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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.)/ U n i v e r s i t y o f B r i t i s h Columbia, 1978 M.Sc, Dalhousie U n i v e r s i t y , 1982  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE  FACULTY OF GRADUATE STUDIES  (Department o f Oceanography)  We accept t h i s t h e s i s as conforming to the r e q u i r e d  THE  standard  UNIVERSITY OF BRITISH COLUMBIA December 198 9  ©  William  P a t r i c k 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 Diel  investigated neritic  i n natural  assemblages  This i s the f i r s t  NH^" ", u r e a )  of phytoplankton  o f fthe coast of  study t o report  and e x t e n s i v e measurements o f ambient  concentrations  uptake  1  -  and oceanic environments  Columbia. rates  (NO3 ,  patterns of nitrogen  i n these waters.  Calculated  from  British  nitrogen NH^  were  uptake  and urea  +  rates  of N  uptake,  15 based  on  course  N incorporation  into  particulate  matter  experiments, were maximal d u r i n g t h e day and minimal a t  night.  Besides the obvious  amplitude  effects  of the periodicity  of irradiance, the  i n uptake  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 community c o m p o s i t i o n , ambient concentration,  forms  of nitrogen  sampling.  Uptake of n i t r o g e n  artificial  darkness  light  uptake  rates,  with  increasing  the  first  study of d i e l  the  field.  the  cycle  The r a t i o s  other reduced Rates  shallow  of NO3  -  over  experiments  similar  N form,  of dark  by marine  t o those  (K  L T  -  over  a  than those of  +  by p h y t o p l a n k t o n i nt h e  layers  of the Strait  a gradient of irradiances be f i t t e d  with  of Georgia  and r e s u l t s o f  a hyperbolic  t o the Michaelis-Menten equation.  constants  phytoplankton i n  NH^ .  and urea uptake  could  This i s  urea uptake  of NO3  and  uptake  N limitation.  of dark t o l i g h t  and deep c h l o r o p h y l l  were measured these  urea uptake  and i n  p r o p o r t i o n s of daytime  increasing  were more s i m i l a r  and depth o f  during the night  t h e importance  with  nitrogen  available,  were measurable  generally  diel  during time  ) f o r light-dependent uptake  function  Half-saturation of urea and NO3  -  ranged was  from  0 t o 14% o f t h e s u r f a c e  a variable,  total  (light The  but often  + dark)  uptake  populations  irradiance  substantial  (> 5 0 % ) p o r t i o n  of nitrate-replete  of the picoflagellate, 1  determined by both  1  urea  medium.  other; range  +  and  NO3  Maximum s p e c i f i c  reported  ± 0.1  v a l u e o f 0.41  uq-at.  f o rsmall,  inhibited  N-L "'"), w h e r e a s u r e a a d d i t i o n -  in  NO3  uptake.  -  variable an  uptake  initial  laboratory and  o f NH^  study of N uptake  measured N-limited  rate.  NO3  i n only  M.  -  uptake  (1-10  was  uq-at.  a 28% r e d u c t i o n  p u s i l l a  exhibited of time,  with  This i s the f i r s t picoplankter  uptake  i t competes  responses  i n the field, are  picoplankter. of nitrogen  uptake  i n N-replete batch cultures cyclostat  Nitrate  for  -  t h a t many o f t h e t r a n s i e n t  periodicity  corresponding  or  N-L "*" i s w i t h i n t h e  by an e u c a r o y o t i c  f o r diatoms, with which  common t o t h i s Diel  response.  -  )  -  and urea as a f u n c t i o n  "surge" uptake  demonstrates  reported  +  of NO3  m a x  N-L -'- o f e a c h  uq-at  resulted of  x  (V  s  NH^"*" a d d i t i o n  Starved cultures  a  rates  constants (K )  oceanic diatoms.  following  m  was  disappearance  uptake  The h a l f - s a t u r a t i o n were w i t h i n  -  t h e average  completely  perturbations  0.13 h ~ ^ , m o r e t h a n 2 t i m e s t h e V -  NH^  -  (Butch.)  p u s i l l a  accumulation and n u t r i e n t  ( c a . 0.05 h ^ ) .  urea,  ofthe  and -starved  Micromonas  M a n t o n e t P a r k e , t o u r e a , NH^" " a n d N O 3  o f NH^" " w e r e  uptake  uptake.  response  from t h e c u l t u r e  and dark  cultures  of  and a s s i m i l a t i o n M.  p u s i l l a  and a l s o i n  (14L:10D) a t t h r e e growth  t o c a . 7 5 , 50 a n d 2 5 % o f i t ' s  were  maximal  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  rates  growth of the  iv  L:D c y c l e  but N O 3 uptake r a t e s  rate, in  the batch  and h i g h e r d i l u t i o n r a t e  t o that  seen i n p r e v i o u s  some d i a t o m s .  D i e l patterns  volume, p o t e n t i a l uptake r a t e s also  observed  nutritional the by  status  Michaelis-Menten of l i g h t  uptake i n c r e a s e d rates,  -  cultures,  studies  a response  of c y c l o s t a t  with  respect  o f NC>3~ were  t o the  p u s i l l a  k i n e t i c s ; with  was a l s o  increasing  N l i m i t a t i o n the  uptake decreased -  at saturating  on  described  and d a r k  f r o m 5-20% t o 21-39% o f N O 3 a n d N H 4 "  respectively,  cell  The e f f e c t o f i r r a d i a n c e  by M.  f o r nitrogen  cultures  d i v i s i o n , mean  and i n t e r n a l p o o l s  of the c e l l s .  1  pronounced p e r i o d i c i t y  in cell  and a r e d i s c u s s e d  u p t a k e o f NH^"" and N O 3  importance  a t the lowest d i l u t i o n  exhibited  -  similar of  i n the c y c l o s t a t cultures  irradiance.  4  uptake  V  T A B L E OF  CONTENTS  ABSTRACT  i  L I S T OF T A B L E S  i i x  L I S T OF F I G U R E S  x i i  ACKNOWLEDGEMENTS  x i x  INTRODUCTION Overview  1  Thesis  objectives  7  Thesis  outline  8  Experimental CHAPTER  1.  9  organism  E F F E C T S OF D I E L P E R I O D I C I T Y ON N I T R O G E N U P T A K E  N A T U R A L A S S E M B L A G E S OF  PHYTOPLANKTON  Introduction Materials  BY  12  and Methods  *  General  15  Sample c o l l e c t i o n  15  Analytical  methods  18  experiments  20  Tracer  Experimental  procedures  21  Results Physical  observations  Biological Nitrogen  observations  uptake  Subarctic Strait  24  rates Pacific  of Georgia  Offshore  31  waters  36 Ocean  36 36 43  vi  Discussion Experimental  considerations  Simultaneous uptake Effects CHAPTER  2.  55  of nitrogen  of l i g h t / d a r k regime  compounds  on n i t r o g e n  u p t a k e ...60  E F F E C T S OF I R R A D I A N C E ON N I T R O G E N U P T A K E  PHYTOPLANKTON:  COMPARISON  OF F R O N T A L AND  58  BY  STRATIFIED  COMMUNITIES Introduction Materials  67  and Methods  General  71  Experimental  73  Kinetic Results  and  General Effect Kinetic  parameters  of nitrogen  uptake  76  Discussion description of stations of light  on n i t r o g e n  parameters  Dark n i t r o g e n  77  uptake  of nitrogen  rates  81  uptake  85  uptake  92  Summary CHAPTER MICROMONAS  3.  100  N I T R O G E N U P T A K E BY THE E U C A R Y O T I C P I C O P L A N K T E R , PUSILLA  AND  THE E F F E C T S OF N D E P R I V A T I O N ON  UPTAKE  RESPONSE Introduction Materials  102  and Methods  Culturing Analytical  106 methods  Experimental Kinetic  107  procedures parameters  f o rN uptake  108  vii Substrate Effect NO3  interaction  o f NH^  +  110  concentration  on  uptake rate  -  I l l  Uptake of n i t r o g e n Estimation  by N 0 3 ~ - s t a r v e d c e l l s  ....112  of k i n e t i c parameters  113  Results Uptake k i n e t i c s  115  Substrate  118  interaction  Nitrogen-starved  cells  121  Discussion Uptake k i n e t i c s Cellular  physiological  Substrate  EFFECTS  state  133  interaction  Ecological C H A P T E R 4.  131  138  significance OF  IRRADIANCE  141 AND  N I T R O G E N U T I L I Z A T I O N I N MICROMONAS  DIEL PERIODICITY PUSILLA  Introduction Materials  ON  143  and Methods  Culturing  146  Analytical  procedures  Experimental  procedures  Diel  cycles  Diel  variation  Effect  148  of uptake  o f PPFD  and growth  14 9  i n maximum N u p t a k e on N 0 ~ 3  and N H  + 4  rates  uptake  ....151 152  Results Nitrate-replete  cultures  . 155  Nitrate-limited  cultures  157  v i i i  Potential  N uptake  rates  170  Influence  of light  on N u p t a k e  rates  175  Discussion  180  GENERAL CONCLUSIONS  190  REFERENCES  193  APPENDIX  1.  Equations  used t o c a l c u l a t e  A P P E N D I X 2.  Growth-irradiance  APPENDIX  Comparison of t h e increases  3.  fluorescence exponential A P P E N D I X 4.  curve  and c e l l  o f Micromonas i n in  concentration  g r o w t h o f Micromonas  Comparisons of t h e rates nitrogen  -^N u p t a k e  production  of  r a t e s ..222 pusilla  vivo during  pusilla  Dissolved curve  APPENDIX  6.  during  Precision  22 9  particulate  and i n o r g a n i c  nitrogen  disappearance A P P E N D I X 5.  .226  232  inorganic  nitrogen  disappearance  g r o w t h o f M.pusilla of analytical  techniques  235 238  ix LIST  OF T A B L E S  Table  1.1  I n i t i a l environmental c o n d i t i o n s of seawater c o l l e c t e d f o r time course experiments of n i t r o g e n uptake by n a t u r a l phytoplankton assemblages. 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 plume o f f southwest coast o f Vancouver I s l a n d ; o f f s h o r e o f western Canadian c o n t i n e n t a l shelf 17  Table  1.2  P l 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 w a t e r o f S t r a i t o f G e o r g i a , B.C., (see F i g . 1.1 B )  Table  1.3  33  R a t i o of dark t o l i g h t uptake r a t e s (V :V ) o f N H , 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 D  +  4  Table  1.4  Chlorophyll and u r e a i n of t h e S t r a i dark period interval  a s p e c i f i c u p t a k e r a t e s o f NH , N0 " f r o n t a l (A5) and s t r a t i f i e d (T4) w a t e r t o f G e o r g i a . , ( s e e F i g . 1.1 B ) . T h e o c c u r s d u r i n g t h e 12 t o 18 h t i m e 45  Table  1.5  I n i t i a l environmental c o n d i t i o n s of seawater c o l l e c t e d f o r n i t r o g e n uptake experiments d u r i n g time course 4 47  Table  1.6  I n i t i a l environmental conditions during time course 5 conducted o f ft h e west c o a s t o f Vancouver I s l a n d o n A u g u s t 25-26, 1986 52  Table  2.1  I n i t i a l environmental c o n d i t i o n s of seawater c o l l e c t e d i n t h e S t r a i t of Georgia f o r N uptake versus irradiance experiments  +  4  3  79  Table  2.2  P h y t o p l 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 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  Table  2.3  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 of 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 phytoplankton assemblages i n t h e S t r a i t o f G e o r g i a , B.C. Definitions are given i n the text, 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  I n d i c e s of N uptake dependency on PPFD f o r phytoplankton i n the S t r a i t of G e o r g i a : 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 *V ) , the PPFD a t which h a l f of t o t a l N uptake occurs (K ', K LT " ) * , r a t i o of uptake under 1% I t o 55% I (V : V ) . The K v a l u e s a r e expressed as PPFD v a l u e s and as a percentage of s u r f a c e PPFD ( I ) which i s shown i n parentheses 88 LT  Q  q  5 5 %  LT  Q  Table 2.5  Comparison of h a l f - s a t u r a t i o n c o n s t a n t s (K ) f o r inorganic n i t r a t e transport i n various aquatic ecosystems 89  Table 2.6  Summary of l i t e r a t u r e v a l u e s of d a r k : l i g h t n i t r o g e n s p e c i f i c ( /V ) o r a b s o l u t e ( P / p ) uptake r a t e s , determined d u r i n g daytime, i n n a t u r a l phytoplankton communities 94  LT  V  D  Table 3.1  L  D  L  K i n e t i c parameters f o r n i t r a t e , urea and ammonium uptake of N - r e p l e t e Micromonas pusilla. Michaelis-Menten parameters, K ( h a l f - s a t u r a t i o n constant) and V (maximum uptake v e l o c i t y ) were estimated from a d i r e c t n o n l i n e a r curve 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 of the data 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 the 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) 117 g  max  1  Table 3.2  2  Average n i t r a t e uptake r a t e s ( h ) f o r N0 ~s t a r v e d Micromonas pusilla. Rates determined from 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 of p a r t i c u l a t e N enrichment or the decrease i n the e x t e r n a l c o n c e n t r a t i o n of N0 ~ + NO " versus time and r e p o r t e d as ± 1 standard d e v i a t i o n ( i n parentheses) of the mean of d u p l i c a t e c u l t u r e s . 123 -1  3  15  3  Table 3.3  Average N uptake r a t e s V ( h ) f o r NO " - s t a r v e d Micromonas pusilla. Rates determined from l e a s t squares l i n e a r r e g r e s s i o n of p a r t i c u l a t e 15 N enrichment o r the decrease i n the e x t e r n a l c o n c e n t r a t i o n of d i s s o l v e d n i t r o g e n versus time and r e p o r t e d as ± 1 standard d e v i a t i o n ( i n parentheses) of the mean of d u p l i c a t e c u l t u r e s . 123  Table 3.4  Summary of c u l t u r e c o n d i t i o n s a t the beginning of each experiment 130  -1  xi Table  4.1  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 ) 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 g r o w n 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 measurements during the l i g h t or dark period are given i n parentheses 168 _ 1  Table Table  4.2 4.3  Summary o f c y c l o s t a t c u l t u r e b e g i n n i n g of each experiment  conditions  at the 171  Nitrogen s p e c i f i c uptake rates ( h ) , determined over 2 h i n l i g h t and d a r k n e s s , and 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 . 2 4 , 0 . 4 9 , 0.74 d " 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 o n 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 on: 0800 h , l i g h t s o f f : 2200 h ) . 174 _ 1  1  Table  4.4  Parameters describing the c h a r a c t e r i s t i c s of N s p e c i f i c u p t a k e ( h " ) , 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 (Fig. 4.11). 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 uptake ( 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 ) 1  T m  i  _ max  .  _  v  LT'  and 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 ) . E s t i m a t e d s t a n d a r d max  errors Table  4.5  of parameters  T m  LT '  are given i n parentheses  177  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: the ratio, of dark t o l i g h t - s a t u r a t e d uptake rate ( V : V ) , 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 occurs (K ', K " ) * and t h e r a t i o o f N uptake a t 1% I t o N u p t a k e a t 1 0 0 % I ( V : V ). S a t u r a t e d P P F D a n d I a r e t h e g r o w t h P P F D ( 1 2 0 uE m ^ s ) . . . 178 D  L  L T  1 %  - 1  xii  L I S T OF FIGURES  Figure  1.1  S t a t i o n l o c a t i o n s f o r time course experiments of n i t r o g e n uptake. (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 85. (B) TC.2 a t s t n A5; TC.3 a t s t n T 4 . 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 by d a s h e d l i n e s i n p a n e l A. 16  Figure  1.2  Depth p r o f i l e s o f 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) O c e a n i c 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, T C . 2 . (C) S t r a t i f i e d s t a t i o n T 4 , T C . 3 . The s h a l l o w t h e r m o c l i n e s t e p i s i n d i c a t e d by t h e a r r o w l a b e l l e d ' s T ' i n panel A 25  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 two 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 drogue-type time course experiments. 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 TC.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 TC.5) 27  Figure  D e p t h p r o f i l e s o f N0 ~ a n d N H d u r i n g time course 4  1.4  F i g u r e 1.5, Figure  1.6  3  Depth p r o f i l e s o f S i 0 and P0 " i n t e r v a l s d u r i n g time course 4 - 4  4  4  F i g u r e 1.8.  at 6 h 29  _  +  3  4  4  1.7  3  A: D e p t h p r o f i l e s o f N 0 (•) a n d N H (C) 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. B: D e p t h p r o f i l e s of Si0 " (•) a n d P 0 " (•) 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. 30 4  Figure  at 6 h intervals 28  + 4  3  4  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 community, A: a t t h e b e g i n n i n g ( s t n 24) a n d e n d ( s t n 49) o f t i m e c o u r s e 4 B: b e g i n n i n g ( s t n 84) a n d e n d ( s t n 98) o f t i m e c o u r s e 5 35 T i m e c o u r s e measurements a t o c e a n i c s t a t i o n F., T i m e C o u r s e 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 during experiment. (B) N atom % excess i n p a r t i c u l a t e matter f o rl 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 b a r s r e p r e s e n t ± 1 S.D. o f t r i p l i c a t e s ) p l o t t e d against elapsed time m e a s u r e d a f t e r t h e a d d i t i o n o f 1.0 p g - a t N-N0 *L . (C) 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 N0 " 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 t h e time 1 5  -1  3  15  3  x i i i i n t e r v a l between i tand t h ep r e v i o u s p o i n t on the curve and p l o t t e d against average i n c u b a t i o n time between sampling 37 F i g u r e 1.9.  Time c o u r s e measurements a t f r o n t a l station ( 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 ) N a t o m % excess i np a r t i c u l a t e matter f o r l i g h t and dark bottle incubations following addition of 6 / j g - a t N - L o f (B) N H , (D) N 0 and (F) urea (error bars represent t h erange 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 ( • ), N0 " ( o ) a n d u r e a ( A ) i n (C) NH , ( E ) N0 ~, a n d (G) u r e a - s p i k e d s a m p l e s . Dashed l i n e i n d i c a t e s no measurements o f d i s s o l v e d urea a t 3 and 6 h;. ( l e f t side of page) 40 1 5  - 1  +  _  4  3  +  4  3  +  4  3  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 (T4), T i m e C o u r s e 3; (right side  F i g u r e 1.11  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 " ( 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.. Rates determined f o r 3 o r 6 h i n t e r v a l s ; each p o i n t i n d i c a t e s a r a t e c a l c u l a t e d over t h etime i n t e r v a l between i t and t h ep r e v i o u s p o i n t on t h ec u r v e . Shaded area on t h eabscissa d e l i m i t s t h edark period. 42  station o f p a g e ) . 40 +  3  F i g u r e 1.12  T i m e c o u r s e measurements a t u p w e l l e d plume s t a t i o n s 24-49, t i m e c o u r s e 4. (A) Daily i n c i d e n t surface i r r a d i a n c e during experiment. ( 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% l " ( • ) a n d 1% I ( 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 a v e r a g e i n c u b a t i o n p e r i o d . . . 49 O  F i g u r e 1.13  1.14  O  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 plume s t a t i o n s 24-49, t i m e c o u r s e 4. (A) Daily i n c i d e n t surface i r r a d i a n c e during experiment. ( 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 100% I ( O ) , 30% I ( • ) a n d 1% I ( 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 a v e r a g e i n c u b a t i o n p e r i o d . . . 50 Q  Figure  o  q  q  T i m e c o u r s e measurements a t s t a t i o n s 85-98, t i m e c o u r s e 5. (A) Daily i n c i d e n t surface i r r a d i a n c e during experiment. (B) Nitrogen 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 and p l o t t e d a g a i n s t a v e r a g e incubation period 54 Q  xiv Figure  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), shallow 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 72  Figure  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 t h e t h r e e s t a t i o n s s a m p l e d (T14: f r o n t a l ; A5: shallow 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  Figure  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 Georgia. The 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 the Michaelis-Menten equation; the 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 were 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 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  Figure  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 ) and DCM (• ) phytoplankton communities of the S t r a i t of Georgia. The 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 the Michaelis-Menten equation; the 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 were 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 ) and T8 (deeply stratified) 84  Figure  3.1.  Nitrogen s p e c i f i c o v e r 10 min a f t e r  -  0.8,  1.6,  2.4,  4.2  u p t a k e r a t e s (V) d e t e r m i n e d t h e a d d i t i o n o f 0.2, 0.4,  and  10 uq-at  N-L"  1  of  N0  _ 3  ( A ) , NH (B) o r u r e a (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,«) o f Micromonas pusilla. Rates (h ) are p l o t t e d versus 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 t h e 10 min interval. C u r v e c a l c u l a t e d by 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 ) 116 +  4  _1  Figure  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 d e t e r m i n e d o v e r 10 and 60 min incubation periods. C u l t u r e s a r e numbered and v a l u e s a r e t h e mean (n = 2) o f 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  Figure  3.3.  N i t r o g e n u p t a k e by r e p l i c a t e c u l t u r e s o f n i t r a t e - r e p l e t e Micromonas pusilla over a 4 h incubation period. A. D i s s o l v e d N0 " + N0 " c o n c e n t r a t i o n (•) and N - a t o m % e x c e s s (O) a f t e r 10 uq-at N - u r e a - L " a d d i t i o n . B. D i s s o l v e d N0 ~ + N0 " 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- N 0 " « L addition, 3  15  1  3  2  _ 1  3  2  XV  respectively. D i s s o l v e d NO ~ + N 0 " c o n c e n t r a t i o n (•,•) a f t e r 10 L / g - a t N*L" a d d i t i o n o f NH and urea, r e s p e c t i v e l y . Dissolved NH 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 " . 120 2  +  4  +  +  Figure  3.4  1  D i s s o l v e d NO " + N 0 " 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 - a t N-L [ N] - NH enrichment; NH atom % 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 (min). Arrows d e s i g n a t e t i m e o f NH addition 122 2  - 1  1 5  +  1 5  4  +  4  +  4  Figure  3.5  N i t r a t e uptake 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 j j g - a t N-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 + NO " (•,•) i n t h e c u l t u r e m e d i u m' ; 15 NO, ~ , - , — " atom % excess 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 " + N0 " disappearance technique. C. [ N] n i t r a t e uptake rate. Values i n A are plotted against elapsed time measured a f t e r enrichment and 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 average incubation time 125 q  3  v  2  3  3  2  1 5  Figure  3.6  Urea uptake 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 / j g - a t N-urea-L" t o d u p l i c a t e c u l t u r e s (O,*). A. 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 elapsed time measured a f t e r addition of urea. B. [ N] u r e a uptake 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 2 6 1  1 5  1 5  Figure  3.7  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 *L to duplicate cultures. A. Dissolved NH 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 ((),•) ; N-NH 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 enrichment. B. Ammonium u p t a k e r a t e , d e t e r m i n e d by NH disappearance technique. C. [ N] NH u p t a k e r a t e . Values i n B and C p l o t t e d against average incubation time. 128 +  _ 1  4  +  4  1 5  4  +  4  4  Figure  4.1  C e l l c o n c e n t r a t i o n as a f u n c t i o n of time f o r nitrate-limited cyclostat cultures of 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 (O) 0 . 7 4 , (©) 0 . 4 9 a n d (A) 0.24 d dilution rates. Experiments were conducted on days 2 , 7 , 8 , 1 1 , 1 3 a n d 16 154 _ 1  Figure  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 o n 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  xvi Micromonas pusilla grown on a 14h:10h L:D illumination cycle. 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 . Growth 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 t i m e between s a m p l i n g  156  Figure  4.3.  Dissolved nitrate concentration i n culture medium ( A ) , 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 ( 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) 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 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 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 . Nitrate concentrations 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 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  Figure  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 o f Micromonas pusilla grown i n a 14h:10h L:D c y c l e a t 0.74 d ( A ) , 0.48 d ( B ) , . a n d 0.24 d (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 elapsed 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 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 159 _ 1  _ 1  _ 1  Figure  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 o f Micromonas pusilla grown i n a 14h:10h L:D c y c l e a t 0.74 d" ( A ) , 0.48 d (B), and 0.24 d" (C) d i l u t i o n r a t e s . Cell division p l o t t e d a g a i n s t average t i m e between s a m p l i n g , 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" . Dashed 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 161 1  _ 1  1  1  Figure  4.6.  Mean c e l l volume 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 grown i n a 14h:10h L:D c y c l e a t 0.74 d ( A ) , 0.48 d" ( B ) , a n d 0.24 d" (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 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 bar 162 _ 1  1  1  Figure  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 t h e medium 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 of Micromonas pusilla grown i n a 14h:10h L:D c y c l e a t 0.74 d ( A ) , 0.48 d" ( B ) , and 0.24 d (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 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 165  - 1  1  Figure  4.8.  _ 1  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 ) :  xvii  0.74 d" ( 0 . 0 3 1 h " ) ; B: 0.48 d " ( 0 . 0 2 0 h ) ; and C: 0.24 d" (O.OlOh ) d i l u t i o n r a t e s . 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 p e r i o d d e n o t e d by d a r k b a r 166 1  1  1  Figure  4.9.  1  I n t r a c e l l u l a r nitrate concentrations 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 g r o w n i n a 1 4 h : 1 0 h L:D cycle a t 0.74 d" ( A ) , 0.48 d " ( B ) , a n d 0.24 d (C) d i l u t i o n rates p l o t t e d against elapsed 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 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 169 1  Figure  4.10.  _ 1  - 1  1  _ 1  Maximum s p e c i f i c u p t a k e r a t e s ( h ) o f n i t r a t e ( A ) , u r e a ( O )/ a n d a m m o n i u m ( • ) d e t e r m i n e d i n 2 h incubations of samples from 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 ( 1 4 h : 1 0 h L:D c y c l e ) g r o w n a t 0.74 d ( A ) , 0.49 d ( B ) , a n d 0.24 d (C) d i l u t i o n rates. S p e c i f i c rates are plotted against average time of incubation period. 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 bar 172 - 1  _ 1  _ 1  Figure  4.11.  _ 1  Nitrogen s p e c i f i c uptake r a t e s , determined over 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 NH (•) or N 0 " (o) to nitrate-limited cyclostat c u l t u r e s o f Micromonas pusilla (14h:10h 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 " ( A ) , 0.52 d" ( B ) , a n d 0.24 d" (C) d i l u t i o n r a t e s . Uptake r a t e s (h ) 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, curved plots are f i t t e d d i r e c t l y to the M i c h a e l i s - M e n t e n e q u a t i o n by c o m p u t e r programme (see t e x t f o r d e t a i l s ) 176 1 5  +  4  1 5  3  1  1  1  _ 1  Figure  A . l .  S p e c i f i c g r o w t h r a t e (p) i n d~^ as a f u n c t i o n o f P P F D f o r M. pusilla grown on NO3 . Bars r e p r e s e n t ± 1 S.D. (n = 2 - 1 3 ) . Error bars are s m a l l e r than symbols where not v i s i b l e 228 -  Figure  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. Semi-log p l o t s of r e l a t i v e i n v i v o fluorescence ( O, • ) a n d c e l l c o n c e n t r a t i o n (• , • ) versus time 231 -  Figure  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. Decrease i n d i s s o l v e d N0o~ + N02~ c o n c e n t r a t i o n i n the culture medium. B. Accumulation of p a r t i c u l a t e organic nitrogen. C. R a t i o of NO3 uptake 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 N03~ + N O 2 disappearance from -  -  xviii  t h e medium. Nitrogen concentrations are p l o t t e d a g a i n s t elapsed time measured a f t e r c u l t u r e i n i t i a t i o n ; uptake rate r a t i o s are p l o t t e d a g a i n s t average elapsed time between successive sampling periods 2 34 Figure  A.4.  D i s s o l v e d NO3 and NC^ concentration i n the c u l t u r e m e d i u m , 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 , Dr. P . J . H a r r i s o n . H i s encouragement 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 personally rewarding. Thanks are a l s o extended t o the o t h e r m e m b e r s 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 . D e n m a n , T.R. P a r s o n s and 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 work. 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 has 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 , and C A . S u t t l e , and 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. T h o m p s o n . 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 the S t r a i t of Georgia 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. Forbes ( O c e a n 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 and 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 ) and 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. Analysts for 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 n d e n u m e r a t i o n w e r e R. W a t e r s and 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 and c r e w o f t h e C.S.S. P a r i z e a u and 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 of Ocean E c o l o g y (I.O.S.) and 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 b y a G r a d u a t e Research, E n g i n e e r i n g and 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 province of B r i t i s h Columbia, a U n i v e r s i t y Graduate F e l l o w s h i p from U . B . C , and s c h o l a r s h i p s from C h e v r o n Canada L t d . and S h e l l Canada L t d . T h e r e s e a r c h was s u p p o r t e d b y g r a n t s awarded t o Dr. P . J . H a r r i s o n from t h e N a t u r a l S c i e n c e s and 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 Canada and t h e Canadian F i s h e r i e s and Oceans S u b v e n t i o n programme.  1 INTRODUCTION  Overview  The g r o w t h  o f marine  on  their  ability  in  an environment  necessarily nutrients  phytoplankton  to assimilate i n which  nutrients  nutrients  o p t i m a l f o r growth.  utilized  by marine  i sdirectly  and photosynthesize  and/or  light  phytoplankton  (nitrogen,  silicon)  nitrogen i sthe nutrient  most  to limit  phytoplankton  (e.g.,  Ryther  Thomas,  o f t e n found  (Perry and Eppley,  is  recycled  however,  the major  slowly  likely  t h e growth  effect  characterized  to limit  (e.g., Nelson  of diatoms  ( i . e .not including + 4  ),  nitrate  i s reduced  dissolved  phytoplankton  growth.  Primary  1977), When  1981).  organic nitrogen,  nitrite  (NO2"")  and, consequently,  model t h a t d i s t i n g u i s h e d of dissolved  Silicon  and t h e r e f o r e  (Walsh,  I n 1967, Dugdale and G o e r i n g  the various sources  1984).  phytoplankton.  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  conceptual  marine  i n the sea that are well-  (N03~),  (CO(NH2)2) a r e t h e most abundant  phytoplankton.  (e.g.,  and Goering,  i s on s p e c i e s c o m p o s i t i o n  DON), ammonium ( N H  coastal  Phosphorus,  1981; Smith,  O f t h e n i t r o g e n (N) s o u r c e s  relevant  1979).  i ti s n o t r e q u i r e d by a l l marine  i s limiting,  i n both  supply i s  i n low concentrations, i s recycled  and t h e r e f o r e i t i s l e s s  generally  whose  1971) a n d o c e a n i c w a t e r s  productivity  it  growth  1966, 1969; Goldman e t a l . ,  although rapidly  and Dunstan,  are not  Of t h e t h r e e m a j o r i n o r g a n i c  phosphorus, likely  dependent  and urea  most  utilization  by  introduced a  the relative  importance  i n o r g a n i c n i t r o g e nf o r  production supported  by  of  2 allochthonous  N  surface waters fixation,  from deep ocean  riverine  production"; sources,  sources, principally  and  reserves,  as u r e a and  and  into  secondarily, was  Numerous f i e l d  from autochthonous  "new  N  was  termed  and  laboratory  documented the p r e f e r e n c e of p h y t o p l a n k t o n f o r the  more  of N  (ammonium a n d  oxidized  forms  (nitrate  Goering,  1967;  Eppley et a l . ,  1982)  presumably  and  because  urea)  nitrite)  reduced  McCarthy  forms  assimilate.  Nevertheless the oxidized  NO3 ,  quantitatively  may  -  be  ( e . g . , C a r p e n t e r and  1985;  1986;  In  the  detection  N-L  - 1  ;  50  Garside,  1985;  seasonal  pattern  Concentrations NO3 .  for  N  Price  t r e n d s and  productive  N-L  and  - 1  )  less  energy  to  particularly  1985;  or below  Probyn,  N-L  1987) Sharp,  - 1  ;  NH  1980; and  t h e most abundant  30  ng-at  Raymont,  1980;  + 4  :  limits  show no  apparent  1983). particularly  form of n i t r o g e n  1983),  exhibits  i s t h e most -important N  areas of the w o r l d ' s ocean  nitrogen  current  higher at depth,  regions (Sharp,  1977,  sources f o r  (McCarthy,  1980;  and  oceanic gyres,  ng-at  Harrison,  more  1989).  near  50  are invariably  )in coastal  seasonal  NO3":  (McCarthy,  Nitrate,  -  2  ng-at  forms,  Dunham,  of c e n t r a l  are consistently  (generally  urea:  for  et a l . ,  surface waters  concentrations of  Piatt  to the  et a l . ,  require  important N  phytoplankton, Cochlan,  relative  (e.g., Dugdale  1973;  and  "regenerated" have  forms  2  termed  studies  reduced  N -  ammonium f r o m a n i m a l e x c r e t i o n  remineralization,  production.  mixed  precipitation,  production resulting  such  microbial  inflow  nitrate  distinctive  source i n  (e.g., Eppley  (except  highly  and  3  Peterson, nitrate during  1979; E p p l e y ,  concentrations  1981; H a r r i s o n  of coastal waters  (> 20 uq-at  the winter  phytoplankton,  are gradually decreased  point  regenerated  t h e s p r i n g and subsequent  phytoplankton  growth  e t a l . , 1977; G l i b e r t  Nitrogen  uptake  or  -  divided  utilization  describes across  1980; Sharp,  into  t o be  tightly little  regions  5  N-L "*" -  1983; A n t i a e t a l . , i n p r e s s ) .  of nitrogen  two s t e p s ;  by p h y t o p l a n k t o n  the f i r s t  the actual transport  step,  termed  "uptake",  of the particular  form of N termed  "assimilation",  r e f e r s t o t h e sequence of metabolic  events  within  i n which the inorganic N ions  + 4  into  the c e l l  of NO3  ( i n the case amino a c i d s  1985).  -  and NC^ )  (Wheeler,  1983; Lobban e t a l . , n u t r i e n t s by  marine  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  N forms,  phytoplankton,  are reduced t o  and i n c o r p o r a t e d p r i m a r i l y  of nitrogenous  ambient N c o n c e n t r a t i o n ,  different  -  and p r o t e i n s  The u t i l i z a t i o n  phytoplankton the  plasmalemma, and t h e second  c a n be  step,  NH  the cell's  N i n coastal  0.5 uq-at  1986).  are of  b u t g e n e r a l l y do n o t e x c e e d  /jg-at N * L ^ and a r e f r e q u e n t l y below  The  gyres,(e.g.,  are envisaged  Forms, o f r e g e n e r a t e d  i n concentration  (McCarthy,  p r i m a r i l y by  1984) and t h e o x i d i z e d N f o r m s  no i m p o r t a n c e .  vary  i s supported  by  limits.  e t a l . , 1982b; C o c h l a n ,  and r e g e n e r a t i o n  (Goldman,  elevated  utilization  to detection  N, a s i n o l i g o t r o p h i c , o c e a n i c  McCarthy  coupled  are usually  -  during  this  Surface  N'L ^) and, f o l l o w i n g  stratification  At  e t a l . , 1987).  the relative  abundance o f  the physiological status  the availability  of l i g h t  of the  and temperature.  4 Uptake the  interactions  subject  McCarthy, range and  between i n o r g a n i c  o f many c u l t u r e  1981;  Syrett,  1981;  of responses which  i t s nutritional  and  vary with  state.  total  suppression (e.g., Syrett  NH  1972)  uptake  + 4  1986).  ( e . g . , Conway,  less  significance natural  1983;  attention  o f u r e a as  Harrison  a source of N  1985;  suppresses the uptake  NH  (e.g., Grant  +  The  effect  et a l . ,  1967;  of c e l l u l a r  Harvey  showed t h a t  (1953)  who  cultures  of  "normal"  cells  the  ability  elevated Harrison, after  -  was  Glibert  t a k e up  NH  +  Goldman,  or urea addition  have has  state  Syrett -  the  of  Kristiansen,  Syrett,  Generally,  on  N uptake  (1953)  uptake  than  1988b). by  and  by  batch  than  by  More r e c e n t s t u d i e s  have  p h y t o p l a n k t o n have  upon e x p o s u r e  ( e . g . , Conway e t a l . , and  1982,  acknowledged  much more r a p i d  4  -  at a lower l e v e l  NO3  N-starved or N-deficient  concentration  a NO3  by  and  + 4  t h a t were N - r e p l e t e .  to rapidly  1977;  NH  and  recently  1986).  M o l l o y and  demonstrated  "N-starved" c e l l s  shown t h a t  1985,  physiological  first  of NO3  forms  1983;  but  -  from and  et a l . ,  been g e n e r a l l y  of NO3 ,  reported  McCarthy  rates  only  a  species  f o r the growth  Turley,  p h y t o p l a n k t o n was  also  because  been  1963;  other N  1972,; Kaufman e t a l . ,  et a l . ,  reveal  ranging  + 4  Maestrini  p h y t o p l a n k t o n assemblages McCarthy,  NH  has  comparable  1977;  partly  which  Morris,  between u r e a and  urea 4  and  by  been  ( r e v i e w s by  1987)  uptake  degrees  t o s i m u l t a n e o u s and  Interactions  attracted  (e.g.,  to different  have  the phytoplankton  Nitrate  inhibited  forms  studies  Ullrich,  t o be  Eppley,  field  N  1982).  197 6; In  to N-deplete c e l l s  to  an  Conway  contrast, enhanced  and  5  u p t a k e may Price  o r may  and The  not  Harrison,  can  concentration  of  and  the  10~  D  1983;  u p t a k e by  nutrient,  has  marine  a h y p e r b o l i c f u n c t i o n of similar  i n form to  (Dugdale  l e d t o numerous  parameters  f o r both  phytoplankton  Goldman and  Glibert,  concentrations  to  10  et  a l . , 1969;  (Carpenter  - /  and  M  (e.g.,  studies  reviews  are  the  which  and  by  I t appears  f o r uptake  the  1967,  laboratory  1983).  regions  constants  than  waters.  for marine phytoplankton Maclsaac  Guillard,  eutrophic  1971)  that  i n the  species  having  explain  species distribution  (Eppley  et  temporal  intensity  and  particularly very  spatial diel  (euphotic  range  (day-night)  the  This  from  and  found  in  oligotrophic low  uptake  advantage  over  to  availability  1969).  a wide  Seasonal,  variations  clones  been used  to N  Dugdale,  Light attenuates  ocean.  has  exhibit  species  half-saturation  a competitive  and  quality  phytoplankton  zone) of  higher  in relation  variation.  pronounced.  limiting  hold  Maclsaac and  1969)  commonly  isolated  constants.  a l . , 1969;  are  that  conditions, species with  constants  higher  and  Dugdale,  which  or clones  Under N - l i m i t i n g  Light  and  show c o n s i s t e n t l y  species  (half-saturation)  depth,  Collos,  7  (Eppley  are  by  f o r enzyme k i n e t i c s  1968)  of  half-saturation fi  of  limiting  kinetic  1981;  review  steady-state N  d e s c r i b e d as  equation  assemblages  McCarthy,  r a t e of  Coatsworth,  have determined natural  be the  Michaelis-Menten  (e.g.,  1988b).  idea that the  phytoplankton  Eppley  occur  range  latitudinal  in overall  of and  intensity  exponentially with  growth to the  surface  Many s t u d i e s h a v e  waters  shown t h a t  N  6  uptake  i s related  saturation 1962;  occurring  Grant,  phytoplankton light of  to light  intensity  at high light  intensities  1967; E p p l e y and Rogers, communities  intensity  has been d e s c r i b e d  1972; F i s h e r  transport  et a l . ,  nor reduction  of oxidized  t h e dependence o f N uptake  one  (Syrett,  reduction and  (Syrett,  of NH  1981) w h i c h  may  + 4  on l i g h t  intensity,  membrane  requires  i s likely  light per  an  f o rNO3  indirect a n d NO2""  -  (Falkowski,  1975a)  f o rthe observations of  rhythms,  light  intensity  fashion  exhibits  every day.  light  assemblages  and i n c l u d e  extreme  Diel  coupled t o fluctuations  have been d e t e c t e d i n c u l t u r e s  phytoplankton  and  uptake.  i n a periodic  physiological  hyperbola  and u r e a depend on p h o t o s y n t h e s i s  account  most e n v i r o n m e n t s ,  variation,  upon  Since neither N forms  Hattori,  In natural  (e.g., Maclsaac  a n d ATP f o r membrane t r a n s p o r t  dependence of N In  1970).  Production of cofactors  the assimilation  (e.g.,  by a r e c t a n g u l a r  1982).  se,  1981).  