Analysis and Applications of Fungal Materials

Analysis and Applications of Novel Fungal Materials
Christopher Perez
Laurent Pilon's Research Group
Mechanical and Aerospace Engineering Department
CARE Scholars, Spring 2016
June 13, 2016

2 | Christopher Perez
Abstract
In an effort to reduce the environmental impact associated with fossil fuel-dependent materials,
fungal materials have been gaining recognition for their potential use in a wide range of
applications such as construction and consumer products. The fungal root structure of
interwoven tissue known as the mycelium, plays a significant role in the fungi’s ability to rapidly
digest a variety of organic materials, acting as a bonding agent for those materials. To generate
novel fungal composite materials, the non-toxic fungal strain Ganoderma lucidum was grown as
a liquid inoculant. This inoculant was used to colonize petri dish samples containing potato
dextrose agar, plastic molds filled with UCLA agricultural waste, and glass jars enclosing a
proven substrate of brown rice flour, vermiculite and water. The agricultural waste consisted of 1
cm and 0.5 cm miscellaneous branch pieces and crushed Liquidambar styraciflua fruit. The
thermal and mechanical properties will be measured and used to assess the fungal materials for
specific applications. Three plastic molds were inoculated with Ganoderma lucidum and
destroyed to reveal that the effectiveness of liquid inoculant for colonizing agricultural waste
substrate. Two glass jars with the proven substrate inoculated with Ganoderma lucidum were
grown, rendered inert by convective heat treatment, and destroyed to gain insight on the type of
growth involved. Four glass jars of the same substrate and inoculant were grown with
temperatures ranging from 23.9 − 28.3 ℃ and were rotated every 12 and 24 hours to notice an
effect on inoculant dispersal and the homogeneity of the resulting material.
Word Count: 250

1. Introduction
It is predicted that sixty percent of the world’s population will live in cities by the year 2030 [1].
From the nature of large metropolitan cities, this translates to a significant amount of municipal
food waste production. In the United States, 25-50% of the food produced is wasted, having a
financial effect totaling a yearly $165 billion [1]. As cities grow larger in population, the need for
renewable resources to control food waste becomes more of an immediate concern. This food
waste alone accounts for 25% of the total yearly freshwater consumption and 2.5% of the total
energy budget of the United States [1]. Much of this waste takes the form of plastics such as
polystyrene, constituting approximately 25 – 30% of the space used up in landfills by volume
[10].
The development of technologies capable of transforming food waste into added-value products
is necessary to ensure sustainable supplies of food, energy, and water for the growing cities of
the world. Not only would such technologies divert food waste from landfills and reduce
greenhouse methane gas production by preventing their decomposition, but would also save both
energy and water by ensuring that this waste is used as a resource.
The growth of fungi on organic waste shows promise as a method to sustainably produce added-
value products. These products include construction materials such as thermal insulation and
particle boards, dinnerware such as disposable cutlery, and packaging material. These materials
can assume desired shapes by being grown in molds, thus avoiding the need for machining. To
even consider the use of mushrooms as a construction material however, the thermophysical
properties of such materials are of paramount importance. The aim of the study presented here is

to establish a comprehensive growth method that will provide insight on the biological
mechanisms that dictate the thermophysical properties of the generated fungal material. The
study is performed in the hopes that this material characterization will provide a basis for the
appropriate applications of future renewable fungi materials.
2. Background
2.1 Mycelium Life Cycle
Fungi are diverse organisms varying tremendously in their environment, form, and nutritional
requirements. These organisms however, are unified by sharing the common trait of consuming
living or lifeless organic matter and are viewed as the key recyclers and decomposers of
materials in the world [7]. Chlorophyll-based organisms use many tightly bound sugars to form
lignin and cellulose from their ability of transforming sunlight into sugars and other molecules.
Through the use of hyphal cells, fungi that nourish themselves with the cellulose from these
organisms colonize their nutrient source as their hyphae develop and propagate. Connections are
made among the hyphae as they secrete digestive enzymes to break down both lignin and
cellulose in order to create chitin, the molecule that comprises their fungal cell walls. Doing so
enables hyphal interweaving, eventually leading to the creation of a fungal root structure known
as mycelium. It is this mycelial cellular fabric that is utilized for material generation for its
strong structure and binding characteristics with the substrate on which it feeds [8].
2.2 Growth Conditions

