Effects of copper concentration in continuous culture on the aa 3-type cytochrome oxidase and respiratory chains of Paracoccus denitrificans

The role(s) of copper in a bacterial cytochrome oxidase of the aa ₃-type was investigated by growth of Paracoccus denitrificans NCIB 8944, in batch and steady state continuous culture, in a medium from which the bulk of the copper had been extracted. In a medium containing approximately 0.02 M copper, cellular copper content, cytochromes a+a ₃ and cytochrome a ₃ were reduced to 55%, 58% and 33% respectively of control values and there were also less marked decreases in cytochromes c+c ₁ (to 85%) and a CO-binding b-type cytochrome, possibly cytochrome o (to 71%). Copper deficiency elicited in reduced minus oxidized difference spectra a shift to shorter wavelengths and narrowing of the band width of the -band of the oxidase, and loss of a (negative) band near 830 nm attributable to Cu_A (the copper functionally associated with haem a in the oxidase complex). The oxidase in copper-deficient cells reacted with oxygen to form the oxy Compound A at rates similar to that in control cells but CO recombination to ferrous haem a ₃ was slowed 4-fold in the copper deficient case. The results are interpreted as indicating loss of Cu_A and changes in the proportions of haems a and a ₃ with retention of catalytic activity. Titrations of respiration rates with antimycin suggested that copper deficiency did not result in diversion of electron flux through an antimycin A-insensitive, cytochrome o-terminated branch of the respiratory chain.     

The effect of copper toxicity on the contents of nitrogen compounds in Silene vulgaris (Moench) Garcke

The effect of copper on the uptake of nitrogen and the tissue contents of inorganic nitrogen, amino acids and proteins were studied in cooper-sensitive Silene vulgaris (Moench) Garcke, grown at different nitrogen sources (NH₄ ⁺ or NO₃ ^-). All the toxic copper levels tested, i.e. 4, 8, 16 M Cu²⁺, strongly inhibited the uptake of nitrogen, especially of NO₃ ^-, and decreased the content of NO₃ ^-, amino acids and proteins. Especially at 4 and 8 M Cu²⁺, NH₄ ⁺ accumulated in the plants, suggesting that the conversion of NH₄ ^- into amino acids was inhibited.     

Copper biosorption by chemically treated Micrococcus luteus cells

In order to clarify the binding states of copper in microbial cells, copper biosorption from aqueous systems using the chemically treated Micrococcus luteus IAM 1056 cells (hot water-treated, diluted NaOH-treated, chloroform–methanol-treated, and chloroform–methanol/concentrated KOH-treated cells) was examined. The intact cells of M. luteus adsorbed 527 mol of copper per g cells, and its copper adsorption was very rapid and was affected by the solution pH. The chloroform–methanol/concentrated KOH-treated cells showed higher copper biosorption capacity than the intact and the other chemically treated cells. The electron paramagnetic resonance (EPR) parameters, g and |A |, of Cu(II) ion in microbial cells indicate that Cu(II) ion in the intact and all the chemically treated cells have coordination environments with nitrogen and oxygen as donor atoms, being similar to those of type II proteins. The parameter g also indicated that the coupling between Cu(II) ion and the cell materials in the CHCl₃–MeOH/concentrated KOH-treated cells is rather more stable than those between Cu(II) ion and the cell materials in the other treated cells.     

Synthesis and antimicrobial activity of copper(II) and manganese(II) α,ω-dicarboxylate complexes

Copper(II) ,-dicarboxylate complexes of general formulae, [Cu(O₂C(CH₂)_nCO₂)]·xH₂O, [Cu(O₂C(CH₂)_nCO₂) (phen)₂]·xH₂O and [Cu(O₂C(CH₂)_nCO₂)(bipy)_y]·xH₂O (n=1–8; y=1, 2; phen = 1,10-phenanthroline; bipy = 2,2-bipyridine) were synthesised. These copper complexes, some related manganese(II) complexes and the metal-free ligands were screened in vitro for their ability to inhibit the growth of Candida albicans. Metal-free 1,10-phenanthroline and all of the copper(II) and manganese(II) phenanthroline complexes were potent growth inhibitors, with only one bipyridine complex, [Cu(O₂C(CH₂)CO₂)(bipy)₂]·2H₂O, having moderate activity. The remaining substances were effectively inactive. Complexes which were active against C. albicans also proved effective against C. glabrata, C. tropicalis and C. kreusi with the manganese complexes retaining superior activity. For the phenanthroline complexes the active drug species is thought to be the dication [M(phen)₂(H₂O)_n]²⁺ (M = Cu, Mn). Escherichia coli and Staphylococcus aureus were resistant to all of the metal complexes and also to metal-free 1,10-phenanthroline. Only the copper phenanthroline complexes showed intermediate activity against Pseudomonas aeruginosa.     

