Impact of a temporal salinity decrease on growth and nitrogen metabolism of the marine diatom Skeletonema costatum in continuous cultures

In 1987 effects of salinity fluctuations on growth of the centric diatom Skeletonema costatum (Greville) Cleve, isolated from the brackish Krammer estuary (SW Netherlands) in 1981, were investigated. Continuous cultures (12 h light: dark cycle) of S. costatum were adapted to constant salinity in natural (16.1) and synthetic (13.5) media. For several days the ammonium-limited cultures were exposed to a salinity fluctuation (minimum 4.8). Decreasing salinity caused an inhibition of photosynthesis, dark respiration and cell growth. Cellular pools of glucose decreased. While the carbohydrate content remained constant, the protein content increased slightly. Net carbon fixation was more inhibited than nitrogen assimilation. Ammonium accumulated during a salinity decrease; a total decline of the overcapacity of ammonium uptake was noticed and nitrogen limitation was relieved. Amino acid pools decreased, probably as a result of excretion (osmoregulation). The enzymes invoilved in ammonium assimilation showed an increased activity. Cellular activities were resumed during a salinity increase. Chlorophyll a increased; photosynthesis, ammonium uptake and growth were stimulated. The ammonium uptake capacity recovered completely; glutamic acid accumulation and increased glutamate-dehydrogenase (GDH) activity indicated supplementary ammonium assimilation via GDH. The activities of glutamine synthetase/glutamate synthase (GS/GOGAT) and GDH stabilized, and the cells returned to steady state under ammonium limitation.Communication no. 426 Delta Institute for Hydrobiological Research, Yerseke, The Netherlands     

Carbohydrate metabolism in the marine diatom Skeletonema costatum

This is a study on the metabolism of the algal biochemical constituents of a marine diatom, Skeletonema costatum. The carbohydrates, protein, and lipid, of the diatom grown under light and dark conditions were analyzed. The composition of the organic compounds did not vary in the alga grown under continuous light conditions throughout the experiment, although a little accumulation of carbohydrate and lipid was observed in the stationary phase of the algal growth. When the diatom, at maximum growth stage, was transferred to darkness, 44.4% of the carbohydrate, 57.8% of the protein, and 27.0% of the lipid were used by respiration within a few days. Detailed analysis of the algal carbohydrate demonstrated that low molecular weight carbohydrates, consisting of glucose, and -1,3-glucan, were readily metabolized by algal respiration, leaving cell-wall carbohydrates consisting of mannan and pentosan which are immune to algal respiration. The respiratory utilization of carbohydrates is discussed in relation to the rapid decay of glucan from marine particulate matter during the course of its sinking to further depths in the ocean, as observed previously by the present author.     

Diel variation in chlorophyll a,carbohydrate and protein content of the marine diatom Skeletonema costatum

Skeletonema costatum (Greville) Cleve isolated from Narragansett Bay, USA, was incubated at 3 light intensities (ca. 0.008, 0.040 and 0.075 ly min^-1) under a 12 h light: 12 h dark (12L:12D) photoperiod at 2°, 10° and 20°C. Cellular chlorophyll a increased at intensities less than ca. 0.040 ly min^-1; increases occured within one photoperiod at temperatures above 10°C. Cellular carbohydrate increased with light intensity at all temperatures; increases during the photophase were due to net production of the dilute acid-soluble fraction. Cellular protein increased during the photoperiod at 10° and 20°C; there was little difference in cellular protein among all cultures after one photoperiod. The rate at which cellular chlorophyll a increased in response to a decrease in light suggests that diel variation in cellular chlorophyll a is temperature-dependent in S. costatum. Protein: carbohydrate ratios ranged from ca. 0.5 to 2.0 over a diel cycle; ratios increased at lower intensities and higher temperatures. The diel range in protein:carbohydrate ratios equals that in cultures developing nitrogen deficiency; thus, use of this ratio as an index to phytoplankton physiological state must account for diel light effects.     

