Aridane G. González

Adsorption of copper on Pseudomonas aureofaciens: protective role of surface exopolysaccharides.

Adsorption of copper on exopolysaccharide (EPS)-rich and (EPS)-poor soil rhizospheric Pseudomonas aureofaciens cells was studied as a function of pH and copper concentration at different exposure time in order to assess the effect of cell exopolysaccharides on parameters of adsorption equilibria. The surface properties of bacteria were investigated as a function of pH and ionic strength using potentiometric acid-base titration and electrophoresis that permitted the assessment of the excess surface proton concentration and zeta-potential of the cells, respectively. For adsorption experiments, wide range of Cu concentration was investigated (0.1-375 microM) in order to probe both weak and strong binding sites at the surface. Experimental results were successively fitted using a Linear Programming Model approach. The groups with pK(a) of 4.2-4.8 and from 5.2 to 7.2, tentatively assigned as carboxylates and phosphoryl respectively, are the most abundant at the surface and thus essentially contribute to the metal binding. The presence of exopolysaccharides on the surface decreases the amount of copper adsorbed on the bacterial cell wall apparently via screening the underlining functional groups of the cell wall. At the same time, dissolved EPS substances do not contribute to Cu binding in aqueous solution. Results of this study allow quantification of the role played by the surface EPS matrix as a protective barrier for metal adsorption on bacterial cell walls.

Metal adsorption on mosses: Toward a universal adsorption model

This study quantifies the adsorption of heavy metals on 4 typical moss species used for environmental monitoring in the moss bag technique. The adsorption of Cu(2+), Cd(2+), Ni(2+), Pb(2+) and Zn(2+) onto Hypnum sp., Sphagnum sp., Pseudoscleropodium purum and Brachytecium rutabulum has been investigated using a batch reactor in a wide range of pH (1.3-11.0) and metal concentrations in solution (1.6μM-3.8mM). A Linear Programming Model (LPM) was applied for the experimental data to derive equilibrium constants and the number of surface binding sites. The surface acid-base titration performed for 4 mosses at a pH range of 3-10 in 0.1M NaNO3 demonstrated that Sphagnum sp. is the most efficient adsorbent as it has the maximal number of proton-binding sites on the surface (0.65mmol g(-1)). The pKa computed for all the moss species suggested the presence of 5 major functional groups: phosphodiester, carboxyl, phosphoryl, amine and polyphenols. The results of pH-edge experiments demonstrated that B. rutabulum exhibits the highest percentage of metal adsorption and has the highest number of available sites for most of the metals studied. However, according to the results of the constant pH Langmuirian isotherm, Sphagnum sp. can be considered as the strongest adsorbent, although the relative difference from other mosses is within 20%. The LPM was found to satisfactorily fit the experimental data in the full range of the studied solution parameters. The results of this study demonstrate a rather similar pattern of five metal adsorptions on mosses, both as a function of pH and as a metal concentration, which is further corroborated by similar values of adsorption constants. Therefore, despite the species and geographic differences between the mosses, a universal adsorption edge and constant pH adsorption isotherm can be recommended for 4 studied mosses. The quantitative comparison of metal adsorption with other common natural organic and inorganic materials demonstrates that mosses are among the most efficient natural adsorbents of heavy metals.

Chemical and structural status of copper associated with oxygenic and anoxygenic phototrophs and heterotrophs: possible evolutionary consequences

