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Journal of Basic and Applied Sciences Vol. 5, No.

2, 55-60, 2009

ISSN: 1814-8085

ISOLATION AND CHARACTERIZATION OF BACTERIAL ISOLATES HAVING HEAVY METAL TOLERANCE


Qurat-ul-Ain Affan*, Erum Shoeb*, Uzma Badar* and Jameela Akhtar** *Department of Genetics, University of Karachi, Karachi-75270, Pakistan. **Centre for Molecular Genetics, University of Karachi, Karachi-75270, Pakistan ABSTRACT Fourteen bacterial strains were isolated from metal contaminated sites close to the automobile and welding workshops. These strains were checked for tolerance against heavy metals both in enriched media and minimal media. All the strains showed tolerance against heavy metals but most promising results were given by strain GESQA002 which showed multiple stress tolerance as its maximum tolerable concentration (MTC) against cadmium chloride (CdCl2) was 6mM in enriched media and 1.4mM in minimal media, against copper sulphate (CuSO4) was 3.5mM in enriched media and 1.8mM in minimal media, and against nickel chloride (NiCl2) it was 1mM in enriched media and 0.8mM in minimal media was observed. GESQA002 was also tested against antibiotics kanamycin (Km), and streptomycin (Sm) and it showed elevated MTCs against Sm (100 g/ml) and Km (25 g/ml). Plasmids were detected in GESQA002 which could be a sign of stress tolerance genes being plasmid borne. Keywords: Heavy metals, bacterial strains, antibiotics kanamycin. INTRODUCTION Earliest living cells were formed in deep oceans which are rich in metals such as arsenic, lead, copper, and zinc (Rasmussen, 2000). Metals are directly or indirectly involve in all aspects of growth, metabolism and differentiation (Beveridge and Doyle, 1989). Many metals are essential, e.g. K, Na, Mg, Ca, Mn, Fe, Co, Ni, Cu, Zn, Mo, whereas others have no known essential biological functions, e.g. Al, Ag, Cd, Sn, Au, Sr, Hg, Tl, Pb (Gadd, 1988). Retaining suitable concentrations of essential metals, such as copper and zinc while rejecting toxic metals, such as arsenic, lead, and cadmium, was probably one of the toughest challenges of the living cells (Gatti et al., 2000). Bacteria are among the most abundant organism that occur every where on earth. Heavy metals are increasingly found in microbial habitats due to several natural and anthropogenic processes; therefore, microbes have evolved mechanisms to tolerate the presence of heavy metals by either efflux, complexation, or reduction of metal ions) or to use them as terminal electron acceptors in anaerobic respiration (Gadd, 1990). Most mechanism studied involve the efflux of metal ions out side the cell, and genes for tolerance mechanisms have been found on both chromosomes and plasmids. Bacteria that are resistant to and grow on metals play an important role in the biogeochemical cycling of those metal ions (Gadd, 1990). Considering the importance of these tolerance mechanisms, in the present study organisms were isolated from contaminated sites of the automobile workshop and welding workshop situated in the residential area of Karachi. MATERIALS AND METHODS Sampling Soil samples were collected from different automobile and welding workshops, in sterilized 20ml sterilized universal bottles. 1gm of soil samples were inoculated in 50ml enriched media incubated at 37C for 24 hours. Media Used For isolation and purification strains were routinely grown in Luria Bertani (LB) medium (Maniatis et al., 1982) at 37C. For antibiotic tolerance LB medium supplemented with 1.8 % agar, solidified plates were utilized. Metal tolerance was analyzed both in LB and Tris minimal media (Mergeay et al., 1985). Study of Colonial Morphology Isolated colonies of purified strains grown on solidified agar plates were observed and data was recorded regarding the form (circular, flamentous and irregular); elevation (flat, convex, and umbonate); margin (entire, undulate, erose and filamentous) ; and optical feature (opaque, translucent,and transparent) of the colonies (Pelczar and Reid, 1958). Cellular Morphology Cells were observed with Gram staining (Duguid 1989) under the microscope (oil immersion, 100 X). Shape of the cell (cocci, bacilli and cocco-bacilli) and arrangement of cells (scattered, bunches and chain) along with the Gram-reaction was observed.

