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Honors Theses

2015

Mechanical Properties of Sheets Comprised of Mycelium: A Paper Mechanical Properties of Sheets Comprised of Mycelium: A Paper

Engineering Perspective Engineering Perspective

Ross Mazur

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Mechanical Properties of Sheets Comprised of Mycelium: A Paper Engineering Perspective

By

Ross Mazur

Candidate for Bachelor of Science

Department of Environmental Resources Engineering

With Honors

May 2015

Approved

Thesis Project Advisor: Dr. Tom Horton

Second Reader: Dr. Stew Diemont

Honors Director: _______________________

William M. Shields, Ph.D.

MECHANICAL PROPERTIES OF SHEETS COMPRISED

OF MYCELIUM: A PAPER ENGINEERING PERSPECTIVE

Upper Division Honors Thesis

Ross Mazur

Department of Environmental Resources Engineering

State University of New York College of Environmental Science and Forestry

1 Forestry Drive

Syracuse, NY 13210 May 4, 2015

Advised by; Dr. Tom Horton, Dr. Stew Diemont, Dr. Ted Endreny, Dr. Gary Scott

ii

ABSTRACT

The global textile industry prides itself on energy and resource efficiency, although arguably has room to

improve. One factor enabling this efficiency is the scale of production, often making it more energy efficient than

alternative fiber sources. One potential alternative fiber source which has the potential to be emergy evaluation

and EROEI (energy returned on energy invested) competitive is fungal mycelium. This could be possible because

rather than the widespread support, harvesting, transport, and physical processing associated with lignocellulosic

fibers, culturing and processing could all occur on site at a textile mill. Fungi could be used to consume what might

otherwise be waste nutrients (the byproduct of an industrial process).

This study sought to compare the properties of mycelial sheets to products of both the paperboard and

office paper classes of textiles in terms of mechanical properties and impact of the manufacturing process. Lab scale

processes for production of mycelium isolates derived from filamentous fungi were explored. Both liquid culture

methods (malt extract broth (MEB) and Sabouraud dextrose broth (SDB)) and solid media culture (malt extract

agar (MEA) and lactose agar media (Desobry and Hardy 1997)) were tested. Fungal species included Pleurotus

pulmonarius (tested in liquid culture), Pleurotus osreatus, and Penicillium camemberti. Had culturing and refining

processes proved successful (mycelial sheets qualitatively resembling textile classes of interest, when desiccated), a

number of physical properties of fungi were to be tested using standardized paper testing equipment owned by

SUNY-ESF’s Department of Paper and Bioprocess Engineering in Walters Hall. Because none of the culturing or

refining procedures following tested culturing methods proved successful, no lab testing of physical characteristics

was carried out. In its absence, I used a modeling approach to analyze the theoretical strength of mycelial sheets.

Model inputs were based on published values.

For future studies, culturing Pleurotus sp. in liquid culture will likely not yield sheets resembling textiles,

whereas Penicillium sp. with surficial solid media methods seem to have potential (other research teams successfully

produced products described as “pliable and elastic in tensile strain” (Van-Horn and Shema 1954)). When using

liquid culture, if no structure (such as a screen) is present (or if the liquid culture vessel is not filled to brim with

media) to prevent mycelium from floating at media-air interface, vegetative mycelium will rapidly be out-grown by

reproductive aerial mycelium. When culturing using liquid media methods, it is recommended to use an apparatus

enabling tight control on environmental variables and to make use of a shaker table. When processing sheets,

steam sterilization followed by lyophilization has proved successful in past studies (Van-Horn and Shema 1954).

Because of such existing precedents, there remains potential for a mycelium-based fiber market.

iii

TABLE OF CONTENTS

Abstract ...................................................................................................................................... ii

Table of Contents ..................................................................................................................... iii

List of Figures ............................................................................................................................ iv

Glossary of Terms ...................................................................................................................... v

Acknowledgements ..................................................................................................................vii

Introduction ................................................................................................................................ 1

Methods ...................................................................................................................................... 3

Production of Sheets ..................................................................................................................................................................... 3

Liquid Culture Methods ............................................................................................................................................................... 3

Solid Media Methods ..................................................................................................................................................................... 7

Results ....................................................................................................................................... 10

Testing of Sheets .......................................................................................................................................................................... 10

Theoretical Analysis .................................................................................................................................................................... 12

Discussion ................................................................................................................................. 14

Comments on Contamination and Sterilization .................................................................................................................. 14

Comments on Growth Rate ..................................................................................................................................................... 14

Discussion of Marketability and Economics .......................................................................................................................... 16

Recommendations for Future Studies .................................................................................................................................... 17

Summary .................................................................................................................................. 19

References ................................................................................................................................ 20

Appendices ............................................................................................................................... 24

Composition of Media and Solutions Prepared ................................................................................................................... 24

Theoretical Strength Analyses .................................................................................................................................................. 24

Additional Graphics ..................................................................................................................................................................... 25

Theoretical Analysis .................................................................................................................................................................... 28

Python Script Used to Generate Theoretical Analysis Output Value ........................................................................... 30

iv

LIST OF FIGURES

Figure 1. Liquid culture experimental culturing apparatus ........................................................................................................... 6

Figure 2. Growth chamber (un-plugged refrigerator) ................................................................................................................... 6

Figure 3. Blender used to process harvested mycelium. .............................................................................................................. 7

Figure 4. Nested aluminum tray solid media testing apparatus (without aluminum foil lid on either the inner or

outer trays). .............................................................................................................................................................................................. 8

Figure 5. Differential growth rates of mycelial mat, leads to discontinuities in sheet. .......................................................... 9

Figure 6. Sheets generated using liquid culture method. Left, CENTER; Sheets produced from short blend time,

right; sheet generated from longer blend time. ............................................................................................................................ 11

Figure 7. harvesting sheet produced using solid media method (sheet highly flexible prior to desiccation). ............... 12

Figure 8. Desiccated rectangular sheets. Left; hole in sheet depicts brittleness of sheet, right; depicts retained

rebounding foam-like consistency (bottom of sheet). ................................................................................................................. 13

Figure 9. Mycelial mat directly after harvesting (prior to processing). Note the thin film of vegetative mycelium on

bottom of mat (visible along edge of lower half of disk, and covering surface of bottom size of top half). ................. 25

Figure 10. Folding aluminum foil onto tray lid, keeping in mind; prevention of air exchange, repeated unfolding

(removing) and re-folding (replacement), and fewest folds. Note ~2” overhang prior to folding. ................................. 26

Figure 11. Examples of contaminated replications. Left; other microbial populations competing with fungi at liquid

media-air interface), right; micrbial or algal colonies outcompeted spores in spore-Saturation solution). .................. 27

Figure 12. Sheet formed using solid media method. Sheet was roll-folded long-ways and short-ways a number of

times at three points during the drying process. Sheet retained slight pliability once fully desiccated. ......................... 27

v

GLOSSARY OF TERMS

Aerial mycelium – Mycelium fraction which grows above the agar (media) surface, can lead to asexual reproductive

(spore-bearing) structures, especially in Ascomycota, hyphae are typically lined with hydrophobins to prevent

water loss (Rao 2006).

Bioburden – Quantification of viables/number of microorganisms with which an organism is contaminated (Salcido

2007)/enumeration of microorganisms following incubation of media (Patel 2010).

Cellulose – The insoluble fraction of plant cell walls. A glucan polymer consisting of linear chains of β-1-4-bonded

anhydroglucose units (Pettersen 1984).

Chitin – The recalcitrant fraction of fungal cell walls. It is the second most abundant natural polymer in nature after

cellulose, and is comprised of β-(1-4)- linked D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine

(acetylated units) . It is synthesized by a wide range of organisms including crustaceans to fungi (Morganti 2012,

Aranaz, et al. 2009).

Electrospinning - A process in which solid fibers are produced from a polymeric fluid stream ~solution or melt!

delivered through a millimeter-scale nozzle (Hohman, et al. 2001).

