Ics 6000

The total carbohydrate content and sugar composition of Extracts 1 and 2 were determined after acidic hydrolysis, with the TFA at 120 °C × 1 h [23 (link)]. The monosaccharides were analysed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD, Dionex ICS 6000, ED40 Electrochemical Detector) equipped with a Thermo Fisher Scientific Dionex CarboPac PA20 column (3 × 150 mm) coupled with a guard column (3 × 30 mm). Samples were filtered (0.2 Ny) before analysis and injected with a Dionex AS-AP autosampler. The eluent flow rate was 0.400 mL/min and the temperature was kept at 30 °C. The mobile phase was made of the following A, H₂O Milli-Q; B, 0.2 M sodium hydroxide; and C, 0.1 M sodium hydroxide, 0.1 M sodium acetate. Elution was performed following the following method: 0–21 min, 1.5% B; 21–33 min, 50% B; 33–49 min, 100% C; 49–53 min, 100% A; and 53–70 min, 1.5% B [24 (link)]. A standard solution of adonitol, mannitol, fucose, arabinose, galactose, glucose, xylose, and mannose was prepared to dissolve 25 mg of each sugar standard in 50 mL of Milli-Q water. Five scalar dilutions of standard solution in a 1–0.0625 ppm range were prepared for calibration. All experiments were carried out in triplicate and results were expressed on a dry weight percentage basis. Data were processed with the Dionex Chromeleon software.

A 5 mg mL⁻¹ solution of ulvan was prepared in 100 mL of 100 mM Tris–HCl pH 8.5 and 0.6 M NaCl. Hydrolysis reactions were carried out overnight at 20 °C and 100 rpm for 24 h with 52 µg of enzyme. The reaction mixture was topped up with the same amount of enzyme after 15 h. Breakdown of the polysaccharide was verified by comparative analysis of unhydrolysed and hydrolysed samples using C-PAGE as described by Zablackis and Perez (1990 ).
Identification of the major breakdown products was achieved by High Pressure Anion Exchange Chromatography (HPAEC). A small portion (25 µL) of the reaction mixture was injected on a Thermo ICS6000 chromatographic system composed of an AG11-HC guard column (4 × 50 mm) and an AS11-HC anion exchange column (4 × 250 mm) connected in series and equilibrated in 8 mM NaOH. Elution was at a flowrate of 0.5 mL min⁻¹ using a multistep NaOH gradient from 8 to 280 mM in 40 min. Charged oligosaccharides were detected using a DC conductivity detector associated to a DRS600 suppressor functioning under a 347 mA current. For comparison, previously purified and characterised oligomers (Δ-Rha3S, Δ-Rha3S-HexA-Rha3S, Δ-Rha3S-Xyl-Rha3S) from ulvan (Reisky et al. 2019 (link)) were also injected.

After centrifugation, the supernatant of 1 mL culture was directly used for NO₃⁻ concentration measurements on an ICS-6000 ion chromatography (IC) system (Thermo Fisher Scientific, Waltham, MA, USA). A Dionex IonPac AS11 with guard column (Thermo Fisher Scientific, Waltham, MA, USA) was set to 30 °C and operated with a flow of 2.0 mL·min⁻¹. Mobile phase A was 100% H₂O and phase B 100 mmol·L⁻¹ NaOH. The binary gradient was from 0 to 1 min 0.2% B, from 1 to 6 min to 5% B, from 6 to 13 min increase to 24% B, from 13 to 13.5 min set to 38% B, and hold until 14.5 min. After this, the initial 0.2% B was reached after 15 min and held until 20 min for equilibration before the next injection. A conductivity detector combined with AERS suppressor (Thermo Fisher Scientific, Waltham, MA, USA) was used for analyte detection. Nitrate standards (Sigma Aldrich, St. Lewis, MO, USA) were prepared and used for quantification of the samples. Data was logged and evaluated by Chromeleon 7.2.8 (Thermo Fisher Scientific, Waltham, MA, USA).

Identification and quantification of polysaccharide neutral sugars were performed by gas-liquid chromatography (GC) after sulfuric acid degradation as described in reference (86 (link)). Briefly, 5 mg of dried alkali insoluble fraction was dispersed in 2 N trifluoroacetic acid and then hydrolyzed 90 min at 121°C. Neutral monosaccharides were converted to alditol acetates and analyzed on a TG-225 GC Column (30 × 0.32 mm ID) using TRACE Ultra Gas Chromatograph (Thermo Scientific; temperature 205°C, carrier gas H₂). Standard sugars’ solution and inositol as internal standard were used for calibration.
Glucosamine residues were quantified after acid hydrolysis [adapted from reference (87 (link))] and HPAEC-PAD analyses [adapted from reference (88 (link))]. Briefly, 5 mg of dried alkali insoluble fraction was dispersed in acetic acid 1% and hydrolyzed in concentrated HCl 10 M at 105°C during 6 h. After dilution in milli-Q water, glucosamine residues were quantified by HPAEC-PAD (ICS-6000, Thermo Scientific) using a CarboPac PA20 column (2 × 250 mm, Thermo Scientific), thermostated at 30°C. An isocratic elution of 1.7 mM sodium acetate (NaOAc) in 1 mM NaOH was used at a 0.25 mL/min flow rate. Standard glucosamine solutions were used for calibration.

IC mobile phase A (MPA; weak) was water, and mobile phase B (MPB; strong) was water containing 100 mM KOH. A Thermo Scientific Dionex ICS-6000+ system included a Thermo IonPac AS11 column (4 μm particle size, 250 × 2 mm) with column compartment kept at 35°C. The autosampler tray was chilled to 4°C. The mobile phase flow rate was 360 μL/min, and the gradient elution program was: 0–5 min, 1% MPB; 5–25 min, 1–35% MPB; 25–39 min, 35–99% MPB; 39–49 min, 99% MPB; 49–50, 99–1% MPB. The total run time was 50 min. To assist the desolvation for better sensitivity, methanol was delivered by an external pump and combined with the eluent via a low dead volume mixing tee.