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Short-term interaction between nitrate and ammonium uptake for cells of a marine diatom grown under different… Yin, Kedong 1988

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SHORT-TERM INTERACTION BETWEEN NITRATE A N D A M M O N I U M UPTAKE FORCELLS OFA MARINE DIATOM GROWN  UNDER  DIFFERENT D E G R E E S O F LIGHT LIMITATION by KEDONG YIN B . S c , T h e Shandong College of Oceanography, C h i n a , 1982  A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E REQUIREMENT FORT H E D E G R E E O F MASTER O F SCIENCE in T H E F A C U L T Y O F G R A D U A T E STUDIES (DEPARTMENT OF OCEANOGRAPHY)  W e accept this thesis as conforming to the required standard  The U N I V E R S I T Y O F B R I T I S H C O L U M B I A January 1988 © K e d o n g Y i n , 1988  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 The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6(3/81)  tAo^rd 3 .  11  ABSTRACT  T h e short-term interaction between nitrate and a m m o n i u m uptake was examined for a marine diatom,  Thalassiosira pseudonana, grown i n the continuous  turbidostat cultures under different degrees of light limitation. Nitrate uptake in the absence and i n the presence of a m m o n i u m and a m m o n i u m uptake in the absence and i n the presence of nitrate were measured during a 6 m i n time course after a solution of both nitrate and a m m o n i u m was passed across the cells trapped on the filter. It was found that the interaction between nitrate and a m m o n i u m uptake occurred immediately and continued for the remainder of the time course. T h e effect of light o n the interaction was apparent. In the less light-limited cultures, nitrate uptake was depressed by a m m o n i u m . In contrast, in the most light-limited culture, the depression of nitrate uptake by a m m o n i u m disappeared. A m m o n i u m uptake was dependent o n the degree of light limitation of the cultures. F o r all the cultures, a m m o n i u m uptake was initially enhanced and then declined with time. However, only the initial (1 min) enhanced uptake of a m m o n i u m was supressed by the presence of nitrate, and the subsequent a m m o n i u m uptake rate was unaffected. Possible explanations and the ecological significance of the interaction are discussed.  iii TABLE O F CONTENTS  ABSTRACT.  ii  LIST O F  TABLES  LIST O F F I G U R E S  ACKNOWLEDGEMENT  INTRODUCTION  viii  ix  xiv  1  Light and nutrients in the ocean  1  Interaction between light and nitrogen  2  E c o l o g i c a l significance of the interaction among nitrate, a m m o n i u m and irradiance  2  Laboratory studies on the interaction among nitrate, a m m o n i u m and irradiance  5  1) Nitrate and a m m o n i u m interaction at saturating light intensity  6  2) Nitrate and a m m o n i u m interaction under light limitation  8  iv  MATERIALS A N DMETHODS Organism  10 10  Culture m e d i u m  10  Culturing  10  Nitrogen analysis  11  Nutrient U p t a k e measurement  12  RESULTS  19  Cultures  19  Interaction experiments  19  A . T i m e course of the interaction between nitrate and a m m o n i u m  uptake  19  1) Nitrate uptake with and without a m m o n i u m  19  2) A m m o n i u m uptake with and without nitrate  23  B . Effects of growth irradiance on the interaction between nitrate and a m m o n i u m uptake  41  1) Nitrate uptake with and without a m m o n i u m  41  2) A m m o n i u m uptake with and without nitrate  54  DISCUSSION  69  Cultures  69  Interaction experiments  70  A . T i m e course of the interaction between nitrate and a m m o n i u m  uptake  71  1) Nitrate uptake with and without a m m o n i u m  72  2) A m m o n i u m uptake with and without nitrate  73  B . Effects of growth irradiance on the interaction between nitrate and  a m m o n i u m uptake  77  1) Nitrate uptake with and without a m m o n i u m  77  2) A m m o n i u m uptake with and without nitrate  80  E C O L O G I C A L SIGNIFICANCE  81  vi  SUMMARY  87  L I T E R A T U R E CITED  89  APPENDIX  96  APPENDIX A. STANDARD ERRORS ASSOCIATED WITH T H E REPLICATION OF NITRATE U P T A K E R A T E M E A S U R E M E N T S FOR DIFFERENT C U L T U R E S  A . l . Culture LO  96  '.  97  A.2. Culture L2  98  A. 3. Culture L3  99  APPENDIX B. VARIATION OF SOME PARAMETERS IN T H E TURBIDOSTATS O V E R A FEW DAYS  100  B. l . Variation of specific growth rate (h"^) based on biovolume (um3 ml"*) •.  101  B.2. Variation of cell density  102  B.3. Variation of biovolume  103  vii  B.4. Variation of particulate nitrogen (uM)  104  B.5. Variation of fluorescence per cell  105  A P P E N D I X C. V A R I A T I O N O F N I T R A T E U P T A K E R A T E O V E R A 2-4 TIME INTERVAL F R O M T H E TIME COURSES  106  viii  LIST OF TABLES  Table I. Outline of experiments. The nitrogen source and the irradiance used to grow steady state cultures are indicated. The additions of NO3 or NH4 singly or in combination to these steady state cells are shown  17  Table II. Standard errors (SE) of the repeated measurements of NO3 uptake rate (mmole (liter cell volume)" * h"*, averaged over 2-4 min in the time course) for the different cultures. For cell number loaded onto the filter each loading and NO3 uptake rate data during each pulse, see appendix A: Al, A2and A3  18  Table III. Steady-state parameters for the turbidostat cultures of Thalassiosira pseudonana representing the starting conditions for the uptake experiments. For each parameter, see Appendix B: Bl, B2, B3, B4, B5 and B6  22  Table IV. Relative enhancement of NH4 uptake rate at 1 min compared to 5 min (i.e. v  l m i n  /V  5 m i n  ) in the presence or absence of NO3  40  ix LIST O F F I G U R E S  Figure 1. A . Apparatus used to measure uptake by cells trapped on an in-line filter (F). Three-way valves  and Vg are used to switch among wash water (W),  culture sample ( C ) a m d a standard solution (S). A four-way valve (VQ) is used to switch between baseline water (B) and the culture sample ( C ) when loading cells onto the filter. B . Cross section through in-line filter assembly. T h e filter is denoted by the dashed line  Figure 2.  14  Specific growth rate vs irradiance for batch cultures. T h e vertical bars represent  +  1 standard error (n > 5)  20  Figure 3. Light saturated culture (150 u E m " s"*) LO: time course of N O 3 uptake 2  in the absence of NH4 (0) and i n the presence of different N H 4 concentrations: 0.25 u M (+), 0.5 u M (a), 1.0 u M (x), 3.0 u M (A) and 5.0 uM  (o)  24  Figure 4. Light limited culture (17 u E m " s"*) L I : time course of N O 3 uptake in 2  the absence of NH4 (O) and in the presence of different N H 4 concentrations: 0.25 u M (+), 0.5 u M (•), 1.0 u M (x), 3.0 u M (A) and 5.0 uM  (O)  26  Figure 5. Light limited culture (9 u E m " s" ) L 2 : time course of N O 3 uptake in the 2  1  absence of N H 4 (0) and in the presence of different NH4 concentrations: 0.25 u M (+), 0.5 u M (•), 1.0 u M  (x), 3.0 u M (A) and 5.0 u M (O). 28  Figure 6 . Light limited culture (2 u E m " s  ) L 3 : time course of N O 3 uptake i n the  z  absence of N H 4 (0) and i n the presence of different N H 4 concentrations: 0.25 u M (+), 0.5 u M (•), 1.0 u M (X), 3.0 u M (A) and 5.0 u M (O)  30  Figure 7. Light saturated culture (150 u E m"^ s"*) L 0 : time course of N H 4 uptake i n the absence of N O 3 ( A ) and i n the presence of N O 3 (B) at different NH  4  concentrations: 0.25 u M (A), 0.5 u M (+), 1.0 u M (•), 3.0 u M (x) and  5.0 u M (O)  32  Figure 8. Light limited culture (17 u E m " s z  ) L l : time course o f N H 4 uptake in  the absence of N O 3 ( A ) and in the presence of N O 3 (B) at different N H 4 concentrations: 0.25 u M (A), 0.5 u M (+), 1.0 u M (•), 3.0 u M (x) and 5.0 u M (O)  34  Figure 9. Light limited culture (9 u E m " s" ) L 2 : time course of N H 4 uptake in the z  1  absence of N O 3 ( A ) and i n the presence of N O 3 (B) at different N H 4 concentrations: 0.25 u M (A), 0.5 u M (+), 1.0 u M (•), 3.0 u M (x) and 5.0 uM  (o)  36  Figure 10. Light limited culture (2 u E m " s z  ) L 3 : time course of N H 4 uptake in  the absence of N O 3 ( A ) and in the presence of N O 3 (B) at different N H 4 concentrations: 0.25 u M (A), 0.5 u M (+), 1.0 u M (•), 3.0 u M (x) and 5.0 u M (O)  Figure 11. Light saturated culture (150 u E m"^ s"*) L 0 : time course of the interaction between N O 3 uptake and N H 4 uptake after a pulse of a  38  xi solution containing 5.0 u M NO3 (O) and 3.0 u M NH4 (+), compared to the uptake of the controls: 5.0 u M NO3 alone (A) and 3.0 u M NH4 alone (•) (from Figs. 3 and 7)  42  Figure 12. Light limited culture (17 u E m " s" ) L l : time course of the interaction z  A  between NO3 uptake and NH4 uptake after a pulse of a solution containing 5.0 u M NO3 (A) and 3.0 u M NH4 (+), compared to the uptake of the controls: 5.0 u M NO3 alone (O) and 3.0 u M NH4 alone (•) (from Figs. 4 and 8)  44  Figure 13. Light limited culture (9 u E m"^ s"*) L 2 : time course of the interaction between NO3 uptake and NH4 uptake after a pulse of a solution containing 5.0 u M NO3 (o) and 3.0 u M NH4 (-f),  compared to the uptake  of the controls: 5.0 u M NO3 alone (A) and 3.0 u M NH4 alone (•) (from Figs. 5 and 9)  46  Figure 14. Light limited culture (2 u E m " s z  ) L 3 : time course of the interaction  between NO3 uptake and NH4 uptake after a pulse of a solution containing 5.0 u M NO3 (O) and 3.0 u M NH4 (+), compared to the uptake of the controls: 5.0 u M NO3 alone (A) and 3.0 u M NH4 alone (•) (from Figs. 6 and 10)  48  Figure 15. NH4 uptake at 3.0 u M concentration in the absence of NO3 ( A ) and in the presence of NO3 (B) for the cultures grown at different irradtances: L 0 (•), L l (+), L 2 (A) and L 3 (O) (from Figs. 7, 8, 9 and 10). 50  xii Figure 16. N H 4 uptake at 5.0 u M concentration i n the absence of N O 3 ( A ) and in the presence of N O 3 (B) for the cultures grown at different irradiances: L O (•), L I (+), 12 (A) and L 3 (O) (from Figs. 7, 8, 9 and 10)  52  Figure 17. A . N O 3 uptake vs growth irradiance in the absence of N H 4 (•) and in the presence of N H at 0.25 u M (A), 0.5 (+), 1.0 u M (•), 3.0 u M (x) and 4  5.0 u M (O). B . N O 3 uptake vs N H 4 concentration for each culture: L 0 (•), L I (+), L 2 (A) and L 3 (O) (replotted from A ) . (The N O 3 uptake rate is averaged over the 2-4 m i n time interval during the time course in Figs. 3, 4, 5 and 6). C . N O 3 uptake rate vs irradiance (in log scale) and N H 4 concentration  55  Figure 18. A . N H 4 uptake vs growth irradiance in the absence of N O 3 at N H  4  concentrations: 0.25 u M (A), 0.5 (+), 1.0 u M (•), 3.0 u M (x) and 5.0 u M (O). B . N H 4 uptake vs N H 4 concentration for cultures grown at four different irradiances: L 0 (•), L I (+), L 2 (A) and L 3 (O) (replotted from A ) . :  58  Figure 19. Light saturated culture (150 u E m " s"^) L 0 : N H 4 concentration effect 2  o n the interaction between N O 3 and N H 4 : N H 4 uptake in the absence of N O 3 (•), N H 4 uptake in the presence of N O 3 (-)-) and N O 3 uptake in the presence of N H (A) (from Figs. 17B and 18B) 4  61  xiii Figure 20. Light limited culture (17 u E m " s"*) L I : N H 4 2  the interaction between N O 3 N O 3  (•), N H 4  and N H 4 : N H 4  uptake in the absence of  uptake in the presence of N O 3  presence of N H 4  (+) and N O 3  2  the interaction between N O 3 (•), N H 4  and N H 4 : N H 4  uptake in the  (A) (from Figs. 17B and 18B)  2  the interaction between N O 3  and N H 4 : N H 4  4  (A) (from Figs. 17B and 18B)  Figure 23. T h e ratio of uptake rate (h"*) of N H 4  65  concentration effect on  uptake in the absence of  uptake in the presence of N O 3  presence of N H  concentration effect o n  (+•) and N O 3  Figure 22. Light limited culture (2 u E m " s"*) L 3 : N H 4  (•), N H 4  63  uptake in the absence of  uptake in the presence of N O 3  presence of N H 4  N O 3  uptake in the  (A) (from Figs. 17B and 18B)  Figure 21. Light limited culture ( 9 u E m " s"^) L 2 : N H 4  N O 3  concentration effect o n  (+) and N O 3  uptake in the 67  alone (avereged over 1-5 min) to  maximal specific growth rate (h"^) at the growth irradiance for the pulses  of N H : .25 u M (A), 0.5 u M (+), 1.0 u M (•), 3.0 u M (x) and 5.0 u M (o)...82 4  xiv ACKNOWLEDGEMENT  I a m very grateful to my supervisor, D r . P . J . Harrison, for his guidance during the whole p e r i o d of my study and research. M y fellow student, P . A . T h o m p s o n , provided valuable practical advice o n the experimental design, techniques and the discussion of the results. I also appreciate the help from other fellow students, W . P . C o c h l a n , G . J . Doucette and P . J . Clifford. I a m thankful for the financial support provided by a scholarship from the International Development Research Center of C a n a d a .  1  INTRODUCTION  Light and nutrients in the ocean  Light and nutrients are two major driving forces i n marine ecosystems. T h e y partially determine primary production and therefore indirectly regulate higher trophic production. However, these parameters vary with time and space in the ocean. T h e r e are diel and seasonal changes in light intensity as well as an attenuation with depth i n the water column. In addition, weather conditions and water turbidity also affect light intensity. Thus, light appears to be one of the most variable factors i n the ocean. Nutrients including nitrogen, phosphorus, silicate, trace metals and vitamins also vary with time and space largely due to biological activities. A l t h o u g h algae require all these nutrients for growth, nitrogen is believed to be the nutrient most likely limiting to primary production in coastal marine waters (Ryther and Dunstan 1971). T h e variation in light and nutrients with time and space results in variable phytoplankton production because light and nutrients can both limit algal production. Light affects both photosynthesis and nutrient uptake. In the upper part of the euphotic zone, light is sufficient for photosynthesis and nutrient uptake, while nutrients often limit primary production. N e a r the bottom of the euphotic zone, light becomes limiting while nutrients are generally present i n saturating concentrations. F o r example, i n field investigations, M a c l s a a c and D u g d a l e (1972) found that near the surface of oligotrophic waters, nitrogen uptake was severely limited by the ambient nitrogen concentration, and maximal nitrogen uptake only occurred deep i n the euphotic zone. In eutrophic areas, however, light, not nutrients, tends to be limiting to primary productivity and the maximal nutrient uptake occurs  2 near the surface. Similarly, observations made by Nelson and Conway (1979) in the Baja Californian and northwest A f r i c a n upwelling systems, indicated that the light regime was more important than the ambient nitrogen concentration in controlling the biological availability of dissolved nitrogen and silicon. Therefore, these two factors, light and nutrients, appear to control primary production i n many cases.  