Flocculation causes inhibitor tolerance in Saccharomyces 1 cerevisiae for 2 nd generation bioethanol production 2 Running title : Inhibitor tolerance through flocculation 3 4 5

12 Yeast has long been considered the microorganism of choice for second generation bioethanol 13 production due to its fermentative capacity and ethanol tolerance. However, tolerance towards 14 inhibitors derived from lignocellulosic materials is still an issue. Flocculating yeast strains often 15 perform relatively well in inhibitory media, but inhibitor tolerance has never been clearly linked 16 to the actual flocculation ability per se. In this study, variants of the flocculation gene FLO1 were 17 transformed into the genome of the otherwise non-flocculating laboratory yeast strain 18 Saccharomyces cerevisiae CEN.PK 113-7D. Three mutants with distinct differences in 19 flocculation properties were isolated and characterised. The degree of flocculation and 20 hydrophobicity of the cells were correlated to the length of the gene variant. The effect of 21 different strength of flocculation on the fermentation performance of the strains was studied in 22 defined medium with and without fermentation inhibitors as well as in media based on dilute acid 23 spruce hydrolysate. Strong flocculation aided against the readily convertible inhibitor furfural, 24 but not against less convertible inhibitors, such as carboxylic acids. During fermentation of dilute 25 acid spruce hydrolysate, the most strongly flocculating mutant with dense cell flocs showed 26 significantly faster sugar consumption. The modified strain with the weakest flocculation showed 27 a hexose consumption profile similar to the non-transformed strain. These findings may explain 28 why flocculation has evolved as a stress response, and can find application in fermentation-based 29 biorefinery processes on lignocellulosic raw materials. 30 31 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom