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Proceedings of the NationalAcademy of Sciences, India Section B:Biological Sciences ISSN 0369-8211Volume 84Number 3 Proc. Natl. Acad. Sci., India, Sect. B Biol.Sci. (2014) 84:657-667DOI 10.1007/s40011-013-0223-5
A Study on Parameters Optimization forDegradation of Endosulfan by BacterialConsortia Isolated from Contaminated Soil
Kaushik Bhattacharjee, Subhro Banerjee,Lalsiamthari Bawitlung, DineshKrishnappa & S. R. Joshi
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RESEARCH ARTICLE
A Study on Parameters Optimization for Degradationof Endosulfan by Bacterial Consortia Isolated from ContaminatedSoil
Kaushik Bhattacharjee • Subhro Banerjee •
Lalsiamthari Bawitlung • Dinesh Krishnappa •
S. R. Joshi
Received: 8 February 2013 / Revised: 24 June 2013 / Accepted: 10 July 2013 / Published online: 24 August 2013
� The National Academy of Sciences, India 2013
Abstract Endosulfan, a non-systemic organochlorine
pesticide used extensively to control the insect pests of a
wide range of crops is of environmental concern because of
its apparent persistence and toxicity to many non-target
organisms. The present study was aimed to find out the
capability of microorganisms to degrade endosulfan, singly
and/or in consortium and optimization of various growth
parameters to achieve optimal degradation. A total of three
isolates showing significantly higher degradation potential
were selected from eight isolates demonstrating substantial
growth. They were identified as Staphylococcus equorum
CM5, Enterobacter sp. MF1 and Bacillus subtilis MF2.
The effect of various parameters (pH, temperature, inocu-
lum size, endosulfan concentration, incubation conditions
and carbon concentration) were also assessed and opti-
mized for degradation of endosulfan. The consortia of
Staphylococcus and Bacillus strain showed near disap-
pearance of endosulfan from the medium after 21 days of
incubation. The maximum degradation percentage of
endosulfan was observed at slightly alkaline pH 8.0 under
shaking conditions of 150 rpm at 30 �C at a concentration
of 1 g l-1 of dextrose and irrespective of the inocula size
used in this study. The isolates identified in this study
present an efficient, economical and ecological alternative
remedy for the removal of endosulfan from contaminated
sites.
Keywords Endosulfan � Persistent organic pollutant �Bioremediation � Bacterial consortium � Biodegradation
Introduction
Endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexa-
hydro-6,9-ethano-2,4,3-benzodioxathiepin-3-oxide), a non-
systemic organochlorine pesticide is of environmental con-
cern due to its apparent persistence and toxicity to many non-
target organisms and is classified under the category of
persistent organic pollutant (POP) by the Stockholm Con-
vention in April 2011. It was first introduced in 1950s and
emerged as a leading chemical used against a broad range of
insects and mites in agriculture and allied sectors [1] under
the trade names like Agrisulfan, Axis, Endofan, Ensure,
Goldenleaf Tobacco Spray, Misulfan, Red Sun, etc. It is used
extensively throughout the world to control the insect pests
of a wide range of crops including cereals, tea, coffee, cotton,
fruits, oil seeds and vegetables [2]. Although ban was
imposed on the production and usage of POPs like endo-
sulfan, it is still produced and widely used in the crop fields in
most of the developing countries including India, due to its
effectiveness and low application cost [3–5]. The levels of
endosulfan in environment have not shown a declining trend
in some regions [6]. Endosulfan and its metabolites have
widely been reported as contaminants in soil, air and water
environments due to their intensive and frequent usage and
potential for atmospheric transport [6, 7]. Its contamination
Electronic supplementary material The online version of thisarticle (doi:10.1007/s40011-013-0223-5) contains supplementarymaterial, which is available to authorized users.
