h e s i s w e r e a s f o l l o w s : (1) To d e t e r m i n e t h e n i t r o g e n d y n a m i c s ( N O 3 - , N H 4 + , a n d u r e a ) o f n a t u r a l p h y t o p l a n k t o n a s s e m b l a g e s f r o m c o a s t a l a n d o c e a n i c w a t e r s o f f t h e c o a s t o f B r i t i s h C o l u m b i a . (2) To d e t e r m i n e i n l a b o r a t o r y s t u d i e s t h e n i t r o g e n ( N O 3 - , N H 4 + , a n d u r e a ) u p t a k e k i n e t i c s o f an e c o l o g i c a l l y i m p o r t a n t e u c a r y o t i c p i c o p l a n k t e r Micromonas pusilla, i s o l a t e d f r o m B r i t i s h C o l u m b i a n c o a s t a l w a t e r s . (3) To d e t e r m i n e i n t e r a c t i o n s b e t w e e n n i t r o g e n u p t a k e a n d 8 l i g h t t h r o u g h d i e l p e r i o d i c i t y a nd i r r a d i a n c e e x p e r i m e n t s i n t h e f i e l d a n d t h e l a b o r a t o r y . Thesis o u t l i n e The u n d e r l y i n g p r e m i s e o f t h i s s t u d y i s t h a t n i t r o g e n u p t a k e a n d a s s i m i l a t i o n a r e a f u n c t i o n o f b o t h t h e e x t e r n a l e n v i r o n m e n t a n d t h e p h y s i o l o g i c a l s t a t e o f t h e p h y t o p l a n k t e r a s a r e s u l t o f p r e v i o u s i n t e r a c t i o n w i t h t h e e n v i r o n m e n t . The f i r s t h a l f o f t h i s d i s s e r t a t i o n e x a m i n e s n i t r o g e n u p t a k e by n a t u r a l p h y t o p l a n k t o n a s s e m b l a g e s a s a f u n c t i o n o f l i g h t i n n e r i t i c a n d o c e a n i c e n v i r o n m e n t s . I n C h a p t e r 1, t i m e c o u r s e e x p e r i m e n t s o f n i t r o g e n u p t a k e a r e d e s c r i b e d f o r a v a r i e t y o f p h y t o p l a n k t o n c o m m u n i t i e s . T h e s e e x p e r i m e n t s w e r e d e s i g n e d t o d i s c e r n t h e e f f e c t ( s ) o f a m b i e n t n i t r o g e n c o n c e n t r a t i o n s on n i t r o g e n u p t a k e o v e r d a y / n i g h t c y c l e s . T h e s e e x p e r i m e n t s w e r e c o n d u c t e d i n a s s o c i a t i o n w i t h p h y t o p l a n k t o n n i t r o g e n o u s n u t r i t i o n s t u d i e s a n d c o n f i r m t h e e x i s t e n c e o f d i e l r h y t h m s a n d t h e n e c e s s i t y o f t i m e c o u r s e e x p e r i m e n t s t o a c c u r a t e l y e s t i m a t e d a i l y r a t e s o f u p t a k e f r o m h o u r l y i n c u b a t i o n s o r v i c e v e r s a . I n C h a p t e r 2 t h e e f f e c t s o f i r r a d i a n c e , d u r i n g t h e d a y t i m e , on t h e u p t a k e o f n i t r o g e n by p h y t o p l a n k t o n f r o m n i t r a t e - r e p l e t e f r o n t a l a nd n i t r a t e - d e p l e t e s t r a t i f i e d , c o a s t a l w a t e r s a r e d e s c r i b e d . T h e s e r e s u l t s p r o v i d e u n i q u e i n f o r m a t i o n r e g a r d i n g u p t a k e o f o x i d i z e d a n d r e d u c e d N f o r m s a s a f u n c t i o n o f i r r a d i a n c e i n two h i g h l y c o n t r a s t i n g e n v i r o n m e n t s . R e c e n t w o r k s h o w i n g t h e u b i q u i t y o f p i c o p l a n k t o n (0.2 - < 2.0 um, S i e b u r t h e t a l . , 1978) ( e . g . , G i e s k e s e t a l . , 9 1979; W a t e r b u r y e t a l . , 1979; J o h n s o n a n d S i e b u r t h , 1982; L i e t a l . , 1983; P i a t t e t a l . , 1983) and t h e i r i m p o r t a n c e a s p h o t o a u t o t r o p h s , p a r t i c u l a r l y i n o l i g o t r o p h i c , o c e a n i c r e g i o n s ( e . g . , L i e t a l . , 1983; T a k a h a s h i a n d B i e n f a n g , 1983; r e v i e w s by J o i n t , 1986; S t o c k n e r a n d A n t i a , 1986) p r o m p t e d my c h o i c e o f a p i c o p l a n k t e r t o e x a m i n e t h e e f f e c t s o f l i g h t on n i t r o g e n u t i l i z a t i o n u n d e r c o n t r o l l e d l a b o r a t o r y c o n d i t i o n s . I n C h a p t e r 3, e x p e r i m e n t s w e r e c o n d u c t e d w i t h b a t c h c u l t u r e s o f t h e e u c a r y o t i c , p i c o p l a n k t e r Micromonas p u s i l l a , a n d d e s i g n e d t o e x a m i n e n i t r o g e n u p t a k e k i n e t i c s , t h e e f f e c t s o f u r e a a n d NH^ + on N O 3 - u p t a k e , a n d t h e t r a n s i e n t u p t a k e r e s p o n s e ( s ) t o N O 3 - s t a r v a t i o n . R e q u i s i t e i n f o r m a t i o n was o b t a i n e d f o r s u b s e q u e n t d i e l s t u d i e s . T h i s i s t h e f i r s t s t u d y o f p i c o p l a n k t o n n i t r o g e n o u s n u t r i t i o n b e s i d e s t h o s e e m p l o y i n g 1 c N t r a c e r s d u r x n g s i z e - f r a c t i o n a t i o n o f n a t u r a l c o m m u n i t i e s ( e . g . , P r o b y n , 1985; H a r r i s o n a n d Wood, 1988) C o n t i n u o u s c u l t u r e s o f M. p u s i l l a grown on a 14:10 l i g h t : d a r k c y c l e ( i . e . c y c l o s t a t ) w e r e u s e d i n t h e e x p e r i m e n t s d e s c r i b e d i n C h a p t e r 4. T h e s e e x p e r i m e n t s w e r e d e s i g n e d t o e x a m i n e t h e e f f e c t o f NC^ - l i m i t a t i o n on d i e l p e r i o d i c i t y o f N u t i l i z a t i o n . I n s i t u N O 3 - u p t a k e r a t e s , p o t e n t i a l r a t e s o f o x i d i z e d a n d r e d u c e d N f o r m s d u r i n g t h e d a y / n i g h t c y c l e , a n d N u p t a k e a s a f u n c t i o n o f i r r a d i a n c e w e r e d e t e r m i n e d . Experimental organism Micromonas p u s i l l a ( B u t c h e r ) M a n t o n e t P a r k e (1960) ( b a s i o n y m : Chromulina p u s i l l a , B u t c h e r , 1952) i s a m i n u t e ( l e n g t h 1 t o 2 um, w i d t h 0.75 t o 1 urn) n a k e d , u n i c e l l u l a r , 10 p h o t o s y n t h e t i c f l a g e l l a t e . Micromonas pusilla i s u s u a l l y c o n s i d e r e d t o be a n a n o m a l o u s member o f t h e P r a s i n o p h y c e a e ( M a n t o n , 1959; M a n t o n and P a r k e , 1960) a n d l a c k s a c e l l w a l l . Micromonas pusilla i s u b i q u i t o u s , o c c u r r i n g i n c o a s t a l a n d o c e a n i c s a m p l e s o f t r o p i c a l , t e m p e r a t e a n d a r c t i c w a t e r s a n d o f t e n a c h i e v i n g n u m e r i c a l d o m i n a n c e ( e . g . , T h r o n d s e n , 1976; J o h n s o n a n d S i e b u r t h , 1982; T a y l o r a n d W a t e r s , 1982; H a l l e g r a e f f , 1983; E s t e p e t a l . , 1984; H a l l e g r a e f f a n d J e f f r e y , 1 9 8 4 ) . I t h a s b e e n r e p o r t e d d e e p e r i n t h e w a t e r c o l u m n t h a n f l a g e l l a t e s i n g e n e r a l , o f t e n w e l l b e l o w t h e e u p h o t i c z o n e ( e . g . , M a n t o n a n d P a r k e , 1960; T h r o n d s e n , 1 9 7 6 ) . Micromonas pusilla i s f r e q u e n t l y e n c o u n t e r e d i n B r i t i s h 7 — 1 C o l u m b i a n c o a s t a l w a t e r s ( e . g . > 2.5*10 c e l l s * L i n J e r v i s I n l e t , J u l y 1977 T a y l o r a nd W a t e r s , 1982; 0.1 - 2 . 2 - 1 0 7 c e l l s « L - 1 i n F r a s e r R i v e r p l u m e , J u l y 1987, C l i f f o r d e t a l . , 1 9 8 9 ) ; s i m i l a r c o n c e n t r a t i o n s a r e o f t e n r e p o r t e d i n N o r w e g i a n w a t e r s ( e . g . , T h r o n d s o n , 1976 a n d r e f e r e n c e s t h e r e i n ) . I t s t o l e r a n c e o f a w i d e r a n g e o f t e m p e r a t u r e a n d s a l i n i t y ( T h r o n d s e n , 1976) o r t h e a b i l i t y t o f o r m s t r a i n s a d a p t e d t o d i f f e r e n t e n v i r o n m e n t a l r e g i m e s , may c o n t r i b u t e t o t h e s u c c e s s o f t h i s p i c o p l a n k t e r i n t h e w o r l d ' s o c e a n s . The u b i q u i t o u s n a t u r e a n d n u m e r i c a l i m p o r t a n c e o f M. pusilla e n h a n c e s t h e p o t e n t i a l f o r e c o l o g i c a l l y r e l e v a n t e x t r a p o l a t i o n o f my l a b o r a t o r y d a t a t o n a t u r a l f i e l d p o p u l a t i o n s . The c u l t u r e u s e d h e r e i n (NEPCC 2 9 - 1 , N o r t h e a s t P a c i f i c C u l t u r e C o l l e c t i o n , D e p t . o f O c e a n o g r a p h y , U n i v e r s i t y o f B r i t i s h C o l u m b i a ) was i s o l a t e d f r o m E n g l i s h B a y , B.C. by R. W a t e r s i n J a n u a r y , 1971 a n d s u b s e q u e n t l y m a i n t a i n e d i n e n r i c h e d , n a t u r a l s e a w a t e r a t 16°C on a 14:10 L:D c y c l e 12 CHAPTER ONE EFFECTS OF D I E L P E R I O D I C I T Y ON NITROGEN UPTAKE BY NATURAL ASSEMBLAGES OF PHYTOPLANKTON INTRODUCTION D a y - n i g h t ( d i e l ) c y c l e s o f b i o l o g i c a l a n d r e l a t e d p a r a m e t e r s i n t h e o c e a n a r e o f t e n t h e m a n i f e s t a t i o n o f t h e e f f e c t s o f s u n l i g h t on b i o l o g i c a l p r o c e s s e s . P e r i o d i c i t y i n b o t h p h o t o s y n t h e t i c c a p a c i t y a n d i n s i t u p h o t o s y n t h e s i s i s t h e most o b v i o u s d i e l c y c l e ( e . g . , S o u r n i a , 1974) a n d b o t h d i e l ( a n d d i u r n a l ) r h y t h m s h a v e b e e n r e p o r t e d f o r many y e a r s ( e . g . , D o t y a n d O g u r i , 1957; V e r d u i n , 1 9 5 7 ) . D i e l f l u c t u a t i o n s i n d i s s o l v e d i n o r g a n i c n u t r i e n t c o n c e n t r a t i o n h a v e b e e n r e p o r t e d i n n a t u r a l p o p u l a t i o n s ( e . g . , L o r e n z e n , 1963; B e e r s a n d K e l l y , 1965) w i t h a c o n c e n t r a t i o n d e c l i n e g e n e r a l l y a s c r i b e d t o t h e a s s i m i l a t o r y a c t i v i t i e s o f p h y t o p l a n k t o n ( a n d b a c t e r i a ) a n d r e g e n e r a t i v e i n c r e a s e s f r o m z o o p l a n k t o n a n d h e t e r o t r o p h i c r e m i n e r a l i z a t i o n . G o e r i n g e t a l . (1964) f i r s t d e m o n s t r a t e d m a r k e d d i e l c y c l e s i n t h e p o t e n t i a l u p t a k e o f N O 3 - a n d N H 4 + by p h y t o p l a n k t o n i n t h e N - d e p l e t e d w a t e r s o f t h e S a r g a s s o S e a . S i n c e t h e n , numerous a c c o u n t s o f n i t r o g e n u p t a k e p e r i o d i c i t y i n i n s i t u ( e . g . , M a c l s a a c , 1 9 7 8 ) , s h i p b o a r d ( e . g . , E p p l e y e t a l . , 1 9 7 1 a ; C o l l o s a n d S l a w y k , 1976) and N - l i m i t e d c y c l o s t a t c u l t u r e s ( e . g . , E p p l e y e t a l . , 1971b; M a l o n e e t a l . , 1975) o f m a r i n e p h y t o p l a n k t o n h a v e shown m a x i m a l u p t a k e d u r i n g t h e d a y l i g h t h o u r s a nd m i n i m a l u p t a k e d u r i n g t h e n i g h t . D a r k u p t a k e o f n i t r o g e n h a s g e n e r a l l y b e e n t h o u g h t t o be a r e s p o n s e t o N l i m i t a t i o n ( e . g . , S y r e t t , 1 9 8 1 ) , d a m p e n i n g d i e l p e r i o d i c i t y by a r e l a t i v e e n h a n c e m e n t o f d a r k u p t a k e c a p a c i t y . 13 C u l t u r e s t u d i e s h a v e d e m o n s t r a t e d t h a t t h e p r e c o n d i t i o n i n g N s u b s t r a t e a f f e c t s t h e u p t a k e r e s p o n s e o f p h y t o p l a n k t o n t o e n r i c h m e n t s o f d i f f e r e n t N s u b s t r a t e s ( e . g . , H o r r i g a n a n d M c C a r t h y , 1 9 8 1 , 1982 ; D o r t c h a n d Conway, 1 9 8 4 ) . A d d i t i o n a l l y , i n N - s t a r v e d p h y t o p l a n k t o n t h e a b i l i t y t o t a k e up N O 3 - may be l o s t a n d must o f t e n be i n d u c e d ( D o r t c h e t a l . , 1982; r e v i e w by C o l l o s , 1983; P a r s l o w e t a l . , 1 9 8 4 b ) . I n i t i a l N H 4 + u p t a k e r a t e s a r e , h o w e v e r , o f t e n e n h a n c e d u p o n e x p o s u r e t o a n e l e v a t e d N H 4 + c o n c e n t r a t i o n i n c u l t u r e ( e . g . , Conway e t a l . , 1976; Conway a n d H a r r i s o n , 1977; Goldman a n d G l i b e r t , 1982; P a r s l o w e t a l . , 1984a,b) a n d n a t u r a l p h y t o p l a n k t o n c o m m u n i t i e s ( e . g . , G l i b e r t a n d Goldman, 1 9 81; W h e e l e r e t a l . , 1982; P r i s c u a n d P r i s c u , 1 9 8 4 ) . I n n i t r o g e n - d e p l e t e w a t e r s , m o s t o f t h e n i t r o g e n demands o f p h y t o p l a n k t o n a r e s u p p l i e d by ammonium a n d u r e a .from i n s i t u r e g e n e r a t i v e p r o c e s s e s , w h e r e a s i n N - r e p l e t e a r e a s , N compounds a p p e a r t o be u t i l i z e d a t r a t e s p r o p o r t i o n a l t o t h e i r a v a i l a b i l i t y ( e . g . , D u g d a l e a n d G o e r i n g , 1967; M c C a r t h y e t a l . , 1 9 7 7 ) . T h e s e o b s e r v a t i o n s s u g g e s t t h a t p h y t o p l a n k t o n c o m m u n i t i e s f r o m N - r e p l e t e a n d N - d e p l e t e w a t e r s may d i f f e r i n t h e i r r e s p o n s e t o p e r t u r b a t i o n s o f 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 , a n d u p t a k e r a t e s o f , 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 a n d t h a t t h e s e d i f f e r e n c e s may be r e f l e c t e d i n d i e l p a t t e r n s o f N u p t a k e . E x p e r i m e n t s i n t h e p r e s e n t s t u d y w e r e d e s i g n e d t o e x a m i n e p e r i o d i c i t y o f n i t r o g e n u p t a k e i n t h r e e c o n t r a s t i n g e n v i r o n m e n t s o f r e l a t i v e b i o m a s s a n d n i t r o g e n c o n c e n t r a t i o n s : t h e o c e a n i c s u b a r c t i c P a c i f i c w i t h l o w b i o m a s s a n d h i g h NC^ -14 c o n c e n t r a t i o n s ; a c o a s t a l u p w e l l i n g plume o f m o d e r a t e b i o m a s s a n d v a r y i n g N O 3 - c o n c e n t r a t i o n s ; a n d c o a s t a l i n s h o r e f r o n t a l a n d s t r a t i f i e d w a t e r s w i t h e l e v a t e d a n d d i m i n i s h e d N O 3 - a n d p h y t o p l a n k t o n c o n c e n t r a t i o n s , r e s p e c t i v e l y . Time c o u r s e e x p e r i m e n t s o f 24 h o r g r e a t e r i n d u r a t i o n , u t i l i z i n g e i t h e r c o n t a i n e d s a m p l e s o r r e p e a t e d s a m p l i n g o f a d r o g u e - t r a c k e d w a t e r p a r c e l , w e r e c o n d u c t e d w i t h N - l a b e l l e d s u b s t r a t e s t o r e l a t e p a t t e r n s o f u p t a k e t o c o n c o m i t a n t c h a n g e s i n i r r a d i a n c e a n d n u t r i e n t c o n c e n t r a t i o n . P r i o r t o t h i s s t u d y o u r k n o w l e d g e o f n i t r o g e n u p t a k e b y p h y t o p l a n k t o n on t h e w e s t c o a s t o f C a n a d a was l i m i t e d t o N O 3 -u p t a k e r a t e s d e t e r m i n e d i n t h r e e f j o r d s on t h e m a i n l a n d c o a s t o f B r i t i s h C o l u m b i a ( C o c h l a n e t a l . , 1 9 8 6 ) . The r a t e s o f NO3"", N H 4 + , a n d u r e a d e s c r i b e d i n t h i s c h a p t e r a r e t h e f i r s t e s t i m a t e s o f t h e u p t a k e c a p a b i l i t y o f n a t u r a l c o m m u n i t i e s o f p h y t o p l a n k t o n on t h e w e s t c o a s t o f C a n a d a . The p r e s e n t s t u d y was t h e f i r s t t o e m p l o y N m e t h o d o l o g y ( D u g d a l e a n d G o e r i n g , 1967) a n d t o r e p o r t a m b i e n t c o n c e n t r a t i o n s o f u r e a a n d f r e s h l y d e t e r m i n e d N H 4 + c o n c e n t r a t i o n s on t h e w e s t c o a s t o f C a n a d a a n d a d j a c e n t o f f s h o r e w a t e r s . 15 MATERIALS AND METHODS General Time c o u r s e e x p e r i m e n t s o f n i t r o g e n u p t a k e w e r e c o n d u c t e d d u r i n g t h r e e c r u i s e s a b o a r d t h e r e s e a r c h v e s s e l s C.S.S. V e c t o r a n d C.S.S. P a r i z e a u . The f i r s t c r u i s e (OE 8 4 - 0 2 ) , p a r t o f P r o j e c t SUPER ( S u b a r c t i c P a c i f i c E c o s y s t e m R e s e a r c h ) i n t h e N o r t h e a s t P a c i f i c O cean was c a r r i e d o u t f r o m 7 May t o 25 May, 1984, a n d one t i m e c o u r s e e x p e r i m e n t i s r e p o r t e d ( T C . l ) . D u r i n g t h e s e c o n d c r u i s e f r o m J u l y t o A u g u s t , 1984 i n t h e S t r a i t o f G e o r g i a , B.C. a c o a s t a l b a s i n on t h e w e s t c o a s t o f C a n a d a b e t w e e n t h e m a i n l a n d a n d V a n c o u v e r I s l a n d , t i m e c o u r s e s t u d i e s w e r e c o n d u c t e d i n f r o n t a l (TC.2) a n d s t r a t i f i e d w a t e r s ( T C . 3 ) . D i e l s t u d i e s on t h e t h i r d c r u i s e (O.E. 86-04) f r o m 18 A u g u s t t o 28 A u g u s t , 1986 w e r e c o n d u c t e d on t h e c o n t i n e n t a l s h e l f o f f t h e s o u t h w e s t c o a s t o f V a n c o u v e r I s l a n d (TC.4) a n d o f f s h o r e o f t h e s h e l f ( T C . 5 ) . S t a t i o n l o c a t i o n s f o r t h e e x p e r i m e n t s a r e p r e s e n t e d i n T a b l e 1.1 a n d shown i n F i g u r e 1.1. Sample collection D i s c r e t e s a m p l e s w e r e c o l l e c t e d f r o m d e p t h s , s e l e c t e d t o c o r r e s p o n d t o 50, 30, a n d 1% o f t h e s u r f a c e i r r a d i a n c e ( I Q ) , u s i n g e i t h e r 2 o r 5 L PVC N i s k i n b o t t l e s ( m o unted on a r o s e t t e o r w i r e ) a n d t h e n t r a n s f e r r e d i n t o d a r k e n e d 10 o r 20 L N a l g e n e c a r b o y s . V e r t i c a l p r o f i l e s o f t e m p e r a t u r e a n d s a l i n i t y w e r e o b t a i n e d f r o m c o n t i n u o u s p r o f i l e s , r u n p r i o r t o b o t t l e s a m p l i n g , u s i n g e i t h e r a I n t e r O c e a n m o d e l 514A CSTD ( c r u i s e 2) o r a G u i l d l i n e m o d e l 8701 d i g i t a l CTD ( c r u i s e s 1 & 16 F i g u r e 1.1. S t a t i o n l o c a t i o n s f o r t i m e c o u r s e e x p e r i m e n t s o f n i t r o g e n u p t a k e . (A) T C . l a t s t n F; TC.4 a t s t n 24; TC.5 a t s t n 8 5. (B) TC.2 a t s t n A5; TC.3 a t s t n T4. P a n e l B i s a n e n l a r g e m e n t o f t h e a r e a d e l i m i t e d b y d a s h e d l i n e s i n p a n e l A. 125° 30' I25°00' I24°30' I24°00' Table 1.1 I n i t i a l environmental co n d i t i o n s of seawater c o l l e c t e d f or time course experiments of nitrogen uptake by natural phytoplankton assemblages. Stations are F: Northeast P a c i f i c Ocean; A5: S t r a i t of Georgia - f r o n t a l ; T4: S t r a i t of Georgia - s t r a t i f i e d ; 24: upwelling plume o f f southwest coast of Vancouver Island; 85: offshore of western Canadian c o n t i n e n t a l s h e l f , (see F i g . 1.1). St a t i o n Time Course Date S t a r t i n g Sample Nitrogen cone. Chi a PON POC and Number time of depth N ° 3 ~ Urea NH + Location incubation (m) (PDT) (ug- -at . N-L" •S (pg-L - 1) {uq-at N-L ) (ug -at C-L - 1) F 49°5?. . 5' N TC. 1 16 May 1984 0245 8 11.99 0. 32* 0. 15* 0.59 1. ,87 19 . 5 145°14. . 6 'W A5 49°53. , 0 ' N TC.2 28 J u l y 1984 1000 0 0.27 4. 55 0. 60 2.12 7. .28 47 . 3 125°05. . 8 ' W T4 49°55, , 5 ' N TC.3 29 J u l y 1984 0800 0 0.19 <. 05 0. 33 0.39 3. .57 31. 4 124°55. .5 'W 24 49°25. . 0 *N TC.4 20 Aug. 1986 1100 2 12.94 2. 38 1. 67 10.35 7, .75 46 . 8 127°32. . 1 'W 5 11.74 0. 82 1. 78 11.74 7. .35 44. 5 14 11.58 1. 02 2 . 63 7.21 6 , . 15 37 . 5 85 48°16. . 7 ' N TC.5 25 Aug. 1986 1140 1 0.09 1. 95 <. 05 1.09 2, .11 17 . 3 128°18. . 9 ' W 28 7 .14 0. 63 1. 25 2.24 2 , .84 12 . 6 • C o l l e c t e d from separate b o t t l e casts at s i m i l a r s t a t i o n s . 18 3 ) . S i m u l t a n e o u s m e a s u r e m e n t s o f c h l o r o p h y l l f l u o r e s c e n c e w e r e d e t e r m i n e d w i t h a V a r i o s e n s I I I i n s i t u f l u o r o m e t e r ( c r u i s e s 1 & 3) o r o b t a i n e d f r o m pumped s a m p l e s a n d m e a s u r e d w i t h a T u r n e r m o d e l 111 f l u o r o m e t e r , e q u i p p e d w i t h a f l o w -t h r o u g h c e l l . 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 m o n i t o r e d c o n t i n u o u s l y w i t h a Lambda I n s t r u m e n t s L i C o r L I - 1 8 5 l i g h t m e t e r e q u i p p e d w i t h a L I - 1 9 0 S B S u r f a c e Quantum S e n s o r a n d r e c o r d e d w i t h a p r i n t i n g i n t e g r a t o r ( m o d e l L I - 5 5 0 D ) o r a c h a r t r e c o r d e r . S u b s u r f a c e i r r a d i a n c e s w e r e d e t e r m i n e d w i t h a L i C o r L I - 1 9 2 S U n d e r w a t e r Quantum S e n s o r (2n, c r u i s e 2) o r a L I - 1 9 3 S B S p h e r i c a l Quantum S e n s o r (4/T, c r u i s e s 1 & 3 ) . Analytical methods S u b s a m p l e s f o r n u t r i e n t a n a l y s e s w e r e f i l t e r e d t h r o u g h p r e w a s h e d , p r e c o m b u s t e d (460°C f o r 4 h) Whatman GF/F f i l t e r s , u s i n g an a c i d - w a s h e d s y r i n g e a n d 25 mm M i l l i p o r e S w i n n e x f i l t e r h o l d e r , i n t o a c i d - w a s h e d , p o l y p r o p y l e n e b o t t l e s . Ammonium ( N H 4 + ) c o n c e n t r a t i o n s w e r e a l w a y s d e t e r m i n e d i m m e d i a t e l y on b o a r d s h i p w i t h a T e c h n i c o n A u t o a n a l y z e r I I f o l l o w i n g t h e m e t h o d o f S l a w y k a n d M a c l s a a c ( 1 9 7 2 ) . S a m p l e s f o r p h o s p h a t e ( P O ^ - ^ ) a n d s i l i c a t e ( S i O ^ - ^ ) w e r e a l s o a n a l y z e d f r e s h f o l l o w i n g t h e a u t o m a t e d p r o c e d u r e s o f H a g e r e t a l . (1968) a n d A r m s t r o n g e t a l . ( 1 9 6 7 ) , r e s p e c t i v e l y . S a m p l e s f o r n i t r a t e ( N O 3 - + NC^ -) a n d u r e a w e r e e i t h e r s t o r e d f r o z e n (-20°C), k e p t d a r k a n d c o o l (< 12 h) o r a n a l y z e d i m m e d i a t e l y f o l l o w i n g t h e a u t o m a t e d p r o c e d u r e s o f Wood e t a l . (1967) a n d P r i c e a n d H a r r i s o n ( 1 9 8 7 ) , r e s p e c t i v e l y . 19 D u p l i c a t e s a m p l e s f o r c h l o r o p h y l l a ( C h i a) w e r e f i l t e r e d (< 125 mm Hg f i l t e r p r e s s u r e d i f f e r e n t i a l ) o n t o Whatman GF/F f i l t e r s w i t h c a . 0.5 m l 1% MgCG^ s u s p e n s i o n a d d e d p r i o r t o c o m p l e t i o n o f f i l t r a t i o n a n d e i t h e r a n a l y z e d i m m e d i a t e l y (< 1 h, c r u i s e 3) o r 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 ( c r u i s e s 1 & 2) u n t i l a n a l y s i s a s h o r e . C h l o r o p h y l l was e x t r a c t e d i n 90% a q u e o u s a c e t o n e a n d a n a l y z e d by i n v i t r o f l u o r o m e t r y ( P a r s o n s e t a l . , 1984) u s i n g a T u r n e r D e s i g n s m o d e l 10 f l u o r o m e t e r , c a l i b r a t e d w i t h c r y s t a l l i n e C h i a ( S i g m a C h e m i c a l C o . ) . D u p l i c a t e s a m p l e s f o r p a r t i c u l a t e o r g a n i c c a r b o n a n d n i t r o g e n (POC & PON) w e r e c o l l e c t e d on c o m b u s t e d Whatman GF/F f i l t e r s , s t o r e d s i m i l a r l y , a n d a n a l y z e d by t h e d r y c o m b u s t i o n m e t h o d o f S h a r p ( 1974) w i t h e i t h e r a P e r k i n - E l m e r m o d e l 240 o r a C a r l o E r b a m o d e l 1106 e l e m e n t a l a n a l y z e r . B o t h i n s t r u m e n t s w e r e c a l i b r a t e d w i t h a c e t a n i l i d e s t a n d a r d s . S a m p l e s f o r p h y t o p l a n k t o n s p e c i e s a n a l y s i s w e r e p r e s e r v e d i n a c i d L u g o l ' s s o l u t i o n ( P a r s o n s e t a l . , 1984) a n d s t o r e d i n t h e d a r k u n t i l a n a l y s i s . T en m l s u b s a m p l e s w e r e s e t t l e d (24 h) a n d c o u n t e d on a n i n v e r t e d m i c r o s c o p e . D u r i n g c r u i s e 1 t o t h e n o r t h e a s t P a c i f i c , p a i r e d s a m p l e s w e r e a l s o p r e s e r v e d i n a l k a l i n e L u g o l ' s s o l u t i o n ( T h r o n d s e n , 1978) f o r e n u m e r a t i o n o f c o c c o l i t h o p h o r i d s a n d t h e r e s u l t s r e p o r t e d a r e c o m b i n e d . S a m p l e s f o r N a n a l y s i s w e r e c o l l e c t e d on p r e c o m b u s t e d Whatman GF/F f i l t e r s , f o l d e d , p l a c e d i n t o a c i d - w a s h e d p e t r i -d i s h e s , a n d i m m e d i a t e l y f r o z e n f o r l a t e r i s o t o p i c a n a l y s e s . N i t r o g e n i n t h e p a r t i c u l a t e s a m p l e s was c o n v e r t e d t o d i n i t r o g e n g a s ( N 2 ) by t h e micro-Dumas d r y c o m b u s t i o n 20 t e c h n i q u e a s o u t l i n e d by C o c h l a n (1982) a n d L a R o c h e (1983) a n d 1 c s u b s e q u e n t l y a n a l y z e d f o r N e n r i c h m e n t w i t h a JASCO m o d e l N-150 e m i s s i o n s p e c t r o m e t e r ( F i e d l e r a nd P r o k s c h , 1 9 7 5 ) . G e n e r a l l y e a c h s a m p l e was s c a n n e d s i x t i m e s (minimum o f 3 t i m e s ) a n d t h e a v e r a g e ^ N / ^ N p e a k h e i g h t r a t i o was u s e d i n t h e c a l c u l a t i o n o f t h e p e r c e n t a g e ( s p e c i f i c a c t i v i t y ) i n t h e p a r t i c u l a t e m a t e r i a l . A u t o m a t i c s e l e c t i o n o f p e a k h e i g h t s d u r i n g s c a n s a n d i s o t o p i c r a t i o c a l c u l a t i o n s w e r e p e r f o r m e d u t i l i z i n g i n - h o u s e s o f t w a r e ( J o n e s , u n p u b l . d o c ) , w i t h an IBM c o m p a t i b l e PC, i n t e r f a c e d w i t h t h e s p e c t r o m e t e r . The e m i s s i o n s p e c t r o m e t e r was r o u t i n e l y c a l i b r a t e d w i t h a s e r i e s o f p u r e N 2 g a s s t a n d a r d s s u p p l i e d by JASCO o f known 1 5 N e n r i c h m e n t t o p r e p a r e c a l i b r a t i o n c u r v e s . The p r e c i s i o n o f t h e a n a l y t i c a l t e c h n i q u e s u s e d a r e p r e s e n t e d i n A p p e n d i x 6. Tracer experiments A l l n i t r o g e n u p t a k e e x p e r i m e n t s w e r e i n i t i a t e d w i t h i n 1 h o f c o l l e c t i o n ; w a t e r was t r a n s f e r r e d i n t o 500 m l 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 , o r d a r k e n e d w i t h b l a c k t a p e : d a r k b o t t l e s ) w i t h t e f l o n - l i n e d c a p s , and e n r i c h e d w i t h e i t h e r 1 5 N H 4 C 1 , N a 1 5 N 0 3 , o r C O ( 1 5 N H 2 ) 2 ( a l l 99 atom % 1 5 N ; K o r I s o t o p e s ) . S a m p l e s w e r e i n c u b a t e d on d e c k i n c l e a r P l e x i g l a s i n c u b a t o r s c o o l e d w i t h c o n t i n u o u s l y - f l o w i n g n e a r s u r f a c e (3 m) s e a w a t e r a n d c o v e r e d w i t h n e u t r a l d e n s i t y s c r e e n i n g t o s i m u l a t e t h e i n s i t u l i g h t r e g i m e a t e a c h s a m p l e d e p t h . A t s e l e c t e d t i m e i n t e r v a l s , r a n d o m l y s e l e c t e d b o t t l e s w e r e f i l t e r e d (< 125 mm H g ) , f o r c o l l e c t i o n o f p a r t i c u l a t e m a t t e r f o r i s o t o p i c a n a l y s i s o f "^N atom % e x c e s s , a n d d i s s o l v e d N 21 c o n c e n t r a t i o n s . L i g h t and d a r k b o t t l e u p t a k e r a t e s o f e a c h n i t r o g e n s u b s t r a t e w e r e m e a s u r e d o v e r t h e t i m e c o u r s e a n d t h e c o n t e n t s o f t h e s a m p l e b o t t l e s m i x e d h o u r l y . N i t r o g e n u p t a k e r a t e s w e r e c a l c u l a t e d a c c o r d i n g t o t h e e q u a t i o n s o f D u g d a l e a n d W i l k e r s o n ( 1 9 8 6) w h i c h a r e p r e s e n t e d i n A p p e n d i x 1. S p e c i f i c u p t a k e r a t e s , V ( n o r m a l i z e d t o PON) w e r e e s t i m a t e d u s i n g a c o n s t a n t s p e c i f i c u p t a k e m o d e l ( V c , e q u a t i o n 6 o f D u g d a l e a n d W i l k e r s o n ) . D i f f e r e n c e s b e t w e e n 1 R 1 R i n i t i a l a n d f i n a l N a t o m % e x c e s s ( N x s ) i n s u c c e s s s i v e s a m p l e s w e r e d i v i d e d by t h e l e n g t h o f t i m e i n t e r v a l t o o b t a i n a v e r a g e u p t a k e r a t e s d u r i n g e a c h i n c u b a t i o n p e r i o d . A b s o l u t e ( t r a n s p o r t ) r a t e s (were c a l c u l a t e d a s t h e p r o d u c t o f n o n -c o n s t a n t s p e c i f i c u p t a k e r a t e s V^, V j and t h e c o n c e n t r a t i o n s o f PON^, PONf, r e s p e c t i v e l y f o r s a m p l e s c o l l e c t e d a t t h e b e g i n n i n g ( i ) a n d e n d ( f ) o f i n c u b a t i o n p e r i o d . D i s a p p e a r a n c e u p t a k e r a t e s (V ) h a v e b e e n c a l c u l a t e d f r o m t h e c h a n g e i n c o n c e n t r a t i o n o f d i s s o l v e d n i t r o g e n p e r u n i t t i m e a n d , l i k e t h e n i t r o g e n - s p e c i f i c a n d a b s o l u t e N r a t e s , a r e r e p o r t e d f o r t h e t i m e i n t e r v a l s o v e r w h i c h t h e y h a v e b e e n c a l c u l a t e d . Experimental procedures O v e r t h e c o u r s e o f t h i s s t u d y , s u b t l e a n d m a j o r c h a n g e s i n e x p e r i m e n t a l d e s i g n w e r e e m p l o y e d , due t o b o t h e n v i r o n m e n t a l a n d l o g i s t i c a l f a c t o r s , i n t h e f i v e d i e l t i m e c o u r s e e x p e r i m e n t s r e p o r t e d . I n T C . l , c o n d u c t e d i n t h e N o r t h e a s t P a c i f i c O c e a n , w a t e r was c o l l e c t e d f r o m t h e 50% I Q l i g h t p e n e t r a t i o n d e p t h ( c a . 8 m) a n d p r e f i l t e r e d t h r o u g h 102 •p um N i t e x n y l o n n e t t i n g t o remove l a r g e r z o o p l a n k t o n b e f o r e 22 t r a c e r e x p e r i m e n t s w e r e i n i t i a t e d . S a m p l e s w e r e i n o c u l a t e d w i t h N a 1 5 N 0 3 a t a t r a c e r l e v e l ( u s u a l l y d e f i n e d a s £ 10% o f t h e a m b i e n t c o n c e n t r a t i o n ) t o b r i n g f i n a l t r a c e r c o n c e n t r a t i o n t o 1.0 uq-at N•L~^ p r i o r t o i n c u b a t i o n . T r i p l i c a t e s a m p l e s w e r e r e m o v e d a n d p a r t i c u l a t e s f i l t e r e d a t 3 h i n t e r v a l s f o r 24 h. N i n e h o u r s a f t e r i s o t o p e e n r i c h m e n t ( T Q = 0245 h PDT) t r i p l i c a t e l i g h t b o t t l e s w e r e d a r k e n e d f o r 3 a n d 5 h d u r i n g m i d - d a y t o e s t i m a t e d a r k u p t a k e r a t e s , d a r k u p t a k e r a t e s w e r e a l s o d e t e r m i n e d f o r t r i p l i c a t e s a m p l e s c o l l e c t e d a n d i n c u b a t e d 24 h i n d a r k n e s s . D u r i n g t h e s e c o n d c r u i s e , i n t h e S t r a i t o f G e o r g i a , s a m p l e s w e r e c o l l e c t e d i n t h e m o r n i n g (07 00-0900 h) a n d 6.0 L i g - a t N - L - 1 o f 1 5 N H 4 + , 1 5 N 0 3 ~ o r C O ( 1 5 N H 2 ) 2 w e r e a d d e d i n TC.2 a n d 3. T i m e - z e r o s a m p l e s f o r d i s s o l v e d n i t r o g e n w e r e w i t h d r a w n i m m e d i a t e l y a n d a n a l y z e d f o r N H 4 + , N O 3 - , a n d u r e a 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 . A t 3 h i n t e r v a l s p a r t i c u l a t e m a t t e r , f r o m d u p l i c a t e s a m p l e s , was c o l l e c t e d by f i l t r a t i o n f o r -^N a n a l y s i s . S a m p l e s 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 w e r e t a k e n c o n c u r r e n t l y a n d t h o s e f o r C h i a a n d POC a n d PON e v e r y 6 h. D u r i n g t h e t h i r d c r u i s e , d r o g u e d d r i f t e r b u o y s ( L o r a n - C d r i f t e r s ; d e s c r i b e d i n M a c k a s e t a l . , 1989) w e r e u s e d t o g u i d e r e p e a t e d s a m p l i n g f r o m a g i v e n w a t e r p a r c e l f o r 48 h (TC.4) a n d 24 h ( T C . 5 ) . The d r o g u e s w e r e c e n t r e d a t 15 m d e p t h a n d t h e i r p o s i t i o n s w e r e r e p o r t e d by r a d i o e v e r y h a l f h o u r ; d r i f t e r t r a c k s a r e r e p o r t e d i n F o r b e s e t a l . ( 1 9 8 7 ) . D u r i n g TC.4, a n u p w e l l i n g p l u m e on t h e w e s t e r n c o a s t o f V a n c o u v e r 23 I s l a n d , was sampled at 3 h i n t e r v a l s f o r p h y s i c a l measurements and at 6 h i n t e r v a l s f o r N uptake experiments and the c o n c e n t r a t i o n s of POC, PON, C h i a and d i s s o l v e d n u t r i e n t s . Samples from 100, 30, and 1% I Q were c o l l e c t e d as d e s c r i b e d p r e v i o u s l y and d u p l i c a t e samples from each depth i n o c u l a t e d w i t h 10 uq-at N - L - 1 of Na 1 5N0 3~ or 1 5NH 4C1 f o r 4 h si m u l a t e d i n s i t u i n c u b a t i o n s . During TC.5, i n the waters o f f s h o r e of the c o n t i n e n t a l s h e l f , a water p a r c e l was r e p e a t e d l y sampled at 2 h i n t e r v a l s f o r b i o l o g i c a l and p h y s i c a l measurements f o r 29 h. At each sampling p e r i o d , d u p l i c a t e samples from j u s t below the s u r f a c e and the 1% I Q were e n r i c h e d w i t h — 1 IS 10 L i g - a t N*L of Na NO3 and xncubated f o r 4 h i n simulated i n s i t u c o n d i t i o n s . Samples f o r enumeration of phytoplankton s p e c i e s were c o l l e c t e d at the beginning and end of both experiments t o determine community composition over the sampling p e r i o d . 24 RESULTS Physical observations The v e r t i c a l p r o f i l e s of temperature, s a l i n i t y and n i t r a t e c o n c e n t r a t i o n f o r TC. 1 ( c r u i s e OE 84 02, s t n F) i n the n o r t h e a s t P a c i f i c Ocean are presented i n F i g u r e 1.2 A. The temperature of the upper water column was 6.5-7.0°C and thoroughly mixed u n t i l c a . 30 m. A shallow t h e r m o c l i n e step (sT, temperature step < 0.5) at 30-40 m e f f e c t i v e l y d i v i d e d the euphotic zone (1% I Q = 70 m) i n t o two l a y e r s w i t h d i f f e r e n t t u r b u l e n c e c h a r a c t e r i s t i c s and dynamics (Denman and G a r g e t t , 1988) above the main p y c n o c l i n e / s e a s o n a l t h e r m o c l i n e at 80-100 m. Denman and Gargett (1988) demonstrated t h a t the shallow t h e r m o c l i n e step p r e s e n t s a s i g n i f i c a n t b a r r i e r t o the v e r t i c a l exchange of phytoplankton and presented two seperate i n d i c a t o r s of the p h y s i o l o g i c a l s t a t e of the phytoplankton which c o n f i r m t h a t t h i s p h y s i c a l b a r r i e r was s u f f i c i e n t t o cause d i f f e r i n g degrees of photoadaptation i n each of the two l a y e r s comprising the euphotic zone. N i t r a t e (plus n i t r i t e ) c o n c e n t r a t i o n s were high (ca. 11 uq-at N'L - 1) i n the s u r f a c e "mixed water" and i n c r e a s e d t o 20-35 uq-at N « L - 1 below the p y c n o c l i n e . 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 i n v i v o f l u o r e s c e n c e and N O 3 - c o n c e n t r a t i o n s f o r the f r o n t a l (TC.2, s t n A5) and s t r a t i f i e d (TC.3, s t n T4) waters of the S t r a i t of Georgia are presented i n F i g u r e 1.2 B & C. 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 t h e r m o c l i n e and h i g h f l u o r e s c e n c e a t the depth of the n i t r a c l i n e (3 t o 7 m). 25 Figure 1 . 2 . Depth profiles of temperature (T), s a l i n i t y ( S ) , in vivo fluorescence (F), and nitrate plus n i t r i t e concentration (N) for three stations sampled for containment time course experiments. (A) Oceanic station F, TC.1. (B) Frontal station A 5, TC . 2 . (C) Str a t i f i e d station T4, TC.3. The shallow thermocline step is indicated by the arrow labelled 'sT' in panel A. 26 Time c o u r s e 3 was c o n d u c t e d i n warm (17°C), s t r a t i f i e d w a t e r a n d t h e d e p t h p r o f i l e d e m o n s t r a t e d a s u b s u r f a c e f l u o r e s c e n c e maximum ( c a . 10m) w h i c h was o v e r l a i n b y n i t r a t e - d e p l e t e d m i x e d w a t e r ( F i g . 1.2 C ) . Time c o u r s e 4 was c o n d u c t e d b y r e p e a t e d s a m p l i n g o f an u p w e l l e d p l u m e o f f V a n c o u v e r I s l a n d ( s t n 24-49) a n d t h e d e p t h s o f s a m p l i n g ( 2 , 6-7,and 14 m) w e r e a l l w i t h i n t h e m i x e d s u r f a c e l a y e r o f n i t r a t e - r e p l e t e ( c a . 10 ^ j g - a t N * L - 1 ) , warm (11°C) s u r f a c e w a t e r a b o v e t h e t h e r m o c l i n e / h a l o c l i n e ( c a . 15 m). The v e r t i c a l p r o f i l e s o f s a l i n i t y a n d t e m p e r a t u r e ( F i g . 1.3 A & B) f o r t h e f i r s t ( s t n 24) a n d l a s t ( s t n 49) p e r i o d o f s a m p l i n g (51 h l a t e r ) d e m o n s t r a t e a s l i g h t d e e p e n i n g o f t h e s u r f a c e m i x e d l a y e r by a w e a k e n i n g o f t h e s h a l l o w e r , f i r s t t h e r m o c l i n e o v e r t h e s a m p l i n g p e r i o d . V e r t i c a l p r o f i l e s o f N O 3 - a n d N H 4 + a t 6 h i n t e r v a l s ( F i g . 1.4) show r e l a t i v e l y l i t t l e c h a n g e o v e r t i m e , a l t h o u g h e l e v a t e d NH^ + c o n c e n t r a t i o n s w e r e i n i t i a l l y o b s e r v e d i n t h e s u r f a c e w a t e r s a t s t n 24; t h e s e d e c r e a s e d d u r i n g t h e n e x t 6 h t o l o w (< 0.5 / j g - a t N ' L - 1 ) b u t v a r i a b l e c o n c e n t r a t i o n s i n t h e s u r f a c e w a t e r s s a m p l e d f o r t h e r e m a i n d e r o f t h e d i e l N u p t a k e e x p e r i m e n t s . V e r t i c a l p r o f i l e s o f S i O ^ - ^ a n d PO^ -^ a m b i e n t c o n c e n t r a t i o n d e m o n s t r a t e d l i t t l e c h a n g e o v e r t h e c o u r s e o f t h e t i m e c o u r s e e x p e r i m e n t ( F i g . 1.5) . Time c o u r s e 5 u t i l i z e d b o t h s u r f a c e (0-2 m) n i t r a t e -d e p l e t e d w a t e r f r o m t h e s h a l l o w ( c a . 7 m) m i x e d l a y e r a n d d e e p (28 m, 1% I Q ) n i t r a t e - r e p l e t e (> 5 / j g - a t N ' L - 1 ) w a t e r f r o m t h e t h e r m o c l i n e d e p t h . P r o f i l e s o f d i s s o l v e d i n o r g a n i c n u t r i e n t s 27 F i g u r e 1.3. D e p t h p r o f i l e s o f t e m p e r a t u r e (T) a n d s a l i n i t y (S) f o r t h e t w o s t a t i o n s r e p e a t e d l y s a m p l e d d u r i n g d r o g u e - t y p e t i m e c o u r s e e x p e r i m e n t s . A: s t n 24 ( b e g i n n i n g o f T C . 4 ) . B: s t n 49 ( e n d o f T C . 4 ) . C: s t n 84 ( b e g i n n i n g o f T C . 5 ) . D: s t n 98 ( e n d o f T C . 5 ) . TEMPERATURE (°C) 12 6 7 8 10 II 12 200 '1 T ] 1 Stn. 49 / ! 1 32 33 34 32 33 SALINITY (%0) TEMPERATURE (°C) 12 14 6 8 10 34 200 31 32 33 34 31 32 SALINITY (%0) 33 34 28 Figure 1.4 Depth profiles of N 0 3 " and NH4+ at 6 h intervals during time course 4. 2 2 O O i CM O 2 + — i ro 1 1 Q 2 NO LJM HN UJ •4— +- o T 9 o o UJ 1 1 i i A (^ ) Hld30 (w) Hld3Q 29 F i g u r e 1.5. D e p t h p r o f i l e s o f S i 0 4 ~ 4 a n d P 0 4 ~ 3 a t 6 h i n t e r v a l s d u r i n g t i m e c o u r s e 4. Q- in a o H±d30 Hld3Q F i g u r e 1.6 A: D e p t h p r o f i l e s o f N0 3" (<») a n d NH 4 + (O) a t 2 i n t e r v a l s d u r i n g t i m e c o u r s e 5. B: D e p t h p r o f i l e s o f S i O (•) a n d P 0 4 - 3 (•) a t 2 h i n t e r v a l s d u r i n g t i m e c o u r s e 5. o o _ Hld30 2 Z CL CO (N0 3~, NH 4 +, S i 0 4 - 4 , P 0 4 - 3 ) are presented i n F i g u r e 1.6 and demonstrated l i t t l e v a r i a t i o n i n c o n c e n t r a t i o n over the 29 h sampling p e r i o d . Biological observations Phytoplankton s p e c i e s were i d e n t i f i e d and counted u s i n g v i s i b l e l i g h t microscopy and thus may be b i a s e d by the e x c l u s i o n of c e l l s < 2 um (picoplankton) , which are d i f f i c u l t t o d i s t i n g u i s h from i n o r g a n i c p a r t i c l e s (e.g., Booth, 1988). In the water used f o r T C . l approximately 83% of the phytoplankton enumerated were smal l haptophytes b e l o n g i n g t o the f o l l o w i n g genera, Imantonia, Phaeocystis or Chrysochromulina (4-10 um). Chrysophytes of the genus Ochromonas were 7% of the t o t a l c e l l c o n c e n t r a t i o n and pennate diatoms, p r i m a r i l y Nitzschia cylindrus and N. closterium v. s t r i a t u l a , composed an a d d i t i o n a l 8%; the Raphidophycean f l a g e l l a t e Heterosigma akashiwo was ca 1%. O c c a s i o n a l d i n o f l a g e l l a t e s , (Gymnodinium spp. and Prorocentrum baltica), c e n t r i c diatoms (Chaetoceros peruvianum and Thalassiosira spp.) and p r a s i n o p h y t e s (Nephroselmis spp.) were observed, but t o t a l l e d < 1% of the t o t a l c e l l c o n c e n t r a t i o n of 4.8 x 10^ c e l l s * L - 1 . Samples were not enumerated f o r p i c o p l a n k t o n , although Booth (1988) observed t h a t i n samples c o l l e c t e d d u r i n g the same time i n the n o r t h e a s t P a c i f i c , 16% of the p l a n t biomass was a t t r i b u t e d t o c e l l s < 2 m^ and 90% of t h i s was composed of the blue-green, c o c c o i d cyanobacterium, Synechococcus spp. The m a j o r i t y of the zooplankton was probably removed by s c r e e n i n g the water samples through N i t e x 32 n e t t i n g ( i n o r d e r t o m i n i m i z e m a c r o z o o p l a n k t o n 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 ) a l t h o u g h s c r e e n e d s a m p l e s w e r e n o t e n u m e r a t e d ; u n f i l t e r e d z o o p l a n k t o n s p e c i e s a n d a b u n d a n c e d a t a a r e . r e p o r t e d i n F o r b e s e t a l . ( 1 9 8 8 ) . The s p e c i e s c o m p o s i t i o n o f t h e p h y t o p l a n k t o n c o m m u n i t y i n t h e f r o n t a l (TC.2) a n d s t r a t i f i e d (TC.3) w a t e r o f t h e S t r a i t o f G e o r g i a was v e r y d i f f e r e n t ( T a b l e 1 . 2 . ) . I n t h e f r o n t a l w a t e r l a r g e , c h a i n -f o r m i n g d i a t o m s o f t h e g e n u s Chaetoceros f o r m e d a g g r e g a t e s (£ 1 mm) w h i c h c o n t a i n e d some p e n n a t e d i a t o m s b e l o n g i n g t o Navicula a n d Nitzschia s p p . The s i z e o f t h e d i a t o m f l o e s p r e v e n t e d s c r e e n i n g p r i o r t o N e x p e r i m e n t a t i o n a n d t h e r e f o r e t o r e m a i n c o n s i s t e n t , none o f t h e f u t u r e w a t e r s a m p l e s w e r e s c r e e n e d . S m a l l f l a g e l l a t e s (< 5 um) w e r e t h e m o s t common p h y t o p l a n k t o n i n t h e s t r a t i f i e d w a t e r . Chaetoceros s p p . , Ch. socialis a n d Skeletonema costatum w e r e t h e m o s t a b u n d a n t d i a t o m s , w h e r e a s d i n o f l a g e l l a t e s w e r e a l m o s t e x c l u s i v e l y Gymnodinium s p p . W a t e r s a m p l e s w e r e n o t o r i g i n a l l y t a k e n f o r z o o p l a n k t o n s p e c i e s e n u m e r a t i o n . However t h e a b u n d a n c e o f t h e s e a n i m a l s , a s s e e n i n t h e p h y t o p l a n k t o n s a m p l e s , s u g g e s t e d t h e y c o u l d h a v e b e e n i m p o r t a n t g r a z e r s a n d N r e m i n e r a l i z e r s . A s a f i r s t a p p r o x i m a t i o n t h e c o n c e n t r a t i o n o f g e n e r a l c a t e g o r i e s o f t h e s e z o o p l a n k t e r s a r e p r e s e n t e d ( T a b l e 1 . 2 ) . The l a r g e , c e n t r i c d i a t o m s , Skeletonema costatum, Thalassiosira nordenskioldii, a n d Chaetoceros s p p . ( p a r t i c u l a r l y Ch. compressum > Ch. radicans > Ch. ceratosporum) d o m i n a t e d i n t e r m s o f r e l a t i v e numbers ( 7 0 % ) i n t h e s a m p l e s c o l l e c t e d a t a l l 3 d e p t h s u s e d i n TC.4. The T a b l e 1.2 Pl a n k t o n community c o m p o s i t i o n i n f r o n t a l and s t r a t i f i e d water of S t r a i t of Georgia, B.C., (see F i g . 