Effects of oxygen and nitrogen conditions on the transformation kinetics of 1,2-dichloroethenes by Methylosinus trichosporium OB3b and its sMMO C mutant

Transformation kinetics of trans- andcis-dichloroethylenes (DCE) by Methylosinus trichosporium OB3b wild type (WT)and PP319, a mutant that expresses soluble methane monooxygenase at copper levels upto 12 M Cu (sMMO^C), were determined to assess theeffects of O₂level and N₂-fixation on degradationcapabilities. Two issues were examined: (1) the influence of O₂level and nitrogen-limitation on DCE degradationkinetics and toxicity in both organisms, and (2) the relative utility of PP319 forcontaminant degradation in bioreactors. When both organisms were grown underhigh O₂conditions (80% saturation in air), maximum transformation rates(V_max) and apparent first-order rate constants(V_max/K_M) were lower compared with organisms grown under low O₂conditions (10% saturation in air)regardless of nitrogen level. Further, V_max values were near zero innitrogen-limited WT cultures when O₂was high (as expected), whereas PP319 retainedmoderate V_max levels even at high O₂levels. In general, elevatedO₂conditions reduced DCE degradation rates in OB3b, although the negative effectsof O₂were less in PP319 than in the WT. Given that PP319 retained moderate DCEdegradation rates under most O₂and copper conditions, the mutant appears to havesome utility for biodegradation applications.     

Chains and channels in polynuclear copper(II) complexes with 2,3-bis(2-pyridyl)pyrazine (dpp) as bridging ligand; syntheses, crystal structures and magnetic properties

The preparation and crystal structures for three Cu(II) polynuclear, chain complexes with 2,3-bis(2-pyridyl)pyrazine (dpp) as bridging ligand are reported, [Cu(dpp)(H₂O)₂]_n(NO₃)_2n·2n/3H₂O (1), [Cu(dpp)(H₂O)₂]_n(CF₃SO₃)_2n (2), and [Cu(dpp)(H₂O)₂]_n(BF₄)_2n·2nH₂O (3). For the latter compound the crystal structure at four different temperatures have been studied. Variable-temperature magnetic susceptibility data and ESR measurements of 1–3 and of the related copper(II) chain [Cu(dpp)(H₂O)₂]_n(ClO₄)_2n·2nH₂O (4) (whose structure was previously reported) have been performed. Compounds 1 and 2 crystallize in the same trigonal space group, R c; 3 is orthorhombic, space group Pbca. Complexes 1 and 2 are built of linear dpp-bridged chains which extend along threefold screw axes. The copper atom has in each case an elongated octahedral geometry with pyrazine nitrogen atoms in axial positions. The prominent feature of the crystal packing is the supramolecular arrangement of six chains around a threefold inversion axis, creating channels housing the counter ions, and in the case of 1, also crystal water. In 3 the chain is zig–zag shaped and extends along a twofold screw axis. Counter ions and crystal water are situated in channels formed between four symmetry related chains. At room temperature (r.t.) the X-ray results show a copper ion with a compressed octahedral coordination geometry, pyrazine and pyridyl nitrogen atoms binding in equatorial and axial positions, respectively. The low temperature X-ray studies of 3 show significant changes in the copper coordination geometry, strongly suggesting that the apparent compressed geometry at r.t. is due to a dynamic Jahn–Teller distortion. The CuCu separations across the bridging dpp at r.t. are, 7.133(1) (1), 7.228(1) (2), 7.005(1) (3) and 7.002(2) Å (4). X-band ESR spectra of 1 and 2 exhibit the pattern of Cu(II) in elongated geometry (g>g>2.0), whereas those of 3 and 4 exhibit inverse (g>g>2.0) patterns with a shoulder in the perpendicular signal. Complexes 1–4 exhibit a Curie law behaviour with very weak intrachain antiferromagnetic coupling, the relevant magnetic parameters being J=−0.27 cm⁻¹, g=2.11 for 1, J=−0.17 cm⁻¹, g=2.09 for 2, J=−1.38 cm⁻¹, g=2.15 for 3, and J=−1.36 cm⁻¹, g=2.14 for 4 (the Hamiltonian being =−JS_AS_B).     

