Halophilic bacterium JAS4 in biomineralisation of endosulfan and its metabolites isolated from Gossypium herbaceum rhizosphere soil

Journal of the Taiwan Institute of Chemical Engineers xxx (2014) xxx–xxx

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JTICE-835; No. of Pages 9

Halophilic bacterium JAS4 in biomineralisation of endosulfan and itsmetabolites isolated from Gossypium herbaceum rhizosphere soil

Sivagnanam Silambarasan, Jayanthi Abraham *

Microbial Biotechnology Laboratory, School of Biosciences and Technology, VIT University, Vellore 632014, Tamil Nadu, India

A R T I C L E I N F O

Article history:

Received 9 October 2013

Received in revised form 16 January 2014

Accepted 19 January 2014

Available online xxx

Keywords:

Biodegradation

Endosulfan

Formulations

Halophilic bacterium JAS4

Kinetic parameters

A B S T R A C T

Bacterial strain, Halophilic bacterium JAS4, capable of degrading endosulfan and its metabolites was

isolated from Gossypium herbaceum rhizosphere soil by enrichment technique, considering the fact that

the microorganism had adapted to exposure in pesticide after having been in contact with pesticide

contaminated soil. The JAS4 isolate had remarkable potential to degrade 1000 mg/l of endosulfan by

catabolic activity and transform them into simpler compounds. The biodegradation experiments

showed that a,b-endosulfan and endosulfan sulfate in the aqueous medium was degraded by JAS4

strain which was characterized by the rate constant (k) of 0.017 d�1, 0.003 d�1, and 1.219 d�1,

respectively. The period within which the initial pesticide concentration was reduced by 50% (DT50)

was 40.7 d (a-endosulfan), 231 d (b-endosulfan) and 0.5 d (endosulfan sulfate). Inoculation of sterile

soil with Halophilic bacterium JAS4 and nutrients enhanced the disappearance rate of pesticide, and

DT50 for a,b-endosulfan and endosulfan sulfate was 0.01 d, 346.5 d and 1.07 d, respectively. In the

present study powder formulations were prepared by two methods; they are less expensive and

handling is also easy.

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jou r nal h o mep age: w ww.els evier . co m/lo c ate / j t i c e

1. Introduction

Over the past decades, the extensive use of pesticide to enhancethe agricultural production has increased tremendously all aroundthe world. There are about numerous of commercial formulationavailable as pesticides, herbicides and fungicides. Though theenvironmental implication of pesticide usage is well documented,pesticide usage is still in practice, there are areas where pesticideshave been still employed to a larger extent thus resulting inwidespread distribution of pesticide. Endosulfan is one of thecontroversial pesticide which has been extensively used inagriculture and is banned from 2011 in India due to the toxiceffects incurred by it in Kasaragod district of Kerala, India. One ofthe world’s worst pesticide disaster endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,3,4-benzo-dioxathiepin-3-oxide, CAS No. 115-29-7) is an organochlorinepesticide, a derivative of hexachlorocyclopentadiene and is amixture of a and b endosulfan at a ratio of 70:30 [1]. It is usedagainst aphids, cabbage worms, white flies, leafhoppers and foundto be neurotoxic to both insects and mammals, which acts as an

* Corresponding author. Tel.: +91 9843580709; fax: +91 416 2243092.

E-mail address: [email protected] (J. Abraham).

Please cite this article in press as: Silambarasan S, Abraham J. Halo

metabolites isolated from Gossypium herbaceum rhizosphere soilj.jtice.2014.01.013

1876-1070/$ – see front matter � 2014 Taiwan Institute of Chemical Engineers. Publis

http://dx.doi.org/10.1016/j.jtice.2014.01.013

endocrine disruptor. Endosulfan is hydrophobic, and persists insoil for more than a year [2]. However, bioremediation of thepesticide by bacteria and fungi are gaining interest as they areecofriendly, economical when compared to other treatmentmethods such as incineration and landfill. The microorganismsespecially bacteria and fungi possessing unique capability todegrade xenobiotic contaminants have been isolated from differ-ent part of the world. Much research has been conducted onpesticide degrading microorganisms. Endosulfan degrading abilitywas recorded in organisms like Klebsiella oxytoca [3], Bacillus

stearothermophilus [4], Mortierella sp. strains W8 and Cm1-45 [5],Trichoderma harzianum [6], Bacillus sp., [7], Aspergillus niger [8],Stenotrophomonas sp. LD-6 [9], Achromobacter xylosoxidans CS5[10], Pseudomonas putida and Pseudomonas aeruginosa [11],Aspergillus sydoni [12] and Phanerochaete chrysosporium [13].

