Inoculum-to-interface distance tunes productivity in static liquid cultures of wood-decay fungi
Static liquid cultures of wood-decay fungi underpin applications in materials, food, and medicine, yet the mechanisms governing pellicle formation remain poorly understood, limiting rational optimisation at scale. We developed a time-lapse scanning and image analysis platform to visualise the process of pellicle formation across five white-rot fungal species, of which Trametes versicolor formed the thickest, heaviest pellicles and completed formation fastest. Kymographs revealed a reproducible four-stage sequence after inoculation and sedimentation of the biomass: recovery of viability, upward growth, pellicle initiation, and thickening. We formalised this as a model in which the time to pellicle establishment is determined by the distance between the settled biomass and the liquid-air interface. Increasing liquid depth relative to a fixed inoculum loading raised biomass and mat thickness, but prolonged formation. The results identify surface proximity as the dominant predictor of initiation timing. By fixing the inoculum at the liquid-air interface with a membrane, we validated that minimising the distance between the sedimented mycelium and the liquid surface accelerated and stabilised formation without compromising on pellicle biomass. By exposing the most relevant control variables, this work provides a quantitative basis for designing reliable liquid-state surface fermentations for emerging applications involving mycelium.