Mycoremediation of hydrocarbon-contaminated brownfield ...
Transcript of Mycoremediation of hydrocarbon-contaminated brownfield ...
![Page 1: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/1.jpg)
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
![Page 2: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/2.jpg)
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.
![Page 3: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/3.jpg)
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
![Page 4: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/4.jpg)
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
![Page 5: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/5.jpg)
1
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.
![Page 6: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/6.jpg)
2
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.
![Page 7: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/7.jpg)
3
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
![Page 8: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/8.jpg)
4
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.
![Page 9: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/9.jpg)
5
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.
![Page 10: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/10.jpg)
6
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).
![Page 11: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/11.jpg)
7
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
![Page 12: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/12.jpg)
8
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
![Page 13: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/13.jpg)
9
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
![Page 14: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/14.jpg)
10
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).
![Page 15: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/15.jpg)
11
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).
![Page 16: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/16.jpg)
12
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).
![Page 17: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/17.jpg)
13
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).
![Page 18: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/18.jpg)
14
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.
![Page 19: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/19.jpg)
15
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
![Page 20: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/20.jpg)
16
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).
![Page 21: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/21.jpg)
17
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/aa2264/img14.gif) Figure 3: molecular structure of lignin (Soygun et al. 2013). Figure 4: molecular structures of selected PAHs
(Burright 1986).
![Page 22: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/22.jpg)
18
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).
![Page 23: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/23.jpg)
19
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.
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
Figure : Phenol molecule (http://ehs.ucsc.edu/lab-safety-manual/specialty-chemicals/phenol.html)
Figure 6: phenol (Djurhuus et al. 2016)
Figure 7: quinone (Djurhuus et al. 2016)
![Page 24: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/24.jpg)
20
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.
![Page 25: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/25.jpg)
21
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.
![Page 26: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/26.jpg)
22
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.
![Page 27: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/27.jpg)
23
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.
![Page 28: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/28.jpg)
24
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
![Page 29: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/29.jpg)
25
(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).
![Page 30: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/30.jpg)
26
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
![Page 31: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/31.jpg)
27
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,
biomounds, compost cells
Involves excavation of soil and placing in heaps or aerated piles, and
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
![Page 32: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/32.jpg)
28
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
In situ bioremediation
Achieved by creating a favorable environment to stimulate the natural or
inoculated population of microorganisms. Biodegradation rate is influenced
by biostimulation, bioaugmentation or cometabolism
Bioventing
In situ bioremediation
Involves injection of air or water to supply
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
attenuation
A strategy of allowing natural processes to reduce contaminant
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.
![Page 33: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/33.jpg)
29
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
![Page 34: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/34.jpg)
30
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).
![Page 35: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/35.jpg)
31
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:
![Page 36: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/36.jpg)
32
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:
![Page 37: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/37.jpg)
33
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
![Page 38: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/38.jpg)
34
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.
![Page 39: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/39.jpg)
35
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
![Page 40: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/40.jpg)
36
(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
![Page 41: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/41.jpg)
37
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).
![Page 42: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/42.jpg)
38
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.
![Page 43: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/43.jpg)
39
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
![Page 44: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/44.jpg)
40
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
![Page 45: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/45.jpg)
41
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.
![Page 46: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/46.jpg)
42
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.
![Page 47: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/47.jpg)
43
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:
![Page 48: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/48.jpg)
44
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
![Page 49: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/49.jpg)
45
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
![Page 50: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/50.jpg)
46
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
![Page 51: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/51.jpg)
47
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.
![Page 52: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/52.jpg)
48
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
![Page 53: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/53.jpg)
49
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).
![Page 54: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/54.jpg)
50
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.
![Page 55: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/55.jpg)
51
Bibliography
Aguilar-rivera, N., A. Moran, and D. Arturo. 2012. “Production of Pleurotus Ostreatus ( Oyster
Mushroom ) Grown on Sugar Cane Biomass ( Trash , Bagasse and Pith ),” no. 1.