K. Bhattacharjee � S. Banerjee � L. Bawitlung � S. R. Joshi (&)
Microbiology Laboratory, Department of Biotechnology and
Bioinformatics, North-Eastern Hill University, Mawlai,
Shillong 793022, Meghalaya, India
e-mail: [email protected]
K. Bhattacharjee
e-mail: [email protected]
D. Krishnappa
Department of Biotechnology, Anna University of Technology,
Tiruchchirappalli 620024, Tamil Nadu, India
123
Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. (July–Sept 2014) 84(3):657–667
DOI 10.1007/s40011-013-0223-5
Author's personal copy
in food and other value-added agro-commodities has also
been widely reported [8, 9]. Endosulfan is extremely toxic to
microbial and enzyme activities in soil and aquatic flora and
fauna with report of severe intoxications to humans as well
[10, 11]. Bioremediation is superior to chemical remediation
process, as the latter one is not applicable to cost-effective
remediation of large-scale sub-surface contamination and on
the other hand the former possesses immense potentiality for
selective removal of toxic compounds [12]. Microbial deg-
radation of endosulfan may play an important role in
detoxifying the endosulfan contaminated sites. Biodegra-
dation of endosulfan has been previously documented in soil
by bacteria like Bacillus sp., Staphylococcus sp., Pseudo-
monas aeruginosa, P. spinosa, Arthrobacter sp., Rhodo-
coccus sp., etc. [13–15]. Although microorganisms could be
used effectively for bioremediation of endosulfan contami-
nated soil and water environments, the environmental factors
that vary in time and space affect the proliferation and
metabolic activities of microorganisms responsible for
degrading toxic compounds in the given environments [10].
Understanding the importance of endosulfan degradation,
the present study was aimed to find out the capability of
microorganisms isolated from hydrocarbon contaminated
soil and pesticide contaminated agricultural soil to degrade
endosulfan singly and/or as consortium. This is one of its first
kind of study on microbes from contaminated sites capable
of biodegradation of endosulfan.
Material and Methods
Soil Samples and Sampling
The soil samples were collected from petroleum contami-
nated sites as well as from different agricultural fields like
that of paddy and maize where endosulfan is used as a
pesticide. For petroleum contaminated sites, soil samples
were collected from 10 m sites adjacent to two different
petrol filling stations. The soil was collected from a depth
of 10 cm in sterilized plastic containers and stored at 4 �C
for further study.
Enrichment of Soil Samples
Fifty grams of each sample was placed in six glass dishes
and stacked one above the other in a closed container to
maintain constant moisture conditions by adding distilled
water every 2 days to maintain their original weight. The
soil samples in the glass dishes were spiked with filter
sterilized endosulfan (dissolved in ethanol) to give a final
concentration of 50 mg kg-1 of soil [16]. The dishes were
kept in a laminar air flow hood till evaporation of ethanol.
The contents were mixed gently and incubated at room
temperature (30 �C) for 4–6 weeks. The pesticide treatment
was repeated three times at bi-weekly intervals.
Isolation of Microorganisms
Pure microbial strains were isolated from the endosulfan
enriched soil samples by serial dilution method followed
by streaking on nutrient agar (NA) plates. Dilution was
made serially till 10-7 and 10-4–10-7 dilutions were used
for inoculating the NA plates by spread plate method. The
plates were then inverted and incubated at 30 �C for
4 days. The mixed cultures obtained were then differenti-
ated on the basis of their colony morphology. Colonies
showing different morphology were selected for obtaining
pure cultures by using streak plate method. The selected
colony was then picked up with a sterilized loop and
streaked on a NA plate and incubated at 30 �C for 4 days.
Based on the ability of the pure culture isolates to grow in
liquid mineral salt medium (MSM) enriched with endo-
sulfan to a concentration of 100 mg l-1, a few microbial
strains were selected for degradation studies.
Biodegradation of Endosulfan by Bacterial Isolates
The isolates demonstrating prolific growth were investigated
for their capability to degrade endosulfan over time (0, 2, 3,
5, 7, 10, 14 and 21 days of incubation). For this purpose,
100 ml Erlenmeyer flasks with MSM were autoclaved at
121 �C for 20 min separately. The medium was adjusted to
pH 8. Fifty milliliters of sterile medium was put into each
flask. These flasks were spiked with endosulfan to a con-
centration of 100 mg l-1. These flasks were inoculated with
100 ll bacterial inocula adjusted to a set optical density of
OD595 = 0.81 and *108 CFU ml-1. These inoculated
flasks were incubated at 30 �C on an orbital shaker at
150 rpm. Uninoculated flasks (control) were also prepared to
check the abiotic degradation under the same conditions.
