J. Bio. & Env. Sci.

2012

Journal of Biodiversity and Environmental Sciences (JBES)

ISSN: 2220-6663 (Print) 2222-3045 (Online)
Vol. 2, No. 6, p. 41-53, 2012
http://www.innspub.net
RESEARCH PAPER OPEN ACCESS

Enrichment and isolation of endosulfan degrading microorganism

from natural resource
Ajit Kumar1,2*, P.J. John1, I. Soni1
1
Environment Toxicology Unit, Centre for Advanced studies in Zoology, University of
Rajasthan, Jaipur – 302 004, India
2
Present address: Centre for Bioinformatics, M.D. University, Rohtak -124 001, India
Received: 18 May 2012
Revised: 18 June 2012
Accepted: 19 June 2012

Key words: Enrichment, isolation, microorganism, endosulfan degradation, soil.

Abstract
The enrichment and isolation of microbes from natural resource for endosulfan degradation was carried out to
search for the potential candidates for development of in situ bioremediation technology for endosulfan. Twelve
soil samples, with history of Endosulfan application, were enriched for microorganisms that can utilize
Endosulfan as carbon source in Carbon-Deficient Medium (CDM) or as sole source of sulfur in Non Sulfur
Medium (NSM). Five monocultures (N1, N2, N3, N4 & N5) were obtained after Round 2 enrichment of 15 days
each, which showed significantly prolific growth on NSM-agar supplemented with Endosulfan as sole sulfur
source. On comparison of these monocultures for amount of pesticide degradation, microbial growth and the
changes in pH of the medium, when grown in NSM with Endosulfan as sole source of sulfur, strain N2 showed
maximum endosulfan degradation (92.2%), microbial growth (0.382) and reduction in media pH (52.78%). The
culture conditions of strain N2 were optimized for maximum pesticide degradation. In an optimized culture
condition, strain N2 degraded endosulfan upto 94.2% within 7 days, when estimated quantitatively by Gas-
Chromatography Electron Capture Detection (GC-ECD) method. The study suggests that strain N2 is worth
investigating for its biochemical and molecular characterization, to develop a valuable candidate for
bioremediation of endosulfan.
*Corresponding Author: Ajit Kumar  akumar.cbt.mdu@gmail.com

41 | Kumar et al.
 J. Bio. & Env. Sci. 2012

Introduction enzymatic degradation of xenobiotics (Hussain et

After worldwide ban/restriction on organochlorine al., 2009). Therefore, the present study was
pesticides like DDT and BHC, the production and designed to isolate the endosulfan degrading MOs
use of endosulfan increased dramatically during from natural resources by enrichment culture
1980s. Presently it is one of the most extensively method. The isolated strains were further studied
used organochlorine pesticides throughout the for their comparative pesticide degradation ability
world and now endosulfan has become a toxic to select the best biodegrader.
environmental menace that needs a thoughtful and
effective address. Endosulfan is used extensively Materials and methods
worldwide as a contact and stomach insecticide for Field site
Colorado potato beetle, flea beetle, cabbageworm, Twelve soil samples were collected, for the study,
peach tree borer, tarnished plant bug and as an from the agricultural-crop fields of Ratangargh
acaricide on field crop like cotton, paddy, sorghum, (Churu), Lakshmangarh (Sikar) and Nawalgarh
oilseeds and coffee (Lee et al, 1995; Kullman and (Jhunjhunu) of Shekhawati region of Rajasthan,
Matsumura, 1996; USEPA, 2002). In addition to India, which have the history of endosulfan
agricultural use, endosulfan is also used in vector- application (Table 1). The collected soil samples
control (tsetse fly), as a wood preservative and for were stored at 40C until they were analyzed.
the control of home garden pests (CNRC, 1975). It is
a highly toxic substance and World Health Chemicals
Organization (WHO) classifies it as Category II Technical grade endosulfan (99% pure), an
(moderately hazardous) while United States organochlorine insecticide with the chemical name
Environment Protection Agency (USEPA) classifies 6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-
it as a Category 1b (highly hazardous) pesticide. It 6,9-methano-2,4,3 benzo-dioxathiepin-3-oxide;
has been reported to be highly toxic to aquatic fauna gifted by Excel India Pvt. Ltd., Ahmedabad, India,
like fish and invertebrates (Sunderam et al., 1992). was used for the present study. The endosulfan
Also, there are reported implications in mammalian isomers (α- and β-isomers) and endosulfan sulfate
gonadal toxicity (Sinha et al., 1997), genotoxicity standards for Gas-Chromatography-Electron
(Chaudhari et al., 1999), teratogenic effects (Yadav, Capture Detection (GC-ECD) were purchased from
2003) and mutagenic effects (U.S. Department of Hewlett Packard Company, Wilmington, Delaware,
Health & Human Services, 1990). These acute and USA. Chloroform for UV-spectroscopy, of
chronic toxicity and environmental concerns have spectroscopic grade and n-Hexane for GC-ECD, of
attracted scientists to search for an effective and chromatographic grade, were used. All other
economically viable option for endosulfan chemicals used for the study were of analytical
degradation. grade.

