Diurnal variations of intracellular nitrate storage by marine diatoms: effects of nutritional state

Nitrate uptake and accumulation were measured in N-sufficient, N-limited, and 24 h N-starved cells of Phaeodactylum tricornutum Bohlin and Skeletonema costatum Grev., growing under a light-dark cycle. In N-sufficient cells the uptakerate was reduced at night and showed possible variation during the light period. In N-limited and N-starved cells such diurnal changes in uptake were absent, except extremely rapid, but short-lived nitrate uptake was observed early in the morning in N-limited cells. The nitrate accumulation inside the cells reflects a transient uncoupling between uptake and reduction mostly due to the light-dark cycle and strongly influenced by the physiological state of the cells. This accumulation is high during the night and at the beginning of the day, but decreases during the light period in N-sufficient cells. On the other hand, nitrate storage in N-sufficient and N-limited cultures shows a strong diurnal pattern, with maximum accumulation, suggesting the greatest uncoupling between uptake and assimilation, in the morning. In N-starved cells, accumulation is high and constant during the entire light period. Consequently, the uncoupling between nitrate uptake and reduction decreases during the light period but increases with N deficiency. These results indicate the importance of light periodicity and nutritional state of the cells on the nitrate utilization. They reveal the need for more systematic studies on N dynamics in relation to nutrient-light regimes.     

Studies on N-limited growth of diatoms in dialysis culture

N-limited growth of Skeletonema costatum (Grev.) Cleve in dialysis culture has been studied. The division rate of exponentially growing cells was independent of the nitrate concentration in the growth medium in the range from 886 down to 0.25 μM N-salt, while no growth beyond one division took place in cultures to which no nitrogen salt was added. The half saturation constant, K₃, for growth must, therefore, be in the range 0–0.13 μM, provided the growth-nutrient relationship is hyperbolic for S. costatum.Contrary to growth rate, cellular chlorophyll and protein were markedly reduced in media poor in nitrogen salts. A dialysis culture chamber was used to demonstrate that the measurement of half saturation constants for S. costatum was influenced by stirring, the stirred culture growing almost twice as fast as the unstirred control under identical conditions. The ability of diatoms to grow rapidly at low nitrogen levels was used to remove nutrients from sewage enriched media. Removal efficiencies corresponding to 80 and 90 % were obtained for nitrate and ammonia, respectively, using the diatom Phaeodactylum tricornutum Bohlin. It was found that both this diatom and S. costatum as well as Thalassiosira pseudonana Hust (Hasle) tolerated ammonia up to at least 450 μM with no deleterious effects on growth rate.     

Nitrate and nitrite uptake by free-living and immobilized N-started cells ofPhormidium laminosum

N-starved free-living and polyvinyl-immobilized cells ofPhormidium laminosum (strain OH-1-pCl₁) have been investigated in relation to their nitrate and nitrite uptake characteristics. N-deficient cells showed higher inorganic N-uptake rates than N-sufficient ones. The photosynthetic activities of the cells decreased progressively with the time of N-starvation. N-starved cells produced high amounts of exopolysaccharides, which appear to assist the immobilization process. Inorganic N-uptake by N-starved cells occurred in both light and dark under aerobic conditions. In anaerobiosis light was required for the uptake, confirming that the necessary energy might perhaps be derived from the respiratory electron transport chain under aerobiosis. Ammonium inhibited nitrate uptake but did not affect the uptake of nitrite. Initial nitrate and nitrite uptake rates were temperature-dependent and yielded hyperbolic curves when plotted against the N source concentration, indicating the existence of saturable transport system(s).     

