Culture medium
Mineral salt (MS1) medium (MM PH 7.0) Contained (g/l): NH4Cl, 2.0; MgSO4.7H2O, 0.2; K2SO4, 0.5; trace elements (mg/l); FeSO4. 7H2O, 2.5; CaCl2. 6H2O, 10.0; CuSO4. 5H2O, 2.0; H3BO3, 0.06; ZnSO4. 7H2O, 20.0; MnSO4. H2O, 1.0; NiCl2. 6H2O, 0.05; Na2Mo4. 2H2O, 0.3; Sodium glutamate was used as the carbon source (20g/l). The same amino acid could serve as a nitrogen source, along with NH4Cl.
Enrichment and isolation of OP compound degrading bacteria
The soil sample of the site, where the synthesis of organophosphorus compound takes places, was collected. Bacteria were isolated by an enrichment culture technique using soil collected from Defence Research Development and establishments (DRDE), Gwalior. The enrichment of biodegrading bacteria for OP compound was carried out in mineral salt (MS1) medium. Which contain 100 ppm of different organophosphorus compound (methyl parathion, DMMP and sarin). One percent weight/volume of soil sample was added to 300 ml medium in a 1.0 L flask for the enrichment at 30 oC in a shaker at 200 rpm for a period of 7 days.
Four enrichment cycles were performed using the same experimental conditions expect the addition of soil was replaced with the enriched culture of previous cycle. Four such transfers were made and every time the enrichment population was plated on MS1 medium plates containing 100 ppm (methyl parathion, DMMP or sarin) of organophosphorus compounds and as sole carbon source and incubated at 30 o C for 24 hours. After fourth transfer a pure isolate capable of growth on different organophosphorus compound was isolated. Bacterial colonies were picked up in the in broth medium and bacteria were subjected to Gram staining. To check the degradation capability of bacterial isolates all isolates were inoculated in MS1 medium using different organophosphorus compound at 30 oC on a shaker at 200 rpm for 24 h. The medium with OP compounds and without bacterial inoculation was used as a control.
Identification of OP degrading gene
Total bacterial DNA was isolated by Qiagen tissue extraction kit (Germany) and used for amplification of 16s rRNA and opd genes. Based on the reported sequence information, primers were designed (F 1: ATG CAA ACG AGA AGG GTT GT, F 2: TCA GTG AAT GAG GCC ATC CC, F 3: TCA TGA CGC CCG GAA GGT CG, F 4: TCT CGG CAG CTC GGC AGG ATT CT and F 5: AGG GCT CAA GCT TCG GAC TCA AAA) from highly conserve region sequence to amplify opd genes. Polymerase chain reaction (PCR) was performed in thermal cycler (C1000TM, Bio-Rad, USA) in a sterile 0.2 ml thin walled PCR tube with the thermal profile; initial denaturation at 94 ºC/ 5 minutes followed by 35 cycles of denaturation at 94 ºC/1 minute, annealing at 50 ºC/1 minute and extension at 72 ºC/1 minute and final extension at 72 ºC/10 minutes.
Phosphate Determination
Concentration of inorganic phosphorus was determined spectrophotometrically by procedure based on a complex formation of phosphomolibdate and malachite green in acidic medium (Zhu et al. 2009). Reagent A was prepared by adding 1.75% ammonium heptamolibdate.4H20 in 6.3 N H2SO4 and reagent B by 0.035% Malachite green and 0.35% poly vinyl alcohol (PVA) dissolving in water at 80 oC, both the reagents are stable at room temperature. To check the concentration of inorganic phosphate 1 ml of sample was mix with 0.02 ml of reagent A and incubated for 10 min at RT after that 0.2 ml of reagent B was added. The solution was mixed again and further incubated at RT for 30 min and absorbance was measured at 610 nm with the help of spectrophotometer. Potassium phosphate in the MS1 medium was used as the standard.
Characterization of bacterial isolates
SDS- PAGE profiling
All bacterial isolates were grown to overnight in MS1 media supplemented with either methyl parathion, dimethyl methylphosphonate (DMMP) or sarin as a sole source of carbon. Cells were harvested by centrifugation at 4000×g at 4 ºC for 10 min. Cell pellet was washed with PBS. Finally the sample preparation involved denaturation of cell lysate by heating with 4X SDS sample lysis buffer for 10 min. 4X sample lysis buffer used for supernatant and 1X lysis buffer used for pellet. Gel Electrophoresis was performed in a Mini protein II Gel Apparatus (BioRad Laboratories, USA) in 10% resolving gel at a constant current of 10 mA per gel in stacking gel and at 20mA per gel in the resolving gel. The gel was stained with 0.8% coomassie blue staining solution for overnight followed by destaining with successive bath of destaining solution. The stained gels were scanned with a high resolution densitometer (GS800, BioRad Laboratories, and USA).