Copper adsorption on the surface and intracellular uptake inside the cells of four representative taxons of soil and aquatic micro-organisms: aerobic rhizospheric heterotrophs (Pseudomonas aureofaciens), anoxygenic (Rhodovulum steppense) and oxygenic (cyanobacteria Gloeocapsa sp. and freshwater diatoms Navicula minima) phototrophs were studied in a wide range of pH, copper concentration, and time of exposure. Chemical status of adsorbed and assimilated Cu was investigated using in situ X-ray absorption spectroscopy. In case of adsorbed copper, XANES spectra demonstrated significant fractions of Cu(I) likely in the form of tri-coordinate complexes with O/N and/or S ligands. Upon short-term reversible adsorption at all four studied micro-organisms cell surface, Cu(II) is coordinated by 4.0 ± 0.5 planar oxygens at an average distance of 1.97 ± 0.02 Å, which is tentatively assigned to the carboxylate groups. The atomic environment of copper incorporated into diatoms and cyanobacteria during long-term growth is similar to that of the adsorbed metal with slightly shorter distances to the first O/N neighbor (1.95 Å). In contrast to the common view of Cu status in phototrophic micro-organisms, XAFS failed to detect sulfur in the nearest atomic environment of Cu assimilated by freshwater plankton (cyanobacteria) and periphyton (diatoms). The appearance of S in Cu 1st coordination shell at 2.27-2.32 Å was revealed only after long-term interaction of Cu with anoxygenic phototrophs (and Cu uptake by soil heterotrophs), suggesting Cu scavenging in the form of sulfhydryl, histidine/carboxyl or a mixture of carboxylate and sulfhydryl complexes. These new structural constraints suggest that adsorbed Cu(II) is partially reduced to Cu(I) already at the cell surface, where as intracellular Cu uptake and storage occur in the form of both Cu(I)-S linked proteins and Cu(II) carboxylates. Obtained results allow to better understand how, in the course of biological evolution, micro-organisms elaborated various mechanisms of Cu uptake and storage, from passive adsorption and uptake to active, protein-controlled surface reduction, and intracellular storage.

Oxidation of Fe(II) in natural waters at high nutrient concentrations.

The Fe(II) oxidation kinetic was studied in seawater enriched with nutrients as a function of pH (7.2-8.2), temperature (5-35 °C), and salinity (10-36.72) and compared with the same parameters in seawater media. The effect of nitrate (0-1.77 × 10(-3) M), phosphate (0-5.80 × 10(-5) M) and silicate (0-2.84 × 10(-4) M) was studied at pH 8.0 and 25 °C. The experimental results demonstrated that Fe(II) oxidation was faster in high nutrient concentrations affecting the lifetime of Fe(II) in nutrient rich waters. Silicate displayed the most significant effects on the Fe(II) oxidation rate with values similar to those determined in seawater enriched with all the nutrients. A kinetic model was applied to the experimental results in order to account for changes in the speciation and to compute the fractional contribution of each Fe(II) species to the total rate constant as a function of pH. FeH(3)SiO(4)(+) played a key role in the Fe(II) speciation, dominating the process at pH over 8.4. At pH 8.0, FeH(3)SiO(4)(+) represented 18% of the total Fe(II) species. Model results show that when the concentration of silicate is 3 × 10(-5) M as in high nutrient low chlorophyll areas, FeH(3)SiO(4)(+) contributed at pH 8.0 by 4% increasing the rate to 11% at 1.4 × 10(-4) M. The effect of nutrients, especially silicate, must be considered in any further study in seawater media cultures and eutrophic oceanic areas.

Phenolic profile of Dunaliella tertiolecta growing under high levels of copper and iron.

The present study investigates the phenolic profile of exudates and extracts of the green algae Dunaliella tertiolecta, harvested in natural seawater in the absence (control) and in the presence of Cu(II) (315 and 790xa0nmolxa0L−1) and Fe(III) (900xa0nmolxa0L−1) in order to identify and quantify the phenolic compounds produced under metallic stress conditions. The presence of metal ions modifies the growth of cells and changes cell metabolism by producing phenolic compounds adapted to the solution. The use of reversed-phase high-performance liquid chromatography (RP-HPLC) permitted the identification of 14 phenolic constituents. The concentration and type of polyphenols detected in cell extracts and in solution are directly related with the metal and its concentration during growth cultures, achieving 1.4 times higher levels of polyphenols under 790xa0nmol Cu(II)u2009L−1 with respect to the control experiments. Microalga excretes polyphenols to be adapted to the environmental conditions. Gentisic acid, (+) catechin and (−) epicatechin, the most prominent phenolic compounds detected in the algae extracts, showed high antioxidant activity in inhibiting 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. This potent activity may be related to its presence in cells and exudates in high concentrations.

