2.2. Shake flask cultivations

250 mL unbaffled shake flasks were placed on an orbital shaker (Climo shaker ISF1‐X, Adolf Kühner AG, Birsfelden, Switzerland) operated at a shaking diameter of 50 mm at 37°C. OTR, carbon dioxide transfer rate (CTR), and RQ were monitored online with an in‐house build Respiration Activity Monitoring System (RAMOS) ²⁴ in duplicates. The measurement principle is explained in detail by Anderlei, et al. ²⁴ and is based on a continuously repeated cycle of actively gassed and ungassed phases. During the ungassed measurement phases, the changes of oxygen and carbon dioxide partial pressures over time are recorded in the headspace of the shake flask. One OTR and CTR value is calculated during each measurement phase, resulting in a measurement interval of 0.5 h. Commercial versions can be purchased from HiTec Zang GMBH (Herzogenrath, Germany) or Adolf Kühner AG (Birsfelden, Switzerland). Offline samples were taken from parallel cultivations in conventional shake flasks under identical cultivation conditions. For each sampling point, one flask was removed for sampling and discarded afterwards. Consequently, each dataset is derived from multiple parallel cultivations as visualized in Supporting Information Figure S1. Thereby, the time course of offline samples and the overlapping information between offline samples and online monitored respiration activity show the consistency and reproducibility of the obtained results. Pre‐cultures were cultivated at 350 rpm after inoculation of 20 mL medium per flask with 20 µL of a glycerol stock. The cells were harvested in the exponential growth phase (determined from the online measured OTR) and a master mix was inoculated to an initial optical density (OD₆₀₀) of 0.1. Each flask of the main culture was then filled from the master mix. Different filling volumes were used as specified in the respective figure legends. At the beginning of the cultivation, a shaking frequency of 350 rpm was applied to enable oxygen unlimited growth conditions. At a defined OTR of 20 mmol/L/h the shaking frequency was reduced to 100 rpm to initiate oxygen limitation.