J. Gen. Appl. Microbiol., 44, 129–132 (1998)

J. Gen. Appl. Microbiol., 44, 129–132 (1998) Screening of heavy metal-tolerant actinomycetes isolated from the Salí River María J. Amoroso,* Guillermo R. Castro, Federico J. Carlino, Nora C. Romero, Russell T. Hill,1 and Guillermo Oliver Instituto de Microbiología, Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Ayacucho 491, 4000 Tucumán, Argentina 1 Center of Marine Biotechnology, University of Maryland, 600 East Lombard Street, Baltimore, Maryland 21202, USA (Received December 24, 1996; Accepted April 21, 1998) Fifty-three strains of actinomycetes resistant to heavy metals were isolated from the Salí River in northwest Argentina. Screening procedures that involve solid and liquid synthetic media containing Cd2ⴙ, Cu2ⴙ, or Hg2ⴙ allowed the selection of six strains. These strains showed a quantitative sorption of Cd 2ⴙ and Cu2ⴙ by more than 98% of the initial metal concentrations (0.1, 0.5, and 1.0 mM) tested. Key Words——actinomycetes; bioremediation; metal resistance Water and soil pollution has become a major concern in the world, as much of the population relies on groundwater as its major source of drinking water as well as on soil as cultivable land. Heavy-metal contamination brings a potential health harzard that can cause metal toxicoses in animals and humans (Volesky and Holan, 1995). Trace elements such as cadmium, copper, and mercury are very toxic heavy metals and have been found in the human environment at increased concentrations, because a wide variety of industrial activities have accelerated the release of these metals at higher rates than natural geochemical cycling processes can tolerate (Nriagu and Pacyma, 1988). Automobile and leather factories, and sugar mills located in Tucumán, a northwestern state of Argentine, are potential sources of effluent contamination of aquifers and rivers. Salí is one of the most important rivers of Tucumán. The Salí River flows to the Río Hondo reservoir in northeast Argentina. This reservoir is a source of drinking water, irrigation and fishing activities, and is considered an important ecological reserve in Argentina. The metal processing capacity of microorganisms can be used to concentrate, remove and recover metals from aqueous streams and enhance the efficiency of wastewater treatment processes (Brown and * Address reprint requests to: Dra. María J. Amoroso, Marcos Paz 360-2° piso, 4000 Tucumán, Argentina. Lester, 1979). Different microorganisms such as fungi, yeast and bacteria were tested for the availability and biosorption potential to bind heavy metals (Volesky and Holan, 1995). However, to our knowledge little information is available about the use of actinomycetes for sequestering heavy metals from solutions (Abbas and Edward, 1989). This study deals with metal-resistant actinomycetes from polluted areas in the Salí River. From the isolates, selected strains were characterized with respect to growth in the presence of different metal ion concentrations (MICs), with the ultimate objective of utilizing these strains in bioremediation processes. Materials and Methods Samples. Sediment samples were collected from the flow of the Salí River in the Río Hondo reservoir using a sediment grab sampler. All of the samples were kept at 0°C until use. The samples were diluted with a sterile 145 mM NaCl solution and spread onto isolation plates in duplicate. Isolation of microorganisms. The isolation and enumeration of microorganisms were carried out in SC medium that contained (per liter): starch 10 g; casein 1.0 g; K2HPO4 0.5 g; and agar 15.0 g. The pH of the medium was adjusted to 7.0 prior to sterilization. The medium was supplemented with 10.0 µg ml⫺1 each of nalidixic acid (NA) and cycloheximide and 25 µg ml⫺1 of nystatin. Plates were incubated at 25°C 130 AMOROSO et al. and colonies were purified by streaking on the same medium without NA. Qualitative assays of metal resistance. Primary qualitative screening assays were carried out in square plates containing SC agar medium. Troughs were made in the center of the plate and filled with 500 µl of metal salt solutions of CdCl2 100 mM; CoCl2 100 mM; CuSO4 100 mM; and HgCl2 10 mM. Microbial growth was used as the qualitative parameter of metal resistance. Semiquantitative screening for metal resistance. Heavy-metal solutions of different concentrations were used to saturate 6 mm-diameter discs. The discs were placed on the surfaces of Petri dishes