Title
Comparative assessment of separate vs. simultaneous saccharification and fermentation using Candida tropicalis–Saccharomyces cerevisiae co-culture for 2G bioethanol production from municipal solid waste
Authors
NAYAB ZAHARA, MUHAMMAD IRFAN JALEES, MUHAMMAD UMAR FAROOQ, MUHAMMAD USMAN SALEEM and SHAMAS TABRAIZ

Received August 27, 2025
Published Volume 60 Issue 1-2 January-February
Keywords Box Behnken Design, bioethanol, enzyme cocktail, microbes, response surface methodology, waste to-energy

Abstract
This study investigates and compares two bioconversion strategies, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF), employing a novel co-culture system of Saccharomyces cerevisiae and thermotolerant Candida tropicalis for efficient fermentation of dilute acid pretreated municipal solid waste. In the SHF process, enzymatic hydrolysis was performed at 50 °C using an optimized enzyme cocktail comprising β-glucosidase, endoglucanase, and total cellulase. Subsequent fermentation resulted in a peak ethanol concentration of 29 g/L at 72 h. In contrast, the SSF process where enzymatic saccharification and microbial fermentation occurred simultaneously at 35 °C achieved an ethanol concentration of 32 g/L under similar conditions, demonstrating enhanced process synergy. Optimization of the SSF parameters using Box–Behnken Design (BBD) further improved ethanol titer to 35.134 g/L at 12% substrate concentration, 35.24 °C, and 72 h, with a desirability score of 0.954. The statistical model exhibited strong predictive capability (R² = 0.984, p < 0.0001), confirming the reliability of the optimized conditions.