Soil sample was taken from pesticide and nonpesticide contaminated field of Chaudhary Charan Singh Haryana Agricultural University, Hisar – (Haryana). The pesticide used was purchased from Sigma or Aldrich, USA. Analytical and spectroscopic grade hexane and acetone was purchased from E. Merck, Germany. All other chemicals were of high purity grade commercially available.

Approximately 10 g of soil sample was suspended in 250 ml minimal medium supplemented with 0.35 mM cypermethrin and incubated at 30 ^o C on plateform shaker at 200 rpm. The MM consisted of Na₂HPO₄, 4 g; KH₂PO₄, 2 g; MgSO₄.7H₂O, 0.8 g; (NH₄)₂SO₄, 0.8 g; trace element solution, 1 ml ( per litre); pH 7.0. The composition of trace element solution was (per litre) Al(OH)₃, 0.10 g; SnCl₂.2H₂O, 0.5 g; KI, 0.05 g; LiCl, 0.05 g; MnSO₄.H₂O, 0.8 g; H₃BO₃, 0.50 g; ZnSO₄.7H₂O, 0.10 g; CoCl₂.6H₂O, 0.10g; NiSO₄.6H₂O, 0.10 g; BaCl₂, 0.05 g; (NH₄)₆MO₇O₂₄.4H₂O, 0.05 g. After 5 days of incubation, 5 ml culture was used to inoculate fresh 0.35 mM cypermethrin containing MM. Subsequently three rounds of enrichment were carried out in MM supplemented with higher concentration of cypermethrin 0.45 mM. Enriched medium was serially diluted and aliquots of 0.1 ml were plated on nutrient agar plates supplemented with 0.45 mM cypermethrin for the isolation of bacterial cultures. Morphologically different types of colonies were streaked for their purification on nutrient agar plates containing 0.45 mM cypermethrin.

The identification of bacterium exhibiting the activity of cypermethrin degradation was carried out by its morphological, physiological and biochemical features using the Bergey’s Manual of Systematic Bacteriology (Krieg, N. R., 1984).

Minimal medium with 0.4 mM cypermethrin was inoculated with 1% seed culture of isolated strain and incubated at 30ºC under shaking conditions (200 rpm). Growth was observed by measuring absorbance (O.D.) at 600 nm.

All isolated strains were tested for their ability to degrade cypermethrin by resting cell studies. These isolates were grown in 300 ml nutrient broth supplemented with 0.45 mM cypermethrin in shaking condition at 30 ^o C up to mid log phase. Isolates were harvested at 4 ^o C and cells were washed twice with MM. These cells were resuspended in 50 ml MM supplemented with 0.4 mM cypermethrin. Aliquots of 10 ml were taken at different time intervals of 0, 12, 24 and 48 hr. Cypermethrin was extracted from cell free supernatant by using a separating funnel with 50 ml hexane (neutral extraction). The pH of the aqueous phase was adjusted to 2.0 with 2N HCl and again extracted with 50 ml hexane: acetone (acidic extraction). The amount of cypermethrin was determined by using gas chromatography (Perkin Elmer Autosystem XL) with a flame ionization detector. The temperature of the injector and detector was 300 ^o C and oven temperature was 250 ^o C.

Soil sample used in this study was taken from the non-pesticide contaminated field. The soil was air-dried and ground to powdered form. Samples (120 g) of the autoclaved and unautoclaved soil were spiked with cypermethrin (40 ppm g ^-1 ) under aseptic conditions. One set of autoclaved and unautoclaved soil was inoculated with strain Cyp19 (10 ⁶ cells g ^-1 ), and another set of autoclaved and unautoclaved soil without inoculation was kept as control. The inoculum was thoroughly mixed in soils under sterile conditions. Soil samples were incubated at 30 ^o C with 45% water-holding capacity in the dark. In this method, 20 g soil sample was taken from both inoculated and uninoculated soils at an interval of 0, 5, 10, 20 and 30 days. Then 0.25 ml of 25% ammonia solution was added to it and mixed well with glass rod. After 30 minutes, when ammonia had evaporated, 0.5 g of activated charcoal and 0.5 g of activated cflorisil was added and mixed thoroughly. The soil was then packed in a 20 mm X 30 cm sintered glass chromatography column containing about 5 cm layer of anhydrous sodium sulphate. The columns were then eluted with 100 ml mixture of acetone: hexane (1: 4). Elutes were dried under nitrogen at 50 ^o C, and finally reconstituted in 20 µl hexane for GC analysis. The population of strain Cyp19 was also checked at different time interval by spread plate method using nutrient agar plates containing 0.45 mM cypermethrin.

Morphologically different bacterial cultures were isolated by enrichment in our laboratory. Out of the twenty bacteria isolated, only one strain Cyp19 utilized cypermethrin as a sole carbon source and energy was selected for further study. The morphological, physiological and biochemical features showed that it was a motile, gram negative, aerobic, short rod, oxidase and catalase positive. It grew on MacConkey agar as lactose fermentating microorganism and utilized citrate as the carbon source. The isolate did not produce acid from sugar such as adonitol, cellobiose, dulcitol, insulin, maltose and rhamnose. On the basis of these results the bacterium was tentatively identified as Pseudomonas sp. The growth of strain Cyp19 in MM supplemented with 0.4 mM cypermethrin is shown in Fig. 1.

To elucidate that strain Cyp19 degrades cypermethrin, resting cell study was performed. After GC analysis, it was found that the initial concentration of cypermethrin 0.4 mM at 0 hr was found to be depleted to 0.1 mM after 24 h. This concentration was further reduced to 0.001 mM after 48 h, indicating almost 100% depletion of cypermethrin as shown in Fig. 2. However, in case of heat killed resting cells no depletion of cypermethrin was observed at any point of time.

The addition of strain Cyp19 to soil resulted in a more rapid degradation of cypermethrin than by indigenous microflora. Degradation of cypermethrin was insignificant in unautoclaved (uninoculated) and autoclaved (uninoculated) soil after 30-days of incubation studies as shown in Fig. 3. Degradation of cypermethrin was significant in unautoclaved (inoculated) and autoclaved (inoculated) soil, whereas 97.5% and 95% of applied cypermethrin degraded respectively in 30-days of incubation studies. Microbial population of strain Cyp19 reduced from 10 ⁶ cells g ^-1 of soil to 10 ⁴ cells g ^-1 of soil in 30 days of incubation study. In the present work the bacterial system successfully degraded cypermethrin in autoclaved and unautoclaved soils indicating that it can survive and compete with the local microflora.

Figure 1

Fig. 1. Growth of strain Cyp19 in minimal medium containing 0.4mM cypermethrin

Figure 2

Fig. 2. Degradation of cypermethrin by strain Cyp19 by using gas chromatographic analysis.

■, Degradation of cypermethrin by heat killed strain Cyp19.

♦, Degradation of cypermethrin by live cells of strain Cyp19.

Figure 3

Fig. 3. Degradation of cypermethrin in soils inoculated with strain Cyp19 at the rate of 10 cells g .

×, autoclaved inoculated soil;

▲, unautoclaved inoculated soil;

■, autoclaved uninoculated soil;

♦,unautoclaved uninoculated soil.