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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 strainsshowed tolerance against heavy metals but most promising results were given by strain GESQA002 which showedmultiple stress tolerance as its maximum tolerable concentration (MTC) against cadmium chloride (CdCl2) was 6mM inenriched media and 1.4mM in minimal media, against copper sulphate (CuSO4) was 3.5mM in enriched media and1.8mM in minimal media, and against nickel chloride (NiCl2) it was 1mM in enriched media and 0.8mM in minimalmedia was observed. GESQA002 was also tested against antibiotics kanamycin (Km), and streptomycin (Sm) and itshowed elevated MTCs against Sm (100 g/ml) and Km (25 g/ml). Plasmids were detected in GESQA002 which couldbe 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 arerich in metals such as arsenic, lead, copper, and zinc(Rasmussen, 2000). Metals are directly or indirectlyinvolve in all aspects of growth, metabolism anddifferentiation (Beveridge and Doyle, 1989). Many metalsare essential, e.g. K, Na, Mg, Ca, Mn, Fe, Co, Ni, Cu, Zn,Mo, whereas others have no known essential biologicalfunctions, e.g. Al, Ag, Cd, Sn, Au, Sr, Hg, Tl, Pb (Gadd,1988). Retaining suitable concentrations of essentialmetals, such as copper and zinc while rejecting toxic

metals, such as arsenic, lead, and cadmium, was probablyone of the toughest challenges of the living cells (Gatti etal., 2000). Bacteria are among the most abundantorganism that occur every where on earth. Heavy metalsare increasingly found in microbial habitats due to severalnatural and anthropogenic processes; therefore, microbeshave evolved mechanisms to tolerate the presence ofheavy metals by either efflux, complexation, or reductionof metal ions) or to use them as terminal electronacceptors in anaerobic respiration (Gadd, 1990). Mostmechanism studied involve the efflux of metal ions outside the cell, and genes for tolerance mechanisms havebeen found on both chromosomes and plasmids. Bacteriathat are resistant to and grow on metals play an importantrole in the biogeochemical cycling of those metal ions(Gadd, 1990). Considering the importance of thesetolerance mechanisms, in the present study organismswere isolated from contaminated sites of the automobileworkshop and welding workshop situated in theresidential area of Karachi.

MATERIALS AND METHODS

Sampling

Soil samples were collected from different automobileand welding workshops, in sterilized 20ml sterilizeduniversal 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 routinelygrown in Luria Bertani (LB) medium (Maniatis et al.,

1982) at 37C. For antibiotic tolerance LB medium

supplemented with 1.8 % agar, solidified plates wereutilized. Metal tolerance was analyzed both in LB andTris minimal media (Mergeay et al., 1985).

Study of Colonial Morphology

Isolated colonies of purified strains grown on solidifiedagar plates were observed and data was recordedregarding 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 MorphologyCells were observed with Gram staining (Duguid 1989)under the microscope (oil immersion, 100 X). Shape ofthe cell (cocci, bacilli and cocco-bacilli) and arrangementof cells (scattered, bunches and chain) along with theGram-reaction was observed.

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

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J. basic appl. sci.56

Metal Tolerance

All the 14 strains were checked for Metal tolerance. Todetermine the maximum tolerable concentration (MTC) ofheavy metals salts such as NiCl2, CuSO4, CdCl2, 1Mstock solution of each of the metal salt was prepared andappropriate dilutions (0.2mM, 0.4mM, 0.6mM, 0.8mMand so on) were prepared. Bacterial strains were streaked

from O/N culture on LB and Tris minimal media agarplates. The plates were incubated at 37C and growth wasobserved 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 fromO/N culture on LB agar plates containing variableconcentration of antibiotic solutions (5, 10, 15, 20, 25, 50,75, 100, 125, 150,175 and 200 g/ml and incubated at37C and growth was observed after 24 hour. Selectedstrains 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 wasinoculated and 0.5mM CuSo4 was added (Test). Culturewas incubated for overnight at 37C on shaker. OD wastaken at 600nm and Growth Curve was studied at 37C inthree conditions: without metal inoculated culture fromControl, with metal (0.5mM CuSo4) inoculate culturefrom Control and with metal (0.5mM CuSo4) inoculatedculture 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 procedurefollowed as described by manufacturer for isolation ofgenomic DNA.