In the present study, JAS4 bacterial strain was isolated usingenrichment method which was able to degrade a-endosulfan, b-endosulfan and major toxic metabolite endosulfan sulfate. To thebest of our knowledge this is the first work reporting on endosulfandegradation by Halophilic bacterium. As bioremediation studieswill be incomplete without application part, in the present workemphasis on employing this efficient strain in degradation hasbeen achieved by preparing appropriate formulation with costeffective fly ash, soil and molasses.

philic bacterium JAS4 in biomineralisation of endosulfan and its. J Taiwan Inst Chem Eng (2014), http://dx.doi.org/10.1016/

hed by Elsevier B.V. All rights reserved.


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2. Materials and methods

2.1. Sample collection

The soil sample used in this study was collected from Gossypium

herbaceum field located in Vellore district, Tamil Nadu, India(12.938 N 79.138 E). The field was previously exposed to endosul-fan in the summer months over a period of 5 years. Topsoil wascollected from 15 cm deep, air dried, and stored for furtheranalysis. The chemical properties of the soil were analyzed fromShri A.M.M Murugappa Chettiar Research Centre, Taramani,Chennai, India (Table 1).

2.2. Chemicals

Analytical standards of a-endosulfan (99% purity), b-endosul-fan (99% purity) and endosulfan sulfate (99% purity) werepurchased from Sigma Aldrich (St. Louis, MO, USA). Technicalgrade endosulfan of 35% emulsified preparation was used in thisstudy was obtained from Hindustan insecticides limited, Kerala,India. Chromatographic grade acetonitrile and ethyl acetate werepurchased from SD Fine Chem Limited (India). All other reagentsused in this study were of analytical grade.

2.3. Enrichment and isolation of bacterial strain

Bacterial isolation was carried out in minimal salt medium(MSM). 20 g of soil sample was inoculated into 50 ml of MSM (g/l:Na2HPO4, 5.8; KH2PO4, 3.0; NaCl, 0.5; NH4Cl, 1; MgSO4, 0.25;distilled H2O, 1000 ml and pH 6.8–7.0) and spiked with 35 mg/lendosulfan. The culture was incubated at room temperature at100 rpm for 7 d. After a week, 5 ml of the sample was transferredinto a freshly prepared MSM containing the same amount ofendosulfan. Three successive transfers were carried out in freshMSM containing endosulfan as the only carbon source. In the lasttransfer, 10-fold dilutions of cultures were prepared and 100 mlof sample was spread on nutrient agar plate containing 35 mg/lendosulfan. Isolated colonies were streaked onto nutrient agarplates containing endosulfan and purified by repeated streaking.Three morphologically different bacterial strains were isolatedand these isolates were initially screened for endosulfantolerance using minimum inhibitory concentration (MIC) meth-od. Out of three isolates, strain JAS4 was selected for furtherstudy due to its ability to tolerate higher concentration ofendosulfan (2100 mg/l).

Table 1Chemical properties of the soil used in the experiment.

