Electrooptical Properties of Cells of the Soil Nitrogen-Fixing Bacterium Azospirillum brasilense: Effects of Copper Ions

The effects of copper ions on the uptake of some essential metals in the biomass and the electrooptical properties of cell suspensions of the nitrogen-fixing soil bacterium Azospirillum brasilensesp. 245 were studied. Copper cations were shown to be effectively taken up by the cell biomass from the culture medium. The addition of copper ions increased the rate of uptake of some other metals present in the culture medium. This was accompanied by changes in the electrooptical characteristics of cell suspension as measured within the orienting electric field frequency range of 10 to 10000 kHz. The effects observed during short-term incubation of A. brasilensein the presence of copper cations were less significant than during long-term incubation. These results can be used for the rapid screening of microbial cultures for the enhanced efficiency of sorption and the uptake of metals.     

Electrophysical characteristics of Azospirillum brasilense Sp245 during interaction with antibodies to various cell surface epitopes

This work was undertaken to examine the electrooptical characteristics of cells of Azospirillum brasilense Sp245 during their interaction with antibodies developed to various cell surface epitopes. We used the dependences of the cell suspension optical density changes induced by electroorientation on the orienting field frequency (740, 1000, 1450, 2000, and 2800kHz). Cell interactions with homologous strain-specific antibodies to the A. brasilense Sp245 O antigen and with homologous antibodies to whole bacterial cells brought about considerable changes in the electrooptical properties of the bacterial suspension. When genus-specific antibodies to the flagellin of the Azospirillum sheathed flagellum and antibodies to the serologically distinct O antigen of A. brasilense Sp7 were included in the A. brasilense Sp245 suspension, the changes caused in the electrooptical signal were slight and had values close to those for the above changes. These findings agree well with the immunochemical characteristics of the Azospirillum O antigens and with the data on the topographical distribution of the Azospirillum major cell surface antigens. The obtained results can serve as a basis for the development of a rapid test for the intraspecies detection of microorganisms.     

Albumin has no role in the uptake of copper by human fibroblasts

The mechanism of copper uptake by cells has been the subject of controversy for some time. This paper examines the possibility of a role for albumin in the uptake of copper by fibroblasts. Although the cells could accumulate copper from a copper-albumin complex, there was no evidence for either copper-albumin or albumin receptors on the cell surface. The possibility of a surface exchange mechanism for copper was examined. While copper uptake showed saturation with increasing concentrations of labelled copper-albumin, adding unlabelled copper to the incubation medium did not inhibit uptake. Adding albumin or histidine to the copper-albumin complex resulted in an inhibition of copper uptake. The results can only be explained by the cell taking up free copper from the incubation medium, with the albumin then releasing its copper to maintain the equilibrium between free and bound metal. Since, in vivo there is essentially no free copper in serum, it is concluded that albumin is most unlikely to play a role in the uptake of copper by fibroblasts.     

Biosorption of lead and copper from aqueous solutions by pre-treated crab and arca shell biomass

Sorption potential of pretreated crab and arca shell biomass for lead and copper from aqueous media was explored. The effects of pH, initial concentration, biosorbent dosage and contact time were studied in batch experiments. Effects of common ions like sodium, potassium, calcium and magnesium on the sorption capacity of pretreated crab and arca biomasses were also studied. At equilibrium, the maximum uptake by crab shell biomass was 19.83+/-0.29 and 38.62+/-1.27 mg/g for lead and copper, respectively. In case of arca shell biomass the maximum uptake capacity was 18.33+/-0.44 mg/g and 17.64+/-0.31 mg/g for lead and copper, respectively. Combined effect of all the common ions up to 50 microg/ml concentration was negligible for both the metals using both biomasses. Sorption isotherms were studied to explain the removal mechanism of both elements by fitting isotherms data into Lagergren, Freundlich and Langmuir equations.     

Insulin action in isolated fat cells. I. Effects of divalent cations on the stimulation by insulin of glucose uptake.

