TECHNIQUES FOR THE BIOREMEDIATION OF PETROLEUM CONTAMINANTS IN THE ENVIRONMENT

石油污染的生物修复技术 ADELEKE OLUKUNLE FRANCIS 奥克陆 02761281

School of Chemical & Environmental Engineering, Shanghai University, China kadeleke@yahoo.com

ABSTRACT Petroleum is a very valuable source of energy and raw material for the petrochemical industry. Compared with coal, it has lower costs, it is liquid at ambient temperature and easy to exploit, transport, utilize; contains less impurity, produces less pollution and it has more vast applications. Among the developed countries, petroleum provides about 60% of their energy source, and in China, petroleum constitutes about 20% of the source of energy (Shen, 2002). With the increasing demand for petroleum products around the world, the exploitation, transportation, loading and unloading and utilization has increased drastically, and also, oil spill and oil pollution incidents has subsequently increased. Biological remediation is an important means of treating petroleum-based contamination of various environmental media including marine environments, soil and groundwater. Compared to other methods, remediation by biological means results in the actual degradation of the organic pollutants rather than the transfer of contaminants from one medium to another. This paper discusses the physical and chemical properties of petroleum, origin and fate of petroleum pollution in the environment and the methods of bioremediation applicable for the treatment of petroleum contaminated environments. 1.0 INTRODUCTION Today, petroleum products and by-products permeate our society, ranging from fuels for transportation and heating to the raw material for thousands of products including plastics, paints, cosmetics, fabrics, pharmaceuticals and medicine, to name a few. The ubiquitous use of petroleum in our society reflects its abundance, and the ease with which it can be produced, transported and converted to other beneficial forms. Worldwide, about 7 million tons of oil and petroleum products are used daily (Testa and Jacobs, 2002). Although much of the world depends on the production or the trade of oil to fuel its economies, these activities can cause severe damage to the environment, either knowingly or unintentionally. Oil production, and/or transportation, can disrupt the human population and the animal and fish life of the region. Oil waste dumping, production pollution, and spills wreak havoc on the surrounding wildlife and habitat. It threatens the extinction of several plants,

and has already harmed many land, air, and sea animal and plant species. A great part of the oil pollution problem results from the fact that the major oil-producing countries are not the major oil consumers. It follows that massive movements of petroleum have to be made from areas of high production to those of high consumption. Other sources of oil pollution are municipal and industrial wastes and industrial wastes and runoffs, leaks in pipelines and underground storage tanks (USTs), and discharge of dirty ballast and bilge waters. Approximately 0.1% (about 35 million tons) of the total world production of petroleum enters the sea per annum (Rosenberg and Ron, 1996). These results mainly from tanker accidents and natural sources (principally seeps). Some recent large oil spills are given in Table 1. In the eastern region of the South China Sea, the total number of oil spill incidents between 1990 and 1997 was ten, with the about 4.4tons of oil spilled. Also, in 1996, there was an accident in which a trawler fishing boat hit an undersea pipeline leading to the spilling of about 1000t of oil (Xu, 1999). Table 1. Recent large oil spills Source Place Date 103 tons of oil Gulf war Exxon Valdez IXTOC I well Amoco Cadiz Torrey Canyon

Iraq/Kuwait Gulf of Alaska Campeche Bay, Mexico Brittany, France Cornwall, England

February 1991 April 1989 June 1979 March 1978 March 1969

1000 33 350 223 117

Source: Rosenberg and Ron, 1996 1.1 COMPOSITION, PHYSICAL AND CHEMICAL PROPERTIES OF

PETROLEUM The Composition of Crude Oil Crude oil is an extremely complex and variable mixture of organic compounds. The majority of compounds in crude oil are hydrocarbons, which can range in molecular weight from the gas methane to the high molecular weight tars and bitumens. These hydrocarbons can also come in a wide range of molecular structures: saturates, olefins, aromatics and polar compounds. The saturates consists primarily of alkanes and cycloalkanes. Larger saturate compounds are often referred to as waxes. The olefins or unsaturated compounds have at least one double carbon-to-carbon bonds. Significant amounts of olefins are found only in refined products (Fingas, 2002). The two major groups of aromatic hydrocarbons are monocyclic, such as benzene, toluene, ethylbenzene and xylene (BTEX), and the polycyclic hydrocarbons (PAHs) such as naphthalene, anthracene and phenanthrene. Polar compounds are those that have significant molecular charge as a result of interaction with compounds such as sulphur, nitrogen or oxygen. In the petroleum

industry, the smallest polar compounds are called resins, which are largely responsible for oil adhesion. The larger polar compounds are called asphaltenes. The proportion of each individual compound can vary greatly between crude oil sources and this variation in composition affects the properties of the oil. Oils with a high proportion of low molecular weight material are known as “light” oils and flow easily, while “heavy oils” are the reverse. In addition to the hydrocarbons, crude oil contains 0.05-3.0% of heterocyclic compounds, containing sulphur, nitrogen and oxygen, and some trace heavy metals (Scragg, 1999). After extraction, crude oil is refined by distillation processes to produce different petroleum products with varying molecular size, boiling point, density, viscosity, etc. The refining process also converts most of the polyaromatic hydrocarbons into monocyclic aromatic compounds and may decrease the levels of alkanes (depentanization), while increasing the level of olefins. Typically naphthalenes can constitute 5-35% of the crude oil, which can be reduced to 1-7% after refining. Refined oil can be split into petroleum, diesel, heating oil and may other products (Scragg, 1999).

petroleum products from crude oil

Petrochemical feedstocks 1.2

Asphalt & road oil 1.3

Coke 1.8

Still gas 1.9

Liguefied refinery gases 1.9

Residual Fuel oil 2.3

Kerosene-type jet fuel 4.1

Distillate fuel oil 9.2

Gasoline 19.5

Others 0.3

Kerosene 0.2

Lubricants 0.5

Fig 1: Products in gallons produced from a barrel of crude oil. (Source: Testa and Jacobs, 2002) The description of some oils and petroleum products are given below:

• Gasoline (petrol): as used in automobiles • Diesel fuel: as used in trucks, trains and buses • Light crude oil: contains a high proportion of low molecular weight material • heavy crude oil: contains a high proportion of low molecular weight material • Intermediate Fuel oil (IFO): a mixture of a heavy residual oil and diesel oil fuel

used primarily as a propulsion fuel for ship

• Bunker fuel: such as Bunker C which is a heavy residual fuel remaining after the production of gasoline and diesel fuel in refineries and often used in heating plants

• Crude oil emulsion: such as an emulsion of water in medium crude oil Typical amounts of hydrocarbons and other compounds found in the different oils are given in Table 2. Table 2: Typical Composition of some Oils and Petroleum Products Group Compound

Class (%) Gasoline

Diesel

Light Crude

Heavy Crude

IFO

Bunker C

Saturates Alkanes Cyclo-alkanes waxes

50-60 45-55 5 -

65-95 35-45 30-50 0-1

55-90 0-20

25-80 0-10

25-35 2-10

20-30 5-15

Olefins 5-10 0-10 Aromatics 25-40 5-25 10-35 15-40 40-60 30-50 Polar Compounds

Resins asphaltenes

0-2 0-2

1-15 0-10 0-10

5-40 2-25 0-20

15-25 10-15 5-10

10-30 10-20 5-20

Metals 30-250 100-500 100-1000 100-2000 Sulfur 0.02 0.1-0.5 0-2 0-5 0.5-2.0 2-4 Source: Fingas, 2002 Characteristics of Petroleum The physical nature and chemical characteristics of petroleum are fundamental to understanding the impacts of its release to the environment. The physical characteristics of petroleum determine how it behaves in the subsurface as well as above ground where it can come in contact with soil, water, and life. The chemistry of petroleum in large part determines how it is dispersed in the environment and impacts life. Both the physical and chemical characteristics of petroleum are important foundations for technology we use to mitigate unwanted environmental consequences of petroleum use (Testa and Jacobs, 2002). Pertinent physical properties for some of the more common petroleum products are presented in Table 3. Some of the common physical properties are viscosity, density, specific gravity, solubility, flash point, pour point, distillation fractions, interfacial tension and vapor pressure.

