Zn2+ biosorption by Oscillatoria anguistissima

Oscillatoria anguistissima showed a very high capacity for Zn²⁺ biosorption (641 mg g⁻¹ dry biomass at a residual concentration of 129·2 ppm) from solution and was comparable to the commmercial ion-exchange resin IRA-400C. Zn²⁺ biosorption was rapid, pH dependent and temperature independent phenomenon. Zn²⁺ adsorption followed both Langmuir and Freundlich models. The specific uptake (mg g⁻¹ dry biomass) of metal decreased with increase in biomass concentration. Pretreatment of biomass did not significantly affect the biosorption capacity of O. anguistissima. The biosorption of zinc by O. anguistissima was an ion-exchange phenomenon as a large concentration of magnesium ions were released during zinc adsorption. The zinc bound to the biomass could be effectively stripped using EDTA (10 mM) and the biomass was effectively used for multiple sorption–desorption cycles with in-between charging of the biomass with tap water washings. The native biomass could also efficiently remove zinc from effluents obtained from Indian mining industries.     

Oscillatoria anguistissima: A Promising Cu2+ Biosorbent

Oscillatoria anguistissima rapidly adsorbs Cu²⁺ from aqueous solution. The adsorption of Cu²⁺ followed Freundlich Isotherm, and the amount of Cu²⁺ removed from solution increased with increasing Cu²⁺ concentration. The adsorption is pH dependent, and maximum Cu²⁺ removal occurs at pH 5. Of the various pretreatments, HCl treatment of the biomass increased the capacity for Cu²⁺ removal. Presence of Mg²⁺ and Ca²⁺ resulted in decline in the Cu²⁺ adsorption capacity of Oscillatoria cells. This species could also effectively remove Cu²⁺ from mine water containing 68.4 μg/ml of Cu²⁺ at pH 3.45. Received: 23 December 1996 / Accepted: 20 February 1997     

Biosorption of Co<Superscript>2+</Superscript> ions by lichen <Emphasis Type="Italic">Hypogymnia physodes</Emphasis> from aqueous solutions

Cobalt is one of the possible contaminants originating from radioactive wastes or from metal mines and refineries. This paper describes sorption of cobalt by the foliose lichen Hypogymnia physodes from CoCl₂ solutions spiked with ⁶⁰Co²⁺ in laboratory experiments. Maximum uptake was reached within 1 hour; the biosorption after 24 hours is not pH-dependent within the range of pH 4–7, negligible at pH 2 and is not dependent on metabolic activity. The process can be described by the Freundlich adsorption isotherm with ln k = 2.77, 1/n = 0.22 and R ² = 0.94. Bivalent metal ions showed a concentration-dependent competitive effect on cobalt biosorption, decreasing in the order: Cu > Ni > Ca > Mg. Monovalent ions, such as K⁺ and Na⁺, showed only very weak competitive effect. Up to 98% of Co taken up by lichen can be removed by washing with 0.1 M NiCl₂ at 20°C. This means that only a small fraction of the cobalt is localized intracellularly. These results can be used for elucidating the behaviour of lichens as bioindicators of cobalt pollution in water systems, including the risk of cobalt leakage from lichen probes under the influence of rain, snow and atmospheric humidity.     

Effect of salinity and pH on cobalt biosorption by the estuarine microalgaChlorella saliva

Summary Accumulation of cobalt (⁶⁰Co) by the estuarine microalgaChlorella salina has been characterized. At cobalt concentrations ranging over 3.125–100 M, a significant amount of cobalt was bound within 1 min. This was metabolism-independent and unaffected by incubation in light or dark conditions. This initial rapid phase of biosorption was followed by a slower phase of uptake which was apparently active and inhibited by incubation in the dark, or by the uncoupler dinitrophenol and the respiratory and photosynthetic inhibitor potassium cyanide in the light. For cells suspended in 10 mM Taps pH 8, cobalt biosorption followed a Freundlich adsorption isotherm. However, in the presence of 0.5 M NaCl, biosorption deviated from the Freundlich model because of competition by Na⁺. Cobalt biosorption was decreased by increasing concentrations of Na⁺, decreasing pH and the presence of Cs⁺, Li⁺, Rb⁺, Zn²⁺. Mn²⁺ and Sr²⁺ (added as chlorides). This was a result of competition between Co²⁺ and the other cations, including H⁺, for available binding sites on the cell wall and was confirmed by increased desorption of cobalt by solutions of low pH or high salinity. Increasing cell density resulted in increased removal of cobalt from solution but decreased the specific amount of cobalt taken up by the cells.     