1.1 B). S t a t i o n P h y t o p l a n k t o n (10 6 c e l l s - L - 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 Zooplankton ( a n i m a l s - L - 1 ) 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 2.3 0.023 1.6 470 50 730 280 (TC.2) S t r a t i f i e d T4 0.43 0.049 1.6 180 60 140 300 (TC.3 ) 34 r e m a i n d e r o f t h e p h y t o p l a n k t o n c o m m u n i t y c o n s i s t e d p r i m a r i l y o f p e n n a t e d i a t o m s (Nitzschia c f . subpacifica > N. delicatissima > N. americana ^ N. longissima), h a p t o p h y t e s (Imantonia a n d Phaeocystis s p p . ) a n d u n i d e n t i f i e d c r y p t o m o n a d s . T h e r e w e r e no o b v i o u s d i f f e r e n c e s i n t h e s p e c i e s c o m p o s i t i o n a n d t o t a l c e l l c o n c e n t r a t i o n a t a n y o f t h e t h r e e d e p t h s b e t w e e n t h e b e g i n n i n g a n d e n d o f t h e d r o g u e s a m p l i n g ( F i g . 1.7 A ) ; s u r f a c e p o p u l a t i o n d e c r e a s e d f r o m 3.0 x 1 0 6 c e l l s - L - 1 t o 1.3 x 1 0 6 c e l l s - L - 1 , t h e m i d - d e p t h ( 3 0 % I Q ) f ft — 1 p o p u l a t i o n f r o m 2.8 x 10 t o 2.3 x 10 c e l l s - L , a n d an i n c r e a s e i n t h e p o p u l a t i o n a t 1% I Q f r o m 3.6 x 10^ t o 5.0 x 1 0 6 c e l l s - L - 1 . The s p e c i e s c o m p o s i t i o n i n t h e s a m p l e s c o l l e c t e d f r o m t h e s u r f a c e (2 m) a n d d e p t h (27 m) f o r TC.5 w e r e v e r y d i f f e r e n t ; a d o m i n a n c e o f d i a t o m s ( 74%) b o t h c e n t r i c ( 5 8%) a n d p e n n a t e ( 1 5 % ) i n t h e d e e p e r w a t e r s . The most common c e n t r i c d i a t o m s w e r e Skeletonema costatum, Thalassiosira conferta, Chaetoceros S P P - ( p r i m a r i l y Ch. compressum a n d Ch. debilis) a n d t h e p e n n a t e s w e r e m o s t l y Thalassionema nitzschioides a n d Nitzschia americana, N. delicatissima a n d N. longissima. I n t h e s u r f a c e s a m p l e s o n l y a b o u t o n e - t h i r d o f t h e c o m m u n i t y was composed o f c e n t r i c d i a t o m s , t h e r e m a i n d e r h a p t o p h y t e s ( 2 1 % ) ( p r i m a r i l y Imantonia a n d Phaeocystis spp.) u n i d e n t i f i e d c r y p t o m o n a d s ( 3 7 % ) a n d d i n o f l a g e l l a t e s ( 8 % ) , i n c l u d i n g g e n e r a o f Gymnodinium a n d Protogonyaulax. S p e c i e s s a m p l e s w e r e o n l y c o l l e c t e d a t m i d - d e p t h (20 m) a t t h e e n d o f t h e t i m e s e r i e s b u t t h i s s a m p l e showed no m a j o r d i f f e r e n c e s i n s p e c i e s 35 Figure 1.7 Composition of the phytoplankton community, A: at the beginning (stn 24) and end (stn 49) of time course 4 B: beginning (stn 84) and end (stn 98) of time course 5. Ld O z: o o LU o < I-o I-| 1 Centric Diatoms § \ Dinoflagellates Pennate Diatoms Chrysophytes Haptophytes Others Cryptophytes 100 -8 0 -6 0 -4 0 2 0 -0 2 m 5 m 14m Stn. 2 4 2 m 6 m 13m S t n . 4 9 2 m 18m 27m 2 0 m Stn. 8 4 Stn .98 36 c o m p o s i t i o n o v e r t i m e , e x c e p t f o r a n i n c r e a s i n g a b u n d a n c e o f h a p t o p h y t e s a n d a d e c r e a s e i n t h e a b u n d a n c e o f d i a t o m s ( F i g . 1.7 B) . Nitrogen uptake rates S u b a r c t i c P a c i f i c O cean D u r i n g T C . l , i n t h e n i t r a t e - r i c h s u b a r c t i c P a c i f i c , t h e 1 c i n c o r p o r a t i o n o f N - l a b e l l e d n i t r a t e i n t o p a r t i c u l a t e m a t t e r was f o l l o w e d f o r 24 h, b e g i n n i n g d u r i n g t h e m i d d l e o f t h e n i g h t ( F i g . 1.8 B ) . A r e l a t i v e l y c l e a r d i e l t r e n d was a p p a r e n t f o r s p e c i f i c NO^ - u p t a k e r a t e s c a l c u l a t e d o v e r 3 h i n t e r v a l s ( F i g . 1.8 C ) ; m a x i m a l u p t a k e r a t e d u r i n g t h e d a y l i g h t p e r i o d (mean = 0.0085, S.D. = 0.0045 h - 1 ) a n d mean r a t e a t n i g h t was l o w e r ( 0.0047 ± 0.0013 h - 1 ) . The mean n i t r a t e u p t a k e a t n i g h t was c a . 5 5 % o f t h e mean d a y t i m e v a l u e . S a m p l e s i n c u b a t e d f o r 24 h i n t h e d a r k ( 0 . 0 0 0 6 3 ± 0.00011 h - 1 ) ha d s i g n i f i c a n t l y l o w e r N O 3 - u p t a k e r a t e s ( p a i r e d t - t e s t , P *s 0.01) t h a n t h o s e i n c u b a t e d i n t h e n a t u r a l l i g h t - d a r k c y c l e ( 0 .0068 ± 0.00046 h - 1 ) . The a r t i f i c i a l d a r k e n i n g o f t r i p l i c a t e s a m p l e s f o r 3 a n d 5 h d u r i n g m i d - d a y h a d no e f f e c t d u r i n g t h e f i r s t 3 h; mean d a r k u p t a k e was 0.0072 h - 1 a n d e q u i v a l e n t t o t h e mean l i g h t u p t a k e ( 0 . 0 0 7 1 h - 1 ) d u r i n g t h i s t i m e , h o w e v e r , d u r i n g t h e s u b s e q u e n t 2 h o f d a r k n e s s , u p t a k e d e c l i n e d 9 2 % t o 0.00058 h - 1 , w h e r e a s t h e l i g h t u p t a k e r a t e o f N O 3 - a t t a i n e d i t s m a x i m a l v a l u e ( 0.0156 h - 1 ) . S t r a i t o f G e o r g i a D u r i n g t h e t i m e c o u r s e e x p e r i m e n t s c o n d u c t e d i n t h e S t r a i t o f G e o r g i a , t h e c h a n g e s i n t h e a m b i e n t c o n c e n t r a t i o n o f Figure 1.8. Time course measurements at oceanic s t a t i o n F., Time Course 1. (A) Daily incident surface i r r a d i a n c e during experiment. (B) 1 5N atom % excess i n p a r t i c u l a t e matter for l i g h t b o t t l e incubations (error bars represent ± 1 S.D. of t r i p l i c a t e s ) p l o t t e d against elapsed time measured a f t e r addition of 1.0 uq-at N-NO -IT1. (C) Nitrogen s p e c i f i c uptake rates of 15N03" cal c u l a t e d for 3 h i n t e r v a l s ; each point i n d i c a t e s a rate ca l c u l a t e d over the time i n t e r v a l between i t and the previous point on the curve and p l o t t e d against average incubation time between sampling. ^ 1500 a1 o.ooo in 8 12. 14 TIME (h) 18 24 38 d i s s o l v e d N H 4 + , N O 3 - a n d u r e a a n d t h e i n c o r p o r a t i o n o f t h o s e 1 5 N - l a b e l l e d s u b s t r a t e s i n t o p a r t i c u l a t e m a t t e r w e r e m e a s u r e d f o r 24 h. B o t h a p p r o a c h e s 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 o n c e r n i n g n i t r o g e n u t i l i z a t i o n b y t h e p h y t o p l a n k t o n . C h a n g e s 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 r e p r e s e n t n e t c o m m u n i t y f l u x o f t h a t n u t r i e n t a n d encompass r e g e n e r a t i v e a n d u p t a k e p r o c e s s e s . By c o n t r a s t , N i s o t o p e a c c u m u l a t i o n i s an e s t i m a t e o f g r o s s u p t a k e b y t h e p h y t o p l a n k t o n p r o v i d i n g t h e r e i s no r e c y c l i n g o f -^N, a n d e n r i c h m e n t i n t h e d i s s o l v e d p h a s e r e m a i n s c o n s t a n t . R e s u l t s f r o m TC.2 ( f r o n t a l w a t e r ) a n d TC.3 ( s t r a t i f i e d w a t e r ) e x p e r i m e n t s a r e shown i n F i g . 1.9 a n d F i g . 1.10, r e s p e c t i v e l y . D a t a f r o m TC.2 d e m o n s t r a t e m u l t i p l e N s u b s t r a t e u p t a k e by p h y t o p l a n k t o n , s p e c i f i c a l l y f o r N H 4 + , N 0 3 ~ a n d u r e a ( F i g . 1.9 C, E) a n d N 0 3 ~ a n d u r e a ( F i g . 1.9 G ) . The e l e v a t e d a m b i e n t N 0 3 ~ c o n c e n t r a t i o n i n t h e f r o n t a l w a t e r s a l l o w e d t h e u p t a k e r a t e s o f N 0 3 ~ i n t h e N H 4 + - a n d u r e a -e n r i c h e d s a m p l e s t o be d e t e r m i n e d b y N d i s a p p e a r a n c e f r o m t h e s e a w a t e r s a m p l e s . U p t a k e r a t e s d e t e r m i n e d by t h e d i s a p p e a r a n c e o f n i t r a t e w e r e s i m i l a r i n t h e p r e s e n c e ( V d Q _ 6 h = 0 . 5 2 1 uq-at N - L - 1 - h _ 1 ) a n d a b s e n c e ( V d 0 _ 9 h = 0 . 5 6 7 uq-at N * L - 1 * h - 1 ) o f u r e a , b u t w e r e r e d u c e d i n t h e NH4"4" e n r i c h e d s a m p l e s ( V d Q _ 9 h = 0 . 2 6 7 uq-at N - L - 1 - h - 1 ) . The 1 5 N - u r e a a tom % a c c u m u l a t i o n r a t e was c o n s t a n t o v e r t h e f i r s t 15 h, b u t p r i o r t o t h e e n d o f t h e d a r k p e r i o d i t i n c r e a s e d a n d r e m a i n e d l i n e a r u n t i l t h e e n d o f t h e i n c u b a t i o n ( F i g . 1.9 F ) . The i n c r e a s e i n t h e u r e a u p t a k e r a t e c o i n c i d e d w i t h t h e d e p l e t i o n o f e x t e r n a l N 0 3 ~ , m o r e o v e r t h e c h a n g e i n u r e a c o n c e n t r a t i o n was m i n i m a l 39 F i g u r e 1.9. Time c o u r s e m e a s u r e m e n t s a t f r o n t a l s t a t i o n ( A 5 ) , T i m e C o u r s e 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 e x p e r i m e n t ( B , D, F) 1 5N atom % e x c e s s i n p a r t i c u l a t e m a t t e r f o r l i g h t a n d d a r k 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 o f 6 / j g - a t N• L - 1 o f (B) NH 4 +, (D) N0 3" a n d ( F ) u r e a ( e r r o r b a r s r e p r e s e n t t h e r a n g e o f d u p l i c a t e s ) . ( C , E, G) C o r r e s p o n d i n g m e a s u r e m e n t s o f d i s s o l v e d NH 4 + ( • ) , N0 3" ( o ) a n d u r e a ( A ) i n (C) NH 4 +, (E) N0 3~, a n d (G) u r e a - s p i k e d s a m p l e s . D a s h e d l i n e i n d i c a t e s no m e a s u r e m e n t s o f d i s s o l v e d u r e a a t 3 a n d 6 h; ( l e f t s i d e o f p a g e ) . F i g u r e 1.10. A s F i g u r e 1.9 e x c e p t a t s t r a t i f i e d s t a t i o n ( T 4 ) , T i m e C o u r s e 3; ( r i g h t s i d e o f p a g e ) . 40 41 ( F i g . 1.9 G) o v e r t h e f i r s t 6 h, when N 0 3 ~ c o n c e n t r a t i o n s w e r e h i g h ( 4 . 5 5 t o 1.4 j v g - a t N * L - 1 ) a n d N O 3 - was b e i n g t a k e n u p. The i n c o r p o r a t i o n o f -^N-NC^ - a n d 1 5 N - N H 4 + was n o n - l i n e a r w i t h t i m e , g e n e r a l l y r e d u c e d d u r i n g t h e n i g h t t i m e a n d s u b s t r a t e e x h a u s t i o n o c c u r r e d d u r i n g t h e 21 t o 24 h t i m e i n t e r v a l . The p a t t e r n o f N u p t a k e b y t h e p h y t o p l a n k t o n i n t h e s t r a t i f i e d w a t e r was s i m i l a r i n t h e NH 4 + , N O 3 - a n d u r e a -e n r i c h e d s a m p l e s ( F i g . 1.10 B, D, F , ) . U p t a k e was c o n s t a n t o v e r t h e f i r s t 9 t o 12 h, t h e n d e c r e a s e d d u r i n g t h e n i g h t a n d i n c r e a s e d a g a i n i n t h e e a r l y m o r n i n g . S u b s t r a t e d e p l e t i o n d i d n o t o c c u r i n t h e s e e x p e r i m e n t s a n d t h e t o t a l u t i l i z a t i o n a f t e r 24 h o f n i t r o g e n i s o t o p e s was m i n i m a l i n t h e NH 4 + , N O 3 - a n d u r e a - e n r i c h e d s a m p l e s ( 2 3 , 18 a n d 10%, r e s p e c t i v e l y ) . C l e a r i n d i c a t i o n s o f u r e a r e g e n e r a t i o n , a n d t o a l e s s e r e x t e n t N H 4 + r e g e n e r a t i o n , w e r e e v i d e n t f r o m i n c r e a s e s i n s u b s t r a t e c o n c e n t r a t i o n s i n TC.2 a n d TC.3 a n d a r e d i s c u s s e d i n d e t a i l by P r i c e e t a l . ( 1 9 8 5 ) . The p a t t e r n o f 1 5 N - l a b e l l e d N H 4 + , NC>3~ a n d u r e a u p t a k e r a t e s s u g g e s t s t h e e x i s t e n c e o f 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 b o t h f r o n t a l a n d s t r a t i f i e d w a t e r ( F i g . 1 . 1 1 ) . The d e c r e a s e i n u p t a k e o f N H 4 + a n d N O 3 - f r o m 21 t o 24 h i n TC.2 was due t o s u b s t r a t e e x h a u s t i o n ( s e e F i g . 1.9 C , E ) . I n t h e f r o n t a l c o m m u n i t y , u p t a k e r a t e s o f N O 3 - w e r e g r e a t e s t t h r o u g h o u t t h e t i m e c o u r s e , i n c o n t r a s t t o t h e s t r a t i f i e d c o m m u n i t y w h e r e NH 4 + u p t a k e r a t e s w e r e h i g h e s t a n d N O 3 - a n d u r e a u p t a k e r a t e s s i m i l a r b u t l o w e r t h a n N H 4 + u p t a k e r a t e s . I n b o t h e x p e r i m e n t s n i t r o g e n u p t a k e r a t e s i n c r e a s e d p r i o r t o t h e o n s e t o f t h e l i g h t p e r i o d 42 Figure 1.11. Nitrogen-specific uptake rates of NH4+ ( • ) , N03 (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 for 3 or 6 h i n t e r v a l s ; each point i n d i c a t e s a rate 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 previous point on the curve. Shaded area on the abscissa d e l i m i t s the dark period. 0 . 0 6 0 0 . 0 0 0 1 1 1 ' • 1 0 3 6 9 1 2 1 5 1 8 2 1 2 4 I N C U B A T I O N P E R I O D ( h ) 43 a n d t h i s was most m a r k e d i n t h e u r e a - e n r i c h e d s a m p l e s . The r a t i o o f d a r k t o l i g h t 1 5 N u p t a k e r a t e ( V D : V L ) f o r N H 4 + , N 0 3 ~ a n d u r e a i s g i v e n i n T a b l e 1.3. I n b o t h 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 , d a r k N H 4 + u p t a k e s w e r e a m a j o r p o r t i o n o f t h e l i g h t u p t a k e r a t e s t h r o u g h o u t t h e e n t i r e t i m e c o u r s e s . The V D : V L f o r N H 4 + i n t h e f r o n t a l w a t e r was c o n s t a n t ( 3 8 % ) a n d l e s s t h e n t h e r a t i o i n s t r a t i f i e d w a t e r (52 t o 1 0 2 % ) . I n i t i a l d a r k r a t e s o f u r e a u p t a k e w e r e 60 t o 66% o f t h e l i g h t r a t e s i n b o t h TCs b u t d a r k u p t a k e d e c l i n e d t o a n e g l i g i b l e p o r t i o n o f l i g h t u p t a k e d u r i n g t h e r e m a i n d e r o f t h e TC.2 a n d 6-24% o f V L i n TC.3. The l i g h t d e p e n d e n c e o f N 0 3 ~ u p t a k e was more s i m i l a r t o t h a t o f u r e a t h a n ammonium i n b o t h s t r a t i f i e d a n d f r o n t a l w a t e r . U p t a k e r a t e s n o r m a l i z e d p e r u n i t C h i a d e m o n s t r a t e d t h a t N H 4 + a n d u r e a u p t a k e r a t e s w e r e on a v e r a g e 2 a n d 2.4 t i m e s g r e a t e r i n t h e s t r a t i f i e d w a t e r t h a n i n f r o n t a l w a t e r , w h e r e a s N 0 3 ~ u p t a k e r a t e s w e r e on a v e r a g e 1.6 t i m e s h i g h e r i n t h e f r o n t a l w a t e r ( T a b l e 1 . 4 ) . C h i a s p e c i f i c u p t a k e r a t e s f o r e a c h s u b s t r a t e , when c o m p a r e d b e t w e e n s t a t i o n s , w e r e most s i m i l a r o v e r t h e d a r k p e r i o d (12 t o 18 h) and t h e g r e a t e s t d i s p a r i t y was f o u n d i n i t i a l l y (0 t o 6 h ) . O f f s h o r e w a t e r s T ime c o u r s e 4 was c o n d u c t e d f r o m s a m p l e s c o l l e c t e d a t 6 h i n t e r v a l s f r o m 3 d e p t h s i n an u p w e l l e d p l u me o f w a t e r on t h e c o n t i n e n t a l s h e l f o f f V a n c o u v e r I s l a n d . The i n i t i a l e n v i r o n m e n t a l c o n d i t i o n s o f t h e w a t e r s a m p l e d d u r i n g TC.4 a r e p r e s e n t e d i n T a b l e 1.5. The s p e c i f i c r a t e s o f N 0 3 ~ u p t a k e f o r 44 1.3. Ratio of dark to l i g h t uptake rates ( V ^ r V ^ ) of Table _ N H 4 + , N O o - and urea for 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 Georgia, B.C., (see F i g . 1.1 B). Station Time i n t e r v a l (h) N H + ( V D = V L ) N0 3~ ( V D : V L ) Urea ( V D : V L ) Frontal A5 0 - 6 0.37 0.08 0.60 (TC.2) 6 - 12 0.39 <.01 <.01 12 - 18 0.37 <.01 <.01 18 - 24 0.39 <.01 <.01 S t r a t i f i e d T4 0 - 9 0.58 0.18 0.66 (TC.3) 9 - 18 1.02 0.60 0.24 18 - 24 0.52 <.01 0.06 45 T a b l e 1.4. C h l o r o p h y l l a s p e c i f i c u p t a k e r a t e s o f N H 4 , NO3" a n d u r e a i n f r o n t a l (A5) a n d s t r a t i f i e d (T4) w a t e r o f t h e S t r a i t o f G e o r g i a , B.C., ( s e e F i g . 1.1 B ) . The d a r k p e r i o d o c c u r s d u r i n g t h e 12 t o 18 h t i m e i n t e r v a l . N i t r o g e n Time i n t e r v a l C h i a s p e c i f i c N - u p t a k e r a t e s u b s t r a t e (h) [ug a t N (ug C h i a ) - 1 h _ 1 ] F r o n t a l S t n S t r a t i f i e d S t n N H 4 + NO3-U r e a 0 - 6 0.091 0.261 6 - 12 0.060 0.133 12 - 18 0.025 0.030 18 - 24 0.028 0.047 0 - 6 0.162 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 0.125 12 - 18 0.026 0.019 18 - 24 0.050 0.053 46 samples c o l l e c t e d from the n e a r - s u r f a c e (1-2 m) and 30% I Q depth (6-7 m) were not s i g n i f i c a n t l y d i f f e r e n t ( p a i r e d t - t e s t , P 2 0.01) and demonstrated pronounced d i e l p e r i o d i c i t y ( F i g . 1.'12 B). Maximum r a t e s of uptake were observed d u r i n g the d a y l i g h t hours, reduced r a t e s i n the e a r l y evening (1900-2300 h) and minimal r a t e s d u r i n g the n i g h t . The mean n i g h t t i m e uptake r a t e was 15-16% of the daytime r a t e . The N O 3 - uptake r a t e s of samples c o l l e c t e d and incubated at the 1% I Q depth (simulated) were v a r i a b l e and d i d not show a c l e a r d i e l p a t t e r n , although the g r e a t e s t v a l u e s were observed d u r i n g d a y l i g h t and the mean nig h t t i m e r a t e was ca. 7 0% of average daytime v a l u e . The s p e c i f i c r a t e s of NH 4 + uptake demonstrated a s i m i l a r p a t t e r n of d i e l p e r i o d i c i t y as the N O 3 - uptake r a t e s . The p o t e n t i a l s p e c i f i c uptake r a t e s of NH 4 + f o r the 2 shallow depths were again not s i g n i f i c a n t l y d i f f e r e n t ( p a i r e d t - t e s t , P 2 0.01), were minimal d u r i n g the n i g h t , maximal d u r i n g daytime and reduced i n the e a r l y evening. The mean nig h t t i m e r a t e was 30-36% of the daytime v a l u e . No d i e l t r e n d was observed i n the uptake r a t e s of the deeper samples and average n i g h t t i m e r a t e s were 120% of daytime r a t e s . A b s o l ute ( t r a n s p o r t ) r a t e s of N0 3~ and NH 4 + r e f l e c t the p a t t e r n s d i s c u s s e d f o r s p e c i f i c r a t e s ( F i g . 1.13). Depth p r o f i l e s of the d i s s o l v e d n u t r i e n t s N O 3 " " , NH 4 +, S i 0 4 3 ~ and P 0 4 3 ~ are presented i n F i g u r e s 1.4 and 1.5. and show l i t t l e change i n ambient c o n c e n t r a t i o n oyer the sampling p e r i o d . Time course 5 was conducted by sampling, at 2 h i n t e r v a l s , the phytoplankton community from the N 0 3 ~ - d e p l e t e Table 1.5 I n i t i a l environmental conditions of seawater c o l l e c t e d f or nitrogen uptake experiments during time course 4. S t a t i o n Date S t a r t i n g Sample Nitrogen cone. Chi a PON POC and time of depth N 0 3 - Urea NH4 l o c a t i o n incubation (PDT) (m) (ug--at N • (pg - lT 1 ) (uq-at N-L - 1) (uq-at C-L - 1) 24 4 9 ° 2 5 .O'N 20 August 1986 1100 1.7 12.90 2.38 1.67 10.35 7.75 46.8 127-32 . 1 'W 5.4 11.80 0.82 1.78 11.74 7.35 44.5 14.4 11.60 1.02 2.63 7.21 6.15 37 .5 28 4 9 ° 2 1 . 2 ' N 20 August 1986 1900 2.0 7.98 0.31 0.12 10.25 11.21 72.8 1 2 7 ° 2 8 . 9 ' W 5.8 7.98 0.26 0.12 12 .89 11.50 74.5 14.4 8.96 0.85 0.14 9 . 16 8.58 60.6 31 4 9 ° 2 0 .3'N 21 August 1986 0134 2.0 8.78 _ 0.82 19 .49 8.18 51.6 1 2 7 ° 2 7 .6 'W 5.8 8.59 0.71 0.60 19 .58 8.27 51.6 14.4 9.07 0.62 0.92 18.54 8.01 52 .9 34 4 9 ° 1 8 .5'N 21 August 1986 0736 2.0 9.84 0.75 0.67 21.90 8.31 53.2 1 2 7 ° 2 7 .7'W 7.3 9 .89 2 .27 0.62 22.73 7.36 45.8 13.7 10.10 0.72 0.75 20.85 7.81 62.6 37 4 9 ° 1 7 . 1' N 21 August 1986 1327 2.0 9.35 0.30 0.20 14.70 9.34 55.3 1 2 7 ° 2 6 .9'W 7.1 9 .16 0.41 0.17 15.04 10.06 58.7 14. 1 9.42 0.36 0.19 16 .12 9.40 56 .1 40 4 9 ° 1 6 . 1' N 21 August 1986 1934 2.0 8.50 0.61 0.11 14.66 9.80 46.7 1 2 7 ° 2 6 .3'W 5.0 8.58 1.26 0.12 15.21 8.57 55.9 14.8 8.58 0.26 0.10 14.83 8.25 57 .6 43 4 9 ° 1 6 .3'N 22 August 1986 0128 2.0 9.09 0.41 0.17 13.03 7.81 46.6 1 2 7 ° 2 3 .2'W 5.0 9.07 0.29 0.17 14.33 7.39 46 . 1 13.4 9.14 0.32 0.18 14.90 6.96 48.7 Table 1.5 continued s t a t i o n Date S t a r t i n g Sample Nitrogen cone. Chi a PON POC and time of depth N ° 3 ~ urea NH4 l o c a t i o n incubation (ug--L~l) (fjg-at N-L - 1) (jjg-at C-L - 1) (PDT) <m) (ug--at N • IT1) 46 49°16 . 0' N 22 August 1986 0730 2.0 8.49 0.33 0.37 15.80 7.20 40.8 127°21 . 4 'W 7.0 8.49 0.32 0.33 15.50 7.22 41.4 14.8 8.50 0.42 0.34 15.38 7.17 50.6 49 49°17 . 4 ' N 22 August 1986 1347 1.5 6 .34 0.33 0.08 16 .10 9.26 58.7 127°21 .3'W 7.2 7.63 0.48 0.26 14.00 8.73 53.7 13.9 8.57 0.61 0.41 13.41 7 .09 50.0 CO 49 F i g u r e 1.12. T i m e c o u r s e m e a s u r e m e n t s a t u p w e l l e d p l u m e s t a t i o n s 2 4 - 4 9 , t i m e c o u r s e 4. (A) D a i l y i n c i d e n t s u r f a c e i r r a d i a n c e d u r i n g e x p e r i m e n t . (B) N i t r a t e a n d ( C ) ammonium s p e c i f i c u p t a k e r a t e s a t 100% I ( O ) , 30% I ( • ) a n d 1% I Q ( A ) c a l c u l a t e d o v e r 4 h i n c u b a t i o n p e r i o d s a n d p l o t t e d a g a i n s t a v e r a g e i n c u b a t i o n p e r i o d . 1500 r CN 'E LU 1000 -- — LU O 500 -< < cr 0 -^ 0.060 -T" "—' 0.050 -LU 0.040 -< h - 0.030 -Q_ Z) 0.020 -1 CO o 0.010 -0.000 -0.060 -"— 0.050 -LU 0.040 -TA 0.030 -OL ZD 0.020 -+ T X 0.010 -0.000 -30 40 50 60 70 TIME (h) 5 0 F i g u r e 1.13. T i m e c o u r s e m e a s u r e m e n t s a t u p w e l l e d p l u m e s t a t i o n s 2 4 - 4 9 , t i m e c o u r s e 4. (A) D a i l y i n c i d e n t s u r f a c e i r r a d i a n c e d u r i n g e x p e r i m e n t . (B) N i t r a t e a n d (C) ammonium a b s o l u t e u p t a k e r a t e s a t 1 0 0 % I Q ( O ) , 3 0 % I O ( • ) a n d 1% I Q ( A ) c a l c u l a t e d o v e r 4 h i n c u b a t i o n p e r i o d s a n d p l o t t e d a g a i n s t a v e r a g e i n c u b a t i o n p e r i o d . 1 5 0 0 ^ 1 0 0 0 o 5 0 0 1 _J 0 . 6 0 0 -0 . 5 0 0 -o 1 0 . 4 0 0 -a. LU 0 . 3 0 0 -< h- 0 . 2 0 0 -0. i r> 0 . 1 0 0 -O 0 . 0 0 0 -0 . 6 0 0 0 . 5 0 0 0 . 4 0 0 0 . 3 0 0 0 . 2 0 0 0 . 1 0 0 0 . 0 0 0 3 0 40 5 0 T IME ( h ) ( 0 . 0 7 - 0 . 3 5 uq-at N-L - h - i ) s u r f a c e w a t e r a n d t h e N 0 3 ~ - r i c h ( 5 . 0 8 - 8 . 8 2 j v g - a t N - L - 1 - h - 1 ) w a t e r f r o m t h e 1% I Q (28 m) d e p t h ( T a b l e 1 . 6 ) . S p e c i f i c u p t a k e r a t e s , d e t e r m i n e d f r o m t h e i n c o r p o r a t i o n o f -^N-NC^ - d u r i n g 4 h i n c u b a t i o n p e r i o d s a r e p l o t t e d a g a i n s t a v e r a g e i n c u b a t i o n i n F i g . 1.14 B. The p a t t e r n o f N O 3 - s p e c i f i c u p t a k e r a t e s s u g g e s t s t h e e x i s t e n c e o f d i e l p e r i o d i c i t y i n t h e s u r f a c e s a m p l e s , s i m i l a r t o t h a t i n TC.4, w i t h m i n i m a l v a l u e s a t n i g h t a n d m a x i m a l v a l u e s d u r i n g t h e d a y t i m e . The mean n i g h t t i m e u p t a k e was 3 7 - 4 7 % o f t h e d a y t i m e r a t e . N O 3 - s p e c i f i c u p t a k e r a t e s o f t h e s a m p l e s c o l l e c t e d a t d e p t h w e r e v a r i a b l e d u r i n g t h e d a y / n i g h t c y c l e b u t s u g g e s t i v e o f a d i e l p a t t e r n a l t h o u g h d i s p l a c e d ( c a . 5 h) l a t e r i n t i m e . The a b s o l u t e u p t a k e r a t e s o f t h e s u r f a c e a n d d e e p c o m m u n i t i e s r e f l e c t t h e p a t t e r n s o b s e r v e d f o r s p e c i f i c u p t a k e r a t e s a n d a r e p r e s e n t e d i n F i g . 1.14 C. D e p t h p r o f i l e s o f d i s s o l v e d _ 1 3 _ 3 n u t r i e n t s NO3 , NH4 , S i 0 4 ~ a n d PO4 a r e p r e s e n t e d x n F i g . 1.6 a n d show l i t t l e c h a n g e o v e r t i m e . Table 1.6. I n i t i a l environmental c o n d i t i o n s during time course 5 conducted o f f the west coast of Vancouver Island on August 25-26, 1986. Stat i o n and l o c a t i o n S t a r t i n g time of incubation (PDT) sample depth (m) Nitrogen cone, Chi a PON POC NO, Urea NH< 1 , (ug-at N-L ) (ug-L (ug-at N-L - 1) (pg-at C-L 84 48°17.5'N 128°19.3'W 0912 2.4 26.6 0.13 8.44 <0.03 0.93 1.44 0.92 85 4 8 °16.7 ' N 128°18.9 W 1140 1.3 28.4 0.09 7 . 14 1.95 <0.03 0.63 1.25 1.09 2.24 2.11 2.84 17 . 3 12.6 86 48°16 .0'N 128°18.9'W 1422 1.5 26.3 0.09 6 .36 0.77 <0.03 1.11 1.79 0.96 3.68 1.98 2. 13 18.3 13.8 87 48°15.8'N 128°18.9'W 1532 1.7 28.4 0.02 6.38 0.41 <0.03 1.59 1.00 3.74 2.00 2.03 25.2 16.7 88 48°15.3'N 128°19.2'W 1736 1.1 27.2 0.09 5.99 0.39 <0.03 0.57 1.19 0.73 3.60 1.52 1.79 14 . 8 15.8 89 48°16.3'N 128°17.0'W 1957 1.1 27.0 0.31 9.39 0.30 <0.03 0.50 0.29 1.02 3.55 1.71 1. 80 18.6 15.8 90 48°16.1*N 128°17.3'W 2128 2.0 28.0 0.11 5. 19 <0.03 0.9.5 0.94 2.88 1.90 1.81 15.4 15.7 91 4 8 ° 1 5 . 4 ' N 1 2 8 ° 1 6 . 4 ' W 2322 1.9 28.4 0.33. 7.66 <0.03 1.25 04 06 1.61 1.75 11.2 13.0 92 48°14.9'N 128°16.8'W 0205 1.9 26.8 0. 13 5.08 0.41 0.11 0.98 1.01 1.21 2.50 1.44 1.77 13.3 15.9 Table 1.6 continued S t a t i o n S t a r t i n g Sample Nitrogen cone. Chi a PON POC and time of depth NO3 Urea NH4 l o c a t i o n incubation (PDT) (m) (pg--at N-L - 1) (/jg-L - 1) (pg-at N'L - 1) (yg-at C-L - 1 93 48° 14 5' W 0340 1.4 0.25 <0.03 0.70 1.72 13.3 128° 16.5' W 27.4 6.48 1.50 2.85 1.69 15.7 94 48° 14.7' ' N 0545 1.7 0.35 <0.03 0.84 1.62 13.5 128° 17.7' 'W 25.8 5.73 1.45 3.10 2.10 16 .0 95 48° 14.6' ' N 0747 1.3 0.33 0.37 0.05 0.83 1.67 13.1 128° 17.0' 'W 28.9 8.50 2.93 1.19 2.70 1.36 12 .9 96 48° 14.6 ' N 0944 2.2 0.08 <0.03 _ 1.14 9.09 128° 16.2 'W 27.7 8.24 0.78 1.30 0.96 9.77 97 48° 13.9 ' N 1154 0.5 0.17 <0.03 0.56 1.14 11.3 128° 16.5 'W 30.1 7.75 1.94 1.72 1.12 11.0 98 48° 13.5 ' N 1457 1.9 0.21 2.27 <0.03 0.74 1.75 15.3 128° 17 .6 'W 29 .1 5.67 1.30 1.25 3.99 1.30 9 .86 * designates cast time; no N uptake experiments at stn 84. 54 F i g u r e 1.14. T i m e c o u r s e m e a s u r e m e n t s a t s t a t i o n s 8 5 - 9 8 , t i m e c o u r s e 5. (A) D a i l y i n c i d e n t s u r f a c e i r r a d i a n c e d u r i n g e x p e r i m e n t . (B) N i t r o g e n s p e c i f i c u p t a k e r a t e s o f n i t r a t e a t 100% I ( o ) a n d 1% ( • ) c a l c u l a t e d o v e r 4 h i n c u b a t i o n p e r i o d s a n d p l o t t e d a g a i n s t a v e r a g e i n c u b a t i o n p e r i o d . (C) A b s o l u t e u p t a k e r a t e s o f n i t r a t e . 0.000 0.020 0.000 1 • • • ' • 1 -0 10 20 30 TIME (h) 55 DISCUSSION Experimental considerations P r e v i o u s d i e l s t u d i e s o f N u p t a k e b y n a t u r a l p h y t o p l a n k t o n a s s e m b l a g e s h a v e t a k e n t h r e e b a s i c a p p r o a c h e s : (1) s a m p l e s a r e c o l l e c t e d a t one l o c a t i o n , t r a n s f e r r e d t o i n c u b a t i o n c o n t a i n e r s , a n d t h e n s u b s a m p l e d o v e r t i m e ( e . g . , E p p l e y e t a l . , 1971b; C o l l o s a n d S l a w y k , 1976; K r i s t i a n s e n a n d L u n d , 1 9 8 9 ) ; (2) s a m p l i n g o c c u r s a t one g e o g r a p h i c l o c a t i o n o v e r t i m e ( e . g . , M a c l s a a c , 1978; T o b i e s e n , 1987; F i s h e r e t a l . , 1 9 8 8 ) ; o r (3) an a t t e m p t i s made t o f o l l o w a w a t e r p a r c e l w h i c h i s s a m p l e d o v e r t i m e ( e . g . , M a c l s a a c , 1 9 7 8 ) . E a c h a p p r o a c h h a s i t s a d v a n t a g e s a n d d i s a d v a n t a g e s . C o n t a i n m e n t h a s t h e a d v a n t a g e o f k n o w i n g t h a t t h e same w a t e r i s b e i n g s u b s a m p l e d o v e r t i m e a n d e l i m i n a t e s 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 s u c h a s a d v e c t i o n , d i e l m i g r a t i o n o f p h y t o p l a n k t o n ( e . g . , B l a s c o , 1978; C u l l e n a n d H o r r i g a n , 1981) a n d d i e l v a r i a b i l i t y i n a m b i e n t N c o n c e n t r a t i o n ( e . g . , L o r e n z e n , 1965; B e e r s a n d K e l l y , 1 9 6 5 ) . The m a j o r d i s a d v a n t a g e i s t h e p r o b l e m a s s o c i a t e d w i t h b o t t l e i n c u b a t i o n t e c h n i q u e s i n g e n e r a l ; t h e s e i n c l u d e c h a n g e s i n p l a n k t o n s p e c i e s c o m p o s i t i o n ( V e n r i c k e t a l . , 1 9 7 7 ) , b o t t l e s i z e e f f e c t s ( G i e s k e s e t a l . , 1 9 7 9 ) , a n d t o x i c i t y due t o t r a c e m e t a l s p r e s e n t a s b o t t l e c o n t a m i n a n t s ( C a r p e n t e r a n d L i v e l y , 1 9 8 0 ) . T h e s e " b o t t l e - e f f e c t s " may s u b s t a n t i a l l y a l t e r t h e c o n t a i n e d w a t e r ; l a r g e , " c l e a n " c o n t a i n e r s w i l l m i n i m i z e t h e s e e f f e c t s . S a m p l i n g a t one g e o g r a p h i c l o c a t i o n h a s t h e a d v a n t a g e o f d e a l i n g w i t h n a t u r a l s a m p l e s , b u t d o e s n o t t a k e i n t o a c c o u n t 56 t h e p r o b l e m o f a d v e c t i o n w h i c h may r e s u l t i n d i f f e r e n t c o m m u n i t i e s b e i n g s a m p l e d a t d i f f e r e n t t i m e s . A d v e c t i o n i s p o t e n t i a l l y a more p r o n o u n c e d p r o b l e m i n t i d a l l y i n f l u e n c e d r e g i o n s s u c h a s t h e S t r a i t o f G e o r g i a w h e r e a p a t t e r n c a u s e d by t i d e s c o u l d be m i s t a k e n f o r a d i e l r h y t h m i n p h y t o p l a n k t o n . S a m p l i n g a p a r t i c u l a r p a r c e l o f w a t e r o v e r t i m e h a s t h e d i s t i n c t a d v a n t a g e t h a t t h e same p l a n k t o n i c c o m m u n i t y i s l i k e l y s a m p l e d e a c h t i m e . However s u c h a n a p p r o a c h r e q u i r e s k n o w l e d g e o f t h e p h y s i c s o f t h e w a t e r p a r c e l a n d a means o f f o l l o w i n g i t , a l o g i s t i c a l l y more d i f f i c u l t a n d e x p e n s i v e e x p e r i m e n t a l a p p r o a c h . B o t h o f t h e l a t t e r m e t h o d s assume t h a t b i o l o g i c a l a c t i v i t y s u c h a s d i e l m i g r a t o r y b e h a v i o u r ( e . g . , C u l l e n a n d H o r r i g a n , 1981; F r e m p o n g , 1984) a n d p h y t o p l a n k t o n s i n k i n g ( e . g . , B i e n f a n g e t a l . , 1982) a r e m i n i m a l i n o r d e r t o a c h i e v e c o n s t a n c y i n p h y t o p l a n k t o n c o m m u n i t y c o m p o s i t i o n . I n t h e c u r r e n t s t u d y b o t h t h e c o n t a i n m e n t a p p r o a c h a n d f o l l o w i n g o f a w a t e r p a r c e l o v e r t i m e w e r e u t i l i z e d . A t t e m p t s w e r e made t o m i n i m i z e z o o p l a n k t o n p r e d a t i o n i n t h e c o n t a i n e d s a m p l e s d u r i n g t h e i n c u b a t i o n p e r i o d b y s c r e e n i n g o u t 1 c m a c r o z o o p l a n k t o n w x t h n e t t i n g p r i o r t o N i n o c u l a t i o n . I n 1 R a l l t i m e c o u r s e e x p e r i m e n t s s a t u r a t i n g a d d i t i o n s o f re-l a b e l l e d s u b s t r a t e s w e r e u s e d s o t h a t t h e e f f e c t s a s s o c i a t e d w i t h i s o t o p e d i l u t i o n o f t h e i s o t o p e e n r i c h m e n t f a c t o r by u n l a b e l e d r e g e n e r a t e d N w o u l d be m i n i m a l ( e . g . G l i b e r t e t a l . , 1 9 8 2 c ; P r i c e e t a l . , 1985) a n d s u b s t r a t e e x h a u s t i o n w o u l d n o t o c c u r d u r i n g t h e i n c u b a t i o n p e r i o d ( e . g . , Goldman e t a l . , 1 9 8 1 ; F i s h e r e t a l . , 1 9 8 1 ) . U p t a k e r a t e s r e p o r t e d a r e 57 t h e r e f o r e n o t n e c e s s a r i l y i n s i t u v a l u e s b u t i n d i c a t i v e o f p o t e n t i a l r a t e s o f N u p t a k e t h a t c a n be r e a l i z e d b y t h e p h y t o p l a n k t o n c o m m u n i t y when p r o v i d e d w i t h s a t u r a t i n g c o n c e n t r a t i o n s o f N, a c o n d i t i o n o f t e n a c h i e v e d f o r N O 3 -u p t a k e i n t h e s u r f a c e , a n d e s p e c i a l l y d e e p e r c o m m u n i t i e s , b u t s e l d o m o b s e r v e d f o r t h e u p t a k e o f r e g e n e r a t e d N i n n a t u r a l s i t u a t i o n s . The i n c u b a t i o n ( s a m p l i n g ) i n t e r v a l s i n t h e t i m e c o u r s e e x p e r i m e n t s w e r e l o n g (3-6 h) r e l a t i v e t o t h e r a p i d u p t a k e r e s p o n s e o f p h y t o p l a n k t o n s e e n i n t h e l a b o r a t o r y ( e . g . , Conway e t a l . , 1976; P a r s l o w e t a l . , 1 9 8 4 a , b) a n d t h e f i e l d ( e . g . , G l i b e r t a n d G o l d m a n , 1981; P r i s c u a n d P r i s c u , 1984) and t h u s I was u n a b l e t o d e t e c t s h o r t t e r m v a r i a t i o n s i n u p t a k e r a t e . E n h a n c e d u p t a k e o f N H 4 + a n d u r e a by N 0 3 ~ - s u f f i c i e n t p h y t o p l a n k t o n h a v e b e e n p r e v i o u s l y r e p o r t e d ( H o r r i g a n a n d M c C a r t h y , 1 9 8 1 , 1982; P a r s l o w e t a l . , 1 9 8 4 b ) . I n l i g h t o f t h e s l o w e r , l o n g t e r m r a t e s o f r e g e n e r a t e d N u p t a k e r e l a t i v e t o N O 3 - u p t a k e i n t h e f r o n t a l s t a t i o n o f S t r a i t o f G e o r g i a (TC.2) a n d t h e u p w e l l e d p l u me (TC.4) o f f V a n c o u v e r I s l a n d i t i s u n l i k e l y t h a t s u c h e n h a n c e d u p t a k e p r o c e s s e s o c c u r r e d on t i m e s c a l e s s h o r t e r t h a n t h e s a m p l i n g i n t e r v a l s e m p l o y e d i n t h e p r e s e n t s t u d y . B o t t l e c o n t a i n m e n t e f f e c t s h a v e b e e n shown t o l e a d t o s e r i o u s u n d e r e s t i m a t e s o f r a t e p r o c e s s e s ( V e n r i c k e t a l . , 1 9 7 7 ) . One w o u l d e x p e c t t h e c o n s e q u e n c e s o f c o n t a i n m e n t t o be most s e v e r e i n h i g h e r b i o m a s s c o m m u n i t i e s b u t t h e c o n s t a n t r a t e s o f C h i a a n d POC a n d PON s y n t h e s i s i n TC.2 a n d TC.3 i n d i c a t e no s u c h a r t i f a c t s i n t h e s e e x p e r i m e n t s . 5 Simultaneous uptake of nitrogen compounds S i m u l t a n e o u s u t i l i z a t i o n o f NH^"1" a n d N O 3 - i s w e l l d o c u m e n t e d i n l a b o r a t o r y ( e . g . , E p p l e y a n d R e n g e r , 1974; B i e n f a n g , 1975; C a p e r o n a n d Z i e m a n n , 1976; DeManche e t a l . , 1979; D o r t c h a n d Conway, 1984) a n d n a t u r a l p h y t o p l a n k t o n a s s e m b l a g e s ( e . g . , C o l l o s a n d L e w i n , 1974; C o n o v e r , 1975; Conway, 1977; M c C a r t h y e t a l . , 1977; M a e s t r i n i e t a l . , 1982, 1986, Q u e g u i n e r e t a l . , 1986; C o l l o s e t a l . , 1 9 8 9 ) . The r e s u l t s o f TC.2 a n d TC.3, c o n d u c t e d i n t h e f r o n t a l a n d s t r a t i f i e d a r e a s o f t h e S t r a i t o f G e o r g i a , r e s p e c t i v e l y n o t o n l y d e m o n s t r a t e d u a l 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 b u t t h a t N H 4 + , N O 3 - , a n d u r e a may be t a k e n up c o n c u r r e n t l y . As f i r s t p o i n t e d o u t by C o l l o s ( 1 9 8 7 ) , 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 w i l l r e s u l t i n a r e d u c t i o n o f t h e n i t r o g e n -1 c s p e c i f i c u p t a k e r a t e o f t h e N - l a b e l l e d compound c o m p a r e d t o t h e N - s p e c i f i c u p t a k e r a t e d e t e r m i n e d when o n l y t h e 1 ^ N -l a b e l l e d compound i s b e i n g t a k e n u p. I n a l l t h e e x p e r i m e n t s 1 5 s a t u r a t i n g a d d i t i o n o f t h e N - l a b e l l e d compound o f i n t e r e s t was u s e d a n d t h e e f f e c t s o f i s o t o p i c d i l u t i o n , due t o u n l a b e l l e d n i t r o g e n b e i n g t a k e n up, a r e p o t e n t i a l l y o n l y a p r o b l e m i n a r e a s o f h i g h a m b i e n t n i t r o g e n l e v e l s ( i . e . N H 4 + u p t a k e i n T C . 4 ) , I n TC.2 a n d TC.3 t h e a b s o l u t e u p t a k e r a t e s w e r e c a l c u l a t e d u s i n g t h e f i n a l PON d e t e r m i n e d a t t h e e n d o f an i n c u b a t i o n ; w h i c h g i v e s an a c c u r a t e m e a s u r e o f u p t a k e r a t e 15 o f t h e N - l a b e l l e d n u t r i e n t i n t o t h e p h y t o p l a n k t o n a n d a v o i d p o t e n t i a l a r t i f a c t s c a u s e d by i n c o r p o r a t i o n o f n o n - ^ N -l a b e l l e d n i t r o g e n f o r m s . 59 M a e s t r i n i e t a l . (1982) d e m o n s t r a t e d t h a t m i c r o a l g a e o f o y s t e r p o n d s t o o k up N H 4 + a n d N O 3 - a t t h e same r a t e o n c e t h e N H 4 + c o n c e n t r a t i o n h a d d e c r e a s e d t o c a . 7 ug-at N » L - 1 . The r e s u l t s f r o m t h e f r o n t a l c o m m u n i t y (TC.2) d e m o n s t r a t e d t h e s i m i l a r i t y o f N H 4 + a n d N O 3 - u p t a k e r a t e s i n t h e N H 4 + e n r i c h e d s a m p l e s . However, t h e N O 3 - u p t a k e r a t e was r e d u c e d b y 5 0 % i n t h e N H 4 + s p i k e d s a m p l e s a s c o m p a r e d t o t h e NC^ - s p i k e d s a m p l e s . S i m i l a r N H 4 + s u p p r e s s i o n o f NC>3~ u p t a k e h a s b e e n r e p o r t e d f o r b o t h l a b o r a t o r y ( e . g . , G r a n t e t a l . , 1967; Conway, 1977; C r e s s w e l l a n d S y r e t t , 1979) a n d n a t u r a l p h y t o p l a n k t o n a s s e m b l a g e s ( e . g . , M c C a r t h y e t a l . , 1977, B l a s c o a n d Conway, 1 9 8 2 ) . The e f f e c t s o f NH 4 + a d d i t i o n on u r e a u p t a k e h a s b e e n l e s s s t u d i e d t h a n i n t e r a c t i o n s b e t w e e n N O 3 -a n d N H 4 + . N e v e r t h e l e s s i t a p p e a r s f r o m t h e f e w a v a i l a b l e s t u d i e s t h a t N H 4 + s u p p r e s s e s u r e a u p t a k e a n d t h e i n h i b i t o r y r e s p o n s e i s e i t h e r i n s t a n t a n e o u s ( W i l l i a m s a n d H o d s o n , 1977; L u n d , 1987) o r m a n i f e s t e d l a t e r ( H o r r i g a n a n d M c C a r t h y , 1982; M o l l o y a n d S y r e t t , 1988a;) I n TC.2 a n d TC.3 t h e e f f e c t o f N H 4 + on u r e a d i s a p p e a r a n c e u p t a k e r a t e i s d i f f i c u l t t o d i s c e r n due t o t h e l o w a m b i e n t c o n c e n t r a t i o n s o f u r e a a n d e v i d e n c e o f u r e a r e g e n e r a t i o n d u r i n g t h e i n c u b a t i o n p e r i o d . I n TC.2 t h e N O 3 - d i s a p p e a r a n c e u p t a k e r a t e was u n a f f e c t e d o r s l i g h t l y e n h a n c e d i n t h e p r e s e n c e o f u r e a i n a g r e e m e n t w i t h t h e l a b o r a t o r y s t u d y o f L u n d (1987) who f o u n d no s u p p r e s s i o n o f N O 3 - u p t a k e i n t h e m a r i n e d i a t o m , Skeletonema costatum. M o l l o y a n d S y r e t t (1988b) f o u n d s i m u l t a n e o u s u p t a k e o f NC"3~ a n d u r e a , b u t u r e a i n h i b i t i o n ( 2 4 - 2 6 % ) o f N O 3 - u p t a k e i n 60 c u l t u r e s o f C h l o r e l l a emersonii a n d Phaeodactylum tricornutum a f t e r p r o l o n g e d (1-2 d) N d e p r i v a t i o n . P a r t i a l i n h i b i t i o n o f u r e a u p t a k e by N O 3 - h a s b e e n r e p o r t e d i n n a t u r a l s e a w a t e r s a m p l e s ( M c C a r t h y a n d E p p l e y , 1972) a n d l a b o r a t o r y c u l t u r e s o f Skeletonema costatum ( L u n d , 1987) a n d P. tricornutum a n d C. emersonii ( M o l l o y a n d S y r e t t , 1988b) . /Ambient c o n c e n t r a t i o n s o f u r e a i n t h e f r o n t a l a n d s t r a t i f i e d s t a t i o n s ( T C . 2 , TC.3) w e r e l o w . The l o w c o n c e n t r a t i o n s o b s e r v e d a n d t h e p o s s i b i l i t y o f u r e a r e g e n e r a t i o n d u r i n g t h e i n c u b a t i o n p e r i o d makes i t d i f f i c u l t t o d i s c e r n any i n h i b i t o r y e f f e c t o f NC^ - on u r e a u p t a k e . I n TC . 4 , a n d TC.5 r e l a t i v e l y h i g h a m b i e n t c o n c e n t r a t i o n s o f u r e a ( 0.75 - 2.38 uq-at N ' L - 1 ) w e r e o c c a s i o n a l l y o b s e r v e d i n t h e u p p e r w a t e r s s a m p l e d (2-7 m) w h i c h may h a v e , e i t h e r b i o l o g i c a l l y o r t h r o u g h i s o t o p i c 1 5 d i l u t i o n o f N i n p a r t i c u l a t e m a t e r i a l , r e d u c e d t h e p o t e n t i a l r a t e s o f s p e c i f i c N O 3 - u p t a k e s a s r e p o r t e d . Effects of light/dark regime on nitrogen uptake D i e l p e r i o d i c i t y i n t h e u p t a k e r a t e s o f NO3 -, NH^ + a n d u r e a w e r e e v i d e n t i n t h e t i m e c o u r s e e x p e r i m e n t s o f t h e v a r i o u s n a t u r a l p h y t o p l a n k t o n a s s e m b l a g e s . I n T C . l , c o n d u c t e d i n t h e N C > 3 - - r e p l e t e w a t e r s o f t h e n o r t h e a s t P a c i f i c O c e a n , n i g h t t i m e N O 3 - u p t a k e r a t e s w e r e a b o u t h a l f t h o s e r e p o r t e d d u r i n g t h e d a y t i m e , w i t h a p a t t e r n o f m a x i m a l r a t e s d u r i n g m i d - d a y a n d l o w e r r a t e s i n t h e m o r n i n g a n d a f t e r n o o n . S i m i l a r s t r o n g , d i e l p a t t e r n s i n N03~ u p t a k e h a v e b e e n o b s e r v e d i n t h e N 0 3 ~ - r i c h A n t a r c t i c w a t e r s by K o i k e e t a l . (1986) w h e r e t h e u p t a k e o f NO3 - a n d N H 4 + d u r i n g t h e n i g h t t i m e a m o u n t e d t o c a . 