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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: srjoshi2006@yahoo.co.in

K. Bhattacharjee

e-mail: kaushik_asm@yahoo.com

D. Krishnappa

Department of Biotechnology, Anna University of Technology,

Tiruchchirappalli 620024, Tamil Nadu, India

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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.

References

1. Kang KH, Zhang L, Zhang Z, Sui Z, Hur J (2013) Acute toxicity

and biodegradation of endosulfan by the polychaeta Perinereis

aibuhitensis in an indoor culture. Chin J Oceanol Limnol

31(1):75–80. doi:10.1007/s00343-013-2051-0

2. Wang B, Huang J, Lu Y, Arai S, Iino F, Morita M, Yu G (2012)

The pollution and ecological risk of endosulfan in soil of Huai’an

city, China. Environ Monit Assess 184(12):7093–7101. doi:

10.1007/s10661-011-2482-z

3. Devi NL, Chakraborty P, Shihua Q, Zhang G (2012) Selected

organochlorine pesticides (OCPs) in surface soils from three

major states from the northeastern part of India. Environ Monit

Assess. doi:10.1007/s10661-012-3055-5

4. Meza-Montenegro MM, Valenzuela-Quintanar AI, Balderas-

Cortes JJ, Yanez-Estrada L, Gutierrez-Coronado ML, Cuevas-

Robles A, Gandolfi AJ (2013) Exposure assessment of organo-

chlorine pesticides, arsenic, and lead in children from the major

agricultural areas in Sonora, Mexico. Arch Environ Contam

Toxicol 64(3):519–527. doi:10.1007/s00244-012-9846-4

5. Murugan AV, Swarnam TP, Gnanasambandan S (2013) Status

and effect of pesticide residues in soils under different land uses

of Andaman Islands, India. Environ Monit Assess. doi:

10.1007/s10661-013-3162-y

6. Weber J, Halsall CJ, Muir D, Teixeira C, Small J, Solomon K,

Hermanson M, Hung H, Bidleman T (2010) Endosulfan, a global

pesticide: a review of its fate in the environment and occurrence

in the Arctic. Sci Total Environ 408(15):2966–2984. doi:10.1016/

j.scitotenv.2009.10.077

7. Kaushik A, Sharma HR, Jain S, Dawra S, Kaushik CP (2010)

Pesticide pollution of river Ghaggar in Haryana, India. Environ

Monit Assess 160:61–69. doi:10.1016/j.biortech.2010.10.005

8. Kelly BC, Gobas FAPC (2003) An Arctic terrestrial food-chain

bioaccumulation model for persistent organic pollutants. Environ

Sci Technol 37(13):2966–2974. doi:10.1021/es021035x

9. Kelly BC, Ikonomou MG, Blair JD, Morin AE, Gobas FAPC (2007)

Food web-specific biomagnification of persistent organic pollutants.

Science 317(5835):236–239. doi:10.1126/science.1138275

10. Hussain S, Siddique T, Saleem M, Saleem M, Arshad M, Khalid

A (2009) Impact of pesticides on soil microbial diversity,

enzymes, and biochemical reactions. In: Sparks DL (ed)

Advances in agronomy, vol 102. Academic Press, San Diego,

pp 159–200. doi:10.1016/S0065-2113(09)01005-0

11. Moses V, Peter JV (2010) Acute intentional toxicity: endosulfan

and other organochlorines. Clin Toxicol (Phila) 48(6):539–544.

doi:10.3109/15563650.2010.494610

12. Sarma B, Acharya C, Joshi SR (2013) Plant growth promoting

and metal bioadsorption activity of metal tolerant Pseudomonas

aeruginosa isolate characterized from uranium ore deposit. Proc

Natl Acad Sci India B. doi:10.1007/s40011-012-0136-8

13. Kumar M, Philip L (2006) Enrichment and isolation of a mixed

bacterial culture for complete mineralization of endosulfan. J Envi-

ron Sci Health B 41(1):81–96. doi:10.1080/03601230500234935

14. Hussain S, Arshad M, Saleem M, Khalid (2007) A biodegradation

of a- and b-endosulfan by soil bacteria. Biodegradation

18(6):731–740. doi:10.1007/s10532-007-9102-1

15. Verma A, Ali D, Farooq M, Pant AB, Ray RS, Hans RK (2011)

Expression and inducibility of endosulfan metabolizing gene in

Rhodococcus strain isolated from earthworm gut microflora for

its application in bioremediation. Bioresour Technol 102(3):

