BIOSORPTION OF SOME HEAVY METALS FROM MEDIA WITH HIGH SALT CONCENTRATIONS BY HALOPHILIC ARCHAEA

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1 BIOSORPTION OF SOME HEAVY METALS FROM MEDIA WITH HIGH SALT CONCENTRATIONS BY HALOPHILIC ARCHAEA G. Popescu and L. Dumitru Institute of Biology Bucharest of the Romanian Academy, Bucharest, Romania, Correspondence to: Gabriela Popescu ABSTRACT Heavy metal pollution represents an important environmental problem due to the toxic effects of metals. Their accumulation throughout the food chain leads to serious ecological and health problems. The remediation of environments metal contaminated through physico-chemical methods present major disadvantages such as high energy consumption, high costs and incomplete removal of metal ions. Biosorption of heavy metals by microorganisms is an alternative method that involves low costs. Active elimination process of heavy metals ions provided by living cells, especially by their components and extracellular products represent a potential way of removing toxic heavy metals from industrial wastewaters. Extremely halophilic archaea require high salt concentrations for growth and inhabit hypersaline environments (solar salterns, salt lakes and salt mines), where the NaCl concentration exceeds g l -1 or reaches saturation. Frequently, hypersaline environments are contaminated with toxic compounds. Therefore, the search of microorganisms able to degrade toxic agents in the presence of high salt concentrations appears to be desirable, especially for biological treatment of highly saline industrial waste effluents. Also, some metal-resistant halophilic microorganisms could be used as indicator organisms in saline polluted environments. The aim of this study was to determine the metal tolerance levels of some collection and wild haloarcheal strains from Haloferax genus. Also, we study the capacity of tested strains to reduce the concentration of some heavy metals from media with high salt concentration by biosorption process involved extracellular polysaccharides. The results showed that the wild strain has a higher susceptibility to Zn ion comparatively with the collection strain. The growth of tested strains and their capacity to removal of metal ions were stimulated by the addition of glucose at culture medium. Thus, the synthesis of exopolysaccharides enhanced the reduction activity of Cr, Zn and Ni concentrations by the haloarcheal investigated strains. Keywords: biosorbtion, haloarchaea, heavy metals, salinity ecological and health problems. Introduction Some of heavy metals, such as cooper, iron and zinc are necessary for microorganism s life, being essential trace elements that are required, at a certain concentration level, for enzymatic activities and bacterial growth. At high concentrations, the most of heavy metal ions become toxic to all branches of life, including microorganisms, by forming toxic compounds within the cells (19, 27). Contamination of the environment by heavy metals is a consequence of technological and industrial processes. This has led to the increasing concern about the effects of toxic metals as environmental contaminants (11). Thus, heavy metal pollution represents an important environmental problem due to the toxic effects of metals, and their accumulation throughout the food chain leads to serious The remediation of environments metal contaminated through common chemical and physical methods present major disadvantages such as high energy consumption and high costs, generation of large amount of sludge containing toxic compounds or incomplete removal of metal ions (1, 14). Biosorption of heavy metals by microorganisms is an attractive and economical alternative method that consists in the removing of toxic metals from aqueous solutions based on the property of certain kinds of biomasses to bind and accumulate these pollutants by different mechanisms such as physical adsorption, complexation, ion exchange and surfacemicro-precipitation (18, 28). The major advantages of biosorption comparatively with conventional treatment methods include: low cost, high efficiency, minimization of chemical sludge, no additional nutrient requirement, 791

