INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 3, No 2, 2012

Copyright by the authors - Licensee IPA- Under Creative Commons license 3.0
Research article

ISSN 0976 4402

Characterization of Endosulfan and Endosulfan sulphate degradation by

strains of Pseudomonas putida

Sarah Sunitha1, Krishna Murthy. V1, Riaz Mahmood2

1- Department of Biotechnology, P.E.S.Institute of Technology, Bangalore-560085, India
2- Department of Biotechnology and Bioinformatics, Kuvempu University, Shimoga, India
sarah@pes.edu
doi:10.6088/ijes.2012030132013
ABSTRACT
In this paper, we describe the characterization of a bacterial culture obtained by the process of
enrichment from an endosulfan contaminated soil sourced from a coffee cultivated area.
When the culture was grown in the presence of endosulfan or endosulfan sulphate as the sole
source of sulphur, more than 70 % of endosulfan and 90% of endosulfan sulphate degradation
was observed. The culture was found to consist of strains of Pseudomonas putida, differing in
their 16srRNA sequence. An increase in biomass up to 1.8g/L and 2.2g/L was observed in the
presence of endosulfan and endosulfan sulphate respectively. Metabolites of endosulfan
degradation like endosulfan diol, endosulfan lactone and endosulfan sulphate were detected
in the cultures. The culture showed higher degradation under aerated conditions compared to
static conditions. With the ability to tolerate high concentrations of endosulfan and
endosulfan sulphate, this culture has the potential for use as a bioremediating agent for
contaminated soil and water.
Keywords: Persistence, degradation, bioremediation, Pseudomonas putida .
1. Introduction
Pesticides have been an indispensable part of modern agriculture. Endosulfan (1,2,3,4,7,7hexachlorobicyclo-(2.2.1)-hepten-2,3-bisoxymethylene-5,6-sulfite) is a cyclodiene pesticide,
which is known by trade names like Thiodan, Endocel, Endosol, and Thiosulfan. It is used
extensively for the control of pests infesting crops like cashew, coffee, tea, cotton and many
other important crops. Commercially available formulations of endosulfan contain the and
steroisomers in the ratio 70:30. Endosulfan is known to be toxic to aquatic organisms (S
Broomhall, 2002; Capkin E et al, 2006). In mammals, it is found to exhibit reproductive
toxicity (Saiyed H et al, 2003; Bharath B K et al, 2011) and neuro toxicity (Ravi Kiran, 1988).
In soil, microbial action converts endosulfan to endosulfan sulphate by oxidation and to
endosulfan diol by hydrolysis. Both of the isomers of endosulfan have different persistence
levels in the environment. The mean half lives of , and total endosulfan in soil are 27.5,
157 and 1336 days respectively under aerobic conditions (GFEA-U ,2007; US EPA 2007c).
Endosulfan sulphate is as toxic as and more persistent than the endosulfan isomers (US EPA,
2007a).
Bacteria like Arthrobacter, Rhodococus, Klebsiella pneumoniae, Pseudomonas aeruginosa
have been reported to degrade endosulfan (K M Weir, 2006; K Verma, 2005; G Kwon 2002;
S Hussain 2009). Very few bacteria have been shown to degrade both endosulfan and
endosulfan sulphate (G Kwon et al, 2002; G Kwon et al, 2005; K M Weir et al, 2006). These
bacteria have the potential to be used as bioremediating agents in endosulfan contaminated

Received on August 2012 Published on September 2012

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Characterization of Endosulfan and Endosulfan sulphate degradation by strains of Pseudomonas putida

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
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Characterization of Endosulfan and Endosulfan sulphate degradation by strains of Pseudomonas putida

inoculated controls in triplicates were also maintained simultaneously. At regular intervals,

the cultures were removed and extracted for analysis. Degradation of endosulfan and
endosulfan sulphate were also studied under static and aerated conditions.
2.6 Extraction of residues and HPLC Analysis
The cultures were extracted twice with equal volumes of ethyl acetate by mixing in an orbital
shaker for 1 hour. The pooled organic phase was concentrated in a rotary evaporator and the
residue was dissolved in 0.5 ml of ethyl acetate. The extracts were filtered through a 0.2m
Syringe Filter (Axiva Biochem) and were analysed by HPLC (Hitachi LaChrome Elite
System) using a C18 RP Column (100 x 5 m). The solvent system used was a mixture of
acetonitrile and water in the ratio 70:30, at a flow rate of 1ml/min. The eluted solutes were
detected by a UV Detector at 214 nm (Arshad et al 2007).
2.7 Identification of the bacterial cultures by 16srRNA sequencing
The culture S3 was found to be made up of three distinct types of colonies, designated as
PES3a, PES3b and PES3c. Genomic DNA was isolated from the pure cultures using GeNei
Ultrapure bacterial genomic DNA purification Kit KT162 (Merck Biosciences). Using
consensus primers (Forward-5 AGAGTTTGATCMTGGCTCAG 3, Reverse -5
TACGGYTACCTTGTTACGACTT 3) the ~1.5 kb 16S rDNA fragment was amplified with
Taq DNA Polymerase. The PCR reaction was set up as follows: 50 ng of DNA in 1 l, 2.5l
of 10x Taq Buffer A, 1l of dNTP mix(2.5mM each), 1l of Forward and reverse primer, and
volume was made upto 25l with distilled water. PCR reaction was carried out as follows in a
Corbett Research thermal cycler; Initial denaturation-940C for 5 min, denaturation- 940C for
1 min, annealing-550C for 45 sec, extension-720C for 1.30 min and final extension at 720C for
10 min. These cycles were repeated 35 times. The PCR product was bi-directionally
sequenced using the forward, reverse and an internal primer. Phylogenetic tree was
constructed using the Neighbour joining method. Distance Matrix based on Nucleotide
sequence homology was obtained using Kimura-2 Parameter.
3. Results and discussion
3.1 Bacterial growth in the presence of endosulfan and endosulfan sulphate

