Ics 5000 dc

The hemicellulose content and amount of acid-insoluble solids were measured according to previous studies [23 (link)]. Ten milliliters of the liquid samples was mixed with 750 µL 72% sulfuric acid and autoclaved (Systec DX 150, Wettenberg, Germany) at 121 °C for 1 h to hydrolyze the polymeric sugars. The samples were then filtered to remove insoluble solids, and the filters were dried overnight in an oven at 105 °C. The acid-insoluble solids content was determined by measuring the dried filters before and after filtration of the samples.
The hemicellulose content was determined by analyzing the liquid fraction after the filtration by high-performance anion-exchange chromatography (HPAEC). The HPAEC system (ICS-5000+ DC, Dionex, Sunnyvale, CA, USA) was a pulsed amperometric detector with a compartment temperature of 30 °C, and the separation was performed on a Carbo Pac PA1 analytical column. Deionized water at 1 mL/min was used as the main eluent, and 200 mM sodium hydroxide solution was used as the postcolumn addition at 0.5 mL/min. The samples were diluted with deionized water, and the injection volume for the HPAEC was 10 µL for all samples. The standards were D-mannose, D-xylose, D-glucose, D-galactose, and L-arabinose (Fluka Chemie AG, Buchs, Switzerland), and the hemicellulose content was determined after anhydro corrections of 0.90 for hexoses and 0.88 for pentoses.

Polar metabolites were extracted, derivatized and run on the GC-TOF-MS as described before (Ribeiro et al. 2014 (link)) but 5 mg of dry material was used instead of 20 mg. All volumes were stoichiometrically adjusted.
In order to confirm the sucrose trend from GC-TOF-MS, soluble carbohydrates were also determined as described previously (Ribeiro et al. 2014 (link)). The supernatant after starch extraction was injected into a Dionex HPLC system (ICS 5000 ⁺ DC) to analyse the soluble carbohydrate content, using a CarboPac PA 1, 4- × 250-mm column preceded by a guard column (CarboPac PA 1, 4 × 50 mm), and a gradient pump module (ICS 5000 Dual Pump, Dionex). Mono-, di-, and tri-saccharides were separated by elution in an increasing concentration of NaOH (20–350 mM) with a flow rate of 1 mL min⁻¹. Peaks were identified by co-elution of soluble carbohydrate standards. Sugar quantity was corrected by mean of the internal standard (melezitose) and transformed to micrograms of sugar per milligram of dry material.

The hemicellulose content was determined according to a standardized National Renewable Energy Laboratory (NREL) method [19 ]. The samples were acid hydrolyzed by adding 750 µl 72% sulfuric acid to 10 mL sample and autoclaved (Systec DX 150, Wettenberg, Germany) at 121 °C for 1 h. The samples were thereafter filtered to remove the acid-insoluble solids. The filter was then dried and weighed to determine the acid insoluble content. The filtrate was diluted with deionized water and analyzed with high-performance anion-exchange chromatography (HPAEC). The HPAEC system consisted of an ICS-5000+ DC (Dionex, Sunnyvale, CA, USA) equipped with pulsed amperometric detection running at a compartment temperature of 30 °C. The different monosaccharides were separated using a Carbo Pac PA1 analytical column where the eluent was deionized water at a flow of 1 and 0.5 mL/min 200 mM sodium hydroxide solution post-column addition. The injection volume was 10 µl and the standards used were L-arabinose, D-galactose, D-glucose, D-xylose, and D-mannose all manufactured by Fluka Chemie AG (Buchs, Switzerland). The amount of hemicellulose was determined after anhydro corrections of 0.90 for hexoses and 0.88 for pentoses.