Team:Stanford-Brown-RISD/Demonstrate

From large scale mycelium growth and modeling, to functionalizing proteins bound to mycelium, our team was able to achieve many of our largest goals for the project. We were also able to coordinate a comprehensive mission architecture that accounts for the harsh and complex task of space travel with mycelium. Below we have demonstrated how we achieved some of our project’s greatest feats.

Mycelium Material & Habitat Development

In growing mycelium, we were able to grow bricks, plates, and stools in only a number of weeks (Figures 1 and 2). We were also able to grow many of these structures using a variety of interesting substrates, like wood chips, yard waste, and sawdust. We also managed to grow Mycelium on Martian regolith, using supplemental nutrients, an idea we believe is novel. Further, we were able to model the growth rate of mycelium in different conditions in order to determine the optimal growth parameters for mycelium (Figures 3 and 4). This information helped inform us as to how the mycelium would fair in Martian conditions. This turn influenced our mission architecture and the design of the habitat itself (finding ways of providing sufficient heat and moisture, etc).


Figure 1: Pile of bricks produced with mycelium and yard waste, wood chips.	Figure 2:Team member Emilia Mann sitting on the stool after it has been baked.


Figure 3: Surface Area of dikaryon, Ganoderma Lucidum mycelia growth over 4 different temperature conditions	Figure 4: Surface Area of monokaryon, Ganoderma Lucidum mycelia growth over 4 different temperature conditions

Mycelium Glue & Mycelium Based Bio-Filtration

In addition to developing mycelium as a material for habitat construction in space, we were also able to functionalize the material by synthesizing fusion proteins that linked a chitin binding domain (CBD) to various different functional proteins. The CBD binds chitin, which makes up the outer cell wall of fungus and our mycelium material, thus allowing CBD fusion proteins to bind to our mycelium. Among our most successful fusion proteins were the CBD fused to three copper binding domains and the CBD fused to the bacterial biofilm adhesion protein csgA.

When the CBD-copper binding fusion protein was applied to our mycelium, it allowed it to act as a scalable, completely biodegradable filtration system for waste metal recovery from aqueous solution. Copper was used as a proof of concept for metal binding, as it is a metal of significant biological and economic importance, as well as a part of many circuit boards and a known contaminant in water. We demonstrated the copper-binding capacity of our mycelium filter system using isothermal titration calorimetry (ITC) and a Phen Green copper concentration assay (Figures 5 and 6). These quantitative tests demonstrated our mycelium filter’s ability to bind 92% of the available copper in 30 minutes.

Another successful CBD fusion protein we synthesized the was CBD-csgA protein, which yielded an effective and mycelium-specific bioadhesive. We demonstrated this by conducting lap shear adhesive strength tests and comparing the relative strength of the adhesive on different materials like mycelium and cardboard (Figure 7).


Figure 5: ITC data for the three HHTC-Re peptides. The binding affinity (K_a) was assessed to determine the strength of the interaction, and the results follow: 1x-HHTC-Re K_a = (1.55 ± 0.21) x 10⁶ M^-1; 2x-HHTC-Re K_a = (3.73 ± 0.53) x 10⁵ M^-1; 3x-HHTC-Re K_a = (1.50 ± 0.05) x 10⁵ M^-1. Figure design credits to Jesica Urbina.	Figure 6: Cu (µM) remaining in solution for n = 3 samples after incubation for 30 minutes while undergoing tangential flow on a flatbed shaker (figure design credits: Jesica Urbina). Experimental conditions were maintained as described previously.

Figure 7: Relative Mycelium Adhesive Strength. After testing the adhesive strength of each bioadhesive candidate on mycelium, we tested each candidate again on cardboard. We then took the ratio of adhesive strength on mycelium to adhesive strength on cardboard to determine the relative efficiency of the two substrates.