The effect of short-term fluctuations in pH on NO−3 uptake and intracellular constituents in Skeletonemacostatum (Grev.) Cleve

Measurements of uptake rates, intracellular nitrogen pools, and other key intracellular constituents were made during exponential growth in Skeletonema costatum (Grev.) Cleve under varying pH levels. An understanding of the overall effects of extracellular pH on the above mentioned cellular parameters is crucial in order to ascertain the degree to which pH must be regulated and monitored in laboratory experiments with marine phytoplankton.It was found that uptake rates and intracellular pool sizes of NO^?₃ were directly influenced by the extracellular pH level, whereas, other cellular compounds remained relatively unchanged. Therefore, nitrogen uptake and intracellular nitrogen storage are dependent on key H⁺ and OH_? ion transport mechanisms that are associated with phytoplankton metabolism. These findings reiterate the fact that investigators examining nitrogen uptake and assimilatory mechanisms in marine phytoplankton must be conscious of cellular H ⁺ and OH^? fluxes that contribute to intracellular pH regulation and changes in extracellular pH levels, both of which interact to affect phytoplankton metabolic processes.     

Temperature-influenced variations in speciation and chemical composition of marine phytoplankton in outdoor mass cultures

Between September, 1976 and July, 1977 Phaeodactylum tricornutum Bohlin was replaced as the dominant species by Skeletonema costatum (Grev) Cleve as temperatures fell below 10°C in the fall in an outdoor pond supplied with a mixture of waste water and sea water. Phaeodactylum tricornutum returned in the spring as the major species when temperatures rose above 10°C. In an adjacent pond in which only nitrogen and phosphorus were added in excess, however, P. tricornutum dominated throughout the entire study period even through the temperature varied between 0 and 25°C. We suspected that the difference inspecies dominance in the two ponds occurred because Skeletonema costatum requires silicon, which was present in sufficient quantities only in the waste-water-enriched pond. whereas Phaeodatylum tricornutum does not have a specififc requirement for this nutrient. The cellular chemical composition of P. tricornutum varied in a U-shaped fashion with changing temperature: minimum values for the cellular carbon, nitrogen, and chlorophyll contents were displayed at 15–20°C and maximum values at 3 and 15°C. Both the cellular carbon: nitrogen and carbon: chlorophyll ratios by weight were invariant with changing temperatures at ≈6: 1 and 50: 1 respectively, indicating nutrient saturation. Only under conditionsof nutrient saturation, which can be established in various ways, can the influence of temperature on phytoplankton physiology be separated from nutrient-related factors.     

Nitrate Utilization by the Diatom Skeletonema costatum: II. Regulation of Nitrate Uptake

Nitrate utilization has been characterized in nitrogen-deficient cells of the marine diatom Skeletonema costatum. In order to separate nitrate uptake from nitrate reduction, nitrate reductase activity was suppressed with tungstate. Neither nitrite nor the presence of amino acids in the external medium or darkness affects nitrate uptake kinetics. Ammonium strongly inhibits carrier-mediated nitrate uptake, without affecting diffusion transfer. A model is proposed for the uptake and assimilation of nitrate in S. costatum and their regulation by ammonium ions.     

Effect of the respiratory activity on photoinhibition of the cyanobacterium Synechocystis sp.

The influence of respiratory activity on photosynthesis in Synechocystis cells that had been exposed to high light intensity was studied using distinct conditions of nitrogen supply. The photoinhibitory rate of N-sufficient cells was not influenced by the presence of different nitrogen sources. In contrast, when N-starved cells were resupplied with ammonium, they were protected from photoinhibition. Although N-starved cells presented a higher rate of dark O₂ uptake than N-sufficient ones, the photoinhibitory rate increased in both cases after addition of sodium azide or sodium azide plus salicylhydroxamic acid in the photoinhibitory treatment. In the absence of the D₁ protein repair mechanism, photodamage to Photosystem II was faster in N-sufficient cells than in N-starved ones. Mitigation of photodamage disappeared when the respiratory activity of N-starved cells was partially suppressed by the addition of sodium azide or sodium azide and salicylhydroxamic acid. Our results suggest that electron flow through cyanobacterial terminal oxidases can assist Photosystem I in removing electrons from the reduced plastoquinone pool, thus contributing to both reopening of Photosystem II reaction centers and avoiding photogeneration of reactive oxygen species under photoinhibitory conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

THE ROLE OF GLUTAMINE SYNTHETASE IN THE INCORPORATION OF AMMONIUM IN SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1

The K_m for ammonia for glutamine synthetase and glutamate dehydrogenase was measured in enzyme extracts from Skeletonema costatum (Grev.) Cleve. At similar physiological pH and temperature the half-saturation constant for glutamine synthetase was 29 μM, whereas for GDH it was 28mM. On the basis of relative enzymic activity, as well as substrate affinity, it is suggested that glutamine synthetase is the enzyme primarily responsible for the incorporation of ammonium into the amino acid pool, when extracellular nitrogen is at ecological concentrations.     

