Biosorption of cadmium,copper and lead by isolated mother cell walls and whole cells of Chlorella fusca

Using a new method for the isolation of released mother cell walls of Chlorella fusca, the biosorption of cadmium, copper and lead by purified cell wall isolates and whole cell suspensions was comparatively characterized. In all cases whole cells accumulated more metal ions than isolated cell walls. Both the Langmuir and Freundlich isotherm models were suitable for describing the short-term adsorption of cadmium, copper and lead by cell walls and the cadmium and copper adsorption by whole cells. However, neither model could sufficiently explain the lead accumulation by whole cells. The feasibility of a practical use of whole cells or isolated cell walls as biosorbents is discussed.     

ORIGINAL PAPERS Biosorption of U, La, Pr, Nd, Eu and Dy by Pseudomonas aeruginosa

Kinetic studies with Pseudomonas aeruginosa using actinides and lanthanides indicated a two-phase metal uptake. Equilibrium uptake data of all the metals studied could be fitted to Langmuir as well as Freundlich models. The Scatchard plots showed that there were mainly two types of receptor sites on the cell walls of P. aeruginosa having different affinities for the metal ions. EDAX studies revealed replacement of calcium and magnesium ions from biomass by sorbed metal. Around 85% of the adsorbed metal could be released using citrate buffer (pH 4.0, 0.2 M). Metal desorption was as high as 95% with HCl. Continuous flow studies using P. aeruginosa immobilized on activated alumina gave 80% enhancement of lanthanum removal efficiency compared to the control column. Regeneration of the column resulted in 80% of its initial capacity in succeeding cycles. Journal of Industrial Microbiology & Biotechnology (2000) 25, 1–7. Received 17 May 1999/ Accepted in revised form 04 April 2000     

The recovery of heavy metals using encapsulated microbial cells

We prepared capsules containingSaccharomyces cerevisiae andZoogloea ramigera cells for the removal of lead (II) and cadmium ions. Microbial cells were encapsulated and cultured in the growth medium. TheS. cerevisiae cells grown in the capsule did not leak through the capsule membrane. The dried cell density reached to 250 g/l on the basis of the inner volume of the 2.0 mm diameter capsule after 36 hour cultivation. The dry whole cell exopolymer density of encapsulatedZ. ramigera reached to 200 g/L. The capsule was crosslinked with triethylene tetramine and glutaric dialdehyde solutions. The cadmium uptake of encapsulated whole cell exopolymer ofZ. ramigera was 55 mg Cd/g biosorbent. The adsorption line followed well Langmuir isotherm. The lead uptake of the encapsulatedS. cerevisiae was about 30 mg Pb/g biomass. The optimum pH of the lead uptake using encapsulatedS. cerevisiae was found to be 6. Freundlich model showed a little better fit to the adsorption data than Langmuir model. 95 percent of the lead adsorbed on the encapsulated biosorbents was desorbed by the 1 M HCl solution. The capsule was reused 50 batches without loosing the metal uptake capacity. And the mechanical strength of the crosslinked capsule was retained after 50 trials.     

Influence of the external redox state on heavy metal adsorption by whole cells and isolated cell walls of Chlorella fusca

Summary A reduction of the external redox potential from 343 mV (atmospheric O₂ saturation) to 143 mV had only a slight effect on heavy metal uptake by whole cells of Chlorella fusca but caused an increased copper adsorption and a threefold increase of lead accumulation by isolated cell walls.     

Flocculation of cell walls of brewer's yeast and effects of metal ions,protein-denaturants and enzyme treatments

Effects of metal ions, protein-denaturants and enzyme treatments on flocculation of cell walls of Beer Yeast IFO 2018 were investigated. Cell walls from flocculent cells grown in a complete medium were able to form flocs as were whole cells, but cell walls from non-flocculent cells, such as “Mg²⁺-deficient” cells, “early-phase” cells and “low-pH” cells, were not. The cell walls dispersed in distilled water reflocculated in solutions containing Ca²⁺ or other metal ions. Of the alkali metal ions tested, only Na⁺ inhibited flocculation of flocculent cell walls at a concentration more than 0.1 M. Ca²⁺ or Sn⁴⁺ was absolutely required for flocculation of cell walls in the physiological saline (NaCl, 150 mM), but the effect of Sn⁴⁺ seems rather non-specific, because it promoted flocculation of non-flocculent cell walls as well. Sr²⁺ and Ba²⁺ were antagonistic to Ca²⁺ and inhibited flocculation. Flocculation of cell walls was also depressed by high concentrations of protein-denaturants, e.g. urea and guanidine·HCl. Treatment with proteolytic enzymes deprived cell walls of floc-forming ability. Effect of metal ions, protein-denaturants and treatment with enzymes on the flocculation of intact cells was investigated as control. Since flocculating properties of cell walls were very similar to those of intact cells, flocculation must be an inherent property of cell walls.     

