Mycoremediation

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Mycoremediation (from ancient Greek μύκης (mukēs), meaning "fungus" and the suffix -remedium, in Latin meaning 'restoring balance') is a form of bioremediation in which fungi-based technology is used to decontaminate the environment. Fungi have been proven to be a very cheap, effective and environmentally sound way for helping to remove a wide array of toxins from damaged environments or wastewater. The toxins include heavy metals, persistent organic pollutants, textile dyes, leather tanning industry chemicals and wastewater, petroleum fuels, polycyclic aromatic hydrocarbon, pharmaceuticals and personal care products, pesticides and herbicide,^[1] in land, fresh water and marine environments. The byproducts of the remediation can be valuable materials themselves, such as enzymes (like laccase^[2]), edible or medicinal mushrooms,^[3] making the remediation process even profitable.

Contents

• 1Pollutants
  • 1.1Metals
  • 1.2Organic pollutants
  • 1.3Pesticides
  • 1.4Dyes
• 2Synergy with phytoremediation
• 3See also
• 4References

Pollutants[edit]

Fungi, thanks to their non-specific enzymes, are able to break down many kinds of substances. They are used for pharmaceuticals and fragrances that normally are recalcitrant to bacteria degradation,^[4] such as paracetamol, the breakdown products of which are toxic in traditional water treatment, using Mucor hiemalis,^[5] but also the phenols and pigments of wine distillery wastewater,^[6] X-ray contrast agents and ingredients of personal care products.^[7]

Mycoremediation is one of the cheaper solutions to remediation, and it doesn't usually require expensive equipment. For this reasons it is often used also in small scale applications, such as mycofiltration of domestic wastewater,^[8] and to help with the decomposition process of a compost toilet.

Metals[edit]

Pollution from metals is very common, as they are used in many industrial processes such as electroplating, textiles,^[9] paint and leather. The wastewater from these industries is often used for agricultural purposes, so besides the immediate damage to the ecosystem it is spilled into, the metals can enter far away creatures and humans through the food chain. Mycoremediation is one of the cheapest, most effective and environmental-friendly solutions to this problem.^[10] Many fungi are hyperaccumulators, that means they are able to concentrate toxins in their fruiting bodies for later removal. This is usually true for populations that have been exposed to contaminants for long time, and have developed a high tolerance, and happens via biosorption on the cellular surface, which means that the metals enter the mycelium in a passive way with very little intracellular uptake.^[11] A variety of fungi, such as Pleurotus, Aspergillus, Trichoderma has proven to be effective in the removal of lead,^[12][13] cadmium,^[14] nickel,^[15][16] chromium,^[17] mercury,^[18] arsenic,^[19]copper,^[20][21] boron,^[22] iron and zinc^[23] in marine environment, wastewater and on land.

Not all the individuals of a species are effective in the same way in the accumulation of toxins. The single individuals are usually selected from an old-time polluted environment, such as sludge or wastewater, where they had time to adapt to the circumstances, and the selection is carried on in the laboratory. A dilution of the water can drastically improve the ability of biosorption of the fungi.^[24]

The capacity of certain fungi to extract metals from the ground also can be useful for bioindicator purposes, and can be a problem when the mushroom is an edible one. For example, the shaggy ink cap (Coprinus comatus), a common edible north-hemisphere mushroom, can be a very good bioindicator of mercury, and accumulate it in its body, which can also be toxic to the consumer.^[25]

The capacity of metals uptake of mushroom has also been used to recover precious metals from medium. VTT Technical Research Centre of Finland reported an 80% recovery of gold from electronic waste using mycofiltration techniques.^[26]

Organic pollutants[edit]

Fungi are amongst the primary saprotrophic organisms in an ecosystem, as they are efficient in the decomposition of matter. Wood-decay fungi, especially white rot, secretes extracellular enzymes and acids that break down lignin and cellulose, the two main building blocks of plant fiber. These are long-chain organic (carbon-based) compounds, structurally similar to many organic pollutants. They do so using a wide array of enzymes. In the case of polycyclic aromatic hydrocarbons (PAHs), complex organic compounds with fused, highly stable, polycyclic aromatic rings, fungi are very effective^[27] also in marine environments.^[28] The enzymes involved in this degradation are ligninolytic and include lignin peroxidase, versatile peroxidase, manganese peroxidase, general lipase, laccase and sometimes intracellular enzymes, especially the cytochrome P450.^[29][30]

