Refrigerated centrivap concentrator

Samples were taken from the -80°C freezer and thawed on ice. After thawing, the samples were vortexed on Vortex QL-901 (Kylin-Bell, China) for 30 s to mix uniformly. 25 μl of the mixture was taken into a centrifuge tube, then 75 μl of pre-cold methanol/acetonitrile (1:1, V/V, Thermo Fisher Scientific, USA) was added, and vortexed for 30 s to mix. The mixture was sonicated in an ice-water bath for 10 min, then incubated at -20°C for 1 h and centrifuged at 13,000 r/min under 4°C for 15 min. After centrifugation, 75 μl of the supernatant was moved into a new centrifuge tube and drained by Refrigerated CentriVap Concentrator (Labconco, USA). 30 μl of methanol/acetonitrile (1:1, V/V) was added into the centrifuge tube, which was vortexed, sonicated, and centrifuged in accordance with the above experimental conditions. Finally, 25 μl of the supernatant was taken into the lining tube of the sample bottle for detection by mass spectrometry.

Metabolites were extracted from culture supernatants using methanol. Three hundred microliters of cold methanol was added to 100 µl of supernatant in a 1.5 ml Eppendorf tube. The sample was vortexed for 1 minute, incubated on ice for 30 minutes and then centrifuged at 8000 g, 4°C for 10 minutes. The supernatant was transferred to a new tube and vacuum-evaporated using a Labconco Refrigerated Centrivap concentrator (Kansas City, MO, USA) at 4°C. The sample was dissolved in mobile phase (95∶5 acetonitrile:water) immediately before analysis.

Biofilm culture was dislodged physically from the wall of the glass bottle and mixed with the planktonic culture before harvesting. As biofilm could readily be observed at the air-medium interface of high biofilm-forming strains cultures, this ensures that both representative low and high biofilm-forming samples were obtained. The bacterial cultures were harvested and washed 3 times at 10,000 × g for 10 minutes at room temperature with BHI broth. The bacterial suspensions were adjusted to OD_{600 nm} of 1.0 in BHI broth and 0.5 ml of bacterial suspensions were used for metabolite extraction. Bacterial pellets were processed according to the modified Bligh and Dyer extraction method^{48 (link)}. In brief, 300 µl methanol-chloroform (2:1, v/v) was added to each bacterial pellet and vortexed for 1 minute followed by incubation for 1 hour at room temperature. Following which, 100 µl of chloroform and 100 µl of water were added to induce phase separation. The extract was left standing for 10 minutes at room temperature. The mixture was then centrifuged at 10,000 × g for 10 minutes at 4 °C. The upper aqueous and lower organic phases were collected and dried in Labconco Refrigerated Centrivap concentrator (Kansas City, MO, USA) at 4 °C. The dried samples were subsequently dissolved in 50 μl of acetonitrile/water (95:5, v/v) and pooled together for LC/MS analysis.