Journal of Environmental Science and Health Part B, 41:81–96, 2006
C Taylor & Francis Inc.
Copyright ISSN: 0360-1234 (Print); 1532-4109 (Online)
DOI: 10.1080/03601230500234935
Enrichment and Isolation
of a Mixed Bacterial Culture
for Complete Mineralization
of Endosulfan
Mathava Kumar and Ligy Philip
Environmental and Water Resources Engineering Division, Department of Civil
Engineering, Indian Institute of Technology Madras, Chennai, India
In the present study, we isolated three novel bacterial species, namely, Staphylococcus
sp., Bacillus circulans–I, and Bacillus circulans–II, from contaminated soil collected
from the premises of a pesticide manufacturing industry. Batch experiments were conducted using both mixed and pure cultures to assess their potential for the degradation
of aqueous endosulfan in aerobic and facultative anaerobic condition. The influence of
supplementary carbon (dextrose) source on endosulfan degradation was also examined.
After four weeks of incubation, mixed bacterial culture was able to degrade 71.82 ± 0.2%
and 76.04 ± 0.2% of endosulfan in aerobic and facultative anaerobic conditions, respectively, with an initial endosulfan concentration of 50 mg l−1 . Addition of dextrose to the
system amplified the endosulfan degradation efficiency by 13.36 ± 0.6% in aerobic system and 12.33 ± 0.6% in facultative anaerobic system. Pure culture studies were carried
out to quantify the degradation potential of these individual species. Among the three
species, Staphylococcus sp. utilized more beta endosulfan compared to alpha endosulfan
in facultative anaerobic system, whereas Bacillus circulans–I and Bacillus circulans–II
utilized more alpha endosulfan compared to beta endosulfan in aerobic system. In any
of these degradation studies no known intermediate metabolites of endosulfan were
observed.
Key Words: Endosulfan; Mixed culture; Biodegradation; Enriched cultures; Staphylococcus sp.; Bacillus circulans; Mineralization.
INTRODUCTION
Endosulfan (6,7,8,9,9a-hexahydro-6,9-methano,3,4-benzo (e. dioxathiepin-3oxide) is a chlorinated cyclodiene insecticide, acaricide widely used throughout
Received March 21, 2005.
Address correspondence to Ligy Philip, Environmental and Water Resources Engineering Division, Department of Civil Engineering, Indian Institute of Technology Madras,
Chennai, India; E-mail: [email protected]
81
82
Kumar and Philip
Figure 1: Molecular structure of endosulfan and its two isomers.
the world to control numerous insects and mites in many food and nonfood crops.
Technical-grade endosulfan is a mixture of two stereo isomers, alpha and beta
endosulfan (Fig. 1), in a ratio of 70:30. Both the isomers are extremely toxic to
aqueous organisms. Because of its widespread usage and potential transport,
endosulfan contamination is frequently found in the environment at considerable distances from the point of its original applications.[1−8]
Detoxification of endosulfan through biological means is receiving serious attention as an alternative to existing methods, such as incineration and
landfill.[8] Numerous studies have been reported regarding the isolation of
pure and mixed cultures of bacteria and fungi [9–15] capable of degrading
endosulfan.
A bacterial coculture of two bacillus spp. was enriched from soil samples collected from a contaminated industrial area for the degradation of endosulfan.[12]
The degradation of endosulfan and the formed metabolites during degradation
by this bacterial coculture were evaluated by the reduction in toxicity against
the test organism Tubifex tubifex Muller.[16] A bacterial strain, Klebsiella pneumoniae (KE-1), capable of degrading endosulfan without the formation of endosulfan sulfate was enriched from endosulfan polluted soil samples.[17] Even
though mortality of the test organism and the reduction in toxicity (converting
the parent compound into less toxic metabolites) is a sensitive end point for
the assessment of ecotoxicological risk by the environmental pollutants, but
none of these microbes were able to mineralize endosulfan to CO2 and other
environmental friendly compounds.