fashion,  t h e dependence o f N uptake  the Michaelis-Menten formulation  Dugdale,  i n a hyperbolic  and  i n light natural  numerous  processes  ( r e v i e w by S o u r n i a , 1974), most n o t a b l y p h o t o s y n t h e s i s ( e . g . , Doty  and O g u r i , 1957; M a c C a u l l and P i a t t ,  "diel", used  used  almost  exclusively  throughout t h i s  whose p e r i o d Circadian describe  i s about  such  environmental  rhythms  to describe  24 h , o b s e r v e d  which  conditions  any  i n natural  as d i e l ,  persist  (i.e.  The  by o c e a n o g r a p h e r s ,  dissertation  h a s t h e same m e a n i n g  1977).  term  will  be  rhythm,  conditions.  but i s used t o  under  constant  endogenous c o n t r o l ) .  The  term  7 "diurnal", 24  being the opposite of "nocturnal"  h cycle,  but only  sunset  (Sournia,  and  rhythms  during  periodicity cultures natural  of N uptake  occurs  during  periodicity incubated rhythm  under  -  and N H  of nitrate  studies  + 4  with  1975) and  1982).  (1964)  Maximum  at night.  observed  by Sargasso  1970 ,  uptake  I n an  uptake  Sea p h y t o p l a n k t o n  which  suggests that the  A dampened a m p l i t u d e i n t h e d i e l uptake  increased  1975) o r N s t a r v a t i o n  Maske,  et al.,  constant illumination  was c i r c a d i a n .  periodicity field  o f NC^  i n cyclostat  (e.g., Eppley e t a l . ,  t h e d a y a n d minimum u p t a k e  study, Goering et a l . ,  t o describe  1971b; Malone e t a l . ,  phytoplankton communities  sunrise  Evidence of the  has been r e p o r t e d  1978; F i s h e r  apply t o a  occurs between  be used  hours.  (e.g., Eppley e t a l . ,  Maclsaac,  al.,  1974) and w i l l  the daylight  1971a,  early  t o an event which  cannot  has been o b s e r v e d N limitation  i n c u l t u r e and  (e.g., Malone e t  (e.g., Harrison,  1976; D o r t c h and  1982).  Objectives  The (1) of  To d e t e r m i n e natural  waters (2) NH  main o b j e c t i v e s  ,  o f fthe coast of B r i t i s h  and urea)  eucaryotic British (3)  the nitrogen  thesis  dynamics  phytoplankton assemblages  To d e t e r m i n e +  4  of this  i n laboratory  uptake  kinetics  To d e t e r m i n e  coastal  (NO3 , -  follows:  NH  + 4  from c o a s t a l  ,  and urea)  and oceanic  Columbia. studies  the nitrogen  o f an e c o l o g i c a l l y  p i c o p l a n k t e r Micromonas  Columbian  were as  pusilla,  (NO3 , -  important  isolated  from  waters.  interactions  between n i t r o g e n  uptake and  8 light the  through d i e l  field  Thesis  and  the  periodicity  and  irradiance  uptake  laboratory.  underlying  and  premise  assimilation  of t h i s  and  as  of previous i n t e r a c t i o n  a result  first  half  the p h y s i o l o g i c a l  of t h i s  dissertation  phytoplankton assemblages  neritic  and  phytoplankton discern  the  nitrogen  uptake  communities.  effect(s)  uptake  of  i n association  nutrition  studies  and  the necessity  versa. daytime,  daily  rates  coastal  waters  information a  In Chapter  These experiments  with  nitrogen  light  in  of  time  2 the  from  effects  of  of n i t r o g e n and  of  were d e s i g n e d  These experiments  to  were  of  irradiance, by  accurately  oxidized  and  highly  vice  during the  phytoplankton  These r e s u l t s  i n two  to  rhythms  hourly incubations or  nitrate-deplete  are described.  irradiance  course  phytoplankton nitrogenous  of uptake  frontal  of  1,  by  c o n c e n t r a t i o n s on  course experiments  r e g a r d i n g uptake  function  a function  The  uptake  confirm the e x i s t e n c e of d i e l  the uptake  nitrate-replete  the environment.  are described f o r a v a r i e t y  ambient  of time  In Chapter on  as  external  of the p h y t o p l a n k t e r  with  over day/night cycles.  conducted  estimate  of both the  state  oceanic environments. of n i t r o g e n  nitrogen  examines n i t r o g e n  natural  experiments  study i s that  are a function  environment  as  in  outline  The  and  experiments  from  stratified, provide reduced  unique N  forms  contrasting  environments. Recent (0.2  - < 2.0  work um,  showing Sieburth  the ubiquity et a l . ,  of p i c o p l a n k t o n  1978)  (e.g., Gieskes et a l . ,  9 1979; et  Waterbury  al.,  1983;  et a l . ,  Piatt  photoautotrophs, (e.g., by  Joint,  of  a picoplankter  utilization  and of  Takahashi  3,  NH^  obtained  NO3  t o NO3  1986)  uptake and  f o r subsequent  diel  light  with  choice nitrogen  batch  Micromonas  p u s i l l a ,  kinetics,  Requisite  picoplankton nitrogenous nutrition  reviews  on  the  the transient  studies.  as  oceanic regions  p r o m p t e d my of  L i  conditions.  picoplankter  starvation.  -  importance  were conducted  uptake,  -  1982;  B i e n f a n g , 1983;  laboratory  experiments  on  +  Sieburth,  their  and  Antia,  controlled  of the e u c a r y o t i c ,  and  and  t o examine t h e e f f e c t s  under  response(s)  1  1983;  and  in oligotrophic,  d e s i g n e d t o examine n i t r o g e n urea  of  1983)  S t o c k n e r and  In Chapter cultures  et a l . ,  Johnson  particularly  L i et a l . , 1986;  1979;  effects  uptake  information  This i s the  was  first  besides those  study  employing  c  N  tracers  (e.g.,  durxng  Probyn,  1985;  Continuous light:dark described  cycle  uptake  and  as  a  Experimental Micromonas  (basionym: (length  of  M.  4.  reduced  NO3  function  N of  natural  grown on  p u s i l l a  -  were used  -  limitation uptake  forms  communities  1988)  These experiments  o f NC^  In s i t u  of  Wood,  ( i . e .c y c l o s t a t )  i n Chapter  utilization.  H a r r i s o n and  cultures  examine the e f f e c t  oxidized  size-fractionation  on  rates,  a  14:10  i n the  experiments  were d e s i g n e d  diel  periodicity  potential  rates  during the day/night cycle,  irradiance  were  to of of and  determined.  organism p u s i l l a Chromulina  1 t o 2 um,  (Butcher) Manton e t Parke p u s i l l a ,  w i d t h 0.75  to  Butcher, 1 urn)  1952)  naked,  i s a  N  (1960) minute  unicellular,  N  10 photosynthetic considered (Manton,  t o be 1959;  Micromonas oceanic often  flagellate.  pusilla  and  Jeffrey, column  numerical  1983;  1984).  than  euphotic Micromonas  and  temperate  Taylor  (e.g.,  pusilla  been r e p o r t e d i n general,  Manton and  Throndsen,  deeper  Inlet,  c o a s t a l waters  July  cells«L  1977  i n Fraser  - 1  1989);  similar  waters  (e.g.,  tolerance  different this  nature  River  Throndson,  Waters,  plume,  1976)  July  and  Throndsen, in  and  potential  numerical  herein  Culture  C o l l e c t i o n , Dept.  British  Columbia)  was  reported  and  in  7  eta l . , Norwegian Its  salinity  s t r a i n s adapted  The  pusilla  to the  to  success  ubiquitous  enhances  e x t r a p o l a t i o n of  the my  populations.  (NEPCC 2 9 - 1 ,  of Oceanography,  isolated  2.2-10  therein).  contribute  i m p o r t a n c e o f M.  field  -  in Jervis  Clifford  i n the world's oceans.  data to natural  c u l t u r e used  1987,  t o form  for ecologically relevant  laboratory  0.1  references  e n v i r o n m e n t a l r e g i m e s , may  1976).  British  cells*L  of temperature  or the a b i l i t y  the  — 1  1982;  are often  1976  range  picoplankter  The  and  water  w e l l below  encountered  ( e . g . > 2.5*10  concentrations  of a wide  (Throndsen,  of  Taylor  1976;  and  i n the  1960;  and  1982;  7  Columbian  and  waters  Hallegraeff  often  wall.  i n coastal  Waters,  Parke,  i s frequently  a cell  arctic  (e.g.,  E s t e p e t a l . , 1984;  I t has  lacks  and  and  i s usually Prasinophyceae  occurring  dominance  1982;  flagellates  zone  1960)  i s ubiquitous,  Sieburth,  Hallegraeff,  Parke,  of t r o p i c a l ,  achieving  pusilla  a n o m a l o u s member o f t h e  Manton and  samples  Johnson  an  Micromonas  Northeast  Pacific  U n i v e r s i t y of  from E n g l i s h Bay,  B.C.  by  R.  Waters  i n January,  enriched,  natural  1971  and  seawater  subsequently at  16°C  on  a  maintained 14:10  L:D  in  cycle  12  CHAPTER  ONE  E F F E C T S OF D I E L P E R I O D I C I T Y ON N I T R O G E N U P T A K E BY A S S E M B L A G E S OF P H Y T O P L A N K T O N  NATURAL  INTRODUCTION Day-night parameters effects both  (diel)  cycles of biological  i n t h e ocean a r e o f t e n  o f s u n l i g h t on b i o l o g i c a l  photosynthetic  most o b v i o u s  diel  capacity  cycle  the manifestation processes.  and i n s i t u  (e.g.,  Sournia,  d i u r n a l ) rhythms have been r e p o r t e d  Doty  and Oguri,  dissolved in  1957).  1965) w i t h  (e.g.,  a concentration  assimilatory  activities  regenerative  increases  f o r many y e a r s  Diel  Lorenzen,  of phytoplankton from  zooplankton  in  then,  i n situ  al.,  numerous a c c o u n t s Maclsaac,  (e.g.,  Eppley  marine phytoplankton daylight uptake  hours  and minimal  of nitrogen  N limitation  periodicity  and K e l l y ,  demonstrated  of NO3  uptake  shipboard  -  and N H  (e.g.,  Eppley et  Malone e t a l . , 1975) o f  shown m a x i m a l u p t a k e d u r i n g t h e uptake during  S y r e t t , 1981),  by a r e l a t i v e  by  periodicity  the night.  has g e n e r a l l y been t h o u g h t t o be a  (e.g.,  + 4  1976) and N - l i m i t e d c y c l o s t a t  e t a l . , 1971b;  have  reported  of t h e Sargasso Sea.  of nitrogen  1978),  1971a; C o l l o s and Slawyk,  cultures  to  e t a l . (1964) f i r s t  i n the N-depleted waters  (e.g.,  have been  and h e t e r o t r o p h i c  i n t h e p o t e n t i a l uptake  Since  (e.g.,  (and b a c t e r i a ) and  marked  phytoplankton  diel  decline generally ascribed to the  Goering  cycles  i sthe  fluctuationsi n  1963; B e e r s  remineralization. diel  Periodicity i n  photosynthesis  inorganic nutrient concentration  natural populations  of the  1974) a n d b o t h  (and  1957; V e r d u i n ,  and r e l a t e d  enhancement  dampening  Dark response  diel  of dark uptake  capacity.  13 Culture  studies  substrate  affects  enrichments McCarthy,  NO3  1982; NH  i n N-starved  may b e l o s t  uptake  +  rates  are,  NH  Parslow  communities 1982;  and P r i s c u ,  N-replete  areas,  proportional 1967;  phytoplankton may d i f f e r their  patterns  of N  environments the  oceanic  Conway e t  phytoplankton  1981; Wheeler  regenerative  eta l . , waters,  a r e s u p p l i e d by processes,  whereas  availability  (e.g.,  Dugdale  These o b s e r v a t i o n s  from N-replete  and  Goering,  suggest  and N-deplete  response t o perturbations rates  at rates  waters  of nitrogen  of,different  that  by  nitrogen  t h e s e d i f f e r e n c e s may b e r e f l e c t e d  i n diel  uptake.  Experiments periodicity  exposure  In nitrogen-deplete  f o r , and uptake  and that  (e.g.,  Initial  N compounds a p p e a r t o be u t i l i z e d  communities  i n their  eta l . ,  1977; Goldman and G l i b e r t ,  1984).  e t a l . , 1977).  preference  substrates  i n culture  demands o f p h y t o p l a n k t o n  to their  McCarthy  t o take  (Dortch  enhanced upon  and Goldman,  ammonium a n d u r e a .from i n s i t u in  and  the ability  be i n d u c e d  however, o f t e n  Glibert  most o f t h e n i t r o g e n  Horrigan  1984).  e t a l . , 1984a,b) a n d n a t u r a l  (e.g.,  Priscu  (e.g.,  N  to  1983; P a r s l o w e t a l . , 1984b).  1976; Conway a n d H a r r i s o n ,  1982;  of phytoplankton  phytoplankton  concentration  + 4  the preconditioning  a n d Conway,  and must o f t e n  by C o l l o s ,  an e l e v a t e d  al.,  N substrates  1 9 8 1 , 1982 ; D o r t c h  review  4  to  -  t h e uptake response  of different  Additionally, up  have demonstrated t h a t  i n the present  of nitrogen of relative  uptake  study  were d e s i g n e d  i n three  contrasting  biomass and n i t r o g e n  subarctic Pacific  with  t o examine  low biomass  concentrations: and high  NC^  -  14 concentrations; NO3  and  varying  and  stratified  experiments  water  samples o r repeated  patterns  Prior  British + 4  ,  sampling  study  determined  Columbia  (Cochlan  utilizing  and  either  drogue-tracked  changes  i n irradiance  described  i nthis  was  t o employ  the first  and t o report NH  + 4  chapter  offshore  -  coast  o f NO3"",  The r a t e s  are the first  o f Canada.  The p r e s e n t  (Dugdale  ambient concentrations  waters.  t o NO3  of n a t u r a l communities of  N methodology  concentrations  uptake by  f j o r d s on t h e m a i n l a n d  e t a l . , 1986).  on t h e west c o a s t  adjacent  -  N-labelled substrates t o  of t h e uptake c a p a b i l i t y  determined  NO3  o f Canada was l i m i t e d  i n three  phytoplankton  1967)  frontal  course  o u r knowledge o f n i t r o g e n  on t h e west c o a s t  and urea  estimates  of a  Time  biomass  concentration.  phytoplankton  NH  i n duration,  o f uptake t o concomitant  to this  uptake rates  and diminished  respectively.  o f 24 h o r g r e a t e r  nutrient  of  elevated  concentrations,  plume o f moderate  and c o a s t a l inshore  p a r c e l , were conducted w i t h  relate and  concentrations;  -  waters with  phytoplankton  contained  a coastal upwelling  and  of urea  on t h e west c o a s t  study  Goering,  and f r e s h l y  o f Canada and  15  M A T E R I A L S 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 during and  three cruises  aboard  C.S.S. P a r i z e a u .  Project  SUPER  (Subarctic  Northeast  Pacific  1984,  one  and  The  cruise  Pacific  the  second  cruise  Strait  o f G e o r g i a , B.C.  carried  from J u l y a coastal  Canada between t h e m a i n l a n d and studies  were conducted  (TC.3).  Diel  August  t o 28  shelf  o f f the  offshore  studies August,  shelf  7 May  i s reported  on  Vancouver  (TC.2) and  Station  are presented i n Table  1.1  i n the  t o 25  May,  (TC.l).  1984  Island,  cruise  of  i n the  the west c o a s t of  on  course waters  86-04) from  the  18  continental  Island  locations and  time  stratified  (O.E.  c o a s t of Vancouver  (TC.5).  part  Research)  from  were conducted  conducted  C.S.S. V e c t o r  84-02),  t o August,  the t h i r d  1986  southwest  of the  experiments  out  basin  in frontal on  (OE  Ecosystem  time course experiment  During  were  the research vessels  first  O c e a n was  uptake  (TC.4)  and  f o r the  shown i n  Figure  1.1.  Sample  collection  Discrete correspond using  samples were c o l l e c t e d  t o 50,  either  o r w i r e ) and  30,  and  2 o r 5 L PVC  1%  of the  Niskin  then transferred  surface  bottles  into  (mounted on  darkened  10  o r 20  carboys.  salinity  were o b t a i n e d from c o n t i n u o u s p r o f i l e s ,  (cruise  sampling, using 2)  profiles  either  or a Guildline  of temperature  a InterOcean model  model  8701  selected  irradiance ( I  Nalgene  bottle  Vertical  from depths,  digital  CTD  a  Q  to ) ,  rosette  L and  run p r i o r 514A  to  CSTD  (cruises  1 &  16  F i g u r e 1.1. S t a t i o n locations f o r time course experiments of nitrogen uptake. ( A ) T C . l a t s t n F ; TC.4 a t s t n 2 4 ; T C . 5 a t s t n 85. ( B ) TC.2 a t s t n A 5 ; TC.3 a t s t n T 4 . P a n e l B i s an 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 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 seawater c o l l e c t e d f o r time c o u r s e experiments of n i t r o g e n uptake by n a t u r a l p h y t o p l a n k t o n assemblages. 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 Ocean; 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 plume o f f southwest c o a s t of Vancouver I s l a n d ; 85: o f f s h o r e 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).  Station and Location  F  Time Course Number  4 9 ° 5 ? .. 5' N  Sample Starting depth time o f i n c u b a t i o n (m) (PDT)  Date  TC. 1  16 May  TC.2  28  TC.3  29  TC.4  20 Aug.  N i t r o g e n cone. Urea NH + °3~ (ug- -at . N-L"•S  POC  (pg-L )  {uq-at N-L  - 1  ) (ug - a t  0. 32*  0. 15*  0.59  1.,87  19 . 5  0.27  4. 55  0. 60  2.12  7..28  47 . 3  0  0.19  <.  05  0. 33  0.39  3..57  31. 4  2  12.94  2. 38  1. 67  10.35  7,.75  46 . 8  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  1  0.09  1. 95  <.  05  1.09  2,.11  17 . 3  7 .14  0. 63  1. 25  2.24  2 .84 ,  12 . 6  0245  8  11.99  J u l y 1984  1000  0  J u l y 1984  0800  1100  1984  PON  Chi a  N  1 4 5 ° 1 4 .. 6 'W A5  4 9 ° 5 3 ., 0 ' N 1 2 5 ° 0 5 .. 8 ' W  T4  4 9 ° 5 5 ,, 5 ' N 1 2 4 ° 5 5 ..5 'W  24  4 9 ° 2 5 .. 0 *N  1986  1 2 7 ° 3 2 .. 1 'W  85  4 8 ° 1 6 .. 7 ' N  TC.5  25 Aug.  1986  1140  1 2 8 ° 1 8 .. 9 ' W  •Collected  from s e p a r a t e b o t t l e c a s t s a t s i m i l a r  28  stations.  C-L ) - 1  18 3).  Simultaneous  were determined (cruises with  with  with  equipped  recorded with recorder. LI-192S  with  a printing  integrator  a  flow-  monitored LI-185  (model  light  LI-550D)  were d e t e r m i n e d  (2n,  Quantum S e n s o r  (4/T, c r u i s e s  f o r nutrient  precombusted  an acid-washed holder, into  Ammonium  (NH  immediately following  + 4  cruise  and  or a  with  2) o r a  a  chart LiCor  LI-193SB  1 & 3).  )  acid-washed,  following  -  kept  a Technicon and Maclsaac  (PO^ ^) and s i l i c a t e -  t h e automated  and Armstrong (NO3  polypropylene  + NC^ ) -  dark  determined Autoanalyzer (1972).  -  and H a r r i s o n (1987),  stored  (< 12 h ) o r a n a l y z e d  procedures  Samples analyzed  Samples f o r frozen immediately  o f Wood e t a l . ( 1 9 6 7 )  respectively.  II  of Hager e t a l .  and u r e a were e i t h e r  t h e automated  Swinnex  ( S i O ^ ^ ) were a l s o  procedures  filters,  bottles.  e t a l . (1967), r e s p e c t i v e l y .  and c o o l  through  GF/F  Millipore  c o n c e n t r a t i o n s were always ship with  filtered  f o r 4 h) Whatman  a n d 25 mm  t h e method o f Slawyk  following  (-20°C),  a n a l y s e s were  (460°C  syringe  on b o a r d  phosphate  Price  with  methods  prewashed,  nitrate  (P.A.R.) was  irradiances  Quantum S e n s o r  Subsamples  (1968)  and measured  a LI-190SB S u r f a c e Quantum S e n s o r  Subsurface  Analytical  fresh  irradiance  a Lambda I n s t r u m e n t s L i C o r  Underwater  Spherical  filter  samples  111 f l u o r o m e t e r , e q u i p p e d  solar  continuously  using  fluorometer  cell.  Incident  meter  a Variosens I I Ii n situ  1 & 3) o r o b t a i n e d f r o m pumped  a Turner model  through  for  measurements 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  and  19 Duplicate (<  1 2 5 mm  filters  Hg f i l t e r  cruise  until  analysis  Duplicate  Sharp Erba  ashore.  with crystalline  model  the  dark  until  solution  Samples  Pacific,  Nitrogen  analyzer.  model  Both  method o f  240 o r a C a r l o  instruments  species analysis  were  were  preserved  e t a l . , 1984) a n d s t o r e d i n  (Throndsen,  N analysis  During  settled cruise  (24 1 to  1978) f o r e n u m e r a t i o n  of  r e p o r t e d a r e combined.  were c o l l e c t e d  folded, placed  into  frozen f o r later  i n the particulate  on precombusted  acid-washed isotopic  petri-  analyses.  s a m p l e s was c o n v e r t e d t o  gas (N ) by t h e micro-Dumas d r y 2  filters,  standards.  and t h e r e s u l t s  for  and n i t r o g e n  p a i r e d samples were a l s o p r e s e r v e d i n  and immediately  dinitrogen  organic carbon  Ten ml subsamples were  solution  Whatman GF/F f i l t e r s , dishes,  (Sigma Chemical C o . ) .  on an i n v e r t e d microscope.  coccolithophorids  1 & 2)  10 f l u o r o m e t e r ,  by t h e d r y combustion  (Parsons  analysis.  Lugol's  (< 1  (Parsons  o n c o m b u s t e d Whatman GF/F  acetanilide  Lugol's  northeast  Chi a  f o rphytoplankton  and counted  alkaline  immediately  fluorometry  model  a Perkin-Elmer  1106 e l e m e n t a l with  Designs  and a n a l y z e d  (1974) w i t h e i t h e r  acid  Whatman GF/F  added p r i o r t o  analyzed  by i n v i t r o  f o rparticulate  similarly,  Samples  h)  onto  filtered  C h l o r o p h y l l was e x t r a c t e d i n 9 0 %  and analyzed  samples  calibrated  the  and e i t h e r  & PON) w e r e c o l l e c t e d  stored  ( C h ia) were  differential)  a l . , 1984) u s i n g a T u r n e r  (POC  a  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  calibrated  in  pressure  of f i l t r a t i o n  aqueous acetone et  f o rchlorophyll  w i t h c a . 0.5 m l 1% MgCG^ s u s p e n s i o n  completion h,  samples  combustion  20 technique  as outlined  by Cochlan 1  subsequently N-150  analyzed for  emission spectrometer  Generally times)  each  and  (1982) and  average  N enrichment  w i t h a JASCO  (Fiedler  Proksch,  the p a r t i c u l a t e  material.  utilizing  a n di s o t o p i c  in-house  compatible  PC,  ratio  software  interfaced  standards  prepare  calibration  techniques  Tracer All of  used  dark  (Jones, unpubl.  were  1  5  heights  doc),  w i t h an IBM The e m i s s i o n o f pure  n i t r o g e n uptake water  (clear:  bottles)  NH C1, Na  1 5  4  incubators  light  experiments  was t r a n s f e r r e d bottles,  o f the  2  analytical  6.  N0 ,  orCO(  1 5  NH ) 2  were i n i t i a t e d into  caps,  2  and  within  500m l Wheaton  o r darkened  with teflon-lined 3  Isotopes).  with black  enriched with  ( a l l 99 a t o m %  Samples were i n c u b a t e d on deck  1  5  N;  i n clear  cooled w i t h c o n t i n u o u s l y - f l o w i n g near  s u r f a c e ( 3 m)  density  screeningt o  simulate  the  in situ  a t each  sample depth.  selected  time  filtered  (< 1 2 5 mm H g ) ,  isotopic  intervals,  analysis  randomly  selected  for collection  bottles  and  At  were  ofparticulate  o f "^N a t o m % e x c e s s ,  either  Plexiglas  covered with neutral regime  glass  Kor  and  light  1 h  tape:  seawater  for  N  N enrichment t o  The p r e c i s i o n i n Appendix  in  performed  with a series  b y JASCO o f known  are presented  o f peak  spectrometer.  calibrated  curves.  was used  experiments  collection;  bottles  1 5  supplied  1975).  activity) i n  selection  calculations  with the  s p e c t r o m e t e r was r o u t i n e l y gas  (specific  Automatic  model  (minimum o f 3  ^ N / ^ N peak h e i g h t r a t i o  o f the percentage  scans  and  s i x times  the c a l c u l a t i o n  during  (1983) a n d  c  sample was scanned  the  LaRoche  matter  dissolved N  21  concentrations.  Light  nitrogen  substrate  contents  of  the  Nitrogen equations in  of  Appendix  1.  and  using  a constant  uptake  (transport)  course  (1986) w h i c h  rates  (normalized  specific  uptake model  Wilkerson).  Differences  rates  (V  concentration  atom % e x c e s s the  length  during  each  (  )  N x s  of  time  incubation  uptake  and  the  rates  (f) of  incubation  environmental  unit  N  rates,  obtain Absolute  of  non-  the  Disappearance change  time are  in  and,  like  reported  for  have been c a l c u l a t e d .  procedures course  of  design and  experiments  Northeast  to  concentrations  period.  per  absolute  i n t e r v a l s over which they  experimental  light  Vj  dissolved nitrogen  time  course  V^,  ) have been c a l c u l a t e d from the  the  in  c  period.  product  n i t r o g e n - s p e c i f i c and  Over the  (V ,  between  interval  the  the  Experimental  PON)  successsive  i n  (were c a l c u l a t e d as  end  of  the  to  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  uptake  to  rates, V  of  ( i ) and  the  presented  specific  beginning  and  are  constant PON^,  each  1 R  N  rates  time  of  hourly.  R  s a m p l e s w e r e d i v i d e d by average  Wilkerson  D u g d a l e and  final  the  rates  r a t e s were c a l c u l a t e d a c c o r d i n g  S p e c i f i c uptake  1  initial  over  sample b o t t l e s mixed  D u g d a l e and  6 of  dark b o t t l e uptake  were measured  uptake  were estimated equation  and  Pacific  penetration  this  study,  were employed,  logistical reported.  (ca.  due  and to  f a c t o r s , i n the In TC.l,  O c e a n , w a t e r was depth  subtle  8 m)  major both  five  conducted  diel in  c o l l e c t e d from the and  changes  prefiltered  time  the 50%  through  I  102  •p  um  Nitex  nylon  netting to  remove l a r g e r z o o p l a n k t o n  Q  before  22  tracer with the to  e x p e r i m e n t s were  Na  1 5  N03  ambient uq-at  1.0  initiated.  at a tracer  level  Nine  N•L~^  hours  triplicate mid-day also 24  prior  after  light  determined  as £ 10% o f  tracer  concentration  Triplicate  samples  at 3 h intervals  enrichment  were darkened  for triplicate  inoculated  (T  = 0245  Q  h  PDT)  f o r 3 and 5 h  rates,  dark uptake  samples  collected  f o r 24  during  rates  were  and i n c u b a t e d  h i n darkness.  samples  3.  the second  cruise,  were c o l l e c t e d  Lig-at N-L  - 1  of  1 5  NH  Time-zero  withdrawn  + 4  ,  1 5  for  analysis.  concentrations a n d PON During drifters;  (07 0 0 - 0 9 0 0  N0 ~  2  3  or CO(  1 5  samples,  nitrogen + 4  were  , NO3-,  and  urea  filtration  collected  Samples f o r d i s s o l v e d  were t a k e n c o n c u r r e n t l y  by  nitrogen  and t h o s e  f o r C h i a and  e v e r y 6 h. the t h i r d  described  cruise,  drogued  positions tracks  The d r o g u e s were r e p o r t e d  are reported  an u p w e l l i n g  drifter  i n Mackas e t a l . ,  (TC.5).  TC.4,  w e r e a d d e d i n TC.2  was  and  drifter  6.0  particulate  sampling from a given water  their  h) a n d  At 3 h intervals  repeated 24 h  NH )2  for dissolved  i n a l lbottles.  from d u p l i c a t e  of Georgia,  i n the morning  samples  matter, -^N  i n the Strait  i m m e d i a t e l y and a n a l y z e d f o r N H  concentrations  POC  final  filtered  isotope  bottles  defined  to incubation.  t o estimate dark uptake  During  and  (usually  concentration) to bring  were removed and p a r t i c u l a t e s h.  Samples were  buoys  1989) w e r e u s e d parcel  f o r 48 h  (Loran-C t o guide (TC.4)  w e r e c e n t r e d a t 15 m d e p t h by r a d i o  i n Forbes  every half  hour;  et a l . (1987).  plume on t h e w e s t e r n  coast of  and  During  Vancouver  23  I s l a n d , was and  sampled  a t 6 h i n t e r v a l s f o r N uptake  concentrations Samples f r o m previously  the at  o f POC,  situ  and d u p l i c a t e N-L  - 1  1 5  3  s h e l f , a water  below t h e s u r f a c e  and t h e 1% I  —1 10 L i g - a t N*L situ  species  or  NH C1 4  duplicate Q  inoculated  for 4 h  simulated  offshore  repeatedly  of  sampled  and p h y s i c a l measurements f o r samples  were e n r i c h e d  from  just  with  IS o f Na  conditions.  NO3 and x n c u b a t e d  for 4 h in  Samples f o r e n u m e r a t i o n  simulated  of phytoplankton  were c o l l e c t e d a t t h e b e g i n n i n g and end o f b o t h  e x p e r i m e n t s t o d e t e r m i n e community sampling  1 5  p a r c e l was  sampling period,  described  from each depth  D u r i n g TC.5, i n t h e w a t e r s  2 h intervals for biological A t each  nutrients.  were c o l l e c t e d a s  Q  N0 ~  measurements  e x p e r i m e n t s and t h e  C h i a and d i s s o l v e d  samples  of N a  incubations.  continental  29 h .  in  PON,  100, 30, and 1% I  10 uq-at  with in  at 3 h i n t e r v a l s f o r physical  period.  composition over the  24  RESULTS  Physical  observations  The nitrate  vertical  Pacific  temperature thoroughly  euphotic  different  at  Ocean a r e p r e s e n t e d  mixed u n t i l  zone  (1% I  Q  A  1988)  s t n F)  i n F i g u r e 1.2  a t 30-40 m e f f e c t i v e l y  = 70 m)  Denman and  i n t o two  Gargett  and  layers  and  state  presented of the  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  cause d i f f e r i n g  of p h o t o a d a p t a t i o n  layers comprising  the  euphotic  c o n c e n t r a t i o n s were h i g h "mixed w a t e r " and  zone.  ( c a . 11  and step  divided  with  dynamics  a significant  exchange of p h y t o p l a n k t o n  degrees  The  (Denman  and  thermocline  (1988) d e m o n s t r a t e d t h a t  step presents  of the p h y s i o l o g i c a l  i n the  A.  6.5-7.0°C  above t h e m a i n p y c n o c l i n e / s e a s o n a l  shallow thermocline  indicators  84 02,  and  shallow thermocline  turbulence characteristics  80-100 m.  vertical  c a . 30 m.  s t e p < 0.5)  salinity  1 ( c r u i s e OE  o f t h e u p p e r w a t e r c o l u m n was  (sT, temperature  Gargett,  of temperature,  c o n c e n t r a t i o n f o r TC.  northeast  the  profiles  N'L  i n c r e a s e d t o 20-35 uq-at  two  to  seperate  sufficient  to  i n each of the  - 1  the  phytoplankton  Nitrate  uq-at  barrier  the  (plus  )  i n the  N«L  - 1  two  nitrite) surface  below  the  pycnocline. Vertical fluorescence  profiles and  NO3  s t n A5)  and  Georgia  are presented  features high  -  stratified  of the  of temperature,  relative  c o n c e n t r a t i o n s f o r the (TC.3, s t n T4) i n F i g u r e 1.2  f l u o r e s c e n c e at the depth  frontal  waters of the  B & C.  f r o n t a l w a t e r were t h e  i n vivo (TC.2, Strait  of  The d i a g n o s t i c  shallow thermocline  of the n i t r a c l i n e  (3 t o 7  and  m).  25  Figure 1 . 2 . Depth p r o f i l e s of temperature (T), s a l i n i t y ( S ) , i n vivo fluorescence (F), and n i t r a t e plus n i t r i t e concentration (N) for three stations sampled for containment time course experiments. (A) Oceanic s t a t i o n F, TC.1. (B) Frontal s t a t i o n A 5 , T C . 2 . (C) S t r a t i f i e d s t a t i o n T 4 , TC.3. The shallow thermocline step i s indicated by the arrow l a b e l l e d 'sT' i n panel A.  26  Time c o u r s e 3 was c o n d u c t e d and  the depth  maximum water  profile  i n warm  demonstrated  plume o f f Vancouver  sampling  surface  ( 2 , 6-7,and  layer  The v e r t i c a l  mixed  sampling  profiles  thermocline and N H  little  layer  ( c a . 10 ^ j g - a t N * L  +  ( s t n 24) a n d l a s t  decreased  demonstrate  a slight  by a weakening  at 6 h intervals  variable  observed  during  the next  concentrations  of the shallower, Vertical  ( F i g . 1.4) s h o w NH^  first  profiles  and PO^ ^  ambient  experiments.  concentration  of  relatively +  concentrations  a t s t n 24; t h e s e  i n the surface waters  of  -  of the  6 h t o l o w (< 0.5 / j g - a t N ' L  of t h e d i e l N uptake  -  warm  ( c a . 15  deepening  i n the surface waters  remainder SiO^ ^  ),  ( s t n 49) p e r i o d o f  change over t i m e , a l t h o u g h e l e v a t e d  were i n i t i a l l y  - 1  o f s a l i n i t y and temperature ( F i g .  over the sampling period. 4  t h e mixed  above t h e t h e r m o c l i n e / h a l o c l i n e  (51 h l a t e r )  s u r f a c e mixed  ( s t n 24-49) and t h e d e p t h s  14 m) w e r e a l l w i t h i n  1.3 A & B ) f o r t h e f i r s t  -  by n i t r a t e - d e p l e t e d  by r e p e a t e d s a m p l i n g o f an  Island  of nitrate-replete  (11°C) s u r f a c e w a t e r  NO3  fluorescence  ( F i g . 1.2 C ) .  upwelled  m).  a subsurface  ( c a . 10m) w h i c h w a s o v e r l a i n  Time c o u r s e 4 was c o n d u c t e d  of  (17°C), s t r a t i f i e d w a t e r  - 1  ) but  sampled  f o r the  Vertical  profiles  demonstrated  little  change over t h e course o f t h e time course experiment ( 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 depleted water ( 2 8 m,  ( 0 - 2 m)  f r o m t h e s h a l l o w ( c a . 7 m) m i x e d  1% I ) n i t r a t e - r e p l e t e  thermocline  surface  Q  depth.  Profiles  (> 5 / j g - a t N ' L of dissolved  - 1  )  nitratelayer  and deep  water  from t h e  inorganic  nutrients  27 F i g u r e 1.3. Depth 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 12  6  (°C) 7  10  8  II  12  '1  T ]  1 /  200 32  33  34  !  6  34  33 (% ) 0  TEMPERATURE 14  1  32  SALINITY  12  Stn. 49  (°C) 8  10  200 31  32  33  34  31  SALINITY  32 (% ) 0  33  34  28  Figure 1.4 Depth p r o f i l e s of N 0 " and NH during time course 4. 3  2  at 6 h i n t e r v a l s  + 4  2 i CM  +-  o1 o 1  2 UJ  oUJ  i  i  LJM  •4—  Q  1  HN  1  2 O + ro  NO  —  T  9  i  A  OO (^)  Hld30  (w)  Hld3Q  29 Figure during  Q-  in  a  o  1.5. time  Depth course  profiles 4.  H±d30  of Si0 ~ 4  4  and  P0 ~ 4  3  at 6 h  Hld3Q  intervals  F i g u r e 1.6 A: D e p t h p r o f i l e s o f N 0 " (<») a n d N H (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 (•) 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. +  3  4  - 3  4  o o _  Hld30 2  Z  CL  CO  (N0 ~, N H 3  + 4  ,  Si0  demonstrated sampling  - 4 4  , P0  little  - 3 4  )  are presented  variation  i n concentration  s p e c i e s were i d e n t i f i e d  and  visible  m i c r o s c o p y and  be  light  exclusion  of c e l l s  to distinguish  phytoplankton  < 2 um  Ochromonas  (4-10  um).  of the  composed an  f l a g e l l a t e Heterosigma dinoflagellates, diatoms  and <  cells*L  .  - 1  1%  total  (Chaetoceros  was spp.  83%  of  1988).  the  cell  same t i m e i n t h e attributed  removed by  The  N.  1%.  and  to c e l l s  majority  v.  Occasional  Prorocentrum  baltica),  and  Thalassiosira  spp.)  were o b s e r v e d , o f 4.8  Pacific, < 2 ^m  coccoid of the  x  but  10^  picoplankton,  i n samples  northeast  pennate  Raphidophycean  enumerated f o r that  genus  closterium  concentration  composed o f t h e b l u e - g r e e n , spp.  ca  (Nephroselmis  total  the  to  or  and  peruvianum  (1988) o b s e r v e d  b i o m a s s was  Synechococcus  difficult  c o n c e n t r a t i o n and  a d d i t i o n a l 8%;  (Gymnodinium  of the  cell  cylindrus  Samples were n o t  Booth  the  probably  h  the  (e.g., Booth,  Phaeocystis  akashiwo  prasinophytes  totalled  plant  29  using  by  Chrysophytes of the  d i a t o m s , p r i m a r i l y Nitzschia  centric  biased  f o r TC.l approximately  were 7%  striatula,  may  counted  (picoplankton) , which are  f o l l o w i n g g e n e r a , Imantonia,  during  the  e n u m e r a t e d were s m a l l h a p t o p h y t e s b e l o n g i n g  Chrysochromulina  was  thus  from i n o r g a n i c p a r t i c l e s  In t h e water used  although  and  observations  Phytoplankton  spp.)  over  1.6  period.  Biological  the  i n Figure  collected 16%  and  of  90%  the of  this  cyanobacterium, zooplankton  was  s c r e e n i n g the water samples t h r o u g h  Nitex  32  netting during  ( i n order t o minimize incubations) although  enumerated;  unfiltered  are.reported of  stratified different  diatoms  (£ 1 mm)  which  Navicula  a n d Nitzschia  of the S t r a i t  spp.  consistent, Small  The  to  (< 5 um)  i n the stratified  water.  a n d Skeletonema  diatoms,  whereas d i n o f l a g e l l a t e s  costatum  Gymnodinium  spp.  zooplankton  species enumeration.  they  animals,  as seen  approximation  categories  of these  aggregates  of the diatom  samples  were t h e most Chaetoceros  were  common spp.,  Ch.  were t h e most abundant were almost  exclusively  However  taken f o r  t h e abundance of samples,  g r a z e r s and N  diatoms,  nordenskioldii,  (particularly  Ch.  ceratosporum)  dominated  compressum  samples c o l l e c t e d  floes  suggested  remineralizers.  the concentration of general  Thalassiosira  the  chain-  belonging to  zooplankters are presented  centric  very  large,  formed  i n the phytoplankton  As a f i r s t  large,  was  Water samples were not o r i g i n a l l y  c o u l d have been important  The  water  none o f t h e f u t u r e w a t e r  flagellates  composition  N e x p e r i m e n t a t i o n and t h e r e f o r e  socialis  these  size  data  (TC.2) and  c o n t a i n e d some p e n n a t e d i a t o m s  to  phytoplankton  species  of Georgia  o f t h e g e n u s Chaetoceros  screening prior  screened.  The  In the frontal  prevented remain  s p e c i e s and abundance  community i n t h e f r o n t a l  1.2.).  forming  predation  samples were not  e t a l . (1988).  (TC.3) w a t e r (Table  screened  zooplankton  i n Forbes  the phytoplankton  macrozooplankton  Skeletonema  costatum,  a n d Chaetoceros > Ch.  spp.  radicans  >  i n terms of r e l a t i v e  a t a l l 3 depths  used  (Table 1.2).  Ch. numbers  i n TC.4.  (70%) i n The  Table  1.2  P l a n k t o n community c o m p o s i t i o n ( s e e F i g . 1.1 B ) .  Station  i n f r o n t a l and s t r a t i f i e d  Phytoplankton (10 c e l l s - L ) Diatoms Dinoflagellates Flagellates 6  w a t e r o f S t r a i t o f G e o r g i a , B.C.,  - 1  Zooplankton (animals-L ) Calanoid ciliates excl. Copepods tintinnids - 1  Tintinnids  Others  F r o n t a l A5 (TC.2)  2.3  0.023  1.6  470  50  730  280  S t r a t i f i e d T4 (TC.3 )  0.43  0.049  1.6  180  60  140  300  34 remainder of  o f t h e p h y t o p l a n k t o n community c o n s i s t e d  pennate  diatoms  delicatissima  6  depths  cells-L  increase 6  surface  -  t o 1.3 x 1 0  1  2.8 x 10  -  1  species  PP-  f  i n t h e deeper  (primarily  pennates  a t 1% I  ( 2 7 m)  waters.  costatum, Ch.  compressum  from  —,  1  3.0 x  (30%I ) Q  and an  3.6 x 1 0 ^ t o 5.0 x  about  one-third  a n d Phaeocystis  (58%) and pennate  conferta, debilis)  diatoms Chaetoceros  and t h e  nitzschioides  and  Nitzschia  In the surface  o f t h e c o m m u n i t y was composed o f haptophytes  (8%), including  a n d Protogonyaulax.  sample  from t h e  f o r TC.5 w e r e v e r y d i f f e r e n t ;  (21%) ( p r i m a r i l y  spp.) u n i d e n t i f i e d  and d i n o f l a g e l l a t e s  a t mid-depth  collected  a n d N. longissima.  diatoms, t h e remainder  this  Q  a n d Ch.  centric  but  , t h e mid-depth  Thalassiosira  only  collected  1  from  T h e m o s t common c e n t r i c  samples  Gymnodinium  -  (74%) both c e n t r i c  N. delicatissima  Imantonia  a t any o f t h e  decreased  ft t o 2.3 x 10 c e l l s - L  w e r e m o s t l y Thalassionema  americana,  (37%)  population  c o m p o s i t i o n i n t h e samples  ( 2 m) a n d d e p t h  w e r e Skeletonema S  concentration  i n the  .  dominance o f diatoms (15%)  cell  cells-L  6  i n the population  The  unidentified  between t h e b e g i n n i n g and end o f t h e drogue  from  cells-L  haptophytes  were no o b v i o u s d i f f e r e n c e s  ( F i g . 1.7 A ) ; s u r f a c e  population  10  spp.) and  c o m p o s i t i o n and t o t a l  sampling 10  There  > N.  ^ N. longissima),  a n d Phaeocystis  cryptomonads.  three  c f . subpacifica  > N. americana  (Imantonia  species  (Nitzschia  primarily  cryptomonads  genera of  S p e c i e s samples  were  ( 2 0 m) a t t h e e n d o f t h e t i m e  showed no m a j o r  differences  only series  i n species  a  35 F i g u r e 1.7 Composition of the p h y t o 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 c o u r s e 4 B: b e g i n n i n g ( s t n 84) and end ( s t n 98) of time c o u r s e 5.  |  Ld O  z:  o o  100  -  80  -  60  -  1 Centric  Diatoms  §\  Dinoflagellates  Pennate Diatoms  Chrysophytes  Cryptophytes  Haptophytes  Others  LU  o  40  < Io I-  20  -  0 2 m 5 m 14m Stn. 2 4  2 m 6 m 13m Stn.49  2 m 18m 2 7 m Stn. 8 4  20m Stn.98  36  composition  over time, except  haptophytes  and a decrease  f o r an i n c r e a s i n g  abundance o f  i n t h e abundance o f diatoms ( F i g .  1.7 B ) .  Nitrogen  uptake  Subarctic  rates  Pacific  During  Ocean  TC.l, i n the nitrate-rich 1  incorporation was  followed  night  daylight rate  of  N-labelled  nitrate  into  A relatively NO^  uptake  -  clear  diel  rates  calculated  ( F i g . 1.8 C ) ; m a x i m a l u p t a k e period  ( m e a n = 0 . 0 0 8 5 , S.D.  a t n i g h t was l o w e r at night  Samples  i n c u b a t e d f o r 24 h i n t h e d a r k  (0.0068  ± 0.00046 h  triplicate during  the first  equivalent time,  -  -  uptake  1  ).  Strait  ).  )  3 h  a n d mean  T h e mean value.  ± 0.00011  (paired  3 h ; mean d a r k  uptake  uptake  -  1  light-dark  - 1  )  cycle  darkening of  was 0.0072 h  (0.0071  h  - 1  )  -  1  (0.0156 h  -  1  effect  and  during  2 h of darkness,  , whereas t h e l i g h t  i t s maximal v a l u e  h  t-test,  f o r 3 a n d 5 h d u r i n g mid-day h a d no  9 2 % t o 0.00058 h  uptake  this  uptake rate of  ) .  of Georgia  During Strait  1  - 1  (0.00063  rates  The a r t i f i c i a l  t o t h e mean l i g h t  attained  -  incubated i n the natural  however, d u r i n g t h e subsequent  declined NO3  samples  -  over  was c a . 5 5 % o f t h e mean d a y t i m e  lower NO3  P *s 0 . 0 1 ) t h a n t h o s e  was  during the  ( 0.0047 ± 0.0013 h  uptake  significantly  rate  matter  of the  trend  = 0.0045 h  nitrate  had  particulate  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  forspecific  intervals  Pacific, the  c  ( F i g . 1.8 B ) .  apparent  subarctic  the time course experiments  o f G e o r g i a , t h e changes  conducted  i n t h e ambient  i nthe  concentrationof  F i g u r e 1.8. 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) N atom % excess i n p a r t i c u l a t e matter f o r l i g h t 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. o f 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 a d d i t i o n o f 1.0 uq-at N-NO -IT . (C) N i t r o g e n s p e c i f i c uptake r a t e s o f N0 " 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 t h e time i n t e r v a l between i t and t h e p r e v i o u s p o i n t on t h e curve 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 time between sampling. 15  1  15  3  ^  a  1  in  1500  o.ooo  8  12.  TIME  14  (h)  18  24  38  dissolved 1 5  NH  + 4  N-labelled  for  24 h .  of that  processes. estimate is  remains  elevated  allowed  Results  t h e uptake  seawater  samples.  Data  3  - 1  *h  samples  - 1  )  (V  Q  accumulation to  3  Uptake  N-L  rates  were s i m i l a r - 1  -h  _ 1  _  9 h  =0.267  uq-at  the N0 ~, 3  )  + 4  -  + 4  ,  ( F i g . 