Clarus 500

Analyses were carried out with a Clarus 500 Perkin Elmer apparatus equipped with two flame ionization detectors (FID), and two fused-silica capillary columns (50 m, 0.22 mm i.d., film thickness 0.25 μm), BP-1 (polydimethylsiloxane), and BP-20 (polyethylene glycol). The oven temperature was programmed from 60 °C to 220 °C at 2 °C/min and then held isothermal at 220 °C for 20 min. The injector temperature was 250 °C. The detector temperature was 250 °C. The carrier gas was hydrogen (1.0 mL/min). The split was 1/60 and the injected volume was 0.5 μL. The relative contents of the oil constituents were expressed as a percentage obtained by peak-area normalization without using correction factors.

The headspace analysis by SPME of samples were carried out using a Clarus 500 model Perkin Elmer (Waltham, MA, USA) gas chromatograph coupled with a mass spectrometer and equipped with a FID (flame ionization detector) [19 (link),29 (link)]. The capillary column used for the separation of compounds, was a Varian Factor Four VF-1. The operative chromatographic and spectrometric conditions were as follows: the oven GC temperature program was: isothermal at 60 °C for 5 min, then ramped to 220 °C at a rate of 6 °C min⁻¹, and finally isothermal at 220 °C for 20 min. The carrier gas was He at flow rate of 1.0 mL min−1 in constant mode. The mass spectra were obtained in the electron impact mode (EI), at 70 eV, in scan mode in the range 35–450 m/z. For the identification of compounds, the matching between their mass spectra with those stored in the Nist 02 mass spectra library database, was performed. Further, the linear retention indices (LRIs), were calculated using a series of alkane standards (C₈–C₂₅n-alkanes) and compared with those available in the literature. Relative amounts of compounds, expressed as percentage, were calculated in relation to the total area of the chromatogram by normalizing the peak area without the use of an internal standard and any factor correction. All analyses were carried out in triplicate.

The sunflower oil was characterized by chemical analysis of its oxidative stability, such as the acidity index (mg KOH/g) Iodine (g/100 g oil) and peroxide (mequiv. O₂/kg) according to standard methods [30] . These analyses provide information about the identity and quality of the oil after processing and marketing. The fatty acid profile was determined as previously described [30] , [31] (link), [32] , [33] employing a Perkin Elmer Clarus 500 gas chromatograph (Norwalk, CT) with an automatic injector and flame ionization detector. Conditions: A 50 m × 0.25 mm WCOT fused silica column (CAPILLARY CP-Sil 88, CP6173, Agilent Technologies, CA, USA), with the injector at 270 °C and a flame ionization detector at 310 °C. Initial oven temperature was 140 °C for 2 min and was increased in 2 °C increments for 60 s to 235 °C, held at 235 °C for 10 min, and finally raised to 270 °C. Hydrogen was used as the carrier gas at a flow of 30 ml per min. Individual fatty acids were identified by comparing their retention times with those of purified standards (fatty acid esters). The peak area was calculated using the chromatographic integrator and chromatography software and expressed as a relative percentage of each fatty acid in relation to the total fatty acids.

The hydrogen and oxygen production potential of A. cylindrica cells was determined as described previously (Leino et al. 2014 (link)). Cell samples cultivated either in ambient air, or in the 100 % CO₂ atmosphere in 100 mbar pressure for 7 days were harvested by pelleting, washed twice with BG-11₀ (BG-medium without combined nitrogen), and adjusted to 5 mg chlorophyll a per ml concentration in BG-11₀ medium. Five milliliter samples of these cell suspensions were taken into air tight 20 ml glass vials. The vials were thoroughly flushed with argon to create anaerobic condition in the head space of the vials. The vials were tightly sealed with rubber septa, supplemented with 10 % CO₂ when needed, and incubated overnight with constant shaking and illumination (150 μmol photons m⁻² s⁻¹) at 25 °C. The following day, 150 μl of air from the head space in each vial was drawn with a gas-tight syringe (Hamilton Co.) and plunged into a gas chromatograph (Perkin Elmer Clarus 500, a Molecular Sieve 5 A, 60/80 Mesh column) to detect and quantify the gases produced, as described earlier (Leino et al. 2012 (link), 2014 (link); Raksajit et al. 2012 ).

Data on potential N₂O production rates (Graf et al. 2016 ) were incorporated as an indication of availability of N₂O. Briefly, rates were determined using 10 g of soil and 1.5 g of root samples placed in 147 and 32 ml flasks, respectively. The soil samples were incubated as a slurry with 20 ml distilled water and a final concentration of 3 mM KNO3, 1.5 mM succinate, 1 mM glucose, and 3 mM acetate at 25°C under anoxic conditions (nitrogen gas in the headspace) during 3.5 h. The roots were incubated with 6 ml water during 7 h, but otherwise under the same conditions. Gas samples from the headspace were taken every 30 min and N₂O concentrations were determined using a gas chromatograph (Clarus-500, Elite-Q PLOT phase capillary column, Perkin-Elmer). Only end-point concentrations of N₂O were above detection level for the root samples.