2.2. Production of α-Humulene and α-Bisabolene and the Analytical Method

Two-phase flask cultivation consisting of the NMS medium as the aqueous phase to cultivate methanotrophs and 20% (v/v) dodecane as the organic overlay to extract α-humulene and α-bisabolene produced from the engineered strains in situ was performed in all of the experiments as described in our previous report [19]. Briefly, precultures were grown in 10 mL NMS medium and then inoculated into 500-mL baffled flasks containing 40 mL fresh media to achieve OD₆₀₀ of 0.1 and 10 mL (20% v/v) dodecane. Sampling was conducted after 96-h cultivation by centrifugation of 50 mL liquid culture at 3220 g for 10 min, and then the upper dodecane layer was collected for α-humulene and α-bisabolene quantification. An Agilent 5977B 5977E GC/MS system (Santa Clara, CA, USA) was used to analyze and quantify α-humulene and α-bisabolene according to previous protocols [23,24]. For quantification of α-humulene and α-bisabolene, standard curves were made from analytical standards dissolved in dodecane which was purchased from Sigma-Aldrich (St. Louis, MO, USA) and Alfa Aesar (Ward Hill, MA, USA). Due to the presence of other bisabolene isomers, the contribution of α-bisabolene to the molarity of 27.52 ± 0.27% in the commercial standard was calculated previously [24].