1.9 G ) . waters  and urea-  - 1  -h  - 1  by t h e  (V  rate  0  ).  The  1 5  9 h  =0.567  uq-at  enriched  N-urea  atom %  15 h , b u t p r i o r  and remained  ( F i g . 1.9 F ) .  coincided with  _ 4  i t increased  t h e end o f t h e incubation  d  i n t h e NH4" "  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  urea uptake  for NH  i n the presence  and absence  N-L  t h e end o f t h e dark period  until  determined  o f u r e a , b u t were reduced d  multiple  t o be d e t e r m i n e d by N d i s a p p e a r a n c e f r o m t h e  uq-at  =0.521  water) and  i nthe frontal  of N0 ~ i n the NH  rates  (V  N*L  (frontal  f r o m TC.2 d e m o n s t r a t e  N0 ~ concentration  of nitrate  6 h  f r o m TC.2  e x p e r i m e n t s a r e s h o w n i n F i g . 1.9 a n d  disappearance _  there  i n the dissolved  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  ambient  samples  Q  enrichment  3  enriched  d  a c c u m u l a t i o n i s an  ( F i g . 1.9 C, E ) a n d N 0 ~ a n d u r e a  and urea  The  and uptake  by 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  water)  uptake  Changes  r e p r e s e n t n e t community  N isotope  constant.  1.10, r e s p e c t i v e l y .  3  by t h e p h y t o p l a n k t o n .  o f -^N, a n d  Fig.  measured  information  and encompass r e g e n e r a t i v e  of gross uptake  substrate  different  nutrient  By c o n t r a s t ,  of those  matter were  concentration  (stratified  N0 ~  yield  utilization  TC.3  N  particulate  nitrogen  no r e c y c l i n g  phase  into  approaches  nitrogen  dissolved  flux  and urea and t h e i n c o r p o r a t i o n  -  substrates  Both  concerning in  , NO3  The i n c r e a s e i n  the depletion  moreover t h e change i n u r e a c o n c e n t r a t i o n  linear  of external  was m i n i m a l  39 F i g u r e 1.9. 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 ) N 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 f o r l i g h t and dark b o t t l e incubations f o l l o w i n g a d d i t i o n of 6 / j g - a t N• L o f ( B ) N H , (D) N 0 " 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 range of 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 measurements o f d i s s o l v e d NH ( • ) , N0 " ( o ) and urea ( A ) i n ( C ) N H , ( E ) N0 ~, a n d (G) u r e a - s p i k e d s a m p l e s . Dashed l i n e i n d i c a t e s no measurements o f d i s s o l v e d u r e a a t 3 and 6 h; ( l e f t side of page). 1 5  - 1  +  4  3  +  4  3  +  4  3  F i g u r e 1 . 1 0 . 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 T i m e C o u r s e 3; (right side of page).  station(T4),  40  41 (Fig.  1.9 G) o v e r t h e f i r s t  high  ( 4 . 5 5 t o 1.4 j v g - a t N * L  The  g e n e r a l l y reduced  exhaustion  occurred  The p a t t e r n stratified enriched  samples  not  i n these  urea-enriched  NH  substrate detail  diel  samples  by P r i c e  NO3  -  water  of urea  constant  t h e night and depletiond i d  utilization  i n t h e NH  regeneration,  were e v i d e n t  , NO3  + 4  after  and  -  from  and t o a  lesser  increases i n  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  uptake  The p a t t e r n  rates  i n nitrogen  suggests  uptake  ( F i g . 1.11).  of NO3  rates  than NH rates  -  of  1 5  N-labelled  the existence of  i n both  The d e c r e a s e  f r o n t a l and i n uptake  + 4  In the frontal  were g r e a t e s t  to the stratified  r a t e s were h i g h e s t  uptake  during  Substrate  was m i n i m a l  i n the  and urea-  -  U p t a k e was  and t h e t o t a l  ( s e e F i g . 1.9 C , E ) .  contrast  lower  interval.  o f NH  + 4  f r o m 2 1 t o 24 h i n T C . 2 w a s d u e t o s u b s t r a t e  exhaustion uptake  morning.  e t a l . (1985).  periodicity  and substrate  , NO3  + 4  with  ( 2 3 , 18 a n d 1 0 % , r e s p e c t i v e l y ) .  concentrations  stratified  in  isotopes  , NC>3~ a n d u r e a  + 4  i n t h e NH  up.  was n o n - l i n e a r  + 4  the nighttime  experiments  regeneration,  +  taken  N uptake by t h e phytoplankton  indications  4  N-NH  t h e 2 1 t o 24 h t i m e  i n the early  24 h o f n i t r o g e n  extent  1 5  9 t o 12 h , t h e n d e c r e a s e d  again  Clear  and  was b e i n g  -  ( F i g . 1.10 B, D, F , ) .  increased occur  and NO3  )  -  w a t e r was s i m i l a r  over t h e f i r s t  and  - 1  during  during  of  were  3  i n c o r p o r a t i o n o f -^N-NC^  time,  NH  6 h, when N 0 ~ c o n c e n t r a t i o n s  and NO3  uptake  increased  -  rates. prior  throughout  community, the time  community where NH  and urea  uptake  I n both  + 4  rates  experiments  t o t h e onset  course,  uptake similar but nitrogen  of the light  period  42 F i g u r e 1.11. N i t r o g e n - s p e c i f i c uptake r a t e s of NH ( • ) , N0 ( O ) and urea ( A ) i n (A) f r o n t a l and (B) s t r a t i f i e d water. Rates determined f o r 3 o r 6 h i n t e r v a l s ; each p o i n t i n d i c a t e s a r a t e c a l c u l a t e d over t h e time i n t e r v a l between i t and 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 . Shaded a r e a on t h e a b s c i s s a d e l i m i t s t h e dark p e r i o d . +  4  0 . 0 6 0  0 . 0 0 0  1  0  1  1  3  6  '  •  9  12  INCUBATION  1 15  PERIOD  18  21  (h)  2 4  3  43  and  this  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 NH  + 4  ,  and  ratio  of dark t o l i g h t  N0 ~ and urea i s given  portion  of the light  courses. (38%)  D  Initial  light  rates  negligible TC.2  uptake for NH  L  dark rates  a n d 6-24% o f V  L  +  3  frontal each  rates  water  Offshore  The l i g h t  rates  60 t o 6 6 % o f  declined  to a ofthe  dependence o f N 0 ~ 3  o f u r e a t h a n ammonium i n b o t h  2 a n d 2.4  than i n frontal  were on average  1.6 t i m e s h i g h e r uptake  initially  that  times  water,  whereas  i n the rates f o r  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  was f o u n d  most  ( 1 2 t o 18 h ) a n d t h e g r e a t e s t (0 t o 6 h ) .  waters  from  continental  3 depths  shelf  environmental presented  (52 t o  t h e remainder  were on average  Time c o u r s e 4 was c o n d u c t e d intervals  during  time  water.  over t h e dark period  disparity  major  w a t e r was c o n s t a n t  were  (Table 1.4). C h i a s p e c i f i c  substrate,  similar  of urea uptake  i n t h e s t r a t i f i e d water  uptake  frontal  uptakes were a  i n s t r a t i f i e d water  t o that  L  normalized per u n i t C h i a demonstrated  and urea uptake  greater N0 ~  rates  D  In both  i nthe frontal  i n TC.3.  and f r o n t a l  Uptake 4  + 4  1.3.  (V :V ) f o r  throughout t h e e n t i r e  of l i g h t uptake  u p t a k e was more s i m i l a r  NH  rates  + 4  rate  i n b o t h TCs b u t d a r k u p t a k e  portion  stratified  uptake  dark NH  and l e s s then t h e r a t i o  102%). the  The V : V  N  i n Table  3  s t r a t i f i e d communities,  1 5  i n Table  1.5.  samples c o l l e c t e d a t 6 h  i n an u p w e l l e d plume o f w a t e r  o f f Vancouver  conditions  from  Island.  The  of t h e water  sampled  The s p e c i f i c  rates  on t h e  initial during  TC.4 a r e  of N0 ~ uptake f o r 3  44 Table 1.3. N H  + 4  ,  R a t i o of dark t o l i g h t uptake r a t e s ( V ^ r V _ ^ ) of and urea f o r f r o n t a l and s t r a t i f i e d water of the of G e o r g i a , B.C., (see F i g . 1.1 B ) .  N O o  Strait  -  Station  Time i n t e r v a l (h)  +  (VD=V )  N0 ~ (V :V )  Urea (V :V )  N H  3  L  D  L  D  F r o n t a l A5 (TC.2)  0 6 12 18  - 6 - 12 - 18 - 24  0.37 0.39 0.37 0.39  0.08 <.01 <.01 <.01  0.60 <.01 <.01 <.01  S t r a t i f i e d T4 (TC.3)  0 - 9 9 - 18 18 - 24  0.58 1.02 0.52  0.18 0.60 <.01  0.66 0.24 0.06  L  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 uptake r a t e s o f NH , NO3" and u r e a i n f r o n t a l (A5) and 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 . 4  Nitrogen substrate  Time i n t e r v a l (h)  C h i a s p e c i f i c N-uptake r a t e [ug a t N (ug C h i a ) h ] Frontal Stn Stratified Stn  NH  0 - 6 6 - 12 12 - 18 18 - 24  0.091 0.060 0.025 0.028  0.261 0.133 0.030 0.047  NO3-  0 - 6 6 - 12 12 - 18 18 - 24  0.162 0.075 0.042 0.068  0.098 0.082 0.019 0.039  Urea  0 - 6 6 - 12 12 - 18 18 - 24  0.040 0.028 0.026 0.050  0.127 0.125 0.019 0.053  + 4  -  1  _ 1  46  samples depth  collected  from t h e n e a r - s u r f a c e  (6-7 m) were n o t s i g n i f i c a n t l y  P 2 0.01) a n d d e m o n s t r a t e d 1.'12 B ) . daylight h)  Maximum r a t e s  uptake rates  different  pronounced  t-test,  periodicity (Fig.  during  i n the early  the night.  r a t e was 15-16% o f t h e d a y t i m e o f samples  diel  (paired  Q  o f u p t a k e were o b s e r v e d d u r i n g t h e  hours, reduced rates  and minimal r a t e s  (1-2 m) a n d 30% I  collected  ( s i m u l a t e d ) were v a r i a b l e  evening  (1900-2300  The mean n i g h t t i m e The N O 3  rate.  a n d i n c u b a t e d a t t h e 1% I a n d d i d n o t show a c l e a r  -  Q  uptake depth  diel  pattern,  although the greatest  daylight  a n d t h e mean n i g h t t i m e r a t e was c a . 7 0% o f a v e r a g e  daytime  value.  a similar rates.  The s p e c i f i c  pattern  of d i e l  The p o t e n t i a l  v a l u e s were o b s e r v e d  rates  of NH  periodicity  specific  uptake  uptake  + 4  during  P 2 0 . 0 1 ) , were m i n i m a l d u r i n g daytime  nighttime  average  n i g h t t i m e r a t e s were  (transport) discussed  rates  the d i s s o l v e d presented ambient  3  forspecific  rates  nutrients NO3"",  i n Figures  concentration  of t h e deeper  reflect  + 4  ( F i g . 1.13). NH  + 4  1.4 a n d 1.5.  ,  S i 0  4  3  ~  The mean No d i e l  trend  samples and  rates.  Absolute  the patterns Depth p r o f i l e s o f and P 0  4  3  a n d show l i t t l e  oyer the sampling  (paired  maximal  evening.  120% o f d a y t i m e  o f N 0 ~ and NH  f o rthe 2  +  different  r a t e was 30-36% o f t h e d a y t i m e v a l u e . rates  uptake  4  the night,  and reduced i n t h e e a r l y  was o b s e r v e d i n t h e u p t a k e  -  o f NH  s h a l l o w d e p t h s were a g a i n n o t s i g n i f i c a n t l y t-test,  demonstrated  as t h e N O 3 rates  during  ~  are  change i n  period.  Time c o u r s e 5 was c o n d u c t e d by s a m p l i n g , a t 2 h intervals,  t h e p h y t o p l a n k t o n community  from t h e N 0 ~ - d e p l e t e 3  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 seawater c o l l e c t e d  Station and location  Date  f o r n i t r o g e n uptake experiments d u r i n g time c o u r s e 4.  Starting Sample time o f depth incubation (PDT) (m)  N i t r o g e n cone. N0 Urea NH 3  PON  Chi a  POC  4  (ug--at N •  (pg-lT ) 1  (uq-at  N - L ) (uq-at - 1  24  4 9 ° 2 5 .O'N 127-32 . 1 'W  20 August  1986  1100  1.7 5.4 14.4  12.90 11.80 11.60  2.38 0.82 1.02  1.67 1.78 2.63  10.35 11.74 7.21  7.75 7.35 6.15  46.8 44.5 37 .5  28  49°21 . 2 ' N 127°28 . 9 ' W  20 August 1986  1900  2.0 5.8 14.4  7.98 7.98 8.96  0.31 0.26 0.85  0.12 0.12 0.14  10.25 12 .89 9 . 16  11.21 11.50 8.58  72.8 74.5 60.6  31  4 9 ° 2 0 .3'N 1 2 7 ° 2 7 .6 'W  21 August  0134  2.0 5.8 14.4  8.78 8.59 9.07  0.71 0.62  0.82 0.60 0.92  19 .49 19 .58 18.54  8.18 8.27 8.01  51.6 51.6 52 .9  34  4 9 ° 1 8 .5'N 1 2 7 ° 2 7 .7'W  21 August  1986  0736  2.0 7.3 13.7  9.84 9 .89 10.10  0.75 2 .27 0.72  0.67 0.62 0.75  21.90 22.73 20.85  8.31 7.36 7.81  53.2 45.8 62.6  37  4 9 ° 1 7 . 1' N 1 2 7 ° 2 6 .9'W  21 August  1986  1327  2.0 7.1 14. 1  9.35 9 .16 9.42  0.30 0.41 0.36  0.20 0.17 0.19  14.70 15.04 16 .12  9.34 10.06 9.40  55.3 58.7 56 .1  40  4 9 ° 1 6 . 1' N 1 2 7 ° 2 6 .3'W  21 August  1986  1934  2.0 5.0 14.8  8.50 8.58 8.58  0.61 1.26 0.26  0.11 0.12 0.10  14.66 15.21 14.83  9.80 8.57 8.25  46.7 55.9 57 .6  43  4 9 ° 1 6 .3'N 1 2 7 ° 2 3 .2'W  22 August  1986  0128  2.0 5.0 13.4  9.09 9.07 9.14  0.41 0.29 0.32  0.17 0.17 0.18  13.03 14.33 14.90  7.81 7.39 6.96  46.6 46 . 1 48.7  1986  _  C-L  - 1  )  Table  1.5 c o n t i n u e d  station and location  Date  Starting Sample time o f depth incubation (PDT) <m)  N i t r o g e n cone. urea NH °3~ N  (ug- -at  Chi a  PON  POC  4  N• •IT ) 1  (ug--L~ ) l  (fjg-at  N - L ) (jjg-at - 1  46  49°16 . 0' N 127°21 . 4 'W  22 August  1986  0730  2.0 7.0 14.8  8.49 8.49 8.50  0.33 0.32 0.42  0.37 0.33 0.34  15.80 15.50 15.38  7.20 7.22 7.17  40.8 41.4 50.6  49  49°17 . 4 ' N 127°21 .3'W  22 August  1986  1347  1.5 7.2 13.9  6 .34 7.63 8.57  0.33 0.48 0.61  0.08 0.26 0.41  16 .10 14.00 13.41  9.26 8.73 7 .09  58.7 53.7 50.0  C-L ) - 1  CO  49  F i g u r e 1.12. Time c o u r s e measurements a t u p w e l l e d plume 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 irradiance during experiment. (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 1 0 0 % I ( O ) , 3 0 % I ( • ) a n d 1% I ( A ) c a l c u l a t e d over 4 h i n c u b a t i o n periods and p l o t t e d against average incubation period. Q  1500 r CN  'E LU -—  1000 -  LU O  < < cr  500 -  0 -  ^  0.060 -  T" "—'  0.050 -  LU  0.040 -  < h-  0.030 -  Z)  0.020 -  Q_  1 CO  o  0.010 0.000 -  "—  0.050 -  LU  0.040 -  TA  0.060 -  0.030 -  OL ZD  0.020 -  + T  X  0.010 0.000  30  TIME  40  50  (h)  60  70  50  F i g u r e 1.13. Time c o u r s e measurements a t u p w e l l e d plume 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 irradiance during experiment. (B) N i t r a t e a n d (C) ammonium a b s o l u t e uptake r a t e s a t 1 0 0 % I (O ), 3 0 % I ( • ) and 1% I (A) c a l c u l a t e d over 4 h incubation periods and p l o t t e d against average incubation period. Q  O  1500  ^  1000  o  500  1  0.600  -  0.500  -  0.400  -  0.300  -  0.200  -  0.100  -  0.000  -  _J  o 1  a. LU  < h-  0.  i r> O  0.600  0.500  0.400  0.300  0.200  0.100  0.000 30  TIME  40  (h)  50  Q  (0.07-0.35  uq-at  N-L  (5.08-8.82 jvg-at N - L (Table  1.6).  - 1  -h  - i  -h  - 1  Specific  incorporation plotted  against  average  pattern  of NO3  specific  of  periodicity  diel  TC.4, the  with  daytime NO3  incubation uptake  i n the surface at night  The  suggests the existence  samples,  similar  to that i n  and maximal values  during  u p t a k e was 3 7 - 4 7 % o f t h e  rate. specific  -  uptake  rates  o f t h e samples  of  displaced  are  depth  from t h e  i n F i g . 1.14 B.  rates  T h e mean n i g h t t i m e  ( 2 8 m)  Q  4 h incubation periods are  the day/night  a diel  pattern  absolute  reflect  3  f r o m t h e 1% I  depth were v a r i a b l e d u r i n g  The  and t h e N 0 ~ - r i c h  water  rates, determined  during  -  minimal values  daytime.  ) water  uptake  o f -^N-NC^  -  ) surface  although  uptake  rates  the patterns  presented  nutrients  NO3  ,  NH4  1.6 a n d s h o w l i t t l e  1  ,  Si0 ~ 4  3  and deep  f o rspecific  i n F i g . 1.14 C. _  cycle but  uptake  Depth p r o f i l e s a n d PO4  change over  suggestive  ( c a . 5 h) l a t e r  of the surface  observed  collected at  _3  time.  i n time.  communities r a t e s and  of dissolved  are presented  xn F i g .  Table  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 time c o u r s e 5 conducted o f f the west c o a s t o f Vancouver I s l a n d on August 25-26, 1986.  Station and location  Starting time o f incubation (PDT)  sample depth  48°17.5'N 128°19.3'W  0912  2.4 26.6  0.13 8.44  <0.03 1.44  0.92  0.93  85  4 8 °16.7 ' N 128°18.9 W  1140  1.3 28.4  0.09 7 . 14  1.95 0.63  <0.03 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  4 8 ° 1 5 . 4 ' N  2322  1.9 28.4  0.33. 7.66  <0.03 1.25  04 06  1.61 1.75  11.2 13.0  0205  1.9 26.8  0. 13 5.08  0.11 1.01  1.21 2.50  1.44 1.77  13.3 15.9  84  91  1 2 8 ° 1 6 . 4 ' W  92  48°14.9'N 128°16.8'W  (m)  N i t r o g e n cone, Urea NH< NO, (ug-at N-L  0.41 0.98  1,  )  Chi a  (ug-L  PON  (ug-at N - L  POC  - 1  )  (pg-at C-L  Table  1.6 c o n t i n u e d  Station and location  Starting time o f incubation (PDT)  Sample depth  N i t r o g e n cone. NO3 Urea NH  Chi a  (pg--at N - L )  (m)  POC  PON  4  - 1  (/jg-L ) -1  (pg-at N ' L ) ( y g - a t C - L - 1  93  48° 128°  14 5' W 16.5' W  0340  1.4 27.4  0.25 6.48  <0.03 1.50  0.70 2.85  1.72 1.69  13.3 15.7  94  48° 128°  14.7'' N 17.7''W  0545  1.7 25.8  0.35 5.73  <0.03 1.45  0.84 3.10  1.62 2.10  13.5 16 .0  95  48° 128°  14.6' ' N 17.0''W  0747  1.3 28.9  0.33 8.50  0.05 1.19  0.83 2.70  1.67 1.36  13.1 12 .9  96  48° 128°  14.6 ' N 16.2 'W  0944  2.2 27.7  0.08 8.24  <0.03 0.78  1.30  1.14 0.96  9.09 9.77  97  48° 128°  13.9 ' N 16.5 'W  1154  0.5 30.1  0.17 7.75  <0.03 1.94  0.56 1.72  1.14 1.12  11.3 11.0  98  48° 128°  13.5 ' N 17 .6 'W  1457  1.9 29 .1  0.21 5.67  2.27 <0.03 1.30 1.25  0.74 3.99  1.75 1.30  15.3 9 .86  * designates  c a s t time; no N uptake e x p e r i m e n t s  a t s t n 84.  0.37 2.93  _  - 1  54 F i g u r e 1.14. Time c o u r s e measurements a t s t a t i o n s 85-98, 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 experiment. (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 periods and p l o t t e d against average incubation p e r i o d . (C) Absolute uptake rates of n i t r a t e .  0.000 0.020  0.000  1  0  •  •  •  10  '  20 TIME  (h)  •  1-  30  55 DISCUSSION Experimental  considerations  Previous  diel  phytoplankton (1)  Eppley Lund,  containers,  et a l . , 1989);  over  time  al.,  1988);  which  (e.g.,  over  time  include  1977),  toxicity  substantially containers  alter  will  Sampling  1978).  Each  Containment  t h e same w a t e r  i s being  potential complicating  and H o r r i g a n , 1981) and  incubation  effects  metals  disadvantage  diel  i s the  as b o t t l e  1979),  large,  these  and  contaminants  "bottle-effects"  the contained water;  may  "clean"  effects.  a t one g e o g r a p h i c samples,  problem  (Venrick et  (Gieskes et a l . ,  These  these  1965;  techniques i n general;  present  1980).  minimize  with natural  parcel  migration of phytoplankton  The m a j o r  size  and L i v e l y ,  et  a water  and d i s a d v a n t a g e s .  and  location  1987; F i s h e r  i n plankton species composition  due t o t r a c e  (Carpenter  dealing  1965).  bottle  (e.g.,  N concentration (e.g., Lorenzen,  with bottle  changes  to  1976; K r i s t i a n s e n  (e.g., Maclsaac,  B l a s c o , 1978; C u l l e n  associated  time  i s made t o f o l l o w  and e l i m i n a t e s  i n ambient  transferred over  1978; T o b i e s e n ,  of knowing t h a t  time  approaches:  o c c u r s a t one g e o g r a p h i c  as a d v e c t i o n , d i e l  and K e l l y ,  subsampled  and Slawyk,  o r (3) an a t t e m p t  such  variability Beers  and then  has i t s advantages  subsampled  natural  a t one l o c a t i o n ,  (e.g., Maclsaac,  over  by  have t a k e n t h r e e b a s i c  (2) s a m p l i n g  t h e advantage  factors  of N uptake  1971b; C o l l o s  i s sampled  approach  al.,  assemblages  samples a r e c o l l e c t e d  incubation  has  studies  location  has t h e advantage  b u t does n o t t a k e  into  of  account  56 the  problem  o f a d v e c t i o n w h i c h may  communities  b e i n g sampled  potentially  a more pronounced  regions by  such  tides  Sampling distinct likely  be m i s t a k e n a particular  advantage  sampled  that  each  it,  experimental biological Cullen  activity  times.  Advection i s  i n tidally  influenced  of G e o r g i a where a p a t t e r n for a diel parcel  rhythm  of water  However  such  of the water  a logistically approach.  problem  i n different  community i s  parcel  of the l a t t e r  such as d i e l  i n phytoplankton.  an approach  more d i f f i c u l t  Both  requires  a n d a means o f and  expensive  methods  assume  migratory behaviour  and H o r r i g a n , 1981; Frempong,  caused  over time has t h e  t h e same p l a n k t o n i c  time.  knowledge of t h e p h y s i c s following  at different  as t h e S t r a i t  could  result  (e.g.,  1984) and p h y t o p l a n k t o n  sinking  (e.g., Bienfang et a l . ,  achieve  c o n s t a n c y i n p h y t o p l a n k t o n community c o m p o s i t i o n .  In the current following  w e r e made t o m i n i m i z e samples d u r i n g  1982) a r e m i n i m a l  study both the containment  of a water  parcel  zooplankton predation period  by  approach  wxth n e t t i n g  prior  and  Attempts  i n the contained  screening 1  macrozooplankton  i n order to  over time were u t i l i z e d .  the incubation  that  out  c  to  N inoculation.  In  1 R  all  time course experiments  labelled with  s u b s t r a t e s were used  isotope  unlabeled  saturating  dilution  so t h a t  of the isotope  regenerated N would  1982c; P r i c e  et al.,  occur  during  the incubation  1981;  Fisher  et a l . ,  additions the effects  enrichment  be m i n i m a l  1985) and s u b s t r a t e  1981).  period  associated  factor  (e.g. G l i b e r t  by et a l . ,  exhaustion would  ( e . g . , Goldman  Uptake r a t e s  re-  of  reported  et a l . , are  not  57  therefore  not  necessarily  potential  rates  phytoplankton  in situ  of N uptake  that  values but can  be  realized  c o m m u n i t y when p r o v i d e d w i t h  concentrations  o f N,  uptake  i n the  seldom  observed  a condition  surface,  and  often  especially  f o r the uptake  indicative by  of  the  saturating NO3  achieved for deeper  -  communities,  of regenerated N  in  but  natural  situations. The  incubation  (sampling) i n t e r v a l s  experiments were long response et  al.,  was  and  unable  Parslow et a l . ,  NO3 and  short  term v a r i a t i o n s  + 4  1982;  that  such  study.  to serious  al.,  1977). most  constant  enhanced  Bottle  station  uptake  would  indicate  o f C h i a and no  such  POC  artifacts  and  ( e . g . , Conway field  1984)  (e.g.,  and  thus  I  rate.  (Horrigan  1984b).  of S t r a i t  and  In l i g h t  of  relative  of Georgia Island  effects  (TC.2)  i t i s  employed  communities PON  i n these  shown  (Venrick  of  to et  containment  but  synthesis  time  i n the  have been  processes  the  to  p r o c e s s e s o c c u r r e d on  expect the consequences  severe i n h i g h e r biomass  uptake  i n uptake  reported  sampling i n t e r v a l s containment  course  N03~-sufficient  underestimates of r a t e  One  rates  Priscu,  (TC.4) o f f V a n c o u v e r  than the  the  of regenerated N uptake  i n the frontal  lead  t o be  u r e a by  Parslow et a l . ,  term rates  shorter  present  and  have been p r e v i o u s l y  t h e u p w e l l e d plume  scales  TC.3  of N H  and  and  to detect  long  unlikely  b)  Priscu  1981,  uptake  -  1984a,  laboratory  1981;  phytoplankton McCarthy,  i n the  to the rapid  Goldman,  Enhanced uptake  slower,  relative  of p h y t o p l a n k t o n seen 1976;  Glibert  ( 3 - 6 h)  i n the time  the  i n TC.2  experiments.  and  5  Simultaneous  uptake of nitrogen  Simultaneous documented Bienfang, 1979;  assemblages  1986,  only + 4  (e.g.,  Queguiner  Eppley  NO3 , -  pointed  and Lewin,  utilization  1974; Conover, 1975;  nitrogen  (1987),  e t a l . , 1989).  The  i n t h e f r o n t a l and  of Georgia, substrate  a n d u r e a may b e t a k e n  will  phytoplankton  e t a l . , 1977; M a e s t r i n i e t a l . ,1982,  of the Strait  o u t by C o l l o s  i s well  1976; DeManche e t a l . ,  e t a l . , 1986; C o l l o s  areas  -  and Renger, 1974;  1984) a n d n a t u r a l  Collos  demonstrate dual ,  (e.g.,  o f TC.2 a n d T C . 3 , c o n d u c t e d  stratified  NH  a n d Conway,  1977; McCarthy  results  1  1975; Caperon and Ziemann,  Dortch  Conway,  o f NH^" " a n d N O 3  utilization  i n laboratory  compounds  respectively not  utilization  but that  up c o n c u r r e n t l y .  multiple nitrogen  result i n a reduction  As  first  substrate  of the nitrogen-  1 c  specific the  uptake  rate  of the  N - s p e c i f i c uptake  labelled  N - l a b e l l e d compound compared t o  r a t e d e t e r m i n e d when o n l y  compound i s b e i n g  taken up.  t h e ^N1  I n a l l the experiments  1 5  saturating was  used  uptake  nitrogen  i n areas i n TC.4),  being  of high  incubation;  taken  ambient n i t r o g e n  which  the final gives  due t o  up, a r e p o t e n t i a l l y  PON d e t e r m i n e d  an accurate  only  a  levels (i.e. NH  I n TC.2 a n d TC.3 t h e a b s o l u t e  were c a l c u l a t e d u s i n g an  N - l a b e l l e d compound o f i n t e r e s t  and t h e e f f e c t s of i s o t o p i c d i l u t i o n ,  unlabelled problem  addition of the  uptake  + 4  rates  a t t h e end of  measure o f uptake  rate  15 of  the  potential labelled  N-labelled artifacts nitrogen  nutrient i n t o t h e phytoplankton caused  forms.  and avoid  by i n c o r p o r a t i o n o f n o n - ^ N -  59 Maestrini oyster NH  e t a l . (1982)  ponds t o o k up N H  concentration  + 4  results  samples. the NH  However, t h e N O 3  -  samples  samples.  Similar  reported  f o r both  Conway,  Conway,  uptake and  NH  +  studies  uptake  r a t e was r e d u c e d  studied  + 4  i seither  and S y r e t t ,  o f NH  due  regeneration  NO3  disappearance uptake  -  enhanced  i n the presence  laboratory -  NC"3~  during  s t u d y o f Lund  uptake  i n t h e marine  Molloy  and S y r e t t  enriched by 50%i n  has been  et al.,  1977, B l a s c o on u r e a  between NO3  than interactions  -  from t h e few a v a i l a b l e and t h e i n h i b i t o r y  (Horrigan  and Hodson, 1977; and McCarthy, 1982;  I n TC.2 a n d TC.3 t h e e f f e c t o f rate  concentrations  urea  + 4  e t a l . , 1967;  instantaneous (Williams  t o t h e l o w ambient  The  spiked  -  addition  + 4  suppresses urea uptake  1988a;)  .  1979) and n a t u r a l  (e.g., McCarthy  Nevertheless i t appears NH  i n t h e NH  (e.g., Grant  on u r e a d i s a p p e a r a n c e uptake  + 4  NO3  rates  1987) o r m a n i f e s t e d l a t e r  Molloy NH  .  uptake  The e f f e c t s  - 1  (TC.2) d e m o n s t r a t e d t h e  and S y r e t t ,  assemblages  once t h e  N»L  s u p p r e s s i o n o f NC>3~ u p t a k e  + 4  laboratory  1982).  that  response Lund,  NH  has been l e s s 4  microalgae of  a t t h e same r a t e  as compared t o t h e NC^  1977; C r e s s w e l l  phytoplankton and  community -  + 4  -  that  h a d d e c r e a s e d t o c a . 7 ug-at  and NO3  of NH  spiked  + 4  and NO3  + 4  from t h e f r o n t a l  similarity  demonstrated  i sdifficult  t o discern  of urea and evidence of  the incubation  period.  r a t e was u n a f f e c t e d  I n TC.2 t h e  or slightly  o f urea i n agreement w i t h t h e (1987)  who f o u n d  no s u p p r e s s i o n o f  d i a t o m , Skeletonema  (1988b)  found  and urea, b u t urea i n h i b i t i o n  costatum.  simultaneous uptake of  (24-26%)  of NO3  -  uptake i n  60 c u l t u r e s o f C h l o r e l l a emersonii after  prolonged  samples  (McCarthy  Skeletonema  has been r e p o r t e d  and Eppley,  (Molloy  low.  occasionally which  may  seawater cultures of  observed  period  effect  o f NC^  high  either  waters  biologically  TC.3)  makes i t on u r e a  -  ambient  ( 0 . 7 5 - 2.38 uq-at N ' L i n t h e upper  (TC.2,  and t h e p o s s i b i l i t y  the incubation  a n d TC.5 r e l a t i v e l y  a n d C.  concentrations  stations  observed  any i n h i b i t o r y  of urea  have,  /Ambient  and s t r a t i f i e d  during  to discern  concentrations  inhibition of  i n natural  1988b) .  The l o w c o n c e n t r a t i o n s  I n TC.4,  uptake.  Partial  1972) and l a b o r a t o r y  and S y r e t t ,  urea regeneration  difficult  tricornutum  ( L u n d , 1 9 8 7 ) a n d P. tricornutum  urea i n the frontal  were of  -  costatum  emersonii of  (1-2 d) N d e p r i v a t i o n .  by NO3  urea uptake  a n d Phaeodactylum  - 1  )  were  sampled  or through  ( 2 - 7 m)  isotopic  1 5  dilution rates  of  N i n particulate  of specific  Effects  NO3  uptakes  -  of light/dark  Diel  regime  periodicity  material, as  reduced  the potential  reported.  on nitrogen  i n the uptake  uptake  rates  o f NO3 ,  urea were e v i d e n t i n t h e time c o u r s e experiments various in  natural  phytoplankton assemblages.  t h e NC>3 -replete -  nighttime during  NO3  -  waters  uptake  mid-day  and lower r a t e s  strong,  diel  patterns  N03~-rich Antarctic uptake  o f NO3  -  a pattern  i n N03~ u p t a k e  and N H  + 4  those  Ocean,  reported  rates  during  and a f t e r n o o n .  Similar  have been o b s e r v e d  i nthe  by K o i k e e t a l . (1986)  during  and  I n T C . l , conducted  o f maximal  i n t h e morning  waters  half  +  of the  of the northeast P a c i f i c  r a t e s were about  the daytime, with  NH^  -  where t h e  t h e n i g h t t i m e amounted t o c a .  61  10-30  and 50%, r e s p e c t i v e l y of the daytime v a l u e s .  Olson  (1980) found i n 2 time course experiments t h a t N O 3 uptake -  ceased d u r i n g the n i g h t t i m e and N H  uptake was e i t h e r 25 o r  + 4  85% of t h e daytime r a t e , whereas G l i b e r t e t a l . (1982a)  found  anomalous r e s u l t s ; they found no d i f f e r e n c e i n N O 3 uptake -  between samples i n c u b a t e d i n t h e dark and those i n c u b a t e d over a normal l i g h t - d a r k regime f o r samples c o l l e c t e d from t h e N O 3 -rich  S c o t i a Sea.  During T C . l when samples which had normally  been exposed t o a n a t u r a l L:D c y c l e were suddenly darkened d u r i n g mid-day, a l a g of > 3 h o c c u r r e d b e f o r e dark uptake r a t e s d e c l i n e d t o the r a t e observed d u r i n g 24 h of darkness. The i n i t i a l  "dark" N O 3 uptake r a t e may have been t h e r e s u l t -  of p r e v i o u s l i g h t  s t i m u l a t i o n and only a f t e r some e l a p s e d time  were the r e d u c t a n t s , c o f a c t o r s and enzymes t h a t a r e necessary f o r N O 3 a s s i m i l a t i o n , and produced -  (or a c t i v a t e d ) d u r i n g t h e  l i g h t p e r i o d , used up (or d e a c t i v a t e d ) d u r i n g t h e a r t i f i c i a l l y imposed darkness. In t h e c o a s t a l waters of the S t r a i t  of G e o r g i a s i m i l a r  p a t t e r n s of d i e l uptake of a l l 3 N s u b s t r a t e s were observed f o r t h e NO-^-replete and NO-^-deplete N i t r a t e and N H of  + 4  s t r a t i f i e d waters.  n i g h t t i m e uptake r a t e s were about  one-third  t h e average daytime r a t e s i n t h e f r o n t a l waters and  d e c l i n e d t o about h a l f t h i s v a l u e i n t h e s t r a t i f i e d waters. Nighttime uptake r a t e s of urea were c a . 75 and 15% of t h e daytime r a t e s f o r f r o n t a l and s t r a t i f i e d waters, r e s p e c t i v e l y . F i s h e r e t a l . (1982) measured s i m i l a r d i e l v a r i a t i o n s i n N H uptake i n an e s t u a r i n e phytoplankton community w h i l e d i e l  + 4  -  62 i n NO3  variation  uptake  -  freshwater  reservoir  periphyton  communities  (1988a) rates  Sargasso replete  (Toetz, 1971). periodicity  collected  constancy of V :V D  bottle)  rates  f o r NH  f o rsaturated  o f phytoplankton exposed  circadian;  i n absence  uptake  areas of the -  running  1981).  by G o e r i n g e t a l . (1964)  variation  i n both potential  phytoplankton  communities  illumination.  variability  urea  i n samples under  collected  who f o u n d  Sea under  -  L  results control.  which  f o r urea and NC^  that  their  is  i ndetail  i n Chapter  phytoplankton  continuous found NH  no or  + 4  have  do n o t s u p p o r t  -  were  dependency  dependence o f uptake  studies  surface  In the Strait of  The l i g h t  Laboratory  (1989)  of NO3 ,  rates  comparable. discussed  by  from t h e N-depleted Barents Sea and  of V :V  other and demonstrate  uptake i s  + 4  rhythmic  and Lund  uptake  endogenous t h e o r y o f uptake D  + 4  t h e rhythm i s  a n d NC>3~ u p t a k e  + 4  constant light,  the results  NH  cycle  of the Sargasso  i n potential  w a t e r , when N H  This conclusion i s  However, K r i s t i a n s e n  diel  incubated  NH  clear  to the natural  of the light/dark  (seeChisholm,  bottle:  suggests that  supported  each  urea  trends i n NO3 -  light/dark  i n the frontal  + 4  c y c l e were p e r i o d i c ,  Georgia  and H a r r i s o n  (dark incubation  L  light/dark  the  Ceratophyllum-  areas.  incubation  free  Price  from N03~-deplete  S e a , b u t no d e f i n i t i v e  The  uptake  have been r e c o r d e d f o r  p l a n k t o n ( T o e t z , 1976) and  observed d i e l  o f samples  rates  similar to  on l i g h t  of these  was  nutrients  2.  shown t h a t  have h i g h e r dark uptake  N-deprived  rates  of nitrogen  than  63  N-replete  phytoplankton  Coatsworth, the  Strait  (e.g., Syrett,  1968; H a r r i s o n ,  1976; Rees and S y r e t t ,  o f Georgia dark N uptake  were h i g h e s t i n t h e N-depleted with  these observations; also  community, dark uptake light  rates  higher  + 4  water  Specifically, s u p p l y most waters  and o f NO3  to the frontal  rates  o f NH  i n frontal  -  (NH  water  important  t o remember  contributed  h a s b e e n shown t o  NO3  (e.g., McCarthy  e t a l . , 1982b; C o c h l a n  that  communities  i n c r e a s e s so  -  e t a l . , 197 7; 1986).  contrasted  t o t h e observed  merely  I t i s  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 markedly  and  likely  variability i n the light  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  based  i n N-depleted  -  ration  1980; G l i b e r t  areas.  (not preference) of NO3 f o r  importance  Harrison,  phytoplankton  are consistent  demand  and as t h e c o n c e n t r a t i o n o f ambient  nitrogen  The  and urea i n  + 4  and urea)  + 4  ofthe  i n s t r a t i f i e d water.  of the phytoplankton nitrogen  phytoplankton  i n agreement  i s envisaged t o support these  regenerated N  does t h e r e l a t i v e  and  relative  In  normalized t o Chi a  s t r a t i f i e d water  and urea  -  uptake  t h e way n i t r o g e n  rates  1979).  r a t e s were a g r e a t e r p r o p o r t i o n NO3  ,  Chi a specific  stratified with  f o r NH  1962; Eppley and  response  of N  uptake  on t h e p h y t o p l a n k t o n communities'  nitrogen  of NO3  observed  status. The upper  rhythmic pattern  waters  indicative  rates  of NH  15-16% o f daytime + 4  uptake  rates  i n the  ( 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  of i n s i t u uptake  v a l u e s were  -  uptake  diel periodicity rates;  was s i m i l a r  where n i g h t t i m e  the pattern  of  potential  b u t n i g h t t i m e r a t e s were  a  64 g r e a t e r p r o p o r t i o n (ca. 30-36% of the daytime S i m i l a r d i e l p e r i o d i c i t y of N O 3 and N H  +  -  4  values).  uptake r a t e s of  n a t u r a l phytoplankton communities i n upwelled r e g i o n s have been observed by o t h e r s .  Eppley e t a l . (1970) found t h a t Peru  C u r r e n t phytoplankton had n i g h t t i m e r a t e s c a . 25 and 62% of daytime v a l u e s f o r N O 3  -  and N H , r e s p e c t i v e l y .  C o l l o s and  +  4  Slawyk (1976) a l s o observed d i e l v a r i a t i o n of NC^  -  uptake i n  shipboard c u l t u r e s of s u r f a c e communities c o l l e c t e d i n t h e u p w e l l i n g area o f f Northwest 20% of daytime  A f r i c a ; n i g h t t i m e v a l u e s were c a .  r a t e s of uptake.  Japan, Mitamura and S a i j o  In e u t r o p h i c Lake Biwa,  (1986) found n i g h t t i m e uptake  of N O 3 t o be o n l y 10% of daytime -  rates  r a t e s and although the phase  f o r urea and NH^ " uptake p e r i o d i c i t y corresponded t o t h a t of 4  NO3  t h e amplitude was lower w i t h n i g h t t i m e v a l u e s 80 and 95%  -  of daytime  rates.  NC>3~ and N H  + 4  The absence  of an apparent d i e l rhythm i n  uptake of the deeper  (1% I ) community of the Q  upwelled plume was a l s o observed by Maclsaac NC>3 -replete -  dominated Mexico.  1% I  Q  (1978) i n t h e  samples of a phytoplankton community  by the d i n o f l a g e l l a t e Gonyaulax  polyedra  D i e l p e r i o d i c i t y i n both p o t e n t i a l N H  + 4  o f f Baja, and NC^  uptake r a t e s was, however, observed down t o t h e 10% I  Q  -  depth.  The amplitude of 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 N O 3 uptake -  r a t e s observed i n t h e N C ^ - d e p l e t e was  s u r f a c e waters used i n TC.5  g r e a t e r than t h a t observed f o r i n s i t u NC>3~ uptake of the  N03~-replete s u r f a c e waters of TC.4; n i g h t t i m e r a t e s were c a . 40% of daytime v a l u e s compared t o 15-16% i n TC.4.  These  r a t e s , however, a r e p o t e n t i a l r a t e s of uptake which r e p r e s e n t  65  rates  t h a t may b e r e a l i z e d  saturating pattern  t o uptake.  f o ri n s i t u  oligotrophic, of  diel  Salhsten  uptake  central  under  (1987)  rates  have been observed f o r n a t u r a l  (Cochlan,  rates  1983a),  the continental  (Whalen of NH  + 4  The  uptake  -  deeper  deplete  d i d n o t show d i e l  rates  no  o f NC^  absence and N H  -  collected  + 4  f r o m N-  Arctic  o f f Nova  Scotia  Toolik  Although  periodicity  diel  i n the  -  Similarly  Canadian  shelf  Lake,  potential  ( C o c h l a n , 1982;  periodicity of  potential  o f t h e f r e s h w a t e r community was o b s e r v e d .  p h y t o p l a n k t o n community  communities  a definitive diel  i n TC.5 was n o t N O 3 -  i n species  o f TC.4.  pattern  uptake were g e n e r a l l y rates  o r NO3  rates  and A l e x a n d e r , 1984a).  a n d more s i m i l a r  upwelled  discern  1982, 1986) and t h e u l t r a o l i g o t r o p h i c  Whalen a n d A l e x a n d e r , 1984a) d i e l NO3  + 4  assemblages  surface waters of the eastern  (Harrison,  Alaska  of NH  uptake  of concentrations  could  North P a c i f i c Gyre.  periodicity i n i n situ  deplete  conditions  composition t othe  Although i t d i dnot demonstrate  of uptake, lower rates  observed a t night with  o f NO^  increased  -  uptake  i n t h e day peaking l a t e r than those o f t h e surface  community.  Miyazaki  e t a l . (1987)  o f u p t a k e maxima f o r N O 3  delay  incubations  -  observed a s i m i l a r  and N H  + 4  o f t h e p h y t o p l a n k t o n o f Lake  They a t t r i b u t e d  this delay  during  dark  Nakanuma,  time bottle  Japan.  t o the cumulative increase i n  stored  energy  during  p h o t o s y n t h e s i s and necessary f o r t h e uptake and  assimilation In  and intermediate  c a r b o n compounds  produced  of nitrogen.  summary i t a p p e a r s  that  diel  periodicity  of nitrogen  uptake (1)  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  t h e amount o f p h y t o p l a n k t o n  composition; nitrogenous variation from  of  assemblages.  the  rhythms  Eppley  by d i e l  phytoplankton. the  need  unialgal effects  1987),  -  The r e s u l t s  that the community  line  of  study  laboratory experiments  reasoning role i n of  demonstrate  utilizing  and determine  on t h e p e r i o d i c i t y  might  occurring at  distribution  to isolate  et  observed  division  of the present  populations i n order  of  Eppley  also play a significant  and temporal  of  phytoplankton  suggested  B y t h e same  may  of spatial  of N l i m i t a t i o n  i n the  of the phytoplankton  of t h e day.  the effect  reductase,  of c e l l  effects  on p e r i o d i c i t y  by n a t u r a l  periodicity  f o r controlled  phytoplankton.  the inhibitory  temperature  e t a l . (1971a)  i n N uptake  regulation  light  Tobiesen,  of n i t r o g e n uptake  times  hence p r e c o n d i t i o n e d  a l s o be r e f l e c t e d  and d i v e r s i t y  affected  diel  ,  and  (3) depth  ( e . g . , NC>3~ a n d N C ^  1 9 7 0 , 1 9 7 1 b ) may  different  + 4  and ambient water  periodicity  be  ambient  (4) t h e  In addition,  (e.g., NH  enzyme a c t i v i t i e s  structure  (irradiance):  i s collected,  of phytoplankton.  other N forms  al.,  biomass and i t s s p e c i e s  i n concentration of  intensity  plankton  irradiance  including:  compounds as t h e s u b s t r a t e f o r N u p t a k e ;  i n light  which  history  (2) v a r i a t i o n  factors  of N uptake  the by  67  CHAPTER  TWO  E F F E C T S OF I R R A D I A N C E ON N I T R O G E N U P T A K E BY P H Y T O P L A N K T O N : C O M P A R I S O N OF F R O N T A L AND S T R A T I F I E D C O M M U N I T I E S  INTRODUCTION In most marine nitrogenous  nutrients  availability 1969;  by  1985)  and  (e.g., Maclsaac  a rectangular  Fisher  photosynthetic Dugdale,  uptake  i n many m a r i n e 1982)  and  Whalen and  Alexander,  uptake  assimilation  and  (e.g. Maclsaac  energy  1972;  to  and  photon  flux  the  density 1984).  The  been d e s c r i b e d  (e.g., Maclsaac  and  Dugdale,  1984b) c o m m u n i t i e s .  1984;  Although  nitrogen  p h y t o p l a n k t o n are dependent  through  p h o t o s y n t h e s i s , the exact b i o c h e m i c a l mechanism(s)  (e.g.  see  activated of  NO3  uptake by  r e v i e w by  1981).  The  a physiological  of N H  + 4  and and  uptake  basis  u r e a as w e l l .  The  reactions  of N H  + 4  (permeases)  presence  (GS/GOGAT) a n d  urea  -  the c e l l  energy  may  NO3 -  of  membranes  1975a,b), between  ATPases e x i s t  f o r the  and  by  remains unresolved  f o r the coupling  probably specific  enzymes  indirectly  phytoplankters (Falkowski  photophosphorylation i s required  these  or  ATPase, a p p a r e n t l y l o c a t e d w i t h i n  uptake  -  nitrogen metabolism  Syrett,  a number o f m a r i n e  provides and  regulates  directly  upon  an  light  source, either  1972;  PPFD as  which  by  to the Michaelis-Menten  freshwater (e.g., Priscu,  by  of  Dugdale,  Priscu,  upon PPFD has  hyperbola similar  et a l . ,  the uptake  phytoplankton i s related  and  dependence of n i t r o g e n  formulation  freshwater systems,  of the n u t r i e n t s  Probyn,  (PPFD)  and  (ATP)  f o r the generated  functioning  also  drive  (UAL-ase)  light  of  the  assimilation.  