Interactions between light and nitrogen  Frequently in areas of mixing, phytoplankton are growing under transient conditions. D u r i n g these conditions the factors limiting phytoplankton growth can change repeatedly or cause some interaction with each other. Recently, it was reported that light and a nutrient could be simultaneously limiting or they might compensate for each other. R h e e and G o t h a m (1981) investigated the effects of simultaneous limitation of light and nitrate o n growth and found that light and the nitrogen cell quota could compensate for each other in maintaining the growth rate within a certain range. Healey (1985) observed the interacting effects of light and nutrient  (NO3) limitation o n the growth rate of Synechococcus linearis  (Cyanophyceae). H e found that the biomass responded to changes i n both the irradiance a n d the limiting nutrient concentration over a certain range of irradiances, indicating simultaneous light and nutrient limitation. Therefore, it appears that light interacts with nitrogen and controls nitrogen uptake and phytoplankton growth.  Ecological significance of the interaction among nitrate, ammonium and irradiance  Nitrogen can be taken up by phytoplankton i n many different forms including nitrate, nitrite, a m m o n i u m , urea and free amino acids. Some of these  3 forms are preferred over others (e.g. the preference of a m m o n i u m over nitrate). T h e utilization of different forms might affect algal growth, species composition and species succession (Harrison and T u r p i n 1982). It is well known that limiting nutrients have selective roles i n determining species competition and succession depending on the nutrient uptake kinetics of the different species. In a nitrogen-limited environment, a species that is capable of migrating into a high nutrient zone such as the nutricline or into microscale patches would have an advantage over species without such an ability. D i e l vertical migration of dinoflagellates down to the nutricline, for example, is suggested to be one mechanism by which dinoflagellates become dominant in the community. A laboratory study on the interacting effects of light, temperature and nitrogen on the migration of the dinoflagellates  Gonyaulaxpolyedra and Ceratium furca provided  evidence for this mechanism (Heaney and E p p l e y 1981). F o r species which cannot swim, the capacity to quickly take up a transient supply of the nutrient (in a patch) would likely be an advantage. F o r example, there is clear evidence for rapid a m m o n i u m uptake by nitrogen-deficient cultures and in particular, fast-growing diatoms (Conway 1976). Thus, the ability to utilize different forms of nitrogen could be very important in nitrogen nutrition since nitrogen is most likely to be limiting in marine waters. O n e of the most important forms of nitrogen is a m m o n i u m . A m m o n i u m i n seawater can be released by biological activities including zooplankton excretion, bacterial decomposition and schooling fish. Regenerated a m m o n i u m has b e e n found to be so substantial that it accounts for a significant portion of the primary production. F o r example, an investigation made by Conway and Whitledge (1979) during a spring b l o o m i n the New Y o r k Bight showed that measurements of a m m o n i u m utilization as a percentage of a m m o n i u m plus nitrate utilization yielded values of 59% for the inshore areas and 70% at the shelf-break. Generally, the more  4  nitrogen-poor the seawater is, the more primary production is supported by regenerated nitrogen. Reviews by E p p l e y and Peterson (1979) and H a r r i s o n (1980) clearly support this generalization. T h e regeneration of a m m o n i u m is sporadic, but its concentrations in marine waters is low, ranging from 0 to 5 u M . T h e kinetics of a m m o n i u m uptake by phytoplankton partially explain these observations. A laboratory study with the diatom  Biddulphia aurita showed that cells removed a m m o n i u m over twice as fast as  nitrate or nitrite ( L u i and Roels 1972). Morever, an observation on growth under different light intensities using different nitrogen sources (NO3, NO2 and NH4) with blue-green algae showed that a m m o n i u m as a nitrogen source always resulted in the highest growth rates under all light regimes ( W a r d and W e t z e l 1980). T h e preference for a m m o n i u m over nitrate and the rapid uptake of a m m o n i u m by algae have been commonly observed both in field investigations (e.g. M c C a r t h y et al. 1977, Takahashi and Saijo 1981 and Glibert et al. 1982) and laboratory studies when the two forms are present simultaneously in the medium. H a n i s a k and H a r l i n (1978) found that i n some macroalgae, maximum uptake rates of a m m o n i u m were higher and the half-saturation constant for a m m o n i u m was lower than those for nitrate or nitrite. It appears that the interaction of a m m o n i u m with nitrate could cause different growth rates of phytoplankton although the detailed mechanisms are not fully understood. In a nitrogen-limited environment, the preferential uptake of a m m o n i u m would enable the species to compete with species which do not have such an ability because a cell of the capable species need only to be exposed to an intermittent pulse of a m m o n i u m in order to acquire its daily ration of nitrogen for growth ( M c C a r t h y and G o l d m a n 1979). Some laboratory studies have shown evidence for such a possibility. It was observed that phytoplankton cells encountering the microscale patches released by swimming animals, accumulated more labelled  5 phosphate as ^ P - P O ^ than cells that did not enter the patches ( L e h m a n and Scavia 1982). E v e n i n nitrogen saturated waters, algae with such physiological properties are able to take up more nitrogen than other algae over the same time period, resulting i n faster growth. Light conditions could also change the availability of a m m o n i u m , nitrate and other nutrients and therefore change the relative requirements between nitrogen and other nutrients by phytoplankton. W y n n e and R h e e (1986) studied the effects of light intensity and quality on the relative nitrogen and phosphorus requirements (the optimum ratio ),of marine phytoplankton and concluded that changes i n the light regime can strongly influence algal nutrient requirements and species interrelationships by altering the optimum cellular N : P ratio. A n o t h e r study with Cyanophyceae showed that only at low irradiance, the availability of a m m o n i u m and CC>2 controlled the buoyancy of the species (Spencer and K i n g 1985). Therefore, interactions between a m m o n i u m and nitrate uptake under different light conditions could change the nutritional regime of algal cells and as a consequence, cause a change i n the growth rates of the cells. Thus, nutrient interactions under different irradiances can affect phytoplankton distribution, species composition and succession, and ultimately regulate the stability of a marine ecosystem. Therefore, it is necessary to examine the kinetics of the interaction between a m m o n i u m and nitrate uptake under different light intensities.  Laboratory studies on the interactions among nitrate, ammonium and light  Nitrate and a m m o n i u m uptake and the interaction between the two nutrients have been well studied. Basically, the uptake rate of a single limiting nutrient by marine phytoplankton is a hyperbolic function of the limiting nutrient concentration.  6 This hyperbolic function also fits the relationship between light and nutrient uptake (e.g. M a c l s a a c and Dugdale 1972). T h e interaction of a m m o n i u m and nitrate with different irradiances could alter these kinetics, but there are few studies on these interactions.  1) Nitrate and ammonium interactions at saturating light intensity  T h e most c o m m o n interaction reported is the inhibition of nitrate uptake by a m m o n i u m or the preference of a m m o n i u m over nitrate in utilization. Syrett and M o r r i s (1963) found that nitrate assimilation by  Chlorella vulgaris growing with  a m m o n i u m plus nitrate ( N H 4 + N O 3 ) ceased completely when a m m o n i u m was added and commenced again as soon as a m m o n i u m had disappeared. Later, T h a c k e r and Syrett (1972) and Florencio and V e g a (1983) obtained similar results from studies with  Chlamydomonas reinhardi. A study with the blue-green alga  Anabaena cylindrica showed an inhibition of nitrate uptake by a m m o n i u m , where the degree of the inhibition was related to the a m m o n i u m concentration ( O h m o r i et al. 1977). Conway (1977) found a m m o n i u m concentration effects o n nitrate uptake both in a lab study with  Dunaliella tertiolecta and Skeletonema costatum and in a  field investigation. H e also observed that approximately 30 minutes were required for a m m o n i u m to exhibit its full inhibitory effect on nitrate uptake. E v e n the macroalgae and  Gracilaria foliifera and Neoagardhiella baileyi ( D ' E l i a and D e B o e r 1978)  Hypnea musciformis and Macrocystis pyrifera (Phaeophyta) (Haines and W h e e l e r  1978) showed preferential utilization of a m m o n i u m over nitrate, but the presence of nitrate had no effect on a m m o n i u m uptake. Serra et al. (1978) studied nitrate utilization characterized in nitrogen-deficient cells of the diatom  Skeletonema  costatum and observed that a m m o n i u m strongly inhibited carrier-mediated nitrate uptake without affecting diffusion. It was reported that a m m o n i u m at low  7 »  concentrations caused a rapid and effective inhibition of nitrate utilization in the light by the cyanobacterium Anacystis  nidulans (Flores et al. 1980). D o r t c h and  Conway (1984) conducted more comprehensive experiments on the interaction between nitrate and a m m o n i u m uptake. T h e y found that the interaction varied with growth rate, nitrogen source and species. After the simultaneous addition of a m m o n i u m and nitrate to the cultures which were preconditioned on nitrate or a m m o n i u m and grown at different growth rates, they found that both nitrate and a m m o n i u m uptake rates decreased in comparison with the rates observed when each nutrient was added alone, although nitrate uptake decreased more than a m m o n i u m uptake. Simultaneous uptake of both nitrate and a m m o n i u m has been frequently reported for other algae such as a red macroalga cyanobacterium,  Gelidium nudifrons (Bird 1976), a  Oscillatoria agardhii ( Z e v e n b o o m and M u r 1981) and natural  oyster-pond algae composed mainly of diatoms (Maestrini et al. 1982, Robert and Maestrini 1986 and Maestrini et al. 1986). T h e later researchers also reported a m m o n i u m thresholds for simultaneous uptake above which nitrate uptake was still inhibited. However, T o p i n k a (1978) reported that high levels of a m m o n i u m did not inhibit nitrate uptake in the macroalga  Fucus spiralis (Phaeophyceae).  T o date little attention has been paid to the effects of nitrate on a m m o n i u m utilization. Stross (1963) observed a preference for nitrate over a m m o n i u m utilization in the green alga  Haematococcus lacustris. However, he used an indirect  method based on a decrease or an increase in p H of the m e d i u m to determine the preference for a m m o n i u m or nitrate. D e M a n c h e and co-workers (1979) used a nitrogen-deficient batch culture of the marine diatom  Skeletonema costatum and  when it was resupplied with a mixture of nitrate and a m m o n i u m , they observed an initial enhanced nitrate uptake rate leading to a large internal pool of nitrate. Following this initial nitrate uptake event, nitrate uptake ceased, and nitrate  8 »  assimilation was inhibited until the ambient a m m o n i u m was taken up. A t this point, nitrate uptake resumed and nitrate assimilation began. In contrast, H a r r i s o n et al. (1986) observed complete supression for 30 minutes of nitrate uptake before the macroalga Laminaria groenlandica (Phaeophyta) took up nitrate and a m m o n i u m equally after NO3 and NH4 were added together. M o r e recently, Collos et al. (1986) reported a long-term elevated NO3 uptake by oyster-pond microalgae in the presence of high a m m o n i u m concentrations. T h e y found a nitrate uptake maximum at NH4 concentrations between 10-30 u M and the maximum coincided either with the maximum NH4 uptake rate or with the start of a decrease in NH4 uptake rate. T h e n NO3 uptake decreased and went through a secondary maximun at lower NH4 concentrations.  2) Nitrate and ammonium interactions under light limitation  T o date most of the studies have focused on nitrogen limitation with only a few studies on light limitation. T h e r e are even fewer studies on the interaction between nitrate and a m m o n i u m uptake under light limitation. In the experiments by Syrett and M o r r i s (1963), two different irradiances did not change the pattern of the inhibition of NO3 by NH4 but at the higher irradiance, NH4 disappeared faster and NO3 uptake commenced earlier in the time course; in other words, the higher irradiance promoted the process. Bates (1976) studied the effects of light and a m m o n i u m on NO3 uptake in a diatom Skeletonema costatum and a chlorophyte. In his experiments, the batch cultures were preconditioned to a low irradiance and a high irradiance. T h e n the cultures were transferred to flasks under a series of light intensities and incubated for two hours to measure uptake rates. H i s results showed that in the presence of NH4, NO3 uptake was depressed at all light intensities, but there was more depression in NO3 uptake by the low light-  9 grown than by the high light-grown cells of both species and more depression in 5.  