Supelcogel column

Sugar concentrations were analyzed via HPLC (Thermo Scientific, Waltham, MA, USA) on a Supelcogel column (Supelco Inc., Bellefonte, Pennsylvania, USA) at a constant flow of 0.5 mL/min at 30 °C. The mobile phase consisted of 0.1% H₃PO₄ and sugars were detected with a Shodex RI-101 refractive index detector (DataApex, Prague, Czech Republic). Analysis of the chromatograms was performed using Chromeleon Software (Dionex, Sunnyvale, California, USA).

Samples were taken at the beginning and end of the batch and the non-induced fed-batch. During induction, sampling was performed every 30 min to analyze DCW and OD₆₀₀. In addition, every 10 min supernatant for sugar analysis was collected via an in-line ceramic 0.2 μm filtration probe (IBA, Heiligenstadt, Germany). DCW was determined by centrifuging (4500 g, 4 °C, 10 min) 1 mL cultivation broth, washing the obtained cell pellet with a 0.9% NaCl solution and subsequent drying at 105 °C for 72 h. Optical density at 600 nm (OD₆₀₀) was determined using a Genesys 20 photometer (Thermo Scientific,Waltham, MA, USA). For staying within the linear range of the photometer (OD₆₀₀ 0.1–0.8) samples were diluted with 0.9% NaCl solution. A calibration correlation OD₆₀₀ to DCW was established. Sugar concentrations were analysed via HPLC (Thermo Scientific, Waltham, MA, USA) on a Supelcogel column (Supelco Inc., Bellefonte, PA, USA) with 0.1% H₃PO₄ as eluent at a constant flow of 0.5 ml/min. The method for sugar analysis lasted 15 min. Analysis of the chromatograms was performed using Chromeleon Software (Dionex, Sunnyvale, CA, USA). In order to examine cell-death during bioreactor cultivations, fluorescence-activated cell sorting (FACS) was conducted via Cube 8 (Sysmex Partec, Görlitz, Germany) according to Langemann et al.37 (link).