Page 177 - IJB-10-5

International Journal of Bioprinting Control nutrients to manipulate fungal growth

machines, they may need to also be compartmentalized guerrilla-type behavior in terms of being fast growers and
with various properties and functions within one ELM, long-range foragers while others such as Tricholomella
calling for material heterogeneity and localized growth constricta and Leucopaxillus genetianeus exhibit phalanx-
of cells. There is therefore a need to be able to control the type behavior as they have the tendency to grow slowly and
22
growth and location of mycelium on materials to enable branch profusely. These species and nutrient-dependent
production of new types of ELMs. variations in mycelium morphology could therefore be
used to tailor the local distribution and morphology of
A common method to create localized properties in mycelium for future fungal-based ELMs.
a material is to use multi-material 3D printing. By using
methods including fused deposition modeling (FDM) In this work, we investigated the growth of mycelium
and direct ink writing (DIW), materials such as plastics from two fungal species Ganoderma lucidum (G. lucidum,
and hydrogels can be precisely positioned in a 3D space, Lingzhi mushroom) and Pleurotus ostreatus (P. ostreatus,
allowing for the fabrication of complex structures. 11,12 With oyster mushroom) in 3D-printed structures containing
multiple nozzles, additional materials and compositions local variations of nutrient content. More specifically,
can be incorporated into the same process, allowing for mycelium-containing inks were developed with varying
the fabrication of a multi-material structure. 13–15 The concentrations of malt and peptone as growth media. These
utilization of 3D printing in fungal-based ELMs with inks were then precisely locally deposited using DIW. The
variations in material, mycelium inoculation techniques, control and tailoring of the mycelium density, growth
and scale has been gaining traction. For example, direct behavior over acellular gaps containing different levels of
inoculation involves the addition of mycelium into the ink nutrients, and mycelium growth in various directions were
prior to the fabrication of the structure. 8,16–18 As for indirect demonstrated. Finally, intentionally patterned structures
inoculation, the structure is first printed before it is placed were fabricated as proof-of-concept examples of what
in direct contact with separately cultured fungi. The fungi this approach can achieve. While fundamental, this work
then grow toward the structure, eventually enveloping it can be leveraged to create more complex types of fungal-
in the process. 19,20 However, the use of multiple nozzles in based ELMs to manipulate properties such as self-healing
fabricating complex, multi-material fungal-based ELMs capabilities and to explore new applications.
has yet to be explored. On the contrary, it is generally
found that mycelium materials grow a homogeneous 2. Materials and methods
fungal skin on their surface that prevents local tailoring of 2.1. Materials
their properties. Exploring ways to create multi-material The following items were purchased from Sigma Aldrich
types of fungal-based ELMs are thus needed. (USA): malt, peptone, agar, carboxymethyl cellulose

In nature, organisms have developed various (CMC), sodium alginate, calcium chloride, and gentamicin
growth, foraging, or translocation strategies to survive sulfate. P. ostreatus spawn bags were obtained from
in ecosystems that may not provide continuous supply Bewilder (Singapore), whereas G. lucidum was acquired
of nutrients. When faced with an environment that is from Malaysian Feedmills Farms (Malaysia).
21
either abundant or lacking in nutrients, filamentous fungi 2.2. Mycelium culture
tend to exhibit two main foraging behaviors: phalanx and In this study, mycelium was cultured on both solid agar
guerrilla. 22,23 Phalanx, also known as exploitative growth, is substrates and in liquid cultures. For solid agar substrates,
characterized by a slow extension rate of hyphae and high malt agar plates were made by dissolving agar (2.5% w/v),
mycelium density. Guerrilla, also known as explorative malt (2.0% w/v), and peptone (0.1% w/v) in deionized
growth, is characterized by a fast extension rate of water. The mixture was then autoclaved (Hiclave HG 80,
hyphae, which are the single filaments in the mycelium Hirayama, Japan) at 121°C for 20 min and poured into
network, but a low mycelium density. In a low-nutrient 90 mm Petri dishes. The mixture was allowed to cool and
environment, guerrilla is the dominant growth pattern set before they were inoculated with a solid agar plug of
whereas guerrilla is usually observed when the fungus is 9 mm diameter containing mycelium from a previously
in a high-nutrient environment. 8,22,24,25 Fungi are able to inoculated plate. For liquid mycelium cultures, malt broth
switch between these foraging behaviors depending on the was made by dissolving malt (1.7% w/v) and peptone (0.3%
environment they encounter. 26,27 While available nutrients w/v) in deionized water and was autoclaved at 121°C for
do dictate the type of growth behavior, the degree to which 20 min. After cooling, the broth was supplemented with
fungi explore or exploit their environment also depends on gentamicin sulfate (50 µg/mL), followed by the addition of
the species. 22,28,29 In a similar environment, species such as a solid agar plug containing mycelium. Liquid mycelium
Coprinus angulatus and Psilocybe cf. subviscida may exhibit cultures were then left to grow under constant magnetic

Volume 10 Issue 5 (2024) 169 doi: 10.36922/ijb.3939