Growth, morphology, and mechanical evolution of Candida albicans and Penicillium rubens fungal biofilms on hydrogels with tunable stiffness

Fungal biofilms are adaptive, soft, three-dimensional composites that exemplify living multiphase matter. While their biological regulation is well studied, the role of ambient environmental mechanics in governing fungal architecture remains underexplored. We examine how substrate stiffness and porosity influence growth, viscoelasticity (G*), and morphology in Candida albicans and Penicillium rubens, clinically and industrially relevant fungi. Agar hydrogels (1–5 % w/v; G* ≈ 10²–10⁵ Pa) served as tunable substrates for 21-day growth. Both species formed radial patterns with a distinct, dense rippled core and a filamentous periphery. Penicillium hyphae elongated fastest on stiff gels, whereas Candida preferred soft ones, reflecting opposite mechanosensitivities. Our results illustrate how substrate stiffness and porosity may fundamentally impact biofilm architecture and matrix organization, hyphal network density, and mechanical properties. Shear rheology revealed region-specific moduli increasing with substrate stiffness, accompanied by decreased pore size and enhanced network density. Intriguingly, preliminary time-resolved analysis of overall biofilm development suggests transformation or remodeling to preferred mechanical states. Overall, our findings establish mechanical interactions between the biofilm and the substrate as a fundamental driver of fungal biofilm development and motivate further studies on the mechanistic underpinnings of fungal biofilm growth.Planned