costatum than in the chlorophyte. However, a field investigation by G a r s i d e (1981) in the N e w Y o r k Bight did not show any apparent trend in the degree of the inhibition of NO3 by NH4 which could be ascribed to light. Definitive data on the interaction between nitrate and a m m o n i u m under different irradiances during short time periods are still lacking. Nitrogen concentrations used in most of the experiments designed to show the interaction were too high to be realistic in terms of nitrogen nutrition and ecology. A l s o , the time scale may not have been short enough to resolve the transient nature of the interactions. However, it is the transient nature that could determine the outcome of species competition and influence species succession since uptake and assimilation of nutrients are frequently uncoupled. In this study, a new cells-on-filter technique (Parslow et al. 1985a) was used to examine the short-term (minutes) interaction between nitrate and a m m o n i u m for continuous cultures of a diatom grown under different irradiances.  10 MATERIALS A N D METHODS  Organism T h e marine diatom Thalassiosira pseudonana (Hust.) H a s l e and H e i m d a l ( W H O I clone 3 H ) was grown in batch and turbidostat cultures. T h e culture used was originally obtained from W H O I and kept in the Northeast Pacific Culture Collection ( N E P C C No.58), Department of Oceanography, University of British C o l u m b i a , B . C . , Canada.  Culture medium T h e culture m e d i u m was artificial seawater with a full enrichment of nutrients (minus nitrate nitrogen), trace metals and vitamins ( E S A W ) . It was modified from H a r r i s o n et al. (1980) by adding 10" u M N a S e 0 8  2  3  which is required for growth by this species (Price et al. 1987). Nitrate was used as the sole nitrogen source for both batch and turbidostat cultures. T h e batch cultures were grown with full nitrate enrichment (550 u M ) and for the turbidostats, the concentration in the reservoir was 100 u M . T h e ambient NO3  NO3  concentration always  remained above 20 u M in the turbidostat culture m e d i u m , ensuring that the cultures were never nitrogen-limited. Culturing Batch cultures were grown in 50 m l testubes in a 1 7 ° C water bath under continuous fluorescent lighting (48T12 U H O , V i t a - L i t e , Tungsten Products) to determine the relationship between light and growth rate. Light was passed through a sheet of blue Plexiglas and a range of irradiances: 76,45, 25,15, 6, 4 and 2 u E m"^ s"l was achieved by covering the cultures with 1,2, 3,4, 5, and 6 layers of neutral density screening. T h e amount of light was measured using a quantum meter ( L i - C o r , m o d e l L l - 1 8 5 ) with a cosine collecter. T h e growth rates i n the batch cultures were determined by measuring  in vivo fluorescence (Turner Designs  F l u o r o m e t e r f ® ) twice a day. T h e batch cultures were grown through several transfers to ensure that the low-light cultures were completely adapted.  11 Turbidostat cultures were grown in 2 L flasks with magnetic stirring bars (120 rpm) at the same temperature (ITC)  as the batch cultures. T h e four turbidostat  cultures were grown under continuous lighting but at different irradiances 150 (LO), 17 ( L I ) , 9 (L2) and 2 (L3) u E m" s~l. These irradiances were chosen from the 2  results of the batch culture experiments and assumed to represent a range of light limitation. O n e of the four turbidostats was the control which was light saturated "7 1 (150 u E m'^s  ) and the other three turbidostats were grown under light-limited  conditions which were achieved by wrapping different layers of screening around the flasks. D i l u t i o n rates of the turbidostats were manually adjusted by measuring in  vivo fluorescence, cell numbers (by Coulter Counter), cell volume (calculated from the channel distribution in cell counts) in order to achieve a steady-state in growth rate. T h e equation U = D + l n ( N 2 / N l ) / t was used to calculate how much the dilution rate should be adjusted in order to keep the growth rate constant, where U is the specific growth rate (h" ), D is dilution rate (h" ) and N l and N 2 are the 1  measured  1  in vivo fluorescence values, or cell numbers, or cell volume on day 1 and  day 2, respectively.  Nitrogen analyses Particulate nitrogen in the turbidostat culture was measured using the mass balance method (Harrison et al. 1976) in which particulate nitrogen was equal to the sum of the inflow concentrations of N O 3 , N O 2 and N H 4 minus the concentrations remaining in the turbidostat culture m e d i u m . T h e s e measurements indicated that N H 4 concentrations in both the reservoir and the m e d i u m were always below 0.05 u M . N H 4 and N O 3 + N O 2 were analyzed with a T e c h n i c o n Autoanalyzer II following standard procedures outlined in Slawyk and M a c l s a a c (1972) and W o o d et al. (1967), respectively. In order to produce nitrogen-free water for washing the cells on the filter, a chemostat culture for the nitrogen-free wash water was set up i n the same manner  12  as the turbidostat, but the concentration of NO3 in the reservoir was only 15 u M . T h e overflow of the chemostat was collected and the cells were removed by filtration, producing N-free wash water.  Nutrient Uptake measurements A cells-on-filter technique (Parslow et al. 1985a) was used to measure nitrogen uptake rates. T h e operating system for this technique is illustrated in F i g . 1. Some modifications were made i n order to avoid smearing the autoanalyzer response to NO3 pulses because of the high NO3 concentration remaining in the culture m e d i u m when the cells from the m e d i u m were loaded onto the filter. Therefore, a 4-way valve was required to get rid of the high NO3 m e d i u m . T h e nitrogen-free wash water was filtrate from the N-limited chemostat culture. T h e standard solutions for uptake experiments were prepared from the same filtrate i n order to keep the standard solutions similar to the turbidostat solutions. W h e n a turbidostat culture reached a steady state, which was determined by observing no significant change in the growth rate over three days, samples were taken and uptake rates were measured. A standard concentration of N a N C ^ (5 u M ) was p u m p e d through the autoanalyzer for 7 m i n and an initial measurement was obtained o n the computer and chart recorder. Next, a known volume of the sample with a known cell density was loaded onto the filter and washed with wash water for 4 min. T h e n the same nitrate standard was p u m p e d across the cells on the filter for 7 m i n and a second measurement was then obtained. T h e difference between the initial measurement (the standard) and the second one (with cells trapped on the filter) is the amount of the nutrient taken up by the cells on the filter. After a new filter was connected, the same procedure as outlined above was followed for a standard a m m o n i u m solution (e.g. 3 u M ) in order to determine an a m m o n i u m uptake rate. Finally, a solution containing both NO3 (5 u M ) and NH4 (3 u M ) was used to measure uptake of NO3 and NH4 during the interaction. D u r i n g the  13 Figure 1. A . Apparatus used to measure uptake by cells trapped on an in-line filter (F). Three-way valves  and V g are used to switch among wash water (W),  culture sample ( C ) amd a standard solution (S). A four-way valve (VQ) is used to switch between baseline water (B) and the culture sample ( C ) when loading cells onto the filter. B . Cross section through in-line filter assembly. T h e filter is denoted by the dashed line.  14  To waste  V  A  B  e  AA  analyzer  W  B  To auto-  21  -7  Plexiglas plates  O-ring - Filter  15  measurements, the nitrite concentration was simultaneously monitored and N O 3 uptake measurements were corrected for nitrite concentration. T h e uptake experiments were performed under the same irradiance and temperature that were used to grow the turbidostat culture. U p t a k e rates were normalized three ways and the formulae that were used to calculate uptake rates were:  (i) o n a per cell basis  V =  (ug-at N c e l l  - 1  h" ) A  v c f — the flow rate of the standard solution ( L h" ). 1  s — the change in the concentration between the standard solution without cells and the standard solution with the cells on the filter (ug-at N L " ) at time t. 1  v — the volume of culture m e d i u m that was passed through the filter (ml), c — the cell concentration (cells ml"^).  (ii) on a cell volume basis  fs V =  (ug-at N u m ° h" ) 1  vl  f s =  1?  x IO  1 , 6  (mmole (liter cell volume)*  1 h" 1)  1  1  vl  1 — biovolume (total cell volume per m l of culture) (um^ ml"^).  16  (iii) specific uptake rate  V =  xlOOO  (h' ) 1  vpn p n — particulate nitrogen in culture m e d i u m (ug-at N L"*).  T h e overall design of the experiments is outlined in T a b l e I. In this study, one N O 3 concentration (5 u M ) and a range of a m m o n i u m concentrations were added as a pulse to cells from the four turbidostat cultures grown under different irradiances. Nitrate uptake rates with and without ammonium and a m m o n i u m uptake rates with and without nitrate were measured. U p t a k e rates were generally expressed on a cell volume basis because the cell volume increased at low irradiances. Before the interaction experiments were conducted, I tested the replication of N O 3 uptake rate measurements with the same amount of cells loaded onto the filter for the light saturated culture (LO). T h e effects of loading different amounts of cells onto the filter for light-limited cultures ( L 2 and L 3 ) o n N O 3 uptake rate measurements during a pulse of N O 3 (5 u M ) across the cells o n the filter were also tested. T h e calculated standard errors of the measurement of N O 3 uptake rates for the three cultures are given in T a b l e II. T h e L l culture was not tested, but the standard errors should lie between LO and L 2 cultures because the replication appears to be better when the culture is less light-limited (Table II). I did not test the replicability for N H 4 uptake rate measurements, but according to P . A . T h o m p s o n (pers. comm.), N H 4 uptake rates replicate m u c h better than N O 3 uptake rate measurements using this technique.  (  Table I. the  Outline  o f experiments. The n i t r o g e n  source and  i r r a d i a n c e used t o grow steady s t a t e c u l t u r e s a r e  i n d i c a t e d . The a d d i t i o n s  o f N0  3  o r NH  4  s i n g l y or i n  combination t o these steady s t a t e c e l l s a r e shown.  C u l - N-source ture  Irradiance  Nitrogen  uE m~^s - Forms  C o n c e n t r a t i o n (uM)  150  5.0  1  LO  N0  3  Additions  N0  3  NH  0.25, 0.5 , 1.0, 3.0, 5. 0  4  N0 +NH 3  4  5.0 (N0 ) + 0.25, 0.5, 3  1.0, 3.0, 5.0 (NH ) 4  LI  N0  3  17  N0  5.0  3  NH  0.25, 0.5 , 1.0, 3.0, 5. 0  4  N0 +NH 3  4  5.0 (N0 ) + 0.25, 0. 5, 3  1.0, 3.0, 5.0 (NH ) 4  L2  NO 3  9  N0  5.0  3  NH  0.25, 0.5 , 1.0, 3.0,5. 0  4  N0 +NH 3  4  5.0 (N0 ) + 0.25, 0.5, 3  1.0, 3.0, 5.0 (NH ) 4  L3  N0  3  2  N0  5.0  3  NH  0.25, 0.5 , 1.0, 3.0, 5. 0  4  N0 +NH 3  4  5.0 (N0 ) + 0.25, 0.5, 3  1.0, 3.0, 5.0 (NH ) 4  18 Table I I . of  Standard e r r o r s  N0  uptake r a t e  3  (SE) o f the repeated measurements  (itunole ( l i t e r c e l l v o l u m e )  - 1  h  - 1  ,  averaged over 2-4 min i n the time course) f o r t h e d i f f e r e n t c u l t u r e s . F o r c e l l number loaded onto t h e f i l t e r each l o a d i n g and N0  3  uptake r a t e data d u r i n g  each p u l s e , see appendix A: A l , A2 and A3.  Culture  N0  3  uptake r a t e  n  SE  C o e f f i c i e n t of Variation  LO  307  3*  24  8 %  L2  122  2**  15  12 %  L3  108  3**  21  20 %  * : an average f o r 3 n u t r i e n t p u l s e s a c r o s s 3 new samples o f s i m i l a r c e l l  cell  number.  **: an average f o r 2 (L2) o r 3 (L3) n u t r i e n t p u l s e s a c r o s s cell  samples o f d i f f e r e n t c e l l  number.  19 RESULTS  Cultures  G r o w t h rates determined in the batch cultures of  Thalassiosira pseudonana  increased with irradiance as a hyperbolic function, reaching a m a x i m u m at 45 u E m" s  (Fig. 2). T h e half-saturation constant (Kj) was estimated graphically to be  approximately 8.7 u E m " s"^ ( H a n e s - W o o l f linear transformation of the equation: 2  U  = U  m  * I/(Kj  + I), where U is the specific growth rate (h" ), U 1  m  is the  m a x i m u m growth rate (h"*), I is irradiance ( u E m " s"*) and Kj is the half2  saturation constant ( u E m "  2  s"*).  In the turbidostat cultures, the growth rates and the steady-state parameters including cell density, cell volume, particulate nitrogen, nitrogen cell quota, fluorescence per cell and nitrite production per cell are given in T a b l e III. T h e cell volume, nitrogen cell quota and fluorescence per cell increased as the irradiance decreased. Nitrite production per cell increased with increasing irradiance.  Interaction experiments  A . Time course of the interaction between nitrate and ammonium uptake  1) NOj uptake with and without NH^  In the light-saturated culture (LO), uptake of NO3 in the absence of NH4 reached a maximum after 1.0 m i n and then fell slightly and remained stable (Fig. 3). However, NO3 uptake in the presence of NH4 yielded negative values for the first 1 or 2 m i n of the time course before reaching stable uptake rates which were always  20 Figure 2.  Specific growth rate vs irradiance for batch cultures. T h e vertical bars represent 1 1 standard error (n > 5).  Irradiance (uE m-2 s-i)  Table I I I .  