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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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S H O R T - T E R M I N T E R A C T I O N B E T W E E N N I T R A T E A N D A M M O N I U M U P T A K E F O R C E L L S O F A M A R I N E D I A T O M G R O W N U N D E R D I F F E R E N T D E G R E E S O F L I G H T L I M I T A T I O N by K E D O N G Y I N B . S c , The Shandong College of Oceanography, China, 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 R E Q U I R E M E N T F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S ( D E P A R T M E N T O F O C E A N O G R A P H Y ) 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 tAo^rd 3 . DE-6(3/81) 11 A B S T R A C T T h e short-term interaction between nitrate and ammonium uptake was examined for a marine diatom, Thalassiosira pseudonana, grown in the continuous turbidostat cultures under different degrees of light limitation. Nitrate uptake in the absence and in the presence of ammonium and ammonium uptake in the absence and in the presence of nitrate were measured during a 6 min time course after a solution of both nitrate and ammonium was passed across the cells trapped on the filter. It was found that the interaction between nitrate and ammonium uptake occurred immediately and continued for the remainder of the time course. The effect of light on the interaction was apparent. In the less light-limited cultures, nitrate uptake was depressed by ammonium. In contrast, in the most light-limited culture, the depression of nitrate uptake by ammonium disappeared. A m m o n i u m uptake was dependent on the degree of light limitation of the cultures. For all the cultures, ammonium uptake was initially enhanced and then declined with time. However, only the initial (1 min) enhanced uptake of ammonium was supressed by the presence of nitrate, and the subsequent ammonium uptake rate was unaffected. Possible explanations and the ecological significance of the interaction are discussed. T A B L E O F C O N T E N T S i i i A B S T R A C T . ii L I S T O F T A B L E S viii L I S T O F F I G U R E S ix A C K N O W L E D G E M E N T xiv I N T R O D U C T I O N 1 Light and nutrients in the ocean 1 Interaction between light and nitrogen 2 Ecological significance of the interaction among nitrate, ammonium and irradiance 2 Laboratory studies on the interaction among nitrate, ammonium and irradiance 5 1) Nitrate and ammonium interaction at saturating light intensity 6 2) Nitrate and ammonium interaction under light limitation 8 i v M A T E R I A L S A N D M E T H O D S 10 Organism 10 Culture medium 10 Culturing 10 Nitrogen analysis 11 Nutrient Uptake measurement 12 R E S U L T S 19 Cultures 19 Interaction experiments 19 A . T ime course of the interaction between nitrate and ammonium uptake 19 1) Nitrate uptake with and without ammonium 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 ammonium uptake 41 1) Nitrate uptake with and without ammonium 41 2) A m m o n i u m uptake with and without nitrate 54 D I S C U S S I O N 69 Cultures 69 Interaction experiments 70 A . T i m e course of the interaction between nitrate and ammonium uptake 71 1) Nitrate uptake with and without ammonium 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 ammonium uptake 77 1) Nitrate uptake with and without ammonium 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 S I G N I F I C A N C E 81 v i SUMMARY 87 LITERATURE CITED 89 APPENDIX 96 APPENDIX A. STANDARD ERRORS ASSOCIATED WITH T H E REPLICATION OF NITRATE UPTAKE R A T E MEASUREMENTS FOR DIFFERENT CULTURES 96 A . l . Culture LO '. 97 A.2. Culture L2 98 A. 3. Culture L3 99 APPENDIX B. VARIATION OF SOME PARAMETERS IN T H E TURBIDOSTATS OVER 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 v i i 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 T I M E I N T E R V A L F R O M T H E T I M E C O U R S E S 106 LIST OF TABLES v i i i 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 L I S T O F F I G U R E S i x 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) 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 Figure 2. Specific growth rate vs irradiance for batch cultures. The vertical bars represent + 1 standard error (n > 5) 20 Figure 3. Light saturated culture (150 u E m" 2 s"*) LO: time course of NO3 uptake in the absence of NH4 (0) and in the presence of different NH4 concentrations: 0.25 u M (+), 0.5 u M (a), 1.0 u M (x), 3.0 u M (A) and 5.0 u M (o) 24 Figure 4. Light limited culture (17 u E m" 2 s"*) L I : time course of NO3 uptake in the absence of NH4 (O) 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) 26 Figure 5. Light limited culture (9 u E m" 2 s"1) L2: time course of NO3 uptake in the absence of NH4 (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 " z s ) L 3 : time course of N O 3 uptake in the absence of N H 4 (0) 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 u M (O) 30 Figure 7. Light saturated culture (150 u E m"^ s"*) L 0 : 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) 32 Figure 8. Light limited culture (17 u E m" z s ) L l : 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) 34 Figure 9. Light limited culture (9 u E m" z s"1) L2: 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) 36 Figure 10. Light limited culture (2 u E m" z s ) 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) 38 Figure 11. Light saturated culture (150 u E m"^ s"*) L0: time course of the interaction between N O 3 uptake and N H 4 uptake after a pulse of a x i 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" z s"A) L l : time course of the interaction 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"*) L2: 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" z s ) 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 (+), L2 (A) and L3 (O) (from Figs. 7, 8, 9 and 10). 50 x i i Figure 16. NH4 uptake at 5.0 u M concentration in the absence of NO3 (A) and in the presence of NO3 (B) for the cultures grown at different irradiances: LO (•), L I (+), 12 (A) and L3 (O) (from Figs. 7, 8, 9 and 10) 52 Figure 17. A . NO3 uptake vs growth irradiance in the absence of NH4 (•) and in 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 . NO3 uptake vs NH4 concentration for each culture: L 0 (•), L I (+), L 2 (A) and L3 (O) (replotted from A ) . (The NO3 uptake rate is averaged over the 2-4 min time interval during the time course in Figs. 3, 4, 5 and 6). C . NO3 uptake rate vs irradiance (in log scale) and NH4 concentration 55 Figure 18. A . NH4 uptake vs growth irradiance in the absence of NO3 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 . NH4 uptake vs NH4 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" 2 s"^ ) L0: NH4 concentration effect on the interaction between NO3 and NH4: NH4 uptake in the absence of NO3 (•), NH4 uptake in the presence of NO3 (-)-) and NO3 uptake in the presence of N H 4 (A) (from Figs. 17B and 18B) 61 x i i i Figure 20. Light limited culture (17 u E m" 2 s"*) L I : N