68 In a d d i t i o n , t h e photogeneration of r e d u c t a n t s NAD(P)H and reduced f e r r e d o x i n w i l l d r i v e t h e r e d u c t i o n of NG^ , NG^ and -  the GOGAT r e a c t i o n of N H  + 4  assimilation.  Other  -  possible  i n t e r a c t i o n s of l i g h t with i n o r g a n i c n i t r o g e n metabolism of phytoplankton are d i s c u s s e d i n d e t a i l by S y r e t t Numerous c u l t u r e s t u d i e s have demonstrated d e p r i v e d phytoplankton have g r e a t e r dark uptake than N - r e p l e t e phytoplankton Coatsworth, 1979)  (e.g., S y r e t t ,  (1981). that nitrogenr a t e s of N  1962; Eppley and  1968; Thacker and S y r e t t , 1972b; Rees and S y r e t t ,  s u g g e s t i n g a l e s s e r l i g h t dependence on N uptake, d u r i n g  N stress.  This together with f i e l d  d e e p - l i v i n g phytoplankton v e l o c i t i e s with l i t t l e Whitledge,  s t u d i e s which show t h a t  s u s t a i n s u b s t a n t i a l N uptake  or no l i g h t  (e.g., Conway and  1979; Nelson and Conway, 1979; P r i s c u , 1984)  suggests t h a t both l i g h t exposure  and n u t r i t i o n a l h i s t o r y of  phytoplankton may be important i n determining t h e i r a b i l i t y t o sequester n i t r o g e n , and t h a t these c o n t r o l l i n g f a c t o r s may d i f f e r f o r the v a r i o u s forms of n i t r o g e n . Shallow sea f r o n t s , l o c a t e d a t t h e 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 and Powell, 1984; LeFevre, primary p r o d u c t i v i t y al.,  (see reviews by Denman  1986) a r e g e n e r a l l y areas of high  (e.g., Pingree e t a l . ,  1981, 1983; H o l l i g a n e t a l . ,  1984).  1975; Parsons e t  These r e g i o n s are  c h a r a c t e r i z e d by having high phytoplankton biomass i n t h e s u r f a c e water w i t h measurable c o n c e n t r a t i o n s of n i t r a t e , and a shallow p y c n o c l i n e which extends t o the s u r f a c e a t t h e 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 normal  to a frontal  boundary  progresses  from  high concentrations of dissolved  well-mixed  side  t o N-deplete  represents  a gradient of both  and  consequently  differ  along  such  physiological  nutrition a transect.  N  such  and urea  + 4  from  whereas,  i n N-rich areas,  utilized  at rates proportional  Dugdale and Goering The the  Strait  presented  of Georgia,  Harrison regions Price  et al.,  et al.,  1985).  regenerative  i n this  by p h y t o p l a n k t o n  study were conducted i n b a s i n on  by L e B l o n d , 1983; tidally-induced  described  The i n f l u e n c e  (e.g.,  1977).  enclosed coastal  where s e v e r a l  and n i t r a t e - d e p l e t e  (Parsons  frontal  e t a l . , 1981;  o f PPFD on t h e u p t a k e  from  nitrate-replete  stratified  water  was e x a m i n e d a n d  dependence o f N uptake  on PPFD b y t h e p h y t o p l a n k t o n  the  subsurface chlorophyll  maximum o f t h e s e  NO3  -  This  uptake  function  i s the first  by n a t u r a l  o f PPFD.  were attempted the  study  true NO3  -  t o measure both  i n situ  response  from areas  urea and as a  experimental conditions  i n order t o obtain a better uptake  two d i s t i n c t  assemblages of phytoplankton  Simulated  of  frontal  the  compared.  reduced  processes  availability  et al.,  (seereviews  have been p r e v i o u s l y  NC>3~ a n d u r e a water  1983),  N-impoverished  a r e s u p p l i e d by  to their  a partially  t h e west c o a s t o f Canada  would  n i t r o g e n compounds a r e g e n e r a l l y  1967; McCarthy  experiments  states.  In the  t h e N demands o f p h y t o p l a n k t o n as N H  availability  of the phytoplankton  waters, forms  on t h e  -  and thus  n i t r o g e n and l i g h t  phytoplankton  Moreover, t h e nitrogenous likely  s t r a t i f i e d waters  NC^  understanding of  t o PPFD i n t h e s e  p h y s i c a l l y and  70  chemically effect(s)  distinct  environments.  o f PPFD on N u p t a k e by p h y t o p l a n k t o n have  saturating  enrichments of i s o t o p i c a l l y  Maclsaac  and D u g d a l e ,  reported  uptake  N  Previous studies of the  concentration.  1972; P r i s c u  r a t e s may r e f l e c t  labelled  N forms  1984; M i t a m u r a the effects  employed (e.g.,  1986) a n d  o f b o t h PPFD and  71  M A T E R I A L S AND  METHODS  General Nitrogen of  Georgia,  August, 1400  uptake  experiments were conducted  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  1984; s t a t i o n  Niskin bottles,  and  from depths corresponding  transfer  •  Millipore  Swinex  polyethylene ammonium  sunlight  and taken  into  f o rnutrient analyses and g e n t l y  + 4  ( 0 - 1 m)  during  removed  through ( m o u n t e d i n 25  R  filter  bottles..  (NH  5 L  the ship's  were  filtered  (460°C f o r 4 h ) W h a t m a n G F / F f i l t e r s  •  July-  t o t h e d e e p c h l o r o p h y l l maximum  carboys  syringe  using  below t h e sea surface  s h i e l d e d from d i r e c t  Subsamples  an acid-washed  combusted  mm  just  t o 10 L N a l g e n e  laboratory. with  from  samples were c o l l e c t e d ,  PVC  Samples were  during  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  a n d 1 5 0 0 h PDT w a t e r  (DCM).  i n the Strait  holders)  Nitrate plus  ) were measured  into  acid-washed  nitrite  immediately  (N03~ + N 0 ~ ) and 2  with  a  Technicon  •p  AutoAnalyzer  I I , f o l l o w i n g the procedures  al.  (1967) and Slawyk and M a c l s a a c  was  determined  technique  described  chlorophyll and  stored  acetone  by t h e d i a c e t y l  a  (1972),  by P r i c e and H a r r i s o n  frozen i n a desiccator.  (Strickland fluorometer.  and analyzed  and Parsons, Particulate  respectively.  (1987).  on Whatman GF/F  filters  C h i a was e x t r a c t e d i n 9 0 %  by i n v i t r o  1972) u s i n g organic  Samples f o r  fluorometry  a Turner  carbon  Designs model  (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 ,  stored  and analyzed  similarly  Urea  monoxime t h i o s e m i c a r b i z i d e  ( C h i a) were c o l l e c t e d  overnight  o u t l i n e d i n Wood e t  later  after  drying  were  (24 h a t  10  72  F i g u r e 2.1. Station locations f o r n i t r o g e n uptake experiments. 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 ,  125°  123°  B.C.  73  < 6 0 ° C ) w i t h a P e r k i n E l m e r model 240 the dry combustion precision At  temperature, prior  method d e s c r i b e d by  of these techniques  each  station  w i t h an  fluorescence  and  pumped s a m p l e s L min  - 1  )  and  + NO2""  514A  (PAR,  plotted  run  were  i n vivo from  meter, equipped  fluorometer  Technicon  T h e s e d a t a were l o g g e d  was  190SB S u r f a c e Quantum S e n s o r ,  light  of  onto  f o r time  l a g s i n pumping  Incident  monitored  continuously with  meter, equipped  and  solar  connected  with  solution  (Parsons  counting. on  a Wild  Ten  ml  to a chart  i r r a d i a n c e s were m e a s u r e d w i t h a L I - 1 8 5 B w i t h a L I - 1 9 2 S U n d e r w a t e r Quantum  samples  (250  ml)  were p r e s e r v e d i n L u g o l ' s  e t a l . , 1984)  and  s t o r e d i n the dark  s u b s a m p l e s were s e t t l e d  i n v e r t e d microscope  following  (24  h)  Utermohl  and  until  counted  (1958).  Experimental Within  a  a LI-  Sensor. Phytoplankton  a  i n r e a l - t i m e u s i n g a custom  LI-185 l i g h t  Subsurface  a  111  ( J o n e s , p e r s . comm.).  400-700 nm)  Lambda I n s t r u m e n t s  and  which compensates  machine a n a l y s e s  recorder.  and  m)  were  -  salinity  CSTD s y s t e m  cell)  II, respectively.  programme  irradiance  (0-20  (mRoy FR162-144 d i a p h r a g m pump, f l o w r a t e c a . 1  p e r s o n a l c o m p u t e r and  and  6.  + NO2  -  The  c o n c e n t r a t i o n s were o b t a i n e d  (equipped with a flow-through  software  NO3  m e a s u r e d w i t h a T u r n e r model  AutoAnalyzer  (1974).  profiles  T e m p e r a t u r e and  InterOcean -  vertical  f l u o r e s c e n c e and  casts.  NO3  Sharp  analyzer, using  i s given i n Appendix  continuous  salinity,  t o the b o t t l e  determined  elemental  1 h of c o l l e c t i o n ,  water samples from  each  depth  74 were t r a n s f e r r e d under reduced Wheaton g l a s s b o t t l e s w i t h  light  c o n d i t i o n s t o 500 m l  teflon-lined  caps.  N i t r a t e and 15  urea  uptake  r a t e s were measured u s i n g  (Kor  Isotopes),  as a t r a c e r  the stable isotope  (Dugdale and Goering,  N  1967).  For  the urea experiments, C O f ^ N ^ ^ ( 9 9 a t o m %) w a s a d d e d t o 15 1 bring the final N c o n c e n t r a t i o n t o 2-4 uq-at N - L . In the nitrate  experiments,  concentrations  ambient NO3  not  always true  -  tracer additions  NO3  the low  enrichments  associated with  screen  PPFDs  Biospherical adapted  clear Plexiglas  w a t e r s were c o o l e d  incubator. filtration  uptake  experiments.  immediately  screening  mixed and  t o simulate the  black  tape.  Whatman GF/F f i l t e r s ,  Incubations  differential into  a  w i t h i n an by  were light  seawater, while  deeper  temperature c o n t r o l l e d  were t e r m i n a t e d  placed  The  Samples from t h e s u r f a c e  surface  i n a separate  Incubations (pressure  flowing  ) .  (± 1.5°C) u n d e r n a t u r a l  deck incubators.  incubated  placed  T h e 0% P P F D w a s a c h i e v e d  temperature  with  Q  was c a l i b r a t e d w i t h  I n s t r u m e n t s Q S L - 1 0 0 4TT s e n s o r  at i n situ  samples were  here t o  from t h e s a t u r a t i n g  i n the incubators  the bottle with  conducted in  the urea  b o t t l e s were  incubation bottle.  wrapping  be used  were  as £ 10% o f  ( 9 5 , 5 5 , 3 1 , 1 0 , 3 . 4 , 1.1 a n d 0 % I  m a t e r i a l used  10% o f  These enrichments  enrichments  within neutral density  following  or less than  —  distinguish  enrichment,  - 1  (usually defined  "tracer" will  15  placed  N-L  + NO2"" c o n c e n t r a t i o n .  but the term  Following  ( 9 9 a t o m %) w a s a d d e d i n  o f e i t h e r 0.05 uq-at  the  ambient),  NO3  Na  after  < 1 2 5 mm plastic  2-4  h by  Hg) o n t o c o m b u s t e d petri  dishes,  and  75  stored  frozen  in a desiccator.  concentration, initial  N  calculated 5.2%  of  that NO3  the  the NO3  the PON.  particulate nitrogen  atom % i n the  incorporated At  the  an  and  of  -  20%  Therefore  experiments  8.5  ±  respectively, in solution  was  SD)  of  this  ±  i t  15.3%  during  the  incubation.  n e v e r more t h a n  were i n c o r p o r a t e d  exhaustion  was  not  (N )  p a r t i c u l a t e s a m p l e s was by  2  with  a  (LaRoche  the  micro-Dumas dry  ,1983) and  J A S C O m o d e l N-150 1975).  equation  7 of  D u g d a l e and  equation  5 of  C o l l o s , 1987)  the  Corrections  were not  remineralization and  Goering,  negligible to  the  1989)  given  bottles.  calculated  Nitrogen  incubation  by  of  beginning may  vary  of with  into  the  converted  to  the  combustion  this large  N  (Fielder  were c a l c u l a t e d  using to  which corrects  f o r changes  in  1,  5).  (see  Appendix  during  the  equation  amount of  the  of  (  Hansell  probably  be  -^N-label led urea  nitrogen  volumetric  transport  rates  Although  PON  from  incubation  c o r r e c t i o n would  experiments. to  and  (equivalent  by  chlorophyll a concentration  d e p t h due  enrichment  (1986)  Specific rates of  c  for  made f o r i s o t o p i c d i l u t i o n  division  the  of  in  spectrometer  Wilkerson  period  as  phaeophytin-corrected  analyzed  uptake rates  ^N-urea  the  then  emission  Proksch,  during  7 0%  a problem  1  technique  was  study.  i n the  gas  concentration  24.1  achieved,  isotope,  substrate  of  Nitrogen dinitrogen  and  rates  urea  nitrogen the  into particulate material uptake  ambient  and  (±  urea,  highest  the  particulate fraction,  average  and  -  B a s e d on  added were  the at  the  c h l o r o p h y l l a per  P P F D d i f f e r e n c e s , i t was  chosen  cell as  76  the  n o r m a l i z a t i o n parameter  necessary specific  to fuel uptake  previously uptake  respect  kinetic  of  curve  the dark  during  value  over  V  V  'max  v  i s made t h a t d a r k  analysis.  data  m  a  x  at saturating  assumption  Only  K  using  a  for light,  showing  f o r dark part of the  follows:  of chlorophyll, average  PPFD a n d K  L T  ,  the  i s a constant  no p h o t o i n h i b i t i o n  were  V  D  PPFD  i s t h e maximum N  i s t h e P P F D a t 0.5  uptake  Corp.).  + I  L T  I i s the integrated  constant  this  +  of N per unit  saturation  levels.  1972) and i s a s  - D  uptake  o f V,  chlorophyll  hyperbola  with  f i tof the  the hyperbolic light  the incubation period, '  unit  uptake  , Laboratories Technologies  and Dugdale,  where V i s t h e t o t a l  per  and urea  -  equation, modified t o account  d e s c r i b e s uptake  (Maclsaac  carbon  non-linear least-squares technique  V  is  f o r NO3  were o b t a i n e d by a d i r e c t  Notebook C u r v e f i t  uptake,  n i t r o g e n and  with  uptake  iterative,  Michaelis-Menten  Chi a  studies.  t o a modified Michaelis-Menten  (Labtec  light  f a c i l i t a t e s the comparison  constants  to irradiance  computerized,  The  irradiance  the  t r a n s p o r t mechanisms.  rates also  parameters  The  data  cellular  i t absorbs  published Chi a normalized  versus  Kinetic  because  V'  uptake halfm a x  .  The  at a l l light used  i n  77  R E S U L T S AND D I S C U S S I O N General  description  The in  vertical  of temperature,  vivo Chi a fluorescence  and N03~ + N O 2  three  features  9 m,  a subsurface which  extended  deeply  stratified  with  upper  depth;  thermocline  extended  throughout (T8),  a weak  from t h e surface t o  maximum  the nitracline  10 m w a s d e v o i d  experiments  The d i a g n o s t i c  t o the surface  station  PPFD  (T14) i n c l u d e d both  fluorescence  + N02~ c o n c e n t r a t i o n s  -  slightly the  water  relative  concentration f o r  -  i n F i g . 2.2.  and h a l o c l i n e which  nitracline  NO3  are presented  of the frontal  thermocline ca.  salinity,  s t a t i o n s a t which N uptake versus  were conducted  the  stations  profiles  the  a  of  layer  ( c a . 5-8  and r e l a t i v e l y  the water  column.  fluorescence occurred  of measurable NO3  -  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  high In  increased  a t c a . 1 2 m, + NO2 .  A  -  t h e deep  strong NO3 -  water  Similar  c o n d i t i o n s were observed  at the shallow-stratified  station  (A5)  thermocline,  developed  but the halocline,  biomass data  station  are given  i n Table  species  composition  the  frontal  2.2).  and environmental  and s t r a t i f i e d  In the frontal  dominant  maximum  species  nitracline  column.  The  c o n d i t i o n s f o r each  of the phytoplankton  community i n  waters v a r i e d considerably  waters,  layer  water.  2.1.  large, chain-forming  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 chlorophyll  and  w i t h i n t h e upper 5 m of t h e water  initial  The  surface  and  replete  all  from t h e N03~-depleted mixed  m),  (DCM).  a t both Chaetoceros  f o l l o w e d i n abundance by  (Table  diatoms  the surface  and t h e  s o c i a l i s  was t h e  Skeletonema  costatum  78 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 t h e t h r e e s t a t i o n s s a m p l e d (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 stratified).  T a b l e 2.1  Initial  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 seawater c o l l e c t e d f o r N-uptake 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 .  Sample Starting time o f depth i n c u b a t i o n (m) (PDT)  N i t r o g e n cone.  27 J u l 1984  1530  6.02 15.05  49°53'02"N 125°05'48"W  Shallow 30 J u l 1984 Stratified  1430  0 15  49°48'36"N 124°50'39"W  Deep Stratified  1500  0 15  station and location  Description  T14  49°53'24"N 125°05'06"W  Frontal  A5  T8  *NH.  concentrations  Date  1 Aug 1984  from s e p a r a t e b o t t l e  N0  3  Chi a (jjg-L )  PON POC (pg-at N - L ) (pg-at  0.23 0.21  1.29 2.28  5.28 6.96  43.1 40.6  0.63 0.72  0.16 0.32  0.33 0.67  2 .57 2.01  22.9 14.5  0.82 0.17  0.17 0.40  0.35 0.99  2.90 3.83  24 . 1 24.1  Urea NH^ (pg-at N - L )  - 1  - 1  N-L ) - 1  - 1  <.05 20.89 <.05 7 .54  casts  vo  80  Table  2.2  P h y t o p l 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 i n the S t r a i t of Georgia, B.C.  Station  Frontal  Depth (m) T14  0 8  Phytoplankton Diatoms  ('10°  cells•L ) Flagellates*  2.3 2.2  0.96 0.76  Shallow Stratified  A5  0 15  0.23 0.73  1.9 0.79  Deeply Stratified  T8  0 15  0.026 0.15  1.5 1.7  * <5%  of  flagellates  were  dinoflagellates  81 and  other diatoms  debilis.  o f t h e genus,  Small pigmented  abundant  costatum  stations;  dominant  a n d Chaetoceros diatoms  appeared small  spp.  (<5 um)  diatoms were  were t h e most  i n small  numbers.  Dinoflagellates  flagellates  Skeletonema  Gymnodinium  i n the relative  and diatoms, but t h e species  genera  flagellates  maximum c o m m u n i t i e s  differed  spp. and  were always a  (<5%) o f t h e t o t a l  The deep c h l o r o p h y l l stations  s t i l l  a n d Nitzschia  and were almost e x c l u s i v e l y  stratified  of both  spp., although Thalassiosira  b e l o n g i n g t o Navicula  numerical fraction  present  flagellates  i n c l u d i n g C.  phytoplankton i n the surface waters  stratified  pennate  Chaetoceros,  or  Amphidinium  o f t h e two  abundance o f  c o m p o s i t i o n was  similar.  Effect  of  light  Maclsaac of  nitrate  could be  under  nitrogen  and Dugdale  passes  consequences  uptake  phytoplankton  at saturating  i n the dark  They  Such (i.e.  For situations  a model  may  dark uptake i s greater  t h e PPFD  response  from t h e l i g h t t h a n c a . 15% o f linear  data are distorted  i n which  assumes  suggested that the  and thus t h e v a l u e s o f d e r i v e d  questionable.  assemblages  h y p e r b o l a ; PPFD  PPFD, c a n be s i g n i f i c a n t ;  of such k i n e t i c  t h e uptake  Michaelis-Menten kinetics  a t z e r o PPFD  of not subtracting  transformations  showed t h a t  stress.  through the o r i g i n ) .  when u p t a k e  usefulness  first  following  o f non u t r i e n t  t h e r e i s no N u p t a k e  uptake,  (1972)  rates  t o PPFD b y a r e c t a n g u l a r  as a substrate,  conditions  curve  uptake  a n d ammonium b y n a t u r a l  be r e l a t e d  treated  that  on  beyond  parameters  dark uptake  i sa  82  substantial proposed  a  portion slightly  study, which and  (>10-15% o f PPFD - s a t u r a t e d m o d i f i e d e q u a t i o n , employed  takes into  describes  N uptake  account  over the hyperbolic  curve, but not p h o t o i n h i b i t i o n .  problems  can  Parker al.  (1974)  (1980)  overcome by  using  or a modification  originally  photosynthesis  i n both marine  Maclsaac  1974;  and  et a l . ,  uptake by  response  of N 0  + 3  1986)  and  NH^  rate  of the  PPFD  Photoinhibition  equation developed  -  response  Priscu,  ( M a c l s a a c and Conway,  communities  1984b; M i t a m u r a ,  portion  f o r the l i g h t  N e l s o n and  freshwater natural  Alexander  an  L e v i n e , 1984;  Numerous s t u d i e s  present  by  of the equation of P i a t t  developed  ( L e w i s and  i n the  a constant dark uptake  response  be  uptake), they  Dugdale,  be  1972;  Slawyk,  1984;  Whalen  have demonstrated can  of  1989).  1979;  (Priscu  et  that  successfully  1979) and the  described  the Michaelis-Menten formulation. In the present study, n i t r a t e  dependent frontal  on  waters  the  natural  DCM  layers  which  PPFD a t b o t h d e p t h s of the S t r a i t  to a gradient  be  adequately described  formulation  up  to inhibiting  Photoinhibition Q  and  was  only  Photoinhibition this  s t u d y due  suffice  t o say,  observed  of N uptake  cannot  to the paucity i t i s not  ( F i g . 2.3 95%  of  and  the  data  and  2.4).  surface  collected  from  PPFD depth.  adequately discussed  o f d a t a a t h i g h PPFD,  likely  i n which  the Michaelis-Menten  and  be  and  surface  i n PPFD y i e l d e d  by  f o r samples  Experiments  from the  PPFD l e v e l s  o c c u r r e d b e t w e e n 55  were  in stratified  of Georgia.  phytoplankton communities were exposed  urea uptake  sampled  could  (I )  and  a problem  f o r the  but surface  in  83 F i g u r e 2.3. N i t r a t e uptake of t h e s u r f a c e ( O ) and DCM ( • ) p h y t o p l a n k t o n 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 ; t h e 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 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 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 ) .  300  PPFD  (jjE-m" -s" ) 2  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 ( • ) phytoplankton communities o f t h eS t r a i t o f Georgia. The curved plots a r ef i t t e d d i r e c t l y t o t h e Michaelis-Menten equation; t h el 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 n o t i n c l u d e d i nt h ec 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- -s" ) 2  2  85  samples,  which  phytoplankton are  are naturally collected  effectively  near  excluded  from  exposed t o high  PPFD;  the bottom of the euphotic the high  PPFD i n t h e m i x e d  surface waters  by t h e p y c n o c l i n e and a r e n o t l i k e l y  encounter  h i g h PPFDs  Kinetic  such  parameters  urea  and n i t r a t e  T h e K-^rp v a l u e s PPFD a t w h i c h  data  response uptake  velocity  n i t r o g e n uptake  i n the present 0.5 V '  remember t h a t t h e s e uptake  from  curve  m a x  data  and f o r c e d t h e i r  through  the origin  50% o f t o t a l i n this  particularly abilities better  a  x  dependent  i n Table  2.3.  representing the  However, i t i s i m p o r t a n t  i n the linear  parameters  only  uptake  represent  dark  (e.g., P r i s c u ,  PPFD r e s p o n s e  to  p o r t i o n o f t h e PPFD  curves was  Half-saturation  N  1984)  transformation of  even though dark N-uptake).  and  have  their t o pass  substantial constants  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 12  a n d s h o u l d be i n t e r p r e t e d  as an i n d i c a t o r  to assimilate  estimate  maximal N uptake (K-^ip')  are those  Some i n v e s t i g a t o r s  kinetic  m  ) for light  and 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  uptake  V = V  m a x  a r e summarized  Michaelis-Menten  dark  which  (K-^rp),  constant  the hyperbolic (or light)  observed.  derived  (V'  study  occurs.  ignored  (ca.  uptake  the half-saturation  maximum n i t r o g e n u p t a k e  to  naturally.  of nitrogen  Dark uptake,  zone,  Michaelis-Menten  of the phytoplankton  specific  of the phytoplankton  equation  communities'  N s u b s t r a t e s a t l o w PPFD.  o f t h e PPFD a t w h i c h  c a n be c a l c u l a t e d  with caution,  by a s i m p l e employed  one-half  the  total  community i s a c h i e v e d rearrangement  i n the present  of the study:  A  Table  2.3  Station  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 , as 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. S t a t i o n s a r e 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 . D e f i n i t i o n s are given i n the text, estimated standard errors of parameters i n parentheses.  Nitrogen substrate  Depth  V  V  D  (m)  ( n g - a t N (jjg C h i a ) "  T14  N0 -  0 8  80 53  (16.8) ( 9.4)  A5  NO3-  0 15  48 55  ( (  Urea  0 15  87 1.8  N0 ~  0 15  Urea  0 15  T8  3  3  K  max 1  h  _ 1  )  LT  (pE•m~  (%I ) 0  225 174  (22 3) ( 12 5)  91 ( 40.1) 53 ( 15.7)  3.2) 8.8)  50 67  ( 4 0) ( 15 3)  ( (  4.2) 2.3)  31 11  ( 4 3) ( 1 9)  8.5 0  (  0.79)  9 . 8 ( 1 02) 55 ( 4 2)  45 18  92 4.0  ( (  14.1) 3.9)  73 24  59 ( 63.4) 72 ( 56.2)  ( 17 6) ( 3 6)  8.2 4. 8  3.6) 1-4)  74 ( 25.9) 156 ( 151 )  6.7 14  2.3) 14 )  140 ( 103 ) 54 ( 40.7)  13 4.9  9.3) 3.7)  4.6 1. 8  1.9) 0.7)  6.7 8.2  7.2) 6.4)  (18.7) ( 6.6)  00  87  K '  =  L T  where V  =  (V - V )  (V  described  by  saturation  at  by  the  realistic N  uptake  The uptake  another  V  PPFD.  is  max  uptake of  t h e maximum  uptake  the  values  more  the  phytoplankton  actual  community  half-saturation study  range  from  constant 0-14%  f o r urea  uptake  maximum as  they  are  similar are  NO3-  for  I ,  which  Q  i s  f o r marine  assemblages  studies f o r urea  (Table  (0-13% I ) .  2.5).  Webb a n d  for  phytoplankton  during 0.02  the  to  L T  uptake  by  (2.44 value  Klux  0.01  l a n g l e y s 'min  the  York  River estuary  summer, a l t h o u g h  0.12  summer K  from  ca.  langleys•min v a l u e was  phytoplankton  i n the  Haas  2  from ). (28  Mitamura  oligotrophic  O  (35*L/E«m in  -1  _  *s  Virginia  autumn ranged  (69-418 uE*m~ •s ).  r e p o r t e d by  = 39 L f E « m -s + K r p f o r NH4 uptake L  - 1  values  -  The  Previously  Q  few.  and  1  r e p o r t a K-^rp o f  N  2.4).  K  ( 1976)  be  of  that are  natural phytoplankton  kinetic  velocity half-  freshwater  published  can  K-^rp",  derived  with previously published values  values  rate.  constant,  consistent  L T  uptake  K^ip i t s h a l f -  PPFD a t o n e - h a l f  (Table the  D  of these  generate  i n the  present  dark  v)  f o r one-half Both  measures of the  i n the  V  half-saturation  substituting  values  V +  i s the  constants w i l l  dark  max -  and  and  taking place  include  /(V  T  rectangular hyperbola,  saturating  saturation  L  D  constant,  calculated  K  + V )/2,  max  Alternatively,  uptake  •  D  A  (1986) f o r Lake Biwa  from  similar urea in  Japan  They a l s o r e p o r t e d a s i m i l a r — 9 — 1 /jE-m *s ) and a g r e a t e r K  L T  I  )  Table  2.4  I n d i c e s o f N uptake dependency to l i g h t - s a t u r a t e d uptake r a t e ratio  o f uptake  a percentage  1% I  substrate  D  L  1% 55% ,V  o f s u r f a c e PPFD  Nitrogen  station  under  o n PPFD f o r p h y t o p l a n k t o n i n t h e s t r a i t o f G e o r g i a : the r a t i o o f dark ( V : V ) , t h e PPFD a t w h i c h h a l f o f t o t a l N u p t a k e o c c u r s (K. K. LT LT The K v a l u e s a r e e x p r e s s e d as PPFD v a l u e s a n d a s t o 55% I (V  Depth (m)  ( I ) which Q  V :V D* L V  V  0.28 0.25  T14  NO-  A5  NO-  0 15  0.51 0.49  Urea  0 15  0.77 0.15  NO-  0 15  0.48 0  Urea  0 15  0.58 0.16  T8  •Definitions  given  )  L T  i s shown i n p a r e n t h e s e s .  L  T  (jiE-m  36 24  -2  (3.2) (2.1)  0 3.0 (0.3)  33  (3.0)  1.9 (0.2) 0 0 39  (4.5)  -S  -1  )  LT  V  1%  : V  55%  (3.9) (2.5)  0.38 0.40  1.6 (0.1) 15 (1.3)  0.60 0.56  0 39  (3.5)  0.80 0.33  3.1 (0.3) 0  0.60 0.37  43 28  0 52  (5.8)  0.65 0.28  i n text.  oo oo  T a b l e 2.5  Comparison o f h a l f - s a t u r a t i o n c o n s t a n t s ( K _ ) f o r i n o r g a n i c n i t r a t e t r a n s p o r t i n v a r i o u s a q u a t i c ecosystems. L  LT< 3~> _ , " % surf l i g h t pE-m -s range (mean) range (mean) K  N0  2  Region  Area  Depth (% I ) Q  Oceanic  E. T r o p i c a l  Upwelling  Peru  Upwelling  N.W.  Africa  Upwelling  N.W.  Africa  Upwelling  Baja C a l i f . ,  Upwelling  Antarctic  Coastal  Peru  Coastal  Strait  Freshwater  T o o l i k L., A l a s k a  Freshwater  L. K i n n e r e t , I s r a e l  Freshwater  L. Biwa, Japan  100  Freshwater  C a s t l e L., c a l i f . , U.S.A.  ca.50 ca. 1  Freshwater  Freshwater  Pacific  25 100 10  Mexico  of Georgia  L. Vanda,  L. F r y x e l l ,  Maclsaac & Dugdale (1972) 14 - 108 ( 63)* 7 - 199 (122)*  Maclsaac & Dugdale (1972)  50  1.5 -  7.0 ( 5.4)  -  Maclsaac e t a l . (1974)  50-0.1  5.5 -  6.2  ( 5.9)  -  Nelson & Conway  (1979)  50-3  3.3 - 32.4 (16.1)  -  Nelson & Conway  (1979)  50-25 7  1.1 1.3  2.3-4.4 2 . 8**  2.3(1.7)  100 10  4.4 1.0  45 14  100 1  4.6 - 8.2 ( 6.5) 1.8 - 14 ( 6.9)  45 18  c a . 10-15  Antarctica  14.0 0.9 - 12.7 ( 5.4) 0.9 - 13.3 ( 8.9)  i.  Antarctica  Reference  10.2 1.0 0.4  6  - 31  (15)  4.29 2.6 - 2.7 (2.65) 0.6 - 3.7 (1.55) 0.5 0.4 0.08  2.0 5.1  7  (3.3)'  Slawyk (1979)  Maclsaac & Dugdale (1972)  - 91 - 156  ( 70) ( 76)  P r e s e n t study  - 29  ( 16)  Whalen & A l e x a n d e r  (1984b)  77  Berman e t a l . (1984)  70. 8***  Mitamura (19 86)  15.1 4.6 2.4 1.7 0.04  - 16.2 - 25.5 - 9.5 - 24.9 (?)  (15.7)+ (10.7)+  Priscu  (1984)  Priscu  (1989)  Priscu  (1989)  Table  2.5  Continued 1  *  Values  calculated  by c o n v e r t i n g  from  ly-min  **  Values  calculated  by  from  quanta-m  converting  using —2—1 -h  1 ly-min using  —1  =  3485 uE-m~  ? -S  7- h — 1=  1 quanta-m  1  4.614  (Richardson x  10  (Richardson  et  7?  et  pE-m  al., -s  P I  1983) (Liining,  1981) —2 ***  Values  calculated  by  +  Values  calculated  from  ?  Probable  error,  converting  correct  total value  from  klux  PPFD d u r i n g from  data  using  1 klux  incubation given  in  =  16.5  periods  Priscu  uE-m (ca.  (1989)  is  -s  12  -1 al.,  1983)  h)  0.4  pE-m~ -s 2  - 1  o  value the  f o rn i t r a t e  small  differences  5 0 uE-m  communities  forward  kinetic  parameter  by comparing  PPFD  Q  represent  A simpler  N uptake  Q  i slikely  two  N-replete communities.  (A5),  respect  At the shallow  t o NC^  uptake  -  response between  dependency:  80 a n d 3 3 % , r e s p e c t i v e l y .  differences  were  urea,  that  has a g r e a t e r  normally the  layers  surface  state  i n both of these  stratified  station  Similar  with  i s a  a n d DCM u r e a  uptake  large  of the deeply s t r a t i f i e d  uptake  c a . 1-3% I  station  of the regenerated N source,  d e p e n d e n c e o n P P F D i n t h e NC>3~-  by t h e strong  received  uptake (38-  are very similar  the surface  DCM c o m m u n i t y w h i c h w a s e f f e c t i v e l y surface  -  f o u n d f o r b o t h NC>3~ ( 6 0 , 3 7 % ) a n d u r e a ( 6 5 ,  i n t h e two communities I t appears  station  ( 6 0 , 56%) b u t t h e r e  difference  (T8).  f o r NO3  c o m p o s i t i o n and t h e p h y s i o l o g i c a l  substantial  28%)  lower percentages  of the similarity  t h e two p h y t o p l a n k t o n communities  c a n be  m a x i m u m (DCM)  40%);  a reflection  straight-  and s a t u r a t i n g  At the frontal  h a v e t h e same P P F D d e p e n d e n c y  species  difficult.  a n d more  at low (1%I )  and deep c h l o r o p h y l l  the  orthe  o f PPFD o n N u p t a k e  communities which  (less  has been i n c l u d e d f o r  PPFD d e p e n d e n c y .  surface  Interpretation of  N substrates  (Conway a n d W h i t l e d g e , 1 9 7 9 ) ;  greater  both  ).  depths, i s rather  index t o assess the effect  (55%I )  -s  between  comparative purposes.  determined  (T14)  ), e i t h e r  taken from d i f f e r e n t  However t h i s literature  «s  ( 6 7 uE*-m~  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  —1  —7  than  uptake  isolated  pycnocline Q  replete  from t h e w e l l - l i t  present and only  . The l e s s e r  PPFD d e p e n d e n c y  p h y t o p l a n k t o n may b e a c o n s e q u e n c e  of their  N-  of  92  depleted  physiological  s t a t e , which  in  PPFD d e p e n d e n c y o f N O 3  of  the s t r a t i f i e d  (38%).  waters  Alternatively  accumulation  uptake  explain the decrease  i n the surface  (60%) r e l a t i v e  populations  to the frontal  waters  i t may be a t t r i b u t e d m e r e l y t o an  of stored  photosynthesis.  -  could  e n e r g y and C s k e l e t o n s p r o d u c e d  One c a n n o t d i r e c t l y  r e s p o n s e s o f u r e a and n i t r a t e  during  compare t h e u p t a k e  i n the surface waters  due t o  1 c  differences the  DCM,  during  in  for NO3  Dark nitrogen Nitrate  DCM  and u r e a uptake  occurred  waters  (A5, T 8 ) , t h e r e l a t i v e  to total  N03~ u p t a k e u n d e r  u p t a k e c o n t r i b u t i o n was o n l y  In the  contribution saturating  of f r o n t a l  s t n ( T 8 ) , t h e r e was no d a r k N O 3  shallow s t r a t i f i e d to their  (49 a n d 2 5 % ) .  (A5) and f r o n t a l  respective  Dark uptake  of t o t a l  dark N O 3  of dark PPFD was c a .  -  At the deeply  u p t a k e by t h e DCM  uptake  at the  (T14) DCM c o m m u n i t i e s  surface phytoplankton o f u r e a was a l s o a  urea uptake  s u r f a c e communities,  -  stratified  s t n (T14) t h e d a r k  28% ( T a b l e 2 . 4 ) .  p o p u l a t i o n whereas t h e r e l a t i v e  and  f o r urea  i n the dark i n both  ( T a b l e 2.3)  50% w h i l e i n t h e s u r f a c e w a t e r s  portion  t o that  uptake  s u r f a c e waters  stratified  d e g r e e o f PPFD  communities.  and s t r a t i f i e d  N03~ u p t a k e  I t appears t h a t the  u p t a k e w h i c h was s i m i l a r  -  At  c o n d i t i o n s were p r e s e n t  i n t h e DCMs had a s i m i l a r  the s t r a t i f i e d  similar  -  a l l t h e uptake experiments.  dependency  frontal  (saturating versus trace).  however, s a t u r a t i n g N O 3  phytoplankton  in  N-enrichment  were  communities  substantial  a v e r a g i n g 16 and 68% f o r t h e DCM  respectively.  Dark N u p t a k e by  93 phytoplankton values for  i s n o t uncommon a n d a summary o f  of the ratio  natural  A review  of dark  phytoplankton  to light  assemblages  of the literature  permits  unity) dark  than  uptake  generally and of  zone.  bynutrient  greater  experiments  (e.g.,  have  degree Syrett,  dark  Eppley  took  diatom  t h e uptake  night This  D  ratio i s  L  PPFD, s u g g e s t i n g a l e s s e r depth  from  dependence  i n the euphotic  spp.  enhances t o a  of N i n the dark and S y r e t t ,  than  i n the light,  1972; H a r r i s o n , 1976;  t o take  species dependent;  coccolithophorid  i n the dark,  i s suggested  the natural  and 2) V :V  showed t h a t a l t h o u g h  up n i t r o g e n i n f o r example  a somewhat N(Emiliana  huxleyi)  a similarly  N-depleted  d i dnot.  A l s o whether  costatum)  i s able t o take  rates,  s u g g e s t i n g t h e enhancement o f  The a b i l i t y  oceanic  o ri n t h e dark  dilution of  1979).  tropical  Chaetoceros  (approaching  t o u n i t y ) i n samples c o l l e c t e d  1962; Thacker  (Skeletonema  species  t obe  i s greater  shown t h a t N d e p r i v a t i o n  e t a l . (1971b)  up n i t r a t e  2.6.  1) i n N-  with increasing  may b e , h o w e v e r ,  depleted  i s shown i n T a b l e  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  Rees and S y r e t t , the  lower  (V^iV^)  rates  f o rN uptake:  ratio  L  stress,  (closer  f o rN uptake  The f i r s t  greater  D  i n N-replete waters,  incubated under light  t h e V :V  waters,  N uptake  two g e n e r a l i z a t i o n s  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 impoverished  literature  up a s i g n i f i c a n t  coastal or not a  amount o f n i t r o g e n a t  may d e p e n d o n i t s d e g r e e o f N d e p l e t i o n .  f o rnitrate-limited (Malone  nitrate  et al.,  uptake  light/dark  continuous  1975);  at the three  was c o n t i n u o u s  cycle,  cultures of  b u t t h e r e was  and  lower  independent  diel  Table 2.6  Summary of l i t e r a t u r e v a l u e s o f d a r k : l i g h t n i t r o g e n s p e c i f i c ( V / V ) or absolute (/p//^) uptake r a t e s , determined d u r i n g daytime, i n n a t u r a l phytoplankton communities.  Area  D  Ambient NO-} cone (pg-at N-L )  L  range (mean) Urea  Reference  (0.30)  (0.59)  Dugdale & Goering (1967)  or  v  NO-  Q/ L NH, V  Oceanic  N. A t l a n t i c Gyre (Sargasso Sea)  N. P a c i f i c C e n t r a l Gyre - 50°N, 155°W - 40°N, 150°W  >10 1.0  0.0 -0.63 (0.30) 0.92  0.38- 2.0 (0.83) 0.78- 1.5 (1.2)  N. P a c i f i c Ocean - northern (J1-J7)  >2  0.0 - 9.7 ( 2.4)  5.7 -27.4 (16.9)  0.0-18.5 ( 9.3)  - tropical/subtropical (J9-J23)  <0.1  7.5 -31.9 (22.4)  16.6 -52.9 (34.0)  12.2-49.8 (25.7)  N . E . P a c i f i c Ocean  12  0.08-1.01 ( 0 . 0 9 )  >10  0.05-0.57 (0.36)  0.00-0.02 (0.07)  Nelson & Conway (1979)  >10  0.10-1.46 (0.49)  0.01-0.67 (0.16)  Nelson & Conway ( 1979 )  0.34  0.02  Maclsaac (1978)  H a t t o r i & Wada ( 1972 )  Kanda e t a l . (1985)  Cochlan  g  (Chap. 1)  Upwelling N.w.  Africa  Baja C a l i f . , Mexico  Baja C a l i f . , Mexico*  Table  2.6 c o n t i n u e d  Area  Ambient NOo NO3 ccoonnce^ ( p g - a t N-L )  ^D^^L NC>2  o  r  V  D^ L NH4 V  r  a  n  9  e  (mean)  Reference Urea  Polar  >20  S c o t i a Sea  0.30-0.47  >20  S c o t i a Sea  (0.38)  13.0-75.0  0.88-1.2  (1.0)  c , g  0.27-1.0  G l i b e r t et a l . (1982a) c  Ronner et a l .  (1983)  Barents Sea  0.3-0.5  0-1.5  'Kristiansen  & Lund  ( 1989)  Coastal Oslofjord  (Norway)  >2 <1 ca.  0.1  0.06-0.57 0.18-1.7 0.2 -1.0  (0.17) (0.47) (0.7)  Paasche  0.5 -1.3  (0.7)  Conway &  New Y o r k B i g h t Gulf  o f Maine  & Erga  (1988) Whitledge  (1979) ca.  1-2  0.00-1.00 (0.26)  0.00-0.20 (0.10)  a , b  Dugdale  & Goering  (1967) Peru*  0.09  0.60-0.86  Dortch  (0.73)  & Maske  (1982) S t r a i t o f G e o r g i a , B.C. - Frontal - stratified  3.0-4.6 <0.05  0.00-0.08 0.00-0.18  (0.03) (0.09)  0.37-0.39 (0.38) 0.52-0.58 (0.55)  0.00-0.81 (0.36) 0.06-0.66 (0.36)  Price et a l . (1985)  Table  2.6  continued  Area  S t r a i t o f G e o r g i a , B.C. - Frontal - Surface s t r a t i f i e d - Bottom s t r a t i f i e d Washington  /^E/ZL  Ambient NO3 conc^ SO-, cone > -1) ( p g - a t N-L  6-15 <0.05 7-20  0.25-0.28 0.48-0.51 0.00-0.49  coast  Western I r i s h Sea - Surface s t r a t i f i e d - Mixed & bottom s t r a t .  O R  V  NO3  D^N HL V  a  n  9  e  (mean) Urea  (0.27) (0.50) (0.25)  0.58-0.77 0.15-0.16  (0.21)  ca. ca.  r  4  (0.43)  2.5 4.5  Reference  (0.38)  0.47-1.1 0.37-1.3  Present  study  Dortch (1989)  & Postel  (0.68) (0.16)  (0.67) (0.72)  1  Turley (1985)  Estuarine P a m l i c o R i v e r , N. S o u t h R i v e r , N.C. Neuse R i v e r , N.C. N e w p o r t R i v e r , N. D e l a w a r e Bay C h e s a p e a k e Bay  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  0.00-0.09  F i s h e r e t a_l. , (1982)  Freshwater L. K i n n e r e t (Israel)  ca.  0.2-0.6  0.40-0.91 0.16-0.33  L. K i n n e r e t (Israel)  <0.05  0.32  L. Nakanuma (Japan)  0.10  (0.56) (0.22)  0.29-1.0  (0.60)  0.13-0.67  (0.34)  McCarthy e t a l .  0.53  (0.53)  0.33-0.44  (0.38)  (1982)  0.59  0.21-1.1  Berman e t a l . (1984) (0.57)  Miyazaki ( 1985)  et a l .  Table  2.6  continued  Area  Ambient NOo cone J -1 (pg-at N - L )  L . Biwa (Japan)  ** L. Kasumigaura (Japan) Shagawa L . (Minnesota,  =0.07  O R  V  D L /V  NH .  r  a  n  9  e  Reference  (mean) Urea  0.26  0.78  0.51  Mitamura (1986)  0.18-0.27  0.60-0.90  0.71  Takamura (1987)  0.27-2.3  (1.0)  ca.  L.  0.0-0.7  Vanda (Antarctica) s f c pop'n d e e p - c h l pop'n  1.0  0.05-0.31  (0.15)  0.27-0.57  (0.41)  0.41 0.24  Czechoslavakian reservoirs  >35  L. C a l a d o (Brazil)  ca.  Amazon R i v e r (Brazil)  11.1  <0.1  (0.11)  0.00-0.32  (0.16)  et a l .  Whalen & A l e x a n d e r (1984b)  0.47 0.83  0.05-0.38  and S a i j o  T o e t z £. C o l e ( 1980)  a , e  U.S.A.)  T o o l i k L. ( A l a s k a , U.S.A.)  "Priscu  ( 1989) Prochazkova e t a l . (1970)  0.11-1.0  (0.49)  Fisher  et a l .  (1988) 0.20  0.54  Fisher  et a l .  ( 1988)  C a s t l e L., (California,  NO-  /V/L  0-ca.  2.5  -  (0.55)  (0.50)  c  U.S.A.)  L. O n t a r i o ( O n t a r i o , Canada)  Priscu  (1984) 0-15  0.02-0.30  (0.14)  0.30-0.60  (0.40)  g  Liao  and Lean  (1978)  Table a  2.6.  Values Values  c  Values  ^Values e  Light  Superscripts.  calculated estimated  1/(V /V ) or L  from  calculated  D  from  r a t e s determined  no  ^Experiments  m  0 D  )  from  reported values  of V / V L  D  or  available. times.  a t ambient  N cone,  dark  r a t e s determined  at s a t u r a t i n g  N  cone.  H  , where N  utilized  24  ^Average v a l u e s  reported.  *Dinoflagellate  bloom.  Microcystis  data  turnover  K  are /^^/^  l/(/^//  figures.  reported i n text,  •f Values  as  m  i s the  chlorophyll  h i n c u b a t i o n s over  specific  t r a n s p o r t r a t e a t o p t i m a l PPFD  natural light/dark  level.  cycle.  bloom  oo  99  periodicity It  i nnitrate  s h o u l d be n o t e d t h a t  present  study and used  determined  rates  course  experiments  study  observed  I observed  frontal  waters  circadian.  uptake  (Chapter  diel  theV :V D  L  for NH  variability. uptake  + 4  estuary (Fisher  -  or dependent comparisons An  bacteria. inhibitors, that  uptake i s  +  i n both NH  + 4  eta l .  and NO3  -  variability  of the daily between  may a l s o  waters  ofthe  Diurnal  (daytime)  both  variability  by f r e s h w a t e r p h y t o p l a n k t o n  et al.,  et al.,  1988) and t h e South  1982) have a l s o  been  were conducted a t  o f t h e day thereby i n N uptake,  light  cycle)  observed.  (either  and thus  compensating independent permitting  stations.  unknown p o r t i o n  experiments  NH^  and urea demonstrated  (Fisher  t h e same t i m e  any d i u r n a l  time  as t h e present  i s supported by G o e r i n g  In t h e present study, a l l t h e experiments  for  24 h  Sea phytoplankton incubated under  o f Lake Calado  approximately  reflect the  uptake i n  + 4  However, i n t h e s t r a t i f i e d  of NH  L  During  suggests that  rhythmic v a r i a t i o n  light.  