Histochemical and atomic absorption demonstration of trace metal mobilization in the central nervous system and liver of the rat.

Histochemical and atomic absorption spectrophotometric analysis of trace metal mobilization caused by the action of ethanol in the central nervous system (CNS) and liver of the rat is described. Histochemically it has been shown that in all neurons examined (motoneurons, pyramidal and Purkinje cells) the trace metals (mainly Zn2+ and Cu2+) are mobilized. Most of the stained materials disappear from the perikaryon of the Purkinje cells, while in both the motoneurons and the pyramidal cells the trace metals are displaced from the perikaryon into the axon and axon hillock. At the same time, some of the glia cells display a high metal content. Quantitative determination of the Zn2+ and Cu2+ by means of atomic absorption spectrophotometry reveals that after 2 hours ethanol treatment both the Zn2+ and the Cu2+ levels are decreased in the archicerebellar cortex, while after 4 hours the Zn2+ levels are increased in the cerebrum and the spinal cord. The present observations on the histochemical localization and the contents of zinc and copper in different parts of the CNS and liver reveal the important role of the effect of ethanol on the trace metal mobilization.     

Role of Vanadium (V) in the Differentiation of C3H10t1/2 Cells Towards Osteoblast Lineage: A Comparative Analysis with Other Trace Elements

In recent time, vanadium compounds are being used as antidiabetic drug and in orthopedic implants. However, the exact role of this incorporated vanadium in improving the quality of bone structure and morphology is not known. The impact of vanadium ion was studied and compared to other trace metal ions with respect to the proliferation and osteoblast differentiation of C3H10t1/2 cells. Toxicity profile of these trace metal ions revealed a descending toxicity trend of Fe²⁺ > Zn²⁺ > Cu²⁺ > Co²⁺ > Mn²⁺ > V⁵⁺ > Cr²⁺. The effect of vanadium and other trace metal ions on osteoblast differentiation was evaluated by culturing the cells for 10 days in osteoblastic medium supplemented with different trace ions at concentrations lower than their cytotoxic doses. The results indicated that vanadium has maximum impact on the induction of osteoblast differentiation by upregulating alkaline phosphatase activity and mineralization by up to 145 and 150 %, respectively (p?<?0.05), over control. Cu²⁺ and Zn²⁺ had a mild inhibitory effect, while Mn²⁺, Fe²⁺, and Co²⁺ demonstrated a clear decrease in osteoblast differentiation when compared to the control. The data as presented here demonstrate that orthopedic implants, if supplemented with trace metals like vanadium, may provide a source of better model for bone formation and its turnover.     

A spectrophotometric method for the determination of zinc, copper, and cobalt ions in metalloproteins using Zincon

Zincon (2-carboxy-2′-hydroxy-5′-sulfoformazylbenzene) has long been known as an excellent colorimetric reagent for the detection of zinc and copper ions in aqueous solution. To extend the chelator’s versatility to the quantification of metal ions in metalloproteins, the spectral properties of Zincon and its complexes with Zn²⁺, Cu²⁺, and Co²⁺ were investigated in the presence of guanidine hydrochloride and urea, two common denaturants used to labilize metal ions in proteins. These studies revealed the detection of metals to be generally more sensitive with urea. In addition, pH profiles recorded for these metals indicated the optimal pH for complex formation and stability to be 9.0. As a consequence, an optimized method that allows the facile determination of Zn²⁺, Cu²⁺, and Co²⁺ with detection limits in the high nanomolar range is presented. Furthermore, a simple two-step procedure for the quantification of both Zn²⁺ and Cu²⁺ within the same sample is described. Using the prototypical Cu²⁺/Zn²⁺-protein superoxide dismutase as an example, the effectiveness of this method of dual metal quantification in metalloproteins is demonstrated. Thus, the spectrophotometric determination of metal ions with Zincon can be exploited as a rapid and inexpensive means of assessing the metal contents of zinc-, copper-, cobalt-, and zinc/copper-containing proteins.     

