ABSTRACT
The bioremediation of petrochemical contaminated soil using indigenous Pseudomonas species was studied. The objective of this study is to monitor the degradation potentials of the consortium of indigenous Pseudomonas spp on petroleum hydrocarbons and their metabolic compounds. This research was carried out for 28 days under designated periods tagged (T0 – T4) at 7 days intermittent sub-sampling. Twelve uniform 3cm X 3cm sterile plastic boxes, each containing100g soil and another plastic box (3cm X 3cm) with 100g soil as control were used. These boxes were in four sets of three boxes each. Three sets were treated with varying concentrations of individual indigenous Pseudomonas spp and the fourth set was treated with their consortium. Each containing plastic box was labeled P1c1, P1c2, P1c3, P2c1, P2c2, P2c3, P3c1, P3c2, P3c3, P1P2P3c1, P1P2P3c2, P1P2P3c3 and control. The indigenous microorganisms were isolated using standard microbiological procedures and molecular identification technique. Optimization of growth conditions for Pseudomonas spp were also carried out under varying conditions of pH, moisture content, temperature and nutrient (N:P) using standard microbiological procedures. The physiochemical properties of the petrochemical contaminated soil and control soil (uncontaminated soil located eighty-five meters away from the petrochemical contaminated area at Umurolu along East-West Road, Eleme in Port Harcourt, Rivers state, Nigeria) were tested and compared for levels of the pollution using standard laboratory procedure of American Public Health Association (APHA). Mineral salt medium (MSM) was used during the bioremediation. The bacterial counts of the Pseudomonas species during the bioremediation was determined using standard microbiological procedures while remediation of total petroleum hydrocarbons (TPH) and assessment of metabolic compounds during biodegradation were carried out by gravimetric technique. The identified microorganisms were P. aeruginosa, P. putida, and P. mendocina. Optimization results showed highest microbial growth of 1.83 x 107, 1.92 x 107, 1.88 x 107(cfu/g) at temperature (30oC), 1.78 x 107, 1.82 x 107, 1.94 x 107(cfu/g) at pH 7, 1.94 x 107, 1.88 x 107, 1.79 x 107 (cfu/g) at moisture content (20%), and 2.01 x 107, 1.94 x 107,1.73 x 107 (cfu/g) at N:P ratio (10:1) by P. aeruginosa, P. putida, and P. mendocina respectively. The physiochemical properties of the soil sample were affected due to the pollution level compared to the control soil. Highest bacterial counts of P. aeruginosa, P. putida, and P. mendocina and their consortium were recorded as 2.90 x 107 (cfu/g), 2.95 x 107 (cfu/g), 2.83 x 107 (cfu/g), and 4.00 x 107 (cfu/g), respectively which increased with increase in time. The results on highest percentage remediation level and rate of remediation of the TPH showed P. putida (74.59% and 0.20 kgTPH/wk), P. aeruginosa (67.57% and 0.18 kgTPH/wk), P. mendocina (61.62% and 0.16 kgTPH/wk) and consortium (80.81% and 0.21 kgTPH/wk) which indicated a decrease in TPH level with increase in time. Assessment on the metabolic compound during the degradation showed the maximum percentage reductions with increase in time which include: saturated hydrocarbons (62%, 75%, 68%, and 81%), phenolic compounds (86.25%, 87.50%, 91%) and 92%), asphaltenes and polar compounds (94.06%, 95.05%, 96.53%, and 97.03%), and aromatic compounds (88.89%, 94.44%, 94.42%, and 97.22%) by P. mendocina, P. putida, P. aeruginosa, and the consortium respectively. This study shows that the petroleum hydrocarbon and their metabolic compounds were largely degraded by the consortium of indigenous Pseudomonas spp and therefore recommended for future bioremediation studies.
TABLE OF CONTENTS
.
Title
Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table
of Contents vi
List of Tables xi
List of Figures xii
Abstract
xiii
CHAPTER 1: INTRODUCTION 1
1.1 Background
of Study 1
1.2 Statement of Problem 2
1.3 Justification of Study 3
1.4 Aim
and Objectives 4
1.5 The
Main Objectives of the Study 4
CHAPTER 2: LITERATURE
REVIEW 5
2.1 Spreading of Polycyclic Aromatic
Hydrocarbons (PAHS) in Nature 5
2.2 Contamination of Soils 6
2.3 Health Impact of Polycyclic Aromatic
Hydrocarbons (PAHS) 7
2.3.1.
Impact of PAHS on human 8
2.3.2.
