Mycoremediation of Hydrocarbon-Contaminated

Natural Sciences International Bachelor

1st semester project

Mycoremediation of hydrocarbon-contaminated

brownfield sites using Pleurotus ostreatus

Group 4 Thor Ekow Sarquah-Djurhuus Torben Callesen Katrine Jakobsen Florin Krijom Signe Skou

Supervisor: Lauren Seaby

Abstract

In urban areas, contamination of soil with petroleum hydrocarbons restricts the amount of land available for use due to the human health hazards posed by the toxic properties of the contaminant compounds. Mycoremediation is a form of bioremediation in which the degradative abilities of fungi are utilized to remove or neutralize harmful contaminants present in the soil and ground water. This project investigates the potential of the white rot fungi Pleurotus ostreatus to remediate hydrocarbon- contaminated brownfield sites, in other words, to clean up oil spills. This is primarily done through literature based research, as well as a review of relevant case studies, and an expert interview with a mycoremediation company. P. ostreatus is relevant for hydrocarbon contamination because it feeds on organic carbon in the form of wood-lignocellulose by degrading lignin using the non-specific extracellular enzymes laccase, manganese peroxidase and versatile peroxidase, which also break down polyaromatic hydrocarbons (PAHs) due to the similarity in molecular structure to lignin. Since most native microflora and simple bacterial remediation techniques are able to degrade lower molecular weight hydrocarbons, the benefit lies primarily in the ability of P. ostreatus to degrade the more recalcitrant, higher molecular weight components, such as >5-ring PAHs, making them more accessible to other decomposers as well as the fungus. This is supported by numerous in situ and in vitro studies, which show that the use of P. ostreatus in cooperation with soil microorganism results in between 40 and 90% of degradation of total hydrocarbons present. Site conditions such as physical environmental factors and the presence of co-contaminants, such as certain heavy metals, play a significant role in the efficiency of remediation. Ex situ mycoremediation is time wise more efficient than in situ methods, but involves higher costs. Compared to bioremediation with bacteria, P. ostreatus needs less attention in regards to maintenance once it has been implemented, decreasing cost and labor requirements. In conclusion, mycoremediation using P. ostreatus has high potential for contribution to the bioremediation of brownfield sites.

Contents Abstract ...... 1. Introduction...... 1 1.1. Research Question: ...... 2 1.1.1. Sub questions: ...... 2 1.2. Hypotheses: ...... 2 2. Method ...... 2 3. Theory ...... 3 3.1. Brownfield sites ...... 3 3.1.1. Definition ...... 3 3.1.2. Urban effects of brownfield sites ...... 4 3.1.3. Occurrence of contaminants ...... 4 3.1.4. Contaminant nature and behavior ...... 5 3.1.5. Toxicity ...... 9 3.2. History: Bioremediation ...... 11 3.3. History: Mycoremediation ...... 12 3.4. Introduction to P. ostreatus ...... 14 3.4.1 Name and taxonomy ...... 14 3.4.2. Anatomy ...... 14 3.4.3. Lifecycle ...... 15 3.5. P. ostreatus as a decomposer ...... 16 3.5.1. Saprotrophic Fungi ...... 16 3.5.2. Degradation of hydrocarbons ...... 17 3.6. Fungal enzymes and the chemical degradation of lignin ...... 18 3.6.1. Laccases vs Peroxidases ...... 18 3.6.2. Laccase ...... 18 3.6.3. Haem-peroxidases ...... 20 3.6.3.1. Manganese peroxidase ...... 21 3.6.3.1. Versatile peroxidase ...... 21 3.7. P. ostreatus cultivation ...... 21 3.7.1. Media for cultivation ...... 23 3.8. Growth conditions and factors ...... 23 3.8.1. Temperature...... 23

3.8.2. Carbon/Nitrogen ratio ...... 24 3.8.3. Heavy Metals ...... 26 3.8.4. Other growth requirements ...... 27 3.9. Other bioremediation technologies ...... 27 3.9.1. Microorganisms: Bacteria ...... 28 3.10. Application of bioremediation ...... 29 3.10.1 In Situ ...... 29 3.10.2. Ex situ ...... 30 3.10.3. Soil Analysis ...... 31 3.11. Application of mycoremediation ...... 33 4. Analysis ...... 34 4.1. Suitability of Mycoremediation ...... 34 4.1.1. Degradative capability of organisms ...... 35 4.1.2. Rate of degradation and bioavailability ...... 35 4.1.3. Harmful byproducts ...... 37 4.1.4. Appropriate site conditions for biodegraders (in situ) ...... 39 4.1.5 Mycoremediation in controlled conditions ...... 41 4.1.6. Economic viability ...... 41 4.2. Email interview: Fungi Perfecti ...... 44 4.2.1. Barriers for implementation ...... 44 4.3. Case study support ...... 46 5. Discussion ...... 47 6. Perspective ...... 49 6.1. Mycorrhizae hyperaccumulation of heavy metals ...... 49 6.2. Mycoremediation in non-urban areas ...... 50 6.3. Bioaccumulation ...... 50 6.4. Gene expression ...... 50 6.5. Alternative fungal species for mycoremediation ...... 50 Bibliography ...... 51 Appendix ...... 57

1. Introduction

Petroleum hydrocarbons have become heavily integrated in modern society, ranging in use from fuels such as gasoline or diesel to construction materials for roads, such as residues like tar. Due to the widespread use of these substances in both industrial and domestic areas of the urban environment, contamination inevitably and frequently occurs, primarily in soil, leading to the pollution of land and the subsequent placement of this land under the category of ‘brownfield site’. This potentially valuable and highly-demanded land in urban environments is made unfeasible or unusable due to the presence of such contamination, which may also potentially threaten the health of both the local ecosystem and human population. To demonstrate a particular case; the percentage of land in Romania which is contaminated is near 4%, or 9000km2 (Oliver et al., 2005), most of which is located within or bordering urban areas, as is the nature of brownfield sites. This corresponds to an area roughly the size of Zealand in Denmark, which at the very least is unavailable for uses such as cultivation or development. Urban areas are also constantly subject to further development and therefore expansion, thus the demand for such land is constantly increasing. Since similar issues can be found in most places worldwide, brownfield sites can be seen as a global issue.

Bioremediation is the use of organisms with degradative capabilities to decontaminate soil, and provides one solution to these issues by remediating brownfield sites. This makes the land available for reuse, which would hold numerous benefits for society in both the short term, by lessening or removing any health hazards, and in the long term by potentially increasing the productivity of an urban area without expanding outwards. Furthermore, this method is relatively environmentally- friendly and non-destructive. However, as with any technology, there are numerous limitations. Several investigations, some included in this report, find that fungi in bioremediation, improve numerous aspects such as time, cost efficiency and effectiveness of remediation in relation to traditional bioremediation methods. This might give bioremediation the advantages needed to make it a more globally recognized and implemented solution than it currently is.

Although many fungal species have been investigated, Pleurotus ostreatus has been selected as the focal species for this project due to a number of its characteristics, such as its ability to degrade high molecular weight aromatic PAHs.

Thus, this project will aim to investigate the following:

1.1. Research Question:

How can mycoremediation using P. ostreatus contribute to bioremediation of soil in hydrocarbon- contaminated brownfield sites?

1.1.1. Sub questions:

1. Is mycoremediation viable in relation to current methods? a. What are the economic costs involved? b. Where can the raw materials needed for mycoremediation be sourced? 2. What makes P. ostreatus particularly suitable for mycoremediation? a. What are the optimal conditions required for the growth of P. ostreatus? b. To what extent does P. ostreatus rely on other organisms for the purpose of remediating soil? c. What makes P. ostreatus capable of degrading hydrocarbons? 3. Why are hydrocarbon-contaminated brownfield sites a problem for society? a. What are the disadvantages of brownfield sites in urban planning? b. What are the harmful effects of oil in soil? c. What chemical contaminants are present in oil?

1.2. Hypotheses:

1. P. ostreatus, if implemented at a brownfield site, can improve the results of bioremediation by degrading recalcitrant hydrocarbon compounds unavailable to most other decomposers.

2. P. ostreatus mycoremediation is relatively economically viable compared to existing methods and applicable under a wide range of conditions.

2. Method

The problem oriented research within the research area and semester constraint is conducted to address the issue of brownfield sites. In order to guide the project report, a hypothesis is established. 2

Relevant scientific literature including textbooks, reports of experiments and papers are consulted to gain a theoretical knowledge on the different aspects of the subject, including brownfield sites, bioremediation in general, the history of mycoremediation, P. ostreatus and its enzymes and chemical process of breaking down hydrocarbons. With this an overview is achieved of the current states of research within the subject, the constraints for implementation and the strengths and weaknesses of the technology of mycoremediation. These are necessary for drawing conclusions and answering the research question while supporting or rejecting the set hypotheses.

Additionally, the comparison of several relevant case-studies provides an overview of results and the effectiveness of P. ostreatus in mycoremediation. Compiling the basic information in tabular form assists in creating a summary of this data for easy reference.

Furthermore, the mycology company, Fungi Perfecti, contributed an email interview, which provides anecdotal answers to various questions that add support in the analysis and discussion of the theory. A comparison and analysis of the results from literature research, answers from the email interview and the case studies together forms a conclusion.

3. Theory

3.1. Brownfield sites

3.1.1. Definition

The term ‘brownfield site’ varies in usage and definition from one country to the next (Oliver et al. 2005). These definitions, while all referring to disused land, can have different implications. For example: The Danish Environmental Protection Agency defines brownfield sites as “Land affected by contamination’’, whereas in Germany the definition “Inner city buildings not under use. Inner city areas for redevelopment and refurbishment” is used. To reduce miscommunication and create a general understanding, CABERNET (Concerned Action on Brownfields and Economic Regeneration) created the following international definition:

“Sites that have been affected by the former uses of the site and surrounding land; are derelict and underused; may have real or perceived

contamination problems; are mainly in developed urban areas; and require intervention to bring them back to beneficial use” (Oliver et al. 2005).

For this project, only brownfield sites which have been confirmed as contaminated with hydrocarbons will be dealt with.

3.1.2. Urban effects of brownfield sites

The core reason for urban sites being decommissioned and classified as unsuitable for development is the toxic effects that arise from the pollutants which are present in many brownfields (Paull, 2008), as can also be inferred from the fact that most definitions of brownfield sites are centered around the issue of contamination (Oliver et al., 2005). The greatest direct effect of the pollutants’, in this case hydrocarbons, toxicity would be ecological and human health hazards (Dindar et al. 2013), and the primary indirect effect, although just one of many, would be the lack of usable urban land as a result of this (Paull 2008). This lack of useable land within urban areas causes cities to expand outwards into undeveloped (greenfield) land, which has a high environmental impact and leads to an increase in urban sprawl. This effect is amplified since inner urban sites tend to be used more intensely than greenfield sites developed for the same purposed, as shown in a study in the US in 2001 (Paull 2008), which found that every 1 acre (4046m2) used in remediated brownfield land had the same efficiency as 4.5 acres (18210.9m2) of newly developed greenfield land (based on urban vs suburban land use tendencies and regulations), therefore for every 1 acre’s worth of inner city space, 4.5 acres of greenfield land need to be developed. As a result, the productivity of a city in relation to its size could be is decreased. Instead of making full use of land within the urban environment by utilizing its full potential for infrastructure and therefore job and investment opportunities within existing city limits, the urban area extends outwards to achieve the same level of productivity at the expense of undeveloped land (Paull 2008).

The potential for human and ecological health hazards is discussed further on in Section 3.1.5.

3.1.3. Occurrence of contaminants

To fully understand the nature of the brownfield sites particular to this project, and subsequently their remediation, one must understand the nature of the pollutants causing them.

