Isolation and Molecular Characterization of Heavy Metal-Resistant Azotobacter chroococcum from Agricultural Soil and Their Potential Application in Bioremediation

Pollution of soil with heavy metals, herbicides, antibiotics and other chemicals is known to have a negative effect on microbial activities. Therefore, the aim of this study was to isolate cultures of Azotobacter sp. from polluted and unpolluted soils and to study the effect of these pollutants on their growth. A total of 120 Azotobacter sp. were isolated from soils irrigated with wastewater (contaminated soils) and groundwater (uncontaminated soils). These isolates were screened for resistance to heavy metals, herbicide and antibiotics. Also, the soils from which the cultures were isolated were analyzed for the concentrations of Zn²⁺, Cd²⁺, Cu²⁺, Pb²⁺ and Mn²⁺ they contained. Contaminated soil showed high levels of heavy metals as compared to uncontaminated soil. The size of the Azotobacter population in contaminated soil was lower than that in uncontaminated soil. Of the Azotobacter isolates, 64 that were recovered from contaminated soil exhibited high resistance to heavy metals (Hg²⁺, Cd²⁺, Cu²⁺, Cr³⁺, Co²⁺, Ni²⁺, Zn²⁺ and Pb²⁺) and herbicide 2,4-D compared to 56 isolates from uncontaminated soil. Also, isolates from contaminated soil showed high resistance to chloramphenicol, nitrofurantoin and co-trimoxazole compared to those isolated from uncontaminated soil. The majority of Azotobacter isolates from contaminated soil showed multiple-resistance to different metal ions and antibiotics. All isolates failed to grow at pH less than 6. Salt concentration (5%) was found to be inhibitory to all isolates. The most potent isolates from contaminated soil that showed multiresistance to all substances tested were identified on the basis of morphological and biochemical characteristics, and 16S rRNA as A. chroococcum. These resistant isolates could be employed in contaminated soils and/or bioremediation.     

Solvent effects on intramolecular hydrophobic ligandligand interactions in binary and ternary complexes

The stability constants of mixed ligand complexes of the type M(Phen)(ACA)⁺, where M = Cu²⁺ or Zn²⁺, Phen = 1,10-phenanthroline and ACA^? = propionate, valerate and 2-cyclohexylacetate, were determined by potentiometric pH titration in 50% (v/v) dioxanewater and were compared with the stabilities of the corresponding ternary complexes formed with formate and acetate. The ternary complexes containing the alkanecar?ylates (ACA^?) are significantly more stable, due to intramolecular hydrophobic interactions between the alkyl residue of the ACAt¯ligands and the 1,10-phenanthroline molecule. For Zn(Phen)(valerate)⁺ this intramolecular ligand-ligand interaction was confirmed by¹H NMR shift measurements. The formation degree of the intramolecular adducts in the ternary Cu²⁺ and Zn²⁺ complexes was calculated and the position of the intramolecular equilibrium between the opened and closed isomer was determined: the closed isomer occurs between about 10 to 35 percent. Comparisons with related data show that the extent of this interaction is about the same in water and in 50% aqueous dioxane; this contrasts with the experience made with simple unbridged adducts, which are destabilized by the addition of dioxane (or other organic solvents). Furthermore, evaluation of the available stability data for the Cu²⁺/leucinate (Leu^?) system shows that addition of some dioxane to an aqueous solution (in which of the closed isomer exists to about 20%) favors the intramolecular interaction between the two isopropyl residues in Cu(Leu)₂ considerably: in 40 to 50% aqueous dioxane the formation degree of the closed isomer reaches about 80%. Higher concentrations of the organic solvent destabilize the hydrophobic interaction. The overall stability of Cu(Leu)⁺ and Cu(Leu)₂, as well of Cu(alaninate)⁺ and Cu(alaninate)₂, is governed by the polarity of the solvent while the extent of the intramolecular ligand-ligand interaction is influenced by the hydrophobic properties of the solvent molecules. Based on the stability data it is shown that intramolecular ligandligand interactions are quite a common feature for many binary and ternary amino acid complexes: e.g., M(norvalinate)₂, M(phenyl-alaninate)₂, M(tyrosinate)₂ [M = Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺] or Cu(tyrptophanate)₂ and M(phenylalaninate)(norvalinate) or M(phenylalaninate)(tyrosinate) [M = Co²⁺, Ni²⁺, Cu²⁺]. In addition, evidence is presented that direct M²⁺-aromatic interactions are of no significance in these amino acid complexes in solution. The relevance of the present results with regard to biological systems is indicated.     

