Factor four vf 23 ms column

For the extraction of PHA, plant tissue from in vitro cultures was lyophilized, weighed and transferred to a glass tube. The dried material was extracted twice with warm methanol (65 °C, 1 h) to remove lipids, while PHA, which is insoluble in methanol, remained associated with the tissue. After centrifugation and removal of the residual methanol, the material was suspended in 1 mL of chloroform, to which 1 mL of methanol containing 15% sulfuric acid and 50 μg/mL of commercial C17:0 (Sigma) as an internal standard were added. The mixture was heated at 100 °C for 2.5 h and immediately cooled down on ice. 1 mL of 0.1% NaCl was added to the tube, and the mixture was vortexed vigorously and centrifuged at 3000g for 5 min. The chloroform phase, containing the methyl-esters of 3-hydroxy acids, was analyzed by a Varian 3900 gas chromatograph equipped with a flame ionization detector and a Varian Factor Four vf-23 ms column with a bleed specification at 260 °C of 3 pA (30 m, 0.25 mm, 0.25 μm). For the extraction of total lipids, exactly the same procedure was followed, omitting the methanol extraction phase.

For dry cell weight (DCW) determination, the culture was washed two times with distilled water and lyophilized in a preweighed tube. The differences in mass corresponded to the mg of cells found in the culture. Lipids were extracted from 10 to 20 mg of dried cells and converted into FA methyl esters (FAMEs) using the procedure described by Browse et al. (1986 (link)) with cyclohexane instead of hexane. Briefly, dried biomass (10–20 mg) was mixed with 1 mL of 2.5% (v/v) sulfuric acid in methanol, which contained 25 μg of commercial dodecanoic acid (Sigma‐Aldrich) as an internal standard. Tubes were vortexed and incubated at 80°C for 120 min to form FAMEs. After transesterification, 1 mL of cyclohexane and 0.5 mL of distilled water were added. The FAME‐containing cyclohexane phase was analyzed by gas chromatography (GC) using a Varian 430 instrument (Varian Inc.) equipped with a flame ionization detector and a Varian FactorFour vf‐23 ms column, where the bleed specification at 260°C was 3 pA (30 m, 0.25 mm, 0.25 μm). The FAMEs were identified by comparison with commercial standards (FAME32; Supelco) and quantified using dodecanoic acid as an internal standard. For each condition, we used three biological replicates and calculated average and standard deviation values. The p‐values were calculated using a Welch t‐test.

Lipids were extracted from 10 to 20 mg of freeze-dried cells and converted into FA methyl esters (FAMEs) using the procedure described by Browse et al. (1986 (link)). The FAMEs were then analyzed using gas chromatography (GC), which was carried out with a Varian 3900 instrument equipped with a flame ionization detector and a Varian FactorFour vf-23ms column, where the bleed specification at 260°C is 3 pA (30 m, 0.25 mm, 0.25 μm). The FAMEs were identified via comparisons with commercial standards (FAME32, Supelco) and quantified using the internal standard method, which involves the addition of 100 μg of commercial dodecanoic acid (Sigma-Aldrich). Commercial odd-chain FAs (Odd Carbon Straight Chains Kit containing 9 FAs, OC9, Supelco) were converted into their FAMEs using the same method employed with the yeast samples. They were then identified using GC and compared with the odd-chain FAs from the yeast samples.
To determine dry cell weight (DCW), 2 mL of the culture was taken from the flasks, washed, and lysophilized in a pre-weighed tube. The differences in mass corresponded to the mg of cells found in 2 mL of culture.