Extraction and quantification of geraniol and geranate in
organic phase
Two hundred millilitres of cell culture (including organic layer) were
collected and transferred to a 1.7 mL centrifuge tube (Axygen) followed
by centrifugation at 10,000 g for 10 min. Two microliters of the
hexadecane phase was taken and diluted with 98 µL ethyl acetate (EA).
One microliter of diluted sample was analyzed by gas chromatography-mass
spectrometry (GC/MS, 5977B, Agilent Technologies) to quantify geraniol
and geranate in the hexadecane phase. HP-5MS capillary column (30 m ×
0.25 mm, 0.25 µm film thickness, Agilent Technologies) was used, with
helium as the carrier gas. The following oven temperature program was
carried out: 50 °C for 1 min, 50–100 °C at a rate of 5 °C/min, 100 °C
for 1 min, 100–200 °C at a rate of 50 °C/min, 200 °C for 1 min,
200–300 °C at a rate of 50 °C/min and 300 °C for 1 min. The reported
geraniol and geranate titer is the sum of the products in the hexadecane
phase and aqueous phase when two phases were used.
Extraction and quantification of isopentenols and geraniol
in aqueous phase Two hundred millilitres of cell culture (including
organic layer) were collected and transferred to a 1.7 mL centrifuge
tube (Axygen) followed by centrifugation at 10,000 g for 10 min. One
hundred microliters of supernatant was mixed with one hundred
microliters of EA and the mixture was vortexed for 30 min. The solution
was then centrifuged at 10,000 g for 10 min. The EA phase (1 μL) was
analyzed by GC/MS to quantify geraniol and isopentenols in the aqueous
phase. The same GC/MS method was used as described above.