Marine Microalga Phaeodactylum Tricornutum Research Articles

Effects of fertilizer-based culture media on the production of exocellular polysaccharides and cellular superoxide dismutase by Phaeodactylum tricornutum (Bohlin)

Microalgae cultures are receiving attention because of increasing biotechnological and biomedical production of active biomolecules. We evaluated various fertilizer-based culture media to scale up production of the marine microalga Phaeodactylum tricornutum for production of exocellular polysaccharides (EPS), soluble proteins, and cellular superoxide dismutase (SOD). The standard source of sodium nitrate was the same as that used in the synthetic f/2 culture medium and ammonium nitrate, urea, ammonium sulfate, and calcium nitrate as alternative sources of nitrogen. The maximum production of EPS was achieved in microalgae cells grown in the culture media containing 63 and 23% nitrogen from ammonium sulfate, and also in microalgae cells grown in the culture media containing 3% nitrogen from ammonium nitrate. The maximum production of cellular SOD was achieved in microalgae cells grown in the culture media containing 35 and 26% nitrogen from ammonium sulfate, and in the culture media containing 17% nitrogen from urea. The results suggest that it is possible to use a source of nitrogen, other than sodium nitrate, to scale up growth of P. tricornutum for production of EPS and SOD at reduced costs.

Biosynthesis of Phytochelatins and Arsenic Accumulation in the Marine Microalga Phaeodactylum tricornutum in Response to Arsenate Exposure

The arsenate-induced synthesis of phytochelatins (PC), intracellular cysteine-rich metal-binding peptides, and its relationship with toxicity and with As accumulation in the cell have been studied in laboratory cultures of the marine microalga Phaeodactylum tricornutum. The time course of cellular PC and As in short-term exposures showed that the involvement of PC in the As detoxification as well as the pathway of cellular As depend on the extent of As accumulation and on the rate of PC synthesis. At arsenate concentrations causing As accumulation at a rate exceeding that of PC synthesis, cells seem to activate a mechanism of release of As mainly in a chemical form not complexed with PC. At arsenate concentrations at which the synthesis of PC occurs at a rate sufficient to allow a significant portion of As accumulated in the cell to be bound, the fate of cellular As seems to be mainly controlled by PC. The occurrence of these different pathways of As detoxification was discussed to explain the pattern of cellular As and PC in cells grown for three days at growth-inhibitory and at no growth-inhibitory concentration of arsenate.

Immobilization of the marine microalga Phaeodactylum tricornutum in alginate for in situ experiments: Bead stability and suitability

Microalgae immobilization in alginate matrixes has been recently used to perform in situ experiments. However, the susceptibility of alginate matrixes to cation chelating agents and to antigelling cations, which can cause bead disruption or dissolution, is a major limitation for in situ exposures in estuarine and marine systems. The ultimate goal of this study was to produce alginate beads stable in seawater and suited for Phaeodactylum tricornutum growth. For this, different concentrations of alginate isolated from Macrocystis pyrifera (1.5, 1.9 and 2.3% [w/v]) and Laminaria hyperborea (4.0, 4.9 and 5.8% [w/v]), two concentrations of the hardening cations calcium and strontium (2.0 and 4.0% [w/v]), and the use of the polycation chitosan were investigated. Only beads found to be more stable after 16 days of exposure in seawater were inoculated with the microalga. P. tricornutum immobilized in beads prepared from 5.8% L. hyperborea alginate and in all beads in which a chitosan hardening treatment was applied showed a weak microalgal growth. Beads prepared using 4.9% of L. hyperborea alginate and a 4% (w/v) strontium solution were found to be the most stable and the most suitable for microalgal growth, and were exposed in the field, under natural fluctuating conditions of light and temperature. In situ growth rates of immobilized P. tricornutum cells demonstrated the potential of these beads for future use in in situ experiments in estuarine and marine systems.

