2.2. Shake Flask Fermentations

Fermentations for biocolorant production were carried out in 500 mL shake flasks with baffles, with a working volume of 100 mL. Effect of initial sugar concentration was evaluated at 30, 60 and 90 g/L glucose supplemented with 2 g/L yeast extract and 50 mM sodium citrate buffer, pH 5. The effect of C:N ratio was evaluated at different glucose to yeast extract concentrations. Glucose was 30 g/L and yeast extract concentrations of 2, 3, 4, 6, 12, 24 and 48 g/L of yeast extract corresponding to C:N ratios of 55:1, 36:1, 27:1, 18:1, 9:1, 5:1 and 2:1, respectively. The combination of the sugar and yeast extract concentrations were selected in order to evaluate a wide range of yeast extract addition in the fermentation medium. Sugar solution, yeast extract solution and citrate buffer solution were sterilized separately to avoid Maillard reactions, at 121 °C for 20 min. Moreover, sugar solution was 200 g/L in order to avoid caramelization of sugars in the autoclave. Inoculum size was 1×10⁷ spores/mL. Agitation and temperature were controlled at 180 rpm and 30 °C in a rotary shaker incubator.