Strain, substrate, and matrix selection for controlled growth of wood-fungi

Fungal fermentation offers a promising approach for the development of engineered living materials (ELMs). The design of the substrate materials to support and enhance fungal growth in both 2D and 3D is essential to realize this potential. We evaluated the mycelium of seven edible mushroom-forming fungi for growth vigor as a function of various abiotic factors. Growth assays using standard malt agar, with varying concentrations of carbohydrates and proteins, revealed that the radial expansion of the fungal is affected by the carbohydrate concentration, showing a maximum expansion rate at mid-low concentrations and a diminishing expansion rate at higher concentrations. In contrast, higher carbohydrate concentrations increased mycelium density. Different plant-based proteins also significantly influenced growth vigor, i.e. the mycelium's thickness and expansion rate. Beyond chemical substrate conditions, we modified the substrate viscoelasticity by increasing agar concentration, which resulted in higher growth proliferation. This was further confirmed using non-standard gelling agents such as guar gum, corn starch, κ-carrageenan, and bacterial cellulose. In a final step to enhance growth for practical applications, we foamed an optimized substrate material for 3D growth, achieving successful growth throughout the entire matrix. This work provides a framework to aid the selection of edible substrate materials for fungal growth, i.e. the design of engineered living materials.