Four different types of soil samples (A, B, C and D) were collected at depth of 10 - 12 cm from rhizoplane of different plants growing around the tannery solid waste of district Kasur Punjab, Pakistan, where pesticides have been used for many years. Collection and transportation of soil samples were done aseptically in labeled plastic bags for further processing in the laboratory.

Enrichment culture techniques were used for the isolation of bifenthrin degrading bacteria from different soil samples using Bushnell-Haas Broth (BHB) [14] [15] . The LB media containing trypton, 10; Yeast extract, 5; NaCl, 5; and agar, 15 (g∙L⁻¹) with pH adjusted to 7.0 was used for the primary cultivation and to obtain discrete colonies. A 20 g of soil sample was added in 100 ml of BHB medium with 24 hours shaking at 25˚C aerobically. After 24 hours of shaking at 200 rpm, solid particles were allowed to settle down for 1 hour. Supernatant (1 ml) was taken from the source flask and mix with 9 ml of BHB media in 50 ml conical flask. Each flask was spiked with 100 µg∙ml⁻¹ bifenthrin and was incubated at 30˚C aerobically for two weeks for the completion of first round of enrichment techniques. After two weeks of incubation 0.1 ml of culture was transferred to 10 ml of fresh BHB media containing 150 µg∙ml⁻¹ of bifenthrin and further incubated for two weeks for the second round of enrichment techniques [16] [17] .

Pure culture of single strain was obtained from second round of enrichment; bacterial culture was centrifuged for 20 minutes at 3500 rpm. The supernatant was discarded and pellet was resuspended in 500 µl BHB media. 50 µl suspensions was taken and spread on plates of BHB media with bifenthrin by adding 2% agar. The plates were incubated at 30˚C till the discrete colonies were appeared. Twelve different strains of bacteria were isolated from four soil samples. The colonies of these strains were streaked on BHB with bifenthrin 100 µg∙ml⁻¹ and the strains that showed growth at this concentration were re-streaked on higher concentration of bifenthrin 200 µg∙ml⁻¹. Maximum growth showing strains (B-B₁, B-B₂) was further streaked on BHB at concentration of 250 µg∙ml⁻¹.

Morphological and biochemical characterization was performed as mentioned by [18] . Microbact Gram-nega- tive 24E system kit (Oxoid, Wade Road, UK) was used for the identification of Gram-negative bacteria. Bacterial suspensions for the Microbact 24E tests were prepared using isolates pre-grown on agar plates. Colonies were suspended in 0.85% sterile normal saline. The suspension was added to wells and change in colour of each well was taken immediately after 24 and 48 hours incubation at 37˚C. Physiological characterization as effects of different antibiotics discs (Chloramphenicol, Ampicillin, Fusidic acid, Carbenicillin, Linomycin and Clarithromycin) (Oxide, Wade Road, UK) as per Clinical Laboratory Standard Institute (CLSI) guidelines and heavy metals solutions (CuSO₄, MnSO₄, ZnSO₄, NiCl₂, CoSO₄, Na₂SO₄, K₂Cr2O₇) with concentration 100 µg∙ml⁻¹ and 300 µg∙ml⁻¹ were checked by incubating plates of L-agar supplemented with antibiotic discs and salt solution at 30˚C for 24 - 48 hours.

Bacterial growth curve was obtained with LB-broth and BHB media along with bifenthrin inoculated with bacteria, incubated at 30˚C on rotatory shaker at 150 rpm. The optical density (O.D) was taken at 600 nm with intervals of 0, 2, 4, 6, 8, 10, 12, 14 and 15 hours using spectrophotometer. Physical growth factors as temperature, pH, Inoculums volume, minimum inhibitory concentration (MIC) were studied by growing strains on different temperatures (25˚C, 30˚C, 37˚C, 42˚C, 56˚C and 57˚C), pH (5, 6, 7, 8, 9, 10), Inoculums volume (125 µl, 250 µl, 500 µl), MIC (50 µg∙ml⁻¹, 100 µg∙ml⁻¹, 150 µg∙ml⁻¹, 200 µg∙ml⁻¹, 250 µg∙ml⁻¹) respectively.