The effects of divalent cations, in particular Ca2+ and Mg2+, on glucose uptake by rat isolated fat cells in the presence and absence of insulin have been studied. EDTA (disodium salt) was used to deplete the bovine serum albumin present in the incubation medium of endogenous divalent cations prior to incubation with the cells, but was not present in the incubation medium during the incubation of the cells. The removal of Ca2+ and Mg2+ from the incubation medium did not affect the basal glucose uptake, but abolished the ability of insulin to stimulate glucose uptake by the cells. Addition of 25 microM MgCl2 or CaCl2 to the incubation medium restored a significant insulin stimulation, and this stimulation was maximal when 0.1 mM MgCl2 or CaCl2 had been added. SrCl2 and BaCl2 were also effective in restoring the insulin stimulation, but did not substitute fully for Ca2+ and Mg2+ in the incubation medium. Possible explanation for these observations are discussed.     

Strain-specific salt tolerance and osmoregulatory mechanisms in Azospirillum brasilense

Salinity stress inhibits the growth and nitrogen fixation ability of the plant growth-promoting rhizobacterium Azospirillum brasilense. Five strains of A. brasilense were isolated from the rhizosphere of Indian cereals and grasses and identified on the basis of their phenotypic features and 16S rRNA gene sequence. The five Indian isolates and two standard strains of A. brasilense, Sp7 and Cd, showed notable differences in growth, acetylene-reducing activity under salt stress, and ability to take up and use glycine betaine for the restoration of growth and acetylene-reducing activity under salt stress. Salt stress also enhanced the production of exopolysaccharides and cell aggregates, the extent of which varied in different strains of A. brasilense at different carbon to nitrogen ratios in the culture medium. It can be concluded that the production of exopolysaccharides and cell aggregates is a more consistent physiological response of A. brasilense to salt stress than is the uptake and osmoprotection by glycine betaine.     

Effect of the Copper Content of Distillery Spent Wash on its Utilization for Single-cell Protein Production by Geotrichum candidum, Hansenula anomala and Candida kruzei

Geotrichum candidum, Hansenula anomala and Candida kruzei were grown in batch and continuous culture on samples of the spent wash from a malt whiskey distillery containing levels of dissolved copper up to 22 mg/l. In batch culture some inhibition of growth at the higher copper concentrations and as much as a sixfold accumulation of copper in the biomass were observed. In Geotrichum candidum this accumulation occurred in three distinct phases during the period of batch growth. Although biomass yields in mixed continuous culture declined with increasing dilution rate there was a corresponding increase in cell copper concentration such that total copper removal from the medium remained approximately constant.     

Cunninghamella echinulata a new biosorbent of metal ions from polluted water in Egypt

Thirty-two fungal species were isolated from a polluted watercourse near the Talkha fertilizer plant, Mansoura Province, Egypt. Aspergillus niger, A. flavus, Cunninghamella echinulata and Trichoderma koningii were isolated frequently. On the basis of its frequency, Cunninghamella echinulata was chosen for biosorption studies. Free and immobilized biomass of C. echinulata sequestered ions in this decreasing sequence is: Pb >Cu >Zn. The effects of biomass concentration, pH and time of contact were investigated. The level of ion uptake rose with increasing biomass. The maximum uptake for lead (45 mg/g), copper (20 mg/g) and zinc (18.8 mg/g) occurred at 200 mg/L biomass. The uptake rose with increasing pH up to 4 in the case of Pb and 5 in the case of Cu and Zn. Maximum uptake for all metals was achieved after 15 min. Ion uptake followed the Langmuir adsorption model, permitting the calculation of maximum uptake and affinity coefficients. Treatment of C. echinulata biomass with NaOH improved biosorbent capacity, as did immobilization with alginate. Immobilized biomass could be regenerated readily by treatment with dilute HCl. The biomass-alginate complex efficiently removed Pb, Zn and Cu from polluted water samples. Therefore,Cunninghamella echinulata could be employed either in free or immobilized form as a biosorbent of metal ions in waste water.     

Changes in electrophysical characteristics of Azospirillum brasilense Sp7 cells during their interaction with polyclonal antibodies

Electrooptical characteristics of Azospirillum brasilense Sp7 cells during their specific interaction with polyclonal rabbit antibodies were studied. Dependence of optical density of cell suspension during electroorientation of cells from frequency of orienting field in interval 10, 100, 250, and 500 kHz was evaluated. Itwas shown that electrooptical (EO) characteristics of bacterial suspensions change during interaction of A. brasilense cells with antibodies, and maximal changes occur when frequency of oriented field amounts 100-250 kHz. During interaction of A. brasilense Sp7 with strain-specific polyclonal antibodies in the presence of Escherichia coli K-12 and Pseudomonas putida C-11 decrease of amplitude of analytic signal was observed but detection of A. brasilense Sp7 cells was possible. Possibility of detection of microorganisms by EO analysis during their interaction with antibodies was shown.     

