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Bioremediation of Contaminated Riparian Zones Using Mycorrhizal Fungi – An Exploration of the Feasibility of Restoration Through Mycoremediation
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2009
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Jones, Gary K
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Environmental Studies
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Bioremediation of Contaminated Riparian Zones Using Mycorrhizal
Fungi – An Exploration of the Feasibility of Restoration Through
Mycoremediation
by
Gary K. Jones
April 2, 2009
A Thesis: Essay of Distinction - Submitted in Partial Fulfillment of the
Requirements for the Degree Master of Environmental Study
The Evergreen State College
Copyright 2009 by Gary K. Jones. All rights reserved.
ii
This Thesis for the Master of Environmental Studies Degree
By
Gary K. Jones
Has been approved for
The Evergreen State College
By
______________________
Linda Moon-Stumpff, PhD
Member of the Faculty
__________
Date
Abstract
Bioremediation of Contaminated Riparian Zones Using Mycorrhizal Fungi – An
Exploration of the Feasibility of Restoration Through Mycoremediation
Gary K. Jones
The author uses an extensive literature review to explore the question “Does current
scientific research support the use of higher order mycorrhizal fungi as a bioremediation
tool in the restoration of riparian zones that have been altered or damaged by
anthropocentric presence and activities?” Emerging scientific literature suggests that
many native fungal species are able to alter or detoxify specific toxins like heavy metals,
petroleum by-products and industrial effluents. Laboratory and field studies indicate the
mycelial networks of these life forms may alter the chemical and molecular structures of
the toxins, rendering them harmless, and reducing their negative impacts on the
environment. The author concludes there is ample data in the research to suggest that
mycorrhizal bioremediation techniques show considerable promise as biomediation
factors in providing and recovering safe and healthy spawning and habitat areas for all
riparian life forms, including fish species, and may also provide a method for reducing
the presence of harmful substances in the food web for the entire planet.
Table of Contents
Introduction
1
Current Ecological Concerns
8
Restoration Ecology
13
About the Fungi and Factors Relevant to Their Selection and Application
In Bioremediation Strategies
21
About the Toxins
29
Fungal Metabolism of Polycyclic Aromatic Hydrocarbons
35
Alternatives to Mycoremediation
39
Challenges
40
Uncontrolled Sources or Recontamination from Ongoing
Efflux of Contaminants
43
Reluctant Liable Party
44
Resources
45
State Owned Aquatic Land Management Policy
45
Potential Superfund Listing
47
Area – wide Contamination
47
Cost Estimates for Site Cleanup
47
Conclusion
50
Bibliography
60
Acknowledgements
I must convey my deepest thanks and appreciation to my primary reader and advisor Dr. Linda
Moon – Stumpff who worked diligently and at length to help me ensure this work appeared in a
logical and consistent manner to its intended audience. Her encouragement and professionalism
were an asset to me throughout this process, including my oral presentation. Equally important
were the contributions of Dr. Paul Przybylowicz, who assisted with some of the more technical
aspects of this research, specifically his vast knowledge in microbiology and the world of fungal
organisms. He helped me get focused on what I knew, and to not exceed my capabilities or
training. Thanks to you both, and to all those incredible people who taught me to love
knowledge for its own sake, and to pursue my interests, wherever they might take me. And to
my mom, Louise Wilson, and stepdad, Robert Wilson, my two best friends, who encouraged and
help support me with this latest intellectual venture. Finally, my thanks to all the staff and other
faculty at The Evergreen State College who helped make this an enjoyable experience. I will
never forget the teachers who teach for the benefit of science and the expansion of knowledge in
its variety of forms. My hat comes off for every teacher I have ever known or heard of. The
civilized planet flows from your fingers.
Gary K. Jones
Introduction
Does current scientific research support the use of higher order mycorrhizal fungi
in the restoration of riparian zones that have been altered or damaged by
anthropocentric presence and activities? There is emerging literature in the
scientific media suggesting that many native fungal species are able to alter or
detoxify specific environmental toxins and other environmental contaminants
including heavy metals, pesticides, effluents, human hair, transmission lubricants,
and certain grades of petroleum, like those spilled in the Exxon-Valdez disaster
(Singh, 2006). Laboratory and field studies indicate the mycelial networks of
these life forms may alter the molecular composition and structures of soil
contaminants and other energy/nutrient sources during their metabolic
breakdown, rendering the toxins either neutral or providing a convenient means to
facilitate their extraction from the affected areas (i.e. harvesting or continued
biodegradation) at reasonable levels of fiscal and natural economy and with
minimal secondary impacts on the environment. This thesis examines the
emerging scientific literature surrounding the feasibility of using fungal
organisms as an applied technology for mediating toxic waste sites, not as a single
solution, but as part of a well engineered and documented bioremediation
strategy; the thesis focuses on how several projects can provide feedback for
research and connect theory and research with practice through application in
various treatment projects.
1
In some cases (Atlas et al, 2008) the introduction of the new fungal species may
actually improve the bio-capabilities of the impacted area by improving soil
conditions, returning the toxic molecules to the carbon cycle/web of life in newly
transformed and benign structural compositions and encouraging the reestablishment of native plant and animal species. In this report the authors define
bioremediation as “…the use of living organisms, especially microorganisms, to
degrade pollutants and restore environmental quality.” The authors go on to
critically assess current contamination problems that can be treated by processes
based on native microorganism introduction. Although most public information
on aquatic oil spills center around images of dead birds and blackened shorelines,
in fact most environmental contaminants come from much smaller hydrocarbon
leakages, like waste motor oils and underground storage tanks (Atlas et al, 2008).
Most of these spills or inputs are readily managed or accommodated “due to the
capacities of microorganisms to biodegrade hydrocarbons.” A common method
for applying this technology, the report asserts, is to “…increase the natural rates
of hydrocarbon biodegradation that produce nontoxic end products. These
treatments may include adding microorganisms with specific enzymatic abilities
to a contaminated site and establishing conditions that favor microbial degradative
activities. In many cases, bioremediation can be carried out in situ. In other
cases, contaminated soils and sediments must be treated in bioreactors to remove
problem pollutants.” Added seed cultures may or may not improve the
biodegradation improvement rates for a given ecological system, as so many
contributing factors affect the metabolic processes. Hydrocarbon degrading fungi
2
and bacteria tend to be widely distributed, and therefore, the report suggests, the
addition of engineered or processed substrates, inoculates, or mulches may not
contribute much to natural processes already underway. It would seem, the
authors continue, that the deposition of starter materials would be most useful in
areas where the active biota systems have been radically altered, like logging
roads, where the sub soils have been eroded away to the mineral layers, and in
this case, straw mulches would definitely assist in building up organic matter and
assist in developing conditions where life can get a fresh start. Prince William
Sound is cited in this report as an example of how these local variants can impact
the natural processes: within this marine area, the compounds pristine and
phytane were rapidly degraded and served as a suitable internal reference marker
for just a few weeks to months. Scientists theorize that this rapid degradation is
due to the fact that microorganisms in Prince William Sound have evolved to
effectively consume terpenes. Pine trees have been dripping terpenes into the sea
for millions of years. Terpenes, which give the pine its distinctive odor, are
isoprenoid hydrocarbons that have similar chemical structure to pristine and
phytane. So, the Prince William Sound hydrocarbon degraders are very good at
consuming these compounds.
Kirk, et al (1991), conducted a survey of several species of authochtonous marine,
filamentous Ascomycetes and Deuteromycetes from southeastern Virgina, species
that have been shown to use specific compounds as sole carbon sources for
growth and to mineralize soils. The intent was to define a field characterization
3
of fungal species that would effectively degrade petroleum byproducts and
compounds in a variety of marine and near shore environments that were
impacted directly and indirectly by the presence of marinas for pleasure and
working craft aquatic vessels. The authors conclude that a need exists for
additional research on the effectiveness of higher order aquatic fungal species as
hydrocarbon degraders: “To date there are no field or microcosm studies to
confirm whether arenicolous or other higher marine fungi degrade hydrocarbons
in their natural environments, or have potential as bioremediation agents for
marine petroleum spills (Kirk et al, 1991).
A second body of literature related to toxicology suggests that native fish species
and higher order predators like bears and human beings, are negatively impacted
by industrial effluents and other toxins that make their way into riparian zones
that are critical in the life cycles of Pacific salmon and other species (Washington
State Pesticide Monitoring Program Reconnaissance Sampling of Fish Tissue and
Sediments, Washington State Department of Ecology, 1994). This paper will not
specifically address the connections or processes that involve bioaccumulation
and the impacts on salmon fry and migrating adults, but these are general areas of
environmental concern that may benefit from work in the area of
mycoremediation and the clean up of environmental toxins.
Most of us think of mushrooms as a sort of strange, fungal life form that appears
in the forest or other dark, damp places following extended periods of rain. We
4
associate them with fairies, toads, and poisonous concoctions like witches brew.
We are happily unaware that the true vegetative body or sustaining structure of
the organism is an invisible, subterranean network of biological cells called
mycelia or mycelium (Singh, 2006). Interlacing membranes of mycelia form a
mosaic of communicating, reactive cells that interact with and respond to changes
in their environments, often with significant impacts. They employ a variety of
enzymatic and chemical responses to complex changes in their habitat and the
terrestrial strata of the earth. Mycelial networks that support the fruiting
mushroom bodies extend from subsoil locations to rain forest canopies. Scientists
started using bacteria and fungi to degrade xenobiotic organic compounds in the
mid-twentieth century. Early successes in research favored bacterial applications
until more recently, when research with so-called “white rot” fungi began to show
more favorable results and increased activity in the area of fungal applications
(Aust 1995, Singh 2006).
In his report to the Conference on Biodegradation: Its Role in Reducing Toxicity
and Exposure to Environmental Contaminants (Aust, 1995), the author covers in
detail a variety of mechanisms employed by white rot fungi to complete the
degradation of lignin and a number of environmental pollutants: “The fungi
secrete a family of peroxidases to catalyze both direct and indirect oxidation of
chemicals. The peroxidases can also catalyze reductions using electron donors to
generate reductive radicals. A cell-surface membrane potential can also be used
to reduce chemicals such as TNT.” White rot fungi are well known for their
5
ability to biodegrade lignin in wood. They also mineralize (oxidize to CO2) some
chemicals that are already highly oxidized. There is some debate over whether the
peroxidases based system of the fungi is sufficient to completely degrade lignin,
and that more research is needed to develop a full understanding of the processes
involved. Several mechanisms, including membrane potentials, are discussed in
an attempt to explain the uniquely effective degradative and reduction properties
associated with the presence of white rot fungal species in soils and woody debris
sites. Aust’s report adds to a wide body of literature that supports the potential for
applied uses of specific fungal species in the treatment of toxic sites in most
terrestrial and near shore environments where these species are able to thrive and
gain an ecological niche as part of a broader bioremediation strategy. There is no
hint of a suggestion that the white rot fungus is a cure all or magic bullet for
applied science or technologies.
Not all scientific evidence points to the same result, and any application of fungal
species that is considered for real world situations must be carefully understood,
and species selection must consider the target result that planners expect to
achieve. The following study suggests that under certain conditions, the presence
of mycorrhizal fungi in the soil or substrate may serve in a detrimental fashion by
facilitating the uptake of heavy metals by plants that are infected by the fungi
(D’Annibale et al, 2006).
6
The authors were particularly concerned with providing data that would help
understand what happens to environments that receive widespread exposure to
contaminants in so called “down wind” conditions – where a manufacturing
process distributes toxins more or less randomly into surface soils, riparian zones,
and other creature habitats. Some good examples are the Hanford Nuclear Site in
south central Washington State, the ASARCO superfund smelter site on
Commencement Bay in the same state, and Bhopal India, where eleven acres of
highly contaminated material sits uncontrolled and aging in tin shacks following
the release of phosphocyanic gas that directly killed thousands of people. Twenty
– four years following the event there has been no direct action to assess the
impacts of the disaster, or follow up on secondary effects on the health of the
local population, or clean-up of the site.
