Ángeles Cid

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.

Purification and characterization of phycocyanin from the marine cyanobacterium Synechococcus sp. IO9201

This paper describes a suitable method for the optimum extraction and isolation of phycocyanin from the cyanobacterium Synechococcus sp. IO9201 isolated from Caribbean waters. Phycocyanin from this microorganism was purified to homogeneity and some of its properties were investigated. The purification steps consisted of extraction, hydrophobic interaction chromatography and ion exchange chromatography. Freezing at −21°C-thawing at 4°C, using an alkaline buffer was the best method for extracting phycocyanin from Synechococcus sp. IO9201. The best extraction was obtained using butyl-sepharose resin for hydrophobic interaction chromatography and 0.05 M Tris-HCl (pH = 7) containing 10% ethanol for phycocyanin elution. Finally, phycocyanin was further purified by ion exchange chromatography using Q-sepharose and eluted with a complex isocratic system. The estimated molecular weight of the phycocyanin purified from Synechococcus sp. IO9201 was 102 000 daltons by gel filtration and the isoelectric point was 4.6. When analyzed by SDS-PAGE, Synechococcus sp. IO9201 phycocyanin migrated as two bands having an apparent molecular weight of 21 360 and 18 980 Da. The first band corresponds to β phycocyanin subunits, whereas the second corresponds to α phycocyanin subunits. So, this phycocyanin was characterized as (αCPCβCPC)3.

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.

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.

Carotenoid accumulation in Haematococcus pluvialis in mixotrophic growth

The microalga Haematococcus pluvialis was cultured with NaNO3 from 0 to 1 g l−1 and optimal growth was obtained at 0.15 g l−1. Sodium acetate and malonate (from 0 to 2% w/v) enhanced the accumulation of astaxanthin three and five times higher, respectively, than in autotrophic control cultures. However, high concentration of those compounds strongly inhibited growth. The ratio chlorophyll a/total carotenoids was a good indicator of the extent of nitrogen deficiency in the cells.

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.

Removal of cadmium ions by the marine diatom Phaeodactylum tricornutum Bohlin accumulation and long-term kinetics of uptake

Abstract The marine diatom Phaeodactylum tricornutum Bohlin was exposed to different cadmium concentrations (1–100 mg l−1) for 4 days. The amount of cadmium removed was recorded, with particular attention paid to long-term uptake kinetics, and to the cellular location of cadmium. Cadmium accumulation occurred at all concentrations assayed. The EC50 of cadmium to P. tricornutum was 22·39 mg l−1 after 4 days of exposure. Cadmium uptake followed a saturation kinetic at cadmium concentrations ≥25 mg l−1. However, at lower cadmium concentrations, the uptake of this metal followed a linear trend for all days of culture. At cadmium concentrations in the medium lower than 25 mg l−1, P. tricornutum removed cadmium mainly within the cell. At higher cadmium concentrations, the amount of cadmium removed by adsorption to the cell surface was higher than intracellular cadmium, because of the toxic effects of cadmium on P. tricornutum cells. This toxicity reduced the cadmium accumulation within the cells.