Yves Collos

Acclimation and toxicity of high ammonium concentrations to unicellular algae

A literature review on the effects of high ammonium concentrations on the growth of 6 classes of microalgae suggests the following rankings. Mean optimal ammonium concentrations were 7600, 2500, 1400, 340, 260, 100 μM for Chlorophyceae, Cyanophyceae, Prymnesiophyceae, Diatomophyceae, Raphidophyceae, and Dinophyceae respectively and their tolerance to high toxic ammonium levels was 39,000, 13,000, 2300, 3600, 2500, 1200 μM respectively. Field ammonium concentrations <100 μM would not likely reduce the growth rate of most microalgae. Chlorophytes were significantly more tolerant to high ammonium than diatoms, prymnesiophytes, dinoflagellates, and raphidophytes. Cyanophytes were significantly more tolerant than dinoflagellates which were the least tolerant. A smaller but more complete data set was used to estimate ammonium EC₅₀ values, and the ranking was: Chlorophyceae>Cyanophyceae, Dinophyceae, Diatomophyceae, and Raphidophyceae. Ammonia toxicity is mainly attributed to NH₃ at pHs >9 and at pHs <8, toxicity is likely associated with the ammonium ion rather than ammonia.

NITROGENOUS NUTRITION OF ALEXANDRIUM CATENELLA (DINOPHYCEAE) IN CULTURES AND IN THAU LAGOON, SOUTHERN FRANCE1

Alexandrium catenella (Whedon et Kofoid) Balech was isolated from Thau lagoon (northern Mediterranean) and its growth and uptake characteristics measured for nitrate, ammonium, and urea. Although affinity constants did not indicate a preference for ammonium over nitrate, there was a strong inhibition of nitrate uptake by ammonium when both nitrogen (N) sources were present. Nitrogen budgets during growth in cultures revealed major imbalances between decreases in dissolved N and increases in particulate N, indicating excretion of dissolved organic N during the early part of the growth phase and uptake during the later part. A quasi‐unialgal bloom in November 2001 (4×106 cells·L−1) allowed measurements of uptake of nitrate, nitrite, ammonium, and urea; net and gross growth rate of A. catenella; and grazing rates on this organism. The affinity constants indicate that it is not a strong competitor for the N nutrients tested when these are in low concentrations (<10 μgat N·L−1), compared with other members of the phytoplankton community. Indirect evidence from cultures indicate that dissolved organic N compounds could be important in triggering those blooms. Finally, the strongly unbalanced growth observed in the field indicates that A. catenella exhibits a storage rather than a growth response to a nutrient pulse and is adapted to low frequency events such as the passage of frontal disturbances. The disappearance of A. catenella was due to grazing that balanced growth at the peak of the bloom.

ACCLIMATION OF NITRATE UPTAKE BY PHYTOPLANKTON TO HIGH SUBSTRATE LEVELS

A literature review of data on nitrate uptake by phytoplankton suggests that nitrate levels above 20 μmol N·L−1 generally stimulated uptake rates in cultured unicellular algae and natural phytoplankton communities. This phenomenon indicates that phytoplankton cells acclimate to elevated nitrate levels by increasing their uptake capacity in a range of concentrations previously considered to be saturating. Cyanobacteria and flagellates were found to present a considerable capacity for acclimation, with low (0.1–2 μmol N·L−1) half‐saturation values (Ks) at low (5–20 μmol N·L−1) substrate levels and high (1–80 μmol N·L−1) Ks values at high (30–100 μmol N·L−1) substrate levels. However, some diatom genera (Rhizosolenia, Skeletonema, Thalassiosira) also appeared to possess a low affinity nitrate uptake system (Ks between 18 and 120 μmol N·L−1), which can help resolve the paradox of their presence in enriched seas. It follows that present models of nitrate uptake can severely underestimate the effects of high nitrate concentrations on phytoplankton dynamics and development. A more adequate approach would be to consider the possibility of multiphasic uptake involving several phase transitions as nitrate concentrations increased. Because it is a nonlinear phenomenon featuring strong thresholds, this effect appears to override that of other variables, such as irradiance, temperature, and cell size. Within the present context of eutrophication and for a range of concentrations that is becoming more and more ecologically relevant, equations are tentatively presented as a first approach to estimate Ks from ambient nitrate concentrations.

