Extracellular polymeric substances (EPS) from algae are complex, secreted, heteropolymers that can represent a significant fraction of biological extracellular carbon. These biopolymers play a tremendously important role in antiviral and antimicrobial bioactivities in vivo influenced by molecular properties like composition, molecular weight (MW), and size. Understanding the structure-function relationship of these polymers remains challenging due to their heterogeneity, high MW, high charge density, and native high-ionic strength environment. Here, we introduce an innovative, non-destructive approach to reducing the complexity and characterizing EPS using a size-based separation technique, asymmetrical flow field-flow fractionation (AF4) with online multi-angle light scattering (MALS) followed by offline compositional analysis using chromatographic techniques.
In this study, AF4-UV-MALS-dRI was used to fractionate and evaluate the different size populations present in Chlorella vulgaris EPS. Fractions were analyzed to probe compositional differences using high-performance anion exchange chromatography (HPAEC), and liquid chromatography-mass spectrometry (LCMS). The study exploits AF4’s flexibility in solvent choice to investigate the effects of the ionic strength and composition of the carrier fluid on the aggregation behavior of these biopolymers to better understand their interactions in their native, higher-salinity, environments. The AF4 results demonstrated that the EPS of C. vulgaris has three populations spanning a molecular weight range from 4 x 10⁴– 3 x 10⁸Daltons, including an ultra-high MW fraction that has not previously been reported for algal EPS. Offline compositional analyses of the primary structures revealed heterogeneity in monosaccharides and amino acids between the different size populations. Distinct ion-mediated behaviour was observed among the fractions in the different ionic environments revealing EPS retention was affected by both ionic strength and the chao-/kosmotropicity of the electrolytes. We report for the first time that Chlorella cultures exhibit different isolated polymers, with distinct primary, secondary, and tertiary structures, and MW distributions, suggesting distinct biological functions among the polymer distributions.

Lieve Laurens

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