Best Mycorestoration Practices for Habitat Restoration of Small Land Parcels

Item

Title

Eng Best Mycorestoration Practices for Habitat Restoration of Small Land Parcels

Date

2013

Creator

Eng Stamets, LaDena Che'

Subject

Eng Environmental Studies

extracted text

Best Mycorestoration Practices for Habitat Restoration of Small Land Parcels

by
La Dena Che’ Stamets

A Thesis
Submitted in partial fulfillment
of the requirements for the degree
Master of Environmental Studies
The Evergreen State College
June 29, 2012

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© 2012 by LaDena Che’ Stamets. All rights reserved.

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Table of Contents
Abstract
List of Figures and Tables
Acronyms used in the thesis

Acknowledgements
Introduction ……………………………………………………………………. 1
Motivating Factors: Mycorestoration in the Pacific Northwest of North America
………………………………………………..………………………… 5

PART I
Defining Mycorestoration: Mycoremediation, Mycofiltration, Mycoforestry…. 15
Pillar I: Defining Mycoremediation; Materials and Methods…………………. 24
Heavy Metal Mobilization Case Studies: Gadd et al. has extensively researched
and tested the relationship between metals and fungal species this section is aimed
to give a brief overview………………………………………………………… 33
First Mycoremediation Example Pilot Project: Mycoremediation, Makah’s Tatoosh
Island Neah Bay, Washington 2009- present………………………….. 42

Second Mycoremediation Example Case Study: Bioremediation of PAHContaminated Soil by Composting: A Case Study, 2003……………………….. 47
Third Mycoremediation Example Case Study: Use of fungal Technology in soil
remediation: Water air and soil pollution, 2003……………………………….. 49
Fourth Mycoremediation Example Case Study: Mycoremediation: WSDOT 19901998……………………………………………………………………… 50

Fifth Mycoremediation Example Case Study: Compost-mediated Removal of
Polycyclic Aromatic Hydrocarbons from Contaminated Soils, 2003…………... 55
Critique of Mycoremediation………………………………………………… 56
Pillar II: Defining Mycofiltration; Materials and Methods……………………... 61
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First Mycofiltration Example: The Evergreen State College Mycofiltration Pilot
Project 2011- present…………………………………………………………… 63
Second Mycofiltration Example Pilot Project: Phase I (Proof-of-Concept) EPA
Bench Study Comprehensive assessment of Mycofiltration Biotechnology to
Remove Pathogens from Urban Storm Water, 2012- present…………………... 65
Third Mycofiltration Example Pilot Project: Fisherville EcoMachine………. 71
Fourth Mycofiltration Example Case Study: Mason County Mycofiltration
Projects, 2008-2011…………………………………………………………….. 86
Fifth Mycofiltration Example Pilot Project: Field Demonstration of
Mycoremediation for Removal of Fecal Coliform Bacteria and Nutrients in the
Dungeness Watershed, Washington 2009………………………………………. 79
Critique of Mycofiltration……………………………………………………. 94
Pillar III: Defining Mycoforestry; Materials and Methods…………………… 100
First Mycoforestry Example Pilot Study: Mycoforestry Research Project on
Cortes Island, Canada 2003- Present………………………………………... 106
Critique of Mycoforestry………………………………………………….. 111

Part II Methodology for Individual Landowners Mycorestoration Projects
High and Low Technology for Cultivation Techniques …………………..….. 112
Simplified method for generating mycoremediation or mycofiltration
mycelium………………………………………………………………………. 121

Part III Conclusion & Future Research…………………………………….. 127
References Cited

Appendices:
i. Glossary of Key Mycorestoration Terms used in the thesis
ii. Scientific Publishers for Researching Mycorestoration
iii. Government agencies website references

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ABSTRACT
Best Mycorestoration Practices for Habitat Restoration of Small Land Parcels
La Dena Che’ Stamets
Environmental pollution emanates from many sources causing harm to natural systems
degrading human health. This thesis examines best mycorestoration (the use of fungi to
prevent, reduce, repair, restore and ameliorate the negative impacts of chemical biological
pollutants), practices, which provides low-cost, low-maintenance, time- conservative,
effective biological solutions to remediate toxins. Throughout my extensive literature
review mycorestoration practices demonstrated significant reductions in biological and
chemical pollutants. The chemical toxins of focus were polycyclic aromatic hydrocarbons
(PAH’s), polychlorinated biphenyls (PCB’s), chlorophenols, dioxins, DDT, trinitrotoluene
and the hyper concentration-uptakes of heavy metals, including but not limited to: lead,
uranium, arsenic from the environment. Fecal coliform bacteria (FCB) populations were
effectively removed within ranges of 87 to 97%, whilst polycyclic aromatic hydrocarbons
(PAHs) reductions ranged from 57 to 97% and total aromatic hydrocarbons (TAH)
reductions were 91 to 99%, respectively
This thesis focuses on the three main pillars of mycorestoration: mycoremediation,
mycofiltration and mycoforestry. I address the need and present an outline for an easy-touse guide for mycorestoration projects that can be utilized at the grass-roots level. By
healing polluted ecosystems with ecological restoration methods, this thesis reaches across
multiple socioeconomic and environmental disciplines. Mycorestoration practices increase
the inherent sustainability of human impacted habitats, reducing the need for remedial
practices while fortifying the ecological services, which healthy habitats provide: clean
water, clean food, clean air, and healthy inhabitants.
This thesis lays the foundation for future work and refinement of applications in this field.
I suggest more development needs to address mycofiltration site design. The number of
potentially useful species should be expanded, especially within white-rot fungi. Creating
and compiling mycorestoration sites on the Geographic Information System (GIS) map
would show locations of projects, including toxins identified and contamination levels
before and after mycorestoration is applied. The implementation of standardization
mycorestoration certification “stamp of approval” authorized by credible mycologists is a
necessary first step before mycorestoration practices are deployed. Later work can focus
on creating a multi-volume guide to Best Mycorestoration Practices specific to each ecoregion to facilitate widespread pollution clean up.

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List of Figures and Tables

Figure 1: Soil Remediation Technologies Costs: Petroleum Hydrocarbons……………. 4
Figure 2: Map of Pacific Northwest …………………………………………………….. 9
Figure 3: Toxins and Their Primary Origins………………………………..…………. 17
Figure 4: PCB Remediation- untrained mycelia………………………………..……… 20
Figure 5: Mushroom with Activity Against Chemical Toxins…………………….……. 21
Figure 6: Mushroom Species’ concentrations of Heavy Metals…………………….…. 22
Figure 7: Mature Fungal Fruit-bodies Commonly Used in Mycorestoration………..…..23
Figure 8: The mushroom life cycle………………………………..…………….……… 27
Figure 9: Parameters of Bioremediation Process……………………………………… 30
Figure 10: Mechanisms of fungal bioweathering of mineral surfaces applicable to mycodeterioration of concrete………………………………………………..………………. 34
Figure 11: Visual Aid Showing Concrete Deterioration……………………………….. 38
Figure 12: Images of Tatoosh Island Neah Bay, Washington………………………….. 45
Figure 13: Native fungi Marasmiellus found on Tatoosh & Salmonberry canes………. 46
Figure 14: Remediation of Aged Oil in Excavated Soil for WSDOT 1998…………….. 54
Figure 15: Simplified Illustration of Mycofiltration Installation process…………….... 62
Figure 16: Mycofiltration Symantec Installation………………………………..……. 118
Figure 17: Fungal Species and Substrates Combinations…………………………..…. 66
Figure18: Mycofilter Resilience Testing Timeline for EPA grant………………………. 71

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Figure 19: Fisherville Mycocells Eco-machine………………………………………... 72
Figure 20: Mycofilter Trough “Trickle-filter”…………………………………………. 73
Figure 21: Eco-machine train…………………………………………………………. 74
Figure 22: Raw Canal Water Prior to Eco-machine Treatment………………………. 80
Figure 23: Water Quality Test 1: Train #1: Comparison of raw water from Blackstone
River Canal to treated water from Fisherville remediation pilot eco-machine w/ reduction
of pollutants (3-5-2007). Water Quality Test 2: (4-2-2007)…………………………….. 80
Figure 24: Water Quality test 1: Train # 2: Comparison of raw water from Blackstone
River Canal to treated water from Fisherville remediation pilot eco-machine w/
reduction of pollutants (3-5-2007). Water Quality test 2: (4-2-2007)………………….. 82
Figure 25: Mycofiltration Sites locations for Hood Canal, Mason County……………. 86
Figure 26: Native plants were used in the biofiltration cells……………………...…… 92
Figure 27: Maple tree mycorrhizal comparisons…………………………………..…..101
Figure 28: Trend Comparisons between Trees inoculated with Mycorrhizae vs. noninoculated trees………………………………………………………………………... 109
Figure 29: Process of Mushroom Cultivation Techniques……………………………. 113
Figure 30: Mycofilters………………………………………………………………… 115
Figure 31: Four ways to inoculate burlap sacks with fungi………………..………… 116
Figure 32: 2012 Mycorestoration Site Description ………………………………….. 125
Figure 33: Ambient Temperature (40-60 F.) Fermentation of Woodchips in Fresh
Water………………………………………………………………………………..…. 122
Figure 34: Small-scale Mycofiltration Installation Examples………………...……… 123

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Acronyms
Cd: cadmium
COD: Chemical Oxygen Demand
Cu: copper
CWA: Clean Water Act of 1972
DNR: Department of Natural Resources
DOE: Department of Ecology
DOH: Department of Health
DNR: Department of Natural Resources
DOT: Department of Transportation
EcM: ectomycorrhizal
EPA: Environmental Protection Agency
ErM: ericoid mycorrhizal
FLT: flouranthene
NOAA: National Oceanic and Atmospheric Administration
NPDES: National Pollutant Discharge Elimination System
PAH: polycyclic aromatic hydrocarbons
Pb: lead
PCB: polychlorinated biphenyls
PHE: phenanthrene
POP: persistent organophosphates
ppm: parts per million
PYR: pyrene
TAH: total aromatic hydrocarbons
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TKN: Total Kjehldahl Nitrogen
TNT: trinitortoluene
TPH: Total Petroleum Hydrocarbon
TSS: Total Suspended Solids
UO3 uranium trioxide
U3O8 triuranium octaoxide
WADOH: Department of Health
WADOT: Department of Transportation
Zn: zinc

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Acknowledgements
I must convey my deepest gratitude and appreciation to my primary reader and advisor;
Judy B. Cushing Ph.D; computer science and Ecology Informatics, who worked
diligently to help me ensure my work, is logical and consistent manner to its targeted
audience. Her encouragement, professionalism, enthusiasms were a great assets to me
throughout this entire process which includes my oral presentation. Equally important
were the contributions of Dr. Paul E. Stamets; Mycologist, who assisted with the more
technical and scientific aspects of this research, specifically his vast knowledge in both
mycology and his ability to break down toxins and uptake heavy metals that are hindering
the health of our environment. In addition to his wealth of knowledge and personal
experiences, he provided creditable scientific research from fellow researchers. As well
as him continued helped with editing this thesis. A special thanks to Fungi Perfecti LLC
for providing charts, diagrams, photographs and other visual aids to help deepen the
understanding for intended audience. In addition to allowing me to learn first had the lab
skill techniques for high-tech methodology and including me in meetings related to
mycorestoration installations and applications. Moreover, I would like to thank my first
Environmental Science and Biology professor Dementria Shew for sparking my interest
in environmental issues; without her enthusiasm I might have gone into business instead.
Also I would like to thank my son, Trevon and husband, Benjamin for having patience
and supporting me through the adventure that comes along with creating a master thesis.
Lastly, I would like to extend my gratitude to all the scientists with their wealth of
knowledge, who have contribute the majority of their lives to research which in turn
contributed to both the health of our environment as well a my research.
La Dena Che’ Stamets, M.E.S.

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Introduction
Environmental pollution emanates from many sources, causing harm to natural
systems and degrading human health. Toxins include oil spills, excess nutrients from farm
animals, and fertilizers affect terrestrial, riparian, and aquatic systems. Human health is
closely linked to the health of these natural systems. Pollutants in our environment taint
food supplies and compromise water quality, which in turn cause increases in the rates of
cancer, asthma, learning disabilities, developmental impairment, and reproductive issues.
For example, pesticides poison farms and wildlife while creating public health issues like
birth defects and cancer (Amaranthus, 2009). Of the tens of thousands of chemicals
currently used, few have been tested for their effects on human health. We know even less
about combined effects of these toxic chemicals. This lack of knowledge leaves us unable
to protect ourselves, let alone our children [1]. Because our country spends billions of
dollars annually to treat illnesses caused by environmental pollutants protecting ourselves
from toxins entering our environment should be a national priority.
The Washington State Department of Ecology (WSDOE) has two mandated ways
to prevent toxic material from entering our soil and water. First, preventing use of toxic
materials averts toxic exposures most effectively, avoiding future health and
environmental costs. Second, the DOE assists businesses in reducing or managing the
toxic chemicals that enter the environment. Should the toxins enter the environment, DOE
will make an effort to clean up the polluted air, land, and water. The DOE’s cleanup
applications are necessary, but often incur extremely costly solutions.
One of DOE’s main strategies is preventing toxic substances from entering
1

http://www.ecy.wa.gov/toxics/index.htm
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stormwater – rain and snow melt that runs off surfaces such as rooftops, paved streets,
highways, parking lots, sidewalks, ditches and other vectors. As water runs off surfaces, it
collects pollution such as oil, fertilizers, pesticides, soil, trash, animal waste, and “rubber”
debris from tires. From these points of pollution, toxins travel down into aquatic systems
(e.g., streams, rivers, bays, lakes, and oceans), or they may be detoured into a storm drain
where toxic substances travel through storm pipes until the pollutants are eventually
discharged, untreated, into our local water ways. A novel application – which is the basis
of this thesis – is to explore the uses of fungal mycelium as a new strategy for dealing with
multiple classes of pollutants that often converge as water carries them into downstream
environments.
The study of fungi is known as mycology and a mycologist is a person who studies
the kingdom of fungi. Mycorestoration is the use of fungi to prevent, reduce, repair,
restore and ameliorate the negative impacts of chemical and biological pollutants.
Myocrestoration methods can also restore and repair habitat whether damaged by human
activity or natural disaster. Saprophytic, endophytic, mycorrhizal, and in some cases
parasitic fungi can aid in this recovery. Saprophytic fungi grow upon dead organic
material. Endophytic fungi join with living plants and the pairing is mutually beneficial,
although each can live independently. Mycorrhizal fungi exist in an obligatory, symbiotic
state. Typically, mushroom-forming mycelium grows on, or in, the roots of trees and other
plants. Mycelium is the body of filamentous fungus; it is composed of a network of
complexly branch hyphae (Trudell et al. 2009). The low-cost (see Figure 1: Soil
Remediation Technologies Costs: Petroleum Hydrocarbons) of implementing

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mycorestoration applications provides an attractive alternative biological solution
compared to Best Management Practice (BMP).

Mycorestoration methods are a potentially low-cost, low-maintenance, biological solution
to remediate toxins and heavy metals from the environment. Many stakeholders and
government agencies, including the Washington State Department of Ecology (DOE) and
the United States Environmental Protection Agency (EPA), may be well served by
examining mycorestoration methods as a Best Management Practices (BMPs). In this
thesis I address the need for an easy-to-use guide for mycorestoration projects that can be
utilized at the grass-roots level.

More specifically, my research targets individual

landowners who own approximately 5-100 acres. Examples of some targeted properties
include small-scale vehicle maintenance yards, agricultural enterprises, managed
timberlands, livestock farms, and residential developments near watersheds or adjacent to
aquatic systems (e.g., rivers, lakes, oceans, bays, creeks, wetlands, and tributaries).
Mycorestoration applications can even be utilized to create buffers to protect endangered
or keystone species from adjacent properties’ contaminants. Though neighbors may not be
consciousness about pollution leaching off their property, individual landowners can
protect their investment by stopping the source of pollution before it negatively affects the
habitat and health of adjacent lands.

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Figure 1: Soil Remediation Technologies Costs: Petroleum Hydrocarbons

Note. Table showing cost comparison of Polycyclic Aromatic Hydrocarbons (PAHs)
remediation techniques. Cost comparisons of remediation methods of polycyclic aromatic
hydrocarbons. Created by Dr. Jack Word, formerly of PNNL/Battelle. (Paul Stamets
2006.)

By protecting our waterways from toxins and heavy metals, we secure
economically significant industries like shellfish harvesting and fishing, which otherwise
stand to suffer devastating economic losses. Polluted sites abound, and even though
mycorestoration is not yet widely used, many parties express interest in small-scale
projects. From the experiences of implementing small-scale projects, larger projects
utilizing mycorestoration methods could become more practical. In addition, by focusing
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on the Pacific Northwest (PNW) as a model, ecologically specific methods will be refined.
Examining PNW pilot projects and case studies have shown positive results can be
achieved by implementing these mycorestoration installations. Later the PNW model can
be expanded to encompass other regions.

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Motivating Factors: Mycorestoration in the Pacific Northwest of North
America
How might individual landowners use mycorestoration to provide a biological
solution to reduce toxins in both soil and water? This thesis constitutes a practical guide
for individual landowners and fits into large-scale environmental and social contexts.
Some aspects related to this topic cannot neatly be categorized into just environmental, or
just social, contexts.
In an environmental context, my thesis topic addresses issues of anthropogenic
toxins entering the environment, harming ecosystems on all three major habitat levels:
terrestrial, aquatic, and riparian. The term “anthropogenic” refers to toxins or pollution
caused by humans. Mycorestoration methods not only involve the study of mycology, but
also have implications for the integration of hydrology, forestry, land-use planning,
conservation, and human health.
In a social context, my thesis connects to political, economic, and historical issues.
Through a political lens, my topic empowers individual landowners demonstrating that
these biological solutions are not just for large-scale government applications. In fact these
solutions can be implemented at the grass-roots level. From an economic perspective,
mycorestoration installations cost less because they work with natural processes, and are
less labor-intensive than other forms of BMP such as bioswales, sand filters, bioretentions
cells (rain gardens) and fabric-filter membranes. BMPs are a compendium of proactive
and often voluntary forest stewardship practices that have been determined to be the most
effective, practical means of preventing or reducing soil and other pollutants from entering
any water; streams ponds, lakes, wetlands, etc. Both fungi and bacteria naturally aid
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wastewater treatment at an estimated ecological service value of $2.4 trillion annually
(Primack, 2010).
The following example fits both into social and environmental contexts. For
instance, excessive amounts of nutrients and toxins entering our environment hinder
aquatic health making seafood unsafe for human consumption, causing both shellfish and
fishing industries to stop harvesting and abate re-planting operations. This coincides with
loss of revenue for employers, which reduces income for employees; the resultant negative
economic consequences trickle throughout the entire larger economic system. This
example has significant, but interrelated environmental and social dimensions.
The goal of small-scale mycorestoration projects such as those outlined within this
thesis will enable and aid recovery of ecosystem health one installation at a time. If
several small-scale mycorestoration projects reduce pollution at the input source or from
adjacent properties the projects will reduce toxins and excessive nutrients, which locally
concentrate. Ecological restoration is altering a site to reestablish – ideally – the original
functioning ecosystem. By healing polluted ecosystems with ecological restoration
methods, this thesis reaches across multiple socioeconomic and environmental disciplines.
The first motivating factor for remediation applications comes from individual
landowners’ realization that the environment health of their land and its surrounding areas
is rapidly degrading. These immediate stakeholders see themselves as stewards of their
own land. The motivation for creating this guide evolved from interest in mycorestoration
projects and the potential for many small individual projects to have a cumulative positive
effect on the entire environment. Stewards of the environment are looking for easy, low

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cost, time- conservative, effective applications to reduce toxins and heavy metals that have
negative impacts on both human and environmental health.
Within the Pacific Northwest (Fig. 2: Map of Pacific Northwest), the Puget
Sound is the nation’s second largest estuary reaching over 100 miles and including 19
river basins, in addition to meandering fresh-water and marine waterways. The Puget
Sound connects to the ocean through the Straits of Juan de Fuca. Puget Sound is a small
portion of what is known as the larger Salish Sea that is one of the most productive and
populated estuary systems in the world.

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Figure 2: Map of Pacific Northwest

Note. Map of Pacific Northwest
http://www.google.com/imgres?q=Pacific+Nw+Map+usgs

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Rich in biodiversity, the Puget Sound supports 211 different fish species, 100
species of waterfowl, 26 species of marine mammals, more than several thousand species
of fungi, in addition to thousands of invertebrates and plant species. Ecosystem diversity
involves different biological communities and their associations with the chemical and
physical environment. Moreover, ecosystem diversity is very rich in the Pacific
Northwest, which classifies it as temperate rainforest (Benedict, 2011).
Archaeological records indicate that people have occupied the PNW since the end
of the last glaciers’ retreat; the Pleistocene era which was about 10,000 years ago (Goble,
1999). Another source states artifacts were found in the Puget Sound at two different
archeological sites one, within the lower Columbia River and the other at the mouth of the
Fraser River dating back 8,000 years ago (Kruckerberg, 1991).
Currently, surrounding the 2,500 miles shoreline, reside 4.4 million people,
approximately 67% of Washington States’ total human population. This includes 15
Native American Tribes (Makah, Nisqually, Puyallup, Quinault, Squaxin, Skokomish,
Suquamish, Snoqualmie, Hoh, Quileute, Muckleshoot etc.) also known as First Peoples
who rely on fishing and shellfish harvest as an economic industry and staple of their diet.
These tribes are commonly referred as the Salish People. To these tribes, the Puget Sound
is culturally and spiritually significant. They have relied on the natural resources of the
Puget Sound for thousands of years. The linear extent of shoreline in the Puget Sound is
comparable to the distance stretching from Washington State all the way to Washington
D.C.!
Ice, water, and wind as well as earthquakes formed the PNW’s unique landscape,
and periodic volcanic rumblings each played a part in creating the Puget Sound and its

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basin, which is commonly known as the Puget Sound Trough (Kruckeberg, 1995). The
Puget Sound Trough reaches as far north as the artificial boundary of the United States
and Canada, as far south as Olympia, Washington, as far east as the Cascade Mountains
and as far west as the Olympic Mountains. Two major land shaping processes were the
continental glaciations following the receding of the glaciers and the stream-cutting
scouring channels which created the Puget Sound, Hood Cannel and Grays Harbor
(Kruckeberg, 1995).
Large underwater formations keep most of the water in the Puget Sound
circulating in the estuary. Approximately 150,000 pounds of untreated toxins go into the
Puget Sound every day, threatening the health of this region’s ecosystem. The Puget
Sound Partnership has identified the main contributing factors to water pollution in the
Puget Sound as human waste, stormwater and industrial discharge. One third of all
shellfish beds show evidence of fecal coliform bacteria contamination in Washington State
(Washington State Department of Health (WSDOH). Furthermore, the US EPA found
30,000 acres of commercial shellfish beds have been forced to close over the last 25 years
because of bacterial pollution.
This pollution impacts human health, causing cancer and birth defects and
reaching across all spectra of income. Within the United States, over 50 % of the nation’s
drinking water wells contain measurable amounts of nitrate and 7 % have detectable
amounts of pesticides. In the U.S. alone approximately 12 billion dollars annually are
spent on health and environmental costs associated with pesticide usage, while estimated
yearly public and environmental health costs related to soil erosion is 45 billion dollars. If
this continues future generations might not be able to produce food for basic survival

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(Amaranthus, 2009).
The scope of this thesis is limited to the most widespread toxins; among these are
polyaromatic hydrocarbons (PAHs), heavy metals, and fecal coliform bacteria
(Escherichia coli). These toxins are widespread and have extremely negative long-term
effects on environmental health (EPA).
For simplicity I have organized this guide into two parts. Part One explains the
different applications of mycorestoration along with case studies and pilot projects. The
purpose of this section is to demonstrate the methods’ effectiveness to potential users.
Case studies are distinguished from pilot projects in that they are completed studies with
reported results. Pilot projects, on the other hand, are promising ongoing projects where
results are not yet available. I will define three major categories of mycorestoration, which
I call “Pillar I, II, and III”: Mycoremediation, Mycofiltration and Mycoforestry. Pillar IV,
Mycopesticide is still highly experimental, and so is not addressed in this thesis which
presents only well understood best mycorestoration practices. I will go through each of the
three pillars in detail, offering for each several scientific case studies and pilot projects.
Part Two focuses on methodology for grass-roots implementation.
1. Mycoremediation centers on the use of fungal mycelium to degrade pollutants in-situ
(i.e., at the place where the original pollution occurred). An example would be
ameliorating an oil spill on land by mixing or layering mycelium onto the polluted soil.
Mycoremediation is a biological solution where saprophytic fungi are used to decompose
toxins in the environment. Saprophytic fungi digest dead organic matter whereas
mycorrhizal fungi live in a symbiotic relationship to most plants and trees. Saprophytic
and mycorrhizal fungi combine to improve plants’ water uptake, eliminate pathogens, and

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make plants’ natural defenses stronger. Mycoremediation has been known to metabolize
petroleum hydrocarbons, and to capture and immobilize heavy metals such as lead,
uranium, and mercury. Fungi have also been known to metabolize chemical pollutants
including, but not limited to: chlorine, dioxins, persistent organophosphates (POPs),
polycyclic aromatic hydrocarbons (PAHs), total aromatic hydrocarbons (TAHs),
polychlorinated biphenyls (PCBs), and trinitortoluene (TNT).

