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34 Seiten, Note: PDF
CHAPTER ONE GENERAL INTRODUCTION
1.1. Background of the Study
1.2. Statement of the Problem
1.3. Research Questions
1.4. Aim and Objectives (Purpose) of the Study
1.5. Significance of the Study
1.6. Scope and Limitation of the Study
1.7. Operational Definition of Terms
CHAPTER TWO LITERATURE REVIEW
2.1. Microbial Techniques for Hydrocarbon Exploration
2.2. Methane Oxidizing Bacteria
2.2.1. Microbial Oxidation of Methane
2.3. Ethane, Propane and Butane Oxidizing Bacteria
2.3.1. Microbial Oxidation of Ethane, Propane and N-Butane
2.4. Sample Collection Method.
2.5. Isolation of Hydrocarbon Oxidizing Bacteria
2.6. Molecular Biology Techniques
CHAPTER THREE MATERIALS AND METHODS
3.1. Collection of Soil Samples
3.2. Isolation of Bacteria from Soil Samples
3.2.1. Serial Dilution
3.2.2. Media Preparation and Sterilization
3.2.3. Spread Plate Method
3.3.0. Gram’s staining
3.4. Application of Oil Utilizing Bacteria
CHAPTER FOUR RESULTS AND DISCUSSION
4.1.1. Colony Characterization
4.1.3. Observation for Application of Oil Utilizing Bacteria
CHAPTER FIVE CONCLUSION AND RECOMMENDATIONS
Author highly acknowledges Yobe State University, Damaturu, Yobe State, Nigeria for providing laboratory facilities for this research work.
Reader & Head
Department of Biological Sciences
Yobe State University
Damaturu, Yobe State, Nigeria
Ancient rocks and natural oils are rich with abundant and hidden information of ancient organic matters responsible for earth formation, including molecular and isotopic signatures of the organisms that existed at the time the organic matter was formed. Considering the above facts of astrobiology, research study was carried out on screening of oil indicating microorganisms from oil contaminated sites of major geo-political zones of Yobe State, Nigeria including Damaturu, Gashua, Potiskum and Nguru local Government areas. Serial dilution was performed for all soil samples collected from the oil contaminated sites of mentioned local government areas followed by culture isolation and identification techniques for bacterial samples only. Nutrient agar was used for the isolation of the bacteria from the soil samples and mixed cultures were obtained. These bacterial cultures were inoculated in petroleum in order to see their oil utilizing capability. Two types of culture showed positive results among all isolated colonies. Rhizoid shaped gram positive rods isolated from soils of Damaturu, Gashua, Potiskum and Nguru & yellow pigmented gram negative bacterial colony isolated from soils of Potiskum and Nguru showed oil utilizing ability by positive growth in the refined crude oil (Petroleum, Diesel, etc.). The oil utilizing ability of those specific bacteria shows their potentiality in detecting the presence of oil and acting as potential oil indicators in Sub-Saharan region of African Continent caring a long history of bio-geo sciences. Findings from the present research work may create an opening for encouraging the geo-biological potentials of West African Countries in astrobiological aspects.
Key words: Geo-political zones, Oil indictor, bacteria, refined crude oil, bio-geo sciences, astrobiology.
Geo-microbial prospecting for hydrocarbons is an exploration method on the premise that the light gaseous hydrocarbons migrate upward from subsurface petroleum accumulations by diffusion and effusion, and are utilized by a variety of microorganisms present in the sub-soil ecosystem. The hydrocarbon oxidizing bacteria exclusively use these gases as carbon source for their metabolic activities and growth. These bacteria are mostly found enriched in the shallow soils/sediments above hydrocarbon-bearing structures and can differentiate between hydrocarbon prospective and non-prospective areas.
A microbial prospecting technique for hydrocarbon research and exploration has been in use for the last five decades. The isolation and enumeration of specific alkane-oxidizing bacteria are used as indirect petroleum prospecting method. Microbial anomalies have been proved to be reliable indicators of oil and gas in the sub-surface (Pareja, 1994). The direct and positive relationships between the microbial population and the hydrocarbon concentration in the soils have been observed in various producing reservoirs worldwide (Miller, 1976; Sealy, 1974; Wagner et al., 2002).
