Jaime Fábregas

Growth of the marine microalga Tetraselmis suecica in batch cultures with different salinities and nutrient concentrations

Abstract The marine microalga Tetraselmis suecica is known for its ability to tolerate a wide range of salt concentrations. Cultures were grown under 48 different nutrient concentration-salinity conditions, ranging from 2 to 64 mM NaNO3 and from 0 to 35‰ S. Salinity was more important for the growth rate of the microalgae when it was related to the nutrient concentration in the culture medium. Optimal growth conditions were between 25 and 35‰ salinity and nutrient concentrations of 2, 4 and 8 mM of NaNO3, resulting in 0.55 doublings/day and a maximum cellular density of 1.3 × 106 cells/ml. Variations in salinity and in nutrient concentration had a greater effect on the final biomass than on the growth velocity. The total protein of the culture and protein per cell increased when the salinity increased for a given nutrient concentration. The total protein of the cultures decreased when the nutrient concentration increased for a given salinity. Protein per cell decreased with increasing salinity up to 20‰ but from this point of the process was reversed. The nitrate-protein transformation rate increased with the salinity and decreased with increasing nutrient concentrations. The maximum rate was 64%.

Two-stage cultures for the production of Astaxanthin from Haematococcus pluvialis

A two-stage culture system was established for the production of astaxanthin from Haematococcus pluvialis. In a first stage green vegetative cells were produced in semicontinuous cultures maintained with daily renewal rates between 10 and 40%. The steady-state cell density decreased with increasing renewal rates. Highest cell productivity, 64 x 10(6) cells l(-1) day(-1) was obtained with a daily renewal rate of 20%. In a second stage the harvested cultures were submitted to high light (240 micromol photon m(-2) s(-1)) under batch conditions for 15 days in order to stimulate the transition to the aplanospore stage and the accumulation of astaxanthin. No decrease in cell density was recorded during the induction period in any of the cultures. Cultures obtained at high renewal rates continued growing during the induction period and no astaxanthin was accumulated until all nitrogen in the media had been consumed. The final concentration of astaxanthin was inversely correlated to the growth rate at which first-stage cultures were maintained. Optimal renewal rate for maximal astaxanthin production depended on the duration of the induction period. After a 12-day induction period the highest astaxanthin production, 5.8 mg l(-1) of semi-continuous culture day -1, was obtained with cultures maintained at a renewal rate of 20%. When the induction period was increased to 15 days maximal astaxanthin productivity, 9.6 mg l(-1) of semi-continuous culture day -1, was obtained from cultures maintained at a renewal rate of 40% despite the much lower astaxanthin concentration achieved in these cultures. Results demonstrate the feasibility of semi-continuous cultivation of H. pluvialis for the two-stage production of astaxanthin.

Optimization of culture medium for the continuous cultivation of the microalga Haematococcus pluvialis.

Abstract The freshwater microalga Haematococcus pluvialis is one of the best microbial sources of the carotenoid astaxanthin, but this microalga shows low growth rates and low final cell densities when cultured with traditional media. A single-variable optimization strategy was applied to 18 components of the culture media in order to maximize the productivity of vegetative cells of H. pluvialis in semicontinuous culture. The steady-state cell density obtained with the optimized culture medium at a daily volume exchange of 20% was 3.77 · 105 cells ml−1, three times higher than the cell density obtained with Bold basal medium and with the initial formulation. The formulation of the optimal Haematococcus medium (OHM) is (in g l−1) KNO3 0.41, Na2HPO4 0.03, MgSO4 · 7H2O 0.246, CaCl2 · 2H2O 0.11, (in mg l−1) Fe(III)citrate · H2O 2.62, CoCl2 · 6H2O 0.011, CuSO4 · 5H2O 0.012, Cr2O3 0.075, MnCl2 · 4H2O 0.98, Na2MoO4 · 2H2O 0.12, SeO2 0.005 and (in μg l−1]) biotin 25, thiamine 17.5 and B12 15. Vanadium, iodine, boron and zinc were demonstrated to be non-essential for the growth of H. pluvialis. Higher steady-state cell densities were obtained by a three-fold increase of all nutrient concentrations but a high nitrate concentration remained in the culture medium under such conditions. The high cell productivities obtained with the new optimized medium can serve as a basis for the development of a two-stage technology for the production of astaxanthin from H. pluvialis.