2. Mycofiltration is the use of mycelium to capture and ameliorate flowing chemical and
biological pollutants, thus preventing them from entering sensitive downstream habitats.
Stamets (2006) describes mycofiltration as the use of fungi as a membrane for filtering out
microorganisms, pollutants and silt. Habitats infused with mycelium reduce downstream
particulate flow, mitigate erosion, filter out bacteria and protozoa, and modulate water
flow through the soil. Chemical pollutant examples include, but are not limited to:
chlorine, dioxins, persistent organophosphates (POPs), polycyclic aromatic hydrocarbons
(PAHs), total aromatic hydrocarbons (TAHs), polychlorinated biphenyls (PCBs), and
trinitortoluene (TNT). Examples of biological pollutants include bacterially rich runoffs.
An example of mycofiltration would be placing mycelium mycofilters below a livestock
farm, which captures fecal coliform bacteria and excessive nitrogen runoff, thus
preventing harmful algal blooms that hinder shellfish harvesting and fishing industries.

3. Mycoforestry is the use of fungi beneficial to trees to aid the regeneration of forests.
For instance, the establishment of a new forest on land devastated by repetitive slash-andburn clear cutting practices could be regenerated by mycoforestry. Mycorrhizae have a
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symbiotic relationship with 90% of the plants on earth (Amaranthus, 2009; Primack,
2010). Mycorrhizae have a close, long term biological relationship with another plant or
tree, and the two species are generally found living concurrently in beneficial symbiosis,
evolving and coexisting together. The plant or tree gives the mycorrhizae sugars and in
exchange the plant is fed minerals and is protected from pathogens, thus increasing uptake
in water and experiencing greater growth as compared to plants without mycorrhizae.
Mycorrhizae can scarify themselves in a time of drought, to save host plants, and regenerate as the plants recover.
At the end of each section, after explaining the three main pillars of mycorestoration, a
critique is given.
Part Two focuses on methodology for grass-roots implementation of small-scale
projects. Low and high tech methods are both given. Lastly, I will offer my conclusions
and suggestions for future research in the emerging field of myco-ecological science. If
pollution sources remain unaltered, pollution continues to compound. Having information
for small-scale mycorestoration projects will help individuals address pollution issues on
their property.

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Part I
Mycorestoration
Mycorestoration is an umbrella term for using mushroom mycelia – fungal
networks of thread like cells or hyphae – as a biological platform for cleaning up toxins
and heavy metals in the environment. Because it’s a biological solution, each
mycorestoration project must be designed specifically for a particular site, commonly
referred to as “site-specificity”. Which kind of mycorestoration is best for your land
(mycoremediation, mycofiltration or mycoforestry) depends on the landscape you are
remediating. All three applications involve the following tasks:
1) Analysis of habitat/inventory of the site needs to be conducted.
2) Toxins, heavy metals, native fungi, plants and animals (both terrestrial and
aquatic) in the studied environment should be determined through sample testing.
3) Once tasks (1) and (2) have been accomplished, bench and mesocosm field
application studies are conducted to see if mycorestoration will likely be effective
at removing toxins or heavy metals from the landscape.
4) Finally, field application is executed at a seasonally appropriate time, commonly
during the spring in the PNW. A spring execution allows for the first flush (i.e.,
fruiting) of mushrooms in following fall.
5) Several months after (4), the extent to which the mycorestoration was effective in
reducing pollutants in contaminated soil or water should be determined. Soil and
water samples are taken both above and below the installation, and subsequently
analyzed.

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How do toxins enter our environment? They enter into our environment via a
variety of different anthropogenic vectors: pesticides, herbicides, fertilizers, munitions,
textile dyes, wastewater treatment plants, illegal dumping and estrogen-based
pharmaceuticals, to name a few. Fig. 3: Toxins and Their Primary Origins shows where
these contaminants came from. All these aforementioned industrial activities and products
contribute to and are susceptible to being broken down by mycelium enzymes. Some
species of fungi have proven to be more effective than others (Stamets 2006).

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Figure 3: Toxins and Their Primary Origins

Not all mushroom species break down toxins and bioaccumulation of heavy
metals. Mushroom species are classified into two fungal subcategories: brown rotters and
white rotters. The focus on hydrocarbon degradation has been to use white rotting fungi.
The manganese-dependent peroxidase enzyme produced by white rot mushrooms breaks
down the wood and bonds of hydrocarbons. Bonds in petroleum products share a similar
type of bond that keeps the backbone of woody plants – the lignin– together. Moreover,
this manganese-dependent peroxidase enzyme can be used to decompose a variety of
hydrocarbons: e.g., different oils, diesel, pesticides, and herbicides. After hydrogen-carbon

27

bonds are broken by mycelium, the leftover non-solid byproducts are primarily water and
carbon dioxide. Once the remediation is completed the organic material loses volumetric
mass, 50% is liberated as gaseous carbon dioxide and a 10-20% is converted into water
(Stamets, 2006).
How do white rotters achieve remediation? White rotters create a ligninase enzyme
that metabolizes brown fiber in wood but leaves the cellulose structure behind, causing the
wood to have a whitish appearance (Cabello 2001, Sasek 2003, Stamets 2006). White
rotters break down toxins through a similar process as the metabolization of wood fiber. A
substrate is rendered light in color from the fungal decomposition of lignin
(delignification), leaving cellulose largely intact. Solid blocks of wood can be utilized for
testing whether fungus causes white rot or brown rot. Most brown rotters, however, are
not as effective in bioremediation of a wide array of toxins typical as are white rotters.
White rotters are also more plentiful in the forest than are brown rotters. White rot fungi
have the fastest degrading rates. White rotter species examples are: the oyster mushrooms
(Pleurotus ostreatus and other Pleurotus species, and subspecies), maitake (Grifola
frondosa), turkey tails (Trametes versicolor and Trametes species), reishi (Ganoderma
lucidum), artist conk (Ganoderma applanatum), and crust fungi (Phanerochaete
chrysosporium). All these species have been proven to degrade POP’s and PAHs. This is
important because white rot mushrooms are mycoremediators of toxins that are joined
together by hydrogen-carbon bonds (Stamets 2006, Thomas, 1999).
Mycoremediation and mycofiltration can capture and metabolize the flow of toxins
such as fecal coliform bacteria (found in wastewater from farming practices or failing
septic systems), organophosphates (found in pesticides, detergents, and fertilizers), and

28

PCBs (e.g., those found in insulating fluids within electrical equipment in power plants,
industries, and large buildings). See Fig. 4: PCB Remediation- untrained mycelia. Specific
mushroom-forming fungi (Basidiomycetes) have been shown to break down certain toxins
more effectively than others. Earlier research by Adinarayana et al. (2001) found in both
laboratory and field scale studies that fungal inoculants successfully detoxified persistent
organophosphates. This process of reducing the toxicities produces a less harmful
compound than other methods that can be naturally absorbed within the environment.
Future studies are needed to focus on analyzing the wide array of fungal species and their
effects on different chemical contaminants (Adinarayana, 2001), and it is clear that
making use of fungi is a challenging, important task that could lead to useful byproducts,
saving energy and preventing pollution (Cohen, 2001).

29

Figure 4: Chart of polychlorinated biphenyls (PCBs) Remediation of Untrained Mycelia
after 11 weeks in sediment.

Note. This chart illustrates reduction in PCBs concentration within soil after 11 weeks of
mycoremediation treatment, data derived from Jack Word, New Fields Washington
personal communication project.
Mycologist Paul Stamets has identified which fungal species influence specific
contaminants. See (Fig. 5: Mushroom with Activity Against Chemical Toxins & 6:
Mushroom Species effects on Heavy Metals) and Fig. 7 for photos of mature fungal fruitbodies commonly used in mycorestoration installations.

30

Figure 5: Mushroom with Activity Against Chemical Toxins

Note. This chart shows Mushrooms species that are able metabolize chemical toxins.

31

Figure 6: Mushroom Species’ concentrations of Heavy Metals (Stamets 2006)

Note. This chart displays different species of fungi against different heavy and their
ability to mobilize heavy metals.

32

Figure 7: Mature Fungal Fruit-bodies Commonly Used in Mycorestoration

Note. From left to right; Pleurotus ostreatus (Pearl Oyster), Stropharia rugosoannulata
(King Storphraia), and Tremetes versicolor (Turkey Tail). Photographs by: La Dena Che’
Stamets 5-18-2012.
The ability of fungi to influence contaminates is generally attributed to the lignindegrading enzyme system of the fungus. Within soil conditions, fungal degradation
capabilities are affected by processes similar to bioremediation such as bioavailability,
temperature and other physical parameters, and pollutant toxicity. Optimal performance of
white-rot fungi introduced into the soil depends on its survival, soil matrix, and relation to
autochthonous soil micro flora. Filamentous fungi grow hyphae that invade soil substrates,
secreting water-laden enzymes that degrade polymeric matter that is then utilized as
nutrients by other plants. Further, fungi are highly adaptive can grow with low moisture,
and many species are valued for human consumption.
33

The development of fungal technologies has been hindered because scientists
initially have generalized to all white-rot fungi results derived only from Phanerochaete
chrysosporium. The physiological and ecological diversity of other white-rot fungi
warranted further investigations. (Sasek, 2003). Cerniglia (2001) observes that numerous
experiments have proven that white rot mushrooms can remove PAHs and other complex
mixtures. Results have been positive in laboratory tests as well as in petroleum
contaminated soils: “… the biotransformation process may be characterized as a
sequestration that can lead to eventual detoxification” (Cerniglia, 2001).

Pillar I: Mycoremediation
Mycoremediation has held promise for cleaning up polluted soils since 1985 when
it was discovered that white rot fungus Phanerochaete chrysosporium could metabolize a
number of important environmental pollutants, including polycylic aromatic hydrocarbons
(PAH’s), polychlorinated biphenyls (PCB’s), chlorophenols, dioxins, DDT,
trinitrotoluene, and synthetic dyes (Eggen et al. 2002, Sasek et al. 2003). This ability to
degrade chemical pollutants of different compounds is caused by the extracellular enzyme
system (lignin peroxidase, manganese-dependent peroxidase and laccase) and production
of free radicals (Eggen et al. 2002, Stamets 2005). Mycoremediation is a type of
bioremediation that utilizes fungal enzymes’ natural ability to break down anthropogenic
contaminants, allowing ecological succession to take place naturally. Succession is the
gradual replacement of one group of organisms by another over time following initial
disturbance. This section will describe the science behind the application of
mycoremediation for restoring contaminated soil.
34

Soil contaminated by PAHs is well documented; the persistence and
compounding of PAHs are tantamount to significant ecological risk because of toxicity,
potential carcinogenicity, and resistance to bioremediation. Therefore, these compounds
are on the US EPA’s Priority Pollutant list, which includes 129 different pollutants.
Below I describe mycoremediation of soils contaminated with PAHs.
Using experiments with fungi and yeasts naturally found in aquatic sediment,
surface waters, and terrestrial environments, Cabello (2001) found that fungi and yeasts
can be used to biodegrade PAHs. Fungi can penetrate the soil whereas bacteria cannot;
fungi accomplish this penetration by sending out infiltrating fungal hyphae that exude
enzymes which reach subsoil PAHs. Other lignin-degrading fungi enzymes can
decompose wood and break down and degrade a variety of toxins (Cabello, 2001).
Eggen and Sasek (2002) describe the capacity of spent substrate from commercial
mushroom production of Pleurotus ostreatus to remove PAHs from weathered creosote in
highly contaminated soil from an abandoned wood preservation site. Adding the spent
fungal compost resulted in reductions from 50% (acenaphthene, anthracene) to 87%
(phenanthrene, flourene) within a twelve-week treatment. The reduction increased to 87%
(anthracene) and 97-99% (phenanthrene, flourene, acenaphthene) after additional reinoculation with fungal substrate and an added three-week incubation period. After a
twelve-week fungal treatment period flouranthene and pyrene decreased by approximately
43% and 34%, respectively.
Spent compost of Agaricus bisporus, commonly known as white button
mushroom, has been utilized by gardeners to fertilize and condition soils on disturbed
commercial sites. Several species of fungi (Pleurotus ostreatus, Trametes versicolor,

35

Agrocybe aegerita, Kuehneromyces mutabilis and Stropharia rugosoannulata), are edible
and medicinal, commonly used to benefit human health, and have been extensively
studied (Eggen et al. 2002). These also enable environmental health by metabolizing
chemical toxins.
After eight weeks of fungal treatment the average degradation was ~ 40% and after
a 16-week period, scientists observed that degradation rates had reached 80%. After
fourteen-months of fungal treatment the residual hydrocarbon concentrations were
approximately 7% of the original level (Eggen et al. 2002).
The success in using fungi for bioremediation is largely determined by which fungi
are selected, and how they interact to abiotic (non-living) and biotic (living) systems. In
addition, the remediation application also needs to take into account the particular natural
biological cycle of the fungi selected. See Fig. 8-Chart3- The mushroom life cycle. Fungi
selected must be able to compete with native soil bacteria because different fungi and
bacteria either work together to destroy the PAHs or compete and thus destroy each other.
One must carefully consider how nutritional factors in soils and environmental factors
influence biodegradation rates. Cerniglia (2001) pointed out that a successful
mycoremediation method will also be reliable enough to meet government regulatory
requirements. Methods for the detoxification of PAH residues in the environment must be
cost-effective, quick and environmentally safe.

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Figure 8: The mushroom life cycle

Note. This illustration shows the Mushroom Life Cycle. Hyphae are long tube-like
elements that make up the body (mycelium) of a fungus, and may or may not be separate.
Starting at spore liberation to spore germination to growing hyphae, creating hyphal
knots (and sometimes sclerotia formation), followed by primordial formation fruitbody
developes which then matures to what we see as a harvestable mushroom. Spores
liberated from the mushroom begin the life cycle anew. Climatic conditions may both
limit and aid growth of cycle. © www.fungi.com

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Anyone utilizing mycoremediation should first try native mushrooms specific to
that location. If local mushrooms cannot be reproduced in the lab or there is no evidence
of mushrooms in that particular location, the next step is to use Pearl Oyster Mushrooms
(Pleurotus ostreatus) local to that bioregion, if not specifically to the toxic waste site.
Certainly, there is no threat of species becoming problematic because the saprophytic
mushrooms will run their natural life cycle and then die off after substrate decomposition
(Fig. xx). Instead of a black, toxic, smelly, lifeless pile of soil, decomposition creates an
oasis for other species such as insect larvae, plants, and birds to inhabitant the area
(Stamets 2006, WSDOT 1998, Battelle 1999).
What is crude oil? Crude oil is complex mixture of hydrocarbons and nonhydrocarbons that are toxic to living systems. It is used for energy and raw material for
industries. Increased demand for energy results in increased production, transportation and
refining of crude oil, which results in increased pollution of the environment. The main
source of petroleum hydrocarbon pollution in the environment is low-level discharge, e.g.,
urban run-off, cleaning operations, and oil treatment. These non-point sources of pollution
combined together account for 90% of the total anthropogenic petroleum pollution. Other
oil pollution comes from oil-well blowouts, seepage, and de-ballasting, sale and usage of
petroleum products, pipeline overflow and breakage, and storage tank spills (Obire, 2003).
Obire reported deliberate discharge of oil field wastewater or effluent (liquid waste or
sewage discharge into a river or the sea) as a source of environmental contaminants. Obire
et al. (2009) later assessed the sources of crude oil pollution and its effects on the
environment and microorganisms. Subsequently, they compared methods using many

38

different species of fungi that are occur in oil-polluted environments and were known to
degrade PAHs.
The level of toxicity of petroleum products and crude oil varies based on
composition and concentrations, biological state, environmental factors like weathering,
and the biological state of the organism when contamination occurs. See Fig 9:
Parameters of Bioremediation Process (Created by LaDena Stamets, reference Obrie
2009).

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Figure 9: Parameters of Bioremediation Process

•
•

•

Oxygen & inorganic material

Ph levels

Adequate supply of oxygen.
Most fungi and bacteria that
degrade PHC require free or
dissolved oxygen.
Oil degradation requires a
mineral: C, Ca, Mg, K, S, Fe, N,
or P.

•

Optimal ph for
biodegrading of
hydrocarbons: 6-8
Biodegradation of
crude oil in acid soil
(ph 4.5) could double
by limiting ph to 7.4.

•

Temperature
Hydrocarbon degradation increases with temperature and peaks around 30-40
0
C, which is 86-104 0F.

Absorption Effects

Water availability

Hydrocarbons are adsorbed
onto organic matter are less
susceptible to microbial
attack. The rate-limiting
process in biodegradation
may be desorption of
contaminants.

•

•

•

Soil with maximum water-holding
capacity of 50-80% has greater
microbial activity.
Below that percentage osmotic
and matrix forces limit water to
microbes.
Above that threshold the
reduction of air space and oxygen
decrease microbial activity.

Note. Created by LaDena Stamets. Data derived from the following references: Obire et
al., Fungi in Bioremediation of Oil Polluted Environments 2009.
In heavily polluted areas the effects are instantaneous and detrimental to the
ecosystem and its inhabitants, including plants, animals life, and agriculture. Species at
different stages in their life cycles will have different susceptibilities to pollution (Obire,
2009).

Fungi are amenable to large-scale production, efficiency, genetic engineering or

manipulation, cost effectiveness, and ease of transportation. Certain fungi are known to
40

possess crude oil biodegradation potential. Besides the classic mushroom-forming fungi
like Pleurotus ostreatus, many other non-mushroom forming fungi have been proven
effective in digesting hydrogen, and include but are not limited to the following twentyfour genera hosting species proven potentially useful for mycoremediation:
Acermonium

Graphium

Aspergillus

Hansenula

Aureobasidium

Mortierella

Candida

Mucor

Cephalosporium

Paecilomyces

Cladosporium

Penicillium

Cunninghamella

Rhodosporidium

Fusarium

Rhodotorula

Geotrichum

Saccharomyces

Giiocladium

Sphaeropsidales

Sporobolomyces

Trichoderma

Torulopsis

Trichosporon

Obire et al. (2009) argue that fungi might have an important role in oil cleanup
within the Niger Delta but further studies are needed to apply those techniques in the
region. Obire concludes fungal mycelia are can penetrate oil and increase surface area
that can then be degraded by other microbes. Fungi have the ability to grow under harsh
environmental conditions, for instance, where low pH and poor nutrient limit bacterial
growth. Moreover, fungi are easy to transport, genetically engineer and can be multiplied

41

into large quantities. Thus, Niger Delta would benefit from this biological technology to
clean up its oil-polluted environment.
Heavy metals are of equal concern to environmental pollution from petroleum
products. Although depleted uranium is less radioactive than natural uranium it has the
same chemotoxicity and is harmful to human health. Uranium is utilized in nuclear power
plants to generate the heat in reactors, and produces material for nuclear weapons.
Uranium is also used as a pigment to color glass to produce orange, yellow, and red hues
and was also used for tinting and shading in early photography.
Lead enters our environment in many ways: lead shots from hunting, fishing
weights or jigs, and industrial waste (e.g., paints printing inks, lead water pipes, lead
glazed pottery, battery casings). Lead in all of its forms is a potentially dangerous
pollutant because of its toxicological effects on humans. In the human food-web toxins
including mercury, lead, dioxins and polychorobiphenyls (PCBs) are passed on throughout
each trophic level becoming more concentrated, in a fashion similar to how toxicity
concentrates in harmful algal blooms (HAB) that are lethal to secondary consumers. HAB
can lead to eutrophication; the process of degradation in aquatic environments caused by
nitrogen and phosphorus pollution, characterized by algal blooms and oxygen depletion.
Biomagnification is the process whereby toxins become more concentrated in animals that
are at the higher levels in the food chain. Organisms that are primary producers include
green plants, alga, or seaweed and obtain their energy directly from the sun via
photosynthesis; such organisms are also known as an autotrophic or photosynthetic
species. Levels of biological communities representing ways in which energy is captured
and moved through the ecosystem by the various types of species is referred to as trophic

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levels. Primary producers are herbivores, secondary consumers are carnivores or
detritivores. Mammals at the top of the food chain experience concentrated levels of
toxins through digestion, resulting in chronic diseases such as neuropathy and cancer.
Mycelia can break down these toxins in soil before they enter our food supply.
Furthermore, saprophytic fungi live on dead organic matter and are among the first
organisms to rejuvenate the food chain after catastrophes (Stamets, 2006).

Heavy Metal Mobilization. The accumulation of heavy metals in soil and water causes
harm to humans because we bioaccumulate heavy metals through consumption.
Geomycology is the scientific study of the roles of fungi in processes of fundamental
importance to geology, and the biogeochemical importance of fungi is significant in
several key areas. These include nutrient and element cycling, rock and mineral
transformation, bioweathering, mycogenic biomineral formation, and interactions of fungi
with clay minerals and metals. These processes can occur in aquatic and terrestrial
environments, but it is within the terrestrial environment that fungi are thought to be the
greatest effective geochemical influence (Gadd, 2011). Geoffrey Gadd and his colleagues
have conducted extensive scientific research on this topic by using fungi to mobilize toxic
metals, lead, and uranium. Please refer to Gadd’s flowchart (fig. 10: Mechanisms of fungal
bioweathering of mineral surfaces applicable to myco-deterioration of concrete) for
clarification.