Copper complexing capacity in fresh-waters using the catechol-cathodic stripping voltammetric method

Abstract

The catechol-cathodic stripping voltammetric (CSV) method for measurement of the copper complexing capacity of marine and estuarine waters, has been modified and applied to fresh-waters. The optimum catechol concentration needed was the major difference between the two methods. It was found necessary to separately optimise the catechol concentration for each of the three fresh-water samples tested, presumably because of the much greater differences in the concentration of complexing ligands in fresh-water compared with marine or estuarine waters. This limits the usefulness of the CSV method for the determination of the complexing capacity of fresh-waters.

The total ligand concentration ([L_t]) and conditional stability constants (*K) derived for the three fresh-waters are compared with similar data obtained using an anodic stripping voltammetric (ASV) method. The set of copper-binding ligands determined using the CSV method were slightly lower in concentration, but stronger binding, than those obtained using the ASV method (CSV: [L_t] 0.04–0.06 μM, log K(pH 6) 8.9–9.4; ASV: [L_t] 0.11–0.17 μM, log *K(pH 6) 7.9–8.1).     

Calcium‐dependent behavioural responses to acute copper exposure in Oncorhynchus mykiss

Using rainbow trout Oncorhynchus mykiss, the present study demonstrated that: (1) calcium (Ca) increased the range of copper (Cu) concentrations that O. mykiss avoided; (2) Ca conserved the maintenance of pre‐exposure swimming activity during inescapable acute (10 min) Cu exposure. Data showed that when presented with a choice of Cu‐contaminated water (ranging from 0 to 454 µg Cu l^?1) and uncontaminated water in a choice tank, O. mykiss acclimated and tested at low Ca concentration (3 mg Ca l^?1) avoided the 10 µg Cu l^?1 only. By contrast, O. mykiss acclimated and tested at high Ca concentration (158 mg Ca l^?1) avoided all the Cu concentrations ≥37 µg l^?1. The Cu avoidance was connected with increased spontaneous swimming speed in the Cu‐contaminated water. When subjected to inescapable Cu exposure (35 µg Cu l^?1), O. mykiss acclimated and tested at low Ca concentration reduced their spontaneous swimming speed, whereas no response was observed in O. mykiss acclimated and tested at high Ca concentration. Collectively, the data support the conclusion that in O. mykiss the behavioural responses to acute Cu exposure are Ca‐dependent.     

Evaluation of Copper Bioaccumulation and Translocation in Jatropha curcas Grown in a Contaminated Soil

Contamination of soils with copper (Cu) has become a serious problem in the environment. Phytoremediation is an emerging green technology that uses green plants to remediate heavy metal contaminated areas. This study was conducted to evaluate the potential of Jatropha curcas for remediation of soils contaminated with Cu. Seedlings were planted in soils spiked with Cu in amount of 0, 50, 100, 200, 300, and 400 mg kg^–1 (Cu_0, Cu₅₀,Cu₁₀₀,Cu₂₀₀,Cu_300, and Cu₄₀₀) for a period of five months. The maximum height and number of leaves were recorded in control (Cu₀) whereas the highest basal stem diameter was found in seedlings exposed to Cu₅₀. Copper concentrations among plant parts were in the following trend: roots > stems > leaves. The highest total Cu concentration (665 ± 1 mg kg^?1) and total Cu removal (1.2 ± 0.2%) based on total plant dry biomass were found in Cu₄₀₀ and Cu₅₀ treatments, respectively. J. curcas exhibited high root concentration factor (RCF > 1) and low translocation factor (TF < 1). Although Cu accumulation by the plant didn't reach the criteria of Cu hyperaccumulators, this species showed a potential to be used in phytostabilization of mildly Cu contaminated areas. However, the plant cannot be used for phytoextraction of Cu-contaminated soils.     