H 4 concentration effect on 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 4 (A) (from Figs. 17B and 18B) 63 Figure 21. Light limited culture ( 9 u E m" 2 s"^ ) L 2 : N H 4 concentration effect on 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 4 (A) (from Figs. 17B and 18B) 65 Figure 22. Light limited culture (2 u E m" 2 s"*) L 3 : N H 4 concentration effect on 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 4 (A) (from Figs. 17B and 18B) 67 Figure 23. T h e ratio of uptake rate (h"*) of N H 4 alone (avereged over 1-5 min) to maximal specific growth rate (h"^) at the growth irradiance for the pulses of N H 4 : .25 u M (A), 0.5 u M (+), 1.0 u M (•), 3.0 u M (x) and 5.0 u M (o)...82 xiv A C K N O W L E D G E M E N T I am very grateful to my supervisor, D r . P . J . Harrison, for his guidance during the whole period of my study and research. M y fellow student, P . A . Thompson, provided valuable practical advice on the experimental design, techniques and the discussion of the results. I also appreciate the help from other fellow students, W . P . Cochlan, G . J . Doucette and P . J . Clifford. I am thankful for the financial support provided by a scholarship from the International Development Research Center of Canada. 1 I N T R O D U C T I O N Light and nutrients in the ocean Light and nutrients are two major driving forces in marine ecosystems. They partially determine primary production and therefore indirectly regulate higher trophic production. However, these parameters vary with time and space in the ocean. There are diel and seasonal changes in light intensity as well as an attenuation with depth in 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 in the ocean. Nutrients including nitrogen, phosphorus, silicate, trace metals and vitamins also vary with time and space largely due to biological activities. Although 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). The 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. Near the bottom of the euphotic zone, light becomes limiting while nutrients are generally present in saturating concentrations. F o r example, in field investigations, Maclsaac and Dugdale (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 in 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 African 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 in many cases. Interactions between light and nitrogen Frequently in areas of mixing, phytoplankton are growing under transient conditions. During 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. Rhee and Gotham (1981) investigated the effects of simultaneous limitation of light and nitrate on 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 on the growth rate of Synechococcus linearis (Cyanophyceae). H e found that the biomass responded to changes in both the irradiance and 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 in many different forms including nitrate, nitrite, ammonium, urea and free amino acids. Some of these 3 forms are preferred over others (e.g. the preference of ammonium over nitrate). The utilization of different forms might affect algal growth, species composition and species succession (Harrison and Turpin 1982). It is well known that limiting nutrients have selective roles in 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 Eppley 1981). For species which cannot swim, the capacity to quickly take up a transient supply of the nutrient (in a patch) would likely be an advantage. For example, there is clear evidence for rapid ammonium 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. One of the most important forms of nitrogen is ammonium. A m m o n i u m in seawater can be released by biological activities including zooplankton excretion, bacterial decomposition and schooling fish. Regenerated ammonium has been found to be so substantial that it accounts for a significant portion of the primary production. For example, an investigation made by Conway and Whitledge (1979) during a spring bloom in the New Y o r k Bight showed that measurements of ammonium utilization as a percentage of ammonium 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 Eppley and Peterson (1979) and Harrison (1980) clearly support this generalization. T h e regeneration of ammonium is sporadic, but its concentrations in marine waters is low, ranging from 0 to 5 u M . The kinetics of ammonium uptake by phytoplankton partially explain these observations. A laboratory study with the diatom Biddulphia aurita showed that cells removed ammonium over twice as fast as nitrate or nitrite (Lui 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 ammonium as a nitrogen source always resulted in the highest growth rates under all light regimes (Ward and Wetzel 1980). T h e preference for ammonium over nitrate and the rapid uptake of ammonium by algae have been commonly observed both in field investigations (e.g. McCarthy 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. Hanisak and Har l in (1978) found that in some macroalgae, maximum uptake rates of ammonium were higher and the half-saturation constant for ammonium was lower than those for nitrate or nitrite. It appears that the interaction of ammonium 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 ammonium 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 ammonium in order to acquire its daily ration of nitrogen for growth (McCarthy and Goldman 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 (Lehman and Scavia 1982). Even in nitrogen saturated waters, algae with such physiological properties are able to take up more nitrogen than other algae over the same time period, resulting in faster growth. Light conditions could also change the availability of ammonium, nitrate and other nutrients and therefore change the relative requirements between nitrogen and other nutrients by phytoplankton. Wynne and Rhee (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 in the light regime can strongly influence algal nutrient requirements and species interrelationships by altering the optimum cellular N:P ratio. Another study with Cyanophyceae showed that only at low irradiance, the availability of ammonium and CC>2 controlled the buoyancy of the species (Spencer and King 1985). Therefore, interactions between ammonium and nitrate uptake under different light conditions could change the nutritional regime of algal cells and as a consequence, cause a change in 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 ammonium and nitrate uptake under different light intensities. Laboratory studies on the interactions among nitrate, ammonium and light Nitrate and ammonium 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. Maclsaac and Dugdale 1972). The interaction of ammonium 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 common interaction reported is the inhibition of nitrate uptake by ammonium or the preference of ammonium over nitrate in utilization. Syrett and Morris (1963) found that nitrate assimilation by