Copper-induced changes of non-protein thiols and antioxidant enzymes in the marine microalga Phaeodactylum tricornutum

The cellular defence mechanisms used by the marine diatom Phaeodactylum tricornutum to cope with copper toxicity were investigated. Copper induced a rapid synthesis of phytochelatins (PC) of different degree of polymerisation, initially formed at the expense of the cellular pool of glutathione. The formation of Cu–PC complexes was demonstrated by using size exclusion chromatography (SEC). These complexes were detected as early as 1 h after Cu exposure, and increased with time. Measurements of the activity of the antioxidant enzymes showed that superoxide dismutase (SOD) and CAT activity increased a few hours after Cu addition up to reach, after 48 h, the 40 and 200% of the control, respectively. Glutathione reductase (GR) activity, after an initial partial inhibition, also enhanced indicating the need to restore the oxidative balance of glutathione. APX and PPX activity was not significantly affected by Cu treatment, suggesting that in P. tricornutum CAT is the major enzyme for scavenging H2O2. The time course of Cu–PC complexes formation suggested that glutathione and its derived peptides (PC) form the first line of defence to combat against Cu-induced reactive oxygen species (ROS) formation. Thereafter, the activity of at least three antioxidant enzymes was enhanced to counteract the oxidative stress induced by Cu. Lipid peroxidation, measured as TBA-rm, enhanced after 24 and 48 h of exposure, indicating that prolonged Cu exposure induced membrane damage.

Copper-induced changes of non-protein thiols and antioxidant enzymes in the marine microalga Phaeodactylum tricornutum

The cellular defence mechanisms used by the marine diatom Phaeodactylum tricornutum to cope with copper toxicity were investigated. Copper induced a rapid synthesis of phytochelatins (PC) of different degree of polymerisation, initially formed at the expense of the cellular pool of glutathione. The formation of Cu–PC complexes was demonstrated by using size exclusion chromatography (SEC). These complexes were detected as early as 1 h after Cu exposure, and increased with time. Measurements of the activity of the antioxidant enzymes showed that superoxide dismutase (SOD) and CAT activity increased a few hours after Cu addition up to reach, after 48 h, the 40 and 200% of the control, respectively. Glutathione reductase (GR) activity, after an initial partial inhibition, also enhanced indicating the need to restore the oxidative balance of glutathione. APX and PPX activity was not significantly affected by Cu treatment, suggesting that in P. tricornutum CAT is the major enzyme for scavenging H 2O 2. The time course of Cu–PC complexes formation suggested that glutathione and its derived peptides (PC) form the first line of defence to combat against Cu-induced reactive oxygen species (ROS) formation. Thereafter, the activity of at least three antioxidant enzymes was enhanced to counteract the oxidative stress induced by Cu. Lipid peroxidation, measured as TBA-rm, enhanced after 24 and 48 h of exposure, indicating that prolonged Cu exposure induced membrane damage.

Speciation of cadmium–γ-glutamyl peptides complexes in cells of the marine microalga Phaeodactylum tricornutum

Phaeodactylum tricornutum responds to Cd exposure by synthesizing phytochelatins (PC n ) [(γ-Glu–Cys) n –Gly] with n values from 2 to 6. Size exclusion chromatography was coupled with reverse-phase HPLC to follow the formation of complexes between Cd and γ-glutamyl peptides in cells exposed to the metal ion during the growth cycle. Five stable complexes (Cd–PC n n=2–6), in which peptides of unique chain length chelate Cd by formation of thiolate bonds, were identified. The formation of Cd–PC n complexes in which acid labile sulfide is incorporated (PC-coated CdS crystallites) was also demonstrated. These complexes exhibited luminescence and UV transition at 280 nm. The apparent MW was 8–12 kDa and the S 2−/Cd ratio 0.4. The 66% of the total PC γ-Glu–Cys units in these complexes were polymerized as PC 4. The incorporation of sulfide into Cd–PC n complexes enhances the Cd/SCys ratio from 0.6 to 1.6. The kinetics of formation of Cd–thiols complexes is consistent with an involvement of the glutathione (GSH) in the Cd sequestration mechanism of P. tricornutum through the initial formation of Cd–GSH complexes. The metal ion is subsequently transferred from GSH to Cd-induced phytochelatins to form more stable Cd–PC n complexes.

