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International Journal of Bioprinting Control nutrients to manipulate fungal growth

the structure. Meanwhile, G. lucidum was observed to better P. ostreatus form thicker mycelium, but the mycelium
conform to the 3D-printed structure. Thus, the curves and sheet formed was only dense near the inoculating agar
contours of the 3D structure remained visible even after plug. The increase growth resulting from the increase in
complete coverage by the mycelium of G. lucidum. This peptone was less pronounced for G. lucidum. However, for
observation demonstrates the different foraging strategies both strains, malt had a significant effect on the mycelium
that each fungus employs in a similar environment. growth regardless of the concentration of peptone with the
Furthermore, while P. ostreatus readily colonized the air- mycelium layer becoming opaque at high levels of malt.
material surfaces, it had a lower tendency to colonize the To quantify these growth behaviors, the growth rate
cramp spaces within the macropores of the structure. This (the area of mycelium present on the agar plate) was
is shown by the sparse mycelium found in the cross-section recorded during the 14 days of growth (Figure 2B). Most
of the macropores (Figure 1E). In contrast, the mycelium growth profiles display a sigmoid curve that is typical
of G. lucidum was equally dense on both the top and inside across different organisms, which was also observed for
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the macropores of the underlying structure (Figure 1E). both fungi in this study. There was an initial lag phase,
However, growth of the mycelium of P. ostreatus in the during which the mycelium from the agar plugs probably
macropores was possible, provided that they are sufficiently initially adjusted to the new environment, followed by an
large (see Figure S6, Supporting Information). exponential growth phase, and a stationary phase that was

Having demonstrated the possibility of preparing inks reached when the mycelium had covered the entire area of
for 3D printing and growing mycelium effectively for two the Petri dish. The growth rate of P. ostreatus was faster at
fungal species with different growth behaviors, the effect low concentrations of malt and peptone as its growth curve
of the nutrient composition present in the inks on their was higher than for the other concentrations and was the
growth was then studied. quickest to plateau (Figure 2B, top). As a fast growth rate is
a known attribute of guerrilla-type behavior, this suggests
3.2. Nutrient concentration controls the foraging that the fungus adopted an explorative foraging behavior
behavior of P. ostreatus and G. lucidum in this environment. When the concentration of malt
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Various media and substrates are suitable for fungal was high, the growth rate became slower but appeared
cultures. Availability and assay types are among the factors to be independent of the concentration of peptone. The
considered when determining the choice of media. Among growth rate was the slowest when the concentration of
the plethora of media used for fungal culture, a mixture of malt was low, and the concentration of peptone was high.
malt and peptone are commonly used in the literature and Surprisingly, for G. lucidum, mycelium grown on agar with
is also chosen here to vary the nutritional content of the high concentrations of malt has a faster rate than those
inks. Malt serves as a source of polysaccharides to provide grown on agar with low concentrations of malt (Figure 2B,
energy for the fungi while peptone acts as a nitrogen bottom). This may seem to contradict the theory that low-
source for the synthesis of proteins and other components nutrient environments lead to a faster growth rate due to
inside the fungal cell. To study the growth of the two fungi the fungus exhibiting guerrilla foraging. However, other
P. ostreatus and G. lucidum when these nutrients are varied, studies in the literature also suggest that this theory still
solid-state mycelium growth on agar plates containing low holds for ranges of malt concentration that are higher than
and high levels of malt and peptone were studied (Figure the one used in this study. This suggests that at low malt
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2, see Figure S7 and time-lapse videos in Videos S1 and S2, concentrations, nutrient deficiency may have hindered
Supporting Information). the growth rate of mycelium. Nevertheless, the mycelium
Agar plates with four levels of nutrient concentrations density remained sparser at low-nutrient conditions, which
were prepared following a two-level full factorial design is another attribute indicating guerrilla-type foraging
where malt and peptones were varied in low and high behavior besides high growth rate. Like P. ostreatus, the
concentrations only. The agar plates were inoculated at growth rate was the slowest when the concentration of
their center by adding an agar plug containing mycelium. peptone was high while the concentration of malt was low.
Differences in the mycelium formed from these four To further quantify the exploitative foraging behavior of
nutrient levels could be readily observed for both species the fungi, the dry biomass of the mycelium sheet formed in
(Figure 2A). At low levels of malt and peptone, both G. each condition was obtained after 14 days of culture (Figure
lucidum and P. ostreatus formed thin and sparse mycelium. 2C). At low malt concentrations, an increase in peptone
From the naked eye, the mycelium was translucent, with caused a non-significant increase in the thickness of the
the underlying agar still visible, suggesting it to be very thin mycelium formed. Malt had a greater influence on the dry
and fragile. Increasing the concentration of peptone helped biomass harvested for both strains of fungi investigated

Volume 10 Issue 5 (2024) 173 doi: 10.36922/ijb.3939