Cell wall characterization of brewery wastewater-cultivated Nannochloropsis: Implications for integrated microalgal wastewater treatment and biorefinery

Microalgae are increasingly cultivated on wastewaters to simultaneously recover nutrients and generate valuable biomass. However, cultivation under wastewater conditions can alter cell-wall physiology and composition, directly influencing downstream processing and bioproduct recovery. Despite their importance, the effects of wastewater cultivation on microalgal cell wall composition and ultrastructure have never been investigated. In this study, the cell wall of lipid-rich Nannochloropsis limnetica cultivated in standard Bold's Basal Medium (BBM), 50% brewery wastewater (BWW), and 100% BWW was characterized using complementary microscopic, chemical, and enzymatic analyses. Electron microscopy showed that the two-layered cell wall ultrastructure was maintained across all conditions, although cells grown in 100% BWW exhibited an 18% increase in wall thickness compared to BBM. Sulfuric acid hydrolysis indicated cultivation-dependent differences in monosaccharide composition, with BBM-grown biomass displaying a glucose-dominated composition. Selective enzymatic hydrolysis showed that glucan-containing polysaccharides represented the dominant enzyme-accessible carbohydrate fraction within the isolated cell wall, with cellulase consistently showing the highest monosaccharide release. Analysis of cell wall-associated amino acids further indicated cultivation-dependent variations in protein-associated components, with considerable differences observed in glycine, histidine, glutamic acid, and aspartic acid under BWW cultivation. Overall, these findings show that wastewater cultivation is associated with compositional changes in the isolated cell wall fraction while the overall cell wall ultrastructure and major chemical functionalities remain largely conserved.