Modification of the nutritive value of Phaeodactylum tricornutum for Artemia sp. in semicontinuous cultures

The biochemical composition of the marine microalga Phaeodactylum tricornutum was modified through semicontinuous culture techniques. Six concentrations of nutrients were combined with six renewal rates to generate 36 different microalgal cultures. The nutritive quality of these cultures was tested by measuring survival, growth and production of nauplii by Artemia sp. There was a wide range of responses generated in Artemia after 23 days of culture, with survival ranging between 17 and 97%, length ranging between 7 and 10 mm, and from 3 to 97 nauplii per spawning, demonstrating that culture conditions can cause dramatic differences in the nutritive value of a microalgal species. Growth, survival and production of nauplii were directly related to growth rate and nutrient concentration in the microalgal culture in the range 0.5–8 mmol N l −1 and renewal rates 10–50%. Microalgal protein content or protein/lipid and protein/carbohydrate ratios could not explain all the differences in the nutritive value of the microalgae. The production of nauplii per spawning was the most sensitive parameter for evaluating the nutritional quality of the microalgae. Results demonstrated that semicontinuous cultures of microalgae provided a simple and useful tool for the study of nutritional requirements of filter-feeding organisms and to manipulate the growth of Artemia nauplii.

Modification of sterol concentration in marine microalgae

Semicontinuous cultures of the marine microalgae Phaeodactylum tricornutum and Tetraselmis suecica were carried out with renewal rates (RR) of 10, 20, 30, 40 and 50%. The concentration of (24S)-24-methylcholesta-5,22E-dien-3β-ol in P. tricornutum followed a progressive fall from a maximum of 29.1 fg cell−1, in the culture with the 10% RR, to a minimum of 20 fg cell−1 in the culture with the 50% RR. In Tetraselmis suecica, 24-methylcholest-5-en-3β-ol ranged from 137 fg cell−1 with a 10% RR to 40 fg cell−1 with a 40% RR and 24-methylcholesta-5,24(28)-dien-3β-ol ranged from 403 fg cell−1 with a 10% RR to 80 fg cell−1 with a renewal rate of 50%. Renewal rate is a good tool to modify the sterol concentration in marine microalgae.

Modification of sterol concentration in marine microalgae

Semicontinuous cultures of the marine microalgae Phaeodactylum tricornutum and Tetraselmis suecica were carried out with renewal rates (RR) of 10, 20, 30, 40 and 50%. The concentration of (24 S)-24-methylcholesta-5,22 E-dien-3β-ol in P. tricornutum followed a progressive fall from a maximum of 29.1 fg cell −1, in the culture with the 10% RR, to a minimum of 20 fg cell −1 in the culture with the 50% RR. In Tetraselmis suecica, 24-methylcholest-5-en-3β-ol ranged from 137 fg cell −1 with a 10% RR to 40 fg cell −1 with a 40% RR and 24-methylcholesta-5,24(28)-dien-3β-ol ranged from 403 fg cell −1 with a 10% RR to 80 fg cell −1 with a renewal rate of 50%. Renewal rate is a good tool to modify the sterol concentration in marine microalgae.

Mixotrophic productivity of the marine diatom Phaeodactylum tricornutum cultured with soluble fractions of rye, wheat and potato

The marine microalga Phaeodactylum tricornutum was cultured semi-continuously with the soluble fractions of wheat, rye and boiled potato flours. Fifteen percent of the culture volume was renewed every 3 d. The cell productivities were 0.9 x 109 cells/l/d, 1.1 x 109 cells/l/d and 2.6 x 109 cells/l/d for wheat, rye and potato respectively. The productivity of the autotrophic control was 1.0 x 109 cell/l/d. When a soluble fraction of raw potato was added, the productivity was enhanced to 4.1 x 109 cells/l/d, 2.4 times higher than the autotrophic culture. The high productivity of P. tricornutum with the soluble fractions of Solanum tuberosum suggests its usefulness as a source of nutrients for the production of microalgal biomass.

