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Mycoremediation of Crude Oil Contaminated Soil Page 1
Introduction
Crude oil is one of the most important resources of energy in the modern
industrial world. Oils are used to run many types of engines, lamps, heaters and
stoves. The invention of the internal combustion engine and its fast adoption in all
transport forms enlarged the employment of this natural resource, thus increasing its
demand, production, transport, stockpiling, and distribution, as well as the raw oil
and its by-products. All these activities involve pollution risks that can be
minimized, but not totally eliminated, causing several problems for the environment
(Pala et al., 2006).
Oil spills have become global problem particularly in industrialized and
developing countries. Contamination of soils and aquifers by oil spills is a persistent
and widespread pollution problem ravaging almost all compartments of the
environment and imposing serious health implications and ecological disturbances
(Bundy et al., 2002; Okoh, 2006). The quality of life on earth is linked, inextricably,
to the overall quality of the environment. Releases of persistent, bioaccumulative
and toxic chemicals have a detrimental impact on human health and the
environment. These contaminants find their way into the tissues of plants, animals
and human beings by the movement of hazardous constituents in the environment
(Vidali, 2001). Petroleum contaminants are typical examples of these hazardous
constituents.
Crude oil possess moderate to high acute toxicity to biota with product
specific toxicity related to the type and concentration of aromatic compounds (Song
and Bartha, 1990). Generally, petroleum contamination results from leakages of
underground and above ground storage tanks, spillage during transport of petroleum
products, tanker accidents, unplanned releases and current industrial processes
(Sarkar et al., 2005). The application of biotechnological processes involving
microorganisms, with the objective of solving environmental pollution problems, is
rapidly growing, in recent decades, where petroleum hydrocarbons and its by-
products are concerned. Mycoremediation, which take advantage of fungal
degradation of organic and inorganic substances, can be defined as the use of fungal
Mycoremediation of Crude Oil Contaminated Soil Page 2
systems to catalyze the destruction or transformation of various chemicals to less
harmful forms (Pinza et al., 1998).
Fungi secrete non specific extracellular enzymes, which are involved in the
degradation of lignin (Barr and Aust, 1994). The same mechanisms that give these
fungi the ability to degrade lignin are also used to degrade a wide range of pollutants
such as total petroleum hydrocarbons (TPHs), dichlorodiphenyltrichloroethane
(DDT), trinitrotoluene (TNT), polychlorinated biphenyl (PCB) and polycyclic
aromatic hydrocarbons (PAHs). Various fungi use some crude oil fractions as a sole
source of carbon and change it to non toxic compounds such as CO2 (Cerniglia,
1992). The aliphatic and some aromatic fractions are the most degradable
components, resins and asphaltenes are believed to be resistant to biodegradation
(Atlas, 1981; Oudot et al., 1993). Mycoremediation is an attractive approach to
cleaning up petroleum hydrocarbons because it is simple to maintain, applicable
over large areas, cost-effective and leads to complete destruction of the contaminant
(Huesemann, 1994).
1.1 Soil environment and oil spills
Soil is a complex system in its structure and function due to intricate
relations between the biotic community and the medium surrounding it. In soil, de
novo material is produced constantly and, at the same time, organic matter is
decomposed, releasing energy and providing nutrients to plants and other organisms
(Paul, 2007). Soil organic matter (SOM) is essential in supporting the chemical and
physical properties of the soil, thus maintaining soil quality and function.
Microorganisms are mainly responsible for SOM dynamics, but the role of
micro-, meso- and macro-fauna is also crucial for assisting microbes in colonizing
and degrading the organic matter by physically and chemically altering the soil
structure (Coleman and Wall, 2007). The most diverse group of microorganisms
living in soil are fungi followed by bacteria and archaea. Representatives of the
traditional phyla of the Fungi Kingdom found in soil are: i) Chytridiomycota is
represented by plant pathogens and parasites; ii) Zygomycota includes parasitic and
saprotrophic fungi; iii) Glomeromycota includes arbuscular mycorrhiza-forming
fungi; iv) Ascomycota is the largest group with approximately 50,000 species and,
Mycoremediation of Crude Oil Contaminated Soil Page 3
thus, with different ecological roles in the soil; v) in Basidiomycota only, the so-
called homobasidiomycetes are found in soils which include wood-decaying and
litter-decomposing fungi, soil-borne pathogens of crops and forest trees, as well as
the ectomycorrhizal fungi of woody plants (Thorn and Lynch, 2007).
Humic substances (HS) constitute the major percentage (up to 80%) of SOM
originating from the transformation of plant and animal residues and from microbial
activity (Senesi and Loffredo, 2001). The exact composition and chemical structure
of HS are not yet known, but lignin-derived structures are the main source of HS
formation (Shevchenko and Bailey, 1996; Senesi and Loffredo, 2001). Another
important aspect is that the chemical structure of HS resembles that of organic
contaminants. Consequently, soil microorganisms, and especially wood-decaying
and litter-decomposing fungi, with the ability to degrade and even mineralize HS are
adapted to degrade contaminants present in the soil (Kastner and Hofrichter, 2001;
Steffen et al., 2002b). This adaptability to contaminants represents an advantage for
soil decontamination by fungi.
The petroleum industry generates a high amount of oily wastes during
storage, refining, drilling and processing operations (Pavlova and Ivanova, 2003).
Accidental and deliberate crude oil spills have been, and still continue to be, a
significant source of environmental pollution, and poses a serious environmental
problem, due to the possibility of air, water and soil contamination (Trindade et al.,
2005). Oil contamination can adversely affect the soil microbes and plant as well as
contaminate groundwater resources for drinking or agriculture (Hong et al., 2005).
Although practically all petroleum constituents can infiltrate the soil the ones that do
it most frequently are petroleum fuels as they have the major share in the turnover of
petroleum products (Snyder and Kalf, 1994; Douaud, 1995). Petroleum products on
the ground surface can penetrate deep into the ground. Their soluble components are
the source of contamination and can reach as far as the underground water table
threatening fauna, flora and underground water reservoir of drinking water
(Schobert, 1990).
Mycoremediation of Crude Oil Contaminated Soil Page 4
1.2 Environmental fate of petroleum hydrocarbons
Petroleum-based products are the major source of energy for industry and
daily life. Petroleum is also the raw material for many chemical products such as
plastics, paints, and cosmetics. The transport of petroleum across the world is
frequent, and the amounts of petroleum stocks in developed countries are enormous.
Petroleum hydrocarbon pollution is one of the main environmental problems, not
only by the important amounts released but also because of their toxicity. When
petroleum is accidentally spilled into the environment, one would like to see an
immediate 100% recovery of the spill to minimize adverse environmental effects.
However, it becomes both impractical and uneconomical to recover all of a
petroleum spill using conventional physical-chemical recovery methods (Cresswell,
1977).
An important area of concern from an oil spill standpoint is the fate of oil
spilled on land, or oil washed ashore from a spill on water. It is generally known that
most soils provide excellent environments for microbial destruction of organic
matter with more than 100 different species of microorganisms found in the soil that
are known to attack and decompose many of the hydrocarbons contained in
petroleum (Dodson et al., 1972).
Petroleum products released into the environment undergo weathering
processes with time. These processes include evaporation, leaching (transfer to the
aqueous phase) through solution and entrainment (physical transport along with the
aqueous phase), chemical oxidation, and microbial degradation (Christensen and
Larsen, 1993). The rate of weathering is highly dependent on environmental
conditions. For example, gasoline, a volatile product, will evaporate readily in a
surface spill; while gasoline released below 10 feet of clay topped with asphalt will
tend to evaporate slowly (weathering processes may not be detectable for years).
Evaporative processes are very important in the weathering of volatile petroleum
products, and may be the dominant weathering process for gasoline. Automotive
gasoline, aviation gasoline, and JP-4 contain 20% to 99% highly volatile (less than 9
carbon atom) components. Figure 1 shows the common routes of hydrocarbons
escaping into environment.
Mycoremediation of Crude Oil Contaminated Soil Page 5
Fig. 1: Contaminants in soil can find their way to other areas of the environment
(Ashman and Puri, 2002; Fragoeiro, 2005)
Some of the common fates of hydrocarbons in environment are:
a) Hydrocarbons dissolved in water
For toxicology studies the part of the hydrocarbons that comes in contact
with organisms or is accumulative in the environment is most important. On the
other hand the molecules that are absorbed in the sediment will remain longer in the
environment because they are less available for degradation. Often water in oil
emulsion is formed in the aquatic environment, due to the increased viscosity of the
oil after evaporation of volatile compounds. This makes degradation less favourable
(Nicodem et al., 1997). In fact bacteria are only able to degrade hydrocarbons
dissolved in water. This explains the persistence of larger PAHs (Wodzinski and
Bertolini, 1972).
Only some fractions are dissolved in water after petroleum spill in the
environment, and this can be as low as only 2% (Nicodem et al., 1997). Other parts
are absorbed in the sediment or soil. Lighter 3- or 4- ring aromatic molecules are
soluble in water (31.7 mg/L), but the PAHs consisting of 5 or more aromatic rings
are insoluble in water (0.003 mg/L) and will become associated with the sediment
(Cerniglia, 1992; Shor et al., 2004). This makes them more persistent. Recent
research revealed that also the presence of humic acids can be very important for
solvability of PAHs insoluble in absence of humic acids.
Mycoremediation of Crude Oil Contaminated Soil Page 6
b) Bioaccumulation
PAHs are known for their bioaccumulation. PAHs accumulate in the lipid
rich tissues of animals. This is especially seen in the liver of fish and in the pancreas
of invertebrates. The hydrophilic PAHs are taken up by aquatic organisms from the
ventilated water. The other uptake route for the hydrophobic PAHs is via food and
sediment. The food uptake route implies accumulation of PAHs in the food chain,
which is of interest because humans are frequently the last part of the food chain.