Alexander, M. 1994. Biodegradation and Bioremediation. 1sted. San Diego: Academic Press Inc.
Anderson, C., and G. Juday. 2016. “Mycoremediation of Petroleum : A Literature Review.” Journal
of Environmental Science and Engineering A (5): 397–405. doi:10.17265/2162-
5298/2016.08.002.
Aust, S. D. 1995. “Mechanisms of Degradation by White Rot Fungi.” Environmental Health
Perspectives 103 (SUPPL. 5): 59–61. doi:10.1289/ehp.95103s459.
Bai, Y.H., Y.Q. Feng, D.B. Mao, and C.P. Xu. 2012. “Optimization for Betulin Production from
Mycelial Culture of Inonotus Obliquus by Orthogonal Design and Evaluation of Its Antioxidant
Activity.” Journal of the Taiwan Institute of Chemical Engineers 43 (5). Taiwan Institute of
Chemical Engineers: 663–69. doi:10.1016/j.jtice.2012.03.004.
Baldrian, P., and J. Gabriel. 2002. “Copper and Cadmium Increase Laccase Activity in Pleurotus
Ostreatus.” FEMS Microbiology Letters 206 (1): 69–74. doi:10.1016/S0378-1097(01)00519-5.
Baldrian, P., C. in der Wiesche, J. Gabriel, F. Nerud, and F. Zadrazil. 2000. “Influence of Cadmium
and Mercury on Activities of Lignolytic Enzymes and Degradation of Polycyclic Aromatic
Hydrocarbons by Pleurotus Ostreatus in Soil.” Applied and Environmental Microbiology 66 (6):
2471–78. doi:10.1128/AEM.66.6.2471-2478.2000.
Bartha, R. 1977. “The Microbiology of Aquatic Oil Spills.” Advances in Applied Microbiology 22 (1):
225–66.
Bhattacharya, S., A. Das, K. Prashanthi, P. Muthusamy, and J. Angayarkanni. 2014.
“Mycoremediation of Benzo[a]pyrene by Pleurotus Ostreatus in the Presence of Heavy Metals
and Mediators.” 3 Biotech 4 (2): 205–11. doi:10.1007/s13205-013-0148-y.
Bogan, B.W., R.T. Lamar, W.D. Burgos, and M. Tien. 1999. “Extent of Humification of Anthracene,
FLuoranthene, andbenz[oAlph]apyrene by Pleurotus Ostreatus during Growth in PAH-
Contaminated Soil.” Letters in Applied Microbiology 28 (1): 250–254.
Bolton, J.L., M.A. Trush, T.M. Penning, G. Dryhurst, and T.J. Monks. 2000. “Role of Quinones in
Toxicology.” Chemical Research in Toxicology 13 (3): 135–60. doi:10.1021/tx9902082.
Bouchez, M., D. Blanchet, V. Bardin, F. Haeseler, and J.P. Vandecasteel. 1999. “Efficiency of
Defined Strains and Soil Consortia in Biodegradation of Polycyclic Aromatic Hydrocarbon
(PAH)mixtures.” Biodegradation 6: 429–35.
Brady, N.C., and R.R. Weil. 2007. The Nature and Properties of Soils. 14e ed. PEARSON Education.
![Page 56: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/56.jpg)
52
Burright, D. 1986. “Coal Tar Pitch Volatiles (CTPV) Coke Oven Emissions (COE) Selected
Polynuclear Aromatic Hydrocarbons (PAHs).” OSHA.
https://www.osha.gov/dts/sltc/methods/organic/org058/org058.html.
Baarschers, W.H., and H.S. Heitland. 1986. “Biodegradation of Fenitrothion and Fenitrooxon by the
Fungus Trichoderma Viride.” Journal of Agricultural and Food Chemistry 34 (4): 707–9.
doi:10.1021/jf00070a029.
