2.3. Fermentations
The five-cycle repeated batch fermentations using the suspended and TSC-immobilized cultures of S. cerevisiae M30 were carried out in duplicate using blackstrap molasses as the main substrate for ethanol fermentation. The experiment was initiated by transferring the suspended or immobilized cultures into 250 mL of culture medium containing 22–24% (w/v) of initial reducing sugar and 0.5 g/L (NH4)2SO4 in a 500 mL Erlenmeyer flask. The flasks were shaken in the incubator at 150 rpm at 33 °C. The duration of each batch was 48 h. The experiment was monitored by removing 5 mL samples every 8 h for cell, sugar and ethanol analysis.
Continuous fermentations were carried out in a 1 L (Ø = 5.7 cm; height = 43.4 cm) packed-bed reactor containing an immobilized cell bed (Fig. 2). The temperature of the system was controlled at 32 ± 1 °C by passing 28 °C cooling water inside the reactor jacket. The initial sugar concentrations of 220 and 240 g/L were continuously fed into the bottom of the reactor at dilution rates at 0.11, 0.16, 0.20 and 0.30 h−1. Five milliliter samples were collected every 8 h. All samples were frozen before sugar, ethanol, and cell concentration analysis to analyze them simultaneously.