Emergy System Evaluation – An evaluation system used to define embodied energy equivalencies (via

transformities) on a systems scale. The system can be described through transformities, emergy storage, empower,

mass emergy, empower density, work and emdollars. Emergy indices have classically been developed to evaluate

alternatives for primary energy resources, environmental impact of a set of processes, and international exchange

of materials. Systems diagrams are used to clarify networks of energy flows, the environment and society, and thus

simplify the emergy evaluation process (Odum 1998).

EROEI (Energy Returned on Energy Invested) – “The energy return on energy investment (EROI) indicator was

introduced to provide a numerical quantification of the benefit that the user gets out of the exploitation of an

energy source”, in terms of “how much energy is gained from an energy production process compared to how

much of that energy (or its equivalent from some other source) is required to extract, grow, etc., a new unit of

the energy in question” –Direct quotes (Raugei, Fullana-i-Palmer and Fthenakis 2012, Murphy, Hall and Powers

2010) respectively.

vi

Lignin – The recalcitrant fraction of wood fiber, a macromolecule with a molecular weight of 950 – 19,650

grams/mole (varying as a function of plant species, preparation/process, and tissue type). A Phenolic substance

consisting of an irregular array of variously bonded hydroxyl- and methoxy- substituted phenylpropane units

(Pettersen 1984, Khunsupat and Ragauskas 2013).

Lignocellulosic Fiber - The most abundant renewable biomass on earth, is composed mainly of cellulose,

hemicellulose and lignin. Both the cellulose and hemicellulose fractions are polymers of sugars and thereby a

potential source of fermentable sugars. Lignin can be used for the production of chemicals, combined heat and

power or other purposes (Harmsen, Huijgen and Bermúdez López 2010).

Liquid Culture- “A submerged culture of microorganisms involves their propagation in a liquid medium inside a vessel commonly called

bioreactor: the bioreactor’s main objective is to provide suitable physical conditions for microorganism growth. These conditions include

optimal pH, dissolved oxygen levels, agitation, temperature, and substrate availability, for both microbial growth and development leading

to high yields of specific metabolites. However, in the bioreactors, the microorganisms are generally subjected to conditions that can differ

significantly from those present on their natural habitats. These changes may lead to microbial stress, and low yields of specific desired

products. As a consequence, the design and operation of bioreactors, along with culture media selection, must be continuously fed by

technological advances and original research.” – Direct quote (García-Soto, et al. 2006).

Solid Media Culture –“solid media used for culture of fungi supply the nutrients required for growth and maintenance”, (Ravimannan,

et al. 2014). “Components include carbohydrates as a source of carbon, peptones as a source of nitrogen, minerals as enzyme activators

and for maintaining osmotic pressure and growth factors (essential amino acids, vitamins etc.), and agar as a binder” (Sneath 1972).

Submerged (vegetative) Mycelium – Mycelium fraction which penetrates the surface of the growth medium and

absorbs nutrients, hyphae are typically hydrophilic (Rao 2006).

vii

ACKNOWLEDGEMENTS

I would like to thank all of members of the ESF community for all of their guidance and assistance which made the

completion of this project possible.

Specifically, I would like to thank Dr. Tom Horton (mycologist, Department of Environmental Forest

Biology), Dr. Theodore Endreny (Chair, Department of Environmental Resource Engineering), Dr. Stewart

Diemont (assistant professor, Department of Environmental Forest Biology), Dr. Gary Scott chair, Department of

Paper and Bioprocess Engineering).

I would also like to thank Gavin McIntyre of Ecovative Design, Dr. Joel Hardy, co-author of mycelial sheet

mechanics study (as a part of food science research team in France), and Dr. William Dschida, author of most

recent Fungal Textile US Patent, for project-steering advice.

1

INTRODUCTION

In order to approach a resource use comparable to that which can be appropriately harvested and

extracted, society is in need of reducing the embodied energy of “every-day products”. The class of products

addressed by this study are textile and fiber-based. A major topic of materials science research focuses on is the

modification of raw fibers for use in fiber products applications. Lignocellulosic; glass; polymer; and carbon fibers

are some of those more commonly studied. All of these materials must be significantly processed to resemble the

final product, some of which require more energy-intensive manufacturing methods than others. Filamentous fungi,

when viewed from the perspective of a raw fiber resource have the potential to out-perform lignocellulosic

textiles in terms of embodied energy and physical properties. It is acknowledged that this is a hefty claim due to

the high efficiency, relatively-low energy inputs, and relatively controlled environmental impacts (where proper

forest management is practiced).

Whereas in lignocellulosic fiber, lignin is the strengthening and stiffening compound within plant cell walls,

fungi have chitin, which although is quite different, serves a fundamentally similar role in the fungal cell wall.

Processes for formation of sheets comprised of filamentous fungi have been studied in the past (Dschida 1997,

Johnson and Carlson 1977, Van-Horn and Shema 1954), although not from a mechanical properties perspective.

There are a handful of articles discussing mechanical properties of mycelial films for niche applications such as

cheese preservation (Bizet, et al. 1997), and exo-biopolymer production (Park and al 2001) which exist. Other

than these publications and a few patents, very little has been studied on the topic of mechanics of myco-materials

as a resource for textiles.

SUNY-ESF serves as a unique institution to carry out this study at because of the intellectual and physical

resources which it possesses. ESF has a Paper and Bioprocess Engineering department with “hundreds of paper

testing apparati” and a number of industry experts. In the department of Environmental and Forest Biology, there

are two renowned mycologists with a wide range of culturing and genetic taxonomy experience. Being trained in

the Environmental Resources Engineering department provides a broad technical background for biological and

chemical systems science.

Fungal mycelium were targeted as a fiber source or textile product feedstock for their physical

resemblance of lignocellulosic fibers, the potential rates at which they can grow, the concept that they could feed

off of what might otherwise be considered waste products, they physical recalcitrance (which turned out to be the

predominant challenge due to the extent of their brittleness when desiccated, and because hyphae are known to

have a high strength to weight ratio (collectively as a mycelial mat) (Allan 2012). Mycelium is generally comprised

2

of a few components in widely varying proportions depending on species and growth substrate (environmental

presence of certain compounds). The common components are raw fats, carbohydrates, cellulose, chitin, various

nitrogen species, vitamins and minerals (Tashpulatov, Baibaev and Shulman 2000). Chitin and cellulose content was

considered when comparing potential species to study, for concern that high content of these compounds (and

other macromolecules) would limit flexibility of mycelium. For practicality, cost and availability of strains (ideally

three or more of the same species were accessible) constrained those chosen. Some species mentioned in the

patent by Dr. William Dshida as used successfully in his trials were heavily considered (Dschida 1997) were

Neurospora crassa, Pisolithus tinctorus, Saprolegnia ferax, and those used in this study. The fungal species used in this

project were; Pleurotus pulmonarius and Pleurotus osreatus (tested in liquid culture), and Penicillium camemberti.

3

METHODS

PRODUCTION OF SHEETS

A number of variations of two main methods were carried out over the course of the study. The first

main method involved culturing of fungal isolates in liquid culture in 2L Erlenmeyer flasks prior to processing or

direct dehydration/desiccation. The second main method involved the culturing of fungal isolates on solid agar

media in aluminum trays. There was concern regarding potential inhibition of mycelial growth should common salts

of aluminum form, but because other apparati were cost-prohibitive, aluminum trays ended up being the chosen

method (Mills et al. 2004). Species used were chosen based upon what was recommended or used in previously

successful studies, and those which had cell wall characteristics deemed ideal for these processes. Pleurotus

pulmonarius and Pleurotus osreatus were grown using the liquid culture, Penicillium camemberti was grown using solid

culture methods. Cultures were purchased in various forms; syringes, plug form, slants, and lyophilized spore

powder. They were sourced from scientific supply companies (Connecticut Valley Biological Supply), hobby edible

mushroom cultivation suppliers (Mushroom Mountain, Spore Works), as well as the USDA-ARS Culture

Collection.