Other toxins fungi are able to degrade into harmless compounds include petroleum fuels,^[31] phenols in wastewater,^[32] polychlorinated biphenyl (PCB) in contaminated soils using Pleurotus ostreatus,^[33] polyurethane in aerobic and anaerobic conditions such as found at the bottom of landfills using two species of the Ecuadorian fungus Pestalotiopsis,^[34] and more.^[35]

The mechanisms of degradation are not always clear,^[36] as the mushroom may be a precursor to subsequent microbial activity rather than individually effective in the removal of pollutants.^[37]

Pesticides[edit]

Pesticide contamination can be long-term and have a significant impact on decomposition processes and thus nutrient cycling^[38] and their degradation can be expensive and difficult. The most used fungi for helping in the degradation of such substances are white rot ones which, thanks to their extracellular ligninolytic enzymes like laccase and manganese peroxidase, are able to degrade high quantity of such components. Examples includes the insecticide endosulfan,^[39] imazalil, thiophanate methyl, ortho-phenylphenol, diphenylamine, chlorpyrifos^[40] in wastewater, and atrazine in clay-loamy soils.^[41]

Dyes[edit]

Dyes are used in many industries, like paper printing or textile. They are often recalcitrant to degradation and in some cases, like some azo dyes, cancerogenic or otherwise toxic.

The mechanism the fungi degrade this dyes is their lignolytic enzymes, especially laccase, so white rot mushrooms are the most commonly used.

Mycoremediation has proven to be a cheap and effective remediation technology for dyes such as malachite green, nigrosin and basic fuchsin with Aspergillus niger and Phanerochaete chrysosporium^[42] and Congo red, a carcinogenic dye recalcitrant to biodegradative processes,^[43] direct blue 14 (using Pleurotus).^[44]

Synergy with phytoremediation[edit]

Phytoremediation is the use of plant-based technologies to decontaminate an area. Most of the plants can form a symbiosis with fungi, from which both the organisms get an advantage. This relationship is called mycorrhiza.

Mycorrhizal fungi, especially arbuscular mycorrhizal fungi (AMF), can greatly improve the phytoremediation capacity of some plants. This is mostly because the stress the plants suffer because of the pollutants is greatly reduced in presence of AMF, so they can grow more and produce more biomass.^[45] The fungi also provide more nutrition, especially phosphorus, and promotes the overall health of the plant. The mycelium quick expansion also can greatly extend the rhizosphere influenze zone (hyphosphere), providing the plant with access to more nutrients and contaminants.^[46] Increasing the rhizosphere overall health also means a rise in the bacteria population, which can also contribute to the bioremediation process.^[47]

This relationship has been proven useful with many pollutants, such as Rhizophagus intraradices and Robinia pseudoacacia in lead contaminated soil,^[48] Rhizophagus intraradiceswith Glomus versiforme incoulated into vetiver grass for lead removal,^[49] AMF and Calendula officinalis in cadmium and lead contaminated soil,^[50] and in general was effective in increasing the plant bioremediation capacity for metals,^[51][52] petroleum fuels,^[53][54] and PAHs.^[55] In wetlands AMF greatly promote the biodegradation of organic pollutants like benzene-, methyl tert-butyl ether- and ammonia from groundwater when inoculated into Phragmites australis.^[56]

Mycoremediation - a potential tool for sustainable management

Sayan Deb Dutta* and Md.Salman Hyder**

*Department of Botany, University of Kalyani, Nadia – 741235

** Department of Botany, University of Kalyani, Nadia – 741235

E-mails: sayan91dutta@gmail.com; hydersalman09@gmail.com

Abstract:

One of the major environmental problems faced by the world today is the contamination of soil,

water and air by toxic chemicals. The distinct and unique role of microorganisms in the

detoxification of polluted soil and environments is well recognized. Mycoremediation systems

basically depend upon microorganisms (fungi) native to the contaminated sites. Fungi belonging

to basidiomycetes (white rot fungi) are also known as mycoremediation tool because of their use

in remediation of different types of pollutants. Mycoremediation relies on the efficient enzymes,

produced by fungus, for the degradation of various types of substrate and pollutants. However,

sometimes they absorb the pollutant in their mycelium (biosorption) and cannot be consumed

due to absorbed toxicants. Examples of fungi used as mycoremediators are - Pleurotus ostreatus,