assemblages River  D  1) w h i c h  This conclusion  and d i u r n a l  2.6 w e r e  i n t h e same w a t e r s i nV :V  rate.  reported i n the  of Table  during the night.  o f G e o r g i a , VptV-^ f o r N O 3  Strait  rates  a n d may n o t n e c e s s a r i l y  conducted  by Sargasso  continuous  uptake  i nthe ratios  a constancy  ( 1 9 6 4 ) who f o u n d  at the highest dilution  the dark  during daylight  .uptake  in  uptake  of t h e dark uptake  be a t t r i b u t e d  Wheeler and Kirchman size-fractionation  7 8 % o f t h e ammonium u p t a k e  i n the present  t o marine  heterotrophic  (1986), u s i n g metabolic and  1 5  N methodology,  estimated  i n the surface waters o f f  100 Sapelo to  Island,  and  NH  pseudomonad and competition  t h e G u l f S t r e a m o f f G e o r g i a was  Brown e t a l . (1975) r e p o r t e d N O 3  bacteria.  reduction  G e o r g i a and  uptake  + 4  by  batch c u l t u r e s  Remsen e t a l . ( 1 9 7 2 )  have  f o r u r e a among b o t h b a c t e r i a  demonstrated and  phytoplankton  of Georgia.  experiments  s t u d y Whatman GF/F  used 1 5  to collect  N-labelled  completely  NO3 ;  between b a c t e r i a  and  and  filters  o r g a n i c N w h i c h may  be  both  greater bacterial  heterotrophic the  activity  stratified  al.,  Sea  (Egan  s i d e of a f r o n t  and  were  incubation with  not d i s c r i m i n a t e and  attributed  (as d e t e r m i n e d  1 9 8 3 ) , i n L i v e r p o o l Bay  Irish  filters  can  (R. K e i l ,  frontal  b i o m a s s and by  i n Saanich  capture  pers. of  to bacteria i s systems  have  relative  glucose uptake) Inlet  (Parsons  on et  ( F l o o d g a t e e t a l . , 1 9 8 1 ) , and  Floodgate,  1985;  Lochte,  of  the  study, the p r o p o r t i o n of uptake  unknown; p r e v i o u s s t u d i e s i n s h a l l o w s e a reported  do  phytoplankton  i n marine systems  In the p r e s e n t  inorganic  these  -  40-50% o f t h e b a c t e r i a comm.).  During  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  u r e a and  and  of a marine  the e s t u a r i e s / c o a s t a l waters reported i n this  uptake  -  due  the  1985).  Summary The frontal can  be  uptake total n o t be  N uptake and  response  stratified  t o PPFD o f t h e p h y t o p l a n k t o n  communities  of the S t r a i t  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 of n i t r a t e uptake  and  overlooked.  In the  PPFD f o r NC>3~ u p t a k e  i s similar  portion  communities,  f r o n t a l waters, f o r both  Georgia  formulation.  urea i s a s u b s t a n t i a l  i n these phytoplankton  of  and  i n the  Dark  of  the  should  the dependency s u r f a c e and  DCM  on  101 communities, whereas i n t h e s t r a t i f i e d phytoplankton DCM,  exhibit  particularly  less  waters,  PPFD d e p e n d e n c y t h a n t h o s e  f o r urea uptake.  The d r a m a t i c  species composition  of the phytoplankton  d o m i n a t e d by l a r g e ,  c h a i n - f o r m i n g diatoms  f r o n t a l waters in  t o one composed p r i m a r i l y  the N-depleted  the observed a simple merely studies  stratified  variability  waters  i n their  on t h e r e s p o n s e  axenic) phytoplankton deficiency, limitation explained.  cultures,  need t o be c o n d u c t e d on t h e N u p t a k e  of m i c r o f l a g e l l a t e s  and p r e c l u d e s  on N u p t a k e more  based  detailed  t o PPFD i n u n i a l g a l (and  a t v a r i o u s degrees  of N  before the e f f e c t ( s )  response  f r o m one  i n the N-replete  Clearly  of N uptake  change i n  communities  PPFD r e s p o n s e  N status.  from t h e  probably contributed t o  e x p l a n a t i o n o f PPFD e f f e c t ( s )  on p h y t o p l a n k t o n  the surface  t o PPFD c a n be  of N  adequately  102 CHAPTER  THREE  N I T R O G E N U P T A K E BY THE E U C A R Y O T I C P I C O P L A N K T E R , MICROMONAS PUSILLA AND THE E F F E C T S OF N D E P R I V A T I O N ON U P T A K E R E S P O N S E INTRODUCTION It is  i s well  established  t h e dominant n u t r i t i o n a l  growth  i n coastal  waters  ( e . g . , T h o m a s , 1966,  focus  o f many  nitrogen  Dugdale  factor  1969;  investigations  uptake  the availability  by marine  (1967) f i r s t  equation  the  concentration  saturation equal  velocity  and adapt  proposed  (h  ),  V  m  of l i m i t i n g  to half  assemblages  1985;  culture  studies  Thomas,  1969;  rates  V = V a  x  nitrogen  m  a  uptake  Eppley et a l . ,  rates  by a  [ S / ( K + S ) ] , where V i s  x  g  t h e maximal  uptake  and K  velocity,  rate  (i.e.,V  S  the half-  s  the value of S a t which =  v m  a  x  V i s  /2).  by n a t u r a l p h y t o p l a n k t o n and Dugdale,  1969;  (e.g., Eppley and Coatsworth,  concentration.  of  t o the Michaelis-Menten  substrate  1969)  of nitrogenous nutrients  nutrient  The  limitation.  Probyn,  W h a l e n a n d A l e x a n d e r , 1986)  function which  and oceanic 1979).  N concentration  t h e maximum u p t a k e  (e.g., Maclsaac  Kanda e t a l . ,  hyperbolic  to nitrogen  relating  constant representing  Measurement o f uptake  uptake  - 1  1971)  phytoplankton i n order t o understand  f o r enzyme k i n e t i c s ,  uptake  nitrogen  phytoplankton  Goldman e t a l . ,  f u n c t i o n w h i c h was s i m i l a r  the  of  has been on t h e k i n e t i c s  phytoplankton t o the external  hyperbolic  regulating  (e.g., Ryther and Dunstan,  how p h y t o p l a n k t o n r e s p o n d  of  that  relates  and numerous  1968;  have demonstrated  c a n be d e s c r i b e d uptake  rate  1985;  by  Eppley and that the this  to the limiting  103  The V  m  a  species  and K  x  involving Button,  the limiting  over  initially  rate  take  elevated  NH  197 6;  and  few minutes  later.  Several  and Goldman,  N-starved  culture  there  i s quite  1982; C o l l o s ,  not  to  exposure t o an (Conway e t  and Goldman,  and G l i b e r t , 1982;  Horrigan  a l a g whose  n i t r a t e uptake  i s observed (Dortch  1 9 8 3 ; P a r s l o w e t a l . , 1984 b ) . 197 9;  1979) i n c r e a s e  phytoplankters  Horrigan  Nitrogen  1988),  have been t e s t e d  ammonium a n d n i t r a t e ,  et  and McCarthy,  urea uptake  i n t e r a c t i o n s between i n o r g a n i c  more p a r t i c u l a r l y  to a  but not always,  or reduced v e l o c i t i e s  (Rees and S y r e t t ,  three  field  1987; S u t t l e and  after a nitrate addition  i s often,  normal  and S y r e t t ,  only  Uptake  upon  more that  have t h e a b i l i t y  e t a l . , 1986; P r i c e and H a r r i s o n ,  (Bekheet  although  and  may  Syrett  elevated  a t a slower,  197 7; M c C a r t h y  v a r i a b l e , before  either elevated,  1981;  i t may b e  have demonstrated  1984, P r i s c u ,  In contrast,  starvation  i s frequently  1981; Wheeler e t a l . , 1982; H a r r i s o n ,  1988).  al.,  x  1982; P a r s l o w e t a l . , 1984a b) a n d i n t h e  Harrison,  at  a  i n t h e c u l t u r e medium  e t a l . , 1982; Goldman  1983a; P r i s c u and P r i s c u ,  duration  studies  Conway a n d H a r r i s o n ,  McCarthy,  m  following enrichment of  phytoplankton  concentration  + 4  1967; T i l m a n , 1977;  uptake proceeding  u p ammonium i n i t i a l l y  Dortch  (Glibert  (Dugdale, V  parameters,  competition  a n d f o r some n u t r i e n t s  or N-starved  rapidly  1979;  time,  kinetic  species  I t i s now r e a l i z e d t h a t  nutrient with  N-deficient  al.,  nutrient  over t h e f i r s t  limiting  constant  n u t r i e n t uptake  may b e u s e d t o e x p l a i n  s  1985).  variable  the  specific  nitrogen  o r may  rates t o date. sources,  have been t h e  104 subject  o f many c u l t u r e s t u d i e s  McCarthy, These on  studies  species  tenets of  1981; S y r e t t ,  s t i l l  nitrate  certain  stops  value.  + 4  severe  simultaneous  field et  and equal  197 6;  (e.g.,  Conway,  Conover,  i sthat  concentration  + 4  on NO3  + 4  literature  phytoplankton estimation  of kinetic  under c o n d i t i o n s  rates  -  picoplankton there  197 7;  uptake  i s often  (e.g.,  -  1975; McCarthy  utilization, parameters,  and N H  ,  + 4  Caperon and  D e M a n c h e e t a l . , 197 9)  and i n t h e  e t a l . , 1977; M a e s t r i n i  studies  e t a l . , 1989).  o n many a s p e c t s  of  i n c l u d i n g : 1) t h e  2) t h e i r  transient  nature  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 of multiple nitrogen  a r e few observations  ( 0 . 2 um - <2.0 um, S i e b u r t h  marine p i c o p l a n k t e r s .  The u b i q u i t o u s  of both Cyanobacteria  size  c l a s s has been r e p o r t e d  (see  reviews  by Fogg,  utilization  studies  on N uptake by  and u s u a l l y  i n offshore  by  e t a l . , 1978) and  and e u c a r y o t i c  1986; J o i n t ,  sources.  of nitrogen  does n o t a p p e a r t o be any k i n e t i c  presence  threshold  of both NO3  of uptake  or N-limited cells  interference/interaction However, t h e r e  a  a r e numerous examples o f  i s replete with  nitrogen  exceeds  uptake causes t h e  a l . , 1982, 1986; P r i c e e t a l . , 1985; C o l l o s The  t h e uptake  t o decrease below t h i s  effect of NH  i n N-deficient  Ziemann,  ecology  a n d resumes when a l g a l  and there  depending  s t a t e , b u t one o f t h e main  i n phytoplankton  The n e g a t i v e  that  mostly  nutritional  concentration  i n press).  a v a r i e t y of responses  when t h e a m b i e n t N H  threshold  ambient N H  not  held  by M o r r i s , 1974;  1981, C o l l o s , 1989, Dortch,  have r e v e a l e d  and t h e i r  (seereviews  abundant  algae  i n this  and nearshore  1986; S t o c k n e r  waters  and A n t i a ,  105 1986; has  Mikheyeva,1988).  been demonstrated  oligotrophic, responsible (e.g.,  oceanic  i n certain  fractionation  techniques  Studies  production  with  size-  and n i t r o g e n t r a c e r s have i s also  10-30% and 30-70% o f t h e t o t a l  respectively  particularly  e t a l , 1983; T a k a h a s h i and  nitrogen uptake  communities  Probyn,  photoautotrophs  environments,  and above r e v i e w s ) .  picoplankton  natural  as  r e g i o n s , where p i c o p l a n k t o n a r e  L i e t e l . , 1983; P i a t t  averaging  importance  f o rt h e majority of photosynthetic  Bienfang,1983;  that  Their  confirmed  substantial, N uptake  o f c o a s t a l and oceanic  of the  waters,  ( G l i b e r t , 1 9 8 2 ; Nalewajko and G a r s i d e , 1983;  1985; Probyn and P a i n t i n g , 1985; H a r r i s o n  a n d Wood,  1988) . In t h e present and  urea  study  by t h e p r a s i n o p h y t e ,  pusilla  (Butch.)  replete  and N-starved  NO^  -  urea.  Therefore,  picoflagellate  cells  of potential  aspects  competitors,  eukaryotic picoflagellate.  t o report  picoplankter.  by u s i n g  on t h e n i t r o g e n u t i l i z a t i o n  rate of  was t o  nutrition  This  N-  ammonium a n d  research  of nitrogenous  ammonium  Micromonas  and measuring t h e uptake  the objective of this  the basic  ubiquitous,  of nitrate,  Manton e t P a r k e was d e m o n s t r a t e d  i n t h e presence  determine  first  the utilization  study  of this  i s the  of a cultured  106 M A T E R I A L S AND METHODS Culturing Stock cultures Northeast  Pacific  o f Micromonas  Culture  filter-sterilized  artificial  of British  t o ESAW i n c l u d e d  concentrations  sodium phosphate 1984a).  of ferric  (Na2HP0 ),  chloride  Sodium m e t a s i l i c a t e by S u t t l e  with  (1988).  the sole  Nitrate,  5 5 0 t o 50 uq-at  preparing  deionized,  salt  thoroughly with  fluorescent  water  DDW,  t o Harrison  e n r i c h m e n t , was r e d u c e d  enrichment  (DDW).  Glass  tubes  through  LI-192SB)  ESAW w e r e 2-3 d a y s ,  and experimental)  side  soaked rinsed  3 mm t h i c k  were  by s i xV i t a - L i t e  of culture blue  2 0 6 9 , Rohm a n d H a a s ) a n d t h e i r r a d i a n c e , (LiCor  and polycarbonate  prior t o use.  from two sides  (3 o n e i t h e r  used  solutions i n  and s t o r i n g  and autoclaved  illuminated  et a l .  Reagent grade c h e m i c a l s were  ( i . e . stock  was f i l t e r e d  collector  (Parslow e t a l . ,  1 0 % HC1 ( v / v ) f o r a t l e a s t  cultures  continuously  .  f o rc u l t u r i n g algae  freshly-made  All  - 1  nitrogen  and n u t r i e n t  distilled  used  N-L  (FeCl3«6H20) a n d  e t a l . ( 1 9 8 6 ) a n d 10 nM S e w a s  (Na2Se03) a c c o r d i n g  light  ammonium  (Na2Si03•9H2O) was p r e p a r e d a n d  added as s e l e n i t e  in  ferrous  respectively  4  added as d e s c r i b e d  flasks  e t a l . , 1980).  4  equimolar  in  nutrient-enriched  (Harrison  replacing  maintained  ( F e N H ^ ( S 0 ) 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  sulfate  from  NEPCC 2 9 - 1  Columbia) were  ( 0 . 2 2 um M i l l i p o r e )  s e a w a t e r b a s e d o n ESAW  Modifications  (culture  C o l l e c t i o n , Department o f  Oceanography, U n i v e r s i t y on  pusilla  vessels).  Plexiglas  R  measured w i t h  from t h e c e n t r e  position  UHO  R  The  (No. a 2n  of the  107  culture of  vessels,  M. pusilla,  17°C  see Appendix  2).  (± 0.5°C) i n a t e m p e r a t u r e  cultures bars  continuously stirred  a t 60 r p m .  2  w a s c a . 1 2 0 uE'm  regulated  employed t o minimize b a c t e r i a l  was  monitored a Turner  Analytical  I I electronic  their  Coulter 2.02  (enabling  prior  and s t e r i l e  stir  technique Cell  growth  a f l u o r e s c e n c e measured  cells  Counter  a Coulter  counter with  with  was c a l i b r a t e d Culture  filter-sterilized,  particle  size  into  with  latex  samples  cell  distribution  16 c h a n n e l s The  were d i l u t e d  (1:20)  with  (gravity  homogenized  ( i . e . mixed)  volumes were computed  based  based  microspheres of  u n e n r i c h e d ESAW  Average  model  a 30 um a p e r t u r e .  Whatman GF/F) a n d g e n t l y  to counting.  Counter  thepopulation  t o be c o u n t e d  volume) and equipped  filtration,  the  particle  um i n d i a m e t e r .  freshly  magnetic  10 f l u o r o m e t e r .  counts were measured w i t h  accessory on  model  bath and  methods  Cell TA  Designs  water  contamination.  chlorophyll  f o r growth  was m a i n t a i n e d a t  by t e f l o n - c o a t e d  C u l t u r e s were u n i a l g a l  by i n v i v o  (saturating  Temperature  was  by  1  *s  from  on e q u i v a l e n t s p h e r i c a l  diameter. Concentrations measured w i t h procedures Maclsaac  of dissolved  i n Wood e t a l . ,  (1972), r e s p e c t i v e l y .  filtered  GF/F  filters  polypropylene  3  a Technicon AutoAnalyzer  outlined  were  N 0 ~ + NO2  through precombusted into  previously  bottles.  and N H  + 4  were  I I following the  (1967)  Samples  -  and Slawyk  f o rnutrient  and analyses  (460°C f o r 4 h ) W h a t m a n  acid-washed,  DDW-rinsed  Ammonium c o n c e n t r a t i o n s w e r e  always  108 determined  occasionally  measured  Duplicate carbon and  and  end  later  samples  nitrogen  of  filters  w h i l e NO3  immediately  after  (POC  and  (<60°C f o r 24  dry  method of  combustion  Equipment Corp. model model Both  were c o l l e c t e d  240)  or  Sharp  a Carlo Erba  instruments  h)  240-XA  organic at the  p r e c o m b u s t e d Whatman  stored frozen i n desiccators.  thawing/drying  (-20°C).  for particulate  PON)  on  c o n c e n t r a t i o n s were  frozen storage  (20-30 ml)  experimentation  and  + NO2""  -  GF/F  After  samples were a n a l y z e d (1974) w i t h e i t h e r  (remanufactured  model  were c a l i b r a t e d  1106  start  a  by  the  Control  Perkin-Elmer  elemental  analyzer.  with acetanilide  standards.  15  Samples Whatman GF/F dishes,  and  Nitrogen  gas  M.  and  Proksch,  parameters kinetic  for N  of  analyses.  converted  to  combustion analyzed  emission  for  1 5  N  spectrometer  outlined  i n Chapter  nitrate,  ammonium a n d  with duplicate N03~-replete  grown i n 2 L Pyrex  with  experiments  petri-  1.  uptake  parameters  were determined  ambient NO3  isotopic  subsequently  as  precombusted  acid-washed,  micro-Dumas d r y  1975)  on  procedures  pusilla  fitted  the  s a m p l e s was  w i t h a J A S C O m o d e l N-150  Experimental  uptake  by  2  into  frozen for later  particulate (N )  were c o l l e c t e d  folded, placed  (LaRoche,1983) a n d  enrichment  The  analysis  filters,  i n the  technique  Kinetic  N  immediately  dinitrogen  (Fiedler  for  silicone -  + NO2""  flat-bottom boiling  stoppers. was  measured  were i n i t i a t e d  Prior at  to  0.5  immediately  urea  c u l t u r e s of flasks,  experimentation, -  1 h  after  intervals. the  nitrate  The  109  c o n c e n t r a t i o n was Less  than  <0.05 ug-at  (0.05 in  they  ug-at  t o growth  had d e c r e a s e d  N«L  - 1  ).  ambient  medium.  concentrations  (>2.5 ^ g - a t N - L  - 1  ) and  to detection limits  The t i m i n g was  critical,  because  nitrate  t h e medium must be d e p l e t e d , b u t t h e c o n d i t i o n o f t h e 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 m i n i m i z e  during  experimentation.  depletion,  Immediately  Erlenmeyer NH4  initial and  f o l l o w i n g ambient  (Nalgene  or urea  R  - 1  ).  incubations  terminated  differential than  s 80 mm  30 s.  experiments  ( 0 . 2 , 0.4,  after  Hg).  The 4.2  The t i t r a t i o n and 10 ug-at  N*L  1.6,  2.4,  4.2  under t h e  were grown and (pressure always  enrichment  - 1  f o r an a d d i t i o n a l  50 min  i f u p t a k e r a t e s were c o n s t a n t w i t h i n c u b a t i o n  rinsed  once w i t h  f i l t r a t e and t h e n  The f i l t e r e d  a m b i e n t NH^"" a n d N O 3  -  used  bottles,  to collect  s a m p l e s were i m m e d i a t e l y  + N02~ concentrations.  s u b s t r a t e c o n c e n t r a t i o n s a t time adding  N-labelled  p e r i o d was  P r e v i o u s l y acid-washed p o l y p r o p y l e n e  1  15  10 m i n by f i l t r a t i o n  time.  filtrate.  0.8,  under which t h e c e l l s  were a l l o w e d t o i n c u b a t e  determine  polycarbonate  I n c u b a t i o n s were c o n d u c t e d  same c o n d i t i o n s a s t h o s e  );  99 atom %) a t a r a n g e o f  substrate concentrations N*L  -  R  (Nalgene  ), and i n o c u l a t e d w i t h  (Kor I s o t o p e s ,  NO3  t o a s e r i e s of  Oak R i d g e t u b e s  200 ml s u b s a m p l e s t o 250 ml  flasks  10 ug-at  less  i n uptake  •  85 ml p o l y c a r b o n a t e  alternatively  NO3,  non-linearity  60 ml s u b s a m p l e s were t r a n s f e r r e d  •  sterile,  to  i n the c u l t u r e  - 1  2 h e l a p s e d between t h e t i m e  were c o n s i d e r e d s a t u r a t i n g the time  N*L  known v o l u m e s o f  15  NO3  —  the  analyzed f o r initial  ( T Q ) were c a l c u l a t e d  zero  and  The  were  1  5  NH4  +  to culture  by  filtrate  110 and  then measuring  rates  (N t a k e n  ambient c o n c e n t r a t i o n s .  up p e r u n i t  PON) w e r e c a l c u l a t e d  constant  s p e c i f i c uptake model  equation  6 of Appendix 1 ) .  Substrate  performed with  of substrate N03~-replete  monitored  every  concentration  decreased  was d i v i d e d  cultures.  not  N0 ~  flasks  (enriched  3  enriched  collected -  1 h prior  + 4  procedures.  Nitrate  from t h e slope concentration rates  expressed obtained  The  following  N-L  by d i v i d i n g  + 4  - 1  linear  - 1  .  of the incubation s p e c i f i c uptake  4-L  A single  flat-  enrichment.  N•L~  1  of  ^N  (99 atom flask  was  of  -  +  uptake  calculated  substrate  time.  or transport  These  rates) rates  and a r e were  r a t e s by t h e e x p o n e n t i a l  (geometric  then  outlined  r a t e s were  against  Specific  mean) o v e r t h e  period.  of urea  %)  F i l t e r e d samples were  regressions  the absolute o f PON  were  1 L Pyrex  previously  (absolute •h  .  and urea  a n d ammonium u p t a k e  a r e termed  levels  were  f o r 4 h f o r immediate NO3  i n t h e medium p l o t t e d  average concentration duration  1986 ;  series,  and t h e remaining  control).  of separate  a s uq-at  - 1  four  NH  of the flasks  analyses  )  t o N substrate  control),  (undisturbed  + 4  w h e n 10 uq-at  a t 20-30 m i n i n t e r v a l s  and NH  uptake  to a  the nitrate  t o 15 uq a t N - L into  experiments  In the first  + N02~ and N H  -  and poured  were added t o t h r e e  NO2  interaction  experiments were i n i t i a t e d  labelled  according  (Dugdale and W i l k e r s o n ,  30 m i n f o r 3 h b e f o r e  bottomed b o i l i n g The  (NO3  inorganic N  culture  uptake  interaction  Two s e r i e s  ambient  Specific  was d e t e r m i n e d  from  a  I l l  constant  specific  equation  8 o f Appendix  effect  uptake model 1).  independent  unlabelled  source Dugdale  uptake  incorrectly The  1987; Lund,  and i s c o r r e c t l y  equation  10 o f D u g d a l e  1) w h i c h  compensates  -  Effect  replete  -  t h e second  series  + 4  culture  of unlabelled  o f PON d u r i n g t h e  on NO3  concentration  concentration  rate  250 m l s u b s a m p l e s ) Erlenmeyer  flasks  (fitted  prior  + NO2  -  - 1  .  N«L  with  t o t h e ambient ^NH^Cl  the initial  t o 5, 2, a n d 1 uq-at  -  were t r a n s f e r r e d t o  r e a c h i n g 15 j u g - a t N - L t o bring  1  -  of NO3  f o r the decline  o f NH^" "  A NO3 -  o f N03~ was e x a m i n e d .  was m o n i t o r e d  (Nalgene  uptake  -  stoppers and sampling tubes)  subsamples  using  of experiments, the effect  on t h e uptake  %) w a s a d d e d t o t h e f l a s k s NH  o f u r e a was c a l c u l a t e d  1.  and W i l k e r s o n ( equation 7 o f Appendix  1  polycarbonate  NO3  i n Appendix  f o r t h e simultaneous uptake  t h e medium a n d f o u r  silicone  sources are present, i s  period.  concentration  in  rate  o f NH^" " c o n c e n t r a t i o n  In  i n the unlabelled  reported  and t h e change i n c o n c e n t r a t i o n  incubation  N  i n the  the equation f o r constant  c  written  1 4  dilution  I t s h o u l d be n o t e d t h a t i n  ( V ) , when u n l a b e l l e d  absolute (transport)  NO3  disappearance  -  from t h e  1987).  a n d W i l k e r s o n (1986)  specific  s u b s t r a t e by  f o r the isotope  matter originating  (Collos,  for the  estimates of t h e absolute uptake of  measurements thus c o r r e c t i n g particulate  of unlabelled  provided from NO3  nitrate  and Wilkerson,1986;  T h i s e q u a t i o n compensates  o f simultaneous uptake  utilizing  ( Dugdale  - 1  .  (99 atom  enrichment Fifty  were removed e v e r y 20-30 m i n f o r 2 h a n d  ml ^N  112  incubations of  terminated  filtrate,  storage  by f i l t r a t i o n .  and analyses  outlined.  S a m p l e s f o r PON  beginning,  middle  NO3  external this  + NO2  -  -  provided  an e s t i m a t e  specific  NH  specific  uptake model  uptake  +  against  1 ) with  particulate  matter  The  a v e r a g e PON NO3  of the specific  time;  -  (Dugdale and W i l k e r s o n ,  estimated  division  uptake  r a t e s were c a l c u l a t e d u s i n g  t h e atom %  of of  concentration  1 C  of Appendix  as p r e v i o u s l y  was c a l c u l a t e d f r o m t h e s l o p e  concentration  -  collection  a n a l y s i s were c o l l e c t e d a t t h e  r a t e by t h e e x p o n e n t i a l  4  were conducted  and end o f t h e i n c u b a t i o n s .  rate of NO3  disappearance  Filtration,  rate. the  The  constant  1986; e q u a t i o n  6  1 c  N excess  (  from t h e slope  N e  x  )  i n the  of the least-  1 5  squares  linear  regression  isotope  depletion.  Uptake of n i t r o g e n Two  + 4  the effect  or urea  In each  the i n i t i a l  of  1 5  were  split  NH C1 4  1  of nitrogen  into  and N source  or CO(  1 5  NH ) 4  both i n  2  (both  The a m b i e n t N H  + 4  flasks  Micromonas  were  50 uq-at  and a l l o w e d  obtained N-N03'L to  N-starved,  a n d e i t h e r 15 uq-at  N«L  - 1  99 a t o m %) w a s a d d e d t o e a c h concentration  i n t h e medium and  c  N accumulation  - 1  the external  A f t e r t h e c u l t u r e s became  separate  to  d e f i c i e n c y on t h e  cells  cultures, started with  concentration  prior  cultures of  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  subculture. the  time,  cells  by N 0 3 ~ - s t a r v e d  n i t r o g e n was d e p l e t e d . they  versus  series nitrogen-starved  from d u p l i c a t e batch  remain  e x  by N 0 3 ~ - s t a r v e d  t o assess  of NH  pusilla.  as  N  s e r i e s o f experiments were conducted,  duplicate, uptake  of  i n the cells  were measured a t time  113 intervals  o f 5-15 m i n f o r 3 h a c c o r d i n g  t o procedures  outlined  above. In the second  s e r i e s of experiments,  duplicate  NC^  -  1 5  starved  c u l t u r e s were e n r i c h e d  with  NO3  Na  (99 atom NO3  samples c o l l e c t e d f o r measurement o f ambient concentration  1 c  and  N accumulation  at time  %) a n d  -  intervals  of  5-30  15  mm  f o r 6 h.  the  particulates,  specific  N accumulation to the  constant,  ( V ) o f Dugdale and W i l k e r s o n  (1986)  c  6 of Appendix  i n  1) f r o m m e a s u r e m e n t s o f a t o m  %  excess  c  of  N i n successive  rates,  estimated  samples d u r i n g  the time of NO3  from disappearance  medium, were c a l c u l a t e d by d i v i d i n g concentration interval; value  i n successive  specific  that  time  medium was  -  intervals or NH  + 4  the difference i n nutrient  samples by t h e l e n g t h  exponential  assuming that  incorporated  Uptake  i n the  of the time  r a t e s were c a l c u l a t e d by d i v i d i n g  by t h e e s t i m a t e d  during the  from  were c a l c u l a t e d a c o r d i n g  uptake model  (equation 1  Uptake r a t e s , estimated  •  average  PON  this  concentration  a l l t h e n u t r i e n t removed  into  from  the p a r t i c u l a t e f r a c t i o n (see  Appendix 4 ) . Estimation  of  The ways:  kinetic  kinetic  a direct  hyperbola squares  using  parameters  parameters,  K  f i t of the data a computerized,  regression technique  g  and V to the  (Labtec  Technologies  regression  a n a l y s i s o f Hanes-Woolf  S) o f t h e d a t a .  a  x  were o b t a i n e d  non-linear,  least-  Notebook C u r v e f i t ,  Corp.) and a l e a s t - s q u a r e s  In the latter  i n two  Michaelis-Menten  iterative,  Laboratory  vs  m  linear  linear  transformation  method, t h e s t a n d a r d  (S/V  errors  114  of  the  kinetic  parameters  method of v a r i a n c e s  were e s t i m a t e d  (Bishop et a l . ,  using the  1975).  t r a n s f o r m a t i o n was  used  transformations  i t gave a b e t t e r spread  as  i n preference  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  and  V  a  (Dowd a n d  x  Riggs,  Michaelis-Menten  1965).  the  of  least-squares regression analysis,  this  variable,  assumption  independent  (Zar,  variable  dependent v a r i a b l e .  problem  correlation  between v a r i a b l e s  transformation fitting  of  Robinson did  (Dowd a n d  not  fitting  data  and  estimated literature  statistically  have the  by  both  1965)  inferior  earlier  enjoy  today,  to  the  in  purposes.  separately f o r the  well  data  The  kinetic  individual  treated together.  the  practice  the  inevitable S appears  in  linear non-linear ( L i , 1983;  investigators non-linear  parameters,  methods, have been i n c l u d e d i n T a b l e  comparative  f o r the  the  equation  same a c c e s s i b i l i t y  g  unweighted,  to direct,  Since  been c a l c u l a t e d as  and  K  the  v a r i a b l e s ) makes a  Michaelis-Menten  c o m p u t e r s a s we  in  to errors i n  utilizing  of  assumption  error  (measured v a r i a b l e  C h a r a c k l i s , 1984).  always by  Riggs,  independent  to the  of  of  i f errors in  The  points  linearization  Although  small relative  data  d e p e n d e n t and  met  data  a basic  lack  are  transformed  both  1974).  is sufficiently  the  determinations  equation violates  S  of  linear  However, any  of  independent  Hanes-Woolf  to other  and  m  The  Delta  parameters cultures  3.1  for have  as  115  RESULTS Uptake  kinetics  Nitrogen average cells of  specific  ambient  uptake rates are p l o t t e d  nitrogen concentration experienced  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  the half  saturation constants  velocities estimated cultures  (V  )  m a x  are presented  standard agreed  estimates  errors.  well,  of urea-V  between t h e l a t t e r paucity  m a x  the  V  m  a  m  a  of NO3  x  f o rNH  x  + 4  .  -  from  and urea  Micromonas  uq-at  N-L  agreed  well  pusilla  w e r e w i t h i n ± 0.1 uq-at  Menten e q u a t i o n parameters of  the experimental  experiments,  N-L  - 1  g  to the  The  and a r e about  half  t h e same  f o rNO3 , -  o f each  uptake  over  the duration  In the present  kinetic  that the non-linearity  t h a t has  f o r n i t r o g e n uptake by N - d e f i c i e n t c e l l s  occur  i n this  Nitrogen-replete incubated  N-replete  c u l t u r e s ( 4 . 2 a n d 9.9  f o r both  + 4  i n the use of the M i c h a e l i s -  incubation.  study  NH  other.  been r e p o r t e d because  i nthe  during the  values  remains constant  i ti s unlikely  discrepancy  enrichment).  - 1  f o rthe estimation of kinetic  i s that uptake  between  demonstrated  and  underlying assumption  f o r separate  partly  occurred  f o r each N s u b s t r a t e as t h e K  An  their  low substrate enrichments  affinity urea  The  list  with  a g r e e m e n t was f o u n d  c a n be a t t r i b u t e d  f o r 0 . 1 , 0 . 2 , a n d 0.4  of V  3.1 a l o n g  a n d NH^-Kg v a l u e s .  A  uptake  g  second c u l t u r e (substrate exhaustion  values  (Fig. 3.1).  Generally the values  estimate  by t h e  ( K ) a n d maximum i n Table  but poorer  of uptake values  incubation  versus the  cells  were  uq-at  10 a n d 60 m i n t o d e t e r m i n e  N-L  would  utilized. - 1  )  i f V  were 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 u p t a k e r a t e s (V) d e t e r m i n e d o v e r 10 m i n a f t e r t h e a d d i t i o n o f 0.2, 0.4, 0.8, 1.6, 2.4, 4 and 10 /jg-at N-L* o f N0 " ( A ) , u r e a (B) o r N H (C) t o duplicate n i t r a t e - r e p l e t e cultures (O , • ) of Micromonas pusilla. Rates (h ) are p l o t t e d versus the average substrat c o n c e n t r a t i o n d u r i n g t h e 10 min i n t e r v a l . Curve c a l c u l a t e d 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 ) . 1  +  3  4  - 1  0.100 0.080  O Z >  0.080 X  z >  0.040 0.000 0.0  2.0  4.0  SUBSTRATE  6.0  8.0 10.0  ( g - a t N-L" ) 1  M  T a b l e 3.1 K i n e t i c p a r a m e t e r s f o r n i t r a t e , u r e a a n d ammonium u p t a k e o f N - r e p l e t e Micromonas pusilla. MichaelisMenten 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 u p t a k e v e l o c i t y ) w e r e e s t i m a t e d f r o m 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 data obtained from r e p l i c a t e c u l t u r e s (1 o r 2) a n d 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 ) .  Substrate  Culture  ^ax* f*  Nitrate  1 2 1 + 2  4.64 (0 118) 5. 07 (0 361) 4.86 (0 183)  0 .44 (0 044) 0 .50 (0 014) 0 .47 (0 069)  4.70 (0 057 ) 5. 32 (0 176) 4.99 (0 125)  0 49 (0 050) 0 60 (0 022) 0 54 (0 108)  Urea " "  1 2 1 + 2  4.70 (0 176) 5. 93 (0 211) 5. 38 (0 257)  0 35 (0 051) 0 40 (0 059 ) 0 38 (0 073)  4.42 (0 098) 5. 64 (0 082 ) 4.95 (0 219)  0 27 (0 093) 0 30 (0 059 ) 0 26 (0 181)  Ammonium  1 2 1 + 2  13 .6 (0 99) 12 . 1 (0 74) 12 .9 (0 61)  0 .49 (0 142) 0 28 (0 104) 0 40 (0 087)  14 .8 (0 56) 12 .8 (0 25) 13 .8 (0 44)  0 .76 (0 165) 0 43 (0 098) 0 62 (0 145)  li  1 0  " * " > 2  1  1  K  1 s  (f 9J  a  t  N-L  - 1  )  V  max  2 (  x  l  0  _  2  h  ~  1  >  K  2 s  <^9-  a t  N  *  L  1  )  118  decreased with v  0-10min  a  n  d  increased  0-60min  v  a  r  incubation  e  p  r  e  S  ented  time.  i n Figure  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 although average  duplicate  during  enriched  + 4  replete  culture  o f M. pusilla 2  - 1  the 3 h experiment l  N-L  i  rate  -  1  cessation  designed NO3  -  of NO3  second  NC^  and  -  o f n i t r a t e t o a NC>3~-  from that  -  1  observed  for 3 h  , 3 d monitoring).  There  between t h e u n d i s t u r b e d (enriched  control  control  = 0.0492  The a d d i t i o n  h  - 1  o f 10  .  resulted  i n a 28% decrease  + NC^  and a s p e c i f i c urea  -  The t o t a l  -  nitrogen  -  (10 ^jg-at N * L + NC^  -  series  - 1  )  uptake  o f 0.0732  of substrate  uptake, were conducted growth rate  no e n r i c h m e n t  resulted  disappearance  t o determine the e f f e c t ( s )  (preconditioned With  of NO3  s p e c i f i c uptake rate The  - 1  h  -  1  - 1  )  alone.  .  + 4  -  1  experiments,  concentration  on a N C ^ - r e p l e t e  -  h  f r o m t h e medium and a  of NH  ( c o n t r o l ) , t h e NO3  uptake  i n the complete  interaction  = 0.0484 h  i n the  (0.0579  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  Ammonium a d d i t i o n  +  of the urea,  (Fig. 3.3).  N] u r e a  rate  o f 0.0260 h  increased  4  was  e  of [  disappearance  )  (0.0446 h  t h e N03~~-enriched c u l t u r e  uq-at  isotope  An  d i d not a l t e r the disappearance  (0.0441 h  t o N enrichment  during  NH  N*L  however a 10% d i f f e r e n c e  and  rates,  respectively.  o f 10 uq-at  + N0 ~  3  was,  between t h e two  t h e 60 m i n i n c u b a t i o n s  addition  of N0 ~  prior  values f o r  x  interaction  The  rate  a  measurements were n o t always p o s s i b l e .  cultures,  Substrate  m  3.2. a n d t h e r e  o f 3 0 , 40 a n d 6 0 % o f t h e a v a i l a b l e  utilized NH  The V  , 4 d  + NO2  -  on  culture monitoring). depletion  rate  )  119 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 d e t e r m i n e d 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 a r e 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.  0.1 4 0  V 0 - 1 Omin V 0-60min  0.1 2 0  > 0.100  <  cn  0,080  LJ  <  Q.  ZD  0.060 0.040  O  o  L±J CL CO  0.020 0.000 1 NITRATE  2  3  4 UREA  5 AMMONIUM  6.  120 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 o f 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 NO ~ + N0 " ( • ) c o n c e n t r a t i o n and N-atom % excess ( O ) a f t e r 10 ^/g-at N-urea-L" a d d i t i o n . B. D i s s o l v e d NO " + NO " c o n c e n t r a t i o n ( O , A ) a f t e r no and 10 /jg-at N- N0 ~*L a d d i t i o n , r e s p e c t i v e l y . D i s s o l v e d NO," + NO~ c o n c e n t r a t i o n ( • , • ) a f t e r 10 p g - a t N-L" a d d i t i o n o f NH ^ and u r e a , respectively. D i s s o l v e d NH 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 ug-at N-NH/'L" . 15  2  1  3  1  4  +  4  1  tn V)  o x  cn a.  E o  I CN  O  < UJ  cr  O  3.  cn 3.  I CN  o  I  I CO  o 120  TIME  180  (min)  240  121  averaged All  the  0.0500 h 1 5  NH  when t h e ^ N H 1  be  NO3  of external  until  mentioned  + NO2"" d i s a p p e a r a n c e  -  the  that  1 5  NH  + NG^  -  of NO3  the uptake  investigators  (e.g., Serra et a l . ,  NO2  -  only  diatoms  1 9 7 7 ) , M. pusilla  trace  M. pusilla  would of NO3  decline apparent  + NO2  -  inhibitory  The  - 1  )  prior  of  of NO3 5-6  (0.0486  -  rate  + 4  1980;  excretion of  (Harrison  and  of NO2  and  -  i n t h e medium d u r i n g 5).  Excretion  of NO2  i n any  measurable  o r u r e a on NO3  -  from  o f M. pusilla  to the depletion with  ± 0.0018 h  by  -  uptake.  -  _  the V 1  ) .  m  a  1.11  d  -  1  i n t h e medium,  f o r NO3  x  The s p e c i f i c  calculated uptake  of N0 ~  material  (Table 3.2).  over the incubation  3  the accumulation  •  and t h e disappearance  t h e medium, a v e r a g e d  period  was  of nitrate  estimated from both  xnto the p a r t i c u l a t e + NO2  other  et al.,  no e x c r e t i o n  found  of NH  •  h incubation  constant  effect  cultures,  -  NO3  )  i n t h e medium and t h u s enhance t h e  -  agrees w e l l  N03~-starved 5  - 1  cells  a value which previously  assemblages  to a reduction  e x p o n e n t i a l growth  (0.0463 h  •h  - 1  I t should  Although  1978a; O l s o n  (Appendix  -  contribute  Nitrogen-starved  1  on NO3  growth  were  -  N*L  was e s t i m a t e d f r o m t h e  -  demonstrated  of NO2  levels  exponential  by  and n a t u r a l  caused  measurably  1984b) have r e c o r d e d s u b s t a n t i a l  by marine  Davis,  -  )  ( F i g . 3.4).  + NO2"" i n t h e m e d i u m .  o f NO^  - 1  i n t h e medium; t h e  d i d not  -  was e x h a u s t e d  + 4  N-L  (< 0.1 uq-at  available  disappearance  Parslow et a l . ,  of the experiment.  ( 5 , 2 , a n d 1 uq-at  i s o t o p e was s t i l l  + 4  concentration decrease  over the 2 h duration  1  enrichments  + 4  of NO3  cessation  -  0.0238 h  This rate  -  1  was  over the roughly  ( 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 " [ N ] - N H enrichment; NH 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 versus time (min). Arrows d e s i g n a t e t i m e o f NH addition. 1  1 5  +  4  +  4  +  4  10 8 6 4 2 0 10  o cn  X CD  E o  6  CN  4  O z:  2  +  1  o  8  1  1  to CD  CO  X I  0 10  o  z:  8 6 4 2 -  0  0  20  40  60  TIME  80  100  (min)  120  140  123 T a b l e 3.2 Average n i t r a t e uptake r a t e s (h ) f o r NC>3 -starved Micromonas pusilla. Rates determined from least-squares l i n e a r regression of p a r t i c u l a t e N enrichment o r t h e decrease 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^" + N G ^ versus time and reported as ± 1 standard deviation ( i n parentheses) of the mean o f d u p l i c a t e c u l t u r e s . -  1 5  -  Time  Interval  N i t r a t e Uptake  (•10~ h 2  1  )  1R  (h) 0 1 2 3 4 5  N  1 2 3 4 5 6*  (* = u p t a k e  2 2 2 2 2 2  0  3  22 10 34 56 44 12  rates  N  0  3  disappearance  (0.0071) (0.184) (0.014) (0.417) (0.099)  1. 1. 2 2 2 2  c a l c u l a t e d from  one  77 86 94 80 66 73  (0.537) (0.233) (0.170) (0.629) (0.438)  culture)  T a b l e 3.3 Average N uptake r a t e s V (h ) f o r NC^'-starved Micromonas pusilla. Rates determined from least-squares l i n e a r regression of p a r t i c u l a t e N enrichment o r t h e decrease in the external concentration of dissolved nitrogen versus time and reported as ± 1 standard d e v i a t i o n ( i n parentheses) 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 . 1 5  N  Substrate  N Uptake v  Urea 1 5  NH  NH  + 4  + 4  (* = u p t a k e  0-60min  v  (•10~  60-120min  h" )  2  1  v  120-180min  4.80  (0.129)  3.72  (0.149)  3.32  (0.158)  7.05  (0.0120)  5.59  (0.0559)  4.23  (0.0288)  6.76  (0.449)  4.85  (0.600)  6.75*(0.739)  rate  calculated  from  2.5 - 60 m i n )  124 that  t h e r e was e l e v a t e d  uptake  (0.0386 ± 0.0012 h  -  1  ) during  1c  the  first  were  5 minafter  not observed  N enrichment.  Elevated  -  possibly  due t o t h e reduced  colourimetric  analysis  a t elevated  less  The average  than t h enitrogen  specific  monitoring probably  period  only  lowNO3 concentrations -  rate  after  starvation  from  either  t h e r e was, no l a g p e r i o d starved The the  cells  exponential  growth  second  urea)  series  averaged  nitrate  1.11 d  -  1  1  Maximum  constant rate  average  hourly  during  the4 h  before  orV  -  1  uptake  calculated m a x  .  However  by t h e previously  cultures  experiments  ) prior  used i n  (NH^  +  and  t odepletion of  [^N] urea uptake  rate  (0.105  t h e 0-5 m i n i n t e r v a l a n d i n t h e next  o f 0.0349 ± 0.0016 h  urea uptake  )  and t h e  1  The average  of duplicate  (0.0463 h  decreased rapidly  roughly  particulate  rate  ) occurred during  subsequently  Average  -  - 1  rate  rate  o f N03~-starved uptake  i n t h e medium.  ± 0.016 h  growth -  3.5).  ) calculated  1  t h e N demand  i nNO3 uptake  (Fig.  1  t o decrease as a consequence  half  thepre-conditioned  nitrate  This reduction i s  i n t h e medium.  i sonly  )  i s c a . 25%  1  -  - 1  N• L ~ • h ~ )  N'L  a minimum e s t i m a t e a s u p t a k e begun  -  (0.0308 h  t oN depletion.  d e p l e t i o n may h a v e a l r e a d y of  rate  o f PON (50.0 uq-at  prior  N-L  o f 0.0238 h  (1.54 uq-at  -  concentration  sensitivity of  (> 15 uq-at  rate  f r o m N O 3 + NO2"" d i s a p p e a r a n c e average  rates  i n t h e N O 3 + NO2"" d i s a p p e a r a n c e  measurements,  concentrations.  uptake  rates,  10-20 m i n t o a -  calculated  1  ( F i g . 3.6)  The  from t h e slope o f  A P E 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.  uptake  rates  during  t h e second  hour  of incubation  were  125  F i g u r e 3.5. N i t r a t e uptake 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 - N 0 " * L to duplicate cultures. A. D i s s o l v e d N 0 " + N 0 " (•,•) i n t h e c u l t u r e medium; N0 ~ 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 ( 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 " + N0 " d i s a p p e a r a n c e technique. C. [ N] n i t r a t e uptake r a t e . Values i n A are 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 enrichment and 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 incubation time. _ 1  3  3  2  1 5  3  3  2  1 5  ~  0.040 -  0.000  1  0  •  1  60  •  1  120  •  1  •  180  TIME  1  240  (min)  •  ' • 300  1  360  126  F i g u r e 3.6. 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 uq-at N - u r e a - L " t o d u p l i c a t e c u l t u r e s (0,»). A. 