Bioremediation has evolved as a very economical Sample collection

and viable process for detoxification of xenobiotics Twelve soil samples, used for enrichment study
in general and pesticides in particular, as an were collected from the crop fields of Ratangarh
alternative to existing methods like incineration and (Churu), Lakshmangarh (Sikar) and Nawalgarh
landfill. Technology development of bioremediation (Jhunjhunu), the three constitutive districts of
process starts with search for potential Shekhawati region of Rajasthan state of India. All
microorganisms (MOs) as a source of xenobiotic- these fields were having the history of endosulfan
degrading enzymes and identification and application. The collected samples wee stored at 40C
establishment of in-situ and ex-situ methods of until further study.

42 | Kumar et al.
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Table 1. Source for soil samples collected for enrichment studies.

Soil sample Sample description
code
S1 Top soil from crop field of Bidasar village, Nawalgarh, Jhunjhunu, Rajasthan.
S2 Top soil from vegetable farm of Nawalgarh, Jhunjhunu, Rajasthan.
S3 Top soil from crop field of Nawalgarh, Jhunjhunu, Rajasthan.
S4 Top soil from agricultural field of Dudwa village, Lakshmangarh, Sikar, Rajasthan.
S5 Top soil from crop field of MIER campus, Lakshmangarh, Sikar, Rajasthan.
S6 Top soil from crop field of Jajod village, Lakshmangarh, Sikar, Rajasthan.
S7 Top soil from agricultural field of Ratangarh, Churu, Rajasthan.
S8 Top soil from crop field of a village, Ratangarh, Churu, Rajasthan.
S9 Top soil from crop field of a village, Ratangarh, Churu, Rajasthan.
S10 6-inch deep soil from vegetable farm of Nawalgarh, Jhunjhunu, Rajasthan.
S11 6-inch deep soil from crop field of MIER campus, Lakshmangarh, Sikar, Rajasthan.
S12 6-inch deep soil from agricultural field of Ratangarh, Churu, Rajasthan.

Experimental design sulfur. These enrichment media were designed for

Endosulfan is an organochlorine pesticide with the study according to earlier reports with certain
sulfate moiety. Therefore, the experiments were modifications (Siddique et al., 2003).
designed to search for MOs that can use it as either
carbon source or as sulfur source. This was achieved Each soil sample (20 g) was first enriched for
by enriching the soil samples collected from the endosulfan degrading MOs by addition of 2mg of
agricultural fields for endosulfan degrading MOs. technical grade endosulfan in 100μL of acetone to
moisten the soil, followed by incubation in dark at
Table 2. Composition of Carbon Deficient Medium room temperature for 1 month. Microbial inocula
(CDM)- pH-7.2. for further enrichment studies were prepared by
S. No. Chemical Amount shaking each soil sample (20 g) overnight in 100 ml
(g/lt) of respective enrichment media (i.e. CDM and
1. Sucrose 1.00
2. NaNO3 3.00 NSM) at 300C and 160 rpm rotatory shaking. The
3. K2HPO4 1.00 solid particles were allowed to settle for one hour
4. MgSO4 0.50
5. KCl 0.50 and aliquots of the supernatant were used to
6. FeSO4 0.01 inoculate the respective medium.

Enrichment of microbes
Table 3. Composition of Non-Sulfur Medium
The whole enrichment process was carried out, in
(NSM)- pH-7.2.
triplicates, with two parallel strategies. On one
S. No. Chemical Amount (g/lt)
hand, the soil samples were enriched for endosulfan
1. K2HPO4 0.225
degrading microbial population in a medium 2. KH2PO4 0.225
deficient in carbon source, Carbon Deficient 3. NH4Cl 0.225
4. MgCl2.6H2O 0.845
Medium (CDM) (Table 2), which was supplemented 5. CaCO3 0.005
with endosulfan as carbon source. On other hand, 6. FeCl2.4H2O 0.005
7. D-Glucose 1.000
enrichment was done in a culture medium without 8. Trace Element 1mg/lt
sulfur, Non Sulfur Medium (NSM) (Table 3), which Solution
was supplemented with endosulfan as sole source of