13C AND 15N UPTAKE BY MARINE PHYTOPLANKTON. I. INFLUENCE OF NITROGEN SOURCE AND CONCENTRATION IN LABORATORY CULTURES OF DIATOMS1

Two species of marine diatoms, Skeletonema costatum (Grev.) Cleve and Phaeodactylum tricornutum Bohlin were grown in batch and continuous cultures on four different nitrogen compounds (nitrate, nitrite, ammonium, urea). Carbon and nitrogen uptake were measured simultaneously with the stable isotopes ¹³C and ¹⁵N. Nitrogen uptake generally increased with N concentration in the medium, but no clear difference existed between the N sources. Carbon fixation was decreased for up to 5 h following the addition of the N compound. Nitrite generally had the greatest inhibitory effect on C uptake. Carbon-to-nitrogen uptake ratios decreased with increasing dissolved N concentration, becoming lower than one in nutrient-limited cultures. In contrast, batch cultures exhibited C:N uptake ratios greater than one. These effects are essentially short-term and differ from long-term influences of the N source on the cellular chemical composition.     

Growth of three plankton diatom species in Oslofjord water in the absence of artificial chelators

Cultures of the plankton diatoms Skeletonema costatum (Greville) Cleve, Thalassiosira nordenskioeldii Cleve, and Lauderia annulata Cleve were grown in sea-water samples collected in the inner Oslofjord on 11 occasions between February, 1976 and January, 1977. The growth experiments were carried out in silicone-coated culture flasks, with nitrate, phosphate, and silicate added in excess, and under standardized conditions of a temperature of 10 °C and a saturating light intensity. Growth rates, as measured during exponential growth over a period of 5–6 days, were, with very few exceptions, close to the maximum rates obtained in a complete medium. The addition of ethylenediaminetetra-acetic acid (EDTA) to the water samples did not lead to improved growth, nor did EDTA have an effect on the initial lag phase. The lack of a seasonal variation in the observed growth responses was in marked contrast to the large fluctuations in the phytoplankton populations in the fjord. These findings give no support to the hypothesis that species successions among marine plankton diatoms are influenced by organic chelators or other extracellular substances produced by planktonic algae.     

Heavy metal tolerance of marine phytoplankton. I. The tolerance of three algal species to zinc in coastal sea water

Tolerance levels to zinc ions of three diatoms (Skeletonema costatum (Grev.) Cleve, Thalassiosira pseudonana (Hust.) Hasle and Phaeodactylum tricornutum (Bohlin) grown in dialysis culture in the local fjord water were studied. Declining relative growth rates were observed by addition of 50, 250 and 25,000μg/l of zinc ions, respectively, for the three algae. Reduced final cell concentrations were found at lower zinc levels. At least for one species a significant increase in zinc uptake by the cells took place at zinc levels which did not seem to influence the growth and development of the alga. Two clones of Skeletonema costatum studied showed significant intraspecific differences regarding the tolerance to zinc pollution. Dialysis bioassay was found suitable for monitoring heavy metal pollution of aquatic recipients.     