Recent interest in the use of mycorrhizal enhancements that may be obtained
through mycoremediation techniques has generated several productive and long
needed studies that examine the exact mechanisms that are at work and how they
might best be employed by scientists and soils/wetland engineers. Killham and
Firestone (1983) investigated the hypothesis that since vesicular mycorrhizal
fungi can stimulate plant metal uptake in soils where the metals are barely
present, they might prove useful in reducing the presence of metals through
uptake in cases where the host plants are growing in soils containing potentially
toxic levels of heavy metals. The authors’ intent was to show the influence of
such fungal infections on heavy metal uptake and growth of a perennial grass in a
7
soil exposed to acidic and heavy metal depositions. This report generated the
following conclusions: The influence of mycorrhizal fungal species infection can
significantly impact the uptake of heavy metals under conditions of high relative
acidity, as is commonly found in smelter effluent conditions. Several factors that
play a role in this process are the specific condition and composition of the soil,
acidity and metal content. Bioavailability of pollutant heavy metals was found to
be more prevalent in sandy soils containing minimal amounts of clay and organic
components. The authors go on to suggest that the processes which result in
increased metal availability, like acidification, can also be expected to produce
less buffering in these soil types. This study identifies potentially adverse
consequences of fungal infection, and calls out the need for stringent guidelines
and more outcome-specific type research that will enable scientists to apply the
right tool to the job. Under conditions of acidic and heavy metal deposition,
mycorrhizal fungal infections can greatly enhance metal uptake and result in
reduced plant growth. This may or may not be a desired outcome, and should be
factored into any planning effort involving these or similar sets of conditions.
Current Ecological Concerns
In a recent report on the effects of pesticides on Pacific Salmon (Ewing 1999),
provides us with the results of a recent review of scientific literature on the effects
of sublethal concentrations of pesticides on salmonids. The report emphasizes
how pesticides can alter the biology of fishes in subtle ways that negatively
impact their chances for survival and reproduction. Pesticides are only one in a
8
variety of classes of toxins, including heavy metals and pharmaceutical byproducts that move into streams and rivers throughout watersheds and have the
potential for posing problems far from the site of application. One significant
problem is that these chemicals do not necessarily disappear over time. They are
transformed into other compounds that may be less toxic, equally toxic, or more
toxic than the original material. Fish and other biological organisms must suffer
the impacts of these toxins and cope as best they can with the results of their
presence, which may be renewed and increased by repeated, uncontrolled,
applications.
Through a process known as bioaccumulation, the concentration of these toxins in
plant and animal tissues may be several times higher than that contained in the
surrounding water or soils. This presents a significant environmental health risk
to subsistence communities whose members rely on these food sources for
survival throughout the year.
One well documented case (Ewing 1999) involved the release of the herbicide
acrolein, which subsequently was determined responsible for the deaths of
approximately 92,000 steelhead, 114 juvenile Coho Salmon, 19 resident Rainbow
Trout, and thousands of non-game fish in Bear Creek, Oregon, a tributary of the
Rogue River. Ewing’s report describes the results of studies which show that, in
contrast to immediate kill effects, many toxins have longer term behavioral
impacts on salmon. These effects include stress in juveniles which may make
9
them more susceptible to predation, effective swimming abilities which may
reduce the ability to feed, avoid predation, defend territories, and maintain
position in the river system. Many pesticides disrupt schooling behavior, a
common observation by scientists, and the ability to seek optimal water
temperatures during their upstream and downstream migrations.
Adult salmon are also known to adjust their migration patterns to avoid areas of
pollution, which may delay spawning, create additional stress in the animal, and
increase the energy consumption needs of the fish. Ewing (1999) makes a final
observation that immune systems of salmon may also be disrupted as the result of
exposure to toxic materials. One of the best documented cases of the immediate
lethal effects of excess quantities of herbicides occurred on the Rogue River,
Oregon in 1977 (Lorz et al, 1979). In this case, a large quantity of treated
irrigation water containing Magnicide H, a gaseous form of acrolein, was released
into the river within 24 hours of treatment instead of the recommended holding
time of 6 days. The Oregon Department of Fish and Wildlife estimates that a ten
mile section of river was affected, that 238,000 fish were killed, including 42,000
salmonids with an estimated value of $284,000. Acrolein is used to control
aquatic vegetation growth, and is highly toxic in its normal applied concentration.
The Washington State Department of Ecology published the results of a pilot
project conducted in the Snohomish River Basin (Ward, 1999). The purpose of
the project was to test a short list of regional salmon habitat indicators using
10
existing data from a pilot watershed located in Washington State. The Snohomish
basin was selected because large amounts of data had previously been collected
compared to other existing watersheds. A work group consisting of select state
agencies identified 15 indicators in 5 functional categories: Fish Abundance,
Water Quality, Water Quantity, Land Use/Cover and Physical Habitat. The study
group broke Water Quality down in to three categories, only one of which of
contained data considered “usable” for inclusion in this report: temperature.
Biological Water Quality was the second subcategory, Chemical Water Quality
was the third subcategory, and it looked at the “Percent of water rated excellent,
good, fair, poor - possible parameters would include temperature, dissolved
oxygen, biological assessment demand, pH, ammonia + nitrate, nitrogen, total
phosphorous, total suspended solids, and bacteria to produce a single number.”
Several plots were developed using existing data from prior reports issued by the
state, however, the authors concluded that due to the low number of assessed
waters, this indicator was not converted into a rating of any kind. In effect, the
Snohomish River Basin report completely overlooks any kind of meaningful
analysis of biological or chemical toxins on the immediate spawning habitat of
Pacific Northwest Salmon. This gigantic northwest watershed presents itself as
an ideal candidate for further studies in applied mycorrhizal mediation techniques,
since the natural terrain is ideal for mushroom habitat, and because a huge number
of returning adult salmon seek these headwaters for spawning. In my literature
research for this thesis, it was common to run across these types of government
reports, that seem to stop just short of making the next step toward taking or
11
recommending really common sense activities to help these natural habitats get
back to pre-human intervention conditions.
The Puget Sound Conservation and Recovery Plan (Office of the Governor, 20052007), was developed by the state legislature and approved by the governor of
the State of Washington to identify and focus on the goals, strategies, funding and
specific measurable results for protecting and conserving Puget Sound. The
following priority areas were identified along with the specific sub-tasks required
to support them and the identification of the responsible agencies.
Improve Water Quality in Hood Canal
Clean Up Contaminated Sites & Sediments
Conserve and Recover Orca, Salmon, Forage and Ground Fish
Prevent Nutrient and Pathogen Pollution Caused by Human and Animal
Wastes
Protect Shorelines and Other Critical Areas That Provide Important
Ecological Functions
Restore Degraded Nearshore and Freshwater Habitats
Reduce the Harm From Storm Water Runoff
Reduce Toxic Contamination and Prevent Future Contamination
The governor’s plan has the impact of providing opportunities for a broad
spectrum approach to managing natural sites, ecological niches, and
12
environmental management and sustainability. In spite of a nearly endless flurry
of scientific studies, discussions, and arguments around how to define or
characterize the natural world we live in, it seems fundamentally obvious that
each day more and more opportunities are presenting them selves for using
ecologically friendly, low cost and low impact methods for helping keep natural
worlds intact and flourishing at every level of biological processing. By
developing more effective methods and applications for introducing target fungal
species to conditions that favor their growth, the possibilities for
mycoremediation to become more prevalent in a variety of mediation and
restoration roles seems reasonable as an ecologically beneficial symbiot. If we
can plant a tree, why not plant a spore as well? As many native fungi have niches
all along the watersheds and food webs of this state and others around the world,
it is easy to envision a comprehensive study that defines the current state and
presence of all known species and their symbiots, and through careful cultivation
of those species arrive at some “optimal” or at least beneficial distribution goal
that meets and supports the condition of so called sustainable agriculture and
environmental management.
Restoration Ecology
Hammel, 1995, conducted laboratory experiments in an effort to isolate and
define the specific metabolic pathways employed by lignolytic fungi that cause
white-rot of wood and also degrade a wide variety of organopollutants. The
ability of these eukaryotes to cleave fused-ring aromatics was generally thought to
13
be impossible, or at least until now, unverified. “Recent lab results have shown
that extracellular peroxidases of these fungi are responsible for the initial
oxidation of PAH’s. Fungal lignin peroxidases oxidize certain PAH’s directly,
and fungal manganese peroxidases co-oxidize them indirectly during enzymemediated lipid peroxidation (Hammel, 1995).” The results of this study were
suggestive of the role played by white-rot fungi in these reactive processes but
work continues on a fully developed, conclusive mapping of these metabolic
processes. The researcher does concede that lignolytic fungi make attractive
candidates for use in low technology bioremediation programs. Although these
organisms are slow reactors, they occur naturally in soil litter and are highly
nonspecific in their action. We still know little about the specific mechanisms
used by white-rots for organopollutants oxidation.
In 1992, article 2 of the Convention on Biological Diversity (Zerbe & Kreyer,
March 2006) defined “biodiversity” as “the variability among living organisms
from all sources including, inter alia, terrestrial, marine and other aquatic
ecosystems, and the ecological complexes of which they are part; this includes
diversity within species, between species and of ecosystems.” Biodiversity has
recently become a common topic in the field of environmental studies and applied
sciences, specifically as it relates to land use, nature conservation, landscape
development and habitat or ecosystem restoration. Human presence and influence
in the environment has had effects that contribute both to the increase of
biodiversity and the decrease of biodiversity. Humans regularly introduce non-
14
native species into some areas, while they destroy species through the destruction
or degradation of their natural habitats in others.
“Ecological restoration is the process of assisting the recovery of an ecosystem
that has been degraded, damaged or destroyed. The goal of restoration is to create
a self supporting ecosystem that is resilient to perturbation without further
assistance” (Ruiz-Jean & Aide, 2005). The criteria for restoration success should
be clearly established to allow meaningful evaluations of restoration projects.
The Society of Ecological Restoration (2004) produced a primer that provides a
list of nine factors that should be used to clearly provide a measure of restoration
success. After evaluating 468 articles that met the study criteria for site
evaluation or restorative success, only 68 were found to have conducted follow-up
studies to evaluate restorative success (Ruiz-Jean & Aide, 2005). Of these 68
studies, none were found to address the full cross section of categories deemed
necessary by the SER criteria established in their 2004 report. They conclude that
ideally, all projects would follow the guidelines set forth in the primer. Cost and
budget were the most common limiting factors. The 9 factors presented were:
(1) similar diversity and community structure in comparison with reference sites;
(2) presence of indigenous species; (3) presence of functional groups necessary
for long-term stability; (4) capacity of the physical environment to sustain
reproducing populations; (5) normal functioning; (6) integration with the
landscape; (7) elimination of potential threats; (8) resilience to natural
disturbances; and (9) self-sustainability. Although measuring these attributes
15
could provide an excellent assessment of restoration success, few studies have the
financial resources to monitor all these attributes. Furthermore, estimates of many
attributes often require detailed long-term studies, but the monitoring phase of
most restoration projects rarely lasts for more than 5 years.
Rivers, streams and riparian zones have been subject to the degrading and toxic
effects of human activities and presence worldwide since the dawn of civilization.
Factors like floodplain inundation, groundwater recharge, and organic matter
exchange adversely affect structural and functional diversity in streams, and
reduce water quality for humans and other biological organisms that depend on
these environments for their success. Where restoration attempts have been
made, they are generally poorly documented, and often ignored, particularly when
projects fail, or reflect poorly upon local, state and federal government. Moerke
& Lamberti (2004), surveyed government, private and non-profit organizations in
the state of Indiana in an attempt to evaluate successes of their designs. Only 10
projects were evaluated and involved relocations, daylighting, and floodplain
reconnection projects. Most involved aesthetic type goals, and were vaguely
defined. The importance of in-stream aquatic biota was considered in only 50%
of the projects.