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For enrichment study, Erlenmeyer flasks (50 ml) Isolation of Endosulfan-Degrading Monocultures
and enrichment media (CDM & NSM) were Cultures after Round 2 enrichment were further
autoclaved separately for 15 minutes at 1210C and 15 sub-cultured 3 times at a time span of 10 days each
lbs pressure. Each sterilized flask was spiked with to encourage the adaptability and degradability of
50 μL of acetone containing 0.5 mg endosulfan the endosulfan degrading cultures. Pure cultures of
under laminar flow, allowing acetone to evaporate. single strains were obtained by centrifuging 1-ml
9ml of enrichment media containing 0.05% Tween aliquots of sub-cultured Round 2 enrichment
80, was added to each respective flask followed by cultures at 8000 rpm for 10 minutes with
inoculation with 1 ml of supernatant solution from microcentrifuge. The supernatant was removed and
the source (inocula) flasks. Uninoculated spiked cell residues were resuspended in 50μL of sterile
flasks were also set up as control to check for any NSM and CDM culture media by vortexing.
chemical, thermal or photo–degradation. The
aerobic culture was incubated at 300C and 160 rpm Aliquots of this suspension were placed on
orbital shaking for two weeks (Round 1 Enrichment respective CDM-endosulfan or NSM-endosulfan
culture). Thereafter, 1ml of the culture was agar media by streaking. The solid medium was
transferred to 9ml of fresh CDM and NSM prepared by adding 2% agar to the enrichment basal
containing 50mg/L (50 ppm) endosulfan and medium followed by autoclaving. Thereafter, 50-
further incubated under same incubation conditions ppm endosulfan dissolved in acetone was
for two weeks (Round 2 Enrichment culture). The aseptically added after cooling the molten agar to
microbial growth was studied by measuring the about 500C. Agar plates were incubated under
optical density (OD) at 600 nm using aerobic conditions at 300C for 15 days and discrete
spectrophotometer. colonies were isolated. Isolates were further
purified by streaking on fresh plates of respective
A sulfur-free medium was also designed because agarified-enrichment culture media.
contaminating sulfur in the enrichment medium
could promote spurious culture growth. A second Screening of isolates for endosulfan degradation
soil culture was initiated for the purpose of The five monocultures (renamed as N1, N2, N3, N4
preparing a medium free of contaminating sulfur. and N5) obtained after enrichment were screened
Sulfur-free medium was prepared by growing soil for their relative endosulfan degrading ability. All
cultures overnight in enrichment medium without these isolates showed prolific growth on NSM as
endosulfan and then removing the cells by compared with CDM. Hence, NSM was selected as
centrifugation (6000 rpm for 10 minutes).The the culture medium for further studies and was
supernatant was then filtered through a 0.22 μm- supplemented with endosulfan as sole source of
pore-size filters using vaccum-filtration assembly. sulfur. This screening was done on the basis of i)
After inoculation of this medium with respective Endosulfan content remaining in the culture (O.D.
endosulfan-degrading cultures and E. coli DH5α (as 248nm), ii) The decrease in pH of the culture medium
negative control), no growth was observed until the during incubation and iii) Increase in microbial
addition of 50μM sodium sulfite as a source of optical density (O.D. 600nm).
sulfur. The microbial growth was studied by
measuring the O.D. at 600 nm. The sterility of Wavelength of maximum absorbance (λmax) for
sulfur-free medium was confirmed by the absence technical grade endosulfan was calculated, using
of growth when the aliquots were incubated on chloroform (spectroscopic grade) as solvent, with
nutrient agar medium plates. the help of double beam UV-Visible
spectrophotometer (Systronic, Type 117). A graph

