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View with images and charts Bioremediation of Metal Contamination – It’ S Future Use In Bangladesh Introduction and Literature Review Metal contamination Metal contamination 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 contribution from a variety of industrial and domestic sources. For example, anthropogenic emission of lead, cadmium, vanadium, and zinc exceed these from natural sources by 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 hypophosphetema, heart disease, central nervous system damage, cancer, neurological and cardiovascular diseases. However, microbial activity in the sediment converted the elemental mercury into the highly toxic and bio-available methylmercury which accumulated in fish. Lead is a second metal of concern especially because lead poisoning of children is common and leads to recantation and semi-parmanent brain damage. Because of the toxic nature, metals are not as amenable to bioremediation as organics. Unlike organics metals are persistent in the environment and can’t be degraded through biological, chemical, or physical means to an innocuous by product . The chemical nature and, thus bioavailability of a metal can be changed through oxidation and reduction; however the chemical nature remains the same because metals are neither thermally decomposable nor microbiologically degradable. Consequently, metals are difficult to remove from the environment. Because metal bioavailability is strongly dependent on environmental components, such as pH, redox potential, and organic content, there is a discrepancy between the total and the bioavailable amount of a metal present. In environmental samples, a bio-available metal is generally soluble and not sorbed to colloids or soil surfaces, and thus mobile. Moreover, because of the toxicity and ubiquity of metals in the environment, microbes have developed unique and sometimes bizarre ways of dealing with unwanted metals. Some microorganisms have mechanisms to sequester and immobilized metals, whereas others actually enhance metal solubility in the environment. Metals most commonly associated with metal pollution include Arsenic (Ar ),Cadmium (Cd), Copper (Cu ), Chromium (Cr), Mercury (Hg), and Zinc (Zn) Bioremeadition In general, the term bioremediation can be defined as technology employing living organisms for removal of pollutant from the environment. Bioremediation can be defined as “ A method of monitoring natural progress of degradation to ensure that the contaminant decreases with sampling time (Bioattenuation)”.

The intentional stimulation of resident xenobiotic-degrading bacteria by electron acceptors, water, nutrient addition or electron donors (Bioaccumulation), or The addition of laboratory grown bacteria that has appropriate degrading abilities (Bioaugmentation). Sources of metals Metal pollution results when human activity disrupts normal biogeochemical activities or results in disposal of concentrated meal waste. Sometimes a single metal is involved but more often mixtures of meals are present. Examples of processes that produce metal by-product include 1. Mining 2. Ore refinement 3. Nuclear processing 4. Industrial manufacturing of batteries, metal alloys, electrical components, paints, preservatives, and pesticides. Examples of specific metal contaminants include Cu and Zn salts that are used extensively as pesticides in agriculture purpose; Pb which is utilized in the production of batteries, cable sheathing and alloys, Hg compounds that’s that are used in utensils electrical equipments, thermometers , and as preservatives in pharmaceuticals and cosmetics. Metal bioavailability in the environment Metal in the environment can be classified into two classes 1. Bio-available (soluble, nonsorbed, and mobile) 2. Nonbio-available ( precipited, sorbed, complexed and nonmotile) The environmental hazards posed by metals are directly linked to their mobility and thus their concentration in the solution. High metal concentrations in the soil solution results in the greater plant uptake, transfer up the food chain, and thus accumulation of toxicity in biological system and/leaching of metals to groundwater; however, metals that are remained in the soil solid phase pose a greatly reduced environmental hazard. Nonetheless, such a metal deposited soil provide a metal sink from which surface waters, groundwater can become contaminated. Metal transformations by microbial activity may also occur at the surface of soil particle or colloid or in the soil solution. Several a biotic and biotic factors can affect the bioavailability and overall toxicity of metals in the environment. These factors include metal chemistry, sorption to clay minerals and organic matter, pH redox potential and the microorganisms present. Metal Chemistry Weather a metal is cationic or anionic in nature determines its fate and bioavailability in the environment. Most metals are cations that mean that they exhibit positive charge when in their free ionic state and are most reactive with negatively charged surfaces. Thus in the soil, cationic metals such as Pb²+ or Ca²+ strongly interact with the negatively charges on clay minerals and with anionic salts, such as phosphates and sulphates. Positively charged metals are additionally attracted to negatively charged functional groups such as hydroxides and thiols on humic residues.