Vorass and Portele (2001), submitted their report to the State of Washington on
contaminated sites and the Endangered Species Act (ESA), pointing out that the
16
state’s regulatory criteria for prioritizing contaminated sites cleanups are based
largely on health risks to humans. Their report to the Department of
Transportation (DOT) was designed to provide an internal assessment tool for the
DOT to assess and prioritize contaminated site risks to the Endangered Species
Act listed aquatic species. The scientists came up with a three tiered evaluation
process to determine relative potential for contaminated sites to affect ESA –
listed fish species. Tier I evaluated sites for their relative distance to the
documented presence of listed fish and their critical habitat. Tier II evaluated the
status of hazardous material releases and the potential for contaminants to impact
surface water and critical habitat areas. Tier III assigned a quantitative site
scoring to rank sites based on risk to ESA – listed fish species. Scores range
between 0 – 100 with higher values assigned to higher risk sites. Out of 103
evaluated sites, 41 were considered to pose potential risk and received
quantitative scores. DOT will seek funding for cleanup of the 16 sites receiving
scores of 75 or more. The remaining sites will receive further assessment to
determine if additional cleanup efforts are warranted. The WSDOT plans to use
this model for future site evaluation and characterization activities, and whether or
not individual sites should become candidates for restoration.
Vorass and Portele (2001) conclude that although there remains some uncertainty
in the area of relative toxicity of individual contaminant levels on fish species, the
model is considered useful as an overall tool for the regulated community and
agencies in establishing supportable cleanup decisions for any controlled site in
17
Washington. Restoration ecology and sciences, including bioremediation and
mycoremediation, provide ideally suited, bio–friendly applications for the kind of
sites that DOT typically encounters. Along the Pacific Coast of North America,
literally from California to Alaska, native fungal species thrive in moist, low light
riparian zones that double as habitat for a variety of marine species, including
migratory salmon, shellfish, etc. The entire web of life becomes candidate for the
naturally enzymatic processes that many plant species employ, particularly as
alternatives to dredging, and other more disruptive methods. Even at the
beginning of new, emerging research on fungal life forms, the use of native life
forms presents a comforting sort of presence and consequence that future research
and practice may eventually illuminate ways for the fungi to be effectively used in
bioremediation, and practical and commercially viable ways.
D’Annibale et al, 2006, investigated the role of autochtonous filamentous fungi in
the bioremediation of sites historically contaminated with aromatic hydrocarbons.
The study looked at nine strains of fungi isolated from a heavily contaminated and
aging soil to assess their potential for degradation. Among these strains were
Allescheriella sp. Strain DABAC 1, Stachybotrys sp. Strain DABAC 3, and
Phlebia sp. Strain DABAC 9 – all were selected for remediation trials and tested
for their abilities to grow under nonsterile conditions and to degrade various
aromatic hydrocarbons in the same contaminated soil. After 30 days, fungal
colonization was obvious to the naked eye and confirmed by ergosterol
determination. Although pH conditions were below normal alkalinity levels, and
18
heavy metals were present in much higher than normal quantities, the fungi still
produced laccase and Mn and lignin peroxidases. All of the selected isolates
resulted in marked removal of naphthalene, dichloroaniline isomers, 0hydrosybiphenyl, and 1,1’-binaphthalene. Overall, these autochthonous fungi led
to a significant decrease in soil toxicity, as assessed by both the Lepidiam sativum
L. germination test and the Collembola mortality test. The study also suggests
that biological treatment technologies for the remediation of soils, groundwater,
and riparian zones are becoming widely known for their ability treat toxic sites
and remain environmentally friendly relative to secondary impacts due to their
presence, whether they are introduced by soils managers, or occur naturally as
part of the plants own spore dispersion mechanisms (D’Annibale et al, 2006):
“Sites contaminated by recalcitrant organic compounds have often been shown to
be characterized by the concomitant presence or heavy metals. In such a difficult
case, the use of a filamentous fungus (white rot fungi, in particular) may give
some advantages over bacterial bioaugmentation. Fungi display a high quality to
immobilize toxic metals by either insoluble metal oxalate formation, biosorption,
or chelation onto melanin like polymers. Moreover, due to the low substrate
specificity of their degradative enzyme machinery (e.g. laccase, lignin
peroxidases, and Mn peroxidases), fungi are able to perform the breakdown of a
wide range of organopollutants in contaminated soils.” (D’Annibale et al, 2006).
Researchers are discovering that lignolytic peroxidases production is no longer
the unique prerogative of white rot basidiomycetes (Field et al, 1993). More and
19
more studies are reporting the presence of these enzymes in other fungal
taxonomic groups, the authors report (Ayed et al, 2004).
Many prior studies in mycoremediation have been performed on artificially
contaminated soils in more or less sterile conditions, suggesting the importance of
investigating the use of fungal remediation in soil from real sites, helping to make
them more transferrable to field scale studies and applications (Pointing, 2001).
In D’ Annibale et al, 2006, the authors study the aged and contaminated soil from
a large, decommissioned chemical industrial site called ACNA, in Savonna Italy.
Large scale production of a variety of industrial chemicals had gone on here for
over 100 years, including the production of many organic chemicals. It was
officially shut down in 1994 and is characterized by the presence of aromatic
hydrocarbons, including chlorinated benzenes and anilines, thiophenes and
polyaromatic hydrocarbons and heavy metals. The authors of this research paper
engaged in a joint study project known as the Sisifo Project, which used various
methods to test remediation strategies and methods that might prove effective at
this specific location. One result of this effort that led D’Annibale and her
colleagues on their quest, was the failure of several indigenous bacterial
prokaryotes who were reported to be highly specialized in the catabolism of
several aromatic compounds. This program failure led the researchers to pursue
special interest in the assessment of indigenous fungi from the site, followed by
their reinoculation as a technically feasible and promising approach to
bioaugmentation. In their own words “The aim of the present work was to screen
20
for the aromatic hydrocarbon degrading potential of fungal strains isolated from
the ACNA soil and to assess the possible use of such selected autochthonous
fungi in an ex situ soil biotreatment via bioaugmentation.“ In this study, all of
the selected isolates were able to colonize the amended ACNA soil under
nonsterile conditions which suggests they have tolerance to high concentrations of
toxic contaminants while at the same time demonstrating their ability to compete
with the indigenous bacterial microflora. The final conclusion of the authors is
that “…this study confirms that the isolation of fungi from a contaminated soil
followed by their reinoculation at the same site can be a valuable remediation
strategy (D’Annibale et al, 2006).” Scientists and other environmental planners
do, however, need to pay attention to many influencing factors in their selection,
or mix, of bioremediation strategies.
About the Fungi and Factors Relevant to Their Selection and Application in
Bioremediation Strategies
In order for mycorrhizal bioremediation techniques to be applied successfully in
the treatment of xenobiotic contaminated soils, a number of factors must be
considered early in the planning stages. These include the identification and
implementation of physiochemical and nutritional conditions that favor the
growth and xenobiotic degradative behaviors of indigenous or inoculated
xenobiotic degrading microbes. One commonly applied bioremediation technique
is biostimulation, which involves the selection of microbes based primarily on
21
their xenobiotic-degrading abilities, using indigenous organisms that are already
resident at the site. Another technique is bioaugmentation, which involves the use
of organisms with superior pollutant degrading qualities and involves inoculating
a contaminated site with organisms from other sites or spawn locations. By
selecting attributes such as higher growth rates, competitive ecological strategies,
tolerances to higher contaminant concentrations, specific nutritional capabilities,
and pH or temperature growth optima, it may become possible to obtain dominant
colonization and remediation outcomes or successes (Lamar et al, 1999).
Extensive laboratory study of a group of wood decay basidiomycetes, collectively
called “white rot fungi” has shown their unique ability to degrade or assist in the
degradation of a wide variety of contaminants and toxic compounds. This ability
makes them ideal candidates for their applied use in contaminated soils,
particularly those contaminated with complex mixtures of hazardous chemicals.
A number of field studies using the bioaugmentation approach have demonstrated
the effectiveness of using fungal treatment of pentachlorophenol (PCP)
contaminated soils (Lamar, 1999). Soils contaminated with both PCP and
creosote, which tends to have high concentrations of polyaromatic hydrocarbons
(PAH’s) have also been successfully treated. Most of the work on pollutant
degradation and soil remediation by white rot fungi has focused on only a few
species, predominantly Phanerochaete chrysosporium (Singh, 2006).
22
Lamar et al. (1999) concluded that identification of the most effective fungus for a
particular set of contaminant-soil conditions are critical to the successful
application of fungal bioaugmentation. Fungi would be selected based on their
biochemical, physiological, and ecological attributes that offer them superior
performance potential under a given set of contaminated media or soil/substrate
conditions.
In their 1996 report, Lestan and Lamar investigated the development of fungal
inocula for bioaugmentation of contaminated soils by inoculating soils
contaminated with hazardous organic compounds. Their study used pelleted solid
substrates coated with a sodium alginate suspension of fungal spores or mycelial
fragments that were incubated until overgrown with the mycelium of selected
lignin-degrading fungi. Evaluations were conducted using Phanerochaete
chrysosporium, P. sordida, Irpex lacteus, Bjerkandera adusta, and Trametes
versicolor. These inocula were selected because they resisted competition and
proliferation from indigenous soil microbes, were lower in moisture content than
current fungal inocula, and possessed enough mechanical strength to allow
handling without a significant change in the mechanical consistency of the pellets.
Following inoculation at a rate of 3% in artificially contaminated nonsterile soils,
I. lacteus, B. adusta, and T. versicolor removed 86, 82, and 90% respectively of
the pentachlorophenol in 4 weeks.
23
Lestan and Lamar, 1996, claim “…the salient feature which makes lignindegrading basidiomycetes attractive as potential microbial agents is their ability to
degrade a wide variety of hazardous compounds.” Many of the obstacles or
impediments that face many users of organic compounds and plant materials face
scientists and technicians involved in the development, production, inoculum
formation, transport, delivery, and application to the soil or site substrate. Getting
the goods to market in a viable condition presents a host of challenges. The
authors cite the presence of extensive literature on the effectiveness and suitability
of different types of carriers of fungal inocula for biological control, fungal
spawn, introduction of mycorrhizal fungi, and bioaugmentation. Most current
methods for delivery involve the use of wheat straw, corn cobs, wood chips and
commercial mushroom spawn that are completely grown through with the
mycelium of the selected fungi. Selection of the proper substrate is important to
ensure sufficient nutrient reserves to support the colonization of the target
contaminated soil or distribution site. Problems that face commercial and
scientific applications of fungi include low inoculum potential, requiring large
quantities by weight of the initial spawn material, and “flashing.” Despite modern
refrigeration techniques, this phenomenon occurs when metabolic temperatures
reach high enough levels, that they may kill the living organism or radically slow
the growth process. Destruction of the fungal biomass also occurs as a result of
this type of temperature rise. Lestan and Lamar 1996, focused on the
effectiveness of inocula in removing pentachlorophenol [PCP], and suggest that
the development of low cost methods of engineering and sustaining the health of
24
the fungal inocula during transport is key to the success of this emerging science
as a practical method of bioaugmentation of soils and other target sites, like
riparian zones.
Lestan and Lamar conclude their 1996 study with the following recommendations
for overcoming some of the logistical and distribution problems that
manufacturers and others interested in scientific application of mycoremediation
techniques. One popular method that is currently used by mushroom growers is
simply to “bulk up” pure cultures using lots and lots of substrate material or
agricultural waste, like corn cobs. These methods tend to be labor intensive, and
the quality of the inoculum produced is often variable and of questionable quality
or consistency. One way of overcoming this problem, and others, is the use of
pelleted fungal inocula: a pellet core, usually made of wooden dowels or plugs,
each of which contains a carrier, nutrient source, binder and some type of
lubricant. This plug is then encapsulated by a layer of mature fungal mycelia.
