Response of marine phytoplankton to nitrogen deficiency: Decreased nitrate uptake vs enhanced ammonium uptake

The uptake of nitrate and ammonium was measured separately in uni-algal, nitrogen-deficient cultures of four species of marine phytoplankton. Nitrogen-deficient phytoplankton took up ammonium at initial rates which greatly exceeded those measured for nitrogen-sufficient phytoplankton. However, nitrate uptake by nitrogendeficient cultures was generally much slower than either nitrate or ammonium uptake by nitrogen-sufficient cultures or ammonium uptake by nitrogen-deficient cultures. Considerable species differences were observed in the degree to which nitrogen deficiency increased ammonium uptake or decreased nitrate uptake. Loss of ability to take up nitrate, but enhanced ability to take up ammonium, as a result of nitrogen deficiency may be an adaptation to the different mechanisms by which nitrate and ammonium are supplied to the euphotic zone. In areas with an intermittent supply of nitrogen, changes in the ability of some species to take up nitrogen as a result of nitrogen starvation will influence species composition and complicate interpretations of measurements of nitrogen uptake.Contribution no. 1249 from the Department of Oceanography, University of Washington, and contribution no. 82006 from the Bigelow Laboratory for Ocean Sciences     

Light stimulation of phosphate uptake in marine phytoplankton

A phosphate uptake system responding positively to light would offer the algae a competitive advantage over bacteria, particularly when phosphorus is limiting. Effects of light on phosphate uptake in phytoplankton were investigated in view of conflicting literature reports, to test the hypothesis that both the P status and the light history of the population controlled the response. Maximum stimulation, to about 2.7x dark uptake, was observed at a Strait of Georgia, British Columbia, Canada station where phytoplankton was phosphorus sufficient but light limited. Stimulation was similar, about 1.8 x the dark rate, in the Northern Sargasso Sea, where phytoplankton was probably limited by phosphate but not by light. Minimal stimulation was observed in the Sheepscot Estuary, Maine, USA, where the population appeared to be neither phosphorus nor light limited.     

Light-dependency of nitrate uptake by phytoplankton over the spring bloom in Auke Bay,Alaska

The effect of light intensity on nitrate uptake by natural populations of phytoplankton was examined by ¹⁵N traceruptake experiments during the spring (March–May 1987) in Auke Bay, Alaska. The data were fit to a rectangular hyperbolic model which included a term for dark uptake. Three types of curves described nitrate uptake as a function of light intensity. The first (Type I) had a low half-saturation light intensity (K _I), low chlorophyll-specific uptakes rates, no dark uptake and occasional photoinhibition. These were observed during a period of biomass decrease, accompanied by low daily light and strong wind, prior to the major bloom. The second type (Type II) had relatively high K _I, high chlorophyll-specific uptake rates, and no dark uptake. Type II curves were observed during most of the period prior to nitrate depletion in the surface waters. Types I and II both appeared prior to nitrate depletion in the water and reflected variations in the light history of the phytoplankton population. The third type (Type III) occurred in nitrate-deplete conditions, when nitrate uptake was less dependent on light intensity (i.e., high rates of dark uptake and lower K _I). Decreased light-dependency during this period was coupled with physiological nitrogen deficiency in the population. Comparing these parameters to those of photosynthetic carbon fixation, K _Ivalues of nitrate uptake were generally higher than those of photosynthesis prior to nitrate depletion, and lower during nutrient-deplete conditions.     

Nitrate uptake in marine phytoplankton: Energy sources and the interaction with carbon fixation

Field studies of whole natural phytoplankton communities from Knight Inlet, B. C., Canada and laboratory cultures of the diatom Skeletonema costatum indicate inorganic carbon fixation may be temporarily suppressed following 10 to 15% enrichment with NO ₃ ^- or NH ₄ ⁺ . (This effect is suggested to be due to competition between inorganic carbon and nitrogen for adenosine triphosphate (ATP), and is reduced when chlorophyll a is increased intracellularly after 6 to 8 h.) Results imply that the source of ATP for nitrate uptake is primarily from Photosystem I (cyclic photophosphorylation) in the presence of light. It would appear that a transient nutrient-adaptive response occurs upon addition of extracellular nitrogen.     

