ISOLATION AND IDENTIFICATION OF HEAVY …

1 , VOL. 5(2) 2015: 150-154 ISSN 2250 3579150 ISOLATION AND IDENTIFICATION OF HEAVY metal (LEAD, ZINC ANDCOPPER) RESISTANT BACTERIA FROM OIL FIELD SOIL COLLECTEDFROM MORAN, DIBRUGARH DISTRICT, ASSAMaPradyut Saikia,a,bSudakshina Das,b*Rajesh Kumar Shah&aSaidul IslamaInstitutional Level Biotech Hub, College, Dibrugarh, Assam, of Zoology, College, Dibrugarh, Assam, India-786001.*Corresponding : present study deals with ISOLATION and IDENTIFICATION of HEAVY metal resistant bacteria from oil field soil collected fromKordoiguri, Moran ofDibrugarh district, Assam. A total of three strains could be isolated from the oil field samples. The isolatedstrains wereKlebselliasp.,Staphylococcussp. the basis of their biochemical and morphological inhibitory concentration (MIC) and antibiotic resistance pattern of the isolates was all three strainsisolated, the maximum resistance was shown for Lead (800 g/ml forthe strainKlebselliasp.)

2 And Zinc (800 g/ml for the strainKlebselliasp.). The minimum resistance was shown for Lead (200 g/ml for the strainBacillussp.).For the antibiotic tolerancetests, maximum resistance was shown for cefotaxime (140 g/ml forthe strainKlebselliasp.) and minimum for Ampicillin(40 g/ml for the strainStaphylococcussp.). The present study reveals that the metal resistant bacteria can be explored in the fieldof : HEAVY metal , Kordoiguri (Moran), Minimum inhibitory concentration, are among the main factors that affect ourenvironment day by day. Among pollutants the HEAVY metalsare the sources of contamination that are observed from theoil wells, petroleum plants etc. as trace HEAVY metals arecommon constituents of crude oil, petroleum derivatives(leaded gasoline, lubricating oils or greases etc).

3 Moreover,metals play a vital role in biological systems as a living cellcannot exist without metal ions. Trace amounts of heavymetals are also required byliving organism including copper,cobalt, iron but excessive levels of essential metals howevercan be toxic to the organism (Frankeet al.,2003). With rapidindustrialization, pollution is also in rapid increase. Pollutionof soil with HEAVY metals is becoming one of the most severeenvironmental and human health hazards. Elevated levels ofheavy metals not only decrease soil microbial activity andcrop production, but also threaten human health through thefood chain (Mclaughlinet al.,1999). HEAVY metals like Zinc,Cadmium, Copper, Lead, Nickel and mercury have beenreported as the most toxic pollutants (Cameron, 1992).

4 Lead(Pb) a major pollutant that is found in soil, water and air is ahazardous waste and is highly toxic to human, animals, plantsandmicrobes (Lowet al.,2000). Excessive levels of heavymetals can be damaging to the organism. Some of them aredangerous tohealth or environment ( , cadmium,lead,chromium) and some may cause corrosion ( zinc,lead(Hogan, 2010). HEAVY metals contaminate theenvironment because of their known accumulation in the foodchain and persistence in nature. HEAVY metal contamination isone of the most important environmental concerns from minetailings. Metals are significant toxic factor to biota in theenvironment. For example, HEAVY metals may decreasemetabolic activity and diversity as well as affect thequalitative and quantitative structure of microbialcommunities (Gilleret al.))

5 ,1998). Each HEAVY metal hasunique toxicity or function. Zinc and copper can enhancemicrobial growth at low concentrations but suppresses growthat high concentrations (Geet al.,2009).Microbial survival inpolluted soils depends on intrinsic biochemical and structuralproperties, physiological, and/or genetic adaptation includingmorphological changes of cells, as well as environmentalmodifications of metal speciation (Wuertz and Mergeayet al.,1997). To survive under metal -stressed conditions, bacteriahave themselves evolved up to a several types of adaptationmechanisms to tolerate the uptake of HEAVY metal ions. Thesemechanisms include the efflux of metal ions outside the cell,bioaccumulation and complexation of the metal ions insidethe cell, and the reduction of the HEAVY metal ions to a lesstoxic state (Montuelleet al.