This technique produces a highly controllable product, as each pellet has a
specific ratio between the amount of fungus and the substrate for growth and
activity.
This allows for easily predicting the amount of growth and pollutant degrading
activity from each inoculum unit [pellet] and provides a direct measure for quality
control and “batch” evaluations. Another measure that factors in the importance
of the transport and distribution of fungal inocula is mechanical strength, which
25
aids in the collection, packing and final application processes. This is especially
critical in soil applications where the rubbing frictions and grinding action of the
soil may break down and reduce the inoculum potential. When the external
coating of mycelium is broken down mechanically, it allows for entry by
competing organisms into the core nutrient and carrier elements of the individual
pellets. This introduces a competitive advantage to local fungi and bacteria that
may significantly reduce or overcome the intended beneficial effects of the
introduced species.
Lestan and Lamar, 1996, conclude that lower moisture content will signigicantly
reduce the costs associated with applied mycoremediation strategies by several
avenues. This not only reduces overall mass and weight of the shipped product,
but can also significantly reduce the volume, and provide enhanced storage
characteristics of the inocula or spawn carrier. Pelleted inoculum was reduced
approximately 50% in volume over the original mixture. On the side of weight or
mass, the report goes on to suggest that a current inoculum formulation using
nutrient-fortified grain-sawdust mix applied at a rate of 10% to a site with
100,000 cubic meters of contaminated soil would require 25,000 metric tons wet
weight of inoculum, at 60% moisture. In this study, the average moisture content
of pelleted inocula varied from 20.7% for an inoculum with I. lacteus to 35.5%
for an inoculum with T. versicolor, about half that present in the fortified grainsawdust mixture.
26
D’Annibale, et al (2006), observed that sites contaminated with persistent organic
compounds are often characterized by the presence of heavy metals. In these
cases, filamentous fungi may have specific advantages over bacterial
bioaugmentation. Fungi display a high ability to immobilize toxic metals by
either insoluble metal oxalate formation, biosorption, or chelation onto melaninlike polymers (D’Annibale, et al 2006). They are also able to break down a wide
range of organopollutants in contaminated soils due to the low substrate
specificity of their degradative enzyme machinery. The investigators in this study
(D’Annibale et al., 2006) attempt to demonstrate the use of fungal remediation
under non-sterile conditions and with soils from real contaminated sites, making
the results potentially transferable on a field scale.
D’Annibale et al. (2006) reviewed and assessed the feasibility of ex situ
bioaugmentation with allochthonous fungi on aged, contaminated soil. The site is
a former industrial area where a wide array of organic chemicals had been
deposited for over 100 years. They included aromatic hydrocarbons, chlorinated
benzenes and anilines, thiophenes, polyaromatic hydrocarbons and heavy metals.
Previous coordinated project attempts to show the success of using indigenous
prokaryotic bacteria specialized in catabolism proved unsuccessful (Duran and
Esposito, 2000). The failures prompted the authors of this study to assess the
eventual presence of yeasts and fungal microbiota adapted to the historical
contaminants of this site for use in its remediation. The aim of this work was to
screen for the aromatic hydrocarbon degrading potential of fungal strains isolated
27
from the site and to assess the possible use of such selected autochthonous fungi
in an ex situ soil biotreatment via bioaugmentation. After an extensive series of
biochemical tests and statistical analyses, the authors confirmed that the isolation
of fungi from a contaminated soil followed by their reinoculation at the same site
can be a valuable remediation strategy.
The use of lignin degrading basidiomycetes for remediation of soils contaminated
with hazardous organic compounds has been studied a great deal (Davis et al,
1993). What makes these organisms attractive is their ability to degrade a wide
variety of compounds (Lamar et al, 1992). In spite of this knowledge, the
development and use of fungal bioaugmentation or remediation on an industrial or
wide-spread scale has been inhibited by inconsistent treatments in the field.
There are many known carriers of fungal inocula that have been demonstrated to
be effective in laboratory and field studies: peat, granular vermiculite, grains,
alginate pellets, wood chips, straw, corn cobs, etc. Problems that have been
identified with large scale production, maintenance and delivery include, cost,
inconsistent quality, drying, flashing (production of excess metabolic heat), and
destruction during the application process.
Zerbe and Kreyer (2006) in their prelude introduction to the fourteenth issue of
The Journal of Society for Ecological Restoration International, point out that the
term “biodiversity” should read more or less as follows, by way of arriving at
some standard and clear definition of the concept: “The variability among living
28
organisms from all sources including, inter alia, terrestrial, marine and other
aquatic ecosystems, and the ecological complexes of which they are part, this
includes diversity within species, between species and of ecosystems.” As an
overall topic then, biodiversity is becoming more important in biological and
ecological research aimed at practical applications like land use, conservation of
natural environments, development and restoration.
About the Toxins
A significant portion of the detailed supporting activities identified in this
literature review include things like cleanup of mercury, poloybrominated
biphenyl ether, (PDBE or flame retardants), and other chemical toxins, oil spill
management, storm water runoff, contaminated sites and sediments, sewage
management, nutrient and pathogen pollution caused by human and animal
wastes. Mycoremediation techniques both in and outside the laboratory have
shown promise as applications for mediation of all these types of impact factors
throughout the world.
In 1994 the Washington State Pesticide Monitoring Report (Davis & Johnson
1994) issued the results of their study of 24 pesticides and breakdown products
detected in fish tissue, and nine compounds detected in sediment samples
collected from seven freshwater sites in 1992. Pesticides detected in high
29
concentrations included forms of DDE, DDT, dieldrin, and heptachlor epoxide.
DDT and metabolites chlordane, and two PCB’s were detected at all sample sites.
High concentrations of DDE were found in whole large scale suckers from the
Yakima River and Kokanee fillets and eggs from Lake Chelan. Lipid normalized
values indicate that the bioavailability of DDT and metabolites to fish in the
Walla Walla River is higher than the other sites tested. EPA human health
screening values, used only to prioritize problem areas, were exceeded for DDT in
fillet samples from Lake Chelan, Crab Creek, and the Yakima River. Screening
values for total PCB’s were exceeded in samples from Lake Chelan, Crab Creek,
Yakima River, and Mercer Slough. Dieldrin in fillet samples from the Yakima
River and heptachlor epoxide from the Walla Walla River were also above
screening values. Sites with high fillet samples exceeding the National Toxics
Rule (NTR) criteria were recommended for addition to the 303(d) water quality
limited list.
Comparisons with historical data indicate that pesticide concentration in fish
tissue have changed little. DDE in samples from the Yakima and Walla Walla
Walla Rivers continue to be above proposed wildlife criteria. Concentrations of
DDE in fish from the Walla Walla and Yakima Rivers, and chlordane in the
Yakima and Mercer Slough are ranked high as compared to national averages.
Pesticide levels in sediment from the Yakima River appear to be decreasing as
compared to samples collected since 1984.
30
Washington State issued its Sediment Cleanup Status Report in June, 2005. The
report summarizes all known or suspected sediment cleanup sites in Washington
State. Sediment is defined by the report as “particles of dirt that are discharged or
washed into and travel through water systems to precipitate out onto sea floors
and lake and river beds. Many contaminants adhere to these solid particles on
their journey so that sediments, by nature, concentrate contamination in the areas
where they collect. It is that muck that squishes up between your toes when you
run out into a lake to go swimming…” The contamination found in many types
of sediment results from the release of harmful contaminants into the aquatic
environment from industrial sources, municipal discharges, commercial activities
and accidents. This report estimates that between 2,940 and 3,340 sites contain
contaminated marine sediment. Several challenges facing cleanup or remediation
are cited by the report: Disposal Capacity and Cost, Uncontrolled Sources,
Reluctant Liable Parties, Resources, State-Owned Aquatic Land Management
Policy, Regulatory Uncertainties, Potential Superfund Listing, Area-wide
Contamination. This document is extensive in its listing of contaminated
sediments sites, but contains little data on what is being done and how to correct
or mediate the presence of these substances in our environment. It is primarily a
database or catalogue of sites and agency links that may or may not reflect
accountability and responsibility for making measurable progress in achieving
results in the actual cleaning or remediation of these areas.
31
Ruckleshaus and McClure (2007), in their ecosystem-scale study of Puget Sound,
conclude the following with respect to toxic contaminants: “Contaminants –
toxins, nutrients, pharmaceuticals and pathogens – entering Puget Sound
accumulate in sediments, marine waters and organisms and negatively impact
biological populations, ecosystem integrity, harvest availability, and human
health.” Toxins introduced in Puget Sound are appearing in high level
concentration in upper level predators like salmon, seals and orcas in spite of
having been banned decades ago. The number of highly contaminated acres has
come down due to clean up efforts, but some contaminants, like PCB’s are
declining slowly, if at all, and PAB’s, polycyclic aromatic hydrocarbons have
increased in long term sediment monitoring stations in Puget Sound. The authors
conclude that reduction of inputs of toxic compounds to the area will benefit
ecosystem and human health in general. Remediation and restoration activities
designed to clean up legacy locations should be applied to help reduce the amount
of toxins that move back into the food web.
An exploratory report issued by the Washington State Department of Ecology
(Seiders et al, 2003) in support off the Washington State Toxics Monitoring
Program, investigated the occurrence and concentration of toxic contaminants in
edible fish tissue and surface waters from freshwater environments in the state.
The program was started in 2001 as a result of increased public and scientific
concerns over the dramatic increase in contaminants in the environment of Puget
Sound. Twenty five samples of fish from eight species were collected from ten
32
separate sites. Contaminants found included mercury, PCB’s, dioxins and furans,
flame retardants (PBDE’s) and chlorinated pesticides like DDT and its
metabolites, chlordane compounds, dieldrin, aldrin, Beta-BHC, chlorpyrifos,
endosulfan sulfate, heptachlor epoxide, hexachlorobenzene, lindane, and mirex.
Tissue samples from eight of the ten sites exceeded National Toxics Rule (NTR)
for the protection of human health. Water samples from ten sites were found to
include six pesticides at low levels and frequencies, including bromacil,
dichlobenil, atrazine, diuron, hexazinone, and terbacil. The report recommended
continued evaluation and monitoring of potential health risks to human health
from consumptions of contaminated fish and the addition of eight sites to
Washington’s 303(d) list. The report suggests that, for many areas of Washington
State, information is not available that shows levels of toxic contamination in
freshwater fish and surface water, as they are both present in riparian zones
throughout the state. These chemicals can be extremely persistent, as they not
easily broken down at the biochemical and molecular levels, resulting in the
presence and accumulation in the environment for decades. This environmental
presence results in biomagnifications and bioaccumulation in organisms that
engage in the food chains, or web of life, thereby expanding and distributing the
impact of their presence.
The harmful effects are carried away from the point source of the pollutant and
find their way into the biochemistry and metabolic pathways of both higher and
lower order life forms. Some of the problems that result on various wildlife and
33
humans involve behavior, neurological, and reproductive abnormalities. The
report cites Washington State Department of Health statistics that list 16 site
specific consumption advisories for finfish and shellfish in Washington due to
contamination by mercury, PCB’s, chlorinated pesticides, and other metals and
inorganic chemicals. The state toxics monitoring program is seen as one method
of relieving an absence of meaningful monitoring and science that is directed at
targeting and managing existing toxins in our environment. Naturally derived
techniques like bioremediation and mycoremediation offer themselves as viable
alternatives, at low cost and low impact, for planning agencies to use when
devising or recommending methods for reducing or eliminating the presence of
these harmful toxins.