44 | Kumar et al.
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was plotted for O.D. against wavelength (236nm to selected for optimization included media-pH,
260nm) to obtain the λmax at 248nm and a standard incubation-temperature and agitation, incubation
curve was prepared by plotting a graph of days and nutritional supplementation. The
O.D.(248nm) against respective concentration range of degradation of endosulfan was estimated by UV-
endosulfan (6.25 ppm – 200 ppm). spectroscopic analysis (O.D.248nm) of residual
endosulfan, extracted by the method described
For the screening study the same NSM medium was earlier. The microbial-growth was estimated by
used as for the enrichment studies, supplemented spectroscopic analysis (O.D.600nm) of the culture
aseptically with 50-ppm of technical-grade medium. Finally, the degradation profile of
endosulfan dissolved in acetone. The monocultures endosulfan was monitored using Gas-
were inoculated onto the sterilized liquid culture Chromatography-Electron Capture Detection (GC-
media (NSM) and were incubated for 15 days at ECD) method under the optimized culture
300C and 160 rpm in orbital shaker incubator. Un- conditions.
inoculated spiked flasks were also set up as control
to check for any chemical degradation. All the The medium used for optimization of degradation
studies were done in triplicate using control as and growth conditions was NSM. Optimal pH for
reference or blank. maximum endosulfan degradation and microbial
growth was calculated by screening the medium pH
For endosulfan extraction and estimation, 5 ml of in a range of 5.5-7.5. Similarly, optimal temperature
culture-broth was acidified to pH 2.0 with 6.0 M was estimated by screening temperature range of
Hydrochloric Acid (HCl) and extracted three times 250C - 370C. Aeration condition was optimized by
with an equal volume of ethyl acetate as per the screening the agitation speed (rpm of orbital shaker
method given by Awasthi et al. (2003). The organic incubator) in a range of 100 rpm – 200 rpm. Effect
phase containing the pesticide was separated using of supplementation of extra source of sulfur was
separatory funnel and was passed through a 6-cm also checked by providing 50 μM NaSO3 to the
MgSO4 column in a Pasteur pipette to remove any culture medium. The incubation time and effect of
residual water (Sutherland et al., 2000). The sub-culturing on reduction of incubation time was
columns were pre-washed with ethyl acetate. The calculated by estimating the degradation of
extracted elutes containing pesticide were gently endosulfan after 10th, 15th and 17th sub-culturing of
evaporated at 500C in oven and were dissolved in isolate N2 under optimized culture conditions.
chloroform (spectroscopic grade) and stored in The degradation profile of isolate N2 was checked
glass vial at 40C for UV-spectroscopic analysis. A under culture conditions optimized as above. The
fortification test was also conducted to check the endosulfan degradation was monitored by UV-
endosulfan recovery from culture with known spectroscopic analysis (O.D.248nm) of residual
concentration of endosulfan and the extraction endosulfan and by GC-ECD method after 3rd and 7th
method was found to be 98.0 + 1.4% accurate, when day of incubation. For GC-ECD, the endosulfan
done in triplicate. residues in the culture medium were extracted by
the method described earlier. After evaporation of
Degradation and culture condition optimization of organic phase from elutes, the resultant dried
best degrading isolate extracts were dissolved in 5ml of n-hexane
On the basis of comparative screening for (GC/HPLC Grade) and were kept in glass vials at
endosulfan degradation, isolate N2 was selected to 40C in refrigerator until analysis.
optimize the growth profile for maximum
endosulfan degradation. The culture condition

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GC-ECD methodology external standard value is reported for microbial

The sample extracts of the N2-culture medium were growth in the given enrichment media. The
analyzed the residues of endosulfan by GC-ECD. individual OD600nm values were compared with the
The analysis was carried out on a Shimadzu Model calculated standard (average) value and cultures
2010 Gas Chromatograph (GC) equipped with 63Ni having growth values above the standard value were
Electron Capture Detector (ECD) and a capillary selected for further investigation.
column HP ultra 2 (US 4293415) 0.52 x 25 x 0.32.
The instrument was supported by Lab Solutions The same statistical tool was applied when the
software for the analysis of endosulfan (α- and β- selected five monocultures (N1, N2, N3, N4 and N5)
isomers) and endosulfan-sulfate. The stock were screened for their microbial growth (OD600nm),
standards (200-ppm) of endosulfan isomers and endosulfan degradation and pH reduction of the
endosulfan sulfate were obtained from Hewlett culture medium after 15 days of incubation. The
Packard Company, USA. Stock standards of 100- monocultures having respective values greater than
ppm were prepared by diluting standard mixture in the calculated standard (average) values were
1:1 solvent mixture of HPLC grade iso-octane and selected for further study.
toluene. These stocks were stored under freezing
conditions. Working standard of the mixture was Results
prepared from 100-ppm stock solution. 0.5 – 1.0 Enrichment
ppm of this mixture of endosulfan isomers and Two rounds of enrichment culture for 15 days each
endosulfan sulfate was used for calibrating the Gas- were carried out with 12 soil samples and their
chromatograph for analyzing residues of endosulfan optical densities (O.D.600nm) were noted. In NSM it
in the sample analyzed. ranged for 0.059 – 0.063 (Fig. 1). 5 (S2, S4, S5, S6
and S9) out of 12 soil samples showed substantial
Statistical analysis microbial growth as evident from their O.D.(600nm).
The experiments in the present investigation were Hence they were selected for further study. All other
carried out in triplicates and the mean values were samples had negligible growth in either enrichment
taken into consideration and the standard errors of medium.
the mean were calculated for each observation.
After the 2nd round of enrichment, Student’s‘t’-test
was used to analyze the significance of difference of
the means of microbial growth (O.D. 600nm) in the
two enrichment media namely, NSM and CDM. A
null hypothesis was generated which stated that
there was no significant difference between the
mean values of O.D.600nm in the two media (NSM Fig. 1. O.D. (600nm) of round 2 enrichment
and CDM). The paired ‘t’-test was conducted with microbial culture.
degree of freedom (df) value of 22 (df = N1+N2-2).
The calculated t-value was compared with the value The microbial growth (O.D.600nm) in NSM and CDM
of‘t’-table at p=0.05 (5% level) to accept or reject were found to have significant difference after the
the hypothesis. statistical analysis by Student’s ‘t’-test. The
difference in the mean values of microbial growth
For selecting the best growing microbial culture (OD600nm) in the two enrichment media was found
after enrichment, the average microbial growth to be statistically significant. On the perusal of the
(OD600nm) was taken as standard value as no microbial growth (Figure 1) in both the enrichment