Heavy metal uptake by intact cells and protoplasts of Aureobasidium pullulans

Protoplasts prepared from yeast-like cells, hyphae and chlamydospores of Aureobasidium pullulans can take up heavy metals such as Zn²⁺, Co²⁺, Cd²⁺ and Cu²⁺. In relation to intact cells, the sensitivity of protoplasts to Cu²⁺ and Cd²⁺ was increased although chlamydospore protoplasts were more tolerant than yeast-like cell protoplasts. Surface binding of metals was reduced in protoplasts as compared with intact cells and this reduction was particularly evident for chlamydospore protoplasts. At the highest concentrations used, uptake of Zn²⁺, Co²⁺ and Cd²⁺ by yeast-like cell protoplasts was greater than that observed in intact cells which may have been due to toxicity, especially for Cd²⁺, resulting in increased membrane permeability, though for Zn²⁺ and Co²⁺ some barrier effect of the cell wall could not be completely discounted. Chlamydospore protoplasts were capable of intracellular metal uptake, unlike intact chlamydospores, and for Zn²⁺, uptake appeared to be via a different system less specific than that of the other cell types. For chlamydospores, the use of protoplasts confirmed the importance of the cell wall in preventing entry of metal ions into the cell.     

Compartments for the Management of Municipal Solid Waste

Despite technological developments and improved liner-material applications, heavy metals in landfill leachate still penetrate the soil profile, polluting the soil and ground-water. An alternative approach therefore must be explored to reduce heavy-metal migration in soil-bentonite landfill liners. By considering the interaction of different heavy metals and their synergetic and antagonistics behaviors, such an approach could be developed. Low mobility metals such as Cu²⁺, and Pb²⁺ inhibit the adsorption of Cd²⁺ which is a moderate-mobility metal and Cu²⁺ sorption is decreased by the presence of Zn²⁺ and Cd²⁺. Therefore, Zn²⁺, a low-mobility metal, cannot be grouped with Cu²⁺. This way, four compatible metal groups have been identified: (1) low mobility: Pb²⁺, Cu²⁺, and Ag, (2) low mobility: Zn²⁺ and Cr³⁺; (3) moderate mobility: As²⁺, Fe²⁺, and Ni²⁺; (4) high mobility: Cd²⁺ and Hg²⁺. Cd²⁺ with a moderate mobility pattern is synergetic to Fe²⁺ and is more mobile with Ni²⁺. Therefore, Cd²⁺ is separated from the moderate-mobility group and is consigned with Hg, a high-mobility metal. The liner materials suitable for Hg²⁺ are assumed to be suitable for Cd²⁺ as well. Based on this concept, and to reduce heavy metal mobility, wastes should be segregated on compatibility basis according to their heavy metal contents before being disposed in different individual compartments. For wastes containing several incompatible heavy metals, sorting should be based on the heavy-metal with the highest concentration. Another solution is the manufacturing of products using compatible heavy metal combinations and then labeling them accordingly. Such waste segregation and landfill compartmentalization lowers risks of groundwater contamination and liner cost.     

Effect of trace metals on ethanol production from synthesis gas by the ethanologenic acetogen, <Emphasis Type="Italic">Clostridium</Emphasis><Emphasis Type="Italic">ragsdalei</Emphasis>

The effect of trace metal ions (Co²⁺, Cu²⁺, Fe²⁺, Mn²⁺, Mo⁶⁺, Ni²⁺, Zn²⁺, SeO₄ ⁻ and WO₄ ⁻) on growth and ethanol production by an ethanologenic acetogen, Clostridium ragsdalei was investigated in CO:CO₂-grown cells. A standard acetogen medium (ATCC medium no. 1754) was manipulated by varying the concentrations of trace metals in the media. Increasing the individual concentrations of Ni²⁺, Zn²⁺, SeO₄ ⁻ and WO₄ ⁻ from 0.84, 6.96, 1.06, and 0.68 μM in the standard trace metals solution to 8.4, 34.8, 5.3, and 6.8 μM, respectively, increased ethanol production from 35.73 mM under standard metals concentration to 176.5, 187.8, 54.4, and 72.3 mM, respectively. Nickel was necessary for growth of C. ragsdalei. Growth rate (μ) of C. ragsdalei improved from 0.34 to 0.49 (day⁻¹), and carbon monoxide dehydrogenase (CODH) and hydrogenase (H₂ase)-specific activities improved from 38.45 and 0.35 to 48.5 and 1.66 U/mg protein, respectively, at optimum concentration of Ni²⁺. At optimum concentrations of WO₄ ⁻ and SeO₄ ⁻, formate dehydrogenase (FDH) activity improved from 32.3 to 42.6 and 45.4 U/mg protein, respectively. Ethanol production and the activity of FDH reduced from 35 mM and 32.3 U/mg protein to 1.14 mM and 8.79 U/mg protein, respectively, upon elimination of WO₄ ⁻ from the medium. Although increased concentration of Zn²⁺ enhanced growth and ethanol production, the activities of CODH, FDH, H₂ase and alcohol dehydrogenase (ADH) were not affected by varying the Zn²⁺ concentration. Omitting Fe²⁺ from the medium decreased ethanol production from 35.7 to 6.30 mM and decreased activities of CODH, FDH, H₂ase and ADH from 38.5, 32.3, 0.35, and 0.68 U/mg protein to 9.07, 7.01, 0.10, and 0.24 U/mg protein, respectively. Ethanol production improved from 35 to 54 mM when Cu²⁺ was removed from the medium. The optimization of trace metals concentration in the fermentation medium improved enzyme activities (CODH, FDH, and H₂ase), growth and ethanol production by C. ragsdalei.     