S t e a d y - s t a t e parameters f o r t u r b i d o s t a t c u l t u r e s o f T h a l a s s i o s i r a pseudonana r e p r e s e n t i n g t h e s t a r t i n g c o n d i t i o n s f o r t h e uptake experiments. For each parameter, see Appendix B: B l , B2, B3, B4, B5 and B6.  Culture Irradiance  P a r t i c u l a t e Nitrogen Cell nitrogen c e l l quotavolume  Growth Cell rate density  (uE nf-^s" ) t h - ) 1  1  (10  5  ml" ) 1  (uM)  Fluorescence per c e l l  (10~ ump l ( u m cell" ) cell" ) 7  3  1  (10-5)  1  Nitrite excrition (fmol cell" ) 1  LO  150  0.061  7.37  58.84  0.80  32.87  1.1  3.25  Ll  17  0.038  4.11  47.31  1.15  36.03  1.8  1.94  L2  9  0.023  6.17  74.93  1.21  39.29  2.2  1.89  L3  2  0.011  2.79  56.09  2.01  59.9  3.5  -0.23  23 lower than the rates without N H 4 (Fig. 3). These negative values were probably due to the excretion of N O 3 (efflux). In the light-limited cultures ( L I and L 2 ) , N O 3 uptake without N H 4 had a similar pattern to the control (LO) except the N O 3 uptake rates were lower (note the different scales); N O 3 uptake i n the presence of N H 4 tended to vary during the first two minutes before it was stable (Figs. 4, 5 and 6). F o r the culture grown at the lowest irradiance (culture L 3 ) , N O 3 uptake without N H 4 was lower than that when N H 4 was present.  2) NH4 uptake with and without NO3  In the light-saturated culture LO, the uptake of N H 4 at higher N H  4  concentrations (3.0 and 5.0 u M ) without N O 3 showed an initial enhanced uptake after 0.5 to 1 m i n (Fig. 7 A ) . T h e N H uptake rates at 1.0 m i n for the 3.0 u M and 5.0 4  u M N H 4 pulse without N O 3 were enhanced 178% and 192%, respectively, compared to the uptake rates for the same two N H 4 pulses at 5.0 m i n . However, in the presence of N O 3 , the enhancement of N H 4 uptake at 1 m i n for the 3 and 5 u M N H 4 pulses was decreased to 133% and 155%, respectively (Fig. 7 B and T a b l e I V ) . T h e light-limited cultures ( L I , L 2 and L 3 ) also showed the initial enhanced N H 4 uptake with and without N O 3 during the pulse of higher N H 4 concentrations (3 and 5 u M ) ( L I : Figs. 8 A and 8B; L 2 : Figs. 9 A and 9B; and L 3 : Figs. 1 0 A and 10B). T h e comparison of the enhancement of the N H 4 uptake rate at 1.0 m i n with the rate at 5.0 m i n i n the absence and the presence of N O 3 is shown i n T a b l e I V for each lightlimited culture. Replotting some of the data from Figs. 3 to 10 showed the dynamic interaction between N O 3 and N H 4 . Nitrate uptake at 5.0 u M without N H 4 was compared to nitrate uptake in the presence of 3.0 u M N H 4 and N H 4 uptake at 3.0 u M N H 4 without N O 3 was compared to N H 4 uptake i n the presence of 5.0 u M  24 Figure 3. Light saturated culture (150 u E m"^ s"*) LO: time course of N O 3 uptake i n the absence of N H 4 (0) and i n the presence of different N H 4 concentrations: 0.25 u M (+), uM  (O).  0.5 u M (•),  1.0 u M (x), 3.0 u M (A) and 5.0  Nitrate Uptake Rate (mmole (liter cell volume)" h" ) 1  1  Figure 4. Light limited culture (17 uE m-2 s-1) L l : time course of NO3 uptake in the absence of NH4 (0) and in the presence of different NH4 concentrations: 0.25 uM (+), 0.5 uM (•), 1.0 uM (x), 3.0 uM (A) and 5.0 uM (O).  Nitrate Uptake Rate (mmole (liter cell volume)' h' ) 1  1  28  Figure 5. Light limited culture (9 uE m" s") L2: time course of NO3 uptake in the z  1  absence of N H 4 ( 0 ) and in the presence of different N H 4 concentrations: 0.25 uM (+), 0.5 uM (•), 1.0 uM  (X),  3.0 uM (A) and 5.0 uM (O).  Nitrate Uptake Rate (mmole (liter cell volume)" h" ) 1  1  Figure 6. Light limited culture (2 u E m " s " ) L 3 : time course of N O 3 uptake in the z  1  absence of N H 4 (0) and in the presence of different N H concentrations: 4  0.25 u M (+), 0.5  u M (•), 1.0 u M  (x), 3 . 0 u M (A) and 5.0 u M (O).  31  Nitrate Uptake Rate (mmole (liter cell volume)' h" ) 1  1  Figure 7. Light saturated culture (150 u E m" s ) LO: time course of N H z  4  uptake  in the absence of N O 3 (A) and in the presence of N O 3 (B) at different NH  4  concentrations: 0.25 u M (A), 0.5 u M (+), 1.0 u M (•), 3.0 u M (X) and  5.0 u M (O).  CO to  34  Figure 8. Light limited culture (17 uE m  s ) LI: time course of N H 4 uptake in  the absence of N O 3 (A) and in the presence of N O 3 (B) at different N H 4 concentrations: 0.25 uM (A), 0.5 uM (+), 1.0 uM (•), 3.0 uM (x) and 5.0 uM (O).  Ammonium Uptake Rate (mmole (liter cell volume)' h' ) 1  1  Ammonium Uptake Rate (mmole (liter cell volume)' h' ) 1  1  36 Figure 9. Light limited culture (9 u E m" s"*) L2: time course of N H 2  4  uptake in the  absence of N O 3 (A) and in the presence of N O 3 (B) at different N H  4  concentrations: 0.25 u M (A), 0.5 u M (+•), 1.0 u M (•), 3.0 u M (x) and 5.0 uM  (O).  A 5003 re  (D ^ 400 re = )  E  f 300  E E «"  o 1200  Ei E <  1  •»  1  1  I I I I I I 1 1  I I I  I I I I  100d I I I I j I I I II I I I I j^l I I I I I I I  1^1  I I I I I I I  Ml  I I I I I I I I j[l I I I I I I  Time (min) 6OO-1  B  Time (min)  38 Figure 10. Light limited culture (2 u E m " s" ) L 3 : time course of N H 4 uptake in z  1  the absence of N O 3 ( A ) and in the presence of N O 3 (B) at different N H 4 concentrations: 0.25 u M (A), 0.5 u M (+), 1.0 u M (•), 3.0 u M (X) and 5.0 u M (O).  400 n  40 T a b l e IV.  R e l a t i v e enhancement o f NH  compared t o 5 min ( i . e .  V  l m i n  /V  4  uptake r a t e a t 1 min  5 i n i n  )  i n t h e presence  or absence o f N0 . 3  Nitrogen Form NH  4  NH  4  NH  4  NH  4  Concentration  + N0  + N0  Cultures  additions  3  3  L0  Ll  L2  L3  (3 uM)  178 %  200 %  211 %  162 %  ( 3 + 5 uM)  133 %  184 %  201 %  168 %  (5 uM)  192 %  227 %  191 %  152 %  ( 5 + 5 uM)  154 %  145 %  215 %  134 %  41 N O 3 (Figs. 11,12, 13 and 14). W h i l e the initial N H uptake rate in the presence of 4  N O 3 increased, N O 3 uptake in the presence of N H 4 was depressed for 1 m i n as shown in Figs. 11,12, 13 and 14 for all the cultures. W h e n N H 4 uptake started to decline after the initial enhanced uptake, N O 3 uptake in the presence of N H 4 increased and it reached a stable uptake rate after 2 min. T h e r e was less initial enhancement of N H 4 uptake in the presence of N O 3 (Figs. 11, 12, 13 and 14). T h e depression of N H 4 uptake by N O 3 gradually decreased and it was almost eliminated in the end of the time course (6 min) i n all the cultures. Replotting the earlier data allows a comparison of how the time course of N H 4 uptake at 3.0 and 5.0 u M with and without N O 3 differs among the cultures grown under different degrees of light limitation (Figs. 15 and 16). A t the higher irradiances (LO and L I ) , the initial enhanced N H 4 uptake with or without N O 3 decreased more slowly from 1 to 5 m i n than the cultures at lower irradiances ( L 2 and L 3 ) (Figs. 1 5 A , B and 16A,B). T h e latter two cultures ( L 2 and L 3 ) even displayed a slight supression of N H 4 uptake at about 2.0 m i n i n the presence of N O 3 following the initial short-lived enhanced uptake (Figs. 15B and 16B). F o r instance, during the pulse of 3.0 u M N H 4 with N O 3 (Fig. 15B), the N H uptake rate 4  at 2 m i n for the culture grown at the lowest irradiance (13) decreased 32% compared to the more stabilized uptake rate at 3.0 min.  B. Effects of growth irradiance on the interaction between nitrate and ammonium uptake  1) NO3 uptake with and without NH4  N O 3 uptake without and with N H 4 was averaged over the 2-4 m i n time interval from the time course. N O 3 uptake in the absence of N H 4 increased with the  42  Figure 11. Light saturated culture (150 u E m"^ s" ) LO: time course of the 1  interaction between NO3 uptake and N H uptake after a pulse of a 4  solution containing 5.0 u M NO3 (O) and 3.0 u M N H (+), compared to the 4  uptake of the controls: 5.0 u M NO3 alone (A) and 3.0 u M N H 4 alone (•) (from Figs. 3 and 7).  43  Uptake Rate of N0 or NH 3  (mmole (liter cell volume)" h" ) 1  1  4  44  Figure 12. Light limited culture (17 u E m " s z  ) L l : time course of the interaction  between N O 3 uptake and N H 4 uptake after a pulse of a solution containing 5.0 u M N O 3 ( A ) and 3.0 u M N H (+), compared to the uptake 4  of the controls: 5.0 u M N O 3 alone (O) and 3.0 u M N H 4 alone (•) (from Figs. 4 and 8).  Uptake Rate of N0 or NH 3  (mmole (liter cell volume)" h" ) 1  1  4  46 Figure 13. Light limited culture (9 u E m " s" ) L 2 : time course of the interaction z  A  between N O 3 uptake and N H 4 uptake after a pulse of a solution containing 5.0 u M N O 3 (O) and 3.0 u M N H 4 (+), compared to the uptake of the controls: 5.0 u M N O 3 alone (A) and 3.0 u M N H 4 alone (•) (from Figs. 5 and 9).  Uptake Rate of N0 or NH 3  (mmole (liter cell volume) h" ) -1  1  4  48 Figure 14. Light limited culture (2 u E m " s" ) L 3 : time course of the interaction z  1  between N O 3 uptake and N H uptake after a pulse of a solution 4  containing 5.0 u M N O 3 (O) and 3.0 u M N H 4 (+), compared to the uptake of the controls: 5.0 u M N O 3 alone (A) and 3.0 u M N H 4 alone (•) (from Figs. 6 and 10).  49  Uptake Rate of N0 or NH 3  (mmole (liter cell volume)" h" ) 1  1  4  50 Figure 15. N H 4 uptake at 3.0 u M concentration i n the absence of N O 3 ( A ) and in the presence of N O 3 (B) for the cultures grown at different irradiances: LO (•), L l (+), L 2 (A) and 13 (O) (from Figs. 7, 8, 9 and 10).  Time (min)  52 Figure 16. N H 4 uptake at 5.0 u M concentration in the absence of N O 3 ( A ) and in the presence of N O 3 (B) for the cultures grown at different irradiances: L O (•), L I (+), L 2 (A) and 13 (O) (from Figs. 7, 8, 9 and 10).  Ammonium Uptake Rate  Ammonium Uptake Rate  cn co  54 irradiance at which they were grown (Fig. 17A). T h e presence of N H 4 , however, changed the pattern. T h e cultures that were grown under the higher irradiances (LO and L l ) showed more of a reduction in N O 3 uptake in the presence of N H  4  than  the cultures ( L 2 and L 3 ) grown at the lower irradiances (Fig. 17A and 17B). In the light-saturated culture (LO), N O 3 uptake decreased as N H 4 concentration increased and 3.0 u M N H 4 decreased N O 3 uptake as much as 5.0 u M (Fig. 17B) (see A p p e n d i x C ) . In the less light-limited cultures ( L l and L 2 ) , smaller N H  4  concentrations (e.g. 0.5 and 1.0 u M ) inhibited N O 3 uptake rate to the same extent as the higher N H 4 concentrations (3 and 5 u M ) (see A p p e n d i x C ) . However, the culture under the lowest irradiance (L3) did not show a depression in N O 3 uptake by N H . 4  T h e s e results above are clearly shown in the three dimensional plot in Fig. 17 C.  2) NH4 uptake with and without NOj  N H 4 uptake without and with N O 3 was averaged over the 1-5 m i n time interval from the time course. In most cases, for all the cultures, N H 4 uptake rates with and without N O 3 except the rates in the pulse of low N H 4 concentration (< 1.0 u M ) were higher than N O 3 uptake rates without N H (Figs. 19, 20, 21 and 22). N H 4  uptake without N O 3 (Fig. 18A) increased with growth irradiance. Plotting N H 4 uptake rate vs N H 4 concentration showed that the N H 4 uptake rate was nearly saturated above 1.0 u M (Fig. 18B). Replotting the data in the earlier figures (Figs. 17 and 18) and N H 4 upake rate in the presence of N O 3 , one can compare N H 4 uptake vs N H 4 concentration for uptake of N H 4 alone, N H uptake in the presence of N O 3 (5.0 u M ) and N O 3 4  uptake in the presence of a range of N H 4 concentrations for each culture  4  55  Figure 17.  A . N O 3 uptake vs growth irradiance i n the absence of N H 4 (•) and i n the presence of N H  4  at 0.25  u M (A), 0.5 (+),  1.0 u M (•),  3.0 u M (x) and  5.0 u M (O). B . N O 3 uptake vs N H concentration for each culture: L0 4  LI (+),  (•),  L2 (A) and L3 (O) (replotted from A ) . ( T h e N O 3 uptake rate is  averaged over the 2-4 m i n time interval during the time course in Figs. 3,  4,  5 and 6). C . N O 3 uptake rate vs irradiance (in log scale) and N H  4  concentration. O n l y the trends are shown i n this computer plot since the plotting program extrapolated between the actual data points.  58 Figure 18. A . N H 4 uptake vs growth irradiance in the absence of N O 3 at N H 4 concentrations: 0.25 u M (A), 0.5 (+), 1.0 u M (•), 3.0 u M ( x ) and 5.0 u M (O). B . N H 4 uptake vs N H 4 concentration for cultures grown at four different irradiances: L 0 (•), L l (+), L 2 (A) and L 3 (O) (replotted from A ) . T h e N H 4 uptake rate is averaged over the 1-5 m i n time interval from the time courses in Figs. 7 A , 8 A , 9 A and 1 0 A  60  (Figs. 19, 20, 21 and 22). T h e presence of N O 3 did not change the typical pattern of N H 4 uptake as i n F i g . 18B, but caused a slight decrease in N H 4 uptake at the higher N H 4 concentrations (3 and 5 u M ) in the less light-limited cultures (Figs. 19, 20 and 21). However, this depression of N H 4 uptake by N O 3 started to occur at the lower N H 4 concentrations for the most light-limited culture ( L 3 ) although the N H 4 uptake rate with and without N O 3 at 5.0 u M N H 4 was similar (Fig. 22).  61  Figure 19. Light saturated culture (150 u E m"^ s" ) LO: N H concentration effect 1  4  o n the interaction between N O 3 and N H 4 : N H 4 uptake i n the absence of N O 3 (•), N H uptake in the presence of N O 3 (+) and N O 3 uptake in the 4  presence of N H  4  (A) (from Figs. 17B and 18B). T h e N H uptake rate is 4  averaged over the 1-5 time interval and N O 3 uptake rate is averaged over the 2-4 time interval from the corresponding time courses.  CM  700  q  0.00  l HH UUI1 I u  1.00  2.00  I I 1| ! J1 1  3.00  4.00  Ammonium Concentration (uM)  63  Figure 20. Light limited culture (17 u E m " s" ) L l : N H 4 concentration effect on z  1  the interaction between N O 3 and N H : N H 4 uptake in the absence of 4  N O 3 (•), N H 4 uptake i n the presence of N O 3 (+) and N O 3 uptake in the presence of N H 4 (A) (from Figs. 17B and 18B). T h e N H 4 uptake rate is averaged over the 1-5 time interval and N O 3 uptake rate is averaged over the 2-4 time interval from the corresponding time courses.  