Unexpected Genetic Diversity among and within Populations of the Toxic Dinoflagellate Alexandrium catenella as Revealed by Nuclear Microsatellite Markers

ABSTRACT Since 1998, blooms of Alexandrium catenella associated with paralytic shellfish poisoning have been repeatedly reported for Thau Lagoon (French Mediterranean coast). Based on data obtained for rRNA gene markers, it has been suggested that the strains involved could be closely related to the Japanese temperate Asian ribotype of the temperate Asian clade. In order to gain more insight into the origin of these organisms, we carried out a genetic analysis of 61 Mediterranean and 23 Japanese strains using both ribosomal and microsatellite markers. Whereas the phylogeny based on ribosomal markers tended to confirm the previous findings, the analysis of microsatellite sequences revealed an unexpected distinction between the French and Japanese populations. This analysis also highlighted great intraspecific diversity that was not detected with the classical rRNA gene markers. The Japanese strains are divided into two differentiated A. catenella lineages: the Sea of Japan lineage and the east coast lineage, which includes populations from the Inland Sea and the Pacific Ocean. A. catenella strains isolated from Thau Lagoon belong to another lineage. These findings indicate that microsatellite markers are probably better suited to investigations of the population genetics of this species that is distributed worldwide. Finally, application of the population genetics concepts available for macroorganisms could support new paradigms for speciation and migration in phytoplankton assemblages.

Ammonium thresholds for simultaneous uptake of ammonium and nitrate by oyster-pond algae

Abstract Natural microalgal populations and axenic algal isolates from oyster ponds have been grown either in situ or in controlled conditions, in the presence of ammonium and nitrate as nitrogen sources. Both ions were added at various concentrations, up to 50 μg-at. N·l −1 ; other nutrients were in excess. In one experiment, urea was also added. Uptake of nitrate was followed by measuring disappearance of nitrate from the medium, and by incorporation of 15 NO 3 ; nitrate reductase (NR) activity and the intracellular nitrate pool were also measured. The uptake of nitrate was prevented by the presence of ammonium above a concentration which varied according to species. The ammonium threshold (in units of μg-at. N·l −1 ) was ≈ 30 for natural populations and the diatom Navicula ostrearia Bory, ≈ 21 for Nitzschia ovalis (sensu Hustedt), and ≈ 44 for Amphora coffeaeformis (sensu Hendey). Nitrate uptake started at a rate which was ≈ 39% of the eventual maximum rate observed for the natural populations, and from 11 to 17% for cultured strains. The initial low rate was maintained until the ambient ammonium concentration had decreased to ≈ 7.5 μ g-at. N·l −1 , except for A. coffeaeformis which shifted from slow to fast nitrate uptake at 23.5μg-at. N·l −1 . The nitrate uptake system then operated at a slightly higher rate than the one for ammonium (ammonium uptake/nitrate uptake = 0.86). Cultures with an initial ammonium concentration lower than the threshold values did not show an initial low rate or a lag phase for uptake of nitrate. NR activity was detectable even in the presence of ≈ 30 μ g-at. NH 4 -N·l −1 in the external medium. When urea and nitrate were presented simultaneously, urea was not taken up preferentially, as reported elsewhere; uptake was initially at a reduced rate until the external nitrate concentration decreased to 3.7 μg-at. N·l −1 . Then the rate of urea uptake increased to a maximum. It is suggested that because oyster-pond algae have evolved in an environment where concentrations of ammonium, nitrate, and organic nitrogen are continuously high, the threshold of inhibition by ammonium of uptake of these compounds is much higher than for similar pelagic and neritic species, so that they are able to assimilate other sources of nitrogen, such as nitrate and urea, simultaneously with ammonium.

Gene Expression in Proliferating Cells of the Dinoflagellate Alexandrium catenella (Dinophyceae)

ABSTRACT Understanding the conditions leading to harmful algal blooms, especially those produced by toxic dinoflagellate species, is important for environmental and health safety. In addition to investigations into the environmental conditions necessary for the formation of toxic blooms, we postulate that investigating gene expression in proliferating cells is essential for understanding bloom dynamics. Expressed sequence tags were produced from cultured cells of the toxic dinoflagellate Alexandrium catenella sampled during the initiation phase of growth using Sangers method and by 454 pyrosequencing. A significant proportion of identified genes (ca. 25%) represented enzymes and proteins that participate in a variety of cellular regulatory mechanisms that may characterize proliferating cells, e.g., control of the cell cycle and division, regulation of transcription, translation and posttranslational protein modifications, signaling, intracellular trafficking, and transport. All of the several genes selected for gene expression assays due to their involvement in metabolism and the cell cycle were overexpressed during exponential growth. These data will be useful for investigating the mechanisms underlying growth and toxin production in toxic Alexandrium species and for studying and monitoring the development of toxic blooms.

