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A Tetraselmis sp. was selected for its antioxidant activity owing to its high lipid peroxidation inhibition capacity. With the aim to monitor culture conditions to improve antioxidant activity, effects of CO2-induced acidification on Tetraselmis growth, elemental composition, photosynthetic parameters and antioxidant activity were determined. Two pH values were tested (6.5 and 8.5) in batch and continuous cultures in photobioreactors. Acidification enhanced cell growth under both culture methods. However, the microalgae physiological state was healthier at pH 8.5 than at pH 6.5. Indeed, photosynthetic parameters measured with pulse amplitude modulated (PAM) fluorometry showed a decrease in the photosystem II (PSII) efficiency at pH 6.5 in batch culture. Yet, with the exception of the PSII recovering capacity, photosynthetic parameters were similar in continuous culture at both pH. These results suggest that lowering pH through CO2-induced acidification may induce a lower conversion of light to chemical energy especially when coupled with N-limitation and/or under un-balanced culture conditions. The highest antioxidant activity was measured in continuous culture at pH 6.5 with an IC50 of 3.44 ± 0.6 μg mL−1, which is close to the IC50 of reference compounds (trolox and α-tocopherol). In addition, the principal component analysis revealed a strong link between the antioxidant activity and the culture method, the photophysiological state and the nitrogen cell quota and C:N ratio of Tetraselmis sp.. These results highlight Tetraselmis sp. as a species of interest for natural antioxidant production and the potential of PAM fluorometry to monitor culture for production of biomass with a high antioxidant activity.

Nitrogen availability often limits biological productivity in marine systems, where inorganic nitrogen, such as ammonium is assimilated into the food web by bacteria and photoautotrophic eukaryotes. Recently, ammonium assimilation was observed in kleptoplast-containing protists of the phylum foraminifera, possibly via the glutamine synthetase/glutamate synthase (GS/GOGAT) assimilation pathway imported with the kleptoplasts. However, it is not known if the ubiquitous and diverse heterotrophic protists have an innate ability for ammonium assimilation. Using stable isotope incubations (15N-ammonium and 13C-bicarbonate) and combining transmission electron microscopy (TEM) with quantitative nanoscale secondary ion mass spectrometry (NanoSIMS) imaging, we investigated the uptake and assimilation of dissolved inorganic ammonium by two heterotrophic foraminifera; a non-kleptoplastic benthic species, Ammonia sp., and a planktonic species, Globigerina bulloides. These species are heterotrophic and not capable of photosynthesis. Accordingly, they did not assimilate 13C-bicarbonate. However, both species assimilated dissolved 15N-ammonium and incorporated it into organelles of direct importance for ontogenetic growth and development of the cell. These observations demonstrate that at least some heterotrophic protists have an innate cellular mechanism for inorganic ammonium assimilation, highlighting a newly discovered pathway for dissolved inorganic nitrogen (DIN) assimilation within the marine microbial loop.

Twelve microalgae species isolated in tropical lagoons of New Caledonia were screened as a new source of antioxidants. Microalgae were cultivated at two light intensities to investigate their influence on antioxidant capacity. To assess antioxidant property of microalgae extracts, four assays with different modes of action were used: 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2&rsquo, azino-bis (3-&eacute, thylbenzothiazoline-6-sulphonique) (ABTS), oxygen radical absorbance capacity (ORAC), and thiobabituric acid reactive substances (TBARS). This screening was coupled to pigment analysis to link antioxidant activity and carotenoid content. The results showed that none of the microalgae studied can scavenge DPPH and ABTS radicals, but Chaetoceros sp., Nephroselmis sp., and Nitzschia A sp. have the capacity to scavenge peroxyl radical (ORAC) and Tetraselmis sp., Nitzschia A sp., and Nephroselmis sp. can inhibit lipid peroxidation (TBARS). Carotenoid composition is typical of the studied microalgae and highlight the siphonaxanthin, detected in Nephroselmis sp., as a pigment of interest. It was found that xanthophylls were the major contributors to the peroxyl radical scavenging capacity measured with ORAC assay, but there was no link between carotenoids and inhibition of lipid peroxidation measured with TBARS assay. In addition, the results showed that light intensity has a strong influence on antioxidant capacity of microalgae: Overall, antioxidant activities measured with ORAC assay are better in high light intensity whereas antioxidant activities measured with TBARS assay are better in low light intensity. It suggests that different antioxidant compounds production is related to light intensity.