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Figure 10: Mechanisms of fungal bioweathering of mineral surfaces applicable to mycodeterioration of concrete

Note. Mechanisms of fungal bioweathering of mineral surfaces applicable to mycodeterioration of concrete. Gadd et al., (2007) Fungal Degradation of Barrier Concrete
used in Nuclear Waste Disposal.
Gadd has extensively explored the ability of fungal species to uptake heavy metals,
and explains how fungi aid metal transformation: “Mobile metals can be either be bound,
accumulated or precipitated by fungal biomass through biosorption to biomass” (Gadd et
al. 2007). Immobilization can result from bio-absorption. The fungi can take up metal
cations into forms that can be made available intracellularly and incorporated into
biogeochemical processes. Although bioabsorption is a relatively new concept, it’s a
promising field. In addition to revealing scientific insight into metal bioabsorption, Gadd
deepens our understanding of how fungi can affect mobility, transfer between biotic and

44

abiotic locations, and their significance in metal cycling in the environment: “The safe
long-term storage of both existing and future nuclear wastes is of vital importance in
protecting the environment” (Gadd, 2007).
Two ways fungi degrade mineral substrates are biomechanical and biochemical.
Biochemical weathering can be direct or indirect. Direct biomechanical degradation of
minerals can occur through penetration by fungal hyphae into decaying rocks by tunneling
into intact mineral matter and can occur along crystal planes, cleavage, and cracks and
grain boundaries, such as sandstone, calcitic, and dolomitic rocks. The fungal hyphae’s
mechanical forces are derived from the osmotically generated turgor pressures inside the
hyphae. The two main mechanisms of solubilization of rocks and minerals by fungi are
acidolysis and complexolysis, which may be enhanced by metal accumulation in and/or
around the fungal biomass. Many fungi can excrete metal-complexing metabolites
(including carboxylic acids, amino acids, siderophores and phenolic compounds) are
associated with complexolysis or ligand-promoted dissolution. Carboxylic acids derived
from fungi with strong chelating properties aggressively attack mineral surfaces (Gadd,
2007). Gadd et al have explored the relationship between heavy metals (e.g., uranium,
lead) and fungi (e.g., mycorrhizal). Below is a list of some of his case studies, each with a
citation followed by a brief one-paragraph summary that could help the reader understand
the relationship between fungi and heavy metals:
1. Fungal transformations of uranium oxides, 2007 (p. 1696-1710). In this
study Gadd et al., revealed that fungi exhibit a high oxide tolerance, possess the ability to
solubilize uranium trioxide (UO3) and triuranium octaoxide (U3O8), and accumulate
uranium within the mycelium. Uranium speciation, biomass showed in most fungi that

45

the uranyl ion was linked to phosphate ligands; however, in the ectomycorrhizal fungi a
mixed phosphate/carboxylate correlation was observed. Abundant uranium precipitates
associated with phosphorus were discovered in the mycelium encrusting the hyphae.
Some fungi caused biomineralization of well-crystallized uranyl phosphate minerals of
the meta-autunite group. They found all fungal cultures were highly tolerant to UO3 and
U3O8; ectomycorrhizal Rhizopogon rubescens and all other fungi tested were completely
tolerant. Beauveria caledonica increased mycelium density while others displayed no
change. The highest uranium levels were observed in treatments with B. caledonica and
S. himantiodes while the least were found in P. simplicissimum treatments. Mycorrhizal
fungi accumulation of uranium was considered intermediate. In general, oxalate excretion
by fungi increased over the first two-months and then decreased, with the exception of S.
himantiodes which maintained high levels of oxalic acid excretion by the end of the
fourth month. “Many free-living and symbiotic fungi are able to tolerate the toxicity of
mobilized metals. Lichens are successful primary colonizers in extreme metalliferous
environments and have been reported to grow directly on the secondary uranium
minerals…” This study was the first experimental evidence that fungi transform uranium
solids as well as they produce secondary mycogenic uranium minerals.

2. Fungal Deterioration of Barrier Concrete used in Nuclear Waste Disposal, 2007
(p. 643-653). Here, Gadd et al. investigated fungi’s ability to biochemically breakdown
barrier concrete used in nuclear waster disposal. They found fungi successfully dissolved
cement with fungal biofilm and its associated microenvironment. Oxalate-excreting
Aspergillus niger was observed forming plentiful calcium oxalate crystals on the concrete

46

and coated the fungi; see fig. 11: Deterioration of concrete used for nuclear waste
treatment. Microorganisms deteriorate all different types of ceramic and building material
including concrete and cement. Fungi can survive in and colonize concrete barriers under
extremely radioactive contamination. For instance, in 1997-1998 extensive fungal growth
was observed on the walls of the “Shelter” and other structures within Chernobly nuclear
power plant building. High radiation pressure inside the No. 4 reactor caused genetically
altered fungal strains of Alternaria, Cladosporium, and Auzreobasidium. Fungi are able
to deteriorate concrete by chemical weathering of rocks and minerals, and the Fusarium
sp. degradation of concrete proceeded more rapidly than bacterium-mediated
Acidithiobacillus sp. degradation with complexolysis as the main mechanism of calcium
mobilization. In this study scientists experimented with the following fungal species
derived from toxic metal or radionuclide-polluted soils in the Ukraine: Aspergillus niger,
A. versicolor, Fennelia flavipes, Euro herbariorum, Paecilomyces lilacinus,
Cladosporium cladosorioriodes, Alternaria alternata. The fungal species listed above
have been commonly reported to cause deterioration of building materials. Analysis of
concrete matrix displayed quartz, feldspar, calcium silicates, calcite, calcium aluminate
and aluminoferrite. Gadd’s results showed colonization of fungi depends on concrete
dimensions. Observed colonization of fungi avoided granite while some lichen hyphae
avoided quartz. They concluded that these phenomena should be taken into account in
exploiting concrete in nuclear waste disposal, in addition to other building contexts.

47

Figure 11: Visual Aid Showing Concrete Deterioration

Note. Simplified illustration of geochemical transformation of concrete by fungi. On
surface of colonized concrete fungi formed a biofilm, which consisted of hyphae and
extracellular polymeric substance (EPS), retaining moisture with exudate containing
fungal metabolites. Mineral composites of quartz, feldspar, calcium aluminate and
aluminoferrite with quartz and feldspar derived from added aggregates proved to be
largely un-reactive. Fungi biochemically attack the concrete surface, excreting protons
and ligands causing dissolution of concrete. First it reacts with cement paste components
Ca (OH)2 and cement paste calcium-silicate hydrate. The major element of cement
mobilized calcium and silicon were leached from the concrete and accumulated within
the biomass and exudates forming complexes with fungi metabolites and re-precipitated.

48

3. Solubilization of toxic metal minerals and metal tolerance of mycorrhizal fungi,
2005 (p. 851-866). This study focused on the ability of ericoid mycorrhizal (ErM) and
ectomycorrhizal (EcM) fungi to solubilize four toxic metals: cadmium (Cd), copper (Cu),
lead (Pb), zinc (Zn). Both the measurement of radical growth and biomass dry weight
provided indications of metal tolerance. Metal accumulation in fungal biomass was
measured using atomic absorption spectrophotometry. Solubilizing and metal tolerance
varied widely between different fungal species and minerals as well as strains derived
from sites with different degrees of metal pollution. Zinc phosphate proved the least toxic
in addition to being the easiest to solubilize by the majority of tested fungal isolates.
Solubiliziation of heavy metals was linked with pH of the medium and growth tolerance
of fungi. It appeared that acidification of the medium was the main process of mineral
dissolution for most of the mycorrhizal fungi used in this study. Scientists observed lethal
effects for ectomycorrhizal isolates for greater than 60% of strains of Pb phosphate,
carbonate, sulphide and tetraoxide. In contrast ErM isolates were able to grow on Pbmineral-amended media. ErM cultures and 70-90% solubilized Cd and Cu phosphate and
cuprite. Neither ErM and EcM produced a clear zone in Pb mineral-containing agar
petridishes. “However, many fungi were able to accumulate mobilized Pb in their
mycelia. Differences in toxic metal mineral tolerance, mineral solubilzation and metal
uptake between populations isolated from metal-polluted and uncontaminated sites were
related to the toxic metal which was the main pollutant in the contaminated environment.
In general, the metal-tolerant fungi grew and solubilized toxic metal minerals better than
non-tolerant isolates,” (p. 851).

49

4. Role of fungi in the biogeochemical fate of depleted uranium (p. 375-377).
Recent war campaigns in Iraq from 1991-2003 and Balkans 1995-1999 have caused
dispersion of thermodynamically unstable depleted uranium (DU) metal into the
environment. Even though DU is less radioactive than natural uranium, both have the
same chemotoxicity and pose a hazard to human health. “Fungi are one of the most
biogeochemically active components of the soil microbiota, particularly in the aerobic
plant-root zone. [Gadd et al.] report[s] free-living and plant symbiotic mycorrhizal fungi
has the ability to colonize DU surfaces and transform metallic DU into uranyl phosphate
minerals” (p. 375). All fungi tested in this study displayed high DU tolerance and were
able to colonize DU surfaces, forming moisture retaining mycelial biofilms. Fungi
formed cord-like mycelial structures through aggregation of vertically aligned hyphae,
which is commonly interpreted as a survival response to metal stress. Oxalic acid, a
strong metal chelator, was produced by the DU-treated fungi and DU was observed to
promote oxalate excretion. Gadd et al. suggests the role of oxalate in ligand promoted DU
dissolution could be more significant than acidification; furthermore fungi Beauveria
caledonic, Hymenoscyphus ericae and Rhizopogon rubescens, where exposed to DU
showed increase accumulation of uranium along with increased amounts of excreted
oxalate. “Metal immobilization can assist mineral dissolution processes and DU-exposed
fungi demonstrated a remarkable ability to accumulate mobilized uranium in their
biomass. Extensive uranium biomineralization occurred in all parts of the fungal
colonies, from those regions adjacent to the DU coupons to even the remote marginal
edges.” (p.376). Fungi are particularly important in metal-rich and acidic soils. Gadd et
al. were the first to show fungi can transform metallic uranium into metallic uranium into

50

meta-autunite minerals that have the ability of long-term uranium retention. Because of
this and the fact that most terrestrial plant species are dependent on symbiotic
mycorrhizal fungi, “this phenomenon could be relevant to the future development of
various remediation and revegetation techniques for uranium-polluted soils” (p.375).

5. Geomycology: metals, actinides and biominerals, 2011 (p. 1-27). Fungi posses
many properties that can effect changes in metal speciation, toxicity and mobility and
mineral formation or mineral dissolution or deterioration. “Some fungal transformations
have beneficial applications in environmental biotechnology, e.g., in metal and
radionuclide leaching, recovery, detoxification and bioremediation, and in the production
or deposition of biominerals or metallic elements with catalytic or other properties” (p.
1). Gadd et al. state, “[g]eomoycological roles of fungi have often been neglected in
wider geomicrobiological contexts but they are of significant importance of in several
key areas” (p. 19). Fungi are also being recognized for their importance within aquatic
habitats specifically in sediments but their importance may be underestimated.
Mutualistic relationships of fungi with phototrophic organisms, lichens and mycorrhizae
are especially important as geoactive agents. Since fungi are found everywhere within the
biosphere these processes emphasize the importance of goemycology as an
interdisciplinary subject area within microbiology and mycology.

6. Lead Transformation to Pyromorphite by fungi, 2012 (p. 1-5). Lead (Pb) in all its
chemical forms is a serious environmental pollutant. Metallic lead is an important
structural and industrial material that is subject to weathering and also enters the soil

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from hunting. Gadd et al. “… examined the influence of fungal [Metarhizium anisopliae
and Paecilomyces javanicus] activity on lead metal and discovered that metallic lead can
be transformed into choloropyromorphite, the most stable lead mineral that exists. This is
of geochemical significance, not only regarding lead fate and cycling in the environment
but also in relation to the phosphate cycle and links to microbial transformations of
inorganic and organic phosphorous” (p.1). Gadd’s report provides insight into mycogenic
choloropyromorphite formation from metallic lead while highlighting its significance as a
biotic component of lead biogeochemistry, along with additional consequences for
microbe survival within lead contaminated environments and bioremedial treatments for
Pb contaminated soils.
In the following sections I detail three mycoremediation case studies and one pilot
project, and aim to provide you with examples of how mycoremediation experiments were
carried out and the results.

First Example: Mycoremediation Pilot Project
Makah Nation’s Tatoosh Island Neah Bay, Washington. 2009- present
The Makah Tribe places great cultural value on Tatoosh Island, and traditionally
this island was a sacred site for burying the deceased. As mentioned earlier,
mycoremediation is a comparatively inexpensive and effective new solution to the
problem of oil contaminated soil and water, which impacts ecosystems health. Restoration
methods first conducted by subcontractors working with the Department of Defense and
the Makah Indians that removed polluted and contaminated soils from the island by
helicopter were simple but cost excessive. They thus searched for a more functional,
inexpensive, time conservative solution to their problems and considered mycorestoration
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as the solution to contamination on Tatoosh Island. Before restoration could begin,
however, more research and proof of concept using native species collected from Tatoosh
Island were needed.
The Makah Environmental Restoration Team (MERT) has therefore been working
for the last nine and a half years, exploring methods for restoring Tatoosh Island to
conditions before Navy occupancy (RIDOLFI, 2009). Cerniglia et al. (2001) stated that
abandoned military sites are usually contaminated by toxins ranging from petroleum, coal,
and chemical residues. Contaminated soils on Tatoosh are extremely toxic. Tatoosh’s
landscape has steep cliffs that make accessing the island by boat or canoe extremely
difficult. Appling clean up and in particular soil removal or replacement have proven
extremely expensive and time consuming. Navigating around the rocks to a small beach
by boat makes access difficult because of strong ocean currents. Thus helicopter transport
is the preferred, although expensive, method of transportation. Removing a single cubic
yard of diesel-contaminated soil off Tatoosh to the mainland a few miles distant costs
approximately $1,000/ton. It is estimated that 300 tons of PCS (Petroleum Contaminated
Soil) are present on Tatoosh Island. In need of a more cost effective solution, MERT
believed solutions using variations of mycoremediation could be the key (RIDOLFI,
2009).
As described earlier, oyster mushrooms are white rot fungi, which are able to
metabolize and digest PHCs “…by breaking apart the hydro carbon bond and releasing
carbon dioxide and water” (Stamets, 2005). The prevailing hypothesis of how this occurs
is that the hydrocarbons are demolecularized and re-assembled into fungal carbohydrates.
Remediation efforts on Tatoosh Island currently focus on using the native fungal species

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to reduce toxicities in the petroleum-contaminated soil. Several species of native
mushrooms were collected for subsequent tissue culture isolation in October 2009. This
approach is intrinsically less expensive because materials transported by helicopter would
be drastically reduced. See Fig. 12 for photographs of Tatoosh Island; the native fungi we
collected for lab analysis along with abundant salmonberry canes as possible substrate for
inoculation while Tatoosh Island in ruins, Fig. 13: Native fungi Marasmiellus found on
Tatoosh & Salmonberry canes.
After flying to Tatoosh Island with Ridolfi, Inc., the author and mycologist Paul
Stamets to help collect native fungi and salmonberry canes, Fungi Perfecti successfully
isolated pure cultures from native wild mushrooms. One species is particularly interesting
because it was found growing on the salmonberry canes, the most abundant source of
cellulose on the island. USDA scientists then showed its effectiveness in degrading PHCs
in contaminated soils, and have documented this fungal species a Marasmiellus candidus
senu lato. Fungi Perfecti proposed to use salmonberry canes as the cellulosic medium for
inoculation. Since salmonberry canes are habitat for many different bird species
participants must be careful not over harvest this resource and displace native bird species.
Before use, salmonberry canes need to be rendered into a form so that mycelium will
colonize. Sterilization by autoclaving is not practical for this site, but submerging canes in
water for anaerobic fermentation or placing the salmon berry canes into .3% solution of
hydrogen peroxide are cost effective methods of rendering this substrate useful for
subsequent colonization by remediating mycelium.
In May 2012, Makah Tribe High School entered a contest through Samsung
Electronics sponsored contest called Solve for Tomorrow and was awarded a $70,000

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grant for the novelty of using mycotechnology. This multiyear project is on-going and will
undoubtedly undergo revisions of methods depending upon results.
Figure 12: Images of Tatoosh Island Neah Bay, Washington

Note. From left to right, view of Tatoosh Island from helicopter, condemned buildings
post navy occupany, imploded cement building, rusted bunker C oil barrel.

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Figure 13: Native fungi Marasmiellus found on Tatoosh & Salmonberry canes
a.)

a.
b.)

Note. a.) Native fungi Marasmiellus found on Tatoosh b.) Abundant salmonberry canes.
Photos by Paul Stamets.

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Second Example: Mycoremediation Case Study
Bioremediation of PAH-Contaminated Soil by Composting: A Case Study
Soil contaminated with PAH is of major concern and significance because of the
toxicity, potential carcinogenicity and resistance to biodegradation. Cajthaml et al. (2002)
Bioremediation of PAH-Contaminated Soil by Composting: A Case Study, a laboratory
and field research project, was carried out to determine the degradability of 3 to 6-ring
unsubstituted PAHs and other organopollutants within a composting system. Composting
relies on the actions of microorganisms to degrade organic materials results in
thermogenesis and the production of production of organic and inorganic compounds.
Metabolically generated heat is concealed within the compost matrix and results in an
increase in temperature.
The aim of this study was to determine the efficacy of composting the PAHcontaminated soil collected from a former tar production plant. Since scientists working
on this case study used the same soil in another study where soil was treated by white-rot
fungi, they were able to compare effectiveness of both composting and fungal treatments
in PAH removal. Because the biodegradation of PAH can result in the production of
metabolites more toxic than the original compounds, they also conducted an ecotoxicity
test using luminescent bacteria and mustard seed.
During the composting soil mixing day, five random samples were taken for
chemical analyses and toxicity testing. In conjunction with five samples from recently
contaminated soil six samples were taken at different intervals within the compost pile.
Samples were then air-dried prior to analysis of PAH and ecotoxicity. Out of the sixteen
PAH samples described as important by the US EPA, eleven were analyzed throughout
the duration of the experiment.
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Study results revealed a 42-68% decrease in concentrations of phenanthrene,
anthracene, flouranthene, and pyrene. The decrease in concentration of the higher-molarmass PAH was lowered to 35-37%. Aside from the anthracene, no future decline in the
concentration of other PAH was observed after maturation. However, in earlier
experiments where the same soil was utilized for bioremedation involving two white-rot
fungal species (Irpex lacteus & Pleurotus ostreatus) results showed that fluorine was
decreased by (41 & 26%), anthracene by (29 & 19%), flouranthene (FLT) by (29 & 29%),
pyrene (PYR) by (24 & 22%), and phenanthrene (PHE), displayed a 20% reduction by
using only Irpex lacteus.
The fungi didn’t degrade higher-molar mass PAH’s. The comparison between
bioremediation efficient of white-rot fungi and composting technology revealed
composting was substantially more efficient in removing PAH, in reference to highermolar mass compared to the fungal treatment.
Guerin (2002) compared two methods for removing PAH from aged soils of a
former tar-contaminated site and found composting to be stronger technique than landfarming processes. Soil composting proved substantially more effective at removing
higher-molar mass PAH. Over a seven-month treatment, removal by composting was 50%
and by the farming technique the removal didn’t surpass 5%. These results gave promise
to Cajthaml et al. that their study would have similar reduction rates.
Comparative analysis of toxicity results obtained after composting and
mycoremediation showed both treatments resulted in a decrease in toxicity of remediated
soils as assayed by bioluminescence test on water elutriates.

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Third Example: Mycoremediation Case Study
Use of fungal Technology in soil remediation: Water, air, and soil pollution,
2003
Sasek et al. (2003) conducted a case study using fungal technology in soil
remediation. Many studies look at removing PAHs either with soil bacteria or with fungi
but none to my knowledge combine these two methods. Moreover, most studies have been
conducted in artificially PAH-contaminated soils. Sasek et al. focused on combining fungi
and bacteria as bioremediation techniques in-situ. They utilized two white rot fungi: Irpex
lactetus and Pleurotus ostreatus; a PAH-degrading bacterial strain of Pseudomonas putida
was used as inoculum for bioremediation of petroleum hydrocarbon-contaminated soil
from a manufactured gas-plant-landscape. After a 10-week duration of experiments, they
found that, out of 12 different PAHs concentrations, four (phenanthrene, anthracene,
flouranthene and pyrene) decreased up to 66%. The eco-toxicity of the soil after fungalbioremediation did not reveal any detectable negative effects on the crustacian Daphina
magna.
Sasek et al. (2003) used biological indicators to assess acute and chronic toxicity
of PAH-contaminated soil before, during, and after remediation. PAHs degradation of soil
toxicity was significant with reductions ranging from 35.2% to 92.3%. Ecotoxicity tests
were performed on three different test organisms: luminescent bacteria, earthworms and
mustard plant seeds (Barassica alba). A two-way analysis of variance (ANOVA) was
utilized to evaluate effects on seed germination and earthworm survival. Soil was
collected from a closed gas plant in Prague, Czech Republic, where total concentrations of
PAHs were 609.8 mg kg -1. The soil was also analyzed for cyanides and several heavy

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metals (e.g., cadmium, cooper, mercury and lead). Fungal cultures were maintained on
malt agar extracts. Finely ground, sieved wheat-straw was used as substrate for the
inoculation of both fungi. The straw was moistened with distilled water and sterilized in
an autoclave; fungi were grown on this substrate for two weeks prior to use for
bioremediation in contaminated soil. In this experiment, Sasek et al. used seven different
combinations with one as control. Fungi did not need to be reapplied but bacteria cultures
were reapplied every 5 weeks.
Sasek et al. (2003) extracted and analyzed soil samples, and showed after 5 weeks
that I. lacteus alone and co-cultured with P. putida were more effective in removing PHE
(63%), FLT (15%), and PYR (36%). Before remediation techniques were applied,
concentration of individual PAHs was respectively 100%. P. ostreatus and its co-cultures
only reduced PAHs by 30%. Sasek et al. (2003) concluded that I. lacteus was more
effective than P. ostreatus and bacterial cultures of P. putida in removing different kinds
of PAHs. Applications of the two individual fungi with P. putida didn’t improve
degradation efficiency compared to fungi alone. Sasek et al remarked: “… this suggests
that co-culture conditions with the respective fungi were not suitable for bacteria to take
part in PAH degradation” (p.13). I suggest this was because the bacteria and fungi were
outcompeting each other instead of metabolizing the PAHs.