Correspondence: Centre for Molecular Genetics, University of Karachi, Karachi-75270, Pakistan

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J. basic appl. sci. paired and scattered, all of the isolated strains were Gram negative. Details for cellular morphology and Gram reaction are given in Table 2. Maximum Tolerance of Heavy Metals Maximum Tolerable concentrations of all the strains against Nicl2, CuSO4, CdCl2 have shown that isolated strains were capable of growing at high concentration of heavy metals in both the media, although it was higher in LB as compare to Tris minimal media. MTCs of metal salts are given in Table 3 and 4. Strains which could not grow at 0.2 mM were taken as 0 MTC. Maximum Tolerance of Antibiotics MTC of selected strains against Km, Sm, and Cm was checked. Results are given in Table 5. Strains which could not grow at 5 g/ml were taken as 0 MTC. Growth Curve of Bacteria with Metal Induction To find out weather metal tolerance mechanism was inducible or not, growth curve of bacterial strains GESQA002 and GESQA013 were studied. It was observed that metal tolerance in GESQA002 and GESQA013 was constitutive since GESQA002 and GESQA013 which were already expose with the metal overnight and the one which was expose to metal just before start of the growth curve did not show any significance difference and no sign of induction of tolerance mechanism was observed with respect to OD600. According to the results, it could be suggested that GESQA002 and GESQA013 have constitutive pathways of tolerance against Copper. Results are shown in Fig. 1 and Table 6.
1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 Control1 Control2 Test 2

Metal Tolerance All the 14 strains were checked for Metal tolerance. To determine the maximum tolerable concentration (MTC) of heavy metals salts such as NiCl2, CuSO4, CdCl2, 1M stock solution of each of the metal salt was prepared and appropriate dilutions (0.2mM, 0.4mM, 0.6mM, 0.8mM and so on) were prepared. Bacterial strains were streaked from O/N culture on LB and Tris minimal media agar plates. The plates were incubated at 37C and growth was observed after 24-48 hour. Antibiotic Tolerance To determine the maximum tolerable concentration (MTC) against different antibiotics such as Sm, Km, Cm. Stock solution of Sm; 50 mg/ml, Km; 25 mg/ml, and Cm; 34 mg/ml were used. Bacterial strains were streaked from O/N culture on LB agar plates containing variable concentration of antibiotic solutions (5, 10, 15, 20, 25, 50, 75, 100, 125, 150,175 and 200 g/ml and incubated at 37C and growth was observed after 24 hour. Selected strains were checked for antibiotic tolerance. Growth Curve of Bacteria with Metal Induction In 100ml of LB broth 100l of culture was inoculated (Control). In 100ml of Luria broth 100l of culture was inoculated and 0.5mM CuSo4 was added (Test). Culture was incubated for overnight at 37C on shaker. OD was taken at 600nm and Growth Curve was studied at 37C in three conditions: without metal inoculated culture from Control, with metal (0.5mM CuSo4) inoculate culture from Control and with metal (0.5mM CuSo4) inoculated culture from Test to check the induction. Isolation of Plasmid DNA Plasmid DNA was isolated by Real Genomics Plasmid Mini Kit of RBC (Cat. No. YPD100). The procedure followed as described by manufacturer for isolation of genomic DNA. RESULTS Isolation and purification of bacterial strains 14 bacterial strains were isolated from the soil samples and designated as GESQA001- GESQA014. Colonial Morphology The colonial morphology of most of the strains was circular, elevation was convex, all colonial margins were entire type; translucent and opaque were equally frequent. A detailed result for colonial morphology has been given in Table 1. Cellular Morphology Cellular morphology such as arrangement, shape and Gram reaction were observed during Gram staining of each strain. Cellular shape of the strains were found to be cocci, whereas cellular arrangement was found to be

0.1 0
0 10 00 12 00 14 00 16 00 40 0 20 0 60 0 80 0

Fig. 1. Induction curve of GESQA002: Control: Bacterial Strains GESQA002 grown in a medium devoid of metal salt Test 1: Induced cells were pre exposed to Cuso4 whereas; Test 2: uninduced cell were not pre exposed to Cuso4.