Growth,photosynthesis and carbon metabolism in the temperate marine diatom Skeletonema costatum adapted to low temperature and low photon-flux density

The temperate diatom Skeletonema costatum (Grev.) Cleve was grown in low temperature and/or low light conditions. The cultures were acclimatized for at least three months before experiments were begun. Our data indicate that the initial slope of the photosynthesis vs irradiance curve () is controlled predominantly by light history and the light-saturated photosynthesis (P _max) by temperature. The number of divisions per day decreased with decreasing light intensity, but was identical for cultures grown at 3° or 18°C. The metabolic pathways of inorganic carbon fixation were not fundamentally affected by low temperature or low light intensity, but both these factors increased labelling of C₃ compounds, synthesized by the Calvin-Benson cycle, and decreased that of phosphoenolpyruvate (PEP) and other metabolites. This indicates an enhancement of ribulose-1,5-bisphosphate (RuBP) carboxylase activity, which is the first step in the C₃ pathway (3-phosphoglycerate and sugar phosphate synthesis); this may optimize cell functions. At low temperatures, a seven-fold increase in RuBP carboxylase activity per cell was observed. S. costatum is able to adapt to low irradiance by increasing and decreasing I _k (the ratio of P _max:, light intensity at onset of light saturation), and to low temperature by increasing its cellular chlorophyll a and RuBP carboxylase content. However, in the latter case, adaptation is not optimal. This study revealed two main features: (1) there is evidence that RuBP carboxylase has a key function in adjustment to high rates of photosynthesis at suboptimal temperatures or irradiances; (2) adaptive mechanisms are dynamic processes and the role of the time scale in physiological adaptation should be considered.     

The rapid response of the marine diatom Skeletonema costatum to changes in external and internal nutrient concentration

A nitrogen-deficient batch culture of the marine diatom Skeletonema costatum, when resupplied with a mixture of nitrate and ammonium, showed an initial enhanced nitrate uptake rate leading to a large internal concentration (pool) of nitrate. Following this initial nitrate uptake event, nitrate uptake ceased, and nitrate assimilation was inhibited until the ammonium present was used. At this point, nitrate uptake resumed and nitrate assimilation began. No internal ammonium pool was observed during nitrate utilization, but a large nitrate pool remained throughout the utilization of external nitrate. The internal nitrate pool decreased rapidly after exhaustion of nitrate from the culture medium, but growth of cellular particulate nitrogen continued for about 24 h. A mathematical simulation model was developed from these data. The model cell consisted of a nitrate pool, ammonium pool, dissolved organic nitrogen pool, and particulate nitrogen. It was found that simple Michaelis-Menten functions for uptake and assimilation gave inadequate fit to the data. Michaelis-Menten functions were modified by inclusion of inhibitory and stimulatory feedback from the internal pools to more accurately represent the observed nutrient utilization.     

Adaptive significance of phytoplankton stickiness with emphasis on the diatom Skeletonema costatum

Diatom aggregate formation was analyzed using coagulation theory. Population dynamics models show that coagulation has an important impact on species succession during diatom blooms. When different species collide and form mixed aggregates this process causes interspecific interference competition within the diatom community. The outcome can be predicted by a set of simple differential equations. For a twospecies system the equations reduce to the Lotka-Volterra two-species competition model. The outcome of this interference competition depends on species-specific growth rates, cell sizes, stickiness and on the species composition of the seeding populations of a bloom. Due to mutual flocculation some species may disappear from the environment. Small and fast growing diatoms are favoured by high stickiness coefficients. The impact of stickiness on species succession was found to be most pronounced in eutrophic and hydrographically isolated environments. The sticking properties of the diatom Skeletonema costatum are discussed in an evolutionary context; we suggest that mutual coagulation increases the abundance of S. costatum relative to other diatom species in coastal areas. The model was tested on field data, and the predicted dynamics of a spring bloom was very similar to that observed.     