Biosorption of copper(II) from aqueous solutions by green alga <Emphasis Type="Italic">Cladophora fascicularis</Emphasis>

Biosorption is an effective means of removal of heavy metals from wastewater. In this work the biosorption behavior of Cladophora fascicularis was investigated as a function of pH, amount of biosorbent, initial Cu²⁺ concentration, temperature, and co-existing ions. Adsorption equilibria were well described by Langmuir isotherm models. The enthalpy change for the biosorption process was found to be 6.86 kJ mol⁻¹ by use of the Langmuir constant b. The biosorption process was found to be rapid in the first 30 min. The presence of co-existing cations such as Na⁺, K⁺, Mg²⁺, and Ca²⁺ and anions such as chloride, nitrate, sulfate, and acetate did not significantly affect uptake of Cu²⁺ whereas EDTA substantially affected adsorption of the metal. When experiments were performed with different desorbents the results indicated that EDTA was an efficient desorbent for the recovery of Cu²⁺ from biomass. IR spectral analysis suggested amido or hydroxy, C=O, and C–O could combine strongly with Cu²⁺.     

Reversible sporicidal action of cuprous ions

At 10 mM, Cu⁺ was highly protective against killing of spores of Bacillus megaterium ATCC 19213 by H₂O₂, while at higher concentrations, from 15–100 mM, killing was augmented. In contrast, Cu²⁺, Fe²⁺, Fe³⁺, Co²⁺ or Co³⁺ ions acted only protectively. Cu⁺ itself was sporicidal in the absence of H₂O₂ or ascorbate, and its sporicidal action did not depend on generation of highly reactive oxygen species. It appeared that killing involved either inhibition of germination or copper toxicity to germinated cells in that Cu⁺-inactivated spores did not germinate readily but chemical decoating of the cells prior to plating on a solid medium resulted in reversal of the sporicidal effect. Received 12 July 1996/ Accepted in revised form 03 November 1996     

Characterization of cadmium removal by Rhodotorula sp. Y11

Experiments were conducted studying the removal of Cd²⁺ from water via biosorption using Rhodotorula sp. Y11. The effects of temperature and initial pH of the solution on biosorption were studied. Caustic and heat treatments showed different influences on the biosorption capacity, and the highest metal uptake value (19.38 mg g⁻¹) was obtained by boiling treated yeast cells. The presence of competing cations, such as Ag⁺, Cu²⁺, and Mg²⁺, except Na⁺ ions, significantly interfered with the metal uptake. Results indicate that the Langmuir model gave a better fit to the experimental data than the Freundlich equation. The q ₁₀ value was 11.38 mg g⁻¹ for Cd²⁺ uptake by Y11. Chemical modifications of the biomass demonstrated that carboxyl and amide groups play an important role in Cd²⁺ biosorption.     

Protein kinase a dependent phosphorylation activates mg2+ efflux in the basolateral region of the liver

Isolated hepatocytes in physiological [Na⁺] ₀ tightly maintain [Mg²⁺] _i . Upon β-adrenergic stimulation or in the presence of permeable cAMP, hepatocytes release 5–10% (1–3 mM Mg²⁺) of their total Mg²⁺ content. However, isolated basolateral liver plasma membranes (bLPM), release Mg²⁺ in the presence of [Na⁺] _o even in the absence of catecholamine stimulation. The data indicate that a physiological brake for Mg²⁺ efflux is present in the hepatocyte and is removed upon cellular signaling. In contrast, this regulation “brake” is absent in purified bLPM thus rendering them fully active. The present study was carried out to reconstruct the missing regulatory component. Activation of Mg²⁺ extrusion in intact cells is consistent with cAMP dependent phosphorylation of the transporter or a regulatory protein. Treatment of bLPM with a non-specific phosphatase such as alkaline phosphatase (AP), decreased Mg²⁺ efflux by 70% compared to untreated bLPM. When AP-treated bLPM were loaded with protein kinase A (PKA), and stimulated with permeable cAMP, Mg²⁺ transport fully recovered. These data suggest that phosphorylation of the Na⁺/Mg²⁺ exchanger or a nearby protein activates the Mg²⁺ transport mechanism in hepatocytes.     