Predicting efficiency of different chemical extraction methods in risk assessment of trace metals in sediment of the Persian Gulf

Different chemical methods have been developed to evaluate the bioavailable fraction of the trace metals. Due to the lack of a universal method for analysis of the bioavailable metal fractions, due to the differences in sediment characteristics, it is necessary to validate an appropriate chemical method for assessing the available fraction of trace metals. For this propose, in this study, different chemical extraction methods including extraction with HCl and desorption test as a single reagent leaching test as well as geochemical fractions method have been evaluated. Bushehr coastal sediments in the Persian Gulf coasts have been selected for this purpose. To validate the efficacy of these methods, a gastropod species (Trochus erithreus), as a bioindicator, has been selected and monitored for trace metals of Fe²⁺, Pb²⁺, Cu²⁺, Zn²⁺, Mn²⁺, and Ni²⁺. According to the one-way ANOVA results, all partial extraction methods for all the trace metals (except for Mn²⁺) showed no significant variation, but linear correlation coefficients were between the results of the selective chemical extraction methods and concentrations of trace metals in organism tissues. It offered that selective extraction with HCl can be used as a simple method for measuring the bioavailable fraction of the metals in the sediments.     

Defining the metal specificity of a multifunctional biofilm adhesion protein

The accumulation associated protein (Aap) of Staphylococcus epidermidis mediates intercellular adhesion events necessary for biofilm growth. This process depends upon Zn²⁺‐induced self‐assembly of G5 domains within the B‐repeat region of the protein, forming anti‐parallel, intertwined protein “ropes” between cells. Pleomorphism in the Zn²⁺‐coordinating residues was observed in previously solved crystal structures, suggesting that the metal binding site might accommodate other transition metals and thereby support dimerization. By use of carefully selected buffer systems and a specialized approach to analyze sedimentation velocity analytical ultracentrifugation data, we were able to analyze low‐affinity metal binding events in solution. Our data show that both Zn²⁺ and Cu²⁺ support B‐repeat assembly, whereas Mn²⁺, Co²⁺, and Ni²⁺ bind to Aap but do not support self‐association. As the number of G5 domains are increased in longer B‐repeat constructs, the total concentration of metal required for dimerization decreases and the transition between monomer and dimer becomes more abrupt. These characteristics allow Aap to function as an environmental sensor that regulates biofilm formation in response to local concentrations of Zn²⁺ and Cu²⁺, both of which are implicated in immune cell activity.     

Changes in lipid composition in the tissues of fresh-water plant <Emphasis Type="Italic">Hydrilla verticillata</Emphasis> induced by accumulation and elimination of heavy metals

Qualitative and quantitative composition of lipids was investigated in fresh-water vascular plant Hydrilla verticillata (L. fil.) Royle in the course of the accumulation and elimination of heavy metals (HM). The plants were incubated in 100μM solutions of metal nitrates for 10 days. The accumulation of Cu²⁺, Zn²⁺, and Pb²⁺ and their elimination from the plants depended on the duration of exposure and chemical nature of the metal. Accumulation of lead and copper salts was the greatest on the 3rd day, and zinc, on the 10th day. It was associated with changes in the composition of total lipids, polar lipids, and fatty acid (FA). Copper ions suppressed lipid metabolism stronger than other metals. Zn²⁺ and Pb²⁺ induced the accumulation of biomass and elevated the content of some phospholipids and glycolipids. The detected changes (decrease or increase) were observed both during the incubation with HM and within an afterstress period when the plants recovered in the medium free of metals. Judging by their effect on the content of lipids and FA, HM form a series: Cu²⁺ > Zn²⁺ > Pb²⁺. The responses of plant lipid metabolism to the metals of various chemical nature are discussed.     