Ecotypic differentiation in the marine diatom Skeletonema costatum: influence of light intensity on the photosynthetic apparatus

Three genetically distinct clones of Skeletonema costatum (Grev.) Cleve were grown at 20°C under high (274 E m^-2 s^-1) and low (27 E m^-2 s^-1) light conditions and their photoadaptive photosynthetic responses compared. When all three clones were grown under low light, pigment analyses and fluorescence excitation spectra demonstrated that the accessory pigments, chlorophyll c and fucoxanthin, became more important in light-harvesting compared to chlorophyll a. Photosynthetic unit sizes increased for Photosystems I and II in low light, but photosynthesis vs irradiance characteristics were not reliable predictors of photosynthetic unit features. Fluorescence excitation spectra and photosynthesis vs irradiance (P-I) relationships indicated that changes in energy transfer occurred independent of changes in pigment content. Large increases in accessory pigment content were not accompanied by large increases in excitation from these pigments. Changes in energy transfer properties were as important as changes in PSU size in governing the photoadaptive responses of S. costatum. When the three clones were grown under identical conditions, each had a separate and distinct pattern of photoadaptation. Significant differences among clones were found for pigment ratios, photosynthetic unit sizes for Photosystems I and II and efficiency of energy transfer between pigments. These strikingly different photoadaptive strategies among clones may partially account for the great ecological success of the diatom species. This is the first quantitative investigation of the importance of both chlorophyll c and fucoxanthin to the adaptive responses of diatoms to light intensity, and represents the most complete characterization of the photoadaptive responses of a single species of marine phytoplankter to differences in light environment.     

Oxidative stress induced by copper: defense and damage in the marine planktonic diatom Ditylum brightwellii,grown in continuous cultures with high and low zinc levels

Light-limited cultures of the planktonic diatom Ditylum brightwellii (West) Grunow were grown at 14 salinity. Cells were subjected to oxidative stress induced by copper, in the presence of zinc. In two continuous cultures with total Zn levels of 40 and 140 nM, respectively, dissolved Cu levels were increased from 3 to 126 nM. This resulted in an increased Cu adsorption capacity of the cell walls, probably due to an increase of surface area and roughness. Sexual reproduction (auxospore formation) was accelerated but was considered as a non-specific stress response. Cu-induced oxidative stress was indicated by a decrease of reduced glutathione (GSH), and a removal of superoxide anions monitored as an increasing activity of superoxide dismutase (SOD). Although Zn has no oxidative potential per se, cell division rates and chlorophyll c contents were lower in the culture with high Zn levels. In both cultures, the pro-oxidant Cu caused a decrease of chlorophyll a, decreasing photosynthetic O₂ evolution and cell devision rates, and a growing number of deformed and broken cells.     

Resting spore formation of the marine planktonic diatom Chaetoceros anastomosans induced by high salinity and nitrogen depletion

The marine planktonic diatom Chaetoceros anastomosans, which was isolated from Sagami Bay, was used for a study of resting spore formation mechanisms in batch culture experiments. Vegetative cells could grow at salinities ranging from 20.7 to 45.5‰, and resting spore formation was enhanced significantly in nitrate-depleted, high salinity media (40.0 to 45.5‰). The rate of resting spore formation (1.9 d⁻¹) was comparable to the specific growth rate (1.8 d⁻¹) of vegetative cells in the exponential growth phase in normal salinity medium. The size of resting spores formed under high salinity conditions was smaller than that of spores formed in normal salinity media. Unlike vegetative cells, resting spores seemed to possess some mechanisms to survive over a wider range of salinities by resisting bacterial attacks on their cell walls. Received: 4 August 1996 / Accepted: 27 August 1996     

Diurnal and circadian rhythms in the turbidity of growing Skeletonema costatum cultures

