Bio-mineralization of Organophosphorous Insecticide-Chlorpyrifos and its Hydrolyzed product 3 , 5 , 6-Trichloro-2-Pyridinol by Staphylococcus sp . ES-2

Application of Chlorpyrifos on agricultural fields to protect crops against pests results in accumulation of it in soil and other environmental samples. The insecticide transform into 3,5,6Trichloro-2-Pyridinol (TCP) through hydrolysis in soil, which has got antimicrobial property and hence resists its degradation in natural condition. In the current findings, a bacterial isolate capable of mineralizing Chlorpyrifos without accumulation of TCP was isolated from agricultural soil by enrichment method. Based on Morphological, Biochemical Characterization and with Bergey’s Manual comparision, the isolate was identified as Staphylococcus sp. The isolate was found to metabolize chlorpyrifos completely in Mineral salt medium with chlorpyrifos as the sole carbon source. No metabolites of chlorpyrifos were detected in Liquid Chromatography-Mass Spectroscopy (LC-MS) analysis after 7 days of incubation. The novelty of the outcome of the experiment relies on Staphylococcus sp.ES-2 in complete mineralization of chlorpyrifos which can be used as a potential bioaugmenting agent in the chlorpyrifos contaminated sites.


INTrOduCTION
Large quantities of pesticides are used in agriculture throughout the world and most of them are toxic to both humans and animals.Once the pesticide is applied in agricultural field, it remains in the soil for longer periods and sometimes it gets transformed into various byproducts.These byproducts may impact on Environment Organophosphorus (OPPs) pesticides are beeing widely used in agricultural practice for more than 40 years.The toxic accumulation of OPPs is achieved by inhibiting acetylcholinesterase, an enzyme necessary for normal nerve impulse transmission.Most of the OPPs have similar general structure, containing three phosphoester linkages, and hydrolysis of one of the phosphor ester bonds dramatically reduce the toxicity of the pesticides by eliminating their acetylcholinesterase-inactivating properties 1 .
OPPs accounts 34% or world's total pesticide application for controlling pests of various crops in different countries.Application of OPPs in the fields or accidental release leads to contamination of surface and ground water.These compounds are being highly mammalian toxicity, has to be removed from the environment 2 .

Chlorpyrifos(CP) O, O-diethyl O-(3,5,6
-trichloro -2n pyridyl phosphorothioate) is a crystalline organophosphate insecticide.It was discovered by Dow chemical company in 1965.CP hydrolyses rapidly in soil into its primary metabolite 3,5,6 trichloro-2-pyridinol (TCP) 3 and this TCP has antimicrobial property, which prevents the degradation of CP by soil microorganisms 4 .The studies on the fate of TCP and its degradation in environment are very much limited.CP is the second most detected insecticide in food and water because of its broad usage in agriculture on various crops.This has raised public concern demand to remove it from the contaminated sites 5 .
Removal of CP in Environment through biotic degradation is one of the most viable option.Several researchers have focused on microbial degradation of CP [6][7][8][9][10][11][12][13][14] .Despite several papers reporting biodegradation of chlorpyrifos by microbial cultures, only few isolates have capable of degrading both chlorpyrifos and its hydrolysis product 3,5,6 trichloro-2-pyridinol.In this context, we framed objective to isolate and degrade both these compounds by enrichment technique.

Sample collection and Chemicals
The soil samples were collected from sugarcane and paddy fields of hosahalli and haniyambadi villages, Mandya Taluk, Karnataka.The collected soil samples were air dried, sieved through a 2mm mesh and stored at 4°C until further use.Analytical grade chlorpyrifos was obtained from Sigma-Aldrich Co., USA.Stock solution (100 ppm) of CP was prepared in hPLC grade Acetonitrile by weighing approximately 1mg of compound into a 10ml volumetric flask and diluting into a volume.Stock standard solutions were stored in the dark at 20°C.Working solutions were prepared as and when required 15 .

Enrichment and isolation of chlorpyrifos degrading bacterial strain
The bacterial strain was screened from soil samples by enrichment technique.For isolation, 1g of soil sample was inoculated into 250 ml Erlenmeyer flask containing 100 ml of MSM medium (Table -1) and 100 mg L -1 chlorpyrifos as sole carbon source at ph 7 and kept for incubation at 120 rpm in shaker at 37°C for 7 days.After 7 days of incubation, 1 ml of supernatant was transferred into fresh MSM medium and incubated for further 7 days.Later, 1 ml of culture aliquot was spread plated on MSM agar plates with Chlorpyrifos as carbon source.The bacterial colony showing luxury growth on agar plates was selected.For biodegradation studies, the pure culture designated as ES-2 was inoculated in MSM medium provided with 100 mg L-1 of insecticide and incubated for 7 days at 37°C.The residues from the medium were then extracted and analyzed by TLC and LC-MS.