Uptake Rate of N0 or NH 3  (mmole (liter cell volume)" h" ) 1  1  4  6 5  Figure 21. Light limited culture (9 u E m " s" ) L 2 : N H concentration effect o n z  1  4  the interaction between N O 3 and N H 4 : N H 4 uptake i n the absence of N O 3 (•), N H uptake i n the presence of N O 3 (+•) and N O 3 uptake i n the 4  presence of N H (A) (from Figs. 17B and 18B). T h e N H uptake rate is 4  4  averaged over the 1-5 time interval and N O 3 uptake rate is averaged over the 2-4 time interval from the corresponding time courses.  66  Uptake Rate of N0 or NH 3  (mmole (liter cell volume)" h" ) 1  1  4  67 Figure 22. Light limited culture (2 u E m " s" ) L 3 : N H 4 concentration effect o n z  1  the interaction between N O 3 and N H 4 : N H 4 uptake i n the absence of N O 3 (o), N H 4 uptake i n the presence of N O 3 (+) and N O 3 uptake i n the presence of N H 4 (A) (from Figs. 17B and 18B). T h e N H uptake rate is 4  averaged over the 1-5 time interval and N O 3 uptake rate is averaged over the 2-4 time interval from the corresponding time courses.  00  300 ?  250 H  °J? 200 \ CO ^  I  os CO §,  CC  o> 100  o  0 E .* E  CO  w  0.00  Ammonium Concentration (uM)  69 DISCUSSION  Cultures  T h e relationship between growth rate of  Thalassiosira pseudonana and  irradiance appears to follow a simple hyperbolic function. T h e saturating irradiance for growth, 45 u E m " s"*, obtained i n this study is low compared to the value (0.04 2  cal c m ' min"*), ca. 140 u E m " s"* for saturating photosynthesis obtained by E p p l e y 2  2  and Renger (1974) with the same species. T h e difference could be due to the differences i n light quality and the photocycle used in the two studies. In my study, continuous irradiance and a blue Plexiglas light filter was used, whereas white light and a light:dark cycle were used in E p p l e y and Renger's (1974) chemostat cultures. In addition to the experimental design differences, it is possible that growth rate becomes saturated at a lower irradiance than does photosynthesis. T h e growth rates i n the turbidostat cultures were lower than the cultures growing under similar irradiances for batch cultures. Since the irradiance measured for the turbidostat culture was not corrected for the light attenuation within the culture flasks, it is possible that self-shading of cells was responsible for the small decreased growth rates. Since fluorescence per cell is thought to reflect the chlorophyll content of a cell, the increase of the chlorophyll with decreasing irradiance indicates that this diatom adapts to low irradiance by increasing its cellular chlorophyll content. Further evidence of adaptation was also shown in the increase i n the cell volume and nitrogen cell quota with decreasing irradiance. This is similar to the observations by Z e v e n b o o m  et al. (1980) who studied Oscillatoria  agardhii, R h e e and G o t h a m (1981) studying Scenedesmus sp and Fragilaria crotonensis and H e a l e y (1985) studying a blue-green alga Synechococcus linearis. In another study o n nutrient- and light-limited growth of  Thalassiosira weissflogii (i.e.7.  70 fluviatilis) in continous culture this species did not show such an increase in nitrogen cell quota with decreasing irradiance (Laws and Bannister 1980). In that study, a light:dark cycle was used. In phytoplankton physiological studies, continuous chemostat cultures have been commonly used to study chemical composition and nutrient uptake related to a steady-state growth rate. However, in the present study, the relationship between chlorophyll or nitrogen cell quota and growth rate is opposite to the results obtained from a chemostat study. Therefore, the same growth rate could be caused by different environmental factors and reflect different physiological conditions. In a turbidostat, for instance, when light is limiting and nutrients are sufficient, cells appear to increase their nutrient content to make up more chlorophyll. In a chemostat, when nutrients are limiting, cells reduce their nutrient content. F o r example, E p p l e y and Renger (1974) observed a decrease i n chlorophyll content per cell and nitrogen cell quota with growth rate for  Thalassiosirapseudonana. These  differences between chemostats and turbidostats indicate that the same growth rate may be a result of different physiological conditions (i.e. light vs nutrient limitation). This must be considered in conducting a field study.  Interaction Experiments  Before the cells-on-filter technique was developed, all uptake rates reported from both laboratory studies and field studies were averaged over a time period. T h e use of a self-cleaning in-line filter to continuously measure the disappearance of a nutrient in a m e d i u m with cells in suspension (Parslow et al. 1984a) enabled us to follow the change i n uptake with time and with a greatly improved time resolution. Now, it is realized that the uptake rates of some nutrients change with both concentration and time, especially when cells are nutrient-deficient (Conway et al.  71 1976). However, the self-cleaning in-line filter method is still subject to a change in nutrient concentration over time and hence it does not resolve the p r o b l e m of whether the uptake rate changes with time or whether it is affected by the changing nutrient concentration when the limiting nutrient falls below a saturating level. This cells-on-filter technique (Parslow et al. 1985a) enables us to obtain an instantaneous uptake rate under a constant nutrient concentration during a very short time course. Such an instantaneous uptake rate may be important i n phytoplankton ecology, because it could aid our understanding of how cells respond to a pulse of the limiting nutrient.  A  Time course of the interaction between nitrate and ammonium uptake  Nitrate and a m m o n i u m uptake rates were observed to increase with time during the first minute before the initial enhanced uptake occurred. T h e s e shorttime ( < 1 min) lower uptake rates were not observed by G o l d m a n and Glibert (1982) or by Parslow et al. (1985a) for their N H ^ l i m i t e d chemostat cultures of  Thalassiosirapseudonana. Parslow et al. (1985a), who used the same technique, found that the enhanced uptake rate occurred sooner (15 s). T h e difference between my results and theirs is that the cultures they used were N H 4 limited while mine were N O 3 saturated and light-limited. Probably i n N H - l i m i t e d cultures, the N H 4 4  uptake system is active and ready to take up available N H 4 quickly. A s soon as N H  4  reaches the cells, surge uptake occurs. However, the cells in my study which were grown at saturating N O 3 concentrations do not appear to have a fully active N H  4  transport system. Therefore, it is possible that the N H 4 uptake system needs to be induced when N H 4 hits the cells during the pulse. This inducing process seems to be reflected i n the acceleration of the uptake rates during the first minute. Different N H 4 concentrations may have a different driving force to accelerate uptake, and  72 light conditions determine the internal power or the accelerating capacity (i.e. how fast the uptake could be accelerated). A s seen in F i g . 16A, for instance, higher N H 4 concentrations appeared to accelerate the uptake faster; and the more light-limited cells had less energy and slower acceleration of the uptake rate ( F i g 16A). Morever, the presence of N O 3 slowed the acceleration of N H 4 uptake, which indicates that the interaction between N O 3 and N H 4 uptake takes place immediately once the molecules of N O 3 and N H 4 contact the cells. This study is one of the first reports of the presence of N O 3 affecting N H 4 uptake rate over a short time period. This inducing process reflected by the acceleration could be very important i n understanding the mechanisms of N O 3 uptake, N H 4 uptake and the interaction between the two. T h e acceleration process may involve complex processes including diffusion, cell membrane potential change and p H change as well as active transport ( R a v e n 1980). Obviously, further studies on the acceleration of N H 4 uptake rate i n response to an a m m o n i u m pulse for N O 3 saturated cells is needed.  1) NO3 uptake with and without NH4  N O 3 uptake without N H 4 appeared to be stable after 1 or 2 min during the time course. However, when the cells were simultaneously exposed to N O 3 and N H 4 , N O 3 uptake rates showed either a fluctuation i n uptake rate or negative uptake rates for c a . l m i n probably resulting from the leakage (efflux) of N O 3 . These transient variation suggests that the interacting effect of N H 4 on N O 3 utilization occurs immediately and directly u p o n N O 3 uptake rather than N O 3 reduction within this short time period. It is not clear, however, whether the interacting effect on the stabilized N O 3 uptake rate i n the remainder of time course is attributed to a decrease i n N O 3 uptake or due to an effect on N O 3 reduction, or both.  73 2) NHj uptake with and without NOj  T h e initial enhanced N H uptake occurred only for larger pulses of N H 4  4  (3.0  and 5.0 u M ) . T h e r e was no initial enhanced N H uptake for pulses less than 1.0 u M . 4  This concentration dependence of the initial enhanced N H uptake was also 4  observed by Parslow et al. (1985a) using the same technique and the same species in N H - l i m i t e d chemostats. Therefore, the occurrence of the time-dependent uptake 4  of N H is also concentration dependent, although the concentration for the 4  dependence can vary somewhat depending on physiological state of the cells. T h e similar time-dependence of N H  4  uptake during a short time course has been shown  for different species. G o l d m a n and Glibert (1982) studied four marine species including  Dunaliella tertiolecta, Phaeodactylum tricornutum, Chaetoceros simplex and  Thalassiosira weissflogii using the  technique and observed different degrees of  the initial N H enhanced uptake among the four species. It has been suggested that 4  this enhanced uptake potential resulted from nitrogen deficiency, the degree of which can be expressed by the nitrogen cell quota. However, i n some cases, it is not necessary for cells to be nitrogen-limited i n order to have this potential. H o r r i g a n and M c C a r t h y (1982) found that before nitrite was depleted in nitrite grown cells in batch cultures of  Thalassiosira pseudonana, the uptake rate i n a 5 m i n incubation  was 78 times the growth rate, and Parslow et al. (1984b) observed that the enhanced NH  4  uptake rate decreased over a 5 m i n time period for N C ^ - g r o w n cells 6 hours  prior to starvation i n batch culture. T h e results obtained here demonstrate a similar p h e n o m e n o n for N O 3 saturated cells in a steady-state turbidostat culture. It appears that the same phenomenon shown by cells grown on different nitrogen forms and different culture regimes (batch or continous) may have the same mechanism. T h e r e are two reasons for this suggestion. First, the preferential utilization for N H over N O 3 has been very commonly observed i n both laboratory 4  74 cultures and natural populations of marine phytoplankton. A l t h o u g h the biochemical mechanisms of the preference for N H 4 are not clear, the physiological and ecological reasons are quite logical, i.e. more expenditure of energy is involved in N O 3 utilization and no reducing power is required for the assimilation of the reduced form, N H 4 , into amino acids. Secondly, the rate-limiting step in the reduction of N O 3 to amino acids is likely to occur when N O 3 is reduced to N H 4 . D o r t c h (1982) studied the effect of growth conditions on the accumulation of internal nitrate, ammonium, amino acids and protein in three marine diatoms and claimed that the pools which formed in  Skeletonema costatum indicated that nitrate  reduction to N H 4 is the slowest step in nitrogen assimilation. T h e synthesis of protein from amino acids is the, next slowest and the incorporation of N H  4  into  amino acids is the fastest. Thus, it is possible that cells grown on oxidized forms such as N O 3 and N O 2 are actually somewhat NF^-deficient. If this is true, the initial N H 4 enhanced uptake which declines quickly with time would occur in both nitrogen-limited cultures and N O 3 - and NO^-replete cultures due to both preferential uptake of N H 4 and the reduced internal pools of N H 4 resulting from the slower N O 3 reduction and the faster N H 4 assimilation. In addition, from a biochemical point of view, when N H 4 is assimilated, the H  +  ion left internally  would lead to an transient drop in p H , which, in turn, favours N H 4 uptake and results in more N H 4 uptake until the other regulatory mechanisms take over (Rithtie and L a r k u m 1987). This transient p H change might have some effect on N O 3 uptake as well. Falkowski (1975) tested the presence of a N C ^ C l - a c t i v a t e d A T P a s e associated with the cell membrane in seven species of phytoplankton. H e found that the o p t i m u m p H for the A T P a s e activity was 7.9 in  Skeletonema costatum  and Chroomonas salina. Therefore it is possible that the transient acid condition resulting from N H 4 assimilation affects the NC^-activated A T P a s e activity and inhibits active N O 3 transport across the membrane. T h e time course experiments  75 appear to reflect such a pH-regulated process, during which the N H uptake was 4  enhanced, the N O 3 uptake was inhibited or fluctuating. It was interesting that the specific uptake rate at 1 m i n i n the pulse of 5 u M NH  without N O 3 is 8.4 d " for the light-saturated culture LO (Fig. 7 A ) . This value 1  4  is remarkably close to the V ^ " l value of 9 d"* (an average over 0-1 min) for the m  pre-N03-starved batch culture (Parslow et al. 1984b) and 10 d  at 1 min for the  N H - l i m i t e d chemostat culture with a growth rate of 0.5 d " (Parslow et al. 1985a). 