Regulation of nitrate and ammonium uptake in the Greenland Sea

The uptake of nitrate and ammonium was investigated experimentally during early spring 1989 in the Greenland Sea, with particular attention placed on the roles of irradiance, nitrogen concentrations and nitrateammonium interactions. The phytoplankton assemblage was dominated by the colonial prymnesiophyte Phaeocystis pouchetii. Nitrate concentrations ranged from undetectable at the end of the cruise to greater than 10 M, and ammonium levels ranged from less than 0.1 to 1.9M. The uptake of both nitrate and ammonium as a function of irradiance was found to be a saturation response. Photoinhibition occurred and was found to be greater for ammonium uptake. Ammonium uptake also saturated at irradiance levels five times lower than those needed to saturate nitrate uptake. Nitrate and ammonium uptake as a function of nitrogen concentration also was characterized by a saturation response, with the estimated half-saturation constant (K _s) value for nitrate uptake being 0.29 M. Elevated ammonium concentrations inhibited nitrate uptake, and the response appeared to be one of exponential decrease with increasing concentrations of ammonium. The most important factor in the Greenland Sea influencing ammonium uptake during the spring was irradiace, while both irradiance and ammonium concentrations played major roles in regulating nitrate uptake and new production.     

Interactions of inorganic nitrogen in the uptake and assimilation by marine phytoplankton

The effect of ambient ammonium concentration on the nitrate uptake rate of marine phytoplankton was investigated. These studies consisted of laboratory experiments using unialgal species and field experiments using natural phytoplankton communities. In laboratory experiments, ammonium suppressed the uptake rates of nitrate and nitrite. Approximately 30 min were required for ammonium to exhibit its fully inhibitory effect on nitrate uptake. At high ammonium concentration (>3 g-at/l), a residual nitrate uptake rate of approximately 0.006 h^-1 was observed. When the ambient ammonium concentration was reduced to a value less than 1 g-at/l, the suppressed nitrate uptake rate subsequently attained a value comparable to that observed before the addition of ammonium. A range of 25 to 60% reduction in the nitrate uptake rate of natural phytoplankton communities was observed at ambient ammonium concentrations of 1.0 g-at/l. A mechanism is proposed for the suppression of nitrate uptake rate by ammonium through feedback control of the nitrate permease system and/or the nitrate reductase enzyme system. The feedback control is postulated to be regulated by the level of total amino acids in the cell.Contribution No. 936 from the Department of Oceanography, University of Washington, Seattle, Washington 98195, USA. This paper represents a portion of a dissertation submitted to the Department of Oceanography, University of Washington, Seattle, in partial fulfillment of the requirements for the Ph.D. degree.     

Questions on the mechanism of temperature adaptation in marine phytoplankton

The rate of light-saturated photosynthesis in 3 marine algae [Phaeodactylum tricornutum Bohlin, Nitzschia closterium (Ehrenberg) Smith and Dunaliella tertiolecta Butcher] varies during growth in batch culture. The photosynthetic rare declines most rapidly during growth at the higher temperatures. Because of these changes in photosynthesis rate, the previously reported enhanced photosynthetic abilities caused by growth at lower temperatures (generally interpreted as evidence for higher enzyme levels) can only be observed when measurements are made late in the exponential phase or after the onset of the stationary phase of growth. When allowance is made for the earlier peak of photosynthetic ability in cultures growing at higher temperatures, there is no evidence for adaptation to lower temperatures being caused by increased levels of the enzymes required for carbon-dioxide fixation. When the changes due to growth in batch culture are taken into account, certain effects of temperature can be recognized. the dry weight: chlorophyll ratio of all 3 algae increases with decreasing growth temperatures. For P. tricornutum and N. closterium, growth at lower temperatures reduces the cellular content of chlorophyll a, but has little effect on the chlorophyll content of D. tertiolecta. The dry weight: cell-number ratio of D. tertiolecta and P. tricornutum increases with lower growth temperatures, but growth temperature has little effect on the cell mass of N. closterium. Growth of the 3 algae at lower temperatures does not increase their ability to photosynthesize at these lower temperatures. Rather, it reduces their ability to assimilate carbon dioxide at the higher temperatures.     

Relationship between phytoplankton cell size and the rate of orthophosphate uptake: in situ observations of an estuarine population