This procedure was carried out in triplicate and results were
mean of three. Efficient isolates among the obtained cultures
were investigated for their consortial degradation ability.
Optimization of Conditions for Maximum Endosulfan
Degradation
In all the cases, conical flasks containing sterile 50 ml MSM
were used for each isolate except otherwise mentioned.
pH
The flasks were spiked with endosulfan to a concentration
of 100 mg l-1 and pH adjusted to 4, 6, 7, 8, and 10 with the
addition of hydrochloric acid (HCl)/sodium hydroxide
(NaOH) solutions.
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Endosulfan Concentration
The flasks were spiked with endosulfan to a concentration
of 5, 10, 50, 100, 200, 300 mg l-1.
Carbon Source
Dextrose was supplied as a supplementary carbon at con-
centrations of 1, 2, 3 and 4 g l-1 to the flasks which were
already spiked with endosulfan (100 mg l-1) and main-
tained at pH 7.
Temperature
Flasks spiked with endosulfan (100 mg l-1) at pH 7 were
inoculated and kept in an orbital shaker at 150 rpm at
different temperatures (20, 25, 30, 35, 40 and 45 �C).
Inoculum Size
Bacterial inocula of 100, 200, 400, 600, 800 and 1000 ll
were inoculated in eight identical conical flasks containing
100 ml of MSM media previously amended with
100 mg l-1 endosulfan. The pH of the medium was adjusted
to 8 and no supplementary carbon was added throughout.
In all the above cases, the flasks were inoculated with
100 ll bacterial inocula adjusted to OD595 = 0.81 (*108
CFU ml-1), except in case of inoculum size study where
inocula were added with varying concentrations. The inocu-
lated flasks were kept in an orbital shaker at 30 �C (except
while studying the effect of temperature where various tem-
peratures were taken into account) at 150 rpm for 21 days.
Blank control reactors were operated simultaneously for all
the cases and each had three replicates per treatment. The
samples were collected from the controlled flasks after
21 days and analyzed for endosulfan concentration.
Microscopic and Biochemical Characterization
of Isolates
Periodical observations on gram staining were recorded
using microscope (Leica DM 5500). Biochemical charac-
terizations were performed according to Bergey’s Manual
of Determinative Bacteriology following the standard
protocols [17, 18].
DNA Isolation, 16S rRNA Gene Amplification
and Sequencing
The isolates showing higher degradation efficiency were
selected and cultured on Luria–Bertani broth at 37 �C for
24 h in an orbital shaking incubator (NBS, USA). Genomic
DNA were isolated by HiPurATM Bacterial and Yeast
Genomic DNA Purification Spin Kit (HiMedia, India) and
used for 16S rDNA gene amplification. Genomic DNA
were amplified with bacterial primers 27F: 50-AGA GTT
TGA TCC TGG CTC AG-30, and 1492R: 50-TAC GGY
TAC CTT GTT ACG ACT T-30 [19]. The reactions were
performed on a GeneAmp PCR system 9700 (Applied
Biosystems, USA). The PCR mixture consisted of 5 ll
109 buffer (with Mg2?), 8 ll dNTP mixture (1.25 mM
each), 0.5 ll of each primer, 1 ll of template DNA, and
1.0 ll of Taq polymerase (Fermentas, USA) in a final
volume of 50 ll. PCR amplification parameters were as
follows: 94 �C for 5 min of initial melting; 30 cycles of
94 �C, 1 min; 55 �C, 1 min; and 72 �C, 2 min; and a final
extension at 72 �C for 5 min [19]. The PCR products were
purified using the QIAquick Gel Extraction Kit (Qiagen,
Germany) according to manufacturer’s protocols. The
DNA content of the PCR products were estimated using a
NanoVue Plus Spectrophotometer (GE Healthcare’s Life
Sciences, Sweden). Sequencing reactions of the 16S rDNA
fragments were performed with the Big Dye Terminator v
3.1 Cycle Sequencing Kit (Applied Biosystems, USA).
Phylogenetic Reconstruction
The 16S rRNA gene sequence of the isolates and their
closely related species were retrieved from EzTaxon-e
server [20] and aligned using the ClustalX2 programme
[21]. The tree topologies were evaluated by bootstrap
analyses based on 1,000 replications with MEGA v4.1
programme [22] and phylogenetic trees were inferred using
the neighbor-joining and maximum likelihood methods
[23]. Gaps in alignment were considered as missing data
for all phylogenetic reconstructions.