6 ,1994). Bacteria have adapted toheavy metals through a variety of chromosomal-, transposon-, and plasmid-mediated resistance systems (Saidet al.,1991).For treating HEAVY metal contaminated tailing and soils,bioremediation is the most efficient and least costly method(Geet al.,2009). Some microorganisms have the resistanceagainst the HEAVY metal and they can grow in the HEAVY metalrich environment most organisms haveIdentification of HEAVY metal resistant bacteria from oil field soil151detoxification abilities( , mineralization, transformationand/or immobilization of pollutants), particularly bacteriaplay a crucial role in biogeochemical cycles and in sustainabledevelopment of the biosphere (Diaz, 2004). Bioremediationmay be employed in order to attack specific contaminantssuch as chlorinated pesticides that are degraded by bacteria,or a more general approach may be taken, such as oil spillsthat are broken down using multiple techniques including theaddition of fertilizer to facilitate the decomposition of crudeoil by bacteria.

7 Bioremediation can be considered as mostuseful technique for environmental cleanup and ecosystemservice provider. The objective of the present study is toisolate and identify the bacteria from oil field soil ofKordoiguri, Moran of Dibrugarh district, Assam. The bacteriawere biochemically identified and their potentiality to resistthe HEAVY metals such as Zinc, Lead and Copper Description and Soil Sampling: Soil samples werecollected from Kordoiguri oil field, Moranof DibrugurahDistrict, contaminated soil sample was collectedfrom Dibrugarhdistrict as control. Soil samples from soilsurface (0-5 cm) and at a depth of approximately 10 cm weretaken in sterilized polyethylene bags using sterilized spatulaand stored at 4 C until examination.

8 To obtain the microbialexamination, top soil wascollected from the oil field and IDENTIFICATION of bacteria: The soil samples werediluted up to a dilution factor 10-4and plated on of dilutions were made to reduce the cells no inthe samples. One ml of dilutedsample was spread onto thesurface of Nutrient Agar medium in the petri dishes andincubated at 37 C and allowed to grow for 24 h. Singledeveloped colony was picked on the Nutrient Agar plates andsubcultured in nutrient broth and plated again on NutrientAgar medium after dilution to obtain pure cultures. Purebacterial strains were obtained after successive transfer ofindividual colony in Nutrient Agar plates and incubated for24 h at 37 C forming units weredetermined after 2 days of incubation at 37 shape andcolors of the colonies were examined under the microscopeafter Gram staining.

9 Isolates were biochemically analyzed forthe activities of Catalase, MR-VP test, Starch hydrolysis,Phenylalanine Agar test, Tryptone test, MSA test, EosineMethylene blue test, Nitrate test, Indole production andCitrate utilization. The tests were used to identify the isolatesaccording to Bergey s Manual of Systematic Bacteriology(Claus and Berkeley, 1986).Determination of MinimumInhibitory Concentration (MIC): For testing HEAVY metalresistance, the metals Cu, Pb and Zn were used as CuSO4,PbNO3and ZnCl2. Stocks of the metal salts were prepared indistilled water and stored at 4 C. The metal salts were thenadded to sterilize nutrient agar medium in concentrationsvarying from range 200 to 800 g/ml with an increment of200 g/ml.

10 Plates were then spot inoculated and incubated at37 C for 2 days. The concentration of the metal whichpermitted growth and beyond which there was no growthwasconsidered as the MIC of the metal against the strain concentration above the MIC value of the control strain wasaccepted as resistant range. All of the experiments werereplicated 3 of Antibiotic Resistance:The isolated strains were tested for antibiotic resistanceagainst Ampicillin and Cefotaxime with concentrationranging from 20 to 200 g/ml. Antibiotic sensitivity andresistance of the isolated HEAVY metal resistant isolates wereassayed according to the Kirby-Bauer disc diffusion method(Baueret al.,1996).RESULT&DISCUSSIONM orphological Characterization: A total of three strains wereisolated from the sample collected from the oil field ofKordoiguri, Moran.