Skeletonema costatum (Grev.) Cleve grown in batch culture at low light intensity under a 14 h light: 10 h dark photocycle showed exponential cell proliferation (1.1 doublings d^-1) without significant phasing of the cell division by the light: dark cycle. The growth in carbon concentration was, however, restricted to the light period. The turbidity of the culture closely followed the carbon oattern, and was not affected by the increase in the cell number during the dark period. It was found that a trustule suspension had only approximately 1% of the turbidity of the corresponding intact algae. Culture turbidity was therefore regarded as a biomass parameter similar to the carbon concentration, without direct correlation to the timing of the cell division. The short-time variations in the turbidity of growing algal cultures were further studied in a cage culture turbidostat. The growth rate (based on turbidity) increased rapidly during the first half of the light period, decreased slightly towards the evening and was zero throughout the dark period. When transformed to continuous light, the growth of the culture continued to show damped oscillations for up to 1 wk, but with a period of 26.7 h instead of 24 h. The same circadian rhythm was observed in chlorophyll content, and is thus possibly a reflection of a freely oscillating internal biological clock. The cage culture turbidostat was found to be a suitable device for studies of the photocycle related regulation of biosynthesis in S. costatum.     

Influence of temperature on the growth rate of Skeletonema costatum in response to variations in daily light intensity

Daily light intensities (I _o) can vary 10-fold during the winter-spring and late-summer diatom blooms in New England, USA, coastal waters. Laboratory cultures and natural populations incubated in dialysis sacs were examined to determine the time course of growth rate in Skeletonema costatum (Greville) Cleve in response to variations in daily light intensity during two bloom periods in Narragansett Bay, Rhode Island, USA. Log-phase cultures of S. costatum require 2 d to attain maximum growth rates at 2°C following transfer to saturating intensities. At 20°C, only 1 d is required. As temperature increases, Detonula confervacea (Cleve) Gran, Thalassiosira nordenskiöldii Cleve and Ditylum brightwellii (West) Grunow also exhibit rapid increases in mean daily division rates (K) following transfer to saturating light intensities. Thalassiosira pseudonana Hustedt, however, did not alter the time required to achieve maximum K as temperature varied. Natural populations of S. costatum did not show a well-defined relationship between K and light. Throughout a winterspring bloom, K was limited by low temperatures and exhibited no clear response to variations in I _o. A change in K in response to variation in I _o may occur on a daily basis during the summer, when temperatures are near 20°C; this has yet to be verified for in situ populations.     

Effect of heat stress on polyamine content and protein pattern in Skeletonema costatum

In the present paper we investigated the polyamine content and protein expression pattern in laboratory-reared Skeletonema costatum (Grev.) Cleve under temperature stress conditions. Putrescine, spermidine and to a lesser extent spermine were present in this alga. Free putrescine and spermidine increased after heat shock while a concomitant decrease in trichloroacetic acid (TCA)-soluble bound putrescine and spermidine occurred. After 40 min of 30 °C stress both were no longer detectable, whereas TCA-soluble bound spermine was present. The appearance of 83, 42, 40.7 and 35 kDa polypeptides was observed when S. costatum cells were subjected to heat treatment. Western blotting experiments revealed the presence in this diatom of ubiquitin and ubiquitin conjugates. Heat stress strongly increased the number of ubiquitin conjugate polypeptides of high molecular weight. No variation in the carbohydrate content was observed in response to stress. A possible role for these different metabolic events in the adaptive response of S. costatum to rapid temperature shifts has been hypothesized.     

Effects of ethyl 2-methyl acetoacetate (EMA) on the growth of Phaeodactylum tricornutum and Skeletonema costatum

We investigated the effects of ethyl 2-methyl acetoacetate (EMA) on growth of the marine diatom algae Phaeodactylum tricornutum (P. tricornutum) and Skeletonema costatum (S. costatum). Growth of P. tricornutum was significantly inhibited by the minimum concentration (3.5 mmol·L ^?1) of EMA at lower initial algal densities (IADs) (3.6×10⁴ and 3.3×10⁵ cells·mL ^?1). However, at the highest IAD, significant growth inhibition was found at above 7 mmol·L ^?1 of EMA exposure. In S. costatum, EMA concentrations of 10.5 mmol·L ^?1 or more significantly inhibited growth at lower IAD (3×10⁴ and 1.8×10⁵ cells·mL ^?1); at the highest IAD, only EMA concentrations above 14 mmol·L ^?1 obviously inhibited the growth of S. costatum. Changes in specific growth rates and pigment were consistent with algal growth, but only at higher EMA concentrations or lower IAD values was the ratio of chlorophyll a (Chla) to carotenoid significantly lower than the control. Medium effective concentration (EC ₅₀) values were in the order 4.07, 8.03 and 12.27 mmol·L ^?1 for P. tricornutum and 7.48, 11.92 and 17.22 mmol·L ^?1 for S. costatum. All these results show that the effect of EMA on the growth of algae was species specific and mainly depended on IAD, which might be an important factor to influence algal growth.     