The culturing methods used were chosen because they would enable rapid production of mycelial biomass

as well as be conducive of physically and geometrically consistent growth. In addition to rapid and consistent

growth, goals of culturing were; to produce a large biomass overall, use low cost and readily available (on a

commercial scale) media reagents, and generate mycelium which was flexible when desiccated. These methods

were also chosen because they would enable “easy” segregation of the mycelium from the culturing media.

LIQUID CULTURE METHODS

Variations of two liquid media broth solutions were used throughout the project; malt extract broth, and

Sabouraud dextrose broth. The procedure for preparing the malt extract media involved cleaning of the 2L

Erlenmeyer flasks with soap and water (for foreign contaminant removal more so than sterilization), rinsing flask

with DI water three times, massing out 20 ± 0.01 (user accuracy) grams of lab-grade malt extract powder (sourced

from MP Biomedicals), addition of 1 ± 0.05 L (user accuracy) of DI water, addition of a scoop (~100 ml) of broken

glass (for mycelium laceration/mixing) (see Figure 1). Making sure the apparatus had the capacity to slice up the

mycelium was important because doing so maintains vigor of culture (growth). The flasks were sealed with a plug

of cotton packed into the neck of the flask (moderately tight, but not too tight such that excess pressure would be

unable to relive itself during elevated temperature and pressure when autoclaved), a sheet of aluminum foil folded

in half twice (effectively 4-ply) then pressed down over the flask opening and hand-clamped tight around the

4

outside of the flask neck. The flasks were shaken for a full minute each to assist with dissolving and even mixing of

the broth powder. Once the flask was prepared, it was labeled with the name of preparer, date of preparation,

contents, dates of handling. The flasks were autoclaved twice, separated by at least 24 hours (with the goal being

sterilization/reduction of bioburden to near zero CFU/L). The first autoclaving run was done using the “liq 15”

(liquid 15 minute at dwell temperature (121˚ C))) setting and the second run was done using the liquid the “liq 30”

(liquid 30 minute at dwell temperature (121˚ C)). Following autoclaving, the flasks were allowed to cool before

placing in the cross-flow hood for media inoculation. The cross-flow hood forced air system was turned on for a

minimum of 10 minutes prior to carrying out any transfer procedures (in order to ensure all airspace in the hood

was replaced with clean air). The work surface was wiped down twice with alcohol, and a Bunsen burner was used

to sterilize any tools which would come into contact with the fungi (*it was later determined that this was an

improper sterilization technique due to deposition of oxides on tool surfaces. Rather than method used, ethyl

alcohol should be sterilizing agent and Bunsen burner, the ignition source), as well as sterilize the air space in the

neck of the flask. The mycelium or spores were transferred into flask, followed by a second heating of the neck

airspace and replacing the cotton plug and the aluminum foil cover.

When preparing Sabouraud dextrose broth, the process was precisely the same other than the malt

extract addition step, which instead was replaced with the addition of 30 grams of Sabouraud dextrose powder.

The growth media were obtained from DB Medical, specifically Difco™ brand. In the case that spores were being

transferred into the flask, a 50 ml beaker was sterilized, then 20 ml of sterilized DI water (autoclaved twice along

with the flasks) was placed in the beaker, then the spore packet contents were rapidly poured into the beaker

outside of the cross-flow hood to prevent the spores from being blown around the lab. The mixture in the beaker

was then quantitatively transferred to the flask using sterilized DI water for subsequent rinses. In the case that

mycelium was being transferred from syringe, the syringe tip was flame-sterilized prior to securing to syringe and

transferring 2.5 ml of syringe solution into each flask. In the case that mycelium was shipped in plug culture, flame-

sterilized tweezers were used pull small sheets of mycelium off of the plugs, two to three of such sheets were

added to each flask. In the case that petri culture was used to maintain the cultures, small square sheets of

mycelium were cut in the agar (using a sterile mini-spatula) then two to three of such squares were transferred to

each flask. Petri dishes were prepared with malt extract agar and three to four small bits of mycelium were

pressed into the surface of the agar (evenly spread out over the surface of the agar).

The inoculated flasks were placed in a growth chamber (unplugged refrigerator, room temperature

approximately 21° C) for storage during culture, were monitored for growth (relative to “completion”) and

5

presence of microbial contamination, and shaken “vigorously” (carefully, such that cotton plug was not wetted by

shaking) for 30 seconds in order to break apart mycelial clumps on a weekly basis. Within the growth chamber,

the flasks were placed in trays which had a ring of Vaseline around the outside of the tray to prevent insects or

other small organisms from crawling into the flasks (potentially attracted by the smell of the broth) (Figure 2). The

flasks were harvested on the third, fourth, fifth, or sixth week, depending on the growth rate of the strain. The

growth chamber was maintained at room temperature, building humidity, and in full darkness.

In order to harvest the sheets, extra-large tweezers were used to pull the large chunks of mycelium from

the surface of the media broth. Some media was decanted from the flask, then remaining mixture of submerged

(vegetative) mycelium and media were poured through a 25 micron filter mesh (made from a circle cut out of a

mesh filter bag) on a vacuum 7.25” (185 mm) diameter Buchner funnel setup. Depending on the processing steps

for the given trial, the mycelium could be further processed according to a few different procedures. Initially, “large

chunks” (fused sheet-like sections of aerial and submerged (vegetative) mycelium) were manually separated using

two standard sized tweezers. For the first few trials, these two fractions were directly desiccated for a minimum of

24 hours in a standard 150°F metal stacking-shelf convection-heated food dehydrator (operated on level “2” (max

heat was a level “6”)), followed by massing on an analytical balance, then placed in a zip-lock bag for storage and

labeled. Subsequent trials involved a size reduction of aerial mycelium chunks using a multi-setting kitchen blender

prior to qualitative transfer to the Buchner funnel setup for vacuum sheet formation (Figure 3). Following a

minimum of four hours on the vacuum filter (or about 30% moisture content), the sheet was desiccated, separated

from the filter mesh, massed, placed in a zip-lock bag and labeled. In attempt to “optimize” the process, various

final mycelium particle sizes were tested by blending at “low” or “high” speeds for varying amounts of time (20

seconds one time, 30 seconds one time, and 15 seconds for six times with a 1:3 (on: off) duty cycle to prevent

foaming over). On two occasions, 10 ml of lactic acid was added to the media-mycelium blend mixture in attempt

to soften the chitin fraction of mycelium.

6

FIGURE 1. LIQUID CULTURE EXPERIMENTAL CULTURING APPARATUS

FIGURE 2. GROWTH CHAMBER (UN-PLUGGED REFRIGERATOR)

4-Ply Aluminum Foil Cap/Cover

Cotton Plug

2L Erlenmeyer flask

1L of Liquid Media

Broken Glass (mixing; break up hyphae

and increase rate of biomass

production)

Vaseline on Tray Rim

(to keep fungus mites

out of flasks)

7

FIGURE 3. BLENDER USED TO PROCESS HARVESTED MYCELIUM.

SOLID MEDIA METHODS

Nested food-grade shallow aluminum trays (10.75” x 6.75” x 0.75” (inner) and 11.75” x 8.50” x 1.25”

(outer)) were used to as a form and culturing vessel for the solid culture trials. The inner tray had a stainless steel

wire bent in an “S” shape such that right and left sides of the “S” would clasp to the rim of the tray (placed to

increase tray rigidity and prevent aluminum foil from sagging into media). Over the bent wire was placed a sheet of

aluminum foil cut such that it had 2” of hangover on all sides of the tray rim (when placed on top of the tray). The

aluminum was folded such that the foil did not leave sides of the tray exposed (a quality standard for sterility)

which did not dramatically change tray outer dimensions (to make sure it would fit flush within the larger tray);

and such that folds were systematic allowing for unfolding and refolding of the sheet (to access the media surface

during transfers of media and fungi). The aluminum foil-encapsulated inner tray was placed into the outer tray and

which itself was covered in a sheet of aluminum foil with roughly 2” of hangover with similar folding technique to

the inner tray (Figure 4). The empty nested-tray sets were autoclaved twice (usually simultaneously with the liquid

culture flasks (thus the same autoclave settings and timing were used)). Along with the trays DI water, spore-

saturation solution, and solid media were autoclaved in autoclavable bottles. The spore-saturation solution (pre-

inoculation) was comprised of 500 ml of DI water, 4.75 grams of bacteriological tryptone salt broth powder, and

0.5 grams of lab-grade anhydrous glucose. The agar media (as per recommendation of Dr. Joel Hardy of France,

who contributed to a publication fundamental to this study (Bizet et al. 1997)) was comprised of; 17.36 grams of

lab-grade agar (MP Biomedicals), 77.2 grams of alpha lactose powder (Fisher Scientific), 17.36 grams of sodium

8

chloride (MP Biomedicals), 0.88 grams of ammonium sulfate (Fisher Scientific), 4.32 grams of sodium lactate

(Bramble Berry Soap Supply), and 13.92 ml of lactic acid (aq) (Northern Brewer Homebrew Supply). A saturated

salt solution was prepared using potassium sulfate (Fisher Scientific) to achieve and maintain a relative humidity of

98% (Greenspan 1976).