Past monitoring efforts in Washington have detected toxic contaminants in
surface water, sediment, and aquatic animal tissues. Efforts to monitor and
manage toxic chemicals in freshwater fish tissue, sediments and water have
unfortunately declined over the last decade due to budget cuts. In 2000, renewed
concerns resulted in the establishment of the Washington State Toxics Monitoring
Program. Among their stated goals are: exploratory monitoring of new instances
and locations of contaminants in freshwater environments; establishment of
communications methods to disseminate date to citizens and resource managers;
trend monitoring; and cooperation with other like groups and agencies.
34
Fungal Metabolism of Polycyclic Aromatic Hydrocarbons
Mycoremediation lends itself readily to the cleanup of many toxins, in particular,
a group known as PAH’s or polycyclic aromatic hydrocarbons. These pollutants
have been detected and observed in many terrestrial and aquatic ecosystems,
including riparian zones. PAH’s find their way into our environment from a
number of sources: incomplete combustion of fossil fuels, shale oil and cigarette
smoke, discharge of petroleum and coal gasification and liquefaction, incineration
of wastes and agricultural and forest residues. The molecular constituents adhere
to suspended particles and are transported into soils, siltation and sediments of
river systems and estuarine zones (Singh 2006). Because they are hydrophobic in
nature, and possess low vapor pressures, PAH’s have a strong affinity for
absorption and accumulation in sediments. They are also very stable
thermodynamically, due to strong negative resonance energy. The half-lives of
the heavier rings are on the order of years in most ecosystems. All these factors
contribute to the persistence and threat posed by these compounds to all living
organisms. PAH’s exhibit toxic, mutagenic, tumorigenic, and carcinogenic
properties. Recent studies have shown that metabolic activation of PAH’s to
electrophilic species form covalent bonding with nucleophilic groups of DNA,
and result in mutations. This poses a significant risk to humans (Singh 2006).
In “The Fungus Among Us” the authors, Aust and Benson, 1993, examine the
potential for practical applications of scientific knowledge that is emerging
throughout the field of toxicology and microbiology from an economic
35
perspective, with some brief background on the biochemistry behind this
economic. The authors argue that with estimates for hazardous waste remediation
and superfund site cleanup ranging from between $0.5 and $1 trillion, that the
application of naturally occurring and native species with bio-degradative
qualities is not only eco-friendly, but cost effective also on a relative scale. One
advantage the authors point out for white rot organisms is that they tend to be
“…nonselective in degrading all the chemical components of complex mixtures.
For example, all the components of Aroclors (PCB), toxaphene, creosote and coal
tars are degraded by fungi. In some cases this is related to the nonspecific nature
of the peroxidases secreted by the fungi, and in other cases it is related to the
variety of mechanisms that the fungi use to degrade chemicals.” The authors
conclude their argument by pointing out that fungi can be grown on very
inexpensive agricultural and forest wastes like sawdust and corn cobs, garbage in
the minds of most people, but with high potential as recyclable carbon materials.
In this case, they would serve as the food source for sustaining, transporting, and
inoculating cultures in select applications. And, relatively cost effective mass
production techniques have already been developed for commercial spawn runs
like common button mushrooms, and other culinary/medicinal varieties. One of
the great advantages of using native species in applied ecological remediation
solutions is they produce many of the secondary and tertiary elements needed for
beneficial symbiots to thrive as part of their natural growth and development
processes.
36
One of the challenges facing successful applications of mycoremediation
techniques in the field is the identification and selection of those organisms that
possess the physiochemical and nutritional needs that will provide optimum
growth and xenobiotic-degrading activities of the target toxin or contaminated soil
site. In addition to selecting organisms with superior pollutant degrading
qualities, the practitioner should also allow for selection of microbes bases on
additional physical, biochemical and ecological characteristics that will lend
themselves to high levels of performance. Simply stated, the preferred approach
is to match the right fungal species with the right toxin, to achieve the desired
result (Lamar, et al 1999). Some things to consider are superior growth rates,
competitive ecological strategies, tolerances to high contaminant concentrations,
specific nutritional capabilities or preferences, pH and temperature optima that
lead to dominant colonization and finally the remediation of the contaminated soil
volume by the introduced or inoculated organism. Recent laboratory studies of
certain wood decay basidiomycetes, commonly referred to as “white-rot” fungi,
have clearly shown that they possess the ability to degrade a wide variety of
contaminants. Most of the work on pollutant degradation and soil remediation
has focused on only a few fungal species (Lamar, et al 1999).
As more and more research is done on the effectiveness of using
mycoremediation as a practical application for removing toxins from
contaminated soil sites and other ecological systems, including riparian zones and
urban or industrial sites contaminated with process wastes, scientists are
37
recognizing the importance of choosing the most effective fungus for a particular
set of conditions. In order to facilitate the optimum growth and xenobiotic
degrading activities of the bioaugmentation, fungi should be selected on the basis
of the “best match” for the site, according to some contaminant remediation
profile. Again, pick the right fungus for the job that lies before you. These
treatability studies can be quite extensive and laborious. For example, tolerance
to contaminant and hyphal extension rate did not prove useful when attempting to
predict the contaminant degrading capability of a fungus for degrading PAH’s in
wood. The most effective fungi for degrading PAH’s in wood turn out to be the
brown-rot basidiomycetes, G. trabeum, and the dueteromycete S. circinatum,
especially for the lower molecular weight toxins. These organisms both exhibit
low hyphal extension rates and are sensitive to creosote. So, it appears that basic
measures of contaminant degrading capability or tolerance to a pollutant can not
alone provide enough information for selecting fungi for use in bioaugmentation
of contaminated soils.
More information on the bioremediation performance of well characterized fungi
on soils that represent a range of soil physical, chemical, and biological
characteristics and pollutants is needed to build a database of information that can
be used to select superior fungal strains to evaluate for application to specific
contaminant-media conditions (Lamar, et al 1999). Forthcoming studies
concerning fungal contaminant degradation in complex media should include in
their design the additional purpose of adding information to such a database.
38
Alternatives to Mycoremediation
How does the state’s primary cleanup agency currently define the known world of
sediment cleanup sites? In their 2005 report, DOE identified 142 specific areas
with enough reliable data to consider them cleanup sites. Of these sites, 115 are
in or near Puget Sound, and 27 are located in freshwater environments. There is a
renewed emphasis on assessing these freshwater “riparian” environments as more
is learned and understood about the accumulation and distribution of toxins
throughout the biosphere. These sites should be considered ideal candidates for
mycoremediation techniques and applications, based on their mutually supportive
habitats for both terrestrial and aquatic fungi, as well as fish species and other
wild organisms that support the carbon chain.
What are some of the methods currently employed by the State of Washington to
cleanup, restore, or mediate sites that have been identified as or meet the
established criteria for cleanup? In their 2005 report, the Washington State
Department of Ecology proposed to answer this question. From a strictly
regulatory perspective based on agency responsibility, the following picture
emerges. Sediment cleanup is primarily under the authority of either federal
Comprehensive Environmental Response Compensation Liability Act [CERCLA]
as controlled by the U.S. Environmental Protection Agency [EPA] “Superfund”
program or via state cleanup laws and rules. The state authorities are the Model
39
Toxics Control Act cleanup regulation, Chapter 173-340 WAC, and the Sediment
Management Standards, Chapter 173-204 WAC.
Challenges
Many of the challenges that are faced by planners and policy makers when
addressing cleanup, restoration, and mediation of marine, freshwater, and
terrestrial environments may be largely overcome someday, or at least to some
beneficial level, by employing mycoremediation techniques. Some of these
obstacles and outcomes are presented below, along with their advantages and
disadvantages. One method that is often used to mitigate the effects of toxins and
other contaminants that appear as the result of human activities is simply to
remove the contaminated material from the site. Once removed, however, an
alternate disposal site must either be selected, or some other method of reducing
the compounds to a non toxic state must be employed. One solution has been to
burn the subject material, which causes a host of secondary problems of its own.
Often the toxins are not destroyed, but simply redistributed as terrestrial and
atmospheric contaminants which may become distributed on a global scale due to
naturally occurring atmospheric and other distribution effects. One of the obvious
advantages of mycoremediation is that the naturally occurring enzymatic
processes employed by certain fungi actually alter or reduce the offending toxins
to benign biochemical compounds and elements that can simply be left in place,
overcoming the need to dredge, remove or otherwise dispose of and manage the
40
original offending compound. This was one of the benefits noted when the
common Oyster Mushroom was used to reduce and breakdown PAH’s that were
introduced into the marine waters of the Pacific Ocean following the Exxon –
Valdez oil spill. The hydrocarbons were actually broken down to into harmless
carbon constituents at rates approaching 75 – 85 percent (Bonaventura & Johnson,
1997).
The importance of selecting an appropriate application method for the problem at
hand is reported on by Sasek, et al (2006) in their comparison of two
biodegradation methods using composting under controlled conditions and
treatment with ligninolytic fungi. The target chemical was synthetic polymers
sourced from used beverage bottles. Two types of copolymers were tested:
polyester-amide and aromatic-aliphatic. Synthetic polymers have the
characteristic of being resistant to microbial attack, and tend to persist as
contaminants in the environment wherever and however they are introduced. The
experiment was run using standard raw materials under controlled conditions to
help eliminate the introduction of confounding factors that are common in field
studies, where control of contaminants and other xenobiotic organisms is difficult.
Ligninolytic fungi were selected for three reasons: they are the only organisms
capable of efficient degradation of lignin, the most resistant biopolymer in the
environment; they have shown the ability to decompose many human derived
organopollutants and; the ability of several fungal strains to attack some resistant
synthetic polymers is documented (Sasek et al, 2006). Composting proved to be
41
the most effective “reducer” in all measures but one in its effect on reducing
molar mass of the original sample. One explanation offered for this result is the
short duration of the inoculation period for the fungi, at 32 days and room
temperature.
Bioaccumulation has been observed as a bioremediation process where living
mycelium and fruiting mushroom bodies take up heavy metals into their flesh to a
considerably higher level than other agricultural crop plants (Oghenekaro et al,
2008). Heavy metals are natural components of the earth and occur, may in fact
be necessary, at trace levels in many higher order organisms, including humans.
They accumulate through the food chain, and may concentrate at high levels that
pose a risk to the proper functioning of cellular and metabolic processes. The
results of their study showed that when heavy metals are present, they influence
the growth of white rot fungi and the subsequent release of enzymes that
biodegrade xenobiotics. In their discussion, the authors conclude that P.
tuberregium in particular, has the ability to accumulate heavy metals, making it a
primary candidate for mycoremediation in polluted environments. Of particular
interest might be follow on processes that can be applied to harvest and deal with
the now highly toxified fruiting bodies.
In their report to the Washington State Department of Transportation, Thomas et
al (1998) provided a comprehensive analysis of a mycoremediation project
conducted in Bellingham Washington. The intent was to examine and contrast
42
the effectiveness of three different biological approaches: mycoremediation,
bioremediation, and enhanced bacterial remediation. The test site consisted of
three excavated, aged, oil-contaminated soils stored at an open WSDOT
maintenance yard in Bellingham Washington. Surprisingly, the results were not
conclusive in distinguishing among the various treatments, as none met the
prescribed criteria for success: attainment of the Method A Cleanup Level of
total petroleum hydrocarbons (TPH) prescribed by the Washington State
Department of Ecology (WSDOE) in Washington Administrative Code (WAC)
173-340 (WAC 1996) and Ecology Publication No. ECY-97-600 (WSDOE 1997)
within the time period prescribed. The authors suggest, however, that the study
program was valuable in understanding the variables involved in moving from a
controlled mesocosm environment to large scale applications of bioremediation
technologies and processes. The inconclusive nature of the results is likely the
result of the vast heterogeneity of the test soils and the weathered condition of the
petroleum hydrocarbons in the soil.