Carlile, M.J., S.C. Watkinson, and G.W. Gooday. 1994. The Fungi. 2nded. Academic Press.
CCME. 1999. “Canadian Water Quality Guidelines for the Protection of Aquatic Life - Polycylic
Aromatic Hydrocarbons.” Canadian Environmental Quality Guidelines, 1–14.
Cerniglia, C.E, and J.B. Sutherland. 2016. “Bioremediation of Polycyclic Aromatic Hydrocarbons by
Ligninolytic and Non-Ligninolytic Fungi.” New Biotechnology 32 (6): 52.
doi:10.1017/CBO9780511541780.008.
Crawford, R.L.C., and D.L. Crawford. 2005. Bioremediation: Principles and Applications. Edited by
Ronald L Crawford and Don L Crawford. Biotechnology Research. 1sted. Vol. 6. University of
Idaho, Moscow, Idaho, USA CAMBRIDGE.
http://assets.cambridge.org/97805210/19156/frontmatter/9780521019156_frontmatter.pdf.
Davis, M.W., J.A. Glaser, J.W. Evans, and R.T. Lamar. 1993. “Field Evaluation of the Lignin-
Degrading Fungus Phanerochaete Sordida to Treat Creosote-Contaminated Soil.” Environ. Sci.
Techno 27 (12): 2572–2576. doi:10.1021/es00048a040.
Diels, L., and K. Vanbroekhoven. 2008. Methods and Techniques for Cleaning-Uo Contaminated
Sites. 1sted. Belgium: NATO Science for Peace and Security Series C: Environmental Security.
http://www.springer.com/us/book/9781402068737.
Dindar, E., F. Olcay, T. Şağban, and H. Savaş Başkaya. 2013. “Bioremediation of Petroleum-
Contaminated Soil.” J. BIOL. ENVIRON. SCI 7 (19): 39–47.
http://jbes.uludag.edu.tr/PDFDOSYALAR/19/mak06.pdf.
Djurhuus, T. S., T. Callensen, F. Krijom, K. Jakobsen, and S. Skou. 2016. “Mushroom Group.”
Roskilde University.
Douben, P.E.T. 2003. PAHs: An Ecotoxicological Perspective. 1sted. Wiley.
http://eu.wiley.com/WileyCDA/WileyTitle/productCd-0471560243.html.
Durr, L. 2016. “Mycoremediation Project: Using Mycelium to Clean up Diesel Contaminated Soil in
Orleans, California.” Orleans,California. http://www.mycoalliance.com/wp-
content/uploads/2016/08/MycoremediationReport_FungaiaFarm_2016.pdf.
Environment Protection Authority. 2005. “Soil Bioremediation,” no. November: 1–9.
http://www.epa.sa.gov.au/xstd_files/Site contamination/Guideline/guide_soil.pdf.
H. McKee, R. 2016. Screening Assessment Petroleum Sector Stream Approach. Government of
![Page 57: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/57.jpg)
53
Canada.
Harris, O., and J. Corcoran. 1999. “TOXICOLOGICAL PROFILE FOR N-HEXANE.”
Haught, R. C., R. Neogy, S. S. Vonderhaar, E. R. Krishnan, S. I. Safferman, and J. Ryan. 1995.
“Land Treatment Alternatives for Bioremediating Wood Preserving Wastes.” Hazardous Waste
and Hazardous Materials 12 (4): 329–44.
Hoa, H.T., C.L. Wang, and C.H. Wang. 2015. “The Effects of Temperature and Nutritional Conditions
on Mycelium Growth of Two Oyster Mushrooms (Pleurotus Ostreatus and Pleurotus
Cystidiosus).” Mycobiology 43 (4): 423–34. doi:10.5941/MYCO.2015.43.4.423.
Hofrichter, M. 2002. “Review: Lignin Conversion by Manganese Peroxidase (MnP).” Enzyme and
Microbial Technology 30 (4): 454–66. doi:10.1016/S0141-0229(01)00528-2.