The physiological responses of freshwater rainbow trout,Oncorhynchus mykiss,during acutely lethal copper exposure

Acutely lethal (24 h) exposure of adult rainbow trout (Oncorhynchus mykiss) to 4.9 mol copper·l^-1 in fresh water (pH 7.9, [Ca²⁺]0.8 mEq·l^-1) caused a rapid decline of plasma Na⁺ and Cl^- and arterial O₂ tension, and initially a pronounced tachycardia. The internal hypoxia probably resulted from histopathologies observed in the gills of fish exposed to copper, such as cell swelling, thickening and curling of the lamellae, and haematomas. Copper cannot therefore be considered purely as an ionoregulatory toxicant during acutely lethal conditions. Mortality during exposure to copper could not simply be explained by the plasma ionic dilution, nor by the internal hypoxia, since arterial O₂ content remained relatively unchanged. Secondary to the ionoregulatory and respiratory disturbances were a number of deleterious physiological responses which included a massive haemoconcentration (haematocrit values as high as 60%) and a doubling of the mean arterial blood pressure. The time-course of these changes suggest that cardiac failure was the final cause of death. In this respect copper exposure resembles low pH exposure in freshwater trout (Milligan and Wood 1982). Copper and H⁺ appear to be similar in both the primary site of their toxic action (the gills) and the secondary physiological consequences which result from acutely lethal exposures. Furthermore, the acute toxicity syndrome observed may be common to many metals which cause ionoregulatory and/or respiratory problems in freshwater fish.Abbreviations C _aO₂ arterial oxygen content - FR water flow rate - Hb haemoglobin - Hct haematocrit - H _m ⁺ net metabolic acid load - IU international unit - MABP mean arterial blood pressure - MCHC mean corpuscular haemoglobin content - MO₂ rate of oxygen consumption - P _aCO₂ arterial carbon dioxide tension - P _aO₂ arterial oxygen partial pressure - T _amm total ammonia (=NH₃+NH ₄ ⁺ ) - TCO₂ total carbon dioxide - TOC total organic carbon - %Hb–O₂ percentage of haemoglobin saturated with oxygen     

Effect of water-borne copper on respiratory and cardiac function during the early ontogeny of the brine shrimp,Artemia franciscana Kellogg 1908 (Branchiopoda: Anostraca)

There was no direct effect of copper on the ontogeny or function of the heart of the brine shrimp Artemia franciscana in sea water (salinity= 36 mg·ml^-1, 25°C). There was, however, an indirect effect as an increase in copper concentration resulted in a reduced growth rate. There was no difference between the critical O₂ tensions of newly hatched (stage 0/1) nauplii of control and treated (<0.32 and 10.11 mol·l^-1 copper, respectively) individuals. However by developmental stages 4–6, when both the heart and thoracic gills are in the process of differentiating, respiratory performance had improved (i.e. critical O₂ tension decreased from 6.27±0.45 to 4.69±0.24 kPa) in control but not in copper-treated individuals. It is suggested that respiratory impairment of stages 4–6 individuals is unlikely to be related to differences in cardiac performance or cellular respiration. Instead it may be related to metal-related damage to newly differentiating gill tissue and/or by copper in some way compromising the normal ontogenic shift in haemoglobin O₂ affinity. Copper-related respiratory impairment develops at a critical point in brine shrimp organogenesis when a good supply of O₂ is essential for normal development and if compromised may reduce the ability of this species to survive copper exposure.Abbreviations BL body length - BW body weight - HR heart rate - HM heavy metals - SW sea water - P _c critical oxygen tension     