Hydrocarbon land-contamination can arise from many different sources and impact various media, such as soil, water or air, which are often interlinked. In urban areas, they are released into the ground mainly through leakage of storage tanks or pipes, accidental oil spillage or improper disposal (Dindar et al. 2013), such as that of used car oil in maintenance areas. A toxicological report done in 1994 shows that the largest source of oil contamination (47.4% in the US) at the time was from storage facilities, such as above and underground tanks, followed by waste from petroleum refineries (17.9%), used motor oil (10.4%) and then fuel station leakage (3.9%), not including the evaporative loss of more volatile components (Todd, Chessin, and Colman 1999). Although these statistics are outdated and can of course vary from country to country, they do demonstrate some major contamination sources.

These modes of contamination release occur mostly in cities (Dindar et al. 2013), in industrial areas, since heavy industry is generally the greatest urban user of petroleum products, such as fuels for heavy machinery or heating applications (H. McKee 2016), or as constituents of actual production processes themselves (e.g.: oil refinery), which often lead to contamination. Thus, industrial areas generally have the highest levels of hydrocarbon soil contamination (Oliver et al. 2005). However, some sources of contamination, like fuel stations, are present in almost all areas of any given city, and there is always some release due to other commercial or private use (Todd, Chessin, and Colman 1999).

As can be inferred from the above processes, petroleum hydrocarbons usually enter soil in liquid or semi-liquid state. The subsequent behavior of the oil depends very much on its composition, which can be incredibly complex and variable (Todd, Chessin, and Colman 1999)

3.1.4. Contaminant nature and behavior

To clarify in broad terms, Table 1 states the general composition of the most common commercial hydrocarbon mixes.

Table 1: description of the 9 groups of petroleum substances (H. McKee 2016)

The following properties detailed in a), b) and c) are relevant to all contaminants and are important for determining their environmental behaviors’ and subsequent handling. a) Water Solubility Despite the fact that most hydrocarbons are strongly hydrophobic, solubility is still a key factor. Due to their lack of polarity or ability to form ions, hydrocarbons do not form dipole- dipole or hydrogen bond interactions with water molecules, thus they are water-repellant (hydrophobic), tending to aggregate into a separate, totally immiscible phase when numerous hydrocarbon molecules are present. This effect is stronger in larger hydrocarbons than those of smaller weight, and in aliphatic hydrocarbons compared to aromatic ones (Todd, Chessin, and Colman 1999). Despite their general hydrophobicity, some components, mainly lighter aromatic compounds like benzene, still enter groundwater as a small percentage usually does manage to dissolve in water or at least be transported in suspension if a high enough concentration of contaminants is present on site (Crawford and Crawford 2005).

b) Octanol/water partition coefficient (LogKoc) The partition coefficient deals with the ratio of a compound's concentration in a mixture of two immiscible phases at the state of equilibrium. These two phases represent the different media that are present in soil and that the compound can attach to or exist in: the soil itself (solid) or groundwater (liquid). The ratio describes the tendency of a compound to exist in each of the two phases at equilibrium and is closely related to solubility. Low values (<10) are hydrophilic. These substances exist mostly in liquid medium. Compounds with high values (>104) are hydrophobic, thus exist mainly in the soil medium. Organic compounds range from 10-3 to 107. A high partition coefficient and low water solubility of a compound results in strong adsorption of solids (sorption), thus low mobility since the compound clings strongly to the soil. The extent of this varies according to soil type. A low partition indicates higher mobility, as the compound is less attracted to the soil more likely to move through groundwater (Crawford and Crawford 2005).

This links strongly to bioavailability, which is discussed in Section 4.1.2.

Vapor pressure and Henry's Law constant (KH) Vapor pressure and Henry´s Law constant measure the liquid-air partitioning of a substance (Crawford and Crawford 2005). Vapor pressure is defined as the pressure exerted by the vapor of a chemical when in equilibrium with its solid or liquid form (Todd, Chessin, and Colman 1999). The higher the vapor pressure, the more likely a substance will volatilize.

In the case that the petroleum liquid contains many components the Henry´s law coefficient indicates the partial vapor pressure which is proportional to a particular fraction in the liquid. In other words: the tendency of an individual compound to volatilize from a mixture.

pA= (cA)(KHA)

pA- partial vapor pressure of the component A

KHA- Henry’s law constant for component A at a given temperature

cA- mole fraction of component A in the liquid

From this it is seen that the higher the concentration of a faction in a mixture, the more likely

it will evaporate. A higher KH also indicates a higher likelihood of evaporation from a mixture. If pressure is exerted on a gas in dynamic equilibrium with a liquid, more gas will liquefy and the solubility increases. If the temperature of both the system increases, more of the substance will volatilize and exist in gas phase, thus the solubility decreases (Crawford and Crawford 2005).

These factors, mainly solubility and volatility, are highly affected by environmental factors and are usually very specific to the conditions of each site.

Table 2 illustrates the trends of the above three properties.

Table 2: Physical properties of petroleum compounds (Todd, Chessin, and Colman 1999).

From this it can be concluded that when a hydrocarbon mix has reached equilibrium with its environment, most of the airborne components will be short-chain aliphatic compounds, e.g.: propane, since they have the highest vapor pressure and KH values, and most waterborne ones will be low-weight aromatics, e.g. benzene. (H. McKee 2016), since these have the highest solubility and lowest LogKoc. The larger molecules with high LogKoc values, low solubility and low vapor pressure 8 will remain relatively immobile in the soil, although they too may undergo a degree of movement in the form of bulk oil migration in some cases due to gravity and capillary action (Todd, Chessin, and Colman 1999). These modes of contaminant migration may to varying extent cause contamination in areas other than where the hydrocarbons were released.

Frequently, in areas of highly concentrated oil-contamination the hydrocarbon mix aggregates into a central subsoil non-aqueous liquid mass just above the groundwater table due to non-polar attraction (van der Waals forces) and hydrophobic interaction with the water-saturated soil. This mass then propagates numerous outwards-moving hydrocarbon plumes. It is from the edges of these plumes that some hydrocarbon components usually enter the actual soil or groundwater (Todd, Chessin, and Colman 1999). This mass will consist primarily of higher-weight compounds as the more mobile ones will mostly have volatilized or migrated through groundwater by the time the mass has reached equilibrium. However, many of these mobile compounds will also remain as part of the mass as solubility is lowered when they are part of a hydrocarbon mix versus when they are isolated (Todd, Chessin, and Colman 1999) presumably due to higher van der Waals forces with larger molecules.

Aside from enlarging brownfield sites through spreading, these pollutant traits greatly influence the threat they pose to human and ecological health.

3.1.5. Toxicity

The details of the hazards posed by hydrocarbon contamination are difficult to assess due to the highly variable composition, and therefore behavior, of any given hydrocarbon mix found in contaminated soil (Todd, Chessin, and Colman 1999). However, a broad overview including the rough toxicological profiles of some representative compounds would serve to give an idea of some direct threats that might be posed by such contamination. Note: the examples given are for singular compounds which may vary greatly in behavior even from other compounds in the same categories. a) The following is a profile of benzene, to give an example of the risk that may be associated with low-weight aromatic compounds. Benzene consists of a single ring of six carbon atoms (EC6) each bonded to a single hydrogen. This compound has a variety of well-categorized adverse effects on both humans and animals even at low levels of exposure. It damages the hematological system by affecting hematopoiesis - the formation of blood cells, as well as components of the lymphoreticular and immune systems (Todd, Chessin, and Colman 1999). It has also been found 9

to have highly carcinogenic effects after long-term exposure, particularly through inhalation, and has been associated with high rates of nonlymphocytic/acute myeloid leukemia in chronically exposed workers as well as general tumor formation (neoplasia) in exposed animals (Todd, Chessin, and Colman 1999). Since benzene is a component of gasoline and is one of the most water soluble of all hydrocarbons found in petroleum products as well as being relatively volatile (see Table 2) at ambient temperatures, brownfield sites contaminated with this petroleum fraction carry high health risk, as groundwater contamination is likely, which could lead to human contact through ingestion of contaminated water. Inhalation, of benzene vapor near the contaminated site could also occur. Both of these could lead to the manifestation of the above-mentioned adverse health effects b) Polyaromatic hydrocarbons (PAHs) are higher-weight aromatic compounds containing benzene rings and sometimes other substituents or ring-structures, and have been shown to have a variety of carcinogenic effects (H. McKee 2016). PAHs are highly recalcitrant: their aromaticity means that electrons delocalize and are “shared” between the carbons of the rings through the pi- orbital(pi-bonding), lending the molecule a high stability and low reactivity. This is expressed by their nonpolar, largely hydrophobic characteristics and insolubility (CCME 1999). PAHs are often produced by incomplete combustion of petroleum fuels (Todd, Chessin, and Colman 1999). Although in the above case they are initially released as particles into the air, they often quickly settle, if >C20 (H. McKee 2016), and can therefore enter soil. They are also found in heavy fuel oils (see Table 1). Although they are not volatile nor highly soluble due to their high molecular weight and stability, they may attach to dust particles in contaminated brownfield sites and then possibly cause respiratory issues following inhalation of that dust, or be ingested following water contamination, although this is less likely than with the more soluble components like benzene (Todd, Chessin, and Colman 1999). One test study performed on rabbits also found that heavy fuel oils cause skin tumor formation after dermal exposure (H. McKee 2016), possibly due to the PAH content. Benzo(a)pyrene is one such PAH, consisting of one benzene ring fused to a pyrene molecule, forming 5 rings in total. It has been shown to have similar adverse effects to benzene (Todd, Chessin, and Colman 1999), primarily carcinogenicity: in humans benzo[a]pyrene has been found to metabolize to diol epoxide, which bonds covalently to the guanine bases in DNA and cause mutations (Mikhail and Gerd 1996).

10 c) To give an example of risk associated with aliphatic compounds, n-hexane is used. This is a molecule consisting of six saturated carbons (EC6) in a straight chain. Although not carcinogenic, moderate concentrations of n-hexane have been found to cause depression of the central nervous system as well as numbness (and even perhaps paralysis) in limbs, due to peripheral neuropathy. In higher concentrations, this can lead to adverse respiratory and renal effects (Todd, Chessin, and Colman 1999). Pure n-hexane is primarily used in laboratories, but this compound also makes up 1-3% of gasoline and is commonly found in solvents such as petroleum naphtha, some of which are used as industrial cleaning agents (Harris and Corcoran 1999). The main method for exposure would be inhalation, since n-hexane is a low-weight aliphatic.

These hazards mentioned above are just some of many that could arise from the various types of hydrocarbon contamination in affected brownfield sites, and clearly demonstrate that these sites pose potentially high risk to human and animal health.

3.2. History: Bioremediation

Bioremediation, classified as an applied microbiology with the use of microorganisms to degrade organic matter and toxic chemicals in different kinds of polluted areas (A. Singh, Kuhad, and Ward 2009), has been used over many years, but without wide recognition.

A change in the application and acceptance of bioremediation occurred when an international appeal for a cleaner environment arose: NATO and NATO partner countries realized that soil and groundwater pollution had become a serious threat to the environment (Diels and Vanbroekhoven 2008), and governmental agencies as well as private industries started to fund technologies that supported a sustainable way for the cleanup of contaminated sites. Thus, bioremediation emerged as an effective and cost efficient solution(Diels & Vanbroekhoven, 2008), especially since it can be executed on contaminated surface water and groundwater as well as in soil and has potential for degrading petroleum pollutants (Crawford and Crawford 2005).

3.3. History: Mycoremediation

Mycoremediation is a relatively new form of bioremediation, and its use only spans a few decades, beginning as early as 1966 (Matsumura and Boush 1966). The problem was that the mycoremediation could not compete with the bacterial remediation, because the research of different fungi strain was lacking, which in turn resulted in mycoremediation lagging behind.

In 1966 a study was commenced with the purpose of removing Malathion, which is an insecticide and a neurotoxin with low toxicity for humans. After using 16 different strains of the soil fungi: Trichoderma viride in combination with one strain of bacterium (Pseudomonas sP.). They were able to breakdown Malathion (Matsumura and Boush 1966).

In 1976 a study found out that yeast, a single celled organism belonging to the Kingdom Fungi, was able to digest and grow in water up to 5 days after an oil spill (Jones 1976).