ADSORPTION AND UPTAKE OF COPPER BY THE GREEN ALGA SCENEDESMUS SUBSPICATUS (CHLOROPHYTA)1

Copper (II) accumulation has been investigated in the green alga Scenedesmus subspicatus G. Brinkmann considering both adsorption and uptake kinetics. Experiments were conducted in a Cu- and PH-buffered medium at different free Cu²⁺ concentrations that were neither growth limiting nor toxic. We distinguished between adsorption on the cell surface and intracellular uptake by extracting copper from the cells with EDTA. Data from short-term experiments were compared with data obtained from experiments under steady state conditions. The accumulation of Cu can be described by two processes, an initial fast adsorption occurring within a minute followed by a slower intracellular uptake. Metal uptake followed Michaelis-Menten kinetics and is mediated by two systems, one with low and the other with high affinity. The maximum uptake rates (1.30 × 10^?-¹⁰ mol·[g dry wt algae]^?1· min^?1, 3.67 × 10^?-⁹ mol·[g dry wt algae]^?1·min^?1), and the half-saturation constants (6.84 × 10^?-¹⁴ M, 2.82 × 10^?-¹² M) for the two uptake systems were determined using the Lineweaver-Burk plot. The calculated maximum concentration of binding sites on the surface of the algae is initially higher (9.0 × 10^?-⁶ mol Cu.[g dry wt algae]^?1) than under steady state conditions (2.9 × 10^?-⁶ mol Cu·[g dry wt algae]^?1). This suggests that the initial binding to the algal surface comprises the binding to specific transport ligands as well as to inert adsorption sites. The conditional stability constant of the Cu binding to surface ligands was calculated as log K_Cu= 11.0 at pH 7.9. This freshwater alga has a high ability to accumulate Cu, reflecting its adaptation to the bioavailable concentration of copper.     

Copper biosorption in an aerobic bioreactor

Abstract

This study evaluates the biosorption of copper by aerobic biomass that was selected from surface waters of the San Pedro River in Sonora, Mexico. Using a batch system, 73% biosorption of copper was obtained in 75 minutes. Continuous biosorption assays were carried out for 133 days in an ascending flow aerobic reactor packed with zeolite (AFAR-PZ) that was inoculated with a bacterial consortium. Strains were grown until 1g L^?1 of biomass was obtained. Tests using continuous biosorption were performed as follows: (i) the addition of 50 mg Cu²⁺ L^?1 without recirculation of biomass; (ii) the addition of 20 mg Cu²⁺ L^?1without recirculation of biomass; and (iii) the biomass were recirculated with the addition of 20 mg Cu²⁺ L^?1 to pH 3 to 4. The fourth and fifth assays varied pH between 4 and 5, with 20 mg Cu²⁺ L^?1and the biomass recirculated. Biosorption capacity of the first and second assays was 96% on the first day of experimentation. During the third trial 97% of biosorption was obtained during 6 days and the process was improved by varying the pH. Copper biosorption equilibrium was investigated under the same operating conditions. Langmuir adsorption isotherms were used to fit experimental data. The biosorption capacity of aerobic biomass was 3.08 mmol g^?1. It was demonstrated that this biomass is capable of biosorbing copper and this method has potential for the treatment of industrial effluents contaminated with heavy metals.     

