formation, hard-soft acids and bases, and, more recently, association with eutrophication and environmental toxicity. Metals have also been classified into three.
Agric. Rev., 31 (2) : 133 - 138, 2010
AGRICULTURAL RESEARCH COMMUNICATION CENTRE
www.arccjournals.com / indianjournals.com
MICROBIAL MECHANISMS OF HEAVY METAL TOLERANCE- A REVIEW M. Gomathy and K.G. Sabarinathan1 Tamil Nadu Agricultural University, Coimbatore - 641 003, India
ABSTRACT Potential cellular mechanisms may be involved in the resistance and tolerance of microorganisms to excess concentrations of heavy metals in the environment. Generally, the strategy adopted by microorganisms aims to avoid the build up of excess metal levels, and thus to prevent the onset of toxicity symptoms. This is achieved by the use of various mechanisms that are present and likely to be employed in general metal homeostasis. It appears likely that specific mechanisms are employed for specific metals in particular species. Research carried out on this area is discussed in this review paper.
Key words: Microbiwal mechanisms, Heavy metal INTRODUCTION Metal pollution is a global concern. The levels of metals in all environments, including air, water and soil, are increasing, in some cases to toxic levels, with contributions from a wide variety of industrial and domestic sources. For example, anthropogenic emissions of lead, cadmium, vanadium, and zinc exceed those from natural sources up to 100 fold. Metal contaminated environments pose serious health and ecological risks. Metals, such as aluminum, antimony, arsenic, cadmium, lead, mercury, and silver, cause conditions including hypophosphatemia, heart disease, liver damage, cancer, neurological and cardiovascular diseases, central nervous system damage, encephalopathy, and sensory disturbances. Because of their toxic nature, metals are not as amenable to bioremediation as organics. The chemical nature and bioavailability of a metal can be changed in nature but remain the same because metals are neither thermally decomposable nor microbiologically degradable. Because of the toxicity and the ubiquity of metals in the environment, bizarre 1
ways of dealing with unwanted metals. Some microorganisms have mechanisms to sequester and immobilize metals, whereas others actually enhance metal solubility in the environment. Classes of metals There are three classes of metals: metals, metalloids, and heavy metals. Metals, in general, are a class of chemical elements that form lustrous solids that are good conductors of heat and electricity. However, not all metals fit this definition, for example mercury is a liquid. Metals such as arsenic, boron, germanium, and tellurium are generally considered metalloids of semimetals in that their properties are intermediate between those of metals and those of nonmetals. Heavy metals are defined in a number of ways based on cationic-hydroxide formation, a specific gravity greater than 5 g / ml, complex formation, hard-soft acids and bases, and, more recently, association with eutrophication and environmental toxicity. Metals have also been classified into three additional classes on the basis of their biological functions and effects: 1) Essential metals with known
Present address: Research and Development, Covalance Inc., Arizona, USA
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biological functions, 2) Toxic metals, and 3) Nonessential, nontoxic metals with no known biological effects. The following metals are currently known to have essential functions in microorganisms: Na, K, Mg, Ca, V, Mn, Fe, Co, Ni, Cu, Zn, Mo, and Cr is also thought to be essential, although this is still in dispute. Metal such as Na, K, Mg, and Ca are required by all organisms. The toxic metals include those with no known biological function. These include Ag, Cd, Sn, Au, Hg, Ti, Pb, Al and the metalloids Ge, As, Sb, and Se. The metalloids exert different toxic effects than the metals because they have different chemistries. Metals are predominantly present as cationic species and metalloids are predominantly present as anionic species. Metal bioavailability in the environment Metals in the environment can be divided into two classes bioavailable (soluble, nonsorbed, and mobile) and nonbioavailble (precipitated, complexed, sorbed, and nonmobile). Much of the research on metal bioavailability has been done in soil systems because understanding