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 elapsed time measured a f t e r a d d i t i o n o f urea. B. [ N] u r e a uptake r a t e p l o t t e d a g a i n s t average incubation time. 1  1 5  1 5  to  O C XD  14.0  A  12.0  cu  10.0  E o o  —  <  LU  fr Z> i  8.0 6.0 4.0 2.0  ~Z.  in i—  •>*  0.0  i  *  ,  .  i  i  0 . 1 2 0  T"rz l±J  0 . 1 0 0  0 . 0 8 0  <  1— Q_  <  0 . 0 6 0  0 . 0 4 0  UJ  cr  0 . 0 2 0  0 . 0 0 0 0  3 0  60  9 0  TIME  1 2 0  (min)  150  180  127  7 8% o f t h e i n i t i a l  hourly  rates  and during  uptake merely  decreased  during  60-120 a n d 120-180 m i n i n t e r v a l s  the to  30-60,  maximal support  exponential  roughly of  1 5  constant  growth observed  uptake rate  4  (0.175  0-5 m i n i n t e r v a l ,  decrease  the first  hourly  NH  the initial  short-term  11%.  (ca.  prior  rates  were < 40% o f  uptake  rate  needed  t o N-depletion.  ± 0.0024 h followed  - 1  )  occurred  by a r a p i d , b u t  80%) i n u p t a k e b e f o r e  i t reached  hour  of incubation  ( F i g . 3.7 C ) .  and averaged  The a v e r a g e  the 3 h of  1 5 0 , 120 a n d 9 0 % o f t h e n i t r o g e n  uptake,  needed t o support  60-120,  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  0 . 0 0 6 0 h~^)  and  rate  disappearance  + 4  growth,  during  i n t h e medium  the  1 5  NH  4  uptake  rate  (0.0561  + 4  disappearance  0-2.5 m i n i n t e r v a l and nearly  uptake  rates  1  However, u n l i k e t h e measurement o f ^ N obtained,  x  greater  i s i n good  ± 0.0010  h  (Table  -  1  ), 3.3 ) .  ± 0 . 1 3 3 h -^) d u r i n g -  greater  than t h e average  than t h e elevated  f o r t h e 0-5 m i n i n t e r v a l  NH  4  ( F i g . 3.7 B ) .  c N-tracer  technique,  i n the unenriched  the concentration  disappearance sample,  e  (0.503  w e r e 7-9 t i m e s  3 times  reported  rates  t h e 0-60,  (0.0612 ±  also declined over t h e 3 h incubation period  Maximal N H the  exponential  ( 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 ) ,  agreement w i t h  a  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  r a t e d e c l i n e d by 20-25% p e r hour over  monitoring  hour,  The u p t a k e  r a t e and 70-80% o f t h e n i t r o g e n  Maximum during  an a d d i t i o n a l  the third  technique  of NH  can only  a n d may b e s u b j e c t  + 4  where an  accurate  particulates  a t time-zero  be e s t i m a t e d  t o measurement  f o r the NH  from a  error.  c a n be + 4  cell-free  128  F i g u r e 3.7. Ammonium u p t a k e b y n i t r a t e - s t a r v e d Micromonas L t o duplicate pusilla a f t e r t h e a d d i t i o n o f 15 uq-at N- NH. cultures. A. D i s s o l v e d NH concentration i nthe culture m e d i u m (•,<">) ; N - N H 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 against elapsed time a f t e r enrichment. B. Ammonium uptake r a t e , determined by NH dissappearance technique. C. [* N] N H u p t a k e r a t e . Values i n B and C p l o t t e d against average incubation time. +  4  1 5  4  +  4  5  4  co co a> o x  E o  3.  X X  0.597 0.409  0.200  B  0.1 60  LU 0.120  OLID  0.080  +* X  0.040 0.000 0.200 -  W  0.160  < £  0.120 -  ZD <*  0.080  n  0.040 0.000  0  30  60  90  TIME  120  (min)  150  180  129  A summary of the culture conditions at the beginning of each series of experiments  i s presented i n Table  3.4.  Table  N0 ~  Experiment and culture  Culture description  3  number  NO ~  sufficient  3  II  N0 ~  sufficient  3  II  NO ~  starved  3  it  NO-j~  starved II  A:  N 0  3~»  N H  4  +  a  n  NH  4  C:  NH  4  D:  NH  4  E:  NO ~ u p t a k e e x p .  *:  +  +  and urea  1  POC (uq-at  Cell 1 1  C-L~ )  (10  density 9 - 1 -L ) L  <0.05 <0.05  49.2 51.9  382.1 382.7  B-1 C-l  13.6  302.3  8.3  35.2 32.5  295.3  D-l D-2  <0.05 <0.05  50.8 54.8  546 .7 549 .6  10.38  E-l E-2  <0.05 <0.05  59 .3 47.1  504.8 526 .7  9.28 10.00  exp.  exp. (Series  exp. (Series  2).  and urea uptake exp.  3  L cell  PON  2  experiment.  A-l A-2  inhibition  inhibition  + N0  ( p g - a t N* L " )  durea uptake k i n e t i c  B:  +  at t h e beginning o f each  Summary o f c u l t u r e c o n d i t i o n s  3.4  volume p e r l i t e r  of culture.  1).  5.71 6.08 _ 4.72  -  Total  cell  Volume* (pL-L ) - 1  13.2 13.3 _ 9.64 14.1  12.3 12.8  Cell (fg-at  Quota N'cell  8.6 8.5 — 6.9 4.9  6.4 4.7  - 1  )  131  DISCUSSION  Uptake  kinetics  Over t h e l a s t determined and  the kinetics  of nitrogen uptake  n a t u r a l assemblages  McCarthy, It  two decades numerous i n v e s t i g a t o r s  1981; Goldman and G l i b e r t ,  i s often d i f f i c u l t  the  of phytoplankton  various  condition  of the phytoplankton  Generally  the values  N-uptake  a r e lower  assemblages  and i s o l a t e d  1969;  Carpenter  values et  size  oligotrophic  and t h e K  1984). V  m  g  value,  a  spring  x  f o rreduced  N forms  This  hypothesis such  a  x  such  as NH  general  (e.g,  +  ,  m a x  -N03~  and Toetz,  depend p r i m a r i l y  (i.e.,  than  during equals  or  1976; D o r t c h , i n  observation i s consistent with the  t h a t t h e l a r g e p l a n k t o n i c forms t h a t bloom  conditions  cell  has been  i s lower  except  V  Eppley  between  (Halterman  where  Dugdale,  e ta l . ,  of t h e ocean.  correlation  4  for  half-saturation  f o rnitrate  blooms o r i n u p w e l l i n g areas  e x c e e d s t h a t o f ammonium press).  m  Eppley  correlation  phytoplankters  s  natural  (e.g.,  no such  (K )  1969; Kanda e ta l . ,  areas  a direct  I t a l s o appears t h a t V  constant  oceanic  1971) t h a n  although  f o rfreshwater  the results of  H a r r i s o n e t a l . , 1989).  clones  from e u t r o p h i c , and n e r i t i c  observed  the  (e.g.,  and Dugdale,  a l . (1969) d e m o n s t r a t e d  i n press).  and t h e p h y s i o l o g i c a l  f o roligotrophic  and G u i l l a r d ,  by  of techniques,  of the half-saturation  (e.g., Maclsaac  1985)  (see reviews  t o compare and i n t e r p r e t  incubation periods  cultured  1983; D o r t c h ,  s t u d i e s due t o t h e v a r i e t y  experimental  i n both  have  NO3  -  on n i t r a t e  i s abundant (Malone,  during  i n the euphotic  1980).  However,  zone)  since  132 nutrient coupled  uptake (i.e.,  chemical  and growth processes balanced  composition  1977; M c C a r t h y , to  on NO^ ,  NH  -  physiology and  (see reviews  1981) p h y t o p l a n k t o n  To d a t e ,  Wetzel  and a l g a l  are adaptable  grow e q u a l l y w e l l  1981).  and equal)  are not necessarily  (eg.,  o n l y a few s t u d i e s (Paasche,  1980; R h e e a n d L e d e r m a n ,  Dugdale,  g e n e r a l l y have t h e a b i l i t y and urea  + 4  by  Syrett,  1971; W a r d a n d  1983; T h o m p s o n e t a l . , 1989)  have p r o v i d e d  good evidence  f o ran i n c r e a s e i n growth  cells  on NH  NO3  growing  versus  + 4  under  -  saturating  rate of  growth  PPFD. In of  NO3  (0.28 by  -  and urea,  - 0.50  Eppley  the  to their  estimated  are twice  Pacific  t h e range  t o date  Ocean;  values  + 4  from  nutrient  diatoms  have been  respectively.  from  and ng-at  N*L ^, -  two p h y t o f l a g e l l a t e s a r e  l o w ammonium e n v i r o n m e n t .  slope  s  f o rN 0  These r e s u l t s  reported  isolated  Platymonas  The  (a) o f t h e M i c h a e l i s 1980; P a r s l o w  work, t h e v a l u e s  ± 8.5  (0.1-0.7  o f 50 a n d 2.9  ( i . e . a = V /K , Healey,  a n d 32.3  reported  a t l o w c o n c e n t r a t i o n s c a n be  the initial m  those  are a l l similar  oceanic  values  s  extremely  In the present  ± 3.4  utilize  within  suggesting that these  f o ra given  Menten p l o t  14.2  North g  adapted  1985).  K  values  s  -  sp. with K -NH  respectively,  best  K  N'L -'") a n d f a l l  The l o w e s t  oligotrophic  affinity  their  values  + 4  e t a l . (1983) f o r t w o m i c r o f l a g e l l a t e s  Mantoniella  well  -NH  m a x  e t a l . (1969) f o r s m a l l ,  -  Koike  work, t h e V  although  uq-at  N-L "*").  uq-at  by  the present  ~ , urea  3  suggest  low concentrations of NH  + 4  that  et a l . ,  o f a a r e 10.3 and M.  NH  + 4  ±  ,  p u s i l l a  more e f f e c t i v e l y  can than  1.9,  133 equivalent kinetic  studies  conducted,  of other  proportions phytoplankton Probyn  that  (Wheeler and Kirchman  of these  reduced  forms  be as c l e a r c u t :  "regenerated"  uptake  the  e t a l . , 1982; Furnas,  r e s u l t s of Koike  phytoplankton  Cellular  between  The  effects of cellular  rates  Syrett  (1953) and Harvey  -  uptake  by phytoplankton  by batch  equally distributed  deficit  pattern.  (1953).  They  cells  quite  that  nitrogen  demonstrated by  showed t h a t  cultures of "nitrogen-starved"  and decreased  was overcome.  habitats  for Antarctica  were f i r s t  F i t z g e r a l d (1968) n o t e d  sustainable  marine  p h y s i o l o g i c a l s t a t e on  much more r a p i d t h a n b y " n o r m a l " replete.  "new" a n d  state  uptake  NO3  distinction not  1983; Ronner e t a l . , 1983) w h i l e  t h e former  physiological  a n d Wood,  o n n e t (> 20 um) a n d  i s nearly  e t a l . , (1986)  confirmed  higher  the larger  1987; H a r r i s o n  between t h e two s i z e - f r a c t i o n s i n v a r i o u s (Sherr  generally  1983; Probyn, 1985;  found t h i s  the partitioning  nitrogen  (1986),  f o r growth than  (Nalewajko and Garside,  (< 20 um) h a v e o f t e n  including  and use r e l a t i v e l y  Similar fractionation studies  nanoplankton  utilizing  tracers or  picoplankton,  forms o f n i t r o g e n  actual  have n o t been  and nitrogen  and P a i n t i n g , 1985; S a h l s t e n ,  1988).  to  techniques  t h e reduced  While  on n a t u r a l assemblages  have demonstrated  microheterotrophs prefer  picoplankters  related studies  size-fractionation analogues  o f u r e a a n d NO3"".  concentrations  that these  were  NH  + 4  and  cells  was  nitrogen-  r a t e s were n o t  r a p i d l y once t h e n i t r o g e n  Conway e t a l . (1976) d e s c r i b e d t h e  134 response  i n considerably  distinguished a  three  phases o f uptake  short-lived period  uptake"  (V ),  of very  a longer,  s  more d e t a i l  (cellularly)  "externally"  (ambient  uptake  demonstrated  "surge"  under c o n d i t i o n s (e.g.,  McCarthy  nutrient  and Goldman,  "surge as  (V^), and  concentration)  studies  o r "enhanced" N H  of N deprivation  nutrient:  phase c h a r a c t e r i z e d  More r e c e n t  e  termed  c o n t r o l l e d uptake  limiting  (V ).  of the limiting  uptake,  sustainable  "internally"  controlled  high  i n marine diatoms and  have  uptake  + 4  also  capabilities  i n numerous c u l t u r e  1979; Dortch  studies  e t a l . , 1982;  Goldman e t a l . , 1981; Goldman and G l i b e r t , 1982; P a r s l o w e t al.,  1984a,b; S y r e t t  communities Harrison, and  and P e p l i n s k a ,  ( G l i b e r t and Goldman,  Harrison,  1988).  N-starved  was  several  7-9  times  uptake  disappearance, rate  greater rates  (Vg°-  rate  growth rate  of N deprivation  These e l e v a t e d ecological  surge N H  + 4  by  with  necessary  observed response  that  allows  uptake  v  0 _ 2 s  rate  -  5 m i n  before i s both  Parslow  controlled  t o maintain  the  N depletion. species  1977) and a f u n c t i o n  (e.g.,  above  and n u t r i e n t  The  specific  ofthe  e t a l . , 1984a).  t r a n s i e n t s have been h y p o t h e s i z e d  adaptation  study  = 2.5-4 t i m e s ,  respectively) than the i n t e r n a l l y  Conway a n d H a r r i s o n ,  duration  5 m i n  obtained  magnitude of surge uptake (e.g.,  1987; S u t t l e  are s i m i l a r t o those described  and t h e uptake  preconditioned  1984:,Priscu,  cultures; the i n i t i a l  fold  e t a l . , 1982;  The r e s u l t s o f t h e p r e s e n t  -  uptake  1981; Wheeler  1983a; P r i s c u and P r i s c u ,  N C > 3 - s t a r v e d M. pusilla with  1988) a n d n a t u r a l  phytoplankton  t o be an to rapidly  =  135 sequester 1979; and  ephemeral micropatches  Glibert  and  m a i n t a i n high growth  (Goldman e t a l . , this and  Goldman,  concept  1979;  i s not  W i l l i a m s and  of N  1981;  Goldman and  Goldman and  Glibert,  w i t h o u t c o n t r o v e r s y as  Muir  (1981) c o n t e n d  of those  patches  them from  existing  l o n g e n o u g h t o be  micropatches reported values  are  ca.  respectively) on  considered 1977;  few.  500, may  300  1980;  areas  of  and  (1986)  60  uM  revised  McCarthy;  produced  ocean;  nutrient  (1979)  P0  they to  topic  ~,  (Allen, with  utilize  (Lehman and  suggestion of r a t e s may  c o m p e t i t i v e advantage  3 4  be  This, together  original  values  were  scales  can  on  (maximum  and  large  spatial  data  that the  2  zooplankton  however, t h i s  prevent  by  upwards as  (1977) t h a t e l e v a t e d uptake  in dictating the  to the  to  , N0 ~  + 4  are too  1980).  by  as  Trent  for NH  (1980)  molecular  suggested  Shanks and  However  Jackson  exploited  showing t h a t phytoplankton  patches  Harrison  important  by  h a v e t o be  1982a,b) l e n d s c r e d e n c e and  Collos  both  rapid  1982)  environments  1982).  that the so  Goldman,  Glibert,  limitations,  relevant to phytoplankton  data  phosphorus  be  sample volumes which  Harris,  empirical  would  to technical  i n micropatches  of  measured  Due  and  rates i n oligotrophic  diffusion  phytoplankton.  (McCarthy  in  Scavia, Conway  be  oligotrophic  is s t i l l  a  subject  of  controversy. A  rare,  but  significant  finding  i n the  p r e s e n t work i s 15  the  appearance of  uptake  following  documented  a rapid, surge  but  uptake.  short-term decrease This response  for freshwater phytoplankton  has  (Suttle  in only  and  NH  4  been  Harrison  136 1988)  and has n o t been r e p o r t e d i n p r e v i o u s t i m e  studies and  of rapid NH  uptake  + 4  i n marine  H a r r i s o n (1988) s u g g e s t e d t h i s  uptake  may be t h e r e s u l t  processed  into  amino a c i d s .  c o u l d be t h e r e s u l t to the i n f l u x Lemna  gibba  phytoplankton.  temporary  of a short  course  decrease i n  l a g b e f o r e NH  Alternatively,  Suttle  + 4  c a n be  they suggest i t  o f a sudden l o s s o f membrane p o t e n t i a l due  of cations,  a phenomenon o b s e r v e d when N - s t a r v e d  (duckweed) was p u l s e d w i t h N H  + 4  (Ullrich  et a l . ,  pusilla  to u r e a  1984 ) . The  response  enrichment elevated  of NC^-starved  was s i m i l a r  surge uptake  to the ^NH  cultures  increase  Price  They c o n t e n d t h a t  uptake  and t h e r a p i d  3  1 5  NH  + 4  i n M. pusilla  .  Others Price  (Rees  was s l i g h t l y  urea uptake  -  a short elevated  of concomitant  controlled  rate  lower than t h e r a t e  also  i n NC>3 -starved l a g period [*^N] u r e a ["^N] u r e a  released  of urea  uptake  needed t o  to N depletion.  1979; H o r r i g a n a n d M c C a r t h y , 1981;  1988b) have f o u n d N d e p r i v a t i o n  i n marine  Rees a n d S y r e t t  after  growth o b s e r v e d p r i o r  and S y r e t t ,  and H a r r i s o n ,  and t h e  reabsorption of previously  The i n t e r n a l l y  s u p p o r t t h e maximum  rate  t h e subsequent  r a t e was t h e c o m b i n e d r e s u l t  NH /  5 m i n was 2-3 f o l d  a n d H a r r i s o n (1988b)  pseudonana  uptake  1 5  response; the  i n ["^N] u r e a u p t a k e  o f Thalassiosira  (5 m i n ) .  uptake  controlled  growth r a t e .  found a s i m i l a r  + 4  during the f i r s t  greater than t h e i n t e r n a l l y preconditioned  Micromonas  diatoms  relative  (1979) s u g g e s t e d t h a t  increased  t o N-replete c e l l s . ammonium  u r e a i n t h e g r o w t h medium may p a r t i a l l y  formed  suppress  from  formation of  137  the  urea uptake  during  nitrogen  contend caused the  mechanism and t h a t  that  deprivation.  the increased  by a r e d u c t i o n  urea-N The  this  repression  P r i c e and H a r r i s o n  n i t r o g e n - s p e c i f i c urea uptake  i n the cell  N quota  importance o f t h e "enhanced" o r "surge" uptake t o growth  depends on t h e c o u p l i n g  of t h e uptake of  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  (Collos,  1986).  incorporate  that  may b e l i m i t e d incorporate  rates  nitrogen nitrogen  on a s i m i l a r a t which  dissolved nitrogen  Nitrate  respond  material  rapidly to brief  but the cells  by t h e r a t e  1982,1983;  urea.  I f uptake  o f ambient  are not able t o  time  scale, then  cellular  growth  metabolism can  i n t o macromolecules  (Wheeler e t  Zar,1988).  was n o t t a k e n up a s r e a d i l y b y t h e N 0 3 ~ ~ - s t a r v e d  o f Micromonas  culture  was  and r e t e n t i o n o f a l l  nitrogen  al.,  (1988b)  by t h e n i t r a t e - s t a r v e d c e l l s .  phytoplankton  pulses  i s removed  pusilla  as t h e reduced N forms, N H  H o w e v e r , u n l i k e many N C ^ - s t a r v e d  phytoplankton  + 4  and  (review  by  C o l l o s , 1983; D o r t c h e t a l . , 1982; P a r s l o w e t a l . , 1984b)  no  previous  commenced The  exposure ( i . e . there  considerably  observed  was r e q u i r e d  uptake  the first  rate  lower than t h e NO3  i n the starved  -  cultures.  controlled rate  required  NO3  before  -  attained, which  5 min a f t e r enrichment,  c u l t u r e s and t h e elevated  internally that  -  uptake  was no l a g p e r i o d ) .  maximum n i t r a t e  measured d u r i n g  replete  t o NO3  rates  was  was  measured  i n NO3 -  of NH  and urea  rates  The l o w e r ,  over t h e next  + 4  -  sustainable  5-6 h w a s o n l y  t o support the pre-conditioned  growth  half  r a t e and  138 comparable t o i n t e r n a l l y  r e d u c t i o n i n N O 3 uptake  This is  n o t uncommon  Syrett,  1980; D o r t c h  I t appears  uptake  -  ability  b e f o r e maximal N O 3  observed  by many o t h e r s  i s possible  that  -  that  uptake  and hence t h e i r  relative  examination  i n N O 3 uptake  decline  c a n be a t t a i n e d ,  -  t o the loss  ability  uptake  enhanced i n i t i a l  NC^  nitrate  pools are o f t e n observed  Dortch,  1982; D o r t c h  -  1983).  reduced  their  capability.  The  was a s s e s s e d by  c o l o u r and m o t i l i t y .  system  reductase  of n i t r a t e  response  e x p e r i e n c e d by  during starvation  prevent  Substrate  cells  of c e l l u l a r  of nitrate  However, i n a c t i v a t i o n  stress  n i t r o g e n uptake  o f an a c t i v e  to inactivation  still  a  by C o l l o s ,  starvation  -  "health" of the N-starved  microscopic  a l t h o u g h M. pusilla  the physiological  viability  The  c o u l d a l s o be  (Falkowski,  1975a)  (e.g., S y r e t t ,  1981).  r e d u c t a s e a l o n e need n o t  uptake  as t r a n s i e n t  i n phytoplankton  internal (e.g.,  e t a l . , 1984).  interaction  Uptake i n t e r a c t i o n s particularly  nitrate  Syrett,  between i n o r g a n i c N s o u r c e s ,  a n d ammonium, have b e e n t h e s u b j e c t o f  many s t u d i e s ( s e e r e v i e w s  reveal  1965; T h a c k e r a n d  (e.g., see review  d u r i n g t h e 48 h o f N O 3  1981;  e t a l . , 1982),  i t may r e q u i r e an " a c c l i m a t i o n  the c e l l s  or  rates.  after N deprivation  (e.g., M o r r i s and S y r e t t ,  t a k e up n i t r a t e .  period"  due  N uptake  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  retained NO3  It  (e.g., C o l l o s ,  others  reduced  capability  -  although  to  controlled  by G u e r r e r o  1981; U l l r i c h ,  a variety  of responses  e t a l . , 1981; M c C a r t h y ,  1983, D o r t c h , depending  i n press)  which  on t h e p h y t o p l a n k t o n  139  species  and i t s n u t r i t i o n a l  reported ranging  t o be i n h i b i t e d from t o t a l  McCarthy  state.  to different  a n d E p p l e y , 1972; C r e s s w e l l  (e.g., Caperon  D o r t c h a n d Conway, communities al.,  stimulation though  1989).  1988;  Dortch e t a l . ,  a n d Ziemann,  Only a f t e r N H  well  inhibit  + 4  submitted).  -  et al.,  (Conover, 1982c;  of nitrate  uptake  and S y r e t t ,  and M o r r i s ,  cannot proceed i n -  does  utilization  i s not  action at  (e.g., E p p l e y and Rogers,  1979; S e r r a e t a l . ,  1979) a n d n i t r a t e  extent the e f f e c t  I t i s now g e n e r a l l y  N-L ^.  of n i t r a t e .  1963; Amy a n d G a r r e t t ,  and t o a c e r t a i n  Yin,  The p r e s e n t s t u d y  There i s evidence f o r r e g u l a t o r y  mechanisms may be i n d e p e n d e n t 1987).  N O 3 uptake  even  c o n c e n t r a t i o n s a s low a s 1.0 uq-at  T i s c h n e r and Lorenzen,  1980)  e t a l . , 1986;  concentrations  mechanism o f d e p r e s s i o n o f n i t r a t e  Cresswell  1977; M a e s t r i n i e t  i s e x h a u s t e d f r o m t h e e x t e r n a l medium  + 4  both t h e l e v e l  Syrett  1979) a n d n a t u r a l  -  understood.  1970;  +  1976; G l i b e r t  p h y t o p l a n k t e r resume i t s u p t a k e The  -  t h a t N O 3 u p t a k e by M. pusilla  the presence of N H  this  and N O 3 uptake i n  1985; Q u e g u i n e r  -  Caperon  to  1976; Conway, 1977;  o f N O 3 u p t a k e a t l o w NH^  1975;  1979)  T h e r e a r e e v e n a few r e p o r t s o f  higher concentrations  demonstrates  + 4  1975; M c C a r t h y ,  et al.,  h a s been  a n d M o r r i s , 1963;  and S y r e t t ,  of NH  a n d Ziemann,  (e.g., Conover,  et al.,  Syrett  1984; DeManche e t a l . ,  1982; 1986; P r i c e  Collos  rate  uptake  d e g r e e s by ammonium  s u p p r e s s i o n (e.g.,  s i m u l t a n e o u s and comparable cultures  Nitrate  reduction  (e.g.,  1974; H i p k i n  et a l . ,  o f ammonium on b o t h  ( B l a s c o a n d Conway  accepted that  1978b;  1982, U l l r i c h  t h e p r i m a r y , and  140  rapidly an  acting  inhibition  effects  effect  of NH  of nitrate  on n i t r a t e activity,  breakdown  (e.g., Hipkin  Pistorius  through i n h i b i t i o n  from e i t h e r  et al.,  Although  (e.g., Florencio  se i n h i b i t s  the  rate  pools  of NO3  -  1 9 6 3 ; Amy  assimilation  -  uptake  are only  however, g e n e r a l l y  McCarthy  believed  and Eppley,  unchanged uq-at  N-L -* -  diatom,  Skeletonema  i n response  the rate  urea,  but i n terms  the  uptake  growth  uptake  -  that  phytoplankton  et al., urea  1985).  I ti s  suppresses the uptake of (Grant e t a l . ,  and S y r e t t ,  (Lund,  communities  1988b).  1987).  1967; An  o f 10 i n t h e marine  In the present  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  of 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 rate  of NO3  -  uptake  alone and  necessary t o maintain the pre-conditioned  n o t measured  + 4  (e.g.,  rate.  Although  NH  t o changes i n  assimilation  i n the presence  than the unaltered rate  that  of simultaneous uptake of  t h a n ammonium  costatum  of NO3  study  greater  -  1974).  1982) a r g u e  o f urea h a s , however, been r e p o r t e d  -  30%  that  1972; M o l l y  of NO3  rate  inactivation  1981).  1972; P r i c e  but a t a lower l e v e l  -  nitrate  proteolytic  and G a r r e t t ,  i s modulated  a few r e p o r t s  and Eppley,  by t h e  most e v i d e n c e s u g g e s t s  and o t h e r N sources by n a t u r a l  (McCarthy  of  reversible  and Vega,  1981,; G u e r r e r o e t a l . ,  There  NO3  1980),  o f some o r g a n i c p r o d u c t o f a m m o n i u m  Syrett,  urea  NO3  i s due t o  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  and S y r e t t ,  per  irreversible  et al.,  (e.g., Morris some  utilization  u p t a k e w h i c h may b e f o l l o w e d  metabolism  reductase  (e.g.,  on n i t r a t e  + 4  i n the present study, i t i s  possible  t h a t NH^  causative addition release  factor  Price  o f NH3/NH  urea.  + 4  Uchida  into  reported  following  Prorocentrum  NH3/NH  rates after the  (1988b) r e p o r t e d t h e  addition  minimum  culture  cultures of  o f 10 uq-at  U-L~^  that the red-tide e x c r e t e d NH3/NH^  medium a n d Rees  r e l e a s e by urea-grown  + 4  a n d was t h e  t h e medium by a x e n i c  (1976) o b s e r v e d  grown i n u r e a - e n r i c h e d  uptake  -  and H a r r i s o n  pseudonana  dinoflagellate  NO3  f o r reduced  of urea.  Thalassiosira of  was e x c r e t e d b y t h e c e l l s  +  when  (1979)  also  Phaeodactylum  tricornutum. Ecological As  significance Dortch  reduction take  i n NO3  up N H  response  + 4  recycled  areas  rapidly  areas  within  which after  t  have t h e a b i l i t y starvation  which  maintain  during N starvation  and u s u a l l y  diffusion  from  Eppley  a r e both  z o n e a n d may b e a d d e d  e x c r e t i o n (e.g., Dugdale,  advantage because n i t r a t e  1967;  ability  may b e a n a d a p t i v e  Ammonium a n d u r e a  the euphotic  where N l i m i t a t i o n  Phytoplankton  scale  and an enhanced  phytoplankton  of t h e ocean.  by animal  Phytoplankton  rapidly  capability  by N-deprived  rapidly  sporadically  in  uptake  -  t o the patterns of nitrogen a v a i l a b i l i t y i n  oligotrophic  urea  e t a l . (1982) have p o i n t e d o u t p r e v i o u s l y , t h e  1967).  to assimilate  may h a v e a s e l e c t i v e  NH  + 4  advantage  i s t h e major environmental the ability confer  t o take  little  up  or  stress  nitrate  competitive  i s r e c y c l e d on a much l o n g e r  time  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 by eddy  deeper n i t r a t e - r i c h  e t a l . , 1979; K i n g  layers  and Devol,  (e.g., 1979).  Dugdale, Physical  142  events such as upwelling (e.g., Walsh et a l . , 1977, 1978) f r o n t a l mixing 1981)  (e.g., Pingree et a l . , 1978; Parsons et a l . ,  i n t e r n a l 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 d i l u t e the -  phytoplankton  concentrations i n the euphotic zone, thereby  decreasing the demand f o r the nutrient, increasing the l i f e t i m e of the pulse and consequently  reducing the benefits  of transient elevated uptake rates; hence the metabolic cost of maintaining high uptake c a p a b i l i t y f o r n i t r a t e during starvation (between pulses) may outweigh the benefits.  143  C H A P T E R FOUR E F F E C T S OF  I R R A D I A N C E AND D I E L P E R I O D I C I T Y ON U T I L I Z A T I O N I N MICROMONAS PUSILLA  NITROGEN  INTRODUCTION In  order  phytoplankton  t o d e s c r i b e the mechanisms i n v o l v e d i n ecology  there  significance  of b i o l o g i c a l  parameters.  I n most marine  availability  of  which  primarily  Dugdale and and  In  interactions  with  Ryther  and  Dugdale and  Goering  partitioned  by  allochthonous N  surface waters  secondarily,  ^-fixation,  "regenerated"  from  sources,  processes  and  the  vertical  1986;  Piatt  result mixing  riverine  ammonium a n d (nutrient  animal  increase  of p h y s i c a l  Maclsaac  "new"  by  urea,  primary  production nitrate and  rainfall;  autochthonous derived  N-  from  r e c y c l i n g ) o c c u r r i n g in euphotic  mechanisms,  which  or a l t e r n a t i v e l y ,  such  biological and  as  situ.  zone  et a l . ,  nitrogen  processes,  Goldman,  nitrogen concentrations.  are  upwelling  Takahashi  i n c r e a s e "new"  e x c r e t i o n (e.g., McCarthy "regenerated"  oceanic  i n p u t s and  ( e . g . , C o d i s p o t i , 1983;  e t a l . , 1989),  concentrations, as  (e.g.,  1971;  principally  Changes 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 the usually  factors  deep ocean r e s e r v e s ,  production i s fuelled  principally  biological  the  the  Dunstan,  is  sources,  and  productivity  according to i t s nitrogen source:  into  environmental  light  production  mixed  the  1972 ) .  1967,  fuelled  understand  shown t o be  regulate phytoplankton 1967;  first  environments,  n i t r o g e n have been  Goering,  Dugdale,  i s a need t o  1979;)  such which  144 The  light  environment  periodic  fashion during  latitude  areas).  fluctuations  Diel  i n light  shows e x t r e m e v a r i a t i o n  24 h , ( e x c e p t  intensity,  processes  such as photosynthesis  and  Prezelin,  1974;  communities (e.g.,  and L e y , 1980; Harding 1988); c e l l u l a r  1989);  i n vivo  Owens e t a l . , 1 9 8 0 ) ; Ricketts,  1977; H i t c h c o c k ,  Sournia,  1974) enzyme a c t i v i t i e s  uptake Terry  (e.g.,  Sournia,  (e.g.,  (e.g.  (e.g.,  e t a l . , 1985);  e t a l . , 1980; Chisholm,  1974; M a r t i n e z  Eppley  (e.g.,  and p r o t e i n content  1980; T e r r y  (e.g.,  and B l a s c o ,  e t a l . , 1981, 1983; P u t t  chlorophyll a fluorescence  division  Packard  and P i a t t ,  e t a l . , 1983; Kohata and  carbohydrate  Chisholm  and i n c l u d e  MacCaull  pigment content  Owens e t a l . , 1 9 8 0 ; H a r d i n g  Watanabe,  i n response t o  have been d e t e c t e d i n  and n a t u r a l phytoplankton  Prezelin  i n high  p h y s i o l o g i c a l rhythms,  cultures  1977;  a t times  and i n a  Eppley  cell  1981;  e t a l . , 1970;  e t a l . , 1987) and n i t r o g e n  e t a l . , 1970 1971a,b; M a c l s a a c ,  1978;  e t a l . , 1985 ) . Previous  s t u d i e s o f c u l t u r e s and n a t u r a l  phytoplankton  communities  suggest that N s t a r v a t i o n (e.g.,  H a r r i s o n , 1976;  Bhovichitra  and S w i f t , 1977) o r N l i m i t a t i o n  (e.g.  al.,  1975; Eppley  of  NO3  NO3  -  -  uptake  and a s s i m i l a t i o n  uptake during  nitrogen studies (e.g.,  e t a l . , 1 9 7 1 b ) may d a m p e n d i e l  the night.  uptake by p i c o p l a n k t o n of size-fractioned  Probyn,  Garside,  by a r e l a t i v e To d a t e  periodicity  enhancement o f  our knowledge o f  have been d e r i v e d  natural phytoplankton  1 9 8 5 ; P r o b y n a n d P a i n t i n g , 1985;  1983) a n d t h e e f f e c t s  Malone e t  of nutrient  from t r a c e r communities  Nalewajko and  limitation,  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  abilities  have not been addressed. The o b j e c t i v e of t h i s study was  t o determine  the  effect(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  potential  N uptake by the u b i q u i t o u s , p i c o f l a g e l l a t e  Micromonas  pusilla  (Butch.) Manton e t Parke.  Nitrate,  and  the N  s u b s t r a t e which supports the most p r o d u c t i v e areas of the world's oceans (e.g., Eppley, 1 9 8 1 ; H a r r i s o n et a l . , 1987) study.  was  the p r i n c i p a l focus of the present  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  r a t e s of urea and N H importance  + 4  of regenerated nitrogenous n u t r i t i o n  environments  uptake  were a l s o examined i n view of the  phytoplankton i n o l i g o t r o p h i c , 1980);  1979;  Eppley and Peterson,  oceanic areas  of  (e.g., H a r r i s o n ,  where p i c o p l a n k t o n have been  demonstrated  to be r e s p o n s i b l e f o r the m a j o r i t y of the p h o t o s y n t h e t i c production  (e.g., L i et a l . , 1983;  r e v i e v s by Fogg, 1986;  Joint,  1986;  P i a t t et a l . , 1 9 8 3 ; Stockner and A n t i a ,  see 1986).  146 M A T E R I A L S AND  METHODS  Culturing Continuous (culture  and  o f Micromonas  batch cultures  NEPCC 2 9 - 1 ,  Northeast  Pacific  Culture  Department of  Oceanography, U n i v e r s i t y of  w e r e g r o w n on  filter-sterilized  enriched 1980).  artificial The  modifications  preparation  and  storage  were m a i n t a i n e d  i n an •  17  ±  0.5°C a n d fluorescent  set  on  14  h  are  the  on  British  ESAW  Columbia)  (Harrison  medium and  described  the  et a l . ,  d e t a i l s of  i n Chapter  air-cooled, walk-in,  3.  Cultures  growth chamber  tubes  light  (4  and  on 10  sides  either side  by  of  h dark cycle.  eight V i t a - L i t e  culture The  vessels)  light  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 , and Haas) and 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 ?  Cor  vessels  LI-185  light  at R  •  culture  i t s  •  i l l u m i n a t e d f r o m two  UHO  a  to  Collection,  um M i l l i p o r e ) n u t r i e n t -  (0.22  seawater based  pusilla  was  ca.  120  uE  m  Rohm the  1  s  , as  meter equipped w i t h  measured w i t h  a LI-190S quantum  a L i sensor  (2TT).  Continuous chemostats Constant  similar to  flow  were used  to  borosilicate vessels.  The  flat-bottom, stopper  so  cultures  o f M.  pusilla  those described  p i s t o n pumps  (Fluid  m e d i u m f r o m 20  glass  silicone  reactor boiling  that  measured before  vessels flasks  a constant  by  Davis  Metering  pump t h e or  were m a i n t a i n e d  L  rubber  volume of  e a c h e x p e r i m e n t ) was  Inc.,  a l . New  Pyrex carboys tubing  consisted  (Pyrex)  et  of  sealed 2.5  L  in (1973).  York) through  i n t o the  reactor  3 L, b o r o s i l i c a t e , with  a  silicone  (accurately  maintained  inside  the  147 reactor. bars  Cultures  a t 60 r p m .  were  stirred  Cultures  by t e f l o n - c o a t e d m a g n e t i c  were u n i a l g a l but not  axenic,  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 medium and c u l t u r e s by u s i n g scrupulously  cleaning  with  acid  1 0 % HC1  water  (DDW)  (v/v), rinsing  a defined  a continuous  culture axenic  using  (e.g.,  t h e above p r e c a u t i o n s  minimized  t o the response(s)  were a l l o w e d before  ±0.9  uq-at  initiated. effluent  daily  N0 ~-L~  by  syringe  were  d "*". -  of i n vivo  i s a common felt  be  that  by  be  negligible  pusilla.  Cultures (4-5)  containing  jjg-at N 0 ~ * L  was  _ 1  2  0.49  a n d 0.74  samples were  cell  of three  g r o w t h c u l t u r e and ±5%  and  period for  counts  and  particle until  w i t h i n each continuous  assumed when c e l l  f o r a minimum  d~^  withdrawn  Experiments were n o t i n i t i a t e d  was  cultures.  keeping  e f f e c t s would  of the l i g h t  fluorescence,  Steady-state  highest  0.24,  at the mid-point  had been achieved  ±10%  ± 0.08  Culture  steady-state  within  i t was  i n  r a t e s , c a l c u l a t e d from t h e volume of  0.01  distribution.  deionized  c u l t u r e s f o r s e v e r a l days  a n d 0.18  1  3  Dilution  less than  periods  e f f e c t s would  o f Micromonas  tubing  and i n  a d d i t i o n o f t h e i n f l o w medium  collected daily,  monitoring size  any b a c t e r i a l  t o grow as b a t c h  continuous  varied  However,  t o t h e p o i n t where t h e i r  relative  50.6  1977).  and  Difficulties  autoclaving  f o r extended  i n the  and  distilled,  t o use.  medium a f t e r  Goldman,  growth  pump f i t t i n g s  with  and a u t o c l a v i n g p r i o r  establishing  problem  aseptic techniques  a l l glassware,  stir  culture.  counts were c o n s t a n t consecutive  days  f o r t h e two lower  to  i n the  growth  rate  148 Analytical  procedures  Cell m o d e l TA  concentrations  II electronic particle  procedures  outlined in detail  volumes were computed based  on  equivalent  Replicate particulate nitrogen  (2-4)  (PON)  desiccators  carbon  were glass  and  samples  analyzed  Equipment Corp. model calibration (cellular  pools)  measured w i t h procedures  a f t e r an frozen pools  filters and  of  culture)  (Whatman GF/F,  subsequently  were 25  are  through  rinsed with  the  internal nitrite  The  presented  the of  i n Appendix  6.  nutrient  p r e c o m b u s t e d Whatman DDW-rinsed filtrate. Internal  a l . , 1982).  Samples  were  dissolved boiling  Phytoplankton  and  50  cells  glass  washed w i t h  boiling  GF/F  polypropylene  onto combusted  previously ml  were  precision  after extraction with  50  in  (Control  II following  R  external  filtered mm,  and  h)  -20°C  analyzer  n i t r a t e and  analyses.  Thoresen et  at  a c e t a n i l i d e as  a l . (1967).  rinse with  (460°C, 6  frozen  (ambient) of  were determined  ( m e t h o d C-2,  (20-50 ml  organic  elemental  a CHN  (-20°C) u n t i l  distribution  particulate  on  acid-washed,  initial  cell  of  stored  measurement of  bottles  the  measurement  filters,  were f i l t e r e d  to  Average  onto precombusted  techniques  into previously  DDW  and  External  filters  nitrogen  f o r the  o u t l i n e d i n Wood e t  f o r the  Counter  diameter.  a Technicon AutoAnalyzer  concentrations  stored  size  concentrations  above a n a l y t i c a l  Samples  particle  240-XA) u s i n g  standards.  according 3.  (POC)  filtered fiber  counter  a Coulter  i n Chapter  from the  spherical  organic  Whatman GF/F  the  were measured w i t h  ml  2N cool  fiber HC1 DDW)  149 with  a low d i f f e r e n t i a l  filtration,  pressure  t h e f i l t e r was r i n s e d w i t h  medium c o n t a i n i n g  no d e t e c t a b l e  from g r a v i t y f i l t r a t i o n Chapter DDW,  3).  The f i l t e r and c e l l s  cells  (-20°C) u n t i l  prior  with  boiling  b o t t l e s , and  B l a n k s were  filters  stored  without  t h e same a s s a m p l e s .  a p p a r a t u s was a c i d - w a s h e d ,  to collection  (obtained  cultures described i n  were e x t r a c t e d  on them and t h e y were t r e a t e d  filtration  nitrogen  i n t o polypropylene  analyses.  After  5-10 m l o f c u l t u r e  inorganic  of N-starved  collected directly  frozen  (< 80 nun H g ) .  The  DDW-rinsed and d r i e d  of samples.  15  Samples  for  N a n a l y s i s were c o l l e c t e d on  Whatman GF/F f i l t e r s enrichment  and s t o r e d  frozen  o f s a m p l e s was a s s a y e d  (JASCO m o d e l N-150) a f t e r c o n v e r s i o n N  2  gas by t h e micro-Dumas  analyses  were conducted  procedures  described  Experimental Diel  cycles  over  by e m i s s i o n  The  spectrometry  of the p a r t i c u l a t e N t o  i n duplicate according i n Chapter  A l l  to the  1.  procedures of uptake  continuous  and growth of c e l l s  and i n o r g a n i c  c u l t u r e s and batch  a 24 h p e r i o d  beginning  were o r i g i n a l l y  inoculated  intervals,  from t h e r e a c t o r concentration  nitrogen  c u l t u r e s was  at the start  These experiments were conducted w i t h  two hour  i n desiccators.  d r y combustion technique.  i n detail  The c o n c e n t r a t i o n three  precombusted  monitored  of the l i g h t  period.  duplicate cultures  f r o m t h e same s t o c k  culture.  samples were drawn by s y r i n g e  vessels  i nthe  f o r determination  and volume and t h e ambient  of  which At  directly  cell  and i n t e r n a l  NC^  -  and  150 NO2""  concentrations.  from the continuous their the  steady-state  rate  was  between  virtually  The  total  1 and 2.5%  The m a t h e m a t i c a l  of substances  identical  t o those  withdrawn  the perturbation  to  volume withdrawn  from  of the c u l t u r e volume.  t h e c y c l o s t a t s were  closed.  of change  (25-60 ml) were  cultures t o minimize  the time that  outflow  volumes  condition.  c y c l o s t a t s was  During  Small  refilling,  equations  the  governing  i n the culture vessels describing overflow  are  conditions,  because the increase  i n c u l t u r e volume due  fresh  t h e c u l t u r e i n a manner s i m i l a r  medium d i l u t e s  resulting (Laws, the of  from overflow  1985; D i T u l l i o  volume  significantly Rates  perturb  of n i t r a t e  calculated  using  by t h e  uptake  of n i t r a t e  -  concentration  NO3  -  is  the dilution  uptake  equation nitrate  volume  that  conditions  Therefore, compared that  of  to  as long  to the  sampling  as  volume  will  not  state condition.  i n the continuous  cultures  o f Caperon and Ziemann concentration  was  = D-(S -S) -  U/V  were (1976).  assumed  i  of NO3  -  i n the chemostat,  o f t h e i n f l o w medium, U i s t h e  t o be  S^ i s total  r a t e , V i s t h e volume o f t h e growth chamber rate.  The  has been g i v e n uptake during  periods i s :  to the input  equation:  Where S i s t h e c o n c e n t r a t i o n NO3  i s small  the steady  dS/dt  the  1986).  i t c a n be assumed  the equations  r a t e of change  described  and Laws,  of sample withdrawn  t h e growth chamber  The  under constant  the  integrated s o l u t i o n of  by C a p e r o n and Ziemann  the interval  and  D  this  (1976) and t h e  ( t ) between two  sampling  151  nX  where  S  a r e NO3  and  Q  = DSj^ + D •  respectively,  n i s t h e PON  (assumed t o be c o n s t a n t uptake rates  rate  with  as  Uptake  rates  specific  samples  I t was  that  intervals,  during  a v e r a g e PON that  periods. from  taken This  f o r M.  removed  -  by i n  value  by t h e  during from  excreted  situation  pusilla  was  cultures  as d i s s o l v e d  observed growing  i n  that  the  into the particulate fraction up was  the  interval;  concentration  a l l the NO3  i s not  are useful  cultures, estimated  of the time  uptake  which  these  c a l c u l a t e d by d i v i d i n g t h i s  assumed  (DON).  v a r i a t i o n i n maximum N u p t a k e 8 separate  conducted  0.49,  rates  specific  and  that  organic  other  under  continuous  (Appendix 4 ) .  