Significance of binary and ternary copper(II) complexes for the promotion and protection of adenosine 5′-di- and triphosphate toward hydrolysis

The dephosphorylation of ADP and ATP was characterized as the first-order rate constant in dependence on pH in the absence and presence of Cu²⁺, and together with Cu²⁺ and a second ligand. The reaction is strongly accelerated by Cu²⁺ and passes through pH optima at about 6.2 and 6.5 for the Cu²⁺ ?ADP and ?ATP systems, respectively (I = 0.1, NaClO₄; 50°C). In the presence of 2,2′-bipyridyl (Bipy), ternary complexes are formed with the nucleotides ADP or ATP (NP), Cu(Bipy)(NP), which are very stable towards dephosphorylation over a large pH range. Similar stabilizing effects were observed in ternary complexes formed with imidazole or OH^?. These results can easily be rationalized by taking into account that in the binary Cu²⁺ complexes macrochelates are formed by the interaction between the adenine moiety and the metal ion. This interaction is crucial for obtaining the labile species and hence, in the mixed-ligand complexes, where the macrophelate can not be formed, the phosphates are protected toward hydrolysis. In agreement with these results is the dephosphorylation behavior of Cu(CDP)^? and Cu(CTP)^2?; they are rather stable. This is in accord with the small coordination tendency of the cytosine moiety.By computing the pH dependence of the distribution of the several species, it is shown that the active species are Cu(ATP)^2? and Cu(ADP)^? and not the hydroxy complexes, [Cu(ATP)(OH)]₂^6? and [Cu(ADP)(OH)₂^4? as were suggested earlier. With the aid of the initial rate, $\text{ν}{}_0\text{=}\text{d}\text{[}\text{PO}{}_4{}^{\mathrm{3?}}\text{]}\text{d}\text{t}$, the rate laws of the ascending side of the pH optima were determined: $\text{ν}{}_0\text{= k}\text{[}\text{Cu(NP)}\text{]}\text{[}\text{H}{}^{\mathrm{+}}\text{]}$. The descending side of the pH optima is attributed to the formation of Cu(NP)(OH), where the metal ion interaction with N-7 of the adenine moiety is inhibited.     

柠檬香蜂草对铜的耐性及其积累特征研究

以柠檬香蜂草(Melissa officinalis)幼苗为材料,设置不同浓度Cu~(2+)胁迫(CK、200、400、800和1 000mg·kg~(-1))盆栽实验,测定胁迫0、7、14、21、28d后植物生物量、叶绿素含量、抗氧化酶活性、可溶性蛋白含量、丙二醛(MDA)含量以及植物体内Cu含量等指标,探讨柠檬香蜂草对Cu~(2+)的耐受性及其积累特征。结果表明:(1)相同处理时间下,柠檬香蜂草除MDA含量外其他所有指标均随着Cu~(2+)胁迫处理浓度的增加呈低浓度促进、高浓度抑制的变化趋势,且高浓度组(800和1 000mg·kg~(-1))与低浓度组(200和400mg·kg~(-1))之间差异显著(P0.05);MDA含量在1 000mg·kg~(-1)浓度下持续增长至第14天后开始下降。(2)柠檬香蜂草体内Cu的积累量随Cu胁迫浓度的升高呈先增加后减少的趋势,并在浓度为400mg·kg~(-1)时达到最高值(0.71mg/盆)。(3)在整个胁迫过程中,柠檬香蜂草植株的铜富集系数及其耐性系数均随Cu浓度的增加而减小,各处理浓度对Cu的耐性系数均大于0.5,富集系数均大于1。研究发现,柠檬香蜂草对Cu胁迫具有一定耐受性和富集能力,具有成为铜污染土壤修复植物的潜力。     

Dynamics of trace metals in the system water – soil – plant – wild rats – tapeworms (Hymenolepis diminuta) in Maglizh area,Bulgaria