Table 3: Typical oil properties Property Units Gasoline diesel Light

crude Heavy crude

IFO Bunker C

Crude oil emulsion

viscosity mPa.s at 15oC

0.5 2 5 to 50 50 to 50,000

1000 to 15,000

10,000 to 50,000

20,000 to 100,000

density g/ml at 15oC

0.72 0.84 0.78 to 0.88

0.88 to 1.00

0.94 to 0.99

0.96 to 1.04

0.95 to 1.0

Flash point

oC -35 45 -30 to 30

-30 to 60

80 to 100

>100 >80

Solubility in water

ppm 200 40 10 to 50

5 to 30 10 to 30

1 to 5 -

Pour point

oC NR -35 to -1

-40 to 30

-40 to 30

-10 to 10

5 to 20 >50

API Gravity

65 35 30 to 50

10 to 30

10 to 20

5 to 15 10 to 15

Interfacial Tension

mN/m at 15oC

27 27 10 to 30

15 to 30

25 to 30

25 to 35

NR

Source: Fingas, 2002 The chemical composition of crude oil can vary significantly, depending on its origin and age. Crude generally rages from 83 to 87 percent carbon (by weight), 11 to 14 percent hydrogen, with lesser amounts of sulfur (o.1 to 5.5 percent), nitrogen (0.05 to 0.08percent), and oxygen (0.1 to 4 percent). Trace constituents constitute less than 1 percent of the total volume and include phosphorus and heavy metals such as vanadium and nickel (Testa and Jacobs, 2002). Crude is classified on the basis of the relative content of three basic hydrocarbon structural types: paraffins (waxy crude), naphthenes, and aromatics. About 85 percents of all crude oil can be classified as asphalt base, paraffin base, or mixed base. Sulfur, oxygen, and nitrogen contents are often relatively high in comparison with paraffin base crude, which contains little to no asphaltic materials. Mixed-base oil is composed of some methane (normal straight chain paraffins), and isoparaffins (branched- chain paraffins) cycloparaffins or naphtenes (ring-structures), aromatics (benzene ring-structures), and asphaltics. The properties of the crude oil from some oil field found in China are given in Table 4. Table 4. The main properties of crude oil found in China ( 中国主要原由的性质) 地点 大庆 胜利 孤岛 辽河 华北 中原 新疆 蜡含量/ % 26.2 14.6 4.9 9.9 22.8 19.7 7.2 沥青质/ % 0 <1 2.9 0 <0.1 0 胶质/ % 8.9 19.0 24.8 13.7 22.0 9.5 10.6 含硫/ % 0.1 0.80 2.09 0.18 0.31 0.52 0.05 原油分类 低硫石蜡

基 含硫中间

基 含硫环烷

中间基 低硫中间

基 低硫石蜡

基 低硫石

蜡基 低硫石蜡

中间基 Source: Shen, 2002.

Hydrocarbon Constituents of Environmental Concern Spilled hydrocarbons can consist of crude, refined petroleum products such as fuels (gasoline, diesel, and aviation and jet fuels), lubricating oil and fluids, and waste oil. These products are of environmental concern if accidentally released in the environment. From an environmental or regulatory perspective, the petroleum product, or a specific constituent may drive the program developed to address the concern. When a specific constituent drives the remedial response, this can present a challenge, since each organic or inorganic compound has specific physical, chemical, and biological properties. The aromatic fraction of petroleum is the most important group of hydrocarbon chemicals. Benzene is known as the parent compound of the aromatic series with toluene, ethlybenzene, and xylenes. Benzene, being a carcinogen, if present, typically drives a remedial effort; however, other constituents, depending on the product released, may drive the remedial effort. These constituents may include certain volatile organic compounds or fuel additives such as methyl tertiary butyl ether (MTBE). MTBE is an octane booster and one of several synthetic fuel oxygenates used to meet regulatory oxygen mandates for reformulated fuels in areas not in compliance with federal standards for ozone pollution (Testa and Jacobs, 2002). Other constituents such as lead, cadmium, chromium, and sulfur are typical constituents of waste oil and can also be of environmental concern. 2.0 BEHAVIOUR AND FATE OF OIL IN THE ENVIRONMENT When oil is spilled, whether on water or land, a number of transformation processes occur that are referred to as the “behavior” of the oil. The most common types of transformation processes can be grouped in to two groups. The first is weathering, a series of processes whereby the physical and chemical properties of change after the spill. The second is a group of processes related to the movement of oil in the environment. The specific behavior processes that occur after an oil spill determine how the oil should be cleaned up and its effect on the environment. Evaporation Evaporation is usually the most important weathering process. It has greatest effect on the oil remaining on water or land after a spill. Over a period of several days, a light fuel such as gasoline evaporates completely at temperatures above freezing, whereas only a small percentage of heavier Bunker C oil evaporates. The rate at which oil evaporates depends primarily on the oil’s composition. The more volatile components an oil or fuel contains, the greater the extent and rate of its evaporation. Many components of heavier oils will not evaporate at all, even over long periods of time and at high temperatures.

Emulsification Emulsification is the process by which one liquid is dispersed into another one in the form of small droplets. Water droplets can remain in an oil layer in a stable form and the resulting material is completely different. The mechanism of emulsion formation is not yet fully understood, but it probably starts with sea energy forcing the entry of small water droplets, about 10 to 25µm in size, into the oil. If the oil is only slightly viscous, these small droplets will not leave the oil quickly. On the other hand, if the oil is very viscous, droplets will not enter the oil to any significant extent. Once in the oil, the droplets slowly gravitate to the bottom of the oil layer. Any asphaltenes or resins in the oil will interact with water droplets to stabilize them. Depending on the quantity of asphaltenes and resins, as well as aromatic compounds which stabilize asphaltenes and resins in solution, an emulsion may be formed. The conditions required for emulsions of any stability to be formed may only be reached after a period of evaporation. Evaporation lowers the amount of low-molecular weight aromatics and increases the viscosity to the critical value. Natural Dispersion Natural dispersion occurs when fine droplets of oil are transferred into the water column by wave action or turbulence. Small oil droplets (less than 20 µm or 0.020 mm) are relatively stable in water and will remain so for long periods of time. Large droplets tend to rise and larger droplets (more than 100 µm) will not stay in the water column for more than a few seconds. Depending on the conditions and amount of sea energy available, natural dispersion can be insignificant or it can remove the bulk of the oil. Natural dispersion is dependent on both oil properties and the amount of sea energy. Heavy oils such as Bunker C or heavy crude will not disperse naturally to any significant extent, whereas light crudes and diesel fuel can disperse significantly if the saturate content is high and the asphaltene and resin contents are low. In addition, significant wave action is needed to disperse oil. Dissolution Through the process of dissolution, some of the most soluble components of the oil are lost to the water under the slick. These include some of the lower molecular weight aromatics and some of the polar compounds, broadly categorized as resins. As only a small amount, usually much less than a fraction of a percent of the oil, actually enters the water column, dissolution does not measurably change the mass balance of the oil. The significance of dissolution is that the soluble aromatic compounds are particularly toxic to fish and other aquatic life. If a spill of oil containing a large amount of soluble aromatic components occurs in shallow water and creates a high localized concentration of compounds, then significant numbers of aquatic organisms can be killed.

