Degradation of Lambdacyhalothrin in Soil Inoculated with Bacillus cereus and Aneurinibacillus migulanus

Indiscriminate application of pesticides in paddy crop field soil to enhance paddy production could cause adverse impact on the both soil fertility and bacterial communities. The present study aimed to isolate bacteria prevalent in Lambdacyhalothrin exposed soil to determine their Lambdacyhalothrin degrading ability and assay the degraded metabolites. The observations registered in this study revealed that the dominant bacteria isolated from paddy crop field soil were Bacillus cereus and Aneurinibacillus migulanus. These bacteria were inoculated in soil spiked with Lambdacyhalothrin. After 36 hours of inocubation, the extract were analysed by GCMS. Lambdacyhalothrin degradation was accelerated by Aneurinibacillus migulanus (100 % of pesticide degraded compared to Bacillus cereus 94.09 % and control 55.2 % ). Thus Aneurinibacillus migulanus could be the used as bioagents to degrade lambdacyhalothrin.

The application of pesticides is a pivotal strategy to control pest.In Tiruchirappalli district of Tamil Nadu, India, commonly applied pesticide in paddy cultivation is Lambda cyhalothrin.The persistence of Lambdacyhalothrin and its degradative products in paddy crop field soil could deteriorate the soil quality.Hence, protocols have to be evolved to eliminate these residues from the soil.In this study, we have aimed to tap the potential of autochthonous bacteria inhabiting the paddy crop field soil to degrade lambdacyhalothrin.

Isolation of pesticide resistant bacteria from paddy crop field soil
1 gm of Lambdacyhalothrin exposed paddy crop field soil were aseptically inoculated in 100 ml of sterile minimal salt media (MSM) into cotton plugged flasks in triplicates.Conical flasks were kept under continuous shaking at room temperature for one week.Minimal salt media containing the following salts: CaCl 2 -0.002 g , MgCl 2 -0.02 g, K 2 HPO 4 -0.1 g, KH 2 PO 4 -0.1 g, NH 4 NO 3 -0.1 g and FeCl 3 trace amount in distilled water (pH 7.2 -7.4) upto 1 L were used for inoculation of soil sample.Total heterotrophic bacteria were isolated and identified following Bergeys manual of Determinative Biology (Sneath, et al., 1994).The dominant bacteria Bacillus cereus and Aneurinibacillus migulanus were selected for pesticide resistant studies.

Bacillus cereus and Aneurinibacillus migulanus
Bacillus cereus was subcultured in autoclaved nutrient broth for 48 hours at 30 ºC in a rotatory shaker.After 48 hours, 1ml (45 x 10 15 cfu/ ml) of Bacillus cereus broth was inoculated in 50 g of sterile paddy crop field soil in 250 ml of cotton plugged conical flask containing 500 ppm Lambdacyhalothrin in triplicates.20 ml of autoclaved minimal salt medium was added to maintain 60 % of humidity.Control (without Bacillus cereus ) was maintained simultaneously.Similar procedure was followed for Aneurinibacillus migulanus ( 29 X 10 15 cfu/ ml).

GC-MS analysis of degraded metabolites of Lambdacyhalothrin
After 36 hours of incubation, the samples were subjected to GC-MS analysis.The control and the Lambdacyhalothrin treated soil were extracted for GCMS analysis based on the method of Malghani et al., (2009) with minor modifications.The pesticides in the control and treatment were extracted using organic solvent extraction three times with acetone and hexane (1:1) mixture, then the extract was concentrated using rotary vaccum evaporator (Buchi R-210, Surkzer ) and cleaned up with silica gel column (1:3 cm dimeter x 243 cm length).The pesticide extract were eluted with n-hexane collected in a glass vial and subjected to gas chromatograph-Mass spectrometer (GC-MS) analysis.

Instrumental Analyses
The qualitative and quantitative determination of Lambdacyhalothrin was performed by GC-MS (45 X GC -44, Bruker) equipped with auto injector (8410).The analyses separation was performed in a 60 mm x 0.25 mm I.D x 0.25 µm film thickness BR 5 ms column (made in USA) and Helium was used as a carrier  gas at a flow rate of 1 ml / min.The column temperature was programmed as 70 ºC to 150 ºC at 10 ºC / min, to 250 ºC at 5 ºC/ min, to 280 ºC at 2 ºC / min, finally to 320 ºC at 5ºC/ min and hold for 10 minutes. 1 µl of the extract was injected into the injection port (at 280 ºC) using auto injector.The mass spectrometer was operated in scan mode and the ion source temperature was kept at 250 ºC.
The electron ionisation (EI) unit was operated at 70 ev and at an emission current of 60 µA.Full scan data was obtained in a mass range of m/ z 50-650.Scanning interval and sample rate were 0.5 and 0.28, respectively.

RESULTS
Bacteria was isolated from paddy crop field soil exposed to lambdacyhalothrin.The 16s rRNA gene sequencing was performed and identified as Bacillus cereus (Gene Bank Accession Number : KY293394) and Aneurinibacillus migulanus (Gene Bank Accession Number : KY293393).
Residual quantification analysis of Lambdacyhalothrin by gas chromatography due to bacterial activity is presented in table 1. Relative to the control (55.2 %), Bacillus cereus and Aneurinibacillus migulanus accelerated the degradation of Lambdacyhalothrin by 94.09 % and 100 %, respectively.These results indicate that Bacillus cereus and Aneurinibacillus migulanus play a vital role in accelerating the degradation of Lambdacyhalothrin.

Fig. 2 .
Fig. 2. Gas chromatogram of 500 ppm of lambdacyhalothrin degraded by Bacillus cereus at 36 th hour in soil As evinced in this study, Geeta et a l ., ( 2 0 1 4 ) h a v e d e m o n s t r a t e d t h a t bacteria accelerated the degradation of pesticides in soil (arbendazim: Bacillus sp., Exiguabacterium, Achromobacter,imidacloprid: Achromobacter;Microbacterium, Pseudomonas sp. and a and b -endosulfan :, Xanthomonas sp., Microbacterium sp., Bacillus sp.,).They have also observed dose -dependent degradation of Carbendazim by Exiguobacterium.In addition, they have reported that comparatively, consortium of bacteria enhanced the degradation of pesticides.The results of the present finding are in corroboration with the work done byAgarry et al., (2013) who have demonstrated that bacterial consortium (Proteus vulgaris, Vibrio sp., Serratia sp., and Acinetobacter sp.,) isolated from agricultural farm soil from Ladike Akintola University of Technology, Nigeria were able to grow in nutrient medium containing Dichlorvos as the only carbon source.Further, they have observed that the percentage removal of Dichlorvos pesticide from the soil was relatively higher in soil amended with NH 4 NO 3 (75%) and KH 2 PO 4 (85%), respectively.Bacillus mediated degradation of Lambdacyhalothrin observed in this study is consistent with the findings of Osama el Gialanielsaid et al., (2010) who have compared the endosulfan degradation potential of mutant

Table 1 .
GCMS result showing the biodegradation of Lambdacyhalothrin by Bacillus cereus andAneurinibacillus migulanus after 36 hours of exposure.

Table 2 .
Biodegraded metabolites of Lambdacyhalothrin in soil detected by GC-MS on inoculation with bacteria R.T -Retention Time PALANIMANICKAM & SEPPERUMAL: DEGRADATION OF LAMBDACYHALOTHRIN