Nephroselmis sp. was previously identified as a species of interest for its antioxidant properties owing to its high carotenoid content. In addition, nitrogen availability can impact biomass and specific metabolites&rsquo, production of microalgae. To optimize parameters of antioxidant production, Nephroselmis sp. was cultivated in batch and continuous culture conditions in stirred closed photobioreactors under different nitrogen conditions (N-repletion, N-limitation, and N-starvation). The aim was to determine the influence of nitrogen availability on the peroxyl radical scavenging activity (oxygen radical absorbance capacity (ORAC) assay) and carotenoid content of Nephroselmis sp. Pigment analysis revealed a specific and unusual photosynthetic system with siphonaxanthin-type light harvesting complexes found in primitive green algae, but also high lutein content and xanthophyll cycle pigments (i.e., violaxanthin, antheraxanthin, and zeaxanthin), as observed in most advanced chlorophytes. The results indicated that N-replete conditions enhance carotenoid biosynthesis, which would correspond to a higher antioxidant capacity measured in Nephroselmis sp. Indeed, peroxyl radical scavenging activity and total carotenoids were higher under N-replete conditions and decreased sharply under N-limitation or starvation conditions. Considering individual carotenoids, siphonaxanthin, neoxanthin, xanthophyll cycle pigments, and lycopene followed the same trend as total carotenoids, while &beta, carotene and lutein stayed stable regardless of the nitrogen availability. Carotenoid productivities were also higher under N-replete treatment. The peroxyl radical scavenging activity measured with ORAC assay (63.6 to 154.9 &micro, mol TE g&minus, 1 DW) and the lutein content (5.22 to 7.97 mg g&minus, 1 DW) were within the upper ranges of values reported previously for other microalgae. Furthermore, contents of siphonaxanthin ere 6 to 20% higher than in previous identified sources (siphonous green algae). These results highlight the potential of Nephroselmis sp. as a source of natural antioxidant and as a pigment of interest.

The demand for natural products isolated from microalgae has increased over the last decade and has drawn the attention from the food, cosmetic and nutraceutical industries. Among these natural products, the demand for natural antioxidants as an alternative to synthetic antioxidants has increased. In addition, microalgae combine several advantages for the development of biotechnological applications: high biodiversity, photosynthetic yield, growth, productivity and a metabolic plasticity that can be orientated using culture conditions. Regarding the wide diversity of antioxidant compounds and mode of action combined with the diversity of reactive oxygen species (ROS), this review covers a brief presentation of antioxidant molecules with their role and mode of action, to summarize and evaluate common and recent assays used to assess antioxidant activity of microalgae. The aim is to improve our ability to choose the right assay to assess microalgae antioxidant activity regarding the antioxidant molecules studied.

Haynesina germanica is a coastal benthic foraminifer known to sequester chloroplasts from benthic pennate diatoms. This study investigates its cellular organization, as well as the oxygen consumption and production rates under dark and light conditions. The implications of the sequestered chloroplasts are subsequently discussed at an individual and ecological level. Living specimens collected in Arcachon Basin showed well-preserved chloroplasts in the cytoplasm in observations using transmission electron microscopy. Microelectrodes were used to measure oxygen consumption and production rates in incubated specimens in the laboratory. In the dark, oxygen consumption rates ranged from 662 ± 12 to 1082 ± 59 pmol O 2 ind -1 d -1 . Under illuminated conditions (i.e., about 300 μmol photon -1 m -2 s -1 ), we observed and quantified substantial photosynthetic activity, up to 199 pmol O 2 ind -1 d -1 . It appears that H. germanica combines autotrophic and heterotrophic nutritional modes, thereby reflecting a mixotrophic strategy. In Arcachon Basin, H. germanica potentially uses chloroplasts acquired from microphytobenthos to carry out photosynthesis and produce oxygen and organic matter. Stored chloroplasts could constitute an additional food source during impoverished periods, providing substantial competitive advantage to this species. This putative behavior could also affect the biogeochemistry of the sediment at a microscale by creating oxygenated microniches around individual foraminifera.