For animals the uptake of PAHs shows a seasonal variation (Meador et al., 1995).
c) Sorption to sediments and soil
Sediment absorption is important for degradation of hydrocarbons because it
makes the hydrocarbons in general less available for degradation. Uptake of
hydrocarbons by microbes was shown to be much slower from the sediment than
when the hydrocarbons are in a solved state (Pignatello and Xing, 1996). The
absorption of organic compounds depends on a lot of factors. First the composition
of the sediment is an important factor. Further the presence of other organic
substances in the soil can have an influence. At last also the environmental
conditions like pH, salinity and temperature play a role in absorption (Meyers and
Quinn, 1973).
The effect of declining ability when hydrocarbons are longer absorbed in the
sediment or soil is called aging. Soil and sediment sorption appears to occur by a
multi-step process, and this is in fact what aging is. The effect of aging was being
determined by extraction experiments. This effect is being caused by the entering of
hydrocarbons in inaccessible parts of the soil matrix. This slows down both biotic
and abiotic degradation. It was found that the aging effect increases with
hydrocarbon molecular weight, and with water partition coefficient (Kow) and soil
organic carbon coefficient (Koc) of the sediment (Northcott and Jones, 2001). Aging
is a process that takes from a week to months and slows down biodegradation.
Aging takes place by diffusion in both organic parts of the soil and through
intraparticle nanopores (Pignatello and Xing, 1996).
Mycoremediation of Crude Oil Contaminated Soil Page 7
d) Formation of non-aqueous phase liquids (NAPLs)
Hydrocarbons absorbed in the soil can also be present in the form of NAPLs.
This is the case when the hydrocarbons are concentrated and are able to form their
own insoluble phase in the soil. This phase contains a mix of a lot of hydrocarbons.
Research on soils with GC/MS revealed that the concentration of harmful PAHs is
high in these NAPLs (Brown et al., 1999). NAPLs are important for bioavailability
because they make just as sediment sorption and aging the hydrocarbons less
available for biodegradation (Salanitro, 2001).
On land, crude oil spills have caused great negative impact on food
productivity. For example, a good percentage of oil spills that occurred on the dry
land between 1978 and 1979 in Nigeria, affected farm-lands in which crops such as
rice, maize, yams, cassava and plantain were cultivated (Onyefulu and Awobajo,
1979). Crude oil affects germination and growth of some plants (Onwurah, 1999a).
It also affects soil fertility but the scale of impact depends on the quantity and type
of oil spilled. Severe crude oil spill in Cross-River state, Nigeria, has forced some
farmers to migrate out of their traditional home, especially those that depend solely
on agriculture. This is because petroleum hydrocarbons ‘sterilize’ the soil and
prevent crop growth and yield for a long period of time. The yield of steroidal
sapogenin from tuber tissues of Dioscorea deltoidea is adversely affected by some
hydrocarbons (Hardman and Brain, 1977). The negative impact of oil spillages
remains the major cause of depletion of the Niger Delta of Nigeria vegetative cover
and the mangrove ecosystem (Odu, 1987). Crude oil contamination of land affects
certain soil parameters such as the mineral and organic matter content, the cation
exchange capacity, redox properties and pH value. As crude oil creates anaerobic
condition in the soil, coupled to water logging and acidic metabolites, the result is
high accumulation of aluminium and manganese ions, which are toxic to plant
growth.
The primary factors affecting the rate of petroleum degradation in soil are
essentially the same factors as for petroleum degradation in a water environment,
namely, petroleum composition, temperature, nutrients, oxygen availability, water,
and pH. For last many years scientists have been studying the effects of oil in the
soil environment by a method called "land farming." By "land farming" mean the
Mycoremediation of Crude Oil Contaminated Soil Page 8
planned, orderly addition of oil or oily wastes to the soil environment in such a
manner as to maximize biodegradation of the oil by the naturally occurring soil
microorganisms. This method could have application for cleanup of accidental
spills, as well as for disposal of oily wastes generated routinely from various
petroleum drilling and processing operations. For spills on land, land farming
approach could be applied directly to degrade the residual oil remaining after
cleanup. Oil-contaminated beach sand, straw, etc., that results after a spill on water
has washed ashore, could be picked up and carried inland to an acceptable area for
land farming (Kincannon, 1972; Raymond et al., 1976).
It is conceivable to say that there is a link between environmental health and
human health. While human health is an established field of science from time of
old, the concept of ‘environmental health’ can be viewed as a modern science,
which is measured as the viability of the inhabitants of a given ecosystem as affected
by ambient environmental factors (Shields, 1990). Practically, environmental health
involves the assessment of the health of the individual organisms and correlating
observed changes in health with changes in environmental conditions. Some
diseases have been diagnosed to be the consequences of crude oil pollution.
The health problems associated with oil spill may be through any or
combinations of the following routes: contaminated food and / or water, emission
and / or vapours. Toxic components in oil may exert their effects on man through
inhibition of protein synthesis, nerve synapse function, and disruption in membrane
transport system and damage to plasma membrane (Prescott et al., 1996). Crude oil
hydrocarbons can affect genetic integrity of many organisms, resulting in
carcinogenesis, mutagenesis and impairment of reproductive capacity (Short and
Heintz, 1997). The risk of drinking water contaminated by crude oil can be
extrapolated from its effect on rats that developed hemorrhagic tendencies after
exposure to water soluble components of crude oil (Onwurah, 2002). Volatile
components of crude oil after a spill have been implicated in the aggravation of
asthma, bronchitis and accelerating aging of the lungs (Kaladumo, 1996). Other
possible health effects of oil spill can be extrapolated from rats exposed to
contaminated sites and these include increased liver, kidney and spleen weights as
well as lipid per-oxidation and protein oxidation (Anozie and Onwurah, 2001).
Mycoremediation of Crude Oil Contaminated Soil Page 9
1.3 Composition and classification of crude oil
1.3.1 Types and composition:
Crude oils range from thin, light coloured oils consisting mainly of gasoline
to thick, black oil similar to melted tar, varying in appearance and composition from
one oil field to another. Crude oil is a viscous liquid mixture that contains thousands
of compounds mainly consisting of carbon and hydrogen. An “average” crude
contains 84% carbon, 14% hydrogen, 1-3% sulphur, and approximately 1.0%
nitrogen, 1.0% oxygen and 0.1% minerals and salts. Crude oil contains a complex
mixture of compounds that can be categorized into four fractions: saturates,
aromatics, asphaltenes and resins (Fig. 2). The saturated fraction consists of straight-
chain alkanes (normal alkanes), branched alkanes (isoalkanes), and cycloalkanes
(naphthenes). The aromatic fraction includes volatile monoaromatic hydrocarbons
such as benzene, toluene, and xylenes; polyaromatic hydrocarbons;
naphthenoaromatics; and aromatic sulfur compounds, such as thiophenes and
dibenzothiophenes. The asphaltene (phenols, fatty acids, ketones, esters, and
porphyrins) and resin (pyridines, quinolines, carbazoles, sulfoxides, and amides)
fractions consist of polar molecules containing N, S, and O. Asphaltenes are large
molecules dispersed in oil in a colloidal manner, whereas resins are amorphous
solids truly dissolved in oil. The relative distribution of these fractions depends on
many factors, such as the source, age, geological history, migration, and alteration of
crude oil (Speight, 1991; Gary and Handwerk, 1993).
Mycoremediation of Crude Oil Contaminated Soil Page 10
SATURATES AROMATICS
n-hexane Toluene
n-heptadecane (n-C17H36) Napthalene
Pristane (n-C19H40) Chrysene
17α(H),21β(H)-hopane benzo[a]pyrene
RESINS ASPHALTENES
(C79
H92
N2S
2O)
3
2-methylpyridine
Dibenzothiophene Fig. 2: Representative organic compounds found in crude oils (Leahy and Colwell, 1990)
Mycoremediation of Crude Oil Contaminated Soil Page 11
1.3.2 Classification
Crude oils are classified by viscosity, density and API gravity. API gravity was
developed as a means to identify the gasoline production potential of a crude oil; the
higher the API gravity, the more valuable the crude. Figure 3 illustrates classification of
crude oil by this density-gravity method.
Fig.3: Classification of crude oil on the basis of viscosity, density and gravity
(Lorraine, 2003)
Characteristics of different types of crude oil on the basis of its density and
gravity are presented in Table-1.
Table-1: Density-gravity characteristics of crude oil (Mackerer and Biggs, 1996;
Platts Oil gram, 2003)
Type of Crude Characteristics
1. Conventional or “light” crude Density-gravity range less that 934 kg/m3
(>33ºAPI)
2. “Heavy” crude oil
Density-gravity range from 1000 kg/m3
to
more than 934kg/m3
(10ºAPI to <28ºAPI)
Maximum viscosity of 10,000mPa.s(cp)
3. “Extra-heavy” crude oil; may
also include atmospheric
residua. (b.p.>340º C; >650ºF)
Density-gravity greater than 1000 kg/m3
(<10ºAPI) Maximum viscosity of
10,000mPa.s(cp)
4. Tar sand bitumen [before
upgrade] or natural asphalt;
may also include vacuum
residua. (b.p.>510° C;>950°F)
Density-gravity greater than 1000 kg/m3
(<10°API) Viscosity greater than
10,000mPa.s(cp)
Mycoremediation of Crude Oil Contaminated Soil Page 12
Heavier crude oils have higher density-gravity values and higher viscosity,
with lower API gravity, making them less suitable for gasoline stocks but better
candidates for lubricant and heavy fuel production (Lorraine, 2003).