IETU. 1999. “Comprehensive Report of Remediation Applications at an Oil Refinery in Southern
Poland, Report Prepared for U.S. DOE, FETC.”
Ipeaiyeda, A.R., G.O. Nwauzor, and S.O. Akporido. 2015. “Biodegradation of Polycyclic Aromatic
Hydrocarbons in Agricultural Soil Contaminated with Crude Oil from Nigeria Refinery Using
Pleurotus Sajor-Caju.” Bioremediation&Biodegradation 6 (4): 4–7. doi:10.4172/2155-
6199.1000301.
Jones, E.B.G. 1976. “Recent Advances in Aquatic Mycology.” Journal of Basic Microbiology 19 (1).
London: Wiley: 70–71.
Kashangura, C. 2008. “Optimisation of the Growth Conditions and Genetic Characterisation of,” no.
September.
Kerem, Z., D. Friesem, and Y. Hadar. 1992. “Lignocellulose Degradation during Solid-State
Fermentation: Pleurotus Ostreatus versus Phanerochaete Chrysosporium.” Appl. Envir.
Microbiol. 58 (4): 1121–27. http://aem.asm.org/cgi/content/long/58/4/1121.
Kummer, P. 1871. Der Führer in Die Pilzkunde. 1sted. Zerbst :Verlag von E. Luppe’s
Buchhandlung,1871. doi:10.5962/bhl.title.50494.
Kurtzman, R. H., and F. Zadrazil. 1982. “Physiological and Taxonomic Considerations for Cultivation
of Pleurotus Mushrooms.” In Tropical Mushrooms: Biological Nature and Cultivation Methods,
1sted., 1:299–348. Hong Kong: The Chinese University of Hong Kong.
https://books.google.dk/books?id=0luzyrBPARgC&pg=PA299&lpg=PA299&dq=Physiological+
and+taxonomic+considerations+for+cultivation+of+Pleurotus+mushrooms+Kurtzman,+R.+H.,
+and+F.+Zadrazil&source=bl&ots=DPrNyCqklG&sig=8tUAiUc37rbnOghgh3UgXuOqLJY&hl=
da&sa=X&ved=.
Marin, S., V. Sanchis, A.J. Ramos, and N. Magan. 1998. “Effect of Water Activity on Hydrolytic
Enzyme Production by Fusarium Moniliforme and Fusarium Proliferatum during Colonisation of
Maize.” International Journal of Food Microbiology 42 (3): 185–94. doi:10.1016/S0168-
![Page 58: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/58.jpg)
54
1605(98)00077-4.
Marzullo, L., R. Cannio, P. Giardina, and G. Sannia. 1995. “Veratryl Alcohol Oxidase from Pleurotus
Ostreatus Participates in Lignin Biodegradation and Prevents Polymerization of Laccase-
Oxidized Substrates.” J Biol Chem 270 (8): 3823–27. doi:10.1074/jbc.270.8.3823.
Matsumura, F., and G.M Boush. 1966. “Malathion Degradation by Trichoderma Viride and a
Pseudomonas Species.” Science 153 (3741): 1278–80.
doi:http://dx.doi.org/10.1126/science.153.3741.1278.
McCartney, M.P., D.K. Yawson, and J. Seshoka. 2004. Hydrology and Water Resources
Development in the Olifants River Catchment. 76thed. International Water Management
Institute, Colombo, Sri Lanka. http://hdl.handle.net/10535/4810.
Mikhail, F., and P. Gerd. 1996. “Preferential Formation of Benzo ( a ) Pyrene Adducts at Lung Cancer
Mutational Hotspots in P53.” Science, New Series 270 (5286): 430–32.
