Estimation and analysis of reducing sugars and ethanol produced

The sugars produced during enzyme hydrolysis were estimated using the 3,5-dinitrosalicylic acid (DNSA) method described by Miller (1959). For ethanol analysis, samples obtained after fermentation were centrifuged to separate the biomass from the product and filtered through a 0.22-μm membrane filter and then analyzed using the Agilent HPLC 1200 Series equipped with an auto injector and isocratic pump. HPLC analyses were carried out using the following conditions: column, HyperSil BDS C18 RP; 10 µl of sample was injected into the HPLC system. The mobile phase was 0.01 M sulphuric acid pH 2; at a flow rate of 1.0 ml/min and the detection was set at a wavelength of 254 nm.