the fate of metals in soil and sediments is crucial in determining metal effects on biota, metal leaching to groundwater, and metal transfer to the food chain. The environmental hazards posed by metals are directly linked to soil solution. High metal concentrations in the soil solution results in greater plant uptake and leaching of metals, while metals that are retained in the soil solid phase pose a greatly reduced environmental hazard. Soils usually exhibit higher concentrations of metals than water because metals are more likely to accumulate in soil versus being diluted or carried elsewhere in water. The cation exchange capacity of soils allows metals to attach to soil particles in response to ionic attractions and accumulate. Several abiotic and biotic factors can affect the chemical speciation of metal in soil, and thus, affect the bioavailability and toxicity of metals to microbial populations. These factors include metal chemistry, sorption to clay minerals and organic
matter, pH, redox potential, and the microorganisms present. All of these factors interact to influence metal speciation, bioavailability, and the overall toxicity of metal in the environment. Thus, it must be emphasized that determination of the total concentration of a metal in a soil is not enough to predict toxicity in biological systems. Mechanisms of microbial metal resistance and detoxification Some microorganisms are believed to have evolved metal resistance because of their exposure to toxic metals shortly after life began. Others are believed to have evolved metal resistance in response to recent exposure to metal pollution over the past 50 years. The pollution of the environment with anthropogenic sources of metals has increased the need for research concerning microbial metal resistance as well as remediation. Microorganisms directly influence the fate of metals in the environment and may provide the key to decreasing already existing contamination. In response to metals in the environment, microorganisms have evolved ingenious mechanisms of metal resistance and detoxification. Some resistance mechanisms are plasmid encoded and tend to be specific for a particular metal. Others are general conferring resistance to a variety of metals. Extracellular polymeric substances (EPSs) Slime layers are composed of carbohydrates, polysaccharides, and sometimes nucleic acids and fatty acids which offer protection against desiccation, phagocytosis, and parasitism. Exopolymers or extracellular polymeric substances (EPSs) are common in natural environments. Many microorganisms produce extra cellular polysaccharides that strongly bind metals. They mobilize or immobilize the toxic metals and processes are important in metal cycling. Microbial exopolymers are particularly efficient in binding heavy metals, such as lead, cadmium and uranium. Interaction with metal ions is generally considered a direct consequence of the presence of negatively
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charged functional groups on the exopolymer. These groups include, phosphate, hydroxyl, succinyl, and uronic acids. The immobilization of lead by exopolymers has been observed in several bacterial genera, including Staphylococcus aureus, Micrococcus luteus, and Azotobacer spp. Siderophores A second extracellular molecule produced microbially that complex metals is the siderophore. Siderophores are iron-complexing, low-molecularweight organic compounds. Their biological function is to concentrate iron in environments were concerntration is low and to facilitate its transport into the cell. Two major types are generally considered, the hydroxamate and catecholate siderophores. Hydroxamate groups strongly bind ferric ion. For example, aluminium, gallium, and chromium form trivalent metal ions of similar size to iron. Aluminium may be a specific competitor for catecholate siderphores. Siderophores have a high affinity (Kb’1030) for ferric iron (Garrison and Crumbliss, 1987). They will also form complexes with metals other than Fe, although with a lower affinity. The effects on metal uptake and toxicity are dependent on this siderophore-metal complex being recognized by an uptake receptor (Huyer and page, 1988). For example, production of the siderophore decreased the toxicity of copper to the cyanobacterium Anabaena sp. but increased the toxicity of copper to Bacillus megaterium. The presence of metals other than iron is known to stimulate siderophore formation in a number of bacteria and fungi (Winkelmann et al., 1973 and Huyer et al., 1988). In addition to aluminum, exposure to copper, chromium, cadmium, and zinc also increase siderophore production in B. megaterium (Byers et al., 1967 and Davis et al., 1971 Siderophore formation in response to heavy metal exposure may have both beneficial and detrimental effects. It may lower the free metal concentration and provide a protective effect if the