RESULTS

Isolation and purification of bacterial strains

14 bacterial strains were isolated from the soil samplesand designated as GESQA001- GESQA014.

Colonial MorphologyThe colonial morphology of most of the strains wascircular, elevation was convex, all colonial margins wereentire type; translucent and opaque were equally frequent.

A detailed result for colonial morphology has been givenin Table 1.

Cellular Morphology

Cellular morphology such as arrangement, shape andGram reaction were observed during Gram staining ofeach strain. Cellular shape of the strains were found to becocci, whereas cellular arrangement was found to be

paired and scattered, all of the isolated strains were Gramnegative. Details for cellular morphology and Gramreaction are given in Table 2.

Maximum Tolerance of Heavy Metals

Maximum Tolerable concentrations of all the strainsagainst Nicl2, CuSO4, CdCl2 have shown that isolated

strains were capable of growing at high concentration ofheavy metals in both the media, although it was higher inLB as compare to Tris minimal media. MTCs of metalsalts are given in Table 3 and 4. Strains which could notgrow at 0.2 mM were taken as 0 MTC.

Maximum Tolerance of AntibioticsMTC of selected strains against Km, Sm, and Cm waschecked. Results are given in Table 5. Strains whichcould not grow at 5 g/ml were taken as 0 MTC.

Growth Curve of Bacteria with Metal InductionTo find out weather metal tolerance mechanism wasinducible or not, growth curve of bacterial strainsGESQA002 and GESQA013 were studied. It wasobserved that metal tolerance in GESQA002 andGESQA013 was constitutive since GESQA002 andGESQA013 which were already expose with the metalovernight and the one which was expose to metal justbefore start of the growth curve did not show anysignificance difference and no sign of induction oftolerance mechanism was observed with respect to OD600.According to the results, it could be suggested thatGESQA002 and GESQA013 have constitutive pathwaysof tolerance against Copper. Results are shown in Fig. 1and Table 6.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0200

400

600

800

100

0

120

0

1400

160

0

Control1

Control2

Test 2

Fig. 1. Induction curve of GESQA002:

Control: Bacterial Strains GESQA002 grown in a mediumdevoid of metal saltTest 1: Induced cells were pre exposed to Cuso4 whereas;Test 2: uninduced cell were not pre exposed to Cuso4.

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Qurat-ul-Ain et al. 57

Isolation of Plasmid DNAPlasmid DNA was successfully isolated. A single band ofplasmid was observed in several strains while somestrains showed two and three plasmids which are shownin Figure 2 and table 7.

L1 L2 L3 L4 L5

Fig. 2. Isolation of Plasmid DNA.Plasmid DNA was extracted and separated by agarosegelelectrophoresis. DNA ladder in lane 1, GESQA006 isloaded in lane2, GESQA007 in lane 3, GESQA011 inlane 4, GESQA014 in lane 5.

DISSCUSSION

Sampling environments that contain elevatedconcentrations of heavy metals are a potential source oftoxic-metal-tolerant bacteria (Clausen, 2000). For thepresent studies bacterial strains were collected from thesoil of most common heavy metal contaminated sites of

our city, the welding shop (sample A) and automobileworkshops (sample B), with the expectation of isolatingmetal tolerant bacteria from the metal contaminated areas.Initially, several bacterial strains were purified out ofwhich few showed consistent behavior and were revivedafter being refrigerated for few weeks. Finally, all thosestrains were selected for characterizations which wereconsistent for their tolerance behavior; the isolated strainswere designated from GESQA001-GESQA014. Glycerolpreserves behaved perfectly and whenever requiredstrains revived within 24 hours.