Identification of the isolated chlorpyrifos degrading bacterial strain
The potent strain isolated was characterized Morphologically and Biochemically and compared with Bergey's manual of systematic bacteriology.

lC-MS analysis
After 7 days of incubation, 35 ml culture aliquot was taken in 50ml centrifuge tube and centrifuged at 10,000 rpm for 5 min.later the supernatant was taken in separate flask and extracted using equal volume of acetonitrile by shake flask method and the organic aqueous layer was separated.The solvent was evaporated by rotary evaporator.The residues was then dissolved in hPLC

Isolation of chlorpyrifos degrading bacteria
In the present study, selective enrichment method was used to isolate chlorpyrifos-degrading bacteria from paddy field.Eight soil samples were screened for isolating potent bacterial strain.From eight samples, three distinct strains were obtained, and among which bacterial strain designated ES-2 was chosen for analysis due to its highest tolerance to chlorpyrifos (100 mg L -1 ) as the sole carbon source on Mineral salt agar medium plates.

Identification of ES-2
The bacterial strain ES-2 was identified as gram-positive facultatively anaerobic coocci.The bacterial strain is non-motile, Catalase positive, oxidasde negative and exhibited creamish white colony morphology.According to the Bergey's Manual of Systematic Bacteriology, the bacterium comes under order Bacillales, family Staphylococcaceae and genus Staphylococcus.Biochemical test results (Table 2) show the close relatedness of strain ES-2 with genus Staphylococcus.

Biodegradation ability of Staphylococcus sp. ES-2
The biodegradation of chlorpyrifos (100 mg L -1 ) was assessed using Staphylococcus sp ES-2.Thin Layer Chromatography (TLC) followed by LC-MS was used to monitor the disappearance of chlorpyrifos and TCP.The result of TLC analyses revealed the formation of unknown metabolites.The LC-MS analysis showed that the bacteria was able to efficiently degrade chlorpyrifos without formation of TCP (based on m/z value obtained).The current findings suggest that the isolate used this compound as carbon and energy source for its growth by forming polar metabolites.In most of the research outcomes on biodegradation of CP, the microorganisms hydrolysis it into TCP, which accumulates in the culture medium or soil and avoids further degradation of the same because of its antimicrobial property [17][18][19] .. Environmental Protection Agency (EPA) of USA has found, TCP has been associated with estrogenic activity and has been listed as potential endocrine disrupting chemical.
Pseudomonas putida was reported to metabolize Chlorpyrifos after the strain immobilized with calcium alginate, the products of degradation detected by LC-MS was found to be TCP and Chlorpyrifos oxon 20 .TCP was accumulated during  the degradation process of 100 mg L -1 of Chlorpyrifos by Sphingomonas sp., which slowed down the degradation process 21 .
The Mechanism of chlorpyrifos degradation in bacteria is fairly understood and a number of degradation products such as diethylthiophosphoric acid, TCP, Chlorodihydro-2-pyridone and Maleamide semialdehyde have been identified 22 .In the present study, it was found that the bacterial isolate ES-2 was able to degrade chlorpyrifos with complete mineralization.The occurrence of unidentified peaks in chromatogram of present study may be byproducts of one of the above said products, which need further investigation.

Fig. 1 :
Fig. 1: Structure of Chlorpyrifos and its hydrolyzed metabolite 3,5,6-trichloro-2-pyridinol Bioremediation, biodegradation and bioaugmentation based techniques are gaining popularity for the purpose of ecological restoration.Potential indigenous microbial strain, isolated from the area where contamination has occurred over a years can be used as successful bioaugmenting agent to remove toxic contaminants.These indigenous microbe give duel benefit, first they detoxify the contaminant and secondly do not pose any serious threat to other native flora and fauna.Many biotic and abiotic factors significantly influence the process of biodegradation.Results from the present study

Fig. 2 :
Fig. 2: Chlorpyrifos standard obtained by lC-MS analysis and its Total Ion Chromatogram (TIC)