2 regeneration of biosorbent and possibility of metal recovery (1). Active elimination process of heavy metals ions provided by living cells, their components and extracellular products represent a potential way of removing toxic heavy metals from industrial wastewaters (17). In this regard, extracellular polysaccharides (EPSs) synthesized by bacteria, archaea, fungi or algae are recommended as surface active agents for heavy metal removal because of their extensive capacity. The adsorption of heavy metal by EPS is a metabolismindependent process, which is attributed to interaction between metal cations and negative charges of acidic functional groups of EPS. Thus, metals may be biosorbed or complexed by carboxyl groups found in microbial polysaccharides (1, 6, 18). The extremely halophilic archaea (also called haloarchaea) are a diverse group of prokaryotes that require high salt concentrations for growth and inhabit hypersaline environments such as solar salterns, natural hypersaline lakes (inclusively alkaline salt lakes) and the Dead Sea. In these habitats the salt concentration exceeds g l -1, or reaches NaCl saturation. Viable halophilic Archaea have also been isolated from rock salt of great geological age ( million years) (4, 10, 13, 23). These extreme halophiles belong to the family Halobacteriaceae that comprise 27 recognized genera with 119 species (5, 8, 9, 12, 26). Beside haloarchaea, the moderately halophilic bacteria are another important group of halophiles that thrive in hypersaline habitats and growing best in media with 3 to 15% NaCl (11, 16). Frequently, saline and hypersaline environments are contaminated with toxic compounds inclusively heavy metals as a result of industrial activities. There is not wide information on heavy-metal resistance in halophilic microorganisms; however, the isolation of microorganisms from polluted saline environments able to tolerate heavy metals in the presence of high salt concentrations appears to be desirable, especially for the decontamination of these ecosystems. Thus, both moderately halophilic bacteria and haloarchaea tolerant to some heavy metals such as Co, Ni, Cd, Cr, Hg, Pb, Ag, Cu and Zn have been isolated from different hypersaline environments (2, 3, 7, 11, 15, 20, 21, 22, 24). Also, some metal-resistant halophilic microorganisms could be potentially used as bioassay indicator organisms in saline aquatic polluted environments or as biological detoxicans (16, 21). Several species of halophilic Archaea, especially those of the genus Haloferax, produce copious amounts of extracellular polysaccharides which have considerable biotechnological potential due to rheological properties of these polymers (high viscosity at low concentrations) and resistance to extremes of salt concentration, temperature and ph (23, 25). The aim of this study was to determine the metal tolerance levels of some collection and wild haloarcheal strains from Haloferax genus. Also, we study the capacity of tested strains to reduce the concentration of some heavy metal ions from media with high salt concentration by biosorption process involved extracellular polysaccharides synthesis. Materials and methods Microorganisms: Haloferax sp. TL5 wild strain and Haloferax meditteranei collection strain were used in the experiments. Haloferax sp. TL5 strain was isolated from a hypersaline water sample taken from Telega salt lake located in Prahova County, Romania (8). The collection strain was obtained by the kindness of Professor A. Oren from The Hebrew University of Jerusalem, Department of Microbial and Molecular Ecology, Israel. Growth conditions: The strains were grown in media containing (g/l): NaCl (175), MgCl 2 6H 2 O (50), K 2 SO 4 (5), CaCl 2 2H 2 O (0.1), yeast extract (Difco) (5), agar (20). The ph medium was Heavy metals: The four heavy metals tested were provided from standard commercial sources like K 2 CrO 4, NiSO 4, Pb(NO 3 ) 2 and ZnSO 4. Stock solutions were made in distilled water and were sterilized by filtration through 0.22 µm pore size membrane filters (Millipore). The range of concentrations for heavy metals tested, similar to the one previously used in studies on metal tolerance of halobacteria (22), was as follows (mm): 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10, 20, 40 and 80. Heavy metals tolerance: Petri dishes containing solidified medium and different concentrations of toxic metals were inoculated with haloarchaeal cultures in exponential growth phase (22). The inocullum was diluted to have an optical density (at 660 nm) between The plates were prepared in triplicate and incubated at 37 0 C for ten days. Agar plates without the antimicrobial agents and inoculated 792

3 with the haloarchaeal strains were used as controls. The minimum concentration of the metal ions that completely inhibited the strain growth was reported as MIC. Metal biosorption experiments: The capacity of haloarchaea to reduce the heavy metals concentration from media with high salt concentration was tested by growing the strains on the same medium containing 2M NaCl and 2.5% glucose for Haloferax sp. TL5 or 3M NaCl and 2% glucose for Haloferax meditteranei. Also, the haloarchaeal strains were cultivated on the same media without the glucose. The heavy metals were added at media in different concentrations: 1.0 mm Pb (331 mg/l), 2.5 mm Cr (485 mg/l), 0.1 mm Zn (16 mg/l) or 0.5 mm Zn (80.5 mg/l) and 1.0 mm Ni (130 mg/l). Erlenmeyer flasks containing 90 ml of culture media were inoculated with 10 ml of haloarchaeal culture in exponential phase. The cultures were incubated at 37 0 C, in stirring conditions (200 rpm) for 240 hours. The strains growth was assessed by spectrophotometric determination of cellular density (at 660 nm) at 72 and 240 hours, comparatively with a blank represented by culture medium without microorganisms. The cultures were centrifuged at 6000 rpm for 30 minutes after 240 hours of incubation. The final heavy metals concentration from the supernatants was determined by using the Merck Aquaquant or Merckoquant kits specific for each metal ion. Polysaccharide assay: The supernatants were precipitated in the cold with ethanol and the dry weight of EPS was determined after drying in an oven at C to constant weight. Results and Discussion MIC determination The response of extremely halophilic archaea to heavy metals action was assessed according to the lowest metal concentration that prevents the growth (MIC). The results showed that the wild strain Haloferax sp. TL 5 has a quite similar behavior with the collection strain Haloferax meditteranei towards to Cr, Ni and Pb ions. Thus, the MICs obtained for the both Haloferax strains tested were 5.0 mm Cr, 2.5 mm Ni and 2.5 mm Pb (table 1). These results allowed to include the Haloferax strains in the category of metal-tolerant microorganisms to Cr, Ni and Pb according to the conclusions of Nieto et al. (20, 22), who proposed to include in this category the strains that are not inhibited by concentrations of 1 mm Cr, Ni, Pb and Zn. The results obtained for zinc showed that the wild strain Haloferax sp. TL 5 has a higher susceptibility to this ion comparatively with the collection strain Haloferax meditteranei which was more resistant (Table 1). TABLE 1 Heavy metal resistance of extremely halophilic archaea tested (expressed as MIC) in culture medium containing 17.5 % NaCl Minimum inhibitory Haloarchaeal strains concentration (MIC) (mm) Cr Ni Pb Zn Haloferax mediterranei Haloferax sp. TL The metal biosorbtion The results obtained revealed the capacity of investigated extremely halophilic archaea from Haloferax genus to reduce the concentration of Pb, Cr, Zn and Ni ions from media with high salinity. The optimum growth of Haloferax sp. TL 5 and Haloferax mediterranei strains was determined in medium with metals ions and glucose addition (as carbon and energy source), the cellular density values being higher than those obtained in the absence of glucose (Table 2). This behavior suggests that exopolysaccharides synthesized in the presence of glucose may have the role to protect the cells against the heavy metals toxicity. The results showed some differences between the wild strain and the collection strain regarding the extracellular polysaccharides synthesis and the biosorption capacity of metal ions. Thus, the Haloferax sp. TL 5 synthesized high amounts of exopolysaccharides (between g %) in the presence of glucose and all metal ions tested. Comparatively, the Haloferax mediterranei synthesized the biopolymer only in the presence of lead ion and glucose 2 % (0.220 g %) (Table 2). In the case of Haloferax sp. TL 5 strain were determined the same concentrations of Pb (5 mg/l) and Ni (0.2 mg/l) ions after 240 hours of growth both in medium with glucose and without the glucide. These results showed that the extracellular polysaccharides synthesized do not influenced the wild strain capacity to reduce the concentration of these ions from media with high salinities (Table 2). In the case of chromium and zinc ions was observed that the addition of glucose to culture medium and implied the exopolysaccharides synthesis enhanced the Haloferax sp. TL 793