BackgroundThe impact of chemical elements on the biosphere is a function of their concentration and chemical form. Elucidation and prognosing of the latters in water basins and soil extracts is of particular significance for the assessment of their bioaccumulation in plants and animals.ObjectivesTrace metals dynamics in the system water – soil–plant–wild rats – Hymenolepis diminuta in two agro-industrial zones (East and West) around Maglizh city, Bulgaria were investigated through experimental studies and thermodynamic modelling of the chemical species.MethodsSamples from surface waters of rivers, their nearby uncultivated soils, meadow uncultivated vegetation (Ranunculus acris and Gramineae) and field rats were collected. In situ measurements and laboratory analyses were performed for the determination of the physico-chemical characteristics and total concentrations of Al, Fe, Mn, Ni, Cu, Zn and Pb. The distribution of their dissolved chemical species in water samples and in the aqueous soil extracts was calculated using a thermodynamic approach. The relationship chemical species - bioaccumulation was discussed.ResultsWaters and soils in the East zone of Maglizh area were found to be more polluted compared to those in the West one, regarding Ni, Mn, Zn, Pb and Cu, while Mn and Cu displayed the highest mobility in West zone soils. Trace metals contents in Ranunculus acris exceed that in Gramineae, since the highest accumulation factors were calculated for Cu and Zn. The highest accumulation in rats was found for Zn followed by Cu, being higher in the West zone. Thermodynamic modelling shows that Mn²⁺ free ions are dominant in both waters and aqueous soil extracts. Ni²⁺ and Zn²⁺ ions followed by metal-organic complexes are dominant in waters of East zone while metal-organic complexes followed by free ions are dominant in waters of West zone and both soil extracts. Metal-organic complexes are dominant for Fe, Cu and Pb in all samples studied, while mainly hydroxy forms (Al(OH)₄⁻) followed by metal-organic complexes are typically for Al depending on pH.ConclusionsExperimentally established bioaccumulation of trace metals in the studied vegetation and rats is a consequence of the total concentration of trace metals in waters and soils, their mobility and chemical species. The dominance of organic complexes of trace metals is a prerequisite for their bioaccumulation in plants. Rats are in direct contact with the soil solution and therefore, of importance is the content of free ions of Mn²⁺, Ni²⁺, Zn²⁺, which are easily absorbed through the skin. The host-helminth system wild rat/H. diminuta could be used as a bioindicator for trace metals pollution.     

Inhibition of bovine plasma amine oxidase by superoxide dismutase active Cu(II) complexes

Aqueous Cu²⁺ and Cu(II) complexes of salicylate, lysine, and tyrosine decrease the rate of benzylamine oxidation by bovine plasma amine oxidase. Bissalicylato Cu(II) and Cu²⁺ inhibit non-competitively with respect to benzylamine. Lysine, tyrosine, Cu(EDTA)^2?, Zn²⁺, and Co²⁺ do not inhibit, and erythrocyte Cu, Zn superoxide dismutase shows only slight inhibition of the amine oxidase. The data are most consistent with an inhibitory mechanism involving dismutation of O₂^? by the Cu(II) complexes within a site relatively inaccessible to the enzyme superoxide dismutase. Excess lysine significantly decreases inhibition by the bis-lysine complex of Cu(II).     

Speciation of copper in relation to its bioavailability

Abstract

Copper speciation and bioavailability for Scenedesmus quadricauda has been studied in natural waters and in synthetic culture media. Other elements were studied simultaneously. When phosphorus and nitrogen limitation were excluded by adding these elements, copper was limiting algal growth in some natural waters. In the toxic range, growth inhibition by copper was highly correlated with copper detected by electrochemical methods and with calculated free copper.

Copper was toxic to S. quadricauda when free copper concentrations roughly exceeded 10^?10.5 M, and was limiting for values somewhere lower than 10^?12.5 M. Because we found copper limitation in some natural water samples, free copper concentration in those water samples therefore must have been lower than 10^?12.5 M.

The hypothesis that the free metal concentration rather than the total concentration determines bioavailability was confirmed for copper, cobalt and zinc.     

