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.