BIODEGRADATION OF BONNYLIGHT CRUDE OIL BY BACTERIA FROM SOIL AND WATER SAMPLES COLLECTED FROM NNPC DEPOT IN ORE TOWN OF ONDO STATE
BIODEGRADATION OF BONNYLIGHT CRUDE OIL BY BACTERIA FROM SOIL AND WATER SAMPLES COLLECTED FROM NNPC DEPOT IN ORE TOWN OF ONDO STATE
CHAPTER ONE
1.1 INTRODUCTION
Petroleum is at present Nigeria’s and indeed the world’s most important derived energy source (Moffat and Linden, 2005). Petroleum in its natural state is referred to as crude oil (Ukoli, 2003). Crude oil is mainly either black or green but it can also be light yellow (Onifade et al., 2007). It varies considerably in density and is described as heavy, average or light (Ojo and Adebusuyi, 1996). Crude oil is one of the most significant pollutants in the environment as it is capable of causing serious damages to humans and the ecosystem (Okpokwasili, 1996).
Crude oil is a complex mixture of thousands of hydrocarbons and non-hydrocarbon compounds including heavy metals. It is the largest and most important source of hydrocarbons (Hunt, 1996). Crude oil varies in appearance and composition from one oil kind to another (Craig, 2003). The varying compositions of one crude oil from the other have diverse effects on different organisms within the same environment (Overton et al., 1994). However, crude oil is not found naturally in every part of the world. It is transported from one place to other for refining. A seemingly inescapable consequence of these transport activities is the accidental spill of the oil into both land and water.
Background to the study: The primary aims of any remediation are reduction of actual or potential environmental threat and reduction of potential risks so that unacceptable risks are reduced to acceptable levels (Atlas, 1995). Consequently, the need for remediation will depend on the degree of actual or potential environmental threat or the level of risk (Ukoli, 2003). It is known that greater degradation of oil pollutants is carried out in situ by a consortium of microorganisms (Okpokwasili, 1996; 2003) and more than 200 species of bacteria, fungi and even algae are capable of degrading hydrocarbons because of their ubiquitous nature. Various genera that have been reported to contain hydrocarbon degrading species include Pseudomonas, Vibrio, Corynebacterium, Arthrobacter, Brevibacterium, Staphylococcus, Bacillus, Thiobacillus, Penicillium, Candida, Fusarium, Aspergillus, Talaromyces and Articulosporium (Snape et al., 2001). These organisms have been isolated in large numbers from many oil polluted waters and soils but are found in less numbers in uncontaminated environments (Okoh, 2003).
Petroleum hydrocarbons are important energy resources used by industry and in our daily life. At the same time, petroleum is a major pollutant of the environment (Mehdi and Giti, 2008). Due to its complicated composition, petroleum has the potential to elicit multiple types of toxic effects. It can cause acute lethal toxicity, sub-lethal chronic toxicity, or both depending on the exposure, dosage, and the organism exposed. Some components of petroleum have the potential to bioaccumulate within susceptible aquatic organisms and can be passed by trophic transfer to other levels of the food chain (Orisakwe et al., 2004). The success of bioremediation technologies applied to hydrocarbon-polluted environments highly depends on the biodegrading capabilities of native microbial populations or exogenous microorganisms used as inoculants (Orisakwe et al, 2004). The presence of microorganisms with the appropriate metabolic capabilities is the most important requirement for oil spill bioremediation (Leahy and Colwell, 1990). The communities which were exposed to hydrocarbons become adapted, exhibiting selective enrichment and genetic changes (Atlas and Bartha, 1998). The adapted microbial communities can respond to the presence of hydrocarbon pollutants within hours and exhibit higher biodegradation rates than communities with no history of hydrocarbon contamination (Leahy and Colwell, 1990). So, the ability to isolate high numbers of certain oil degrading microorganisms from an environment is commonly taken as evidence that those microorganisms are the most active oil degraders of that environment (Atlas and Bartha, 1998) and can be used in the bioremediation of petroleum oil polluted sites. Since crude oil is made of a mixture of compounds, and since individual microorganisms metabolize only a limited range of hydrocarbon substrates, biodegradation of crude oil requires mixture of different bacterial groups or consortia functioning to degrade a wider range of hydrocarbons (Bordenave et al, 2007).
Pollution is an undesirable change in the physical, chemical and biological characteristics of all the components of an environment (Aboriba, 2001). The greatest single environmental problem connected with crude oil exploration in Nigeria is oil spillage at both onshore and offshore. The rate of oil spillage reported in the country has been rising with a corresponding increase in petroleum production (Onifade et al., 2007). Bacteria and fungi make the major contribution to mineralization of oil pollutants (Abed et al., 2001). Bacteria most commonly encountered are the Gram-negative of the alpha-proteobacteria group such as species of Pseudomonas, Sphingomonas, Moraxella, Acinetobacter, Alcaligenes, and Proteus. Also important are the low G+C Gram-positives such as Bacillus, Micrococcus and the high G+C Gram-positives, particularly the Actinomycetes (Amund, 2000; Wackett and Hershberger, 2001; Parales et al., 2003). Pseudomonas species are often isolated from hydrocarbon contaminated sites and hydrocarbon degrading cultures. Members of this genus have broad affinity for hydrocarbon and can degrade selected alkanes, alycyclics, thiophenes and aromatics (Vankateswaran et al., 1995; Allen et al., 1997). Polycyclic aromatic hydrocarbons (PAHs) are among the most recalcitrant components of crude oil (Kanaly and Harayama, 2000).
