Visualization of extracellular polymeric substances in Aspergillus niger biofilms using fluorescent-labeled lectins

Aspergillus niger is a filamentous fungus that adheres to different substrate surfaces and form biofilms consisting of dense hyphal networks embedded in a self-produced gelatinous matrix composed of extracellular polymeric substance (EPS). The EPS mainly contains exopolysaccharides. The objective of this study was to visualize and assess the different exopolysaccharides structure in this extracellular polymeric substance using a combination of two different fluorescent-labeled lectin probes, Erythrina cristagalli (ECA) and Canavalia ensiformis (Con A). Each lectin is a unique protein that binds to specific a carbohydrate moiety and is classified based on these terminal sugars to which they are binding. Exopolysaccharides are imaged according to their distinct fluorescence color with the help of confocal laser scanning microscopy (CLSM). The biomass, average thickness, and roughness of biofilms were calculated from the Z-stack images using the computer program COMSTAT. A statistically significant difference was observed in the biomass (μm ³ /μm ² ), average thickness (μm), and roughness of the biofilm treated with the two different lectin probes indicating the presence of a higher amount of galactose and β-1,4 N-acetylgalactosamine (β4GalNAc) when compared to the mannose and glucose moieties in the EPS of A.niger biofilm. The combination of two lectin-fluorescent probe method staining should help better characterization of A. niger biofilms in terms of their heterogeneity with regards to the EPS production.