This study investigated bioethanol production from rice straw (RS) and sugarcane bagasse (SCB) which containing 72.8 and 73.2% holocellulose, 56.8 and 58.6% α-cellulose, and 14.9 and 25.1% lignin for RS and SCB, respectively. To eliminate the lignin content, different pretreatment conditions, such as hot water, dilute acid, and acid-alkali, were designed. Acid-alkali was characterized as the best pretreatment for removing ∼79 and 70% of lignin, α-cellulose increased 91.4 and 91%, and holocellulose reached 90.8 and 90% for RS and SCB, respectively. The results revealed that acid-alkali was highly efficient than other pretreatment used for both RS and SCB. After enzymatic hydrolysis of acid-alkali-treated RS and SCB with cellulase, glucose concentrations reached 45 and 42 g/l, respectively. Pichia occidentalis AS.2 was isolated and identified based on 18S rRNA sequencing as a bioethanol producer. Maximization of bioethanol production by P. occidentalis AS.2 using the resulting glucose as a carbon source from RS and SCB was studied using an experimental design. The pH, incubation period, and inoculum size were optimized using Box-Behnken designs (BBD), the final conditions for bioethanol production used 100 g/l acid-alkali-treated fibers, 10 ml cellulase enzyme at 50°C for 5 days at 75 rpm for enzymatic hydrolysis. After time consumed and adjusting the pH to 6, the mixture was inoculated with 2.5% P. occidentalis AS.2 and incubated at 35°C for 24 h at 200 rpm to increase the bioethanol yield by 1.39-fold to 23.7 and 21.4 g/l compared to initial production (17 and 15.3 g/l) between RS and SCB, respectively.