Silver nanoparticles impact phototrophic biofilm communities to a considerably higher degree than ionic silver

Due to the significant increase in nanoparticle production and especially that of silver nanoparticles over the past decade, the toxicity of silver in both ionic (Ag+) and nanoparticulate (AgNPs) form must be studied in detail in order to understand their impact on natural ecosystems. A comparative study of the effect of AgNPs and ionic silver on two independent phototrophic biofilms was conducted in a rotating annular bioreactor (RAB) operating under constant conditions. The concentration of dissolved silver in the inlet solution was progressively increased every 4xa0days of exposure, from 0.1 to 100xa0μgxa0L−1. In the course of the 40-day experiment, biofilm samples were collected to determine the evolution of biomass, chlorophyll-a, as well as photosynthetic and heterotrophic enzymatic activities in response to silver addition. Analysis of both dissolved and particulate silver allowed quantification of the distribution coefficient and uptake rate constants. The presence of both AgNPs and Ag+ produced significant changes in the biofilm structure, decreasing the relative percentage of Diatomophyceae and Cyanophyceae and increasing the relative percentage of Chlorophyceae. The accumulation capacity of the phototrophic biofilm with respect to ionic silver and the corresponding distribution coefficients were an order of magnitude higher than those of the phototrophic biofilm with respect to AgNPs. Higher levels of AgNPs decreased the biomass from 8.6u2009±u20090.2xa0mgxa0cm−2 for 0–10xa0μgxa0L−1 AgNPs to 6.0u2009±u20090.1xa0mgxa0cm−2 for 100xa0μgxa0L−1 added AgNPs, whereas ionic silver did not have any toxic effect on the biofilm growth up to 100xa0μgxa0L−1 of added Ag+. At the same time, AgNPs did not significantly affect the photosynthetic activity of the biofilm surface communities compared to Ag+. It can thus be hypothesized that negatively charged AgNPs may travel through the biofilm water channels, thereby affecting the whole biofilm structure. In contrast, positively charged Ag+ is bound at the cell surfaces and EPS, thus blocking its further flux within the biofilm layers. On the whole, the phototrophic biofilm demonstrated significant capacities to accumulate silver within the surface layers. The main mechanism to avoid the toxic effects is metal complexation with exopolysaccharides and accumulation within cell walls, especially pronounced under Ag+ stress. The significant AgNPs and Ag+ uptake capacities of phototrophic biofilm make it a highly resistant ecosystem in silver-polluted river waters.

Effect of Dunaliella tertiolecta organic exudates on the Fe(II) oxidation kinetics in seawater.

The role played by the natural organic ligands excreted by the green algae Dunaliella tertiolecta on the Fe(II) oxidation rate constants was studied at different stages of growth. The concentration of dissolved organic carbon increased from 2.1 to 7.1 mg L(-1) over time of culture. The oxidation kinetics of Fe(II) was studied at nanomolar levels and under different physicochemical conditions of pH (7.2-8.2), temperature (5-35 °C), salinity (10-37), and dissolved organic carbon produced by cells (2.1-7.1 mg L(-1)). The experimental rate always decreased in the presence of organic exudates with respect to that in the control seawater. The Fe(II) oxidation rate constant was also studied in the context of Marcus theory, where ΔG° was 39.31-51.48 kJ mol(-1). A kinetic modeling approach was applied for computing the equilibrium and rate constants for Fe(II) and exudates present in solution, the Fe(II) speciation, and the contribution of each Fe(II) species to the overall oxidation rate constant. The best fit model took into account two acidity equilibrium constants for the Fe(II) complexing ligands with pKa,1=9.45 and pKa,2=4.9. The Fe(II) complexing constants were KFe(II)-LH=3×10(10) and KFe(II)-L=10(7), and the corresponding computed oxidation rates were 68±2 and 36±8 M(-1) min(-1), respectively.

Metal and proton adsorption capacities of natural and cloned Sphagnum mosses.

