Enzymatic Hydrolysis of Starchy Waste for Bio-Ethanol Production

Abstract

Amylase activity in the cell free supernatant of starch degrading bacteria Bacillus licheniformis NA11 was studied using 1% starch. The protein precipitated up to 20-80% saturation with ammonium sulphate and further partially purified was found stable between pH 5-7 and temperature between 40-60° C with optimal activity at pH 7.0 and temperature 60°C. The Km value of the partially purified enzyme for starch substrate was 0.621 mg/ml and Vmax=0.0354 µmole/min/ml. Amylase activity of partially purified enzyme from Bacillus licheniformis showed maximum activity of 1.12 U/ml/min, with production of 0.467 mg/ml reducing sugar when 1% starch was used as substrate. When starchy wastes were used as substrate the enzyme activity in 160 min at 60°C was 1.048 U/ml/min for orange peel waste (OPW) with production of 0.435 mg/ml of reducing sugar, 0.99 U/ml/min for banana peel waste (BPW) with production of 0.411 mg/ml of reducing sugar and 0.959 U/ml/min for potato peel waste (PPW) with production of 0.398 mg/ml of reducing sugar. Iodine assay in test tube and micro-plates, showed blue colour in the presence of starch- iodine and at 60-70°C, all blue color disappeared after 60 min. Statistical optimization using response surface methodology predicted amylase activity of 0.47 U/mL, which coincided with the experimental amylase activity of 0.46 U/mL using starch (10 g/L), yeast extract (6 g/L), and MnCl2.4H2O (1.0 g/L). Starchy waste such as Potato peel waste (PPW), orange peel waste (OPW), banana peel waste were (BPW) biomass were washed to remove adhering debris, dried and powdered (0.3- 0.5 mm) to investigate its physico-chemical properties. Potato peel waste (PPW) was found to contain 6.64% moisture, 5.48% ash and 75.79 % volatile matter, similarly orange peel waste (OPW) was found to contain 5.451 % moisture, 9.717 % ash, 84.52 % volatile matter and banana peel waste (BPW) was found to contain 5.01 % moisture, 7.35 % ash, 81.40 % volatile matter. Pre-treatment of starchy waste using dilute acid-alkali and enzymatic hydrolysis were done to enhance the yield of reducing sugar for ethanol production. Enzymatic pre-treatment was found to be the best pre-treatment strategy with increase in amylose subunits, while decrease in starch content. Yield of reducing sugar was enhanced from 0.195mg/ml to 0.467 mg/ml. Scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared analysis (FTIR) analysis of native and enzyme pre-treated biomass was done to study physical and chemical changes. Enzymatically pre-treated starchy wastes (PPW, OPW and BPW) were used as substrate for ethanol production using cell free supernatant from Bacillus licheniformis NA11 followed by ethanol fermentation using Saccharomyces cerevisiae (NCIM 3215) via SSF. Ethanol production using starch as substrate was 2.51%, with orange peel waste it was 1.33%, with banana peel waste it was 1.03% and with potato peel waste it was 0.89%. The high catalytic activity and its stability to temperature, pH, indicated that the amylase enzyme from Bacillus licheniformis NA11 is a good candidate for hydrolysis of starchy waste into bio-refinery processes.