Py-GC/MS of bacterial isolates
Pyrolysis-GC/MS analysis of bacterial isolates was performed on a Frontier Laboratories model PY-2020iD double-shot pyrolyser. The samples were pyrolyzed for 0.5 min at 500 °C. The pyrolyser was directly coupled to the inlet port of Agilent 6890N gas chromatograph interfaced to Agilent 5973 inert mass selective detector. The Agilent HP-5MS metal capillary column (30 m x 25 mm ID) were used for analysis. Ten µg of the cultivated bacteria from the pure samples were placed on the platinum foil of the pyrolyser instrument using a sterile inoculating loop. The sample was then dried using a warm stream of air for five minutes. The pyrolyser is based on a technique in which a small deactivated stainless steel cup loaded with sample into a small sized vertical micro-furnace by gravitational free-fall with push button mechanism. This system provides precise temperature control and minimal condensation of pyrolysates in the system. Because the pyrolysation process works best with volatile compounds, the sample needs to be methylized in order to have the methyl esters of the fatty acids. The fatty acid methyl ester (FAME) is the same fatty acid with the proton in the carboxylic acid group -C-OOH change by a methyl group -C-OOCH3. This makes the fatty acid more volatile and easier to measure in the chromatogram. To achieve this, around 1 µl of tetra methyl ammonium hydroxide (TMAH) was applied to the platinum foil. The sample was dried again for five minutes and placed in the pyrolyser chamber. In the pyrolyser chamber, the sample was decomposed under heat treatment at 500 oC in an inert atmosphere of helium gas. The helium gas was used also as a carrier gas for the pyrolysed sample throughout the chromatographic column and to the mass spectrometer. The program used begins at 50 oC and holds this temperature for 2 minutes. The temperature was then increased at 10 oC/min to 250 oC. This temperature was hold for five minutes. The GC injector temperature was kept at 250 °C with the injector split ratio set to 50:1. Helium was used as a carrier gas with a constant flow rate of 1.0 ml/min. The separated pyrolyzates from the GC were transferred to mass spectrometer (MS) via a heated interface maintained at 280 °C temperature. The ion source and quadrupole mass analyzer temperatures were kept at 230 °C and 150 °C, respectively. The total running time of the program is 27 minutes. Mass spectrometer was tuned at 70 eV in EI mode with the mass range of 50–550 amu. Post-experimental data analysis (peak integration) was done by the Chemstation software (Agilent Technologies, USA).
Identification of bacterial isolates by 16S rRNA sequencing
Based on reported conserve 16S rRNA sequence forward primer (5-GAGTTTGATCCTGGCTCA-3) and reverse primer (5-CGGCTACCTTGTTACGACTT-3) were designed to amplify 16S rRNA gene from total genomic DNA of bacterial isolates. For amplification of 16S rRNA gene, thermal profile was; initial denaturation at 94 ºC/ 5 minutes followed by 35 cycles of denaturation at 94 ºC/1 min, annealing at 50 ºC/1 min and extension at 72 ºC/1 min and final extension at 72 ºC/10 min. Taxonomic characterization of these strains was defined by direct sequencing of amplicons of 16S rRNA gene.
Characterization of metabolites by GC-MS analysis
The degradation products of methyl parathion, DMMP, sarin were extracted from sample after 24 hours of incubation with bacterial culture at 30 °C. The samples were separated into two major fractions before analysis by GC/MS. The sample was first separated into solid and aqueous fractions by centrifugation (4000×g, 5 min RT). The supernatant clear solution from centrifugation was extracted with equal volume of ethyl acetate with vortexing for 15 minutes. In present study we used, an Agilent 7890A GC system coupled with 5976 C inert mass detectors was used for analysis. The GC separation was performed on a C-18 column (30 m2×50 µm×0.25 µm) with the helium carrier gas at the flow rate of 1 ml/min. The injector temperature was kept at 250 °C and detector temperature at 300 °C. The degradation product of methyl parathion, DMMP and sarin was identified by comparing retention times and MS data those with known compounds.