1.3.3 Total petroleum hydrocarbons (TPHs):
TPHs are a term used to describe a large family of several chemical
compounds that originally come from crude oil. Crude oil is used to make petroleum
products, which can contaminate the environment. Because there are many different
chemicals in crude oil and in other petroleum products, evaluation of hundreds to
thousands of compounds can be impractical. Evaluations for overall TPH are
common and generally accepted. TPHs are mixture of chemicals, but they are all
made mainly from hydrogen and carbon, called hydrocarbons. TPH are divided into
groups of petroleum hydrocarbons that act alike in soil or water and are called
petroleum hydrocarbon fractions. Each fraction contains many individual chemicals.
TPHs are carbon chains in the range of C6 to C35. Some chemicals that may be
found in TPHs are hexane, jet fuels, mineral oils, benzene, toluene, xylenes,
naphthalene, and fluorene, as well as other petroleum products and gasoline
components. However, it is likely that samples of TPHs will contain only some, or a
mixture, of these chemicals. Some hydrocarbon mixtures may also contain priority
pollutants including volatile organic compounds (VOCs), semi-volatile organic
compounds (SVOCs), and metals, each of which have their own specific toxicity
information (ATSDR, 1999).
Department of Environmental Quality (DEQ), Oklahoma (2012) defines
three ranges of TPHs:
Gasoline range organics (GRO) > C6-C10
Diesel range organics (DRO) > C11-C28
Lube oil compounds > C28-C35
1.3.4 Poly-cyclic aromatic hydrocarbons (PAHs):
PAHs, also known as poly-aromatic hydrocarbons or poly-nuclear aromatic
hydrocarbons, are potent pollutants that consist of fused aromatic rings and do not
contain heteroatoms or carry substituents. Naphthalene is the simplest example of a
PAH. PAHs occur in oil, coal, and tar deposits, and are produced as by products of
Mycoremediation of Crude Oil Contaminated Soil Page 13
fuel burning (whether fossil fuel or biomass). As a pollutant, they are of concern
because some compounds have been identified as carcinogenic, mutagenic,
and teratogenic (Fetzer, 2000).
PAHs are chemicals that are often found together in groups of two or more.
PAHs are found naturally in the environment but they can also be man-made. In
their purest form, PAHs are solid and range in appearance from colourless to white
or pale yellow green. PAHs are created when products like coal, oil, gas, and
garbage are burned but the burning process is not complete. Although PAHs can
exist in over 100 different combinations, the National Waste Minimization Program
defines this group using the Toxic Release Inventory Reporting Category for
polycyclic aromatic compounds (ATSDR, 1996).
Polycyclic aromatic hydrocarbons are lipophilic, meaning they mix more
easily with oil than water. The larger compounds are less water-soluble and
less volatile. Because of these properties, PAHs in the environment are found
primarily in soil, sediment and oily substances, as opposed to in water or air.
However, they are also a component of concern in particulate matter suspended in
air (Roy, 1995).
List of PAHs:
The United States Environmental Protection Agency (EPA) has designated
32 PAH compounds as priority pollutants. The original 16 listed are:
naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene,
anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene,
benzo[b]fluoranthene, benzo[k]flouranthene, benzo[a]pyrene,
dibenz(ah)anthracene, benzo[ghi]perylene, and indeno(1,2,3-cd) pyrene.
This list of the 16 EPA priority PAHs is often targeted for measurement in
environmental samples.
Chemical structures of some priority pollutants (PAHs) and its toxicity
equivalent factors are shown in Table-2 and 3.
Mycoremediation of Crude Oil Contaminated Soil Page 14
Table-2: Chemical structures of PAH compounds
Chemical compound Chemical compound
Anthracene
Benzo[a]pyrene
Chrysene
Coronene
Corannulene
Tetracene
Naphthalene
Pentacene
Phenanthrene
Pyrene
Triphenylene
Ovalene
(Source: Wikipedia-http://en.wikipedia.org/wiki/Polycyclic_aromatic_hydrocarbon)
Mycoremediation of Crude Oil Contaminated Soil Page 15
Table-3: Toxic equivalent factors for PAHs (Nisbet and LaGoy, 1992)
1.4 Physico-chemical properties of crude oil and oil products
Crude oil and petroleum products are very complex and variable mixtures of
thousands of individual compounds that exhibit a wide range of physical properties.
Understanding these properties is important in determining behaviour of spilled oil
and the appropriate response option. The composition and properties of various
petroleum hydrocarbons have been described in detail by Clark and Brown (1977)
(Table-4) and the National Academy of Sciences (NAS, 1985). Large oil property
databases also exist such as the one posted on the internet by Environment Canada
(www.etcenttre.org/spills), which contains information on over 400 oils (Jokuty et
al., 2000).
1.4.1 Physical properties of oil
Important physical properties of oil that affect its behaviour in the
environment and spill cleanup responses include:
a) Density: Two types of density expressions for oils are often used: specific gravity
and American Petroleum Institute (API) gravity. Specific gravity is the ratio of the
mass of a substance to the mass of the equivalent volume of water at a specified
temperature. The API gravity arbitrarily assigns a value of 10° to pure water at 10°C
(60°F). The API gravity can be calculated from the specific gravity using the
formula:
141.5 -131.5 API Gravity (º) = Specific Gravity
Mycoremediation of Crude Oil Contaminated Soil Page 16
Oils with low densities or low specify gravities have high API gravities.
Crude oils have specific gravities in the range of 0.79 to 1.00 (equivalent to API
Gravities of 10 to 48) (Clark and Brown, 1977). Oil density is an important index of
oil composition that is frequently used to predict its fate in water.
b) Viscosity: Viscosity is the property of a fluid that describes how it resists a
change in shape or movement. The lower the viscosity a fluid has, the more easily it
flows. The viscosity of petroleum is related to oil compositions and the ambient
temperature. It is an important index of the spreading rate of spilled oil.
c) Pour Point: The pour point of an oil is the temperature at which it becomes semi-
solid or stops flowing. The pour point of crude oils varies from –57°C to 32°C. It is
another important characteristic with respect to oil fate and cleanup strategies.
d) Solubility in water: The solubility of oil in water is extremely low and depends
on the chemical composition of the petroleum hydrocarbon in question and
temperature. For a typical crude oil, solubility is around 30 mg/L (NAS, 1985). The
most soluble oil components are the low molecular weight aromatics such as
benzene, toluene and xylene. This property is important with respect to oil fate, oil
toxicity and bioremediation processes.
Other important physical properties of oils include flash point, vapour
pressure, surface tension, and adhesion.
1.4.2 Chemical properties of crude oils and oil products
Crude Oil is comprised of both hydrocarbon compounds (accounting for 50–
98% of total composition) and non-hydrocarbon compounds (containing sulphur,
nitrogen, oxygen, and various trace metals) in a wide array of combinations (Clark
and Brown, 1977). Hydrocarbons differ in their susceptibility to microbial attack
and ranked in the following order of decreasing susceptibility : n-alkanes > branched
alkanes > low molecular weight aromatics > cyclic alkanes (Perry, 1984). Table-4
presents the chemical and physical properties of crude oil from different
geographical locations.
Mycoremediation of Crude Oil Contaminated Soil Page 17
Table-4: Chemical composition and physical properties of representative crude oils
(adapted from Clark and Brown, 1977)
Characteristics or
component
Prudhoe Bay South
Louisiana
Kuwait
API gravity (20°C)
Sulphur (wt %)
Nitrogen (wt %)
Nickel (ppm)
Vanadium (ppm)
Naphtha fraction (wt %)
Saturates
Aromatics
Resins & Asphaltenes
High-boiling fraction (wt %)
Saturates
Aromatics
Resins & Asphaltenes
27.8
0.94
0.23
10
20
23.2
19.9
3.2
-
76.8
47.7
25
4.1
34.5
0.25
0.69
2.2
1.9
18.6
16.5
2.1
-
81.4
56.3
16.5
8.6
31.4
2.44
0.14
7.7
28
28.0
20.3
2.4
-
77.3
34.0
21.9
21.4
These analyses represent values for one typical crude oil from three distinct geographical regions; variations in
composition can be expected for oils produced from different formations or fields within each region. a Fraction
boiling from 20° to 205°
Cb Fraction boiling above 205°C.
1.4.3 Refined oil products
Refined petroleum products, such as gasoline, kerosene, jet fuels, fuel oils,
and lubricating oils, are derived from crude oils through processes such as catalytic
cracking and fractional distillation. These products have physical and chemical
characteristics that differ according to the type of crude oil and subsequent refining
processes. They contain components of crude oil covering a narrow range of boiling
points. In addition, during catalytic cracking operations, unsaturated compounds, or
olefins (alkenes and cycloalkenes), which are not present in crude oils, can be
formed. The concentrations of olefins are as high as 30% in gasoline and about 1%
in jet fuel (NAS, 1985). A list of chemical compositions of the fractions of crude
oils and the refined products is shown in Table-5.
Mycoremediation of Crude Oil Contaminated Soil Page 18
Table-5: Chemical compositions of refined petroleum products (adapted from Clark
and Brown, 1977)
Distillation
fraction
Hydrocarbon
types
Range of carbon
atoms
Typical Refined
products
Gasoline & naptha Saturates
Olefins
Aromatics
4-12 Gasoline
Middle distillate Saturates
Olefins
Aromatics
10-20 Kerosene
Jet fuel
Heating Oils
Diesel Oils
Wide-cut gas oil Saturates
Aromatics
18-45 Wax
Lubricating oil
Residum Resins
Asphaltenes
>40 Residual Oil
Asphalt
1.5 Mycoremediation: Fungal bioremediation
Fungi have been harnessed by humans in many diverse applications for
thousands of years. In any ecosystem, fungi are among the major decomposers of
plant polymers such as cellulose, hemicellulose, and lignin. Fungi have the ability to
mineralize, release, and store various elements and ions and accumulate toxic
materials. They can facilitate energy exchange between the aboveground and
belowground systems. Fungi have proven to modify soil permeability and soil ion
exchange and to detoxify contaminated soil. Edible and/or medicinal fungi also play
a role as natural environmental remediators (Pletsch et al., 1999), as do aquatic fungi
(Hasija, 1994). Fungi are usually slow in growth and often require substrates for co-
metabolism. The mycelial growth habit is responsible for the rapid colonization of
substrates. The process of fungal biotransformation of compounds, wastes, or
wastewaters is termed mycotransformation.