Biosorption of uranium and lead by Streptomyces longwoodensis

Biosorption of uranium and lead by lyophilized cells of Streptomyces longwoodensis was examined as a function of metal concentration, pH, cell concentration, and culture age. Cells harvested from the stationary growth phase exhibited an exceptionally high capacity for uranium (0.44 g U/g dry weight) at pH 5. Calculated values of the distribution coefficient and separation factor indicated a strong preference of the cell mass for uranyl ions over lead ions. The specific uranium uptake was similar for the cell wall and the cytoplasmic fraction. Uranium uptake was associated with an increase in hydrogen ion concentration, and phosphorus analysis of whole cells indicated a simple stoichiometric ratio between uranium uptake and phosphorus content. It is proposed that metal ions are bound to phosphodiester residues present both in the cell wall and cytoplasmic fractions. Based on this model, it was shown that uranium accumulation exhibits a maximum at pH 4.6 that is supported by experimental data from previous investigations.     

Characteristics of lead adsorption byUndaria pinnatifida

Summary We reportUndaria pinnatifida as a potential biosorbent for lead removal.U. pinnatifida exhibited approximately 350 mg of lead uptake capacity per gram of dry biomass within a pII range of 3 to 4. The major cellular component ofU. pinnatifida in lead binding was thought as alginic acid existing in the cell wall.U. pinnatifida adsorbed lead ion selectively over other alkaline metal ions such as K⁺, Na⁺, Mg⁺⁺ and Ca⁺⁺. It was confirmed by using instrumental analysis including EDX and XPS thatU. pinnatifida adsorbed lead ions on the surface of itself.This research was supported by Bioprocess Engineering Research Center and Kyunggi Chemical Industrial Company.     

Cell surface-engineered yeast displaying a histidine oligopeptide (hexa-His) has enhanced adsorption of and tolerance to heavy metal ions

A histidine oligopeptide (hexa-His) with the ability to chelate divalent heavy metal ions was displayed on the yeast cell surface for the purpose of enhanced adsorption of heavy metal ions. We genetically fused a hexa-His-encoding gene with the gene encoding the C-terminal half of alpha-agglutinin that includes a glycosylphosphatidylinositol anchor attachment signal sequence and attached the hexa-His peptide on the cell wall of Saccharomyces cerevisiae. This surface-engineered yeast adsorbed three to eight times more copper ions than the parent strain and was more resistant to copper (4 mM) than the parent (below 1 mM at pH 7.8). It was possible to recover about a half of the copper ions adsorbed by whole cells with EDTA treatment without disintegrating the cells. Thus, we succeeded in constructing a novel yeast cell with both tolerance to toxic contaminants and enhanced adsorption of metal ions onto the cell surface.     

Contribution of peptidoglycan to the binding of metal ions by the cell wall ofBacillus subtilis

Equilibrium dialysis techniques were utilized in assessing the interaction between the cell walls ofBacillus subtilis and metal ions. The results show that cell wall teichoic acid molecules are not required for wall-metal ion complex formation. The numbers of combining sites for metals on unmodified cell walls were 0.7–0.9 μmol metal bound per mg wall. Thus, it is likely that the metals are combining with identical sites in the cell walls. The geometry of the sites may be responsible for the changes in affinity of the walls for various metals.     

The distribution of lead in duckweed (Lemna minor L.) root tip

While considerable information on lead distribution in the cells of terrestrial plants has been collected, little is known about lead localization in the cells of the aquatic plant. Lemna minor L. (duckweed) roots were examined using X-ray microanalysis. After 1-h treatment with lead, its concentration was the highest in small vacuoles. After 6 and 12 h, the lead content of cell walls gradually increased. The changes of lead level between vacuoles and cell walls may result from redistribution of this metal from symplast to cell walls or it may reflect increased apoplastic transport. Lead was not found in the ground cytoplasm of any variants of the experiments. This fact and presence of lead in small vesicles suggests that endocytosis may play the role in lead uptake in Lemna.     