Uncontrolled Sources or Recontamination from Ongoing Efflux of
Contaminants
This potential and very real problem is faced regardless of the mediation
technique that is employed, and should be managed more from a policy and
regulatory perspective. The idea here is that it makes little sense to engage in
costly and complex methods for mitigating contamination of a site when it is
known that some pollution source will continue to introduce contaminants that
43
will require more action in the future. In these cases, it makes logical sense of
course to control the efflux prior to placing money into the cleanup effort. Even
in this type of situation, however, there can be specific advantages to employing
naturally occurring fungal species to a site while the lengthy machineries of
government and policy making grind slowly to some permanent solution.
The introduction of a toxin specific fungal species can provide benefit simply by
being allowed to run its natural bio-enzymatic course following an initial spawn
introduction. As the mycelia spread, and the plant flourishes, the “mediation”
advantage could be simply keeping the accumulation at bay, or at some reduced
level, as politics runs its course. This can involve several years, or even decades
as we all know, during which time the mushroom in its vegetative state quietly
grows beneath our feet and alters or breaks down the harmful compounds. In
theory, at some future point, more final and comprehensive plans can be
implemented. This could be thought of as a tactical response to a known threat
that costs very little to get started.
Reluctant Liable Party
In cases where there is reluctance by the liable party in a clean up or mediation
event, time can be a factor. Negotiations are often stalled by an entity that does
not see any advantage to cooperating with the cleanup, and may be fighting the
outlay of large sums of working capital that they would prefer to see go into some
other account or distribution. This may provide another window of opportunity
for employing mycoremediation science up front, at low cost, that even the most
44
intolerant party may agree to, since it can be viewed as good faith, kicked off with
minimal or relatively low start up costs, and capable of providing real benefit to
the environment. This can be viewed as beneficial to the offending or liable
party, as their may be a public image promoted to reflect favorably upon them. In
other words, we are taking some action, while discussions continue.
Resources
Lack of resources, or more accurately, not enough resource to go around, is a
problem faced by nearly every entity in government, business, and virtually any
agency of any type or make up. This presents another opportunity for employing
simpler, low cost, front end activities that may become part of a host of
multifaceted actions designed to recover or mediate disturbed habitats and recover
from the introduction of toxic chemicals. This site would be an excellent place to
test the practical application of biological uptake: the simple inoculation of a
fungus that has an affinity for picking up arsenic and other pollutants that were
generously spread around Puget Sound for decades on end, may have highly
beneficial results for this abused ecosystem. If this fill were effectively
impregnated with a mushroom spore and food source, of a variety that targets
these local toxins, the downstream rewards could be significant.
State Owned Aquatic Land Management Policy
As more and more agencies become players in the field of environmental
management of aquatic lands, sediments sites, disposal areas and long term
45
remedies, the opportunity for mycoremediation applications presents the potential
for an effective, eco-friendly technology for mitigation and sustainability of
resources. Increased interagency coordination has re-emphasized the scrutiny and
detail required to provide long term planning solutions for state owned aquatic
lands and ecosystems. According to this report, Washington State has recently
introduced a formalized methodology they call “collaborative management”
which, in theory, results in more efficient cross functional communication
between departments and agencies within the state’s broad administrative
network. Again, this should provide more opportunities for introducing
mycoremediation techniques into the comprehensive planning efforts of
legislators, policy and other decision makers.
As sustainability acquires an increased importance and focus at all levels of
government administration, more and more research projects aimed at how to
apply or most effectively employ processes in mycoremediation and
bioremediation techniques will naturally lend themselves to planning efforts.
Scientists and land use planners are recognizing that less is better. The natural
balance that ecosystems strike for themselves is the “way of the way” for keeping
the environment clean and efficient in terms of keeping the world green, and
developing so called sustainable processes for all of our manufacturing and
agricultural endeavors.
46
Potential Superfund Listing
Department of Energy site managers often delay or postpone actions on a given
site when they anticipate the possibility of the site eventually becoming added to
some broader and better funded cleanup activity like the federal Superfund. This
was exactly the case for the Lower Duwamish Waterway and the East Waterway
of Harbor Island. A more positive approach might be to implement in stages, as
many larger programs are, clearly recognizing and identifying the later phases of
the activity will be tied to out-year funding. For example, a pilot study using
native fungal species in a confined site could be carried out using volunteers and
scientific resources like graduate students or interns from forestry, fishery and
other agencies with potentially common interests and strategic goals.
Area-wide contamination
Sites that have been identified as contaminated and treated as isolated cases, may
be found to be part of a broader contamination source that is being included under
a broader management program. If planners and site managers sit and wait for
some potential action that rolls their site up into a broader funded program, much
like the Superfund situation discussed above, then valuable time may lost, and a
potential health hazard is left more or less unattended. Mycoremediation offers
its self as a frugal means for intervening on a known hazard situation, and
possibly defusing the threat potential in the near term.
47
Cost Estimates for Site Cleanup
In his book review of Biodegradation and Bioremediation by Martin Alexander,
(Gealt, 1995) the author provides a broad variety of links to how humans might
manage the disposal of wastes and ecological contamination using naturally
occurring biotic organisms, like fungi and bacteria. At press time at least, they
claim, some 32,000 hazardous waste sites existed in the United States alone, as of
1989 specifically.
In their 2005 report on toxics cleanup, the Washington State DOE developed a
rough methodology for estimating costs associated with contaminated sediment
cleanup. Out of 90 known contaminated sites, cost information was limited to or
screened to include only the cost of construction and post-cleanup remedial action
monitoring. The report confesses that these figures should be treated as order–of–
magnitude estimates, since some information was either not directly available, or
the sites were in varying stages of cleanup or construction. Other variances are
introduced by the uncertainty of site boundaries, as yet undetermined or imprecise
mediation remedies of strategies, and progress toward completion. In cases where
the project has been completed in its entirety, actual data on costs experienced are
easier to obtain and more reliable, since they are after the fact book recordings of
real world cost experiences.
Remedies range from natural or enhanced natural recovery with monitoring,
capping, dredging, in situ bioremediation and active treatment. Disposal options
varied from near shore placement and confined aquatic disposal at regulated
48
landfills. The report concludes that the options chosen in the various management
action plans can significantly alter or impact the final cost estimates, and actual
cost experience overall. Another factor that should be considered in this type of
estimating, are the extended or subsequent costs and impacts, regardless of
whether the current project or program budget is the cited or responsible
authority. For example, simply moving contaminated material from one location
to another, does not present a truly final solution to the toxic presence.
The contaminated material still exists, it is just in another physical location, and
this may or may not be an overall improvement or remedy to the situation. This is
one argument that was presented when the ASARCO smelter in Tacoma,
Washington was originally tagged as a prime contributor to the presence of heavy
metals in the soils and marine/freshwater environments of Tacoma and several
neighboring counties. Many planners and politicians argued that, since much of
the contamination is due to heavy metals, just leave them and they will sink to the
bottom of the soil or marine substrates. Left undisturbed, covered over, or
whatever, they pose no threat to the environment. This is similar to the argument
that non-friable asbestos should just be left undisturbed, rather than attempt to
remove or dispose of safely and cleanly.
The Department of Energy report presents the following cost categories in a
tabled format: Out of the 90 sites that have associated cost information available,
56 are estimated at $5 million or less. Total cost for the 90 sites is roughly
49
between $400 million and $1.2 billion. The variances, lack of good data, and
errors in estimating make it clear that far better effort and attention needs to be
applied to the area of mitigating the generation of toxins and other pollutants
associated with any project, whether large or small. On the other hand, this
knowledge begins to illustrate opportunities for many potential market and
business niches that deal with waste management as a revenue source, and for
newly applied science and technology in the field of bioremediation and
mycoremediation.
Conclusion
In field settings, the ecologist or environmental planner is faced with the
challenge of a diverse presence of arbuscular-mycorrhizal (AM) fungi that occur
naturally, or without the intentional behavior of terrestrial modifiers like humans
and other organisms. Bever, et al, 2001, examine this diversity and some
implications of its presence as an agent in site restoration activities, whether
planned for or not. Their study was aimed at finding multiple species of AM
fungi in the field. Their initial examination of field soil was collected in 1992,
and 11 species were identified, recognizing some margin of error based on
sampling bias. Using a variety of entrapment techniques to refine their samples
and obtain better counts, they arrived at a final count of 37 different species, onethird of which had not been described before. Recognizing that the presence of
one species may promote the growth, health and well being of one plant, another
50
may have just the opposite effect. For example, the presence of Ac. Collosica
was negatively associated with soil phosphorous concentration while the opposite
was true for Gi.gigantea.
The authors suggest that the entire plant and fungal community combine as a
“superorganism” with complex interactions that support, enhance and evolve as
an interrelated biotic system, with a web or network of communication and
transport dynamics. The authors conclude by emphasizing the importance of
reevaluations away from past understandings of plant succession, that include
such process as even “below ground” organisms as dynamic participants to
provide us a complete or better understanding of the many consequences of
mycorrhizal mediation or intervention techniques in ecology planning and the
intentional alteration of agricultural and riparian species that impact plant and
animal life at virtually every level of the carbon cycle. Fungal species affect the
ecosystem in the way they break down, build up, bioaccumulate, absorb and
sterilize or destroy other organisms in the environment.
Bever et al, 2001, propose a model for viewing certain root symbiots, arbuscular
mycorrhizal (AM) fungi, as key promoters of the general health of plant
communities across the terrestrial biosphere. Specifically, they are critical in
facilitating the ability of most plants to uptake phosphorous. This newly rendered
version of plant ecology suggests many creative ways of employing these curious
biota in landscape gardening, forestry, agriculture, and remediation of
51
contaminated sites around the world. The authors performed a research study on
a one-hectare grassland that had been abandoned as an agricultural site some 60
years. Their goal was to examine the presence of AM species in the field. 37
species were eventually identified, half of which have not previously been
described. This number was much higher than the researchers expected.
Speculation is made that the high number of species may contribute to overall
trophic responses of the native or host plants, as each will have differing
requirements in a symbiotic relationship. The authors also suggest the total
number plays some role in successional dynamics, as new plants migrate in over
time from conditions of disturbance like fire. The paper concludes with another
call for more research on the complex interactions that occur within soils and
between plant ecologies. One confounding factor may have been the use of
“trapping” methods – this involves the capture and return of small pots to the lab
for examination over time, reasoning that certain species may not appear until
seasonal changes alter environmental factors like humidity, etc. This allows for
the introduction of error through cross contamination, and other vectors.
Another aspect of the benefits provided by the presence of mycorrhizal fungi in
soils and marine systems relate to the trophic benefits that so called host plants
experience. Rai, 2001 examined the use of microbial inoculants as a replacement
for chemical fertilizers and pesticides. Estimates place the symbiotic partnership
of plants and fungi at 90% of all known species. Among the established and
52
theorized benefits are processes that result in the uptake of nutrients, reduced
stress, improved nutrition, improved aeration, and soil structure. The author
identifies several aspects of the technology that could be improved and
recommends screening bioassays as an effective way to identify the most
effective strain. The report concludes in general that the “hairy-root” technology
has great promise for cultivation of target species and that the enzymatic
processes that these organisms deliver to ecological planners are beneficial in not
only breaking down toxins, but also as highly beneficial support members in most
plant communities. This becomes more true as more data is collected and
recorded on species interactions at the subsoil level.
Lamar et al (1999), noted that for the application of bioremediation techniques
such as mycoremediation to be useful for mediating the impacts of xenobiotic
contaminated soils, scientists and project managers must be able to identify and
select for the introduction of species that will thrive in the physiochemical and
nutritional conditions that are present in the target site or contaminated zone. The
ultimate goal of this identification process is to obtain dominant colonization and
ultimately remediation of a contaminated soil volume by the inoculated organism.
