Analyses
Determination of acid soluble lignin was carried out using the liquid part after acid hydrolysis and by reading the sample at 240 nm wavelength using UV–VIS spectrophotometer Lambda 35 by PerkinElmer, Massachusetts, USA. The blank solution was 4% H2SO4. Acid insoluble lignin, on the other hand, was determined by using the dry weight difference of the filter paper before and after vacuum filtration [29].
Sugars (glucose, sucrose, fructose, xylose and arabinose) were analyzed using Agilent 1200 HPLC system (California, USA) equipped with Rezex RPM (Phenomenex, USA) 300 mm × 7.8 mm column and Refractive Index Detector (RID) [30]. Column temperature was 60 °C. Water was isocratically eluted at a flow rate of 0.6 mL/min. The same method was applied for the analysis of ethanol by using a different column which is Rezex ROA column (Phenomenex, USA). Soluble metabolites such as succinic acid, formic acid and acetic acid were analyzed using Agilent 1100 HPLC system (California, USA) equipped with Rezex ROA column (Phenomenex, USA) and Ultraviolet detector (UVD) at 210 nm. The column was eluted isocratically at a rate of 0.5 mL/min with 0.0025 M H2SO4 at 40 °C.