Engineering of microorganisms towards recovery of rare metal ions

The bioadsorption of metal ions using microorganisms is an attractive technology for the recovery of rare metal ions as well as removal of toxic heavy metal ions from aqueous solution. In initial attempts, microorganisms with the ability to accumulate metal ions were isolated from nature and intracellular accumulation was enhanced by the overproduction of metal-binding proteins in the cytoplasm. As an alternative, the cell surface design of microorganisms by cell surface engineering is an emerging strategy for bioadsorption and recovery of metal ions. Cell surface engineering was firstly applied to the construction of a bioadsorbent to adsorb heavy metal ions for bioremediation. Cell surface adsorption of metal ions is rapid and reversible. Therefore, adsorbed metal ions can be easily recovered without cell breakage, and the bioadsorbent can be reused or regenerated. These advantages are suitable for the recovery of rare metal ions. Actually, the cell surface display of a molybdate-binding protein on yeast led to the enhanced adsorption of molybdate, one of the rare metal ions. An additional advantage is that the cell surface display system allows high-throughput screening of protein/peptide libraries owing to the direct evaluation of the displayed protein/peptide without purification and concentration. Therefore, the creation of novel metal-binding protein/peptide and engineering of microorganisms towards the recovery of rare metal ions could be simultaneously achieved.     

Bioadsorption of cadmium ion by cell surface-engineered yeasts displaying metallothionein and hexa-His

The Cd²⁺-chelating abilities of yeast metallothionein (YMT) and hexa-His displayed on the yeast-cell surface were compared. Display of YMT and hexa-His by -agglutinin-based cell-surface engineering was confirmed by immunofluorescent labeling. Surface-engineered yeast cells with YMT and hexa-His fused in tandem showed superior cell-surface adsorption and recovery of Cd²⁺ under EDTA treatment on the cell surface than hexa-His-displaying cells. YMT was demonstrated to be more effective than hexa-His for the adsorption of Cd²⁺. Yeast cells displaying YMT and/or hexa-His exhibited a higher potential for the adsorption of Cd²⁺ than Escherichia coli cells displaying these molecules. In order to investigate the effect of the displayed YMT and hexa-His on sensitivity to toxic Cd²⁺, growth in Cd²⁺-containing liquid medium was monitored. Unlike hexa-His-displaying cells, cells displaying YMT and hexa-His fused in tandem induced resistance to Cd²⁺ through active and enhanced adsorption of toxic Cd²⁺. These results indicate that YMT-displaying yeast cells are a unique bioadsorbent with a functional chelating ability superior to that of E. coli.     

Copper Tolerance and Biosorption of Saccharomyces cerevisiae during Alcoholic Fermentation

At high levels, copper in grape mash can inhibit yeast activity and cause stuck fermentations. Wine yeast has limited tolerance of copper and can reduce copper levels in wine during fermentation. This study aimed to understand copper tolerance of wine yeast and establish the mechanism by which yeast decreases copper in the must during fermentation. Three strains of Saccharomyces cerevisiae (lab selected strain BH8 and industrial strains AWRI R2 and Freddo) and a simple model fermentation system containing 0 to 1.50 mM Cu²⁺ were used. ICP-AES determined Cu ion concentration in the must decreasing differently by strains and initial copper levels during fermentation. Fermentation performance was heavily inhibited under copper stress, paralleled a decrease in viable cell numbers. Strain BH8 showed higher copper-tolerance than strain AWRI R2 and higher adsorption than Freddo. Yeast cell surface depression and intracellular structure deformation after copper treatment were observed by scanning electron microscopy and transmission electron microscopy; electronic differential system detected higher surface Cu and no intracellular Cu on 1.50 mM copper treated yeast cells. It is most probably that surface adsorption dominated the biosorption process of Cu²⁺ for strain BH8, with saturation being accomplished in 24 h. This study demonstrated that Saccharomyces cerevisiae strain BH8 has good tolerance and adsorption of Cu, and reduces Cu²⁺ concentrations during fermentation in simple model system mainly through surface adsorption. The results indicate that the strain selected from China’s stress-tolerant wine grape is copper tolerant and can reduce copper in must when fermenting in a copper rich simple model system, and provided information for studies on mechanisms of heavy metal stress.     