At  +  assumes  but the c a l c u l a t e d rates uptake  incorporated  experiments  4  equation  by t h e l e n g t h  were  o f t h e N03~  nitrogen  NH  and X i s t h e  i n t h e medium, were c a l c u l a t e d  -  exponential  medium was  Diel  of the culture  state)  the sampling  i n the batch  of NO3  rates  estimated  light  This  0 and t ,  the difference i n the nutrient concentration  successive  time.  true,  of average  disappearance dividing  during  at times  concentration  at steady  = U/V.  always  estimates  none  nX  are constant  necessarily  concentrations  -  times  t o determine  and urea a n d 0.24  from d ^". -  over  rates  a 17 d p e r i o d ,  the.saturated  the continuous  uptake  cultures  These experimental  experiments rates  grown  times  of at  were  NO3 , -  0.74,  corresponded  to  152  the beginning, middle periods  a n d a r e g i v e n i n T a b l e 4.3.  procedure first  and end o f t h e 14 h l i g h t  duplicate  f o r t h e measurement o f POC, PON,  nitrate  and n i t r i t e  samples  were t r a n s f e r r e d  and c e l l  tubes with t e f l o n - l i n e d  with  16.6 /jg-at N ' L  of  - 1  99 atom % ) .  concentrations.  to sterile,  test  Isotopes,  The e x p e r i m e n t a l  a t e a c h t i m e was a s f o l l o w s :  collected  and 10 h d a r k  1 5  4  Na  T h i r t y ml  50 ml b o r o s i l i c a t e  1 5  N0  or C O (  3  were  ambient  caps and d u p l i c a t e s  NH C1,  samples  glass  inoculated 1 5  NH ) 4  I n c u b a t i o n s were c o n d u c t e d  (Kor  2  i n the dark —2  and  a t t h e same PPFD a s p r e v i o u s l y  and  then terminated a f t e r  differential  < 80 mm  Hg).  r a t e s were d e t e r m i n e d of Dugdale  15%  ( c a . 120 uE m  2 h by f i l t r a t i o n The N s p e c i f i c  (1986)  saturated  model 1).  sample volume w i t h d r a w n r e p r e s e n t s o n l y 10-  never  monitored  o v e r t h e 16 d e x p e r i m e n t a l p e r i o d  less  that  1 d ^.  Cell  -  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  4.6  variations  at  c o n c e n t r a t i o n was by w i t h d r a w i n g  at the mid-point of the l i g h t - d a r k cycle  t h e s t u d y and d a i l y and  uptake  o f t h e g r o w t h chamber v o l u m e , t h e c u l t u r e s were sampled  samples  )  uptake  ( e q u a t i o n 6 o f Appendix  intervals  Cell  s  (pressure  from t h e c o n s t a n t s p e c i f i c  and W i l k e r s o n  Although the t o t a l  grown  —1  stable  averaged  (Fig.  4.1).  over the course of  8.4  ( ± 3 . 7 ) % i n t h e 0.73, 0.49 and 0.24 d  ( ± 7 . 3 ) , 3.7 -  1  (±3.2)  cyclostats,  respectively, Effect  o f PPFD on N 0 ~  Three harvested  3  and N H  + 4  continuous cultures just  prior  uptake (0.77,  t o the middle  0.52 and 0.24 d ^ ) were  of the l i g h t  -  period  (6 h  light)  t o d e t e r m i n e t h e e f f e c t ( s ) o f PPFD  uptake  rate.  collected N0 ~  culture Na  1 5  -  N0 ~ 3  or  samples were conditions, with  —7  (KorIsotopes,  4  immediately to sterile  t o simulate  -  99 a t o m % ) .  50 m l b o r o s i l i c a t e and placed  Forty ml  glass  from  light  test  within neutral  a r a n g e o f PPFDs  ofthe  15 uq-at N ' L ^  t r a n s f e r r e d , under reduced  caps,  ambient  The r e m a i n d e r  i n h a l f and i n o c u l a t e d w i t h  NH C1  teflon-lined  screening  uE m  1 5  cells.  4  initially  m e a s u r e m e n t s o f PON, POC,  and phytoplankton  was s p l i t  3  (3 o r 4) s a m p l e s w e r e  f o r concentration  and NO2  3  of  Duplicate  on N 0 ~ and NH  tubes,  density  1 4 0 t o 3.5  1  —  s  and darkness.  due t o d i s t a n c e Biospherical  Irradiances, achieved  and screening,  by  attenuation  were measured w i t h  I n s t r u m e n t s QSL-100  in s e n s o r  a  placed  within the  screening  i n the incubation position.  Incubations  conducted  a t t h e growth temperature and terminated  were after  2 h 1 5  by  l o w vacuum  labelled with  filtration  80 mm  particulate material.  N - l a b e l l e d N 0 ~ and NH 3  Hg) f o r c o l l e c t i o n  Duplicate  cultures  N-  enriched  were a l s o i n c u b a t e d  4  of  f o r4 and  6 h i n t h e dark. Nitrogen equations  described  experiments. respect modified  r a t e s were c a l c u l a t e d a c c o r d i n g previously f o r the saturated  Kinetic  t o PPFD  constants  were o b t a i n e d  Michaelis-Menten  least-squares Chapter  uptake  2.  technique  f o r N0 ~ and N H 3  by a d i r e c t  hyperbola  using  and f o r m u l a t i o n  N +  4  t othe  uptake uptake  f i t of t h e data  with to a  the non-linear  described  earlier i n  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 as a f u n c t i o n of time 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 grown i n a 1 4 h : 1 0 h L:D c y c l e a t (O) 0 . 7 4 , (•) 0.49 a n d (A) 0.24 d " dilution rates. E x p e r i m e n t s were c o n d u c t e d on days 2,7,8,11,13 and 16. 1  12  10  -A-A.  A  .A-A  —A A-A  O 6 00  4  0- -o-o.  ~o-o-o'  -O-o  o. •o-  -o- •o.  UJ  o  2 0 0  2  4  10  6  TIME  ( d )  12  4  6  155 RESULTS  Nitrate-replete The cultures 1.13  the  specific  based  averaged  on c e l l  h  fluorescence,  under  interval  division  during  resulted  (um  The i n c r e a s e  -cell  i n cell  i n d e c r e a s e d mean c e l l  ; F i g . 4.2C) a t e a c h cycle.  the late  i n cell  period  concentration  i n cell  volume d u r i n g  by  cell  s i z e and  the dark  period  volume o f d u p l i c a t e N C ^ - r e p l e t e  g r o w n o n a 1 4 : 1 0 L:D c y c l e w a s 1.96 ± 0.02 um^; increased  3  from  2  Cell  light  concentration  by a r e d u c t i o n  T h e mean c e l l  v o l u m e o f M. pusilla  period  — 1  increase  resulted  cell  -  and p r i m a r i l y throughout t h e dark p e r i o d and  was a c c o m p a n i e d  cultures  from t h e  ( c e l l s •L -'-; F i g . 4 . 2 A )  occurred during  division  4.2C).  prior to  (1.11 d~^, Chapter  the light-dark illumination  i n a ca. two-fold  4.2A).  This  v o l u m e p e r L c u l t u r e medium)  concentration  volume  o f M. pusilla  h light)  measured  continuous light  (L;L c e l l  volume  e s t i m a t e mean c e l l  (Fig.  cell  r a t e , c a l c u l a t e d by  -  d i v i d e d by t h e c e l l  (Fig.  and t o t a l  ( 1 . 0 3 d "^) a n d i s n o t d i f f e r e n t  r a t e measured  Total cell  (>10  -  -  growth  3  to  NC>3 -replete  1.08 ± 0 . 0 1 3 d ^ a n d  concentration  t h e average  i n in vivo  experimentation  was  pusilla  i s consistent with  growth  rate u of duplicate  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 .  increase  3).  growth  o f Micromonas  ± 0.022d~l  volume, rate  cultures  86% d u r i n g  mean  the light  3  1.35 L m r t o 2.57 um  a t the onset of the dark  period. Nitrate marked d i e l  uptake  and i n t r a c e l l u l a r  pool  a c c u m u l a t i o n showed  v a r i a t i o n s i n t h e exponentially growing c u l t u r e s ,  156 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), growth r a t e (B) and mean c e l l volume (C) v e r s u s e l a p s e d time s i n c e l i g h t s on i n d u p l i c a t e ((},•) b a t c h 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 b a r . Growth r a t e p l o t t e d a g a i n s t average time between sampling. - •  <  6.000  or  5.000  UJ  4.000  o o o  i  •  1  •  •  r  — i  r  1  i • •  A  •  -  3.000 2.000  _j  1.000  o  0.000  o  j 1  ^ 8 - 8 = 8 ^ ®  UJ i  i  i  i  i  i  i  i  i  i  UJ r-  < or x  o tr  I  I  UJ Z5 _l  o  > 1 1  UJ  2.50  2.00  1.50  o  1.00  < UJ  0.50  /  ro  £  0.00 8  10  12  TIME  14  16  (h)  18  20  22  24  157  although  there  concentration uptake light  rates period  was c o n s i d e r a b l e between r e p l i c a t e  averaged  cell  v o l u m e s h o w e d t h e same d i e l  cell the  Rates  daytime values  of NG^ uptake -  uptake  concentrations  during  other  period  i n parallel  The l o w e s t the f i r s t  markedly  internal  4 h of darkness  concentrations  with  time  specific  than  nighttime  (mg-at  N«litre  of the light were increased  slightly  i n the  period.  a strong  c u l t u r e t h e r a t e o f change (dx/dt)  i s given  a s u(t).  population  An increase  t h e 1 4 h : 1 0 h L:D p h o t o p e r i o d ,  by:  - D  rate of the c u l t u r e , which  same u n i t s o f t i m e  diel  c y c l o s t a t c u l t u r e s o f M.  l n x / d t = u{t)  u(t) i s t h e i n s t a n t a n e o u s  time during  to total  and subsequently  In continuous  d  the dilution  during the  concentrations  of the l i g h t  i n the NC^-limited  in  is  1  a c t i v e phaseo f  the remainder  ( c y t o k i n e s i s ) showed  (Fig. 4.4).  where  -  at the beginning o f  i n one c u l t u r e and o n l y  pusilla cell  -  -  cultures  division  periodicity  asN  -  t h e 14 h  normalized  t h e most  nitrate  t h e commencement  Nitrate-limited  Cell  with  throughout  (ca. 7-fold)  before  during  o f NO3  -  and decreased  period.  1  variation  volume "' ; F i g . 4.3C) w e r e g r e a t e s t light  S p e c i f i c NO3  were c a . 2 - f o l d g r e a t e r  The i n t e r n a l -  -  i n i n t e r n a l NO3  a n d d e c r e a s e d t o 0.0168 ± 0.0028 h  ( F i g . 4.3B).  values.  cultures.  0.0360 ± 0.0010 h  dark  rates;  variability  growth  rate  and D  i s expressed  i n cell  numbers  i . e .a positive  i n the with  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 elapsed 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. f-  < cc I-  z  Ul  o o o  a.  UJ  < cc  r—  Ul  <  r— CL Ul f— < CC  Ul •  r-  <  a> E  CC  r-  O >  ua>  cc <  a> Ul  o < cc  o i  e 4  6  8  10  12  TIME  14  16  (h)  18  20  22  24  F i g u r e 4.4. C e l l concentrations of duplicate nitrate-limited 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 " ( A ) , 0.48 d " ( B ) , a n d 0.24 d " ( 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 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 o f dark p e r i o d denoted by d a r k bar. 1  1  10.0  1  -  a.ol  1  6.0  -  4.0  -  2.0  -  0.0  -  .  _ l  o  •  , 10.0  8.0  < tr hLd O o o  6.0  4.0  2.0  B  0.0 -i  1  1  r  -o- -o -•-—•  10.0  UJ o  8.0  6.0  4.0  2.0  0.0 0  2  4  6  8  10  12  TIME  14  16  (h)  18  20  22  24  160 dlnx/dt,  indicates that  during  which dlnx/dt  period  o f maximum c e l l  the  dilution  rates  u(t)  > D.  Thus t h e t i m e  i s both p o s i t i v e and g r e a t e s t division.  ( h e r e a f t e r r e f e r r e d t o as  -  division  -  occurred  during  period  (4-8 h a f t e r onset o f d a r k n e s s )  although  values  of dlnx/dt  light  lights  o n ) a n d 2-4 h a f t e r d a r k n e s s  h at  occurred  division  a f t e r onset  of darkness)  0.4 8 d "*" d i l u t i o n values  of dlnx/dt  after  lights  (0.24  d ^) t h e period  after  the onset of dln/dt  including culture darkness  were  light  lower  (10-12 h  growing continuous division  grown  cultures  occurred  6-8  h  throughout  the dark  throughout  period,  i n the replicate until  the night  a f t e r 2 h of  and t h e f i r s t  light.  Mean c e l l  volume  (um  in  a l l cyclostat cultures  at  a dilution  average  the late  were n o t observed  and continued  of  there  2 h of darkness, while  p o s i t i v e values  cultures  i n one c u l t u r e l o w e r p o s i t i v e  were observed  the first  The p e r i o d o f  i n t h e d a r k p e r i o d (1-6  again  o f maximum c e l l  of darkness;  lower p o s i t i v e  (10-14 h a f t e r  f o rthe continuous  during  the mid-dark  (Fig. 4.5).  earlier  I n the slowest  -  values  hours  on).  late  rate, although  -  positive  during  occurred  i st h e  F o r t h e c u l t u r e s grown a t  0.73 a n d 0.75 d ^  0.74 d -*-) m a x i m u m c e l l  maximum c e l l  interval  rate  showed marked d i e l v a r i a t i o n  (Fig. 4.6).  o f 0.74  o f 85% during  1 . 1 2 - 1 . 2 0 um  -cell)  the light  at the beginning  the cell period  In the cultures  grown  volume i n c r e a s e d (0800 h - 2200  of the light  period  h)  by an from  to a  3  m a x i m u m s i z e o f 2 . 0 8 um then decreased  during  , achieved  the night  after 6 h of light,  (2200 h t o 0800  and  h) t o t h e  161  F i g u r e 4.5. Cell division rate of duplicate nitrate-limited 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 ( A ) , 0.48 d " ( B ) , a n d 0.24 d (C) d i l u t i o n rates. 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 rate i n h . Dashed v e r t i c a l l i n e i n d i c a t e s onset o f d a r k p e r i o d denoted by d a r k bar, 2.50 _ 1  1  _ 1  - 1  2.00 .50 1.00  0.50 0.00  UJ  -0.50  < cr  i  O  A : •A/ \ \/ \ AA/ .  r—  1  1  1  1  A  I  1—'  —1  2.00 .  1  A  o  I  1  1  1  1  1  1.50  >  1.00 0.50 0.00  UJ O  -0.50  *  1  A  B  o  c o  1  a  oV 1  v/\//V^» 1  1  1  1  8  1  10  T I M E  1  L  12  14  16  (h)  18  20  1  A. \ o  22  24  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 nitrate-limited 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 cycl a t 0.74 d ( A ) , 0.48 d ( B ) , a n d 0.24 d (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 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 . _ 1  LU  _ 1  _ 1  0.50 -  o  <  0.00  _i  i  i  -l  i  i  r  1  UJ  2.00 1.50 1.00 0.50 0.00  1  0  ' 2  1  4  '  1  6  '  1  8  10  12  TIME  14  ' 16  (h)  '  1  18  ' 20  1  22  24  163 original diurnal was  minimum s i z e by e n l a r g e m e n t and  1.15  um  to  3  4.6). 1.69  The  of  over the  mean  0.74,  nitrogen,  0.48  concentration variation period  and  estimated  of  and  of  26%  0.24  from the  d "*" grown  to  1.38  67%  um )  (1.11-  ,  3  illumination cycle  1.23  ± 0.01  d~^,  respectively.  /jm  grown a t  3  mass b a l a n c e o f  reactor  (Fig.  the  night  Cellular  the  NO^  NC^  the  f o r a l l the  +  -  +  -  rhythmic  during  was  dilution  external  chamber and  s t e a d i l y increasing values during  volume  cultures  -  average of  (1.08  This  in cellular  i n c o m i n g medium, showed t h i s  reduction  period.  volume o f d u p l i c a t e c u l t u r e s  and  i n the  0.24 an  c o u r s e of the  ± 0.03  concentration  -  and  -  and  light  reduction  d "^  (daily) c e l l  1.50  of the  volume i n c r e a s e d 3  ± 0.01,  rates  0.48  1.83-1.92 um )  respectively,  start  nocturnal  a l s o observed i n the  where mean c e l l u l a r  NC^  the  NC^  -  diel  light continuous  cultures. The  0.74  external  NO3  with the  rapid reduction  period for  -  -  concentration  i n the  during  second h a l f of the  of darkness the  started  to  A  -  concentrations: period  and  0.48  variation  external  similar diel  d ^ -  period  dilution  in external  steady  beginning  h a l f of the  was  the  increase  NO3 )  -  -  observed  first during  With  2  the  dark  a l s o demonstrated  NO3  concentration  and  NC^  -  7.0  N0 ~  for  h a l f of the  (ca.  rate cultures -  the  immediately  " s u n r i s e " maximum  trend  light  N*L ^  ( F i g . 4.7A).  concentration  a decrease during  subsequent  first  (0.2-1.0 uq-at  to a  variations in  c u l t u r e medium,  the  light  steadily increase  j j g - a t N'L "*").  The  rate resulted i n d i e l  to minimal concentrations  the  onset  d "*" d i l u t i o n  light  period. diel  beginning  1  with  rapid reduction  concentrations NO2  during  NO3  -  concentrations  increased  maximum  (ca.  reached  a maximal concentration  to  lights  0.24  -  on ( F i g . 4.7B).  No d i e l -  equations  of nitrate  same d i e l  nitrate  greater  nocturnal 0.74  d  -  (0.0309  h  (1976); time  (h~^)  specific  rates  i n t e r v a l ) showed t h e  to total time  cellular  - 1  -  1  dilution  ) dilut ion rate -  1  period.  period  and demonstrated  cultures.  c u l t u r e s , maximal  were o b t a i n e d  thefirst  t o half this  maximal dark uptake  rates  both  for the  I n t h e 0.74 d ~ uptake  value  rates of  t h eremaining  came  by t h e end o f t h e i nuptake  2 h o f darkness with  increasing during  were  4-6 h a f t e r t h e l i g h t s  An a d d i t i o n a l 50% r e d u c t i o n  during  subsequently  rate  volume  i n F i g u r e 4.8.  of thelight  of t h edark period  and s t e a d i l y declined  occurred  detection  and d i u r n a l v a r i a t i o n i nuptake v e l o c i t i e s  0.0530 ± 0.0007 h  light  concentrations  ( F i g . 4.7C).  normalized  uptake rates  a n d 0.48 d  1  for either of the  uptake were c a l c u l a t e d u s i n g t h e  as rates  than those  prior  variation i nthe external  c u l t u r e , and a r ep l o t t e d against  Specific  -  of analytical  t o a v e r a g e PON d u r i n g  trends  uq-at N'L "'") 2 h  c u l t u r e s and ambient  o f Caperon and Ziemann  (normalized  "sunrise  concentrations  -  was o b s e r v e d  thelight-dark cycle  Rates  on  (0.30  a t o r near t h e l i m i t s  throughout  of  rate  -  remained  -  o f NC>3~ o r N C ^  d ^ dilution  0.01  -  steadily to a  NO2  1.7 ug-at N'L "' ) w h i l e  concentration  (NO3 :  W i t h t h e commencement o f d a r k n e s s ,  -  external  2 h of light t o  a t o r near t h e l e v e l s o f d e t e c t i o n  0 . 0 1 ug-at N-L ''-).  -  thefirst  rate  rates  dark period t o  o f 0.0183 ± 0.0003 h ^ d u r i n g -  20-22  165  F i g u r e 4.7. D i s s o l v e d n i t r a t e ( 0 , « ) and n i t r i t e (A,A) concentrations 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 at 0.74 d" (A), 0.48 d" (B), and 0.24 d (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 elapsed time since l i g h t s on. Dashed l i n e i n d i c a t e s onset o f dark p e r i o d denoted by dark bar. 1  1  _ 1  V7.0  O  <  - 6.0  - 4.0 - 3.0  o  - 2.0  I  cn  1  _I•  -at  - 5.0  cn  - 1.0  =1.  L\ J  1  1  1  1  1  0.0  1  B - 3.0  3.0  <  < cr  2.0  LJJ  1.0  o  o o  - 2.0  CT  T  0.0  X <>\ -\\  1.0  LU o  o  0.0  o  LU I< cr  cr I-  8  10  12  TIME  14  16  (h)  18  20  22  4)0.0  24  166  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: 0.74 d (0.031 h " ) ; B: 0.48 d" (0.020h ); and C: 0.24 d" (O.OlOh ) d i l u t i o n r a t e s . Rates p l o t t e d a g a i n s t average time between sampling. Dashed l i n e i n d i c a t e s onset o f dark p e r i o d denoted by dark b a r . _ 1  _1  1  1  1  1  1  1  1  -1  1  1  0.050  A  0.040  0.030  0.020  UJ  0.010  I— < cr  0.000  LU  < lQ_ Z> o  *  i  i  i  i  i  i  i  i  i  i  i  1  1  1  1  0.050  B 0.040  o 0.030  0.020  0.010 o  LU Q_ LU (3  O  cr  0.000  1  1  1  1  1  1  1  I  1  1  1  1  c  0.050  0.040  0.030  0.020  0.000  t  0  2  1  4 - 6  1  1  1  1  8  10  12  TIME  -.  14  16  (h)  18  20  22  24  167 h  internal.  nocturnal  increase  dilution (0.0331 of  rate  light)  light  rate  increased  occurred  h  - 1  )  value  22 h i n t e r v a l . dilution cycle  uptake  light  rate  increased  growing cultures dilution  rate  (during  2-4  rates  - 1  )  of 2 h  relatively  2 h of darkness the  o f 22% and then  o f t h e 0.24 throughout  rates  4.1.  steadily  reaching  during d  -  1  t h e 20-  (0.0100  h  )  - 1  cultures.  f o r the cyclostat of dark:light  o f 0.4 0 f o r t h e f a s t e s t  t o 0.78  t o a value  a  the light-dark  The r a t i o  from an average  cultures  h to a  -  d  )  - 1  rate  ± 0.0001 h ^ f o r d u p l i c a t e  i n Table  (D=0.74  h  the f i r s t  ± 0 . 0 0 1 3 h~^)  and dark uptake  are presented  uptake  of the dark period  (0.0195  0.0100  (0.0200  1  -  by an average  rate  -  and  ± 0 . 0 0 0 1 h "*" o v e r t h e r e m a i n d e r  were c o n s t a n t  and averaged  cultures  over the next  The u p t a k e  cultures  The a v e r a g e  earlier  the remainder  maximum d a r k u p t a k e  d  light  During the f i r s t  decreased  during  The m a x i m a l  o f 0.202  period.  of diurnal decline  i n t h e 0.48  occurred  and d e c l i n e d  the light  uptake  similar pattern  cultures.  ± 0.0040  constant of  A very  f o r t h e 0.40  of unity  d  -  1  f o r t h e 0.24  d ^ -  cultures. Internal part  of the night  attaining = 0-2  pools  maximal  cultures  uptake,  + NO2  -  increased  size at the beginning Although there  i n the magnitude of pool  both  cultures,  -  t h e 0.74  which  during  the  ( t = 20-24 h) i n a l l t h e c y c l o s t a t  h; F i g . 4 . 9 ) .  variability  of NO3  also  d "^ a n d 0.48 -  was  increase  cultures period  (t  considerable  s i z e s between d ^" d i l u t i o n -  demonstrated d i e l  showed a c a . 2 - f o l d  of the l i g h t  last  replicate  rate  variability i n  i n NO3  -  NO^  + NC>2~ p o o l  -  size  Table  4.1  Mean l i g h t  and d a r k  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 g r o w n o n 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 period are given i n parentheses.  Micromonas pusilla  NO3- u p t a k e Growth  rate  (h  - 1  )  Light  rate  (h- ) Dark 1  Dark:Light  0.0470 * 0.0449 *  0.0353 (0.0071) 0.0366 (0.0053)  0.0187 (0.0046) 0.0149 (0.0042)  0.53 0.41  0.0314 0.0304  0.0400 (0.0092) 0.0409 (0.0106)  0.0161 (0.0020) 0.0164 (0.0020)  0.39 0.41  0.0200 0.0200  0.0226 (0.0060) 0.0216 (0.0039)  0.0171 (0.0020) 0.0175 (0.0008)  0.76 0.81  0.0100 0.0100  0.0100 (0.0002) 0.0100 (0.0001)  0.0100 (0.0001) 0.0099 (0.0004)  1.00 0.99  J.  \J -/  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 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" (A), 0.48 d" ( B ) , and 0.24 d" (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 b a r . 1  0.0  1  0  ' 2  1  1  4  1  6  1  1  8  1  •  10 • 12  TIME  1  14'  1  1  16  '  1  18  (h)  20  1  22  24  170  after  the f i r s t  2 h of darkness i n p a r a l l e l  reduction  i n NC>3~ u p t a k e d u r i n g t h i s  Potential  nitrogen  At  the beginning,  10 h d a r k p e r i o d s urea  middle,  presented  from 2 h i n c u b a t i o n s  f o r these  i n Table  4.2.  over  batch  style  i n the i n i t i a l  t o the c y c l i c  cycle.  -  -  + NC^  uptake r a t e s of N H mid-point  of t h e i r  -  + 4  c o n c e n t r a t i o n observed. , NO3  discussed  c y c l o s t a t s sampled a t 2 h i n t e r v a l s  grown c u l t u r e (D = 0.74 d ^ ) was d i e l  external N O 3  cell  N quota of the  patterns  t h e 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  fastest  conditions of  incubations are  volume a n d c e l l u l a r  f o r the replicate  and  i n the dark or  The i n i t i a l  Diel variability  c u l t u r e s were s i m i l a r  earlier  + 4  f r o m t h e 0.74, 0.49, a n d 0.24  cultures.  c o n c e n t r a t i o n , mean c e l l three  a n d e n d o f t h e 14 h l i g h t a n d -  rate cyclostat  c u l t u r e s used  time.  rates  f o r samples c o l l e c t e d  dilution  the rapid  s a t u r a t e d uptake r a t e s o f N O 3 , N H  were d e t e r m i n e d ,  light,  the  uptake  with  -  and u r e a  In only the  variation i n The N  specific  are p l o t t e d versus the  incubation periods  i n Figure  4.10.  Rates  o f ammonium u p t a k e were c o n s i s t e n t l y 2-3 f o l d  greater  than  N03~  t o each  other.  It  and u r e a  should  culture,  be n o t e d estimated  underestimated substrate sampling  u p t a k e r a t e s w h i c h were s i m i l a r that the urea  u p t a k e r a t e s o f t h e 0.74 d ^  u s i n g t h e "^N t e c h n i q u e ,  due t o s i m u l t a n e o u s  (Collos,  -  1987; L u n d ,  During  p e r i o d s , NG"3~ c o n c e n t r a t i o n s (Chapter  the  uptake o f u n l a b e l e d  potential  slightly  uptake of u n l a b e l e d  1987).  by M. pusilla  may be  5 of the 6  s a t u r a t i n g f o r uptake  3) were p r e s e n t  i n t h e e x t e r n a l medium;  NO3 will -  dilute the  Table  4.2  Day  Culture  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  Dilution  description  rate (d ) _ 1  13 7  i n i t i a l dark mid-dark  8  end dark  )  (pg-at  C-L  - 1  )  Average  (10 -L 9  - 1  )  cell  Volume (M~i ) 3  Cell  Quota  (fg-at  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  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  mid-dark end dark light  - 1  density  6.9 9.6 8.7 9.2 8.5 6.5  initial light mid-light end l i g h t i n i t i a l dark  mid-light end l i g h t  N-L  Cell  1.4 1.8 2.0 2.2 1.8 1.3  16 2 11 13  initial  (^g-at  POC  .56 .73 .84 .04 .86 .87  0.76  2 11  PON  2  experiment.  244 321 406 425 340 273  initial light mid-light end l i g h t i n i t i a l dark mid-dark end dark  16  3  o f each  31.6 45.5 50.6 46.2 41.2 38.1  16 2 11 13 7 8  7 8  N 0 ~ + NO ~  a t the beginning  19 . 9. 0. 3. 9. 11.  N-cell  - 1  )  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 ) 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 of samples from 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 " ( A ) , 0.49 d " ( B ) , a n d 0.24 d " ( C ) d i l u t i o n r a t e s . Specific rates are p l o t t e d against average time 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 onset o f dark p e r i o d denoted by dark b a r . - 1  1  1  1  0.0  1  1  1  1  1  1  L  TIME  (h)  173  isotopic uptake  ratio  i n t h e p a r t i c u l a t e m a t t e r and t h u s d e c r e a s e t h e I n N 0 3 ~ - r e p l e t e c u l t u r e s o f M. pusilla,  rates.  under c o n t i n u o u s l i g h t , in  uptake  uptake  -  ( C h a p t e r 3 ) , however, t h e  of urea suppression of N O 3  chemostat  in  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  a 28% d e c r e a s e i n N O 3  degree  cultures  grown  i s unknown.  uptake  -  i n N-limited  I t i s unlikely that  NH  + 4  NO3  r a t e s were a f f e c t e d by t h e p r e s e n c e o f u n l a b e l e d  t h e medium.  -  The p r e s e n c e o f ammonium, a t c o n c e n t r a t i o n s a s  low a s 1.0 pq-at  N*L ^, -  r e p l e t e M. pusilla  c o m p l e t e l y i n h i b i t e d NC>3~ u p t a k e  i n N-  ( C h a p t e r 3) t h u s , t h e p o s s i b i l i t y o f  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 -  unlikely. A marked d i e l substrates 0.74  d~^ d i l u t i o n  r a t e c u l t u r e , average  (V^) o f u r e a , N H  respectively,  rates)  dark uptake  uptake  rates  (V  L T  ).  cultures  respectively  the  were 65, 52, and 40%,  -  rates  (0.49 and 0.24  +  and N O 3  o f t h e mean l i g h t  of urea, N H  -  (Table d ^ -  4.3).  dilution  averaged  rate  cultures,  106, 83, and 72%,  v a l u e s and i n t h e 0.24  d ^ -  v a l u e s were 93, 71, and 80%, + 4  and NC>3~ p o t e n t i a l l i g h t  uptake  Although i n both t h e slower growing c y c l o s t a t  mean l i g h t  In  very s i m i l a r to l i g h t  I n t h e 0.49 d ~ ^ d i l u t i o n  rate cultures  respectively  uptake  r a t e s were g e n e r a l l y  V^j v a l u e s o f u r e a , NH^  rates.  of a l l three N  (n = 3) p o t e n t i a l d a r k  and N O 3  + 4  o f t h e mean l i g h t  more N - d e f i c i e n t  dilution  i n t h e uptake  was o b s e r v e d f o r 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  uptake r a t e s  the  variability  and d a r k r a t e s were o f a c o m p a r a b l e  a d i u r n a l v a r i a t i o n i n V-^rp was a p p a r e n t f o r N O 3  -  cultures  magnitude,  and u r e a ;  Table  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 ~ ), d e t e r m i n e d 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 i n N0 ~-limited cyclostat c u l t u r e s o n 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) . -  1  3  N  Starting time of I n c u b a t i o n Day:time (h)  0.74 d L  D  1  D/L  s p e c i f i c uptake r a t e (h 1 0.49 d " L D D/L  - 1  )  0.24 d L  D  _  1  D/L  Urea  16 2: 11 : 13 : 7 8  0845 1425 2007 2200 0210 0615  0.0140 0.0099 0.0213 0.0155 0.0137 0.0160  0.0019 0.0030 0.0029 0.0055 0.0104 0.0136  0.13 0.30 0.14 0.35 0.75 0.85  0.0239 0.0208 0.0191 0.0314 0.0209 0.0313  0.0058 0.0025 0.0103 0.0234 0.0203 0.0232  0.24 0.12 0.54 0.75 0.97 0.74  0 .0150 0 .0151 0 .0140 0 .0242 0 .0142 0 .0263  0.0076 0.0054 0.0057 0.0129 0.0113 0.0168  0.51 0.36 0.41 0.53 0.80 0.64  0.0133 0.0142 0.0280 0.0291 0.0291 0.0390  0.26 0.21 0.43 0.53 0.74 0.58  0.0584 0.0554 0.0566 0.0718 0.0534 0.0735  0.0247 0.0181 0.0329 0.0542 0.0327 0.0546  0.42 0.33 0.58 0.75 0.61 0.74  0 .0516 0 .0541 0 .0517 0 . 0550 0 .0369 0 .0616  0.0277 0.0234 0.0298 0.0348 0.0306 0.0455  0.54 0.43 0.58 0.63 0.83 0.74  0.0037 0.0036 0.0068 0.0067 0.0105 0.0109  0.31 0.16 0.19 0.32 0.66 0.40  0.0355 0.0219 0.0300 0.0456 0.0292 0.0490  0.0028 0.0018 0.0110 0.0234 0.0194 0.0199  0.08 0.08 0.37 0.51 0.66 0.41  0 .0190 0 .0183 0 . 0096 0 .0300 0 .0212 0 .0383  0.0056 0.0019 0.0076 0.0103 0.0110 0.0162  0.30 0.10 0.79 0.34 0.52 0.42  Ammonium  16 2 11 13 7 8  0847 1435 2010 2202 0212 0616  0.0520 0.0682 0.0658 0.0555 0.0394 0.0668 Nitrate  16 2 11 13 7 8  0851 1445 2014 2203 0215 0618  0.0119 0.0223 0.0356 0.0214 0.0160 0.0270  175  maximal  r a t e s were observed  beginning rates  of the light  were r e l a t i v e l y  Dark uptake collected both  Influence  while  potential  NH4"**  the night,  of light  specific  uptake  rates,  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  L  T  4.11).  + 4  and NO3  i s important t o note that  are derived portion  rate  a r e t h e PPFD a t w h i c h maximal v e l o c i t y which  uptake  of the cells  total  0.5 V '  constant  curve, thus K m a x  These two l a t t e r  inversely  i n T a b l e 4.4.  L  T  with  dilution  a  D  constant  c a n be  Another  PPFD  similar  half-  f o rhalf the  ( 1 2 0 juE m ~ s 2  _ 1  constants include the  velocities rate  2  i s the  m a x i m u m N-  + V )/ ) ,  x  c a n be c a l c u l a t e d  half-saturation  dark uptake  m  reported  of the Michaelis-Menten  2 f o rd e t a i l s ) . T  m a x  The h a l f - s a t u r a t i o n  i s achieved, ( V '  (K-^ ")  (or light)  values  o c c u r s where V '  N-uptake achieved a t t h e growth  substantial  for light  m a x  i s t h e PPFD when o n e - h a l f t h e t o t a l  (seeChapter  constant  these Michaelis-Menten parameters  by simple rearrangement  saturation  cyclostats  V'  a r e summarized  i n the light.  (K-^rp' )  equation  velocity  2 h, were  previously  from data o b t a i n e d from t h e h y p e r b o l i c  o f t h e PPFD r e s p o n s e  calculated  uptake  uptake  -  rates  (V-p), t h e h a l f - s a t u r a t i o n  a n d maximum n i t r o g e n  ' ) ,  dependent N H It  Dark uptake  from  equitable.  determined over  -  ( K  rates  and ammonium uptake  uptake  period.  lower f o r samples  however l i g h t  on nitrate  atthe uptake  constant throughout the l i g h t  g r o w n i n 0 . 2 4 , 0.52 a n d 0.77 d ^ d i l u t i o n (Fig.  collected  and dark p e r i o d s were g e n e r a l l y  Nitrogen plotted  period,  r a t e s were c o n s i s t e n t l y  during  light  i n t h e samples  which  appear  (Table 4.5).  t o vary  The d a r k  NO3  -  ).  F i g u r e 4.11. N i t r o g e n s p e c i f i c uptake r a t e s , determined over 2 h, a f t e r s a t u r a t i n g enrichment of NH (•) o r N 0 (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 of Micromonas pusilla (14h:10h L:D c y c l e ) p r e v i o u s l y grown a t 0.77 d ( A ) , 0.52 d (B), and 0.24 d (C) d i l u t i o n r a t e s . Uptake r a t e s ( h ) 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, curved 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 by computer programme (see t e x t f o r d e t a i l s ) . 15  +  15  4  _  3  _ 1  - 1  80.0  ro •  o  LU <  cc  < hO  O LU CL  (J)  0  _ 1  -1  20  40  PPFD  60  80  100 120 140  (pE-m- -s- ) 2  2  Table  4.4  Parameters cyclostat (V'  m a x  curve  ),  describing cultures  the characteristics of N specific  Micromonas pusilla  the h a l f - s a t u r a t i o n constant  (a =  V  ' - , / m  K  X  Dilution rate (d )  PON cone. (AJg-at N - L  0.77  28.0  - 1  of  T  T  ) •  Estimated  L T  standard  Nitrogen substrate - 1  (K  ( F i g . 4.11). ),  uptake  errors  V  o f parameters  (h  NH/  0. 0017 0. 0137  - 1  D  portion  light  uptake  o f N u p t a k e v s PPFD  are given i n parentheses.  v D  o f PPFD f o r  ( V " ) , maximum s p e c i f i c  and t h e s l o p e o f i n i t i a l  )  NO-  ( h ~ ), as a f u n c t i o n  Dark uptake  'max  K  )  LT  a  (uE m ~ s 2  _ 1  )  (0 .00160) (0 .00255)  0 .0323 (0. 00203) 0 .0656 (0. 00411)  13 (3 .3) 26 (5 .8)  2 .6 2 .8  0.52  53.5  NO-  NH"  0. 0048 (0 .00155) 0. 0243 (0 .00370)  0 .0460 (0. 00198) 0 .0693 (0. 00496)  15 (2 •7) 16 (4 •5)  3 .2 4 .2  0.24  47.8  NONH\  0. 0076 0. 0276  0 .0310 0 .0499  13 (2 •9) 18 (2 •4)  2 .5 2 .8  (0 .00134) (0 .00126)  (0. 00164) (0. 00173)  Table  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 o n 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 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 : V ) , 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 ' , 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 ( i% ioo%** 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 piE m ~ s ) . D  L  L T  v  :V  L T  2  Dilution (d )  rate  - 1  - 1  Nitrogen Substrate  0.77  N0  0.77  NH  0.52  V  D  : V  L  K  LT'  K  LT"  V  1%  : V  0 . 05  11.3  9.6  0 .15  0.20  15.4  11.3  0.25  N0 -  0. 10  11.9  9.8  0.18  0.52  NH  0.29  7.9  7.3  0.34  0.24  NO3-  0.21  7.6  6.4  0.29  0.24  NH  0.39  5.1  3.5  0.43  * Definitions  3  + 4  3  given  i n text  + 4  + 4  179  uptake 0.77,  rate  i s 5, 1 0 , a n d 2 1 % o f t h e t o t a l  0 . 5 2 , a n d 0.24 d ~ * d i l u t i o n r a t e  Dark N H  uptake  + 4  rates,  consistently  uptake  rates,  uptake  f o r t h e same c u l t u r e s .  portion  o f t h e PPFD r e s p o n s e  V  max  b  v  LT  K  (  H e a l e  Y'  and NO3  NH  d !  d i l u t i o n rate  -  +  uptake  NH^ , i n response +  increases with  was  observed  + 4  and  3  results  cultures  appear  o f NC^ , and -  but  (light  + dark) can  a t 1 and 100% o f t h e  o f PPFD o n t o t a l  dilution rate, the light  pusilla.  t a k e n up a t g r e a t e r  which  NH  + 4  rates  c a . 40% l e s s  N  PPFD uptake  suggests  light  , which  than NO3  that  of NO3  dependency  Although t h i s  f o r both N substrates,  t h e dark, demonstrated  than N0 ~.  + 4  growing  ability  N uptake  The e f f e c t  increasing  b y Micromonas  consistently  growing  o f uptake  N l i m i t a t i o n lessens  NH  These  for NH  lower percentages represent greater  (Table 4.5).  and  greater  and slow  t o increasing,  o f PPFD on t o t a l  irradiance;  increased  by d i v i s i o n o f  PPFDs.  effects  dependency  dark)  of both N-substrates t o  t h e i r uptake  e s t i m a t e d by a comparison  growth  +  -  (a) o f t h e i n i t i a l  culture.  PPFDs by f a s t  most c a p a b l e o f i n c r e a s i n g  The  NO3  f r o m t h e 0.77 a n d 0.24  response  whereas t h e i n t e r m e d i a t e l y  subsaturating  (light  but substantially  uptake  subsaturating  particularly  respectively.  than dark  curve, calculated  by t h e c e l l s  cultures,  similar light  cultures  forthe  1980; P a r s l o w e t a l . , 1985) a r e s i m i l a r  uptake  -  The s l o p e  -  increasing  be  greater  b y t h e 0.52 d ^ " d i l u t i o n r a t e  suggest  cultures,  a r e 2 0 , 29 a n d 3 9 % o f t h e t o t a l  for  4  uptake  -  effect  was -  i n both  dependency  light  o n PPFD  180  DISCUSSION The  cell  partially  on  cycle.  natural  u,  (see  cycles,  varies  division  Evidence  communities  light:dark  rate,  o f most p h y t o p l a n k t o n c e l l s i s  with  of phasing i n both  h a s shown t h a t ,  a 24 h p e r i o d i c i t y  o f Micromonas  pusilla,  phasing of c e l l  division  rates  dark  decreased  dilution  appeared  division  was s h i f t e d  d "*" d i l u t i o n -  often  was e v i d e n t .  With  rate)  t o decrease  rate  1980; Sweeney,  later  of synchronous  cell  periodicity  reason  the majority  of the results  been n o r m a l i z e d t o t o t a l of c e l l  (or size)  steadily  a t the onset  i n uptake  cellular  division  and t h e  limitation  of the light of the dark i n t h e 0.24 uptake  basis,  can rates.  seriously For this  volume o r p a r t i c u l a t e  was r e f l e c t e d cells.  during the light of the dark period  period  i n t h e mean  Cell  rates  b u t any  i n the present study  o f t h e M. pusilla  increased  period  i n the night  division  cycle,  Maximum  N i t r o g e n and carbon  the apparent  synchrony  (cyclostat)  nitrate  and t h e t i m i n g  slightly  cultures.  increased  the importance  modify  size  and s p e c i e s  et al.,  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  co-occurrence  volume  the timing of  g r o w n i n a 1 4 : 1 0 L:D  division  period.  division  The  and t h a t  o c c u r r e d a t t h e end of t h e l i g h t  to late  cultures  s p e c i e s grown  I n both N - r e p l e t e (batch) and N - l i m i t e d  partial  (i.e.  i n most  depends 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  cultures  middle  algal  the instantaneous population division  r e v i e w s by S o u r n i a , 1974; C h i s h o l m  1983).  are  cycle  s y n c h r o n i z e d o r phased by t h e e n v i r o n m e n t a l  light:dark and  division  have N. cell  volume  attaining  and subsequently  maximal declined  181 steadily onset  throughout the night  of the l i g h t  limitation, light  period  rate  maximal s i z e a t t a i n e d mean (r  reduction  been r e p o r t e d  limited Meyer, and  u and i m p r o v e d N O 3  in cell  volume w i t h  gravida  -  supply).  (Harrison  Dunaliella  tertiolecta,  Coccochloris in  cell  stagnina  cyclostat  cultures  weisflogii,  and  Riley  nutrients.  function  increase  Using  of  (1980) s t u d i e d  Thalassiosira the e f f e c t of  and c o n c l u d e d t h a t of average c e l l  the c e l l ' s  i n mean c e l l  growth  volume.  the larger the surface  (SA/V) t h e g r e a t e r The r e d u c t i o n  and  1972) o r an  d i f f e r e n t clones  (1952) s t a t e t h a t  volume r a t i o  chemostat c u l t u r e s of  (Fuhs e t a l . , 1 9 7 2 ) .  s i z e on g r o w t h r a t e  r a t e was an i n c r e a s i n g  with  P - l i m i t a t i o n i n chemostat  C h i s h o l m and C o s t e l l o  average c e l l  However,  volume v a r i a b i l i t y  ( C a p e r o n and Meyer,  of four  -  costatum,  e t a l . , 1977).  T h a l a s s i o s i r a pseudonana  o f T. pseudonana  + 4  ( C a p e r o n and  Skeletonema  i n N-limited  volume w i t h i n c r e a s i n g  cultures  Similar  1979) a n d N H  lutheri  o t h e r s have documented e i t h e r no c e l l ( o r growth) r a t e  dilution  nutrient limitation  (Burmaster,  debilis,  The  significantly  steady-state  o f Pavlova  1972), and Chaetoceros  dilution  by t h e  c a . 50% o f t h e  increased  increasing  f o rP-limited  chemostat c u l t u r e s  Thalassiosira  volumes a t t a i n e d  by t h e N 0 3 ~ - r e p l e t e b a t c h c u l t u r e s .  ( d a i l y ) c e l l .volume o f M. pusilla  (increasing  -  volume d u r i n g t h e  c u l t u r e s were o n l y  = 0.99, P ^ 0.01) w i t h i n c r e a s i n g  rate  has  i n average c e l l  was r e d u c e d ; m a x i m a l c e l l  0.24 d ~ l d i l u t i o n  size at the  However, w i t h i n c r e a s i n g N O 3  period.  the increase  a t t a i n i n g minimal  capacity  v o l u m e o f M.  Munk  area t o t o absorb pusilla  182 and  t h e subsequent i n c r e a s e  i n r e l a t i v e surface  area a v a i l a b l e  for  N O 3 uptake i s perhaps an a d a p t i v e response t o N0-}~ -  limitation. In the n i t r a t e - r e p l e t e batch c u l t u r e s and the 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 , where growth r a t e i s l i m i t e d by the r a t e of supply of N O 3 , obvious d i e l p a t t e r n s  for N O 3  -  uptake were observed f o r M. p u s i l l a d e f i c i e n t c y c l o s t a t populations  -  i n a l l but the most N O 3 -  (D = 0.24 d "^).  The n i t r a t e  -  uptake r a t e s , f o r the c u l t u r e s grown i n batch or i n c y c l o s t a t s at 0.74 and 0.42 d ^ d i l u t i o n r a t e s , were maximal d u r i n g the -  l i g h t p e r i o d and decreased d u r i n g  the dark p e r i o d .  In  a d d i t i o n t o the d i e l p a t t e r n , the e a r l y l i g h t n i t r a t e uptake maximum ( e a r l y morning) and i n c r e a s e d night  dark uptake d u r i n g  (pre-dawn) suggests d i u r n a l and n o c t u r n a l  0.74 and 0.42 d ^ d i l u t i o n r a t e c u l t u r e s . -  late  c y c l e s f o r the  Malone e t a l .  (1975) found s i m i l a r c y c l i c v a r i a t i o n s i n N O 3 uptake by an -  outdoor c u l t u r e of Chaetoceros (2.0  sp. grown a t high d i l u t i o n  rate  d ^ ) under n a t u r a l s u n l i g h t , but uptake independence of -  the  l i g h t - d a r k c y c l e f o r three  slower d i l u t i o n r a t e s  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 -  (0.5, 1.0  -  a l s o observed i n axenic c y c l o s t a t c u l t u r e s of the same Chaetoceros  sp. (STX-105) grown a t 6 low d i l u t i o n r a t e s  1.2 d""*") by P i c a r d  Dark N O 3 uptake c a p a c i t y of  ( 1976).  -  c y c l o s t a t c u l t u r e s of the marine prymnesiophyte, l u t h e r i and c h l o r o p h y t e ,  (0.3 -  Pavlova  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 r a t e of N O 3 a t high d i l u t i o n r a t e s -  (> c a . 0.5  d "'"), but no d i e l p e r i o d i c i t y was observed a t lower d i l u t i o n -  183 rates the  (Laws a n d C a p e r o n ,  diatom,  Thalassiosira  similar  lack of d i e l  limited  cyclostat  greater  NG^  lesser  rates  cyclostat  -  species  periodicity i n nitrogen-(NO3 -  a n d NH^**" u p t a k e r a t e s i n t h e dark p e r i o d  o f Emiliana  for a similarly  dark uptake c a p a c i t y  t h e degree o f N a n d hence  diel  p e r i o d i c i t y o f N O 3 u p t a k e by n i t r o g e n  s u g g e s t e d by t h e r e s u l t s o f numerous f i e l d (see Chapter  d i n o f l a g e l l a t e blooms d o m i n a t e d  splendens  (= G. sanguineum)  at N « L ~ 1 )  waters  night  In contrast, averaged only  Gonyaulax the  polyedra  ambient  Maclsaac  -  c a . 1 uq-at  shallower  than o f f Peru.  