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�Wm� 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

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[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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