Mass culture and biochemical variability of the marine microalga Tetraselmis suecica Kylin (Butch) with high nutrient concentrations

Abstract Mass cultures of Tetraselmis suecica were carried out with four nutrient concentrations, ranging from 2 to 16 mM of NaNO3 and salinity 35‰. An air flow of 15 l/min maintained a CO2 transference rate sufficient to keep the pH below 8.4. Using these cultural conditions equations were calculated, by a multiple non-linear least squares regression of order four, enabling predictions to be made of growth kinetics and chemical composition. Maximum cellular densities of 7.83 × 106 and 7.15 × 106 cells/ml were obtained with 8 and 16 mM of NaNO3, respectively. Growth velocity ranged between 0.53 and 0.63 doublings (dbl)/day, although 0.98 dbl/day were reached with 16 mM of NaNO3. Volume increased with nutrient concentration from 252 to 905 μm3. Protein content reached maximum values of 306 μg/ml or 59.8 pg/cell. In the logarithmic phase, protein was regulated by nutrient concentration and decreased according to this concentration. Maximum efficiency of transformation from nitrate to protein was 108%, obtained at 2 mM of NaNO3. Efficiency decreased, to 14%, when nutrient concentration increased. This fact indicates that the lowest cost of harvesting is obtained with a nutrient concentration of 2 mM NaNO3. Chlorophyll a cell reached values between 3.1 and 3.8 pg/cell in the stationary phase. There was a relationship between nutrient concentration and chlorophyll a cell in the logarithmic phase, with an increase from 2.15 pg/cell to 3.74 pg/cell. Changes in chlorophyll a level are related to nitrogen depletion. Carbohydrate/cell was constant at values of 19.84–28.68 pg/cell in the logarithmic and stationary phases and was not related to nitrogen depletion. RNA/cell ranged from 4.17 to 5.48 pg/cell, except at 2 mM of NaNO3 when it was 2.77 pg/cell, probably due to nitrogen depletion. The level of DNA/cell was constant in all the nutrient concentrations assayed and ranged from 0.1 to 1.09 pg/cell. Great variability in the chemical composition of T. suecica has been shown. Growth in mass cultures is closely coupled to changes in nutrient concentrations and variations occur in protein, chlorophyll a and RNA content, showing differences of 215%, 190% and 203%, respectively, in the stationary phase. This biochemical variability, mainly in protein content, must have a marked effect on the nutritive value of this microalga as feed in mariculture.

Induction of astaxanthin accumulation by nitrogen and magnesium deficiencies in Haematococcus pluvialis

Haematococcus pluvialis was cultured under N– and Mg+2-deficient conditions with two light intensities: 40 and 230 μmol m2 s−1. Highest astaxanthin concentration, 49.5 μg·ml−1, was obtained when high light was applied under N-deficient conditions. N-deficiency has a greater effect than high light intensity on astaxanthin synthesis by exerting a stronger blocking effect on cell division. The effect of high light was synergetic with the other stress conditions in stimulating the synthesis of astaxanthin. Mg+2 deficiency also stimulated the synthesis of astaxanthin but produced lower concentrations: 7 and 26 μg·ml−1 for low and high light intensities respectively. When both N and Mg+2 were absent from the culture media the concentration of astaxanthin was lower than with N-deficiency alone but higher than with Mg+2-deficiency.

