Ics 5000

Approximately 5 mg of the sample was hydrolyzed using trifluoroacetic acid (2 M) at 121 °C for 2 h in a sealed tube, and the sample was dried with nitrogen. Methanol was added to wash, blow dry, and repeat methanol wash 2–3 times. For evaluation, the residue was redissolved in deionized water and filtered through 0.22 μm microporous filtering film. The sample extracts were analyzed using high-performance anion-exchange chromatography (HPAEC) over a CarboPac PA-20 anion-exchange column (3 by 150 mm; Dionex) through a pulsed amperometric detector (PAD; Dionex ICS 5000 system). Flow rate, 0.5 mL/min; injection volume, 5 μL; solvent system, B: (0.1 M NaOH, 0.2 M NaAc); gradient program, 95:5 v/v at 0 min, 80:20 v/v at 30 min, 60:40 v/v at 30.1 min, 60:40 v/v at 45 min, 95:5 v/v at 45.1 min, and 95:5 v/v at 60 min. The data were acquired on the ICS5000 (Thermo Scientific, Waltham, MA, USA) and processed using chameleon 7.2 CDS (Thermo Scientific, Waltham, MA, USA). The quantified data were output into the Excel format.

The branch chain-length distribution of amylopectin was analyzed by high-performance anion-exchange chromatography (HPAEC) on a CarboPac PA-100 anion-exchange column (4.0*250 mm, Dionex) using a pulsed amperometric detector (PAD, Dionex ICS 5000 system). Data were acquired on the ICS5000 (Thermo Fisher Scientific, Waltham, MA, United States), and processed using chromeleon 7.2 CDS (Thermo Fisher Scientific, Waltham, MA, United States).

The general composition of the bark residues, which comprised wood particles, ash, extractives, lignin, cellulose, and hemicellulose content, was determined using the National Renewable Energy Laboratory (NREL) protocols: TP‐510‐42618, TP‐510‐42619, TP‐510‐42620, and TP‐510‐42622 (Hames et al., 2008; Sluiter, Hames, et al., 2005; Sluiter et al., 2008b; Sluiter, Ruiz, Scarlata, Sluiter, & Templeton, 2005). Briefly, the method for determining the ash composition was based on the percentage of residue remaining after dry oxidation at 550–600°C. All results are reported relative to the 105°C oven dry weight of the sample. The extractive composition was carried out using a Soxhlet apparatus with ethanol at reflux for 16–24 hr. The lignin content was measured by UV‐Vis spectroscopy (Hach DR6000) after a two‐step acid hydrolysis to fractionate the biomass into forms that are more easily quantified. The monomeric sugars constituting cellulose and hemicellulose were quantified by ion chromatography (Dionex ICS‐5000) with the Dionex CarboPac SA‐10 column and the electrochemical detector.

Lichenase-treated extracts of S. viridis vegetative tissues and grain samples were purified using solid phase extraction (SPE) cartridges packed with graphitized carbon (Varian Bond Elut Carbon 50 mg ml⁻¹ columns) according to Ermawar et al. [61 (link)]. The ratio of DP3:DP4 oligosaccharides in the vegetative tissue extracts were analysed using either high-performance liquid chromatography (HPLC) [91 (link)] or HPAEC-PAD on a Dionex ICS-5000 chromatograph. The grain extracts were analysed using HPAEC-PAD following Ermawar et al. [61 (link)].

The calcium ion concentration of column effluent was measured using an Ion chromatograph (IC). Dionex ICS 5000+ Cation analysis was conducted using 20 mM methanesulfonic acid eluent starting concentration, on a Dionex CS12A column, using 112 mA suppressor output. The first 5 mL effluent from each column was discarded at this stage, since this may contain some unreacted substrates that had been held within the column outlet, outlet tubing had otherwise been drained after each treatment. Beyond this point, up to 20 mL the effluent was mixed for each column to obtain a representative average concentration of calcium ions for each column. The effluent beyond this point was not tested, since this may contain the new CM being injected at later stages in the treatment process as pore volume decreases. Effluent samples were tested daily within two hours of collection to ensure accuracy of results.

The contents of main components in raw materials (bamboo or another two kinds of wood) and pretreated substrates were measured according to the method developed by National Renewable Energy Laboratory [63 ]. In general, glucan, xylan and Klason lignin contents were determined by a two-step acid hydrolysis method. First, 0.3 g of the raw biomass powder or the pretreated substrate powder was placed in a pressure-resistant glass vessel containing 3 mL of 72% (w/w) sulfuric acid solution and stirred every 15 min at 30 °C for 1 h. After the treatment, 84 mL of deionized water was added to adjust the final sulfuric acid concentration to 4% (w/w). The vessel was sealed and autoclaved at 121 °C for 1 h. Upon completion, the pressure-resistant vessel was immediately taken out of the autoclave and cooled to room temperature in the air. The solid–liquid mixture was then filtered on a Büchner funnel with filter paper. The filtrate was analyzed by IC (Dionex ICS-5000, Carbopac PA20, US) for the concentrations of five monosaccharides (arabinose, galactose, glucose, xylose, and mannose) [62 (link)]. The contents of glucan and xylan in the pretreated solid substrates or raw materials were calculated based on the concentrations of glucose and xylose in the filtrate, respectively. The residual solid was weighed to determine the content of Klason lignin.