Improvement of the purification method for retaining the activity of the particulate methane monooxygenase from Methylosinus trichosporium OB3b

The purification method of particulate methane monooxygenase (pMMO) from Methylosinus trichosporium OB3b was improved, and purified pMMO retained its activity with duroquinol as a reductant. n-Dodecyl-,d-maltoside was used for the solubilization of pMMO and Brij 58 was used for the purification for anion exchange chromatography. Compared to the original pMMO activity in the membrane fraction, 88% of the activity was now retained in the purified material. The purified pMMO monomer (94 kDa) contained only two copper atoms and did not contain iron. Both copper ions showed only a typical type II copper EPR signal with a superhyperfine structure at the g region, indicating that the type II copper ions play an important role as the active site of methane hydroxylation in pMMO.     

Effects of copper on the photosynthesis of intact chloroplasts: interaction with manganese

Highly purified, intact chloroplasts were prepared from pea (Pisum sativum L.) and spinach (Spinacia oleracea L.) following an identical procedure, and were used to investigate the cupric cation inhibition on the photosynthetic activity. In both species, copper inhibition showed a similar inhibitor concentration that decreases the enzyme activity by 50% (IC₅₀≈ 1.8 µM) and did not depend on the internal or external phosphate (Pi) concentration, indicating that copper did not interact with the Pi translocator. Fluorescence analysis suggested that the presence of copper did not facilitate photoinhibition, because there were no changes in maximal fluorescence (F_m) nor in basal fluorescence (F_o) of copper‐treated samples. The electron transport through the photosystem II (PSII) was also not affected (operating efficiency of PSII— similar in all conditions). Yet, under Cu²⁺ stress, the proportion of open PSII reaction centers was dramatically decreased, and the first quinone acceptor (Q_A) reoxidation was fully inhibited, as demonstrated by the constant photochemical quenching (q_P) along experiment time. The quantum yield of PSII electron transport (Φ_PSII) was also clearly affected by copper, and therefore reduced the photochemistry efficiency. Manganese, when added simultaneously with copper, delayed the inhibition, as measured by oxygen evolution and chlorophyll fluorescence, but neither reversed the copper effect when added to copper‐inhibited plastids, nor prevented the inhibition of the Hill activity of isolated copper‐treated thylakoids. Our results suggest that manganese competed with copper to penetrate the chloroplast envelope. This competition seems to be specific because other divalent cations e.g. magnesium and calcium, did not interfere with the copper action in intact chloroplasts. All results do suggest that, under these conditions, the stroma proteins, such as the Calvin–Benson cycle enzymes or others are the most probable first target for the Cu²⁺ action, resulting in the total inhibition of chloroplast photosynthesis and in the consequent unbalanced rate of production and consumption of the reducing power.     

Copper-binding proteins and copper tolerance in Pisam sativum L.

The effect of high nutrient levels of copper on the low-molecular-weight copper-proteins of leaves from plants of two cultivars of Pisum sativum L., with different sensitivity to copper, was investigated. Gel-filtration chromatography of leaf extracts from Cu-tolerant and Cu-sensitive plants grown with 1 M Cu(II), showed the presence of only two copper peaks (I and II), but growth of plants with 240 M Cu(II) induced two additional copper fractions (III and IV). Fractions II and III were purified by solvent extraction, gel-filtration and ion-exchange chromatography, and their molecular weights, subunit sizes, absorption spectra, metalprotein stoichiometry and amino-acid contents were determined. Fraction II was a polypeptide of M_r 15000 composed of a single chain. The purification of fraction III produced a copper-containing fraction (III-1) of M_r 3700, and a copper-protein (III-2) with an M_r, by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis, of 66000. The metal contents of fractions III-1 and III-2 were higher in Cu-tolerant than in Cu-sensitive plants. On the basis of amino-acid analyses, fraction III-1 appeared to be complexes of Cu(II)-poly-isoleucine and Cu(II)-poly-leucine. The results rule out the existence, in pea leaves, of any protein similar to either animal metallothioneins or to any of the low-molecularweight metal-binding proteins or peptides described in other plants and reported to be involved in metal tolerance. In the mechanism of copper tolerance at the leaf level, fractions III-1 (M_r 3700), III-2 (M_r 66000), and IV (M_r 2000) appear to have a role, fraction IV being specifically induced in the tolerant cultivar by Cu(II). Fractions III-1 and III-2 could participate in a different mechanism, adaptive in character, involving an enhanced capacity to bind copper in Cu-tolerant plants.Abbreviations DEAE diethylaminoethyl - M_r relative molecular mass - SDS sodium dodecyl sulfate - PAGE polyacrylamide-gel electrophoresis J.M. Palma was the recipient of a research fellowship from the Caja General de Ahorros y Monte de Piedad de Granada and CSIC. We are grateful to Dr. J. Moreno-Carretero, R + D Department, UNIASA, Granada, for conducting the amino-acid analyses. This work was supported by grant 603/275 from CAICYT-CSIC (Spain).     