For Metropolis–Hastings coupled Markov chain Monte
Carlo phylogenetic analysis, DNA sequences were aligned
using ClustalX2 and the interleaved NEXUS file was edited
manually in order for it to be recognized by Mr. Bayes v3.1.2
programme. The evolutionary model that best fits the data was
determined by Modeltest 3.6 9 [24] and analyzed under the
Akaike’s Information Criterion. Bayesian phylogenetic
reconstructions were done using Mr. Bayes v3.1.2 adopting
the General Time Reversible (GTR) model with gamma dis-
tributed rates and invariant sites (GTR ? I ? G) of nucleotide
substitution and run until the mean standard deviation (SD) of
split frequencies was below 0.01. A consensus tree was con-
structed following a visually determined burn-in of 25 %.
Nucleotide Sequence Accession Numbers
The 16S rRNA gene sequences determined for the three
representative isolates in this study were deposited in the
GenBank database with the accession numbers JN230520
to JN230522.
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Data Analysis
Percentage of biodegradation was calculated on the basis of
the difference between residual endosulfan in treated
samples and uninoculated controls. The experimental data
were presented as mean ± SD of three replicates. Post-hoc
comparisons (Fisher’s least significant difference p \ 0.05)
were performed to examine the significant differences
among means. Statistical analyses of differences among the
obtained results were performed using one-way analysis of
variance (ANOVA) and Kruskal–Wallis one-way
ANOVA. In the observed data, the hypotheses of variance
normality (tested with Shapiro–Wilk test, Jarque–Bera test,
Anderson–Darling test, Lilliefors test) and homogeneity of
variances were tested successfully with at least one sig-
nificant result. All analysis was conducted using XLStat
v7.5.2 (Addinsoft, USA) and Graph Pad Prism 5.00
(GraphPad, San Diego, CA, USA).
Results and Discussion
Enrichment
Isolation of many endosulfan-degrading bacteria have been
reported using enrichment cultures. Generally two types of
enrichment cultures have been used, with and without
using endosulfan as the sole sulfur source. Sulfur-limited
conditions have been used in enrichment cultures because
many microorganisms can use sulfonates and sulfate esters
as a source of sulfur for growth, even when they are unable
to use the carbon skeleton of the compounds [25]. Endo-
sulfan as the only source of available sulfur in enriched
culture medium for the isolation of endosulfan degrading
bacterium is also reported by some researchers [26]. Eight
microbial strains are selected for further studies with sub-
stantial growth in the enrichment. During the biodegrada-
tion process of toxic compounds, certain enzymes of
bacteria are induced which are available for taking part in
the process which is at large a metabolic reaction. In this
study, microorganisms were selected by enrichment for
their ability to release the sulfite group from endosulfan
and to use this as a source of sulfur for growth. This
selection procedure enriches a culture capable of either the
direct hydrolysis of endosulfan or the oxidation of the
insecticide followed by its hydrolysis.
Identification of Selected Isolates
Microscopic observation revealed two organisms to be
gram positive (CM5 and MF2) and one gram negative
(MF1) with distinct biochemical characteristics (Table 1).
Out of eight bacterial isolates, three potent forms based on
their activity were identified up to species level using 16S
rRNA phylogenetic analysis. PCR amplification of total
genomic DNA of the isolates using 16S rRNA bacterial
universal primers yielded a band of the expected size
of *1,500 bp. An almost-complete 16S rDNA sequence
containing less than 1 % undetermined positions were
obtained for all the isolates. The isolates belonged to the
respective genus, supported by the tested treeing algo-
rithms showing a high bootstrap value in the neighbour-
joining analysis (Fig. 1) and also higher degree of taxon
separation in Bayesian phylogeny (Online Resource 1).