Effective display of metallothionein tandem repeats on the bioadsorption of cadmium ion

To increase the level of adsorption of heavy metal ions in surface-engineered yeasts, a yeast metallothionein (YMT) was tandemly fused and displayed by means of an α-agglutinin-based display system. The display of the YMT and its tandem repeats was examined by immunofluorescent labeling. The adsorption and recovery of Cd²⁺ on the cell surface was increasingly enhanced with increasing number of tandem repeats. All Cd²⁺-binding sites in the YMT tandem repeats were suggested to be completely occupied. To investigate the relationship between cell-surface adsorption and protection against heavy metal ion toxicity, the tolerance of these surface-engineered yeasts to Cd²⁺ was examined by growing in Cd²⁺-containing liquid medium. The rate of growth was found to be dependent on the number of displayed tandem repeats of YMT. These results suggest that the characteristics of surface-engineered yeasts as a bioadsorbent were dependent on the ability of the displayed proteins to bind metal ions, and the adsorption of heavy metal ions on the cell surface plays a major role in the ability of the cells to resist the toxic effects of metal ions.     

Comparative studies of the heavy metal uptake of whole cells and different types of cell walls from Chlorella fusca

Summary Short-term uptake of cadmium, copper and lead by whole cells, mother cell walls (mcw) and cell walls from cell homogenates (hcw) of Chlorella fusca were similar for all biosorbents: Adsorption equilibrium was reached five minutes after metal addition. Analogous, over 90% of the readily adsorbed metal ions could be desorbed by a 1 minute EDTA treatment. In all cases, short-term or long-term uptake and single ion solution or metal mixtures, whole cells showed the highest metal accumulation while hcw adsorbed more metal ions than mcw. The role of the cell wall for metal ion adsorption is discussed.     

Characteristics of copper tolerance in Yarrowia lipolytica

We discovered that a mutant strain of the dimorphic yeast Yarrowia lipolytica could grow in the yeast form in high concentrations of copper sulfate. The amount of metal accumulated by Y. lipolytica increased with increasing copper concentrations in the medium. Washing with 100 mM EDTA released at least 60% of the total metal from the cells, but about 20–25 μmol/g DW persisted, which represented about 30% of the soluble fraction of cultured cells. The soluble fraction (mainly cytosol) contained only about 10% of the total metal content within cells cultured in medium supplemented with 6 mM copper. We suggest that although a high copper concentration induces an efflux mechanism, the released copper becomes entrapped in the periplasm and in other parts of the cell wall. Washing with EDTA liberated not only copper ions, but also melanin, a brown pigment that can bind metal and which located at the cell wall. These findings indicated that melanin participates in the mechanism of metal accumulation. Culture in medium supplemented with copper obviously enhanced the activities of Cu, Zn-SOD, but not of Mn-SOD.     

Influence of heavy metal ions on the electrophysical properties of Anacystis nidulans and Escherichia coli cells]

The influence of heavy metal ions (Ag+, Cu2+, Cd2+, Pb2+, Mn2+, Zn2+, Gd3+, 1 microM-1 mM) on Anacystis nidulans and Escherichia coli cells has been studied by means of electrophoresis and electro-orientation spectroscopy methods. It has been shown that changes of cell electrophoretic mobility (EM) and low-frequency (20 Hz) electro-orientation effect (EOE) observed with the increase of metal cation concentration characterize the adsorption of these ions on surface layers of cell envelopes. The degree and the character of these changes depend on cation valency and the initial value of cell EM. At the same time different changes of EM and EOE as a result of the multivalent cation adsorption allows to conclude that in that case the anisotropy of the cell surface increases. Cell damages were determined by changes in high-frequency EOE of cells which indicated the disturbance of barrier properties of their cytoplasmic membrane. Toxic effects of Ag+, Cu2+, Cd2+ ions on cells of both species and of Pb2+ on E.coli cells were observed. By toxic effects on the cytoplasmic membrane these ions could be placed in the order: for A.nidulans cells--Ag+ greater than Cu2+ greater than Cd2+; for E.coli cells Ag+ greater than Cu2+ greater than Cd2+ greater than Pb2+. Higher toxicity of heavy metals on E.coli cells seems to be connected with the more negative charge of deep layers of the cell surface.     

