New insights into the effects of growth phase and enzymatic treatment on the cell-wall properties of Chlorella vulgaris microalgae

Green microalgae are a natural source of oil for commercial biodiesel production. However, their cell-wall barrier remains a major obstacle for effective intracellular lipid extraction. Solution to this issue includes the use of surface-digesting enzymes and the control of the so-far poorly understood changes in structural and mechanical properties of the microalgal envelops during growth and upon action of enzymes. Here, we used a combination of atomic force microscopy (AFM) and confocal microscopy to decipher the variation of cell-wall ultrastructure, composition and nanomechanics of Chlorella vulgaris upon culture ageing and lysozyme treatment. AFM imaging revealed the presence of a fibrillated mesh at the surface of cells harvested in the stationary phase, hardly distinguishable on cells from younger culture (mid-log phase). The fibrils form a chitin-like network containing the N-acetyl-D-glucosamine unit, and this structured network is severely damaged upon lysozyme treatment, a property we identified at the cellular and molecular scales by fluorescent-lectin staining and AFM-based force spectroscopy using lectin-modified tips, respectively. The enzyme was also found to act on algal physiology, to trigger oxidative stress and changes in cell lipid content. Detailed single-cell mapping of the nanomechanical properties of C. vulgaris further indicated that microalgal cells soften upon ageing and it confirmed that lysozyme affects both their surface and intracellular compartments. Altogether, our results emphasize that AFM-based multi-parametric analysis in combination with confocal microscopy allows accurate evaluation of physico-chemical surface properties of microalgae and offers an exciting perspective on cell culture condition optimization for facilitated oil extraction.