FIGURE 4. NESTED ALUMINUM TRAY SOLID MEDIA TESTING APPARATUS (WITHOUT ALUMINUM FOIL LID ON

EITHER THE INNER OR OUTER TRAYS).

After autoclaving all of the components twice (using same settings as for the liquid culture experiments),

the agar media was re-melted and transferred to the inner aluminum tray using sterile technique in the cross-flow

hood. Before replacing the inner tray into the outer tray, ~100 ml of the saturated salt solution was transferred

into the outer tray (Figure 4). The spore-saturation solution was allowed to cool, Penicillium camemberti was

transferred into it using the same method as described for the lyophilized spores into liquid culture. The inoculated

spore-saturation solution was placed in a refrigerator for 10-12 hours in order to hydrate the spores before

transferring equal portions of it to the surface of the agar. The trays containing media and hydrated spore-

saturation solution were then placed in zip-lock bags (to prevent air exchange between building air and sterile air

in headspace of the trays). The trays were stored on the top of a shelf (that which runs along the top center of the

lab benches) such that the inner trays were all level with the floor (to maintain even distribution of spore-

saturation solution across the media surface). The trays were allowed to culture (not harvested) until a dense

mycelial mat developed on the surface of the tray (which was continuous, see Figure 5 for trays which continuity

was problematic).

Inner Tray (10.75” x 6.75” x

0.75”) Wire Foil-Support-Structure

Outer Tray (11.75” x 8.5” x

1.25”)

Solid Media Goes Here

Saturated Salt Solution Goes

Here

9

FIGURE 5. DIFFERENTIAL GROWTH RATES OF MYCELIAL MAT, LEADS TO DISCONTINUITIES IN SHEET.

10

RESULTS

TESTING OF SHEETS

None of the sheets retained flexibility, when “fully” desiccated, but instead were extremely brittle. As a

result no paper testing was able to be carried out on them. There are various methods proposed later on in this

report which have potential for yielding textile-like products.

For the first three liquid culture trials, the mass of the aerial and submerged (vegetative) mycelium was

recorded. The average mass of aerial mycelium per flask was 3.17 grams (84.5% of total (by mass)) and the average

mass of submerged (vegetative) mycelium per flask was 0.53 grams (15.4% of total (by mass)). After drying, the

liquid culture methods generally yielded brittle disk-shaped sheets (Figure 6). It turned out that the malt extract

broth became contaminated more often than the Sabouraud dextrose broth. This conclusion was arrived at

because every one of the first 16 flasks prepared with malt extract broth became contaminated, whereas four of

the first 16 flasks prepared with Sabouraud dextrose broth did not become contaminated. There were much lower

contamination rates in subsequent trials, as a result of better sterile practice, although proportionally more malt

extract broth flasks became contaminated than did Sabouraud dextrose broth-containing flasks. On March 24th

2014 (about halfway through the liquid culture experiment, a new container of Sabouraud dextrose powder

starting being used (the previous one had run out). The new powder had a brighter hue and dissolved more readily

than did the older powder, this could have affected growth rates and occurrence of contamination on early trials

containing the older powder. The various blending schemes all yielded a highly brittle product, although when

moist, those sheets associated with the longest blend-time were most flexible, and physically resembled translucent

crepes. The sheets generated from shorter blend times fell apart if they were removed from the filter mesh before

desiccation. The addition of lactic acid to the mixture in the blender did not noticeably affect the sheets generated.

11

FIGURE 6. SHEETS GENERATED USING LIQUID CULTURE METHOD. LEFT, CENTER; SHEETS PRODUCED FROM

SHORT BLEND TIME, RIGHT; SHEET GENERATED FROM LONGER BLEND TIME.

The separation of mycelium from the solid media turned out to be a time intensive process, requiring use

of tweezers and small spatula tools in spots where the mycelium had begun substantially growing into the media,

effectively anchoring it to the media. Once the sheets were fully separated, they exhibited superb flexibility (Figure

7), moderate strength, were elastic (relative to lignocellulosic textiles), and had a rebounding compressive foam-

like consistency. These statements were made based only upon qualitative observations. The solid media method

generally yielded brittle rectangular sheets (as the mycelial mats took on the shape of the trays which they were

cultured in) once dried (Figure 8). Although they became highly brittle upon desiccation, they retained rebounding

compressive foam-like consistency (orthogonal to the plane in which the sheet lay). One of the sheets generated

using this method was repeatedly fatigued in bending along its entire length at three points during the air-drying

process. This sheet was able to be bent slightly without shattering when fully dry.

12

FIGURE 7. HARVESTING SHEET PRODUCED USING SOLID MEDIA METHOD (SHEET HIGHLY FLEXIBLE PRIOR TO

DESICCATION).

THEORETICAL ANALYSIS

A Theoretical tensile stress analysis was carried out on Saprolegnia ferax (Appendix: Theoretical Analysis).

The analysis involved the application of a “fiber-web theory” tensile strength model published by Baker and

Peterson in 1960. The model outputs the tensile strength of a sheet of non-unidirectional fibers (with known fiber

parameters). The model requires inputs of fiber (hyphal) tensile strength, fiber (hyphal) cross-sectional area, and

fiber (hyphal) Poisson’s ratio (ratio of longitudinal to lateral elastic strain which the material exhibits). The model

predicts that a sheet of Saprolegnia ferax mycelium (with a hyphal diameter of 11.5 micrometers (Santiago et al.

2006), a hyphal tensile strength of 20 MPa (Money et al. 1997), and a Poisson’s Ratio of 0.6 (Silberstein et al. 2012))

would have an ultimate tensile strength of 41N (as compared to 528N for a standard copy paper, giving it roughly

1/10th the tensile strength of standard weight (60g/m2) copy paper). Because of variability of input parameters and

the application of a lignocellulose fiber model to mycelium, there is not much certainty in the realism of the results.

13

FIGURE 8. DESICCATED RECTANGULAR SHEETS. LEFT; HOLE IN SHEET DEPICTS BRITTLENESS OF SHEET, RIGHT;

DEPICTS RETAINED REBOUNDING FOAM-LIKE CONSISTENCY (BOTTOM OF SHEET).

14

DISCUSSION

COMMENTS ON CONTAMINATION AND STERILIZATION

Due to poor cultural practice, during the first dozen or so trials, all but a single flask became

contaminated (biologically), and thus no mycelial product was able to be grown or experimented with. Factors

which were potential sources of biological contaminant introduction include; poor seal formed by combination of

loose, single ply/layer aluminum foil seal and too little cotton used for the flask neck plug; insufficient or incomplete

autoclaving, poor fungal transfer technique, poorly isolated (contaminated) fungal isolates. Even though the cotton

plug could have generated a visually seal (no holes visible from outside flask to inside flask) and the aluminum foil

physically covered the top of the flask, there could have been microscopic tears in the foil, additionally

contaminated air could have been pulled into the flask through the base of folds of aluminum foil around flask neck

and through non-visible continuous pores, allowing passage of microbes. Once the microbes were on the surface

of the liquid media they would exponentially reproduce outcompeting the culture which the flask was inoculated

with. Poor transfer technique could have been caused by lack of thoroughness of ensuring everything was either

flame or alcohol sterilized before coming in contact with the flask neck or the inoculant. The strains purchased for

testing came in a variety of forms (slants, syringes, mycelium on wood dowel plugs, and lyophilized (freeze dried)

spore packets), some more intended for home and hobby use than sterile lab use. Because strains, such as those

shipped on wood plugs, could have been cultured under the principal of outcompeting foreign microbes with

quantity of spores used to inoculate the media (wood plugs), the mycelium which was scrapped from the plugs

upon transferring could have carried over contaminants.