Chlorella vulgaris growing with ammonium plus nitrate ( N H 4 + N O 3 ) ceased completely when ammonium was added and commenced again as soon as ammonium had disappeared. Later, Thacker and Syrett (1972) and Florencio and Vega (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 ammonium, where the degree of the inhibition was related to the ammonium concentration (Ohmori et al. 1977). Conway (1977) found ammonium concentration effects on 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 ammonium to exhibit its full inhibitory effect on nitrate uptake. Even the macroalgae Gracilaria foliifera and Neoagardhiella baileyi ( D ' E l i a and D e B o e r 1978) and Hypnea musciformis and Macrocystis pyrifera (Phaeophyta) (Haines and Wheeler 1978) showed preferential utilization of ammonium over nitrate, but the presence of nitrate had no effect on ammonium uptake. Serra et al. (1978) studied nitrate utilization characterized in nitrogen-deficient cells of the diatom Skeletonema costatum and observed that ammonium strongly inhibited carrier-mediated nitrate uptake without affecting diffusion. It was reported that ammonium 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). Dortch and Conway (1984) conducted more comprehensive experiments on the interaction between nitrate and ammonium uptake. They found that the interaction varied with growth rate, nitrogen source and species. After the simultaneous addition of ammonium and nitrate to the cultures which were preconditioned on nitrate or ammonium and grown at different growth rates, they found that both nitrate and ammonium uptake rates decreased in comparison with the rates observed when each nutrient was added alone, although nitrate uptake decreased more than ammonium uptake. Simultaneous uptake of both nitrate and ammonium has been frequently reported for other algae such as a red macroalga Gelidium nudifrons (Bird 1976), a cyanobacterium, Oscillatoria agardhii (Zevenboom 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). The later researchers also reported ammonium thresholds for simultaneous uptake above which nitrate uptake was still inhibited. However, Topinka (1978) reported that high levels of ammonium 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 ammonium utilization. Stross (1963) observed a preference for nitrate over ammonium 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 medium to determine the preference for ammonium or nitrate. DeManche 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 ammonium, 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 ammonium was taken up. A t this point, nitrate uptake resumed and nitrate assimilation began. In contrast, Harrison et al. (1986) observed complete supression for 30 minutes of nitrate uptake before the macroalga Laminaria groenlandica (Phaeophyta) took up nitrate and ammonium equally after NO3 and NH4 were added together. More recently, Collos et al. (1986) reported a long-term elevated NO3 uptake by oyster-pond microalgae in the presence of high ammonium concentrations. They 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. There are even fewer studies on the interaction between nitrate and ammonium uptake under light limitation. In the experiments by Syrett and Morris (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 ammonium 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. Then the cultures were transferred to flasks under a series of light intensities and incubated for two hours to measure uptake rates. His 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 Garside (1981) in the New 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 ammonium 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. Also, 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 ammonium for continuous cultures of a diatom grown under different irradiances. 10 M A T E R I A L S A N D M E T H O D S Organism The marine diatom Thalassiosira pseudonana (Hust.) Hasle and Heimdal ( W H O I clone 3H) was grown in batch and turbidostat cultures. The 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 Columbia, B . C . , Canada. Culture medium The culture medium was artificial seawater with a full enrichment of nutrients (minus nitrate nitrogen), trace metals and vitamins ( E S A W ) . It was modified from Harrison et al. (1980) by adding 10"8 u M N a 2 S e 0 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. The batch cultures were grown with full nitrate enrichment (550 u M ) and for the turbidostats, the NO3 concentration in the reservoir was 100 u M . The ambient NO3 concentration always remained above 20 u M in the turbidostat culture medium, ensuring that the cultures were never nitrogen-limited. Culturing Batch cultures were grown in 50 ml testubes in a 17° C water bath under continuous fluorescent lighting (48T12 U H O , Vita-Lite, 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. The amount of light was measured using a quantum meter (Li -Cor , model Ll-185) with a cosine collecter. The growth rates in 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. The 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. The four turbidostat cultures were grown under continuous lighting but at different irradiances 150 (LO), 17 (LI) , 9 (L2) and 2 (L3) u E m" 2s~l. These irradiances were chosen from the results of the batch culture experiments and assumed to represent a range of light limitation. One 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. Dilution 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. The 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"1), D is dilution rate (h"1) and N l and N2 are the measured 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 medium. These measurements indicated that N H 4 concentrations in both the reservoir and the medium were always below 0.05 u M . N H 4 and N O 3 + N O 2 were analyzed with a Technicon Autoanalyzer II following standard procedures outlined in Slawyk and Maclsaac (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 in the same manner 12 as the turbidostat, but the concentration of NO3 in the reservoir was only 15 u M . T he 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. The operating system for this technique is illustrated in Fig. 1. Some modifications were made in order to avoid smearing the autoanalyzer response to NO3 pulses because of the high NO3 concentration remaining in the culture medium when the cells from the medium were loaded onto the filter. Therefore, a 4-way valve was required to get rid of the high NO3 medium. The nitrogen-free wash water was filtrate from the N-limited chemostat culture. The standard solutions for uptake experiments were prepared from the same filtrate in 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 pumped through the autoanalyzer for 7 min and an initial measurement was obtained on 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 pumped across the cells on the filter for 7 min and a second measurement was then obtained. The 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 ammonium solution (e.g. 3 u M ) in order to determine an ammonium 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. During 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. The filter is denoted by the dashed line. 14 To waste AA W V A B 21 e To auto-analyzer B -7 Plexiglas plates O-ring - Filter 1 5 measurements, the nitrite concentration was simultaneously monitored and N O 3 uptake measurements were corrected for nitrite concentration. The uptake experiments were performed under the same irradiance and temperature that were used to grow the turbidostat culture. Uptake rates were normalized three ways and the formulae that were used to calculate uptake rates were: (i) on 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 " 1 ) at time t. v — the volume of culture medium that was passed through the filter (ml), c — the cell concentration (cells ml"^). (ii) on a cell volume basis f s V = (ug-at N u m ° h"1) v l f s 1? 