2.3. Analytical methods
Four OD units of liquid yeast cell was harvested and subsequently was
pelleted by centrifugation at 14,000 rpm for 5min. Water was completely
withdrawn and 500 uL 0.5 M sodium methoxide (sodium hydroxide dissolved
in pure methanol) was used to resuspend the cell pellet. The mixture was
kept at room temperature with shaking for 2 hours at 1,200 rpm with a
high-duty vortex to allow complete saponification of lipids and
extraction of squalene. Then 400 uL hexane was added to extract
squalene. The mixture was vortexed at room temperature for 10 min and
hexane phase was directly injected to gas chromatography-FID (GC-FID)
for squalene analysis. Gas chromatography–flame ionization detector
(GC-FID) system (Agilent 7820A) equipped with HP-5 column (30 m × 320 μm
× 0.25 μm) was used to detect squalene, using helium as the carrier gas
with a linear velocity of 2 ml/min. The column temperature profile was
175 ℃ for 3 min, 20 ℃/min ramping to 200 ℃ and holding for 3 min, and
then 20 ℃/min ramping to 260 ℃ and holding for 4 min.
The cell growth was monitored by measuring the optical density at 600 nm
(OD600) with a UV-vis spectrophotometer that could also be converted to
dry cell weight (DCW) according to the calibration curve DCW : OD600 =
0.33:1 (g/L). The fermentation broth was centrifuged at 14,000 rpm for 5
min and the supernatant was used for analyzing the concentration of
glucose, mannitol, and acetic acid by HPLC with a refractive index
detector and Supelcogel TM Carbohydrate column. The
column was eluted with 10 mM H2SO4 at a
column temperature of 50 ℃, a refractive index detector temperature of
50 ℃, and a flow rate of 0.4 mL/min.