Photooxidation of wetland and riverine dissolved organic matter: altered copper complexation and organic composition

In natural waters, the uptake of transition metals such as copper (Cu) by aquatic biota depends on the activity of the free cupric ion ({Cu²⁺}) rather than on total Cu concentration. Thus, an important ecological function of dissolved organic matter (DOM) in aquatic ecosystems is Cu–DOM complexation, which greatly decreases the {Cu²⁺}. However, Cu bioavailability is greatly modified by source and environmental history of DOM because DOM affinity for Cu varies by orders of magnitude among DOM sources; moreover, DOM is photochemically unstable. During 72-h irradiation experiments at intensities approximating sunlight with DOM from a palustrine wetland and a third-order river, we investigated photooxidative effects on DOM complexation of Cu as well as spectral and chemical changes in DOM that might explain altered Cu complexation. Irradiation decreased Cu complexation by riverine DOM, but unexpectedly increased Cu complexation by wetland DOM, resulting in 150% greater {Cu²⁺} in riverine DOM at the same dissolved organic carbon concentrations. The specific ultraviolet absorption (SUVa) and humic substances tracked photochemical changes in the conditional stability constants of Cu–DOM complexes, suggesting that the aromaticity of DOM influences its affinity for Cu. Carbonyl concentration in ¹³C nuclear magnetic resonance spectra (¹³C-NMR) covaried directly with Cu binding-site densities in DOM. However, no aspect of Cu–DOM complexation consistently covaried with fluorophores (i.e., the fluorescence index) or low molecular weight organic acids. Our results suggest that global increases in UV radiation will affect Cu–DOM complexation and subsequent Cu toxicity depending on light regime as well as DOM source. Handling editor: K. Martens     

Self-association phenomenon specific to the copper complexes with ADP,dADP, and IDP

Circular dichroism spectra between 200 and 800 nm are presented for solutions of nucleotides with pyrophosphate group derived from adenine and inosine in the presence of the Cu₂₊ cation. A study as a function of the pH, temperature, and concentration has shown the existence of a self-association, which implies a strong interaction between the bases of the two nucleotides. The interpretation of the dichroic spectra allows us to show that two oligomer species are formed successively in low concentrations in the presence of the Cu²⁺ cation. The first, constituted by Cu(ADP)^? molecules, appears at pH 5.3 and gives rise to a nonconservative excitonic term. The second, responsible for a conservative excitonic term, is a heterooligomer formed at pH 7.3 by the assembling of a Cu(ADP)^? and a Cu(ADP)(OH)^2? molecule. Furthermore, this self-association is destabilized if the nucleotide derived from adenine is replaced by the corresponding deoxyribonucleotide or by IDP.     

Effect of copper on the content and the biosynthesis of indole glucosinolates glucobrassicin and neoglucobrassicin in etiolated rape seedlings (Brassica napus var.arvensis (Lam.) Thell.)

The influence of Cu²⁺ ions (in the form of CuCl₂) in the concentration range 10^?3 to 10^?6 M on the content and biosynthesis of indole glucosinolates glucobrassicin and neoglucobrassicin has been studied on etiolated seedlings of rape (Brassica napus var.arvensis (Lam.) Thell.). Ions Cu²⁺ acted on the seedlings either chronically from the beginning of the germination or acutely, during 3 to 72 h, on seven days old seedlings. The biosynthesis of both glucosinolates was followed by the incorporation of³⁵S from Na₂ ³⁵SO₄ into them in hypocotyl segments from seven days old intact etiolated seedlings. After the entry of small amounts of Cu²⁺ ions into the plants, stimulation of the glucosinolates formation occurs, as was found after three h action of Cu²⁺ ions. After the entry of a greater amount of Cu²⁺ ions into the plant, harmful effects appear, as was found after chronic two days action or after 24 and 48 hours acute action of Cu²⁺ ions. Later further stimulation of glucosinolate formation occurs, probably due to enhanced metabolism during reparation processes, as was manifested after chronic action of Cu²⁺ ions lasting four and eight days. The optimal effect of copper was found mainly in the concentration range 5×10^?4 M to 10^?5 M. Ions Cu²⁺ in higher concentration increased the uptake of sulphate ions by hypocotyl segments, and in lower concentrations increased the incorporation of³⁵S from³⁵SO₄ ^2? into the proteins.     