Impact of PAHS on animal 11
2.3.3 Impact of PAHS on plant 13
2.4 Polycyclic Aromatic Hydrocarbons as
Constituent of Petroleum
Contaminants 13
2.4.1 Number of rings in PAHs compounds 18
2.4.2 Interactions between soil and hydrocarbons 23
2.5 Biodegradation of Hydrocarbons 25
2.5.1 Role of indigenous microorganisms in
remediation of contaminated soils 25
2.5.2 Factors affecting the rate of
biodegradation of polyaromatic hydrocarbons 27
2.5.2.1 Temperature 27
2.5.2.2 Soil Characteristics 28
2.5.2.3 pH 28
2.5.2.4 Oxygen 29
2.4.2.5 Nutrient availability 30
2.5.2.6 Microorganisms number and catabolism evolution 30
2.5.2.7 Consortium of microorganisms 31
2.5.2.8 Bioavailability 32
2.5.2.9 Contaminant characteristics 33
2.5.2.10 Toxicity of end-products 34
2.5.2.11 Moisture 35
2.5.2.12 Organic matter 35
2.5.2.13 Oil surface and
concentration 35
2.5.2.14 Salinity 36
2.6 Bioremediation 36
2.6.1 Bioremediation
techniques 38
2.6.1.1 Biological analysis 39
2.6.1.1.1 Soil respirometry 39
2.6.1.1.2 Luminescence techniques 39
2.6.1.1.3 Dehydrogenase activity 40
2.6.1.2 Chemical analysis 40
2.6.1.2.1 Gas chromatography (GC) 40
2.6.1.2.2 Gas chromatography/mass
spectroscopy (GC/MS) 41
2.6.1.2.3 Gas chromatography/flame
ionization detection (GC/FID) 41
2.6.1.2.4 Fluorescence analysis 41
2.6.1.2.5 Use of internal petroleum
biomarkers 42
2.6.1.2.6
Total petroleum hydrocarbon/infrared spectroscopy (TPH/IRS) –
Total petroleum hydrocarbon/gas
chromatography (TPH/GC) 42
2.6.1.2.7 Gravimetric analysis 43
2.7 Pseudomonas spp. Involved in Biodegradation 44
2.7.1 Pseudomonads classification 45
2.7.2 General characteristics 45
2.8 Response to oil
Contamination 48
2.8.1 Metabolism of polycyclic aromatic hydrocarbons 49
2.8.2 Catabolic
pathways of PAHs degradation :-(Naphthalene) 49
2.8.3 Naphthalene 51
CHAPTER 3: MATERIALS AND
METHODS 57
3.1 Experimental Layout 57
3.2 Study Area 58
3.3 Sample
Collection 58
3.4 Isolation and Identification of Pseudomonas spp from the Petrochemical
Contaminated
Soil 59
3.4.1 Morphological and colony characterization of
bacterial isolates 59
3.4.2 Biochemical
characterization of the bacterial isolates 59
3.4.3
Molecular characterization of
the bacterial isolates 63
3.5 Determination of Physicochemical
Properties of Petrochemical
Contaminated
Soil 65
3.5.1 The
pH and electrical conductivity 66
3.5.2
Moisture content (%)
66
3.5.3 The
determination of soil temperature 66
3.5.4 Bulk density of the Soil 67
3.5.5 Porosity (%) 67
3.5.6 Soil texture 67
3.5.7 Water holding capacity (WHC) 68
3.5.8 Organic carbon (%) 68
3.5.9 Total nitrogen (mg/kg) 69
3.5.10 Total phosphorus (mg/kg) 69
3.5.11 Exchangeable
acids 70
3.5.12 Potassium (mg/kg) 70
3.5.13 Calcium and magnesium (mg/kg) 71
3.5.14 Effective cation exchange capacity
(ECEC)
71
3.6 Assessment
of the Conditions Optimum for/Factors Affecting Catabolic 72
Activity
of Pseudomonas spp by Monitoring the
Soil Temperature,
Soil
pH, Soil Moisture and Nutrient Availability
3.7 Determination of Bacterial Count of Pseudomonas Species 73
3.8 Analysis
of Total Petroleum Hydrocarbons (TPH) by Gravimetry 73
3.9 Assessment of Catabolic Activity (rate of
biodegradation) by Monitoring
the
Metabolic Compounds; Saturated Hydrocarbons Oil, Aromatic
Hydrocarbons, Asphaltenes and Polar
Compounds, Phenolics 74
3.9.1 Extraction
of residual saturated
hydrocarbons
75
3.9.2 Determination of aromatic
hydrocarbons 75
3.9.3 Determination of asphaltenes and
polar compounds 76
3.9.4 Determination of phenolic compounds 76
3.9 Statistical Analysis of the Results 77
CHAPTER 4: RESULTS AND
DISCUSSION 78
4.0. Results and Discussion 78
4.1 Isolation and
identification of Pseudomonas
species
78
4.1.1 Cultural
characterization of bacterial isolates and biochemical test
results
of the indigenous Pseudomonas
isolates. 78
4.1.2 Molecular
identification of the Pseudomonas
isolates
82
4.2 Determination
of Physicochemical Properties of Petrochemical
Contaminated
Soil. 88
4.3 Optimization