Although there are numerous studies focusing on mycelium growth dynamics, they tend to view
this growth in an ecological or nutritional sense. From this, various growth conditions have been
identified and much is known about the growth of fungi specifically for the harvesting of their
fruiting body. Chief among the requirements for optimal fungal growth are carbon sources such
as sugars, polysaccharides, and organic acids and alcohols. These carbon sources may take the
form of agricultural or food waste known as the fungal substrate. In their natural environments,
fungi can also be exposed to a wide range of temperatures depending on seasonal variations. As
such, most fungi generally have a maximum growth temperature of around 30 − 40℃ [3]. Light
has also been commonly observed to reduce the rate at which fungi spread either by
photochemical destruction of components of the medium or having a direct effect on metabolism
[3].
2.3 Fungi as a Building Material
Rigorous research on fungal materials and their characterization is sparse and generally lacking
due to their relatively novel nature. Commercially, there exist businesses for producing,
developing, and marketing these renewable biomaterials. Ecovative Design® is one such
company as it uses this mushroom technology to create alternatives for plastics in areas such as
packaging, thermal insulation, and engineered wood. Due to the possibility of copyright
infringement, many of these companies prevent the full disclosure of the exact processes by
which their products were derived, let alone their material characteristics. Few academic reviews
on such a field exist that are specifically concerned with producing fungal materials and
measuring their material properties for added-value products.

Holt et al [4] set out to characterize fungal materials derived from six different size blends of
agricultural residue from cotton plant material processing and two inoculation methods using
liquid or solid Ganoderma lucidum fungus inoculant. The material generation process, developed
by Ecovative Design®, involved sterilizing the substrate, inoculating said substrate, packing the
mixture into a sealed mold, waiting until the substrate is fully colonized, and subsequently
heating the material to render it inert. The resulting physical and thermal characteristics of the
material confirmed the viability of cotton-based fungal mycelium packaging material as an
alternative to polystyrene packaging. Pelletier et al [5] examined the use of these fungal
materials in acoustic absorption applications. Produced in a similar method to that of Holt et al
described earlier, the significant relationship between the generated fungal material density and
machining quality with respect to substrate selection was noted as various types of substrate were
used. Travaglini et al [9] modeled mycelium material as a composite material comprised of a
mycelium foam matrix with reinforcing substrate fibers and the mechanical properties of the
material were tested. The process of creating the fungal material was developed by Mycoworks®
and shares many similarities with that of Ecovative Design®, with the addition of a nutrient
solution during colonization. The mechanical measurements revealed fungal material properties
closest to expanded polystyrene foam and its robustness was predicted to be improved by means
of substrate control post-processing.
Overall, the few studies in the literature for organically derived fungal materials delve into
preliminary detail. Therefore, it remains clear that more research is needed to properly
characterize fungal materials if they are to be considered a dependable renewable resource to
rival and mitigate the use of fossil fuel-dependent plastics.