Interactions between irradiance and nutrient availability during astaxanthin accumulation and degradation in Haematococcus pluvialis

Fully synchronised germination of Haematococcus pluvialis astaxanthin-replete aplanospores was induced by transfer to nitrogen-sufficient conditions under either high or low light intensities, and growth, pigment content and nitrogen consumption were monitored during the cell cycle. No germination of the aplanospores was achieved in the absence of nitrate, even when cells were transferred at low light intensities. On the other hand, cell density and chlorophyll concentration increased dramatically and astaxanthin concentration decreased in N-sufficient cultures due to the germination of 100% of the aplanospores, as demonstrated by flow cytometry. No significant effect of light intensity was observed on the degradation of astaxanthin during germination. In germinated cultures, nitrogen was depleted more rapidly under high light conditions, which resulted in earlier entry into the aplanospore stage and accumulation of astaxanthin. Germination of aplanospores accompanied by astaxanthin degradation could also be obtained in the dark in nutrient-sufficient conditions although at a much lower efficiency. The results demonstrate that nutrient availability is the main factor controlling the transition between red and green stages of H. pluvialis, with astaxanthin being accumulated only when cell division has ceased. High light levels accelerate the process by increasing the rate of nutrient depletion and providing more energy for astaxanthin synthesis.

Vitamin content of four marine microalgae. Potential use as source of vitamins in nutrition

SummaryCertain marine microalgae contain water-and lipid-soluble vitamins and can be used as food supplements or food ingredients. A number of vitamins are present in higher concentrations in the microalgae than in conventional foods traditionally considered rich in them. Ingestion of relatively small quantities of microalgae can cover the requirements for some vitamins in animal nutrition, including human nutrition, while supplementing others. Marine microalgae can thus be considered to represent a non-conventional source of vitamins or a vitamin supplement for animal or human nutrition.

Changes in the cell composition of the marine microalga, Nannochloropsis gaditana, during a light:dark cycle

Nannochloropsis gaditana was grown in semicontinuous culture with a circadian light:dark cycle in a flat-panel photobioreactor. The microalga had a maximal protein content (3 pg cell−1) after 6 h light and then only storage compounds were accumulated that were consumed during the dark phase. Carbohydrates reached their maximum value after 8 h (0.8 pg cell−1) and lipids after 12 h light (2.5 pg cell−1). The results demonstrated that young or adult microalgae might be obtained according to the time of day.

Changes in the nutrient composition of Tetraselmis suecica cultured semicontinuously with different nutrient concentrations and renewal rates

Abstract The marine microalga Tetraselmis suecica was cultured semicontinuously with two nutrient concentrations: 2 and 4 mmol N l −1 and five rates of daily renewal of the culture media in the range 10–50%, in order to study the changes in the nutrient composition of the cells produced under such conditions. An increase in the renewal rate produced an increase in protein and lipid content of the biomass to the detriment of carbohydrate content, the only fraction accumulated by T. suecica as energetic reserve under nitrogen deficient conditions. Cellular content of these three fractions did not accurately reflect the relative composition, since total organic content of the cells decreased with increasing renewal rates attenuating the increase in protein and lipid contents. As a result, cell caloric content remained almost constant. The degree of unsaturation of fatty acids increased with increasing renewal rate, with a two-fold increase of the percentage of total n − 3 fatty acids. On the other hand, the percentage of eicosapentaenoic acid (EPA) decreased with increasing renewal rate indicating a different control of the metabolism of this long-chain polyunsaturated fatty acid in T . suecica . Results demonstrate the efficiency of the use of the semicontinuous culture technique for the production of microalgal biomass of optimal biochemical composition being especially useful for the study of the nutritional requirements of filter-feeding marine organisms.

β-carotene, vitamin C and vitamin E content of the marine microalga Dunaliella tertiolecta cultured with different nitrogen sources

This work was supported by a grant from the Conselleria de Educacion y Ordenacion Universitaria, Xunta de Galicia.