GoKawiilChemicals and reagents
Fucoidan from F. vesiculosus (Sigma-Aldrich, F8190, lot no. 0000485452) was used as the primary substrate throughout this study. Additional F. vesiculosus fucoidans were obtained from Marinova (FVF2021547) and Biosynth (YF57714). Fucoidans from other species were sourced from Fucus serratus (Biosynth, YF09360), Fucus evanescens (OceanBasis and extracted as described previously60), Cladosiphon okamuranus (Biosynth, YF146834), Durvillaea potatorum (Biosynth, YF157165), Ecklonia maxima (Biosynth, YF157166) and Laminaria hyperborea (TheFucoidanStore, LowEndo Fucoidan). For derivatization, 1-phenyl-3-methyl-5-pyrazolone (PMP) was obtained from Sigma-Aldrich (M70800). Internal standards for LC–MS analysis included d-galactose-13C 6 (Sigma-Aldrich, 605379), d-mannose-13C 6 (Sigma-Aldrich, 592994) and PMP-[d 5 ] (CAS 1228765-67-0), custom-synthesized by BOC Sciences (Shirley). LC–MS-grade solvents and reagents were acetonitrile (Honeywell), methanol (Honeywell), ethanol (Sigma-Aldrich), formic acid (Sigma-Aldrich) and ammonium formate (Merck). Ultrapure water was produced using a Q-POD system (Merck). Unless otherwise stated, all other chemicals were of analytical grade and sourced from Sigma-Aldrich.
Bacterial growth media
Throughout this study, three distinct media detailed in Supplementary Table 1 were used for (1) enrichment of bacteria, (2) isolation of bacteria on solid medium and (3) routine cultivation of bacterial isolates in MBL medium52. All media mimicked the ionic composition of coastal seawater and contained 340 mM NaCl, 15 mM MgCl 2 , 6.75 mM KCl and 1 mM CaCl 2 . The pH was buffered to 8.0 with either bicarbonate in the enrichment and plate media or 50 mM HEPES in the MBL medium. Nutrients were ammonium chloride, sodium phosphate, sodium sulfate, trace metal mix and vitamin mix52. Enrichment medium contained 0.02% (w/v) fucoidan from F. vesiculosus. Solid medium was prepared with 2% (w/v) carrageenan (Sigma C1013) and a mix of carbon sources (acetate, citrate, xylose, galactose, mannose, glucose, fucose, cellobiose and tryptone) at 0.002% (w/v) each. Bacterial isolates were routinely cultured in MBL medium with carbon sources specific to their metabolic requirements. Verrucomicrobiota degraders were routinely grown with 0.2% (w/v) fucoidan from F. vesiculosus. Other degraders (Flavobacteriia and Gammaproteobacteria) and exploiters were grown on a mix of 0.2% (w/v) l-fucose and 0.2% (w/v) fucoidan from F. vesiculosus. Scavengers were grown in a mix of carbon sources (acetate, citrate, xylose, galactose, mannose, glucose, fucose, cellobiose and tryptone) at 0.02% (w/v) each.
Enrichment of fucoidan-degrading communities
Surface seawater was collected on 30 March 2019 from a rocky shoreline covered with the brown algae F. vesiculosus, Ascophyllum nodosum and Saccharina latissima near the Marine Science Center of Northeastern University (Canoe Beach, Nahant, MA, USA; 42° 25′ 10.8732″ N, 70° 54′ 25.686″ W). The seawater was pre-filtered through a 10-μm PTFE membrane filter (Millipore, JCWP04700) and diluted 1:100 to inoculate three replicate enrichment cultures (25 ml each) containing 0.02% (w/v) F. vesiculosus fucoidan in 150 ml glass bottles sealed with rubber stoppers. Cultures were incubated at 20 °C in the dark and agitated at 50 rpm.
Enrichment cultures were monitored every 24–48 h for microbial growth (OD 600 ) and fucoidan degradation, quantified via the phenol–sulfuric acid method61. For each measurement, 200 µl of culture was mixed with 1 ml of concentrated sulfuric acid and 200 µl of 5% (v/v) phenol, then incubated at 50 °C for 20 min. Absorbance was measured at 490 nm and fucoidan concentration was determined using an external standard curve of fucose. After 10 days, cultures reached 40–60% degradation of the initial fucoidan, at which point serial growth–dilution cycles were initiated by transferring 1:50 into freshly prepared medium every 2 days for a total of 12 cycles. At every second time point, 10 ml of culture was filtered onto a 0.22 μm Sterivex filter (Millipore, SVGPB1010) for DNA extraction using the DNeasy Blood & Tissue Kit (Qiagen) and subsequent metagenomic sequencing. Final enrichment communities were cryopreserved at −80 °C with 15% (v/v) glycerol.
Isolation of bacterial strains
From each enrichment culture, cells were enumerated using a counting chamber by light microscopy. Aliquots corresponding to 102, 103 and 104 cells were plated in triplicate onto solid medium in 150 mm Petri dishes (VWR 391-0616) and incubated for 14 days at ambient temperature in the dark. A total of 768 colonies were picked and re-streaked at least 3 times until only a single colony morphotype was observed. Colonies were lysed in 0.1% (v/v) Triton X-100 in TE buffer for Sanger sequencing of the 16S rRNA gene. Pure isolates were grown in MBL medium with a mix of carbon source and cryopreserved at −80 °C in 15% (v/v) glycerol. To de-replicate repeatedly isolated strains, we selected 96 isolates based on their 16S sequences for genomic DNA extraction using the DNAadvance kit (Beckman Coulter) and draft genome sequencing. To identify redundant isolates, pairwise average nucleotide identity (ANI) was computed using OrthoANIu v1.262 yielding 28 unique bacterial strains. We additionally included the characterized degrader ‘Lentimonas’ sp. CC4 as Verruco4 in the isolate collection3 resulting in a total of 29 strains in the isolate collection.
Sequencing of metagenomes and isolate genomes
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