Dionex carbopac pa10 column

High molecular weight DOM (HMWDOM) was sampled using tangential flow filtration in parallel to OMICs sampling from the same water body. Samples were processed as described previously [75 (link)] with slight modifications (see Additional file 1). In brief, polysaccharides from HMWDOM samples were extracted and analyzed using carbohydrate microarrays in combination with monoclonal antibodies specific for various polysaccharides (Additional file 15). Antibody-binding signal intensities were quantified with Array-Pro Analyzer 6.3 (Media Cybernetics Inc., Rockville, MD, USA).
Aliquots of the HMWDOM samples were also used for monosaccharide analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) with a Dionex CarboPac PA10 column (ThermoFisher Scientific, Waltham, MA, USA) as described elsewhere [19 (link), 75 (link)].

The mono and oligosaccharide profiles from the supernatants of C. vulgaris after control and Mix treatments were analysed by High Performance Liquid Chromatography (HPLC) on an Agilent system (Agilent 1200 Series, Agilent Technologies Inc., Palo Alto, CA), equipped with an electrochemical detector (Coulochem III, ESA Dionex Thermo Fisher Scientific Inc, USA). The HPLC analysis was performed using a Dionex CarboPac PA10 column (4 × 250 mm, Thermo Fisher Scientific Inc, USA) fitted to a CarboPac PA10 guard column (4 × 50 mm), following the procedure described by Thermo Fisher Scientific⁴⁶ with slight modifications. The separation of mono and oligosaccharides was achieved using a mobile phase with a flow rate of 1 mL/min for 60 min at 25 °C, as follows: isocratic elution with 18 mM NaOH (eluent A) during 18 min, gradient with 100–0 mM NaOH (eluent B) and 0–75 mM sodium acetate in 100 mM NaOH (eluent C) from 18–40 min, and re-equilibration to 18 mM NaOH during 20 min. The quantification of total oligosaccharides was based on a standard curve, using a range of concentrations from 0.025 mM to 0.2 mM of glucose. The results were expressed as equivalent moles of glucose released per gram of microalgae.

The ulvan, UHA and UHB raw media (Additional file 1: Fig. S1) were chemically hydrolysed (1 M HCl for 24 h at 100 °C). Afterwards, the samples were filtered (0.2 µm Spin-X filter) prior to HPAEC-PAD analyses using a Dionex CarboPac PA10 column (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and monosaccharide mixtures as standards for column calibration [57 ].