Fungal treatment of wastes in nature has been known for centuries. The
ubiquitous presence of fungi has allowed acclimation of some types of wastes, if not
most. Most of our knowledge related to interactions between fungi and wastes is
based on studies performed in the laboratory. However, during the last two decades,
fungi have been used in the treatment of a wide variety of wastes and wastewaters,
and the role of fungi in the bioremediation of various hazardous and toxic
compounds in soils and sediments has been established. Fungi have also
Mycoremediation of Crude Oil Contaminated Soil Page 19
demonstrated the removal of metals and the degradation and mineralization of
phenols and chlorinated phenolic compounds, petroleum hydrocarbons, polycyclic
aromatic hydrocarbons, polychlorinated biphenyls, chlorinated insecticides and
pesticides, dyes, biopolymers, and other substances in various matrices. The role of
fungi in the treatment of various wastes and wastewaters has been discussed by
Singh (1991).
Soil contamination is more or less common phenomenon in most of the oil
exploration regions in the world. In oil industry, oil spill during various drilling and
production operations is a problem of serious concern. The intensified oil activity
has increased the risk of on land spill, which cannot be eliminated totally, but can
only be minimized. Bioremediation is an emerging biotechnological approach used
to degrade and detoxify the hydrocarbon contaminants in a cost effective manner
with minimum threat to environment.
Bioremediation, a process defined as a managed treatment process that uses
microorganisms to degrade and transform chemicals in contaminated soil, aquifer
material, sludge’s and residues, is an important tool to mitigate environmental
contamination. By means of exploiting catalytic abilities of microorganisms, the rate
of extent of pollutant destruction can be enhanced. Bioremediation exploits the
genetic diversity and metabolic versatility of microorganisms for the transformation
of contaminants into less harmful end-products, which are then integrated into
natural biogeochemical cycles. This is an attractive process due to its cost
effectiveness and the benefit of pollutant mineralization to CO2 and H2O (Mills et
al., 2004).
Mycoremediation is a form of bioremediation, the process of using fungi to
return an environment (usually soil) contaminated by pollutants to a less
contaminated state. The concept of mycoremediation was explored in the 1984 film
‘Nausicaa of the Valley of the Wind’, where vast tracts of fungal forest rehabilitate
the planet after catastrophic human polluting and apocalypse. The term
mycoremediation was coined by Paul Stamets and refers specifically to the use of
fungal mycelia in bioremediation. One of the primary roles of fungi in the ecosystem
is decomposition, which is performed by the mycelium. The mycelium secretes
extracellular enzymes that break down lignin and cellulose, the two main building
Mycoremediation of Crude Oil Contaminated Soil Page 20
blocks of plant fiber. These are organic compounds composed of long chains of
carbon and hydrogen, structurally similar to many organic pollutants. The key to
mycoremediation is determining the right fungal species to target a specific pollutant
(Stamets, 2005).
Mycoremediation is an economically and environmentally sound alternative
to extracting, transporting and storing toxic waste. It restores depleted land into
valuable land. The current policy concerning toxic waste removal/clean up
prescribes burning, hauling, and/or burying the waste. The results of these actions do
not really get rid of the waste or restore the ecology, but cripple it and leave it
lifeless. Toxins in our food chain (including mercury, polychlorinated biphenyl, and
dioxins) become more concentrated at each step, with those at the top being
contaminated by ingesting toxins consumed by those lower on the food chain.
Mycelia can destroy these toxins in the soil before they enter our food supply. They
remove heavy metals from land by channelling them to fruit bodies for removal.
They essentially use and digest these toxins as nutrients. Mycelial enzymes can
decompose some of the most resistant materials made by humans or nature, because
many of the bonds that hold plant material together are similar to the bonds found in
petroleum products including diesel, oil, and many herbicides and pesticides
(Stamets, 2005).
1.5.1 Cleanup technologies – Bioremediation versus other methods
For the past decades, the method of choice for ground water cleanup, for
example, involves the pump-and-treat systems. These systems consist of a series of
wells used to pump water to the surface and the surface treatment facility used to
clean up the extracted water. This method is used to control contaminant migration,
and if recovery wells are located in the heart of the plume, it can easily remove
contaminant mass. However, since many common contaminants become trapped in
the subsurface, complete flushing out may require the pumping of extremely large
volumes of water over very long period of time. Because it treats contaminants in
place instead of requiring their extraction, in situ bioremediation takes care of these
shortcomings in a cleanup process. Consequently, bioremediation is likely to yield
faster results, take a few to several years compared to a few to several decades for
the pump-and-treat technology (Testa and Winegardner, 1991).
Mycoremediation of Crude Oil Contaminated Soil Page 21
The microbiological decontamination of oil-polluted soils has been assessed
to be an efficient, economic and versatile alternative to physiochemical treatment
(Bartha, 1986) even though the rate of hydrocarbon biodegradation in soils is
affected by other physiochemical and biological parameters. While capital and
annual operating cost may be higher for bioremediation, its shorter operating time
should compensate in a reduction of total cost. Other factors that may contribute to
cost reduction in bioremediation compared to pump-an-treat method include reduced
time required for site monitoring, reporting and management, as well as reduced
need for maintenance, labour, and supplies (NRC, 1993).
Furthermore, the surface treatment methods that are part of pump-and-treat
systems typically use air stripping and/or carbon treatment to remove contaminants
from the water. The process is mainly that of transferring the contaminant to another
medium (the air or the land) instead of destroying it. Bioremediation on the other
hand, can completely destroy contaminants, converting them to carbon dioxide,
water, and new cell mass, or at least convert them to non-toxic products some of
which may even be useful to the ecosystem. For cleanup of contaminated soils, in
situ bioremediation is only one of several possible technologies. Alternatives
include:
(1) Excavation followed by sea disposal or incineration.
(2) On-site bioremediation using land-farming or fully enclosed soil cell techniques.
(3) Low temperature desorption.
(4) In situ vapour recovery.
(5) Containment using slurry walls and caps.
In situ methods (desorption, vapour recovery, containment, and
bioremendiation) have the advantages of being minimally disruptive to the site and
are potentially less expensive. Because ex situ methods require excavation, they
disrupt the landscape, expose the contaminants, and require replacement of soils. For
these reasons, ex situ methods are sometimes impracticable. Potential advantages of
bioremediation compared to other in situ methods include destruction rather than
transfer of the contaminant to another medium; minimal exposure of the on-site
workers to the contaminant; long time protection of public health; and possible
Mycoremediation of Crude Oil Contaminated Soil Page 22
reduction in the duration of the remedial process. These advantages of the
bioremediation systems over the other technologies have been summarised (Leavin
and Gealt, 1993) as follows: can be done on site i.e. in situ application; keeps site
destruction to a minimum; eliminates transportation, costs and liabilities; eliminates
long-term liability; biological systems are involved, hence often less expensive; and
can be coupled with other treatment techniques to form a treatment train.
1.5.2 Fungi utilizing petroleum hydrocarbons/ fungi in bioremediation
The first time that fungi were proposed as specific contaminant degraders
was in 1973 when Cerniglia and Perry (1973) published a study on the potential of
the non-ligninolytic fungus Cunninghamella elegans to degrade crude oil. One
decade later, the same authors concluded that C. elegans used a similar mechanism
as mammals to metabolize PAHs, which involved the intracellular enzymes
cytochrome P450 monoooxygenase and epoxide hydrolase and yielded the
formation of trans-dihydrodiols, phenols, quinones, and dihydrodiol-epoxides
(reviewed by Cerniglia, 1997). The ability to degrade not only PAHs but also other
recalcitrant pollutants was extended later to the white-rot fungus Phanerochaete
chrysosporium (Bumpus et al., 1985). From that moment on, a considerable number
of studies have been published on the potential of other white rot fungi to degrade a
wide range of contaminants. The most studied fungi in addition to P. chrysosporium
are Trametes versicolor (Logan et al., 1994; Johannes et al., 1996; Novotný et al.,
1997; Majcherczyk et al., 1998; Tuomela et al., 1999), Pleurotus ostreatus (Bezalel
et al., 1996a; Novotný et al., 1997; Beaudette et al., 1998), Bjerkandera adusta
(Field et al., 1992; Kotterman et al., 1994; Beaudette et al., 1998), Irpex lacteus
(reviewed by Novotný et al., 2009) and Phlebia spp. (Van Aken et al., 1999; Mori
and Kondo, 2002a; Mori and Kondo, 2002b; Mori et al., 2003; Kamei et al., 2005;
Kamei et al., 2009). All of these studies linked the degradation of contaminants to
the production of lignin-modifying enzymes (LMEs; Field et al., 1992; Sack and
Gunther, 1993). Later, several studies extended the fungal degradation capability of
PAHs (Gramss et al., 1999a; Steffen et al., 2002a; Steffen et al., 2003), TNT
(Scheibner et al., 1997a) and dyes (Baldrian and Šnajdr, 2006) to litter-decomposing
fungi, which mainly oxidize contaminants using MnP or laccase (Carrera, 2010).