Biosorption of lead(II), cadmium(II), copper(II) and nickel(II) by anaerobic granular biomass

Biosorption is potentially an attractive technology for treatment of wastewater for retaining heavy metals from dilute solutions. This study investigated the feasibility of anaerobic granules as a novel type of biosorbent, for lead, copper, cadmium, and nickel removal from aqueous solutions. Anaerobic sludge supplied from a wastewater treatment plant in the province of Quebec was used. Anaerobic granules are microbial aggregates with a strong, compact and porous structure and excellent settling ability. After treatment of the biomass with Ca ions, the cation exchange capacity of the biomass was approximately 111 meq/100 g of biomass dry weight which is comparable to the metal binding capacities of commercial ion exchange resins. This work investigated the equilibrium, batch dynamics for the biosorption process. Binding capacity experiments using viable biomass revealed a higher value than those for nonviable biomass. Binding capacity experiments using non-viable biomass treated with Ca revealed a high value of metals uptake. The solution initial pH value affected metal sorption. Over the pH range of 4.0-5.5, pH-related effects were not significant. Meanwhile, at lower pH values the uptake capacity decreased. Time dependency experiments for the metal ions uptake showed that adsorption equilibrium was reached almost 30 min after metal addition. It was found that the q(max) for Pb2+, Cd2+, Cu2+, and Ni2+ ions, were 255, 60, 55, and 26 mg/g respectively (1.23, 0.53, 0.87, and 0.44 mmol/g respectively). The data pertaining to the sorption dependence upon metal ion concentration could be fitted to a Langmiur isotherm model. Based on the results, the anaerobic granules treated with Ca appear to be a promising biosorbent for removal of heavy metals from wastewater due to its optimal uptake of heavy metals, its particulate shape, compact porous structure, excellent settling ability, and its high mechanical strength.     

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 Cu^{2 +} . 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.     

The use of flocculating brewer's yeast for Cr(III) and Pb(II) removal from residual wastewaters

The use of inexpensive biosorbents to sequester heavy metals from aqueous solutions, is one of the most promising technologies being developed to remove these toxic contaminants from wastewaters. Considering this challenge, the viability of Cr(III) and Pb(II) removal from aqueous solutions using a flocculating brewer's yeast residual biomass from a Portuguese brewing industry was studied. The influence of physicochemical factors such as medium pH, biomass concentration and the presence of a co-ion was characterised. Metal uptake kinetics and equilibrium were also analysed, considering different incubation temperatures. For both metals, uptake increased with medium pH, being maximal at 5.0. Optimal biomass concentration for the biosorption process was determined to be 4.5?g dry weight/l. In chromium and lead mixture solutions, competition for yeast binding sites was observed between the two metals, this competition being pH dependent. Yeast biomass showed higher selectivity and uptake capacity to lead. Chromium uptake kinetic was characterised as having a rapid initial step, followed by a slower one. Langmuir model describes well chromium uptake equilibrium. Lead uptake kinetics suggested the presence of mechanisms other than biosorption, possibly including its precipitation.     

In vitro effects of islet-activating protein on cultured rat pancreatic islets. Enhancement of insulin secretion, adenosine 3':5'-monophosphate accumulation and 45Ca flux

Pancreatic islets were maintained in culture with or without islet-activating protein (IAP), which is a new protein purified from culture medium of Bordetella pertussis. These cultured islets (IAP-treated or control) were then incubated for 30 min in IAP-free medium with various insulin secretagogues. During incubation, much more insulin was released from IAP-treated islets than control islets in response to glucose, arginine, glucagon, and sulfonylurea. IAP was effective in this regard when added to cultures at concentrations higher than 0.01 ng/ml; the effect was dependent on concentration up to 100 ng/ml. Enhanced insulin secretion was associated with accumulation of cyclic AMP when breakdown of the nucleotide was prevented by a methylxanthine. Epinephrine caused marked inhibitions, via alpha-adrenergic receptors, of glucose-induced insulin release, cyclic AMP accumulation and 45Ca uptake in control islets but did not in IAP-treated islets during incubation. None of these effects of IAP pretreatment were observed unless the medium for incubation was supplemented with Ca ions. 45Ca ion flux through the islet cell membrane was accelerated by the IAP treatment; conceivably, IAP was effective in causing sustained activation of native calcium ionophores on the membrane, which would be responsible for the enhanced insulin and cyclic AMP responses characteristic of IAP-treated islets.     