The effect of some micronutrients and heavy metals on phosphate absorption by maize root cortex segments

The effect of salts (nitrates, chlorides, and sulfates) of microelements, Cd²⁺, Ni²⁺, and Co²⁺ and the effect of boric acid and ammonium molybdate on phosphate uptake by maize root cortex segments were tested. Higher concentration (0.1 mM) of Cu²⁺ salts caused enhancement of phosphate efflux to the extent that efflux was higher than influx. Inhibitory action on phosphate uptake by maize root cortex segments was exerted by following salts: 0.01 mM Cu²⁺ salts (20–30% inhibition), 0.5 mM ZnSO₄ (9.7%), 0.5 and 0.05 mM ZnCl₂ (34.3% and 20.8%), 0.1 mM salts of Cd²⁺, Ni²⁺, Co²⁺ (35–78%). 1 mM FeS_O4 had significant stimulatory effect (92%) on phosphate uptake. Much weaker stimulatory effect was exerted by 1 mM FeCl₃ (14%), 0.05 mM ZnS_O4 (9.6%), 0.005 mM ZnCla and ZnS_O4 (8.4 and 18.5%) and 0.001 mM CdCl2 and CdS_O4 (20.8 and 12.4%). All other tested salts-salts of Mn²⁺ (0.1 and 0.01 mM), 0.01 and 0.001 mM salts of Co²⁺ and Ni²⁺, 0.001 mM salts of Cu²⁺, 0.001–10 mM boric acid, and 0.001–0.1 mM ammonium molybdate left phosphate uptake unaffected.     

Removing heavy metals from synthetic effluents using “kamikaze” Saccharomyces cerevisiae cells

One key step of the bioremediation processes designed to clean up heavy metal contaminated environments is growing resistant cells that accumulate the heavy metals to ensure better removal through a combination of biosorption and continuous metabolic uptake after physical adsorption. Saccharomyces cerevisiae cells can easily act as cation biosorbents, but isolation of mutants that are both hyperaccumulating and tolerant to heavy metals proved extremely difficult. Instead, mutants that are hypersensitive to heavy metals due to increased and continuous uptake from the environment were considered, aiming to use such mutants to reduce the heavy metal content of contaminated waters. In this study, the heavy metal hypersensitive yeast strain pmr1∆ was investigated for the ability to remove Mn²⁺, Cu²⁺, Co²⁺, or Cd²⁺ from synthetic effluents. Due to increased metal accumulation, the mutant strain was more efficient than the wild-type in removing Mn²⁺, Cu²⁺, or Co²⁺ from synthetic effluents containing 1–2 mM cations, with a selectivity $ {\text{Mn}}^{{{\text{2}} + }} > {\text{Co}}^{{{\text{2}} + }} ~ > {\text{Cu}}^{{{\text{2}} + }} $ {\text{Mn}}^{{{\text{2}} + }} > {\text{Co}}^{{{\text{2}} + }} ~ > {\text{Cu}}^{{{\text{2}} + }} and also in removing Mn²⁺ and Cd²⁺ from synthetic effluents containing 20–50 μM cations, with a selectivity Mn²⁺ > Cd²⁺.     

Purification and characterization of a novel β-galactosidase from Bacillus sp MTCC 3088

