60 g of the substrate was used in batch pretreatment and hydrolysis process in 600 ml dilute sulfuric acid (H2SO4), to enhance the rate of conversion from polysaccharides into monosaccharides. For this study, diluted H2SO4 (2% for pretreatment and 4% for hydrolysis process) was mixed with the substrate at 1 : 10 solid-to-liquid ratio to be treated inside an autoclave at 121°C for 50 minutes and hydrolysed at 150°C for 1 hour. 1 N of sodium hydroxide and hydrochloric acid were used to adjust the pH [21]. Batch fermentation of the experiment was designed by a factorial design with three factors (inoculum size, pH, and dilution factor) and three replications for each treatment of the two varieties and one positive control group for both varieties. This fermentation was carried out using baker's yeast Saccharomyces cerevisiae. The negative control was yeast mixed with water, while positive control was yeast served with glucose [22]. After adjusting the pH at 3, 4, and 5 and dilution for 10 ml, 20 ml, and 30 ml and inoculum size of 5%, 10%, and 15%, the impact of those variables on the ethanol yield was evaluated. Fermentation media was incubated at a temperature of 30°C and stired at 180 rpm for 72 hours [3]. The independent variables were pH, inoculum size, and dilution. The dependent variable was ethanol yield.

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