Regulation of l-Glutamate Biosynthesis by Copper Ions

A specific regulatory effect of copper ions on the microbiological synthesis of l-glutamate from acetate was found. The minimal concentration of copper ions necessary for the maximal production of l-glutamate was about 0.025 µg/ml at which the yield of l-glutamate was four times greater than that in the absence of copper ions. This effect of copper was demonstrated only when acetate was the substrate; it was not observed when the substrate was glucose ethanol, lactate or n-paraffin.

The physiological features of the l-glutamate production from acetate were examined in the presence or absence of copper ions. The most striking features of the culture without added copper ions were the increase in Q_O2 and NADH oxidase and the marked reduction of succinate oxidase accompanied with the reduction of l-glutamate formation. In addition, the regulation of l-glutamate synthesis by copper ions proved to have no relation to the wellknown regulatory factor, cell permeability. These facts suggest that the l-glutamate biosynthesis from acetate is regulated through unknown factors related to the respiratory activities.     

Probable involvement of sulfhydryl groups and a metal as essential components of the cellulase of Clostridium thermocellum

The crude extracellular cellulase from Clostridium thermocellum was oxidatively inactivated by air and inhibited by sulfhydryl reagents. Activity-loss was prevented and reversed by the addition of a high concentration (10 mM) dithiothreitol (DDT) at zero time and up to 24 h respectively. In the presence of a low concentration (0.4 mM) of DTT, the enzyme was more rapidly inactivated than in air alone. This was probably due to autoxidation of the low DTT concentration to H₂O₂ as shown by its prevention by a high DTT concentration, exclusion of air, or catalase; and by the oxidative inactivation of the enzyme by H₂O₂. The inactivation by H₂O₂ could be prevented by a high concentration of DTT but not by air exclusion. EDTA protected the enzyme from inactivation in air by a low concentration of DTT or by H₂O₂. This is presumably due to the role of metals in oxidation of SH groups. Furthermore, copper (5 M) also caused inactivation and this was prevented by the presence of a high DTT concentration. Even in the protective atmosphere of a high DTT concentration, cellulase was inactivated by certain apolar chelating agents such as o-phenanthroline and -¹-dipyridyl, such inactivation being preventable by the prior incubation of the chelator with a mixture of Fe²⁺ and Fe³⁺. These data suggest that the clostridial cellulase, unlike the enzyme from aerobic fungi, contains essential sulfhydryl groups and is stimulated by iron. The endo--glucanase component of the cellulase complex was not susceptible to oxidative inactivation.Abbreviations DTT dithiothreitol - CMC carboxymethylcellulose - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - NEM N-ethylmaleimide - p-CMB p-chloromercuribenzoic acid     

The influence of copper on alkaline protease stability toward autolysis and thermal inactivation