There were a number of incidents of contamination on subsequent trials (Figure 11). Although it was

possible that poor sterile technique was causal, it is also possible that the autoclave settings chosen were not

sufficient for neutralizing viables (fertile spores/microbial components). Additionally, it was possible that the

autoclave was malfunctioning. There is discrepancy among the literature about what autoclave settings are

“sufficient”. One publication recommends a liquid-35 minute (dwell time) autoclave setting for flasks containing 500

ml of liquid and liquid-40 minute setting for flasks containing 1000 ml of liquids (PhytoTechnology Laboratories®

2014) while another study recommends a liquid-30 minute setting for 500-1000 ml (Judelson 2004).

COMMENTS ON GROWTH RATE

Culturing method ended up being a minor factor constraining growth rate, relative to other process

variables such as; media composition, temperature, dissolved oxygen content, headspace air-refresh rate, and

15

strain vigor. These statements were made based only upon qualitative observations. Although liquid culture in

theory is supposed to support rapid growth of many fungal species, it turned out to be the slower of the methods

employed, for the species used in this experiment.

The liquid culture trials could have possibly been the slowest growth method because of the media

composition. This might have been the case if the various media were lacking some minerals or compounds which

might be considered “required” for growth and reproduction, similarly to the way micronutrients (in addition to

the macronutrients commonly found in fertilizers) are “required” for plant growth. The culture might have gained

biomass, but might not have growing in a “healthy” or “vigorous” manner. Many liquid cultures call for the addition

of mineral salts, peptone, and on occasion antibiotics to reduce bacterial pressure (Booth 1971, Romero-Rangel et

al. 2012). Another potential source of slow growth was the lack of physical disturbance of the mycelium. Often

rotary shaker tables (used with Fernback baffled flasks) are employed to break apart the mycelium, and maintain a

state of highly-consistent hyphal geometry and vigorous growth (Lomberh M. et al. 2002). Rotary shaker table

access was not obtained for this experiment, had more time been available experimenting usage of this device

would be sought after in attempt to improve control over process variables. Shaker tables also are also useful for

increasing dissolved oxygen content in the media. Dissolved oxygen content was another possible parameter with

relative lack of optimization control, which if was increased could have supported more vigorous growth.

The temperature of the liquid culture vessels could have further constrained growth by being too low

(~20˚C) for the species tested. According to the literature, optimal temperatures for; Pleurotus pulmonarius: 25-

35˚C (Kong 2004) , Pleurotus ostreatus: 28˚C (Nwokoye et al. 2010), Penicillium camemberti: 25˚C (Bockelmann et al.

1999). Had the “incubation chamber” (un-powered refrigerator) been slightly heated, the mycelium might have

grown faster.

In later experimental trials, physical age of the culture (time stored in the container shipped prior to use

(transferring)) could have detracted from vigor of growth. This might have occurred because once the mycelium

starts to physically reach the extents of the media which it was shipped in, and thus have an effectively

exponentially decreasing “food supply”, it might start the transition from the exponential growth phase and the

deceleration growth phase (Trinci 1969).

The solid-media sterile aluminum tray culture method was chosen because it generated a mycelial mass

shaped like a sheet that according to experiences in previous experiments was “easily separated from the surface

of the media”. One possible source of separation difficulties with the solid media was the sophistication of the

culturing apparatus itself. A food science team from France advised using an apparatus which could both effectively

16

circulate and replenish air with low-bioburden filtered air (Maekawa and Intabon 2002). Additionally the apparatus

controlled temperature, light levels and relative humidity (Bizet et al. 1997).

It was apparent that P. camemberti strain had transitioned to the deceleration phase (as mentioned

previously) during the last trial when the culture took more than two months to put on roughly the same amount

of biomass which it had in a week during previous trials (in terms of time after transfer from petri culture).

DISCUSSION OF MARKETABILITY AND ECONOMICS

The economics of a mycelium textile product might be even more challenging to sort out (such that such

a manufacturing corporation or industry is profitable) than the materials science itself. What makes the alternative

fiber textile market so difficult to enter is the scale of feedstock, manufacturing, and distribution; markets, existing

infrastructure, and amount of research and process refinement which has taken place over the past few hundred

years associated with the lignocellulosic fiber industry. Because of its scale and history, the textile industry is highly

efficient and highly industrial, enabling it to generate a large volume of product at very low prices. Another

challenge preventing long term success in the market is the volatility of the pulp and fiber markets (Barr and

Cossalter 2006). A success story is the group; Marlow Foods corporation, which cultures Fusarium venenatum as a

“myco-protein” (vegetarian protein food source) on an industrial scale and is tremendously profitable doing so,

albeit they sell the product in tiny quantities giving them large profit margins. Marlow Foods myco-protein

production facility is likely (they are quite secretive about specific processing procedures and associated

economics) operating under a continuous-harvesting fermentation reactor set up (or possibly, a large batch

fermentation reactor setup if it enables tighter food safety control). A similar production operation who shared

some process economics, the Finnish Pulp and Paper Research Institute (FPRI) became involved with the in-vitro

protein synthesis market, and began commercially culturing Paecilomyces varioti (with the goal of optimizing protein

yield). It cost the FPPRI $660-$1000/metric ton at a production capacity of 50k–100k metric tons/year (1970), and

it is noted that this cost would be roughly double ($1320-$2000/metric ton) “today” (1989) (Litchfield H. 1989).

The FPPRI took interest in this synthetic protein production industry because they were able use spent sulfite

waste liquor (a byproduct of the sulfite pulping process) as a nutrient feedstock for the fungi they were culturing,

and were looking to reduce the quantity of waste as well as generate an additional value added product (Tyler and

Gunter 1948, Litchfield H. 1989). It is an interesting potential irony to plan to make paper from waste products of the

conventional paper manufacture process… It should be noted that the Sulfite pulping process is becoming less and

less common (less than 15 mills domestically remaining in operation after 2000) as the Kraft process has become

17

the universally accepted modern method (Smith, Rice and Ince 2003). As this market dwindles, so does the

potential availability of spent sulfite waste liquor.

Kraft pulp is a crude pulp (wood/lignocellulosic material which has gone through a series of;

chippers/grinders/mills, digesters, a blow tank, and has been screened and cleaned, (i.e. has not yet gone through

brown stock washers, oxygen delignification, or pulper processes (in which case it would be called fluff pulp)))

which could be considered an industrial equivalent to harvested, harvested washed and lyophilized mycelium (Tran

and Vakkilainnen K. 2008). The commercial pulp industry sells Kraft pulp at market for $800-$1100 per ton

(McAuley 2013). There is clearly an inequality between FPPRI’s protein production costs and Kraft pulp market

rates. This has the implication of requiring mycelium as a fiber source to enter a niche or value added product

market in order to be profitable… unless a plant is creative enough to make production costs less than market

values. One such example of a value-added product market would be that of medical manufacturing. Because of the

potential to sterilize the mycelium and due to it being bio-based one patent describes medical wound dressings

such as gauze and surgical tape, (Lemmons and Eugene 2009).

RECOMMENDATIONS FOR FUTURE STUDIES

A number of methods were discovered through the course of this study which there was not adequate

time to test. Saprolegnia ferax is a fish entomopathogenic fungus (oomycete, not in kingdom fungi) which has

interesting potential because chitin is “virtually absent” in the cell walls of Saprolegnia sp., (Van Den Berg et al.