Enrichment and Isolation of Bacterial Culture
Endosulfan can be degraded by attacking the sulfide group by oxidation
and/or hydrolysis to form the toxic metabolite endosulfan sulfate and less toxic
endosulfan diol, respectively.[15] But the formation of intermediate compounds
(metabolites) is mainly based on the metabolic activity of the specific culture
and the environmental conditions. Many species were isolated and checked
for the endosulfan degradation potential, but in no case endosulfan was completely mineralized. Also, not many studies were carried out on the behavior of
individual pure cultures of a mixed consortium on endosulfan and its isomers
degradation.
This paper describes the enrichment and isolation of endosulfan mineralizing microbial cultures. The endosulfan degradation capacities of these microbes
individually and as a group were evaluated under various conditions.
MATERIALS AND METHODS
Chemicals
High purity (99.4%) endosulfan, endosulfan sulfate, endosulfan ether,
and endosulfan lactone were purchased from Sigma Aldrich Ltd., USA, and
technical-grade endosulfan of 96% purity was purchased from EID Parry India
Ltd., Chennai, India. Other chemical reagents and solvents used were of HPLC
grade purchased from Ranbaxy Ltd., Chennai, India. The stock endosulfan solution of 1% was prepared in methanol and used for all the experiments. All the
glassware used was supplied by Borosil, India, and before every experiment,
all glassware was cleaned with distilled water and dried at 110◦ C for 5 h.
Sample Collection for Enrichment Studies
Soil samples used in this study were collected from the premises of an endosulfan processing industry (EID Parry India Ltd., Chennai, India). Topsoil was
collected from the first 15 cm and preserved at 4◦ C (at the site).The preserved
soil samples were brought to the laboratory on the same day, and the microbial
enrichment was started immediately.
Enrichment of Mixed Bacterial Culture
A soil suspension (10 g /50 ml) was made in a nutrient broth (NB). The
composition of NB is as follows: KH2 PO4 1.0 g, K2 HPO4 ; 1.0 g, NH4 NO3 ; 1.0 g,
NaCl; 1.0 g, MgSO4 ·7H2 O; 0.2 g, CaCl2 ; 0.02 g, Fe (SO4 )3 ; 0.02 g, trace metal
solution[12] 1 ml dissolved in 1 liter of distilled water; final pH of the minimal
medium was adjusted to 7 using HCl and/or NaOH. The soil suspension was
kept in an orbital shaker (Remi Instruments Ltd., India) set for 28 ± 2◦ C for
24 h at 150 rpm. The solid particles were allowed to settle for 1 h, and 1 ml of
83
84
Kumar and Philip
the supernatant was inoculated into a fresh 100 ml Erlenmeyer flask containing 10 ml of fresh NB spiked with 50 mg l−1 of endosulfan. The contents were
incubated at 28 ± 2◦ C and 150 rpm for one week. Thereafter, 1 ml of the suspension was transferred into a fresh Erlenmeyer flask and cultured as above
(at each culturing step, the concentration of endosulfan was increased, and at
the end, the concentration of endosulfan was around 500 mg l−1 in the NB). After six transfers, a loopful of this inoculum was streaked onto agar plates and
incubated for 24 h at 28 ± 2◦ C. Dilution and streaking of colonies with same
morphology was carried out three times. The colonies with distinct morphology
were streaked on agar slants for 24 h at 28 ± 2◦ C and refridgerated 4◦ C for
further use.
Endosulfan Degradation Studies with Mixed Bacterial Culture
To study the degradation of endosulfan, the mixed bacterial culture was
grown in NB at 28 ± 2◦ C and 150 rpm for two to three days. After this, the
cells were centrifuged at 5000 × g for 10 min and suspended in physiological
saline water. The bacterial concentration in the system was estimated in terms
of optical density using a UV spectrophotometer at a wavelength of 550 nm.
