ABSTRACT
This study investigated the use of snail (Achatina achatina) shell; an alternative adsorbent to natural matrices (such as polyvinyl alcohol and polyurethane) and agricultural wastes (such as sugarcane bagasse, orange peels, spent tea leaves, corn cobs and cotton seed hull) as a carrier to immobilize Pseudomonas putida and Bacillus subtilis for the degradation of naphthalene in synthetic wastewater. The hydrocarbon-degrading bacteria (HDB) used for the study were isolated from a refinery effluent and were further cultured in a nutrient medium. Naphthalene concentrations of 10, 20, 30, 40 and 50 mg/dm3 were studied at constant temperature of 30 oC, pH of 5, 7 and 9 and adsorbent dosage of 2, 3 and 5 g for 72 hours in batch mode. The naphthalene concentrations were determined using UV-spectrophotometer (Agilent CARY 300, USA) and the products of the degradation were determined using Fourier Transform Infra-red spectroscopy (CARY 630, USA) to assess the adsorption and simultaneous adsorption-biodegradation capacity and equilibrium. The results showed that optimal adsorption and biodegradation occurred at naphthalene concentration of 50 mg/dm3 and adsorbent dosage of 2 g. For the effect of pH, optimal adsorption took place at basic pH of 9, while the optimal biodegradation occurred at neutral pH of 7. The ranges of the percentage removal for the Pseudomonas putida and Bacillus subtilis immobilized on treated snail shell were: 73.23 – 82.12 % and 66.33 – 76.79 % respectively while on untreated snail shell were: 63.26 – 73.11 % and 60.17 – 74.46 % respectively. These values were significantly higher (P<0.05) than the values obtained from the adsorption of naphthalene which ranges were: 50.62 – 65.20 % and 57.05 – 67.10 % respectively for untreated and treated snail shell. The results of the equilibrium study indicated favourable adsorption and biodegradation, the R2 values obtained were: 0.9933,
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0.9997, 1.0000, 0.9973, 0.9947 and 1.0000. This indicated that the sorbent systems were well fitted to both the Langmuir and the Freundlich models. Therefore, snail shell can be employed as low-cost adsorbent and solid support matrix for the immobilization of microorganisms for the remediation of hydrocarbon contaminants.
TABLE OF CONTENTS
Cover Page i
Fly Leaf ii
Title Page iii
Declaration iv
Certification v
Dedication vi
Acknowledgement vii
Abstract viii
Table of Contents x
List of Tables xv
List of Figures xvi
List of Appendices xvii
Abbreviations xxi
CHAPTER ONE 1
1.0 INTRODUCTION 1
1.1 Background Information 1
1.2 Statement of Problem 2
1.3 Justification 4
1.4 Aim 4
1.5 Objectives 4
CHAPTER TWO 6
2.0 REVIEW OF RELATED LITERATURE 6
2.1 Environmental Pollution 6
2.2 Crude Oil (Petroleum) 7
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2.3 Hydrocarbons 7
2.3.1 Polycyclic aromatic hydrocarbons (PAHs) 8
2.3.2 Naphthalene 9
2.4 Biodegradation 9
2.5 Factors Affecting Biodegradation 10
2.5.1 Temperature 10
2.5.2 Nutrients 11
2.5.3 Oxygen 11
2.5.4 pH 12
2.5.5 Concentration of petroleum hydrocarbon 12
2.6 Enhancement of Biodegradation 13
2.6.1 Biostimulation 13
2.6.2 Bioaugumentation 13
2.7 Microbial Degradation of Petroleum Hydrocarbons 14
2.8 Africa Giant Snail (Achatina achatina) Shell 18
2.8.1 Solid support 19
2.9 Equilibrium studies 19
2.9.1 Adsorption and adsorption-biodegradation isotherm 19
2.9.1.1 Langmuir isotherm model 20
2.9.1.2 Freundlich isotherm model 21
CHAPTER THREE 23
3.0 MATERIALS AND METHODS 23
3.1 Materials 23
3.1.1 Snail shell collection and identification 23
3.1.2 Collection of refinery effluents samples 23
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3.2 Chemicals and Reagents 23
3.3 Preparation and Pre-treatment of Adsorbent (Snail Shell) 24
3.3.1 Carbonization process 24
3.3.2 Activation process 24
3.4 Determination of Physicochemical Properties of the Snail Shell 25
3.4.1 pH 25
3.4.2 Bulk density 26
3.4.3 Moisture content 26
3.4.4 Ash content 26
3.5 Analysis of Snail shell 27
3.6 Isolation of Bacillus subtilis and Pseudomonas Putida from the Refinery Effluent 27
3.6.1 Inoculation 27