Properties Soil sample

pH 7.84

EC 0.46

Organic carbon 0.46 kg/acre

Organic carbon 0.51%

Nitrogen 137.07 kg/acre

Phosphorus 8.16 kg/acre

Potassium 101.14 kg/acre

Calcium 314.19 mg/kg

Magnesium 155.4 mg/kg

Sodium 103.51 mg/kg

Iron 7.44 mg/kg

Manganese 6.72 mg/kg

Copper 1.01 mg/kg

Zinc 0.34 mg/kg

Sulfate 18.56 mg/kg

Humus 82.95 kg/acre

Total minerals 239.37 kg/acre



2.4. Identification of highly efficient bacterial strain

Strain JAS4 was identified according to the Bergey’s Manual ofSystematic Bacteriology [14] and sequence analysis of 16S rRNAgene was performed. Pure culture of strain JAS4 was grown innutrient broth for 24 h, and genomic DNA was extracted using theAMpurE Bacterial gDNA Mini Spin kit (Amnion Biosciences Pvt.Ltd., Bangalore, India). For genetic affiliation analysis, 16S rRNAgene fragments were amplified by polymerase chain reaction (PCR)using the universal forward primer 50-CWG RCC TAN CAC ATG SAAGTC-30 and reverse primer 50-GRC GGW GTG TAC NAG GC-30. PCRreaction mix of 50 ml final volume contained: 50 ng sample gDNA,100 ng forward primer, 100 ng reverse primer, 2 ml dNTP’s mixture(10 mM), 5 ml 10� Taq polymerase buffer, 3 U Taq polymeraseenzyme and PCR grade water to make up the volume. AmplifiedPCR product was sequenced by using ABI3730xl genetic analyzer(Amnion Biosciences Pvt. Ltd., Bangalore, India). The sequencingresult was submitted to the GenBank National Center forBiotechnology Information (NCBI) database.

2.5. Growth kinetics of strain JAS4 in different media

Growth of strain JAS4 in different media, MSM and nutrientbroth with endosulfan were studied in terms of optical density. Toinvestigate the growth of strain JAS4 with endosulfan as solesource of carbon, 100 ml of strain JAS4 was inoculated into 20 ml ofthe MSM and 20 ml of nutrient broth medium with endosulfan(1000 mg/l) in 100 ml Erlenmeyer flask and the control flasks wasmaintained without endosulfan. The culture was incubated at30 � 2 8C on a rotary shaker at 120 rpm. The bacterial growth wasregularly monitored by spectrophotometer at 600 nm.

2.6. Studies on degradation of endosulfan in MSM

The degradation studies were performed in 250 ml Erlenmeyerflasks containing 100 ml of sterile MSM supplemented with1000 mg/l of endosulfan. One milliliter of bacterial suspensionwas transferred to the MSM to give a final concentration ofapproximately 3 � 106 cells ml�1. All the flasks were incubated at30 � 2 8C on a rotary shaker at 120 rpm. Samples of MSM wereperiodically removed aseptically to determine endosulfan and itsmajor metabolite concentration by high performance liquid chroma-tography (HPLC).

2.7. Studies on degradation of endosulfan in soil

The two soil microcosm treatments were carried out withisolated JAS4 strains: (1) addition of endosulfan, JAS4 and amendedwith nutrients (carbon, nitrogen and phosphorus) and (2) additionof endousulfan and JAS4 devoid of nutrients (control). Before usingthe soil for degradation studies, it was sterilized three fold byautoclaving for 30 min at 121 8C. 30 ml of solution containing JAS4strain, nutrients and 1000 mg/kg of endosulfan were added to250 ml Erlenmeyer flask which contained 100 g of sterilized soil.The sources of carbon, nitrogen and phosphorus were glucose,ammonium sulphate and dipotassium hydrogen phosphate,respectively. The amounts of carbon, nitrogen and phosphoruswere calculated by the relationship C/N/P (100:10:1) [15,16]. Allthe flasks were incubated at 30 8C and soil samples were analyzedat one day interval regularly for the determination of endosulfanand its major metabolites.

2.8. Analytical methods

Endosulfan and its major metabolites in the aqueous mediumwas extracted by addition of equal volume of acetonitrile in a




Table 2Morphological and biochemical characteristics of bacterial strain JAS4.

Characteristics JAS4

Colony morphology Small colonies and nonpigmented

Gram stain �ve bacilli

Motility �Indole test �Methyl red �Voges-Proskauer �Citrate utilization �Catalase +

Oxidase +

Urease +

Nitrate Reduction +

H2S production �Glucose +

Sucrose �Lactose �Arabinose �Sorbitol �Mannitol �Rhamnose �Phenylalanine deamination �

S. Silambarasan, J. Abraham / Journal of the Taiwan Institute of Chemical Engineers xxx (2014) xxx–xxx 3

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whole flask and thoroughly mixed for 1 h with rotary shaker andthen centrifuged. And for extraction from soil microcosm, 10 g ofsoil sample was extracted with 50 ml of acetonitrile, which waskept in rotary shaker for 1 h at 200 rpm followed by centrifugation.The supernatant was decanted into a glass beaker and the organicsolvent was concentrated in a water bath at 35 8C [10]. Theextracted samples were analyzed by HPLC (Waters 1525 binaryHPLC pump, Milford, USA) on a Symmetry C18 column (Waters5 mm, 4.6 mm � 150 mm). The isocratic mobile phase composed amixture of acetonitrile:water (65:35, V:V), which was pumpedthrough the column at a flow rate of 1 ml/min, duration of cycle20 min, detector wavelength 214 nm at an injection volume 25 ml[17].