Orthophosphate uptake by a natural estuarine phytoplankton population was estimated using two methods: (1) ³²P uptake experiments in which filters of different pore sizes were used to separate plankton size-fractions; (2) ³³P autoradiography of phytoplankton cells. Results of the first method showed that plankton cells larger than 5 m were responsible for 2% of the total orthophosphate uptake rate. 98% of the total uptake rate occurred in plankton composed mostly of bacteria, which passed the 5 m screen and were retained by the 0.45 m pore-size filter. There was no orthophosphate absorption by particulates in a biologically inhibited control containing iodoacetic acid. Orthophosphate uptake rates of individual phytoplankton species were obtained using ³³P autoradiography. The sum of these individual rates was very close to the estimated rate of uptake by particulates larger than 5 m in the ³²P labelling experiment. Generally, smaller cells were found to have a faster uptake rate per m³ biomass than larger cells. Although the nannoplankton constituted only about 21% of the total algal biomass, the rate of phosphate uptake by the nannoplankton was 75% of the total phytoplankton uptake rate. Results of the plankton autoradiography showed that the phosphate uptake rate per unit biomass is a power function of the surface: volume ratio of a cell; the relationship is expressed by the equation Y=2x10^-11 X ^1.7, where Y is gP m^-3 h^-1 and X is the surface: volume ratio. These results lend support to the hypothesis that smaller cells have a competitive advantage by having faster nutrient uptake rates.     

Cadmium uptake by a phytoplankton species in the presence of different inhibitors&

The uptake of cadmium by the Haptophyceae Hymenomonas elongata was studied as regards the energetic processes and Ca‐transport. For this purpose, experiments were carried out in the presence of an uncoupler of the phosphorylation : CCCP and of an inhibitor of calcium channels and also of Ca²⁺‐ATPase : verapamil. To reduce the number of assays, a factorial experimental design was used, in which all variables:— concentration of Cd in the medium—incubation time and—the presence of the inhibitor, are at two levels. In the absence of inhibitor, Cd uptake was found to vary as a function of metal concentration in the medium and of incubation time. CCCP significantly increased Cd uptake by H. elongata at sufficient incubation time. Therefore Cd uptake seemed linked to an energy‐dependent process, involving an ATPase. Verapamil immediately increased Cd uptake, implying an interaction between Cd and Ca.     

Interactions between nitrate and ammonium uptake: variation with growth rate,nitrogen source and species

The interaction between nitrate and ammonium uptake was examined as a function of preconditioning growth rate and nitrogen source by adding nitrate, ammonium, or both to nitrogen-sufficient,-deficient, and-starvedSkeletonema costatum (Grev.) Cleve and nitrogen-deficientChaetoceros debilis Cleve. By simultaneously measuring the internal accumulation of intermediates of nitrogen assimilation and the rates of nitrogen assimilation, the metabolic control of nitrogen uptake could be assessed. After the simultaneous addition of nitrate and ammonium to culture, both nitrate and ammonium uptake rates were decreased in comparison with the rates observed when each was added alone, although nitrate uptake was usually decreased more than ammonium uptake. Since both nitrate and ammonium uptake rates vary with time, preconditioning growth conditions, nitrogen sources present, and species, it was necessary to use several different indices to quantify inhibition. In general, ammonium inhibition of nitrate uptake inS. costatum was greatest in cultures preconditioned to ammonium and those at low growth rates, whereas ammonium uptake was inhibited most in cultures preconditioned to nitrate. In nitrogen-deficientC. debilis, nitrate uptake was more inhibited by ammonium, but uptake returned to normal rates more quickly than inS. costatum, whereas inhibition of ammonium uptake was similar. These results explain why the interaction between nitrate and ammonium uptake in the field can be so variable. Inhibition of uptake is not controlled by internal ammonium or total amino acids, nor is it related to the inability to reduce nitrate. Instead, inhibition must be determined in part by the external concentration of nitrogen compounds and in part by some intermediate(s) of nitrogen assimilation present inside the cell.Bigelow Laboratory Contribution No. 82022     

The ammonium/methylammonium uptake system of Symbiodinium microadriaticum

The substrate analogue [¹⁴C]-methylammonium was used to study ammonium/methylammonium uptake by Symbiodinium microadriaticum (zooxanthellae). The value of the Michaelis constant (K _m) for the uptake system was approximately 35 M with methylammonium as substrate; ammonium was a competitive inhibitor of methylammonium uptake, and the K _m for ammonium uptake (determined as the inhibition constant, K _i, for methylammonium) was 6.6 M. Methylammonium uptake by zooxanthellae was light-dependent. Methylammonium uptake rates of zooxanthellae which had been freshly isolated from the hermatypic coral Acropora formosa (0.85±0.05x10^-10 mol min^-1 cell^-1) were lower than those of axenic cultures of the zooxanthellae from Montipora verrucosa (Acroporidae) grown under various nitrogen regimes (1.6 to 12x10^-10 mol min^-1 cell^-1). Maximum uptake rates were found for ammonium-starved cultured M. verrucosa zooxanthellae (10.2 to 12x10^-10 mol min^-1 cell^-1); M. verrucosa zooxanthellae growing with ammonium as nitrogen source and zooxanthellae which had been freshly isolated from A. formosa gave similar and considerably lower uptake rates (0.85 to 1.6x10^-1 mol min^-1 cell^-1). These results suggest that either coral tissue contains sufficient ammonium to repress synthesis of the uptake system of the algal symbionts or, alternatively, there are additional barriers to ammonium transport for zooxanthellae in vivo.     