Growth experiments on bacteria were carried out by bifenthrin as sole carbon source in 50 ml BHB media with 100 µg∙ml⁻¹ bifenthrin in 100 ml Erlenmeyer flasks. The BHB media was aseptically inoculated with seed suspension and incubated for one week at 30˚C with shaking at 160 rpm on orbital shaker OS-752 (Optima Japan). Each treatment was set with control samples in which no bifenthrin was added. Samples were withdrawn periodically from cultures to examine the growth by recording the O. D values at 600 nm using spectrophotometer.

Bifenthrin residues were extracted by mixing ethyl acetate in sample culture (1:1 by volume) in 50 ml conical flask and flask was kept on shaker at 160 rpm for 1hour. The organic phase was carefully separated and passed through anhydrous sodium sulphate column (6 cm) to remove water contents. The organic phase was allowed to elute drop wise by gravity. The column was made in pasture pipette stopped with glass wool. The organic solvent (ethyl acetate) was evaporated on rotary evaporator. The dried sample was dissolved in methanol and then applied to TLC plate.

Pre-coated silica gel plates (silica gel 60 F254 0.25 mm thicknesses, 20 × 20 cm, Merck Ltd., Germany) were used for TLC of bifenthrin [13] . TLC plates were spotted with 5 µl sample volume at 1cm apart with micropipette with same volume of standard bifenthrin in lane 1 for comparison of RF values. The plates were dried and chromatogram was developed in pre-saturated tank with Benzene: Ethyl acetate (6:1 by volume) as solvent system. After developing the plates, the solvent front was immediately marked and extra solvent was evaporated in fume hood. The plates were kept under U.V at 245 nm for 20 minutes. Three spots were clearly visible upon exposure to UV. The spots were marked and RF values were calculated.

Two isolates B-B₁ Bacillus sp. and B-B₂ Xanthomonas sp. that were able to grow with bifenthrin as sole source of carbon were identified (Figure 1 and Figure 2) (Table 1 and Table 2).

No growth (−), Less (+), Good (++), Excellent (+++).

Bacillus sp. showed resistance to almost all the antibiotics and heavy metals whereas Xanthomonas sp. were sensitive to FD and CLR and ZnSO₄ and K₂CrO₇ salts at 300 μg∙ml⁻¹ (Table 3 and Table 4).

The optimum conditions for bifenthrin utilization by Bacillus sp. B-B₁ were at 25˚C, pH 7, with 500 μL inoculum and MIC 150 μg∙ml⁻¹, whereas for Xanthomonas strain B-B₂ were at 30˚C, pH 7 with inoculum 500 μL and MIC 50 μg∙ml⁻¹. These conditions were determined by taking mean O.D at 600 nm in triplicates (Figure 3).

It was observed that there was increase in cell biomass in both media up to 12 hours (O.D_600nm = 2) and after 12 hours, cells entered into decline phase (Figure 4).

Comparative growth response of two strains Xanthomonas sp. B-B₂ and Bacillus sp. B-B₁ in presence of two different concentrations of bifenthrin (100 µg∙ml⁻¹, 250 µg∙ml⁻¹) is shown (Figure 5(a) and Figure 5(b)) along with control (without bifenthrin). The O.D were 1.115 to 1.50 in experimental and no growth appeared in control flask. The increase in O.D might be due to utilization of bifenthrin by bacteria as carbon and energy source. Degradation of pesticide was also determined by TLC. The RF value obtained for standard was 0.87 while the RF value of B-B₁ spot was 0.91 and for B-B2, it was 0.90 in lane No. 2 and 3 (Table 5). The intensity of color of B-B₂ was lighter as compared to B-B1 that represented greater rate of degradation in B-B₂ as com- pared to B-B1. The RF values of all the tested samples are in close agreement with that of standard one (Figure 6).