Liquid-state surface fermentation as a platform for pure mycelium materials production

Liquid-state surface fermentation (LSSF) is emerging as an alternative to submerged and solid-state fermentation for producing pure mycelium materials (PMM), yet a systematic framework linking culture geometry, substrate consumption, inoculum preparation and material properties is lacking. Here, we characterised LSSF with Trametes versicolor across culture volumes and inoculation strategies. The biomass accumulation model described a trade-off between colonisation speed and biomass accumulation. Smaller volumes, with a higher specific interfacial area, colonised the surface earlier and converted glucose more efficiently (up to 0.64 g g -1 ), but depleted it prematurely and produced less biomass. After pellicle formation, glucose was consumed at a common volumetric rate (~1.0 g L -1 d -1 ), having glucose availability as a limiting factor for pellicle densification. In deeper cultures, much of the glucose consumed early was not recovered in the pellicle, indicating competition from submerged growth. Backlit transmittance imaging provided a non-destructive estimate of pellicle biomass and of its spatial heterogeneity. Inoculum source and processing determined material performance: liquid-derived inocula and a shaken incubation step increased biomass, an additional blending step produced more homogeneous and stronger pellicles. These results provide process-level design rules for LSSF and a basis for its application to PMM production.