1 1 = x I O 1 , 6 (mmole (liter cell volume)* 1 h"1) v l 1 — biovolume (total cell volume per ml of culture) (um^ ml"^). 16 (iii) specific uptake rate V = xlOOO (h' 1 ) v p n pn — particulate nitrogen in culture medium (ug-at N L"*). The overall design of the experiments is outlined in Table I. In this study, one N O 3 concentration (5 u M ) and a range of ammonium 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 ammonium uptake rates with and without nitrate were measured. Uptake 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). The effects of loading different amounts of cells onto the filter for light-limited cultures (L2 and L3) on N O 3 uptake rate measurements during a pulse of N O 3 (5 u M ) across the cells on the filter were also tested. The calculated standard errors of the measurement of N O 3 uptake rates for the three cultures are given in Table II. The 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 . Thompson (pers. comm.), N H 4 uptake rates replicate much better than N O 3 uptake rate measurements using this technique. ( Table I. Outline of experiments. The nitrogen source and the irradiance used to grow steady state cultures are indicated. The additions of N0 3 or NH4 s i n g l y or i n combination to these steady state c e l l s are shown. Cul- N-source Irrad- Nitrogen Additions ture iance uE m~^s -1 Forms Concentration (uM) LO N0 3 150 N0 3 5.0 NH4 0.25, 0.5 , 1.0, 3.0, 5. 0 N03+NH4 5.0 (N03) 1.0, 3.0, + 0.25, 0. 5.0 (NH4) 5, LI N0 3 17 N0 3 5.0 NH4 0.25, 0.5 , 1.0, 3.0, 5. 0 N03+NH4 5.0 (N03) 1.0, 3.0, + 0.25, 0. 5.0 (NH4) 5, L2 NO 3 9 N0 3 5.0 NH4 0.25, 0.5 , 1.0, 3.0, 5. 0 N03+NH4 5.0 (N03) 1.0, 3.0, + 0.25, 0. 5.0 (NH4) 5, L3 N0 3 2 N0 3 5.0 NH4 0.25, 0.5 , 1.0, 3.0, 5. 0 N03+NH4 5.0 (N03) 1.0, 3.0, + 0.25, 0. 5.0 (NH4) 5, 18 Table I I . Standard errors (SE) of the repeated measurements of N0 3 uptake rate (itunole ( l i t e r c e l l volume) - 1 h - 1 , averaged over 2-4 min i n the time course) for the d i f f e r e n t cultures. For c e l l number loaded onto the f i l t e r each loading and N0 3 uptake rate data during each pulse, see appendix A: A l , A2 and A3. Culture N0 3 uptake rate n SE C o e f f i c i e n t of Var i a t i o n LO 307 3* 24 8 % L2 122 2** 15 12 % L3 108 3** 21 20 % * : an average for 3 nutrient pulses across 3 new c e l l samples of s i m i l a r c e l l number. **: an average for 2 (L2) or 3 (L3) nutrient pulses across c e l l samples of d i f f e r e n t c e l l number. 19 R E S U L T S Cultures Growth rates determined in the batch cultures of Thalassiosira pseudonana increased with irradiance as a hyperbolic function, reaching a maximum at 45 u E m" s (Fig. 2). The half-saturation constant (Kj) was estimated graphically to be approximately 8.7 u E m " 2 s"^  (Hanes-Woolf linear transformation of the equation: U = U m * I/(Kj + I), where U is the specific growth rate (h"1), U m is the maximum growth rate (h"*), I is irradiance ( u E m" 2 s"*) and Kj is the half-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 Table III. The 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 min 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 min 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 . Steady-state parameters for turbidostat cultures of Thalassiosira pseudonana representing the s t a r t i n g conditions for the uptake experiments. For each parameter, see Appendix B: B l , B2, B3, B4, B5 and B6. Culture Irradiance Growth rate C e l l density Particulate Nitrogen C e l l Fluorescence nitrogen c e l l quotavolume per c e l l N i t r i t e e x c r i t i o n (uE nf-^s" 1) th- 1) (10 5 ml" 1) (uM) (10~7ump c e l l " 1 l(um 3 (10-5) ) c e l l " 1 ) (fmol c e l l " 1 ) LO 150 0.061 7.37 58.84 0.80 32.87 1.1 3.25 L l 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 L2), 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 in the presence of N H 4 tended to vary during the first two minutes before it was stable (Figs. 4, 5 and 6). For the culture grown at the lowest irradiance (culture L3), 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 min (Fig. 7A) . The N H 4 uptake rates at 1.0 min for the 3.0 u M and 5.0 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 min. However, in the presence of N O 3 , the enhancement of N H 4 uptake at 1 min for the 3 and 5 u M N H 4 pulses was decreased to 133% and 155%, respectively (Fig. 7B and Table IV) . The light-limited cultures (LI , L 2 and L3) 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; L2: Figs. 9 A and 9B; and L 3 : Figs. 10A and 10B). The comparison of the enhancement of the N H 4 uptake rate at 1.0 min with the rate at 5.0 min in the absence and the presence of N O 3 is shown in Table I V for each light-limited 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 in 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 in the absence of N H 4 (0) 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 u M (O). Nitrate Uptake Rate (mmole (liter cell volume)"1 h"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)'1 h'1) 28 Figure 5. Light limited culture (9 uE m"z s"1) L2: time course of NO3 uptake in the 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)"1 h"1) Figure 6. Light limited culture (2 u E m" z s" 1 ) L 3 : time course of N O 3 uptake in the absence of N H 4 (0) 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 u M (O). 