2979–2984. doi:10.1016/j.biortech.2010.10.005

16. Martens R (1976) Degradation of [8,9-14C] endosulfan by soil

microorganisms. Appl Environ Microbiol 31(6):853–858

17. Holt JG (1994) Bergey’s manual of determinative bacteriology,

9th edn. Lippincott Williams and Wilkins, Baltimore

18. Harley JP, Prescott LM (1996) Laboratory exercises in microbi-

ology, 3rd edn. WCB McGraw-Hill Company Inc., Boston

19. Kumar R, Nongkhlaw M, Acharya C, Joshi SR (2013) Uranium

(U)-tolerant bacterial diversity from U ore deposit of Domiasiat in

North-East India and its prospective utilisation in bioremediation.

Microbes Environ 28(1):33–41. doi:10.1264/jsme2.ME12074

20. Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon

YS, Lee JH, Yi H, Won S, Chun J (2012) Introducing EzTaxon-e:

a prokaryotic 16S rRNA gene sequence database with phylotypes

that represent uncultured species. Int J Syst Evol Microbiol 62(Pt

3):716–721. doi:10.1099/ijs.0.038075-0

21. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan

PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R,

Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and

Clustal X version 2.0. Bioinformatics 23(21):2947–2948. doi:

10.1093/bioinformatics/btm404

22. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular

evolutionary genetics analysis (MEGA) software version 4.0.

Mol Biol Evol 24(8):1596–1599. doi:10.1093/molbev/msm092

23. Felsenstein J (1985) Confidence limits on phylogenies: an

approach using the bootstrap. Evolution 39(4):783–791

24. Posada D, Crandall KA (1998) MODELTEST: testing the model

of DNA substitution. Bioinformatics 14(9):817–818. doi:

10.1093/bioinformatics/14.9.817

25. Kataoka R, Takagi K (2013) Biodegradability and biodegradation

pathways of endosulfan and endosulfan sulfate. Appl Microbiol

Biotechnol 97(8):3285–3292. doi:10.1007/s00253-013-4774-4

26. Sutherland TD, Weir KM, Lacey MJ, Horne I, Russell RJ,

Oakeshott JG (2002) Enrichment of a microbial culture capable

of degrading endosulfate, the toxic metabolite of endosulfan.

J Appl Microbiol 92:541–548

27. Tolan V, Ensari Y (2006) Effect of endosulfan on growth, alpha

amylase activity and plasmids amplification in Bacillus subtilis.

Indian J Biochem Biophys 43(2):123–126

28. Khalid A, Kausar F, Arshad M, Mahmood T, Ahmed I (2012)

Accelerated decolorization of reactive azo dyes under saline

conditions by bacteria isolated from Arabian seawater sediment.

Appl Microbiol Biotechnol. doi:10.1007/s00253-012-3877-7

29. Kamei I, Takagi K, Kondo R (2011) Degradation of endosulfan

and endosulfan sulfate by white-rot fungus Trameteshirsuta.

J Wood Sci 57:317–322. doi:10.1007/s10086-011-1176-z

666 K. Bhattacharjee et al.

123

Author's personal copy

30. Rand GM, Carriger JF, Gardinali PR, Castro J (2010) Endosulfan

and its metabolite, endosulfan sulfate, in freshwater ecosystems of

South Florida: a probabilistic aquatic ecological risk assessment.

Ecotoxicology 19(5):879–900. doi:10.1007/s10646-010-0469-0

31. Arshad M, Hussain S, Saleem M (2008) Optimization of envi-

ronmental parameters for biodegradation of alpha and beta

endosulfan in soil slurry by Pseudomonas aeruginosa. J Appl

Microbiol 104(2):364–370

32. Kwon G, Kim J, Kim T, Sohn H, Koh S, Shin K, Kim D (2002)

Klebsiella pneumoniae KE-1 degrades endosulfan without for-

mation of the toxic metabolite, endosulfan sulfate. FEMS

Microbiol Lett 215(2):255–259. doi:10.1111/j.1574-6968.2002.

tb11399.x

33. Awasthi N, Ahuja R, Kumar A (2000) Factors influencing the

degradation of soil applied endosulfan isomers. Soil Biol Bio-

chem 32(11):1697–1705. doi:10.1016/S0038-0717(00)00087-0

A study on parameters optimization 667

123

Author's personal copy