Unfortunately, cationic metals are also attracted to negatively charged cell surfaces where they can be taken up and cause toxicity. Cationic metals adsorb to both soil particles and cell surfaces with with various strength, termed absorption affinity. For example, of the common soil cations, aluminum binds more strongly calcium or magnesium. Al 3+> Ca 2+ = Mg 2+> K +> Na + The size and charge of the cationic metal determines the strength of adsorption. For example, Al 3+ has much strong affinity for clay particles that it is primarily found as Al(OH) 3, and therefore has extremely low bioavailability. In contrast, negatively charged or anionic metals such as AsO43-(arsenate) are attracted to positively charged surfaces Cation Exchange Capacity/Sorption to clay minerals and organic matters One of the most important factors affecting metal bioavailability is the soil cation exchange capacity (CEC), which is dependent on both the organic matter and clay content of the soil. Cation exchange reflects the capacity for a soil to adsorb metals. Thus, the toxicity of metals within soils high CEC (organic and clay soils) is often low even at high metal concentrations. In contrast, sandy soils with low CEC, and therefore low metal binding capacity, show decreased microbial activity at comparatively low total metal concentrations, indicative of metal toxicity. Redox Potential Metal bioavailability changes in response to changing redox conditions. Under oxidizing or aerobic conditions(+800 to 0 mV), metals are usually found as soluble cationic forms, for example Cu2+, Cd2+, Pb2+, and Ca2+ . In contrast, reduced or anerobic condition (0 to – 400 mV), such as those found in sediments or saturated soils, often results in metal precipitation. pH For cationic metals, the pH of a system can have an influence on metal solubility, and, hence, metal bioavailability. At high pH, metals are predominantly found as insoluble metal mineral phosphates and carbonates: while at the low pH they are more commonly found as free ionic species or as soluble organometals. The pH of a system also affects metal sorption to soil surfaces. The effect of increased soil pH is to decrease metal bioavailability. In contrast, as soil pH decreases, metal solubility is increased, enhancing metal bioavailability. Microorganisms Present Most microorganisms are negatively charged. Metals can attach with negatively charged microbes. The metals can be taken up by microorganisms. Many microorganisms can neutralize the toxicity of metals. In response to metal toxicity, overall community number and diversity decreases. Mechanism of microbial metal detoxification In response to metals in the environment, many microorganisms have evolved unique mechanisms to resist and detoxify harmful metals. Some resistance mechanisms are plasmid mediated and tend to be specific for a particular metal. Others are general conferring resistance to a variety of metals.