International audience; Ammonia tepida is a dominant benthic foraminifer colonizing intertidal mudflat sediments. Horizontal locomotion speeds were monitored using time-lapse image analysis over 6 and 24h. Experimental conditions were based on foraminifera exposed to dry sediment re-suspended in artificial sea water (ASW) without any nutrient addition (condition DS), to combusted sediment re-suspended in in ASW also without any nutrient addition (condition CS), or to combusted sediment re-suspended in ASW enriched with either: nitrate, urea, glucose, soil extract (SE), extracellular polymeric substances (EPS), benthic diatoms (Entomoneis paludosa) or natural microphytobenthic assemblages (MPB). Significant differences were already measured after 6h between A. tepida mean locomotion speeds at the different experimental conditions. However, differences were clearer after 24h where the slowest A. tepida mean locomotion speed was measured in specimens placed in CS (1.00±0.30mmh−1) and the highest mean locomotion speed in DS (2.99±0.22mmh−1). Three different groups were defined according to their locomotion speed, (1) foraminifera exposed to DS had a locomotion speed significantly higher than all other conditions, (2) foraminifera placed in conditions enriched in SE, Glucose, Urea and EPS had intermediary locomotion speeds (1.8–2.5mmh−1), and (3) conditions with foraminifera showing the lowest locomotion speeds (1–1.6mmh−1) were CS, nitrate, MPB and E. paludosa. Thus, foraminifera exposed to organic matter (DS, SE, Glucose and Urea) showed faster locomotion speeds than foraminifera exposed to inorganic matter (CS, nitrate) or live preys (E. paludosa, MPB).Dissolved organic matter enrichment enhanced foraminifera locomotion speed, which might be a behavioural response to satisfy their carbon and/or nitrogen requirements, and the lowest locomotion speed observed when feeding on live preys might be a consequence of longer time required for live prey phagocytosis.

This study presents a new gel based technique to describe the pore water ammonium distribution through the sediment-water interface in two dimensions at a millimeter scale. The technique is an adaptation of the classical colorimetric method based on the Berthelot's reaction. After the thin film of the gel probe was equilibrated by diffusion either in standard solutions or in pore waters, a colorimetric reagent gel was set on the gel probe, allowing development of the characteristic green color. A flatbed scanner and subsequent densitometry image analysis allowed to determine the concentration distribution of ammonium. The gel probe was tested in the laboratory for two media, deionized water and seawater, within the range 0–3000 μM in NH4+. Detection limit is about 20 μM and accuracy about ±25 μM. The field validation was realized in a tidal mudflat of the French Atlantic coast by comparison with conventional pore water extraction and colorimetric analysis. The large range of concentrations and its applicability in continental and marine sediments suggest a wide range of applications of the technique for a reasonable cost., Graphical abstract Image 1, Highlights • Fast and inexpensive ammonium 2D imaging through the sediment water interface. • Millimetric spatial resolution. • Applicable in marine and fresh aquatic systems.

Haynesina germanica, an ubiquitous benthic foraminifer in intertidal mudflats, has the remarkable ability to isolate, sequester, and use chloroplasts from microalgae. The photosynthetic functionality of these kleptoplasts has been demonstrated by measuring photosystem II quantum efficiency and O2 production rates, but the precise role of the kleptoplasts in foraminiferal metabolism is poorly understood. Thus, the mechanism and dynamics of C and N assimilation and translocation from the kleptoplasts to the foraminiferal host requires study. The objective of this study was to investigate, using correlated TEM and NanoSIMS imaging, the assimilation of inorganic C and N (here ammonium, NH4+) in individuals of a kleptoplastic benthic foraminiferal species. H. germanica specimens were incubated for 20 h in artificial seawater enriched with H13CO3− and 15NH4+ during a light/dark cycle. All specimens (n = 12) incorporated 13C into their endoplasm stored primarily in the form of lipid droplets. A control incubation in darkness resulted in no 13C-uptake, strongly suggesting that photosynthesis is the process dominating inorganic C assimilation. Ammonium assimilation was observed both with and without light, with diffuse 15N-enrichment throughout the cytoplasm and distinct 15N-hotspots in fibrillar vesicles, electron-opaque bodies, tubulin paracrystals, bacterial associates, and, rarely and at moderate levels, in kleptoplasts. The latter observation might indicate that the kleptoplasts are involved in N assimilation. However, the higher N assimilation observed in the foraminiferal endoplasm incubated without light suggests that another cytoplasmic pathway is dominant, at least in darkness. This study clearly shows the advantage provided by the kleptoplasts as an additional source of carbon and provides observations of ammonium uptake by the foraminiferal cell.

