Fungal mycelium processing: heat pressing and additive manufacturing for enhanced material properties

Fungi play a crucial role in the ecosystem for nutrient cycling, symbiotic relationship with plants, and decomposing lignocellulose. Fungal mycelium is the vegetative part of the fila-mentous fungus, which consists of an intricate network of hyphae. In recent years, fungal mycelium has attracted large attention for its excellent material properties and for being a promising and sustainable alternative in various applications. It can be cultivated either on nutrient abundant liquid and agar-based media in laboratory settings, or on agricultural and forestry side streams, or even on food wastes like coffee ground. One of its most promising applications is in the development of fungal mycelium-based composites. They can be manufactured using filamentous fungi that colonize lignocellulosic substrates such hemp shives, sawdust, rice husk, and rice straw in shape-defined molds. Following cultivation under mild conditions and a subsequent drying process, the resulting composites are light-weight and biodegradable with good insulating properties. Pure fungal mycelium material can be obtained by separating the aerial mycelium layer from the fungal mycelium-based composites, or be prepared from liquid cultivation media. Due to its poor mechanical prop-erties, it requires post-processing to make it suitable for products like leather substitutes. Furthermore, dependence on molds limits design flexibility. This dissertation explores mechanisms and strategies to improve the mechanical properties of fungal mycelium-based materials derived from the basidiomycete Fomes fomentarius, as well as methods for the flexible fabrication of fungal mycelium-based composites with re-spect to cultivation, material fabrication and post-processing methods. It examines the in-fluence of heat pressing on pure fungal mycelium mats grown on different cultivation me-dia; the improvement of mechanical properties through material extrusion additive manu-facturing (MEX AM) by depositing bio-based polymers onto fungal mycelium mats; and the enhancement of manufacturing flexibility through extrusion-based additive manufacturing (AM) using both inactive and living fungal mycelium. Heat pressing is one of the simplest and the most effective post-processing steps to im-prove the mechanical properties of fungal mycelium-based materials. The mechanisms un-derlying heat pressing, however, remain poorly understood, especially for pure fungal myce-lium mats. To study the effects of heat pressing on fungal mycelium, as well as the influ-ence of the cultivation substrate, pure fungal mycelium mats from lignocellulose-based sol-id state and glucose-based liquid cultivation of F. fomentarius were heat-pressed at 160 °C at 100 MPa for 5 min. Heat pressing significantly increased the tensile strength, with notable differences observed between fungal mycelium mats cultivated in liquid medium and solid substrate. Liquid-cultivated fungal mycelium showed a denser hyphal network with higher chitin content and thus higher tensile strength after heat pressing. In contrast, solid-cultivated mycelium led to a looser hyphal network, higher β-glucan content, and lower tensile strength after heat pressing. These differences might be due to variations in gene expression stimulated by the nutrient supply. Besides heat pressing, a new approach to enhance the mechanical strength of grown fungal mycelium mats was proposed in this study. Biodegradable polymers poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) and polylactic acid (PLA) were deposit-ed onto the surface of fungal mycelium mats in a specific arrangement via MEX AM. These load-path optimized depositions of PHBH and PLA increased the tensile strength of the pure fungal mycelium mats along the direction of the printed lines by a factor of 2.8 and 4.8, respectively, without sacrificing the biodegradability of the fungal mycelium material. Surface roughness characterization also revealed distinct behaviors of different polymers on the fungal myce-lium mat surface. To improve the design flexibility of fungal mycelium materials and to reduce the depend-ence on molds, extrusion-based AM was also applied on both inactive and living fungal mycelium. Extrudable pastes from inactive F. fomentarius mycelium and alginate were de-veloped, with rheology and homogeneity tests identifying a solid content of 71 wt% mycelium and 29 wt% alginate as optimal. This formulation showed shear-thinning behavior, good viscosity recovery, and high stackability without collapse. Complex geometries were printed with nozzle diameters as small as 0.58 mm. The crosslinking of alginate by calcium chloride was confirmed by Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS), enhancing water and thermal stability of the printed structure. To maintain a sustainable and a bio-based approach for extrusion-based AM with living fungal mycelium, rapeseed straw and sodium alginate were used as nutrient supply and rhe-ological tuner respectively. F. fomentarius mycelium showed good growth on the extruded three-dimensional structure, forming a thick layer of aerial hyphae that was able to be peeled off from the scaffold for use as a pure three-dimensional mycelium shell, as needed. The predefined shapes were well maintained when combining manual compressing of the mycelium during the growth stage with a subsequent freeze-drying step for dehydration and deactivation. The resulting compact fungal mycelium composites and pure fungal mycelium shell were ultra-lightweight, with compressive or tensile strength comparable to, or even exceeding, those of fungal mycelium-based materials prepared using other methods. In short, this work demonstrates that altering the cultivation medium can change the com-position of fungal mycelium from the same strain, and that the mechanical properties of pure fungal mycelium mats can be further improved by heat pressing as well as through combination with bio-based polymers via MEX AM. This dissertation provides procedures for producing lightweight fungal mycelium composites from F. fomentarius, and for produc-ing pure fungal mycelium mats in complex shapes via extrusion-based AM. These findings further broaden the application of fungal mycelium in the field of sustainable materials, leading to increased performance and greater design flexibility.