An extracellular β-galactosidase which catalyzed the production of galacto-oligosaccharide from lactose was harvested from the late stationary-phase of Bacillus sp MTCC 3088. The enzyme was purified 36.2-fold by ZnCl₂ precipitation, ion exchange, hydrophobic interaction and gel filtration chromatography with an overall recovery of 12.7%. The molecular mass of the purified enzyme was estimated to be about 484 kDa by gel filtration on a Sephadex G-200 packed column and the molecular masses of the subunits were estimated to be 115, 86.5, 72.5, 45.7 and 41.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the native enzyme, determined by polyacrylamide gel electrofocusing, was 6.2. The optimum pH and temperature were 8 and 60°C, respectively. The Michaelis–Menten constants determined with respect to o-NO₂-phenyl-β-D-galactopyranoside and lactose were 6.34 and 6.18 mM, respectively. The enzyme activity was strongly inhibited (68%) by galactose, the end product of lactose hydrolysis reaction. The β-galactosidase was specific for β-D anomeric linkages. Enzyme activity was significantly inhibited by metal ions (Hg²⁺, Cu²⁺ and Ag⁺) in the 1–2.5 mM range. Mg²⁺ was a good activator. Catalytic activity was not affected by the chelating agent EDTA. Journal of Industrial Microbiology & Biotechnology (2000) 24, 58–63. Received 09 February 1999/ Accepted in revised form 24 September 1999     

Zinc biosorption from aqueous solution by a halotolerant cyanobacterium Aphanothece halophytica

We have investigated the characteristics of zinc biosorption by Aphanothece halophytica. Zinc could be rapidly taken up from aqueous solution by the cells with an equilibrium being reached within 15 min of incubation with 100 mg L⁻¹ ZnCl₂. The adsorbed zinc was desorbed by treatment with 10 mM EDTA. The presence of glucose, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and N,N′-dicyclohexylcarbodiimide (DCCD) did not affect the uptake of zinc. The specific uptake of zinc increased at low cell concentration and decreased when cell concentration exceeded 0.2 g L⁻¹. The binding of zinc followed Langmuir isotherm kinetics with a maximum zinc binding capacity of 133 mg g⁻¹ and an apparent zinc binding constant of 28 mg L⁻¹. The presence of an equimolar concentration of Mn²⁺, Mg²⁺, Co²⁺, K⁺, or Na⁺ had no effect on zinc biosorption, whereas Ca²⁺, Hg²⁺, and Pb²⁺ showed an inhibitory effect. The biosorption of zinc was low at a pH range from 4 to 6, but increased progressively at pH 6.5 and 7. Received: 12 December 2001 / Accepted: 11 January 2002     

Purification of His6–organophosphate hydrolase using monolithic supermacroporous polyacrylamide cryogels developed for immobilized metal affinity chromatography

Organophosphate hydrolase containing hexahistidine tag at the N-terminus of recombinant protein (His₆-OPH) and expressed in Escherichia coli cells was purified using supermacroporous polyacrylamide-based monolith columns with immobilized metal affinity matrices [Me²⁺-iminodiacetic acid (IDA)–polyacrylamide cryogel (PAA) and Me²⁺-N,N,N’-tris (carboxymethyl) ethylendiamine (TED)–PAA]. Enzyme preparation with 50% purity was obtained by direct chromatography of nonclarified cell homogenate, whereas the combination of addition of 10 mM imidazole to buffers for cell sonication and sample loading, the use of precolumn with IDA–PAA matrix noncharged with metal ions, and the application of high flow rate provided the 99% purity of enzyme isolated directly from crude cell homogenate. Co²⁺-IDA–PAA provided the highest level of selectivity for His₆-OPH. Comparative analysis of purification using Co²⁺-IDA–PAA and Ni-nitrilotriacetic acid–agarose showed obvious advantages of the former in process time, specific activity of purified enzyme, and simplicity of adsorbent regeneration.     

Bioaccumulation and biosorption of Co2+ by Neurospora crassa

Summary Mycelial biomass of wild type and a Co²⁺-resistant N.crassa (cor) was used to remove Co²⁺ from aqueous media. Mycelia obtained from growth in nitrate N-medium (NaNO₃) was more effective than ammonium N-medium (NH₄NO₃), in removing Co²⁺. Co²⁺-resistant N.crassa cor was more efficient than wild type in removing Co²⁺ from medium containing higher concentrations (500 mg/L). Metal removal was linear up to 12 h at which 35–60% Co²⁺ is depleted from medium, reaching near saturation by 24 h (90% removal). Co²⁺ removal was as efficient even from pure solutions and sodium azide inhibited the process up to 60%. Cell walls prepared from nitrate N-medium grown mycelia bound 3–5 fold more Co²⁺ when compared to ammonium N-medium. The importance of bioaccumulation and biosorption in bioremediating toxic metal ions from effluents is discussed.     