31 Nitrate Uptake Rate (mmole (liter cell volume)'1 h"1) Figure 7. Light saturated culture (150 uE m" z s ) LO: time course of NH 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 uM (A), 0.5 uM (+), 1.0 uM (•), 3.0 uM (X) and 5.0 uM (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)'1 h'1) Ammonium Uptake Rate (mmole (liter cell volume)'1 h'1) 36 Figure 9. Light limited culture (9 u E m" 2 s"*) L2: 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). A 5003 re (D ^  400 re E = ) f 300 E E «" o 1200 E i E < 100d 1 •» 1 1 I I I I I I 1 1 I I I I I I I 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 I 1^  Time (min) 6OO-1 B Time (min) 38 Figure 10. Light limited culture (2 u E m" z s"1) L 3 : time course of N H 4 uptake in the absence of NO3 (A) and in the presence of NO3 (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 Table IV. Relative enhancement of NH4 uptake rate at 1 min compared to 5 min ( i . e . V l m i n / V 5 i n i n ) i n the presence or absence of N0 3. Nitrogen additions Cultures Form Concentration L0 L l L2 L3 NH4 (3 uM) 178 % 200 % 211 % 162 % NH4 + N0 3 ( 3 + 5 uM) 133 % 184 % 201 % 168 % NH4 (5 uM) 192 % 227 % 191 % 152 % NH4 + N0 3 ( 5 + 5 uM) 154 % 145 % 215 % 134 % 41 N O 3 (Figs. 11,12, 13 and 14). While the initial N H 4 uptake rate in the presence of N O 3 increased, N O 3 uptake in the presence of N H 4 was depressed for 1 min as shown in Figs. 11,12, 13 and 14 for all the cultures. When 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. There was less initial enhancement of N H 4 uptake in the presence of N O 3 (Figs. 11, 12, 13 and 14). The 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) in 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 min than the cultures at lower irradiances (L2 and L3) (Figs. 15A,B and 16A,B). The latter two cultures (L2 and L3) even displayed a slight supression of N H 4 uptake at about 2.0 min in the presence of N O 3 following the initial short-lived enhanced uptake (Figs. 15B and 16B). For instance, during the pulse of 3.0 u M N H 4 with N O 3 (Fig. 15B), the N H 4 uptake rate at 2 min 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 min 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"1) LO: time course of the interaction between NO3 uptake and N H 4 uptake after a pulse of a solution containing 5.0 u M NO3 (O) and 3.0 u M N H 4 (+), 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). 43 Uptake Rate of N03 or NH4 (mmole (liter cell volume)"1 h"1) 4 4 Figure 12. Light limited culture (17 u E m" z s ) 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 4 (+), compared to the uptake 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 N03 or NH4 (mmole (liter cell volume)"1 h"1) 46 Figure 13. Light limited culture (9 u E m " z s"A) L 2 : 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 (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 N03 or NH4 (mmole (liter cell volume)-1 h"1) 48 Figure 14. Light limited culture (2 u E m" z s"1) L 3 : 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 (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 N03 or NH4 (mmole (liter cell volume)"1 h"1) 50 Figure 15. N H 4 uptake at 3.0 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: 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). The presence of N H 4 , however, changed the pattern. The 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 (L2 and L3) 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 Appendix C) . In the less light-limited cultures ( L l and L2), 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 Appendix C) . However, the culture under the lowest irradiance (L3) did not show a depression in N O 3 uptake by N H 4 . These 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 min 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 4 (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 4 uptake in the presence of N O 3 (5.0 u M ) and N O 3 uptake in the presence of a range of N H 4 concentrations for each culture 5 5 Figure 17. A . N O 3 uptake vs growth irradiance in the absence of N H 4 (•) and in 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 4 concentration for each culture: L0 (•), LI (+), L2 (A) and L3 (O) (replotted from A ) . (The N O 3 uptake rate is averaged over the 2-4 min 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. Only the trends are shown in 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 ) . The N H 4 uptake rate is averaged over the 1-5 min time interval from the time courses in Figs. 7A, 8A, 9 A and 1 0 A 60 (Figs. 19, 20, 21 and 22). Th e presence of N O 3 did not change the typical pattern of N H 4 uptake as in Fig. 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 (L3) 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"1) LO: N H 4 concentration effect on 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 4 (A) (from Figs. 17B and 18B). The 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. CM 700 q 0.00 l H H U U I 1 I u 1.00 2.00 I I 1 | ! J 1 1 3.00 4.00 Ammonium Concentration (uM) 63 Figure 20. Light limited culture (17 u E m " z s"1) L l : N H 4 concentration effect on 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 4 (A) (from Figs. 17B and 18B). The 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 N03 or NH4 (mmole (liter cell volume)"1 h"1) 6 5 Figure 21. Light limited culture (9 u E m" z s"1) L 2 : N H 4 concentration effect on 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 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. 66 Uptake Rate of N03 or NH4 (mmole (liter cell volume)"1 h"1) 67 Figure 22. Light limited culture (2 u E m " z s"1) L 3 : N H 4 concentration effect on the interaction between N O 3 and N H 4 : N H 4 uptake in the absence of N O 3 (o), N H 4 uptake in the presence of N O 3 (+) and N O 3 uptake in the presence of N H 4 (A) (from Figs. 17B and 18B). The 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. 00 300 ? 250 H °J? 200 \ CO ^ I o s CO §, CC o> 100 o 0 E .* E CO w 0.00 Ammonium Concentration (uM) 69 D I S C U S S I O N Cultures The relationship between growth rate of Thalassiosira pseudonana and irradiance appears to follow a simple hyperbolic function. The saturating irradiance for growth, 45 u E m" 2 s"*, obtained in this study is low compared to the value (0.04 cal c m ' 2 min"*), ca. 140 u E m" 2 s"* for saturating photosynthesis obtained by Eppley and Renger (1974) with the same species. The difference could be due to the differences in 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 Eppley 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 in 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 in the cell volume and nitrogen cell quota with decreasing irradiance. This is similar to the observations by Zevenboom et al. (1980) who studied Oscillatoria agardhii, Rhee and Gotham (1981) studying Scenedesmus sp and Fragilaria crotonensis and Healey (1985) studying a blue-green alga Synechococcus linearis. In another study on 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. For example, Eppley and Renger (1974) observed a decrease in 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 medium with cells in suspension (Parslow et al. 1984a) enabled us to follow the change in 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 problem 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 in 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 ammonium uptake rates were observed to increase with time during the first minute before the initial enhanced uptake occurred. These short-time ( < 1 min) lower uptake rates were not observed by Go ldman 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). The difference between my results and theirs is that the cultures they used were NH4 limited while mine were NO3 saturated and light-limited. Probably in N H 4 - l i m i t e d cultures, the NH4 uptake system is active and ready to take up available NH4 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 NO3 concentrations do not appear to have a fully active N H 4 transport system. Therefore, it is possible that the NH4 uptake system needs to be induced when N H 4 hits the cells during the pulse. This inducing process seems to be reflected in 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 Fig . 