2013). If attempting to culture on the surface of a solid media, using methods outlined above, the American Type

Culture Collection (ATCC) recommends using cornmeal agar, (American Type Culture Collection 2014). The

volume of spore-saturation solution would need to be great enough such that the mycelium can grow submerged

for an extended duration, as it requires being submerged in order to maintain state of vegetative growth (if

exposed to air, will likely sporulate) as it is an aquatic organism, (Kiziewicz 2004). Saprolegnia ferax “mycelium” as a

fiber feedstock would provide an interesting set of physical properties because of its lack of chitin, and because the

hyphal diameters can approach 1 mm, which would likely generate a sheet of different form and feel (Malloch

1981).

Had more experimentation been carried out with the solid media method, there likely would have been

more successful films generated. In order to generate more pliable films using the solid media method, an

apparatus which pumps filtered air over the media surface and which controls temperature and relative humidity

could be used. The team in France was able to repeatedly generate pliable mycelial films of P. camemberti using this

18

method. If this could be successfully replicated, there is much potential to test out different fungi, and carry out the

testing which this study intended to address (physical properties from a paper perspective).

The biomass generated in all of the liquid culture trials which did not become contaminated with other

microbial colonies was predominantly aerial mycelium, when the goal was to generate submerged (vegetative)

mycelium. Had there been a structure (such as a screen) in place preventing the cultures from growing at the air-

liquid media interface, or just using different flasks with very little air space, or more ideally a small air-media

interface area, aerial mycelial growth could have been minimized. In the textile patents, the enforcement of

submerged growth could have enabled the inventor’s success. The patent describes the use of jugs or carboys (full

to the top) for initial growth which were then transferred into troughs for final growth into sheet form (Dschida

1997).

A team from the Institute of Paper Chemistry in Appleton, Wisconsin published their methods of making

“handsheets” comprised of aspen pulp and varying amounts (0-50%) of cultured and processed mycelium. Their

methods involved liquid culture in a 40% glucose, 0.1% yeast media with a pH of 6.0. The cultures were grown in

400 ml of the media (which had been autoclaved for 20 minutes) in 3L Erlenmeyer flasks placed on a rotary table

shaker at 50 rpm in the dark in a temperature-controlled space held at 29˚ C. Rather than glass shards, they used

glass beads to break apart mycelium prior to harvesting. The cultures in the flasks were neutralized by placing in a

steam oven for an hour, followed by qualitatively transferring to a Buchner funnel, re-suspension in 300 ml of DI

water, blending “beaten” for 30 seconds, again transferred to the Buchner funnel for a final washing with DI water

before being lyophilized (Johnson and Carlson 1977). The specific parts of their process which were possibly

involved with enabled them to generate a physically different product were; the rotary shaker table culturing,

steam sterilization, and lyophilization steps. The handsheets exhibited “an increase in stretch” (when compared

with the 100% aspen pulp handsheets), indicating that the final mycelial product retained elastic strain properties).

An additional species which would have been valuable to experiment with would be Ganoderma lucidum.

Ganoderma sp. are often trimitic, meaning they grow generative (vegetative hyphae which is moderately branched

and septate with clamp connections), skeletal (aseptate, non-branching thick-walled hyphae) and binding hyphae

(hyphae which is aseptate, highly branched and thick-walled). Being trimitic, and thus the combination of these

three mycelial morphologies, gives Ganoderma interesting physical properties, notably its malleability, durability, and

toughness (even when desiccated). There are numerous groups, artists, architects who have created small

structures, furniture, wearables from Ganoderma sp. mycelium.

19

SUMMARY

The generation of textile products from commonly available fungi using low-cost apparatus’s and

processes is challenging. Culturing, blending, and desiccation of P. pulmonarius, P. ostreatus, and as well as the

culturing and desiccation of P. camemberti yield brittle sheets which do not resemble textile products. No

quantitative testing was carried out because of the lack of applicability of physical paper tests to the products

generated. Longer blending times (2 minutes as compared with 20 seconds) generates a more pliable (when moist)

sheet. Blending yields a sheet which (when moist) physically resembles a thin crepe, solid media culturing,

harvesting and drying yields a sheet physically resembling a foam-covered very-thin sheet of caramelized sugar.

Even though this project did not proceed as intended, there remains potential for myco-based textile

products. Because patents have been filed on the topic since the mid 1950’s documenting successful textile

generation (Van-Horn and Shema 1954); because some authors of relevant patents are still alive today (Dschida

1997); because there are companies which specialize in the commercial scale batch production of mycelium

products (Marlow Foods Ltd. 2009) as well as companies which specialize in the myco-materials research and

development (Diana 2015); and because of the capacity of today’s biotechnology industry to genetically modify

microbes to generate specific enzymes (such as chitinase) enabling metabolization of specific compounds (such as

chitin, the mycelial fraction responsible for rigidity of desiccated sheets), which could enable bio-pre-processing of

mycelial fibers (Neeraja et al. 2010), a myco-textile industry appears to be a physically feasible. Future studies

should focus on identifying species or strains which might be optimal for this purpose. The economics of an

industry such as this are quite uncertain, but if niche or specialty markets such as medical manufacturing were

sought out, there is the possibility of making a competitive product.

20

REFERENCES

Allan, Michael. Testing and Characterization of Mycelium Composites. Capstone Poster, Mechanical Engineering, Union

College, Schenectady: Bucinell, Ronald, 2012.

American Type Culture Collection. "Saprolegnia ferax (Gruithuisen) Thuret (ATCC® 48236™); Documentation."

ATCC. ATCC. 2014. file:///C:/Users/Ross%20Mazur/Downloads/201886.pdf (accessed March 13, 2015).

Aranaz, Inmaculada, Marian Mengíbar, Ruth Harris, Inés Paños, Beatriz Miralles, and Niuris Acosta. "Functional

Characterization of Chitin and Chitosan." Current Chemical Biology (Bentham Science Publishers Ltd.) 3, no.

2 (2009): 203-230.

Barr, Christopher, and Christian Cossalter. How Cost Competitive is Wood Pulp Production in South China? Market

Research Presentation, Beijing Branch, Center for International Forestry Research (CIFOR), CIFOR, 2006,

1-29.

Bizet, C, S Desobry, J Fanni, and J Hardy. "Composition and Physical Properties of the Penicillium camemberti

mycelium." Lait (ELSEVIER/Inra) 1997, no. 77 (March 1997): 1-6.

Bockelmann, W., S. Portius, S. Lick, and KJ Heller. "Sporulation of Penicillium camemberti in submerged batch

culture." Systematic and Applied Microbiology (Urban and Fischer Verlag) 22, no. 3 (December 1999): 479-

485.

Booth, C. Vol. 4, chap. IV in Methods in Microbiology, by C. Booth, 35-39. London, England: Academic Press Inc.

LTD. (ELSEVIER), 1971.

Diana, Chelsea. "Ecovative Design to move manufacturing operations from Iowa to Troy." Albany Buisness Review,

February 2, 2015: 1.

Dschida, William J. Fungal Cell Wall Production and Utilization As a Raw Resource For Textiles. Ed. David A.

Redding. United States Patent 5,854,056. November 28, 1997.

García-Soto, Mariano J., Enrique Botello-Álvarez, Hugo Jiménez-Islas, José Navarrete-Bolaños, Eloy Barajas-Conde,

and Ramiro Rico-Martínez. "Growth morphology and hydrodynamics of filamentous fungi in submerged

cultures." Advances in Agricultural and Food Biotechnology (Research Signpost) 2006, no. 1 (2006): 17-34.

Greenspan, Lewis. "Humidity Fixed Points of Binary Saturated Aqueous Solutions." JOURNAL OF RESEARCH of the

National Bureau of Standards- A. Physics and Chemistry (National Bureau of Standards) 81A, no. 1 (October

1976): 90-96.