3. Methods and Materials
3.1 Substrate Selection and Preparation
The chosen substrates were rye grain, a mixture of organic brown rice flower and vermiculite,
and UCLA agricultural waste. Figure 1 shows how the agricultural waste was divided into three
groups: 1cm and 0.5cm miscellaneous branch pieces, and crushed “gum tree” (Liquidambar
styraciflua) fruits.. The UCLA agricultural waste substrates were sized according to the
aforementioned dimensions and contained in separate glass jars. They were then sterilized with a
pressure cooker at 15 psi for 30 minutes, reaching temperatures of up to 121℃ or 250℉ . The
organic brown rice flower/vermiculite mix was purchased pre-sterilized from
EverythingMushrooms®, a mycological vendor based in Knoxville Tennessee. Figure 2
illustrates how the organic brown rice flower/vermiculite mix was contained in glass half pint
jars.
3.2 Growth and Culture Conditions
The isolated strain, Ganoderma lucidum was used for all of the experiments detailed in this
paper. The Ganoderma lucidum strain was kept as both a liquid and petri dish culture. For
inoculation purposes however, strictly liquid culture was used to better disperse the mushroom
spores for more effective and uniform substrate colonization. By transferring a pure petri dish
culture of the Ganoderma lucidum strain, more cultures were created and used to provide a
constant fungal reserve. These petri dishes however, would be subsequently transformed into a
liquid culture by blending a fully colonized petri dish with 400mL of de-ionized water as
illustrated in Figure 3. All cultures are kept in the dark at approximately 20℃ while waiting to
be used in inoculation. After sample inoculation, all of the samples, with the exception of the

four samples for homogeneity experiments, were kept in the same environment as the culture
itself.
The four samples for the homogeneity experiments were kept in environments with hotter-than-
ambient temperatures. These temperatures ranged from 23.9 − 28.3 ℃ (75 − 83 ℉) and were
achieved by using a Phillips® 100 watt heat bulb. This higher temperature environment was
found by Jayasinghe et al [2] to be the most ideal temperature range to promote mycelium
growth. The samples were shielded using a cardboard container with hole cut outs to
accommodate gas exchange. A K-type thermocouple was calibrated, affixed to the side of the
cardboard enclosure, and used to measure the temperatures within. Figure 4 illustrates this
experimental setup to produce this higher temperature environment.
3.3 Created Sample Molds
Several molds were created specifically for fitment around the guarded hot plate for thermal
conductivity measurements, discussed later in this study. The molds are made of high
temperature Teflon® PTFE tubing and are able to withstand temperatures of up to
260℃ or 500℉ for sterilization purposes. The tubing is 2 inches inner diameter and cut to 3
inches long and plugged on one end using high temperature silicone able to handle temperatures
of up to 302℃ or 575℉. The silicon plugs slide 1.5 inches into one end of the cylindrical mold
while the rest of the mold is filled with substrate. One end of the cylindrical mold was sealed
with Parafilm® plastic film to facilitate gas exchange and prevent contamination. Figure 5
displays the created molds.

3.4 Inoculation Processes
To prevent the possibility of airborne contamination, a still-air environment was created by
modifying a 66 quart plastic storage container to resemble a glovebox. All agar petri dish
transfers, substrate handling, and inoculations were performed in this still-air environment. The
fungal liquid cultures were stored in a syringe where a controlled amount could be easily
dispensed. The created molds were filled with their respective substrates, 30.2 grams of 1 cm
miscellaneous branches, 25.83 grams of 0.5 cm miscellaneous branches, and 7.64 grams of
crushed “gum tree” (Liquidambar styraciflua) fruits. The molds were then inoculated by
dispersing 10 mL of liquid Ganoderma lucidum culture made from a transferred agar petri dish
into each mold.
The three glass jars containing the organic brown rice flower/vermiculite substrate were
inoculated using liquid Ganoderma lucidum culture. For inoculation, 8 mL of inoculant was
injected into each of the jars, using the adhesive tape included on the jar covers to inoculate the
substrate. Since there are four inoculation ports on the top of the jars, 2mL of inoculant was used
for each hole. After inoculation, the samples are kept in the dark and allowed to be observed for
when full colonization occurs.
The four glass jars containing the organic brown rice flower/vermiculite substrate were
inoculated, again, using liquid Ganoderma lucidum culture. These samples were subsequently
“rolled” for 15 minutes; the jars placed on their sides and spun laterally to further disperse the
inoculant inside the jars. Again, 2mL of inoculant was used for each inoculation port. After
inoculation, the samples were kept in the environment described in the previous section 3.2,
“Culture Conditions”.