Utilization of glutamate and glucose for heterotrophic growth by the marine pennate diatom Nitzschia laevis

Nitzschia laevis Hustedt grew in the dark in the presence of either glutamate or glucose as substrate. Complex mixtures of yeast extract or tryptone plus lactate also supported good heterotrophic growth, while tryptone alone only supported very slow growth in the dark. The observed growth rates of N. laevis in the dark at different concentrations of glutamate or glucose could be accounted for by the measured uptake rates of these compounds. The affinity of the uptake systems for glutamate and glucose (K _s =0.03 mM for each) was quite high, and similar for dark- and light-grown cells. The lack of a lag-phase when cells were transferred from photoautotrophic to heterotrophic growth conditions can be explained by the presence of uptake systems for glutamate and glucose in ligh--grown cells, as well as in dark-grown cells. However, the uptake capacity was generally higher in the latter than the former. N. laevis also took up alanine and lactate according to Michaelis-Menten kinetics, with a K _s for alanine of 0.02 mM and for lactate of 0.4 mM. Malate and glycerol were not taken up to a significant extent by the cells. Cells grown in continous light had a doubling time of 18 h. The shortest doubling time observed in the dark on glutamate was 48 h and on glucose 24 h. Glutamate was used for heterotrophic growth with an efficiency of 43% and glucose with an efficiency of 48%.Contribution No., 945 from the Department of Oceanography, University of Washington, Seattle, Washington 98195, USA.     

Partitioning of nitrogen and carbon in cultures of the marine diatom Thalassiosira fluviatilis supplied with nitrate,ammonium, or urea

Small or negligible differences in growth rates, average cell size, yields in cell numbers and total cell volumes were found in cultures of Thalassiosira fluviatilis inriched with nitrate, ammonium, or urea. Intracellular pools of unassimilated nitrate, nitrate, and ammonium were found in nutrient-rich conditions, but urea was not accumlated internally. Nitrogen assimilation into organic combination rather than nitrogen nutrient uptake was a critical rate-limiting step in nitrogen utilization. The free amino acid pool, protein, lipid-associated nitrogen, pigments, and total cell nitrogen were all highest in young or mature phase cells and decreased with age in senescent cells, whereas chitan, lipid, carbohydrate, and total cellular carbon all continued to increase during senescence. Dissolved organic nitrogen compounds accumulated in the medium only during senescence. C:N and lipid:protein were sensitive indicators of nitrogen depletion and age in T. fluviatilis.     

Variations in the biochemical composition of the diatom Coscinodiscus eccentricus with culture age and salinity

Cultures of the marine diatom Coscinodiscus eccentricus Ehrenberg were grown at four salinities, namely 20, 25, 30 and 35%. The changes in the carbohydrate, protein, silicon and pigment concentrations of the cells were studied throughout the growth cycles in these salinities. The carbohydrate content, expressed as a percentage of the cell dry weight, increased in all cultures during the lag and early log phases. It later fell, sharply at first, as it was used up faster than it was produced. It was not stored during the stationary phase. The protein percentage composition built up to a peak at different stages during the exponential or stationary growth stages, depending on the salinity, but was later respired. The silicon percentage of the dry weight usually decreased during the log phase, as the cells were dividing before becoming fully silicified, and remained low in the stationary phase due to an increase in the volume of the cells. The changes in the frustular thickness were also calculated. The values ranged from 0.24 to 0.35 μm (at the beginning and end of the experiments) to 0.15 μm during the log growth phase. The pigment content was always low. The lipid composition of the cells was calculated by difference. It was usually low during the exponential growth phase, but was accumulated during the stationary phase. The point at which this accumulation began was associated with the time when protein respiration started.     