Fourth Example: Mycoremediation Case Study
Mycoremediation: WSDOT 1996-1998 Case Study
In 1996, Paul Stamets in partnership with Battelle Pacific Northwest Marine
Sciences Laboratories (MSL) conducted mycoremediation experiments using Pleurotus
ostreatus to break down toxicities in diesel saturated soil and bunker C oil which was 30
60

years old (Stamets, 2006). As oil ages, it becomes more difficult to remove from soils.
The oil and diesel contamination in these experiments was 20,000 parts per million (ppm)
of total aromatic hydrocarbons (TAH), or about 2% of total mass. Concentrated
contamination of this spill was comparable to the 1989 Exxon Valdez event where 11
million gallons of crude oil spilled into Prince William Sound. In 1998 the Washington
Department of Ecology gave the Washington State Department of Transportation
(WSDOT) the authority to conduct an experiment using mycoremediation. Researchers at
the MSL in Sequim, Washington, teamed with Paul Stamets of Fungi Perfecti to test
mycoremediation on this contaminated site (WSDOT, 1998, Stamets 2006).
WSDOT put four piles of diesel-contaminated soil – approximately 50 cubic yards
each – onto individual 6mm black plastic polyethylene tarps. Piles were all of the same
size, mounds sloping up to 4 feet high, 8 feet wide and 20 feet long. Into one of the four
piles they mixed 3 cubic yards of cultured Pleurotus ostreatus sawdust spawn (Fig. 14),
which is approximately equal to 20% percent of the pile’s volume. A layering method was
used, called parallel sheet spawning. This proved more effective as the mycelial fragments
seek connection to one another. Two of the other piles were given bacterial and enzyme
treatments while the fourth pile was left alone as an untreated control. The pile inoculated
with mycelium had a shade cloth draped over it. The other piles were covered with black
plastic tarps to avoid contact with rainfall. After a four-week period the piles were
examined. The bacteria-treated and untreated piles of dirt were still black, smelled of
diesel fuel and showed no signs of life, while the myceliated oil pile was lighter in color,
lacked diesel fumes, and showed signs of myceliation. (Stamets 2006, WDOT 1998,
Battelle 1999).

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Upon examination, the scientists were astonished to see a flush of hundreds of
Pleurotus ostreatus (Pleurotus ostreatus) mushrooms, some as large as twelve inches in
diameter (Fig. 14). Stamets’ text states this result can only be obtained if the substrate
within the medium is nutritionally supportive for mushroom formation and development.
Moreover, the soil that was once black had been transformed and become light brown and
lacked the oil and diesel smells (Stamets 2006, WDOT 1998, Battelle 1999).
Another important aspect to this study is that the mycoremediated pile didn’t
require any additional maintenance; in contrast the WSDOT and sub contractor had to
maintain the bioremediation- and enhanced-bacterial- applications in the other test piles.
WSDOT applied 12 pounds of nitrogen fertilizers to the 50 cubic yards of contaminated
soils under the assumption that the level of contamination was approximately 20,000
mg/kg TPH. Maintenance required monthly turning and additional fertilizers. WSDOT
and PSCI Tank services applied enhanced bacterial treatments, which require biweekly
and/or monthly applications of liquid fertilizer and bacterial inoculums along with
biweekly or monthly rotations of the soil.
These experiments took place within a 16-week period from March to July 1998
(WDOT 1998, Battelle 1999, Stamets 2006). Scientists, however, speculated colder
months during the experiment could have hindered the growth of the fungi. MSL cautions
that variables to be considered are humidity, temperature of substrate, distribution of
contamination within soil, and duration of treatment along with biological availability
(Battelle, 1999).
After eight weeks the vascular plants (Fig. 14: Remediation of Aged Oil in
Excavated Soil for WSDOT 1998) were growing and a community of insects and birds

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were feeding off the mycoremediated pile (Stamets 2006, WDOT 1998, Battelle 1999).
The fruitbodies of Pleurotus ostreatus sporulated at maturity, and thereafter began to
decompose, primarily from bacteria and other fungi. (Stamets 2006, WDOT 1998). During
decomposition many forms of life were attracted to this habitat, including insects, which
in turn attracted birds. Birds feeding off insect larvae presumably left seeds, which could
also have been windblown. The pile soon harbored a complex diversity of plants, bacteria,
insect and fungal species (Stamets 2006, WDOT 1998).
Even though the insects and animals consumed the mushrooms, the researchers
advised that humans, out of an abundance of caution, should not consume mushrooms
from contaminated soils. Although there were no detectable petroleum residues in the
mushrooms tested, analysis has shown an increase in heavy metals, which is cause for
concern. The primary by-products from the mycelium were water and carbon dioxide
because they are heterotrophic. The physical volume of the pile substantially shrunk in
comparison to the other piles. Although the soil was not recommended for agriculture, nor
were the mushrooms considered safe for human consumption, the biomass was approved
for use in landscaping.
Battelle and WSDOT reported TPHs were reduced from 20,000 ppm to less than
200 ppm after a period of sixteen weeks, thus demonstrating mycoremediation effective in
small-scale habitat recovery with the potential for habitat recovery on global scales. It is
common knowledge in environmental studies that the more weathered and aged are
petroleum-contaminated soils, the harder they are to remediate (WADOT 1998). Fig. 14
shows the mycoremediation process in chronological order from left to right; note the
color and texture change over time.

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Figure 14: Remediation of Aged Oil in Excavated Soil for WSDOT 1998

Note. From left to right, soils contaminated with 30-year-old Bunker C oil, inocluated
pearl oyster mushroom sawdust spawn, mycoremedaiton pile control and bacteria enzyme
treated piles, pearl oyster mushroom flush (fruiting), remediated pile. Notice other plants
have begun to take hold. Photos by Susan Thomas.

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Fifth Example: Mycoremediation
Compost-mediated Removal of Polycyclic Aromatic Hydrocarbons from
Contaminated Soils, 2003
In this experiment Sasek et al. (2003) examined compost-assisted remediation for a
manufactured-gas plant soil contaminated with polycyclic aromatic hydrocarbons within a
thermally insulated composting chamber using mushroom compost that consisted of wheat
straw, chicken manure, and gypsum.
Composting is a widely used practice to degrade solid waste materials like
agricultural wastes, sewage sludge, and food waste. In more recent times, composting has
been studied as a remediation technology for hazardous waste. Both laboratory and field
scale studies have been carried out to determine the ability to degrade of PAHs along with
other organopollutants within the composting system. PAHs are of especial concern at
many sites, which includes wood-treatment facilities and manufactured-gas plants. Soil
that is contaminated with PAHs is of major concern because its toxicity and potential of
being carcinogenic in addition to being resistance to biodegradation. PAHs pose
significant ecological risk therefore these compounds are listed on the EPA priority
pollutants list which includes 129 different pollutants2.
In this study initial degradation of individual PAHs was from 20-60%. At the end
of the 54-day period they observed an additional reduction of 37-80% of PAH. Chemical
analysis of the contaminated soil for PAHs, ecotoxicity tests on bioluminescent bacteria,
earthworms, and plants seeds were measured both before and after decomposition. After
decomposition scientists noticed inhabitants of bioluminescence declined, and no
significant change in toxicity was observed for earthworm survival and seedling
2

http://water.epa.gov/scitech/methods/cwa/pollutants.cfm
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germination. Genotoxicity tests were performed on samples taken from different parts of
the composted pile. Only after composting were decreases in genotoxicity observed in
samples from the top of the composted pile.

Critiques of Mycoremediation Studies
The following section gives my personal critique of mycoremediation in reference
to the case studies and pilot projects discussed in the above sections.

First example. Makah Nation’s Tatoosh Island Neah Bay, Washington 2009- present
mycoremediation pilot project. Unfortunately this project has another obstacle to
overcome: upon conducting a test for measuring heavy metals Ridolfi found abundant
amounts of lead, which cannot be degraded but could be captured and moved into another
insoluble form by using fungi, thus preventing it from leaching back into the environment.
This is a good example of why it is important to test for a variety of possible toxins on
your site during the early phases of a remediation project. Since funds are usually tight
and testing expensive, any new discoveries of toxins complicate remediation and increases
expenses. Luckily other fungi have the ability to uptake heavy metals. For instance, Gadd
et al. studied the influence of fungal Metarhizium anisopliae and Paecilomyces javanicus
activity on lead metal and discovered that metallic lead can be transformed into
choloropyromorphite, the most stable lead mineral that exists. I suggest that if no native
fungi on the island have the ability to uptake heavy metals scientists need to consider nonnative fungal species, like those listed in the above studies. That said, participants in the

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Tatoosh mycoremediation project need to be cautious about introducing non-native
species so not to disturbed the natural ecosystem of this small island.
For removal of petroleum in contaminated soils a Marasmiellus candidus senu lato
could be utilized; USDA scientists documented its effectiveness in degrading PHCs in
contaminated soils. However if this species cannot give the Tribe the results it wants then
Pleurotus ostreatus or Irpex lacteus species are promising candidate fungi to be
implemented.

Second example. Cajthaml et al. (2002) Bioremediation of PAH-Contaminated Soil by
Composting: A Case Study. The goal of this study was to determine the efficiency of
composting the degradation of PAH in contaminated soil collected from a former tarproduction plant. Since scientists used the same soil in another study where soil was
treated by white-rot fungi, they were able to compare the PAH-removal effectiveness by
both composting and fungal treatment. The biodegradation of PAH may result in the
production of metabolites more toxic than the original compounds; ecotoxicity tests were
conducted using luminescent bacteria and mustard seed.
Out of the sixteen PAH’s described to be important by the US EPA, eleven were
analyzed throughout the duration of the experiment. They compared this study to control
samples PAH analysis of composted material revealed a 42-68% decrease in the
concentrations of phenanthrene , anthracene, flouranthene, and pyrene. The decrease in
concentration of the higher-molar-mass PAH was to 35-37%. Aside from the anthracene
no future decline in the concentration of other PAH was observed after maturation.

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The fungi didn’t degrade higher-molar mass PAH as readily as lower-molar mass
chemicals. However, in their earlier experiments where the same soil was utilized for
bioremediation, white-rot fungal species (Irpex lacteus and Pleurotus ostreatus) were able
to remove fluorine (41 & 26%), anthracene (29 & 19%), flouranthene (FLT) (29 & 29%),
pyrene (PYR) (24 & 22%), and phenanthrene (PHE) (20% by only Irpex lacteus). Whiterot species were effective at removing PAHs in other past and future experiments
conducted by Sasek, who was one of the leading scientists working on this study and
concluded reduction in PAHs using fungi to be 35.2 -92.3%. I ask whether the mushrooms
were utilized at a seasonally appropriate time and whether other environmental conditions
like moisture and temperature were considered. How often was the compost pile turned?
How substantial is it to have a reduction in high molar-mass when other PAHs reductions
are apparent? Without this information, it is hard to determine how they can improve the
study in the future.
Comparative analysis of toxicity results obtained after composting and
mycoremediation showed both treatments resulted in a decrease in toxicity of remediated
soils as assayed by bioluminescence test on water elutriates. They found using seedgermination tests that the actual soil toxicity was lowered only after mycoremediation.

Third example. Sasek (2003), Use of fungal Technology in soil remediation: Water air
and soil pollution, utilized two white rot fungi Irpex lactetus and Pleurotus ostreatus with
a PAH-degrading bacterial strain of Pseudomonas putida used as inoculum for
bioremediation of petroleum hydrocarbon-contaminated soil from a manufactured gasplant-landscape. After a 10-week duration of experiments, they found that, out of 12

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different PAHs concentrations, 4 were decreased up to 66%. PAH degradation of soil
toxicity was significant with reductions between 35.2% and 92.3%.
Fungi did not need to be reapplied when bacteria cultures were reapplied every 5
weeks. Extraction and analysis of soil samples showed after 5 weeks that I. lacteus alone
and co-cultured with P. putida were more effective in removing PHE (63%), FLT (15%),
and PYR (36%). P. ostreatus and its co-cultures only reduced PAHs by 30%. Sasek et al.
(2003) concluded that I. lacteus was more effective than P. ostreatus and bacterial cultures
of P. putida in removing different kinds of PAHs in this particular circumstance.
Applications of the two individual fungi with P. putida didn’t improve degradation
efficiency compared to fungi by itself. Sasek et al remarked: “… this suggests that coculture conditions with the respective fungi were not suitable for bacteria to take part in
PAH degradation” (p.13).
I suggest fungi were not suitable to use with bacteria for PAH degradation because
the bacteria and fungi were outcompeting each other instead of metabolizing the PAHs.
Variables for fungi growth should be considered including humidity, temperature of
substrate, distribution of contamination within soil, and duration of treatment along with
biological availability. These variables could have had positive outcomes on degradation
of PAHs. In future work I suggest they try remediation using fungi first and then apply
bacteria subsequently. Other fungi I suggest utilizing for PAHs removal include
Bjerkandera adusta, Naematoloma (Hypholoma) frowardii, Serpula lacrymans, and/or
Stropharia rugosoannulata.

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Fourth example. Washington State Department of Transportation 1998 mycoremediation
case study. After mycoremediation of PAHs with Pleurotus ostreatus mushrooms – if
analysis has shown there to be an increase in or presence of heavy metals, even though
there may be no detectable petroleum residues – I suggest using a fungi species can
mobilize heavy metals out of the soil. To determine which species to use depends largely
on which heavy metals are present. Please refer to Figure xx: Mushroom Species effects
on Heavy Metals for more information about which species are active against arsenic,
cadmium, copper, lead, mercury, and radioactive cesium. This case study was
fundamental to understanding that Pleurotus ostreatus mushroom mycelium has the
ability to remove 30 year old aged PAHs out of contaminated soils. I would like to see
another study similar to this but with the added factor of high concentrations of heavy
metals, comparing and contrasting concentration levels both before and after
mycoremediation. I recommend utilizing the natural symbioses of Pleurotus ostreatus
and another fungal species that has ability to mobilize the heavy metal of concern.

Fifth example. Compost-mediated Removal of Polycyclic Aromatic Hydrocarbons from
Contaminated Soils; in this experiment Sasek et al. found degradation of individual PAHs
was from 20-60%. At the end of the 54 day period they observed an additional reduction
of 37-80% of PAH. Total PAHs reduction in this study was considerable. Both laboratory
and field scale studies have been carried out to determine the ability to degrade of PAHs
along with other organopollutants within the composting system. This study showed
fungi’s ability to reduce PAHs by a substantial amount. Future studies by Sasek should be
referenced if one is interested in PAHs degradation by white-rot fungi. Had their testing

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period been at least a year long, a degradation curve would have provided much more
information about the duration of active decomposition beyond the confines of two month
observations.

Pillar II: Mycofiltration
Mycofiltration (fig. 15: Simplified Illustration of Mycofiltration Installation
process and fig. 16: Mycofiltration Symantec Installation) is a relatively new concept that
could be installed into streams or ditches to filter harmful anthropogenic contaminants out
of watersheds. The mycofiltration installation process is achieved by first identifying the
contaminants at the site. Next, the remediating fungal species and delivery vehicle is
chosen. For streams, ditches, and swales, burlap sacks filled with woodchips, and then
inoculated with fungi, are preferred. Next, mycofiltration bags are strategically placed
downstream. Four different options are illustrated in fig. 16 to help clarify installation can
be found on page 118. Then mycofilter bags are secured by nails and stakes to prevent
movement. Installation time is estimated at 45 minutes for 20-30 units. Cost of the
mycofiltration installation is about $600 to $800 per site, which includes labor, equipment,
and other materials not including inoculated burlap sacks donated by Fungi Perfecti
(which would be $8/per mycofilter bag, with each weighing approximately 22 pounds).

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Figure 15: Simplified Illustration of Mycofiltration Installation process

© Paul Stamets from Mycelium Running, 2005
Note. The above illustration shows a simplified version of the mycofiltration process
capturing toxins from both industrial and residential pollution.

The Clean Water Act (CWA) of 1972 established regulations relating to discharge
of pollutants into U.S waters, and also regulates quality standards for surface waters.
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Under the CWA the EPA has the authority to set effluent limits on an industry-wide basis
and has implemented pollution control programs as they relate to water quality, e.g.,
setting wastewater standards for industry. The EPA has also set water quality standards for
all contaminants in surface waters. The CWA made it illegal to discharge pollutants from
a point source into navigable waters unless a National Pollutant Discharge Elimination
System (NPDES) permit is obtained. Point sources are discrete conveyances and include
pipes or man-made ditches, individual homes that are connected to the municipal system,
and septic systems. Industrial, municipal and other facilities must have a permit if their
discharge goes directly into surface waters (Clean Water Act 1972). Alternative
approaches for filtering water are bioretention cells (rain gardens), retention ponds,
bioswales, water harvesting, sand filters, contour line infiltration trenches, small check
dams, and straw bales. Bioswales are commonly found in urban areas to filter stormwater
runoff.
First Example: Mycofiltration Pilot Project
The Evergreen State College Mycofiltration Pilot Project 2011- present
Tim Benedict, a Master of Environmental Studies graduate student, conducted
bench test studies in The Evergreen State College (TESC) laboratory to see if a
mycofiltration installation pilot project could be implemented in Snyder Creek on the
Evergreen campus. Snyder Creek is commonly used to teach students how to conduct
water quality testing. Benedict’s paper examines fungi’s ability to avert waterborne
pollutants from entering the Puget Sound and its tributaries by using mycofiltration as a
biological solution.

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This study was conducted in collaboration with Fungi Perfecti, a company that is
developing new concepts and methods of using fungi to degrade and sequester
contaminates in our waterways. Benedict conducted controlled laboratory experiments
with the Nisqually strain of Pleurotus ostreatus to assess the capability of using
mycofiltration as a possible BMP. He found that filtering fecal coliform bacteria (FCB)
contaminated water with nothing but woodchips reduced FCB concentration by 12%.
However, when filtering through a mycofilter of sterilized alder wood chips inoculated
with Nisqually strain of Pleurotus ostreatus, the fecal coliform count was reduced by
63%. While using non-sterilized alder wood chips inoculated with Nisqually strain of
Pleurotus ostreatus the fecal coliform count was reduced by 87%. Benedict concluded that
there was a 26% increase in effectiveness when using unsterilized woodchips colonized
with Pleurotus ostreatus mycelium.
The second set of mycofiltration tests used two different substrates that were
inoculated with same fungal species: straw and a sawdust/straw blend. In this portion of
the experiment only sterilized mycofilters were utilized. Benedict found that FCB
concentration levels fell below detectable levels and visually showed a significant
reduction in fecal coliform bacteria. Benedict concluded that a “… mycelium network of
Pleurotus ostreatus Nisqually strain efficiently filters and metabolically consumes fecal
coliform bacteria. Mycofiltration has substantial promise for helping address fecal
coliform contamination in the Puget Sound.” He concluded that there are many advantages
to mycofiltration: it is an effective, low cost, biological solution that requires minimal
maintenance and is flexible for installation at a variety of sites. In addition, only
decomposable byproducts are left by this application. The total costs of the lab supplies

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were $300 for 200 mycofiltration tests (Benedict, 2011). He suggests further research to
address flow-rates parameters and installation design, to both maximize filtration while
allowing for appropriate oxygen levels. Evergreen’s sustainability office showed interest
in a mycofiltration installation at Synder Creek but felt it needed several more months of
research. Benedict concluded that mycofiltration should be an ongoing project and should
be applied as a BMP.

Second Example: Mycofiltration Pilot Project
Phase I (Proof-of-Concept) EPA Bench Study Comprehensive Assessment of
Mycofiltration Biotechnology to Remove Pathogens from Urban Stormwater,
2012- present
In February 2012, the EPA awarded Fungi Perfecti LLC a Phase I EPA SBIR
$80,000 grant entitled “Comprehensive Assessment of Mycofiltration Biotechnology to
remove Pathogens from Urban Stormwater”. The research outlined in this grant will “…
seek to identify which fungal species and cultivation methods can filter pathogens from
stormwater while meeting the physical and temporal demands required for
commercialization. These objectives will be accomplished through a university-industry
collaboration that will mandate permeability and resiliency requirements for stormwater
treatment. This research is anticipated to confirm that fungal mycelium can remove E. coli
from flowing water, and that mycofilters can be developed to meet design requirements to
treat municipal stormwater runoff.” In addition to determining which species are optimal
for mycofiltration in urban areas, Fungi Perfecti will experiment with both different
combinations of species and substrate combinations. The following chart illustrates the
different species and substrates for evaluation as mycofilters: 30 different combinations
will be tested (Fig. 17: Fungal Species and Substrates Combinations). Note there are 17
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mycofilters for each combination, 13 will be inoculated, while four will remain as controls
in each batch.