Microbial metal resistance may be divided into three categories. These include resistance mechanisms that (1) are general and do not require metal stress; (2) are dependent on a specific metal for activation; and (3) are are general and are activated by metal stress. General mechanisms of metal resistance Slime Layer Production Binding of metals to extracellular materials immobilizes the metal and prevents its entry into the cell. Metal binding to anionic cell surfaces occurs with a large number of cationic metals, including Cd, Pb, Zn and Fe. For example, algal surface contain carboxylic, amino, thio, hydroxyl and hydroxycarboxylic groups that strongly bind metals. Phosporyl groups and phospholipids in bacterial lipopolysaccharides in the outer membrane also strongly interact with cationic metals. Microbial Exopolymer Microbial exopolymers are particularly efficient in binding heavy metals, such as Pb, Cd and U. For example, the immobilization of Pb by exopolymers has been demonstrated in several bacterial genera, including Staphylococcus aureus, Micrococcus luteus, and Azotobacter spp In fact, extracellular polymeric metal binding is the most common resistance mechanism against Pb. Siderophore A second extracellular molecule produced microbially those complexes metals is the siderophore. Siderophores are iron-complexing, low molecular weight compounds that concencntrate irons into the cell. Siderophores may interact with other metals that have chemistry similar to that of iron, such as Al, Ga, and Cr (which form trivalent ions similar in size to iron). By binding metals, siderophores can reduce metal bioavailability and there by metal toxicity. For example, siderophore complexation reduces Cu toxicity in cyanobacteria. Biosurfactants Biosurfactants are a class of metal compounds produced by many microbes. Biosurfactant complexation with metal increases the apparent solubility of metals. Moreover, biosurfactantcomplexed metal is not toxic to cells, and that’s why it is assumed that microorganisms produce biosurfuctants as a means of reducing metal toxicity. Metabolic by Product 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. For example, toxic Cr(IV)is reduced to a less toxic form Cr(III) by cellular reductases. Under aerobic condition Citrobacter spp. can enzymatically produce phosphate which results in the precipitation of Pb and Cu. Under anerobic conditions, high H 2S concentrations from sulfate reducing bacteria, e.g Desulfovibrio spp. readily cause metal precipitation. Metal dependent mechanisms of resistence Metallothioneins Intracellular metal resistance mechanisms in bacteria involve binding or sequestration by metallothioneins or similar proteins. These are low molecular weight cysteine rich proteins with a high affinity for Cd, Zn, Cu, Ag, and Hg metals. Their function is metal

detoxification .These proteins have been isolated from Cyanobacteria, Syneehococeus spp, Escheriachia coli, Pseudomonas putida. Energy Dependent metal Efflux Systems These systems involve ATPase and others are chemi-osmotic ion/proton pumps. Arsenate (AsO43-, Cr, and Cd are the three metals most commonly associated with efflux resistance in both gram-positive and gram-negative bacteria. Future use of bioremediation of contaminating metals in Bangladesh Bangladesh is located in an area with a high population density, poor sanitation, limited natural resource and industries developed without proper effluent treatment. Fortunately enough we have a good collection of microbes that can be used for effluent treatment, specially metal removal. Some Microorganisms Involved in Metal Removal/Recovery from Industrial waste water Microorganism removed/ recovered


Pseudomonas spp, Pediococcus spp.


Saccharomyces cerevisiae

Uranium and other metals

Rhizopus arrhizus


Chlorella vulgaris

Gold, Zinc, Copper, mercury

Aspergillus oryzae


Aspergillus niger

Copper, Cadmium, Zinc

Pecicillium spinulosum


The benefit of metal-microbial interaction

Although microorganisms can sometimes increase metal toxicity, solubility, and mobilization, microorganisms can also can be used to reduce metal waste bioavailability, which can be exploited for mining purpose. Certain microorganigms such as Thiobacillus ferrooxidans, can facilitate the removal of metals from soil through metal solubilization or leaching. Bioleaching is used in the commercial recovery of economically valuable metals (Cu, Pb and U) from low grade ores. Metals recovery by leaching can be concentrated by complexation with chelating agents or precipitation with lime. There are several types of leaching such as Heap leaching Heapleaching usually involves excavating a long narrow ditch about 30cm in depth and lining it with polyethylene. Air lines are evenly placed along the ditch to provide aeration. The ditch is filled with crushed ore to a height of 2 to 3 cm and then saturated with acidified water (pH ranging from 2 to 3) containing an acidophilic bacterial inoculums of T. ferrooxidans. The leachate is collected at the bottom, metal is separated and the inoculums is recycled through the heap. Dump leaching Dump leaching is carried on an impermeable surface. There is no artificial aeration, and that’s why the set up costs are lower. However, lack of artificial aeration, results in slower microbial growth and reduced efficiency of metal extraction. Vat leaching Vat leaching involves continuous stirring of low-grade slurry in large tanks. The ore is mixed with acidified water and T. ferrooxidans. The advantage of vat leaching over heap leaching is the ability to control the leaching conditions effectively. In Place leaching In-place leaching is carried out in underground spent mines. The mine is flooded with the acidified bacterial solution, resulting in an acid-rich, metal-rich leachate several months later from which metal is extracted and recovered. Advantages of in-place leaching: 1. Lower associated energy cost 2. Production and collection of leachate is properly controlled 3. Has a less negative environmental impact. Disadvantages of in-place leaching: Uncontrolled acid-mine drainage cause environmental pollution of metals. Use The in place leaching of uranium has received considerable attention as it involves less risk of human exposure, and thus better control of radiation exposure. Uranium leaching relies on the bacterial oxidation of insoluble UO 2 to acid-soluble U6+ in the presence of Fe3+ which was added in the bacterial inoculums. T. ferrooxidans reoxidizes Fe2+ to Fe3+ which is then recycled through the processes. UO2 + 2 Fe3+ = 6UO2+ 2Fe2+