Photooxidation Photooxidation can change the composition of oil. It occurs when the sun’s action on an oil slick causes oxygen and carbons to combine and form new products that may be resins. The resins may be somewhat soluble and dissolve into the water or they may cause water-in-oil emulsions to form. It is not well understood how photooxidation specifically affects oils, although some oils are susceptible to the process while others are not. For most oils, photooxidation is not an important process in terms of changing their fate or mass balance after a spill. Sedimentation, Adhesion to Surfaces, and Oil-Fines Interaction Sedimentation is the process by which oil is deposited on the bottom of the sea or other water body. While the process itself in not well understood, certain facts about it are. Most sedimentation noted in the past has occurred when oil droplets reached a higher density than water after interacting with mineral matter in the water column. This interaction sometimes occurs on the shoreline or very close to the shore. Once oil is in the bottom, it is usually covered by other sediment and degrades very slowly. In a few well-studied spills, a significant amount (about 10%) of the oil was sedimented on the sea floor. Such amounts can be very harmful to biota that inevitably comes in contact with the oil on the sea bottom. Because or the difficulty of studying this, data are limited (Fingas, 2002). Biodegradation A large number of microorganisms are capable of degrading petroleum hydrocarbons. Many species of bacteria, fungi, and yeasts metabolize petroleum hydrocarbons as a food and energy source. Bacteria and other degrading organisms are most abundant on land in areas where there have been petroleum seeps, although these microorganisms are found every where in the environment. As each species can utilize only a few related compounds at most, however, broad-spectrum degradation does not occur. Hydrocarbons metabolized by microorganisms are generally converted to an oxidized compound, which may be further degraded, may be soluble, or may accumulate in the remaining oil. The aquatic toxicity of the biodegradation products is sometimes greater that of the parent compounds. Formation of Tar Balls Tar balls are agglomerations of thick oil less than about 10cm in diameter. Large accumulations of the material ranging from about 10cm to 1m in diameter are called tar mats. Tar mats are pancake-shaped, rather than round. Their formation is still not completely understood, but it is known that they are formed from the residuals of heavy crudes and Bunker C. After these oils weather at sea and slicks are broken down, the residuals remain in tar balls or tar mats.

3.0 BIODEGRADATION OF PETROLEUM CONTAMINANTS It has been known for about 80 years that certain microorganisms are able to degrade petroleum hydrocarbons and use them as a sole source of carbon and energy for growth (Rosenberg and Ron, 1996). The use of hydrocarbons as substrates for bacterial growth presents special problems to both the microorganisms using them as a source of carbon and energy (Table 5) and to investigators in the field of hydrocarbon microbiology. They are two essential characteristics that define hydrocarbon-oxidizing microorganisms: 1. Membrane-bound, group-specific oxygenases, and 2. Mechanism for optimizing contacts between the microorganisms and the water-

insoluble hydrocarbon. The localization of hydrocarbon-oxidizing bacteria in natural environments has received considerable attention because of the possibility of utilizing their biodegradation potential in the treatment of oil spills. Because of the enormous quantities of crude and refined oils transported over long distances and consumed in large amounts, the hydrocarbons have now become a very important class of potential substrates for microbial oxidation. Table 5. Requirements for biodegradation of petroleum A. Microorganisms with

1. Hydrocarbon-oxidizing enzymes 2. Ability to adhere to hydrocarbons 3. Emulsifier-producing potential 4. Mechanisms for desorption from hydrocarbons

B. Water C. Oxygen D. Phosphorus E. Utilizable nitrogen source Hydrocarbon-oxidizing microorganisms exist in a wide variety of natural aquatic and terrestrial natural environments, and several investigators have demonstrated an increase in the number of hydrocarbon-oxidizing bacteria in areas that suffer from oil pollution. Thus the presence of hydrocarbons in the environment brings about a selective enrichment in situ for hydrocarbon–utilizing microorganisms (Rosenberg and Ron, 1996). Thus, seeding an oil-polluted area with microorganisms may not always be necessary. However, under some circumstances, seeding may be advantageous to ensure uniformity of the hydrocarbon breakdown pattern and to encourage the degradation of the toxic PAH fraction.

PHYSICAL INTERACTION OF MICROORGANISMS WITH HYDROCARBONS The low solubility of hydrocarbons in water, coupled with the fact that the first step in hydrocarbon degradation involves a membrane-bound oxygenase, makes it essential for bacteria to come into direct contact with the hydrocarbon substrates. This takes place through specific adhesion/desorption mechanisms and emulsification of the hydrocarbon. Adhesion/desorption Adhesion of microorganisms to the hydrocarbon/water interface is the first step in the growth cycle of microorganisms on water-insoluble hydrocarbons. Adhesion is caused by hydrophobic interactions. Desorption fro the hydrocarbon is a critical part of the growth cycle of petroleum-degrading bacteria. Petroleum is a mixture of thousands of different hydrocarbon molecules. Any particular bacterium is only able to use part of the petroleum. As the multiply at the hydrocarbon/water interface of a droplet, the relative amount of nonutilizable hydrocarbon continually increases until the cells can no longer grow. For bacteria to be able to continue to multiply, they must be able to move from the depleted droplet to another one. Emulsifiers Hydrocarbon-degrading microorganisms produce a wide variety of surface-active agents. Microbiologically derived surfactants can be divided into low and high molecular weight products. The low molecular weights are a mixture of glycolipids, fatty acids, phospholipids and lipopeptides. The high molecular weight surfactants are amphipatic polymers and complexes of hydrophobic and hydrophilic polymers. The natural role of emulsans is to enhance the growth of bacteria on petroleum by two mechanisms: (1) increasing the hydrocarbon surface area and (2) desorbing the bacteria from ‘used’ oil droplets. The growth of microorganisms on water-insoluble hydrocarbons is restricted to the hydrocarbon/water interface because the hydrocarbon oxygenases are always membrane-bound, never extracellular. After the bacteria adhere to the hydrocarbon surface, they begin to multiply on the surface becomes saturated with bacteria, and growth becomes limited by the available surface. If the bacteria can split the oil droplets (emulsification), new surfaces become available for growth. Nutrient requirements As mentioned above, microorganisms that have the genetic potential to bind, emulsify, transport and degrade hydrocarbons are widely distributed in nature. Thus, the rate-limiting step in biodegradation of hydrocarbons is generally not the lack of appropriate microorganisms. Depending upon the particular environmental situation, the extent of degradation depends on the availability of moisture, oxygen, and utilizable sources of nitrogen and phosphorus. Thus, the effectiveness of a bioremediation program depends on defining the limitations and overcoming them-in a practical way.