Figure 17: Fungal Species and Substrates Utilized in EPA Phase I
Fungal Species

Substrate used in Inoculations

Pleurotus ostreatus (Pearl
Oyster)

100% alder chips

Pleurotus ostreatus var.
columbinus

50% alder chips, 50% alders sawdust

Ganoderma applanatum

50% alder chips, 50% rice straw

Ganoderma oregonense

50% alder chips, 25% alder sawdust, 25%
rice straw

Trametes versicolor
(Turkey Tail)

25% alder chips, 50% alder sawdust, 25%
rice straw

Laetiporus sulphureus

Note. Thirty different combinations of substrate and fungal species used in EPA grant
Phase I. Table created by LaDena Stamets, reference Fungi Perfecti’s EPA Grant 2012.
Fungi Perfect LLC has found three conditions that need to be effectively
implemented in the field when utilizing mycofiltration; the installation must:
1.

remain bactericidal for six-month duration of normal stormwater infrastructure
maintenance cycles,

2.

be capable of removing pathogens during high flow events, and

3.

discharge treated stormwater with low pathogen concentrations.
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To meet the three conditions above two key technical objectives need to be met. The first
technical condition is to identify which species and filtration media combinations can
maintain biological activity and appropriate permeability throughout the cycles of
saturation, drying, heating, as well as freezing that will be encountered in mycofiltration
installations. The second technical objective is to quantify the effects of mycofilters on
bacteria. Then used as a model for pathogen filtration, the E. coli removal of the most
viable fungal filter combinations is the first objective and will be evaluated at an average
coliform runoff concentration (400 cfu/100mL) under an average size-adjusted
Washington State six-month design storm hydraulic rate (2.2 L/min).
This research will also evaluate the presence of non-fecal coliform Klebsiella
species bacteria, which is commonly found on wood. For example, the alder chips that
will be utilized in this experiment either alone or in combination with other substrates will
create the bulk of the mycofilters (EPA Grant 2012). Though testing for coliform is often
considered by scientists an outdated, 20th century method to test if waters and shellfish
harvest are safe, Fungi Perfecti will be able to eliminate “false positives” caused by this
method of testing coliform bacteria as opposed to fecal coliform bacteria. If total
coliforms are counted without discriminating between fecal and non-fecal coliforms, then
the data could be skewed.
Mycofiltration is low-cost, low-impact and low-footprint technology. A recent
analysis by Clary (2008) cited in the above grant proposal states “…analysis of
stormwater treatment Best Management Practices (BMPs) has documented that coliform
bacteria levels in treated effluent generally do not meet water quality standards,” (EPA
Grant no. SOL-NC-11-00012). This biological technology is competitive with other BMP

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(Best Management Practice) currently utilized. For instance, the cost of sandfilters is
$10,000-$20,000 with an annual maintenance cost of $3,000, while “mycofilters” only
cost $8 each and maintenance cost are minimal. To determine how many mycofilters
should be used in the field is largely a function of site characteristics. Throughout my
research I saw that ten or more mycofilters are typically needed for the average site
installation.
Once each mycofilter is fully colonized the three least colonized from each batch
will be discarded. Of the remaining ten mycofilters six from each batch will be selected
for a series of testing that includes: resilience, saturation, and permeability. These tests are
aimed to simulate different climatic and environmental conditions. Resilience testing
consists of cycles of saturation, drying, heating, and freezing as a way of evaluating their
ability to maintain biological activity under field conditions. In saturation testing each
mycofilter is submerged in water for thirty minute; mycofilters are then refrigerated at 4
0

C for a two-day period. Mycofilters will then be kept at -20 0C for a 24-hour period,

which will cause the mycofilters to dry. The next 24-hour period will induce a hot spell by
raising the temperature up to 40 0C. For the next 24-hour period the mycofilters’
temperature will be dropped by 10 0C. After cooling, the mycofilters will be submerged in
water for twenty minutes. Lastly, mycofilters will be stored for 24-hours at 20 0C and 80%
humidity. This will be the end of a 15-day testing period that concludes the resiliency
testing process. From that point Fungi Perfecti will analyze one mycofilter from each
batch for permeability and the other five will be evaluated for resiliency before fieldtesting. The strongest species substrate combination will be sent to Dr. Mark Beutel at
Washington State University (WSU) for bacteria filtration efficacy testing. At this point

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the controls will also endure the same test. Refer to figure 18 Mycofilter Resilience
Testing Timeline for further explanation of this process:
Development of mycofiltration technology will result in a wide array of
implications, commercial applications and benefits to society. Phase I, successful
laboratory proof-of-concept data, will verify mycofiltration works. Phase II will advance
development and field efficiency and support commercial development of mycofiltration
as a low-cost, low-impact, and low-footprint application for removing pathogens from
stormwater. The expected result of this research effort is a stormwater treatment system
that will enhance the ability of municipalities both to improve quality of stormwater and to
support a small innovative business. This biological technology will benefit society by
providing cleaner water for commercial fishing and recreation. Moreover, this research
could mobilize worldwide interest, engagement, and understanding in mycology, and
provide innovation in solving complex anthropogenic contamination issues of non-point
water pollution.
In Spring 2012 all companies awarded Phase I Small Business Innovation
Research (SBIR) grants attended Phase I meeting in Washington D.C. Of 400 grant
proposals, only 25 (6.25%) were selected. The SBIR grant program is aimed to enhance
programs, open new markets and help existing businesses. In March 23, 2012 meetings
with the EPA discussed the first phase of mycofiltration work. Many companies with
promising technology will compete for the limited funding allocated in Phase II of the
grant. If third-party investments totaling $100,000 are secured, EPA will include an
additional $70,000 for successful proposals, which could potentially receive up to
$300,000 for two years to develop the product further and bring it to market. Only 7 of 13

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applicants will be funded for Phase II. The workshop focused on how to get the product to
market, how to utilize EPA resources and meet deadlines as well as how get matching
funds for Phase II. The following list shows potential target markets or consumers of
vested interest for mycofilters installations:
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•

Life stock farms in violation
Individual landowners of leaking septic tank violations
“Buffers” or “riparian buffers” in National Forests where endangered
or key-stone species live
Native Tribal lands
Commercial Fishing Industries
State Agencies
Watershed buffers
Community garden buffers
Individual landowners with adjacent neighbors who create pollution
that travels onto their properties
Housing developments with stormwater regulations or violations
Landscape companies
Horse boarders, horse pastures
Agricultural runoff (cow, pig, etc.)
Municipalities
Conservation organizations
Restoration organizations
Land-use developers

The list above may help you the reader broaden your understanding of who may benefit
from this biological technology in the future.

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Figure 18: Mycofilter Resilience Testing Timeline for EPA grant.
1 hr

Several
day
period
2 wk
duration

4-6 wk
duration

4-6 wk
duration

2days

30
minutes

• Hydrogen Peroxide solution (0.3%)
• Substrate inoculation all 30 combinations
• Inoculated bags sit in 20 degrees celsius, until 30% colonization
• Temp drops to 10 degrees celsius
• Fully colonized mycofilters
• Mycofilter put into grow rooms 20 degrees celsius, "Simulation of Fall season"
• Re-submerge then drained

2 days

• Mycofilter refrigerated @ 4 degrees celsius

24 hrs

• -20 degrees celsius (dry out mycofilters by freezing period)

24 hrs

• Hot Spell 40 degrees celsius

24 hrs

• 30 degrees celsius

20 mins

• Submerge in water

24 hrs

End of
Testing
Period

• Store in 20 degrees celsius and 80% humidity.
• Conclude Resiliency Testing Process

Note. Mycofilter Resilience Testing Timeline for EPA grant. This timeline shows the
sequential process Fungi Perfecti used during resilience testing in order to find the
strongest combinations between the thirty. This timeline was created by LaDena Stamets
in April 2012 to aid clarification of resiliency testing.
Third Example: Mycofiltration coupled with other ecological process
Fisherville Eco-Machine Pilot Project 2006- Present
This project aimed to develop a complex ecological design that naturally evolves
to maximize dynamic and diverse biological surface areas, to treat wastewater by
converting contaminated water and sediment. In this project John Todd, who owns John
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Todd Ecological Design, Inc., wanted to develop an “eco-machine” (fig 19: Fisherville
Mycocells Eco-machine) that would rapidly biodegrade Bunker C oil. Bunker C oil is a
toxic, tar-like, thick viscosity, residual material from the manufacture of petroleum
products, and often used as a fuel source for ships and electrical power plants. When
spilled into the environment it can create environmental damage, which persists for years
in both sediments and soils.
Figure 19: Fisherville Mycocells Eco-machine

Note. Notice the transparent solar penetrated eco-machine cells with marsh plants and
Algae. Photograph from John Todd and Eugene Bernat.

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Todd, in conjunction with Eugene Bernat, designed and constructed a greenhouse
based pilot system where a pair of eco-machines operated in parallel. To maximize both
surface area and ecological elements, each system has four ecologically different cell
types, which include:
1. Solar based cells which support algal turf communities, and Physa gyrins
(pouch snails) inhabitants,
2. Higher plant based cells with marsh plants growing on rafts,
3. Open water fish dominate cells, and
4. Fungi dominated “trickle filter” cells designed to support rapid growth of fungi.
mycelia (fig. 20: Mycofilter Trough “Trickle-filter”).
Figure 20: Mycofilter Trough “Trickle-filter”

Note. Fourth eco-machine cell inoculated with fungi. Photograph from John Todd and
Eugene Bernat
Cell types 1-3 are contained inside clear transparent tanks to optimize solar penetration
while the fourth cell type is covered to protect the fungi dominated system from light
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penetration. These pilot systems are operated in a continuous recycle method. The system
was inoculated with organisms from local salt marshes and ponds. In one of the Ecomachines a microbial chemostat was added to provide additional bacterial biomass on a
consistent basis. A “train” is several eco-machines lined up to work in unison. See Figure
21: Eco-machine train.
Figure 21: Eco-machine train

Note. There are two Eco-machine Trains, with four eco-machines in each Train: (1) Solar
based cells which support algal turf communities, (2) Higher plant based cells with marsh
plants growing on rafts, (3) Open water fish dominate cells, (4) Fungi dominated “trickle
filter” cells designed to support rapid growth of fungi mycelia. Photograph from John
Todd and Eugene Bernat.

Ten pounds of sediment were extracted from the Fisherville Canal, divided equally
into each of the two trains and added to the fungal cells. Over the next three months 573
gallons of water from the canal were divided between the two eco-machines. Each ecomachine’s hydraulic capacity was approximately 200 gallons.
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Before canal water and sediment were treated, scientists conducted chemical
analysis. They found detectable measurements of chemical oxygen demand (COD), total
suspended solids (TSS), alkalinity, ammonia, total kjehldahl nitrogen (TKN), and
conductivity. The petroleum measurements included total organic carbon (TOC) in canal
water and total petroleum hydrocarbon (TPH) found in both sediment and canal water.
The first water tests in March 2007 revealed that TPH had dropped from 110,000 to nondetectable level and TSS decreased from 1,700 mg/l to a non-detectable level. Train #1
dropped from 1500 mg/l to 50 mg/l and train #2 dropped to 51 mg/l. Dilution alone had a
87% reduction of COD in both treatment train systems.
Approximately one month later, in April 2007, despite added contaminated canal
water, COD dropped to 21 milligrams per liter (mg/l) in test one and 18 mg/l in test two.
TPH water measured at 0.5 mg/l and 0.6 mg/l, thus the data for April showed a 92%
decrease of TPH in the water. The sediment showed a significant reduction in volume. In
Train #1 the sediment was reduced by 89% and in Train #2 by 57% reduction. Todd et al.
observed that snails had begun to eat the Bunker C sediment attached to the side of the
transparent tanks. A sample of the sediment revealed TPH of 66,000 mg/kg (a 40%
reduction in Train #1), and 49,000 mg/kg (a 56% reduction in Train #2). “These numbers
combined with the reduction in Bunker C sediment volume, indicate that the overall
concept of the Fisherville Eco-Machine is valid and that the system is working” (as cited
in Fisherville Eco-machine 2007 pp. 5).

Eco-Machine Design and Functions. How the infrastructure of the system is designed
directly influences the ecological conditions of the eco-machine (fig. 19: Fisherville

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Mycocells Eco-machine). Diversity in the physical characteristics of the eco-machine
creates many different gradients of environmental factors such as light, oxygen, and
turbulence and creates different niches for speciation that support a broad range of
ecological communities. By having diverse ecologies, the eco-machine’s functionality
becomes both resilient and resistant to disturbance. This is significant because of the
constant influx of highly polluted sediments and water into the eco-machines. The
physical design was to maximize surface area to aid establishment of ecological
communities, which are crucial to the function of the system. Transition zones are
commonly referred to in the scientific community as “ecotones.” Transitional zones within
the eco-machines allowed the different ecological communities to interact. The pilot
project design to include clear tanks, floating plant racks, screens, and mycofiltration
troughs influenced the structure, composition, function and processes in the system that
produced beneficial ecotones.
Floating racks suspend plants at the water-air interface. Growing vegetation
created habitat niches for insects and for other organisms; the biomass of from the plants
functions as a sink for nutrients derived from breakdown of the pollutants. The plants are
self-organizing and self-repairing, their root matrices hosting communities of microbes
that in turn either metabolize pollutants or externally degrade pollutants with enzymes.
Microbial communities excrete simple compounds, which are then absorbed by the plant’s
root hairs and sequestered as plant biomass.
The mycofiltration troughs were housed within opaque materials to achieve a lowlight environment and mimic a terrestrial environment, which creates a habitat that
facilitate fungal growth. The substrate within the cells, coupled with different water

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delivery systems, resulted in a “trickle-filter” corridor for water to pass through substrate
colonized with diverse fungi and microorganisms, while maintaining high oxygen levels.
Participants of the pilot project allowed for a period of ‘ramp up’ during which the
inoculated biology self-organized and established itself before being exposed to pollutants.
After establishment, pollutants were gradually introduced and their concentration
increased to give the ecological communities time to acclimate to the waste stream.
Species were selected that tolerate the pollutants within the canal sediment and water. The
project continues to adapt and evolve chemical conditions created by increased
concentration of polluted canal water. At the time the 2007 report, the system was
observed approaching, but has not yet reached, biological capacity. Additional
inoculations of fungi will be necessary to introduce species of varying seasonality, and to
increase both mineral and biological diversity within the system.
“As the biological capacity and full concentration of polluted water are reached,
the ability to assess the design and operation variables influencing long term system
functioning increases, providing key information for optimizing efficiency and
minimizing costs of further efforts” (as cited in Fisherville Eco-machine Project, pp. 9).
Initial positive results of this project demonstrated the need to continue the experiment and
subsequent results confirm that this method offers a unique solution to a suite of
chemically induced eco-challenges.

Ecological Development. Biological components of the system have self-organized into
ecological communities that include food chains and symbiotic relationships. When
interspecies interactions are considered it was found that species “x” only achieves high

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population growth in the presence of species “y” even though species “y” may not break
down pollutants alone. Managing the species that doesn’t break down pollutants achieves
better pollution reduction, higher adaptability, and increased resistance to disturbance (as
cited in Fisherville Eco-machine Project).
Algae and snails. Biological cycles of algae blooms followed by pouch snail (Physa
gyrins) population climax have been observed. At the same time that algae attached to the
tank walls and continued to grow, the sediment contaminated with TPH suspended within
the water and then became trapped where algal colonies experienced expositional growth
covering most of the container surface area available. Scientists observed after several
weeks that pouch snails began to scrub the tank walls by eating algae and sediment (as
cited in Fisherville Eco-machine Project).
The EPA classify the pouch snail as a bioindicator generally associated with high
nutrient levels. The snail population boomed initially with the available food source and
then declined after the attached algae communities were consumed. Scientists hypothesize
that this relationship is playing a major role in the reduction of TSS and TPH in the
system, and suggest that this phenomenon opens a promising avenue of research options.

Fungi. Fungi species Pleurotus ostreatus and Trametes versicolor, which have the ability
to degrade petroleum hydrocarbons, were inoculated within the substrate. As the
mycelium mass expands it colonizes more substrate. The mycelium surface area was
exposed to contaminated water and sediment inputs. Most white rot fungi secrete
extracellular enzymes that can catalyze oxidation processes. Several mechanisms for
enzyme secretion are utilized by Pleurotus ostreatus to oxidize PAH. Mycofilters

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originally weighed five pounds each, and later, when mycofilters were excavated,
sediment mass was only .549 lbs. from Train # 1 and 2.174 pounds from Train #2,
equating to a reduction of respectively, 89% and 57%. Scientists also noticed that
characteristics of the soil had changed in that it became more pliable and less sticky.

Plants. Plants have multiple cycling pathways that help influence microclimatic
conditions, which vary by species. Plant roots are able to secrete enzymes that promote
particular microbial diversity and activity depending on plant species and incumbent
environmental conditions. Scientists suggest that “[h]aving these roots in an aqueous
solution may have a similar enhancing effect on enzyme production as noted by Lenz and
Holker for fungi. Matching plant adaptive strategies and chemical pathways to the
challenge of degrading complex hydrocarbon chains poses great opportunities for further
research. Plants are key to the photosynthetic base of the system and further diversify the
system with habitats for terrestrial and flying insects, which play a yet-to-be understoodrole in overall balance and health of system.” (as cited in Fisherville Eco-machine Project
pp. 11).

Water Quality Data. Raw canal water, before treatment, was tested on several water
quality parameters with (TPAH) in the bunker C oil as a measurement of particular
significance and interest. Alkalinity, ammonia, chemical oxygen demand (COD), total
suspended solids (TSS), total kjehldahl nitrogen (TKN), conductance, as well as total
phosphorous were measured. Refer to Figure XX: Water quality measurements from the
Blackstone River Canal in Fisherville, MA (2006) below.

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Figure 22: Raw Canal Water Prior to Eco-machine Treatment

Figure 23: Water Quality Test 1: Train #1: Comparison of raw water from Blackstone
River Canal to treated water from Fisherville remediation pilot eco-machine w/ reduction
of pollutants (3-5-2007). Water Quality Test 2: (4-2-2007)

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91

Figure 24: Water Quality test 1: Train # 2: Comparison of raw water from Blackstone
River Canal to treated water from Fisherville remediation pilot eco-machine w/ reduction
of pollutants (3-5-2007). Water Quality test 2: (4-2-2007)
a)

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b)

Sediment Data. Sediment from the Blackstone River Canal was tested for both TPH and
TOC. TPH in the sediment was 110,000 mg/kg while the TOC was 130,000 mg/kg. Of the
five pounds initial sediment added to train #1 and train #2, scientists observed mass
reduction. At the end of test period 2, the sediment in Train #2 weighed 89.02% of the
original weight, leaving only 0.549 lbs. sediment. The texture and viscosity of the
sediment displayed considerable change from the original sediments. Treated sediment
was softer, less sticky, and lost some of the metallic luster characteristic of petroleum. The
small quantity of sediment that settled out in the aquatic cells was tested for TPH in both
trains #1 and #2. A mixed sampling from all aquatic cells revealed a TPH concentration of

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60,000 mg/kg in train #1 and 49,000 mg/kg in train #2. When these data were compared to
the raw canal sediments, the reduction was approximately 45.5% and 56%.

Fisherville Eco-machine Conclusion & Further Recommendations. Fisherville Ecomachine Pilot project contributed measureable empirical data towards establishing the
efficacy of the living system approach to treatment of oil contaminated canal sediment and
poor river water quality. This project also revealed insight into appropriate optimal design
of eco-machines to treat contaminants in the Blackstone River Basin.
The eco-machine project demonstrated significant reductions in both sediment
mass and pollutant concentrations within sediment and water. Significant biomass was
produced in an environment poor in nutrients excluding inputs of polluted water and
sediment, and validated the research hypothesis that a complex ecology designed in
conjunction with natural principles of evolution to maximize dynamic biological surface
area and including waste water/sediment contact is capable of utilizing and converting
contaminated water and sediment to an energy source readily available for biomass
production. During the duration of the study scientists observed life colonization in all
areas of the eco-machine trains. They noted that the thick algal mats that had covered
almost all of the aquatic tank’s walls were entirely consumed and metabolized by snails.
Plant shoots grew to three-feet tall while their plant roots masses were thick and reached
to the bottom of the five-foot enclosures. Even though the scientists didn’t achieve
completed colonization of the mushroom substrate they did observe significant patches of
Trametes versicolor beginning to take hold in the system. Suggested fungi could have

94

played a more significant role in pollutant degradation through optimizing the mycofilters
if they were well established before installation.
Future investigation of how pollutants were converted could explain exactly what
paths the contamination was taking but scientists acknowledge that carbon isotope tracing
testing is expensive and somewhat unnecessary since the process can be observed directly.
Scientists measured an 89% reduction of sediment with a TPH concentrations of
110,000 mg/kg in Train #1, and a 56% reduction in Train #2. Sediment characteristics
changed, indicating some degraded of TPH but unfortunately they didn’t have enough
funding at the time of this study to conduct a chemical analysis of the remaining sediment.
In addition, sediment that settled out of the water column had significantly lower
concentrations of TPH, a reduction of 45.5% and 55.5 % compared to the sediment that
settled in Fisherville and coated the Blackstone River Canal channel.
Water quality tests showed even greater removal of pollutants. TPH reductions
were measured at less than 74.2% in both Trains #1 and #2 for test period one. For test
period two approximately 92.5% and 91% reductions of TPH while COD and TSS were
reduced by 99%. These large reductions in targeted contaminants of interest demonstrated
the efficacy of Eco-machine technology to treat polluted waterways. Eco-machines’ team
of scientists attributes much of these reductions in toxins to the combination of algae-snail
cycles, fungi, and the interaction of the dynamic root surfaces of the higher plants and
their associated microbial communities with the water.
This study provided information on how to implement either direct or full-scale
systems. The project showed how the technology using native organisms from all five
kingdoms of life works, removing contaminants from both water and sediment. The

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insights gained from this study will be utilized to optimize the technology and quantify
appropriate scale and treatment rate for a full-scale system.
This technology is adaptable and can be scaled up or down to adapt to changing
conditions or needs with minimal expenditure. It is also applicable to large-scale
watershed restoration because of its ability to be moved. The study authors envision that
the Eco-machine hybrid can be broken down to small functional units, which can be
located up and down the stream of the watershed in situ to deliver treatment where it is
most needed. Units can be linked to handle high loads where pollution is abundant, then
split into independent, smaller units to clean less polluted areas in unison to neutralize
pollution hot spots. This approach would keep costs low, while also making treatment
adaptable to conditions in the field. Furthermore, once the restored area reaches desired
reduction in contaminates, these units can be transferred to other restoration efforts and
continue to clean polluted waters beyond the scope of a single project.

Fourth Example: Mycofiltration Case Study
Mason County Mycofiltration Projects, 2008 -2011
In 2011, Mason County had several ongoing mycofiltration installations (see fig.
25: Mycofiltration Sites locations for Hood Canal, Mason County) but all except one were
discontinued due to lack of funding. At the time federal funding had been allocated to
Squaxin Tribe’s marine biologist John Konovsky for a mycorestoration project in a
polluted waterway in Allyn, Washington. The Allyn site has become a high priority for
Mason County and lessons learned from the prior sites will be applied to this and new
mycofiltration installations (Book et al., 2009).