BRACKISH WATER AND FRESHWATER SPECIES OF THE DIATOM GENUS SKELETONEMA. II. SKELETONEMA POTAMOS COMB. NOV.1

Electron microscope investigations of the siliceous frustule show that the diatom described by Hustedt as Stephanodiscus subsalsus (A. Cleve) Hust. is not Skeletonema subsalsum (A. Cleve) Bethge (Melosira subsalsa A. Cleve) but is Microsiphona potamos Weber. This species is so similar to Skeletonema costatum (Grev.) Cleve and Skeletonema subsalsum that the combination Skeletonema potamos (Weber) Hasle is suggested. Present records classify Skeletonema potamos as a freshwater species of lakes and rivers. In Sandusky Bay, Lake Erie (U.S.A.) and in River Wümme, a tributary of the River Weser (Germany) it grows with Skeletonema subsalsum. In nature, and when grown in cultures at a salinity of 0%, the processes are extremely short; when grown at salinities of 2% or more, the processes are much longer.     

Heavy metal tolerance of marine phytoplankton. II. Copper tolerance of three species in dialysis and batch cultures

The tolerance to copper ions of three diatoms, namely, Skeletonema costatum, (Grev.) Cleve, Thalassiosira pseudonana (Hust.) Hasle and Phaeodactylum tricornutum Bohlin grown in dialysis and batch cultures in the local fjord water has been established. Reduction of growth rates was observed by the addition of 10, 25 and 400 μg/1 of copper ions, respectively for the three species investigated. At the higher levels of copper addition (400 and 700 MS/1) cells of P. tricornutum in dialysis culture increased their copper content to more than 200 times over those of the controls, the ratio of copper to chlorophyll in the cells increasing 150 times.All three species showed marked increases in copper content when a copper salt was added to batch cultures of the algae. The two clones of Skeletonema costatum tested showed nearly identical sensitivity to copper ions, but they differed markedly in their zinc tolerance.     

INTRASPECIFIC COMPARISONS OF NITRATE UPTAKE IN THREE MARINE DIATOMS1

Short-term (within 5 min) and long-term (≤2 h) rates of nitrate uptake were determined for the marine diatoms, Nitschiella longissima (Cleve), Skeletonema costatum (Greville) Cleve and Asterionella japonica (Cleve). Pigment levels, cell carbon, nitrogen and cell volume were also determined for cells in the logarithmic and stationary phases of growth. For each species, one clone isolated from oligotrophic coastal water and one clone isolated from eutrophic coastal water were compared. Long-term NO₃^? uptake typically followed saturation kinetics describable by the Michaelis-Menten expression. Under experimental conditions, half-saturation constants ranged from 0.6 to 2.2 μM NO₃^?. Generally, the oligotrophic clones had lower Ks and V_max (on a per cell basis) than their eutrophic counterparts, though this was only statistically significant in one pair of clones. Eutrophic and oligotrophic clones also differed in their short-term response to nutrient addition; oligotrophic clones showed greatest rate of uptake at the lowest nitrate addition while uptake by eutrophic clones increased with increasing nitrate concentration. However, all clones had very similar V_max values expressed on a dry weight basis. Under N-starvation, cellular C and pigment levels (and N to a lesser extent) generally declined more in eutrophic than in oligotrophic clones. While the differences between inshore and offshore clones were not great, the results are consistent with the hypothesis that eutrophic waters support algae which grow faster and are less conservative biochemically than cells in oligotrophic waters.     

Biochemical compositions of two dominant bloom- forming species isolated from the Yangtze River Estuary in response to different nutrient conditions

Variations of cellular total lipid, total carbohydrate and total protein content of two dominant bloom-forming species (Skeletonema costatum and Prorocentrum donghaiense) isolated from the Yangtze River Estuary were examined under six different nutrient conditions in batch cultures. Daily samples were collected to estimate the cell growth, nutrient concentration and three biochemical compositions content during 7 days for S. costatum and the same sampling procedure was done every other day during 10 days for P. donghaiense. Results showed that for S. costatum, cellular total lipid content increased under phosphorus (P) limitation, but not for nitrogen (N) limitation; cellular carbohydrate were accumulated under both N and P limitation; cellular total protein content of low nutrient concentration treatments were significantly lower than that of high nutrient concentration treatments. For P. donghaiense, both cellular total lipid content and total carbohydrate content were greatly elevated as a result of N and P exhaustion, but cellular total protein content had no significant changes under nutrient limitation. In addition, the capability of accumulation of three biochemical constituents of P. donghaiense was much stronger than that of S. costatum. Pearson correlation showed that for both species, the biochemical composition of three constituents (lipid, carbohydrate and protein) had no significant relationship with extracellular N concentration, but had positive correlation with extracellular and intracellular P concentration. The capability of two species to accumulate cellular total lipid and carbohydrate under nutrient limitation may help them accommodate the fluctuating nutrient condition of the Yangtze River Estuary. The different responses of two species of cellular biochemical compositions content under different nutrient conditions may provide some evidence to explain the temporal characteristic of blooms caused by two species in the Yangtze River Estuary.     