of Growth Conditions for Pseudomonas
Isolates Used in
Remediation 94
4.4 Determination
of Bacterial Counts of Pseudomonas spp
99
4.5
Analysis of Total Petroleum
Hydrocarbons (TPH) by Gravimetry 103
4.6
Assessment of Catabolic
Activity (Rate of Biodegradation) by Monitoring
the Metabolic
Compounds; Residual Oils, Aromatic
Hydrocarbons, Phenolic
Compounds, and Asphaltenes and Polar Compounds. 109
CHAPTER
5: CONCLUSION AND RECOMMENDATIONS 119
5.1 Conclusion 119
5.2 Recommendations 119
References
Appendices
LIST OF TABLES
4.1: Cultural characterization of bacterial
isolates
79
4.2: The
morphological and biochemical test results of the indigenous
Pseudomonas
isolates.
80
4.3: Physicochemical properties of contaminated
soil sample and control soil 90
4.4: Optimization
of growth conditions for Pseudomonas
isolates (x
107) 97
4.5: Bacterial
count of Pseudomonas and their consortium isolates (x 107) 101
4.6: Percentage remediation level of petrochemical polluted soil by each
Pseudomonas
species and their
consortium.
105
4.7: Remediation rate per week of petrochemical polluted soil by each
Pseudomonas spp and their consortium
106
4.10: Catabolic activities of aromatics by each Pseudomonas
sp and their
consortium 113
4.11: Catabolic activity of saturated hydrocarbon by each Pseudomonas spp
and
their consortium
114
4.12: Catabolic activities of phenolics by each Pseudomonas
spp and their
consortium
115
4.13. Catabolic
activities of asphaltene and polar by each Pseudomonas spp and
their consortium.
116
LIST OF FIGURES
2.1 Structure of naphthalene
18
2.2 Structure of phenanthrene
19
2.3 Structure
of anthracene 20
2.4 Structure of fluorene
20
2.5 Structure of pyrene
21
2.6 Structure
of fluoranthene
structure of benzo(a) anthracene 21
2.7 Structure of benzo(a) anthracene
22
2.8 Bacterial
community composition
22
2.9 The proposed pathway for naphthalene biodegradation by Pseudomonas 46
2.10 Ortho-cleavage of catechol in the TCA
(tricarboxylic acid) cycle 52
2.11 Meta-cleavage of catechol in the TCA
(tricarboxylic acid) cycle
53
2.12 The structure of Pseudomonas putida NAH7 plamid 54
4.1 Molecular
identification of the Pseudomonas
aeruginosa 84
4.2 Molecular
identification of the Pseudomonas
mendocina
85
4.3 Molecular
identification of the Pseudomonas
putida
86
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND OF STUDY
Petrochemical
hydrocarbons are one of the resident threats for entire ecosystem. These are
considered as the most significant environmental pollutants. Besides petroleum,
other sources of petrochemicals could be fossil fuels such as coal or natural
gas, or renewable sources, e.g., corn or sugar cane (Lee et al., 2014 and Soccol et al., 2011). The enormous use of
petroleum products such as engine oil, due to rapid expansions in different
types of automobiles and machinery, is the major cause of used engine oil
contamination (Mandri and Lin, 2007). The spillages of used motor oils such as
diesel oil or jet fuel are also the major sources of hydrocarbons contamination
that adversely affect the natural habitats (Husaini
et al., 2008). Likewise, the
illegal dumping of used engine oil is also an environmental hazard with global
implications (Blodgett, 1997).
In
general, the petrochemicals belong to the group of polyaromatic hydrocarbons
(PAHs) consisting of two or more benzene rings
fused in a linear, angular, or cluster arrangement. PAHs are characterized by
their high hydrophobicity, and resistance to natural degradation and
carcinogenic properties. PAH releases to soils and other wider environment have
led to higher concentrations of these contaminants that would not be expected
from natural processes alone. They are known soil and aquatic contaminants (Piskonen
and Itävaara, 2004). Polyaromatic hydrocarbons (PAHs) are highly
toxic and may have mutagenic and carcinogenic effects (Rubio-Clemente et al.,
2014; Cerniglia et al., 1980; Lee et al., 1992 and Boonchan et al., 2000).