Terrestrial mosses are commonly used as bioindicators of atmospheric pollution. However, there is a lack of standardization of the biomonitoring preparation technique and the efficiency of metal adsorption by various moss species is poorly known. This is especially true for in vitro-cultivated moss clones, which are promising candidates for a standardized moss-bag technique. We studied the adsorption of copper and zinc on naturally grown Sphagnum peat moss in comparison with in vitro-cultivated Sphagnum palustre samples in order to provide their physico-chemical characterization and to test the possibility of using cloned peat mosses as bioindicators within the protocol of moss-bag technique. We demonstrate that in vitro-grown clones of S. palustre exhibit acid-base properties similar to those of naturally grown Sphagnum samples, whereas the zinc adsorption capacity of the clones is approx. twice higher than that of the samples from the field. At the same time, the field samples adsorbed 30-50% higher amount of Cu(2+) compared to that of the clones. This contrast may be related to fine differences in the bulk chemical composition, specific surface area, morphological features, type and abundance of binding sites at the cell surfaces and in the aqueous solution of natural and cloned Sphagnum. The clones exhibited much lower concentration of most metal pollutants in their tissues relative to the natural samples thus making the former better indicators of low metal loading. Overall, in vitro-produced clones of S. palustre can be considered as an adequate, environmentally benign substitution for protected natural Sphagnum sp. samples to be used in moss-bags for atmospheric monitoring.

Oxidation of Cu(I) in Seawater at Low Oxygen Concentrations

The oxidation of nanomolar copper(I) at low oxygen (6 μM) concentrations was studied as a function of pH (6.7-8.2), ionic strength (0.1-0.76 M), total inorganic carbon concentration (0.65-6.69 mM), and the added concentration of hydrogen peroxide, H(2)O(2) (100-500 nM) over the initial 150 nM H(2)O(2) concentration in the coastal seawater. The competitive effect between H(2)O(2) and O(2) at low O(2) concentrations has been described. Both the oxidation of Cu(I) by oxygen and by H(2)O(2) had a reaction order of one. The reduction of Cu(II) back to Cu(I) in the studied seawater by H(2)O(2) and other reactive oxygen intermediates took place at both high and low O(2) concentrations. The effect of the pH on oxidation was more important at low oxygen concentrations, where δlog k/δpH was 0.85, related to the presence of H(2)O(2) in the initial seawater and its role in the redox chemistry of Cu species, than at oxygen saturation, where the value was 0.6. A kinetic model that considered the Cu speciation, major ion interactions, and the rate constants for the oxidation and reduction of Cu(I) and Cu(II) species, respectively, was applied. When the oxygen concentration was lower than 22 μM and under the presence of 150 nM H(2)O(2), the model showed that the oxidation of Cu(I) was controlled by its reaction with H(2)O(2). The effect of the pH on the oxidation rate of Cu(I) was explained by its influence on the oxidation of Cu(I) with O(2) and H(2)O(2), making the model valid for any low oxygen environment.

Chemical and structural characterization of copper adsorbed on mosses (Bryophyta).

The adsorption of copper on passive biomonitors (devitalized mosses Hypnum sp., Sphagnum denticulatum, Pseudoscleropodium purum and Brachythecium rutabulum) was studied under different experimental conditions such as a function of pH and Cu concentration in solution. Cu assimilation by living Physcomitrella patents was also investigated. Molecular structure of surface adsorbed and incorporated Cu was studied by X-ray Absorption Spectroscopy (XAS). Devitalized mosses exhibited the universal adsorption pattern of Cu as a function of pH, with a total binding sites number 0.05-0.06 mmolg(dry)(-1) and a maximal adsorption capacity of 0.93-1.25 mmolg(dry)(-1) for these devitalized species. The Extended X-ray Absorption Fine Structure (EXAFS) fit of the first neighbor demonstrated that for all studied mosses there are ∼4.5 O/N atoms around Cu at ∼1.95 Å likely in a pseudo-square geometry. The X-ray Absorption Near Edge Structure (XANES) analysis demonstrated that Cu(II)-cellulose (representing carboxylate groups) and Cu(II)-phosphate are the main moss surface binding moieties, and the percentage of these sites varies as a function of solution pH. P. patens exposed during one month to Cu(2+) yielded ∼20% of Cu(I) in the form of Cu-S(CN) complexes, suggesting metabolically-controlled reduction of adsorbed and assimilated Cu(2+).