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 (Fig 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 in understanding the mechanisms of N O 3 uptake, N H 4 uptake and the interaction between the two. Th e acceleration process may involve complex processes including diffusion, cell membrane potential change and p H change as well as active transport (Raven 1980). Obviously, further studies on the acceleration of N H 4 uptake rate in response to an ammonium 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 in uptake rate or negative uptake rates for ca . l min 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 upon 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 in the remainder of time course is attributed to a decrease in N O 3 uptake or due to an effect on N O 3 reduction, or both. 73 2) NHj uptake with and without NOj The initial enhanced N H 4 uptake occurred only for larger pulses of N H 4 (3.0 and 5.0 u M ) . There was no initial enhanced N H 4 uptake for pulses less than 1.0 u M . This concentration dependence of the initial enhanced N H 4 uptake was also observed by Parslow et al. (1985a) using the same technique and the same species in N H 4 - l i m i t e d chemostats. Therefore, the occurrence of the time-dependent uptake of N H 4 is also concentration dependent, although the concentration for the dependence can vary somewhat depending on physiological state of the cells. The similar time-dependence of N H 4 uptake during a short time course has been shown for different species. Goldman 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 4 enhanced uptake among the four species. It has been suggested that this enhanced uptake potential resulted from nitrogen deficiency, the degree of which can be expressed by the nitrogen cell quota. However, in some cases, it is not necessary for cells to be nitrogen-limited in order to have this potential. Horrigan and McCarthy (1982) found that before nitrite was depleted in nitrite grown cells in batch cultures of Thalassiosira pseudonana, the uptake rate in a 5 min incubation was 78 times the growth rate, and Parslow et al. (1984b) observed that the enhanced N H 4 uptake rate decreased over a 5 min time period for NC^-grown cells 6 hours prior to starvation in batch culture. The results obtained here demonstrate a similar phenomenon 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. There are two reasons for this suggestion. First, the preferential utilization for N H 4 over N O 3 has been very commonly observed in both laboratory 74 cultures and natural populations of marine phytoplankton. Although 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 . Dortch (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. The 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 Larkum 1987). This transient p H change might have some effect on N O 3 uptake as well. Falkowski (1975) tested the presence of a NC^Cl-act ivated A T P a s e associated with the cell membrane in seven species of phytoplankton. H e found that the optimum 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. The time course experiments 75 appear to reflect such a pH-regulated process, during which the N H 4 uptake was enhanced, the N O 3 uptake was inhibited or fluctuating. It was interesting that the specific uptake rate at 1 min in the pulse of 5 u M N H 4 without N O 3 is 8.4 d" 1 for the light-saturated culture LO (Fig. 7A). This value is remarkably close to the V m ^ " l value of 9 d"* (an average over 0-1 min) for the pre-N03-starved batch culture (Parslow et al. 1984b) and 10 d at 1 min for the N H 4 - l i m i t e d chemostat culture with a growth rate of 0.5 d" 1 (Parslow et al. 1985a). After this 1 min point, the rates started to decline. The similarity in values may be explained by recent observations in the literature. Ohmori and Hattori (1978) observed a transient change in the A T P pool oiAnabaem cylindrica when N H 4 was added to the cells. They found that the A T P pool dropped dramatically within the first minute and recoverd at 6 min. The 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 in this study. W h e n the N H 4 uptake rate increased to the highest rate at 1 min and declined with time until it was stable at 6 min, the A T P pool decreased to a lowest level at 1 min and then recovered during the remaining 1-6 min. Morever, a study by Flores et al. (1980) on short-term ammonium inhibition of N O 3 utilization by Anacystis nidulans and other cyanobacteria showed that the increase in glutamine reached a maximum at 1 min while glutamate reached a minimum at 1 min indicating the maximum assimilation of N H 4 in this initial 1 min 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 from the time dependence of enhanced N H 4 uptake alone and the simultaneous interaction with N O 3 uptake. This study is the first one to clearly show a short-term depression of N H 4 uptake by N O 3 . This 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 on the other hand, N H 4 uptake is still exerting an inhibiting effect on N O 3 uptake during the same time period. The 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 4 utilization via the N H 4 transport system, and on the other hand, the feedback regulation gradually takes over in nitrogen assimilation, resulting in a slow down of N H 4 uptake and an inhibition of N O 3 uptake simultaneously. But it is not clear whether the inhibition of N O 3 uptake by N H 4 occurs at the site of N O 3 transport or the reduction of N O 3 . T h e enhanced N H 4 uptake and its time dependence is similar to a model proposed by Conway et al.