The effect of amphotericin B on the permeability of lipid bilayers to divalent trace metals

In this study amphotericin B released the divalent trace metals Zn²⁺, Co²⁺, Cu²⁺, Ni²⁺, Mn²⁺, Fe²⁺, Cd²⁺ and Pb²⁺ from multilamellar liposomes containing cholesterol. This observation is consistent with amphotericin B channels being permeable to these metals, and it is proposed, therefore, that the antibiotic may be useful in investigating the metabolism of these elements.     

Enhanced heavy metal removal from waste water by viable, glucose pretreated Saccharomyces cerevisiae cells

Summary The bioaccumulation of metals (Cu²⁺, Cr⁶⁺, Cd²⁺, Ni²⁺ and Zn²⁺) from three electroplating effluents by viable Saccharomyces cerevisiae, and the effect of glucose treatment on accumulation was determined. Pretreatment of the yeast cells with glucose increased the amount of metal removed, whilst direct addition of glucose to the yeast-effluent solution had no effect on the amount of metal accumulated.     

径流雨水中溶解性有机质特征演化及其对典型污染物迁移和生物有效性的影响

地表径流污染已经逐渐成为城市面源污染的重要组成部分。其中溶解性有机质DOM (Dissolved organic matter)是有机污染物的主要组成部分。DOM中因为含有大量不饱和结构、官能团,其中包括羟基、羧基、羰基、胺基等,这些结构容易与径流中重金属结合发生络合反应,改变重金属的赋存形态,从而对其迁移转化及其生物有效性产生很大的影响。本文以北京市地表径流为研究对象,研究城市地表径流在冬、夏季不同功能区不同下垫面中溶解性有机质特征及其与典型重金属的作用机制,通过荧光淬灭滴定实验,研究夏季径流雨水中DOM的不同组分与重金属Cu~(2+)、Pb~(2+)、Zn~(2+)之间的结合机制。研究结果显示PARAFAC将获得的样品都分解成2类6个不同的组分,1种腐殖酸,1种类蛋白;类蛋白物质的荧光强度与Cu~(2+)、Pb~(2+)的淬灭率要强于腐殖酸,而Zn~(2+)则呈现相反趋势;通过使用二维相关同步光谱发现DOM对重金属Cu~(2+)、Pb~(2+)、Zn~(2+)的敏感性呈现递减趋势,二维相关异步光谱发现Cu~(2+)、Pb~(2+)会先与位于270—300nm附近类蛋白光谱带反应,Zn~(2+)则会先与330nm附近的腐殖酸光谱带反应。     

Effect of metal Ions (Ni<Superscript>2+</Superscript>, Cu<Superscript>2+</Superscript> and Zn<Superscript>2+</Superscript>) and water coordination on the structure of L-phenylalanine,L-tyrosine,L-tryptophan and their zwitterionic forms

Methods of quantum chemistry have been applied to double-charged complexes involving the transition metals Ni²⁺, Cu²⁺ and Zn²⁺ with the aromatic amino acids (AAA) phenylalanine, tyrosine and tryptophan. The effect of hydration on the relative stability and geometry of the individual species studied has been evaluated within the supermolecule approach. The interaction enthalpies, entropies and Gibbs energies of nine complexes Phe•M, Tyr•M, Trp•M, (M = Ni²⁺, Cu²⁺ and Zn²⁺) were determined at the Becke3LYP density functional level of theory. Of the transition metals studied the bivalent copper cation forms the strongest complexes with AAAs. For Ni²⁺and Cu²⁺ the most stable species are the NO coordinated cations in the AAA metal complexes, Zn²⁺cation prefers a binding to the aromatic part of the AAA (complex II). Some complexes energetically unfavored in the gas-phase are stabilized upon microsolvation.     