Gunasekaran, Sundaram, and M. Mehmet Ak. Cheese Rheology and Texture. Boca Raton, Florida: Taylor and Francis

Group, 2002.

Harmsen, P.F.H., W.J.J. Huijgen, and L.M. Bermúdez López. Literature Review of Physical and Chemical Pretreatment

Processes for Lignocellulosic Biomass. Technical Report Literature Review, Food and Biobased Research,

Energy Research Center of the Netherlands, Wageningen: WUR-FBR, ECN, ABNT, 2010, 1-49.

Hohman, Moses, Michaell Shin, Gregory Rutledge, and Michael P. Brenner. "Electrospinning and electrically forced

jets. I. Stability theory." Physics of Fluids (American Institute of Physics) 13, no. 8 (May 2001): 2201-2220.

Johnson, Morris A., and John A. Carlson. Mycelial Paper: A Potential Resorce Recovery Process. Product Overview,

Appleton: Institute of Paper Chemistry, 1977.

Judelson, Howard. Operation of Autoclaves. .doc. Riad El-Solh, Beirut: American University of Beirut, June 28, 2004.

Kaminskyj, Susan G., Ashley Garrill, and I. Brent Heath. "The Relation Between Turgor and Tip Growth in

Saprolegnia ferax: Turgor is Necessary, but not sufficient to explain Apical Extension Rates." Experimental

Mycology (Academic Press) 1992, no. 16 (October 1992): 64-75.

21

Khunsupat, Ratayakorn, and Arthur J. Ragauskas. Molecular Weight Distribution of Lignin. Literature Review/Database

Compilation, School of Chemistry and Biochemistry, Georgia Institue of Technology, Atlanta, GA: GIT,

2013, 1-26.

Kiziewicz, B. "Aquatic Fungi Growing on Seeds of Plants in Various Types of Water Bodies of Podlasie Province."

Polish Journal of Environmental Studies (PJOES) 14, no. 1 (May 2004): 49-55.

Kong, Won-Sik. Chapter 4: Descriptions of Commercially Important Pleurotus Species. Vol. 1, chap. 4 in Mushroom

Growers Handbook 1; Oyster Mushroom Cultivation, by Evaristo A. Abella, et al., edited by Korea Rural

Development Administration, 54-61. Jeonju-si, Jeollabuk-do: Heinheart Inc., 2004.

Lemmons, and Kel Eugene. Antimicrobial compositions and methods of use thereof. US Patent WO 2009099419

A2. August 13, 2009.

Litchfield H., John. Single-Cell Proteins. Vol. 1, in A Revolution in Biotechnology, by Jean Marx L., 42-55. Cambridge,

Massachusetts: International Council of Scientific Unions, 1989.

Lomberh M., L., F. Solomko E., S. Buchalo A., and B. Kirchhoff. "Studies of Medicinal Mushrooms in Submerged

Cultures." Mushroom Biology and Mushroom Products (UAEM) 2002, no. 1 (2002): 367-377.

Lopez-Franco, Rosamaria, Salomon Bartnicki-Garcia, and Charles E. Bracker. "Pulsed Growth of Fungal Hyphal

Tips." Developmental Biology (Proc. Nati. Acad. Sci. USA) 91, no. 1 (June 1994): 12228-12232.

Maekawa, Takaaki, and Keo Intabon. Method for culturing a basidiomycetous fungus in a liquid culture medium. Ed.

Charles T. Jordan. Japan/ US Patent Patent 6490824. December 10, 2002.

Malloch, David. Moulds Their Isolation, Cultivation and Identification. Vol. 1. 1 vols. Toronto: University of Toronto

Press, 1981.

Marlow Foods Ltd. "Nutritional Profile of Quorn Mycoprotein." Mycoprotein.org What is Mycoprotein: Resources.

Quorn. July 1, 2009. http://www.mycoprotein.org/assets/Nutritional_profile_of_Quorn.pdf (accessed

March 11, 2015).

McAuley, Kathy. Weyerhaeuser Analyst Meeting; May 10, 2013. Annual Report Presentation, Investor Relations,

Weyerhaeuser, Longview: Weyerhaeuser, 2013, 42.

Mills, A.A.S., H.W. Platt, and R.A.R. Hurta. "Effect of salt compounds on mycelial growth, sporulation and spore

germination of various potato pathogens." Postharvest Biology and Technology (ELSEVIER, Science Direct)

2004, no. 34 (May 2004): 341-350.

Money, Nicholas P., and Terry W. Hill. "AmericaCorrelation between Endoglucanase Secretion and Cell Wall

Strength in Oomycete Hyphae: Implications for Growth and Morphogenesis." Mycologia (Mycological

Society of America, JSTOR) 89, no. 5 (March 1997): 777-785.

Morganti, Pierfrancesco. "Nanoparticles and Nanostructures Man-Made or Naturally Recovered: The Biomimetic

Activity of Chitin Nanofibrils." Nanomaterials & Molecular Nanotechnology (SciTechnol) 2012, no. 2

(December 2012).

Murphy, David J., Charles A. S. Hall, and Bobby Powers. "New perspectives on the energy return on (energy)

investment (EROI) of corn ethanol." Environ Dev Sustain (Springer Science+Business Media B.V. ) 2011, no.

13 (July 2010): 179-202.

Neeraja, Chilukoti, Kondreddy Anil, Pallinti Purushotham, Katta Suma, Bruno M. Moerschbacher, and Appa Rao

Podile. "Biotechnological approaches to develop bacterial chitinases as a bioshield against fungal diseases

of plants." Critical Reviews in Biotechnology (Informa Healthcare) 30, no. 3 (April 2010): 231-241.

22

NWOKOYE A., I., O.O. KUFORIJI, and P.I. ONI. "Studies on Mycelial Growth Requirements of Pleurotus

Ostreatus (Fr.) Singer." International Journal of Basic & Applied Sciences (IJBAS-IJENS) 10, no. 2 (April 2010):

47-53.

Odum, Howard T. "Emergy Evaluation." International Workshop on Advances in Energy Studies: Energy Flows in Ecology

and Economy. Porto Venere: IWAES, 1998. 1-12.

Park, Jong, and et. al. "Effect of aeration rate on the mycelial morphology and exo-biopolymer production in

Cordyceps militaris." Process Biochemistry (Elsevier) 37, no. 1 (December 2001): 1257-1262.

Patel, Alpa. Microbial Reduction Testing Procedures for Tissue Process Validations. pdf. Nelson Laboratories. Salt Lake

City, Utah, April 7, 2010.

Pettersen, Roger C. "Chapter 2 ." Chap. Chapter 2 in The Chemical Composition of Wood, by Roger C. Pettersen,

57-126. Madison, Wisconsin: American Chemical Society, 1984.

PhytoTechnology Laboratories®. "Sterilizing Nutrient Media." PhytoTechnology Laboratories Technical Information.

Prod. PhytoTechnologies Laboratories. Shawnee Mission, Kansas, June 1, 2014.

Rao, Sridhar. Introduction to Mycology. pdf. Davangere, Karnataka, June 2006.

Raugei, Marco, Pere Fullana-i-Palmer, and Vasilis Fthenakis. "The Energy Return on Energy Investment (EROI) of

Photovoltaics: Methodology and Comparisons with Fossil Fuel Life Cycles." VMF (National Photovoltaic

Environmental Research Center, Brookhaven National Laboratory), 2012: 2.

Ravimannan, Nirmalal, Revathie Arulanantham, Sevvel Pathmanathan, and Kularajani Niranjan. "Alternative culture

media for fungal growth using different formulation of protein sources." Annals of Biological Research

(Scholars Research Library) 5, no. 1 (2014): 36-39.

Rawal, Amit. "A Modified Micromechanical Model for the Prediction of Tensile Behavior of Nonwoven Structures."

Journal of Industrial Textiles (Sage Publications) 77, no. 6 (October 2006): 133-149.