(1916). For a time course of N H 4 uptake by N H 4 - l i m i t e d cultures, they defined three distinct phases of uptake: surge uptake (Vs), internally controlled uptake (Vi) and externally controlled uptake (Ve). In this study, I found that low N H 4 concentrations (< 1.0 u M ) did not result in an initial enhanced uptake. This would represent a range under the external control in the Conway's model (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 4 uptake rate for the 1 u M pulse which did not have an initial enhanced uptake rate regardless of the degree of light limitation of culture. The initial enhanced uptake of N H 4 was also affected by light. W h e n light limitation increased, the initial enhancemant of N H 4 uptake was less and lasted a shorter time. Obviously, the enhancement is light-dependent through the effect of light on 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 pool was fast. In 77 contrast, in the most light-limited culture, the cells were less active, the transfer of nitrogen between each pool was slower and the accumulation of nitrogen in the internal pool 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. Th 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 in Fig. 17, the lowest irradiance during growth appeared to result in no inhibition of N O 3 uptake by the increase in N H 4 concentration. This result is in 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). Major differences exist between his study and my study, besides the different species used in each study. First, batch cultures were used in his study, while steady-state turbidostat cultures were used in this study. Bates stated that in his batch cultures, an 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 had 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 on 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 pool 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. The existence of a light-dependent energy pool has been demonstrated by Falkowski and Stone (1975). The problem is that we do not know the mechanisms of N O 3 and N H 4 uptake, let alone their interaction. Morever, 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 on 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 4 are directly 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) (Candau et al. 1976). It has been claimed that such a mechanism exists at least in 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. For 79 example, Serra et al. (1978) have shown that ammonium 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). Dark 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 Raimbault 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 Fig . 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 4 , at least during a short period of time (e.g. 6 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 Candau et al. (1976), while the feedback regulation is slow in 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 This continuing flow into the N H 4 pool could affect N H 4 uptake rates. This will be discussed in the next section. 2) NH4 uptake with and without NOj A s shown in Fig. 18A, N H 4 uptake is also dependent on light limitation of the cultures. This 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. Nor did it change the concentration where the uptake rate is nutrient saturated (1.0 u M ) . A s discussed in 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. Thus, it is likely that N O 3 uptake affects N H 4 uptake before the feedback regulation inhibits N O 3 uptake and reduction. Funkhousor and Ramados (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 in the cell would induce N 0 3 reductase activity or synthesis. However, a regulated inhibition by N H 4 assimilation is still exerted on these activities. Therefore, the competition between utilization and inhibition of N O 3 may occur. Conway (1977) observed a delay of approximately 30 min for N H 4 to fully inhibit N O 3 uptake. This 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 in utilizing different nitrogen forms could be significant, since more pathways are open for nitrogen uptake. 81 Ecological significance Although 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 Turpin and Harrison (1978), McCarthy and Goldman (1979), Glibert and Goldman (1981), Horrigan and McCarthy (1982), Goldman and Glibert (i982) Quarmby 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 from the low light conditions. McCarthy and Goldman (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 light-limited growth. It has been shown that such an enhanced uptake appeared to be dependent on 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. The ratio of uptake rate (h"A) of N H 4 alone to maximal specific growth 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). The N H 4 uptake rate is averaged over the 1-5 min 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. The increase in nitrogen requirement with decreasing irradiance has been suggested by Rhee and Gotham (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. The 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. The 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. The rate of the inducing process depends on the response time of the cells. Maclsaac et al. (1985) distinguished four zones of physiological condition along the axis of the upwelling plume for the coast of Peru. They proposed that in Zone 1, phytoplankton upwelled with nutrient-rich water were initially shifted-down (low nutrient uptake); in Zone 2 the phytoplankton undergo light induced shift-up to 85 increased nutrient uptake, photosynthesis and synthesis of macromolecules. Z immerman 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. The 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. These 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. When the cells are upwelled to the saturating-light zone, they will reach maximum uptake or complete their shift up. This 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. During the inducing process (from slow growth to fast growth), the intermittent pulse of N H 4 produced by other organisms could frequently stimulate cell division. N o inhibition of N O 3 uptake by N H 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 S U M M A R Y 1. Growth 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. The short-term time course of the interaction between nitrate and ammonium uptake was followed. a. Comparison of nitrate uptake in the absence and presence of ammonium showed that nitrate uptake was affected immediately by ammonium 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. The 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 on the interaction between nitrate and ammonium uptake was apparent. a. For the light-saturated culture and the less light- limited cultures, the depression of nitrate uptake by ammonium increased with ammonium concentration. However, for the most light-limited culture, the depression of nitrate by ammonium 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 . 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M U R . 1980. Effects of light on nitrate-limited Oscillatoria aghardii in chemostat cultures. A r c h . Microbiol . 125: 59-65. Zimmerman, R . C , J .N. Kremer 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. 