The requirements for oxygen and moisture are not a problem in oil spill in aquatic environments. However, on land, oxygen and water are often rate-limiting. If oil has not penetrated too deeply into the ground, then watering and tilling are often advantageous. In some circumstances, hydrogen peroxide has been used as a source of oxygen. Nitrate has also been tested as an alternative electron acceptor (Rosenberg and Ron, 1996). The major limitation in the biodegradation of hydrocarbons on land and water is available sources of nitrogen and phosphorus. In theory, approximately 150 mg of nitrogen and 30 mg of phosphorus are consumed in the conversion of 1g of hydrocarbon to cell material. The nitrogen and phosphorus requirements for maximum growth of hydrocarbon oxidizers can generally be satisfied by ammonium phosphate. Alternatively, these requirements can be met by a mixture of other salts, such as ammonium sulfate, ammonium nitrate, ammonium chloride, Potassium phosphate, sodium phosphate and calcium phosphate. Recently, commercial nitrogen- and phosphorus-containing fertilizers that have affinity for hydrocarbons are being developed for treating oil pollution (Rosenberg and Ron, 1996). PATHWAYS OF DEGRADATION Petrochemicals, PAHs and BTEX compounds are degraded by soil microorganisms, which use them as a source of both energy and carbon compounds for cell synthesis. Hydrocarbons are stable reduced compounds and therefore degradation generally proceeds by oxidation under either aerobic or anaerobic conditions. Oxidation of n-alkanes proceeds, in general via terminal oxidation to the corresponding alcohol, aldehydes and fatty acids. The group specificity of alkane oxygenase system is different in various bacterial species. For example, Pseudomaonas putida grows on alkanes of six to ten carbons in length, whereas Acinetobacter is capable of growing on long-chain alkanes. The oxidation of branched and cyclic alkanes is probably responsible for the formation of long-chain secondary alcohols and ketones. Some bacteria species are capable of this type of oxidation, but in some cases, especially in the case of cyclohexane and cyclopentane, mixed bacteria cultures first convert the cyclic alkanes to cyclic ketones, which are then oxidized by specific bacteria (Rosenberg and Ron, 1996). Both prokaryotic and eukaryotic microorganisms have enzymatic potential to oxidize aromatic hydrocarbons that range in size from a single ring (e.g BTEX), to polycyclic aromatics (PAHs), such as naphthalene, anthracene, phenanthrene, etc. However the molecular mechanisms by which bacteria and higher microorganisms degrade aromatic compounds are fundamentally different. A list of the microorganisms responsible for the degradation of aromatic compounds is given in Table 6. The recalcitrance of PAHs to biodegradation is directly proportional to molecular weight (Table 7). The slow degradation of high molecular weight PAHs is probably due to low solubility and less availability for biological uptake (Rosenberg and Ron, 1996). Monocyclic aromatics, as in the case of benzene, are first hydroxylated by a dioxygenase enzyme to cis-1,2-dihydroxy-1,2-dihydrobenzene, which is then converted to catechol.

The subsequent metabolism of catechol can take one of two pathways: ortho cleavage yields cis,cis-muconate, whereas meta cleavage yields 2-hydroxymuconic semialdehyde. Both pathways lead to compounds which can enter the Krebs cycle. The first two stages of the degradation of benzene are common for the breakdown of many other monocyclic and polycyclic hydrocarbons (Scragg, 1999).

The pathway for the biodegradation of Benzene. In general aromatic ring hydroxylation is followed by ring cleavage, and both of these reactions are carried out by oxygenases. The incorporation of two oxygen molecules causes the introduction of two hydroxyl groups which can undergo either meta or ortho cleavage. Incorporation of a single oxygen molecule is catalyzed by monooxygenases and both enzymatic systems can be used to degrade polycyclic aromatic hydrocarbons. Many of the monocyclic aromatic hydrocarbon-degrading bacteria have chromosomal genes coding for the enzymes of the pathway for the degradation of hydrocarbons, but plasmids have been found that code for the degradation of compounds such as camphor, octane, toluene, naphthalene, and some herbicides and pesticides. Some plasmids code only for some part of the path. It is clearly an advantage in the soil to be able to transfer the degradative ability between bacteria, as this allows the rapid adaptation of the population to a particular compound.

Fig. 2. The pathway for the degradation of PAH by fungi, bacteria and algae The bioremediation of hydrocarbon-contaminated soil cannot always be maintained under aerobic conditions due to water logging, the fine particle structure of the soil and blocking of the soil pores with the biomass itself. However, aliphatic, monocyclic and polycyclic aromatic hydrocarbons can be degraded anaerobically provided oxygen can be obtained from water under methanogenic conditions, from nitrate under nitrifying conditions and from sulphate under sulphur-reducing conditions. The hydrocarbons are converted to central metabolic intermediates by hydration, dehydration, reductive dehydroxylation, nitroreduction and carboxylation. The central intermediates are benzoyl CoA and sometimes resorcinol can enter the Krebs cycle. The only disadvantage with anaerobic degradation is that the process is much slower than the aerobic pathway (Scragg, 1999). On of the features of the degradation of both hydrocarbons and other organic molecules is the ability of some enzymes to function with compounds other than their normal substrate. This condition, often known as gratuitous metabolism, is probably a result of broad enzyme specificity. Another feature required for growth in which describes the benefit is derived by the organism regards co-metabolism as an undefined term describing the imprecise specificity of systems and their indication systems.

Table 6. Microorganisms that metabolize aromatic hydrocarbons Organisms Organisms Bacteria Pseudomonas Aeromonas Moraxella Bierjerinckia Flavobacteria Achrobacteria Nocardia Corynbacteria Acinnetobacter Alcaligenes Mycobacteria Rhodococci Streptomyces Bacilli Arthrobacter Aeromonas Cyanobacteria

Fungi Chytridomycetes Oomycetes Zygomycota Ascomycota Basidiomycota Deuteromycota Microalgae Porphyridium Petalonia Diatoms Chlorella Dunaliella Chlamydomonas Ulva

Source: Rosenberg and Zon, 1996

Table 7. Half-lives for the microbial degradation of PAHs in soil. Aromatic compound Molecular weight Half-life (weeks) Naphtalene Phenanthrene 2-Methylnaphtalene Pyrene 3-Methylcholanthrene Benzo[a]pyrene

128 178 142 202 226 252

2.4-4.4 4-18 14-20 34-90 87-200 200-300

Source: Rosenberg and Zon, 1996 4.0 BIOREMEDIATION IN DIFFERENT ENVIRONMENTAL MEDIA Bioremediation of marine oil spills Crude oil when released at sea will not mix with sea water and will float on the surface, allowing the escape of the volatile components, those of carbons and below. The floating oil, if it does not reach the shore, will be dispersed due to the action of waves. The dispersion will allow naturally occurring hydrocarbon-degrading organisms to break down the oil. Oil breakdown will occur at the interface between the oil and water and therefore the better the oil dispersion, the greater the area, the faster the degradation. Crude oil is a naturally occurring product and as such is biodegradable, and it is perhaps no surprise that hydrocarbon-degrading microorganisms are distributed widely in nature.