Romero-Rangel, Oscar, María Guadalupe Maldonado-Blanco, Cynthia Carolina Aguilar-López, Myriam Elías-Santos,

Raúl Rodríguez-Guerra, and José Isabel López-Arroyo. "Production of mycelium and blastospores of

Hirsutella sp. in submerged culture." African Journal of Biotechnology (Academic Journals) 11, no. 87 (June

2012): 15336-15340.

Salcido, Richard "Sal". "Advances in Skin & Wound Care: What is Bioburden? The Link to Chronic Wounds - See

more at: http://www.nursingcenter.com/lnc/JournalArticle?Article_ID=727985#sthash.CsxNOJdt.dpuf."

The Journal for Prevention and Healing ( Lippincott Williams & Wilkins) 20, no. 1 (July 2007): 368-369.

Schutyser, M. A. I., P. de Pagter, F.J. Weber, W.J. Briels, R. M. Boom, and A. Rinzema. "Substrate Aggregation Due

to Aerial Hyphae During Discontinuously Mixed Solid-State Fermentation With Aspergillus oryzae:

Experiments and Modeling." Biotechnology and Bioengineering (Wiley Periodicals 2003) 83, no. 5 (February

2003): 503-513.

Silberstein, Meredith N., Chia-Ling Pai, Gregory C. Rutledge, and Mary C. Boyce. "Elastic–plastic behavior of non-

woven fibrous mats." Journal of the Mechanics and Physics of Solids (ELSEVIER) 60, no. 1 (October 2011):

295-318.

Smith, Brett R., Robert W. Rice, and Peter J. Ince. Pulp Capacity in the United States, 2000. Forest Products

Laboratory - General Technical Report (FPL-GTR), Forest Service, USDA, Madison: USDA Forest Service,

2003, 1-23.

Sneath, P. H. A. Miniturization in Microbiological Methods. Vol. 7A, in Methods in Microbiology, by J.R. Norris, & D.W.

Ribbons, edited by S. Lapage, J. Shelton, & T. Mitchell, 62-96. Kent, England and Miami, Florida: Academic

Press, 1972.

23

Tashpulatov, Zh., B. G. Baibaev, and T. S. Shulman. "Chemical Composition of Mycelium of the Thermotolerant

Fungus Penicillium atrovenetum." Chemistry of Natural Compounds (Plenum Publishing Corporation) 36, no.

5 (July 2000): 518-520.

Tran, Honghi, and Esa Vakkilainnen K. "The Kraft Chemical Recovery Process." TAPPI Manuscripts (TAPPI) 2008,

no. 1 (2008): 1.1 - 1.8.

Trinci, A.P.J. "A Kinetic Study of the Growth of Aspergillus nidulans and Other Fungi." Journal of General

Microbiology (SGM Journals) 57, no. 1 (March 1969): 11-24.

Tyler, Richard G., and Shirley Gunter. "Biochemical Oxygen Demand of Sulfite Waste Liquor." Sewage Works

Journal (Water Environment Federation) 20, no. 4 (July 1948): 709-719.

Van Den Berg, Albert Hendrik, Debbie McLaggan, Javier Dieguez-Uribeondo, and Pieter Van West. "The impact of

the water moulds Saprolegnia diclina and Saprolegnia parasitica on natural ecosystems and the aquaculture

industry." Edited by British Mycological Society. Fungal Biological Reviews (ELSEVIER) 2013, no. 27 (May

2013): 33-42.

Van-Horn, Willis M., and Bernard F. Shema. Sheets Comprising of Filamentous Fungi. United States Patent

2,811,442. October 29, 1954.

24

APPENDICES

COMPOSITION OF MEDIA AND SOLUTIONS PREPARED

Malt Extract Agar

30 grams Malt Extract Powder

20 grams Agar Powder

1 L DI Water

Malt Extract Broth

20 grams Malt Extract Powder

1 L DI Water

Sabouraud Dextrose Broth

30 grams Malt Extract Powder

1 L DI Water

Tryptone Spore-Saturation Solution

1.9 grams Tryptone Salts

0.2 grams Anhydrous Glucose

200 ml DI Water

1.6E9 Spores P. camemberti

Saturated Salt Solution

200 grams potassium sulfate

400 ml DI water

THEORETICAL STRENGTH ANALYSES

CITATIONS FOR THEORETICAL STRENGTH ANALYSES

Poisson Ratio for the P. camemberti mycelial sheet, assumed to act the same way as the non-woven fiber

mat which the model is based off of (Silberstein et al. 2011).

Source for Non-woven Tensile Strength Model (Rawal 2006).

25

ADDITIONAL GRAPHICS

FIGURE 9. MYCELIAL MAT DIRECTLY AFTER HARVESTING (PRIOR TO PROCESSING). NOTE THE THIN FILM OF

VEGETATIVE MYCELIUM ON BOTTOM OF MAT (VISIBLE ALONG EDGE OF LOWER HALF OF DISK, AND

COVERING SURFACE OF BOTTOM SIZE OF TOP HALF).

26

FIGURE 10. FOLDING ALUMINUM FOIL ONTO TRAY LID, KEEPING IN MIND; PREVENTION OF AIR EXCHANGE,

REPEATED UNFOLDING (REMOVING) AND RE-FOLDING (REPLACEMENT), AND FEWEST FOLDS. NOTE ~2”

OVERHANG PRIOR TO FOLDING.

27

FIGURE 11. EXAMPLES OF CONTAMINATED REPLICATIONS. LEFT; OTHER MICROBIAL POPULATIONS

COMPETING WITH FUNGI AT LIQUID MEDIA-AIR INTERFACE), RIGHT; MICRBIAL OR ALGAL COLONIES

OUTCOMPETED SPORES IN SPORE-SATURATION SOLUTION).

FIGURE 12. SHEET FORMED USING SOLID MEDIA METHOD. SHEET WAS ROLL-FOLDED LONG-WAYS AND

SHORT-WAYS A NUMBER OF TIMES AT THREE POINTS DURING THE DRYING PROCESS. SHEET RETAINED

SLIGHT PLIABILITY ONCE FULLY DESICCATED.

28

THEORETICAL ANALYSIS

29

30

Sources; (Money and Hill 1997, Kaminskyj et al. 1992, Schutyser et al. 2003, Lopez-Franco et al1994). Method

makes use of the electrospun web tensile strength theory.

PYTHON SCRIPT USED TO GENERATE THEORETICAL ANALYSIS OUTPUT VALUE

#Ross Mazur Upper Division Honors Thesis SUNY-ESF Theoretical Model Testing for Mycelial Sheet Stress Analysis

#Tensile Strength Analysis

import math

#Constants

StressMax = 30.0 #Mpa

Vxy = 0.6

YoungsModulus = 110 #Mpa

StrainYield = StressMax/YoungsModulus

StressXComponentArray = []

StressYComponentArray = []

StrainYComponentArray = []

for i in range(0,180):

StrainYieldComponent = StrainYield*((math.cos(i*(3.1415926/180))**2)-Vxy*(math.sin(i*(3.1415926/180))**2))

Constant = YoungsModulus*StrainYield

IntegralXVal=abs((math.cos(i*(3.1415926/180))**2)-

(Vxy*(math.sin(i*(3.1415926/180))**2)))*(math.cos(i*(3.1415926/180))**2) IntegralYVal=abs((math.cos(i*(3.1415926/180))**2)-

(Vxy*(math.sin(i*(3.1415926/180))**2)))*(math.sin(i*(3.1415926/180))**2)

StressXComponent = Constant*IntegralXVal/360

StressYComponent = Constant*IntegralYVal/360

StressXComponentArray.append(StressXComponent)

StressYComponentArray.append(StressYComponent)

StrainYComponentArray.append(StrainYieldComponent/360)

YieldStressYDir = sum(StressYComponentArray)

YieldStressXDir = sum(StressXComponentArray)

YieldStrain = sum(StrainYComponentArray)

print("Longitudinal Yield Stress (perpendicular to direction stress is applied): ", YieldStressYDir, "MPa")

print

print("Lateral Yield Stress (parallel to direction stress is applied: ", YieldStressXDir, "MPa")

print

print("Yield Strain: ", YieldStrain, "meters/(meter length)")