Alkaline proteases are one of the most important group of enzymes that are indispensable in a number of different industrial sectors. In this work, the effect of copper ions (Cu²⁺) was investigated for improving the thermostability and hydrolytic performance of Bacillus clausii GMBE 42 alkaline protease at different temperatures (45–65°C). Maximal residual activity was observed in the presence of 5 mM CuCl₂. The enzyme was thermoinactivated according to first‐order kinetics. A stabilization effect caused by copper ions was the result of a decrease in both autolysis and thermoinactivation rates. Thermodynamic analysis of the thermoinactivation process showed that E_a,i, ΔG_i, and ΔH_i values of the enzyme were higher in the presence of copper ions, but there was no measurable change in ΔS_i values. These results show the thermostabilizing potential of copper ions on the enzyme. Lower K_m values and higher k_cat and k_cat/K_m values were obtained in the presence of copper ions, which is an indication of the nonessential activation of the enzyme by copper ions. Thermodynamic analysis of casein hydrolysis showed that in presence of copper ions E_a, ΔG^≠, ΔH^≠, , and values of enzyme were lower, but there was no change in ΔS^≠ values. This is so far the first study that investigates the effect of cations on the basic catalytic and thermodynamic properties of an alkaline serine protease, which may be used to remove protein wastes from various industries such as food and leather processing.     

Copper(II) nitrato and chloro complexes with sterically hindered tridentate ligands: Influence of ligand framework and charge on their structure and physicochemical properties

Copper(II) coordination complexes of the neutral ligand, tris(3-tert-butyl-5-methyl-1-pyrazolyl)methane (L2′), i.e. the copper(II) nitrato complexes [Cu(L2′)(NO₃)][Cu(NO₃)₄]_1/2 (1) and [Cu(L2′)(NO₃)](ClO₄) (2) and the copper(II) chloro complex [Cu(L2′)(Cl)](ClO₄) (3), and its anionic borate analogue, hydrotris(3-tert-butyl-5-methyl-1-pyrazolyl)borate (L2⁻), i.e. the copper(II) nitrato complex [Cu(L2)(NO₃)] (4) and the copper(II) chloro complex [Cu(L2)(Cl)] (5), were synthesized in order to investigate the influence of ligand framework and charge on their structure and physicochemical properties. While X-ray crystallography did not show any definitive trends in terms of copper(II) atom geometry in four-coordinate copper(II) chloro complexes 3 and 5, different structural trends were observed in five-coordinate copper(II) nitrato complexes 1, 2, and 4. These complexes were also characterized by spectroscopic techniques, namely, UV-Vis, ESR, IR/far-IR, and X-ray absorption spectroscopy.     

Oxidative stress by menadione affects cellular copper and iron homeostasis

Menadione produces DNA strand breaks (DNA sb) in cultured Chinese hamster fibroblasts which are, to a great extent, mediated by OH radical. A reasonable hypothesis is that H₂O₂, a product of menadione metabolism, reacts with nuclear iron and produces OH radicalin situ. Consistent with that, 1,10-phenanthroline (PHEN) prevents menadione-induced DNA sb at low (<200 M) concentrations of the chelator. However, at higher PHEN concentrations, the effect is reversed and an enhancement of DNA sb is observed. The PHEN-induced enhancement of DNA sb becomes more evident at high (>60 M) menadione concentrations and is strongly prevented by neocuproine (NEO), an efficient copper chelator. However, NEO offers only a slight protection against DNA sb caused by menadione alone. The results are consistent with the following events: (i) the products of menadione metabolism causes copper ion release from some cellular compartment; (ii) in the presence of PHEN, a Cu(PHEN)₂ complex is formed; (iii) the Cu(PHEN)₂ complex is known to be very clastogenic, inducing DNA damage in a reducing environment.Evidence is also presented that menadione metabolism causes an increase in intracellular chelatable iron: in the presence of a constant 2,2-dipyridyl concentration, the DNA sb produced by increasing concentrations of menadione become progressively less susceptible to inhibition by the chelator.Therefore the DNA damage originated from menadione metabolism seems to be caused by two conjugated and synergistic events, viz., the production of reactive oxygen species and the release of copper and iron from a cellular storage site into a free form pool, capable of catalyzing DNA damaging reactions.Abbreviations sb strand breaks - PHEN 1,10-phenanthroline - PBS phosphate-buffered saline (8.1 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 1.68 mM KCl, 137 mM NaCl, pH 7.0) - HP Hydrogen Peroxide - Md Menadione - NEO Neocuproine     

Plant uptake and leaching of copper during EDTA-enhanced phytoremediation of repacked and undisturbed soil