About 75 mg l−1 (0.15 OD) of bacterial concentration was added to seven identical conical flasks (triplicate) containing 100 ml of NB that was amended with a
technical grade endosulfan concentration of 50 ppm. The studies were carried
out in aerobic and facultative anaerobic conditions for 28 days at 28 ± 2◦ C with
occasional shaking. To maintain the facultative anaerobic condition, nitrogen
gas was flushed to the conical flask and sealed with an air-tight septum. At
the end of 2, 5, 7, 10, 14, 21, and 28 days, 5 ml of sample was collected using a
syringe from each conical flask and analyzed for endosulfan concentration using gas chromatography (PerkinElmer Clarus 500) with ECD (electron capture
detector).
Bacterial Cell Density Measurement
The mixed bacterial culture was grown to mid log phase in NB, and the cells
were centrifuged at 5000 × g for 10 min. The bacterial pellets were washed twice
and suspended in 100 ml of saline water (0.8% NaCl). About 0.1, 0.2, 0.3, 0.4,
and 0.5 ml of above set solution was diluted and made up to 10 ml using fresh
saline water. From each dilution, 3 ml of sample was filtered through Millipore
filter paper (0.45 µm) in triplicate, and the filter paper was kept in the oven
at 104◦ C for 3 h. The difference in weight (average weight) of the filter paper
was recorded as bacterial cell density. The optical density of the corresponding
dilutions was found by UV digital spectrometer in fixed-point measurement at
a wavelength of 550 nm. Using bacterial cell density and the optical density, a
standard graph was prepared, and thereafter the bacterial cell density in the
samples was calculated using the standard graph.
Enrichment and Isolation of Bacterial Culture
Figure 2: Chromatograph of endosulfan and its metabolites at standard operating
condition: 1. n-hexane, 2. endosulfan ether, 3. endosulfan lactone, 4. alpha endosulfan,
5. beta endosulfan, 6. endosulfan sulfate.
Endosulfan Degradation Studies with Pure Cultures
The pure cultures were grown separately in nutrient medium at 28 ± 2◦ C
and 150 rpm for two to three days. The grown cells were centrifuged, quantified
as above, and the same bacterial concentration (75 mg l−1 , OD 0.15 at 550 nm)
was added to nine identical conical flasks (triplicate) containing 100 ml of NB
amended with 5 ppm of technical-grade endosulfan. The study was conducted
in aerobic condition at 28 ± 2◦ C with occasional shaking, and the samples were
collected at 0, 2, 5, 7, 10, and 14 days. The collected samples were analyzed
for endosulfan and bacterial cell concentrations. Occasionally, the endosulfan
concentration in bacterial cells was also measured by digesting and extracting
the endosulfan using n-hexane.
Analysis of Endosulfan
Endosulfan containing sample (5 ml) was extracted with 10 ml of n-hexane
and shaken vigorously for 15 min in a standard separating funnel with Teflon
stopper. The water layer was decanted carefully, and the supernatant was extracted with 5 ml of n-hexane two more times. Finally, the extracted sample
was dehydrated by passing through anhydrous sodium sulfate and analyzed
in PerkinElmer Clarus 500 gas chromatograph with electron capture detection
(GC/ECD) equipped with autosampler, an on-column, split/splitless capillary
injection system, and with PerkinElmer (PE)-35 capillary column (30 m × 0.53
mm × 0.5 µm film thickness). The operating conditions were as follows. The
column was held initially at a temperature of 120◦ C for 1 min, then at 30◦ C
min−1 to 180◦ C, then at 20◦ C min−1 to 240◦ C, and finally held at that temperature for 3 min. The temperature of injector and detector were maintained at
260◦ C and 300◦ C, respectively. Nitrogen was used as a carrier gas at a flow
rate of 30 ml min−1 , and the injections were made in the split mode with a
split ratio of 1:10. Under these conditions the retention time for alpha endosulfan, beta endosulfan, endosulfan sulfate, endosulfan ether, and endosulfan
lactone was 6.31 min, 9.36 min, 11.95 min, 3.72 min, and 6.42 min, respectively
(Fig. 2).