Microbial degradation of crude oil often occur by attack on alkanes or light aromatic fractions, while the high molecular weight aromatics, resins and asphalthenes are considered recalcitrant (Lal and Khanna, 1996). Low molecular weight PAHs such as naphthalene and phenanthrene are degraded rapidly in sediments, whereas higher molecular weight PAHs such as benzo[a]anthracene, pyrene, chrysene, benzo[a]pyrene are quite resistant to microbial attack (Cerniglia, 1992). It is uncommon to find organisms which could degrade both aliphatics and aromatics effectively. However, such findings portend well for the future of bioremediation. The difficulty may not be unconnected with the difference in the pathways for biodegradation of the two classes of hydrocarbons. But long periods of exposure to mixture of hydrocarbon and preponderance of enabling intrinsic and extrinsic factors could lead to acquisition of such rare ability.
Bacterial species with broad substrate specificity for both alkanes and polycyclic aromatic hydrocarbons have been reported by Amund et al. (1987). It is fair guess, therefore, that given the ubiquitous nature of hydrocarbon degraders and the reckless pollution of the environment with oils and PAHs, organisms capable of degrading wide range of hydrocarbons could abound and only need to be sought out and properly identified and characterized.
Objective of the study: This present investigation was conducted to determine the biodegradation of bonny light crude oil by bacteria from Ore, Ondo State.
1.2 LITERATURE REVIEW
1.2.1 Petroleum
Petroleum (L. petroleum, from early 15c. "petroleum, rock oil" (mid-14c. in Anglo-French), from Medieval Latin petroleum, from Latin: petra "rock" + oleum "oil" is a naturally occurring, yellow-to-black liquid found in geological formations beneath the Earth's surface, which is commonly refined into various types of fuels (Concise Oxford English Dictionary).
It consists of hydrocarbons of various molecular weights and other organic compounds (Guerriero et al., 2011).The name petroleum covers both naturally occurring unprocessed crude oil and petroleum products that are made up of refined crude oil. A fossil fuel, petroleum is formed when large quantities of dead organisms, usually zooplankton and algae, are buried underneath sedimentary rock and subjected to intense heat and pressure.
Petroleum is recovered mostly through oil drilling (natural petroleum springs are rare). This comes after the studies of structural geology (at the reservoir scale), sedimentary basin analysis, and reservoir characterization (mainly in terms of the porosity and permeability of geologic reservoir structures). It is refined and separated, most easily by distillation, into a large number of consumer products, from gasoline (petrol) and kerosene to asphalt and chemical reagents used to make plastics and pharmaceuticals. Petroleum is used in manufacturing a wide variety of materials, and it is estimated that the world consumes about 90 million barrels each day (Guerriero et al., 2011).
Concern over the depletion of the earth's finite reserves of oil, and the effect this would have on a society dependent on it, is a concept known as peak oil. The use of fossil fuels, such as petroleum, has a negative impact on Earth's biosphere, damaging ecosystems through events such as oil spills and releasing a range of pollutants into the air including ground-level ozone and sulfur dioxide from sulfur impurities in fossil fuels.
1.2.2 Crude Oil Composition and Its Classification
In its strictest sense, petroleum includes only crude oil, but in common usage it includes all liquid, gaseous, and solid hydrocarbons. Under surface pressure and temperature conditions, lighter hydrocarbons methane, ethane, propane and butane occur as gases, while pentane and heavier ones are in the form of liquids or solids. However, in an underground oil reservoir the proportions of gas, liquid, and solid depend on subsurface conditions and on the phase diagram of the petroleum mixture (Hyne, 2001).
An oil well produces predominantly crude oil, with some natural gas dissolved in it. Because the pressure is lower at the surface than underground, some of the gas will come out of solution and be recovered (or burned) as associated gas or solution gas. A gas well produces predominantly natural gas. However, because the underground temperature and pressure are higher than at the surface, the gas may contain heavier hydrocarbons such as pentane, hexane, and heptane in the gaseous state. At surface conditions these will condense out of the gas to form natural gas condensate, often shortened to condensate. Condensate resembles petrol in appearance and is similar in composition to some volatile light crude oils.
The proportion of light hydrocarbons in the petroleum mixture varies greatly among different oil fields, ranging from as much as 97 percent by weight in the lighter oils to as little as 50 percent in the heavier oils and bitumen. The hydrocarbons in crude oil are mostly alkanes, cycloalkanes and various aromatic hydrocarbons while the other organic compounds contain nitrogen, oxygen and sulfur, and trace amounts of metals such as iron, nickel, copper and vanadium. Many oil reservoirs contain live bacteria (Ollivier and Magot, 2005). The exact molecular composition varies widely from formation to formation but the proportion of chemical elements varies over fairly narrow limits as follows: carbon (83-85%), hydrogen (10-14%), nitrogen (0.1 – 2%), oxygen (0.05 – 1.5%), sulfur (0.05-6.0%) and metals (<0.1%) (Speight, 1999).
Four different types of hydrocarbon molecules appear in crude oil. The relative percentage of each varies from oil to oil, determining the properties of each oil. These are alkanes (30%), naphthenes (49%), aromatics (15%) and asphaltics (6%) (Hyne, 2001).