3.5 Glass Jar Sample Preparation and Destruction
The three glass jars containing the organic brown rice flower/vermiculite substrate and
inoculated using liquid Ganoderma lucidum culture were used to create the very first round of
fungal materials. Only two of these samples achieved 100% colonization after six weeks and
three days and were subsequently rendered inert via convective heating. This protocol for heating
was followed closely from that of Travaglini et al [9], and included removing the samples from
their jars and placing them in a convection oven at 220℃ (428℉) for 120 minutes [10]. After
cooling, the samples were then cut in half using a small hand saw and the resulting fragments
collected. The samples were then observed under an optical microscope at 250x magnification to
analyze the type of mycelium growth, the prevalence of edge effects, the homogeneity of the
colonization, and the degree of substrate binding within the sample.
3.6 Methods for Improving Homogeneity
The four glass jar samples used for homogeneity experiments were grown very similarly when
compared with the three glass jar samples described previously. These samples differed in two
ways from the three glass jar samples. First, after inoculation, the samples were kept in the
environment described in the previous section 3.2, “Culture Conditions”. Second, in an effort to
improve inoculant dispersal generate a more homogenous material, half of the samples were
rotated 180° upside down every 12 and the other half every 24 hours. Figure 6 describes the
general protocol for this homogeneity experiment.
3.7 Measuring Thermal Properties
To measure one of the most important thermal properties of interest, thermal conductivity, a
guarded hot plate apparatus will be used. The device was designed to comply with ASTM C177-

13 [6]. The guarded hot plate approach for measuring thermal conductivity requires two identical
samples to be secured between hot and cold plates. By strategically inserting thermocouples in
the sample at known distances and observing the linear function of voltage for each
thermocouple. The steady-state thermal energy dissipated in the hot plate is then ensured to be
evenly distributed between the two identical samples. Modifying Fourier’s law, the thermal
conductivity may be evaluated using the following expression
k𝑖𝑖(T�) =
q𝑖𝑖L𝑖𝑖
A�T2,𝑖𝑖 − T1,𝑖𝑖�
with 𝑖𝑖 = A or B
Where qi is the heat transfer rate�
W
m
� through the sample 𝑖𝑖 and L𝑖𝑖 is the distance the
thermocouples measuring T1,𝑖𝑖 and T2,𝑖𝑖 [6]. A schematic for this guarded hot plate setup is shown
in Figure 7.
4. Results and Discussion
4.1 Material Generation Process
The general protocol for creating these fungal materials was closely modeled after the processes
detailed by commercial entities such as Ecovative Design® and Mycoworks®. This process
involves sterilizing the desired substrate, as previously described. Under very sterile conditions,
achieved by a still-air environment to prevent airborne contamination, the sterilized substrate is
placed in the desired mold as liquid inoculant is carefully dispersed inside via a syringe. For the
created molds, 10 mL of liquid inoculant was used for approximately 50 grams of substrate. The
pre-sterilized substrates possess their own containers while 10 mL of inoculant was used for the
glass jars containing organic brown rice flower/vermiculite mix. The inoculated plastic mold

sample is then sealed with Parafilm® plastic film to prevent contamination. The samples are then
kept in the dark until full colonization is achieved. When this occurs, the sample will be
subjected to high temperature convective heating to make sure no further growth occurs. The
sample will be post-processed by making sure the desired geometry is attained, thus
accommodating density measurements by providing reliable dimensions.
4.2 Destructive Investigation of Created Molds
After implementing the material generation process described in the preceding section, three
samples were inoculated using each of the agricultural waste substrates (1 cm miscellaneous
branch pieces, 0.5 cm miscellaneous branch pieces, and crushed Liquidambar styraciflua fruits).
The samples were left to colonize for 2 weeks and 5 days. Since this was the first trial and the
samples showed little signs of life, a destructive investigation was conducted to confirm the
effectiveness of liquid inoculant on agricultural waste. It was revealed that the liquid inoculant
was only effective in one out of the three inoculated samples: the sample inoculated with crushed
Liquidambar styraciflua fruits. Although this sample showed signs of life, the fungus was
characterized by abnormally slow growth. As seen in Figure 8, it is apparent that the mycelium
had just barely begun to grow after almost 3 weeks. This slow growth, and the complete lack of
growth from the other two samples, was attributed to excessive water and blending when
creating the liquid inoculant. The fully colonized plate/400 mL de-ionized water mix was
deemed too saturated with water and the blending process too harsh. Despite the faults in
preparing the liquid inoculant, mycelium growth was still achieved and demonstrated the
extreme adaptability of fungi as they grew on what was once considered waste.