The three highly efficient bacterial isolates CM5, MF1 and
MF2 were identified as Staphylococcus equorum CM5,
Enterobacter sp. MF1 and Bacillus subtilis MF2. These
bacterial isolates have already been documented as excel-
lent degraders of a wide range of xenobiotic compounds
both in soil and water environment [27, 28].
Biodegradation of Endosulfan
Endosulfan has been shown to be microbially degraded by
two separate pathways, viz., hydrolytic and oxidative. Oxi-
dation of endosulfan yields endosulfan sulfate which is
reported to be as toxic and as persistent as its parent com-
pound. But, as per some reports during biodegradation pro-
cess, endosulfan sulfate also decreases with the parent
compound with the passage of time [29]. Chemical hydro-
lysis of endosulfan at alkaline pH (pH [7) generates non-
toxic endosulfan diol [30]. As per present experimental
design, the media pH was maintained at pH 8, which nullifies
the presence of toxic endosulfan sulfate as an end product of
biodegradation process. The reduction in concentration of
endosulfan from the spiked and inoculated broth varied
significantly among the bacterial strains (F = 5.89,
p \ 0.001). Biodegradation of endosulfan revealed marked
variation in the final concentration of endosulfan after
21 days (Fig. 2; Table 2).The descriptive statistical analysis
infers a normal distribution and randomization of the
experimental data (Online Resource 2). The biodegradation
potential of endosulfan by isolates and consortium signifi-
cantly varied (Levene’s F = 16.108, p \ 0.0001) depend-
ing on days of incubation and with valid equal variance
(Online Resource 3). The variance of normality and homo-
geneity were tested successfully with at least one significant
result (Online Resource 3). Among the eight isolates
obtained using the enrichment technique, three isolates, viz.,
CM5, MF1 and MF2 degraded endosulfan to reach a con-
centration of almost 16 mg l-1 from 100 mg l-1. It is
striking to note that the same observations were not recip-
rocated while performing the biodegradation studies with a
consortium of the above three isolates. The combination of
CM5 ? MF1, MF1 ? MF2 and CM5 ? MF1 ? MF2
yielded almost negligible degradation of endosulfan while
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Table 1 Biochemical
characteristics observed for the
isolates under study
Characteristics Isolate CM5 Isolate MF1 Isolate MF2
Colony size 0.5–1.5 lm 9 0.5–1.5 lm 1.2–3.0 lm 9
0.5–1.1 lm
1.2–3 lm 9
0.7–0.8 lm
Colony shape Circular, convex with an entire
margin
Irregular, smooth,
moist
Undulate, circular
and flat
Colony color on nutrient
agar media
Cream-colored Grey Cream-colored
Cell shape Cocci, in cluster Straight rods Rod
Cell motility Non-motile Motile Motile
Capsule -ve -ve ?ve
Gram character ?ve -ve ?ve
Pigmentation -ve -ve ?ve, yellow
Sporulation -ve -ve ?ve, central spore
Indole -ve -ve -ve
Methyl red ?ve -ve ?ve
Voges–Proskauer -ve ?ve ?ve
Citrate utilization -ve ?ve ?ve
Urease ?ve -ve ?ve
H2S production -ve -ve -ve
Oxidase -ve -ve -ve
Catalase ?ve ?ve ?ve
Lipase ?ve -ve ?ve
Coagulase -ve -ve -ve
Nitrate reduction ?ve ?ve ?ve
Litmus milk ND ND -ve, alkaline
Hydrolysis of
Gelatin ?ve -ve ?ve
Casein -ve ?ve ?ve
