Investigating the Nanoscale Dynamics of Flocculation with Pyridinium-Modified Cellulose Nanocrystals.

Microalgae show significant potential as a sustainable resource for the production of food, animal feed, biofuels, and several high-value products. However, the lack of effective harvesting techniques limits the large-scale production of microalgae. A strategy to enhance the separation of microalgae from their growth media is to flocculate the microalgae cells into larger particles that can then be separated from water by sedimentation or flotation. Understanding the flocculation mechanism is crucial for developing a more efficient separation methodology. To this end, we applied atomic force microscopy (AFM) to probe and localize the interactions between negatively charged AFM tips and the surface of cells in contact with varying concentrations of cationic cellulose nanocrystals (CNCs), a novel type of biobased flocculant. AFM force spectroscopy experiments performed at high speed and with high spatial resolution allowed to detect the presence of the cationic nanoparticles on the cell surface. While the height images recorded proved the presence of an added material on the cell surface, which was further quantified by roughness measurements, adhesive maps correlated these structures with cationic charges detected by the anionic tips. These results allowed to determine that the flocculation occurs when the CNCs form cationic patches on an otherwise homogeneous anionic cell surface and represent the first direct evidence of the patch mechanism. These insights provide a foundation for the rational and dose-efficient design of biobased flocculants for large-scale microalgae harvesting. Additionally, this work establishes the AFM methodology developed here for probing nanoscale flocculant-cell interactions at high resolution, enabling a deeper understanding of the molecular mechanisms driving flocculation.