Crude oil varies greatly in appearance depending on its composition. It is usually black or dark brown (although it may be yellowish, reddish, or even greenish). In the reservoir it is usually found in association with natural gas, which being lighter forms a gas cap over the petroleum, and saline water which, being heavier than most forms of crude oil, generally sinks beneath it. Crude oil may also be found in semi-solid form mixed with sand and water, as in the Athabasca oil sands, where it is usually referred to as crude bitumen (Speight, 1999).
Bonny Light Crude
Bonny Light oil is a high grade of Nigerian crude oil with high API gravity (low specific gravity), produced in the Niger Delta basin and named after the prolific region around the city of Bonny. The very low sulfur content of Bonny Light crude makes it a highly desired grade for its low corrosiveness to refinery infrastructure and the lower environmental impact of its byproducts in refinery effluent.
1.2.3 Environmental Pollution due to Oil Spillage in Nigeria
The economic life of the people in the affected area is usually adversely affected, such as farm lands, navigation activities and fishing efforts as well as the disruption of the ecobalance in the affected ecosystem (Dibble, 1979). Oil spillage is a release of a liquid petroleum hydrocarbon into the environment due to human activity, and is a form of pollution. The term often refers to marine oil spills, where oil is released into the ocean or coastal waters. Oil spills include releases of crude oil from tankers, offshore platforms, drilling rigs and wells, as well as spills of refined petroleum products (such as gasoline, diesel) and their by-products, and heavier fuels used by large ships such as bunker fuel, or the spill of any oily white substance refuse or waste oil. Spills may take months or even years to clean up. Oil also enters the marine environment from natural oil seeps. Public attention and regulation has tended to focus most sharply on seagoing oil tankers (Morgan and Watkinson, 1993).
Oil spills may occur for numerous reasons such as equipment failure, disasters, deliberate acts, or human error (Anderson and LaBelle, 2000). Crude oils are exclusively natural products, most of which are produced from artificial wells. Natural seepage of crude oils occurs in various parts of the world, not only on land, but also on the seabed. Seeps emerge through fractures in the crests of folds in rock formations beneath the sea floor that contain oil and gas deposits. Oil and gas tend to rise and become trapped in anticlinal folds in sub -sea rock strata. Seepage occurs through fracture zones where the folds are truncated at the sea floor. Seeps may emanate from a single point or as many as 3 x 104 individual seepage signals may be merged onto a high resolution profile record (Clark et al., 2000).
1.2.4 Causes of oil spillage in Nigeria
Oil spills are a common event in Nigeria and occur due to a number of causes, including: corrosion of pipelines and tankers (accounting for 50% of all spills), sabotage (36%), and oil production operations (6.5%), with 1% of the spills being accounted for by inadequate or non-functional production equipment. The largest contributor to the oil spill total, corrosion of pipes and tanks, is the rupturing or leaking of production infrastructures that are described as, "very old and lack regular inspection and maintenance" (Adelana et al., 2011).
A reason that corrosion accounts for such a high percentage of all spills is that as a result of the small size of the oilfields in the Niger Delta, there is an extensive network of pipelines between the fields, as well as numerous small networks of flow lines—the narrow diameter pipes that carry oil from wellheads to flow stations—allowing many opportunities for leaks. In onshore areas most pipelines and flow lines are laid above ground. Pipelines, which have an estimate life span of about fifteen years, are old and susceptible to corrosion. Many of the pipelines are as old as twenty to twenty-five years. Even Shell admits that "most of the facilities were constructed between the 1960s and early 1980s to the then prevailing standards. SPDC [Shell Petroleum and Development Company] would not build them that way today.” Sabotage is performed primarily through what is known as "bunkering", whereby the saboteur attempts to tap the pipeline. In the process of extraction sometimes the pipeline is damaged or destroyed. Oil extracted in this manner can often be sold (Adelana et al., 2011).
Sabotage and theft through oil siphoning has become a major issue in the Niger River Delta states as well, contributing to further environmental degradation. Damaged lines may go unnoticed for days, and repair of the damaged pipes takes even longer. Oil siphoning has become a big business, with the stolen oil quickly making its way onto the black market (Adelana et al., 2011).
Consequences of oil spillage in Nigeria: Oil spillage has a major impact on the ecosystem into which it is released. Immense tracts of the mangrove forests, which are especially susceptible to oil, have been destroyed. An estimated 5 to 10% of Nigerian mangrove ecosystems have been wiped out either by settlement or oil. The rainforest which previously occupied some 7,400 km² of land has disappeared as well (Adelana et al., 2011).
Spills in populated areas often spread out over a wide area, destroying crops and aquacultures through contamination of the groundwater and soils. The consumption of dissolved oxygen by bacteria feeding on the spilled hydrocarbons also contributes to the death of fish. In agricultural communities, often a year's supply of food can be destroyed instantaneously. Because of the careless nature of oil operations in the Delta, the environment is growing increasingly uninhabitable (Adelana et al., 2011).
People in the affected areas complain about health issues including breathing problems and skin lesions; many have lost basic human rights such as health, access to food, clean water, and an ability to work.
Loss of mangrove forests: Vegetation in the Niger River Delta consists of extensive mangrove forests, brackish swamp forests, and rainforests. The large expanses of mangrove forests are estimated to cover approximately 5,000 to 8,580 km² of land. Mangroves remain very important to the indigenous people of Nigeria as well as to the various organisms that inhabit these ecosystems.
