2.5. Methods of Bioethanol Production

Dilute sulfuric acid 2% was used for the treated solution inside an autoclave and heated at a temperature of 121°C, for 30 minutes by 1 : 10 solid-to-liquid ratio pretreated inside an autoclave at 140°C for reaction time of 50 minutes [26, 27]. After cooling and filtered through a filter paper (Whatman no. 42), the filtrated contents were preserved in another conical flask and were kept in a refrigerator at 4°C for further use. The pretreated solid residues were washed twice by distilled water to remove sulfuric acid from it and dried before keeping for hydrolysis purpose. Stalk powder was fed as batches, and every batch contains 60 g of screened stalks powder with a ratio of 1 : 10 (w/v) sample to the water [12].

Acid hydrolysis procedure was started by adding 4% diluted sulfuric acid to the nonsoluble component from pretreatment steps of 60 g. The stalk residues were hydrolysed inside an autoclave and heated at the temperature of 150°C, for 90 minutes. After cooling, the liquid was separated from solid particles by filtering through a filter paper (Whatman no. 42). After separation, the solid parts were washed with distilled water two times. Finally, pretreated and hydrolysate filtered solutions were mixed together and preserved in a refrigerator [28].

Saccharomyces cerevisiae was collected from Ethiopian Health and Nutrition Research Institute (EHNRI) and transported to a laboratory in ice box and culture in yeast extract broth. The conical flask was placed on a shaking incubator adjusted at 180 rpm and incubated at 30°C for 24 hours [29].

Fermentation media were prepared by placing 100 ml of pretreated and hydrolysed solution into 250 ml flasks. Pretreated and hydrolysed solution was mixed before addition of any microorganism to the prepared samples. The pH of these samples was adjusted between 3.0 and 5.0 before shaking the substrate by using a digital pH meter. The reactor and all the equipment that were used for fermentation purposes were autoclaved at the temperature of 121°C for 15 minutes. After it was cooled in the hood, activated yeast was added into the fermentation medium. Batch types of fermentation were conducted in incubators preset at 180 rpm for 72 hours at 30°C.

The effect of yeast inoculum size on ethanol yield was determined by varying the yeast inoculum size to 5%, 10%, and 15% [3]. Also, the effect of pH on the ethanol yield was determined by varying the pH level of the fermentation broth for 3.0, 4.0 and 5.0 [3]. Finally, the effect of dilution rate on ethanol production during fermentation was determined by varying the addition of 10 ml, 20 ml, and 30 ml water into the prepared solution [30].