EDTA-enhanced phytoremediation of copper contaminated soil was evaluated. Up to 740 g g^–1 of Na₂H₂ EDTA in solution was added to repacked soil columns, and intact cores of a sandy loam of volcanic origin, that was growing Agrostis tenuis. The soil contained up to 400 g g^–1 of copper due to a history of fungicide spraying. EDTA application increased the herbage copper concentration of the grass growing in repacked soil from 30 to 300 g g^–1, but the same application to an intact core only brought about an increase from 10 to 60 g g^–1. More copper accumulated in the herbage when the EDTA was applied in numerous small doses than in just one or two larger amounts. Calculation of the concentration of copper in the water taken up by the grass revealed this to be two orders of magnitude lower than that in the soil solution. As a result of the EDTA applications, about 100 times more copper was leached than was taken up by the herbage. This means that a strategy for managing leaching losses needs to be part of any plan for EDTA-enhanced phytoremediation.     

Reusability of immobilised Saccharomyces cerevisiae with successive copper adsorption-desorption cycles

Summary Immobilised Saccharomyces cerevisiae in batch reactors effectively removed copper from solution with a binding equilibrium of 70 % being attained within 20 minutes of contact. Maximum uptake was between pH 3 to 5 (V_max = 24.1 mol/g) and was substantially reduced at pH 2. Bound copper was readily recovered by addition of 1.0 M HCl ( % v/v). In adsorption-desorption studies metal removal and recovery was high, and uptake was increased with repeated use. Electron microscopy confirmed that no morphological changes occur to the cells during repeated adsorption-desorption.     

Copper supply and the leaf emergence rate of spring wheat

Copper deficiency can delay flowering and plant maturity. However, the effect of copper deficiency on the rate of leaf emergence has not been quantified. We tested the hypothesis that low copper supply decreases the rate of leaf emergence of wheat (Triticum aestivum L. cv Gamenya). Copper foliar sprays are commonly applied to wheat. We examined the response of the rate of leaf emergence to a foliar application of copper sulphate.Wheat was grown in root cooling tanks (20°C) in the glasshouse. Soil copper treatments were applied as solutions of CuSO₄.5H₂O at three rates: Cu₀=no added Cu, Cu₄₀₀=400 g Cu per 3 kg pot, and Cu₁₆₀₀=1600 g Cu per pot. An additional treatment of a foliar spray of CuSO₄.5H₂O (0.4 mg Cu per plant) was applied to Cu₀ and Cu₄₀₀ plants 45 days after sowing (5.5 leaves on the main stem). Leaves on the main stem were counted and the rate of leaf emergence was estimated from the regression of number of emerged leaves against thermal time (base 0°C). The phyllochron was calculated as 1/rate of emergence.Leaves on Cu₀ and Cu₄₀₀ plants took longer to emerge than on Cu₁₆₀₀ plants, with the phyllochron of Cu₁₆₀₀ plants being 130 compared to 137 for the Cu₄₀₀ plants and 158 for the Cu₀ plants. The foliar application of CuSO₄ at the 5–6 leaf stage resulted in a decrease in the phyllochron of the Cu₀ plants to 127, but no change in that of the Cu₄₀₀ plants.     

Technical communication: Determination of cuprous ions in bacterial leachates and for environmental monitoring

A sensitive and precise spectrophotometric method has been developed for the determination of copper(I) in bacterial leach liquors produced by the action of Thiobacillus ferrooxidans and T. thiooxidans on copper ores. In this method bicinchoninic acid (BCA) has been used as the chromogenic reagent which produces a stable purple complex with Cu(I) which was found to obey Beer's Law and with _max at 560 nm. The coloured complex has a molar extinction coefficient () value of 6.6 × 10³ l mol^–1 cm^–1; specific absorptivity () value of 0.104 ml^–1 g cm^–1 and the Sandell sensitivity (S) value was 0.0096 g cm². Optimal conditions for development of coloration/sensitivity were determined. Interferences due to cations and anions were investigated and various masking agents for alleviating their inhibition were studied. The method has been found very useful in determining ratios of Cu(I) to Cu(II) in bacterial leach liquors and should play a significant role in determining the reaction mechanisms of biological leaching and for environmental monitoring.