20 ml headspace vial

Volatile compounds were analyzed by headspace solid phase microextraction gas chromatography mass spectrometry (HS-SPME GC-MS) following the method described by Pongsetkul et al. [18 ] with some modifications. Fish meat was ground using liquid nitrogen and a blender. Ground samples (5 g) were placed in a 20 mL headspace vial (Supelco, Bellefonte, PA, USA), then tightly capped with a PTFE septum and heated at 60 °C with an equilibrium time of 10 h. The SPME fiber (50/30 lm DVB/Carboxen™/PDMS StableFlex™) (Supelco, Bellefonte, PA, USA) was heated at 270 °C for 15 min before being exposed to the headspace. The 20 mL vials (Agilent Technologies, Palo Alto, CA, USA) containing the extracts were allowed to absorb into the SPME fiber at 60 °C for 1 h. The volatile compounds were then desorbed in the GC injector port for 15 min at 270 °C. After that, GC-MS analysis was performed in a HP 5890 series II gas chromatography (GC) coupled with HP 5972 mass-selective detector equipped with a splitless injector and coupled with a quadrupole mass detector (Hewlett Packard, Atlanta, GA, USA). The identified volatile compounds were presented in terms of abundance.

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).

Before first use, the fiber equipped with a manual SPME holder (both purchased from Supelco, Bellefonte, PA, USA) was conditioned according to the manufacturer’s recommendation in the GC injector, and blank analyses were conducted. Fresh individual flowers collected at random were excised carefully and sealed in a 20-mL headspace vial (Supelco). After 20 min at room temperature (22 ± 3 °C), the fiber was exposed to the headspace of the vial for sampling. Subsequently, the fiber was transferred to the injection port for GC–MS analysis (GC-MS QP2010 coupled with single quadrupole triple-axis detector, Shimadzu, Kyoto, Japan). The HS-SPME procedure was set to the optimal conditions established in the orthogonal test (Table 1) and GC–MS analysis was conducted according to the conditions in Table 6. A blank sample was used as a control. Linear retention indices (LRI) of the volatile compounds were calculated using an alkane series standard (C7-C33) (Restek, Bellefonte, PA, USA) under the same conditions.