Trypsin activity in the small intestine of rats after application of copper and zinc

In vivo experiments with Sprague-Dawley rats were conducted in order to explore the influence of Cu²⁺, Zn²⁺ as well as of the combinations of both on the activity of trypsin. The solutions of the trace elements were given per os, the animals were killed 30 min after the applications, and the activity of trypsin was determined in the juice of the small intestine by usingN ^α-benzoyl-L-arginine-p-nitroanilide (L-BAPA) as the substrate. The activity of trypsin depends on the concentration of the trace elements. When Cu²⁺ ions are applied, there is a minimum activity at 10⁻⁵ mol Cu²⁺/L and a maximum at 10⁻⁴ mol Cu²⁺/L. When giving Zn²⁺ ions, a minimum of trypsin activity is found at 10⁻⁵ mol Zn²⁺/L and a maximum at 5×10⁻⁶ mol Zn²⁺/L. On the whole, the trypsin activity is lower when the Cu²⁺/Zn²⁺ combinations are applied compared to the addition of the single trace elements. On principle, a good conformity of the in vivo results was found with in vitro results.     

Heavy metal ions affect the activity of DNA glycosylases of the Fpg family

Prokaryotic enzymes formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei) and their eukaryotic homologs NEIL1, NEIL2, and NEIL3 define the Fpg family of DNA glycosylases, which initiate the process of repair of oxidized DNA bases. The repair of oxidative DNA lesions is known to be impaired in vivo in the presence of ions of some heavy metals. We have studied the effect of salts of several alkaline earth and transition metals on the activity of Fpg-family DNA glycosylases in the reaction of excision of 5,6-dihydrouracil, a typical DNA oxidation product. The reaction catalyzed by NEIL1 was characterized by values K _m = 150 nM and k _cat = 1.2 min⁻¹, which were in the range of these constants for excision of other damaged bases by this enzyme. NEIL1 was inhibited by Al³⁺, Ni²⁺, Co²⁺, Cd²⁺, Cu²⁺, Zn²⁺, and Fe²⁺ in Tris-HCl buffer and by Cd²⁺, Zn²⁺, Cu²⁺, and Fe²⁺ in potassium phosphate buffer. Fpg and Nei, the prokaryotic homologs of NEIL1, were inhibited by the same metal ions as NEIL1. The values of I₅₀ for NEIL1 inhibition were 7 μM for Cd²⁺, 16 μM for Zn²⁺, and 400 μM for Cu²⁺. The inhibition of NEIL1 by Cd²⁺, Zn²⁺, and Cu²⁺ was at least partly due to the formation of metal-DNA complexes. In the case of Cd²⁺ and Cu²⁺, which preferentially bind to DNA bases rather than phosphates, the presence of metal ions caused the enzyme to lose the ability for preferential binding to damaged DNA. Therefore, the inhibition of NEIL1 activity in removal of oxidative lesions by heavy metal ions may be a reason for their comutagenicity under oxidative stress.     

Enzymatic activity of metal-binding proteins in epidermal cells

Summary Enzymatic activity was investigated in metal-binding proteins from rat epidermal cells. Tris-HCl buffer soluble and KSCN solubilized proteins were extracted stepwise from granular and cornified cells of 2-day old rat epidermis. Each extract was separately applied to a Cu²⁺ or Zn^2– chelate Sepharose 6B column and the proteins were eluted with buffers of different pHs and finally with EDTA solution. Metal chelate-binding proteins were found in both soluble and solubilized proteins but there was a larger amount in the latter. Affinity of the proteins to bind with Cu²⁺ chelate was greater than that with Zn²⁺ chelate. In Tris-HCl buffer extract, histidase activity was detected in Cu²⁺ chelate-binding proteins, but not in Zn²⁺ chelate-binding proteins. Acid phosphatase, cysteine proteinase, dipeptidase, cathepsin D, -galactosidase, gelatin hydrolase, and superoxide dismutase did not bind to metal chelates although these enzymes, except acid phosphatase, were inhibited by Cu²⁺, but not by Zn²⁺. In contrast, KSCN solubilized metal chelate-binding proteins showed plasminogen activator, acid phosphatase, and gelatin and casein hydrolases while histone hydrolase did not bind to either chelate column. Since metal-binding proteins in rat epidermal cells have been shown previously to be histidine- and cysteine-rich proteins concentrated in keratohyalin granules, interaction of metals and the structural proteins with certain enzymes may be involved in the regulation of epidermal cell functions.     