The following year after another study was able to identify several fungi species that were able to metabolize hydrocarbons (Bartha 1977).

Two years after Bartha's discovery, a study found out that sixty different fungi isolates were able to survive a harsher environment than bacteria would be able to. By harsher environments it is meant that the fungi isolates were able to grow in more acidic conditions with scarcer nutrients (J. Davis and Westlake 1979).

In 1986 a study used the same fungi species as Matsumara and Boush did to test its effect on other insecticide such as fenitrothion and fenitrooxon (Baarschers and Heitland 1986).

A year after Baarschers and Heitlands affirmation of Matsumara and Boush's experiment, another group of scientist commenced a study that researched which factors that had an effect on biodegradation in soil. Factors like chemical nature, contaminant concentration, the bacterial community were tested to be affecting biodegradation (Winterlin and Schoen 1987).

Later a study found out that several aquatic yeast species were able to degrade oil (Obiro 1988). In 1993 a group of scientists commenced a study of P. sordida's ability to degrade PAH's. they found that P. sordida is able to degrade PAH's with three and four rings but not above 5 rings (Davis et al. 1993). 12

In 1994 a study found out that when one is degrading pesticides in soil, the process starts out with a high degradation rate, and ends up with a very slow dissipation, which means that the remaining residues are somewhat resistant to further degradation (Alexander 1994).

The following year a study demonstrated that the fungi species Phanerochaete chrysosphorium and P. Sordida had potential regarding PAH degradation. Unfortunately, these two species of white rot fungi showed no ability to degrade PAHs above 5 rings (Haught et al. 1995), although this is not true for all white rot fungi as shown by a later study (Ipeaiyeda, Nwauzor, and Akporido 2015).

A year later, in 1996, a study compared two different fungi, Aspergillus niger and T. viride, in how they were able to degrade the insecticide chlorpyrifos. A. niger was able to degrade the chlorpyrifos with 95.7 % and T. viride with 72.3 % after 14 days. After 14 days, the toxic component of chlorpyrifos was not detected, which means it was degraded completely (Mukherjee and Gopal 1996).

In 1998 a study found out that, between the different factors that affect mycoremediation, water in the soil is one of the more important one (Marin et al. 1998).

In 1999 Lentinus edodes also known as the shiitake mushroom was found to have the ability to remove over 60 % pentachlorophenol which is an insecticide and is highly toxic to humans (Pletsch, de Araujo, and Charlwood 1999).

That year a group of scientist commenced a study where they used the fungi strain P. ostreatus. They found that the P. ostreatus was able to degrade 80 % of the PAH's in soil within 35 days (Bogan et al. 1999).

That same year, another group of scientists compared 3 different mushrooms ability to degrade PAHs: The P. ostreatus, P. chrysosphorium and the Trametes versicolor. Looking at the production of ligninolytic enzymes P. ostreatus stood out as the better strain. Both P. ostreatus and T. versicolor produced similar rates of manganese peroxidase and laccase, while P. chrysosporium produced very low rates of these enzymes (Novotný et al. 1999).

Also in same year IETU grew several fungal strains in an oil-contaminated site near an oil refinery in Poland (IETU 1999).

3.4. Introduction to P. ostreatus

3.4.1 Name and taxonomy

P. ostreatus was first described in 1774 by the Dutch Nikolaus Joseph von Jacquin and in 1871 classified by Paul Kummer in the present genus (Kummer 1871). The taxonomy of the P. ostreatus is showed in Table 3.

Table 3: Taxonomy of P. ostreatus (Kummer 1871)

Kingdom Fungi

Division Basidiomycota

Class Agaricomycetes

Order Agaricales

Family Pleurotaceae

Genus Pleurotus

Species ostreatus

The P. ostreatus , commonly known by its popular name Oyster Mushroom, has a large variety of subspecies, varieties and strains (Stamets 2005), and is mainly known for culinary uses. The genus Pleurotus represents 20% of the world’s annual market for cultivated fungi (Spooner and Roberts, 2005). Due to its popularity throughout the world as a culinary mushroom, the P. ostreatus has many different popular names in English. In this report, it is consistently called by the binominal name P. ostreatus.

3.4.2. Anatomy

Fungi are built up by cells called Hyphae. The hyphae contain the nuclei and the organelles and are divided by cross-walls called septa. The septa have microscopic pores allowing a flow of water and nutrients.

Under the soil the hyphae build up a larger thread-like network just one cell thick called mycelium. This is the main part of the fungi. The mycelium exists in both mono-, di-caryote forms. The fruiting bodies consist of a cluster of hyphae threads and are what are commonly called mushrooms (Spooner and Roberts 2005). The mycelium and fruiting body is illustrated in Figure 1.

Figure 1: General basidiomycetes anatomy (Sharp 1943)

3.4.3. Lifecycle

As stated in Table 3, the P. ostreatus belongs to the division Basidiomycota. The sexual reproduction of Basidiomycota involves basidiospores borne on a club shaped structure called the basidium. In the basidium the diploid cell undergoes meiosis and thereby creates four haploid basidiospores. When the basidiospore is launched from the basidium to favorable conditions in the soil or an old tree stump, it will germinate and form primary mycelium.

Primary mycelium is build up by haploid hyphae that are homokaryon and monokaryon. When the primary mycelium meets with the primary mycelium of a different mating type, they will via plasmogamy fuse to create secondary dikaryotic mycelium. The secondary mycelium continues to

15 divides by mitosis, ensuring that each cell will have a nucleus of each mating type. Secondary mycelium is thicker, about 7µm instead of 4µm, and advances faster. Here, chlamydospores, resting spores that can survive dry and hot conditions and start to germinate when conditions again become favorable, might be created.

From the secondary mycelium, the first stage of the fruiting bodies, called primordia, are formed. From there a young fruiting body evolves containing all the parts of the mature fruiting body. During 6-9 hours’ elongation of the cell will occur and the fruiting body will double its size. From now on most growth occurs from cell elongation, and only very little cell division will take place. The diploid state of the lifecycle is very short, since meiosis occurs right after karyogamy. When the fruiting body reaches maturity the four haploid nuclei move into the four newly developed basidiospores on the basidium (Carlile, Watkinson, and Gooday 1994).

3.5. P. ostreatus as a decomposer

As for all species of the kingdom Fungi, P. ostreatus is heterotrophic. Contrary to autotrophs, like green plants, that are capable of producing their nutrition from inorganic compounds via photosynthesis, fungi have to obtain all their nutrition from their surroundings (H. Singh 2006). This is why fungi function as the ecosystem’s decomposers and recyclers of nutrients and is the essential reason for using Fungi in bioremediation.

3.5.1. Saprotrophic Fungi

P. ostreatus is a saprotroph, meaning that it obtains its organic carbon from discarded and dead material like old logs and dead trees.

In wood the cells are hardened by the polymer lignin that covers cellulose, creating lignocellulose, which is highly resistant to degradation. The lignin is formed of cross linked phenolic polymers and is responsible for the formation of cell walls as they give it rigidity (Sánchez 2009). Fungi absorb their nutrition through the chitinous wall of the hyphae. They can absorb small molecules like amino acids and simple sugars like glucose, but not more complex substances like cellulose. For these substances, the mycelium produces extracellular enzymes that break them down to smaller component that can be absorbed by the hyphae (Brian Spooner 2005).

White rot fungi like the P. ostreatus, produce enzymes to ‘attack’ the lignin structure and force a depolymerization of the organic compound to release the cellulose from the lignin. This makes both the lignin-metabolites and cellulose available as sources of nutrition for the fungus. White-rot fungi vary in their preference for either the cellulose or lignin components of lignocellulose as an energy source. P. ostreatus has been shown to preferentially utilize lignin, degrading cellulose through a variety of celluloses only to a lesser extent (Kerem, Friesem, and Hadar 1992).

The enzymes for breaking down lignin, produced by the mycelium of the P. ostreatus, are manganese peroxidase, versatile peroxidase and laccase, (Marzullo et al. 1995). The chemical process of breaking down lignin is described in section 3.6.

3.5.2. Degradation of hydrocarbons

The molecular structure of the lignocellulosic plant materials is similar to those of PAHs found in petroleum products like diesel and oil (Pozdnyakova 2012), which means that white rot fungi can potentially break down these complex hydrocarbons into simpler organic substances.

Figure : examples of PAHs (http://www.aanda.org/articles/aa/full/2002/30/aa22 Figure 3: molecular structure of lignin (Soygun et al. 2013).64/img14.gif) Figure 4: molecular structures of selected PAHs (Burright 1986).

Figure 3 is an exemplary structure of lignin and Figure 4 shows different examples of heavier polycyclic aromatic hydrocarbons, by comparing the molecular structure from figure 3 and 4 a similar structure can be seen.

Many studies have indicated the P. ostreatus’ ability to break down hydrocarbon in oil-contaminated soil, some of which are shown in Section 4.3.

3.6. Fungal enzymes and the chemical degradation of lignin

The white rot fungi use lignin-modifying enzymes to degrade any suitable recalcitrant organic compounds. The goal is the mineralization of the lignin: the conversion of the organic compound to its inorganic mineral constituents, e.g. CO2 from carbon-containing compounds. The most important characteristic of these enzymes is their low substrate specificity, enabling the depolymerization of some xenobiotics such as PAHs as well as lignin. In Section 3.5.2 examples of both the lignin structure and exemplary polyaromatic hydrocarbons are shown, displaying their structural similarities. The PAHs are discussed in more detail in Section 3.1.5.

As mentioned in the section 3.5.1, P. ostreatus has three major lignin-modifying enzymes to catalyze the breakdown of lignin or similar compounds by oxidative mechanisms: manganese peroxidase, versatile peroxidase and laccase (Aust 1995).

3.6.1. Laccases vs Peroxidases

Peroxidases catalyze the oxidation of various aromatic substrates to cation radicals and laccases oxidize phenolic substrates to their respective radicals. Phenolic compounds are those containing one or more hydroxyl groups (-OH) attached directly to aromatic hydrocarbon groups. These radicals spontaneously rearrange themselves, leading to fission of the carbon-carbon or carbon-oxygen bonds of the alkyl side chains or the cleavage of aromatic rings (Marzullo et al. 1995).

3.6.2. Laccase

Laccase is an oxidase enzyme containing copper in its catalytic center, which is why they are also called multicopper oxidases. In order to perform its catalytic process, laccase needs oxygen as the oxidizing agent (Plácido and Capareda 2015).

Figure 5: Mechanism of laccase (Plácido and Capareda 2015)

Figure 5 illustrates a simple diagram of the laccase redox mechanism (SH = reduced substrate, S* = oxidized substrate). The laccase utilizes oxygen and hydrogen from the substrate to form water, thereby oxidizing the substrate.

It radicalizes a wide range of phenol like compounds by extracting one electron, used to reduce the oxygen to water.

Figure 7: quinone (Djurhuus et al. Figure :6: Phenol phenol molecule (Djurhuus et al. 2016) (http://ehs.ucsc.edu/lab2016) -safety- manual/specialty- chemicals/phenol.html) The oxidized version of a phenol is a Quinone, see Figure 6 & 7. Quinones are cyclic compounds with an oxygen attached by a double bond, thereby breaking the aromaticity. This has increased the redox potential which was needed to break the recalcitrance. After it is reduced, the Quinone can either re-aromatize, but mostly it breaks the conjugation.

Laccase has 4 copper molecules in the active site which participate in the oxygen reduction and water production. Those 4 copper (Cu) molecules are divided up in 2 metallic active sites: 1 Cu in on location and the other 3 in the second location, where copper 3 and 4 are bonded together. The Cu 1 has the highest redox potential in the enzyme thereby enabling the catalysis by oxidizing the substrate

19 to its corresponding radical, although its oxidative power is also affected by the amino acids surrounding the first location. The Cu 3 and 4 which are bonded together participate in the substrate oxidation as electron acceptors. The enzyme’s redox potential varies between 300-800 mV, depending on factors such as the distances between the Cu molecules.