Azadinium spinosum, a small dinoflagellate isolated from the North Sea, is a producer of azaspiracids (AZAs), a group of biotoxins associated with human illness following ingestion of contaminated shellfish. Using batch and continuous cultures of A. spinosum, the present study investigated the effects of different environmental and nutritional factors (salinity, temperature, photon flux density, aeration, culture media, nitrogen sources, phosphate source, and N/P ratios) on growth, maximum cell concentration, and AZA cell quota. Azadinium spinosum grew in a wide range of conditions; from 10 ̊C to 26 ̊C and salinities from 30 to 40, under irradiances ranging from 50 μmol m−2 s−1 to 250 μmol m−2 s−1, with or without aeration. Growth and maximum cell concentration were highest at a salinity of 35, at temperatures between 18 ̊C and 22 ̊C, and with aeration. Concerning AZA cell quota, the most significant effect was observed at low temperature; the AZA cell quota was more than 20 times higher at 10 ̊C (220 fg cell−1) than at temperatures between 18 ̊C and 26 ̊C. A. spinosum grew on all media tested with only slight differences in growth rate and AZA cell quota. In continuous culture, lowering the concentration of nutrients (0.5 strength of a modified K-medium) in the inflow improved AZA cell quota whereas higher concentration (doubling the normal strength of K-medium) improved maximal cell concentration. A. spinosum grew on different sources of nitrogen tested (nitrate, urea, ammonium) with almost no effect on toxin cell quota and growth, except that adding ammonium caused a decrease in growth. These first experiments on Azadinium spinosum increased our knowledge on factors affecting its growth and toxin production; furthermore, these results allowed and improved particularly A. spinosum production in pilot scale photobioreactors for AZA isolation.

Benthic diatoms are dominant primary producers in intertidal flats. This study investigated the effect of different nitrogen sources and concentrations on Entomoneis paludosa growth and physiological responses. Six nitrogen sources either inorganic (nitrate and ammonium) or organic (urea, arginine, glutamine and glycine) supplied at two concentrations (40 and 400 µM-N) induced significant effects on growth, carbon, nitrogen, pigment content and maximum PSII quantum efficiency (F v /F m). Entomoneis paludosa grew under all nitrogen sources albeit showing differences in lag phase, growth rate and cell yield. Inorganic nitrogen, urea and arginine induced higher growth, whereas glycine did not support high biomass. F v /F m showed variability dependent on nitrogen source and C/N ratio. F v /F m varied between 0.55 and 0.65 at 400 μM-N with the highest values observed in glycine, glutamine and urea, whereas nitrate, ammonium and arginine induced lower F v /F m. All cellular components decreased in the 40 µM-N treatments, with nitrogen and pigments being lower than carbon content. Light-harvesting pigment ratios Chl c/Chl a and photoprotective pigment ratios (diatoxanthin + diadinoxanthin)/Chl a increased, while fucoxanthin/Chl a ratios were unaffected by N-limitations. Entomoneis paludosa was capable of quickly adapting and using a wide variety of nitrogen sources. This adaptability may contribute to microphytobenthos diatom ecological success in mudflat ecosystems.

