Ana Teresa Lombardi

MUCILAGINOUS CAPSULE ADSORPTION AND INTRACELLULAR UPTAKE OF COPPER BY KIRCHNERIELLA APERTA (CHLOROCOCCALES)1

The purpose of the present investigation was to evaluate possible ecological and physiological functions of mucilaginous capsules produced by the freshwater algae Kirchneriella aperta Teiling (Chlorococcales) as related to copper ions. All experiments were performed using synthetic media under laboratory‐controlled conditions. Copper interactions were investigated by distinguishing between adsorption onto the mucilaginous material present at the surface of the cells, intracellular uptake, and differentiation between total dissolved copper and free copper ions in the culture medium. Kirchneriella aperta is sensitive to copper, as revealed by a 96‐h EC50 value of 10−9.22 M Cu2+. We demonstrated that the mucilaginous capsules were able to sequester copper ions from the medium through a passive mechanism, thus providing the cell with a mechanism able to postpone the toxic effects of copper. The organic material that diffuses into the test medium as well as the mucilaginous capsules produced by K. aperta both effectively complex copper; thus, toxicity must be related to free copper ions and not the total dissolved copper concentration in the medium.

Lipid composition of Chlorella vulgaris (Trebouxiophyceae) as a function of different cadmium and phosphate concentrations

Fatty acids are the fundamental structural components of membrane lipids, and the degree of saturation of the long hydrocarbon chains in microalgae contributes to regulation of growth, biomass production and reproduction of aquatic consumers. This research aimed at evaluating the effects of cadmium (2×10(-8); 10(-7) mol L(-1) Cd) on lipid class and fatty acid composition of the microalga Chlorella vulgaris under varying phosphate (PO(4)(3-)) concentrations (6.0×10(-7) to 2.3×10(-4) mol L(-1)). Under PO(4)(3-) limitation and Cd stress, the storage lipid class triacylglycerol (TAG) was the most accumulated among the lipid classes. Fatty acid composition revealed that the degree of saturation increased with increasing Cd stress and PO(4)(3-) limitation. Decreasing PO(4)(3-) and increasing Cd concentrations resulted in higher saturated fatty acid (SAFA) and monounsaturated FA (MUFA) concentrations. Total polyunsaturated FA (PUFA) and ω3 PUFA, and PUFA:SAFA ratios were higher in the control (2.3×10(-4) mol L(-1) PO(4)(3-)) cells than in either PO(4)(3-) limitation or Cd stress, or in the combination of both stresses. Contrasting with all the other PUFAs, 18:2n - 6 increased as PO(4)(3-) limitation increased. A significant positive relationship of PUFAs, acetone mobile polar lipids (AMPL) and phospholipids (PL) with phosphate concentration in the culture media was obtained, while TAG concentrations had a positive association with total MUFA and SAFA. Total SAFA, 14:0, 18:1n - 9 and 18:2n - 6 were positively correlated with Cd and negatively with PO(4)(3-) concentrations. The microalga responded to combined PO(4)(3-) limitation and Cd exposure by increasing its total lipid production and significantly altering its lipid composition. The FA 18:2n - 6 may be considered a stress biomarker for PO(4)(3-) limitation and Cd stress in C. vulgaris.

Effects of cadmium and nitrogen on lipid composition of Chlorella vulgaris (Trebouxiophyceae, Chlorophyta)

Investigations were carried out to evaluate lipid class and fatty acid composition of Chlorella vulgaris in relation to several combinations of cadmium (2 × 10−8; 10−7 M Cd) and nitrogen (2.9 × 10−6 to 1.1 × 10−3 M N) concentrations. Triacylglycerols (TAG), acetone mobile polar lipids (AMPL) and phospholipids (PL) were the major lipid classes of C. vulgaris under all the tested conditions. TAG was the lipid class accumulated in the highest amount in N-limited C. vulgaris. The AMPL lipid class was lowest at the lowest N and highest Cd treatment. High proportions of saturated fatty acids (SAFA) and monounsaturated fatty acids (MUFA) in the total lipid extracts were recorded under N limitation in the presence of Cd. 16:1(n-11) was an exception among the MUFA as its levels decreased at low N. Total polyunsaturated FA (PUFA), ω3 PUFA levels, and PUFA:SAFA ratios increased with increasing N concentrations, but when exposed to Cd, their levels were significantly reduced. Unlike the other PUFA, 18:2(n-6) increased with increasing N limitation. Significant negative relationships between TAG, MUFA and SAFA with N were recorded. Cd had a positive correlation with TAG, while N was with AMPL, PL and PUFA. In conclusion, a combination of N limitation and Cd stress significantly altered the lipid composition of C. vulgaris, and N limitation had the most significant overall effect on lipid class and fatty acid composition of the microalga.