4.3 Post Mortem Analysis of Glass Jar Samples
After six weeks and three days, 100% colonization was achieved and the jars were emptied of
their contents. The immediate observation from the generated materials upon releasing them
from their jars was that they was extremely weak and lacked rigidity. This was attributed to the
granular choice of substrate that provided a light mycelial bonding. Another observation was the
presence of what seemed to be edge effects. These edge effects were characterized by heavy
colonization at the glass/substrate interface. The interior of the sample however, was highly
granular and seemed to exhibit light bonding as pieces of the samples would tear off. After the
convective heat treatment described in section 3.5, “Glass Jar Sample Preparation and
Destruction”, the samples were observed to be calcined. Figure 9 shows the two samples before
and after heat treatment.
Upon destruction of the sample, the edge effects were confirmed when cutting the sample in
half. A dense mycelial mat was seen to encapsulate the sample and the transition from the center
substrate colonization to the mat edge was not gradual, but sudden. This was further established
with the use of an optical microscope at 250x magnification. In addition to this, observing the
colonization, it was noticed that the mycelium occupied much less volume than the host
substrate. This is attributed to the hard grain composition of the substrate that likely made it
more difficult to be fully consumed by the mycelium. The heat treatment was deemed too
extreme since the calcination was seen on a microscopic level. Figure 10 demonstrates how the
mycelium was burned off and left residue on the substrate, while the substrate seemed to only
have lost moisture content.

5 Future Work
Thus far, a superficial investigation of fungal materials has been conducted. Although there has
been much trial and error when growing the fungi, the use of pre-sterilized substrates has vastly
expedited the material generation. This technique however, does not allow for complete control
over substrate selection. This is especially detrimental to the theme of renewable materials as
substrates comprising of agricultural waste are likely to be used. For the immediate future
however, the samples utilizing pre-sterilized substrates will be used to gain a familiarity with the
post processing of fungal materials as well as to possess a physical sample. Doing so will
simplify the measuring of the material properties as different substrates and growing conditions
will be explored. The conditions explored thus far include hotter temperature environments and
intermittently changing the orientation of the sample to ensure a more even inoculant dispersal to
aid in the pursuit of a more homogenous fungal material.
More complex post-processing of the generated fungal material will also be investigated in an
attempt to produce a less dense material: a desirable quality for thermal insulation applications.
This includes creating a process for infiltrating the developing stages of mycelium growth by
including non-organic, less dense material as part of the substrate. Initially, non-organic material
is expected to be expanded polystyrene due to very small density and mycelium’s inability to
digest it. Varying amounts of this non-organic material will be mixed with the substrate to notice
an effect in the density of the resultant material. Although the use of expanded polystyrene goes
against the theme of renewable sustainability, it will be experimented with until an organic or
less prolific substitute is found.

Figures
Figure 1: UCLA Agricultural waste substrate used for cylindrical molds.( From left to right) 1 cm miscellaneous branch pieces,
crushed “gum tree” (Liquidambar styraciflua) fruits, and 0.5 cm miscellaneous branch pieces.
Figure 2: Half pint glass jars containing the organic brown rice flower/vermiculite substrate that were inoclated with liquid
Ganoderma lucidum culture.