Toxic effects caused by heavy metals in the yeast Saccharomyces cerevisiae: a comparative study

The decreasing order of toxicity of select heavy metals on the yeast Saccharomyces cerevisiae, in 10 mM MES (2-(N-morpholino)ethanesulfonic acid) pH buffer at pH 6.0, was found to be copper, lead, and nickel. Heavy metal (200 microM) induced a decrease in the number of viable cells by about 50% in the first 5 min for copper and in 4 h for lead, while nickel was not toxic up to a 200 microM concentration over a period of 48 h. Glucose (25 mM) strongly enhanced the toxic effect of 50 microM copper but had little or no effect on the toxicity of 200 microM lead or nickel. Copper, lead, and nickel induced the leakage of UV260-absorbing compounds from cells with different kinetics. The addition of 0.5 mM calcium, before addition of 200 microM copper, showed a protective action against cell death and decreased the release of UV-absorbing compounds, while no effect was observed against lead or nickel toxic effects. Copper complexation capacities of the filtrates of cells exposed for 2 h in 200 microM copper and 24 h in 200 microM lead were 51 and 14 microM, respectively. The implication of the complexation shown by these soluble compounds in the bioavailability of heavy metals is discussed.     

ROLE OF PROLINE ACCUMULATION IN RESPONSE TO TOXIC COPPER IN CHLORELLA SP. (CHLOROPHYCEAE) CELLS

The green alga Chlorella sp. (strain 2350) was found to accumulate proline, up to a maximum of 0.027 fmol·cell⁻¹, under stressful concentrations of cupric ions. The function of the accumulated proline was studied with respect to its effect on copper (Cu) uptake. By induction with salt stress, cells with various levels of intracellular proline were used for this study. It is shown that the amount of Cu taken up by the cells was reduced when the intracellular proline levels were enhanced. When proline was exogenously supplied prior to Cu treatment, the adsorption of Cu was markedly reduced. When exogenous proline was supplied after Cu treatment, it resulted in a remarkable desorption of the adsorbed Cu immediately after the addition of proline. The results of the present study indicate that proline may exert some action on the cell surface and suggest that one function of accumulated proline is to reduce the uptake of metal ions. The accumulation of proline may be related to a tolerance mechanism for dealing with Cu stress.     

Adsorption of sphingomyelinase of Bacillus cereus onto erythrocyte membranes

Sphingomyelinase of Bacillus cereus proved to be specifically adsorbed onto mammalian erythrocyte membranes in the presence of either Ca2+ or Ca2+ plus Mg2+ in the order of sphingomyelin content; i.e., sheep, bovine greater than porcine greater than rat erythrocytes. No appreciable adsorption was observed in the presence of Mg2+ alone nor in the absence of divalent metal ions. The enzyme adsorption onto bovine erythrocytes was dependent upon the incubation temperature. By shifting the temperature from 37 to 0 degrees C, sphingomyelinase once adsorbed onto the surface of bovine erythrocytes was released into the supernatant. Ca2+ proved to be an essential factor for the enzyme adsorption: The addition of 1 mM Ca2+ enhanced the adsorptive process, but inhibited sphingomyelin hydrolysis and hot or hot-cold hemolysis of erythrocytes, while the addition of 1 mM Ca2+ plus 1 mM Mg2+ enhanced sphingomyelin breakdown and hemolysis as well as the enzyme adsorption. However, when the amount of sphingomyelin fell off to 0.2-0.7 nmol/ml or less by the action of sphingomyelinase, the enzyme once adsorbed was completely released from the surface of erythrocytes. The result indicates that the major binding site for sphingomyelinase is sphingomyelin. In the presence of 1 mM Mg2+ alone, the enzymatic hydrolysis of sphingomyelin and hemolysis proceeded whereas the enzyme adsorption was not encountered during 60 min incubation at 37 degrees C. The change in the molar ratio of Ca2+ to Mg2+ affected the enzyme adsorption and sphingomyelin breakdown; the higher Ca2+ enhanced the adsorption whereas the higher Mg2+ stimulated sphingomyelin hydrolysis.     