46 | Kumar et al.
 J. Bio. & Env. Sci. 2012

media, NSM was found to promote higher growth of ppm) as sole source of sulfur; b). Amount of
enriched microbes than CDM. Therefore, NSM was endosulfan degraded; c). Decrease in medium pH
selected as culture medium for further study. after 15 days of incubation.

Endosulfan degrading monocultures

The selected microbial cultures i.e., S2, S4, S5, S6
and S9, were sub-cultured thrice for 10 days each
for 1st two sub-culturing and 15 days for 3rd sub-
culturing. There occurred a substantial increase in
the microbial densities of the respective microbial
cultures, due to better adaptation in the culture
medium (Fig. 2). These microbial cultures produced Fig. 4. Endosulfan recovered from selected
pure discrete colonies when streaked over solid monocultures.
NSM-medium and significant colony growth was
observed. The respective monocultures were used Isolate N2 was found to have the maximum
for the rest of the investigations and were renamed microbial density (OD600nm) of 0.382 after 15 days
as N1, N2, N3, N4 and N5 in further study. when compared to control taken as blank (Fig. 3).
Amount of endosulfan recovered from culture broth
was minimum for isolate N2 and was found to be
3.9-ppm with about 92.2% degradation as observed
from spectroscopic estimation at O.D.248nm (Fig. 4).
The control had the abiotic-endosulfan degradation
of 5.7%. pH of the culture medium was found to
reduce from 7.2 (initial) to 3.4 for the isolate N2
culture with about 52.78% pH reduction as
Fig. 2. O.D. (600nm) of selected monocultures compared to 6.9 (4.17%) of that of the control (Fig.
after 4 days. 5).

Fig. 5. pH reduction of culture medium by selected

Fig. 3. O.D. (600nm) of selected monocultures. monocultures.

Screening of selected isolates for endosulfan Looking into these comparative profiles, isolate N2
degradation was found to be the most potential candidate for the
Selected monocultures (N1, N2, N3, N4 and N5) endosulfan bioremediation and hence was selected
were screened for their relative ability of endosulfan for further investigations regarding degradation and
degradation. The screening was based on three growth optimization.
parameters a) The relative microbial growth of
selected monocultures in NSM with endosulfan (50-

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Degradation and growth profile optimization of of 140 – 150 rpm and was maximum of 11.68% at
isolate N2 200 rpm. To verify the biological degradation of
The different culture and incubation parameters endosulfan by the isolate N2, additional source of
were optimized for maximum degradation of sulfur (Na2SO4) was supplied in the culture medium
endosulfan and efficient microbial growth. A pH of (NSM) along with endosulfan. There occurred
6.5 was found to be optimal as it elicited a almost no biological degradation till 5th day of
maximum microbial density of 0.398 and 92.40% incubation while after 10th and 15th day; endosulfan
endosulfan degradation after 15 days of incubation. degradation was 11.69% and 22.08% as compared
Abiotic degradation of endosulfan was 6.5% at pH to 6.5% degradation in control. The culture medium
5.5, 6.0 and 6.5 while it was 19.48% and 37.66% at with endosulfan as sole source of sulfur showed
pH 7.0 and 7.5. Thus, pH 6.5 was found to be degradation of 87.02%, 92.22% and 94.8% after 5th,
optimal for maximum microbial growth and 10th and 15th day of incubation as compared to 6.5%
biological degradation of endosulfan. Out of three of abiotic degradation in control (Table 4).
incubation temperatures (250C, 300C and 370C)
selected for maximum microbial growth and While observing the effect of incubation days and
biological degradation of endosulfan, 300C was sub-culturing of isolate N2 on endosulfan
found to be the best promoting the maximum degradation, it was found that there was 92.2%
microbial growth (OD 600nm = 0.397) and 92.22% degradation of endosulfan after 15 days of
endosulfan degradation. incubation when inoculated with 10th sub-cultured
inocula. The degradation increased to 97.42% in
The agitation of 130 rpm was found to show just 10 days for 15th sub-cultured inoculum while it
maximum microbial density (OD 600nm = 0.414) and remained 97.42% in 7 days for the 17th sub-cultured
also the maximum endosulfan degradation of inoculum. The degradation profile remained at the
94.8%. The stationary culture showed the lowest maximum of 97.42% in 7 days, even after 20th sub-
microbial growth of OD 600nm of 0.093 and 53.24% culturing.
endosulfan degradation. The abiotic degradation of
endosulfan was found to be 3.9% for stationary
phase while it was 6.5% for the agitation speeds of
100 – 130 rpm. It increased to 9.1% for the speeds