85
86
Kumar and Philip
Identification of Bacterial Cultures
The bacterial strains were isolated from the mixed culture by repeated
streaking on agar plates. The isolates were separated based on their morphology. Each colony was carefully separated and streaked on fresh agar plates,
and this was repeated three times. Thereafter, the cultures were streaked on
agar slants and preserved at 4◦ C for further experiments. The agar slants in
triplicate were sent to Microbial Type Culture Collection (MTCC), Institute of
Microbial Technology (IMTECH), Chandigarh, India, for identification.
RESULTS AND DISCUSSION
Degradation Studies in Aerobic and Facultative Anaerobic
Conditions with Mixed Culture
Endosulfan degradation studies were conducted at an initial endosulfan
concentration of 50 mg l−1 amended in 200 ml of NB with 75 mg l−1 (OD 0.15
at 550 nm) of mixed bacterial culture in a batch system for a period of 28 days.
At regular intervals, samples were collected from the aerobic and facultative
anaerobic system and analyzed for endosulfan concentration in GC/ECD. The
decrease in concentration of endosulfan in the system was considered as microbial degradation. The GC analysis and optical density measurements confirmed substantial removal of endosulfan with simultaneous increase in bacterial mass. Previous researchers reported that the removal of endosulfan took
place simultaneously with the formation of intermediate metabolites like endosulfan sulfate, endosulfan lactone, endosulfan ether, endosulfan hydroxy ether,
and endosulfan monoaldehyde. In the present study, none of the reported endosulfan metabolites were accumulated in the system. The metabolites might
have formed and immediately degraded by the microbial consortium, or the
pathway of endosulfan degradation might be entirely different. In the initial
stages of the study, degradation of endosulfan in facultative anaerobic and aerobic systems were almost the same, and around 50% of the initial concentration
was degraded within the first 7 to 10 days. The enriched mixed bacterial culture
was able to degrade/mineralize alpha and beta isomers of endosulfan.
At the end of 28 days, 10.01 mg l−1 of alpha endosulfan and 4.08 mg l−1 of
beta endosulfan were remaining in the aerobic system, which corresponds to a
total endosulfan degradation efficiency of 71.82 ± 0.2% with a maximum bacterial cell density of 355 mg l−1 at OD550 . The concentrations of alpha and beta
endosulfan remaining and their percentage degradation in both aerobic and
facultative anaerobic systems are shown in Figures 3a and 3b. The endosulfan
degradation efficiency of the mixed culture was 71.4 ± 0.15% and 72.8 ± 0.05%
for alpha and beta isomers, respectively, at the end of four weeks of incubation.
The rate of alpha and beta endosulfan degradation was more in the first week,
Enrichment and Isolation of Bacterial Culture
Figure 3: Kinetics of alpha and beta endosulfan degradation in aerobic system (a) and
facultative anaerobic system (b).
87
88
Kumar and Philip
and it reduced considerably in the second and third week of incubation. After
that, the rate of degradation was insignificant, and the residual alpha and beta
endosulfan concentration remained almost the same (Figs. 3a and 3b).
