Mycelium Scaffolds for Improved Viability and Control over Interior Microarchitecture of Biomineralized Engineered Living Materials

Engineered living materials (ELMs) offer new strategies for manufacturing more sustainable building materials. ELMs that are biomineralized by leveraging the metabolism of microbes can generate simple, low-load bearing structures. Biomineralized ELMs may be useful for more complex applications if they surmount two major limitations. The first limitation is transient cell viability. Cells in previous biomineralized ELMs did not survive more than a few days to weeks limiting limits how long these cells could perform desirable functions (self-healing or environmental sensing). A second limitation is a lack of control over internal microarchitecture. Biomineralized composites in nature can optimize their strength while remaining lightweight due to specialized microarchitecture (bone, coral, nacre). Fungal mycelium used as a scaffold for biomineralization might address both limitations. Some fungi, including Neurospora crassa, can perform biomineralization. N. crassa scaffolds may have excellent viability characteristics, since N. crassa is able to survive a wide range of environmental conditions. The scaffold may also protect the viability of other biomineralizing microorganisms, such as the bacterium Sporosarcina pasteurii. Fungal scaffolds may also introduce new strategies to design the internal geometries of biomineralized materials, as these scaffolds can be grown and then shaped.
We created biomineralized, fungally-scaffolded ELMs using two strategies. The first strategy employed a living mycelium scaffold that mineralizes itself. The second uses a non-viable mycelium scaffold that is mineralized by S. pasteurii. The viability of living components (N. crassa or S. pasteurii) within these biomineralized ELMs was analyzed after drying for 4-weeks at either room temperature or elevated temperature (30°C). Both biomineralized ELMs showed abundant culturable cells after drying for 4-weeks at either temperature condition. Mineralization efficiency, determined by comparing calcium and urea usage over time, was greater for the bacterially-mineralized fungal scaffolds. Biomineral microscale morphology and moduli were analyzed using scanning electron microscopy with elemental dispersive spectroscopy and nanoindentation, respectively. Bacterially-mineralized ELMs had more mineral accumulation and both larger and stiffer biomineral crystals than fungally-mineralized scaffolds. These results demonstrate that biomineralized ELMs with excellent viability can be created using fungal scaffolds mineralized either by the fungus or by bacteria, but bacterial biomineralization generates mineralized material more efficiently.
Bacterial biomineralization of mycelium was then used to create mineralized fungal scaffolds with a complex interior microarchitecture inspired by osteons within cortical bone. Osteons were chosen as a model since this structure optimizes mechanical and biological performance in the skeleton. Mycelium scaffolds were shaped and then mineralized with S. pasteurii to form artificial osteons from columns with concentric rings and a central canal. Sand was used as an interphase between the osteons and the entire structure was biomineralized using S. pasteurii. This strategy created biocemented specimens (4”x1”x1”) with a designed internal microarchitecture, as demonstrated by electron microscopy and microCT.
This work establishes the potential of fungal scaffolds for the manufacturing of biomineralized ELMs with improved viability and designability. Both types of ELMs (self-mineralized living fungal scaffolds or bacterially-mineralized killed fungal scaffolds) have potential as useful ELMs with excellent viability characteristics. The improved viability characteristics of these scaffolds, together with the formability of mycelium into useful shapes that confer control over internal microarchitecture, may enable the design of new materials that surmount design limitations of previous biomineralized ELMs.