3.3. Determination of Cellulose, Hemicellulose, and Lignin Content in Corn Straw

The method is two-step acid hydrolysis [22,27]. The samples were straw treated with ozone after 0, 1, 2, 3, 4, 5, and 6 cycles of alkaline black liquor treatment. The first step: Accurately weighed 0.300 g of the sample into a test tube, added 3.0 mL of 72% sulfuric acid solution, and mixed by vortexing. The tube was placed in a 30 °C water bath for 60 min and mixed once every 5–10 min. The second step: We diluted the sulfuric acid concentration to 4% after the water bath was completed and put it in a retort at 121 °C for 1 h. After filtration, the filtrate was passed through a 0.22 μm filter and the glucose and xylose content in the filtrate was determined by HPLC to calculate the cellulose and hemicellulose content in the sample. Furthermore, the absorbance of the filtrate was measured at 320 nm with 4% sulfuric acid as the control by UV–Vis spectrophotometry to calculate the content of acid soluble lignin (ASL). The filter residue was dried to constant weight and used to determine the content of acid insoluble lignin (AIL). The acid-insoluble lignin determined by this method contains acid-insoluble ash and acid-insoluble protein, which were negligible due to their small content. We repeated the measurement three times for each sample and used the average as the result of the calculation.

where C₁ was glucose concentration (mg/mL), C₂ was xylose concentration (mg/mL), 0.9 and 0.88 are glucose and xylose conversion coefficients, respectively, V was the total reaction system volume (mL), m was the test sample dry matter content (mg), n was the dilution times, ε was the absorbance coefficient of corn stover at wavelength of 320 nm (30 L/g·cm), m₁ was the quality of the core funnel (mg), and m₂ was the total mass of the sand funnel and filter residue (mg).