By use of a mediator, Laccase is able to indirectly oxidize phenol-like substrates. Redox mediators are utilized by enzymes to achieve the desired depolymerization process. They are chemical compounds, which act as electron carriers between the enzyme and the substrate. It’s redox potential increase when it gets oxidized by the enzyme, and in order to recuperate the lost electrons they react with the substrate. So it splits the recalcitrant compounds which the laccase wouldn’t have been able to alone (Plácido and Capareda 2015).

3.6.3. Haem-peroxidases

The following two oxidases fall in the group of haem-peroxidases. These kinds of peroxidases contain haem attached to their enzyme, acting as a cofactor. A cofactor is a non-protein chemical compound or metallic ion which the enzyme needs in order to activate the enzymatic process. In this case the cofactor is the haem, an iron ion (Fe) centered in a porphyrin (multiple 5-carbon ring connected)

The following simplifies the redox reaction generally happening in peroxidases:

1. reduced peroxidase + H2O2 -> modified enzyme (compound 1) + H2O through electron-transfer from the reduced substrate (SH) (S* = radicalizes substrate)

2. compound 1 + SH -> compound 2 + S* compound 2 reacts with a second substrate

3. compound 2 + SH -> reduced peroxidase + S* + H2O (Plácido & Capareda, 2015)

The peroxide required for ligninolytic peroxidases is produced by oxidases, such as glyoxal oxidase, a copper radical enzyme.

3.6.3.1. Manganese peroxidase

The manganese peroxidase is the most common peroxidase secreted by organisms to degrade lignin or lignin-like structure. It is made up out of approximately 350 amino acid residues, water molecules and haem group as the active site.

When the heminic group connects with its cofactor peroxide the catalytic cycle begins with a transfer of 2 electrons from the haem to H2O2. The products are compound 1 and water, where the compound 1 then as a second step can oxidize one substrate molecule to a radical and itself gets further processed to compound 2. This compound 2 then oxidizes its Mn2+, which must be present for this reaction to occur, to produce Mn3+. In general manganese is always present in wood and soil. Mn3+ is the actual cation capable of oxidizing phenolic compounds. It is stabilized by fungal chelators, such as oxalic acid and can thereby act as a redox-mediator. It’s small size and high redox-potential enables it to diffuse into the organic compound and remove an electron and proton from the substrate, forming unstable free radicals that tend to disintegrate spontaneously resulting in depolymerization of the substrate (Hofrichter 2002).

3.6.3.1. Versatile peroxidase

The second peroxidase involved is the versatile peroxidase; it is defined by its capabilities to oxidize substrates using the same mechanism as Manganese-peroxidase, utilizing an oxidized manganese ion to react with the phenol-like structures. Aside from that it is also able to oxidize veratryl alcohol, the typical lignin peroxidase substrate and simple phenols.

These hybrid properties are caused by the coexistence of a protein in the catalytic sites similar to those present in the peroxidase families, although a difference in its Mn-oxidation results in efficient Mn2+ oxidation with only two out of three acidic residues forming the binding site (Ruiz-Dueñas et al. 2009).

3.7. P. ostreatus cultivation

“By far the easiest and least expensive [mushroom] to grow” (Stamets 2005). Some main factors affecting the mycelium growth for processing spawn are; cultural media, temperature, carbon and nitrogen presence and lignolytic substrate source (Hoa, Wang, and Wang 2015). The requirements for growth are discussed in section 3.8. 21

A P. ostreatus culture can be started from either spores or tissue. Tissue can come from stem buds or mycelium. Using the tissue would create a clone of the original mushroom, whereas spores create new individuals which are genetically differentiated from the original fungus. The great genetic variability in spores gives them a high adaptively for media and method for cultivation. Some examples for media (see Section 3.7.1) for germination of spores are sugar-salt broth, oil, cardboard, straw, and burlap (Stamets 2005). All these examples are relatively inexpensive and easily available.

The mycelium germinating from spores will form haploid infertile primary mycelium. As described in Section 3.4.3, primary mycelium from spores needs primary mycelium from a different mating type to form secondary mycelium and fruiting bodies. The critical part of cultivating mycelium from spores is that when there is a high concentration of the protein rich spores, they provide a fertile breeding ground for bacteria, which can easily outcompete and devour them. Once the mycelium has matured it produces antibiotics to defend itself, targeting particular bacteria which pose a threat to it (Stamets 2005), thus this is not an issue when using spawn.

Spawn is material impregnated with the mycelium. The material is used to inoculate more massive substrates with the wanted mycelium. Commercial spawn of various fungi species can be bought or spawn can be prepared from wild fungi.

A form of spawn could potentially be sourced from commercial gourmet oyster mushroom farms: for each metric ton of P. ostreatus commercially produced for the food marked at least a metric ton of spent compost is produced as a waste product. This spent compost already contains mycelium and lignolytic enzymes capable of breaking down hydrocarbons (Aguilar-rivera, Moran, and Arturo 2012).

Wild spawn has the advantage of already being adapted to the habitat with high competitors from other species of fungi and bacteria, whereas pure culture spawn, such as from commercial waste, must first slowly be adapted to the complex microbial ecosphere found where it is going to be implemented. The pure spawn grows faster to start with, but have low chances for survival in wild nature, if not slowly adapted before the implementation (Stamets 2005). Natural spawn will take longer to impregnate the substrate, but has greater chances of survival when implemented in wild habitants, (Stamets 2005) such as brownfields sites.

3.7.1. Media for cultivation

P. ostreatus Mycelium can colonize all cellulose containing substrates, including a wide variety of agricultural wastes. Because the lignolytic enzymes of P. ostreatus are nonspecific it is highly adaptable in relation to its media for growth (Hoa, Wang, and Wang 2015). Examples of possible substrates are paper, straw, wood, seeds, (Stamets 2005). One study found that although P. ostreatus grows to some extent over a wide range of carbon sources, glucose, sucrose and molasses were the most favorable for the mycelium growth (Hoa, Wang, and Wang 2015), most likely because the sugars present in these are monomers and therefore more easily available to the fungus. Other isolates of the same species might give different results, due to physiological differences among the species (Kurtzman and Zadrazil 1982).

The fact that such a wide range of easily-available substances can be used is advantageous for P. ostreatus mycoremediation, since it is unnecessary import specialized substrates. This makes it possible to cultivate mycelium almost anywhere with regards to the substrate and therefore also close the brownfield site in need for mycoremediation.

3.8. Growth conditions and factors

3.8.1. Temperature

Temperature is a very important factor concerning mycelium growth. One experiment from Department of Tropical Agriculture and International Cooperation, National Pingtung University of Technology, Pingtung, Taiwan, tested the optimal temperature for mycelium growth of two species of P. ostreatus and P. cystidosus, by inoculation of mycelium in a petri dish and measuring the diameter of the mycelium every day. Figure 8 shows the petri dishes at day 8. Both strains had the fastest growing mycelium at 28 °C, followed by 32°C and 24°C.

Figure 8: The influence of temperature on mycelium growth. 8 days after inoculation in a petri dish with 20 mL sterilized potato dextrose agar the diameters of mycelium were measured. A: P. ostreatus , B: P. cystidosus (Hoa, Wang, and Wang 2015).

Another experiment has shown an optimal temperature of 25-30°C and that the particular strain of P. ostreatus studied grows optimally in Summer and Autumn in sub-tropical regions (Hoa, Wang, and Wang 2015). It has also been found that a strain of P. ostreatus grows well during the summer season in the tropics (Kashangura 2008). The minimum and maximum temperature for the formation of the fruiting bodies of P. ostreatus was found to be 15-33°C in another study (Aguilar-rivera, Moran, and Arturo 2012). This indicates that in terms of temperature mycoremediation with various strains of P. ostreatus could take place at brownfield sites in temperate, sub-tropical and tropical climates, and if not all year round, then for the majority of the year.

3.8.2. Carbon/Nitrogen ratio

Nitrogen is essential to fungi for growth and synthesis of organic compounds containing nitrogen, such as the nitrogenous bases of DNA/RNA, amino acids for proteins and for the cell wall component chitin (Hoa, Wang, and Wang 2015).

A too high Carbon/Nitrogen –ratio (C/N-ratio) can inhibit the mycelium growth. One experiment (Hoa, Wang, and Wang 2015) gives an example of a C/N-ratio being too high: The greatest mycelium growth was found at 1-3% sucrose as a carbon source, with more than 5% sucrose as a carbon source proving to have no improvement of the mycelial growth. This is supported by another experiment 24

(Bai et al. 2012), which showed that the lower carbon source concentration (30g glucose/L) gave the highest mycelium growth and yield of mushroom.

The C/N ratio at brownfield sites can be regulated by the addition of Nitrogen. In the experiment from Department of Tropical Agriculture and International Cooperation, National Pingtung University of

Technology, Pingtung, Taiwan, it was found that ammonium chloride (NH4Cl) is the nitrogen source promoting the greatest mycelium growth. The second greatest mycelium growth was as a result of

Ammonium sulfate (N2H8SO4) fertilization. The growth change upon adding nitrogenous fertilizers was not very significant compared to the control for this strain of Oyster Mushroom (Hoa, Wang, and Wang 2015).

An excessively low C/N-ration is also restrictive for the mycelium growth. A concentration of ammonium higher than 0,09%, in the experiment decreased the growth of mycelium compared to the control.

Figure 9: Effect of different NH3CL concentrations on mycelium growth. PO= P. ostreatus, PC= P. cystidosus. On the y- axis is diameter of the mycelium in cm and on the x-axis is the percent of Ammonium chloride in the substrate. A concentration higher than 0.09% has a restricting effect on the mycelium growth compared to the control (Hoa, Wang, and Wang 2015).

Fungi in general require a C/N ratio of 10, whereas most soil bacteria require a C/N ratio of 5 (Brady and Weil 2007)., meaning that fungi generally require half the amount of nitrogen in relation to carbon sources that bacteria do. This might explain why the growth of P. ostreatus did not change significantly between the fertilized and unfertilized specimens (Hoa, Wang, and Wang 2015).

3.8.3. Heavy Metals

Soil contaminated with hydrocarbons is often also contaminated with a high concentration of heavy metals. This might be a restriction for in situ mycoremediation with P. ostreatus at these brownfield sites (Baldrian and Gabriel 2002). Examples of heavy metals often found in brownfield sites contaminated with hydrocarbons are cadmium, copper and mercury (Baldrian et al. 2000).

Some metals have shown to increase P. ostreatus’ ability to degrade hydrocarbons: Copper and manganese increase the activity of the enzymes laccase and manganese peroxidase as well as the transcription of genes associated with them respectively (Baldrian et al. 2000), likely due to the fact that laccase and manganese peroxidase contain copper and manganese respectively, as mentioned in Section 3.6.2 and 3.6.3.1.

Other heavy metals are known to be toxic for the organism like white rot fungus and bacteria used for bioremediation. The present of these heavy metals can constrict both the growth of the mycelium of the P. ostreatus and the efficiency of its ligninolytic enzymes. The concentration of the different heavy metals and whether the fungus is exposed in a liquid culture or in the soil greatly influences the extent of the adverse effects. Cadmium and mercury have proven especially toxic for the P. ostreatus in liquid culture (Baldrian et al. 2000). For heavy metal exposure in soil the outcome is different; An experiment carried out in 2000 in the institute of Microbiology, Academy of science of Czech Republic (Baldrian et al. 2000), shows that at concentrations of 10 -100 ppm, cadmium or mercury in soil cause no decrease in the breakdown of substrates. Cadmium at the concentration of 500 ppm in soil restricted P. ostreatus’ ability to break down the hydrocarbons. Soil bacteria were not affected. Mercury concentrations of 50-100ppm or cadmium 100-500ppm were found to limit the extent of the mycelium’s penetration of the soil. After the mycelium’s colonization of the soil enzyme activities were the same in all different concentrations of mercury, and for cadmium 10 -100 ppm (Baldrian et al. 2000). This shows that Mercury concentrations up to 100 ppm give a slower start for the implementation of the mycelium and enzyme activity, but do not decrease the degradation of the

26 hydrocarbons once mycelium is established. This study also showed that the activity of soil bacteria was not affected, so for bioremediation of brownfield sites that besides hydrocarbon contain high concentrations of Mercury and cadmium, heavy metal-resistant species of white-rot fungi might be a solution.