Infrared (IR) spectra of the parent compound (endosulfan) andsample after degradation of endosulfan with fungal strains wererecorded in the frequency range of 4000–500 cm�1 with a Fouriertransform infrared (FTIR) spectrophotometer (8400 Shimadzu,Japan, with Hyper IR-1.7 software for Windows) with a helium-neon laser lamp as a source of IR radiation. Pressed pellets wereprepared by grinding the extracted samples with potassiumbromide in a mortar with 1:100 ratio and immediately analyzed inthe region of 4000–400 cm�1 at a resolution of 4 cm�1.

2.9. Kinetic studies

Degradation of pesticide in aqueous medium and soil has beenapplied to the various kinetic models such as zero order, first order,pseudo first order, second order and pseudo second order todetermine the rate constant (k). The times in which the pesticideconcentration in MSM or soil was reduced by 50% (DT50 values)which was calculated from the linear equation obtained from theregression between Ct and Co (zero order model), ln(Ct/Co) (firstorder model), lnCt (pseudo first order model), 1/C (second ordermodel), t/Ct (pseudo second order model) of the chemical data andtime. Kinetic model equations were described by:

(i) Ct � Co = kt (zero order model)(ii) Ct/Co = e�kt (first order model)

(iii) ln Ct = �kt + lnCo (pseudo first order)(iv) 1/C = kt + 1/Co (second order kinetic)(v) t/Ct = t/Ce + 1/kCe2 (pseudo second order)

Whereas Co, Ce is the amount of pesticide in MSM or soil at timezero and Ct, C, Ct is the amount of pesticide in MSM or soil at time t.k and t are the rate constant (d�1) and degradation time in days,respectively.

2.10. Formulations

Liquid culture of bacterial strain was prepared in nutrient broth.The saw dust and molasses were procured from the local market.Halophilic bacterium JAS4 was mass produced by using theagricultural waste material such as Saw dust–soil–5% molassesin the ratio of 15:5:1 and Saw dust–soil–nutrients (carbon,nitrogen and phosphorus) in the ratio of 15:5:1. The carriermaterial such as fly ash was used for immobilization of bacterialstrain. The carrier material was improved by incorporation of soiland finally a mixture of fly ash, soil and 5% molasses in the ratio of15:3:1 plus 75 mg cycloheximide/kg material whereas in the othermethod a mixture of fly ash, soil and nutrients C/N/P (100:10:1) inthe ratio of 15:3:1 plus 75 mg cycloheximide/kg material wereused as a carrier to immobilize the bacterial strain. The mixturewas filled in heat resistant polybags and autoclaved at 121 8C for20 min. Thereafter, 1 part stock culture was added to the bagscontaining carriers and shaken for uniform distribution. To test theshelf life of the formulations with regard to CFU load and



contamination by other microorganisms, the packets were sealedand stored at room temperature for 12 weeks. After incubation,CFU load was determined using dilution plate method.

3. Results and discussion

3.1. Isolation and characterization of strain JAS4

The endosulfan degrading bacterium JAS4 was isolated from G.

herbaceum field soil sample through selective enrichment tech-nique which was able to use endosulfan as the sole carbon andenergy source. The JAS4 strain was found to be Gram-negative rodshaped bacterium and the detailed biochemical analysis ispresented in Table 2. The molecular characterization of 16S rRNAgene sequence of strain JAS4 illustrated high similarity with thosespecies of genus Halophilic and it was named as Halophilic

bacterium JAS4. Phylogenetic analysis of 16S rRNA gene alsoclustered strain JAS4 within the clade of Halophilic bacterium in thephylogenetic tree (Fig. 1). The GenBank accession number for the16S rRNA sequence of strain JAS4 is KC509575.