Mycoremediation of Crude Oil Contaminated Soil Page 23
The information gained during the last year’s permits experts to draw a list
of the main facts about fungal degradation of contaminants:
i) Among all fungi, white-rot and litter-decomposing fungi are the most
efficient degraders of recalcitrant compounds, an ability attributed to
the production of lignin modifying enzymes.
ii) Fungi may also exhibit other enzymatic or non-enzymatic mechanisms
involved in the degradation process.
iii) Due to their low substrate specificity, lignin modifying enzymes degrade
organic compounds with molecular structures similar to lignin.
iv) Degradation of contaminants occurs during secondary metabolism and
thus, generally under nutrient-starvation conditions (i.e., low levels of
nitrogen content; Glenn and Gold, 1983; Reddy, 1995; Pointing, 2001;
Gao et al., 2010).
v) The extracellular nature of lignin modifying enzymes enables fungi to
degrade molecules larger than the ones degradable by bacteria.
vi) Fungi are able to mineralize organic contaminants or to form low-
molecular mass metabolites which may be co-metabolized by bacteria.
vii) Fungi tolerate high concentrations of organic contaminants and heavy
metals without detrimental effects to their enzyme activity (Baldrian et
al., 2000; Baldrian, 2003; Tuomela et al., 2005).
viii) In soil, fungi can cause the humification of organic contaminants,
meaning that the compound is bound to humic substances, thereby
reducing availability and thus toxicity (Bollag, 1992; Bogan et al., 1999).
1.5.3 Degradation of PAHs by fungi
The mechanism for lignin modifying enzymes to degrade PAHs is thought to
be similar to that of lignin degradation. The breakdown of PAHs yields quinones,
free radical intermediates and carboxyl radicals that can undergo further oxidation to
form carbon dioxide (Cerniglia and Sutherland, 2001; Singh, 2006). Fungal
peroxidases oxidize PAHs with an ionization potential (IP) lower than 8.0 eV in the
case of Lignin peroxidase (LiP) and 7.8 eV in the case of Manganese peroxidase
(MnP). Unlike peroxidases, laccases are able to oxidize PAHs with an IP lower than
7.55 eV (Singh, 2006; Farnet et al., 2009). However, several authors disagree with
the correlation between IP values and the oxidation of PAHs (Majcherczyk et al.,
Mycoremediation of Crude Oil Contaminated Soil Page 24
1998; Cañas et al., 2007; Wu et al., 2008). They have proposed that other
mechanisms involving the intracellular cytochrome P450 enzyme (Bezalel et al.,
1996b) and the MnP-mediated lipid peroxidation play a role in PAH degradation
(Kapich et al., 2005; Steffen et al., 2007), especially in the initial attack of the ring.
The enzymatic strategy of fungi to degrade PAHs as well as other contaminants
depends upon the fungal species and nutrients or the addition of mediators. For
instance, the white rot fungus Irpex lacteus is able to simultaneously produce MnP
and LiP, but only MnP seems to be responsible for PAH degradation, regardless of
the nitrogen concentration in the medium (Novotný et al., 2009). In the studies by
Johannes et al. (1996) and Majcherczyk et al. (1998), the laccase of Trametes
versicolor was able to oxidize PAHs independent of the PAH’s IP and in the
presence of different mediators, such as 2,2'-azino-bis 3-ethylbenzothiazoline-6-
sulphonic acid (ABTS) and 1-hydroxybenzotriazole (HBT).
1.5.4 Role of white rot fungi in mycoremediation
White-rot fungi for lignin degradation have been examined for more than
half a century, after the discovery of the extracellular oxidative ligninolytic enzymes
of the white-rot fungus Phanerochaete chrysosporium. Bumpus et al. (1985)
proposed the use of this fungus for bioremediation. Enzymes involved in the
degradation of wood are also responsible for the degradation of a wide variety of
persistent organic pollutants. The white-rot fungus P. chrysosporium has emerged as
an archetypal model system for fungal bioremediation or mycoremediation. P.
chrysosporium has the ability to degrade toxic or insoluble compounds more
efficiently than other fungi or microorganisms. The numerous oxidative and
reductive mechanisms of degradation make its application attractive in different
matrices. In addition to P. chrysosporium, several other white-rot fungi (e.g.,
Bjerkandera adusta, Irpex lacteus, Lentinula edodes, Pleurotus ostreatus, Trametes
versicolor) are known to degrade these compounds.
Based on the literature of the past two decades, it appears that the white-rot
fungi account for at least 30% of the total research on fungi used in bioremediation.
White-rot fungi have added a new dimension to the already complex system of
fungal bioremediation. During the 1980s, marketing attempts by unskilled persons
resulted in failures in white-rot fungal technology. Successful use depends on a
Mycoremediation of Crude Oil Contaminated Soil Page 25
comprehensive knowledge of fungal physiology, biochemistry, enzymology,
ecology, genetics, molecular biology, engineering, and several related disciplines.
The field conditions and factors that induce fungal biodegradation are taken into
consideration before development of the final design. Lamar and White (2001)
advocated the use of four phases in their approach: bench-scale treatability studies,
on-site pilot testing, production of inoculum, and full-scale treatment.
A variety of substrates, such as wood chips, wheat straw, peat, corncobs,
sawdust, a nutrient-fortified mixture of grain and sawdust, bark, rice, annual plant
stems and wood, fish oil, alfalfa, spent mushroom compost, sugarcane bagasse,
coffee pulp, sugar beet pulp, okra, canola meal, cyclodextrins, and surfactants, can
be employed in inoculum production off-site or on-site or mixed with contaminated
soils to enhance degradation. Care is required to balance the carbon and nitrogen
ratio in the substrates, which have a significant influence on the degradative
performance of white-rot fungi. Pelleted fungal inocula coated with alginate, gelatin,
agarose, carrageenan, chitosan, and so on, are used by several researchers and offer
several advantages over inocula produced using bulk substrates. This strategy,
adapted from the mushroom spawn industry, is known as encapsulation.
Encapsulation sustains the viability of the inoculum and provides sources of
nutrition for the maximum degradation of pollutants (Bennett et al., 2001). This also
enhances the survival and effectiveness of the introduced species. Solid-state
fermentation (SFF) is another method for producing fungal inoculum. However,
fungi secrete several by-products during conversion of agricultural waste under SFF
conditions (Cohen and Hadar, 2001).
Three phases of strategies are envisioned for the successful implementation
of mycoremediation. Inoculum preparation techniques and their improvements lead
to success in the first phase of the use of white-rot fungi in mycoremediation. The
second phase includes clear technical protocols for the final design and associated
engineering processes. The remediation protocols for the monitoring, adjustment,
continuity, and maintenance of the engineering system dictate the success of the
third and final phase of the mycoremediation process. Competition from native
microbial populations contributes to the outcome of mycoremediation, but protocols
to eliminate such variability have yet to be developed. Processes using white-rot
fungi have been patented. A few companies, including EarthFax Development
Mycoremediation of Crude Oil Contaminated Soil Page 26
Corporation in Utah and Gebruder Huber Bodenrecycling in Germany, employ these
fungi for soil bioremediation, but a broader use is not known at the present time
(Singh, 2006). A list of bacteria and fungi degrading oil is given in Table-6.
Table-6: Major genera of oil-degrading bacteria and fungi (Floodgate, 1984)
Bacteria Fungi
Achrornobacter
Acinetobacter
Actinomyces
Aeromonas
Alcaligenes
Arthrobacter
Bacillus
Beneckea
Brevebacterium
Coryneforms
Erwinia
Flavobacterium
Klebsiella
Lactobaoillus
Leumthrix
Moraxella
Nocardia
Peptococcus
Pseudomonas
Sarcina
Spherotilus
Spirillum
Streptomyces
Vibrio
Xanthomyces
Allescheria
Aspergillus
Aureobasidium
Botrytis
Candida
Cephalosporium
Cladosporium
Cunninghamella
Debaromyces
Fusarium
Gonytrichum
Hansenula
Helminthosporium
Mucor
Oidiodendrum
Paecylomyces
Phialophora
Penicillium
Rhodosporidium
Rhodotorula
Saccharomyces
Saccharomycopisis
Scopulariopsis
Sporobolomyces
Torulopsis
Trichoderma
Trichosporon
1.5.5 Mechanism of degradation
Fig. 4: Degradation mechanism for bacteria and fungi (Boonchan et al., 2000)
Mycoremediation of Crude Oil Contaminated Soil Page 27
Fungal and bacterial degradation of PAHs is presented in Fig. 4, which
shows the influence of different enzymes in degradation of complex molecules to
convert them into less harmful end product. Petroleum hydrocarbons are subject to
microbial degradation under both aerobic and anaerobic conditions (IPCS, 1993;
ATSDR, 2007); the former is typically much more rapid (EA, 2003). Biodegradation
rates are dependent on several factors including: the presence of sunlight; the type
and population of microbes present; initial concentration of benzene; soil
temperature; soil oxygen content; and the potential presence of other electron
receptors (EA, 2000a, 2003). When mixtures of benzene, toluene, xylene and
ethylbenzene (BTEX) are present in an anaerobic environment, there is a sequential
utilisation of substrate hydrocarbons, with toluene usually being the first to be
degraded, followed by the isomers of xylene in varying order. Benzene and
ethylbenzene tend to be degraded last (ATSDR, 2007).