6 1 10-30 and 50%, respectively of the daytime values. Olson (1980) found i n 2 time course experiments that N O 3 - uptake ceased during the nighttime and NH 4 + uptake was either 25 or 85% of the daytime rate, whereas G l i b e r t et a l . (1982a) found anomalous r e s u l t s ; they found no difference i n N O 3 - uptake between samples incubated i n the dark and those incubated over a normal light-dark regime for samples c o l l e c t e d from the N O 3 -- r i c h Scotia Sea. During TC.l when samples which had normally been exposed to a natural L:D cycle were suddenly darkened during mid-day, a lag of > 3 h occurred before dark uptake rates declined to the rate observed during 24 h of darkness. The i n i t i a l "dark" N O 3 - uptake rate may have been the re s u l t of previous l i g h t stimulation and only a f t e r some elapsed time were the reductants, cofactors and enzymes that are necessary for N O 3 - assimilation, and produced (or activated) during the l i g h t period, used up (or deactivated) during the a r t i f i c i a l l y imposed darkness. In the coastal waters of the S t r a i t of Georgia si m i l a r patterns of d i e l uptake of a l l 3 N substrates were observed for the NO-^-replete and NO-^-deplete s t r a t i f i e d waters. Nitrate and NH 4 + nighttime uptake rates were about one-third of the average daytime rates i n the f r o n t a l waters and declined to about half t h i s value i n the s t r a t i f i e d waters. Nighttime uptake rates of urea were ca. 75 and 15% of the daytime rates for f r o n t a l and s t r a t i f i e d waters, respectively. Fisher et a l . (1982) measured sim i l a r d i e l v a r i a t i o n s i n NH 4 + uptake i n an estuarine phytoplankton community while d i e l 62 v a r i a t i o n i n N O 3 - u p t a k e r a t e s h a v e b e e n r e c o r d e d f o r f r e s h w a t e r r e s e r v o i r p l a n k t o n ( T o e t z , 1976) a n d Ceratophyllum-p e r i p h y t o n c o m m u n i t i e s ( T o e t z , 1 9 7 1 ) . P r i c e a n d H a r r i s o n ( 1 9 8 8 a ) o b s e r v e d d i e l p e r i o d i c i t y f o r s a t u r a t e d u r e a u p t a k e r a t e s o f s a m p l e s c o l l e c t e d f r o m N 0 3 ~ - d e p l e t e a r e a s o f t h e S a r g a s s o S e a , b u t no d e f i n i t i v e l i g h t / d a r k t r e n d s i n N O 3 - -r e p l e t e a r e a s . The c o n s t a n c y o f V D : V L ( d a r k i n c u b a t i o n b o t t l e : c l e a r i n c u b a t i o n b o t t l e ) f o r NH 4 + i n t h e f r o n t a l w a t e r , when N H 4 + u p t a k e r a t e s o f p h y t o p l a n k t o n e x p o s e d t o t h e n a t u r a l l i g h t / d a r k c y c l e w e r e p e r i o d i c , s u g g e s t s t h a t N H 4 + u p t a k e i s c i r c a d i a n ; i n a b s e n c e o f t h e l i g h t / d a r k c y c l e t h e r h y t h m i s f r e e r u n n i n g ( s e e C h i s h o l m , 1 9 8 1 ) . T h i s c o n c l u s i o n i s s u p p o r t e d b y G o e r i n g e t a l . (1964) who f o u n d r h y t h m i c v a r i a t i o n i n b o t h p o t e n t i a l N H 4 + a n d NC>3~ u p t a k e by s u r f a c e p h y t o p l a n k t o n c o m m u n i t i e s o f t h e S a r g a s s o S e a u n d e r c o n t i n u o u s i l l u m i n a t i o n . However, K r i s t i a n s e n a n d L u n d (1989) f o u n d no d i e l v a r i a b i l i t y i n p o t e n t i a l u p t a k e r a t e s o f N O 3 - , N H 4 + o r u r e a i n s a m p l e s c o l l e c t e d f r o m t h e N - d e p l e t e d B a r e n t s S e a a n d i n c u b a t e d u n d e r c o n s t a n t l i g h t , r e s u l t s w h i c h do n o t s u p p o r t t h e e n d o g e n o u s t h e o r y o f u p t a k e c o n t r o l . I n t h e S t r a i t o f G e o r g i a t h e r e s u l t s o f V D : V L f o r u r e a a n d NC^ - w e r e s i m i l a r t o e a c h o t h e r a n d d e m o n s t r a t e t h a t t h e i r d e p e n d e n c y on l i g h t was c o m p a r a b l e . The l i g h t d e p e n d e n c e o f u p t a k e o f t h e s e n u t r i e n t s i s d i s c u s s e d i n d e t a i l i n C h a p t e r 2. L a b o r a t o r y s t u d i e s h a v e shown t h a t N - d e p r i v e d p h y t o p l a n k t o n h a v e h i g h e r d a r k u p t a k e r a t e s o f n i t r o g e n t h a n 63 N - r e p l e t e p h y t o p l a n k t o n ( e . g . , S y r e t t , 1962; E p p l e y a n d C o a t s w o r t h , 1968; H a r r i s o n , 1976; Rees a n d S y r e t t , 1 9 7 9 ) . I n t h e S t r a i t o f G e o r g i a d a r k N u p t a k e r a t e s n o r m a l i z e d t o C h i a w e r e h i g h e s t i n t h e N - d e p l e t e d s t r a t i f i e d w a t e r i n a g r e e m e n t w i t h t h e s e o b s e r v a t i o n s ; a l s o r e l a t i v e t o t h e f r o n t a l c o m m u n i t y , d a r k u p t a k e r a t e s w e r e a g r e a t e r p r o p o r t i o n o f t h e l i g h t r a t e s f o r N H 4 + , N O 3 - a n d u r e a i n s t r a t i f i e d w a t e r . The h i g h e r C h i a s p e c i f i c u p t a k e r a t e s o f N H 4 + a n d u r e a i n s t r a t i f i e d w a t e r a n d o f N O 3 - i n f r o n t a l w a t e r a r e c o n s i s t e n t w i t h t h e way n i t r o g e n i s e n v i s a g e d t o s u p p o r t t h e s e a r e a s . S p e c i f i c a l l y , r e g e n e r a t e d N ( N H 4 + a n d u r e a ) h a s b e e n shown t o s u p p l y most o f t h e p h y t o p l a n k t o n n i t r o g e n demand i n N - d e p l e t e d w a t e r s a n d a s t h e c o n c e n t r a t i o n o f a m b i e n t N O 3 - i n c r e a s e s s o d o e s t h e r e l a t i v e i m p o r t a n c e ( n o t p r e f e r e n c e ) o f N O 3 - f o r p h y t o p l a n k t o n n i t r o g e n r a t i o n ( e . g . , M c C a r t h y e t a l . , 197 7; H a r r i s o n , 1980; G l i b e r t e t a l . , 1982b; C o c h l a n 1 9 8 6 ) . I t i s i m p o r t a n t t o remember t h a t t h e s p e c i e s c o m p o s i t i o n o f t h e two p h y t o p l a n k t o n c o m m u n i t i e s c o n t r a s t e d m a r k e d l y a n d l i k e l y c o n t r i b u t e d t o t h e o b s e r v e d v a r i a b i l i t y i n t h e l i g h t r e s p o n s e a n d p r e c l u d e s an e x p l a n a t i o n o f d i e l p e r i o d i c i t y o f N u p t a k e b a s e d m e r e l y on t h e p h y t o p l a n k t o n c o m m u n i t i e s ' n i t r o g e n s t a t u s . The r h y t h m i c p a t t e r n o f N O 3 - u p t a k e r a t e s o b s e r v e d i n t h e u p p e r w a t e r s (1-7 m) o f t h e u p w e l l e d N 0 3 ~ - r i c h p l u m e a r e i n d i c a t i v e o f i n s i t u u p t a k e d i e l p e r i o d i c i t y w h e r e n i g h t t i m e v a l u e s w e r e 15-16% o f d a y t i m e r a t e s ; t h e p a t t e r n o f p o t e n t i a l r a t e s o f N H 4 + u p t a k e was s i m i l a r b u t n i g h t t i m e r a t e s w e r e a 64 greater proportion (ca. 30-36% of the daytime values). Similar d i e l p e r i o d i c i t y of N O 3 - and NH 4 + uptake rates of natural phytoplankton communities i n upwelled regions have been observed by others. Eppley et a l . (1970) found that Peru Current phytoplankton had nighttime rates ca. 25 and 62% of daytime values for N O 3 - and NH 4 +, respectively. Collos and Slawyk (1976) also observed d i e l v a r i a t i o n of NC^- uptake i n shipboard cultures of surface communities c o l l e c t e d i n the upwelling area off Northwest A f r i c a ; nighttime values were ca. 20% of daytime rates of uptake. In eutrophic Lake Biwa, Japan, Mitamura and Saijo (1986) found nighttime uptake rates of N O 3 - to be only 10% of daytime rates and although the phase for urea and NH^4" uptake p e r i o d i c i t y corresponded to that of N O 3 - the amplitude was lower with nighttime values 80 and 95% of daytime rates. The absence of an apparent d i e l rhythm i n NC>3~ and NH 4 + uptake of the deeper (1% I Q) community of the upwelled plume was also observed by Maclsaac (1978) i n the NC>3--replete 1% I Q samples of a phytoplankton community dominated by the d i n o f l a g e l l a t e Gonyaulax polyedra off Baja, Mexico. D i e l p e r i o d i c i t y i n both pote n t i a l NH 4 + and NC^-uptake rates was, however, observed down to the 10% I Q depth. The amplitude of d i e l p e r i o d i c i t y of poten t i a l N O 3 - uptake rates observed i n the NC^-deplete surface waters used i n TC.5 was greater than that observed for i n s i t u NC>3~ uptake of the N03~-replete surface waters of TC.4; nighttime rates were ca. 40% of daytime values compared to 15-16% i n TC.4. These rates, however, are potential rates of uptake which represent 65 r a t e s t h a t may be r e a l i z e d u n d e r c o n d i t i o n s o f c o n c e n t r a t i o n s s a t u r a t i n g t o u p t a k e . S a l h s t e n (1987) c o u l d d i s c e r n no d i e l p a t t e r n f o r i n s i t u u p t a k e r a t e s o f N H 4 + o r N O 3 - i n t h e o l i g o t r o p h i c , c e n t r a l N o r t h P a c i f i c G y r e . S i m i l a r l y a b s e n c e o f d i e l p e r i o d i c i t y i n i n s i t u u p t a k e r a t e s o f NC^ - a n d N H 4 + h a v e b e e n o b s e r v e d f o r n a t u r a l a s s e m b l a g e s c o l l e c t e d f r o m N-d e p l e t e s u r f a c e w a t e r s o f t h e e a s t e r n C a n a d i a n A r c t i c ( H a r r i s o n , 1 9 8 3 a ) , t h e c o n t i n e n t a l s h e l f o f f Nova S c o t i a ( C o c h l a n , 1982, 1986) a n d t h e u l t r a o l i g o t r o p h i c T o o l i k L a k e , A l a s k a ( W h a l e n a n d A l e x a n d e r , 1 9 8 4 a ) . A l t h o u g h p o t e n t i a l r a t e s o f N H 4 + d i d n o t show d i e l p e r i o d i c i t y ( C o c h l a n , 1982; W h a l e n a n d A l e x a n d e r , 1984a) d i e l p e r i o d i c i t y o f p o t e n t i a l N O 3 - u p t a k e r a t e s o f t h e f r e s h w a t e r c o m m u n i t y was o b s e r v e d . The d e e p e r p h y t o p l a n k t o n c o m m u n i t y i n TC.5 was n o t N O 3 - -d e p l e t e a n d more s i m i l a r i n s p e c i e s c o m p o s i t i o n t o t h e u p w e l l e d c o m m u n i t i e s o f TC.4. A l t h o u g h i t d i d n o t d e m o n s t r a t e a d e f i n i t i v e d i e l p a t t e r n o f u p t a k e , l o w e r r a t e s o f NO^ -u p t a k e w e r e g e n e r a l l y o b s e r v e d a t n i g h t w i t h i n c r e a s e d u p t a k e r a t e s i n t h e d a y p e a k i n g l a t e r t h a n t h o s e o f t h e s u r f a c e c o m m u n i t y . M i y a z a k i e t a l . (1987) o b s e r v e d a s i m i l a r t i m e d e l a y o f u p t a k e maxima f o r N O 3 - a n d N H 4 + d u r i n g d a r k b o t t l e i n c u b a t i o n s o f t h e p h y t o p l a n k t o n o f L a k e Nakanuma, J a p a n . T hey a t t r i b u t e d t h i s d e l a y t o t h e c u m u l a t i v e i n c r e a s e i n s t o r e d e n e r g y a n d i n t e r m e d i a t e c a r b o n compounds p r o d u c e d d u r i n g p h o t o s y n t h e s i s a n d n e c e s s a r y f o r t h e u p t a k e a n d a s s i m i l a t i o n o f n i t r o g e n . I n summary i t a p p e a r s t h a t d i e l p e r i o d i c i t y o f n i t r o g e n u p t a k e i s i n f l u e n c e d by s e v e r a l c o n f o u n d i n g f a c t o r s i n c l u d i n g : (1) t h e amount o f p h y t o p l a n k t o n b i o m a s s a n d i t s s p e c i e s c o m p o s i t i o n ; (2) v a r i a t i o n i n c o n c e n t r a t i o n o f a m b i e n t n i t r o g e n o u s compounds a s t h e s u b s t r a t e f o r N u p t a k e ; (3) v a r i a t i o n i n l i g h t i n t e n s i t y ( i r r a d i a n c e ) : a n d (4) t h e d e p t h f r o m w h i c h p l a n k t o n i s c o l l e c t e d , h e n c e p r e c o n d i t i o n e d l i g h t h i s t o r y o f p h y t o p l a n k t o n . I n a d d i t i o n , t h e i n h i b i t o r y e f f e c t s o f o t h e r N f o r m s ( e . g . , N H 4 + , T o b i e s e n , 1 9 8 7 ) , t h e e f f e c t o f i r r a d i a n c e a n d a m b i e n t w a t e r t e m p e r a t u r e on p e r i o d i c i t y o f enzyme a c t i v i t i e s ( e . g . , NC>3~ a n d NC^ - r e d u c t a s e , E p p l e y e t a l . , 1970, 1971b) may a l s o be r e f l e c t e d i n t h e o b s e r v e d p e r i o d i c i t y o f n i t r o g e n u p t a k e by n a t u r a l p h y t o p l a n k t o n a s s e m b l a g e s . E p p l e y e t a l . ( 1 9 7 1 a ) s u g g e s t e d t h a t t h e s t r u c t u r e a n d d i v e r s i t y o f t h e p h y t o p l a n k t o n c o m m u n i t y m i g h t be a f f e c t e d b y d i e l p e r i o d i c i t y o f c e l l d i v i s i o n o c c u r r i n g a t d i f f e r e n t t i m e s o f t h e d a y . By t h e same l i n e o f r e a s o n i n g d i e l r h y t h m s i n N u p t a k e may a l s o p l a y a s i g n i f i c a n t r o l e i n t h e r e g u l a t i o n o f s p a t i a l a n d t e m p o r a l d i s t r i b u t i o n o f p h y t o p l a n k t o n . The r e s u l t s o f t h e p r e s e n t s t u d y d e m o n s t r a t e t h e n e e d f o r c o n t r o l l e d l a b o r a t o r y e x p e r i m e n t s u t i l i z i n g u n i a l g a l p o p u l a t i o n s i n o r d e r t o i s o l a t e a n d d e t e r m i n e t h e e f f e c t s o f N l i m i t a t i o n on t h e p e r i o d i c i t y o f N u p t a k e by p h y t o p l a n k t o n . 67 CHAPTER TWO EFFECTS OF IRRADIANCE ON NITROGEN UPTAKE BY PHYTOPLANKTON: COMPARISON OF FRONTAL AND S T R A T I F I E D COMMUNITIES INTRODUCTION I n m o s t m a r i n e a n d f r e s h w a t e r s y s t e m s , t h e u p t a k e o f n i t r o g e n o u s n u t r i e n t s by p h y t o p l a n k t o n i s r e l a t e d t o t h e a v a i l a b i l i t y o f t h e n u t r i e n t s ( e . g . M a c l s a a c a n d D u g d a l e , 1969; P r o b y n , 1985) a n d p h o t o s y n t h e t i c p h o t o n f l u x d e n s i t y (PPFD) ( e . g . , M a c l s a a c a n d D u g d a l e , 1972; P r i s c u , 1 9 8 4 ) . The d e p e n d e n c e o f n i t r o g e n u p t a k e u p o n PPFD h a s b e e n d e s c r i b e d b y a r e c t a n g u l a r h y p e r b o l a s i m i l a r t o t h e M i c h a e l i s - M e n t e n f o r m u l a t i o n i n many m a r i n e ( e . g . , M a c l s a a c a n d D u g d a l e , 1972; F i s h e r e t a l . , 1982) a n d f r e s h w a t e r ( e . g . , P r i s c u , 1984; W h a l e n a n d A l e x a n d e r , 1984b) c o m m u n i t i e s . A l t h o u g h n i t r o g e n u p t a k e a n d a s s i m i l a t i o n by p h y t o p l a n k t o n a r e d e p e n d e n t u p o n PPFD a s a n e n e r g y s o u r c e , e i t h e r d i r e c t l y o r i n d i r e c t l y t h r o u g h p h o t o s y n t h e s i s , t h e e x a c t b i o c h e m i c a l m e c h a n i s m ( s ) by w h i c h l i g h t r e g u l a t e s n i t r o g e n m e t a b o l i s m r e m a i n s u n r e s o l v e d ( e . g . s e e r e v i e w by S y r e t t , 1 9 8 1 ) . The p r e s e n c e o f N O 3 - -a c t i v a t e d A T P a s e , a p p a r e n t l y l o c a t e d w i t h i n t h e c e l l membranes o f a number o f m a r i n e p h y t o p l a n k t e r s ( F a l k o w s k i 1 9 7 5 a , b ) , p r o v i d e s a p h y s i o l o g i c a l b a s i s f o r t h e c o u p l i n g b e t w e e n l i g h t a n d N O 3 - u p t a k e a n d p r o b a b l y s p e c i f i c A T P a s e s e x i s t f o r t h e u p t a k e o f N H 4 + a n d u r e a a s w e l l . The e n e r g y (ATP) g e n e r a t e d by p h o t o p h o s p h o r y l a t i o n i s r e q u i r e d f o r t h e f u n c t i o n i n g o f t h e s e u p t a k e enzymes ( p e r m e a s e s ) a n d may a l s o d r i v e t h e r e a c t i o n s o f N H 4 + (GS/GOGAT) a n d u r e a ( U A L - a s e ) a s s i m i l a t i o n . 68 In addition, the photogeneration of reductants NAD(P)H and reduced ferredoxin w i l l drive the reduction of NG^-, NG^- and the GOGAT reaction of NH 4 + a s s i m i l a t i o n . Other possible interactions of l i g h t with inorganic nitrogen metabolism of phytoplankton are discussed i n d e t a i l by Syrett (1981). Numerous culture studies have demonstrated that nitrogen-deprived phytoplankton have greater dark uptake rates of N than N-replete phytoplankton (e.g., Syrett, 1962; Eppley and Coatsworth, 1968; Thacker and Syrett, 1972b; Rees and Syrett, 1979) suggesting a lesser l i g h t dependence on N uptake, during N stress. This together with f i e l d studies which show that deep-living phytoplankton sustain substantial N uptake v e l o c i t i e s with l i t t l e or no l i g h t (e.g., Conway and Whitledge, 1979; Nelson and Conway, 1979; Priscu, 1984) suggests that both l i g h t exposure and n u t r i t i o n a l history of phytoplankton may be important i n determining t h e i r a b i l i t y to sequester nitrogen, and that these c o n t r o l l i n g factors may d i f f e r for the various forms of nitrogen. Shallow sea fronts, located at the boundary between s t r a t i f i e d and v e r t i c a l l y mixed regimes (see reviews by Denman and Powell, 1984; LeFevre, 1986) are generally areas of high primary productivity (e.g., Pingree et a l . , 1975; Parsons et a l . , 1981, 1983; Holligan et a l . , 1984). These regions are characterized by having high phytoplankton biomass i n the surface water with measurable concentrations of n i t r a t e , and a shallow pycnocline which extends to the surface at the f r o n t a l boundary (e.g., Simpson and Pingree 1978). 69 A s u r f a c e t r a n s e c t n o r m a l t o a f r o n t a l b o u n d a r y p r o g r e s s e s f r o m h i g h c o n c e n t r a t i o n s o f d i s s o l v e d NC^ - on t h e w e l l - m i x e d s i d e t o N - d e p l e t e s t r a t i f i e d w a t e r s a n d t h u s r e p r e s e n t s a g r a d i e n t o f b o t h n i t r o g e n a n d l i g h t a v a i l a b i l i t y a n d c o n s e q u e n t l y p h y t o p l a n k t o n p h y s i o l o g i c a l s t a t e s . M o r e o v e r , t h e n i t r o g e n o u s n u t r i t i o n o f t h e p h y t o p l a n k t o n w o u l d l i k e l y d i f f e r a l o n g s u c h a t r a n s e c t . I n t h e N - i m p o v e r i s h e d w a t e r s , t h e N demands o f p h y t o p l a n k t o n a r e s u p p l i e d by r e d u c e d N f o r m s s u c h a s N H 4 + a n d u r e a f r o m r e g e n e r a t i v e p r o c e s s e s w h e r e a s , i n N - r i c h a r e a s , n i t r o g e n compounds a r e g e n e r a l l y u t i l i z e d a t r a t e s p r o p o r t i o n a l t o t h e i r a v a i l a b i l i t y ( e . g . , D u g d a l e a n d G o e r i n g 1967; M c C a r t h y e t a l . , 1 9 7 7 ) . The e x p e r i m e n t s p r e s e n t e d i n t h i s s t u d y w e r e c o n d u c t e d i n t h e S t r a i t o f G e o r g i a , a p a r t i a l l y e n c l o s e d c o a s t a l b a s i n on t h e w e s t c o a s t o f C a n a d a ( s e e r e v i e w s by L e B l o n d , 1983; H a r r i s o n e t a l . , 1 9 8 3 ) , w h e r e s e v e r a l t i d a l l y - i n d u c e d f r o n t a l r e g i o n s h a v e b e e n p r e v i o u s l y d e s c r i b e d ( P a r s o n s e t a l . , 1 9 8 1 ; P r i c e e t a l . , 1 9 8 5 ) . The i n f l u e n c e o f PPFD on t h e u p t a k e o f NC>3~ a n d u r e a by p h y t o p l a n k t o n f r o m n i t r a t e - r e p l e t e f r o n t a l w a t e r a n d n i t r a t e - d e p l e t e s t r a t i f i e d w a t e r was e x a m i n e d a n d t h e d e p e n d e n c e o f N u p t a k e on PPFD by t h e p h y t o p l a n k t o n f r o m t h e s u b s u r f a c e c h l o r o p h y l l maximum o f t h e s e t w o d i s t i n c t a r e a s c o m p a r e d . T h i s s t u d y i s t h e f i r s t t o m e a s u r e b o t h u r e a a n d N O 3 - u p t a k e b y n a t u r a l a s s e m b l a g e s o f p h y t o p l a n k t o n a s a f u n c t i o n o f PPFD. S i m u l a t e d i n s i t u e x p e r i m e n t a l c o n d i t i o n s w e r e a t t e m p t e d i n o r d e r t o o b t a i n a b e t t e r u n d e r s t a n d i n g o f t h e t r u e N O 3 - u p t a k e r e s p o n s e t o PPFD i n t h e s e p h y s i c a l l y a n d 70 c h e m i c a l l y d i s t i n c t environments. P r e v i o u s s t u d i e s of the e f f e c t ( s ) of PPFD on N uptake by phytoplankton have employed s a t u r a t i n g enrichments of i s o t o p i c a l l y l a b e l l e d N forms (e.g., Maclsaac and Dugdale, 1972; P r i s c u 1984; Mitamura 1986) and r e p o r t e d uptake r a t e s may r e f l e c t the e f f e c t s of both PPFD and N c o n c e n t r a t i o n . 71 MATERIALS AND METHODS General N i t r o g e n u p t a k e e x p e r i m e n t s w e r e c o n d u c t e d i n t h e S t r a i t o f G e o r g i a , B.C., C a n a d a a b o a r d t h e C.S.S. V e c t o r d u r i n g J u l y -A u g u s t , 1984; s t a t i o n l o c a t i o n s a r e shown i n F i g . 2.1. B e t w e e n 1400 a n d 1500 h PDT w a t e r s a m p l e s w e r e c o l l e c t e d , u s i n g 5 L PVC N i s k i n b o t t l e s , f r o m j u s t b e l o w t h e s e a s u r f a c e ( 0 - 1 m) a n d f r o m d e p t h s c o r r e s p o n d i n g t o t h e d e e p c h l o r o p h y l l maximum (DCM). S a m p l e s w e r e s h i e l d e d f r o m d i r e c t s u n l i g h t d u r i n g t r a n s f e r t o 10 L N a l g e n e c a r b o y s a n d t a k e n i n t o t h e s h i p ' s l a b o r a t o r y . S u b s a m p l e s f o r n u t r i e n t a n a l y s e s w e r e r e m o v e d w i t h a n a c i d - w a s h e d s y r i n g e a n d g e n t l y f i l t e r e d t h r o u g h c o m b u s t e d (460°C f o r 4 h) Whatman GF/F f i l t e r s ( m o u n t e d i n 25 • • R mm M i l l i p o r e S w i n e x f i l t e r h o l d e r s ) i n t o a c i d - w a s h e d p o l y e t h y l e n e b o t t l e s . . N i t r a t e p l u s n i t r i t e ( N 0 3 ~ + N 0 2 ~ ) a n d ammonium ( N H 4 + ) w e r e m e a s u r e d i m m e d i a t e l y w i t h a T e c h n i c o n •p A u t o A n a l y z e r I I , f o l l o w i n g t h e p r o c e d u r e s o u t l i n e d i n Wood e t a l . ( 1 9 6 7 ) a n d S l a w y k a n d M a c l s a a c ( 1 9 7 2 ) , r e s p e c t i v e l y . U r e a was d e t e r m i n e d b y t h e 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 t e c h n i q u e d e s c r i b e d by P r i c e a n d H a r r i s o n ( 1 9 8 7 ) . S a m p l e s f o r c h l o r o p h y l l a ( C h i a) w e r e c o l l e c t e d on Whatman GF/F f i l t e r s a n d 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 . C h i a was e x t r a c t e d i n 90% a c e t o n e o v e r n i g h t a n d a n a l y z e d by i n v i t r o f l u o r o m e t r y ( S t r i c k l a n d a n d P a r s o n s , 1972) u s i n g a T u r n e r D e s i g n s m o d e l 10 f l u o r o m e t e r . P a r t i c u l a t e o r g a n i c c a r b o n (POC) a n d n i t r o g e n (PON), c o l l e c t e d on c o m b u s t e d Whatman GF/F f i l t e r s , w e r e s t o r e d s i m i l a r l y a n d a n a l y z e d l a t e r a f t e r d r y i n g (24 h a t 72 F i g u r e 2.1. S t a t i o n l o c a t i o n s f o r n i t r o g e n u p t a k e e x p e r i m e n t s . F r o n t a l ( T 1 4 ) , s h a l l o w s t r a t i f i e d (A5) and d e e p l y s t r a t i f i e d (T8) s t a t i o n s i n t h e S t r a i t o f G e o r g i a , B.C. 125° 1 2 3 ° 73 <60°C) wi t h a P e r k i n Elmer model 240 elemental a n a l y z e r , u s i n g the dry combustion method d e s c r i b e d by Sharp (1974). The p r e c i s i o n of these techniques i s g i v e n i n Appendix 6. At each s t a t i o n continuous v e r t i c a l p r o f i l e s (0-20 m) of temperature, s a l i n i t y , f l u o r e s c e n c e and N O 3 - + N O 2 - were run p r i o r t o the b o t t l e c a s t s . Temperature and s a l i n i t y were determined w i t h an InterOcean 514A CSTD system and i n v i v o f l u o r e s c e n c e and N O 3 - + NO2"" c o n c e n t r a t i o n s were o b t a i n e d from pumped samples (mRoy FR162-144 diaphragm pump, flow r a t e ca. 1 L m i n - 1 ) and measured wi t h a Turner model 111 f l u o r o m e t e r (equipped w i t h a flow-through c e l l ) and a Technicon AutoAnalyzer I I , r e s p e c t i v e l y . These data were logged onto a p e r s o n a l computer and p l o t t e d i n r e a l - t i m e u s i n g a custom software programme which compensates f o r time l a g s i n pumping and machine analyses (Jones, p e r s . comm.). I n c i d e n t s o l a r i r r a d i a n c e (PAR, 400-700 nm) was monitored c o n t i n u o u s l y with a Lambda Instruments LI-185 l i g h t meter, equipped w i t h a L I -190SB Sur f a c e Quantum Sensor, and connected t o a c h a r t r e c o r d e r . Subsurface i r r a d i a n c e s were measured wi t h a LI-185B l i g h t meter, equipped with a LI-192S Underwater Quantum Sensor. Phytoplankton 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 (Parsons et a l . , 1984) and s t o r e d i n the dark u n t i l c o u n t i n g . Ten ml subsamples were s e t t l e d (24 h) and counted on a W i l d i n v e r t e d microscope f o l l o w i n g Utermohl (1958). Experimental W i t h i n 1 h of c o l l e c t i o n , water samples from each depth 74 w e r e t r a n s f e r r e d u n d e r r e d u c e d l i g h t c o n d i t i o n s t o 500 m l Wheaton g l a s s b o t t l e s w i t h t e f l o n - l i n e d c a p s . N i t r a t e a n d 1 5 u r e a u p t a k e r a t e s w e r e m e a s u r e d u s i n g t h e s t a b l e i s o t o p e N ( K o r I s o t o p e s ) , a s a t r a c e r ( D u g d a l e a n d G o e r i n g , 1 9 6 7 ) . F o r t h e u r e a e x p e r i m e n t s , C O f ^ N ^ ^ (99 atom %) was a d d e d t o 1 5 1 b r i n g t h e f i n a l N c o n c e n t r a t i o n t o 2-4 uq-at N-L . I n t h e n i t r a t e e x p e r i m e n t s , Na N O 3 (99 a t o m %) was a d d e d i n c o n c e n t r a t i o n s o f e i t h e r 0.05 uq-at N - L - 1 o r l e s s t h a n 10% o f t h e a m b i e n t N O 3 - + NO2"" c o n c e n t r a t i o n . T h e s e e n r i c h m e n t s w e r e n o t a l w a y s t r u e t r a c e r a d d i t i o n s ( u s u a l l y d e f i n e d a s £ 10% o f a m b i e n t ) , b u t t h e t e r m " t r a c e r " w i l l be u s e d h e r e t o 1 5 — d i s t i n g u i s h t h e l o w NO3 e n r i c h m e n t s f r o m t h e s a t u r a t i n g e n r i c h m e n t s a s s o c i a t e d w i t h t h e u r e a u p t a k e e x p e r i m e n t s . F o l l o w i n g e n r i c h m e n t , b o t t l e s w e r e i m m e d i a t e l y m i x e d a n d p l a c e d w i t h i n n e u t r a l d e n s i t y s c r e e n i n g t o s i m u l a t e t h e f o l l o w i n g PPFDs ( 9 5 , 5 5 , 3 1 , 10, 3.4, 1.1 a n d 0 % I Q ) . The s c r e e n m a t e r i a l u s e d i n t h e i n c u b a t o r s was c a l i b r a t e d w i t h a B i o s p h e r i c a l I n s t r u m e n t s QSL-100 4TT s e n s o r p l a c e d w i t h i n a n a d a p t e d i n c u b a t i o n b o t t l e . The 0% PPFD was a c h i e v e d b y w r a p p i n g t h e b o t t l e w i t h b l a c k t a p e . I n c u b a t i o n s w e r e c o n d u c t e d a t i n s i t u t e m p e r a t u r e (± 1.5°C) u n d e r n a t u r a l l i g h t i n c l e a r P l e x i g l a s d e c k i n c u b a t o r s . S a m p l e s f r o m t h e s u r f a c e w a t e r s w e r e c o o l e d w i t h f l o w i n g s u r f a c e s e a w a t e r , w h i l e d e e p e r s a m p l e s w e r e i n c u b a t e d i n a s e p a r a t e t e m p e r a t u r e c o n t r o l l e d i n c u b a t o r . I n c u b a t i o n s w e r e t e r m i n a t e d a f t e r 2-4 h by f i l t r a t i o n ( p r e s s u r e d i f f e r e n t i a l < 125 mm Hg) o n t o c o m b u s t e d Whatman GF/F f i l t e r s , p l a c e d i n t o p l a s t i c p e t r i d i s h e s , a n d 75 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 . B a s e d on t h e a m b i e n t n i t r o g e n c o n c e n t r a t i o n , t h e 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 n d t h e i n i t i a l N a t o m % i n t h e p a r t i c u l a t e f r a c t i o n , i t was c a l c u l a t e d t h a t a n a v e r a g e (± SD) o f 24.1 ± 1 5 . 3 % a n d 8.5 ± 5.2% o f t h e N O 3 - a n d u r e a , r e s p e c t i v e l y , i n s o l u t i o n was 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 m a t e r i a l d u r i n g t h e i n c u b a t i o n . A t t h e h i g h e s t u p t a k e r a t e s a c h i e v e d , n e v e r more t h a n 7 0% o f t h e N O 3 - a n d 2 0 % o f u r e a i s o t o p e , w e r e i n c o r p o r a t e d i n t o t h e PON. T h e r e f o r e s u b s t r a t e e x h a u s t i o n was n o t a p r o b l e m i n t h e e x p e r i m e n t s o f t h i s s t u d y . N i t r o g e n i n t h e p a r t i c u l a t e s a m p l e s was c o n v e r t e d t o d i n i t r o g e n g a s ( N 2 ) by t h e m i c r o - Dumas d r y c o m b u s t i o n 1 c t e c h n i q u e ( L a R o c h e ,1983) a n d t h e n a n a l y z e d f o r N e n r i c h m e n t w i t h a JASCO m o d e l N-150 e m i s s i o n s p e c t r o m e t e r ( F i e l d e r a n d P r o k s c h , 1 9 7 5 ) . N i t r o g e n u p t a k e r a t e s w e r e c a l c u l a t e d u s i n g e q u a t i o n 7 o f D u g d a l e and W i l k e r s o n (1986) ( e q u i v a l e n t t o e q u a t i o n 5 o f C o l l o s , 1987) w h i c h c o r r e c t s f o r c h a n g e s i n PON d u r i n g t h e i n c u b a t i o n p e r i o d ( s e e A p p e n d i x 1, e q u a t i o n 5 ) . C o r r e c t i o n s w e r e n o t made f o r i s o t o p i c d i l u t i o n f r o m r e m i n e r a l i z a t i o n o f ^ N - u r e a d u r i n g t h e i n c u b a t i o n ( H a n s e l l a n d G o e r i n g , 1989) a s t h i s c o r r e c t i o n w o u l d p r o b a b l y be n e g l i g i b l e g i v e n t h e l a r g e amount o f - ^ N - l a b e l l e d u r e a a d d e d t o t h e b o t t l e s . S p e c i f i c r a t e s o f n i t r o g e n t r a n s p o r t w e r e c a l c u l a t e d by d i v i s i o n o f t h e v o l u m e t r i c r a t e s b y t h e p h a e o p h y t i n - c o r r e c t e d c h l o r o p h y l l a c o n c e n t r a t i o n a t t h e b e g i n n i n g o f t h e e x p e r i m e n t s . A l t h o u g h c h l o r o p h y l l a p e r c e l l may v a r y w i t h d e p t h due t o PPFD d i f f e r e n c e s , i t was c h o s e n a s 76 t h e n o r m a l i z a t i o n p a r a m e t e r b e c a u s e i t a b s o r b s t h e l i g h t n e c e s s a r y t o f u e l c e l l u l a r t r a n s p o r t m e c h a n i s m s . C h i a s p e c i f i c u p t a k e r a t e s a l s o f a c i l i t a t e s t h e c o m p a r i s o n w i t h p r e v i o u s l y p u b l i s h e d C h i a n o r m a l i z e d n i t r o g e n a n d c a r b o n u p t a k e v e r s u s i r r a d i a n c e s t u d i e s . Kinetic parameters of uptake The k i n e t i c c o n s t a n t s f o r N O 3 - a n d u r e a u p t a k e w i t h r e s p e c t t o i r r a d i a n c e w e r e o b t a i n e d b y a d i r e c t f i t o f t h e d a t a t o a m o d i f i e d M i c h a e l i s - M e n t e n h y p e r b o l a u s i n g a c o m p u t e r i z e d , i t e r a t i v e , n o n - l i n e a r l e a s t - s q u a r e s t e c h n i q u e ( L a b t e c N o t e b o o k C u r v e f i t , L a b o r a t o r i e s T e c h n o l o g i e s C o r p . ) . The M i c h a e l i s - M e n t e n e q u a t i o n , m o d i f i e d t o a c c o u n t f o r d a r k u p t a k e , d e s c r i b e s u p t a k e o v e r t h e h y p e r b o l i c l i g h t p a r t o f t h e c u r v e ( M a c l s a a c a n d D u g d a l e , 1972) a n d i s a s f o l l o w s : V - V D + V'max K L T + I w h e r e V i s t h e t o t a l u p t a k e o f N p e r u n i t o f c h l o r o p h y l l , V D i s t h e d a r k v a l u e o f V, I i s t h e i n t e g r a t e d a v e r a g e PPFD d u r i n g t h e i n c u b a t i o n p e r i o d , v ' m a x i s t h e maximum N u p t a k e p e r u n i t c h l o r o p h y l l a t s a t u r a t i n g PPFD a n d K L T , t h e h a l f -s a t u r a t i o n c o n s t a n t f o r l i g h t , i s t h e PPFD a t 0.5 V ' m a x . The a s s u m p t i o n i s made t h a t d a r k u p t a k e i s a c o n s t a n t a t a l l l i g h t l e v e l s . O n l y d a t a s h o w i n g no p h o t o i n h i b i t i o n w e r e u s e d i n t h i s a n a l y s i s . 77 RESULTS AND DISCUSSION General description of stations The v e r t i c a l p r o f i l e s o f t e m p e r a t u r e , s a l i n i t y , r e l a t i v e i n v i v o C h i a f l u o r e s c e n c e a n d N03~ + N O 2 - c o n c e n t r a t i o n f o r t h e t h r e e s t a t i o n s a t w h i c h N u p t a k e v e r s u s PPFD e x p e r i m e n t s w e r e c o n d u c t e d a r e p r e s e n t e d i n F i g . 2.2. The d i a g n o s t i c f e a t u r e s o f t h e f r o n t a l w a t e r (T14) i n c l u d e d b o t h a weak t h e r m o c l i n e a n d h a l o c l i n e w h i c h e x t e n d e d f r o m t h e s u r f a c e t o c a . 9 m, a s u b s u r f a c e f l u o r e s c e n c e maximum l a y e r ( c a . 5-8 m), a n i t r a c l i n e w h i c h e x t e n d e d t o t h e s u r f a c e a n d r e l a t i v e l y h i g h N O 3 - + N02~ c o n c e n t r a t i o n s t h r o u g h o u t t h e w a t e r c o l u m n . I n t h e d e e p l y s t r a t i f i e d s t a t i o n ( T 8 ) , f l u o r e s c e n c e i n c r e a s e d s l i g h t l y w i t h d e p t h ; t h e n i t r a c l i n e o c c u r r e d a t c a . 1 2 m, a n d t h e u p p e r 10 m was d e v o i d o f m e a s u r a b l e N O 3 - + N O 2 - . A s t r o n g t h e r m o c l i n e a n d h a l o c l i n e a t 5-15 m s e p a r a t e d t h e d e e p N O 3 - -r e p l e t e w a t e r f r o m t h e N 0 3 ~ - d e p l e t e d m i x e d s u r f a c e w a t e r . S i m i l a r c o n d i t i o n s w e r e o b s e r v e d a t t h e s h a l l o w - s t r a t i f i e d s t a t i o n (A5) b u t t h e h a l o c l i n e , t h e r m o c l i n e , a n d n i t r a c l i n e a l l d e v e l o p e d w i t h i n t h e u p p e r 5 m o f t h e w a t e r c o l u m n . The i n i t i a l b i o m a s s d a t a a n d e n v i r o n m e n t a l c o n d i t i o n s f o r e a c h s t a t i o n a r e g i v e n i n T a b l e 2.1. The s p e c i e s c o m p o s i t i o n o f t h e p h y t o p l a n k t o n c o m m u n i t y i n t h e f r o n t a l a n d s t r a t i f i e d w a t e r s v a r i e d c o n s i d e r a b l y ( T a b l e 2 . 2 ) . I n t h e f r o n t a l w a t e r s , l a r g e , c h a i n - f o r m i n g d i a t o m s w e r e t h e most common p h y t o p l a n k t o n a t b o t h t h e s u r f a c e a n d t h e c h l o r o p h y l l maximum l a y e r (DCM). Chaetoceros s o c i a l i s was t h e d o m i n a n t s p e c i e s f o l l o w e d i n a b u n d a n c e by Skeletonema costatum 78 F i g u r e 2.2. D e p t h p r o f i l e s o f t e m p e r a t u r e ( T ) , s a l i n i t y ( S ) , i n v i v o f l u o r e s c e n c e ( F ) a n d n i t r a t e p l u s n i t r i t e c o n c e n t r a t i o n (N) f o r t h e t h r e e s t a t i o n s s a m p l e d ( T 1 4 : f r o n t a l ; A 5 : s h a l l o w s t r a t i f i e d ; a n d T8: d e e p l y s t r a t i f i e d ) . Table 2.1 I n i t i a l environmental conditions of seawater c o l l e c t e d f or N-uptake versus irr a d i a n c e experiments. s t a t i o n and l o c a t i o n D e s c r i p t i o n Date S t a r t i n g time of depth incubation (m) (PDT) Sample Nitrogen cone. N0 3 Urea NH^  (pg-at N-L - 1) Chi a PON POC (jjg-L - 1) (pg-at N-L - 1) (pg-at N-L - 1) T14 49°53'24"N F r o n t a l 125°05'06"W 27 J u l 1984 1530 6.02 15.05 0.23 0.21 1.29 2.28 5.28 6.96 43.1 40.6 A5 49°53'02"N Shallow 30 J u l 1984 125°05'48"W S t r a t i f i e d 1430 0 15 <.05 20.89 0.63 0.72 0.16 0.32 0.33 0.67 2 .57 2.01 22.9 14.5 T8 49°48'36"N Deep 1 Aug 1984 124°50'39"W S t r a t i f i e d 1500 0 15 <.05 7 .54 0.82 0.17 0.17 0.40 0.35 0.99 2.90 3.83 24 . 1 24.1 *NH. concentrations from separate b o t t l e casts vo 80 T a b l e 2.2 P h y t o p l a n k t o n c o m m u n i t y c o m p o s i t i o n i n f r o n t a l a n d s t r a t i f i e d w a t e r i n t h e S t r a i t o f G e o r g i a , B.C. S t a t i o n D e p t h P h y t o p l a n k t o n ('10° c e l l s • L ) (m) D i a t o m s F l a g e l l a t e s * F r o n t a l T14 0 2.3 0.96 8 2.2 0.76 S h a l l o w 0 0.23 1.9 S t r a t i f i e d A5 15 0.73 0.79 D e e p l y 0 0.026 1.5 S t r a t i f i e d T8 15 0.15 1.7 * <5% o f f l a g e l l a t e s w e r e d i n o f l a g e l l a t e s 81 a n d o t h e r d i a t o m s o f t h e g e n u s , Chaetoceros, i n c l u d i n g C. debilis. S m a l l p i g m e n t e d f l a g e l l a t e s (<5 um) w e r e t h e most a b u n d a n t p h y t o p l a n k t o n i n t h e s u r f a c e w a t e r s o f b o t h s t r a t i f i e d s t a t i o n s ; d o m i n a n t d i a t o m s w e r e s t i l l Skeletonema costatum a n d Chaetoceros s p p . , a l t h o u g h T h a l a s s i o s i r a s p p . a n d p e n n a t e d i a t o m s b e l o n g i n g t o Navicula a n d Nitzschia g e n e r a a p p e a r e d i n s m a l l n u m b e rs. D i n o f l a g e l l a t e s w e r e a l w a y s a s m a l l n u m e r i c a l f r a c t i o n (<5%) o f t h e t o t a l f l a g e l l a t e s p r e s e n t a n d w e r e a l m o s t e x c l u s i v e l y Gymnodinium o r Amphidinium s p p . The d e e p c h l o r o p h y l l maximum c o m m u n i t i e s o f t h e t w o s t r a t i f i e d s t a t i o n s d i f f e r e d i n t h e r e l a t i v e a b u n d a n c e o f f l a g e l l a t e s a n d d i a t o m s , b u t t h e s p e c i e s c o m p o s i t i o n was s i m i l a r . E f f e c t of light on nitrogen uptake rates M a c l s a a c a n d D u g d a l e (1972) f i r s t showed t h a t t h e u p t a k e o f n i t r a t e a n d ammonium by n a t u r a l p h y t o p l a n k t o n a s s e m b l a g e s c o u l d be r e l a t e d t o PPFD by a r e c t a n g u l a r h y p e r b o l a ; PPFD may be t r e a t e d a s a s u b s t r a t e , f o l l o w i n g M i c h a e l i s - M e n t e n k i n e t i c s u n d e r c o n d i t i o n s o f no n u t r i e n t s t r e s s . S u c h a m o d e l assumes t h a t t h e r e i s no N u p t a k e a t z e r o PPFD ( i . e . t h e PPFD r e s p o n s e c u r v e p a s s e s t h r o u g h t h e o r i g i n ) . T h ey s u g g e s t e d t h a t t h e c o n s e q u e n c e s o f n o t s u b t r a c t i n g d a r k u p t a k e f r o m t h e l i g h t u p t a k e , when u p t a k e i n t h e d a r k i s g r e a t e r t h a n c a . 15% o f u p t a k e a t s a t u r a t i n g PPFD, c a n be s i g n i f i c a n t ; l i n e a r t r a n s f o r m a t i o n s o f s u c h k i n e t i c d a t a a r e d i s t o r t e d b e y o n d u s e f u l n e s s a n d t h u s t h e v a l u e s o f d e r i v e d p a r a m e t e r s q u e s t i o n a b l e . F o r s i t u a t i o n s i n w h i c h d a r k u p t a k e i s a 82 s u b s t a n t i a l p o r t i o n (>10-15% o f PPFD - s a t u r a t e d u p t a k e ) , t h e y p r o p o s e d a s l i g h t l y m o d i f i e d e q u a t i o n , e m p l o y e d i n t h e p r e s e n t s t u d y , w h i c h t a k e s i n t o a c c o u n t a c o n s t a n t d a r k u p t a k e r a t e a n d d e s c r i b e s N u p t a k e o v e r t h e h y p e r b o l i c p o r t i o n o f t h e PPFD r e s p o n s e c u r v e , b u t n o t p h o t o i n h i b i t i o n . P h o t o i n h i b i t i o n p r o b l e m s c a n be o v e r c o m e by u s i n g an e q u a t i o n d e v e l o p e d by P a r k e r ( 1 974) o r a m o d i f i c a t i o n o f t h e e q u a t i o n o f P i a t t e t a l . ( 1 9 8 0 ) o r i g i n a l l y d e v e l o p e d f o r t h e l i g h t r e s p o n s e o f p h o t o s y n t h e s i s ( L e w i s a n d L e v i n e , 1984; P r i s c u , 1 9 8 9 ) . Numerous s t u d i e s i n b o t h m a r i n e ( M a c l s a a c a n d D u g d a l e , 1972; M a c l s a a c e t a l . , 1974; N e l s o n a n d Conway, 1979; S l a w y k , 1979) a n d f r e s h w a t e r n a t u r a l c o m m u n i t i e s ( P r i s c u 1984; W h a l e n a n d A l e x a n d e r 1984b; M i t a m u r a , 1986) h a v e d e m o n s t r a t e d t h a t t h e u p t a k e r e s p o n s e o f N 0 3 + a n d NH^ - c a n be s u c c e s s f u l l y d e s c r i b e d b y t h e M i c h a e l i s - M e n t e n f o r m u l a t i o n . I n t h e p r e s e n t s t u d y , n i t r a t e a n d u r e a u p t a k e w e r e d e p e n d e n t on PPFD a t b o t h d e p t h s s a m p l e d i n s t r a t i f i e d a n d f r o n t a l w a t e r s o f t h e S t r a i t o f G e o r g i a . E x p e r i m e n t s i n w h i c h t h e n a t u r a l p h y t o p l a n k t o n c o m m u n i t i e s f r o m t h e s u r f a c e a n d t h e DCM l a y e r s w e r e e x p o s e d t o a g r a d i e n t i n PPFD y i e l d e d d a t a w h i c h c o u l d be a d e q u a t e l y d e s c r i b e d by t h e M i c h a e l i s - M e n t e n f o r m u l a t i o n up t o i n h i b i t i n g PPFD l e v e l s ( F i g . 2.3 a n d 2 . 4 ) . P h o t o i n h i b i t i o n o c c u r r e d b e t w e e n 55 a n d 9 5 % o f s u r f a c e PPFD ( I Q ) a n d was o n l y o b s e r v e d f o r s a m p l e s c o l l e c t e d f r o m d e p t h . P h o t o i n h i b i t i o n o f N u p t a k e c a n n o t be a d e q u a t e l y d i s c u s s e d i n t h i s s t u d y due t o t h e p a u c i t y o f d a t a a t h i g h PPFD, b u t s u f f i c e t o s a y , i t i s n o t l i k e l y a p r o b l e m f o r t h e s u r f a c e 83 Figure 2.3. N i t r a t e uptake of the surface ( O ) and DCM ( • ) phytoplankton communities of the S t r a i t of Georgia. The curved p l o t s are f i t t e d d i r e c t l y to the Michaelis-Menten equation; the l i n e a r (dashed l i n e ) PPFD-inhibited portions were not included i n the c a l c u l a t i o n s . Stations are T14 ( f r o n t a l ) , A5 (shallow s t r a t i f i e d ) and T8 (deeply s t r a t i f i e d ) . 3 0 0 PPFD (jjE-m"2-s"2) F i g u r e 2.4. U r e a u p t a k e o f t h e s u r f a c e ( O ) a n d DCM ( • ) p h y t o p l a n k t o n c o m m u n i t i e s o f t h e S t r a i t o f G e o r g i a . T he c u r v e d p l o t s a r e f i t t e d d i r e c t l y t o t h e M i c h a e l i s - M e n t e n e q u a t i o n ; t h e l i n e a r ( d a s h e d l i n e ) P P F D - i n h i b i t e d p o r t i o n s w e r e n o t i n c l u d e d i n t h e c a l c u l a t i o n s . S t a t i o n s a r e A5 ( s h a l l o w s t r a t i f i e d ) a n d T8 ( d e e p l y s t r a t i f i e d ) . PPFD (pE-m-2-s"2) 85 s a m p l e s , w h i c h a r e n a t u r a l l y e x p o s e d t o h i g h PPFD; p h y t o p l a n k t o n c o l l e c t e d n e a r t h e b o t t o m o f t h e e u p h o t i c z o n e , a r e e f f e c t i v e l y e x c l u d e d f r o m t h e h i g h PPFD i n t h e m i x e d s u r f a c e w a t e r s b y t h e p y c n o c l i n e a n d a r e n o t l i k e l y t o e n c o u n t e r s u c h h i g h PPFDs n a t u r a l l y . Kinetic parameters of nitrogen uptake D a r k u p t a k e , t h e h a l f - s a t u r a t i o n c o n s t a n t (K-^rp), a n d maximum n i t r o g e n u p t a k e v e l o c i t y ( V ' m a x ) f o r l i g h t d e p e n d e n t u r e a a n d n i t r a t e n i t r o g e n u p t a k e a r e s u m m a r i z e d i n T a b l e 2.3. The K-^ rp v a l u e s i n t h e p r e s e n t s t u d y a r e t h o s e r e p r e s e n t i n g t h e PPFD a t w h i c h 0.5 V ' m a x o c c u r s . H owever, i t i s i m p o r t a n t t o remember t h a t t h e s e M i c h a e l i s - M e n t e n p a r a m e t e r s o n l y r e p r e s e n t u p t a k e d a t a f r o m t h e h y p e r b o l i c ( o r l i g h t ) p o r t i o n o f t h e PPFD r e s p o n s e c u r v e a n d do n o t i n c l u d e t h e s u b s t a n t i a l d a r k N u p t a k e o b s e r v e d . Some i n v e s t i g a t o r s ( e . g . , P r i s c u , 1984) h a v e i g n o r e d d a r k u p t a k e i n t h e l i n e a r t r a n s f o r m a t i o n o f t h e i r k i n e t i c d a t a a n d f o r c e d t h e i r PPFD r e s p o n s e c u r v e s t o p a s s t h r o u g h t h e o r i g i n e v e n t h o u g h d a r k u p t a k e was s u b s t a n t i a l ( c a . 5 0 % o f t o t a l N - u p t a k e ) . H a l f - s a t u r a t i o n c o n s t a n t s d e r i v e d i n t h i s manner a r e n o t an a c c u r a t e m e a s u r e o f PPFD a t w h i c h V = V m a x 12 a n d s h o u l d be i n t e r p r e t e d w i t h c a u t i o n , p a r t i c u l a r l y a s a n i n d i c a t o r o f t h e p h y t o p l a n k t o n c o m m u n i t i e s ' a b i l i t i e s t o a s s i m i l a t e s p e c i f i c N s u b s t r a t e s a t l o w PPFD. A b e t t e r e s t i m a t e o f t h e PPFD a t w h i c h o n e - h a l f t h e t o t a l m a x i m a l N u p t a k e o f t h e p h y t o p l a n k t o n c o m m u n i t y i s a c h i e v e d (K-^ip') c a n be c a l c u l a t e d by a s i m p l e r e a r r a n g e m e n t o f t h e M i c h a e l i s - M e n t e n e q u a t i o n e m p l o y e d i n t h e p r e s e n t s t u d y : Table 2.3 Parameters d e s c r i b i n g the c h a r a c t e r i s t i c s of n i t r o g e n uptake, as a f u n c t i o n of PPFD, f o r phytoplankton assemblages i n the S t r a i t o f Georgia, B.C. S t a t i o n s are T14: f r o n t a l ; A5: shallow s t r a t i f i e d ; and T8: deeply s t r a t i f i e d . D e f i n i t i o n s are given i n the t e x t , estimated standard e r r o r s of parameters i n parentheses. S t a t i o n N i t r o g e n s u b s t r a t e Depth VD V max K L T (m) (ng-at N (jjg C h i a ) " 1 h _ 1 ) (pE•m~ (%I 0) T14 N0 3- 0 80 (16.8) 225 (22 3) 91 ( 40.1) 8.2 3.6) 8 53 ( 9.4) 174 ( 12 5) 53 ( 15.7) 4 . 