Generation of oxidant response to copper and iron nanoparticles and salts: Stimulation by ascorbate

The present work describes a two-stage approach to analyzing combustion-generated samples for their potential to produce oxidant stress. This approach is illustrated with the two commonly encountered transition metals, copper and iron. First, their abilities to generate hydroxyl radical were measured in a cell-free, phosphate-buffered saline solution containing ascorbate and/or citrate. Second, their abilities to induce heme oxygenase-1 in cultured human epidermal keratinocytes were assessed in cell culture. Combustion-generated copper oxide nanoparticles were active in both assays and were found to be soluble in culture medium. Depletion of glutathione in the cells or loading the cells with ascorbate greatly increased heme oxygenase-1 induction in the presence of copper. By contrast, iron oxide nanoparticles were active in the phosphate-buffered saline but not in cell culture, and they aggregated in culture medium. Soluble salts of copper and iron exhibited the same contrast in activities as the respective combustion-generated particles. The results suggest that the capability of combustion-generated environmental samples to produce oxidant stress can be screened effectively in a two step process, first in phosphate-buffered saline with ascorbate and subsequently in epithelial cell culture for those exhibiting activity initially. The results also point to an unanticipated interaction in cells of oxidant stress-generating metals with an antioxidant (ascorbate) that is usually missing in culture medium formulations. Thus, ascorbate supplementation of cultured human cells is likely to improve their ability to model the in vivo effects of particulate matter containing copper and other redox-active metals.     

Effects of heavy metals on plant-associated rhizobacteria: comparison of endophytic and non-endophytic strains of Azospirillum brasilense.

The plant-associated nitrogen-fixing rhizobacterium Azospirillum brasilense attracts world-wide attention owing to its plant growth-promoting activities. Among hundreds of its strains known up to date, wild-type strain Sp245 has been proved to be capable of colonising both the plant-root interior and exterior (i.e. a facultative endophyte), whereas others are non-endophytes colonising the root surface only. Thus, the different ecological niches occupied by these strains in the rhizosphere suggest that their responses to environmental conditions might differ as well. In this study, responses of A. brasilense strains Sp245 and Sp7 to several heavy metals (Co2+, Cu2+, Zn2+), present in the medium at tolerable concentrations (up to 0.2 mmol/l) and taken up by the bacteria, were compared. Fourier transform infrared (FTIR) spectroscopy was used for controlling the compositional features of whole cells. The results obtained show that in strain Sp7 (non-endophyte) the heavy metals induced an enhanced accumulation of polyester compounds (poly-3-hydroxybutyrate; PHB). In contrast, the response of the endophytic strain Sp245 to heavy metal uptake was found to be much less pronounced. These dissimilarities in their behaviour may be caused by different adaptation abilities of these strains to stress conditions owing to their different ecological status. It was also found that adding 0.2 mmol/l Cu2+ or Cd2+ in the culture medium resulted in noticeably reducing the levels of indole-3-acetic acid (IAA, auxin) produced by both the strains of the bacterium. This can directly affect the efficiency of associative plant-bacterial symbioses involving A. brasilense in heavy-metal-contaminated soil.     

The effect of copper on the growth of wood-rotting fungi and a blue-stain fungus

The effect of copper (II) ions on the growth of three brown-rot fungi, six white-rot fungi and one blue-stain fungus in solid medium was evaluated. The fungi were grown in malt extract agar with different concentrations of copper added, and the radial growth rate was determined. At the end of the incubation period, the mycelial biomass and the media pH were determined. The white-rot and blue-stain fungus grew up to 3 mM and 6 mM copper, respectively and the brown-rot fungi were the only ones that grew up to 10 mM, with higher growth rates than those shown by the other fungi. In general, the brown-rot fungi produced greater acidification in the culture media than the white-rot fungi and blue-stain fungus, and the acidification increased when the amount of copper was increased. The biomass production for the different species, in the absence or presence of copper, was not related to the radial growth rate, and the fungal species that produced the greatest biomass amounts did not correspond to those that presented the highest growth rates. The brown-rot fungi Wolfiporia cocos and Laetiporus sulfureus and blue-stain fungus Ophiostoma sp. demonstrated greater tolerance to high copper concentrations in solid medium than the white-rot fungi, determined as radial growth rate. On the other hand, the highest biomass producers in solid medium with copper added were the white-rot fungi Ganoderma australe and Trametes versicolor and the brown-rot fungus Gloeophyllum trabeum.     