Human impact from poor land management upstream coupled with the constant pollution of petroleum has caused five to ten percent of these mangrove forests to disappear. The volatile, quickly penetrating, and viscous properties of petroleum have wiped out large areas of vegetation. When spills occur close to and within the drainage basin, the hydrologic force of both the river and tides force spilled petroleum to move up into areas of vegetation.
Mangrove forests are included in a highly complex trophic system. If oil directly affects any organism within an ecosystem, it can indirectly affect a host of other organisms. These floral communities rely on nutrient cycling, clean water, sunlight, and proper substrates. With ideal conditions they offer habitat structure, and input of energy via photosynthesis to the organisms they interact with. The effects of petroleum spills on mangroves are known to acidify the soils, halt cellular respiration, and starve roots of vital oxygen (Adelana et al., 2011).
An area of mangroves that has been destroyed by petroleum may be susceptible to other problems. These areas may not be suitable for any native plant growth until bacteria and microorganisms can remediate the conditions. A particular species of mangrove, Rhizophora racemosa lives higher in the delta system. As the soils supporting R. racemosa become too toxic, a non-native invasive species of palm, Nypa fruticans, quickly colonizes the area. This invasive species has a shallower root system that destabilizes the banks along the waterways, further impacting sediment distribution lower in the delta system. N. fruticans also impedes navigation and decreases overall biodiversity. In places where N. fruticans has invaded, communities are investigating how the palm can be used by local people (Adelana et al., 2011).
The loss of mangrove forests is not only degrading life for plants and animals, but for humans as well. These systems are highly valued by the indigenous people living in the affected areas. Mangrove forests have been a major source of wood for local people. They also are important to a variety of species vital to subsistence practices for local indigenous groups, who unfortunately see little to none of the economic benefits of petroleum. Mangroves also provide essential habitat for rare and endangered species like the manatee and pygmy hippopotamus. Poor policy decisions regarding the allocation of petroleum revenue has caused political unrest in Nigeria. This clash among governing bodies, oil corporations, and the people of Nigeria has resulted in sabotage to petroleum pipelines, further exacerbating the threat to mangrove forests (Adelana et al., 2011).
The future for mangrove forests and other floral communities is not all negative. Local and outside groups have provided funds and labor to remediate and restore the destroyed mangrove swamps. The federal government of Nigeria established the Niger Delta Development Commission (NDDC) in 2000 which aims to suppress the environmental and ecological impacts petroleum has had in the region. Governmental and nongovernmental organizations have also utilized technology to identify the source and movement of petroleum spill.
Depletion of fish populations: The fishing industry is an essential part of Nigeria’s sustainability because it provides much needed protein and nutrients for people, but with the higher demand on fishing, fish populations are declining as they are being depleted faster than they are able to restore their number. Fishing needs to be limited along the Niger River and aquacultures should be created to provide for the growing demand on the fishing industry. Aquaculture allows for fish to be farmed for production and provide more jobs for the local people of Nigeria (Leahy and Colwell, 1990).
Overfishing is not the only impact on marine communities. Climate change, habitat loss, and pollution are all added pressures to these important ecosystems. The banks of the Niger River are desirable and ideal locations for people to settle. The river provides water for drinking, bathing, cleaning, and fishing for both the dinner table and trading to make a profit. As the people have settled along the shores of the rivers and coasts, marine and terrestrial habitats are being lost and ecosystems are being drastically changed. The shoreline along the Niger River is important in maintaining the temperature of the water because the slightest change in water temperature can be fatal to certain marine species. Trees and shrubs provide shade and habitat for marine species, while reducing fluctuation in water temperature (Leahy and Colwell, 1990).
The Niger River is an important ecosystem that needs to be protected, for it is home to 36 families and nearly 250 species of fish, of which 20 are endemic, meaning they are found nowhere else on Earth. With the loss of habitat and the climate getting warmer, prevention of temperature increase is necessary to maintain some of the marine environments. Other than restoring habitat, pollution can also be reduced. Problems such as pesticides from agricultural fields could be reduced if a natural pesticide was used, or the fields were moved farther away from the local waterways (Amund, 2000; Okoh, 2003).
Oil pollution can be lowered as well; if spills were reduced then habitat and environmental impacts could be minimized. By limiting the devastation caused by disturbances to the marine environment, such as pollution, overfishing, and habitat loss, the productivity and biodiversity of the marine ecosystems would increase (Amund, 2000; Okoh, 2003).
1.2.5 Oil Pollution and Some Cases of Oil Spillage in Nigeria
The Department of Petroleum Resources estimated 1.89 million barrels of petroleum were spilled into the Niger Delta between 1976 and 1996 out of a total of 2.4 million barrels spilled in 4,835 incidents (approximately 220 thousand cubic metres) (Adelana et al., 2011).
The Nigerian National Petroleum Corporation places the quantity of petroleum jettisoned into the environment yearly at 2,300 cubic metres with an average of 300 individual spills annually. However, because this amount does not take into account "minor" spills, the World Bank argues that the true quantity of petroleum spilled into the environment could be as much as ten times the officially claimed amount. The largest individual spills include the blowout of a Texaco offshore station which in 1980 dumped an estimated 400,000 barrels (64,000 m3) of crude oil into the Gulf of Guinea and Royal Dutch Shell's Forcados Terminal tank failure which produced a spillage estimated at 580,000 barrels (92,000 m3). Baird (2010) reported that between 9 million and 13 million barrels have been spilled in the Niger Delta since 1958.