Combined nitrogen limitation and cadmium stress stimulate total carbohydrates, lipids, protein and amino acid accumulation in Chlorella vulgaris (Trebouxiophyceae)

Metals have interactive effects on the uptake and metabolism of nutrients in microalgae. However, the effect of trace metal toxicity on amino acid composition of Chlorella vulgaris as a function of varying nitrogen concentrations is not known. In this research, C. vulgaris was used to investigate the influence of cadmium (10(-7) and 2.0×10(-8)molL(-1) Cd) under varying nitrogen (2.9×10(-6), 1.1×10(-5) and 1.1×10(-3)molL(-1)N) concentrations on its growth rate, biomass and biochemical composition. Total carbohydrates, total proteins, total lipids, as well as individual amino acid proportions were determined. The combination of Cd stress and N limitation significantly inhibited growth rate and cell density of C. vulgaris. However, increasing N limitation and Cd stress stimulated higher dry weight and chlorophyll a production per cell. Furthermore, biomolecules like total proteins, carbohydrates and lipids increased with increasing N limitation and Cd stress. Ketogenic and glucogenic amino acids were accumulated under the stress conditions investigated in the present study. Amino acids involved in metal chelation like proline, histidine and glutamine were significantly increased after exposure to combined Cd stress and N limitation. We conclude that N limitation and Cd stress affects the physiology of C. vulgaris by not only decreasing its growth but also stimulating biomolecule production.

Toxicity of ionic copper to the freshwater microalga Scenedesmus acuminatus (Chlorophyceae, Chlorococcales)

A.T. Lombardi, T.M.R. Hidalgo, A.A.H. Vieira and A.L. Sartori. 2007. Toxicity of ionic copper to the freshwater microalga Scenedesmus acuminatus (Chlorophyceae, Chlorococcales). Phycologia 46: 74–78. DOI: 10.2216/06-40.1 Metal ecotoxicity testing with microalgae is subject to interferences such as change in bioavailable metal fraction by excreted metabolites. Moreover, high metal loads are usually necessary for obtaining 50% effect concentration (EC50) values based on cell number or chlorophyll a. Thus, sensitive parameters, which promptly respond to low dissolved metal variations at environmentally relevant concentrations, are much needed. The genus Scenedesmus, a coenobium-forming freshwater microalga, is cosmopolitan and inhabits pristine and contaminated waters. The present investigation addressed cellular phenotypic alterations in Scenedesmus acuminatus (Largerheim) Chodat 1902 caused by the presence of copper. Copper toxicity to the alga was investigated under laboratory-controlled conditions using a concentration range of 10−8 mol l−1 to 10−3 mol l−1 total dissolved Cu2+, thus including environmentally relevant concentrations. Free Cu2+ concentrations were determined using an ion-selective electrode calibrated with a metal ion buffer. The phenotypic plasticity of S. acuminatus was measured as the number of cells per coenobia and found to be readily affected at low copper concentrations. Under healthy conditions (10−8 mol l−1 total dissolved Cu2+, 10−11 mol l−1 free Cu2+) S. acuminatus formed eight-celled coenobia, whereas at 10−7 mol l−1 total dissolved Cu2+ (3 × 10−10 mol l−1 free Cu2+) these were absent and at 10−5 mol l−1 total dissolved Cu2+ (5 × 10−8 mol l−1 free Cu2+) free cells were predominant. Chlorophyll a synthesis was affected by 5 × 10−8 mol l−1 free Cu2+ or higher. EC50 (3.3 × 10−5 mol l−1 free Cu2+) was obtained using chlorophyll a values after 96-h exposure to the toxicant. The number of cells per coenobia was a more sensitive parameter than chlorophyll a to assess Cu toxicity, and at concentrations that may be encountered in the environment.

