Fed-batch fermentation

To test the feasibility of producing 2-FL at high titers, fed-batch fermentation was performed. To prepare a seed culture, the engineered yeast, D452-2_LFF was cultivated overnight in a 50-mL flask containing 10 mL of the modified Verduyn medium with 20 g/L glucose in 50 mM KHP buffer (pH 5.5) at 30 °C and 250 rpm. Cells from the seed culture were harvested by centrifugation at 1789×g for 5 min at 4 °C. For the fed-batch fermentation, the cells obtained by the centrifugation were inoculated into a 125-mL baffled flask containing 25 mL of the modified Verduyn medium containing 20 g/L glucose, 2 g/L fucose, and 2 g/L lactose in 50 mM KHP buffer (pH 5.5). The initial cell density was set at OD₆₀₀ = 1, and the fermentation was performed at 30 °C and 250 rpm. The fed-batch fermentation process was divided into the glucose batch phase and the ethanol feeding phase. In the glucose batch phase, 20 g/L glucose was added as the initial carbon source. After the initially added glucose and the produced ethanol from glucose during cultivation were depleted, 20 g/L ethanol was added into the flask as a carbon source. When the added ethanol was depleted, additional 20 g/L ethanol was fed into the flask until 120 h.