MICROBES IN AREA CONTAMINATED WITH CRUDE OIL AND MICROBES ASSOCIATED WITH THE AREA

1 MICROBES IN AREA CONTAMINATED WITH CRUDE OIL AND MICROBES ASSOCIATED WITH THE AREA BY OHANYEREM UCHECHUKWU HENRILYN 14/24362 A PROJECT WORK SUBMITTED TO THE DEPARTMENT OF MEDICAL LABORATORY SCIENCE, FACULTY OF HEALTH SCIENCES, IMO STATE UNIVERSITY IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF BACHELOR OF MEDICAL LABORATORY SCIENCE (B.MLS) DEGREE OF IMO STATE UNIVERSITY, OWERRI. SUPERVISOR DR (MRS) J.N. DIKE JANUARY, 2020 2 DEDICATION This research work is dedicated to God Almighty 3 CERTIFICATION This is to certify that this project titled ‘microbes in area contaminated with crude oil and microbes associated with the area” was carried out by Ohanyerem Uchechukwu Henrilyn with matriculation number 14/24362 in Medical Microbiology option, Department of Medical Laboratory Science, Faculty of Health Sciences, Imo State University, under the supervision of Dr. J.N. Dike …………………. ………………….. Dr. J.N. Dike Date (Supervisor) …………………. ………………….. Prof. D.C. Nwosu Date (H.O.D) …………………. ………………….. Prof. H.U. Nwanjo Date (Dean of faculty of Health Sciences) …………………… …………………… ………………… Signature External Supervisor Date 4 ACKNOWLEDGEMENT My immeasurable gratitude goes to God Almighty who made it possible for me to be alive this moment and who supplied me with his grace to complete this project. I also wish to express profound gratitude to my hardworking supervisor, Dr. J.N. Dike for her guidance which was essential for the completion of this work. I am equally indebted to all my lecturers in Medical Laboratory Science Department, Imo State University, Owerri. Most especially my H.O.D, Prof. D.C. Nwosu and my Dean Professor H.U Nwanjo for their contribution towards my academics. My sincere gratitude goes to my family Eze Ben Ohanyerem, Ugoeze Chinyere Ohanyerem and Dr Wendy Amah for their support, financially and otherwise. May Almighty God bless you all. 5 ABSTRACT Petrochemical hydrocarbons are valuable source of energy around the world for our day to day activities and industrial developments. Nigerians use petrol as one of its world’s most important derived energy source. Nevertheless, civilization and activities of petroleum and petroleum associated industries in Nigeria and other parts of the world had led to increase in oil pollution in our environment. This study was carried out to identify microbial populations of crude petroleum- polluted soil and water in selected areas of Ohaji/Egbema, Imo State. Soil samples collected from 3 different polluted sites in Ohaji/Egbema local government area of Imo state, Nigeria were collected at a depth of 0 – 15cm with the aid of auger. Standard microbiological procedures were employed in collection and handling of the soil and water samples and they were analyzed within 24hours of collections. Different bacteria and fungi were isolated, identified and their populations estimated in CFU/g, CFU/ml, SFU/g and SFU/ml respectively. Ekugba had the highest soil bacteria load (32.4±2.5 CFU/g) followed by Abaezi (29±3.5 CFU/g) and Abacheke (28.5±2.8 CFU/g) respectively. The bacterial load of Abaezi water was the highest (34±3.2 CFU/ml) followed by Abacheke (33±1.0 CFU/ml) and then Ekugba (24.2±3 CFU/ml). Fungal counts (x106) of oil polluted soil and water collected from Ohaji Egbema are presented in table 2, fig 2. The highest soil fungal load was obtained from Abacheke (26.4±2.8 SFU/g) followed by Abaezi (21±3.2 SFU/g) and Ekugba (21±2.2 SFU/g ) respectively. Ekugba had the highest fungal populationin water (25.2±1.0 SFU/ml) followed by Abacheke (25±2.0 SFU/ml ) and then Abaezi (24±2.4 SFU/ml). In conclusion, this study has shown that oil-degrading bacteria and fungi can be isolated from oil polluted sites. These bacteria and fungi are indigenous in the polluted sites and they are responsible for the degradation of oil and therefore bioremediation. 6 TABLE OF CONTENTS Title page i Certification ii Dedication iii Acknowledgements iv Table of Contents v List of Tables vi List of Figures vii Abstract viii CHAPTER ONE: INTRODUCTION 1.1 Background of the Study 1 1.2 Justification of Study 2 1.3 Study Aim and Objectives 3 CHAPTER TWO: LITERATURE REVIEW 2.1 Petroleum 4 2.1.1 Etymology of Petroleum 5 2.1.2 Early History of Petroleum 6 2.1.3 Composition of Petroleum 12 2.1.4 Formation of Petroleum 18 2.2 Bioremediation 28 7 CHAPTER THREE: MATERIALS AND METHODS 3.1 Sample collection 3.2 Materials and instruments 37 3.3 Preparation of Media 37 3.4 Methods 38 3.5 Statistical Analysis 40 CHAPTER FOUR: RESULT 4.1 Microbial population of environmental samples from 41 oil polluted sites 4.2 Cultural, microscopic and biochemical characteristics 46 of bacteria and fungi isolated from oil polluted sites in Ohaji Egbema CHAPTER FIVE DISCUSSION 5.1 Discussion of Findings 49 5.2 Conclusion 51 References 55 Appendices 62 8 LIST OF TABLES Table Title Page 4.1 Bacterial counts (x104) of oil polluted soil and water collected 42 from Ohaji Egbema 4.2 Fungal counts (x106) of oil polluted soil and water collected from 43 Ohaji Egbema 4.3 Morphological and biochemical characteristics of bacteria isolated 47 from oil polluted sites in Ohaji Egbema. 4.4 Microscopic and cultural characteristics of fungi isolated from oil 48 polluted areas in Ohaji egbema. 9 CHAPTER ONE 1.0 INTRODUCTION 1.1 Background of the Study Petroleum-based products are the major source of energy for industry and daily life. Leaks and accidental spills occur regularly during the exploration, production, refining, transport, and storage of petroleum and petroleum products. The amount of natural crude oil seepage was estimated to be 600,000 metric tons per year with a range of uncertainty of 200,000 metric tons per year (Kvenvolden and Cooper, 2003). Release of hydrocarbons into the environment whether accidentally or due to human activities is a main cause of water and soil pollution. Soil contamination with hydrocarbons causes extensive damage of local system since accumulation of pollutants in animals and plant tissue may cause death or mutations. The technology commonly used for the soil remediation includes mechanical, burying, evaporation, dispersion, and washing. However, these technologies are expensive and can lead to incomplete decomposition of contaminants. The process of bioremediation, defined as the use of microorganisms to detoxify or remove pollutants owing to their diverse metabolic capabilities is an evolving method for the removal and degradation of many environmental pollutants including the products of petroleum industry (Medina- Bellver et al., 2005). In addition, bioremediation technology is believed to be non- invasive and relatively cost-effective (April et al., 2000). Biodegradation by 10 natural populations of microorganisms represents one of the primary mechanisms by which petroleum and other hydrocarbon pollutants can be removed from the environment (Ulrici, 2000) and is cheaper than other remediation technologies. The success of oil spill bioremediation depends on one’s ability to establish and maintain conditions that favour enhanced oil biodegradation rates in the contaminated environment. Numerous scientific review articles have covered various factors that influence the rate of oil biodegradation. One important requirement is the presence of microorganisms with the appropriate metabolic capabilities. If these microorganisms are present, then optimal rates of growth and hydrocarbon biodegradation can be sustained by ensuring that adequate concentrations of nutrients and oxygen are present and that the pH is between 6 and 9 (Ulrici, 2000). The physical and chemical characteristics of the oil and oil surface area are also important determinants of bioremediation success. There are the two main approaches to oil spill bioremediation: bioaugmentation, in which known oildegrading bacteria are added to supplement the existing microbial population, and biostimulation, in which the growth of indigenous oil degraders is stimulated by the addition of nutrients or other growth-limiting cosubstrates. 1.2 Justification The success of bioremediation efforts in certain oil spills has increased the interest in the potential of biodegradation and bioremediation technology. Most existing studies have concentrated on evaluating the factors affecting oil bioremediation 11 or testing favoured products and methods through laboratory studies. Only limited numbers of pilot scale and field trials have provided the most convincing demonstrations of this technology which have been reported in the peer-reviewed literature (Venosa et al., 2002). The scope of current understanding of oil bioremediation is also limited because the emphasis of most of these field studies and reviews has been given on the evaluation of bioremediation technology for dealing with large-scale oil spills on marine shorelines. 