Gc ms instrument

Samples for total fatty acid analysis were taken as 5 mL of culture at the end of shake flask or bioreactor cultivations. The 5-mL culture volume was kept at −80 °C and then freeze dried. The total lyophilized culture was processed for fatty acid extraction and derivatization to methyl esters (FAMEs), using the method described before [29 (link)]. In short, 10 mg of biomass was mixed with 2 mL of solvent solution: 2.5% H₂SO₄, 97.5% methanol, 50 μg/mL of C17:0 as an internal standard, in Pyrex glass tubes (Sigma-Aldrich, Saint Louis, MI, USA). Then all samples were thoroughly mixed and incubated at 80 °C overnight to form FAMEs. FAMEs were extracted by hexane and 0.9% NaCl. The organic phase was collected and stored at −20 °C until use. FAME analysis was performed by gas chromatography on a GC-MS instrument (Shimadzu, Kyoto, Japan) equipped with a Zebron ZB-FAME capillary column (30 m × 0.25 mm × 0.20 µm). The samples (1 µL at 250 °C) were injected in splitless mode using helium (1 mL min⁻¹). The identification of fatty acids was carried out by the comparison of retention times with reference compounds (Supelco 37 Component FAME Mix, Sigma-Aldrich).

Raffinose was extracted using the protocol employed for hydrophilic compounds, and the extracts were injected into the GC–MS instrument (Shimadzu). The operating conditions were as follows: carrier gas = helium (1.00 mL/min); injection volume = 1 μL; split mode ratio = 25:1; injection temperature = 290 °C; column = Rtx-5MS (30 m × 0.25 mm, 0.25 μm; Restek); temperature program = start at 150 °C, hold for 2 min, increase at 15 °C/min to 320 °C, hold for 25 min; ion source and interface temperatures = 230 and 280 °C, respectively. Ions with m/z 361 and 217 were used for SIM. Data were processed by Labsolutions GCMSsolution software (version 4.20, Shimadzu), and calibration curves for absolute quantification were plotted in the range of 0.10–50.00 μg.