2.5 Chemical composition analysis of untreated and pretreated biomass
The chemical composition of untreated and pretreated biomass was analyzed in accordance with the NREL procedure.29 In brief, 3 ml of 72% sulfuric acid and 0.3 g of biomass were mixed in a glass tube, and the mixture was stirred for 1 h at 30 °C in a water bath. After the 1 h reaction, the mixture was diluted with 84 ml of distilled water and autoclaved at 121 °C for 1 h. The mixture was filtered through medium pore-size crucible glass filters, and the filtrate was collected to determine the content of acid-soluble lignin and sugar. Acid-soluble lignin content was measured by a UV-vis spectrometer at 320 nm wavelength, while the sugar composition was analyzed by a high-performance liquid chromatography (HPLC, Agilent Technologies 1260 Infinity) equipped with a Biorad Aminx HPX-87H column. The crucible was dried, weighed, heated to 575 °C in a muffle furnace, and weighed again to determine the content of acid-insoluble lignin. All untreated and pretreated biomass were analyzed in duplicates.