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Figure 25: Mycofiltration Site Locations for Mason County Installations

Mycofiltration Sites Locations for Mason
County
Annas Bay
Hwy 101 near Finch Creek, Hoodsport
Two sites on State Route 106 near Skokomish
River on Warren Drive 1/2 mile apart
Main Street
Oakland Bay
251 Sunset Road
State Route 3
520 Eckler Road (SR3 ROW) at creek
Belfair
Roessel Road
Hwy 3 Log Plaza
Hwy 3 HC Auto Sales
Old Belfair Hwy
Other sites

Chapman Cove
18280 SR3, Allyn
221 E. 4th Street, Union
Note. Table created by LaDena Stamets reference Quality Assurance Project Plan for
Mason County Mycoremediation Investigation.
The Squaxin Tribe was interested in this biological solution for capturing and
removing bacteria from running water. Mycelia acts as a biological filter capturing fecal
bacteria from human and livestock waste in stormwater runoff, which is one of the most
widespread contributors of water pollution. It was hoped that mycofiltration could be
utilized to protect the rich shellfish heritage of the Puget Sound, which is historically,

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culturally and economically important to Salish Tribal communities. Runoff pollution
into Puget Sound from upland transportation each winter is a common cause of closing
shellfish harvest. Shellfish growers fear that this yearly cycle of pollution will negatively
impact economic development; as Konovsky states, “We need innovative and costeffective methods to solve the problem.” Squaxin’s benchmark for a cleanup Puget Sound
is whether the Natives can eat its shellfish and harvest healthy populations of salmon. If
so, then mycofiltration can be a valuable tool to aid the clean up of Puget Sound (Squaxin
mushroom water quality solution, 2011).
Due to hydrological characteristics of the design, influent water traveling pooled
behind mycofiltration bags and then slowly filtered through the bags as it flows down to
lower elevations. Mycelium filters toxins out of the water by capturing them in its cellular
net and then digesting them as a nutrient source (Book et al., 2009).
In February 2010 a mycofiltration meeting of State Public Works, Conservation
District officials, Squaxin’s Fish Biologists, and Fungi Perfecti included discussions about
the limitations and concerns with the data collected. One issue with the burlap bags was
that the mycelium, at times, dies off; another was that extra debris and sediment prevents
water from flowing. It is important that the sacks be lifted out of waterways without
falling apart. Heavy water flows have caused bags to blow out. Options to ameliorate
these issues include hemp sacks, thicker threading on burlap sacks and use of the plastic
netting commonly used in the shellfish industry. Introducing plastic netting into the
environment is not favored because the plastic might fragment into smaller pieces that
could later consumed by riparian creatures and fish. Since mycofiltration is a biological
solution, it is important that personnel pay attention to natural life cycles and climatic

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conditions. Data show that while mushrooms thrive between the months of September and
December, the mycelium grows well from March through November, so the incubation of
substrate inoculated with mycelium can be without climatic controls 75% of the time in
the PNW (Book et al., 2009). This is important to note because mycofiltration installations
need to take place during seasonally appropriate times to maximize effectiveness.
From the meeting, Stamets’ team focused on two issues concerning how the
mycoremediation was being carried out: 1) the way the bags were being stored before
installation hindered their effectiveness, and 2) bags were not being replaced within the
prescribed time period. Study participants agreed that these several pilot studies needed to
be better documented, and that more controls needed to be in place. Several fungal species
were included in some bags, and data suggested that the strongest strain was Pleurotus
ostreatus (the Nisqually Oyster strain). Some results showed a reduction in fecal coliform
bacteria (FCB) while others showed an increase in fecal matter. Study participants
speculated that animals such as mice were leaving fecal matter on bags. Other suggestions
were to have a few control bags with just sterilized wood chips to provide an in-situ
control, against which toxicity data could be compared. Thus methods used in this case
study need to be further researched and developed before being designated as a Best
Management Practice (BMP) by the EPA (Book et al., 2009).
Fifth Example: Mycofiltration Pilot Project
Field Demonstration of Mycoremediation for Removal of Fecal Coliform
Bacteria and Nutrients in the Dungeness Watershed, Washington 2009
Battelle Pacific Northwest National Laboratory in partnership with the U.S. EPA
and DOE integrated fungi into bioretention cells in efforts to reduce FCB contamination in
the Dungeness Watershed. Mycofiltration reduced FCB by 90-97% when using native

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vegetation and natural microbial assemblages, while the control bioretention cell without
fungi reduced FCB by only 66-73%. Thus, it can be seen that fungi significantly increased
the effectiveness of the bioretention cell by 24% (Thomas et al. 2009).
The Dungeness Watershed is located within the Olympic Peninsula of the northern
part of the Puget Sound. Rivers originate from the Olympic Mountains and flow 32 miles
downstream through wilderness, forested, agricultural and residential landscapes into
Dungeness Bay. The 200 square mile watershed is habitat for over 200 fish and wildlife
species and is an important waypoint for migratory waterfowl.
Dungeness Bay is located within the Dungeness National Wildlife Refuge and
provides refuge and nursery grounds for native birds, fish and shellfish species. For over
20 years, a collaborative effort between local and regional institutions as well as other
collaborative partnerships worked to maintain ecosystem function. Ecosystem
functionality is dependent upon the interactions between organisms and the physical
environment, such as nutrient cycling, soil development, water budgeting, and
flammability in the Dungeness Watershed. In recent years, anthropogenic impacts
impaired natural function of both river and bay causing multiple health problems,
including the listing of salmonid species under the Endangered Species Act and, since
2000, closure of Dungeness Bay shellfish harvesting due to high levels of fecal coliform
bacteria. Although some improvements have been made, failing septic systems, impaired
in-stream flows, pollutant inputs caused by stormwater runoff, and flood plain
development continue to threaten habitat health. This study took place on a residential
property in an agricultural setting in the lower Dungeness Watershed. At one point, the
site had been utilized as an irrigation overflow pond. This site was adjacent to pasture land

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and tidal wetland that is contiguous to the Strait of Juan de Fuca.
The study Field Demonstration of Mycoremediation for Removal of Fecal
Coliform Bacteria and Nutrients in the Dungeness Watershed, Washington is technically,
a mycofiltration study, not a mycoremediation study, as mycofiltration captures pathogens
flowing with water whereas mycoremediation which degrades arrested pollutants in situ. .
Thomas et al. focused on mycofiltration used in conjunction with bioretention cells as a
possible Best Management Practice to remove fecal coliform bacteria. They used a
bioretention cell (rain garden) as a control and a bioretention cell coupled with fungal
mycelium and mycorrhizae as the treatment. Their design protocol was to see if adding the
bioretention cell infused with fungal species Pleurotus ostreatus and mycorrhizae
increased the removal of fecal coliform bacteria. The study site received runoff from an
irrigation ditch. Scientists spiked the experiment with fecal coliform, which allowed a
comparison of treatment with control. This study is a part of the larger body of research
conducted under the funding of the EPA Targeted Watershed Initiative within the
Dungeness Watershed and Bay to encourage community based solutions to protect and
restore clean surface water.
The mycofiltration application consisted of a layer of oyster mushroom (Pleurotus
ostreatus) mycelium to inoculate alder (Alnus rubra) chips mulch and mycorrhizal fungi
applied to plants. Refer to Figure 26: Native plants were used in the biofiltration cells,
which includes a list of species of trees, shrubs, emergent, and herbaceous plants used in
this study.

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Figure 26: Native Plants used in Biofiltration Cells
Native plants used in biofiltration Cells, Dungeness Watershed
Plant Type
Genus/Species
Common Name
Trees
Malus fusca
Pacific crab apple
Salix lucida
Shining willow
Crataegus douglasii
Black hawthron
Shrub

Cornus sericea
Lonicera involucrata
Myrica gale
Physocarpus capitatus
Oemleria cerasiformis
Symphoricarpos albus
Ribes lacustre
R. sanguineum
Crataegus douglasii
Spiraea densiflora
S. betulifolia

red-osier dogwood
twinberry honeysuckle
Sweetgale
Pacific ninebark
Indian plum
common snowberry
black swamp gooesberry
red-flowering currant
black hawthron
rosy spiraea
white sporaea

Emergent

Carex lyngbyei
C. mertensii
C. obnupta
C. pachystachya
C. pansa
C. sitchensis
C. spectabilis
Eleocharis palustris
Juncus effusus
J. tenuis
Scirpus microcarpus

Lyngbye's sedge
Mertens' sedge
slough sedge
chamisso sedge
sanddune sedge
sitka sedge
showy sedge
common spikerush
common rush
proverty rush
panicled bulrush

Herbaceous

Aster chilensis
Iris tenax
Sisyrinchium angustifolium
Fragria chiloensis
Potentilla fruitcosa

Pacific aster
thoughleaf iris
narrowleaf blue-eyed grass
beach strawberry
shrubby cinquefoil

Note. Table created by LaDena Stamets. Reference: Field Demonstration of
Mycoremediation for Removal of Fecal Coliform Bacteria and Nutrients in the
Dungeness Watershed, Washington 2009.

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Study authors found the bioretention cell alone decreased fecal coliform countforming-units (CFU’s) by 66%, but the bioretention cell infused with mycorrhizae reduced
CFU by a additional 29%, for a total 90% reduction. The bioretention cell outflow
revealed an initial spike of 376 CFU/100 ml at one hour, and then consistently dropped
over time. In contrast the mycoremediation outflow showed concentrations no greater than
10 CFU/100 ml and remained relatively constant through the duration of the experiment
with an average mean of 5 CFU/100 ml. Thomas et al. (2009) concluded: “While the
bioretention cell itself performed well at reducing fecal coliform bacteria, the
mycoremediation (sic) treatment provided a greater reduction of bacteria. This was
particularly evident during the spike experiment where a higher concentration of bacteria
and nutrients were introduced into the cells.” Thomas et al. outlined the benefits of the
mycofiltration treatment application to a bioretention cell or other type of site (e.g., stream
bank, riparian buffer) which include:
• A technologically based natural systems,
• Only native fungal species used; can locally source all materials (plants and
fungi),
• Minimal handling and low maintenance,
• Visible improvement to a site,
• Non toxic byproducts; no secondary waste streams produced,
• Local water quality protected,
• Mobile and flexible; no structures, no minimum batch size,
• Economical,
• Effective at reducing fecal coliform and nutrients when properly designed, and

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• Applicable to a variety of other contaminants (e.g., PAHs, PCBs, metals).

For more information about this study I encourage you to read this field demonstration
study.

Critiques of Mycofiltration Studies
The following section aims to give my personal critique of mycofiltration
reference to case studies and pilot projects examples discussed in detail in the above
sections.

First example. The Evergreen State College Mycofiltration Pilot Project. I found this
study helpful in highlighting that mycofiltration was more effective when woodchips were
not sterilized. I suggest this was because other naturally occurring microbial organisms
were established and either aided in remediation or became food source for Pleurotus
ostreatus mushrooms. This is helpful information for individual land-owners who might
not have the ability or funding to sterilize would chips before inoculation.
I recommend that The Evergreen State College implement this mycotechnology at
Snyder Creek as a field study – especially since students are already conducting water
quality tests regularly in Snyder Creek. Students would take samples above and below
mycofiltration installation for multiple quarters, then compile data into a useable form.

Second example. Phase I (Proof-of-Concept) EPA Bench Study Comprehensive
assessment of Mycofiltration Biotechnology to Remove Pathogens from Urban
Stormwater. The studies being carried out by Fungi Perfecti in collaboration with WSU
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and EPA are fundamental to understanding which species and substrates will provide the
most effective product for cleaning up fecal coliform bacteria in both urban and
agricultural settings. The value of this research effort is a potential stormwater treatment
system that would both enhance the ability of municipalities to improve quality of
stormwater and supporting a small innovative business. A recent analysis of stormwater
treatment BMPs has documented that coliform bacteria levels in treated effluent generally
do not meet water quality standards. This research will also evaluate the use of non-fecal
Klebsiella species bacteria, which are commonly found on wood, but are not harmful to
humans. For example, alder chips will be utilized in this experiment, either alone or in
combination with other substrates to create the bulk of mycofilters. Although testing for
total coliforms, without distinguishing between fecal versus non-fecal coliform, is often
considered by scientists an outdated, 20th century method to test if waters and shellfish
harvest are safe, Fungi Perfecti approach was able to eliminate “false positives” caused by
this antiquated testing method. Measurements of total coliforms, without distinguishing
fecal and non-fecal coliforms used in mycofilter effluent skewed efficacy data. False
positives have been a major obstacle in past mycofiltration water quality assessment with
local state governments, particularly Washington State DOE, which has historically
depended and currently depend upon on a flawed testing methodology. WSDOE needs to
conform to the practices recommended by the EPA, to which the majority of states have
also complied by updating their testing protocols.
Mycofiltration is low-cost, low-impact and low-footprint technology. Moreover,
this research could mobilize worldwide interest, engagement, and understanding in

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mycology, and provide innovation in solving complex anthropogenic contamination issues
of non-point water pollution.
I suggest participants seek private investors for Phase II of the EPA grant and that
a Mycorestoration Certification Training Program be established to establish standard
protocols, hands-on training, and documentation. If private investors – especially
stakeholders – shared financial responsibility for cleanup, then the EPA will match that
funding for implementation of mycofiltration for urban stormwater. The Puget Sound area
would be an optimal place to carry out this study because it has a massive human
population living on thousands of miles of waterfront properties, especially considering
that about 150,000 pounds of untreated toxins go into the Puget Sound each day.

Third example. Fisherville Eco-Machine Pilot Project is of considerable importance to
bioremediation technologies that are coupled with mycofiltration’s ability to ameliorate
toxins in contaminated waterways. A complex ecology designed in conjunction with
natural principles of evolution to maximize dynamic biological surface area and including
waste water/sediment contact can utilize and convert contaminated water and sediment to
an energy source readily available for biomass production. The Eco-machine Pilot project
contributed empirical data demonstrating the efficacy of the living system approach to
treatment of oil-contaminated canal sediment and poor river water quality.
Even though the scientists didn’t achieve complete colonization of the mushroom
substrate they did observe significant patches of Trametes versicolor beginning to take a
strong hold in the system. I suggest in their next experiments to let fungi within the “filter
trickle” feeder establish itself before introducing contaminated canal water. One major

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benefit of this project is that its technology could have wide-scale application throughout
watershed ecosystems. Eco technologies can be built at appropriate scales in the river or
canal water, based on sediment volumes needing treatment to restore water quality;
custom designs for each site can be easily scaled up or down to adapt to different
conditions or needs with minimal expenditure. This technology is ideal for implantation in
large-scale watershed restoration because it can be moved relatively easily.
Pleurotus ostreatus and Trametes versicolor, both of which can degrade petroleum
hydrocarbons, were inoculated throughout the substrate. As the mycelium mass expands,
the mycelium’s surface area exposed to contaminated water and sediment increases. Most
white rot fungi secrete extracellular enzymes that can catalyze oxidation processes via
mechanisms that utilize similar pathways as Pleurotus ostreatus uses to oxidize PAH’s.
During this project the sediment characteristics changed, thus indicating some
degradation but unfortunately the project did not have enough funding to conduct a
chemical analysis of the remaining sediment. Their next step is to seek additional funding
so they can conduct proper analysis.
This study provided beneficial information for implementation of either direct or
full-scale systems. The project has shown how technology using native organisms from all
five kingdoms of life work well to remove contaminants from both water and sediment.
The second round of pilot studies will seek to optimize the technology to quantify
appropriate scale and treatment rate for a full-scale system. EPA has awarded the Ecomachine project another $700,000 to develop this technology.

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Fourth example. Mason County Mycofiltration Projects. If Mason County reinitiates its
prior projects, I suggest they implement mycofiltration installations at: Oakland Bay,
Daniels Rd., Beaver Ave, Wiley Lane, and Eckler Rd. Four other possible sites include 1)
Annas Bay area: 1/8 of a mile from the existing installation at Finch Creek, junction of
Hill Creek and Hwy 101, 2) two at the junction of Main Street and Warren Drive, as well
as 3) Hwy 101 between No Name Creek and Valley Rd.
One issue that this project experienced with burlap sacks is that the mycelium, at
times, dies off. This issue could be resolved if mycofilters were appropriately stored when
not being used. Another issue was that extra debris and sediment prevented water from
flowing. I suggest using a type of screen upland from the installation to prevent leaf litter
from restricting the flow of water. It is important to elevate the sacks slightly above
waterways to capture contaminants. Heavy water flows have caused bags to blow out; to
prevent this from happening in the future other materials to encompass the mycelium and
substrate need to be explored.
We see two additional ways to improve the effectiveness of this study. First, the
way the bags were being stored before installation hindered their effectiveness. Second,
bags were not replaced within the ideal time periods. This method shows that there is a
limited window of efficacy, but considering its effectiveness versus costs, mycofilters still
appear economically feasible given the more costly alternatives.
Scientists working on this project concluded that there are some limitations to this
application and that it needs to be further researched and developed before being
designated as a BMP by the EPA. I believe that the suggestions I articulate above in
conjunction with recommendations by Paul Stamets’ team would create a more effective

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mycofiltration FCB reduction database. Most mistakes that occur in these types of
installations are human induced. Also, with each experience, methods will be better
refined.
Fifth example. Field Demonstration of Mycoremediation for Removal of Fecal Coliform
Bacteria and Nutrients in the Dungeness Watershed, Washington. Results of this study
revealed mycofiltration reduced FCB by 90-97% when using native vegetation and natural
microbial assemblages in unison, compared to the control bioretention cell without fungi
(which reduced FCB by 66-73%). Battelle scientists concluded that fungi increased the
effectiveness of the bioretention cell by an additional 24%. They used a bioretention cell
(rain garden) as a control and a bioretention cell coupled with fungal mycelium and
mycorrhizae as the treatment. Their design protocol was to see if adding the bioretention
cell infused with fungal species Pleurotus ostreatus and mycorrhizae increased the ability
of removing fecal coliform bacteria.
Biological technologies that can remove harmful toxins are undeniably needed, but
many believe that their advantages are touted in an overly optimistic way. I suggest that
even more biological technology efforts need to be employed to prevent pollutants from
entering the environment in the first place. Stricter regulations for landowners with failing
septic tanks need to be enforced by Department of Health by costly fines, if preventive
measures prove unsuccessful. In addition to being fined, polluters need to be reeducated
on the negative environmental impact of their practices.
Using mycorestoration in conjunction with other biological solutions for removing
chemical toxins and heavy metals, i.e., synergistically rather than individually applied
biological remediators to achieve ecosystem health is a promising area. I predict this

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approach will become more commonly utilized because of its combined effectiveness.
Scientists in this study found the bioretention cell alone decreased fecal coliform count
forming units (CFU) by 66% but the bioretention cell infused with mycorrhizae was able
to reduce CFU by an additional 29%, totaling 90% reduction, thus showing that adding
fungi significantly enhanced the remediation methods in practice.

Pillar III: Mycoforestry
Mycoforestry is the use of fungi beneficial to trees to aid in the regeneration of
forests. An example is the re-establishment of a new forest on land devastated by
repetitive slash-and-burn clear cutting practices. The interaction of fungi and trees allow
for healthy, sustainable fertile soils. In the following section, I focus on mycorrhizal
species.
Mycorrhizal fungi have been coevolving with over ninety percent of the plants on
earth through a symbiotic association, exchanging important nutrients. One single
mycorrhizal fungus can help many different plant species fight off pathogens, compensate
for soil nutrient loss and ameliorate the effects of drought. These associations have
evolved over millions of years creating a mutuality where vital elements are shared for
survival. The word “mycorrhizal” originated from the Greek words “mykes” meaning
fungus, and “rhiza” meaning root (Stamets, 2005; Amaranthus, 2009). There are two
major types of mycorrhizae: endomycorrhizae and ectomycorrhizae. When mycorrhizae
develop in the cortical cells, resulting in swelling of roots, usually black in color, they are
termed “endomycorrhizal”, “endotropic”, or “Vesicular-arbuscular mycorrhizae” or
“VAM”. After inhabiting the roots, the fungi project their filaments, known as “mycelia”
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into the soil, extending the plant’s roots and root absorbing capacity from approximately
ten to a thousand fold, significantly more than what the plant would be able to hold alone.
Mycorrhizal feeder roots give plants the ability to up-take nutrients and water from natural
soils. See fig. 27: Maple tree mycorrhizal comparisons. The mycorrhizal fungal symbiont
receives both shelter and sugars from the plant. The host plant receives phosphorus and
nitrogen, and both aids the plant in times of drought and increases its salt tolerance.
Overall increase in plant growth and development is achieved when mycorrhizal fungal
communities are well established. These mycostructures are essential to plants, especially
trees, that grow under stressful conditions (Marx et al., 1989) such as drought, sudden
climate change, fire, pathogens.

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Figure 27: Maple tree mycorrhizal comparisons

Note. Bigleaf Maple (Acer macrophyllum); left Maple tree without mycorrhizae, right
Maple tree with mycorrhizae (Stamets, 2006).
Similarly, food crops joined with mycorrhizae can increase the effective surface
absorption rate from their roots by several hundred to several thousand fold. In some
cases, farmers are able to increase their crops by 30% or more (Amaranthus, 2009).
Although “Degraded Soils, Food Storage and Eating Oil” (Adinarayana, 2001) is
largely focused on agricultural crops and its symbioses with mycorrhizal fungi,
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mycorrhizae clearly has benefits that can be exploited in mycoremediation, mycoforestry
and possibly utilized in future mycofiltration installations.
Scientists believe that it is necessary to monitor and assess long term results after
contaminated soils are remediated. Mycorrhizae can be used to treat large quantities of
surface contaminated soils, and in addition are low in cost. Adinarayana et al. suggest only
larger scale projects can “… demonstrate the ease and viability of the inoculation methods
suggested in the literature.”
Plants symbiotic with mycorrhizae are known to tolerate higher temperatures, a
variety of soil-based and root-borne pathogens, and increase in heavy metals. Since crops
inoculated with mycorrhizae are able to better uptake nitrogen resulting in a reduction in
the need for synthesized fertilizers. Fertilizers cannot maintain healthy roots, improve soil
structure or aid in water up-take whereas mycorrhizae can. In fact fertilizers negatively
affect the factors listed above. Fertilizers can lead to deteriorated water quality, soil
structure and salinity (Amaranthus, 2009).
Even though 80% of our atmosphere is nitrogen, plants cannot uptake nitrogen in
gas form; they can, however, absorb nitrogen indirectly in its form fixed by bacteria
cooperating with mycorrhizal fungi. Rhizobium bacteria are a classic example.
Amaranthus, a mycorhizzologist, argues that mycorrrhizal fungi can help plants take up
nitrogen, without chemical fertilizers, pesticides, or extensive irrigation. The use of
organic amendments and biological inoculants like mycorrhizae fungi has been widely
studied and proven beneficial by several scientists. Also, the use of mycorrhizal fungi is
more economically feasible than chemical soil supplements. In North America, largescale conventional and organic farmers are utilizing mycorrhizal fungi for common food

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crops, e.g., wheat, soybeans, corn and flax. In India, farmers are utilizing mycorrhizal
fungi to reduce their use of chemical fertilizers by 50% without any loss in crop yield of
crops. “Clearly we stand at a crossroads. We must feed the world today without destroying
future generations’ ability to produce enough food. We need an approach that maximizes
agricultural production while restoring clean water, protecting the environment and
building soils, and sustaining soil resources.” (Amaranthus, 2009, pp.3). I highly concur
with Amaranthus’s assessment on focusing upon an organic biologically based strategy for
managing soils. The following section describes each type of Mycorrhizal associations.

Ectomycorrhizae. ectomycorrhizae form a structure called Hartig Net between the plant’s
root cells, or cover the surface of the feeder roots (Marx, 1989). Ectomycorrhizae are a
mutualistic, often obligatory, symbiosis between the hyphae of certain basidiomycetes and
ascomycetes and the fine roots of certain plants. The ectomycorrhizally endowed feeder
roots develop a swollen appearance and in pines they usually appear as forking habit.
Ectomycorrhizae

are

normally

found

on

the

following

tree

species:

alder (Alnus), beech (Fagus), Douglas fir (Psedudotsuga menziesii), eucalyptus
(Eucalyptus), fir (Abies), hickory (Carva), oak (Quercus), pine (Pinus), and spruce
(Picea).
Marx et al. suggest that Thelephora terrestris is the most common ectomycorrhizal
fungus used by bare-root seedling nurseries in the United States. This mycorrhizal species
is well adapted to growing conditions in nursery beds but is not equipped for adverse
conditions of many reforestation sites (Marx et al, 1989). Where Thelephora terrestris
may not be effective, another fungus, Pisolithus tinctorius (“Pt”), often will be. Research

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showed that when tree seedlings are inoculated with Pt the number of culled seedlings is
reduced while survival and growth in field planting are increased. These benefits are most
apparent in adverse forestation sites such as strip-mining banks.