8 1-4) A5 NO3- 0 48 ( 3.2) 50 ( 4 0) 74 ( 25.9) 6.7 2.3) 15 55 ( 8.8) 67 ( 15 3) 156 ( 151 ) 14 14 ) Urea 0 87 ( 4.2) 31 ( 4 3) 140 ( 103 ) 13 9.3) 15 1 . 8 ( 2.3) 11 ( 1 9) 54 ( 40.7) 4 . 9 3.7) T8 N0 3~ 0 8.5 ( 0.79) 9 . 8 ( 1 02) 45 (18.7) 4.6 1.9) 15 0 55 ( 4 2) 18 ( 6.6) 1 . 8 0.7) Urea 0 92 ( 14.1) 73 ( 17 6) 59 ( 63.4) 6.7 7.2) 15 4.0 ( 3.9) 24 ( 3 6) 72 ( 56.2) 8.2 6.4) 00 87 K L T ' = (V - V D ) • K L T / ( V max - V + v D ) w h e r e V = ( V max + V D ) / 2 , a n d V max i s t h e maximum u p t a k e d e s c r i b e d by t h e r e c t a n g u l a r h y p e r b o l a , K^ip i t s h a l f -s a t u r a t i o n c o n s t a n t , a n d i s t h e d a r k u p t a k e r a t e . A l t e r n a t i v e l y , a n o t h e r h a l f - s a t u r a t i o n c o n s t a n t , K-^rp", c a n be c a l c u l a t e d by s u b s t i t u t i n g V f o r o n e - h a l f t h e v e l o c i t y o f N u p t a k e a t s a t u r a t i n g PPFD. B o t h o f t h e s e d e r i v e d h a l f -s a t u r a t i o n c o n s t a n t s w i l l g e n e r a t e v a l u e s t h a t a r e more r e a l i s t i c m e a s u r e s o f t h e PPFD a t o n e - h a l f t h e a c t u a l maximum N u p t a k e t a k i n g p l a c e i n t h e p h y t o p l a n k t o n c o m m u n i t y a s t h e y i n c l u d e d a r k u p t a k e ( T a b l e 2.4). The v a l u e s o f t h e h a l f - s a t u r a t i o n c o n s t a n t f o r N O 3 -u p t a k e i n t h e p r e s e n t s t u d y r a n g e f r o m 0-14% I Q , w h i c h i s c o n s i s t e n t w i t h p r e v i o u s l y p u b l i s h e d v a l u e s f o r m a r i n e a n d f r e s h w a t e r n a t u r a l p h y t o p l a n k t o n a s s e m b l a g e s ( T a b l e 2.5). The K L T v a l u e s f o r u r e a u p t a k e a r e s i m i l a r (0-13% I Q ) . P r e v i o u s l y p u b l i s h e d k i n e t i c s t u d i e s f o r u r e a a r e f e w . Webb a n d Haas 1 O _ I ( 1976) r e p o r t a K-^rp o f c a . 0.01 l a n g l e y s 'min - (35*L/E«m *s ) f o r p h y t o p l a n k t o n f r o m t h e Y o r k R i v e r e s t u a r y i n V i r g i n i a d u r i n g t h e summer, a l t h o u g h v a l u e s i n t h e autumn r a n g e d f r o m 0.02 t o 0.12 l a n g l e y s • m i n - 1 (69-418 uE*m~2•s-1). A s i m i l a r summer K L T v a l u e was r e p o r t e d by M i t a m u r a (1986) f o r u r e a u p t a k e by p h y t o p l a n k t o n f r o m o l i g o t r o p h i c L a k e B i w a i n J a p a n (2.44 K l u x = 39 LfE«m -s ). They a l s o r e p o r t e d a s i m i l a r + — 9 — 1 v a l u e K L r p f o r N H 4 u p t a k e (28 /jE-m *s ) a n d a g r e a t e r K L T T a b l e 2.4 I n d i c e s o f N uptake dependency on PPFD f o r p h y t o p l a n k t o n i n the s t r a i t of G e o r g i a : the r a t i o o f dark t o l i g h t - s a t u r a t e d uptake r a t e ( V D : V L ) , the PPFD a t which h a l f of t o t a l N uptake o c c u r s (K. r a t i o o f uptake under 1% I t o 55% I LT K. LT (V 1% , V55% ) a p e r c e n t a g e o f s u r f a c e PPFD ( I Q ) which i s shown i n p a r e n t h e s e s . The K L T v a l u e s are e x p r e s s e d as PPFD v a l u e s and as s t a t i o n N i t r o g e n s u b s t r a t e Depth (m) V : V VD* V L L T -2 -1 (jiE-m -S ) LT V 1 % : V 5 5 % T14 NO- 0.28 0.25 36 (3.2) 24 (2.1) 43 28 (3.9) (2.5) 0.38 0.40 A5 NO- 0 15 0.51 0.49 0 3.0 (0.3) 1.6 (0.1) 15 (1.3) 0.60 0.56 Urea 0 15 0.77 0.15 33 (3.0) 0 39 (3.5) 0.80 0.33 T8 NO- 0 15 0.48 0 1.9 (0.2) 0 3.1 (0.3) 0 0.60 0.37 Urea 0 15 0.58 0.16 0 39 (4.5) 0 52 (5.8) 0.65 0.28 • D e f i n i t i o n s g i v e n i n t e x t . oo oo Table 2.5 Comparison of h a l f - s a t u r a t i o n constants (K L_) for inorganic n i t r a t e transport i n various aquatic ecosystems. KLT< N 03~> _ 2 , " Region Area Depth % surf l i g h t pE-m -s Reference (% I Q ) range (mean) range (mean) Oceanic Upwelling Upwelling Upwelling Upwelling Upwelling Coastal Coastal Freshwater Freshwater Freshwater Freshwater Freshwater Freshwater E. T r o p i c a l P a c i f i c Peru N.W. A f r i c a N.W. A f r i c a Baja C a l i f . , Mexico A n t a r c t i c Peru S t r a i t of Georgia 25 14.0 100 0.9 - 12.7 ( 5.4) 10 0.9 - 13.3 ( 8.9) 14 - 108 ( 63)* 7 - 199 (122)* 50 1.5 - 7.0 ( 5.4) -50-0.1 5.5 - 6.2 ( 5.9) -50-3 3.3 - 32.4 (16.1) -50-25 7 100 10 100 i . 1 T o o l i k L., Alaska ca. 10-15 L. Kinneret, I s r a e l L. Biwa, Japan 100 Ca s t l e L., c a l i f . , U.S.A. L. Vanda, A n t a r c t i c a L. F r y x e l l , A n t a r c t i c a ca.50 ca. 1 10.2 1.0 0.4 1.1 - 2 . 3 ( 1 . 7 ) 2.3-4.4 (3.3)' 1.3 4.4 1.0 4.6 - 8.2 ( 6.5) 1.8 - 14 ( 6.9) 6 - 31 (15) 4.29 2 . 8** 45 14 45 18 - 91 - 156 7 77 70. 8*** - 29 ( 70) ( 76) ( 16) 2.6 - 2.7 (2.65) 15.1 - 16.2 (15.7)+ 0.6 - 3.7 (1.55) 4.6 - 25.5 (10.7)+ 0.5 -0.4 -0.08 2.0 5.1 2.4 - 9.5 1.7 - 24.9 0.04 (?) Maclsaac & Dugdale (1972) Maclsaac & Dugdale (1972) Maclsaac et a l . (1974) Nelson & Conway (1979) Nelson & Conway (1979) Slawyk (1979) Maclsaac & Dugdale (1972) Present study Whalen & Alexander (1984b) Berman et a l . (1984) Mitamura (19 86) Pr i s c u (1984) Pri s c u (1989) Pri s c u (1989) T a b l e 2 . 5 C o n t i n u e d 1 — 1 ? 1 * V a l u e s c a l c u l a t e d by c o n v e r t i n g f r o m l y - m i n u s i n g 1 l y - m i n = 3485 uE-m~ -S ( R i c h a r d s o n e t a l . , 1983) — 2 — 1 7 — 1 7 ? P I * * V a l u e s c a l c u l a t e d by c o n v e r t i n g f r o m q u a n t a - m -h u s i n g 1 q u a n t a - m -h = 4 .614 x 10 pE-m - s ( L i i n i n g , 1981) — 2 -1 * * * V a l u e s c a l c u l a t e d by c o n v e r t i n g f r o m k l u x u s i n g 1 k l u x = 16 .5 uE-m -s ( R i c h a r d s o n e t a l . , 1983) + V a l u e s c a l c u l a t e d f rom t o t a l PPFD d u r i n g i n c u b a t i o n p e r i o d s ( c a . 12 h) ? P r o b a b l e e r r o r , c o r r e c t v a l u e f r o m d a t a g i v e n i n P r i s c u (1989) i s 0.4 p E - m ~ 2 - s - 1 o v a l u e f o r n i t r a t e u p t a k e (67 uE*-m~ -s ). I n t e r p r e t a t i o n o f t h e s m a l l d i f f e r e n c e s i n t h e K^-p's o f t h e p r e s e n t s t u d y ( l e s s — 7 — 1 t h a n 50 uE-m «s ) , e i t h e r b e t w e e n N s u b s t r a t e s o r t h e c o m m u n i t i e s t a k e n f r o m d i f f e r e n t d e p t h s , i s r a t h e r d i f f i c u l t . H owever t h i s k i n e t i c p a r a m e t e r h a s b e e n i n c l u d e d f o r l i t e r a t u r e c o m p a r a t i v e p u r p o s e s . A s i m p l e r a n d more s t r a i g h t -f o r w a r d i n d e x t o a s s e s s t h e e f f e c t o f PPFD on N u p t a k e c a n be d e t e r m i n e d by c o m p a r i n g N u p t a k e a t l o w ( 1 % I Q ) a n d s a t u r a t i n g ( 5 5 % I Q ) PPFD (Conway a n d W h i t l e d g e , 1 9 7 9 ) ; l o w e r p e r c e n t a g e s r e p r e s e n t g r e a t e r PPFD d e p e n d e n c y . A t t h e f r o n t a l s t a t i o n (T14) b o t h s u r f a c e a n d deep c h l o r o p h y l l maximum (DCM) c o m m u n i t i e s h a v e t h e same PPFD d e p e n d e n c y f o r N O 3 - u p t a k e ( 3 8 -4 0 % ) ; w h i c h i s l i k e l y a r e f l e c t i o n o f t h e s i m i l a r i t y i n b o t h t h e s p e c i e s c o m p o s i t i o n a n d t h e p h y s i o l o g i c a l s t a t e o f t h e s e t w o N - r e p l e t e c o m m u n i t i e s . A t t h e s h a l l o w s t r a t i f i e d s t a t i o n ( A 5 ) , t h e t w o p h y t o p l a n k t o n c o m m u n i t i e s a r e v e r y s i m i l a r w i t h r e s p e c t t o NC^ - u p t a k e r e s p o n s e ( 6 0 , 56%) b u t t h e r e i s a s u b s t a n t i a l d i f f e r e n c e b e t w e e n t h e s u r f a c e a n d DCM u r e a u p t a k e d e p e n d e n c y : 80 a n d 3 3 % , r e s p e c t i v e l y . S i m i l a r l a r g e d i f f e r e n c e s w e r e f o u n d f o r b o t h NC>3~ ( 6 0 , 37%) a n d u r e a ( 6 5 , 28%) i n t h e t w o c o m m u n i t i e s o f t h e d e e p l y s t r a t i f i e d s t a t i o n ( T 8 ) . I t a p p e a r s t h a t u p t a k e o f t h e r e g e n e r a t e d N s o u r c e , u r e a , h a s a g r e a t e r d e p e n d e n c e on PPFD i n t h e NC>3~- r e p l e t e DCM c o m m u n i t y w h i c h was e f f e c t i v e l y i s o l a t e d f r o m t h e w e l l - l i t s u r f a c e l a y e r s b y t h e s t r o n g p y c n o c l i n e p r e s e n t a n d o n l y n o r m a l l y r e c e i v e d c a . 1-3% I Q . The l e s s e r PPFD d e p e n d e n c y o f t h e s u r f a c e p h y t o p l a n k t o n may be a c o n s e q u e n c e o f t h e i r N-92 d e p l e t e d p h y s i o l o g i c a l s t a t e , which c o u l d e x p l a i n the decrease i n PPFD dependency of N O 3 - uptake i n the s u r f a c e p o p u l a t i o n s of the s t r a t i f i e d waters (60%) r e l a t i v e t o the f r o n t a l waters (38%). A l t e r n a t i v e l y i t may be a t t r i b u t e d merely t o an accumulation of s t o r e d energy and C s k e l e t o n s produced d u r i n g p h o t o s y n t h e s i s . One cannot d i r e c t l y compare the uptake responses of urea and n i t r a t e i n the s u r f a c e waters due t o 1 c d i f f e r e n c e s i n N-enrichment ( s a t u r a t i n g v ersus t r a c e ) . At the DCM, however, s a t u r a t i n g N O 3 - c o n d i t i o n s were pres e n t d u r i n g a l l the uptake experiments. I t appears t h a t the phytoplankton i n the DCMs had a s i m i l a r degree of PPFD dependency f o r N O 3 - uptake which was s i m i l a r t o t h a t f o r urea i n the s t r a t i f i e d DCM communities. Dark nitrogen uptake N i t r a t e and urea uptake o c c u r r e d i n the dark i n both f r o n t a l and s t r a t i f i e d waters (Table 2.3) In the s t r a t i f i e d s u r f a c e waters (A5, T8), the r e l a t i v e c o n t r i b u t i o n of dark N03~ uptake t o t o t a l N03~ uptake under s a t u r a t i n g PPFD was ca. 50% w h i l e i n the s u r f a c e waters of f r o n t a l s t n (T14) the dark uptake c o n t r i b u t i o n was onl y 28% (Table 2.4). At the deeply s t r a t i f i e d s t n (T8), th e r e was no dark N O 3 - uptake by the DCM p o p u l a t i o n whereas the r e l a t i v e dark N O 3 - uptake at the shallow s t r a t i f i e d (A5) and f r o n t a l (T14) DCM communities were s i m i l a r t o t h e i r r e s p e c t i v e s u r f a c e phytoplankton communities (49 and 25%). Dark uptake of urea was a l s o a s u b s t a n t i a l p o r t i o n of t o t a l urea uptake averaging 16 and 68% f o r the DCM and s u r f a c e communities, r e s p e c t i v e l y . Dark N uptake by 93 p h y t o p l a n k t o n i s n o t uncommon a n d a summary o f l i t e r a t u r e v a l u e s o f t h e r a t i o o f d a r k t o l i g h t N u p t a k e r a t e s (V^iV^) f o r n a t u r a l p h y t o p l a n k t o n a s s e m b l a g e s i s shown i n T a b l e 2.6. A r e v i e w o f t h e l i t e r a t u r e p e r m i t s t w o g e n e r a l i z a t i o n s t o be made c o n c e r n i n g d e p e n d e n c e o f l i g h t f o r N u p t a k e : 1) i n N-i m p o v e r i s h e d w a t e r s , t h e V D :V L r a t i o i s g r e a t e r ( a p p r o a c h i n g u n i t y ) t h a n i n N - r e p l e t e w a t e r s , s u g g e s t i n g t h e e n h a n c e m e n t o f d a r k u p t a k e b y n u t r i e n t s t r e s s , a n d 2) V D :V L r a t i o i s g e n e r a l l y g r e a t e r ( c l o s e r t o u n i t y ) i n s a m p l e s c o l l e c t e d f r o m a n d i n c u b a t e d u n d e r l o w e r PPFD, s u g g e s t i n g a l e s s e r d e p e n d e n c e o f l i g h t f o r N u p t a k e w i t h i n c r e a s i n g d e p t h i n t h e e u p h o t i c z o n e . The f i r s t s u g g e s t i o n i s n o t new a s many l a b o r a t o r y e x p e r i m e n t s h a v e shown t h a t N d e p r i v a t i o n e n h a n c e s t o a g r e a t e r d e g r e e t h e u p t a k e o f N i n t h e d a r k t h a n i n t h e l i g h t , ( e . g . , S y r e t t , 1962; T h a c k e r a n d S y r e t t , 1972; H a r r i s o n , 1976; R e e s a n d S y r e t t , 1 9 7 9 ) . The a b i l i t y t o t a k e up n i t r o g e n i n t h e d a r k may b e , h o w e v e r , s p e c i e s d e p e n d e n t ; f o r e x a m p l e E p p l e y e t a l . ( 1 9 7 1 b) showed t h a t a l t h o u g h a somewhat N-d e p l e t e d t r o p i c a l o c e a n i c c o c c o l i t h o p h o r i d (Emiliana huxleyi) t o o k up n i t r a t e i n t h e d a r k , a s i m i l a r l y N - d e p l e t e d c o a s t a l d i a t o m (Skeletonema costatum) d i d n o t . A l s o w h e t h e r o r n o t a s p e c i e s i s a b l e t o t a k e up a s i g n i f i c a n t amount o f n i t r o g e n a t n i g h t o r i n t h e d a r k may d e p e n d on i t s d e g r e e o f N d e p l e t i o n . T h i s i s s u g g e s t e d f o r n i t r a t e - l i m i t e d c o n t i n u o u s c u l t u r e s o f Chaetoceros s p p . ( M a l o n e e t a l . , 1 9 7 5 ) ; a t t h e t h r e e l o w e r d i l u t i o n r a t e s , n i t r a t e u p t a k e was c o n t i n u o u s a n d i n d e p e n d e n t o f t h e n a t u r a l l i g h t / d a r k c y c l e , b u t t h e r e was d i e l Table 2.6 Summary of l i t e r a t u r e values of dark:light nitrogen s p e c i f i c (V D/V L) or absolute (/p//^) uptake rates, determined during daytime, i n natural phytoplankton communities. Area Ambient NO-} cone (pg-at N-L ) or vQ/ VL range (mean) NO- NH, Urea Reference Oceanic N. A t l a n t i c Gyre (Sargasso Sea) (0.30) (0.59) Dugdale & Goering (1967) N. P a c i f i c Central Gyre - 50°N, 155°W >10 - 40°N, 150°W 1.0 0.0 -0.63 (0.30) 0.38- 2.0 (0.83) 0.92 0.78- 1.5 (1.2) Hattori & Wada ( 1972 ) N. P a c i f i c Ocean - northern (J1-J7) >2 - t r o p i c a l / s u b t r o p i c a l <0.1 (J9-J23) N . E . P a c i f i c Ocean 12 0.0 - 9.7 ( 2.4) 5.7 -27.4 (16.9) 0.0-18.5 ( 9.3) Kanda et a l . (1985) 7.5 -31.9 (22.4) 16.6 -52.9 (34.0) 12.2-49.8 (25.7) 0.08-1.01 (0.09) g Cochlan (Chap. 1) Upwelling N.w. A f r i c a >10 0.05-0.57 (0.36) 0.00-0.02 (0.07) Nelson & Conway (1979) Baja C a l i f . , Mexico >10 0.10-1.46 (0.49) 0.01-0.67 (0.16) Nelson & Conway ( 1979 ) Baja C a l i f . , Mexico* 0.34 0.02 Maclsaac (1978) Table 2.6 c o n t i n u e d Area Ambient ^D^^L o r V D ^ V L r a n 9 e (mean) Reference NOo cone (pg-at N-L ) 3 c^ NC>2 NH4 Urea P o l a r S c o t i a Sea S c o t i a Sea Barents Sea C o a s t a l O s l o f j o r d (Norway) New York B i g h t Gulf of Maine Peru* >20 >20 0-1.5 0.30-0.47 (0.38) 0.88-1.2 (1.0) 13.0-75.0 0.27-1.0 >2 <1 0.09 0.06-0.57 (0.17) 0.18-1.7 (0.47) c a . 0.1 0.2 -1.0 (0.7) 0.5 -1.3 (0.7) ca . 1-2 0.00-1.00 (0.26) 0.00-0.20 (0.10) 0.60-0.86 (0.73) 0.3-0.5 c , g G l i b e r t e t a l . (1982a) cRonner e t a l . (1983) ' K r i s t i a n s e n & Lund ( 1989) Paasche & Erga (1988) Conway & Whitledge (1979) a , b D u g d a l e & Goer i n g (1967) Dortch & Maske (1982) S t r a i t of G e o r g i a , B.C. - F r o n t a l - s t r a t i f i e d 3.0-4.6 0.00-0.08 (0.03) <0.05 0.00-0.18 (0.09) 0.37-0.39 (0.38) 0.00-0.81 (0.36) P r i c e e t a l . 0.52-0.58 (0.55) 0.06-0.66 (0.36) (1985) Table 2.6 c o n t i n u e d Area Ambient /^E/ZL O R VD^ VL r a n 9 e (mean) Reference SO-, cone -> -1 (pg-at N-L ) NO3 conc^ NO3 NH 4 Urea S t r a i t o f G e o r g i a , B.C. - F r o n t a l 6-15 - S u r f a c e s t r a t i f i e d <0.05 - Bottom s t r a t i f i e d 7-20 0.25-0.28 (0.27) 0.48-0.51 (0.50) 0.00-0.49 (0.25) 0.58-0.77 (0.68) 0.15-0.16 (0.16) P r e s e n t study Washington c o a s t Western I r i s h Sea - S u r f a c e s t r a t i f i e d - Mixed & bottom s t r a t . c a . c a . 2.5 4.5 (0.21) (0.43) 0.47-1.1 0.37-1.3 (0.38) (0.67) (0.72) Dortch & P o s t e l (1989) 1 T u r l e y (1985) E s t u a r i n e Pamlico R i v e r , N. South R i v e r , N.C. Neuse R i v e r , N.C. Newport R i v e r , N. Delaware Bay Chesapeake Bay 0.00-0.09 0.71-0.82 0.18-1.01 (0.57) 0.04-0.95 (0.61) 0.02-0.11 (0.06) 0 . 06-1.02 0.26 F i s h e r e t a_l. , (1982) Freshwater L. K i n n e r e t ( I s r a e l ) c a . 0.10 0.2-0.6 0.40-0.91 (0.56) 0.16-0.33 (0.22) 0.29-1.0 (0.60) 0.13-0.67 (0.34) 0.53 (0.53) 0.33-0.44 (0.38) McCarthy e t a l . (1982) L. K i n n e r e t ( I s r a e l ) <0.05 0.32 0.59 Berman e t a l . (1984) L. Nakanuma (Japan) 0.21-1.1 (0.57) M i y a z a k i e t a l . ( 1985) Table 2.6 c o n t i n u e d Area Ambient NOo cone J -1 (pg-at N - L ) / V / L O R V D / V L r a n 9 e (mean) NO- NH . Urea Reference L. Biwa (Japan) ** L. Kasumigaura (Japan) Shagawa L. (Minnesota, U.S.A.) T o o l i k L. (Alaska, U.S.A.) L. Vanda ( A n t a r c t i c a ) s f c pop'n dee p - c h l pop'n C z e c h o s l a v a k i a n r e s e r v o i r s L. Calado ( B r a z i l ) 0.26 =0.07 0.18-0.27 0.0-0.7 >35 0.78 0.51 0.60-0.90 0.71 0.27-2.3 (1.0) c a . 1.0 0.05-0.31 (0.15) 0.27-0.57 (0.41) 0.41 0.24 0.05-0.38 (0.11) 0.47 0.83 c a . <0.1 0.00-0.32 (0.16) 0.11-1.0 (0.49) Mitamura and S a i j o (1986) Takamura e t a l . (1987) a , e T o e t z £. Cole ( 1980) Whalen & Alexander (1984b) " P r i s c u ( 1989) Prochazkova e t a l . (1970) F i s h e r e t a l . (1988) Amazon R i v e r ( B r a z i l ) C a s t l e L., ( C a l i f o r n i a , U.S.A.) L. O n t a r i o ( O n t a r i o , Canada) 11.1 0.20 0-ca. 2.5 - (0.55) 0.54 (0.50) 0-15 0.02-0.30 (0.14) 0.30-0.60 (0.40) F i s h e r e t a l . ( 1988) c P r i s c u (1984) g L i a o and Lean (1978) Table 2.6. S u p e r s c r i p t s . a V a l u e s c a l c u l a t e d as 1/(V L/V D) o r l / ( / ^ / / 0 D ) from r e p o r t e d v a l u e s of V L / V D or Values e s t i m a t e d from f i g u r e s . c V a l u e s r e p o r t e d i n t e x t , no data a v a i l a b l e . ^Values c a l c u l a t e d from t u r n o v e r t i m e s . e L i g h t r a t e s determined a t ambient N c o n e , dark r a t e s determined at s a t u r a t i n g N cone. •f K H Values are /^^/^ m , where N m i s the c h l o r o p h y l l s p e c i f i c t r a n s p o r t r a t e a t o p t i m a l PPFD l e v e l . ^Experiments u t i l i z e d 24 h i n c u b a t i o n s over n a t u r a l l i g h t / d a r k c y c l e . ^Average v a l u e s r e p o r t e d . * D i n o f l a g e l l a t e bloom. Microcystis bloom oo 99 p e r i o d i c i t y i n n i t r a t e u p t a k e a t t h e h i g h e s t d i l u t i o n r a t e . I t s h o u l d be n o t e d t h a t t h e d a r k u p t a k e r a t e s r e p o r t e d i n t h e p r e s e n t s t u d y a n d u s e d i n t h e r a t i o s o f T a b l e 2.6 w e r e d e t e r m i n e d d u r i n g d a y l i g h t a n d may n o t n e c e s s a r i l y r e f l e c t t h e .uptake r a t e s o b s e r v e d d u r i n g t h e n i g h t . D u r i n g 24 h t i m e c o u r s e e x p e r i m e n t s c o n d u c t e d i n t h e same w a t e r s a s t h e p r e s e n t s t u d y I o b s e r v e d a c o n s t a n c y i n V D : V L f o r N H 4 + u p t a k e i n f r o n t a l w a t e r s ( C h a p t e r 1) w h i c h s u g g e s t s t h a t N H^ + u p t a k e i s c i r c a d i a n . T h i s c o n c l u s i o n i s s u p p o r t e d by G o e r i n g e t a l . (1964) who f o u n d r h y t h m i c v a r i a t i o n i n b o t h N H 4 + a n d N O 3 -u p t a k e b y S a r g a s s o S e a p h y t o p l a n k t o n i n c u b a t e d u n d e r c o n t i n u o u s l i g h t . H owever, i n t h e s t r a t i f i e d w a t e r s o f t h e S t r a i t o f G e o r g i a , V p t V - ^ f o r N O 3 - a n d u r e a d e m o n s t r a t e d b o t h d i e l a n d d i u r n a l v a r i a b i l i t y . D i u r n a l ( d a y t i m e ) v a r i a b i l i t y i n t h e V D : V L o f N H 4 + u p t a k e by f r e s h w a t e r p h y t o p l a n k t o n a s s e m b l a g e s o f L a k e C a l a d o ( F i s h e r e t a l . , 1988) a n d t h e S o u t h R i v e r e s t u a r y ( F i s h e r e t a l . , 1982) h a v e a l s o b e e n o b s e r v e d . I n t h e p r e s e n t s t u d y , a l l t h e e x p e r i m e n t s w e r e c o n d u c t e d a t a p p r o x i m a t e l y t h e same t i m e o f t h e d a y t h e r e b y c o m p e n s a t i n g f o r a n y d i u r n a l v a r i a b i l i t y i n N u p t a k e , ( e i t h e r i n d e p e n d e n t o r d e p e n d e n t o f t h e d a i l y l i g h t c y c l e ) a n d t h u s p e r m i t t i n g c o m p a r i s o n s b e t w e e n s t a t i o n s . An unknown p o r t i o n o f t h e d a r k u p t a k e i n t h e p r e s e n t e x p e r i m e n t s may a l s o be a t t r i b u t e d t o m a r i n e h e t e r o t r o p h i c b a c t e r i a . W h e e l e r a n d K i r c h m a n ( 1 9 8 6 ) , u s i n g m e t a b o l i c i n h i b i t o r s , s i z e - f r a c t i o n a t i o n a n d 1 5 N m e t h o d o l o g y , e s t i m a t e d t h a t 7 8% o f t h e ammonium u p t a k e i n t h e s u r f a c e w a t e r s o f f 100 Sapelo I s l a n d , Georgia and the G u l f Stream o f f Georgia was due t o b a c t e r i a . Brown et a l . (1975) r e p o r t e d N O 3 - uptake and r e d u c t i o n and N H 4 + uptake by batch c u l t u r e s of a marine pseudomonad and Remsen e t al.(1972) have demonstrated c o m p e t i t i o n f o r urea among both b a c t e r i a and phytoplankton of the e s t u a r i e s / c o a s t a l waters of Georgia. During the experiments r e p o r t e d i n t h i s study Whatman GF/F f i l t e r s were used t o c o l l e c t the p a r t i c u l a t e m a t e r i a l a f t e r i n c u b a t i o n with 1 5 N - l a b e l l e d urea and N O 3 - ; these f i l t e r s do not d i s c r i m i n a t e completely between b a c t e r i a and phytoplankton and can capture 40-50% of the b a c t e r i a i n marine systems (R. K e i l , p e r s . comm.). In the present study, the p r o p o r t i o n of uptake of i n o r g a n i c and o r g a n i c N which may be a t t r i b u t e d t o b a c t e r i a i s unknown; p r e v i o u s s t u d i e s i n shallow sea f r o n t a l systems have r e p o r t e d both g r e a t e r b a c t e r i a l biomass and r e l a t i v e h e t e r o t r o p h i c a c t i v i t y (as determined by glucose uptake) on the s t r a t i f i e d s i d e of a f r o n t i n Saanich I n l e t (Parsons et a l . , 1983), i n L i v e r p o o l Bay (Floodgate e t a l . , 1981), and the I r i s h Sea (Egan and Floodgate, 1985; Lochte, 1985). Summary The N uptake response t o PPFD of the phytoplankton i n the f r o n t a l and s t r a t i f i e d communities of the S t r a i t of Georgia can be d e s c r i b e d by the Michaelis-Menten f o r m u l a t i o n . Dark uptake of n i t r a t e and urea i s a s u b s t a n t i a l p o r t i o n of the t o t a l uptake i n these phytoplankton communities, and should not be overlooked. In the f r o n t a l waters, the dependency on PPFD f o r NC>3~ uptake i s s i m i l a r f o r both s u r f a c e and DCM 101 communities, whereas i n the s t r a t i f i e d waters, the s u r f a c e phytoplankton e x h i b i t l e s s PPFD dependency than those from the DCM, p a r t i c u l a r l y f o r urea uptake. The dramatic change i n s p e c i e s composition of the phytoplankton communities from one dominated by l a r g e , c h a i n - f o r m i n g diatoms i n the N - r e p l e t e f r o n t a l waters t o one composed p r i m a r i l y of m i c r o f l a g e l l a t e s i n the N-depleted s t r a t i f i e d waters probably c o n t r i b u t e d t o the observed v a r i a b i l i t y i n t h e i r PPFD response and p r e c l u d e s a simple e x p l a n a t i o n of PPFD e f f e c t ( s ) on N uptake based merely on phytoplankton N s t a t u s . C l e a r l y more d e t a i l e d s t u d i e s on the response of N uptake t o PPFD i n u n i a l g a l (and axenic) phytoplankton c u l t u r e s , at v a r i o u s degrees of N d e f i c i e n c y , need t o be conducted b e f o r e the e f f e c t ( s ) of N l i m i t a t i o n on the N uptake response t o PPFD can be adequately e x p l a i n e d . 102 CHAPTER THREE NITROGEN UPTAKE BY THE EUCARYOTIC PICOPLANKTER, MICROMONAS PUSILLA AND THE EFFECTS OF N DEPRIVATION ON UPTAKE RESPONSE INTRODUCTION I t i s w e l l e s t a b l i s h e d t h a t t h e a v a i l a b i l i t y o f n i t r o g e n i s t h e d o m i n a n t n u t r i t i o n a l f a c t o r r e g u l a t i n g p h y t o p l a n k t o n g r o w t h i n c o a s t a l ( e . g . , R y t h e r a n d D u n s t a n , 1971) a n d o c e a n i c w a t e r s ( e . g . , Thomas, 1966, 1969; Goldman e t a l . , 1979). The f o c u s o f many i n v e s t i g a t i o n s h a s b e e n on t h e k i n e t i c s o f n i t r o g e n u p t a k e by m a r i n e p h y t o p l a n k t o n i n o r d e r t o u n d e r s t a n d how p h y t o p l a n k t o n r e s p o n d a n d a d a p t t o n i t r o g e n l i m i t a t i o n . D u g d a l e (1967) f i r s t p r o p o s e d r e l a t i n g n i t r o g e n u p t a k e r a t e s o f p h y t o p l a n k t o n t o t h e e x t e r n a l N c o n c e n t r a t i o n b y a h y p e r b o l i c f u n c t i o n w h i c h was s i m i l a r t o t h e M i c h a e l i s - M e n t e n e q u a t i o n f o r enzyme k i n e t i c s , V = V m a x [ S / ( K g + S ) ] , w h e r e V i s t h e u p t a k e v e l o c i t y ( h - 1 ) , V m a x t h e m a x i m a l u p t a k e v e l o c i t y , S t h e c o n c e n t r a t i o n o f l i m i t i n g s u b s t r a t e a n d K s t h e h a l f -s a t u r a t i o n c o n s t a n t r e p r e s e n t i n g t h e v a l u e o f S a t w h i c h V i s e q u a l t o h a l f t h e maximum u p t a k e r a t e ( i . e . , V = v m a x / 2 ) . M e a s u r e m e n t o f u p t a k e r a t e s b y n a t u r a l p h y t o p l a n k t o n a s s e m b l a g e s ( e . g . , M a c l s a a c a n d D u g d a l e , 1969; P r o b y n , 1985; K a n d a e t a l . , 1985; W h a l e n a n d A l e x a n d e r , 1986) a n d numerous c u l t u r e s t u d i e s ( e . g . , E p p l e y a n d C o a t s w o r t h , 1968; E p p l e y a n d Thomas, 1969; E p p l e y e t a l . , 1969) h a v e d e m o n s t r a t e d t h a t t h e u p t a k e o f n i t r o g e n o u s n u t r i e n t s c a n be d e s c r i b e d b y t h i s h y p e r b o l i c f u n c t i o n w h i c h r e l a t e s u p t a k e r a t e t o t h e l i m i t i n g n u t r i e n t c o n c e n t r a t i o n . 103 The s p e c i e s s p e c i f i c n u t r i e n t u p t a k e k i n e t i c p a r a m e t e r s , V m a x a n d K s may be u s e d t o e x p l a i n s p e c i e s c o m p e t i t i o n i n v o l v i n g t h e l i m i t i n g n u t r i e n t ( D u g d a l e , 1967; T i l m a n , 1977; B u t t o n , 1 9 8 5 ) . I t i s now r e a l i z e d t h a t V m a x i s f r e q u e n t l y v a r i a b l e o v e r t i m e , a n d f o r some n u t r i e n t s i t may be e l e v a t e d i n i t i a l l y o v e r t h e f i r s t f e w m i n u t e s f o l l o w i n g e n r i c h m e n t o f t h e l i m i t i n g n u t r i e n t w i t h u p t a k e p r o c e e d i n g a t a s l o w e r , more c o n s t a n t r a t e l a t e r . S e v e r a l s t u d i e s h a v e d e m o n s t r a t e d t h a t N - d e f i c i e n t o r N - s t a r v e d p h y t o p l a n k t o n h a v e t h e a b i l i t y t o r a p i d l y t a k e up ammonium i n i t i a l l y u p o n e x p o s u r e t o a n e l e v a t e d N H 4 + c o n c e n t r a t i o n i n t h e c u l t u r e medium (Conway e t a l . , 197 6; Conway a n d H a r r i s o n , 197 7; M c C a r t h y a n d G o l d m a n , 1979; D o r t c h e t a l . , 1982; Goldman a n d G l i b e r t , 1982; H o r r i g a n a n d M c C a r t h y , 1982; P a r s l o w e t a l . , 1984a b) a n d i n t h e f i e l d ( G l i b e r t a n d Goldm a n , 1 9 81; W h e e l e r e t a l . , 1982; H a r r i s o n , 1 9 8 3 a ; P r i s c u a n d P r i s c u , 1984, P r i s c u , 1987; S u t t l e a n d H a r r i s o n , 1 9 8 8 ) . I n c o n t r a s t , a f t e r a n i t r a t e a d d i t i o n t o a N - s t a r v e d c u l t u r e t h e r e i s o f t e n , b u t n o t a l w a y s , a l a g whose d u r a t i o n i s q u i t e v a r i a b l e , b e f o r e n i t r a t e u p t a k e i s o b s e r v e d a t e i t h e r e l e v a t e d , n o r m a l o r r e d u c e d v e l o c i t i e s ( D o r t c h e t a l . , 1982; C o l l o s , 1983; P a r s l o w e t a l . , 1984 b ) . N i t r o g e n s t a r v a t i o n may (R e e s a nd S y r e t t , 197 9; H o r r i g a n a n d M c C a r t h y , 1981; S y r e t t e t a l . , 1986; P r i c e a n d H a r r i s o n , 1 9 8 8 ) , o r may n o t ( B e k h e e t a n d S y r e t t , 1979) i n c r e a s e u r e a u p t a k e r a t e s a l t h o u g h o n l y t h r e e p h y t o p l a n k t e r s h a v e b e e n t e s t e d t o d a t e . U p t a k e i n t e r a c t i o n s b e t w e e n i n o r g a n i c n i t r o g e n s o u r c e s , a n d more p a r t i c u l a r l y ammonium a n d n i t r a t e , h a v e b e e n t h e 104 s u b j e c t o f many c u l t u r e s t u d i e s ( s e e r e v i e w s b y M o r r i s , 1974; M c C a r t h y , 1 9 8 1; S y r e t t , 1 9 8 1 , C o l l o s , 1989, D o r t c h , i n p r e s s ) . T h e s e s t u d i e s h a v e r e v e a l e d a v a r i e t y o f r e s p o n s e s d e p e n d i n g on s p e c i e s a n d t h e i r n u t r i t i o n a l s t a t e , b u t one o f t h e m a i n t e n e t s s t i l l h e l d i n p h y t o p l a n k t o n e c o l o g y i s t h a t t h e u p t a k e o f n i t r a t e s t o p s when t h e a m b i e n t N H 4 + c o n c e n t r a t i o n e x c e e d s a c e r t a i n t h r e s h o l d a n d r e s u m e s when a l g a l u p t a k e c a u s e s t h e a m b i e n t N H 4 + c o n c e n t r a t i o n t o d e c r e a s e b e l o w t h i s t h r e s h o l d v a l u e . The n e g a t i v e e f f e c t o f N H 4 + on N O 3 - u p t a k e i s o f t e n n o t t h a t s e v e r e a n d t h e r e a r e numerous e x a m p l e s o f s i m u l t a n e o u s a n d e q u a l r a t e s o f u p t a k e o f b o t h N O 3 - and N H 4 + , m o s t l y i n N - d e f i c i e n t o r N - l i m i t e d c e l l s ( e . g . , C a p e r o n a n d Z i e m a n n , 197 6; Conway, 197 7; DeManche e t a l . , 197 9) a n d i n t h e f i e l d ( e . g . , C o n o v e r , 1975; M c C a r t h y e t a l . , 1977; M a e s t r i n i e t a l . , 1982, 1986; P r i c e e t a l . , 1985; C o l l o s e t a l . , 1 9 8 9 ) . The l i t e r a t u r e i s r e p l e t e w i t h s t u d i e s on many a s p e c t s o f p h y t o p l a n k t o n n i t r o g e n u t i l i z a t i o n , i n c l u d i n g : 1) t h e e s t i m a t i o n o f k i n e t i c p a r a m e t e r s , 2) t h e i r t r a n s i e n t n a t u r e u n d e r c o n d i t i o n s o f p h y s i o l o g i c a l s t r e s s a n d 3) t h e i n t e r f e r e n c e / i n t e r a c t i o n o f m u l t i p l e n i t r o g e n s o u r c e s . H o wever, t h e r e a r e few o b s e r v a t i o n s o f n i t r o g e n u t i l i z a t i o n by p i c o p l a n k t o n (0.2 um - <2.0 um, S i e b u r t h e t a l . , 1978) a n d t h e r e d o e s n o t a p p e a r t o be any k i n e t i c s t u d i e s on N u p t a k e by m a r i n e p i c o p l a n k t e r s . The u b i q u i t o u s a n d u s u a l l y a b u n d a n t p r e s e n c e o f b o t h C y a n o b a c t e r i a a n d e u c a r y o t i c a l g a e i n t h i s s i z e c l a s s h a s b e e n r e p o r t e d i n o f f s h o r e a n d n e a r s h o r e w a t e r s ( s e e r e v i e w s b y F o g g , 1986; J o i n t , 1986; S t o c k n e r a n d A n t i a , 105 1986; M i k h e y e v a , 1 9 8 8 ) . T h e i r i m p o r t a n c e a s p h o t o a u t o t r o p h s h a s b e e n d e m o n s t r a t e d i n c e r t a i n e n v i r o n m e n t s , p a r t i c u l a r l y o l i g o t r o p h i c , o c e a n i c r e g i o n s , w h e r e p i c o p l a n k t o n a r e r e s p o n s i b l e f o r t h e m a j o r i t y o f p h o t o s y n t h e t i c p r o d u c t i o n ( e . g . , L i e t e l . , 1983; P i a t t e t a l , 1983; T a k a h a s h i a n d B i e n f a n g , 1 9 8 3 ; a n d a b o v e r e v i e w s ) . S t u d i e s w i t h s i z e -f r a c t i o n a t i o n t e c h n i q u e s a n d n i t r o g e n t r a c e r s h a v e c o n f i r m e d t h a t p i c o p l a n k t o n n i t r o g e n u p t a k e i s a l s o s u b s t a n t i a l , a v e r a g i n g 1 0 - 3 0 % a n d 3 0 - 7 0 % o f t h e t o t a l N u p t a k e o f t h e n a t u r a l c o m m u n i t i e s o f c o a s t a l a n d o c e a n i c w a t e r s , r e s p e c t i v e l y ( G l i b e r t , 1 9 8 2 ; N a l e w a j k o a n d G a r s i d e , 1983; P r o b y n , 1985; P r o b y n a nd P a i n t i n g , 1985; H a r r i s o n a n d Wood, 1988) . I n t h e p r e s e n t s t u d y t h e u t i l i z a t i o n o f n i t r a t e , ammonium a n d u r e a by t h e p r a s i n o p h y t e , p i c o f l a g e l l a t e Micromonas pusilla ( B u t c h . ) M a n t o n e t P a r k e was d e m o n s t r a t e d b y u s i n g N-r e p l e t e a n d N - s t a r v e d c e l l s a n d m e a s u r i n g t h e u p t a k e r a t e o f NO^ - i n t h e p r e s e n c e o f p o t e n t i a l c o m p e t i t o r s , ammonium a n d u r e a . T h e r e f o r e , t h e o b j e c t i v e o f t h i s r e s e a r c h was t o d e t e r m i n e t h e b a s i c a s p e c t s o f n i t r o g e n o u s n u t r i t i o n o f t h i s u b i q u i t o u s , e u k a r y o t i c p i c o f l a g e l l a t e . T h i s s t u d y i s t h e f i r s t t o r e p o r t on t h e n i t r o g e n u t i l i z a t i o n o f a c u l t u r e d p i c o p l a n k t e r . 106 MATERIALS AND METHODS Culturing S t o c k c u l t u r e s o f Micromonas pusilla ( c u l t u r e NEPCC 29-1 N o r t h e a s t P a c i f i c C u l t u r e C o l l e c t i o n , D e p a r t m e n t o f O c e a n o g r a p h y , U n i v e r s i t y o f B r i t i s h C o l u m b i a ) w e r e m a i n t a i n e d on 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 ) 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 s e a w a t e r b a s e d on ESAW ( H a r r i s o n e t a l . , 1 9 8 0 ) . M o d i f i c a t i o n s t o ESAW i n c l u d e d r e p l a c i n g f e r r o u s ammonium s u l f a t e ( F e N H ^ ( S 0 4 ) 2 *6H2O) a n d s o d i u m g l y c e r o p h o s p h a t e w i t h e q u i m o l a r c o n c e n t r a t i o n s o f f e r r i c c h l o r i d e (FeCl3«6H20) a n d s o d i u m p h o s p h a t e ( N a 2 H P 0 4 ) , r e s p e c t i v e l y ( P a r s l o w e t a l . , 1 9 8 4 a ) . S o d i u m m e t a s i l i c a t e (Na2Si03•9 H 2 O) was p r e p a r e d a n d a d d e d a s d e s c r i b e d by S u t t l e e t a l . ( 1 9 8 6) a n d 10 nM Se was a d d e d a s s e l e n i t e (Na2Se03) a c c o r d i n g t o H a r r i s o n e t a l . ( 1 9 8 8 ) . N i t r a t e , t h e s o l e n i t r o g e n e n r i c h m e n t , was r e d u c e d f r o m 550 t o 50 uq-at N - L - 1 . R e a g e n t g r a d e c h e m i c a l s w e r e u s e d i n p r e p a r i n g s a l t a n d n u t r i e n t e n r i c h m e n t s o l u t i o n s i n d e i o n i z e d , d i s t i l l e d w a t e r (DDW). G l a s s a n d p o l y c a r b o n a t e f l a s k s u s e d f o r c u l t u r i n g a l g a e a n d s t o r i n g ESAW w e r e s o a k e d i n f r e s h l y - m a d e 10% HC1 ( v / v ) f o r a t l e a s t 2-3 d a y s , r i n s e d t h o r o u g h l y w i t h DDW, a n d a u t o c l a v e d p r i o r t o u s e . A l l c u l t u r e s ( i . e . s t o c k a n d e x p e r i m e n t a l ) w e r e c o n t i n u o u s l y i l l u m i n a t e d f r o m two s i d e s by s i x V i t a - L i t e R UHO f l u o r e s c e n t t u b e s (3 on e i t h e r s i d e o f c u l t u r e v e s s e l s ) . The l i g h t was f i l t e r e d t h r o u g h 3 mm t h i c k b l u e P l e x i g l a s R (No. 20 6 9 , Rohm a n d Haas) a n d t h e i r r a d i a n c e , m e a s u r e d w i t h a 2n c o l l e c t o r ( L i C o r L I - 1 9 2 S B ) f r o m t h e c e n t r e p o s i t i o n o f t h e 107 2 1 c u l t u r e v e s s e l s , was c a . 120 uE'm *s ( s a t u r a t i n g f o r g r o w t h o f M. pusilla, s e e A p p e n d i x 2 ) . T e m p e r a t u r e was m a i n t a i n e d a t 17°C (± 0.5°C) i n a t e m p e r a t u r e r e g u l a t e d w a t e r b a t h a n d c u l t u r e s c o n t i n u o u s l y s t i r r e d b y t e f l o n - c o a t e d m a g n e t i c s t i r b a r s a t 60 rpm. C u l t u r e s w e r e u n i a l g a l a n d s t e r i l e t e c h n i q u e was e m p l o y e d t o m i n i m i z e b a c t e r i a l c o n t a m i n a t i o n . C e l l g r o w t h was m o n i t o r e d b y 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 m e a s u r e d by a T u r n e r D e s i g n s m o d e l 10 f l u o r o m e t e r . Analytical methods C e l l c o u n t s w e r e m e a s u r e d w i t h a C o u l t e r C o u n t e r m o d e l TA I I e l e c t r o n i c p a r t i c l e c o u n t e r w i t h t h e p o p u l a t i o n a c c e s s o r y ( e n a b l i n g c e l l s t o be c o u n t e d i n t o 16 c h a n n e l s b a s e d on t h e i r v o l u m e ) a n d e q u i p p e d w i t h a 30 um a p e r t u r e . The C o u l t e r C o u n t e r was c a l i b r a t e d w i t h l a t e x m i c r o s p h e r e s o f 2.02 um i n d i a m e t e r . C u l t u r e s a m p l e s w e r e d i l u t e d ( 1 : 2 0 ) w i t h f r e s h l y f i l t e r - s t e r i l i z e d , u n e n r i c h e d ESAW ( g r a v i t y f i l t r a t i o n , Whatman GF/F) a n d g e n t l y h o m o g e n i z e d ( i . e . m i x e d ) p r i o r t o c o u n t i n g . A v e r a g e c e l l v o l u m e s w e r e c o m p u t e d f r o m t h e p a r t i c l e s i z e d i s t r i b u t i o n b a s e d on e q u i v a l e n t s p h e r i c a l d i a m e t e r . C o n c e n t r a t i o n s o f d i s s o l v e d N 0 3 ~ + NO2 - a n d N H 4 + w e r e m e a s u r e d w i t h a T e c h n i c o n A u t o A n a l y z e r I I f o l l o w i n g t h e p r o c e d u r e s o u t l i n e d i n Wood e t a l . , ( 1967) a n d S l a w y k a n d M a c l s a a c ( 1 9 7 2 ) , r e s p e c t i v e l y . S a m p l e s f o r n u t r i e n t a n a l y s e s w e r e f i l t e r e d t h r o u g h p r e c o m b u s t e d (460°C f o r 4 h) Whatman GF/F f i l t e r s i n t o p r e v i o u s l y a c i d - w a s h e d , D D W - r i n s e d p o l y p r o p y l e n e b o t t l e s . Ammonium c o n c e n t r a t i o n s w e r e a l w a y s 108 d e t e r m i n e d i m m e d i a t e l y w h i l e N O 3 - + NO2"" c o n c e n t r a t i o n s w e r e o c c a s i o n a l l y m e a s u r e d l a t e r a f t e r f r o z e n s t o r a g e (-20°C). D u p l i c a t e s a m p l e s (20-30 m l ) f o r p a r t i c u l a t e o r g a n i c c a r b o n a n d n i t r o g e n (POC a n d PON) w e r e c o l l e c t e d a t t h e s t a r t a n d e n d o f e x p e r i m e n t a t i o n on p r e c o m b u s t e d Whatman GF/F f i l t e r s a n d s t o r e d f r o z e n i n d e s i c c a t o r s . A f t e r t h a w i n g / d r y i n g (<60°C f o r 24 h) s a m p l e s w e r e a n a l y z e d by t h e d r y c o m b u s t i o n m e t h o d o f S h a r p ( 1974) w i t h e i t h e r a C o n t r o l E q u i p m e n t C o r p . m o d e l 240-XA ( r e m a n u f a c t u r e d P e r k i n - E l m e r m o d e l 240) o r a C a r l o E r b a m o d e l 1106 e l e m e n t a l a n a l y z e r . B o t h i n s t r u m e n t s w e r e c a l i b r a t e d w i t h a c e t a n i l i d e s t a n d a r d s . 