Gc chemstation software

Concentrations of fecal SCFAs were determined by gas chromatography/mass spectrometry (GC/MS). Briefly, 600 mg of feces was homogenized with 1.2 mL of phosphate buffer (pH 7.3) and centrifuged at 4 °C at 16,000× g for 15 min. The supernatants were filtered through a 0.22 µm nylon filter (EMD Millipore, St. Louis, MO, USA). An aliquot (200 µL) of the supernatant was acidified by adding 0.1 mL of 50% (v/v) sulfuric acid. After vortexing and standing for 2 min, the organic acids were extracted by adding 0.4 mL of diethyl ether, and the supernatants were measured by GC/MS on a 7 Agilent 6890 (Agilent Technologies, Santa Clara, CA, USA) equipped with flame ionization, thermal conductivity detectors, capillary columns, and GC Chem Station software (version E.02.00.493, Agilent Technologies, JSB, Eindhoven, The Netherlands).

Fatty acid methyl esters were analyzed by GC using a HP 5890 GC System (Agilent, Santa Clara, CA, USA) equipped with a flame-ionization detector. The chromatography utilized an Omegawax capillary column (L × I.D. 30 m × 0.25 mm; Sigma-Aldrich). Fatty acids were identified by matching their GC retention time with those in a PUFA standard (Nu-Chek-Prep) and further analyzed using GC ChemStation Software (Agilent). The concentration of fatty acids was calculated using the internal standard curve in relation to the weighed-in quantity. Standard curves of the assayed fatty acids are provided in the supplements (Figure S1).

B. japonicum cells were cultured as indicated above for NO consumption experiments, except that in addition to 2% (v/v) initial O₂, the headspace of the cultures also contained 10% (v/v) acetylene in order to inhibit N₂O reductase activity. After 96 h growth, gaseous samples were taken from the headspace of cultures. N₂O was measured using an HP 4890D gas chromatography instrument equipped with an electron capture detector (ECD). The column was packed with Porapak Q 80/100 MESH and the carrier gas was N₂ at a flow rate of 23 ml·min⁻¹. The injector, column and detector temperatures were 125, 60 and 375°C respectively. The samples were injected manually using a Hamilton® Gastight syringe. Peaks corresponding to N₂O were integrated using GC ChemStation Software (Agilent Technologies^©) and the concentrations of N₂O in each sample were calculated using N₂O standards (Air Liquid).

B. diazoefficiens cells were cultured in serum tubes under microoxic conditions as indicated above. Cultures were incubated at 30 °C and 170 rpm for 7 days. N₂O was measured using a Hewlett Packard HP-4890D gas chromatography instrument equipped with an electron capture detector (ECD) (Hewlett Packard, San Jose, CA, USA), as essentially described by Torres et al. (2014) [72 (link)]. The column was packed with Porapak Q 80/100 mesh. N₂ was used as the carrier gas circulating at a flow rate of 23 mL min⁻¹. The injector, column and detector temperatures were 125, 60 and 375 °C, respectively. Gas samples were taken from the headspace of the cultures after 1, 2, 3, 6 and 7 days and injected manually using luer-lock gas-tight syringes BD Microlance^TM 3. Peaks corresponding to N₂O were integrated using GC ChemStation Software (Agilent Technologies, Santa Clara, CA, USA) and the values obtained were used to calculate the N₂O concentration in each sample through extrapolation from a standard curve, performed using 2% (v/v) N₂O standard (Air Liquid, Paris, France) and including the following gas volumes: 0, 0.2, 0.4, 0.6, 0.8 and 1 mL. Total N₂O concentration was determined taking into account N₂O in headspace and dissolved N₂O, applying the Bunsen solubility coefficient (47.2% at 30 °C).

E. meliloti cells were incubated in MMN with an initial O₂ concentration of 2% in the headspace or anoxically. After 18 or 36 h of incubation, 500-μl gaseous aliquots were taken from the culture headspaces to determine the N₂O level. In anoxic cultures (filled tubes), headspace was created by transferring 10 ml of liquid culture into a 20-ml headspace vial (Supelco®). Gas–liquid phase equilibration was performed by incubating the vials for 2 h at 30°C and at 185 rpm. To stop cell growth, 200 μl of 1 mg · ml^-1 HgCl₂ was added to each vial. The N₂O production in liquid cultures was corrected using the dissolved N₂O Bunsen solubility coefficient (47.2% at 30°C). Then, N₂O was measured with a gas chromatograph type HP 4890D equipped with an electron capture detector (ECD). The column was packed with Porapak Q 80/100 MESH (6 ft), and the carrier gas was N₂ at a flow rate of 23 ml/min. The injector, column and detector temperatures were 125, 60 and 375°C, respectively. The N₂O peaks were integrated using GC ChemStation Software (Agilent Technologies^© 1990–2003). The samples were injected manually through a Hamilton® Gastight syringe. The concentrations of N₂O in each sample were calculated from pure nitrous oxide standards (Air Liquid, France).