Eicosapentaenoic acid (20∶5n‐3) from the marine microalgaPhaeodactylum tricornutum

AbstractEicosapentaenoic acid (EPA, 20∶5n‐3) was obtained from the marine microalgaePhaeodactylum tricornutum by a three‐step process: fatty acid extraction by direct saponification of biomass, polyunsaturated fatty acid (PUFA) concentration by formation of urea inclusion compounds, and EPA isolation by semipreparative high‐performance liquid chromatography (HPLC). Alternatively, EPA was obtained by a similar two‐step process without the PUFA concentration step by the urea method. Direct saponification of biomass was carried out with two solvents that contained KOH for lipid saponification. An increase in yield was obtained because the problems associated with emulsion formation were avoided by separating the biomass from the soap solution before adding hexane for extraction of insaponifiables. The most efficient solvent, ethanol (96%) at 60°C for 1 h, extracted 98.3% of EPA. PUFA were concentrated by the urea method with a urea/fatty acid ratio of 4∶1 at a crystallization temperature of 28°C and by using methanol and ethanol as urea solvents. An EPA concentration ratio of 1.73 (55.2∶31.9) and a recover yield of 78.6% were obtained with methanol as the urea solvent. This PUFA concentrate was used to obtain 93.4% pure EPA by semipreparative HPLC with a reverse‐phase, C18, 10 mm i.d.×25‐cm column and methanol/water (1% acetic acid), 80∶20 w/w, as the mobile phase. Eighty‐five percent of EPA loaded was recovered, and 65.7% of EPA present inP. tricornutum biomass was recovered in highly pure form by this three‐step downstream process. Alternatively, 93.6% pure EPA was isolated from the fatty acid extract (without the PUFA concentration step) with 100% EPA recovery yield. This two‐step process increases the overall EPA yield to 98.3%, but it is only possible to obtain 20% as much EPA as that obtained by three‐step downstream processing.

Discrepancies between cell volume and organic content in semi-continuous cultures of a marine microalga

J. FÁBREGAS, A. CID, E. MORALES, B. CORDERO AND A. OTERO. 1996. Changes in average cell volume, measured by flow cytometry, and cell organic content were studied in light/dark synchronized semi-continuous cultures of the marine microalga Phaeodactylum tricornutum. Cell volume and organic content both increased with nutrient concentration at all the renewal rates tested. Cell volume against renewal rate, at each nutrient concentration, followed a U-shaped curve with smallest cells at intermediate renewal rates. In contrast, cell organic content decreased continuously with increasing renewal rate. The variation in cell volume and organic content, related to culture conditions, should be taken into account if biochemical composition and productivity of microalgal cultures are assessed on the basis of cell counts.

Use of agricultural surpluses for production of biomass by marine microalgae

The marine microalga Phaeodactylum tricornutum was cultivated in semi-continuous culture under mixotrophic conditions with the soluble fractions of potato, rye and wheat flours that had been naturally fermented, at 2% or 4% (w/v). The rye flour produced the highest microalgal cellular density of 90×10(6) cells.ml(-1) when supplemented with NaNO3 and NaH2PO4. The autotrophic control only gave 57×10(6) cells.ml(-1). The value of agricultural surpluses, such as rye flour, can therefore be increased by its use in the production of valuable, microalgal biomass which is rich in protein, pigments and fatty acids.

Optimal Renewal Rate and Nutrient Concentration for the Production of the Marine Microalga Phaeodactylum tricornutum in Semicontinuous Cultures

A factorial experiment comprising six nutrient concentrations and six renewal rates was set to optimize semicontinuous cultures of Phaeodactylum tricornutum. Maximal cell density, 85 x 10(sup6) cells ml(sup-1), was obtained with 8 and 16 mmol of N per liter and a renewal rate of 10%. Protein stabilized at about 60% of the organic fraction for nitrogen-sufficient cultures. Both lipids and carbohydrates were stored as energetic reserves.

Light and nitrogen deficiency effects on the growth and composition ofPhaeodactylum tricornutum

The marine microalgaePhaeodactylum tricornutum, with a high lipid content constituting 20–60% of its dry weight under controlled growth conditions (1), has recently come into focus as a potentially rich source of dietary marine vegetable oil. In particular, this species has a characteristic high content of eicosapentenoic acid (EPA), which has potential benefits in human nutrition, since it cannot be synthesized in vivo in the human body. Some factors that could alter the biochemical composition ofPhaeodactylum tricornutum in favor of lipid production have been examinated in this study.