Separate hydrolysis and fermentation was performed to determine the pretreated substrates. Firstly, the Saccharomyces cerevisiae yeast and 40 mL of 2% glucose solution were added to a 10-mL glass flask and placed in a 38 C water bath for 15–20 min, and then moved to a 33 C water bath for 1.5 h to preculture preparation of the yeast. Secondly, the ethanol fermentation solutions were prepared from enzymatic hydrolyzates solutions supplementing with the nutrients peptone (5.0 g/L), KH2PO4 (2.0 g/L), MgSO4 (1.0 g/L), and CaCl2 (0.25 g/L).After the pH of the fermentation solutions were adjusted to 5.5 ± 0.1, they were sterilized in an autoclave at 121 C for 20 min.Thirdly, the former precultured yeast solution was then inoculated into the prepared hydrolyzates for ethanol fermentation and placed in a shaker at 150 rpm and 37 C for 24 h.The flasks were sealed with plastic wrap to provide the anaerobic conditions.The solution were sampled
and stored at 4 C for glucose and ethanol determination