Transformation reactions under aerobic conditions during mycoremediation
are: In presence of oxygen, aerobic microorganisms oxidize organic carbon
completely to carbon dioxide using oxygen as terminal electron acceptor (oxygen is
reduced to water) in a series of oxidation-reduction reactions used to produce energy
for cell maintenance and growth. The vast majority of the organic carbon available
to microorganisms in the vadose zone is material which has been photosynthetically
fixed (plant material). However, in some instances authropogenic activity results in
addition of organic carbon in the form of industrial or agricultural chemicals such as
petroleum products, organic solvents, or pesticides. Many of these chemicals are
readily degraded in the environment because of their structural similarity to
naturally occurring organic carbon. However, some chemical structures may require
long periods of adaptation, or have low bioavailability, or steric or electronic
characteristics that result in slow to nonexistent biodegradation rates. Aliphatic
hydrocarbons include straight chain and branched chain structures. Industrial
solvents wastes and petroleum industry by-products and spills are the primary
sources of aliphatic hydrocarbon contaminants introduced into the environment
(U.S. EPA, 1984; Plumb, 1985). Many microorganisms in the environment can
utilize aliphatic hydrocarbons as carbon sources (Britton, 1984; Singer and Finnerty,
1984b; Leahy and Colwell, 1990; Pitter and Chudoba, 1990; Prince, 1993).
Mycoremediation of Crude Oil Contaminated Soil Page 28
The following generalizations can be made about biodegradation of
aliphatics by microorganisms. 1) Mid sized straight-chain aliphatics (n-alkanes C10-
C18 in length) are utilized more readily than n-alkanes with either shorter or longer
chains. Long-chain n-alkanes are utilized more slowly, due to low bioavailability
resulting from extremely low water solubilities (Miller and Bartha, 1989). For
example, the reported water solubility of decane is 0.052 mg/L, while the solubility
of octadecane (C18) is 10-fold less (0.006 mg/L) (Singer and Finnerty, 1984b).
Solubilty continues to decrease with increasing chain length. In contrast, short-chain
n-alkanes have higher aqueous solubility, e.g., the water solubility of butane (C4) is
61.4 mg/L, but they are toxic to cells. Toxicity is caused by disruption of the cell
membrane through interaction with membrane- bound proteins that function in
transport and oxidation of aliphatics (Britton, 1984). In some cases it has been
shown that the toxicity of short chain n-alkanes can be reduced by the presence of
long chain n-alkanes. The protective effect is attributed to partitioning of the toxic
hydrocarbon from the aqueous phase into the long chain alkane, thereby reducing
the concentration (Britton, 1984). Therefore, degradation rates may differ depending
on whether the substrate is a pure compound or a mixture of compounds. 2)
Saturated aliphatics are hydrocarbons with a carbon skeleton which is saturated with
hydrogen, or contain only single carbon-carbon bonds. Unsaturated hydrocarbons
contain one or more double (alkenes) or triple (alkynes) carbon-carbon bonds. In
general, saturated aliphatics are degraded more readily than unsaturated ones
(Britton, 1984). 3) Biodegradability of aliphatics is negatively influenced by
branching in the hydrocarbon chain (Pitter and Chudoba, 1990). The degree of
resistance to biodegradation depends on both the number of branches and on the
positions of methyl groups in the molecule. Compounds with a quaternary carbon
atom (4 carbon-carbon bonds) are extremely stable due to steric effects. Terminal
quaternary carbons particularly inhibit biodegradation.
Alicyclic hydrocarbons are saturated carbon chains which form a ring
structure. There is a great variety of naturally occurring alicyclic hydrocarbons. For
example, alicyclic hydrocarbons are major component of crude oil, comprising 20%
to 67% by volume. The various components range from simple such as cyclopentane
and cyclohexane, to complex such as trimethylcyclopentane and various
cycloparaffins (Perry, 1984). Cyclopentane and cyclohexane derivates that contain
Mycoremediation of Crude Oil Contaminated Soil Page 29
one or two OH, C=O, or COOH groups are readily metabolized. Aromatic
compounds contain at least one unsaturated ring system with the general structure
C6R6, where R is any functional group. Benzene is the parent hydrocarbon of this
family of unsaturated cyclic compounds and is unique in that it does not exhibit the
high reactivity of typical polyenes. It is remarkably inert to many oxidising reagent,
stable to air, and tolerates many free radical initiators. This stability is due to the
resonance energy which comes from the delocalization of electrons around. PAHs of
two or three condensed rings are transformed rapidly and often completely
mineralized, whereas PAHs of four or more condensed rings are transformed much
more slowly, often as a result of co-metabolic attack (Gibson and Subramanian,
1984; Cerniglia and Heitkamp, 1989; Wilson and Everett, 1994).
1.6 Laboratory studies on mycoremediation
1.6.1 Natural attenuation (soil’s natural ability to degrade the contaminant):
The term Natural Attenuation (NA) is a process which includes a variety of
physical, chemical, or biological processes that, under favourable conditions, act
without human intervention to reduce the mass, toxicity, mobility, volume, or
concentration of contaminants in soil or ground water. These processes include
biodegradation; dispersion; dilution; sorption; volatilization; and chemical or
biological stabilization, transformation, or destruction of contaminants. Spills and
leaks of petroleum hydrocarbons such as gasoline, diesel, motor oils, and similar
materials have caused widespread contamination in the environment. Generally
these contaminants are present both in NAPL form (non-aqueous phase liquid; the
bulk liquid petroleum hydrocarbon) and also as dissolved contaminants in the
ground water. Cleanup of both the NAPL and dissolved contamination in soils and
ground water using many common remedial techniques is often expensive and slow.
However, under the proper conditions at some sites, natural attenuation can
contribute significantly to remediation of dissolved petroleum hydrocarbon
contamination and may accomplish site remediation goals at a lower cost than
conventional remediation technologies, within a similar time frame (U.S. EPA,
1999).
Mycoremediation of Crude Oil Contaminated Soil Page 30
1.6.2 Biostimulation (adding nutrients to improve the natural biodegradation
rate):
Biostimulation involves the addition of rate-limiting nutrients to accelerate
the biodegradation process. In most shoreline ecosystems that have been heavily
contaminated with hydrocarbons, nutrients are likely the limiting factors in oil
biodegradation. The main purpose of bench-scale treatability studies is to determine
the type, concentration, and frequency of addition of amendments needed for
maximum stimulation in the field (Venosa, 1998). Most laboratory experiments have
shown that addition of growth limiting nutrients, namely nitrogen and phosphorus,
has enhanced the rate of oil biodegradation. However, the optimal nutrient types and
concentrations vary widely depending on the oil properties and the environmental
conditions. Wrenn et al. (1994) studied the effects of different forms of nitrogen on
biodegradation of light Arabian crude oil in respirometers. They found that in poorly
buffered seawater, nitrate is a better nitrogen source than ammonia because acid
production associated with ammonia metabolism may inhibit oil biodegradation.
When the culture pH was controlled, the performance of oil biodegradation was
similar for both amendments with a shorter lag time for ammonia addition. The
nutrient concentration should be maintained at a level high enough to facilitate
fungal growth. Using nitrate as a biostimulation agent, (Venosa et al., 1994)
determined that approximately 1.5 to 2.0 mg N/L supported near maximal
biodegradation of heptadecane immobilized onto sand particles in a microcosm
study (Zhu et al., 2001).
1.6.3 Bioaugmentation (addition of a microbial consortium from selected species
isolated from a contaminated soil plus nutrients):
Bioaugmentation is the term used to describe the addition of cultured
microorganisms that are capable of biodegrading or transforming specific
soil/groundwater contaminants. Bioaugmentation is the addition of microorganisms
that specifically degrade the oil at the site of the oil spill. The oil-degradation
organisms were collected from other sites and commercially cultivated. They are
selected to withstand harsh environmental conditions such as high salt and variable
temperature combined with a superior ability to use the resources such as oxygen,
nitrogen, phosphorus and others sources available. They are also able to compete
from indigenous microorganisms, so they can clean up the site rapidly (Campo et
Mycoremediation of Crude Oil Contaminated Soil Page 31
al., 2007; Basharudin, 2008). The rationale for adding oil-degrading microorganisms
is that indigenous microbial populations may not be capable of degrading the wide
range of potential substrates present in complex mixtures such as petroleum (Leahy
and Colwell, 1990). Other conditions under which bioaugmentation may be
considered are when the indigenous hydrocarbon-degrading population is low, the
speed of decontamination is the primary factor, and when seeding may reduce the
lag period to start the bioremediation process (Forsyth et al., 1995; Zhu et al., 2001).
1.7 Oxidation of petroleum hydrocarbons by fungal enzymes:
Little is known of the enzymatic oxidation of petroleum hydrocarbons, and
this topic is emerging due to regio- and stereo-selectivity and mild physiological
conditions. Enzymatic oxidation can take the upper hand when success is not
achieved with chemical catalysts. Faber (1997) discussed biocatalytic oxidation
reactions of alkanes, alkenes, aromatics, and heteroatoms. In general, these
biotransformations are carried out by microbial cultures. Choloroperoxidase (CPO)
has been employed for the enantioselective epoxidation of alkenes and olefins
(Dexter et al., 1995). Laccase is well documented with a mediator to oxidize certain
aromatic compounds. This is called mediated oxidation. Laccase from the white rot
fungus Trametes hirsuta is employed for the oxidation of alkenes (Niku-Paavola and
Viikari, 2000). This oxidation is a two-step process: (1) the enzyme catalyzes the
oxidation of primary substrate, the mediator; and (2) the oxidized mediator oxidizes
the secondary substrate, the alkene. All alkenes are oxidized, and the extent of
transformation depends on the alkene and the mediator. The highest degrees of
conversion are obtained using hydroxylbenzotriazole (HBT) as a mediator.
Treatment at 20°C for 20 hours results in a 45 to 50% oxidation rate of α-pinene
and, cis-2- and cis-3-hexenols and a 90 to 100% oxidation rate of linalool, geraniol,
nerol, and cinnamyl alcohol. Other alkenes, such as allyl ether, cis-2-heptene, and
cyclohexene, are oxidized less than 25%, even with all the mediators. The main
reaction products of alkenes are aldehydes and ketones, but other products are also
identified.