4Fe2+ + O2 + 4H+ = 4 Fe3+ + H2O Each of these approaches produces a metal-containing leachate. A number of methods can be used to recover the metal from leachate, including Solvent extraction, increasing the Ph to precipitate the metals, Electrolysis, in which an electrical current is passed through the metal solution to accumulate cationic metals at the anode and anionic metals at the cathode. Bangladesh can use this approach for U removal which can protect public health and U can be used for grenerating electrical energy. Innovative microbial approaches in the remediation of metal-contaminated soils and sediments The goals of microbial remediation of metal-contaminated soils and sediments are to immobilize the metal in situ to reduce metal bioavailability and mobility, or remove the metal from the soil In metal immobilization as a remediation technology, it is difficult to predict whether the metals will remain immobilized indefinitely, and hence soil reuse is limited because of the continued potential risk exposure. Metal removal is ideal because following treatment the soil is available for re-use. However, metal removal is also difficult because the heterogenous nature of the soil and is expensive. There are several proposed methods for microbial remediation of metal contaminated soils including microbial leaching, microbial surfactants, microbially induced metal volatilization, and microbial immobilization and complexation.

Microbial remediation of metal-contaminated soil In-situ/ex-situ metal removal -metal oxidation -metal leaching e.g., acid production, chelation, surfactant production, volatilization

In-situ metal immobilization -microbial binding e.g., EPS production and complexation with metals, metal reduction

Increased metal solubility and enhanced recovery

Decreased metal solubility and decreased toxicity

Treated soil available for re-use

Treated soil can’t be disturbed; potential risk of metal exposure remains; requires regular monitoring

Figure:- Microbial metal remediation approaches for metal-contaminated soils. Certain microorganisms, such as T. ferrooxidans, can facilitate the removal of metals from soil through metal solubilization. Microorganisms can also increase metal solubility for recovery through the production of surfactants. Bacterial surfactants are water-soluble, low-molecular weight compounds that have a high affinity for metals; and that’s why, once complexed, contaminating metals can be removed from the soil by flushing. Some surfactants, such as rhamnolopid produced by Pseudomonas aeruginosa, show specificity for certain metals, such as Cd and Pb. Like leaching, volatilization of metals, specifically their methylation, increases metal bioavailability and hence toxicity. Methylated metals are more liporhilic than their nonmethylated counterparts. In spite of increased toxicity, many microorganisms volatilize metals to facilitate their removal from the immediate environment. Because methylation enhances metal removal, methylation of certain metals has been used as a remediation strategy. The most famous example is the removal of selenium from contaminated soil by selenium-volatilizing microorganisms. Metal Sequestration Metal sequestration provides an alternative approach, which relies on the ability of some microorganisms to produce metal-complexing, polymer (both extracellular and intra cellular). These polymeric substances have high affinities for various metals facilitating their removal from the environment. Innovative microbial approaches in the remediation of metal contaminated aquatic systems Microbially facilited removal of metals from water is based on the ability of microorganisms to complex and precipitate metals, resulting in both detoxification and removal from the water column. Specific interactions for metal removal include: Metal binding to microbial cell surfaces and exo-polymer layers  Intracelular uptake  Metal volatilization  Metal precipitation Althoough these microbial mechanisms can effectively remove metals from contaminated aquatic systems, it is important to note that the metals not destroyed, just transformed into non-available form, and still have to be properly disposed of. Available metal removal treatments for metal-polluted waters include  Wetland treatment  Microbial biofilms Wetland treatment is a cost effective and efficient method for removal of metals from contaminated waters, such as acid mine drainage. Metal reductions are often greater than 90%. Wetland remediation is based on microbial adsorption of metals, metal bioaccumulation, bacterial metal oxidation, and sulfate reduction. The high organic metal