Variation in cadmium uptake by estuarine phytoplankton and transfer to the copepod Eurytemora affinis

The pathways of cadmium (Cd) uptake and transfer within an estuarine planktonic community from the Patuxent River, Maryland, USA, were investigated using an assemblage of natural phytoplankton and the copepod Eurytemora affinis Poppe. The experiment was carried out in October 1992 in replicated 500-liter, flow-through, fiberglass tanks. Growth rate, species composition, and Cd loading affected the accumulation of Cd by the phytoplankton. Uptake of Cd by phytoplankton was proportional to the amount of Cd available in the water column. Partition coefficients (K _d) for phytoplankton uptake averaged 4.4 × 10⁴. As metal loading rates and phytoplankton species composition changed during the 12-d experiment, Cd partitioning declined. Transfer of Cd to E. affinis occurred from Cd-laden phytoplankton, with levels in the copepods being approximately the same as, or somewhat less, than in the phytoplankton. Some Cd uptake occurred in copepods exposed to dissolved Cd only; however, the uptake was considerably less than that seen from food. Thus, Cd content of higher trophic levels, such as copepods, can be affected by the degree of Cd incorporation in their food source, and by ecological factors regulating phytoplankton ingestion. Received: 13 September 1995 / Accepted: 29 October 1998     

Determination of ammonium uptake and regeneration rates using the seawater dilution method

We have developed a method for the determination of ammonium uptake and regeneration rates applying the principle of the seawater dilution technique. The method is based on the separation of uptake and regeneration processes in the dilution series. A model is used to estimate ammonium uptake and regeneration rates simultaneously, in addition to phytoplankton growth and grazing rates. The method was applied to dilution experiments conducted during a two-year study of the upwelling region off Oregon, USA. Ammonium uptake and regeneration rates determined with our method ranged from 0.5 to 3 mol l^-1d^-1 and from 0.2 to 2.9 mol l^-1d^-1, respectively. These values agree well with those from other studies applying ¹⁵N tracer techniques in the same or similar environments. We found a close coupling between ammonium uptake and regeneration, and a strong relationship between ammonium regeneration and grazing rates. In addition, the nutritional status of the phytoplankton community could be assessed by comparing instantaneous ammonium uptake rates with the specific phytoplankton growth rates. Using the dilution technique to determine ammonium uptake and regeneration rates of the plankton community is a promising alternative to the application of tracer techniques conventionally used to determine these rates.     

Pathways of arsenic uptake and incorporation in estuarine phytoplankton and the filter-feeding invertebrates Eurytemora affinis,Balanus improvisus and Crassostrea virginica

Arsenic uptake from water and from phytoplankton was followed in the copepod Eurytemora affinis and the barnacle Balanus improvisus collected from the Patuxent River estuary, Chesapeake Bay, eastern coast of the USA in 1987, and in the oyster Crassostrea virginica obtained from a hatchery on the shore of Chesapeake Bay in 1987. Dissolved arsenic was readily taken up by phytoplankton and by shell material of B. improvisus and C. virginica; however, no dissolved arsenic was incorporated into the invertebrate tissues. When E. affinis, B. improvisus and C. virginica were fed phytoplankton containing elevated arsenic contents, significant arsenic incorporation occurred. Juvenile B. improvisus incorporated relatively more arsenic than adults of all three species. Compared to the 100 to 200% increase in arsenic content by phytoplankton exposed to dissolved arsenic, the 25 to 50% increase in these invertebrate species via trophic transfer is relatively small. Even though the trophic pathway for arsenic transfer is the major one for higher trophic levels within an ecosystem, the potential for direct arsenic impact to trophic levels other than phytoplankton appears to be minimal.     

Saturated uptake kinetics: transient response of the marine diatom Thalassiosira pseudonana to ammonium,nitrate, silicate or phosphate starvation

Changes in the saturated uptake kinetics of the limiting nutrient were followed as Thalassiosira pseudonana (Clone 3 H) batch cultures entered ammonium, nitrate, silicate and phosphate starvation. Cultures starved of ammonium or phosphate developed very high specific uptake capacities over a 24 to 48 h starvation period, due to both decreases in cell quota and increases in uptake rates per cell. In particular, the cell phosphorus quota decreased ca. 8-fold during phosphate starvation and specific uptake rates exceeded 100 d^-1. In contrast, cultures entering nitrate or silicate starvation underwent little or no further cell division, and the uptake capacity declined during starvation. After 24 to 48 h starvation, an induction requirement for uptake of nitrate or silicate was apparent. These responses are consistent with adaptation to the pattern of supply of these nutrients in the field.     