1.3 Aim and Objectives Aim To identify microbial populations of crude petroleum-polluted soil in selected areas of Ohaji/Egbema, Imo State. Objectives i. To isolate and identify bacteria associated with petroleum polluted soil in selected areas of Ohaji/ Egbema, Imo State. ii. To isolate and identify fungi associated with petroleum polluted soil in selected areas of Ohaji/ Egbema, Imo State. iii. To determine chemical composition of soil in crude oil polluted areas. 12 CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 Petroleum Petroleum (/pəˈtroʊliəm/) is a naturally occurring, yellowish-black liquid found in geological formations beneath the Earth's surface. It is commonly refined into various types of fuels. Components of petroleum are separated using a technique called fractional distillation, i.e. separation of a liquid mixture into fractions differing in boiling point by means of distillation, typically using a fractionating column. It consists of naturally occurring hydrocarbons of various molecular weights and may contain miscellaneous organic compounds (EIA, 2018). 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, mostly zooplankton and algae, are buried underneath sedimentary rock and subjected to both intense heat and pressure. Petroleum has mostly been recovered by oil drilling (natural petroleum springs are rare). Drilling is carried out after studies of structural geology (at the reservoir scale), sedimentary basin analysis, and reservoir characterisation (mainly in terms of the porosity and permeability of geologic reservoir structures) have been completed (Guerriero et al., 2011). It is refined and separated, most easily by distillation, into numerous consumer products, from gasoline (petrol) and kerosene to asphalt and chemical reagents used to make plastics, pesticides 13 and pharmaceuticals. Petroleum is used in manufacturing a wide variety of materials, and it is estimated that the world consumes about 95 million barrels each day. The use of petroleum as fuel is controversial due to its impact on global warming and ocean acidification. According to the UN's Intergovernmental Panel on Climate Change, fossil fuel phase-out, including petroleum, needs to be completed by the end of 21st century to avoid "severe, pervasive, and irreversible impacts for people and ecosystems" (IPCC, 2014). 2.1.1 Etymology of Petroleum The word petroleum comes from Medieval Latin petroleum (literally "rock oil"), which comes from Latin petra', "rock", (from Ancient Greek: πέτρα, romanized: petra, "rock") and Latin oleum, "oil", (from Ancient Greek: ἔλαιον, romanized: élaion, "oil"). The term was used in the treatise De Natura Fossilium, published in 1546 by the German mineralogist Georg Bauer, also known as Georgius Agricola. In the 19th century, the term petroleum was often used to refer to mineral oils produced by distillation from mined organic solids such as cannel coal (and later oil shale) and refined oils produced from them; in the United Kingdom, storage (and later transport) of these oils were regulated by a series of Petroleum Acts, from the Petroleum Act 1863 onwards. 14 2.1.2 Early History of Petroleum Petroleum, in one form or another, has been used since ancient times, and is now important across society, including in economy, politics and technology. The rise in importance was due to the invention of the internal combustion engine, the rise in commercial aviation, and the importance of petroleum to industrial organic chemistry, particularly the synthesis of plastics, fertilisers, solvents, adhesives and pesticides. More than 4000 years ago, according to Herodotus and Diodorus Siculus, asphalt was used in the construction of the walls and towers of Babylon; there were oil pits near Ardericca (near Babylon), and a pitch spring on Zacynthus (Zhiguo, 1998). Great quantities of it were found on the banks of the river Issus, one of the tributaries of the Euphrates. Ancient Persian tablets indicate the medicinal and lighting uses of petroleum in the upper levels of their society. The use of petroleum in ancient China dates back to more than 2000 years ago. In I Ching, one of the earliest Chinese writings cites that oil in its raw state, without refining, was first discovered, extracted, and used in China in the first century BCE. In addition, the Chinese were the first to record the use of petroleum as fuel as early as the fourth century BCE (Deng, 2011). By 347 AD, oil was produced from bamboo-drilled wells in China. Crude oil was often distilled by Persian chemists, with clear descriptions given in Arabic handbooks such as those of Muhammad ibn Zakarīya Rāzi (Rhazes). The streets of Baghdad were paved with tar, derived from petroleum that became accessible from natural fields in the 15 region. In the 9th century, oil fields were exploited in the area around modern Baku, Azerbaijan. These fields were described by the Arab geographer Abu al- Hasan 'Alī alMas'ūdī in the 10th century, and by Marco Polo in the 13th century, who described the output of those wells as hundreds of shiploads (Salim. 2008). Arab and Persian chemists also distilled crude oil in order to produce flammable products for military purposes. Through Islamic Spain, distillation became available in Western Europe by the 12th century. It has also been present in Romania since the 13th century, being recorded as păcură (Joseph et al., 2008). Early British explorers to Myanmar documented a flourishing oil extraction industry based in Yenangyaung that, in 1795, had hundreds of hand-dug wells under production. Pechelbronn (Pitch fountain) is said to be the first European site where petroleum has been explored and used. The still active Erdpechquelle, a spring where petroleum appears mixed with water has been used since 1498, notably for medical purposes. Oil sands have been mined since the 18th century. In Wietze in lower Saxony, natural asphalt/bitumen has been explored since the 18th century. Both in Pechelbronn as in Wietze, the coal industry dominated the petroleum technologies. Chemist James Young noticed a natural petroleum seepage in the Riddings colliery at Alfreton, Derbyshire from which he Proven world oil reserves, 2013. Unconventional reservoirs such as natural heavy oil and oil sands are included.distilled a light thin oil suitable for use as lamp oil, at the same time obtaining a more viscous oil suitable for lubricating machinery. In 1848, Young set up a small business refining the crude oil (Russel, 2003). Young 16 eventually succeeded, by distilling cannel coal at a low heat, in creating a fluid resembling petroleum, which when treated in the same way as the seep oil gave similar products. Young found that by slow distillation he could obtain a number of useful liquids from it, one of which he named "paraffine oil" because at low temperatures it congealed into a substance resembling paraffin wax (Russel, 2003). The production of these oils and solid paraffin wax from coal formed the subject of his patent dated 17 October 1850. In 1850 Young & Meldrum and Edward William Binney entered into partnership under the title of E.W. Binney & Co. at Bathgate in West Lothian and E. Meldrum & Co. at Glasgow; their works at Bathgate were completed in 1851 and became the first truly commercial oil-works in the world with the first modern oil refinery (Frank, 2005). The world's first oil refinery was built in 1856 by Ignacy Łukasiewicz. His achievements also included the discovery of how to distill kerosene from seep oil, the invention of the modern kerosene lamp (1853), the introduction of the first modern street lamp in Europe (1853), and the construction of the world's first modern oil well (1854). The demand for petroleum as a fuel for lighting in North America and around the world quickly grew (Maugeri, 2005). Edwin Drake's 1859 Shale bings near Broxburn, 3 of a total of 19 in West Lothian. well near Titusville, Pennsylvania, is popularly considered the first modern well. Already 1858 Georg Christian Konrad Hunäus had found a significant amount of petroleum while drilling for 17 lignite 1858 in Wietze, Germany. Wietze later provided about 80% of the German consumption in the Wilhelminian Era. The production stopped in 1963, but Wietze has hosted a Petroleum Museum since 1970. Drake's well is probably singled out because it was drilled, not dug; because it used a steam engine; because there was a company associated with it; and because it touched off a major boom (Vassiliou, 2018). However, there was considerable activity before Drake in various parts of the world in the mid-19th century. A group directed by Major Alexeyev of the Bakinskii Corps of Mining Engineers hand-drilled a well in the Baku region in 1848. There were engine-drilled wells in West Virginia in the same year as Drake's well. An early commercial well