To our knowledge, this is the first report on biodegradation ofendosulfan and its metabolite endosulfan sulfate by Halophilic

bacterium. Siddique et al. [18] have reported the isolation ofendosulfan degrading microorganisms through enrichment cul-ture technique by providing the carbon or sulfur source for theirgrowth. Singh and Singh [17] reported the selective enrichmenttechnique in sulfur free medium with endosulfan as source ofsulfur, those microorganisms which were able to release the sulfitegroup from endosulfan and use it as a sulfur source for growth.However, in the present study endosulfan was used as sole carbonand energy source in MSM for the isolation of Halophilic bacteriumJAS4 and interesting results pertaining to degradation has beenobtained.

3.2. Minimum inhibitory concentration of endosulfan for JAS4 strain

MIC was determined with various concentration of endosulfan.The supplemented concentration was either too low to thrive on ortoo high to be survived for the isolated strain JAS4. The Halophilic

bacterium JAS4 showed maximum growth at 1000 mg/l concen-tration in endosulfan-MSM and tolerates upto 2100 mg/l ofendosulfan.




Fig. 1. Phylogenetic tree based on the 16S rRNA gene sequences of strain JAS4 and related species. The GenBank accession number for each microorganism used in the analysis

is shown after the species name.


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3.3. Growth kinetics of Halophilic bacterium JAS4

The growth of Halophilic bacterium JAS4 was monitored byrecording the OD value of the culture at 600 nm following differenttime intervals. The JAS4 strain was grown in MSM and nutrientbroth with and without 1000 mg/l of endosulfan for 10 days. Thegrowth of JAS4 was found to be absent in the MSM withoutendosulfan, but in the MSM spiked with endosulfan there wasactive growth of JAS4 strain (Fig. 2a). The JAS4 strain acclimatizedin the medium quickly and utilized endosulfan as a carbon source.A gradual increase in growth was observed upto 10 days and thisincubation period was sufficient for JAS4 strain to thrive onexposure to stress. The growth of JAS4 strain in the nutrient brothwith addition of endosulfan was monitored; there was a significantincrease in growth of Halophilic bacterium JAS4 at 1000 mg/l ofendosulfan supplemented with nutreint broth when compared tonutrient broth without addition of endosulfan (Fig. 2b). This couldbe due to the availability of additional carbon source for the growthof JAS4 and subsequent degradation of endosulfan in the medium.

3.4. Degradation of endosulfan and its metabolites by strain JAS4 in

liquid culture and soil

Several bacterial and fungal species which degrade endosulfanisomers under aerobic conditions have been isolated andcharacterized. Endosulfan sulfate, endosulfan diol, endosulfanether, endosulfan hydroxyether, endosulfan lactone, endosulfanmonoaldehyde and endosulfan dialdehyde have been reported asthe major metabolites formed during the microbial degradation ofendosulfan isomers [13,19–21]. However, in the present study,degradation of endosulfan by JAS4 strain with production ofisomers viz. a-endosulfan, b-endosulfan and its metaboliteendosulfan sulfate were obtained in both MSM as well as soilamended with nutrients. The degradation experiment was



conducted in soil with addition and without addition of nutrientsfor 10 d. JAS4 strain failed to degrade endosulfan in the absence ofnutrients. Pino and Penuela [16] reported, the addition of nutrientsto the soil is necessary to support the develpment and mainte-nance of the microbial population responsible for the degradation.Therefore in the present investigation, degradation of endosulfanisomers and the toxic metabolite endosulfan sulfate was recordedin soil ammended with nutrients.

According to previous report, there are two types of endosulfandegradation by soil microorganisms. Endosulfan transformed intoendosulfan sulfate through oxidation or into endosulfan diol byhydrolysis. The major biotransformation products of endosulfandiol and endosulfan sulfate are more toxic and more persistentthan the parent compound [22]. Therefore, it can be inferred in thepresent investigation that Halophilic baterium converted endosul-fan into endosulfan sulfate by oxidative pathway.