15 S a m p l e s f o r N a n a l y s i s w e r e c o l l e c t e d on p r e c o m b u s t e d Whatman GF/F f i l t e r s , f o l d e d , p l a c e d i n t o a c i d - w a s h e d , p e t r i -d i s h e s , a n d i m m e d i a t e l y f r o z e n f o r l a t e r i s o t o p i c a n a l y s e s . N i t r o g e n i n t h e p a r t i c u l a t e s a m p l e s was c o n v e r t e d t o d i n i t r o g e n g a s ( N 2 ) by t h e m i c r o - Dumas d r y c o m b u s t i o n t e c h n i q u e (LaRoche,1983) an d s u b s e q u e n t l y a n a l y z e d f o r 1 5 N e n r i c h m e n t w i t h a JASCO m o d e l N-150 e m i s s i o n s p e c t r o m e t e r ( F i e d l e r a n d P r o k s c h , 1975) a s o u t l i n e d i n C h a p t e r 1. Experimental procedures K i n e t i c p a r a m e t e r s f o r N u p t a k e The k i n e t i c p a r a m e t e r s o f n i t r a t e , ammonium a n d u r e a u p t a k e w e r e d e t e r m i n e d w i t h d u p l i c a t e N 0 3 ~ - r e p l e t e c u l t u r e s o f M. pusilla grown i n 2 L P y r e x f l a t - b o t t o m b o i l i n g f l a s k s , f i t t e d w i t h s i l i c o n e s t o p p e r s . P r i o r t o e x p e r i m e n t a t i o n , a m b i e n t N O 3 - + NO2"" was m e a s u r e d a t 0.5 - 1 h i n t e r v a l s . The e x p e r i m e n t s w e r e i n i t i a t e d i m m e d i a t e l y a f t e r t h e n i t r a t e 109 c o n c e n t r a t i o n was <0.05 ug-at N * L - 1 i n the c u l t u r e medium. Less than 2 h e l a p s e d between the time ambient c o n c e n t r a t i o n s were c o n s i d e r e d s a t u r a t i n g t o growth (>2.5 ^g-at N-L - 1) and the time they had decreased t o d e t e c t i o n l i m i t s (0.05 ug-at N « L - 1 ) . The t i m i n g was c r i t i c a l , because n i t r a t e i n the medium must be d e p l e t e d , but the c o n d i t i o n of the c e l l s had t o be n e a r l y N r e p l e t e t o minimize n o n - l i n e a r i t y i n uptake d u r i n g e x p e r i m e n t a t i o n . Immediately f o l l o w i n g ambient NO3 -d e p l e t i o n , 60 ml subsamples were t r a n s f e r r e d t o a s e r i e s of • • R s t e r i l e , 85 ml polycarbonate Oak Ridge tubes (Nalgene ); a l t e r n a t i v e l y 200 ml subsamples t o 250 ml pol y c a r b o n a t e R 1 5 Erlenmeyer f l a s k s (Nalgene ), and i n o c u l a t e d w i t h N - l a b e l l e d NO3, NH4 or urea (Kor Isotopes, 99 atom %) at a range of i n i t i a l s u b s t r a t e c o n c e n t r a t i o n s (0.2, 0.4, 0.8, 1.6, 2.4, 4.2 and 10 ug-at N * L - 1 ) . Incubations were conducted under the same c o n d i t i o n s as those under which the c e l l s were grown and i n c u b a t i o n s terminated a f t e r 10 min by f i l t r a t i o n ( pressure d i f f e r e n t i a l s 80 mm Hg). The t i t r a t i o n p e r i o d was always l e s s than 30 s. The 4.2 and 10 ug-at N * L - 1 enrichment experiments were allowed t o incuba t e f o r an a d d i t i o n a l 50 min to determine i f uptake r a t e s were constant w i t h i n c u b a t i o n time. P r e v i o u s l y acid-washed po l y p r o p y l e n e b o t t l e s , were r i n s e d once wi t h f i l t r a t e and then used t o c o l l e c t the f i l t r a t e . The f i l t e r e d samples were immediately analyzed f o r ambient NH^ "1" and N O 3 - + N 0 2 ~ c o n c e n t r a t i o n s . The i n i t i a l s u b s t r a t e c o n c e n t r a t i o n s at time zero ( T Q ) were c a l c u l a t e d by 1 5 — 1 5 + adding known volumes of NO3 and NH4 t o c u l t u r e f i l t r a t e 110 a n d t h e n m e a s u r i n g a m b i e n t c o n c e n t r a t i o n s . S p e c i f i c u p t a k e r a t e s (N t a k e n up p e r u n i t PON) w e r e c a l c u l a t e d a c c o r d i n g t o a c o n s t a n t s p e c i f i c u p t a k e m o d e l ( D u g d a l e a n d W i l k e r s o n , 1986 ; e q u a t i o n 6 o f A p p e n d i x 1 ) . S u b s t r a t e i n t e r a c t i o n Two s e r i e s o f s u b s t r a t e i n t e r a c t i o n e x p e r i m e n t s w e r e p e r f o r m e d w i t h N 0 3 ~ - r e p l e t e c u l t u r e s . I n t h e f i r s t s e r i e s , a m b i e n t i n o r g a n i c N ( N O 3 - + N02~ a n d N H 4 + ) l e v e l s w e r e m o n i t o r e d e v e r y 30 m i n f o r 3 h b e f o r e t h e n i t r a t e c o n c e n t r a t i o n d e c r e a s e d t o 15 uq a t N - L - 1 . A s i n g l e 4-L c u l t u r e was d i v i d e d a n d p o u r e d i n t o f o u r 1 L P y r e x f l a t -b o t t o m e d b o i l i n g f l a s k s 1 h p r i o r t o N s u b s t r a t e e n r i c h m e n t . The e x p e r i m e n t s w e r e i n i t i a t e d when 10 uq-at N • L ~ 1 o f ^ N l a b e l l e d N 0 3 ~ ( e n r i c h e d c o n t r o l ) , N H 4 + and u r e a (99 atom %) w e r e a d d e d t o t h r e e o f t h e f l a s k s a n d t h e r e m a i n i n g f l a s k was n o t e n r i c h e d ( u n d i s t u r b e d c o n t r o l ) . F i l t e r e d s a m p l e s w e r e t h e n c o l l e c t e d a t 20-30 m i n i n t e r v a l s f o r 4 h f o r i m m e d i a t e N O 3 - + N O 2 - a n d NH 4 + a n a l y s e s f o l l o w i n g p r e v i o u s l y o u t l i n e d p r o c e d u r e s . N i t r a t e a n d ammonium u p t a k e r a t e s w e r e c a l c u l a t e d f r o m t h e s l o p e o f s e p a r a t e l i n e a r r e g r e s s i o n s o f s u b s t r a t e c o n c e n t r a t i o n i n t h e medium p l o t t e d a g a i n s t t i m e . T h e s e u p t a k e r a t e s a r e t e r m e d ( a b s o l u t e o r t r a n s p o r t r a t e s ) a n d a r e e x p r e s s e d a s uq-at N - L - 1 • h - 1 . S p e c i f i c u p t a k e r a t e s w e r e o b t a i n e d b y d i v i d i n g t h e a b s o l u t e r a t e s b y t h e e x p o n e n t i a l a v e r a g e c o n c e n t r a t i o n o f PON ( g e o m e t r i c mean) o v e r t h e d u r a t i o n o f t h e i n c u b a t i o n p e r i o d . The s p e c i f i c u p t a k e o f u r e a was d e t e r m i n e d f r o m a I l l c o n s t a n t s p e c i f i c u p t a k e m o d e l ( D u g d a l e a n d W i l k e r s o n , 1 9 8 6 ; e q u a t i o n 8 o f A p p e n d i x 1 ) . T h i s e q u a t i o n c o m p e n s a t e s f o r t h e e f f e c t o f s i m u l t a n e o u s u p t a k e o f u n l a b e l l e d s u b s t r a t e b y u t i l i z i n g i n d e p e n d e n t e s t i m a t e s o f t h e a b s o l u t e u p t a k e o f u n l a b e l l e d n i t r a t e p r o v i d e d f r o m N O 3 - d i s a p p e a r a n c e m e a s u r e m e n t s t h u s c o r r e c t i n g f o r t h e i s o t o p e d i l u t i o n i n t h e p a r t i c u l a t e m a t t e r o r i g i n a t i n g f r o m t h e 1 4 N i n t h e u n l a b e l l e d s o u r c e ( C o l l o s , 1987; L u n d , 1 9 8 7 ) . I t s h o u l d be n o t e d t h a t i n D u g d a l e a n d W i l k e r s o n (1986) t h e e q u a t i o n f o r c o n s t a n t s p e c i f i c u p t a k e ( V c ) , when u n l a b e l l e d s o u r c e s a r e p r e s e n t , i s i n c o r r e c t l y w r i t t e n a n d i s c o r r e c t l y r e p o r t e d i n A p p e n d i x 1. The a b s o l u t e ( t r a n s p o r t ) r a t e o f u r e a was c a l c u l a t e d u s i n g e q u a t i o n 10 o f D u g d a l e a n d W i l k e r s o n ( e q u a t i o n 7 o f A p p e n d i x 1) w h i c h c o m p e n s a t e s f o r t h e s i m u l t a n e o u s u p t a k e o f u n l a b e l l e d N O 3 - a n d t h e c h a n g e i n c o n c e n t r a t i o n o f PON d u r i n g t h e i n c u b a t i o n p e r i o d . E f f e c t o f NH^"1" c o n c e n t r a t i o n on N O 3 - u p t a k e r a t e I n t h e s e c o n d s e r i e s o f e x p e r i m e n t s , t h e e f f e c t o f NH^"1" c o n c e n t r a t i o n on t h e u p t a k e o f N03~ was e x a m i n e d . A N O 3 - -r e p l e t e c u l t u r e was m o n i t o r e d f o r t h e d e c l i n e o f N O 3 - + N O 2 -i n t h e medium a n d f o u r 250 m l s u b s a m p l e s w e r e t r a n s f e r r e d t o p o l y c a r b o n a t e ( N a l g e n e ) E r l e n m e y e r f l a s k s ( f i t t e d w i t h s i l i c o n e s t o p p e r s a n d s a m p l i n g t u b e s ) p r i o r t o t h e a m b i e n t N O 3 - c o n c e n t r a t i o n r e a c h i n g 15 jug-at N - L - 1 . ^ N H ^ C l (99 atom %) was a d d e d t o t h e f l a s k s t o b r i n g t h e i n i t i a l e n r i c h m e n t N H 4 + c o n c e n t r a t i o n t o 5, 2, a n d 1 uq-at N « L - 1 . F i f t y m l s u b s a m p l e s w e r e r e m o v e d e v e r y 20-30 m i n f o r 2 h a n d ^ N 112 i n c u b a t i o n s t e r m i n a t e d by f i l t r a t i o n . F i l t r a t i o n , c o l l e c t i o n o f f i l t r a t e , s t o r a g e a n d a n a l y s e s w e r e c o n d u c t e d a s p r e v i o u s l y o u t l i n e d . S a m p l e s f o r PON a n a l y s i s w e r e c o l l e c t e d a t t h e b e g i n n i n g , m i d d l e a n d e n d o f t h e i n c u b a t i o n s . The d i s a p p e a r a n c e r a t e o f N O 3 - was c a l c u l a t e d f r o m t h e s l o p e o f e x t e r n a l N O 3 - + N O 2 - c o n c e n t r a t i o n a g a i n s t t i m e ; d i v i s i o n o f t h i s r a t e b y t h e e x p o n e n t i a l a v e r a g e PON c o n c e n t r a t i o n p r o v i d e d a n e s t i m a t e o f t h e s p e c i f i c N O 3 - u p t a k e r a t e . The s p e c i f i c N H 4 + u p t a k e r a t e s w e r e c a l c u l a t e d u s i n g t h e c o n s t a n t s p e c i f i c u p t a k e m o d e l ( D u g d a l e a n d W i l k e r s o n , 1986; e q u a t i o n 6 1 C 1 c o f A p p e n d i x 1 ) w i t h t h e atom % N e x c e s s ( N e x ) i n t h e p a r t i c u l a t e m a t t e r e s t i m a t e d f r o m t h e s l o p e o f t h e l e a s t -1 5 s q u a r e s l i n e a r r e g r e s s i o n o f N e x v e r s u s t i m e , p r i o r t o i s o t o p e d e p l e t i o n . U p t a k e o f n i t r o g e n by N 0 3 ~ - s t a r v e d c e l l s Two s e r i e s o f e x p e r i m e n t s w e r e c o n d u c t e d , b o t h i n d u p l i c a t e , t o a s s e s s t h e e f f e c t o f n i t r o g e n d e f i c i e n c y on t h e u p t a k e o f N H 4 + o r u r e a by N 0 3 ~ - s t a r v e d c u l t u r e s o f Micromonas pusilla. I n e a c h s e r i e s n i t r o g e n - s t a r v e d c e l l s w e r e o b t a i n e d f r o m d u p l i c a t e b a t c h c u l t u r e s , s t a r t e d w i t h 50 uq-at N - N 0 3 ' L - 1 a s t h e i n i t i a l c o n c e n t r a t i o n a n d N s o u r c e a n d a l l o w e d t o r e m a i n i n n i t r o g e n - f r e e medium f o r 2 d a f t e r t h e e x t e r n a l n i t r o g e n was d e p l e t e d . A f t e r t h e c u l t u r e s became N - s t a r v e d , t h e y w e r e s p l i t i n t o s e p a r a t e f l a s k s a n d e i t h e r 15 uq-at N « L - 1 o f 1 5 N H 4 C 1 o r C O ( 1 5 N H 4 ) 2 ( b o t h 99 atom %) was a d d e d t o e a c h s u b c u l t u r e . The a m b i e n t N H 4 + c o n c e n t r a t i o n i n t h e medium a n d 1 c t h e N a c c u m u l a t i o n i n t h e c e l l s w e r e m e a s u r e d a t t i m e 113 i n t e r v a l s o f 5-15 m i n f o r 3 h a c c o r d i n g t o p r o c e d u r e s o u t l i n e d a b o v e . I n t h e s e c o n d s e r i e s o f e x p e r i m e n t s , d u p l i c a t e N C ^ -1 5 s t a r v e d c u l t u r e s w e r e e n r i c h e d w i t h Na NO3 (99 a t o m %) a n d s a m p l e s c o l l e c t e d f o r me a s u r e m e n t o f a m b i e n t N O 3 -1 c c o n c e n t r a t i o n a n d N a c c u m u l a t i o n a t t i m e i n t e r v a l s o f 5-30 1 5 • m m f o r 6 h. U p t a k e r a t e s , e s t i m a t e d f r o m N a c c u m u l a t i o n i n t h e p a r t i c u l a t e s , w e r e c a l c u l a t e d a c o r d i n g t o t h e c o n s t a n t , s p e c i f i c u p t a k e m o d e l ( V c ) o f D u g d a l e a n d W i l k e r s o n ( 1 9 8 6) ( e q u a t i o n 6 o f A p p e n d i x 1) f r o m m e a s u r e m e n t s o f a t o m % e x c e s s 1 c o f N i n s u c c e s s i v e s a m p l e s d u r i n g t h e t i m e i n t e r v a l s U p t a k e r a t e s , e s t i m a t e d f r o m d i s a p p e a r a n c e o f N O 3 - o r N H 4 + i n t h e medium, w e r e c a l c u l a t e d by d i v i d i n g t h e d i f f e r e n c e i n n u t r i e n t c o n c e n t r a t i o n i n s u c c e s s i v e s a m p l e s by t h e l e n g t h o f t h e t i m e i n t e r v a l ; s p e c i f i c r a t e s w e r e c a l c u l a t e d by d i v i d i n g t h i s v a l u e b y t h e e s t i m a t e d e x p o n e n t i a l a v e r a g e PON c o n c e n t r a t i o n d u r i n g t h a t t i m e a s s u m i n g t h a t a l l t h e n u t r i e n t r e m o v e d f r o m t h e medium was i n c o r p o r a t e d i n t o t h e p a r t i c u l a t e f r a c t i o n ( s e e A p p e n d i x 4 ) . Estimation of kinetic parameters The k i n e t i c p a r a m e t e r s , K g a n d V m a x w e r e o b t a i n e d i n t w o w a y s : a d i r e c t f i t o f t h e d a t a t o t h e M i c h a e l i s - M e n t e n h y p e r b o l a u s i n g a c o m p u t e r i z e d , i t e r a t i v e , n o n - l i n e a r , l e a s t -s q u a r e s r e g r e s s i o n t e c h n i q u e ( L a b t e c N o t e b o o k C u r v e f i t , L a b o r a t o r y T e c h n o l o g i e s C o r p . ) a n d a l e a s t - s q u a r e s l i n e a r r e g r e s s i o n a n a l y s i s o f H a n e s - W o o l f l i n e a r t r a n s f o r m a t i o n (S/V v s S) o f t h e d a t a . I n t h e l a t t e r m e t h o d , t h e s t a n d a r d e r r o r s 114 o f t h e k i n e t i c p a r a m e t e r s w e r e e s t i m a t e d u s i n g t h e D e l t a m e t h o d o f v a r i a n c e s ( B i s h o p e t a l . , 1 9 7 5 ) . The H a n e s - W o o l f t r a n s f o r m a t i o n was u s e d i n p r e f e r e n c e t o o t h e r l i n e a r t r a n s f o r m a t i o n s a s i t g a v e a b e t t e r s p r e a d o f t h e d a t a p o i n t s a n d g e n e r a l l y p r o v i d e d t h e most a c c u r a t e d e t e r m i n a t i o n s o f K g a n d V m a x (Dowd a n d R i g g s , 1 9 6 5 ) . H owever, an y l i n e a r i z a t i o n o f t h e M i c h a e l i s - M e n t e n e q u a t i o n v i o l a t e s a b a s i c a s s u m p t i o n o f l e a s t - s q u a r e s r e g r e s s i o n a n a l y s i s , l a c k o f e r r o r i n t h e i n d e p e n d e n t v a r i a b l e , S ( Z a r , 1 9 7 4 ) . A l t h o u g h i n p r a c t i c e t h i s a s s u m p t i o n i s s u f f i c i e n t l y met i f e r r o r s i n t h e i n d e p e n d e n t v a r i a b l e a r e s m a l l r e l a t i v e t o e r r o r s i n t h e d e p e n d e n t v a r i a b l e . The p r o b l e m o f u t i l i z i n g u n w e i g h t e d , t r a n s f o r m e d d a t a (Dowd a n d R i g g s , 1965) a n d t h e i n e v i t a b l e c o r r e l a t i o n b e t w e e n v a r i a b l e s ( m e a s u r e d v a r i a b l e S a p p e a r s i n b o t h d e p e n d e n t a n d i n d e p e n d e n t v a r i a b l e s ) makes a l i n e a r t r a n s f o r m a t i o n s t a t i s t i c a l l y i n f e r i o r t o d i r e c t , n o n - l i n e a r f i t t i n g o f d a t a t o t h e M i c h a e l i s - M e n t e n e q u a t i o n ( L i , 1983; R o b i n s o n a n d C h a r a c k l i s , 1 9 8 4 ) . S i n c e e a r l i e r i n v e s t i g a t o r s d i d n o t a l w a y s h a v e t h e same a c c e s s i b i l i t y t o n o n - l i n e a r f i t t i n g by c o m p u t e r s a s we e n j o y t o d a y , t h e p a r a m e t e r s , e s t i m a t e d by b o t h m e t h o d s , h a v e b e e n i n c l u d e d i n T a b l e 3.1 f o r l i t e r a t u r e c o m p a r a t i v e p u r p o s e s . The k i n e t i c p a r a m e t e r s h a v e b e e n c a l c u l a t e d s e p a r a t e l y f o r t h e i n d i v i d u a l c u l t u r e s a s w e l l a s f o r t h e d a t a t r e a t e d t o g e t h e r . 115 RESULTS Uptake kinetics N i t r o g e n s p e c i f i c u p t a k e r a t e s a r e p l o t t e d v e r s u s t h e a v e r a g e a m b i e n t n i t r o g e n c o n c e n t r a t i o n e x p e r i e n c e d b y t h e c e l l s d u r i n g t h e 10 m i n i n c u b a t i o n p e r i o d ( F i g . 3 . 1 ) . A l i s t o f t h e h a l f s a t u r a t i o n c o n s t a n t s ( K g ) a n d maximum u p t a k e v e l o c i t i e s ( V m a x ) a r e p r e s e n t e d i n T a b l e 3.1 a l o n g w i t h t h e i r e s t i m a t e d s t a n d a r d e r r o r s . G e n e r a l l y t h e v a l u e s f o r s e p a r a t e c u l t u r e s a g r e e d w e l l , b u t p o o r e r a g r e e m e n t was f o u n d b e t w e e n e s t i m a t e s o f u r e a - V m a x a n d NH^-Kg v a l u e s . The d i s c r e p a n c y b e t w e e n t h e l a t t e r e s t i m a t e c a n be a t t r i b u t e d p a r t l y t o t h e p a u c i t y o f u p t a k e v a l u e s f r o m l o w s u b s t r a t e e n r i c h m e n t s i n t h e s e c o n d c u l t u r e ( s u b s t r a t e e x h a u s t i o n o c c u r r e d d u r i n g t h e i n c u b a t i o n f o r 0.1, 0.2, a n d 0.4 uq-at N - L - 1 e n r i c h m e n t ) . The v a l u e s o f V m a x o f N O 3 - a n d u r e a a g r e e d w e l l a n d a r e a b o u t h a l f t h e V m a x f o r N H 4 + . Micromonas pusilla d e m o n s t r a t e d t h e same a f f i n i t y f o r e a c h N s u b s t r a t e a s t h e K g v a l u e s f o r N O 3 - , N H 4 + , and u r e a w e r e w i t h i n ± 0.1 uq-at N - L - 1 o f e a c h o t h e r . An u n d e r l y i n g a s s u m p t i o n i n t h e u s e o f t h e M i c h a e l i s -M e n t e n e q u a t i o n f o r t h e e s t i m a t i o n o f k i n e t i c u p t a k e p a r a m e t e r s i s t h a t u p t a k e r e m a i n s c o n s t a n t o v e r t h e d u r a t i o n o f t h e e x p e r i m e n t a l i n c u b a t i o n . I n t h e p r e s e n t k i n e t i c e x p e r i m e n t s , i t i s u n l i k e l y t h a t t h e n o n - l i n e a r i t y t h a t h a s b e e n r e p o r t e d f o r n i t r o g e n u p t a k e b y N - d e f i c i e n t c e l l s w o u l d o c c u r b e c a u s e i n t h i s s t u d y N - r e p l e t e c e l l s w e r e u t i l i z e d . N i t r o g e n - r e p l e t e c u l t u r e s (4.2 a n d 9.9 uq-at N - L - 1 ) w e r e i n c u b a t e d f o r b o t h 10 and 60 m i n t o d e t e r m i n e i f V m = v F i g u r e 3.1. N i t r o g e n s p e c i f i c uptake r a t e s (V) determined over 10 min a f t e r the a d d i t i o n of 0.2, 0.4, 0.8, 1.6, 2.4, 4 and 10 /jg-at N-L*1 of N03" (A), urea (B) or NH 4 + (C) t o d u p l i c a t e n i t r a t e - r e p l e t e c u l t u r e s ( O , • ) of Micromonas pusilla. Rates (h - 1) are p l o t t e d v e r s u s the average s u b s t r a t c o n c e n t r a t i o n d u r i n g the 10 min i n t e r v a l . Curve c a l c u l a t e d computer programme (see t e x t f o r d e t a i l s ) . O Z > X z > 0.100 0.080 0.080 -0.040 0.000 0.0 2.0 4.0 6.0 8.0 10.0 S U B S T R A T E ( M g-at N-L" 1) T a b l e 3.1 K i n e t i c parameters f o r n i t r a t e , u r e a and ammonium uptake o f N - r e p l e t e Micromonas pusilla. M i c h a e l i s -Menten p a r a m e t e r s , K ( h a l f - s a t u r a t i o n c o n s t a n t ) and V (maximum uptake v e l o c i t y ) were e s t i m a t e d from a d i r e c t n o n - l i n e a r c u r v e f i t t i n g model and Hanes-Woolf l i n e a r t r a n s f o r m a t i o n o f the dat a o b t a i n e d from r e p l i c a t e c u l t u r e s (1 o r 2) and t h e c u l t u r e s t r e a t e d t o g e t h e r (1 + 2). S u b s t r a t e C u l t u r e ^ a x * f * 1 0 " 2 * 1 " 1 > K s 1 ( f J 9 - a t N - L - 1 ) Vmax 2 ( x l 0 _ 2 h ~ 1 > K s 2 < ^ 9 - a t N * L 1 ) N i t r a t e 1 4. 64 (0 118) 0 .44 (0 044) 4. 70 (0 057 ) 0 49 (0 050) 2 5. 07 (0 361) 0 .50 (0 014) 5. 32 (0 176) 0 60 (0 022) li 1 + 2 4. 86 (0 183) 0 .47 (0 069) 4. 99 (0 125) 0 54 (0 108) Urea 1 4. 70 (0 176) 0 35 (0 051) 4. 42 (0 098) 0 27 (0 093) " 2 5. 93 (0 211) 0 40 (0 059 ) 5. 64 (0 082 ) 0 30 (0 059 ) " 1 + 2 5. 38 (0 257) 0 38 (0 073) 4. 95 (0 219) 0 26 (0 181) Ammonium 1 13 .6 (0 99) 0 .49 (0 142) 14 .8 (0 56) 0 .76 (0 165) 2 12 . 1 (0 74) 0 28 (0 104) 12 .8 (0 25) 0 43 (0 098) 1 + 2 12 .9 (0 61) 0 40 (0 087) 13 .8 (0 44) 0 62 (0 145) 118 d e c r e a s e d w i t h i n c r e a s e d i n c u b a t i o n t i m e . The V m a x v a l u e s f o r v 0 - 1 0 m i n a n d v 0 - 6 0 m i n a r e p r e S e n t e d i n F i g u r e 3.2. a n d t h e r e a p p e a r s t o be no s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e t w o r a t e s , a l t h o u g h d u p l i c a t e m e a s u r e m e n t s w e r e n o t a l w a y s p o s s i b l e . An a v e r a g e o f 30, 40 a n d 60% o f t h e a v a i l a b l e i s o t o p e was u t i l i z e d d u r i n g t h e 60 m i n i n c u b a t i o n s o f t h e u r e a , N C ^ - a n d N H 4 + e n r i c h e d c u l t u r e s , r e s p e c t i v e l y . Substrate interaction The a d d i t i o n o f 10 uq-at N * L - 1 o f n i t r a t e t o a NC>3~-r e p l e t e c u l t u r e o f M. pusilla d i d n o t a l t e r t h e d i s a p p e a r a n c e r a t e o f N 0 3 ~ + N 0 2 ~ (0. 0 4 4 1 h - 1 ) f r o m t h a t o b s e r v e d f o r 3 h p r i o r t o N e n r i c h m e n t (0.0446 h - 1 , 3 d m o n i t o r i n g ) . T h e r e was, h o w e v e r a 10% d i f f e r e n c e b e t w e e n t h e u n d i s t u r b e d c o n t r o l a n d t h e N 0 3 ~ ~ - e n r i c h e d c u l t u r e ( e n r i c h e d c o n t r o l = 0.0492 h - 1 ) d u r i n g t h e 3 h e x p e r i m e n t ( F i g . 3 . 3 ) . The a d d i t i o n o f 10 l i e uq-at N-L o f [ N] u r e a r e s u l t e d i n a 2 8 % d e c r e a s e i n t h e d i s a p p e a r a n c e r a t e o f N O 3 - + NC^ - a n d a s p e c i f i c u r e a u p t a k e r a t e o f 0.0260 h - 1 . The t o t a l n i t r o g e n u p t a k e ( 0 . 0 5 7 9 h - 1 ) i n c r e a s e d by c a . 30% o v e r t h e n i t r a t e e n r i c h m e n t a l o n e . Ammonium a d d i t i o n (10 ^ j g - a t N * L - 1 ) r e s u l t e d i n t h e c o m p l e t e c e s s a t i o n o f N O 3 - + NC^ - d i s a p p e a r a n c e f r o m t h e medium a n d a N H 4 + s p e c i f i c u p t a k e r a t e o f 0.0732 h - 1 . The s e c o n d s e r i e s o f s u b s t r a t e i n t e r a c t i o n e x p e r i m e n t s , d e s i g n e d t o d e t e r m i n e t h e e f f e c t ( s ) o f N H 4 + c o n c e n t r a t i o n on N O 3 - u p t a k e , w e r e c o n d u c t e d on a N C ^ - r e p l e t e c u l t u r e ( p r e c o n d i t i o n e d g r o w t h r a t e = 0.0484 h - 1 , 4 d m o n i t o r i n g ) . W i t h no e n r i c h m e n t ( c o n t r o l ) , t h e N O 3 - + N O 2 - d e p l e t i o n r a t e 119 Figure 3.2. Comparison of nitrogen s p e c i f i c uptake rates for n i t r a t e - r e p l e t e c u l t u r e s of Micromonas pusilla determined over 10 and 60 min incubation periods. Cultures are numbered and values are the mean (n = 2) of duplicate incubations, * designates no r e p l i c a t e . Bar represents ± 1 S.D. > < cn L J < Q. ZD O o L±J CL CO 0.1 4 0 0.1 2 0 0 . 1 0 0 0 , 0 8 0 0 . 0 6 0 0 . 0 4 0 0 . 0 2 0 0 . 0 0 0 V 0 - 1 O m i n V 0 - 6 0 m i n 1 2 N I T R A T E 3 4 U R E A 5 6. A M M O N I U M 120 Figure 3.3. Nitrogen uptake by r e p l i c a t e c u l t u r e s of n i t r a t e -r e p l e t e Micromonas pusilla over a 4 h incubation period. A. Dissolved NO ~ + N02" ( • ) concentration and 1 5N-atom % excess ( O ) a f t e r 10 ^/g-at N-urea-L"1 addition. B. Dissolved NO " + NO " concentration ( O , A ) a f t e r no and 10 /jg-at N- N03~*L ad d i t i o n , r e s p e c t i v e l y . Dissolved NO," + NO~ concentration ( • , •) a f t e r 10 pg-at N-L"1 addition of NH4^ and urea, r e s p e c t i v e l y . Dissolved NH4+ concentration ( • ) a f t e r addition of 10 ug-at N-NH/'L"1. cn a. I CN O O 3 . I CN o I CO o tn V) o x E o < UJ cr cn 3 . I 120 180 TIME (min) 240 121 a v e r a g e d 0.0500 h - 1 o v e r t h e 2 h d u r a t i o n o f t h e e x p e r i m e n t . A l l t h e 1 5 N H 4 + e n r i c h m e n t s ( 5 , 2, a n d 1 uq-at N - L - 1 ) c a u s e d c e s s a t i o n o f N O 3 - + NO2"" d i s a p p e a r a n c e (< 0.1 uq-at N * L - 1 • h - 1 ) when t h e 1 ^ N H 4 + i s o t o p e was s t i l l a v a i l a b l e i n t h e medium; t h e c o n c e n t r a t i o n o f e x t e r n a l N O 3 - + NG^ - d i d n o t m e a s u r a b l y d e c r e a s e u n t i l t h e 1 5 N H 4 + was e x h a u s t e d ( F i g . 3 . 4 ) . I t s h o u l d be m e n t i o n e d t h a t t h e u p t a k e o f N O 3 - was e s t i m a t e d f r o m t h e d i s a p p e a r a n c e o f NO^ - + NO2"" i n t h e medium. A l t h o u g h o t h e r i n v e s t i g a t o r s ( e . g . , S e r r a e t a l . , 1 9 7 8 a ; O l s o n e t a l . , 1980; P a r s l o w e t a l . , 1984b) h a v e r e c o r d e d s u b s t a n t i a l e x c r e t i o n o f N O 2 - by m a r i n e d i a t o m s a n d n a t u r a l a s s e m b l a g e s ( H a r r i s o n a n d D a v i s , 1 9 7 7 ) , M. pusilla d e m o n s t r a t e d no e x c r e t i o n o f N O 2 - a n d o n l y t r a c e l e v e l s o f N O 2 - w e r e f o u n d i n t h e medium d u r i n g e x p o n e n t i a l g r o w t h on N O 3 - ( A p p e n d i x 5 ) . E x c r e t i o n o f N O 2 - by M. pusilla w o u l d c o n t r i b u t e t o a r e d u c t i o n i n a n y m e a s u r a b l e d e c l i n e o f N O 3 - + N O 2 - i n t h e medium a n d t h u s e n h a n c e t h e a p p a r e n t i n h i b i t o r y e f f e c t o f N H 4 + o r u r e a on N O 3 - u p t a k e . Nitrogen-starved cells The e x p o n e n t i a l g r o w t h r a t e o f M. pusilla was 1.11 d - 1 ( 0 . 0 4 6 3 h - 1 ) p r i o r t o t h e d e p l e t i o n o f n i t r a t e i n t h e medium, a v a l u e w h i c h a g r e e s w e l l w i t h t h e V m a x f o r NO3 c a l c u l a t e d p r e v i o u s l y ( 0 .0486 ± 0.0018 h _ 1 ) . The s p e c i f i c u p t a k e o f N 0 3 ~ by N 0 3 ~ - s t a r v e d c u l t u r e s , e s t i m a t e d f r o m b o t h t h e a c c u m u l a t i o n 1 5 - • • o f NO3 x n t o t h e p a r t i c u l a t e m a t e r i a l a n d t h e d i s a p p e a r a n c e o f N O 3 - + N O 2 - f r o m t h e medium, a v e r a g e d 0.0238 h - 1 o v e r t h e 5-6 h i n c u b a t i o n p e r i o d ( T a b l e 3 . 2 ) . T h i s r a t e was r o u g h l y c o n s t a n t o v e r t h e i n c u b a t i o n ( F i g . 3.5) a l t h o u g h i t a p p e a r s F i g u r e 3.4. D i s s o l v e d NO " + NO " c o n c e n t r a t i o n w i t h o u t ( O ) a n d w i t h ( • ) , 5 ( A ) , 2 ( B ) , a n d 1 ( C ) uq-at N-L" 1 [ 1 5 N ] - N H 4 + e n r i c h m e n t ; NH 4 + a t o m % e x c e s s i n p a r t i c u l a t e s (•) p l o t t e d v e r s u s t i m e ( m i n ) . A r r o w s d e s i g n a t e t i m e o f NH 4 + a d d i t i o n . 10 8 6 4 2 0 10 o 1 8 cn 6 1 CN 4 O z : 2 + 1 CO o 0 z : 10 8 6 4 2 -0 to CD o X CD E o X I 0 20 40 60 80 100 120 140 TIME (min) 123 T a b l e 3.2 A v e r a g e n i t r a t e u p t a k e r a t e s ( h ) f o r N C > 3 - - s t a r v e d Micromonas pusilla. R a t e s d e t e r m i n e d f r o m l e a s t - s q u a r e s l i n e a r r e g r e s s i o n o f p a r t i c u l a t e 1 5N e n r i c h m e n t o r t h e d e c r e a s e i n t h e e x t e r n a l c o n c e n t r a t i o n o f NO^" + NG^ - v e r s u s t i m e a n d r e p o r t e d a s ± 1 s t a n d a r d d e v i a t i o n ( i n p a r e n t h e s e s ) o f t h e mean o f d u p l i c a t e c u l t u r e s . T i me I n t e r v a l N i t r a t e U p t a k e (•10~ 2h 1 ) 1 R (h) N 0 3 N 0 3 d i s a p p e a r a n c e 0 1 2 3 4 5 1 2 3 4 5 6* 2 2 2 2 2 2 22 10 34 56 44 12 ( 0 . 0 0 7 1 ) ( 0 . 1 8 4 ) ( 0 . 0 1 4 ) ( 0 . 4 1 7 ) ( 0 . 0 9 9 ) 1. 1. 2 2 2 2 77 86 94 80 66 73 ( 0 . 5 3 7 ) ( 0 . 2 3 3 ) ( 0 . 1 7 0 ) ( 0 . 6 2 9 ) ( 0 . 4 3 8 ) (* = u p t a k e r a t e s c a l c u l a t e d f r o m one c u l t u r e ) T a b l e 3.3 A v e r a g e N u p t a k e r a t e s V ( h ) f o r N C ^ ' - s t a r v e d Micromonas pusilla. R a t e s d e t e r m i n e d f r o m l e a s t - s q u a r e s l i n e a r r e g r e s s i o n o f p a r t i c u l a t e 1 5N e n r i c h m e n t o r t h e d e c r e a s e i n t h e e x t e r n a l c o n c e n t r a t i o n o f d i s s o l v e d n i t r o g e n v e r s u s t i m e a n d r e p o r t e d a s ± 1 s t a n d a r d d e v i a t i o n ( i n p a r e n t h e s e s ) o f t h e mean o f d u p l i c a t e c u l t u r e s . N S u b s t r a t e N U p t a k e ( • 1 0 ~ 2 h " 1 ) v 0 - 6 0 m i n v 6 0 - 1 2 0 m i n v 1 2 0 - 1 8 0 m i n U r e a 4.80 (0. 1 2 9 ) 3.72 ( 0 . 1 4 9 ) 3.32 ( 0 . 1 5 8 ) 1 5 N H 4 + 7.05 (0 . 0 1 2 0 ) 5.59 ( 0 . 0 5 5 9 ) 4.23 ( 0 . 0 2 8 8 ) N H 4 + 6 . 7 5 * ( 0 . 7 3 9 ) 6.76 ( 0 . 4 4 9 ) 4.85 ( 0 . 6 0 0 ) (* = u p t a k e r a t e c a l c u l a t e d f r o m 2.5 - 60 m i n ) 124 t h a t t h e r e was e l e v a t e d u p t a k e (0.0386 ± 0.0012 h - 1 ) d u r i n g 1 c t h e f i r s t 5 m i n a f t e r N e n r i c h m e n t . E l e v a t e d u p t a k e r a t e s w e r e n o t o b s e r v e d i n t h e N O 3 - + NO2"" d i s a p p e a r a n c e m e a s u r e m e n t s , p o s s i b l y due t o t h e r e d u c e d s e n s i t i v i t y o f c o l o u r i m e t r i c a n a l y s i s a t e l e v a t e d (> 15 uq-at N - L - 1 ) n i t r a t e c o n c e n t r a t i o n s . The a v e r a g e r a t e o f 0.0238 h - 1 i s c a . 25% l e s s t h a n t h e n i t r o g e n s p e c i f i c r a t e (0.0308 h - 1 ) c a l c u l a t e d f r o m N O 3 - + NO2"" d i s a p p e a r a n c e (1.54 uq-at N• L ~ 1 • h ~ 1 ) a n d t h e a v e r a g e c o n c e n t r a t i o n o f PON (50.0 uq-at N ' L - 1 ) d u r i n g t h e 4 h m o n i t o r i n g p e r i o d p r i o r t o N d e p l e t i o n . T h i s r e d u c t i o n i s p r o b a b l y o n l y a minimum e s t i m a t e a s u p t a k e r a t e b e f o r e d e p l e t i o n may h a v e a l r e a d y b e g u n t o d e c r e a s e a s a c o n s e q u e n c e o f l o w N O 3 - c o n c e n t r a t i o n s i n t h e medium. The a v e r a g e u p t a k e r a t e a f t e r s t a r v a t i o n i s o n l y h a l f t h e N demand c a l c u l a t e d f r o m e i t h e r t h e p r e - c o n d i t i o n e d g r o w t h r a t e o r V m a x . However t h e r e was, no l a g p e r i o d i n N O 3 - u p t a k e b y t h e p r e v i o u s l y s t a r v e d c e l l s ( F i g . 3.5). The e x p o n e n t i a l g r o w t h r a t e o f d u p l i c a t e c u l t u r e s u s e d i n t h e s e c o n d s e r i e s o f N 0 3 ~ - s t a r v e d u p t a k e e x p e r i m e n t s ( N H ^ + and u r e a ) a v e r a g e d 1.11 d - 1 (0.0463 h - 1 ) p r i o r t o d e p l e t i o n o f n i t r a t e i n t h e medium. Maximum [ ^ N ] u r e a u p t a k e r a t e (0.105 ± 0.016 h - 1 ) o c c u r r e d d u r i n g t h e 0-5 m i n i n t e r v a l a n d s u b s e q u e n t l y d e c r e a s e d r a p i d l y i n t h e n e x t 10-20 m i n t o a r o u g h l y c o n s t a n t r a t e o f 0.0349 ± 0.0016 h - 1 ( F i g . 3.6) The a v e r a g e h o u r l y u r e a u p t a k e r a t e s , c a l c u l a t e d f r o m t h e s l o p e o f p a r t i c u l a t e APE v e r s u s t i m e , a r e p r e s e n t e d i n T a b l e 3.3. A v e r a g e u p t a k e r a t e s d u r i n g t h e s e c o n d h o u r o f i n c u b a t i o n w e r e 125 F i g u r e 3.5. N i t r a t e u p t a k e by n i t r a t e - s t a r v e d Micromonas pusilla a f t e r t h e a d d i t i o n o f 15 uq-at N-N0 3"*L _ 1 t o d u p l i c a t e c u l t u r e s . A. D i s s o l v e d N0 3" + N0 2" (•,•) i n t h e c u l t u r e medium; 1 5N0 3~ a t o m % e x c e s s i n p a r t i c u l a t e m a t t e r ( O , * ) . B. N i t r a t e u p t a k e r a t e d e t e r m i n e d f r o m N0 3" + N0 2" d i s a p p e a r a n c e t e c h n i q u e . C. [ 1 5N] n i t r a t e u p t a k e r a t e . V a l u e s i n A a r e p l o t t e d a g a i n s t e l a p s e d t i m e m e a s u r e d a f t e r e n r i c h m e n t a n d u p t a k e r a t e s (B,C) a r e p l o t t e d a g a i n s t a v e r a g e i n c u b a t i o n t i m e . ~ 0.040 -0.000 1 • 1 • 1 • 1 • 1 • ' • 1 0 60 120 180 240 300 360 TIME (min) 1 2 6 F i g u r e 3.6. U r e a u p t a k e by n i t r a t e - s t a r v e d Micromonas pusilla a f t e r t h e a d d i t i o n o f 10 uq-at N - u r e a - L " 1 t o d u p l i c a t e c u l t u r e s ( 0 , » ) . A. 1 5 N - u r e a atom % e x c e s s i n p a r t i c u l a t e m a t t e r i s p l o t t e d a g a i n s t e l a p s e d t i m e m e a s u r e d a f t e r a d d i t i o n o f u r e a . B. [ 1 5N] u r e a u p t a k e r a t e p l o t t e d a g a i n s t a v e r a g e i n c u b a t i o n t i m e . 1 4 . 0 to CD OX 1 2 . 0 cu 1 0 . 0 E o 8 . 0 o — 6 . 0 < LU 4 . 0 fr Z> i 2 . 0 ~Z. in i— 0 . 0 0 . 1 2 0 T" rz 0 . 1 0 0 l±J 0 . 0 8 0 < 1— Q_ 0 . 0 6 0 < UJ cr A •>* * i i , . i 0 . 0 4 0 0 . 0 2 0 0 . 0 0 0 0 3 0 6 0 9 0 1 2 0 1 5 0 1 8 0 TIME (min) 127 7 8% o f t h e i n i t i a l h o u r l y r a t e s a n d d u r i n g t h e t h i r d h o u r , u p t a k e m e r e l y d e c r e a s e d an a d d i t i o n a l 1 1 % . The u p t a k e r a t e s d u r i n g 3 0 - 6 0 , 60-120 a n d 120-180 m i n i n t e r v a l s w e r e < 4 0 % o f t h e m a x i m a l r a t e a n d 7 0 - 8 0 % o f t h e n i t r o g e n u p t a k e r a t e n e e d e d t o s u p p o r t e x p o n e n t i a l g r o w t h o b s e r v e d p r i o r t o N - d e p l e t i o n . Maximum 1 5 N H 4 u p t a k e r a t e ( 0.175 ± 0.0024 h - 1 ) o c c u r r e d d u r i n g t h e i n i t i a l 0-5 m i n i n t e r v a l , f o l l o w e d b y a r a p i d , b u t s h o r t - t e r m d e c r e a s e ( c a . 80%) i n u p t a k e b e f o r e i t r e a c h e d a r o u g h l y c o n s t a n t r a t e o f 0.0668 ± 0.0073 h ~ ^ f o r t h e r e m a i n d e r o f t h e f i r s t h o u r o f i n c u b a t i o n ( F i g . 3.7 C ) . The a v e r a g e h o u r l y r a t e d e c l i n e d by 2 0 - 2 5 % p e r h o u r o v e r t h e 3 h o f m o n i t o r i n g a n d a v e r a g e d 150, 120 a n d 90% o f t h e n i t r o g e n u p t a k e , n e e d e d t o s u p p o r t e x p o n e n t i a l g r o w t h , d u r i n g t h e 0-60, 6 0 - 1 2 0 , a n d 120-180 m i n i n c u b a t i o n p e r i o d s r e s p e c t i v e l y . The r a t e o f NH 4 + d i s a p p e a r a n c e i n t h e medium (0.0612 ± 0.0060 h~^) ( e x c l u d i n g t h e 0-2.5 m i n i n t e r v a l ) , i s i n g o o d a g r e e m e n t w i t h t h e 1 5 N H 4 u p t a k e r a t e ( 0 . 0 5 6 1 ± 0.0010 h - 1 ) , a n d a l s o d e c l i n e d o v e r t h e 3 h i n c u b a t i o n p e r i o d ( T a b l e 3.3 ). M a x i m a l N H 4 + d i s a p p e a r a n c e r a t e s ( 0 .503 ± 0.133 h--^) d u r i n g t h e 0-2.5 m i n i n t e r v a l w e r e 7-9 t i m e s g r e a t e r t h a n t h e a v e r a g e r a t e a n d n e a r l y 3 t i m e s g r e a t e r t h a n t h e e l e v a t e d NH 4 u p t a k e r a t e s r e p o r t e d f o r t h e 0-5 m i n i n t e r v a l ( F i g . 3.7 B ) . 1 c However, u n l i k e t h e N - t r a c e r t e c h n i q u e , w h e r e an a c c u r a t e m e a s u r e m e n t o f ^ N e x i n t h e u n e n r i c h e d p a r t i c u l a t e s c a n be o b t a i n e d , t h e c o n c e n t r a t i o n o f N H 4 + a t t i m e - z e r o f o r t h e N H 4 + d i s a p p e a r a n c e t e c h n i q u e c a n o n l y be e s t i m a t e d f r o m a c e l l - f r e e s a m p l e , a n d may be s u b j e c t t o meas u r e m e n t e r r o r . 128 F i g u r e 3.7. Ammonium u p t a k e by n i t r a t e - s t a r v e d Micromonas pusilla a f t e r t h e a d d i t i o n o f 15 uq-at N- NH. L t o d u p l i c a t e c u l t u r e s . A. D i s s o l v e d NH 4 + c o n c e n t r a t i o n i n t h e c u l t u r e medium (•,<">) ; 1 5N-NH 4 a t o m % e x c e s s i n p a r t i c u l a t e m a t t e r (•,•; p l o t t e d a g a i n s t e l a p s e d t i m e a f t e r e n r i c h m e n t . B. Ammonium u p t a k e r a t e , d e t e r m i n e d b y NH 4 + d i s s a p p e a r a n c e t e c h n i q u e . C. [*5N] NH 4 u p t a k e r a t e . V a l u e s i n B a n d C p l o t t e d a g a i n s t a v e r a g e i n c u b a t i o n t i m e . co co a> o x 3. X E o X LU OL-ID + * X 0.200 0.1 60 0.120 0.080 0.040 0.000 0.597 0.409 B 0.200 -W 0.160 < £ 0.120 -ZD <* 0.080 0.040 -0.000 n 0 30 60 90 120 150 180 TIME (min) 129 A summary of the culture conditions at the beginning of each series of experiments i s presented in Table 3 .4. T a b l e 3.4 Summary o f c u l t u r e c o n d i t i o n s a t t h e b e g i n n i n g of each experiment. C u l t u r e Experiment N0 3 ~ + N0 2 PON POC C e l l d e n s i t y T o t a l c e l l C e l l Quota d e s c r i p t i o n and c u l t u r e Volume* number (p g - a t N* L " 1 ) (uq-at C-L~ 1 9 - 1 1 ) (10 -L L) ( p L - L - 1 ) ( f g - a t N ' c e l l - 1 ) NO3~ s u f f i c i e n t A - l <0.05 49.2 382.1 5.71 13.2 8.6 II A-2 <0.05 51.9 382.7 6.08 13.3 8.5 N0 3 ~ s u f f i c i e n t B-1 13.6 35.2 302.3 _ _ — II C - l 8.3 32.5 295.3 4.72 9.64 6.9 NO3~ s t a r v e d D - l <0.05 50.8 546 .7 10.38 14.1 4.9 it D-2 <0.05 54.8 549 .6 - - -NO-j~ s t a r v e d E - l <0.05 59 .3 504.8 9.28 12.3 6.4 II E-2 <0.05 47.1 526 .7 10.00 12.8 4.7 A: N 0 3 ~ » N H 4 + a n d u r e a uptake k i n e t i c exp. B: NH 4 + and u r e a i n h i b i t i o n exp. ( S e r i e s 1) . C: NH 4 + i n h i b i t i o n exp. ( S e r i e s 2 ) . D: NH 4 + and u r e a uptake exp. E: NO3~ uptake exp. *: L c e l l volume p e r l i t e r o f c u l t u r e . 131 DISCUSSION Uptake kinetics O v e r t h e l a s t t w o d e c a d e s numerous i n v e s t i g a t o r s h a v e d e t e r m i n e d t h e k i n e t i c s o f n i t r o g e n u p t a k e i n b o t h c u l t u r e d a n d n a t u r a l a s s e m b l a g e s o f p h y t o p l a n k t o n ( s e e r e v i e w s by M c C a r t h y , 1 9 8 1; Goldman a n d G l i b e r t , 1983; D o r t c h , i n p r e s s ) . I t i s o f t e n d i f f i c u l t t o co m p a r e a n d i n t e r p r e t t h e r e s u l t s o f t h e v a r i o u s s t u d i e s due t o t h e v a r i e t y o f t e c h n i q u e s , e x p e r i m e n t a l i n c u b a t i o n p e r i o d s a n d t h e p h y s i o l o g i c a l c o n d i t i o n o f t h e p h y t o p l a n k t o n ( e . g . , H a r r i s o n e t a l . , 1 9 8 9 ) . G e n e r a l l y t h e v a l u e s o f t h e h a l f - s a t u r a t i o n c o n s t a n t ( K s ) f o r N - u p t a k e a r e l o w e r f o r o l i g o t r o p h i c o c e a n i c n a t u r a l a s s e m b l a g e s ( e . g . , M a c l s a a c a n d D u g d a l e , 1969; K a n d a e t a l . , 1985) a n d i s o l a t e d o l i g o t r o p h i c c l o n e s ( e . g . , E p p l e y e t a l . , 1969; C a r p e n t e r a n d G u i l l a r d , 1971) t h a n h a l f - s a t u r a t i o n v a l u e s f r o m e u t r o p h i c , a n d n e r i t i c a r e a s o f t h e o c e a n . E p p l e y e t a l . ( 1 9 6 9) d e m o n s t r a t e d a d i r e c t c o r r e l a t i o n b e t w e e n c e l l s i z e a n d t h e K g v a l u e , a l t h o u g h no s u c h c o r r e l a t i o n h a s b e e n o b s e r v e d f o r f r e s h w a t e r p h y t o p l a n k t e r s ( H a l t e r m a n a n d T o e t z , 1 9 8 4 ) . I t a l s o a p p e a r s t h a t V m a x f o r n i t r a t e i s l o w e r t h a n t h e V m a x f o r r e d u c e d N f o r m s s u c h a s N H 4 + , e x c e p t d u r i n g s p r i n g b l o o m s o r i n u p w e l l i n g a r e a s w h e r e V m a x - N 0 3 ~ e q u a l s o r e x c e e d s t h a t o f ammonium ( e . g , D u g d a l e , 1976; D o r t c h , i n p r e s s ) . T h i s g e n e r a l o b s e r v a t i o n i s c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t t h e l a r g e p l a n k t o n i c f o r m s t h a t b l o o m d u r i n g s u c h c o n d i t i o n s ( i . e . , N O 3 - i s a b u n d a n t i n t h e e u p h o t i c z o n e ) d e p e n d p r i m a r i l y on n i t r a t e ( M a l o n e , 1 9 8 0 ) . However, s i n c e 132 n u t r i e n t u p t a k e a n d g r o w t h p r o c e s s e s a r e n o t n e c e s s a r i l y c o u p l e d ( i . e . , b a l a n c e d a n d e q u a l ) a n d a l g a l p h y s i o l o g y a n d c h e m i c a l c o m p o s i t i o n a r e a d a p t a b l e ( s e e r e v i e w s b y D u g d a l e , 1977; M c C a r t h y , 1981) p h y t o p l a n k t o n g e n e r a l l y h a v e t h e a b i l i t y t o g row e q u a l l y w e l l on NO^ -, NH 4 + a n d u r e a ( e g . , S y r e t t , 1981). To d a t e , o n l y a f e w s t u d i e s ( P a a s c h e , 1971; Ward a n d W e t z e l 1980; Rhee a n d L e d e r m a n , 1983; Thompson e t a l . , 1989) h a v e p r o v i d e d g o o d e v i d e n c e f o r a n i n c r e a s e i n g r o w t h r a t e o f c e l l s g r o w i n g on N H 4 + v e r s u s N O 3 - u n d e r s a t u r a t i n g g r o w t h PPFD. I n t h e p r e s e n t w o r k , t h e V m a x - N H 4 + v a l u e s a r e t w i c e t h o s e o f N O 3 - a n d u r e a , a l t h o u g h t h e i r K s v a l u e s a r e a l l s i m i l a r (0.28 - 0.50 uq-at N'L--'") a n d f a l l w i t h i n t h e r a n g e r e p o r t e d by E p p l e y e t a l . (1969) f o r s m a l l , o c e a n i c d i a t o m s (0.1-0.7 uq-at N-L -"*"). The l o w e s t K s v a l u e s t o d a t e h a v e b e e n r e p o r t e d by K o i k e e t a l . (1983) f o r two m i c r o f l a g e l l a t e s i s o l a t e d f r o m t h e o l i g o t r o p h i c N o r t h P a c i f i c O c e a n ; Platymonas a n d Mantoniella s p . w i t h K g - N H 4 + v a l u e s o f 50 a n d 2.9 n g - a t N * L - ^ , r e s p e c t i v e l y , s u g g e s t i n g t h a t t h e s e two p h y t o f l a g e l l a t e s a r e w e l l a d a p t e d t o t h e i r e x t r e m e l y l o w ammonium e n v i r o n m e n t . The a f f i n i t y f o r a g i v e n n u t r i e n t a t l o w c o n c e n t r a t i o n s c a n be b e s t e s t i m a t e d f r o m t h e i n i t i a l s l o p e (a) o f t h e M i c h a e l i s -M e n t e n p l o t ( i . e . a = V m / K s , H e a l e y , 1980; P a r s l o w e t a l . , 1985). I n t h e p r e s e n t w o r k , t h e v a l u e s o f a a r e 10.3 ± 1.9, 14.2 ± 3.4 a n d 32.3 ± 8.5 f o r N 0 3 ~ , u r e a a n d N H 4 + , r e s p e c t i v e l y . T h e s e r e s u l t s s u g g e s t t h a t M. p u s i l l a c a n u t i l i z e l o w c o n c e n t r a t i o n s o f N H 4 + more e f f e c t i v e l y t h a n 133 e q u i v a l e n t c o n c e n t r a t i o n s o f u r e a a n d NO3"". W h i l e a c t u a l k i n e t i c s t u d i e s o f o t h e r p i c o p l a n k t e r s h a v e n o t b e e n c o n d u c t e d , r e l a t e d s t u d i e s on n a t u r a l a s s e m b l a g e s u t i l i z i n g s i z e - f r a c t i o n a t i o n t e c h n i q u e s a n d n i t r o g e n t r a c e r s o r a n a l o g u e s h a v e d e m o n s t r a t e d t h a t p i c o p l a n k t o n , i n c l u d i n g m i c r o h e t e r o t r o p h s ( W h e e l e r a n d K i r c h m a n ( 1 9 8 6 ) , g e n e r a l l y p r e f e r t h e r e d u c e d f o r m s o f n i t r o g e n a n d u s e r e l a t i v e l y h i g h e r p r o p o r t i o n s o f t h e s e r e d u c e d f o r m s f o r g r o w t h t h a n t h e l a r g e r p h y t o p l a n k t o n ( N a l e w a j k o a n d G a r s i d e , 1983; P r o b y n , 1985; P r o b y n a n d P a i n t i n g , 1985; S a h l s t e n , 1987; H a r r i s o n a n d Wood, 1 9 8 8 ) . S i m i l a r f r a c t i o n a t i o n s t u d i e s on n e t (> 20 um) a n d n a n o p l a n k t o n (< 20 um) h a v e o f t e n f o u n d t h i s d i s t i n c t i o n n o t t o be a s c l e a r c u t : t h e p a r t i t i o n i n g b e t w e e n "new" a n d " r e g e n e r a t e d " n i t r o g e n u p t a k e i s n e a r l y e q u a l l y d i s t r i b u t e d b e t w e e n t h e t w o s i z e - f r a c t i o n s i n v a r i o u s m a r i n e h a b i t a t s ( S h e r r e t a l . , 1982; F u r n a s , 1983; R o n n e r e t a l . , 1983) w h i l e t h e r e s u l t s o f K o i k e e t a l . , ( 1 9 8 6) f o r A n t a r c t i c a p h y t o p l a n k t o n c o n f i r m e d t h e f o r m e r p a t t e r n . Cellular physiological state The e f f e c t s o f c e l l u l a r p h y s i o l o g i c a l s t a t e on n i t r o g e n u p t a k e r a t e s b y p h y t o p l a n k t o n w e r e f i r s t d e m o n s t r a t e d b y S y r e t t ( 1 9 5 3) a n d H a r v e y ( 1 9 5 3 ) . T hey showed t h a t N H 4 + a n d N O 3 - u p t a k e by b a t c h c u l t u r e s o f " n i t r o g e n - s t a r v e d " c e l l s was much more r a p i d t h a n by " n o r m a l " c e l l s t h a t w e r e n i t r o g e n -r e p l e t e . F i t z g e r a l d (1968) n o t e d t h a t t h e s e r a t e s w e r e n o t s u s t a i n a b l e a n d d e c r e a s e d q u i t e r a p i d l y o n c e t h e n i t r o g e n d e f i c i t was o v e r c o m e . Conway e t a l . ( 1 9 7 6) d e s c r i b e d t h e 134 r e s p o n s e i n c o n s i d e r a b l y more d e t a i l i n m a r i n e d i a t o m s a n d d i s t i n g u i s h e d t h r e e p h a s e s o f u p t a k e o f t h e l i m i t i n g n u t r i e n t : a s h o r t - l i v e d p e r i o d o f v e r y h i g h u p t a k e , t e r m e d " s u r g e u p t a k e " ( V s ) , a l o n g e r , s u s t a i n a b l e p h a s e c h a r a c t e r i z e d a s " i n t e r n a l l y " ( c e l l u l a r l y ) c o n t r o l l e d u p t a k e ( V ^ ) , a n d " e x t e r n a l l y " ( a m b i e n t l i m i t i n g n u t r i e n t c o n c e n t r a t i o n ) c o n t r o l l e d u p t a k e ( V e ) . More r e c e n t s t u d i e s h a v e a l s o d e m o n s t r a t e d " s u r g e " o r " e n h a n c e d " N H 4 + u p t a k e c a p a b i l i t i e s u n d e r c o n d i t i o n s o f N d e p r i v a t i o n i n numerous c u l t u r e s t u d i e s ( e . g . , M c C a r t h y a n d Goldm a n , 1979; D o r t c h e t a l . , 1982; Goldman e t a l . , 1 9 8 1 ; Goldman a n d G l i b e r t , 1982; P a r s l o w e t a l . , 1 9 8 4 a ,b; S y r e t t a n d P e p l i n s k a , 1988) a n d n a t u r a l c o m m u n i t i e s ( G l i b e r t a n d Goldm a n , 1 9 81; W h e e l e r e t a l . , 1982; H a r r i s o n , 1 9 8 3 a ; P r i s c u a n d P r i s c u , 1 9 8 4 : , P r i s c u , 1987; S u t t l e a n d H a r r i s o n , 1 9 8 8 ) . The r e s u l t s o f t h e p r e s e n t s t u d y w i t h N C > 3 - - s t a r v e d M. pusilla a r e s i m i l a r t o t h o s e d e s c r i b e d a b o v e w i t h N - s t a r v e d c u l t u r e s ; t h e i n i t i a l s u r g e N H 4 + u p t a k e r a t e was s e v e r a l f o l d g r e a t e r ( V g ° - 5 m i n = 2.5-4 t i m e s , v s 0 _ 2 - 5 m i n = 7-9 t i m e s u p t a k e r a t e s o b t a i n e d by a n d n u t r i e n t d i s a p p e a r a n c e , r e s p e c t i v e l y ) t h a n t h e i n t e r n a l l y c o n t r o l l e d u p t a k e r a t e a n d t h e u p t a k e r a t e n e c e s s a r y t o m a i n t a i n t h e p r e c o n d i t i o n e d g r o w t h r a t e o b s e r v e d b e f o r e N d e p l e t i o n . The m a g n i t u d e o f s u r g e u p t a k e r e s p o n s e i s b o t h s p e c i e s s p e c i f i c ( e . g . , Conway a n d H a r r i s o n , 1977) a n d a f u n c t i o n o f t h e d u r a t i o n o f N d e p r i v a t i o n ( e . g . , P a r s l o w e t a l . , 1 9 8 4 a ) . T h e s e e l e v a t e d t r a n s i e n t s h a v e b e e n h y p o t h e s i z e d t o be an e c o l o g i c a l a d a p t a t i o n t h a t a l l o w s p h y t o p l a n k t o n t o r a p i d l y 135 s e q u e s t e r e p h e m e r a l m i c r o p a t c h e s o f N ( M c C a r t h y a n d G o l d m a n , 1979; G l i b e r t a n d G o l d m a n , 1981; Goldman a n d G l i b e r t , 1982) a n d m a i n t a i n h i g h g r o w t h r a t e s i n o l i g o t r o p h i c e n v i r o n m e n t s ( G o l d m an e t a l . , 1979; Goldman a n d G l i b e r t , 1 9 8 2 ) . However t h i s c o n c e p t i s n o t w i t h o u t c o n t r o v e r s y a s b o t h J a c k s o n ( 1 9 80) a n d W i l l i a m s a n d M u i r (1981) c o n t e n d t h a t t h e m o l e c u l a r d i f f u s i o n o f t h o s e p a t c h e s w o u l d be s o r a p i d a s t o p r e v e n t t h e m f r o m e x i s t i n g l o n g e n o u g h t o be e x p l o i t e d by p h y t o p l a n k t o n . Due t o t e c h n i c a l l i m i t a t i o n s , n u t r i e n t d a t a on m i c r o p a t c h e s a r e f e w . C o l l o s (1986) s u g g e s t e d t h a t t h e v a l u e s r e p o r t e d i n m i c r o p a t c h e s by S h a n k s a n d T r e n t ( 1 9 7 9 ) (maximum v a l u e s o f c a . 