was hand dug in Poland in 1853, and another in nearby Romania in 1857. At around the same time the world's first, small, oil refinery was opened at Jasło in Poland, with a larger one opened at Ploiești in Romania shortly after. Romania is the first country in the world to have had its annual crude oil output officially recorded in international statistics: 275 tonnes for 1857 (Thomas, 2017). The first commercial oil well in Canada became operational in 1858 at Oil Springs, Ontario (then Canada West). Businessman James Miller Williams dug several wells between 1855 and 1858 before discovering a rich reserve of oil four metres below ground. Williams extracted 1.5 million litres of crude oil by 1860, refining much of it into kerosene lamp oil. Williams's well became commercially viable a year before Drake's Pennsylvania operation and could be argued to be the first commercial oil well in North America (ICLMG, 2013). The discovery at Oil Springs touched off an oil 18 boom which brought hundreds of speculators and workers to the area. Advances in drilling continued into 1862 when local driller Shaw reached a depth of 62 metres using the spring-pole drilling method. On January 16, 1862, after an explosion of natural gas Canada's first oil gusher came into production, shooting into the air at a recorded rate of 3,000 barrels per day. By the end of the 19th century the Russian Empire, particularly the Branobel company in Azerbaijan, had taken the lead in production (Akiner, 2004). Access to oil was and still is a major factor in several military conflicts of the twentieth century, including World War II, during which oil facilities were a major strategic asset and were extensively bombed.[42] The German invasion of the Soviet Union included the goal to capture the Baku oilfields, as it would provide much needed oil-supplies for the German military which was suffering from blockades.[43] Oil exploration in North America during the early 20th century later led to the US becoming the leading producer by mid-century. As petroleum production in the US peaked during the 1960s, however, the United States was surpassed by Saudi Arabia and the Soviet Union. In 1973, Saudi Arabia and other Arab nations imposed an oil embargo against the United States, United Kingdom, Japan and other Western nations which supported Israel in the Yom Kippur War of October 1973. The embargo caused an oil crisis with many short- and long-term effects on global politics and the global economy. 19 Today, about 90 percent of vehicular fuel needs are met by oil. Petroleum also makes up 40 percent of total energy consumption in the United States, but is responsible for only 1 percent of electricity generation (EIA, 2017). Petroleum's worth as a portable, dense energy source powering the vast majority of vehicles and as the base of many industrial chemicals makes it one of the world's most important commodities. Viability of the oil commodity is controlled by several key parameters: number of vehicles in the world competing for fuel; quantity of oil exported to the world market (Export Land Model); net energy gain (economically useful energy provided minus energy consumed); political stability of oil exporting nations; and ability to defend oil supply lines. The top three oil producing countries are Russia, Saudi Arabia and the United States. In 2018, due in part to developments in hydraulic fracturing and horizonal drilling, the United States became the world's largest producer. About 80 percent of the world's readily accessible reserves are located in the Middle East, with 62.5 percent coming from the Arab 5: Saudi Arabia, United Arab Emirates, Iraq, Qatar and Kuwait. A large portion of the world's total oil exists as unconventional sources, such as bitumen in Athabasca oil sands and extra heavy oil in the Orinoco Belt. While significant volumes of oil are extracted from oil sands, particularly in Canada, logistical and technical hurdles remain, as oil extraction requires large amounts of heat and water, making its net energy content quite low relative to conventional crude oil. Thus, Canada's oil sands are not expected to provide more than a few million barrels per day in the foreseeable future. 20 2.1.3 Composition of Petroleum Petroleum includes not only crude oil, but all liquid, gaseous and solid hydrocarbons. Under surface pressure and temperature conditions, lighter hydrocarbons methane, ethane, propane and butane exist as gases, while pentane and heavier hydrocarbons 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 (Norman, 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 gasoline 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 bitumens. The hydrocarbons in crude oil are mostly alkanes, cycloalkanes and various aromatic 21 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 (Olliver and Magot, 2005). The exact molecular composition of crude oil varies widely from formation to formation but the proportion of chemical elements varies over fairly narrow limits as follows: Composition by weight Element Percent range Carbon 83 to 85% Hydrogen 10 to 14% Nitrogen 0.1 to 2% Oxygen 0.05 to 1.5% Sulfur 0.05 to 6.0% Metals < 0.1%. 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. Composition by weight Hydrocarbon Average Range Alkanes (paraffins) 30% 15 to 60% Naphthenes 49% 30 to 60% Aromatics 15% 3 to 30% Asphaltics 6% remainder. Unconventional resources are much larger than conventional ones (Alboudwarej et al., 2006). 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 a semisolid form mixed with sand and water, as in the Athabasca oil sands in Canada, where it is usually referred to as crude bitumen. In Canada, bitumen is considered a 22 sticky, black, tar-like form of crude oil which is so thick and heavy that it must be heated or diluted before it will flow. Venezuela also has large amounts of oil in the Orinoco oil sands, although the hydrocarbons trapped in them are more fluid than in Canada and are usually called extra heavy oil. These oil sands resources are called unconventional oil to distinguish them from oil which can be extracted using traditional oil well methods. Between them, Canada and Venezuela contain an estimated 3.6 trillion barrels (570×109 m3) of bitumen and extra-heavy oil, about twice the volume of the world's reserves of conventional oil. Petroleum is used mostly, by volume, for refining into fuel oil and gasoline, both important "primary energy" sources. 84 percent by volume of the hydrocarbons present in petroleum is converted into energy-rich fuels (petroleum-based fuels), including gasoline, diesel, jet, heating, and other fuel oils, and liquefied petroleum gas. The lighter grades of crude oil produce the best yields of these products, but as the world's reserves of light and medium oil are depleted, oil refineries are increasingly having to process heavy oil and bitumen, and use more complex and expensive methods to produce the products required. Because heavier crude oils have too much carbon and not enough hydrogen, these processes generally involve removing carbon from or adding hydrogen to the molecules, and using fluid catalytic cracking to convert the longer, more complex molecules in the oil to the shorter, simpler ones in the fuels. 23 Due to its high energy density, easy transportability and relative abundance, oil has become the world's most important source of energy since the mid-1950s. Petroleum is also the raw material for many chemical products, including pharmaceuticals, solvents, fertilizers, pesticides, and plastics; the 16 percent not used for energy production is converted into these other materials. Petroleum is found in porous rock formations in the upper strata of some areas of the Earth's crust. There is also petroleum in oil sands (tar sands). Known oil reserves are typically estimated at around 190 km3 (1.2 trillion (short scale) barrels) without oil sands (EIA, 2010), or 595 km3 (3.74 trillion barrels) with oil sands.[58] Consumption is currently around 84 million barrels (13.4×106 m3) per day, or 4.9 km3 per year, yielding a remaining oil supply of only about 120 years, if current demand remains static. Chemistry of Petroleum Petroleum is a mixture of a very large number of different hydrocarbons; the most commonly found molecules are alkanes (paraffins), cycloalkanes (naphthenes), aromatic hydrocarbons, or more complicated chemicals like asphaltenes. Each petroleum variety has a unique mix of molecules, which define its Octane, a hydrocarbon found in petroleum. Lines represent single bonds; black spheres represent carbon; white spheres represent hydrogen. physical and chemical properties, like color and viscosity. 