Tables 3 and 4 depict the values such as degradation rateconstants (k) and DT50 of zero order, first order, pseudo first order,second order, pseudo second order equations for degradation ofpesticides in MSM and soil with addition of nutrients, respectively.The degradation dynamics of a,b-endosulfan and endosulfansulfate from MSM showed a very good compliance with pseudosecond order (Fig. 3a), second order (Fig. 3b) and first order model(Fig. 3c), respectively. The a,b-endosulfan and endosulfan sulfatein the aqueous medium was degraded by JAS4 strain which wascharacterized by the rate constant (k) of 0.017 d�1, 0.003 d�1, and1.219 d�1, respectively. The period within which the initialpesticide concentration was reduced by 50% (DT50) was 40.7 d(a-endosulfan), 231 d (b-endosulfan) and 0.5 d (endosulfansulfate). The two endosulfan isomers (a,b-endosulfan) andmetabolite endosulfan sulfate appeared in the soil microcosmstudy was degraded by JAS4 strain and found to fit well in thedegradation kinetics of zero order (Fig. 4a), second order (Fig. 4b)and first order model (Fig. 4c), respectively. The rate constant for




Fig. 2. (a) Halophilic bacterium JAS4 growth in MSM and (b) Halophilic bacterium JAS4 growth in nutrient broth. Each value is the mean of three replicates with error bars

representing the standard deviation of the mean.


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a,b-endosulfan and endosulfan sulfate was 44.61 d�1, 0.002 d�1

and 0.646 d�1; DT50 was 0.01 d, 346.5 d and 1.07 d, respectively.Previously Kwon et al. [23] reported 65 mg/l of a-endosulfan and28 mg/l of b-endosulfan with degradation rate of 6.17 mg/l/d and2.55 mg/l/d, respectively. Fusarium ventricosum degraded 70 mg/land 30 mg/l of a- and b-endosulfan with degradation rate of14.2 mg/l/d and 6.6 mg/l/d, respectively. Pandoraea sp. degraded70 mg/l and 30 mg/l of a- and b-endosulfan with degradation rateof 8.19 mg/l/d and 3.18 mg/l/d, respectively [18]. Chlorococcum sp.degraded a-endosulfan and endosulfan sulfate 4.24 mg/l and8.44 mg/l with degradation rate of 0.13 mg/l/d and 0.12 mg/l/d,respectively. Scenedesmus sp. degraded a-endosulfan and endo-sulfan sulfate 4.24 mg/l and 8.44 mg/l with degradation rate of0.14 mg/l/d and 0.05 mg/l/d, respectively [24]. Awasthi et al. [25]described the degradation of 388 mg/l of a-endosulfan and386 mg/l of b-endosulfan by Bacillus sp. with a rate of degradationwas 29.7 mg/l/d and 27.7 mg/l/d. Bhalerao and Puranik [26]



reported A. niger could degraded 350 mg/l of endosulfan. To ourknowledge Halophilic bacterium JAS4 is the first reported strainthat can tolerate or degrade endosulfan 1000 mg/l.

The FTIR analysis showed the various structural changes ofendosulfan. Comparison of FTIR spectrum of control (Fig. 5a) withextracted metabolites after complete degradation (Fig. 5b) clearlyindicated that the biodegradation of endosulfan and its metabo-lites. The infrared spectrum of endosulfan degraded sample in theaqueous medium showed bands at 3441, 1629 and 1402 cm�1

were represented N–H stretch, C55O stretch and C–N stretch amidegroups, respectively. Peak at 1082 cm�1 represent the C–N bandand the acid dimer band was seen at 989 cm�1. The overall changesin the FTIR spectrum and formation of acid dimer in the finaldegraded samples confirms the degradation of endosulfan. Thisresult was well in accordance with the observation of Guerin [27]who reported that major metabolite of endosulfan sulfate whichwas converted to non-recoverable acidic forms.




Table 3Kinetic parameters for the degradation of endosulfan and its metabolites in MSM by

Halophilic bacterium JAS4.