Rhizopus arrhizus, Phanerochaete chrysosporium, Tramates hirsute, T. versicolor, Lentinus

edodes, Cladosporium resinae, Aspergillus niger, A. flavus, A. terrus and Trichoderma

longibrachiatum. In situ mycoremediation treats the contaminated soil in the location in which it

was found, whereas, ex situ processes require excavation of contaminated soil before they can be

treated. However, despite being the living dominating biomass in soil, fungi have not yet been

significantly exploited for mycoremediation of such polluted environments. More extensive

research needs to be carried out on the use of fungi in mycoremediation.

Keywords: Detoxification, Mycoremediation, Biosorption, Excavation.

Introduction:

Soil pollution has significant deleterious consequences for ecosystem. In the past few years the

soil is getting more and more polluted. Remediation of these polluted soils is a challenging job.

Bioremediation is a treatment process that uses naturally occurring microorganisms to break

down, or degrade, hazardous substances into less toxic or nontoxic substances. The microbes

used to perform the function of bioremediation are known as bioremediators. To “bioremediate”,

means to use living things to solve an environmental problem such as contaminated soil or

groundwater. The introduction of exogenous microorganisms into environments –

bioaugmentation, has been used as an attempt to accelerate bioremediation (Watanabe, 2001).

Some microorganisms that live in soil and groundwater naturally degrade certain chemicals that

are harmful to people and the environment. These microorganisms are also able to change these

chemicals into water and harmless gases, such as carbon dioxide etc. Plants can also be used to

clean up soil, water or air; this is called phytoremediation. Watanabe (2001) reported that

naturally occurring microbial consortia (bacteria / fungi) have been utilized in a variety of

bioremediation processes.

Bioremediation is an attractive technology that utilizes the metabolic potential of

microorganisms in order to clean up the environmental pollutants to the less hazardous or non-

hazardous forms with less input of chemicals, energy and time. During last two decades, many

mycologists have tried the use of various fungal species in the degradation of organic

compounds. The discovery of the white rot fungi (Phanerochaete chrysosporium) in

bioremediation has brought greater success and thus initiated the research throughout the world

on mycoremediation, establishing the fact that fungi can be successfully used in bioremediation

(Singh, 2006).

The term mycoremediation can be broken down as myco (fungus) and remediation (to

clean, resolve, or correct), and indeed, mycoremediation is the use of fungi, specifically

mushrooms, for creating simple yet effective biomass capable of breaking down environmental

and industrial pollutants. The mycelium is a sort of self-healing filter that targets specific organic

compounds and pollutants. Research has proven the efficacy of using fungi to degrade

contamination such as PCBs, aromatic hydrocarbons, and oil spills. Biological pollutants,

especially E. coli, have been of special interest in recent years, and a wealth of data now supports

the benefits of mycoremediation in reducing or eliminating such pathogenic organisms. Jagtap et

al. (2003) discussed mycoremediation as a form of bioremediation in which more contaminated

sites are converted into less contaminated sites by the use of fungi. Mycelium stimulates

microbial and enzyme activity and thus, reduces in-situ production of toxins. The potential

applications for mycoremediation technologies have been reported from time to time. Fungi have

been shown to accumulate toxic metals and even rare earth elements. Fungi are great

biodegrades and the resultant compost has been used to enhance the growth of plants as well as

bioremediation activity in the environment. Mycelia of fungi are unique among microorganism

having the ability to enhance plant growth. They secrete variety of extracellular enzymes

involved in pollutants degradation. Some fungi are hyperaccumulators and are capable of

absorbing and concentrating heavy metals in the fruiting bodies of mushrooms (Jagtap et al.,

2003).Mycoremediation practices involve mixing of mycelium (the vegetative part of a fungus)

into contaminated soil; placing mycelial mats over toxic sites; or and even the combination of

these two techniques. Mycoremediation has been applied to oil spills, contaminated and polluted

soil, industrial chemicals, contaminated water and even farm waste (Bennet et al., 2001). They

reported that bioremediation technology leads to degradation of pollutants and may be a lucrative

and environmentally beneficial alternative. Many reports have published to emphasize the role of

mushroom in bioremediation of wastes by the process of biodegradation, biosorption and

bioconversion. Mushroom can produce extracellular peroxidases, ligninase (lignin peroxidase,

manganese dependent peroxidase and laccase), cellulases, pectinases, xylanases and oxidases