The rate of dispersion of the oil will depend on the wave action which in turn is dependent on the weather. The more complex and less soluble oil components will be degraded much more slowly than the lighter oils, and it is these high molecular weight components that will persist on the sand and rocks if the spill reaches shore. The first stage in the recovery and clean-up of an oil spill is to stop the release and contain the spill. Once this has been done the surface oil can be removed mechanically with skimmers and other machines. The process of recovery of oil will depend on the location, the volume of the spill, the weather conditions, and the nature of the oil. If the oil reaches the shore mechanical removal is possible on sandy areas, but on rocky shores washing the oil back into the sea is usually attempted. Chemical dispersants can be used on both floating oil and oil which has reached a rocky shore but care has to be taken as the detergents can be as harmful to the environment as the oil. Although both physical and chemical methods are efficient, not all the oil can be removed and it is the remaining high molecular weight oil that may need to be removed by some form of bioremediation. Potential bioremediation approaches for marine oil spills fall into three major categories: 1) Stimulation of indigenous microorganisms through addition of nutrients (fertilization), 2) Introduction of special assemblages of naturally occurring oil-degrading microorganisms (seeding), and 3) introduction of genetically engineered microorganisms (GEMs) with special oil-degrading properties. Stimulation of indigenous organisms by the addition of nutrients is the approach that has been tested most rigorously. This approach is viewed by many researchers as the most promising one for responding to most types of marine spills. It is generally agreed that rates of biodegradation in most marine environments are constrained by lack of nutrients rather than by the absence of oil-degrading microbes. Thus, to encourage degradation slow-release fertilizers have been added to oil slicks at a nitrogen to oil ratio of 1:100 (Scragg, 1999). . A better approach has been to treat the oil slick with a nitrogen- and phosphate-containing dispersant, which also contains a surfactant which directs the salts to the oil droplet surfaces. In the case of the Exxon Valdez spill, the Alaskan shore was so free of nitrogen and phosphate compounds that addition of fertilizers was particularly effective (Scragg, 1999). In coastal areas which receive waste streams, such as sewage outfall, there is little effect on the additional of further nitrogen and phosphate compounds as the levels are already high. The Exxon Valdez oil tanker spillage provided the opportunity to study the effect of nutrient addition on oil removal from the rocky shore, and the different forms of nitrogen and phosphate fertilizer were tried. The first was a soluble fertilizer with a ratio of 23:2 nitrogen to phosphorus, he second was a slow-release encapsulated fertilizer, and the last was an oleophilic fertilizer, which would concentrate in the oil droplets. All three fertilizers were tired on the oil-contaminated rocky shore, where the oleophilic fertilizer gave the best results, cleaning the rocks of oil after only 10 days’ treatment (Scragg, 1999).

Bioremediation of Salt Marsh

Build-up of petroleum products (e.g. crude oil, gasoline, diesel, No.2 or No.6 fuel oil) in coastal and estuarine environments result from a variety of human activities. These include spills from shipping accidents, bilge cleaning, runoff from land and municipal and industrial wastes, recreational boating, and atmospheric deposition. Of all estuarine and coastal environments, salt marshes are often the most ecologically sensitive areas impacted by oil spills and leaks. Oil is swept into salt marsh environments by tidal currents and wind and is trapped by marsh grass and organic-rich sediment. Once trapped in the marsh, the contaminants are available to organisms through the water column, suspended matter, sediments, and other organisms. A research carried out to evaluate four methods of bioremediation to clean up these salt marshes: natural attenuation (no amendment – control); nutrient-enhanced remediation (applying nitrogen and phosphorus as a liquid); bioventing (air addition); and nitrate addition (NCNERR). The researchers found that the majority of the oil was within the top 3 cm of sediment, and thus focused their injection and monitoring system efforts in this zone. The marsh was divided into 4 separate plots. The control plot (no amendments) measured natural attenuation. The other three plots acted as experimental plots in which horizontal wells and connecting tubing were installed. This was accomplished by driving 3m long hollow steel pipes with bolts loosely fitted on one end into the sediments. The pipe was pushed into the sediment approximately 16-20 cm below and parallel to the marsh surface. A perforated plastic tube was inserted into the pipe and then the pipe was twisted out of the sediment leaving the plastic tube and bolt in place. Based on the data collected from these field seasons, air and nitrate additions can significantly improve the rate of petroleum hydrocarbon degradation when compared with natural attenuation (NCNERR). Bioremediation of soils Soils contain a very large number of microorganisms which can include a number of hydrocarbon-utilizing bacteria and fungi, representing 1% of the total pollution of some 104-106 cells per gram of soil. In addition, cyanobacteria and algae have also been found to degrade hydrocarbons. Hydrocarbon-contaminated soils have been found to contain more microorganisms than uncontaminated soils, but the diversity of microorganisms was reduced (Scragg, 1999). The biodegradation of the hydrocarbons is associated with microbial growth and metabolism and therefore any of the factors affecting microbial growth will influence degradation. If the microorganisms cannot use the hydrocarbons as their sole source of energy and carbon skeletons, some other growth substrate will be needed. In some cases if another substrate is present the microorganisms my use this in preference to the hydrocarbons. The microorganisms may also require supplementation with nitrogen-and

phosphorus- containing compounds as demonstrated in marine conditions. Aerobic degradation of hydrocarbons is considerably faster than the anaerobic process, so that a supply of oxygen will be needed to maintain aerobic conditions if rapid degradation is required. A soil with an open structure will encourage oxygen transfer and a waterlogged soil will gave the revere affect. The temperature affects microbial growth, so that al low temperature the rate of degradation will be slow. Nutrient addition to soils at temperatures of 4-10oC has been shown to have little effect as the low temperature has reduced growth to such a low level. The pH of the soil will affect both the growth and the solubility of the compound to be degraded. The presence of large numbers of hydrocarbon-degrading microorganisms in the soil will clearly be if advantage at the start, but as most soils contains these types of organisms growth will soon increase the numbers, so that seeding with specific hydrocarbon-degrading organisms will probably not needed. Hydrocarbon contamination may also be associated with high levels of heavy metal which may inhibit growth, depending on the concentration and type of metals. The rate of degradation of the hydrocarbon will also be dependent on the structure of the compound. The simpler aliphatics and monocyclic aromatics are readily degradable, but more complex structures such as PAHs are not easily degrades and may persist for some time. The persistence will be increased if the compound is also toxic or is its breakdown products are toxic. Another crucial factor is the availability of the compound for degradation within the soil. Availability will be affected by the soil structure, its porosity and composition, and solubility of the compound itself. Some compounds can be adsorbed to clays and are thus rendered invulnerable to degradation. To overcome this problem surfactants have been added to contaminated soils to improve the availability of hydrocarbons (Scragg, 1999). 5.0 TECHNIQUES FOR BIOREMEDIATION OF PETROLEUM

