Bio one cellstar cellreactor tubes

IMS0408, IMS0556, IMS0558, IMS0559, IMS0617, IMX1824, IMX1825, and IMX1826 were grown in triplicate during 72 h in vented 50 mL Bio-One Cellstar Cellreactor tubes (Sigma-Aldrich) on 20 mL YPD at 30°C and 200 RPM until stationary phase. For each sample, the biomass was resuspended by vigorous vortexing and 1 mL was sampled immediately after vortexing from right underneath the meniscus. After 60 s of stationary incubation, another sample was taken by the same procedure. Biomass sedimentation was quantified as the ratio of the OD₆₆₀ values of the two samples.

Microaerobic cultivation was performed in 250 mL airlock-capped Neubor infusion bottles (38 mm neck, Dijkstra, Lelystad, Netherlands) containing 200 mL 3-fold diluted wort supplemented with 0.4 mL L^-1 Pluronic antifoam (Sigma-Aldrich). Bottle caps were equipped with a 0.5 mm x 16 mm Microlance needle (BD Biosciences) sealed with cotton to prevent pressure build-up. Sampling was performed aseptically with 3.5 mL syringes using a 0.8 mm x 50 mm Microlance needle (BD Biosciences). Microaerobic cultures were inoculated at an OD₆₆₀ of 0.1 from stationary-phase precultures in 50 mL Bio-One Cellstar Cellreactor tubes (Sigma-Aldrich) containing 30 mL of the same medium, grown for 4 days at 12°C. Bottles were incubated at 12°C and shaken at 200 RPM in a New Brunswick Innova43/43R shaker. At regular intervals, 3.5 mL samples were collected in 24 deep-well plates (EnzyScreen BV, Heemstede, Netherlands) using a LiHa liquid handler (Tecan, Männedorf, Switzerland) to measure OD₆₆₀ and external metabolites. 30 μL of each sample was diluted 5 fold in demineralized water in a 96 well plate and OD₆₆₀ was measured with a Magellan Infinite 200 PRO spectrophotometer (Tecan, Männedorf, Switzerland). From the remaining sample, 150 μL was vacuum filter sterilized using 0.2 μm Multiscreen filter plates (Merck, Darmstadt, Germany) for HPLC measurements.