Over the last century, human activities have altered the global nitrogen cycle, and anthropogenic inputs of both inorganic and organic nitrogen species have increased around the world, causing significant changes to the functioning of aquatic ecosystems. The increasing frequency of Pseudo-nitzschia spp. in estuarine and coastal waters reinforces the need to understand better the environmental control of its growth and domoic acid (DA) production. Here, we document Pseudo-nitzschia spp. growth and toxicity on a large set of inorganic and organic nitrogen (nitrate, ammonium, urea, glutamate, glutamine, arginine and taurine). Our study focused on two species isolated from European coastal waters: P. multiseries CCL70 and P. australis PNC1. The nitrogen sources induced broad differences between the two species with respect to growth rate, biomass and cellular DA, but no specific variation could be attributed to any of the inorganic or organic nitrogen substrates. Enrichment with ammonium resulted in an enhanced growth rate and cell yield, whereas glutamate did not support the growth of P. multiseries. Arginine, glutamine and taurine enabled good growth of P. australis, but without toxin production. The highest DA content was produced when P. multiseries grew with urea and P. australis grew with glutamate. For both species, growth rate was not correlated with DA content but more toxin was produced when the nitrogen source could not sustain a high biomass. A significant negative correlation was found between cell biomass and DA content in P. australis. This study shows that Pseudo-nitzschia can readily utilize organic nitrogen in the form of amino acids, and confirms that both inorganic and organic nitrogen affect growth and DA production. Our results contribute to our understanding of the ecophysiology of Pseudo-nitzschia spp. and may help to predict toxic events in the natural environment.

Intertidal mudflats are important feeding areas for migrating and wintering shorebird species. Partial release of organic and inorganic nutrients through bird excretion may enrich the nutrients flow of mudflat ecosystems, especially in areas with a low flushing rate. The objective of the present work was to experimentally evaluate the effect of Dunlin droppings on the growth and biochemical composition of the microphytobenthic diatom, Entomoneis paludosa (W. Smith) Reimer. Different nutritional conditions were tested to investigate the impact on E. paludosa growth rate, biomass, elemental (CHN) and biochemical composition (lipids, proteins, carbohydrates, pigments). Various culture media were used going from plain artificial seawater to F/2 culture media containing variable nitrate concentrations (0; 50 or 882 μM-NO3) to which bird dropping extract was added or not. The faeces extracts contained inorganic nitrogen (9.1 μM-NH4), inorganic phosphorus (8.2 μM-PO4), traces of silicate (0.2 μM-Si), organic nitrogen in the form of urea (16 μM-N) and other dissolved organic nitrogen (120 μM-N). Faeces extract inartificial seawater was sufficient to sustain E. paludosa growth (up to 6.8 cell divisions in 9 days). A significant growth rate increase (+ 20%) and higher biomasses were observed when faeces extract was added to inorganic media enriched with 50 μM-NO3. Bird droppings had a significant effect in E. paludosa final biochemical composition with the addition of faeces extract to a culture medium containing 50 μM-NO3 increasing E. paludosa protein content and decreasing carbohydrate content. Pigment content per cell increased with the addition of bird dropping extract but ratios of light-harvesting and photo-protective pigments to chlorophyll a were unaffected. E. paludosa grown with faeces extract showed high cellular nitrogen and carbon contents, close to those obtained when cells were grown in F/2 medium. This study showed that shorebird droppings, through the addition of dissolved material, can significantly affect microphytobenthic diatom growth and biochemical composition.

Azadinium spinosum, a small dinoflagellate has recently been discovered and identified as the primary producer of azaspiracid-1 (AZA) and -2. Since AZA poisoning has been reported following consumption of contaminated shellfish it is important to have these toxins available for toxicological studies, and a sustainable production of AZAs as calibrants in monitoring programs without having to rely on natural events. In order to address this concern, continuous pilot scale cultures were carried out to evaluate the feasibility of AZA production from A. spinosum. Algae were cultured using two 100 L chemostats in series (R1 and R2), with agitation and pH control. Four different dilution rates were tested (0.15, 0.2, 0.25 and 0.3 day−1) to evaluate chemostat bioreactors in terms of cell and toxin productivity. Algae were collected in a 300 L transparent cylindro-conical tank and harvested with a tangential flow filtration device. Subsequently, toxins were extracted from the algal retentate and separately from the permeate using solid phase adsorption procedures. The cell concentration at steady state remained stable using different dilution rates (190,000 and 210,000 cells · mL−1 in R1 and R2 respectively). However, the AZA cell quota decreased as the dilution rate increased, consequently an optimum production was obtained at 0.25 day−1 under the studied conditions. After filtration, 50–70 % of the toxin was contained in the retentate and 30–50 % was released into the permeate. After optimization, the procedures for solid phase extraction of toxins from the retentate and permeate allowed for the recovery of 80 ± 5 % of original toxins produced. This work demonstrated the feasibility of producing AZAs from A. spinosum produced in a bioreactor for purification and production of certified standards.