Figure 1(a): Growth of S3 culture in the presence of

-Endosulfan,
-Endosulfan,
+ Endosulfan,
Endosulfan sulphate,
Control
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Characterization of Endosulfan and Endosulfan sulphate degradation by strains of Pseudomonas putida

Figure 1(b): Biomass yield of culture S3 grown with 20mg/L Endosulfan

The S3 mixed culture showed maximum growth in medium supplemented with 20 mg/L
endosulfan sulphate. There was also considerable increase in the optical density at 600nm for
cultures supplemented with , and mixture of + isomers. In the control medium where no
sulphur source was included, very little growth was seen. Initially, the growth could be
sustained by small amount of sulphur compounds present as contaminants in media
components. When these were exhausted, there was no further increase in bacterial growth.
A mixed bacterial culture (Sutherland et al, 2000) grown under similar conditions showed
very little growth in the presence of Endosulfan sulphate, compared to the isomers of
Endosulfan. The biomass of the S3 culture increased steadily during the first 5 days and
reached a maximum of 1.8mg/L with endosulfan. There was no marked increase in biomass
after the 5th day. Endosulfan sulphate produced a higher yield of biomass compared to
endosulfan.

Figure 1 (c): Growth of culture S3 in different concentrations of endosulfan (mg/L)

The culture also exhibited good growth in the presence of high concentrations of endosulfan
sulphate while higher concentrations of endosulfan were inhibitory. In the case of endosulfan,
maximum growth was seen in 50mg/L concentration while 200mg/L of endosulfan sulphate.
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Characterization of Endosulfan and Endosulfan sulphate degradation by strains of Pseudomonas putida

Figure 1 (d): Growth of culture S3 in different concentrations of endosulfan sulphate (mg/L)

Table 2: Metabolites detected in the mixed culture S3 when grown with different isomers of
Endosulfan and endosulfan sulphate
Sl.
No

Spiked
component

Endosulfan

Endosulfan
+
Endosulfan
Endosulfan
sulphate

2
3
4

Endosulfa
n
+

Detected isomers and metabolites

Endosulfa Endosulf Endosulf
an ether an
Endosulfa n
n
Sulphate
lactone
+
+
+

Endosulfa
n
Diol
+

Figure 2: HPLC chromatogram of Endosulfan isomers and metabolites.

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Characterization of Endosulfan and Endosulfan sulphate degradation by strains of Pseudomonas putida

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

Figure 3(a): Residual Endosulfan concentration and percentage degradation

Figure 3(b): Residual Endosulfan sulphate concentration and percentage degradation

Maximum degradation of endosulfan occurred during the first three days of the incubation
and 70 % of the pesticide was degraded. In the uninoculated control, abiotic process resulted
in degradation of 20% of endosulfan. The increase in degradation corresponded with an
increase in cell density.
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Characterization of Endosulfan and Endosulfan sulphate degradation by strains of Pseudomonas putida

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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Characterization of Endosulfan and Endosulfan sulphate degradation by strains of Pseudomonas putida

3.3 Identification of the bacterial culture S3

The mixed culture S3 was found to contain three types of bacterial strains designated as
PES3a, PES3b and PES3c, based on their distinct colony morphology. All the three were
found to be gram negative rods. Based on their 16srRNA sequence analysis, all the three
were identified as Pseudomonas putida. The similarity between PESS3a and PESS3b
16srRNAsequences was found to be 98%, when analysed by the Needleman-Wunsch
algorithm. 100% similarity was observed with the 16srRNA sequences of PES3b and PES3c.
3.3.1 Genbank Accession numbers
Pseudomonas putida PES3a and Pseudomonas putida PES3b 16srRNA sequences have been
deposited in the GenBank database, under the accession numbers JX081247 and JX112653
respectively.
Pseudomonas spinosa, Pseudomonas aeruginosa, (Hussain, 2007), Pseudomonas KS-2P (Lee
et al, 2006) and Pseudomonas IITR01 (A.Bajaj et al, 2010) are some of the Pseudomonas
strains that have been reported so far, which can metabolize endosulfan. Some of the strains
reported, which can degrade endosulfan sulphate are Arthrobacter sps (Weir et al, 2006),
Pseudomonas KS-2P (Lee et al, 2006), Klebsiella oxytoca KE-8 (G Kwon et al, 2005) and
Pseudomonas IITR01. Both Pseudomonas KS-2P and Pseudomonas IITR01 was capable of
degrading 70% of Endosulfan sulphate. Pseudomonas putida has the advantage of being a
non-pathogenic organism, while Pseudomonas aeruginosa is an opportunistic pathogen.
Hence, the former can be an ideal candidate for use in bioremediation studies where this
organism can be introduced into the environment without safety concerns.

Figure 4: Structures of endosulfan isomers and metabolites

4. Conclusion
The mixed culture composed of Pseudomonas putida strains PESS3a and PES3b were
capable of degrading endosulfan and its toxic metabolite endosulfan sulphate. The next

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Characterization of Endosulfan and Endosulfan sulphate degradation by strains of Pseudomonas putida

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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