Lignocellulose-degrading enzymes
The mechanism of contaminant degradation by fungi (wood- and litter-
decomposing fungi) is based on the production of the oxidoreductases and
Mycoremediation of Crude Oil Contaminated Soil Page 32
hydrolytic enzymes involved in the degradation of lignin and polysaccharides,
respectively (Carrera, 2010).
1.7.1 Lignin modifying enzymes
Fungal oxidoreductase laccase and the peroxidases lignin peroxidise (LiP),
manganese peroxidase (MnP), and versatile peroxidise (VP), a hybrid form of MnP
and LiP, are responsible for the degradation of lignin (Hatakka, 2001; Hofrichter,
2002; Martínez et al., 2005; Baldrian, 2006). In addition, other enzymes are
indirectly involved in lignin modification. For example, the hydrogen peroxide-
generating enzymes glyoxal oxidase (GLOX) and aryl alcohol oxidase (AAO) are
essential in the catalytic cycle of peroxidases since they require H2O2 as an electron
acceptor (Hatakka, 2001; Lundell et al., 2010). Moreover, cellobiose-oxidizing
enzymes, that is to say, cellobiose dehydrogenase and cellobiose:quinone
oxidoreductase, are also proposed to be involved in the degradation of non-phenolic
substructures of lignin by the formation of reactive hydroxyl radicals •OH (Hildén et
al., 2000).
1.7.1.1 Laccases
Laccases are mostly extracellular blue glycosylated multi-copper-containing
oxidases that are larger than peroxidases and have a molecular weight of 60 to 80
kDa. Laccases are widely distributed in many white-rot basidiomycetes. They are
also found in higher plants and in several fungi belonging to ascomycetes and
deuteromycetes. They are involved in lignin degradation and also serve other
functions: in fungal pigmentation, pathogenicity, fructification formation,
sporulation, and detoxification. Laccases contain four coppers per enzyme and are of
three different types: type I, type II, and type III. Each type has a distinct role in the
oxidation of laccase substrates (Singh, 2006). Laccase catalyzes the oxidation of the
phenolic substructures of lignin via one molecular oxygen reduction to water
(Hatakka, 2001; Baldrian, 2006). Other non-phenolic compounds with high redox
potential, including PAHs or other recalcitrant compounds, may also be oxidized by
laccase (Camarero et al., 2005).
1.7.1.2 Manganese peroxidases (MnPs)
Manganese peroxidases (MnPs) are also glycosylated heme-containing
extracellular peroxidase. Its catalytic cycle is similar to those of LiP and horseradish
Mycoremediation of Crude Oil Contaminated Soil Page 33
peroxidase (HRP), but it uses absolute Mn(II) as a substrate that is widespread in
lignocelluloses and soil. Manganese peroxidase is secreted in multiple forms in
microenvironments by white-rot fungi and certain soil litter–decomposing fungi. A
list of 56 fungi that produce MnP in liquid and/or solid-state fermentation has been
compiled by Hofrichter (2002). MnP is secreted by a distinct group of
basidiomycetes, such as the families Coriolaceae, Meruliaceae, Polyporaceae, and
the soil litter families Strophariaceae and Tricholomataceae. The molecular weight
of MnP ranges between 38 and 62.5 kDa, and the molecular weight of the most
purified MnP is 45 kDa. About 11 isozymes of MnP are known to be produced by
Ceriporiopsis subvermispora (Lobos et al., 1994; Urzua et al., 1995). Five isozymes
in P. chrysosporium MP-1 have been detected to date (Kirk and Cullen, 1998).
1.7.1.3 Lignin peroxidase (LiPs)
Lignin peroxidase (LiPs) are glycosylated heme proteins secreted during
secondary metabolism in nutrient-limited cultures. LiPs have a molecular weight of
about 40 kDa. Lignin peroxidase was discovered in Phanerochaete chrysosporium
(Glenn and Gold, 1983; Tien and Kirk, 1984) and has become the most thoroughly
studied peroxidase. LiPs are produced by most white-rot fungi, such as
Phanerochaete flavido-alba (Ben Hamman et al., 1999), Trametes trogii (Vares and
Hatakka, 1997), Phlebia ochraceofulva (Vares et al., 1993), and Phlebia tremellosa
(Vares et al., 1994). Several isozyme forms have been detected in P. chrysosporium
cultures and a number of other white-rot fungi (e.g., Trametes versicolor,
Bjerkandera adusta and Phlebia radiata).
In addition, a hybrid enzyme possessing the catalytic properties of LiP and
MnP, namely versatile peroxidase (VP), is also a lignin-modifying enzyme
(Camarero et al., 1999). Unlike MnP and LiP, VP oxidizes both low and high redox
potential compounds with or without Mn3+ mediation (Ruíz-Dueñas and Martínez,
2009). The versatility to degrade directly a wide variety of substrates, which LiP or
MnP have enabled, makes VP an enzyme with a large potential for industrial
applications including in the field of contaminant degradation (Pozdnyakova et al.,
2010). VP has only been found in Bjerkandera and Pleurotus species (Hammel and
Cullen, 2008; Ruíz-Dueñas and Martínez, 2009).
Mycoremediation of Crude Oil Contaminated Soil Page 34
A considerable number of studies have been published on the potential of WRF to
degrade a wide range of contaminants (Table-7).
Table-7: The most studied fungal species for bioremediation and their enzymes
involved in the degradation of contaminants (Carrera, 2010).
Fungus
(ecophysiological group)a
Order
(Family)c
Contaminantd Lignin
modifying enzymes
References
Agrocybe praecox
(LDF)
Agaricales
(Strophariaceae)
PAHs, TNT Lacc., MnP Scheibner et al., 1997a; Gramss
et al., 1999a; Steffen et al., 2000;
Steffen et al., 2002a.
Bjerkandera adusta
(WRF)
Polyporales PAHs, PCBs (LiP)e, MnP,
VP
Field et al., 1992; Kotterman et
al., 1994; Beaudette et
al., 1998; Kotterman et al., 1998.
Irpex lacteus (WRF)
Polyporales Dyes, PAHs, lindane, TNT,
bisphenol A,
nonylphenol, dimethyl phthalate
Lacc., LiP, MnP, VP
Reviewed by Novotný et al., 2009.
Phanerochaete
chrysosporium (WRF)
Polyporales Synthetic dyes,
PAHs, lindane, DDT, PCP, PCBs
LiP, MnP Glenn and Gold, 1983; Bumpus
et al., 1985; Field et al., 1992; Cerniglia, 1997;
Novotný et al., 1997;
Beaudette et al., 1998.
Phlebia spp.(WRF) Polyporales PAHs, TNT,
AmDNT, coal
humic acids
Lacc. LiP,
MnP
Hofrichter and Fritsche, 1996;
Hofrichter and Fritsche,
1997a; Hofrichter and Fritsche, 1997b; Sack et al.,
1997; Scheibner et al., 1997a;
Scheibner et al., 1997b.
Phlebia sp. b19b (WRF)
Polyporales PCDD/Fs, TNT Lacc. LiP, MnP
van Aken et al., 1999; Mori and Kondo, 2002a; Mori
and Kondo, 2002b; Mori et al.,
2003; Kamei et al., 2005; Kamei et al., 2009.
Pleurotus ostreatus
(WRF)
Agaricales
(Pleurotaceae)
PAHs, PCBs, TNT Lacc., (LiP)e,
(MnP)e, VP
Bezalel et al., 1996a; Novotný et
al., 1997; Scheibner et al., 1997a; Beaudette et al., 1998;
Axtell et al., 2000.
Stropharia
rugosoannulata (LDF)
Agaricales
(Strophariaceae)
PAHs, TNT,
synthetic dyes
Lacc., MnP Scheibner et al., 1997a; Gramss
et al., 1999a; Steffen et al., 2000; Steffen et al., 2002a; Baldrian
and Šnajdr,
2006.
Trametes versicolor
(WRF)
Polyporales PAHs, PCP, PCBs Lacc., (LiP)e,
MnP
Field et al., 1992; Logan et al.,
1994; Johannes et al.,
1996; Novotný et al., 1997; Scheibner et al., 1997a;
Beaudette et al., 1998;
Majcherczyk et al., 1998; Tuomela et al.,1999.
a WRF = white-rot fungus; LDF = litter-decomposing fungus.
b Former Nematoloma frowardii b19 (Hildén et al., 2008).
c International Mycological Assosiation, 2010. Family classification for Polyporales is not as straightforward as
for Agaricales.
d PAHs = polycyclic aromatic hydrocarbons; TNT = 2,4,6-trinitrotoluene; PCBs = polychlorinated biphenyls;
DDT = 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane; PCP =
pentachlorophenol; AmDNT = amino-dinitrotoluene; PCDD/Fs = dibenzo-p-dioxins and -furans.
e Enzyme not detected and/or not directly involved in degradation.
Mycoremediation of Crude Oil Contaminated Soil Page 35
1.8 Mycoremediation: Potential advantages and disadvantages (Source:
USC, 1991).
Advantages:
Usually involves only minimal physical disruption of a site.
No significant adverse effects when used correctly.
Maybe helpful in removing some of the toxic components of oil.
Offers a simpler and more thorough solution than mechanical technologies.
Possibly less costly than other approaches.
Disadvantages:
Of undetermined effectiveness for many types of spills.
May not be appropriate at sea.
Takes time to work.
Approach must be specifically tailored for each polluted site.
Optimization requires substantial information about spill site and oil
characteristics.
