Detection and quantification of microcystins in the mucilage of colonial cyanobacteria

This study investigated the detection and quantification of microcystins (MCs) within the mucilage of a Microcystis-dominated bloom, employing methods to separate and quantify attached extracellular polymeric substances (EPS) from cyanobacterial cells. The lowest MCs concentration was measured on the filter by classical filtration (112.44 ± 4.94 μg L^-1) and methanol extraction, representing free MCs (intracellular and associated with EPS of the mucilage) and MCs adsorbed onto large environmental particles. Centrifugation followed by lyophilization of the pellets significantly elevated particulate MC concentrations (153.98 ± 15.65 μg L^-1), likely due to the inclusion of more environmental particles on which MCs can be adsorbed. However, fixation and extended solubilization (10-60 min) by heating further increased particulate MC concentrations, yielding values 1.6 to 1.9 times higher than those obtained via classical filtration, depending on the solubilization duration. It demonstrated a significant contribution (up to 72 %) of MCs associated with attached EPS probably via non-covalent binding. These results indicated that alternative methods such as centrifugation, fixation, lyophilization and heating allow for the quantification of MC forms not captured by simple filtration. Additionally, a shift in MC variants between intracellular and attached EPS was observed. We discussed the implications of these findings, particularly the role of heating in detecting mucilage-associated MCs, its impact on MC quotas and bioavailability, and the degradation of MCs associated with EPS by heterotrophic bacteria.