Biophysical Properties of Material‑Colonizing Fungal Biofilms: Multiscale Structural and Mechanical Characterization

Abstract Black extremotolerant fungi form persistent biofilms on a wide range of natural and engineered substrates. Able to weather minerals, affect stone monument surfaces, and colonize solar panels, they demonstrate a strong capacity to interact with and modify material surfaces, even under most extreme conditions. In this study, we establish a methodological workflow for the structural and mechanical characterization of melanized biofilms formed by the black fungus Knufia petricola. This species represents a broader group of resilient surface colonizers and provides a model for in‑depth investigation. When grown on the solid agar/air interface, this species predominantly forms a compact biofilm, composed of spherical cells, while retaining the capacity for filamentous growth, providing a suitable framework to explore morphology-dependent biomechanical responses. The proposed toolbox combines complementary analytical techniques spanning multiple spatial scales, including shear rheology to quantify bulk viscoelastic behavior, microindentation to resolve local stiffness of the biofilm surface, microcomputed tomography for nondestructive three-dimensional visualization of biofilm architecture, and cryogenic preparation methods and electron microscopy for high-resolution ultrastructural analysis. As a methodological case study, we applied this workflow to compare biofilms grown on two different nitrogen sources (NO3− vs NH4+). Our results reveal nitrogen-associated differences in biofilm organization across multiple hierarchical levels, ranging from biofilm morphology and bulk mechanical behavior to cellular morphology and EPS distribution. NO3−-grown biofilms presented more filaments with cells exhibiting budding-like morphologies and higher bulk viscoelastic moduli, whereas NH4+-grown biofilms were thicker with meristematic-like morphologies and a softer, more shear-tolerant mechanical response. The successful application and combination of complementary methods originally developed for bacterial biofilm research demonstrates that quantitative multiscale characterization can be extended to melanized fungal biofilms. Ultimately, this multiscale toolbox will help in advancing the mechanistic understanding of fungal biofilms and biofilm−material interactions, with implications for geomicrobiology, material biodeterioration, and the design of bioinspired functional materials.