Production of Amylolytic Enzymes by Bacillus spp. and Rhizopus spp. and their Use in Ethanol Production

Abstract

Twenty- eight strains of bacteria and fungi were isolated from soil and soil and bread, respectively. Preliminary screening for amylotic bacteria and fungi was performed on starch media. Out of twenty – eight bacterial and fungal isolates, 61% and 86% of isolates showed positive result with iodine solution, respectively. Identification of bacteria and fungi was carried out and it found that all the isolates of bacteria belonged to the genus Bacillus and nine isolates of fungi belonged to the genus Aspergillus and the other isolates belonged to the genus Rhizopus. Three strains of Bacillus (B1, B2, and B11) and Rhizopus (F16, F22, F23) were chosen for this study according to the amylotic activity. Optimum conditions for enzyme production were studied, solid state fermentation method was used and wheat bran was used as the main substrate and it was analysed. The incubation time 48 hours and incubation temperatures 25 and 350C were found the best for Rhizopus and Bacillus strains,respectively except Rhizopus F23 with the best at 300C. pH 7.0 was found the best for all the Rhizopus and Bacillus strains except Bacillus B1 with the best at pH 8.0. Optimum conditions for enzyme activity were studied. Temperatures 40, 50, 600C and pH 6.0 and 7.0 and incubation times of 30, 40, 50 minutes were found the best for activity of Rhizopus and Bacillus strains enzymes. Thermal inactivation of enzyme for Rhizopus and Bacillus strains was also studied. Incubation at low temperatures for enzyme did not show any decrease in activity and incubation at medium temperatures showed decrease in activity and increased with increase in high incubation temperatures. pH inactivation of enzyme for Rhizopus and Bacillus strains was detected. The activity of enzyme for Rhizopus and Bacillus strains was increased above pH 4.0 and enzymes were inactivated above pH 8.0. Storage inactivation of enzyme for Rhizopus and Bacillus strains was detected as well. High activity was observed at the beginning of storage at room temperature and refrigerator and slight decrease in activity was found with increase in time. Identification of hydrolytic products of soluble starch by Rhizopus and Bacillus strains enzymes was investigated on paper chromatography. Glucose and maltose were produced by Rhizopus strains enzymes and maltose was produced by Bacillus strains enzymes. Different yeasts were isolated from different sources, two isolates of yeast were identified to Shizosaccharomyces octosporus and Saccharomyces cerevisiae. Saccharomyces cerevisiae was used in this study for fermentation in ethanol production. The best combination of Rhizopus and Bacillus enzymes for high level of activity was found in combination of Bacillus B1 and Rhizopus F22. Two samples of sorghum and corn starch were used in ethanol production, sorghum starch was found the best for producing sugars. High level of ethanol was produced by sorghum starch (81.13%) compared with corn starch (60.33%).