24 The alkanes, also known as paraffins, are saturated hydrocarbons with straight or branched chains which contain only carbon and hydrogen and have the general formula CnH2n+2. They generally have from 5 to 40 carbon atoms per molecule, although trace amounts of shorter or longer molecules may be present in the mixture. The alkanes from pentane (C5H12) to octane (C8H18) are refined into gasoline, the ones from nonane (C9H20) to hexadecane (C16H34) into diesel fuel, kerosene and jet fuel. Alkanes with more than 16 carbon atoms can be refined into fuel oil and lubricating oil. At the heavier end of the range, paraffin wax is an alkane with approximately 25 carbon atoms, while asphalt has 35 and up, although these are usually cracked by modern refineries into more valuable products. The shortest molecules, those with four or fewer carbon atoms, are in a gaseous state at room temperature. They are the petroleum gases. Depending on demand and the cost of recovery, these gases are either flared off, sold as liquefied petroleum gas under pressure, or used to power the refinery's own burners. During the winter, butane (C4H10), is blended into the gasoline pool at high rates, because its high vapour pressure assists with cold starts. Liquified under pressure slightly above atmospheric, it is best known for powering cigarette lighters, but it is also a main fuel source for many developing countries. Propane can be liquified under modest pressure, and is consumed for just about every application relying on petroleum for energy, from cooking to heating to transportation. 25 The cycloalkanes, also known as naphthenes, are saturated hydrocarbons which have one or more carbon rings to which hydrogen atoms are attached according to the formula CnH2n. Cycloalkanes have similar properties to alkanes but have higher boiling points. The aromatic hydrocarbons are unsaturated hydrocarbons which have one or more planar six-carbon rings called benzene rings, to which hydrogen atoms are attached with the formula CnH2n-6. They tend to burn with a sooty flame, and many have a sweet aroma. Some are carcinogenic. These different molecules are separated by fractional distillation at an oil refinery to produce gasoline, jet fuel, kerosene, and other hydrocarbons. For example, 2,2,4trimethylpentane (isooctane), widely used in gasoline, has a chemical formula of C8H18 and it reacts with oxygen exothermically: 2 C 8H 18(l) + 25 O 2(g) → 16 CO 2(g) + 18 H 2O(g) (ΔH = −5.51 MJ/mol of octane) The number of various molecules in an oil sample can be determined by laboratory analysis. The molecules are typically extracted in a solvent, then separated in a gas chromatograph, and finally determined with a suitable detector, such as a flame ionization detector or a mass spectrometer (Temanord, 2003). Due to the large number of co-eluted hydrocarbons within oil, many cannot be resolved by traditional gas chromatography and typically appear as a hump in the chromatogram. This Unresolved Complex Mixture (UCM) of hydrocarbons is particularly apparent when analysing weathered oils and extracts from tissues of organisms exposed to oil. Some of the component of oil will mix with water: the water associated fraction of the oil. 26 Incomplete combustion of petroleum or gasoline results in production of toxic byproducts. Too little oxygen during combustion results in the formation of carbon monoxide. Due to the high temperatures and high pressures involved, exhaust gases from gasoline combustion in car engines usually include nitrogen oxides which are responsible for creation of photochemical smog. 2.1.4 Formation of Petroleum Structure of a vanadium porphyrin compound (left) extracted from petroleum by Alfred E. Treibs, father of organic geochemistry. Treibs noted the close structural similarity of this molecule and chlorophyll a (right) (Kvenvolden, 2006). Petroleum is a fossil fuel derived from ancient fossilized organic materials, such as zooplankton and algae. Vast amounts of these remains settled to sea or lake bottoms where they were covered in stagnant water (water with no dissolved oxygen) or sediments such as mud and silt faster than they could decompose aerobically. Approximately 1 m below this sediment or water oxygen concentration was low, below 0.1 mg/l, and anoxic conditions existed. Temperatures also remained constant (Kvenvolden, 2006). As further layers settled to the sea or lake bed, intense heat and pressure built up in the lower regions. This process caused the organic matter to change, first into a waxy material known as kerogen, found in various oil shales around the world, and then with more heat into liquid and gaseous hydrocarbons via a process known as catagenesis. Formation of petroleum occurs from hydrocarbon pyrolysis in a 27 variety of mainly endothermic reactions at high temperature or pressure, or both. These phases are described in detail below. First phase of diagenesis: anaerobic decay In the absence of plentiful oxygen, aerobic bacteria were prevented from decaying the organic matter after it was buried under a layer of sediment or water. However, anaerobic bacteria were able to reduce sulfates and nitrates among the matter to H2S and N2 respectively by using the matter as a source for other reactants. Due to such anaerobic bacteria, at first this matter began to break apart mostly via hydrolysis: polysaccharides and proteins were hydrolyzed to simple sugars and amino acids respectively. These were further anaerobically oxidized at an accelerated rate by the enzymes of the bacteria: e.g., amino acids went through oxidative deamination to imino acids, which in turn reacted further to ammonia and α-keto acids. Monosaccharides in turn ultimately decayed to CO2 and methane. The anaerobic decay products of amino acids, monosaccharides, phenols and aldehydes combined to fulvic acids. Fats and waxes were not extensively hydrolyzed under these mild conditions (Schobert, 2013). Second phase of diagenesis: kerogen formation Some phenolic compounds produced from previous reactions worked as bactericides and actinomycetales order of bacteria produced antibiotic 28 compounds (e.g., streptomycin). Thus the action of anaerobic bacteria ceased at about 10 m below the water or sediment. The mixture at this depth contained fulvic acids, unreacted and partially reacted fats and waxes, slightly modified lignin, resins and other hydrocarbons. As more layers of organic matter settled to the sea or lake bed, intense heat and pressure built up in the lower regions (Schobert, 2013). As a consequence, compounds of this mixture the began to combine in poorly understood ways to kerogen. Combination happened in a similar fashion as phenol and formaldehyde molecules react to ureaformaldehyde resins, but kerogen formation occurred in a more complex manner due to a bigger variety of reactants. The total process of kerogen formation from the beginning of anaerobic decay is called diagenesis, a word that means a transformation of materials by dissolution and recombination of their constituents (Schobert, 2013). Catagenesis: transformation of kerogen into fossil fuels Kerogen formation continued to the depth of about 1 km from the Earth's surface where temperatures may reach around 50 °C. Kerogen formation represents a halfway point between organic matter and fossil fuels: kerogen can be exposed to oxygen, oxidize and thus be lost or it could be buried deeper inside the Earth's crust and be subjected to conditions which allow it to slowly transform into fossil fuels like petroleum. The latter happened through catagenesis in which the reactions were mostly radical rearrangements of kerogen. These reactions took thousands to millions of years and no external reactants were involved. Due to 29 radical nature of these reactions, kerogen reacted towards two classes of products: those with low H/C ratio (anthracene or products similar to it) and those with high H/C ratio (methane or products similar to it); i.e., carbon-rich or hydrogen-rich products. Because catagenesis was closed off from external reactants, the resulting composition of the fuel mixture was dependent on the composition of the kerogen via reaction stoichiometry. 