Their research identifies a need for further investigations that will advance the
application of this laboratory knowledge into the field, and eventually contribute
to the development of physical processes that enhance mycoremediation
techniques on a commercial scale. As this knowledge broadens, and more
53
reference data is available to planners, the more practical and cost effective these
specialty techniques in bioremediation will become.
Lamar’s 1999 paper examined 20 fungal strains comprising six species of whiterot fungi on specific performance parameters. This report identified the most
effective fungus for a given set of contaminant and soil conditions that one might
encounter in the field, giving optimal growth and degrading activities, all of
which are essential to the successful application of fungal bioaugmentation. Their
work sets the stage for development of a universal, comprehensive reference
database that would allow engineers and planners to apply the best available
fungus and promotes its ultimate success as a mitigation technique that meets all
the favorable attributes of being eco-friendly, benign, and transparent to users and
other members of the biotic community, at all scales of magnitude and diversity.
This study also illustrates, as the authors reflect in their conclusion, that simple
measures of contaminant degrading ability or tolerance to a pollutant/s do not
provide the complete picture for selection of fungi in bioaugmentation of
contaminated soils or riparian zones.
As demonstrated by the case of P. sordida, one fungus may achieve superior
performance under one set of conditions, and remain mediocre under another set
of contaminant – media conditions. This statement is key to their work as well as
the underlying thesis of this author’s study: “More information on the
bioremediation performance of well characterized fungi on soils that represent a
54
range of soil physical, chemical, and biological characteristics and pollutants is
necessary to build a database of information from which to base selection of
superior fungal strains to evaluate for application to specific contaminant – media
conditions.” A database of this kind would include at least fungal characteristics
like growth rates, growth temperature ranges and optima, sensitivities to and
abilities to degrade specific contaminants, bioremediation performance in well
characterized media and relative competitive abilities. This final factor is critical,
and has been largely ignored in the scientific literature. A fungus must be able to
survive a given set of conditions as well as degrade target toxins and pollutants.
In their report on environmental contamination, Bonaventura and Johnson, 2008,
the authors examine environmental contamination sites that are challenging our
“…global society to find effective measures of remediation to reverse the
negative conditions that severely threaten human and environmental health.”
They define bioremediation as the use of “…microbes (bacteria, fungi, yeast and
algae, although higher plants are used in some applications. New bioremediation
approaches are emerging based on advances in molecular biology and process
engineering.” The report goes on to suggest that these methods are favored from
the perspective of environmental impact as the outcomes of these processes avoid
the production or regeneration of microbial pathogenesis. As a relatively benign
and inexpensive contributor to a entire spectrum of mediation approaches to
contaminated ecosystems, the report concludes that “Bioremediation…will play
an increasingly important role as a result of new and emerging techniques and
55
processes.” A specific finding pointed out in this report is that enzymes found in
the fungus Phanerochaete chrysosporium or white rot fungus effectively degrade
some wastes that prove resistant to most bacterial action (e.g. DDT and 2, 4, 5trichlorophenyoxyacetic acid).”
Bonaventura et al, 2008 warn us, that “As the population of the planet continues
to rise at alarming rates, natural checks and balances in the earths biochemistry
may play more important roles, as waste materials like feces and urine begin to
compete with and contaminate long standing human food sources.” Is the day of
the microbe upon us, or has it always been thus? The authors examine a
collection of bioabsorption, biodegradation, and bioremediation processes before
concluding generally that this emerging science provides a well demonstrated
method for developing applied strategies in dealing with a wide spectra of
ecologically degraded environments and areas that are at risk due to current and
past activities of humans. No “proofs” are offered here, only a series of rational
approaches to dealing with real problems that have potentially negative impacts
on the human biosphere. In one of their summary statements, the may have said it
best, “Bioremediation is a technological attempt to exploit the abilities of
microbes and other members of the biosphere to restore and maintain
environmental quality for all forms of life in the ecosystem, especially humans.”
Skipper, et al, 1996 report on their attempt to “…document the existing status of
the microbiology of environmental fate studies with pesticides.” The authors
56
assert that verification of data from laboratory studies to the field environment is
needed, and that better field studies are also necessary to complete our
understanding of the processes that ultimately breakdown and deposit these
potentially lethal compounds. There is merit to this investigation, as the use of
chemicals and pesticides seems to remain the preferred method in organized
commercial farming and agriculture.
These authors go into significant detail
examining some of the more prevalent methods for determining the fate of
pesticides as they move through their natural progression in the various
environments of the world. There work seems through in its scope and critical
examination, but in the end the conclusions they draw are somewhat banal and
rather mainstream. They simply restate what we all knew, that the challenges
here are complicated, and better practices need to come forth for deriving
meaningful and accurate representation of the micro-compounds. Perhaps the
“unknown” nature of their discussion can be seen as erring on the side of caution
as we plan for current and so called sustainable forestry and agricultural processes
in the future. That is, we should employ natural, benign processes whenever and
wherever it is practical, to avoid adding fuel to the fire so to speak. If there is an
implicit argument or finding to this study, perhaps this is it. Do not burn your
maple leaves each fall because they make the sidewalk slippery, but mulch them
lightly over your garden and lawn areas, as an alternative to dumping processed
nitrates and the like into your rose beds?
57
In his recent book on mycoremediation, Singh (2006) notes that mycorrhizas
provide a nutrient transport from soils to plant roots. Their role in the formation
of soil aggregates and the protection of plants against drought and root pathogens
is well established in the literature. These mediation processes occur over time,
and depend on the successful development of a host root system associated with
the fungal biomass. By selecting compatible host-fungus-substrate combinations
these relationships can be exploited through careful study and inoculation of
plant, riparian and other ecosystems. Future experiments should be designed to
determine the precise fungal degradations in symbiotic field associations with a
host plant or environment. Current knowledge on the functional diversity of
mycorrhizal communities is fragmentary (Singh, 2006). Study of the effects of
pollutants on the growth of extrametrical mycelium of these fungi is necessary in
understanding their stability and sustainability in the ecosystems.
A large body of research is available that shows the potential for using
mycorrhizal biomediation and bioremediation techniques in removing toxins from
soils and groundwater, as well as in providing symbiotic and trophic benefits to a
broad spectrum of environments and host organisms like trees, grasses, and
riparian zones. This paper presents a sampling of this scientific data, but is not
intended to be a complete listing of all the known works. The number is simply
too large. In addition, there is clearly a gap between what is being suggested in
the research, and the application of techniques in the real world that would benefit
contaminated and otherwise degraded ecosystems.
58
Subsequent application of mushroom mycelia might employ naturally occurring
plants that form beneficial symbiotic relationships in the environment, based on
thorough study and organized planning methods. Lamar, et al (1999), have
shown that a large variety of fungal species may be effective in remediating the
presence of potentially harmful compounds in a variety of terrestrial and riparian
zones including salmon spawning habitat locations. These include products like
heavy metals, pesticides, petroleum, fuel oils, and others. The costs to introduce
the methods should be fairly inexpensive and bio-friendly compared to practices
like dredging, or introducing other chemicals that may be just as harmful. This
author feels there is ample data in the research to suggest that mycorrhizal
bioremediation techniques show considerable promise as biomediation factors.”
Considering the need to recover safe, healthy spawning and habitat areas for all
fish species, further research on these processes may also eventually provide
understanding of applied methods to reduce the presence of harmful substances in
the food web for the entire planet. The potential benefits of this new branch of
physical and organic science are enormous as we posture on the edge of an
overgrown and polluted biosphere. The lowly mushroom is actually performing a
multi-faceted role in its varied capacities as a primary decomposer of organics, a
beneficial symbiot to other plants, an antibiotic toward harmful biota, an
environmental architect at the biochemical level as it combines with and
restructures toxic elements. Did we mention food sources and medicinal benefits?
Is there a single organism, in its inherent complexity and pervasiveness that
59
contributes as much to the sustainable architecture of the planet, and every carbon
based life form it supports?
60
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White Rot Fungi to Biodegrade Environmental Pollutants, Environmental Health
Perspectives, Vol. 101, No. 3 (Aug., 1993), pp. 232-233.
Ayed, L., Assas, N., Sayadi, S., and Hamdi, M., (2004). Involvement of lignin
peroxidases in the decolorization of black olive mill wastewaters by Geotrichum
candidum. Lett. Appl. Microbiol. 40:7 – 11.
Bever, James D., Schultz, Peggy A., Pringle Anne, Morton, Joseph B., [2001],
Arbuscular Mycorrhizal Fungi: More Diverse Than Meets the Eye, and the
Ecological Tale of Why, BioScience, Vol. 51, No. 11 (Nov. 2001) pp. 923-931.
Bonaventura, Celia and Johnson, Franklin M. [2008], Healthy Environments for
Healthy People: Bioremediation Today and Tomorrow, Environmental Health
Perspectives, Vol. 105, Supplement 1 (Feb., 1997), pp. 5-20.
Davis, M.W., Glaser J.A., Evans J.W. & Lamar R.T. (1993). Field evaluation of
the lignin-degrading fungus Phanerochaete sordida to treat creosote-contaminated
soil Environmental Science Technology 27: 2572-2576.
Davis, Mark A. & Slobodkin, Lawrence B. (2004). The Science and Values of
Restoration Ecology. Restoration Ecology 12 (1), 1-3.
Davis, M. A., & Slobodkin, L. B. (March 2004). The Science and Values of
Restoration Ecology, Restoration Ecology 12 (1), 1.
Duran, N., and Esposito, E., 2000. Potential applications of oxidative enzymes
and phenoloxidase-like compounds in wastewater and soil treatment. Appl. Catal.
B 28c83-89.
D’Annibale, A., Rosetto, F., Leonardi, V. Federici, F., & Petruccioli, M. 2006.
Role of Autochthonous Filamentous Fungi in Bioremediation of a Soil
Historically Contaminated with Aromatic Hydrocarbons. Applied and
Environmental Microbiology, 72 (1): 28-36.
Field, J. A., de Jong, E., Costa, G. F. and de Bont, A.M., 1993. Screening of
ligninolytic fungi applicable to biodegradation of xenobiotics. Trends
Biotechnology 11:44-49.
Gealt, Michael A., [1995], Getting Rid of Wastes, BioScience, Vol 45, No. 9
(Oct., 1995), pp. 635-638.
Hammel, Kenneth E., [1995], Mechanisms for Polycyclic Aromatic Hydrocarbon
Degradation by Lignolytic Fungi, Environmental Health Perspectives, Vol. 103,
Supplement 5: Biodegradation (Jun., 1995), pp. 41-43.
Hobbs, Richard J. (2005). The Future of Restoration Ecology: Challenges and
Opportunities.” Restoration Ecology 13 (2), 239-241.
Killham, K. & Firestone, M.K. (1983). Vesicular Arbuscular Mycorrhizal
Mediation of Grass Response to Acidic and Heavy Metal Depositions. Plant and
Soil (72), 39-48.
Kirk, P.W., Dyer, B.J., Noe, J., [1991], Hydrocarbon Utilization by Higher
Marine Fungi from Diverse Habitats and Localities, Mycologia, Vol. 83, No. 2
(Mar. – Apr., 1991), pp. 227 – 230.
Lamar, R. T., Glaser J.A., & Kirk T.K. (1992). White rot fungi in the treatment of
hazardous chemicals and wastes. Frontiers in Industrial Mycology, G. F.
Leatham (ed.). Chapman & Hall, New York, p. 127-143.
Lamar, Richard T., Main, Laura M., Dietrich, Diane M., & Glaser, John A.
(1999). Screening of Fungi for Soil Remediation Potential. American Society of
Agronomy, Bioremediation of Contaminated Soils, Agronomy Monograph No. 37.
437-456.
Lestan, D. and Lamar, Richard T., (Jun. 1996). Development of Fungal Inocula
for Bioaugmentation of Contaminated Soils. Applied and Environmental
Microbiology, 62, 2045-2052.