Figure 3: The creation of the liquid inoculant. (Left) Modified jar to facilliate the blending of the colonized agar plate and water.
(Center) The blending attachment affixed to electric drill press. (Right) the final liquid inoculant pruduct stored in plastic
syringe.
Figure 4: The setup to achieve a hotter temperature environment for the homogeneity experiment samples. (Left) the Phillips®
100 W heat lamp bulb shining on top of the cardboard sample enclosure. (Right) the K-type thermocouple, enclosed in a blue
circle, affixed to the wall of the enclosure to measure temperatures within.

Figure 5: The design of the plastic molds made from 2” ID high temperature Teflon® PTFE tubing and high temperature silicon
plugs.The un-plugged end is to be sealed with Parafilm® plastic filmfor gas exchange and contamination prevention.
Figure 6: The general protocol for attempting to create a more homogenous fungal sample consisting of inoculating the
substrate and rotating the sample every 12 and 24 hrs.

Figure 7: The setup used to measure the eﬀective thermal conductivity of composite based materials using the guarded hot
plate method [6].
Figure 8: Destructive investigation of the inoculated mold sample containing crushed Liquidambar styraciflua fruits.Although
very little mycelium growth took place, the ability of fungi to grow on this substrate was demonstrated as the sample was left to
colonize for 2 weeks and 5 days.

Figure 9: The glass jar samples after six weeks and three days of growth and subsequent convective heat treatment

Figure 10: (Top left) Half cut sample after convective heat treatment. (Top right) Peeling of the dense mycelial mat that
encapsulated the interior substrate. (Bottom left) Microscopic view of sudden mycelial density due to edge effecfs. (Bottom
center) Microscopic view of calcined mycelium on the granular substrate. (Bottom right) Microscopic view of the degree of
mycelium colonization in relation to substrate quantity. NOTE: All microscopic pictures are 250x magnification.
76 μm 76 μm 76 μm

References
[1] Bouton, S., Cis, D., Mendonca, L., Pohl, H., Remes, J., Ritchie, H., Woetzel, J. (2013)
How to Make a City Great. McKinsey and Company.
[2] C. Jayasinghe, A. Imtiaj, H. Hur, G. Woo Lee, T. Soo Lee, U. Youn Lee. (2008)
Favorable Culture Conditions for Mycelial Growth of Korean Wild Strains in
Ganoderma lucidum. Microbiology. Vol.36 , pp. 28 - 33.
[3] Carlile, M., Watkinson, S., Gooday, Graham. (2001) The fungi (San Diego: Academic
Press).
[4] G. A. Holt, G. McIntyre, D. Flagg, E. Bayer, J. D. Wanjura1, M. G. Pelletier.(2012)
Fungal Mycelium and Cotton Plant Materials in the Manufacture of Biodegradable
Molded Packaging Material: Evaluation Study of Select Blends of Cotton Byproducts.
Journal of Biobased Materials and Bioenergy. Vol.6 , pp. 431 - 439.
[5] M.G. Pelletier, G.A. Holt, J.D. Wanjura, E. Bayer, G. McIntyre. (2013) An evaluation
study of mycelium based acoustic absorbers grown on agricultural by-product substrates.
Industrial Crops and Products. Vol.51, pp. 480–485.
[6] Rickleffs, A., Thiele, A., Falzone, G., Sant., G., Pilon, L. (2016) Thermal Conductivity of
Cementitious Composites Containing Microencapsulated Phase Change Materials.
University of California, Los Angeles.
[7] Ross, Phillip. (2012) Method for Producing Fungal Stuctures. U.S. patent 0135504 A1.
Filed November 28, 2011. Issued May 31, 2012.
[8] Stamets, Paul. (2005) Mycelium Running, How Mushrooms Can Help Save the World
(New York: Ten Speed Press).
[9] Travaglini, S., Noble, J., PG Ross, CKH Dharan. (2013) Mycological Matrix Composites.
American Society for Composites, 28th
Conference.
[10] Polystyrene Fact Sheet. Foundation for Advancements in Science and Education, Los
Angeles, California.

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