content of wetlands provided by high plant and algal growth encourages both the growth of sulfate-reducing microorganisms and metal sorption to the organic material Microbiological remediation of metal-contaminated water In-situ/ex-situ metal removal

Metal oxidation e.g. Volatilization

Microbial binding e.g.EPS

Metal reduction e.g. precipitation

Release of gaseous metal

Recovery of metals bound by microorganisms

Recovery of metal containing precipitate via sedimentation or filtration

Treated water available for reuse

Treated water available for re use

Treated water available for reuse

Figure:- Microbial metal remediation approaches for metal-contaminated waters. In each method, the treated water is safe to release into the environment. Both metals and microorganisms can easily be recovered during treatment for proper disposal. However, the toxic metals are not destroyed, just removed from the water column. Consequently, wetlands are constantly monitored for environmental change that may adversely affect metal removal. For example, a decrease in pH may solubilize, precipited metals, or a disturbance of the wetland sediment may change the redox conditions and oxidize reduced metals, thus making them soluble and bioavailable. The most common treatment for metal-contaminated water is with microbial biofilms. Many microorganisms including, Bacillus, Cirtobacter, Arthrobacter, Streptomyces and the yeast Saccharomyces and Candida produce exopolymersand tend to form biofilms. Metals have high affinities for these anionic exo-polymers and adsorb to them. Often, a mixure of biofilm-producing microorganisms is grown on a support, providing a constant supply of fresh biofilms, and then contaminated water is passed through the support. This may results in a continuous removal of metals from contaminated water by sorption to biofilms. For example, live Citrobacter spp biofilms are used to remove uranium from contaminated water. Both Arthrobacter spp biofilms and biomass (non-living) are used in the recovery of Cd, Pb, Cr, Cu, and Zn from wastewaters. Non-living Bacillus spp. biomass are also used in the treatment of metal contaminated marine waters in which marine bacteria such as Deleya venustas and Moraxella spp are used. In domestic waste treatment,the important biofilmproducing organisms include Zoogolea, klebsiella and Pseudomonas spp. Complexed metals

are removed from the wastewater via sedimentation before release from the sewage treatment plant. DISCUSSION Metals that are proven threat to environment and health include Aluminum (Al), Cadmium (Cd), Cobalt (Co), Chromium (Cr), Copper (Cu), Mercury (Hg), Nickel (Ni), Lead (Pb), Zinc (Zn), Uranium (U). Some of these are required in our body as trace elements, used as co-factor such as Zn, Cu, Ni, Al. If these metals remain higher level in the environment, they are toxic and hence bioremediation of these metals are necessary. Remediation can also be done by physical and chemical methods also but physical or chemical methods are costly, limited area can be treated and physical excavation increases metal toxicity. Metals can’t be degraded by incineration. Incineration also destroys soil properties and structure and soil biota. It is already established that Pseudomona spp., Alkaligenes, Pediococcu spp., Escheriachia coli, Staphylococcus aureus, Zooglea ramigera, Saccharomyces cerevisiae, Rhizopus arrhizus, Chorella vulgaris, Aspergillus oryzae, Asp. Niger, Penicillum spinulosum, Trichoderma viridae are very efficient in bioremediation and these organisms are found in Bangladesh. Bioremediation can be used for metal recovery such as U and Cu. The role of U to produce ececticity is well known. It can also help to solve scarcity of electricity. Metal microbial interactions can help us to leach valuable metals by various methods such as heap leaching, vat leaching, in-place leaching, dump leaching. Metal immobilization applicable only for soil but metal removal can be applied for both land and water. In many of our districts, drinking water contain unacceptable high amount of arsenic that can be treated by arsenic oxidizing bacteria. In Hazaribugh, Conabari, Naryaganj contain nearly all types of toxic metals in high levels. Hazaribug containing high tannery effluents rich in chromium. It is found that Pseudomonas spp and Pediocuccos sp. able to detoxify it. FUTURE TRENDS 1. Use of microbes for removal of contaminating water. 2. Use of biosensors for detection of toxic metals. 3. Increasing specificity and sensivity of existing probes. 4. Use of microbes (Zooglea ramigera) to concentrate uranium. 5. Effective arsenic reducer construction for Arsenic bioremediation. 6. Cloning the metal detoxifying genes in existent natural population for metal detoxification. 7. Antibiograms of the organisms to identify antiobiotic resistant strain. 8. Evaluating effectiveness of organisms in various seasons. 9. Determination of biofilm formation capabilities of the isolates which can trap contaminating metal 10. Purification of enzymes and proteins. 11. Multidisciplinatory approach aiming to improve these strains along with introducing sustainable technologies.