Kinetic model Parameters Treatments

MSM + alpha

endosulfan +

JAS4

MSM + beta

endosulfan +

JAS4

MSM + endosulfan

sulfate + JAS4

Zero order k (d�1) 8.134 60.57 129.6

DT50 0.0852 0.0114 0.0053

R2 0.755 0.864 0.924

First order k (d�1) 0.843 0.439 1.219

DT50 0.8222 1.5789 0.5686

R2 0.784 0.912 0.943

Pseudo

first order

k (d�1) 0.786 0.379 1.219

DT50 0.8818 1.8288 0.5686

R2 0.620 0.934 0.942

Second order k (d�1) 0.003 0.003 0.025

DT50 231.0 231.0 27.72

R2 0.657 0.940 0.855

Pseudo

second order

k (d�1) 0.017 0.021 0.102

DT50 40.77 33.00 6.7955

R2 0.981 0.910 0.812

Table 4Kinetic parameters for the degradation of endosulfan and its metabolites in soil by

Halophilic bacterium JAS4.

Kinetic model Parameters Treatments

Soil + N + alpha

endosulfan +

JAS4

Soil + N + beta

endosulfan +

JAS4

Soil + N +

endosulfan

sulfate + JAS4

Zero order k (d�1) 44.61 90.54 101.5

DT50 0.015 0.007 0.006

R2 0.951 0.781 0.879

First order k (d�1) 0.167 0.360 0.646

DT50 4.150 1.925 1.072

R2 0.889 0.963 0.961

Pseudo

first order

k (d�1) 0.167 0.360 0.646

DT50 4.150 1.925 1.072

R2 0.889 0.963 0.961

Second order k (d�1) 0.000 0.002 0.011

DT50 – 346.57 63.01

R2 0.778 0.977 0.590

Pseudo

second order

k (d�1) 0.006 0.015 0.081

DT50 115.5 46.20 8.557

R2 0.870 0.908 0.558


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3.5. Shelf life and contamination in the formulation

Shelf life of the two bacterial formulations and microbialcontamination in the formulations were tested at room temperature

Fig. 3. Degradation kinetics of a,b-endosulfan and endosulfan sulfate in MSM showed a

model (c), respectively.



for 12 weeks (Fig. 6). The bacterial strain was not only remainedviable during the storage but also multiplied maintaining a higherCFU load/g formulation. Contamination by other microorganismswas below the detection limit. A microorganism other than the

very good compliance with pseudo second order (a), second order (b) and first order




Fig. 4. Degradation kinetics of zero order (a), second order (b), first order model (c) for a,b-endosulfan and endosulfan sulfate in the soil, respectively.

Fig. 5. (a) FTIR spectra of endosulfan at standard condition, (b) FTIR spectra for the degradation of endosulfan and its metabolites in aqueous medium by Halophilic bacterium

JAS4.


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Please cite this article in press as: Silambarasan S, Abraham J. Halophilic bacterium JAS4 in biomineralisation of endosulfan and itsmetabolites isolated from Gossypium herbaceum rhizosphere soil. J Taiwan Inst Chem Eng (2014), http://dx.doi.org/10.1016/j.jtice.2014.01.013



Fig. 6. (a) The CFU of Halophilic bacterium JAS4 in a mixture of fly ash, soil and 5% molasses and (b) CFU of Halophilic bacterium JAS4 in a mixture of fly ash, soil and nutrients.


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bacterial strain JAS4 was rarely recorded; contamination whendetected was 1 or 2 colonies in all petri plates of a treatments. Theliquid fermentation technology is costly and thereby the resultingformulations become expensive. In the present study, a method isdescribed that used low cost solid materials: sawdust, soil, fly ash,molasses and nutrients to achieve greater multiplication andsurvival of the bacterial strain JAS4. However, the use of formulatedJAS4 strain for removal of endosulfan from the contaminated sitesrequires an understanding of ecological requirements.

4. Conclusions

In this study, JAS4 bacterial strain obtained from the soilcontaminated with endosulfan was capable of utilizing endosulfanas the only carbon source. Based on 16S rRNA gene sequence, theywere identified as Halophilic bacterium. To our knowledge, this isthe first report on endosulfan degrading bacteria from the genusHalophilic. As indicated by the kinetic constants, they showeddifferent capabilities for the degradation of endosulfan and itsmetabolites. This ability of Halophilic bacterium JAS4 makes it asuitable strain for bioremediation of endosulfan polluted environ-ments.



Acknowledgements

We are grateful to the DST (Department of Science andTechnology, Govt of India, New Delhi) for financial support(research grant, sanction no. DST/TSG/NTS/2009/67).

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