Studies on the Mode of Action of the Phenolic Antibacterial Agent Fentichlor against Staphylococcus aureus and Escherichia coli I. The Adsorption of Fentichlor by the Bacterial Cell and its Antibacterial Activity

Fentichlor is adsorbed in fairly large amounts by Staphylococcus aureus and Escherichia coli and, according to the quantity adsorbed, is either bacteriostatic or bactericidal. The observed pattern of uptake, measured under various conditions, indicates that uptake involves reversible adsorption of the neutral molecule on to the cell. The drug is taken up by the cell wall and cell membrane, the latter probably being the main site of adsorption and main site of action. Although both whole cells and cell walls of E. coli have a higher affinity for Fentichlor than those of Staph. aureus , the former is less susceptible to its antibacterial action. Results indicate that this may be due to the lipid-rich nature of the cell walls of E. coli which act as an adsorbing barrier preventing the access of the drug to its site of action.     

On the evidence of a diffusion barrier in the outer cortex apoplast of cress-roots (Lepidium sativum), demonstrated by analytical electron microscopy

To mark the apoplastic pathway of ions in the root of the dicotyledonous plant Lepidium sativum we used the heavy element lanthanum, which can be identified by analytical electron microscopy (EELS and ESI). In the front root tip, the primary walls of all meristematic cells contained lanthanum. 10-15 mm behind the root apex, lanthanum was found in the cortex cell walls up to the endodermis, but not in the stele. 20-25 mm from the tip, lanthanum was accumulated in the radial cell walls of the hypodermis, which, however, is not a complete diffusion barrier for ions, so that traces of lanthanum also were found in the cortex cell walls up to the endodermis. This study provides evidence for the presence of two apolastic diffusion barriers in the region of highest water uptake in cress roots.     

Uptake of cobalt and cesium by microalgal- and cyanobacterial-clay mixtures

Accumulation of cobalt and cesium by the microalga Scenedesmus obliquus and the cyanobacterium Synechocystis PCC 6803 has been characterized at metal concentrations ranging from 1–100 µM in the presence of three clay minerals, montmorillonite, illite, and kaolinite. The majority of metal uptake over a 4 h period consisted of rapid binding to the clay mineral-cell aggregates, and was unaffected by incubation in the dark or by the presence of the metabolic inhibitor carbonyl cyanide-3-chlorophenyl hydrazone (CCCP). This was followed by a slower, energy-dependent uptake of metal by the cell components of the mixtures, which was inhibited by incubation in the dark or in the presence of CCCP. The initial phase of uptake by the clay mineral-cell mixtures and mixture components alone conformed to a Freundlich adsorption isotherm, the order of uptake for both cobalt and cesium being montmorillonite-cells > illite-cells > kaolinite-cells. S. obliquus-clay mineral mixtures accumulated more cobalt and cesium than Synechocystis PCC 6803-clay mineral mixtures. On a dry weight basis, clay minerals alone accumulated greater amounts of metals than clay mineral-cell mixtures, which accumulated more than the cells alone. However, when the same data was expressed as amount of metal adsorbed per unit surface area, S. obliquus, in most cases, adsorbed greater amounts of cobalt and cesium than the clay minerals or Synechocystis PCC 6803. As the proportion of clay in a cell-clay mineral mixture was increased, the amount of metal accumulated also increased. Reduced accumulation of cobalt and cesium by cell-clay mineral mixtures, exhibited by equal amounts of the individual components added together, indicated that the formation of clay-cell aggregates had masked some of the binding sites normally available to metal ions. Accumulation of cobalt and cesium by all clay mineral-cell mixtures was dependent on the external pH and NaCl concentration, and decreased with decreasing pH and increasing external NaCl concentration. Offprint requests to: G. M. Gadd.     

The regulation of ionic nickel uptake and cytotoxicity by specific amino acids and serum components

