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Research article
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soil and water. The degradation of endosulfan produces metabolites like endosulfan sulphate,
endosulfan alcohol, endosulfan ether and endosulfan lactone. Endosulfan sulphate is mainly
produced by microbial hydrolysis and is also detected in plants (Beard.J.E and Wara, 1969).
We had earlier reported the derivation of endosulfan degrading bacterial cultures from
endosulfan contaminated soils by the process of enrichment (S Sunitha et al, 2012). In this
paper, we report the kinetics of endosulfan and endosulfan degradation, and the identification
of the bacterial cultures by 16srRNA sequencing.
2. Materials and methods
2.1 Chemicals: All the chemicals used were of analytical Grade. HPLC grade solvents were
obtained from Merck India. Analytical standards of endosulfan mixture(70:30), and
isomers of Endosulfan, Endosulfan Sulphate, Endosulfan Ether, Endosulfan Lactone and
Endosulfan Diol were obtained from Sigma Aldrich Chemicals India.
2.2 Maintenance of the bacterial cultures
The bacterial culture designated as S3 was maintained on agar slants of minimal medium (T
S Sutherland et al, 2000) containing 20 mg/L Endosulfan as the sole source of sulphur.
Endosulfan and Endosulfan sulphate were dissolved in small amounts of acetone and added
to the medium after sterilization by autoclaving. The culture was sub cultured every fifteen
days by transferring a loopful of cells on fresh agar medium and incubating at 28+20C for 48
hours and stored at 40C. Glycerol stocks of the cultures were prepared for long term storage
at -200C.
2.3 Growth with different Endosulfan isomers
50 ml of the minimal medium containing 20 mg/L each of , , + and Endosulfan sulphate
were taken in 250 ml conical flasks and inoculated with 1 % (v/v) log phase culture of S3.
Before inoculation, the cells were washed in plain medium to remove any traces of
Endosulfan or its metabolites and were resuspended in the original volume of medium. Plain
medium with cells but without endosulfan was also maintained in parallel. The flasks were
incubated at 28+20C for 12 days in a shaking incubator at 120 rpm and absorbance at 600 nm
was recorded in a spectrophotometer (Jenway, UK) at regular intervals. Similarly, the growth
of the cells in the presence of 25, 50, 100 and 200 mg/L of endosulfan and endosulfan
sulphate was studied.
2.4 Estimation of Biomass
100 ml of minimal medium containing 20 mg/L of Endosulfan (+) and 20mg/L endosulfan
sulphate separately were inoculated with 1% (v/v) of S3 culture and incubated on a shaker
incubator at 120 rpm at 28+20C. 10 ml of the culture was withdrawn at 24 hour intervals and
was filtered under suction through a preweighed 0.22m mixed cellulose ester membrane of
47mm diameter (GSWPO4700, Millipore). The membrane was then dried in a hot air oven at
700C for 2 hours, weighed and the biomass was calculated.
2.5 Rate of degradation of Endosulfan
250 ml conical flasks in triplicates, containing 50 ml of minimal medium with 50 mg/L
Endosulfan (+) and 40 mg/L endosulfan sulphate were inoculated separately with 1% (v/v)
of log phase S3culture and incubated on a shaking incubator at 120 rpm at 28+20C. UnSarah Sunitha, Krishna Murthy. V, Riaz Mahmood
International Journal of Environmental Sciences Volume 3 No.2, 2012
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Spiked
component
Endosulfan
Endosulfan
+
Endosulfan
Endosulfan
sulphate
2
3
4
Endosulfa
n
+
Endosulfa
n
Diol
+
863
2.50 min-Acetone, 4.13 min-Endosulfan diol, 5.25 min-Endosulfan lactone, 6.62 minEndosulfan sulphate, 8.19 min-Endosulfan ether, 9.56 min- Endosulfan, 11.81min-
Endosulfan
The degradation of endosulfan occurs through the oxidative or the hydrolytic pathway.
Endosulfan sulphate is the predominant metabolite produced by oxidation of endosulfan.
Hydrolysis of endosulfan produces the less toxic endosulfan diol (Kumar et al, 2006). In
aqueous environments, hydrolysis is the dominant pathway. Some microbial strains are also
specific to the isomer of endosulfan. For example, the Mycobacterium tuberculosis strain
degrades the beta isomer to yield monoaldehyde and hydroxyl ether but degrades the alpha
isomer to give endosulfan sulphate (Sutherland et al, 2002a). In some mixed cultures, though,
none of the metabolites were detected (Kumar and Philip 2006).
3.2 Rate of Endosulfan and Endosulfan sulphate degradation
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In similar experiments, an enrichment bacterial culture derived from a tropical acid soil
showed about 70 % degradation of endosulfan after 20 days (Surya Kalyani S, 2009).
Endosulfan sulphate degradation gradually increased over the period of 21 days and the
culture degraded 90% of the added endosulfan sulphate at the end of 21 days while close to
20% degradation was observed in the un-inoculated controls.
Figure 3 (c): Comparison of endosulfan degradation under static and aerated conditions
Figure 3 (d): Comparison of degradation of endosulfan sulphate under static and aerated
conditions
Degradation of both endosulfan and endosulfan sulphate decreased under static conditions. In
some mixed cultures, increased degradation in facultatively anaerobic systems have been
observed though the cell density was higher in the aerobic system (Kumar and Philip 2006).
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obvious direction is to evaluate these cultures as a consortium as well as individually for their
ability to degrade endosulfan and endosulfan sulphate in soil and water.
Acknowledgements
The authors would like to thank the management of P.E.S Institute of Technology for the
facilities. This research was supported by financial assistance to the corresponding author
from University Grants Commission, Government of India (Grant number: MRP(S)-704/1011/KABA037/UGC-SWRO).
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