METABOLIC SEGREGATION OF INTRACELLULAR FREE AMINO ACIDS IN PLATYMONAS (CHLOROPHYTA)1

¹⁴C-amino acids were supplied to Platymonas subcordiformis (Wille) Hazen and the incorporation of radioactivity into protein and other compounds was followed. Alanine was rapidly metabolized by both N-limited and N-sufficient cells. Arginine and lysine were metabolized rapidly by N-limited cells, but were sequestered from metabolism in N-sufficient cells. This suggests the existence of two functionally distinct pools; a “metabolic” pool that is rapidly metabolized and preferentially used for incorporation into protein, and a “storage” pool rich in basic amino acids that is sequestered from metabolism.     

DIVERSITY IN THE GENUS SKELETONEMA (BACILLARIOPHYCEAE): III. PHYLOGENETIC POSITION AND MORPHOLOGICAL VARIABILITY OF SKELETONEMA COSTATUM AND SKELETONEMA GREVILLEI,WITH THE DESCRIPTION OF SKELETONEMA ARDENS SP. NOV.1

Skeletonema costatum (Grev.) Cleve emend. Zingone et Sarno and S. grevillei Sarno et Zingone were known only from the type material collected from Hong Kong waters more than a century ago. Both species have now been collected as live material, and their morphology and phylogenetic position are investigated in this study. Eight Skeletonema strains isolated from Florida, USA; Uruguay; and Brazil are attributed to S. costatum, while one strain from Oman is ascribed to S. grevillei based on morphological similarity to the type material of these species. In addition, a new Skeletonema species, S. ardens Sarno et Zingone, is described for a strain from Singapore and two from northern Australian waters. Skeletonema ardens has terminal fultoportula processes ending in a tapered, undulate protrusion and long intercalary fultoportulae with 1:1 junctions. The rimoportula of terminal valves is located at the margin of the valve face. No major morphological variations were observed within S. grevillei and S. ardens along a salinity gradient, whereas in S. costatum, the processes shortened and the valves came into close contact at low salinities, as already described for S. subsalsum (Cleve) Bethge. Consistent with their morphology, Skeletonema costatum and Skeletonema subsalsum also had similar rDNA sequences. Skeletonema grevillei and S. ardens were distinct in the large subunit (LSU) phylogeny. Skeletonema ardens exhibited consistent intraspecific genetic differences in both the LSU and small subunit (SSU) rDNA.     

Alkaline and acid phosphatases of the marine diatoms Chaetoceros affinis var. willei (Gran) Hustedt and Skeletonema costatum (Grev.) Cleve

Alkaline phosphatase activity in cultures of the marine diatom Chaetoceros affinis var. willei (Gran) Hustedt was higher than in Skeletonema costatum (Grev.) Cleve. The enzyme activity was localized in coarse cell particles. Acid phosphatase activity was found in the cytoplasmic fraction. Induction of alkaline phosphatase depended on the $\text{N}\text{P}$ ratio in the culture medium. A $\text{N}\text{P}$ ratio > 40 in dilution/batch culture and > 30 in large scale batch culture, respectively, induced alkaline phosphatase.Cell phosphorus showed a critical value below which alkaline phosphatase was induced. Alkaline phosphatase in natural phytoplankton from the Trondheimsfjord is unlikely to occur except possibly in special situations.