Resistance systems not only protect the organism in aharsh environment, but they also play an important role inthe cycling of toxic metals in the biosphere. In some casesbacterial metal resistances have been shown to be due todifferences in uptake and/or transport of the toxic metal,while in other cases the metal is enzymaticallytransformed, by oxidation, reduction, methylation ordemethylation, into chemical species which is either lesstoxic or more volatile than the parent compound(Williams and Silver, 1984). Sampling sites wereselected with the aim to isolate metal tolerating bacteriafor which the best option was to locate metalcontaminated sites. Instead of concentrating on remoteindustrial areas, it was preferred to explore inner-cityenvironment with respect to heavy metal tolerance in

bacteria, specially the automobile workshops locatedwithin heavily populated residential areas of Buffer zonenorth Karachi.

Table 1. Colonial Morphology of the Isolates

Strain Codes Colour Size Shape Margin Elevation Opacity Surface

GESQA001 Pale Yellow Small Circular Entire Convex Opaque Smooth & Shiny

GESQA002 Pale Yellow Large Circular Entire Convex Opaque Smooth & Shiny



GESQA005 Pale Yellow Extra Large Circular Entire Convex Opaque Smooth & Shiny





GESQA010 Off White Pin Head Circular Entire Convex Translucent Smooth & Shiny


GESQA012 Off White Pin Head Circular Entire Convex Translucent Smooth & Shiny



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Sampling, isolation and purification of several bacterialstrains has been done and variability among all theisolated strains was observed with respect to all thestudied parameters. Metal tolerance and antibiotictolerance behavior of all the strains have revealed a veryinteresting pattern, which indicate multiple stresstolerance i.e., the strains which showed tolerance againstmetal have also shown tolerance against antibiotics.

Table 3. MTC of Heavy Metals (LB)

Strain CodesHeavy Metals

NiCl2 CuSO4 CdCl2.

GESQA001 1.0mM 3.5mM 3.5mM














Using bacterial tolerance against widespread metalcompounds one can minimize the effect of heavy metalson total biological activity of the ecosystem. Heavy metaltolerance of bacteria can play an important role inbioremediation of metal pollution in the environment by

either reducing the more toxic metals into less toxic formsor by accumulating the metal ions thus removing it fromthe effluent or areas containing concentration of particularmetals.

Table 4. MTC of Heavy Metals (Tris Minimal Media)

Strain CodesHeavy Metals

NiCl2 CuSO CdCl2.










GESQA010 0 1.8mM 1.4mM





Table 5. MTC of Antibiotics

Strain CodesAntibiotics g/ml

Sm Km

GESQA002 100 25

GESQA004 125 50

GESQA007 75 50

GESQA012 100 0

GESQA014 75 75

Table 2. Cellular morphology of bacterial isolates

Strain Codes Shape Arrangement Type Colour Gram Reaction

GESQA001 Rod Paired Diplobacillus Pink Gram negative

GESQA002 Round Scattered Streptococcus Pink Gram negative

GESQA003 Round Paired Diplococcus Pink Gram negative












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Table 6. Growth Curve with metal induction of GESQA002