Vascular Endo-mycorrhizal (VAM). Endomycorrhizae are normally found on the
following tree species: ash (Fraxinus), cedar (Thuja, Chamaecyparis), cypress
(Cyparissus), gum, (Eucalyptus), maple (Acer), poplars (e.g., Cottonwood), sycamore
(Platanus), walnut (Juglans),and other conifers. Endo-mycorrhizal relationships also
occur on agronomic crops, such as corn, sorghum, millet, sudex and grasses utilized as
cover crops in tree nurseries. Such mycorrhizal crops add organic biomass to the soil
while reducing water and wind erosion.
Most major hardwood species form endomycorrhizal (EM) relationships and need
these for normal development in forest plantings. In nursery settings, adequate
development of endomycorrhizae has two major benefits. First, seedling quality and
success is improved. Second, hardwoods that have good lateral roots and endomycorrhizal
development will have a higher rate of survival than without. Hardwoods armed with
endomycorrhizae are more capable of competing within harsher environments and
undesired vegetation on the site (Marx, 1989).
The forest floor usually lacks phosphorus in a form available to trees.
Endomycorrhizae can help phosphorus uptake. Without the help of these phosphorus
transporters, some trees would dwindle, sicken and likely die. Despite the fact that these
mycorrhizae occur naturally in the forest, they need to be managed in nursery soils (Marx,
1989). In nursery beds, phosphorus fertilization must be closely monitored or it could

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hinder mycorrhizal development. Soil phosphorus should be kept between 75-100 parts
per million (ppm). If seedlings without mycorrhizae are exposed to phosphorus levels
below 50 ppm, development will be hindered. If artificially applied phosphorus raises
levels above 100 ppm, endomycorrhizal development will be adversely affected (Marx,
1989). The cost of one pound of standardized mycorrhizae is approximately $59.95 with a
suggested rate of 1-3 teaspoons application to each plant. For example, 2 teaspoons of
mycorrhizae mixture is recommended per plant for inoculating ferns.
First Example: Mycoforestry Pilot Study
Mycoforestry Research Pilot Project on Cortes Island, Canada 2003- Present
In 2003, Fungi Perfecti purchased 160 acres near Mary’s Point, on Cortes Island,
British Columbia, Canada, of which approximately 60 acres were clear-cut by the
previous owner. Where some might have seen a devastated landscape, Paul Stamets and
his partner Dusty Yao saw an opportunity for demonstrating mycoforestry strategies that
would reestablish a healthy forest, ultimately exhibiting old growth characteristics.
While traditional forest practices prescribe burning the debris after clear cutting,
this was an opportunity to show how other practices could be successful. This multilifetime experiment will compare the effectiveness of introducing mycorrhizal fungi with
top-dressing; the use of mulching through chipping trees and limbs that were left after
clear cutting or which came down by wind throw. Top-dressing results in delayed release
nutrients and aids in retaining moisture.
In forests, terrestrial ecosystems adjacent to wetlands and riparian zones can
regulate retention and the release of nutrients and carbon into the aquatic systems.
Hydrochemistry is a subdivision of hydrology that deals with the chemical characteristics

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of the water on and beneath the surface of the earth. Water – in all forms – is affected
chemically by the materials with which it comes into contact, and it can dissolve many
elements in significant quantities. Chemical hydrology is concerned with the processes
involved and thus includes the study of phenomena such as the transport of salts from land
to sea (by erosion of rocks and surface runoff) and from sea to land (by evaporation, cloud
formation, and precipitation), and the age and origin of groundwater in desert regions and
of ice sheets and glaciers. Water moving through shallow soil horizons as an important
contributor to stream hydrochemistry. Studies like these provided empirical data that is
utilized to determine relationships between soil or surface water chemistry and soil
chemical properties, soil physical properties and terrestrial basin topographic
characteristics. Analyzing these relationships can be used predict ecosystem water
response and is important in identifying the influences of natural or anthropogenic factors.
Understanding natural soil processes is vital to decision-making when implementing
regulations [3].
Canada has a strict view of management and harvesting timber on land within 200
feet of waterfront. On Cortes Island, it is illegal to harvest timber without appropriate
permits. On the 160 acres testing mycoforestry, due to environmental covenants, all the
wood that is harvested must be acquired from natural processes like wind throw during
harsh winter season. If timber should fall within a leave-zone it must be left to aid the
ecological health of that habitat to allow natural succession.
Approximately 35,000 Douglas fir (Pseudotsuga menziesii) and western red cedar
(Thuja plicata) seedlings were planted in November 2003. Half the trees were root-dipped

3

http://cfs.nrcan.gc.ca/projects/118
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with mycorrhizae while the other half were not; in other words, the roots of half the cedar
were each covered in approximately half a million spores of the endomycorrhizal Glomus
intradices while half the Douglas firs were each exposed to similar amount of
ectomycorrhizal Rhizopogon parksii. In the summer of 2004, volunteers distributed a
gallon of wood chips around half the spore-treated and half the non-spore-treated trees to
test if decomposing woodchips further benefited tree growth.
Immediate benefit received by top-dressing was the lowering of the soil
temperature for newly planted seedlings. While native fungi decomposed the wood chips,
a slow release of nutrients travels into rhizopshere. Mushroom mycelium de-molecularizes
plant fibers (lignin and cellulose), adding healthy soil as a positive direct consequence
(Stamets, 2004).
The goal of this project is to increase the soil depth sequentially resulting in greater
carrying capacity for the tree and natural successions. Mulching reduces fuel load in
forests while increasing moisture retention. Stamets sees wood chips as valuable
ecological currency that should be re-invested into forests to enhance sustainability. There
are many different interpretations and definitions of sustainability, so it is important to
define this term to eliminate confusion. Here, sustainability is the characteristic by which a
process or state can be indefinitely maintained to meet or exceed a certain level whose
metrics included increased timber mass, biodiversity, humus accumulation, aquifer
protection and hydrological dynamics.
In September of 2004, 700 trees were tagged and measured for height and girth.
The data initially revealed a net increase of 9-10% in both height and girth for 10-month
duration. Annual measurement will be gathered, and after 10 years a chi square data

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analysis will be completed. At 8 years, statistical analysis showed significance (P<.05) in
the mycorrhizally treated trees increasing in biomass (as measured by height and girth)
over non-mycorrhizally treated trees.
Figure 28: Trend Comparisons between Trees inoculated with Mycorrhizae vs.
non-inoculated trees
450.0#

400.0#

350.0#

300.0#

Mycorrhizae#8#Height#(cm)#

250.0#

Control#8#Height#(cm)#

200.0#

Mycorrhizae#8#Basal#Diameter#at#
Ground#Level#(mm)#

150.0#

Control#8#Basal#Diameter#at#Ground#
Level#(mm)#

100.0#

50.0#

0.0#
1/2006#

1/2007#

1/2008#

1/2009#

1/2010#

1/2011#

1/2012#

Trend comparisons between mycorrhizally
inoculated vs. non-inoculated trees
6 years: Significance P < .05
Note. This chart shows comparisons between trees that are inoculated with
Mycorrhizae compared to those trees that were not inoculated with mycorrhizae.
In the summer of 2011, I visited the site and observed healthy trees with
approximate heights averaging 9-10 feet; where there was once a scared devastated forest
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is now an oasis of life, with increasing species biodiversity as a new ecological
community emerges. As of December 2012, the mycoforestry project continues requiring
low maintenance, periodically requiring the Vemar cones to be removed once the trees are
above the browsing height of deer.
Logging companies could follow this application by chipping woody debris and
leaving them within the cut forestland, dispersing the nutrient feedstock over the
landscape to help refuel the carbon cycle. Chipping will add to cost in short term but the
long-term benefits from mycologically enhanced forests will result in ecological health
and economic returns.
Approximately half of Canada’s land surface is covered in trees: 400 million
hectares from coast to coast. The benefits provided by this natural wealth extend to
ecological, economic, recreational, social, cultural, traditional, as well as spiritual.
Canada’s federal government has made caring for its forests a top priority [4]. The criteria
and indicators for Canada’s framework include biological diversity, ecosystem condition
and productivity, soil and water health, role in global ecological cycles, economic and
social benefits and society’s responsibility.
Canada’s forests purify water, stabilize soil and cycle nutrients, creating habitat for
wildlife, as well as nurturing environments rich biological diversity. About 77% of
Canada forests are publicly owned, while 16 % are federally owned and only 7% privately
owned. Federally owed land can be managed better because of recourses and policy
guidelines, therefore they have more control over protecting the land. Canada sustains a
national-wide forest products industry, which supports hundreds of thousands of jobs and

4

http://cfs.nrcan.gc.ca
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contributes billions of dollars to the country’s economy. Canada is committed to
Sustainable Forest Management (SFM).

Critique of Mycoforestry Study
The following section is aimed to give my personal critique of mycoforestry
reference to the pilot project discussed in detail in the above section.
First example. Mycoforestry Research Pilot Project on Cortes Island, Canada. Although
within the last decade copious data have been collected from this particular site, the data
have not yet been fully quantified into a usable form for data analysis. The next step for
scientists working on this project is to complete the measurements and then analyze results
for statistical significance after a decade of data collection. However, when conducting a
study like this in the field, it is hard to determine if the trees received beneficial
mycorrhizae naturally from the soil or from inoculation. I suggest before starting a field
study like this that several soil samples be analyzed under microscopy in specific locations
where experiments will take place. This way soil samples can be compared later in the
study to determine if they naturally occurred or were introduced by scientists. We can
analyze trees introduced with and without mycorrhizae in a controlled indoor setting but
once in the natural environment, is much harder to distinguish introduced from native
mycorrhizae. Since data for establishing significance do not necessarily need to include
hundreds of trees, I recommend comparing the trees that were planted directly onto
logging roads, where native mycorrhizae would be absent or extremely low. Comparing
trees placed into these mineral soils, lacking humus and native mycorrhizae, could give
significant results without a great expenditure of time and labor.

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Part II
Methodology for Individual Landowner Mycorestoration Projects
High and Low Technology for Cultivation Techniques
This section is focused on high-tech and low-tech methods for cultivation; its
purpose is to offer information that can help you generate mycelium spawn and implement
mycorestoration installation.
The process by which mushrooms are harvested from nature and then cultivated in
the laboratory (fig. 29: Process of mushroom cultivation techniques) is important but it is
not critical to fully understand it for implementing mycorestoration at the grass roots level.
If you harvest from nature keep in mind the concept of “sustainable harvest”: only take a
few mature mushrooms and don’t devastate the population by over harvesting or taking
the under developed mushrooms. The Precautionary Principle should be applied through
research and application of mycorestoration, emphasizing that new methods are carefully
considered before implementation to prevent collateral damage to other components
essential for environmental equilibrium and health. The Precautionary Principle states that
it may be better to avoid taking a particular action if there is a possibility of causing
unexpected harm; a classic example here in the PNW was choosing to introduce a nonnative species like scotch broom to promote slope stability, which is now considered an
invasive weed, requiring laborious efforts for eradication.

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Figure 29: Process of Mushroom Cultivation Techniques

Note. In this simplified illustration you can follow the process of cultivating mushrooms
in lab setting. This process to expand your mycelium with substrates results in variety of
different end products. © Property of Paul Stamets
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High-tech, in vitro propagation methods of growing mycelium to create spawn is fully
covered in-depth in several books such as The Mushroom Cultivator by Paul Stamets
(1983) and Growing Gourmet and Medicinal Mushrooms by Paul Stamets (1997). The
end-user will be purchasing “spawn”, i.e., mycelium grown out on a carrier substrate such
as grain, woodchips, sawdust, etc., from a reputable, and preferably local, certified organic
spawn producing company. The section that follows illustrates how use the spawn, once
acquired.
I will explain low technology which most of you will be utilizing. However this
section is not intended to teach you how to cultivate mushrooms – for more information
about cultivating fungi please refer to the aforementioned books. This part of the guide is
focused on how to implement mycorestoration that can be utilized on small-scale projects.
You have two convenient avenues or pathways to achieve mycofiltration installation. The
easiest is purchase mycofilter bags already made and inoculated with mycelium (see Fig
30: Mycofilters). The second is to make your own mycobags by expanding your
commercially acquired spawn.

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Figure 30: Mycofilters
a.)

b.)

Note. a.) New Mycofilters b.) Blake Westman holding colonized mycofilters made for
the EPA grant pilot project. Photos by Paul Stamets 2012.
Once you have your mycelium for mycofilter bags or for mycofilter sheetmulches, chose a method of grounding the mycelium in a tributary, or for soil remediation
chose a parallel sheet inoculation approach also called ‘lasagna layering’ technique. Below
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is a diagram that may be helpful for anchoring sacks. Also refer to Mycofiltration Project
Installation fig. 31: Four ways to inoculate burlap sacks with fungi. This simplified
illustration shows four different ways of that the installation of anchoring burlap sacks can
also inoculate the sacks with specific species of fungi: galvanized nails and stakes, stakes
through bags, stakes and twine, bags installed opposite each other with galvanized nails
and stakes. As water percolates through the burlap sacks, mycelium breaks down toxins.

Figure 31: Four Ways to Inoculate Burlap Sacks with Fungi

Mycofiltration
Installation Site
Evaluation and
Implemenation of Best
Methods

Mycofilter bags

Gravlanized nails &
stakes

Stakes through bags

Layer lasana "sheet
mulching" of substrate
and myclium (e.g. King
Strophia or Pearl
Oyster)

Stake & twine

Bags installed opposite
each other w/
galvanized nails &
Stakes

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Figure 16: Mycofiltration Symantec Installation

Note. Illustrations of anchoring systems utilized for Mycofiltration installation within
tributary. Property of Paul Stamets.

For an overview of higher technology methods for mycelial spawn production, see
Growing Gourmet & Medicinal Mushrooms or The Mushroom Cultivator. These books
can guide you to successful cultivation and eliminate the need to purchase mycofilters.
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A synopsis of essential steps for spawn production are listed here:
1. Preparation and pouring of agar media into petri dishes.
2. Germination of spore and isolation of pure mushroom mycelium.
3. Expansion of mycelial mass on agar media.
4. Preparation of grain media.
5. Inoculation of grain media with pure mycelium grown on agar media.
6. Incubation of inoculated grain media (spawn).
7. Laying out grain spawn onto trays, or inoculation of grain spawn into bulk
substrates.
8. Casing – covering substrate with a moist mixture of peat and other materials.
9. Initiation – lowering temperature, increasing humidity to 95%, increasing air
circulation, decreasing carbon dioxide and or light levels.
10. Cropping – maintaining temperature, lowering humidity to 85-92%,
maintaining air circulation, carbon dioxide and/or light levels.
Raw material that can be utilized from agriculture or the forestry industry to create
“fruiting substrate” are listed below:
•
•
•
•
•
•
•
•
•
•
•
•
•

Wood waste paper products
Cereal straws and grain hulls
Coconut fibers
Corncobs
Coffee plants and waste (e.g. used coffee grounds)
Tea leaves
Sugarcane bagasse
Bananas fronds
Seed hulls (cotton seeds, sunflower seeds, and oil-rich seeds)
Almond, walnuts, pecan, peanut hulls
Soybean meal, roughage (Okara), soy waste
Artichoke waste
Cactus waste: saguaro and prickly pear, yucca, agave

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Cost of mycelium is ~ $125-325 per test-tube, depending on the species. One
could eliminate this cost by harvesting species from the forest using sustainable harvesting
principles. If you would like to expand your mycelium to make mycofilter or mix in
mycoremediation in situ, below is a checklist that includes all the material one should
consider:
High-Technology Check-list
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

Autoclave
Petri-dishes
Para-film
Glass jars
Malt agar
Micron (HEPA) filtered
laminar flow bench
Digital thermohygrometer
Synthetic filter disks
Scalpels
Bacticinerator® electric
scalpels sterilizers

11.
12.
13.
14.
15.
16.
17.
18.

Spawn bags with filter
patch
Impulse sealer
Erlenmeyer media flasks
Cost of electricity
High-sensitive digital
scale
Organic rye
Space and time in lab.
Labor: Skilled Lab
technicians & educated
scientist

Low-Technology check-list expanding medium (for individual landowners):
1.
2.
3.
4.
5.
6.
7.
8.

Spawn from mycologist
Cardboard
Hydrogen peroxide (0.3%)
Zip-lock plastic bag
Medium (alder woodchips, straw, rice, ect.)
Burlap sacks (non petroleum treated)
Labor
Soaking tank

For constructing mycofilters you will need the following:
1. Substrate (alder wood, chips, sawdust, straw ect.)
2. Tools (shovels, pitch fork)
3. Rubbing alcohol

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4. Burlap sacks
5. Twine or hemp string
6. Plastic bags
7. Rent or borrow a tractor if you don’t have one.
8. Wooden stakes

After sending soil or/and water samples to a local environmental toxicity lab for
analysis and receiving the results, the landowner or stakeholder will complete the 2012
Mycorestoration Site Description (refer to fig. 32: 2012 Mycorestoration Site Description
at the end of this section) and identify which toxins and heavy metals are present on the
land. After careful analysis of the many parameters of concern, he or she will then choose
a fungal species that has the ability to ameliorate the identified chemical toxins or
accumulated heavy metals of concern. Please refer back to Figures 5: Mushrooms with
Activity Against Chemical Toxins (page 21) and Figure 6: Mushroom Species’
concentrations of Heavy Metals (page 22). The following section explains low-tech
methods of implementation of mycoremediation and mycofiltration projects.
The simplest method for making a substrate like woodchips or straw ready for the
introduction of spawn is ambient temperature fermentation. This method, although now
readily articulated, was only recently refined for use in mycoremediation and reduces
costs by several orders of magnitude because high heat pressure sterilization, the use of
caustic chemicals, or much of the traditional infrastructure for incubation of mycelium are
now deemed unnecessary.

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Simplified Method for Generating Mycoremediation or -filtration Mycelium
STEP 1: Acquire freshly chipped hardwood or conifer (not fungally resistant tree
species like cedar, walnut, or redwood).
STEP 2: Place wood chips and/or straw into containment vessel such as an open
tank, farm watering trough, seafood “tote” or plastic lined pond.
STEP 3: Fill container with wood chips until submerged with fresh or salt water at
ambient temperature, preferably 40-600 F. Cold fermentation of wood chips could
be utilized (see fig. 33: Ambient Temperature (40-60 F.) Fermentation of
Woodchips in Fresh Water ). Cold fermentation works because an anaerobic
environment with multiple species of bacteria Klebsiella followed by exposure to
oxygen kills anaerobic microflora thus the substrate exposable by the oxygen
loving mushroom mycelium.
STEP 4: Allow the container to sit undisturbed for 1-3 weeks. A biofilm will form
and fine gaseous bubbles will be emitted after a few days.
STEP 5: Drain container completely.
STEP 6: Wait ~2-4 hours then inoculate with 2-10% spawn by volume.
STEP 7a: Allow spawn to incubate within the container where soaking occurred.
STEP 7b: Fill burlap sacks with inoculated mycelium (optional if doing
mycofiltration).
STEP 8: Place into incubation environment at 50-700 F for one week. Lower
temperature to 35-550 F for an additional 4-8 weeks.
STEP 9: Move myceliated substrate to mycoremediation site.
STEP 10: Position myceliated substrate for optimal configuration according to site
characteristics.

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Figure 33: Ambient Temperature (40-60 F.) Fermentation of Woodchips in Fresh Water

Note. This photograph shows the fermentation process of wood chips before they
are used to create mycofilters. Barrels are used to keep substrate submerged. Photo by
Paul Stamets, 2012.
Depending on contaminant load characteristics, slope, weather, and other
environmental factors, mycofilters may have to be renewed for optimal performance based
on results determined by periodic-testing. Mycofilters need to be installed at a weatherappropriate time, since fungi can tolerate colder temperatures better than hot temperatures.
Such care constitutes being sensitive to the biological cycles and environmental conditions
mushrooms thrive in. Good luck to you and your mycorestoration endeavors!
The below photographs are examples of small-scale mycofiltration installations in
Washington State. Refer to fig.34: Small-scale Mycofiltration Installation Examples
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Figure 34: Small-scale Mycofiltration Installation Examples in Washington State
a.)

Note. Small-scale mycofiltration installation on Oyster Bay Road at Pat Labine’s farm,
Thurston County, Washington. Photos a-c by Paul Stamets
b.)

Note. Small-scale mycofiltration installation on Oyster Bay Road at Pat Labine’s farm.
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c.)

Note. LaDena Stamets with triangular mycofiltration mycobag design. a-c Photographs
by Paul Stamets
d. - e.)

Note. Small-scale mycofiltration installation example, at DNR Webster Tree Nursery,
Washington State. Photo by John Trobaugh

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Figure XX: 2011 Mycoremediation Site Description

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Note. Site Description outline helps property owners qualify the site they are considering
using mycorestoration. © Paul Stamets 2011.

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Part III
Conclusion and Future Research
This thesis has documented the ability of mycorestoration to counteract
anthropogenic pollutants the environment. Such pollutants cause degradation to human
health as well as to other natural systems, ultimately increasing the cost of living in these
ecosystems. Using mycorestoration increases the inherent sustainability of habitats,
reducing the need for remedial practices while fortifying the ecological benefits healthy
habitats provide: clean water, clean food, clean air, and healthy inhabitants.
Mycoremediation and mycofiltration are low-cost, time conservative, low
maintenance biological solutions, which decrease toxicity in soil better than other
methods. Moreover, the cost of removing petroleum hydrocarbons using mycoremediation
techniques is significantly less than other methods such as soil washing and
bioremediation.
Many attempts have been made to develop methods to remove oil spills and
toxins from water and soils. Mycoremediation and mycofiltration are worthwhile
techniques for reducing petroleum-based pollutants in our environment. Research has
shown mycoremediation can remove hydrocarbons such as oil, petroleum based products,
pharmaceuticals, pesticides, and farm wastewater. For example, TAH reduction
highlighted in this thesis averaged 95.5%. In most cases reduction of PAHs was 86-92%.
FCB reductions averaged 92%, with the highest reduction being 97%. Through my
literature review, I found that reductions in contamination levels of fecal coliform
bacteria (FCB) ranged from 87 to 97%, while polycyclic aromatic hydrocarbons (PAHs)
reduction ranged from 57 to 97%. Total aromatic hydrocarbons (TAH) reductions were
91 to 99%. Mycoremediation scientists agree more research needs to be conducted to
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further our understanding of species that will be the most effective against specific toxins.
Species specificity factors are critical to match before executing installation for achieving
the best results.
While there are hundreds if not thousands of enzymes secreted by fungi, many of
which can break down PAHs, only a few have been thoroughly researched for the
purposes of mycoremediation. Since so many fungi have not yet been thoroughly tested
for enzymes, there are opportunities for future research to discover new enzymatic
systems that can metabolize contaminants detrimental to ecosystem health. Manganesedependent peroxidase enzymes in particular are strong catalysts of oxidation processes.
Fungi excrete metal-binding and up-take metabolites associated with complexolysis or
ligand-promoted dissolution, including carboxylic acids, amino acids, siderophores and
phenolic compounds.
Clearly, mycorestoration can be documented as effective under some
circumstances, but the range of applicability needs to be expanded. By building upon the
synergistic relationships fungi have in nature, mycorestoration applications can be become
more effective and have a wider range of applicability.
Increasing awareness for environmental scientists and public policy makers is
essential. Policies and funding limitations have been major obstacles that have prevented
the use of wide spread mycoremediation to clean up of chemical toxins. Research and
publications about mycorestoration are available and continue to compile as does public
interest. Politicians and other environmental sectors should push to make this innovation
available globally for the health of our environment that directly affects our well-being.
The US EPA is investing tens of thousands of dollars in mycorestoration grants. Once
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mycorestoration is adopted as a Best Management Practice (BMP), these applications can
be commercialized and utilized for global contamination clean up of a variety of chemical
toxins. Toxins and pollution know no borders and what we do affects our neighbors
locally and internationally. For example, a paper mill in Tacoma, Washington, caused
high levels of arsenic in contaminated soils on Vashon Island. Such situations could use
mycorestoration techniques but landowners usually don’t know how to get started. Future
research will emphasize the best practices for implementation of mycoremediation,
mycofiltration and mycoforestry methods as well as applicability to site specificities.
Biological systems are inherently different from mechanical systems. Given this,
we need to work within the biorhythms of ecosystems. The number of individuals or
biomass of a species that an ecosystem can support is known as its carrying capacity.
Reducing our ecological footprint, which is the influence that people's patterns of
consumption and life style has on the surrounding ecosystem and across the globe, enables
us to live within the carrying capacity of the environments that support us. My intention of
this thesis has been to bridge the gap between government funded research studies and
individual landowners with small-scale installations, with the goal of empowering
landowners to take control and protect their land from chemical toxins and heavy metals.
This thesis has defined the parameters for designing a private, individual site-specific
mycorestoration installation. Projects with the Makah First Peoples, the Squaxin First
Peoples, the Mason County Departments of Health, the Washington State Department of
Transportation (WSDOT), and private companies such as Ridolfi, Inc., and Ecological
Design, Inc., help build awareness and develop data that will illustrate proof-of -concept.