Growth and biochemical composition of Chlorella vulgaris in different growth media

The need for clean and low-cost algae production demands for investigations on algal physiological response under different growth conditions. In this research, we investigated the growth, biomass production and biochemical composition of Chlorella vulgaris using semi-continuous cultures employing three growth media (LC Oligo, Chu 10 and WC media). The highest cell density was obtained in LC Oligo, while the lowest in Chu medium. Chlorophyll a, carbohydrate and protein concentrations and yield were highest in Chu and LC Oligo media. Lipid class analysis showed that hydrocarbons (HC), sterol esthers (SE), free fatty acids (FFA), aliphatic alcohols (ALC), acetone mobile polar lipids (AMPL) and phospholipids (PL) concentrations and yields were highest in the Chu medium. Triglyceride (TAG) and sterol (ST) concentrations were highest in the LC Oligo medium. The results suggested that for cost effective cultivation, LC Oligo medium is the best choice among those studied, as it saved the cost of buying vitamins and EDTA associated with the other growth media, while at the same time resulted in the best growth performance and biomass production.

Microalgae population dynamics in photobioreactors with secondary sewage effluent as culture medium

Nitrogen and phosphorus present in sewage can be used for microalgae growth, possibiliting cost reduction in the production of microalgae at the same time that it decreases the eutrophication potential of the effluent. This research aimed at monitoring the native community of microalgae and coliform bacteria in a secondary effluent from anaerobic municipal sewage treatment. Two treatments (aerated and non-aerated) were performed to grow microalgae under semi-controlled conditions in semi-closed photobioreactors in a greenhouse. The results showed no significant pH and coliforms (total and Escherichia coli ) variation between treatments. Nutrient concentrations were reduced supporting microalgae growth up to 10 7 cells.mL −1 independent of aeration. Exponential growth was obtained from the first day for the non-aerated, but a 5 day lag phase of growth was obtained for the aerated. Chlorella vulgaris was the dominant microalgae (99.9%) in both treatments. In the aerated, 5 algae classes were detected (Chlorophyceae, Cyanophyceae, Chrysophyceae, Bacillariophyceae and Euglenophyceae), with 12 taxa, whereas in the non-aerated, 2 classes were identified (Chlorophyceae and Cyanophyceae), with 5 taxa. We concluded that effluent is viable for microalgae growth, especially Chlorella vulgaris, at the same time that the eutrophication potential and coliforms are decreased, contributing for better quality of the final effluent.

The effects of solar irradiation on copper speciation and organic complexation

Este trabalho teve como objetivo avaliar o efeito da irradiacao solar na especiacao do cobre em amostras de agua coletadas em um reservatorio eutrofizado e contaminado por metais. Voltametria de redissolucao anodica e eletrodo de ion seletivo foram utilizados para determinacao de cobre nas amostras irradiadas e nao irradiadas. Os resultados demonstraram que apos 7 h de irradiacao solar, a materia orgânica dissolvida (DOM) foi degradada promovendo aumento de cobre ionico livre, labil e de carbono inorgânico dissolvido (DIC), e diminuicao da concentracao de ligantes disponiveis (CL) e de carbono orgânico dissolvido (DOC). Os valores da constante de estabilidade condicional (logK’) variaram em duas amostras, permanecendo constantes nas demais. Os resultados demonstraram que as reacoes fotoquimicas afetaram a especiacao do cobre e as interacoes Cu-DOM, ressaltando implicacoes ecologicas da fotoxidacao, como o aumento do cobre biodisponivel e, desta forma, contribuindo para o entendimento do ciclo biogeoquimico desse metal. The aims of this research were to evaluate the effects of solar irradiation on copper speciation in water samples obtained in eutrophic and metal contaminated reservoir. Anodic stripping voltammetry and ion selective electrode were used for metal determinations of solar irradiated and non-irradiated samples. The results showed that after 7 h of solar irradiation, dissolved organic matter (DOM) was degraded promoting the increase of labile, free Cu 2+ ions and dissolved inorganic carbon (DIC). Decreases of ligand concentration (CL) and dissolved organic carbon (DOC) were also observed. Conditional stability constant (logK’) values varied for two sampling sites, but remained unchanged for the others. The present results demonstrated that photochemical reactions affected copper speciation and Cu-DOM interactions. This research is a contribution to the understanding of biogeochemical cycle of copper and highlights some ecological implications of photooxidation in freshwater ecosystems, such as the increase of copper bioavailability.