1.9 Factors affecting metabolism of petroleum hydrocarbons and PAHs
The biodegradation of petroleum hydrocarbons in the environment is
determined largely by abiotic factors. Factors affecting microbial degradation of
petroleum hydrocarbons have been the subject of considerable interest during the
past two decades. Fungi can withstand fairly wide fluctuations in environmental
conditions. The various factors that influence the growth rates and enzymatic
activities of yeasts and fungi also affect the rates of petroleum degradation. Various
factors influencing the fungal degradation of petroleum hydrocarbons have been
recognized. These factors are divided into three categories: (1) physicochemical
(physical nature, solubility, size and concentration, oil–water interface, volatility,
etc.), (2) environmental (temperature, pH, light, salinity, oxygen level, nutrients,
soil/sediment type, etc.), and (3) fungal (distribution in an area, population density
adaptation, uptake, genetic composition, microbial interactions, etc.). Important
factors affecting the fungal metabolism of petroleum hydrocarbons are mentioned
below:
Mycoremediation of Crude Oil Contaminated Soil Page 36
a) Physical nature: The physical nature of hydrocarbons has a great effect on the
process of biodegradation. The hydrocarbon-degrading fungi act primarily at the
oil–water interface. However, fungi can be found growing over the entire surface
of an oil droplet, and growth does not occur within oil droplets in the absence of
entrained water. The movement of emulsion droplets through a water column
allows the uptake of oxygen, nutrients, and oil to fungi.
b) Temperature: Based on temperature, hydrocarbon degradation can occur under
three conditions: psychrophilic, mesophilic, and thermophilic. In general, most
fungi are mesophilic in isolation, growth, and reproduction. Temperature is
essential for the growth requirements of certain fungi along with petroleum as a
substrate. Low temperatures generally retard the rates of volatilization of low-
molecular-weight hydrocarbons, some of which are toxic to fungi. Fungi also
show a propensity to withstand dry environments and high temperatures and thus
appear to be suitable for the remediation of these contaminated areas.
Temperature influences diesel oil biodegradation by the psychrotrophic yeast
Yarrowia lipolytica in a mineral medium and in soil (Margesin and Schinner,
1997). Abiotic loss of diesel oil increases with incubation time and with
temperature and is lower in a mineral medium than in soil. This amounts to a
loss of 20 to 45% (5000 mg/kg soil dry weight) in soil and 15 to 27% in liquid
media after 30 days at 4 to 30°C. BTEX degradation by Phanerochaete
chrysosporium was higher at 25°C than at 37°C (Yadav and Reddy, 1993).
c) pH: Several fungi grow well at pH levels of 4 to 5 and yeasts at 3 to 4 and are
more tolerant of acidic conditions, where it is difficult for bacteria to thrive.
Cladosporium resinae grows slowly in seawater and requires organic stimulation
for growth (Neihof and May, 1983). In certain cases, BTEX degradation by P.
chrysosporium is little affected by pH variations between 4.5 and 7.0 (Yadav and
Reddy, 1993).
d) Oxygen: Fungi are both aerobic and anaerobic but grow well under aerobic
conditions. Oxygen is necessary for the mineralization of hydrocarbons in
estuarine sediments. The rates of hydrocarbon degradation are reduced with
decreasing oxygen reduction potential. Hydrocarbons persist in reduced
Mycoremediation of Crude Oil Contaminated Soil Page 37
sediments for longer periods than in aerated surface layers. The initial steps in
the catabolism of aliphatic, cyclic, and aromatic hydrocarbons by fungi involve
oxidation of the substrate by oxygenases and molecular oxygen. Thus, aerobic
conditions are necessary for the oxidation of hydrocarbons in the environment.
Substantially greater degradation of all BTEX compounds occurs in static than
in shaken liquid cultures (Yadav and Reddy, 1993). Negligible rates of
biodegradation of hydrocarbons occur in anaerobic environments.
e) Nutrients, Dispersants, and biosurfactants: Little is known about petroleum
degradation in the presence of nutrients by fungi. Low nitrogen levels, low pH,
low moisture content, and inadequacy of certain nutrients favour the
development of fungi. In oil slicks, a proportion of carbon is readily available for
fungi growth within a limited area. Since nitrogen and phosphorus components
are essential for incorporation into fungal biomass, the availability of these
nutrients within the hydrocarbon location is important. In many cases, the supply
of nitrogen and phosphorus depends on the diffusion in the oil slick. The
degradation of hydrocarbons can be accelerated by the addition of specific urea-
phosphate, N-P-K fertilizers, and so on, for fungal growth. Fungal cells usually
contain less nitrogen than bacterial cells and thus fungi can act favourably in
ecosystems that have low nitrogen content. Many fungal isolates grow equally
well in laboratory diesel–water systems with or without an additive (Bento and
Gaylarde, 2001).
However, the composition of fungal cells can be represented empirically by
C10H17O6N. Dispersants have demonstrated a positive effect on rates of degradation
by dissolution and emulsification of hydrocarbons. Fungal levels in analytical
freshwater ponds are enhanced significantly after the addition of oil–dispersant
mixtures (Sherry, 1984). Some dispersants are toxic and inhibitory to yeasts and
fungi. The use of natural biosurfactants produced by yeasts or fungi has a marked
potential in such biospheres (Lindley, 1991, 1994). The chemical nature of
biosurfactants produced by yeasts appears to be that of glycolipids. The physical
properties of such compounds play an important role in petroleum degradation. The
major action of these biosurfactants is to increase the available surface area of the
Mycoremediation of Crude Oil Contaminated Soil Page 38
hydrocarbon phase for uptake transport by fungi. Yeast extract and malt extract
enhance cell growth and overall n-alkane degradation by the polyethylene-degrading
fungus Penicillium simplicissimum YP (Yamada-Onodera et al., 2002).
Squalane is more favorable than pristane to long-chain n-alkane degradation
when the cell density is higher. The degradation efficiency is enhanced further using
Plysurf A210G as the dispersant and supplementing with a high concentration
(0.3%) of malt extract. The fungus can also grow in the presence of pristane,
squalane, and n-alkanes with a chain 20 to 50 carbons long. This fungus has a
potential for application in bioremediation of contaminated areas containing
recalcitrant long-chain alkanes (Singh, 2006).
1.10 Economic importance
The fermentation strategies for a potential biotechnological process on
petroleum hydrocarbons as substrates are well known and have tremendous
biotechnology applications. These biotechnology applications are summarized
below.
a) Single-cell protein: Much of the work on single-cell protein (SCP) was
accomplished in the 1970s and later abandoned due to marketing problems.
Protein extracted from petroleum by some yeasts and fungi has been described
(Humphrey, 1970). Candida lipolytica is used in the processing of alkane as a
substrate for SCP (Whiteworth, 1974). Of 67 potential yeasts, Candida tropicalis
and Yarrowia lipolytica are found to be excellent for SCP production on diesel
oil as the sole source of carbon. Maximum yield has been shown to occur for a
diesel oil concentration of 40 to 60 ml/l after 168 hours (Ashy and Abou-Zeid,
1982). A good source of protein was achieved through fermentation of
hydrocarbon-derived SCP, and additional processing was required to remove
undesirable components (Scrimshaw, 1984). Cloning and recombinant DNA
techniques can be used to improve SCP production.
b) Surfactant production: Fungi and yeasts produce a wide variety of surfactants,
ranging from simple fatty acids and phospholipids by filamentous species to
complex polymers by various yeasts. The extracellular phospholipids of
Mycoremediation of Crude Oil Contaminated Soil Page 39
Cladosporium resinae are dodecanoic acid–substituted (Kan and Cooney, 1975).
The most suitable substrate for phospholipid formation by Aspergillus sp. is n-
alkane C16 (Miyazima et al., 1985). Yeasts produce surfactants that exhibit
highly active emulsifying properties. Species of Torulopsis produce glycolipids
that show similarities to bacterial rhamnolipids. Acetyl-substituted disaccharide
linked to the hydroxyl function of a hydroxycarboxylic acid is found in
sophoroselipids of Torulopsis bombicola (Inoue and Ito, 1982). Sophorolipid
production can be increased from 5 to 150 g/l in the presence of glucose and
hexadecane (Linton, 1990). Species of Candida produce surfactants that are
polysaccharide-lipid or lipopeptide in nature, produced either within the
infrastructure of the cell wall or in the medium.
c) Metabolite overproduction: Several yeasts and filamentous fungi produce a
wide range of metabolities of industrial importance on hydrocarbons in the
presence of optimum growth conditions. These products include organic acids,
amino acids, antibiotics, sterols, and others. The use of products derived from
hydrocarbons in the food industry must still obtain public approval. Various
strains of C. lipolytica are established to produce organic acids. Citric acid
production exceeding 200 g/l is achieved by C. lipolytica grown on alkanes
(Ikeno et al., 1975). Despite high production, the commercial production of
citric acid employs Aspergillus niger and carbohydrate substrates. Several yeasts
excrete small amounts of dicarboxylic acids during growth on alkanes due to ω-
oxidation involving diterminal oxidation. By selection techniques and
nonspecific mutagenesis, specific strains can result that show perturbance in β-
oxidation. These strains accumulate dicarboxylic acids >100 g/l during growth
on a mixture of alkane–acetate substrates.
The future direction of long-term strategies of economic importance on
alkane substrates is unclear. The potential of hydrocarbon-degrading yeasts and
fungi has been largely ignored due to public unacceptance of the products.
Harnessing the full potential of advances in molecular technology will open the door
for correct assessment of the economic importance of alkane degrading yeasts and
filamentous fungi in the future (Singh, 2006).
Mycoremediation of Crude Oil Contaminated Soil Page 40
Aims and Objectives:
Screening and selection of potential fungi/white rot fungi for their
adaptability in crude oil contaminated soil.
To test the efficacy of selected fungi/white rot fungus for removal of total
petroleum hydrocarbons and polycyclic aromatic hydrocarbons from crude
oil contaminated soil.