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This collective effort will provide the basis for more funding to demonstrate that
mycorestoration is a low-cost method practice for cleaning up pollutants.
Mycoremediation and bioremediation eliminate the cost of removing thousands of
tons of soil from toxic waste storage sites. Costs for storing toxic waste is expensive and
continues indeterminately – much toxic waste must be guarded in a secure area for the
unforeseeable future, incurring an unpredictable expense for future generations. Some
toxic waste will take thousands of years to degrade. Consequently policies that allow
burning, hauling, and burying of toxic waste leave the environment unhealthy and unable
to achieve a healthy ecological equilibrium. A natural myco-biological solution can turn
contaminated soils into useable medium for soil generation and landscaping.
Further research needs to be conducted to see what other fungal species will prove
useful for mycoremediation. For instance, Marasmiellus candidus sensu lato is a dominant
white rot species proliferating where cane berries thrive, and could be utilized for
mycoremediation to clean up environments characterized by overgrowth of blackberries,
salmonberries, etc., as suggested in this thesis for implementation on Tatoosh Island for
PAH clean up. Another esoteric species is the recently discovered underwater mushroom
Psathyrella aquatica found in fresh water river systems. If this species has the potential to
break down toxins like PAHs or capture pathogenic bacteria it could aid in recovering
fresh water environments from anthropogenic contamination.
Throughout the literature research I conducted there has been general agreement
among the scientific community that there are many advantages to mycoremediation and
mycofiltration: these are effective, low cost, biological solutions that require minimal
maintenance and are flexible for installation in a variety of sites. That said, mycofiltration

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has some limitations: First, scientists need to find stronger netting/mesh material to hold
the substrates together and won’t decompose before fungi have completed their life cycles
or blow out from heavy water flows. Running parallel controls in several locations along
each site would eliminate data errors and provide comparison of contamination levels.
Another issue that needs to be addressed is using different species in interspersing
mycobags; data could be collected specific to the ability of one fungal species to another.
Currently, the oyster mushroom, in particular the Pleurotus ostreatus strain from
Nisqually Delta, has become the primary species for mycofiltration installations within
Mason County, Washington, but it is possible that other resilient species are more
appropriate for other sites. Keep this in mind while selecting a fungal species for
remediation.
Future funding and research needs to be focused on testing applications of mycoenhanced constructed wetlands, buffer zones, mycobooms and in-line wastewater
treatment systems in urban and rural areas. More studies like those using eco-machines at
the Fisherville project, which take a multi-kingdom approach, hold great promise. Dr.
Susan Thomas’ Dungeness Watershed project was helpful in understanding fungi’s ability
to metabolize E. coli.
Fungi Perfecti’s EPA Bench Study “Comprehensive Assessment of Mycofiltration
Biotechnology to Remove Pathogens from Urban Stormwater” identifies which
combinations of substrate and fungal species are the best for treating stormwater. The
EPA grant “Mycofiltration in Urban Landscapes” will tentatively enter phase II in June
2012 with field applications at numerous locations.
Mycoforestry is another new concept showing promise. The interaction of fungi

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and trees allows for healthy fertile forests and regenerating soils. Mycorrhizal fungi have
been coevolving with over ninety percent of the plants on earth through a symbiotic
association, exchanging important nutrients. Even though it is well known the presence of
mycodiversity in soils is an indicator that soils are healthy, fungal strategies to restore
devastated forests are not commonly applied. However, government agency sites, like the
Washington State Department of Natural Resources at Webster’s Nursery, use
mycorrhizal species every few years to help establish tree saplings and improve outplanting. Mycoforestry is not just about applying mycorrhizae to one tree. Vast fungal
networks of fungi link trees together by bridging root systems through hyphae, exchanging
nutrients with each other, and helping assure habitat health through biodiversity support.
One single mycorrhizal fungus can aid many different plant species to fight off
pathogens, compensate for soil nutrients loss and ameliorate the effects of drought. The
fungi project mycelia into the soil, extending the plant’s roots and root absorbing capacity
many fold, significantly more than what the plant would achieve alone. Crops with
mycorrhizae present can increase the effective surface absorption area of the roots by
several hundreds to several thousands fold. Commercialization of methods for applications
is being developed as demand increases. Adinarayana et al. (2001) suggest that only larger
scale projects can “… demonstrate the ease and viability of the inoculation methods
suggested in the literature.”
Mycorrhizae can maintain healthy roots, improve soil structure and aid in water
up-take whereas fertilizers cannot. In fact, fertilizers negatively affect the factors listed
above, often leading to deteriorated water quality, soil structure and salinity. The use of
organic amendments and biological inoculants like mycorrhizal fungi has been widely

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studied and proven effective by dozens of universities. Also mycorrhizal applications are
economically feasible at a fraction of the cost of chemical soil supplements. In India, some
farmers utilize mycorrhizal fungi to reduce their use of chemical fertilizers by 50%
without any production loss.
In nursery settings, endomycorrhizae have two major benefits. First, seedling
quality and success is improved. Second, hardwoods that have good lateral roots and
endomycorrhizal development will have a higher success of survival than without.
Hardwoods enhanced with endomycorrhizae are more capable of competing with
undesired vegetation and/or in harsher environments. Forest floors are usually lacking
phosphorus in a form that is available to trees. Endomycorrhizae can naturally help in
phosphorus uptake. Without these mycohelpers, the trees wood die. However, these
mycorrhizae need to be managed in ‘constructed’ nursery soils which are often chemically
treated to reduce possible pathogens, and in doing so, the beneficial fungi are also
adversely affected.
Canada’s sustainable forest practices should be echoed across the globe. Future
research should explore implementation of large-scale field studies of using mycorrhizae
to regenerate devastated forests. However, scientific comparisons of forests planted with
and without introduced mycorrhizae are difficult experiments to manage because of
naturally occurring mycorrhizae in soils and the fact that mycorrhizal species easily
distribute themselves via spore transportation.
Planted Plant Production is a company created to produce a product called “Life
Box.” A Life Box is a corrugated cardboard box infused with mycorrhizal fungi and the
seeds of several different tree species. The idea is that people receive a Life Box through

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the mail then germinate the seeds contained in the box, and then plant the resulting trees to
regreen the Earth. This innovative technology is aimed at sequestering carbon from the
environment. Once Life Box trees are planted one can use a hand held device application
to geographically pinpoint the GPS coordinates where one’s trees are and calculate how
much carbon has been sequestered.
Traditional methods for removing oil spills from water, e.g., skimmers, booms,
dispersants, and controlled burning, are not effective. The Gulf of Mexico’s April 20,
2010, event where the BP Deepwater Horizon oil rig blew up in the Gulf of Mexico
resulted in the death of eleven people and the dumping of more that 200 million gallons
of oil into the ocean. Only 8% of the oil was recovered. In 2011, Exxon Mobil pipeline
ruptured, causing 42,000 gallons of oil to spill into the Yellowstone River that runs
through one of our national parks. In 2011, the Fukushima nuclear power plant disaster
was yet another example of an environmental catastrophe where mycoremediation
methods might be employed.
Future work in mycorestoration will likely focus on investigating other white-rot
fungi for remediation abilities – including those that could be used to counteract
environmental catastrophes such as those mentioned above. I suggest more development
needs to address mycofiltration site design by creating and compiling mycorestoration
sites using a Geographic Information System (GIS) map showing locations of projects,
including identifying toxins and contamination levels before and after mycorestoration is
applied. A mycorestoration “stamp of approval” from credible and experienced
mycologists could certify that sites are being managed appropriately. Further work could

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create a multi-volume guide to Best Mycorestoration Practices specific to each ecoregion to induce widespread pollution cleanup.

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Glossary of Key Mycorestoration Terms
anthropogenic

Caused by humans.

abiotic

Non-living.

aerobic

Living or occurring only in the presence of oxygen.

Best Management Practice
(BMP)

Proactive and often voluntary forest stewardship practices
that have been determined to be the most effective, practical
means of preventing or reducing soil and other pollutants
from entering any water; streams ponds, lakes, wetlands,
ect.
Biological indicators are species used to monitor the health
of an environment or ecosystem. They are any biological
species or group of species whose function, population, or
status can be used to determine ecosystem or environmental
integrity.

bioindicator

biomagnifications

process where toxins become more concentrated in animals
higher levels in the food chain.

biomass

The total weight of living material in a place often
expressed as weight per unit.

bioswale

Use vegetation and gentle gradients to slow and infiltrate
water. Common for holding storm water.

bioventing

Bioventing is an in situ remediation technology that utilizes
microorganisms to biodegrade organic constituents
absorbed within the ground water. Bioventing will enhance
the activity of native bacteria as well as simulates natural in
situ biodegradation of hydrocarbons by inducing air or
oxygen flow.

biotic

Of or having to do with life or living organisms. Produced
or caused by living organisms.

bole

A trunk or main stem of a tree.

brown rot

A condition caused by the degradation of cellulose by fungi.
It leaves the substrate a brown color largely due to
undecomposed lignin. Soild blocks of wood are used for
testing wheather a dungus causes brown rot or white rot.

carrying capacity

The number of individuals or biomass of a species that an
ecosystem can support.

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conifer

A cone-bearing tree with needles (e.g. pine, spruce, fir,
larch).

deciduous

A tree that loses its leaves or needles during the fall and
winter (e.g. Maple, Alder and Oak).

dilution

The process of making a weaker or less concentrated.

ecological footprint

The influence that people's patterns of consumption and life
style have on the surrounding ecosystem and across the
globe.

ecological restoration

Altering a site to reestablish the original ecosystem.

ecosystem diversity

The different biological communities and their associations
with the chemical and physical environment.

ecosystem function

The interactions between organisms and the physical
environment, such as nutrient cycling, soil development,
water budgeting, and flammability.

ecotone

A transition area between two biomes or different patches
of the landscape. An ecotone may appear on the ground as a
gradual blending of the two communities across a area or it
may be a sharp boundary line (e.g., grass land to forest).

ectomycorrhiza

A mutialistic symbiosis between the hyphae of certain
basidiomycetes and ascomycetes and the fine roota of
certain plants.

effluent

Liquid waste or sewage discharge into a river or the sea
(e.g., the bay was contaminated with the effluent from an
industrial plant).

euthrophication

Process of degradation in aquatic environments caused by
nitrogen and phospurus pollution and characterized by algal
blooms and oxygen depletion.

global climate change

Climate characteristics that are changing now and will
continue to change in the future, resulting in part from
human activity.

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Goemycology

The scientific study of the roles of fungi in processes of
fundamental importance to geology and the biogeochemical
importance of fungi is significant in several key areas.
Which include nutrient and element cycling, rock and
mineral transformation, bioweathering, mycogenic
biomineral formation and interactions of fungi with clay
minerals and metals. These processes can occur in aquatic
and terrestrial environments.

hydrochemistry

A subdivision of hydrology that deals with the chemical
characteristics of the water on and beneath the surface of
the Earth. Water in all forms is affected chemically by the
materials with which it comes into contact, and it can
dissolve many elements in significant quantities. Chemical
hydrology is concerned with the processes involved and
thus includes study of phenomena such as the transport of
salts from land to sea (by erosion of rocks and surface
runoff) and from sea to land (by evaporation, cloud
formation, and precipitation) and the age and origin of
groundwater in desert regions and of ice sheets and glaciers.

hypha (-ae)

Long tube-like elements that make up the body (mycelium)
of a fungus; may or may not be separate.

in-situ

In the original position.

interdisciplinary

Of relating to, or involving two or more academic
disciplines that are usually considered distinct.

infiltrate

To cause a liquid to permeate by passing through interstices
or pores.

keystone species

A species that has a disproportionate impact (relative to its
numbers or biomass) on the organization of an ecosystem.
Loss of keystone species has far reached consequences for
the ecosystem.

lignin

The organic substance that, with cellulose, forms the
structural basis of most wood tissues.

mycelia

Fungal network of thread like cells.

mycelium

The body of filamentous fungus, Composed of a network of
complexly branch hyphae.

148

mycofiltration

The use of fungi as a membrane for filtering out
microorganisms, pollutants and silt. Habitat infused with
mycelium reduce downstream particulate flow, mitigate
erosion, filter out bacteria and protozoa, and modulate water
flow through the soil (Stamets 2006).

mycoflora

All species of fungi that in habit a given area (=mycota).

mycoforestry

The use of fungi beneficial to trees to aid the regeneration
of forests (e.g., the establishment of a new forest on land
devastated by repetitive slash-and-burn clear cutting
practices).

mycologist

A person whom studies the kingdom of fungi.

mycology

The study of fungi.

mycopesticides

The use of fungi like, Cordyceds to infect and then kill
insects like carpenter ants from investaion and deteration of
your home.

mycoremediation

Mycoremediation centers on the use of fungal mycelium to
degrade pollutants in situ. (e.g. to degrade an oil spill on
land by mixing or layering mycelium onto the polluted
soil.)
The use of fungi to repair or restore the weakened immune
system of the environments. Whether the habitat is
damaged by human activity or natural disaster saprophytic,
endophytic, mycorrhizal and in some cases parasitic fungi
can aid recovery.

mycorestoration

mycorrhizal (-ae)

A symbiotic state wherein mushroom mycelium forms on or
in the roots of trees and other plants. Mycorrhizae are either
endo or ecto.

niche

The function of position of an organism or population with
in a ecological community. The particular area within a
habitat occupied by an organism.

Phytoremediation

The use of plants to remove or neutralize contaminants as in
polluted soil or water.

Precautionary Principle

Principle stating that it may be better to avoid taking a
particular action due or the possibility of causing
unexpected harm (e.g. choosing not introduce a non-native
species like scotch broom to promote slope stability because
it will out compete native plants).
149

primary producers

An organism such as green plants, alga, or seaweed that
obtains its energy directly from the sun via photosynthesis.
Also known as an autotrophy or photosynthetic species.

resilience

The ability of an ecosystem to return to its original state
following disturbance.

saprophyte

A fungus that lives on dead organic matter. [These fungi]
are among the first organisms to rejuvenate the food chain
after catastrophe.

Species diversity

all the species on earth, including single-celled bacteria and
protisits as well as the species of the multicellular kingdoms
(plants, fungi, animals).

species richness

The number of species found in a community. Species
richness increase with decrease in elevation, increasing
solar radiation increasing precipitation, hot raining low
lands have the most species.

succession

The gradual replacement of one group of organisms by
another over time following initial disturbance.

sustainability

Characteristics by which a process or state can be
maintained at a certain level indefinitely.

sustainable harvest level

Level of wood, plant, or fungi harvesting that can be
sustained indefinitely. In forestry this is often calculated as
annual allowable cut on per-year basis for any specific
region.

sustained yield

Amount of natural resource, such as fungi, that can be
harvests without reducing the inventory or production
potential.

symbiotic relationship

A close long term biological relationship in which two
species are always found living together.

trophic levels

Levels of biological communities representing ways in
which energy is captured and moved through the ecosystem
by the various types of species. Primary producer;
herbivore, secondary consumers are carnivore and
detritivore.
A ridge of high land dividing two areas that are drained by
different river systems. The region draining into a river,
river system, or other body of water.

watershed

150

white rot

A condition whereby a substrate is rendered light in color
from the fungal decomposition of lignin (delignification),
leaving cellulose largely intact. Solid blocks of wood can be
utilized for testing whether fungus causes white rot or
brown rot.

151

References
Adinarayana, R., Mathew, Z. (2001). Bioremediation potential of white rot fungi.
Cambridge University Press. NY. Pp. 52-78
Amaranthus, M., Anderson, J., Amaranthus, Z., Perry, D., (2009). Building soil organic
matter biologically: the benefits of mycorrhizal seed inoculation. Acres USA. 40,
(1), Pp. 1-3
Amaranthus, M., Anderson, J., Marler, J., Degraded soils, food storage & eating oil:
restoring soil life through biological agriculture. Acres USA. 40 (3), Pp. 1-3
Benedict, T., (2011). Mycofiltration for the Puget Sound: Pleurotus ostreatus & fecal
coliform bacteria. Pp. 1-10
Cabello, M.N. (2001). Mycorrhizas and hydrocarbons. Cambridge University Press. NY.
Pp. 456-471
Cajthaml, T., Bhatt, M., Sasek, V., Mateju, V. (2002). Bioremediation of PAHContaminated Soil by Composting: A Case Study. Folia Microbiol. Praque
Czechia. Pp. 696-700
Cerniglia, C., Sutherland, J.B. (2001). Bioremediation of polycyclic aromatic hydro
carbons by ligninolytic and non-ligninolytic fungi. Cambridge University Press.
NY. Pp.136-187
Cohen, R., Yitzhak, H. (2001). The roles of fungi in agricultural waste conversion.
Cambridge University Press. NY. Pp. 305-334
Eggen, T., Sasek, V. (2002). Use of Edible and Medicinal Oyster Mushrooms; Pleurotus
ostreatus Spent Compost in Remediation of Chemically Polluted Soils.
International Journal of Medicinal Mushrooms. 4. Pp. 255-261
Gadd, G.M., Rhee, Y.J., Hillier, S. (2012). Lead Transformation to Pyromorphite by
fungi. Current Biology. 22. Pp. 1-5
Gadd, G.M., Rhee, Y.J., Stephenson, K., Wei, Z. (2011). Geomycology: metals, actinides
and biominerals. Pp. 1-27
Gadd, G.M., Fomina, M., Charnock, J., Hiller, S., Alvarez, R., Livens, F. (2008) Role of
fungi in the biogeochemical fate of depleted uranium. Current Biology. 19. Pp.
375-377
Gadd, G.M., Fomina, M., Alexander, I.J., Colpaert, J.V. (2005) Solubilization of toxic
152

metal minerals and metal tolerance of mycorrhizal fungi, Soil Biology &
Biochemistry. 37. Pp. 851-866
Gadd, G.M., Fomina, M., Podgorsky, V.S., Olishevska, S.V., Kadoshnikov, V.M.,
Pisanska, I.R., Hillier, S. (2007) Fungal Deterioration of Barrier Concrete used
in Nuclear Waste Disposal. Geomicrobiology Journal. 24. Pp. 643-653.
Gadd, G.M., Fomina, M., Charnock. J.M., Hillier, S., Alvarez, R. (2007) Fungal
transformations of uranium oxides, Environmental Microbiology. 9 Pp. 16961710
Kruckeberg, A. (1995). The natural history of Puget Sound Country. Weyerhaeuser
Environmental Books. Pp. 3-4, 385-389
Marx, D.H., Cordell, C.E., Kormanik, P. (1989). Mycorrhizae: benefits and practical
application in Forest Tree Nurseries. USDA Forest Service, Agriculture Handbook,
680, Pp. 1-5
Obire, O., Putheti, R.R. (2009). Fungi in bioremediation of oil polluted Environments.
Pp. 1-11
Primack, R. (2010). Essentials of conservation biology. Massachusetts: Sinauer
Associates Inc.
Sasek, V., Cajthaml, T., Bhatt, M. (2003). Use of fungal Technology in soil remediation:
A case study. Archives of Environmental Contamination and Toxicology. 3. Pp. 514
Sasek, V., Bhatt, M., Cajthaml, T., Malachova, K., Lednicka, D. (2003). Compostmediated Removal of Polycyclic Aromatic Hydrocarbons from Contaminated Soils.
Archives of Environmental Contamination and Toxicology. 44. Pp. 336-342
Sasek, V., (2003). Why mycoremediations have not yet come into practice. Kluwer
Academic Publishers. Netherlands. Pp. 247-266
Stamets, P. (2005). Mycelium running: how mushrooms can help save the world. Pp. 51109. Ten Speed Press. California.
Stamets, P. (2000). Growing Gourmet and Medicinal Mushrooms (3rd ed.). Pp. 45-385.
California: Ten Speed Press.
Stamets, P., (2004, November). Cortes Ecoforestry Society: Healthy forests for Healthy
Communities; Stamets Mycoforestry Project, 17, Pp. 5

153

Todd, J., Bernat, E., Adams, J., Beam, M., Christy, O., (2007). Fisherville Ecomachine
Pilot Final Report. John Todd Ecological Design, INC. Pp.1-31
Native American Lands Environmental Mitigation Program 2008. (2008)
Mycoremediation pilot study Tatoosh Island for Makah tribe of Indians. By Ridolfi
Inc. Pp. 1-18
Thomas, S., Aston, L., Woodruff, D., Cullinan, V. (2009). Field Demonstration of
Mycoremediation for removal of fecal coliform Bacteria and nutrients in the
Dungeness watershed, Washington State. U.S. Department of Energy. Pp. 1-32
Trudell, S., and Ammirati J. (2009). Mushrooms of the Pacific Northwest. Timber Press:
Oregon.
RIDOLFI Inc. (2009). Mycoremediation Pilot Study. Native American Lands
Environmental Mitigation Program. Pp 1-17
Washington State’s Department of Transportation. (1998). Pp. 1-55
(2004) The American Heritage College dictionary Fourth Edition. 4, New York:
Houghton Mifflin Company

154

Web References
(2011). Squaxin mushroom water quality solution. Shelton, Washington, USA. Retrieved
from http://nwifc.org/2011/01/squakin-island-tribe-further-testing -mushrooms as
water quality solution
Washington Department of Health (WADOH) http://www.doh.wa.gov
Washington Department of Ecology (WADOE) http://www.ecy.wa.gov
Washington Department of Transportation (WADOT) http://www.wsdot.wa.gov
Environmental Protection Agency (EPA) http://www.epa.gov
EPA. Priority Pollutants. http://water.epa.gov/scitech/methods/cwa/pollutants.cfm
Clean Water Act of 1972 http://www.epa.gov/lawsregs/laws/cwa.html
Washington Dept of Natural Resources (WADNR) http://www.dnr.wa.gov
Dept of Fish and Wildlife (WADFW) http://wdfw.wa.gov
Arwyn R. (2012, February, 7). Neah Bay students finalists in national project; already win $70,000 prize.
Peninsula Daily News. Retrieved February 10, 2012, from
http://www.peninsuladailynews.com/article/20120208/news/302089988/neah-baystudents-finalists-in-national-project-already-win-70000
http://cfs.nrcan.gc.ca
National Resources Canada. http://cfs.nrcan.gc.ca/projects/118. Retrieved February 5,
2012.
WADOE. http://www.ecy.wa.gov/toxics/index.htm. Retrieved January 20, 2012.
Map of the Pacific Northwest. http://soundwaves.usgs.gov/2007/10/outreach3.html.
Retrieved June 2, 2012

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