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uptake receptor discriminates against the metalsiderophore complex. Biosurfactants Biosurfactants are a class of compounds produced by many microbes that in some cases are excreted. Recently biosurfactants have been investigated for their ability to complex metals such as cadmium, lead, and zinc. Biosurfactant complexation can actually increase the apparent solubility of metals, however, the biosurfactantcomplexed metal is not toxic to cells. Evidence showed that biosurfactant-producing microorganisms can be isolated in greater diversity from metal-contaminated environments than from uncontaminated ones (Miller, 1995). Finally, metal bioavailability can be influenced by common metabolic by-products that result in metal reduction. In this case, soluble metals are reduced to less soluble metal salts, including sulfidic and phosphidic precipitates. For example, under aerobic conditions Citrobacter spp, can enzymatically produce phosphate which results in the precipitation of lead and copper. Under anaerobic conditions, high H2S concentrations from sulfate reducing bacteria, e.g., Desulfovibrio sp., readily cause metal precipitation. Metal dependent mechanisms of resistance Metallothioneins Metallothioneines, discovered about 45 years ago, play a central role in heavy metal metabolism and in the management of various forms of stress. Metallothioneines (MTs) are low molecular weight (6-7 kDa), cystine-rich proteins, divided into three different classes on the basis of their cystine content and structure. The Cys-Cys, Cys-X-Cys and Cys-XX-Cys, motifs are characteristic and invariant for metallothioneine. Metal regulatory transcriptional factor-1 (MTF-1) was essential for basal and heavy metal induced transcription of the stress- responsive metallothionein-I and metallothionein-II. Metallothionein like proteins have been isolated from the Cyanobacterium, Syneococcus spp as well as
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E. coli and Pseudomonas putida . Primarily documented in higher microorganisms, plants, algae, yeast, and some fungi, metallothioneins are low molecular weight, cysteine-rich proteins with a high affinity for cadmium, zinc, copper, silver and mercury metals. Their production is induced by the presence of metals, and their primary function is meal detoxification. Metal binding by metallothioneins can result in cellular accumulations visible as electron dense areas within the cell matrix. Suspected deposits are confirmed using electron dispersive spectroscopy which can identify the metal. Roane and Pepper (2000) reported that plasmid-encoded energy dependent metal efflux systems involving ATPases and chemiosmotic ion/ proton pumps are associated to Ar, Cr and Cd resistance in Staphylococcus aureus, Bacillus subtilis, Listeria spp, E.coli, Alcaligenes eutroplnts P.putida, some cyanobacteria, fungi and algae. Methylation of metals The methylation of metals is considered a metal dependent mechanism of resistance because only certain metals are involved. Methylation generally increases metal toxicity as a result of increased lipophilicity, thus increased permeation across cell membranes. However, metal volatilization facilitates metal diffusion away from the cell, and in this way effectively decreased metal toxicity. Metal volatilization has been observed with lead, m e r c u r y, t i n , s e l e n i u m a n d a r s e n i c . Fo r example, mercury (Hg 2+) is readily oxidized to the volatile and very toxic forms methylmercury and dimethylmercury, which can then diffuse away from the cell. Methylation of metals has been known to remove significant amounts of metal from contaminated surface waters, sewage, and soils. Mercury resistance may involve the enzymatic reduction of Hg 2+ to elemental mercury (Hg 0) in both gram-positive and gram-negative bacteria.