The effects of serum components and amino acids on the uptake and cytotoxicity of NiCl₂ were examined in cultured Chinese hamster ovary (CHO) cells. CHO cells maintained in a minimal salts/glucose medium accumulated 10-fold more⁶³Ni than did cells maintained in complete medium supplemented with 10% fetal bovine serum. Cell-surface binding of⁶³Ni appeared to account for the majority of this increased accumulation of cell-associated nickel observed in the simple maintenance medium since such increases were reduced 70% by trypsin treatment. The addition of the Ni²⁺-binding amino acids cysteine or histidine to the salts/glucose medium markedly decreased⁶³Ni accumulations, an effect not observed following addition of any of several amino acids that do not bind Ni²⁺. Supplementation of the salts/glucose medium with fetal bovine serum decreased in a concentration dependent fashion both the⁶³Ni²⁺ uptake and cell detachment caused by Ni²⁺, while dialyzed (amino acid-free) serum was 3–5-fold less effective than undialyzed serum at reducing⁶³Ni²⁺ uptake and similarly exhibited only a slight protective effect against nickel-induced cytotoxicity. Supplementation of dialyzed serum with cysteine at levels approximating those in whole serum partially restored its inhibitory activity toward nickel uptake by cells and restored completely its inhibition of nickel's cytotoxicity, indicating the predominant role of specific amino acids over serum proteins in regulating the uptake and subsequent cytotoxicity of Ni²⁺. Addition of cysteine to the salts/glucose medium during a 2 h exposure of cells to either 100 μM HgCl₂ or 1 mM NiCl₂ masked the cytotoxic effects of these metal ions. These results demonstrate the importance of extracellular small molecular weight metal ion chelators in altering the biological effects of metal ions at the level of metal uptake.     

锰对植物毒害及植物耐锰机理研究进展

含锰矿渣的排放造成了严重的土壤锰污染。揭示锰毒害和植物的耐锰机制对于污染土壤治理具有重要意义。研究表明, 高浓度的Mn²⁺能够抑制根系Ca²⁺、Fe²⁺和Mg²⁺等元素的吸收及活性, 引起氧化性胁迫导致氧化损伤, 使叶绿素和Rubisco含量下降、叶绿体超微结构破坏和光合速率降低。而锰超累积植物则具有多种解毒或耐性机制, 如区域化、有机酸螯合、外排作用、抗氧化作用和离子交互作用等。根系主要通过有机酸的螯合作用促进植物对Mn²⁺的转运解毒, 同时能够将过量的Mn²⁺区域化在根细胞壁中; 叶片可通过酚类物质或有机酸螯合Mn²⁺, 并将其区域化在叶片表皮细胞和叶肉细胞的液泡中(或通过表皮毛将Mn²⁺排出体外)。其中, 金属转运蛋白在植物对Mn²⁺的吸收、转运、累积和解毒过程中发挥着重要作用。     

Biotechnological potential of Chlorella vulgaris for accumulation of Cu and Ni from single and binary metal solutions

This study provides information on the mechanism(s) of Cu and Ni ion biosorption by C. vulgaris, distinguishing adsorption from intracellular accumulation under various conditions. Surface adsorption was found to contribute maximally (>70%) to total Cu/Ni ion accumulation by the test alga (total accumulation efficiencies were 60 and 53 g metal ion mg protein^–1, respectively for Cu and Ni). Maximum intracellular uptake was reported at a pH range of 6.5–7.5, whereas adsorption reached its maximum at pH 3.5 for Cu, and pH 3.5 and 6.5 in the case of Ni. 35 °C was found to be the best temperature for maximum adsorption, whereas intracellular uptake was highest at 25 °C. Though exponentially grown C. vulgaris registered maximum metal ion uptake, adsorption maxima reached the highest values in the declining phase of the culture. Heat-killed and air-dried C. vulgaris accumulated Cu and Ni at about 80% of the values for viable samples, whereas formaldehyde-treated and immobilized biomasses depicted better accumulating potential than the control cells. Na, K, Mn and Zn caused competitive inhibition, whereas for Ca a mixed-type inhibition was evident. Thus, the present study suggests that the general concept that cations inhibit metal ion accumulation by competing with them for the same binding sites on the cell surface is not absolutely valid. As these results also demonstrate that a large amount of the bound metal (>70%) is in the adsorbed fraction, it is advantageous in the sense that it could be recovered by a suitable desorbing agent, especially in case of precious metals and the biomass could be exploited for repeated use in reactors.     