Serial No. Time (mins) Time (hrs) Actual time Control 1 Control 2 Test

1 0 0 9:40 0.1 0.052 0.016

2 15 0.25 9:55 0.15 0.049 0.012

3 30 0.5 10:10 0.11 0.046 0.011

4 45 0.75 10:25 0.13 0.052 0.02

5 60 1 10:40 0.16 0.055 0.0136 90 1.5 11:10 0.17 0.066 0.094

7 120 2 11:40 0.18 0.085 0.094

8 150 2.5 12:10 0.19 0.121 0.094

9 180 3 12:40 0.201 0.2 0.195

10 210 3.5 13:10 0.292 0.279 0.263

11 240 4 13:40 0.398 0.364 0.382

12 270 4.5 14:10 0.459 0.452 0.441

13 300 5 14:40 0.5 0.485 0.487

14 330 5.5 15:10 0.56 0.538 0.522

15 360 6 15:40 0.589 0.571 0.56

16 390 6.5 16:10 0.615 0.616 0.587

17 420 7 16:40 0.644 0.645 0.625

18 450 7.5 17:10 0.692 0.688 0.661

19 480 8 17:40 0.722 0.715 0.699

20 510 8.5 18:10 0.729 0.722 0.729

21 540 9 18:40 0.732 0.736 0.731

22 570 9.5 19:10 0.745 0.775 0.753

23 600 10 19:40 0.755 0.768 0.776

24 630 10.5 20:10 0.768 0.772 0.771

25 660 11 20:40 0.795 0.783 0.775

26 690 11.5 21:10 0.825 0.797 0.782

27 720 12 21:40 0.85 0.835 0.796

28 750 12.5 22:10 0.862 0.851 0.811

29 780 13 22:40 0.869 0.858 0.815

30 810 13.5 23:10 0.871 0.861 0.81931 840 14 23:40 0.873 0.864 0.821

32 870 14.5 0:10 0.875 0.866 0.825

33 900 15 0:40 0.874 0.867 0.823

34 930 15.5 1:10 0.871 0.865 0.822

35 960 16 1:40 0.869 0.861 0.819

36 990 16.5 2:10 0.866 0.855 0.806

37 1020 17 2:40 0.852 0.852 0.799

38 1050 17.5 3:10 0.846 0.842 0.781

39 1080 18 3:40 0.822 0.836 0.769

40 1110 18.5 4:10 0.816 0.823 0.753

41 1140 19 4:40 0.788 0.811 0.746

42 1170 19.5 5:10 0.766 0.789 0.738

43 1200 20 5:40 0.752 0.773 0.7244 1230 20.5 6:10 0.741 0.761 0.716

45 1260 21 6:40 0.732 0.753 0.689

46 1290 21.5 7:10 0.709 0.733 0.665

47 1320 22 7:40 0.688 0.715 0.649

48 1350 22.5 8:10 0.669 0.689 0.631

49 1380 23 8:40 0.643 0.658 0.622

50 1410 23.5 9:10 0.625 0.629 0.609

51 1440 24 9:40 0.514 0.57 0.541

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Table 7. Plasmid DNA Isolation (Mini Prep):

Strain Codes Conc.g/ml Sm Plasmid Present

GESQA001 50 Not Present

GESQA002 50 Present

GESQA003 50 Not Present

GESQA004 50 Present

GESQA005 50 PresentGESQA006 50 Present

GESQA007 50 Present

GESQA008 50 Present

GESQA009 50 Present

GESQA010 50 Present

GESQA011 50 Present

GESQA012 50 Present

GESQA013 50 Present

GESQA014 50 Present

REFERENCES

Beveridge, TJ. and Doyle, RJ. 1989. Metal ions andBacteria. Wiley, New York.

Clausen, CA. 2000. Isolation metal-tolerant bacteriacapable of removing copper, chromium and arsenic fromtreated 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 ComprehensiveTreatise, (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 ofBiological Chemistry, 275(44): 34009-34012.

Maniatis T, Fritsch EF. and Sambrook, J. 1982. Molecularcloning: A Laboratory Manual Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY.

Mergeay, M., Nies, DH., Schlegel, HG., Gerits, J.,Charles, P. and & van Gijsegem, F. 1985. Alcaligeneseutrophus CH34 is a facultative chemolithotroph withplamid-bond resistance to heavy metas. J. Bateriol.162:328-334.

Pelczar, MJJ. and Reid, RD. 1958. Pure cultures andgrowth characteristics. In: Microbiology, McGraw-HillBook Company, New York, pp.76-84.

Rasmussen, B. 2000. Filamentous microfossils in a 3,235-million-year-old volcanogenic massive sulphide deposite.Nature, 405(6787):677-679.

Williams, JW. and Silver, S.1984. Bacterial resistance anddetoxification of heavy metals. Enzyme Microb Technol,6:30-37.

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