Improved inhibitor tolerance and simultaneous utilisation of hexoses and pentoses during fermentation of inhibitory lignocellulose hydrolysates by yeast at high local cell density
Conference contribution, 2014
Issues still creating a barrier for successful commercialization of second generation bioethanol are the inhibitory compounds present in the lignocellulose derived media and the inability of Saccharomyces cerevisiae to efficiently utilise pentoses. Both of the issues have been addressed by construction of recombinant yeast strains, often in combination with evolutionary engineering. However, hexoses and pentoses are mainly fermented sequentially by these yeasts, prolonging the total fermentation time. In our research, we have shown that encapsulation of S. cerevisiae cells in semi-permeable alginate-chitosan liquid core gel capsules increased the tolerance to lignocellulose hydrolysates and specifically furan aldehydes. The potential formation of concentration gradients of these convertible inhibitors through the cell pellet inside the capsule has been given as explanation to the increased tolerance . Gradients of carbohydrates in the capsules were further hypothesised to lead to an improvement in the simultaneous utilisation of hexose and pentose sugars by the cells. To verify this hypothesis we constructed and encapsulated the xylose fermenting S. cerevisiae strain CEN.PK XXX. We found that encapsulation of the strain not only increased the inhibitor tolerance of the yeast, but also promoted simultaneous utilisation of glucose and xylose. Furthermore, during the 96 hour fermentations of a medium with glucose and xylose, the encapsulated yeast consumed at least 50% more xylose compared to the suspended cells. This led to approximately 15% higher final ethanol titres in batch fermentations. As proof of concept, an inhibitory spruce hydrolysate was fermented by suspended and encapsulated cells. The suspended cells fermented the hexoses and pentoses mainly sequentially, after a long lag phase. The encapsulated yeast, on the other hand, did not display a lag phase, and consumed glucose, mannose, galactose and xylose simultaneously from the start of the batch. However, encapsulation of yeast cells in an alginate membrane would likely not be economically permissible in an industrial setting. We therefore investigated whether keeping the cells tight together would be sufficient, even without a membrane. To this end we constructed a set of flocculating yeast strains with different flocculation strengths by expression of different variants of a flocculation gene. We found that the strongest flocculating strain, forming large dense cell flocs in the batch reactor, increased the tolerance towards furfural and increased the fermentation rate in an inhibitory spruce hydrolysate, compared to the non-flocculating strain. Overall, yeast at high local cell density comes out as a promising option for production of second generation bioethanol.
 J.O. Westman et al., Int. J. Mol. Sci., 13, 11881-11894, 2012.