Table 4. Microbial Growth & Endosulfan Degradation on Adding Additional Sulfur Source to Isolate N2
Sample NSM with Endosulfan as Sole Source of Sulfur NSM with Endosulfan (50ppm) Supplemented
(Days) with Na2SO4 as Sources of Sulfur
O.D. (600nm) O.D. Endosulfan Endosulfan O.D. O.D. Endosulfan Endosulfan
(248nm) (ppm) Degradation (600nm) (248nm) (ppm) Degradation
Control (15) 0.000 0.036+0.005 46.75 6.5 % 0.000 0.036+0.004 46.75 6.5 %
N2 (5) 0.102+0.005 0.005+0.001 6.49 87.02 % 0.849+0.011 0.036+0.005 46.75 6.5 %
N2 (10) 0.247+0.008 0.003+0.002 3.89 92.22 % 1.513+0.013 0.034+0.004 44.16 11.69 %
N2 (15) 0.416+0.004 0.002+0.001 2.60 94.80 % 1.832+0.015 0.030+0.003 38.96 22.08 %
Values of O.D. (600nm) and O.D. (248nm) are the mean + Standard Error of the triplicate samples
Culture condition
Temperature : 30+ 10C
pH : 6.5
Agitation : 130 + 10 rpm
Incubation time : 15 Days

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Table 5. Gas Chromatography – ECD Data of Endosulfan isomers & Endosulfan sulfate after degradation by
Isolate N2.
Sample Peak Retention Area Height Area (%) Compound Concentration Endosulfan
# time (min) (ppm) degradation
(%)
Control 13.818 154053207.4 10321065.0 53.46 α–Endosulfan 24.82 18.74
(Day 3) 24 15.751 98608717.8 10132336.4 33.38 β-Endosulfan 15.81
32 17.207 6966260.7 1197855.2 2.29 Endosulfan 6.42
37 sulfate

Control 13.800 141291680.7 10335461.7 57.81 α–Endosulfan 23.93 22.58

(Day 7) 25 15.729 76248397.4 9776147.1 31.14 β-Endosulfan 14.78
32 17.205 6230901.6 1049637.3 2.56 Endosulfan 6.98
37 sulfate
N2 13.757 98072452.1 10341409.6 69.71 α–Endosulfan 8.65 71.0
(Day 3) 26 15.708 2979152.8 4726282.5 21.16 β-Endosulfan 5.85
32 17.207 2464016.1 413903.2 1.74 Endosulfan 2.98
38 sulfate
N2 13.709 8457622.4 1443946.9 60.10 α–Endosulfan 1.79 94.0
(Day 7) 27 15.697 3256538.5 562847.6 23.30 β-Endosulfan 1.21
32 17.207 184822.8 33147.8 1.25 Endosulfan 0.32
33 sulfate
Values of O.D. (600nm) and O.D. (248nm) are the mean + Standard Error of the triplicate samples
Culture Condition:
Media : NSM with 50-ppm technical Endosulfan as sole source of Sulfur.
pH : 6.5
Temperature : 300C
Agitation : 130 rpm

GC-ECD estimation of endosulfan degradation by Discussion

isolate N2 The present investigation was carried out to enrich
After optimization of microbial growth the microbes from natural resources for Endosulfan
endosulfan degradation profile of the isolate N2 was degradation and to search for the potential
verified quantitatively by GC-ECD method. After candidates for development of in-situ
GC-separation and ECD analysis, it was found that bioremediation of Endosulfan. The observations are
8.65 ppm of α-endosulfan, 5.85 ppm of β- in accordance with several previously reported
endosulfan and 2.98 ppm of endosulfan sulfate findings (Hussain et al., 2007; Siddique et al.,
remained in the N2-culture system, accounting for 2003; Sutherland et al., 2000). All these authors
about 71.0% degradation after 3 days of incubation, have reported about enrichment of microbial
as compared to 24.82 ppm of α-endosulfan, 15.81 populations capable of utilizing endosulfan as sulfur
ppm of β-endosulfan and 6.42 ppm of endosulfan source. This finding also support the fact that
sulfate detected in the control sample accounting endosulfan is a poor biological energy source as it
for 18.74% abiological degradation. After 7 days of contains six potential reducing electrons and has a
incubation, the degradation of endosulfan in N2- relatively reactive cyclic sulfite diester group
culture system was found to be 94.0% with 1.79 (Sutherland et al., 2000; Guerin, 1999; Van
ppm of α-endosulfan, 1.21 ppm of β-endosulfan and Woerden, 1963). On the contrary, there are findings
0.32 ppm of endosulfan sulfate detected by GC-ECD stating that the enriched microbes have utilized
as compared to 23.93 ppm of α-endosulfan, 14.78 endosulfan as carbon source (Siddique et al., 2003;
ppm of β-endosulfan and 6.98 ppm of endosulfan Shetty et al, 2000; Awasthi et al., 2000; 1997).
sulfate detected in the control sample that accounts
for 22.58% abiological degradation (Table 5). The findings of the present investigation also
suggest that the microbial growth (OD600nm) in NSM

