Surface colonization of Aureobasidium pullulans: a multimodal microscopy study toward living coating development.

Recent advances in engineered living materials (ELMs), which integrate living cells into functional structural and protective systems, have accelerated interest in understanding microbial surface interactions at fundamental scales. In this context, Aureobasidium pullulans, a polymorphic and polyextremotolerant black yeast fungus, has emerged as a promising candidate for diverse biotechnological applications, including engineered living coatings. However, its microscale surface colonization dynamics remain insufficiently characterized, limiting predictive control over colonization of various surfaces. Complementary optical approaches, including VHX digital imaging, fluorescence observation, and quantitative fluorescence measurements, were used to characterize temporal colonization of fungus on plastic coverslips and pine wood, representing two contrasting substrates used in the building sector. On plastic substrates, initial attachment involved dispersed cells that rapidly proliferated and merged into a continuous layer. Quantitative fluorescence revealed a significant increase in signal from Day 1 to Day 3 (from 25,328 to 42,510 RFU; P = 5.4 × 10⁻⁴) followed by a decrease by Day 6 (10,555 RFU). This decline may be associated with reduced dye penetration into the compact, melanized matrix rather than a reduction in biomass. On wood, colonization followed the native fiber orientation and progressed into cohesive multicellular structures. The results provide a new understanding of substrate-dependent colonization of A. pullulans and highlight methodological limitations in quantifying biomass on porous, heterogeneous materials. The multimodal microscopy framework established a robust comparative platform for analysing fungal-material interactions and enabled the rational development of fungal-based living coatings for protective and functional applications.