(Kulshrestha et al. 2014). These are able to oxidize recalcitrant pollutants in vitro. These

enzymes are typically induced by their substrates. These enzymes have also been found to

degrade non-polymeric, recalcitrant pollutants such as nitrotoluenes (Kulshrestha et al. 2014),

PAHs (Kulshrestha et al. 2014), organic and synthetic dyes, and pentachlorophenol under in vitro

conditions (Kulshrestha et al. 2014). Recently, it is reported that mushroom species are able to

degrade polymers such as plastics (Kulshrestha et al. 2014). The biodegradation mechanism is

very complex. The reason is the influence of other biochemical systems and interactions of

lignolytic enzymes with cytochrome P450 monooxygenase system, hydroxyl radicals and the

level of H₂O₂ which are produced by the mushroom.

Role of fungi in mycoremediation: Fungi have been harnessed in many diverse applications

since thousands of years ago. In an ecosystem, they are among the major decomposers of various

complex polymers as - cellulose, hemicelluloses and lignin etc. Fungi have the ability to store,

release various elements and ions and they can even accumulate toxic elements (D‟Annible et

al., 2006). An edible and medicinal fungus also plays an important role as natural environment

remediator (Pletsch et al., 1999). The goal of mycoremediation is to stimulate microorganisms

with nutrients and other chemicals that will enable them to destroy the contaminants.

Mycoremediation is an innovative biotechnological application that uses living fungus for in situ

and ex situ cleanup and management of contaminated sites (Thomas et al., 2009).

Mycoremediation is not widely used at present, but the above applications suggest its broader

potential. Fungi perform a wide variety of functions in ecosystem and may be a clean, simple and

relatively inexpensive method of environmental remediation, especially if species native to each

site are used. Mycoremediation is a form of bioremediation that uses native fungi and fungal

mycelium applied to surface soils to remove and degrade contaminants (Thomas et al., 2009).

Mycoremediation and xenobiotics: Contamination of soil and water by toxic pollutants is a

worldwide problem. These contaminants include Petroleum hydrocarbons, polycyclic aromatic

hydrocarbons (PAHs), halogenated organic compounds, dyes, nitrogen containing xenobiotics,

pesticides and inorganic pollutants (heavy metals). These chemicals are called „Xenobiotics‟ [G.

xenos = foreigner, stranger; bios = life] since these compounds differ substantially in chemical

structure from natural organic compounds and these are relatively recalcitrant to biodegradation.

Certain substituents such as halogen, sulfo-, azo- or nitro-groups, particularly the accumulation

of such groups and specific substitution patterns, confer xenobiotic character to a synthetic

compound. Moreover, the electron-withdrawing character of these substituents generates an

electron deficiency and thus makes the compounds less susceptible to oxidative catabolism. Loske

et al. (1992) reported the main contaminants of polluted soils as follows:

1. Petroleum hydrocarbons: Petroleum hydrocarbons contain a complex mixture of

compounds that can be categorized into four fractions: saturates, aromatics, asphaltenes, and

resins. The saturated fraction consists of straight-chain alkanes (normal alkanes), branched

alkanes (isoalkanes), and cycloalkanes (naphthenes). The aromatic fraction includes volatile

monoaromatic hydrocarbons such as benzene, toluene, and xylenes; polyaromatic hydrocarbons

(PAH) such as naphthenoaromatics; and aromatic sulfur compounds, such as thiophenes and

dibenzothiophenes. The asphaltene (phenols, fatty acids, ketones, esters, and porphyrins) and

resin (pyridines, quinolines, carbazoles, sulfoxides, and amides) fractions consist of polar

molecules containing N, S, and O₂. Asphaltenes are large molecules dispersed in oil in a colloidal

manner, whereas resins are amorphous solids truly dissolved in oil.

2. Polycyclic aromatic hydrocarbons (PAHs): PAHs are released into the environment as a

result of a variety of activities, such as incomplete combustion of fossil fuels, shale oil, and

cigarette smoke; accidental discharge of petroleum or during the use and disposal of petroleum

products; and coal gasification and liquefaction.