Biosorption mechanisms The complex structure of microorganisms implies that there are many ways for the metal to be taken up by the microbial cell. According to the dependence on the cell’s metabolism, biosorption mechanisms can be divided into: Metabolism dependent and Non -metabolism dependent. Transport of the metal across the cell membrane yields intracellular accumulation, which is dependent on the cell’s metabolism. It is often associated with an active defense system of the microorganism, which reacts in the presence of toxic metal. During non-metabolism dependent biosorption, metal uptake is by physico-chemical interaction between the metal and the functional groups present on the microbial cell surface. This is based on physical adsorption, ion exchange and chemical sorption, which is not dependent on the cells’ metabolism. Cell walls of microbial biomass, mainly composed of polysaccharides, proteins and lipids have abundant metal binding groups such as carboxyl, sulphate, phosphate and amino groups. This type of biosorption, i.e., non-metabolism dependent is relatively rapid and can be reversible (Kuyucak and Volesky (1988) and Ercole et al. (1994)). Extracellular Processes Organic or inorganic acids produced by microorganisms, including Thiobacillus, Serratia, Pseudomonas, Bacillus, Penicillium, and Aspergillus, are able to extract metals from solid substrates. Toxic metals can be mobilized under anaerobic conditions as co precipitates with iron oxides. This may be an important process, since co precipitation of toxic metals with ferric iron is a widely used treatment for high metal content waste streams. Metals closely associated with iron (Cd and Zn) were solubilized enzymatic reduction of ferric iron, whereas others (particularly Pb) are solubilized by the indirect action of bacterial metabolites. Many metal salts are insoluble and their formation results in immobilization of toxic metals by sequestering to
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sediments or adsorption to soil particles. Frequently metals form insoluble complexes with, for example, hydroxides, carbonates, phosphates, and sulfides. Probably the best known microbial immobilization process is sulfide production by the sulfate reducing bacteria. Intracellular Accumulation Concentration of metals within bacterial and other microbial cells can result from interaction with surface ligands followed by slow transport into the cell. This may be an important form of detoxification or a means of incorporating specific metals into enzymes (e.g., Cu and Zn). Bacterial assimilation of metals may be important in detoxification, enzyme function, and physical characteristics of the cell. Extra cellular or cell wall- attached ligands are thought to bind toxic metals. These ligands transport the complexed metals through the cell wall in a slow transport step. The metals are released inside the cell, incorporated into biochemical pathways, or trapped in an inactive form by complexation with another high-affinity ligand. The protoplasm of E. coli typically contains 0.3% trace elements, including manganese, cobalt, copper, zinc, and molybdenum. The ability of certain bacterial cells to accumulate metals intracellular has been exploited in mining practices, particularly in management of effluent treatment lagoons. Uranium has been shown to accumulate rapidly in cells of Saccharomyces cerevisiae and P. aeruginosa. Ion Exchange Cell walls of microorganisms contain polysaccharides and bivalent metal ions exchange with the counter ions of the polysaccharides. For example, the alginates of marine algae occur as salts of K+, Na+, Ca2+, and Mg2+ (Rubinelli et al., 2002). These ions can exchange with counter ions such as C02+, Cu2+, Cd2+and Zn2+ resulting in the biosorptive uptake of heavy metals (Kuyucak and Volesky 1988). The metal removal from solution may also take place by complex formation on the cell surface after the interaction between the metal and the active groups.