1  4  After this 1 m i n point, the rates started to decline. T h e similarity i n values may be explained by recent observations i n the literature. O h m o r i and Hattori (1978) observed a transient change in the A T P p o o l oiAnabaem  cylindrica when N H 4 was  added to the cells. T h e y found that the A T P pool dropped dramatically within the first minute and recoverd at 6 min. T h e change in the A T P pool with time in their study shows the opposite pattern to the pattern of N H 4 uptake rate i n this study. W h e n the N H 4 uptake rate increased to the highest rate at 1 m i n and declined with time until it was stable at 6 min, the A T P pool decreased to a lowest level at 1 m i n and then recovered during the remaining 1-6 min. Morever, a study by Flores et al. (1980) o n short-term a m m o n i u m inhibition of N O 3 utilization by Anacystis nidulans and other cyanobacteria showed that the increase in glutamine reached a maximum at 1 m i n while glutamate reached a m i n i m u m at 1 m i n indicating the maximum assimilation of N H 4 in this initial 1 m i n period. It appears that 1 minute is adequate time for cells to start regulating nitrogen assimilation at the cellular level. This regulation likely functions via feedback mechanism (Raven 1980). This is also inferred f r o m the time dependence of enhanced N H 4 uptake alone and the simultaneous interaction with N O 3 uptake. T h i s study is the first one to clearly show a short-term depression of N H  4  uptake by N O 3 . T h i s depression occurs only during the initial enhanced N H 4 uptake, and it is almost eliminated during the remainder of the time course, while  76 o n the other hand, N H uptake is still exerting an inhibiting effect on N O 3 uptake 4  during the same time period. T h e elimination of the supression of N H  4  uptake by  N O 3 indicates that, on the one hand, N O 3 uptake affects N H utilization v i a the 4  NH  4  transport system, and on the other hand, the feedback regulation gradually  takes over i n nitrogen assimilation, resulting in a slow down of N H uptake and an 4  inhibition of N O 3 uptake simultaneously. But it is not clear whether the inhibition of N O 3 uptake by N H occurs at the site of N O 3 transport or the reduction of N O 3 . 4  T h e enhanced N H uptake and its time dependence is similar to a model 4  proposed by Conway et al.(1916). F o r a time course of N H  4  uptake by N H - l i m i t e d 4  cultures, they defined three distinct phases of uptake: surge uptake (Vs), internally controlled uptake ( V i ) and externally controlled uptake ( V e ) . In this study, I found that low N H concentrations (< 1.0 u M ) did not result i n an initial enhanced 4  uptake. T h i s would represent a range under the external control i n the Conway's m o d e l (1976). T h e initial enhanced uptake at the higher N H  4  concentrations  reflects a surge uptake, and the following decline with time probably suggests a gradual shift from surge uptake to the internally controlled uptake, indicating a feedback regulatory process (Conway et al. 1976). A s shown in Figs. 7-10, for example, all the initial enhanced uptake eventually declined towards the same rate as the N H uptake rate for the 1 u M pulse which did not have an initial enhanced 4  uptake rate regardless of the degree of light limitation of culture. T h e initial enhanced uptake of N H was also affected by light. W h e n light 4  limitation increased, the initial enhancemant of N H uptake was less and lasted a 4  shorter time. Obviously, the enhancement is light-dependent through the effect of light o n the growth rates. Light limits the growth of cells and determines the cellular activities and responses including the extent of uptake and the speed of uptake regulation to external nutrient pulses. In the light saturated culture, the cells were actively growing and the transfer of nitrogen between each p o o l was fast. In  77 contrast, in the most light-limited culture, the cells were less active, the transfer of nitrogen between each p o o l was slower and the accumulation of nitrogen i n the internal p o o l may have occurred. Thus, at the higher growth irradiances, the initial enhancement of N H 4 uptake was greater and longer. A t the lower growth irradiances, the initial enhancement of N H 4 uptake was less and shorter in duration.  B. Effects of growth irradiance on the interaction between nitrate and ammonium uptake  1) NO3 uptake with and without NH4  In the light saturated culture, the inhibition of N O 3 uptake was a function of N H 4 concentration. T h e higher the N H 4 concentration was, the more the N O 3 uptake was inhibited. However, the severe light limitation changed the interaction between N O 3 and N H 4 uptake. A s shown i n F i g . 17, the lowest irradiance during growth appeared to result i n no inhibition of N O 3 uptake by the increase i n N H 4 concentration. T h i s result is i n contrast to the observation by Bates (1976). Bates found that the shade-adapted cells (grown under the limiting irradiance) showed a greater depression of N O 3 uptake by N H 4 than the sun-adapted cells (grown under the high irradiance). M a j o r differences exist between his study and my study, besides the different species used i n each study. First, batch cultures were used i n his study, while steady-state turbidostat cultures were used i n this study. Bates stated that i n his batch cultures, a n initial N O 3 concentration of 0.1 u M in the culture medium was necessary to have active N O 3 reductase. In that case, the cells h a d been under N O 3 limitation for a while before uptake rates were measured. H o w much effect the N O 3 limitation had o n the interaction between N O 3 and N H 4 uptake was not clear. Next, he used a light:dark cycle for his batch cultures. This makes the interpretation  78 of the interaction more complicated since diel variation of the intracellular accumulation of N O 3 probably occurred. Recently Raimbault and Mingazzini (1987) studied such a diurnal variation in  Skeletonema costatum, the same species  that Bates used, and found that N O 3 accumlation inside the N-sufficient cells was high during the night and early in the morning. Finally, the last difference (and perhaps the most important one) was that uptake rates were measured over 2 h. In my study, uptake rates were measured over 6 min. It can be argued that the cells grown under the lowest irradiance should show more depression of N O 3 uptake than the cells grown under higher irradiances, because N O 3 uptake and reduction require more energy than N H 4 utilization on one hand, and on the other hand, the rapidly enhanced N H 4 uptake consumes a larger part of the present energy pool for cells grown at the lowest irradiance and the recovery of the p o o l or the regulation may be slower. In comparison, the rapid N H 4 uptake may need a smaller part of the energy pool in the less light-limited cells and the recovery is probably faster. T h e existence of a light-dependent energy pool has been demonstrated by Falkowski and Stone (1975). T h e p r o b l e m is that we do not know the mechanisms of N O 3 and N H 4 uptake, let alone their interaction. M o r e v e r , we have little knowledge on the mechanism of direct light reactions on N O 3 uptake or N H 4 uptake and their assimilation. However, from evidence at present, there are some possible explanations for the lack of an inhibiting effect of N H 4 o n N O 3 uptake. First, it has been demonstrated that N O 3 reduction to N H 4 is light-dependent (i.e. electrons required for N O 3 reduction to N H are directly 4  derived from ferridoxin which is the first electron acceptor from photosystem I rather than from N A D ( P ) H supplied throughth the carbon cycle) ( C a n d a u et al. 1976). It has been claimed that such a mechanism exists at least i n cyanobacteria, and nitrite reductase has such an dependence in photosynthetic eukaryotes including algae and higher plants. Next, algae possess more or less diffusive N O 3 uptake. F o r  79 example, Serra et al. (1978) have shown that a m m o n i u m did not affect N O 3 diffusion although it strongly inhibited carrier-mediated nitrate uptake. Finally, dark N O 3 uptake has been frequently reported (e.g.Bates 1976, Serra et al. 1978 and Collos 1982). D a r k N O 3 uptake indicates either diffusive transport or continuing active transport supported by respiration that is less affected by light, or possibly both depending on the concentration of nutrients and the length of the incubation. In the observation by Serra et al. (1978), darkness did not even affect N O 3 uptake kinetics including active tranport, while Collos (1982) simply suggested for his results that only diffusive processes were involved in dark N O 3 uptake which declined with time over the 3 h incubation. A more recent study by R a i m b a u l t and Mingazzini (1987) on diurnal variations of intracellular nitrate storage in marine diatoms showed that the N O 3 accumulation inside the N-sufficient cells was high during the night and at the beginning of the day but decreased during the light period, indicating dark N O 3 uptake. Thus, under the lowest irradiance, if the rate of N O 3 reduction to N H 4 is limited by irradiance, the uptake rate of N O 3 into the cells in either ways (diffusive or active) could be enough to meet the nitrogen requirement. In fact, as shown in F i g . 17B, N O 3 uptake inhibited by the high N H 4 concentrations for the cultures grown under high irradiances appears to decrease towards the same rates as the cells growing under the lowest irradiance. This probably implies a background N O 3 uptake rate that is not affected by N H , at least during a short period of time (e.g. 6 4  min). Therefore, the cells grown under the lowest irradiance did not show an inhibition of N O 3 uptake by N H 4 . Furthermore, if N O 3 reduction to N H 4 is directly light-dependent, as proposed by C a n d a u et al. (1976), while the feedback regulation is slow i n inhibiting N O 3 reduction, the continuing flow of N O 3 reduction would drive N O 3 transport across the membrane continuously in the presence of N H 4 .  80 T h i s continuing flow into the N H 4 p o o l could affect N H uptake rates. This will be 4  discussed in the next section.  2) NH4 uptake with and without NOj  A s shown i n F i g . 18A, N H 4 uptake is also dependent on light limitation of the cultures. T h i s light-dependence is a response of cells to a nitrogen pulse when they were grown under a range of irradiance. Irradiance did not appear to change the half-saturation constant for N H 4 significantly. N o r did it change the concentration where the uptake rate is nutrient saturated (1.0 u M ) . A s discussed i n the last section, if intracellular N H 4 derived from N O 3 uptake and reduction continues to flow into the N H 4 pool, from which the removal of N H 4 is limited by its assmilation into amino acids, the accumulation of N H 4 in the pool would affect N H 4 uptake into the pool. T h u s , it is likely that N O 3 uptake affects N H uptake before the feedback regulation inhibits N O 3 uptake and 4  reduction. Funkhousor and R a m a d o s (1980) found the continuing synthesis and degradation of the inactive nitrate reductase precursor protein by the N H 4 - g r o w n cells and stated that this is a wasteful process. If N O 3 uptake continues when a cell is utilizing N H 4 , N O 3 i n the cell would induce N 0 3 reductase activity or synthesis. However, a regulated inhibition by N H 4 assimilation is still exerted o n these activities. Therefore, the competition between utilization and inhibition of N O 3 may occur. Conway (1977) observed a delay of approximately 30 m i n for N H to fully 4  inhibit N O 3 uptake. T h i s delay may indicate that the synthesis of certain amino acids are required to inhibit N O 3 uptake, rather than N H 4 itself. Nevertheless, the ecological roles i n utilizing different nitrogen forms could be significant, since more pathways are open for nitrogen uptake.  81  Ecological significance  A l t h o u g h the mechanisms involved in N O 3 and N H 4 utilization and the interaction between the two are not yet understood, their ecological significance is quite apparent. Since Conway et al. (1976) and Conway and Harrison (1977) interpreted the enhanced rapid N H 4 uptake as a nutritional strategy for marine phytoplankton to adapt to low nitrogen environments and compete with other species which are not capable of quickly taking up N H 4 , similar observations have been reported subsequently from both laboratory and field studies, for example, by T u r p i n and H a r r i s o n (1978), M c C a r t h y and G o l d m a n (1979), Glibert and G o l d m a n (1981), H o r r i g a n and M c C a r t h y (1982), G o l d m a n and Glibert (i982) Q u a r m b y et al. (1982) and Parslow et al. (1984a,b and 1985a,b). This is the first study to report the enhanced N H 4 uptake rate relative to growth rate and its rapid decline with time (< 6 min) for light-limited cells grown in steady-state turbidostats at saturating N O 3 concentrations. It is not clear why cells under light limitation possess or develop such a potential since temporal N H 4 utilization would compete for the already limited energy derived f r o m the low light conditions. M c C a r t h y and G o l d m a n (1979) observed an enhanced N H 4 uptake rate that was higher than the growth rates and related it to the relative growth rate, U : U m , or nitrogen cell quota. However, such a relationship does not hold for lightlimited growth. It has been shown that such an enhanced uptake appeared to be dependent o n light-limited growth rates, i.e. the enhancement increases with growth rate. However, the relative enhancement (i.e. V m / U m ) for N H 4 uptake increases with decreasing irradiance (Fig. 23). This implies that the relative potential for the enhanced N H 4 uptake or uncoupling between uptake and assimilation is increased  82 Figure 23. T h e ratio of uptake rate (h" ) of N H 4 alone to maximal specific growth A  rate (h"*) at the growth irradiance for the pulses of N H 4 : 0.25 u M (A), 0.5 u M (+), 1.0 u M (•), 3.0 u M (x) and 5.0 u M (O). T h e N H uptake rate is 4  averaged over the 1-5 m i n time interval from the corresponding time course.  