3.6.2 Identification of the isolates 28
3.6.3 Screening the capacity of Bacillus subtilis and Pseudomonas putida to degrade hydrocarbons 29
3.7 Preparation of Inoculum for Biodegradation 29
3.8 Batch Adsorption and Biodegradation Studies 30
3.8.1 Effect of initial naphthalene concentration 32
3.8.2 Effect of adsorbent dosage 32
3.8.3 Effect of pH 32
3.9 Sample Analysis 33
3.10 Statistical Analysis of Data 33
CHAPTER FOUR 35
4.0 RESULTS 35
4.1 Physicochemical Properties of the Snail (Achatina achatina) Shell 35
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4.2 Composition of the Snail (Achatina achatina) Shell 35
4.3 Identification of Bacillus subtilis and Pseudomonas putida Isolates based on
Microgen kit 35
4.4 Crude Oil Utilization by the Isolates of Bacillus subtilis and Pseudomonas putida 39
4.5 Adsorption and Biodegradation of Naphthalene 39
4.5.1 Effect of initial naphthalene concentration 39
4.5.2 Effect of adsorbent dosage 42
4.5.3 Effect of pH 42
4.6 FTIR Analysis of Naphthalene and its Metabolites 42
4.7 Comparisons of Adsorption of Naphthalene and its Metabolites 47
4.8 Adsorption and Biodegradation Isotherms 47
CHAPTER FIVE 63
5.0 DISCUSSIONS 63
5.1 Physicochemical Properties of Achatina achatina 63
5.1.1 pH 63
5.1.2 Moisture content 63
5.1.3 Ash content 63
5.1.4 Bulk density 64
5.2 Composition of Snail Shell 64
5.3 Utilization of Crude Oil as Source of Carbon by the Wild Strains of Bacillus
subtilis and Pseudomonas putida 64
5.4 Adsorption and Biodegradation of Naphthalene 65
5.4.1 Effect of initial concentration 65
5.4.2 Effect of adsorbent dosage 66
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5.4.3 Effect of pH 67
5.5 FTIR Spectra of Naphthalene and its Metabolite 67
5.6 Comparison of Adsorption of Naphthalene and its Biodegradation 68
5.7 Equilibrium Studies 68
5.7.1 Langmuir isotherm model 68
5.7.2 Freundlich isotherm model 69
CHAPTER SIX 70
6.0 CONCLUSION AND RECOMMENDATIONS 70
6.1 Conclusion 70
6.2 Recommendations 71
REFERENCES 72
APPENDICES 84
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List of Tables
Table 4.1: Physicochemical Properties of Snail hell 36
Table 4.2: Composition of Snail Shell 37
Table 4.3: Identity of Bacillus subtilis and Pseudomonas putida isolates Based on Microgen 38
Table 4.4: Growth of Bacillus subtilis and Pseudomonas putida isolates on
Mineral medium containing Crude Oil 40
Table 4.5: Comparison of Adsorption of Naphthalene and its Metabolites 48
Table 4.6: Correlation Parameters of Langmuir and Freundlich Isotherms 55
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background Information
Petroleum hydrocarbons and their other derivatives are majorly the energy source of various industrial, automobiles and domestic use. Petroleum (crude oil) is a complex mixture of several organic compounds such as aromatic cyclic hydrocarbons, complex branched aliphatic and cyclic alkanes as well as residual substances. Some of these do not degrade easily, and as such are termed recalcitrant (Nilanjana and Preethy, 2011). The increasing soil, surface and groundwater pollution through the release of petroleum hydrocarbons has been seen as one of the major issues in environmental pollution (Shah and Bhatt, 2006). Petroleum products have a long-term impact on the aquatic, soil, natural resources, and human health. Most of these contaminants have been found to be carcinogenic and toxic, and they easily find their way into the food chain (Farrington and McDowell, 2004).
Hydrocarbons produced by natural activities are usually present at trace levels, whereas hydrocarbons originating from different anthropogenic activities are present at higher levels, especially in industrial and transportation associated areas (Okedeyi et al., 2013).
In most cases, the pollutant released through industrial emissions are deliberate and well regulated, while cases such as chemical spills are mostly accidental and largely unavoidable (Akpofure et al., 2000). Also, anthropogenic activities from several auto mechanic workshops increasingly contaminate the environment with various petroleum products and motor spent oil such as diesel, engine oil and petrol (Yahaya, 2016).