However, the total endosulfan degradation at the end of 28 days was more
in facultative anaerobic system compared to aerobic system, even though the
bacterial cell density in the facultative anaerobic system was less compared to
aerobic system. It is reported that the occurrence of dehalogenation is faster
in anaerobic process. Almost 76.04 ± 0.2% of the initial endosulfan concentration disappeared from facultative anaerobic system (Fig. 4), which is 5.9 ±
0.6% more than that of aerobic system. Also in facultative anaerobic system,
half of the initial endosulfan concentration was degraded within the first 7 to
10 days. Degradation of alpha and beta endosulfan was almost the same in
aerobic system, but in the case of facultative anaerobic system the percentage of alpha endosulfan degradation (76.51 ± 0.15%) was slightly more compared to degradation of beta endosulfan (74.91 ± 0.05%). This may be due to
stereoisomerism, where the enzymes released from bacterial system may be active toward one of the stereo isomers. While studying endosulfan degradation
by various microbes, it is reported that fungal cultures removed more alpha endosulfan compared to beta endosulfan, while bacteria degraded more beta endosulfan compared to alpha endosulfan.[8] But Awasthi, Manickam, and Kumar[12]
reported that degradation of beta endosulfan was less compared to alpha endosulfan. These observations reflect that stereoisomerism and the release of
enzymes from the bacterial/fungal systems play a major role in degrading
endosulfan.
Sutherland et al.[15] reported that degradation of endosulfan can be
achieved via oxidation and hydrolysis pathways, but it leads to the formation of toxic endosulfan sulfate and less toxic endosulfan diol, respectively.
But throughout the present study, the intermediate metabolites (endosulfan
sulfate, endosulfan lactone, endosulfan diol, endosulfan ether, endosulfan hydroxy ether, and endosulfan monoaldehyde) reported by previous researchers
[11,15,16,18,19]
were not observed. In this study, the loss due to dissipation was
checked by the help of control reactors (without bacterial culture and with endosulfan). It was observed that the abiotic loss in the system was negligible
(Fig. 5).
During the study, samples were collected at regular intervals from aerobic and facultative anaerobic systems, and the bacterial culture was separated
by centrifugation. The bacterial pellets were suspended in distilled water and
mixed thoroughly in a vortex mixer, sonicated, extracted with n-hexane, and analyzed for endosulfan concentration. The endosulfan concentration in bacterial
cell extracts was negligible (0.01 mg l−1 ). This result shows that endosulfan was
used completely by the bacterial culture for metabolic activity, and the chance
of bioaccumulation of endosulfan as the reason for disappearance of endosulfan
from the NB can be ruled out.
Enrichment and Isolation of Bacterial Culture
Figure 4: Degradation of endosulfan in aerobic and facultative anaerobic systems without
supplementary carbon.
Endosulfan is highly immiscible in water. In order to get higher concentration in water, methanol was used as a solvent. The addition of methanol
to the system created more chemical oxygen demand and also acted as a carbon source for the microbes. But methanol is a less preferred substrate for the
microbial consortia compared to dextrose, which is evident from the growth
kinetics (Fig. 5).
Influence of Supplementary Carbon on Endosulfan
Biodegradation
The degradation studies were repeated with the addition of dextrose (supplementary carbon) to check the influence of auxiliary carbon on endosulfan
degradation. Awasthi, Manickam, and Kumar[12] reported that the addition of
glucose did not increase the degradation efficiency of endosulfan. Hence, dextrose was used as an auxiliary carbon source in the present study. The addition
of dextrose (1 g l−1 ) increased the concentration of bacterial cells and the degradation efficiency of endosulfan from 71.82 ± 0.2% to 81.42 ± 0.2% in aerobic
system and from 76.04 ± 0.2% to 85.42 ± 0.2% in facultative anaerobic system
(Fig. 5). Significant increase in bacterial cell density was observed in the first
89
90
Kumar and Philip
Figure 5: Degradation of endosulfan in aerobic and facultative anaerobic systems with
supplementary carbon source.
two days due to the addition of dextrose (Fig. 5), which increased the endosulfan degradation efficiency from the initial stages itself. The enhancement in
the degradation efficiency due to supplementary carbon was 13.36 ± 0.6% in
aerobic system and 12.33 ± 0.6% in facultative anaerobic system, respectively.