Harrison  by  Gymnodinium  rates  (Dortch  (1978) r e p o r t e d  N'L ''" a n d t h e n i t r a c l i n e -  were  a n d Maske, that  rates  i n the surface  (<0.1 uq-  rates  -  o f f Baja C a l i f o r n i a .  N O 3 concentrations  generally  F o r example, i n  N O 3 uptake  10-20% o f t h e d a y l i g h t bloom  of natural  i n the nitrate-depleted  uptake  -  may  limitation  studies  1).  extensive  1982).  the night  stress i s  -  50% o f d a y l i g h t N C ^  It  A dampening e f f e c t on  -  o f f Peru, nighttime  but  the presence or lack of  p e r i o d i c i t y i n N O 3 uptake r a t e s .  p h y t o p l a n k t o n communities  )  grown  costatum.  -  and t h a t  + 4  p e r i o d and  t o t a k e up N O 3 d u r i n g  the a b i l i t y  + NH  -  huxleyi,  i n the light  diel  ca.  f r o m 0.1-1.4  E p p l e y e t a l . (1971b) f o u n d a  (0.78 d-'") c u l t u r e s  dependent  diel  rates  (0.73 d~^) c u l t u r e o f Skeletonema  appears t h a t  affects  n e v e r showed  -  (Laws a n d Wong, 1 9 7 8 ) .  - 1  be  allenii  i n N O 3 uptake a t 6 d i l u t i o n  periodicity d  1976; Laws a n d Wong, 197 8) a l t h o u g h  uptake a t  for a  However, waters  here  were  was much  S i m i l a r r e s u l t s were o b s e r v e d by  (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; but  NO3  nighttime  dark uptake  starved  u p t a k e was  -  increased  c u l t u r e s and  d o m i n a t e d by  compare the  r e s u l t s of  (e.g.,  as  the  or  1976;  due  field  studies  of  1982,  1986;  (e.g.  Whalen and  clear  evidence  to  et  nitrogen  Intracellular marked  the  nocturnal and  period  pusilla.  are  uptake and  are  low  increase  (see  to N  the  and  Mingazzini  m o r n i n g maximum i n N O 3 c u l t u r e s of  Skeletonema singular  costatum nighttime  dark period. culture  of  Only  of  sp.  highest  NO3  -  NH  of  no + 4  ambient  a  p o r t i o n of  at  the  N-limited  the  beginning  NO3  i n the  dilution  pool  M.  an  size for  In-  and pool  -  first rate  in  h a l f of  their the  cyclostat  a l . (1975)  size,  of  c u l t u r e s of  pool  internal  d i d Malone et  internal  or  -  tricornutum  measurement t a k e n i n the  and  demonstrated  intracellular  a minimal  few  (1987) a l s o o b s e r v e d  Phaeodactylum  and  Chaetoceros  periodicity  -  Cochlan  Discussion).  latter  f o r N - s u f f i c i e n t and  Diel  environments  i n e i t h e r NO3  the  ambient  (e.g.,  relatively  concentrations during  substrate  added.  freshwater  1  cyclostat  investigations  i n marine  Chapter  N-  to  generally  of  are  periodicity  nitrate  Raimbault  sufficient  diel  in field  concentratxon  1984)  in  tide  laboratory  a t t a i n e d maximal c o n c e n t r a t i o n s  light  early  to  1978)  a l . , 1986)  diel  daytime values  enhanced r e l a t i v e  -  uptake,  It is difficult  reported  NO3  daytime  -  studies  Alexander,  of  of  of  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  inorganic  night  the  20%  NC>3 -depleted r e d  Maclsaac,  in situ  Koike  40%  polyedra.  field  considerably  concentration  uptake  G.  uptake rates  Harrison,  saturated  ca.  similarly  populations  studies  to  ca.  while  observe internal  185 NO3  concentrations  -  dilution  independence of pools  cyclostats shipboard an  Bay,  w e r e o b s e r v e d by Chaetoceros  c u l t u r e s of region, NO3  intracellular light  period  and  grown a t  communities  no  C o l l o s and  Dortch  Slawyk  et  at  and  night.  pools  t o my  J u l y and  the  maximal  of  the  In  Dabob  surface  results, for  surface  from  diel  for  rhythm apparent  In  community  a l . (1985) r e p o r t e d  clear diel  rates.  beginning  the  in  similar  dilution  the  nitrate  uptake  -  (1976) o b s e r v e d  during  July, similar  i n May  low  lower  trends  (1976) i n  a natural phytoplankton  in intracellular  with  i n the  clear diel  Picard  minimal values  communities during results  constant  No  concentrations  -  Washington,  variability  and  light-dark cycle.  with  upwelled  low  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 NO3  rate  internal  remained  but  variable  deepwater  community  during  May. The  increase  pusilla  pools  were constant  equal  the  (or both  rate  uptake night and  reduction  of  diel  cycles Early  et  zero),  of  are  a l . , 1974)  not  nitrate  studies and  of  and  of  uncoupling  of  uptake  1976).  and  reduction  i n p h a s e , and reductase Peru  pools  greater  processes are  (NR)  -  decreased  than  likely  rate the  NO3  of  would  -  the  of  late uptake  result  of  activity.  (Eppley  northwest A f r i c a n  NO3  intracellular  -  Clearly during  the  Micromonas  between  i f intracellular  periods,  the  i n the  i f NO3  time;  NC"3~would be  Slawyk,  early light  reduction  this  then rates  reduction  ( C o l l o s and and  during  observed  -  c u l t u r e s d e m o n s t r a t e s an  u p t a k e and  be  NO3  in internal  et  a l . , 1970;  (Packard  and  Packard  Blasco  1974)  186 upwelling and  cycles  intensity:  Packard  reported  around  insufficient  unialgal  1974),  al.,  1971b) w i t h  A similar  coincident with i n NR a c t i v i t y  minimal  rhythm  1976),  e t a l . ,1970; the sampling  carterae  Emiliana  activity  and  huxleyi  reported  i n M. pusilla  the increased  morning  observed  NO3  uptake  -  -  during leakage  late  A reduction night  of NO3  -  during  late of diel  the light-dark of  Gonyaulax  1975; H a r r i s o n ,  1976),  during  night  the late  night.  combined and e a r l y  i n t h e t w o f a s t e r g r o w n 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 internally.  during  et  account  t h e absence  as i n N-starved n a t u r a l populations  with  o r no v a r i a t i o n  late  could  NC>3~ o b s e r v e d  Alternatively,  and Harrison,  a  Cachonina  (Eppley  during  cycle,  (Eppley  o f such  has a l s o been observed i n  with  polyedra  failed  had been  NR a c t i v i t y such  little  They  c o n c l u s i v e l y t h e presence  i n NR a c t i v i t y  and e a r l y morning.  that  variation  minimum a n d  dawn. that  diel  s t u d i e s was  a s Amphidinium  and S w i f t ,  the diel  (Eppley  but suggested  the accumulation of internal  night  areas  periodicity  c u l t u r e s , such  (Hershey  followed  dawn i n t h o s e e a r l i e r  Diel  niei  activity  observed  maximum, n i g h t t i m e  i n upwelling  t o determine  pre-dawn r i s e .  c y c l e s o f NR  e t a l . (1987)  clearly  a daytime  and Blasco,  frequency  that  a pre-dawn r i s e  previously  diel  Martinez  o f NR a c t i v a t i o n  observe  for  system,  i n NR a c t i v i t y  onset  suggested  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  upwelling  light  the to  have  i n a similar,  African  in  system  (e.g.,  + NC^  -  lead t o accumulation of NO3  i n nitrite Eppley  reductase  (NiR) a c t i v i t y  e t a l . , 1971b) a n d  from t h e c e l l s  -  may a c c o u n t  subsequent  f o r the  187  increase  i n e x t e r n a l NC^  -  concentrations  the  0.74 a n d 0.48 d""* d i l u t i o n  the  rise  i n e x t e r n a l NC^  resulting cell  from c e l l u l a r  NC>3~, N H  cyclostat  concentration  loss during  diel  rhythm  similar  nighttime uptake  night  uptake  as  o f maximal  averaged there  cyclostat  from  -  capacity.  -  although they  allenni  a r e no o t h e r  c u l t u r e s growing  nighttime  daylight NO3  comparable at various  observed  over a range of  120% o f t h e r e s p e c t i v e  diel  uptake  degrees  of N  -  uptake.  data f o r limitation  c a n b e m a d e b e t w e e n my r e s u l t s a n d  example,  t h e d i n o f l a g e l l a t e Gyrodinium  N-replete  and N-starved c u l t u r e s . aureolum  does  For not take  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 w h e n N - s t a r v e d f o r 24 h n i g h t t i m e  ability  of potential  ( 0 . 0 0 5 7 3 - 0 . 0 2 4 6 4 h ^") i n  lutheri,  work w i t h  light  importance  to potential daylight  rate  previous  -  L a w s a n d Wong  0.00474 t o 0.05937 h ~ * , i n f a c t  a r e v e a l i n g comparison  NO3  growing  (D = 0.48 a n d 0.24 d ^ )  decreased r e l a t i v e  f o r Thalassiosira  capacity  (D = 0.74 d ~ ^ ) ,  the relative  c u l t u r e s o f Pavlova  rates  Although  cultures  increasing dilution  such trend  dilution  of  period  i n the fastest  pusilla  and day N uptake  observed that  uptake  with  cyclostat  up  Alternatively,  i n t h e p o t e n t i a l uptake  c u l t u r e s o f Micromonas  (1978) a l s o  no  night f o r  may b e e x p l a i n e d  this  a n d u r e a was o b s e r v e d  + 4  whereas t h e more N - l i m i t e d had  cultures.  late  division. A pronounced  of  -  rate  during  uptake  (Paasche e t a l . , 1984).  t o t a k e up NC^  considerably  uptake  -  and N H  + 4  during  They  became a b o u t  half  found that t h e  the nighttime  varied  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  relative  dark uptake  of  similar  NG^ , -  Similarly, uptake  of NH  t o that  Bhovichitra  capacity  Pyrocystis  It  was g e n e r a l l y  observed here  and S w i f t  greater  f o r M.  (1977)  than  pusilla.  showed t h a t  a n d Dissodinium  lunula,  were  that  N starvation or limitation  potential  dark uptake  resulting  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  more t h a n  light  uptake  enhances  of  nitrogen  of nitrogen i n  phytoplankton, including the picoplankter  Micromonas  pusilla.  versus  decreasing  dark  i s also  capacity  N0 ~ uptake  increased  A comparison  o f samples  reduction  incubated  1 2 0 L(E*m  N limitation  decreased l i g h t  adaptive  response  optimize  i t s uptake  the  relatively  picoflagellate  from  With  the relative  5 t o 21% of  uptake  NH  (light  + 4  +  a t 1 and 100% o f t h e growth  —1  *s  ) reveals  an approximate -  and N H  dependence o f N uptake which  two-fold  uptake  + 4  i n cyclostat cultures  to N limitation  high  uptake  20 t o 3 9 % o f t o t a l  dependence o f NO3  i n light  increasing The  (  N  mid-day.  rate)  f o u r - f o l d from  of calculated total  —2  irradiance  increasing  during  (decreased d i l u t i o n  and two-fold  3  uptake. dark)  dependence w i t h  suggested by t h e r e s u l t s o f t h e N  N limitation  uptake  total  light  i r r a d i a n c e experiments conducted  increased  -  virtually  N-depleted  limitation  the NO3  of the light-dark cycle.  appears  The  that  of N-starved oceanic d i n o f l a g e l l a t e s ,  noctiluca  independent  + 4  with  o f M.  i s perhaps  allows  pusilla. an  the cell  to  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 metabolic costs  (Raven,  of migration  1986) t o a more s u i t a b l e  f o ra light  189  environment  s a t u r a t i n g t o N uptake.  Despite 1960;  i t s pronounced p h o t o t a x i s  T h r o n d s e n , 1973)  ability  and i t s r e l a t i v e l y  ( K n i g h t - J o n e s and Walne,  Micromonas  pusilla  flagellates euphotic  1952;  good  Throndsen,  and i t i s o f t e n  pusilla  Throndson,  1973)  —1 c a n swim a t c a . 90 jjm*s which would e n a b l e t h i s  (PPFD) d u r i n g d a y t i m e v e r t i c a l m i g r a t i o n  the  (75-100  o f G e o r g i a , B r i t i s h C o l u m b i a , M. pusilla  p h y t o p l a n k t o n biomass  density  c a n be  i n the euphotic  a c c o u n t s f o r <7%  that  t h e 3.9  m t h a t M.  (12 h) swimming  i n c r e a s e t h e c e l l ' s mean i n c i d e n t  PPFD was cell  (attenuation c o e f f i c i e n t  value.  This could  specific limiting.  from a r e g i o n  PPFD b u t s t i l l  nutrient  o f c a . 0.42  saturating  From  a day's m ^) -  PPFD by 5 t i m e s t h e  be a d v a n t a g e o u s , i n t e r m s o f and g r o w t h r a t e ,  i f the  initial  C o n v e r s e l y , downward swimming c o u l d of high,  of  pusilla  c o u l d move v e r t i c a l l y i n t h e s e N - r e p l e t e w a t e r s d u r i n g  increased  ,  to  (Harrison et a l . , submitted).  B e e r ' s l a w one c a n c a l c u l a t e  original  1  i n a s t r a t i f i e d water  ( C l i f f o r d e t a l . , 1989), b u t i t o n l y  would  1976).  jjm«s  photon f l u x  n u m e r i c a l l y t h e most a b u n d a n t p h y t o p l a n k t e r  the  Throndsen,  I n t h e F r a s e r R i v e r plume, i n t h e s o u t h e r n p o r t i o n o f  Strait  zone  below t h e  picoflagellate  a c h i e v e m e a n i n g f u l changes i n i t s i n c i d e n t  column.  1973)  found w e l l  ( e . g . , Manton and P a r k e , 1960;  Micromonas  swimming  has b e e n f o u n d d e e p e r i n t h e s e a t h a n  i n general,  zone  (Manton and P a r k e ,  inhibitory  PPFD t o one w i t h  f o r nitrogen uptake.  take a lower  190  CONCLUSIONS This  dissertation  nutrients and  by b o t h n a t u r a l  unialgal  pusilla  1.  cultures  as a f u n c t i o n  specific  findings  phytoplankton  maximal  periodicity  assemblages  of marine  phytoplankton Micromonas  of light  history.  and n u t r i t i o n a l  NH  -  communities with  during  appeared  + 4  demonstrated  minimal uptake the daytime.  intensity  species  c o m p o s i t i o n , t h e ambient N substrate  diel  a t night and  by a number o f  and these i n c l u d e d  utilized,  and oceanic  The a m p l i t u d e o f u p t a k e  t o be i n f l u e n c e d  light  pronounced rates  The  below.  and urea by c o a s t a l  besides  actual  of nitrogenous  o f t h e r e s e a r c h a r e summarized  rhythms, uptake  t h e uptake  of the picoflagellate,  of NO3 ,  The u p t a k e  periodic  examined  nitrogen  and t h e depth  factors  the phytoplankton concentrations, the i n the water  column. 2. of  In both the frontal  G e o r g i a t h e dependence o f n i t r a t e  irradiance similar  could  be d e s c r i b e d  of NO3  of the Strait  and urea uptake  by a r e c t a n g u l a r  upon  hyperbola The  light  u p t a k e w a s t h e same f o r b o t h t h e s u r f a c e  -  DCM c o m m u n i t i e s  of the frontal  stratified  waters  dependence  f o rNO3 ,  those  waters  t o the Michaelis-Menten formulation.  dependency and  and s t r a t i f i e d  surface -  f r o m t h e DCM.  water, whereas  p h y t o p l a n k t o n showed  and p a r t i c u l a r l y  i nthe  less  light  f o rurea uptake,  than  191  3.  Uptake  rates  o f NC>3~, N H  + 4  ,  and urea  artificial  darkness  by  phytoplankton communities;  coastal  nitrogen  limitation  increased 4.  relative  ( l o w ambient to light  cultures  inhibitory  effect  i npartial of NH  5.  Uptake  take  up N H  Although  kinetic  uptake  -  experiments  the half-saturation  slope  of the Michaelis-Menten plot  effectively 6.  than  pusilla.  after  Nitrate  uptake  the i n i t i a l  NO3  -  7.  p a t t e r n s were observed  and  o f M. pusilla  internal  uptake.  -  the greater uptake  + 4  of NH  + 4  (25-50%)  of starved  -  suggests + 4  more  and NO3  -  uptake o f M.  i n N-starved  cells;  uptake  i n both continuous and batch  division,  pools of NO3  initial  and urea  g r o w n o n a L:D i l l u m i n a t i o n  i ncell  f o r the  of N-starved cultures  enrichment  immediately.  periodicity  or urea  b u t t h e r e was no l a g i n u p t a k e  commenced  Diel  -  for NH  was s l o w e r  after  even a t  can  low concentrations of NH  N enrichment  than N-replete c e l l s ,  cultures  -  The  -  N-L "' ),  (surge) rates  cells  Diel  was c o m p l e t e  equivalent concentrations of urea  Transient elevated  were observed  uptake.  constants are similar  uq-at  can u t i l i z e  -  N'L "'".  of NO3  substrates  b y N-  -  s h o w e d t h a t M. pusilla  three  t h a t M. pusilla  (0.3-0.5  of NO3  of NO3  inhibition  at twice the rates  + 4  uptake  whereas t h e a d d i t i o n o f  c o n c e n t r a t i o n s a s l o w a s 1 uq-at  + 4  uptake  conditions of  t h e uptake  pusilla  on NO3  + 4  under  of total  uptake.  o f Micromonas  urea only resulted  portion  N concentrations) dark  /Ammonium c o m p l e t e l y s u p p r e s s e d  replete  NH  were a s u b s t a n t i a l  i n t h e night and  mean c e l l  were observed.  cycle.  volume, N  With  uptake  decreased  192  dilution  rate  NC>3~ u p t a k e  (decreased u and slower NO3  periodicity  were c o n s i s t e n t l y rates  regardless  variability  was a b s e n t .  2-3 f o l d  o f t h e degree  i n potential  supply) i n s i t u  Potential than NC^  rates  rates  i n the fastest  8.  The e f f e c t  _ 1  grown c y c l o s t a t  Dark uptake  of irradiance  could rates  increased  -  + 4  (0.49 and  rates,  N limitation  of NH  portion  and t h e r e l a t i v e increased  the light  and NO3  + 4  by M i c h a e l i s - M e n t e n  were a g r e a t e r  increased with  was l e s s e n e d .  on t h e uptake  be d e s c r i b e d  of NH  t h a n d a r k NO^  dark N uptake  uptake  culture  )  b y M. pusilla  uptake  diel  of a l lthree N  -  d  + 4  uptake  A marked  d -*-), b u t n o t i n t h e s l o w e r g r o w n c y c l o s t a t s  0.24  o f NH  or urea  -  of N limitation.  uptake  s u b s t r a t e s was a p p a r e n t (0.74  greater  -  -  kinetics.  of total  importance of  N limitation.  dependency o f NO3  -  With and N H  + 4  193  REFERENCES A l l e n , T.F.H. 1977. Scale i n microscopic a l g a l ecology: neglected dimension. P h y c o l o g i a 16: 253-257 Amy,  a  N.K. and R.H. G a r r e t t . 1974. 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F u r t h e r t r a n s i t i o n s t a t e s o f t h e Baja C a l i f o r n i a u p w e l l i n g ecosystem. L i m n o l . Oceanogr. 22: 264-280. Ward, A.K. a n d R.G. W e t z e l . 1980. I n t e r a c t i o n s o f l i g h t a n d n i t r o g e n s o u r c e among p l a n k t o n i c b l u e - g r e e n a l g a e . Arch. H y d r o b i o l . 90: 1-25. W a t e r b u r y , J . B . , S.W. Watson, R.R.L. G u i l l a r d a n d L . E . B r a n d . 1979. Wide-spread occurrence of a u n i c e l l u l a r , marine, p l a n k t o n i c , cyanobacterium. N a t u r e (Lond.) 277: 293294 . Webb, K.L. a n d L.W. Haas. 1976. The s i g n i f i c a n c e o f u r e a f o r p h y t o p l a n k t o n n u t r i t i o n i n t h e York R i v e r , V i r g i n i a , p. 90-102 I n : M. W i l e y ( e d . ) E s t u a r i n e p r o c e s s e s , V o l . 1. A c a d e m i c P r e s s , New Y o r k , N.Y. Whalen, S.C. a n d V. A l e x a n d e r . 1984a. Diel variations i n i n o r g a n i c c a r b o n and n i t r o g e n u p t a k e by p h y t o p l a n k t o n i n an a r c t i c l a k e . J . P l a n k t o n R e s . 6: 571-590. Whalen, S . C a n d V. A l e x a n d e r . 1984b. Influence of t e m p e r a t u r e a n d l i g h t on r a t e s o f i n o r g a n i c n i t r o g e n t r a n s p o r t by a l g a e i n an a r c t i c l a k e . Can. J . F i s h . A q u a t . S c i . 41: 1310-1318. Whalen, S . C a n d V. A l e x a n d e r . 1986. S e a s o n a l i n o r g a n i c c a r b o n a n d n i t r o g e n t r a n s p o r t by p h y t o p l a n k t o n i n an arctic lake. C a n . J . F i s h . A q u a t . S c i . 43: 1177-1186. W h e e l e r , P.A. 1983. P h y t o p l a n k t o n n i t r o g e n m e t a b o l i s m , p.309-346. I n : E . J . C a r p e n t e r a n d D.G. Capone ( e d s . ) N i t r o g e n i n t h e marine environment. A c a d e m i c P r e s s , New Y o r k , N.Y.  221  W h e e l e r , P.A. a n d D.L. K i r c h m a n . 1986. U t i l i z a t i o n o f i n o r g a n i c a n d o r g a n i c n i t r o g e n by b a c t e r i a i n m a r i n e systems. L i m n o l . O c e a n o g r . 31: 998-1009. W h e e l e r , P.A., P.M. G l i b e r t a n d J . J . M c C a r t h y . 1982. Ammonium u p t a k e a n d i n c o r p o r a t i o n by C h e s a p e a k e Bay phytoplankton: Short-term uptake k i n e t i c s . Limnol. O c e a n o g r . 27: 1113-1128. W h e e l e r , P.A., R . J . O l s o n a n d S.W. C h i s h o l m . 1983. E f f e c t s o f p h o t o c y c l e s a n d p e r i o d i c ammonium s u p p l y on t h r e e marine phytoplankton s p e c i e s . I I . Ammonium u p t a k e a n d assimilation. J . P h y c o l . 19: 528-533. W i l l i a m s , P . J . L . a n d L.R. M u i r . 1981. D i f f u s i o n a s a c o n s t r a i n t on t h e b i o l o g i c a l i m p o r t a n c e o f m i c r o z o n e s i n t h e s e a , p . 209-218. I n : J . C . J . N i h o u l (ed.) Ecohydrodynamics. E l s e v i e r O c e a n o g r a p h y 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 Y o r k . W i l l i a m s , S.K. a n d R.C. Hodson. 1977. low c o n c e n t r a t i o n s i n Chlamydomonas B a c t e r i o l . 130: 266-273.  Transport of urea a t reinhardii. J.  Wood, E.D., F . A . J . A r m s t r o n g a n d F.A. R i c h a r d s . 1967. D e t e r m i n a t i o n o f n i t r a t e i n s e a w a t e r by cadmium-copper reduction to n i t r i t e . J . Mar. B i o l . A s s . U.K. 47: 2331. Yin,  K. 1988. S h o r t - t e r m i n t e r a c t i o n between n i t r a t e a n d ammonium u p t a k e f o r c e l l s o f a m a r i n e d i a t o m grown u n d e r d i f f e r e n t degrees o f 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 o f B r i t i s h Columbia, V a n c o u v e r , B.C., 106 p .  Z a r , J.H. Inc.,  1974. B i o s t a t i s t i c a l a n a l y s i s . E n g l e w o o d C l i f f s , N . J . , 620 p .  Prentice-Hall  Z a r , J . P . , P.G. F a l k o w s k i , J . F o w l e r a n d D.G. Capone. 1988. C o u p l i n g between ammonium u p t a k e a n d i n c o r p o r a t i o n i n a marine diatom: Experiments with the s h o r t - l i v e d radioisotope N. L i m n o l . O c e a n o g r . 33: 518-527. 1 3  222 APPENDIX 1 E q u a t i o n s used t o c a l c u l a t e  Once the percentage o f particulate material  (  N) s  1 5  N  uptake  rates,  1 5  N  ( s p e c i f i c a c t i v i t y ) i n the  has been e x p e r i m e n t a l l y determined  by e m i s s i o n spectrometry (procedures reviewed by F i e d l e r and Proksch, 1975;  H a r r i s o n , 1983a) the a p p r o p r i a t e e q u a t i o n t o  c a l c u l a t e n i t r o g e n uptake must be chosen t o correspond w i t h the experimental p r o t o c o l employed. The atom %  1 5  N  excess (  1 5  N  X S  ) o f the p a r t i c u l a t e m a t e r i a l  i s c a l c u l a t e d by s u b t r a c t i n g the n a t u r a l abundance o f  1 5  N  (F)  which i s g e n e r a l l y taken as ca. 0.365% ( n a t u r a l atmospheric enrichment o f  15  N)  f o r f i e l d samples  d i r e c t l y i n culture  1 5 N  XS  =  1 5 N  S  "  and can be measured  samples:  (!)  F  The s p e c i f i c uptake r a t e  (N taken up per u n i t p a r t i c u l a t e  N)  i s c a l c u l a t e d as V^. and a r i s e s from a c o n s t a n t t r a n s p o r t model based on the i s o t o p i c r a t i o o f the p a r t i c u l a t e sample taken a t the end o f the i n c u b a t i o n : 15  v  t  =  N  (R - F ) • T  (2)  where T i s the i n c u b a t i o n time and R i s the i n i t i a l atom % enrichment o f the N s u b s t r a t e c a l c u l a t e d as  223 100-[(S^-A + S F ) / ( S ^ a  + S )] a  where  i s the concentration of  added s u b s t r a t e , A t h e s p e c i f i c a c t i v i t y o f t h e i s o t o p e (always 99 atom % i n p r e s e n t c o n c e n t r a t i o n o f unenriched and  accurate determination  s t u d y ) , and S N substrate.  absolute  t h e ambient  Careful  chemistry  o f ambient N c o n c e n t r a t i o n a r e  e s s e n t i a l f o r an a c c u r a t e d e t e r m i n a t i o n The  a  (or transport) r a t e  o f R. ( ) 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 o f V^. and t h e PON c o l l e c t e d a t t h e end o f t h e incubation period  p  t  = V  (PONj.).:  • PON  t  Another equation  (3)  t  f o r the c a l c u l a t i o n o f s p e c i f i c uptake r a t e  (Vj.) a r i s e s from t h e c o n s t a n t  t r a n s p o r t model based on PON  c o n c e n t r a t i o n c o l l e c t e d at the beginning (P0N ):  period  Q  15 V  o  N  TJ—  -  (R - H ) ib  s  (4)  •T  A l t e r n a t i v e l y an e q u i v a l e n t e x p r e s s i o n denominator w i t h rate p  of t h e i n c u b a t i o n  Q  [(R - F) - ~ N 5  x g  ]  • T.  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  Po " o V  *  P 0 N  replaces the The a b s o l u t e of V  Q  by PON :  o  S i n c e both e q u a t i o n s  uptake Q  (5)  2 and 4 a r e d e r i v e d from a c o n s t a n t  t r a n s p o r t model they do not a l l o w  f o r changes i n PON  224  concentration yielding  an  during  c o u r s e of  underestimate  mean s p e c i f i c D u g d a l e and achieve  the  (V )  and  t  uptake rate during  Wilkerson  a better  the an  the  incubation,  thus  overestimate  (V^)  incubation  (1986) s u g g e s t t h e  estimate  i s t o use  the  of  the  period.  most o b v i o u s way mean o f  the  to  two  values:  V  The  m  =  ( o v  constant  •  In  the  equivalent  t o the  can  are  of  1987). rate  estimated  F)  (  1  -  R  [(R  5  n x  s  to  the  cell  sum V  of  can  c  be  :  - F)  unlabelled N  result the  * N-labelled 5  (R -  be  - ^ N  substituted with  5  X S  shown i n h i s F i g . 3  uptake of  uptake r a t e  transport  equally  ].  Equation  u p t a k e r a t e c a l c u l a t e d as  (1987) and  incubation  Lund,  as  i s c o n t r i b u t i n g and  d e n o m i n a t o r can  expression  the  contributes  e a c h new  (6)  equivalent  The  u p t a k e model assumes t h a t  as:  Alternatively  Collos  (5')  1 2  incubation  = T  c  t>  each p r e - e x i s t i n g c e l l  calculated  V  V  specific  added d u r i n g u p t a k e as  +  forms d u r i n g  i n a reduction  ^N-labelled  (P^)  o  r  from the  3 y i e l d s an t  n  e  of  the  6 is  In(PON /PON )/T t  Q  labelled  same sample  in  8'). the  course  nitrogen  t a k e n up  (Collos,  accurate  estimate  of  compound  since  and  ( D u g d a l e and  V  t  of  an  specific  compound d e t e r m i n e d when  compound i s b e i n g  Equation  (Eq.  the  only  1987;  PON  Wilkerson,  t  225  1986;  Collos,  unlabelled  estimates  ( p j j are  disappearance  15 N V  o  The  =  dilution  over  15  compensated  of  +  compensated  the  of  the  uptake  for V ,  transport rate  of  provided  Q  for unlabelled  ( i . e . ambient n u t r i e n t  sum  r Pi  r  i  L PON J  constant  f o r uptake R  -  R  -  of  15 N  •  - R  Q  f o r the  1  effect  time):  T  equation  be  available  xs  (R  The  s u b s t r a t e can  independent sources  1987).  specific  uptake  xs  -  (V) 1 5  N  model  unlabelled N i s :  F  sum  •  T  In T  It  should  equation curly as and  be  noted  parenthesis  of  estimates  N  that  i s incorrectly  shown a b o v e . 11  1 5  Both  Dugdale of  concentration incubation.  V  should  and  J  i n Dugdale written; not  be  equations Wilkerson,  according added  s  (8  PON,  to the  and  the  Wilkerson  placement  placed 7 and 1986)  before 8  (1986)  of the  the  Eq.  increased  p r o p o r t i o n of  from u n l a b e l l e d sources  left  In term  (equivalent to yield  this  initial  during  the  PON  but 10  226  APPENDIX Growth-irradiance Objective:  The  irradiance necessary  o f Micromonas  e x p e r i m e n t was  curve  f o r M.  pusilla.  conducted  pusilla  Methods:  to obtain  f o r determination  t o s u s t a i n maximal growth  utilization  in  curve  2  o f M.  50 m l b o r o s i l i c a t e  pusilla  were  glass test  tubes with  teflon-lined  section  techniques  of Chapter work;  3.  Sterile  examination.  15 L - E - m - s - 2  • Continuous tubes and  • light  filtered  attenuated  Incident  was  quantum meter  - 1  (Lambda  blue  was  calibrated with  4rc s e n s o r  placed  ca.  • • R by V i t a - L i t e UHO • R  Plexiglas  by (± 34,  fluorescent  ( N o . 2 0 6 9 , Rohm &  and n e u t r a l d e n s i t y  measured w i t h  a L i C o r model L I a 2n  collector  fluorescence)  by i n s e r t i n g  10 f l u o r o m e t e r ,  mixing  185 and t h e  QSL-100 tubes.  w e r e made a t  the whole tube after  Haas)  screening.  a B i o s p h e r i c a l Instruments  of biomass ( i n v i v o  Designs model  inversions.  confirmed  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  12 o r 24 h i n t e r v a l s  Turner  f o ra l l  145, 120, 89, 71, 55, 44,  Instruments) with  screening  Determinations  Culturing  (2 - 13 c u l t u r e s p e r P P F D ) .  by d i s t a n c e  irradiation  only  caps.  A l l c u l t u r e s w e r e g r o w n a t 17°C  • provided  through  i n the  were used  t h e a b s e n c e o f b a c t e r i a was  0.5°C) a t t h e f o l l o w i n g P P F D s : 19 a n d  nitrogen  g r o w n i n 40 m l o f m e d i u m  are described  27,  o f t h e PPFD  i n subsequent  The medium and i t sp r e p a r a t i o n  microscopic  growth-  studies.  Cultures  culturing  a  into  a  by m u l t i p l e  227  Cultures were was  were t r a n s f e r r e d p r i o r  never  nutrient-limited.  obtained  between over  per  each  the  t r a n s f e r by  successive  4-6  day  where  F-^  and  F  are  2  One  =  calculating  Growth  In  depletion  estimate  measure of  period.  u  to N  the  (u)  (F /F )/(t -t 1  fluorescence  2  at  time  that  the  they  growth  growth  fluorescence  rates  2  of  so  rate  rate  and  averaging  were c a l c u l a t e d as:  1  )  1  (tj_) and  time  2  (t ), respectively. 2  Results  and  Conclusions:  calculated plotted  from  against  Specific  increases  growth  in in vivo  PPFD i n F i g u r e  A.l.  rates  (d "*") w e r e -  fluorescence These  and  results  indicate  2  that no  growth  pusilla  saturated  p h o t o i n h i b i t i o n apparent  ^E*m~ • s 2  would as  o f M.  be  _ 1  .  I t was  employed  i t was  difficulty 14:10  )  at  concluded  <100  the  that  and  continuous  l i g h t - d a r k (Chapter  4)  uE'm~  greatest a  the  could  light  experimental  be  1  *s  ,  with  PPFD e x a m i n e d  PPFD o f  e x c l u s i v e l y throughout  s a t u r a t i n g f o r growth i n both  the  at  are  120  JJE-m*" • s 2  current  3)  chambers.  -  1  research  achieved  (Chapter  (145  and  without the  228  Figure A . l . S p e c i f i c growth rate (u) i n d as a function of PPFD f o r 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 visible. - i  1 .00 h "O  0.80 -  =1 t— <  0.60  o  0.40 h  cr: O  0.20  0  30  60  90  PPFD ( /j,E • m  120 2  • s ) 1  150  APPENDIX 3  Comparison  of the increases  concentration  Objective: vivo  during  This  growth  exponential  experiment  fluorescence  i n in vivo  could  growth  Cultures  according  to the conditions  Culturing  s e c t i o n of Chapter  were c o l l e c t e d  cell pusilla.  t o determine  routinely  o f b a t c h c u l t u r e s o f M.  and  o f Micromonas  was c o n d u c t e d  be used  Methods:  fluorescence  i f i n  t o monitor the  pusilla.  o f M. pusilla  were grown i n d u p l i c a t e  and procedures 3.  outlined i nthe  A t 12 h i n t e r v a l s  f o rdetermination  of both c e l l  samples  concentration,  p  with  a Coulter  population  Counter  m o d e l TA I I e q u i p p e d  accessory, and i n v i v o  Designs  model  10 f l u o r o m e t e r  Methods  f o radditional  were c a l c u l a t e d between each fluorescence  and c e l l  fluorescence,  (see Chapter  details).  successive  time  ( t ) and time  the  2  mean  Results  1  2  a n d F-^ a r e t h e f l u o r e s c e n c e 2  Turner  growth  rates  (d "^) -  measure o f  -  t) x  or cell  concentration at  1 (t-jj , r e s p e c t i v e l y and a r e r e p o r t e d  ( n = 2 ) ± 1 S.D.  & Conclusions:  a  concentration as:  2  2  with  3, M a t e r i a l s a n d  Specific  u = In (F /F )/(t  where F  with the  as  of duplicate cultures.  The i n c r e a s e s  i n i n vivo  fluorescence  230  and  cell  plotted  concentration i n Figure  A.2.  o f M. pusilla At time  as a f u n c t i o n of time a r e  zero  ( t = 0) t h e N 0 ~  concentration  i n the c u l t u r e s averaged  and  t o < 1.0 L!g-at N * L ^  decreased  exponential N-replete and and  growth  after  90 a n d 95 h o f  c o n d i t i o n s t h e growth r a t e , averaged  that  M. pusilla  ( n = 2) f r o m i n v i v o  that  i n vivo  cell  growth  degree of accuracy.  fluorescence o f M. pusilla  c a n be used  During  0.835 ± 0.011 fluorescence  This  b o t h methods measure t h e i n c r e a s e  to a similar  -  -  count measurements, r e s p e c t i v e l y .  indicates  NC^  5 0 . 1 ± 0.5 / j g - a t N-L "'"  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 .  0.832 ± 0.001  cell  monitor  -  +  3  result i n biomass of  I t was  decided  to accurately  measure  and w o u l d be employed t o r o u t i n e l y  t h e growth of batch  cultures prior  to  experimentation.  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 grown on NC^ u n d e r s a t u r a t i n g PPFD. Semi-log plots of 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 ) and c e l l concentration (•,•) versus time. -  232 APPENDIX  Comparison inorganic  of  the  rates  nitrogen  Two  particulate  organic  NO3  over time,  + N02~  measures of  efficiency  of  N  estimate  and  loss  thus during  the  Duplicate  according  to  Culturing  s e c t i o n of  Details are  the  described  Results the  of  these  by  intervals  the  production  of  disappearance  and  low  At  12  N  (DON)  were  grown  outlined  in  the  intervals,  50  ml  (2  vacuum f i l t r a t i o n Whatman GF/F with  procedure  The  increase -  Hg) for  Equipment  filtrate  was R  and  analytical  Methods of  disappearance  i n PON  NO3  mm  filters  a Control The  80  a Technicon AutoAnalyzer  M a t e r i a l s and  presented  pusilla  h  analyzer.  with  organic  pusilla.  procedures  3.  of  concentration  uptake  i n Figure  of  the  •  + NO3  measures of are  and  dissolved organic  —  & Conclusions:  two  production  to particulate  c u l t u r e s o f M.  Chapter  filtration  medium, t h e  of  concentration  i n the  the  g r o w t h o f M.  240-XA e l e m e n t a l  f o r NO3  and  (460°C f o r 4 h)  PON  uptake,  inorganic N  conditions  —  analyzed  of  batch  samples were c o l l e c t e d  measurement of  (PON)  extent  exponential  onto precombusted  nitrogen  were compared t o determine  conversion  the  nitrate  nitrogen  Methods:  Corp. model  particulate  disappearance.  Objectivei  -  of  4  over A.3.  II.  techniques  Chapter NO3  -  and  successive  +  3.  N02~  the  from  ratio  of  sampling  These r e s u l t s  indicate  233  that  during  neither less of  measure  o f NO3  than the other; t o N0 ~  PON  for  the period  3  of N-replete  the ratio  2  o f t h e change  averaged  0.96  c u l t u r e 1 a n d 2, r e s p e c t i v e l y .  there  was no s i g n i f i c a n t  disappearance  a n d PON  (0-ca.  90  h)  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  -  + N0 ~  growth  i n concentration  (± 0 . 0 8 ) a n d 0.97  (±  In both N-replete  cultures  o f N0 "  difference i n rates  accumulation  or  +  3  (paired t-test,  0.13)  NO2""  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 w h e n t h e cells  became N - l i m i t e d  average  o f 54% o f t h e n i t r a t e  accounted during NO2""  f o r i n t h e PON.  conditions  Past  o f DON  estimates  phytoplankton  e x p e r i m e n t s may  Fuhrman  and B e l l ,  passage  (e.g.,  filtration.  suggest  i n similar  excretion typically  cells  Sharp,  ranged  3  from  1977).  and Dennett,  20-40%  However, i t i n these  breakage 1985) o r  et a l . , i n press)  +  i n PON  and 5-10% i n  e x c r e t i o n measured  L i , 1986; S t o c k n e r  i n N0 "  t o t h e medium.  have r e s u l t e d from c e l l  1985; Goldman  only  increases  i n N-limited cells (e.g.,  was  that  the decreases  e x c r e t i o n o f DON  p o s s i b l e t h a t m u c h o f t h e DON  early  f r o m t h e medium  These r e s u l t s  the assimilated nitrogen  healthy is  removed  an  -  3  f r o m t h e medium n o t r e f l e c t e d suggesting  + NO2 )  -  o f N - l i m i t a t i o n were  concentration,  of  N-L -'- o f N0 ~  (< 1 uq-at  (e.g., cell  during  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. Accumulation of particulate organic nitrogen. 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 from NO3 + N02~ d i s a p p e a r a n c e f r o m t h e medium. Nitrogen concentrations are p l o t t e d a g a i n s t elapsed time measured a f t e r c u l t u r e i n i t i a t i o n ; uptake r a t e r a t i o s are p l o t t e d against average elapsed time between successive sampling periods. -  -  -  0.4  1  0  •  '  20  •  '  •  •  40  60 TIME  (h)  •  '  80  .  1  100  235 APPENDIX 5  Dissolved o f M.  inorganic nitrogen disappearance  To d e t e r m i n e i f n i t r i t e  c u l t u r e medium by M. pusilla,  Methods:  A batch  constant  blue  conditions  analyzed  light  approaches  filtered  (NO3 ) a n d n i t r i t e -  AutoAnalyzer  respective ;  N02~  analytical  N0 ":  limits  uq-at  & Discussion:  s a m p l e s were  (NO2 ) w i t h a -  N-L  Sampling c o n t i n u e d  and N C ^  -  of d e t e c t i o n - 1  outlined  -  reached  (NO3 : 0.03 -  declined  f r o m 5.76 pq-at  concomittant  decreases  a n d NO2".  was o b s e r v e d  c o n c e n t r a t i o n o f N0 " 3  N-L  o f NO3  - 1  +  t o <0.05 j j g - a t N - L  -  and  Over a  NO2"" - 1  with  i n t h e ambient c o n c e n t r a t i o n o f both  No e v i d e n c e  with  uq-at  ).  The a m b i e n t c o n c e n t r a t i o n s  o f 4.5 h t h e t o t a l  until  their  a r e p l o t t e d as a f u n c t i o n o f t i m e i n F i g u r e A.4.  period  NO3-  0.02  2  Results  o f NO3  concentrations  3.  t o the s e c t i o n of  II following the techniques  i n Wood e t a l . (1967) and C h a p t e r ambient  was grown, u n d e r  o u t l i n e d i n t h e Culturing  A t 30 min i n t e r v a l s ,  as t h e  zero.  a t a s a t u r a t i n g PPFD, a c c o r d i n g  for nitrate  Technicon  g r o w t h on n i t r a t e ,  c u l t u r e o f M. pusilla  and p r o c e d u r e s  C h a p t e r 3.  - 1  growth  i s released into the  during  ambient c o n c e n t r a t i o n o f n i t r a t e  N-L  during  pusilla.  Objective:  the  curve  o f NO2  i n c r e a s i n g NO3  -  -  e x c r e t i o n by M.  limitation.  pusilla  The a m b i e n t  236  concentration ug-at N0 ~  N03 'L ^ -  -  of N0 ~ i n five 2  enriched  + N0 ~ concentration  which  i s consistent with  during  the first  pusilla, Thalassiosira al.,  ( 0 . 2 0 ± 0.07 L i g - a t the i n i t i a l  hour of sampling.  unlike other  phytoplankton  pseudonana  20 L r e s e r v o i r s o f 50  ESAW a v e r a g e d < 0 . 0 5 % o f t h e t o t a l  3  2  separate  (e.g.,  values  species  PPFD.  reported  _ 1  ,  n = 5)  here that  M.  such as  e t a l . , 1980; Parslow  1984b), does n o t r e l e a s e N 0 ~ d u r i n g  saturating  2  I t i s concluded  Olson 2  N0 ~-L  growth  et  on N03~ under  237  F i g u r e A.4. D i s s o l v e d NO3 and NO?" c o n c e n t r a t i o n c u l t u r e medium, d u r i n g batch growth of M. p u s i l l a , a g a i n s t time of sampling. -  120 TIME ( min  180 )  240  i n the plotted  300  238  APPENDIX 6  Precision  The  of a n a l y t i c a l  precision  throughout  this  techniques.  of the a n a l y t i c a l  techniques  employed  s t u d y a r e r e p o r t e d a s t h e mean c o e f f i c i e n t o f  variation  ( C V . = S.D./x  collected  and p r o c e s s e d a c c o r d i n g t o t h e p r o c e d u r e s  in  Chapters  (i.e.  1-4.  filtration)  • 100) o f r e p l i c a t e  The e s t i m a t e d e r r o r and a n a l y t i c a l  "1 c  excess  (  N  i n c l u d e s both  errors,  except  outlined collection  i n t h e atom %  1 c e x  )  measurements o f  N i n particulates.  d u p l i c a t e measurements were d e t e r m i n e d sample  s a m p l e s (n)  (collected  by f i l t r a t i o n ) ,  a n a l y z e d by e m i s s i o n s p e c t r o m e t r y  Measure  Here  f r o m t h e same o r i g i n a l  but evacuated,  combusted and  s e p a r a t e l y (n = 150 p a i r s ) .  Number o f R e p l i c a t e s (n)  C o e f f i c i e n t of Variation  (%) N0 ~  + N0 ~  2-3  1.2  2-3  1.5  2  0.3  2  4.4  POC  2-3  5.2  PON  2-3  3.8  2  3.5  2  1.5  3  NH  2  + 4  Urea Chi  a  cell 1  5  N  e  concen. x  Refereed  Publications  Parsons, T.R., H.M. Dovey, W.P. Cochlan, R.I. P e r r y and P.B. Crean. 1984. F r o n t a l zone a n a l y s i s a t the mouth of a f j o r d - J e r v i s I n l e t , B r i t i s h Columbia. S a r s i a 69.: 133137. P r i c e , N.M., W.P. Cochlan and P.J. H a r r i s o n . 1985. Time course of uptake of i n o r g a n i c and o r g a n i c n i t r o g e n by phytoplankton i n the S t r a i t of G e o r g i a : comparison of f r o n t a l and s t r a t i f i e d communities. Mar. E c o l . Prog. Ser. 27.: 39-53. Cochlan, W.P. 1986. Seasonal study of uptake and r e g e n e r a t i o n of n i t r o g e n on the S c o t i a n S h e l f . S h e l f Res. 5: 555-577.  Cont.  Cochlan, W.P., P.J. H a r r i s o n , P.A. Thompson and T.R. Parsons. 1986. P r e l i m i n a r y o b s e r v a t i o n s of the summer p r o d u c t i o n of t h r e e B r i t i s h Columbian c o a s t a l i n l e t s . S a r s i a 71 :• 161-168. H a r r i s o n , 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 e f f e c t s of crude o i l and C o r e x i t 9527 on marine phytoplankton i n an experimental e n c l o s u r e . Mar. E n v i r . 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 c o n t r i b u t i o n of phytoplankton t o ocean d e n s i t y g r a d i e n t s . Deep-Sea Res. 3_6: 1277-1282. Stockner, J.G., M.E. K l u t and W.P. Cochlan. (In press) Leaky f i l t e r s : a warning t o a q u a t i c ecologists'. Can. J . F i s h . Aquat. S c i . 47.  

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