The prolonged exposure of petrochemicals may cause severe effects and numerous
health problems including liver or kidney diseases and possible damage to the
bone marrow (Mishra et al., 2001; Hadibarata and Tachibana, 2009, and Lloyd and Cackette, 2001).
The
microorganisms are ubiquitous in environment playing key role in the
detoxification of such petroleum hydrocarbons while using them as sole source
of carbon and energy. Due to the complex nature of hydrocarbon contaminants,
the soil microorganisms have evolved complex metabolic pathways (Medina-Bellver et
al., 2005). The discovery of such metabolic pathways of the
on-degrading bacteria is supposed to be very essential for the eradication of
environmental hazards of oil spills as well as to tackle the factors associated
with in situ microbial catalyses (Kostka et
al., 2014). Bacteria are the
most active agents in petroleum biodegradation and there is evidence of their
fundamental role as primary degraders of spilled oil (Head et al., 2006; Da Cruz et al.,
2011 and Oliveira et al., 2012).
Several factors, both physico-chemical and biological, affect the rate of
microbial degradation of hydrocarbons in soil. Recently, growing interest in
the use of several Pseudomonads
during degradation of crude oil have been reported (Toledo et al., 2006; Song et al.,
2006; Ueno et al., 2006; Das and
Mukherjee, 2007, and Mittal and Singh, 2009). The fuel eating bacteria known as
Pseudomonas sp. have
evolved a taste for hydrocarbons are the major components of fossil fuels.
1.2 STATEMENT OF THE PROBLEM
Reports have indicated that worldwide industrial and
agricultural developments have released a large number of petrochemical
contaminants into the environment and accidental spill which can cause damage
to aquatic flora, soil ecosystem, human health and natural resources (Wilson
and Jones, 1993). Over the years, numerous studies have adopted a costly and
non-ecofriendly techniques, and also described the application of microbial
consortia for hydrocarbons degradation throughout the world (Rahman et al., 2002; Plaza et al., 2008; Sathishkumar et al., 2008; Bao et al., 2012). But studies on
degradation of petroleum hydrocarbons by employing indigenous bacterial
consortia from this petro-chemically important geographical region are very
limited (Das and Mukherjee, 2007). With relation to that, this study focuses on
the approach to elevate the level of petroleum hydrocarbons degradation using a
legitimate native Pseudomonas spp
consortium
1.3 JUSTIFICATION OF STUDY
Bioremediation of complex hydrocarbons mixture usually
necessitates the cooperation of more than a single species, because an
individual microorganism can generally metabolize only a limited range of
hydrocarbon substrates. Therefore, conglomerations of mixed populations
equipped with broad enzymatic capacities are entailed to increase the rate and
extent of petroleum biodegradation further (Calvo et al., 2009; Joutey et
al., 2013).
Application of an indigenous consortium of Pseudomonas
spp
by adopting gravimetric analysis will enhance the rate of petrochemical degradation.
Kaustuvmani et al., (2016) investigated the development of an efficient bacterial
consortium for the potential remediation of hydrocarbons from contaminated
sites where consortium comprising two Bacillus strains
namely, Bacillus pumilus KS2 and B. cereusR2
showed degradation up to 84.15% of TPH after 5 weeks of incubation, as revealed
from gravimetric analysis.
1.4 AIM AND OBJECTIVES
The overall aim of this study is to monitor the degradation potentials of the consortium of
indigenous Pseudomonas spp on petroleum hydrocarbons
and their metabolic compounds.
1.5 THE MAIN
OBJECTIVES OF THE STUDY
The main objectives of the study are:
·
Isolation and
identification of Pseudomonas spp
biochemically and molecularly from the petrochemical contaminated soil.
·
Determination of
physicochemical properties of petrochemical contaminated soil.
·
Assessment of the
conditions optimum for/factors affecting catabolic activity of Pseudomonas spp by monitoring the soil
temperature, soil pH, soil moisture and nutrient availability.
·
Determination of bacterial growth.
·
Assessment of catabolic
activity by monitoring the metabolic compounds; saturated hydrocarbons, aromatic
hydrocarbons, asphaltene and polar compounds,
phenolics using each Pseudomonas sp
and their consortium.
·
Analysis of Total
Petroleum Hydrocarbons (TPH) by gravimetry.
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