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).

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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).

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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

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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.