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Abstract
The small scale industries located at Mira Road, Mumbai, are engaged in various processes/operations which
generate the wastes. The waste generated are collected and disposed of at the dumping ground near the industrial
estate. In the present study, the physico-chemical and microbial status of the contaminated sites has been carried
out. The heavy metal with special reference to copper was exposed to microbial consortium at increasing
concentrations viz 5, 25, 50, 75, 100 up to 800ppm to isolate potential microorganism for bioremediation. Citrobacter
freundii has been identified by 16SrDNA technique as potential microorganism for bioaccumulation/bioremediation of
copper. This organism can be used for remediation of copper from contaminated environment.
according to a standard procedure (Kumar, shaker at37°C, 200rpm. pH was adjusted to 7.0
2004). Briefly, 1gm of soil was mixed with 10ml before inoculation. pH and optical density at
of sterile distilled water. An aliquot of 0.1 ml of 600nm were measured daily to analyze bacterial
dilutions for each soil samples was spread growth. Further, 1ml. bacterial culture was
plated onto agar plates on to agar from the transferred to metal concentration of 25mg/l, and
appropriate dilution tubes and incubated at room subsequently to 50mg/l, 100mg/l upto 800mg/l to
temperature (Collins, 1985). The bacterial get potential microorganism. 100µl of samples of
colonies were counted after every 24 hrs. Only each concentration were plated on minimal
the plates showing between 25 to 300 colonies media containing metal solutions of respective
were tallied, and the results were averaged for concentration. Single colony was isolated at
each soil samples. The fungal colonies were higher concentration and identified as potential
counted after 48-72 h. Samples were preserved microorganism for bioaccumulation copper.
at 4°C for further microbial analysis (Chao et al.
2003). Isolated colonies were further analyzed Identification of isolated microorganism by
using specialized agar / 16SrDNA sequencing. 16SrDNA analysis
The isolates were then identified based on the 16SrDNA analysis was done by using
morphological, cultural and biochemical predetermined universal primers of 16SrDNA.
characteristics following Bergey’s Manual of DNA isolated from pure culture was used as
Determinative Bacteriology (Holt et al. 1994). template. PCR was performed with a 50μl
The specialized agar (from Hi media) was used reaction mixture containing primer 16S, DNA
for identifying E. coli, Salmonella, Shigella, template buffer, MgCl2, dXTPs, Taq polymerase.
Vibriocholerae , Yeast and Mould, S. aureus, PCR products were analyzed by electrophoresis
Clostridium, Pseudomonas , Streptococcus in 1.8% agarose gel. PCR program was carried
faecalis, Serratia (Klinge 1960). Other out in PTC-200 Peltier thermocycler which
microorganisms present were identified using comprises of three steps; 1) Denaturation at
16SrDNA sequencing. 94ºC for 1minute; 2) Annealing at 55ºC for
1minute; 3) Extension at 72ºC for 1minute
Metal analysis (Bosshard et al. 2003).
Each soil sample was digested with 10ml of a DNA sequences were compared with
mixture perchloric acid: nitric acid already submitted sequence in database using
(HCLO4:HNO3-1:5 v/v) (Lone et al. 2008). Acid BLAST software. Further, most similar
digestion was carried out on a hot plate at 70- sequences were aligned by ClustalW and
100C until yellow fumes of HNO3 and white ClustalX software and phylogenetic tree was
fumes of HClO4 were observed. The digestion drawn using PHYLIP software to analyze
process was continued until a clear solution evolutionary relationships among sequences of
remained after volatilization of acids, and was isolated microorganism and nearest neighbors
stopped when the residue in the flask was clear (Fulekar 2008).
and white. The digested sample was dissolved
in distilled water, filtered through Whatman no.1 Results and Discussion
filter paper to remove impurities and made up to The present research study has been carried out
the desired volume (APHA, 1979). at the waste disposal site located at Mira Road,
Bhayander, Mumbai. The small scale industries
Isolation of potential microorganism carried out the various processes and
Soil and sediments were serially diluted to operations which generates wastes containing
10,000 folds and plated on nutrient agar. 1ml metals. The potential microorganism has been
bacterial culture was inoculated in nutrient broth identified from the microbial consortium present
and further, in 250ml erlenmeyer flasks in the waste disposal area for remediation of the
containing 100ml minimal media with a metal metals. The physico-chemical parameters
concentration of 5mg/l. Minimal media studied include: -pH, Temperature, Moisture
comprised of Na2HPO4, NH4Cl, glucose blended content, Bulk density, Phosphate, Sulfate, Total
with 0.6ml of trace elements solution. Trace Organic Carbon, Biological Oxygen Demand,
elements solution contains CaCl2.2H2O, MgSO4, Chemical Oxygen Demand, Total Nitrogen,
MnSO4.7H2O, and FeSO4.7H2O. Copper was Alkalinity, Total Organic Matter. The
added from 1000mg/l stock solution of concentrations of each parameter obtained are
CuSO4.5H2O. Flasks were kept in incubator presented in table 1.
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Research Article Biology and Medicine, 1 (3): 7-14, 2009
2. Temperature 30 28 30 32 30
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Research Article Biology and Medicine, 1 (3): 7-14, 2009
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Research Article Biology and Medicine, 1 (3): 7-14, 2009
Metals Samples
I II III IV Avg.
Copper (mg/l) 9.54 8.54 10.4 13.92 10.6
Iron(mg/l) 10.6 7.6 9.9 15.8 10.98
Cadmium 20.02 19.87 21.3 26.81 22.0
(mg/l)
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Research Article Biology and Medicine, 1 (3): 7-14, 2009
GGGTCGCGGGCTAAAACATGCAAGTCGAACGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGTGGCGGACGGGTGA
GTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGAC
CAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCA
CCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGG
GAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGG
GTTGTAAAGTACTTTCAGCGGGGAGGAAGGTGTTGTGGTTAATAACCACAGCAATTGACGTTACCCGCAGAAGAAGC
ACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGC
ACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTGGA
GTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGC
GGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACGGATTAAAACGCGGGGGGTCCACAAA
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Research Article Biology and Medicine, 1 (3): 7-14, 2009
Gene Bank Entry Domain Phylum Class Order Family Genus Species
Sample
AJ233408 Bacteria Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Citrobacter freundii
BI 359
AF025368 Bacteria Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Citrobacter braakii
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