Mukherjee, I., and M. Gopal. 1996. “Degradation of Chlorpyrifos by Two Soil Fungi Aspergillus Niger
and Trichoderma Viride.” Toxicological & Environmental Chemistry 57 (1–4): 145–51.
doi:10.1080/02772249609358383.
Novotný, Č., P. Erbanová, V. Šašek, A. Kubátová, T. Cajthaml, E. Lang, J. Krahl, and F. Zadražil.
1999. “Extracellular Oxidative Enzyme Production and PAH Removal in Soil by Exploratory
Mycelium of White Rot Fungi.” Biodegradation 10 (1): 159–68. doi:10.1023/A:1008324111558.
Nzengung, V.D. 2005. “Case Studies of Phytoremediation of Petrochemicals and Chlorinated
Solvents in Soil and Groundwater.” Proceedings of the 2005 Georgia Water Resources
Conference. https://smartech.gatech.edu/bitstream/handle/1853/47319/Nzengungv -
GWRCPAPER March23.pdf?sequence=1&isAllowed=y.
Obiro, O. 1988. “Studies on the Biodegradation Potentials of Some Microorganisms Isolated from
Water Systems of Two Petroleum Producing Areas in Nigeria.” Nigeria. Nig. J. Bot. 1 (1): 81–
90.
Oliver, L., U. Ferber, D. Grimski, K. Millar, and P. Nathanail. 2005. “The Scale and Nature of
European Brownfields.” Berlin, Germany.
https://www.researchgate.net/publication/228789048_The_Scale_and_Nature_of_European_
Brownfield.
Ouyang, W., H. Liu, V. Murygina, Y. Yu, Z. Xiu, and S. Kalyuzhnyi. 2005. “Comparison of Bio-
Augmentation and Composting for Remediation of Oily Sludge: A Field-Scale Study in China.”
Process Biochemistry 40 (12): 3763–68.
http://www.sciencedirect.com/science/article/pii/S1359511305002564.
Paull, E. 2008. “The Environmental and Econonic Impacts of Brownfields Redevelopment.”
http://www.nemw.org/wp-content/uploads/2015/06/2008-Environ-Econ-Impacts-Brownfield-
![Page 59: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/59.jpg)
55
Redev.pdf.
Petavy, F, V Ruban, P Conil, J.Y Viau, and J.C Auriol. 2009. “Two Treatment Methods for Stormwater
Sediments--Pilot Plant and Landfarming--and Reuse of the Treated Sediments in Civil
Engineering.” Environ Technol 30 (8): 825–30. doi:10.1080/09593330902990113.
Plácido, J., and S. Capareda. 2015. “Ligninolytic Enzymes: A Biotechnological Alternative for
Bioethanol Production.” Bioresources and Bioprocessing 2 (1). Springer: 23.
doi:10.1186/s40643-015-0049-5.
Pletsch, M., B.S. de Araujo, and B.V. Charlwood. 1999. “Novel Biotechnological Approaches in
Environmental Remediation Research.” Biotechnology Advances 17 (8): 679–87.
doi:10.1016/S0734-9750(99)00028-2.
Pozdnyakova, N. N., V. E. Nikitina, and O. V. Turovskaya. 2008. “Bioremediation of Oil-Polluted Soil
with an Association Including the Fungus Pleurotus Ostreatus and Soil Microflora.” Applied
Biochemistry and Microbiology 44 (1): 60–65. doi:10.1007/s10438-008-1010-6.
Pozdnyakova, N.N. 2012. “Involvement of the Ligninolytic System of White-Rot and Litter-
Decomposing Fungi in the Degradation of Polycyclic Aromatic Hydrocarbons.” Biotechnology
Research International 2012 (243217): 1–20. doi:10.1155/2012/243217.
Reece, J.B., L.A. Urry, M.L. Cain, S.A. Wasserman, P.V. Minorsky, and R.B. Jackson. 2013.
Campbell Biology. 10thed. Pearson.