International audience; In aquaculture, particularly in bivalve hatcheries, the biochemical composition of algal diets has a strong influence on larval and post-larval development. Biochemical composition is known to be related to culture conditions, among which light represents a major source of variation. The effects of blue light on biochemical composition and photosynthetic rate of Isochrysis sp. (T-iso) CCAP 927/14 were assessed in chemostat at a single irradiance (300 μmol photons m−2 s−1) and compared with white light. Two different dilution (renewal) rates were also tested: 0.7 and 0.2 d−1. Relative carbohydrate content was lower under blue light than under white light at both dilution rates, whereas chlorophyll a and photosynthesis activity were higher. In contrast, carbon quota was lower and protein content higher under blue light than under white light, but only at 0.7 d−1. Despite these metabolic differences, cell productivity was not significantly affected by the spectrum. However, the nitrogen to carbon ratio and photosynthetic activity were higher at 0.7 d−1 than at 0.2 d−1, while carbon quota and carbohydrate content were lower. Our results show that blue light may influence microalgal metabolism without reducing productivity for a given growth rate, a result that should be of great interest for microalgal production in aquaculture.

The presence of dinoflagellates of the genus Ostreopsis along Mediterranean coasts was first observed in 1972, in the bay of Villefranche-sur-Mer. However, over the past ten years, harmful events related to this benthic dinoflagellate have been reported in Italian, Spanish, Greek, French, Tunisian and Algerian coastal areas. In France, during a hot period in August 2006, cases of dermatitis and respiratory problems were registered in Marseille area. At that time, a link to the proliferation of Ostreopsis was highlighted for the first time in that area. A specific monitoring was designed and implemented in the summer 2007. Two strains of Ostreopsis cf. ovata, collected in 2008 from Villefranche-sur-Mer and Morgiret coastal waters and grown in culture, were identified by molecular analysis and studied to characterise their growth and toxin profile. Liquid chromatography-mass spectrometry (LC-MS/MS) indicated that both strains produced ovatoxin-a (OVTX-a) as the major component (ca. 90%), and traces of palytoxin (PLTX). Toxin content was determined at the end of the exponential growth phase with highest concentration of 55 pg.cell(-1) of OVTX-a and 2.5 pg.cell(-1) of PLTX.

The original publication is available at http://www.springerlink.com/content/ft63475038857t41/, peer-reviewed, Azaspiracids (AZAs) are secondary metabolites of Azadinium spinosum that can accumulate in shellfish and cause food poisoning when consumed. We describe here an analytical procedure for the determination of AZAs in cultures of A. spinosum with a focus on the formation of AZA methyl esters as artefacts during extraction and sample pre-treatment. A. spinosum cells were collected from bioreactor cultures using centrifugation or filtration. Different extraction procedures were evaluated for formation of methyl ester artefacts, yield, and matrix effects. Filtration of cultures using glass-fibre filters led to increased formation of methyl esters, and centrifugation is recommended for recovery of cells. The extraction solvent (methanol (MeOH), acetone, and acetonitrile (MeCN)) did not significantly affect the yield of AZAs as long as the organic content was 80% or higher. However, the use of MeOH as extraction solvent led to increased formation of methyl esters. AZA1 recovery over two successive extractions was 100% at the 95% confidence level for acetone and MeOH. In standard-addition experiments, no significant matrix effects were observed in extracts of A. spinosum or Azadinium obesum up to a sample size of 4.5 × 109 μm3. Moreover, experiments carried out to clarify the formation and structure of methylated AZA analogues led to the description of two AZA methyl esters and to the correction of the chemical structures of AZAs29–32.