5 0 0 , 300 a n d 60 uM f o r N H 4 + , N 0 2 ~ a n d P 0 4 3 ~ , r e s p e c t i v e l y ) may h a v e t o be r e v i s e d u p w a r d s a s t h e y w e r e m e a s u r e d on s a m p l e v o l u m e s w h i c h a r e t o o l a r g e t o be c o n s i d e r e d r e l e v a n t t o p h y t o p l a n k t o n s p a t i a l s c a l e s ( A l l e n , 1977; H a r r i s , 1980; M c C a r t h y ; 1 9 8 0 ) . T h i s , t o g e t h e r w i t h e m p i r i c a l d a t a s h o w i n g t h a t p h y t o p l a n k t o n c a n u t i l i z e p h o s p h o r u s p a t c h e s p r o d u c e d by z o o p l a n k t o n (Lehman a n d S c a v i a , 1982a,b) l e n d s c r e d e n c e t o t h e o r i g i n a l s u g g e s t i o n o f Conway an d H a r r i s o n (1977) t h a t e l e v a t e d u p t a k e r a t e s may be i m p o r t a n t i n d i c t a t i n g c o m p e t i t i v e a d v a n t a g e i n o l i g o t r o p h i c a r e a s o f t h e o c e a n ; h o w e v e r , t h i s t o p i c i s s t i l l a s u b j e c t o f c o n t r o v e r s y . A r a r e , b u t s i g n i f i c a n t f i n d i n g i n t h e p r e s e n t w o r k i s 15 t h e a p p e a r a n c e o f a r a p i d , b u t s h o r t - t e r m d e c r e a s e i n NH 4 u p t a k e f o l l o w i n g s u r g e u p t a k e . T h i s r e s p o n s e h a s o n l y b e e n d o c u m e n t e d f o r f r e s h w a t e r p h y t o p l a n k t o n ( S u t t l e a n d H a r r i s o n 136 1988) and has not been r e p o r t e d i n pr e v i o u s time course s t u d i e s of r a p i d N H 4 + uptake i n marine phytoplankton. S u t t l e and H a r r i s o n (1988) suggested t h i s temporary decrease i n uptake may be the r e s u l t of a sh o r t l a g b e f o r e NH 4 + can be processed i n t o amino a c i d s . A l t e r n a t i v e l y , they suggest i t c o u l d be the r e s u l t of a sudden l o s s of membrane p o t e n t i a l due to the i n f l u x of c a t i o n s , a phenomenon observed when N-starved Lemna gibba (duckweed) was p u l s e d w i t h NH 4 + ( U l l r i c h e t a l . , 1984 ) . The response of N C ^ - s t a r v e d Micromonas pusilla to urea enrichment was s i m i l a r t o the ^ N H 4 + uptake response; the e l e v a t e d surge uptake d u r i n g the f i r s t 5 min was 2-3 f o l d g r e a t e r than the i n t e r n a l l y c o n t r o l l e d r a t e and the p r e c o n d i t i o n e d growth r a t e . P r i c e and H a r r i s o n (1988b) a l s o found a s i m i l a r i n c r e a s e i n ["^N] urea uptake i n NC>3 --starved c u l t u r e s of Thalassiosira pseudonana a f t e r a sh o r t l a g p e r i o d (5 min). They contend t h a t the subsequent e l e v a t e d [*^N] urea uptake r a t e was the combined r e s u l t of concomitant ["^N] urea uptake and the r a p i d r e a b s o r p t i o n of p r e v i o u s l y r e l e a s e d 1 5 N H 3 / 1 5 N H 4 + . The i n t e r n a l l y c o n t r o l l e d r a t e of urea uptake i n M. pusilla was s l i g h t l y lower than the r a t e needed t o support the maximum growth observed p r i o r t o N d e p l e t i o n . Others (Rees and S y r e t t , 1979; Hor r i g a n and McCarthy, 1981; P r i c e and H a r r i s o n , 1988b) have found N d e p r i v a t i o n i n c r e a s e d urea uptake i n marine diatoms r e l a t i v e t o N - r e p l e t e c e l l s . Rees and S y r e t t (1979) suggested t h a t ammonium formed from urea i n the growth medium may p a r t i a l l y suppress f o r m a t i o n of 137 t h e u r e a u p t a k e m e c h a n i s m a n d t h a t t h i s r e p r e s s i o n i s r e m o v e d d u r i n g n i t r o g e n d e p r i v a t i o n . P r i c e a n d H a r r i s o n ( 1 9 8 8 b ) c o n t e n d t h a t t h e i n c r e a s e d n i t r o g e n - s p e c i f i c u r e a u p t a k e was c a u s e d b y a r e d u c t i o n i n t h e c e l l N q u o t a a n d r e t e n t i o n o f a l l t h e u r e a - N by t h e n i t r a t e - s t a r v e d c e l l s . The i m p o r t a n c e o f t h e " e n h a n c e d " o r " s u r g e " u p t a k e t o p h y t o p l a n k t o n g r o w t h d e p e n d s on t h e c o u p l i n g o f t h e u p t a k e o f n i t r o g e n t o t h e i n c o r p o r a t i o n i n t o new c e l l u l a r m a t e r i a l ( C o l l o s , 1 9 8 6 ) . I f u p t a k e r a t e s r e s p o n d r a p i d l y t o b r i e f p u l s e s o f a m b i e n t n i t r o g e n b u t t h e c e l l s a r e n o t a b l e t o i n c o r p o r a t e t h a t n i t r o g e n on a s i m i l a r t i m e s c a l e , t h e n g r o w t h may be l i m i t e d b y t h e r a t e a t w h i c h c e l l u l a r m e t a b o l i s m c a n i n c o r p o r a t e d i s s o l v e d n i t r o g e n i n t o m a c r o m o l e c u l e s ( W h e e l e r e t a l . , 1 9 8 2 , 1 9 8 3 ; Z a r , 1 9 8 8 ) . N i t r a t e was n o t t a k e n up a s r e a d i l y by t h e N 0 3 ~ ~ - s t a r v e d c u l t u r e o f Micromonas pusilla a s t h e r e d u c e d N f o r m s , N H 4 + and u r e a . H owever, u n l i k e many N C ^ - s t a r v e d p h y t o p l a n k t o n ( r e v i e w by C o l l o s , 1 983; D o r t c h e t a l . , 1982; P a r s l o w e t a l . , 1984b) no p r e v i o u s e x p o s u r e t o N O 3 - was r e q u i r e d b e f o r e N O 3 - u p t a k e commenced ( i . e . t h e r e was no l a g p e r i o d ) . The maximum n i t r a t e u p t a k e r a t e a t t a i n e d , w h i c h was m e a s u r e d d u r i n g t h e f i r s t 5 m i n a f t e r e n r i c h m e n t , was c o n s i d e r a b l y l o w e r t h a n t h e N O 3 - r a t e s m e a s u r e d i n N O 3 - -r e p l e t e c u l t u r e s a n d t h e e l e v a t e d r a t e s o f N H 4 + a n d u r e a o b s e r v e d i n t h e s t a r v e d c u l t u r e s . The l o w e r , s u s t a i n a b l e i n t e r n a l l y c o n t r o l l e d r a t e o v e r t h e n e x t 5-6 h was o n l y h a l f t h a t r e q u i r e d t o s u p p o r t t h e p r e - c o n d i t i o n e d g r o w t h r a t e a n d 138 comparable t o i n t e r n a l l y c o n t r o l l e d reduced N uptake r a t e s . T h i s r e d u c t i o n i n N O 3 - uptake c a p a b i l i t y a f t e r N d e p r i v a t i o n i s not uncommon (e.g., C o l l o s , 1980; Dortch et a l . , 1982), although others (e.g., M o r r i s and S y r e t t , 1965; Thacker and S y r e t t , 1972a; H a r r i s o n , 1976) have r e p o r t e d i n c r e a s e d a b i l i t y t o take up n i t r a t e . I t appears t h a t although M. pusilla s t i l l r e t a i n e d N O 3 - uptake a b i l i t y i t may r e q u i r e an " a c c l i m a t i o n p e r i o d " b e f o r e maximal N O 3 - uptake can be a t t a i n e d , a response observed by many others (e.g., see review by C o l l o s , 1983). I t i s p o s s i b l e t h a t the p h y s i o l o g i c a l s t r e s s e x p e r i e n c e d by the c e l l s d u r i n g the 48 h of N O 3 - s t a r v a t i o n reduced t h e i r v i a b i l i t y and hence t h e i r n i t r o g e n uptake c a p a b i l i t y . The r e l a t i v e " h e a l t h " of the N-starved c e l l s was assessed by m i c r o s c o p i c examination of c e l l u l a r c o l o u r and m o t i l i t y . The d e c l i n e i n N O 3 - uptake a b i l i t y d u r i n g s t a r v a t i o n c o u l d a l s o be due t o the l o s s of an a c t i v e uptake system (Falkowski, 1975a) or t o i n a c t i v a t i o n of n i t r a t e reductase (e.g., S y r e t t , 1981). However, i n a c t i v a t i o n of n i t r a t e reductase alone need not prevent enhanced i n i t i a l NC^ - uptake as t r a n s i e n t i n t e r n a l n i t r a t e p o o ls are o f t e n observed i n phytoplankton (e.g., Dortch, 1982; Dortch et a l . , 1984). Substrate interaction Uptake i n t e r a c t i o n s between i n o r g a n i c N sources, p a r t i c u l a r l y n i t r a t e and ammonium, have been the s u b j e c t of many s t u d i e s (see reviews by Guerrero e t a l . , 1981; McCarthy, 1981; S y r e t t , 1981; U l l r i c h , 1983, Dortch, i n press) which r e v e a l a v a r i e t y of responses depending on the phytoplankton 139 s p e c i e s and i t s n u t r i t i o n a l s t a t e . N i t r a t e uptake has been r e p o r t e d t o be i n h i b i t e d t o d i f f e r e n t degrees by ammonium rang i n g from t o t a l s u p p r e s s i o n (e.g., S y r e t t and M o r r i s , 1963; McCarthy and Eppley, 1972; C r e s s w e l l and S y r e t t , 1979) t o simultaneous and comparable r a t e of N H 4 + and N O 3 - uptake i n c u l t u r e s (e.g., Caperon and Ziemann, 1976; Conway, 1977; Dortch and Conway, 1984; DeManche e t a l . , 1979) and n a t u r a l communities (e.g., Conover, 1975; McCarthy, 1977; M a e s t r i n i et a l . , 1982; 1986; P r i c e et a l . , 1985; Queguiner et a l . , 1986; C o l l o s et a l . , 1989). There are even a few r e p o r t s of s t i m u l a t i o n of N O 3 - uptake at low NH^ + c o n c e n t r a t i o n s even though h i g h e r c o n c e n t r a t i o n s i n h i b i t N O 3 - uptake (Conover, 1975; Caperon and Ziemann, 1976; G l i b e r t e t a l . , 1982c; Y i n , 1988; Dortch e t a l . , submitted). The present study demonstrates t h a t N O 3 - uptake by M. pusilla cannot proceed i n the presence of N H 4 + c o n c e n t r a t i o n s as low as 1.0 uq-at N-L -^. Only a f t e r N H 4 + i s exhausted from the e x t e r n a l medium does t h i s p h y t o p l a n k t e r resume i t s uptake of n i t r a t e . The mechanism of d e p r e s s i o n of n i t r a t e u t i l i z a t i o n i s not w e l l understood. There i s evidence f o r r e g u l a t o r y a c t i o n a t both the l e v e l of n i t r a t e uptake (e.g., Eppley and Rogers, 1970; C r e s s w e l l and S y r e t t , 1979; S e r r a et a l . , 1978b; T i s c h n e r and Lorenzen, 1979) and n i t r a t e r e d u c t i o n (e.g., S y r e t t and M o r r i s , 1963; Amy and G a r r e t t , 1974; H i p k i n e t a l . , 1980) and t o a c e r t a i n extent the e f f e c t of ammonium on both mechanisms may be independent (Blasco and Conway 1982, U l l r i c h 1987). I t i s now g e n e r a l l y accepted t h a t the primary, and 140 r a p i d l y a c t i n g e f f e c t o f N H 4 + o n n i t r a t e u t i l i z a t i o n i s due t o an i n h i b i t i o n o f n i t r a t e u p t a k e w h i c h may be f o l l o w e d b y t h e e f f e c t s on n i t r a t e m e t a b o l i s m t h r o u g h i n h i b i t i o n o f n i t r a t e r e d u c t a s e a c t i v i t y , f r o m e i t h e r i r r e v e r s i b l e p r o t e o l y t i c b r e a k d o w n ( e . g . , H i p k i n e t a l . , 1 9 8 0 ) , r e v e r s i b l e i n a c t i v a t i o n ( e . g . , P i s t o r i u s e t a l . , 1978) o r s u p p r e s s i o n o f i t s s y n t h e s i s ( e . g . , M o r r i s a n d S y r e t t , 1963; Amy a n d G a r r e t t , 1 9 7 4 ) . A l t h o u g h some ( e . g . , F l o r e n c i o a n d V e g a , 1982) a r g u e t h a t N H 4 +  p e r s e i n h i b i t s N O 3 - a s s i m i l a t i o n m o s t e v i d e n c e s u g g e s t s t h a t t h e r a t e o f N O 3 - u p t a k e i s m o d u l a t e d i n r e s p o n s e t o c h a n g e s i n p o o l s o f some o r g a n i c p r o d u c t o f ammonium a s s i m i l a t i o n ( e . g . , S y r e t t , 1 9 8 1 , ; G u e r r e r o e t a l . , 1 9 8 1 ) . T h e r e a r e o n l y a f e w r e p o r t s o f s i m u l t a n e o u s u p t a k e o f u r e a a n d o t h e r N s o u r c e s b y n a t u r a l p h y t o p l a n k t o n c o m m u n i t i e s ( M c C a r t h y a n d E p p l e y , 1972; P r i c e e t a l . , 1 9 8 5 ) . I t i s h o w e v e r , g e n e r a l l y b e l i e v e d t h a t u r e a s u p p r e s s e s t h e u p t a k e o f N O 3 - b u t a t a l o w e r l e v e l t h a n ammonium ( G r a n t e t a l . , 1967; M c C a r t h y a n d E p p l e y , 1972; M o l l y a n d S y r e t t , 1 9 8 8 b ) . An u n c h a n g e d r a t e o f N O 3 - u p t a k e i n t h e p r e s e n c e o f 10 uq-at N-L--*- o f u r e a h a s , h o w e v e r , b e e n r e p o r t e d i n t h e m a r i n e d i a t o m , Skeletonema costatum ( L u n d , 1 9 8 7 ) . I n t h e p r e s e n t s t u d y t h e r a t e o f N O 3 - u p t a k e was l o w e r i n t h e p r e s e n c e o f u r e a , b u t i n t e r m s o f t o t a l N t a k e n up i t was a p p r o x i m a t e l y 30% g r e a t e r t h a n t h e u n a l t e r e d r a t e o f N O 3 - u p t a k e a l o n e a n d t h e u p t a k e r a t e n e c e s s a r y t o m a i n t a i n t h e p r e - c o n d i t i o n e d g r o w t h r a t e . A l t h o u g h n o t m e a s u r e d i n t h e p r e s e n t s t u d y , i t i s p o s s i b l e t h a t NH^ was e x c r e t e d b y t h e c e l l s a n d was t h e c a u s a t i v e f a c t o r f o r r e d u c e d N O 3 - u p t a k e r a t e s a f t e r t h e a d d i t i o n o f u r e a . P r i c e a n d H a r r i s o n ( 1 9 8 8 b) r e p o r t e d t h e r e l e a s e o f N H 3 / N H 4 + i n t o t h e medium b y a x e n i c c u l t u r e s o f Thalassiosira pseudonana f o l l o w i n g a d d i t i o n o f 10 uq-at U-L~^ o f u r e a . U c h i d a (1976) o b s e r v e d t h a t t h e r e d - t i d e d i n o f l a g e l l a t e Prorocentrum minimum e x c r e t e d NH3/NH^ + when gr o w n i n u r e a - e n r i c h e d c u l t u r e medium a n d Rees ( 1 9 7 9 ) a l s o r e p o r t e d N H 3 / N H 4 + r e l e a s e b y u r e a - g r o w n Phaeodactylum tricornutum. Ecological significance A s D o r t c h e t a l . (1982) h a v e p o i n t e d o u t p r e v i o u s l y , t h e r e d u c t i o n i n N O 3 - u p t a k e c a p a b i l i t y a n d a n e n h a n c e d a b i l i t y t t a k e up N H 4 + by N - d e p r i v e d p h y t o p l a n k t o n may be a n a d a p t i v e r e s p o n s e t o t h e p a t t e r n s o f n i t r o g e n a v a i l a b i l i t y i n o l i g o t r o p h i c a r e a s o f t h e o c e a n . Ammonium a n d u r e a a r e b o t h r e c y c l e d r a p i d l y w i t h i n t h e e u p h o t i c z o n e a n d may be a d d e d s p o r a d i c a l l y by a n i m a l e x c r e t i o n ( e . g . , D u g d a l e , 1 9 6 7 ) . P h y t o p l a n k t o n w h i c h h a v e t h e a b i l i t y t o a s s i m i l a t e N H 4 + o r u r e a r a p i d l y a f t e r s t a r v a t i o n may h a v e a s e l e c t i v e a d v a n t a g e i n a r e a s w h e r e N l i m i t a t i o n i s t h e m a j o r e n v i r o n m e n t a l s t r e s s P h y t o p l a n k t o n w h i c h m a i n t a i n t h e a b i l i t y t o t a k e up n i t r a t e r a p i d l y d u r i n g N s t a r v a t i o n c o n f e r l i t t l e c o m p e t i t i v e a d v a n t a g e b e c a u s e n i t r a t e i s r e c y c l e d on a much l o n g e r t i m e s c a l e a n d u s u a l l y s u p p l i e d c o n t i n u o u s l y a t l o w r a t e s b y e d d y d i f f u s i o n f r o m d e e p e r n i t r a t e - r i c h l a y e r s ( e . g . , D u g d a l e , 1967; E p p l e y e t a l . , 1979; K i n g a n d D e v o l , 1 9 7 9 ) . P h y s i c a l 142 events such as upwelling (e.g., Walsh et a l . , 1977, 1978) frontal mixing (e.g., Pingree et a l . , 1978; Parsons et a l . , 1981) internal waves (e.g., McGowan and Hayward, 1978; Cullen et a l . , 1983) may supply N O 3 - at elevated concentrations to the euphotic zone at intervals of days, weeks or longer. However, as Parslow et a l . (1984b) pointed out these physical mechanisms of sporadic N O 3 - supply also dilute the phytoplankton concentrations in the euphotic zone, thereby decreasing the demand for the nutrient, increasing the lifetime of the pulse and consequently reducing the benefits of transient elevated uptake rates; hence the metabolic cost of maintaining high uptake capability for nitrate during starvation (between pulses) may outweigh the benefits. 143 CHAPTER FOUR EFFECTS OF IRRADIANCE AND D I E L P E R I O D I C I T Y ON NITROGEN U T I L I Z A T I O N I N MICROMONAS PUSILLA INTRODUCTION I n o r d e r t o d e s c r i b e t h e m e c h a n i s m s i n v o l v e d i n p h y t o p l a n k t o n e c o l o g y t h e r e i s a n e e d t o f i r s t u n d e r s t a n d t h e s i g n i f i c a n c e o f b i o l o g i c a l i n t e r a c t i o n s w i t h e n v i r o n m e n t a l p a r a m e t e r s . I n most m a r i n e e n v i r o n m e n t s , l i g h t a n d t h e a v a i l a b i l i t y o f n i t r o g e n h a v e b e e n shown t o be t h e f a c t o r s w h i c h p r i m a r i l y r e g u l a t e p h y t o p l a n k t o n p r o d u c t i v i t y ( e . g . , D u g d a l e a n d G o e r i n g , 1967; R y t h e r a n d D u n s t a n , 1971; M a c l s a a c a n d D u g d a l e , 1972 ) . I n 1967, D u g d a l e a n d G o e r i n g p a r t i t i o n e d o c e a n i c p r i m a r y p r o d u c t i o n a c c o r d i n g t o i t s n i t r o g e n s o u r c e : "new" p r o d u c t i o n i s f u e l l e d by a l l o c h t h o n o u s N s o u r c e s , p r i n c i p a l l y n i t r a t e m i x e d i n t o s u r f a c e w a t e r s f r o m d e e p o c e a n r e s e r v e s , a n d s e c o n d a r i l y , ^ - f i x a t i o n , r i v e r i n e i n p u t s a n d r a i n f a l l ; " r e g e n e r a t e d " p r o d u c t i o n i s f u e l l e d by a u t o c h t h o n o u s N-s o u r c e s , p r i n c i p a l l y ammonium a n d u r e a , d e r i v e d f r o m b i o l o g i c a l p r o c e s s e s ( n u t r i e n t r e c y c l i n g ) o c c u r r i n g in situ. C h a n g e s i n n i t r o g e n c o n c e n t r a t i o n s i n t h e e u p h o t i c z o n e a r e u s u a l l y t h e r e s u l t o f p h y s i c a l m e c h a n i s m s , s u c h a s u p w e l l i n g a n d v e r t i c a l m i x i n g ( e . g . , C o d i s p o t i , 1983; T a k a h a s h i e t a l . , 1986; P i a t t e t a l . , 1 9 8 9 ) , w h i c h i n c r e a s e "new" n i t r o g e n c o n c e n t r a t i o n s , o r a l t e r n a t i v e l y , b i o l o g i c a l p r o c e s s e s , s u c h a s a n i m a l e x c r e t i o n ( e . g . , M c C a r t h y a n d G o l d m a n , 1979;) w h i c h i n c r e a s e " r e g e n e r a t e d " n i t r o g e n c o n c e n t r a t i o n s . 144 The l i g h t e n v i r o n m e n t shows e x t r e m e v a r i a t i o n a n d i n a p e r i o d i c f a s h i o n d u r i n g 24 h, ( e x c e p t a t t i m e s i n h i g h l a t i t u d e a r e a s ) . D i e l p h y s i o l o g i c a l r h y t h m s , i n r e s p o n s e t o f l u c t u a t i o n s i n l i g h t i n t e n s i t y , h a v e b e e n d e t e c t e d i n c u l t u r e s a n d n a t u r a l p h y t o p l a n k t o n c o m m u n i t i e s a n d i n c l u d e p r o c e s s e s s u c h a s p h o t o s y n t h e s i s ( e . g . , M a c C a u l l a n d P i a t t , 1977; P r e z e l i n a n d L e y , 1980; H a r d i n g e t a l . , 1 9 8 1 , 1983; P u t t a n d P r e z e l i n , 1 9 8 8 ) ; c e l l u l a r p i g m e n t c o n t e n t ( e . g . , S o u r n i a , 1974; Owens e t a l . , 1980; H a r d i n g e t a l . , 1 983; K o h a t a a n d W a t a n a b e , 1 9 8 9 ) ; i n 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 ( e . g . , Owens e t a l . , 1 9 8 0 ) ; c a r b o h y d r a t e a n d p r o t e i n c o n t e n t ( e . g . , R i c k e t t s , 1977; H i t c h c o c k , 1980; T e r r y e t a l . , 1 9 8 5 ) ; c e l l d i v i s i o n ( e . g . , C h i s h o l m e t a l . , 1980; C h i s h o l m , 1 9 8 1; S o u r n i a , 1974) enzyme a c t i v i t i e s ( e . g . E p p l e y e t a l . , 1970; P a c k a r d a n d B l a s c o , 1974; M a r t i n e z e t a l . , 1987) a n d n i t r o g e n u p t a k e ( e . g . , E p p l e y e t a l . , 1970 1971a,b; M a c l s a a c , 1978; T e r r y e t a l . , 1985 ) . P r e v i o u s s t u d i e s o f c u l t u r e s a n d n a t u r a l p h y t o p l a n k t o n c o m m u n i t i e s s u g g e s t t h a t N s t a r v a t i o n ( e . g . , H a r r i s o n , 1976; B h o v i c h i t r a a n d S w i f t , 1977) o r N l i m i t a t i o n ( e . g . M a l o n e e t a l . , 1975; E p p l e y e t a l . , 1971b) may dampen d i e l p e r i o d i c i t y o f N O 3 - u p t a k e a n d a s s i m i l a t i o n by a r e l a t i v e e n h a n c e m e n t o f N O 3 - u p t a k e d u r i n g t h e n i g h t . To d a t e o u r k n o w l e d g e o f n i t r o g e n u p t a k e b y p i c o p l a n k t o n h a v e b e e n d e r i v e d f r o m t r a c e r s t u d i e s o f s i z e - f r a c t i o n e d n a t u r a l p h y t o p l a n k t o n c o m m u n i t i e s ( e . g . , P r o b y n , 1985; P r o b y n a n d P a i n t i n g , 1985; N a l e w a j k o a nd G a r s i d e , 1983) a n d t h e e f f e c t s o f n u t r i e n t l i m i t a t i o n , 145 irradiance, or l i g h t p e r i o d i c i t y on t h e i r N uptake a b i l i t i e s have not been addressed. The objective of t h i s study was to determine the ef f e c t ( s ) of N l i m i t a t i o n on the p e r i o d i c i t y of in s i t u and po t e n t i a l N uptake by the ubiquitous, p i c o f l a g e l l a t e Micromonas p u s i l l a (Butch.) Manton et Parke. Nitr a t e , the N substrate which supports the most productive areas of the world's oceans (e.g., Eppley, 1981; Eppley and Peterson, 1979; Harrison et a l . , 1987) was the p r i n c i p a l focus of the present study. However, the d i e l p e r i o d i c i t y of p o t e n t i a l uptake rates of urea and NH 4 + were also examined i n view of the importance of regenerated nitrogenous n u t r i t i o n of phytoplankton i n oligotrophic, oceanic areas (e.g., Harrison, 1 9 8 0 ) ; environments where picoplankton have been demonstrated to be responsible for the majority of the photosynthetic production (e.g., L i et a l . , 1983; P i a t t et a l . , 1983; see revievs by Fogg, 1986; Joint, 1986; Stockner and Antia, 1 9 8 6 ) . 146 MATERIALS AND METHODS Culturing C o n t i n u o u s a n d b a t c h c u l t u r e s o f Micromonas pusilla ( c u l t u r e NEPCC 2 9 - 1 , N o r t h e a s t P a c i f i c C u l t u r e C o l l e c t i o n , D e p a r t m e n t o f O c e a n o g r a p h y , U n i v e r s i t y o f B r i t i s h C o l u m b i a ) w e r e grown on 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 ) 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 s e a w a t e r b a s e d on ESAW ( H a r r i s o n e t a l . , 1 9 8 0 ) . The m o d i f i c a t i o n s t o t h e medium a n d t h e d e t a i l s o f i t s p r e p a r a t i o n a n d s t o r a g e a r e d e s c r i b e d i n C h a p t e r 3. C u l t u r e s w e r e m a i n t a i n e d i n a n a i r - c o o l e d , w a l k - i n , g r o w t h c h a m b e r a t • • R 17 ± 0.5°C a n d i l l u m i n a t e d f r o m two s i d e s by e i g h t V i t a - L i t e UHO f l u o r e s c e n t t u b e s (4 on e i t h e r s i d e o f c u l t u r e v e s s e l s ) s e t on a 14 h l i g h t a n d 10 h d a r k c y c l e . The l i g h t was • R f i l t e r e d t h r o u g h 3 mm t h i c k b l u e P l e x i g l a s (No. 2 0 6 9 , Rohm an d Haas) a n d t h e i r r a d i a n c e a d j a c e n t t o t h e c e n t r e o f t h e ? 1 c u l t u r e v e s s e l s was c a . 120 uE m s , a s m e a s u r e d w i t h a L i -C o r L I - 1 8 5 l i g h t m e t e r e q u i p p e d w i t h a L I - 1 9 0 S q u a n t u m s e n s o r ( 2 T T ) . C o n t i n u o u s c u l t u r e s o f M. pusilla w e r e m a i n t a i n e d i n c h e m o s t a t s s i m i l a r t o t h o s e d e s c r i b e d by D a v i s e t a l . ( 1 9 7 3 ) . C o n s t a n t f l o w p i s t o n pumps ( F l u i d M e t e r i n g I n c . , New Y o r k ) w e r e u s e d t o pump t h e medium f r o m 20 L P y r e x c a r b o y s t h r o u g h b o r o s i l i c a t e g l a s s o r s i l i c o n e r u b b e r t u b i n g i n t o t h e r e a c t o r v e s s e l s . The r e a c t o r v e s s e l s c o n s i s t e d o f 3 L, b o r o s i l i c a t e , f l a t - b o t t o m , b o i l i n g f l a s k s ( P y r e x ) s e a l e d w i t h a s i l i c o n e s t o p p e r s o t h a t a c o n s t a n t v o l u m e o f 2.5 L ( a c c u r a t e l y m e a s u r e d b e f o r e e a c h e x p e r i m e n t ) was m a i n t a i n e d i n s i d e t h e 147 r e a c t o r . C u l t u r e s w e r e s t i r r e d by t e f l o n - c o a t e d m a g n e t i c s t i r b a r s a t 60 rpm. C u l t u r e s w e r e u n i a l g a l b u t n o t a x e n i c , h o w e v e r a t t e m p t s w e r e made t o m i n i m i z e b a c t e r i a l g r o w t h i n t h e medium a n d c u l t u r e s b y u s i n g a s e p t i c t e c h n i q u e s a n d s c r u p u l o u s l y c l e a n i n g a l l g l a s s w a r e , pump f i t t i n g s a n d t u b i n g w i t h 10% HC1 a c i d ( v / v ) , r i n s i n g w i t h d i s t i l l e d , d e i o n i z e d w a t e r (DDW) a n d a u t o c l a v i n g p r i o r t o u s e . D i f f i c u l t i e s i n e s t a b l i s h i n g a d e f i n e d medium a f t e r a u t o c l a v i n g a n d i n k e e p i n g a c o n t i n u o u s c u l t u r e a x e n i c f o r e x t e n d e d p e r i o d s i s a common p r o b l e m ( e . g . , G o l d m a n , 1 9 7 7 ) . However, i t was f e l t t h a t b y u s i n g t h e a b o v e p r e c a u t i o n s any b a c t e r i a l e f f e c t s w o u l d be m i n i m i z e d t o t h e p o i n t w h e r e t h e i r e f f e c t s w o u l d be n e g l i g i b l e r e l a t i v e t o t h e r e s p o n s e ( s ) o f Micromonas pusilla. C u l t u r e s w e r e a l l o w e d t o grow a s b a t c h c u l t u r e s f o r s e v e r a l d a y s ( 4 - 5 ) b e f o r e c o n t i n u o u s a d d i t i o n o f t h e i n f l o w medium c o n t a i n i n g 50.6 ± 0 . 9 uq-at N 0 3 ~ - L ~ 1 a n d 0.18 ± 0.08 j j g - a t N 0 2 ~ * L _ 1 was i n i t i a t e d . D i l u t i o n r a t e s , c a l c u l a t e d f r o m t h e v o l u m e o f e f f l u e n t c o l l e c t e d d a i l y , w e r e 0.24, 0.49 a n d 0.74 d~^ a n d v a r i e d l e s s t h a n 0.01 d-"*". C u l t u r e s a m p l e s w e r e w i t h d r a w n d a i l y b y s y r i n g e a t t h e m i d - p o i n t o f t h e l i g h t p e r i o d f o r m o n i t o r i n g o f i n v i v o f l u o r e s c e n c e , c e l l c o u n t s a n d p a r t i c l e s i z e d i s t r i b u t i o n . E x p e r i m e n t s w e r e n o t i n i t i a t e d u n t i l s t e a d y - s t a t e h a d b e e n a c h i e v e d w i t h i n e a c h c o n t i n u o u s c u l t u r e . S t e a d y - s t a t e was assumed when c e l l c o u n t s w e r e c o n s t a n t t o w i t h i n ±10% f o r a minimum o f t h r e e c o n s e c u t i v e d a y s i n t h e h i g h e s t g r o w t h c u l t u r e a n d ±5% f o r t h e t w o l o w e r g r o w t h r a t e c u l t u r e s . 148 Analytical procedures C e l l 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 w i t h a C o u l t e r C o u n t e r m o d e l TA I I e l e c t r o n i c p a r t i c l e c o u n t e r a c c o r d i n g t o t h e p r o c e d u r e s o u t l i n e d i n d e t a i l i n C h a p t e r 3. A v e r a g e c e l l v o l u m e s w e r e c o m p u t e d f r o m t h e p a r t i c l e s i z e d i s t r i b u t i o n b a s e d on e q u i v a l e n t s p h e r i c a l d i a m e t e r . R e p l i c a t e ( 2 - 4 ) s a m p l e s f o r t h e m e a s u r e m e n t o f p a r t i c u l a t e o r g a n i c c a r b o n (POC) a n d p a r t i c u l a t e o r g a n i c n i t r o g e n (PON) w e r e f i l t e r e d o n t o p r e c o m b u s t e d (460°C, 6 h) Whatman GF/F g l a s s f i b e r f i l t e r s , s t o r e d f r o z e n a t -20°C i n d e s i c c a t o r s a n d a n a l y z e d on a CHN e l e m e n t a l a n a l y z e r ( C o n t r o l E q u i p m e n t C o r p . m o d e l 240-XA) u s i n g a c e t a n i l i d e a s t h e c a l i b r a t i o n s t a n d a r d s . E x t e r n a l ( a m b i e n t ) a n d i n t e r n a l ( c e l l u l a r p o o l s ) c o n c e n t r a t i o n s o f n i t r a t e a n d n i t r i t e w e r e m e a s u r e d w i t h a T e c h n i c o n A u t o A n a l y z e r R I I f o l l o w i n g t h e p r o c e d u r e s o u t l i n e d i n Wood e t a l . ( 1 9 6 7 ) . The p r e c i s i o n o f t h e a b o v e a n a l y t i c a l t e c h n i q u e s a r e p r e s e n t e d i n A p p e n d i x 6. S a m p l e s f o r t h e m e a s u r e m e n t o f e x t e r n a l n u t r i e n t c o n c e n t r a t i o n s w e r e f i l t e r e d t h r o u g h p r e c o m b u s t e d Whatman GF/F f i l t e r s i n t o p r e v i o u s l y a c i d - w a s h e d , D D W - r i n s e d p o l y p r o p y l e n e b o t t l e s a f t e r an i n i t i a l r i n s e w i t h f i l t r a t e . S a m p l e s w e r e s t o r e d f r o z e n (-20°C) u n t i l a n a l y s e s . I n t e r n a l d i s s o l v e d n i t r o g e n p o o l s w e r e d e t e r m i n e d a f t e r e x t r a c t i o n w i t h b o i l i n g DDW ( m e t h o d C-2, T h o r e s e n e t a l . , 1 9 8 2 ) . P h y t o p l a n k t o n c e l l s ( 20-50 m l o f c u l t u r e ) w e r e f i l t e r e d o n t o c o m b u s t e d g l a s s f i b e r f i l t e r s (Whatman GF/F, 25 mm, p r e v i o u s l y w a s h e d w i t h 2N HC1 a n d s u b s e q u e n t l y r i n s e d w i t h 50 m l b o i l i n g a n d 50 m l c o o l DDW) 149 w i t h a l o w d i f f e r e n t i a l p r e s s u r e (< 80 nun H g ) . A f t e r f i l t r a t i o n , t h e f i l t e r was r i n s e d w i t h 5-10 m l o f c u l t u r e medium c o n t a i n i n g no d e t e c t a b l e i n o r g a n i c n i t r o g e n ( o b t a i n e d f r o m g r a v i t y f i l t r a t i o n o f N - s t a r v e d c u l t u r e s d e s c r i b e d i n C h a p t e r 3 ) . The f i l t e r a n d c e l l s w e r e e x t r a c t e d w i t h b o i l i n g DDW, c o l l e c t e d d i r e c t l y i n t o p o l y p r o p y l e n e b o t t l e s , a n d s t o r e d f r o z e n (-20°C) u n t i l a n a l y s e s . B l a n k s w e r e f i l t e r s w i t h o u t c e l l s on them a n d t h e y w e r e t r e a t e d t h e same a s s a m p l e s . The f i l t r a t i o n a p p a r a t u s was a c i d - w a s h e d , D D W - r i n s e d a n d d r i e d p r i o r t o c o l l e c t i o n o f s a m p l e s . 15 S a m p l e s f o r N a n a l y s i s w e r e c o l l e c t e d on p r e c o m b u s t e d Whatman GF/F f i l t e r s a n d s t o r e d f r o z e n i n d e s i c c a t o r s . The e n r i c h m e n t o f s a m p l e s was a s s a y e d by e m i s s i o n s p e c t r o m e t r y (JASCO m o d e l N-150) a f t e r c o n v e r s i o n o f t h e p a r t i c u l a t e N t o N 2 g a s by t h e m i c r o - D u m a s d r y c o m b u s t i o n t e c h n i q u e . A l l a n a l y s e s w e r e c o n d u c t e d i n d u p l i c a t e a c c o r d i n g t o t h e p r o c e d u r e s d e s c r i b e d i n d e t a i l i n C h a p t e r 1. Experimental procedures D i e l c y c l e s o f u p t a k e a n d g r o w t h The c o n c e n t r a t i o n o f c e l l s a n d i n o r g a n i c n i t r o g e n i n t h e t h r e e c o n t i n u o u s c u l t u r e s a n d b a t c h c u l t u r e s was m o n i t o r e d o v e r a 24 h p e r i o d b e g i n n i n g a t t h e s t a r t o f t h e l i g h t p e r i o d . T h e s e e x p e r i m e n t s w e r e c o n d u c t e d w i t h d u p l i c a t e c u l t u r e s w h i c h w e r e o r i g i n a l l y i n o c u l a t e d f r o m t h e same s t o c k c u l t u r e . A t t w o h o u r i n t e r v a l s , s a m p l e s w e r e d r a w n by s y r i n g e d i r e c t l y f r o m t h e r e a c t o r v e s s e l s f o r d e t e r m i n a t i o n o f c e l l c o n c e n t r a t i o n a n d v o l u m e a n d t h e a m b i e n t a n d i n t e r n a l NC^ - a n d 150 NO2"" c o n c e n t r a t i o n s . S m a l l v o l u m e s (25-60 m l ) w e r e w i t h d r a w n f r o m t h e c o n t i n u o u s c u l t u r e s t o m i n i m i z e t h e p e r t u r b a t i o n t o t h e i r s t e a d y - s t a t e c o n d i t i o n . The t o t a l v o l u m e w i t h d r a w n f r o m t h e c y c l o s t a t s was b e t w e e n 1 a n d 2.5% o f t h e c u l t u r e v o l u m e . D u r i n g t h e t i m e t h a t t h e c y c l o s t a t s w e r e r e f i l l i n g , t h e o u t f l o w was c l o s e d . The m a t h e m a t i c a l e q u a t i o n s g o v e r n i n g t h e r a t e o f c h a n g e o f s u b s t a n c e s i n t h e c u l t u r e v e s s e l s a r e v i r t u a l l y i d e n t i c a l t o t h o s e d e s c r i b i n g o v e r f l o w c o n d i t i o n s , b e c a u s e t h e i n c r e a s e i n c u l t u r e v o l u m e due t o t h e i n p u t o f f r e s h medium d i l u t e s t h e c u l t u r e i n a manner s i m i l a r t o t h a t r e s u l t i n g f r o m o v e r f l o w u n d e r c o n s t a n t v o l u m e c o n d i t i o n s ( L a w s , 1985; D i T u l l i o a n d L a w s , 1 9 8 6 ) . T h e r e f o r e , a s l o n g a s t h e v o l u m e o f s a m p l e w i t h d r a w n i s s m a l l c o m p a r e d t o t h e v o l u m e o f t h e g r o w t h chamber i t c a n be assumed t h a t s a m p l i n g w i l l n o t s i g n i f i c a n t l y p e r t u r b t h e s t e a d y s t a t e c o n d i t i o n . R a t e s o f n i t r a t e u p t a k e i n t h e c o n t i n u o u s c u l t u r e s w e r e c a l c u l a t e d u s i n g t h e e q u a t i o n s o f C a p e r o n a n d Z i e m a n n ( 1 9 7 6 ) . The r a t e o f c h a n g e o f n i t r a t e c o n c e n t r a t i o n was assumed t o be d e s c r i b e d by t h e e q u a t i o n : d S / d t = D - ( S i - S ) - U/V Where S i s t h e c o n c e n t r a t i o n o f N O 3 - i n t h e c h e m o s t a t , S^ i s t h e N O 3 - c o n c e n t r a t i o n o f t h e i n f l o w medium, U i s t h e t o t a l N O 3 - u p t a k e r a t e , V i s t h e v o l u m e o f t h e g r o w t h c h a m b e r a n d D i s t h e d i l u t i o n r a t e . The i n t e g r a t e d s o l u t i o n o f t h i s e q u a t i o n h a s b e e n g i v e n by C a p e r o n a n d Z i e m a n n (1976) a n d t h e n i t r a t e u p t a k e d u r i n g t h e i n t e r v a l ( t ) b e t w e e n two s a m p l i n g p e r i o d s i s : 151 nX = DSj^ + D • w h e r e S Q a n d a r e N O 3 - c o n c e n t r a t i o n s a t t i m e s 0 a n d t , r e s p e c t i v e l y , n i s t h e PON c o n c e n t r a t i o n o f t h e c u l t u r e ( a s s u m e d t o be c o n s t a n t a t s t e a d y s t a t e ) a n d X i s t h e s p e c i f i c u p t a k e r a t e w i t h nX = U/V. T h i s e q u a t i o n a s s u m e s t h a t u p t a k e r a t e s a r e c o n s t a n t d u r i n g t h e s a m p l i n g i n t e r v a l s , w h i c h i s n o t n e c e s s a r i l y a l w a y s t r u e , b u t t h e c a l c u l a t e d r a t e s a r e u s e f u l a s e s t i m a t e s o f a v e r a g e u p t a k e r a t e s d u r i n g t h e s e p e r i o d s . U p t a k e r a t e s i n t h e b a t c h c u l t u r e s , e s t i m a t e d f r o m t h e d i s a p p e a r a n c e o f N O 3 - i n t h e medium, w e r e c a l c u l a t e d b y d i v i d i n g t h e d i f f e r e n c e i n t h e n u t r i e n t c o n c e n t r a t i o n i n s u c c e s s i v e s a m p l e s b y t h e l e n g t h o f t h e t i m e i n t e r v a l ; s p e c i f i c r a t e s w e r e c a l c u l a t e d b y d i v i d i n g t h i s v a l u e b y t h e e s t i m a t e d e x p o n e n t i a l a v e r a g e PON c o n c e n t r a t i o n d u r i n g t h a t t i m e . I t was a s s u m e d t h a t a l l t h e N O 3 - r e m o v e d f r o m t h e medium was i n c o r p o r a t e d i n t o t h e p a r t i c u l a t e f r a c t i o n a n d t h a t n one o f t h e N03~ t a k e n up was e x c r e t e d a s 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). T h i s s i t u a t i o n was o b s e r v e d i n o t h e r e x p e r i m e n t s f o r M. pusilla c u l t u r e s g r o w i n g u n d e r c o n t i n u o u s l i g h t ( A p p e n d i x 4 ) . D i e l v a r i a t i o n i n maximum N u p t a k e r a t e s A t 8 s e p a r a t e t i m e s o v e r a 17 d p e r i o d , e x p e r i m e n t s w e r e c o n d u c t e d t o d e t e r m i n e t h e . s a t u r a t e d u p t a k e r a t e s o f N O 3 - , N H 4 + a n d u r e a f r o m t h e c o n t i n u o u s c u l t u r e s g r o w n a t 0.74, 0.49, a n d 0.24 d -^". T h e s e e x p e r i m e n t a l t i m e s c o r r e s p o n d e d t o 152 the b e g i n n i n g , middle and end of the 14 h l i g h t and 10 h dark p e r i o d s and are gi v e n i n Table 4.3. The experimental procedure a t each time was as f o l l o w s : d u p l i c a t e samples were f i r s t c o l l e c t e d f o r the measurement of POC, PON, ambient n i t r a t e and n i t r i t e and c e l l c o n c e n t r a t i o n s . T h i r t y ml samples were t r a n s f e r r e d t o s t e r i l e , 50 ml b o r o s i l i c a t e g l a s s t e s t tubes w i t h t e f l o n - l i n e d caps and d u p l i c a t e s i n o c u l a t e d w i t h 16.6 /jg-at N ' L - 1 of 1 5NH 4C1, N a 1 5 N 0 3 or C O ( 1 5 N H 4 ) 2 (Kor Isotopes, 99 atom % ) . Incubations were conducted i n the dark — 2 — 1 and at the same PPFD as p r e v i o u s l y grown (ca. 120 uE m s ) and then t e r m i n a t e d a f t e r 2 h by f i l t r a t i o n ( p ressure d i f f e r e n t i a l < 80 mm Hg). The N s p e c i f i c s a t u r a t e d uptake r a t e s were determined from the con s t a n t s p e c i f i c uptake model of Dugdale and Wilkerson (1986) (equation 6 of Appendix 1). Although the t o t a l sample volume withdrawn r e p r e s e n t s only 10-15% of the growth chamber volume, the c u l t u r e s were sampled at i n t e r v a l s never l e s s t h a t 1 d - ^ . C e l l c o n c e n t r a t i o n was monitored over the 16 d experimental p e r i o d by withdrawing samples a t the mid-point of the l i g h t - d a r k c y c l e ( F i g . 4.1). C e l l c o n c e n t r a t i o n s were r e l a t i v e l y s t a b l e over the course of the study and d a i l y v a r i a t i o n s averaged 8.4 (±7.3), 3.7 (±3.2) and 4.6 (±3.7)% i n the 0.73, 0.49 and 0.24 d - 1 c y c l o s t a t s , r e s p e c t i v e l y , E f f e c t of PPFD on N0 3~ and NH 4 + uptake Three continuous c u l t u r e s (0.77, 0.52 and 0.24 d -^) were har v e s t e d j u s t p r i o r t o the middle of the l i g h t p e r i o d (6 h l i g h t ) t o d e t e r m i n e t h e e f f e c t ( s ) o f PPFD on N 0 3 ~ a n d N H 4 u p t a k e r a t e . D u p l i c a t e (3 o r 4) s a m p l e s w e r e i n i t i a l l y c o l l e c t e d f o r c o n c e n t r a t i o n m e a s u r e m e n t s o f PON, POC, a m b i e n t N 0 3 ~ a n d NO2 - a n d p h y t o p l a n k t o n c e l l s . The r e m a i n d e r o f t h e c u l t u r e was s p l i t i n h a l f a n d i n o c u l a t e d w i t h 15 uq-at N ' L - ^ o f N a 1 5 N 0 3 ~ o r 1 5 N H 4 C 1 ( K o r I s o t o p e s , 99 a t o m % ) . F o r t y m l s a m p l e s w e r e i m m e d i a t e l y t r a n s f e r r e d , u n d e r r e d u c e d l i g h t c o n d i t i o n s , t o s t e r i l e 50 m l b o r o s i l i c a t e g l a s s t e s t t u b e s , w i t h t e f l o n - l i n e d c a p s , a n d p l a c e d w i t h i n n e u t r a l d e n s i t y s c r e e n i n g t o s i m u l a t e a r a n g e o f PPFDs f r o m 140 t o 3.5 — 7 — 1 uE m s a n d d a r k n e s s . I r r a d i a n c e s , a c h i e v e d b y a t t e n u a t i o n due t o d i s t a n c e a n d s c r e e n i n g , w e r e m e a s u r e d w i t h a B i o s p h e r i c a l I n s t r u m e n t s QSL-100 in s e n s o r p l a c e d w i t h i n t h e s c r e e n i n g i n t h e i n c u b a t i o n p o s i t i o n . I n c u b a t i o n s w e r e c o n d u c t e d a t t h e g r o w t h t e m p e r a t u r e a n d t e r m i n a t e d a f t e r 2 h 1 5 by l o w vacuum f i l t r a t i o n 80 mm Hg) f o r c o l l e c t i o n o f N-l a b e l l e d p a r t i c u l a t e m a t e r i a l . D u p l i c a t e c u l t u r e s e n r i c h e d w i t h N - l a b e l l e d N 0 3 ~ a n d NH 4 w e r e a l s o i n c u b a t e d f o r 4 a n d 6 h i n t h e d a r k . N i t r o g e n u p t a k e r a t e s w e r e c a l c u l a t e d a c c o r d i n g t o t h e e q u a t i o n s d e s c r i b e d p r e v i o u s l y f o r t h e s a t u r a t e d N u p t a k e e x p e r i m e n t s . K i n e t i c c o n s t a n t s f o r N 0 3 ~ a n d N H 4 + u p t a k e w i t h r e s p e c t t o PPFD w e r e o b t a i n e d by a d i r e c t f i t o f t h e d a t a t o a m o d i f i e d M i c h a e l i s - M e n t e n h y p e r b o l a u s i n g t h e n o n - l i n e a r l e a s t - s q u a r e s t e c h n i q u e a n d f o r m u l a t i o n d e s c r i b e d e a r l i e r i n C h a p t e r 2. 154 F i g u r e 4.1. C e l l c o n c e n t r a t i o n a s a f u n c t i o n o f t i m e f o r n i t r a t e - l i m i t e d c y c l o s t a t c u l t u r e s o f Micromonas pusilla g r o w n i n a 14h:10h L:D c y c l e a t (O) 0.74, (•) 0.49 a n d (A) 0.24 d" 1 d i l u t i o n r a t e s . E x p e r i m e n t s w e r e c o n d u c t e d on d a y s 2,7,8,11,13 a n d 16. 1 2 1 0 O 00 U J o 6 0-4 2 0 0 -A-A. A — A .A-A A - A o. •o- -o-•o. -o-o. ~o-o-o' -O-o 2 4 6 10 12 4 6 T I M E ( d ) 155 RESULTS Nitrate-replete cultures The s p e c i f i c g r o w t h r a t e u o f d u p l i c a t e N C > 3 - - r e p l e t e c u l t u r e s o f Micromonas pusilla a v e r a g e d 1.08 ± 0.013 d - ^ a n d 1.13 ± 0 . 0 2 2 d ~ l b a s e d on c e l l c o n c e n t r a t i o n a n d t o t a l c e l l v o l u m e , r e s p e c t i v e l y , o v e r t h e 22 h m o n i t o r i n g p e r i o d . T h i s r a t e i s c o n s i s t e n t w i t h t h e a v e r a g e g r o w t h r a t e , c a l c u l a t e d b y t h e i n c r e a s e i n in vivo f l u o r e s c e n c e , m e a s u r e d p r i o r t o e x p e r i m e n t a t i o n ( 1 . 03 d-"^) a n d i s n o t d i f f e r e n t f r o m t h e g r o w t h r a t e m e a s u r e d u n d e r c o n t i n u o u s l i g h t ( 1 . 1 1 d ~ ^ , C h a p t e r 3 ) . T o t a l c e l l v o l u m e (L ; L c e l l v o l u m e p e r L c u l t u r e medium) was d i v i d e d b y t h e c e l l c o n c e n t r a t i o n ( c e l l s •L--'-; F i g . 4.2A) 3 — 1 t o e s t i m a t e mean c e l l v o l u m e (um - c e l l ; F i g . 4.2C) a t e a c h 2 h i n t e r v a l d u r i n g t h e l i g h t - d a r k i l l u m i n a t i o n c y c l e . C e l l d i v i s i o n o f M. pusilla o c c u r r e d d u r i n g t h e l a t e l i g h t p e r i o d (>10 h l i g h t ) a n d p r i m a r i l y t h r o u g h o u t t h e d a r k p e r i o d a n d r e s u l t e d i n a c a . t w o - f o l d i n c r e a s e i n c e l l c o n c e n t r a t i o n ( F i g . 4 . 2 A ) . The i n c r e a s e i n c e l l c o n c e n t r a t i o n b y c e l l d i v i s i o n was a c c o m p a n i e d b y a r e d u c t i o n i n c e l l s i z e a n d r e s u l t e d i n d e c r e a s e d mean c e l l v o l u m e d u r i n g t h e d a r k p e r i o d ( F i g . 4 . 