In aerobic system, without dextrose, alpha and beta endosulfan degradation
efficiency was 71.4 ± 0.15% and 72.8 ± 0.05%, respectively. The addition of
dextrose (aerobic co-metabolic system) to the aerobic system amplified the alpha
endosulfan degradation by 15.56 ± 0.5% (71.4 ± 0.15% to 82.51 ± 0.15%) and
beta endosulfan degradation by 8.3 ± 0.15% (72.8 ± 0.05% to 78.86 ± 0.05%).
On the other hand, in facultative anaerobic system the degradation of both the
isomers was reached around 85.5%, and the amplification in alpha and beta
endosulfan degradation efficiency was 11.58 ± 0.5% (76.51 ± 0.15% to 85.37
± 0.15%) and 14.15 ± 0.15% (74.93 ± 0.05% to 85.53 ± 0.05%), respectively
(Fig. 6). From the results it is clear that the addition of supplementary carbon
increased the degradation rate of both alpha and beta endosulfan in aerobic
and facultative anaerobic systems.
Several researchers have observed that the addition of auxiliary carbon to the system having xenobiotic compounds increased the biodegradation
Enrichment and Isolation of Bacterial Culture
Figure 6: Degradation of alpha and beta endosulfan in aerobic and facultative anaerobic
systems with supplementary carbon.
potential of bacterial and fungal cultures. But some of the researchers observed
no such increase in degradation efficiency due to the addition of secondary
carbon.[12,20] The addition of methanol to the system (as a solvent) would create
more chemical oxygen demand and also act as a carbon source for the microbes.
In such cases, the addition of supplementary carbon source to the system may
not be useful to the microbes if the microbes are able to use methanol effectively, and obviously in such cases, no increase in degradation efficiency can be
observed. The mixed bacterial consortium used in the present study was preferring dextrose over methanol as the substrate, which was evident from the
growth kinetics (results not shown). The maximum specific growth rate of the
mixed culture was 0.0395 h−1 while using dextrose, whereas the specific growth
rate was only 0.0137 h−1 when methanol was the auxiliary carbon source. The
use of multiple substrates did not change the metabolism of the cultures, and
during degradation of endosulfan no intermediate metabolites were observed.
Isolation of relevant enzymes and genes from the mixed bacterial culture and
comparison with those of other endosulfan degrading bacteria may give insight
into the pathway/mechanism of endosulfan degradation. The investigations are
in progress for determining the pathway of endosulfan degradation by the isolated pure strains as well as the mixed bacterial consortium.
91
92
Kumar and Philip
Bacterial Identification
The bacterial cultures isolated from the mixed microbial consortium were
identified as Staphylococcus sp., Bacillus circulans–I, and Bacillus circulans–
II through Microbial Type Culture Collection (MTCC), Chandigarh, India,
as MTCC 6801, MTCC 6802, and MTCC 6803. Also, these cultures are preserved in the gene bank of MTCC. All cultures were Gram-positive endosporeforming rods (except Staphylococcus sp. (non-endospore-forming cocci) and
round shaped. All the cultures can hydrolyze starch and grow in the presence
of 2.5% NaCl. Negative tests include indole formation, citrate utilization, and
growth in 10% NaCl. The favorable growth temperature lies between 25◦ C and
42◦ C and can grow favorably in a range of pH between 5 and 11. Some of the
other characteristics of the cultures are given in Table 1.
Degradation Studies with Pure Cultures Under Aerobic Condition
From the mixed culture studies it was observed that the utilization of alpha
endosulfan was more in aerobic condition, and utilization of beta endosulfan
was more in facultative anaerobic condition. The main objective of pure culture study was to investigate the endosulfan degradation ability of individual
cultures and their rate of alpha and beta endosulfan degradation.
Table 1: Characteristics of bacterial culture.