12. To design a cation exchange filter with some selective absorbent of toxic metals and transformation of biofilm to detoxify trapped contaminating metals. 13. Determination of the the bioremediation capabilities of the isolates. REFERENCES Anderson, G.L., Williams, J., and Hille R. ( 1992) The purification and characterization of arsenite oxidase from Alcaligenes faecalis, a molybdenum-containing hydroxide. J. boil, chem., 267, 23674-23682 Avery, S.V., Codd, G. A., and Gadd, G,M, (1991) Caesium accumulation and interactions with other monovalent cations in the cyanobacteriaum Synechocystis PCC6893, J, General microbial,137, 405-413 Beveridge, T. J., Hughes, M. N., Lee, h., Leung, K. R.,Savvaidis, I., Silver,S., and Trevors, J. T. (1997)Metal-microbe in teractions; contemporary approaches.Adv.Micbiol, Phys, 38, 177-243 Eorgen, F. D. (1971) Soil geochemistry in the Canadian Shield. Can, min:Metall, 64;37-42 Gadd, G. M. (1988) Accumulation of metals by microorganisms and algae, In “Biothechnology- A Comprenhensive Treatise. Special Microbial Processes”(H. J. Rehma, ed.)Verlagasesellschaft, Weinheim, pp 401-433 Goldman, , J. R., and Horne, A.J. “limnology” McGraw-Hill, New York. Gupta, A., Morby, A. P.,Turner, J.S., Whitton, B.A.,and Robinson, N. J. (1993). Deletion within metallothionein locus of cadmium-tolerant Syneehococcus PCC 6304 involving a highly iterated palindrome (HIPI). Mol. Microbiol, 7, 189-195 Huges, M. N., and Poole, R.K. (1989) “Metals and Microorganisms.”Chapman & Hill, New York. Leppard, G. (1981) ‘ trace element speciation in surface Waters”Plenum Press, New York. Lindsay, W. L (1979) “Chemical Equilibrium in soils.”John Whiley and Sons Inc., New York. Nilsson, J.R.(1981) Effect of copper on phagocytosis in Tetrahymena, Protoplasma 109, 359-370 Sigg, L. (1985)Metal transfer mechanisms in lakes: the role of settling particles. In”Chemical Processes in Lakes” (W. Stumm, ed.) John Wiley, New York. Torrens, J. L., D. Herman , D. C. and Miller-Maier, R. M. 1998, Biosurfactant (rhamnolipid)sorption and impact on rhamnplipid-facilitated removal of cadmium from various soils. Environ, Sci. Technol. 32, 776-681 Warren, H.v., Delevault, R. E., and Barakso, J.(1966) Some observations on the geochemistry of mercury as applied to prospecting. Econ. Geol. Ser. Can 61, 1010 1028

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