2.1. Collection of Soil Sample, Pesticide and Isolation of Bacteria Samples of diazinon (amcozinon) treated soil were collected from rice cultivating fields (Khorkhori bypass, Rajshahi), which had 10 years of histories of diazinon uses. The soil samples were collected about 15 cm below the soil surface using a sterile spatula in sterile polythene bags from different sites of the rice field and were collected 10 days after the application of pesticides. The collected soil samples were ground and passed through 2 mm sieve. Collected soil samples were used as a source of inoculum for enrichment culture and isolation of bacteria capable of degrading diazinon. Commercial grade diazinon (Amcozinon 60EC) was procured from the local pesticide shop from Katakhali, Rajshahi and used for the experiment.
One gram diazinon treated soil sample was suspended to a 250 ml Erlenmayer flask containing 100 ml of minimal salts (MS) media supplemented with 2 µg/ml concentration of diazinon. Control flasks without an inoculum were also prepared to take account of any abiotic disappearance of diazinon. The primary enrichment was incubated for three days at 37oC with shaking at 120 rpm on a temperature controlled orbital shaker (RivoTEK, TC344, India). When the cultures reached adequate turbidity, they were plated on minimal salts agar containing diazinon. Well grown bacterial colonies were picked and further purified by streaking. The isolated strain (isolate A) was maintained by weekly passage in liquid mineral salts medium containing diazinon. Bacteria from soil sample grown in the MS media supplemented with diazinon was considered to be capable of degrading diazinon and was used as a source of inocula in subsequent experiments.
2.2. Characterization of Bacterial Isolate Colonial morphology (e.g., shape, size and color of the bacterial colony), Gram’s staining, motility test, viable cell counting, etc. morphological tests were conducted and bacterial morphology was observed under a light microscope (LABOMED, CXL, USA). Methyl red, catalase, macconkey, starch agar, mannitol salt agar, simmon citrate agar, TSI agar, urea agar, etc. tests were performed to characterize the isolate.
Effects of pH, temperatures, carbon sources and different salt concentrations on bacterial growth were performed to study the stability of the bacterial isolates for the biodegradation of pesticides and these experiments were conducted in an Erlenmeyer flask containing 2 µg/ml of pesticides in 100 ml minimal salt broth. After sterilization by autoclaving the flasks were cooled to room temperature and inoculated with the bacterial cultures and maintained at different temperatures (25°C, 30°C, 35°C and 40°C) for the test of temperature effects; maintained at different pH (6.5, 7, 7.5, 8, 8.5 and 9.5) for checking the pH effects; added 1g of various carbon sources (glucose, sucrose, peptone and glycerol) for examining the effects of carbon sources and maintained salt concentrations (1%, 5%, 10%, 15%, 20% and 25%) for observing the salt tolerance of the bacterial isolate and incubated at 37°C. The bacterial growth was measured by observing the optical density (OD) at 660 nm using UV– spectrophotometer (ANALYTIK JENA AG, SPOKOL 1500/1, GERMANY) after different time intervals.
Molecular identification and characterization of the isolated bacteria was performed through the following steps: extraction of chromosomal DNA, amplification of 16S rRNA gene, purification of PCR products, cycle sequencing, purification of cycle sequencing products, detection of nucleotides and sequence analysis. The genomic DNA of the isolated diazinon degrading bacterium was extracted (about 1465bp) using phenol/chloroform method (indicated by the ‘L1’ in the Figure 3). The 16S rRNA genes were amplified by PCR using 16S rDNA specific primer forward primer 27F 5′-AGAGTTTGATCMTGGCTCAG-3′and reverse primer 1492R 5′-GGTTACCTTGTTACGACTT-3′. The PCR reactions were carried out in thermal cycler (Applied Biosystem 9700, USA) using following amplification conditions: an initial denaturation step at 95°C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 1 min and the final extension at 72°C for 10 min. The PCR products were purified and were sequenced on both strands on genetic analyzer (Prism 310, USA). The sequences were then edited by bioinformatics software Chromas.
2.3. Degradation Efficacy, Cytotoxicity and Resistance Pattern of Isolated Bacteria Diazinon degradation efficiency of the isolated bacterial strain was observed in MS media supplemented with different doses of diazinon (2 µg/ml to 50 µg/ml) at optimized conditions. Cytotoxic activity of diazinon was evaluated through brine shrimp (A. salina) lethality assay (Ramesh et al., 2008). The antimicrobial susceptibility test of the isolate was performed against seven antibiotics following the Kirby-Bauer (1966) disc diffusion susceptibility test protocol. Again, the MIC (minimal inhibitory concentration) values of gentamicin and amoxicillin were determined by broth tube dilution method. Furthermore, the antagonistic effect of the isolated bacteria was tested against some pure culture strains of environmental and pathogenic bacteria.