Starch -ve -ve ?ve
Esculin -ve ?ve ?ve
ONPG ?ve ?ve -ve
Growth at
10 �C -ve -ve -ve
25 �C ?ve ?ve ?ve
30 �C ?ve ?ve ?ve
35 �C ?ve ?ve ?ve
45 �C -ve -ve -ve
50 �C -ve -ve -ve
pH 5 -ve -ve -ve
pH 6 ?ve ?ve ?ve
pH 7 ?ve ?ve ?ve
pH 8 ?ve ?ve ?ve
pH 9.6 -ve -ve ?ve
Growth with
2 % NaCl ?ve ?ve ?ve
4 % NaCl ?ve ?ve ?ve
6.5 % NaCl ?ve -ve ?ve
10 % NaCl -ve -ve -ve
40 % Bile -ve -ve -ve
Crystal violet -ve -ve -ve
0.1 % phenol -ve -ve ?ve
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the degradation was prominent when the same isolates were
used singly. Contrary to this effect, the third combination of
CM5 ? MF2 showed almost near disappearance of endo-
sulfan from the broth after 21 days which is noteworthy. The
reason for the deviation of this consortium from the other two
needs evaluation. The findings hypothesized that a consortia
of gram positive and negative bacteria showed better deg-
radation of endosulfan. Whereas, when the consortia of
Fig. 1 Phylogenetic tree showing inter-relationship of bacterial
strains CM5, MF1 and MF2, which was reconstructed by using the
neighbor-joining (NJ) method analysis of 16S rRNA gene sequences
of selected strains and their closest phylogenetic neighbours. Vibrio
cholerae and Xanthomonas sp. were included as outgroups. Bootstrap
values, expressed as percentage of 1,000 replications and are
indicated at the nodes (bar represents 5 % sequence divergence and
filled circles represent the studied isolates)
Table 1 continued
CM5, Staphylococcus equorum
CM5; MF1, Enterobacter sp.
MF1; MF2, Bacillus subtilis
MF2; ?ve, positive; -ve,
negative; ND, not detected
Characteristics Isolate CM5 Isolate MF1 Isolate MF2
Acid from (aerobically)
D-glucose ?ve ?ve ?ve
D-galactose ?ve ?ve ?ve
D-mannose ?ve ?ve ?ve
a-D-lactose ?ve ?ve -ve
Maltose ?ve ?ve ?ve
Sucrose ?ve ?ve ?ve
Beta-D-fructose ?ve ?ve ?ve
Turanose ?ve -ve -ve
D-trehalose ?ve ?ve ?ve
D-xylose ?ve ?ve ?ve
L-arabinose ?ve ?ve -ve
D-cellobiose -ve ?ve ?ve
Salicin ?ve ?ve -ve
D-ribose ?ve ?ve ?ve
D-arabitol -ve ?ve -ve
D-raffinose ?ve ?ve ?ve
D-sorbitol -ve ?ve ?ve
D-mannitol ?ve ?ve ?ve
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isolates of same gram character are employed, almost neg-
ligible degradation of endosulfan takes place (Fig. 2;
Table 2). An in-depth understanding of the enzymes and
mechanism of the reaction can further help in understanding
this riddle.
Effect of Various Parameters on Endosulfan
Degradation
The biodegradation process in soil is affected by many
environmental factors and therefore, it is important to
Fig. 2 Time course of in situ
biodegradation pattern of
endosulfan in mineral salt
medium (MSM) by the bacterial
isolates alone and in
consortium. Each point is the
mean of three replicates and
represents the endosulfan
concentration (mg l-1) of each
isolate/consortium at the
respective days of incubation
Table 2 Degradation of endosulfan by bacterial isolates and consortium at different time intervals (mean ± SD, n = 3)
Days of incubation Control CM5 CM6 MF1 MF2 PM10 PM11
0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0
2 99 ± 0.005 97.57 ± 0.23 96.91 ± 0.16 97.69 ± 0.02 96.27 ± 0.11 95.62 ± 0.15 98.08 ± 0.06
