A green biomass pretreatment strategy to produce 3-hydroxypropionic acid and ethanol with engineered Saccharomyces cerevisiae, Rhodosporidium toruloides, and Issatchenkia orientalis.

Chemical and water overconsumption involved in solid-liquid separation and post-washing after biomass pretreatment pose significant challenges to scaling up the production of platform chemicals and biofuels. This study explored a novel one-pot biomass valorization strategy that merges CaO pretreatment of corn stover (CS) or brewer's spent grain (BSG) with CO2 carbonation. The pretreated biomass slurry was used for 3-hydroxypropionic acid (3-HP) or ethanol production without solid-liquid separation or post-washing. Results showed that carbonation reduced the pH to around 6 of BSG slurries while facing challenges in CS slurries at 20 wt% solid loading. For BSG, the highest glucan (54 %) and xylan (34 %) conversions were achieved at 20 wt% solid loading with 32 g/L of glucose and 17 g/L xylose in the hydrolysate. For CS, glucan (51 %) and xylan (88 %) conversions were impressive at 5 wt% solid loading with 10.6 g/L glucose and 8.2 g/L xylose in hydrolysate but significantly decreased due to incomplete carbonation at higher solid loadings. The highest ethanol concentration of 18.4 g/L was achieved at 20 wt% BSG solid loading using engineered Saccharomyces cerevisiae with ethanol yields higher than 70 % for all tested hydrolysates. Most importantly, the resulting hydrolysates can be fermented to 3-HP by both engineered Rhodosporidium toruloides and Issatchenkia orientalis, as the highest 3-HP concentration of 6.9 g/L was achieved at 20 wt% BSG solid loading with I. orientalis. These findings suggest that the CO2-assisted strategy for CaO pretreatment offers a carbon-neutral pathway to valorize biomass to platform chemicals and biofuels.