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Precipitation Precipitation may be either dependent on the cellular metabolism or independent of it. In the first case, the metal removal from solution is often associated with active defense system of the microorganisms. They react in the presence of toxic metal producing compounds, which favour the precipitation process. In the case of independent precipitation it may be a consequence of the chemical interaction between the metal and the cell surface. Complexation The metal removal from solution may also take place by complex formation on the cell surface after the interaction between the metal and the active groups. Aksu et al .( 1992) hypothesized that biosorption of copper by C. vulgaris and Z. ramigera takes place through both adsorption and formation of coordination bonds between metals and amino and carboxyl groups of cell wall polysaccharides. Complexation was found to be the only mechanism responsible for calcium, magnesium, cadmium, zinc, copper and mercury by Pseudomonas syringae. Microorganisms may also produce organic acids (e.g., citric, oxalic,gluonic, fumaric, lactic and malic acids), which may chelate toxic metals resulting in the formation of metallo-organic molecules. These organic acids help in the solubilization of metal compounds and their leaching from their surfaces (Nies, 1999). Metals may be biosorbed or complexed by carboxyl groups found in microbial polysaccharides and other polymers. Transformation and volatilization of metals Pure culture experiments have shown that many bacteria, including Clostridium, Neurospora, Pseudomonas, Bacillus, Mycobacterium, E. coli, Aerobacter aerogenes, B. megaterium, and a number of fungi have the capability to methylate mercury. However, the sulfate - reducing bacteria (SRB) are the most significant. Methylation of arsenic by fungi has been studied extensively as a result of human poisoning by fungal transformations of arsenic in
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paints. More recent work has been concerned with transformation and mobilization in sediments and soils. The importance of biomethylation cannot be overstressed because of the potential health consequences of high methylation rates. CONCLUSION These processes involved in reducing toxicity are of considerable current interest because an understanding of the means of manipulating metal tolerance could be important for identifying
microorganisms for bioremediation purposes, particularly for highly contaminated sites. However, the evidence reviewed strongly suggests that there is no single mechanism that can account for tolerance to a wide range of metals. It is also possible that more than one mechanism may be involved in reducing the toxicity of a particular metal. Although adaptive tolerance appears to be under relatively simple genetic control, tolerance to individual metals involves distinct metal-specific mechanisms.
REFERENCES Aksu, Z.(1992)The biosorption of copper (II) by C. Vulgaris and Zramigera. Environ. Technol., 13: 579-586 Byers, B. R., Powell M.V., and Lankford C.E. (1967) Iron-chelating hydroxamic acid (schizokinen) active in initiation of cell division in Bacillus megaterium. J. Bacteriol., 93:286–294. Davis, W. B., McCauley M.J. and Byers B.R.. (1971) Iron requirements and aluminum sensitivity of a hydroxamic acid-requiring strain of Bacillus megaterium.J. Bacteriol., 105:589–594. Ercole, C. VegIio, F. Toro L., Ficara, G and. Lepidi, A. (1994). Immobilisation of microbial cells for metal adsorptin and desorption. In: Mineral Bioprocessing II. Snowboard. Utah Gadd, G.M. (1988) Heavy metal and radionucIide by fungi and yeasts. In: P.R. Norris and D.P. Kelly (Editors), Biohydrometallurgy. A. Rowe, Chippenham, Wilts., U.K. Garrison, J. M., and Crumbliss A.L.. (1987). Kinetics and mechanism of aluminum(III)/siderophore ligand exchange: mono(deferriferrioxamine B) aluminum(III) formation and dissociation in aqueous acid solution. Inorg. Chim. Acta., 138:61–65. Huyer, M., and Page W.J. (1988) Zn21 increases siderophore production in Azotobacter vinelandii. Appl. Environ. Microbiol., 54:2625–2631. Kuyucak, N. and Volesky, B. (1988).Biosorbents for recovery of metals from industrial solutions. Biotechnol. Left., 10 (2): 137-142 Miller, R.. (1995) Biosurfactant-facilitated remediation of metal contaminated soils, Environ, health perspec., 103:59-62. Nies, D. H. (1999) Microbial heavy metal resistance. Appl. Microbial. Biotechnol. 51:730-750 Roane, T. M. and Pepper, I. L. (2000).Microorganisms and metal pollution, In environmental Microbiology, edited by Maier R M, Pepper I L and Gerba. C B (Academic Press, London, NW 1 7BY.UK), 55. Rubinelli, P., Siripornadulsil, S., Gao-Rubinelli, F., and Sayre, R.T. (2002). Cadmium- and iron-stress-inducible gene expression in the green alga Chlamydomonas reinhardtii: Evidence for H43 protein function in iron assimilation. Planta 215: 1–13. Winkelmann, G., A. Barnekow, D. Ilgner, and H. Za¨hner. (1973) Stoffwechselprodukte von Mikroorganismen. 120. Mitteilung. Eisenaufnahme bei Neurospora crassa. Arch. Mikrobiol. 92:285–300.