Influence of phosphorus on copper toxicity to Selenastrum gracile (Reinsch) Korshikov

Microalgae need a variety of nutrients for optimal growth and health. However, this rarely occurs in nature, and if nutrient proportions vary, biochemical changes can occur in phytoplankton community. This may result in modifications of zooplankton food quality, affecting aquatic food chains. Our aim was to investigate the toxicity of copper (Cu) to Selenastrum gracile, a common freshwater Chlorophyceae, at different physiological status induced by varying phosphorus (P) concentration in culture medium. Phosphorus was investigated at 2.3×10(-4), 1.1×10(-4), 2.3×10(-5), 4.6×10(-6) and 2.3×10(-6) mol L(-1) and Cu at six concentrations, ranging from 6.9×10(-9) mol L(-1) to 1.0×10(-7) mol L(-1) free Cu(2+) ions. To guarantee the cells would be in a physiological status that reflected the external P concentration, they were previously acclimated up to constant growth rate at each P concentration. Phosphorus acclimated cells were then exposed to Cu and toxicity was evaluated through population density, growth rates and chlorophyll a content. Free Cu(2+) ions concentrations were calculated through the chemical equilibrium model MINEQL(+). The results showed that higher Cu toxicity was obtained in P-limited than in P-replete cells, and that chlorophyll a/cell was higher in P-limited cells and excess Cu than in P-replete cells. This confirms the importance of microalgae nutritional status to withstand the negative effects of the trace metal.

Chemical behavior of Cu, Zn, Cd, and Pb in a eutrophic reservoir: speciation and complexation capacity

This research aimed at evaluating cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn) speciation in water samples as well as determining water quality parameters (alkalinity, chlorophyll a, chloride, conductivity, dissolved organic carbon, dissolved oxygen, inorganic carbon, nitrate, pH, total suspended solids, and water temperature) in a eutrophic reservoir. This was performed through calculation of free metal ions using the chemical equilibrium software MINEQL+ 4.61, determination of labile, dissolved, and total metal concentrations via differential pulse anodic stripping voltammetry, and determination of complexed metal by the difference between the total concentration of dissolved and labile metal. Additionally, ligand complexation capacities (CC), such as the strength of the association of metals–ligands (logK′ML) and ligand concentrations (CL) were calculated via Ruzic’s linearization method. Water samples were taken in winter and summer, and the results showed that for total and dissolved metals, Zn > Cu > Pb > Cd concentration. In general, higher concentrations of Cu and Zn remained complexed with the dissolved fraction, while Pb was mostly complexed with particulate materials. Chemical equilibrium modeling (MINEQL+) showed that Zn2+ and Cd2+ dominated the labile species, while Cu and Pb were complexed with carbonates. Zinc was a unique metal for which a direct relation between dissolved species with labile and complexed forms was obtained. The CC for ligands indicated a higher CL for Cu, followed by Pb, Zn, and Cd in decreasing amounts. Nevertheless, the strength of the association of all metals and their respective ligands was similar. Factor analysis with principal component analysis as the extraction procedure confirmed seasonal effects on water quality parameters and metal speciation. Total, dissolved, and complexed Cu and total, dissolved, complexed, and labile Pb species were all higher in winter, whereas in summer, Zn was mostly present in the complexed form. A high degree of deterioration of the reservoir was confirmed by the results of this study.