CONTAMINANTS Bioremediation applications fall into two broad categories: in situ or ex situ. In situ bioremediation treats the contaminated soil and/or groundwater in the location in which it is found. Thus, neither excavation of the soil nor pumping of the water is required. Ex situ bioremediation processes require excavation of contaminated soil or pumping of groundwater before they can be treated. In situ techniques may be less expensive, create less dust, cause less release of contaminants than ex situ techniques, and it is possible to treat a large volume of soil at once. In situ techniques, however, may be slower than ex situ techniques and may be difficult to manage. 5.1 EX-SITU BIOLOGICAL REMEDIATION Biodegradation in ‘pile’ This tem covers the methods of composting, landfarming and biopile. These methods are applied on site to soil contaminated by petroleum products or other organic pollutants. The basic process of biodegradation in pile consists of excavating the soil and bringing it

to a nearby treatment unit designed to facilitate natural aerobic degradation. If acceleration of the process is desired, the three influencing factors to be considered, irrespective of the technique used are aeration, level of humidity and supply of nutrient. The source of microorganisms is usually the bacterial flora present in the soil but microorganisms of other origin may be added. Composting is the simplest technique. In composting, the excavated soil is mixed together in a regularly spaced swathes (small heaps) a few meters in circumference and about one meter high. The soil is mixed with solid organic matter that is readily degraded and supplemented with nutrient, air and microorganisms (inoculums) if required. Heat is normally generated during the composting which is favorable to biodegradation. In a humid temperate climate, natural conditions suffice for humidification of the swatches, however, under this conditions, the process of degradation is very slow and not cost effective (Lecomte, 1998). Excavation, sieving and piling also facilitates the aeration of materials for the starting process. The efficiency obtained is generally low and the technique is used for easily biodegradable contaminants like petroleum-based hydrocarbons. The three major types of composting are open windrow, static windrow and reactor system. The reactor system is often more efficient because the environmental conditions can be controlled. In an experiment to determine the appropriate mix ratio of organic amendments for enhancing the degradation of diesel oil-contaminated soil with composting (Namkoong et. al., 2002), sewage sludge or compost was added organic amendment to act as easily degradable organic matter. The soil was spiked with diesel at 10,000mg/kg of soil on dry weight basis. The temperature was maintained at 20oC ad the moisture content of the sample was controlled at 70% of the sample field capacity before input of sample in the reactor. An aeration rate of 100ml/min (200l/min m3) was maintained in the reactor. Degradation rates of total petroleum hydrocarbons (TPH) and n-alkanes were greatest at ratio 1:0.5 of contaminated soil to organic amendments on wet weight basis. Preferential degradation of n-alkanes over TPH was observed regardless of the type and amount of organic amendments. Landfarming involves treating the material as an agricultural soil to facilitate its remediation. Landfarming is also known as land treatment. This technology usually involves spreading excavated contaminated soils in a thin layer (a few tens of cm) on the ground surface, the addition of chemical fertilizers (or organic manure) and moisture which is kneaded together by use of agricultural equipment. The addition of fertilizer improves the balance between the nutrients and the source of carbon and, in case of manure, augments the quantity of available microorganisms. The soil is tilled and/or ploughed at regular intervals to facilitate aeration and microbial activity. The enhanced microbial activity results in degradation of adsorbed petroleum product constituents through microbial respiration (Lecomte, 1998). To support bacterial growth, the soil pH should be within the 6 to 8 range, with a value of about 7 (neutral) being optimal. Soil pH within the landfarm can be raised through the addition of lime and lowered by adding elemental sulfur. The ideal range for soil

moisture is between 40 and 85 percent of the water-holding capacity (field capacity) of the soil or about 12 percent to 30 percent by weight (USEPA, 1995). Microorganisms require inorganic nutrients such as nitrogen and phosphorus to support cell growth and sustain biodegradation processes. The techniques of composting and landfarming are particularly suitable for the biodegradation of hydrocarbons which are not very volatile such as diesel fuel oils, etc. The major advantage of landfarming is the low cost-equipment construction and operation. However, the methods require a large land area may be environmentally unacceptable due to the possibility of groundwater contamination, volatile emissions and the long-term accumulation of heavy metals in the soil. In a study conducted by Loehr and Webster, 1996, land treatment was used to treat oily waste from petroleum industry operations. The treatment was conducted nine years after operations has ceased on the site. Test plots preparation comprises diking, tilling, liming and fertilizing. Bioremediation relied on indigenous microorganisms with no seed or specialty microorganisms added. The results shows that land-treatment successfully reduced the concentration of the organics in the wastes considerably, and the accumulation of the organic and metal constituents of the wastes in surface soils. Even though, residual chemical concentrations were above background concentrations, the mobility of the chemicals in the land-treatment residues was limited, and simulated weathering did not increase the mobility of these constituents. Thus, biodegradation and biostabilization of the residues was achieved. Prepared bed reactor. Is similar to land farming but includes irrigation water systems, nutrient addition, a liner at the bottom of the soil and leachate collection system. Clay or synthetic material is used as liner. The method is often used for contaminants like PAHs, and BTEX (benzene, toluene, ethylbenzene and xylene) which are also found in petroleum-contaminated sites. In a two-phased treatment of contaminated soil from Liaohe Oil Field in China (Li Peijun et. al., 2003) using the prepared bed method, the initial concentration of total petroleum hydrocarbons (TPH) consisting of thin oil, high condensation oil, viscous oil and high viscous oil were in the range of 25.8 – 77.2 g/kg of dry soil. The treatment period was divided into two phases with a total time of 210 days. The operating conditions were temperature (20 – 40oC), O2>14%, pH range 6-8, moisture content 10–25%. After the first phases comprising 53 days, petroleum removal rate was between 38 and 57 %; and in the second phase comprising 156 days, the removal rates reached 67 – 81%. The result showed that the easily degraded hydrocarbons have been removed in the first phase as the removal efficiency dropped in the second phase. Soil piles or Biopiles. As in composting and landfarming, the excavated contaminated soil is stack in piles. The pile is placed on an impermeable layer (asphalt or concrete) and is covered with an impermeable membrane and any inflow and outflow of liquid and gaseous phases is carefully monitored. When the contaminated soil contains a significant fraction of volatile pollutant, the technique is more suitable than composting or

landfarming. While landfarms are aerated by tilling or plowing, biopiles are aerated most often by forcing air to move by injection or extraction through slotted or perforated piping placed throughout the pile. At the base of the pile, a system of pipes is installed in a drainage layer (gravel); this facilitates efficient aeration of the ensemble of materials through a uniform distribution. At the top of the pile, a sprayer is installed; its purpose is to humidify the material and when necessary to add nutrients and/or microorganisms. A network of channels is provided to enable the recovery and recycling of effluents leaching from the pile and flowing over the impermeable slab. Finally, the ensemble is covered with a plastic membrane, isolating it from the exterior. The greenhouse benefits obtained from the system is keeping the atmosphere humid and especially increasing the temperature, thereby helping the growth of biomass even in cold weather. Biopiles, like landfarms, have been proven effective in reducing concentrations of nearly all the constituents of petroleum products typically found at underground storage tank (UST) sites (USEPA, 1995). Lighter (more volatile) petroleum products (e.g., gasoline) tend to be removed by evaporation during aeration processes (i.e., air injection, air extraction, or pile turning) and, to a lesser extent, degraded by microbial respiration. The typical height of biopiles varies between 3 and 10 feet. Additional land area around the biopile(s) will be required for sloping the sides of the pile, for containment berms, and for access. The length and width of biopiles is generally not restricted unless aeration is to occur by manually turning the soils. In general, biopiles which will be turned should not exceed 6 to 8 feet in width. The bioreactor The bioreactor represents a technique of remediation by biological means which can be used for numerous applications; the operation consists of biodegrading the contaminant in a container installed on site by addition of the other ingredients necessary for the reaction. It is normally ex situ and often more expensive than in situ processes. Through a bioreactor one remediate:

• Contaminated water pumped out beforehand, • Soil, treated in the form of slurry, or sludge, • A gaseous phase by the use of biofilter.