2 C ) . The mean c e l l v o l u m e o f d u p l i c a t e N C ^ - r e p l e t e c u l t u r e s g rown on a 14:10 L:D c y c l e was 1.96 ± 0.02 um^; mean c e l l v o l u m e o f M. pusilla i n c r e a s e d 86% d u r i n g t h e l i g h t 3 3 p e r i o d f r o m 1.35 L m r t o 2.57 um a t t h e o n s e t o f t h e d a r k p e r i o d . N i t r a t e u p t a k e a nd i n t r a c e l l u l a r p o o l a c c u m u l a t i o n showed m a r k e d d i e l v a r i a t i o n s i n t h e e x p o n e n t i a l l y g r o w i n g c u l t u r e s , 156 Figure 4.2. C e l l concentration (A), growth rate (B) and mean c e l l volume (C) versus elapsed time since l i g h t s on i n duplicate ((},•) batch cultures of Micromonas pusilla grown on a 14h:10h L:D i l l u m i n a t i o n cycle. Dashed l i n e i n d i c a t e s onset of dark period denoted by dark bar. Growth rate p l o t t e d against average time between sampling. < or UJ o o o _j UJ o 6 . 0 0 0 5 . 0 0 0 4 . 0 0 0 3 . 0 0 0 2 . 0 0 0 1 1.000 0 . 0 0 0 - • i • 1 • • r — i 1 r i • • A • -^ 8 - 8 = 8 ^ ® j i i i i i i i i i i o UJ r -< or x o tr UJ 2 .50 Z5 _ l o 2 . 00 > 1 1.50 1 UJ o 1.00 < 0 . 50 UJ 0 . 00 I I / ro £ 8 10 12 14 16 18 2 0 2 2 2 4 TIME (h) 157 a l t h o u g h t h e r e was c o n s i d e r a b l e v a r i a b i l i t y i n i n t e r n a l NO3 -c o n c e n t r a t i o n b e t w e e n r e p l i c a t e c u l t u r e s . S p e c i f i c NO3 -u p t a k e r a t e s a v e r a g e d 0.0360 ± 0.0010 h - 1 d u r i n g t h e 14 h l i g h t p e r i o d a n d d e c r e a s e d t o 0.0168 ± 0.0028 h - 1 d u r i n g t h e d a r k ( F i g . 4 . 3 B ) . R a t e s o f NG^ - u p t a k e n o r m a l i z e d t o t o t a l c e l l v o l u m e showed t h e same d i e l v a r i a t i o n a s N s p e c i f i c r a t e s ; d a y t i m e v a l u e s w e r e c a . 2 - f o l d g r e a t e r t h a n n i g h t t i m e v a l u e s . The i n t e r n a l c o n c e n t r a t i o n s o f NO3 - (mg-at N«litre c e l l volume -"' -; F i g . 4.3C) w e r e g r e a t e s t a t t h e b e g i n n i n g o f t h e l i g h t p e r i o d i n p a r a l l e l w i t h t h e most a c t i v e p h a s e o f u p t a k e a n d d e c r e a s e d t h r o u g h o u t t h e r e m a i n d e r o f t h e l i g h t p e r i o d . The l o w e s t i n t e r n a l n i t r a t e c o n c e n t r a t i o n s w e r e d u r i n g t h e f i r s t 4 h o f d a r k n e s s a n d s u b s e q u e n t l y i n c r e a s e d m a r k e d l y ( c a . 7 - f o l d ) i n one c u l t u r e a n d o n l y s l i g h t l y i n t h e o t h e r b e f o r e t h e commencement o f t h e l i g h t p e r i o d . N i t r a t e - l i m i t e d c u l t u r e s C e l l d i v i s i o n ( c y t o k i n e s i s ) showed a s t r o n g d i e l p e r i o d i c i t y i n t h e N C ^ - l i m i t e d c y c l o s t a t c u l t u r e s o f M. pusilla ( F i g . 4 . 4 ) . I n c o n t i n u o u s c u l t u r e t h e r a t e o f c h a n g e i n c e l l c o n c e n t r a t i o n s w i t h t i m e ( d x / d t ) i s g i v e n b y : d l n x / d t = u{t) - D wh e r e u(t) i s t h e i n s t a n t a n e o u s p o p u l a t i o n g r o w t h r a t e a n d D i s t h e d i l u t i o n r a t e o f t h e c u l t u r e , w h i c h i s e x p r e s s e d i n t h e same u n i t s o f t i m e a s u(t). An i n c r e a s e i n c e l l numbers w i t h t i m e d u r i n g t h e 14h:10h L:D p h o t o p e r i o d , i . e . a p o s i t i v e Figure 4.3. Dissolved nit r a t e concentration i n culture medium (A), s p e c i f i c n i t r a t e uptake rate (B), and i n t r a c e l l u l a r n i t r a t e concentration (C) of duplicate, batch cultures of Micromonas pusilla grown on a 14h:10h L:D illumination cycle. Dashed l i n e indicates onset of dark period denoted by dark bar. Nitrate concentrations plotted against elapsed time since l i g h t s on; nitra t e uptake plotted against average time between sampling. f -< cc I-z Ul o o o UJ < cc r— Ul < r— CL Ul f— < CC Ul • r-< CC r-cc < Ul o < cc a. a> E O > a> u a> o i e 4 6 8 10 12 14 16 18 2 0 22 2 4 TIME (h) F i g u r e 4.4. C e l l c o n c e n t r a t i o n s o f d u p l i c a t e n i t r a t e - l i m i t e d c y c l o s t a t s o f Micromonas pusilla g r o w n i n a 1 4 h : 1 0 h L:D c y c l e a t 0.74 d" 1 ( A ) , 0.48 d" 1 (B) , a n d 0.24 d" 1 (C) d i l u t i o n r a t e . C o n c e n t r a t i o n p l o t t e d a g a i n s t e l a p s e d t i m e s i n c e l i g h t s o n . D a s h e d l i n e i n d i c a t e s o n s e t o f d a r k p e r i o d d e n o t e d b y d a r k b a r . 10.0 -a . o l 6.0 -1 . 4.0 -_ l 2.0 -o • 0.0 -, < tr h-Ld O o o U J o 10.0 8.0 6.0 4.0 2.0 0.0 10.0 8.0 6.0 4.0 2.0 0.0 B - i 1 1 r -o - -o - • - — • 0 2 4 6 8 10 12 14 16 18 2 0 2 2 2 4 TIME (h) 160 d l n x / d t , i n d i c a t e s t h a t u(t) > D. Thus t h e t i m e i n t e r v a l d u r i n g w h i c h d l n x / d t i s b o t h p o s i t i v e a n d g r e a t e s t i s t h e p e r i o d o f maximum c e l l d i v i s i o n . F o r t h e c u l t u r e s g rown a t t h e d i l u t i o n r a t e s 0.73 a n d 0.75 d - ^ ( h e r e a f t e r r e f e r r e d t o a s 0.74 d--*-) maximum c e l l d i v i s i o n o c c u r r e d d u r i n g t h e m i d - d a r k p e r i o d (4-8 h a f t e r o n s e t o f d a r k n e s s ) a l t h o u g h l o w e r p o s i t i v e v a l u e s o f d l n x / d t o c c u r r e d d u r i n g l a t e l i g h t (10-14 h a f t e r l i g h t s on) a n d 2-4 h a f t e r d a r k n e s s ( F i g . 4 . 5 ) . The p e r i o d o f maximum c e l l d i v i s i o n o c c u r r e d e a r l i e r i n t h e d a r k p e r i o d (1-6 h a f t e r o n s e t o f d a r k n e s s ) f o r t h e c o n t i n u o u s c u l t u r e s g rown a t 0.4 8 d-"*" d i l u t i o n r a t e , a l t h o u g h a g a i n t h e r e w e r e l o w e r p o s i t i v e v a l u e s o f d l n x / d t d u r i n g t h e l a t e l i g h t ( 10-12 h a f t e r l i g h t s o n ) . I n t h e s l o w e s t g r o w i n g c o n t i n u o u s c u l t u r e s (0.24 d - ^ ) t h e p e r i o d o f maximum c e l l d i v i s i o n o c c u r r e d 6-8 h a f t e r t h e o n s e t o f d a r k n e s s ; i n one c u l t u r e l o w e r p o s i t i v e v a l u e s o f d l n / d t w e r e o b s e r v e d t h r o u g h o u t t h e d a r k p e r i o d , i n c l u d i n g t h e f i r s t 2 h o f d a r k n e s s , w h i l e i n t h e r e p l i c a t e c u l t u r e p o s i t i v e v a l u e s w e r e n o t o b s e r v e d u n t i l a f t e r 2 h o f d a r k n e s s a n d c o n t i n u e d t h r o u g h o u t t h e n i g h t a n d t h e f i r s t h o u r s o f l i g h t . Mean c e l l v o l u m e (um -cell) showed m a r k e d d i e l v a r i a t i o n i n a l l c y c l o s t a t c u l t u r e s ( F i g . 4 . 6 ) . I n t h e c u l t u r e s g rown a t a d i l u t i o n r a t e o f 0.74 t h e c e l l v o l u m e i n c r e a s e d by an a v e r a g e o f 8 5 % d u r i n g t h e l i g h t p e r i o d (0800 h - 2200 h) f r o m 1.12-1.20 um a t t h e b e g i n n i n g o f t h e l i g h t p e r i o d t o a 3 maximum s i z e o f 2.08 um , a c h i e v e d a f t e r 6 h o f l i g h t , a n d t h e n d e c r e a s e d d u r i n g t h e n i g h t (2200 h t o 0800 h) t o t h e 161 F i g u r e 4.5. C e l l d i v i s i o n r a t e o f d u p l i c a t e n i t r a t e - l i m i t e d c y c l o s t a t s o f Micromonas pusilla g r o w n i n a 14h:10h L:D c y c l e a t 0.74 d _ 1 ( A ) , 0.48 d" 1 ( B ) , a n d 0.24 d _ 1 (C) d i l u t i o n r a t e s . C e l l d i v i s i o n p l o t t e d a g a i n s t a v e r a g e t i m e b e t w e e n s a m p l i n g , d a s h e d h o r i z o n t a l l i n e i n d i c a t e s d i l u t i o n r a t e i n h - 1 . D a s h e d v e r t i c a l l i n e i n d i c a t e s o n s e t o f d a r k p e r i o d d e n o t e d by d a r k b a r , 2.50 2.00 .50 1.00 0.50 0.00 UJ - 0 . 5 0 i r— 1 1 1 1 1 . A : • A O A / \ A A / I A I \/ \ o 1 1 1 1 < cr o c o > UJ O 2.00 1.50 1.00 0.50 0.00 -0.50 —1 1— ' 1 1 1 1 . B * a A oV A. v/\//V 1 1 1 1 1 1 L 1 \»^ o 8 10 12 14 16 18 20 22 24 T I M E ( h ) F i g u r e 4.6. Mean c e l l v o l u m e o f d u p l i c a t e n i t r a t e - l i m i t e d c y c l o s t a t s o f Micromonas pusilla g r o w n i n a 1 4 h : 1 0 h L:D c y c l a t 0.74 d _ 1 ( A ) , 0.48 d _ 1 ( B ) , a n d 0.24 d _ 1 (C) d i l u t i o n r a t e s p l o t t e d a g a i n s t e l a p s e d t i m e s i n c e l i g h t s o n . D a s h e d l i n e i n d i c a t e s o n s e t o f d a r k p e r i o d d e n o t e d b y d a r k b a r . LU 0.50 -o < U J 0.00 2.00 1.50 _i i i i i 1.00 - l 1 r 0.50 -0.00 1 ' 1 1 ' 1 ' ' 1 ' ' 1 0 2 4 6 8 10 12 14 16 18 20 22 24 TIME (h) 163 o r i g i n a l minimum s i z e by the s t a r t of the l i g h t p e r i o d . T h i s d i u r n a l enlargement and n o c t u r n a l r e d u c t i o n i n c e l l u l a r volume was a l s o observed i n the 0.48 d-"^ and 0.24 d-"*" grown c u l t u r e s where mean c e l l u l a r volume i n c r e a s e d an average of 67% (1.11-1.15 um3 t o 1.83-1.92 um3) and 26% (1.08 t o 1.38 um3) , r e s p e c t i v e l y , over the course of the i l l u m i n a t i o n c y c l e ( F i g . 4.6). The mean ( d a i l y ) c e l l volume of d u p l i c a t e c u l t u r e s was 1.69 ± 0.01, 1.50 ± 0.03 and 1.23 ± 0.01 /jm3 grown at d i l u t i o n r a t e s of 0.74, 0.48 and 0.24 d~^, r e s p e c t i v e l y . C e l l u l a r n i t r o g e n , estimated from the mass balance of e x t e r n a l NC^ - + NC^ - c o n c e n t r a t i o n i n the r e a c t o r chamber and the NO^ - + NC^ -c o n c e n t r a t i o n of incoming medium, showed t h i s rhythmic d i e l v a r i a t i o n of s t e a d i l y i n c r e a s i n g v a l u e s d u r i n g the l i g h t p e r i o d and r e d u c t i o n d u r i n g the n i g h t f o r a l l the continuous c u l t u r e s . The 0.74 d-"*" d i l u t i o n r a t e r e s u l t e d i n d i e l v a r i a t i o n s i n e x t e r n a l N O 3 - c o n c e n t r a t i o n i n the c u l t u r e medium, begin n i n g w i t h the r a p i d r e d u c t i o n d u r i n g the f i r s t h a l f of the l i g h t p e r i o d t o minimal c o n c e n t r a t i o n s (0.2-1.0 uq-at N*L -^ N O 3 - ) f o r the second h a l f of the l i g h t p e r i o d ( F i g . 4.7A). With the onset of darkness the e x t e r n a l c o n c e n t r a t i o n immediately s t a r t e d t o s t e a d i l y i n c r e a s e t o a " s u n r i s e " maximum (ca. 7.0 jjg-at N'L-"*"). A s i m i l a r d i e l t r e n d was observed f o r N 0 2 ~ c o n c e n t r a t i o n s : a decrease d u r i n g the f i r s t h a l f of the l i g h t p e r i o d and subsequent steady i n c r e a s e d u r i n g the dark p e r i o d . The 0.48 d - ^ d i l u t i o n r a t e c u l t u r e s a l s o demonstrated d i e l v a r i a t i o n i n e x t e r n a l N O 3 - and NC^ - c o n c e n t r a t i o n b e g i n n i n g 1 w i t h r a p i d r e d u c t i o n d u r i n g t h e f i r s t 2 h o f l i g h t t o c o n c e n t r a t i o n s a t o r n e a r t h e l e v e l s o f d e t e c t i o n ( N O 3 - : 0.01 N O 2 - 0.01 ug-at N-L -''-). W i t h t h e commencement o f d a r k n e s s , e x t e r n a l N O 3 - c o n c e n t r a t i o n s i n c r e a s e d s t e a d i l y t o a " s u n r i s e maximum ( c a . 1.7 ug-at N'L-"'-) w h i l e N O 2 - c o n c e n t r a t i o n s r e a c h e d a m a x i m a l c o n c e n t r a t i o n (0.30 uq-at N'L-"'") 2 h p r i o r t o l i g h t s on ( F i g . 4 . 7 B ) . No d i e l v a r i a t i o n i n t h e e x t e r n a l c o n c e n t r a t i o n o f NC>3~ o r NC^ - was o b s e r v e d f o r e i t h e r o f t h e 0.24 d - ^ d i l u t i o n r a t e c u l t u r e s a n d a m b i e n t c o n c e n t r a t i o n s r e m a i n e d a t o r n e a r t h e l i m i t s o f a n a l y t i c a l d e t e c t i o n t h r o u g h o u t t h e l i g h t - d a r k c y c l e ( F i g . 4 . 7 C ) . R a t e s o f n i t r a t e u p t a k e w e r e c a l c u l a t e d u s i n g t h e e q u a t i o n s o f C a p e r o n a n d Z i e m a n n ( 1 9 7 6 ) ; s p e c i f i c r a t e s ( n o r m a l i z e d t o a v e r a g e PON d u r i n g t i m e i n t e r v a l ) showed t h e same d i e l t r e n d s a s r a t e s n o r m a l i z e d t o t o t a l c e l l u l a r v o l u m e o f c u l t u r e , a n d a r e p l o t t e d a g a i n s t t i m e i n F i g u r e 4.8. S p e c i f i c n i t r a t e u p t a k e r a t e s ( h ~ ^ ) o f t h e l i g h t p e r i o d w e r e g r e a t e r t h a n t h o s e o f t h e d a r k p e r i o d a n d d e m o n s t r a t e d b o t h n o c t u r n a l a n d d i u r n a l v a r i a t i o n i n u p t a k e v e l o c i t i e s f o r t h e 0.74 d - 1 a n d 0.48 d - 1 d i l u t i o n r a t e c u l t u r e s . I n t h e 0.74 d~ (0.030 9 h - 1 ) d i l u t i o n r a t e c u l t u r e s , m a x i m a l u p t a k e r a t e s o f 0.0530 ± 0.0007 h - 1 w e r e o b t a i n e d 4-6 h a f t e r t h e l i g h t s came on a n d s t e a d i l y d e c l i n e d t o h a l f t h i s v a l u e by t h e e n d o f t h e l i g h t p e r i o d . An a d d i t i o n a l 5 0 % r e d u c t i o n i n u p t a k e r a t e o c c u r r e d d u r i n g t h e f i r s t 2 h o f d a r k n e s s w i t h r a t e s s u b s e q u e n t l y i n c r e a s i n g d u r i n g t h e r e m a i n i n g d a r k p e r i o d t o m a x i m a l d a r k u p t a k e r a t e s o f 0.0183 ± 0.0003 h - ^ d u r i n g 20-22 165 Figure 4.7. Dissolved nitrate ( 0 , « ) and n i t r i t e (A,A) concentrations i n the medium of duplicate n i t r a t e - l i m i t e d cyclostats of Micromonas pusilla grown i n a 14h:10h L:D cycle at 0.74 d"1 (A), 0.48 d"1 (B), and 0.24 d _ 1 (C) d i l u t i o n rates plotted against elapsed time since l i g h t s on. Dashed l i n e indicates onset of dark period denoted by dark bar. O o I cn =1. V 7 . 0 < - 6.0 - 5.0 1 _I - 4.0 • - 3.0 - 2.0 -at - 1.0 cn L \ J 0 . 0 < cr LJJ o o o 3.0 2.0 1.0 0.0 1 1 1 1 1 1 B -T -<>\ -\\ X 3.0 2.0 1.0 0.0 < CT LU o o o I-< cr 4)0.0 LU cr I-8 10 12 14 16 18 2 0 22 2 4 TIME (h) 166 Figure 4.8. S p e c i f i c n i t r a t e uptake rates of duplicate n i t r a t e - l i m i t e d c y c l o s t a t s of Micromonas pusilla grown i n a 14h:10h L:D cycle at A: 0.74 d _ 1 (0.031 h" 1); B: 0.48 d"1 (0.020h _ 1); and C: 0.24 d"1 (O.OlOh-1) d i l u t i o n rates. Rates p l o t t e d against average time between sampling. Dashed l i n e i n d i c a t e s onset of dark period denoted by dark bar. U J I— < cr LU < l -Q_ Z> o o LU Q_ LU (3 O cr 0 . 0 5 0 0 . 0 4 0 0 . 0 3 0 0 . 0 2 0 0 . 0 1 0 0 . 0 0 0 0 . 0 5 0 0 . 0 4 0 0 . 0 3 0 0 . 0 2 0 0 . 0 1 0 0 . 0 0 0 0 . 0 5 0 0 . 0 4 0 0 . 0 3 0 0 . 0 2 0 0 . 0 0 0 1 1 1 1 1 1 * i i i i i A 1 1 1 1 i i i i i i o 1 1 1 1 1 1 B 1 I 1 1 1 1 t 1 1 1 1 1 -. c 0 2 4 - 6 8 10 12 14 16 18 2 0 22 2 4 T I M E ( h ) 167 h i n t e r n a l . A v e r y s i m i l a r p a t t e r n o f d i u r n a l d e c l i n e a n d n o c t u r n a l i n c r e a s e o c c u r r e d i n t h e 0.48 d - 1 ( 0 . 0 2 0 0 h - 1 ) d i l u t i o n r a t e c u l t u r e s . The m a x i m a l l i g h t u p t a k e r a t e o f ( 0 . 0 3 3 1 ± 0.0040 h - 1 ) o c c u r r e d e a r l i e r ( d u r i n g t h e f i r s t 2 h o f l i g h t ) a n d d e c l i n e d o v e r t h e n e x t 2-4 h t o a r e l a t i v e l y c o n s t a n t l i g h t v a l u e o f 0.202 ± 0.0001 h-"*" o v e r t h e r e m a i n d e r o f t h e l i g h t p e r i o d . D u r i n g t h e f i r s t 2 h o f d a r k n e s s t h e u p t a k e r a t e d e c r e a s e d b y an a v e r a g e o f 2 2 % a n d t h e n s t e a d i l y i n c r e a s e d d u r i n g t h e r e m a i n d e r o f t h e d a r k p e r i o d r e a c h i n g a maximum d a r k u p t a k e r a t e ( 0 .0195 ± 0.0013 h~^) d u r i n g t h e 20-22 h i n t e r v a l . The u p t a k e r a t e s o f t h e 0.24 d - 1 (0.0100 h - 1 ) d i l u t i o n c u l t u r e s w e r e c o n s t a n t t h r o u g h o u t t h e l i g h t - d a r k c y c l e a n d a v e r a g e d 0.0100 ± 0.0001 h - ^ f o r d u p l i c a t e c u l t u r e s . The a v e r a g e l i g h t a n d d a r k u p t a k e r a t e s f o r t h e c y c l o s t a t c u l t u r e s a r e p r e s e n t e d i n T a b l e 4.1. The r a t i o o f d a r k : l i g h t u p t a k e r a t e i n c r e a s e d f r o m a n a v e r a g e o f 0.4 0 f o r t h e f a s t e s t g r o w i n g c u l t u r e s (D=0.74 d - 1 ) t o 0.78 f o r t h e 0.40 d - 1 d i l u t i o n r a t e c u l t u r e s t o a v a l u e o f u n i t y f o r t h e 0.24 d - ^ c u l t u r e s . I n t e r n a l p o o l s o f N O 3 - + N O 2 - i n c r e a s e d d u r i n g t h e l a s t p a r t o f t h e n i g h t ( t = 20-24 h) i n a l l t h e c y c l o s t a t c u l t u r e s a t t a i n i n g m a x i m a l s i z e a t t h e b e g i n n i n g o f t h e l i g h t p e r i o d ( t = 0-2 h; F i g . 4 . 9 ) . A l t h o u g h t h e r e was c o n s i d e r a b l e v a r i a b i l i t y i n t h e m a g n i t u d e o f p o o l s i z e s b e t w e e n r e p l i c a t e c u l t u r e s b o t h t h e 0.74 d -"^ a n d 0.48 d -^" d i l u t i o n r a t e c u l t u r e s , w h i c h a l s o d e m o n s t r a t e d d i e l v a r i a b i l i t y i n NO^ -u p t a k e , showed a c a . 2 - f o l d i n c r e a s e i n N O 3 - + NC>2~ p o o l s i z e T a b l e 4.1 Mean l i g h t and dark s p e c i f i c n i t r a t e uptake r a t e s (h~ ) and t h e i r r a t i o s ( d a r k : l i g h t ) f o r Micromonas pusilla grown on a 14:10 l i g h t - d a r k c y c l e i n b a t c h (*) and c y c l o s t a t c u l t u r e s . The s t a n d a r d d e v i a t i o n s o f s e p a r a t e (5-7) r a t e measurements d u r i n g the l i g h t or dark p e r i o d a r e g i v e n i n p a r e n t h e s e s . NO3- uptake r a t e ( h - 1 ) Growth r a t e ( h - 1 ) L i g h t Dark D a r k : L i g h t 0.0470 * 0.0353 (0.0071) 0.0187 (0.0046) 0.53 0.0449 * 0.0366 (0.0053) 0.0149 (0.0042) 0.41 0.0314 0.0400 (0.0092) 0.0161 (0.0020) 0.39 0.0304 0.0409 (0.0106) 0.0164 (0.0020) 0.41 0.0200 0.0226 (0.0060) 0.0171 (0.0020) 0.76 0.0200 0.0216 (0.0039) 0.0175 (0.0008) 0.81 0.0100 0.0100 (0.0002) 0.0100 (0.0001) 1.00 0.0100 0.0100 (0.0001) 0.0099 (0.0004) 0.99 J. \J -/ Figure 4.9. I n t r a c e l l u l a r n i t r a t e concentrations of duplicate n i t r a t e - l i m i t e d c y c l o s t a t s of Micromonas pusilla grown i n a 14h:10h L:D cycle at 0.74 d"1 (A), 0.48 d"1 (B), and 0.24 d"1 (C) d i l u t i o n rates p l o t t e d against elapsed time since l i g h t s on. Dashed l i n e i ndicates onset of dark period denoted by dark bar. 0.0 1 ' 1 1 1 1 1 • 1 1 1 ' 1 0 2 4 6 8 10 • 12 1 4 ' 16 18 20 22 2 4 TIME (h) 170 a f t e r the f i r s t 2 h of darkness i n p a r a l l e l w i t h the r a p i d r e d u c t i o n i n NC>3~ uptake d u r i n g t h i s time. Potential nitrogen uptake rates At the beginning, middle, and end of the 14 h l i g h t and 10 h dark p e r i o d s s a t u r a t e d uptake r a t e s of N O 3 - , N H 4 + and urea were determined, from 2 h i n c u b a t i o n s i n the dark or l i g h t , f o r samples c o l l e c t e d from the 0.74, 0.49, and 0.24 d i l u t i o n r a t e c y c l o s t a t c u l t u r e s . The i n i t i a l c o n d i t i o n s of the c u l t u r e s used f o r these batch s t y l e i n c u b a t i o n s are presented i n Table 4.2. D i e l v a r i a b i l i t y i n 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 , mean c e l l volume and c e l l u l a r N quota of the th r e e c u l t u r e s were s i m i l a r t o the c y c l i c p a t t e r n s d i s c u s s e d e a r l i e r f o r the r e p l i c a t e c y c l o s t a t s sampled a t 2 h i n t e r v a l s over the 14 h l i g h t : 1 0 h dark i l l u m i n a t i o n c y c l e . In onl y the f a s t e s t grown c u l t u r e (D = 0.74 d -^) was d i e l v a r i a t i o n i n e x t e r n a l N O 3 - + NC^ - c o n c e n t r a t i o n observed. The N s p e c i f i c uptake r a t e s of NH 4 +, N O 3 - and urea are p l o t t e d v e r s u s the mid-point of t h e i r i n c u b a t i o n p e r i o d s i n F i g u r e 4.10. Rates of ammonium uptake were c o n s i s t e n t l y 2-3 f o l d g r e a t e r than N03~ and urea uptake r a t e s which were s i m i l a r t o each o t h e r . I t should be noted t h a t the urea uptake r a t e s of the 0.74 d - ^ c u l t u r e , estimated u s i n g the "^N technique, may be s l i g h t l y underestimated due t o simultaneous uptake of u n l a b e l e d s u b s t r a t e ( C o l l o s , 1987; Lund, 1987). During 5 of the 6 sampling p e r i o d s , NG"3~ c o n c e n t r a t i o n s s a t u r a t i n g f o r uptake by M. pusilla (Chapter 3) were present i n the e x t e r n a l medium; the p o t e n t i a l uptake of u n l a b e l e d N O 3 - w i l l d i l u t e the T a b l e 4.2 Summary o f c y c l o s t a t c u l t u r e c o n d i t i o n s a t the b e g i n n i n g o f each experiment. Day C u l t u r e D i l u t i o n N 0 3 ~ + NO 2~ PON POC C e l l d e n s i t y Average c e l l C e l l Quota d e s c r i p t i o n r a t e Volume ( d _ 1 ) (^g-at N - L - 1 ) (pg-at C - L - 1 ) ( 1 0 9-L - 1) (M~i3) ( f g - a t N - c e l l - 1 ) 16 i n i t i a l l i g h t 2 m i d - l i g h t 11 end l i g h t 13 i n i t i a l dark 7 mid-dark 8 end dark 0.76 19 . 9 . 0. 3. 9. 11. 31.6 45.5 50.6 46.2 41.2 38.1 244 321 406 425 340 273 .56 .73 .84 .04 .86 .87 1.4 1.8 2.0 2.2 1.8 1.3 6.9 9.6 8.7 9.2 8.5 6.5 16 i n i t i a l l i g h t 2 m i d - l i g h t 11 end l i g h t 13 i n i t i a l dark 7 mid-dark 8 end dark 0.49 53. 49 . 52. 48. 51. 53. 386 419 429 513 468 399 10.14 6.98 7.90 7.65 8.13 9.52 1.1 1.5 1.6 1.8 1.5 1.1 5.2 7.1 6.6 6.4 6.3 5.6 16 i n i t i a l l i g h t 2 m i d - l i g h t 11 end l i g h t 13 i n i t i a l dark 7 mid-dark 8 end dark 0 .24 51.1 50.3 51.6 47 .1 53.8 51.9 411 443 416 468 470 429 9.86 9.68 8.55 8.70 8.69 9 .56 1.1 1.2 1.3 1.3 1.3 1.1 5.2 5.2 6.0 5.4 6.2 5.4 172 F i g u r e 4.10. Maximum s p e c i f i c u p t a k e r a t e s ( h - 1 ) o f n i t r a t e (A), u r e a (o), a n d ammonium (•) d e t e r m i n e d i n 2 h i n c u b a t i o n s o f s a m p l e s f r o m n i t r a t e - l i m i t e d c y c l o s t a t c u l t u r e s o f Micromonas pusilla (14h:10h L:D c y c l e ) grown a t 0.74 d" 1 ( A ) , 0.49 d" 1 ( B ) , and 0.24 d" 1 (C) d i l u t i o n r a t e s . S p e c i f i c r a t e s a r e p l o t t e d a g a i n s t a v e r a g e t i m e o f i n c u b a t i o n p e r i o d . Dashed l i n e i n d i c a t e s o n s e t o f d a r k p e r i o d d e n o t e d by d a r k b a r . 0.0 1 1 1 1 1 1 L TIME (h) 173 i s o t o p i c r a t i o i n the p a r t i c u l a t e matter and thus decrease the uptake r a t e s . In N03~-replete c u l t u r e s of M. pusilla, grown under continuous l i g h t , a s a t u r a t i n g a d d i t i o n of urea r e s u l t e d i n a 28% decrease i n N O 3 - uptake (Chapter 3), however, the degree of urea s u p p r e s s i o n of N O 3 - uptake i n N - l i m i t e d chemostat c u l t u r e s i s unknown. I t i s u n l i k e l y t h a t N H 4 + uptake r a t e s were a f f e c t e d by the presence of u n l a b e l e d N O 3 -i n the medium. The presence of ammonium, at c o n c e n t r a t i o n s as low as 1.0 pq-at N*L -^, completely i n h i b i t e d NC>3~ uptake i n N-r e p l e t e M. pusilla (Chapter 3) thus, the p o s s i b i l i t y of simultaneous uptake of l a b e l l e d N H 4 + and u n l a b e l e d N O 3 - i s u n l i k e l y . A marked d i e l v a r i a b i l i t y i n the uptake of a l l t h r e e N s u b s t r a t e s was observed f o r the samples c o l l e c t e d from the 0.74 d~^ d i l u t i o n r a t e c u l t u r e , average (n = 3) p o t e n t i a l dark uptake r a t e s (V^) of urea, N H 4 + and N O 3 - were 65, 52, and 40%, r e s p e c t i v e l y , of the mean l i g h t uptake r a t e s (Table 4.3). In the more N - d e f i c i e n t c u l t u r e s (0.49 and 0.24 d - ^ d i l u t i o n r a t e s ) dark uptake r a t e s were g e n e r a l l y v ery s i m i l a r t o l i g h t uptake r a t e s ( V L T ) . In the 0.49 d~^ d i l u t i o n r a t e c u l t u r e s , V^j v a l u e s of urea, NH^ + and N O 3 - averaged 106, 83, and 72%, r e s p e c t i v e l y of the mean l i g h t v a l u e s and i n the 0.24 d - ^ d i l u t i o n r a t e c u l t u r e s v a l u e s were 93, 71, and 80%, r e s p e c t i v e l y of urea, N H 4 + and NC>3~ p o t e n t i a l l i g h t uptake r a t e s . Although i n both the slower growing c y c l o s t a t c u l t u r e s the mean l i g h t and dark r a t e s were of a comparable magnitude, a d i u r n a l v a r i a t i o n i n V-^ rp was apparent f o r N O 3 - and urea; T a b l e 4.3 N i t r o g e n s p e c i f i c uptake r a t e s (h~ ), determined o v e r 2 h i n l i g h t and darkness, and t h e t h e i r r a t i o s (D/L) f o r Micromonas pusilla p r e v i o u s l y grown a t 0.24, 0.49, 0.74 d - 1 i n N 0 3 ~ - l i m i t e d c y c l o s t a t c u l t u r e s on a 14 h l i g h t : 1 0 h dark i l l u m i n a t i o n c y c l e ( l i g h t s on: 0800 h, l i g h t s o f f : 2200 h) . S t a r t i n g t i m e of I n c u b a t i o n Day:time (h) 0.74 d 1 N s p e c i f i c uptake 0.49 d" r a t e ( h - 1 ) 1 0.24 d _ 1 L D D/L L D D/L L D D/L Urea 16 0845 0.0140 0.0019 0.13 0.0239 0.0058 0.24 0 .0150 0.0076 0.51 2 : 1425 0.0099 0.0030 0.30 0.0208 0.0025 0.12 0 .0151 0.0054 0.36 11 : 2007 0.0213 0.0029 0.14 0.0191 0.0103 0.54 0 .0140 0.0057 0.41 13 : 2200 0.0155 0.0055 0.35 0.0314 0.0234 0.75 0 .0242 0.0129 0.53 7 0210 0.0137 0.0104 0.75 0.0209 0.0203 0.97 0 .0142 0.0113 0.80 8 0615 0.0160 0.0136 0.85 0.0313 0.0232 0.74 0 .0263 0.0168 0.64 Ammonium 16 0847 0.0520 0.0133 0.26 0.0584 0.0247 0.42 0 .0516 0.0277 0.54 2 1435 0.0682 0.0142 0.21 0.0554 0.0181 0.33 0 .0541 0.0234 0.43 11 2010 0.0658 0.0280 0.43 0.0566 0.0329 0.58 0 .0517 0.0298 0.58 13 2202 0.0555 0.0291 0.53 0.0718 0.0542 0.75 0 . 0550 0.0348 0.63 7 0212 0.0394 0.0291 0.74 0.0534 0.0327 0.61 0 .0369 0.0306 0.83 8 0616 0.0668 0.0390 0.58 0.0735 0.0546 0.74 0 .0616 0.0455 0.74 N i t r a t e 16 0851 0.0119 0.0037 0.31 0.0355 0.0028 0.08 0 .0190 0.0056 0.30 2 1445 0.0223 0.0036 0.16 0.0219 0.0018 0.08 0 .0183 0.0019 0.10 11 2014 0.0356 0.0068 0.19 0.0300 0.0110 0.37 0 . 0096 0.0076 0.79 13 2203 0.0214 0.0067 0.32 0.0456 0.0234 0.51 0 .0300 0.0103 0.34 7 0215 0.0160 0.0105 0.66 0.0292 0.0194 0.66 0 .0212 0.0110 0.52 8 0618 0.0270 0.0109 0.40 0.0490 0.0199 0.41 0 .0383 0.0162 0.42 175 m a x i m a l r a t e s w e r e o b s e r v e d i n t h e s a m p l e s c o l l e c t e d a t t h e b e g i n n i n g o f t h e l i g h t p e r i o d , w h i l e NH4"** p o t e n t i a l u p t a k e r a t e s w e r e r e l a t i v e l y c o n s t a n t t h r o u g h o u t t h e l i g h t p e r i o d . D a r k u p t a k e r a t e s w e r e c o n s i s t e n t l y l o w e r f o r s a m p l e s c o l l e c t e d d u r i n g t h e n i g h t , h o w e v e r l i g h t u p t a k e r a t e s f r o m b o t h l i g h t a n d d a r k p e r i o d s w e r e g e n e r a l l y e q u i t a b l e . Influence of light on nitrate and ammonium uptake rates N i t r o g e n s p e c i f i c u p t a k e r a t e s , d e t e r m i n e d o v e r 2 h, w e r e p l o t t e d v e r s u s t h e PPFD e x p e r i e n c e d b y t h e c e l l s p r e v i o u s l y g r own i n 0.24, 0.52 a n d 0.77 d - ^ d i l u t i o n r a t e c y c l o s t a t s ( F i g . 4 . 1 1 ) . D a r k u p t a k e (V-p), t h e h a l f - s a t u r a t i o n c o n s t a n t ( K L T ' ) , a n d maximum n i t r o g e n u p t a k e v e l o c i t y V ' m a x f o r l i g h t d e p e n d e n t N H 4 + a n d N O 3 - u p t a k e a r e s u m m a r i z e d i n T a b l e 4.4. I t i s i m p o r t a n t t o n o t e t h a t t h e s e M i c h a e l i s - M e n t e n p a r a m e t e r s a r e d e r i v e d f r o m d a t a o b t a i n e d f r o m t h e h y p e r b o l i c ( o r l i g h t ) p o r t i o n o f t h e PPFD r e s p o n s e c u r v e , t h u s K L T v a l u e s r e p o r t e d a r e t h e PPFD a t w h i c h 0.5 V ' m a x o c c u r s w h e r e V ' m a x i s t h e m a x i m a l v e l o c i t y i n t h e l i g h t . The h a l f - s a t u r a t i o n c o n s t a n t (K-^rp' ) w h i c h i s t h e PPFD when o n e - h a l f t h e t o t a l maximum N-u p t a k e o f t h e c e l l s i s a c h i e v e d , ( V ' m a x + V D ) / 2 ) , c a n be c a l c u l a t e d b y s i m p l e r e a r r a n g e m e n t o f t h e M i c h a e l i s - M e n t e n e q u a t i o n ( s e e C h a p t e r 2 f o r d e t a i l s ) . A n o t h e r s i m i l a r h a l f -s a t u r a t i o n c o n s t a n t ( K - ^ T " ) c a n be c a l c u l a t e d f o r h a l f t h e t o t a l N - u p t a k e a c h i e v e d a t t h e g r o w t h PPFD (120 juE m ~ 2 s _ 1 ) . T h e s e t w o l a t t e r h a l f - s a t u r a t i o n c o n s t a n t s i n c l u d e t h e s u b s t a n t i a l d a r k u p t a k e v e l o c i t i e s w h i c h a p p e a r t o v a r y i n v e r s e l y w i t h d i l u t i o n r a t e ( T a b l e 4 . 5 ) . The d a r k N O 3 -Figure 4.11. Nitrogen s p e c i f i c uptake rates, determined over 2 h, a f t e r saturating enrichment of 1 5NH 4 + (•) or 1 5N0 3 _ (O) to n i t r a t e - l i m i t e d c y c l o s t a t cultures of Micromonas pusilla (14h:10h L:D cycle) previously grown at 0.77 d _ 1 (A), 0.52 d _ 1 (B), and 0.24 d - 1 (C) d i l u t i o n rates. Uptake rates (h - 1) are plo t t e d against incident PPFD, curved pl o t s are f i t t e d d i r e c t l y to the Michaelis-Menten equation by computer programme (see text for d e t a i l s ) . 80.0 r o • o LU < cc < h-O O LU CL (J) 0 20 40 60 80 100 120 140 P P F D (pE-m-2-s-2) T a b l e 4.4 Parameters d e s c r i b i n g t h e c h a r a c t e r i s t i c s o f N s p e c i f i c uptake (h~ ), as a f u n c t i o n o f PPFD f o r c y c l o s t a t c u l t u r e s o f Micromonas pusilla ( F i g . 4.11). Dark uptake (V" D), maximum s p e c i f i c l i g h t uptake ( V ' m a x ) , t h e h a l f - s a t u r a t i o n c o n s t a n t ( K L T ) , and the s l o p e o f i n i t i a l p o r t i o n o f N uptake vs PPFD cu r v e (a = V ' m - , X / K T T ) • E s t i m a t e d s t a n d a r d e r r o r s o f parameters are g i v e n i n p a r e n t h e s e s . D i l u t i o n PON N i t r o g e n V D v'max K L T a r a t e cone. s u b s t r a t e ( d - 1 ) (AJg-at N - L - 1 ) ( h - 1 ) (uE m ~ 2 s _ 1 ) 0.77 0.52 0.24 28.0 53.5 47.8 NO-NH/ NO-NH" NO-NH\ 0. 0017 (0 .00160) 0 .0323 (0. 00203) 13 (3 .3) 2 .6 0. 0137 (0 .00255) 0 .0656 (0. 00411) 26 (5 .8) 2 .8 0. 0048 (0 .00155) 0 .0460 (0. 00198) 15 (2 •7) 3 .2 0. 0243 (0 .00370) 0 .0693 (0. 00496) 16 (4 •5) 4 .2 0. 0076 (0 .00134) 0 .0310 (0. 00164) 13 (2 •9) 2 .5 0. 0276 (0 .00126) 0 .0499 (0. 00173) 18 (2 •4) 2 .8 Table 4.5 I n d i c e s o f N uptake dependency on PPFD f o r c y c l o s t a t c u l t u r e s o f Micromonas pusilla: the r a t i o of dark t o l i g h t - s a t u r a t e d uptake r a t e ( V D : V L ) , the PPFD a t which h a l f the t o t a l N uptake o c c u r s ( K L T ' , K L T " ) * and the r a t i o o f N uptake a t 1% I t o N uptake at 100% I ( vi% : Vioo%** S a t u r a t e d PPFD and I are the growth PPFD (120 piE m ~ 2 s - 1 ) . D i l u t i o n r a t e N i t r o g e n VD : VL KLT' KLT" V 1 % : V ( d - 1 ) S u b s t r a t e 0.77 N0 3 0 . 05 11.3 9.6 0 .15 0.77 NH 4 + 0.20 15.4 11.3 0.25 0.52 N0 3- 0. 10 11.9 9.8 0.18 0.52 NH 4 + 0.29 7.9 7.3 0.34 0.24 NO3- 0.21 7.6 6.4 0.29 0.24 NH 4 + 0.39 5.1 3.5 0.43 * D e f i n i t i o n s g i v e n i n t e x t 179 u p t a k e r a t e i s 5, 10, a n d 2 1 % o f t h e t o t a l u p t a k e f o r t h e 0.77, 0.52, a n d 0.24 d ~ * d i l u t i o n r a t e c u l t u r e s , r e s p e c t i v e l y . D a r k N H 4 + u p t a k e r a t e s , c o n s i s t e n t l y g r e a t e r t h a n d a r k N O 3 -u p t a k e r a t e s , a r e 20, 29 a n d 39% o f t h e t o t a l ( l i g h t + d a r k ) u p t a k e f o r t h e same c u l t u r e s . The s l o p e (a) o f t h e i n i t i a l p o r t i o n o f t h e PPFD r e s p o n s e c u r v e , c a l c u l a t e d b y d i v i s i o n o f V m a x b v K L T ( H e a l e Y ' 1980; P a r s l o w e t a l . , 1985) a r e s i m i l a r f o r N H 4 + a n d N O 3 - u p t a k e b y t h e c e l l s f r o m t h e 0.77 a n d 0.24 d - ! d i l u t i o n r a t e c u l t u r e s , b u t s u b s t a n t i a l l y g r e a t e r f o r N H 4 + u p t a k e b y t h e 0.52 d -^" d i l u t i o n r a t e c u l t u r e . T h e s e r e s u l t s s u g g e s t s i m i l a r l i g h t u p t a k e r e s p o n s e o f b o t h N - s u b s t r a t e s t o i n c r e a s i n g s u b s a t u r a t i n g PPFDs by f a s t a n d s l o w g r o w i n g c u l t u r e s w h e r e a s t h e i n t e r m e d i a t e l y g r o w i n g c u l t u r e s a p p e a r m o s t c a p a b l e o f i n c r e a s i n g t h e i r u p t a k e a b i l i t y o f NC^ -, a n d p a r t i c u l a r l y NH^ +, i n r e s p o n s e t o i n c r e a s i n g , b u t s u b s a t u r a t i n g P P F D s . The e f f e c t s o f PPFD on t o t a l N u p t a k e ( l i g h t + d a r k ) c a n be e s t i m a t e d b y a c o m p a r i s o n o f u p t a k e a t 1 a n d 100% o f t h e g r o w t h i r r a d i a n c e ; l o w e r p e r c e n t a g e s r e p r e s e n t g r e a t e r PPFD d e p e n d e n c y ( T a b l e 4 . 5 ) . The e f f e c t o f PPFD on t o t a l N u p t a k e i n c r e a s e s w i t h i n c r e a s i n g d i l u t i o n r a t e , w h i c h s u g g e s t s t h a t i n c r e a s e d N l i m i t a t i o n l e s s e n s t h e l i g h t d e p e n d e n c y o f N O 3 -a n d N H 4 + by Micromonas pusilla. A l t h o u g h t h i s l i g h t e f f e c t was o b s e r v e d f o r b o t h N s u b s t r a t e s , N H 4 + , w h i c h was c o n s i s t e n t l y t a k e n up a t g r e a t e r r a t e s t h a n N O 3 - i n b o t h l i g h t a n d t h e d a r k , d e m o n s t r a t e d c a . 4 0 % l e s s d e p e n d e n c y on PPFD t h a n N 0 3 ~ . 180 DISCUSSION The c e l l d i v i s i o n c y c l e o f mo s t p h y t o p l a n k t o n c e l l s i s p a r t i a l l y s y n c h r o n i z e d o r p h a s e d b y t h e e n v i r o n m e n t a l l i g h t : d a r k c y c l e . E v i d e n c e o f p h a s i n g i n b o t h a l g a l c u l t u r e s a n d n a t u r a l c o m m u n i t i e s h a s shown t h a t , i n most s p e c i e s grown on l i g h t : d a r k c y c l e s , t h e i n s t a n t a n e o u s p o p u l a t i o n d i v i s i o n r a t e , u, v a r i e s w i t h a 24 h p e r i o d i c i t y a n d t h a t t h e t i m i n g o f d i v i s i o n d e p e n d s on b o t h e x p e r i m e n t a l c o n d i t i o n s a n d s p e c i e s ( s e e r e v i e w s b y S o u r n i a , 1974; C h i s h o l m e t a l . , 1 9 80; Sweeney, 1 9 8 3 ) . I n b o t h N - r e p l e t e ( b a t c h ) a n d N - l i m i t e d ( c y c l o s t a t ) c u l t u r e s o f Micromonas pusilla, grown i n a 14:10 L:D c y c l e , p a r t i a l p h a s i n g o f c e l l d i v i s i o n was e v i d e n t . Maximum d i v i s i o n r a t e s o c c u r r e d a t t h e e n d o f t h e l i g h t p e r i o d a n d t h e m i d d l e t o l a t e d a r k p e r i o d . W i t h i n c r e a s e d n i t r a t e l i m i t a t i o n ( i . e . d e c r e a s e d d i l u t i o n r a t e ) t h e i m p o r t a n c e o f t h e l i g h t d i v i s i o n a p p e a r e d t o d e c r e a s e a n d t h e t i m i n g o f t h e d a r k d i v i s i o n was s h i f t e d s l i g h t l y l a t e r i n t h e n i g h t i n t h e 0.24 d-"*" d i l u t i o n r a t e c u l t u r e s . N i t r o g e n a n d c a r b o n u p t a k e r a t e s a r e o f t e n p r e s e n t e d on a n o r m a l i z e d c e l l u l a r b a s i s , b u t a n y c o - o c c u r r e n c e o f s y n c h r o n o u s c e l l d i v i s i o n c a n s e r i o u s l y m o d i f y t h e a p p a r e n t p e r i o d i c i t y i n u p t a k e r a t e s . F o r t h i s r e a s o n t h e m a j o r i t y o f t h e r e s u l t s i n t h e p r e s e n t s t u d y h a v e b e e n n o r m a l i z e d t o t o t a l c e l l u l a r v o l u m e o r p a r t i c u l a t e N. The s y n c h r o n y o f c e l l d i v i s i o n was r e f l e c t e d i n t h e mean c e l l v o l u m e ( o r s i z e ) o f t h e M. pusilla c e l l s . C e l l v o l u m e s t e a d i l y i n c r e a s e d d u r i n g t h e l i g h t p e r i o d a t t a i n i n g m a x i m a l s i z e a t t h e o n s e t o f t h e d a r k p e r i o d a n d s u b s e q u e n t l y d e c l i n e d 181 s t e a d i l y throughout the n i g h t a t t a i n i n g minimal s i z e at the onset of the l i g h t p e r i o d . However, w i t h i n c r e a s i n g N O 3 -l i m i t a t i o n , the i n c r e a s e i n average c e l l volume d u r i n g the l i g h t p e r i o d was reduced; maximal c e l l volumes a t t a i n e d by the 0.24 d ~ l d i l u t i o n r a t e c u l t u r e s were onl y c a . 50% of the maximal s i z e a t t a i n e d by the N03~-replete batch c u l t u r e s . The mean ( d a i l y ) c e l l .volume of M. pusilla i n c r e a s e d s i g n i f i c a n t l y (r = 0.99, P ^ 0.01) wit h i n c r e a s i n g s t e a d y - s t a t e d i l u t i o n r a t e ( i n c r e a s i n g u and improved N O 3 - s u p p l y ) . S i m i l a r r e d u c t i o n i n c e l l volume with i n c r e a s i n g n u t r i e n t l i m i t a t i o n has been r e p o r t e d f o r P - l i m i t e d (Burmaster, 1979) and NH 4 +-l i m i t e d chemostat c u l t u r e s of Pavlova lutheri (Caperon and Meyer, 1972), and Chaetoceros debilis, Skeletonema costatum, and Thalassiosira gravida ( H a r r i s o n e t a l . , 1977). However, oth e r s have documented e i t h e r no c e l l volume v a r i a b i l i t y with d i l u t i o n (or growth) r a t e i n N - l i m i t e d chemostat c u l t u r e s of D u n a l i e l l a t e r t i o l e c t a , T h a l a s s i o s i r a pseudonana and Coccochloris stagnina (Caperon and Meyer, 1972) or an i n c r e a s e i n c e l l volume wi t h i n c r e a s i n g P - l i m i t a t i o n i n chemostat c u l t u r e s of T. pseudonana (Fuhs et a l . , 1972). Using c y c l o s t a t c u l t u r e s of fou r d i f f e r e n t c l o n e s of Thalassiosira weisflogii, Chisholm and C o s t e l l o (1980) s t u d i e d the e f f e c t of average c e l l s i z e on growth r a t e and concluded t h a t growth r a t e was an i n c r e a s i n g f u n c t i o n of average c e l l volume. Munk and R i l e y (1952) s t a t e t h a t the l a r g e r the s u r f a c e area t o volume r a t i o (SA/V) the g r e a t e r the c e l l ' s c a p a c i t y t o absorb n u t r i e n t s . The r e d u c t i o n i n mean c e l l volume of M. pusilla 182 and the subsequent increase i n r e l a t i v e surface area available for N O 3 - uptake i s perhaps an adaptive response to N0-}~ l i m i t a t i o n . In the ni t r a t e - r e p l e t e batch cultures and the n i t r a t e -l i m i t e d cyclostat cultures, where growth rate i s li m i t e d by the rate of supply of N O 3 - , obvious d i e l patterns for N O 3 -uptake were observed for M. p u s i l l a i n a l l but the most N O 3 - -d e f i c i e n t cyclostat populations (D = 0.24 d -"^). The n i t r a t e uptake rates, for the cultures grown i n batch or i n cyclostats at 0.74 and 0.42 d - ^ d i l u t i o n rates, were maximal during the l i g h t period and decreased during the dark period. In addition to the d i e l pattern, the early l i g h t n i t r a t e uptake maximum (early morning) and increased dark uptake during late night (pre-dawn) suggests diurnal and nocturnal cycles for the 0.74 and 0.42 d - ^ d i l u t i o n rate cultures. Malone et a l . (1975) found s i m i l a r c y c l i c variations i n N O 3 - uptake by an outdoor culture of Chaetoceros sp. grown at high d i l u t i o n rate (2.0 d -^) under natural sunlight, but uptake independence of the light-dark cycle for three slower d i l u t i o n rates (0.5, 1.0 and 1.5 d -^"); absence of d i e l p e r i o d i c i t y i n N O 3 - uptake was also observed i n axenic cyclostat cultures of the same Chaetoceros sp. (STX-105) grown at 6 low d i l u t i o n rates (0.3 -1.2 d""*") by Picard ( 1976). Dark N O 3 - uptake capacity of cyclostat cultures of the marine prymnesiophyte, Pavlova l u t h e r i and chlorophyte, D u n a l i e l l a t e r t i o l e c t a was exceeded by the supply rate of N O 3 - at high d i l u t i o n rates (> ca. 0.5 d-"'"), but no d i e l p e r i o d i c i t y was observed at lower d i l u t i o n 183 r a t e s (Laws and Caperon, 1976; Laws and Wong, 197 8) although the diatom, Thalassiosira a l l e n i i never showed d i e l p e r i o d i c i t y i n N O 3 - uptake a t 6 d i l u t i o n r a t e s from 0.1-1.4 d - 1 (Laws and Wong, 1978). Eppley e t a l . (1971b) found a s i m i l a r l a c k of 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 - ( N O 3 - + NH 4 +) l i m i t e d c y c l o s t a t (0.78 d--'") c u l t u r e s of Emiliana huxleyi, but g r e a t e r NG^ - and NH^ **" uptake r a t e s i n the l i g h t p e r i o d and l e s s e r r a t e s i n the dark p e r i o d f o r a s i m i l a r l y grown c y c l o s t a t (0.73 d~^) c u l t u r e of Skeletonema costatum. I t appears t h a t the a b i l i t y t o take up N O 3 - d u r i n g the n i g h t may be s p e c i e s dependent and t h a t the degree of N l i m i t a t i o n a f f e c t s dark uptake c a p a c i t y and hence the presence or l a c k of d i e l p e r i o d i c i t y i n N O 3 - uptake r a t e s . A dampening e f f e c t on d i e l p e r i o d i c i t y of N O 3 - uptake by n i t r o g e n s t r e s s i s suggested by the r e s u l t s of numerous f i e l d s t u d i e s of n a t u r a l phytoplankton communities (see Chapter 1). For example, i n e x t e n s i v e d i n o f l a g e l l a t e blooms dominated by Gymnodinium splendens (= G. sanguineum) i n the n i t r a t e - d e p l e t e d (<0.1 uq-at N«L~1) waters o f f Peru, ni g h t t i m e N O 3 - uptake r a t e s were ca . 50% of d a y l i g h t NC^ - uptake r a t e s (Dortch and Maske, 1982). In c o n t r a s t , Maclsaac (1978) r e p o r t e d t h a t uptake at n i g h t averaged o n l y 10-20% of the d a y l i g h t r a t e s f o r a Gonyaulax polyedra bloom o f f Baja C a l i f o r n i a . However, here the ambient N O 3 - c o n c e n t r a t i o n s i n the s u r f a c e waters were g e n e r a l l y c a . 1 uq-at N'L-''" and the n i t r a c l i n e was much shallower than o f f Peru. S i m i l a r r e s u l t s were observed by H a r r i s o n (1976) f o r N - s u f f i c i e n t c u l t u r e s of Gonyaulax 184 polyedra; n i g h t t i m e N O 3 - u p t a k e was c a . 20% o f d a y t i m e u p t a k e , b u t d a r k u p t a k e i n c r e a s e d t o c a . 