3 main types of kerogen exist: type I (algal), II (liptinic) and III (humic), which were formed mainly from algae, plankton and woody plants (this term includes trees, shrubs and lianas) respectively (Schobert, 2013). Catagenesis was pyrolytic despite of the fact that it happened at relatively low temperatures (when compared to commercial pyrolysis plants) of 60 to several hundred °C. Pyrolysis was possible because of the long reaction times involved. Heat for catagenesis came from the decomposition of radioactive materials of the crust, especially 40K, 232Th, 235U and 238U. The heat varied with geothermal gradient and was typically 1030 °C per km of depth from the Earth's surface. Unusual magma intrusions, however, could have created greater localized heating (Schobert, 2013). Geologists often refer to the temperature range in which oil forms as an "oil window" (Schobert, 2013). Below the minimum temperature oil remains trapped in the form of kerogen. Above the maximum temperature the oil is converted to natural gas through the process of thermal cracking. Sometimes, oil formed at extreme depths may migrate and become trapped at a much 30 shallower level. The Athabasca Oil Sands are one example of this (Schobert, 2013). Abiogenic Petroleum An alternative mechanism to the one described above was proposed by Russian scientists in the mid-1850s, the hypothesis of abiogenic petroleum origin (petroleum formed by inorganic means), but this is contradicted by geological and geochemical evidence (Glasby, 2006). Abiogenic sources of oil have been found, but never in commercially profitable amounts. "The controversy isn't over whether abiogenic oil reserves exist," said Larry Nation of the American Association of Petroleum Geologists. "The controversy is over how much they contribute to Earth's overall reserves and how much time and effort geologists should devote to seeking them out." Three conditions must be present for oil reservoirs to form: Reservoirs ; Hydrocarbon trap.which is a source rock rich in hydrocarbon material buried deeply enough for subterranean heat to cook it into oil, a porous and permeable reservoir rock where it can accumulate, a caprock (seal) or other mechanism to prevent the oil from escaping to the surface. Within these reservoirs, fluids will typically organize themselves like a three-layer cake with a layer of water below the oil layer and a layer of gas above it, although the different layers vary in size between reservoirs. Because most hydrocarbons are less dense than rock or water, they often migrate upward through adjacent rock layers until either reaching the surface or becoming trapped within porous rocks 31 (known as reservoirs) by impermeable rocks above. However, the process is influenced by underground water flows, causing oil to migrate hundreds of kilometres horizontally or even short distances downward before becoming trapped in a reservoir. When hydrocarbons are concentrated in a trap, an oil field forms, from which the liquid can be extracted by drilling and pumping. The reactions that produce oil and natural gas are often modeled as first order breakdown reactions, where hydrocarbons are broken down to oil and natural gas by a set of parallel reactions, and oil eventually breaks down to natural gas by another set of reactions. The latter set is regularly used in petrochemical plants and oil refineries. Wells are drilled into oil reservoirs to extract the crude oil. "Natural lift" production methods that rely on the natural reservoir pressure to force the oil to the surface are usually sufficient for a while after reservoirs are first tapped. In some reservoirs, such as in the Middle East, the natural pressure is sufficient over a long time. The natural pressure in most reservoirs, however, eventually dissipates. Then the oil must be extracted using "artificial lift" means. Over time, these "primary" methods become less effective and "secondary" production methods may be used. A common secondary method is "waterflood" or injection of water into the reservoir to increase pressure and force the oil to the drilled shaft or "wellbore." Eventually "tertiary" or "enhanced" oil recovery methods may be used to increase the oil's flow characteristics by injecting steam, carbon dioxide and other gases or chemicals into the reservoir. In the United States, primary production methods account for less than 40 percent of the oil 32 produced on a daily basis, secondary methods account for about half, and tertiary recovery the remaining 10 percent. Extracting oil (or "bitumen") from oil/tar sand and oil shale deposits requires mining the sand or shale and heating it in a vessel or retort, or using "in-situ" methods of injecting heated liquids into the deposit and then pumping the liquid back out saturated with oil. Unconventional Oil Reservoirs Oil-eating bacteria biodegrade oil that has escaped to the surface. Oil sands are reservoirs of partially biodegraded oil still in the process of escaping and being biodegraded, but they contain so much migrating oil that, although most of it has escaped, vast amounts are still present— more than can be found in conventional oil reservoirs. The lighter fractions of the crude oil are destroyed first, resulting in reservoirs containing an extremely heavy form of crude oil, called crude bitumen in Canada, or extra-heavy crude oil in Venezuela. These two countries have the world's largest deposits of oil sands. On the other hand, oil shales are source rocks that have not been exposed to heat or pressure long enough to convert their trapped hydrocarbons into crude oil. Technically speaking, oil shales are not always shales and do not contain oil, but are fined-grain sedimentary rocks containing an insoluble organic solid called kerogen. The kerogen in the rock can be converted into crude oil using heat and pressure to simulate natural processes. The method has been known for centuries and was patented in 1694 under British Crown Patent No. 330 covering, "A way to extract 33 and make great quantities of pitch, tar, and oil out of a sort of stone." Although oil shales are found in many countries, the United States has the world's largest deposits (Lambertson, 2008). Classification of Crude oil Some marker crudes with their sulfur content (horizontal) and API gravity (vertical) and relative production quantity. The petroleum industry generally classifies crude oil by the geographic location it is produced in (e.g., West Texas Intermediate, Brent, or Oman), its API gravity (an oil industry measure of density), and its sulfur content. Crude oil may be considered light if it has low density or heavy if it has high density; and it may be referred to as sweet if it contains relatively little sulfur or sour if it contains substantial amounts of sulfur. The geographic location is important because it affects transportation costs to the refinery. Light crude oil is more desirable than heavy oil since it produces a higher yield of gasoline, while sweet oil commands a higher price than sour oil because it has fewer environmental problems and requires less refining to meet sulfur standards imposed on fuels in consuming countries. Each crude oil has unique molecular characteristics which are revealed by the use of Crude oil assay analysis in petroleum laboratories. Barrels from an area in which the crude oil's molecular characteristics have been determined and the oil has been classified are used as pricing references throughout the world. Some of the common reference crudes are: 34 West Texas Intermediate (WTI), a very high-quality, sweet, light oil delivered at Cushing, Oklahoma for North American oil Brent Blend, consisting of 15 oils from fields in the Brent and Ninian systems in the East Shetland Basin of the North Sea. The oil is landed at Sullom Voe terminal in Shetland. Oil production from Europe, Africa and Middle Eastern oil flowing West tends to be priced off this oil, which forms a benchmark Dubai-Oman, used as benchmark for Middle East sour crude oil flowing to the Asia-Pacific region Tapis (from Malaysia, used as a reference for light Far East oil) Minas (from Indonesia, used as a reference for heavy Far East oil) The OPEC Reference Basket, a weighted average of oil blends from various OPEC (The Organization of the Petroleum Exporting Countries) countries Midway Sunset Heavy, by which heavy oil in California is priced. Western Canadian Select the benchmark crude oil for emerging heavy, high TAN (acidic) crudes (NRC, 2011). There are declining amounts of these benchmark oils being produced each year, so other oils are more commonly what is actually delivered. While the reference price may be for West Texas Intermediate delivered at Cushing, the actual oil being traded may be a discounted Canadian heavy oil—Western Canadian Select— delivered at Hardisty, Alberta, and for a Brent Blend delivered at Shetland, it may be a discounted Russian Export Blend delivered at the port of Primorsk (NYMEX, 2006). Petroleum Industry 35 The petroleum industry is involved in the global processes of exploration, extraction, refining, transporting (often with oil tankers and pipelines), and marketing petroleum products. The largest volume products of the industry are fuel oil and gasoline. Petroleum is also the raw material for many chemical products, including pharmaceuticals, solvents, fertilizers, pesticides, and plastics. The industry is usually divided into three major components: upstream, midstream and downstream. Midstream operations are usually included in the downstream category. Petroleum is vital to many industries, and is of importance to the maintenance of industrialized civilization itself, and thus is a critical concern to many nations. Oil accounts for a large percentage of the world's energy consumption, ranging from a low of 32 percent for Europe and Asia, up to a high of 53 percent for the Middle East, South and Central America (44%), Africa (41%), and North America (40%). The world at large consumes 30 billion barrels (4.8 km3) of oil per year, and the top oil consumers largely consist of developed nations. In fact, 24 percent of the oil consumed in 2004 went to the United States alone, though by 2007 this had dropped to 21 percent of world oil consumed. 