83  Ratio (Vm/Um)  on-i o  84 by light limitation. In another words, the relative capacity for N H 4 uptake increases with nitrogen cell quota. O n one hand, it is apparent that light limitation is distinctly different from nutrient limitation in terms of the physiological performance at a given growth rate, and on the other hand, the enhanced relative capacity induced by light limitation indicates that the nitrogen requirement increased as irradiance decreased. T h e increase in nitrogen requirement with decreasing irradiance has been suggested by R h e e and G o t h a m (1981) based on increasing cell quota with decreasing irradiance. Therefore, the enhanced relative capacity ( V m / U m ) which increased with decreasing irradiance indicates the existence of this physiological strategy in terms of uptake kinetics. Obviously, it could be expected that phytoplankton cells under both nitrogen limitation and light limitation would have such a potential. T h e N H 4 taken into the cells via the rapid enhanced uptake in a short time would be utilized either to synthesize more chlorophyll apparatus for deriving more light energy by nitrogen-limited cells or to make up new cellular macromolecules for division by nitrogen-saturated cells. T h e relative enhanced potential for rapid nitrogen uptake and the increased nitrogen cell quota resulting from light limitation could have ecological siginificance in an eutrophic area. In an eutrophic area such as an upwelling area, light is limiting to phytoplankton growth near the bottom of the euphotic zone while nitrogen concentrations are high. Thus, the light-limited cells develop a large nitrogen cell quota. A s the water is brought up to shallower zone by upwelling, the cells with slower growth respond to the increase in irradiance and they are induced to grow faster. T h e rate of the inducing process depends on the response time of the cells. M a c l s a a c et al. (1985) distinguished four zones of physiological condition along the axis of the upwelling plume for the coast of Peru. T h e y proposed that i n Z o n e 1, phytoplankton upwelled with nutrient-rich water were initially shifted-down (low nutrient uptake); in Z o n e 2 the phytoplankton undergo light induced shift-up to  85 increased nutrient uptake, photosynthesis and synthesis of macromolecules. Z i m m e r m a n et al. (1987) developed a model in which the rate of shift-up (maximum uptake rate) appears to be related to irradiance and the ambient concentration of the limiting nutrient at the time of upwelling. T h e results presented in this study appear to support the proposal from these field investigations. A s the deep water near the bottom of euphotic zone is brought up by upwelling, the cells in nutrient rich water represented by the most light-limited cells in this study will respond to the increase in irradiance, resulting in a gradually increasing growth rate. T h e s e cells are enriched with nitrogen and have a large cell volume. It is no longer necessary to keep this larger cell volume after they are induced to divide by increasing irradiance. It takes a shorter time for these new cells to divide since they are moving up in the water column and can take up nutrients more rapidly under increased irradiance but require less nitrogen for their cell quota. Thus, during this process, growth and uptake rate are accelerated. W h e n the cells are upwelled to the saturating-light zone, they will reach maximum uptake or complete their shift up. T h i s is because they have the smallest cell volume giving the best advantage (highest surface:volume ratio) for nutrient uptake, and the lowest nitrogen cell quota; they can take up nitrogen the fastest at saturating irradiances. These cells could be represented by the light-saturated cells in this study. D u r i n g the inducing process (from slow growth to fast growth), the intermittent pulse of N H produced by other 4  organisms could frequently stimulate cell division. N o inhibition of N O 3 uptake by NH  4  uptake leading to the increase in total nitrogen uptake by the most light-  limited cells as well as the maximum uncoupling, result in more nitrogen content which provides a potential basis for the light induced process. Often, an upwelling area is also characterized by great production in higher trophic organisms and large standing stock as well. N H 4 regeneration is proportional to both the production and the standing stock. However, N H 4 concentration is not high in these area. It appears  86 that preferential and rapid N H 4 uptake are partially responsible for the low concentration in the water. Although the regenerated N H 4 may not make as much contribution to primary production as N O 3 in those upwelling areas, the most important aspects of the interaction between N O 3 and N H 4 uptake by cells under different light limitation lies in species-specific interacting kinetics, resulting in the coexistence of different species and regulating the stability of an ecosystem.  87  SUMMARY  1. G r o w t h rates of a marine diatom, Thalassiosirapseudonana  were determined for  batch cultures and turbidostat cultures under different irradiances. 2. Physiological parameters such as growth rates, cell density, particulate nitrogen, nitrogen cell quota, cell volume, fluorescence per cell and nitrite excretion were determined for the turbidostat cultures. 3. T h e short-term time course of the interaction between nitrate and a m m o n i u m uptake was followed. a. C o m p a r i s o n of nitrate uptake in the absence and presence of a m m o n i u m showed that nitrate uptake was affected immediately by a m m o n i u m and depressed in the remainder of the time course regardless of the different growth irradiances. b. A m m o n i u m uptake in the absence and presence of nitrate showed an initial enhanced uptake and declined with time. T h e presence of nitrate depressed the initial enhanced N H 4 uptake, but did not affect the N H 4 uptake during the remainder of the time course for all the cultures. 4. Effects of irradiance o n the interaction between nitrate and a m m o n i u m uptake was apparent. a. F o r the light-saturated culture and the less light- limited cultures, the depression of nitrate uptake by a m m o n i u m increased with a m m o n i u m concentration. However, for the most light-limited culture, the depression of nitrate by a m m o n i u m disappeared. b. A m m o n i u m uptake was dependent on the growth irradiance of the cultures and was saturated at approximately 1.0 u M . T h e presence of nitrate slightly affected a m m o n i u m uptake.  88 5. T h e s e results on the turbidostat cultures and uptake dynamics during the interaction imply some physiological regulation in nitrogen incorporation and suggest ecological significance in an upwelling system.  89 LITERATURE CITED  Bates, S.S. 1976. Effects of light and a m m o n i u m on nitrate uptake by two species of estuarine phytoplankton. L i m n o l . Oceanogr. 21: 212-218.  B i r d , K . T . 1976. Simultaneous assimilation of N H 4 and N C H by (Gelidiales: Rhodophyta). J . Phycol. 12: 238-241.  Gelidium nudifrons  C a n d a u , P., C . M a n z a n o and M . L o s o d a . 1976. Bioconvension of light energy into chemical energy through reduction with water to a m m o n i u m . Nature 262: 716-717.  Collos, Y . , S . Y . Maestrini and J . M . Robert. 1986. (in press).  Collos, Y . 1982. Transient situations i n nitrate assimilations by marine diatom. 2. Changes in nitrate and nitrite following a nitrate perturbation. L i m n o l . Oceanogr. 27: 528-536.  Conway, H . L . , and P.J. Harrison and C O . Davis. 1976. M a r i n e diatoms grown in chemostats under silicate or a m m o n i u m limitation. II. Transient response of Skeletonema costatum to a single addition of the limiting nutrient. M a r . B i o l . 35: 187-199.  Conway, H . L . 1977. Interactions of inorganic nitrogen in the uptake and assimilation by marine phytoplankton. M a r . B i o l . 39: 221-232.  Conway, H . L . and P . J . Harrison. 1977. M a r i n e diatoms grown i n chemostats under silicate or a m m o n i u m limitation. I V . Transient response of Chaetoceros debilis, Skeletonema costatum and Thalassiosira gravida to a single addition of the limiting nutrient. M a r . B i o l . 43: 33-43.  Conway, H . L . and T . E . R . Whitledge. 1979. Distribution of fluxes and biological utilization of inorganic nitrogen during a spring b l o o m i n the N e w Y o r k Bight. J . M a r . R e s . 37: 657-668.  D'elia, C F . and J . A . D e B o e r . 1978. Nutritional studies of two red algae II. Kinetics of a m m o n i u m and nitrate uptake. J . Phycol. 14: 266-272.  D e m a n c h e , J . M . and H . C C u r l .  1979. T h e rapid response of the marine diatom  Skeletonema costatum to changes in external and internal nutrient concentration. M a r . B i o l . 53: 323-333.  90  D o r t c h , Q . 1982. Effect of growth conditions on accumulation of internal pools of nitrate, ammonium, amino acids and protein i n three marine diatoms. J . E x p . M a r . B i o l . E c o l . 61: 243-264.  D o r t c h , Q . and H . L . Conway. 1984. Interactions between nitrate and a m m o n i u m uptake: variations with growth rate, nitrogen source and species. M a r . B i o l . 79: 151-164.  E p p l e y , R . W . and E . H . Renger. 1974. Nitrogen assimilation of an oceanic diatom in nitrogen limited continuous culture. J . Phycol. 10: 15-23.  Eppley, R . W . and B J . Peterson. 1979. Particulate organic matter flux and planktonic new production in the deep ocean. Nature 282: 677-680.  Falkowski, P . G . 1975. Nitrate uptake in marine phytoplankton: comparison of halfsaturation constants from seven species. L i m n o l . Oceanogr. 20: 412-417.  Falkowski, P . G . and D . P . Stone. 1975. Nitrate uptake in marine phytoplantkon: energy sources and the interaction with carbon fixation. M a r . B i o l . 32: 7784.  Florencio, F J . and J . M . V e g a . 1983. Utilization of nitrate, nitrite and a m m o n i u m by Chlamydomonas reinhardii. Planta 158: 288-293.  Flores, E . , M . G . G u e r r e r o and M . L o s o d a . 1980. Short-term a m m o n i u m inhibition of nitrate utilization by Anacystis nidulans and other cyanobacteria. A r c h . M i c r o b i o l . 128: 137-144.  Funkhousor, E . A . and C . S . Ramados. 1980. Synthesis of Nitrate reductase in Chlorella. II. evidence for synthesis in a m m o n i u m grown cells. Plant Physiol. 65: 944-948.  Garside, E C . 1981. Nitrate and a m m o n i u m uptake in the apex of the N e w Y o r k Bight. L i m n o l . Oceanogr. 26: 731-739.  Glibert, P . M . , D . C . Biggs and J.J. M c C a r t h y . 1982. Utilization of a m m o n i u m and nitrate during austral summer in the Scotia Sea. D e e p - S e a Res. 29: 837850.  Glibert, P . M . and J . C . G o l d m a n . 1981. R a p i d a m m o n i u m uptake by marine phytoplankton. M a r . B i o l . Lett. 2: 25-31.  91  G o l d m a n , J . C . and P . M . Glibert. 1982. Comparative rapid a m m o n i u m uptake by four species of marine phytoplankton. L i m n o l . Oceanogr. 27: 814-827.  Haines, K . C . and P . A . Wheeler. 1978. A m m o n i u m and nitrate uptake by the marine macrophytes Hypnea musciformis (Rhodophyta) and Macrocystis pyrifera (Phaeophyta). J . Phycol. 14: 319-324.  Hanisak, D . M . and M . M . H a r l i n . 1978. U p t a k e of inorganic nitrogen by Codium fragile subsp. tomentosoides (Chlorophyte). J . Phycol. 14: 450-454.  Harrison, P.J., H . L . Conway and C . Dugdale. 1976. M a r i n e diatoms grown in chemostats under silicate or a m m o n i u m limitation. I. Cellular chemical composition and steady-state growth kinetics of Skeletonema costatum. M a r . B i o l . 35:177-186. Harrison, P.J., R . E . Waters and F . J . R . Taylor. 1980. A broad spectrum artificial seawater m e d i u m for coastal and open ocean phytoplankton. J . Phycol. 16: 28-35.  Harrison, P . J . and D . H . T u r p i n . 1982. T h e manipulation of physical, chemical, and biological factors to select species from natural phytoplankton communities. In: M a r i n e Mesocosms: Biological and chemical research in experimental ecosystems, G . D . G r i c e and M . R . R e e v e (ed.), Springer-Verlag New Y o r k . , p.275-289.  H a r r i s o n , P . J . , L . D . D r e u h l , K . E . L l o y d and P . A . T h o m p s o n . 1986. Nitrogen uptake kinetics in three-year classes of Laminaria groenlandica (Laninariales: Phaeophyta). M a r . B i o l . 93: 29-35.  H a r r i s o n , W . G . 1980. Nutrient regeneration and primary production in the sea. In: Primary Productivity in the Sea, P . G . Falkowski (ed.), P l e n u m Press, New Y o r k . , p.433-460.  Healey, F . P . 1985. Interacting effects of light and nutrient limitation o n growth rate of Synechococcus linearis (Cyanophyceae). J . Phycol. 21: 30: 134-146.  Heaney, H . I . and R . W . E p p l e y . 1981. Light, temperature and nitrogen as interacting factors affecting diel vertical migration of dinoflagellates in culture. J . Plankton Res. 3: 331-344.  H o r r i g a n , S . G . and J J . M c C a r t h y . 1982. Phytoplankton uptake of a m m o n i u m and urea during growth on oxidized forms of nitrogen. J . Plankton Res. 4: 379389.  92  Laws, E . A . and T . T . Bannister. 1980. Nutrient and light-limited growth of Thalassiosira fluviatilis in continuous culture with implications for phytoplankton growth in the ocean. L i m n o l . Oceanogr. 25: 457-473.  L e h m a n , J . T . and D . Scavia. 1982. Microscale patchiness of nutrients in plankton communities. Science 216: 729-730.  L u i , N . S . T . and O . A . Roels. 1972. Nitrogen metabolism of aquatic organisms. II. T h e assimilation of nitrate, nitrite and a m m o n i u m by Biddulphia aurita. J . Phycol. 8: 259-264.  Maclsaac, J . J . and R . C . Dugdale. 1972. Interaction of light and inorganic nitrogen in controlling nitrogen uptake i n the sea. D e e p - S e a Res. 19: 209-232.  M a c l s a a c , J.J., R . C . Dugdale, R . T . Barber, D . Blasco and T . T . Packard. 1985. Primary production cycle in an upwelling center. D e e p - S e a Res. 32: 503530.  Maestrini, S . Y . , J . Robert and I. Truauet. 1982. Simultaneous uptake of nitrate and a m m o n i u m by oyster pond algae. M a r . B i o l . Lett. 3: 143-153.  Maestrini, S . Y . , J . Robert, J . W . Leftley and Y . Collos. 1986. A m m o n i u m thresholds for simultaneous uptake of a m m o n i u m and nitrate by oysterp o n d algae. J . E x p . M a r . B i o l . E c o l . 102: 75-98.  