containing media previously inoculated with spores of the strain to be tested. The diameter of the growth inhibition was measured after incubation at room temperature for 7 days. Determination of metal toxicity. Spore suspensions of the selected strains were inoculated in a liquid-defined medium (MM) containing (per liter): L-asparagine 0.5 g; K2HPO4 0.5 g; MgSO4 · 7H2O 0.2 g; FeSO4 · 7H2O 0.01 g; and glucose 10.0 g. The MM medium was supplemented with the following metal ion solutions: 0.1 to 1.0 mM CuSO4; 0.1 to 1.0 mM CdCl2; and 0.01 to 0.1 mM HgCl2. Cultures were incubated by shaking (100 rpm) at 28°C for 48 h and centrifuged (3,000⫻g, 10 min). After washing the resulting pellets with 25 mM Tris-EDTA buffer (pH 8.0), the biomass was estimated by drying the pellets to constant weight at 105°C. Analysis of metals. Cadmium and copper residuals of the supernatants were determined by atomic absorption spectrophotometry. Results Qualitative analysis of inhibition by heavy metals Fifty-three colonies of actinomycetes were isolated and tested for qualitative metal resistance by plate analysis (Table 1). Measures of growth showed very low incidence (5.7%) of resistance to cobalt (100 mM) and a high incidence (88.7%) of resistance to copper (100 mM). The incidence of resistance to mercury (10 mM) was intermediate (43.4%) as compared with other metals, although the mercury concentration assayed was ten-fold lower than other heavy metals because it is very toxic. Semiquantitative analysis of inhibition by heavy metals Ten strains were selected for these experiments considering their multiple metal resistance to Cd2⫹, Cu2⫹, and Hg2⫹. Increasing the metal concentration in plate diffusion experiments resulted in a marked inhi- Vol. 44 Table 1. Qualitative screening of metal resistance. Metal resistance Total Strain % 53 100 Cd2⫹ Co2⫹ (100 mM) (100 mM) 10 18.9 3 5.7 Cu2⫹ (100 mM) Hg2⫹ (10 mM) 47 88.7 23 43.4 bition of microbial growth. This effect was strong, in the order of Hg2⫹, Cd2⫹, and Cu2⫹. An inhibition zone of 10 mm in diameter was arbitrarily designated as a semiquantitative criterion to determine the metal tolerance of the tested strains. As the result, all the strains but R10 turned out to be sensitive to Cd2⫹ at 10 mM or higher. Strain R10 was inhibited at 20 mM Cd2⫹. Interestingly Streptomyces lividans TK24, used as the control, was tolerant to 10 mM Cd2⫹ but not to 20 mM. The growth inhibition profiles at 100 mM concentrations revealed two sensitive strains; R06 and R25 of the ten selected wild-type strains (results not shown). Strain R25, the most Cu2⫹-sensitive isolate, exhibited a similar profile to S. lividans TK24 at concentrations higher than 50 mM Cu2⫹. Eight of ten selected isolates were resistant to 1 mM Hg2⫹ in the plate assay (results not shown), but R25 was considered to be sensitive at this concentration, indicating higher sensitivity than S. lividans. Quantitative analysis of inhibition by heavy metals The toxicities of cadmium, copper, and mercury were evaluated in MM medium, in terms of growth inhibition in the six promising strains. The relative growth curves showed a hyperbolic response with the increase of Cd2⫹ concentration in the medium. Relative growth on the medium containing cadmium ion higher than 0.1 mM was inhibited by almost 60% in all selected strains (data not shown). The typical curves of one strain (R25) are shown in Fig. 1. The uptake analysis of cadmium by the cells showed that the uptake, defined as metal concentration consumed per biomass, increased with the initial cadmium fed in the medium with the exception of S. lividans TK24 (Table 2). However, at the highest Cd2⫹ concentration (1 mM), the range of relative growth was 2 to 15% of the control growth without metal solution. Selected strains in MM medium containing Cu2⫹ showed higher resistance to Cd2⫹ (data not shown). However, the hyperbolic profiles of relative growth with the copper concentration are similar to those with cadmium. The growth of strains in MM medium containing increased copper concentrations is shown in Fig. 1. The growth of the R25 strain was not inhibited 1998 Metal-resistant actinomycetes from the Salí River Fig. 1. Effect of heavy-metal concentrations on the growth of R25 strain. Metals: 䊉, Cu2⫹; 䊏, Cd2⫹; 䉱, Hg2⫹; ——, relative growth; – – –, residual ion concentrations. Table 2. Consumption of cadmium and copper by the selected actinomycete isolate. Specific consumption (µmol mg⫺1) of different