Concepción Herrero

Effects of nitrogen source and growth phase on proximate biochemical composition, lipid classes and fatty acid profile of the marine microalga "Isochrysis Galbana"

The marine microalga Isochrysis galbana was cultured in nitrate, nitrite or urea media to examine changes in the gross biochemical composition, with special emphasis on the growth phase associated changes in the compositions of the lipid classes and fatty acids. The gross biochemical composition was affected more by the growth phase than by the nitrogen source. Protein was higher during exponential growth (about 37–45% AFDW) but the pattern changed as the cultures aged, and lipids were the main algal constituents on all nitrogen sources in the late stationary phase. In all cultures, the relative abundance of neutral lipid increased in the late stationary phase concomitant with a proportional reduction in phospholipids, whereas galactolipids only slightly changed during the growth of the cultures. Total fatty acid content was affected by nitrogen source and growth phase. Maximal PUFA values were obtained at the early stationary phase and decreased throughout the stationary phase. The highest fatty acid contents in the early stationary phase were produced in urea cultures; these cultures also had higher PUFA content, with EPA and DHA contents of 27.66% and 14.13% of total fatty acids, respectively.

Copper toxicity on the marine microalga Phaeodactylum tricornutum: effects on photosynthesis and related parameters

Abstract Essential heavy metals, as copper, can be toxic for microalgae at high concentrations. Copper affected growth and other parameters closely related to photosynthesis of the marine diatom Phaeodactylum tricornutum. A copper concentration of 0.10 mg l−1 provoked about 50% growth reduction and 1 mg l−1 inhibited the growth. Copper also interfered with photosynthesis and ATP production. A copper concentration of 0.5 mg l−1 reduced in a 50% the photosynthetic rate. Therefore, growth is more affected by copper than photosynthesis. Results of chlorophyll a fluorescence obtained by flow cytometry showed that coppers inhibitory effect on PS II activity is located on its oxidizing side. The lower copper concentration assayed provoked a significant decrease in the cellular pool of ATP. Pigment analysis by HPLC showed that copper affected the pigment pattern of P. tricornutum. Important changes were observed for chlorophyll a and its allomer: chlorophyll a proportion decreased while its allomer increased with the copper concentration, being maximum at 1 mg Cu l−1. The study of the intracellular pH by flow cytometry revealed that P. tricornutum cells exposed to 0.5 and 1 mg Cu l−1 showed an intracellular pH higher than control cultures cells, explaining the high proportion of the chlorophyll a allomer in these cells.

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%.

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.

Potential use of flow cytometry in toxicity studies with microalgae.

Cytotoxic effects of aquatic pollutants on microalgae are very heterogeneous, and they are influenced by environmental conditions and the test species. Stress produced by copper or paraquat addition to the culture medium of two microalgae was analysed by flow cytometry. Parameters assayed were: cell volume, chlorophyll a fluorescence and cell viability. The variety of results obtained in the present study reveals that flow cytometry is a useful tool in the toxicity tests with microalgae, both marine and freshwater species, and for different kind of pollutants.

Comparison of the accumulation of astaxanthin in Haematococcus pluvialis and other green microalgae under N-starvation and high light conditions

Haematococcus pluvialis gave the highest astaxanthin accumulation rate (2.7 mg l−1 day−1) and total astaxanthin content ( 22.7 mg g−1 biomass). Astaxanthin accumulation in Neochloris wimmeri, Protosiphon botryoides, Scotiellopsis oocystiformis, Chorella zofingiensis and Scenedesmus vacuolatus was, respectively, 19.2, 14.3, 10.9, 6.8 and 2.7 mg astaxanthin g−1 biomass, respectively.

Physiological response of freshwater microalga (Chlorella vulgaris) to triazine and phenylurea herbicides.