M c C a r t h y , J.J., W . R . Taylor, and J . L . Taft. 1977. Nitrogenous nutrition of the phytoplankton i n the Chesapeake Bay. I. Nutrient availability and phytoplankton preferences. L i m n o l . Oceanogr. 22: 996-1011.  M c C a r t h y , J . J . and J . C . G o l d m a n . 1979. Nitrogenous nutrition of marine phytoplankton in nutrient depleted waters. Science. 203: 670-672.  Nelson, D . M . and H . L . Conway. 1979. Effects of the light regime o n nutrient assimilation by phytoplankton i n the Baja California and northwest A f r i c a upwelling systems. J . M a r . Res. 37: 301-318.  O h m o r i , M . , K . O h m o r i and H . Strotmann. 1977. Inhibition of nitrate uptake in a blue-green alga, Anabaena cylindrica. A r c h . M i c r o b i o l . 114: 225-229.  O h m o r i , M . and A . Hattori. 1978. Transient change i n the A T P p o o l of Anabaena cylindrica associated with ammonia assimilation. A r c h . M i c r o b i o l . 117: 1720.  93  Parslow, J.S., P . J . H a r r i s o n and P . A . T h o m p s o n . 1984a. U s e of a self-cleaning inline filter to continuously monitor phytoplankton nutrient uptake rates. C a n . J . Fish. A q u a t . Sci. 41: 540-544.  Parslow, J.S., P . J . H a r r i s o n and P . A . T h o m p s o n . 1984b. Saturated uptake kinetics: transient response of the marine diatom Thalassiosira pseudonana to a m m o n i u m , nitrate, silicate or phosphate starvation. M a r . B i o l . 83: 51-59.  Parslow, J.S., P . J . H a r r i s o n and P . A . T h o m p s o n . 1985a. A m m o n i u m uptake by phytoplankton cells on a filter: a new high-resolution technique. M a r . E c o l . Prog. Ser. 25: 121-129.  Parslow, J.S., P . J . H a r r i s o n and P . A . T h o m p s o n . 1985b. Interpreting rapid changes in uptake kinetics in the marine diatom thalassiosira pseudonana (Hustedt). J . E x p . M a r . B i o l . E c o l . 91: 53-64.  Price, N . M . P . A T h o m p s o n and P . J . Harrison. 1987. Selenium: an essential element for growth of the coastal marine diatom Thalassiosira pseudonana (Bacillariophyceae). J . Phycol. 23: 1-9.  Quarmby, L . M . , D . H . T u r p i n and P . J . Harrison. 1982. Physiological responses of two marine diatoms to pulsed additions of ammonium. J . E x p . M a r . B i o l . E c o l . 63: 173-181.  Raimbault, P. and M . mingazzini. 1987. D i u r n a l variations of intracellular nitrate storage by marine diatoms: effects of nutritional state. J . E x p . M a r . B i o l . E c o l . 112: 217-232.  R a v e n , J . A 1980. Nutrient transport in microalgae. A d v . M i c r o b i o l , physiol. 21: 47-226.  R h e e , G . Y . , and I J . G o t h a m . 1981. T h e effect of environmental factors on phytoplankton growth: light and interactions of light with nitrate limitation. L i m n o l . Oceanogr. 26: 649-659.  Ritchie, R J . and A . W . D . L a r k u m . 1987. T h e ionic relations of small-celled marine algae. Progress in Phyiological Research 5: 179-222.  Robert, J . - M . et S . Y . Maestrini. 1986. Absorptions simultanees des ions N O 3 at N H par trois diatomees de claires a huitres, en culture axenique. Phycologia 25:152-159. 4  94  Rigano, C , G . A l i o t t a and U . Violante. 1974. Reversible inactivation by a m m o n i u m of assimilatory nitrate reductase in G y a n i d i u m caldarium. M i c r o b i o l . 99: 81-90.  Ryther, J . H . and W . M . Dunstan. 1971. Nitrogen, phosphorus and eutrophication in the coastal environment. Science 171: 1008-1013.  Serra, J . L . , M . J . L l a m a , and E . Codenas. 1978. Nitrate utilization by the diatom Skeletonema costatum. 2. Regulation of nitrate uptake. P l . Physiol. 62: 991994.  Slawyk, G . and J J . Maclsaac. 1972. C o m p a r i s o n of two automated a m m o n i u m methods in a region of coastal upwelling. D e e p Sea Res. 19: 521-524.  Spencer, C . N . and D . L . K i n g . 1985. Interactions between light, N H , and CO2 in buoyancy regulation of A n a b a e n a flos-aquae (Cyanophyceae). J . Phycol. 21: 194-199. 4  Stross, R . G . 1963. Nitrate preference in Haematococcus as controlled by strain, age of inoculum and p H of the medium. Canadian J . of M i c r o b i o l . 9: 33-40.  Syrett, P J . and I. Morris. 1963. T h e inhibition of nitrate assimilation by a m m o n i u m in chlorella. Biochimica et Biophysica A c t a 67: 566-575.  Takahashi, M . and Y . Saijo. 1981. Nitrogen metabolism in L a k e K i z a k i Japan: A m m o n i u m and nitrate uptake by phytoplankton. A r c h . H y d r o b i o l . 91: 393-407.  Thacker, A . and P J . Syrett. 1972. Assimilation of Nitrate and a m m o n i u m by Chlamydomonas reinhardi. New Phytol. 71: 423-434.  T o p i n k a , J . A . 1978. Nitrogen uptake by 14: 241-247.  Fucus spiralis (Phaeophyceae). J . Phycol.  T u r p i n , D . H . and P J . Harrison. 1978. Fluctuations in free amino acid pools of Gymnodinium simplex (Dinophyceae) in response to a m m o n i a perturbation: evidence for glutamine synthetase pathway. J . Phycol. 14: 461-464.  W a r d , A . K . and R . G . Wetzel. 1980. Interaction of light and nitrogen source among planktonic blue-green algae. A r c h . H y d r o b i o l . 90: 1-25.  95 W o o d , E . D . , F.A.J. Armstrong and F . A . richards. 1967. Determination of nitrate in seawater by cadmium-copper reduction to nitrite. J. M a r . B i o l . Assoc. U . K . 47: 23-31.  Wynne, D . and G - Y . R h e e . 1986. Effects of light intensity and quality on the relative N and P requirement (the optimum N : P ratio) of marine planktonic algae. J. Plankton Res. 8: 91-103.  Z e v e n b o o m , W . and L . R . M u r . 1981. Simultaneous short-term uptake of nitrate and a m m o n i u m by Oscillatoria agardhii grown in nitrate-or light-limited continuous culture. J. G e n . M i c r o b i o l . 126: 355-363.  Z e v e n b o o m , W . , G.J. D E G R O O T and L . R . M U R . 1980. Effects of light on nitrate-limited Oscillatoria aghardii in chemostat cultures. A r c h . M i c r o b i o l . 125: 59-65.  Z i m m e r m a n , R . C , J . N . K r e m e r and R . C . Dugdale. 1987. Acceleration of nutrient uptake by phytoplankton in a coastal upwelling ecosystem. A modelling analysis. Lirnno. Oceanogr. 32: 359-367.  96  APPENDIX  APPENDIX A. of N 0  Standard e r r o r s a s s o c i a t e d w i t h the r e p l i c a t i o n 3  uptake r a t e measurements f o r d i f f e r e n t  cultures  A.l.  C u l t u r e LO  A.2.  C u l t u r e L2  A.3.  C u l t u r e L3  97 A.l.  C u l t u r e LO: S t a n d a r d e r r o r s r e p l i c a t i o n of N0  3  (SE) a s s o c i a t e d w i t h t h e  uptake r a t e measurements  ( 5 . 0 uM  amounts l o a d e d o n t o t h e f i l t e r i s t h e  N 0 ) . The c e l l 3  same ( 4 . 3 x 1 0  6  m l " ) :f o r t h e t h r e e p u l s e s . Mean i s 1  the v a l u e averaged the time course  Uptake r a t e  o v e r t h e 2--4 m i n t i m e  (along the  i n t e r v a l from  column).  (mmole ( l i t e r c e l l v o l u m e ) -  1  h" ) 1  Pulsing  C e l l number l o a d e d (10 ml" )  time  1st  2nd  3rd  (min)  4.3  4.3  4.3  2.00 2.17 2 . 34 2 . 50 2.67 2.83 3.00 3.17 3.34 3.50 3 .67 3.83 4.00  318 315 307 311 315 311 315 318 318 307 318 318 326  308 312 316 326 323 319 326 326 341 337 337 330 323  291 277 281 281 281 277 277 277 274 274 277 281 277  300 297 297 302 301 300 304 306 309 306 310 310 308  17 19 17 21 21 19 22 22 28 26 25 21 22  5.6 6.5 5.9 6.8 6.9 6.3 7.1 7.1 9.1 8.5 8.2 6.8 7.2  315  325  279  306  24  8.0  Mean  6  -  Average  1  SE  Coefficient of  variation (%)  98 A.2.  C u l t u r e L2: Standard e r r o r s r e p l i c a t i o n o f N0 N0 ). 3  The c e l l  different  3  (SE) a s s o c i a t e d w i t h the  uptake r a t e measurements (5.0 uM  amounts loaded onto the f i l t e r were  f o r the two p u l s e s . Mean i s t h e v a l u e  averaged over the 2-4 min time i n t e r v a l from t h e time course  (along the column).  Uptake r a t e Pulsing  (mmole ( l i t e r  C e l l number loaded (10° ml"*--) 1  time  1st  2nd  (min)  3.2  12.6  2.00 2.17 2.34 2.50 2.67 2.83 3 . 00 3 .17 3.34 3.50 3 . 67 3.83 4 . 00  182 161 140 140 136 136 136 131 136 127 110 68 114  118 116 114 111 113 112 112 110 111 111 107 105 106  Mean  132  111  c e l l. v o l u m e )  Average  SE  - 1  h  variation (%)  Q  )  Coefficient of  X  - 1  150 139 127 125 124 124 124 121 123 119 109 87 110  45 32 18 21 16 17 17 15 18 12 2 27 6  30 23 14 16 17 13 14 13 14 10 2 31 5  122  15  12  99 A.3.  C u l t u r e L3: Standard e r r o r s r e p l i c a t i o n o f N0 N0 ). 3  The c e l l  different averaged course  3  (SE) a s s o c i a t e d w i t h t h e  u p t a k e r a t e m e a s u r e m e n t s ( 5 . 0 uM  number l o a d e d o n t o t h e f i l t e r i s  f o r t h e t h r e e p u l s e s . Mean i s t h e v a l u e o v e r t h e 2-4 m i n t i m e i n t e r v a l f r o m t h e t i m e  (along t h e column).  Uptake r a t e  (mmole ( l i t e r c e l l  volume)  h  x  )  Pulsing  C e l l number l o a d e d (10 ml )  time  1st  2nd  3rd  (min)  1.7  3.7  6.6  2.00 2.17 2.34 2.50 2.67 2 .83 3.00 3.17 3.34 3.50 3 . 67 3.83 4.00  168 162 151 133 128 110 99 99 87 93 151 110 109  87 76 79 79 87 92 87 92 76 76 84 90 84  118 121 118 118 117 120 121 118 121 118 121 108 120  124 120 116 110 110 107 102 103 95 96 119 102 104  41 43 36 28 21 14 17 13 24 21 33 11 18  33 36 31 26 19 13 17 13 25 22 28 11 17  Mean  123  84  118  108  21  20  6  Average  - 1  SE  Coefficient of  variation (%)  APPENDIX B.  Variation  cultures  o f some p a r a m e t e r s  i n the turbidostat  o v e r a few d a y s  B.l.  Variation  of s p e c i f i c  growth  rate  B.2.  Variation  of c e l l  B.3.  Variation  o f biovolume  B.4.  Variation  of particulate  B.5.  Variation  of fluorescence per c e l l  density  nitrogen  B.l.  V a r i a t i o n o f s p e c i f i c growth r a t e biovolume i n the t u r b i d o s t a t s  (h  - 1  ) based on  over a few days  b e f o r e the experiments were conducted  Date  June June June June June June June June June  Culture  4 5 6 7 8 9 10 11 12  Average  LO  Ll  L2  L3  0.061 0.058 0.057 0.061 0.061* 0.060 0.062 0.066 0.060  0.036 0.032 0.038 0.032 0.035 0.034 0.041* 0.035  0.024 0.024* 0.023 0.024  0.012 0.012 0.012 0.012* 0.010  0.061  0.035  0.024  0.012  0.003  0.003  0.000  0.001  4.2%  8.7%  2.1%  9.5%  Standard error Coefficient of v a r i a t i o n *  r e p r e s e n t s t h e day on which t h e samples from t h a t c u l t u r e were taken f o r the experiments.  B.2.  V a r i a t i o n of c e l l  density  (10° m l ~ ) x  i n the  t u r b i d o s t a t s o v e r a few d a y s b e f o r e t h e e x p e r i m e n t s were Date  Culture LO 4 5 6 7 8 9 10 11 12 13  June June June June June June June June June June  7.1 7.8 7.8 7.6 7.4* 7.4 7.6 7.5 7.8 6.9  LI  3.8 3.9 4 . 1* 4.7 4.4  L2 6.0 6.0 5.5 5.4 5.8 6.2* 6.1 6.3 6.8 6.2  L3  3.2 2.9 2.8 3.1 2.8* 2.6  Aver?ige (h" )  7.5  4.2  6.0  2.9  Standard error  0.3  0.3  0.4  0.2  Coefficient of v a r i a t i o n  4.4%  8.2%  6.7%  7.1%  5  *  conducted.  r e p r e s e n t s t h e d a y on w h i c h t h e s a m p l e s f r o m c u l t u r e were t a k e n f o r t h e e x p e r i m e n t s .  that  103 B.3.  V a r i a t i o n of biovolume ( 1 0 um 7  turbidostats  3  ml ) - 1  i n the  over a few days b e f o r e t h e experiments  were conducted Date  Culture LO  June June June June June June June June June June  4 5 6 7 8 9 10 11 12 13  Aver?ige  1.7 1.6 1.5* 1.7 1.6  L2  L3  2.1 2.2 2.2 2. 3 2.4* 2.4  1.6 1.5 1.7 1.5 1.5 1.6 1.6 1.7 1.6 1.7  2.5  1.6  2.3  1.6  Standard error  0.1  0.1  0.0  0.1  Coefficient of v a r i a t i o n  3.7%  4.8%  5.5%  4.1%  )  *  2.4 2.7 2.6 2.4 2.4* 2.5 2.5 2.5  LI  r e p r e s e n t s the day on which t h e samples from t h a t c u l t u r e were taken f o r the experiments.  104 B.4.  V a r i a t i o n of p a r t i c u l a t e n i t r o g e n turbidostats  (uM) i n t h e  over a few days b e f o r e t h e experiments  were conducted Date  Culture Ll  L2  L3  62.4 64.6 62.9 58.8* 61.4 61.6 62.7  52 . 3 50.5 47.3* 47.6  75.7 75. 3 72.2 72.7 74 .9* 76.1 81.8  53 .9 55. 1 53.9 50.0 53 . 6  62.1  49.4  75.5  53.8  Standard error  1.8  2.2  3.2  1.9  Coefficient of v a r i a t i o n  2.9 %  4.5 %  4.2 %  3.5 %  LO June June June June June June June  5 6 7 8 9 10 11  Average  *  56.1*  r e p r e s e n t s the day on which t h e samples from t h a t c u l t u r e were taken f o r t h e experiments.  105 B.5.  V a r i a t i o n of f l u o r e s c e n c e per c e l l turbidostats  (10~ ) i n the D  over a few days b e f o r e t h e experiments  were conducted Culture  Date LO June June June June June June June June June June  *  4 5 6 7 8 9 10 11 12 13  Ll  L2 2.09 2.08 2.24 2.31 2.15 2 .19* 1.99 2.15 1.99  L3  0.99 0.94 1.05 1.05 1.06* 0. 98 0.98 0.96  2.37 2.08 1.78* 1.83  Average )  0.99  2 . 01  2. 13  3.06  Standard error  0. 06  0.27  0.11  0.23  Coefficient of v a r i a t i o n  6.3 %  13.5 %  5.1 %  7.4 %  3. 07 2.83 3 . 07 3.18 2.81 3 . 51* 3.10 2.91  r e p r e s e n t s t h e day on which the samples from t h a t c u l t u r e were taken f o r the experiments.  106 APPENDIX C.  V a r i a t i o n o f N 0 uptake r a t e  c e l l volume)"  (mmole  3  1  (liter  h " ) over a 2-4 min time i n t e r v a l 1  from  a time course, i n c l u d i n g standard e r r o r and c o e f f i c i e n t o f v a r i a t i o n (Coeff. V a r . ) -  The r e d u c t i o n  of N O 3 uptake r a t e i n the presence o f NH the r a t e i n t h e absence  o f NH  NH,,  4  4  i s included  relative to (N0H/N0)  additions  0.0  0.25  0.5  1.0  3.0  5.0  C u l t u r e LO  312  255  212  153  80  103  NOH/NO (%)  100  82  70  49  26  33  Standard E r r o r  16  13  4  7  16  9  C o e f f . V a r . (%)  5  5  2  5  20  9  Culture LI  204  138  70  67  72  65  NOH/NO (%)  100  68  34  33  36  32  Standard E r r o r  24  9  16  7  9  10  C o e f f . Var. (%)  12  7  23  11  12  16  C u l t u r e L2  98  87  46  70  42  43  NOH/NO (%)  100  88  47  71  42  44  Standard E r r o r  5  30  no  19  4  4  C o e f f . Var. (%)  5  34  no  28  10  8  C u l t u r e L3  105  92  149  103  126  159  NOH/NO (%)  100  88  141  98  120  151  Standard  8  7  8  13  13  8  8  8  6  13  11  5  Error  C o e f f . Var. (%)  no means "not c a l c u l a t e d " .  

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