Phenotypic seedling responses of a metal-tolerant mutant line of sunflower growing on a Cu-contaminated soil series: potential uses for biomonitoring of Cu exposure and phytoremediation

Background and aims

The potential use of a metal-tolerant sunflower mutant line for both biomonitoring and phytoremediating a Cu-contaminated soil series was investigated.

Methods

The soil series (21–1,170 mg Cu kg^?1) was sampled in field plots at control and wood preservation sites. Sunflowers were cultivated 1 month in potted soils under controlled conditions.

Results

pH and dissolved organic matter influenced Cu concentration in the soil pore water. Leaf chlorophyll content and root growth decreased as Cu exposure rose. Their EC₁₀ values corresponded to 104 and 118 μg Cu L^?1 in the soil pore water, 138 and 155 mg Cu kg^?1 for total soil Cu, and 16–18 mg Cu kg^?1 DW shoot. Biomass of plant organs as well as leaf area, length and asymmetry were well correlated with Cu exposure, contrary to the maximum stem height and leaf water content.

Conclusions

Physiological parameters were more sensitive to soil Cu exposure than the morphological ones. Bioconcentration and translocation factors and distribution of mineral masses for Cu highlighted this mutant as a secondary Cu accumulator. Free Cu²⁺ concentration in soil pore water best predicted Cu phytoavailability. The usefulness of this sunflower mutant line for biomonitoring and Cu phytoextraction was discussed.     

Effect of linear alkylbenzene sulfonate on Cu2+ removal by Spirulina platensis strain (FACHB‐834)

The removal efficiency of Cu²⁺ by Spirulina platensis (strain FACHB‐834), in viable and heat‐inactivated forms, was investigated in the presence and absence of linear alkylbenzene sulfonate (LAS). When the initial Cu²⁺ concentration was in the range of 0.5–1.5 mg · L^?1, a slight increase in growth rate of FACHB‐834 was observed. In contrast, when Cu²⁺ or LAS concentrations were at or higher than 2.0 or 6.0 mg · L^?1, respectively, the growth of FACHB‐834 was inhibited and displayed yellowing and fragmentation of filaments. The presence of LAS improved Cu²⁺ removal by ~20%, and accelerated attainment of Cu²⁺ retention equilibrium. For the 2‐ mg · L^?1 Cu²⁺ treatments, retention equilibrium occurred within 2 d and showed maximum Cu²⁺ removal of 1.83 mg · L^?1. In the presence of LAS, the ratio of extracellular bound Cu²⁺ to intracellular Cu²⁺ taken up by the cells was lower (1.05–2.26) than corresponding ratios (2.46–7.85) in the absence of LAS. The percentages of extracellular bound Cu²⁺ to total Cu²⁺ removal (both bound and taken up by cells) in the presence of LAS ranged from 51.2% to 69.3%, which was lower than their corresponding percentages (71.1%–88.7%) in the absence of LAS. LAS promoted biologically active transport of the extracellular bound form of Cu²⁺ into the cell. In contrast, the addition of LAS did not increase the maximum removal efficiency of Cu²⁺ (61.4% ± 5.6%) by heat‐inactivated cells compared to that of living cells (59.6% ± 6.0%). These results provide a theoretical foundation for designing bioremediation strategies using FACHB‐834 for use in surface waters contaminated by both heavy metals and LAS.     