Qurat-ul-Ain et al. Isolation of Plasmid DNA Plasmid DNA was successfully isolated. A single band of plasmid was observed in several strains while some strains showed two and three plasmids which are shown in Figure 2 and table 7. L1 L2 L3 L4 L5 DISSCUSSION

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Sampling environments that contain elevated concentrations of heavy metals are a potential source of toxic-metal-tolerant bacteria (Clausen, 2000). For the present studies bacterial strains were collected from the soil of most common heavy metal contaminated sites of our city, the welding shop (sample A) and automobile workshops (sample B), with the expectation of isolating metal tolerant bacteria from the metal contaminated areas. Initially, several bacterial strains were purified out of which few showed consistent behavior and were revived after being refrigerated for few weeks. Finally, all those strains were selected for characterizations which were consistent for their tolerance behavior; the isolated strains were designated from GESQA001-GESQA014. Glycerol preserves behaved perfectly and whenever required strains revived within 24 hours. Resistance systems not only protect the organism in a harsh environment, but they also play an important role in the cycling of toxic metals in the biosphere. In some cases bacterial metal resistances have been shown to be due to differences in uptake and/or transport of the toxic metal, while in other cases the metal is enzymatically transformed, by oxidation, reduction, methylation or demethylation, into chemical species which is either less toxic or more volatile than the parent compound (Williams and Silver, 1984). Sampling sites were selected with the aim to isolate metal tolerating bacteria for which the best option was to locate metal contaminated sites. Instead of concentrating on remote industrial areas, it was preferred to explore inner-city environment with respect to heavy metal tolerance in bacteria, specially the automobile workshops located within heavily populated residential areas of Buffer zone north Karachi.

Fig. 2. Isolation of Plasmid DNA. Plasmid DNA was extracted and separated by agarose gelelectrophoresis. DNA ladder in lane 1, GESQA006 is loaded in lane2, GESQA007 in lane 3, GESQA011 in lane 4, GESQA014 in lane 5.

Table 1. Colonial Morphology of the Isolates Strain Codes GESQA001 GESQA002 GESQA003 GESQA004 GESQA005 GESQA006 GESQA007 GESQA008 GESQA009 GESQA010 GESQA011 GESQA012 GESQA013 GESQA014 Colour Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow Pale Yellow Off White Pale Yellow Off White Pale Yellow Pale Yellow Size Small Large Large Large Extra Large Extra Large Large Small Extra Large Pin Head Extra Large Pin Head Small Large Shape Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Margin Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Elevation Convex Convex Convex Convex Convex Convex Convex Convex Convex Convex Convex Convex Convex Convex Opacity Opaque Opaque Opaque Opaque Opaque Opaque Opaque Opaque Opaque Translucent Opaque Translucent Opaque Opaque Surface Smooth & Shiny Smooth & Shiny Smooth & Shiny Smooth & Shiny Smooth & Shiny Smooth & Shiny Smooth & Shiny Smooth & Shiny Smooth & Shiny Smooth & Shiny Smooth & Shiny Smooth & Shiny Smooth & Shiny Smooth & Shiny

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J. basic appl. sci. Table 2. Cellular morphology of bacterial isolates Strain Codes GESQA001 GESQA002 GESQA003 GESQA004 GESQA005 GESQA006 GESQA007 GESQA008 GESQA009 GESQA010 GESQA011 GESQA012 GESQA013 GESQA014 Shape Rod Round Round Round Round Round Round Round Round Round Round Round Round Round Arrangement Paired Scattered Paired Scattered Scattered Paired Scattered Paired Scattered Scattered Scattered Scattered Scattered Scattered Type Diplobacillus Streptococcus Diplococcus Streptococcus Streptococcus Diplococcus Streptococcus Diplococcus Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus Colour Pink Pink Pink Pink Pink Pink Pink Pink Pink Pink Pink Pink Pink Pink Gram Reaction Gram negative Gram negative Gram negative Gram negative Gram negative Gram negative Gram negative Gram negative Gram negative Gram negative Gram negative Gram negative Gram negative Gram negative