3 98.97 ± 0.005 89.47 ± 0.15 93.69 ± 0.19 89.33 ± 0.59 89.51 ± 0.17 93.79 ± 0.04 95.17 ± 0.05
5 98.97 ± 0.01 67.53 ± 0.17 91.38 ± 0.13 57.08 ± 0.22 65.54 ± 0.18 92.82 ± 0.02 90.81 ± 0.01
7 98.96 ± 0.005 40.41 ± 0.13 90.92 ± 0.16 41.48 ± 0.12 36.90 ± 0.27 91.61 ± 0.05 79.54 ± 0.02
10 98.94 ± 0.01 28.28 ± 0.14 88.88 ± 0.09 30.18 ± 0.59 23.71 ± 0.21 89.56 ± 0.04 79.05 ± 0.02
14 98.93 ± 0.005 19.36 ± 0.05 85.62 ± 0.09 18.25 ± 0.40 17.50 ± 0.05 85.05 ± 0.11 79.04 ± 0.01
21 98.93 ± 0.005 18.55 ± 0.62 81.51 ± 0.15 17.13 ± 0.11 16.31 ± 0.11 83.93 ± 0.03 78.53 ± 0.03
Days of incubation PM14 PF CM5 ? MF1 CM5 ? MF2 MF1 ? MF2 CM5 ? MF1 ? MF2
0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0
2 98.04 ± 0.01 96.38 ± 0.50 98.58 ± 0.04 97.16 ± 0.05 98.89 ± 0.05 98.93 ± 0.098
3 95.11 ± 0.005 93.82 ± 0.01 98.13 ± 0.05 90.60 ± 0.20 97.65 ± 0.07 97.55 ± 0.22
5 90.82 ± 0.01 91.37 ± 0.01 97.84 ± 0.05 63.31 ± 0.06 96.99 ± 0.05 96.89 ± 0.11
7 79.62 ± 0.05 90.73 ± 0.06 97.79 ± 0.02 47.26 ± 0.02 96.75 ± 0.18 96.87 ± 0.04
10 79.11 ± 0.014 87.75 ± 0.02 97.82 ± 0.01 51.36 ± 0.03 96.38 ± 0.05 96.57 ± 0.09
14 79.04 ± 0.01 84.01 ± 0.005 97.81 ± 0.02 6.38 ± 0.03 95.88 ± 0.04 85.62 ± 0.09
21 79.01 ± 0.005 79.42 ± 0.01 97.83 ± 0.01 2.71 ± 0.01 95.64 ± 0.07 81.51 ± 0.15
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understand the appropriate environmental conditions to
achieve optimal degradation [25]. A significant difference
(F1,6 = 11.14, p \ 0.01) was noted between biodegradation
of endosulfan by the selected bacterial strains under static
incubation versus incubation with agitation (Fig. 3a). A
decrease of 65–70 % in degradation was observed under
static condition in all the cases. This observation is also
supported by some previous studies [15, 31]. The pH of the
solution or culture media may affect the adsorption process
as well as microbial activity. Batch experiments were con-
ducted in aerobic conditions to study the influence of pH on
endosulfan degradation by the microbial consortium. In the
present study, endosulfan was amended directly into MSM
and endosulfan degradation experiments were conducted at
various pH levels, i.e., 4, 6, 7, 8 and 10. The degradation
efficiency showed a gradual increase from 6 to 8 with almost
negligible degradation at pH 4 (F4,24 = 10.94, p \ 0.0001).
An alkaline pH of more than 8 abruptly decreased the deg-
radation efficiency (Fig. 3b). Similar trends were observed
by Arshad et al. [31] while studying the endosulfan degra-
dation by enriched bacterial strains. This may be due to the
decreased growth of microbes at extreme pH. It is reported
that at high pH, hydrolysis of endosulfan was faster [32].
Testing the parameters for different concentration of endo-
sulfan, about 70–72 % decrease at 50 mg l-1, 85–96 % at
10 mg l-1 and 90–97 % at 5 mg l-1 were observed when
compared with maximum degradation of endosulfan (i.e., at
100 mg l-1). But further increase in the concentration of
endosulfan showed a decrease in the rate of biodegradation.
This decrease in degradation might be due to cytotoxicity of
endosulfan. All the strains and consortia responded similarly
to the change in the concentration of endosulfan (Fig. 4a)
with a considerable variance (F5,30 = 8.773, p \ 0.0001).