Production,purification and characterization of a thermostable laccase from a tropical white-rot fungus

A thermostable laccase was isolated from a tropical white-rot fungus Polyporus sp. which produced as high as 69,738 units of laccase l⁻¹ in an optimized medium containing 20 g of malt extract l⁻¹, 2 g of yeast extract l⁻¹, 1.5 mM CuSO₄. The laccase was purified to electrophoretic purity with a final purification of 44.70-fold and a recovery yield of 21.04%. The purified laccase was shown to be a monomeric enzyme with a molecular mass of 60 kDa. The optimum temperature and pH value of the laccase were 75°C and pH 4.0, respectively, for 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS). The Michaelis–Menten constant (K _m ) of the laccase was 18 μM for ABTS substrate. The laccase was stable at pH values between 5.5 and 7.5. About 80% of the initial enzyme activity was retained after incubation of the laccase at 70°C for 2 h, indicating that the laccase was intrinsically highly thermostable and with valuable potential applications. The laccase activity was promoted by 4.0 mM of Mg²⁺, Mn²⁺, Zn²⁺ and Ca²⁺, while inhibited by 4.0 mM of Co²⁺, Al³⁺, Cu²⁺, and Fe²⁺, showing different profiles of metal ion effects.     

Response of the carotenoidless mutant Rhodobacter sphaeroides growing cells to cobalt and nickel exposure

The interaction of cobalt (Co²⁺) and nickel (Ni²⁺) ions with whole cells of the photosynthetic purple bacterium Rhodobacter sphaeroides strain R26 was investigated. Active and passive uptakes were examined in cells grown in the presence of increasing amounts of Co²⁺ and Ni²⁺. Inductively coupled plasma atomic emission spectroscopy (ICP-AES), pH titration, and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy were used to assess the role of cell envelope and metabolism in accumulating the two heavy metals. The chosen microorganism was able to uptake cobalt and nickel up to 2.2 and 0.25 mg per gram of dried cells respectively, with the largest part found bound to the cell surface. Carboxylate groups lying on the cell wall of this Gram-negative bacterium proved to be the major candidates for binding protons and metal cations. Co²⁺ was found to interfere with Mg²⁺ extracellular immobilization and transport across the membrane, indicating that these ions share binding sites on the cell envelope and ion transport systems. According to the presence of a competition mechanism, bacterial growth experiments showed that high Mg²⁺ concentrations are able to rescue R. sphaeroides from Co²⁺ toxicity.     

Conjugative plasmid mediated inducible nickel resistance in<Emphasis Type="Italic"> Hafnia alvei</Emphasis> 5-5

Hafnia alvei 5-5, isolated from a soil-litter mixture underneath the canopy of the nickel-hyperaccumulating tree Sebertia acuminata (Sapotaceae) in New Caledonia, was found to be resistant to 30 mM Ni²⁺ or 2 mM Co²⁺. The 70-kb plasmid, pEJH 501, was transferred by conjugation to Escherichia coli, Serratia marcescens, and Klebsiella oxytoca. Transconjugant strains expressed inducible nickel resistance to between 5 and 17 mM Ni²⁺, and cobalt resistance to 2 mM Co²⁺. A 4.8-kb Sal–EcoRI fragment containing the nickel resistance determinant was subcloned, and the hybrid plasmid was found to confer a moderate level of resistance to nickel (7 mM Ni²⁺) even to E. coli. The expression of nickel resistance was inducible by exposure to nickel chloride at a concentration as low as 0.5 mM Ni²⁺. By random TnphoA′-1 insertion mutagenesis, the fragment was shown to have structural genes as well as regulatory regions for nickel resistance. Southern hybridization studies showed that the nickel-resistance determinant from pEJH501 of H. alvei 5-5 was homologous to that of pTOM9 from Alcaligenes xylosoxydans 31A. Electronic Publication     

An intracellular aminopeptidase from Streptomyces rimosus that prefers basic amino acids

