The present study provides solutions to the bioethanol industry’s main issues of the absence of production of maximum fermentable sugars from lignocellulosic biomass, the unsuitability of a single microorganism to convert the fermentable sugars (xylose and glucose) together to bioethanol, and the unavailability of fermentation technique to run the process unlimitedly with consistent yield and productivity. A novel fractional acid hydrolysis technology was demonstrated in this study, which produced more than 90% (w/w) of available glucose in wheat straw as glucose-rich hydrolysate (GRH). This hydrolysate was then made acid-free using a membrane-based acid separation unit to reuse the separated acid to make the hydrolysis process environmentally safe and chemically inexpensive. Thereafter, this acid-free GRH was fermented to bioethanol using a unique constant volume fed-batch fermentation technique under an optimum glucose-feeding strategy. Remarkably, this fermentation technique yielded optimal average bioethanol productivity (g/Lh), yield (g/g), and titer (g/L) of 7.147±0.533, 0.508±0.002, and 58.835±0.766; 5.722±0.529, 0.501±0.006, and 33.748±0.322, respectively, with more than 99% glucose utilization during ten fed-batch cycles of synthetic and GRH glucose. Notably, the high production of GRH from lignocellulosic biomass, acid separation and reuse, and continuous and consistent bioethanol production sets this study apart as a pioneering endeavor. Furthermore, the bioethanol profit of USD 0.019/kg biomass from the second batch onwards at a bioethanol selling price of USD 0.745/L suggests the feasibility of the current study for industrial-scale bioethanol production from lignocellulosic biomass.
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