Increasing the tolerance of the non-conventional yeast Candida intermedia to ethanol and lignocellulose-derived inhibitors

Conference poster, 2016

The necessity of providing ‘robust microorganisms’ – defined as the ability to efficiently ferment all the available sugars (both hexoses and pentoses) and to cope with the main stressors present during the fermentation process (including biomass-derived products and ethanol) – represents one of the major challenges for a cost-effective lignocellulosic bioethanol production. The yeast Saccharomyces cerevisiae is the preferred fermentative microorganism in the current bioethanol industry due to its superior fermentation capacity of hexose sugars and its tolerance to several inhibitory compounds. The main disadvantage of S. cerevisiae is, however, its inability to ferment xylose, the second most abundant sugar in lignocellulose (>30% of the total sugar). Metabolic and evolutionary engineering methods have been applied to allow xylose fermentation in S. cerevisiae. Still, xylose-fermenting S. cerevisiae strains lack an efficient xylose-to-ethanol conversion system and issues such as low xylose uptake rates and conversion yields, redox imbalance and the lack of simultaneous use of glucose and xylose are important parameters that still need to be optimized. As an alternative to genetically modified S. cerevisiae strains, non-conventional, native xylose-utilizing yeasts such as the Scheffersomyces species S. stipitis and S. shehatae, Spathaspora passalidarum and various Candida species (C. tropicalis, C. guilliermondii or C. intermedia) have been considered for the fermentation of pentose sugars. These yeasts have, however, a modest tolerance to lignocellulose-derived inhibitors and ethanol, which limits their applicability. Candida intermedia is a xylose-fermenting yeast species that encompasses a high capacity xylose transport system. This trait makes C. intermedia attractive for being a non-GMO alternative in the lignocellulosic bioethanol industry. In the present work, the ethanol tolerance and the fermentation capacity in the presence of lignocellulose-derived inhibitors of an in-house isolated C. intermedia strain was evaluated. The isolated strain showed a medium-tolerance towards lignocellulose-derived inhibitors, being more sensitive when using xylose as a carbon source. The ethanol concentration above which there is no growth was estimated to be 42 g/L when growing in glucose and 55 g/L when growing in xylose. The isolated strain was subjected to evolutionary engineering with the aim of increasing its tolerance towards both lignocellulose-derived inhibitors and ethanol. The obtained evolved population was able to ferment a lignocellulosic hydrolysate (steam-exploded wheat straw), not fermentable by the isolated strain. Furthermore, the evolved population produced higher biomass concentration (7.5-fold higher OD600nm values) when growing in the presence of 36 g/L ethanol, compared to the parental strain. These results highlight the potential of C. intermedia to become a robust yeast microorganism for the lignocellulose-to-ethanol conversion.