Implementation of the technique remains the same for these three types of media; only the equipment may differ. Two types of reactors are used in which:

1) The micro-organisms grow in suspended state in the contaminated environment and floc,

2) The micro-organisms grow in the reactor attached to a support.

In the suspended system, the contaminated water circulates in a basin in which a population of micro-organisms degrades the organic matter (comprising the pollutants and grows by feeding on it. The floc formed is subsequently settled out the biomass is

recycled to the reactor. The operation is similar to activated–sludge process and the biodegradation is extensive because microorganisms’ population, temperature, nutrient, oxygen and other conditions can be controlled. In the system with support, the micro-organisms are attached on an inert material or structure provided or installed in the reactor. For soil and water, this technique poses the same major drawbacks as do all other on-site treatments: water has to be pumped out or soil excavated beforehand. But the bioreactor has certain significant advantages:

• It enables precise control and management of the process of biodegradation; including pH, humidity, concentration of nutrients, etc.

• Mixing the material under treatment and micro-organisms on the one hand, and nutrients on the others, is readily and effectively accomplished; aeration of the ensemble is likewise generally easy.

• Optimal conditions of biodegradation ca be quickly attained and hence high operational efficiency; reaction times can be continuously readjusted, depending on concentration of residual contaminant and metabolites an even the biomass present in the reactor.

• Depending on the pollutant and the material, the micro-organism best suited for the treatment can be determined (either part of the bacterial flora from the contaminated medium itself, or alien stump added in the reactor).

For treatment of gas, the use of biofilter increases the effectiveness and simplicity of the system. The biological filter is usually made of compost or peat. It can be installed in two alternative forms; the filter ‘in bed’ or the filter ‘in packet’. The latter consists of a layer of humidified compost held between two grills, in a closed tank; air loaded with pollutants is injected from below at low pressure, through a layer of coarse porosity (gravel, plastic stand), which facilitates the uniform distribution in the tank. A tap is provided at the bottom to recover the water of condensation and a sprinkler above the compost to humidify as per requirement. The efficiency of the biofilter depends directly on the thickness of the compost filter and the velocity of the injected gas. The major factors for the success of the compost bed are 50 to 70% humidity, porosity between 80 and 90%, pH 7 to 8 and temperature varying from 15 to 45oC (Lecomte, 1998). 5.2 IN-SITU BIOLOGICAL REMEDIATION Bioventing and biosparging These two terms refers to techniques that combine the two mechanisms of remediation: biodegradation and ventilation. Bioventing involve introducing air into contaminated soil above the water table, thereby providing the oxygen needed for the aerobic bacteria to biodegrade the pollutant. The air is introduced by vacuum extraction method, air injection wells, etc. Biosparging is

similar to bioventing, but the air is introduced below water table (i.e. saturation zone). The purpose is to use the air to transfer the volatile pollutants into overlying unsaturated zone with higher microorganism population. Also, some biodegradation will occur in the aquifer (Alexander, 1999). The supply of oxygen aids the growth of the biomass by inducing the consumption of the carbon of the organic compounds present in the soil and consequently their degradation. Supply of nutrients is generally necessary to balance the proportions of carbon, nitrogen and phosphorus. These, mixed with water are either injected in the groundwater upstream of the zone under treatment, or infiltrated directly on the soil of the zone by spraying or lagooning. In bioventing, the volatile compounds of the soil are mobilized by the air current and thi proceeds simultaneously with biodegradation. In addition, as the gaseous compound degrade the injection of air aids volatilization of the liquid phase. In biosparging, the injected air mobilizes the contaminants dissolved in water or trapped by capillary action in the pores, by vaporizing them. This vaporized phase is carried upward by the rising air bubbles and is biologically degraded in the unsaturated soil. The airflow injected in the subsoil is recovered by suction. The flow rate of injection/suction is maintained sufficiently low to provide the bacterial flora of the soil sufficient time to degrade the contaminating compounds volatilized and entrained by the air current. The major difficulty lies in the follow up and control process, access to which is only indirect and punctilious. The low permeability of soils as well as layers or strata of clay between more permeable zones is a constraining factor. The presence of such layers can cause lateral dispersion of pollutants in the unsaturated zones or aquifers and thus extend the polluted zone. Besides, injection of compressed air in the subsoil can lead to formation of preferential channels of circulation which may drastically reduce the efficiency of the process (particularly in the case of biosparging). Bioventing is attractive because it operates in situ and because little equipment is required. It has been used for hydrocarbon remediation. It is however not suitable for compounds with high volatility, and soils of low permeability. Biosparging with vapor extraction has been used in sites contaminated with JP-4 jet fuel, and BTEX in soil and groundwater (Headley et al., 2000). Excellent results have been obtained, with higher than 90% rate of biodegradation in a few months when biosparging was used in the degradation of soils contaminated by petroleum hydrocarbon (Lecomte, 1998). Biobarriers This is a recently developed technology for the in-situ treatment of contaminated groundwater. It involves creating downstream of the pollution (and on route of travel of the groundwater) a zone rich in microorganisms well suited for removing the contamination to be treated. This zone of intense bacterial action constitutes a real biological barrier, acting as a screen against the propagation of pollution. Very often, it primarily involves only bacterial microflora, indigenous to the soil of that area.

The design of biological screens or barriers is similar to that of the physiochemical barriers allowing the precipitation of toxic metals by redox reaction. Phytoremediation The term involves several processes which use vegetation for cleaning the environment by the removal or degradation pollutants. This is achieved by the uptake of the contaminants by the plant, or by biodegradation by microorganisms in or near the root system of the plant in the rhizosphere (immediate surface of the root and adjacent soil). The reasons for the enhanced biodegradation in the rhizosphere is not yet known but may be due to the larger bacterial mass near the root zone than farther down in the soil. The pollutants that can be handled are primarily heavy metals and hydrocarbons. The methods of phytotransformation and phytostimulation are particularly applicable to the degradation of organic pollutants. Phytotransformation helps in the degradation of complex organic molecules into simpler compounds which are integrated in the vegetal tissues. Phytostimulation does not really bring the pollutants and plants into contact with each other. It involves only stimulation of the microbial and fungal degradation by the exudants and enzymes released in the root rhizosphere, thereby entraining the destruction of organic pollutants. These techniques are better suited to sites with pollution at low level of concentration but spread over large volume and where contaminants are near the surface (1-2m deep). The decontaminating efficiency of this technique is rather low, and the treatment has to spread over several seasons or years. Phytoremediation has nevertheless the advantage of low cost, less cumbersome and allowing a landscaped management of zones in the course of treatment (Lecomte, 1998). Its use may be limited where pollutants are strongly sorbed or have become aged or sequestrated, where phytotoxicity prevents the plant from rooting extensively, where contaminants leach quickly out of rooting zone, or if the site is oxygen deficient (Alexander, 1999). A greenhouse experiment was to demonstrate the use of plants (clover) to increase the degree of degradation of pollutants in a petroleum contaminated soil obtained from a refinery area (Malachowska-Jutsz and Miksh, 2003). Initial concentration of TPHs was 19.11g/kg dry soil while heavy fractions were 159.11g/kg dry soil. Some samples were treated with inoculated with microorganisms obtained from top 15cm layer of the contaminated soil; some were treated with the clover plant only while others were treated with both the plant and microorganisms. After 12 weeks of treatment, the following were observed:

PAHs with two and three rings shows the highest removal rates of 81.96% (clover alone), 89.47% (microorganisms alone), and 90.11% (clover and microorganisms). Concentration of naphthalene decreased from 1.41mg/kg to 0.23mg/kg.

TPHs removal was highest in soils treated with clover alone (62.64%), although the concentration actually increased above the initial in some samples including the

control sample. Also, the concentration of the four and higher rings PAHs were found to increase above the initial concentrations.

The removal of heavy oil fractions was found to be significantly greater in vegetated soil than in unvegetated soil.

All the tested PAHs were detected in the plant roots, with the adsorption of the PAHs with four and five rings being highest.

The increase in the concentration of the higher ring PAHs and TPHs in some of the samples may be due to desorption of compounds from soil by the biosurfactants exuded by the microorganisms which might have dissolved the hydrophobic contaminants from the soil.

Thus, the experiment shows that the presence of vegetation and microorganisms enhances the removal of TPHs, PAHs and heavy fractions from petroleum contaminated soil. This is most likely as a result of exudation of organic substances from plant roots into the rhizosphere, which permits the growth of microbial population.

6.0 CONCLUSIONS

1. The main factors that influence the bioremediation of petroleum contaminated sites are presence of hydrocarbon-degrading microorganisms, moisture, nutrients addition, oxygen, pH and temperature. Hydrocarbon-degrading microorganisms are widely found in nature and have higher concentration in environments polluted by petroleum products.

2. The environment in which the pollution takes place will determine the

bioremediation approach to be taken. For example, the method of bioremediation in a marine environment will be different from that for soil, groundwater, salt marsh, etc.

3. The physical and chemical properties of the petroleum contaminants will also

determine the bioremediation technique to be employed. The approach for pollutants with a high proportion of volatile compounds will be different from that with less volatile compounds. Also, the biodegradability of the compound will also influence the method to be used.

4. The initial concentration of the contaminant in the environmental medium will

also determine the method of choice for bioremediation. Sometimes, in the case of high concentration, other physical, chemical or biological means might first be employed before bioremediation is commenced. Bioremediation processes are often slower than physical or chemical remediation processes, but they have the advantage of actual destruction or degradation of the pollutant as compared to transfer from one medium to another as is often the case in other processes.

5. In situ or ex situ techniques may be employed for the bioremediation process.

Ex situ bioremediation processes require excavation of contaminated soil or pumping of groundwater before they can be treated. In situ techniques may be less expensive, create less dust, cause less release of contaminants than ex situ techniques, and it is possible to treat a large volume of soil at once. In situ techniques, however, may be slower than ex situ techniques and may be difficult to manage.

REFERENCES Alexander, M. 1999. Biodegradation and Bioremediation. 2nd. Ed. Academic Press. N.Y. USA. Chapters 16 and 17. Brebbia. C. A. 2002. Coastal Environment: Environmental Problems in Coastal Regions IV. WIT Press. 139-175 Crocetti C. A., Head C. L., and Riccardelli A. J. Aeration-enhanced bioremediation of oil-contaminated soils: a laboratory treatability study. GZA GeoEnvironmental Inc. www.bioremediationgroup.org/BioReferences/Tier1Papers/Aeration.htm Fingas M. 2002. The Basics of Oil Spill Cleanup. 2nd Edition. Edited by Jennifer Charles. Lewis Publishers. USA. pp 2-51 Headley, J.V. et al. Removal of Heavy oil Sludge Contamination by Composting. In Bioremediation of Contaminated Soils. Ed. Wise, D.L. and others. Marcel Dekker Inc. USA. 2000. Chapter 32. Lecomte P. 1999. Polluted Sites: Remediation of Soils and Groundwater. A.A.Balkema Publishers. USA.134-175 Li Peijun et. al. 2003. 李培军等. 2003.辽河油田石油污染土壤的 2 阶段生物修复. 环境科学.第 24 卷.第 3 期. Loehr R. C. and Webster M. T. 1996. Performance of long-term field bioremediation processes. Journals of Hazardous Materials. 50. 105-128 Malachowska-Jutsz A. and Miksh K. 2003. Accumulation of organic contaminants in plant roots and influence of plant rhizosphere on removal of PAH, TPH and heavy oil fractions from soil. In The Utilization of Bioremediation to Reduce Soil Contamination, problems and Solutions. NATO Science Series: Series IV: Earth and Environmental Science. Vol. 19. Kluwer Academic Publishers. The Netherlands. Namkoong W. et al. 2002. Bioremediation of diesel-contaminated soil with composting. Environmental Pollution. 119. 23-31.

NCNERR. North Carolina National Estuarine Research Reserve (NCNERR). Solving Problems of Oil Pollution in a Salt Marsh. Technical Paper Series: No. 3 www.ncnerr.org Rosenberg E. and Ron E. Z. 1996. Bioremediation of Petroleum Contamination. In Bioremediation: Principles and Applications. Edited by Ronald L. Crawford and Don L. Crawford. Cambrigde University Press. Great Britain. Pgs 100 – 119. Scragg A. 1999. Environmental Biotechnology. Pearson Education Limited. 1999. Chapter 5, pg 105-137 Shen De Zhong. 2002. 沈德中. 2002.污染环境的生物修复. 化学工业出版社. 北京. 268-279 Testa S. M. and Jacobs J. A. 2002. Oil Spills and Leaks. In Handbook of Complex Environmental Remediation Problems. Ed. Lehr et. al. McGraw Hill Handbooks. Chapter 9. USEPA. 1995. In-Situ Groundwater Bioremediation: How to Evaluate Alternative Cleanup Technologies for Underground Storage Tank Sites: A Guide for Corrective Action Plan Reviewers. (EPA 510-B-95-007). http://www.epa.gov/swerust1/pubs/tums.htm. Xu Wei Dong. 1999. 徐卫东. 1999.油气田环境保护. 9 (2): 26-29 Bioremediation for Marine Oil Spills http://www.wws.princeton.edu/cgi-bin/byteserv.prl/~ota/disk1/1991/9109/910904.PDF http://www.wws.princeton.edu/cgi-bin/byteserv.prl/~ota/disk1/1991/9109/910905.PDF