Sampling, isolation and purification of several bacterial strains has been done and variability among all the isolated strains was observed with respect to all the studied parameters. Metal tolerance and antibiotic tolerance behavior of all the strains have revealed a very interesting pattern, which indicate multiple stress tolerance i.e., the strains which showed tolerance against metal have also shown tolerance against antibiotics. Table 3. MTC of Heavy Metals (LB) Strain Codes GESQA001 GESQA002 GESQA003 GESQA004 GESQA005 GESQA006 GESQA007 GESQA008 GESQA009 GESQA010 GESQA011 GESQA012 GESQA013 GESQA014 Heavy Metals CuSO4 3.5mM 3.5mM 3.5mM 3.5mM 3.5mM 3.5mM 3.5mM 3.5mM 3.0mM 3.0mM 3.0mM 3.0mM 3.0mM 3.0mM

either reducing the more toxic metals into less toxic forms or by accumulating the metal ions thus removing it from the effluent or areas containing concentration of particular metals. Table 4. MTC of Heavy Metals (Tris Minimal Media) Strain Codes GESQA001 GESQA002 GESQA003 GESQA004 GESQA005 GESQA006 GESQA007 GESQA008 GESQA009 GESQA010 GESQA011 GESQA012 GESQA013 GESQA014 Heavy Metals CuSO4 1.8mM 1.8mM 1.8mM 1.8mM 1.8mM 1.8mM 1.8mM 1.8mM 1.8mM 1.8mM 1.8mM 1.8mM 1.8mM 1.8mM

NiCl2 1.0mM 1.0mM 1.0mM 1.0mM 1.0mM 1.0mM 1.0mM 1.0mM 1.0mM 1.0mM 1.0mM 1.0mM 1.0mM 1.0mM

CdCl2. 3.5mM 6.0mM 3.5mM 4.0mM 3.5mM 3.5mM 3.0mM 3.0mM 3.0mM 3.0mM 3.0mM 3.5mM 3.5mM 3.0mM

NiCl2 0.8mM 0.8mM 0.8mM 0.8mM 0.8mM 0.8mM 0.8mM 0.6mM 0.8mM 0 0.8mM 0.8mM 0.8mM 0.8mM

CdCl2. 1.4mM 1.4mM 1.4mM 1.4mM 1.4mM 1.4mM 1.4mM 1.4mM 1.4mM 1.4mM 1.4mM 1.4mM 1.4mM 1.4mM

Table 5. MTC of Antibiotics Strain Codes GESQA002 GESQA004 GESQA007 GESQA012 GESQA014 Antibiotics g/ml Sm Km 100 25 125 50 75 50 100 0 75 75

Using bacterial tolerance against widespread metal compounds one can minimize the effect of heavy metals on total biological activity of the ecosystem. Heavy metal tolerance of bacteria can play an important role in bioremediation of metal pollution in the environment by