The Effect of Copper and Lead Ions on Growth and Lipid Composition of the Fern Matteuccia sthruthiopteris

In the present study, the effect of copper (Cu²⁺) and lead (Pb²⁺) ions on the growth and lipid composition of various parts of the fern, Matteuccia sthruthiopteris, was examined. Plants were incubated in the presence or absence of 1, 10, 100 μM of Cu(NO₃)₂ or Pb(NO₃)₂. Cu²⁺ and Pb²⁺ ions at concentrations of 1 and 10 μM caused an increased growth of the roots and leaves. A higher concentration of Pb²⁺ did not show any effect on growth, whereas that of Cu²⁺ slowed down the growth of the whole plants. The roots accumulated more than 700 μg of Cu²⁺ and 400 μg of Pb²⁺ per 1 g dry weight when the plants were incubated with the higher concentrations of metals, whereas in the leaves the concentration of Cu²⁺ was much lower and did not exceed 12 μg/g dry weight. No accumulation of Pb²⁺ ions by leaves was detected. The lipid composition of photosynthetic leave tissues was shown to be affected by the presence of metal ions in the root medium at either concentration studied. Various changes in lipid classes were noted as responsive reactions of M. sthruthiopteris to the heavy metal ions in nutrient medium. Cu²⁺ ions decreased the content of total lipids, total phospholipids, and individual phosphatidylcholines and phosphatidylethanolamines, whereas Pb²⁺ ions caused a decrease in the content of total lipids and glycolipids. Changes in the lipid composition were more pronounced in the mature leaves than in the scrolls of the studied fern.     

Mouse divalent metal transporter 1 is a copper transporter in HEK293 cells

Divalent Metal Transporter 1 (DMT1) is an apical Fe transporter in the duodenum and is involved in endosomal Fe export. Four protein isoforms have been described for DMT1, two from mRNA with an iron responsive element (IRE) and two from mRNA without it. The sets of two begin in exon 1A or 2. We have characterized copper transport using mouse 2/?IRE DMT1 during regulated ectopic expression. HEK293 cells carrying a TetR:Hyg element were stably transfected with pDEST31 containing a 2/?IRE construct. ⁶⁴Cu¹⁺ incorporation in doxycycline treated cells exhibited 18.6 and 30.0-fold increases in Cu content, respectively when were exposed to 10 and 100 μM of extracellular Cu. Cu content was ~4-fold above that of parent cells or cells carrying just the vector. ⁶⁴Cu uptake in transfected cells pre-incubated with 5 μM of Cu-His revealed a Vmax and Km of 11.98 ± 0.52 pmol mg protein^?1 min^?1 and 2.03 ± 0.03 μM, respectively. Doxycycline-stimulated Cu uptake was linear with time. The rates of apical Cu uptake decreased and transepithelial transport increased when intracellular Cu increased. The optimal pH for Cu transport was 6.5; uptake of Cu was temperature dependent. Silver does not inhibit Cu uptake in cells carrying the vector. In conclusion, Cu uptake in HEK293 cells that over-expressed the 2/?IRE isoform of DMT1 transporter supports our earlier contention that DMT1 transports Cu as Cu¹⁺.     

Spectroscopic and equilibrium dialysis studies of poly(α-L-glutamic acid)–Cu(II) complexes in the pH range 4–7

The formation of complex between the Cu²⁺ ion and poly(α-L -glutamic acid) [poly(Glu)] in 150 mM NaCl solutions was studied by uv–visible absorption and equilibrium dialysis methods at the mixing ratios of Glu residues to Cu²⁺, R, of 32, 16, and 8 and in the pH range 4–7. The results showed that more than 90% of Cu²⁺ ions bind to the poly(Glu) at pH > 4.9, but the bound Cu(II) begins to dissociate with a decrease in pH. The absorption spectra of bound Cu(II) varied with pH and R in a complicated manner. Three different component spectra were disclosed from the analysis of the pH dependence of the bound spectra. We concluded that poly(Glu)–Cu(II) complexes fall into three classes in the pH range 4–7, with the proportions of these complexes varying with both pH and R. The three complexes predominate either in the helix or extended-coil region, in the helix–coil transition region, or in the helix-aggregate region. The stability constant and binding mode of each Cu(II)–Glu complex were estimated from the dialysis data. With these results, the possible structure of each complex is discussed.