Isotopic labeling of cultured macroalgae and isolation of 13C-labeled cell wall polysaccharides for trophic investigations

Abstract

Macroalgae are considered as major primary producers in coastal environments, acting as a global carbon sink. This abundant biomass contains up to 50% of storage or cell wall polysaccharides, which therefore represent a reservoir of organic matter for potential algal consumers. Tracking of natural isotopic abundance (δ 13 C vs. δ 15 N) in macroalgae-colonized habitats such as kelp forests and rocky shores previously evidenced the importance of the algal resource to support local and adjacent trophic webs mostly via the microbial detrital pathway. However, such bulk isotopic measures of natural abundances cannot inform precisely on the microbial actors and processes at play for the degradation of selected algal compounds. To overcome these limitations, we developed a stable isotope labelling procedure for cultures of the brown alga Laminaria digitata and tested its ability to yield labelled polysaccharides. Sporophytes of L. digitata were grown in controlled conditions for three months in seawater regularly amended with 13 C-labelled sodium bicarbonate. Elemental analysisisotope ratio mass spectrometry of algal specimens showed a significant enrichment after 10 days of treatment, reaching a maximum of At% 13 C = 3.5321% after 80 days. Sequential polysaccharide extraction from this labelled algal biomass allowed retrieving both alginate and fucose-containing sulphated polysaccharide fractions with high isotopic enrichment (At% C = 3.6279% and 3.5868%, respectively). This labelling protocol opens the way for future studies combining coastal ecosystems trophic interactions and microbial activities towards macroalgal biomass degradation.

Isotopic tracing reveals single-cell assimilation of a macroalgal polysaccharide by a few marine Flavobacteria and Gammaproteobacteria

“…Natural ( 12 C-natural) and 13 C-enriched alginates were prepared as described previously [ 40 ]. Briefly, the kelp Laminaria digitata was cultivated with either natural (98.9 12 C%) or 13 C-labeled (99 13 C%) NaHCO 3 .…”

“…The absence of inhibitors was confirmed by checking the growth of the alginolytic strain Zobellia galactanivorans Dsij T in minimum medium supplemented with 12 C-natural or 13 C-enriched alginate (1 g l −1 ). Alginate isotopic ratio was measured by elemental analysis coupled to isotope ratio mass spectrometry (EA-IRMS) as described previously [ 40 ]. 12 C-natural and 13 C-enriched alginate had 1.1337 ± 0.0004 and 3.6279 ± 0.0009 13 C atom percent, respectively (mean ± s.d.…”

Production of isotopically enriched high molecular weight hyaluronic acid and characterization by solid-state NMR

“…Thus, isotopic enrichment with 13 C and 15 N is essential to allow application of advanced NMR experiments. While 13 C and 15 N isotopic enrichment is a well-established strategy for structure determination of proteins, fewer methods are known to produce other labelled biomacromolecules, including polysaccharides (Pomin et al, 2010; Thomas et al, 2020). Efficient isotope enrichment strategies for HA (and other polysaccharides) promise to facilitate the deployment of similarly advanced NMR methods to this important and growing field of research, spanning biology, pharmaceutical and other (industrial) applications.…”

Probing rapid carbon fixation in fast-growing seaweed Ulva meridionalis using stable isotope 13C-labelling

“…After the hydrolysis of ulvan, the radioactivity of Rha was higher than that of GlcA, indicating that Rha deposition occurs earlier than GlcA deposition. Recent advances in analytical instruments such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) allow for a more comprehensive analysis of fluxes of metabolites and material exchanges between organisms by stable isotope labelling [26][27][28] . Natural 13 C distribution is only 1.07%, but concentrating 13 C makes structural analysis of metabolites by 13 C-NMR spectroscopy easier.…”