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There are several methods for removing petroleum hydrocarbons such as physical and chemical methods, but these methods have limitations and are very expensive. However, biodegradation as a method involves the decomposition of organic pollutants by microorganisms into products that are non-toxic and environmentally friendly, which can enter into trophic levels of food chain without posing any challenges to live. This is an adequate method for remediation (Plaza et al., 2005), and it is considered as a more sustainable method than excavation (Penn et al., 2002; Plaza et al., 2005). The biodegradation of petroleum hydrocarbons in the environment is found to be a slow complex process that depends mainly on the nature and amount of the hydrocarbons present in the contaminants. It is also influenced by factors such as degrading microbial community, temperature, and nutrients (Ekpo and Udofia, 2008). The addition of nutrients like nitrogen and phosphorus are essential for the enhancement of biodegradation process (Wang, 2011). The success of this technology depends on the ability to establish and maintain conditions that favour enhanced oil biodegradation rates in the polluted environment (Nilanjana and Preethy, 2011).
1.2 Statement of Problem
Petroleum hydrocarbons are recalcitrant pollutants that do not degrade easily and are deleterious to human health, animals, and plants due to their possible carcinogenic and mutagenic actions. The release of oil or hydrocarbons into the environment either by accident or through various anthropogenic activities is a major cause of water and soil pollution (Nilanjana and Preethy, 2011). In most mechanic workshops and automobiles, several liters of waste oil are generated daily and discharged carelessly into the environment (Adegoroye, 1997; Fagboya, 1997; Adelowo et al., 2006). Oil spillage during
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exploration and pipelines vandalization without effective clean-up is capable of destroying the natural habitat of an oil-rich region(United States Environmental Protection Agency,1996), especially as had been witnessed in the Niger Delta region of Nigeria (Oluyege and Oluyemi, 2005). Adelowo et al. (2006) reported that one litre of petroleum hydrocarbon is capable of contaminating a million litres of fresh water.
Several technologies like mechanical and physicochemical processes such as evaporation, burying, dispersion, and washing are the commonly employed remediation for an oil spill contamination. However, the problem of incomplete decomposition or breakdown of contaminants can occur, and moreover the technologies are comparatively expensive when compared to biodegradation (Nilanjana and Preethy, 2011). Biodegradation is the most efficient method for hydrocarbon degradation but the process is very slow. Combining biodegradation with adsorption makes degradation to be completed within a short period of time. Several studies on adsorption-biodegradation method have been reported, and these have drawn great attention (Agarry and Aremu, 2012; Lin et al., 2015). Solid media such as activated carbon, kaolin, periwinkle shell, orange peel, groundnut shell and sugarcane bagasse have been extensively used for mineralizing hydrocarbons (Agarry and Aremu, 2012; Lin et al., 2015), but so far there is little or no report on snail shell as solid media (adsorbent) for immobilizing microorganisms for degradation of hydrocarbons. Thus, this study sought to evaluate the removal of mineralization of petroleum hydrocarbon by simultaneous adsorption-biodegradation using snail shell incorporated bacteria to achieve synergistic effects.
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1.3 Justification
Biodegradation is an eco-friendly and cost-effective alternative to all other methods of degradation. The conventional physicochemical remedies have high expenses and can generate products or deposits that are toxic to the biota (Bidoia et al., 2010). However, biodegradation is non-invasive as it involves complete transformation of the pollutants into carbon dioxide, inorganic or simpler inorganic compounds (Nilanjana and Preethy, 2011). Also, combining high effectiveness and ease; bioremediation processes represent a highly important way of recovering polluted areas among a few other cleaning-up techniques (Bidoia et al., 2010). Moreover, the remediation technique adopted in this study is simple and cheap, as the snail shell to be used is available locally and in abundance.
1.4 Aim
This research aimed at investigating the removal of naphthalene by adsorption and simultaneous adsorption-biodegradation using snail shell.
1.5 Objectives
The aim of this research will be achieved through the following objectives:
i. Determination of the physicochemical properties of the snail shell;
ii. Composition of the snail shell using x-ray fluorescence (XRF);
iii. Isolation and identification of bacteria capable of utilizing hydrocarbon from petroleum contaminated wastewater;
iv. Optimization of process parameters (initial hydrocarbon concentration, adsorbent dosage, and pH) for the mineralization of hydrocarbon;
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v. Comparison of adsorption of naphthalene on snail shell and its biodegradation using snail shell incorporated with bacteria; and
vi. Evaluation of the performance of the snail shell using some selected models (Langmuir and Freundlich isotherms).
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