Bioethanol production from shrub willow (Salix spp.) using a low severity pretreatment and detoxification-free fermentation by engineered Saccharomyces cerevisiae

The growing demand for renewable fuels has intensified interest in lignocellulosic biomass as a feedstock for bioethanol production. Shrub willow (Salix spp.), a fast-growing woody crop, offers favorable composition with high carbohydrates, high yields, and short rotation cycles, making it an attractive resource for bioenergy applications. This study evaluates a chemical-free, two-step pretreatment approach - hot water pretreatment followed by disk refining - to improve ethanol production from willow biomass. Pretreatment temperatures ranging from 140 to 200 °C were tested to assess their effects on sugar release, inhibitor formation, and fermentation performance. A genetically engineered Saccharomyces cerevisiae SR8 strain, capable of co-fermenting glucose and xylose, was used for co-fermentation. Importantly, the fermentation was carried out without any washing or detoxification of the pretreated biomass. Compared to untreated biomass, which released only 18.8 g/L glucose and 2.8 g/L xylose, the two-step pretreatment at 180 °C increased glucose and xylose concentrations to 47.5 g/L and 17.5 g/L, respectively, corresponding to a 2.5-fold and 6.2-fold improvement. Cellulose conversion during enzymatic hydrolysis reached 95.6 %. Simultaneous saccharification and co-fermentation of biomass pretreated at these conditions resulted in the highest ethanol titer of 26.9 g/L, with over 50 % of the released xylose successfully fermented. However, pretreatment at 200 °C led to high inhibitor levels, completely halting ethanol production. These findings demonstrate a robust, scalable, and detoxification-free process for bioethanol production from willow biomass, reinforcing its promise as a sustainable lignocellulosic feedstock.