2.2 Bioremediation Bioremediation is a process used to treat contaminated media, including water, soil and subsurface material, by altering environmental conditions to stimulate growth of microorganisms and degrade the target pollutants. In many cases, 36 bioremediation is less expensive and more sustainable than other remediation (EPA, 2011). Biological treatment is a similar approach used to treat wastes including wastewater, industrial waste and solid waste. Most bioremediation processes involve oxidation-reduction reactions where either an electron acceptor (commonly oxygen) is added to stimulate oxidation of a reduced pollutant (e.g. hydrocarbons) or an electron donor (commonly an organic substrate) is added to reduce oxidized pollutants (nitrate, perchlorate, oxidized metals, chlorinated solvents, explosives and propellants).In both these approaches, additional nutrients, vitamins, minerals, and pH buffers may be added to optimize conditions for the microorganisms. In some cases, specialized microbial cultures are added (bioaugmentation) to further enhance biodegradation. Some examples of bioremediation related technologies are phytoremediation, mycoremediation, bioventing, bioleaching, landfarming, bioreactor, composting, bioaugmentation, rhizofiltration, and biostimulation. Chemistry of Bioremediation Most bioremediation processes involve oxidation-reduction (Redox) reactions where a chemical species donates an electron (electron donor) to a different species that accepts the electron (electron acceptor). During this process, the electron donor is said to be oxidized while the electron acceptor is reduced. Common electron acceptors in bioremediation processes include oxygen, nitrate, manganese (III and IV), iron (III), sulfate, carbon dioxide and some pollutants 37 (chlorinated solvents, explosives, oxidized metals, and radionuclides). Electron donors include sugars, fats, alcohols, natural organic material, fuel hydrocarbons and a variety of reduced organic pollutants. The redox potential for common biotransformation reactions is shown in the table. Process Reaction Redox potential (Eh in mV) aerobic O2 + 4e− + 4H+ → 2H2O 600 ~ 400 anaerobic denitrification 2NO3− + 10e− + 12H+ → N2 + 6H2O 500 ~ 200 manganese IV reduction MnO2 + 2e− + 4H+ → Mn2+ + 2H2O 400 ~ 200 iron III reduction Fe(OH)3 + e− + 3H+ → Fe2+ + 3H2O 300 ~ 100 sulfate reduction SO42− + 8e− +10 H+ → H2S + 4H2O 0 ~ −150 fermentation 2CH2O → CO2 + CH4 −150 ~ −220 Aerobic bioremediation is the most common form of oxidative bioremediation process where oxygen is provided as the electron acceptor for oxidation of petroleum, polyaromatic hydrocarbons (PAHs), phenols, and other reduced pollutants. Oxygen is generally the preferred electron acceptor because of the higher energy yield and because oxygen is required for some enzyme systems to initiate the degradation process (Norris, 1993). Numerous laboratory and field studies have shown that microorganisms can degrade a wide variety of hydrocarbons, including components of gasoline, kerosene, diesel, and jet fuel. Under ideal conditions, the biodegradation rates of the low- to moderate-weight aliphatic, alicyclic, and aromatic compounds can be very high. As the molecular weight of the compound increases, so does the resistance to biodegradation 38 (Norris, 1993). Common approaches for providing oxygen above the water table include landfarming, composting and bioventing. During landfarming, contaminated soils, sediments, or sludges are incorporated into the soil surface and periodically turned over (tilled) using conventional agricultural equipment to aerate the mixture. Composting accelerates pollutant biodegradation by mixing the waste to be treated with a bulking agent, forming into piles, and periodically mixed to increase oxygen transfer. Bioventing is a process that increases the oxygen or air flow into the unsaturated zone of the soil which increases the rate of natural in situ degradation of the targeted hydrocarbon contaminant (Frutos et al., 2010). Approaches for oxygen addition below the water table include recirculating aerated water through the treatment zone, addition of pure oxygen or peroxides, and air sparging. Recirculation systems typically consist of a combination of injection wells or galleries and one or more recovery wells where the extracted groundwater is treated, oxygenated, amended with nutrients and reinjected. However, the amount of oxygen that can be provided by this method is limited by the low solubility of oxygen in water (8 to 10 mg/L for water in equilibrium with air at typical temperatures). Greater amounts of oxygen can be provided by contacting the water with pure oxygen or addition of hydrogen peroxide (H2O2) to the water. In some cases, slurries of solid calcium or magnesium peroxide are injected under pressure through soil borings. These solid peroxides react with water releasing H2O2 which then decomposes releasing oxygen. Air sparging involves the injection of air under 39 pressure below the water table. The air injection pressure must be great enough to overcome the hydrostatic pressure of the water and resistance to air flow through the soil (Leeson, 2002). Anaerobic bioremediation can be employed to treat a broad range of oxidized contaminants including chlorinated ethenes (PCE, TCE, DCE, VC), chlorinated ethanes (TCA, DCA), chloromethanes (CT, CF), chlorinated cyclic hydrocarbons, various energetics (e.g., perchlorate,[6] RDX, TNT), and nitrate (TX, 2004). This process involves the addition of an electron donor to: 1) deplete background electron acceptors including oxygen, nitrate, oxidized iron and manganese and sulfate; and 2) stimulate the biological and/or chemical reduction of the oxidized pollutants. Hexavalent chromium (Cr[VI]) and uranium (U[VI]) can be reduced to less mobile and/or less toxic forms (e.g., Cr[III], U[IV]). Similarly, reduction of sulfate to sulfide (sulfidogenesis) can be used to precipitate certain metals (e.g., zinc, cadmium). The choice of substrate and the method of injection depend on the contaminant type and distribution in the aquifer, hydrogeology, and remediation objectives. Substrate can be added using conventional well installations, by directpush technology, or by excavation and backfill such as permeable reactive barriers (PRB) or biowalls. Slow-release products composed of edible oils or solid substrates tend to stay in place for an extended treatment period. Soluble substrates or soluble fermentation products of slow-release substrates can potentially migrate via advection and diffusion, providing broader but shorterlived treatment zones. The added organic substrates are first fermented to 40 hydrogen (H2) and volatile fatty acids (VFAs). The VFAs, including acetate, lactate, propionate and butyrate, provide carbon and energy for bacterial metabolism (EPA, 2013). Heavy Metals Heavy metals including cadmium, chromium, lead and uranium are elements so they cannot be biodegraded. However, bioremediation processes can potentially be used to reduce the mobility of these material in the subsurface, reducing the potential for human and environmental exposure. The mobility of certain metals including chromium (Cr) and uranium (U) varies depending on the oxidation state of the material (Ford et al., 2007). Microorganisms can be used to reduce the toxicity and mobility of chromium by reducing hexavalent chromium, Cr(VI) to trivalent Cr (III). Uranium can be reduced from the more mobile U(VI) oxidation state to the less mobile U(IV) oxidation state (Wiliams et al., 2013). Microorganisms are used in this process because the reduction rate of these metals is often slow unless catalyzed by microbial interactions. Research is also underway to develop methods to remove metals from water by enhancing the sorption of the metal to cell walls. This approach has been evaluated for treatment of cadmium, chromium, and lead (Tripathi et al., 2005). Phytoextraction processes concentrate contaminants in the biomass for subsequent removal. Additives 41 In the event of biostimulation, adding nutrients that are limited to make the environment more suitable for bioremediation, nutrients such as nitrogen, phosphorus, oxygen, and carbon may be added to the system to improve effectiveness of the treatment (Adams, 2015). Many biological processes are sensitive to pH and function most efficiently in near neutral conditions. Low pH can interfere with pH homeostasis or increase the solubility of toxic metals. Microorganisms can expend cellular energy to maintain homeostasis or cytoplasmic conditions may change in response to external changes in pH. Some anaerobes have adapted to low pH conditions through alterations in carbon and electron flow, cellular morphology, membrane structure, and protein synthesis (Slonczewski, 2009). Bioremediation can be used to completely mineralize organic pollutants, to partially transform the pollutants, or alter their mobility. Heavy metals and radionuclides are elements that cannot be biodegraded, but can be bio-transformed to less mobile forms. Limitations In some cases, microbes do not fully mineralize the pollutant, Limitations potentially producing a more toxic compound (Wexler, 2014). For example, under anaerobic conditions, the reductive dehalogenation of TCE may produce dichloroethylene (DCE) and vinyl chloride (VC), which are suspected or known carcinogens. However, the microorganism Dehalococcoides can further reduce DCE and VC to the non-toxic product ethane (Boopathy, 2000). Additional 42 research is required to develop methods to ensure that the products from biodegradation are less persistent and less toxic than the original contaminant. Thus, the metabolic and chemical pathways of the microorganisms of interest must be known (Juwarkar et al, 2010)). In addition, knowing these pathways will help develop new technologies that can deal with sites that have uneven distributions of a mixture of contaminants.Also, for biodegradation to occur, there must be a microbial population with the metabolic capacity to degrade the pollutant, an environment with the right growing conditions for the microbes, and the right amount of nutrients and contaminants. The biological processes used by these microbes are highly specific, therefore, many environmental factors must be taken into account and regulated as well. Thus, bioremediation processes must be specifically made in accordance to the conditions at the contaminated site. Also, because many factors are interdependent, small-scale tests are usually performed before carrying out the procedure at the contaminated site. However, it can be difficult to extrapolate the results from the small-scale test studies into big field operations. In many cases, bioremediation takes more time than other alternatives such as land filling and incineration (Vidali, 2001)). Genetic engineering The use of genetic engineering to create organisms specifically designed for bioremediation is under preliminary research (Lovely, 2003). Two category of genes can be inserted in the organism: degradative genes which encode proteins 43 required for the degradation of pollutants, and reporter genes that are able to monitor pollution levels. Numerous members of Pseudomonas have also been modified with the lux gene, but for the detection of the polyaromatic hydrocarbon naphthalene. A field test for the release of the modified organism has been successful on a moderately large scale (Ripp et al., 2000)). There are concerns surrounding release and containment of genetically modified organisms into the environment due to the potential of horizontal gene transfer (Davison, 2005). Genetically modified organisms are classified and controlled under the Toxic Substances Control Act of 1976 under United States Environmental Protection Agency (Sayler and Ripp, 2000). Measures have been created to address these concerns. Organisms can be modified such that they can only survive and grow under specific sets of environmental conditions (Davison, 2005). In addition, the tracking of modified organisms can be made easier with the insertion of bioluminescence genes for visual identification (Shanker et al 2008). 44 CHAPTER THREE 3.0 MATERIALS AND METHODS 3.1 Sample collection The soil samples were collected from a flow station owned by a major upstream operator in an oil polluted site in Okashikpa village, Abacheke community Ohaji/Egbema local government area of Imo state, Nigeria. The soil samples were collected at a depth of 0 – 15cm with the aid of auger. Standard microbiological procedures were employed in collection and handling of the soil samples and they were analyzed within 24hours of collections. 3.2 Materials and instruments Soil samples, Nutrient Agar, fungizone, Sabourad agar, Petri dishes, antibiotics (penicillin G, 0.05 g/l; chloramphenicol, 0.5 g/l and streptomycin, 0.025 g/l) Gloves, cotton, incubator, Aluminium foil, Forceps, electronic balance, Saline (0.85% NaCl), beaker, conical flask, distilled water 3.3 Preparation of Media The mineral salt Agar (MSA) comprised of NaCl, 10.0gram; MgSO4.7H2O, 0.29g; KCl, 0.29g; K2HPO4 ,1.25g;KH2PO4,0.83g; NaNO3,0.42g, Agar, 15g and deionized water, 1L . The pH of the medium was adjusted to 7.2 and 4.0 for bacteria and fungi respectively (Mills et al, 1988; Benker-coker and Ekundayo, 1996). Nutrient agar and Sabourad dextrose agar were prepared following the 45 agar manufacturer’s instructions. The medium was sterilized by autoclaving at 15 psi and 1210C for 15 minutes, allowed to cool, and dispensed into sterile Petri dishes. Solid media were incubated at 30 + 2 0C for 48 h to check for sterility. 3.4 Methods A Enumeration of total heterotrophic bacteria and fungi Total counts of heterotrophic bacteria in soil samples were estimated by spreading aliquots (0.1ml) of decimal serial dilutions of the soil samples onto duplicate nutrient agar plates (pH 7.2) into which were incorporated 10 mg/ml fungizone to inhibit fungal growth. Fungal counts were determined using Sabourad agar (pH 4.5) containing antibiotics (penicillin G, 0.05 g/l; chloramphenicol, 0.5 g/l and streptomycin, 0.025 g/l) to suppress bacterial growth. All plates were incubated at 30+ 2 0C for 72 h (Benker-coker and Ekundayo, 1996). The means (duplicate readings) of colony counts were used to compute number of heterotrophic bacteria and fungi in the original sample (Benker-coker and Ekundayo, 1996) One gram of soil samples was added aseptically to 9 mls of sterile mineral salts medium. Decimal serial dilutions of the soil samples were subsequently made with sterile mineral salts medium and aliquots (0.1 ml) inoculated on the mineral salts agar surface by the spread plate technique. A set of inoculated plates were 46 inverted onto their lids into which each of a Whatman No. 1 filter paper had been placed and saturated with sterile Nigerian Bonny Light blend crude oil as carbon source. The saturated filter paper supplied the crude oil by vapour transfer to the inoculum. Duplicate plates containing no inoculum but carbon source or no carbon source but inoculum served as controls. All plates were incubated at 30 + 2 0C for 7 days. Microorganisms growing on the mineral salts agar plates were counted, purified and isolated. The bacterial isolates were examined for colonial, morphological and biochemical characteristics according to methods described by Carpenter (1977) and Cruickshank et al. (1975). Identification involved the use of the determinative schemes of Buchanan and Gibbons (1974) and Cowan (1974). Fungal isolates were examined macroscopically and microscopically using the needle mounts method. The identification of petroleum hydrocarbon utilizing fungi was made by reference to the Illustrated Genera of Imperfect Fungi (Barnett and Hunter, 1972). B. Identification and confirmation of isolates This was done for each sample by Gram staining followed by examination under the microscope (oil immersion). Further confirmation was by biochemical testing using catalase and coagulase (Cheesbrough, 2005). Gram staining: A colony was picked with a sterile (flamed) inoculating loop and emulsified on a slide to make a smear. It was then air dried and heat fixed by passing over the flame. The slide was then floated with 5 drops of crystal violet 47 and allowed for 1min and then washed off. Five drops of iodine was placed on the slide and allowed for 1min and then washed. Next it was decolorized by tilting in alcohol for 5secs. After these, 5 drops of safranin were placed on the slide and then washed off gently. The slide was allowed to dry and examined under the microscope (x1000). Catalase test: Catalase test was done by putting 2ml of H2O2 in a test tube then, a colony was placed in it to observe for effervescence. Coagulase test: Coagulase test was done by collecting 5ml of venous blood and placed in an EDTA tube and then centrifuged to obtain plasma. A drop of distilled water was dropped on a slide using a dropper and using a flamed wire loop, a colony of the organism that has been checked for positive results of Gram stain was emulsified to make a thick suspension. A loopful of plasma was then added to the suspension and mixed gently (Cheesbrough, 2005). 