CHAPTER ONE
1.1 INTRODUCTION
Petroleum is at present Nigeria’s and indeed the world’s most important derived energy source (Moffat and Linden, 2005). Petroleum in its natural state is referred to as crude oil (Ukoli, 2003). Crude oil is mainly either black or green but it can also be light yellow (Onifade et al., 2007). It varies considerably in density and is described as heavy, average or light (Ojo and Adebusuyi, 1996). Crude oil is one of the most significant pollutants in the environment as it is capable of causing serious damages to humans and the ecosystem (Okpokwasili, 1996).
Crude oil is a complex mixture of thousands of hydrocarbons and non-hydrocarbon compounds including heavy metals. It is the largest and most important source of hydrocarbons (Hunt, 1996). Crude oil varies in appearance and composition from one oil kind to another (Craig, 2003). The varying compositions of one crude oil from the other have diverse effects on different organisms within the same environment (Overton et al., 1994). However, crude oil is not found naturally in every part of the world. It is transported from one place to other for refining. A seemingly inescapable consequence of these transport activities is the accidental spill of the oil into both land and water.
Background to the study: The primary aims of any remediation are reduction of actual or potential environmental threat and reduction of potential risks so that unacceptable risks are reduced to acceptable levels (Atlas, 1995). Consequently, the need for remediation will depend on the degree of actual or potential environmental threat or the level of risk (Ukoli, 2003). It is known that greater degradation of oil pollutants is carried out in situ by a consortium of microorganisms (Okpokwasili, 1996; 2003) and more than 200 species of bacteria, fungi and even algae are capable of degrading hydrocarbons because of their ubiquitous nature. Various genera that have been reported to contain hydrocarbon degrading species include Pseudomonas, Vibrio, Corynebacterium, Arthrobacter, Brevibacterium, Staphylococcus, Bacillus, Thiobacillus, Penicillium, Candida, Fusarium, Aspergillus, Talaromyces and Articulosporium (Snape et al., 2001). These organisms have been isolated in large numbers from many oil polluted waters and soils but are found in less numbers in uncontaminated environments (Okoh, 2003).
Petroleum hydrocarbons are important energy resources used by industry and in our daily life. At the same time, petroleum is a major pollutant of the environment (Mehdi and Giti, 2008). Due to its complicated composition, petroleum has the potential to elicit multiple types of toxic effects. It can cause acute lethal toxicity, sub-lethal chronic toxicity, or both depending on the exposure, dosage, and the organism exposed. Some components of petroleum have the potential to bioaccumulate within susceptible aquatic organisms and can be passed by trophic transfer to other levels of the food chain (Orisakwe et al., 2004). The success of bioremediation technologies applied to hydrocarbon-polluted environments highly depends on the biodegrading capabilities of native microbial populations or exogenous microorganisms used as inoculants (Orisakwe et al, 2004). The presence of microorganisms with the appropriate metabolic capabilities is the most important requirement for oil spill bioremediation (Leahy and Colwell, 1990). The communities which were exposed to hydrocarbons become adapted, exhibiting selective enrichment and genetic changes (Atlas and Bartha, 1998). The adapted microbial communities can respond to the presence of hydrocarbon pollutants within hours and exhibit higher biodegradation rates than communities with no history of hydrocarbon contamination (Leahy and Colwell, 1990). So, the ability to isolate high numbers of certain oil degrading microorganisms from an environment is commonly taken as evidence that those microorganisms are the most active oil degraders of that environment (Atlas and Bartha, 1998) and can be used in the bioremediation of petroleum oil polluted sites. Since crude oil is made of a mixture of compounds, and since individual microorganisms metabolize only a limited range of hydrocarbon substrates, biodegradation of crude oil requires mixture of different bacterial groups or consortia functioning to degrade a wider range of hydrocarbons (Bordenave et al, 2007).
Pollution is an undesirable change in the physical, chemical and biological characteristics of all the components of an environment (Aboriba, 2001). The greatest single environmental problem connected with crude oil exploration in Nigeria is oil spillage at both onshore and offshore. The rate of oil spillage reported in the country has been rising with a corresponding increase in petroleum production (Onifade et al., 2007). Bacteria and fungi make the major contribution to mineralization of oil pollutants (Abed et al., 2001). Bacteria most commonly encountered are the Gram-negative of the alpha-proteobacteria group such as species of Pseudomonas, Sphingomonas, Moraxella, Acinetobacter, Alcaligenes, and Proteus. Also important are the low G+C Gram-positives such as Bacillus, Micrococcus and the high G+C Gram-positives, particularly the Actinomycetes (Amund, 2000; Wackett and Hershberger, 2001; Parales et al., 2003). Pseudomonas species are often isolated from hydrocarbon contaminated sites and hydrocarbon degrading cultures. Members of this genus have broad affinity for hydrocarbon and can degrade selected alkanes, alycyclics, thiophenes and aromatics (Vankateswaran et al., 1995; Allen et al., 1997). Polycyclic aromatic hydrocarbons (PAHs) are among the most recalcitrant components of crude oil (Kanaly and Harayama, 2000).
