P139: Pseudo-continuous operation: an effective way to improve ethanol tolerance of Pichia stipitis in hexose/pentose fermentation

Many studies have indicated that ethanol tolerance of yeast strains can be enhanced by cell immobilization due to the enhanced hydration layer stability resulted from attaching cells to the carrier. However, the fact that the cells maintain their enhanced ethanol tolerance after being removed from the carrier, suggests that the ethanol tolerance enhancement associated with cell immobilization may be caused by adaptation. Therefore, it is expected that adaptation can and perhaps may significantly improve the ethanol tolerance of P. stipitis.

In this work, we propose to ferment different sugars (glucose and xylose) using P. stipitis under a pseudo-continuous operation, characterized by continuous nutrient feeding and continuous cell-free broth withdrawal. We hypothesize that pseudo-continuous fermentation can significantly improve P. stipitis’ ethanol tolerance. In order to validate our hypothesis, we performed fermentation experiments using a modified New Brunswick Bioflo110 fermentor. During the experiments, P. stipitis CBS 6054 has been adapted to 20~23 g/L ethanol by pseudo-continuous fermentation with glucose or xylose as carbon source. After adaptation, the ethanol tolerance of P. stipitis CBS 6054 improved significantly. The cell viability under 2h 60 g/L ethanol shock increased by 8 folds on glucose and 7 folds on xylose. The influence of ethanol to plasma membrane permeability and ATPase activity also confirmed the improvement of ethanol tolerance. At the same time, the ethanol limit concentration of cell growth increased 28% (glucose) and 32% (xylose) respectively. The results showed that pseudo-continuous fermentation could be used as an effective approach for cell adaptation.