TABLE OF CONTENTS
Title page
Certification
Dedication
Acknowledgment
Table of Contents
List of Figures
List of Tables
Abstract
CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW
1.1 Introduction
1.1.1 Statement of the problem
1.1.2 Aim of the study
1.1.3 Research objectives
1.2 Literature Review
1.2.1 Crude oil and kerosene
1.2.2 Nitrification
1.2.3 Nitrifying bacteria
1.2.4 Isolation of nitrifying bacteria
1.2.5 Utilization of crude oil and kerosene by the autotrophic nitrifying bacteria: Nitrosomonas sp and Nitrobacter sp
1.3 Effects of Environmental Factors on the Nitrification Process and on the Growth of Nitrifying Bacteria
1.4 Characteristics of Nitrifying Bacteria (Cell and Colonial Morphology)
1.4.1 Ammonia-oxidizing bacteria
1.4.2 Nitrite oxidizing bacteria
1.4.3 Nitrosomonas sp and Nitrobacter sp.
1.4.4 Nitrosomonas sp
1.4.5 Nitrobacter sp
1.5 Toxicity of Crude Oil and Kerosene
1.6 Toxicity Indices
1.7 Enzyme Activities and Substrate Degradation
1.8 Bioavailability
CHAPTER TWO: MATERIALS AND METHODS
2.1 Sample Collection
2.2 Preparation of Samples
2.3 Preparation of Media
2.3.1Preparation of Winogradsky broth media
2.3.2Preparation of Winogradsky agar media
2.3.3 Preparation of Griess – Ilosvay reagent
2.4 Isolation of Nitrifying Bacteria
2.4.1 Purification of isolates
2.5 Identification of isolates
2.5.1 Morphological Identification
2.5.2 Colonial morphology
2.5.3 Cell morphology
2.5.4 Motility test
2.5.5 Gram staining
2.6 Physiological Characterization
2.6.1 Ammonia oxidation test
2.6.2 Nitrite oxidation test
2.7 Biochemical Characterization
2.7.1 Catalase test
2.7.2 Urease test
2.7.3 Sugar fermentation test
2.7.4 Oxidase test
2.7.5 Indole test
2.7.6 Coagulase test
2.7.7 Citrate utilization test
2.7.8 Nitrate reduction test
2.7.9 Methyl red test
2.7.10 Voges-Proskauer test
2.8 Preparation and standardization of inoculum
2.9 Effect of Crude Oil and Kerosene on the Growth of the Isolates.
2.10 Determination of the Effective Concentrations (EC50) and the lethal Time ( LT50 ) of Crude Oil and Kerosene for the Isolates
2.11 Effects of Crude Oil and Kerosene on the Oxidation of Nitrite to Nitrate
2.12 Analysis of Results
CHAPTER THREE: RESULTS
3.1Characterization and identification of isolates
3.2 Effect of different concentrations of crude oil on the growth of Nitrosomonas sp
3.3 Effect of different concentrations of kerosene on the growth of Nitrosomonas sp.
3.4 Effect of different concentrations of crude oil on the growth of Nitrobacter sp
3.5 Effect of different concentrations of kerosene on the growth of Nitrobacter sp.
3.6 The effective concentrations (EC50) of crude oil and kerosene for Nitrosomonas sp.
3.7 The effective concentrations (EC50) of crude oil and kerosene for Nitrobacter sp.
3.8 The lethal time ( LT50) at different concentrations of crude oil and kerosene for Nitrosomonas sp
3.9 The lethal time ( LT50 ) at different concentrations of crude oil and kerosene for Nitrobacter sp.
3.10 Effects of different concentrations of crude oil on the oxidation of nitrite to nitrate.
3.11 Effecst of different concentrations of kerosene on the oxidation of nitrite to nitrate.
CHAPTER FOUR: DISCUSSION AND CONCLUSION
4.1 Discussion
4.2 Conclusion
REFERENCES
ABSTRACT
Crude oil and kerosene have useful applications in the society and are, therefore, used as sources of income and energy. However, the spills arising from crude oil and kerosene in our environment are becoming a visible problem in our ecosystem. Crude oil and kerosene pollute our farmlands and other arable lands and may negatively affect the growth and activities of nitrifying bacteria responsible for nitrification process, leading to loss of soil fertility. Considering the important roles played by nitrifying bacteria in soil fertility in our ecosystem, the assessment of the response of the nitrifying bacteria to pollution stress exerted by crude oil and kerosene on them was investigated. Two genera of nitrifying bacteria were isolated from the soil and were identified as Nitrosomonassp and Nitrobacter sp based on their morphological, biochemical and physiological characteristics.Nitrosomonas sp was isolated using Winogradsky medium for nitrification phase I whileNitrobacter sp was isolated using Winogradsky medium for nitrification phase II. A range of crude oil and kerosene concentrations (in triplicates) were prepared and introduced into sterile Winogradsky salt broth to arrive at final concentrations of 0.5%, 1%, 2%, 5% and 10% (v/v) respectively. The flasks were thereafter inoculated with standardized pure cultures of Nitrosomonassp and Nitrobacter sp using a uniform inoculum size of 1ml of 1.5 x106 cells for Nitrosomonas sp and 1.2 x106 cells for Nitrobacter sp and incubated for 120 hours. The result of the toxicity test carried out on these isolates showed that the sensitivity of each test organism was a function of both the contact time and concentrations of crude oil and kerosene. The effect of different concentrations of crude oil and kerosene on the oxidation of nitrite to nitrate showed that oxidation of nitrite to nitrate was hindered at higher concentrations of the two pollutants, but, at lower concentrations, crude oil and kerosene did not hinder oxidation of nitrite to nitrate. Information generated from various tests can be of use in the management of pollution for the purpose of prediction of environmental effects of crude oil and kerosene, comparison of toxicants or organisms or test conditions and regulation of crude oil and kerosene spills. For instance this study will help us to provide information for regulatory purposes that a soil contaminated with certain percentage of crude oil or kerosene should not be used for agricultural purposes or practices.
CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
1.1 Introduction
Crude oil and kerosene have useful applications in our society and are, therefore, used as
source of income and energy. However, the spill arising from crude oil and kerosene dumped into our environment is becoming a visible problem in our ecosystems ( Njoku et al., 2009). The environment is increasingly exposed to changes resulting from both crude oil and refined petroleum products contamination. These changes could be drastic and as such affect the ecosystem substantially (Ikhajiagbe and Anoliefo, 2010). Crude oil and its refined products can be released into the environment from a number of sources, which can pollute both land and water (Jane, 2010). Refined petroleum products result when crude oil is split into fractions such as petrol, kerosene, diesel oil, engine oil and many other petroleum products by a process called fractional distillation. Crude oil, which has accumulated underground, can reach the surface if not contained by impermeable rock.
Apart from this release of crude oil, the main sources of crude oil and refined petroleum products such as petrol, kerosene, diesel and motor engine oil, released on land comes from the disposal of waste motor oil, the leaking of storage tanks, and other spillages and accidents during the transportation of crude oil and other petroleum-based products (Raina et al., 2009). The spill arising from crude oil and refined petroleum products dumped into our environment is becoming a visible problem in our ecosystem. Crude oil and kerosene can pollute our farmlands and other arable lands and may, negatively, affect the growth and activities of nitrifying bacteria responsible for nitrification process, leading to loss of soil fertility (John et al., 2011). These contaminants/pollutants may contain components that may be toxic to soil microorganisms including the nitrifying bacteria and as a result may display some levels of toxicity on the growth of the nitrifying bacteria indigenous to the soil thereby affecting their activities in the soil
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