Fig1: Initial steps in the pathways of degradation of PAHs by fungi

and bacteria (Loske et al. 1990)

3. Halogenated organic compounds: The relatively great electronegativity of halogens confers

chemical stability of the compound, making these recalcitrant to biodegrading. Halogen

substituents can increase the hydrophobicity of the compounds, increasing their tendency to

bioaccumulate in food chains as well as to sorb to soil. Finally the halogen substituents can

contribute to harmful biological effects of the compounds, increasing their toxicity, mutagenicity

and other detrimental capacities. Some xenobiotic halogenated organic compounds are

pentachlorophenol (PCP), trichloroethene (TCE), 2, 4-dichlorophenoxyacetic acid (2, 4-D),

polychlorinated biphenyl (PCB), dioxins.

4. Dyes: Synthetic dyes are employed increasingly in the textile, paper, cosmetic,

pharmaceutical, and food industries, due to high stability, wide variety of color, and good cost-

effectiveness in synthesis compared to natural dyes. These are also used in printing industries,

color photography, and as additives in petroleum products. Environmental control of dyes is

important due to their possible toxicity and carcinogenicity.

5. Pesticides: Synthetic pesticides have been known since 1939, when the insecticidal properties

of DDT were discovered (Tessier, 1982). Pesticides are used extensively related to agriculture,

animals, and humans to protect the public health. The extensive use of pesticides has contributed

to the contamination of many terrestrial and aquatic global ecosystems due to their extreme

toxicity and persistence in the environment. Based on their applications in agriculture, animals,

and humans, pesticides are divided into three categories: insecticides, herbicides, and fungicides.

Insecticides, herbicides, and fungicides are further classified into different categories based on

chemical composition and organic grouping.

6. Heavy metal toxicity and its sources: The term „heavy metals‟ strictly refers to metallic

elements which have a specific mass > 5 gcm-³ and able to form sulphides. Zn, Cu, Mn, Ni and

Co are essential nutrients and are toxic at high concentration; Cd, Pb, As and Hg are non-

essential with no known biological function and are toxic at low concentration. After entering

within the cell through specific uptake system, heavy-metal cations, such as Hg²+ and Cd²+ tend

to bind to SH groups, and inhibit the activity of sensitive enzymes. Other heavy-metal cations

may interact with physiological ions, thereby inhibiting the function of the respective

physiological cation. The sources of the metals in the soil are diverse, including burning of fossil

fuels, mining and smelting of metalliferous ores, municipal wastes, fertilizers, pesticides, sewage

sludge amendments, effluents from industries like electroplating, leather tanning, wood

preservation, pulp processing, steel manufacturing, etc. To survive under metal-stressed

environment, microorganisms have evolved several mechanisms. They can change or reduce the

toxicity of metallic contaminants through pH change, biosorption and bioaccumulation.

Biosorption consists of a metabolism-independent binding of metal ions to negatively charged

free groups in several biopolymers that form the microbial cell wall whereas bioaccumulation

employs an energy-dependent metal influx. They produce intracellular / extracellular enzymes to

resist the metal concentration or they possess the processes of active transport of metal ions

outside the cell, masking metals by chelating, enzymatic transformation of metal ions, creating

vacuoles in which metal ions are gathered and immobilization in the form of polyphosphates,

increased production of melanin and other pigments, and production of specific metal binding

compounds (e.g. metallothienins) inside the cell. Biovolatilization is an enzymatic conversion of

organic and inorganic compounds of metal (loid) s into their volatile derivatives by an

intracellular biochemical reaction (biomethylation).

How does mycoremediation work?

In order for the fungal cultures to do their work, the extrinsic and intrinsic growth factors viz.,

right temperature, nutrients and amount of oxygen must be present in the soil and groundwater.

The right combinations of these cultures can eat the pollutants until they disappear. After the

process of remediation is over, the fungal mycelia themselves disappear because there‟s no more

pollution for them to eat. Fungi are proficient bioremediators by breaking down long chained

toxins into simpler less toxic chemicals. They remove metals from land by channeling them to

mushroom fruiting bodies for removal. They essentially use and digest these toxins as nutrients.

Even the enzymes secreted from mycelium can decompose some of the most resistant hazardous

toxin materials made by humans or nature. These toxins are vulnerable to enzymes secreted by

the mycelia. Fungi possess the biochemical and ecological capacity to degrade environmental

organic chemicals and to decrease the risk associated with metals, metalloids and radionuclides,

either by chemical modification or by influencing chemical bioavailability. Furthermore, the

ability of these fungi to form extended mycelial networks, the low specificity of their catabolic

enzymes and their independence from using pollutants as a growth substrate makes them well

suited for bioremediation processes (Hauke et al., 2011).