The effects of two herbicides used wide-spread, isoproturon (phenylurea) and terbutryn (triazine), on growth, dry weight, elemental composition, photosynthetic pigments and protein content, and cell volume assayed by flow cytometry in the freshwater microalgae Chlorella vulgaris were studied. Different parameters for algal activity show widely different sensitivities to these aquatic pollutants. After 96 h of herbicide exposure, terbutryn was the strongest inhibitor of growth, giving an EC50 value for growth twice lower than that for isoproturon cultures (EC50 terbutryn=0.097 microM; EC50 isoproturon=0.199 microM). However, lower concentrations of the triazine herbicide provoked an increase in the cellular density and growth rate of this microalga, not observed in the phenylurea-treated cultures. Cellular volume and dry weight of C. vulgaris cells were increased strongly in the presence of isoproturon and terbutryn. Other cellular parameters, such as pigment and protein content, were stimulated with both herbicides at higher concentrations.

Comparison of the sensitivity of different toxicity test endpoints in a microalga exposed to the herbicide paraquat.

The use of herbicides constitutes the principal method of weed control but the introduction of these compounds into the aquatic environment can provoke severe consequences for non-target organisms such as microalgae. Toxic effects of these pollutants on microalgae are generally evaluated using phytotoxicity tests based on growth inhibition, a population-based parameter. However, physiological cellular endpoints could allow early detection of cell stress and elucidate underlying toxicity mechanisms. Effects of the herbicide paraquat on the freshwater microalga Chlamydomonas moewusii were studied to evaluate growth rate and cellular parameters such as cellular viability and metabolic activity assayed by flow cytometry and DNA damage assayed by the comet assay. Sensitivity of growth and parameters assayed by flow cytometry were similar, showing a significant effect in cultures exposed to a paraquat concentration of 0.1 microM or higher, although in cultures exposed during 48 h to 0.05 microM, a significant stimulation of cellular fluorescein fluorescence was observed, related to cellular metabolic activity. After only 24 h of herbicide exposure significant DNA damage was observed in microalgal cells exposed to all paraquat concentrations assayed, with a 23.67% of comets in cultures exposed to 0.05 microM, revealing the genotoxicity of this herbicide. Taking into account the results obtained, comet assay provides a sensitive and rapid system for measuring primary DNA damage in Chlamydomonas moewusii, which could be an important aspect of environmental genotoxicity monitoring in surface waters.

Long-chain class III metallothioneins as a mechanism of cadmium tolerance in the marine diatom Phaeodactylum tricornutum Bohlin

Abstract Cadmium tolerance of the marine diatom Phaeodactylum tricornutum Bohlin was studied. P. tricornutum growth was significantly reduced by cadmium concentrations of 5 mg l −1 or higher. The EC 50 value was 22.39 mg l −1 of cadmium after four days of exposure. Therefore, P. tricornutum is more tolerant to cadmium than other microalgal cells. In response to exposure to cadmium, P. tricornutum synthesize class III metallothioneins which sequester the metal in a harmless form. However, cadmium tolerance of P. tricornutum cells is not only due to the ability of this microalga to synthesize class III metallothioneins (γ-Glu-Cys) n -Gly, but also because these metallothioneins are composed of long-chain polypeptides. The cadmium-metallothionein complexes of P. tricornutum cells contain predominantly polypeptides of n = 4−9. Long-chain metallothioneins are more efficient in binding heavy metals. Capillary electrophoresis was used to separate class III metallothioneins. This is a recent technique used to separate many molecules. With this technique, the length of the separated polypeptides can also be estimated.

Toxic action of copper on the membrane system of a marine diatom measured by flow cytometry

Flow cytometric measurements were used to investigate the toxic action of copper on some Phaeodactylum tricornutum membrane systems. Throughout the time of metal exposure, the percentage of viable cells decreased as copper concentration increased. The forward scatter signal increased as a result of copper exposure. After 72 h of metal exposure, cultures with 0.5 and 1 mg l-1 of copper showed an important increase in the peroxidase activity in comparison with control cells. Cells cultured with copper presented alterations in the membrane potential, increasing as copper concentration increased, after 96 h of metal exposure. Results obtained in this work showed that copper induced a degenerative process in P. tricornutum cells, closely related with alternations or disorders in membrane systems.