3.8.4. Other growth requirements

Moisture level is an important factor influencing whether it is possible for fungi to grow. One study the found the optimal relative humidity for that particular strain of P. ostreatus to be > 70% (Aguilar- rivera, Moran, and Arturo 2012). Since direct sunlight often reduces moisture levels, in soils without excessive moisture content fungi often need shade to prevent evaporation of water from the surrounding soil.

Fungi also need a moderate level of oxygen, since they perform aerobic respiration and oxygen is needed for the extracellular enzymes to function, see section 3.6.

3.9. Other bioremediation technologies

This section gives an overview of different bioremediation technologies which might be used in conjunction with mycoremediation.

Table 4: Soil remediation technologies (A. Singh, Kuhad, and Ward 2009). Specific technologies Description

Landfarming Involves excavation of soil and by placing on lined landfarms and stimulation of natural microbial population by providing nutrients, water, bulking agents and tilling

Biopile, biocells, bioheaps, Involves excavation of soil and placing in heaps or aerated piles, and biomounds, compost cells stimulating microbial activity by providing nutrients, water and oxygen

Slurry bioreactor Involves excavation of soil and treatment in a contained environment such as tanks/reactors by providing oxygen, water and nutrients under controlled conditions for accelerated biodegradation

Bioleaching Clean up of heavy metal contaminated soil using acidophilic bacteria that oxidize reduced sulfur compounds to sulfuric acid. Performed either in slurry or by heap leaching system

Enhanced bioremediation Achieved by creating a favorable environment to stimulate the natural or In situ bioremediation inoculated population of microorganisms. Biodegradation rate is influenced by biostimulation, bioaugmentation or cometabolism

Bioventing Involves injection of air or water to supply In situ bioremediation oxygen and nutrients into the underground contaminated mass

Biosparging Addition of air/oxygen and nutrients to enhance biodegradation of groundwater contaminants. Also potentially improves biodegradation in the unsaturated zone

Anaerobic biodegradation Anaerobic degradation of polychlorinated organic pollutants in sediments. Generally followed by an aerobic process for further dechlorination of the pollutants

Phytoremediation Higher plants are used either to degrade contaminants, to fix them in the ground, to accumulate them within plant tissue or to release them to the atmosphere

Monitored natural A strategy of allowing natural processes to reduce contaminant attenuation concentrations over time, involving physical, chemical and biological processes with continuous monitoring

3.9.1. Microorganisms: Bacteria

In bioremediation, microorganisms need special attention to make sure that they adapt to the contaminated area of treatment. In some cases, the site can be remediated with indigenous microorganisms, known as natural attenuation, while on other sites inoculation with foreign microorganisms is necessary (Crawford and Crawford 2005).

In bioremediation with focus on microbes, a steady supply of nutrition should be ensured to feed heterotrophic bacteria, which need a high supply of nitrogen in addition to the carbon used for energy (Crawford and Crawford 2005). The carbon is provided by the hydrocarbons in the case of brownfields sites dealt with in this project.

In cellular respiration, organic molecules break down in 3 metabolic stages, namely; Carbon source, Pyruvate Oxidation and Citric Acid Cycle (in the Cytoplasm of the Bacteria), Oxidative Phosphorylation (ETC and ATP Synthase takes place in Plasma membrane), synthesizing ATP, and + Heat (NADH and FADH2 move H and electrons from Cytoplasm to Plasma membrane to generate Energy production) (Reece et al. 2013). Degradation is highly efficient with a high growth and microbial mass (Crawford and Crawford 2005), since there is a higher overall demand for energy which degradation of the hydrocarbons would provide. For maintaining cell viability, cell growth, or rather degradation rates it might be necessary to inoculate the soil with sugars (Crawford and Crawford 2005), boosting the bacterial population which would then need to metabolize the hydrocarbons to sustain itself.

Bioremediation can be executed anaerobically or aerobically. Efficient degradation of hydrocarbons is shown with aerobic bacteria consuming oxygen, which can be a rate-limiting factor, thus it is necessary to apply an aeration system to the place of treatment to ensure a steady oxygen supply. Anaerobic bacterial processes in some cases match or even exceed aerobic methods in terms of efficiency, making this a potentially viable alternative.

3.10. Application of bioremediation

Bioremediation can be divided into ex situ and in situ.

3.10.1 In Situ

In situ remediation allows a treatment without excavation and transport. In situ processes, can last for a long time as extensive analysis is often needed prior to remediation to investigate variables and soil characteristics. In addition to mycoremediation, examples for this microbial bioremediation are bioventing and phytoremediation (A. Singh, Kuhad, and Ward 2009). This type of treatment involves creating a specific environment that stimulates the natural or inoculated population of microorganisms, affecting their catabolic potential to grow with the consumption of contaminated substances as a food and energy source (Suthersan 1999).

Bioventing is a method of aerobic stimulation. It stimulates the natural in situ biodegradation of compounds in soil by providing oxygen to existing aerobic soil microorganisms. The cleanup process can range from some months to many years. Bioventing techniques have been successfully used to 29 remediate soils contaminated by petroleum hydrocarbons and some other organic chemicals (Whitacre 2016).

Phytoremediation, remediation with the use of plants, can be used to extract, contain, immobilize, or degrade contaminants from soil and water. Some plants have the affinity to take up contaminated substances and subsequently transform them, accumulating the non-phytotoxic products (Whitacre 2016). Associations of plants and microbes can enhance the cleanup of inorganic and organic pollutants. Phytoremediation is generally used for large areas with low to moderately contaminated soils. This technology is adaptable to many site conditions and has potential to remediate surface water and leachate or soils, sediments, and sludge’s contaminated with heavy metals, hydrocarbons, or other toxic chemicals (United States Environmental Protection Agency 2000). In 1998 a case study treated petroleum spills with willow trees planted over the spill. As a result, 90% of the contamination was removed from the site after three growing seasons (Nzengung 2005).

3.10.2. Ex situ

Ex situ remediation involves excavates the soil and transportation to a place of treatment. This process requires a shorter time period and allows controlled composition of the soil, but also includes higher transport and engineering costs. Ex situ technologies include landfilling, biopiling, composting and treatment with slurry bioreactors (A. Singh, Kuhad, and Ward 2009). Ex situ will be used in cases in situ treatments cannot take place at a polluted site. Reasons for relocating the biological degradation treatment can be either land-site regulations or unavailability of sufficient land, threat to groundwater or air pollution (A. Singh, Kuhad, and Ward 2009).

In landfarming the contaminated substances are placed in isolated soil beds and periodically tilled to aerate the soil. Nutritions or microorganisms can improve the biodegradation process of the polluted soil. In landfarming a leachate collection system prevents the off-site migration of water-soluble- hydrocarbons, thus it prevents the leaching of hydrocarbons into the ground water. This technology shows successful approaches in decontaminating lighter petroleum hydrocarbons (A. Singh, Kuhad, and Ward 2009), although in this case vapors that can cause air pollution may be involved. In a pilot study (Petavy et al. 2009) where this method was used on storm water sediments which were contaminated with a mix of hydrocarbons including PAHs, total degradation reached 60 to 95 % of PAHs and 53 to 97 % of other hydrocarbons (Whitacre 2016).

In composting, microorganisms decompose organic contaminants into smaller byproducts which are less harmful to the environment than before. The process can be aerobic or anaerobic. In composting a steadily growing microbe population is advantageous to obtain a sufficient degradation rate (Whitacre 2016). The soil gets embedded with hay, vegetative wastes, wood chips and manure creating a thermophilic environment for microbial activity. Limiting factors are solid space and the excavation of the contaminated soil. Furthermore, it produces scent and leachates that needs to be managed.

A field study from (Ouyang et al. 2005) compared bio-augmentation, the cultivation of microbes at a contaminated site, and composting, inferring that bio-augmentation could degrade oil and soil sludge by 45-53%, whereas composting removed 31% of all hydrocarbons in the soil after 30 days (Whitacre 2016).

Bioreactors combine landfarming with composting commonly in the treatment of petroleum hydrocarbons. The polluted soil is piled and microbes are stimulated by aeration followed by additional water and nutrient, with controlling pH and heat (A. Singh, Kuhad, and Ward 2009). This technology uses aeration systems to stimulate microorganisms. With bioreactors, optimum levels of moisture, temperature, pH, aeration and nutrients for microbes and its activity and survival rates can be controlled and leads to faster biodegradation (McCartney, Yawson, and Seshoka 2004). It can be a closed system controlling vapor emission, and can be engineered for petro-chemicals and physical settings. (Roldán-Martín et al. 2006)described the utilization of biopile technology for remediating oil sludge with TPH (total petroleum hydrocarbon) concentration up to 300 mg/kg sludge, where 60 % degradation was achieved after 3 months of treatment (Whitacre 2016).

3.10.3. Soil Analysis

Before a process option can be selected, analysis of site characteristics and viability studies of chemical, physical and microbiological properties of the contaminated area must take place. These studies are necessary to identify possible rate-limiting factors for later applications on the field and for kinetic or rather equilibrium data for process design (Crawford and Crawford 2005). In soil bioremediation, three steps must generally be performed before actual remediation takes place:

1. An off-site laboratory-based study determines the biodegradability of chemical substances and observes the degradation ability of indigenous microorganisms, or alternatively, introduced organisms. 2. Pilot projects provide data important for process designs to insure an efficient treatment. 3. A full- scale bioremediation takes place on site or at an EPA-licensed facility. In cases of sufficient analysis, it can provide data about the type of contaminants, their concentration, and the extension of contamination (Environment Protection Authority 2005).

The figure 10 is an example of the analysis process, provided by The Environment Protection Authority(EPA) from South Australia (this organization is controlling and assisting in remediation processes of polluted sites):

Figure 10: Outline of Bioremediation management, in this case for bio-piles and landfarming (Environment Protection Authority 2005).

The Australian NEPM (National Environmental Protection Measures) outlines remediation methods ordered from likely to less likely:

1. on-site treatment of the chemical substances to reduce risk to an acceptable level 2. off-site treatment of excavated soil to reduce risk to an acceptable level, after which the treated soil is returned to the site 3. containment of soil on site with a properly designed barrier 4. disposal of affected soil to an approved landfill. 5. Furthermore, the need of other remediation technologies in addition to the available bioremediation method must be considered, as well as the disposal of treated soil.

3.11. Application of mycoremediation

There are two ways in which mycoremediation can be applied in brownfield sites, with each of them having its own strengths and weaknesses. These two methods are the use of ‘bunker spawn’ or burlap mats and application of the fungi directly to the area of contamination. Both of these can be done in situ, but for the purpose of this project the focus will be on the bunker spawn method.

Since there is no ideal method for applying them to the brownfield site, besides the requirement that the burlap sacks are in direct contact with the soil, it is common practice to dig down the sacks right below earth surface without covering them (Durr 2016), or lay them upon the ground (Stamets 2005).

Creating bunker spawn involves filling burlap sacks or burlap mats with spawn, using various possible substrates as mentioned in section 3.7.1. These sacks are then embedded in the contaminated soil, allowing nutrients and contaminants it be absorbed through the netlike structure of the sack or mat, and subsequently be degraded by the fungal mycelium.

These nutrients allow the mycelium to grow and consume the burlap sack, extending into the surrounding soil while still having the primary mass restricted by the sack. The burlap sack or mats consists of mainly Jute but can also be constructed by linen or hemp (Woolley 2000). Since jute, linen or hemp are organic materials consisting of starch, they are easily degraded by mycelium. This makes burlap sacks or mats a prime candidate to use for remediating in an area while keeping the consumed contaminants in place and allowing the fungus to more easily establish a subsoil mycelial mass.