Moerke, Ashley H. & Lamberti, Gary A. (2004). Restoring Stream Ecosystems:
Lessons from a Midwestern State. Restoration Ecology 12 (3), 327-334.
Office of the Governor, State of Washington. 2005 – 2007 Puget Sound
Conservation & Recovery Plan Highlights. Puget Sound Action Team.
Oghenekaro A.O., Okhuoya J.A., and Akpaja E.O. (October 2008). Growth of
Pleurotus tuberregium (Fr) Singer on some heavy metal-supplemented substrates.
African Journal of Microbiology Research Vol. (2), 268-271.
Pointing, S.B., 2001. Feasibility of bioremediation by white-rot fungi. Applied
Microbiology Biotechnoogy. 57:20 – 33.
Rai, M.K. [2001], Current Advances in Micropropagation, In Vitro Cellular &
Developmental Biology. Plant, Vol. 37, No. 2 (Mar. – Apr., 2001), pp. 158 – 167.
Ruckleshaus, M.H. & McClure, M.M. (2007). Sound Science: Synthesizing
Ecological & Socio-economic Information About the Puget Sound Ecosystem.
Sound Science Collaborative Team, U.S. Dept. of Commerce, National Oceanic
& Atmospheric Administration (NMFS) Northwest Fisheries Science Center,
Seattle Washington.
Ruiz-Jaen M.C., & Aide T.M. (Sep. 2005). Restoration Success: How Is It Being
Measured? Restoration Ecology, 13 (3), 569.
Sasek V., Vitasek J., Chromcova D., Prokopova I., Bozek J., Nahlik J. (2006).
Biodegradation of Synthetic Polymers by Composting and Fungal Treatment.
Folia Microbiol. 51 (5), 425-430.
Seiders, K., Deligeannis, C., & Kinney, K. (May 2006). Toxic Contaminants in
Fish Tissue and Surface Water in Freshwater Environments, 2003. Washington
State Toxics Monitoring Program, Washington State Department of Ecology,
Pub. No. 06-03-019.
Singh, H. (2006). Mycoremediation: Fungal Bioremediation. New Jersey: John
Wiley & Sons, Inc.
Skipper, Horace D., Wollum, Arthur G., Turco, Ronald F., Wolf, Duane C.
(1996). Microbiological Aspects of Environmental Fate Studies of Pesticides,
Weed Technology, Vol. 10, No. 1 (Jan. – Mar., 1996), pp. 174 – 190.
Summers J. & Serdal D. (Jun 2000). Washington State Department of Ecology.
Program for Monitoring Salmon Recovery in Index Watersheds: Water Quality
and Quantity. Quality Assurance Program Plan.
Thomas S., Becker P., Pinza M.R., and Word J.Q., (November 1998).
Mycoremediation of Aged Petroleum Hydrocarbon Contaminants in
Soil. Prepared for the Washington State Department of Transportation
Vorass M., & Portele G.J. (2001). Washington State Department of
Transportation Contaminated Sites and Endangered Species Act Risk Reduction,
John Muir Institute of the Environment, Road Ecology Center, Paper: Vorass
2001a.
Washington State Department of Ecology (1999). Pacific Northwest Salmon
Habitat Indicators: Pilot Project Snohomish River Basin. (Publication No. 99301). Olympia WA: Ward W.J.
Washington State Department of Ecology ( 1994). Washington State Pesticide
Monitoring Program Reconnaissance Sampling of Fish Tissue and Sediments.
(Publication No. 94-194). Olympia WA: Davis, D. & Johnson, A.
Washington State Department of Ecology (2005). Sediment Cleanup Status
Report (Publication No. 05-09-092). Olympia WA.
Zerbe, Stefan & Kreyer, Daria (2006). “Introduction to Special Section on
Ecosystem Restoration and Biodiversity: How to Assess and Measure Biological
Diversity?” Restoration Ecology 14 (1), 103-104.
Bibliography
Aust, Steven D., and Benson, John T. [1993], The Fungus Among Us: Use of
White Rot Fungi to Biodegrade Environmental Pollutants, Environmental Health
Perspectives, Vol. 101, No. 3 (Aug., 1993), pp. 232-233.
Ayed, L., Assas, N., Sayadi, S., and Hamdi, M., (2004). Involvement of lignin
peroxidases in the decolorization of black olive mill wastewaters by Geotrichum
candidum. Lett. Appl. Microbiol. 40:7 – 11.
Bever, James D., Schultz, Peggy A., Pringle Anne, Morton, Joseph B., [2001],
Arbuscular Mycorrhizal Fungi: More Diverse Than Meets the Eye, and the
Ecological Tale of Why, BioScience, Vol. 51, No. 11 (Nov. 2001) pp. 923-931.
Bonaventura, Celia and Johnson, Franklin M. [2008], Healthy Environments for
Healthy People: Bioremediation Today and Tomorrow, Environmental Health
Perspectives, Vol. 105, Supplement 1 (Feb., 1997), pp. 5-20.
Davis, M.W., Glaser J.A., Evans J.W. & Lamar R.T. (1993). Field evaluation of
the lignin-degrading fungus Phanerochaete sordida to treat creosote-contaminated
soil Environmental Science Technology 27: 2572-2576.
1
Davis, Mark A. & Slobodkin, Lawrence B. (2004). The Science and Values of
Restoration Ecology. Restoration Ecology 12 (1), 1-3.
Davis, M. A., & Slobodkin, L. B. (March 2004). The Science and Values of
Restoration Ecology, Restoration Ecology 12 (1), 1.
Duran, N., and Esposito, E., 2000. Potential applications of oxidative enzymes
and phenoloxidase-like compounds in wastewater and soil treatment. Appl. Catal.
B 28c83-89.
D’Annibale, A., Rosetto, F., Leonardi, V. Federici, F., & Petruccioli, M. 2006.
Role of Autochthonous Filamentous Fungi in Bioremediation of a Soil
Historically Contaminated with Aromatic Hydrocarbons. Applied and
Environmental Microbiology, 72 (1): 28-36.
Field, J. A., de Jong, E., Costa, G. F. and de Bont, A.M., 1993. Screening of
ligninolytic fungi applicable to biodegradation of xenobiotics. Trends
Biotechnology 11:44-49.
Gealt, Michael A., [1995], Getting Rid of Wastes, BioScience, Vol 45, No. 9
(Oct., 1995), pp. 635-638.
2
Hammel, Kenneth E., [1995], Mechanisms for Polycyclic Aromatic Hydrocarbon
Degradation by Lignolytic Fungi, Environmental Health Perspectives, Vol. 103,
Supplement 5: Biodegradation (Jun., 1995), pp. 41-43.
Hobbs, Richard J. (2005). The Future of Restoration Ecology: Challenges and
Opportunities.” Restoration Ecology 13 (2), 239-241.
Killham, K. & Firestone, M.K. (1983). Vesicular Arbuscular Mycorrhizal
Mediation of Grass Response to Acidic and Heavy Metal Depositions. Plant and
Soil (72), 39-48.
Kirk, P.W., Dyer, B.J., Noe, J., [1991], Hydrocarbon Utilization by Higher
Marine Fungi from Diverse Habitats and Localities, Mycologia, Vol. 83, No. 2
(Mar. – Apr., 1991), pp. 227 – 230.
Lamar, R. T., Glaser J.A., & Kirk T.K. (1992). White rot fungi in the treatment of
hazardous chemicals and wastes. Frontiers in Industrial Mycology, G. F.
Leatham (ed.). Chapman & Hall, New York, p. 127-143.
Lamar, Richard T., Main, Laura M., Dietrich, Diane M., & Glaser, John A.
(1999). Screening of Fungi for Soil Remediation Potential. American Society of
Agronomy, Bioremediation of Contaminated Soils, Agronomy Monograph No. 37.
437-456.
3
Lestan, D. and Lamar, Richard T., (Jun. 1996). Development of Fungal Inocula
for Bioaugmentation of Contaminated Soils. Applied and Environmental
Microbiology, 62, 2045-2052.
Moerke, Ashley H. & Lamberti, Gary A. (2004). Restoring Stream Ecosystems:
Lessons from a Midwestern State. Restoration Ecology 12 (3), 327-334.
Office of the Governor, State of Washington. 2005 – 2007 Puget Sound
Conservation & Recovery Plan Highlights. Puget Sound Action Team.
Oghenekaro A.O., Okhuoya J.A., and Akpaja E.O. (October 2008). Growth of
Pleurotus tuberregium (Fr) Singer on some heavy metal-supplemented substrates.
African Journal of Microbiology Research Vol. (2), 268-271.
Pointing, S.B., 2001. Feasibility of bioremediation by white-rot fungi. Applied
Microbiology Biotechnoogy. 57:20 – 33.
Rai, M.K. [2001], Current Advances in Micropropagation, In Vitro Cellular &
Developmental Biology. Plant, Vol. 37, No. 2 (Mar. – Apr., 2001), pp. 158 – 167.
Ruckleshaus, M.H. & McClure, M.M. (2007). Sound Science: Synthesizing
Ecological & Socio-economic Information About the Puget Sound Ecosystem.
Sound Science Collaborative Team, U.S. Dept. of Commerce, National Oceanic
4
& Atmospheric Administration (NMFS) Northwest Fisheries Science Center,
Seattle Washington.
Ruiz-Jaen M.C., & Aide T.M. (Sep. 2005). Restoration Success: How Is It Being
Measured? Restoration Ecology, 13 (3), 569.
Sasek V., Vitasek J., Chromcova D., Prokopova I., Bozek J., Nahlik J. (2006).
Biodegradation of Synthetic Polymers by Composting and Fungal Treatment.
Folia Microbiol. 51 (5), 425-430.
Seiders, K., Deligeannis, C., & Kinney, K. (May 2006). Toxic Contaminants in
Fish Tissue and Surface Water in Freshwater Environments, 2003. Washington
State Toxics Monitoring Program, Washington State Department of Ecology,
Pub. No. 06-03-019.
Singh, H. (2006). Mycoremediation: Fungal Bioremediation. New Jersey: John
Wiley & Sons, Inc.
Skipper, Horace D., Wollum, Arthur G., Turco, Ronald F., Wolf, Duane C.
(1996). Microbiological Aspects of Environmental Fate Studies of Pesticides,
Weed Technology, Vol. 10, No. 1 (Jan. – Mar., 1996), pp. 174 – 190.
5
Summers J. & Serdal D. (Jun 2000). Washington State Department of Ecology.
Program for Monitoring Salmon Recovery in Index Watersheds: Water Quality
and Quantity. Quality Assurance Program Plan.
Thomas S., Becker P., Pinza M.R., and Word J.Q., (November 1998).
Mycoremediation of Aged Petroleum Hydrocarbon Contaminants in
Soil. Prepared for the Washington State Department of Transportation
Vorass M., & Portele G.J. (2001). Washington State Department of
Transportation Contaminated Sites and Endangered Species Act Risk Reduction,
John Muir Institute of the Environment, Road Ecology Center, Paper: Vorass
2001a.
Washington State Department of Ecology (1999). Pacific Northwest Salmon
Habitat Indicators: Pilot Project Snohomish River Basin. (Publication No. 99301). Olympia WA: Ward W.J.
Washington State Department of Ecology ( 1994). Washington State Pesticide
Monitoring Program Reconnaissance Sampling of Fish Tissue and Sediments.
(Publication No. 94-194). Olympia WA: Davis, D. & Johnson, A.
6
Washington State Department of Ecology (2005). Sediment Cleanup Status
Report (Publication No. 05-09-092). Olympia WA.
Zerbe, Stefan & Kreyer, Daria (2006). “Introduction to Special Section on
Ecosystem Restoration and Biodiversity: How to Assess and Measure Biological
Diversity?” Restoration Ecology 14 (1), 103-104.
7