Microbial degradation of crude oil often occur by attack on alkanes or light aromatic fractions, while the high molecular weight aromatics, resins and asphalthenes are considered recalcitrant (Lal and Khanna, 1996). Low molecular weight PAHs such as naphthalene and phenanthrene are degraded rapidly in sediments, whereas higher molecular weight PAHs such as benzo[a]anthracene, pyrene, chrysene, benzo[a]pyrene are quite resistant to microbial attack (Cerniglia, 1992). It is uncommon to find organisms which could degrade both aliphatics and aromatics effectively. However, such findings portend well for the future of bioremediation. The difficulty may not be unconnected with the difference in the pathways for biodegradation of the two classes of hydrocarbons. But long periods of exposure to mixture of hydrocarbon and preponderance of enabling intrinsic and extrinsic factors could lead to acquisition of such rare ability.
Bacterial species with broad substrate specificity for both alkanes and polycyclic aromatic hydrocarbons have been reported by Amund et al. (1987). It is fair guess, therefore, that given the ubiquitous nature of hydrocarbon degraders and the reckless pollution of the environment with oils and PAHs, organisms capable of degrading wide range of hydrocarbons could abound and only need to be sought out and properly identified and characterized.
Objective of the study: This present investigation was conducted to determine the biodegradation of bonny light crude oil by bacteria from Ore, Ondo State.
1.2 LITERATURE REVIEW
1.2.1 Petroleum
Petroleum (L. petroleum, from early 15c. "petroleum, rock oil" (mid-14c. in Anglo-French), from Medieval Latin petroleum, from Latin: petra "rock" + oleum "oil" is a naturally occurring, yellow-to-black liquid found in geological formations beneath the Earth's surface, which is commonly refined into various types of fuels (Concise Oxford English Dictionary).
It consists of hydrocarbons of various molecular weights and other organic compounds (Guerriero et al., 2011).The name petroleum covers both naturally occurring unprocessed crude oil and petroleum products that are made up of refined crude oil. A fossil fuel, petroleum is formed when large quantities of dead organisms, usually zooplankton and algae, are buried underneath sedimentary rock and subjected to intense heat and pressure.
Petroleum is recovered mostly through oil drilling (natural petroleum springs are rare). This comes after the studies of structural geology (at the reservoir scale), sedimentary basin analysis, and reservoir characterization (mainly in terms of the porosity and permeability of geologic reservoir structures). It is refined and separated, most easily by distillation, into a large number of consumer products, from gasoline (petrol) and kerosene to asphalt and chemical reagents used to make plastics and pharmaceuticals. Petroleum is used in manufacturing a wide variety of materials, and it is estimated that the world consumes about 90 million barrels each day (Guerriero et al., 2011).
Concern over the depletion of the earth's finite reserves of oil, and the effect this would have on a society dependent on it, is a concept known as peak oil. The use of fossil fuels, such as petroleum, has a negative impact on Earth's biosphere, damaging ecosystems through events such as oil spills and releasing a range of pollutants into the air including ground-level ozone and sulfur dioxide from sulfur impurities in fossil fuels.
1.2.2 Crude Oil Composition and Its Classification
In its strictest sense, petroleum includes only crude oil, but in common usage it includes all liquid, gaseous, and solid hydrocarbons. Under surface pressure and temperature conditions, lighter hydrocarbons methane, ethane, propane and butane occur as gases, while pentane and heavier ones are in the form of liquids or solids. However, in an underground oil reservoir the proportions of gas, liquid, and solid depend on subsurface conditions and on the phase diagram of the petroleum mixture (Hyne, 2001).
An oil well produces predominantly crude oil, with some natural gas dissolved in it. Because the pressure is lower at the surface than underground, some of the gas will come out of solution and be recovered (or burned) as associated gas or solution gas. A gas well produces predominantly natural gas. However, because the underground temperature and pressure are higher than at the surface, the gas may contain heavier hydrocarbons such as pentane, hexane, and heptane in the gaseous state. At surface conditions these will condense out of the gas to form natural gas condensate, often shortened to condensate. Condensate resembles petrol in appearance and is similar in composition to some volatile light crude oils.
The proportion of light hydrocarbons in the petroleum mixture varies greatly among different oil fields, ranging from as much as 97 percent by weight in the lighter oils to as little as 50 percent in the heavier oils and bitumen. The hydrocarbons in crude oil are mostly alkanes, cycloalkanes and various aromatic hydrocarbons while the other organic compounds contain nitrogen, oxygen and sulfur, and trace amounts of metals such as iron, nickel, copper and vanadium. Many oil reservoirs contain live bacteria (Ollivier and Magot, 2005). The exact molecular composition varies widely from formation to formation but the proportion of chemical elements varies over fairly narrow limits as follows: carbon (83-85%), hydrogen (10-14%), nitrogen (0.1 – 2%), oxygen (0.05 – 1.5%), sulfur (0.05-6.0%) and metals (<0.1%) (Speight, 1999).
Four different types of hydrocarbon molecules appear in crude oil. The relative percentage of each varies from oil to oil, determining the properties of each oil. These are alkanes (30%), naphthenes (49%), aromatics (15%) and asphaltics (6%) (Hyne, 2001).
