Db 1701

Nitrogen-fixing ability of all isolate was tested by using ARA previously described by Hardy et al. (1968 (link)). All bacterial isolate was inoculated in a 25 mL flask containing 10 mL semi solid JNFb medium (Table S1) and bacteria were grown at 30 ± 2°C for 3 days. Five Percent air from the tubes was replaced by acetylene through a syringe, incubated for 12 h, 0.5 mL gas was withdrawn from the tube, and ethylene formation was analyzed through a gas chromatograph (GC-17A, Shimadzu, Kyoto, Japan) with a flame ionization detector and a column filled with DB-1701 (Agilent, Santa Clara, USA).

The milled lignins (1.7 mg) were pyrolysed in a EGA/Py-3030D micro-furnace pyrolyzer (Frontier Laboratories Ldt.), connected to an Agilent 7820A GC system equipped with a DB-1701 fused-silica capillary column (60 m × 0.25 mm i.d. × 0.25 μm film thickness), and to a Agilent 5975 Mass detector (EI at 70 eV). The pyrolysis was performed at 500°C (1 min). The oven temperature was programmed from 45°C (4 min) to 280°C at a heating rate of 4°C min⁻¹, and held at 280°C during 10 min. The GC/MS interface was kept at 280°C and the injector at 250°C. The carrier gas was Helium with a flow of 2 mL min⁻¹. The compounds were identified using the literature (Faix et al., 1990 (link); Ralph and Hatfield, 1991 (link)) and the Wiley and NIST libraries. Peak molar areas were calculated for each compound (by dividing the peak area by the respective molecular weights), the summed molar areas were normalized and the data expressed as percentage.

For the quantitation of the analytes identified by GC-MS, the experiments were performed using a GC system (Thermo Trace GC Ultra) equipped with a Flame Ionisation Detector (FID). Separation of the compounds was conducted on a column DB-1701 30 m × 0.25 mm (0.25 µm film thickness; Agilent Technologies, Basel, Switzerland), using helium as the carrier gas. The injection volume was 1 µL, and the flow rate 2.5 mL/min. The injector temperature was 200 °C with a split ratio of 1:10 and the FID temperature was 250 °C. The oven temperature was programmed as follows: the column was held initially at 40 °C for 5 min, then increased to 190 °C at 7 °C/min, then increased to 280 °C at 50 °C/min. For the GC-FID quantitation, standards and blanks were prepared. For blank, 7 mL of acetonitrile +70 µL of stock solution of injection standard at 1 g/L, then prepare as described above.
For calibration, three different concentrations of each analyte were prepared by diluting 30, 70 and 140 µL of the standard stock solution (1 g/L) in 7 mL of acetonitrile and 70 µL of stock solution of injection standard (1 g/L), then prepared as described above. The determined calculations showed a calibration range from 0.1 to 92 mg/kg and a coefficient of determination (R²) ≥ 0.999.

A 100-µL aliquot from the HPLC fractions was spotted onto filter paper in a 35 mm petri dish in a rectangular plate containing half-strength MS medium. The volatilized compound (atmospheric pressure at 23 °C) was trapped during overnight incubation with 2 discs of monolithic MonoTrap DCC18 (GL Science, Tokyo, Japan), which were placed at a distance of 2 cm from the 35 mm petri dish. The trapped compounds were extracted with methylene chloride, dried with anhydrous Na₂SO₄, and concentrated to 10 µL. We used 1 µL for the GC–MS analysis via a 6890 A with 5973 network mass selective detector (Agilent Technologies) equipped with a GC column DB1701 (model 122-0732) (Agilent Technologies, Santa Clara, CA, USA) under split mode (split ratio 1:10), with the oven temperature program starting from 10 min holding at 40 °C, followed by a gradient temperature increase to 220 °C in 30 min at a rate of 5 °C/min. The identity of the compounds was evaluated from retention time and fragmentation patterns using the Wiley 275 library. A GC column Cyclodex-B (model 112-2532) (Agilent Technologies) was used for the chiral GC–MS analysis.

OPPs in the samples were quantified by an Agilent 7890 Gas Chromatography (Agilent Technologies, Inc, Santa Clara, CA, USA) with a capillary column (DB-1701, 30 m × 0.25 mm × 0.25 mm) and a flame photometric detector. Flow rate of the carrier gas (nitrogen gas) was 3 mL/min. The temperatures of injection, column and detector were set at 200, 90 and 250 °C, respectively. The purified sample (1.0 μL) was detected under programmed temperature gradient from initial temperature of 90 °C for 1 min, heating from 100 to 260 °C at 10 °C/min, holding for 3 min at 260 °C. Quantification of the pesticides was carried out by comparison of their peak areas with a calibrated standard curve with multitude-point calibration.
The relative decreasing levels of OPPs was calculated as the decreased concentrations of OPPs at the last week compared with the beginning ones.