Inorganic nitrogen metabolism in marine bacteria: Nitrate uptake and reduction in a marine pseudomonad

Growth experiments in batch cultures indicated that the uptake of nitrate by the marine pseudomonad PL1 was inhibited in the presence of ammonia provided that the ammonia concentration was higher than 1 mM. At ammonia concentrations of less than about 1 mM, however, both nitrate and ammonia were utilised simultaneously. The saturation constants for nitrate and ammonia uptake were both 2.6x10^-4 M, and similar to the Michaelis constants of nitrate reductase for nitrate (2.9x10^-4 M) and glutamine synthetase for ammonia (2x10^-4 M). Nitrate reductase activity linked to NADH was detected in chemostat-grown cultures with nitrate as nitrogen source, and in cultures containing limiting concentrations of nitrate and ammonia, ammonia or glutamate. Enzyme synthesis appeared to be repressed in cultures containing an excess of ammonia or glutamate. Chemostat cultures utilised ammonia or glutamate in preference to nitrate, while there was no marked preference between ammonia and glutamate.     

Marine diatoms grown in chemostats under silicate or ammonium limitation. I. Cellular chemical composition and steady-state growth kinetics of Skeletonema costatum

Skeletonema costatum was grown in chemostats under ammonium or silicate limitation to examine its growth kinetics and changes in cellular chemical composition at different steady-state growth rates. When the relationship between the effluent limiting substrate concentration and steady-state growth rates was examined, deviations from the simple hyperbolic form of the Monod growth equation were noted at low and high dilution rates. The data from the plot of growth rate and substrate concentration were divided into 4 regions and the relationship of these region to cell quota is discussed. Two physiological states were identified. All populations grown at D<0.05 h^-1, regardless of the size of the cells or the magnitude of Q, exhibited a maximal growth rate of approximately 0.05 h^-1, while populations grown at higher dilution rates (D>0.06 h^-1 to 0.14 h^-1). The maximal value of growth rate is obtained only in cultures grown at very high dilution rates where nutrient shift-up appears to occur, the cell quota approaches a maximum and the heterogeneous cell population becomes more homogeneous.Contribution No. 881 from the Department of Oceanography, University of Washington, Seattle, Washington 98195, USA. This paper represents a portion of two dissertations submitted by P.J.H. and H.L.C. to the Department of Oceanography, University of Washington, Seattle, in partial fulfillment of the requirements for the Ph.D. degree.     

Autecology and clonal variability of the marine centric diatom Thalassiosira rotula (Bacillariophyceae) in response to light,temperature and salinity

The influence of 49 combinations of salinity (10–40 S, at 5 S intervals) and temperature (0°–30°C, at 5C° intervals) on the maximum daily division rate (K) and 18 combinations of light intensity (six levels) and temperature (5°, 15°, and 25°C) on photosynthesis, cell division, and chlorophyll a was examined using two clones of Thalassiosira rotula Meunier isolated from the upwelling area of Baja California (clone C8) and from Narragansett Bay, Rhode Islands (clone A8). Physiological differences appear to characterize these to clones with regard to their temperature tolerance (C8 5°–30°C, A8 0°–25°C), maximum growth rate (C8 K=2.9, A8 K=2.4), chlorophyll a content, and in the rates of growth and photosynthesis in response to light intensity and temperature. Optimum salinity for both clones (25–30 S) was generally independent of temperature, while chlorophyll a content decreased with temperature. T. rotula is a cosmopolitan paractic species; experimental studies indicate that it is eurythermal and moderately euryhaline. Comparison of five additional Narragansett Bay isolates of T. rotula reveal minimal spacial or temporal variability in genetically determined physiological characteristics within this local population.     

The origin of nitrogen and phosphorus for growth of the marine angiosperm Thalassia testudinum

Marine Biology - How are high rates of production by Thalassia testudinum König maintained in notably nutrient-poor tropical waters? Yield-nutrient supply correlations indicate that a...