The microbial survey was first proposed and applied in the U.S.S.R. Early use of bacterial soil flora as a means of detecting gas as a means of detecting gas seepages was a development stemming from oil gas surveys performed in the U.S.S.R (Mogilevskii, 1959). The initial microbial investigations of Mogilevskii and his associates in the field of petroleum prospecting incited the interest of petroleum geochemist worldwide.
It has been shown that out of 20 microbial anomalies, 16 were proved by successful drilling. Sealy(1974a) in U.S.A, carried out microbial prospecting surveys and showed a positive correlation of 85.7%. Miller(1976) reported microbial survey carried out in the oil fields of the U.S.A, in which the microbial activity profile indicated a good contrast between oil field and nearby dry area.Beghtel (1987) predicted hydrocarbon potential of 18 wildcat wells in the Kansas; out of which, 13 have proved to be commercial produces oil and gas. As a result, microbial methods for detecting petroleum gas in the soils and waters have been tested in Europe, U.S.A and elsewhere. A sustained effort for the presence of efficiently utilizing microbiological prospecting methods had been underway since 1938 including independent surveys, surveys coordinated with geochemical and geophysical operation, microbiological tests of both soils and subsurface waters and analysis for methane oxidizing as well as higher hydrocarbon-oxidizing bacteria. However, oil and gas fields also build up micro-seepages at the subsurface soils, and these micro-seepages are detectable using a variety of analytical techniques that have been developed the basis for these new surface prospecting methods. The pioneers were Laubmeyer (1993) in Germany, Rosaire (1939) and Horvitz (1939) in the United States. Using methods such as extraction of adsorbed hydrocarbon gases from surface samples, they documented a correlation between higher hydrocarbon concentrations and oil and gas fields. At almost at the same time, the microbiologists Mogilevskii (1938, 1940) in the U.S.S.R. and Taggart (1941) and Blau (1942) in the United States described the use of hydrocarbon-oxidizing bacteria, when measured in surface soil samples, as an indicator for oil and gas fields in the deeper subsurface. In the 1950s and early 1960s, many relevant publications came from the United States (Updegraff et al., 1954; Davis, 1956). Several microbiological methods for detecting the distribution and activity of hydrocarbon-oxidizing bacteria were developed, such as enumeration of cell content in soil samples, measuring gas-consumption rates and radioautography. The field trials conducted by Sealy (1974) in U.S.A., using microbiological techniques, showed that out of 89 locations tested in West Texas, Wildcats predicted as productive and non-productive correlation of 54% and 92% respectively.
Microbial prospecting survey has been widely used in Germany since 1961 and a total of 17 oil and gas fields were identified. The success rate of Microbial Prospecting for Oil and Gas (MPOG) method has been reported to be 90%.
Most exploration depends on highly sophisticated technology to detect and determine the extent of hydrocarbon deposits using exploration geophysics. Areas thought to contain hydrocarbons are initially subjected to a gravity survey, magnetic survey, passive seismic reflection or regional seismic reflection surveys to detect large scale features of the subsurface geology. Features of interest (known as leads) are subjected to more detailed seismic surveys which work on the principle of the time it takes for reflected sound waves to travel through matter (rock) of varying densities and using the process of depth conversion to create a profile of the substructure. Finally, where a prospect has been identified and evaluated and passes the oil company’s selection criteria an exploration well is drilled in attempt to conclusively determine the presence or absence of oil and gas.
This exploration technique is an expensive, high risk operation. Offshore and remote area exploration is generally only undertaken by very large corporations or national governments.
In 1956, Shell British petroleum discovered crude oil at village Oloibiri in Bayelsa state in the Niger Delta within Nigeria (Anifowose, 2008; Onuhua, 2008) and since then oil exploitation and exploration have been on-going for several decades. However, that area has seen increasing militant actions by locals unhappy with both their exploitation and the environmental destruction caused by the exploitation. Recently, it was reported according to some geological observations, that the northern part of Nigeria has massive quantities of oil and there is need for modern technology to explore this oil in the rich areas. Nevertheless, as the northern part of Nigeria is in the savannah region, the geophysical data is hardly obtained.
i) Why do microorganisms oxidize/utilize light gaseous hydrocarbons in contaminated soil?
ii) How do hydrocarbon-oxidizing bacteria have positive relationship with hydrocarbon concentration in the soil?
iii) Why are hydrocarbon-oxidizing bacteria used as indicators for finding oil and gas reservoirs.
