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International Journal of Bioprinting Control nutrients to manipulate fungal growth

3. Results and discussion its addition also decreased the rigidity of the overall ink
(see Figures S2–S4, Supporting Information). Thus, high
3.1. Ink formulation and 3D printing strategies for concentrations of agar and CMC were incorporated to
fungal-based ELMs provide the bulk of the viscosity and rigidity of the ink
To enable the 3D printing via DIW of aqueous inks while sufficient alginate was added to enable the structure
containing fungal cells to create ELMs, it is important to be rigid after printing and crosslinking using calcium
to first design an ink system that simultaneously allows
the safe growth of the fungi and satisfies the 3D printing chloride.
requirements. The ink should therefore contain the The composite inks exhibited a shear-thinning behavior,
nutrients necessary for the fungus to grow and develop its which made them suitable for 3D printing via DIW (Figure
mycelium while the ink should also exhibit the rheological 1B). The viscosity profile of the ink was also independent
properties necessary for their extrusion through the nozzle of the concentration of malt and peptone incorporated. As
and their shape retention after 3D printing (Figure 1). G. such, the printing parameters did not need to be adjusted
lucidum and P. ostreatus were two fungal species selected when modifying the concentration of malt and peptone,
for this investigation, owing to their extensive use in the which made it convenient for multi-material 3D printing.
fabrication of mycelium-bound composites and fungal- Furthermore, the inks had solid-like properties as the
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based ELMs, and their availability. Indeed, G. lucidum is storage modulus was greater than the loss modulus (G’ >
known for its medicinal properties in traditional Chinese G’’) in the linear viscoelastic region for all inks (Figure 1C).
medicine and P. ostreatus is a popular mushroom used in Therefore, the inks did not spontaneously flow under the
various cuisines. influence of gravity after the printing process, allowing for

The ink for producing the fungal-based ELMs was buildability and high shape fidelity. Indeed, various well-
formulated to contain agar, alginate, and CMC (Figure 1A). defined shapes could be printed using the finalized ink
Agar is a polysaccharide known to be suitable for fungal compositions (see Figure S5, Supporting Information).
growth. For this ink, it provides necessary stiffness and The 3D-printed structures can be inoculated with
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strength for achieving the desired rheological properties mycelium either before the printing process (i.e., mycelium
of the ink. As the ink cannot be printed at elevated spawn or liquid culture is incorporated into the ink before
temperatures owing to the presence of mycelium in the printing) 8,16–18,38 or after printing (i.e., mycelium spawn is
ink during printing, 33,34 the agar was present as solid gel embedded into the 3D-printed structure or the structure
particulates, which tend to form discontinuous filaments is placed in direct contact with mycelium that was cultured
when extruded. To remedy this, CMC was added to act separately). 19,39 In this study, the ink was inoculated directly
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as a rheological modifier, allowing for a smooth extrusion with the liquid mycelium culture being incorporated
of the ink at room temperature (see Supplementary File, into the ink. Compared to direct inoculation using solid
Figure S1). Alginate was also supplemented to act as a spawns, the risk of clogging was lower when using the
crosslinking agent that undergoes gelation in the presence liquid culture (due to the heterogeneity of the ink caused
of calcium chloride. 35,36 After printing, calcium chloride by the added spawns), and the homogeneity of the ink was
solution was sprayed onto the construct to fix the printed maintained. This is especially important in this study as a
shape to prevent sagging or deformation until the mycelium 22G nozzle (0.41 mm internal diameter) was used. Based
fully colonized the structure. Conveniently, the calcium on Figure 1D, the ink containing medium levels of malt
ions from calcium chloride have also been reported in the and peptone can effectively support the growth of both P.
literature to stimulate mycelium growth, which provides ostreatus and G. lucidum. After 4 days, white, fluffy spots
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an additional benefit to the process. The moisture from were seen at various parts of the surface of the structure as
the calcium chloride spray also helped provide a moist the mycelium began to grow. The mycelium then eventually
environment for mycelium growth and gentamicin was enveloped the entire surface of the structure. These
incorporated into the spray to prevent contamination preliminary experiments that were meant to demonstrate
before the mycelium was fully grown. This is especially the 3D printing of the inks have already revealed the
beneficial when the structure is printed in a non-sterile different growth or foraging behaviors from the two fungi.
environment, away from biosafety cabinets, as conducted
in this work. The ratio of each component was tailored Indeed, P. ostreatus had a greater tendency to form
based on each component’s contribution to the rheology hyphae that spread away from the structure and extend into
of the overall ink. Agar had the greatest contribution to the air once the surface was fully covered by the mycelium.
the viscosity and rigidity of the ink, followed by CMC. The lack of any specific direction in the tip extension of
While alginate was essential as the crosslinking agent, it the hyphae resulted in the underlying structure being less
had the least contribution to the rheology of the ink and pronounced after the mycelium had completely enveloped

Volume 10 Issue 5 (2024) 171 doi: 10.36922/ijb.3939