Applying mycoremediation to a brownfield sites require several things. First the knowledge or expertise needed to analyze the target area and determine the contaminants is needed. That means

33 that an expert with extensive knowledge regarding certain fungal strains and their interactions with different contaminants is required. Workers are required for the initial setup, as well as the following:

 Burlap sacks or mats o Fungi strain(s) prepared beforehand  Shielding against macro-fauna  Removal of burlap sacks or mats

Should some of the bunker spawn become contaminated by heavy metals during the remediation period, removal and replacement of the bunker spawn may be required. The reason for this is to limit the contaminants available for fauna to come in contact with. By replacing the heavy metal filled sacks or mats one takes into the account the effect of the contaminated sacks on the local ecosystem.

4. Analysis

4.1. Suitability of Mycoremediation

Five major points should be considered in assessing whether a process is suitable for bioremediation in any particular case (A. Singh, Kuhad, and Ward 2009):

1. The catabolic activity and capacity of organisms involved to transform the target compound(s) and bring the concentrations to levels that meet regulatory standards 2. The rate of bioremediation 3. The possible production of toxic byproducts at dangerous levels during the remediation process 4. Adaptability of the process to site conditions (environmental and anthropogenic) 5. Economic viability of the process Mycoremediation with P. ostreatus will analyzed in relation to each of these points in the following sections.

4.1.1. Degradative capability of organisms

Intrinsic microorganisms that can degrade aliphatic compounds are in most cases present in soil (Cerniglia and Sutherland 2016), but seldom those which can degrade larger, more recalcitrant and persistent molecules such as PAHs without additional assistance. Many indigenous soil bacteria have been shown as capable of degrading PAHs with fewer rings, such as anthracene, which has 3, but few have shown any sign of breaking down PAHs of 5 rings and above (Pozdnyakova, Nikitina, and Turovskaya 2008), such as benzo(a)pyrene with 5 rings. As mentioned before in section 3.1.5, such PAHs can still pose significant societal issues.

P. ostreatus represents a possible solution to this problem, as this fungus has shown significant ability to degrade and metabolize both high-weight aliphatic and aromatic petroleum hydrocarbons, including >5 ring PAHS, in both laboratory and on-site conditions, as described in section 3.2 and supported by all the studies in Section 4.2.

This makes P. ostreatus a very suitable candidate for contributing to the overall catabolic activity and the ability to degrade soil to concentrations where it can be reused for other purposes.

The degree of reduction in pollutant concentration levels that can be considered “remediated” depends largely on the specific compounds present and the intended use of the land/soil, as well as the surroundings. For example: A brownfield site next to a school, needing to be remediated so it can be used as a recreational park, would need a much lower pollution levels, than an isolated site intended as the construction-site of a coal power station. Added to the fact that minimum safety levels vary greatly according to national and local regulations, this makes the level of remediation required very case specific, and as such it should be determined during site assessment.

4.1.2. Rate of degradation and bioavailability

The degradation order of hydrocarbon compounds in soil is generally as follows (H. McKee 2016):

(1) n-alkanes, especially in the C10–C25 range (degraded readily)

(2) isoalkanes

(3) alkenes

(4) benzene, toluene, ethylbenzene and xylenes (BTEX) (when present in concentrations that are not toxic to microorganisms)

(5) monoaromatics

(6) polycyclic aromatic hydrocarbons (PAHs)

(7) higher molecular weight cycloalkanes (which may degrade very slowly)

This is primarily due to the recalcitrance of molecules and their bioavailability, as well as the relative abundance of the organisms mentioned in Section 4.1.1. As described in Section 3.1.4, higher weight compounds, especially PAHs, are far more recalcitrant and stable than lower weight aliphatic compounds, thus are more difficult for most organisms to degrade. In the case of PAHs, highly branched isoalkanes and cycloalkanes, it is the convoluted complex structures which make these substances difficult for enzymes to access. The lignolytic enzymes of P. ostreatus, as in Section 3.6, negate this issue. The recalcitrant compounds also have longer, more complex breakdown patterns. Therefore, contamination containing lighter hydrocarbons will be degraded swiftly, whereas the more recalcitrant variety will be degraded more slowly and only if certain less-common organisms are present. The difference in physical behavior as detailed in Section 3.1.4 also accounts for the differences in persistence. From this one can conclude that remediation of sites containing can vary vastly according to which fractions are present.

Bioavailability is the accessibility of pollutants to microorganisms. It also largely affects the ease and rate of biodegradation (Cerniglia and Sutherland 2016). The higher the bioavailability, the higher the rate of degradation, since the pollutants need to be accessible to the decomposer organisms before they can be broken down. The tendency of insoluble and immobile higher weight molecules, like PAHs, to remain in semi-liquid masses makes them less likely to be exposed to microorganisms that might be able to degrade them (Bhattacharya et al. 2014). These components also cling tightly to the soil media as they have high Koc values, further making them inaccessible. Many of the water-soluble fractions tend to be degraded as they go into solution (Todd, Chessin, and Colman 1999). This would include mainly monoaromatics, as described in section 3.1.4. Particles trapped in micropores in soils, especially dense ones such as clay, are also often inaccessible to microorganisms (Cerniglia and Sutherland 2016), and large particles are more likely to be trapped than smaller ones. This depends largely on the clay type present in the soil. By breaking up the larger, less bioavailable molecules into smaller components, pollutants are made more accessible to other organisms, such as bacteria as 36 well as the fungal mycelium itself, as smaller molecules are more mobile due to their higher solubility and lower LogKoc values.

By acting as a sole decomposer of hydrocarbon contaminants, P. ostreatus is able to access and degrade a very wide range of compounds, but the absorption and final metabolism of the extracellular metabolites seems to be a bottleneck in the overall mineralization process. Many studies have been found suggesting that soil bacteria work in conjunction with fungi for effective degradation of large compounds, see Case Study C in Section 4.3. One current model for mycoremediation suggests that once fungal extracellular enzymes have attacked the recalcitrant compounds, such as >5 ring PAHs, and produced smaller compounds, these metabolites are then not all utilized by the fungi. Instead, some are then absorbed and metabolized by the far more prolific native soil bacteria before they are taken up by the fungal hyphae. While not directly benefiting the fungus, this does increase the overall rate of biodegradation.

As mentioned in Section 4.1.1, bacteria, capable of degrading smaller, simpler compounds such as light aliphatics are common in most places and are more prolific than organisms, either introduced or intrinsic, capable of degrading PAHs and other heavy complex compounds. This is another reason for remediation of contamination with lighter petroleum factions being much faster.

Numerous environmental factors, such as temperature and soil moisture content, can also significantly impact degradation rate, as written in section 4.1.4.

4.1.3. Harmful byproducts

When PAHs are metabolized, the products are predominantly quinones and dihydrodiol (Pozdnyakova 2012). Although the toxicity of the soil with these metabolites is generally lower in comparison to the parent compounds, they often show a higher bioavailability. This is due to the fact that they gain a polar character, which allows them to bind to the water, hence the higher solubility (Bolton et al. 2000). Increased bioavailability allows the metabolites to easily be degraded further, but also brings them more into contact with organisms to which they may be toxic.

The study from Haeseler (Bouchez et al. 1999), showed a short increase of toxicity from the soil, due to the forming of intermediates, which show a higher mobility than the parent compound. Although this formation of intermediates occurs, at the end of the study the overall result shows a drastic drop in toxicity (Douben 2003). 37

Quinones, see Figure 10, are the most common metabolites forming in the degradation process of hydrocarbons.

They undergo a range of rearrangements during the oxidation process, the important aspect being the heightened redox potential (Bolton et al. 2000). This strong reactivity leads to autooxidization of the compound, where it undergoes different stages. It oxidizes both itself and as well as helping with the formation of peroxide, which in turn initiates another cycles of degrading process (Bolton et al. 2000). The Quinone’s then can get further metabolized via ring fission (Pozdnyakova 2012).

The reactivity in terms of being harmful to humans is variable. Depending on the place of the quinones in the compound it can have a low reactivity when they for example lay in the bay region, which is at the edges of the compound. The high redox potential oxidizes the cells in human as well, radicalizing them and thereby damaging the macromolecules, leading for example to cell death.

Table 4 below depicts different types of quninones, stating the various potential health risks to humans.

Table 5: Cytotoxic effects of various quinones (Bolton et al. 2000).

4.1.4. Appropriate site conditions for biodegraders (in situ)

The results of degradation of hydrocarbons can be highly varied, as seen in the case studies analyses in Section 4.3, where the degree of degradation varied between 40-90% in the different experiments. The rate of degradation using white rot fungi is highly dependent on the specific conditions at the site, such as geographic location, climate, and the presence of co-contaminants.

As detailed in Section 3.8.1, in terms of temperature, mycoremediation with the various strains of P. ostreatus could take place at brownfield sites in temperate, sub-tropical and tropical climates, for the majority of the year. Research in with specific strain of P. ostreatus with potential for growth for each specific brownfield site would be necessary. This means it could essentially be a potentially viable

39 solution to some extent in a very wide range of locations and climates, and in most seasons, although as mentioned in Section 3.8.4, other conditions such as moisture are also limiting factors.

Furthermore, P. ostreatus itself remains ultimately unaffected by indigenous microorganisms in soil (Bhattacharya & Das, 2014), as mentioned in Section 3.7, if introduced as spawn, by which stage the mycelium is capable of defending itself against hostile bacteria through the production of antibiotics. This leaves the fungus free to operate with or alongside neutral bacteria as described in Section 4.1.2.

As discussed in section 3.7, when a brownfield site has to be colonized with mycelium, spawn produced from wild mushroom has a great advantage over spawn produced under sterile conditions, because it is already adapted to soil environments inhabited with a great variety of competing bacteria and other microorganisms.

P. ostreatus is advantageous in that it requires a higher C/N ratio than most soil bacteria, as stated in Section 3.8.2, thus its nitrogen requirements are lower. This is particularly beneficial in the remediation of hydrocarbons, which have high carbon content and significantly raise the C/N ratio of contaminated soil (Dindar et al. 2013). This is why bacterial bioremediation frequently requires fertilization, as mentioned in Section 3.9.1, specifically with nitrogenous compounds. This need would be reduced through the utilization of P. ostreatus.

However, the native soil bacteria are still an essential part of the degradation process in mycoremediation, thus although the fungi might be resilient to certain conditions, the fact that the bacterial population is impaired might still slow or reduce the effectiveness of the overall process.

As described in Section 3.8.3, the presence of heavy metals, which frequently accompanies that of hydrocarbons, significantly impacts degradation: copper and manganese up to certain concentrations can improve degradation rate by affecting the synthesis of laccase and manganese peroxidase enzymes respectively, whereas mercury and cadmium have displayed toxicity towards P. ostreatus , impeding mycelial growth at low concentrations and hindering enzyme activity at higher levels, in the case of cadmium. Therefore, the presence of these metals is an important factor in determining whether mycoremediation will be suitable for a particular brownfield site or not.

Some of the factors influencing the implementation of P. ostreatus can be regulated in situ if not ideal for mycoremediation: The moisture content of soil could be raised by irrigation or providing shade to prevent evaporation, nitrogenous fertilizer such as ammonium chloride, see section 3.8.2, could be

40 added if the C/N ratio in the soil is too high for the fungi, and pH can be offset either by finding a strain of P. ostreatus more suitable to that present at the site or by regulating the soil pH through techniques such as liming. In extreme cases the oxygen content of the soil could be boosted by aeration to increase the catabolic activity of the fungi. These measures, especially aeration and adding nitrogenous fertilizers, likely also increase the activity of the soil’s microbial population, although they may incur significant additional financial costs.

Some factors, such as the presence of heavy metal co-contaminants and temperature, cannot be easily controlled, although the latter is usually compensated for by finding a local strain of P. ostreatus suited to that particular climate.