Qurat-ul-Ain et al. Table 6. Growth Curve with metal induction of GESQA002 Serial No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 Time (mins) 0 15 30 45 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 900 930 960 990 1020 1050 1080 1110 1140 1170 1200 1230 1260 1290 1320 1350 1380 1410 1440 Time (hrs) 0 0.25 0.5 0.75 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5 20 20.5 21 21.5 22 22.5 23 23.5 24 Actual time 9:40 9:55 10:10 10:25 10:40 11:10 11:40 12:10 12:40 13:10 13:40 14:10 14:40 15:10 15:40 16:10 16:40 17:10 17:40 18:10 18:40 19:10 19:40 20:10 20:40 21:10 21:40 22:10 22:40 23:10 23:40 0:10 0:40 1:10 1:40 2:10 2:40 3:10 3:40 4:10 4:40 5:10 5:40 6:10 6:40 7:10 7:40 8:10 8:40 9:10 9:40 Control 1 0.1 0.15 0.11 0.13 0.16 0.17 0.18 0.19 0.201 0.292 0.398 0.459 0.5 0.56 0.589 0.615 0.644 0.692 0.722 0.729 0.732 0.745 0.755 0.768 0.795 0.825 0.85 0.862 0.869 0.871 0.873 0.875 0.874 0.871 0.869 0.866 0.852 0.846 0.822 0.816 0.788 0.766 0.752 0.741 0.732 0.709 0.688 0.669 0.643 0.625 0.514 Control 2 0.052 0.049 0.046 0.052 0.055 0.066 0.085 0.121 0.2 0.279 0.364 0.452 0.485 0.538 0.571 0.616 0.645 0.688 0.715 0.722 0.736 0.775 0.768 0.772 0.783 0.797 0.835 0.851 0.858 0.861 0.864 0.866 0.867 0.865 0.861 0.855 0.852 0.842 0.836 0.823 0.811 0.789 0.773 0.761 0.753 0.733 0.715 0.689 0.658 0.629 0.57 Test 0.016 0.012 0.011 0.02 0.013 0.094 0.094 0.094 0.195 0.263 0.382 0.441 0.487 0.522 0.56 0.587 0.625 0.661 0.699 0.729 0.731 0.753 0.776 0.771 0.775 0.782 0.796 0.811 0.815 0.819 0.821 0.825 0.823 0.822 0.819 0.806 0.799 0.781 0.769 0.753 0.746 0.738 0.72 0.716 0.689 0.665 0.649 0.631 0.622 0.609 0.541

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60 Table 7. Plasmid DNA Isolation (Mini Prep): Strain Codes GESQA001 GESQA002 GESQA003 GESQA004 GESQA005 GESQA006 GESQA007 GESQA008 GESQA009 GESQA010 GESQA011 GESQA012 GESQA013 GESQA014 REFERENCES Conc.g/ml Sm 50 50 50 50 50 50 50 50 50 50 50 50 50 50

J. basic appl. sci. Rasmussen, B. 2000. Filamentous microfossils in a 3,235million-year-old volcanogenic massive sulphide deposite. Nature, 405(6787):677-679. Williams, JW. and Silver, S.1984. Bacterial resistance and detoxification of heavy metals. Enzyme Microb Technol, 6:30-37.

Plasmid Present Not Present Present Not Present Present Present Present Present Present Present Present Present Present Present Present

Beveridge, TJ. and Doyle, RJ. 1989. Metal ions and Bacteria. Wiley, New York. Clausen, CA. 2000. Isolation metal-tolerant bacteria capable of removing copper, chromium and arsenic from treated wood. Waste manages. Res. 18:264-268. Duguid, JP. 1989. Staining Methods. In: JG. Collee, JG., JP. Duguid, AG. Fraser, and BP. Marmion, eds., Eds., Mackie & McCartney Practical Medical Microbiology, Churchill Livingstone, New York., 41-51. Gadd, GM 1990. In: Microbial Mineral Recovery (Ehrlich, HL and Brierley, CL., Eds.), McGraw-Hill, New York, pp.249-275. Gadd, GM. 1988. In: Biotechnology, A Comprehensive Treatise, (Rehm, HJ., Ed), VCH Verlagsgesellschaft, Weinheim Vol.6b, pp.401-433. Gatti D., Mitra, B. and Rosen, BP. 2000. Mini-review: E. coli Soft Metal Iontranslocating ATPases. The Journal of Biological Chemistry, 275(44): 34009-34012. Maniatis T, Fritsch EF. and Sambrook, J. 1982. Molecular cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Mergeay, M., Nies, DH., Schlegel, HG., Gerits, J., Charles, P. and & van Gijsegem, F. 1985. Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plamid-bond resistance to heavy metas. J. Bateriol. 162:328-334. Pelczar, MJJ. and Reid, RD. 1958. Pure cultures and growth characteristics. In: Microbiology, McGraw-Hill Book Company, New York, pp.76-84.

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