Total lignin content was determined by the two-step acid hydrolysis method according to Laboratory Analytical Procedure of the National Renewable Energy Laboratory. The lignin includes acid-insoluble and -soluble lignin. The acid-insoluble lignin was calculated gravimetrically after correction for ash, and the acid-soluble lignin was measured by UV spectroscopy.
For acid-insoluble lignin determination, a 0.5-g sample was recorded as W1. The sample was extracted with benzene-ethanol (2:1, v/v) in a Soxhlet for 4 h, and then air-dried in a hood overnight. The sample was hydrolyzed with 10 mL 72% H2SO4 (v/v) in a shaker at 30°C for 1.5 h. After hydrolysis, the acid was diluted to a concentration of 2.88%, and then placed in the autoclave for 1 h at 121°C (15 psi). The autoclaved hydrolysis solution was vacuum-filtered through the previously weighed filtering crucible. The filtrate was captured in a filtering flask for acid-soluble lignin. The lignin was washed free of acid with hot distilled water and the crucible and acid-insoluble residue was dried in an oven at 80°C until constant weight was achieved. Then, the samples were removed from the oven and cooled in a dry container. The weight of the crucible and dry residue was recorded to the nearest 0.1 mg (W2). Finally the dried residue was ashed in the muffle furnace at 200°C for 30 minutes and at 575°C for 4 h. The crucibles and ash were weighed to the nearest 0.1 mg and we recorded the weight (W3). The acid-insoluble lignin (AIL) of the original sample was calculated as follows:
Each sample was tested in biological triplicate. For the acid-soluble lignin determination, the acid-soluble lignin was solubilized during the hydrolysis process, and was measured by UV spectroscopy. The hydrolysis liquor obtained previously was transferred into a 250-mL volumetric flask and brought up to 250 mL with 2.88% sulfuric acid. The absorbance of the sample was read at 205 nm using UV–vis spectroscopy (Beckman Coulter Inc., Du800), and 2.88% sulfuric acid was used as blank. The method of calculation for the amount of acid-soluble lignin was as follows:
Where A is the absorption value, D is the dilution ratio of the sample, and K (the absorptivity constant) = 110 L/g/cm.
All experiments were carried out in triplicate.
For acid-insoluble lignin determination, a 0.5-g sample was recorded as W1. The sample was extracted with benzene-ethanol (2:1, v/v) in a Soxhlet for 4 h, and then air-dried in a hood overnight. The sample was hydrolyzed with 10 mL 72% H2SO4 (v/v) in a shaker at 30°C for 1.5 h. After hydrolysis, the acid was diluted to a concentration of 2.88%, and then placed in the autoclave for 1 h at 121°C (15 psi). The autoclaved hydrolysis solution was vacuum-filtered through the previously weighed filtering crucible. The filtrate was captured in a filtering flask for acid-soluble lignin. The lignin was washed free of acid with hot distilled water and the crucible and acid-insoluble residue was dried in an oven at 80°C until constant weight was achieved. Then, the samples were removed from the oven and cooled in a dry container. The weight of the crucible and dry residue was recorded to the nearest 0.1 mg (W2). Finally the dried residue was ashed in the muffle furnace at 200°C for 30 minutes and at 575°C for 4 h. The crucibles and ash were weighed to the nearest 0.1 mg and we recorded the weight (W3). The acid-insoluble lignin (AIL) of the original sample was calculated as follows:
Each sample was tested in biological triplicate. For the acid-soluble lignin determination, the acid-soluble lignin was solubilized during the hydrolysis process, and was measured by UV spectroscopy. The hydrolysis liquor obtained previously was transferred into a 250-mL volumetric flask and brought up to 250 mL with 2.88% sulfuric acid. The absorbance of the sample was read at 205 nm using UV–vis spectroscopy (Beckman Coulter Inc., Du800), and 2.88% sulfuric acid was used as blank. The method of calculation for the amount of acid-soluble lignin was as follows:
Where A is the absorption value, D is the dilution ratio of the sample, and K (the absorptivity constant) = 110 L/g/cm.
All experiments were carried out in triplicate.
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