Study of the biosorption of different heavy metal ions onto Kraft lignin

The ability of Kraft lignin, a waste product of paper production, for removing copper, zinc, cadmium and chromium ions from water was investigated. The studies were conducted by a batch method to determine equilibrium parameters. The adsorbed heavy metal ions followed the order: Cr(VI) ? Cd(II) > Cu(II) > Zn(II). The influence of other ions such as Ni(II), Cd(II) and Pb(II), on Cu(II) adsorption by Kraft lignin was evaluated. Obtained results support the idea that adsorption behaviour of heavy metal ions have to be perceived from the aspect of possible influence of interfering ion species.     

Recovery and removal of uranium by using plant wastes

The uranium-adsorbing abilities of seven plant wastes were investigated. High abilities to adsorb uranium from non-saline water containing 10 mg dm⁻³ of uranium were observed with a number of plant wastes tested. However, with seawater supplemented with 10 mg dm⁻³ of uranium, similar results were found only with chestnut residues. When the plant wastes were immobilized with formaldehyde, their ability to adsorb uranium was increased. Uranium and copper ions were more readily adsorbed by all plant wastes tested than other metal ions from a solution containing a mixture of seven different heavy metals. The selective adsorption of heavy metal ions differs with different species of plant wastes. The immobilization of peanut inner skin, orange peel and grapefruit peel increased the selectivity for uranium.     

Efficient expression of the yeast metallothionein gene in Escherichia coli.

The yeast metallothionein gene CUP1 was cloned into a bacterial expression system to achieve efficient, controlled expression of the stable, unprocessed protein product. The Escherichia coli-synthesized yeast metallothionein bound copper, cadmium, and zinc, indicating that the protein was functional. Furthermore, E. coli cells expressing CUP1 acquired a new, inducible ability to selectively sequester heavy metal ions from the growth medium.     

Identification of a gene conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae

Summary A DNA fragment conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae was isolated from a library of yeast genomic DNA. Its nucleotide sequence revealed the presence of a single open reading frame (ORF; 1326 bp) having the potential to encode a protein of 442 amino acid residues (molecular mass of 48.3 kDa). A frameshift mutation introduced within the ORF abolished resistance to heavy metal ions, indicating the ORF is required for resistance. Therefore, we termed it the ZRC1 (zinc resistance conferring) gene. The deduced amino acid sequence of the gene product predicts a rather hydrophobic protein with six possible membrane-spanning regions. While multiple copies of the ZRC1 gene enable yeast cells to grow in the presence of 40 mM Zn²⁺, a level at which wild-type cells cannot survive, the disruption of the chromosomal ZRC1 locus, though not a lethal event, makes cells more sensitive to zinc ions than are wild-type cells.     

The removal of Cu(II) from aqueous solutions by Ulothrix zonata

In this work, adsorption of copper(II) ions on alga has been studied by using batch adsorption techniques. The equilibrium biosorption level was determined as a function of contact time at several initial metal ion concentrations. The effect of adsorbent concentration on the amount adsorbed was also investigated. The experimental adsorption data were fitted to the Langmuir adsorption model. The free energy change (deltaG0) for the adsorption process was found to be -12.60 kJ/mol. The results indicated that the biomass of Ulothrix zonata is a suitable biosorbent for both the removal and recovery of heavy metals from wastewater.     

The requirement for yeast superoxide dismutase is bypassed through mutations in BSD2, a novel metal homeostasis gene.

Oxygen toxicity in Saccharomyces cerevisiae strains lacking superoxide dismutase can be suppressed through mutations in either the BSD1 or BSD2 gene. In this report, we demonstrate that the BSD2 gene normally functions in the homeostasis of heavy metal ions. A mutation in BSD2 not only reverses the aerobic defects of yeast strains lacking superoxide dismutase but also is associated with an increased sensitivity to copper and cadmium toxicity and an elevation in copper ion accumulation. The BSD2 gene was cloned by functional complementation and is predicted to encode a novel 37.5-kDa protein with three potential transmembrane domains. The mutant bsd2-1 allele was isolated and found to contain a single C-to-T transition changing a centrally located proline to a serine. This substitution results in total inactivation of BSD2, since the bsd2-1 mutation is identical to a bsd2 delta gene deletion in phenotype. BSD2 is expressed in yeast cells as a 1.5-kb mRNA. Although the gene functions in copper detoxification, BSD2 is not induced by copper ions, as is the case with S. cerevisiae metallothioneins. A probable role for copper ions in the bsd2 reversal of oxidative damage is discussed.