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Monosaccharides quantified in porewater samples from coastal vegetated ecosystems

Porewater was taken from 30 to 50 cm depths in saltmarsh, seagrass and unvegetated areas around the German Bight in 2022 and 2023. Up to 9 points per ecosystem were sampled along a transect. Polysaccharides >5kDa were upconcentrated using AMICON-filtration devide and afterwards freeze dried. Dired samples were resuspended in MilliQ-water and acid hydrolysed (1 M HCl, 24 h, 100°C). Monosaccharides were analysed using anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD, ThermoFisher Dionex ICS-5000+ system equipped with a CarboPac PA10 analytical column (2 x 250 mm) and a CarboPac PA10 guard column (2 x 50 mm)), according to Engel et al. (2011).

Polysaccharide fucoidan BAM1 antibody binding in porewater samples from coastal vegetated ecosystems

Porewater of saltmarsh, seagrass and unvegetated areas around the German Bight in 2022 and 2023 was sampled at 50cm depth. A transect of up to 9 points per ecosystem were sampled. Polysaccharides >5kDa were upconcentrated using AMICON-filtration device, freeze dried and dried samples were resuspended in MilliQ-water. Polysaccharides were screened for fucoidan BAM1 antibody binding using ELISA method, according to Cornuault et al. (2014).

Total carbohydrates quantified in sediment cores from coastal vegetated ecosystems

50-cm deep sediment cores were taken in saltmarsh, seagrass, mangroves and unvegetated areas around the German Bight, Malaysia and Columbia in 2022 and 2023. Up to 3 points per ecosystem were sampled along a transect, in total 93 cores were analysed. Carbohydrates were sequentially extracted using MilliQ-water and 0.3 M EDTA for later analyses. The total carbohydrate content was assessed using the phenol-sulfuric acid assay (Dubois et al., 1956). Briefly, 100 µL of resuspended samples or extracts were mixed with 100 µL of 5% phenol solution, followed by the addition of 500 µL of concentrated sulfuric acid. The reaction mixture was incubated at room temperature for 10 minutes, then further incubated at 30°C for 20 minutes. Absorbance at 490 nm was measured using a Spectramax Id3 plate reader (Molecular Devices) and quantified against a glucose standard curve.

Monosaccharides quantified in sediment cores from coastal vegetated ecosystems

50-cm deep sediment cores were taken in saltmarsh, seagrass, mangroves and unvegetated areas around the German Bight, Malaysia and Columbia in 2022 and 2023. Up to 3 points per ecosystem were sampled along a transect, in total 93 cores were analysed. Carbohydrates were sequentially extracted using MilliQ-water and 0.3 M EDTA for later analyses. Extracts were acid hydrolysed (1 M HCl, 24 h, 100°C) and monosaccharides were analysed using anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), according to Engel et al., 2011. Briefly, sample analysis was performed using a Dionex ICS-5000+ system with a CarboPac PA10 analytical column (2 × 250 mm) and a CarboPac PA10 guard column (2 × 50 mm). Neutral and amino sugars were separated under isocratic conditions with 18 mM NaOH, while acidic monosaccharides were separated using a gradient up to 200 mM NaCH₃COO.

Polysaccharide fucoidan BAM1 and arabinogalactan-protein glycan JIM13 antibody binding in sediment cores from coastal vegetated ecosystems

50-cm deep sediment cores were taken in saltmarsh, seagrass, mangroves and unvegetated areas around the German Bight, Malaysia and Columbia in 2022 and 2023. Up to 3 points per ecosystem were sampled along a transect, in total 93 cores were analysed. Carbohydrates were sequentially extracted using MilliQ-water and 0.3 M EDTA for later analyses. For more specific analysis enzyme-linked immunosorbent assay (ELISA) was used for detection of fucoidan and arabinogalactan-protein glycan as described in the following studies (Vidal-Melgosa et al., 2021 and Cornuault et al., 2014). In short, 100 µL of sediment extracts were added to a pre-coated 96-well plate and incubated overnight at 4°C. The signal was developed using primary antibodies, BAM1 (fucoidan) and JIM13 (arabinogalactan-protein glycan), diluted 1:10 in skim milk PBS solution, followed by anti-rat antibody at a 1:1000 dilution in the same solution. Absorbance was measured at 450 nm using a Spectramax Id3 plate reader (Molecular Devices).

Polysaccharides quantified in sediment cores from coastal vegetated ecosystems

50-cm deep sediment cores were taken in saltmarsh, seagrass, mangroves and unvegetated areas around the German Bight, Malaysia and Columbia in 2022 and 2023. Up to 3 points per ecosystem were sampled along a transect, in total 93 cores were analysed. Carbohydrates were sequentially extracted using MilliQ-water and 0.3 M EDTA for later analyses. Polysaccharides were screened using microarray analysis following the method described by Vidal-Melgosa et al. (2022). Briefly, sediment extracts from MilliQ-water and EDTA were combined in equal volumes, and 30 µL of the mixture was transferred into wells of 384-microwell plates. Two consecutive two-fold dilutions were performed using a printing buffer (55.2% glycerol, 44% water, 0.8% Triton X-100). The plates were then centrifuged at 3,500 × g for 10 minutes at 15 °C. Each microarray was individually probed with a monoclonal antibody (mAb), and binding was detected using a secondary antibody conjugated to alkaline phosphatase. In the presence of its substrate, this reaction produced a colorimetric signal. Developed arrays were scanned at 2400 dots per inch, and binding signal intensity was quantified using Array-Pro Analyzer 6.3 software (Media Cybernetics).