4.1.5 Mycoremediation in controlled conditions

When mycoremediation cannot be applied in situ, due to unsuitable site conditions, ex situ treatments can be chosen. Ex situ treatments, as described in section 3.10.2, are designed to provide optimal conditions, but also carry additional expenses, such as transport of soil and equipment. This may lead to faster and potentially more effective degradation than seen in equivalent in situ methods. An example is using P. ostreatus as a pre-decomposer for higher weight hydrocarbons in a bioreactor, where optimum levels of moisture, temperature, pH, aeration and nutrients can be provided and monitored, and allowing smaller fractions to be degraded by a predetermined cultivation of bacteria. Such methods also have the advantage of not allowing leachates or intermediate compounds to contaminate groundwater or surrounding soils.

4.1.6. Economic viability

Using mycoremediation as a remediation technology has the potential to lower the total cost of a remediation project. The main reason being that other remediation technologies usually require machines, structures, facilities, maintenance and power. At the same time the resources required for initiating mycoremediation of an area are less than traditional remediation techniques.

Mycoremediation does not leave any waste products after the remediation process. In most cases the fungi decompose or integrate with the soil ecosystem after breaking down the contaminants, resulting in less money lost since there is no disposal of the treatment material afterwards.

Additionally, a wide variety of substrates can be used for mycelium cultivation, section 3.7.1, meaning that this can be locally and cheaply acquired from whatever source is best available in each case.

Figure 11 below show a collection of prices of each treatment technique (Anderson and Juday 2016).

Figure 11: Graphical plot of the data found in (Anderson and Juday 2016).

As shown in the Figure 11 which is an overview of the prices of the different types of remediation, Phytoremediation is the remediation technique whose price can vary greatly. Phytoremediation can involve relative low costs, but can also range to high costs. From a financial perspective, this method involves taking a risk, whereas using biopilling or mycoremediation as a method of remediation would pose a lower financial risk. Each technique has its strength and weaknesses but mycoremediation is the technique that has the most consistent price pr. US Ton which means that there is a lower financial risk involved in using this technique.

Figure 12: Soil Remediation Technologies Cost: Petroleum Hydrocarbons (Stamets 2005).

Another graph as seen in Figure 12 from Mycelium Running (Stamets 2005) shows mycoremediation to be 50$/ton this conflicts with the information given in (Anderson and Juday 2016). This can be due to different reasons, such as the fact that the Mycelium Running graph is from 2006 and the Figure 14 above contains data from 2016. The 10 years between could be enough time for the biotechnology’s price to be reduced due to more available knowledge and more experts on the subject that is mycoremediation, but one thing which is certain is that the graph created by Stamets also show that using mycoremediation as a remediation technology is more efficient finance wise than using bioremediation and by far more efficient than incineration of the contaminated soil.

From these, one is able to deduce that mycoremediation may have fewer steps but also each step may cost less than e.g. incineration or bioremediation. To sum up what the mycoremediation cost contains:

 Initial setup o Analysis of the contaminated site o (This step is required for all remediation processes) o Cultivating the fungi-strain, see Section 3.7 o Constructing Fungi filled burlap sacks o Transport of burlap sacks o Applying burlap sacks to the site  Maintenance o Removing sacks filled with too much heavy metal o Testing for degree of remediation o Adding nutrients if necessary

Furthermore, the cost of fungi cultivation could be almost negated if spawn were sourced as spent mycelium-bearing compost from a gourmet oyster mushroom farm, as described in section 3.7, especially in proximity to the brownfield site in question.

Most of the above steps require little manpower and the greatest cost of the initial setup is the analysis of a contaminated site, considering the time, money and equipment required to do these. But this step is required of every remediation technique, therefore it is a common denominator and can be excluded when comparing these. There is also no end step in mycoremediation since the burlap sacks will decompose by themselves, which means that the cost of cleaning the contaminated area is reduced.

4.2. Email interview: Fungi Perfecti

See transcription of interview(Ronnebaum 2016) in appendix B. Fungi Perfecti is optimistic to the idea of implementing the P. ostreatus for mycoremediation on brownfields sites.

4.2.1. Barriers for implementation

According to Fungi Perfecti the main reasons that mycoremediation is not a widely used remediation technique on the large scale are not the lack of information of this technique or proof of the fungi’s

44 ability to degrade contaminants, but rather regulative and logistically constrictions. The following are a few legal points Fungi Perfecti point out as barriers for implementing large scale mycoremediation.

Environmental remediation company wanting to use mycoremediation on a site in the US need:

1. To obtain permits from governing state or federal agencies. 2. A plan for how to address all pollutants in the specific site not degraded by the specific fungi chosen. For example, brownfield sites contaminated by hydrocarbon also has heavy metal co- contaminants not degradable by P. ostreatus, a plan for how to deals with them would be necessary before the mycoremediation could take place. 3. A plan for controlling the quality of the remediation for each project. 4. A professional licensed environmental engineer in their team to secure professional liability.

Financially, in order to produce the mycelium, need for commercial scale remediation sites a farm/factory would require a laboratory and heavy equipment. Fungi Perfecti estimate that a company would need at least 2 million US dollars for initial capital costs. This indicates that although running costs of mycoremediation as written in Section 4.1.6. are low, the initial costs are considerable. Fungi Perfecti suggested that a solution to this might be using spent mycelium-bearing compost from oyster mushroom farms, and that it would be further advantageous if the farms were local as this would reduce transportation costs of the heavy mycelium, supporting the idea mentioned in Sections 4.1.6 and 3.7

Internationally they point to one of the bigger restrictions before implementing large scale mycoremediation as researching in specific native mushroom species and available substrates to find combinations with robust growth in the local climate and the ability to degrade the present contaminant(s). This is site specific work that needs to be done for each particular brownfield site, as described in Section 3.10.3.

Fungi Perfecti confirms the finding in section 4.1.2 and Case Study A in section 4.3 that bioremediation with mycelium works more efficiently than bacteria or plants alone, but, in accordance with section 3.8, efficiency is highly depending on environmental factors.

Fungi Perfecti writes in the interview, that P. ostreatus has global distribution and spores travel vast distances, so relocating mycelium is generally not thought to pose a problem. This confirms the conclusion in section 4.1.4 that a local strain of P. ostreatus adapted to the specific climate, would be

45 found in most cases, and also indicates that the introduction of P. ostreatus would not cause an ecological problem.

4.3. Case study support

Four experiments have been briefly analyzed to compare to other findings in Section 4.1 and potentially give insight into other important details. See Appendix A for the tabulated summary.

Studies B (Young 2012), C (Pozdnyakova, Nikitina, and Turovskaya 2008) and D (Zitte, Awi-Waadu, and John 2012) all demonstrated quantitatively and in a controlled environment that P. ostreatus does degrade petroleum hydrocarbons, thus supporting proving the assertion in Section 4.1.1 that it has suitable catabolic capabilities for hydrocarbon degradation. Although variation in equivalent degradation percentages is seen, this can be explained by the specific method and time period over which the experiment was performed, natural variation and other factors not given in the reports or too detailed to explore here.

Study B indicates that P. ostreatus most effectively degrades PAHs and shorter aliphatic hydrocarbons. This partly contradicted by study C, which shows that the degradation of the shorter aliphatic compounds may largely be due to the natural soil bacteria, but that the PAH compounds could only be effectively broken down if P. ostreatus mycelium was present, from which we can infer P. ostreatus is primarily responsible for this faction. This links to and supports the model described in Section 4.1.2.

Study C also shows that the association between mycelium and soil microflora is essential to the overall breakdown, as neither seems to be able to work as effectively alone as they do together. The fact that microflora alone was able to degrade 34.53% whereas P. ostreatus only managed 9.39% could be due to the fact that the majority of factions in most petroleum mixes (although as mentioned before this can vary greatly) are lighter and therefore could be dealt with by bacteria present in the soil. If P. ostreatus mycelia deal preferentially with PAHs due to their similarity in structure with lignin (see Figures 3 and 4), then this would account for a smaller portion of the overall oil. This also supports the model in Section 4.1.2.

Study A (Thomas et al. 1998) was important in terms of testing mycoremediation in an open environment instead of laboratory conditions, with factors involved similar to those which might be present during actual remediation of a brownfield site. Although very thorough analyses of soil were 46 performed post and during remediation, there was an error in the mixing of the soil to ensure homogeneity of petroleum contaminants so concentrations varied according to where on the sample soil they were taken, and the gasoline-contaminated soil sample actually contained a large amount of heavy oil fractions meaning it did not accurately represent the lighter hydrocarbon spectrum. This led to inconsistencies and overall unusable analytical results. However, simple observation of the soil samples does lend support to the idea that mycoremediation is an effective method since the mycelium fully penetrated the soil, produced fruiting bodies, changed the soil texture to that more resembling uncontaminated soil and removed the odor of oil. The samples treated with the other methods, by contrast, showed little change in this regard, meaning that the methods were either far less effective of much slower. The study report (Thomas et al. 1998) suggested that this may be due to the fact that these bioremediation methods typically take a much longer period of time than was allocated in the study, supporting the findings in Section 4.1.2 that the introduction of P. ostreatus could greatly speed the remediation process.

5. Discussion

The research on mycoremediation with the use of P. ostreatus to decompose hydrocarbons in brownfield sites, has yielded several conclusive points.

Although existing bioremediation methods, primarily using bacteria, have shown positive results in some cases, specifically regarding the degradation of lighter petroleum compounds, if successfully applied, mycoremediation with P. ostreatus has shown a number of potential advantages in remediation of brownfields contaminated with hydrocarbons:  Highly recalcitrant petroleum compounds such as PAHs, which are accessible to relatively few native microorganisms, can be effectively degraded by the fungus with minimal production of harmful byproducts. By making these compounds more bioavailable, the rate of degradation is increased.  Due to their high C/N ratio compared to bacteria, fungi such as P. ostreatus can be effective remediators with less supplementary resources, such as the nitrogenous fertilizers, lowering the overall need for these.  The association between P. ostreatus and native soil bacteria is an essential part of mycoremediation and more efficiently degrades overall pollutants than either does alone.

This shows that in theory mycoremediation using P. ostreatus would definitely contribute to solving the issue of hydrocarbon contamination in urban areas.

Functionality would be optimal in sites with the following conditions, some of which can be altered accordingly if not within acceptable parameters:  Relatively high moisture content: can be raised by shading or irrigation to increase water content of soil  Moderate concentrations of copper and manganese  High oxygen content: can be increased by aeration (bioventing)  C/N ratio between 10 and 5: can be altered by addition of nitrogenous fertilizer if C/N ratio is too high (usually unnecessary due to low N requirement of fungi, but may be needed for native bacteria)  An average temperature of roughly 25 degrees Celsius Although these corrective measures can be performed, they may raise costs to the point where mycoremediation is no longer the most suitable method. These measures are frequently unnecessary if a strain of P. ostreatus adapted to the local conditions can be sourced.

If in situ mycoremediation is unfeasible due to conditions or the required remediation time frame, ex situ methods such as landfarming or bioreactors can be used, resulting in faster, and potentially more effective soil remediation with the added advantage of preventing the spread of more mobile pollutants, such as benzene. This has the disadvantage of much higher costs and intensive labor requirements. Therefore, if there is an acute need for remediation, such as after the spillage of highly toxic and mobile contaminants like benzene or similar compounds, or time is a higher priority than cost, ex situ methods might be more suitable. Otherwise, in situ methods would be more suitable in cases where the need is not as urgent, as it is less costly, labor intensive and environmentally disruptive, since it does not involve excavation.

Mycoremediation using P. ostreatus also shows promise as a potentially more cost-effective solution compared to other non-biological and bioremediation methods, since its application involves relatively little labor if applied in situ and the raw materials, primarily the spawn and additional growth substrate, can in most cases be sourced locally since P. ostreatus can use a wide range of cellulosic substances for nutrition and has a global distribution, with a local strain existing in most 48 temperate, subtropical and tropical regions. It is even more cost-effective if the brownfield site is within transport range of a gourmet oyster mushroom farm, as the spent compost can be used as spawn.