4 5 capacity to reduce these heavy metals concentration (0.8 mg/l) comparatively with the strain activity grown in medium without the glucide (1.6 mg Cr/L and 8 mg Zn/L) (table 2). These results suggested that extracellular polysaccharides may have an important role in the biosorption process of metal ions by haloarchaeal strain Haloferax sp. TL 5. The Haloferax meditteranei strain has a different behavior comparatively with the wild strain. Thus, the determining of the metals concentration after the 240 hours of cultivation showed the same values of Pb (5 mg/l) and Cr (1.6 mg/l) ions in both variants of medium (with and without the glucose). Some differences were observed in the case of zinc and nickel ions. The collection strain grown optimal and significant reduced these ions concentration in the presence of glucose, the amounts of metals assessed from medium with the glucose ( 0.1 mg Zn/L and 0.2 mg Ni/L) being lower than those determined in medium without the glucide (0.4 mg/l) (Table 2). The investigated Haloferax strains showed the same capacity to reduce the concentration of lead ion, after 240 hours of growth being determined the same amount of metal (5 mg/l). The Haloferax sp. TL5 wild strain has a higher capacity to biosorption of chromium ion from medium with glucose comparatively with the collection strain. The Haloferax meditteranei strain presented a higher biosorption activity of zinc ion from the both variants of medium (with glucose 2% and without the glucide) comparatively with the wild Haloferax TL 5 strain. The Haloferax TL 5 strain has a higher capacity to remove the Ni ion from medium without the glucose comparatively with the collection strain (Table 2). The results revealed that the synthesis of exopolysaccharides enhanced the reduction activity of Cr, Zn and Ni concentrations by the investigated haloarchaeal strains. The anionic nature of exopolysaccharides synthesized by Haloferax strains, based on their high sulphate and uronicacid contents (16, 25), similar with EPSs synthesized by other halophilic microorganisms, may be responsible for the capacity of these strains to bind and remove heavy metals from solutions with high NaCl concentrations. This property would make these biopolymers a viable alternative to more aggressive physical and chemical methods as a biosorbent in polluted hipersaline environments. TABLE 2 Testing the haloarchaeal capacity to reduce the heavy metals concentration from media with high salinity Strains Heavy NaCl (M) and glucose Growth (O. D. 660 nm) EPS ** Heavy metals concentration (mg / L) metals concentration (%) T0 * 72 h 240 h (g %) Initial After 240 h Pb 2M NaCl, 2.5 % glucose M NaCl Cr 2M NaCl, 2.5 % glucose Haloferax sp. TL 5 Zn Haloferax mediterranei Ni Pb Cr Zn Ni 2M NaCl M NaCl, 2.5 % glucose M NaCl M NaCl, 2.5 % glucose M NaCl M NaCl, 2 % glucose M NaCl M NaCl, 2 % glucose M NaCl M NaCl, 2 % glucose M NaCl M NaCl, 2 % glucose M NaCl T0 * = optical density determined at the moment of strains inoculation; EPS ** = exopolysaccharides synthesized by Haloferax strains tested; - = EPS not produced by tested strain 794

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