49 | Kumar et al.
 J. Bio. & Env. Sci. 2012

with endosulfan as sole source of sulfur, increased (Sutherland et al., 2000). Contrary to this, Marten
by 57% to 186%, when the selected microbial (1976) and Miles and Moy (1979) reported about
cultures were sub-cultured consecutively three increase in pH of the culture medium with
times. This may be attributed to the better increased endosulfan degradation. But this
adjustment of microbial metabolism and the gene- degradation could not be differentiated as biological
expression system in a nutritionally stressed or chemical degradation because the latter is
environment. Similar results were observed by favored at alkaline pH.
Sutherland et al (2000), where it was found that
after approximately six rounds of successive sub- Isolate N2 was found to be the most efficient
culturing in enrichment media, there was a biodegrader of endosulfan and hence was selected
substantial disappearance of endosulfan with a for further study of optimizing the culture condition
simultaneous increase in microbial biomass. parameters for maximum endosulfan degradation.
While optimizing the culture conditions, isolate N2
A quick and relatively easier method of endosulfan showed maximum growth and endosulfan
content estimation was formulated using UV-Visible degradation at a pH of 6.5. Almost similar
spectroscopic method. The λmax for technical grade degradation was observed at pH 7.0 but the abiotic
endosulfan in chloroform was calculated as 248nm. degradation of endosulfan was found to be more
The finding is similar to the observations of Guha et (6.5%) as compared to culture at pH 6.5 (5.26%). A
al (1999). While screening the selected high abiotic degradation of 19.48% was observed at
monocultures (N1, N2, N3, N4 & N5), strain N2 higher pH of 7.5. This observation finds its
showed the maximum endosulfan degradation similarity with the earlier findings (Sutherland et
(92.2%) and microbial growth (0.382). This is in al., 2000; Guerin, 1999). Sutherland et al (2002), in
accordance with the findings of Awasthi et al. their findings, reported that endosulfan is
(1997), Siddique et al. (2003), Sutherland et al. susceptible to alkaline hydrolysis with
(2000) and Hussain et al. (2007) where approximately 10-fold increase in hydrolysis
biodegradation of endosulfan was observed to be occurring with each increase in pH units. They
accompanied with substantial increase in microbial suggested buffering the enrichment medium at pH
biomass. 6.6 (below 7.0) to minimize non-biological
hydrolysis of endosulfan. Guerin (1999) also had the
While screening the monocultures for pH reduction same opinion regarding the endosulfan-
of the culture medium, the isolate N2 showed biodegradation.
52.78% reduction. Earlier, Siddique et al. (2003)
and Hussain et al. (2007; 2006) also reported a A temperature of 300C was found to promote
reduction in the pH of culture medium. This maximum microbial growth and endosulfan
reduction in medium-pH has been attributed either degradation (92.22%) followed by 250C with
to the dehalogenation of endosulfan resulting in the 87.02% degradation. At incubation temperature of
formation of hydrochloric acid (HCl) or organic 370C, higher abiotic degradation of the pesticide
acids produced by microorganism during their was noted in the control sample. This may be
metabolic activities (Siddique et al., 2003). It has attributed to spontaneous thermolysis of endosulfan
also been found that the proton-transfer chemical isomers to endosulfan sulfate. Thus, the isolate N2
ionization (PCI) mass spectrum of the metabolites may be suggested to be a potential candidate for in-
of culture medium displayed fragment ions situ bioremediation of endosulfan with an active
indicating consecutive loss of the two molecules of degradation profile in the normal temperature
HCl from the molecular parent ions [M+H]+ range of 250C – 300C.