Character
Parameter
Configuration
Gram’s reaction
Shape
Size
Arrangements
Endospore
Shape
Motility
Growth under anaerobic condition
Gas production from glucose
Casein hydrolysis
Starch hydrolysis
Urea hydrolysis
Nitrate reduction
Nitrite reduction
H2 S production
Cytochrome oxidase
Catalase test
Oxidation/fermentation (O/F)
Gelatin hydrolysis
Arginine dihydrolase
Staphylococcus
sp.
Bacillus
circulans–I
Bacillus
circulans–II
Round
+ ve
Cocci
Round
+ ve
Rods
Long
Single
+
Oval
+
−
−
±
+
−
+
+
−
−
+
−
+
+
Round
+ ve
Rods
Long
Single
+
Oval
+
−
−
±
+
−
+
+
−
+
+
−
+
+
Groups
−
±
+
−
±
+
−
−
−
−
−
+
F
−
+
Enrichment and Isolation of Bacterial Culture
Figure 7: Degradation of total endosulfan by pure cultures.
The degradation of endosulfan was almost the same with all three pure
cultures studied. At the end of 14 days of incubation, total endosulfan degradation efficiency of 87.8 ± 0.2%, 88.1 ± 0.2%, and 88.4 ± 0.2%, was achieved by
Staphylococcus sp., Bacillus circulans I and II with corresponding final bacterial cell density of 285 mg l−1 , 275 mg l−1 , and 280 mg l−1 , respectively (Fig. 7).
Alpha endosulfan was utilized up to 93.3 ± 0.15%, and 93.4 ± 0.15% by Bacillus
circulans I and II , respectively, at the end of 14 days (Fig. 8a), which was 3.74
± 0.5% and 3.84 ± 0.5% more compared to the degradation efficiency achieved
by Staphylococcus sp. (89.95%). On the other hand, beta endosulfan was utilized more by Staphylococcus sp. that was 9.14 ± 0.15% and 8.04 ± 0.15% more
compared to the degradation efficiency achieved by Bacillus circulans I and II.
At the end of 14 days of incubation 82.9 ± 0.05%, 75.96 ± 0.05% and 76.73 ±
0.05% of beta endosulfan degradation efficiency was achieved by Staphylococcus sp., Bacillus circulans I and II, respectively (Fig. 8b). These results were in
good agreement with our earlier observations. It is evident from the bacterial
identification tests that the growth of Staphylococcus sp. is more favorable in
facultative anaerobic condition compared to other Bacillus cultures. Staphylococcus sp. might be mainly responsible for the degradation of endosulfan in
93
94
Kumar and Philip
Figure 8: Degradation of alpha endosulfan (a) and beta endosulfan (b) by pure cultures.
Enrichment and Isolation of Bacterial Culture
facultative anaerobic system whereas Bacillus circulans I and II degraded majority of endosulfan in aerobic system.
CONCLUSION
The isolated mixed bacterial culture was able to mineralize endosulfan without the formation of any intermediate metabolites reported by previous researchers. Also, the culture was able to work in both aerobic and facultative
anaerobic environments. Staphylococcus sp. utilized more beta endosulfan in
facultative anaerobic system, whereas Bacillus circulans I and II preferred
more alpha endosulfan in aerobic system. The addition of dextrose increased
the degradation efficiency of endosulfan in both aerobic and facultative anaerobic systems. These microbial consortiums can effectively be used to degrade
endosulfan from contaminated soils, sediments, and wastewaters.
REFERENCES
1. Kannan, S.T.; Sengupta, R. Organochlorine residues in zooplankton of Saurashtra
coast, India. Mar. Pollut. Bull. 1987, 18, 92–104.
2. Rajendran, R.B.; Karunagaran, V.M.; Babu, S.; Subramanian, A.N.; Mohan, D. Levels of chlorinated insecticide in fishes from the Bay of Bengal. Mar. Pollut. Bull. 1992,
24, 567–570.
3. Mansingh, A.; Wilson, A. Insecticide contamination of Jamaican environment. Baseline studies on the status of Kingston Harbour. Mar. Pollut. Bull. 1995, 30, 640–645.