Potential of mushrooms in mycoremediation: Although, bioremediation by bacterial agents

has received attention of workers, the role of fungi has been inadequately studied. The ability of

fungi to transform a wide variety of hazardous chemicals has aroused interest in using them for

bioremediation. Mushroom forming fungi (mostly basidiomycetes) are amongst nature‟s most

powerful decomposers, secreting strong extra cellular enzymes due to their aggressive growth

and biomass production (Elekes and Busuioc, 2010). These enzymes include lignin peroxidases

(LiP), manganese peroxidase (MnP) and laccase, etc. Thus, carbon sources such as sawdust,

straw and corn cob can be used to enhance the degradation rates by these organisms at polluted

sites (Adenipekun and Lawal, 2012). Based on literature and studies, white rot fungus accounts

for at least 30 per cent of the total research on fungi used in bioremediation (Adenipekun and

Lawal, 2012).

Advantages of mycoremediation: Mycoremediation technologies assist fungal growth and

increase its population by creating optimum environmental conditions for them to detoxify the

maximum amount of contaminants. A fungus produces various enzymes which are non-specific,

means that they can act on various environment pollutants. There are numerous advantages of

using mycoremediation over commercialized technologies, including the following:

1. It is a natural system, and does not introduce any corrosives or harmful chemicals for

cleanup.

2. The process is environmental friendly and works on a variety of organic and inorganic

compounds.

3. Mycoremediation is expected to be safer than most other alternatives of bioremediation.

It does not require digging up contaminated products, and disposing of it at waste sites.

4. The technology is simple than many other alternatives.

5. Low maintenance and reusable of end products.

6. The cost of using mycoremediation is relatively low in comparison to other technologies

and treatment methods, as it does not require building of new structures.

7. The technology shows immediate results. There is immediate mitigation of odor and

visible improvement to a site. For end results, mycoremediation is quicker than other

technologies, such as phytoremediation and bacterial bioremediation.

Constraints for mycoremediation: The use of higher fungi like mushrooms has been known in

the remediation of polluted soil for some years only. Research has shown that mushroom species

like P. ostreatus and P. chrysosoporium have emerged as model systems for studying

bioremediation. But, a great deal still remains to be learned about the basic knowledge of how

this white-rot fungus removes pollutants. Mycoremediation is a very important process but still

there are various problems that are hindering the potential of mycoremediation. Boopathy (2005)

discussed some of the factors limiting bioremediation technologies.

Future prospects and challenges: Recent advancements - the addition of required potential

fungal strains to the soil and the enhancement of the indigenous microbial population and its

ability to break down various contaminants have proven successful. Whether the fungal mycelia

are native or newly introduced to the site, the process of destroying contaminants is important

and critical for understanding mycoremediation. Further, the application of this technology in

large scale projects will demand much more work to streamline the methodologies. Once the

research and development gets started, the technology must pass evaluations at the local, state

and federal levels, which requires funding and also the time to do so. With appropriate funding,

certain products could be developed and made available for licensing and commercialization.

However, current funding has been limited. But extensive research needs to be pursued as the

technology has proven successful. Researchers feel that this technology is expected to be faster

and more cost effective than other remediation technologies once it is commercialized. The use

of fungi for remediation would allow commercial concern to offer inexpensive, safe products to

their customers. If the underexploited potential of fungus mycelium is further exploited, it will

go a long way in eradicating pollution from soil (Thakur, 2014).