Calcium and A23187-induced cytolysis of mouse thymocytes

The cytotoxic effects of ionophore A₂₃₁₈₇ were studied in parallel with its action on calcium uptake in isolated mouse thymocytes. Under conditions where the cells were preincubated in a calcium-containing medium prior to ionophore treatment a close relationship could be observed between the extent of cell lysis and the stimulation of calcium uptake in the presence of A₂₃₁₈₇. In addition, increasing concentrations of calcium ions in the incubation medium lead to a pronounced decrease of cell viability and to a stimulation of calcium uptake suggesting that calcium is critical for cell survival.     

Effects of Metals on Streptomyces coelicolor Growth and Actinorhodin Production

Actinorhodin production by Streptomyces coelicolor was used as a model system to study the effects of metals on growth and polyketide synthesis in a streptomycete. Numerous metals were tested in cultures grown in liquid media. Mercury and cadmium were highly toxic, and copper, nickel, and lead were less so, but all tended to inhibit both growth and antibiotic synthesis to a similar extent. Unexpectedly, manganese, cobalt, zinc, and, to a lesser extent, chromium caused complex effects that in general resulted in some enhancement of growth yield but a reduction in antibiotic titers. These complex effects meant that cobalt, manganese, and zinc had lower 50% inhibitory concentrations for antibiotic yields compared with those for biomass. The physiologically active divalent cations calcium and magnesium were also tested. Calcium at high concentrations was particularly effective in reducing antibiotic titers and enhancing growth yields. By adding calcium at different phases of growth, it could be demonstrated that it was most effective in reducing the antibiotic yield when added during the early growth phase. Addition during the antibiotic-producing phase resulted in little reduction of final actinorhodin titers.     

Intracellular Copper Accumulation Enhances the Growth of Kineococcus radiotolerans during Chronic Irradiation

The actinobacterium Kineococcus radiotolerans is highly resistant to ionizing radiation, desiccation, and oxidative stress, though the underlying biochemical mechanisms are unknown. The purpose of this study was to explore a possible linkage between the uptake of transition metals and extreme resistance to ionizing radiation and oxidative stress. The effects of six different divalent cationic metals on growth were examined in the absence of ionizing radiation. None of the metals tested were stimulatory, though cobalt was inhibitory to growth. In contrast, copper supplementation dramatically increased colony formation during chronic irradiation. K. radiotolerans exhibited specific uptake and intracellular accumulation of copper, compared to only a weak response to both iron and manganese supplementation. Copper accumulation sensitized cells to hydrogen peroxide. Acute-irradiation-induced DNA damage levels were similar in the copper-loaded culture and the age-synchronized no-copper control culture, though low-molecular-weight DNA was more persistent during postirradiation recovery in the Cu-loaded culture. Still, the estimated times for genome restoration differed by only 2 h between treatments. While we cannot discount the possibility that copper fulfills an unexpectedly important biochemical role in a low-radioactivity environment, K. radiotolerans has a high capacity for intracellular copper sequestration and presumably efficiently coordinated oxidative stress defenses and detoxification systems, which confers cross-protection from the damaging effects of ionizing radiation.     

Cell Wall Composition of Neurospora crassa Under Conditions of Copper Toxicity

The mycelia of Neurospora crassa grown in the presence of high concentrations of copper were blue in color, but only on a medium containing inorganic nitrate and phosphate as the nitrogen and phosphate sources, respectively. The cell wall isolate of the blue mycelia contained large amounts (12%) of copper and higher amounts of chitosan, phosphate, and amino groups, with a 42% decrease in the chitin content. Although all the glucosamine of the cell wall of control cultures could be released within 6 h of hydrolysis with acid, that of the blue mycelium required prolonged hydrolysis for 24 h. On removal of copper, the cell wall of the blue mycelium could quantitatively bind again to copper as well as to zinc. Although zinc binding was fivefold greater, copper alone was preferentially bound from a mixture of the two metal ions. Supplementation of iron along with copper in the culture medium resulted in the disappearance of the blue color of the mycelium and restoration of normal growth and composition of the cell wall, probably by limiting the uptake of copper from the medium. The possibility of the cell wall being a specific site of lesion in copper toxicity in the mold is discussed.