Nitrogen Uptake and Assimilation by Marine Phytoplankton

The introduction of the 15N tracer technique into ocean research (1) has allowed the direct measurement of growth-limiting nutrient salts such as nitrate or ammonium, and has led to new insights into the mechanisms of control of primary productivity in the marine environment.

Ocean urea fertilization for carbon credits poses high ecological risks

The proposed plan for enrichment of the Sulu Sea, Philippines, a region of rich marine biodiversity, with thousands of tonnes of urea in order to stimulate algal blooms and sequester carbon is flawed for multiple reasons. Urea is preferentially used as a nitrogen source by some cyanobacteria and dinoflagellates, many of which are neutrally or positively buoyant. Biological pumps to the deep sea are classically leaky, and the inefficient burial of new biomass makes the estimation of a net loss of carbon from the atmosphere questionable at best. The potential for growth of toxic dinoflagellates is also high, as many grow well on urea and some even increase their toxicity when grown on urea. Many toxic dinoflagellates form cysts which can settle to the sediment and germinate in subsequent years, forming new blooms even without further fertilization. If large-scale blooms do occur, it is likely that they will contribute to hypoxia in the bottom waters upon decomposition. Lastly, urea production requires fossil fuel usage, further limiting the potential for net carbon sequestration. The environmental and economic impacts are potentially great and need to be rigorously assessed.

Transient situations in nitrate assimilation by marine diatoms. III. short-term uncoupling of nitrate uptake and reduction

The response of nitrate uptake, storage, and reduction by nitrogen-limited cultures of three marine diatoms has been investigated following the addition of nitrate. During perturbation experiments, uncoupling between uptake and reduction was extensive in Skeletonema costatum (Grev.) Cleve and Phaeodactylum tricomutum Bohlin, as internal nitrate could reach 15% of cell nitrogen compared with 1 to 2% under steady-state conditions. This capacity for nitrate storage allowed these species to maintain high rates of nitrate reduction at low external nitrate levels. In contrast, Chaetoceros affinis Lauder accumulated much less nitrate during a perturbation, indicating a closer coupling between uptake and reduction, a phenomenon which was also observed in the field for nitrogen sufficient and nitrogen deficient phytoplankton. Nitrate reduction was related to external nitrate by a hyperbola when data was obtained by either the graded addition or the perturbation method. The relationship to internal nitrate clearly showed saturation kinetics only when data were obtained by the latter method. The sudden decrease in nitrate reduction upon external nitrate depletion, however, made it necessary to separate the data into two groups and to fit them to two hyperbolas with different kinetic characteristics.

Patterns in nutrient limitation and chlorophyll a along an anthropogenic eutrophication gradient in French Mediterranean coastal lagoons

A cross-ecosystem comparison of data obtained from 20 French Mediterranean lagoons with contrasting eutrophication status provided the basis for investigating the variables that best predict chlorophyll a (Chl a) concentrations and nutrient limitation of phytoplankton biomass along a strong nutrient enrichment gradient. Summer concentrations of dissolved inorganic nitrogen (DIN) and phosphorus (DIP) comprised only a small fraction of total nitrogen (TN) and total phosphorus (TP). On the basis of inorganic nutrient concentrations, the most oligotrophic lagoons appeared to be phosphorus-limited, with a tendency towards the development of nitrogen limitation as eutrophication increased, as evidenced by decreasing DIN:DIP ratios. A weak but significantly positive relationship was found between dissolved silicate (DSi) and Chl a, reflecting DSi accumulation in the water column along the trophic state gradient and implying a progressive shift away from potential Si limitation of phytoplankton growth. Observed concentrations of Chl a were far better explained by TN and TP than by DIN and DIP concentrations, suggesting that a total nutrient based approach is likely to be the most appropriate for managing eutrophication in Mediterranean lagoons and other coastal waters. These results give credence to the idea that marine and freshwater environments respond in a similar fashion to nutrient enrichment.