Association of aureolic acid antibiotic, chromomycin A3 with Cu2+ and its negative effect upon DNA binding property of the antibiotic

Here we have examined the association of an aureolic acid antibiotic, chromomycin A3 (CHR), with Cu²⁺. CHR forms a high affinity 2:1 (CHR:Cu²⁺) complex with dissociation constant of 0.08 × 10⁻¹⁰ M² at 25°C, pH 8.0. The affinity of CHR for Cu²⁺ is higher than those for Mg²⁺ and Zn²⁺ reported earlier from our laboratory. CHR binds preferentially to Cu²⁺ in presence of equimolar amount of Zn²⁺. Complex formation between CHR and Cu²⁺ is an entropy driven endothermic process. Difference between calorimetric and van’t Hoff enthalpies indicate the presence of multiple equilibria, supported from biphasic nature of the kinetics of association. Circular dichroism spectroscopy show that [(CHR)₂:Cu²⁺] complex assumes a structure different from either of the Mg²⁺ and Zn²⁺ complex reported earlier. Both [(CHR)₂:Mg²⁺] and [(CHR)₂:Zn²⁺] complexes are known to bind DNA. In contrast, [(CHR)₂:Cu²⁺] complex does not interact with double helical DNA, verified by means of Isothermal Titration Calorimetry of its association with calf thymus DNA and the double stranded decamer (5′-CCGGCGCCGG-3′). In order to interact with double helical DNA, the (antibiotic)₂ : metal (Mg²⁺ and Zn²⁺) complexes require a isohelical conformation. Nuclear Magnetic Resonance spectroscopy shows that the Cu²⁺ complex adopts a distorted octahedral structure, which cannot assume the required conformation to bind to the DNA. This report demonstrates the negative effect of a bivalent metal upon the DNA binding property of CHR, which otherwise binds to DNA in presence of metals like Mg²⁺and Zn²⁺. The results also indicate that CHR has a potential for chelation therapy in Cu²⁺ accumulation diseases. However cytotoxicity of the antibiotic might restrict the use.     

Enhanced heavy metal immobilization by a bacterial-chitosan complex in soil

Soil contaminated with heavy metals (e.g., Pb²⁺, Cu²⁺, and Zn²⁺) was treated with aminopoly-saccharide chitosan alone or a strain of the bacterium Bacillus subtilis to determine their effects on metal ion accumulations. Phosphatase and Chitosanase production were also assayed. The combined bacterial-chitosan treatment showed the greater accumulation of Zn²⁺ followed by Cu²⁺ as compared to single treatments, while Pb²⁺ did not show a marked accumulation by either single or combined treatments.     

The Effects of Metal Ions on the Cytotoxicity and Selectivity of a Histidine-Containing Lytic Peptide

The histidine-containing peptide L5C (PAWRHAFHWAWHMLHKAA) is a histidine-rich lytic peptide. Interactions of some divalent metal ions with peptide L5C and their effects on the cell lysis activity of the peptide were studied. The presence of Cu²⁺ caused a secondary structure change (from random coil to α-helix) which resulted in the loss of cell lysis activity in peptide L5C. Binding of Zn²⁺ to peptide L5C also reduced the lytic activity of the peptide but Zn²⁺ did not affect the secondary structure of the peptides. Instead, Zn²⁺ induced peptide L5C aggregation. Unlike Zn²⁺ and Cu²⁺, Mg²⁺ had no significant effect on the activity of peptide L5C. Further experiments revealed that formed ion-peptide L5C complexes were sensitive to pH and dissociated in acidic solutions. Peptide L5C demonstrated improved pH-selectivity in the presence of trace amount of Zn²⁺. This property of histidine-containing lytic peptides can be used to improve their therapeutic effectiveness in the treatment of cancers.     

Enhanced heavy metal immobilization by a bacterial-chitosan complex in soil

Soil contaminated with heavy metals (e.g., Pb²⁺, Cu²⁺, and Zn²⁺) was treated with aminopoly-saccharide chitosan alone or a strain of the bacterium Bacillus subtilis to determine their effects on metal ion accumulations. Phosphatase and Chitosanase production were also assayed. The combined bacterial-chitosan treatment showed the greater accumulation of Zn²⁺ followed by Cu²⁺ as compared to single treatments, while Pb²⁺ did not show a marked accumulation by either single or combined treatments.