There are some drawbacks to the mycoremediation process:  Heavy metals such as cadmium and mercury, which can often be found as co-contaminants to petroleum waste, can severely hinder the degradative abilities of P. ostreatus.  There are numerous legal requirements in countries such as the US, which make mycoremediation difficult to implement due to the complex legal requirements  The effectiveness and speed of the process are still very site specific, thus mandating a lengthy and possibly expensive site analysis and excluding remediation of more extreme environments such as arid or polar climates. This is true for all bioremediation methods, meaning that non- biological remediation such as incineration may be advantageous in this regard.

In conclusion, mycoremediation with P. ostreatus is a very promising form of bioremediation which could potentially be applied with high success to many of the world’s brownfield sites contaminated by various hydrocarbons. This confirms Hypothesis 1. Additionally, based on the information available, it is strongly indicated that mycoremediation with P. ostreatus is a cost effective method with a wide range of applicable situations, thus partially confirming Hypothesis 2. However, it may still face significant limitations in terms of legislature and environmental conditions with resulting impacts on cost and effectiveness.

6. Perspective

6.1. Mycorrhizae hyperaccumulation of heavy metals

Further research could look into solving the problem of high concentrations of heavy metals that restrict the mycelium growth of white rot fungi like P. ostreatus. From the interview with Fungi Perfecti we learned about Mycorrhizal fungi that tend to hyper accumulate metals. Relevant research could be looking into which plants and correlating endo/ecto fungi that associate with those plants could be used to target the heavy metals that are found in oil-contaminated soils, and remove them from the area by disposing the plant like toxic waste (Incineration).

6.2. Mycoremediation in non-urban areas

The technique of using white rot fungi for soil remediation does not only apply to brownfield sites. There are currently also studies going on, using mycoremediation in more inaccessible areas, such as the rainforests, where oil spills from leaking pipelines contaminate the ecosystem and water sources for both animal life and native people highly dependent on the forest they live in. The theory that P. ostreatus not only breaks down pollutants but also rehabilitates the local ecosystem to an extent and helps life flourish at the site (Stamets 2005), would be an interesting aspect for further research relating to contaminated areas intended to be restored to their natural conditions.

6.3. Bioaccumulation

PAHs and other hydrocarbons are nonpolar and therefore likely to be lipid-soluble. This means they could potentially accumulate in the fatty tissue of organisms that ingest or come into contact with them, and then be passed up the food chain as these poisoned organisms are consumed. The ecological impacts of this, as well as the potential for spreading to humans, could be a very relevant aspect to research further.

6.4. Gene expression

Current research on the P. ostreatus is, amongst others, addressing the monitoring of the gene expression responsible for the enzyme production. The goal is to be able to enhance the enzyme production by modifying the genome, ensuring a more productive degradation process. This was however not the angle of this report.

6.5. Alternative fungal species for mycoremediation

Although P. ostreatus was selected for this project and has shown numerous advantages in mycoremediation, several other species, such as A. niger have also shown strong degradative capabilities and other unique characteristics, but also drawbacks. A more thorough comparison of the capabilities of different species which might be used for mycoremediation might give a more comprehensive view of the remediation method, and possibly shed light on new possibilities or issues which P. ostreatus alone is not able to solve.

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Appendix

Appendix A:

Tabular comparison of case studies on P. ostreatus ability to degrade hydrocarbons.

Title Reference Description Degradation Findings time

A) (Thomas, Mycoremediation (using P. 4 months All mycoremediated soil Mycoremediation Becker, Pinza, ostreatus ) was compared showed flourishing fungal of aged petroleum & Word, with bioremediation and growth. Vascular plant and hydrocarbons in 1998), enhanced bacterial wild fungi communities soil (Stamets, remediation of diesel and began to develoP. The soil 2005) heavy-oil contaminated changed texture and oil soil, gasoline contaminated odour was no longer soil, and soil from a present. Soil in Bellingham truck repair bioremediated, enhanced yard containing weathered bacteria remediated and oil contaminants control groups remained unchanged.

B) Bioremediation (Young & Investigating the ability of 6 Months (+ P. ostreatus reduced the with White-Rot Young, 2012) P. ostreatus and 5 other 8 months presence of phenathrene( Fungi at fungal species (Irpex prior a representative PAH) by Fisherville Mill: lacteus, Phlebia radiata, adapting to 75-95%, C14 alkane by 35- Analyses of Gene Punctularia strigosozonata, oil in agar) 48%, and C10 alkane by 97- Expression and Trametes versicolor and 98% Number 6 Fuel Oil Trichaptum biforme) Degradation spawns to degrade Bunker.C oil obtained from Fisherville Mill brownfield site in laboratory conditions

C) Bioremediation (Pozdnyakova, The ability of 2 strains of P. 4 weeks After 2 weeks the P. of Oil-polluted Soil Nikitina, & ostreatus (D1 and 339) as ostreatus mycelium in with an Turovskaya, well as L. edodes 0779 to sterilized soil showed poor Association 2008) grow on petroleum oil degradation (9.39%

Including contaminated soil and with D1 strain and 6.35 the Fungus degrade the oil was with strain 339), but was P. ostreatus investigated. This was done able to degrade 60.45% and Soil using both sterilised and (D1) and 14.5%(336) in Microflora non-sterilised soil as well as unsterilized soil. Soil a culture without fungi to microflora alone degraded determine the role of the 34.53% of oil. After an soil microflora in the additional 2 weeks, it was process. found that the microflora alone could degrade a maximum of 9% of PAHs, but in association with P. ostreatus (D1) mycelium degraded 31%

D) Effect of Oyster (Zitte, Awi- The effectiveness of 3 4 weeks The amount of crude oil Myshroom (P. Waadu, & identical samples of present in the mushroom ostreatus ) John, 2012) sawdust P. ostreatus spawn mixtures reduced by 90%, mycelia on at degrading 20ml, 40ml 87% and 85% respectively petroleum and 60ml of crude oil hydrocarbon (added directly to the contaminated spawn mix) was tested. substrate

Appendix B:

Transcription of email interview with mycologist company Fungi Perfecti.

For this interview, the interviewer is the mushroom group and the interviewees are:

Loni Jean Ronnebaum Retail Office Manager at Fungi Perfecti

Paul Stamets Doctor of science and owner of Fungi Perfecti

Interviewer ”Paul Stamets wrote in his book ‘Mycelium Running’ that one of the main reasons mycoremediation is not widely used in the US is patents on the technology, but what might the main obstacles preventing its use elsewhere in the world be?”

Loni Ronnebaum: There are actually many reasons why Mycoremediation is still not widely used in the US or internationally. The main reason for this is that while there is a wealth of information on how, why, and with what efficiency fungi are capable of degrading hydrocarbons, effective large-scale delivery systems for mycoremediation are still considered experimental by regulatory authorities tasked with permitting remediation plans as they have yet to be performed. Legally - In order to do hands on remediation a group/company would need to have a certified environmental professional (like a licensed environmental engineer) on their team in order to be able to be secure the professional liability insurance necessary to practice environmental remediation. These policies are pretty costly, in addition to general liability insurance. All environmental cleanup companies must also secure permits from governing state and/or federal agencies and in turn need to prepare quality assurance/quality control plans for each project. Contaminated land often has a mixture of pollutants. You need to plan on how you will address any pollutants that are not remediated using the fungi you select. Logistically - Facilities/farms need to produce mycelium by the cubic yard, or hundreds of cubic yards. This would require a laboratory and heavy equipment. A company would need to start with at least 2 million dollars for capital costs if intended to practice at a commercial scale.

Paul Stamet: " Every community should have a gourmet mushroom farm — to help build carbon in the soil, to provide local healthy food and to be able to recycle very proximate sources of debris and waste. Every gourmet mushroom farm (they should all be certified organic) should be reinvented as an environmental healing center so that the mycelium can be used for remediation locally. Moist mycelium weighs a lot; so shipping tons of mycelium across country does not make any sense for remediation. With the debris fields that are close to the problems, you want to keep that distance as short as possible and site the farms in close proximity. My dream is that there would thousands upon thousands of small mushroom farms spread across the world that would be tied in to healing art centers, schools, to teaching environmental sciences, to teaching basic biology and the role of fungi in nature."

Loni Ronnebaum: There are many, many variables to consider in large-scale remediation projects internationally. To give you an idea of the complexity, factors to consider include screening the appropriate native mushroom species and available substrates to find combinations with the capacity for robust growth in the local climate as well as species

59 with the targeted metabolic activity to degrade the contaminants of concern. As you can see there is a lot more standing in the way than patents. You might be happy to know that Paul has since released his Mycoremediation patents to public domain.

Interviewer: How effective is the mycoremediation process compared to other soil remediation techniques (eg: phytoremediation/incineration) in terms of...

a.time taken? b.the removal of pollutants (specifically hydrocarbons) from soil?

Loni Ronnebaum: “In my email I have attached the WSDOT study for you to review and compare. Mycelium seems to work faster and better than plants or bacteria alone on the remediation of hydrocarbons from soil. That said - a lot of this depends on environmental factors.”

Interviewer: “Which types of bioremediation are generally combined with mycoremediation and why?”

Loni Ronnebaum: Phytoremediation is a common companion. One of the most valuable restoration tools that we have as mycologists for helping ecosystem recovery is by using mycorrhizal fungi. In some cases adding mycorrhizal fungi to the roots can improve plants growth factors and help with immune responses to infections. Mycorrhizal soil drench or root inoculation of the to be planted trees is a good first line of defense to reestablish a viable forest.

Interviewer:” Since P. ostreatus doesn’t produce lignin-peroxidase, why is it still equally or more effective at degrading pollutants than fungi which do produce this enzyme?”

Loni Ronnebaum: “There are many key players in the lignolytic system of white rot fungi. Lignin peroxidase, manganese peroxidase, other H2O2 producing enzymes, and laccases can all act upon molecules that are broadly similar to lignin.”

Interviewer: “Is P. ostreatus capable of degrading long-chain aliphatic hydrocarbons as well as aromatic ones, since these are less similar to the structure of lignin than polyaromatic hydrocarbons?”

Loni Ronnebaum: “Yes, P. ostreatus and many other fungi have been studied for their ability to degrade these compounds.”

Interviwer: “Would mixed hydrocarbons like diesel, for example, be toxic to P. ostreatus at high concentrations? and If so, what is the approximate maximum concentration that it can be expected to endure and subsequently degrade”

Loni Ronnebaum: “We are not aware of the maximum concentration of petroleum tolerated by P. ostreatus . Certain fungi can use aliphatic or aromatic hydrocarbons as a sole carbon source, indicating that it is not toxic to those organisms. That said, there are often other compounds present in the petroleum that can exhibit other toxic effect.”

Interviewer: “Supposing that a spawn of P. ostreatus was introduced to a hydrocarbon-contaminated site and then proceeded to successfully grow, degrade the pollutants and flourish on the contaminated soil...Is there a possibility that the presence of P. ostreatus might disrupt the local ecosystem if it is not native species there ? If so, how?”

Loni Ronnebaum: “P. ostreatus has global distribution and spores travel vast distances, so relocating mycelium is generally not thought to pose a problem. For that matter we suggest working with local strains when finding a suitable candidate for remediation of a particular site.”

Interviewer: “Would the mushrooms be likely to accumulate contaminants from the soil, such as heavy metals, which might bioaccumulate in organisms of an ecosystem if the mushroom were then consumed? If so, how might these mushrooms be dealt with?”

Loni Ronnebaum: “It is important to note which species bioaccumulate, or hyperaccumulate which toxins. Metals accumulate within many fungi to varying degrees, so there is not a straightforward answer to this question. P. ostreatus is not a known hyperaccumulator, but other mushrooms are. Also, the bioaccumulation factor varies depending on the specific metal. Any mushrooms grown on contaminated materials should be met with caution in terms of their edibility. See Mycelium Running for details.

Most species that tend to hyper accumulate metals are Mycorrhizal so a mix of plants and correlating endo/ecto fungi that associate with those plants could be used to target those toxins. They would need to be removed from the area and disposed of like toxic waste (Incineration). A similar plan of attack was developed by Paul regarding the Fukushima meltdown.