Roldán-Martín, A., F. Esparza-García, G. Calva-Calva, and R.R. Vazquez. 2006. “Effects of Mixing
Low Amounts of Orange Peel ( Citrus Reticulata) with Hydrocarbon-Contaminated Soil in Solid
Culture to Promote Remediation.” Journal of Environmental Science and Health 41(A) (10):
2373–85. doi:10.1080/10934520600873548.
Ronnebaum, L.J. 2016. “Email Correspondence with Loni Jean Ronnebaum from Fungi Perfecti.”
Ruiz-Dueñas, F.J., M. Morales, E. García, Y. Miki, M.J. Martínez, and A.T. Martínez. 2009.
“Substrate Oxidation Sites in Versatile Peroxidase and Other Basidiomycete Peroxidases.”
Journal of Experimental Botany 60 (2): 441–52. doi:10.1093/jxb/ern261.
Sánchez, C. 2009. “Lignocellulosic Residues: Biodegradation and Bioconversion by Fungi.”
Biotechnology Advances 27 (2). Elsevier Inc.: 185–94. doi:10.1016/j.biotechadv.2008.11.001.
Sharp, L. W. 1943. Fundamentals of Cytology. New York & London, McGraw-Hill Book Company,
Inc.
Singh, A., R.C. Kuhad, and O.P. Ward. 2009. Advances in Applied Bioremediation. Soil Biology, Vol.
17. Vol. 17. doi:10.1007/978-3-540-89621-0.
Singh, H. 2006. Mycoremediation : Fungal Bioremediation. New York: Wiley-Interscience. WIley.
http://fmedicine.ajums.ac.ir/_fmedicine/documents/fungal
bioremediation_20130415_112807.pdf.
![Page 60: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/60.jpg)
56
Soygun, K., S. Şimşek, E. Yılmaz, and G. Bolayır. 2013. “Investigation of Mechanical and Structural
Properties of Blend Lignin-PMMA.” Advances in Materials Science and Engineering 2013
(Article ID 435260,): 1–6. doi:10.1155/2013/435260.
Spooner, B., and P. Roberts. 2005. Fungi. Edited by Derek A. Ratcliffe Sarah A. Corbet, S. M.
Walters, Richard West, David Streeter. 1sted. N N Collins.
Stamets, P. 2005. Mycelium Running: How Mushrooms Can Save the World. 1sted. Ten Speed
Press. https://www.amazon.com/Mycelium-Running-Mushrooms-Help-World/dp/1580085792.
Suthersan, S.S. 1999. “In Situ Bioremediation.” In Remediation Engineering: Desing Concepts,
1sted., 123–58. CRC Press. doi:978-1-56670-137-2.
Thomas, S.A., P. Becker, M.R. Pinza, and J.Q. Word. 1998. “Mycoremediation of Aged Petroleum
Hydrocarbon Contaminants in Soil.” Washington. doi:WA-RD 464.1.
Todd, D.G., R.L. Chessin, and J. Colman. 1999. “TOXICOLOGICAL PROFILE FOR TOTAL
PETROLEUM HYDROCARBONS (TPH).” Atlanta, Georgia.
https://www.atsdr.cdc.gov/ToxProfiles/tp123.pdf.
United States Environmental Protection Agency. 2000. “Treatment Experiences at RCRA Corrective
Actions.” www.epa.gov/tio.
Whitacre, David M. 2016. Reviews of Environmental Contamination and Toxicology Volume 236.
Reviews of Environmental Contamination and Toxicology. Vol. 236. doi:10.1007/978-3-319-
20013-2.
Winterlin, W., and S. R. Schoen. 1987. “The Effects of Various Soil Factors and Amendments on the
Degradation of Pesticide Mixtures.” J. Env. Sci. and Health B 22 (3): 347–77.
doi:10.1080/03601238709372561.
Woolley, Tom. 2000. Green Building Handbook: A Companion Guide to Building Products and Their
Impact on the Environment. 1sted. Vol. 2. Taylor & Francis Ltd.