50 | Kumar et al.
 J. Bio. & Env. Sci. 2012

The rotatory speed of 130 rpm was observed to is in good accordance with the report of Sutherland
result in maximum microbial growth and et al (2000) who found that continuous sub-
endosulfan degradation (94.8%). Both the factors culturing in sulfur-free medium led to an increase
decreased on the either side of the rotatory speed. in rate of endosulfan disappearance, with no
The degradation reduced to 63.64% at a speed of detectable levels of pesticide remaining after 4 days
200 rpm while it was 53.24% under stationary by the 20th subculture as compared to 8 days after
condition. These observations suggest that isolate 10th sub-culturing. In the present investigation, a
N2 utilizes endosulfan as nutrient source under maximum of 97.42% endosulfan degradation was
suitably agitated condition (130rpm) while higher achieved in just 7 days after 17th repeated sub-
agitation and aeration slows down microbial growth culturing. No further increment in either rate or
and endosulfan degradation. extent of pesticide degradation was observed after
17th sub-culturing.
To confirm the utilization of endosulfan as sulfur
source by the isolate N2, the culture medium (NSM) Thus, the culture-conditions optimized for the
with 50-ppm endosulfan was supplemented Na2SO4 isolate N2 for maximum microbial growth and
as readily assimilable sulfur source. No biodegradation of endosulfan includes non-sulfur
biodegradation of endosulfan was observed till 5th medium (NSM) with endosulfan as the sole source
day of incubation, while significant microbial of sulfur, having pH maintained at 6.5. The optimal
growth was observed for the same. The temperature, agitation and time of incubation are
biodegradation was observed after 10th day of 300C, 130 rpm and 7 days, respectively.
incubation with about 22.08% endosulfan being
degraded on 15th day as compared to 6.5% abiotic Finally the endosulfan degradation by isolate N2
degradation. But when the isolate N2 was cultured under optimal culture condition was quantified by
in NSM with endosulfan as the sole source of sulfur, Gas Chromatography with Electron Capture
94.8% degradation of the pesticide was observed Detection (GC-ECD) method. The residual levels of
after 15 days of incubation as compared to 6.5% of α- and β- endosulfan were estimated to be 1.79-ppm
abiotic degradation. These results confirm that the and 1.21-ppm respectively, after 7 days of
isolate N2 is capable of utilizing endosulfan as the incubation with the initial combined endosulfan
sulfur source and hence is a potential candidate for isomers’ concentration of 50-ppm. This residual
endosulfan bioremediation. The results are in amount corresponds to about 94.0% degradation of
accordance with the report of Weir et al (2006). the pesticide. The results obtained were in
They isolated an Arthrobacter sp. from soil concomitance with the previous estimation by UV-
microbial population that was enriched with spectroscopic method. This gives further
continuous nutritional pressure to use endosulfan confirmation about endosulfan degradation ability
sulfate as the sole source of sulfur. They found that of the isolate N2. A proportionate decrease of
the organochlorine-degrading activity was absent in endosulfan sulfate with both the isomers of
the presence of sodium sulfite as an alternative endosulfan was observed during GC-ECD. This
sulfur source. This suggested that the activity was observation suggests that the isolate N2 is also
part of the sulfur starvation response of the strain. capable of degrading endosulfan-sulfate along with
its parent compound.
It was also observed that on repeated sub-culturing,
the rate and extent of endosulfan biodegradation Conclusion
increased upto 97.42% with about 91% of net In the present work, a soil bacterium (N2) was
biodegradation by the isolate N2. This observation enriched and isolated by the application of strong

51 | Kumar et al.
 J. Bio. & Env. Sci. 2012

selection pressure on the bacterium to release the on Scientific Criteria for Environmental Quality,
sulfur moiety from endosulfan and use it as sole Canada National Research (CNRC), Report No. 11,
source of sulfur. The isolated bacterium showed an Subcommittee of pesticides on related compounds,
extensively high biotic degradation of the pesticide Subcommitte Report No. 3, Publication No. NRCC
of about 94%. Therefore, the isolate N2 holds a 14098 of the Environmental Secretariat.
strong potential to be studied further as a candidate
for developing in-situ bioremediation process for Guerin TF. 1999. The anaerobic degradation of
endosulfan degradation. The strain N2 is being endosulfan by indigenous microorganisms from low
presently studied in our laboratory for its oxygen soils and sediments. Environ. Pollut. 106,
identification as well as biochemical and molecular 13 – 21.
characterization to develop a technically and
economically viable bioremediation technology for Guha A, Kumari B, Bora TC, Roy MK. 1999.
combating endosulfan menace. Degradation of endosulfan by Micrococcus sp. and
Acknowledgements partial characterization of metabolites. Asian J.
Microbiol. Biotech. & Env. Sci. 1, 29 – 32.
The authors wish to thank Faculty of Arts, Science,
Commerce & Law, Mody Institute of Technology & Hussain S, Arshad M, Saleem M, Zahir ZA.
Science, Lakshmangarh, Sikar, Rajasthan, India for 2006. Screening of soil fungi for in-vitro
providing laboratory facilities for carrying out the degradation of endosulfan. World J Microbiol.
present study. Biotechnol. ISSN: 1573 – 0972 (Online).

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