Crude oil varies greatly in appearance depending on its composition. It is usually black or dark brown (although it may be yellowish, reddish, or even greenish). In the reservoir it is usually found in association with natural gas, which being lighter forms a gas cap over the petroleum, and saline water which, being heavier than most forms of crude oil, generally sinks beneath it. Crude oil may also be found in semi-solid form mixed with sand and water, as in the Athabasca oil sands, where it is usually referred to as crude bitumen (Speight, 1999).
Bonny Light Crude
Bonny Light oil is a high grade of Nigerian crude oil with high API gravity (low specific gravity), produced in the Niger Delta basin and named after the prolific region around the city of Bonny. The very low sulfur content of Bonny Light crude makes it a highly desired grade for its low corrosiveness to refinery infrastructure and the lower environmental impact of its byproducts in refinery effluent.
1.2.3 Environmental Pollution due to Oil Spillage in Nigeria
The economic life of the people in the affected area is usually adversely affected, such as farm lands, navigation activities and fishing efforts as well as the disruption of the ecobalance in the affected ecosystem (Dibble, 1979). Oil spillage is a release of a liquid petroleum hydrocarbon into the environment due to human activity, and is a form of pollution. The term often refers to marine oil spills, where oil is released into the ocean or coastal waters. Oil spills include releases of crude oil from tankers, offshore platforms, drilling rigs and wells, as well as spills of refined petroleum products (such as gasoline, diesel) and their by-products, and heavier fuels used by large ships such as bunker fuel, or the spill of any oily white substance refuse or waste oil. Spills may take months or even years to clean up. Oil also enters the marine environment from natural oil seeps. Public attention and regulation has tended to focus most sharply on seagoing oil tankers (Morgan and Watkinson, 1993).
Oil spills may occur for numerous reasons such as equipment failure, disasters, deliberate acts, or human error (Anderson and LaBelle, 2000). Crude oils are exclusively natural products, most of which are produced from artificial wells. Natural seepage of crude oils occurs in various parts of the world, not only on land, but also on the seabed. Seeps emerge through fractures in the crests of folds in rock formations beneath the sea floor that contain oil and gas deposits. Oil and gas tend to rise and become trapped in anticlinal folds in sub -sea rock strata. Seepage occurs through fracture zones where the folds are truncated at the sea floor. Seeps may emanate from a single point or as many as 3 x 104 individual seepage signals may be merged onto a high resolution profile record (Clark et al., 2000).
1.2.4 Causes of oil spillage in Nigeria
Oil spills are a common event in Nigeria and occur due to a number of causes, including: corrosion of pipelines and tankers (accounting for 50% of all spills), sabotage (36%), and oil production operations (6.5%), with 1% of the spills being accounted for by inadequate or non-functional production equipment. The largest contributor to the oil spill total, corrosion of pipes and tanks, is the rupturing or leaking of production infrastructures that are described as, "very old and lack regular inspection and maintenance" (Adelana et al., 2011).
A reason that corrosion accounts for such a high percentage of all spills is that as a result of the small size of the oilfields in the Niger Delta, there is an extensive network of pipelines between the fields, as well as numerous small networks of flow lines—the narrow diameter pipes that carry oil from wellheads to flow stations—allowing many opportunities for leaks. In onshore areas most pipelines and flow lines are laid above ground. Pipelines, which have an estimate life span of about fifteen years, are old and susceptible to corrosion. Many of the pipelines are as old as twenty to twenty-five years. Even Shell admits that "most of the facilities were constructed between the 1960s and early 1980s to the then prevailing standards. SPDC [Shell Petroleum and Development Company] would not build them that way today.” Sabotage is performed primarily through what is known as "bunkering", whereby the saboteur attempts to tap the pipeline. In the process of extraction sometimes the pipeline is damaged or destroyed. Oil extracted in this manner can often be sold (Adelana et al., 2011).
Sabotage and theft through oil siphoning has become a major issue in the Niger River Delta states as well, contributing to further environmental degradation. Damaged lines may go unnoticed for days, and repair of the damaged pipes takes even longer. Oil siphoning has become a big business, with the stolen oil quickly making its way onto the black market (Adelana et al., 2011).
Consequences of oil spillage in Nigeria: Oil spillage has a major impact on the ecosystem into which it is released. Immense tracts of the mangrove forests, which are especially susceptible to oil, have been destroyed. An estimated 5 to 10% of Nigerian mangrove ecosystems have been wiped out either by settlement or oil. The rainforest which previously occupied some 7,400 km² of land has disappeared as well (Adelana et al., 2011).
Spills in populated areas often spread out over a wide area, destroying crops and aquacultures through contamination of the groundwater and soils. The consumption of dissolved oxygen by bacteria feeding on the spilled hydrocarbons also contributes to the death of fish. In agricultural communities, often a year's supply of food can be destroyed instantaneously. Because of the careless nature of oil operations in the Delta, the environment is growing increasingly uninhabitable (Adelana et al., 2011).
People in the affected areas complain about health issues including breathing problems and skin lesions; many have lost basic human rights such as health, access to food, clean water, and an ability to work.
Loss of mangrove forests: Vegetation in the Niger River Delta consists of extensive mangrove forests, brackish swamp forests, and rainforests. The large expanses of mangrove forests are estimated to cover approximately 5,000 to 8,580 km² of land. Mangroves remain very important to the indigenous people of Nigeria as well as to the various organisms that inhabit these ecosystems.