Surface chemical properties of purified root cell walls from two tobacco genotypes exhibiting different tolerance to manganese toxicity

Surface chemical characteristics of root cell walls extracted from two tobacco genotypes exhibiting differential tolerance to Mn toxicity were studied using potentiometric pH titration and Fourier transform infrared spectroscopy. The Mn-sensitive genotype KY 14 showed a stronger interaction of its cell wall surface with metal ions than did the Mn-tolerant genotype Tobacco Introduction (T.I.) 1112. This observation may be attributed to the relatively higher ratio of COO⁻ to COOH in KY 14 cell walls than that found in the cell walls of T.I. 1112 in the pH range of 4 to 10. For both genotypes, the strength of binding between metal ions and cell wall surface was in the order of Cu > Ca > Mn > Mg > Na. However, a slightly higher preference of Ca over Mn was observed with the T.I. 1112 cell wall. This may explain the high accumulation of Mn in the leaves of Mn-tolerant genotype T.I. 1112 rather than the high accumulation of Mn in roots, as occurred in Mn-sensitive KY 14. It is concluded that surface chemical characteristics of cell walls may play an important role in plant metal ion uptake and tolerance.     

Electron paramagnetic resonance studies and effects of vanadium in Saccharomyces cerevisiae

Vanadium uptake by whole cells and isolated cell walls of the yeast Saccharomyces cerevisiae was studied. When orthovanadate was added to wild-type S. cerevisiae cells growing in rich medium, growth was inhibited as a function of the VO₄ ^3- concentration and the growth was completely arrested at a concentration of 20 mM of VO₄ ^3- in YEPD. Electron paramagnetic resonance (EPR) spectroscopy was used to obtain structural and dynamic information about the cell-associated paramagnetic vanadyl ion. The presence of EPR signals indicated that vanadate was reduced by whole cells to the vanadyl ion. On the contrary, no EPR signals were detected after interaction of vanadate with isolated cell walls. A mobile and an immobile species associated in cells with small chelates and with macromolecular sites, respectively, were identified. The value of rotational correlation time _r indicated the relative motional freedom at the macromolecular site. A strongly immobilized vanadyl species bound to polar sites mainly through coulombic attractions was detected after interaction of VO²⁺ ions with isolated cell walls.     

65Zn2+ transport by isolated gill epithelial cells of the American lobster, Homarus americanus

Gills are the first site of impact by metal ions in contaminated waters. Work on whole gill cells and metal uptake has not been reported before in crustaceans. In this study, gill filaments of the American lobster, Homarus americanus, were dissociated in physiological saline and separated into several cell types on a 30, 40, 50, and 80% sucrose gradient. Cells from each sucrose solution were separately resuspended in physiological saline and incubated in ⁶⁵Zn²⁺ in order to assess the nature of metal uptake by each cell type. Characteristics of zinc accumulation by each kind of cell were investigated in the presence and absence of 10 mM calcium, variable NaCl concentrations and pH values, and 100 μM verapamil, nifedipine, and the calcium ionophore A23187. ⁶⁵Zn²⁺ influxes were hyperbolic functions of zinc concentration (1–1,000 μM) and followed Michaelis–Menten kinetics. Calcium reduced both apparent zinc binding affinity (K _m) and maximal transport velocity (J _max) for 30% sucrose cells, but doubled the apparent maximal transport velocity for 80% sucrose cells. Results suggest that calcium, sodium, and protons enter gill epithelial cells by an endogenous broad-specificity cation channel and trans-stimulate metal uptake by a plasma membrane carrier system. Differences in zinc transport observed between gill epithelial cell types appear related to apparent affinity differences of the transporters in each kind of cell. Low affinity cells from 30% sucrose were inhibited by calcium, while high affinity cells from 80% sucrose were stimulated. ⁶⁵Zn²⁺ transport was also studied by isolated, intact, gill filament tips. These intact gill fragments generally displayed the same transport properties as did cells from 80% sucrose and provided support for metal uptake processes being an apical phenomenon. A working model for zinc transport by lobster gill cells is presented.     

Probing metal ion substrate-binding to the E. coli ZitB exporter in native membranes by solid state NMR

Metal ion homeostasis is important for healthy cell function and is regulated by metal ion transporters and chaperones. To explore metal ion binding to membrane transport proteins we have used cadmium-113 as a solid state NMR probe of the Escherichia coli zinc exporter ZitB present in native membrane preparations. Competition experiments with other metal ions indicated that nickel and copper are also able to bind to this protein. Metal ion uptake studies were also performed using ZitB-reconstituted into proteoliposomes for a well established fluorescence assay. The results of both the solid state NMR and the uptake studies demonstrate that ZitB is potentially capable of transporting not only zinc but also cadmium, nickel and copper. The solid state NMR approach therefore offers great potential for defining the substrate spectrum of metal ion transporter proteins in their native membrane environments. Further, it should be useful for functional dissection of transporter mechanisms by facilitating the identification of functional residues by mutational studies.