Agilent 7000b

Because FPP is difficult to detect, we firstly used calf intestine alkaline phosphatase (CIAP; Catalog: CP8531-1000U; Coolaber, Beijing, China) to dephosphorylate FPP into farnesol. A 6 µL volume of CIAP Reaction Buffer, 6 µL CIAP, and 48 µL ddH₂O were mixed to 60 µL [42 (link)]. After addition of 20 µL of the above mixture, incubation was performed for 30 min. The experiment was repeated twice. Finally, reaction products were extracted and analyzed by GC–MS using an Agilent 7000B instrument (Agilent, California, USA). The products of MrBBS catalysis were also analyzed by GC–MS. Extracts from Escherichia coli BL21 (DE3) pLysS containing empty vector or deactivated enzyme were used as controls. The flow rate of nitrogen was 1.0 mL/min, the injector temperature was 250 °C, and the oven temperature was programmed to increase from 40 °C to 250 °C at 10 °C/min [4 ]. Volatile components were identified by comparing with spectra in the NIST (National Institute of Standards and Technology) database.

ST-TJ4 strain was inoculated into 250 mL Erlenmeyer flask containing 100 mL of liquid KB medium at a rate of 1% and cultured at 28 °C, 180 rpm for 60 h. To avoid the escape of VOCs, the conical flask was sealed with aluminium foil. Uninoculated KB medium was performed as the control. A 65 μm PDMS/DVB fiber tip was selected for the determination of bacterial VOCs, and the extraction tip was first aged when used for the first time [16 (link)]. The aging temperature of the extraction head selected in this experiment was 250 °C, and the time was 30 min. The cultured bacteria sample was shaken and placed in a 40 °C water bath. The needle of the SPME was inserted through the tin foil and extracted for 30 min. The fiber head was inserted into the gasification chamber of the gas chromatograph (Agilent 7000B, Santa Clara, CA, USA). The sample was analyzed at high temperature in the gasification chamber for 3 min.
GC-MS conditions: using Rtx–5 quartz capillary column; He as the carrier gas; 230 °C as the inlet temperature; 40 °C as the initial temperature, keep it for 3 min, increase temperature at 10 °C/min to 95 °C, and then raise temperature at 30 °C/min to 230 °C. Keep 230 °C for 5 min, ion source is EI source; electron energy is 70 eV; spectrum search is searched by Nist 05 and Nist 05 s library [16 (link)].

The dried extract was mixed with 50 μl methoxyamine hydrochloride (15 mg/ml in pyridine, Sigma Aldrich) and incubated at 30 °C for 90 min. After adding 100 μl N,O-Bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylsilyl chloride (Sigma Aldrich), the mixture was further incubated at 60 °C for 1 h. The derivatized sample was analysed using a GC (Agilent 7890A), coupled with a triple quadrupole mass analyser (Agilent 7000B). The ion source temperature, transfer line temperature and injector temperature were set at 230 °C, 290 °C and 250 °C, respectively. The oven temperature ramp was set as follows: initially at 60 °C for 2 min, increased to 300 °C at a rate of 7.5 °C/min and held for 6 min. The carrier gas was helium at a flow rate of 1.0 ml/min. Derivatized samples of 1 μl were injected via splitless mode, and analyzed under full scan mode. Metabolites were separated on a DB-5MS capillary column (30 m X 0.25 mm i.d., 0.25 μm film thickness) consisting of a stationary phase of 5% phenyl 95% methylpolysiloxane, and detected in electron impact ionization mode at 70 eV. Acquired mass range was 50 – 650 Da. Samples were randomized and analysed within 24 h after derivatization.