The aim of this study is to isolate oil degrading microorganisms from soils of major areas of Yobe State.The objectives of this study are stated below:
i) To provide efficient, less expensive method of oil prospecting in area, thus reducing the risk of operation.
ii) To provide method that is helpful in the quick evaluation of hydrocarbon prospects and generation of exploratory leads.
iii) To provide a means of identification for prospective oil and oil and gas in areas where no geophysical data is available or where such investigation is difficult.
iv) To provide a means that can be integrated with geological, geophysical and geochemical methods to prioritize drilling locations thereby reducing drilling risks and achieving higher success in petroleum exploration.
This study will be of great importance to oil explorers since it will help to provide a cost effective, low-risk means of oil prospecting in an area. It will also help them in identification of oil and gas in areas where no geographical data is available or where such investigation is difficult. The study will also help in the identification/discovery of oil in rich areas. It will serve as a reference point for future researchers who will want to research more on the topic.
This research tends to detect the presence of oil degrading microorganisms in contaminated sites in Yobe State. However, the research will be restricted to four selected towns in the major zones of Yobe State. The towns are Damaturu, Potiskum, Gashua and Nguru.
i. Exploration: examination of something in order to find out about it.
ii. Geochemistry: derived from Geology and Chemistry describing the nature and appearance of matter together with its chemical composition.
iii. Geo-microbial prospecting: derived from geology and microbes, describing the use of microorganisms for prospecting oil.
iv. Geophysics: scientific study of the earth’s atmosphere, oceans and climate.
v. Hydrocarbons: chemicals made of hydrogen and carbon only found in petroleum and gas.
vi. Hydrocarbon-oxidizing bacteria: the bacteria responsible for utilizing/oxidizing hydrocarbons.
vii. Isolation of microbes: the growing a pure culture of (a microbe), usually as individual colonies on a solid medium. Or, it is the separation of pure strain of a microorganism or virus.
viii. Oil seepage: the process by which oil flows slowly and in small quantities through the soil.
Bacteria are ubiquitous in distribution and their exceptionally high adaptability to grow on different nutrient sources form the basis of microbial prospecting (Rasheed et al, 2013). Several investigators have used bacteria that degrade hydrocarbons used as indicators for finding oil and gas reservoirs.
Sealy (1974) in U.S.A, carried out microbial prospecting surveys and showed a positive correlation of 85.7%. Miller (1976) reported microbial survey carried out in the oil fields of the U.S.A, in which the microbial activity profile indicated a good contrast between oil fields and nearby dry area. Beghtel (1987) predicted hydrocarbon potential of 18 wildcat wells in the Kansas; out of which, 13 have proved to be commercial producers of oil and gas.
Rasheed et al., (2013) carried out geo-microbial prospecting method for hydrocarbon exporation in Vengannapalli village, Cuddapah Basin India, with a view to investigate whether the leakage of natural gas is associated with some geo-microbial anomalies that can establish an upward migration of hudrocarbons from deep subsurface source, where they report high bacterial counts for methane (5.4 × 105cfu/g) and propane-oxidizing bacteria (4.6 × 104cfu/g) in soil samples collected from Vengannapalli village. The adsorbed light gaseous hydrocarbon analysis shows the presence of moderate to low concentrations methane, ethane, propane and butane in the soil samples. Carbon isotope analysis of few soil samples show enriched δ13 C, values of methane ranging from -35.0 to 10.6‰, indicative of methane oxidation. Geo-microbial prospecting method coupled with adsorbed soil gas and carbon isotope studies, suggest that hydrocarbon micro-seepage of subsurface origin is present in the area and indicate that the area is worth visiting for conventional petroleum exploration. Similar technique was used by Rasheed et al., in Deccan Syncline, Maharashta, India. Subsurface soil samples were collected in an interval of 5 × 5 km, and analysed for propane oxidizing bacterial concentration. The propane oxidizing bacterial count in the soil samples of the studied area ranged from 1 × 102 to 6.7 × 105 cfu/gmof soil sample. Two microbial blooms of high concentration of propane oxidizing bacteria were identified and mapped in the study area.
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