concentrations of metals (mM) Strain Cd2⫹ S. lividans TK24 R06 R10 R16 R22 R25 R27 Cu2⫹ 0.1 0.5 1.0 0.1 0.5 1.0 0.06 0.24 0.19 0.18 0.24 0.42 0.50 0.06 1.35 1.79 1.46 1.55 3.84 3.84 0.06 3.70 3.33 — 4.34 9.99 — 0.07 0.12 0.18 0.07 0.21 0.11 0.28 0.07 1.35 2.38 0.62 1.19 0.93 — 0.08 2.37 — 1.99 2.22 4.16 6.24 Specific consumption is defined as metal consumption (µmol) per biomass (mg). significantly at 0.1 mM Cu2⫹, but by approximately 40% at 0.5 mM. Discussion The results obtained in this study indicate that metal resistance and metal consuming capability may be widespread amongst actinomycetes growing in contaminated environments. The sensitivity of six selected strains to the heavy metals tested was Hg2⫹⬎Cd2⫹⬎ Cu2⫹ at all concentrations assayed in MM medium. Mercury concentrations higher than 10 mM could not be assayed by the plate diffusion technique, because no microbial growth was observed under our experimental conditions. This can be attributed to the high toxicity of Hg2⫹ (Duxbury, 1981). MM medium supple- 131 mented with Hg2⫹ concentrations higher than 0.01 mM showed high toxicity in the six strains tested; however, the tolerance of these strains expressed as relative growth was 1.5–3-fold higher than S. lividans TK24 that presented a relative growth of 0.1 at 0.01, 0.5, and 1.0 mM Hg2⫹ under the same experimental conditions. Low mercury (II) resistance, as compared with the selected strains, has been reported in Pseudomonas fluorescens (Farrel et al., 1993), Streptomyces coelicolor (Abbas and Edward, 1989), Saccharomyces cerevisiae and Candida albicans (Yannai et al., 1991). High mercury resistance has been reported in three heavy metal-resistant strains of Pseudomonas syringae with MICs of 75 and 10 mM (Kidambi et al., 1995). However, the tolerance to high mercury concentrations by P. syringae species could be attributed to the low availability of Hg2⫹ ion because of the complex organic compounds in the supplemented medium used in the assays (Volesky and Holan, 1995). As shown in Table 2, the six selected strains showed at least 50-fold higher specific consumption of Cd2⫹ and Cu2⫹ as compared to that of the control strain, S. lividans TK24, which showed higher Cd2⫹ resistance than the isolated actinomycete strains in a semi-quantitative analysis. The toxic level of cadmium to Pseudomonas aeruginosa and Aeromonas sp. in a synthetic medium was reported to be 6.45 and 2.00 µM, respectively (Walker and Houston, 1981). Abbas and Edward (1989) reported that the growth of S. coelicolor was inhibited by 50% after 16 h of culturing in the presence of 0.14 mM Cd2⫹. Although cadmium has been reported as a very toxic metal for microorganisms, in a selected strain and at all Cd2⫹ concentrations used in the assays, percentages of cadmium remnant in the supernatants were below 0.2% of the initial concentrations (Fig. 1). Abbas and Edward (1989) reported that S. coelicolor could tolerate Cu2⫹ concentrations no higher than 0.047 mM, and that its growth was reduced by 50% when the strain was cultured in a complex medium containing starch and yeast extract. It is well known that medium composition may influence metal sensitivity (Duxbury, 1981). It was for this reason that a quantitative evaluation of metal resistance was performed in a defined and minimal medium. In concordance with cadmium biosorption, the residual cooper in the supernatant of the culture medium became lower than 0.1% of the initial Cu2⫹ concentration (0.1 to 1.0 mM). In this regard, it was reported that Gaeumannomyces graminis gave a cooper uptake of 92.6, 83.5, and 71.0% when the fungal culture was exposed to 0.04, 0.06, and 0.08 mM of CuSO4 (Caesar-Tonthat et 132 AMOROSO et al. al., 1995). Isolation of the six metal-resistant actinomycetes described here opens up opportunities to investigate their mechanisms of metal resistance. The metal-resistant actinomycetes or genes encoding metal resistance isolated from these organisms may be useful in the bioremediation of contaminated sediments. The authors gratefully acknowledge the financial support of CONICET and COCYTUC, Argentina. Metal determinations were performed by Mr. Alberto Durán, Estación Exp. Agro Ind. O. Colombres, whose assistance is gratefully acknowledged. References Abbas, A. and Edward, C. (1989) Effects of metals on a range of Streptomyces species. Appl. Environ. Microbiol., 55, 2030– 2035. Brown, M. J. and Lester, J. N. 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