4. Miles, C.J.; Pfeuffer, R.J. Pesticides in canals of south Florida. Arch. Environ. Contam. Toxicol. 1997, 32, 337–345.
5. Shailaja, M.S.; Nair, M. Seasonal differences in organochlorine pesticide concentrations of zooplankton and fish in the Arabian Sea. Mar. Pollut. Bull. 1997, 44, 263–274.
6. Sujatha, C.H.; Nair, S.M.; Chacko, J. Determination and distribution of endosulfan
and malathion in an Indian estuary. Water Res. 1999, 33(1), 109–114.
7. Bhattacharya, B.; Sarkar, S.; Mukherjee, N. Organochlorine pesticide residues in
sediments of a tropical mangrove estuary, India: Implications for monitoring Environ.
Int. 2003, 29, 587–592.
8. Siddique, T.; Okeke, B.C.; Arshad, A.; Frankenberger, W.T., Jr. Enrichment and isolation of endosulfan-degrading microorganisms. J. Environ. Qual. 2003, 32, 47–54.
9. Miles, J.R.W.; Moy, P. Degradation of endosulfan and its metabolites by a mixed
culture of soil microorganisms. Bull. Environ. Contam. Toxicol. 1979, 23, 13–19.
10. Mukherjee, I.; Gopal, M. Degradation of beta-endosulfan by Aspergillus niger. Toxicol. Environ. Chem. 1994, 46, 217–221.
11. Kullman, S.W.; Matsumura, F. Metabolic pathway utilized by Phenerochete
chrysosporium for degradation of the cyclodine pesticide endosulfan. Appl. Environ.
Microbiol. 1996, 62, 593–600.
12. Awasthi, N.; Manickam, N.; Kumar, A. Biodegradation of endosulfan by a bacterial
co-culture. Bull. Environ. Contam. Toxicol. 1997, 59, 928–934.
95
96
Kumar and Philip
13. Guerin, T.F. The anaerobic degradation of endosulfan by indigenous microorganisms from low-oxygen soils and sediments. Environ. Pollut. 1999, 106, 13–21.
14. Shetty, P.K.; Mitra, J.; Murthy, N.B.K.; Namitha, K.K.; Savitha, K.N.; Raghu, K.
Biodegradation of cyclodiene insecticide endosulfan by mucor-thermo-hyalospora MTCC
1384. Current Sci. 2000, 79(9), 1381–1383.
15. Sutherland, T.; Horne, I.; Lacey, M.; Harcourt, R.; Russell, R.; Oakeshott, J. Enrichment of an endosulfan-degrading mixed bacterial culture. Appl. Env. Microbiol. 2000,
66(7), 2822–2828.
16. Awasthi, N.; Singh, A.K.; Jain, R.K.; Khangarot, B.S.; Kumar, A. Degradation and
detoxification of endosulfan isomers by a defined co-culture of two Bacillus strains. Appl.
Microbiol. Biotechnol. 2003, 62(2–3):279–283.
17. Kwon, G.; Kim, J.; Kim, T.; Sohn, H.; Koh, S.; Shin, K.; Kim, D. Klebsiella pneumoniae KE-1 degrades endosulfan without formation of the toxic metabolite, endosulfan
sulfate. FEMS Microbiol. Lett. 2002, 215, 255–259.
18. Martens, R. Degradation of (8–9
Environ. Microbiol. 1976, 31, 853–858.
14
C) endosulfan by soil microorganisms. Appl.
19. Katayama, A.; Matsumura, F. Degradation of organochlorine pesticides, particularly endosulfan by Trichoderma harzianum. Environ. Toxicol. Chem. 1993, 12, 1059–
1065.
20. Awasthi, N.; Ahuja, R.; Kumar, A. Factors influencing the degradation of soil applied endosulfan isomers. Soil. Biol. Biochem. 2000, 32 (11–12), 1697–1705.