Young, Darcy. 2012. “Bioremediation with White-Rot Fungi at Fisherville Mill: Analyses of Gene
Expression and Number 6 Fuel Oil Degradation.” Process Biochemistry 1 (12): 89.
http://commons.clarku.edu/mosakowskiinstitute/12/.
Zitte, L.F., G.D.B. Awi-Waadu, and A.U. John. 2012. “Effect of Oyster Mushroom (Pleurotus
Ostreatus) Mycelia on Petroleum Hydrocarbon Contaminated Substrate.” Journal of Agriculture
and Social Research 12 (2): 115–21. doi:10.1073/pnas.0703993104.
![Page 61: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/61.jpg)
57
Appendix
Appendix A:
Tabular comparison of case studies on P. ostreatus ability to degrade
hydrocarbons.
Title Reference Description Degradation
time
Findings
A) Mycoremediation of aged petroleum hydrocarbons in soil
(Thomas, Becker, Pinza, & Word, 1998), (Stamets, 2005)
Mycoremediation (using P. ostreatus ) was compared with bioremediation and enhanced bacterial remediation of diesel and heavy-oil contaminated soil, gasoline contaminated soil, and soil from a Bellingham truck repair yard containing weathered oil contaminants
4 months
All mycoremediated soil showed flourishing fungal growth. Vascular plant and wild fungi communities began to develoP. The soil changed texture and oil odour was no longer present. Soil in bioremediated, enhanced bacteria remediated and control groups remained unchanged.
B) Bioremediation with White-Rot Fungi at Fisherville Mill: Analyses of Gene Expression and Number 6 Fuel Oil Degradation
(Young & Young, 2012)
Investigating the ability of P. ostreatus and 5 other fungal species (Irpex lacteus, Phlebia radiata, Punctularia strigosozonata, Trametes versicolor and Trichaptum biforme) spawns to degrade Bunker.C oil obtained from Fisherville Mill brownfield site in laboratory conditions
6 Months (+ 8 months prior adapting to oil in agar)
P. ostreatus reduced the presence of phenathrene( a representative PAH) by 75-95%, C14 alkane by 35-48%, and C10 alkane by 97-98%
C) Bioremediation of Oil-polluted Soil with an Association
(Pozdnyakova, Nikitina, & Turovskaya, 2008)
The ability of 2 strains of P. ostreatus (D1 and 339) as well as L. edodes 0779 to grow on petroleum
4 weeks
After 2 weeks the P. ostreatus mycelium in sterilized soil showed poor oil degradation (9.39%
![Page 62: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/62.jpg)
58
Including the Fungus P. ostreatus and Soil Microflora
contaminated soil and degrade the oil was investigated. This was done using both sterilised and non-sterilised soil as well as a culture without fungi to determine the role of the soil microflora in the process.
with D1 strain and 6.35 with strain 339), but was able to degrade 60.45% (D1) and 14.5%(336) in unsterilized soil. Soil microflora alone degraded 34.53% of oil. After an additional 2 weeks, it was 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 Myshroom (P. ostreatus ) mycelia on petroleum hydrocarbon contaminated substrate
(Zitte, Awi-Waadu, & John, 2012)
The effectiveness of 3 identical samples of sawdust P. ostreatus spawn at degrading 20ml, 40ml and 60ml of crude oil (added directly to the spawn mix) was tested.
4 weeks
The amount of crude oil present in the mushroom mixtures reduced by 90%, 87% and 85% respectively
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?”
![Page 63: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/63.jpg)
59
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
![Page 64: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/64.jpg)
60
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.”
![Page 65: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/65.jpg)
61
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.
![Page 66: Mycoremediation of hydrocarbon-contaminated brownfield ...](https://reader030.fdocuments.us/reader030/viewer/2022040518/624a73c70e3f881e400c4496/html5/thumbnails/66.jpg)
62