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Second generation bioethanol represents an important biofuel, but innovations on biomass pre-treatment and fermentation are needed to improve efficiency and cost-effectiveness in its production. In this work, beech wood chips were treated using an acetone-based organosolv fractionation process at pilot scale, resulting in the isolation of lignin, a high-purity cellulosic pulp and a liquid stream containing hemicellulose sugars. The hemicellulose stream (C5 stream) contained xylose as major sugar and also contained fermentation inhibitors such as furanics, organic acids and phenolics. The cellulosic pulp was enzymatically hydrolysed producing a glucose-rich stream (C6 stream). These streams were used as fermentation substrates by the yeasts Saccharomyces cerevisiae and Spathaspora passalidarum , in separate cultures. Addition of ammonium phosphate or of yeast extract and peptone (YP) as nitrogen sources improved yeast growth. The C6 stream was readily fermentable. When at a sugar concentration of 192 g L−1 and supplemented with YP, the ethanol titre reached 91 g L−1, with a yield of 0.49 g ethanol per g consumed sugars. The C5 stream required detoxification to achieve fermentability. In detoxified C5 stream, S. passalidarum produced 0.46 g ethanol per gram consumed sugars. Fermentation of the resulting streams at 10-L scale confirmed the results obtained at laboratory scale. As alternative approach, sequential fermentation of the C5 stream by S. passalidarum followed by C6 stream addition and inoculation with S. cerevisiae resulted in almost complete sugar utilization and an ethanol yield of 0.41 g per gram consumed sugar albeit with a lower ethanol productivity. [Display omitted] • Fermentable sugar streams produced from beech wood chips by upscaled acetone fractionation. • Fermentation of glucose-rich and xylose-rich streams validated at 10-L scale. • High ethanol titre (10 %, w/v) and yield (95 %) were obtained from lignocellulosic beech wood C6 sugars. • Ethanol was produced from detoxified beech wood C5 sugar stream at a titre of 3 % (w/v) and 70 % yield. [ABSTRACT FROM AUTHOR]