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 of N0 3 uptake r a t e measurements f o r d i f f e r e n t c u l t u r e s 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 (SE) a s s o c i a t e d w i t h t h e r e p l i c a t i o n o f N0 3 uptake r a t e measurements (5.0 uM N0 3) . The c e l l amounts l o a d e d onto t h e f i l t e r i s t h e same (4.3 x 1 0 6 m l " 1 ) : 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 averaged o v e r t h e 2--4 min t i m e i n t e r v a l from t h e t i m e c o u r s e ( a l o n g t h e column). Uptake r a t e (mmole ( l i t e r c e l l volume) - 1 h" 1) C e l l number l o a d e d P u l s i n g ( 1 0 6 m l " 1 ) Average SE C o e f f i c i e n t t i m e 1 s t 2nd 3rd o f v a r i a t i o n (min) 4.3 4.3 4.3 (%) 2.00 318 308 291 300 17 5.6 2.17 315 312 277 297 19 6.5 2 . 34 307 316 281 297 17 5.9 2 . 50 311 326 281 302 21 6.8 2.67 315 323 281 301 21 6.9 2.83 311 319 277 300 19 6.3 3.00 - 315 326 277 304 22 7.1 3.17 318 326 277 306 22 7.1 3.34 318 341 274 309 28 9.1 3.50 307 337 274 306 26 8.5 3 .67 318 337 277 310 25 8.2 3.83 318 330 281 310 21 6.8 4.00 326 323 277 308 22 7.2 Mean 315 325 279 306 24 8 . 0 98 A.2. Culture L2: Standard errors (SE) associated with the r e p l i c a t i o n of N0 3 uptake rate measurements (5.0 uM N0 3). The c e l l amounts loaded onto the f i l t e r were d i f f e r e n t for the two pulses. Mean i s the value averaged over the 2-4 min time i n t e r v a l from the time course (along the column). Uptake rate (mmole ( l i t e r c e l l . volume) - 1 h - 1 ) Pulsing C e l l number loaded (10° ml"*-1-) Average SE C o e f f i c i e n t time 1st 2nd of v a r i a t i o n (min) 3.2 12.6 (%) 2.00 182 118 150 45 30 2.17 161 116 139 32 23 2.34 140 114 127 18 14 2.50 140 111 125 21 16 2.67 136 113 124 16 17 2.83 136 112 124 17 13 3 . 00 136 112 124 17 14 3 .17 131 110 X Q 121 15 13 3.34 136 111 123 18 14 3.50 127 111 119 12 10 3 . 67 110 107 109 2 2 3.83 68 105 87 27 31 4 . 00 114 106 110 6 5 Mean 132 111 122 15 12 99 A.3. C u l t u r e L3: S t a n d a r d e r r o r s (SE) a s s o c i a t e d w i t h t h e r e p l i c a t i o n o f N0 3 u p t a k e r a t e measurements (5.0 uM N 0 3 ) . The c e l l number l o a d e d onto t h e f i l t e r i s d i f f e r e n t 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 averaged o v e r t h e 2-4 min t i m e i n t e r v a l from t h e t i m e c o u r s e ( a l o n g t h e column). Uptake r a t e (mmole ( l i t e r c e l l volume) x h ) C e l l number l o a d e d P u l s i n g ( 1 0 6 m l - 1 ) Average SE C o e f f i c i e n t t i m e 1 s t 2nd 3 r d o f v a r i a t i o n (min) 1.7 3.7 6.6 (%) 2.00 168 87 118 124 41 33 2.17 162 76 121 120 43 36 2.34 151 79 118 116 36 31 2.50 133 79 118 110 28 26 2.67 128 87 117 110 21 19 2 .83 110 92 120 107 14 13 3.00 99 87 121 102 17 17 3.17 99 92 118 103 13 13 3.34 87 76 121 95 24 25 3.50 93 76 118 96 21 22 3 . 67 151 84 121 119 33 28 3.83 110 90 108 102 11 11 4.00 109 84 120 104 18 17 Mean 123 84 118 108 21 20 APPENDIX B. V a r i a t i o n of some parameters i n the t u r b i d o s t a t c u l t u r e s over a few days B . l . V a r i a t i o n of s p e c i f i c growth r a t e B.2. V a r i a t i o n o f c e l l d e n s i t y B.3. V a r i a t i o n of biovolume B.4. V a r i a t i o n o f p a r t i c u l a t e n i t r o g e n B . 5 . V a r i a t i o n o f f l u o r e s c e n c e per c e l l B . l . V a r i a t i o n of s p e c i f i c growth rate ( h - 1) based on biovolume i n the turbidostats over a few days before the experiments were conducted Date Culture LO L l L2 L3 June 4 0.061 0.036 June 5 0.058 0.032 June 6 0.057 0.038 June 7 0.061 0.032 June 8 0.061* 0.035 0.024 0.012 June 9 0.060 0.034 0.024* 0.012 June 10 0.062 0.041* 0.023 0.012 June 11 0.066 0.035 0.024 0.012* June 12 0.060 0.010 Average 0.061 0.035 0.024 0.012 Standard error 0.003 0.003 0.000 0.001 Co e f f i c i e n t of v a r i a t i o n 4.2% 8.7% 2.1% 9.5% * represents the day on which the samples from that culture were taken for the experiments. B.2. V a r i a t i o n o f c e l l d e n s i t y (10° m l ~ x ) i n t h e t u r b i d o s t a t s o v e r a few days b e f o r e t h e e x p e r i m e n t s were conducted. Date C u l t u r e LO L I L2 L3 June 4 7.1 6.0 June 5 7.8 6.0 June 6 7.8 5.5 June 7 7.6 5.4 3 . 2 June 8 7.4* 3.8 5.8 2.9 June 9 7.4 3.9 6.2* 2.8 June 10 7.6 4 . 1* 6.1 3 . 1 June 11 7.5 4.7 6.3 2.8* June 12 7.8 4.4 6.8 2.6 June 13 6.9 6.2 Aver? ige ( h " 5 ) 7.5 4.2 6.0 2.9 S t a n d a r d e r r o r 0.3 0.3 0.4 0.2 C o e f f i c i e n t o f v a r i a t i o n 4 . 4 % 8 . 2 % 6 . 7 % 7 . 1 % * 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 t a k e n f o r t h e e x p e r i m e n t s . 103 B.3. Va r i a t i o n of biovolume (10 7 um3 ml - 1) i n the turbidostats over a few days before the experiments were conducted Date Culture LO LI L2 L3 June 4 2.4 1.6 June 5 2.7 1.5 June 6 2.6 2.1 1.7 June 7 2.4 2.2 1.5 June 8 2.4* 1.7 2.2 1.5 June 9 2.5 1.6 2 . 3 1.6 June 10 2.5 1.5* 2.4* 1.6 June 11 2.5 1.7 2.4 1.7 June 12 1.6 1.6 June 13 1.7 Aver? ige ) 2.5 1.6 2.3 1.6 Standard error 0.1 0.1 0.0 0.1 Co e f f i c i e n t of v a r i a t i o n 3.7% 4.8% 5.5% 4.1% * represents the day on which the samples from that culture were taken for the experiments. 104 B.4. Vari a t i o n of p a r t i c u l a t e nitrogen (uM) i n the turbidostats over a few days before the experiments were conducted Date Culture LO L l L2 L3 June 5 62.4 75.7 53 .9 June 6 64.6 75. 3 55. 1 June 7 62.9 72.2 53.9 June 8 58.8* 52 . 3 72.7 50.0 June 9 61.4 50.5 74 .9* 53 . 6 June 10 61.6 47.3* 76.1 June 11 62.7 47.6 81.8 56.1* Average 62.1 49.4 75.5 53.8 Standard error 1.8 2.2 3.2 1.9 C o e f f i c i e n t of v a r i a t i o n 2.9 % 4.5 % 4.2 % 3.5 % * represents the day on which the samples from that culture were taken for the experiments. 105 B.5. Vari a t i o n of fluorescence per c e l l (10~ D) i n the turbidostats over a few days before the experiments were conducted Date Culture LO L l L2 L3 June 4 0.99 2.09 June 5 0.94 2.08 June 6 1.05 2.24 3. 07 June 7 1.05 2.31 2.83 June 8 1.06* 2.37 2.15 3 . 07 June 9 0. 98 2.08 2 .19* 3.18 June 10 0.98 1.78* 1.99 2.81 June 11 0.96 1.83 2.15 3 . 51* June 12 1.99 3.10 June 13 2.91 Average ) 0.99 2 . 01 2. 13 3.06 Standard error 0. 06 0.27 0.11 0.23 C o e f f i c i e n t of v a r i a t i o n 6.3 % 13.5 % 5.1 % 7.4 % * represents the day on which the samples from that culture were taken for the experiments. 106 APPENDIX C. Variation of N 0 3 uptake rate (mmole ( l i t e r c e l l volume)" 1 h" 1) over a 2-4 min time i n t e r v a l from a time course, including standard error and c o e f f i c i e n t of v a r i a t i o n (Coeff. Var.)- The reduction of N O 3 uptake rate i n the presence of NH4 r e l a t i v e to the rate i n the absence of NH4 i s included ( N 0 H / N 0 ) NH,, additions 0.0 0.25 0.5 1.0 3.0 5.0 Culture LO 312 255 212 153 80 103 NOH/NO (%) 100 82 70 49 26 33 Standard Error 16 13 4 7 16 9 Coeff. Var. (%) 5 5 2 5 20 9 Culture LI 204 138 70 67 72 65 NOH/NO (%) 100 68 34 33 36 32 Standard Error 24 9 16 7 9 10 Coeff. Var. (%) 12 7 23 11 12 16 Culture L2 98 87 46 70 42 43 NOH/NO (%) 100 88 47 71 42 44 Standard Error 5 30 no 19 4 4 Coeff. Var. (%) 5 34 no 28 10 8 Culture L3 105 92 149 103 126 159 NOH/NO (%) 100 88 141 98 120 151 Standard Error 8 7 8 13 13 8 Coeff. Var. (%) 8 8 6 13 11 5 no means "not calculated". 

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