Objective: This study aims to evaluate the effects of pH, ammonium sulfate supplementation, and refrigeration on fermentation performance in sugarcane molasses, with the goal of optimizing ethanol production under simulated industrial conditions. Theoretical Framework: The research builds on key concepts in biofuel production, emphasizing the role of pH, nutrient supplementation, and temperature control in influencing yeast metabolism and fermentation efficiency. Theories on enzymatic activity and nutrient absorption (Vidal et al., 2013; Gutierrez, 1993) provide the foundation for understanding the interactions between these factors. Method: A full 2³ factorial design was employed, evaluating the effects of pH (3.5 and 5.0), supplementation (0.0 and 1.0 g/L), and refrigeration (with or without) on fermentation efficiency, process efficiency, ethanol productivity, and substrate-to-cell conversion in a microdistillery. Analytical methods included spectrophotometry, refractometry, and chromatography. Results and Discussion: The results indicate that fermentation conditions with pH 3.5, without supplementation, and with refrigeration yielded the best performance, with significant increases in ethanol productivity and fermentation efficiency. The interaction between variables suggests that these factors should be considered jointly to optimize the process. Research Implications: The findings offer practical insights for improving fermentation in industrial biofuel production, particularly in optimizing conditions for ethanol yield and process efficiency under near-industrial conditions. Originality/Value: This study contributes by using a microdistillery to simulate industrial conditions, providing more accurate data for scaling ethanol production. It adds value by highlighting how specific combinations of pH, supplementation, and refrigeration can improve biofuel sustainability.
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