Human impact from poor land management upstream coupled with the constant pollution of petroleum has caused five to ten percent of these mangrove forests to disappear. The volatile, quickly penetrating, and viscous properties of petroleum have wiped out large areas of vegetation. When spills occur close to and within the drainage basin, the hydrologic force of both the river and tides force spilled petroleum to move up into areas of vegetation.
Mangrove forests are included in a highly complex trophic system. If oil directly affects any organism within an ecosystem, it can indirectly affect a host of other organisms. These floral communities rely on nutrient cycling, clean water, sunlight, and proper substrates. With ideal conditions they offer habitat structure, and input of energy via photosynthesis to the organisms they interact with. The effects of petroleum spills on mangroves are known to acidify the soils, halt cellular respiration, and starve roots of vital oxygen (Adelana et al., 2011).
An area of mangroves that has been destroyed by petroleum may be susceptible to other problems. These areas may not be suitable for any native plant growth until bacteria and microorganisms can remediate the conditions. A particular species of mangrove, Rhizophora racemosa lives higher in the delta system. As the soils supporting R. racemosa become too toxic, a non-native invasive species of palm, Nypa fruticans, quickly colonizes the area. This invasive species has a shallower root system that destabilizes the banks along the waterways, further impacting sediment distribution lower in the delta system. N. fruticans also impedes navigation and decreases overall biodiversity. In places where N. fruticans has invaded, communities are investigating how the palm can be used by local people (Adelana et al., 2011).
The loss of mangrove forests is not only degrading life for plants and animals, but for humans as well. These systems are highly valued by the indigenous people living in the affected areas. Mangrove forests have been a major source of wood for local people. They also are important to a variety of species vital to subsistence practices for local indigenous groups, who unfortunately see little to none of the economic benefits of petroleum. Mangroves also provide essential habitat for rare and endangered species like the manatee and pygmy hippopotamus. Poor policy decisions regarding the allocation of petroleum revenue has caused political unrest in Nigeria. This clash among governing bodies, oil corporations, and the people of Nigeria has resulted in sabotage to petroleum pipelines, further exacerbating the threat to mangrove forests (Adelana et al., 2011).
The future for mangrove forests and other floral communities is not all negative. Local and outside groups have provided funds and labor to remediate and restore the destroyed mangrove swamps. The federal government of Nigeria established the Niger Delta Development Commission (NDDC) in 2000 which aims to suppress the environmental and ecological impacts petroleum has had in the region. Governmental and nongovernmental organizations have also utilized technology to identify the source and movement of petroleum spill.
Depletion of fish populations: The fishing industry is an essential part of Nigeria’s sustainability because it provides much needed protein and nutrients for people, but with the higher demand on fishing, fish populations are declining as they are being depleted faster than they are able to restore their number. Fishing needs to be limited along the Niger River and aquacultures should be created to provide for the growing demand on the fishing industry. Aquaculture allows for fish to be farmed for production and provide more jobs for the local people of Nigeria (Leahy and Colwell, 1990).
Overfishing is not the only impact on marine communities. Climate change, habitat loss, and pollution are all added pressures to these important ecosystems. The banks of the Niger River are desirable and ideal locations for people to settle. The river provides water for drinking, bathing, cleaning, and fishing for both the dinner table and trading to make a profit. As the people have settled along the shores of the rivers and coasts, marine and terrestrial habitats are being lost and ecosystems are being drastically changed. The shoreline along the Niger River is important in maintaining the temperature of the water because the slightest change in water temperature can be fatal to certain marine species. Trees and shrubs provide shade and habitat for marine species, while reducing fluctuation in water temperature (Leahy and Colwell, 1990).
The Niger River is an important ecosystem that needs to be protected, for it is home to 36 families and nearly 250 species of fish, of which 20 are endemic, meaning they are found nowhere else on Earth. With the loss of habitat and the climate getting warmer, prevention of temperature increase is necessary to maintain some of the marine environments. Other than restoring habitat, pollution can also be reduced. Problems such as pesticides from agricultural fields could be reduced if a natural pesticide was used, or the fields were moved farther away from the local waterways (Amund, 2000; Okoh, 2003).
Oil pollution can be lowered as well; if spills were reduced then habitat and environmental impacts could be minimized. By limiting the devastation caused by disturbances to the marine environment, such as pollution, overfishing, and habitat loss, the productivity and biodiversity of the marine ecosystems would increase (Amund, 2000; Okoh, 2003).
1.2.5 Oil Pollution and Some Cases of Oil Spillage in Nigeria
The Department of Petroleum Resources estimated 1.89 million barrels of petroleum were spilled into the Niger Delta between 1976 and 1996 out of a total of 2.4 million barrels spilled in 4,835 incidents (approximately 220 thousand cubic metres) (Adelana et al., 2011).
The Nigerian National Petroleum Corporation places the quantity of petroleum jettisoned into the environment yearly at 2,300 cubic metres with an average of 300 individual spills annually. However, because this amount does not take into account "minor" spills, the World Bank argues that the true quantity of petroleum spilled into the environment could be as much as ten times the officially claimed amount. The largest individual spills include the blowout of a Texaco offshore station which in 1980 dumped an estimated 400,000 barrels (64,000 m3) of crude oil into the Gulf of Guinea and Royal Dutch Shell's Forcados Terminal tank failure which produced a spillage estimated at 580,000 barrels (92,000 m3). Baird (2010) reported that between 9 million and 13 million barrels have been spilled in the Niger Delta since 1958.
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