3.5 Statistical Analysis Statistical analysis was then done using student’s t test with the aid of SPSS version 2.0 48 CHAPTER FOUR RESULTS 4.1 Microbial population of environmental samples from oil polluted sites Bacterial counts (x104) of oil polluted soil and water collected from Ohaji Egbema are presented in table 1, fig 1. Ekugba had the highest soil bacteria load (32.4±2.5 CFU/g) followed by Abaezi (29±3.5 CFU/g) and Abacheke (28.5±2.8 CFU/g) respectively. The bacterial load of Abaezi water was the highest (34±3.2 CFU/ml) followed by Abacheke (33±1.0 CFU/ml) and then Ekugba (24.2±3 CFU/ml). Fungal counts (x106) of oil polluted soil and water collected from Ohaji Egbema are presented in table 2, fig 2. The highest soil fungal load was obtained from Abacheke (26.4±2.8 SFU/g) followed by Abaezi (21±3.2 SFU/g) and Ekugba (21±2.2 SFU/g ) respectively. Ekugba had the highest fungal populationin water (25.2±1.0 SFU/ml) followed by Abacheke (25±2.0 SFU/ml ) and then Abaezi (24±2.4 SFU/ml) 49 Table 4.1: Bacterial counts (x104) of oil polluted soil and water collected from Ohaji Egbema Abacheke Abaezi Ekugba Control F value p value Soil (CFU/g) 28.5±2.8 29±3.5 32.4±2.5 7.2±2.3 83.81 0.000 water (CFU/mL) 33±1.0 34±3.2 24.2±3 10±2.7 89.63 0.000 Table 4.2: Fungal counts (x106) of oil polluted soil and water collected from Ohaji Egbema Abacheke Abaezi Ekugba Control F value p value Soil (SFU/g) 26.4±2.8 21±3.2 21±2.2 8.2±2.1 43.75 0.000 Water (SFU/ml) 25±2.0 24±2.4 25.2±1.0 10.6±2.5 59.06 0.000 50 Fig 1: Bacterial counts (x104) of oil polluted soil and water collected from Ohaji Egbema Fig 2: Fungal counts (x106) of oil polluted soil and water collected from Ohaji Egbema 0 5 10 15 20 25 30 Abacheke Abaezi Ekugba Control soil water 0 5 10 15 20 25 30 35 40 Abacheke Abaezi Ekugba Control soil water 51 4.2 Cultural, microscopic and biochemical characteristics of bacteria and fungi isolated from oil polluted sites in Ohaji Egbema The morphological and biochemical characteristics of bacteria isolated from Ohaji Egbema are presented in Table 4.3. These include Staphylococcus aureus, Micrococcus species, Acinetobacter species, Pseudomonas stutzeri and Bacillus pumillus. The genera of Staphylococcus, Micrococcus, Acinetobacter and Bacillus are gram positive while pseudomonas is gram negative. The microscopic and cultural characteristics of fungi isolated from oil polluted areas in Ohaji egbema are presented in Table 4.4. The fungi appeared in different colours in SDA. The fungi identified are Geniculosporangium serpens, Pleurophrangium species, Haplosporangium species, Penicilium italicum, Aspergillus niger, Lagellospora penicilloides, Penicillium oxalicum, Streptothrix atra, Articulosporium inflate, Aspergillus flavus, Halosporangium pavum, Aspergillus fumigatus, Aspergillus rapens and Geotrichum albidan. 52 Table 4.3: Morphological and biochemical characteristics of bacteria isolated from oil polluted sites in Ohaji Egbema. Characteristics Isolate A Isolate B Isolate C Isolate D Isolate E Cultural Colony shape Irregular Circular Oval Circular Irregular Elevation Flat Raised Raised Raised Flat Edge Entire Entire Entire Entire Undulate Pigmentation Yellow Cream Cream Yellow White Gram stain + + - - + Cell shape Rod Spherical Rod Rod Rod Spore stain + - - - + Catalase + + + + + Motility - - - + + Fermentation Lactose + + - - + 53 Probable bacteria Staphylococcus aureus Micrococcus sp. Acinetobacter sp. Pseudomonas stutzeri Bacillus pumillus Table 4.4: Microscopic and cultural characteristics of fungi isolated from oil polluted areas in Ohaji egbema. 54 CHAPTER FIVE DISCUSSION Microbiological study carried out on the environmental samples revealed the presence of both bacteria and fungi in the soil and water tested. This confirms that microorganisms are ubiquitous, diverse in nature and are able to adapt in extreme environments (Watanabe et al., 2002). Oil polluted sites do harbour a vast array of microbial flora, hence diversity in all the types of bacteria and fungi identified in this study. The existence of the microorganisms in the oil polluted environment suggests that the isolated bacteria could utilize the oil as energy and carbon source (Das and Mukherjee, 2007). The high loads of the microbes indicate that the indigenous microbes in the polluted soil and water have catabolized part of the oil and used it to grow. According to Ojo (2006), the activities of the indigenous microbes could be responsible for thee bioremediation of the polluted sites. The unpolluted samples had the least population of bacteria and fungi in this study. This low population means that natural unpolluted water and soil environment do not contain abundant microorganisms because there was no carbon and energy source in the environment that could encourage them to multiply. Similar reports were published by Boboye et al. (2010). They concluded that hhigh numbers of certain oil-degrading microorganisms from an environment implies that those organisms are the active degraders of that oil. 55 Bacterial counts (x104) of oil polluted soil and water collected from Ohaji Egbema showed high load of hydrocarbon utilizing bacteria such as bacillus and pseudominas. Soil bacteria load ranged from 32.4±2.5 CFU/g in soil from Abaezi to 28.5±2.8 CFU/g in Abacheke soil. The water bacteria load ranged between 34±3.2 CFU/ml in water from Abaezi to 24.2±3 CFU/ml in water from Ekugba. Fungal counts (x106) of oil polluted soil and water collected from Ohaji Egbema ranged from 26.4±2.8 SFU/g in Abacheke soil to 21±2.2 SFU/g in Ekugba soil and 25.2±1.0 SFU/ml in water from Ekugba 24±2.4 SFU/ml in Abaezi. These populations were significantly higher than those of non-polluted areas which served as control. The microbial populations obtained during this study showed that bacteria counts were higher than fungi counts. This corroborates the findings of Onafide and Abubakar (2007) and Olukunle et al. (2012) and this could be attributed to the nutrient status of the soils and water. The bacteria identified in oil polluted soil and water collected from Ohaji egbema include the gram positive bacteria: Staphylococcus aureus, Micrococcus species, Acinetobacter species, Bacillus pumillus and the gram negative Pseudomonas stutzeri. Bacillus pumillus was the only spore forming bacteria isolated. Micrococcus and Pseudomonas species are the most extensively studied among oil degraders because these bacteria have the ability to degrade so many different contaminants (Wackett, 2003). 56 The fungi identified are Geniculosporangium serpens, Pleurophrangium species, Haplosporangium species, Penicilium italicum, Aspergillus niger, Lagellospora penicilloides, Penicillium oxalicum, Streptothrix atra, Articulosporium inflate, Aspergillus flavus, Halosporangium pavum, Aspergillus fumigatus, Aspergillus rapens and Geotrichum albidan. Some of the fungi are similar to those identified in a previous study that was conducted to assay for degrading activity of fungi on kerosene, diesel and petrol using classical selective enrichment method (Olukunle et al., 2011). Techniques for identifying and characterizing such organisms for bioremediation are important and further research to assay for the degradative ability of the microorganisms is necessary for effective and efficient ex situ bioremediation process. CONCLUSION The results obtained in this study have revealed that oil-degrading bacteria and fungi can be isolated from oil polluted sites. These bacteria and fungi are indigenous in the polluted sites and they are responsible for the degradation of oil. The traditional techniques of identification however enable microbiologists to understand the basic principles of identification on microorganisms which are based on morphological, metabolic and physiologic traits. Traditional techniques are useful especially in laboratories where there are no molecular biology 57 facilities. 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Current Opinion in Biotechnology. 24 (3): 489–97. Raw data Soil bacteria count (x10 4) CFU/mL S/N Abacheke Abaezi Ekugba Control 1 29.58 32.15 33.02 6.58 2 30.5 23.18 36 4.49 3 25.28 30.7 29.29 10.65 4 31.39 28.55 32.69 8.04 5 25.77 30.45 31.02 6.26 65 water bacteria count (x10 4) CFU/mL S/N Abacheke Abaezi Ekugba Control 1 32.31 30.9 22.33 11.97 2 33.76 38.24 23.45 6.33 3 31.72 31.86 21.08 12.84 4 33.09 36.55 28.76 10.7 5 34.14 32.48 25.39 8.19 Soil fungal count (x10 6 ) SFU/g S/N Abacheke Abaezi Ekugba Control 1 24.02 24.66 18.59 9.32 2 23.21 21.18 20.91 11.31 3 28.83 19.38 22.71 6.13 4 26.47 23.25 23.68 6.87 5 29.5 16.55 19.14 7.41 water fungal count (x10 6 ) SFU/g S/N Abacheke Abaezi Ekugba Control 1 24.07 21.24 26.49 10.32 2 27.56 24.7 24.96 6.55 3 23.68 25.83 24.77 11.29 4 26.7 26.53 25.88 11.59 5 23.02 21.72 23.92 13.26