The effects of ammonium and phosphate enrichments on clorophyll a,pigment ratio and species composition of phytoplankton of Vineyard Sound

Seawater containing natural phytoplankton populations from Vineyard Sound, USA was enriched in the laboratory with three levels each of ammonium and phosphate and with a combination of ammonium and phosphate which provided three different N:P ratios. The addition of ammonium produced more cells and chlorophyll a than the control or the phosphate enrichments. However, enrichment with ammonium and phosphate, regardless of the N:P ratio, yielded the most cells and chlorophyll a. Thus, nitrogen seems to be the primary limiting nutrient, with phosphate showing secondary limiting effects. The ratios of photosynthetic pigments decreased with the increased chlorophyll a production in the enriched cultures. There were no significant changes in the species composition within the cultures, so that the observed changes in pigment ratio and chlorophyll a content were due to physiological responses.     

Nitrogen fixation by blue-green algae associated with the siphonous green seaweed Codium decorticatum: effects on ammonium uptake

Nitrogen fixation (acetylene reduction) at rates of up to 1.2 g N₂ g dry wt^-1 h^-1 was measured for the siphonous green seaweed Codium decorticatum. No nitrogenase activity was detected in C. isthmocladum. The nitrogenase activity was light sensitive and was inhibited by the addition of DCMU and triphenyl tetrazolium chloride. Additions of glucose did not stimulate nitrogen fixation. Blue-green algae (Calothrix sp., Anabaena sp., and Phormidium sp.) were implicated as the organisms responsible for the nitrogenase activity. They occurred in a reduced microzone within the C. decorticatum thallus where nitrogen fixation was optimized. Nitrogen fixation did not affect the kinetic constants for ammonium uptake in C. decorticatum (K_s=12.0 M, V_max=13.4 mol NH₃ g dry wt^-1 h^-1) determined using the perturbation method. Nevertheless, C. decorticatum thalli which fixed nitrogen had internal dissolved nitrogen concentrations which were over 1.4 times higher than in non-fixing thalli. This suggests that if C. decorticatum does derive part of its nitrogen requirement from the blue-green algae which it harbors, the transfer does not involve competition between this process and the uptake of ambient ammonium.     

Seaweed diversity enhances nitrogen uptake via complementary use of nitrate and ammonium

The consequences of declining biodiversity remain controversial, in part because many studies focus on a single metric of ecosystem functioning and fail to consider diversity's integrated effects on multiple ecosystem functions. We used tide pool microcosms as a model system to show that different conclusions about the potential effects of producer diversity on ecosystem functioning may result when ecosystem functions are measured separately vs. together. Specifically, we found that in diverse seaweed assemblages, uptake of either nitrate or ammonium alone was equal to the average of the component monocultures. However, when nitrate and ammonium were available simultaneously, uptake by diverse assemblages was 22% greater than the monoculture average because different species were complementary in their use of different nitrogen forms. Our results suggest that when individual species have dominant effects on particular ecosystem processes (i.e., the sampling effect), multivariate complementarity can arise if different species dominate different processes. Further, these results suggest that similar mechanisms (complementary nutrient uptake) may underlie diversity-functioning relationships in both algal and vascular-plant-based systems.     

DCMU-enhanced fluorescence as an index of photosynthetic activity in phytoplankton

Tests have been carried out, both on phytoplankton cultures and in the field, on the relation between the ratio of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU)-enhanced to normal fluorescence (F _D /F _N ) and the specific photosynthetic rate (P/B). In all cases observed, significant linear correlations were found between these two values. Differences in this relation were observed according to species and physiological conditions. Nutrient stress that occurs in batch cultures decreases both F _D /F _N and P/B, while lowered light intensity has a different effect on both, increasing F _D /F _N and decreasing P/B. This is interpreted as indicating that F _D /F _N is an index of photosynthetic efficiency at different light levels and an indicator of the specific photosynthetic rate (P/B) when physiological conditions vary at a given light intensity. The practical use of DCMU-enhanced fluorometry to estimate instantaneous P/B values in the field is discussed, stressing the frequent calibrations needed, as in all in situ fluorometry studies.