40% o f d a y t i m e v a l u e s i n N-s t a r v e d c u l t u r e s a n d s i m i l a r l y N C > 3 - - d e p l e t e d r e d t i d e p o p u l a t i o n s d o m i n a t e d by G. polyedra. I t i s d i f f i c u l t t o c o m p a r e t h e r e s u l t s o f f i e l d s t u d i e s t o l a b o r a t o r y c y c l o s t a t s t u d i e s a s t h e u p t a k e r a t e s r e p o r t e d i n f i e l d i n v e s t i g a t i o n s ( e . g . , H a r r i s o n , 1976; M a c l s a a c , 1978) a r e g e n e r a l l y s u b s t r a t e s a t u r a t e d o r c o n s i d e r a b l y e n h a n c e d r e l a t i v e t o t h e a m b i e n t c o n c e n t r a t i o n due t o t h e c o n c e n t r a t x o n o f N a d d e d . D i e l f i e l d s t u d i e s o f i n s i t u N O 3 - u p t a k e i n m a r i n e ( e . g . , C o c h l a n 1982, 1986; K o i k e e t a l . , 1986) a n d f r e s h w a t e r e n v i r o n m e n t s ( e . g . W h a l e n a n d A l e x a n d e r , 1984) a r e r e l a t i v e l y f e w a n d no c l e a r e v i d e n c e o f d i e l p e r i o d i c i t y i n e i t h e r N O 3 - o r N H 4 + u p t a k e r a t e s w e r e o b s e r v e d when c o n c e n t r a t i o n s o f a m b i e n t i n o r g a n i c n i t r o g e n a r e l o w ( s e e C h a p t e r 1 D i s c u s s i o n ) . I n t r a c e l l u l a r n i t r a t e c o n c e n t r a t i o n s d e m o n s t r a t e d a m a r k e d n o c t u r n a l i n c r e a s e d u r i n g t h e l a t t e r p o r t i o n o f t h e n i g h t a n d a t t a i n e d m a x i m a l c o n c e n t r a t i o n s a t t h e b e g i n n i n g o f t h e l i g h t p e r i o d f o r N - s u f f i c i e n t a n d N - l i m i t e d c u l t u r e s o f M. pusilla. R a i m b a u l t a n d M i n g a z z i n i (1987) a l s o o b s e r v e d an e a r l y m o r n i n g maximum i n N O 3 - i n t r a c e l l u l a r p o o l s i z e f o r In-s u f f i c i e n t c u l t u r e s o f Phaeodactylum tricornutum a n d Skeletonema costatum a n d a m i n i m a l i n t e r n a l N O 3 - p o o l i n t h e i r s i n g u l a r n i g h t t i m e m e a s u r e m e n t t a k e n i n t h e f i r s t h a l f o f t h e d a r k p e r i o d . O n l y i n t h e h i g h e s t d i l u t i o n r a t e c y c l o s t a t c u l t u r e o f Chaetoceros s p . d i d M a l o n e e t a l . (1975) o b s e r v e d i e l p e r i o d i c i t y o f i n t e r n a l N O 3 - p o o l s i z e , w h i l e i n t e r n a l 185 N O 3 - c o n c e n t r a t i o n s r e m a i n e d l o w a n d c o n s t a n t i n t h e l o w e r d i l u t i o n r a t e c u l t u r e s w h i c h a l s o d e m o n s t r a t e d N O 3 - u p t a k e i n d e p e n d e n c e o f l i g h t - d a r k c y c l e . No c l e a r d i e l t r e n d s i n i n t e r n a l p o o l s w e r e o b s e r v e d by P i c a r d (1976) i n s i m i l a r c y c l o s t a t s w i t h Chaetoceros g r o w n a t l o w d i l u t i o n r a t e s . I n s h i p b o a r d c u l t u r e s o f a n a t u r a l p h y t o p l a n k t o n c o m m u n i t y f r o m an u p w e l l e d r e g i o n , C o l l o s a n d S l a w y k ( 1976) o b s e r v e d m a x i m a l i n t r a c e l l u l a r N O 3 - c o n c e n t r a t i o n s a t t h e b e g i n n i n g o f t h e l i g h t p e r i o d a n d m i n i m a l v a l u e s d u r i n g t h e n i g h t . I n Dabob B a y , W a s h i n g t o n , D o r t c h e t a l . (1985) r e p o r t e d d i e l v a r i a b i l i t y i n i n t r a c e l l u l a r n i t r a t e p o o l s f o r s u r f a c e c o m m u n i t i e s d u r i n g J u l y , s i m i l a r t o my r e s u l t s , b u t v a r i a b l e r e s u l t s w i t h no c l e a r d i e l r h y t h m a p p a r e n t f o r d e e p w a t e r c o m m u n i t i e s i n May a n d J u l y a n d t h e s u r f a c e c o m m u n i t y d u r i n g May. The i n c r e a s e i n i n t e r n a l N O 3 - o b s e r v e d i n t h e Micromonas pusilla c u l t u r e s d e m o n s t r a t e s an u n c o u p l i n g b e t w e e n N O 3 -u p t a k e a n d r e d u c t i o n d u r i n g t h i s t i m e ; i f N O 3 - i n t r a c e l l u l a r p o o l s w e r e c o n s t a n t t h e n r a t e s o f u p t a k e a n d r e d u c t i o n w o u l d be e q u a l ( o r b o t h z e r o ) , a n d i f i n t r a c e l l u l a r p o o l s d e c r e a s e d t h e r a t e o f r e d u c t i o n o f NC " 3~would be g r e a t e r t h a n r a t e o f u p t a k e ( C o l l o s a n d S l a w y k , 1 9 7 6 ) . C l e a r l y d u r i n g t h e l a t e n i g h t a n d e a r l y l i g h t p e r i o d s , t h e p r o c e s s e s o f N O 3 - u p t a k e a n d r e d u c t i o n a r e n o t i n p h a s e , a n d a r e l i k e l y t h e r e s u l t o f d i e l c y c l e s o f n i t r a t e r e d u c t a s e (NR) a c t i v i t y . E a r l y s t u d i e s o f t h e P e r u ( E p p l e y e t a l . , 1970; P a c k a r d e t a l . , 1974) a n d n o r t h w e s t A f r i c a n ( P a c k a r d a n d B l a s c o 1974) 186 u p w e l l i n g s y s t e m h a v e s u g g e s t e d d i e l c y c l e s o f NR a c t i v i t y a n d i n a s i m i l a r , b u t more d e t a i l e d s t u d y o f t h e n o r t h w e s t A f r i c a n u p w e l l i n g s y s t e m , M a r t i n e z e t a l . (1987) o b s e r v e d d i e l c y c l e s i n NR a c t i v i t y t h a t c l e a r l y f o l l o w e d t h e d i e l v a r i a t i o n i n l i g h t i n t e n s i t y : a d a y t i m e maximum, n i g h t t i m e minimum a n d t h e o n s e t o f NR a c t i v a t i o n c o i n c i d e n t w i t h dawn. T h e y f a i l e d t o o b s e r v e a p r e - d a w n r i s e i n NR a c t i v i t y t h a t h a d b e e n p r e v i o u s l y r e p o r t e d i n u p w e l l i n g a r e a s ( E p p l e y e t a l . , 1970; P a c k a r d a n d B l a s c o , 1 9 7 4 ) , b u t s u g g e s t e d t h a t t h e s a m p l i n g f r e q u e n c y a r o u n d dawn i n t h o s e e a r l i e r s t u d i e s was i n s u f f i c i e n t t o d e t e r m i n e c o n c l u s i v e l y t h e p r e s e n c e o f s u c h a p r e - d a w n r i s e . D i e l p e r i o d i c i t y h a s a l s o b e e n o b s e r v e d i n u n i a l g a l c u l t u r e s , s u c h a s Amphidinium carterae a n d Cachonina niei ( H e r s h e y a n d S w i f t , 1 9 7 6 ) , Emiliana huxleyi ( E p p l e y e t a l . , 1971b) w i t h m i n i m a l a c t i v i t y r e p o r t e d d u r i n g l a t e n i g h t . A s i m i l a r r h y t h m i n NR a c t i v i t y i n M. pusilla c o u l d a c c o u n t f o r t h e a c c u m u l a t i o n o f i n t e r n a l NC>3~ o b s e r v e d d u r i n g l a t e n i g h t a n d e a r l y m o r n i n g . A l t e r n a t i v e l y , t h e a b s e n c e o f d i e l NR a c t i v i t y w i t h l i t t l e o r no v a r i a t i o n d u r i n g t h e l i g h t - d a r k c y c l e , s u c h a s i n N - s t a r v e d n a t u r a l p o p u l a t i o n s o f Gonyaulax polyedra ( E p p l e y a n d H a r r i s o n , 1975; H a r r i s o n , 1 9 7 6 ) , c o m b i n e d w i t h t h e i n c r e a s e d N O 3 - u p t a k e d u r i n g t h e l a t e n i g h t a n d e a r l y m o r n i n g o b s e r v e d i n t h e t w o f a s t e r g rown c y c l o s t a t c u l t u r e s (D = 0.74 a n d 0.48 d-"*") , c o u l d l e a d t o a c c u m u l a t i o n o f N O 3 -i n t e r n a l l y . A r e d u c t i o n i n n i t r i t e r e d u c t a s e ( N i R ) a c t i v i t y d u r i n g l a t e n i g h t ( e . g . , E p p l e y e t a l . , 1971b) a n d s u b s e q u e n t l e a k a g e o f N O 3 - + NC^ - f r o m t h e c e l l s may a c c o u n t f o r t h e 187 i n c r e a s e i n e x t e r n a l NC^ - c o n c e n t r a t i o n s d u r i n g l a t e n i g h t f o r t h e 0.74 a n d 0.48 d""* d i l u t i o n r a t e c u l t u r e s . A l t e r n a t i v e l y , t h e r i s e i n e x t e r n a l NC^ - c o n c e n t r a t i o n may be e x p l a i n e d a s r e s u l t i n g f r o m c e l l u l a r l o s s d u r i n g t h i s p e r i o d o f m a x i m a l c e l l d i v i s i o n . A p r o n o u n c e d d i e l r h y t h m i n t h e p o t e n t i a l u p t a k e c a p a c i t y o f NC>3~, N H 4 + a n d u r e a was o b s e r v e d i n t h e f a s t e s t g r o w i n g c y c l o s t a t c u l t u r e s o f Micromonas pusilla (D = 0.74 d ~ ^ ) , w h e r e a s t h e more N - l i m i t e d c u l t u r e s (D = 0.48 a n d 0.24 d - ^ ) h a d s i m i l a r n i g h t a n d d a y N u p t a k e c a p a c i t y . Laws a n d Wong (1978) a l s o o b s e r v e d t h a t t h e r e l a t i v e i m p o r t a n c e o f p o t e n t i a l n i g h t t i m e u p t a k e d e c r e a s e d r e l a t i v e t o p o t e n t i a l d a y l i g h t u p t a k e w i t h i n c r e a s i n g d i l u t i o n r a t e ( 0 . 0 0 5 7 3 - 0 . 0 2 4 6 4 h-^") i n c y c l o s t a t c u l t u r e s o f Pavlova lutheri, a l t h o u g h t h e y o b s e r v e d no s u c h t r e n d f o r Thalassiosira allenni o v e r a r a n g e o f d i l u t i o n r a t e s f r o m 0.00474 t o 0.05937 h ~ * , i n f a c t n i g h t t i m e u p t a k e a v e r a g e d 120% o f t h e r e s p e c t i v e d a y l i g h t N O 3 - u p t a k e . A l t h o u g h t h e r e a r e no o t h e r c o m p a r a b l e d i e l u p t a k e d a t a f o r c y c l o s t a t c u l t u r e s g r o w i n g a t v a r i o u s d e g r e e s o f N l i m i t a t i o n a r e v e a l i n g c o m p a r i s o n c a n be made b e t w e e n my r e s u l t s a n d p r e v i o u s w o r k w i t h N - r e p l e t e a n d N - s t a r v e d c u l t u r e s . F o r e x a m p l e , t h e d i n o f l a g e l l a t e Gyrodinium aureolum d o e s n o t t a k e up N O 3 - i n t h e d a r k when i n a s t a t e o f n i t r o g e n s u f f i c i e n c y , b u t when N - s t a r v e d f o r 24 h n i g h t t i m e u p t a k e became a b o u t h a l f o f l i g h t u p t a k e ( P a a s c h e e t a l . , 1 9 8 4 ) . They f o u n d t h a t t h e a b i l i t y t o t a k e up NC^ - a n d N H 4 + d u r i n g t h e n i g h t t i m e v a r i e d c o n s i d e r a b l y among N - s u f f i c i e n t d i n o f l a g e l l a t e s , b u t t h a t 188 r e l a t i v e d a r k u p t a k e o f N H 4 + was g e n e r a l l y g r e a t e r t h a n t h a t o f NG^ -, s i m i l a r t o t h a t o b s e r v e d h e r e f o r M. pusilla. S i m i l a r l y , B h o v i c h i t r a a n d S w i f t (1977) showed t h a t t h e N O 3 -u p t a k e c a p a c i t y o f N - s t a r v e d o c e a n i c d i n o f l a g e l l a t e s , Pyrocystis noctiluca a n d Dissodinium lunula, w e r e v i r t u a l l y i n d e p e n d e n t o f t h e l i g h t - d a r k c y c l e . I t a p p e a r s t h a t N s t a r v a t i o n o r l i m i t a t i o n e n h a n c e s p o t e n t i a l d a r k u p t a k e more t h a n l i g h t u p t a k e o f n i t r o g e n r e s u l t i n g i n a more o r l e s s c o n t i n u o u s u p t a k e o f n i t r o g e n i n N - d e p l e t e d p h y t o p l a n k t o n , i n c l u d i n g t h e p i c o p l a n k t e r Micromonas p u s i l l a . The d e c r e a s i n g l i g h t d e p e n d e n c e w i t h i n c r e a s i n g N l i m i t a t i o n i s a l s o s u g g e s t e d b y t h e r e s u l t s o f t h e N u p t a k e v e r s u s i r r a d i a n c e e x p e r i m e n t s c o n d u c t e d d u r i n g m i d - d a y . W i t h i n c r e a s e d N l i m i t a t i o n ( d e c r e a s e d d i l u t i o n r a t e ) t h e r e l a t i v e d a r k u p t a k e c a p a c i t y i n c r e a s e d f o u r - f o l d f r o m 5 t o 2 1 % o f t o t a l N 0 3 ~ u p t a k e a n d t w o - f o l d f r o m 20 t o 39% o f t o t a l N H 4 + u p t a k e . A c o m p a r i s o n o f c a l c u l a t e d t o t a l u p t a k e ( l i g h t + d a r k ) o f s a m p l e s i n c u b a t e d a t 1 a n d 100% o f t h e g r o w t h — 2 — 1 i r r a d i a n c e ( 120 L(E*m *s ) r e v e a l s a n a p p r o x i m a t e t w o - f o l d r e d u c t i o n i n l i g h t d e p e n d e n c e o f N O 3 - a n d N H 4 + u p t a k e w i t h i n c r e a s i n g N l i m i t a t i o n i n c y c l o s t a t c u l t u r e s o f M. pusilla. The d e c r e a s e d l i g h t d e p e n d e n c e o f N u p t a k e i s p e r h a p s a n a d a p t i v e r e s p o n s e t o N l i m i t a t i o n w h i c h a l l o w s t h e c e l l t o o p t i m i z e i t s u p t a k e c a p a b i l i t y a t l o w PPFDs w i t h o u t i n c u r r i n g t h e r e l a t i v e l y h i g h m e t a b o l i c c o s t s o f m i g r a t i o n f o r a p i c o f l a g e l l a t e ( R a v e n , 1986) t o a more s u i t a b l e l i g h t 189 environment s a t u r a t i n g t o N uptake. Des p i t e i t s pronounced p h o t o t a x i s (Manton and Parke, 1960; Throndsen, 1973) and i t s r e l a t i v e l y good swimming a b i l i t y (Knight-Jones and Walne, 1952; Throndsen, 1973) Micromonas pusilla has been found deeper i n the sea than f l a g e l l a t e s i n g e n e r a l , and i t i s o f t e n found w e l l below the euphotic zone (e.g., Manton and Parke, 1960; Throndsen, 1976). — 1 1 Micromonas pusilla can swim at c a . 90 jjm*s (75-100 jjm«s , Throndson, 1973) which would enable t h i s p i c o f l a g e l l a t e t o achieve meaningful changes i n i t s i n c i d e n t photon f l u x d e n s i t y (PPFD) d u r i n g daytime v e r t i c a l m i g r a t i o n i n a s t r a t i f i e d water column. In the F r a s e r R i v e r plume, i n the southern p o r t i o n of the S t r a i t of Georgia, B r i t i s h Columbia, M. pusilla can be n u m e r i c a l l y the most abundant p h y t o p l a n k t e r i n the euphotic zone ( C l i f f o r d e t a l . , 1989), but i t only accounts f o r <7% of the phytoplankton biomass ( H a r r i s o n et a l . , s ubmitted). From Beer's law one can c a l c u l a t e t h a t the 3.9 m t h a t M. pusilla c o u l d move v e r t i c a l l y i n these N - r e p l e t e waters d u r i n g a day's (12 h) swimming ( a t t e n u a t i o n c o e f f i c i e n t of c a . 0.42 m-^) would i n c r e a s e the c e l l ' s mean i n c i d e n t PPFD by 5 times the o r i g i n a l v a l u e . T h i s c o u l d be advantageous, i n terms of i n c r e a s e d s p e c i f i c n u t r i e n t and growth r a t e , i f the i n i t i a l PPFD was l i m i t i n g . Conversely, downward swimming c o u l d take a c e l l from a r e g i o n of high, i n h i b i t o r y PPFD t o one w i t h lower PPFD but s t i l l s a t u r a t i n g f o r n i t r o g e n uptake. 190 CONCLUSIONS T h i s d i s s e r t a t i o n e x a m i n e d t h e u p t a k e o f n i t r o g e n o u s n u t r i e n t s by b o t h n a t u r a l a s s e m b l a g e s o f m a r i n e p h y t o p l a n k t o n a n d u n i a l g a l c u l t u r e s o f t h e p i c o f l a g e l l a t e , Micromonas pusilla a s a f u n c t i o n o f l i g h t a n d n u t r i t i o n a l h i s t o r y . The s p e c i f i c f i n d i n g s o f t h e r e s e a r c h a r e s u m m a r i z e d b e l o w . 1. The u p t a k e o f N O 3 - , N H 4 + a n d u r e a b y c o a s t a l a n d o c e a n i c p h y t o p l a n k t o n c o m m u n i t i e s d e m o n s t r a t e d p r o n o u n c e d d i e l p e r i o d i c r h y t h m s , w i t h m i n i m a l u p t a k e r a t e s a t n i g h t a n d m a x i m a l u p t a k e d u r i n g t h e d a y t i m e . The a m p l i t u d e o f u p t a k e p e r i o d i c i t y a p p e a r e d t o be i n f l u e n c e d by a number o f f a c t o r s b e s i d e s l i g h t i n t e n s i t y a n d t h e s e i n c l u d e d t h e p h y t o p l a n k t o n s p e c i e s c o m p o s i t i o n , t h e a m b i e n t n i t r o g e n c o n c e n t r a t i o n s , t h e a c t u a l N s u b s t r a t e u t i l i z e d , a n d t h e d e p t h i n t h e w a t e r c o l u m n . 2. I n b o t h t h e f r o n t a l a n d s t r a t i f i e d w a t e r s o f t h e S t r a i t o f G e o r g i a t h e d e p e n d e n c e o f n i t r a t e a n d u r e a u p t a k e u p o n i r r a d i a n c e c o u l d be d e s c r i b e d by a r e c t a n g u l a r h y p e r b o l a s i m i l a r t o t h e M i c h a e l i s - M e n t e n f o r m u l a t i o n . The l i g h t d e p e n d e n c y o f N O 3 - u p t a k e was t h e same f o r b o t h t h e s u r f a c e a n d DCM c o m m u n i t i e s o f t h e f r o n t a l w a t e r , w h e r e a s i n t h e s t r a t i f i e d w a t e r s s u r f a c e p h y t o p l a n k t o n showed l e s s l i g h t d e p e n d e n c e f o r N O 3 - , a n d p a r t i c u l a r l y f o r u r e a u p t a k e , t h a n t h o s e f r o m t h e DCM. 191 3. U p t a k e r a t e s o f NC>3~, N H 4 + , a n d u r e a i n t h e n i g h t a n d a r t i f i c i a l d a r k n e s s w e r e a s u b s t a n t i a l p o r t i o n o f t o t a l u p t a k e by c o a s t a l p h y t o p l a n k t o n c o m m u n i t i e s ; u n d e r c o n d i t i o n s o f n i t r o g e n l i m i t a t i o n ( l o w a m b i e n t N c o n c e n t r a t i o n s ) d a r k u p t a k e i n c r e a s e d r e l a t i v e t o l i g h t u p t a k e . 4 . /Ammonium c o m p l e t e l y s u p p r e s s e d t h e u p t a k e o f N O 3 - by N-r e p l e t e c u l t u r e s o f Micromonas pusilla w h e r e a s t h e a d d i t i o n o f u r e a o n l y r e s u l t e d i n p a r t i a l i n h i b i t i o n o f N O 3 - u p t a k e . The i n h i b i t o r y e f f e c t o f N H 4 + on N O 3 - u p t a k e was c o m p l e t e e v e n a t N H 4 + c o n c e n t r a t i o n s a s l o w a s 1 uq-at N'L-"'". 5. U p t a k e k i n e t i c e x p e r i m e n t s showed t h a t M. pusilla c a n t a k e up N H 4 + a t t w i c e t h e r a t e s o f N O 3 - o r u r e a u p t a k e . A l t h o u g h t h e h a l f - s a t u r a t i o n c o n s t a n t s a r e s i m i l a r f o r t h e t h r e e s u b s t r a t e s ( 0 . 3 - 0 . 5 uq-at N-L -"' -), t h e g r e a t e r i n i t i a l s l o p e o f t h e M i c h a e l i s - M e n t e n p l o t f o r N H 4 + u p t a k e s u g g e s t s t h a t M. pusilla c a n u t i l i z e l o w c o n c e n t r a t i o n s o f N H 4 + more e f f e c t i v e l y t h a n e q u i v a l e n t c o n c e n t r a t i o n s o f u r e a a n d N O 3 -6 . T r a n s i e n t e l e v a t e d ( s u r g e ) r a t e s o f N H 4 + a n d u r e a u p t a k e w e r e o b s e r v e d a f t e r N e n r i c h m e n t o f N - s t a r v e d c u l t u r e s o f M. pusilla. N i t r a t e u p t a k e was s l o w e r ( 2 5 - 5 0 % ) i n N - s t a r v e d c e l l s t h a n N - r e p l e t e c e l l s , b u t t h e r e was no l a g i n u p t a k e a f t e r t h e i n i t i a l N O 3 - e n r i c h m e n t o f s t a r v e d c e l l s ; u p t a k e commenced i m m e d i a t e l y . 7. D i e l p a t t e r n s w e r e o b s e r v e d i n b o t h c o n t i n u o u s a n d b a t c h c u l t u r e s o f M. pusilla grown on a L:D i l l u m i n a t i o n c y c l e . D i e l p e r i o d i c i t y i n c e l l d i v i s i o n , mean c e l l v o l u m e , N u p t a k e a n d i n t e r n a l p o o l s o f N O 3 - w e r e o b s e r v e d . W i t h d e c r e a s e d 192 d i l u t i o n r a t e ( d e c r e a s e d u a n d s l o w e r N O 3 - s u p p l y ) i n s i t u NC>3~ u p t a k e p e r i o d i c i t y was a b s e n t . P o t e n t i a l r a t e s o f NH 4 + w e r e c o n s i s t e n t l y 2-3 f o l d g r e a t e r t h a n N C ^ - o r u r e a u p t a k e r a t e s r e g a r d l e s s o f t h e d e g r e e o f N l i m i t a t i o n . A m a r k e d d i e l v a r i a b i l i t y i n p o t e n t i a l u p t a k e r a t e s o f a l l t h r e e N s u b s t r a t e s was a p p a r e n t i n t h e f a s t e s t g r o w n c y c l o s t a t c u l t u r e (0.74 d --*-), b u t n o t i n t h e s l o w e r grown c y c l o s t a t s ( 0 . 4 9 a n d 0.24 d _ 1 ) 8 . The e f f e c t o f i r r a d i a n c e on t h e u p t a k e o f N H 4 + a n d N O 3 -by M. pusilla c o u l d be d e s c r i b e d b y M i c h a e l i s - M e n t e n k i n e t i c s . D a r k u p t a k e r a t e s o f N H 4 + w e r e a g r e a t e r p o r t i o n o f t o t a l u p t a k e t h a n d a r k NO^ - r a t e s , a n d t h e r e l a t i v e i m p o r t a n c e o f d a r k N u p t a k e i n c r e a s e d w i t h i n c r e a s e d N l i m i t a t i o n . W i t h i n c r e a s e d N l i m i t a t i o n t h e l i g h t d e p e n d e n c y o f N O 3 - a n d N H 4 + u p t a k e was l e s s e n e d . 193 REFERENCES A l l e n , T.F.H. 1977. S c a l e i n m i c r o s c o p i c a l g a l ecology: a n e g l e c t e d dimension. P h y c o l o g i a 16: 253-257 Amy, N.K. and R.H. G a r r e t t . 1974. P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of the n i t r a t e reductase from the diatom Thalassiosira pseudonana. P l a n t P h y s i o l . 54: 629-637. A n t i a , N.J., P.J. H a r r i s o n and L. O l i v e i r a (In p r e s s ) . 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D i f f u s i o n as a c o n s t r a i n t on the b i o l o g i c a l importance of microzones i n the sea, p. 209-218. In: J.C.J. N i h o u l (ed.) Ecohydrodynamics. E l s e v i e r Oceanography S e r i e s 32. E l s e v i e r / N o r t h - H o l l a n d , New York. W i l l i a m s , S.K. and R.C. Hodson. 1977. T r a n s p o r t of urea a t low c o n c e n t r a t i o n s i n Chlamydomonas reinhardii. J . B a c t e r i o l . 130: 266-273. Wood, E.D., F.A.J. Armstrong and F.A. R i c h a r d s . 1967. Determination of n i t r a t e i n seawater by cadmium-copper r e d u c t i o n t o n i t r i t e . J . Mar. B i o l . Ass. U.K. 47: 23-31. Y i n , K. 1988. Short-term i n t e r a c t i o n between n i t r a t e and ammonium uptake f o r c e l l s of a marine diatom grown under d i f f e r e n t degrees of l i g h t l i m i t a t i o n . M.Sc. T h e s i s , Dept. Oceanogr., U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B.C., 106 p. Zar, J.H. 1974. B i o s t a t i s t i c a l a n a l y s i s . P r e n t i c e - H a l l Inc., Englewood C l i f f s , N.J., 620 p. Zar, J.P., P.G. Falkowski, J . Fowler and D.G. Capone. 1988. Co u p l i n g between ammonium uptake and i n c o r p o r a t i o n i n a marine diatom: Experiments w i t h the s h o r t - l i v e d r a d i o i s o t o p e 1 3N. Limnol. Oceanogr. 33: 518-527. 222 APPENDIX 1 Equations used to calculate 1 5 N uptake rates, Once the percentage of 1 5 N ( s p e c i f i c a c t i v i t y ) i n the p a r t i c u l a t e material ( Ns) has been experimentally determined by emission spectrometry (procedures reviewed by F i e d l e r and Proksch, 1975; Harrison, 1983a) the appropriate equation to ca l c u l a t e nitrogen uptake must be chosen to correspond with the experimental protocol employed. The atom % 1 5 N excess ( 1 5N X S) of the p a r t i c u l a t e material i s calculated by subtracting the natural abundance of 1 5 N (F) which i s generally taken as ca. 0.365% (natural atmospheric enrichment of 1 5N) for f i e l d samples and can be measured d i r e c t l y i n culture samples: 1 5 N X S = 1 5 N S " F (!) The s p e c i f i c uptake rate (N taken up per unit p a r t i c u l a t e N) i s calculated as V^ . and arises from a constant transport model based on the i s o t o p i c r a t i o of the p a r t i c u l a t e sample taken at the end of the incubation: v t = 15 N (R - F ) • T (2) where T i s the incubation time and R i s the i n i t i a l atom % enrichment of the N substrate calculated as 223 100-[(S^-A + S a F ) / ( S ^ + S a ) ] where i s the concentration of added substrate, A the s p e c i f i c a c t i v i t y of the isotope (always 99 atom % i n present study), and S a the ambient concentration of unenriched N substrate. Careful chemistry and accurate determination of ambient N concentration are es s e n t i a l f o r an accurate determination of R. The absolute (or transport) rate ( ) i - s c a l c u l a t e d by m u l t i p l i c a t i o n of V^ . and the PON c o l l e c t e d at the end of the incubation period (PONj.).: p t = V t • PONt (3) Another equation for the c a l c u l a t i o n of s p e c i f i c uptake rate (Vj.) a r i s e s from the constant transport model based on PON concentration c o l l e c t e d at the beginning of the incubation period ( P 0 N Q ) : 15N V o - TJ— (4) (R - ibHs) • T A l t e r n a t i v e l y an equivalent expression replaces the denominator with [(R - F) - ~ 5N x g] • T. The absolute uptake rate p Q i s c a l c u l a t e d by m u l t i p l i c a t i o n of V Q by PONQ: Po " V o * P 0 N o (5) Since both equations 2 and 4 are derived from a constant transport model they do not allow f o r changes i n PON 2 2 4 c o n c e n t r a t i o n d u r i n g the course of the i n c u b a t i o n , thus y i e l d i n g an u n d erestimate (V t) and an o v e r e s t i m a t e (V^) of the mean s p e c i f i c uptake r a t e d u r i n g the i n c u b a t i o n p e r i o d . Dugdale and W i l k e r s o n (1986) suggest the most obvious way t o a c h i e v e a b e t t e r e s t i m a t e i s t o use the mean of the two v a l u e s : Vm = ( vo + V t > 1 2 (5') The c o n s t a n t s p e c i f i c uptake model assumes t h a t each new c e l l added d u r i n g i n c u b a t i o n c o n t r i b u t e s as e q u a l l y t o the sum of uptake as each p r e - e x i s t i n g c e l l i s c o n t r i b u t i n g and V c can be c a l c u l a t e d as: V c = - • In T (R - F) ( R - 1 5 n x s : (6) A l t e r n a t i v e l y the denominator can be s u b s t i t u t e d w i t h the e q u i v a l e n t e x p r e s s i o n [(R - F) - ^ 5 N X S ] . E q u a t i o n 6 i s e q u i v a l e n t t o the uptake r a t e c a l c u l a t e d as In(PON t/PON Q)/T i n C o l l o s (1987) and shown i n h i s F i g . 3 (Eq. 8'). The uptake of u n l a b e l l e d N forms d u r i n g the c o u r s e of an i n c u b a t i o n can 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 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 determined when o n l y the * 5 N - l a b e l l e d compound i s being taken up ( C o l l o s , 1987; Lund, 1987). E q u a t i o n 3 y i e l d s an a c c u r a t e e s t i m a t e of t r a n s p o r t r a t e (P^) o r t n e l a b e l l e d compound s i n c e V t and PON t are e s t i m a t e d from the same sample (Dugdale and W i l k e r s o n , 2 2 5 1986; C o l l o s , 1987). The d i l u t i o n e f f e c t o f t h e u p t a k e o f u n l a b e l l e d s u b s t r a t e c a n be c o m p e n s a t e d f o r V Q , p r o v i d e d i n d e p e n d e n t e s t i m a t e s o f t h e t r a n s p o r t r a t e f o r u n l a b e l l e d s o u r c e s ( p j j a r e a v a i l a b l e ( i . e . a m b i e n t n u t r i e n t d i s a p p e a r a n c e o v e r t i m e ) : 15 N V o = x s (R 15 r P i i + sum • T L P O N Q J -r 15 N x s R - 1 5 N ( V ) The e q u a t i o n f o r t h e c o n s t a n t s p e c i f i c u p t a k e m o d e l c o m p e n s a t e d f o r u p t a k e o f u n l a b e l l e d N i s : 1 T I n R - F R - 1 5 N s J sum • T PON, (8 I t s h o u l d be n o t e d t h a t i n D u g d a l e a n d W i l k e r s o n (1986) t h i s e q u a t i o n i s i n c o r r e c t l y w r i t t e n ; t h e p l a c e m e n t o f t h e l e f t c u r l y p a r e n t h e s i s s h o u l d n o t be p l a c e d b e f o r e t h e I n t e r m b u t a s shown a b o v e . B o t h e q u a t i o n s 7 a n d 8 ( e q u i v a l e n t t o E q . 10 a n d 11 o f D u g d a l e a n d W i l k e r s o n , 1986) y i e l d i n c r e a s e d e s t i m a t e s o f V a c c o r d i n g t o t h e p r o p o r t i o n o f i n i t i a l PON c o n c e n t r a t i o n a d d e d f r o m u n l a b e l l e d s o u r c e s d u r i n g t h e i n c u b a t i o n . 226 APPENDIX 2 G r o w t h - i r r a d i a n c e c u r v e o f Micromonas pusilla. Objective: The e x p e r i m e n t was c o n d u c t e d t o o b t a i n a g r o w t h -i r r a d i a n c e c u r v e f o r M. pusilla f o r d e t e r m i n a t i o n o f t h e PPFD n e c e s s a r y t o s u s t a i n m a x i m a l g r o w t h i n s u b s e q u e n t n i t r o g e n u t i l i z a t i o n s t u d i e s . Methods: C u l t u r e s o f M. pusilla were grown i n 40 m l o f medium i n 50 m l b o r o s i l i c a t e g l a s s t e s t t u b e s w i t h t e f l o n - l i n e d c a p s . The medium a n d i t s p r e p a r a t i o n a r e d e s c r i b e d i n t h e Culturing s e c t i o n o f C h a p t e r 3. S t e r i l e t e c h n i q u e s w e r e u s e d f o r a l l c u l t u r i n g w o r k ; t h e a b s e n c e o f b a c t e r i a was o n l y c o n f i r m e d by m i c r o s c o p i c e x a m i n a t i o n . A l l c u l t u r e s w e r e g r o w n a t 17°C (± 0.5°C) a t t h e f o l l o w i n g P P F D s : 145, 120, 89, 7 1 , 5 5 , 44, 34, 27, 19 a n d 15 L - E - m - 2 - s - 1 (2 - 13 c u l t u r e s p e r P P F D ) . • • • • • R C o n t i n u o u s l i g h t was p r o v i d e d b y V i t a - L i t e UHO f l u o r e s c e n t • R t u b e s f i l t e r e d t h r o u g h b l u e P l e x i g l a s (No. 2 0 6 9 , Rohm & Haas) a n d a t t e n u a t e d b y d i s t a n c e a n d n e u t r a l d e n s i t y s c r e e n i n g . I n c i d e n t i r r a d i a t i o n was m e a s u r e d w i t h a L i C o r m o d e l L I 185 q u a n t u m m e t e r (Lambda I n s t r u m e n t s ) w i t h a 2n c o l l e c t o r a n d t h e s c r e e n i n g c a l i b r a t e d w i t h a B i o s p h e r i c a l I n s t r u m e n t s QSL-100 4rc s e n s o r p l a c e d i n t h e same p o s i t i o n a s t h e c u l t u r e t u b e s . D e t e r m i n a t i o n s o f b i o m a s s ( i n v i v o f l u o r e s c e n c e ) w e r e made a t c a . 12 o r 24 h i n t e r v a l s by i n s e r t i n g t h e w h o l e t u b e i n t o a T u r n e r D e s i g n s m o d e l 10 f l u o r o m e t e r , a f t e r m i x i n g b y m u l t i p l e i n v e r s i o n s . 227 C u l t u r e s w e r e t r a n s f e r r e d p r i o r t o N d e p l e t i o n s o t h a t t h e y w e r e n e v e r n u t r i e n t - l i m i t e d . One e s t i m a t e o f t h e g r o w t h r a t e was o b t a i n e d p e r t r a n s f e r b y c a l c u l a t i n g t h e g r o w t h r a t e b e t w e e n e a c h s u c c e s s i v e m e a s u r e o f f l u o r e s c e n c e a n d a v e r a g i n g o v e r t h e 4-6 d a y p e r i o d . G r o w t h r a t e s (u) w e r e c a l c u l a t e d a s : u = I n ( F 2 / F 1 ) / ( t 2 - t 1 ) w h e r e F-^  a n d F 2 a r e f l u o r e s c e n c e a t t i m e 1 ( t j _ ) a n d t i m e 2 ( t 2 ) , r e s p e c t i v e l y . Results and Conclusions: S p e c i f i c g r o w t h r a t e s (d-"*") w e r e c a l c u l a t e d f r o m i n c r e a s e s i n i n v i v o f l u o r e s c e n c e a n d a r e p l o t t e d a g a i n s t PPFD i n F i g u r e A . l . T h e s e r e s u l t s i n d i c a t e 2 1 t h a t g r o w t h o f M. pusilla s a t u r a t e d a t <100 uE'm~ *s , w i t h no p h o t o i n h i b i t i o n a p p a r e n t a t t h e g r e a t e s t PPFD e x a m i n e d (145 ^ E*m~ 2 • s _ 1 ) . I t was c o n c l u d e d t h a t a PPFD o f 120 JJE-m*"2 • s - 1 w o u l d be e m p l o y e d e x c l u s i v e l y t h r o u g h o u t t h e c u r r e n t r e s e a r c h a s i t was s a t u r a t i n g f o r g r o w t h a n d c o u l d be a c h i e v e d w i t h o u t d i f f i c u l t y i n b o t h t h e c o n t i n u o u s l i g h t ( C h a p t e r 3) a n d t h e 14:10 l i g h t - d a r k ( C h a p t e r 4) e x p e r i m e n t a l c h a m b e r s . 228 Figure A . l . Specific growth rate (u) in d - i as a function of PPFD for M. p u s i l l a grown on NOo". Bars represent ± 1 S.D. (n = 2-13). Error bars are smaller than symbols where not v i s i b l e . "O =1 t— < o cr: O 1 .00 h 0.80 -0.60 0.40 h 0.20 0 30 60 90 120 150 PPFD ( /j,E • m 2 • s 1) APPENDIX 3 C o m p a r i s o n o f t h e i n c r e a s e s i n in vivo f l u o r e s c e n c e a n d c e l l  c o n c e n t r a t i o n d u r i n g e x p o n e n t i a l g r o w t h o f Micromonas pusilla. Objective: T h i s e x p e r i m e n t was c o n d u c t e d t o d e t e r m i n e i f i n v i v o f l u o r e s c e n c e c o u l d be u s e d r o u t i n e l y t o m o n i t o r t h e g r o w t h o f b a t c h c u l t u r e s o f M. pusilla. Methods: C u l t u r e s o f M. pusilla w e r e grown i n d u p l i c a t e a c c o r d i n g t o t h e c o n d i t i o n s a n d p r o c e d u r e s o u t l i n e d i n t h e Culturing s e c t i o n o f C h a p t e r 3. A t 12 h i n t e r v a l s s a m p l e s w e r e c o l l e c t e d f o r d e t e r m i n a t i o n o f b o t h c e l l c o n c e n t r a t i o n , p w i t h a C o u l t e r C o u n t e r m o d e l TA I I e q u i p p e d w i t h t h e p o p u l a t i o n a c c e s s o r y , a n d i n v i v o f l u o r e s c e n c e , w i t h a T u r n e r D e s i g n s m o d e l 10 f l u o r o m e t e r ( s e e C h a p t e r 3, M a t e r i a l s a n d M e t h o d s f o r a d d i t i o n a l d e t a i l s ) . S p e c i f i c g r o w t h r a t e s (d -"^) w e r e c a l c u l a t e d b e t w e e n e a c h s u c c e s s i v e m e a s u r e o f f l u o r e s c e n c e a n d c e l l c o n c e n t r a t i o n a s : u = I n ( F 2 / F 1 ) / ( t 2 - tx) w h e r e F 2 a n d F-^  a r e t h e f l u o r e s c e n c e o r c e l l c o n c e n t r a t i o n a t t i m e 2 ( t 2 ) a n d t i m e 1 ( t - j j , r e s p e c t i v e l y a n d a r e r e p o r t e d a s t h e mean (n = 2) ± 1 S.D. o f d u p l i c a t e c u l t u r e s . Results & Conclusions: The i n c r e a s e s i n i n v i v o f l u o r e s c e n c e 230 a n d c e l l c o n c e n t r a t i o n o f M. pusilla a s a f u n c t i o n o f t i m e a r e p l o t t e d i n F i g u r e A.2. A t t i m e z e r o ( t = 0) t h e N 0 3 ~ + NC^ -c o n c e n t r a t i o n i n t h e c u l t u r e s a v e r a g e d 50.1 ± 0.5 / j g - a t N-L-"'" a n d d e c r e a s e d t o < 1.0 L!g-at N * L - ^ a f t e r 90 a n d 95 h o f e x p o n e n t i a l g r o w t h i n c u l t u r e s 1 a n d 2, r e s p e c t i v e l y . D u r i n g N - r e p l e t e c o n d i t i o n s t h e g r o w t h r a t e , a v e r a g e d 0.835 ± 0.011 a n d 0.832 ± 0.001 (n = 2) f r o m i n v i v o f l u o r e s c e n c e a n d c e l l c o u n t m e a s u r e m e n t s , r e s p e c t i v e l y . T h i s r e s u l t i n d i c a t e s t h a t b o t h m e t h o d s m e a s u r e t h e i n c r e a s e i n b i o m a s s o f M. pusilla t o a s i m i l a r d e g r e e o f a c c u r a c y . I t was d e c i d e d t h a t i n v i v o f l u o r e s c e n c e c a n be u s e d t o a c c u r a t e l y m e a s u r e c e l l g r o w t h o f M. pusilla a n d w o u l d be e m p l o y e d t o r o u t i n e l y m o n i t o r t h e g r o w t h o f b a t c h c u l t u r e s p r i o r t o e x p e r i m e n t a t i o n . 231 F i g u r e A . 2 . G r o w t h c u r v e s o f d u p l i c a t e b a t c h c u l t u r e s o f M. pusilla g r o w n o n N C ^ - u n d e r s a t u r a t i n g PPFD. S e m i - l o g p l o t s o f r e l a t i v e i n v i v o f l u o r e s c e n c e ( 0 , t ) a n d c e l l c o n c e n t r a t i o n (•,•) v e r s u s t i m e . 232 APPENDIX 4 C o m p a r i s o n o f t h e r a t e s o f p a r t i c u l a t e n i t r o g e n p r o d u c t i o n and  i n o r g a n i c n i t r o g e n d i s a p p e a r a n c e . Objectivei Two m e a s u r e s o f n i t r a t e u p t a k e , t h e p r o d u c t i o n o f p a r t i c u l a t e o r g a n i c n i t r o g e n (PON) a n d t h e d i s a p p e a r a n c e o f NO3 - + N02~ o v e r t i m e , w e r e c o m p a r e d t o d e t e r m i n e t h e e f f i c i e n c y o f c o n v e r s i o n o f i n o r g a n i c N t o p a r t i c u l a t e o r g a n i c N a n d t h u s e s t i m a t e t h e e x t e n t o f d i s s o l v e d o r g a n i c N (DON) l o s s d u r i n g e x p o n e n t i a l g r o w t h o f M. pusilla. Methods: D u p l i c a t e b a t c h c u l t u r e s o f M. pusilla w e r e grown a c c o r d i n g t o t h e c o n d i t i o n s a n d p r o c e d u r e s o u t l i n e d i n t h e Culturing s e c t i o n o f C h a p t e r 3. A t 12 h i n t e r v a l s , 50 m l s a m p l e s w e r e c o l l e c t e d b y l o w vacuum f i l t r a t i o n (2 80 mm Hg) o n t o p r e c o m b u s t e d (460°C f o r 4 h) Whatman GF/F f i l t e r s f o r m e a s u r e m e n t o f PON c o n c e n t r a t i o n w i t h a C o n t r o l E q u i p m e n t C o r p . m o d e l 240-XA e l e m e n t a l a n a l y z e r . The f i l t r a t e was — — • R a n a l y z e d f o r NO3 + NO3 w i t h a T e c h n i c o n A u t o A n a l y z e r I I . D e t a i l s o f t h e f i l t r a t i o n p r o c e d u r e a n d a n a l y t i c a l t e c h n i q u e s a r e d e s c r i b e d i n t h e M a t e r i a l s a n d M e t h o d s o f C h a p t e r 3. Results & Conclusions: The d i s a p p e a r a n c e o f NO3 - + N02~ f r o m t h e medium, t h e i n c r e a s e i n PON c o n c e n t r a t i o n a n d t h e r a t i o o f t h e s e t w o m e a s u r e s o f NO3 - u p t a k e o v e r s u c c e s s i v e s a m p l i n g i n t e r v a l s a r e p r e s e n t e d i n F i g u r e A . 3 . T h e s e r e s u l t s i n d i c a t e 233 t h a t d u r i n g t h e p e r i o d o f N - r e p l e t e g r o w t h ( 0 - c a . 90 h) n e i t h e r m e a s u r e o f NO3- u p t a k e was c o n s i s t e n t l y g r e a t e r o r l e s s t h a n t h e o t h e r ; t h e r a t i o o f t h e c h a n g e i n c o n c e n t r a t i o n o f PON t o N03~ + N02~ a v e r a g e d 0.96 (± 0.08) a n d 0.97 (± 0.13) f o r c u l t u r e 1 a n d 2, r e s p e c t i v e l y . I n b o t h N - r e p l e t e c u l t u r e s t h e r e was no s i g n i f i c a n t d i f f e r e n c e i n r a t e s o f N0 3" + NO2"" d i s a p p e a r a n c e a n d PON a c c u m u l a t i o n ( p a i r e d t - t e s t , P £ 0.05, n = 7 p a i r s i n c u l t u r e 1, n = 8 p a i r s i n c u l t u r e 2) b u t when t h e c e l l s became N - l i m i t e d (< 1 uq-at N-L--'- o f N03~ + NO2-) a n a v e r a g e o f 54% o f t h e n i t r a t e r e m o v e d f r o m t h e medium was a c c o u n t e d f o r i n t h e PON. T h e s e r e s u l t s s u g g e s t t h a t o n l y d u r i n g c o n d i t i o n s o f N - l i m i t a t i o n w e r e t h e d e c r e a s e s i n N03" + NO2"" f r o m t h e medium n o t r e f l e c t e d i n s i m i l a r i n c r e a s e s i n PON c o n c e n t r a t i o n , s u g g e s t i n g e x c r e t i o n o f DON t o t h e medium. P a s t e s t i m a t e s o f DON e x c r e t i o n t y p i c a l l y r a n g e d f r o m 2 0 - 4 0 % o f t h e a s s i m i l a t e d n i t r o g e n i n N - l i m i t e d c e l l s a n d 5-10% i n h e a l t h y p h y t o p l a n k t o n c e l l s ( e . g . , S h a r p , 1 9 7 7 ) . However, i t i s p o s s i b l e t h a t much o f t h e DON e x c r e t i o n m e a s u r e d i n t h e s e e a r l y e x p e r i m e n t s may h a v e r e s u l t e d f r o m c e l l b r e a k a g e ( e . g . , F u hrman a n d B e l l , 1985; Goldman a n d D e n n e t t , 1985) o r c e l l p a s s a g e ( e . g . , L i , 1986; S t o c k n e r e t a l . , i n p r e s s ) d u r i n g f i l t r a t i o n . 234 F i g u r e A.3. N i t r a t e u p t a k e by d u p l i c a t e b a t c h c u l t u r e s o f Micromonas pusilla. A. D e c r e a s e i n d i s s o l v e d NO3- + NO9-c o n c e n t r a t i o n i n t h e c u l t u r e medium. B. A c c u m u l a t i o n o f p a r t i c u l a t e o r g a n i c n i t r o g e n . C. R a t i o o f NO3" u p t a k e r a t e _ c a l c u l a t e d f r o m PON a c c u m u l a t i o n t o r a t e c a l c u l a t e d f r o m NO3-+ N02~ d i s a p p e a r a n c e f r o m t h e medium. N i t r o g e n c o n c e n t r a t i o n s a r e p l o t t e d a g a i n s t e l a p s e d t i m e m e a s u r e d a f t e r c u l t u r e i n i t i a t i o n ; u p t a k e r a t e r a t i o s a r e p l o t t e d a g a i n s t a v e r a g e e l a p s e d t i m e b e t w e e n s u c c e s s i v e s a m p l i n g p e r i o d s . 0 . 4 1 • ' • ' • • • ' . 1 0 20 40 60 80 100 TIME (h) 235 APPENDIX 5 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 disappearance curve d u r i n g growth  of M. p u s i l l a . O b j e c t i v e : To determine i f n i t r i t e i s r e l e a s e d i n t o the c u l t u r e medium by M. pusilla, d u r i n g growth on n i t r a t e , as the ambient c o n c e n t r a t i o n of n i t r a t e approaches zero . Methods: A batch c u l t u r e of M. pusilla was grown, under constant blue l i g h t at a s a t u r a t i n g PPFD, a c c o r d i n g t o the c o n d i t i o n s and procedures o u t l i n e d i n the Culturing s e c t i o n of Chapter 3. At 30 min i n t e r v a l s , f i l t e r e d samples were ana l y z e d f o r n i t r a t e (NO3-) and n i t r i t e (NO2-) w i t h a Technicon AutoAnalyzer II f o l l o w i n g the techniques o u t l i n e d i n Wood e t a l . (1967) and Chapter 3. Sampling c o n t i n u e d u n t i l the ambient c o n c e n t r a t i o n s of NO3- and NC^ - reached t h e i r r e s p e c t i v e a n a l y t i c a l l i m i t s of d e t e c t i o n (NO3-: 0.03 uq-at N-L - 1; N0 2": 0.02 uq-at N - L - 1 ) . Results & Discussion: The ambient c o n c e n t r a t i o n s of NO3- and N02~ are p l o t t e d as a f u n c t i o n of time i n F i g u r e A.4. Over a p e r i o d of 4.5 h the t o t a l c o n c e n t r a t i o n of N03" + NO2"" d e c l i n e d from 5.76 pq-at N - L - 1 t o <0.05 jjg-at N - L - 1 w i t h concomittant decreases i n the ambient c o n c e n t r a t i o n of both NO3- and NO2". No evidence of NO2- e x c r e t i o n by M. pusilla was observed w i t h i n c r e a s i n g NO3- l i m i t a t i o n . The ambient 236 c o n c e n t r a t i o n o f N 0 2 ~ i n f i v e s e p a r a t e 20 L r e s e r v o i r s o f 50 ug-at N 0 3 - ' L - ^ e n r i c h e d ESAW a v e r a g e d < 0.05% o f t h e t o t a l N 0 3 ~ + N 0 2 ~ c o n c e n t r a t i o n (0.20 ± 0.07 L i g - a t N 0 2 ~ - L _ 1 , n = 5) w h i c h i s c o n s i s t e n t w i t h t h e i n i t i a l v a l u e s r e p o r t e d h e r e d u r i n g t h e f i r s t h o u r o f s a m p l i n g . I t i s c o n c l u d e d t h a t M. pusilla, u n l i k e o t h e r p h y t o p l a n k t o n s p e c i e s s u c h a s Thalassiosira pseudonana ( e . g . , O l s o n e t a l . , 1980; P a r s l o w e t a l . , 1 9 8 4 b ) , d o e s n o t r e l e a s e N 0 2 ~ d u r i n g g r o w t h on N03~ u n d e r s a t u r a t i n g PPFD. 237 Figure A.4. Dissolved NO3- and NO?" concentration i n the c u l t u r e medium, during batch growth of M. p u s i l l a , plotted against time of sampling. 120 180 TIME ( min ) 240 300 238 APPENDIX 6 P r e c i s i o n of a n a l y t i c a l t e c h n i q u e s . The p r e c i s i o n of the a n a l y t i c a l techniques employed throughout t h i s study are r e p o r t e d as the mean c o e f f i c i e n t of v a r i a t i o n ( C V . = S.D./x • 100) of r e p l i c a t e samples (n) c o l l e c t e d and processed a c c o r d i n g t o the procedures o u t l i n e d i n Chapters 1-4. The estimated e r r o r i n c l u d e s both c o l l e c t i o n ( i . e . f i l t r a t i o n ) and a n a l y t i c a l e r r o r s , except i n the atom % "1 c 1 c excess ( N e x ) measurements of N i n p a r t i c u l a t e s . Here d u p l i c a t e measurements were determined from the same o r i g i n a l sample ( c o l l e c t e d by f i l t r a t i o n ) , but evacuated, combusted and analyzed by emission spectrometry s e p a r a t e l y (n = 150 p a i r s ) . Measure Number of R e p l i c a t e s C o e f f i c i e n t of (n) V a r i a t i o n (%) N0 3~ + N0 2~ 2-3 1.2 N H 4 + 2-3 1.5 Urea 2 0.3 Chi a 2 4.4 POC 2-3 5.2 PON 2-3 3.8 c e l l concen. 2 3.5 1 5 N e x 2 1.5 Refereed Publications Parsons, T.R., H.M. Dovey, W.P. Cochlan, R.I. Perry and P.B. Crean. 1984. Frontal zone analysis at the mouth of a f jord-Jervis Inlet, B r i t i s h Columbia. Sarsia 69.: 133-137. Price, N.M., W.P. Cochlan and P.J. Harrison. 1985. Time course of uptake of inorganic and organic nitrogen by phytoplankton i n the S t r a i t of Georgia: comparison of f r o n t a l and s t r a t i f i e d communities. Mar. Ecol. Prog. Ser. 27.: 39-53. Cochlan, W.P. 1986. Seasonal study of uptake and regeneration of nitrogen on the Scotian Shelf. Cont. Shelf Res. 5: 555-577. Cochlan, W.P., P.J. Harrison, P.A. Thompson and T.R. Parsons. 1986. Preliminary observations of the summer production of three B r i t i s h Columbian coastal i n l e t s . Sarsia 71 :• 161-168. Harrison, P.J., W.P. Cochlan, J.C. Acreman, T.R. Parsons, P.A. Thompson, H.M. Dovey and Chen X i a o l i n . 1986. The ef f e c t s of crude o i l and Corexit 9527 on marine phytoplankton i n an experimental enclosure. Mar. Envir. Res. 18: 93-109. M i t c h e l l , J.G., A. Okubo, J.A. Fuhrman and W.P. Cochlan. 1989. The contribution of phytoplankton to ocean density gradients. Deep-Sea Res. 3_6: 1277-1282. Stockner, J.G., M.E. Klut and W.P. Cochlan. (In press) Leaky f i l t e r s : a warning to aquatic ecologists'. Can. J. F i s h . Aquat. S c i . 47. 

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