Mycoremediation, the process of detoxifying environmental contaminants, is a technique of bioremediation. Bioremediation includes two different approaches, biostimulation and bioaugmentation. Biostimulation is the stimulation of pollutant-degrading microbes that already exist in the environment by aeration, nutrient addition, or changing the environmental conditions in other ways to optimize degradation. Bioaugmentation is the addition of microbes, fungi, or plants to the contaminated area to enhance degradation of target compounds. Bioaugmentation is used when the organisms required to degrade the contaminant are not present, or when they are too sparse to be effective [1]. Culturing and reinoculating the contaminated area with microbes that are already naturally present is thought to be more effective than adding non-native cultivars [2], because the native organisms are presumed to be well suited to other components of their environment. In the context of mycoremediation, fungi isolated
from the environment may be tested to determine whether they are capable of degrading the contaminant, and following studies of autochthonous contaminant-degrading microbes could reveal whether bioaugmentation is required. 2. Methods This review was conducted as part of a project to examine the potential for mycoremediation of soil contaminated by leaking diesel storage tanks in rural Alaska off the road system. Unique challenges to diesel spill cleanup in such circumstances include cold soils and the impracticality or extreme cost of the use of heavy equipment. The review served as a basis for the design of a multifactorial experiment involving the requirements of the commercially available fungal inoculant Pleurotus ostreatus on different substrates at a range of temperatures. The search began with a review of bioremediation experience in general, especially in circumstances with limited options for large scale mechanical alteration of soil. Lessons learned from previous experience of the Leigh Lab at the University of Alaska Fairbanks in bioremediation of a diesel-contaminated site in Alaska

provided themes that served as useful guides. The search first examined mycoremediation in general, and then the degradation activity of white rot fungi. Finally, the search focused on available experience with Pleurotus ostreatus, and temperature influence, particularly its activity at cold temperatures. Specific searches were conducted for the terms “petroleum”, “diesel”, “degradation” and “bioremediation”. To investigate mycoremediation of diesel with Pleurotus ostreatus, the keywords “Pleurotus”, “mycoremediation”, and “white-rot fungi” were entered into Web of Science. Combinations of keywords and the addition of “temperature”, “cold” or “arctic” were used to search for temperature effects. Finally, useful leads were obtained in this fast-developing field from the Alaska Oil Spill Technology Symposium.

3. Results and Discussion 3.1 History The ability of fungi to break down phenols and aromatic hydrocarbons in wood has been known for decades. In the 1960s, Dr. Horst Lyr described ligninolytic degradation by white rot fungi [3]. White rot fungi are wood decomposers that preferentially degrade lignin, leaving behind pulpy, white cellulose and hemicellulose. Bumpus [4] expanded on these initial findings, hypothesizing that white rot fungi could degrade persistent xenobiotics, which have carbon skeletons similar to lignin. Bumpus evaluated the chemical breakdown of polychlorinated biphenyls, trinitrotoluene (TNT), dioxins, and lindane by Phanerochaete chrysosporium. Phanerochaete chrysosporium was the first popular white rot fungal species used to degrade pollutants. Most ensuing mycoremediation research initially evaluated Phanerochaete chrysosporium and its closely related species. White rot fungi are believed to be the only group of organisms capable of completely mineralizing lignin [5]. However, further research has revealed that many different groups of fungi are

capable of at least partially degrading lignin and similar hydrocarbons, including the coprophilic Agaricus bisporus [6], the mycorrhizal Glomus caledonium [7], the airborne Aspergillus fumigatus [8], the black yeast Exophiala xenobiotica [9], a lichen [10] and many other species [5, 10-13]. As investigations of mycoremediation have proceeded, the breadth of pollutants fungi known to degrade has also increased. In addition to petroleum and polycyclic aromatic hydrocarbons, white rot fungi were also found to degrade explosives, pesticides, polychlorinated biphenyls, synthetic dyes, and creosote [14]. Fungi that could live on the pollutant alone, without co-metabolism, were discovered subsisting on volatile organic hydrocarbons [9] and polyurethane [15]. As documentation of additional fungal species and pollutants they degraded grew, a new field emerged, and the term “mycoremediation” was coined in 2005 by Paul Stamets. Mycoremediation refers to any method of detoxifying pollutants using fungi. The concept of mycoremediation came to popular public attention as a result of several TED talks by Stamets and the creation of the website and book “Radical Mycology” by Paul McCoy [16]. 3.2 Mycoremediation Potential Many oil-degrading fungi have been isolated from the environment, some of which are more effective than bacteria in completely degrading crude oil [11, 17]. Studies that are more recent have found that microfungi such as ascomycetes [8], Cladosporium sp. [18], Penicillium sp. and other deuteromycetes [19] isolated from the environment can degrade hydrocarbons. However, in nearly all of these studies of fungal degradation of petroleum products, the species examined were microfungi. Most white rot fungi are basidiomycetes, which consist of a mycelial network that can draw water and nutrients throughout the substrate. In addition, the structure of