Spectroscopic Insights into Nutrient-Controlled Biofilm Formation in Aureobasidium pullulans as a Basis for...

Fungal biofilms represent complex extracellular systems with potential application in engineered living materials (ELMs), yet their chemical composition and structural variability under defined environmental conditions remain poorly understood. Biofilms of Aureobasidium pullulans strains grown on different solid media were investigated using a multimodal spectroscopic approach, including FT-IR, FT-NIR, and Raman spectroscopy. Nutrient-rich media (PDA, SDA, YNB) produced biofilms with largely conserved chemical profiles across strains, whereas nutrient-poor SNA medium induced pronounced changes in polysaccharide, protein, and lipid components, as well as in matrix organization and hydration. Principal component analysis and multiblock data fusion revealed that growth medium is the primary driver of spectral variation, with strain identity exerting a secondary influence. Vibrational bands corresponding to hydroxyl (-OH) and aliphatic (-CH) groups were identified as key contributors to differentiation, reflecting changes in the composition of Extracellular Polymeric Substances (EPS). Raman spectroscopy provided complementary molecular information, such as the presence of characteristic protein and polysaccharide vibrations, which helped confirm the biochemical composition of the biofilms and supported the observations from IR and NIR analyses. Results show that the control of nutrient composition and growth conditions enables precise tuning of biofilm properties, including hydration, matrix density, and functional polysaccharide content. Biofilm composition is primarily influenced by nutrient composition rather than by the strain's morphological features. This is important because it indicates that research on living coating formulation can focus on supplying nutrients that promote EPS production and biofilm development, rather than on selecting strains with particular morphologies. These findings provide a foundation for understanding the environmental control of A. pullulans biofilm formation and are relevant to the future development of engineered living coatings, where environmental conditions can be leveraged to influence biofilm composition, structure, and its functional properties.