Efficient 2G Ethanol Production via Optimized Dilute Acid Pretreatment, High-Solids Enzymatic Hydrolysis, and High-Temperature Fermentation

The growing demand for 2G ethanol emphasizes the need to improve its economic sustainability through different approaches, including optimized pretreatments, high-solids enzymatic hydrolysis, and high-temperature fermentation. This study evaluated the dilute acid pretreatment of deacetylated rice straw using the response surface methodology. Under optimal conditions (85 mg of H₂SO₄/g biomass, for 10 min at a constant temperature of 150 °C), the process was scaled up to an 80 L reactor, obtaining a cellulose-rich (58.2% w/w) pretreated solid. Then, high-solids enzymatic hydrolysis at 24% w/v solids loading was performed in a vertical ball mill (VBM) reactor in fed-batch mode, resulting in a hydrolysate of 129 g/L of fermentable sugars, which corresponded to a cellulose conversion yield (CCY) of 78.6%. Finally, the effect of nutritional supplementation of the obtained slurry and hydrolysate on the fermentability of <i>Kluyveromyces marxianus</i> at 43 °C was studied in conical flasks under orbital shaking. The higher <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msub><mml:mi>Y</mml:mi> <mml:mrow><mml:mi>P</mml:mi> <mml:mo>/</mml:mo> <mml:mi>S</mml:mi></mml:mrow> </mml:msub> </mml:math> (0.46 g/g), <i>Q</i> _P (1.74 g/L/h), and η (90%) were achieved in the hydrolysate + nutrients medium. Additionally, the effect of the VBM reactor on ethanol production was evaluated, further increasing <i>Q</i> _P (3.04 g/L/h) with an ethanol titer of 37 g/L. Therefore, the processing sequential steps and conditions efficiently produced a glucose-rich hydrolysate, which was successfully fermented into ethanol at high temperatures and could support the process feasibility at a large scale.