One-way ANOVA analysis of the experimental data repre-
sents a significant variance between the concentrations of
carbon sources (F3,18 = 85.50, p \ 0.0001). MSM when
supplemented with 1 g l-1 dextrose as a sole source of car-
bon showed maximum degradation efficiency with a slight
decrease in case of 2 g l-1. But further increase in the con-
centration of dextrose showed no change (Fig. 4b) and these
findings are in conformity with the work done by Kumar and
Philip [13]. The findings indicate that co-metabolic pro-
cesses increased the endosulfan degradation and 1 g l-1 of
dextrose can be used as an optimum supplementary carbon
dose. Endosulfan was amended directly from stock solution,
which is prepared in methanol. Methanol also acted as a
carbon source for the microbes, though it is a less preferred
substrate for the microbial isolate and consortia compared to
dextrose. Temperature is undoubtedly the most fundamental
factor for all living organisms as it affects metabolic pro-
cesses and biochemical composition of cells. The optimal
growth temperature and tolerance to the extreme values
usually vary from strain to strain while sudden temperature
changes exert stress on the organisms. At high temperature,
deficiency of oxygen, which is much less soluble in hot than
in cold water, may be the proximal cause of stress. Endo-
sulfan degradation efficiency varies significantly
(F5,30 = 9.754, p \ 0.0001) and maximum was found at
30 �C and thereafter it was found to decrease up to 45 �C
(Fig. 5a). Very little growth was observed at 20 and 25 �C.
The obtained results are also in aggregation with the results
obtained in other studies [15, 31]. Assessment of biodegra-
dation of endosulfan at different inocula sizes incubated
under optimal temperature (30 �C) and pH (8) conditions
revealed that the various inocula sizes used in the study
Fig. 3 In situ endosulfan biodegradation (%) of endosulfan in
mineral salt medium (MSM) by the bacterial isolates alone and in
consortium after a 21-days incubation period: (a) under shaking and
static conditions, (b) at different pH conditions (bars are the means of
three replicates)
664 K. Bhattacharjee et al.
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(100–1,000 ll) have very less effect on biodegradation
process with a variation of *0.04 % (Fig. 5b) which is in
accordance with the report that the increase in inoculum size
had no considerable effect on the degradation of endosulfan
[31, 33]. In all the above cases the only exception is with the
consortia of isolates CM5 ? MF1 and MF1 ? MF2 where
the changes in parameters have almost no effect on degra-
dation efficiency of endosulfan. On the other hand in all the
above parameters, the consortia of CM5 ? MF2 always
displayed high efficiency of endosulfan degradation. But
when the consortia of all the three isolates were used, the
efficiency was low as compared to the optimum efficiency
(though not negligible as of CM5 ? MF1 and
MF1 ? MF2). The difference in enzyme system and/or
difference in their growth rate alone or in consortia may be
responsible for the difference in degradation capabilities.
Conclusion
In the light of findings of this study, it could be concluded
that the maximum degradation percentage of endosulfan
was observed at slightly alkaline pH 8.0 under shaking
conditions of 150 rpm at 30 �C at a concentration of
1 g l-1 of dextrose and irrespective of the inocula size used
in this study. To the authors’ knowledge, this is the first
Fig. 4 In situ endosulfan biodegradation (%) of endosulfan in
mineral salt medium (MSM) by the bacterial isolates alone and in
consortium after a 21-days incubation period: (a) at different
concentrations of endosulfan, (b) at different concentrations of
dextrose (bars are the means of three replicates)
Fig. 5 In situ endosulfan biodegradation (%) of endosulfan in
mineral salt medium (MSM) by the bacterial isolates alone and in
consortium after a 21-days incubation period: (a) at different
incubation temperatures, (b) at different inocula sizes (bars are the
means of three replicates)
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report on endosulfan biodegradation by the isolates S.
equorum CM5, Enterobacter sp. MF1 and B. subtilis MF2
characterized from soils of north-east India. The enriched
bacterial consortium of Gram positive and Gram negative
bacteria used in the study can be effectively used for the
treatment of endosulfan contaminated water and soil as the
mixed bacterial consortium was able to mineralize endo-
sulfan under all the parameters tested. This treatment
technology should have useful application for cleanup of
contaminated soils and sediments, particularly in soils that
are highly contaminated with concentrations that can sup-
port the growth and activity of the inoculated bacterium.
Acknowledgments Authors acknowledge the research support
received from the Department of Electronics and Information Tech-
nology, Ministry of Communications and Information Technology,
Government of India. K. Dinesh is thankful to The Indian Academies
of Science Summer Fellowship Programme for the SRF award. Part
support received form DST Inspire programme is duly acknowledged.
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