An aminopeptidase from the mycelia of Streptomyces rimosus was isolated in an electrophoretically homogeneous form. It was shown to be a monomeric, acidic protein (pI = 4.4, mol. wt. approx. 83,000), with optimal activity at pH 7.1–7.8 and at 35–41° C. The enzyme was fully inhibited by 0.1 mM EDTA or 1 mM o-phenanthroline; the activity was restored upon addition of 0.05 mM Co²⁺, Zn²⁺, or Ni²⁺. Amastatin, bestatin, and puromycin also inhibited the enzyme. The aminopeptidase hydrolyzed amino-acid-2-naphthylamides and various di- to heptapeptides. The highest catalytic coefficients (23 and 19 μM^–1 s^–1) were obtained with Arg- and Lys-2-naphthylamide, followed by Leu-, Phe- and Met-derivatives with one order of magnitude lower catalytic coefficients. Basic or bulky hydrophobic amino acids at the P₁ and/or P₁′ position of peptide substrates were preferred. Acidic amino acids and proline were not accepted. The affinity of the enzyme increased with the length of peptide. According to these properties, S. rimosus intracellular aminopeptidase is distinct from the extracellular leucine aminopeptidase of the same organism and can be classified as an Arg(Lys)-preferring metalloaminopeptidase. Received: 18 January 1996 / Accepted: 19 March 1996     

Purification and characterization of thermostable xylose(glucose) isomerase from Bacillus thermoantarcticus

Xylose isomerase produced by Bacillus thermoantarcticus was purified 73-fold to homogeneity and its biochemical properties were determined. It was a homotetramer with a native molecular mass of 200 kDa and a subunit molecular mass of 47 kDa, with an isoelectric point at 4.8. The enzyme had a K _m of 33 mM for xylose and also accepted D-glucose as substrate. Arrhenius plots of the enzyme activity of xylose isomerase were linear up to a temperature of 85°C. Its optimum pH was around 7.0, and it had 80% of its maximum activity at pH 6.0. This enzyme required divalent cations for its activity and thermal stability. Mn²⁺, Co²⁺ or Mg²⁺ were of comparable efficiency for xylose isomerase reaction, while Mg²⁺ was necessary for glucose isomerase reaction. Journal of Industrial Microbiology & Biotechnology (2001) 27, 234–240. Received 18 March 2001/ Accepted in revised form 03 July 2001     

Intracellular Mg<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Influences Both Open and Closed Times of a Native Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript>-activated BK Channel in Cultured Human Renal Proximal Tubule Cells

Effects of intracellular Mg²⁺ on a native Ca²⁺-and voltage-sensitive large-conductance K⁺ channel in cultured human renal proximal tubule cells were examined with the patch-clamp technique in the inside-out mode. At an intracellular concentration of Ca²⁺ ([Ca²⁺]_i) of 10⁻⁵–10⁻⁴ M, addition of 1–10 mM Mg²⁺ increased the open probability (P_o) of the channel, which shifted the P_o –membrane potential (V_m) relationship to the negative voltage direction without causing an appreciable change in the gating charge (Boltzmann constant). However, the Mg²⁺-induced increase in P_o was suppressed at a relatively low [Ca²⁺]_i (10^−5.5–10⁻⁶ M). Dwell-time histograms have revealed that addition of Mg²⁺ mainly increased P_o by extending open times at 10⁻⁵ M Ca²⁺ and extending both open and closed times simultaneously at 10^−5.5 M Ca²⁺. Since our data showed that raising the [Ca²⁺]_i from 10⁻⁵ to 10⁻⁴ M increased P_o mainly by shortening the closed time, extension of the closed time at 10^−5.5 M Ca²⁺ would result from the Mg²⁺-inhibited Ca²⁺-dependent activation. At a constant V_m, adding Mg²⁺ enhanced the sigmoidicity of the P_o–[Ca²⁺]_i relationship with an increase in the Hill coefficient. These results suggest that the major action of Mg²⁺ on this channel is to elevate P_o by lengthening the open time, while extension of the closed time at a relatively low [Ca²⁺]_i results from a lowering of the sensitivity to Ca²⁺ of the channel by Mg²⁺, which causes the increase in the Hill coefficient. M. Kubokawa and Y. Sohma contributed equally to this work.